Compounds

Abstract

The invention provides compounds for use in a method of treating and/or preventing a bacterial infection in a human or non-human mammal, said method comprising administration of said compound in combination with (either simultaneously, separately, or sequentially) a β-lactam antibiotic, wherein said compound has the general formula I: ##STR00001##
(wherein: Q is a lipophilic, zinc chelating moiety which is selective for Zn.sup.2+ ions and which comprises at least one, preferably two or more (e.g 2, 3 or 4), optionally substituted, unsaturated heterocyclic rings, e.g. 5 or 6-membered heterocyclic rings (such rings preferably include at least one heteroatom selected from N, S and O, preferably N); wherein any optional substituents may be selected from C.sub.1-6 alkyl, C.sub.1-6 alkoxy, halogen, nitro, cyano, amine, and substituted amine; each L, which may be the same or different, is a covalent bond or a linker; each W, which may be the same or different, is a non-peptidic hydrophilic group which comprises one or more hydroxy groups; and x is an integer from 1 to 3)
or a stereoisomer, pharmaceutically acceptable salt or prodrug thereof.

Claims

1. A compound of the general formula I, or a stereoisomer, pharmaceutically acceptable salt or prodrug thereof: ##STR00221## wherein: Q is a lipophilic, zinc chelating moiety which is selective for Zn.sup.2+ ions selected from the group consisting of: ##STR00222## ##STR00223## ##STR00224## wherein * denotes the point (or points) of attachment to the linker group L; and R′, where present, is H or C.sub.1-6 alkyl; each L, which may be the same or different, is a covalent bond or a linker; each W, which may be the same or different, is a non-peptidic hydrophilic group which comprises a cyclic boronic acid selected from the group consisting of: ##STR00225##  and x is an integer from 1 to 3.

2. A compound according to claim 1, wherein the compound has a formula
Q-L-W wherein Q, L and W are as defined in claim 1.

3. A compound according to claim 1, wherein for each occurrence, the cyclic boronic acid is independently selected from cyclic boronic acids having a structure: ##STR00226##

4. A compound as claimed in claim 1, having a structure ##STR00227## or a stereoisomer or pharmaceutically acceptable salt thereof.

5. A pharmaceutical composition comprising a compound as defined in claim 1, together with one or more pharmaceutically acceptable carriers or excipients, and optionally including one or more antioxidants.

6. A method of treating a bacterial infection in a human or non-human mammal, the method comprising administering to the human or non-human mammal an effective amount of a compound according to claim 1 or a pharmaceutical composition thereof in combination with a β-lactam antibiotic.

7. The method according to claim 6, wherein the infection is associated with Gram positive or Gram negative bacteria which are resistant to one or more antibiotics.

8. The method according to claim 6, wherein the infection is associated with Gram negative bacteria which are resistant to one or more antibiotics.

9. The method according to claim 6, wherein the infection is associated with Gram negative bacteria which are resistant to a β-lactam antibiotic.

10. The method according to claim 8, wherein the bacteria comprise metallo-β-lactamases.

11. The method according to claim 6, wherein the infection is associated with gram negative multi-resistant bacteria harboring extended spectrum metallo-β-lactamases (ESBL).

12. The method according to claim 6, wherein the compound is administered together with a carbapenem antibiotic agent and the infection is associated with gram negative carbapenem-resistant bacteria harboring metallo-β-lactamases, or with gram negative carbapenem-resistant bacteria harboring Klebsiella pneumoniae carbapenemases, KPC and/or metallo-β-lactamases.

13. The method according to claim 6, wherein the compound and β-lactam antibiotic are provided in the same formulation.

14. The method according to claim 6, wherein the compound and β-lactam antibiotic are provided in different formulations.

15. The method according to claim 6, wherein the β-lactam antibiotic is selected from the following: penams, cephems, monobactams, penems, carbapenems, and clavams.

16. The method according to claim 15, wherein the β-lactam antibiotic is a carbapenem.

17. A pharmaceutical formulation comprising a compound according to claim 1 together with a β-lactam antibiotic, and optionally one or more pharmaceutically acceptable carriers or excipients.

18. The pharmaceutical formulation according to claim 17, wherein the β-lactam antibiotic is selected from the following: penams, cephems, monobactams, penems, carbapenems, and clavams.

19. The pharmaceutical formulation according to claim 18, wherein the β-lactam antibiotic is a carbapenem.

20. A kit comprising: (i) a first container containing a compound according to claim 1 or a pharmaceutical composition thereof; and (ii) a second container containing a β-lactam antibiotic.

21. The kit according to claim 20, wherein the β-lactam antibiotic is selected from the following: penams, cephems, monobactams, penems, carbapenems, and clavams.

22. The kit according to claim 21, wherein the β-lactam antibiotic is a carbapenem.

Description

EXAMPLES

(1) The invention will be described in more detail in the following non-limiting examples and with reference to the accompanying figures, in which:

(2) FIG. 1 shows the HPLC purity of the compound of Example 26;

(3) FIG. 2 shows the in vitro toxicity of compounds according to the invention in human hepG2 cells;

(4) FIG. 3 shows the in vitro toxicity of compounds according to the invention in human hepG2 cells;

(5) FIG. 4 shows the results of a time-kill study of the compound of Example 26 with meropenem;

(6) FIG. 5 shows the protein binding of the compound of Example 26 at two different concentrations;

(7) FIG. 6 shows the results of an irreversibility study of the compound of Example 26 with purified VIM-2. The left hand side of the figure shows the experimental set-up; the right hand side shows the enzyme activity with the compounds of Examples 207a-c;

(8) FIG. 7 shows the results of an enzyme restoration study of the compound of Example 207c with Zn.sup.2+;

(9) FIG. 8A shows the zinc restorability of the NDM VIM-2 after in vitro incubation with the compound of Example 26 compared to incubation with standards TPEN and EDTA;

(10) FIG. 8B shows the effect of increasing concentrations of Example 26 on VIM-2 enzyme compared to the PAC chelator EDTA;

(11) FIG. 9 shows the in vitro interaction of the compound of Example 26 with zinc-containing human enzymes—comparison to the APC-chelator EDTA;

(12) FIG. 10 shows the MIC values of a peptide-based inhibitor (Example 195) compared to a non-peptide inhibitor after storage in solution;

(13) FIG. 11 shows blood and peritoneal fluid CFU of KP #50752504 as a function of MEM concentration; and

(14) FIG. 12 shows colony counts in blood and peritoneal fluid in neutropenic mice at 1 and 5 hours after inoculation. Detection limit 1.0 log.sub.10 CFU/ml. ** p<0.01, *** p<0.001 vs vehicle Group.

(15) General Procedures

(16) All reagents and solvents used are of commercial grade and were used without further purifications prior to use. NMR (.sup.1H, .sup.13C) spectra were recorded on a Bruker AVI-600 MHz, AVII-400 MHz, a DPX-300 MHz or a DPX-200 MHz spectrometer. Coupling constants (J) are reported in hertz, and chemical shifts are reported in parts per million (ppm) relative to CDCl.sub.3 (7.26 ppm for .sup.1H and 77.16 ppm for .sup.13C) and [D.sub.6]DMSO (2.50 ppm for .sup.1H and 39.52 ppm for .sup.13C). IR spectra were obtained on a Perkin-Elmer Spectrum BX series FT-IR spectrometer and only selected peaks are reported. Mass spectra were recorded at 70 eV on Waters Prospec Q spectrometer using E, ES or CJ as the methods of ionization. High resolution mass spectra were recorded on Waters Prospec Q spectrometer using EI or ESI as the methods of ionization.

Example 1—Synthesis of tert-butyl (4-(2-azidoacetamido)phenethyl)carbamate

(17) ##STR00052##

(18) Tert-butyl (4-aminophenethyl)carbamate (2.33 g, 9.89 mmol, 1.0 eq) was dissolved in DCM (100 mL) and mixed with HBTU (3.75 g, 9.89 mmol, 1.0 eq) and cooled to 0° C. using an ice bath. Azidoacetic acid (1.0 g, 9.89 mmol, 1.0 eq) was added to the stirring mixture followed by NMM (2.18 mL, 19.79 mmol, 2.0 eq). The mixture was stirred 1 hour at 0° C. and 23 hours at room temperature. The mixture was then diluted with 0.5 M NaHCO.sub.3 (100 mL) and the organic phase was separated. The aqueous phase was then extracted two times with 50 mL DCM, the combined organic phases dried over MgSO.sub.4, filtered and concentrated in vacuo. The crude material was further purified using column chromatography on SiO.sub.2 with 50-100% EtOAc in heptane as eluent. This gave a pale yellow solid.

Example 2—Synthesis of tert-butyl (4-(2-bromoacetamido)phenethyl)carbamate

(19) ##STR00053##

(20) Tert-butyl (4-aminophenethyl)carbamate (4.5 g, 21.83 mmol, 1.0 eq) was dissolved in DCM (150 mL) and cooled to 0° C. using an ice bath. DMAP (1.6 eq) was added in one portion to the stirring mixture followed by dropwise addition of bromoacetyl bromide (1.2 eq) in 50 mL DCM. The mixture was stirred for 30 minutes at 0° C. and then 1.5 hours at room temperature before it was concentrated in vacuo. The crude material was purified using column chromatography on SiO.sub.2 with 75-100% EtOAc in heptane as eluent. This gave a colorless solid which was used directly in the next example.

Example 3—Synthesis of tert-butyl (4-(2-((2-(bis(pyridin-2-ylmethyl)amino) ethyl)(pyridin-2-ylmethyl)amino)acetamido)phenethyl)carbamate

(21) ##STR00054##

(22) The tert-butyl (4-(2-bromoacetamido)phenethyl)carbamate prepared in Example 2 (3.57 g, 9.99 mmol, 1.0 eq) was dissolved in acetonitrile (350 mL). Potassium iodide (1.0 g, 6 mmol, 0.6 eq) and TEA (13.3 mL, 100 mmol, 10 eq) was then added, followed by N1,N1,N2-tris(pyridin-2-ylmethyl)ethane-1,2-diamine (4.0 g, 12 mmol, 1.2 eq) dissolved in 50 mL acetonitrile. The mixture was heated to reflux and stirred overnight. After cooling the mixture to room temperature, it was filtered on a glass filter to remove inorganic salts. The solution was then concentrated in vacuo to give a red-brown oil. This crude oil was further purified by column chromatography on neutral Al.sub.2O.sub.3 with 0-5% MeOH in DCM as eluent. This gave the title product as a red oil.

(23) .sup.1H NMR (600 MHz, CDCl.sub.3) δ 10.40 (s, 1H), 8.51-8.48 (m, 1H), 8.47-8.44 (m, 2H), 7.59 (d, J=8.4 Hz, 2H), 7.57-7.49 (m, 3H), 7.39 (d, J=7.8 Hz, 2H), 7.14-7.10 (m, 4H), 7.10-7.06 (m, 2H), 3.72 (s, 6H), 3.33 (d, J=6.8 Hz, 2H), 3.27 (s, 2H), 2.81-2.71 (m, 4H), 2.68 (t, J=6.4 Hz, 2H), 1.40 (s, 9H). .sup.13C NMR (151 MHz, CDCl.sub.3) δ 169.92 (s), 159.14 (s), 158.18 (s), 155.93 (s), 149.55 (s), 149.08 (s), 136.94 (s), 136.59 (s), 136.47 (s), 134.40 (s), 129.17 (s), 123.15 (s), 123.11 (s), 122.56 (s), 122.11 (s), 119.99 (s), 61.01 (s), 60.47 (s), 58.70 (s), 52.09 (s), 51.59 (s), 43.50 (s), 41.90 (s), 35.66 (s), 28.48 (s). HRMS: (TOF MS ES+): calculated for C.sub.35H.sub.43N.sub.7O.sub.3 [M+H].sup.+: 610.3505, found 610,3511.

Example 4—Synthesis of tert-butyl (2-(bis(pyridin-2-ylmethyl)amino)ethyl)carbamate

(24) ##STR00055##

(25) Tert-butyl (2-aminoethyl)carbamate (75.0 g, 465.3 mmol, 1.0 eq) was suspended in 1.5 L dest. H.sub.2O. Chloromethyl pyridine hydrochloride (168 g 1.0 mol, 2.2 eq) was then added to the stirring suspension followed by ice cold 5 M NaOH (1.5 L). The suspension became dark red and slightly hot. The mixture was stirred at room temperature overnight. The deep red solution was then extracted with DCM (3×750 mL) and the combined organic phases were dried over K.sub.2CO.sub.3, filtered and concentrated in vacuo to yield a deep red oil which needed no further purification. NMR were in accordance with published data. See Kikuchi et al., Inorg. Chem., 2009, 48 (16), pp 7630-7638.

Example 5—Synthesis of N1,N1-bis(pyridin-2-ylmethyl)ethane-1,2-diamine

(26) ##STR00056##

(27) The tert-butyl (2-(bis(pyridin-2-ylmethyl)amino)ethyl)carbamate prepared in Example 4 was dissolved in 600 mL DCM and cooled to 0° C. in an ice bath. Trifluoroacetic acid (600 mL) was then added slowly to the stirring mixture at 0° C. After complete addition, the mixture was allowed to warm to room temperature and stirred for an additional 18 hours. The mixture was concentrated in vacuo, dissolved in 2M NaOH (500 mL) and extracted three times with DCM (3×500 mL). The combined organic phases were dried over K.sub.2CO.sub.3, filtered and concentrated in vacuo to give 92 grams (379 mmol, 82% yield over two steps) as a deep red oil which needed no further purification. NMR were in accordance with published data. See Kikuchi et al., Inorg. Chem., 2009, 48 (16), pp 7630-7638.

Example 6—Synthesis of N1,N1,N2-tris(pyridin-2-ylmethyl)ethane-1,2-diamine

(28) ##STR00057##

(29) The N1,N1-bis(pyridin-2-ylmethyl)ethane-1,2-diamine prepared in Example 5 (500 mg, 2.06 mmol, 1.0 eq) was dissolved in absolute ethanol (10 mL), followed by about 500 mg 4 Å molecular sieves and 2-pyridinecarboxaldehyde (196 μL, 2.06 mmol, 1.0 eq). The mixture was placed under nitrogen atmosphere and heated to reflux for 90-120 minutes before it was cooled to room temperature. Then NaBH.sub.4 (243 mg, 6.42 mmol) was added with the aid of absolute ethanol (4 mL). The mixture was stirred at room temperature for 16 hours before it was concentrated under reduced pressure. The crude mixture was suspended in DCM (25 mL) and washed with 1M NaOH (3×25 mL). The organic phase was concentrated under reduced pressure and further purified by column chromatography on neutral alumina using 1-5% MeOH in DCM as eluent to afford the titled compound as a pale yellow oil (315 mg, 46%).

(30) 1H NMR (600 MHz, DMSO) δ 8.50-8.42 (m, 3H), 7.77-7.66 (m, 3H), 7.54 (d, J=7.8 Hz, 2H), 7.34 (d, J=7.8 Hz, 1H), 7.25-7.17 (m, 3H), 3.75 (s, 4H), 3.70 (s, 2H), 2.67-2.58 (m, 4H). 13C NMR (151 MHz, DMSO) δ 160.84 (s), 159.88 (s), 149.16 (d, J=3.2 Hz), 136.82 (d, J=12.1 Hz), 123.10 (s), 122.47 (s), 122.20 (s), 122.10 (s), 60.45 (s), 54.92 (s), 54.06 (s), 46.89 (s).

Example 7—Synthesis of N1-(prop-2-yn-1-yl)-N1,N2,N2-tris(pyridin-2-ylmethyl)ethane-1,2-diamine

(31) ##STR00058##

(32) The N1,N1,N2-tris(pyridin-2-ylmethyl)ethane-1,2-diamine prepared in Example 6 (943 mg, 2.83 mmol, 1.0 eq) was dissolved in THF (7 mL), mixed with K.sub.2CO.sub.3 (1.56 g, 11.32 mmol, 4.0 eq) and cooled to 0° C. Propagyl bromide solution (80% in toluene) (3.15 μL, 2.83 mmol, 1.0 eq) was then added and the flask was sealed with a rubber septum. The mixture was stirred for 1 hour at 0° C. followed by 15 hours at room temperature. The mixture was then filtered through a pad of K.sub.2CO.sub.3 with the aid of DCM (100 mL). The liquid was concentrated in vacuo and purified by column chromatography on a neutral Al.sub.2O.sub.3 column using 50-100% DCM in EtOAc as eluent. This afforded the titled compound in 508 mg (48%) as an orange oil.

(33) .sup.1H NMR (400 MHz, DMSO-d6) δ 8.66-8.27 (m, 3H), 7.87-7.58 (m, 3H), 7.49 (d, J=7.8 Hz, 2H), 7.34 (d, J=7.8 Hz, 1H), 7.28-7.15 (m, 3H), 3.75 (s, 4H), 3.68 (s, 2H), 3.32 (d, J=2.1 Hz, 2H), 3.10 (t, J=2.1 Hz, 1H), 2.82-2.55 (m, 4H). .sup.13C NMR (101 MHz, DMSO) δ 159.21, 158.83, 148.68, 136.39, 122.55, 122.52, 122.07, 122.01, 78.93, 75.72, 59.84, 59.45, 51.44, 50.55, 42.02.

Example 8—Synthesis of N-(4-(2-aminoethyl)phenyl)-2-((2-(bis(pyridin-2-yl methyl)amino)ethyl)(pyridin-2-ylmethyl)amino)acetamide

(34) ##STR00059##

(35) The carbamate prepared in Example 3 (135 mg, 0.22 mmol, 1.0 eq) was dissolved in DCM (10 mL) and cooled to 0° C. TFA (1.0 mL, 13.06 mmol, 59 eq) was added dropwise to the stirring solution over 5 minutes. The mixture was allowed to warm to room temperature and was then stirred an additional 2 hours at room temperature. The mixture was then concentrated under reduced pressure, dissolved in DCM (20 mL) and washed with 1M K.sub.2CO.sub.3 (3×20 mL). The organic phase was dried over K.sub.2CO.sub.3, filtered and concentrated under reduced pressure to give 70 mg (62%) of the title compound as a pale green oil.

(36) .sup.1H NMR (600 MHz, CDCl.sub.3) δ 10.39 (s, 1H), 8.54-8.51 (m, 1H), 8.50-8.46 (m, 2H), 7.63-7.59 (m, 2H), 7.59-7.52 (m, 3H), 7.41 (d, J=7.8 Hz, 2H), 7.17-7.07 (m, 6H), 3.75-3.72 (m, 6H), 3.29 (s, 2H), 2.95 (t, J=6.9 Hz, 2H), 2.79 (t, J=6.5 Hz, 2H), 2.74-2.67 (m, 4H), 2.16 (s, 2H). .sup.13C NMR (151 MHz, CDCl3) δ 169.94 (s), 159.27 (s), 158.31 (s), 149.66 (s), 149.18 (s), 136.82 (s), 136.66 (s), 136.54 (s), 135.34 (s), 129.29 (s), 123.23 (s), 123.17 (s), 122.62 (s), 122.18 (s), 119.99 (s), 61.09 (s), 60.59 (s), 58.80 (s), 52.20 (s), 51.68 (s), 43.77 (s), 39.68 (s). HRMS: (TOF MS ES+): Calculated for C.sub.30H.sub.35N.sub.7O [M+H]+: 510.2981, found: 510.2987.

Example 9—Synthesis of N1-(2-(2-(2-ethoxyethoxy)ethoxy)ethyl)-N1,N2,N2-tris(pyridin-2-ylmethyl)ethane-1,2-diamine

(37) ##STR00060##

(38) The amine prepared in Example 6 (568 mg, 1.70 mmol, 1.0 eq) was dissolved in THF (10 mL). K.sub.2CO.sub.3 (470 mg, 3.40 mmol, 2.0 eq) was added along with the tosylate (623 mg, 1.87 mmol, 1.1 eq). The mixture was heated to reflux and stirred for 16 hours. The mixture was then cooled to room temperature, concentrated under reduced pressure and purified by column chromatography on neutral alumina using 0-5% MeOH in DCM as eluent to afford 468 mg (56%) of the titled compound as a pale orange oil.

(39) .sup.1H NMR (400 MHz, DMSO-d6) δ 8.64-8.30 (m, 3H), 7.84-7.59 (m, 3H), 7.59-7.44 (m, 2H), 7.37 (d, J=7.8 Hz, 1H), 7.31-7.08 (m, 3H), 3.87-3.64 (m, 6H), 3.62-3.34 (m, 12H), 2.74-2.54 (m, 6H), 1.14-0.99 (m, 3H). .sup.13C NMR (101 MHz, DMSO) δ 159.81, 159.40, 148.64, 148.49, 136.35, 136.17, 122.46, 121.96, 121.80, 69.81, 69.73, 69.67, 69.16, 68.95, 65.48, 60.58, 60.04, 53.49, 52.22, 51.63, 15.07. HRMS: (ES+): Calculated for C.sub.28H.sub.40N.sub.5O.sub.3 [M+H]+: 493.3126. Found: 494.3127.

Example 10—Synthesis of tert-butyl N-(2-(bis(pyridin-2-ylmethyl)amino)ethyl)-N-(pyridin-2-ylmethyl)glycinate

(40) ##STR00061##

(41) The amine prepared in Example 6 (1 g, 3 mmol, 1.0 eq) was dissolved in MeCN (10 mL) along with NaHCO.sub.3 (0.5 g, 6 mmol, 2 eq). Tert-butyl bromoacetate (0.5 mL, 3.4 mmol, 1.1 eq) was then added and the mixture heated to reflux for 16 hours. The mixture was cooled to room temperature, mixed with Et.sub.2O (10 mL) and filtered. The remaining solids were washed with Et.sub.2O (2×10 mL) and DCM (10 mL). The combined organic phases were concentrated under reduced pressure to give a dark red oil. The crude material was purified using column chromatography by neutral alumina eluting with 1-2.5% MeOH in DCM. This gave 894 mg (67%) of the titled product as a red oil.

(42) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.55-8.43 (m, 3H), 7.60 (td, J=7.7, 1.8 Hz, 2H), 7.55 (td, J=7.7, 1.8 Hz, 1H), 7.49 (d, J=7.8 Hz, 2H), 7.44 (d, J=7.8 Hz, 1H), 7.16-7.05 (m, 3H), 3.89 (s, 2H), 3.80 (s, 4H), 3.28 (s, 2H), 2.88 (dd, J=8.1, 5.9 Hz, 2H), 2.77-2.65 (m, 2H), 1.42 (s, 9H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 170.82, 159.98, 149.13, 136.46, 123.00, 122.95, 121.99, 80.99, 60.91, 60.83, 56.42, 52.86, 52.23, 28.35. HRMS: (ES+): Calculated for C.sub.26H.sub.34N.sub.5O.sub.2 [M+H]+: 448.2707. Found: 448.2706.

Example 11—Synthesis of ethyl N-(2-(bis(pyridin-2-ylmethyl)amino)ethyl)-N-(pyridin-2-ylmethyl)glycinate

(43) ##STR00062##

(44) The amine prepared in Example 6 (2 g, 6 mmol, 1.0 eq) was dissolved in MeCN (20 mL) along with NaHCO.sub.3 (1 g, 12 mmol, 2 eq). Ethyl bromoacetate (1 mL, 6.5 mmol, 1.1 eq) was then added and the mixture heated to reflux for 16 hours. The mixture was cooled to room temperature, mixed with Et.sub.2O (10 mL) and filtered. The remaining solids were washed with Et.sub.2O (2×10 mL) and DCM (10 mL). The combined organic phases were concentrated under reduced pressure to give a dark red oil. The crude material was purified using column chromatography by neutral alumina eluting with 1-5% MeOH in DCM. This gave 768 mg (31%) of the title product as a red oil.

(45) .sup.1H NMR (400 MHz, DMSO-d6) δ 8.65-8.30 (m, 3H), 7.84-7.58 (m, 3H), 7.46 (d, J=7.8 Hz, 2H), 7.36 (d, J=7.8 Hz, 1H), 7.28-7.12 (m, 2H), 4.03 (q, J=7.1 Hz, 2H), 3.79 (s, 2H), 3.71 (s, 4H), 3.35 (app. s, 2H), 2.77 (t, J=6.7 Hz, 2H), 2.57 (t, J=6.7 Hz, 2H), 1.14 (t, J=7.1 Hz, 3H). .sup.13C NMR (101 MHz, DMSO) δ 170.84, 159.31, 159.29, 148.69, 148.61, 136.40, 136.36, 122.56, 122.53, 122.03, 59.91, 59.70, 54.64, 51.80, 51.21, 14.11. HRMS: (APCI+): Calculated for C.sub.24H.sub.30N.sub.5O.sub.2 [M+H]+: 420.2394. Found: 420.2393.

Example 12—Synthesis of methyl 6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinate

(46) ##STR00063##

(47) The nicotinate (1 g, 4.34 mmol, 1.0 eq) was suspended in THF (50 mL). Dipicolylamine (935 μL, 5.21 mmol, 1.2 eq) and DIPEA (1.23 mL, 7.38 mmol, 1.7 eq) was added to the pink mixture. A precipitate began forming on the side of the flask after about 2 hours. The reaction mixture was stirred at room temperature for an additional 22 hours before the slurry was filtered through a plug of celite with the aid of THF. The yellow filtrate was concentrated under reduced pressure to give a pale orange semisolid. This was suspended in Et.sub.2O (50 mL) and filtered through celite with the aid of an additional 25 mL Et.sub.2O. The filtrate was concentrated under reduced pressure to about 30 mL where a white precipitate began to form. The flask was placed in the freezer (−20° C.) to facilitate further precipitation. The crystals formed were filtered off to give 860 mg (57%) the title compound as a white powder. .sup.1H NMR and .sup.13C NMR were in accordance with reported data. See K. J. Humphreys, K. D. Karlin, S. E. Rokita, J. Am. Chem. Soc. 2002, 124, 6009-6019.

Example 13—Synthesis of 6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinic acid

(48) ##STR00064##

(49) The ester prepared in Example 12 (200 mg) was dissolved in MeOH (0.65 mL) and 2M KOH solution (0.45 mL) was added. The mixture was stirred for 2 hours at room temperature before 1M HCl (0.97 mL) was added and the mixture was concentrated under reduced pressure to a sticky solid. The crude material was suspended in MeOH (1.5 mL) and filtered first through a paper filter and then through a syringe filter. The filtrate was concentrated under reduced pressure to give 197 mg (>99%) of the titled compound as a foamy white solid. NMR was in accordance with the reported data (Natsuho Yamamoto et al, J. Med. Chem., (2012), 11013-11021.

Example 14—Synthesis of (6-((bis(pyridin-2-ylmethyl)amino)methyl)pyridin-3-yl) methanol

(50) ##STR00065##

(51) The ester prepared in Example 12 (330 mg, 0.95 mmol, 1.0 eq) was dissolved in absolute ethanol (10 mL) and placed under argon. NaBH.sub.4 pellets (250 mg, 6.95 mmol, 7.0 eq) was added to the stirring mixture and the slurry was heated to 50° C. for 48 hours. The mixture was then quenched by the addition of NH.sub.4Cl solution and concentrated under reduced pressure to give a white sticky solid. The crude material was suspended in 1M K.sub.2CO.sub.3 (25 mL) and extracted with DCM (3×25 mL). The combined organic phases were dried over K.sub.2CO.sub.3, filtered and concentrated under reduced pressure to give 221 mg (69%) of the title product as a pale yellow oil. NMR was in accordance with published data. See K. J. Humphreys, K. D. Karlin, S. E. Rokita, J. Am. Chem. Soc. 2002, 124, 6009-6019.

Example 15—Synthesis of 1-(5-(chloromethyl)pyridin-2-yl)-N,N-bis(pyridin-2-yl methyl)methanamine

(52) ##STR00066##

(53) The alcohol prepared in Example 14 (500 mg, 1.5 mmol, 1.0 eq) was dissolved in CHCl.sub.3 (15 mL) and cooled to 0° C. before SOCl.sub.2 (1 mL, 13.8 mmol, 9 eq) was added dropwise. The mixture was stirred for 30 minutes at 0° C. before it was warmed to room temperature and stirred for an additional 16 hours. The solution was then concentrated under reduced pressure to give a dark green paste. The crude material was dissolved in DCM (25 mL) and washed with 1M K.sub.2CO.sub.3 (3×25 mL). The organic phase was dried over K.sub.2CO.sub.3, filtered and concentrated under reduced pressure. The material was further purified by column chromatography using neutral Al.sub.2O.sub.3 as stationary phase and EtOAc as mobile phase to give 395 mg (74%) the title compound as a pale yellow solid. NMR was in accordance with published data. See K. J. Humphreys, K. D. Karlin, S. E. Rokita, J. Am. Chem. Soc. 2002, 124, 6009-6019.

