PHARMACEUTICAL COMPOUNDS
20220348565 · 2022-11-03
Assignee
Inventors
- Robert George Boyle (Cambridge, GB)
- Meriel Ruth Major (Cambridge, GB)
- Stuart TRAVERS (Cambridge, GB)
- David Winter Walker (Cambridge, GB)
- Michal CZYZEWSKI (Dunham, GB)
- Derek John Londesbrough (Hartlepool, GB)
- Julian Scott Northen (South Shields, GB)
- Stefania SANTONI (Newcastle uponTyne, GB)
Cpc classification
C07D207/33
CHEMISTRY; METALLURGY
C07D231/12
CHEMISTRY; METALLURGY
C07D401/04
CHEMISTRY; METALLURGY
C07D409/04
CHEMISTRY; METALLURGY
C07D261/08
CHEMISTRY; METALLURGY
International classification
Abstract
The invention provides a composition of matter which:
(i) consists of at least 90% by weight of an atropisomer (2A) and 0-10% by weight of an atropisomer of formula (2B); or
(ii) consists of at least 90% by weight of an atropisomer (2B) and 0-10% by weight of an atropisomer of formula (2A);
wherein the atropisomer of formula (2A) and the atropisomer of formula (2B) are represented by:
##STR00001##
or are pharmaceutically acceptable salts or tautomers thereof, wherein ring X is a benzene or pyridine ring; ring Y is selected from a benzene ring, a pyridine ring and a thiophene ring; R.sup.1 is trifluoromethyl; R.sup.2 is hydrogen; R.sup.3 is hydrogen; m is 0 or 1; n is 0, 1 or 2; Ar.sup.1 is a monocyclic aromatic ring selected from benzene and pyridine; each monocyclic aromatic ring being unsubstituted or substituted with 1 or 2 substituents R.sup.5 as defined herein; and R.sup.4; R.sup.5 when present, R.sup.6 and R.sup.7 independently selected from various substituents as defined herein.
Also provided are individual atropisomers, pharmaceutical compositions and the uses of the atropisomers and compositions are inhibitors of PLK1- and PLK4 kinases, for example in the treatment of cancers.
Claims
1. A composition of matter which: (i) consists of at least 90% by weight of an atropisomer (2A) and 0-10% by weight of an atropisomer of formula (2B); or (ii) consists of at least 90% by weight of an atropisomer (2B) and 0-10% by weight of an atropisomer of formula (2A); wherein the atropisomer of formula (2A) and the atropisomer of formula (2B) are represented by: ##STR00176## or are pharmaceutically acceptable salts or tautomers thereof, wherein: ring X is a benzene or pyridine ring; ring Y is selected from a benzene ring, a pyridine ring and a thiophene ring; R.sup.1 is trifluoromethyl; R.sup.2 is hydrogen; R.sup.3 is hydrogen; m is 0 or 1; n is 0, 1 or 2; R.sup.4 is selected from: fluorine; chlorine; bromine; and a C.sub.1-4 alkyl group where 0 or 1 of the carbons in the alkyl group are replaced with a heteroatom 0, the alkyl group being optionally substituted with one or more fluorine atoms; Ar.sup.1 is a monocyclic aromatic ring selected from benzene and pyridine; each monocyclic aromatic ring being unsubstituted or substituted with 1 or 2 substituents R.sup.5; R.sup.5 when present is selected from bromine; fluorine; chlorine; and cyano; R.sup.7 is independently selected from R.sup.4; R.sup.6 is a group Q.sup.1-R.sup.a—R.sup.b; Q.sup.1 is absent or is selected from CH.sub.2, CH(CHs), C(CH.sub.3).sub.2, cyclopropane-1,1-diyl and cyclobutane-1,1-diyl; R.sup.a is absent or is selected from O; C(O); C(O)O; CONR.sup.c; N(R.sup.c)CO; N(R.sup.c)CONR.sup.c; NR.sup.c; and SO.sub.2; R.sup.b is selected from: a C.sub.1-4 non-aromatic hydrocarbon group where 0 or 1 but not all of the carbon atoms in the hydrocarbon group are replaced with a heteroatom selected from N and O, the C.sub.1-4 non-aromatic hydrocarbon group being optionally substituted with one or more substituents selected from fluorine and a group Cyc.sup.1; and a group Cyc.sup.1; R.sup.c is selected from hydrogen and a C.sub.1-4 non-aromatic hydrocarbon group; Cyc.sup.1 is a non-aromatic 4-7 membered heterocyclic ring group containing a nitrogen ring member and optionally second heteroatom ring member selected from N and O; the non-aromatic 4-7 membered heterocyclic ring group being optionally substituted with one or more substituents selected from hydroxyl; amino; mono-C.sub.1-4 alkylamino; di-C.sub.1-4 alkylamino; and a C.sub.1-4 saturated hydrocarbon group where 0 or 1 but not all of the carbons in the hydrocarbon group are replaced with a heteroatom selected from N and O.
2. A composition of matter according to claim 1 wherein m is 0.
3. A composition of matter according to claim 1 wherein Ar.sup.1 is a benzene ring optionally substituted with one or more substituent R.sup.5.
4. A composition of matter according to claim 3 wherein the benzene ring Ar.sup.1 is unsubstituted or is substituted with 1 substituent R.sup.5.
5. A composition of matter according to claim 1 wherein R.sup.5, when present, is selected from fluorine, chlorine and cyano.
6. A composition of matter according to claim 1 wherein the ring Y is a benzene ring or a pyridine ring.
7. A composition of matter according to claim 1 wherein R.sup.6 is a group Q.sup.1-R.sup.a—R.sup.b; and Q.sup.1 is absent or is selected from CH.sub.2, CH(CH.sub.3), C(CH.sub.3).sub.2, cyclopropane-1,1-diyl and cyclobutane-1,1-diyl.
8. A composition of matter according to claim 1 wherein R.sup.a is CONR.sup.c.
9. A composition of matter according to claim 1 wherein R.sup.b is selected from: a C.sub.1-8 non-aromatic hydrocarbon group wherein 1 of the carbon atoms in the hydrocarbon group is replaced with a nitrogen heteroatom.
10. A composition of matter according to claim 1 wherein R.sup.c, when present is hydrogen.
11. A composition of matter according to claim 1 wherein R.sup.6 is selected from the groups in the table below: TABLE-US-00037
12. A single atropisomer having a chemical structure as defined in claim 1, said single atropisomer being unaccompanied by any other atropisomer, or being accompanied by no more than 0.5% by weight relative to the single atropisomer of any other atropisomer.
13. A single atropisomer according to claim 12 which has an R-configuration about the bond linking ring X to the pyrrole nitrogen atom.
14. A single atropisomer according to claim 13, which has the R configuration represented by formula (1), or is a salt thereof: ##STR00210##
15. A (+)-L-tartaric acid salt of 2,4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2 (dimethylamino)ethyl]benzamide having the formula (2): ##STR00211##
16. A pharmaceutical composition comprising a composition of matter according to claim 1 and a pharmaceutically acceptable excipient.
17. A method of treating a subject suffering from cancer, which method comprises administering to the subject a therapeutically effective amount of a composition of matter according to claim 1.
18. An invention as defined in any one of Embodiments 1.1 to 1.211, 2.1 to 2.15, 3.1 to 3.38, 4.1 to 4.12 and 5.1 to 5.9 herein.
19. A method of inhibiting PLK1 kinase or PLK4 kinase, which method comprises contacting the PLK1 kinase or the PLK4 kinase with a kinase inhibiting amount of a composition of matter according to claim 1.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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EXAMPLES
[0685] The invention will now be illustrated, but not limited, by reference to the specific embodiments described in the following examples.
[0686] In the examples, the following abbreviations are used. [0687] aq aqueous [0688] CaCl.sub.2 calcium chloride [0689] DCM dichloromethane [0690] DEA diethylamine [0691] DIPEA N,N-diisopropylethylamine [0692] DMF dimethylformamide [0693] DMP Dess-Martin periodinane [0694] DMSO dimethylsulfoxide [0695] Et.sub.2O diethyl ether [0696] EtOAc ethyl acetate [0697] EtOH ethanol [0698] h hour(s) [0699] HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxid hexafluorophosphate) [0700] HCl hydrogen chloride [0701] HPLC high performance liquid chromatography [0702] H.sub.2SO.sub.4 sulfuric acid [0703] IPA iso-propanol [0704] LC liquid chromatography [0705] LCMS liquid chromatography-mass spectrometry [0706] UOH lithium hydroxide [0707] MeCN acetonitrile [0708] MeOH methanol [0709] min minute(s) [0710] MTBE methyl tert-butyl ether [0711] NaBH.sub.4 sodium borohydride [0712] NaHCO.sub.3 sodium hydrogen carbonate [0713] NaOH sodium hydroxide [0714] Na.sub.2SO.sub.4 sodium sulfate [0715] NH.sub.4Cl ammonium chloride [0716] NMR nuclear magnetic resonance [0717] PTSA p-toluenesulfonic acid [0718] TEA triethylamine [0719] THF tetrahydrofuran
COMPOUND DETAILS AND EXPERIMENTAL
[0720] Atropisomers A-1 to A-8
TABLE-US-00002 Chemical name Atropisomer Structure (IUPAC via ISIS draw) Notes A-1
[0721] Proton magnetic resonance (1H NMR) spectra were recorded on a Bruker 400 instrument operating at 400 MHz, in DMSO-d6 or MeOH-d4 (as indicated) at 27° C., unless otherwise stated and are reported as follows: chemical shift 6/ppm (multiplicity where s=singlet, d=doublet, dd=double doublet, dt—double triplet, t=triplet, q=quartet, m=multiplet, br=broad, number of protons). The residual protic solvent was used as the internal reference.
[0722] Liquid chromatography and mass spectroscopy analyses were carried out using the system and operating conditions set out below. Where atoms with different isotopes are present and a single mass quoted, the mass quoted for the compound is the monoisotopic mass (i.e. .sup.35Cl; .sup.79Br etc.)
[0723] LCMS Conditions
[0724] The LCMS data given in the following examples were obtained using one of the methods described below.
[0725] LCMS Method 1
[0726] LCMS was carried out on UPLC AQUITY with PDA photodiode array detector and QDa mass detector. The column used was a C18, 2.1×50 mm, 1.9 μm. The column flow was 1.2 mL/min and the mobile phase used was: (A) 0.1% Formic acid in MilliQ water (pH=2.70) (B) 0.1% Formic acid in water:MeCN (10:90), the injection volume was between 4 and 7 μL. The sample was prepared in MeOH:MeCN to achieve an approximate concentration of 250 ppm.
[0727] The following gradient was used for the elution:
TABLE-US-00003 Time (min) Flow (ml/min) % A % B 0.00 0.8 97 3 0.20 0.8 97 3 2.70 0.8 2 98 3.00 1.0 00 100 3.50 1.0 00 100 3.51 0.8 97 3 4.00 0.8 97 3
[0728] Mass Parameters
Probe: ESI capillary
Source Temperature: 120° C.
Probe Temperature: 600° C.
Capillary Voltage: 0.8 KV (+Ve and −Ve)
Cone Voltage: 10 & 30 V
[0729] Mode of Ionization: Positive and negative
[0730] LCMS Method 2
[0731] LCMS was carried out on Agilent Infinity II G6125C LCMS. The column used was an XBridge C18, 50×4.6 mm, 3.5 μm. The column flow was 1.0 mL/min and the mobile phase used was: (A) 5 mM Ammonium Bicarbonate in Milli-Qwater and (B) MeOH. The injection volume was 5 μL. The sample was prepared in water MeCN to achieve an approximate concentration of 250 ppm.
[0732] The following gradient was used for the elution.
TABLE-US-00004 Time (min) % A % B 0.00 92 8 0.75 92 8 3.00 30 70 3.70 5 95 4.20 0 100 5.20 0 100 5.21 92 8 7.00 92 8
[0733] Mass Parameter
Ion Source: MMI
[0734] Fragmentation voltage: 70V
Mode of Ionization: Positive and negative
Gas Temperature: 250° C.
Vaporizer 160° C.
[0735] Gas flow: 10 L/min
Nebulizer Pressure: 45 psi
[0736] HPLC Method 1
[0737] HPLC analysis was carried out on an Agilent Technologies 1100/1200 series HPLC system. The column used was an ACE 3 C18; 150×4.6 mm, 3.0 μm particle size (Ex: Hichrom, Part number ACE-111-1546). The flow rate was 1.0 mL/min. Mobile phase A was Water:Trifluoroacetic acid (100:0.1%) and mobile phase B was Acetonitrile:Trifluoroacetic acid (100:0.1%). The injection volume was 5 μL and the following gradient was used:
TABLE-US-00005 Time (mins) % A % B 0 80 20 35 5 95 39.5 5 95 40 80 20
[0738] Chiral HPLC Analysis
[0739] The chiral HPLC data reported were obtained using one of the methods described below.
[0740] Chiral HPLC Method 1
[0741] Chiral HPLC was analysis was carried out on an Agilent Technologies 1200 series HPLC system. The column used was a CHIRAL PAK IG, 250×4.6 mm, 5 μm. The column flow rate was 1.0 mL/min and the mobile phase was: (A) 0.1% v/v DEA in n-heptane and (B) IPA:MeOH (70:30). The injection volume was 25 μL. Samples were prepared in IPA:MeOH to achieve an approximate concentration of 250 ppm and with the following isocratic method:
TABLE-US-00006 Time Flow % A % B 0.01 1.0 mL/min 90 10 45 1.0 mL/min 90 10
[0742] Chiral HPLC Method 2 Chiral HPLC was analysis was carried out on an Agilent Technologies 1200 series HPLC system. The column used was a CHIRALPAK IG SFC, 21×250 mm, 5 μm. The column flow rate was 1.0 mL/min and the mobile phase was: (A) 0.1% v/v DEA in n-heptane and (B) IPA:MeOH (70:30). The injection volume was 20 μL. Samples were prepared in IPA:MeOH to achieve an approximate concentration of 250 ppm and with the following isocratic method:
TABLE-US-00007 Time Flow % A % B 0.01 1.0 mL/min 85 15 30 1.0 mL/min 85 15
[0743] Chiral HPLC Method 3
[0744] Chiral HPLC was carried out on an Agilent Technologies 1200 series HPLC system. The column used was a CHIRAL PAK IG, 250×4.6 mm, 5 μm. The column flow rate was 1.0 mL/min and the mobile phase was: (A) 0.1% v/v DEA in n-heptane and (B) IPA:MEOH (70:30). The injection volume was 10 μL Samples were prepared in IPA:MeCN to achieve an approximate concentration of 250 ppm and with the following isocratic method:
TABLE-US-00008 Time Flow % A % B 0.01 1.0 mL/min 85 15 25 1.0 mL/min 85 15
[0745] Chiral HPLC Method 4
[0746] Identical conditions to chiral method 3 except using the following isocratic method:
TABLE-US-00009 Time Flow % A % B 0.01 1.0 mL/min 70 30 25 1.0 mL/min 70 30
[0747] Chiral HPLC Method 5
[0748] Identical conditions to chiral method 3 except using the following isocratic method:
TABLE-US-00010 Time Flow % A % B 0.01 1.0 mL/min 90 10 25 1.0 mL/min 90 10
[0749] Chiral HPLC Method 7
[0750] Chiral HPLC was analysis was carried out on an Agilent Technologies 1100/1200 series HPLC system. The column used was a CHIRALPAK IA; 250×4.6 mm, 5.0 μm. The column flow rate was 1.0 mL/min and the mobile phase was: Hexane:EtOH:Ethanolamine (90:10:0.1%). The injection volume was 5 μL. Samples were prepared in 100% EtOH to achieve an approximate concentration of 0.5 mg/mL.
