Antibiotic resistance breakers
11746116 · 2023-09-05
Assignee
Inventors
- Khondaker Mirazur Rahman (London, GB)
- Shirin Jamshidi (London, GB)
- Mark Benjamin Laws (London, GB)
- Kazi Nahar (London, GB)
- John Mark Sutton (Salisbury, GB)
- Charlotte Hind (Salisbury, GB)
Cpc classification
A61K45/06
HUMAN NECESSITIES
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
International classification
A61K45/06
HUMAN NECESSITIES
Abstract
The invention relates to antibiotic compounds of formula (A1) and pharmaceutically acceptable salts, solvates, tautomers and combinations thereof, wherein X and L are optional linkers and one of R.sub.A or R.sub.1 comprises Ar.sub.1, wherein Ar.sub.1 is an antibiotic resistance breaker moiety which comprises an optionally substituted C.sub.6-10 aryl, C.sub.7-13 aralkyl, C.sub.5-10 heteroaryl, C.sub.6-13 heteroaralkyl, C.sub.5-10 heterocyclyl, C.sub.6-13 heterocyclalkyl, C.sub.3-10 carbocyclyl, C.sub.4-13 carbocyclalkyl, —C(═NR′)—NR′R″ or —CH.sub.2—CH═CH.sub.2 group; wherein after administration of the compound to a bacterial infection this moiety reduces or prevents efflux. The invention also discloses pharmaceutical compositions comprising compounds of formula (A1) and the use of such compounds as medicaments, in particular, to treat bacterial infections, such as drug-resistant bacterial infections.
Claims
1. An antibiotic compound of formula (A1): ##STR00340## or a pharmaceutically acceptable salt, solvate, tautomer, or combination thereof, wherein: X.sub.1 is C—R.sub.B, and R.sub.A and R.sub.B together with the atoms to which they are attached form a 6-membered ring wherein from R.sub.A to R.sub.B is a —CH(CH.sub.3)—CH.sub.2—O— linking group; R* is H or NH.sub.2; either Z.sub.1 is CH and X* is —NR′—; or Z.sub.1 is N and X* is absent; L.sub.1 is —NH— or —N(CH.sub.3)—; n is 1; Z is N or C—H; R′.sub.1 is H, C.sub.1-6 alkyl or —(CH.sub.2).sub.t—C(═O)—OR′; R.sub.1 is Ar.sub.1; Ar.sub.1 is a six-membered ring; and the Ar.sub.1 group may be optionally substituted with 1, 2 or 3 substituents selected from the group consisting of —C.sub.1-6 alkyl, -halo, —(CH.sub.2).sub.t—OR′, —(CH.sub.2).sub.r C(═O)—OR′, oxo, —(CH.sub.2).sub.t—NR′R″, —NO.sub.2, —NR′-(cyclopropyl), -(cyclopropyl), —NR′—(CH.sub.2).sub.t—NR′R″, —C(═NR′)—NR′R″, —(CH.sub.2).sub.t—NR′—C(═NR′)—NR′R″, —CH.sub.2—CH═CH.sub.2, —CH═CH—(C.sub.1-6 alkyl), —CH═CH—CN, —SO.sub.2—NR′R″ and —SO.sub.2NR′—(CH.sub.2).sub.t—Ar.sub.2; each t is independently 0, 1, 2, 3, 4 or 5; each Ar.sub.2 is independently C.sub.5-9 heteroaryl; and each R′ and R″ is independently H or C.sub.1-6 alkyl.
2. The compound of claim 1, wherein the compound is a compound of formula (A2); ##STR00341## or a pharmaceutically acceptable salt, solvate, tautomer, or combination thereof.
3. The compound of claim 1, wherein Ar.sub.1 is a ring structure selected from the group consisting of cyclohexyl; morpholinyl; phenyl; piperazinyl; piperidinyl; pyrazinyl; pyridinyl; pyrimidinyl; thiomorpholinyl; and triazinyl.
4. The compound of claim 1, wherein Ar.sub.1 is a ring structure selected from the group consisting of morpholinyl; piperazinyl; pyrazinyl; pyridinyl; pyrimidinyl; thiomorpholinyl; and triazinyl; wherein the ring structure is optionally substituted with 1, 2 or 3 substituents selected from the group consisting of —C.sub.1-6 alkyl, -halo, —(CH.sub.2).sub.t—OR′, —(CH.sub.2).sub.t—C(═O)—OR′, oxo, —(CH.sub.2).sub.t—NR′R″, —NO.sub.2, —NR′-(cyclopropyl), -(cyclopropyl), —NR′—(CH.sub.2).sub.t—NR′R″, —C(═NR′)—NR′R″, —(CH.sub.2).sub.t—NR′—C(═NR′)—NR′R″, —CH.sub.2—CH═CH.sub.2, —CH═CH—(C.sub.1-6 alkyl), —CH═CH—CN, —SO.sub.2—NR′R″ and —SO.sub.2NR′—(CH.sub.2).sub.t—Ar.sub.2.
5. The compound of claim 1, wherein the structure: ##STR00342## is: ##STR00343## or ##STR00344##
6. The compound of claim 1, wherein the compound is selected from the group consisting of: ##STR00345## ##STR00346## ##STR00347## ##STR00348## ##STR00349## or a pharmaceutically acceptable salt, solvate, tautomer, or combination thereof.
7. A pharmaceutical composition comprising a compound of claim 1 or a pharmaceutically acceptable salt, solvate, tautomer, or combination thereof and a pharmaceutically acceptable carrier or diluent.
8. The pharmaceutical composition of claim 7, further comprising an agent for increasing the permeability of a bacterial membrane.
9. The pharmaceutical composition of claim 7, wherein the compound is selected from the group consisting of: ##STR00350## ##STR00351## ##STR00352## ##STR00353## ##STR00354## or a pharmaceutically acceptable salt, solvate, tautomer, or combination thereof.
10. A kit comprising: (i) a compound of claim 1, or a pharmaceutically acceptable salt, solvate, tautomer, or combination thereof, and at least one of: an agent for increasing permeability of a bacterial membrane; and an efflux pump inhibitor.
11. The kit of claim 10, wherein the compound is selected from the group consisting of: ##STR00355## ##STR00356## ##STR00357## ##STR00358## ##STR00359## or a pharmaceutically acceptable salt, solvate, tautomer, or combination thereof.
12. A method of treating a bacterial infection in a patient, the method comprising a step of administering to the patient a therapeutically effective amount of a compound of claim 1 or a pharmaceutically acceptable salt, solvate, tautomer, or combination thereof.
13. The method of claim 12, wherein the bacterial infection is caused by at least one bacterium of a genera selected from the group consisting of Acinetobacter, Bacillus, Brucella, Burkholderia, Campylobacter, Coxiella, Enterococcus, Enterobacter, Escherichia, Francisella, Klebsiella, Neisseria, Pseudomonas, Staphylococcus, Streptococcus and Yersina.
14. The method of claim 12, wherein the bacterial infection is a caused by at least one bacterium selected from the group consisting of Campylobacter jejuni, Neisseria gonorrhoea, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter cloacae and Escherichia coli; or at least one bacterium selected from Bacillus anthracis, Burkholderia mallei, Burkholderia pseudomallei, Brucella melitensis, Coxiella bumettii, Francisella tularensis, Proteus mirabilis and Yersinia pestis.
15. The method of claim 12, wherein the bacterial infection is anthrax, bronchitis, pneumonia, prostatitis, pyelonephritis, sinusitis, skin and skin structure infections, sexually transmitted disease or urinary tract infections.
16. The method of claim 12, wherein the bacterial infection is a multi-drug resistant bacterial infection.
17. The method of claim 12, wherein the compound is selected from the group consisting of: ##STR00360## ##STR00361## ##STR00362## ##STR00363## ##STR00364## or a pharmaceutically acceptable salt, solvate, tautomer, or combination thereof.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) Embodiments of the present invention will now be described further, with reference to the accompanying drawings, in which:
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EXPERIMENTAL
(38) Methods and Materials
(39) Reagent Sources
(40) Synthetic building blocks and reagents were purchased from a number of suppliers including Sigma-Aldrich (Merck KGaA, USA), Thermo Fisher Scientific (UK, including Acros Organics, Maybridge and Alfa Aesar), Fluorochem (USA), Insight Biotechnology (UK), Activate Scientific (UK), Enamine (Ukraine), VWR International (USA), Oxchem (USA), Apollo
(41) Scientific (UK), Combi-Blocks (USA) and Ark Pharm Inc (USA). Solvents were purchased from Sigma-Aldrich and Thermo Fisher Scientific. SCX-2 solid phase extraction cartridges were purchased from Biotage (Sweden).
(42) Microwave Reactions
(43) Microwave reactions were performed in a Biotage Initiator+Microwave Synthesiser fitted with a pressurised air supply for cooling. Vessels were stirred at 600 RPM and cooled to below 40° C. before finishing a reaction.
(44) Bacterial Strains
(45) The bacterial strains used in the biological tests were obtained from type culture collections, in particular, ATCC, the National Collection of Culture Types (NCTC) and the Belgium Coordinated Collection of Microorganisms. In some cases the strains have been previously described 25-27).
(46) Thin Layer Chromatography (TLC)
(47) Thin-layer chromatography (TLC) analysis was performed using silica gel plates (Merck silica gel 60 F.sub.254 plates) and visualised using ultraviolet (UV) light (254 nm wavelength) and/or staining with potassium permanganate solution.
(48) Flash Column Chromatography
(49) Manual flash column chromatography was performed using silica gel (Merck 9385, 230-400 mesh ASTM, 40-63 μM) as the stationary phase. TLC was employed to discern solvent systems (mobile phases) with appropriate separation profiles and were comprised of hexanes, ethyl acetate, dichloromethane and methanol. Purification of tertiary amine-containing compounds was expedited through pre-neutralisation of the stationary phase via the addition of 3% triethylamine to the initial non-polar solvent wash, i.e. in the non-polar mobile phase component prior to running the column.
(50) Automated flash column chromatography is an air pressure-driven hybrid of medium pressure and short column chromatography, optimized for rapid separations on the basis of UV and ELSD detection. It was performed using a Reveleris® X2 Flash Chromatography System. Normal phase separations were carried out on Grace™ Reveleris™ Silica Flash Cartridges. Reverse phase separations were carried out on Biotage® SNAP Ultra C18 Cartridges. This technique was employed for the separation of particularly difficult mixtures of compounds with similar Rf values. This technique was employed for the separation of particularly difficult mixtures of compounds, such as tertiary amine-containing compounds, with similar Rf values.
(51) Mass-Directed Reverse-Phase High Performance Liquid Chromatography (HPLC)
(52) Mass-Directed Reverse-Phase High Performance Liquid Chromatography (HPLC) was performed on an Agilent 1290 Infinity II Preparative LC/MSD System fitted with the following subunits; 1290 MS Flow Modulator, 1290 Prep Fraction Collector, 1290 Prep Column Compartment, 1290 Prep Bin Pump, 1260 Prep Autosampler, 1260 DAD WR, 1260 Quat Pump and InfinityLab LC/MSD. The column used was a Phenomenex Luna® 5 μm C18(2) 100 Å LC column, 100×21.2 mm. Mobile phases were water (A) and acetonitrile (B); formic acid (0.1%) was added through a separate channel to ensure acidic conditions throughout the purification method. The following methods were employed for compound purification;
(53) Method 1 (10 min): Flow rate 20 mL/min. i) 95% A/5% B for one minute; ii) from 95% A/5% B to 70% A/30% B over a further minute; iii) from 70% A/30% B to 50% A/50% B over 3.5 minutes; iv) from 50% A/50% B to 10% A/90% B over 1.5 minutes; v) from 10% A/90% B to 80% A/20% B over 30 seconds; vi) held constant at 80% A/20% B for a further minute.
Purification Via Recrystallisation
(54) Purification of compounds via recrystallisation was achieved by dissolving the crude compound in the minimum volume of a hot solvent of choice, then covering the container and leaving it to cool to room temperature gradually until crystal formation was observed. Any insoluble contaminants or byproducts were removed by employing a hot filtration step prior to cooling. Slow crystallisations were further cooled to −20° C. from room temperature to expedite crystal formation.
(55) Liquid Chromatography-Mass Spectrometry (LC-MS)
(56) Liquid chromatography-mass spectrometry (LC-MS) was employed to monitor reaction progression and compound identification. All LC-MS analysis was performed on a Waters Alliance 2695 with water (A) and acetonitrile (B) comprising the mobile phases. Formic acid (0.1%) was added to both acetonitrile and water to ensure acidic conditions throughout the analysis. Function type: Diode array (535 scans). Column type: Monolithic C18 50×4.60 mm. Mass spectrometry data (both ESI+ and ESI− modes) were collected using a Waters Micromass ZQ instrument coupled to a Waters 2695 HPLC with a Waters 2996 PDA. Waters Micromass ZQ parameters used were: Capillary (kV), 3.38; Cone (V), 35; Extractor (V), 3.0; Source temperature (° C.), 100; De-solvation Temperature (° C.), 200; Cone flow rate (L/h), 50; De-solvation flow rate (L/h), 250. LC-MS gradient conditions are described as follows;
(57) Method A (5 min): (i) from 95% A/5% B to 50% A/50% B over 3 min. (ii) Then from 50% A/50% B to 20% A/80% B over 2 min. (iii) Then from 20% A/80% B to 5% A/95% B over 1.5 min and (iv) held constant at 5% A/95% B for 1.5 min. (v) This was then reduced from 5% A/95% B to 95% A/5% B over 0.2 min and (iv) maintained to 95% A/5% B for 1.8 min. The flow rate was 0.5 mL/min, 200 μL was split via a zero dead volume T piece which passed into the mass spectrometer. The wavelength range of the UV detector was 220-400 nm.
(58) Method B (5 min): (i) from 95% A/5% B to 10% A/90% B over 3 min. (ii) Then from 10% A/90% B to 5% A/95% B over 0.5 min and (ii) held constant at 5% A/95% B for 1 min. (iv) This was then reduced from 5% A/95% B to 95% A/5% B over 0.5 min. The flow rate was 1.0 mL/min, 100 μL was split via a zero dead volume T piece which passed into the mass spectrometer. The wavelength range of the UV detector was 220-500 nm.
(59) High Resolution Mass Spectrometry (HRMS)
(60) High resolution mass spectra (HRMS) were obtained on a Thermo Navigator mass spectrometer coupled with liquid chromatography (LC) using electrospray ionisation (ES) and time-of-flight (TOF) mass spectrometry.
(61) Infrared Spectroscopy (IR)
(62) Infrared spectra (IR) were recorded on a Perkin Elmer spectrum 1000 instrument. Compounds were analysed in solid form.
(63) Nuclear Magnetic Resonance Spectroscopy (NMR)
(64) All NMR spectra were obtained at room temperature using a Bruker DPX400 spectrometer. Chemical shifts (δ H) are expressed in parts per million (ppm) relative to deuterated chloroform (CDCl.sub.3 or CHLOROFORM-d, residual signal .sup.1H δ=7.26, .sup.13C δ=77.2) or deuterated dimethyl sulfoxide (DMSO-d.sub.6, residual signal .sup.1H δ=2.54, .sup.13C δ=40.45) or deuterated methanol (METHANOL-d.sub.4, residual signal .sup.1H δ=3.31, .sup.13C δ=49.0). Coupling constants are expressed in Hz. Multiplicities in .sup.1H NMR spectra are quoted as s=singlet, d=doublet, t=triplet q=quartet, m=multiplet, dd=doublet of doublets, ddd=doublet of doublet of doublets, dt=doublet of triplets, td=triplet of doublets, spt=septet and br=broad. The code (o) in .sup.13C NMR spectra denotes the presence of a quaternary carbon.
(65) Solid Phase Extraction
(66) SCX-2 resin cartridges, purchased from Biotage (Uppsala, Sweden), contain propylsulfonic acid-functionalised silica, a strong cation exchange sorbent primarily used for basic drug extraction. Cartridges (1 g, 2 g, 10 g) were selected based on reaction scale; sorbent mass should be 10 times that of the calculated mass of the product. Cartridges were first activated by an initial wash with 2 column volumes of dichloromethane and 4 column volumes of methanol. The reaction mixtures were then poured onto the cartridge and the solvent allowed to pass through the cartridges under gravity. The cartridges were then washed with dichloromethane (3 times), dimethylformamide (3 times) and methanol (1 time) and this cycle was repeated three times under vacuum to remove impurities. Products were eluted using 2M ammonia solution in methanol and concentrated in vacuo.
(67) Purification Via Trituration
(68) Trituration is the process of purifying a compound from a mixture based on the different solubility profiles of the mixture's constituents. A solvent (either polar or non-polar) was selected in which the desired product was poorly soluble and the unwanted by-products were highly soluble. The crude material was suspended in the solvent, filtered and washed again, leaving the purified product in solid form and any impurities in solution.
(69) Lyophilisation
(70) Lyophilisation (freeze-drying, cryodesiccation) was carried out on a Frozen in Time Lablyo bench top freeze drier connected to an Edwards RV vacuum pump. Samples, dissolved in water or mixtures of water and acetonitrile, were frozen solid in a dewar of dry ice before being connected to the lyophiliser unit
(71) Organic Synthesis
Synthesis of Levofloxacin Based Fluoroquinolone Core for ARB Fragment Attachment
Synthesis of ethyl (R,Z)-3-((1-hydroxypropan-2-yl)amino)-2-(2,3,4,5-tetrafluorobenzoyl) acrylate (A1.2)
(72) Ethyl 2,3,4,5-tetrafluorobenzoylacetate (A1.1; 5 g, 18.9 mmol, 1 eq) was dissolved in triethyl orthoformate (6.30 mL, 37.9 mmol, 2 eq) and heated at 140° C. for 30 minutes. Acetic anhydride (5.37 mL, 56.8 mmol, 3 eq) was then added and the mixture refluxed at 140° C. for another 40 hours and monitored by TLC (10% ethyl acetate/90% hexanes). Upon completion, the reaction was cooled to room temperature, dichloromethane (15 mL) was added and the mixture stirred at room temperature for 5 minutes. Then L-alaninol (3.01 mL, 37.9 mmol, 2 eq) was added and the reaction was stirred for 48 hours at room temperature. Then a further 2 eq. of L-alaninol was added, and the reaction stirred for a further 48 hours. The crude was concentrated in vacuo and purified by flash column chromatography (1:1 ethyl acetate/hexanes rising to 3:1 ethyl acetate/hexanes) to give A1.2 (5.768 g, 87.3% yield) as a yellow oil.
(73) ##STR00096##
(74) .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 10.93 (br. s., 0.75H, H5), 9.58 (br. s., 0.25H, H5), 8.21 (d, J=14.21 Hz, 1H, H1), 7.10 (br. s., 0.25H, H4), 6.98 (br. s., 0.75H, H4), 3.93-4.19 (m, 2H, H2), 3.73-3.85 (m, 1H, H6), 3.56-3.72 (m, 2H, H8), 2.44 (br. s., 1H, H9), 1.31-1.42 (m, 3H, H7), 1.10 (t, J=7.06 Hz, 2.25H, H3), 0.98 (t, J=6.24 Hz, 0.75H, H3); .sup.13C NMR (100 MHz, CHLOROFORM-d) δ 186.9, 185.1, 168.3, 166.6, 159.9, 159.4, 148.4, 145.6, 145.5, 142.9, 141.4, 138.8, 127.2, 127.1, 110.7, 110.5, 109.9, 109.7, 101.0, 66.1, 60.0, 59.7, 57.9, 57.4, 17.0, 14.0, 13.6; LC-MS (Method B) Retention time 3.35 minutes, purity=100%, Found 350.1 [M+H].sup.+; calculated for C.sub.15H.sub.15F.sub.4NO.sub.4 350.29 [M+H].sup.+; R.sub.f 0.81 (100% ethyl acetate).
Synthesis of ethyl (S)-9,10-difluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylate (A1.3)
(75) Compound A1.2 (5.933 g, 16.99 mmol, 1 eq) was dissolved in dimethyl acetamide (40 mL) and stirred for 5 mins to dissolve. The solution was then divided evenly into two 20 mL capacity microwave vessels fitted with magnetic stirrer bars and potassium carbonate (7.043 g, 50.96 mmol, 3 eq.) was divided evenly and added to the two vessels. Each microwave vessel was then, in turn, microwaved at 160° C. for 20 minutes. Upon cooling, the contents of each vessel was added to dichloromethane (200 mL) and washed with distilled water (300 mL). The organic layers were combined and washed two further times with distilled water (2×100 mL). The organics were dried over Na.sub.2SO.sub.4, decanted and concentrated in vacuo to afford crude A1.3 (4.651 g, 88.5% yield) as an off white solid. This material was used in subsequent reactions without further purification.
(76) ##STR00097##
(77) .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 8.36 (s, 1H, H4), 7.76 (dd, J=8.07, 10.18 Hz, 1H, H1), 4.40-4.52 (m, 3H, H5+6), 4.36 (q, J=7.00 Hz, 2H, H2), 1.60 (d, J=6.51 Hz, 3H, H7), 1.39 (t, J=7.02 Hz, 3H, H3); .sup.13C NMR (100 MHz, DMSO-d.sub.6) δ 171.1, 164.2, 149.7, 149.4, 149.3, 149.1, 146.6, 135.4, 135.2, 124.4, 124.4, 123.7, 123.7, 109.8, 103.8, 103.6, 68.8, 59.9, 53.8, 17.6, 14.3; 19F NMR (400 MHz, CHLOROFORM-d) δ −136.4 (d, J=21.46 Hz, 1F), −151.3 (d, J=21.45 Hz, 1F); LC-MS (Method B) Retention time 2.85 minutes, purity=75%, Found 310.1 [M+H].sup.+; calculated for C.sub.15H.sub.13F.sub.2NO.sub.4 310.28 [M+H].sup.+; [α].sup.25.3D, −43° (c=0.117, CH.sub.2Cl.sub.2)
Synthesis of (S)-9,10-difluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (A1.4)
(78) To compound A1.3 (2.018 g, 6.53 mmol, 1 eq) was added ethanol (20 mL) and a 15% w/v aqueous solution of sodium hydroxide (20 mL) and the suspension stirred at room temperature for one hour. The mixture was then acidified to pH 3 using a 1M hydrochloric acid and a few drops of 37% hydrochloric acid, vacuum filtered and washed with distilled water (3×100 mL). Powder was collected and dried for 1 hour more on a Schlenk line to afford the crude A1.4 (1.599 g, 87.2% yield) as an off white solid. This material was used in subsequent reactions without further purification.
(79) ##STR00098##
(80) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 14.80 (s, 1H, H2), 9.09 (s, 1H, H3), 7.80 (dd, J=7.79, 10.36 Hz, 1H, H1), 5.02 (q, J=6.48 Hz, 1H, H5), 4.64-4.73 (m, 1H, H4), 4.44-4.55 (m, 1H, H4), 1.47 (d, J=6.79 Hz, 3H, H6); .sup.13C NMR (100 MHz, DMSO-d.sub.6) δ 176.4, 176.4, 165.6, 150.2, 150.1, 147.7, 147.6, 147.1, 143.0, 142.8, 140.5, 140.3, 136.0, 135.9, 135.9, 135.8, 125.3, 125.3, 121.4, 121.4, 121.3, 121.3, 107.7, 103.6, 103.4, 68.9, 55.0, 17.8; .sup.19F NMR (400 MHz, DMSO-d.sub.6) δ−135.9 (d, J=22.47 Hz, 1F), −151.0 (d, J=22.48 Hz, 1F); LC-MS (Method B) Retention time 3.11 minutes, purity=81%, Found 282.1 [M+H].sup.+; calculated for C.sub.13H.sub.9F.sub.2NO.sub.4 282.22 [M+H].sup.+; R.sub.f 0.30, streaks (100% acetone); [α].sup.25.9D, −20° (c=0.088, CH.sub.2Cl.sub.2)
(81) Compound 1.4 was used in the synthesis of all of the ARB-linked fluoroquinolones.
Synthesis of (S)-9,10-difluoro-3-methyl-8-nitro-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (A1.5)
(82) Compound A1.4 (818.9 mg, 2.91 mmol, 1 eq) was dissolved in 96% sulphuric acid (5 mL) and cooled to 0° C. over 10 minutes. Then potassium nitrate (589 mg, 5.82 mmol, 2 eq) was added in small portions with stirring over 15 minutes. The reaction was subsequently allowed to warm to room temperature over 15 more minutes. The reaction was quenched over 200 mL of ice/water slurry, then extracted with dichloromethane (3×30 mL washes). Organic layers were combined, dried over MgSO.sub.4, filtered and concentrated in vacuo to afford the crude A1.5 (760.7 mg, 80.1% yield) as a pink solid. This material was used in subsequent reactions without further purification.
(83) ##STR00099##
(84) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 13.80 (br. s., 1H, H1), 9.16 (s, 1H, H2), 5.09 (q, J=6.66 Hz, 1H, H4), 4.77 (dd, J=1.33, 11.42 Hz, 1H, H3), 4.52 (dd, J=2.06, 11.42 Hz, 1H, H3), 1.47 (d, J=6.79 Hz, 3H, H5); .sup.13C NMR (100 MHz, DMSO-d.sub.6) δ 173.8, 173.8, 164.6, 148.1, 143.3, 143.2, 142.1, 142.0, 140.8, 140.7, 139.6, 139.4, 137.8, 137.7, 137.6, 137.6, 128.9, 128.8, 125.2, 125.1, 113.1, 113.1, 109.2, 69.1, 55.4, 17.8; .sup.19F NMR (400 MHz, DMSO-d.sub.6) δ −148.1 (d, J=23.16 Hz, 1F), −149.7 (d, J=22.82 Hz, 1F); LC-MS (Method B) Retention time 3.27 minutes, purity=97%, Found 326.9 [M+H].sup.+; calculated for C.sub.13H.sub.8F.sub.2N.sub.2O.sub.6 327.22 [M+H].sup.+
Synthesis of (S)-8-amino-9,10-difluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (A1.6)
(85) Compound A1.5 (711.6 mg, 2.18 mmol, 1 eq) was dissolved in N,N-dimethylformamide (12 mL) and added to a hydrogenation vessel. Palladium on carbon (233 mg, 0.22 mmol, 0.1 eq) was subsequently added to the vessel in N,N-dimethylformamide (1 mL). The mixture was then hydrogenated at 30 psi for 2 hours, whereupon the pressure stabilized, indicating reaction completion. The mixture was vacuum filtered through two successive celite plugs, washing each time with dichloromethane (3×20 mL per plug). The combined filtrate was concentrated in vacuo to afford crude A1.6 (250.9 mg, 38.8% yield) as a yellow solid. This material was used in subsequent reactions without further purification.
(86) ##STR00100##
(87) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 14.70 (br. s., 1H, H2), 8.89 (s, 1H, H3), 7.26 (br. s., 2H, H1), 4.79-4.97 (m, 1H, H5), 4.48 (d, J=11.19 Hz, 1H, H4), 4.22 (d, J=11.37 Hz, 1H, H4), 1.41 (d, J=6.60 Hz, 3H, H6); .sup.13C NMR (100 MHz, DMSO-d.sub.6) δ 180.4, 180.3, 165.6, 147.2, 144.2, 144.1, 141.7, 141.6, 136.3, 136.1, 134.2, 134.2, 134.1, 134.1, 133.9, 133.8, 124.5, 124.5, 124.5, 124.5, 122.7, 122.6, 122.5, 107.0, 107.0, 106.9, 106.3, 67.4, 55.6, 17.8; .sup.19F NMR (400 MHz, DMSO-d.sub.6) δ −149.8 (d, J=21.80 Hz, 1F), −162.8 (d, J=21.80 Hz, 1F); LC-MS (Method B) Retention time 3.08 minutes, purity=97%, Found 297.0 [M+H].sup.+; calculated for C.sub.13H.sub.10F.sub.2N.sub.2O.sub.4 297.24 [M+H].sup.+.
Synthesis of (S)-9-fluoro-3-methyl-7-oxo-10-(4-(pyrimidin-4-yl)piperazin-1-yl)-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (A1.7)
(88) Compound A1.4 (1.37 g, 4.87 mmol, 1 eq) was dissolved in N,N-dimethylformamide (20 mL) with 4-(piperazin-1-yl)pyrimidine (1.00 g, 6.09 mmol, 1.25 eq) and stirred at 140° C. for 90 hours. Upon cooling, the mixture was added to 50 mL dichloromethane and washed with 100 mL brine (back extracted with 50 mL more dichloromethane) and 100 mL distilled water. Organic layers were combined, dried over MgSO.sub.4, filtered and concentrated in vacuo to yield the crude product. Purification was achieved via flash column chromatography (100% dichloromethane to 100% acetonitrile to 1% water/acetonitrile; product subsequently eluted with 2M ammonia in methanol). Pure fractions were concentrated in vacuo, re-dissolved in dichloromethane, filtered and concentrated again to afford A1.7 (234.4 mg, 18.1% yield) as an orange solid.
(89) ##STR00101##
(90) .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 14.92 (br. s., 1H, H2), 8.66 (s, 1H, H3), 8.62 (s, 1H, H13), 8.24 (d, J=6.29 Hz, 1H, H11), 7.71 (d, J=12.09 Hz, 1H, H1), 6.56 (d, J=6.29 Hz, 1H, H12), 4.53-4.64 (m, 1H, H5), 4.49 (d, J=11.33 Hz, 1H, H4), 4.41 (d, J=11.33 Hz, 1H, H4), 3.75-3.89 (m, 4H, H7+8), 3.36-3.50 (m, 4H, H9+10), 1.63 (d, J=6.80 Hz, 3H, H6); .sup.19F NMR (400 MHz, CHLOROFORM-d) δ −119.1 (s, 1F); LC-MS (Method B) Retention time 2.70 minutes, purity=100%, Found 426.0 [M+H].sup.+; calculated for C.sub.21H.sub.20FN.sub.5O.sub.4 426.43 [M+H].sup.+; R.sub.f 0.31 (5% methanol in dichloromethane)
Synthesis of (S)-9-fluoro-10-(4-(2-isopropyl-6-methylpyrimidin-4-yl)piperazin-1-yl)-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (A1.8)
(91) (S)-9,10-difluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinolone-6-carboxylic acid (A1.4; 128 mg, 0.45 mmol, 1 eq) and 2-isopropyl-4-methyl-6-(piperazin-1-yl)pyrimidine (100 mg, 0.45 mmol, 1 eq) were added to DMF (3 mL) and stirred at 140° C. for 1.5 hours. The mixture was allowed to cool and the crude concentrated in vacuo, then re-suspended in 3:1 distilled water:MeOH (20 mL) and filtered hot. Purification was achieved via automated flash column chromatography of the crude solid (see Flash Column Chromatography; 0%-50%-100% DCM/Acetone) to afford A1.8 (22.0 mg, 10.0%) as a light brown solid.
(92) ##STR00102##
(93) .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 14.92 (br. s., 1H, H2), 8.64 (s, 1H, H3), 7.76 (d, J=12.09 Hz, 1H, H1), 6.26 (s, 1H, H11), 4.44-4.58 (m, 2H, H4+5), 4.36-4.43 (m, 1H, H4), 3.85 (m, 4H, H7+8), 3.45 (td, J=5.07, 9.76 Hz, 4H, H9+10), 3.04-3.13 (m, 1H, H14), 2.41 (s, 3H, H12), 1.64 (d, J=6.55 Hz, 3H, H6), 1.29 (d, J=7.05 Hz, 6H, H13+15); .sup.19F NMR (400 MHz, CHLOROFORM-d) δ −119.2 (s, 1F); LC-MS (Method B) Retention time 30.07 minutes, purity=100%, Found 482.0 [M+H].sup.+; calculated for C.sub.25H.sub.28FN.sub.5O.sub.4 482.53 [M+H].sup.+
Synthesis of (S)-9-fluoro-3-methyl-7-oxo-10-(4-(pyrazin-2-yl)piperazin-1-yl)-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (A1.9)
(94) Compound A1.4 (100.4 mg, 0.36 mmol, 1 eq) was dissolved in dimethyl sulfoxide (3 mL) with 2-(piperazin-1-yl)pyrazine (175.9 mg, 1.07 mmol, 3 eq) and stirred at 140° C. for 48 hours. Upon cooling, the mixture concentrated in vacuo before being purified via automated flash column chromatography (see Flash Column Chromatography; 5%-95% acetonitrile in water) to afford A1.9 as an orange solid.
(95) ##STR00103##
(96) .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 14.92 (br. s., 1H, H2), 8.64 (s, 1H, H3), 8.21 (d, J=1.10 Hz, 1H, H11), 8.07-8.14 (m, 1H, H12), 7.90 (d, J=2.57 Hz, 1H, H13), 7.76 (d, J=12.10 Hz, 1H, H1), 4.44-4.58 (m, 2H, H4+5), 4.36-4.44 (m, 1H, H4), 3.69-3.84 (m, 4H, H7+8), 3.41-3.58 (m, 4H, H9+10), 1.64 (d, J=6.60 Hz, 3H, H6); .sup.19F NMR (400 MHz, CHLOROFORM-d) δ −119.10 (s, 1F); LC-MS (Method B) Retention time 30.23 minutes, purity=95%, Found 426.1 [M+H].sup.+; calculated for C.sub.21H.sub.20FN.sub.5O.sub.4 426.43 [M+H].sup.+
Synthesis of (S)-9-fluoro-3-methyl-7-oxo-10-(4-(pyrimidin-2-yl)piperazin-1-yl)-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (A1.10)
(97) Compound A1.4 (100 mg, 0.36 mmol, 1 eq) was dissolved in dimethyl sulfoxide (2 mL) with 2-(piperazin-1-yl)pyrimidine (146 mg, 0.89 mmol, 2.5 eq) in a 5 mL capacity microwave vessel fitted with a magnetic stirrer bar and microwaved at 200° C. for 20 minutes. Upon cooling, the mixture was filtered through a Mini-UniPrep™ polypropylene filter (0.45 μm pore size). Recrystallisation occurred upon leaving overnight; crystals were vacuum filtered and further concentrated in vacuo to afford A1.10 (37.2 mg, 24.6% yield) as an orange solid.
(98) ##STR00104##
(99) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 15.11 (br. s., 1H, H2), 8.97 (s, 1H, H3), 8.39 (d, J=4.68 Hz, 2H, H11+13), 7.58 (d, J=12.20 Hz, 1H, H1), 6.65 (t, J=4.72 Hz, 1H, H12), 4.93 (q, J=6.63 Hz, 1H, H5), 4.56-4.63 (m, 1H, H4), 4.35-4.44 (m, 1H, H4), 3.81-3.95 (m, 4H, H7+8), 3.33-3.41 (m, 4H, H9+10), 1.46 (d, J=6.69 Hz, 3H, H6); .sup.19F NMR (400 MHz, DMSO-d.sub.6) δ −120.70 (s, 1F);); LC-MS (Method A) Retention time 7.30 minutes, purity=100%, Found 426.1 [M+H].sup.+; calculated for C.sub.21H.sub.20FN.sub.6O.sub.4 426.43 [M+H].sup.+; LC-MS (Method B) Retention time 3.34 minutes, purity=100%, Found 426.1 [M+H].sup.+; calculated for C.sub.21H.sub.20FN.sub.5O.sub.4 426.43 [M+H].sup.+
Synthesis of (S)-9-fluoro-10-(4-(5-fluoropyrimidin-2-yl)piperazin-1-yl) methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (A1.11)
(100) Compound A1.4 (71 mg, 0.25 mmol, 1 eq) was dissolved in dimethyl sulfoxide (2 mL) with 5-fluoro-2-(piperazin-1-yl)pyrimidine (100 mg, 0.50 mmol, 2 eq) in a 5 mL capacity microwave vessel fitted with a magnetic stirrer bar and microwaved at 160° C. for 1.5 hours. Upon cooling, methanol (10 mL) was added and the mixture filtered. The crude solid was recrystallized from hot methanol to afford A1.11 (69.6 mg, 62.13% yield) as an orange solid.
(101) ##STR00105##
(102) 1H NMR (400 MHz, DMSO-d.sub.6) δ 8.98 (s, 1H, H3), 8.49 (s, 2H, H11+12), 7.62 (d, J=12.10 Hz, 1H, H1), 4.89-4.98 (m, 1H, H5), 4.60 (d, J=11.37 Hz, 1H, H4), 4.39 (d, J=11.19 Hz, 1H, H4), 3.84 (t, J=5.23 Hz, 2H, H7+8), 3.73-3.80 (m, 2H, H7+8), 3.34-3.41 (m, 2H, H9+10), 2.95-3.02 (m, 2H, H9-1-10), 1.46 (d, J=6.60 Hz, 3H, H6); LC-MS (Method B) Retention time 3.57 minutes, purity=98%, Found 444.1 [M+H].sup.+; calculated for C.sub.21H.sub.19F.sub.2N.sub.5O.sub.4 444.42 [M+H].sup.+
Synthesis of (S)-10-(4-(4,6-dimethylpyrimidin-2-yl)piperazin-1-yl)-9-fluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (A1.12)
(103) Compound A1.4 (71 mg, 0.25 mmol, 1 eq) was dissolved in dimethyl sulfoxide (2 mL) with 4,6-dimethyl-2-(piperazin-1-yl)pyrimidine (91.8 mg, 0.50 mmol, 2 eq) in a 5 mL capacity microwave vessel fitted with a magnetic stirrer bar and microwaved at 160° C. for 1.5 hours. Upon cooling, methanol (10 mL) was added and the mixture filtered. The crude solid was recrystallized from hot methanol to afford A1.12 as a yellow solid,
(104) ##STR00106##
(105) 1H NMR (400 MHz, DMSO-d.sub.6) δ 14.99 (br. s., 1H, H2), 8.97 (s, 1H, H3), 7.61 (d, J=12.01 Hz, 1H, H1), 6.44 (s, 1H, H12), 4.93 (q, J=7.00 Hz, 1H, H5), 4.60 (d, J=11.55 Hz, 1H, H4), 4.39 (d, J=12.20 Hz, 1H, H4), 3.83-3.90 (m, 4H, H7+8), 3.29-3.36 (m, 4H, H9+10), 2.25 (s, 6H, H11+13), 1.46 (d, J=6.60 Hz, 3H, H6); LC-MS (Method B) Retention time 3.34 minutes, purity=84%, Found 454.1 [M+H].sup.+; calculated for C.sub.23H.sub.24FN.sub.5O.sub.4 454.48 [M+H].sup.+
Synthesis of ARB-Antibiotics with ARB Fragment Containing 5-Membered Pyrrolidine with Exocyclic Nitrogen Linked to a Pyrimidine Ring or Substituted Pyrimidine Rings
Synthesis of (3S)-9-fluoro-3-methyl-7-oxo-10-(3-(pyrimidin-2-ylamino)pyrrolidin-1-yl)-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (A1.13, KSN-82-L7)
(106) Compound A1.4 (80 mg, 0.284 mmol, 1 eq) was dissolved in dimethyl sulfoxide (2 mL) with N-(pyrrolidin-3-yl)pyrimidin-2-amine (93.43 mg, 0.568 mmol, 2 eq) in a 5 mL capacity microwave vessel fitted with a magnetic stirrer bar and microwaved at 160° C. for 1.5 hours. The DMSO was evaporated and the compound was crystalized using hot ethanol to afford compound A1.13.