Example 16—Synthesis of tert-butyl 4-(6-((bis(pyridin-2-ylmethyl)amino)methyl) nicotinamido)phenethylcarbamate

(54) ##STR00067##

(55) The 6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinic acid prepared in Example 13 was dissolved in 20 mL dry DMF at room temperature and filtered into a 50 mL round bottomed flask prior to reaction to remove the insoluble salts. To this solution was added tert-butyl 4-aminophenethylcarbamate (1.76 g, 7.45 mmol, 1.5 eq.), followed by EDCl (1.428 g, 7.45 mmol, 1.5 eq.), HOAt (1.014 g, 7.45 mmol, 1.5 eq.) and NMM (0.821 mL, 7.45 mmol, 1.5 eq.). The reaction mixture was stirred at room temperature for 16 h and then concentrated under reduced pressure. The residual crude mixture was dissolved in 100 mL CHCl.sub.3, transferred into a separation funnel and washed with 100 mL sat. aq. K.sub.2CO.sub.3 solution and 100 mL brine. The organic phase was separated, dried oved Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. Purification of the product was performed by column chromatography on neutral Al.sub.2O.sub.3 using 1% MeOH in DCM giving the product with minor impurities, followed by C18-SPE using gradient elution (10% MeOH to 90% MeOH in H.sub.2O) affording 1.697 g (3.07 mmol, 62%) of the product as a yellow oil.

(56) .sup.1H NMR (300 MHz, MeOH) δ 8.96 (d, J=1.8 Hz, 1H), 8.46-8.39 (m, 2H), 8.23 (dd, J=8.2, 2.3 Hz, 1H), 7.76 (td, J=7.7, 1.5 Hz, 3H), 7.62 (dd, J=13.5, 8.1 Hz, 4H), 7.24 (ddd, J=7.3, 5.0, 1.1 Hz, 2H), 7.17 (d, J=8.5 Hz, 2H), 3.89 (s, 2H), 3.85 (s, 4H), 3.24 (t, J=7.3 Hz, 2H), 2.72 (t, J=7.3 Hz, 2H), 1.40 (s, 9H). .sup.13C NMR (101 MHz, MeOD) δ 166.10, 163.40, 159.85, 158.28, 149.56, 148.92, 138.55, 137.76, 137.41, 137.15, 130.88, 130.16, 124.83, 124.14, 123.81, 122.23, 79.86, 61.10, 60.84, 42.95, 36.59, 28.79. APCI-HRMS e/z calc. for C.sub.32H.sub.36N.sub.6O.sub.3: 552.2849, found: 553.2920 [M+H].

Example 17—Synthesis of N-(4-(2-aminoethyl)phenyl)-6-((bis(pyridin-2-ylmethyl) amino)methyl)nicotinamide

(57) ##STR00068##

(58) The tert-butyl 4-(6-((bis(pyridin-2-ylmethyl)amino)methyl)-nicotinamido) phenethylcarbamate (1.697 g, 3.07 mmol, 1 eq.) prepared in Example 16 was dissolved in 10 mL DCM at room temperature. To this solution was added TFA (5 mL) and the mixture stirred at room temperature until TLC (Al.sub.2O.sub.3, 5% MeOH in DCM) or NMR indicated full conversion. The mixture was then concentrated under reduced pressure, the residue dissolved in a mixture of CHCl.sub.3/dest. H.sub.2O/sat. aq. K.sub.2CO.sub.3 (100 mL/10 mL/100 mL) and transferred into a separation funnel. The organic phase was separated, the aq. phase extracted twice with 50 mL CHCl.sub.3 and the combined organics washed with 100 mL brine, dried over K.sub.2CO.sub.3/Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to afford N-(4-(2-aminoethyl)phenyl)-6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinamide in quantitative yield.

(59) .sup.1H NMR (300 MHz, MeOH) δ 8.97 (d, J=1.7 Hz, 1H), 8.45 (ddd, J=5.0, 1.7, 0.9 Hz, 2H), 8.26 (dd, J=8.2, 2.3 Hz, 1H), 7.80 (td, J=7.6, 1.6 Hz, 3H), 7.68 (d, J=7.8 Hz, 2H), 7.62 (d, J=8.5 Hz, 2H), 7.28 (ddd, J=7.4, 5.0, 1.2 Hz, 2H), 7.23 (d, J=8.5 Hz, 2H), 3.94 (s, J=4.3 Hz, 2H), 3.90 (s, 4H), 2.88 (t, J=6.7 Hz, 2H), 2.76 (t, J=6.9 Hz, 2H). .sup.13C NMR (101 MHz, MeOD) δ 163.55, 159.96, 149.62, 148.93, 138.69, 137.51, 131.02, 130.19, 124.99, 124.32, 123.92, 122.54, 79.46, 61.24, 60.96, 44.03, 39.22. APCI-HRMS e/z calc. for C.sub.27H.sub.28N.sub.6O: 452.2325, found: 453.2396 [M+H].

Example 18—Synthesis of methyl 2-(4-aminophenyl)acetate hydrochloride

(60) ##STR00069##

(61) 2-(4-aminophenyl)acetic acid (5.176 g, 34.2 mmol, 1 eq.) was suspended in 100 mL methanol and cooled to 0° C. in an ice bath. To this mixture SOCl.sub.2 (2.89 mL, 41.07 mmol, 1.2 eq.) was added dropwise over a period of 10 minutes. The mixture was stirred at 0° C. for another 30 minutes, 1 h at room temperature and then refluxed for 14 h. The reaction mixture was then concentrated under reduced pressure to afford the title compound as a pale brown solid in quantitative yield that was taken to the next step without further purification. .sup.1H NMR was in accordance with published data. See Threadgill et al., Bioorganic & Medicinal Chemistry, 2003, 11, 4189-4206.

Example 19—Synthesis of methyl 2-(4-(2-bromoacetamido)phenyl)acetate or methyl 2-(4-(2-chloroacetamido)phenyl)acetate

(62) ##STR00070##

(63) Methyl 2-(4-aminophenyl)acetate hydrochloride prepared in Example 18 (6.89 g, 34.2 mmol, 1 eq.) was suspended in 100 mL DCM and cooled to 0° C. in an ice bath. A solution of DMAP (7.51 g, 61.56 mmol, 1.8 eq.) and NMM (3.75 mL, 34.2 mmol, 1 eq.) in 50 mL DCM was added dropwise over 30 minutes via a dropping funnel, followed by dropwise addition of bromoacetyl bromide or chloroacetyl chloride (1.8 eq.) over 30 minutes at 0° C. The mixture was stirred at 0° C. for another 30 minutes and at room temperature for 12 h. The mixture was then washed with 0.1 M HCl (3×50 mL) and brine (50 mL) and the organic phase dried over anhydrous MgSO.sub.4, filtered and concentrated under reduced pressure. The product was isolated via column chromatography on SiO.sub.2 using isocratic elution with a 3:1 mixture of n-heptane and EtOAc to afford methyl 2-(4-(2-bromoacetamido)phenyl)acetate or methyl 2-(4-(2-chloroacetamido)phenyl)acetate in 82% (Br) or 73% (C1) yield as white solids which could be used directly in the next step. Optional additional purification could be obtained by recrystallization from EtOAc.

Example 20—Synthesis of methyl 2-(4-(2-((2-(bis(pyridin-2-yl methyl)amino)ethyl)(pyridin-2-ylmethyl)amino)acetamido)phenyl)acetate

(64) ##STR00071##

(65) N1,N1,N2-tris(pyridin-2-ylmethyl)ethane-1,2-diamine described in Example 6 (1 eq.) was dissolved in 50 mL CH.sub.3CN at room temperature. To this solution was added K.sub.2CO.sub.3 (4 eq.) and KI (0.6 eq.), followed by methyl 2-(4-(2-bromoacetamido)phenyl)acetate or methyl 2-(4-(2-chloroacetamido)phenyl)acetate prepared in example 18 (1.1 eq.). The mixture was heated to reflux for 16 h until TLC indicated full conversion of the amine. The mixture was then passed through a pad of celite using CH.sub.3CN as eluent and concentrated under reduced pressure. The product was isolated via column chromatography on neutral Al.sub.2O.sub.3 using 1-2% MeOH in DCM as eluent to afford the titled compound in 76% yield as a brown oil. .sup.1H NMR (400 MHz, DMSO-d6) δ 10.29 (s, 1H), 8.52 (dd, J=4.8, 0.8 Hz, 1H), 8.44 (dd, J=4.8, 0.9 Hz, 2H), 7.71 (td, J=7.6, 1.8 Hz, 1H), 7.64 (td, J=7.6, 1.8 Hz, 2H), 7.56 (d, J=8.5 Hz, 2H), 7.41 (d, J=7.8 Hz, 2H), 7.33 (d, J=7.7 Hz, 1H), 7.29-7.23 (m, 1H), 7.22-7.16 (m, 4H), 3.75 (s, 2H), 3.69 (s, 4H), 3.62 (s, 2H), 3.61 (s, 3H), 3.28 (s, 2H), 2.76 (t, J=6.4 Hz, 2H), 2.61 (t, J=6.4 Hz, 2H). .sup.13C NMR (101 MHz, DMSO-d6) δ 171.74, 169.46, 158.98, 158.53, 149.03, 148.72, 137.41, 136.66, 136.35, 129.64, 129.17, 123.07, 122.75, 122.39, 122.05, 119.11, 60.16, 59.65, 58.15, 51.74, 51.65, 51.14, 30.67.

Example 21—Synthesis of 2-(4-(2-((2-(bis(pyridin-2-ylmethyl)amino)ethyl)(pyridin-2-yl methyl)amino)acetamido)phenyl)acetic acid

(66) ##STR00072##

(67) Methyl 2-(4-(2-((2-(bis(pyridin-2-ylmethyl)amino)ethyl)(pyridin-2-ylmethyl)amino)acetamido)phenyl)acetate prepared in example 20 (922 mg, 1.7 mmol, 1 eq.) was dissolved in 10 mL THF at 0° C. To this solution was added LiOH.H.sub.2O (144 mg, 3.4 mmol, 2.0 eq.) in 7 mL dest. H.sub.2O and the solution stirred at 0° C. until TLC indicated full conversion (Al.sub.2O.sub.3, 5% MeOH in DCM). The mixture was then concentrated under reduced pressure to remove the THF, and the residual aq. solution adjusted to pH 7 with 0.5 M HCl. The solvent was removed under reduced pressure to afford the product in quantitative yield. .sup.1H NMR (600 MHz, DMSO) δ 10.20 (s, 1H), 8.51 (dd, J=4.7, 0.7 Hz, 1H), 8.44 (dd, J=4.8, 0.8 Hz, 2H), 7.71 (td, J=7.6, 1.8 Hz, 1H), 7.65 (td, J=7.6, 1.8 Hz, 2H), 7.45 (t, J=9.3 Hz, 2H), 7.42 (t, J=9.4 Hz, 2H), 7.33 (d, J=7.7 Hz, 1H), 7.27-7.23 (m, 1H), 7.23-7.18 (m, 2H), 7.15 (d, J=8.4 Hz, 2H), 3.74 (s, 2H), 3.68 (s, 4H), 3.26 (s, 2H), 3.22 (s, 2H), 2.75 (t, J=6.5 Hz, 2H), 2.60 (t, J=6.5 Hz, 2H). .sup.13C NMR (151 MHz, DMSO) δ 174.40, 169.17, 158.98, 158.57, 149.04, 148.76, 136.69, 136.45, 136.00, 134.36, 129.43, 123.09, 122.79, 122.43, 122.14, 118.65, 60.20, 59.65, 58.18, 51.76, 51.13, 44.97.

Example 22a—Synthesis of (3R,4R,5S,6R)-6-(acetoxymethyl)-3-(2-(4-(2-((2-(bis(pyridin-2-ylmethyl)amino)ethyl)(pyridin-2-ylmethyl)amino)acetamido)phenyl)acetamido) tetrahydro-2H-pyran-2,4,5-triyltriacetate

(68) ##STR00073##

(69) The acid prepared in Example 21 (220.8 mg, 0.442 mmol, 1 eq.) was suspended in 5 mL dry CH.sub.3CN under N.sub.2 atmosphere cooled to 0° C. in ice bath. HATU (168 mg, 0.442 mmol, 1.05 eq.), 1,3,4,6-tetra-O-acetyl-β-D-glucosamine hydrochloride (170 mg, 0.442 mmol, 1.05 eq.) and NMM (139 μL, 3 eq.) was then added to the mixture. The mixture was stirred at 0° C. for 1 h, then at room temperature for 16 h and was concentrated under reduced pressure. The residue was dissolved in a minimum amount of 5% MeOH in DCM and passed through a plug of neutral Al.sub.2O.sub.3 eluting with 10% MeOH in DCM. The resulting solution was concentrated under reduced pressure and the product isolated via column chromatography on neutral Al.sub.2O.sub.3 using 1-5% MeOH in DCM as eluent to afford 168.8 mg (48%) of the product as a yellow foamy semi solid.

(70) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 10.53 (s, 1H), 8.47 (dd, J=5.0, 1.6 Hz, 1H), 8.44 (dd, J=4.9, 0.8 Hz, 2H), 7.62 (d, J=8.5 Hz, 2H), 7.53 (ddd, J=15.9, 7.9, 1.8 Hz, 3H), 7.36 (d, J=7.8 Hz, 2H), 7.08 (ddd, J=8.4, 7.4, 4.7 Hz, 6H), 6.26 (d, J=9.3 Hz, 1H), 5.68 (d, J=8.8 Hz, 1H), 5.16 (dd, J=10.5, 9.4 Hz, 1H), 5.02 (t, J=9.6 Hz, 1H), 4.24-4.11 (m, 2H), 4.04 (dd, J=12.5, 2.2 Hz, 1H), 3.76-3.69 (m, 7H), 3.38 (s, 2H), 3.29 (s, J=10.6 Hz, 2H), 2.81-2.73 (m, 2H), 2.73-2.63 (m, 2H), 2.01 (s, 3H), 1.95 (s, 3H), 1.92 (s, 3H), 1.85 (s, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 171.39, 170.66, 170.64, 170.04, 169.32, 169.23, 158.93, 158.07, 149.52, 149.09, 137.76, 136.63, 136.49, 129.96, 129.50, 123.17, 122.60, 122.17, 120.17, 92.33, 72.77, 72.24, 68.09, 61.74, 60.82, 60.33, 58.70, 52.99, 51.96, 51.44, 43.27, 20.74, 20.71, 20.57, 20.53. APCI-HRMS e/z calc. for C.sub.44H.sub.51N.sub.7O.sub.11: 853.3647, found 854.3726 [M+H].

Example 22b—Synthesis of 2-((2-(bis(pyridin-2-ylmethyl)amino)ethyl)(pyridin-2-ylmethyl)amino)-N-(4-(2-oxo-2-(((3R,4R,5S,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)ethyl)phenyl)acetamide

(71) ##STR00074##

(72) The title compound can be obtained from Example 22a as described in Example 81.

Example 23—Synthesis of (R)-2-((2-(bis(pyridin-2-ylmethyl)amino)ethyl)(pyridin-2-yl methyl)amino)-N-(4-(2-(2,3-dihydroxypropylamino)-2-oxoethyl)phenyl)acetamide

(73) ##STR00075##

(74) The acid prepared in Example 21 (124.3 mg, 0.237 mmol, 1 eq.) was dissolved in 2 mL dry DMF under N2 atmosphere at room temperature. (R)-3-Amino-1,2-propanediol (32 mg, 0.355 mmol, 1.5 eq.), EDCl (68 mg, 0.355 mmol, 1.5 eq.), HOAt (48 mg, 0.355 mmol, 1.5 eq.) and NMM (39 μL, 1.5 eq.) were then added and the mixture was stirred at room temperature for 16 h. The reaction mixture was concentrated under reduced pressure and the product isolated via chromatography on reversed phase silica-C18 using 20-75% MeOH in H.sub.2O affording 61 mg (50%) of the title product as a yellow glassy oil.

(75) .sup.1H NMR (400 MHz, MeOD) δ 8.51 (d, J=4.8 Hz, 1H), 8.43 (d, J=4.9 Hz, 2H), 7.75 (t, J=7.7 Hz, 1H), 7.70 (t, J=7.7 Hz, 2H), 7.56 (dd, J=19.5, 8.1 Hz, 4H), 7.39 (d, J=7.7 Hz, 1H), 7.34-7.22 (m, 5H), 3.81 (s, 2H), 3.77 (s, 4H), 3.75-3.70 (m, 1H), 3.56 (s, 2H), 3.54-3.48 (m, 2H), 3.42 (dd, J=13.8, 4.8 Hz, 1H), 3.33 (s, 2H), 3.26 (dd, J=13.8, 6.7 Hz, 1H), 2.83 (t, J=6.4 Hz, 2H), 2.73 (t, J=6.4 Hz, 2H). .sup.13C NMR (101 MHz, MeOD) δ 174.60, 172.23, 160.20, 159.47, 150.20, 149.50, 138.58, 138.53, 138.15, 132.84, 130.57, 125.00, 124.88, 124.02, 123.79, 121.38, 71.96, 65.05, 62.08, 61.27, 59.78, 53.72, 53.10, 43.56, 43.26. APCI-HRMS e/z calc. for C.sub.33H.sub.39N.sub.7O.sub.4: 597.3064, found 598.3135 [M+H].

Example 24—Synthesis of 2-((2-(bis(pyridin-2-ylmethyl)amino)ethyl)(pyridin-2-yl methyl)amino)-N-(4-(2-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-ylamino)-2-oxoethyl)phenyl)acetamide

(76) ##STR00076##

(77) The acid prepared in Example 21 (185.4 mg, 0.354 mmol, 1 eq.) was dissolved in 3 mL dry DMF under N2 atmosphere at room temperature. Tris-(hydroxymethyl)-aminomethane (Trizma base, 64 mg, 0.53 mmol, 1.5 eq.), EDCl (101 mg, 0.53 mmol, 1.5 eq.), HOAt (72 mg, 0.53 mmol, 1 eq.) and NMM (58 μL, 1.5 eq.) were then added and mixture was stirred at room temperature for 16 h. The reaction mixture was concentrated under reduced pressure and the product isolated via chromatography on reversed phase silica-C18 using 10-90% MeOH in H.sub.2O affording 59.2 mg (27%) of the title product as a yellow glassy oil.

(78) .sup.1H NMR (400 MHz, MeOD) δ 8.49 (d, J=4.4 Hz, 1H), 8.42 (d, J=4.5 Hz, 2H), 7.71 (dt, J=20.2, 7.6 Hz, 3H), 7.59 (d, J=7.7 Hz, 2H), 7.52 (d, J=7.8 Hz, 2H), 7.37 (d, J=7.7 Hz, 1H), 7.32 (d, J=7.9 Hz, 2H), 7.26 (dd, J=13.2, 7.0 Hz, 3H), 3.80 (s, 2H), 3.77 (s, 6H), 3.75 (s, 4H), 3.61 (s, 2H), 3.32 (s, 2H), 2.82 (t, J=6.1 Hz, 2H), 2.71 (t, J=6.1 Hz, 2H). .sup.13C NMR (101 MHz, MeOD) δ 175.00, 172.18, 160.17, 159.43, 150.20, 149.51, 138.56, 138.49, 138.18, 132.73, 130.73, 124.97, 124.86, 123.99, 123.77, 121.36, 63.56, 62.54, 62.06, 61.23, 59.73, 53.65, 53.06, 49.00, 43.74. APCI-HRMS e/z calc. for C.sub.34H.sub.41N.sub.7O.sub.5: 627.3169, found 628.3236 [M+H].

Example 25—Synthesis of 2-(4-(2-((2-(bis(pyridin-2-ylmethyl)amino)ethyl)(pyridin-2-yl methyl)amino)acetamido)phenyl)-N-methyl-N-(2,3,4,5,6-pentahydroxyhexyl)acetamide

(79) ##STR00077##

(80) The acid prepared in Example 21 (203.2 mg, 0.387 mmol, 1 eq.) was dissolved in 2 mL dry DMF under N2 atmosphere at room temperature. N-Methyl-D-glucamine (113 mg, 0.581 mmol, 1.5 eq.), EDCl (111 mg, 0.581 mmol, 1.5 eq.), HOAt (79 mg, 0.581 mmol, 1.5 eq.) and NMM (64 μL, 1.5 eq.) was then added and the mixture stirred at room temperature for 30 min, then heated to 50° C. for 12 h. The reaction mixture was concentrated under reduced pressure and the product isolated via chromatography on reverse phase silica-C18 using 10-90% MeOH in H.sub.2O affording 109.6 mg (40%) of the product as a yellow oil. The product appears as a syn/anti mixture regarding the amide bond.

(81) .sup.1H NMR (600 MHz, MeOD) δ 8.48-8.45 (m, 1H), 8.39 (d, J=4.1 Hz, 2H), 7.71 (td, J=7.7, 1.3 Hz, 1H), 7.67 (tt, J=7.7, 2.1 Hz, 2H), 7.55 (dd, J=11.3, 8.5 Hz, 2H), 7.50 (d, J=8.0 Hz, 2H), 7.35 (d, J=7.8 Hz, 1H), 7.24 (dd, J=14.8, 8.0 Hz, 5H), 4.05-3.98 (m, 1H), 3.94-3.78 (m, 2H), 3.78-3.74 (m, 4H), 3.72 (s, 5H), 3.70-3.60 (m, 4H), 3.44 (ddd, J=8.0, 5.2, 1.4 Hz, 1H), 3.28 (d, J=3.1 Hz, 2H), 3.15, 3.00 (2×s, 3H), 2.79 (t, J=6.5 Hz, 2H), 2.68 (t, J=6.5 Hz, 2H). .sup.13C NMR (201 MHz, MeOD) δ 174.65, 174.54, 172.19, 172.16, 160.14, 159.46, 150.23, 149.54, 138.61, 138.54, 138.03, 137.93, 132.89, 132.22, 130.54, 130.45, 125.00, 124.89, 124.02, 123.81, 121.45, 121.34, 74.09, 73.74, 73.07, 73.01, 72.82, 72.32, 71.54, 71.24, 64.77, 64.74, 62.03, 61.20, 59.69, 54.12, 53.59, 53.03, 52.72, 49.00, 41.07, 40.65, 38.31, 34.81. APCI-HRMS e/z calc. for C.sub.37H.sub.47N.sub.7O.sub.7: 701.3537, found 702.3607 [M+H].

Example 26—Synthesis of 6-((bis(pyridin-2-ylmethyl)amino)methyl)-N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)nicotinamide

(82) ##STR00078##

(83) The 6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinic acid prepared in Example 13 (194 mg, 0.58 mmol, 1 eq.) was dissolved in 5 mL dry DMF at room temperature. N-Methyl-D-glucamine (170 mg, 0.87 mmol, 1.5 eq.), EDCl (167 mg, 0.87 mmol, 1.5 eq.), HOAt (118 mg, 0.87 mmol, 1.5 eq.) and NMM (96 μL, 0.87 mmol, 1.5 eq.) was then added. Upon addition of the HOAt the mixture turned from colorless to yellow. The mixture was heated to 50° C. for 16 h with stirring and then concentrated under reduced pressure. The product was isolated via chromatography on reverse phase silica-C18 using 10-90% MeOH in H.sub.2O affording 99 mg (33%) of product as a yellow glassy oil. The product appears as a syn/anti mixture regarding the amide bond.

(84) .sup.1H NMR (300 MHz, MeOH) δ 8.57 (d, J=11.3 Hz, 1H), 8.44 (d, J=4.7 Hz, 2H), 7.89 (dd, J=20.9, 7.9 Hz, 1H), 7.79 (t, J=7.7 Hz, 2H), 7.72 (d, J=7.6 Hz, 1H), 7.67 (d, J=7.9 Hz, 2H), 7.34-7.22 (m, 2H), 4.08 (dt, J=18.6, 6.6 Hz, 1H), 3.87 (s, J=3.1 Hz, 6H), 3.84-3.46 (m, 7H), 3.14, 3.07 (2×s, 3H). .sup.13C NMR (101 MHz, MeOD) δ 170.57, 169.95, 160.31, 159.73, 158.51, 148.12, 147.03, 146.52, 137.21, 136.23, 135.55, 131.20, 130.93, 123.50, 122.84, 122.60, 122.43, 72.52, 72.02, 71.59, 71.51, 70.97, 70.24, 70.10, 69.65, 63.27, 59.88, 59.73, 59.60, 59.43, 53.78, 51.00, 38.54, 32.37. APCI-HRMS e/z calc. for C.sub.26H.sub.33N.sub.5O.sub.6: 511.2431, found 512.2503 [M+H].

Example 27—Synthesis of N-(4-(2-((2-(bis(pyridin-2-ylmethyl)amino)ethyl)(pyridin-2-yl methyl)amino)acetamido)phenethyl)pyrazine-2-carboxamide

(85) ##STR00079##

(86) The amine prepared in Example 8 (87.7 mg, 0.172 mmol, 1 eq.) was dissolved in 6 mL acetone cooled to 0° C. in an ice bath. To this solution was added 2,3-pyrazinedicarboxylic acid anhydride (25.8 mg, 0.172 mmol, 1 eq.). The yellow solution turned into a suspension and was then stirred room temperature for 10 min. The precipitate was filtered off with suction, washed with acetone, dissolved in methanol and concentrated under reduced pressure to afford 54 mg (51%) of the title product as an orange solid.

(87) .sup.1H NMR (400 MHz, DMSO-d6) δ 10.23 (s, 1H), 8.92 (t, J=5.9 Hz, 1H), 8.80 (d, J=2.4 Hz, 1H), 8.76 (d, J=3.4 Hz, 1H), 8.75 (d, J=2.5 Hz, 1H), 8.51 (d, J=4.0 Hz, 1H), 8.44 (d, J=4.0 Hz, 2H), 7.71 (td, J=7.7, 1.7 Hz, 1H), 7.64 (td, J=7.7, 1.7 Hz, 2H), 7.53 (d, J=8.4 Hz, 2H), 7.42 (t, J=9.2 Hz, 2H), 7.33 (d, J=7.8 Hz, 1H), 7.28-7.15 (m, 5H), 3.76 (s, 2H), 3.72 (s, 4H), 3.49 (dd, J=14.2, 6.5 Hz, 3H), 3.28 (s, 2H), 2.81 (dt, J=13.7, 8.2 Hz, 4H), 2.65 (t, J=6.2 Hz, 2H). .sup.13C NMR (101 MHz, DMSO-d6) δ 169.28, 166.90, 165.62, 163.12, 158.66, 158.42, 149.03, 148.75, 147.71, 146.48, 145.66, 144.90, 143.82, 143.43, 136.84, 136.72, 136.46, 134.16, 128.88, 123.13, 122.87, 122.44, 122.16, 119.27, 60.09, 59.53, 58.08, 54.89, 51.65, 51.16, 48.62, 34.30. APCI-HRMS e/z calc. for C.sub.35H.sub.37N.sub.9O.sub.2: 615.3070, found 616.3141 [M+H].

Example 28—Synthesis of 2-((6-((bis(pyridin-2-ylmethyl)amino)methyl)pyridin-2-yl) methyl)isoindoline-1,3-dione

(88) ##STR00080##

(89) The title compound was prepared according to a slightly modified published literature procedure, see Z. Guo, G.-H. Kim, J. Yoon, I. Shin, Nat. Protocols 2014, 9, 1245-1254. 2-((6-(chloromethyl)pyridin-2-yl)methyl)isoindoline-1,3-dione (0.842 g, 3.16 mmol, 1 eq.) was suspended in 10 mL CH.sub.3CN at room temperature. Bis(2-pyridylmethyl)amine (0.625 mL, 3.48 mmol, 1.1 eq.) and K.sub.2CO.sub.3 (0.917 g, 6.63 mmol, 2.1 eq.) were added and the mixture heated to reflux for 16 h. After cooling to room temperature, the mixture was passed through a plug of celite using CH.sub.3CN as eluent and the solution concentrated under reduced pressure. The product was isolated via column chromatography on neutral Al.sub.2O.sub.3 using 3-5% MeOH in DCM as eluent to afford 952 mg (66%) of a yellow oil that was pure enough for the next step. Further purification from the obtained yellow oil was achieved by addition of Et.sub.2O and precipitation of a pale beige solid (614 mg). The NMR data obtained for the product corresponded to the reported data in the reference.

Example 29—Synthesis of 1-(6-(aminomethyl)pyridin-2-yl)-N,N-bis(pyridin-2-yl methyl)methanamine

(90) ##STR00081##

(91) The title compound was prepared according to a slightly modified published literature procedure from the phthalimide protected amine in example 28, see Z. Guo, G.-H. Kim, J. Yoon, I. Shin, Nat. Protocols 2014, 9, 1245-1254. The 2-((6-((bis(pyridin-2-ylmethyl)amino)methyl)pyridin-2-yl)methyl)isoindoline-1,3-dione prepared in Example 28 (0.499 g, 1.11 mmol, 1 eq.) was dissolved in 10 mL MeOH at room temperature. Hydrazine hydrate solution (35%, 1.11 mL, 12.24 mmol, 11 eq.) was added and the mixture was heated to reflux for 5 h until TLC (Al.sub.2O.sub.3, 3% MeOH in DCM) indicated full conversion of the starting material and a new spot developed that stained with ninhydrine solution. The mixture was concentrated under reduced pressure at 30° C. and the residual white solid dissolved in 150 mL CHCl.sub.3 and washed with 100 mL H.sub.2O. The organic phase was separated and the aqueous phase extracted with CHCl.sub.3 (3×30 mL). The combined organics were washed with 50 mL 1 M NaOH solution and 50 mL brine, dried over anhydrous MgSO.sub.4, filtered and concentrated under reduced pressure at 30° C. to afford a pale yellow oil. The product was isolated via column chromatography on neutral Al.sub.2O.sub.3 using 5-10% MeOH in DCM as eluent to afford 241 mg (68%) of the title product as a pale yellow oil. The product was directly used for the next step without prolonged storage.