[0751] Preparative HPLC Methods
[0752] Final compounds were purified using one of the following preparative HPLC methods.
[0753] Preparative HPLC Method 1
[0754] Preparative HPLC was carried out using a SUNFIRE Prep C18 OBD, 19×250 mm, 5 μm column with (A) 0.05% HCl in water and (B) 100% MeCN as mobile phase and a flow rate of 17 mL/min and with the following isocratic system for the elution:
TABLE-US-00011 Time (min) Flow % A % B 00.01 17 70 30 16.00 17 57 43 16.01 17 2 98 18.00 17 2 98 18.01 17 70 30 20.00 17 70 30
[0755] Preparative HPLC Method 2
[0756] Preparative HPLC was carried out using an X-bridge prep, C18, 30×250 mm, 5 μm column with (A) 0.05% HCl in water and (B) 100% MeCN as mobile phase and a flow rate of 25 mL/min with the following isocratic system for the elution:
TABLE-US-00012 Time (min) Flow % A % B 00.01 25 80 20 15.00 25 20 80 15.01 25 2 98 17.00 25 2 98 17.01 25 80 20 19.00 25 80 20
[0757] Preparative Chiral HPLC Methods:
[0758] The atropisomers were isolated using one of the following preparative chiral HPLC methods.
[0759] Preparative Chiral HPLC Method 1
[0760] Preparative chiral HPLC was carried out using a CHIRALPAK IG SFC, 21×250 mm, 5 μm column, eluting with (A) 0.1% DEA in heptane and (B) IPA as mobile phase, with the flow rate of 30 mL/min and the following isocratic system:
TABLE-US-00013 Time (min) % A % B 0.01 94 6 50.00 94 6
[0761] Preparative Chiral HPLC Method 2
[0762] Preparative chiral HPLC was carried out using a CHIRALPAK IG SFC column, 21×250 mm, 5 μm eluting with (A) 0.1% DEA in heptane and (B) IPA:MeOH (90:10) as mobile phase and a flow rate of 22 mL/min and with the following isocratic system was used for the elution:
TABLE-US-00014 Time (min) % A % B 0.01 93 7 35.00 93 7
[0763] Chiral Analysis Specific Optical Rotation Protocol
Instrumentation: Optical Activity AA-10 Automatic Polarimeter
Wavelength: 589 nm
Temperature: 23° C.
[0764] Pathlength of cell: 1 dm
Solvent: Chloroform (Fisher, HPLC grade)
Concentration: 1.0 g/100 mL
[0765] Sampling Technique
[0766] The instrument was switched on and allowed to stabilize for 30 minutes before calibration was checked using an Optical Activity Quartz Control Plate (S/N 00049). The angular rotation at 23° C. using sodium yellow D line was measured at 34.16° (after firstly zeroing the instrument without any sample tube). The sample tube quality was then checked by zeroing the instrument, then filling the sample tube with chloroform and checking the instrument was still reading 0.00 (+/−0.02). The instrument was zeroed with the chloroform blank in place.
[0767] The sample was dissolved in CHCl.sub.3 (2 mg in 2 mL), filtered and 2 mL was pipetted into the cell to measure α.
[0768] The specific optical rotation was calculated from the following equation: [α]Tλ=(α×100)/(cl)
Synthesis of Intermediates
Intermediate A: 1-(4-chlorophenyl)-3-(dimethylamino) propan-1-one hydrochloride
[0769] ##STR00067##
[0770] To a solution of 4′-chloroacetophenone (10 g, 65 mmol) in absolute EtOH (50 mL) at room temperature were added paraformaldehyde (1.94 g, 64 mol), N,N-dimethylamine hydrochloride (5.27 g, 64.68 mmol) and conc. HCl (2 mL). The resulting reaction mixture was stirred at between 80-90° C. for 30 h. The reaction mixture was concentrated under reduced pressure and the resulting residue was purified by column chromatography with silica gel (60-120 mesh) eluting with 2% EtOAc/hexane) and trituration with Et.sub.2O (100 mL) to afford the title compound (10 g, 40 mmol, 62%).
Intermediate B: 3-(dimethylamino)-1-(4-fluorophenyl) propan-1-one hydrochloride
[0771] ##STR00068##
[0772] Intermediate B was prepared using the same method as described for intermediate A except that 4′-fluoroacetophenone (20 g, 144.87 mmol) was used and the resulting residue was purified by column chromatography with silica gel (60-120 mesh) eluting with 4% MeOH/DCM) followed by trituration with Et.sub.2O (400 mL) to afford the title compound (15 g, 77 mmol, 53%).
Intermediate C: 4-(3-(dimethyl amino) propanol) benzonitrile hydrochloride
[0773] ##STR00069##
[0774] Intermediate C was prepared using the same method as described for intermediate A except that 4-acetylbenzonitrile (25 g, 172 mmol) was used and the resulting residue was purified by column chromatography with silica gel (60-120 mesh) eluting with 5% MeOH/DCM followed by trituration with Et.sub.2O (400 mL) to afford the title compound (20 g, 99 mmol, 57%).
Example 1
Preparation of Atropisomers A-1 and A-2
[0775] Atropisomers A-1 and A-2 can be prepared by following Synthetic Route A as shown below.
##STR00070##
Step 1: 4-[4-(4-chlorophenyl)-4-oxo-butanoyl]benzonitrile
[0776] ##STR00071##
[0777] Zinc chloride (30.5 g, 223 mmol) was heated to melting under vacuum then cooled to room temperature. Toluene (100 mL), tert-butanol (16.5 mL, 172 mmol) and TEA (24 mL, 172 mmol) and the mixture stirred at room temperature for 2 h under a nitrogen atmosphere at which point the zinc chloride had fully dissolved. 4-Cyanoacetophenone (25 g, 172 mmol) and 4-chlorophenacylbromide (40.2 g, 172 mmol) were added and the reaction mixture was stirred at room temperature for 48 h. The reaction mixture was diluted with EtOAc (300 mL) and washed with water (5×100 mL). The combined organic extracts were dried (Na.sub.2SO.sub.4) and evaporated under reduced pressure. The resulting residue was purified by trituration using MTBE (400 mL) to afford the title compound (30 g, 101 mmol, 59%).
Step 2: 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl) phenyl)-1H-pyrrol-2-yl) benzonitrile
[0778] ##STR00072##
[0779] A stirred solution of 4-(4-(4-chlorophenyl)-4-oxobutanoyl) benzonitrile (30 g, 101 mmol), 2-trifluoromethyl aniline (48.79 g, 303 mmol) and PTSA (1.92 g, 10.099 mmol) in dioxane (300 mL) was heated at 150° C. for 16 h. The reaction mixture concentrated under reduced pressure and the resulting residue was purified by column chromatography on silica gel (60-120 mesh) using 8% EtOAc/hexane as the eluent to afford the title compound (30 g, 71 mmol, 70%).
Step 3: 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl) phenyl)-1H-pyrrol-2-yl) benzoic acid
[0780] ##STR00073##
[0781] To a solution of 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl) phenyl)-1H-pyrrol-2-yl) benzonitrile (2 g, 4.739 mmol) in MeOH (20 mL) was added NaOH (1.89 g, 47 mmol) in water (10 mL) and the resulting mixture was stirred at 90° C. for 24 h. The mixture was concentrated under reduced pressure and the resulting residue was purified by trituration by using Et.sub.2O (10 mL) to afford the title compound (1.8 g, 4.1 mmol, 86%).
Step 4: 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl) phenyl)-1H-pyrrol-2-yl)-N-(2-(dimethylamino) ethyl) benzamide
[0782] ##STR00074##
[0783] To a stirred solution of 4(5-(4-chlorophenyl)-1-(2-(trifluoromethyl)phenyl)-1H-pyrrol-2-yl)benzoic acid (1.8 g, 4.0 mmol) in DMF (12 mL) was added DIPEA (2.13 mL, 22 mmol) followed by HATU (4.65 g, 12 mmol). The reaction mixture was stirred at room temperature for 30 min followed by the addition of N,N′-dimethylethylenediamine (1.08 g, 12 mmol) dropwise and stirring continued at room temperature for 4 h. The mixture was poured into ice-cold water (150 mL) and extracted with EtOAc (3×100 mL). The combined organic layers were dried (Na.sub.2SO.sub.4) and concentrated under reduced pressure. The resulting residue was purified by column chromatography on neutral Alumina eluting with 6% MeOH/DCM to afford the title compound (1.2 g, 2.3 mmol, 57%) as a mixture of atropisomers.
[0784] Separation of Atropisomers
[0785] The atropisomers (A-1 and A-2) of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2-(dimethylamino)ethyl]benzamide may be resolved by chiral HPLC using preparative chiral HPLC method 1.
[0786] Two peaks were isolated: [0787] Peak 1: Atropisomer A-1, 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2 (dimethylamino)ethyl]benzamide-atropisomer1 (0.3 g, 0.58 mmol, 38%, >99% ee), and: [0788] Peak 2: Atropisomer A-2, 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2 (dimethylamino)ethyl]benzamide-atropisomer2 (0.31 g, 0.606 mmol, 39%, 98% ee).
[0789] The compounds can also be isolated as their hydrochloride salts.
Example 2
[0790] Further purification and characterisation of the atropisomers
Atropisomer A-1: 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2 (dimethylamino)ethyl]benzamide hydrochloride salt
[0791] Peak 1 (0.31 g, 0.606 mmol) was further purified by stirring in HPLC grade water (30 mL) followed by sonication for 10 min and extraction with EtOAc (3×30 mL). The combined organic layers were dried (Na.sub.2SO.sub.4), filtered and concentrated under reduced pressure followed by lyophilisation to afford an amorphous solid (0.290 g, 0.567 mmol, 94%) which was dissolved in DCM (7.12 mL). The resulting solution was cooled to 0° C. and 4N HCl in dioxane (1.42 mL) was added. The reaction mixture was stirred at room temperature for 3 h. The mixture was concentrated and dried under high vacuum. Purification by trituration using Et.sub.2O (10 mL) and lyophilisation afforded the title compound (0.3 g, 0.56 mmol, 98%) as an off-white solid.
[0792] .sup.1H NMR (DMSO-d.sub.6) δ 10.03, (brs, 1H), 8.62 (s, 1H), 7.81-7.68 (m, 6H), 7.25 (d, J=8.4 Hz, 2H), 7.10-7.03 (m, 4H), 6.67-6.58 (m, 2H), 3.56-3.54 (m, 2H), 3.20-3.18 (m, 2H), 2.76 (s, 6H). LCMS (Method 1)—RT 2.54, MH+ 512.4
Atropisomer A-2: 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2 (dimethylamino)ethyl]benzamide hydrochloride salt
[0793] The hydrochloride salt of atropisomer A-2 was prepared using the same method as used for atropisomer A-1 starting from peak 2 to afford the title compound (0.31 g, 0.56 mmol, 99%) an off-white solid.
[0794] .sup.1H NMR (DMSO-d.sub.6) δ 9.91 (brs, 1H), 8.69 (s, 1H), 7.81-7.68 (m, 6H), 7.25 (d, J=8.0 Hz, 2H), 7.10-7.03 (m, 4H), 6.67-6.58 (m, 2H), 3.56-3.54 (m, 2H), 3.20-3.18 (m, 2H), 2.77 (s, 6H). LCMS (Method 1)—RT 2.56, MH+ 512.4
[0795] Single crystal X-ray crystallographic analysis of atropisomer A-2 (see Example 3 below) indicated that atropisomer A-2 is the R-isomer (Compound (1)) and hence atropisomer A-1 must be the S-isomer.
[0796] Chiral Analysis
[0797] Analysis of the chiral properties of the Atropisomers A-1 and A-2 was carried out by measuring their optical rotations and their retention times obtained by chiral HPLC using the methods described above to give the results shown in the table below.
TABLE-US-00015 Chiral HPLC RT Chiral HPLC Specific Optical Atropisomer (min) Method Rotation A-1 (S-atropisomer) 17.063 1 +12.39° A-2 (R-atropisomer) 20.553 1 −11.76°
[0798] Atropisomer Classification
[0799] Stability studies were carried out on the isolated atropisomers, atropisomers A-1 and A-2.
[0800] To assess the interconversion of atropisomer A-1 and atropisomer A-2 chiral stability was monitored at 40° C. and 80° C. As shown by the results set out below, no interconversion was observed on heating for 10 days at either temperature.
TABLE-US-00016 % ee of sample @ 40° C. % ee sample @ 80° C. Time/h A-1 A-2 A-1 A-2 0 100 97.20 100 97.20 24 100 97.34 100 95.62 48 100 97.30 nd nd 72 100 97.24 nd nd 96 100 97.14 100 96.62 10 days 100 97.46 100 97.12
[0801] Protocol:
[0802] 1. 2×1 mg of pure atropisomer was dissolved in 1 mL of EtOH in a sealed-dram vial.
[0803] 2. One set of vials was heated at 40° C. and another set at 80° C.