(107) ##STR00107##
(108) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.89 (br. s., 1H), 8.30 (d, J=4.65 Hz, 2H), 7.54 (d, J=14.43 Hz, 1H), 7.45 (d, J=5.62 Hz, 1H), 6.61 (t, J=4.52 Hz, 1H), 4.86 (br. s., 1H), 4.52 (d, J=11.25 Hz, 1H), 4.44-4.40 (m, 1H), 4.28 (d, J=11.25 Hz, 1H), 4.00 (br. s., 1H), 4.04-3.87 (m, 2H), 3.82-3.60 (m, 2H), 2.19-1.94 (m, 2H), 1.45 (d, J=6.36 Hz, 3H); Formula C.sub.21H.sub.20FN.sub.5O.sub.4;
(109) LC-MC Retention time 2.946 min, Found 426.1 [M−H].sup.+
Synthesis of (S)-9-fluoro-3-methyl-7-oxo-10-((S)-3-(pyrimidin ylamino)pyrrolidin-1-yl)-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline carboxylic acid (A1.14/KSN-82-L19)
(110) Compound A1.4 (50 mg, 0.177 mmol, 1 eq) was dissolved in dimethyl sulfoxide (2 mL) with (S)—N-(pyrrolidin-3-yl)pyrimidin-2-amine (58.39 mg, 0.355 mmol, 2 eq) in a 5 mL capacity microwaveA vessel fitted with a magnetic stirrer bar and microwaved at 160° C. for 1.5 hours. The DMSO was evaporated the compound was crystalized using hot ethanol to afford compound A1.14.
(111) ##STR00108##
(112) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.89 (s, 1H), 8.33 (d, J=4.65 Hz, 2H), 7.54 (d, J=14.43 Hz, 1H), 6.65-6.63 (m, 1H), 4.87 (d, J=6.36 Hz, 1H), 4.52 (d, J=10.76 Hz, 1H), 4.43 (br. s., 1H), 4.28 (d, J=10.03 Hz, 1H), 4.04-3.90 (m, 2H), 3.77-3.63 (m, 3H), 2.20-1.96 (m, 2H), 1.45 (d, J=6.60 Hz, 3H); Formula C.sub.21H.sub.2OFN.sub.5O; LC-MC Retention time 2.945 min, Found 426.1 [M+H].sup.+
Synthesis of (S)-10-((S)-3-((6-(cyclopropylamino)pyrimidin-4-yl)amino)pyrrolidin-1-yl)-9-fluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (KSN-L21/A1.15)
(113) Compound A1.4 (50 mg, 0.177 mmol, 1 eq) was dissolved in dimethyl sulfoxide (2 mL) (S)—N4-cyclopropyl-N6-(pyrrolidin-3-yl)pyrimidine-4,6-diamine (77.98 mg, 0.355 mmol, 2 eq) in a 5 mL capacity microwave vessel fitted with a magnetic stirrer bar and microwaved at 160° C. for 1.5 hours. The DMSO was evaporated the compound was crystalized using hot ethanol to afford compound A1.15.
(114) ##STR00109##
(115) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 15.42 (br. s., 1H), 8.89 (s, 1H), 7.92 (s, 1H), 7.54 (d, J=13.94 Hz, 1H), 7.03 (d, J=4.65 Hz, 1H), 6.83 (br. s., 1H), 5.64 (s, 1H), 4.88 (d, J=7.09 Hz, 1H), 4.53 (d, J=10.76 Hz, 1H), 4.41 (br. s., 1H), 4.28 (d, J=10.03 Hz, 1H), 4.03-3.90 (m, 2H), 3.77-3.55 (m, 2H), 2.38 (br. s., 1H), 2.17-1.90 (m, 2H), 1.45 (d, J=6.60 Hz, 3H), 0.66 (d, J=4.65 Hz, 2H), 0.43 (br. s., 2H); Formula C.sub.24H.sub.25FN.sub.6O.sub.4; LC-MC Retention time 2.453 min, Found 481.2 [M+H].sup.+
Synthesis of (S)-9-fluoro-3-methyl-7-oxo-10-aR)-3-(pyrimidin ylamino)pyrrolidin-1-yl)-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (KSN-82-L22/A1.16)
(116) Compound A1.4 (50 mg, 0.177 mmol, 1 eq) was dissolved in dimethyl sulfoxide (2 mL) with (R)—N-(pyrrolidin-3-yl)pyrimidin-2-amine (58.39 mg, 0.355 mmol, 2 eq) in a 5 mL capacity microwave vessel fitted with a magnetic stirrer bar and microwaved at 160° C. for 1.5 hours. The DMSO was evaporated the compound was crystalized using hot ethanol to afford compound A1.16.
(117) ##STR00110##
(118) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 15.41 (br. s., 1H), 8.89 (s, 1H), 8.30 (d, J=4.58 Hz, 2H), 7.54 (d, J=14.12 Hz, 1H), 7.47 (d, J=6.24 Hz, 1H), 6.61 (t, J=4.77 Hz, 1H), 4.87 (d, J=6.42 Hz, 1H), 4.52 (d, J=11.55 Hz, 1H), 4.44-4.40 (m, 1H), 4.28 (d, J=10.82 Hz, 1H), 4.0-3.86 (m, 2H), 3.84-3.61 (m, 2H), 2.19-1.96 (m, 2H), 1.45 (d, J=6.60 Hz, 3H); Formula C.sub.21H.sub.2OFN.sub.5O.sub.4; LC-MC Retention time 2.932 min, Found 426.2 [M+H].sup.+
Synthesis of (3S)-9-fluoro-3-methyl-10-(3-(4-methylpiperazin-1-yl)pyrrolidin-1-yl)-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (KSN-82-L31/A1.17)
(119) Compound A1.4 (50 mg, 0.177 mmol, 1 eq) was dissolved in dimethyl sulfoxide (2 mL) 1-methyl-4-(pyrrolidin-3-yl)piperazine (60.19 mg, 0.355 mmol, 2 eq) in a 5 mL capacity microwave vessel fitted with a magnetic stirrer bar and microwaved at 160° C. for 1.5 hours. The DMSO was evaporated the compound was crystalized using hot ethanol to afford compound A1.17.
(120) ##STR00111##
(121) .sup.1H NMR (400 MHz, Chloroform-d) δ 15.04 (br. s., 1H), 8.58 (s, 1H), 7.67 (d, J=4.58 Hz, 2H), 4.49 (dd, J=2.29, 6.69 Hz, 1H), 4.37-4.45 (m, 1H), 4.26-4.35 (m, 1H), 4.07-3.95 (m, 1H), 3.89-3.67 (m, 8H), 2.94-2.88 (m, 1H), 2.58-2.67 (m, 4H), 2.22-2.16 (m, 1H), 1.92-1.77 (m, 2H), 1.62 (d, J=6.60 Hz, 3H); Formula C.sub.22H.sub.27FN.sub.4O.sub.4; LC-MC Retention time 2.031 min, Found 431.1 [M−H].sup.+
Synthesis of (3S)-9-fluoro-3-methyl-10-(3-morpholinopyrrolidin-1-yl)-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (KSN-82-L33/A1.18)
(122) Compound 1.4 (50 mg, 0.177 mmol, 1 eq) was dissolved in dimethyl sulfoxide (2 mL) with 4-(pyrrolidin-3-yl)morpholine (55.56 mg, 0.355 mmol, 2 eq) in a 5 mL capacity microwave vessel fitted with a magnetic stirrer bar and microwaved at 160° C. for 1.5 hours. The DMSO was evaporated the compound was crystalized using hot ethanol to afford compound A1.18.
(123) ##STR00112##
(124) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.90 (s, 1H), 7.54 (d, J=14.12 Hz, 1H), 4.87 (t, J=6.88 Hz, 1H), 4.57-4.51 (m, 1H), 4.32-4.24 (m, 1H), 3.92-3.84 (m, 1H), 3.69 (dd, J=2.66, 7.61 Hz, 2H), 3.67-3.55 (m, 5H), 2.86-2.76 (m, 1H), 2.49-2.35 (m, 4H), 2.16-2.07 (m, 1H), 1.78-1.67 (m, 1H), 1.45 (dd, J=4.86, 6.51 Hz, 3H); Formula C.sub.21H.sub.24FN.sub.3O.sub.5; LC-MC Retention time 2.032 min, Found 418.2 [M−H].sup.+
Synthesis of (S)-9-fluoro-10-((R)-3-((5-fluoropyrimidin-2-yl)amino)pyrrolidin-1-yl)-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (KSN-82-L34/A1.19)
(125) Compound A1.4 (48 mg, 0.170 mmol, 1 eq) was dissolved in dimethyl sulfoxide (2 mL) with (R)-5-fluoro-N-(pyrrolidin-3-yl)pyrimidin-2-amine (62.20 mg, 0.341 mmol, 2 eq) in a 5 mL capacity microwave vessel fitted with a magnetic stirrer bar and microwaved at 160° C. for 1.5 hours. The DMSO was evaporated the compound was crystalized using hot ethanol to afford compound A1.19.
(126) ##STR00113##
(127) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 15.42 (s, 1H), 8.89 (s, 1H), 8.40 (d, J=0.92 Hz, 2H), 7.57 (d, J=6.24 Hz, 1H), 7.54 (d, J=14.12 Hz, 1H), 4.90-4.84 (m, 1H), 4.52 (dd, J=1.47, 11.37 Hz, 1H), 4.40-4.32 (m, 1H), 4.30-4.26 (m, 1H), 4.00-3.86 (m, 2H), 3.82-3.62 (m, 2H), 2.20-1.93 (m, 2H), 1.45 (d, J=6.79 Hz, 3H); Formula C.sub.21H.sub.19F.sub.2N.sub.5O.sub.4; LC-MC Retention time 3.332 min, Found 444.1 [M−H].sup.+
Synthesis of (S)-10-((R)-3-(cyclopropyl(methyl)amino)pyrrolidin-1-yl)-9-fluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (KSN-82-L36/A1.20)
(128) Compound A1.4 (48 mg, 0.170 mmol, 1 eq) was dissolved in dimethyl sulfoxide (2 mL) with Cyclopropyl-methyl-(R)-pyrrolidin-3-yl-amine (47.87 mg, 0.341 mmol, 2 eq) in a 5 mL capacity microwave vessel fitted with a magnetic stirrer bar and microwaved at 160° C. for 1.5 hours. The DMSO was evaporated the compound was crystalized using hot ethanol to afford compound A1.20.
(129) ##STR00114##
(130) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.90 (s, 1H), 7.54 (d, J=14.12 Hz, 1H), 4.91-4.85 (m, 1H), 4.57-4.49 (m, 1H), 4.29-4.26 (m, 1H), 3.93-3.85 (m, 1H), 3.76-3.68 (m, 1H), 3.63 (t, J=8.80 Hz, 1H), 3.15-3.04 (m, 1H), 2.49 (s, 3H), 2.14-2.12 (In, 1H), 1.76-1.88 (m, 1H), 1.84-1.78 (m, 1H), 1.74-1.70 (m, 1H), 1.42-1.48 (m, 3H), 0.29-0.53 (m, 4H); Formula C.sub.21H.sub.24FN.sub.3O.sub.4; LC-MC Retention time 2.123 min, Found 402.2 [M−H].sup.+
Synthesis of (S)-9-fluoro-3-methyl-7-oxo-10-((R)-3-(3-(pyridin-2-yl)ureido)pyrrolidin-1-yl)-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline carboxylic acid (KSN-82-L37/A1.21)
(131) Compound A1.4 (50 mg, 0.177 mmol, 1 eq) was dissolved in dimethyl sulfoxide (2 mL) (R) (pyridin-2-yl)-3-(pyrrolidin-3-yl)urea (73.34 mg, 0.355 mmol, 2 eq) in a 5 mL capacity microwave vessel fitted with a magnetic stirrer bar and microwaved at 160° C. for 1.5 hours. The DMSO was evaporated the compound was crystalized using hot ethanol to afford compound A1.21.
(132) ##STR00115##
(133) Formula C.sub.23H.sub.22FN.sub.5O.sub.5; LC-MC Retention time 2.23 min, Found 468.3 [M+H].sup.+
Synthesis of (S)-8-amino-9-fluoro-3-methyl-7-oxo-10-((R)-3-(pyrimidin-2-ylamino)pyrrolidin-1-yl)-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (KSN-82-L40/A 1.22)
(134) Compound A1.6 (34 mg, 0.114 mmol, 1 eq) was dissolved in dimethyl sulfoxide (2 mL) with (S)—N-(pyrrolidin-3-yl)pyrimidin-2-amine (37.70 mg, 0.229 mmol, 2 eq) in a 5 mL capacity microwave vessel fitted with a magnetic stirrer bar and microwaved at 160° C. for 1.5 hours. The DMSO was evaporated the compound was crystalized using hot ethanol to afford compound A1.22.
(135) ##STR00116##
(136) Formula C.sub.21H.sub.21FN.sub.6O.sub.4; LC-MC Retention time 2.47 min, Found 441.36 [M+H].sup.+
Synthesis of (S)-10-((R)-3-aminopyrrolidin-1-yl)-9-fluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (KSN-82-L44-D/A1.23)
(137) Compound A1.4 (44 mg, 0.156 mmol, 1 eq) was dissolved in dimethyl sulfoxide (2 mL) with (3S)-(−)-3-(tert-Butoxycarbonylamino)pyrrolidine (58.28 mg, 0.312 mmol, 2 eq) in a 5 mL capacity microwave vessel fitted with a magnetic stirrer bar and microwaved at 100° C. for 1 hour. The DMSO was evaporated and the compound was crystalized. The crystalized product was dissolved in methanol and 2M HCl in dioxane (2 mL) was added. Reaction was left at room temperature for 3 hours so that the boc protected group was removed. Solvent was evaporated and compound 1.22 was isolated as a free base to afford compound A1.23 (KSN-82-L44-D) was isolated as a free base.
(138) ##STR00117##
(139) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.89 (s, 1H), 7.54 (d, J=14.12 Hz, 1H), 4.91-4.84 (m, 1H), 4.53 (d, J=9.72 Hz, 1H), 4.28 (d, J=9.35 Hz, 1H), 4.05 (d, J=5.87 Hz, 1H), 3.92-3.76 (m, 2H), 3.76-3.64 (m, 1H), 3.53-3.46 (m, 1H), 2.09-1.98 (m, 1H), 1.89-1.74 (m, 1H), 1.45 (d, J=6.79 Hz, 3H); Formula C.sub.17H.sub.18FN.sub.3O.sub.4; LC-MC Retention time 1.916 min, Found 348.1 [M+H].sup.+
Synthesis of (S)-10-(((R)-1-(5-aminopyrimidin-2-yl)pyrrolidin-3-yl)amino)-9-fluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (KSN-82-L46/A1.24)
(140) (S)-9-fluoro-3-methyl-7-oxo-10-(((R)-pyrrolidin-3-yl)amino)-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (39 mg, 0.112 mmol, 1 eq) was dissolved in dimethyl sulfoxide (2 mL) 2-Chloropyrimidin-5-amine (43.64 mg, 0.336 mmol, 3 eq) in a 5 mL capacity microwave vessel fitted with a magnetic stirrer bar and microwaved at 160° C. for 1.5 hours. The DMSO was evaporated the compound was crystalized using hot ethanol to afford compound A1.24.
(141) ##STR00118##
(142) Formula C.sub.21H.sub.21FN.sub.6O.sub.4; LC-MC Retention time 2.12 min, Found 441.27 [M+H].sup.+
Synthesis of (S)-9-fluoro-3-methyl-10-((R)-3-((5-nitropyrimidin-2-yl)amino)pyrrolidin-1-yl)-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (KSN-82-L52/A1.25)
(143) Compound A1.4 (190 mg, 0.675 mmol, 1 eq) was dissolved in dimethyl sulfoxide (2 mL) with (R)-5-nitro-N-(pyrrolidin-3-yl)pyrimidin-2-amine (282.70 mg, 1.35 mmol, 2 eq) in a 5 mL capacity microwave vessel fitted with a magnetic stirrer bar and microwaved at 160° C. for 1.5 hours. The DMSO was evaporated the compound was crystalized using hot ethanol to afford compound A1.25.
(144) ##STR00119##
(145) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.15 (d, J=3.30 Hz, 1H), 9.08 (d, J=3.30 Hz, 1H), 8.83 (s, 1H), 7.51 (d, J=14.49 Hz, 1H), 4.86-4.78 (m, 1H), 4.61-4.54 (m, 1H), 4.51 (d, J=9.90 Hz, 1H), 4.27 (d, J=9.17 Hz, 1H), 4.04-3.85 (m, 2H), 3.78-3.61 (m, 2H), 2.35-2.32 (m, 1H), 2.25-2.20 (m, 1H), 2.09-2.03 (m, 1H), 1.43 (d, J=6.60 Hz, 3H); Formula C.sub.21H.sub.19FN.sub.6O.sub.6; LC-MC Retention time 3.365 min, Found 471.1 [M−H].sup.+
Synthesis of (S)-9-fluoro-3-methyl-10-((S)-3-((5-nitropyrimidin-2-yl)amino)pyrrolidin-1-yl)-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (KSN-82-L56/A1.26)
(146) Compound A1.4 (40 mg, 0.142 mmol, 1 eq) was dissolved in dimethyl sulfoxide (2 mL) with (S)-5-nitro-N-(pyrrolidin-3-yl)pyrimidin-2-amine (59.52 mg, 0.284 mmol, 2 eq) in a 5 mL capacity microwave vessel fitted with a magnetic stirrer bar and microwaved at 160° C. for 1.5 hours. The DMSO was evaporated the compound was crystalized using hot ethanol to afford compound A1.26.
(147) ##STR00120##
(148) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ15.39 (br. s., 1H), 9.14 (d, J=3.30 Hz, 1H), 9.08 (d, J=3.30 Hz, 1H), 8.90 (s, 1H), 7.54 (d, J=14.12 Hz, 1H), 4.90-4.85 (m, 1H), 4.62-4.48 (m, 2H), 4.32-4.24 (m, 1H), 4.05-3.89 (m, 2H), 3.82-3.68 (m, 2H), 2.27-2.19 (m, 1H), 2.08-2.01 (m, 1H), 1.45 (d, J=6.60 Hz, 3H); Formula C.sub.21H.sub.19FN.sub.6O.sub.6; LC-MC Retention time 3.372 min, Found 471.1 [M-1-1]+
Synthesis of (3S)-9-fluoro-3-methyl-10-(3-((5-nitropyrimidin yl)amino)pyrrolidin-1-yl)-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (KSN-82-L57/A1.27)
(149) (3S)-10-(3-aminopyrrolidin-1-yl)-9-fluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (40 mg, 0.115 mmol, 1 eq) was dissolved in dimethyl sulfoxide (2 mL) with 2-Chloro-5-nitropyrimidine (36.74 mg, 0.230 mmol, 2 eq) in a 5 mL capacity microwave vessel fitted with a magnetic stirrer bar and microwaved at 160° C. for 1.5 hours. The DMSO was evaporated the compound was crystalized using hot ethanol to afford compound A1.27.
(150) ##STR00121##
(151) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 15.38 (br. s., 1H), 9.15 (d, J=3.30 Hz, 1H), 9.08 (d, J=3.30 Hz, 1H), 8.90 (s, 1H), 7.56 (d, J=14.12 Hz, 1H), 4.90-4.85 (m, 1H), 4.60-4.56 (m, 1H), 4.53 (d, J=11.74 Hz, 1H), 4.28 (d, J=11.19 Hz, 1H), 4.05-3.90 (m, 2H), 3.75-3.83-3.68 (m, 2H), 2.22 (td, J=6.53, 12.79 Hz, 1H), 2.05 (dd, J=6.51, 12.56 Hz, 1H), 1.45 (d, J=6.79 Hz, 3H); Formula C.sub.21H.sub.19FN.sub.6O.sub.6; LC-MC Retention time 3.362 min, Found 471.1 [M+H].sup.+
Synthesis of (S)-10-((S)-3-((5-aminopyrimidin-2-yl)amino)pyrrolidin-1-yl)-9-fluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (KSN-82-L62/A1.28)
(152) Compound A1.4 (50 mg, 0.177 mmol, 1 eq) was dissolved in dimethyl sulfoxide (2 mL) with (S)—N2-(pyrrolidin-3-yl)pyrimidine-2,5-diamine (47.80 mg, 0.266 mmol, 2 eq) in a 5 mL capacity microwave vessel fitted with a magnetic stirrer bar and microwaved at 160° C. for 1.5 hours. The DMSO was evaporated the compound was crystalized using hot ethanol to afford compound A1.28.
(153) ##STR00122##
(154) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.88 (s, 1H), 70.83 (s, 2H), 7.54 (d, J=14.12 Hz, 1H), 6.51 (d, J=6.42 Hz, 1H), 4.82-4.91 (m, 1H), 4.52 (d, J=9.90 Hz, 1H), 4.45 (s, 2H), 4.24-4.33 (m, 1H), 3.96-3.93 (m, 1H), 3.74-3.79-3.86 (m, 2H), 3.62-3.57 (m, 1H), 2.35 (s, 1H), 2.13 (td, J=6.19, 12.20 Hz, 1H), 1.88-1.98 (m, 1H), 1.45 (d, J=6.79 Hz, 3H); Formula C.sub.21H.sub.21FN.sub.6O.sub.4; LC-MC Retention time 2.357 min, Found 441.1 [M+H].sup.+
Synthesis of (S)-10-((R)-3-((5-aminopyrimidin-2-yl)amino)pyrrolidin-1-yl) fluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline carboxylic acid (KSN-82-L65/A1.29)
(155) Compound A1.4 (40 mg, 0.142 mmol, 1 eq) was dissolved in dimethyl sulfoxide (2 mL) with (R)—N2-(pyrrolidin-3-yl)pyrimidine-2,5-diamine (38.24 mg, 0.213 mmol, 1.5 eq) in a 5 mL capacity microwave vessel fitted with a magnetic stirrer bar and microwaved at 160° C. for 1.5 hours. The DMSO was evaporated the compound was crystalized using hot ethanol to afford compound A1.29.
(156) ##STR00123##
(157) Formula C.sub.21H.sub.21FN.sub.6O.sub.4; LC-MC Retention time 2.37 min, Found 441.1 [M+H].sup.+
Synthesis of (S)-9-fluoro-3-methyl-7-oxo-10-((R)-3-(pyrazin-2-ylamino)pyrrolidin-1-yl)-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (BL-1/A1.30)
(158) Compound A1.4 (107.1 mg, 0.381 mmol, 1 eq) was dissolved in dimethyl sulfoxide (2 mL) with (R)-4-(pyrrolidin-3-yl)pyrimidine (126 mg, 0.767 mmol, 2 eq) in a 5 mL capacity microwave vessel fitted with a magnetic stirrer bar and microwaved at 160° C. for 1.5 hours. The DMSO was evaporated the compound was crystalized using hot ethanol to afford compound A1.30.
(159) ##STR00124##
(160) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.89 (s, 1H), 7.96 (s, 1H), 7.69 (d, J=2.38 Hz, 1H), 7.54 (d, J=14.12 Hz, 1H), 7.38 (d, J=6.24 Hz, 1H), 4.89-4.84 (m, 1H), 4.56-4.48 (m, 1H), 4.45-4.35 (m, 1H), 4.32-4.24 (m, 1H), 4.07-3.99 (m, 1H), 3.95-3.75 (m, 3H), 3.65-3.57 (m, 1H), 2.27-2.16 (m, 1H), 1.98-1.87 (m, 1H), 1.45 (d, J=6.79 Hz, 3H); Formula C.sub.21H.sub.20FN.sub.5O.sub.4; LC-MC Retention time 30.002 min, 426 Found [M−H].sup.+
Synthesis of (S)-10-((3R,4S)-3-carboxy-4-(pyridin-4-yl)pyrrolidin-1-yl) fluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (BL-4/A1.31)
(161) Compound A1.4 (50 mg, 0.177 mmol, 1 eq) was dissolved in dimethyl sulfoxide (2 mL) (3R,4S)-4-(pyridin-3-yl)pyrrolidine-3-carboxylic acid (68.43 mg, 0.356 mmol, 2 eq) in a 5 mL capacity microwave vessel fitted with a magnetic stirrer bar and microwaved at 160° C. for 1.5 hours. The DMSO was evaporated the compound was crystalized using hot ethanol to afford compound A1.31.
(162) ##STR00125##
(163) Formula C.sub.23H.sub.20FN.sub.3O.sub.6; LC-MC Retention time 2.148 min, Found 451.9 [M−H].sup.+
Synthesis of (S)-9-fluoro-3-methyl-7-oxo-10-((R)-3-(quinazolin-4-yl)amino)pyrrolidin-1-yl)-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (BL-5/A1.32)
(164) Compound A1.4 (50 mg, 0.177 mmol, 1 eq) was dissolved in dimethyl sulfoxide (2 mL) (R)—N-(pyrrolidin-3-yl)quinazolin-4-amine (76.3 mg, 0.356 mmol, 2 eq) in a 5 mL capacity microwave vessel fitted with a magnetic stirrer bar and microwaved at 160° C. for 1.5 hours. The DMSO was evaporated the compound was crystalized using hot ethanol to afford compound A1.32.
(165) ##STR00126##
(166) Formula C.sub.25H.sub.22FN.sub.5O.sub.4; LC-MC Retention time 2.439 min, Found 476 [M+H].sup.+
Synthesis of (S)-9-fluoro-3-methyl-10-((R)-3-((6-methylpyrimidin yl)amino)pyrrolidin-1-yl)-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (BL-6/A1.33)
(167) Compound A1.4 (50 mg, 0.177 mmol, 1 eq) was dissolved in dimethyl sulfoxide (2 mL) (R)-6-methyl-N-(pyrrolidin-3-yl)pyrimidin-4-amine (63.45 mg, 0.356 mmol, 2 eq) in a 5 mL capacity microwave vessel fitted with a magnetic stirrer bar and microwaved at 160° C. for 1.5 hours. The DMSO was evaporated the compound was crystalized using hot ethanol to afford compound A1.33.
(168) ##STR00127##
(169) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.90 (s, 1H), 8.33 (s, 1H), 7.57-7.50 (m, 2H), 6.36 (br. s., 1H), 4.88 (q, J=6.91 Hz, 1H), 4.54-4.47 (m, 2H), 4.32-4.25 (m, 1H), 4.04-3.97 (m, 1H), 3.91-3.76 (m, 2H), 3.59 (dd, J=2.57, 5.50 Hz, 1H), 2.24-2.15 (m, 4H), 1.91 (dd, J=6.05, 11.92 Hz, 1H), 1.45 (d, J=6.60 Hz, 3H); Formula C.sub.22H.sub.22FN.sub.5O.sub.4; LC-MC Retention time 2.262 min, Found 440.1 [M+H].sup.+
Synthesis of (S)-10-((R)-3-((4,6-dimethylpyrimidin-2-yl)amino)pyrrolidin-1-yl)-9-fluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (BL-7/A1.34)
(170) Compound A1.4 (50 mg, 0.177 mmol, 1 eq) was dissolved in dimethyl sulfoxide (2 mL) (R)-4,6-dimethyl-2-(pyrrolidin-3-yl)pyrimidine (68.5 mg, 0.356 mmol, 2 eq) in a 5 mL capacity microwave vessel fitted with a magnetic stirrer bar and microwaved at 160° C. for 1.5 hours. The DMSO was evaporated the compound was crystalized using hot ethanol to afford compound A1.34.
(171) ##STR00128##
(172) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 15.43 (s, 1H), 8.89 (s, 1H), 7.54 (d, J=14.12 Hz, 1H), 7.25 (d, J=6.60 Hz, 1H), 6.39 (s, 1H), 4.87 (d, J=6.97 Hz, 1H), 4.52 (d, J=10.27 Hz, 1H), 4.49-4.41 (m, 1H), 4.28 (d, J=9.72 Hz, 1H), 3.99-3.92 (m, 1H), 3.87-3.80 (m, 2H), 3.64-3.56 (m, 1H), 2.23-2.18 (m, 6H), 2.18-2.09 (m, 1H), 2.02-1.90 (m, 1H), 1.45 (d, J=6.60 Hz, 3H); Formula C.sub.23H.sub.24FN.sub.5O.sub.4; LC-MC Retention time 2.686 min, Found 454.1 [M+H].sup.+
Synthesis of Parent Fluoroquinolones
Synthesis of ethyl (Z)-3-(cyclopropylamino)-2-(2,4,5-trifluorobenzoyl)-acrylate (1.2)
(173) Ethyl 2,4,5-trifluorobenzoylacetate (1.1; 1 g, 4.06 mmol, 1 eq) was dissolved in triethyl orthoformate (1.15 mL, 6.90 mmol, 1.7 eq) and heated at 140° C. for 30 minutes. Ac.sub.2O (1.15 mL, 12.19 mmol, 3 eq) was added and the mixture refluxed at 140° C. for another 3 hours and monitored by TLC (90% Hex/10% EtOAc). Upon completion, the reaction was cooled to room temperature, DCM (3 mL) was added and the mixture was stirred at room temperature for 10 minutes. Then cyclopropylamine (703 μL, 10.15 mmol, 2.5 eq) was added and the reaction was stirred at room temperature until completion. The crude was concentrated under reduced pressure and the resulting solid was dissolved in DCM (2 mL) and purified by flash chromatography (50% Hex/50% EtOAc) to yield compound 1.2 (1.158 g, 91.0% yield) as a pale yellow solid.
(174) ##STR00129##
(175) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 10.87 (d, J=14.10 Hz, 1H), 8.14-8.22 (m, 1H), 7.18 (td, J=6.29, 9.32 Hz, 1H), 6.87 (td, J=6.29, 9.57 Hz, 1H), 4.05 (q, J=7.13 Hz, 2H), 2.97 (qt, J=3.27, 6.88 Hz, 1H), 1.08 (t, J=7.18 Hz, 3H), 0.88-0.94 (m, 2H), 0.82-0.87 (m, 2H); IR (υ.sub.max/cm.sup.−1) 1686, 1623, 1569, 1508, 1426, 1406, 1357, 1330, 1294, 1244, 1228, 1174, 1136, 1088, 1058, 1033, 1015, 890, 877, 809, 797, 773, 754, 734, 660, 588
Synthesis of ethyl 1-cyclopropyl-6,7-difluoro-4-oxo-1,4-dihydroquinoline-3-carboxylate (1.3)
(176) Compound 1.2 (1 g, 3.13 mmol, 1 eq) was dissolved in DCM (7 mL). DBU (573 μL, 3.83 mmol, 1.2 eq) and LiCl (270 mg, 6.38 mmol, 2 eq) were subsequently added and the reaction was stirred at 45° C. for 2.5 hours, then at room temperature for 15 hours. Upon completion, the mixture was extracted with DCM (2×20 mL) and washed with water (15 mL) with the aqueous phase neutralized using a 1M solution of citric acid (3 mL). The combined organic phases were dried over magnesium sulphate and concentrated under reduced pressure to give the crude product 1.3 (1.022 g, >95% crude yield) as a pale yellow solid which was used in the successive reaction without further purification.
(177) ##STR00130##
(178) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.55 (s, 1H), 8.20 (dd, J=8.69, 10.45 Hz, 1H), 7.72 (dd, J=6.29, 11.33 Hz, 1H), 4.37 (q, J=7.05 Hz, 2H), 3.42-3.48 (m, 1H), 1.40 (t, J=7.18 Hz, 3H), 1.21-1.37 (m, 2H), 1.13-1.18 (m, 2H); IR (υ.sub.max/cm.sup.−1) 1723, 1617, 16.02, 1490, 1479, 1454, 1446, 1424, 1396, 1386, 1379, 1335, 1314, 1287, 1228, 1209, 1202, 1167, 1121, 1094, 1054, 1033, 1018, 899, 855, 849, 826, 802, 781, 748, 729, 717, 619, 607, 595, 548, 540; LC-MS Retention time 3.28 minutes, found 294.0 [M+H].sup.+; calculated for C.sub.15H.sub.13F.sub.2NO.sub.3 294.27 [M+H].sup.+.
Synthesis of 1-cyclopropyl-6,7-difluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (1.4)
(179) Crude compound 1.3 (700 mg, 2.39 mmol, 1 eq) was refluxed with concentrated HCl (3.5 mL) and concentrated AcOH (13 mL) for 2.5 hours. The mixture was allowed to cool to room temperature, and the resulting precipitate was filtered, washed with water (3 mL) and dried to give the crude product 1.4 (573 mg, 90.4% yield) as a white solid which was used in the successive reaction without further purification.
(180) ##STR00131##
(181) .sup.1H NMR (400 MHz, TFA-d) δ 9.34 (d, J=3.53 Hz, 1H), 8.32-8.40 (m, 2H), 3.99-4.06 (m, 1H), 1.54-1.62 (m, 2H), 1.35 (br. s., 2H); IR (υ.sub.max/cm.sup.−1) 1719, 1614, 1556, 1421, 1332, 1303, 1289, 1231, 1204, 1056, 1033, 1020, 891, 806, 778, 748, 719, 606; LC-MS Retention time 3.37 minutes, found 265.9 [M+H].sup.+; calculated for C.sub.13H.sub.9F.sub.2NO.sub.3 266.22[M+H].sup.+.
Synthesis of 7-(4-(tert-butoxycarbonyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1-4-dihydroquinoline-3-carboxylic acid (1.5)
(182) A mixture of compound 1.4 (500 mg, 1.89 mmol, 1 eq), tert-butyl piperazine-1-carboxylate (1.06 g, 5.67 mmol, 3 eq) and potassium carbonate (552 mg, 3.78 mmol, 2 eq) was stirred in DMF (18 mL) at 140° C. for 15 hours. Upon completion (as monitored by LC-MS), the mixture was extracted with DCM (2×15 mL) and washed with water (10 mL) with the aqueous layer neutralized using a 1M solution of citric acid. The combined organic layers were dried over magnesium sulfate and concentrated under reduced pressure. The crude compound was recrystallized from DMF (5 mL) to give pure compound 1.5 (285 mg, 35.0% yield) as a pale orange solid.
(183) ##STR00132##
(184) IR (υ.sub.max/cm.sup.−1) 1729, 1687, 1627, 1502, 1462, 1417, 1383, 1340, 1286, 1246, 1217, 1168, 1124, 1083, 1045, 1034, 940, 891, 856, 828, 805, 782, 770, 746, 703, 667, 625; LC-MS Retention time 3.95 minutes, found 432.0 [M+H].sup.+; calculated for C.sub.22H.sub.26FN.sub.3O.sub.5 432.46 [M+H].sup.+
Synthesis of 1-cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid (2.1)
(185) Compound 1.5 (200 mg, 0.46 mmol, 1 eq) was dissolved in dry DCM (7 mL) at room temperature, then the mixture was cooled to 0° C. and TFA (710 μL, 9.27 mmol, 20 eq) added. The mixture was allowed to warm to room temperature while stirring over 2 hours. Upon completion (monitored by LC-MS), the solution was concentrated under reduced pressure and washed with toluene (4×3 mL). The crude solid was washed with EtOAc (5 mL) and MeOH (5 mL) to give pure compound 2.1 (150 mg, 98.7% yield) as a light orange solid.
(186) ##STR00133##
(187) .sup.1H NMR (400 MHz, TFA-d) δ 9.29 (s, 1H), 8.24 (d, J=12.34 Hz, 1H), 7.90 (d, J=6.80 Hz, 1H), 4.03-4.12 (m, 1H), 3.90-3.99 (m, 4H), 3.70-3.78 (m, 4H), 1.60-1.68 (m, 2H), 1.34-1.43 (m, 2H); IR (υ.sub.max/cm.sup.−1) 1685, 1627, 1612, 1490, 1454, 1341, 1272, 1259, 1184, 1138, 1107, 1056, 1034, 941, 894, 886, 829, 807, 793, 785, 749, 723, 708, 665, 637, 609; LC-MS Retention time 2.48 minutes, found 332.0 [M+H].sup.+; calculated for C.sub.17H.sub.18FN.sub.3O.sub.3 332.35 [M+H].sup.+.
Synthesis of 1-cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (1.6)
(188) Compound 2.1 (150 mg, 0.45 mmol, 1 eq) was stirred in DCM (7 mL) for 5 minutes. Then 4M HCl in dioxane (2.26 mL, 9.05 mmol, 20 eq) was added dropwise and the mixture stirred for 1 hour. Upon completion, the mixture was washed with hexane (3×1 mL) and lyophilized overnight to give compound 1.6 (>95%) as a pale brown solid.