(92) .sup.1H NMR (400 MHz, CD.sub.2Cl.sub.2) δ 8.49 (d, J=4.4 Hz, 2H), 7.69-7.55 (m, 5H), 7.41 (d, J=7.7 Hz, 1H), 7.17-7.10 (m, 3H), 3.92 (s, 2H), 3.84 (s, 4H), 3.82 (s, 2H). .sup.13C NMR (101 MHz, CD.sub.2Cl.sub.2) δ 160.25, 159.46, 149.58, 137.39, 136.83, 123.45, 122.48, 121.49, 119.86, 60.77, 60.63, 48.02.

Example 30—Synthesis of methyl 2-(4-(2-((6-((bis(pyridin-2-yl methyl) amino)methyl)pyridin-2-yl)methylamino)acetamido)phenyl)acetate

(93) ##STR00082##

(94) The amine prepared in Example 29 (214 mg, 0.67 mmol, 1 eq.) is dissolved in 10 mL CH.sub.3CN at room temperature. To this mixture is added methyl 2-(4-(2-chloroacetamido)phenyl)acetate prepared in example 19 (170 mg, 0.7 mmol, 1.05 eq.), KI (66.7 mg, 0.402 mmol, 0.6 eq.) and K.sub.2CO.sub.3 (185 mg, 1.34 mmol, 2 eq.). The mixture is heated to reflux for 16 h or until all starting material has been consumed as evident by a TLC analysis. The mixture is then passed through a plug of celite using CH.sub.3CN as solvent. The solution is concentrated under reduced pressure and the product can be isolated via column chromatography on neutral Al.sub.2O.sub.3 or reverse phase C18 silica using an appropriate solvent system.

Example 31—Synthesis of (1-(((3aR,5R,5aS,8aS,8bR)-2,2,7,7-Tetramethyltetrahydro-5H-bis([1,3]dioxolo)[4,5-b:4′,5′-d]pyran-5-yl)methyl)-1H-1,2,3-triazol-4-yl)methanol

(95) ##STR00083##

(96) To a solution of 6-azido-6-deoxy-1,2:3,4-di-O-isopropyliden-α-D-galactose (286 mg, 1.0 mmol) in 3 mL acetonitrile, propargyl alcohol (0.12 mL, 2.1 mmol) was added, followed by copper(I)iodide (40.5 mg, 0.21 mmol). The reaction was stirred over night at room temperature. After removal of solvent, the product was purified on a silica column, eluting with a gradient 3:1 ethyl acetate/heptane to pure ethyl acetate. Fractions containing pure product were collected and the solvent removed under reduced pressure to afford 247.7 mg product (73%) which was used directly in the next example.

Example 32—Synthesis of (1-(((3aR,5R,5aS,8aS,8bR)-2,2,7,7-Tetramethyltetrahydro-5H-bis([1,3]dioxolo)[4,5-b:4′,5′-d]pyran-5-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl methanesulfonate

(97) ##STR00084##

(98) The parent alcohol described in Example 31 (246.3 mg, 0.722 mmol) was dissolved in 5 mL DCM and cooled to 0° C. Triethylamine (0.15 mL, 1.08 mmol) was added, followed by methanesulfonyl chloride (0.07 mL, 0.9 mmol) and the reaction stirred at room temperature for 1 h. The reaction mixture was immediately loaded onto a plug of silica and eluted with ethyl acetate. Removal of solvent gave quantitative conversion to crude product, which was used in the subsequent step (Example 34) without further purification.

Example 33—Synthesis of N.SUP.1.,N.SUP.1.,N.SUP.2.-Tris(pyridin-2-ylmethyl)-N.SUP.2.-((1-(((3aR,5R,5aS,8aS,8bR)-2,2,7,7-tetramethyltetrahydro-5H-bis([1,3]dioxolo)[4,5-b:4′,5′-d]pyran-5-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)ethane-1,2-diamine

(99) ##STR00085##

(100) To an ice cold solution of N.sup.1,N.sup.1,N.sup.2-tris(pyridin-2-ylmethyl)ethane-1,2-diamine described in Example 6 (241.2 mg, 0.723 mmol) in 10 mL acetonitrile, K.sub.2CO.sub.3 (208 mg, 1.50 mmol) was added, and subsequently a solution of (1-(((3aR,5R,5aS,8aS,8bR)-2,2,7,7-tetramethyltetrahydro-5H-bis([1,3]dioxolo)[4,5-b:4′,5′-d]pyran-5-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl methanesulfonate as described in Example 32 (0.722 mmol) in 5 mL acetonitrile was then added dropwise, after which the mixture was allowed to warm to room temperature to further react overnight. After filtration through celite and subsequent removal of solvent under reduced pressure, the crude product was purified on an alumina column, with a 1%-1.5% gradient of methanol in DCM as eluent. Further purification of the resulting brown oil was achieved by way of dry column vacuum chromatography on Bondesil C18-OH material, using a stepwise elution from 30% to 70% methanol in water. Evaporation of solvents gave the product as an orange gum (93.8 mg, 20%).

(101) .sup.1H NMR (400 MHz, methanol-d.sub.4) δ 8.40 (m, 3H), 7.90 (s, 1H), 7.69-7.76 (m, 3H), 7.50-7.56 (m, 3H), 7.24-7.27 (m, 3H), 5.41 (d, J=4.9 Hz, 1H), 4.67 (dd, J=2.5 Hz, 7.9 Hz, 1H), 4.61 (dd, J=3.2 Hz, 14.2 Hz, 1H), 4.46 (dd, J=9.3 Hz, 14.2 Hz, 1H), 4.35 (dd, J=2.5 Hz, 4.9 Hz, 1H), 4.30 (dd, J=1.9 Hz, 7.9 Hz, 1H), 4.19 (ddd, J=1.9 Hz, 3.2 Hz, 9.3 Hz, 1H), 3.76 (s, 6H), 3.68 (s, 2H), 2.66-2.72 (m, 4H), 1.47 (s, 3H), 1.36 (s, 3H), 1.29 (s, 3H), 1.21 (s, 3H). .sup.13C NMR (100 MHz, methanol-d.sub.4) δ 160.7, 149.39, 149.37, 145.3, 138.64, 138.60, 126.0, 124.9, 124.8, 123.73, 123.69, 110.9, 110.0, 97.7, 72.6, 72.2, 71.8, 68.8, 61.5, 60.8, 53.4, 52.7, 51.8, 50.2, 26.3, 26.2, 25.1, 24.6.

Example 34—Synthesis of tert-butyl (4-(2-(4-(bromomethyl)-1H-1,2,3-triazol-1-yl)acetamido)phenethyl)carbamate

(102) ##STR00086##

(103) Tert-butyl (4-(2-bromoacetamido)phenethyl)carbamate (1 eq) is suspended in a mixture of tert-butanol and water or another appropriate solvent mixture, and mixed with potassium iodide (0.1-1.0 eq) and copper sulfate (0.1-1.0 eq). Propagyl bromide solution (1.0-100 eq) is then added to the stirring mixture and it is stirred for 1-72 hours at a temperature between 20-100° C. or until all starting material is consumed as monitored by TLC or HPLC. The reaction mixture is then diluted with water and extracted with an appropriate organic solvent (e.g. DCM) three times. The combined organic phases are dried over MgSO.sub.4, filtered and concentrated in vacuo. If further purification is necessary, the crude material is purified by column chromatography using an appropriate combination of stationary phase and solvent mixture or recrystallization from an appropriate solvent or solvent mixture.

Example 35—Synthesis of tert-butyl (4-(2-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)acetamido)phenethyl)carbamate

(104) ##STR00087##

(105) Tert-butyl (4-(2-bromoacetamido)phenethyl)carbamate as described in Example 2 (1 eq) is suspended in a mixture of tert-butanol and water or another appropriate solvent mixture, and mixed with potassium iodide (0.1-1.0 eq) and copper sulfate (0.1-1.0 eq). Propargyl alcohol solution (1.0-100 eq) is then added to the stirring mixture and it is stirred for 1-72 hours at a temperature between 20-100° C. or until all starting material is consumed as monitored by TLC or HPLC. The reaction mixture is then diluted with water and extracted with an appropriate organic solvent (e.g. DCM) three times. The combined organic phases are dried over MgSO.sub.4, filtered and concentrated in vacuo. If further purification is necessary, the crude material is purified by column chromatography using an appropriate combination of stationary phase and solvent mixture or recrystallization from an appropriate solvent or solvent mixture.

Example 36—Synthesis of tert-butyl (4-(2-(4-((bis(pyridin-2-ylmethyl)amino)methyl)-1H-1,2,3-triazol-1-yl)acetamido)phenethyl)carbamate

(106) ##STR00088##

(107) Tert-butyl (4-(2-azidoacetamido)phenethyl)carbamate as prepared in Example 1 (1 eq) is suspended in a mixture of tert-butanol and water or another appropriate solvent mixture, and mixed with copper sulfate (0.1-1.0 eq). N,N-bis(pyridin-2-ylmethyl)prop-2-yn-1-amine (1.0-100 eq) is then added to the stirring mixture and it is stirred for 1-72 hours at a temperature between 20-100° C. or until all starting material is consumed as monitored by TLC or HPLC. The reaction mixture is then either diluted with water and extracted with an appropriate organic solvent (e.g. DCM) three times, the combined organic phases dried over K.sub.2SO.sub.4, filtered and concentrated in vacuo or directly purified by column chromatography using an appropriate combination of stationary phase and solvent mixture or recrystallization from an appropriate solvent or solvent mixture to give the titled compound.

Example 37—Synthesis of N-(4-(2-aminoethyl)phenyl)-2-(4-((bis(pyridin-2-ylmethyl) amino)methyl)-1H-1,2,3-triazol-1-yl)acetamide

(108) ##STR00089##

(109) The carbamate prepared in Example 36 is dissolved in DCM and cooled to 0° C. Trifluoroacetic acid (5-100 eq) is then added slowly to the stirring mixture and the solution is kept cold using an ice bath. The mixture is left at 0° C. until all trifluoroacetic acid is added, then stirred at room temperature until all starting material is consumed. The mixture is then concentrated in vacuo, dissolved in 2M NaOH and extracted three times with an appropriate solvent (e.g. DCM). The combined organic phases are dried over K.sub.2CO.sub.3, filtered and concentrated in vacuo. If further purification is needed, the material can be subjected to column chromatography using an appropriate combination of stationary phase and eluent, preparative HPLC, recrystallization or combinations thereof.

Example 38—Synthesis of 2-((bis(pyridin-2-ylmethyl)amino)methyl)tetrahydro-2H-pyran-2,3,4,5-tetraol

(110) ##STR00090##

(111) Dipicolylamine (1.0 eq) and D-glucose (1.0-10.0 eq) are suspended in absolute ethanol. Glacial acetic acid (3.0-20.0 eq) is then added and the mixture is stirred at a temperature between room temperature and reflux for 1-72 hours or until all starting material has been consumed. The mixture is then cooled to room temperature and purified by extraction, column chromatography, recrystallization, preparative HPLC or combinations thereof.

Example 39—Synthesis of N-(2-(bis(pyridin-2-ylmethyl)amino)ethyl)-N-(pyridin-2-yl methyl)glycine

(112) ##STR00091##

(113) The ester prepared in Example 10 is dissolved in DCM and stirred rapidly. H.sub.3P04 (85%) is then added and the mixture is stirred at room temperature for 1-72 hours or until all starting material has been consumed. The organic solvent is then removed in vacuo and the aqueous phase is neutralized with NaHCO.sub.3 or K.sub.2CO.sub.3 to pH 7. The solution is then concentrated under reduced pressure, the solids washed with absolute ethanol and filtered to remove inorganic salts. The ethanolic solution is then concentrated under reduced pressure to give the titled acid.

Example 40—Synthesis of N-(2-(bis(pyridin-2-ylmethyl)amino)ethyl)-N-(pyridin-2-yl methyl)glycine

(114) ##STR00092##

(115) The ester prepared in Example 11 is suspended in 2M KOH and is stirred at room temperature for 1-72 hours or until all starting material has been consumed. The aqueous phase is neutralized with 1M HCl until pH 7. The solution is then concentrated under reduced pressure, the solids washed with absolute ethanol and filtered to remove inorganic salts. The ethanolic solution is then concentrated under reduced pressure to give the titled acid.

Example 41—Synthesis of 2-((2-(bis(pyridin-2-ylmethyl)amino)ethyl)(pyridin-2-yl methyl)amino)-N-methyl-N-(2,3,4,5,6-pentahydroxyhexyl)acetamide

(116) ##STR00093##

(117) The acid described in Example 40 is dissolved in an appropriate solvent (e.g. DMF, DCM or EtOH) and cooled to 0° C. before megluamine (1.0-10.0 eq). A coupling agent (e.g. HBTU, HATU, CDI, EDC, DCC, COMU) (1.0-10.0 eq) and additive if necessary (HOAt, Oxyma) (0.5-20.0 eq) is then added. The mixture is stirred for 0-15 minutes before the base (e.g. NMM or DIPEA) (1.0-40.0 eq) is added. The mixture is stirred for 0-72 hours at 0° C. and an additional 1-72 hours at room temperature or until all starting material is consumed. The mixture is then either first diluted with 1M K.sub.2CO.sub.3 and extracted repeatedly with EtOAc, the organic phases pooled, dried over K.sub.2CO.sub.3, filtered and concentrated under reduced pressure, or concentrated under reduced pressure directly. The crude material can then be further purified by column chromatography using an appropriate combination of stationary phase and eluent, preparative HPLC, recrystallization or combinations thereof.

Example 42—Synthesis of 3-((2-(bis(pyridin-2-ylmethyl)amino)ethyl)(pyridin-2-yl methyl)amino)propanoic acid

(118) ##STR00094##

(119) The amine prepared in Example 6 (1.0 eq) is dissolved in an appropriate solvent (e.g. EtOH (abs)) and mixed with 3-bromopropionic acid (1.1-5.0 eq) and TEA (1.2-10.0 eq). The mixture is heated to reflux and stirred for 1-72 hours or until all starting material has been consumed. The mixture is then concentrated under reduced pressure to an absolute minimum amount of solvent, filtered and diluted with Et.sub.2O. The mixture is then filtered again and the remaining solution is concentrated under reduced pressure. The crude material is then purified by column chromatography using an appropriate combination of stationary phase and eluent, preparative HPLC, recrystallization or combinations thereof.

Example 43—Synthesis of 2-((2-(bis(pyridin-2-ylmethyl)amino)ethyl)(pyridin-2-yl methyl)amino)-N-((2S,3R,4R,5S,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide

(120) ##STR00095##

(121) The acid described in Example 40 is dissolved in an appropriate solvent (e.g. DMF, DCM or EtOH) and cooled to 0° C. before D-glucosamine hydrochloride (1.0-10.0 eq) is added. A coupling agent (e.g. HBTU, HATU, CDI, EDC, DCC, COMU) (1.0-10.0 eq) and additive if necessary (HOAt, Oxyma) (0.5-20.0 eq) is then added. The mixture is stirred for 0-15 minutes before the base (e.g. NMM or DIPEA) (1.0-40.0 eq) is added. The mixture is stirred for 0-72 hours at 0° C. and an additional 1-72 hours at room temperature or until all starting material is consumed. The mixture is then either first diluted with 1M K.sub.2CO.sub.3 and extracted repeatedly with EtOAc, the organic phases pooled, dried over K.sub.2CO.sub.3, filtered and concentrated under reduced pressure, or concentrated under reduced pressure directly. The crude material can then be further purified by column chromatography using an appropriate combination of stationary phase and eluent, preparative HPLC, recrystallization or combinations thereof.

Example 44—Synthesis of 3-((2-(bis(pyridin-2-ylmethyl)amino)ethyl)(pyridin-2-yl methyl)amino)-N-((2S,3R,4R,5S,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)propanamide

(122) ##STR00096##

(123) The acid described in Example 42 is dissolved in an appropriate solvent (e.g. DMF, DCM or EtOH) and cooled to 0° C. before D-glucosamine hydrochloride (1.0-10.0 eq) is added. A coupling agent (e.g. HBTU, HATU, CDI, EDC, DCC, COMU) (1.0-10.0 eq) and additive if necessary (HOAt, Oxyma) (0.5-20.0 eq) is then added. The mixture is stirred for 0-15 minutes before the base (e.g. NMM or DIPEA) (1.0-40.0 eq) is added. The mixture is stirred for 0-72 hours at 0° C. and an additional 1-72 hours at room temperature or until all starting material is consumed. The mixture is then either first diluted with 1M K.sub.2CO.sub.3 and extracted repeatedly with EtOAc, the organic phases pooled, dried over K.sub.2CO.sub.3, filtered and concentrated under reduced pressure, or concentrated under reduced pressure directly. The crude material can then be further purified by column chromatography using an appropriate combination of stationary phase and eluent, preparative HPLC, recrystallization or combinations thereof.

Example 45—Synthesis of tert-butyl (4-(((6-((bis(pyridin-2-ylmethyl)amino)methyl) pyridin-3-yl)methyl)amino)phenethyl)carbamate

(124) ##STR00097##

(125) The chloride described in Example 15 (1.0 eq) is dissolved in an appropriate solvent (e.g. MeCN) and mixed with KI (0.1-5.0 eq) and TEA (1.0-20.0 eq). The aniline (0.5-10.0 eq) is then added. The mixture is heated to reflux and stirred for 1-72 hours or until all starting material has been consumed. The mixture is then cooled to room temperature and concentrated under reduced pressure. The crude material is then either suspended in 1M K.sub.2CO.sub.3 and extracted with DCM three times, the organic phases pooled, dried over K.sub.2CO.sub.3, filtered and concentrated under reduced pressure or directly subjected to column chromatography using an appropriate combination of stationary phase and mobile phase (e.g. neutral alumina with 0-5% MeOH in DCM). If further purification is necessary the compound can be subjected to preparative HPLC, recrystallization or combinations thereof.

Example 46—Synthesis of 6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinaldehyde

(126) ##STR00098##

(127) The alcohol prepared in Example 14 is dissolved in EtOAc. A mild oxidating agent (e.g. DMP, IBX or TEMPO/H.sub.2O.sub.2) is then added and the mixture is stirred for 1-72 hours at a temperature between 20° C. and reflux or until all starting material is consumed. The mixture is then cooled to room temperature and then either first diluted with 1M K.sub.2CO.sub.3 and extracted repeatedly with EtOAc, the organic phases are pooled, dried over K.sub.2CO.sub.3, filtered and concentrated under reduced pressure, or concentrated under reduced pressure directly. The crude material can then be further purified by column chromatography using an appropriate combination of stationary phase and eluent, preparative HPLC, recrystallization or combinations thereof.

Example 47—Synthesis of tert-butyl (4-(((6-((bis(pyridin-2-ylmethyl) amino)methyl)pyridin-3-yl)methyl)amino)phenethyl)carbamate

(128) ##STR00099##

(129) The aldehyde described in Example 46 (1.0 eq) is dissolved in absolute ethanol under argon and the aniline (0.5-5.0 eq) is added. 3 Å or 4 Å molecular sieves can be added in order to increase ratio or yield of the imine formation. The mixture is heated to reflux for 1-72 hours or until all starting material has been consumed. The mixture is then cooled to room temperature and NaBH.sub.4 (1-20 eq) is added. The mixture is stirred for 1-72 hours at 20-78° C. before NH.sub.4Cl-solution is added. The mixture is then concentrated under reduced pressure, suspended in 1M K.sub.2CO.sub.3 and extracted with DCM three times. The combined organic phases are dried over K.sub.2CO.sub.3, filtered and concentrated under reduced pressure. The crude material can then be further purified by column chromatography using an appropriate combination of stationary phase and eluent, preparative HPLC, recrystallization or combinations thereof.

Example 48—Synthesis of methyl 2-(4-(((6-((bis(pyridin-2-ylmethyl)amino)methyl) pyridin-3-yl)methyl)amino)phenyl)acetate

(130) ##STR00100##

(131) The chloride described in Example 15 (1.0 eq) is dissolved in an appropriate solvent (e.g. MeCN) and mixed with KI (0.1-5.0 eq) and TEA (1.0-20.0 eq). The aniline (0.5-10.0 eq) is then added. The mixture is heated to reflux and stirred for 1-72 hours or until all starting material has been consumed. The mixture is then cooled to room temperature and concentrated under reduced pressure. The crude material is then either suspended in 1M K.sub.2CO.sub.3 and extracted with DCM three times, the organic phases pooled, dried over K.sub.2CO.sub.3, filtered and concentrated under reduced pressure or directly subjected to column chromatography using an appropriate combination of stationary phase and mobile phase (e.g. neutral alumina with 0-5% MeOH in DCM). If further purification is necessary the compound can be subjected to preparative HPLC, recrystallization or combinations thereof.

Example 49—Synthesis of tert-butyl (4-(2-((6-((bis(pyridin-2-ylmethyl)amino)methyl) pyridin-3-yl)methoxy)acetamido)phenethyl)carbamate

(132) ##STR00101##

(133) The alcohol prepared in Example 14 (1.0 eq) is dissolved in an appropriate solvent (e.g. MeCN) and mixed with KI (0.1-5.0 eq) and a base (e.g. DIPEA) (1.0-20.0 eq). The bromo amide described in Example 2 (0.5-10.0 eq) is then added. The mixture is heated to reflux and stirred for 1-72 hours or until all starting material has been consumed. The mixture is then cooled to room temperature and concentrated under reduced pressure. The crude material is then either suspended in 1M K.sub.2CO.sub.3 and extracted with DCM three times, the organic phases pooled, dried over K.sub.2CO.sub.3, filtered and concentrated under reduced pressure or directly subjected to column chromatography using an appropriate combination of stationary phase and mobile phase (e.g. neutral alumina with 0-5% MeOH in DCM). If further purification is necessary the compound can be subjected to preparative HPLC, recrystallization or combinations thereof.

Example 50—Synthesis of methyl 2-(4-(2-((6-((bis(pyridin-2-ylmethyl)amino)methyl) pyridin-3-yl)methoxy)acetamido)phenyl)acetate

(134) ##STR00102##

(135) The alcohol prepared in Example 14 (1.0 eq) is dissolved in an appropriate solvent (e.g. MeCN) and mixed with KI (0.1-5.0 eq) and a base (e.g. DIPEA) (1.0-20.0 eq). The chloroamide described in example 18 (0.5-10.0 eq) is then added. The mixture is heated to reflux and stirred for 1-72 hours or until all starting material has been consumed. The mixture is then cooled to room temperature and concentrated under reduced pressure. The crude material is then either suspended in 1M K.sub.2CO.sub.3 and extracted with DCM three times, the organic phases pooled, dried over K.sub.2CO.sub.3, filtered and concentrated under reduced pressure or directly subjected to column chromatography using an appropriate combination of stationary phase and mobile phase (e.g. neutral alumina with 0-5% MeOH in DCM). If further purification is necessary the compound can be subjected to preparative HPLC, recrystallization or combinations thereof.

Example 51—Synthesis of 2-(4-(2-((6-((bis(pyridin-2-ylmethyl)amino)methyl)pyridin-3-yl)methoxy)acetamido)phenyl)acetic acid

(136) ##STR00103##

(137) The ester described in Example 50 is dissolved in THF and placed under argon and 2M LiOH solution is added at 0° C. The mixture is stirred for 0-72 hours at 0° C. and 0-72 hours at room temperature before 1M HCl is to neutralize the mixture to pH 7. The mixture is then concentrated under reduced pressure and the crude material is suspended in MeOH and filtered. The filtrate is concentrated under reduced pressure and used directly without any further purification.

Example 52—Synthesis of N-(4-(2-aminoethyl)phenyl)-2-((6-((bis(pyridin-2-yl methyl)amino)methyl)pyridin-3-yl)methoxy)acetamide

(138) ##STR00104##

(139) The carbamate described in Example 49 is dissolved in DCM under argon and cooled to 0° C. TFA (1-200 eq) is then added slowly to the stirring mixture and it is stirred for 0-72 hours at 0° C. and 0-72 hours at room temperature. The mixture is then concentrated under reduced pressure, suspended in 1M K.sub.2CO.sub.3 and extracted three times with DCM. The combined organic phases are dried over K.sub.2CO.sub.3, filtered and concentrated under reduced pressure. The crude material can then be further purified by column chromatography using an appropriate combination of stationary phase and eluent, preparative HPLC, recrystallization or combinations thereof.

Example 53—Synthesis of 2-(4-(2-((6-((bis(pyridin-2-ylmethyl)amino)methyl)pyridin-3-yl)methoxy)acetamido)phenyl)-N-methyl-N-(2,3,4,5,6-pentahydroxyhexyl)acetamide

(140) ##STR00105##

(141) The acid described in Example 51 is dissolved in an appropriate solvent (e.g. DMF, DCM or EtOH) and cooled to 0° C. before megluamine (1.0-10.0 eq) is added. A coupling agent (e.g. HBTU, HATU, CDI, EDC, DCC, COMU) (1.0-10.0 eq) and additive if necessary (HOAt, Oxyma) (0.5-20.0 eq) is then added. The mixture is stirred for 0-15 minutes before the base (e.g. NMM or DIPEA) (1.0-40.0 eq) is added. The mixture is stirred for 0-72 hours at 0° C. and an additional 1-72 hours at room temperature or until all starting material is consumed. The mixture is then either first diluted with 1M K.sub.2CO.sub.3 and extracted repeatedly with EtOAc, the organic phases pooled, dried over K.sub.2CO.sub.3, filtered and concentrated under reduced pressure, or concentrated under reduced pressure directly. The crude material can then be further purified by column chromatography using an appropriate combination of stationary phase and eluent, preparative HPLC, recrystallization or combinations thereof.

Example 54—Synthesis of 2-((6-((bis(pyridin-2-ylmethyl)amino)methyl)pyridin-3-yl) methoxy)-N-(4-(2-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)-2-oxoethyl)phenyl)acetamide

(142) ##STR00106##

(143) The acid described in Example 51 is dissolved in an appropriate solvent (e.g. DMF, DCM or EtOH) and cooled to 0° C. before Tris(hydroxymethyl)aminomethane hydrochloride (1.0-10.0 eq) is added. A coupling agent (e.g. HBTU, HATU, CDI, EDC, DCC, COMU) (1.0-10.0 eq) and additive if necessary (HOAt, Oxyma) (0.5-20.0 eq) is then added. The mixture is stirred for 0-15 minutes before the base (e.g. NMM or DIPEA) (1.0-40.0 eq) is added. The mixture is stirred for 0-72 hours at 0° C. and an additional 1-72 hours at room temperature or until all starting material is consumed. The mixture is then either first diluted with 1M K.sub.2CO.sub.3 and extracted repeatedly with EtOAc, the organic phases pooled, dried over K.sub.2CO.sub.3, filtered and concentrated under reduced pressure, or concentrated under reduced pressure directly. The crude material can then be further purified by column chromatography using an appropriate combination of stationary phase and eluent, preparative HPLC, recrystallization or combinations thereof.

Example 55—Synthesis of Chitosan Functionalized Zinc Chelators by Amide Bond Formation

(144) ##STR00107##

(145) The acid described in Example 13 (1.0-100 eq) is dissolved in an appropriate solvent (e.g. DMF, DCM or EtOH) and cooled to 0° C. a coupling agent (e.g. HBTU, HATU, CDI, EDC, DCC, COMU) (1.0-200.0 eq) an additive if necessary (HOAt, Oxyma) (0.5-400.0 eq) and a base (e.g. NMM or DIPEA) (2.0-800.0 eq) is added. The mixture is stirred for 0-1 hours at 0° C. and an additional 0-1 hours at room temperature before chitosan is added (1.0 eq). The mixture is then stirred at room temperature for 1-72 hours or until all of the starting material has been consumed. The mixture is then concentrated under reduced pressure. The crude material can then be further purified by column chromatography using an appropriate combination of stationary phase and eluent, preparative HPLC, recrystallization or combinations thereof.

Example 56—Synthesis of Chitosan Functionalized Zinc Chelators by Reductive Amination

(146) ##STR00108##

(147) The aldehyde described in Example 46 (1.0-100 eq) is dissolved in an appropriate solvent (e.g. EtOH) and cooled to 0° C. before chitosan is added (1.0 eq). The mixture is then stirred at a temperature between 20-80° C. for 1-72 hours or until all of the starting material has been consumed. The mixture is then cooled to room temperature before NaBH.sub.4 (3-300 eq) is added and the mixture is stirred for an additional 1-72 hours at room temperature. 1M K.sub.2CO.sub.3 or NH.sub.4Cl is then added slowly to quench the reaction. The mixture is then concentrated under reduced pressure. The crude material can then be further purified by column chromatography using an appropriate combination of stationary phase and eluent, preparative HPLC, recrystallization or combinations thereof.