[0804] 3. At specified time-points a 20 μL aliquot from each stock solution (1 mL) was taken and quenched into a HPLC vial containing a 80 μL solution of hexane:EtOH; 80:20 to afford a final concentration of 200 ppm and the sample was analysed by chiral HPLC
[0805] 4. Analysis was carried out at the following timepoints: 0 h, 24 h, 48 h, 72 h, 96 h and 240 h for the samples kept at 40° C. and 24 h, 96 h and 240 h for the samples kept at 80° C. using Chiral HPLC method 5
[0806] The stabilities of the isolated atropisomers, Example A-1 and A-2, confirmed that they are Class 3 atropisomers (LaPlante et al., J. Med. Chem., 54:7005-7022 (2011))).
Example 3
[0807] X-Ray Crystallographic Analysis of Atropisomer A-2
[0808] Atropisomer A-2 free base was prepared, and a single crystal was subjected to X-ray crystallographic studies as described below.
EXPERIMENTAL
[0809] Single non-defined morphology crystals of atropisomer A-2 were obtained by recrystallisation from methyl isobutyl ketone (MIBK). A suitable crystal 0.19×0.13×0.04 mm.sup.3 was selected and, using MiTiGen MicroMount, mounted on a Rigaku XtaLAB Syngery-S diffractometer equipped with a HyPix-6000HE detector. The crystal was kept at a steady T=123(2) K during data collection.
[0810] Data were generated using CuKα radiation. The maximum resolution that was achieved was Θ=74.263° (0.80 Å). Data reduction, scaling and absorption corrections were performed. The final completeness was 100.00% out to 74.263° in Θ. The absorption coefficient μ of the compound was determined as being 1.761 mm.sup.−1 at the wavelength (λ=1.542 Å).
[0811] The data were collected and processed using CrysAlisPro software and the structure was solved with the SheIXT (Sheldrick, 2015) structure solution program using the Intrinsic Phasing solution method and by using Olex2 (Dolomanov et al., 2009) as the graphical interface. The model was refined with version 2018/3 of SheIXL-2018/3 (Sheldrick, 2018) using Least Squares minimisation.
[0812] The crystal structure was found to be monoclinic and was assigned the space group P21 (#4).
[0813] All non-hydrogen atoms were refined anisotropically. Hydrogen atom positions were calculated geometrically and refined using the riding model. [0814] References: O. V. Dolomanov and L. J. Bourhis and R. J. Gildea and J. A. K. Howard and H. Puschmann, Olex2: A complete structure solution, refinement and analysis program, J. Appl. Cryst., (2009), 42, 339-341. [0815] Sheldrick, G. M., Crystal structure refinement with SheIXL, Acta Cryst., (2015), C71, 3-8. [0816] Sheldrick, G. M., SheIXT-Integrated space-group and crystal-structure determination, Acta Cryst., (2015), A71, 3-8.
[0817] The results of the studies are set out below in Tables 1-7.
TABLE-US-00017 TABLE 1 Data for crystal of Atropisomer A-2 Free Base Formula C.sub.28H.sub.25CIF.sub.3N.sub.3O D.sub.calc./g cm.sup.−3 1.350 m/mm.sup.−1 1.761 Formula Weight 511.96 Colour n/a Shape n/a Size/mm.sup.3 0.19 × 0.13 × 0.04 T/K 123(2) Crystal System monoclinic Flack Parameter −0.03(2) Hooft Parameter −0.020(6) Space Group P2.sub.1 a/Å 10.1964(3) b/Å 8.6349(4) c/Å 14.3398(6) a/° 90 b/° 93.955(4) g/° 90 v/Å.sup.3 1259.53(9) Z 2 Z′ 1 Wavelength/Å 1.54184 Radiation type CuK.sub.a Q.sub.min/° 3.089 Q.sub.max/° 74.263 Measured Refl. 17242 Independent Refl. 4929 Reflections with I > 2(I) 4544 R.sub.int 0.0357 (3.57 %) Parameters 327 Restraints 1 Largest Peak 0.381 Deepest Hole −0.188 GooF 1.032 wR.sub.2 (all data) 0.1228 wR.sub.2 0.1174 R.sub.1 (all data) 0.0474 R.sub.1 0.0433 Reflections d min (Cu) = 0.80; I/σ = 35.2; Complete 10% (IUCR) = 99% Refinement Shift = 0.000; Max. Peak = 0.4; Min peak = −0.2
TABLE-US-00018 TABLE 2 Fractional Atomic Coordinates (×104) and Equivalent Isotropic Displacement Parameters (Å.sup.2 × .sup.103) for Atropisomer A-2. U.sub.eq is defined as ⅓ of the trace of the orthogonalised U.sub.ij. Atom x y z U.sub.eq CI36 7764.8(9) 10484.3(12) 2175.1(6) 58.8(3) F19 8576.0(19) 5604(3) 7787.5(13) 47.5(5) F20 9300.0(18) 6815(3) 6631.6(14) 56.0(6) F21 8706(2) 8065(3) 7827.5(18) 61.5(6) 035 6134(2) -381(3) 9646.9(18) 44.1(5) N29 4745(2) 1641(3) 9788.3(18) 36.4(5) N32 1997(2) 656(3) 9424.4(17) 36.5(5) N7 7412(2) 4971(3) 5673.0(16) 32.3(5) C28 5700(3) 889(3) 9377(2) 34.9(6) C12 6589(3) 6128(3) 6040(2) 31.4(6) C11 7755(3) 3554(4) 6084(2) 35.0(6) C9 8822(3) 3840(4) 4773(2) 39.7(7) C17 7018(3) 7016(4) 6817(2) 34.2(6) C13 5360(3) 6390(4) 5597(2) 36.7(6) C26 7530(3) 1442(4) 8352(2) 36.1(6) C2 7880(3) 8040(4) 4542(2) 37.9(6) C8 8077(3) 5149(4) 4861(2) 35.3(6) C1 7975(3) 6504(4) 4243(2) 36.4(6) C24 5413(3) 2530(4) 7922(2) 35.7(6) C18 8397(3) 6877(4) 7264(2) 37.7(6) C22 7203(3) 2948(4) 6928(2) 34.9(6) C3 7834(3) 9267(4) 3916(2) 41.2(7) C25 6226(3) 1670(3) 8545(2) 33.5(6) C31 2911(3) -109(4) 10106(2) 38.8(7) C6 8027(3) 6246(4) 3276(2) 40.6(7) C23 5889(3) 3149(4) 7118(2) 36.8(6) C30 4005(3) 939(4) 10513(2) 38.6(7) C27 8013(3) 2085(4) 7566(2) 36.1(6) C10 8632(3) 2863(4) 5533(2) 39.2(7) C16 6185(3) 8123(4) 7156(2) 42.2(7) C15 4949(4) 8367(4) 6710(3) 46.4(8) C4 7882(3) 8953(5) 2972(2) 42.5(7) C14 4541(3) 7517(4) 5926(3) 41.6(7) C5 7986(3) 7463(5) 2643(2) 43.4(8) C34 1206(3) 1831(5) 9855(2) 47.0(8) C33 1136(4) -484(5) 8950(3) 49.5(8)
TABLE-US-00019 TABLE 3 Anisotropic Displacement Parameters (×10.sup.4) SOL_686_i42-5 Hz. The anisotropic displacement factor exponent takes the form: −2π.sup.2[h.sup.2a*.sup.2 × U.sub.11 + . . . + 2hka* × b* × U.sub.12] Atom U.sub.11 U.sub.22 U.sub.33 U.sub.23 U.sub.13 U.sub.12 Cl36 57.6(5) 68.4(6) 49.6(4) 19.0(4) −1.6(4) −1.6(4) F19 46.7(10) 46.1(11) 48.0(10) 7.6(9) −10.0(8) −6.8(9) F20 31.1(9) 89.9(17) 46.4(10) 9.7(11) −1.4(7) −10.4(10) F21 62.3(13) 47.5(12) 70.7(14) −13.8(11) −24.9(11) −5.3(11) O35 43.0(12) 35.0(11) 54.6(13) 7.3(10) 4.8(10) 3.5(10) N29 33.7(12) 32.3(12) 43.3(13) 2.5(11) 2.4(10) −1.1(10) N32 33.3(12) 36.4(13) 39.4(12) −0.1(11) −0.2(10) −1.4(11) N7 25.9(11) 36.3(13) 34.6(11) −1.4(10) 1.9(8) 0.6(9) C28 31.2(13) 29.4(14) 43.5(14) 0.1(12) −1.6(11) −2.7(11) C12 28.5(12) 31.1(14) 35.0(13) 0.7(11) 4.2(10) −1.3(10) C11 25.8(13) 38.8(16) 39.9(15) −2.0(12) −1.8(11) 0.4(11) C9 29.5(14) 48.7(18) 41.1(15) −9.2(14) 4.5(11) 1.8(13) C17 34.6(14) 34.6(14) 33.4(13) −1.3(12) 1.8(10) −4.7(12) C13 31.9(13) 38.9(16) 39.1(14) 0.3(13) 0.0(11) −1.3(12) C26 31.2(13) 30.9(13) 45.5(16) −0.7(12) −1.8(11) 1.3(11) C2 30.7(13) 47.2(17) 36.0(14) −3.7(13) 2.2(11) −1.6(13) C8 24.1(12) 45.1(17) 37.0(14) −7.5(12) 3.5(10) −3.1(12) C1 24.4(12) 46.7(17) 38.2(15) −2.2(13) 3.1(10) −2.5(12) C24 25.2(12) 36.6(16) 45.0(16) −2.3(12) 0.3(11) −1.3(11) C18 36.8(15) 39.8(16) 36.0(14) −0.6(13) −2.0(11) −7.1(13) C22 31.7(13) 32.7(14) 40.0(15) −3.5(12) −0.1(11) −0.6(12) C3 34.0(15) 44.6(17) 44.6(16) 0.8(14) −1.6(12) −0.8(13) C25 30.8(13) 27.1(13) 42.0(15) −2.2(12) −0.5(11) −1.1(11) C31 37.1(15) 37.8(15) 41.7(15) 7.4(13) 3.3(12) 2.1(12) C6 30.4(14) 51.6(19) 39.8(15) −6.2(14) 2.7(11) −5.0(13) C23 28.7(13) 37.2(15) 43.7(16) 1.4(13) −3.1(11) 1.8(12) C30 36.7(15) 41.7(16) 37.5(14) 3.1(13) 2.6(11) 0.0(13) C27 26.8(13) 34.6(15) 46.4(16) −2.8(13) −0.2(11) 2.8(11) C10 29.3(13) 40.0(16) 47.9(16) −7.4(14) 0.8(11) 5.6(13) C16 47.9(18) 35.6(16) 43.5(16) −4.9(13) 5.1(13) −3.5(14) C15 42.7(18) 35.7(17) 62(2) −5.8(15) 12.1(15) 6.5(13) C4 28.8(14) 56(2) 41.9(16) 8.1(15) −0.9(11) −2.0(14) C14 28.7(14) 39.9(17) 55.8(19) 4.1(14) 0.9(12) 2.1(12) C5 30.8(14) 64(2) 35.2(14) −0.8(14) 2.5(11) −2.9(14) C34 42.7(17) 52(2) 45.6(17) 0.8(15) 0.3(13) 8.4(15) C33 47.8(18) 48.9(19) 51.0(18) 0.3(16) −1.6(15) −9.8(16)
TABLE-US-00020 TABLE 4 Bond Lengths in A for Atropisomer A-2 Atom Atom Length/A CI36 04 1.746(4) F19 C18 1.336(4) F20 C18 1.338(4) F21 C18 1.331(4) 035 C28 1.234(4) N29 C28 1.340(4) N29 C30 1.457(4) N32 C33 1.456(4) N32 C34 1.459(5) N32 C31 1.461(4) N7 C11 1.392(4) N7 C8 1.396(4) N7 C12 1.429(4) 028 C25 1.501(4) C12 C13 1.384(4) C12 C17 1.397(4) C11 C10 1.370(4) C11 C22 1.466(4) 09 C8 1.372(5) 09 C10 1.403(5) C17 C16 1.388(5) C17 C18 1.510(4) C13 C14 1.386(5) C26 C27 1.377(5) 026 C25 1.391(4) 02 C3 1.388(5) 02 C1 1.400(5) 08 C1 1.468(5) C1 C6 1.409(4) 024 C23 1.389(5) C24 C25 1.390(4) C22 C23 1.396(4) C22 C27 1.404(4) C3 C4 1.385(5) C31 C30 1.521(5) C6 C5 1.387(5) C16 C15 1.390(5) C15 C14 1.383(5) C4 C5 1.377(6)
TABLE-US-00021 TABLE 5 Bond Angles in ° for Atropisomer A-2 Atom Atom Atom Angle/° C28 N29 030 122.6(3) C33 N32 034 109.6(3) C33 N32 031 110.1(3) C34 N32 031 112.1(2) C11 N7 08 109.1(3) C11 N7 012 126.6(2) 08 N7 012 124.2(3) 035 028 N29 123.2(3) 035 028 025 120.5(3) N29 028 025 116.2(3) 013 012 017 120.0(3) 013 012 N7 118.8(3) 017 012 N7 121.2(3) 010 011 N7 107.1(3) 010 011 022 128.6(3) N7 011 022 124.3(3) 08 09 010 108.4(3) 016 017 012 119.5(3) 016 017 018 118.7(3) C12 C17 C18 121.8(3) C12 C13 C14 120.4(3) C27 C26 C25 120.5(3) C3 C2 C1 121.7(3) C9 C8 N7 106.9(3) C9 C8 C1 128.1(3) N7 C8 C1 125.0(3) C2 C1 C6 117.3(3) C2 C1 C8 125.0(3) C6 C1 C8 117.6(3) C23 C24 C25 120.8(3) F21 C18 F19 106.0(2) F21 C18 F20 107.3(3) F19 C18 F20 105.9(3) F21 C18 C17 111.8(3) F19 C18 C17 113.1(2) F20 C18 C17 112.3(2) C23 C22 C27 117.9(3) C23 C22 C11 123.1(3) C27 C22 C11 119.0(3) C4 C3 C2 118.8(3) C24 C25 C26 118.9(3) C24 C25 C28 121.4(3) C26 C25 C28 119.6(3) N32 C31 C30 113.9(3) C5 C6 C1 121.6(3) C24 C23 C22 120.6(3) N29 C30 C31 112.1(2) C26 C27 C22 121.3(3) C11 C10 C9 108.5(3) C17 C16 C15 120.0(3) C14 C15 C16 120.4(3) C5 C4 C3 121.8(3) C5 C4 CI36 119.1(3) C3 C4 CI36 119.1(3) C15 C14 C13 119.6(3) C4 C5 C6 118.9(3)