(189) ##STR00134##
(190) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 15.20 (br. s., 1H), 9.46 (br. s., 2H), 8.67 (s, 1H), 7.94 (d, J=13.09 Hz, 1H), 7.61 (d, J=7.30 Hz, 1H), 3.86 (br. s., 1H), 3.57 (br. s., 4H), 3.31 (br. s., 4H), 1.29-1.35 (m, 2H), 1.15-1.24 (m, 3H); HRMS Observed 332.1404 [M+H].sup.+; theoretical value 332.1405 [M+H].sup.+; IR (υ.sub.max/cm.sup.−1) 1701, 1624, 1491, 1458, 1383, 1341, 1272, 1142, 1106, 1034, 941, 909, 889, 853, 829, 804, 774, 749, 703, 665, 636, 619; LC-MS Retention time 4.70 minutes, found 332.0 [M+H].sup.+; calculated for C.sub.17H.sub.18FN.sub.3O.sub.3 332.35 [M+H].sup.+.
Synthesis of ethyl (Z)-3-(ethylamino)-2-(2,4,5-trifluorobenzoyl)acrylate (1.7)
(191) Ethyl 2,4,5-trifluorobenzoylacetate (1.1; 5 g, 20.31 mmol, 1 eq) was dissolved in triethyl orthoformate (5.74 mL, 34.53 mmol, 1.7 eq) and heated at 140° C. for 30 minutes. Ac.sub.2O (5.76 mL, 60.93 mmol, 3 eq) was then added and the mixture refluxed at 140° C. for another 3 hours and monitored by TLC (90% Hex/10% EtOAc). Upon completion, the reaction was cooled, DCM (8 mL) added and the mixture stirred at room temperature for 10 minutes. Then ethylamine (20.31 mL, 40.62 mmol, 2 eq) was added and the reaction was stirred for 15 hours at room temperature. The crude was concentrated under reduced pressure and purified by flash chromatography (50% Hex/50% EtOAc) to give compound 1.7 (4.916 g, 80.3% yield) as a pale yellow solid.
(192) ##STR00135##
(193) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 10.89 (br. s., 1H), 8.13 (d, J=14.10 Hz, 1H), 7.19 (dd, J=9.32, 15.61 Hz, 1H), 6.82-6.91 (m, 1H), 4.05 (q, J=7.13 Hz, 2H), 3.46-3.54 (m, 2H), 1.36 (t, J=7.18 Hz, 3H), 1.07 (t, J=7.18 Hz, 3H); IR (υ.sub.max/cm.sup.−1) 1678, 1622, 1560, 1510, 1428, 1415, 1380, 1364, 1330, 1310, 1283, 1244, 1219, 1175, 1156, 1136, 1092, 1050, 1036, 884, 1015, 863, 829, 800, 774.
Synthesis of ethyl 1-ethyl-6,7-difluoro-4-oxo-1,4-dihydroquinoline carboxylate (1.8)
(194) Compound 1.7 (5 g, 16.60 mmol, 1 eq) was dissolved in DCM (35 mL). DBU (2.98 mL, 19.92 mmol, 1.2 eq) and LiCl (1.41 g, 33.20 mmol, 2 eq) were subsequently added and the reaction was stirred at 45° C. for 2.5 hours, then at room temperature for 15 hours. Upon completion, the mixture was extracted with DCM (2×15 mL) and washed with water (20 mL) with the aqueous neutralized using a 1M solution of citric acid (5 mL). The crude compound 1.8 was dried over magnesium sulphate, concentrated under reduced pressure to give a pale yellow solid (4.55 g, >95% crude yield) which was used in the successive reaction without further purification.
(195) ##STR00136##
(196) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.47 (s, 1H), 8.27 (dd, J=8.81, 10.32 Hz, 1H), 7.27 (dd, J=6.17, 11.20 Hz, 1H), 4.38 (q, J=7.05 Hz, 2H), 4.21 (q, J=7.22 Hz, 2H), 1.55 (t, J=7.30 Hz, 3H), 1.40 (t, J=7.05 Hz, 3H); IR (υm/cm.sup.−1) 1720, 1617, 1566, 1466, 1449, 1375, 1369, 1311, 1288, 1228, 1217, 1209, 1173, 1157, 1137, 1094, 1071, 1049, 1016, 902, 863, 829, 814, 802; LC-MS Retention time 3.18 minutes, Found 281.9 [M+H].sup.+; calculated for C.sub.14H.sub.13F.sub.2NO.sub.3 282.26 [M+H].sup.+
Synthesis of 1-ethyl-6-7-difluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (1.9)
(197) Crude compound 1.8 (4.5 g, 16.00 mmol, 1 eq) was refluxed in 2M NaOH (40 mL, 80.00 mmol, 5 eq) for 2 hours. The mixture was then allowed to cool to room temperature and acidified with a 1M solution of acetic acid (15 mL). The solid was filtered, washed with hexane (3×20 mL) and concentrated under reduced pressure. The solid was recrystallised from DMF (60 mL) to give pure compound 1.9 (2.964 g, 73.2% yield) as a pale yellow solid.
(198) ##STR00137##
(199) .sup.1H NMR (400 MHz, TFA-d) δ 9.36 (s, 1H), 8.39 (t, J=8.43 Hz, 1H), 7.99 (dd, J=6.17, 9.95 Hz, 1H), 4.79 (q, J=7.13 Hz, 2H), 1.66 (t, J=7.05 Hz, 3H); IR (υ.sub.max/cm.sup.−1) 1719, 1617, 1484, 1396, 1385, 1361, 1306, 1289, 1231, 1213, 1094, 1042, 948, 900, 874, 808; LC-MS Retention time 30.28 minutes, found 253.8 [M+H].sup.+; calculated for C.sub.12H.sub.9F.sub.2NO.sub.3 254.2 [M+H].sup.+
Synthesis of 7-(4-(tert-butoxycarbonyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (1.10)
(200) A mixture of compound 1.9 (200 mg, 0.79 mmol, 1 eq), tert-butyl piperazine-1-carboxylate (442 mg, 2.37 mmol, 3 eq) and potassium carbonate (218 mg, 1.58 mmol, 2 eq) was stirred in DMF (8 mL) at 140° C. for 20 hours. Upon completion (monitored by LC-MS), the mixture was extracted with DCM (2×15 mL) and washed with water (10 mL) with the aqueous phase neutralized using a 1M solution of citric acid (1 mL). The combined organic phases were dried over magnesium sulphate and concentrated under reduced pressure. The crude solid was recrystallized from DMF (2 mL) to give pure compound 1.10 (160 mg, 48.3% yield) as a pale brown solid.
(201) ##STR00138##
(202) IR (υ.sub.max/cm.sup.−1) 1727, 1696, 1681, 1629, 1614, 1518, 1478, 1470, 1446, 1424, 1380, 1362, 1332, 1287, 1268, 1245, 1199, 1168, 1126, 1107, 1093, 1055, 1033, 1005, 925, 906, 860, 834, 822, 803, 761; LC-MS Retention time 3.88 minutes, found 420.0 [M+H].sup.+; calculated for C.sub.21H.sub.26FN.sub.3O.sub.5 420.45 [M+H].sup.+.
Synthesis of 1-ethyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid (3.1)
(203) Compound 1.10 (85 mg, 0.20 mmol, 1 eq) was dissolved in dry DCM (5 mL), then the solution cooled to 0° C. and TFA (233 μL, 3.04 mmol, 15 eq) added. The mixture was allowed to warm to room temperature while stirring over 4 hours. Upon completion (monitored by LC-MS), the mixture was concentrated under reduced pressure and washed with toluene (3×2 mL). The crude solid was recrystallised from DMF (600 μL) and washed with EtOAc (5 mL) and MeOH (5 mL) to give pure compound 3.1 (44 mg, 68.9% yield) as a pale orange solid.
(204) ##STR00139##
(205) .sup.1H NMR (400 MHz, TFA) δ 9.30 (s, 1H), 8.29 (d, J=12.09 Hz, 1H), 7.48 (d, J=6.55 Hz, 1H), 4.85 (q, J=7.22 Hz, 2H), 3.91-3.99 (m, 4H), 3.71-3.79 (m, 4H), 1.74 (t, J=7.30 Hz, 3H); IR (υ.sub.max/cm.sup.−1) 1696, 1624, 1610, 1508, 1474, 1453, 1421, 1399, 1382, 1366, 1310, 1265, 1202, 1125, 1104, 1088, 1053, 1033, 990, 934, 916, 900, 828, 808, 797, 748, 720; LC-MS Retention time 2.38 minutes, found 320.0 [M+H].sup.+; calculated for C.sub.16H.sub.18FN.sub.3O.sub.3 320.33 [M+H].sup.+.
Synthesis of 1-ethyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (1.11)
(206) Compound 3.1 (39 mg, 0.12 mmol, 1 eq) was stirred in the minimum amount of DCM (3 mL) for 5 minutes. Then 4M HCl in dioxane (611 μL, 2.44 mmol, 20 eq) was added dropwise and the mixture stirred for 1 hour. Upon completion, the mixture was washed with hexane (3×1 mL), concentrated under reduced pressure and lyophilized overnight to give compound 1.11 (41 mg, >95% yield) as a pale orange solid.
(207) ##STR00140##
(208) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 15.31 (br. s., 1H), 9.37 (br. s., 2H), 8.97 (s, 1H), 7.96 (d, J=13.09 Hz, 1H), 7.26 (d, J=7.30 Hz, 1H), 4.62 (q, J=7.13 Hz, 2H), 3.52-3.59 (m, 4H), 3.30 (br. s., 4H), 1.41 (t, J=7.18 Hz, 3H), 1.22 (d, J=6.55 Hz, 1H); HRMS Observed 320.1404 [M+H].sup.+; theoretical value 320.1405 [M+H].sup.+; Ir (υ.sub.max/cm.sup.−1) 1701, 1696, 1626, 1507, 1454, 1345, 1340, 1332, 1273, 1130, 1053, 1033, 933, 899, 859, 829, 804, 746, 665; LC-MS Retention time 4.67 minutes, found 320.0 [M+H].sup.+; calculated for C.sub.16H.sub.18FN.sub.3O.sub.3 320.34 [M+H].sup.+.
Synthesis of Ciprofloxacin-ARB Hybrid Compounds
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-(naphthalen-1-ylmethyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.2)
(209) Ciprofloxacin (2.1; 1 g, 3.02 mmol, 1 eq) was added to DMF (50 mL total) and stirred for 30 minutes at reflux. Then 1-(bromomethyl)naphthalene (647 mg, 2.92 mmol, 1 eq) was pre-dissolved in DMF (2 mL) at 115° C. and added via syringe. Potassium carbonate (1251 mg, 9.05 mmol, 3 eq) was subsequently added and the mixture stirred for a further 1 hour at reflux. The mixture was allowed to cool, then extracted with ethyl acetate (2×100 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (100 mL) and brine (100 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved via flash column chromatography using ethyl acetate to afford compound 2.2 (433.27 mg, 30.4% yield) as an off white solid.
(210) ##STR00141##
(211) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.07 (s, 1H), 8.77 (s, 1H), 8.34 (d, J=8.56 Hz, 1H), 8.03 (d, J=13.09 Hz, 1H), 7.89 (d, J=7.30 Hz, 1H), 7.83 (d, J=7.55 Hz, 1H), 7.49-7.58 (m, 2H), 7.41-7.49 (m, 2H), 7.33 (d, J=7.05 Hz, 1H), 4.02 (s, 2H), 3.49 (br. s., 1H), 3.34 (br. s., 4H), 2.76 (br. s., 4H), 1.31-1.39 (m, 2H), 1.14-1.22 (m, 2H); LC-MS Retention time 30.08 minutes, found 472.1 [M+H].sup.+; calculated for C.sub.28H.sub.26FN.sub.3O.sub.3 472.53 [M+H].sup.+.
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-(naphthalen-1-ylmethyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (2.3)
(212) 2.2 (780 mg, 1.65 mmol, 1 eq) was added to dichloromethane (15 mL) and stirred for 5 minutes. Then 4M HCl in dioxane (8.3 mL, 33.08 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 2.3 (771.55 mg, 91.8% yield) as a pale yellow solid.
(213) ##STR00142##
(214) .sup.1H NMR (400 MHz, TFA-d) δ 9.27 (s, 1H), 8.22 (d, J=12.09 Hz, 1H), 8.12 (d, J=8.31 Hz, 1H), 8.05 (d, J=8.06 Hz, 1H), 7.97 (d, J=80.6 Hz, 1H), 7.93 (d, J=6.55 Hz, 1H), 7.78 (d, J=7.05 Hz, 1H), 7.65-7.71 (m, 1H), 7.54-7.65 (n, 2H), 5.03 (s, 2H), 4.19 (d, J=13.09 Hz, 2H), 4.10 (br. s., 1H), 3.83-3.98 (m, 4H), 3.65 (t, J=10.95 Hz, 2H), 1.64 (d, J=5.54 Hz, 2H), 1.38 (br. s., 2H); .sup.13C NMR (100 MHz, DMSO-d.sub.6) δ 176.3, 165.8, 152.8 (C-6, .sup.1J(C-F)=249 Hz), 148.2, 143.6 (C-7, .sup.2J(C-F)=12 Hz), 139.0, 133.4, 132.2, 131.7, 130.4, 128.8, 127.1, 126.3, 125.6, 125.4, 124.1, 119.3, 111.2 (C-5, .sup.2J(C-F)=25 HZ), 106.8, 62.8, 55.0, 50.4, 46.2, 35.9, 7.6; IR (υ.sub.max/cm.sup.−1) 3370, 1718, 1628, 1506, 1436, 1399, 1340, 1266, 1038, 939, 792; LC-MS Retention time 6.00 minutes, found 472.0 [M+H].sup.+; calculated for C.sub.28H.sub.26FN.sub.3O.sub.3 472.53 [M+H].sup.+; HRMS Observed 472.2023 [M+H].sup.+; theoretical value 472.2031 [M+H].sup.+.
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-(naphthalen-2-ylmethyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.4)
(215) Ciprofloxacin (2.1; 200 mg, 0.60 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and water (10 mL total). After stirring for 5 minutes, potassium carbonate (250 mg, 1.81 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 2-(bromomethyl)naphthalene (127 mg, 0.57 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 24 hours. Upon completion, the product was extracted with dichloromethane (2×30 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (30 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford compound 2.4 (219.68 mg, 81.2% yield) as a pale yellow solid.
(216) ##STR00143##
(217) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.05 (br. s., 1H), 8.72 (s, 1H), 7.96 (d, J=13.09 Hz, 1H), 7.81-7.86 (m, 3H), 7.78 (s, 1H), 7.54 (dd, J=1.64, 8.44 Hz, 1H), 7.45-7.52 (m, 2H), 7.34 (d, J=7.05 Hz, 1H), 3.77 (s, 2H), 3.52 (br. s., 1H), 3.34-3.40 (m, 4H), 2.71-2.77 (m, 4H), 10.32-1.39 (m, 2H), 1.14-1.21 (m, 2H); LC-MS Retention time 2.95 minutes, found 472.0 [M+H].sup.+; calculated for C.sub.28H.sub.26FN.sub.3O.sub.3 472.53 [M+H].sup.+.
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-(naphthalen-2-ylmethyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (2.5)
(218) 2.4 (30 mg, 0.063 mmol, 1 eq) was added to equal parts methanol and dioxane (60 mL total) and stirred for 10 minutes. Then 4M HCl in dioxane (32 μL, 0.126 mmol, 2 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 2.5 (22.18 mg, 68.6% yield) as a white solid.
(219) ##STR00144##
(220) .sup.1H NMR (400 MHz, TFA-d) δ 9.29-9.35 (m, 1H), 8.28 (dd, J=30.65, 12.21 Hz, 1H), 7.99-8.05 (m, 2H), 7.88-7.97 (m, 3H), 7.59-7.67 (m, 2H), 7.55 (d, J=6.04 Hz, 1H), 4.72 (br. s., 2H), 4.26 (d, J=12.34 Hz, 2H), 4.08-4.16 (m, 1H), 3.99 (d, J=11.33 Hz, 2H), 3.78 (t, J=11.08 Hz, 2H), 3.60 (t, J=11.83 Hz, 2H), 1.68 (br. s., 2H), 1.42 (br. s., 2H); IR (υm/cm.sup.−1) 3428, 2923, 2282, 1728, 1629, 1500, 1449, 1387, 1267, 1104, 941, 804; LC-MS Retention time 5.93 minutes, found 472.1 [M+H].sup.+; calculated for C.sub.28H.sub.26FN.sub.3O.sub.3 472.53 [M+H].sup.+; HRMS Observed 472.2020 [M+H].sup.+; theoretical value 472.2031 [M+H].sup.+.
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-benzylpiperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.6)
(221) Ciprofloxacin (2.1; 100 mg, 0.30 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and water (10 mL total). After stirring for 5 minutes, potassium carbonate (125 mg, 0.91 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, bromomethylbenzene (49 mg, 0.29 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 24 hours. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford compound 2.6 (112.69 mg, 93.3% yield) as a pale yellow solid.
(222) ##STR00145##
(223) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.05 (br. s., 1H), 8.76 (s, 1H), 8.00 (d, J=13.09 Hz, 1H), 7.32-7.39 (m, 5H), 7.28-7.32 (m, 1H), 3.62 (s, 2H), 3.53 (br. s., 1H), 3.32-3.40 (m, 4H), 2.65-2.73 (m, 4H), 1.38 (q, J=6.38 Hz, 2H), 1.19 (br. s., 2H); LC-MS Retention time 2.83 minutes, found 422.0 [M+H].sup.+; calculated for C.sub.24H.sub.24FN.sub.3O.sub.3 422.47 [M+H].sup.+.
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-benzylpiperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (2.7)
(224) 2.6 (30 mg, 0.07 mmol, 1 eq) was added to methanol (10 mL) and stirred for 10 minutes. Then 4M HCl in dioxane (36 μL, 0.14 mmol, 2 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 2.7 (30.68 mg (91.7% yield) as an off white solid.
(225) ##STR00146##
(226) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 15.13 (br. s., 1H), 11.55 (br. s., 1H), 8.68 (s, 1H), 7.96 (d, J=13.09 Hz, 1H), 7.68 (dd, J=2.77, 6.29 Hz, 2H), 7.60 (d, J=7.55 Hz, 1H), 7.46-7.51 (m, 3H), 4.42 (d, J=5.04 Hz, 2H), 3.81-3.92 (m, 3H), 3.44-3.52 (m, 4H), 3.29 (q, J=10.74 Hz, 2H), 1.28-1.35 (m, 2H), 1.15-1.21 (m, 2H); IR (υ.sub.max/cm.sup.−1) 2922, 2286, 1729, 1628, 1502, 1468, 1335, 1270, 1104, 941, 803, 702; LC-MS Retention time 5.38 minutes, found 422.1 [M+H].sup.+; calculated for C.sub.24H.sub.24FN.sub.3O.sub.3 422.47 [M+H].sup.+; HRMS Observed 422.1864 [M+H].sup.+; theoretical value 422.1874 [M+H].sup.+.
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-(benzo[d][1,3]dioxol-4-ylmethyl)-piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.8)
(227) Ciprofloxacin (2.1; 53 mg, 0.16 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and water (5 mL total). After stirring for 5 minutes, potassium carbonate (66 mg, 0.48 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 4-(bromomethyl)benzo[d][1,3]dioxole (33 mg, 0.15 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 24 hours. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford compound 2.8 (47.78 mg, 67.6% yield) as an off white solid.
(228) ##STR00147##
(229) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.04 (br. s., 8.76 (s, 1H), 8.01 (d, J=13.09 Hz, 1H), 7.35 (d, J=70.05 Hz, 1H), 6.76-6.88 (m, 3H), 5.98 (s, 2H), 3.63 (s, 2H), 3.53 (br. s., 1H), 3.33-3.40 (m, 4H), 2.69-2.76 (m, 4H), 1.38 (m, 2H), 1.19 (br. s., 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 177.1, 167.1, 155.0, 152.5, 147.4, 147.3, 146.3, 146.1, 146.0, 139.1, 123.5, 121.4, 119.8, 119.7, 118.6, 112.5, 112.3, 108.1, 107.8, 104.7, 100.8, 56.2, 52.5, 49.8, 49.8, 35.3, 8.3; LC-MS Retention time 2.87 minutes, found 466.0 [M+H].sup.+; calculated for C.sub.25H.sub.24FN.sub.3O.sub.5 466.48 [M+H].sup.+
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-(benzo[d][1,3]dioxol-4-ylmethyl)-piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (2.9)
(230) 2.8 (50 mg, 0.11 mmol, 1 eq) was added to methanol (25 mL) and stirred for 10 minutes. Then 4M HCl in dioxane (54 μL, 0.21 mmol, 2 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 2.9 (38.23 mg, 70.9% yield) as a pale yellow solid.
(231) ##STR00148##
(232) .sup.13C NMR (100 MHz, DMSO-d.sub.6) δ 176.3, 165.8, 152.8 (C-6, .sup.1J(C-F)=254 Hz), 148.3, 147.5, 147.3, 143.6 (C-7, .sup.2J(C-F)=15 Hz), 139.0, 125.1, 121.9, 119.3 ((C-F)=7 Hz), 111.2 (C-5, .sup.2J(C-F)=17 HZ), 110.3, 109.9, 107.0, 106.8, 101.3, 52.7, 50.1, 46.1, 35.9, 7.6; IR (υ.sub.max/cm.sup.−1) 3378, 2913, 2571, 2362, 1719, 1627, 1507, 1452, 1399, 1253, 1035, 952, 804, 725; LC-MS Retention time 5.48 minutes, found 466.1 [M+H].sup.+; calculated for C.sub.25H.sub.24FN.sub.3O.sub.5 466.48 [M+H].sup.+; HRMS Observed 466.1762 [M+H].sup.+; theoretical value 466.1773 [M+H].sup.+.
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-(benzo[b]thiophen-7-ylmethyl)-piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.10)
(233) Ciprofloxacin (2.1; 77 mg, 0.23 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and water (5 mL total). After stirring for 5 minutes, potassium carbonate (96 mg, 0.70 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 7-(bromomethyl)benzo[b]thiophene (50 mg, 0.22 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 24 hours. Upon completion, extraction using dichloromethane (2×20 mL), using a 1M solution of citric acid to neutralise the aqueous phase, resulted in formation of a white precipitate. The precipitate was filtered, washed with distilled water (3×20 mL), re-suspended in dichloromethane (2 mL) and purified via automated column chromatography (see Flash Column Chromatography method) to afford compound 2.10 (34.67 mg, 32.9% yield) as an off white solid.
(234) ##STR00149##
(235) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.09 (br. s., 1H), 8.78 (s, 1H), 8.03 (d, J=13.09 Hz, 1H), 7.79 (d, J=70.81 Hz, 1H), 7.47 (d, J=5.29 Hz, 1H), 7.34-7.41 (m, 3H), 7.28 (s, 1H), 3.89 (s, 2H), 3.48-3.56 (m, 1H), 3.36-3.42 (m, 4H), 2.73-2.79 (m, 4H), 1.37 (q, J=6.71 Hz, 2H), 1.17-1.23 (m, 2H); LC-MS Retention time 3.15 minutes, found 477.9 [M+H].sup.+; calculated for C.sub.26H.sub.24FN.sub.3O.sub.3S 478.55 [M+H].sup.+.
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-(benzo[b]thiophen-7-ylmethyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (2.11)
(236) 2.10 (20 mg, 0.042 mmol, 1 eq) was added to methanol (10 mL) and stirred for 10 minutes. Then 4M HCl in dioxane (21 μL, 0.084 mmol, 2 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 2.11 (16.35 mg, 73.3% yield) as a pale yellow solid.
(237) ##STR00150##
(238) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ15.13 (br. s, 1H), 11.45 (br. s., 1H), 8.68 (s, 1H), 7.86-8.05 (m, 4H), 7.51-7.61 (m, 3H), 4.71 (br. s., 2H), 3.90 (br. s., 2H), 3.82 (br. s., 2H), 3.51 (br. s., 6H), 1.30 (d, J=5.79 Hz, 2H), 1.18 (d, J=4.03 Hz, 3H); IR (υ.sub.max/cm.sup.−1) 3380, 1715, 1628, 1457, 1339, 1265, 1042, 943, 803; LC-MS Retention time 6.12 minutes, found 478.0 [M+H].sup.+; calculated for C.sub.26H.sub.24FN.sub.3O.sub.3S 478.55 [M+H].sup.+; HRMS Observed 478.1584 [M+H].sup.+; theoretical value 478.1595 [M+H].sup.+.
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-((4-fluoronaphthalen yl)methyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.12)
(239) Ciprofloxacin (2.1; 100 mg, 0.30 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and water (10 mL total). After stirring for 5 minutes, potassium carbonate (125 mg, 0.91 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 1-(bromomethyl)-4-fluoronaphthalene (69 mg, 0.29 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 24 hours. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford compound 2.12 (74.68 mg, 53.2% yield) as a pale yellow solid.
(240) ##STR00151##
(241) LC-MS Retention time 3.18 minutes, found 490.0 [M+H].sup.+; calculated for C.sub.28H.sub.25F.sub.2N.sub.3O.sub.3 490.52 [M+H].sup.+.
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-((4-fluoronaphthalen-1-yl)methyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (2.13)
(242) 2.12 (80 mg, 0.16 mmol, 1 eq) was added to methanol (40 mL) and stirred for 10 minutes. Then 4M HCl in dioxane (82 μL, 0.33 mmol, 2 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 2.13 (75.76 mg, 88.1% yield) as a light brown solid.
(243) ##STR00152##
(244) .sup.1H NMR (400 MHz, TFA-d) δ 9.24 (d, J=3.78 Hz, 1H), 8.25 (d, J=8.56 Hz, 1H), 8.19 (d, J=13.35 Hz, 1H), 8.06 (d, J=7.55 Hz, 1H), 7.87 (br. s., 1H), 7.62-7.75 (m, 3H), 7.20 (t, J=8.44 Hz, 1H), 4.96 (br. s., 2H), 4.16 (d, J=12.34 Hz, 2H), 4.05 (br. s., 1H), 3.90 (d, J=11.58 Hz, 2H), 3.75 (t, J=11.71 Hz, 2H), 3.59 (t, J=10.95 Hz, 2H), 1.60 (br. s., 2H), 1.34 (br. s., 2H); IR (υ.sub.max/cm.sup.−1) 3373, 1715, 1628, 1457, 1395, 1340, 1266, 1042, 943, 804; LC-MS Retention time 6.08 minutes, found 490.1 [M+H].sup.+; calculated for C.sub.28H.sub.25F.sub.2N.sub.3O.sub.3 490.52 [M+H].sup.+; HRMS Observed 490.1929 [M+H].sup.+; theoretical value 490.1937 [M+H].sup.+.
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-(2-oxo-1,2-dihydroquinolin yl)methyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.14)
(245) Ciprofloxacin (2.1; 1 g, 3.02 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and water (50 mL total). After stirring for 5 minutes, potassium carbonate (1251 mg, 9.05 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 4-(bromomethyl)quinolin-2(1H)-one (683 mg, 2.87 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 24 hours. Upon completion, extraction using dichloromethane (2)(100 mL), using a 1M solution of citric acid to neutralise the aqueous phase, resulted in formation of a white precipitate. The precipitate was filtered, washed with distilled water (100 mL) and methanol (100 mL) then re-dissolved in excess DMSO. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford compound 2.14 (1.06 g, 75.7% yield) as a pale yellow solid.
(246) ##STR00153##
(247) LC-MS Retention time 2.92 minutes, found 489.0 [M+H].sup.+; calculated for C.sub.27H.sub.25FN.sub.4O.sub.4 489.52 [M+H].sup.+.
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-(2-oxo-1,2-dihydroquinolin-4-yl)methyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (2.15)
(248) 2.14 (50 mg, 0.10 mmol, 1 eq) was added to methanol (15 mL) and stirred for 10 minutes. Then 4M HCl in dioxane (524, 0.20 mmol, 2 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 2.15 (42.09 mg, 78.3% yield) as a white solid.
(249) ##STR00154##
(250) .sup.1H NMR (400 MHz, TFA-d) δ 9.32 (s, 1H), 8.20-8.31 (m, 2H), 7.97-8.03 (m, 1H), 7.94 (br. s., 1H), 70.86 (d, J=8.56 Hz, 1H), 7.77 (br. s., 2H), 5.14 (br. s., 2H), 4.25 (br. s., 2H), 4.12 (br. s., 3H), 30.86 (br. s., 4H), 1.67 (br. s., 2H), 1.41 (br. s., 2H); IR (υ.sub.max/cm.sup.−1) 3446, 2826, 2362, 1710, 1664, 1625, 1558, 1473, 1439, 1357, 1257, 1038, 958, 887, 805, 747; LC-MS Retention time 5.67 minutes, found 489.0 [M+H].sup.+; calculated for C.sub.27H.sub.25FN.sub.4O.sub.4 489.52 [M+H].sup.+; HRMS Observed 489.1924 [M+H].sup.+; theoretical value 489.1933 [M+H].sup.+.
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-(quinolin-8-ylmethyl)piperazin-1-yl) oxo-1,4-dihydroquinoline-3-carboxylic acid (2.16)
(251) Ciprofloxacin (2.1; 100 mg, 0.30 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and water (10 mL total). After stirring for 5 minutes, potassium carbonate (125 mg, 0.91 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 8-(bromomethyl)quinoline (64 mg, 0.29 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 24 hours. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford compound 2.16 as a tan solid.
(252) ##STR00155##
(253) LC-MS Retention time 2.88 minutes, found 473.0 [M+H].sup.+; calculated for C.sub.27H.sub.25FN.sub.4O.sub.3 473.52 [M+H].sup.+.
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-(quinolin-8-ylmethyl)piperazin-1-34)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (2.17)
(254) 2.16 (50 mg, 0.11 mmol, 1 eq) was added to methanol (25 mL) and stirred for 10 minutes. Then 4M HCl in dioxane (53 μL, 0.21 mmol, 2 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 2.17, as a light brown solid.
(255) ##STR00156##
(256) .sup.1H NMR (400 MHz, TFA-d) δ 9.42 (d, J=5.29 Hz, 1H), 9.37 (s, 1H), 9.35 (d, J=8.56 Hz, 1H), 8.66 (d, J=7.55 Hz, 1H), 8.60 (d, J=8.06 Hz, 1H), 8.29-8.36 (m, 2H), 8.24 (t, J=7.30 Hz, 1H), 8.01 (d, J=5.79 Hz, 1H), 5.48 (s, 2H), 4.34 (d, J=11.83 Hz, 2H), 4.08-4.20 (m, 3H), 3.99 (t, J=10.70 Hz, 2H), 3.81-3.92 (m, 2H), 1.73 (d, J=5.04 Hz, 2H), 1.47 (br. s., 2H); IR (υ.sub.max/cm.sup.−1) 3393, 2355, 1726, 1628, 1473, 1333, 1257, 943, 832, 804, 746; LC-MS Retention time 5.62 minutes, found 473.1 [M+H].sup.+; calculated for C.sub.27H.sub.25FN.sub.4O.sub.3 473.52 [M+H].sup.+; HRMS Observed 473.1974 [M+H].sup.+; theoretical value 473.1983 [M+H].sup.+.
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-((5,6,7,8-tetrahydronaphthalen-1-yl)methyl)-piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.18)
(257) Ciprofloxacin (2.1; 100 mg, 0.30 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and water (10 mL total). After stirring for 5 minutes, potassium carbonate (125 mg, 0.91 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 5-(bromomethyl)-1,2,3,4-tetrahydronaphthalene (65 mg, 0.29 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 24 hours. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford compound 2.18 (96.47 mg, 70.8% yield) as a pale brown solid.
(258) ##STR00157##
(259) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.06 (br. s., 1H), 8.75 (s, 1H), 7.98 (d, J=13.09 Hz, 1H), 7.34 (d, J=6.80 Hz, 1H), 7.02-7.14 (m, 3H), 3.52 (s, 3H), 3.31-3.37 (m, 4H), 2.79-2.88 (m, 4H), 2.66-2.71 (m, 4H), 1.76-1.88 (m, 4H), 1.34-1.41 (m, 2H), 1.20 (m, 2H); LC-MS Retention time 30.17 minutes, found 476.1 [M+H].sup.+; calculated for C.sub.28H.sub.30FN.sub.3O.sub.3 476.56 [M+H].sup.+.
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-((5,6,7,8-tetrahydronaphthalen-1-yl)methyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (2.19)
(260) 2.18 (40 mg, 0.084 mmol, 1 eq) was added to methanol (10 mL) and stirred for 10 minutes. Then 4M HCl in dioxane (42 μL, 0.17 mmol, 2 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 2.19 (259.01 mg, 98.6% yield) as a yellow solid.
(261) In an alternative method, 2.18 (244 mg, 0.51 mmol, 1 eq) was added to dichloro-methane (5 mL) and stirred for 10 minutes. Then 4M HCl in dioxane (2.6 mL, 10.26 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 2 hours. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford 2.19 as a yellow solid.
(262) ##STR00158##
(263) .sup.1H NMR (400 MHz, DMSO-d.sub.6+TFA) δ 8.69 (s, 1H), 7.97 J=13.35 Hz, 1H), 7.59 (d, J=7.30 Hz, 1H), 7.52 (d, J=6.55 Hz, 1H), 7.14-7.22 (m, 2H), 4.39 (br. s., 2H), 3.79-3.94 (m, 3H), 3.36-3.53 (m, 4H), 2.89 (t, J=5.54 Hz, 2H), 2.76 J=5.92 Hz, 2H), 1.67-1.83 (m, 4H), 1.31 (d, J=6.55 Hz, 1H), 1.19 (br. s., 2H); IR (υ.sub.max/cm.sup.−1) 3382, 2928, 1716, 1628, 1452, 1388, 1265, 941, 831, 804; LC-MS Retention time 6.07 minutes, found 476.1 [M+H].sup.+; calculated for C.sub.28H.sub.30FN.sub.3O.sub.3 476.56 [M+H].sup.+; HRMS Observed 476.2334 [M+H].sup.+; theoretical value 476.2344 [M+H].sup.+.
Synthesis of 1-cyclopropyl-7-(4-(4-(dimethylamino)benzyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.20)
(264) Ciprofloxacin (2.1; 100 mg, 0.30 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and water (5 mL total). After stirring for 5 minutes, potassium carbonate (125 mg, 0.91 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 4-(bromomethyl)-N,N-dimethylanilene (65 mg, 0.30 mmol, 1 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 24 hours. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford compound 2.20 (67.25 mg, 50.5% yield) as an off white solid.
(265) ##STR00159##
(266) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.07 (br. s., 1H), 8.77 (s, 1H), 8.02 (d, J=13.09 Hz, 1H), 7.35 (d, J=7.30 Hz, 1H), 7.19-7.23 (m, J=8.56 Hz, 2H), 6.70-6.75 (m, J=8.56 Hz, 2H), 3.52 (s, 3H), 3.31-3.37 (m, 4H), 2.96 (s, 6H), 2.64-2.70 (m, 4H), 1.34-1.40 (m, 2H), 1.16-1.22 (m, 2H); LC-MS Retention time 50.48 minutes, found 465.0 [M+H].sup.+; calculated for C.sub.26H.sub.29FN.sub.4O.sub.3 465.54 [M+H].sup.+.
Synthesis of 1-cyclopropyl-7-(4-(4-(dimethylamino)benzyl)piperazin-1-yl) fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (2.21)
(267) 2.20 (61.14 mg, 0.13 mmol, 1 eq) was added to dichloromethane (5 mL) and stirred for 5 minutes. Then 4M HCl in dioxane (658 μL, 2.63 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 2.21 (61.72 mg, 93.6% yield) as an off white solid.
(268) ##STR00160##
(269) .sup.1H NMR (400 MHz, TFA-d) δ 9.26 (d, J=3.02 Hz, 1H), 8.22 (d, J=12.09 Hz, 1H), 7.89-7.96 (m, 3H), 7.82 (d, J=5.54 Hz, 2H), 4.62 (br. s., 2H), 4.18 (d, J=13.60 Hz, 2H), 4.08 (br. s., 1H), 3.78-3.90 (m, 4H), 3.57 (t, J=11.20 Hz, 2H), 3.43 (br. s., 6H), 1.63 (br. s., 2H), 1.37 (br. s., 2H); 13C NMR (100 MHz, DMSO-d.sub.6) δ 176.0 (C═O), 169.0 (CO.sub.2H), 153.3 (C-6, .sup.1J(C-F)=250 Hz), 148.4, 144.4, 144.0 (C-7, .sup.2J(C-F)=10 Hz), 138.9, 133.4, 128.9, 120.7, 120.4, 118.9 ((C-F)=8 Hz), 110.7 (C-5, .sup.2J(C-F)=23 Hz), 106.8, 1051, 59.3, 51.0, 46.3, 45.6, 36.1, 7.4; IR (υ.sub.max/cm.sup.−1) 3500, 3438, 3384, 2553, 2459, 1718, 1631, 1478, 1390, 1267, 1184, 1103, 1027, 946, 894, 801, 594; LC-MS Retention time 50.53 minutes, found 465.0 [M+H].sup.+; calculated for C.sub.26H.sub.29FN.sub.4O.sub.3 465.54 [M+H].sup.+; HRMS Observed 465.2285 [M+H].sup.+; theoretical value 465.2296 [M+H].sup.+.
Synthesis of tert-butyl 4-(2-(naphthalen-1-yl)ethyl)piperazine-1-carboxylate (2.23)
(270) 1-(2-bromoethyl)naphthalene (2.22; 1 g, 4.25 mmol, 1 eq), tert-butyl piperazine-1-carboxylate (1 g, 50.37 mmol, 1.25 eq) and potassium carbonate (2.23 g, 16.1 mmol, 3.8 eq) were added to a 1:1 mixture of acetonitrile and water (10 mL total) and stirred for 72 hours at room temperature. Upon completion, the mixture was extracted with dichloromethane (3×50 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (50 mL) followed by brine (50 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Flash column chromatography (0%-3%-10% DCM/MeOH) was used to afford compound 2.23 (1.093 g, 75.5% yield) as a brown oil.