Example 57—Synthesis of Chitosan Functionalized Zinc Chelators by Amide Bond Formation 2

(148) ##STR00109##

(149) The acid described in Example 40 (1.0-100 eq) is dissolved in an appropriate solvent (e.g. DMF, DCM or EtOH) and cooled to 0° C. a coupling agent (e.g. HBTU, HATU, CDI, EDC, DCC, COMU) (1.0-200.0 eq) an additive if necessary (HOAt, Oxyma) (0.5-400.0 eq) and a base (e.g. NMM or DIPEA) (2.0-800.0 eq) is added. The mixture is stirred for 0-1 hours at 0° C. and an additional 0-1 hours at room temperature before chitosan is added (1.0 eq). The mixture is then stirred at room temperature for 1-72 hours or until all of the starting material has been consumed. The mixture is then concentrated under reduced pressure. The crude material can then be further purified by column chromatography using an appropriate combination of stationary phase and eluent, preparative HPLC, recrystallization or combinations thereof.

Example 58—Synthesis of 2-((2-(bis(pyridin-2-ylmethyl)amino)ethyl)(pyridin-2-yl methyl)amino)ethan-1-ol

(150) ##STR00110##

(151) The ester prepared in Example 11 (1.0 eq) is dissolved in an appropriate solvent (e.g. absolute ethanol) and placed under argon. LiAlH.sub.4 pellets or solution (1.0-10.0 eq) is added to the stirring mixture and the slurry is stirred at a temperature between −20-80° C. for 1-78 hours or until all starting material has been consumed. The mixture is then quenched by the addition of NH.sub.4Cl solution and concentrated under reduced pressure. The mixture is then either first diluted with 1M K.sub.2CO.sub.3 and extracted repeatedly with EtOAc, the organic phases pooled, dried over K.sub.2CO.sub.3, filtered and concentrated under reduced pressure, or concentrated under reduced pressure directly. The crude material can then be further purified by column chromatography using an appropriate combination of stationary phase and eluent, preparative HPLC, recrystallization or combinations thereof.

Example 59—Synthesis of 2-((2-(bis(pyridin-2-ylmethyl)amino)ethyl)(pyridin-2-yl methyl)amino)acetaldehyde

(152) ##STR00111##

(153) The alcohol prepared in Example 58 is dissolved in EtOAc. A mild oxidating agent (e.g. DMP, IBX or TEMPO/H.sub.2O.sub.2) is then added and the mixture is stirred for 1-72 hours at a temperature between 20° C. and reflux or until all starting material is consumed. The mixture is then cooled to room temperature and then either first diluted with 1M K.sub.2CO.sub.3 and extracted repeatedly with EtOAc, the organic phases pooled, dried over K.sub.2CO.sub.3, filtered and concentrated under reduced pressure, or concentrated under reduced pressure directly. The crude material can then be further purified by column chromatography using an appropriate combination of stationary phase and eluent, preparative HPLC, recrystallization or combinations thereof.

Example 60—Synthesis of Chitosan Functionalized Zinc Chelators by Reductive Amination 2

(154) ##STR00112##

(155) The aldehyde described in Example 59 (1.0-100 eq) is dissolved in an appropriate solvent (e.g. EtOH) and cooled to 0° C. before chitosan is added (1.0 eq). The mixture is then stirred at a temperature between 20-80° C. for 1-72 hours or until all of the starting material has been consumed. The mixture is then cooled to room temperature before NaBH.sub.4 (3.0-300.0 eq) is added and the mixture is stirred for an additional 1-72 hours at room temperature. 1M K.sub.2CO.sub.3 or NH.sub.4Cl is then added slowly to quench the reaction. The mixture is then concentrated under reduced pressure. The crude material can then be further purified by column chromatography using an appropriate combination of stationary phase and eluent, preparative HPLC, recrystallization or combinations thereof.

Example 61—Synthesis of 2-isothiocyanato-N,N-bis(pyridin-2-ylmethyl)ethan-1-amine

(156) ##STR00113##

(157) The amine prepared in Example 5 is dissolved in an appropriate solvent (e.g. DCM, DMF or MeCN) under argon before CS.sub.2 is added. The mixture is then heated to reflux and stirred for 1-72 hours or until all starting material has been consumed. The mixture is then cooled to room temperature and Pb(NO.sub.3).sub.2 is added. The mixture is stirred at room temperature for 1-72 hours or until full product conversion is observed either by TLC, HPLC, NMR or GC. The mixture is then concentrated under reduced pressure. The crude material can then be further purified by column chromatography using an appropriate combination of stationary phase and eluent, preparative HPLC, recrystallization or combinations thereof.

Example 62—Synthesis of 2-isocyanato-N,N-bis(pyridin-2-ylmethyl)ethan-1-amine

(158) ##STR00114##

(159) The amine prepared in Example 5 is dissolved in an appropriate solvent (e.g. DCM, DMF or MeCN) under argon and cooled to 0° C. before COCl.sub.2 is added. The mixture is then stirred at 0° C. for 0-2 hours before the mixture is then heated to reflux and stirred for 1-72 hours or until all starting material has been consumed. The mixture is then concentrated under reduced pressure. The crude material can then be further purified by column chromatography using an appropriate combination of stationary phase and eluent, preparative HPLC, recrystallization or combinations thereof.

Example 63—Synthesis of methyl 2-(4-(2-((6-((bis(pyridin-2-yl methyl)amino)methyl)pyridin-2-yl)methylamino)acetamido)phenyl)acetate

(160) ##STR00115##

(161) The amine prepared in Example 29 (1 eq.) is dissolved in 10 mL CH.sub.3CN at room temperature. To this mixture is added methyl 2-(4-(2-chloroacetamido)phenyl)acetate prepared in Example 19 (1.05 eq.), KI (0.6 eq.) and K.sub.2CO.sub.3 (2 eq.). The mixture is heated to reflux for 16 h until TLC control reveals full conversion of the amine. The mixture is then passed through a plug of celite using CH.sub.3CN and the solution is concentrated under reduced pressure. The product isolated via column chromatography on an appropriate stationary phase using an appropriate solvent mixture as eluent.

Example 64—Synthesis of 2-(4-(2-((6-((bis(pyridin-2-ylmethyl)amino)methyl)pyridin-2-yl)methylamino)acetamido)phenyl)acetic acid

(162) ##STR00116##

(163) Methyl 2-(4-(2-((6-((bis(pyridin-2-ylmethyl)amino)methyl)pyridin-2-yl)methylamino) acetamido)phenyl)acetate prepared in Example 63 (1 eq.) is dissolved in an appropriate amount of THF cooled to 0° C. in an ice bath. To this solution is added LiOH.H.sub.2O (2.0 eq.) in an appropriate amount of distilled H.sub.2O and the solution is stirred at 0° C. until TLC indicates full conversion. The mixture is then concentrated under reduced pressure to remove the THF, and the residual aq. solution adjusted to pH 7 with 0.5 M HCl. The water is removed under reduced pressure to afford the product acid.

Example 65—Synthesis of 2,2′-((2-(Bis(pyridin-2-ylmethyl)amino)ethyl)azanediyl) diacetic Acid

(164) ##STR00117##

(165) Chloroacetic acid (2 mmol), dissolved in 2 mL water, is neutralized by an equimolar amount of sodium hydroxide in 2 mL water, and N.sup.1,N.sup.1-bis(pyridin-2-ylmethyl)ethane-1,2-diamine as described in Example 5 (1 mmol) is added. After heating to reflux, more sodium hydroxide is added dropwise (2.1 mmol in 2 mL water) and the solution kept on reflux for 1 h. After neutralization with 4M HCl (aq.) and further addition to acidic pH, the compound is purified using a pH-gradient on a strong cation exchange column, or by crystallization from methanol-pyridine mixture.

Example 66—Synthesis of 4-(2-(Bis(pyridin-2-ylmethyl)amino)ethyl)morpholine-2,6-dione

(166) ##STR00118##

(167) 2,2′-((2-(Bis(pyridin-2-ylmethyl)amino)ethyl)azanediyl)diacetic acid (described in Example 65) is heated to reflux in excess acetic anhydride and an appropriate solvent. After removal of volatiles, the crude product is used as is for subsequent steps, or purified by crystallization/precipitation methods from appropriate solvents or solvent mixtures.

Example 67—Synthesis of (2S,3R,4S,5R,6R)-6-((4-(((2-(Bis(pyridin-2-ylmethyl)amino)ethyl)(pyridin-2-ylmethyl)amino)methyl)-1H-1,2,3-triazol-1-yl) methyl)tetrahydro-2H-pyran-2,3,4,5-tetraol

(168) ##STR00119##

(169) Treatment of N.sup.1,N.sup.1,N.sup.2-Tris(pyridin-2-ylmethyl)-N.sup.2-((1-(((3aR,5R,5aS,8aS,8bR)-2,2,7,7-tetramethyltetrahydro-5H-bis([1,3]dioxolo)[4,5-b:4′,5′-d]pyran-5-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)ethane-1,2-diamine as described in Example 33 with 50% trifluoracetic acid in water for 1h-24h affords, after removal of solvents, the title compound. The crude product may be further purified with reversed phase chromatographic methods, or cation exchange materials, or combinations thereof.

Example 68a—Synthesis of N.SUP.1.-methyl-N.SUP.2.,N.SUP.2.-bis(pyridin-2-ylmethyl)-N-((1-(((3aR,5R,5aS,8aS,8bR)-2,2,7,7-tetramethyltetrahydro-5H-bis([1,3]dioxolo)[4,5-b:4′,5′-d]pyran-5-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)ethane-1,2-diamine

(170) ##STR00120##

(171) To a solution of N-methyl-N′,N′-bis(pyridin-2-ylmethyl)ethane-1,2-diamine as described in the literature (G. Berggren et al, Dalton Trans., (2009), 10044-10054) 2 mmol) in 30 mL acetonitrile or other suitable non-nucleophilic solvents, K.sub.2CO.sub.3 (4 mmol) is added and the mixture is then cooled down on an ice bath. A solution of (1-(((3aR,5R,5aS,8aS,8bR)-2,2,7,7-tetramethyltetrahydro-5H-bis([1,3]dioxolo) [4,5-b:4′,5′-d]pyran-5-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl methanesulfonate as described in Example 32 (2 mmol) in the same solvent is then added dropwise, and the mixture is allowed to heat to room temperature to further react for 4h-48h. After filtration and removal of solvent, product may be further purified using either chromatography on alumina, silica or reversed phase materials, and/or with SPE methods, both cationic exchange and reversed phase packing materials.

Example 68b—Synthesis of (2S,3R,4S,5R,6R)-6-((4-(((2-(Bis(pyridin-2-yl methyl)amino)ethyl)(methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)methyl)tetrahydro-2H-pyran-2,3,4,5-tetraol

(172) ##STR00121##

(173) Treatment of the compound as described in Example 68a with 50% trifluoracetic acid in water for 1h-24h affords, after removal of solvents, the title compound. The crude product may be further purified with reversed phase chromatographic methods, or cation exchange materials, or combinations thereof.

Example 69—Synthesis of 2-((2-(bis(pyridin-2-ylmethyl)amino)ethyl)(methyl)amino)-N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)acetamide

(174) ##STR00122##

(175) The title compound was prepared as described below for Example 96e.

Example 70—Synthesis of N.SUP.1.,N.SUP.1.-Bis(pyridin-2-ylmethy)-N.SUP.2.,N.SUP.2.-bis((1-(((3aR,5R,5aS,8aS,8bR)-2,2,7,7-tetramethyltetrahydro-5H-bis([1,3]dioxolo)[4,5-b:4′,5′-d]pyran-5-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)ethane-1,2-diamine

(176) ##STR00123##

(177) To a solution of N.sup.1,N.sup.1-bis(pyridin-2-ylmethyl)ethane-1,2-diamine as described in Example 5 (2 mmol) in 30 mL acetonitrile or other suitable non-nucleophilic solvents, K.sub.2CO.sub.3 (8 mmol) is added and the mixture was then cooled down on an ice bath. A solution of (1-(((3aR,5R,5aS,8aS,8bR)-2,2,7,7-tetramethyltetrahydro-5H-bis([1,3]dioxolo) [4,5-b:4′,5′-d]pyran-5-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl methanesulfonate as described in Example 32 (4 mmol) in the same solvent is then added dropwise, and the mixture is allowed to heat to room temperature to further react for 4h-48h. After filtration and removal of solvent, product may be further purified using either chromatography on alumina, silica or reversed phase materials, and/or with SPE methods, both cationic exchange and reversed phase packing materials.

Example 71—Synthesis of (2S,2'S,3R,3′R,4S,4'S,5R,5′R,6R,6′R)-6,6′-(((((2-(Bis(pyridin-2-ylmethyl)amino)ethyl)azanediyl)bis(methylene))bis(1H-1,2,3-triazole-4,1-diyl))bis(methylene))bis(tetrahydro-2H-pyran-2,3,4,5-tetraol)

(178) ##STR00124##

(179) Treatment of N.sup.1,N.sup.1-Bis(pyridin-2-ylmethyl)-N.sup.2,N.sup.2-bis((1-(((3aR,5R,5aS,8aS,8bR)-2,2,7,7-tetramethyltetrahydro-5H-bis([1,3]dioxolo)[4,5-b:4′,5′-d]pyran-5-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)ethane-1,2-diamine as described in Example 70 with 50% trifluoracetic acid in water for 1h-24h affords, after removal of solvents, the title compound. The crude product may be further purified with reversed phase chromatographic methods, or cation exchange materials, or combinations thereof.

Example 72—Synthesis of Bis-Pentaacetylated Gluconate According to a Published Literature Procedure Described by I.-H. Paik, D. Tapriyal, R. M. Enick, A. D. Hamilton, Angew. Chem. Int. Ed. 2007, 46, 3284-3287

(180) ##STR00125##

(181) A solution of pentaacetylated gluconic acid (2.08 eq.) in anhydrous DCM is treated with EDCl (2.2 eq.) and pyridine (3.4 eq.). N-Boc-serinol (1.0 eq.) is added into the reaction mixture followed by addition of DMAP (catalytic amount). The reaction mixture is stirred for 12 hours and is poured into saturated aq. ammonium chloride solution. The organic phase is separated and extracted with DCM, dried over MgSO.sub.4 and concentrated under reduced pressure to give a sticky solid. Flash column chromatography on silica eluting with 2% methanol/DCM gives N-Boc-protected bis-acetylated gluconate ester as a colorless dense oil.

Example 73—Boc-Deprotection of Bis-Pentaacetylated Gluconate According to a Published Literature Procedure Described by I.-H. Paik, D. Tapriyal, R. M. Enick, A. D. Hamilton, Angew. Chem. Int. Ed. 2007, 46, 3284-3287

(182) ##STR00126##

(183) To a solution of N-Boc-protected bis-acetylated gluconate ester prepared in Example 72 in anhydrous dichloromethane is added large excess trifluoroacetic acid (50-100 eq.) slowly dropwise at room temperature. The reaction mixture is stirred for 3 hours and concentrated under reduced pressure and completely dried under high vacuum to afford the deprotected bis-pentaacetylated gluconate amine.

Example 74—General Procedure for the Coupling of Chelator-Linker-Acid or Chelator-Acids to the Bis-Pentaacetylated Gluconate Amine

(184) ##STR00127##

(185) The chelator-linker acid or chelator-acid prepared in e.g. Example 13 or 21 (1 eq.) is dissolved in an appropriate solvent (e.g., but not limited to DMF, CH.sub.3CN, EtOAc, CH.sub.2Cl.sub.2). To this mixture is added the coupling reagent (e.g., but not limited to HATU, COMU, PyBOP, EDCl), (1-1.5 eq.), optional activator (e.g., but not limited to HOAt, HOBt), (1-1.5 eq.) and appropriate non-nucleophilic base (e.g., but not limited to NMM, DIPEA), (2-3 eq.) at a temperature between 0° C. and room temperature. The amine in Example 73 (1-1.2 eq.) is added. Alternatively, an activated acid can be employed (1 eq.) and the amine in example 73 (1-1.2 eq.) and base (1-1.2 eq.) are added to the mixture. The reaction mixture is stirred at a temperature between 0° C. and reflux until full conversion of the acid or active ester indicated by TLC. The solvent is removed under reduced pressure and the product isolated via column chromatography on an appropriate stationary phase using an appropriate solvent mixture as eluent.

Example 75—General Procedure for the Synthesis of Alkyladenosines with Halo Chelators According to an Appropriately Modified Literature Procedure Described by V. E. Oslovsky, M. S. Drenichev, S. N. Mikhailov, Nucleosides, Nucleotides and Nucleic Acids 2015, 34, 475-499

(186) ##STR00128##

(187) To a solution of triacetyladenosine (1 eq.) in DMF barium carbonate (2.5 eq.), potassium iodide (2 eq.) and the chelator-chloride prepared in Example 15 (2 eq.) are added, and the mixture is stirred at 65° C. for 24 hours. Then the mixture is cooled to room temperature, diluted with ethyl acetate and filtered through celite from BaCO.sub.3. Celite is washed with ethyl acetate and the combined filtrate evaporated in vacuum to the volume ca. 2 mL. Then 25% aqueous ammonia is added, and the mixture is left to stay at r.t. during a week. The mixture is evaporated under reduced pressure, evaporated with EtOH, and the product is isolated via column chromatography, prep. HLPC, crystallization or distillation using the appropriate conditions.

Example 76—Synthesis of Amides from N2-(1-Carboxyethyl)-Guanosine 5′-Monophosphate According to an Appropriately Modified Literature Procedure Described in D. Festring, A. Brockhoff, W. Meyerhof, T. Hofmann, J. Agric. Food Chem. 2011, 59, 8875-8885

(188) ##STR00129##

(189) A mixture of guanosine 5′-monophosphate (1 eq.) and 1,3-dihydroxyacetone dimer (1.5 eq.)) in phosphate buffer (1 mol/L, pH 7.0; 5 mL) is heated for 24 h at 70° C. The crude mixture is diluted with water and purified via column chromatography, prep. HLPC, crystallization or distillation using the appropriate conditions. A solution of the carboxyethyl (1 eq.), EDCl (5 eq.) and the chelator amine, e.g. the amine prepared in Example 29 (20 eq.) in H.sub.2O or an alternative appropriate solvent is adjusted to pH 5 with 1M HCl or 1M NaOH. The mixture stirs at room temperature maintaining pH 5 until full conversion is detected. The product is isolated and purified via column chromatography, prep. HLPC, crystallization or distillation using the appropriate conditions.

Example 77—General Procedure for the N-Acylation of Guanosine with Activated Chelator Acid According to an Appropriately Modified Literature Procedure, See: Y. Fan, B. L. Gaffney, R. A. Jones, Org. Lett. 2004, 6, 2555-2557

(190) ##STR00130##

(191) To dried guanosine hydrate (1 eq.) in dry dichloromethane under N.sub.2 cooled in an ice bath, TMSCl (9 eq.) is added over 2 min, and the mixture allowed to stir for 2 h at room temperature. The flask is then cooled again in an ice bath, and the activated chelator acid, e.g. the activated version of the acid described in example 13 (1.1 eq.) dissolved in an appropriate solvent is added over 10 min. The mixture is stirred at a temperature between 0° C. and room temperature until full conversion (TLC, Ninhydrine). Methanol in excess is then added, and the solution is stirred at room temperature until complete desilylation. The mixture is evaporated, and the product is isolated via column chromatography, prep. HLPC, crystallization or distillation using the appropriate conditions.

Example 78—General Procedure for the Synthesis of 5′ Esterificated Nucleosides Using the Chelator-Acids According to Appropriately Adopted Literature Procedures, See: W. Wei, W.-K. Shi, P.-F. Wang, X.-T. Zeng, P. Li, J.-R. Zhang, Q. Li, Z.-P. Tang, J. Peng, L.-Z. Wu, M.-Q. Xie, C. Liu, X.-H. Li, Y.-C. Wang, Z.-P. Xiao, H.-L. Zhu, Bioorg. Med. Chem. 2015, 23, 6602-6611; E. Hernández-Vázquez, V. Chagoya, Med. Chem. Res. 2014, 24, 2325-2335

(192) ##STR00131##

(193) To a suspension of nucleoside (1 eq.) and p-toluensulfonic acid (1.1 eq.) in dry acetone, 2,2-dimethoxypropane (4 eq.) is added. After 3 days of vigorous stirring, the solution is neutralized with a saturated solution of Na.sub.2CO.sub.3 and then extracted with chloroform. The organic layers are joined and dried using Na.sub.2SO.sub.4. The solvent is removed under reduced pressure and the remainder solid is suspended with ether and washed with cold water obtaining a crude product that is purified and isolated via column chromatography, prep. HLPC, crystallization or distillation using the appropriate conditions. The 2′,3′-O-isopropylidenadenosine prepared above (1 eq.) and the chelator-acid, e.g. the one described in Example 13, (1 eq.) are dissolved in an appropriate dry solvent (e.g., but not limited to DMF, CH.sub.3CN, EtOAc, CH.sub.2C2) at room temperature. To this mixture is added the coupling reagent (e.g., but not limited to HATU, COMU, PyBOP, EDCl), (1-1.5 eq.), optional activator (e.g., but not limited to HOAt, HOBt), (1-1.5 eq.) and appropriate non-nucleophilic base (e.g., but not limited to NMM, DIPEA), (2-3 eq.) at a temperature between 0° C. and room temperature. The mixture is stirred for 1 h-20 h until full conversion and worked up by extraction, celite filtration, or direct concentration under reduced pressure, followed by purification and isolation via column chromatography, prep. HLPC, crystallization or distillation using the appropriate conditions.

Example 79—General Procedure for the Synthesis of Chelator Functionalized Guanosine Triphosphates According to an Appropriately Modified Literature Procedure, See: S. Masuda, T. Tomohiro, S. Yamaguchi, S. Morimoto, Y. Hatanaka, Bioorg. Med. Chem. Lett. 2015, 25, 1675-1678

(194) ##STR00132##

(195) A solution of the chelator-amine described in Example 8 in water (50 mM) or a mixture of water and e.g. methanol, DMF, acetonitrile, THF is added to a buffered solution (pH 6.8) of ATP (50 mM). An aqueous solution of EDCl and a buffered solution (pH 6.8) of NEt3 (600 mM) are added at room temperature and the mixture is stirred for 1-8 h. The mixture is then concentrated under reduced pressure and the product isolated and purified via column chromatography using the appropriate stationary phase and eluent, prep. HPLC, crystallization or distillation.

Example 80—General Procedure for the Synthesis of Chelator Functionalized Adenosine Monophosphates According to an Appropriately Modified Literature Procedure, See: H. Fu, B. Han, Y.-F. Zhao, Chem. Commun. 2003, 134-135

(196) ##STR00133##

(197) Under nitrogen atmosphere at room temperature trimethylsilyl chloride (10 eq.) is added dropwise to a mixture of ADP (adenosine 5A-diphosphate disodium salt) and a chelator-amine, e.g. the amine described in example 29 (2 eq.) in pyridine and stirred for two days. The solvent is removed under reduced pressure, the residue hydrolyzed in 2 M NH.sub.3 (aq), and extracted with diethyl ether four times, and the remaining solution is evaporated to dryness. The product is isolated via column chromatography, prep. HLPC, crystallization or distillation using the appropriate conditions.

Example 81—General Procedure for the Acetate-Deprotection of the Coupled Products Prepared in the Previous Example According to an Appropriately Modified Literature Procedure, See: L. Zeng, G. Xu, P. Gao, M. Zhang, H. Li, J. Zhang, Eur. J. Med. Chem. 2015,93, 109-120

(198) ##STR00134##

(199) CH.sub.3ONa (15 eq.) is added to a solution of the bis-pentaacetylated gluconate amide prepared in example 74 (1 eq.) in methanol at 0° C. The mixture was stirred at 0° C. for 4 h and its pH was adjusted to 7 with 4 mol/L HCl in EtOAc. The reaction mixture was subject to evaporation to remove solvent and purified and isolated via column chromatography, prep. HLPC, crystallization or distillation using the appropriate conditions.

Example 82—General Procedure for the Reaction of Poly-Phosphateguanidine Salts Linked to a Chelator-Linker-Alcohol Using an Appropriately Modified Literature Procedure, See: C. J. McKinlay, R. M. Waymouth, P. A. Wender, J. Am. Chem. Soc. 2016, 138, 3510-3517

(200) ##STR00135##

Example 83a—1-(pyridin-2-yl)-N-(pyridin-2-ylmethyl)-N-((1-(((3aR,5R,5aS,8aS,8bR)-2,2,7,7-tetramethyltetrahydro-5H-bis([1,3]dioxolo)[4,5-b:4′,5′-d]pyran-5-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)methanamine

(201) ##STR00136##

(202) The title compound was prepared by reacting the product from Example 32 with di-(2-pyridyl-methyl)amine (DPA) as described in Example 68a.

Example 83b—(3R,4S,5R,6R)-6-((4-((bis(pyridin-2-ylmethyl)amino)methyl)-1H-1,2,3-triazol-1-yl)methyl)tetrahydro-2H-pyran-2,3,4,5-tetraol

(203) ##STR00137##

(204) The title compound was prepared by deprotecting the product from Example 83a as described in Example 68b.

Example 84—(3R,4R,5S,6R)-3-(4-((bis(pyridin-2-ylmethyl)amino)methyl)-1H-1,2,3-triazol-1-yl)-6-(hydroxymethyl)tetrahydro-2H-pyran-2,4,5-triol

(205) ##STR00138##

(206) The title compound is prepared from commercially available 2-Azido-2-deoxy-D-glucose (712795 Aldrich) using a modification of the procedure in Example 83.

Example 85—6,6′,6″-(nitrilotris(methylene))tris(N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)nicotinamide)

(207) ##STR00139##

(208) The title compound can be prepared using modifications of the methods described in Example 106.

Examples 86-94

(209) The compounds given in Schemes 10-20 above can be prepared using modifications of the methods described above.

Example 95a—tert-Butyl(4-(4-((bis(pyridin-2-ylmethyl)amino)methyl)-1H-1,2,3-triazol-1-yl)phenethyl)carbamate

(210) ##STR00140##

(211) Sodium ascorbate (10.15 g, 51.24 mmol) and CuSO.sub.4.5H.sub.2O (6.40 g, 25.62 mmol) was mixed rapidly in 60 mL water, and the resulting yellow solution transferred to a flask containing N-propargyl-di(2-picolyl)amine (6.08 g, 25.62 mmol), prepared as described by Simmons et al., Inorg. Chem., 2013, 52, 5838-5850, dissolved in 60 mL tert-butanol. After rapid stirring, the resulting green solution was transferred to a flask containing tert-butyl 4-azidophenethylcarbamate (5.60 g, 21.35 mmol), prepared according to Murai et al., Eur. J. Org. Chem., 2013, 2428-2433, and allowed to stir for 15 h. The reaction mixture was diluted with ethyl acetate, and washed with 1:1 water/brine. The water phase was then alkalized to pH 9-10 and extracted with ethyl acetate. Combined organic extracts was washed with a 0.025 M EDTA/0.5 M NaHCO.sub.3 mixture and dried over sodium sulfate. Filtration and subsequent removal of solvent under reduced pressure gave a crude product as a brown oil which could be purified on neutral alumina, gradient 0-1% methanol in dichloromethane, giving the product as a thick orange oil. Purification was performed on three combined batches of different sizes, with a combined yield of 14.09 g (73%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.73 (s, 1H), 8.51 (m, 2H), 7.83-7.77 (m, 4H), 7.63 (d, J=7.9 Hz, 2H), 7.41 (m, 2H), 7.27 (m, 2H), 6.92 (br t, NH, 1H), 3.89 (s, 2H), 3.85 (s, 4H), 3.19 (m, 2H), 2.78 (t, J=7.3 Hz, 2H), 1.37 (s, 9H). MS (ESI, positive mode) m/z 500.5 [M+H].sup.+.

Example 95b—2-(4-(4-((Bis(pyridin-2-ylmethyl)amino)methyl)-1H-1,2,3-triazol-1-yl)phenyl)ethan-1-amine

(212) ##STR00141##

(213) To a solution of tert-butyl (4-(4-((bis(pyridin-2-ylmethyl)amino)methyl)-1H-1,2,3-triazol-1-yl)phenethyl)carbamate (12.70 g, 25.4 mmol) in 96 mL dioxane, 4 M HCl in dioxane (46 mL) was added slowly. After stirring under Ar overnight, the volatiles were removed under reduced pressure. The crude product was redissolved in 50 mL saturated NaHCO.sub.3 solution (aq.) and extracted with 250 mL dichloromethane. This first extract contained product in a rather low purity. Repeated extractions with 100 mL dichloromethane (>10 repetitions) afforded, after removal of solvent under reduced pressure, pure product as an orange, thick oil (8.98 g, 88.5%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.70 (s, 1H), 8.50 (m, 2H), 7.82-7.75 (m, 4H), 7.63 (d, J=7.6 Hz, 2H), 7.42 (m, 2H), 7.25 (m, 2H), 3.86 (s, 2H), 3.82 (s, 4H), 2.83 (m, 2H), 2.73 (m, 2H), 1.74 (br, 2H). MS (ESI, positive mode) m/z 400.4 [M+H].sup.+.