TABLE-US-00022 TABLE 6 Torsion Angles in ° for Atropisomer A-2 Atom Atom Atom Atom Angle/° C30 N29 C28 O35 −8.5(4) C30 N29 C28 C25 170.8(2) C11 N7 C12 C13 −110.3(3) C8 N7 C12 C13 74.5(4) C11 N7 C12 C17 71.3(4) C8 N7 C12 C17 −103.9(3) C8 N7 C11 C10 0.3(3) C12 N7 C11 C10 −175.5(3) C8 N7 C11 C22 −177.3(3) C12 N7 C11 C22 6.8(4) C13 C12 C17 C16 2.1(4) N7 C12 C17 C16 −179.5(3) C13 C12 C17 C18 −174.1(3) N7 C12 C17 C18 4.3(4) C17 C12 C13 C14 −0.8(5) N7 C12 C13 C14 −179.2(3) C10 C9 C8 N7 −0.8(3) C10 C9 C8 C1 179.0(3) C11 N7 C8 C9 0.3(3) C12 N7 C8 C9 176.2(3) C11 N7 C8 C1 −179.4(3) C12 N7 C8 C1 −3.5(4) C3 C2 C1 C6 0.1(4) C3 C2 C1 C8 177.6(3) C9 C8 C1 C2 −141.6(3) N7 C8 C1 C2 38.1(4) C9 C8 C1 C6 35.8(4) N7 C8 C1 C6 −144.5(3) C16 C17 C18 F21 −11.9(4) C12 C17 C18 F21 164.2(3) C16 C17 C18 F19 107.6(3) C12 C17 C18 F19 −76.2(4) C16 C17 C18 F20 −132.6(3) C12 C17 C18 F20 43.6(4) C10 C11 C22 C23 −139.7(3) N7 C11 C22 C23 37.4(5) C10 C11 C22 C27 38.3(5) N7 C11 C22 C27 −144.6(3) C1 C2 C3 C4 0.2(5) C23 C24 C25 C26 0.2(5) C23 C24 C25 C28 −175.1(3) C27 C26 C25 C24 1.3(5) C27 C26 C25 C28 176.7(3) O35 C28 C25 C24 144.1(3) N29 C28 C25 C24 −35.3(4) O35 C28 C25 C26 −31.2(4) N29 C28 C25 C26 149.5(3) C33 N32 C31 C30 169.7(3) C34 N32 C31 C30 −68.1(4) C2 C1 C6 C5 0.0(4) C8 C1 C6 C5 −177.6(3) C25 C24 C23 C22 −1.3(5) C27 C22 C23 C24 0.9(5) C11 C22 C23 C24 179.0(3) C28 N29 C30 C31 −80.9(4) N32 C31 C30 N29 −55.5(4) C25 C26 C27 C22 −1.7(5) C23 C22 C27 C26 0.6(5) C11 C22 C27 C26 −177.6(3) N7 C11 C10 C9 −0.8(3) C22 C11 C10 C9 176.8(3) C8 C9 C10 C11 0.9(3) C12 C17 C16 C15 −1.7(5) C18 C17 C16 C15 174.6(3) C17 C16 C15 C14 −0.1(5) C2 C3 C4 C5 −0.7(5) C2 C3 C4 CI36 177.7(2) C16 C15 C14 C13 1.5(5) C12 C13 C14 C15 −1.0(5) C3 C4 C5 C6 0.8(5) CI36 C4 C5 C6 −177.5(2) C1 C6 C5 C4 −0.5(4)
TABLE-US-00023 TABLE 7 Hydrogen Fractional Atomic Coordinates (×10.sup.4) and Equivalent Isotropic Displacement Parameters (Å.sup.2 × 10.sup.3) for Atropisomer A-2. U.sub.eq is defined as ⅓ of the trace of the orthogonalised U.sub.ij. Atom x y z U.sub.eq H29 4556.28 2597.1 9613.5 44 H9 9373.45 3631.45 4279.86 48 H13 5075.26 5792.63 5065.04 44 H26 8091.21 838.52 8764.29 43 H2 7844.85 8248.03 5190.71 46 H24 4521.78 2695.77 8048.38 43 H3 7771.85 10303.2 4130.77 49 H31A 3309.1 −1006.73 9802.84 47 H31B 2411.76 −508.61 10623.33 47 H6 8091.06 5215.49 3052.71 49 H23 5315.8 3715.16 6693.61 44 H30A 3620.15 1767.17 10885.52 46 H30B 4611.45 327.4 10937.24 46 H27 8912.55 1941.11 7453.43 43 H10 9042.29 1887.07 5647.28 47 H16 6460.32 8712.19 7693.92 51 H15 4381.36 9123.95 6944.76 56 H14 3703.76 7704.45 5613.91 50 H5 8029.04 7271.37 1993.52 52 H34A 665.96 1339.38 10310.64 71 H34B 634.49 2342.88 9371.1 71 H34C 1786.66 2599.59 10171.65 71 H33A 1667.2 −1246.13 8637.31 74 H33B 539.51 36.17 8485.32 74 H33C 622.37 −1010.97 9408.31 74
[0818] On the basis of the data set out below, atropisomer A-2 is believed to have the R configuration as shown in
Example 4
[0819] Preparation of Atropisomers A-3 and A-4
[0820] Atropisomers A-3 and A-4 were prepared by following Synthetic Route B, as shown below.
##STR00075## ##STR00076##
Step 1: Diethyl pyridine-2,5-dicarboxylate
[0821] To a suspension of 2, 5-pyridinedicarboxylic acid (20 g, 120 mmol) in absolute EtOH (120 mL) was added conc. H.sub.2SO.sub.4 (25.6 mL, 0.048 mmol) dropwise over a period of 30 min. The resulting reaction mixture was refluxed for 48 h. The reaction mixture was concentrated, and the resulting residue basified to pH 8 (sat. aq. NaHCO.sub.3). The resulting aqueous layer was extracted with EtOAC (4×200 mL). The combined organic layers were washed with brine, washed, dried (Na.sub.2SO.sub.4) and concentrated. Four other 20 g batches were reacted in parallel and the resulting crude material from each reaction was combined and purified by column chromatography on silica gel (60-120 mesh) eluting with 5% EtOAC/hexane to afford the title compound (65 g, 291 mmol, 49%).
Step 2: Ethyl 6-(hydroxymethyl)pyridine-3-carboxylate
[0822] To a cooled (ice-bath) solution of diethyl pyridine-2, 5-dicarboxylate (10 g, 45 mmol) in a mixture of absolute EtOH (40 mL) and THF (3.5 mL) under nitrogen were added NaBH.sub.4 (4.26 g, 112 mmol) and anhydrous CaCl.sub.2 (7.86 g, 71 mmol) portion wise over 30 min. The resulting reaction mixture was stirred at 0° C. for 5 h. The reaction mixture was poured in sat. aq. NH.sub.4Cl (150 mL) and extracted with EtOAc (4×150 mL). The combined organic extracts were dried Na.sub.2SO.sub.4) and concentrated. Six other 10 g batches and one 5 g batch were reacted in parallel and the resulting crude material from each reaction was combined and purified by column chromatography with silica gel (60-120 mesh) eluting with 20% EtOAc/hexane to afford the title compound (55 g, 320 mmol, 100%).
Step 3: Ethyl 6-formylpyridine-3-carboxylate
[0823] To a cooled (ice-bath) solution of ethyl 6-(hydroxymethyl)pyridine-3-carboxylate (30 g, 166 mmol) in DCM (360 mL) under nitrogen was added DMP (84.32 g, 199 mmol) portion wise over 20 min. The reaction was stirred at rt for 3 h. The reaction mixture was poured into ice-cold water (1.5 L) and the resulting mixture basified to ˜pH 8 (sat. aq. NaHCO.sub.3) and extracted with EtOAc (4×1000 mL). The combined organic layers were washed with brine, dried (Na.sub.2SO.sub.4) and concentrated. The resulting residue was purified by column chromatography with silica gel (60-120 mesh) eluting with 12% EtOAc/hexane to afford the title compound (19 g, 106 mmol, 33%).
Step 4: Ethyl 6-[4-(4-chlorphenyl)-4-oxo-butanoyl]pyridine-3-carboxylate
[0824] To a stirred solution of intermediate A (1.17 g, 5.6 mmol) and TEA (1.56 mL, 11.2 mmol) in 1,2-dimethoxyethane (10 mL) were added ethyl 6-formylpyridine-3-carboxylate (1 g, 5.6 mmol) and 3-ethyl-5-(2-hydroxyethyl)-4-methylthiazol-3-ium bromide (0.28 g, 11.2 mmol) at room temperature. The resulting solution was heated at 80-90° C. for 5 h. The reaction was diluted with ice-cold water (400 mL) and extracted with EtOAc (3×200 mL). The combined organic layers were dried (Na.sub.2SO.sub.4) and concentrated. The resulting residue was purified by column chromatography with silica gel (60-120 mesh) eluting with 8% EtOAc/hexane to afford the title compound (5.5 g, 15.9 mmol, 17%).
Step 5: Ethyl 6-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]pyridine-3-carboxylate
[0825] To a solution of ethyl 6-[4-(4-chlorophenyl)-4-oxo-butanoyl]pyridine-3-carboxylate (2.5 g, 7.2 mmol) in 1,4-dioxane (25 mL) were added 2-aminobenzotrifluoride (3.5 g, 21.7 mmol) and PTSA (0.14 g, 0.72 mmol) at room temperature. The resulting solution was heated at 150° C. for 48 h. The reaction mixture was concentrated and purified by column chromatography with silica gel (60-120 mesh) eluting with 6% EtOAc/hexane to afford the title compound (2.5 g, 5.3 mmol, 64%).
Step 6: 6-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]pyridine-3-carboxylic acid
[0826] To a solution of ethyl 6-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]pyridine-3-carboxylate (2.2 g, 4.7 mmol) in mixture of THF (10 mL) and water (10 mL) at room temperature was added UOH (0.59 g, 14 mmol). The resulting solution was stirred at 80° C. for 16 h. The reaction mixture was concentrated, diluted with water (150 mL) and extracted with EtOAc (4×150 mL). The combined organic extracts were dried (Na.sub.2SO.sub.4) and concentrated. The resulting material was triturated with n-pentane (15 mL) and Et.sub.2O (15 mL) to afford the title compound (2 g, 4.5 mmol, 97%).
Step 7: 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2-(dimethylamino)ethyl]benzamide
[0827] To a solution of 6-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]pyridine-3-carboxylic acid (2.8 g, 6.33 mol) in DMF (20 mL) was added HATU (7.22 g, 19 mol) and the reaction mixture was stirred at room temperature for 20 min. Unsym-N, N-dimethyl ethylenediamine (1.11 g, 12.7 mol) and DIPEA (3.31 mL, 19 mol) were added and the reaction mixture was stirred at room temperature for 4 h. The reaction mixture was diluted with ice-cold water (200 mL) and extracted with EtOAc (4×100 mL). The combined organic extracts were dried (Na.sub.2SO.sub.4) and concentrated. The resulting residue was purified by column chromatography with silica gel (60-120 mesh) eluting with 30% EtOAc/hexane) to afford the title compound (2.4 g, 4.7 mmol, 74%).
Step 8: Separation of Atropisomers A-3 and A-4
[0828] The atropisomers of 6-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2-(dimethylamino)ethyl]pyridine-3-carboxamide may be resolved by chiral HPLC using preparative chiral HPLC method 2.
[0829] Two peaks were isolated: [0830] Peak 1: Atropisomer A-3, 6-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2-(dimethylamino)ethyl]pyridine-3-carboxamide-atropisomer 1 (70 mg, 0.14 mmol, 355%), brown solid. [0831] Peak 2: Atropisomer A-4, 6-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2-(dimethylamino)ethyl]pyridine-3-carboxamide-atropisomer 2 (75 mg, 0.15 mmol, 38%), brown solid.
[0832] Both peaks were purified further to remove aliphatic impurities:
[0833] Peak 1: (A-3) (57 mg, 0.11 mmol) was diluted with HPLC grade water (25 mL) followed by sonication for 10 min and extraction with EtOAc (3×20 mL). The combined organic extracts were dried (Na.sub.2SO.sub.4), filtered, concentrated and lyophilised to afford atropisomer A-3 (56 mg, 0.11 mmol, 98%, >99% ee).
[0834] .sup.1H NMR (DMSO-d.sub.6) δ 8.45-8.43 (m, 2H), 8.01 (d, J=6.8 Hz, 1H), 7.74-7.68 (m, 2H), 7.65-7.60 (m, 3H), 7.25 (d, J=8.4 Hz, 2H), 7.11-7.04 (m, 3H), 6.60 (d, J=4 Hz, 1H), 3.32 (m, 2H, obscured by residual water peak), 2.30 (m, 2H, obscured by residual solvent peak), 2.19 (s, 6H). LCMS (Method 1)—RT 2.41, MH+ 513.4
[0835] Peak 2: (A-4): (60 mg, 0.117 mmol) was diluted with HPLC grade water (25 mL) followed by sonication for 10 min and extraction with EtOAc (3×20 mL). The combined organic extracts were dried (Na.sub.2SO.sub.4), filtered, concentrated and lyophilised to afford Example A-4 (60 mg, 0.12 mmol, 99%, 95% ee).
[0836] .sup.1H NMR (DMSO-d.sub.6) δ 8.47-8.43 (m, 2H), 8.02 (d, J=7.2 Hz, 1H), 7.74-7.68 (m, 2H), 7.65-7.60 (m, 3H), 7.25 (d, J=8.4 Hz, 2H), 7.11-7.04 (m, 3H), 6.60 (d, J=4 Hz, 1H), 3.32 (m, 2H, obscured by residual water peak), 2.30 (m, 2H, obscured by residual solvent pea), 2.20 (s, 6H). LCMS (Method 1)—RT 2.41, MH+ 513.4
[0837] Chiral Analysis
[0838] Analysis of the chiral properties of the Atropisomers A-3 and A-4 was carried out by measuring their optical rotations and their retention times obtained by chiral HPLC using the methods described above to give the results shown in the table below.