(271) ##STR00161##
(272) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.05 (dd, J=1.13, 8.43 Hz, 1H), 7.87 (dd, J=1.26, 8.31 Hz, 1H), 7.74 (d, J=8.06 Hz, 1H), 7.46-7.55 (m, 2H), 7.34-7.43 (m, 2H), 3.49-3.54 (m, 4H), 3.26-3.32 (m, 2H), 2.71-2.77 (m, 2H), 2.52-2.59 (m, 4H), 10.49 (s, 9H); LC-MS Retention time 2.97 minutes, found 341.1 [M+H].sup.+; calculated for C.sub.21H.sub.28N.sub.2O.sub.2 341.47 [M+H].sup.+.
Synthesis of 1-(2-(naphthalen-1-yl)ethyl)piperazine (2.24)
(273) 2.23 (1.093 g, 3.21 mmol, 1 eq) was added to dichloromethane (10 mL) followed by 4M HCl in dioxane (16 mL, 64.3 mmol, 20 eq), the flask sealed and stirred for 3 hours at room temperature. Upon completion, the mixture was extracted with dichloromethane (2×50 mL) using a saturated solution of sodium hydrogencarbonate to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (50 mL) followed by brine (50 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product 2.24 as an off white solid which was used in successive reactions without further purification.
(274) ##STR00162##
(275) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.99 (d, J=8.56 Hz, 1H), 7.78 (dd, J=1.64, 7.93 Hz, 1H), 7.65 (d, J=7.81 Hz, 1H), 7.38-7.47 (m, 2H), 7.26-7.35 (m, 2H), 3.19-3.25 (m, 2H), 2.90 (t, J=4.91 Hz, 4H), 2.62-2.67 (m, 2H), 2.52 (br. s., 4H); LC-MS Retention time 2.27 minutes, found 241.0 [M+H].sup.+; calculated for C.sub.16H.sub.20N.sub.2 241.35 [M+H].sup.+.
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-(2-(naphthalen-1-yl)ethyl)-piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.25)
(276) 1-cyclopropyl-6,7-difluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (1.4; 100 mg, 0.38 mmol, 1 eq) and 1-(2-(naphthalen-1-yl)ethyl)piperazine (2.24; 200 mg, 0.83 mmol, 2.2 eq) were added to DMSO (5 mL) and stirred until full dissolution of both compounds was achieved. The reaction was subsequently heated to 140° C. for one and a half hours. Upon completion, the mixture was allowed to cool, then extracted with dichloromethane (25 mL). Combined organic fractions were washed with distilled water (2×200 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo to remove any residual DMSO. The crude solid was purified via trituration; the crude was washed with methanol (5×10 mL), then the remaining powder collected and re-filtered using dichloromethane. This second filtrate was concentrated in vacuo to afford compound 2.25 (45 mg, 24.6% yield) as a light brown solid.
(277) ##STR00163##
(278) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.05 (br. s., 1H), 8.79 (s, 1H), 8.08 (d, J=8.31 Hz, 1H), 8.04 (d, J=13.09 Hz, 1H), 7.88 (dd, J=1.01, 8.06 Hz, 11-1), 7.76 (d, J=7.81 Hz, 1H), 7.48-7.57 (m, 2H), 7.37-7.45 (m, 3H), 3.53-3.60 (m, 1H), 3.40-3.46 (m, 4H), 3.31-3.37 (m, 2H), 2.81-2.88 (m, 6H), 1.38-1.44 (m, 2H), 1.19-1.25 (m, 2H); .sup.19F NMR (400 MHz, CDCl.sub.3) δ −120.66; LC-MS Retention time 3.15 minutes, found 486.2 [M+H].sup.+; calculated for C.sub.29H.sub.28FN.sub.3O.sub.3 486.56 [M+H].sup.+.
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-(2-(naphthalen-1-yl)ethyl)-piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (2.26)
(279) 2.25 (25.8 mg, 0.053 mmol, 1 eq) was added to dichloromethane (5 mL total) and stirred for 10 minutes. Then 4M HCl in dioxane (265 μL, 1.06 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 2.26 as a light brown solid.
(280) ##STR00164##
(281) LC-MS Retention time 6.08 minutes, found 486.2 [M+H].sup.+; calculated for C.sub.29H.sub.28FN.sub.3O.sub.3 486.56 [M+H].sup.+.
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-(naphthalen-1-yl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.28)
(282) Firstly, 1-(naphthalen-1-yl)piperazine dihydrochloride (2.27; 100 mg, 0.35 mmol, 1 eq) was converted to its freebase form through workup with dichloromethane (3×20 mL) and water (10 mL) using a saturated solution of sodium hydrogencarbonate to neutralise the aqueous phase. Combined organic fractions were dried over MgSO.sub.4, filtered and concentrated in vacuo. Secondly, 1-(naphthalen-1-yl)piperazine freebase (74.4 mg, 0.35 mmol, 1 eq) and 1-cyclopropyl-6-7-difluoro-4-oxo-1-4-dihydroquinoline-3-carboxylic acid (1.4; 83.6 mg, 0.32 mmol, 0.9 eq) were added to DMSO (5 mL) and stirred until full dissolution of both compounds was achieved. The reaction was subsequently heated to 140° C. for one and a half hours. Upon completion, the mixture was allowed to cool and added to an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to remove the DMSO solvent. The eluted crude was purified via trituration; the crude was washed with methanol (5×10 mL), then the remaining powder collected and re-filtered using dichloromethane. This second filtrate was concentrated in vacuo to afford compound 2.28 (67.05 mg, 41.8% yield) as a brown solid.
(283) ##STR00165##
(284) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.05 (br. s., 1H), 8.80 (s, 1H), 8.24-8.29 (m, 1H), 8.07 (d, J=12.84 Hz, 1H), 7.86-7.90 (m, 1H), 7.63 (d, J=8.06 Hz, 1H), 7.47-7.54 (m, 3H), 7.45 (d, J=8.06 Hz, 1H), 7.19 (dd, J=0.88, 7.43 Hz, 1H), 3.63 (br. s., 4H), 3.50 (br. s., 1H), 3.37 (br. s., 4H), 1.45 (br. s., 2H), 1.26 (br. s., 2H); .sup.19F NMR (400 MHz, CDCl.sub.3) δ −120.50; LC-MS Retention time 8.78 minutes, found 458.1 [M+H].sup.+; calculated for C.sub.27H.sub.24FN.sub.3O.sub.3 458.51 [M+H].sup.+.
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-(naphthalen-1-yl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (2.29)
(285) 2.28 (32.94 mg, 0.072 mmol, 1 eq) was added to dichloromethane (5 mL total) and stirred for 10 minutes. Then 4M HCl in dioxane (360 μL, 1.44 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 2.29. (35.27 mg, 99.2% yield) as a grey solid.
(286) ##STR00166##
(287) LC-MS Retention time 8.77 minutes, found 458.1 [M+H].sup.+; calculated for C.sub.27H.sub.24FN.sub.3O.sub.3 458.51 [M+H].sup.+
Synthesis of 1-cyclopropyl-7-(4-(3,5-dimethylbenzyl)piperazin-1-yl)-6-fluoro oxo-1,4-dihydroquinoline-3-carboxylic acid (2.30)
(288) Ciprofloxacin (2.1; 100 mg, 0.30 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and distilled water (5 mL total). After stirring for 5 minutes, potassium carbonate (125 mg, 0.91 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 3,5-dimethylbenzyl bromide (60 mg, 0.29 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 43 hours. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford 2.30 as an off-white solid.
(289) ##STR00167##
(290) LC-MS Retention time 3.03 minutes, found 450.0 [M+H].sup.+; calculated for C.sub.26H.sub.28FN.sub.3O.sub.3 450.53 [M+H].sup.−1.
Synthesis of 1-cyclopropyl-7-(4-(3,5-dimethylbenzyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (2.31)
(291) 2.30 (61.14 mg, 0.13 mmol, 1 eq) was added to dichloromethane (5 mL) and stirred for 5 minutes. Then 4M HCl in dioxane (658 μL, 2.63 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford 2.31 as an off-white solid.
(292) ##STR00168##
(293) LC-MS Retention time 50.92 minutes, found 450.1 [M+H].sup.+; calculated for C.sub.26H.sub.28FN.sub.3O.sub.3 450.53 [M+H].sup.+.
Synthesis of 7-(4-(4-(1H-pyrrol-1-yl)benzyl)piperazin-1-yl)-1-cyclopropyl fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.32)
(294) Ciprofloxacin (2.1; 100 mg, 0.30 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and distilled water (5 mL total). After stirring for 5 minutes, potassium carbonate (125 mg, 0.91 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 1-(4-(bromomethyl)phenyl)-1H-pyrrole (68 mg, 0.29 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 7 days. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford 2.32 (56.90 mg, 40.8% yield) as an off-white solid.
(295) ##STR00169##
(296) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 14.95 (br. s., 1H), 8.71 (s, 1H), 7.94 (d, J=13.09 Hz, 1H), 7.31-7-45 (m, 5H), 7.10 (t, J=2.27 Hz, 2H), 6.36 (t, J=2.27 Hz, 2H), 3.63 (s, 2H), 3.50-3.57 (m, 1H), 3.34-3.41 (m, 4H), 2.66-2.74 (m, 4H), 1.34-1.41 (m, 2H), 1.15-1.22 (m, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 177.0, 167.1, 154-9, 152.4, 147.4, 146.0, 145.9, 139.9, 139.1, 135.1, 130.3, 120.4, 119.7, 119.6, 119.3, 112.4, 112.2, 110.4, 110.1, 108.0, 104.8, 62.3, 52.7, 49.8, 35.3, 8.2; LC-MS Retention time 6.05 minutes, found 487.2 [M+H].sup.+; calculated for C.sub.28H.sub.27FN.sub.4O.sub.3 487.2 [M+H].sup.+.
Synthesis of 7-(4-(4-(1H-pyrrol-1-yl)benzyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (2.33)
(297) 2.32 (40.98 mg, 0.08 mmol, 1 eq) was added to dichloromethane (3 mL) and stirred for 5 minutes. Then 4M HCl in dioxane (421 μL, 1.68 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford 2.33 as an off-white solid.
(298) ##STR00170##
(299) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ15.12 (br. s., 1H), 11.44 (s, 1H), 8.69 (s, 1H), 7.97 (d, J=12.96 Hz, 1H), 7.73 (m, 4H), 7.61 (d, J=7.41 Hz, 1H), 7-45 (t, J=2.31 Hz, 2H), 6.30 (t, J=2.21 Hz, 2H), 4.44 (d, J=5.07 Hz, 2H), 3.93-3.79 (m, 3H), 3.47 (m, 4H), 3.30 (m, 2H), 1.37-1.27 (m, 2H), 1.19 (m, 2H); LC-MS Retention time 5.97 minutes, found 487.1 [M+H].sup.+; calculated for C.sub.28H.sub.27FN.sub.4O.sub.3 487.2 [M+H].sup.+.
Synthesis of 7-(4-(4-(1H-pyrazol-1-yl)benzyl)piperazin-1-yl)-1-cyclopropyl fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.34)
(300) Ciprofloxacin (2.1; 100 mg, 0.30 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and distilled water (5 mL total). After stirring for 5 minutes, potassium carbonate (125 mg, 0.91 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 1-(4-(bromomethyl)phenyl)-1H-pyrazole (68 mg, 0.29 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 96 hours. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford 2.34 (131.70 mg, 94.2% yield) as an off-white solid.
(301) ##STR00171##
(302) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.03 (br. s., 1H), 8.75 (s, 1H), 7.99 (d, J=13.09 Hz, 1H), 7.94 (dd, J=0.50, 2.52 Hz, 1H), 7.72-7.74 (m, 1H), 7.65-7.70 (m, 2H), 7.43-7.47 (m, 2H), 7.35 (d, J=6.55 Hz, 1H), 6.46-6.50 (m, 1H), 3.64 (s, 2H), 3.54 (br. s., 1H), 3.33-3.40 (m, 4H), 2.66-2.74 (m, 4H), 1.38 (m, 2H), 1.19 (br. s., 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 177.1, 167.1, 147.4, 141.1, 139.4, 139.1, 136.1, 130.2 (2C), 126.7, 119.8, 119.2 (2C), 112.5, 112.3, 108.1, 107.7, 104.8, 62.3, 52.7, 49.8, 35.3, 8.2; LC-MS Retention time 2.83 minutes, found 488.1 [M+H].sup.+; calculated for C.sub.27H.sub.26FN.sub.3O.sub.3 488.54 [M+H].sup.+.
Synthesis of 7-(4-(4-(1H-pyrazol-1-yl)benzyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (2.35)
(303) 2.34 (73.78 mg, 0.15 mmol, 1 eq) was added to dichloromethane (3 mL) and stirred for 5 minutes. Then 4M HCl in dioxane (757 μL, 30.03 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford 2.35 as an off-white solid.
(304) ##STR00172##
(305) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 15.11 (s, 1H), 11.73 (s, 1H), 8.67 (s, 1H), 8.58 (d, J=2.52 Hz, 1H), 8.00-7.91 (m, 3H), 7.84-7.76 (m, 3H), 7.60 (d, J=7.41 Hz, in), 6.59-6.57 (m, 1H), 4.45 (s, 2H), 3.93-3.79 (m, 3H), 3.56-3.43 (m, 4H), 3.31 (m, 2H), 1.31 (dd, J=5.51, 7.46 Hz, 2H), 1.21-1.14 (m, 2H); LC-MS Retention time 5.48 minutes, found 488.1 [M+H].sup.+; calculated for C.sub.27H.sub.26FN.sub.3O.sub.3 488.54 [M+H].sup.+.
Synthesis of 7-(4-(4-(1H-1,2,4-triazol-1-yl)benzyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.36)
(306) Ciprofloxacin (2.1; 100 mg, 0.30 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and distilled water (5 mL total). After stirring for 5 minutes, potassium carbonate (125 mg, 0.91 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 1-(4-(bromomethyl)phenyl)-1H-1,2,4-triazole (68 mg, 0.29 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 116 hours. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford 2.36 as an off-white solid.
(307) ##STR00173##
(308) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.02 (br. s., 1H), 8.72 (s, 1H), 8.56 (s, 1H), 8.11 (s, 1H), 7.95 (d, J=13.09 Hz, 1H), 7.63-7.69 (m, 2H), 7.50-7.55 (m, 2H), 7.35 (d, J=7.05 Hz, 1H), 3.67 (s, 2H), 3.50-3.57 (m, 1H), 3.33-3.40 (m, 4H), 2.67-2.75 (m, 4H), 1.34-1.41 (m, 2H), 1.16-1.22 (m, 2H); .sup.13C NMR (1.00 MHz, CDCl.sub.3) δ 177.0, 167.0, 154.9, 152.6, 152.4, 147.4, 145.9, 140.9, 139.1, 138.2, 136.2, 130.4, 120.1, 119.8, 112.5, 112.2, 108.0, 104.8, 62.1, 52.7, 49.8, 35.3, 8.2; LC-MS Retention time 2.73 minutes, found 489.0 [M+H].sup.+; calculated for C.sub.26H.sub.25FN.sub.6O.sub.3 489.52 [M+H].sup.+.
Synthesis of 7-(4-(4-(1H-1,2,4-triazol-1-yl)benzyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (2.37)
(309) 2.36 (19.76 mg, 0.04 mmol, 1 eq) was added to dichloromethane (3 mL) and stirred for 5 minutes. Then 4M HCl in dioxane (202 μL, 0.81 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 18 hours. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford 2.37 as an off-white solid.
(310) ##STR00174##
(311) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.76 (s, .sup.1H), 9.39 (s, 1H), 8.69 (s, 1H), 8.29 (s, .sup.1H), 8.04 (m, 3H), 7.91-7.84 (m, 2H), 7.61 (d, J=7.40 Hz, 1H), 4.48 (s, 2H), 3.93-3.79 (m, 3H), 3.48 (d, J=12.46 Hz, 4H), 3.32 (d, J=11.27 Hz, 2H), 1.31 (dd, J=5.49, 7.53 Hz, 2H), 1.25-1.14 (m, 2H); LC-MS Retention time 5.30 minutes, found 489.1 [M+H].sup.+; calculated for C.sub.26H.sub.23FN.sub.6O.sub.3 489.52 [M+H].sup.+.
Synthesis of 7-(4-(4-(1,2,3-thiadiazol-4-yl)benzyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.38)
(312) Ciprofloxacin (2.1; 100 mg, 0.30 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and distilled water (5 mL total). After stirring for 5 minutes, potassium carbonate (125 mg, 0.91 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 4-(4-(bromomethyl)phenyl)-1,2,3-thiadiazole (73 mg, 0.29 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 42 hours. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford 2.38 (108.9 mg, 75.1% yield) as an off-white solid.
(313) ##STR00175##
(314) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.02 (br. s., 1H), 8.76 (s, 1H), 8.67 (s, 1H), 8.03-80.6 (m, 2H), 8.01 (d, J=13.09 Hz, 1H), 7.53 (d, J=8.31 Hz, 2H), 7.36 (d, J=7.30 Hz, 1H), 3.69 (s, 2H), 3.50-3.57 (m, .sup.1H), 3.35-3.41 (m, 4H), 2.71-2.76 (m, 4H), 1.35-1.41 (m, 2H), 1.17-1.23 (m, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 177.1, 167.1, 162.7, 147.4, 146.0, 145.9, 139.3, 139.1, 129.9, 127.5, 112.6, 112.3, 108.1, 104.8, 62.6, 52.7, 49.9, 35.3, 8.3; LC-MS Retention time 2.87 minutes, found 506.0 [M+H].sup.+; calculated for C.sub.26H.sub.24FN.sub.5O.sub.3S 506.57 [M+H].sup.+.
Synthesis of 7-(4-(4-(1,2,3-thiadiazol-4-yl)benzyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (2.39)
(315) 2.38 (32.49 mg, 0.06 mmol, 1 eq) was added to dichloromethane (3 mL) and stirred for 5 minutes. Then 4M HCl in dioxane (320 μL, 1.28 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford 2.39 as an off white solid.
(316) ##STR00176##
(317) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 15.12 (br. s., 11.60 (br. s., 9.73 (s, 1H), 8.68 (s, 1H), 8.24-8.28 (m, J=8.31 Hz, 2H), 7.96 (d, J=12.84 Hz, 1H), 7.83-7.88 (m, J=8.31 Hz, 2H), 7.61 (d, J=7.30 Hz, 1H), 4.50 (br. s., 2H), 3.81-3.94 (m, 3H), 3.65-3.73 (m, 1H), 3.45-3.54 (m, 5H), 1.29-1.35 (m, 2H), 1.16-1.21 (m, 2H); LC-MS Retention time 5.12 minutes, found 506.0 [M+H].sup.+; calculated for C.sub.26H.sub.24FN.sub.5O.sub.3S 506.57 [M+H].sup.+.
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-(4-(5-methyl-1,2,4-oxadiazol-3-yl)benzyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.40)
(318) Ciprofloxacin (2.1; 100 mg, 0.30 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and distilled water (5 mL total). After stirring for 5 minutes, potassium carbonate (125 mg, 0.91 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 3-(4-(bromomethyl)phenyl)-5-methyl-1,2,4-oxadiazole (73 mg, 0.29 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 96 hours. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford (113.21 mg, 78.4% yield) 2.40 as an off white solid.
(319) ##STR00177##
(320) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.01 (br. s., 1H), 8.73 (s, 1H), 8.01-8.06 (m, J=8.31 Hz, 2H), 7.97 (d, J=13.09 Hz, 1H), 7.46-7.51 (m, J=8.06 Hz, 2H), 7.35 (d, J=6.80 Hz, 1H), 3.66 (s, 2H), 3.54 (br. s., 1H), 3.37 (m, 4H), 2.71 (m, 4H), 2.67 (s, 3H), 1.38 (d, J=5.04 Hz, 2H), 1.19 (br. s., 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 177.1, 176.6, 168.2, 167.1, 147.4, 145.9, 141.2, 139.1, 129.5 (2C), 127.4 (2C), 125.9, 119.7, 112.5, 112.2, 108.1, 104.8, 62.6, 52.8, 49.9, 49.8, 35.3, 12.5, 8.2; LC-MS Retention time 2.87 minutes, Found 504.0 [M+H].sup.+; calculated for C.sub.24H.sub.26FN.sub.5O.sub.4 504.53 [M+H].sup.+
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-(4-(5-methyl-1,2,4-oxadiazol-3-yl)benzyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (2.41)
(321) 2.40 (93.48 mg, 0.19 mmol, 1 eq) was added to dichloromethane (3 mL) and stirred for 5 minutes. Then 4M HCl in dioxane (928 μL, 3.71 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford 2.41 as an off white solid.
(322) ##STR00178##
(323) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.99 (s, 1H), 8.67 (s, 1H), 8.08 (d, J=7.94 Hz, 2H), 7.95 (d, J=13.00 Hz, 1H), 7.89 (d, J=7.98 Hz, 2H), 7.60 (d, J=7.35 Hz, 1H), 4.51 (s, 2H), 3.93-3.81 (m, 3H), 3.60-3.42 (m, 4H), 3.33 (m, 2H), 2.69 (s, 3H), 1.36-1.27 (m, 2H), 1.25-1.13 (m, 2H); LC-MS Retention time 5.58 minutes, Found 504.2 [M+H].sup.+; calculated for C.sub.27H.sub.26FN.sub.5O.sub.4 504.53 [M+H].sup.+
Synthesis of 7-(4-([1,1′-biphenyl]-4-ylmethyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.42)
(324) Ciprofloxacin (2.1; 100 mg, 0.30 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and distilled water (5 mL total). After stirring for 5 minutes, potassium carbonate (125 mg, 0.91 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 4-(bromomethyl)-1,1′-biphenyl (71 mg, 0.29 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 7 days. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford (107.1 mg, 75.1% yield) 2.42, as an off white solid.
(325) ##STR00179##
(326) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.00 (br. s., 1H), 8.75 (s, 1H), 8.00 (d, J=13.09 Hz, 1H), 7.56-7.63 (m, 4H), 7.41-7.48 (m, 4H), 7.33-7.39 (m, 2H), 3.66 (s, 2H), 3.53 (tt, J=3.75, 7.08 Hz, 1H), 3.35-3.41 (m, 4H), 2.70-2.76 (m, 4H), 1.35-1.41 (m, 2H), 1.17-1.22 (m, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 177.1, 167.1, 155.0, 152.5, 147.4, 146.1, 146.0, 140.8, 140.3, 139.1, 136.7, 129.6, 128.8, 127.3, 127.1, 127.1, 119.8, 119.7, 112.5, 112.3, 108.1, 104.8, 62.6, 52.7, 49.9, 49.9, 35.3, 8.2; LC-MS Retention time 6.23 minutes, Found 498.1 [M+H].sup.+; calculated for C.sub.30H.sub.28FN.sub.3O.sub.3 498.57 [M+H].sup.+
Synthesis of 7-(4-([1,1′-biphenyl]-4-ylmethyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (2.43)
(327) 2.42 (85.37 mg, 0.17 mmol, 1 eq) was added to dichloromethane (3 mL) and stirred for 5 minutes. Then 4M HCl in dioxane (858 μL, 3.43 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford 2.43 as an off white solid.
(328) ##STR00180##
(329) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 15.13 (br. s., 1H), 11.32 (br. s., 1H), 8.69 (s, 1H), 7.97 (d, J=13.09 Hz, 1H), 7.69-7.82 (m, 6H), 7.61 (d, J=7.30 Hz, 1H), 7.47-7.53 (m, 2H), 7.38-7.43 (m, 1H), 4.47 (d, J=4.03 Hz, 2H), 3.90 (d, J=12.59 Hz, 2H), 3.81-3.86 (m, 1H), 3.42-3.54 (m, 4H), 3.32 (br. s., 2H), 1.28-1.35 (m, 2H), 1.15-1.22 (m, 2H); LC-MS Retention time 6.18 minutes, Found 498.1 [M+H].sup.+; calculated for C.sub.30H.sub.28FN.sub.3O.sub.3 498.57 [M+H].sup.+
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-(4-(hydroxymethyl)benzyl)piperazin yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.44)
(330) Ciprofloxacin (2.1; 100 mg, 0.30 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and distilled water (5 mL total). After stirring for 5 minutes, potassium carbonate (125 mg, 0.91 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, (4-(bromomethyl)phenyl)methanol (61 mg, 0.30 mmol, 1 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 7 days. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford 2.44 as an off white solid.
(331) ##STR00181##
(332) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.05 (br. s., 1H), 8.75 (s, 1H), 7.99 (d, J=13.09 Hz, al), 7.36 (m, 5H), 4.71 (s, 2H), 3.61 (s, 2H), 3.50-3.56 (m, 1H), 3.32-3.38 (m, 4H), 2.65-2.71 (m, 4H), 1.34-1.40 (m, 2H), 1.16-1.22 (m, 2H).sup.13C NMR (100 MHz, CDCl.sub.3) δ 177.1, 167.1, 155.0, 152.5, 147.4, 146.1, 146.0, 140.0, 139.1, 137.0, 129.4, 128.2, 127.1, 119.8, 119.7, 112.5, 112.3, 108.1, 104.8, 65.1, 62.6, 53.5, 52.6, 49.9, 49.8, 35.3, 8.2; LC-MS Retention time 50.03 minutes, Found 452.2 [M+H].sup.+; calculated for C.sub.25H.sub.26FN.sub.3O.sub.4 452.50 [M+H].sup.+
(333) Synthesis of 1-cyclopropyl-6-fluoro-7-(4-(4-(hydroxymethyl)benzyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (2.45) 2.44 (61.14 mg, 0.13 mmol, 1 eq) was added to dichloromethane (5 mL) and stirred for 5 minutes. Then 4M HCl in dioxane (658 μL, 2.63 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 30 minutes. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford 2.45 as an off white solid.
(334) ##STR00182##
(335) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.66 (br. s., 1H), 8.67 (s, 1H), 70.94 (d, J=13.09 Hz, 1H), 7.63 (d, J=80.6 Hz, 2H), 7.59 (d, J=7.55 Hz, 1H), 7.41 (d, J=7.81 Hz, 2H), 4.54 (s, 2H), 4.39 (d, J=4.53 Hz, 2H), 3.81-3.91 (m, 3H), 3.39-3.54 (m, 4H), 3.21-3.33 (m, 2H), 1.28-1.35 (m, 2H), 1.15-1.20 (m, 2H); LC-MS Retention time 5.10 minutes, Found 452.1 [M+H].sup.+; calculated for C.sub.25H.sub.26FN.sub.3O.sub.4 452.50 [M+H].sup.+
Synthesis of (4-(methoxymethyl)phenyl)methanol (2.47)
(336) (4-(bromomethyl)phenyl)methanol (2.46; 306 mg, 1.52 mmol, 1 eq) was added to excess methanol (20 mL) at room temperature and stirred for 2 minutes. To the resulting brown suspension, potassium carbonate (631 mg, 4.57 mmol, 3 eq) was added and the suspension stirred for a further 5 minutes at room temperature. The mixture was then heated to reflux, with distilled water (5 mL) added during heating. After 15 minutes at reflux, complete dissolution of 2.46 furnished a yellow solution. TLC indicated reaction was complete after 15 hours; product was extracted using ethyl acetate (10 mL) washed with distilled water (3×20 mL). Combined aqueous fractions were back extracted using dichloromethane (20 mL). Combined organic fractions were then dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved via automated flash column chromatography of the crude oil (see Flash Column Chromatography method; 0%-10% DCM/ethyl acetate) to afford pure (56.95 mg, 24.6% yield) 2.47 as a yellow oil.
(337) ##STR00183##
(338) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.31 (s, 4H), 4.62 (d, J=5.54 Hz, 2H), 4.44 (s, 2H), 3.37 (s, 3H), 2.60 (t, J=5.67 Hz, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 140.5, 137.3, 128.0, 127.0, 74.5, 64.9, 58.1
Synthesis of 1-(chloromethyl)-4-(methoxymethyl)benzene (2.48)
(339) (4-(methoxymethyl)phenyl)methanol (2.47; 56.95 mg, 0.37 mmol, 1 eq) was added to anhydrous dichloromethane (1 mL) and the solution cooled to 0° C. Then thionyl chloride (30 μL, 0.41 mmol, 1.1 eq) was added and the reaction stirred at 0° C. for 1 hour, then warmed to room temperature over a second hour. After 3 hours, a catalytic amount of DMF (3 drops) was added. TLC indicated reaction was complete after 20 hours; reaction was quenched by the addition of a saturated solution of NaHCO.sub.3 (1 mL) dropwise, then the product was extracted using dichloromethane (10 mL) washed with distilled water (3×10 mL). TLC confirmed reaction was spot-10-spot; (43.76 mg, 68.5% yield) 2.48 as a colourless oil, 2.48 was characterised and used in subsequent reactions without further purification.
(340) ##STR00184##
(341) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.39 (d, 2H), 7.35 (d, 2H), 4.60 (s, 2H), 4.47 (s, 2H), 3.40 (s, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 138.6, 136.9, 128.7, 128.0, 74.2, 58.2, 46.1
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-(4-(methoxymethyl)benzyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.49)
(342) Ciprofloxacin (2.1; 29 mg, 0.09 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and distilled water (2 mL total). After stirring for 5 minutes, potassium carbonate (36 mg, 0.26 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 1-(chloromethyl)-4-(methoxymethyl)benzene (14 mg, 0.08 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 17 hours. Reaction was subsequently heated to reflux and stirred for 24 hours. Upon completion, the product was extracted with dichloromethane (3×10 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (10 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Flash column chromatography (0%-100% DCM/acetone) was employed to afford pure (35.05 mg, 90.6% yield) 2.49 as an off white solid.
(343) ##STR00185##
(344) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.06 (br. s., 1H), 8.76 (s, 1H), 8.00 (d, J=13.09 Hz, 1H), 7.30 7.38 (m, 5H), 4.46 (s, 2H), 3.61 (s, 2H), 3.50-3.56 (m, 1H), 3.42 (s, 3H), 3.33-3.38 (m, 4H), 2.66-2.71 (m, 4H), 1.34-1.39 (m, 2H), 1.16-1.22 (m, 2H); LC-MS Retention time 2.87 minutes, Found 466.1 [M+H].sup.+; calculated for C.sub.26H.sub.28FN.sub.3O.sub.4 466.53 [M+H]F
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-(4-(methoxymethyl)benzyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (2.50)
(345) 2.49 (11.14 mg, 0.02 mmol, 1 eq) was added to dichloromethane (2 mL) and stirred for 5 minutes. Then 4M HCl in dioxane (120 μL, 0.48 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×10 mL), concentrated in vacuo and lyophilised for 24 hours to afford 2.50 as an off white solid.
(346) ##STR00186##
(347) LC_MS Retention time 4.87 minutes, Found 466.2 [M+H].sup.+; calculated for C.sub.26H.sub.28FN.sub.3O.sub.4 466.53 [M+H].sup.+
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-(3-(methoxymethyl)benzyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.51)
(348) Ciprofloxacin (2.1; 100 mg, 0.30 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and distilled water (5 mL total). After stirring for 5 minutes, potassium carbonate (125 mg, 0.91 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 1-(bromomethyl)-3-(methoxymethyl)benzene (62 mg, 0.29 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 88 hours. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Flash column chromatography (0%-20% DCM/acetone, silica deactivated via 3% triethylamine in DCM wash) was employed to afford pure 2.51 as an off white solid.
(349) ##STR00187##
(350) LC-MS Retention time 5.45 minutes, Found 466.1 [M+H].sup.+; calculated for C.sub.26H.sub.28FN.sub.3O.sub.4 466.53 [M+H].sup.+
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-(3-(methoxymethyl)benzyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (2.52)
(351) 2.51 (16 mg, 0.03 mmol, 1 eq) was added to dichloromethane (3 mL) and stirred for 5 minutes. Then 4M HCl in dioxane (172 μL, 0.69 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 40 minutes. The mixture was then washed with hexane (3×20 mL), concentrated in vacuo and lyophilised for 24 hours to afford 2.52 as an off white solid.
(352) ##STR00188##
(353) LC-MS Retention time 4.83 minutes, Found 466.2 [M+H].sup.+; calculated for C.sub.26H.sub.28FN.sub.3O.sub.4 466.53 [M+H].sup.+
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-(3-(methoxymethyl)benzyl)-piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.53)
(354) Ciprofloxacin (2.1; 100 mg, 0.30 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and distilled water (5 mL total). After stirring for 5 minutes, potassium carbonate (125 mg, 0.91 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, N-(2-(1H-indol-3-yl)ethyl)-4-(bromomethyl)benzenesulfonamide (119 mg, 0.30 mmol, 1 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 7 days. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford 2.53 as an off white solid.
(355) ##STR00189##
(356) LC-MS Retention time 3.13 minutes, Found 644.0 [M+H].sup.+; calculated for C.sub.34H.sub.34FN.sub.5O.sub.5S 644.73 [M+H].sup.+
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-(3-(methoxymethyl)benzyl)-piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (2.54)
(357) 2.53 (61.14 mg, 0.13 mmol, 1 eq) was added to dichloromethane (5 mL) and stirred for 5 minutes. Then 4M HCl in dioxane (658 μL, 2.63 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford 2.54 as an off white solid.
(358) ##STR00190##
(359) LC-MS Retention time 6.03 minutes, Found 644.2 [M+H].sup.+; calculated for C.sub.34H.sub.34FN.sub.5O.sub.5S 644.73 [M+H].sup.+
Synthesis of Norfloxacin-ARB Hybrid Compounds
Synthesis of 1-ethyl-6-fluoro-7-(4-(naphthalen-1-ylmethyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3.2)
(360) Norfloxacin (3.1; 1 g, 3.13 mmol, 1 eq) was added to DMF (10 mL total) and stirred for 105 minutes at 115° C. Then 1-(bromomethyl)naphthalene (685 mg, 30.13 mmol, 1 eq) and potassium carbonate (1250 mg, 9.04 mmol, 2.9 eq) were added and the mixture stirred for a further 1 hour at reflux. The mixture was allowed to cool, then extracted with ethyl acetate (2×50 mL). Combined organic fractions were washed with distilled water (20 mL) and brine (20 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford compound 3.2 (1.00 g, 69.5% yield) as a tan solid.
(361) ##STR00191##
(362) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.13 (s, 1H), 8.66 (s, 1H), 8.33 (d, J=8.06 Hz, 1H), 8.07 (d, J=13.09 Hz, 1H), 7.89 (d, J=7.05 Hz, 1H), 7.82 (d, J=7.30 Hz, 1H), 7.49-7.57 (m, 2H), 7.45 (q, J=7.22 Hz, 2H), 6.81 (d, J=7.05 Hz, 1H), 4.23-4.32 (m, 2H), 4.02 (s, 2H), 3.33 (br s., 4H), 2.76 (br. s., 4H), 1.56 (t, J=6.92 Hz, 3H); LC-MS Retention time 3.05 minutes, found 460.0 [M+H].sup.+; calculated for C.sub.27H.sub.26FN.sub.3O.sub.3 460.52 [M+H].sup.+.
Synthesis of 1-ethyl-6-fluoro-7-(4-(naphthalen-1-ylmethyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (3.3)
(363) 3.2 (30 mg, 0.07 mmol, 1 eq) was added to methanol (10 mL total) and stirred for 10 minutes. Then 4M HCl in dioxane (33 μL, 0.13 mmol, 2 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 3.3 (42.06 mg, 95.7% yield) as a light brown solid.
(364) ##STR00192##
(365) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 15.25 (br. s., 1H), 11.17 (br. s., 1H), 8.96 (s, 1H), 8.49 (d, J=8.56 Hz, 1H), 80.8 (d, J=8.31 Hz, 1H), 8.04 (d, J=7.05 Hz, 2H), 7.95 (d, J=13.35 Hz, 1H), 7.68 (ddd, J=1.38, 6.86, 8.37 Hz, 1H), 7.59-7.65 (m, 2H), 7.23 (d, J=7.30 Hz, 1H), 4.93 (hr. s., 2H), 40.59 (q, J=7.05 Hz, 2H), 3.83-3.94 (m, 2H), 3.48 (br. s., 6H), 1.40 (t, J=7.18 Hz, 3H); .sup.13C NMR (100 MHz, DMSO-d.sub.6) δ 176.1 (C═O), 166.0 (CO.sub.2H), 152.6 (C-6, .sup.1J(C-F)=247 Hz), 148.7 (C-2), 143.8 (C-7, .sup.2J(C-F)=11 Hz), 137.1, 133.4, 132.2, 131.7, 130.4, 128.8, 127.1, 126.3, 125.3, 124.1, 119.8 (C-4a, .sup.3J(C-F)=8 Hz), 111.4 (C-5, .sup.2J(C-F)=23 Hz), 107.1, 106.3, 54.9, 50.4, 49.1, 46.3, 30.7, 14.4; IR (υ.sub.max/cm.sup.−1) 3389, 2921, 1700, 1628, 1457, 1267, 1195, 1104, 937, 802; LC-MS Retention time 5.85 minutes, found 460.1 [M+H].sup.+; calculated for C.sub.27H.sub.26FN.sub.3O.sub.3 460.52 [M+H].sup.+; HRMS Observed 460.2020 [M+H].sup.+; theoretical value 460.2031 [M+H].sup.+.
Synthesis of 1-ethyl-6-fluoro-7-(4-(naphthalen-1-ylmethyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3.4)
(366) Norfloxacin (3.1; 200 mg, 0.63 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and water (10 mL total). After stirring for 5 minutes, potassium carbonate (260 mg, 1.88 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 2-(bromomethyl)naphthalene (132 mg, 0.59 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 24 hours. Upon completion, the product was extracted with dichloromethane (2×30 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (30 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford compound 3.4 (126.00 mg, 46.1% yield) as a pale yellow solid.
(367) ##STR00193##
(368) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.16 (br. s., 1H), 8.64 (s, 1H), 8.00 (d, J=13.09 Hz, 1H), 7.80-7.86 (m, 3H), 7.77 (s, 1H), 7.53 (dd, J=1.64, 8.44 Hz, 1H), 7.45-7.51 (m, 2H), 6.81 (d, J=6.80 Hz, 1H), 4.29 (q, J=7.05 Hz, 2H), 3.77 (s, 2H), 3.32-3.39 (m, 4H), 2.69-2.77 (m, 4H), 1.55 (t, J=7.18 Hz, 3H); LC-MS Retention time 2.95 minutes, found 460.1 [M+H].sup.+; calculated for C.sub.27H.sub.26FN.sub.3O.sub.3 460.52 [M+H].sup.+.