Example 96—2-(4-(4-((bis(pyridin-2-ylmethyl)amino)methyl)-1H-1,2,3-triazol-1-yl)phenyl)-N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)acetamide

Example 96a—Ethyl 2-(4-(4-((bis(pyridin-2-ylmethyl)amino)methyl)-1H-1,2,3-triazol-1-yl)phenyl)acetate

(214) ##STR00142##

(215) To a solution of ethyl 4-azidophenylacetate (5.00 g, 24.4 mmol), prepared according to Rao et al.,.sup.3 in 58 mL acetonitrile, propargyl alcohol (2.73 g, 48.7 mmol) and copper(I)iodide (0.928 g, 4.87 mmol) was added. After stirring for 24 h, volatiles were removed under reduced pressure and the crude product purified using column chromatography on silica, using a gradient of ethyl acetate in dichloromethane. Removal of solvent gave the product as a yellow solid (6.14 g, 96%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.66 (s, 1H), 7.86 (m, 2H), 7.48 (m, 2H), 5.33 (t, J=5.6 Hz, 1H), 4.61 (d, J=5.6 Hz, 2H), 4.11 (q, J=7.12 Hz, 2H), 3.78 (s, 2H), 1.21 (t, J=7.1 Hz, 3H). MS (ESI, positive mode) m/z 262.3 [M+H]+.

Example 96b—Ethyl 2-(4-{4-[(methanesulfonyloxy)methyl]-1H-1,2,3-triazol-1-yl}phenyl)acetate

(216) ##STR00143##

(217) Ethyl 2-(4-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)phenyl)acetate (6.100 g, 23.35 mmol) was dissolved in 58 mL dichloromethane and kept on an ice bath. Triethylamine (3.544 g, 35.02 mol) was added, followed by methanesulfonyl chloride (3.209 g, 28.02 mmol). The reaction mixture was kept stirring on ice bath for 3 h, after which volatiles were removed under reduced pressure. Purification of the crude product on a silica column, using a gradient of 5%-10% ethyl acetate in dichloromethane, gave, after removal of solvent, the product as a colourless solid (5.11 g, 64%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.99 (s, 1H), 7.87 (m, 2H), 7.51 (m, 2H), 5.44 (s, 2H), 4.11 (q, J=7.12 Hz, 2H), 3.79 (s, 2H), 3.29 (s, 3H), 1.21 (t, J=7.1 Hz, 3H).

Example 96c—Ethyl 2-[4-(4-{[bis(pyridin-2-ylmethyl)amino]methyl}-1H-1,2,3-triazol-1-yl)phenyl]acetate

(218) ##STR00144##

(219) Di-(2-picolyl)amine (2.83 g, 14.2 mmol) was dissolved in acetonitrile. Potassium carbonate (3.93 g, 28.4 mmol) was added and the mixture was cooled down on an ice bath. Ethyl 2-(4-{4-[(methanesulfonyloxy)methyl]-1H-1,2,3-triazol-1-yl}phenyl)acetate (4.82 g, 14.2 mmol), dissolved in acetonitrile (total volume of acetonitrile in the reaction was 310 mL), was subsequently added dropwise, and after removal of the ice bath, allowed to react at room temperature for 12 hours. Filtration through celite, and with subsequent wash of the celite with dichloromethane, gave, after removal of solvents, a crude product as a dark orange thick oil. Purification on a silica column, using a gradient of methanol in dichloromethane, with additional ammonia added to the mobile phase, gave the product as a dark orange oil. Purification was performed on three combined batches of different sizes, with a combined yield of 6.02 g (80%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.75 (s, 1H), 8.50 (m, 2H), 7.87 (m, 2H), 7.78 (td, J=7.6 Hz, 1.9 Hz, 2H), 7.63 (d, J=7.9 Hz, 2H), 7.49 (m, 2H), 7.25 (m, 2H), 4.11 (q, J=7.1 Hz, 2H), 3.86 (s, 2H), 3.82 (s, 4H), 3.78 (s, 2H), 1.20 (t, J=7.1 Hz, 3H). MS (ESI, positive mode) m/z 443.2 [M+H]+.

Example 96d 2-(4-(4-((bis(pyridin-2-ylmethyl)amino)methyl)-1H-1,2,3-triazol-1-yl) phenyl)acetic acid

(220) ##STR00145##

(221) Ethyl 2-(4-(4-((bis(pyridin-2-ylmethyl)amino)methyl)-1H-1,2,3-triazol-1-yl)phenyl)acetate (323.1 mg, 0.73 mmol, 1.0 eq.) from Example 96a was dissolved in 10 mL THF and cooled to 0° C. in an ice bath. A solution of LiOH hydrate (61 mg, 1.46 mmol, 2.0 eq.) in 5 mL dest. H.sub.2O was added and the solution stirred at 0° C. until TLC (Alumina, 5% MeOH/CH.sub.2Cl.sub.2) indicated full conversion. The THF was removed under reduced pressure and the residual aqueous solution was adjusted to pH=6 using 4 N HCl. The solvent was removed under reduced pressure affording the product in quantitative yield, used in the next step without further purification.

Example 96e—2-(4-(4-((bis(pyridin-2-ylmethyl)amino)methyl)-1H-1,2,3-triazol-1-yl) phenyl)-N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)acetamide

(222) ##STR00146##

(223) 2-(4-(4-((bis(pyridin-2-ylmethyl)amino)methyl)-1H-1,2,3-triazol-1-yl)phenyl) acetic acid from example 96d (302 mg, 0.73 mmol, 1.0 eq.) was dissolved in 3 mL dry DMF at room temperature. N-Methyl-D-Glucamine (213 mg, 1.059 mmol, 1.5 eq.), EDCl (209.9 mg, 1.095 mmol, 1.5 eq.), HOAt (149 mg, 1.095 mmol, 1.5 eq.) and NMM (120 μL, 1.095 mmol, 1.5 eq.) were then added. The mixture was heated to 50° C. for 16 h with stirring and then concentrated under reduced pressure. Purification of the product was achieved by way of dry column vacuum chromatography on C18 material, using a stepwise elution from 10% to 90% methanol in water affording 321.1 mg (0.542 mmol, 74%) of product as a pale yellow foam. The product appears as a syn/anti mixture regarding the amide bond. .sup.1H NMR (400 MHz, MeOD) δ 8.50-8.39 (m, 3H), 7.78 (dd, J=13.8, 7.2 Hz, 4H), 7.69 (d, J=7.8 Hz, 2H), 7.47 (d, J=8.0 Hz, 2H), 7.32-7.20 (m, 2H), 4.08-3.96 (m, 2H), 3.93 (s, 2H), 3.87 (s, 5H), 3.83-3.57 (m, 6H), 3.46 (dd, J=13.3, 6.5 Hz, 1H), 3.19, 3.02 (2×s, 3H). .sup.13C NMR (101 MHz, MeOD) δ 174.01, 173.91, 160.20, 149.49, 146.06, 138.69, 138.29, 137.62, 137.13, 137.04, 131.81, 131.70, 124.99, 123.82, 123.51, 121.66, 121.54, 74.11, 73.69, 73.11, 73.04, 72.76, 72.22, 71.61, 71.24, 64.77, 64.75, 60.63, 54.09, 52.73, 40.92, 40.49, 38.25, 34.79. APCI-HRMS e/z calc. for C.sub.3H.sub.37N.sub.7O.sub.6: 591.2805, found: 592.2878 [M+H].

Example 97—(R)-6-((bis(pyridin-2-ylmethyl)amino)methyl)-N-(2,3-dihydroxypropyl)nicotinamide

(224) ##STR00147##

(225) The 6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinic acid from example 13 (135.7 mg, 0.406 mmol, 1.0 eq.) was dissolved in 3 mL dry DMF at room temperature. (R)-3-aminopropane-1,2-diol (55 mg, 0.609 mmol, 1.5 eq.), EDCl (117 mg, 0.609 mmol, 1.5 eq.), HOAt (83 mg, 0.609 mmol, 1.5 eq.) and NMM (67 μL, 0.609 mmol, 1.5 eq.) were then added and the mixture stirred at room temperature for 16 h. The reaction mixture was concentrated under reduced pressure and the purification of the product was achieved by way of dry column vacuum chromatography on C18 material, using a stepwise elution from 10% to 90% methanol in water affording 61 mg (0.162 mmol, 40%) of product. .sup.1H NMR (300 MHz, MeOH) δ 8.88 (dd, J=2.2, 0.6 Hz, 1H), 8.44 (ddd, J=5.0, 1.6, 0.9 Hz, 2H), 8.17 (dd, J=8.2, 2.3 Hz, 1H), 7.78 (ddd, J=11.4, 8.7, 5.2 Hz, 3H), 7.66 (d, J=7.8 Hz, 2H), 7.27 (ddd, J=7.4, 5.0, 1.2 Hz, 2H), 3.91 (s, 2H), 3.88 (s, 4H), 3.86-3.78 (m, 1H), 3.61-3.51 (m, 3H), 3.41 (dd, J=13.7, 7.0 Hz, 1H). .sup.13C NMR (101 MHz, MeOD) δ 168.32, 163.39, 159.94, 149.59, 148.72, 138.68, 137.26, 130.28, 124.97, 124.30, 123.90, 71.95, 65.22, 61.25, 60.95, 44.12. APCI-HRMS e/z calc. for C.sub.22H.sub.25N.sub.5O.sub.3: 407.1957, found 408.2029 [M+H].

Example 98—2-(4-(4-((bis(pyridin-2-ylmethyl)amino)methyl)-1H-1,2,3-triazol-1-yl)phenyl)-N-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)acetamide

(226) ##STR00148##

(227) 2-(4-(4-((bis(pyridin-2-ylmethyl)amino)methyl)-1H-1,2,3-triazol-1-yl)phenyl)acetic acid from example 96d (132.5 mg, 0.3199 mmol, 1.0 eq.) was dissolved in 2 mL dry DMF at room temperature. 2-amino-2-(hydroxymethyl)propane-1,3-diol (58 mg, 0.479 mmol, 1.5 eq.), EDCl (91.6 mg, 0.479 mmol, 1.5 eq.), HOAt (65 mg, 0.479 mmol, 1.5 eq.) and NMM (53 μL, 0.479 mmol, 1.5 eq.) were then added. The mixture was stirred at room temperature for 16 h and then concentrated under reduced pressure. Purification of the product was achieved by way of dry column vacuum chromatography on C18 material, using a stepwise elution from 10% to 90% methanol in water affording 76.3 mg (0.147 mmol, 46%) of product. .sup.1H NMR (400 MHz, MeOD) δ 8.45 (s, 1H), 8.43 (d, J=3.7 Hz, 2H), 7.78 (t, J=7.9 Hz, 4H), 7.68 (d, J=7.8 Hz, 2H), 7.51 (d, J=7.8 Hz, 2H), 7.25 (t, J=5.7 Hz, 2H), 3.92 (s, 2H), 3.86 (s, 4H), 3.75 (s, 6H), 3.68 (s, 2H). .sup.13C NMR (101 MHz, MeOD) δ 174.24, 160.15, 149.47, 146.05, 138.67, 138.03, 137.17, 131.78, 124.96, 123.81, 123.47, 121.54, 63.68, 62.49, 60.62, 49.96, 43.54. APCI-HRMS e/z calc. for C.sub.27H.sub.31N.sub.7O.sub.4: 517.2438, found: 518.2509 [M+H].

Example 99—6-((bis(pyridin-2-ylmethyl)amino)methyl)-N-(4-(2-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-2-oxoethyl)phenyl)nicotinamide

Example 99a—Methyl 2-(4-(6-((bis(pyridin-2-ylmethyl)amino)methyl) nicotinamido)phenyl)acetate

(228) ##STR00149##

(229) The 6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinic acid from example 13 (976.3 mg, 2.92 mmol, 1.0 eq.) was dissolved in 20 mL dry DMF, cooled to 0° C. in an ice bath. Methyl 2-(4-aminophenyl)acetate hydrochloride (883 mg, 4.38 mmol, 1.5 eq.), EDCl (839 mg, 4.38 mmol, 1.5 eq.) and HOAt (596 mg, 4.38 mmol, 1.5 eq.) were added, followed by NMM (740 μL, 6.71 mmol, 2.3 eq.) dropwise over a period of 30 min at 0° C. The mixture was allowed to warm to room temperature and stirred for 16 h and was concentrated under reduced pressure. The residue was dissolved in 100 mL CHCl.sub.3 and transferred into a separation funnel. The organic phase was washed with a mixture of sat. aq. K.sub.2CO.sub.3 solution and H2O (50 mL each) followed by brine (50 mL). The organic phase was separated, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure and the product purified by dry column vacuum chromatography on C18 material, using a stepwise elution from 10% to 90% methanol in water affording 723.9 mg (1.5 mmol, 51%) of product as a yellow oil. .sup.1H NMR (200 MHz, MeOD) δ 8.97 (d, J=2.0 Hz, 1H), 8.49-8.41 (m, 2H), 8.27 (dd, J=8.2, 2.3 Hz, 1H), 7.81 (td, J=7.5, 1.8 Hz, 3H), 7.73-7.61 (m, 4H), 7.28 (ddd, J=6.5, 4.8, 2.3 Hz, 4H), 3.94 (s, 2H), 3.90 (s, 4H), 3.68 (s, 3H), 3.65 (s, 2H). .sup.13C NMR (101 MHz, MeOD) δ 173.93, 166.05, 161.46, 157.71, 157.66, 149.15, 148.63, 140.22, 138.46, 137.69, 132.12, 131.28, 130.80, 125.48, 124.72, 124.52, 122.21, 60.53, 60.17, 52.47, 41.08. APCI-HRMS e/z calc. for C.sub.28H.sub.27N.sub.3O.sub.3: 481.2114, found: 482.2184 [M+H].

Example 99b—2-(4-(6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinamido) phenyl)acetic acid

(230) ##STR00150##

(231) Methyl 2-(4-(6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinamido)phenyl)acetate from example 99a (287.7 mg, 0.59 mmol, 1.0 eq.) was dissolved in 10 mL THF and cooled to 0° C. in an ice bath. A solution of LiOH hydrate (49.5 mg, 1.18 mmol, 2.0 eq.) in 10 mL dest. H.sub.2O was added and the solution stirred at 0° C. until TLC (Alumina, 5% MeOH in CH.sub.2CO.sub.2) indicated full conversion. The THF was removed under reduced pressure and the residual aqueous solution was adjusted to pH=6 using 4 N HCl. The solvent was removed under reduced pressure affording the product in quantitative yield which was used in the next step without further purification.

Example 99d—6-((bis(pyridin-2-ylmethyl)amino)methyl)-N-(4-(2-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-2-oxoethyl)phenyl)nicotinamide

(232) ##STR00151##

(233) The 2-(4-(6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinamido)phenyl)acetic acid from example 99b (276 mg, 0.59 mmol, 1.0 eq.) was dissolved in 2.5 mL dry DMF at room temperature. N-Methyl-D-Glucamine (173 mg, 0.885 mmol, 1.5 eq.), EDCl (169 mg, 0.885 mmol, 1.5 eq.), HOAt (120 mg, 0.885 mmol, 1.5 eq.) and NMM (98 μL, 0.885 mmol, 1.5 eq.) were then added. The mixture was heated to 50° C. for 16 h with stirring and then concentrated under reduced pressure. Purification of the product was achieved by way of dry column vacuum chromatography on C18 material, using a stepwise elution from 10% to 90% methanol in water affording 207.4 mg (0.322 mmol, 54%) of product as a white foam. The product appears as a syn/anti mixture regarding the amide bond. .sup.1H NMR (400 MHz, MeOD) δ 9.00-8.93 (m, 1H), 8.44 (dd, J=4.9, 0.7 Hz, 2H), 8.25 (dd, J=8.2, 1.7 Hz, 1H), 7.79 (td, J=7.7, 1.8 Hz, 3H), 7.65 (dd, J=16.4, 7.7 Hz, 4H), 7.27 (dd, J=9.5, 4.5 Hz, 4H), 4.06-3.97 (m, 1H), 3.93 (s, 2H), 3.89 (s, 4H), 3.82-3.58 (m, 7H), 3.43 (ddd, J=7.9, 5.1, 1.4 Hz, 1H), 3.15, 3.00 (2×s, 3H). .sup.13C NMR (101 MHz, MeOD) δ 174.60, 174.50, 166.30, 163.54, 163.52, 159.93, 149.60, 148.94, 138.66, 138.32, 138.22, 137.51, 133.40, 132.71, 130.98, 130.55, 130.45, 124.95, 124.28, 123.89, 122.40, 122.30, 74.12, 73.78, 73.09, 73.04, 72.80, 72.33, 71.56, 71.24, 64.76, 64.74, 61.21, 60.94, 54.14, 52.71, 41.09, 40.66, 38.29, 34.82. ESI-HRMS e/z calc. for C.sub.34H.sub.40N.sub.6O.sub.7: 644.2958, found: 707.2166 [(M−H)Zn].sup.+.

Example 100—6-((bis(pyridin-2-ylmethyl)amino)methyl)-N-(4-(2-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-ylamino)-2-oxoethyl)phenyl)nicotinamide

(234) ##STR00152##

(235) The 2-(4-(6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinamido)phenyl)acetic acid from Example 99b (99.5 mg, 0.213 mmol, 1.0 eq.) was dissolved in 3 mL dry DMF at room temperature. 2-amino-2-(hydroxymethyl)propane-1,3-diol (38.7 mg, 0.3195 mmol, 1.5 eq.), EDCl (61 mg, 0.3195 mmol, 1.5 eq.), HOAt (43 mg, 0.3195 mmol, 1.5 eq.) and NMM (35 μL, 0.3195 mmol, 1.5 eq.) were then added. The mixture was stirred at room temperature for 16 h and then concentrated under reduced pressure. Purification of the product was achieved by way of dry column vacuum chromatography on C18 material, using a stepwise elution from 10% to 90% methanol in water affording 91.4 mg (0.16 mmol, 75%) of product. .sup.1H NMR (400 MHz, MeOD) δ 8.97 (s, 1H), 8.45 (s, 2H), 8.26 (d, J=8.2 Hz, 1H), 7.79 (t, J=7.8 Hz, 3H), 7.66 (t, J=8.2 Hz, 4H), 7.30 (dd, J=13.2, 6.6 Hz, 4H), 3.94 (s, 2H), 3.89 (s, 4H), 3.72 (s, 6H), 3.58 (s, 2H). .sup.13C NMR (101 MHz, MeOD) δ 174.99, 166.34, 163.56, 159.94, 149.61, 148.94, 138.69, 138.50, 137.52, 133.28, 131.00, 130.75, 124.97, 124.31, 123.91, 122.33, 63.58, 62.51, 61.23, 60.95, 43.73. APCI-HRMS e/z calc. for C.sub.31H.sub.34N.sub.6O.sub.5: 570.2591, found: 571.2664 [M+H].

Example 101—(2S,3R,4S,5R,6S)—N-(4-(4-((Bis(pyridin-2-ylmethyl)amino)methyl)-1H-1,2,3-triazol-1-yl)phenethyl)-3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2-carboxamide

Example 101a—(3aR,5S,5aR,8aS,8bR)—N-(4-(4-((Bis(pyridin-2-ylmethyl)amino)methyl)-1H-1,2,3-triazol-1-yl)phenethyl)-2,2,7,7-tetramethyltetrahydro-5H-bis([1,3]dioxolo)[4,5-b:4′,5′-d]pyran-5-carboxamide

(236) ##STR00153##

(237) To an ice-cooled flask under nitrogen, containing 2-(4-(4-((Bis(pyridin-2-ylmethyl)amino)methyl)-1H-1,2,3-triazol-1-yl)phenyl)ethan-1-amine (207.5 mg, 0.519 mmol) from Example 95b 1,2:3,4-di-O-isopropylidene-αiD-galactouronide (141.3 mg, 0.515 mmol) and HATU (201.5 mg, 0.530 mmol), dissolved in 3 mL DMF, N-methylmorpholine (0.07 mL, 0.6 mmol) was added. After stirring for 30 min, the flask was left at room temperature and stirred overnight. Solvent was removed under reduced pressure and the crude product loaded onto a plug of Bondesil C-18 OH SPE material. The product was eluted using portion-wise additions of methanol/water mixtures, ranging from 50% to 75% methanol. Pure fractions were collected and removal of solvent gave the title compound as a pale yellow film (195.1 mg, 57.8%). .sup.1H NMR (400 MHz, methanol-d.sub.4) δ (app. dt, J=1.8 Hz, 7.8 Hz, 2H), 7.75 (m, 2H), 7.70 (d, J=7.8 Hz, 2H), 7.46 (m, 2H), 7.27 (m, 2H), 5.61 (d, J=4.9 Hz, 1H), 4.69 (dd, J=7.8 Hz, 2.5 Hz, 1H), 4.58 (dd, J=7.8 Hz, 2.1 Hz, 1H), 4.41 (dd, J=4.9 Hz, 2.5 Hz, 1H), 4.23 (d, J=2.1 Hz, 1H), 3.93 (s, 2H), 3.87 (s, 4H), 3.57 (m, 1H), 3.47 (m, 1H), 2.91 (app. t, J=7.2 Hz, 2H), 1.47 (s, 3H), 1.38 (s, 3H), 1.34 (s, 3H), 1.32 (s, 3H). .sup.13C NMR (100 MHz, methanol-d.sub.4) δ 136.8, 131.4, 125.0, 123.9, 123.5, 121.5, 110.6, 110.3, 97.8, 73.0, 72.1, 72.0, 70.0, 60.6, 50.0, 41.3, 36.0, 26.34, 26.26, 25.0, 24.5. MS (APCI, positive mode) m/z 656.3 [M+H].sup.+, HR-MS (APCI, pos. mode) m/z 656.3191 calculated for C.sub.35H.sub.42N.sub.7O.sub.6, found m/z 656.3189.

Example 101b—(2S,3R,4S,5R,6S)—N-(4-(4-((Bis(pyridin-2-ylmethyl)amino)methyl)-1H-1,2,3-triazol-1-yl)phenethyl)-3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2-carboxamide

(238) ##STR00154##

(239) From the doubly isopropylidene protected sugar in Example 101a (186.6 mg, 0.285 mmol), the deprotected product was obtained by stirring in room temperature with 20 mL trifluoroacetic acid and 15 mL water for 4 h. The volatile materials were removed after repeated additions of 10 mL toluene followed by evaporation on a rotary evaporator between each addition. The crude product was loaded onto a small SPE cartridge with C18 material and eluted with a methanol in water gradient, going from 25% to 45%. Only limited retention was observed on this material. Final purification was achieved after removal of eluent and re-loading the material onto a cartridge containing strong cationic exchange SPE material, using 1:1 methanol/water and a pH-gradient from pH 3 to pH 10 with formic acid and ammonia as additives to elute the product. The product eluted at pH 9, and removal of solvent under reduced pressure afforded the title compound as a colourless oil or foam (128.9 mg, 78.6%). The NMR spectra have a very complex appearance overall due to presence of a and R anomers, and also additional minor peaks, likely from furanose forms. For 6H below 6 ppm, peaks from the sugar moiety are reported with shift only. In the multiplicity edited HSQC spectrum, 20 methine resonances, some of which are partly overlapping, are clearly visible. However, we have only reported 6c data for resonances clearly visible in the 1D spectrum. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.46-8.43 (m, 3H), 7.80 (app. dt, J=1.8 Hz, 7.7 Hz, 2H), 7.75 (m, 2H), 7.70 (d, J=7.8 Hz, 2H), 7.47 (m, 2H), 7.28 (m, 2H), 5.24, 5.14, 4.49, 4.41, 4.36, 4.21, 4.15, 4.10, 4.08, 4.02, 3.94 (s, 2H), 3.91, 3.87 (s, 4H), 3.83, 3.76, 3.53 (m, 2.8H (methylene overlapping with sugar resonances), 2.92 (m, 2H). .sup.13C NMR (100 MHz, DMSO-d.sub.6) δ 174.8, 172.1, 171.3, 160.2, 149.5, 145.94, 145.93, 141.74, 141.69, 138.8, 136.8, 131.44, 131.43, 131.36, 131.34, 125.0, 123.9, 123.5, 121.66, 121.64, 121.62, 98.7, 94.4, 76.6, 74.7, 73.2, 72.4, 71.6, 70.9, 70.0, 60.6, 50.0, 41.47, 41.44, 41.37, 36.1, 36.0.

(240) Compound 102 given in Schemes 10-20 above can be prepared using modifications of the methods described above.

Example 103—6-((bis(pyridin-2-ylmethyl)amino)methyl)-N-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)nicotinamide

(241) ##STR00155##

(242) The 6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinic acid from Example 13 (121 mg, 0.363 mmol, 1.0 eq.) was dissolved in 3 mL dry DMF at room temperature. 2-amino-2-(hydroxymethyl)propane-1,3-diol (66 mg, 0.545 mmol, 1.5 eq.), EDCl (104 mg, 0.545 mmol, 1.5 eq.), HOAt (74 mg, 0.545 mmol, 1.5 eq.) and NMM (60 μL, 0.545 mmol, 1.5 eq.) were added and the mixture stirred at room temperature for 16 h. The reaction mixture was concentrated under reduced pressure and the purification of the product was achieved by way of dry column vacuum chromatography on C18 material, using a stepwise elution from 10% to 90% methanol in water affording 97.2 mg (0.222 mmol, 61%) of product. .sup.1H NMR (300 MHz, MeOH) δ 8.92-8.81 (m, 1H), 8.44 (ddd, J=5.0, 1.6, 0.8 Hz, 2H), 8.15 (dd, J=8.2, 2.3 Hz, 1H), 7.78 (ddd, J=14.2, 10.1, 5.0 Hz, 3H), 7.66 (d, J=7.8 Hz, 2H), 7.27 (ddd, J=7.4, 5.0, 1.2 Hz, 2H), 3.90 (s, 2H), 3.87 (s, 6H), 3.87 (s, 4H). .sup.13C NMR (101 MHz, MeOD) δ 168.80, 163.25, 159.93, 149.59, 148.78, 138.67, 137.36, 131.11, 124.94, 124.16, 123.89, 64.23, 62.38, 61.15, 60.89. APCI-HRMS e/z calc. for C.sub.23H.sub.27N.sub.5O.sub.4: 437.2063, found 438.2134 [M+H].

Example 104—(S)-2-(4-(4-((bis(pyridin-2-ylmethyl)amino)methyl)-1H-1,2,3-triazol-1-yl)phenyl)-N-(2,3-dihydroxypropyl)acetamide

(243) ##STR00156##

(244) 2-(4-(4-((bis(pyridin-2-ylmethyl)amino)methyl)-1H-1,2,3-triazol-1-yl)phenyl) acetic acid from Example 96d (152 mg, 0.367 mmol, 1.0 eq.) was dissolved in 2 mL dry DMF at room temperature. (R)-3-aminopropane-1,2-diol (50 mg, 0.55 mmol, 1.5 eq.), EDCl (105 mg, 0.55 mmol, 1.5 eq.), HOAt (75 mg, 0.55 mmol, 1.5 eq.) and NMM (60 μL, 0.55 mmol, 1.5 eq.) were then added. The mixture was stirred at room temperature for 16 h and then concentrated under reduced pressure. Purification of the product was achieved by way of dry column vacuum chromatography on C18 material, using a stepwise elution from 10% to 90% methanol in water affording 126.8 mg (0.26 mmol, 71%) of product. .sup.1H NMR (300 MHz, MeOH) δ 8.46 (s, 1H), 8.43 (ddd, J=5.0, 1.6, 0.8 Hz, 2H), 7.84-7.71 (m, 4H), 7.69 (t, J=7.7 Hz, 2H), 7.49 (d, J=8.6 Hz, 2H), 7.25 (ddd, J=7.3, 5.0, 1.2 Hz, 2H), 3.92 (s, 2H), 3.86 (s, 4H), 3.80-3.66 (m, 1H), 3.62 (s, 2H), 3.50 (d, J=0.6 Hz, 1H), 3.48 (d, J=1.5 Hz, 1H), 3.40 (dd, J=13.8, 4.8 Hz, 1H), 3.24 (dd, J=13.8, 6.8 Hz, 1H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 173.80, 160.12, 149.46, 146.03, 138.67, 138.03, 137.16, 131.60, 124.94, 123.80, 123.45, 121.53, 71.93, 65.07, 60.58, 43.62, 43.10. APCI-HRMS e/z calc. for C.sub.26H.sub.29N.sub.7O.sub.3: 487.2332, found: 488.2403 [M+H].

Example 105—(6-((bis(pyridin-2-ylmethyl)amino)methyl)pyridin-3-yl)(piperazin-1-yl)methanone

Example 105a—tert-Butyl 4-(6-((bis(pyridin-2-ylmethyl)amino)methyl) nicotinoyl)piperazine-1-carboxylate

(245) ##STR00157##

(246) The 6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinic acid from Example 13 (179.5 mg, 0.537 mmol, 1.0 eq.) was dissolved in 3 mL dry DMF at room temperature. Tert-butyl piperazine-1-carboxylate (150 mg, 0.806 mmol, 1.5 eq.), EDCl (134 mg, 0.806 mmol, 1.5 eq.), HOAt (109.6 mg, 0.806 mmol, 1.5 eq.) and NMM (89 μL, 0.806 mmol, 1.5 eq.) were added and the mixture was stirred at room temperature for 16 h. The reaction mixture was concentrated under reduced pressure and the purification of the product was achieved by way of dry column vacuum chromatography on C18 material, using a stepwise elution from 10% to 90% methanol in water affording 244 mg (0.486 mmol, 91%) of product. .sup.1H NMR (300 MHz, MeOH) δ 8.52 (dd, J=2.1, 0.7 Hz, 1H), 8.44 (ddd, J=4.9, 1.6, 0.9 Hz, 2H), 7.80 (ddd, J=9.4, 7.9, 2.0 Hz, 3H), 7.74 (d, J=7.9 Hz, 1H), 7.68 (d, J=7.8 Hz, 2H), 7.27 (ddd, J=7.4, 5.0, 1.2 Hz, 2H), 3.91 (s, 2H), 3.89 (s, 4H), 3.82-3.62 (m, 2H), 3.46 (s, 6H), 1.46 (s, 9H). .sup.13C NMR (101 MHz, MeOD) δ 169.81, 162.16, 159.99, 156.19, 149.59, 148.15, 138.64, 137.18, 131.40, 124.97, 124.38, 123.87, 81.71, 61.32, 61.02, 28.59. APCI-HRMS e/z calc. for C.sub.28H.sub.34N.sub.6O.sub.3: 502.2692, found 503.2765 [M+H].