TABLE-US-00024 Chiral Chiral Specific HPLC RT HPLC Optical Example (min) Method Rotation A-3 10.400 2 +9.92° A-4 12.090 2 −4.89°
Example 5
Preparation of Atropisomers A-5 and A-6: N-[2-(dimethylamino)ethyl]-6-[5-(4-fluorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]pyridine-3-carboxamide
[0839] Atropisomers A-5 and A-6 were prepared as a racemic mixture using the same method as described above in Example 4 for atropisomers A-3 and A-4 with the following exceptions: (a) Intermediate B (3.23 g, 16.58 mmol) was used in step 4 and 3-benzyl-5-(2-hydroxyethyl)-4-methylthiazol-3-ium bromide (0.678 g, 2.51 mmol) and purification was carried out using 10% EtOAc/hexane as eluent (b) step 5 purification used 1.3% EtOAc/hexane as eluent (c) MeOH was used instead of THF in step 6 and purification was trituration with Et.sub.2O (d) In step 7 the isolated residue was purified by chromatography with basic alumina gel eluting with DCM to afford the title compound (0.16 g, 0.32 mmol, 55%) (e) Purification by preparative HPLC method 1 afforded the title compound (61 mg, 0.12 mmol, 38%) (racemic mixture of atropisomers) as its hydrochloride salt, a light yellow solid.
[0840] .sup.1H NMR (DMSO-d.sub.6) δ 10.09 (bs, 1H), 8.85 (m, 1H), 8.49 (s, 1H), 8.11 (d, J=8.0 Hz, 1H), 7.73-7.70 (m, 2H), 7.69-7.61 (m, 3H), 7.13-7.10 (m, 3H), 7.06-7.02 (m, 2H), 6.56 (d, J=4.0 Hz, 1H), 3.56 (m, 2H), 3.20 (m, 2H), 2.76 (d, J=4.4 Hz, 6H).
[0841] LCMS (Method 2)—RT 5.06, MH+ 497.2
[0842] Chiral HPLC analysis with chiral HPLC method 3 indicated a mixture of atropisomers, RT peak 1, 9.95 min, 49.8% area (Atropisomer A-5) and peak 2, 11.52 min, 50.2% area (Atropisomer A-6).
Example 6
Preparation of Atropisomers A-7 and A-8 of 6-[5-(4-cyanophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2-(dimethylamino)ethyl]pyridine-3-carboxamide
[0843] Atropisomers A-7 and A-8 were prepared as a racemic mixture using the same method as described above in Example 4 for atropisomers A-3 and A-4 with the following exceptions: (a) Intermediate C (0.28 g, 1.39 mmol) was used in step 4 and 3-benzyl-5-(2-hydroxyethyl)-4-methylthiazol-3-ium bromide (0.04 g, 0.14 mmol) and purification was carried out using 10% EtOAc/hexane as eluent (b) step 5 purification used 7% EtOAc/hexane as eluent (c) In step 7 the isolated residue was purified by chromatography with basic alumina gel eluting with 10% EtOAc/hexane to afford the title compound (0.13 g, 0.25 mmol, 75%) (d) Purification by preparative HPLC method 2 afforded the title compound (54 mg, 0.11 mmol, 36%) as its hydrochloride salt, a light yellow solid.
[0844] .sup.1H NMR (DMSO-d.sub.6) δ 9.83 (brs, 1H), 8.80 (t, J=5.2 HZ, 1H), 8.50 (d, J=1.6 Hz, 1H), 8.11 (dd, J=8.4, 2.0 Hz, 1H), 7.79-7.64 (m, 6H), 7.32-7.07 (m, 4H), 6.83 (d, J=4.0 Hz, 1H), 3.56-3.46 (m, 2H), 3.22-3.18 (m, 2H), 2.78 (d, J=4.8 Hz, 6H).
[0845] LCMS (Method 1)—RT 2.05, MH+ 504.1
[0846] Chiral HPLC analysis with chiral HPLC method 4 indicated a mixture of atropisomers, RT peak 1, 8.82 min, 50.2% area (Example A-7) and peak 2, 10.10 min, 49.8% area (Example A-8).
Example 7
[0847] Preoaration of Compounds B-2 to B-107
[0848] Further examples of atropisomer compounds of the present invention can be prepared by preparing racemic mixtures of the compounds shown in the table below, and then separating the individual atropisomers using the chiral HPLC methods described above or methods similar thereto. In the table, the Compound numbers given correspond to the Example numbers in our earlier International patent application WO2018/197714 but with the prefix B- added. Thus, Compound B-2 corresponds to Example 2 in WO2018/197714, Compound B-3 corresponds to Example 3 in WO2018/197714 and so on. The NMR, LCMS and other characterising data for the racemic compounds and their biological activity data are as given in WO02018/197714.
TABLE-US-00025
Example 8
Alternative Method for Preparing (R)-4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2 (dimethylamino)ethyl]benzamide (Atropisomer A-2)
[0849] ##STR00175##
[0850] The title compound was prepared by following Steps 1, 2, 3, 4a and 5a of the synthetic routes shown in Scheme 1 above. In this route, chiral resolution is carried out on the carboxylic acid intermediate (8) rather than on the dimethylamino-ethyl amide (9).
Step 1: 4-[4-(4-chlorophenyl)-4-oxo-butanoyl]benzonitrile (6)
[0851] A flask was charged with tetrahydrofuran (4 mL/g) and zinc chloride (1.222 g/g, 1.3 eq.) was added in portions to afford a white mobile suspension which was stirred for 15 min. tert-butanol (0.66 mL/g, 1 eq) was added followed by triethylamine (0.96 mL/g, 1 eq) in portions keeping the temperature below 40° C. The reaction was stirred for 2 h. 4-Cyanoacetophenone (1 g/g, 1 eq) and 4-chlorophenacyl bromide (1.61 g/g, 1 eq) were added and the reaction mixture was stirred at 20° C. (±5) for 48 h or until reaction was complete. The product was isolated by precipitation with aqueous HCl and slurry in aqueous HCl and methanol. The resulting solid was dried under vacuum (45° C.) to afford the title compound as a pale yellow solid.
Step 2: 4-(5-(4-chlorophenyl-1-(2-(trifluoromethyl) phenyl)-1H-pyrrol-2-yl) benzonitrile (7)
[0852] 4-(4-(4-chlorophenyl)-4-oxobutanoyl) benzonitrile (1 g/g, 1 eq) was charged to a flask and dioxane (10 mL/g) was added to afford a yellow suspension. 2-Trifluoromethyl aniline (1.269 mL/g, 3 eq) was added in a single portion followed by p-toluenesulfonic acid (0.06399 g/g, 0.1 eq) and the reaction mixture was heated at 101° C. for 40-72 h (additional portions of p-toluenesulfonic acid (0.1 eq) were added if required every 8 hours to push the reaction to completion). The reaction mixture was cooled to room temperature and concentrated under vacuum. The resulting oily residue was purified by slurring in methanol (10 mL/g). The solid was isolated by filtration and dried under vacuum (45° C.) to afford the title compound as a yellow solid.
Step 3: 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl) phenyl)-1H-pyrrol-2-yl) benzoic acid (8)
[0853] To 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl) phenyl)-1H-pyrrol-2-yl) benzonitrile (1 g/g, 1 eq) in methanol (10.9 mL/g) was added sodium hydroxide (0.948 g/g, 10 eq) in water (5 mL/g) dropwise over 15 minutes and the resulting mixture was stirred at 70-76° C. for 18 hours or until complete. The reaction mixture was cooled to room temperature, acidified and the product isolated by filtration, washing with water (5 mL/g) and acetonitrile (3 mL/g). The product was slurried in acetone/water (20 vols, 75:25) at 50-55° C. and dried under vacuum (60° C.) to afford the title compound as a yellow solid.
Step 4a: (R) 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl) phenyl)-1H-pyrrol-2-yl) benzoic acid (3) by chiral resolution of (8)
[0854] 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl) phenyl)-1H-pyrrol-2-yl) benzoic acid (1 g/g, 1 eq) was added to a flask followed by tetrahydrofuran (2 mL/g) and acetonitrile (0.75 mL/g). (S)-1-(4-methoxyphenyl)-ethylamine (0.335 mL/g, 1 eq) was added dropwise over 5 min and the resulting reaction mixture was stirred at 40-50° C. for 15 min then cooled to room temperature. Acetonitrile (7.25 mL/g) was added and the reaction seeded (0.0001 g/g, 99% ee, (S)-1-(4-methoxyphenyl)-ethylamine salt of desired atropisomer). The reaction mixture was stirred for 16 h and the resulting solids were isolated by filtration washing with acetonitrile. Hot (75-80° C.) slurry in acetonitrile afforded the chiral salt as a white solid (40% yield, 98.16% ee). Salt break was achieved in THF/water (2/2 vols) using 1M HCl (2.2 eq) to afford the acid which was further purified by slurry in water affording the title compound (90.52 g, salt break yield 97%, overall yield 39%, 98.06% ee). .sup.1H NMR (DMSO-d6) δ 12.83 (brs, 1H), 7.77-7.67 (m, 6H), 7.23-7.10 (m, 2H), 7.08-7.01 (m, 4H), 6.68 (d, J=4.0 Hz, 1H), 6.59-6.58 (d, J=4.0 Hz, 1H). Chiral HPLC with chiral HPLC method 6 showed a single atropisomer, RT 6.083 min, 99.02% area (minor atropisomer RT 7.07 min, 0.98% area).
[0855] Chiral resolution can also be achieved using (S)-(−)-1-phenylethylamine.
Step 5a: (R)-4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2 (dimethylamino)ethyl]benzamide (1)
[0856] 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl) phenyl)-1H-pyrrol-2-yl) benzoic acid (single atropisomer) (1 g/g, 1 eq) was dissolved in THF (5 mL/g) and N,N-dimethylethylenediamine (0.75 mL/g, 3 eq) was added dropwise followed by DIPEA (1.58 mL/g, 4 eq). 50% T3P in THF (2.72 mL/g, 2 eq) was added dropwise and the reaction mixture stirred at 20° C. for 15 min. Additional portions of 50% T3P in THF were added until reaction was complete. The reaction mixture was diluted with 10% brine (2 mL/g) and sodium hydroxide solution (2 mL/g) until pH8-10. The layers were separated, and the aqueous layer extracted with ethyl acetate (2×5 mL/g). The combined organic layers were washed with brine, dried (MgSO4) and concentrated to afford the title compound (80 g, 156 mmol, 71%) as a white triboluminescent solid. Chiral HPLC with chiral HPLC method 7 showed a single atropisomer, RT 12.62 min, 99.32% area (minor atropisomer, RT 10.58 min, 0.67% area)
Example 9
Preoaration and characterisation of (R)-4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2 (dimethylamino)ethyl]benzamide tartrate
[0857] Method 1: Small Scale Preparation of Tartrate Salt
[0858] Atropisomer A-2 free base (904.2 mg) was suspended in acetone (9.042 mL, 10 vols) and stirred at 25° C. for 40 minutes. When the solution was free of visible particulates, it was split into 12 equal aliquots (603 μL), giving an approximate active content of 60.3 mg per sample.
[0859] An aliquot of 247 μL (1.05 eq) of a 0.5 M solution of tartaric acid in ethanol was added to an aliquot of the free base solution at 25° C. The mixture was stirred at 25° C. for 18 hours after which time a white suspension formed, and the resulting solids were then isolated by filtration (PTFE 10 micron fritted cartridge) and the resulting solids were then isolated and dried in vacuo at 40° C. for ca. 72 hours.
[0860] The resulting salt was labelled as Tartrate Pattern A (solvate).
[0861] Method 2: Preparation of Tartrate Salt Using an Isopropyl Acetate Solution of Atropisomer A-2
[0862] Atropisomer A-2 (749.8 mg) was suspended in isopropyl acetate (15 mL, 20 vols) and the suspension was heated to 40° C. with agitation. When the solution was free of visible particulates, it was split into 12 equal aliquots (1 ml), giving an approximate active content of 50 mg per sample. An aliquot of 195.3 μL of a 1 M solution of atropisomer A-2 in ethanol was added to an aliquot of the free base solution at 40° C. The resulting mixture was cooled to 25° C. at a cooling rate of approximately 10° C./hour. A white suspension formed and the resulting solids were then isolated by filtration (PTFE 10 micron fritted cartridge) and dried in vacuo at 40° C. for ca. 18 hours. The resulting salt was labelled as Tartrate Pattern B.
[0863] Method 3: Preparation of Tartrate Salt Using an Isopropyl Alcohol Solution of Atropisomer A-2
[0864] By following Method 2, except that atropisomer A-2 (750.1 mg) was initially suspended in isopropyl alcohol (15 ml, 20 vols), Tartrate Pattern A salt was prepared.
[0865] Method 4: Preparation of Tartrate Salt Using a 2-Methyl-Tetrahydrofuran Solution of Atropisomer A-2
[0866] Method 1 was repeated, except that atropisomer A-2 (913.9 mg) was initially suspended in 2-methyl-tetrahydrofuran (15 ml, 20 vols), (9.139 mL, 10 vols) and stirred at 25° C. for ca. 40 minutes, and then a 250 μl (1.05 eq) aliquot of 1 M tartaric acid in ethanol was added to an aliquot of the A-2 free base solution, to give Tartrate Pattern A salt.
[0867] Method 5: 500 mg Scale Preparation of Atropisomer A-2 Tartrate Pattern B Salt
[0868] Atropisomer A-2 free base (521.5 mg) was weighed into a glass vial and charged with isopropyl acetate (20 vols, 10.430 ml). The mixture was heated to 40° C. and stirred for 15 minutes to give a clear solution. The solution was then charged with tartaric acid (1.05 eq, 162.5 mg) dissolved in 3 mL of tetrahydrofuran. The resulting mixture was seeded with atropisomer A-2.tartrate pattern B, which caused the salt to immediately precipitate at 40° C. forming a mobile suspension.
[0869] The mixture was cooled to 25° C. and stirred for 20 hours. The resulting solid was isolated by filtration and dried at 40° C. in vacuo to afford the atropisomer A-2 Tartrate Pattern B salt in 84% yield.