Synthesis of 1-ethyl-6-fluoro-7-(4-(naphthalen-1-ylmethyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (3.5)
(369) 3.4 (30 mg, 0.065 mmol, 1 eq) was added to equal parts methanol and dioxane (40 mL total) and stirred for 10 minutes. Then 4M HCl in dioxane (33 μL, 0.131 mmol, 2 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 3.5 (23.95 mg, 74.0% yield) as an off white solid.
(370) ##STR00194##
(371) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 15.28 (br. s., 1H), 11.52 (br. s., 1H), 8.97 (s, 1H), 8.17 (s, 1H), 8.03 (d, J=8.56 Hz, 1H), 7.93-8.01 (m, 3H), 7.84 (d, J=7.05 Hz, 1H), 7.60 (m, 2H), 7.26 (d, J=7.05 Hz, 1H), 4.56-4.65 (m, 4H), 3.88 (d, J=12.34 Hz, 2H), 3.44-3.54 (m, 4H), 3.27-3.32 (m, 2H), 1.40 (t, J=7.18 Hz, 3H); .sup.13C NMR (100 MHz, DMSO-d.sub.6) δ 176.2, 166.0, 152.6 (C-6, .sup.1J(C-F)=248 Hz), 148.7, 137.1, 133.1, 132.5, 131.3, 128.4, 128.2, 128.0, 127.7, 127.1, 127.0, 126.7, 119.9, 115.6, 111.4 (C-5, .sup.2J(C-F)=24 Hz), 107.2, 106.5, 58.8, 50.2, 49.1, 46.3, 14.4; IR (υ.sub.max/cm.sup.−1) 2922, 2500, 2399) 1718, 1627, 1516, 1456, 1414, 1279, 1099 961, 802, 746; LC-MS Retention time 5.95 minutes, found 460.1 [M+H].sup.+; calculated for C.sub.27H.sub.26FN.sub.3O.sub.3 460.52 [M+H].sup.+; HRMS Observed 460.2019 [M+H].sup.+; theoretical value 460.2031 [M+H].sup.+.
Synthesis of 1-ethyl-6-fluoro-7-(4-benzylpiperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3.6)
(372) Norfloxacin (3.1; 100 mg, 0.31 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and water (10 mL total). After stirring for 5 minutes, potassium carbonate (130 mg, 0.94 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, bromomethylbenzene (51 mg, 0.30 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 24 hours. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford compound 3.6 (770.00 mg, 63.2% yield) as an off white solid.
(373) ##STR00195##
(374) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.14 (br. s., 1H), 8.66 (s, 1H), 8.02 (d, J=13.09 Hz, 1H), 7.32-7.38 (m, 4H), 7.28-7.32 (m, 1H), 6.82 (d, J=6.80 Hz, 1H), 4.31 (q, J=7.05 Hz, 2H), 3.61 (s, 2H), 3.31-3.37 (m, 4H), 2.66-2.72 (m, 4H), 1.57 (t, J=6.92 Hz, 3H); LC-MS Retention time 2.95 minutes, found 410.0 [M+H].sup.+; calculated for C.sub.23H.sub.24FN.sub.3O.sub.3 410.46 [M+H].sup.+.
Synthesis of 1-ethyl-6-fluoro-7-(4-benzylpiperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (3.7)
(375) 3.6 (30 mg, 0.073 mmol, 1 eq) was added to methanol (25 mL) and stirred for 10 minutes. Then 4M HCl in dioxane (37 μL, 0.15 mmol, 2 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 3.7 as a yellow solid.
(376) ##STR00196##
(377) .sup.1H NMR (400 MHz, TFA) δ 9.21 (s, 1H), 8.22 (d, J=12.59 Hz, 1H), 7.43-7.53 (m, 3H), 7.37-7.43 (m, 3H), 4.77 (q, J=7.47 Hz, 2H), 4.45 (s, 2H), 4.14 (d, J=13.60 Hz, 2H), 3.82 (d, J=12.09 Hz, 2H), 3.56-3.66 (m, 2H), 3.38-3.48 (m, 2H), 1.67 (t, J=7.18 Hz, 3H); IR (υ.sub.max/cm.sup.−1) 3390, 2358, 1700, 1628, 1457, 1420, 1271, 1104, 961, 804, 747, 699; LC-MS Retention time 5.37 minutes, found 410.0 [M+H].sup.+; calculated for C.sub.23H.sub.24FN.sub.3O.sub.3 410.46 [M+H].sup.+; HRMS Observed 410.1863 [M+H].sup.+; theoretical value 410.1874 [M+H].sup.+.
Synthesis of 1-ethyl-6-fluoro-7-(4-(benzo[d][1,3]dioxol-4-ylmethyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3.8)
(378) Norfloxacin (3.1; 157 mg, 0.49 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and water (10 mL total). After stirring for 5 minutes, potassium carbonate (203 mg, 1.47 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 4-(bromomethyl)benzo[d][1,3]dioxole (100 mg, 0.47 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 24 hours. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford compound 3.8 (178.48 mg, 84.3% yield) as a white solid.
(379) ##STR00197##
(380) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.13 (br. s., 1H), 8.66 (s, 1H), 8.03 (d, J=13.35 Hz, 1H), 6.76-6.87 (m, 4H), 5.98 (s, 2H), 4.27-4.35 (m, 2H), 3.63 (s, 2H), 3.32-3.38 (m, 4H), 2.69-2.75 (m, 4H), 1.57 (t, J=6.80 Hz, 3H); LC-MS Retention time 2.87 minutes, found 454.1 [M+H].sup.+; calculated for C.sub.24H.sub.24FN.sub.3O.sub.5 454.47 [M+H].sup.+.
Synthesis of 1-ethyl-6-fluoro-7-(4-(benzo[d][1,3]dioxol-4-ylmethyl)-piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (3.9)
(381) 3.8 (20 mg, 0.044 mmol, 1 eq) was added to equal parts methanol and dioxane (60 mL total) and stirred for 10 minutes. Then 4M HCl in dioxane (22 μL, 0.088 mmol, 2 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 3.9 (15.58 mg, 70.4% yield) as a white solid.
(382) ##STR00198##
(383) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 15.27 (br. s., 1H), 11.47 (br. s., 1H), 8.98 (s, 1H), 7.96 (d, J=13.35 Hz, 1H), 7.26 (d, J=7.30 Hz, 1H), 7.18 (d, J=7.55 Hz, 1H), 7.03 (d, J=7.81 Hz, 1H), 6.94 (t, J=7.93 Hz, 1H), 6.11 (s, 2H), 4.62 (q, J=7.22 Hz, 2H), 4.36 (br. s., 2H), 3.89 (d, J=12.59 Hz, 2H), 3.44-3.56 (m, 4H), 3.22-3.34 (m, 2H), 1.40 (t, J=7.18 Hz, 3H); .sup.13C NMR (100 MHz, DMSO-d.sub.6) δ 176.1, 166.0, 151.4, 147.5, 147.3, 137.1, 125.0, 121.9, 120.0, 119.9, 111.5, 111.3, 110.3, 109.8, 107.2, 106.5, 101.3, 52.5, 50.1, 49.1, 46.3, 14.4; IR 2906, 2358, 1700, 1628, 1464, 1378, 1251, 1051, 958, 807, 725; LC-MS Retention time 5.48 minutes, found 454.0 [M+H].sup.+; calculated for C.sub.24H.sub.24FN.sub.3O.sub.5 454.47 [M+H].sup.+; HRMS Observed 4540.1763 [M+H].sup.+; theoretical value 4540.1773 [M+H].sup.+.
Synthesis of 1-ethyl-6-fluoro-7-(4-(benzo[b]thiophen-7-ylmethyl)piperazin yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3.10)
(384) Norfloxacin (3.1; 174 mg, 0.54 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and water (5 mL total). After stirring for 5 minutes, potassium carbonate (226 mg, 1.63 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 7-(bromomethyl)benzo[b]thiophene (118 mg, 0.52 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 24 hours. Upon completion, extraction using dichloromethane (2×30 mL), using a 1M solution of citric acid to neutralise the aqueous phase, resulted in formation of a white precipitate. The precipitate was filtered, washed with distilled water (3×20 mL), re-suspended in dichloromethane (2 mL) and purified via automated column chromatography (see Flash Column Chromatography method) to afford compound 3.10 (84.50 mg, 35.1% yield) as a yellow solid.
(385) ##STR00199##
(386) LC-MS Retention time 3.13 minutes, found 466.0 [M+H].sup.+; calculated for C.sub.25H.sub.24FN.sub.3O.sub.3S 466.54 [M+H].sup.+.
Synthesis of 1-ethyl-6-fluoro-7-(4-(benzo[b]thiophen-7-ylmethyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (3.11)
(387) 3.10 (20 mg, 0.043 mmol, 1 eq) was added to methanol (10 mL) and stirred for 10 minutes. Then 4M HCl in dioxane (21 μL, 0.086 mmol, 2 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 3.11 (11.19 mg, 76.9% yield) as a pale yellow solid.
(388) ##STR00200##
(389) IR (υ.sub.max/cm.sup.−1) 3394, 1718, 1624, 1457, 1257, 943, 803; LC-MS Retention time 6.03 minutes, found 466.0 [M+I-1]+; calculated for C.sub.25H.sub.24FN.sub.3O.sub.3S 466.54 [M+H].sup.+; HRMS Observed 466.1584 [M+H].sup.+; theoretical value 466.1595 [M+H].sup.+.
Synthesis of 1-ethyl-6-fluoro-7-(4-((4-fluoronaphthalen-1-yl)methyl)-piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3.12)
(390) Norfloxacin (3.1; 100 mg, 0.31 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and water (5 mL total). After stirring for 5 minutes, potassium carbonate (130 mg, 0.94 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 1-(bromomethyl)-4-fluoronaphthalene (71 mg, 0.30 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 24 hours. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford compound 3.12 (64.91 mg, 45.7% yield) as a white solid.
(391) ##STR00201##
(392) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.13 (s, 1H), 8.65 (s, 1H), 8.31-8.35 (m, 1H), 8.12-8.17 (m, 1H), 8.04 (d, J=13.09 Hz, 1H), 7.55-7.62 (m, 2H), 7.38 (dd, J=5.41, 7.68 Hz, 1H), 7.09 (dd, J=7.81, 10.32 Hz, 1H), 6.80 (d, J=7.05 Hz, 1H), 4.28 (q, J=7.05 Hz, 2H), 3.97 (s, 2H), 3.29-3.34 (m, 4H), 2.71-2.76 (m, 4H), 1.55 (t, J=7.05 Hz, 3H); IR Retention time 3.13 minutes, found 478.0 [M+H].sup.+; calculated for C.sub.27H.sub.23F.sub.2N.sub.3O.sub.3 478.51 [M+H].sup.+.
Synthesis of 1-ethyl-6-fluoro-7-(4-((4(4-fluoronaphthalen-1-yl)methyl)-piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (3.13)
(393) 3.12 (50 mg, 0.10 mmol, 1 eq) was added to methanol (25 mL) and stirred for to minutes. Then 4M HCl in dioxane (52 μL, 0.21 mmol, 2 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 3.13 (43.22 mg, 77.5% yield) as a white solid.
(394) ##STR00202##
(395) .sup.1H NMR (400 MHz, DMSO-d.sub.6+TFA) δ 8.98 (s, 1H), 8.55 (d, J=7.55 Hz, 1H), 8.17 (d, J=8.56 Hz, 1H), 7.95-8.03 (m, 2H), 7.72-7.83 (m, 2H), 7.48-7.55 (m, 1H), 7.24 (d, J=7.55 Hz, 1H), 4.89-4.94 (m, 2H), 4.60 (q, J=6.88 Hz, 2H), 3.90 (d, J=11.58 Hz, 2H), 3.51 (br. s., 4H), 3.39 (br. s., 2H), 1.40 (t, J=7.05 Hz, 3H); IR (υ.sub.max/cm.sup.−1) 3385, 2928, 1715, 1628, 1457, 1387, 1266, 1044, 942, 805; LC-MS Retention time 6.07 minutes, found 478.1 [M+H].sup.+; calculated for C.sub.27H.sub.23F.sub.2N.sub.3O.sub.3 478.51 [M+H].sup.+; HRMS Observed 478.1925 [M+H].sup.+; theoretical value 478.1937 [M+H].sup.+.
Synthesis of 1-ethyl-6-fluoro-7-(4-((2-oxo-1,2-dihydroquinolin-4-yl)-methyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3.14)
(396) Norfloxacin (3.1; 1 g, 3.13 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and water (50 mL total). After stirring for 5 minutes, potassium carbonate (1298 mg, 9.39 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 4-(bromomethyl)quinolin-2(1H)-one (708 mg, 2.97 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 24 hours. Upon completion, extraction using dichloromethane (2×100 mL), using a 1M solution of citric acid to neutralise the aqueous phase, resulted in formation of a white precipitate. The precipitate was filtered, washed with distilled water (100 mL) and methanol (100 mL) then re-dissolved in excess DMSO. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford compound 3.14 (1.26 g, 88.9% yield) as an off white solid.
(397) ##STR00203##
(398) LC-MS Retention time 2.87 minutes, found 477.0 [M+H].sup.+; calculated for C.sub.26H.sub.25FN.sub.4O.sub.4 477.51 [M+H].sup.+.
Synthesis of 1-ethyl-6-fluoro-7-(4-((2-oxo-1,2-dihydroquinolin-4-yl)-methyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (3.15)
(399) 3.14 (too mg, 0.21 mmol, 1 eq) was added to methanol (25 mL) and stirred for to minutes. Then 4M HCl in dioxane (105 μL, 0.42 mmol, 2 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 3.15 (94.53 mg, 87.8% yield) as an off white solid.
(400) ##STR00204##
(401) .sup.1H NMR (400 MHz, TFA-d) δ 9.23 (s, 1H), 8.23 (d, J=12.84 Hz, 1H), 8.19 (d, J=7.55 Hz, 1H), 7.89-7.95 (m, 1H), 7.77 (d, J=7.30 Hz, 1H), 7.69 (br. s., 2H), 7.45 (br. s., 1H), 5.05 (br. s., 2H), 4.79 (br. s., 2H), 4.16 (d, J=9.06 Hz, 2H), 4.00-4.10 (m, 2H), 3.70-3.89 (m, 4H), 1.68 (t, J=6.04 Hz, 3H); IR (υ.sub.max/cm.sup.−1) 2975, 2365, 1700, 1663, 1628, 1517, 1477, 1437, 1374, 1273, 957, 805; LC-MS Retention time 5.52 minutes, found 477.0 [M+H].sup.+; calculated for C.sub.26H.sub.25FN.sub.4O.sub.4 477.51 [M+H].sup.+; HRMS Observed 477.1923 [M+H].sup.+; theoretical value 477.1933 [M+H].sup.+.
Synthesis of 1-ethyl-6-fluoro-7-(4-(quinolin-8-ylmethyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3.16)
(402) Norfloxacin (3.1; 100 mg, 0.31 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and water (10 mL total). After stirring for 5 minutes, potassium carbonate (130 mg, 0.94 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 8-(bromomethyl)quinoline (66 mg, 0.30 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 24 hours. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford compound 3.16 (67.52 mg, 49.3% yield) as an off white solid.
(403) ##STR00205##
(404) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.15 (br. s., 1H), 8.96 (dd, J=1.76, 4.28 Hz, 1H), 8.67 (s, 1H), 8.19 (dd, J=1.76, 8.31 Hz, 1H), 8.06 (d, J=13.35 Hz, 1H), 7.90 (d, J=7.05 Hz, 1H), 7.78 (dd, J=1.26, 8.06 Hz, 1H), 7.57 (dd, J=7.05, 80.6 Hz, 1H), 7.44 (dd, J=4.15, 8.18 Hz, 1H), 6.84 (d, J=6.80 Hz, 1H), 4.40 (s, 2H), 4.31 (q, J=7.55 Hz, 2H), 3.38-3.44 (m, 4H), 2.84-2.90 (m, 4H), 1.58 (t, J=7.18 Hz, 3H); LC-MS Retention time 2.85 minutes, found 461.0 [M+H].sup.+; calculated for C.sub.26H.sub.25FN.sub.4O.sub.3 461.51 [M+H].sup.+.
Synthesis of 1-ethyl-6-fluoro-7-(4-(quinolin-8-ylmethyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (3.17)
(405) 3.16 (50 mg, 0.11 mmol, 1 eq) was added to methanol (25 mL) and stirred for 10 minutes. Then 4M HCl in dioxane (54 μL, 0.22 mmol, 2 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 3.17 as a brown solid.
(406) ##STR00206##
(407) .sup.1H NMR (400 MHz, TFA-d) δ 9.31 (d, J=4.78 Hz, 1H), 9.21-9.27 (m, 2H), 8.55 (d, J=7.30 Hz, 1H), 8.49 (d, J=80.6 Hz, 1H), 8.18-8.29 (m, 2H), 8.13 (t, J=7.55 Hz, 1H), 7.44-7.51 (m, 1H), 5.37 (br. s., 2H), 4.76-4.86 (m, 2H), 4.21 (d, J=11.58 Hz, 2H), 3.96-4.05 (m, 2H), 3.88 (t, J=10.32 Hz, .sup.2H), 3.77 (t, J=13.09 Hz, 2H), 1.70 (t, J=6.42 Hz, 3H); IR (υ.sub.max/cm.sup.−1) 3393, 2377, 1700, 1624, 1457, 1388, 1270, 1195, 953, 935, 834, 805, 750; LC-MS Retention time 5.48 minutes, found 461.0 [M+H].sup.+; calculated for C.sub.26H.sub.25FN.sub.4O.sub.3 461.51 [M+H].sup.+; HRMS Observed 461.1973 [M+H].sup.+; theoretical value 461.1973 [M+H].sup.+.
Synthesis of 1-ethyl-6-fluoro-7-(4-((5,6,7,8-tetrahydronaphthalen-1-yl)methyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3.18)
(408) Norfloxacin (3.1; 100 mg, 0.31 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and water (10 mL total). After stirring for 5 minutes, potassium carbonate (130 mg, 0.94 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 5-(bromomethyl)-1,2,3,4-tetrahydronaphthalene (67 mg, 0.30 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 24 hours. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford compound 3.18 (59.70 mg, 43.2% yield) as a pale brown solid.
(409) ##STR00207##
(410) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.15 (br. s., 1H), 8.67 (s, 1H), 8.04 (d, J=13.09 Hz, 1H), 7.01-7.14 (m, 3H), 6.83 (d, J=6.29 Hz, 1H), 4.31 (d, J=6.80 Hz, 2H), 3.52 (s, 2H), 3.28-3.35 (m, 4H), 2.78-2.87 (m, 4H), 2.65-2.71 (m, 4H), 1.75-1.87 (m, 4H), 1.58 (t, J=6.55 Hz, 3H); LC-MS Retention time 3.18 minutes, found 4640.1 [M+H].sup.+; calculated for C.sub.27H.sub.30FN.sub.3O.sub.3 464.55 [M+H].sup.+.
Synthesis of 1-ethyl-6-fluoro-7-(4-((5,6,7,8-tetrahydronaphthalen yl)methyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (3.19)
(411) 3.18 (30 mg, 0.065 mmol, 1 eq) was added to methanol (10 mL) and stirred for 10 minutes. Then 4M HCl in dioxane (32 μL, 0.13 mmol, 2 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 3.19 (32.57 mg, 100% yield) as a pale brown solid.
(412) ##STR00208##
(413) 13C NMR (100 MHz, DMSO-d.sub.6) δ 176.1, 166.0, 148.8, 137.9, 137.4, 137.1, 130.6, 129.7, 127.8, 125.3, 107.2, 55.7, 50.5, 49.1, 46.2, 29.5, 25.8, 22.6, 22.0, 14.4; IR (υ.sub.max/cm.sup.−1) 2929, 2363, 1718, 1700, 1628, 1473, 1261, 1043, 1007, 946, 891, 804, 750; LC-MS Retention time 6.00 minutes, found 4640.0 [M+H].sup.+; calculated for C.sub.27H.sub.30FN.sub.3O.sub.3 464.55 [M+H].sup.+; HRMS Observed 464.2333 [M+H].sup.+; theoretical value 464.2344 [M+H].sup.+.
Synthesis of 7-(4-(4-(dimethylamino)benzyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3.20)
(414) Norfloxacin (3.1; 100 mg, 0.31 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and water (5 mL total). After stirring for 5 minutes, potassium carbonate (130 mg, 0.94 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 4-(bromomethyl)-N,N-dimethylanilene (64 mg, 0.30 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 24 hours. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product.
(415) Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford compound 3.20 (75.04 mg, 55.7% yield) as a yellow solid.
(416) ##STR00209##
(417) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.14 (br. s., 1H), 8.67 (s, 1H), 8.05 (d, J=13.09 Hz, 1H), 7.19-7.23 (m, 2H), 6.82 (d, J=7.05 Hz, 1H), 6.70-6.74 (m, 2H), 4.31 (q, J=6.88 Hz, 2H), 3.52 (s, 2H), 3.29-3.37 (m, 4H), 2.96 (s, 6H), 2.63-2.70 (m, 4H), 1.57 (t, J=6.80 Hz, 3H); LC-MS Retention time 5.43 minutes, found 453.1 [M+H].sup.+; calculated for C.sub.25H.sub.29FN.sub.4O.sub.3 453.53 [M+H].sup.+.
Synthesis of 7-(4-(4-(dimethylamino)benzyl)piperazin-1-yl)-1-ethyl-6-fluoro oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (3.21)
(418) 3.20 (56.26 mg, 0.12 mmol, 1 eq) was added to dichloromethane (5 mL) and stirred for 5 minutes. Then 4M HCl in dioxane (622 μL, 2.49 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 3.21 (59.94 mg, 98.6% yield) as a yellow solid.
(419) ##STR00210##
(420) .sup.1H NMR (400 MHz, DMSO-d.sub.6+TFA) δ 8.94 (s, 1H), 7.92 (d, J=13.09 Hz, 1H), 7.72-7.77 (m, J=8.56 Hz, 2H), 7.45-7.51 (m, J=8.31 Hz, 2H), 7.25 (d, J=7.30 Hz, 1H), 4.60 (q, J=6.97 Hz, 2H), 4.40 (s, 2H), 3.86 (d, J=11.33 Hz, 2H), 3.53 (br. s., 2H), 3.35-3.42 (m, 2H), 3.25 (br. s., 2H), 3.06 (s, 6H), 1.35-1.42 (m, 3H); 13C NMR (100 MHz, DMSO-d.sub.6) δ 176.1, 166.0, 152.6 (C-6, .sup.1J(C-F)=248 Hz), 148.6, 146.9, 143.9 (C-7, .sup.2J(C-F)=10 Hz), 137.1, 132.9, 119.9 ((C-F)=8 Hz), 117.8, 111.4 (C-5, .sup.2J(C-F)=23 Hz), 107.1, 106.4, 66.3, 57.8, 49.8, 49.1, 46.2, 43.2, 14.4; IR (υm/cm.sup.−1) 3411, 2507, 2433, 1718, 1627, 1517, 1472, 1417, 1276, 1100, 961, 897, 803, 746, 591; LC-MS Retention time 5.50 minutes, found 453.1 [M+H].sup.+; calculated for C.sub.25H.sub.29FN.sub.4O.sub.3 453.53 [M+H].sup.+; HRMS Observed 453.2286 [M+H].sup.+; theoretical value 4530.2296 [M+H].sup.+.
Synthesis of 1-ethyl-6-fluoro-7-(4-(2-(naphthalen-1-yl)ethyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3.22)
(421) 1-ethyl-6,7-difluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (1.9; 100 mg, 0.39 mmol, 1 eq) and 1-(2-(naphthalen-1-yl)ethyl)piperazine (2.24; 190 mg, 0.79 mmol, 2 eq) were added to DMSO (5 mL) and stirred until full dissolution of both compounds was achieved. The reaction was subsequently heated to 140° C. for one and a half hours. Upon completion, the mixture was allowed to cool for 10 minutes and formation of a precipitate was observed.
(422) The crude precipitate was purified via trituration; the crude was filtered, then washed with methanol (5×10 mL), then the remaining powder collected and re-filtered using dichloromethane. This second filtrate was concentrated in vacuo to afford compound 3.22 (75 mg, 40.1% yield) as a light brown solid.
(423) ##STR00211##
(424) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.12 (br. s., 1H), 8.69 (s, 1H), 8.05-8.11 (m, 2H), 7.86-7.90 (m, 1H), 7.76 (d, J=7.55 Hz, 1H), 7.48-7.58 (m, 2H), 7.37-7.45 (m, 2H), 6.86 (d, J=7.05 Hz, 1H), 4.34 (q, J=7.30 Hz, 2H), 3.37-3.44 (m, 4H), 3.31-3.37 (m, 2H), 2.81-2.88 (m, 6H), 1.61 (t, J=7.30 Hz, 3H); .sup.19F NMR (400 MHz, CDCl.sub.3) δ −120.46; LC-MS Retention time 6.05 minutes, found 474.1 [M+H].sup.+; calculated for C.sub.28H.sub.28FN.sub.3O.sub.3 474.55 [M+M].sup.+.
Synthesis of 1-ethyl-6-fluoro-7-(4-(2-(naphthalen-1-yl)ethyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (3.23)
(425) 3.22 (40.28 mg, 0.085 mmol, 1 eq) was added to dichloromethane (5 mL total) and stirred for 10 minutes. Then 4M HCl in dioxane (425 μL, 1.70 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 3.23, as a light brown solid.
(426) ##STR00212##
(427) LC-MS Retention time 6.15 minutes, found 474.1 [M+H].sup.+; calculated for C.sub.28H.sub.28FN.sub.3O.sub.3 474.55 [M+H].sup.+.
Synthesis of 1-ethyl-6-fluoro-7-(4-(naphthalen-1-yl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3.24)
(428) Firstly, 1-(naphthalen-1-yl)piperazine dihydrochloride (2.27; 1 g, 3.51 mmol, 1 eq) was converted to its freebase form through workup with dichloromethane (3×50 mL) and water (50 mL) using a saturated solution of sodium hydrogencarbonate to neutralise the aqueous phase. Combined organic fractions were dried over MgSO.sub.4, filtered and concentrated in vacuo. Secondly, 1-(naphthalen-1-yl)piperazine freebase (115.3 mg, 0.54 mmol, 1 eq) and 1-ethyl-6-7-difluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (1.9; 124.0 mg, 0.49 mmol, 0.9 eq) were added to DMSO (5 mL) and stirred until full dissolution of both compounds was achieved. The reaction was subsequently heated to 140° C. for one and a half hours. Upon completion, the mixture was allowed to cool for to minutes and formation of a precipitate was observed. The crude precipitate was purified via trituration; the crude was washed with methanol (5×10 mL), then the remaining powder collected and re-filtered using dichloromethane. This second filtrate was concentrated in vacuo to afford compound 3.24 (145.09 mg, 60.0% yield) as a yellow solid.
(429) ##STR00213##
(430) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.13 (s, 1H), 8.71 (s, 1H), 8.24-8.28 (m, 1H), 8.11 (d, J=13.09 Hz, 1H), 7.85-7.90 (m, 1H), 7.63 (d, J=80.6 Hz, 1H), 7.49-7.55 (m, 2H), 7.44-7.49 (m, 1H), 7.19 (dd, J=1.01, 7.30 Hz, 1H), 6.97 (d, J=6.80 Hz, 1H), 4.37 (q, J=7.30 Hz, 2H), 3.62 (br. s., 4H), 3.37 (br. s., 4H), 1.65 (t, J=7.30 Hz, 3H); .sup.19F NMR (400 MHz, CDCl.sub.3) δ −120.32; LC-MS Retention time 8.75 minutes, found 446.2 [M+H].sup.+; calculated for C.sub.26H.sub.24FN.sub.3O.sub.3 446.49 [M+H].sup.+.
Synthesis of 1-ethyl-6-fluoro-7-(4-(naphthalen-1-yl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (3.25)
(431) 3.24 (61.14 mg, 0.137 mmol, 1 eq) was added to dichloromethane (5 mL total) and stirred for 10 minutes. Then 4M HCl in dioxane (686 μL, 2.74 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 3.25 (67.50 mg, 98.0% yield) as an off white solid.
(432) ##STR00214##
(433) LC-MS Retention time 8.75 minutes, found 446.1 [M+H].sup.+; calculated for C.sub.26H.sub.24FN.sub.3O.sub.3 446.49 [M+H].sup.+.
Synthesis of 7-(4-(3,5-dimethylbenzyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3.26)
(434) Norfloxacin (3.1; 100 mg, 0.31 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and distilled water (5 mL total). After stirring for 5 minutes, potassium carbonate (130 mg, 0.94 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 1-(bromomethyl)-3,5-dimethylbenzene (62 mg, 0.31 mmol, 1 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 42 hours. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford 3.26 as an off white solid.
(435) ##STR00215##
(436) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.12 (br. s., 1H), 8.67 (s, 1H), 8.06 (d, J=13.35 Hz, 1H), 6.97 (s, 2H), 6.94 (s, 1H), 6.83 (d, J=7.30 Hz, 1H), 4.27-4.36 (m, 2H), 3.55 (s, 2H), 3.36 (br. s., 4H), 2.69 (br. s., 4H), 2.33 (s, 6H), 1.58 (t, J=6.92 Hz, 3H); LC-MS Retention time 3.05 minutes, Found 438.0 [M+H].sup.+; calculated for C.sub.25H.sub.28FN.sub.3O.sub.3 438.52 [M+H].sup.+
Synthesis of 7-(4-(3,5-dimethylbenzyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (3.27)
(437) 3.26 (61.14 mg, 0.13 mmol, 1 eq) was added to dichloromethane (5 mL) and stirred for 5 minutes. Then 4M HCl in dioxane (658 μL, 2.63 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford 30.27 as an off white solid.
(438) ##STR00216##
(439) LC-MS Retention time 5.88 minutes, Found 438.2 [M+H].sup.+; calculated for C.sub.25H.sub.28FN.sub.3O.sub.3 438.52 [M+H].sup.+
Synthesis of 7-(4-(4-(1H-pyrrol-1-yl)benzyl)piperazin-1-yl)-1-ethyl-6-fluoro oxo-1-4-dihydroquinoline-3-carboxylic acid (3.28)
(440) Norfloxacin (3.1; 100 mg, 0.31 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and distilled water (5 mL total). After stirring for 5 minutes, potassium carbonate (130 mg, 0.94 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 1-(4-(bromomethyl)phenyl)-1H-pyrrole (70 mg, 0.30 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 7 days. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford (470.20 mg, 34.7% yield) 3.28 as an off white solid.
(441) ##STR00217##
(442) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.10 (br. s., 1H), 8.68 (s, 1H), 8.08 (d, J=13.09 Hz, 1H), 7.33-7.44 (m, 4H), 7.10 (t, J=2.14 Hz, 2H), 6.84 (d, J=6.80 Hz, 1H), 6.37 (t, J=2.14 Hz, 2H), 4.31 (q, J=7.22 Hz, 2H), 3.63 (s, 2H), 3.33-3.38 (m, 4H), 2.68-2.74 (m, 4H), 1.55-1.59 (m, 3H); LC-MS Retention time 50.92 minutes, and 475.1 [M+H].sup.+; calculated for C.sub.27H.sub.27FN.sub.4O.sub.3 475.54 [M+H].sup.+
Synthesis of 7-(4-(4-(1H-pyrrol-1-yl)benzyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (3.29)
(443) 3.28 (42.96 mg, 0.09 mmol, 1 eq) was added to dichloromethane (3 mL) and stirred for 5 minutes. Then 4M HCl in dioxane (453 μL, 1.81 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford 30.29 as an off white solid.
(444) ##STR00218##
(445) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 15.29 (br. s., 1H), 11.45 (s, 1H), 8.98 (s, 1H), 7.98 (d, J=13.07 Hz, 1H), 7.79-7.66 (m, 4H), 7.46 (t, J=2.23 Hz, 2H), 7.28 (d, J=7.22 Hz, 1H), 6.30 (t, J=2.20 Hz, 2H), 4.62 (q, J=7.05 Hz, 2H), 4.47-4.40 (m, 2H), 3.90 (d, J=13.31 Hz, 2H), 3.55-3.42 (m, 4H), 3.34-3.22 (m, 2H), 1.41 (t, J=7.10 Hz, 3H); LC-MS Retention time 5.95 minutes, Found 4750.1 [M+H].sup.+; calculated for C.sub.27H.sub.27FN.sub.4O.sub.3 475.54 [M+H].sup.+
Synthesis of 7-(4-(4-(1H-pyrazol-1-yl)benzyl)piperazin-1-yl)-1-ethyl-6-fluoro oxo-1-4-dihydroquinoline-3-carboxylic acid (3.30)
(446) Norfloxacin (3.1; 100 mg, 0.31 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and distilled water (5 mL total). After stirring for 5 minutes, potassium carbonate (130 mg, 0.94 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 1-(4-(bromomethyl)phenyl)-1H-pyrazole (71 mg, 0.30 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 7 days. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford 30.30 as an off white solid.
(447) ##STR00219##
(448) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.12 (br. s., 1H), 8.65 (s, 1H), 8.03 (d, J=13.09 Hz, 1H), 7.93 (d, J=2.27 Hz, 1H), 7.73 (d, J=1.76 Hz, 1H), 7.67 (d, J=8.31 Hz, 2H), 7.45 (d, J=8.56 Hz, 2H), 6.83 (d, J=6.80 Hz, 1H), 6.45-6.50 (m, 1H), 4.31 (q, J=7.22 Hz, 2H), 3.64 (s, 2H), 3.31-3.38 (m, 4H), 2.66-2.73 (m, 4H), 1.57 (t, J=7.30 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 177.0, 167.3, 154.8, 152.3, 147.1, 146.2, 146.1, 141.1, 139.4, 137.1, 136.0, 130.2, 126.8, 120.5, 120.4, 119.2, 112.8, 112.6, 108.3, 107.7, 103.8, 62.2, 52.7, 49.9, 49.9, 49.8, 14.5; LC-MS Retention time 2.85 minutes, Found 476.0 [M+H].sup.+; calculated for C.sub.26H.sub.26FN.sub.5O.sub.3 476.52 [M+H].sup.+
Synthesis of 7-(4-(4-(1H-pyrazol-1-yl)benzyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (3.31)
(449) 3.30 (37.21 mg, 0.08 mmol, 1 eq) was added to dichloromethane (3 mL) and stirred for 5 minutes. Then 4M HCl in dioxane (391 μL, 1.57 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford 3.31 as an off white solid.
(450) ##STR00220##
(451) LC-MS Retention time 50.53 minutes, Found 476.1 [M+H].sup.+; calculated for C.sub.26H.sub.26FN.sub.5O.sub.3 476.52 [M+H].sup.+
Synthesis of 7-(4-(4-(1H-1,2,4-triazol-1-yl)benzyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3.32)
(452) Norfloxacin (3.1; 100 mg, 0.31 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and distilled water (5 mL total). After stirring for 5 minutes, potassium carbonate (130 mg, 0.94 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 1-(4-(bromomethyl)phenyl)-1H-1,2,4-triazole (71 mg, 0.30 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 116 hours. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford 3.32 as an off white solid.
(453) ##STR00221##
(454) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.11 (br. s., 8.65 (s, 1H), 8.57 (s, 1H), 8.11 (s, 1H), 8.00 (d, J=13.09 Hz, 1H), 7.63-7.68 (m, J=8.56 Hz, 2H), 7.49-7.54 (m, J=8.56 Hz, 2H), 6.83 (d, J=6.80 Hz, 1H), 4.32 (q, J=7.22 Hz, 2H), 3.66 (s, 2H), 3.31-3.38 (m, 4H), 2.65-2.74 (m, 4H), 1.57 (t, J=7.30 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 17.0, 167.2, 154.8, 152.6, 152.3, 147.1, 146.2, 146.1, 140.9, 138.2, 137.1, 136.1, 130.4, 120.5, 120.4, 120.1, 112.8, 112.6, 108.3, 103.8, 62.1, 52.7, 49.9, 49.8, 14.5; LC-MS Retention time 2.68 minutes, Found 477.0 [M+H].sup.+; calculated for C.sub.25H.sub.25FN.sub.16O.sub.3 477.51 [M+H].sup.+
Synthesis of 7-(4-(4-(1H-1,2,4-triazol-1-yl)benzyl)piperazin-1-yl)-1-ethyl fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3.33)
(455) 3.32 (28.07 mg, 0.06 mmol, 1 eq) was added to dichloromethane (3 mL) and stirred for 5 minutes. Then 4M HCl in dioxane (295 μL, 1.18 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 17 hours. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford 3.33 as an off white solid.
(456) ##STR00222##
(457) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.75 (s, 1H), 9.39 (s, 1H), 8.98 (s, 1H), 8.29 (s, 1H), 8.03-7.93 (m, 3H), 7.93-7.84 (m, 2H), 7.27 (d, J=7.28 Hz, 1H), 4.62 (q, J=7.09 Hz, 2H), 4.48 (d, J=4.57 Hz, 2H), 3.88 (d, J=13.28 Hz, 2H), 3.58-3.40 (m, 4H), 3.29 (d, J=11.35 Hz, 2H), 1.40 (t, J=7.09 Hz, 3H); LC-MS Retention time 50.20 minutes, Found 4770.1 [M+H].sup.+; calculated for C.sub.25H.sub.25FN.sub.6O.sub.3 477.51 [M+H].sup.+
Synthesis of 7-(4-(4-(1,2,3-thiadiazol-4-yl)benzyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3.34)
(458) Norfloxacin (3.1; 100 mg, 0.31 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and distilled water (5 mL total). After stirring for 5 minutes, potassium carbonate (130 mg, 0.94 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 4-(4-(bromomethyl)phenyl)-1,2,3-thiadiazole (76 mg, 0.30 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 96 hours. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford (117.85 mg, 80.3% yield) 3.34 as an off white solid.