Example 105b—(6-((bis(pyridin-2-ylmethyl)amino)methyl)pyridin-3-yl)(piperazin-1-yl) methanone

(247) ##STR00158##

(248) The tert-butyl 4-(6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinoyl)piperazine-1-carboxylate prepared in the previous reaction (233.5 mg, 0.465 mmol, 1.0 eq.) was dissolved in 10 mL CH.sub.2C2 at room temperature. To this solution was added TFA (2.84 mL, 80 eq.) and the mixture stirred at room temperature until NMR indicated full conversion. The mixture was concentrated under reduced pressure, the residue dissolved in dest. H.sub.2O, neutralized with sat. aq. K.sub.2CO.sub.3 solution and concentrated under reduced pressure. Purification of the product was achieved by way of dry column vacuum chromatography on C18 material, using a stepwise elution from 10% to 90% methanol in water affording 79.6 mg (0.35 mmol, 76%) of product. .sup.1H NMR (300 MHz, MeOH) δ 8.50 (dd, J=2.1, 0.7 Hz, 1H), 8.44 (ddd, J=5.0, 1.7, 0.9 Hz, 2H), 7.79 (ddd, J=9.6, 6.2, 2.3 Hz, 3H), 7.75-7.70 (m, 1H), 7.67 (d, J=7.8 Hz, 2H), 7.27 (ddd, J=7.4, 5.0, 1.2 Hz, 2H), 3.90 (s, 2H), 3.89 (s, 4H), 3.72 (s, J=15.5 Hz, 2H), 3.40 (s, J=18.3 Hz, 2H), 2.84 (d, J=17.0 Hz, 4H). .sup.13C NMR (101 MHz, MeOD) δ 169.56, 162.02, 160.00, 149.58, 148.01, 138.64, 137.10, 131.57, 124.98, 124.41, 123.87, 61.36, 61.06, 45.67, 44.01. APCI-HRMS e/z calc. for C.sub.23H.sub.26N.sub.6O: 402.2168, found 403.2240 [M+H].

Example 106-6,6′,6″-nitrilotris(methylene)tris(N—((S)-2,3-dihydroxypropyl) nicotinamide)

(249) ##STR00159##

Example 106a—6,6′,6″-nitrilotris(methylene)tris(N—((S)-2,3-dihydroxypropyl) nicotinamide)

(250) ##STR00160##

(251) Commercially available trimethyl 6,6′,6″-nitrilotris(methylene)trinicotinate (0.222 g, 0.478 mmol, 1.0 eq.) was dissolved in a mixture of 10 mL THF and 10 mL H2O at room temperature. To this solution was added LiOH.H2O (0.2 g, 4.87 mmol, 10.0 eq.) and the reaction progress monitored by TLC on alumina using 5% MeOH in CH2Cl2. Upon full conversion, the crude reaction mixture was concentrated under reduced pressure and the residue dissolved in 5 mL dest. H2O. The pH of the basic solution was adjusted to 6 with 2M HCl and the mixture concentrated under reduced pressure. The obtained 6,6′,6″-nitrilotris(methylene)trinicotinic acid was used for the next reaction without further purification. .sup.1H NMR (300 MHz, D.sub.2O) δ 8.76 (d, J=2.0 Hz, 1H), 8.09 (dd, J=8.1, 2.0 Hz, 1H), 7.55 (d, J=8.1 Hz, 1H), 3.94 (s, 1H).

Example 106b—6,6′,6″-nitrilotris(methylene)tris(N—(((S)-2,2-dimethyl-1,3-dioxolan-4-yl) methyl)nicotinamide)

(252) ##STR00161##

(253) The 6,6′,6″-nitrilotris(methylene)trinicotinic acid (0.202 g, 0.478 mmol, 1.0 eq.) was dissolved in 10 mL dry DMF and filtered into an oven-dried 25 mL round bottomed flask to remove insoluble salts from the neutralization in the previous reaction. To this solution (R)-3-aminopropane-1,2-diol (0.376 g, 2.87 mmol, 6.0 eq.), EDCl (0.550 g, 2.87 mmol, 6.0 eq.), HOAt (0.390 g, 2.87 mmol, 6.0 eq.) and NMM (0.316 ml, 2.87 mmol, 6.0 eq.) were added and the mixture was stirred at room temperature for 16 h. The reaction mixture was then concentrated under reduced pressure and purification of the product was achieved by way of dry column vacuum chromatography on C18 material, using a stepwise elution from 10% to 90% methanol in water affording 0.177 g (0.23 mmol, 49%) of product. .sup.1H NMR (400 MHz, MeOD) δ 8.87 (d, J=1.9 Hz, 3H), 8.15 (dd, J=8.2, 2.3 Hz, 3H), 7.73 (d, J=8.2 Hz, 3H), 4.31 (p, J=5.8 Hz, 3H), 4.07 (dd, J=8.5, 6.3 Hz, 3H), 3.95 (s, 6H), 3.74 (dd, J=8.5, 6.0 Hz, 3H), 3.53 (dd, J=5.5, 1.6 Hz, 6H), 1.40 (s, 9H), 1.32 (s, 9H). .sup.13C NMR (101 MHz, MeOD) δ 168.12, 163.13, 148.77, 137.30, 130.29, 124.48, 110.56, 75.89, 68.20, 61.14, 43.51, 27.16, 25.57. APCI-HRMS e/z calc. for C.sub.39H.sub.51N.sub.7O.sub.9: 761.3748, found 762.3814 [M+H].

Example 106c—the title compound 6,6′,6″-nitrilotris(methylene)tris(N—((S)-2,3-dihydroxypropyl)nicotinamide)

(254) ##STR00162##

(255) The 6,6′,6″-nitrilotris(methylene)tris(N—(((S)-2,2-dimethyl-1,3-dioxolan-4-yl)methyl)nicotinamide) (50.5 mg, 0.066 mmol, 1.0 eq.) was dissolved in 1.5 mL MeOH at room temperature in a screw-capped vial. To this solution was added 1 mL of H2O and 3 drops conc. HCl. The reaction mixture was sealed and stirred at room temperature for 16 h and then concentrated under reduced pressure. The obtained crude was dissolved in 1 mL H2O, the pH adjusted to 7 with 0.1 M NaOH and concentrated under reduced pressure to dryness. The resulting semi-solid was treated with ice-cooled MeOH (2 mL) and filtered into a new flask. Removal of the solvent under reduced pressure resulted in 36.1 mg (0.056 mmol, 85%) of product. .sup.1H NMR (400 MHz, D.sub.2O) δ 8.61 (d, J=1.8 Hz, 3H), 7.94 (dd, J=8.2, 2.1 Hz, 3H), 7.47 (d, J=8.2 Hz, 3H), 3.93 (ddd, J=11.3, 6.7, 4.7 Hz, 3H), 3.84 (s, 5H), 3.68 (dd, J=11.8, 4.1 Hz, 3H), 3.58 (dd, J=11.9, 6.3 Hz, 3H), 3.52 (dd, J=14.0, 4.7 Hz, 3H), 3.41 (dd, J=14.0, 7.3 Hz, 3H). .sup.13C NMR (101 MHz, D.sub.2O) δ 168.05, 161.27, 146.78, 136.06, 128.24, 124.17, 70.26, 63.33, 61.10, 42.37. ESI-HRMS e/z calc. for C.sub.30H.sub.39N.sub.7O.sub.9: 641.2809, found 664.2701 [M+Na].

Examples 107-144

(256) Compounds 107-144 given in Schemes 10-20 above can be prepared using modifications of the methods described above.

Example 145—(6-((bis(pyridin-2-ylmethyl)amino)methyl)pyridin-3-yl)methyl methanesulfonate

(257) ##STR00163##

(258) (6-((bis(pyridin-2-ylmethyl)amino)methyl)pyridin-3-yl)methanol prepared in Example 14a (2.822 g, 8.81 mmol, 1.0 eq.) was dissolved in 150 mL dry THF under Ar and cooled to 0° C. in an ice bath. To this solution was added NEt3 (2.45 mL, 17.62 mmol, 2.0 eq.), followed by a solution of mesyl chloride (1.363 mL, 17.62 mmol, 2.0 eq.) in 30 mL dry THF dropwise. A precipitate formed and the suspension was stirred at 0° C. for 30 min, until TLC (Alumina, 3% MeOH in CH2Cl2) indicated full conversion. The mixture was filtered into a new flask, concentrated under reduced pressure to a volume of ca. 100 mL at 40° C., and DMF (80 mL) was added. The remaining THF was removed under reduced pressure and the obtained solution of (6-((bis(pyridin-2-ylmethyl)amino)methyl)pyridin-3-yl)methyl methanesulfonate in DMF used in the next reaction without further treatment under assumption of quantitative conversion. .sup.1H NMR (300 MHz, Chloroform-d) δ 10.59 (s, 1H), 8.70 (ddd, J=5.5, 1.6, 0.8 Hz, 2H), 8.50 (dd, J=2.2, 0.8 Hz, 1H), 8.05 (td, J=7.8, 1.7 Hz, 2H), 7.80 (d, J=7.9 Hz, 1H), 7.69 (dd, J=8.1, 2.3 Hz, 1H), 7.61-7.37 (m, 2H), 4.47 (s, 2H), 4.42 (s, 4H), 4.16 (s, 2H), 3.06 (s, 3H).

Example 146—1-(5-(azidomethyl)pyridin-2-yl)-N,N-bis(pyridin-2-ylmethyl) methanamine

(259) ##STR00164##

(260) To the solution of the (6-((bis(pyridin-2-ylmethyl)amino)methyl)pyridin-3-yl)methyl methanesulfonate (3.51 g, 8.81 mmol, 1.0 eq.) in 80 mL DMF, obtained in Example 145, was added NaN.sub.3 (2.864 g, 44.04 mmol, 5.0 eq.) at room temperature. The mixture was stirred at room temperature for 20 h, then filtered into a new flask and concentrated in vacuo to a volume of approximately 30 mL. The mixture was diluted with 100 mL H.sub.2O, transferred into a separation funnel and extracted with EtOAc (2×100 mL). The combined organics were washed with sat. aq. K.sub.2CO.sub.3 solution (50 mL), brine (50 mL), dried over K.sub.2CO.sub.3, filtered and concentrated under reduced pressure. The obtained compound was used in the next reaction without further treatment.

Example 147—1-(5-(aminomethyl)pyridin-2-yl)-N,N-bis(pyridin-2-ylmethyl) methanamine hydrochloride

(261) ##STR00165##

(262) The 1-(5-(azidomethyl)pyridin-2-yl)-N,N-bis(pyridin-2-ylmethyl)methanamine, obtained in Example 146 (2.783 g, 8.06 mmol, 1.0 eq.) was dissolved in 50 mL THF. To this solution was added 5 mL dest. H.sub.2O and PPh.sub.3 (4.228 g, 16.12 mmol, 2.0 eq.) in one portion. The mixture was heated to 50° C. and stirred for 3 h until TLC (Alumina, 3% MeOH in CH2C2) indicated full conversion. The mixture was concentrated under reduced pressure, the residue was treated with CH2C2 and H2O (100 mL each) and the pH of the aqueous phase adjusted to 1 with conc. HCl with stirring. The mixture was transferred into a separation funnel, the aq. phase washed with CH2C2 (50 mL) and concentrated under reduced pressure to afford 2.825 g (7.93 mmol, 98%) of 1-(5-(aminomethyl)pyridin-2-yl)-N,N-bis(pyridin-2-ylmethyl)methanamine as the hydrochloride salt. .sup.1H NMR (300 MHz, DMSO) δ 9.00 (s, 3H), 8.91 (d, J=1.6 Hz, 1H), 8.81 (dd, J=5.7, 0.9 Hz, 2H), 8.48 (ddd, J=9.3, 8.3, 1.5 Hz, 3H), 8.16 (d, J=7.9 Hz, 2H), 8.11 (d, J=8.2 Hz, 1H), 7.94-7.84 (m, 2H), 4.38 (s, 4H), 4.30 (s, 2H), 4.17 (d, J=5.5 Hz, 2H). .sup.13C NMR (101 MHz, DMSO) δ 153.22, 152.46, 145.43, 142.25, 131.67, 126.96, 125.90, 125.82, 56.20, 55.59.

Example 148—(3aS,5S,5aR,8aS,8bR)—N-((6-((bis(pyridin-2-ylmethyl)amino)methyl) pyridin-3-yl)methyl)-2,2,7,7-tetramethyltetrahydro-3aH-bis[1,3]dioxolo[4,5-b:4′,5′-d]pyran-5-carboxamide

(263) ##STR00166##

(264) 1-(5-(aminomethyl)pyridin-2-yl)-N,N-bis(pyridin-2-ylmethyl)methanamine hydrochloride from example 147 (0.276 g, 0.775 mmol, 1.0 eq.), was suspended in 5 mL dry DMF and cooled to 0° C. in an ice bath. To this suspension was added 1,2:3,4-Di-O-isopropylidene-a-D-galacturonide (0.213 g, 0.775 mmol, 1.0 eq.), HATU (0.295 g, 0.775 mmol, 1.0 eq.) and NMM (0.256 mL, 2.325 mmol, 3.0 eq.) after which the mixture turned into a solution. The mixture was stirred at 0° C. for 30 min, then at room temperature for 16 h and concentrated under reduced pressure. Purification of the product was achieved by way of dry column vacuum chromatography on C18 bondesil material, using a stepwise elution from 10% to 70% methanol in water affording 156.1 mg (0.271 mmol, 35%) of product. .sup.1H NMR (400 MHz, MeOD) δ 8.42 (dd, J=7.1, 3.3 Hz, 3H), 7.77 (ddd, J=16.5, 8.3, 1.9 Hz, 3H), 7.66 (d, J=7.8 Hz, 2H), 7.60 (d, J=8.1 Hz, 1H), 7.30-7.24 (m, 2H), 5.62 (d, J=4.9 Hz, 1H), 4.71 (dd, J=7.7, 2.5 Hz, 1H), 4.63 (d, J=15.4 Hz, 1H), 4.59 (dd, J=7.8, 2.1 Hz, 1H), 4.43 (dd, J=4.9, 2.6 Hz, 1H), 4.30 (d, J=2.0 Hz, 1H), 4.25 (d, J=15.4 Hz, 1H), 3.84 (s, 6H), 1.49 (s, 3H), 1.34 (s, 3H), 1.33 (s, 3H), 1.30 (s, 3H). .sup.13C NMR (101 MHz, MeOD) δ 171.51, 160.15, 158.85, 149.62, 148.80, 138.76, 137.99, 134.94, 124.94, 124.47, 123.95, 110.79, 110.45, 97.89, 73.28, 72.07, 72.04, 70.30, 61.21, 60.97, 40.81, 26.39, 26.29, 25.06, 24.61. APCI-HRMS e/z calc. for C.sub.31H.sub.37N.sub.5O.sub.6: 575.2744, found 576.2817 [M+H].

Example 149—(2S,3R,4S,5R,6R)—N-((6-((bis(pyridin-2-ylmethyl)amino)methyl)pyridin-3-yl)methyl)-3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2-carboxamide

(265) ##STR00167##

(266) The protected starting material (0.1339 mg, 0.232 mmol, 1.0 eq.) was dissolved in 10 mL THF and 5 mL H2O at room temperature. To this solution was added 5 drops of conc. HCl and the mixture stirred at room temperature for 16 h in a tightly sealed vessel. The pH was adjusted to 9 with sat. aq. K.sub.2CO.sub.3 solution, and the mixture concentrated under reduced pressure. The residue was treated with MeOH (30 mL) and filtered into a new flask, and concentrated under reduced pressure. Purification of the product was achieved by way of dry column vacuum chromatography on C18 bondesil material, using a stepwise elution from 10% to 50% methanol in water affording 45.7 mg (0.092 mmol, 40%) of product.

Example 150—S-((6-((bis(pyridin-2-ylmethyl)amino)methyl)pyridin-3-yl)methyl) ethanethioate

(267) ##STR00168##

(268) The mesylate from Example 145 (64 mg, 0.16 mmol) was dissolved in 10 mL MeCN before potassium thioacetate (100 mg, 0.88 mmol) was added. The mixture was stirred for 3 hours at room temperature before it was concentrated under reduced pressure. The crude material was purified on a neutral alumina column using 0-2% MeOH in DCM as eluent giving the product. .sup.1H NMR (400 MHz, Chloroform-d) δ 8.51 (ddd, J=5.0, 1.8, 1.0 Hz, 2H), 8.43 (dd, J=2.4, 0.9 Hz, 1H), 7.63 (td, J=7.6, 1.9 Hz, 2H), 7.59-7.42 (m, 4H), 7.12 (ddd, J=7.5, 4.9, 1.4 Hz, 2H), 4.04 (s, 2H), 3.89 (app. d, J=5.3 Hz, 6H), 2.33 (s, 3H).

Example 151—tert-Butyl 2-((tert-butoxycarbonyl)oxy)-3-methylbenzoate

(269) ##STR00169##

(270) The title compound was prepared from 3-methylsalisylic acid (19.1 g, 0.1255 mol) as described in Reddy et al WO 2016/003929. Purification was done by way of dry column vacuum chromatography using silica as adsorbent, giving 9.85 g product (25.5%). .sup.1H NMR (400 MHz, chloroform-d) δ 7.74 (m, 1H), 7.35 (m, 1H), 7.15 (app. t, J=7.7 Hz, 1H), 2.26 (s, 3H), 1.57 (s, 9H), 1.56 (s, 9H).

Example 152—tert-Butyl 3-(bromomethyl)-2-((tert-butoxycarbonyl)oxy)benzoate

(271) ##STR00170##

(272) Free radical bromination of tert-butyl 2-((tert-butoxycarbonyl)oxy)-3-methylbenzoate (8.72 g, 28.3 mmol) was performed with N-bromosuccinimide (5.29 g, 29.7 mmol) in tetrachloromethane (100 mL) as described by Hecjer et al in WO 201614939. Azobisisobutyronitrile (463 mg, 2.82 mmol) was employed as radical initiator. After recrystallization from hexane, the product was obtained as a colourless solid (8.18 g, 74.6%). .sup.1H NMR (400 MHz, chloroform-d) δ 7.83 (dd, J=7.8 Hz, 1.7 Hz, 1H), 7.55 (dd, J=7.7 Hz, 1.7 Hz, 1H), 7.24 (app. t, J=7.8 Hz, 1H), 4.50 (s, 2H), 1.57 (s, 18H).

Example 153—tert-Butyl 2-((tert-butoxycarbonyl)oxy)-3-(((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)methyl)benzoate

(273) ##STR00171##

(274) Miyaura-borylation of tert-butyl 3-(bromomethyl)-2-((tert-butoxycarbonyl)oxy)benzoate (4.71 g, 12.2 mmol) was performed as described in Reddy et al WO 2016/003929, and the title compound was obtained as a colourless oil after purification on a silica column eluted with a gradient of ethyl acetate in n-heptane (3.18 g, 53.8%). .sup.1H NMR (400 MHz, chloroform-d) δ 7.70 (dd, J=7.8 Hz, 1.8 Hz, 1H), 7.40 (dd, J=7.7 Hz, 1.8 Hz, 1H), 7.14 (app. t, J=7.7 Hz, 1H), 4.25 (dd, J=8.7 Hz, 2.0 Hz, 1H), 2.31-2.24 (m, 3H), 2.19 (m, 1H), 2.02 (m, 1H), 1.90-1.81 (m, 2H), 1.554 (s, 9H), 1.548 (s, 9H), 1.38 (s, 3H), 1.27 (s, methyl overlapping with lipid impurities), 1.18 (d, J=11.0 Hz, 1H), 0.82 (s, 3H). MS (ESI, positive mode) m/z 509.3 [M+Na].sup.+, HR-MS (ESI, pos. mode) m/z 509.2681 calculated for C.sub.27H.sub.39O.sub.7.sup.11BNa, found m/z 509.2682.

Example 154—tert-Butyl 2-((tert-butoxycarbonyl)oxy)-3-((S)-2-chloro-2-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)benzoate

(275) ##STR00172##

(276) Matteson-homologation of tert-butyl 2-((tert-butoxycarbonyl)oxy)-3-(((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)methyl)benzoate (3.03 g, 6.24 mmol) was performed as described in Reddy et al WO2016003929. Purification of the product was achieved by way of column chromatography, with silica as adsorbent and a gradient of ethyl acetate in n-heptane as eluent. After removal of solvent, the product was obtained as a pale yellow oil (2.10 g, 62.9%). .sup.1H NMR (400 MHz, chloroform-d) δ 7.81 (dd, J=7.8 Hz, 1.8 Hz, 1H), 7.48 (dd, J=7.6 Hz, 1.8 Hz, 1H), 7.19 (app. t, J=7.7 Hz, 1H), 4.36 (dd, J=8.8 Hz, 1.8 Hz, 1H), 3.67 (dd, J=9.0 Hz, 6.9 Hz, 1H), 3.23 (dd, J=14.2 Hz, 6.9 Hz, 1H), 3.05 (dd, J=14.2 Hz, 9.0 Hz, 1H), 2.33 (m, 1H), 2.19 (m, 1H), 2.06 (m, 1H), 1.92-1.85 (m, 2H), 1.56 (s, 9H), 1.55 (s, 9H), 1.37 (s, 3H), 1.28 (s, 3H), 1.10 (d, J=11.0 Hz, 1H), 0.83 (s, 3H). .sup.13C NMR (100 MHz, chloroform-d) 6164.1, 151.4, 149.1, 135.2, 132.4, 130.5, 125.7, 125.4, 87.0, 83.7, 81.6, 78.7, 51.3, 41.4 (br), 39.5, 38.4, 35.3, 35.1, 28.5, 28.3, 27.9, 27.2, 26.4, 24.1. Minor peaks from the diastereomer were visible in .sup.1H and .sup.13C spectrum. MS (ESI, positive mode) m/z 557.2 [M+Na].sup.+, HR-MS (ESI, pos. mode) m/z 557.2448 calculated for C.sub.28H.sub.40.sup.35ClO.sub.7.sup.11BNa, found m/z 557.2450.

Example 155—tert-Butyl 3-((R)-2-(bis(trimethylsilyl)amino)-2-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)-2-((tert-butoxycarbonyl)oxy)benzoate

(277) ##STR00173##

(278) A flask containing tert-butyl 2-((tert-butoxycarbonyl)oxy)-3-((S)-2-chloro-2-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)benzoate (113.5 mg, 0.212 mmol) under a nitrogen atmosphere was dissolved in 5 mL tetrahydrofuran and cooled down to −78° C. A solution of lithium bis(trimethylsilyl)amide, 1.0 M in tetrahydrofuran was added (0.21 mL, 0.21 mmol), and the solution was then allowed to reach room temperature. Stirring was continued over night, after which volatiles were removed under reduced pressure, and the resulting crude mixture used directly in the next step.

Example 156a—tert-Butyl 3-((R)-2-(2-(4-(4-((bis(pyridin-2-ylmethyl)amino)methyl)-1H-1,2,3-triazol-1-yl)phenyl)acetamido)-2-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)-2-((tert-butoxycarbonyl)oxy)benzoate

(279) ##STR00174##

(280) 2-(4-(4-((Bis(pyridin-2-ylmethyl)amino)methyl)-1H-1,2,3-triazol-1-yl)phenyl)acetic acid (0.248 mmol) from example 96d, was dissolved in 5 mL dimethylformamide and cooled down on an ice bath. To this solution, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride salt (59.7 mg, 0.311 mmol) and 1-hydroxy-7-azabenzotriazole (34.4 mg, 0.253 mmol) was added, and allowed to stir on ice bath for 30 min. A solution of the bis(trimethylsilyl)amino compound, described above, in 2 mL dimethylformamide, was added (0.212 mmol assumed), followed by N-methylmorpholine (0.08 mL, 0.7 mmol). The mixture was allowed to stir over night. After being concentrated on a rotary evaporator, the reaction mixture was partitioned between dichloromethane and a mixture of 0.5 M K.sub.2CO.sub.3 (aq.) and saturated NaCl solution. The phases were separated and the aqueous phase extracted with more dichloromethane. The combined organic extracts were then dried over Na.sub.2SO.sub.4, filtered and solvents removed under reduced pressure. The crude product was loaded onto a 2 g SPE plug with C18 material. Pure product was eluted by running through 25 mL portions of methanol/water mixture, going stepwise from 70% to 90% methanol, giving after removal of solvents under reduced pressure, 70.9 mg product (36.7% over two steps). .sup.1H NMR (400 MHz, chloroform-d) δ 8.55 (br d, 2H), 8.05 (s, 1H), 7.73 (dd, J=7.8 Hz, 1.8 Hz, 1H), 7.68-7.64 (m, 4H), 7.59 (br d, J=7.8 Hz, 2H), 7.34 (m, 2H), 7.27 (m, 1H), 7.17-7.10 (m, 3H), 4.31 (dd, J=8.9 Hz, 2.1 Hz, 1H), 3.96 (s, 2H), 3.90 (s, 4H), 3.62 (d, J=3.0 Hz, 2H), 2.95-2.84 (m, 3H), 2.35 (m, 1H), 2.19 (m, 1H), 2.02 (app t, J=5.5 Hz, 1H), 1.92-1.84 (m, 2H), 1.67 (s, 3H (exchanging protons)), 1.54 (s, 9H), 1.53 (s, 9H), 1.44-1.40 (m, 4H), 1.29 (s, 3H), 0.87 (s, 3H). Minor peaks from the diastereomer were visible in .sup.1H spectrum. .sup.13C NMR (100 MHz, chloroform-d) 6173.5 (only visible from HMBC), 164.0, 159.3, 152.1, 149.3, 148.8, 145.3, 136.64, 136.58, 134.9, 134.4, 130.9, 130.1, 125.9, 125.7, 123.5, 122.2, 121.3, 121.0, 84.6, 84.1, 81.8, 77.3, 59.9, 52.2, 48.8, 42.1 (only visible in HSQC spectrum), 40.1, 39.9, 38.3, 36.5, 31.4, 29.1, 28.3, 27.9, 27.5, 26.7, 24.3. MS (APCI, positive mode) m/z 912.5 [M+H].sup.+, HR-MS (APCI, pos. mode) m/z 911.4862 calculated for Cs.sub.1H.sub.63N.sub.7O.sub.8.sup.10B, found m/z 911.4863.

Example 156b—(R)-3-(2-(4-(4-((Bis(pyridin-2-ylmethyl)amino)methyl)-1H-1,2,3-triazol-1-yl)phenyl)acetamido)-2-hydroxy-3,4-dihydro-2H-benzo[e][1,2]oxaborinine-8-carboxylic acid hydrochloride

(281) ##STR00175##

(282) Global deprotection and cyclisation was achieved by treatment of a solution of the triply protected precursor (64.1 mg, 0.0703 mmol) in 2 mL dioxane with 1 mL 4 M HCl in dioxane. After heating to reflux for 2 h, a solid precipitate was formed, which after cooling was collected by way of suction filtration. This gave after drying an ochre powder (58.3 mg, >100%), containing an unknown amount of HCl. Purification was attempted using SPE plugs with respectively cation exchange, anion exchange and reverse phase (C18) materials, without any success. Chromatographic purity of the precipitate was 80%, using a hybrid C18 reversed phase column with high pH tolerance, a methanol gradient and buffered with potassium phosphate adjusted with HCl (aq.) to pH 11.5. .sup.1H NMR (400 MHz, mixture trifluoroacetic acid-d and water-d.sub.2) δ (uncalibrated) 8.55 (br d, J=6.0 Hz, 2H), 8.33 (app br t, J=8.0 Hz, 2H), 8.13 (s, 1H), 7.89 (d, J=8.1 Hz, 2H), 7.75 (app br t, 2H), 7.11 (m, 4H), 6.75 (d, J=8.2 Hz, 2H), 6.67 (app t, J=7.6 Hz, 1H), 4.23 (s, 4H), 3.88 (s, 2H). The remaining signals were broadened beyond identification, possibly due to the numerous equilibria exhibited by the compound. MS (ESI, negative mode, dissolved in water/methanol) m/z 648.3 (100%, “base peak”, tetrahedral boron doubly esterified with methanol), 634.3 (23%, tetrahedral boron, with one methanol), 616.2 (31%, trigonal planar boron, with one methanol).