[0870] Method 6: Scaled-Up Preparation of Atropisomer A-2 Tartrate Pattern B Salt (Anhydrous Form)
[0871] Atropisomer A-2 free base (10.0497 g) was weighed into a Buchi flask and charged with isopropyl acetate (20 vols, 200 ml). The mixture was heated to 40° C. to afford a clear solution, free of particulates, and stirred for 30 minutes. The solution was charged with tartaric acid (3.1954 g, 1.08 eq.) dissolved in tetrahydrofuran (50 mL), the acid being was added in portions as follows: 15 mL at 40° C.; seeded with atropisomer A-2 tartrate pattern B salt and stirred for 30 minutes; 10 mL and stirred for 1 hour; 10 mL and stirred for 30 minutes; 15 mL and stirred for 30 minutes. The white suspension was then cooled to RT at a cooling rate of 10° C./h and stirred for 18 hours. The resulting solid was isolated by filtration in vacuo and washed with isopropyl acetate (2×2 vols) and dried in vacuo at 40° C. for 20 hours to afford the A-2 Tartrate Pattern B salt (anhydrous) in a yield of 97%; HPLC purity 99.74% (HPLC method 1), chiral purity 99.27% (Chiral HPLC method 7).
[0872] Method 7: Alternative Scaled-Up Preparation of Atropisomer A-2 Tartrate Pattern B Salt (Anhydrous Form) by Cooling Crystallisation from Butanol/Water 96:4
[0873] Atropisomer A-2 free base (36.79 g) was weighed into a flask and charged with butanol (282.57 ml, 7.68 vols). The mixture was heated to 80° C. (pale yellow, hazy solution) and stirred for 30 minutes before clarification into a Mya* vessel, pre-heated at 80° C. The solution was then charged with L-(+)-tartaric acid (1.023 eq, 11.0806 g) as a solution in water (11.77 mL, 0.32 vols of the initial API charge). The addition was made dropwise at 80° C. with clarification of the acid solution. The mixture was then cooled to 68° C. over a period of 30 minutes, seeded with 0.1% of ground atropisomer A-2 tartrate Pattern B salt seed crystals (32.6 mg) and held for 1 hour. The mixture was then cooled to 5° C. at a cooling rate of 5° C./hour and stirred at 5° C. for 6 hours before isolation of the solid. The solid was filtered in vacuo, washed twice with butanol and dried for 15 minutes on the filter and then at 40° C. for 20 hours to afford atropisomer A-2 Tartrate Pattern B salt (anhydrous) in a yield of 83%; HPLC purity 99.84% (HPLC method 1), chiral purity 99.66% (Chiral HPLC method 7).
*Note: In the foregoing equilibrations or crystallisaions that required temperature control and/or defined heating/cooling profiles, a Radley's Mya4 Reaction Station was used. The Radley's Mya4 Reaction Station is a 4-zone reaction station with magnetic and overhead stirring capabilities and a temperature range of −30 to 180° C. on 2 to 400 mL scale mixtures. The reaction conditions required were programmed via the Mya 4 Control Pad.
[0874] Characterisation of the Atropisomer A-2 Tartrate Salts
[0875] The identities of the salts as 1:1 (molar ratio of free base:tartaric acid) stoichiometric salts were confirmed from their .sup.1H NMR spectra which were collected using a JEOL ECX 400 MHz spectrometer equipped with an auto-sampler. The samples were dissolved in a suitable deuterated solvent for analysis. The data were acquired using Delta NMR Processing and Control Software version 4.3.
[0876] The tartrate salts were characterised using X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), gravimetric solubility tests and gravimetric vapour sorption tests using the techniques described below.
[0877] X-Ray Powder Diffraction (XRPD)
[0878] X-Ray Powder Diffraction patterns were collected on a PANalytical diffractometer using Cu Kα radiation (45 kV, 40 mA), θ-θ goniometer, focusing mirror, divergence slit (½″), soller slits at both incident and divergent beam (4 mm) and a PiXcel detector. The software used for data collection was X'Pert Data Collector, version 2.2f and the data was presented using X'Pert Data Viewer, version 1.2d. XRPD patterns were acquired under ambient conditions via a transmission foil sample stage (polyimide—Kapton, 12.7 μm thickness film) under ambient conditions using a PANalytical X'Pert PRO. The data collection range was 2.994-35°2θ with a continuous scan speed of 0.202004° s-1.
[0879] Differential Scanning Calorimetry (DSC)
[0880] DSC data were collected on a PerkinElmer Pyris 6000 DSC equipped with a 45-position sample holder. The instrument was verified for energy and temperature calibration using certified indium. A predefined amount of the sample, 0.5-3.0 mg, was placed in a pin-holed aluminium pan and heated at 20° C.min—from 30 to 350° C. or varied as experimentation dictated. A purge of dry nitrogen at 20 ml min.sup.−1 was maintained over the sample. The instrument control, data acquisition and analysis were performed with Pyris Software v11.1.1 revision H.
[0881] Thermo-Gravimetric Analysis (TGA)
[0882] TGA data were collected on a PerkinElmer Pyris 1 TGA equipped with a 20-position auto-sampler. The instrument was calibrated using a certified weight and certified Alumel and Perkalloy for temperature. A predefined amount of the sample, 1-5 mg, was loaded onto a pre-tared aluminium crucible and heated at 20° C.min.sup.−1 from ambient temperature to 400° C. A nitrogen purge at 20 ml.Math.min.sup.−1 was maintained over the sample. Instrument control, data acquisition and analysis were performed with Pyris Software v11.1.1 revision H.
[0883] Gravimetric Solubility
[0884] The solubility in water of the salts was measured using a gravimetric solubility protocol.
[0885] 1 ml of water was charged into crystallisation tubes. The solid was weighed into a tared glass vial, added in portions to the solutions and the vial weighed after each addition until a hazy solution was observed. The amount in mg was then calculated to give the solubility in mg/ml.
[0886] The results obtained from the characterisation studies are set out in Table 8 below.
TABLE-US-00026 TABLE 8 Solubility XRPD XRPD in water Salt pattern FIG. DSC TGA (mg/mL) Free Pattern A FIG. 4 FIG. 6 line 6A FIG. 6 <5 base onset 157° C. line 6B peak 159° C. Tartrate Pattern A FIG. 5 FIG. 7 line 7A FIG. 7 6.7 (EtOH onset~173° C. line 7B solvate) peak~175° C., 78-157° C. thermal event loss 6% 117° C. Tartrate Pattern B FIG. 5 FIG. 8 line 8A FIG. 8 7.5 (anhydrous) onset~172° C. line 8B peak at~174° C.
[0887] Gravimetric Vapour Sorption (GVS)
[0888] GVS studies were carried out on atropisomer A-2 Tartrate Pattern B salt using the protocol set out below:
[0889] Sorption isotherms were obtained using a Hiden Isochema moisture sorption analyser (model IGAsorp), controlled by IGAsorp Systems Software V6.50.48. The sample was maintained at a constant temperature (25° C.) by the instrument controls. The humidity was controlled by mixing streams of dry and wet nitrogen, with a total flow of 250 ml.Math.min-1. The instrument was verified for relative humidity content by measuring three calibrated Rotronic salt solutions (10-50-88%). The weight change of the sample was monitored as a function of humidity by a microbalance (accuracy +/−0.005 mg). A defined amount of sample was placed in a tared mesh stainless steel basket under ambient conditions. A full experimental cycle typically consisted of three scans (sorption, desorption and sorption) at a constant temperature (25° C.) and 10% RH intervals over a 0-90% range (60 minutes for each humidity level). This type of experiment should demonstrate the ability of samples studied to absorb moisture (or not) over a set of well-determined humidity ranges
[0890] GVS analysis (see
[0891] It can therefore be concluded that the atropisomer A-2 Tartrate Pattern B salt exists as a stable solid, only absorbing surface moisture with no change in form.
Example 10
Preoaration and characterisation of other salts of (R)-4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2 (dimethylamino)ethyl]benzamide
[0892] The hydrochloride, mesylate, maleate, malate, tosylate, sulfate and phosphate salts of (R)-4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2 (dimethylamino)ethyl]benzamide have been prepared and characterised. Their X-ray powder diffraction patterns (XRPD), thermal profiles (DSC and TGA) and solubilities in water are set out in the table below.
[0893] For all of the salts, .sup.1H NMR showed that there was a 1:1 ratio between free base and counterion.
TABLE-US-00027 Solubility XRPD Thermal Profile in water Salt FIG. DSC/TGA (mg/mL) Hydrochloride FIG. 19 DSC events: 12.9 (XRPD Pattern A) top 2 traces onset~204° C. peak~206° C. TGA events: 30-207° C. loss 0.4% then decomposition Hydrochloride FIG. 19 DSC events: n/a (XRPD Pattern B) bottom trace onset~200° C. peak~205° C. Mesylate FIG. 20 DSC events: 21.6 (XRPD Pattern A) onset~162° C. peak~164° C. Maleate FIG. 21 DSC events: n/a (XRPD Pattern A top trace onset~115° C. (amorphous peak at~119° C., 2.sup.nd content)) endotherm 132° C. thermal event~151° C. Maleate FIG. 21 DSC events: 9.8 (XRPD Pattern B- bottom trace onset~130° C. an hydrate) peak~132° C. Malate FIG. 22 DSC events: n/a (XRPD Pattern A) bottom trace onset 176° C. peak 211° C. glass transition~139° C., “shoulder” at 178° C. Malate FIG. 22 DSC events: 2 (XRPD Pattern B- top trace onset~184° C. solvate) peak~211° C., thermal events 64° C., 107° C. TGA events: 24-85° C. loss 1.5% 88-197° C. loss 6.1% Tosylate FIG. 23 DSC events: <5 (XRPD Pattern A) onset 176° C. peak 179° C. Phosphate FIG. 24 DSC events: n/a (XRPD Pattern A- top trace onset~162-164° C., anhydrous) peak~166° C.-167° C. thermal event 246° C. Phosphate FIG. 24 DSC events n/a (XRPD Pattern B) bottom trace onset~162-164° C. peak at~166-167° C., thermal events~137° C. and 225° C. Sulfate FIG. 25 DSC events: n/a (XRPD Pattern A) top and onset~176° C. middle traces peak at~183° C. decomposition after 300° C. Sulfate FIG. 25 DSC events: n/a (XRPD Pattern B) bottom trace onset~112° C. peak 118° C. melt/decomposition after 300° C. TGA events: 28-84° C. loss 0.9% 86-154° C. loss 2.7%
[0894] The solubility in water of the salts was measured using a gravimetric solubility protocol. Thus, 1 ml of water was charged into crystallisation tubes. The solid was weighed into a tared glass vial, added in portions to the solutions and the vial weighed after each addition until a hazy solution was observed. The amount in mg was then calculated to give the solubility in mg/mL.
[0895] Proton-NMR
[0896] .sup.1H NMR spectra were collected using a JEOL ECX 400 MHz spectrometer equipped with an auto-sampler. The samples were dissolved in a suitable deuterated solvent for analysis. The data was acquired using Delta NMR Processing and Control Software version 4.3.
[0897] Preoaration of the Salts
[0898] Low Scale Preparation of Example A-2 Salts
[0899] Method 1: Acetone Mediated
[0900] Example A-2 free base (904.2 mg) was suspended in acetone (9.042 mL, 10 vols) and stirred at 25° C. for 40 minutes. When the solution was free of visible particulates, it was split into 12 equal aliquots (603 μL), giving an approximate active content of 60.3 mg per sample.
[0901] 0.5 M or 1 M acid stock solutions (247 μL or 124 μL, 1.05 eq) in EtOH were charged to the solutions at 25° C. The mixtures were stirred at 25° C. for 18 hours. If required, the samples were manipulated further (e.g. by trituration of the solids and addition of anti-solvent) to recover solids for analysis, which were isolated and dried in vacuo at 40° C. for ca. 72 hours.
[0902] The amounts of acid used, the anti-solvent, and the resulting crystalline form are set out in the table below. Alternative methods can be used to isolate the salts.
TABLE-US-00028 Amount charged Acid (μl) Anti-solvent Crystalline Form Hydrochloric 124 Et.sub.2O Hydrochloride Pattern A Methane sulfonic 124 Heptane Mesylate Pattern A Maleic 124 Heptane Maleate Pattern A (−)-L-Malic 124 Gum isolated Malate Pattern A and dried to afford solid (+)-L-Tartaric 247 Not required Tartrate Pattern A (solvate) p-Toluenesulfonic 124 Not required Tosylate Pattern A Sulfuric 124 Et.sub.2O Sulfate Pattern A
[0903] Method 2: Isopropyl acetate mediated
[0904] Example A-2 (749.8 mg) was suspended in iPrOAc (15 mL, 20 vols) heated to 40° C. with agitation. When the solution was free of visible particulates, it was split into 12 equal aliquots (1 ml), giving an approximate active content of 50 mg per sample. 0.5 M or 1 M acid stock solutions (195.3 μL or 97.7 μL, 1 eq) in EtOH were charged to the solutions at 40° C. The mixtures were cooled to 25° C. at approximately 10° C./h. If required, the samples were manipulated further (e.g. by trituration of the solids and addition of anti-solvent) to recover solids for analysis, which were isolated and dried in vacuo at 40° C. for ca. 18 h.
[0905] HCl pattern A (TBME anti-solvent), tartrate pattern B (1 M acid stock solution (195.3 μL) in EtOH), tosylate pattern A and phosphate pattern B can be isolated by Method 2
[0906] Method 3: IPA Mediated
[0907] Method identical to method 2 except that Example A-2 (750.1 mg) was suspended in IPA (15 mL, 20 vols).
[0908] HCl pattern A (TBME anti-solvent), tartrate pattern A (1 M acid stock solution (195.3 μL) in EtOH), tosylate pattern A and phosphate pattern A can be isolated by method 3.
[0909] Method 4: 2-Methyl THF Mediated
[0910] Method identical to method 1 except that Example A-2 (913.9 mg) was suspended in 2-Methyl THF (9.139 mL, 10 vols) and stirred at 25° C. for ca. 40 min and 0.5 M or 1 M acid stock solutions (250 μl or 125 μl, 1.05 eq) in EtOH were used.
[0911] HCl pattern B (heptane as anti-solvent), maleate pattern A (heptane as anti-solvent), tartrate pattern A (1 M acid (250 μl, 1.05 eq) in EtOH) and tosylate pattern A can be isolated by method 4.
[0912] A sub-set of salts were scaled up and more fully characterised.