(459) ##STR00223##
(460) LC-MS Retention time 2.92 minutes, Found 494.0 [M+H].sup.+; calculated for C.sub.25H.sub.24FN.sub.5O.sub.3S 494.56 [M+H].sup.+
Synthesis of 7-(4-(4-(1,2,3-thiadiazol-4-yl)benzyl)piperazin-1-yl)-1-ethyl fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (3.35)
(461) 3.34 (90 mg, 0.18 mmol, 1 eq) was added to dichloromethane (3 mL) and stirred for 5 minutes. Then 4M HCl in dioxane (912 μL, 3.65 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford 3.35 as an off white solid.
(462) ##STR00224##
(463) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 15.26 (s, 1H), 11.77 (s, 1H), 9.73 (s, 1H), 8.97 (s, 1H), 8.25 (d, J=8.34 Hz, 2H), 7.96 (d, J=13.08 Hz, 1H), 7.87 (d, J=8.20 Hz, 2H), 7.27 (d, J=7.25 Hz, 1H), 4.61 (q, J=7.10 Hz, 2H), 4.50 (d, J=4.74 Hz, 2H), 3.89 (d, J=13.31 Hz, 2H), 3.62-3.43 (m, 4H), 3.37-3.26 (m, 2H), 1.41 (t, J=7.08 Hz, 3H); LC-MS Retention time 5.62 minutes, Found 4940.1 [M+H].sup.+; calculated for C.sub.25H.sub.24FN.sub.5O.sub.38 494.56 [M+H].sup.+
Synthesis of 1-ethyl-6-fluoro-7-(4-(4-(5-methyl-1,2,4-oxadiazol-3-yl)-benzyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinone-3-carboxylic acid (3.36)
(464) Norfloxacin (3.1; 100 mg, 0.31 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and distilled water (5 mL total). After stirring for 5 minutes, potassium carbonate (130 mg, 0.94 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 3-(4-(bromomethyl)phenyl)-5-methyl-1,2,4-oxadiazole (75 mg, 0.30 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 7 days. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford 3.36 as an off white solid.
(465) ##STR00225##
(466) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.13 (br. s., 1H), 8.63 (s, 1H), 8.02 (d, J=8.31 Hz, 2H), 7.98 (d, J=13.09 Hz, 1H), 7.48 (d, J=80.6 Hz, 2H), 6.82 (d, J=6.80 Hz, 1H), 4.31 (q, =7.05 Hz, 2H), 3.65 (s, 2H), 3.32-3.38 (m, 4H), 2.67-2.73 (m, 4H), 2.66 (s, 3H) 1.56 (t, J=7.18 Hz, 3H); .sup.13C NMR (too MHz, CDCl.sub.3) δ 176.9, 176.6, 168.2, 167.2, 152.3, 147.1, 146.2, 146.1, 141.1, 137.1, 129.5 (2C), 127.4 (2C), 125.9, 120.4, 120.3, 112.7, 112.5, 108.2, 103.8, 62.5, 52.7 (2C), 49.9, 49.9, 49.8, 14.4, 12.4; LC-MS Retention time 2.88 minutes, Found 492.0 [M+H].sup.+; calculated for C.sub.26H.sub.26FN.sub.5O.sub.4 492.52 [M+1-1]
Synthesis of 1-ethyl-6-fluoro-7-(4-(4-(5-methyl-1,2,4-oxadiazol yl)benzyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (3.37)
(467) 3.36 (17.34 mg, 0.04 mmol, 1 eq) was added to dichloromethane (3 mL) and stirred for 5 minutes. Then 4M HCl in dioxane (176 μL, 0.71 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 17 hours. The mixture was then washed with hexane (3×20 mL), concentrated in vacuo and lyophilised for 24 hours to afford 3.37 as an off white solid.
(468) ##STR00226##
(469) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.54 (s, 1H), 8.98 (s, 1H), 8.12-8.06 (m, 2H), 7.98 (d, J=13.06 Hz, 1H), 7.89-7.82 (m, 2H), 7.27 (d, J=7.23 Hz, 1H), 4.62 (q, J=7.07 Hz, 2H), 4.50 (s, 2H), 3.89 (d, J=13.27 Hz, 2H), 3.48 (m, 4H), 3.31 (d, J=11.54 Hz, 2H), 1.40 (t, J=7.07 Hz, 3H); LC-MS Retention time 5.58 minutes, Found 492.1 [M+H].sup.+; calculated for C.sub.26H.sub.26FN.sub.5O.sub.4 492.52 [M+H].sup.+
Synthesis of 7-(4-([1,1′-biphenyl]-4-ylmethyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3.38)
(470) Norfloxacin (3.1; 100 mg, 0.31 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and distilled water (5 mL total). After stirring for 5 minutes, potassium carbonate (130 mg, 0.94 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 4-(bromomethyl)-1,1′-biphenyl (74 mg, 0.30 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 24 hours. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford (123.74 mg, 85.7% yield) 3.38 as an off white solid.
(471) ##STR00227##
(472) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.12 (br. s., 1H), 8.66 (s, 1H), 8.04 (d, J=13.09 Hz, 1H), 7.56-7.63 (m, 4H), 7.41-7.48 (m, 4H), 7.33-7.39 (m, 1H), 6.83 (d, J=6.29 Hz, 1H), 4.26-4.37 (m, 2H), 3.66 (s, 2H), 3.37 (br. s., 4H), 2.73 (br. s., 4H), 1.58 (t, J=6.17 Hz, 3H); LC-MS Retention time 6.18 minutes, Found 486.2 [M+H].sup.+; calculated for C.sub.29H.sub.28FN.sub.3O.sub.3 486.56 [M+H].sup.+
Synthesis of 7-(4-([1,1′-biphenyl]-4-ylmethyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1-4-dihydroquinoline-3-carboxylic acid hydrochloride (3.39)
(473) 3.38 (123.74 mg, 0.25 mmol, 1 eq) was added to dichloromethane (5 mL) and stirred for 5 minutes. Then 4M HCl in dioxane (1.27 mL, 5.10 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford 3.39 as an off white solid.
(474) ##STR00228##
(475) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 15.27 (br. s., 1H), 11.43 (br. s., 1H), 8.98 (s, 1H), 7.97 (d, J=13.09 Hz, 1H), 7.73-7.81 (m, 4H), 7.69-7.73 (m, 2H), 7.47-7.52 (m, 2H), 7.38-7.43 (m, 1H), 7.27 (d, J=7.55 Hz, 1H), 4.62 (q, J=6.88 Hz, 2H), 4.44-4.49 (m, 2H), 3.90 (d, J=12.59 Hz, 2H), 3.44-3.54 (m, 4H), 3.24-3.32 (m, 2H), 1.41 (t, J=7.05 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 177.0, 167.3, 154.8, 152.3, 147.1, 146.3, 146.2, 140.8, 140.3, 137.1, 136.7, 129.6, 128.8, 127.3, 127.1, 127.1, 120.5, 120.4, 112.9, 112.6, 108.3, 103.8, 62.6, 52.7, 50.0, 49.9, 49.8, 49.8, 14.5; LC-MS Retention time 6.17 minutes, Found 486.1 [M+H].sup.+; calculated for C.sub.29H.sub.28FN.sub.3O.sub.3 486.56 [M+H].sup.+
Synthesis of 1-ethyl-6-fluoro-7-(4-(4-(hydroxymethyl)benzyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3.40)
(476) Norfloxacin (3.1; 100 mg, 0.31 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and distilled water (5 mL total). After stirring for 5 minutes, potassium carbonate (130 mg, 0.94 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, (4-(bromomethyl)phenyl)methanol (63 mg, 0.31 mmol, 1 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 7 days. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford (133.70 mg, 97.1% yield) 3.40 as an off white solid.
(477) ##STR00229##
(478) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.13 (br. s., 1H), 8.64 (s, 1H), 7.98 (d, J=13.09 Hz, 1H), 7.35 (s, 4H), 6.82 (d, J=6.80 Hz, 1H), 4.70 (s, 2H), 4.31 (q, J=7.13 Hz, 2H), 3.59 (s, 2H), 3.30-3.36 (m, 4H), 2.64-2.70 (m, 4H), 1.56 (t, J=7.18 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 176.9, 167.3, 152.3, 147.1, 146.3, 146.2, 140.1, 137.1, 136.9, 129.4 (2C), 127.1 (2C), 120.4, 120.3, 112.7, 112.5, 108.2, 103.8, 65.0, 62.6, 53.5, 52.6 (2C), 49.9, 49.8, 14.4; LC-MS Retention time 50.03 minutes, Found 440.1 [M+H].sup.+; calculated for C.sub.24H.sub.26FN.sub.3O.sub.4 440.49 [M+H].sup.+
Synthesis of 1-ethyl-6-fluoro-7-(4-(4-(hydroxymethyl)benzyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (3.41)
(479) 3.40 (35.61 mg, 0.8 mmol, 1 eq) was added to dichloromethane (3 mL) and stirred for 5 minutes. Then 4M HCl in dioxane (405 μL, 1.62 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 30 minutes. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford 3.41 as an off white solid.
(480) ##STR00230##
(481) 1H NMR (400 MHz, DMSO-d.sub.6) δ 15.26 (s, 1H), 11.32 (s, 1H), 8.98 (s, 1H), 7.98 (d, J=13.07 Hz, 1H), 7.61 (d, J=8.06 Hz, 2H), 7.42 (d, J=7.83 Hz, 2H), 7.27 (d, J=7.23 Hz, 1H), 4.62 (q, J=7.09 Hz, 2H), 4.55 (s, 2H), 4.40 (d, J=5.09 Hz, 2H), 3.88 (d, J=13.30 Hz, 2H), 3.53-3.41 (m, 4H), 3.33-3.19 (m, 2H), 1.41 (t, J=7.07 Hz, 3H); LC-MS Retention time 5.15 minutes, Found 440.0 [M+H].sup.+; calculated for C.sub.24H.sub.26FN.sub.3O.sub.4 440.49 [M+H]+
Synthesis of 1-ethyl-6-fluoro-7-(4-(4-(methoxymethyl)benzyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3.42)
(482) Norfloxacin (3.1; 28 mg, 0.09 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and distilled water (2 mL total). After stirring for 5 minutes, potassium carbonate (36 mg, 0.26 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 1-(chloromethyl)-4-(methoxymethyl)benzene (14 mg, 0.08 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently heated to reflux and stirred for 24 hours. Upon completion, the product was extracted with dichloromethane (2×10 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (10 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Flash column chromatography (0%-100% DCM/acetone) was employed to afford pure 3.42.
(483) ##STR00231##
(484) TABLE-US-00001 3.42 .sup.1H .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.14 (br. s., 1H), 8.67 Off NMR (s, 1H), 8.04 (d, J = 13.09 Hz, 1H), 7.30-7.38 (m, 4H), white 6.82 (d, J = 6.80 Hz, 1H), 4.46 (s, 2H), 4.31 (q, J = solid 7.30 Hz, 2H), 3.61 (s, 2H), 3.42 (s, 3H), 3.31-3.36 (m, 4H), 2.66-2.71 (m, 4H), 1.58 (t, J = 7.18 Hz, 3H) .sup.13C .sup.13C NMR (100 MHz, CDCl.sub.3) δ 177.0, 167.3, 154.8, NMR 152.3, 147.1, 146.3, 146.2, 137.4, 137.1, 137.0, 129.3, 127.9, 120.5, 120.5, 112.9, 112.6, 108.3, 103.7, 74.5, 62.6, 58.3, 52.6, 49.9, 49.9, 49.8, 14.4 Yield 23.31 mg (61.7% yield)
Synthesis of 1-ethyl-6-fluoro-7-(4-(4-(methoxymethyl)benzyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (3.43)
(485) 3.42 (12.01 mg, 0.03 mmol, 1 eq) was added to dichloromethane (2 mL) and stirred for 5 minutes. Then 4M HCl in dioxane (1324, 0.53 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×20 mL), concentrated in vacuo and lyophilised for 24 hours to afford 3.43.
(486) ##STR00232##
(487) TABLE-US-00002 3.43 LC-MS Retention time 4.79 minutes Off Found 454.2 [M + H].sup.+; calculated for C.sub.25H.sub.28FN.sub.3O.sub.4 white 454.51 [M + H].sup.+ solid
Synthesis of 1-ethyl-6-fluoro-7-(4-(3-(methoxymethyl)benzyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3.44)
(488) Norfloxacin (3.1; 100 mg, 0.31 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and distilled water (5 mL total). After stirring for 5 minutes, potassium carbonate (130 mg, 0.94 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 1-(bromomethyl)-3-(methoxymethyl)benzene (64 mg, 0.30 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 88 hours. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved via automated flash column chromatography of the crude solid (see Flash Column Chromatography method; 0%-30% DCM/acetone) to afford pure 3.44.
(489) ##STR00233##
(490) TABLE-US-00003 3.44 LC-MS Retention time 5.33 minutes Off Found 454.1 [M + H].sup.+; calculated for C.sub.25H.sub.28FN.sub.3O.sub.4 white 454.51 [M + H].sup.+ solid Yield 119.00 mg (88.2% yield)
Synthesis of 1-ethyl-6-fluoro-7-(4-(3-(methoxymethyl)benzyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (3.45)
(491) 3.44 (36.59 mg, 0.09 mmol, 1 eq) was added to dichloromethane (3 mL) and stirred for 5 minutes. Then 4M HCl in dioxane (403 μL, 1.61 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 40 minutes. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford 3.45.
(492) ##STR00234##
(493) TABLE-US-00004 3.45 LC-MS Retention time 4.78 minutes Off Found 454.2 [M + H].sup.+; calculated for C.sub.25H.sub.28FN.sub.3O.sub.4 white 454.51 [M + H].sup.+ solid
Synthesis of 1-ethyl-6-fluoro-7-(4-(3-(methoxymethyl)benzyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3.46)
(494) Norfloxacin (3.1; 100 mg, 0.31 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and distilled water (5 mL total). After stirring for 5 minutes, potassium carbonate (130 mg, 0.94 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, N-(2-(1H-indol-3-yl)ethyl)-4-(bromomethyl)benzenesulfonamide (123 mg, 0.31 mmol, 1 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 42 hours. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see 1.1.8 Solid Phase Extraction) to afford 3.46.
(495) ##STR00235##
(496) TABLE-US-00005 3.46 LC-MS Retention time 3.12 minutes Off Found 632.1 [M + H].sup.+; calculated for C.sub.33H.sub.34FN.sub.5O.sub.5S white 632.72 [M + H].sup.+ solid
Synthesis of 1-ethyl-6-fluoro-7-(4-(3-(methoxymethyl)benzyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (3.47)
(497) 3.46 (61.14 mg, 0.13 mmol, 1 eq) was added to dichloromethane (5 mL) and stirred for 5 minutes. Then 4M HCl in dioxane (658 μL, 2.63 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford 3.47.
(498) ##STR00236##
(499) TABLE-US-00006 3.47 LC-MS Retention time 6.07 minutes Off Found 632.2 [M + H].sup.+; calculated for C.sub.33H.sub.34FN.sub.5O.sub.5S white 632.72 [M + H].sup.+ solid
Synthesis of Enoxacin-ARB Hybrid Compounds
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-(naphthalen-1-ylmethyl)piperazin-1-yl)-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid (4.2)
(500) Enoxacin (4.1; 250 mg, 0.78 mmol, 1 eq) was added to DMF (15 mL) and stirred for 20 minutes at 120° C. Then 1-(bromomethyl)naphthalene (173 mg, 0.78 mmol, 1 eq) and potassium carbonate (324 mg, 2.34 mmol, 3 eq) were added and the mixture stirred for a further 30 minutes at reflux. The mixture was allowed to cool, then extracted with dichloromethane (2×50 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (50 mL) followed by brine (50 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford compound 4.2 (113.86 mg, 79.2% yield) as a pale brown solid.
(501) ##STR00237##
(502) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.11 (br. s., 8.69 (s, 1H), 8.33 (dd, J 1.64, 7.93 Hz, 1H), 8.09 (d, J=13.35 Hz, 1H), 7.86-7.91 (m, 7.80-7.86 (m, 1H), 7.49-7.58 (m, 2H), 7.41-7.46 (m, 2H), 4.38 (q, J=7.05 Hz, 2H), 3.99 (s, 2H), 3.84-3.90 (m, 4H), 2.65-2.71 (m, 4H), 1.48 (t, J=7.18 Hz, 3H); LC-MS Retention time 3.05 minutes, found 461.1 [M+H].sup.+; calculated for C.sub.26H.sub.25FN.sub.4O.sub.3 461.51 [M+H].sup.+.
Synthesis of 1-cyclopropyl-6-fluoro-7-(4-(naphthalen-1-ylmethyl)piperazin-1-yl)-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid hydrochloride (4.3)
(503) 4.2 (30 mg, 0.07 mmol, 1 eq) was added to methanol (10 mL) and stirred for 10 minutes. Then 4M HCl in dioxane (33 μL, 0.13 mmol, 2 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 4.3 (18.64 mg, 86.4% yield) as a pale brown solid.
(504) ##STR00238##
(505) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 15.21 (br. s., 1H), 11.07 (br. s., 1H), 9.03 (s, 1H), 8.41 (d, J=8.31 Hz, 1H), 8.21 (d, J=12.84 Hz, 1H), 8.08 (d, J=8.56 Hz, 1H), 8.04 (d, J=80.6 Hz, 1H), 7.97 (d, J=7.30 Hz, 1H), 7.59-7.69 (m, 3H), 4.87 (br. s., 2H), 4.58 (d, J=12.84 Hz, 2H), 4.51 (q, J=6.88 Hz, 2H), 3.66 (t, J=12.09 Hz, 2H), 3.47 (br. s., 4H), 1.37 (t, J=7.05 Hz, 3H); IR (υ.sub.max/cm.sup.−1) 3388, 1715, 1628, 1457, 1396, 1340, 1265, 1042, 943, 804; LC-MS Retention time 5.85 minutes, found 461.1 [M+H].sup.+; calculated for C.sub.26H.sub.25FN.sub.4O.sub.3 461.51 [M+H].sup.+; HRMS Observed 461.1974 [M+H].sup.+; theoretical value 461.1983 [M+H].sup.+.
Synthesis of Levofloxacin-ARB Hybrid Compounds
Synthesis of (S)-9-fluoro-3-methyl-10-(4-(naphthalen-1-ylmethyl)piperazin-1-yl)-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (5.2)
(506) (S)-9,10-difluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinolone carboxylic acid (A1.4, 5.1; 100 mg, 0.36 mmol, 1 eq) and 1-(naphthalen-1-ylmethyl)piperazine (322 mg, 1.42 mmol, 4 eq) were added to DMSO (5 mL) and stirred until full dissolution of both compounds was achieved. The reaction was subsequently heated to 100° C. for 1 hour, then 140° C. for another hour. The mixture was allowed to cool, then extracted with dichloromethane (2×20 mL). Combined organic fractions were washed with distilled water (3×100 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo to remove any residual DMSO. The crude solid was purified via trituration; the crude was washed with methanol (5×10 mL), then the remaining powder collected and re-filtered using dichloromethane. This second filtrate was concentrated in vacuo to afford compound 5.2 (75 mg, 43.3% yield) as a pale yellow solid.
(507) ##STR00239##
(508) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 14.73 (s, 1H), 13.16 (br. s., 1H), 8.62 (s, 1H), 8.20 (d, J=6.55 Hz, 1H), 8.16 (d, J=80.6 Hz, 1H), 8.01 (d, J=8.06 Hz, 1H), 7.97 (d, J=8.06 Hz, 1H), 7.57-7.74 (m, 4H), 4.79 (br. s., 2H), 4.48-4.53 (m, 1H), 4.45 (d, J=11.83 Hz, 1H), 4.33 (d, J=12.59 Hz, 1H), 4.17-4.29 (m, 2H), 3.49 (br. S., 2H), 3.41 (d, J=12.84 Hz, 2H), 3.10 (br. s., 2H), 1.59 (d, J=6.55 Hz, 3H); .sup.19F NMR (400 MHz, CDCl.sub.3) δ −106.9, −119.5; LC-MS Retention time 3.00 minutes, found 488.1 [M+H].sup.+; calculated for C.sub.28H.sub.26FN.sub.3O.sub.4 488.53 [M+H].sup.+.
Synthesis of (S)-9-fluoro-3-methyl-10-(4-(naphthalen-1-ylmethyl)piperazin-1-yl)-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid hydrochloride (5.3)
(509) 5.2 (20 mg, 0.04 mmol, 1 eq) was added to dichloromethane (2 mL) and stirred for 10 minutes. Then 4M HCl in dioxane (205 μL, 0.82 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 5.3 (18 mg, 83.7% yield) as a yellow solid.
(510) ##STR00240##
(511) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.92 (br. s., 1H), 9.00 (s, 1H), 8.50 (d, J=8.31 Hz, 1H), 8.08 (d, J=8.31 Hz, 1H), 8.04 (dd, J=4.66, 7.43 Hz, 2H), 7.66-7.71 (m, 1H), 7.59-7.66 (m, 3H), 4.90-4.97 (m, 3H), 4.56-4.61 (m, 1H), 4.36-4.41 (m, 1H), 3.63-3.74 (m, 2H), 3.49-3.57 (m, 2H), 3.41 (br. s., 4H), 1.44 (d, J=6.55 Hz, 3H); .sup.19F NMR (400 MHz, CDCl.sub.3) δ −106.9, −120.5; LC-MS Retention time 5.92 minutes, found 488.1 [M+H].sup.+; calculated for C.sub.28H.sub.26FN.sub.3O.sub.4 488.53 [M+H].sup.+.
Synthesis of (S)-9-fluoro-10-(4-(2-isopropyl-6-methylpyrimidin-4-yl)piperazin-1-yl)-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (5.4)
(512) (S)-9,10-difluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinolone-6-carboxylic acid (A1.4, 5.1; 128 mg, 0.45 mmol, 1 eq) and 2-isopropyl-4-methyl-6-(piperazin-1-yl)pyrimidine (100 mg, 0.45 mmol, 1 eq) were added to DMF (3 mL) and stirred at 140° C. for 1.5 hours. The mixture was allowed to cool and the crude concentrated in vacuo, then re-suspended in 3:1 distilled water:MeOH (20 mL) and filtered hot. Purification was achieved via automated flash column chromatography of the crude solid (see Flash Column Chromatography method); 0%-50%-100% DCM/Acetone) to afford compound 5.4 (22 mg, 10.0%) as a pale yellow solid.
(513) ##STR00241##
(514) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 14.92 (br. s., 1H), 8.64 (s, 1H), 7.76 (d, J=12.09 Hz, 1H), 6.26 (s, 1H), 4.53 (d, J=5.54 Hz, 1H), 4.49 (d, J=11.33 Hz, 1H), 4.36-4.43 (m, 1H), 3.85 (br. s., 4H), 3.45 (td, J=5.07, 9.76 Hz, 4H), 3.04-3.13 (m, 1H), 2.41 (s, 3H), 1.64 (d, J=6.55 Hz, 3H), 1.29 (d, J=7.05 Hz, 6H); .sup.19F NMR (400 MHz, CDCl.sub.3) δ −106.9, −119.2; LC-MS Retention time 3.07 minutes, found 482.0 [M+H].sup.+; calculated for C.sub.25H.sub.28FN.sub.5O.sub.4 482.53 [M+H].sup.+.
Synthesis of (S)-9-fluoro-10-(4-(2-isopropyl-6-methylpyrimidin-4-yl)piperazin-1-yl)-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline carboxylic acid hydrochloride (5.5)
(515) 5.4 (22 mg, 0.05 mmol, 1 eq) was added to dichloromethane (2 mL) and stirred for 2 minutes. Then 4M HCl in dioxane (228 μL, 0.91 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 15.5 hours. The mixture was then washed with hexane (3×10 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 5.5 as a pale yellow solid.
(516) ##STR00242##
(517) LC-MS Retention time 5.92 minutes, found 482.1 [M+H].sup.+; calculated for C.sub.23H.sub.28FN.sub.3O.sub.4 482.53 [M+H].sup.+.
Synthesis of (S)-9-fluoro-3-methyl-7-oxo-10-(4-(pyrimidin-4-yl)piperazin-1-yl)-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (5.6)
(518) (S)-9,10-difluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinolone-6-carboxylic acid (A1.4, 5.1; 1.37 g, 4.87 mmol, 1 eq) and 4-(piperazin-1-yl)pyrimidine (1 g, 6.09 mmol, 1.25 eq) were added to DMF (20 mL) and stirred at 140° C. for 90 hours. The mixture was allowed to cool, then DMF removed using a 10 g SCX-2 catch and release cartridge (see Solid Phase Extraction method) followed by extraction with dichloromethane (2×50 mL, washed with 100 mL distilled water, 100 mL brine). Purification was achieved via flash column chromatography (gradient elution; 0-100% DCM/acetonitrile, then 1% water in acetonitrile, then 100% NH.sub.3 in MeOH) to afford compound 5.6 (234.4 mg, 11.3% yield) as a pale yellow solid.
(519) ##STR00243##
(520) LC-MS Retention time 2.72 minutes, found 425.9 [M+H].sup.+; calculated for C.sub.21H.sub.20FN.sub.5O.sub.4 426.42 [M+H].sup.+.
Synthesis of (S)-9-fluoro-3-methyl-7-oxo-10-(4-(pyrimidin-4-yl)piperazin-1-yl)-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid hydrochloride (5.7)
(521) 5.6 (5.07 mg, 0.01 mmol, 1 eq) was added to dichloromethane (1 mL) and stirred for 2 minutes. Then 4M HCl in dioxane (60 μL, 0.24 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×10 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 5.7 as a pale yellow solid.
(522) ##STR00244##
(523) LC-MS Retention time 5.25 minutes, found 426.0 [M+H].sup.+; calculated for C.sub.21H.sub.20FN.sub.5O.sub.4 426.42 [M+H].sup.+.
Synthesis of (S)-9-fluoro-3-methyl-7-oxo-w-(4-(pyridin-2-yl)piperazin-1-yl)-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (5.8)
(524) (S)-9,10-difluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinolone-6-carboxylic acid (A1.4, 5.1; 100 mg, 0.36 mmol, 1 eq) and 1-(pyridin-2-yl)piperazine (108 μL, 0.71 mmol, 2 eq) were added to DMSO (3 mL) and stirred at 140° C. for 2 hours. The mixture was allowed to cool, then DMSO removed using a 1 g SCX-2 catch and release cartridge (see Solid Phase Extraction method). Trace solvent was subsequently removed by extraction with dichloromethane (20 mL) and washing with saturated brine (3×100 mL). Combined organic fractions were dried over MgSO.sub.4, filtered and concentrated in vacuo. Purification was achieved via automated flash column chromatography (see Flash Column Chromatography method; 0%-5%-10%-20%-50% DCM/Acetone) to afford compound 5.8 (69.19 mg, 45.8% yield) as a yellow solid.
(525) ##STR00245##
(526) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 14.99 (s, 1H), 8.64 (s, 1H), 8.23 (dd, J=1.64, 4.91 Hz, 1H), 7.74 (d, J=12.09 Hz, 1H), 7.52-7.58 (m, 1H), 6.73 (d, J=8.81 Hz, 1H), 6.69 (dd, J=50.04, 6.80 Hz, 1H), 4.51-4.57 (m, 1H), 4.48 (dd, J=2.14, 11.46 Hz, 1H), 4.39 (dd, J=2.39, 11.46 Hz, 1H), 3.72 (br. s., 4H), 3.43-3.56 (m, 4H), 1.64 (d, J=6.80 Hz, 3H); .sup.19F NMR (400 MHz, CDCl.sub.3) δ −107.0, −134.8; LC-MS Retention time 5.43 minutes, found 425.0 [M+H].sup.+; calculated for C.sub.22H.sub.21FN.sub.4O.sub.4 425.43 [M+H].sup.+.
Synthesis of (S)-9-fluoro-3-methyl-7-oxo-10-(4-(pyridin-2-yl)piperazin-1-yl)-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid hydrochloride (5.9)
(527) 5.8 (20.27 mg, 0.05 mmol, 1 eq) was added to dichloromethane (2 mL) and stirred for 2 minutes. Then 4M HCl in dioxane (239 μL, 0.96 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1.5 hours. The mixture was then washed with hexane (3×10 mL), concentrated in vacuo and lyophilised for 24 hours to afford compound 5.9 as a pale yellow solid.
(528) ##STR00246##
(529) LC-MS Retention time 50.33 minutes, found 425.0 [M+H].sup.+; calculated for C.sub.22H.sub.21FN.sub.4O.sub.4 425.43 [M+H].sup.+.
Synthesis of (S)-10-(4-(1H-pyrazol-1-yl)piperidin-1-yl)-9-fluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (5.10)
(530) (S)-9,10-difluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinolone-6-carboxylic acid (A1.4, 5.1; 100 mg, 0.36 mmol, 1 eq) and 4-(1H-pyrazol-1-yl)piperidine (108 mg, 0.71 mmol, 2 eq) were added to DMF (3 mL) and stirred at 140° C. for 95 hours. The mixture was allowed to cool, then concentrated in vacuo. The crude was then dissolved in minimal 1:1 DCM/methanol (3 mL) and added dropwise to 200 mL of ice water. Following stirring for 10 minutes, the brown bottom layer was pipetted out and concentrated in vacuo. Purification was achieved via automated flash column chromatography (see Flash Column Chromatography method; 0%-100% DCM/acetone) to afford 5.10.
(531) ##STR00247##
(532) TABLE-US-00007 5.10 .sup.1H .sup.1H NMR (400 MHz, CDCl.sub.3) δ 15.01 (s, 1H), 8.64 White NMR (s, 1H), 7.73 (d, J = 12.09 Hz, 1H), 7.54-7.57 (m, 1H), 7.51 (d, J = 2.27 Hz, 1H), 6.30 (t, J = 1.76 Hz, 1H), 4.51-4.58 (m, 1H), 4.46-4.51 (m, 1H), 4.39 (dd, J = 2.27, 11.58 Hz, 1H), 4.31-4.37 (m, 1H), 3.61 (d, J = 13.35 Hz, 2H), 3.32-3.47 (m, 2H), 2.16-2.29 (m, 4H), 1.63 (d, J = 6.80 Hz, 3H) solid .sup.19F .sup.19F NMR (400 MHz, CDCl.sub.3) δ −119.1 NMR LC-MS Retention time 3.53 minutes Found 413.0 [M + H].sup.+; calculated for C.sub.21H.sub.21FN.sub.4O.sub.4 413.42 [M + H].sup.+
Synthesis of (S)-10-(4-(1H-pyrazol-1-yl)piperidin-1-yl)-9-fluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid hydrochloride (5.11)
(533) 5.10 (13.30 mg, 0.03 mmol, 1 eq) was added to dichloromethane (3 mL) and stirred for 2 minutes. Then 4M HCl in dioxane (161 μL, 0.64 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×20 mL), concentrated in vacuo and lyophilised for 24 hours to afford 5.11.
(534) ##STR00248##
(535) TABLE-US-00008 5.11 LC-MS Retention time 7.11 minutes White Found 413.2 [M + H].sup.+; calculated for C.sub.21H.sub.21FN.sub.4O.sub.4 solid 413.42 [M + H].sup.+
Synthesis of (S)-9-fluoro-3-methyl-7-oxo-10-(piperazin-1-yl)-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (5.12)
(536) (S)-9,10-difluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinolone-6-carboxylic acid (A1.4, 5.1; 1 g, 3.56 mmol, 1 eq) and piperazine (613 mg, 7.11 mmol, 2 eq) were added to DMSO (5 mL) and stirred at 125° C. for 16 hours. The mixture was allowed to cool, then ice cold acetone (50 mL) was added. The resulting brown precipitate was filtered and triturated with further cold acetone (5×10 mL), air dried for 30 minutes then dried under vacuum for a further 1 hour. This cycle of trituration and drying was then repeated once more to afford 5.12.
(537) ##STR00249##
(538) TABLE-US-00009 5.12 LC-MS Retention time 1.86 minutes Light Found 348.1 [M + H].sup.+; calculated for C.sub.17H.sub.18FN.sub.3O.sub.4 brown 348.35 [M + H].sup.+ solid Yield 931.63 mg (75.4 % yield)
Synthesis of (S)-9-fluoro-3-methyl-7-oxo-10-(piperazin-1-yl)-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid hydrochloride (5.13)
(539) 5.12 (147.42 mg, 0.42 mmol, 1 eq) was added to dichloromethane (5 mL) and stirred for 2 minutes. Then 4M HCl in dioxane (2.12 mL, 8.49 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford 5.13.
(540) ##STR00250##
(541) TABLE-US-00010 5.13 LC-MS Retention time 3.14 minutes solid Found 348.1 [M + H].sup.+; calculated for C.sub.17H.sub.18FN.sub.3O.sub.4 White 348.35 [M + H].sup.+
Synthesis of (3S)-9-fluoro-3-methyl-7-oxo-10-(3-(pyrimidin-2-ylamino)-pyrrolidin-1-yl)-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (5.14)
(542) ##STR00251##
(543) TABLE-US-00011 5.14 LC-MS Retention time 3.28 minutes White Found 426.2 [M + H].sup.+; calculated for C.sub.21H.sub.20FN.sub.5O.sub.4 solid 426.42 [M + H].sup.+
Synthesis of (3S)-9-fluoro-3-methyl-7-oxo-10-(3-(pyrimidin-2-ylamino)-pyrrolidin-1-yl)-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid hydrochloride (5.15)
(544) 5.14 (6.81 mg, 0.02 mmol 1 eq) was added to dichloromethane (1 mL) and methanol (1 mL) and stirred for 2 minutes. Then 4M HCl in dioxane (80 μL, 0.32 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×10 mL), concentrated in vacuo and lyophilised for 24 hours to afford 5.15.
(545) ##STR00252##
(546) TABLE-US-00012 5.15 LC-MS Retention time 6.32 minutes White Found 426.2 [M + H].sup.+; calculated for C.sub.21H.sub.20FN.sub.5O.sub.4 solid 426.42 [M + H].sup.+
Synthesis of (S)-9-fluoro-3-methyl-7-oxo-10-(4-(pyrazin-2-yl)-1,4-diazepan-1-yl)-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (5.16)
(547) (S)-9,10-difluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinolone carboxylic acid (5.1; 80 mg, 0.284 mmol, 1 eq) and 1-(pyrazin-2-yl)-1,4-diazepane (101.41 mg, 0.568 mmol, 2 eq) were added to DMSO (3 mL) and stirred at 140° C. for 18 hours. The mixture was allowed to cool, then added dropwise to ice cold water. The resulting precipitate was filtered and dried in vacuo to produce pure 5.16.
(548) ##STR00253##
(549) TABLE-US-00013 5-16 LC-MS Retention time 3.57 minutes White Found 440.2 [M + H].sup.+; calculated for C.sub.22H.sub.22FN.sub.5O.sub.4 solid 440.45 [M + H].sup.+ Yield 84 mg (68%)
Synthesis of (S)-9-fluoro-3-methyl-7-oxo-10-(4-(pyrazin-2-yl)-1,4-diazepan-1-yl)-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid hydrochloride (5.17)
(550) 5.16 (20.29 mg, 0.05 mmol, 1 eq) was added to dichloromethane (2 mL) and methanol (2 mL) and stirred for 2 minutes. Then 4M HCl in dioxane (231 μL, 0.92 mmol, 20 eq) was added dropwise and the flask sealed and stirred for 1 hour. The mixture was then washed with hexane (3×10 mL), concentrated in vacuo and lyophilised for 24 hours to afford 5.17.
(551) ##STR00254##
(552) TABLE-US-00014 5-17 LC-MS Retention time 7.00 minutes White Found 440.2 [M + H].sup.+; calculated for C.sub.22H.sub.22FN.sub.5O.sub.4 solid 440.45 [M + H].sup.+
Synthesis of Moxifloxacin-ARB Hybrid Compounds
Synthesis of 1-cyclopropyl-6-fluoro-8-methoxy-7-((4aS-7aS)-1-(naphthalen-1-ylmethyl)octahydro-6H-pyrrolo[.SUB.3,4.-b]pyridine-6-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (6.2)
(553) Moxifloxacin hydrochloride (6.1; 100 mg, 0.23 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and distilled water (10 mL total). After stirring for 5 minutes, potassium carbonate (95 mg, 0.69 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 1-(bromomethyl)naphthalene (48 mg, 0.22 mmol, 0.95 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 24 hours. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the crude product. Purification was achieved using an SCX-2 catch and release cartridge (see Solid Phase Extraction method) to afford 6.2.
(554) ##STR00255##
(555) TABLE-US-00015 6.2 LC-MS Retention time 3.23 minutes Pale Found 542.1 [M + H].sup.+; calculated for C.sub.32H.sub.32FN.sub.3O.sub.4 yellow 542.62 [M + H].sup.+ solid Yield 104.65 mg (84.6 % yield)
Synthesis of 1-cyclopropyl-6-fluoro-8-methoxy-7-((4aS,7aS)-1-(naphthalen-1-ylmethyl)octahydro-6H-pyrrolo[.SUB.3,4.-b]pyridine-6-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (6.3)
(556) 6.2 (30.65 mg, 0.06 mmol, 1 eq) was added to methanol (25 mL) and stirred for 10 minutes. Then 4M HCl in dioxane (28 μL, 0.11 mmol, 2 eq) was added dropwise and the flask sealed and stirred for 6 hours. The mixture was then washed with hexane (3×30 mL), concentrated in vacuo and lyophilised for 24 hours to afford 6.3.
(557) ##STR00256##
(558) TABLE-US-00016 6.3 LC-MS Retention time 6.30 minutes Yellow Found 542.1 [M + H].sup.+; calculated for C.sub.32H.sub.32FN.sub.3O.sub.4 solid 542.62 [M + H].sup.+ Yield 18.30 mg (57.2 % yield)
Synthesis of 7-((4aS,7aS)-1-benzyloctahydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (6.4)
(559) Moxifloxacin hydrochloride (6.1; 80 mg, 0.199 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and distilled water (5 mL total). After stirring for 5 minutes, potassium carbonate (82.63 mg, 0.58 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, bromomethylbenzene (30.68 mg, ° AB mmol, 0.90 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 18 hours. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the pure product 6.4.