Example 157—(R)-3-(2-(2-(4-(4-((bis(pyridin-2-ylmethyl)amino)methyl)-1H-1,2,3-triazol-1-yl)phenyl)acetamido)-2-boronoethyl)benzoic acid

(283) ##STR00176##

(284) The title compound can be prepared analogously using the methods described in Example 156.

Example 158—6-((bis(pyridin-2-ylmethyl)amino)methyl)-N-(2-mercaptoethyl) nicotinamide

(285) ##STR00177##

(286) The 6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinic acid prepared in Example 13 was dissolved in 20 mL dry DMF at room temperature and filtered into a 50 mL round bottomed flask prior to reaction to remove the insoluble salts. To this solution was added 2-aminoethane-1-thiol (575 mg, 7.45 mmol, 1.5 eq.), followed by EDCl (1.428 g, 7.45 mmol, 1.5 eq.), HOAt (1.014 g, 7.45 mmol, 1.5 eq.) and NMM (0.821 mL, 7.45 mmol, 1.5 eq.). The reaction mixture was stirred at room temperature for 16 h and then concentrated under reduced pressure. The residual crude mixture was dissolved in 100 mL CHCl.sub.3, transferred into a separation funnel and washed with 100 mL sat. aq. K.sub.2CO.sub.3 solution and 100 mL brine. The organic phase was separated, dried oved Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. Purification of the product was performed by column chromatography on neutral Al.sub.2O.sub.3 using 1% MeOH in DCM giving the product with minor impurities, followed by C18-SPE using gradient elution (10% MeOH to 90% MeOH in H.sub.2O) affording the product as a yellow oil without visible impurities, that was used without further purification.

Example 159—tert-Butyl(3S,6R)-6-((2-(6-((bis(pyridin-2-ylmethyl)amino)methyl) nicotinamido)ethyl)thio)-3-((tert-butyldimethylsilyl)oxy)-6-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)hexanoate

(287) ##STR00178##

(288) To a flask containing (3S,6S)-tert-butyl 3-(tert-butyldimethylsilyloxy)-6-chloro-6-[(2S,6R)-2,9,9-trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.0.sup.2,6]-decan-4-yl)hexanoate (293.8 mg, 0.570 mmol), prepared as described in Hecker et al. J. Med. Chem., 2015, 58, 3682-3692, 6-((bis(Pyridin-2-ylmethyl)amino)methyl)-N-(2-mercaptoethyl)nicotinamide (298 mg, 0.757 mmol) from example 158 was added as a solution in 15 mL dichloromethane. After dropwise addition of triethylamine (0.21 mL, 1.5 mmol), the mixture was stirred at room temperature overnight. The mixture was worked up by transfer to a separatory funnel with 50 mL 0.5 M NaHCO.sub.3 (aq.) and extracted twice with 50 mL ethyl acetate. The extract was washed with sat. NaCl (aq.) and dried over Na.sub.2SO.sub.4. Filtration and subsequent removal of solvents under reduced pressure gave a crude product which was purified initially by loading onto a plug of Bondesil-C18 OH SPE material and eluted by running through portions of methanol/water mixture, going stepwise from 50% to pure methanol. This procedure was repeated once, and final purification was achieved by loading onto a plug of strong cationic exchange SPE material, using 1:1 methanol/water and a pH-gradient from pH 3 to pH 10 with formic acid and ammonia as additives. Fractions containing product was collected and the solvent removed under reduced pressure to give the desired product (136.3 mg, 27.4%). .sup.1H NMR (400 MHz, chloroform-d) δ 8.91 (m, 1H), 8.54 (m, 2H), 8.08 (dd, J=8.1 Hz, 2.3 Hz, 1H), 7.68-7.63 (m, 3H), 7.55 (d, J=7.8 Hz, 2H), 7.15 (m, 2H), 6.90-6.84 (br m, 1H), 4.30-4.26 (m, 1H), 4.08 (m, 1H), 3.93 (s, 2H), 3.88 (s, 4H), 3.66 (m, 2H), 2.83 (m, 2H), 2.41-2.27 (m, 3H), 2.23-2.12 (m, 2H), 2.03-2.00 (m, 1H), 1.89-1.79 (m, 2H), 1.76-1.57 (m, 4H), 1.42 (s, 2.5H (diastereomer)), 1.41 (s, 6.5H), 1.36 (s, 3H), 1.25 (s, 3H), 1.13 (d, J=10.9 Hz, 1H), 0.85 (s, 9H), 0.80 (s, 3H), 0.05 (s, 3H), 0.04 (s, 3H). The product appears to be a 5:2 mixture of diastereomers, most readily apparent by the split chemical shifts of the tert-butyl ester protons. .sup.13C NMR (100 MHz, chloroform-d) 6171.1, 165.7, 162.9, 159.2, 149.3, 147.7, 136.7, 135.7, 128.8, 123.2, 122.7, 122.3, 86.4, 80.5, 78.3, 69.1, 60.3, 60.1, 51.4, 43.9, 39.5, 39.2, 38.3, 36.2, 35.6, 32.0, 28.7, 28.3, 27.1, 26.7, 26.6, 26.0 (overlapping with severely broadened resonance 6 to boron, only visible in HSQC), 24.1, 18.2, −4.3, −4.5. Shifts only given for major diastereomer. MS (APCI, pos. mode) m/z 872.5 [M+H].sup.+, HR-MS (APCI, pos. mode) m/z 871.5018 calculated for C.sub.47H.sub.71N.sub.5O.sub.6SSi.sup.10B, found m/z 871.5015.

Example 160—2-((3R,6S)-3-((2-(6-((bis(pyridin-2-ylmethyl)amino)methyl) nicotinamido)ethyl)thio)-2-hydroxy-1,2-oxaborinan-6-yl)acetic acid

(289) ##STR00179##

(290) A mixture of the triply protected precursor from Example 159 (84.4 mg, 0.0967 mmol), and p-toluenesulfonic acid monohydrate (1.9 g, 10 mmol) was mixed with 20 mL acetonitrile and heated to reflux for two hours. After removal of solvent under reduced pressure, the residue was dissolved in 15 mL 0.5 M HCl (aq.) and washed with diethyl ether. The aqueous phase was loaded onto a plug of strong cationic exchange SPE material, and eluted using 1:1 methanol/water and a pH-gradient from pH 3 to pH 9 with formic acid and ammonia as additives. The acid eluted in the acidic fractions, whereas the desired product eluted at pH 9. Purity was difficult to assess by way HPLC due to presence of diastereomers (methanol in water, gradient eluted, with 0.5% trifluoroacetic acid in the eluent). .sup.1H NMR (400 MHz, methanol-d.sub.4) δ 8.88 (m, 1H), 8.44 (m, 2H), 8.18 (m, 1H), 7.81-7.65 (m, 5H), 7.28 (m, 2H), 4.20 (m, 1H), 3.92 (s, 2H), 3.90 (s, 4H), 3.65 (m, 1H), 3.43 (m, 1H), 2.74-2.55 (m, 3H), 2.19 (m, 1H), 2.09 (m, 1H), 1.84-1.74 (m, 2H), 1.58 (br d, 1H), 1.45 (m, 1H). Reported peaks and integrals correspond to main peaks from the edited HSQC spectrum. There are also several minor, unreported peaks; either arising from equilibrium forms and/or diasteromers of product or said forms, and possibly also from impurities. .sup.13C NMR (100 MHz, methanol-d.sub.4) δ 178.5, 168.2, 163.1, 159.8, 149.6, 148.9, 138.8, 137.3, 130.5, 125.0, 124.3, 124.0, 68.1, 61.3, 61.0, 40.8, 37.1, 35.3, 29.8, 28.9. Shifts from 1D .sup.13C spectrum only given for the major diastereomer, as interpreted from the additional HSQC and HMBC spectra. Shift of carbon 6 to boron not visible in any spectrum. MS (ESI, neg. mode) m/z 548.2 [M−H].sup.−, HR-MS (ESI, neg. mode) m/z 547.2181 calculated for C.sub.27H.sub.31N.sub.5O.sub.5S.sub.10B, found m/z 547.2180.

Example 161—tert-Butyl (3S,6R)-6-(bis(trimethylsilyl)amino)-3-((tert-butyldimethylsilyl)oxy)-6-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)hexanoate

(291) ##STR00180##

(292) A flask containing (3S,6S)-tert-butyl 3-(tert-butyldimethylsilyloxy)-6-chloro-6-[(2S,6R)-2,9,9-trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.0.sup.26]-decan-4-yl)hexanoate (307.9 mg, 0.598 mmol), prepared as described in Hecker et al. J. Med. Chem., 2015, 58, 3682-3692, was put under a nitrogen atmosphere, dissolved in 10 mL dry tetrahydrofuran and cooled down on a dry ice/acetone bath to −78° C. Lithium bis(trimethylsilyl)amide was added (1.0 M solution in THF, 0.60 mL, 0.60 mmol), after which the cooling bath was removed and the mixture allowed to reach room temperature and stir overnight. At the end of the reaction, 5 mL dry dimethylformamide was added, and the tetrahydrofuran solvent removed under reduced pressure. The unstable product was transferred to the next reaction step as a solution in DMF without any additional purification or characterisation.

Example 162—tert-Butyl (3S,6R)-6-(6-((bis(pyridin-2-ylmethyl)amino)methyl) nicotinamido)-3-((tert-butyldimethylsilyl)oxy)-6-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)hexanoate

(293) ##STR00181##

(294) To a flask containing 6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinic acid (0.718 mmol), prepared in situ by hydrolysis, neutralization and subsequent solvent removal from the corresponding methyl ester, dimethylformamide (5 mL) was added, and the resulting solution cooled down on an ice bath under nitrogen. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride salt (177.2 mg, 0.924 mmol), followed by 1-hydroxy-7-azabenzotriazole (103.4 mg, 0.760 mmol), was then added, followed by a solution of tert-butyl (3S,6R)-6-(bis(trimethylsilyl)amino)-3-((tert-butyldimethylsilyl)oxy)-6-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)hexanoate (0.598 mmol, prepared as described above) in 5 mL dimethylformamide. Finally, N-methylmorpholine (0.20 mL, 1.8 mmol) was added and the mixture was allowed to stir for 3 h at room temperature. The reaction mixture was transferred to a separatory funnel containing a mixture of sat. NaCl (aq.) and 0.5 M K.sub.2CO.sub.3 (aq.). After extracting twice with 40 mL dichloromethane, the combined organic phase was dried over MgSO.sub.4 (s), filtered and then solvent was removed under reduced pressure. The crude product was loaded onto a plug of Bondesil-C18 SPE material and eluted by running through portions of methanol/water mixture, going stepwise from 70% up to pure methanol. Fractions containing pure product were collected, and solvent removed under reduced pressure to afford 191.4 mg product (39.4%). .sup.1H NMR (400 MHz, chloroform-d) δ 8.93 (m, 1H), 8.53 (m, 2H), 8.10 (m, 1H), 7.67-7.62 (m, 3H), 7.52 (d, J=7.8 Hz, 2H), 7.15 (m, 2H), 4.31 (m, 1H), 4.12 (m, 1H), 3.93 (s, 2H), 3.88 (s, 4H), 3.20 (m, 1H), 2.46-2-31 (m, 3H), 2.17 (m, 1H), 2.02 (m, 1H), 1.92-1.57 (m, should be 6H, but integrates to 9H, probably due to overlap from diastereomeric shifts), 1.43 (s, 3H), 1.41 (s, 9H), 1.34 (d, J=10.8 Hz, 1H), 1.28 (s, 3H), 0.85 (s, 9H), 0.84 (s, 3H), 0.054 (s, 3H), 0.048 (s, 3H). The product appears to be a 5:2 mixture of diastereomers, most easily determined from the multiplet pattern at SH 8.93 ppm. Of the same reason most peaks appear split. .sup.13C NMR (100 MHz, chloroform-d) δ 172.0, 167.4, 163.6, 159.2, 149.3, 148.0, 136.6, 135.9, 126.1, 123.2, 122.7, 122.3, 85.2, 81.0, 77.6, 68.2, 60.4, 60.0, 51.9, 43.6, 40.0, 39.5 (only visible from HSQC spectrum), 36.2, 34.7, 29.0, 28.3, 27.4, 26.7, 26.4, 26.0, 24.3, 18.1, −4.4, −4.5. Shifts only given for major diastereomer. MS (APCI, pos. mode) m/z 812.5 [M+H].sup.+, HR-MS (APCI, pos. mode) m/z 811.4984 calculated for C.sub.45H.sub.67N.sub.5O.sub.6Si.sup.10B, found m/z 811.4981.

Example 163a—2-((3R,6S)-3-(6-((Bis(pyridin-2-ylmethyl)amino)methyl)nicotinamido)-2-hydroxy-1,2-oxaborinan-6-yl)acetic acid

(295) ##STR00182##

(296) From the triply protected compound described above (109 mg, 0.132 mmol), global deprotection was achieved by dissolving the starting material in 4 mL dioxane, and adding 2 mL 4 M HCl in dioxane and heating to 70° C. for 30 min. After cooling down, a pale yellow hygroscopic precipitate deposited on the glass surface, which was isolated by way of suction filtration. The precipitate was washed on the filter with diethyl ether and redissolved in water. Final purification was achieved by way of preparative HPLC, with multiple injections, on a preparative scale hybrid silica C18 reversed phase column eluted with 28:72 water/methanol mixture containing 0.5% trifluoroacetic acid. Fractions were collected as timeslices and analysed with analytical HPLC using a similar column and eluent. The fractions containing product in satisfactory purity was loaded directly onto a plug of strong cation exchange material, and excess acid washed out using 1:1 methanol/water. The product was eluted using the same 1:1 mixture, with addition of NH.sub.3 (aq.). Removal of volatiles under reduced pressure gave 14.8 mg of the title compound as a colourless solid, with approximately 95% purity according to HPLC (27.7%). .sup.1H NMR (300 MHz, methanol-d.sub.4) δ 8.90 (m, 1H), 8.48 (m, 2H), 8.18 (dd, J=8.2 Hz, 2.3 Hz, 1H), 7.82 (app dt, J=1.8 Hz, 7.7 Hz, 2H), 7.70-7.63 (m, 3H), 7.31 (m, 2H), 4.21 (m, 1H), 3.98 (s, 2H), 3.97 (s, 4H), 3.07 (dd, J=10.4 Hz, 5.7 Hz, 1H), 2.68 (dd, J=17.4 Hz, 7.2 Hz, 1H), 2.26 (dd, J=17.2 Hz, 1.5 Hz, 1H), 2.11 (m, 1H), 1.87 (m, 1H), 1.53 (m, 1H), 1.36 (m, 1H). The spectrum shows no signs of diastereomers and no excessive broadening due to equilibria. .sup.13C NMR (100 MHz, methanol-d.sub.4) δ 179.0, 162.7 (only visible from HMBC spectrum), 159.8, 149.6, 149.5, 148.7, 138.8, 138.7, 137.0, 125.0, 124.2, 123.9, 68.3, 61.3, 61.0, 43.3 (only visible from HSQC spectrum), 37.4 (br), 33.6 (br), 27.3. MS (ESI, neg. mode) m/z 488.2 [M−H]-.

Example 163b—Hydrochloride Salt of the Compounds of Example 163a

(297) ##STR00183##

(298) In an alternative preparation, the compound in example 162 (41.2 mg, 0.0507 mmol) was dissolved in 2 mL dioxane, 1 mL 4 M HCl in dioxane added, and the mixture heated on reflux for 30 min. After transfer to a separatory funnel using 20 mL water, and washing the aqueous phase two times with 20 mL diethyl ether, the water phase was evaporated to yield 30.0 mg product (quantitative, with unknown amount of HCl present). Purity by way of HPLC was less than 70%, and NMR showed some evidence of partial deprotection. MS (ESI, neg. mode) m/z 488.2 [M−H].sup.−, HR-MS (ESI, neg. mode) m/z 487.2147 calculated for C.sub.25H.sub.27N.sub.5O.sub.5.sup.10B, found m/z 487.2150.

Example 164—tert-Butyl 3-((R)-2-(6-((bis(pyridin-2-ylmethyl)amino)methyl) nicotinamido)-2-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)-2-((tert-butoxycarbonyl)oxy)benzoate

(299) ##STR00184##

(300) To a flask containing 6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinic acid (0.471 mmol), prepared in situ by hydrolysis, neutralization and subsequent solvent removal from the corresponding methyl ester, dimethylformamide (5 mL) was added, and the resulting solution cooled down on an ice bath under nitrogen. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride salt (115.2 mg, 0.601 mmol), followed by 1-hydroxy-7-azabenzotriazole (65.5 mg, 0.481 mmol), was then added, followed by a solution of tert-butyl 3-((R)-2-(bis(trimethylsilyl)amino)-2-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)-2-((tert-butoxycarbonyl)oxy)benzoate (0.387 mmol, prepared in an adapted procedure from Hecker et al. J. Med. Chem., 2015, 58, 3682-3692 and Reddy et al WO2016003929A1, in 5 mL dimethylformamide. Finally, N-methylmorpholine (0.13 mL, 1.2 mmol) was added and the mixture was allowed to stir for 4 h at room temperature. The reaction mixture was transferred to a separatory funnel containing a mixture of sat. NaCl (aq.) and 0.5 M K.sub.2CO.sub.3 (aq.). After extracting twice with 40 mL dichloromethane, the combined organic phase was dried over MgSO.sub.4 (s), filtered and then solvent was removed under reduced pressure. The crude product was loaded onto a plug of Bondesil-C18 SPE material and eluted by running through portions of methanol/water mixture, going stepwise from 60% up to pure methanol. Fractions containing pure product were collected, and solvent removed under reduced pressure to afford 168.4 mg product (52.3%). .sup.1H NMR (400 MHz, chloroform-d) δ 8.89 (br d, 1H), 8.53 (m, 2H), 8.03 (dd, J=8.2 Hz, 2.3 Hz, 1H), 7.79 (dd, J=7.8 Hz, 1.7 Hz, 1H), 7.67-7.62 (m, 3H), 7.54-7.48 (m, 3H), 7.24 (m, 1H), 7.14 (m, 2H), 4.34 (m, 1H), 3.92 (s, 2H), 3.86 (s, 4H), 3.12 (m, 1H), 3.00 (m, 2H), 2.38 (m, 1H), 2.21 (m, 1H), 2.05 (t, J=5.5 Hz, 1H), 1.93-1.87 (m, 2H), 1.57-1.54 (m, 10H), 1.46 (s, 3H), 1.34 (br s, 7-8H, possibly some in situ hydrolysis), 1.30 (s, 3H), 0.89 (s, 3H). .sup.13C NMR (100 MHz, chloroform-d) δ 168.8, 164.6, 163.9, 159.1, 149.4, 148.8, 148.7, 136.6, 136.1, 135.2, 130.2, 126.2, 125.5, 123.2, 123.1, 122.6, 122.3, 84.3, 84.2, 81.8, 77.4, 60.4, 60.0, 52.4, 44.2 (only visible in HSQC spectrum), 40.2, 38.4, 36.7, 31.3 (br), 29.2, 28.3, 27.7, 27.5, 26.9, 24.4. Additional unreported minor peaks visible in .sup.13C spectrum, probably from a diastereomer. Two carbonyl signals could not be identified. MS (APCI, pos. mode) m/z 832.4 [M+H].sup.+, HR-MS (APCI, pos. mode) m/z 831.4488 calculated for C.sub.47H.sub.9N.sub.5O.sub.8.sup.10B, found m/z 831.4483.

Example 165—(R)-3-(6-((Bis(pyridin-2-ylmethyl)amino)methyl)nicotinamido)-2-hydroxy-3,4-dihydro-2H-benzo[e][1,2]oxaborinine-8-carboxylic acid

(301) ##STR00185##

(302) Global deprotection of the precursor described above was achieved by dissolving the precursor (90.8 mg, 0.109 mmol) in 8 mL dry dioxane, followed by addition of 2 mL 4 M HCl in dioxane. After heating to 70° C. for 1h, and subsequent cooling to room temperature, an inhomogenous, hygroscopic precipitate was isolated by way of suction filtration, washed with diethyl ether, and redissolved in water prior to loading onto a plug of strong cation exchange material. The plug was washed with 1:1 methanol/water and the product eluted with the same solvents, with the addition of some aqueous ammonia. Removal of solvent under reduced pressure gave the product as a film which could be scraped off the glass wall to provide a yellow powder (35.4 mg, 62.1%). No meaningful NMR data could be recorded. HPLC purity was estimated to 70-80%, using phosphate buffering to pH 11.5, as described above. MS (ESI, neg. mode) m/z 522.2 [M−H]-, HR-MS (ESI, neg. mode) m/z 521.1991 calculated for C.sub.28H.sub.25N.sub.5O.sub.5.sup.10B, found m/z 521.1989.

Example 166—tert-butyl 3-(3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)benzoate

(303) ##STR00186##

(304) (3-(tert-Butoxycarbonyl)phenyl)boronic acid (978 mg, 4.40 mmol) was suspended in 40 mL Et.sub.2O and mixed with (+)-pinanediol (750 mg, 4.40 mmol) and anhydrous MgSO.sub.4 (1 gram, 8.30 mmol). The mixture was stirred at room temperature under argon for 3 hours before the inorganic material was filtered off and the filtrate concentrated under reduced pressure. This gave a sticky clear oil (1568 mg, 4.40 mmol, >99%) which solidified overnight at room temperature. .sup.1H NMR (600 MHz, Chloroform-d) δ 8.41 (t, J=1.5 Hz, 1H), 8.07 (dt, J=7.8, 1.6 Hz, 1H), 7.95 (dt, J=7.4, 1.4 Hz, 1H), 7.42 (t, J=7.6 Hz, 1H), 4.47 (dd, J=8.8, 1.9 Hz, 1H), 2.42 (ddt, J=14.8, 8.8, 2.4 Hz, 1H), 2.30-2.20 (m, 1H), 2.16 (t, J=5.5 Hz, 1H), 2.05-1.89 (m, 2H), 1.60 (s, 10H), 1.49 (s, 3H), 1.31 (s, 3H), 1.19 (s, 1H), 0.89 (s, 3H). .sup.13C NMR (151 MHz, CDCl.sub.3) δ 166.04, 138.84, 135.78, 132.22, 131.60, 127.79, 86.62, 81.12, 78.57, 77.37, 77.16, 76.95, 66.00, 51.53, 39.67, 38.35, 35.64, 28.84, 28.37, 27.25, 26.65, 24.20, 15.43. MS (ESI positive mode): m/z 379.2 (M+Na.sup.+)

Example 167—6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinic Acid

(305) ##STR00187##

Example 167a—tert-Butyl 4-(6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinamido) phenethylcarbamate

(306) ##STR00188##

(307) 6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinic acid from Example 13 (1.66 g, 4.97 mmol, 1.0 eq.) was dissolved in 20 mL dry DMF at room temperature and filtered into a 50 mL round bottomed flask prior to reaction to remove the insoluble inorganics. To this solution was added tert-butyl 4-aminophenethylcarbamate (1.76 g, 7.45 mmol, 1.5 eq.), followed by EDC (1.428 g, 7.45 mmol, 1.5 eq.), HOAt (1.014 g, 7.45 mmol, 1.5 eq.) and NMM (0.821 mL, 7.45 mmol, 1.5 eq.). The reaction mixture was stirred at room temperature for 16 h and then concentrated under reduced pressure. The residual crude mixture was dissolved in 100 mL CHCl.sub.3, transferred into a separation funnel and washed with sat. aq. K.sub.2CO.sub.3 solution (100 mL) and brine (100 mL). The organic phase was dried oved Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. Purification of the product was performed by column chromatography on Alumina using 1% MeOH in CH.sub.2C2 giving the product with minor impurities, followed by C18-SPE using gradient elution from 10% to 90% methanol in water affording 1.697 g (3.07 mmol, 62%) of the product as a yellow oil. .sup.1H NMR (300 MHz, MeOH) δ 8.96 (d, J=1.8 Hz, 1H), 8.46-8.39 (m, 2H), 8.23 (dd, J=8.2, 2.3 Hz, 1H), 7.76 (td, J=7.7, 1.5 Hz, 3H), 7.62 (dd, J=13.5, 8.1 Hz, 4H), 7.24 (ddd, J=7.3, 5.0, 1.1 Hz, 2H), 7.17 (d, J=8.5 Hz, 2H), 3.89 (s, 2H), 3.85 (s, 4H), 3.24 (t, J=7.3 Hz, 2H), 2.72 (t, J=7.3 Hz, 2H), 1.40 (s, 9H). .sup.13C NMR (101 MHz, MeOD) δ 166.10, 163.40, 159.85, 158.28, 149.56, 148.92, 138.55, 137.76, 137.41, 137.15, 130.88, 130.16, 124.83, 124.14, 123.81, 122.23, 79.86, 61.10, 60.84, 42.95, 36.59, 28.79. APCI-HRMS e/z calc. for C.sub.32H.sub.36N.sub.6O.sub.3: 552.2849, found: 553.2920 [M+H].

Example 167b—N-(4-(2-aminoethyl)phenyl)-6-((bis(pyridin-2-ylmethyl)amino) methyl)nicotinamide

(308) ##STR00189##

(309) The tert-butyl 4-(6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinamido)phenethylcarbamate from example 167a (1.697 g, 3.07 mmol, 1.0 eq.) was dissolved in 10 mL CH.sub.2Cl.sub.2 at room temperature. To this solution was added TFA (5 mL) and the mixture stirred at room temperature until TLC (Alumina, 5% MeOH in CH.sub.2C2) or NMR indicated full conversion. The mixture was concentrated under reduced pressure, the residue dissolved in a mixture of CHCl.sub.3/dest. H.sub.2O/sat. aq. K.sub.2CO.sub.3 (100 mL/10 mL/100 mL) and transferred into a separation funnel. The organic phase was separated, the aq. phase extracted with CHCl.sub.3 (2 times 50 mL) and the combined organics washed with brine (100 m), dried over K.sub.2CO.sub.3/Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to afford N-(4-(2-aminoethyl)phenyl)-6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinamide in quantitative yield. .sup.1H NMR (300 MHz, MeOH) δ 8.97 (d, J=1.7 Hz, 1H), 8.45 (ddd, J=5.0, 1.7, 0.9 Hz, 2H), 8.26 (dd, J=8.2, 2.3 Hz, 1H), 7.80 (td, J=7.6, 1.6 Hz, 3H), 7.68 (d, J=7.8 Hz, 2H), 7.62 (d, J=8.5 Hz, 2H), 7.28 (ddd, J=7.4, 5.0, 1.2 Hz, 2H), 7.23 (d, J=8.5 Hz, 2H), 3.94 (s, J=4.3 Hz, 2H), 3.90 (s, 4H), 2.88 (t, J=6.7 Hz, 2H), 2.76 (t, J=6.9 Hz, 2H). .sup.13C NMR (101 MHz, MeOD) δ 163.55, 159.96, 149.62, 148.93, 138.69, 137.51, 131.02, 130.19, 124.99, 124.32, 123.92, 122.54, 79.46, 61.24, 60.96, 44.03, 39.22. APCI-HRMS e/z calc. for C.sub.27H.sub.28N.sub.6O: 452.2325, found: 453.2396 [M+H].

Example 168—6-((bis(pyridin-2-ylmethyl)amino)methyl)-N-(4-(2-(6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinamido)ethyl)phenyl)nicotinamide

(310) ##STR00190##

(311) 6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinic acid from example 13 (70.9 mg, 0.212 mmol, 1.05 eq.) and N-(4-(2-aminoethyl)phenyl)-6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinamide from example 17 (91.3 mg, 0.202 mmol, 1.0 eq.) were dissolved in 3 mL dry DMF cooled to 0° C. in an ice bath. HATU (80.6 mg, 0.212 mmol, 1.05 eq.) and NMM (49 μL, 0.444 mmol, 2.1 eq.) were added and the mixture stirred at 0° C. for 1h, then at room temperature for 16 h. The mixture was concentrated under reduced pressure and the product purified by dry column vacuum chromatography on C18 material, using a stepwise elution from 10% to 90% methanol in water affording 84 mg (0.11 mmol, 55%) of product. .sup.1H NMR (300 MHz, MeOH) δ 8.96 (d, J=1.7 Hz, 1H), 8.80 (d, J=1.6 Hz, 1H), 8.43 (tdd, J=2.5, 1.6, 0.8 Hz, 4H), 8.25 (dd, J=8.2, 2.3 Hz, 1H), 8.09 (dd, J=8.2, 2.3 Hz, 1H), 7.78 (dtd, J=7.9, 6.3, 1.8 Hz, 5H), 7.73-7.58 (m, 7H), 7.27 (ddd, J=8.7, 6.1, 3.0 Hz, 6H), 3.93 (s, 2H), 3.89 (s, 2H), 3.88 (s, 4H), 3.86 (s, 4H), 3.61 (t, J=7.2 Hz, 2H), 2.91 (t, J=7.2 Hz, 2H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 167.80, 166.28, 163.50, 163.27, 159.91, 149.60, 149.56, 148.97, 148.58, 138.67, 138.65, 137.96, 137.49, 137.12, 137.07, 130.99, 130.43, 130.28, 124.96, 124.28, 123.91, 123.88, 122.38, 61.27, 61.20, 60.95, 42.50, 35.88.