[0913] 500 Ma Scale Preparation of Example A-2 Salts
[0914] Hydrochloride Salt
[0915] Example A-2 free base (524.9 mg) was weighed into a glass vial and charged with IPA (20 vols, 10.498 ml) and heated to 40° C. The solution was stirred at 40° C. for 40 min and then charged with HCl (4.4 M in IPA, 1.2 eq, 280 μl). The mixture was then seeded with HCl salt pattern B and stirred at 40° C. for 15 min before being cooled down to 25° C. The mixture was concentrated in vacuo to afford a pale-yellow oil residue. The oil was suspended in 10 vols of TBME and stirred at 25° C. for 72 h, obtaining a white suspension. The solid was isolated and dried at 40° C. in vacuo for 18 h to afford the title salt pattern A in 73% yield.
[0916] Mesylate Salt
[0917] Example A-2 free base (503.9 mg) was weighed into a glass vial and charged with 2-Me THF (10 vols, 5.039 ml). The mixture was stirred at RT for 30 min. The solution was then charged with Methanesulfonic acid (1 M solution in EtOH, 1.05 eq, 1.033 ml), seeded with Example A-2.MsOH pattern A and stirred at 25° C. for 30 min. The mixture became a hazy solution and then formed a white suspension which was stirred at 25° C. for 72 h. The solid was isolated by filtration and dried in vacuo at 40° C. for 18 h to afford the title salt pattern A in 46% yield.
[0918] Tartrate Salt
[0919] Example A-2 free base (521.5 mg) was weighed into a glass vial and charged with iPrOAc (20 vols, 10.430 ml). The mixture was heated to 40° C. and stirred for 15 min to deliver a clear solution. The solution was then charged with Tartaric acid (1.05 eq, 162.5 mg) dissolved in 3 mL of THF. The mixture was then seeded with Example A-2.tartrate pattern B, which caused the salt to immediately precipitate at 40° C. forming a mobile suspension. The mixture was cooled to 25° C. and stirred for 20 h. The solid was isolated by filtration and dried at 40° C. in vacuo to afford the title salt pattern B in 84% yield.
[0920] Tosylate Salt
[0921] Example A-2 free base (504.5 mg), was weighed into a glass vial charged with iPrOAc (20 vols, 10.090 ml) and heated to 40° C. The solution was stirred at 40° C. for 40 min and then charged with p-toluenesulfonic acid (1 M in EtOH, 1.05 eq, 1.04 ml). The mixture was then seeded with a small amount of Example A-2.tosylate pattern A and stirred at 40° C. for 15 min before being cooled to 25° C. The mixture quickly became a white suspension and it was stirred at 25° C. for 72 h. The solid was isolated and dried at 40° C. in vacuo for 18 h to afford the title salt pattern A in 82% yield.
[0922] Maleate Salt
[0923] Example A-2 free base (523.9 mg) was weighed into a glass vial and charged with 2-Me THF (10 vols, 5.239 mL). The mixture was stirred at RT for 30 min, to give a clear solution. To the solution was then added Maleic acid (0.5 M in THF, 1.05 eq, 2.149 mL), seeded with a small amount of Example A-2.maleate pattern A and stirred at 25° C. for 30 min. The mixture was reduced in vacuo to yield a white gum. The gum was suspended in 10 vols of heptane and stirred at 25° C. for 72 h. The solid was isolated and dried in vacuo at 40° C. for 18 h to afford the title salt pattern B. 1H NMR conforms to structure but indicates ˜1:0.8 stoichiometry.
[0924] Malate Salt
[0925] Example A-2 free base (524.9 mg) was weighed into a glass vial, charged with IPA (20 vols, 10.618 ml) and heated to 40° C. The solution was stirred at 40° C. for 40 min and then charged with Malic acid (1 M solution in EtOH, 1.05 eq, 1.09 ml). The mixture was then stirred at 40° C. for 15 min before being cooled down to 25° C. The mixture, which remained as a solution at 25° C., was reduced in vacuo leaving an oil residue. The oil was suspended in 10 vols of heptane and stirred at 25° C. for 70 h obtaining a white suspension. The solid was isolated and dried at 40° C. in vacuo for 18 h to afford the title salt pattern B.
[0926] Sulfate Salt
[0927] Example A-2 free base (520 mg) was weighed into a glass vial charged with acetone (10 vols, 5.2 mL). The mixture was stirred at RT for 30 min, to yield a clear solution.
[0928] The solution was charged with Sulphuric acid (1 M in EtOH, 1.05 eq, 1.066 ml), seeded with Example A-2.Sulfate pattern A and stirred at 25° C. for 30 min. The mixture remained as a solution, so it was reduced in vacuo with a gentle stream of Nitrogen, which left a white gum.
[0929] The gum was suspended in 10 vols of diethyl ether and stirred at 25° C. for 70 h. The solid was then isolated and dried in vacuo at 40° C. for 18 h to afford the title salt pattern A sim (similar but not identical to previously isolated sulfate salt pattern A).
Example 11
[0930] Biological Activity
[0931] A. Assay to Measure the Effects of Compounds of the Invention on U87MG Human Glioblastoma Cancer Cell Viability
[0932] The following protocol was used to measure the effects of compounds of the invention on U87MG cell viability.
[0933] U87MG cells were grown in their recommended growth media/supplements (ATCC). Cells were seeded at a concentration of 5000 cells per well into 96 well plates overnight at 37° C., 5% CO.sub.2. Cells were treated with relevant concentrations of test compound for 72 hours. After 72 hours incubation, viability was established using sulforhodamine B (SRB) colorimetric assay. Percentage viability was calculated against the mean of the DMSO treated control samples, and IC.sub.50 values for inhibition of cell growth were calculated using GraphPad Prism software by nonlinear regression (4 parameter logistic equation).
[0934] From the results obtained by following the above protocol, the IC.sub.50 values against the U87MG cell line of the atropisomers of the Examples were determined as shown in Table 9 below.
TABLE-US-00029 TABLE 9 IC.sub.50 Atropisomer (μM) A-1 4.6 A-2 0.22 A-3 0.15 A-4 3.94 A-5/A-6 0.73* A-7/A-8 1.48* *Although separate atropisomers A-5 and A-6, and A-7 and A-8, were identified by chiral chromatography, the racemic mixtures were tested in the U87MG cell viability assay.
[0935] B. Assay to Measure the Effects of Atropisomers A-1 and A-2 on Cancer Cell Viability of a Diverse Cancer Cell Line Panel
[0936] Screening against diverse cancer cell lines was performed to identify tumour types displaying sensitivity to atropisomers A-1 and A-2. A panel of 48 cancer-derived cell lines was screened in a high-throughput proliferation assay using dilutions of atropisomers A-1/A-2. Cell lines that were screened included those representing cancer of the pancreas, large intestine/colorectum, lung, brain and nerves, and lymphoma and leukaemia cell lines. Cell lines were treated with serial half-log dilutions of compound and assayed 72 hours later for proliferation using CellTiter-Glo Assay (Promega). IC.sub.50 values were calculated by fitting the dose-response data using a nonlinear regression model. The IC.sub.50 values in micromolar for atropisomers A-1 and A-2 are shown in Table 10 below.
TABLE-US-00030 TABLE 10 Cell Line Tissue Origin A-1 A-2 MIA PaCa-2 Pancreas 9.17 0.33 PANC-1 Pancreas >10 0.83 AsPC-1 Pancreas >10 2.9 Capan-1 Pancreas >10 1.23 Panc 10.05 Pancreas 9 1.14 BxPC-3 Pancreas 6.97 0.3 HOT 116 Large intestine/Colorectum 7.36 0.23 LoVo Large intestine/Colorectum 8.36 0.54 SVV620 Large intestine/Colorectum 8.35 0.19 SW480 Large intestine/Colorectum 7.55 0.87 COLO 205 Large intestine/Colorectum 6.75 0.38 HT-29 Large intestine/Colorectum 4.53 0.45 RKO Large intestine/Colorectum 6.17 0.16 A549 Lung 9.19 0.3 NCI-H460 Lung 8.13 0.3 HCC44 Lung 8.26 0.48 NCI-H1373 Lung >10 3.21 NCI-H1792 Lung 7.73 0.25 NCI-H1299 Lung 8.25 0.37 NCI-H1975 Lung 5.14 0.63 SK-MES-1 Lung 7.78 0.36 U118 MG Brain & Nerves 7.6 0.47 A-172 Brain & Nerves 7.07 0.34 LN-229 Brain & Nerves 7.37 0.31 SW1088 Brain & Nerves 8.49 0.77 T98G Brain & Nerves 5.26 0.28 D283 Med Brain & Nerves 9.26 0.31 Daoy Brain & Nerves 7 0.18 DOHH-2 Blood/Lymphoma 5.64 0.23 HBL-1 Blood/Lymphoma 9.94 0.66 OCI-LY-19 Blood/Lymphoma 6.67 0.22 SU-DHL-6 Blood/Lymphoma 3.98 0.26 U-2932 Blood/Lymphoma 4.47 0.28 WSU-DLCL2 Blood/Lymphoma 6.74 0.36 SU-DHL-2 Blood/Lymphoma 7.01 0.2 Toledo Blood/Lymphoma >10 0.88 JeKo-1 Blood/Lymphoma 7.94 0.19 Z-138 Blood/Lymphoma 7.04 0.21 GRANTA-519 Blood/Lymphoma 7.42 0.22 JVM-2 Blood/Lymphoma 4.71 0.38 Daudi Blood/Lymphoma 7.17 0.39 NAMALWA Blood/Lymphoma 7.05 0.25 Raji Blood/Lymphoma 3.91 0.36 Ramos Blood/Lymphoma 3.99 0.34 ML-2 Blood/Leukaemia 4.01 0.18 KG-1 Blood/Leukaemia >10 0.46 MV-4-11 Blood/Leukaemia 6.49 0.28 Kasumi-1 Blood/Leukaemia 5.13 0.33
[0937] As can be seen from the data, atropisomer A-2 was a significantly more active cell growth inhibitor than atropisomer A-1 against all of the cell lines
[0938] C. Assay to Measure the Effects of Compounds of the Invention on Cells in Mitosis
[0939] Inhibiting the ability of PLK1 and PLK4 to bind to their partners through their PBDs is known to cause cells to arrest in mitosis. Experimentally, this can be measured by assessing the number of cells which are in mitosis at a certain time after treatment with a test compound by immunofluorescent detection of phosphorylated Histone H3 (pH3), a mark which is only present in mitotic cells. PLK1/4-PBD inhibitors are expected to cause a dose-dependent increase in pH3-positive cells, which is reported as Mitotic Index (MI)—the percentage of cells which, at a given time, are positive for this mitotic mark.
[0940] Distinct mitotic phenotypes are induced following inhibition of PLK1 and PLK4 in cells. Disruption of the PBD domain of PLK1 has been demonstrated to trigger mitotic arrest with non-congressed chromosomes, a distinct phenotype from the monopolar spindle phenotype induced by ATP-competitive PLK1 inhibitors (Hanisch et al., 2006 Mol. Biol. Cell 17, 448-459). Centriole assembly is controlled by PLK4, with inhibitors inducing a multipolar spindle phenotype due to centrosome defects which results in abnormal cyokinesis (Wong et al., 2015. Science 348(6239); 1155-1160).
[0941] The following protocol was used to measure the effects of atropisomer A-2 and atropisomer A-3 on arresting cells in mitosis.
[0942] Cells were plated at 10 000/well in 96-well plates and incubated overnight. The following day atropisomer A-2 stocks in DMSO were diluted in medium then added to cells with a maximum final DMSO concentration on cells of 0.2%. Cells were incubated with the compound for 24 hours then fixed in 3.7% formaldehyde. Cells were permeabilised with 0.1% Triton X-100 then incubated with anti-phospho-histone H3 (Ser10) antibody (Abcam). The cells were washed with PBS then incubated with AlexaFluor 488 labelled goat anti-rabbit IgG (Invitrogen) in the presence of 4 ug/mL Hoechst 33342 (Invitrogen). Cells were washed in PBS then imaged on an Arrayscan VTi HCS instrument using the Target Activation V4 Bioapplication. A user-defined threshold was applied to identify mitotic cells based on the intensity of phospho-histone H3 staining.
[0943] GraphPad Prism was used to plot % mitotic cells against compound concentration using log(inhibitor) vs response variable slope with least squares fitting and no constraints.
[0944] From the results obtained by following the above protocol, the EC.sub.50 values and the percentages of cells in mitosis against the HeLa and U87MG cell lines were obtained for atropisomer A-2 and atropisomer A-3. The EC.sub.50 values are shown in Table 11 below.
TABLE-US-00031 TABLE 11 U87MG HeLa EC.sub.50 EC.sub.50 Example (μM) (μM) A-2 0.09 0.03 A-3 0.10 0.03
[0945] Phenotype Study
[0946] In a separate study, following the above protocol and using single compound concentrations of 0.03 μM for each of atropisomer A-1 and atropisomer A-2, the frequency of observed mitotic phenotypes in U87MG cells was manually assessed and classified into the following phenotypes: non-congressed chromosomes, multipolar spindles/abnormal cytokinesis, monopolar spindles, normal prometaphase, normal metaphase for each of A-1 and A-2. The results are shown in
[0947] Results
[0948] The results presented in
[0949] D. Assay to Measure the Effects of Atropisomer A-2 on Centrosomes
[0950] The results of study C above show that atropisomer A-2 causes mitotic effects which are characteristic of dysregulated centrosome function. The effects of A-1 and A-2 on centrosome function were therefore investigated further. HeLa cells stably expressing a Centrin1-GFP fusion protein were seeded into 96-well plates overnight. Cells were treated with atropisomer A-1 or atropisomer A-2 (at concentrations of 0.02 μM in DMSO) or DMSO control for 72 hours and then imaged using a fluorescence microscope. Multiple cell fields were captured for each treatment condition, and the images were subsequently analysed manually. Centrin1-GFP specifically marks centrioles as discrete foci, and therefore can be used to quantitate centriole number per cell. Thus, for each treatment condition, 100 cells were analysed and the number of centrioles present in each cell was recorded. The data were then separated into bins (no centrioles, 1 centriole, 2 centrioles, and greater than 2 centrioles) and are shown in
[0951] From the data, it can be concluded that atropisomer A-2 exhibits evidence of PLK4 inhibition phenotypes on HeLa cells.