(560) ##STR00257##
(561) TABLE-US-00017 6.4 LC-MS Retention time 2.90 minutes Brown Found 492.0 [M + H].sup.+; calculated for C.sub.32H.sub.32FN.sub.3O.sub.4 solid 492.56 [M + H].sup.+
Synthesis of 1-cyclopropyl-6-fluoro-7-((4aS-7aS)-1-(4-(hydroxymethyl)benzyl)octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (6.6)
(562) Moxifloxacin hydrochloride (6.1; 80 mg, 0.199 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and distilled water (5 mL total). After stirring for 5 minutes, potassium carbonate (82.63 mg, 0.58 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, ((4-(bromomethyl)phenyl)methanol (36.06 mg, 0.18 mmol, 0.90 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 18 hours. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the pure product 6.6.
(563) ##STR00258##
(564) TABLE-US-00018 6.6 LC-MS Retention time 2.85 minutes Brown Found 522.1 [M + H].sup.+; calculated for C.sub.32H.sub.32FN.sub.3O.sub.4 solid 522.59 [M + H].sup.+
Synthesis of 1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-7-((4aS,7aS)-1-(4-(pyrrolidin-1-yl)benzyl)octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-1,4-dihydroquinoline-3-carboxylic acid (6.8)
(565) Moxifloxacin hydrochloride (6.1; 80 mg, 0.199 mmol, 1 eq) was added to a 1:1 mix of acetonitrile and distilled water (5 mL total). After stirring for 5 minutes, potassium carbonate (82.63 mg, 0.58 mmol, 3 eq) was added and the mixture stirred for a further 5 minutes. Once fully dissolved, 1-(4-(bromomethyl)phenyl)pyrrolidine (43 mg, 0.18 mmol, 0.90 eq) was added slowly over the course of 1 hour and the mixture subsequently stirred for 18 hours. Upon completion, the product was extracted with dichloromethane (2×20 mL) using a 1M solution of citric acid to neutralise the aqueous phase. Combined organic fractions were washed with distilled water (20 mL) and dried over MgSO.sub.4, filtered and concentrated in vacuo to give the pure product 6.8.
(566) ##STR00259##
(567) TABLE-US-00019 6.8 LC-MS Retention time 6.30 minutes Yellow Found 558.0 [M − H].sup.+; calculated for C.sub.32H.sub.32FN.sub.3O.sub.4 solid 559.67 [M − H].sup.+
Biological Testing
Minimum Inhibitory Concentration Protocol
(568) Minimal inhibitory concentrations (MICs) were determined using the microdilution broth method. Briefly, compounds were added to the first two columns of a 96-well plate and diluted two-fold down the plate in tryptic soy broth (TSB). Overnight cultures of bacteria were then adjusted to an optical density (OD) of 0.01 in TSB, which is equivalent to 1×10.sup.6 CFU/mL, and added to each well. Untreated controls and blank wells were included. Compounds were initially dissolved in DMSO prior to dilution in water and in broth. Equivalent concentrations of solvent had no effect on bacterial growth. The MIC was defined as the lowest concentration of compound which resulted in no visible growth at an optical density of 600 nm after 20 hours incubation at 37° C.
(569) Reserpine Assay
(570) A simple plate based method was used as described by Beyer et al. (24). The compound was added at a final concentration of 0.25×MIC (made up at 1×MIC in water, 50 μL/well). Reserpine was added at a final concentration of 10 μg/mL (made up at 40 μg/mL in TSB, 50 μL per well). The bacteria were added at an OD of 0.01 in TSB (100 μL/well). The plate was a normal clear plate as used for MICs. OD was read every 30 minutes for 9 hours (the literature states 7 hours, but this is strain-dependent). Blanks of TSB and water were added to ensure no infections were present.
(571) Galleria Assay
(572) Wax moth larvae (Galleria mellonella) were purchased from Livefood UK Ltd (Rooks Bridge, Somerset, UK) and were maintained on wood chips in the dark at 15° C. until used. Bacteria from overnight cultures were adjusted to a known concentration in PBS and a Hamilton syringe was used to inject 10 μL aliquots of this suspension into G. mellonella larvae. Injections were performed into haemocoel of 10 larvae per bacterial strain via the foremost leg proleg. Control larvae were either injected with 10 μL PBS in order to measure any potential lethal effects of the injection process, or not injected to measure the effects of the incubation procedure. After injection, larvae were incubated statically at 37° C. inside petri dishes and the number of dead larvae was scored periodically. Larvae were considered dead when they displayed no movement in response to gentle prodding with a pipette tip. All experiments were carried out at least in triplicate. Data were analysed using the Mantel-Cox method using Prism Software Version 6 109 (Graphpad, San Diego, Calif., USA).
(573) Computational Modelling
(574) Homology Modelling
(575) A 3D structure for the Staphylococcus aureus NorA efflux pump protein was generated by homology modelling through its corresponding amino acid sequence (FASTA format) through use of the I-TASSER web server. PDB crystal structure 3WDO was used as the template. A C-score (confidence score; an estimate of the quality of models predicted by I-TASSER) of 1.27 was obtained for the model generated. C-scores are typically in the range of −5 to 2, with higher C-scores indicating higher quality models. Compound structures were generated using Chem3D 15.0 software and minimised using both the AMBER 12 package program and SYBYL software.
(576) Molecular Docking
(577) Molecular docking protocols were used in order to predict compound binding sites and affinities. The relationship between the binding affinity of the compounds under study and the docking score was used for comparison of the binding energies and affinities of the ligands for NorA. Molecular docking was performed to generate several distinct binding orientations and binding affinities for each binding mode. Subsequently, the binding modes that showed the lowest binding free energy were considered as the most favorable binding mode for each compound.
(578) In the first step, AutoDock SMINA was used for molecular docking of the compounds to the efflux pump structure for finding the best binding pocket by exploring all probable binding cavities in the proteins. All the parameters were set in their default values. Then GOLD molecular docking was applied for docking of the compounds into the SMINA-located best binding site for performing flexible molecular docking and determining more precise and evaluated energies and scores. Based on the fitness function scores and ligand binding position, the best-docked poses for each ligand were selected; the pose with the smallest fitness function score and highest GOLD fitness energy was considered the best-docked pose for each system.
(579) Genetic algorithm (GA) is used in GOLD ligand docking to thoroughly examine the ligand conformational flexibility along with partial flexibility of the protein. The maximum number of runs was set to 20 for each compound and the default parameters were selected (100 population size, 5 for the number of islands, 100,000 number of operations and 2 for the niche size). Default cutoff values of 2.5 Å (dH-X) for hydrogen bonds and 4.0 Å for van-der-Waals distance were employed. When the top solutions attained the RMSD values within 1.5 A°, the GA docking was terminated.
(580) Molecular Hydrophobicity Potential (MHP)
(581) Molecular Hydrophobicity Potential (MHP) was performed for the compounds in free form and complex states with NorA by using the Protein-Ligand Attractions Investigation Numerically (PLATINUM) web server. Molecular Hydrophobicity Potential (MHP) parameters for rescoring results of GOLD were set as follows: dotdensity=High, MHPoffset=0.03, MHPshift-lig=0.5, MHPshift-rec=0.5. After docking and hydrophobic complementarity calculations, the part of ligand responsible for non-optimal hydrophobic or hydrophilic contact was investigated and the overall distribution of ligand hydrophobicity analysed. To this end, visualization of hydrophobic/hydrophilic properties and their complementarity between ligand and receptor molecules was performed by Jmol.
(582) Modelling Experiment
(583) Two example fragments, see ML-77-005 and ML-77-076 entries in Table 1, interact with the MFS-type pumps that operate in many clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and a large number of pathogens that are resistant to ciprofloxacin (20,21). The resistance in MRSA and VRE is commonly mediated by up-regulation of the NorA efflux pump (22,23) and this also contributes to subsequent mutations giving high level resistance to ciprofloxacin (e.g. gyrA, parC).
(584) The identified ARB-fragment was linked to the fluoroquinolone (initially ciprofloxacin was used as the model fluoroquinolone) and advanced MD simulations were carried out to ensure the ARB-linked fluoroquinolone still occupied the same binding domain of DNA gyrase (
(585) Molecular Models of Key Efflux Pumps in ESKAPE Pathogens and Binding Pockets of Ligands that were Utilized to Identify the ARB Fragments
(586) Gram-Negative Species:
(587) Acinetobacter baumannii AYE, NCTC13424 Klebsiella pneumoniae MGH78578, NTUH K2044 Pseudomonas aeruginosa PAO1, PA14 Escherichia coli
Gram-Positive Species: Enterococcus faecalis (Streptococcus faecalis) Enterococcus faecium (Streptococcus faecium) Staphylococcus aureus
(588) TABLE-US-00020 TABLE 1 Details of multidrug efflux pumps in ESKAPE pathogens utilized to identify the ARB fragments Multidrug Species Strain Efflux Pump Type Template Acinetobacter AYE AdeB RND 3aoa baumannii — NorM MATE 3mkt Klebsiella MGH 78578 AcrB RND 2j8s pneumoniae MGH 78578 MdtK MATE 3mku Pseudomonas PAO1 MexB RND 3w9i aeruginosa PAO1 PmpM MATE 3mku PA14 MexB RND 3w9i PA14 MexF RND 3w9j Escherichia coli K12 AcrB RND 3aoc GM4792 MdtK MATE 3mku Enterococcus — EmeA MFS 3wdo faecalis — OqxD RND 2v5o (Streptococcus — EfrB ABC 3qf4 faecalis) Enterococcus — EfmA MFS 1pw4 faecium E980 EfmE MATE 5c6o faecium) (Streptococcus Staphylococcus N315 SdrM MFS 4w6v aureus N315 MepA MATE 4lz6 NCTC 8325 MepA MATE 3wbn — QacA MFS 4zpo — NorA MFS 3wdo
(589) Molecular models of various of the above key efflux pumps in ESKAPE pathogens and binding pockets of ligands that were utilized to identify the ARB fragments are shown in
(590) Biological Data
(591) NorA-Targeting Series
(592) MIC Data
(593) With regard to the Gram positive bacteria in Table 1, MSSA 9144 is methicillin sensitive S. aureus ATTC 9144 (NCTC 6571); EMRSA15 is a strain endemic to UK hospitals (NCTC 13616, HO 5096 0412); EMRSA16 is a representative of the epidemic EMRSA16 lineage endemic in UK hospitals (NCTC 13277, MRSA252); VSE 775 is E. faecalis VSE NCTC 775; VRE 12201 is E. faecalis VRE NCTC 12201 and VRE 12204 is E. faecium VRE NCTC 12204.
(594) MIC Tests
(595) The synthesized conjugate ML-77-005 was tested against MDR Gram-positive strains that over-express NorA efflux pump. ML-77-005 showed a 128 to 64 fold reduction in MIC compared to ciprofloxacin in EMRSA-15 and EMRSA-16. This is highly surprising as compositions comprising combinations of an efflux pump inhibitors and an antibiotic usually result in 2-8 fold potentiation of MIC without any significant effect of antimicrobial resistance. Retention of activity in strains which are susceptible to fluoroquinolones (e.g. Ab17978, VSE/VRE strains) suggests that the molecule is fully functional. This is a very significant observation as for the first time an efflux pump-targeting ARB has re-sensitised resistant bacteria to an antibacterial agent (see MIC data in Table 2).
(596) A range of further ARB-linked ciprofloxacin compounds were prepared and tested. It was possible to establish a well-defined SAR profile and the hydrophobicity of the ARB-linked fluoroquinolones played an important role in reversing the multiple drug resistance and re-sensitising the bacteria (see Table 2).
(597) The complete structure of the ciprofloxacin derivatives shown in Table 2 are arrived at by replacing the hydrogen of the NH in ciprofloxacin with the bond indicated by the zig-zag line to the fragment structure. The complete structure of the fragment ML-77-005 can also be seen, for example, as compound (2.2) in the organic synthesis section, and the complete structure of the HCl salt ML-77-023 can be seen as compound (2.3).
(598) TABLE-US-00021 TABLE 2 MIC Data for Ciprofloxacin and Ciprofloxacin Derivatives Freebase Code Ciprofloxacin ML-77-005 (2.2) ML-77-036 (2.4) ML-77-048 (2.12) HCl Salt Code ML-77-023 (2.3) ML-77-044 (2.5) ML-77-061 (2.13) Structure
(599) The same ARB-fragment as ML-77-005 was covalently linked to norfloxacin (see ML-77-021 in Table 3), another 4-fluoroquinolone antibiotic, as norfloxacin suffers from the same efflux-mediated resistance. Again, the ARB-linked norfloxacin was able to re-sensitise the resistant EMRSA-15 and EMRSA-16 strains without any loss of activity against sensitive strains. A range of further ARB-linked norfloxacin compounds were prepared and tested and the results are also give in Table 3. The complete structure of the norfloxacin derivatives shown in Table 3 are arrived at by replacing the hydrogen of the NH in norfloxacin with the bond indicated by the zig-zag line to the fragment structure.
(600) TABLE-US-00022 TABLE 3 MIC Data for Norfloxacin and Norfloxacin Derivatives Freebase Norfloxacin ML-77-021 (3.2) ML-77-031 ML-77-049 Code (3.4) (3.12) HCl Salt Code ML-77-024 (3.3) ML-77-037 (3.5) ML-77-062 (3.13) Structure
(601) The same ARB-fragment as used for (ML-77-005) was linked to enoxacin, which is another 4-fluoroquinolone antibiotic hampered by efflux-mediated resistance, and testing against the same panel of bacteria. The ARB-linked enoxacin (ML-77-025) similarly re-sensitised the resistant EMRSA strains with 64-128 fold potentiation of MIC (see Table 4). The complete structure of the enoxacin derivatives shown in Table 3 are arrived at by replacing the hydrogen of the NH in enoxacin with the bond indicated by the zig-zag line to the fragment structure.
(602) TABLE-US-00023 TABLE 4 MIC Data for Enoxacin and a Enoxacin Derivative Freebase Code Enoxacin ML-77-025 (4.2) HCl Salt Code ML-77-034 (4.3) Structure
(603) The same ARB-fragment as used for (ML-77-005) was linked to levofloxacin, which is another 4-fluoroquinolone antibiotic, and testing against the same panel of bacteria. The ARB-linked levofloxacin (ML-77-140) similarly re-sensitised the resistant EMRSA strains (see Table 5). The complete structure of the levofloxacin derivatives shown in Table 3 are arrived at by replacing the methyl group of the N—CH.sub.3 in enoxacin with the bond indicated by the zig-zag line to the fragment structure.
(604) TABLE-US-00024 TABLE 5 MIC Data for Levofloxacin and a Levofloxacin Derivative Freebase Code Levofloxacin ML-77-140 (5.2) HCl Salt Code ML-77-144 (5.3) Structure
(605) ARB-linked ciprofloxacin (ML-77-05) and ARB-linked norfloxacin (ML-7-021) were subsequently tested against a wider panel of 4-fluoroquinolone-resistant Streptococcus and Enterococcus strains. Both conjugate compounds were able to re-sensitise resistant bacterial strains including cases where resistance is due to mutations in DNA gyrases (Table 6). This was surprising and suggests that reversing efflux-mediated resistance can also reverse other associated resistance mechanisms including mutations due to the presence of high intracellular concentrations of ARB-linked antibiotic.
(606) TABLE-US-00025 TABLE 6 ML-77-005/023 and ML-77-021/024 Extended Gram-positive MIC Panel Chromosomal ML-77-005 Fold ML-77-021 Fold Species Strain mutations CIP (2.2) decrease NOR (3-2) decrease S. epidermidis SE1 N/A 0.5-1 0.25-2 0.25-4 1-4 1 1-4 S. aureus SA1 gyrA, 84:S => L; 64-128 2 32-63 >128 4 ≥32 grlA, 80:S => F S. aureus SA2 N/A 1 0.5 0.5 8-64 1 8-64 S. aureus SA3 gyrA, 84:S => L; 32 2 16 64 2 32 grlA, 80:S => F S. haemolyticus SH1 N/A 16 4 4 128 4 32 S. aureus SA4 gyrA, 84:S => L; 16 2 8 64 2 32 grlA, 80:S => F S. aureus SA5 gyrA, 84:S => L; 32-64 2 16-32 >128 2 ≥64 grlA, 80:S => F S. aureus SA6 gyrA, 84:S => V; 128 2 64 >128 2 ≥64 grlA, 80:S => F E.faecalis EF1 N/A 64 2 32 >128 4 ≥32 E.faecium EF2 N/A 128 2 64 >128 2-4 ≥32 S. aureus SA7 gyrA, 84:S => L; 64 2 32 >128 2 ≥64 grlA, 80:S => F S. aureus SA8 gyrA, 84:S => L; 64 2 32 128 2 64 grlA, 80:S => F S. aureus SA9 gyrA, 84:S => L; 128 2 64 >128 2 ≥64 grlA, 80:S => F S. aureus SA10 gyrA, 84:S => L; 32-64 2 16-32 128 2 64 grlA, 80:S => F
AdeB-Targeting Series
MIC Data
(607) Further ciprofloxacin linked compounds ML-77-147 and ML-77-138 were prepared and tested against a wider panel of bacteria. The results for these two compounds are provided below in Table 7.
(608) TABLE-US-00026 TABLE 7 MIC Data for Ciprofloxacin and Ciprofloxacin Derivatives Against AdeB-targeting Series Freebase Code Ciprofloxacin ML-77-147 (2.44) ML-77-138 (2.53) HCl Salt Code ML-77-158 (2.45) ML-77-153 (2.54) Structure
(609) Further norfloxacin linked compounds ML-77-148 and ML-77-146 were prepared and tested against a wider panel of bacteria. The results for these two compounds are provided below in Table 8.
(610) TABLE-US-00027 TABLE 8 MIC Data for Norfloxacin and Norfloxacin Derivatives Against AdeB- targeting Series Freebase Code Norfloxacin ML-77-148 (3.40) ML-77-146 (3.46) HCl Salt Code ML-77-159 (3.41) ML-77-154 (3.47) Structure
(611) The HCl salt ciprofloxacin linked compound ML-77-158 (structure shown in table 7 above) was also tested against an extended A. baumannii panel of bacteria. The results for this panel are provided below in Table 9.
(612) TABLE-US-00028 TABLE 9 ML-77-147/158 A. baumannii Extended MIC Panel Species Strain CIP ML-77-158 Fold decrease A. baumannii W1 128 32 4 A. baumannii OXA23.1 128 64 2 A. baumannii UKA7 >512 64 >8 A. baumannii UKA10 256 128 2 A. baumannii UKA12 512 32 16 A. baumannii UKA16 128 32 8 A. baumannii 13423 256 128 2 A. baumannii UKA13 512 32 16 A. baumannii 13302 128 64 2 A. baumannii UKA1 32 16 2 A. baumannii A601 32 8 4 A. baumannii 12156 1 0.5 2
MexB-Targeting Series
(613) Two further levofloxacin-linked compounds were prepared and tested against a wider panel of bacteria. The results for these compounds against this wider panel of bacteria are shown in Table 10 below.
(614) TABLE-US-00029 TABLE 10 MIC Data for Levofloxacin and Levofloxacin Derivatives Against MexB-targeting Series Freebase Levofloxacin ML-83-019 ML-83-0018 ML-83-001 (5.6) ML-83-011 Code (5.8) (5.4) HCl Salt ML-83-025 ML-83-009 (5.7) ML-83-012 Code (5.9) (5.5) Structure
(615) TABLE-US-00030 TABLE 11 Evidence of reversal of efflux mediated resistance observed using ARB technology in fluoroquinolones Chromosomal Strain mutations Fold Species Name Levofloxacin Ciprofloxacin KSN-L22 (quinolones) change S. aureus SA238 32 >64 1 gyrA, 84:S => L; 32-64 grlA, 80:S => F S. aureus SA215 32 64-128 1 gyrA, 84:S => L; 32-128 grlA, 80:S => F S. aureus SA454 8 >64 1 gyrA, 84:S => L; 8-64 grlA, 80:S => F S. aureus SA282 16 >64 1 gyrA, 84:S => L; 16-64 grlA, 80:S => F S. aureus SA275 8 128 1 gyrA, 84:S => V; 8-128 grlA, 80:S => F S. aureus SA046 16 >64 1 gyrA, 84:S => L; 16-64 grlA, 80:S => F S. aureus SA275 16 128 1 gyrA, 84:S => L; 16-128 grlA, 80:S => F S. aureus EMRSA15 16 128 0.5-1 gyrA, 84:S => L; 32-256 grlA, 80:S => F S. aureus EMRSA16 16 128 1 gyrA, 84:S => L; 16-128 grlA, 80:S => F
(616) Using a unique approach of antibiotic resistance breakers, that has never been adopted or tested by researchers working in the field, has resulted in surprising potentiation of the modified antibiotic by up to 128 fold which has never been observed before. A traditional approach of combining an efflux pump inhibitors with antibiotics typically result in 4-8 fold potentiation, which is not enough to reverse efflux mediated resistance. We have been able to use this unique approach to reverse efflux mediated resistance in a number of multiple drug resistance pathogens with multiple target mutations using fluoroquinolones as model antibiotics (Table 11). The ability of these molecules to reverse efflux mediated resistance was studied using MIC testing against MDR pathogens, and the inability of bacteria to efflux these molecules was tested using a reserpine growth assay.
(617) ##STR00328##
(618) Modification of ML-83-009 with six membered piperazine ring to 5-membered pyrrolidine ring to develop ARB s.
(619) One of the identified ARB fragments contained a six-membered piperazine ring connected directly to a pyrimidine ring via N—C coupling. This first generation ARB-modified ML-83-009 was weakly inhibiting efflux. Further MD simulations revealed its contact with the key residues were not sufficiently strong to prevent its efflux. Molecular modelling revealed that the molecule cannot fit snugly within the binding pocket and interact with key residues due to its linear shape and relative rigidity of the piperazine ring containing ARB. This information was used to convert the 6-membered piperazine ring to a 5-membered pyrrolodine ring, and take the second nitrogen outside the ring. Finally, the pyrimidine ring was connected with the amine group that was placed outside the ring. This provided additional flexibility to the ARB fragment and allowed the terminal pyrimidine ring to form to rotate and form curved structure that allowed optimum contact with the key residues which was not possible with the rigid linear six-membered piperzine ring linked ARB fragment. This has been illustrated below with NorA efflux pump in S. aureus as an example (
(620) This flexibility of the terminal ARB fragment with the key residues were only observed when the amine group was placed outside the five membered ring, as the molecule with a seven membered diazepine ring did not provide adequate contact due to relatively linear and inflexible nature of the molecule.
(621) TABLE-US-00031 TABLE 12 Superiority of 5-member pyrrolidine ring with exocyclic amine as part of ARB fragment. ML-83-009 KSN-L22 ML-83-034 6-member ring 5-member ring with 7-member ring exocyclic amine
(622) This flexible ARB fragment which can adopt different curvature, helped to obtain the critical and stable contact with the key residues within the binding pocket (
(623) This was further evident when the 6-member ring containing ML-83-009 was tested against an extended MDR panel with known multiple target mutations (Table 13). The compound did show 4-8 fold potentiation but was significantly less active than KSN-L22 which showed between 16 to 266 fold potentiation (Table 13) due to the superiority of the ARB fragment.
(624) TABLE-US-00032 TABLE 13 Activity of ML-83-009 and KSN-L22 against extended panel of MDR strains. Fold Fold decrease decrease Strain ML83-009 KSN-L22 name Species ML83-009 Levofloxacin KSN-L22 Mutations v Levo v Levo SA 215 S. aureus 4 16 1 gyrA, 84:S => L; 4 16 grlA, 80:S => F SA 105 S. aureus 4 8-16 1 gyrA, 84:S => L; 2-4 16 grlA, 80:S => F SA 275 S. aureus 4 32 1 gyrA, 84:S => V; 8 32 grlA, 80:S => F SA 046 S. aureus 4 16 1 gyrA, 84:S => L; 4 16 grlA, 80:S => F SA 318 S. aureus 4 16 1 gyrA, 84:S => L; 4 16 grlA, 80:S => F SA 388 S. aureus 4 16 1 gyrA, 84:S => L; 4 16 grlA, 80:S => F EF 205 E. faecium 16-32 >32 0.12 Not done >2 266 EF 602 E. faecalis 16 >32 0.25 Not done >2 64
(625) Four pairs of molecules were synthesized to rationalise the observation and demonstrate the flexibility of the ARB units, and in each case the 5-membered pyrrolidine ring with exocyclic amine containing ARB-Fluoroquinolone showed superior MIC against efflux resistant MDR strains with multiple target mutations compared to corresponding six-membered or seven-membered analogues (see below and
(626) ##STR00332## ##STR00333##
(627) Four pair of compounds as shown above were synthesized to show the superiority of ARB fragments with five membered pyrrolidine ring with an exocyclic amine group compared to six membered piperazine ring containing ARB fragments.
(628) TABLE-US-00033 TABLE 14 MIC comparison of compounds (KSN-coded) containing 5-membered pyrrolidine ring with exocyclic amino group with 6-membered piperazine ring containing compounds (ML-coded). KSN-L22 ML-83-009 KSN-BL1 ML-83-012 KSN-L34 ML-83-010 KSN-BL7 ML-83-011 MIC (μg/mL) Gram-Negative Klebsiella 4 64 8 64 32 32 16 64 pneumoniae (KP13368) Klebsiella 1 8 0-5 16 2 4 2 16-64 pneumoniae (M6) Acinetobacter 4 128 8 128 4 128 8 128 baumannii (AYE) Acinetobacter 0.125 4 0.25 8 ≤0.125 2 ≤0.125 16 baumannii (AB17978) Pseudomonas 4 32 4 32 4 64 8 128 aeruginosa (PA01) Escherichia 0.5 1-4 0.25 4 0.125 2 1 1-8 coli (EC12923) Gram-Positive Staphylococcus ≤0.03 0.125 ≤0.125 0.25 ≤0.125 ≤0.125 ≤0.0039 0.25 aureus (MSSA9144) Staphylococcus 0.5 4 1 2 0.5 2 0.125 16 aureus (EMRSA15) Staphylococcus 1 4 1 2 1 2 0.25 16 aureus (EMRSA16) Enterococcus 0.06 0.25 ≤0.125 0-5 ≤0.125 0.5 0.06 1 (VRE775) Enterococcus 0.06 0.25 ≤0.125 ≤0.125 ≤0.125 ≤0.125 0.03 0.5-1 (VRE12201)
(629) TABLE-US-00034 TABLE 15 MIC comparison of KSN-BL-7 containing a 5-membered pyrrolidine ring with three known antibiotics against an extended bacterial panel MIC, μg/ml PT#1218400 No. Batch No. Assay # Species Strain ID Resistance KSN-BL-7 Linezolid Tigecycline Vancomycin 1 421779 602050 Enterococcus ATCC 29212 — 0.25 0.125 faecalis 2 421780 602100 Enterococcus ATCC 51575 VanB 0.25 0.125 faecalis 3 421781 602200 Enterococcus ATCC 51299 VanB 0.125 0.0625 faecalis 4 421782 602201 Enterococcus CCUG 47775 Van A 0.5 0.125 faecalis 5 421783 602202 Enterococcus ATCC 700802 VanB 0.125 0.125 faecalis 12 421790 602374 Enterococcus TUH44-29, Van A 1 0.0625 faecium CCUG 59167 13 421791 602380 Enterococcus ATCC 49608 VanC 0.25 0.125 gallinarum 14 421792 603100 Streptococcus ATCC 12386 — 0.25 0.5 agalactiae 15 421793 603200 Streptococcus ATCC 9811 — 0.25 0.5 oralis 16 421794 603900 Streptococcus ATCC 6301 — 0.125 0.25 pneumoniae 17 421795 603910 Streptococcus S. Africa — 0.0625 0.25 pneumoniae 6B-8, ATCC 700675 18 421796 603920 Streptococcus ATCC 49619 — 0.125 0.25 pneumoniae 19 421797 603930 Streptococcus Tenessee 23F, MDR 0.125 0.25 pneumoniae ATCC 51916 20 421798 603940 Streptococcus CSR 14-10, PRSP 0.0625 0.25 pneumoniae ATCC 700677 21 421799 603941 Streptococcus SP264, ST 23F 0.0625 0.25 pneumoniae ATCC 700669 22 421800 603942 Streptococcus Hungary 19A, MDR 0.0625 0.25 pneumoniae ATCC 700673 23 421801 603943 Streptococcus England ST14-9 ERY-R 0.125 0.25 pneumonia ATCC 700676 24 421802 603950 Strepotococcus ATCC 14289 — 0.125 0.25 pyogenes 25 421803 603960 Strepotococcus ATCC 19615 — 0.125 0.25 pyogenes 26 421804 604000 Staphylococcus 6538P — ≤0.03125 0.125 aureus 27 421805 604010 Staphylococcus ATCC 33592 MRSA ≤0.03125 0.5 aureus 28 421806 604020 Staphylococcus ATCC 33594 — ≤0.03125 0.5 aureus 29 421807 604030 Staphylococcus ATCC 27660 — ≤0.03125 0.5 aureus 30 421808 604035 Staphylococcus MW2, USA400 ≤0.03125 0.5 aureus BAA-1707 MRSA 31 421809 604040 Staphylococcus Mu50, MRSA/VISA 2 2 aureus ATCC 700699 32 421810 604045 Staphylococcus TCH1516, USA300 ≤0.03125 0.5 aureus BAA-1717 MRSA 33 421811 604050 Staphylococcus ATCC 13709 MRSA ≤0.03125 0.5 aureus 34 421812 604055 Staphylococcus FPR3757, USA300 0.5 0.5 aureus BAA-1556 MRSA 35 421813 604060 Staphylococcus ATCC 49230 — ≤0.03125 0.5 aureus 36 421814 604070 Staphylococcus R136 MRSA 0.5 0.5 aureus 37 421815 604100 Staphylococcus ATCC 10390 — ≤0.03125 1 aureus 38 421816 604110 Staphylococcus ATCC 29213 — ≤0.03125 0.5 aureus 39 421817 604111 Staphylococcus ATCC 29213 — 1 1 aureus with 50% human serum 40 421818 604112 Staphylococcus ECL 2963621 MRSA 0.25 1 aureus DAP-NS 41 421819 604113 Staphylococcus ECL 2963646 VRSA 0.5 2 aureus 42 421820 604114 Staphylococcus ECL 2963666 MRSA 1 1 aureus DAP-NS 43 421821 604115 Staphylococcus ECL 2963667 MRSA 1 1 aureus DAP-NS 44 421822 604116 Staphylococcus ECL 2963743 MRSA 0.5 1 aureus DAP-NS 45 421823 604117 Staphylococcus COL, NRS100 MRSA ≤0.03125 2 aureus 46 421824 604118 Staphylococcus NRS101 — ≤0.03125 1 epidermidis 47 421825 604119 Staphylococcus NRS119 MRSA 1 1 aureus LZD-NS 48 421826 604120 Staphylococcus NRS12 VISA 0.0625 4 aureus 49 421827 604121 Staphylococcus NRS123 USA400 ≤0.03125 0.5 aureus MRSA 50 421828 604122 Staphylococcus NRS127 MRSA 4 1 aureus LZD-NS 51 421829 604123 Staphylococcus NRS157 — ≤0.03125 0.5 aureus 52 421830 604124 Staphylococcus NRS17 MRSA/VISA 1 2 aureus 53 421831 604126 Staphylococcus NRS22 USA600 4 4 aureus MRSA/VISA 54 421832 604127 Staphylococcus NRS269 MRSA 2 2 aureus TGC-NS 55 421833 604128 Staphylococcus E-MRSA 15, MRSA 0.5 0.5 aureus NRS271 LZD-NS 56 421834 604129 Staphylococcus NRS3 MRSA/VISA 0.5 1 aureus 57 421835 604130 Staphylococcus NRS382 USA100 0.5 1 aureus MRSA 58 421836 604131 Staphylococcus NRS383 USA200 1 0.5 aureus MRSA TIG-NS 59 421837 604132 Staphylococcus NRS384 USA300 ≤0.03125 0.5 aureus MRSA 60 421838 604133 Staphylococcus NRS385 USA500 0.5 0.5 aureus MRSA 61 421839 604134 Staphylococcus NRS386 USA700 0.25 0.5 aureus MRSA 62 421840 604135 Staphylococcus NRS387 USA800 ≤0.03125 1 aureus MRSA 63 421841 604136 Staphylococcus NRS402 MRSA/VISA 1 1 aureus DAP-NS 64 421842 604137 Staphylococcus NRS483 USA1000 ≤0.03125 0.5 aureus MRSA 65 421843 604138 Staphylococcus NRS484 USA1100 ≤0.03125 1 aureus MRSA 66 421844 604139 Staphylococcus NRS56 MRSA/VISA 1 2 aureus 67 421845 604140 Staphylococcus NRS60 VISE 0.0625 2 epidermidis 68 421846 604141 Staphylococcus NRS7 VISE 0.5 4 epidermidis 69 421847 604142 Staphylococcus NRS71 MRSA 1 0.25 aureus 70 421848 604143 Staphylococcus Sanger-476, — ≤0.03125 0.5 aureus NRS72 71 421849 604144 Staphylococcus NRS8 VISE 1 2 epidermidis 72 421850 604145 Staphylococcus VRS1 VanA 1 2 aureus MRSA/VRSA 73 421851 604146 Staphylococcus VRS11b VanA 2 2 aureus MRSA/VRSA 74 421852 604147 Staphylococcus VRS2 VanA 0.5 2 aureus MRSA/VRSA 75 421853 604148 Staphylococcus VRS3a VanA 0.5 4 aureus MRSA/VRSA 76 421854 604149 Staphylococcus FDA-CDC MRSA/VISA 16 4 aureus AR-BANK# 0215 77 421855 604150 Staphylococcus FDA-CDC MRSA/VISA 0.5 2 aureus AR-BANK# 0216 78 421856 604151 Staphylococcus FDA-CDC MRSA/VISA 1 1 aureus AR-BANK# 0217 79 421857 604152 Staphylococcus FDA-CDC MRSA/VISA 1 1 aureus AR-BANK# 0218 Mupirocin-R 80 421858 604153 Staphylococcus FDA-CDC MRSA/VISA 4 2 aureus AR-BANK# 0219 81 421859 604154 Staphylococcus FDA-CDC MRSA/VISA 1 2 aureus AR-BANK# 0220 82 421860 604155 Staphylococcus FDA-CDC MRSA/VISA 0.5 2 aureus AR-BANK# 0221 83 421861 604156 Staphylococcus FDA-CDC VISA ≤0.03125 2 aureus AR-BANK# 0222 84 421862 604157 Staphylococcus FDA-CDC MRSA/VISA 1 2 aureus AR-BANK# 0223 85 421863 604158 Staphylococcus FDA-CDC MRSA/VISA 1 4 aureus AR-BANK# 0224 Mupirocin-R 86 421864 604159 Staphylococcus FDA-CDC MRSA/VISA 1 2 aureus AR-BANK# 0225 87 421865 604160 Staphylococcus FDA-CDC MRSA/VISA ≤0.03125 2 aureus AR-BANK# 0226 88 421866 604161 Staphylococcus FDA-CDC MRSA/VISA 1 4 aureus AR-BANK# 0227 89 421867 604162 Staphylococcus FDA-CDC MRSA/VISA 8 2 aureus AR-BANK# 0228 Mupirocin-R 90 421868 604250 Staphylococcus ATCC 29663 — ≤0.03125 1 intermedius 91 421869 604300 Staphylococcus ATCC 12228 — ≤0.03125 0.5 epidermidis 92 421870 604310 Staphylococcus CCF 15990, — ≤0.03125 1 epidermidis ATCC 51625 93 421871 604400 Staphylococcus ATCC 29970 — ≤0.03125 1 haemolyticus 94 421872 604450 Staphylococcus ATCC 15305 — 0.0625 0.5 saprophyticus 95 421873 604500 Streptococcus ATCC 25175 — ≤0.03125 2 mutans 96 421874 604600 Streptococcus ATCC 13419 — 0.25 0.5 salivarius 97 421875 604700 Streptococcus ATCC 10556 — 0.25 0.5 sanguinis 98 421876 605000 Staphylococcus ATCC 33591 MRSA ≤0.03125 0.5 aureus 99 421877 606000 Staphylococcus Smith, — ≤0.03125 0.5 aureus ATCC 19636 100 421878 661000 Streptococcus TM532 PRSP 0.0625 0.25 pneumoniae
(630) The role of the ARB fragment in reversing efflux mediated resistance was further studied using a SAR study, and the absence of the terminal pyrimidine, which provides critical contact, showed no potentiation in KSN-L44 against EMRSA-15 and EMRSA-16, in which NorA efflux pump is upregulated (Table 16). Similarly, removing the complete ARB fragment also resulted in loss of activity against MDR strains, showing the importance of the ARB fragment in conferring activity against MDR strains and reversing efflux mediated resistance.
(631) TABLE-US-00035 TABLE 16 Role of the terminal pyrimidine ring in reversing resistance against resistance strains. EMRSA15 EMRSA16 KSN-L22
(632) Finally, the superiority of the five-membered pyrrolidine ring with exocyclic amine group containing ARB was further demonstrated in the in vivo thigh infection model in which KSN-L22 showed statistically significant reduction of bacterial load while six-membered piperazine ring containing ML-83-009 wasn't able to reduce the bacterial load significantly. Treatment with ML-83-009 at 20 or 50 mg/kg/dose slightly decreased bacterial burden, by 0.26 and 1.12 log.sub.10 CFU/g compared to vehicle group, respectively, which was not statistically significant (
(633) The current patent application covers these 5-member pyrrolidine ring with exocyclic amine containing ARB-fragment linked antibiotics, which have not been synthesized before using this approach or any other synthetic approach.