[0952] E. Assay to Measure the Effects of Compounds of the Invention on Wild-Type Versus KRAS HeLa Cell Viability
[0953] Atropisomers A-1, A-2, A-3 and A-4 were tested on HeLa cells engineered to inducibly express wild-type or oncogenic KRasG12V transgenes using the FLP-in/T-Rex system (Invitrogen). Cells were plated, and then treated with or without Doxycycline to induce transgene expression, and then treated with serially-diluted PBD inhibitors. After 72 hours of incubation, cell viability was assessed using the Cell Titre Blue reagent (Promega) and a BMG Pherastar plate reader. The effect of PBD inhibition on cell viability with either wild-type or oncogenic G12V KRAS was assessed using GraphPad Prism.
[0954] From the results obtained by following the above protocol, the GI.sub.50 values against the wild-type and KRAS G12V HeLa cell line of each of the atropisomers were determined as shown in Table 12.
TABLE-US-00032 TABLE 12 WT G12V GI.sub.50 GI.sub.50 Atropisomer (nM) (nM) A-1 NA NA A-2 3.01 2.08 A-3 4.68 2.6 A-4 254 153
[0955] F. Kinase Selectivity Assay
[0956] Compounds of the invention bind to the PBD domain of PLK1 and PLK4 but not to the catalytic domains of PLK1 and PLK4 and should exhibit good selectivity over other kinases. Atropisomer A-2 has been tested for off-target activity against a panel of ninety-seven kinases distributed across the kinome at a concentration of 3 μM using the DiscoverX KinomeScreen assay. The results are shown in Table 13 below.
[0957] The DiscoverX KinomeScreen assay is a site-directed competition binding assay which measures the binding affinity of a compound to a kinase, by use of a solid supported control compound which can bind or capture the kinases in solution. In the absence of a kinase-inhibitor test compound, all of the kinase will bind to the solid support. If a kinase-inhibitor test compound is added to the assay mix, the amount of kinase binding to the solid support will be reduced, the extent of reduction being dependent on the potency of the test compound as a kinase inhibitor. The potencies of the test compounds against the kinases can be expressed as the percentage (Percent Control) of the kinase binding to the solid support at a given concentration of the test compound, the lower the percentage the more potent the kinase-binding capability of the test compound. Thus, a Percent Control value of 100% would indicate that the test compound does not bind to the kinase at all, since all of the kinase has bound to the solid support. Conversely, a Percent Control value of 0% would indicate that the test compound has bound all of the kinase since none is bound to the solid support.
[0958] Protocol:
[0959] For most assays, kinase-tagged T7 phage strains were grown in parallel in 24-well blocks in an E. coli host derived from the BL21 strain.
[0960] E. coli were grown to log-phase and infected with T7 phage from a frozen stock (multiplicity of infection=0.4) and incubated with shaking at 32° C. until lysis (90-150 minutes). The lysates were centrifuged (6,000×g) and filtered (0.2 μm) to remove cell debris. The remaining kinases were produced in HEK-293 cells and subsequently tagged with DNA for qPCR detection. Streptavidin-coated magnetic beads were treated with biotinylated small molecule ligands for 30 minutes at room temperature to generate affinity resins for kinase assays. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific phage binding. Binding reactions were assembled by combining kinases, liganded affinity beads, and test compounds in 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). Test compounds were prepared as 40× stocks in 100% DMSO and directly diluted into the assay. All reactions were performed in polypropylene 384-well plates in a final volume of 0.02 ml. The assay plates were incubated at room temperature with shaking for 1 hour and the affinity beads were washed with wash buffer (1×PBS, 0.05% Tween 20). The beads were then re-suspended in elution buffer (1×PBS, 0.05% Tween 20, 0.5 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The kinase concentration in the eluates was measured by qPCR.
[0961] Compounds that bind the kinase active site and directly (sterically) or indirectly (allosterically) prevent kinase binding to the immobilized ligand, will reduce the amount of kinase captured on the solid support. Conversely, test molecules that do not bind the kinase have no effect on the amount of kinase captured on the solid support.
[0962] The strength of binding of the test molecule to the kinase can be expressed as the percent control (% Ctrl)
[0963] Percent Control (% Ctrl)
[0964] The compound(s) were screened at 3000 nM concentration, and results for primary screen binding interactions are reported as ‘% Ctrl’, where lower numbers indicate stronger hits in the matrix on the following page(s).
[0965] % Ctrl Calculation
[0968] The % Ctrl values for atropisomer A-2 against the panel kinases are set out in Table 13 below.
TABLE-US-00033 TABLE 13 Gene % Ctrl @ Symbol 3 μM ABL1 100 ABL1 100 ABL1 100 ABL1 97 ACVR1B 96 CABC1 63 AKT1 98 AKT2 100 ALK 89 AURKA 98 AURKB 84 AXL 93 BMPR2 82 BRAF 96 BRAF 100 BTK 94 CDK19 81 CDK2 94 CDK3 100 CDK7 100 CDK9 97 CHEK1 100 CSF1R 100 CSNK1D 91 CSNK1G2 100 DCLK1 87 DYRK1B 97 EGFR 100 EGFR 100 EPHA2 100 ERBB2 66 ERBB4 99 MAPK3 99 PTK2 98 FGFR2 85 FGFR3 92 FLT3 100 GSK3B 88 IGF1R 100 CHUK 100 IKBKB 96 INSR 100 JAK2 80 JAK3 100 MAPK8 77 MAPK9 80 MAPK10 93 KIT 100 KIT 100 KIT 100 STK11 81 MAP3K4 93 MAPKAPK2 75 MARK3 100 MAP2K1 84 MAP2K2 81 MET 100 MKNK1 92 MKNK2 85 MAP3K9 96 MAPK14 100 MAPK11 99 PAK1 87 PAK2 100 PAK4 100 CDK16 100 PDGFRA 100 PDGFRB 100 PDPK1 96 PIK3C2B 100 PIK3CA 100 PIK3CG 100 PIM1 99 PIM2 100 PIM3 98 PRKACA 100 PLK1 97 PLK3 87 PLK4 100 PRKCE 99 RAF1 92 RET 95 RIOK2 100 ROCK2 88 RPS6KA3 100 NUAK2 99 SRC 100 SRPK3 71 TGFBR1 95 TEK 99 NTRK1 100 TSSK1B 100 TYK2 100 ULK2 93 KDR 100 STK32C 100 ZAP70 92
[0969] The results against ninety-seven kinases demonstrate that atropisomer A-2 has poor or non-existent binding activity against a wide range of kinases and therefore is unlikely to suffer from problems associated with off-target kinase inhibition.
[0970] In the case of PLK1 and PLK4, atropisomer A-2 showed little or no binding affinity for the catalytic domains of these kinases (% Control values of 97% and 100% respectively). It is concluded therefore that the activity profiles indicative of PLK1/PLK4 inhibitory activity demonstrated in the examples above is a consequence of to the non-catalytic polo box domains of PLK1 and PLK4.
[0971] G. Determination of Oral Bioavailability and Brain Exposure in Mouse PK
[0972] Atropisomers A-2 and A-3 were evaluated in an in vivo mouse model to determine brain and plasma concentrations following p.o. and i.v. dosing.
[0973] The following protocol was followed:
[0974] Male CD-1 mice were dosed with the compounds of Examples A-2 and A-3, either by i.v. administration (2 mg/kg) or by p.o. administration (10 mg/kg). Eight samples were taken for analysis in the i.v. leg at 2, 10, 30 min, 1, 2, 4, 8, and 24 (for i.v) and 9 samples in the p.o. leg at 15, 30 min, 1, 2, 4, 8, 24, 48 and 72 hrs.
[0975] The compounds of Examples A-2 and A-3 were both formulated in 10% DMSO/90% hydroxypropyl-beta-cyclodextrin (20% w/v in water) for i.v. and p.o. dosing. N =3 mice per time point.
[0976] Post dosing, terminal blood samples were taken from individual animals and delivered into labelled polypropylene tubes containing anticoagulant (EDTA). The samples were held on wet ice for a maximum of 30 min while sampling of all the animals in the cohort was completed. The blood samples were centrifuged for plasma (4° C., 21100 g for 5 min) and the resulting plasma transferred into corresponding labelled tubes. Terminal brains from each PO dosed animal were excised, rinsed with saline and placed into pre-weighed labelled polypropylene tubes and the samples re-weighed prior to storage.
[0977] Quantitative bioanalysis was carried out using liquid chromatography—mass spectroscopy was performed. The results are shown in Tables 14, 15 and 16 below and in
[0978] Oral Bioavailability
TABLE-US-00034 TABLE 14 2 mq/Kq IV Atropisomer Atropisomer Parameter Units A-2 A-3 T ½ Hr 9.0 8.4 CI mL/min/kg 20.5 16.8 Cmax ng/mL 278 274 AUCinf ng .Math. hr/mL 1624 1987
TABLE-US-00035 TABLE 15 10 mq/Kq PO Atropisomer Atropisomer Parameter Units A-2 A-3 T ½ Hr 10.7 8.3 Cmax ng/mL 265 322 AUCinf ng .Math. hr/mL 6131 6925 F % 76 70
[0979] The results demonstrate that Atropisomers A-2 and A-3 are highly absorbed following oral dosing in mice.
[0980] Brain Exposure
TABLE-US-00036 TABLE 16 Atropisomer Atropisomer Parameter Units A-2 A-3 T ½ Hr 12.1 8.2 Tmax Hr 8.0 8.0 Cmax ng/mL 693 604 AUClast plasma ng .Math. hr/mL 6131 6925 AUClast brain ng .Math. hr/mL 20528 14103
[0981] The results of the brain exposure studies presented in Table 16 demonstrate that atropisomers A-2 and A-3 both have high brain exposure. In the case of atropisomer A-2, the results demonstrate that atropisomer A-2 has high brain exposure with an AUC B:P ratio of 3.3 following oral dosing in mice.
[0982] H. In Vivo Efficacy
[0983] Atropisomer A-2 shows efficacy in glioblastoma mouse models when tumours are implanted subcutaneously and orthotopically, as indicated by the studies described below.
[0984] (i) In Vivo Anti-Cancer Activity in U87MG Subcutaneous Xenograft Model
[0985] Male athymic nude mice bearing U87MG tumours were given an oral dose of 100 mg/kg of atropisomer A-2 on days 1, 4 and 7 and the tumour volumes were measured over 20 days. Tumour volumes in a control group of tumour-bearing mice, who had received vehicle only at the same time points were also measured. The treated group showed significantly decreased tumour volume compared to control (3.85% T/C at day 13), as shown in
[0986] (ii) In Vivo Anti-Cancer Activity in U87-Luc Orthotopic Xenograft Model
[0987] U87-Luc cells were intracerebrally implanted into the brains of male athymic nude mice and tumour growth was monitored by bioluminescent signal. In the treatment group animals were given an oral dose of 100 mg/kg of atropisomer A-2 on days 1, 4, 7, 10 and 13. The control group animals were given vehicle only. The results, shown in
[0988] (iii) In Vivo Anti-Cancer Activity in Mice Bearing HCT116 Tumours
[0989] Atropisomer A-2 has shown efficacy in a KRAS mutated colorectal cancer model, as described below.
[0990] Male athymic nude mice bearing HCT116 xenograft tumours were give an oral dose of 100 mg/kg atropisomer A-2 on days 1, 8 and 15 and the tumour volumes were measured over 3 weeks. Tumour volumes in a control group of tumour-bearing mice, who had received vehicle only at the same time points were also measured.
[0991] The results, shown in
[0992] Pharmaceutical Formulations
[0993] (i) Tablet Formulation
[0994] A tablet composition containing a composition of matter or an atropisomer of the invention is prepared by mixing 50 mg of the compound with 197 mg of lactose (BP) as diluent, and 3 mg magnesium stearate as a lubricant and compressing to form a tablet in known manner.
[0995] (ii) Capsule Formulation
[0996] A capsule formulation is prepared by mixing 100 mg of a composition of matter or an atropisomer of the invention with 100 mg lactose and filling the resulting mixture into standard opaque hard gelatin capsules.
[0997] (iii) Injectable Formulation I
[0998] A parenteral composition for administration by injection can be prepared by dissolving a composition of matter or an atropisomer of the invention (e.g. in a salt form) in water containing 10% propylene glycol to give a concentration of active compound of 1.5% by weight. The solution is then sterilised by filtration, filled into an ampoule and sealed.
[0999] (iv) Injectable Formulation II
[1000] A parenteral composition for injection is prepared by dissolving in water a composition of matter or an atropisomer of the invention (e.g. in salt form) (2 mg/ml) and mannitol (50 mg/ml), sterile filtering the solution and filling into sealable 1 ml vials or ampoules.
[1001] (v) Injectable formulation III
[1002] A formulation for i.v. delivery by injection or infusion can be prepared by dissolving a composition of matter or an atropisomer of the invention (e.g. in a salt form) in water at 20 mg/ml. The vial is then sealed and sterilised by autoclaving.
[1003] (vi) Injectable formulation IV
[1004] A formulation for i.v. delivery by injection or infusion can be prepared by dissolving a composition of matter or an atropisomer of the invention (e.g. in a salt form) in water containing a buffer (e.g. 0.2 M acetate pH 4.6) at 20 mg/ml. The vial is then sealed and sterilised by autoclaving.
[1005] (vii) Subcutaneous Injection Formulation
[1006] A composition for sub-cutaneous administration is prepared by mixing a composition of matter or an atropisomer of the invention with pharmaceutical grade corn oil to give a concentration of 5 mg/ml. The composition is sterilised and filled into a suitable container.
[1007] (viii) Lyophilised formulation
[1008] Aliquots of formulated a composition of matter or atropisomer of the invention are put into 50 ml vials and lyophilized. During lyophilisation, the compositions are frozen using a one-step freezing protocol at (−45° C.). The temperature is raised to −10° C. for annealing, then lowered to freezing at −45° C., followed by primary drying at +25° C. for approximately 3400 minutes, followed by a secondary drying with increased steps if temperature to 50° C. The pressure during primary and secondary drying is set at 80 millitor.
EQUIVALENTS
[1009] The foregoing examples are presented for the purpose of illustrating the invention and should not be construed as imposing any limitation on the scope of the invention. It will readily be apparent that numerous modifications and alterations may be made to the specific embodiments of the invention described above and illustrated in the examples without departing from the principles underlying the invention. All such modifications and alterations are intended to be embraced by this application.