(634) Reserpine Assay Experiments
(635) The ability of a napthyl-linked ciprofloxacin (ML-77-005 referred to as ML005 in the figures) and napthyl-linked norfloxacin (ML-77-021 referred to as ML021 in the figures) to prevent efflux was investigated through a reserpine growth assay (see
(636) Galleria mellonella Challenge Model
(637) G. mellonella larvae were challenged with 107 colony forming units of either S. aureus strains USA300 (
(638) The data is the average of 3 independent experiments each with 10 larvae.
(639) Pharmacokinetic Study of KSN-L22
(640) Mouse Strain
(641) ICR breed Mice (CD-1® IGS mice) were used in these studies and was supplied by Charles River (Margate UK) and. The mice were allowed to acclimatise for at least 7 days before starting the study.
(642) Animal Housing
(643) Mice were housed in sterilised individual ventilated cages that expose the mice at all times to HEPA filtered sterile air. Mice had free access to food and water (sterile) and will have aspen chip bedding.
(644) The room temperature was 22° C.+/−1° C., with a relative humidity of 60% and maximum background noise of 56 dB. Mice was exposed to 12 hour light/dark cycles.
(645) WP1: Single Dose Pharmacokinetics Study
(646) Treatments was administered by oral route or intravenous route according to the table below.
(647) For the PK study, 12 mice for each compound for oral route and 12 mice per compound for the intravenous route was treated.
(648) Mice was sequentially bled from a caudal vein into a 20 μL capillary (by agreement containing an anticoagulant) at different time as indicated in table below and a terminal bleed by cardiac puncture at 8 h after administration. Samples collected by capillary was directly transferred into a 96 well and mixed with 20 μl, of water, then frozen and store at −80° C. until bioanalysis.
(649) Mice for urine collection was housed singly in metabolic cages and urine collected at 0-1, 1-2, 2-4 h and 4-8 h post administration. Urine removed from the collection vessels in the metabolic cages and were frozen and store at −80° C. until bioanalysis. 12 mice were used for urine collection.
(650) TABLE-US-00036 TABLE 17 Summary of treatments Gp. Dose Blood Collection No Mice Urine No Mice No. Treatment (mg/kg) Route (h after administration) per group collection per group 1 KSN-82-L22 20 PO 0.25, 0.5, 1, 2, 4 and 3.sup. 0-1 h, 1-2 h, 3 8 h 2-4 h, 4-8 h, 8-24 h 2 KSN-82-L22 5 IV 0.083, 0.25, 0.5, 1, 2, 6.sup.b 0-1 h, 1-2 h, 3 4 and 8 h 2-4 h, 4-8 h, 8-24 h 3 Levofloxacin 20 PO 0.25, 0.5, 1, 2, 4 and 3.sup. 0-1 h, 1-2 h, 3 8 h 2-4 h, 4-8 h, 8-24 h 4 Levofloxacin 5 IV 0.083, 0.25, 0.5, 1, 2, 6.sup.b 0-1 h, 1-2 h, 3 4 and 8 h 2-4 h, 4-8 h, 8-24 h .sup.aTotal number of mice: 30 mice .sup.bOnly 3 mice per time point was bleed .sup.cSamples for bioanalysis 69 per test article - 39 plasma and 30 urine, 138 in total for 2 test articles
(651) WP2: Bio-Analysis and Determination of PK Parameters
(652) Analysis of up to 39 blood samples and up to 30 urine samples per test compound. A compound specific LC-MS/MS method was developed for each test compound and samples was quantified using matrix matched calibrators. Samples was prepared for bio-analysis via protein precipitation.
(653) Concentration vs time data and non-compartmental analysis was utilised to determine relevant PK parameters e.g. CL, Vss, t.sub.1/2, AUC.sub.inf AUCo-t, Cmax, tmax, % F, % dose in urine and CLR. Levofloxacin was used as a control in all cases for comparison purpose.
(654) TABLE-US-00037 TABLE 18 Result of IV PK Study with key parameters KSN-L-22 Levofloxacin PK Parameter (iv) Composite Mean Composite Mean Dose (mg/kg) 5.0 5.0 Dose (μmol/kg) 11.8 13.8 C.sub.o/C.sub.max (ng/mL) 9389 3890 C.sub.o/C.sub.max (nM) 22071 10764 T.sub.max (h) — — T½ (h) 0.6 0.5 MRT (h) 1.8 30 Vdss (L/kg) 1-4 4-5 Blood CL (ml/min/kg) 12.5 25.2 CL_F (ml/min/kg) — — Liver Blood Flow (%) 10.4% 21.0% AUG.sub.inf (ng.hr/mL) 6691 3313 AUC.sub.inf (nM.hr) 15728 9186 AUC.sub.o.t (ng.hr/mL) 6425 3091 AUC.sub.o.t (nM.hr) 15103 8553 Fraction Absorbed — — C.sub.last (ng/mL) 286 281.6 Bioavailability (%) — — Using AUC.sub.inf Bioavailability (%) — — Using AUC.sub.o.t
(655) A graph showing the mean total blood concentrations of KSN-82-L22 and Levofloxacin following iv administration to Male CD1 mouse at 5 mg/kg is shown in
(656) TABLE-US-00038 TABLE 19 Result of Oral PK Study with key parameters KSN-L-22 Levofloxacin Mean/Median Mean/Median PK Parameter (PO) (T.sub.max) (T.sub.max) Dose (mg/kg) 20.0 20.0 Dose (μmol/kg) 47-0 55-3 C.sub.o/C.sub.max (ng/mL) 2965 2947 C.sub.o/C.sub.max (nM) 4007 8156 T.sub.max (h) 1.00 0.50 T½ (h) 5-1 5-7 MRT (h) — — Vdss (L/kg) — — Blood CL (ml/min/kg) — — CL_F (ml/min/kg) 26.4 65.4 Liver Blood Flow (%) — — AUG.sub.inf (ng.hr/mL) 13203 5388 AUC.sub.inf (nM.hr) 31036 14909 AUC.sub.o.t (ng.hr/mL) 8231 4264 AUC.sub.o.t (nM.hr) 19347 11800 Fraction Absorbed — — C.sub.last (ng/mL) 628 115 Bioavailability (%) 49-3% 40.7% Using AUC.sub.inf Bioavailability (%) 32.0% 34-5% Using AUC.sub.o.t
(657) A graph showing the mean total blood concentrations of KSN-82-L22 and Levofloxacin following PO administration to Male CD1 mouse at 20 mg/kg is shown in
(658) In Vivo Efficacy Data of KSN-L22 and ML-83-009
(659) KSN-L22 which has the ARB Fragment Containing the 5-Members Pyrrolodine Ring Showed Notably Superior Activity Compared to Six-Member Piperazine Ring Containing ML-83-009.
(660) Executive Summary
(661) The aim of the study was to assess the efficacy of ML-83-009 and KSN-82-L22 at 20 mg/kg and 50 mg/kg in murine model of thigh infection with Staphylococcus aureus (S. aureus) ATCC29213 in comparison to levofloxacin as reference at the same dose. At 18 h post infection, after dosing at 2 h, 8 h and 14 h post infection, bacterial burden was unchanged by ML-83-009 treatment at 20 and 50 mg/kg. However, KSN-82-L22 at 20 and 50 mg/kg significantly decreased bacterial burden in comparison to vehicle and to pre-treatment group demonstrating bactericidal activity. This efficacy was similar to the Levofloxacin
(662) Study Aim
(663) The aim of the study was to assess the efficacy of two test articles in murine thigh infection model with S. aureus ATCC29213, 26 h post infection. The primary objectives was to assess the efficacy at 2 doses for each articles at 26 h, after dosing 2 h, 8 h and 14 h post infection. Moreover their efficacy were compared to the Levofloxacin efficacy with same doses and same regimen.
(664) Methods
(665) Regulatory
(666) The experiment was performed under UK Home Office Licences and with local ethical committee clearance. The experiment was performed by technicians that have completed parts A, B and C of the Home Office Personal License course and hold a current personal license. The experiment was performed in dedicated Biohazard 2 facilities.
(667) Animal Strain and Housing
(668) 40 Male mice used in these study were supplied by Charles River UK and were specific pathogen free. The strain of mouse used was Hsd:ICR (CD-1®), which is a well characterized outbred strain. Mice were 11-15 g on receipt at Evotec's facility and were allowed to acclimatize for minimum of 7 days prior to infection. Mice were approximately 28 g at the start of the study. Mice were housed in sterile individual ventilated cages exposing animals at all times to HEPA filtered sterile air. Mice had free access to food and water (sterile) and had sterile aspen chip bedding. The room temperature was 22° C.±1° C., with a relative humidity of 50-60% and maximum background noise of 56 dB. Mice were exposed to 12 hour light/dark cycles with dawn/dusk phases.
(669) Immunosuppression
(670) All mice were rendered neutropenic by immunosuppression with cyclophosphamide at 150 mg/kg 4 days before infection and then 100 mg/kg 1 day before infection administered by intraperitoneal injection. The immunosuppression regime leads to neutropenia starting 24 hours post administration of the first injection, which continues throughout the study. Mice were infected approximately 24 hours after the second dose of immunosuppressive agent.
(671) Preparation of Organism and Infection
(672) Staphylococcus aureus ATCC29213 was used for the study. Mice were infected with an inoculum prepared from a frozen stock diluted with phosphate buffered saline (PBS) to obtain 105 CFU/mL. 50 μL of this solution was injected to administer 5×10.sup.3 CFU/thigh. The actual concentration of organism was 2.65×10.sup.5 CFU/mL corresponding to 1.33×10.sup.4 CFU/thigh.
(673) Mice were infected by intramuscular injection of 50 μL inoculum into both lateral thigh muscles under inhaled anaesthesia using 2.5% isofluorane in 97.5% oxygen. Whilst still under anaesthesia mice were administered a single dose of buprenorphine (0.03 mg/kg) subcutaneously for pain relief.
(674) Preparation of Test Articles
(675) Test articles and comparator were prepared in stock solutions at 10× concentration as described in Table 20. The stock solutions were prepared in aliquots (i aliquot per time of dosing) and frozen at −20° C. The solutions were thawed and then diluted in water just before dosing to obtain the appropriate concentration of 5 mg/mL or 2 mg/mL for oral administration at 10 mL/kg of the dose of 50 mg/kg or 20 mg/kg respectively (Table 20). 10% DMSO in water was used as the vehicle
(676) TABLE-US-00039 TABLE 20 test article stock solutions Quantity of Corr- active Stock Stock Test Quantity ection molecule solution solution article (mg) factor (mg) 50 mg/mL 20 mg/mL ML-83-009 40 1 40 0.8 mL 0.21 mL of of 50 mg/mL DMSO stock added solution + 0.31 mL DMSO KSN-82-L22 41-30 1-17 41-30 0826 mL 0.21 mL of of 50 mg/mL DMSO stock added solution + 0.31 mL DMSO Levofloxacin 45 1 45 0.9 mL 0.21 mL of (Sigma ref of 50 mg/mL 28266) DMSO stock added solution + 0.31 mL DMSO
Study Design and Schedule
(677) Two hour post infection animals from the pre-treatment groups were euthanised and the remainder of animals were treated at 2 h, 8 h and 14 h post infection. Eighteen hours post infection, vehicle group displayed clinical signs of infection beyond the ethical limit, and thus the experiment was stopped and all remaining animals were euthanised. The study schedule is summarised in
(678) TABLE-US-00040 TABLE 21 Study design Dose per Total Route Test Concentration Treatment Dose of Treatments End of Experiment Group # Article (mg/mL) (mg/kg) (mg/kg) admin. (h post-infection) (h post infection) 1 Pre-Treatment NA NA NA NA 2 h 2 Vehicle — — — PO 2 h, 8 h, 14 h 18 h 3 ML-83-009 2 20 60 PO 2 h, 8 h, 14 h 18 h 4 ML-83-009 5 50 150 PO 2 h, 8 h, 14 h 18 h 5 KSN-82-L7 2 20 60 PO 2 h, 8 h, 14 h 18 h 6 KSN-82-L7 5 50 150 PO 2 h, 8 h, 14 h 18 h 7 Levofloxacin 2 20 60 PO 2 h, 8 h, 14 h 18 h 8 Levofloxacin 5 50 150 PO 2 h, 8 h, 14 h 18 h
(679) For all of the above groups the No. mice was 5
(680) For all of the above groups 2 to 8 the dose volume was 10 mL/kg. For the pre-treatment (group 1) the dose volume is not applicable.
(681) General Health Monitoring
(682) General health of animal were monitored in particular after 14 h post infection. Where the clinical deterioration of mice exceeded the ethically agreed limits, they were immediately euthanized using a pentobarbitone overdose. Once animals from the vehicle group exceeded ethical limits all animals in the experiment were euthanized.
(683) Endpoints
(684) After sacrifice of animal, both posterior thighs were removed, weighed, then homogenised in 2 mL PBS using a Precellys bead beater and the homogenates individually quantitatively cultured on CLED agar to determine the bacterial burdens.
(685) Statistics
(686) The data from the culture burdens were analysed using appropriate non-parametric statistical models (Kruskal-Wallis using Conover-Inman to make all pairwise comparisons between groups) with StatsDirect software v. 2.7.8., and compared to pre-treatment and vehicle controls.
(687) Results
(688) The aim of the study was to assess the efficacy of two client's test articles in murine thigh infection model with S. aureus ATCC29213, 26 h post infection. 2 doses for each articles were assesses with dosing 2 h, 8 h and 14 h post infection. Moreover their efficacy was compared to the one of levofloxacin with same doses and same regimen.
(689) Body Weights
(690) Animal weights as group averages throughout the study are shown in
(691) Following infection, Vehicle and ML-83-009 treated animals lost weight. The body weights of animals of treated groups with levofloxacin were unchanged and slightly increased for the animals of the KSN-82-L22 treated groups.
(692) Clinical Observations
(693) The experiment was terminated at 18 h post infection due to vehicle treated animals reaching the clinical end point for the model. No adverse effect of treatment was observed in treated groups.
(694) Thigh Bacteria Burden
(695) The infection was well established with the bacterial burden increasing by about 3.5 log.sub.10 CFU/g between 2 h and 18 h post infection. (Table 22 and
(696) Levofloxacin administered at 20 and 50 mg/kg/dose reduced thigh bacterial burden by ˜5 log.sub.10 cfu/g (P<0.0001).
(697) Treatment with ML-83-009 at 20 or 50 mg/kg/dose slightly decreased bacterial burden, by 0.26 and 1.12 log.sub.10 CFU/g compared to vehicle group, respectively, which was not statistically significant (
(698) TABLE-US-00041 TABLE 22 Thigh bacterial burden Group Standard Log.sub.10 Group Log.sub.10 reduction Group Geometric Deviation Geometric from vehicle No Treatment mean (cfu/g) (cfu/g) mean (cfu/g) control n 1 Pre-treatment 6.67 × 10.sup.4 2.36 × 10.sup.4 4.82 5 2 Vehicle 2.01 × 10.sup.8 1.33 × 10.sup.8 8.30 — 5 3 ML-83-009 20 mg/kg 1.11 × 10.sup.8 3.06 × 10.sup.8 8.05 0.26 5 4 ML-83-009 50 mg/kg 1.51 × 10.sup.7 8.29 × 10.sup.7 7.18 1.12 5 5 KSN-82-L22 20 mg/kg 2.01 × 10.sup.3 3.27 × 10.sup.3 3.30 5.00 5 6 KSN-82-L22 50 mg/kg 1.37 × 10.sup.3 7.50 × 10.sup.2 3.14 5.16 5 7 Levofloxacin 20 mg/kg 3.39 × 10.sup.3 2.02 × 10.sup.3 3.53 4.77 5 8 Levofloxacin 50 mg/kg 1.68 × 10.sup.3 1.00 × 10.sup.3 3.23 5.08 5
(699) TABLE-US-00042 TABLE 23 Statistic analysis ML-83-009 ML-83-009 KSN-82-L22 KSN-82-L22 Levofloxacin Levofloxacin Vehicle 20 mg/kg 50 mg/kg 20 mg/kg 50 mg/kg 20 mg/kg 50 mg/kg Pre treatment P = 0.0153 P = 0.0359 NS P = 0.0139 P = 0.0026 NS P = 0.0084 Vehicle NS NS P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 ML-83-009 NS P < 0.0001 P < 0.0001 P = 0.0003 P < 0.0001 20 mg/kg ML-83-009 P = 0.0002 P < 0.0001 P = 0.0058 P < 0.0001 50 mg/kg KSN-82-L22 NS NS NS 20 mg/kg KSN-82-L22 NS NS 50 mg/kg Levofloxacin NS 20 mg/kg
Summary
(700) A robust infection of S. aureus ATCC29213 was achieved in the thigh of ICR mice with −4.5 log.sub.10 CFU/g of tissue increase between pre-treatment group and vehicle group and the vehicle group reaching a terminal burden of 2×10.sup.8 CFU/g ML-83-009 at 20 and 50 mg/kg reduced thigh burden by 0.26 and 1.12 log.sub.10 CFU/g compared to vehicle treated animals, respectively, which was not statistically significant.
(701) KSN-82-L22 at 20 and 50 mg/kg significantly decreased bacterial burden by >5 Log.sub.10 CFU/g compared to vehicle group level and by >1.6 log.sub.10 CFU/g compared to the pre-treatment group suggesting bactericidal activity.
(702) The efficacy of KS-82-L22 at 20 and 50 mg/kg was similar to groups treated with Levofloxacin at 20 and 50 mg/kg.
(703) Raw Data
(704) TABLE-US-00043 TABLE 24 Bodyweight raw data Group No. Mouse No. −4 −1 0 1 1 Pretreatment 1 26.9 26.3 26.4 2 27.9 28.2 28.7 3 29.1 28.6 28.8 4 28.1 28.4 28.5 5 27.9 28.4 28.7 2 Vehicle 6 29.4 30 31 29.2 7 31.3 31.4 29.9 28.9 8 31.0 30.9 30.9 29.2 9 25.8 26.7 31.4 24 10 29.7 29.8 26.6 27.5 3 ML-83-009 11 29.4 29.6 29.7 27.4 20 mg/kg 12 25.4 25.9 25.9 23.3 13 25.7 25.9 26.1 23.8 14 29.1 30.0 29.7 27.4 15 27.2 27.6 27.6 26.4 4 ML-83-009 16 27.9 27.6 27 24.4 50 mg/kg 17 27.5 27.4 27.9 25.5 18 30.1 30.3 30.7 28.1 19 26.5 26.8 26.2 23.6 20 28.7 27.9 27.7 25.8 5 KSN-82-L22 21 28.7 29.1 28.4 29.3 20 mg/kg 22 30.2 29.2 29.4 29.5 23 26.9 27-3 27.2 27.5 24 29.8 30.2 30.5 31.3 25 29.7 29.8 29.7 31.0 6 KSN-82-L22 26 27.8 27.9 28.2 29 50 mg/kg 27 29.2 29.5 27.8 28.1 28 28.9 29.1 29.3 30.1 29 27.0 26.5 26.6 26.1 30 27.9 26.8 25.6 26.4 7 Levofloxacin 31 30.2 30.5 30.1 29.1 20 mg/kg 32 27.8 28.2 28.2 28.9 33 28.9 29.2 29.3 29.5 34 29.3 30.0 29.4 29.7 35 29.9 30.0 30.3 30.7 8 Levofloxacin 36 27.1 28.1 27.5 27 50 mg/kg 37 27.7 27.5 27.1 27.2 38 29.5 30.2 30.3 29.8 39 31.2 30.7 30.9 30.2 40 28.5 29.5 29.6 28.4
(705) TABLE-US-00044 TABLE 25 Bacterial burden data ML-83-009 ML-83-009 KSN-82-L7 KSN-82-L7 Levofloxacin Levofloxacin Pretreatment Vehicle 20 mg/kg 50 mg/kg 20 mg/kg 50 mg/kg 20 mg/kg 50 mg/kg 8.67E+04 2.72E+08 9.25E+08 1.25E+07 2.48E+03 2.58E+03 5.82E+03 1.32E+03 8.39E+04 4.93E+08 1.01E+08 2.30E+07 3.92E+03 1.98E+03 6.89E+03 1.59E+03 1.15E+05 4.01E+08 3.48E+07 2.62E+08 5.78E+02 5.78E+02 1.86E+03 8.41E+02 7.04E+04 1.75E+08 3.27E+07 1.82E+07 1.80E+03 1.70E+03 3.76E+03 2.19E+03 4.28E+04 1.05E+08 1.77E+07 1.33E+08 6.42E+02 5.94E+02 2.36E+03 9.47E+02 5.55E+04 1.24E+08 5.48E+07 4.12E+07 1.94E+03 2.63E+03 2.68E+03 3.73E+03 6.94E+04 2.90E+08 3.93E+08 2.96E+06 1.60E+03 2.00E+03 1.23E+03 3.09E+03 8.68E+04 2.15E+08 3.15E+07 1.05E+05 3.05E+03 1.44E+03 4.80E+03 2.01E+03 5.94E+04 9.82E+07 4.35E+08 2.60E+07 1.51E+03 9.34E+02 2.90E+03 2.64E+03 3.49E+04 1.42E+08 5.32E+08 1.04E+07 1.18E+04 1.14E+03 6.60E+03 7.99E+02
(706) TABLE-US-00045 TABLE 26 Interaction of the lead compound KSN-L22 with cytochrome P450 enzymes at 10 μM concentration CLIENT ASSAY STUDY COMPOUND % NUMBER ASSAY NAME NUMBER ID inhibition 1770 CYP3A inhibition (HLM, midazolam US034- KSN-82-L22 25.1969 substrate) 0002914 4481 CYP2C8 inhibition (HLM, US034- KSN-82-L22 3.05344 amodiaquine substrate) 0002914 1769 CYP3A inhibition (HLM, testosterone US034- KSN-82-L22 6.52493 substrate) 0002914 2065 CYP2B6 inhibition (HLM, bupropion US034- KSN-82-L22 −1.31579 substrate) 0002914 1838 CYP2D6 inhibition (HLM, US034- KSN-82-L22 0 dextromethorphan substrate) 0002914 2066 CYP2C9 inhibition (HLM, diclofenac US034- KSN-82-L22 12.8205 substrate) 0002914 2064 CYPiA inhibition (HLM, phenacetin US034- KSN-82-L22 9.55985 substrate) 0002914 1772 CYP2C19 inhibition (HLM, US034- KSN-82-L22 0.631579 omeprazole substrate) 0002914
(707) TABLE-US-00046 TABLE 27 Safetyscreen44 data for the lead compound KSN-L22: This assay tests potential off target toxicity. CLIENT ASSAY STUDY COMPOUND % NUMBER ASSAY NAME NUMBER ID inhibition 933 AR (h) (agonist radioligand) FR095-0004874 KSN-82-L22 4.24138 39 CCK1 (CCKA) (h) (agonist FR095-0004874 KSN-82-L22 −2.03813 radioligand) 18 beta 1 (h) (agonist radioligand) FR095-0004874 KSN-82-L22 1.92061 469 GR (h) (agonist radioligand) FR095-0004874 KSN-82-L22 3.45577 118 mu (MOP) (h) (agonist FR095-0004874 KSN-82-L22 6.53465 radioligand) 471 5-HT2A (h) (agonist FR095-0004874 KSN-82-L22 −8.88055 radioligand) 132 5-HT1B (antagonist FR095-0004874 KSN-82-L22 −16.7063 radioligand) 4173 COX1 (h) FR095-0004874 KSN-82-L22 30.3185 439 5-HT transporter (h) FR095-0004874 KSN-82-L22 −16.9663 (antagonist radioligand) 2338 alpha 1A (h) (antagonist FR095-0004874 KSN-82-L22 11.5312 radioligand) 131 5-HT1A(h) (agonist FR095-0004874 KSN-82-L22 −3.77016 radioligand) 93 M2 (h) (antagonist FR095-0004874 KSN-82-L22 1.06575 radioligand) 4 A2A (h) (agonist radioligand) FR095-0004874 KSN-82-L22 53.628 54 ETA (h) (agonist radioligand) FR095-0004874 KSN-82-L22 −5.23641 1208 H2 (h) (antagonist radioligand) FR095-0004874 KSN-82-L22 −40.1709 166 KV channel (antagonist FR095-0004874 KSN-82-L22 −4-64385 radioligand) 28 BZD (central) (agonist FR095-0004874 KSN-82-L22 −44.8289 radioligand) 1322 D2S (h) (agonist radioligand) FR095-0004874 KSN-82-L22 −7.80669 20 beta 2 (h) (antagonist FR095-0004874 KSN-82-L22 11.3086 radioligand) 52 dopamine transporter (h) FR095-0004874 KSN-82-L22 10.0257 (antagonist radioligand) 13 alpha 2A (h) (antagonist FR095-0004874 KSN-82-L22 4-4791 radioligand) 4186 COX2(h) FR095-0004874 KSN-82-L22 8.11129 1971 kappa (KOP) (agonist FR095-0004874 KSN-82-L22 27.3513 radioligand) 95 M3 (h) (antagonist FR095-0004874 KSN-82-L22 2.27343 radioligand) 114 delta (DOP) (h) (agonist FR095-0004874 KSN-82-L22 2.15892 radioligand) 37 CB2 (h) (agonist radioligand) FR095-0004874 KSN-82-L22 0 161 Ca2+ channel (L, FR095-0004874 KSN-82-L22 6.04874 dihydropyridine site) (antagonist radioligand) 159 V1a (h) (agonist radioligand) FR095-0004874 KSN-82-L22 −12.5662 1333 5-HT2B (h) (agonist FR095-0004874 KSN-82-L22 5.37361 radioligand) 4077 PDE4D2 (h) FR095-0004874 KSN-82-L22 14.8003 44 D1 (h) (antagonist radioligand) FR095-0004874 KSN-82-L22 −154033 870 H1 (h) (antagonist radioligand) FR095-0004874 KSN-82-L22 5.56418 3029 N neuronal alpha 4beta 2 (h) FR095-0004874 KSN-82-L22 −41.8108 (agonist radioligand) 66 NMDA (antagonist FR095-0004874 KSN-82-L22 12.6124 radioligand) 443 MAO-A (antagonist FR095-0004874 KSN-82-L22 −9.12972 radioligand) 4072 PDE3A (h) FR095-0004874 KSN-82-L22 7.50221 2906 Lck kinase (h) FR095-0004874 KSN-82-L22 −2.89115 4094 Potassium Channel hERG FR095-0004874 KSN-82-L22 −0.246078 (human)-[3H] Dofetilide 169 Na+ channel (site 2) FR095-0004874 KSN-82-L22 23.6539 (antagonist radioligand) 363 acetylcholinesterase (h) FR095-0004874 KSN-82-L22 32.9861 411 5-HT3 (h) (antagonist FR095-0004874 KSN-82-L22 −4.70826 radioligand) 36 CB1 (h) (agonist radioligand) FR095-0004874 KSN-82-L22 1.85658 355 norepinephrine transporter (h) FR095-0004874 KSN-82-L22 −6.47059 (antagonist radioligand) 91 M1 (h) (antagonist radioligand) FR095-0004874 KSN-82-L22 3.12943
(708) TABLE-US-00047 TABLE 28 MIC Data of ARB compounds against ESKAPE panel of pathogens Freebase Code Levofloxacin ML-83-009 ML-83-011 ML-83-010 ML-83-012 Code in patent Commercial 1-9 1.11 1.10 1.6 document KP13368 1-2 64 64 32 64 M6 0.125 8 16-64 4 16 AYE 8 128 128 128->128 128 Ab17978 0.125 4 16 2 8 PA01 1-4 32 128 64 32 PA13437 64-128 >128 128 >128 >128 EC12923 N/A 1-4 1-8 2 4 MSSA9144 0.125 0.06 0.25 2 0.125 0.25 EMRSA15 16 4 16 2 16 EMRSA16 16 4 16 2 16 VSE775 1 0.25 1 < 0.125 - 0.5 1 VRE12201 0.5-1 0.25 0.5-1 2 0.125 0.5-1 VRE12204 1-2 2 8 1-4 8 Freebase Code KSN-L7 KSN-L14 KSN-L19 KSN-L21 KSN-L22 KSN-L31 KSN-L33 KSN-L34 Code in patent 1.12 1.13 1.14 1.15 1.16 1.17 1.18 document Gram-negative KP13368 16 16 >32 >32 4 64 32 32 M6 4-8 2 4 16 1 4 8 2 AYE 16-32 >32 >32 >32 4 >128 128 4 Ab17978 0.5 0.5 4 2 0.125 4 2 ≤0.125 PA01 8-32 32 >32 >32 4 64 32-64 4 PA13437 >32 >32 >32 >32 >32 >128 128 >32 EC12923 1 1 4 0.5 0.5 2 2 0.125 Gram-positive MSSA9144 0.06 0.125 0.25 0.5 ≤0.03 0.5-1 0.25 ≤0.125 EMRSA15 4 8 8 32 0.5 64 16 0.5 EMRSA16 4 8-32 8 >32 1 64 16-32 1 VSE775 0.5 1 2 4 0.06 4 4 ≤0.125 VRE12201 0.5 0.5 1 1 0.06 2 2 ≤0.125 VRE12204 4 4 4 16 1 8 4 1 Freebase Code KSN-L36 KSN-L44-D KSN-L62 KSN-L65 Figure Number 1-19 1.22 1.27 1.28 Gram Negative KP13368 32 4 16 M6 2-4 ≤0.125 1 AYE 64 128 4 Ab17978 0.5-1 2 0.25 PA01 16 0.25-0.5 8 PA13437 >64 32 128 EC12923 0.5-1 ≤0.125 1 Gram Positive MSSA9144 0.25-0.5 0.25 ≤0.125 EMRSA15 32 32 1 EMRSA16 32 32 4 VSE775 4 1-4 ≤0.125 VRE12201 2 0.5-1 ≤0.125 VRE12204 8 1 1 Freebase Code KSN-BL-1 KSN-BL-3 KSN-BL-6 KSN-BL-7 Figure Number 1.29 1-34 1.32 1.33 Gram-negative KP13368 8 8 16 16 M6 0.5 1-4 1-4 2 AYE 8 32 16 8 A17978 0.25 0.25 0.5 ≤0.125 PA01 4 8 8 8 PA13437 >128 >128 >128 >128 EC12923 0.25-0.5 0.5 0.5 1 Gram-positive MSSA9144 ≤0.125 0.0156 0.0156 ≤0.0039 EMRSA15 1 0.5 2 0.125- EMRSA16 1 0.5 1 0.25 VSE775 ≤0.125 ≤0.125 0.0625-0.25 0.0625 VRE12201 ≤0.125 0.0625 0.0625 0.0313 VRE12204 0.5 1 1 1
(709) The ARB compound KSN-82-L22 was tested against a broad spectrum of Neisseria gonorrhoeae strains and the MIC data was determined (see Table 29).
(710) TABLE-US-00048 TABLE 29 MIC Data of ARB compound KSN-82-L22 against a broad spectrum of Neisseria gonorrhoeae strains MIC, μg/ml PT #1218401 Assay KSN-82- No. # Strain ID Resistance L22 1 612500 ATCC 49226 PEN-I TET-I <0.03125 2 612501 ATCC 700825 50.03125 3 612502 CCUG 57595 50.03125 4 612503 CCUG 57596 CIP-I PEN-I TET-R 0.125 5 612504 CCUG 57597 FEP-NS FOX-I CAZ-NS CIP-R 2 OFX-R PEN-R TET-R 6 612505 CCUG 57598 FOX-R CAZ-NS CRO-NS CIP- 2 R OFX-R PEN-R TET-R 7 612506 CCUG 57599 CIP-R OFX-R PEN-R TET-R 0-5 8 612507 CCUG 57600 CIP-R OFX-R PEN-R TET-R 1 9 612508 CCUG 57601 PEN-R TET-R <0.03125 10 612509 CCUG 57602 PEN-I TET-I 0.0625 11 612568 NCTC 13817 PEN-R TET-I 0-5 12 612569 NCTC 13818 CIP-R OFX-R PEN-R TET-R 2 13 612570 NCTC 13819 FEP-NS CAZ-NS CIP-R OFX-R 2 PEN-R TET-I 14 612571 NCTC 13820 FEP-NS CFM-NS CAZ-NS 2 CRO-NS CIP-R OFX-R PEN-R TET-R 15 612572 NCTC 13821 FEP-NS CFM-NS CAZ-NS 2 CRO-NS CIP-R PEN-I TET-I 16 612573 NCTC 13822 FEP-NS CFM-NS CAZ-NS CIP- 2 R OFX-R PEN-R TET-I 17 612510 BAA-1833 PEN-I TET-I 50.03125 18 612511 BAA-1838 50.03125 19 612512 TCDC-NG08107 PEN-R CFM-R CPD-R CIP-R 2 20 612513 2007NG046 PEN-R CFM-R CPD-R CIP-R 4 21 612514 2008NG057 PEN-R CFM-R CPD-R CIP-R 2 22 612515 2008NG097 PEN-R CFM-R CPD-R CIP-R 2 23 612516 2009NG514 PEN-R CPD-R CIP-R 2 24 612517 2010NG122 PEN-R CFM-R CIP-R 2 25 612518 FDA-CDC AR-BANK#0165 CIP-R PEN-I TET-R 2 26 612519 FDA-CDC AR-BANK#0166 CIP-R PEN-I TET-R 2 27 612520 FDA-CDC AR-BANK#0167 CIP-S PEN-S TET-I <0.03125 28 612521 FDA-CDC AR-BANK#0168 CIP-R PEN-I TET-R 1 29 612522 FDA-CDC AR-BANK#0169 CIP-R PEN-I TET-R 2 30 612523 FDA-CDC AR-BANK#0170 CIP-R PEN-I TET-R 2 31 612524 FDA-CDC AR-BANK#0171 CIP-R PEN-I TET-R 1 32 612525 FDA-CDC AR-BANK#0172 CIP-R PEN-R TET-R 1 33 612526 FDA-CDC AR-BANK#0173 CIP-R PEN-R TET-R 2 34 612527 FDA-CDC AR-BANK#0174 CIP-R PEN-R TET-R 2 35 612528 FDA-CDC AR-BANK#0175 CIP-S PEN-I TET-I <0.03125 36 612529 FDA-CDC AR-BANK#0176 CIP-R PEN-R TET-R 2 37 612530 FDA-CDC AR-BANK#0177 CIP-S PEN-I TET-R <0.03125 38 612531 FDA-CDC AR-BANK#0178 CIP-R PEN-R TET-R 2 39 612532 FDA-CDC AR-BANK#0179 CIP-S PEN-S TET-I <0.03125 40 612533 FDA-CDC AR-BANK#0180 CIP-R PEN-I TET-R 2 41 612534 FDA-CDC AR-BANK#0181 CIP-S PEN-I TET-R <0.03125 42 612535 FDA-CDC AR-BANK#0182 CIP-R PEN-I TET-R 2 43 612536 FDA-CDC AR-BANK#0183 CIP-R PEN-R TET-R 2 44 612537 FDA-CDC AR-BANK#0184 CIP-R PEN-R TET-R 2 45 612538 FDA-CDC AR-BANK#0185 CIP-R PEN-I TET-R 2 46 612539 FDA-CDC AR-BANK#0186 CIP-R PEN-R TET-R 1 47 612540 FDA-CDC AR-BANK#0187 CIP-R PEN-I TET-R 2 48 612541 FDA-CDC AR-BANK#0188 CIP-R PEN-I TET-R 1 49 612542 FDA-CDC AR-BANK#0189 CIP-R PEN-R TET-R 2 50 612543 FDA-CDC AR-BANK#0190 CIP-R PEN-R TET-R 2 51 612544 FDA-CDC AR-BANK#0191 CIP-R PEN-R TET-R 2 52 612545 FDA-CDC AR-BANK#0192 CIP-R PEN-R TET-R 2 53 612546 FDA-CDC AR-BANK#0193 CIP-S PEN-R TET-R <0.03125 54 612547 FDA-CDC AR-BANK#0194 CRO-NS CIP-S PEN-I TET-R <0.03125 55 612548 FDA-CDC AR-BANK#0195 CIP-R PEN-I TET-R 2 56 612549 FDA-CDC AR-BANK#0196 CIP-R PEN-R TET-R 2 57 612550 FDA-CDC AR-BANK#0197 CIP-R PEN-I TET-I 2 58 612551 FDA-CDC AR-BANK#0198 CIP-R PEN-R TET-R 2 59 612552 FDA-CDC AR-BANK#0199 CIP-S PEN-R TET-R <0.03125 60 612553 FDA-CDC AR-BANK#0200 CIP-R PEN-R TET-R 2 61 612554 FDA-CDC AR-BANK#0201 CIP-R PEN-R TET-R 2 62 612555 FDA-CDC AR-BANK#0202 CIP-S PEN-S TET-I <0.03125 63 612556 FDA-CDC AR-BANK#0203 CIP-R PEN-R TET-R 2 64 612557 FDA-CDC AR-BANK#0204 CIP-R PEN-I TET-R 2 65 612558 FDA-CDC AR-BANK#0205 CIP-R PEN-R TET-R 2 66 612559 FDA-CDC AR-BANK#0206 CIP-R PEN-R TET-R 2 67 612560 FDA-CDC AR-BANK#0207 CIP-R PEN-R TET-R 2 68 612561 FDA-CDC AR-BANK#0208 CIP-R PEN-I TET-R 2 69 612562 FDA-CDC AR-BANK#0209 CIP-R PEN-I TET-R 2 70 612563 FDA-CDC AR-BANK#0210 CIP-R PEN-I TET-I 2 71 612564 FDA-CDC AR-BANK#0211 CIP-R PEN-R TET-R 2 72 612565 FDA-CDC AR-BANK#0212 CIP-R PEN-I TET-R 2 73 612566 FDA-CDC AR-BANK#0213 CIP-R PEN-I TET-I 1 74 612567 FDA-CDC AR-BANK#0214 CIP-R PEN-R TET-R 2
(711) All publications mentioned in the above specification are herein incorporated by reference. Although illustrative embodiments of the invention have been disclosed in detail herein, with reference to the accompanying drawings, it is understood that the invention is not limited to the precise embodiment and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope of the invention as defined by the appended claims and their equivalents.
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