(312) APCI-HRMS e/z calc. for C.sub.46H.sub.44N.sub.10O.sub.2: 768.3649, found: 769.3719 [M+H].

Example 169—6-((bis(pyridin-2-ylmethyl)amino)methyl)-N-(2-(tritylthio)ethyl) nicotinamide—Examples 169 and 170 are an Alternative Method to Prepare the Compound of Example 158

(313) ##STR00191##

(314) 6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinic acid (0.763 g, 2.343 mmol, 1.0 eq.), prepared in example 13, and 2-(tritylthio)ethanamine (0.823 g, 2.577 mmol, 1.1 eq.) were dissolved in 15 mL dry DMF cooled to 0° C. in an ice bath. HATU (0.98 g, 2.577 mmol, 1.1 eq.) and NMM (0.284 mL, 2.577 mmol, 2.0 eq) were added. The mixture was stirred at 0° C. for 30 min, then at room temperature for 16 h and concentrated under reduced pressure. Purification of the product was achieved by way of dry column vacuum chromatography on C18 material, using a stepwise elution from 10% to 90% methanol in water affording 1.053 g (1.65 mmol, 71%) of product.

(315) .sup.1H NMR (400 MHz, MeOD) δ 8.82 (d, J=2.0 Hz, 1H), 8.41 (d, J=4.9 Hz, 2H), 8.09 (dd, J=8.2, 2.1 Hz, 1H), 7.75 (td, J=7.7, 1.3 Hz, 2H), 7.70 (d, J=8.2 Hz, 1H), 7.63 (d, J=7.8 Hz, 2H), 7.36 (d, J=7.5 Hz, 6H), 7.22 (dd, J=9.9, 4.8 Hz, 8H), 7.16 (t, J=7.2 Hz, 3H), 3.89 (s, J=7.4 Hz, 2H), 3.86 (s, 4H), 3.28 (d, J=6.8 Hz, 2H), 2.45 (t, J=6.9 Hz, 2H). .sup.13C NMR (101 MHz, MeOD) δ 148.18, 147.30, 137.28, 135.85, 129.31, 127.54, 126.44, 123.57, 122.93, 122.51, 59.88, 59.58, 38.56, 31.31.

Example 170—6-((bis(pyridin-2-ylmethyl)amino)methyl)-N-(2-mercaptoethyl) nicotinamide (Same as Example 158)

(316) ##STR00192##

(317) 6-((bis(pyridin-2-ylmethyl)amino)methyl)-N-(2-(tritylthio)ethyl)nicotinamide (1.053 g, 1.65 mmol, 1.0 eq.) was dissolved in 25 mL CH.sub.2C2 at room temperature. To this solution was added TFA (25 mL, 330 mmol, 200 eq.) and HSiEt.sub.3 (0.527 mL, 3.3 mmol, 2.0 eq.). The mixture was stirred at room temperature for 30 min, then concentrated under reduced pressure to remove as much TFA as possible. The residue was dissolved in 100 mL CH.sub.2C2 and transferred into a separation funnel. The organic phase was extracted with 100 mL H2O, the aqueous phase washed with CH.sub.2Cl.sub.2 (5 times 50 mL), and the pH adjusted to 10 with sat. aq. K.sub.2CO.sub.3 solution. The aqueous phase was then extracted with CH.sub.2C2 (3 times 100 mL), the combined organics dried over Na.sub.2SO.sub.4 and K.sub.2CO.sub.3, filtered and concentrated under reduced pressure to afford 0.549 g (1.397 mmol, 85%) of the thiol which was used immediately for the next step without further purification or stored in a tightly sealed vial in the freezer. .sup.1H NMR (300 MHz, MeOH) δ 8.87 (d, J=1.8 Hz, 1H), 8.44 (d, J=4.2 Hz, 2H), 8.16 (dd, J=8.2, 2.3 Hz, 1H), 7.78 (ddd, J=12.1, 9.0, 5.0 Hz, 3H), 7.66 (d, J=7.8 Hz, 2H), 7.36-7.19 (m, 2H), 3.92 (s, 2H), 3.88 (s, 4H), 3.54 (t, J=7.0 Hz, 2H), 2.72 (t, J=6.9 Hz, 2H). APCI-HRMS e/z calc. for C.sub.21H.sub.23N.sub.5OS: 393.1623, found 394.1695 [M+H].

Example X (OAHA-VII-75)

Example 171—tert-Butyl 2-(3-((S)-2-chloro-2-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)benzoate

(318) ##STR00193##

(319) Matteson-homologation of tert-butyl (3-(((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)methyl)benzoate (3.03 g, 6.24 mmol) was performed as described in Example 154 and in Reddy et al WO2016003929 to yield the title compound.

Example 172—tert-butyl 3-(2-((2-(6-((bis(pyridin-2-ylmethyl)amino)methyl) nicotinamido)ethyl)thio)-2-(3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)benzoate

(320) ##STR00194##

(321) The thiol (254 mg, 0.64 mmol) from Example 174 was dissolved in 8 mL THF and placed under argon and cooled to 0° C. DIPEA (250 μL) was then added followed by the chloride from Example 175 (52 mg, 0.12 mmol) with the aid of 2 mL THF. The mixture was stirred for 30 minutes at 0° C. and then at room temperature overnight. The mixture was then concentrated under reduced pressure and the crude material was purified on neutral Al.sub.2O.sub.3 using 0-5% MeOH in DCM giving 32 mg of a pale yellow oil. MS (APCI positive mode) m/z 776.401 (M+H). C.sub.44H.sub.54BN.sub.5O.sub.5S calculated to 775.39.

Example 173—3-(2-((2-(6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinamido) ethyl)thio)-2-boronoethyl)benzoic Acid

(322) ##STR00195##

(323) Global deprotection and cyclisation was achieved as described in Example 157 by treatment of a solution of the doubly protected precursor from Example 172 (64.1 mg, 0.0703 mmol) in 2 mL dioxane with 1 mL 4 M HC in dioxane.

Example 174—3-(2-((2-(6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinamido) ethyl)thio)-2-boronoethyl)benzoic Acid Sodium Salt

(324) ##STR00196##

(325) A solution of the sodium salt of Example 173 is prepared by adding 1 molar equivalent NaOH to a solution of Example 173 in 50:50 dioxane/water.

Example 175—N-allyl-6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinamide

(326) ##STR00197##

(327) The ester (1043 mg, 2.99 mmol) was dissolved in THF (5 mL) and LiOH hydrate (376 mg, 8.97 mmol) was added with the aid of 5 mL water. The mixture was stirred for 3 hours at room temperature before 10 mL 1M HCl was added and the mixture was concentrated under reduced pressure. The crude acid was taken up into 20 mL DMF and HATU (1140 mg, 2.99 mmol), the amine (751 μL, 9.99 mmol) and NMM (751 μL, 4.00 mmol) was added. The mixture was stirred at room temperature overnight before it was concentrated under reduced pressure. The crude material was suspended in 250 mL 1M K.sub.2CO.sub.3 and extracted with 5×25 mL EtOAc. The combinder organic fractions were pooled, dried over K.sub.2CO.sub.3, filtered and concentrated under reduced pressure to give a pale brown oil. The material was purified on neutral Al.sub.2O.sub.3 using 0-5% MeOH in DCM as eluent. A total of 726 mg (65%) of clean product was obtained. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.93 (dd, J=2.3, 0.8 Hz, 1H), 8.77 (t, J=5.7 Hz, 1H), 8.49 (ddd, J=4.9, 1.8, 0.9 Hz, 2H), 8.17 (dd, J=8.1, 2.3 Hz, 1H), 7.77 (td, J=7.6, 1.9 Hz, 2H), 7.69 (dd, J=8.2, 0.8 Hz, 1H), 7.58 (dt, J=7.8, 1.1 Hz, 2H), 7.25 (ddd, J=7.4, 4.8, 1.2 Hz, 2H), 5.89 (ddt, J=17.2, 10.4, 5.3 Hz, 1H), 5.27-5.01 (m, 2H), 3.91 (tt, J=5.5, 1.7 Hz, 2H), 3.85 (s, 2H), 3.80 (s, 4H). .sup.13C NMR (101 MHz, DMSO) δ 164.52, 161.79, 158.75, 148.82, 147.70, 136.52, 135.41, 135.09, 128.29, 122.59, 122.14, 121.95, 115.27, 59.37, 59.15, 41.40. MS (APCI positive mode) m/z 374.20 [M+H]-, HRMS (APCI positive mode) m/z 373.1904 calculated for C.sub.22H.sub.23N.sub.5O, found m/z 374.1975.

Example 176—Methyl 5-(hydroxymethyl)thiophene-2-carboxylate

(328) ##STR00198##

(329) Methyl 5-formylthiophene-2-carboxylate (1.0 g, 5.87 mmol, 1.0 eq.) was dissolved in 50 mL MeOH cooled to 0° C. in an ice bath. To this solution was added NaBH4 (0.445 g, 11.75 mmol, 2.0 eq.) slowly in portions and the reaction was monitored by TLC (SiO2, 9:1 n-hexanes:EtOAc). Upon full conversion of the aldehyde, the reaction was quenched by slow addition of ice cooled water (10 mL). The mixture was then transferred into a separation funnel, diluted with 50 mL H2O end extracted with diethyl ether (3 times 100 mL). The combined organics were washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The product was obtained pure (0.935 g, 5.4 mmol, 92%) and used for the next step without further purification.

(330) .sup.1H NMR (300 MHz, CDCl.sub.3) δ 7.67 (d, J=3.8 Hz, 1H), 6.98 (dt, J=3.8, 0.8 Hz, 1H), 4.85 (d, J=0.7 Hz, 2H), 3.87 (s, 3H), 1.95 (s (br), 1H).

Example 177—Methyl 5-(chloromethyl)thiophene-2-carboxylate

(331) ##STR00199##

(332) The methyl 5-(hydroxymethyl)thiophene-2-carboxylate prepared in the previous reaction (0.935 g, 5.4 mmol, 1.0 eq.) was dissolved in 50 mL CH2C2 cooled to 0° C. in an ice bath. To this was added a solution of SOCl.sub.2 (0.787 mL, 10.8 mmol, 2.0 eq.) in 10 mL CH2C12 dropwise via a dropping funnel. The mixture was stirred at 0° C. for 1 h, then at room temperature for 16 h. TLC control indicated full conversion of the alcohol (SiO2, 5:1 n-hexanes:EtOAc) and the mixture was concentrated under reduced pressure. The residue was dissolved in CH2C2 (100 mL), transferred into a separation funnel and washed with sat. aq. K2CO3 solution (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. Column chromatography on silica using gradient elution (n-hexanes to 9:1 n-hexanes:EtOAc) afforded 0.754 g (3.9 mmol, 73%) of methyl 5-(chloromethyl)thiophene-2-carboxylate as a pale yellow oil. .sup.1H NMR (300 MHz, CDCl.sub.3) δ 7.65 (d, J=3.8 Hz, 1H), 7.07 (dt, J=3.8, 0.7 Hz, 1H), 4.76 (d, J=0.6 Hz, 2H), 3.88 (s, J=1.6 Hz, 3H).

Example 178—Methyl 5-((bis(thiophen-2-ylmethyl)amino)methyl)thiophene-2-carboxylate

(333) ##STR00200##

(334) The methyl 5-(chloromethyl)thiophene-2-carboxylate obtained in the previous reaction (0.405 g, 2.12 mmol, 1.0 eq.) was dissolved in 25 mL dry CH3CN at room temperature. To this solution was added bis(thiophen-2-ylmethyl)amine hydrochloride (0.575 g, 2.34 mmol, 1.1 eq.), KI (0.352 g, 2.12 mmol, 1.0 eq.) and K2CO3 (0.877 g, 6.3 mmol, 3.0 eq.). The mixture was heated to reflux and stirred for 16 h, then filtered into a new flask and concentrated under reduced pressure. The oily residue was treated with 50 mL EtOAc, transferred into a separation funnel and washed with H2O (30 mL), sat. aq. K2CO3 (30 mL) and brine (30 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. Column chromatography on silica using gradient elution (n-hexanes to 9:1 n-hexanes:EtOAc) afforded 0.488 g (1.34 mmol, 63%) of methyl 5-((bis(thiophen-2-ylmethyl)amino)methyl)thiophene-2-carboxylate. .sup.1H NMR (300 MHz, CDCl.sub.3) δ 7.26 (d, J=2.0 Hz, 1H), 7.25 (d, J=1.9 Hz, 1H), 7.00-6.93 (m, 5H), 3.89 (s, 4H), 3.88 (s, 3H), 3.85 (s, 2H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 162.92, 151.22, 141.95, 133.48, 132.57, 126.63, 126.26, 126.12, 125.30, 52.16, 51.85, 51.81. APCI-HRMS e/z calc. for C.sub.17H.sub.17NO.sub.2S.sub.3: 363.0421, found 364.0494 [M+H].

Example 179—Methyl 6-((bis(thiophen-2-ylmethyl)amino)methyl)nicotinate

(335) ##STR00201##

(336) Methyl 6-(bromomethyl)nicotinate (1.044 g, 4.53 mmol, 1.0 eq.) was suspended in 60 mL THF at room temperature. To this mixture was added bis(thiophen-2-ylmethyl)amine hydrochloride (1.222 g, 5.0 mmol, 1.1 eq.) and DIPEA (2.13 mL, 12.23 mmol, 2.7 eq.) and stirred at room temperature for 16 h. The mixture was filtered into a new flask and concentrated under reduced pressure, resulting in a brownish semi-solid. The residue was treated with diethyl ether (100 mL), transferred into a separation funnel and washed with H2O (50 mL) and brine (50 mL). The organic phase was dried over Na2SO4, filtered and concentrated under reduced pressure. Column chromatography on silica using gradient elution (n-hexanes to 5:1 n-hexanes:EtOAc) afforded 0.184 g (0.51 mmol, 11%) of methyl 6-((bis(thiophen-2-ylmethyl)amino)methyl)nicotinate. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.10 (d, J=1.6 Hz, 1H), 8.30 (dd, J=8.2, 2.2 Hz, 1H), 7.83 (d, J=8.1 Hz, 1H), 7.24 (dd, J=4.9, 1.3 Hz, 2H), 7.01-6.91 (m, 4H), 3.94 (s, 3H), 3.90 (s, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 166.02, 150.29, 137.83, 126.73, 126.20, 125.25, 52.54, 52.45, 31.07. APCI-HRMS e/z calc. for C.sub.18H.sub.18N.sub.2O.sub.2S.sub.2: 358.0810, found 359.0882 [M+H].

Example 180—Methyl 5-((bis(pyridin-2-ylmethyl)amino)methyl)thiophene-2-carboxylate

(337) ##STR00202##

(338) The aldehyde (851 mg, 5.0 mmol) was added in one portion to a stirring solution of the amine (996 mg, 5.0 mmol) in 20 mL dichloroethane. The mixture was stirred for 3 hours at room temperature before NaBH(OAc).sub.3 (5.3 grams, 25 mmol) was added. The mixture was then stirred overnight before it was concentrated under reduced pressure to a sticky pale orange solid. This was diluted with 100 mL 0.5M K2CO3 and extracted with 3×25 mL DCM. The combined organic fractions were pooled, dried over K2CO3, filtered and concentrated under reduced pressure. The crude mixture was purified on neutral Al.sub.2O.sub.3 using 10-100% EtOAc in DCM which gave 885 mg (50%) of a brown solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.50 (ddd, J=4.8, 1.8, 0.9 Hz, 2H), 7.81 (td, J=7.7, 1.8 Hz, 2H), 7.67 (d, J=3.7 Hz, 1H), 7.57 (dt, J=7.8, 1.1 Hz, 2H), 7.27 (ddd, J=7.5, 4.9, 1.2 Hz, 2H), 7.14-7.04 (m, 1H), 3.93-3.86 (m, 2H), 3.80 (s, 3H), 3.78 (s, 4H). .sup.13C NMR (101 MHz, DMSO) δ 161.87, 158.51, 151.56, 148.87, 136.70, 133.66, 131.34, 126.85, 122.40, 122.29, 58.85, 52.21, 52.07.

Example 181—5-((bis(pyridin-2-ylmethyl)amino)methyl)thiophene-2-carboxylic acid

(339) ##STR00203##

(340) The methyl 5-((bis(pyridin-2-ylmethyl)amino)methyl)thiophene-2-carboxylate (0.325 g, 0.919 mmol, 1.0 eq.), prepared in the previous example, was dissolved in a mixture of 10 mL THF and 10 mL H2O at room temperature. To this solution was added LiOH.H2O (0.193 g, 4.6 mmol, 5 eq.) and the reaction progress monitored by TLC on alumina using 5% MeOH in CH2C12. Upon full conversion, the crude reaction mixture was concentrated under reduced pressure and the residue dissolved in 5 mL dest. H2O. The pH of the basic solution was adjusted to 4 with 2M HCl and the mixture concentrated under reduced pressure. The obtained 5-((bis(pyridin-2-ylmethyl)amino)methyl)thiophene-2-carboxylic acid was used for the next reaction without further purification after confirmation of purity by 1H NMR.

(341) .sup.1H NMR (300 MHz, D.sub.2O) δ 8.37 (d, J=4.7 Hz, 2H), 7.76 (t, J=7.7 Hz, 2H), 7.52 (d, J=7.9 Hz, 2H), 7.38 (d, J=3.7 Hz, 1H), 7.34-7.23 (m, 2H), 6.93 (d, J=3.5 Hz, 1H), 3.88 (s, 2H), 3.80 (s, 4H).

Example 182—5-((bis(pyridin-2-ylmethyl)amino)methyl)-N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)thiophene-2-carboxamide

(342) ##STR00204##

(343) The 5-((bis(pyridin-2-ylmethyl)amino)methyl)thiophene-2-carboxylic acid obtained in the previous reaction (0.312 g, 0.919 mmol, 1.0 eq.) was dissolved in 5 mL dry DMF at room temperature. N-Methyl-D-Glucamine (0.269 g, 1.378 mmol, 1.5 eq.), EDCl (0.264 g, 1.378 mmol, 1.5 eq.), HOAt (0.187 g, 1.378 mmol, 1.5 eq.) and NMM (0.152 mL, 1.378 mmol, 1.5 eq.) were then added. The mixture was heated to 50° C. for 16 h with stirring and then concentrated under reduced pressure. Purification of the product was achieved by way of dry column vacuum chromatography on C18 bondesil material, using a stepwise elution from 10% to 90% methanol in water affording 0.357 g (0.691 mmol, 75%) of product.

(344) .sup.1H NMR (400 MHz, MeOD) δ 8.44 (d, J=4.5 Hz, 2H), 7.83 (t, J=7.6 Hz, 2H), 7.72 (d, J=7.9 Hz, 2H), 7.39 (d, J=7.2 Hz, 1H), 7.32-7.26 (m, 2H), 6.98 (s, 1H), 4.16-4.06 (m, 1H), 3.90 (s, 2H), 3.84 (s, 4H), 3.81-3.55 (m, 6H), 3.34 (d, J=8.2 Hz, 1H), 2.15 (s, 3H).

(345) .sup.13C NMR (101 MHz, MeOD) δ 149.21, 138.53, 131.04, 126.94, 124.36, 123.67, 72.73, 64.47, 60.16, 53.80, 30.39.

(346) APCI-HRMS e/z calc. for C.sub.25H.sub.32N.sub.4O.sub.6S: 516.2043, found 517.2115 [M+H].

Example 183—methyl 6-((dibenzylamino)methyl)nicotinate

(347) ##STR00205##

(348) Dibenzylamine (986 mg, 5 mmol) was dissolved in 100 mL THF and mixed with methyl bromomethyl nicotinate (1150 mg, 5 mmol) and DIPEA (1,275 mL, 7.5 mmol). The mixture was stirred at room temperature for 24 hours before it was filtered through a plug of celite. The filtrate was concentrated under reduced pressure, re-dissolved in 50 mL Et2O and filtered through a plug of celite. The filtrate was concentrated under reduced pressure to give 1.66 grams (96%) of material. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.98 (d, J=2.2 Hz, 1H), 8.27 (dd, J=8.1, 2.2 Hz, 1H), 7.72 (d, J=8.1 Hz, 1H), 7.39 (d, J=7.1 Hz, 4H), 7.33 (t, J=7.5 Hz, 4H), 7.24 (t, J=7.2 Hz, 2H), 3.86 (s, 3H), 3.71 (s, 2H), 3.57 (s, 4H). .sup.13C NMR (101 MHz, DMSO) δ 165.19, 164.31, 149.38, 138.61, 137.33, 128.57, 128.29, 127.04, 123.99, 122.25, 58.66, 57.35, 52.29.

Example 184—N-benzyl-1-(pyridin-2-yl)methanamine

(349) ##STR00206##

(350) Benzylamine (1000 mg, 9.33 mmol) was dissolved in 50 mL absolute ethanol at room temperature and mixed with 2-pyridinecarboxaldehyde (1000 mg, 9.34 mmol). The solution turned read and it was heated to 50° C. and stirred for 24 hours before NaBH4 (2500 mg, 66.09 mmol) was added in portions. The mixture was then stirred for 4 days at 50° C. before it was concentrated under reduced pressure to give a yellow solid. The crude solid was dissolved in 1M K2CO3 (100 mL) and extracted with DCM (3×50 mL). The combined organic fractions were pooled, dried over K2CO3, filtered and concentrated under reduced pressure to give 1.826 grams (99%) of a pale yellow oil. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.70-8.21 (m, 1H), 7.75 (td, J=7.7, 1.8 Hz, 1H), 7.46 (d, J=7.8 Hz, 1H), 7.39-7.28 (m, 5H), 7.27-7.19 (m, 2H), 3.78 (s, 2H), 3.72 (s, 2H), 2.70 (s, 1H). .sup.13C NMR (101 MHz, DMSO) δ 160.29, 148.70, 140.69, 136.40, 128.10, 127.92, 126.54, 121.81, 121.74, 53.81, 52.41.

Example 185—6-((dibenzylamino)methyl)-N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)nicotinamide

(351) ##STR00207##

(352) The ester (1.73 grams, 5.0 mmol) was dissolved in 10 mL THF and LiOH monohydrate (1049 mg, 25.0 mmol) was added with the aid of 2 mL water. The mixture was stirred for 2 hours at room temperature before 20 mL 1M HCl (20.0 mmol) was added to neutralize the reaction. The mixture was then concentrated under reduced pressure to give a white powder which was dissolved in 15 mL DMF. EDC hydrochloride (959 mg, 5.0 mmol), meglumine (976 mg, 5.0 mmol) and HOAT (681 mg, 5.0 mmol) was added before NMM (1.10 mL, 10.0 mmol) was added. The mixture was heated to 50° C. and left for 18 hours before it was cooled to room temperature and concentrated under reduced pressure. Purification of the product was achieved by way of dry column vacuum chromatography on C18 bondesil material, using a stepwise elution from 10% to 80% methanol in water affording 0.457 g (0.9 mmol, 18%) of product.

(353) .sup.1H NMR (400 MHz, MeOD) δ 8.54 (d, J=21.2 Hz, 1H), 7.91 (dd, J=36.5, 7.6 Hz, 1H), 7.73 (d, J=8.1 Hz, 1H), 7.40 (d, J=7.4 Hz, 4H), 7.35-7.25 (m, 4H), 7.22 (t, J=7.0 Hz, 2H), 4.24-3.98 (m, 1H), 3.87-3.45 (m, 12H), 3.14, 3.07 (2×s, 3H). .sup.13C NMR (101 MHz, MeOD) δ 172.09, 171.47, 162.93, 162.35, 148.23, 147.65, 140.21, 137.80, 137.05, 132.52, 132.24, 130.01, 129.37, 128.23, 124.04, 123.75, 73.96, 73.49, 73.01, 72.91, 72.42, 71.62, 71.51, 71.01, 64.75, 60.21, 59.96, 59.64, 59.37, 55.24, 52.44, 40.02, 33.75. APCI-HRMS e/z calc. for C.sub.28H.sub.35N.sub.3O.sub.6: 509.2526, found 510.2599 [M+H].

Example 186—methyl 6-((benzyl(pyridin-2-ylmethyl)amino)methyl)nicotinate

(354) ##STR00208##

(355) N-benzyl-1-(pyridin-2-yl)methanamine (991 mg, 5 mmol) was dissolved in 30 mL THF and mixed with methyl bromomethyl nicotinate (1150 mg, 5 mmol) and DIPEA (1.36 mL, 8.0 mmol). The mixture was stirred at room temperature for 24 hours before it was filtered through a plug of celite. The filtrate was concentrated under reduced pressure, re-dissolved in 50 mL Et20 and filtered through a plug of celite. The filtrate was concentrated under reduced pressure to give 1.66 grams (96%) of material.

Example 187—6-((benzyl(pyridin-2-ylmethyl)amino)methyl)-N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)nicotinamide

(356) ##STR00209##

(357) The ester (973 mg, 2.8 mmol) was dissolved in 10 mL THF and LiOH monohydrate (588 mg, 14.0 mmol) was added with the aid of 2 mL water. The mixture was stirred for 2 hours at room temperature before 11.2 mL 1M HCl (11.2 mmol) was added to neutralize the reaction. The mixture was then concentrated under reduced pressure to give a white powder which was dissolved in 10 mL DMF. EDC hydrochloride (537 mg, 2.8 mmol), meglumine (547 mg, 2.8 mmol) and HOAT (381 mg, 2.8 mmol) was added before NMM (616 μL, 5.6 mmol) was added. The mixture was heated to 50° C. and left for 18 hours before it was cooled to room temperature and concentrated under reduced pressure. Purification of the product was achieved by way of dry column vacuum chromatography on C18 bondesil material, using a stepwise elution from 10% to 70% methanol in water affording 0.72 g (1.4 mmol, 50%) of product. .sup.1H NMR (400 MHz, MeOD) δ 8.56 (d, J=17.6 Hz, 1H), 8.43 (dd, J=4.9, 0.7 Hz, 1H), 7.91 (ddd, J=33.7, 8.0, 1.8 Hz, 1H), 7.80 (t, J=7.6 Hz, 1H), 7.72 (dd, J=7.7, 5.8 Hz, 1H), 7.68 (d, J=7.9 Hz, 1H), 7.40 (d, J=7.4 Hz, 2H), 7.35-7.15 (m, 4H), 4.21-3.97 (m, 1H), 3.88-3.46 (m, 12H), 3.14, 3.07 (2×s, 3H). .sup.13C NMR (101 MHz, MeOD) δ 172.03, 171.41, 162.28, 161.70, 160.43, 149.42, 148.37, 147.83, 139.69, 138.72, 137.78, 137.06, 132.58, 132.31, 130.10, 129.42, 128.36, 124.79, 124.15, 123.84, 73.93, 73.45, 73.00, 72.90, 72.40, 71.63, 71.51, 71.02, 64.70, 60.92, 60.70, 60.62, 60.40, 60.02, 59.83, 57.57, 55.22, 52.43, 44.78, 40.01, 33.77. APCI-HRMS e/z calc. for C.sub.27H.sub.34N.sub.4O.sub.6: 510.2478, found 511.2551 [M+H].

Example 188—(2R,3R,4R,5S)-6-(methylamino)hexane-1,2,3,4,5-pentayl pentaacetate hydrochloride

(358) ##STR00210##

(359) Angew. Chem. Int. Ed. 2007, 46, 3284-3287.

(360) N-Methyl-D-Glucamine (2.6 g, 13.3 mmol, 1.0 eq.) was dissolved in 15 mL conc. acetic acid and cooled to 0° C. in an ice bath. To this solution was added acetyl chloride (21.8 mL, 306 mmol, 23 eq.) dropwise over a period of approximately 1 h. The mixture was left stirring for 16 h while the ice bath expired. The colorless solution was then concentrated under reduced pressure to dryness. The obtained oily residue was dissolved in a mixture of methanol (8 mL) and ethanol (5 mL) and diluted with EtOAc (50 mL). Diethyl ether was added with stirring until the mixture became cloudy and was then placed in the freezer for 12 h. The white precipitate formed was filtered off with suction, though appeared to be hygroscopic and turned into an oil. The oily compound was then dissolved in MeOH, collected and concentrated under reduced pressure to afford a colorless semi-solid that was used for the next reaction without further purification.

Example 189—(2R,3R,4R,5S)-6-(6-((bis(pyridin-2-ylmethyl)amino)methyl)-N-methylnicotinamido)hexane-1,2,3,4,5-pentayl pentaacetate

(361) ##STR00211##

(362) 6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinic acid (0.275 g, 0.823 mmol, 1.0 eq.) was dissolved in 5 mL dry DMF and cooled to 0° C. in an ice bath. To this solution was added (2R,3R,4R,5S)-6-(methylamino)hexane-1,2,3,4,5-pentayl pentaacetate hydrochloride (0.655 g, 1.48 mmol, 1.8 eq.), prepared in the previous reaction, HATU (0.328 g, 0.864 mmol, 1.05 eq.) and NMM (0.272 mL, 2.47 mmol, 3.0 eq.) dropwise. The mixture was stirred at 0° C. for 30 min, then at room temperature for 16 h and concentrated under reduced pressure. Purification of the product was achieved by way of dry column vacuum chromatography on C18 material, using a stepwise elution from 10% to 90% methanol in water, followed by column chromatography on basic alumina using 1% MeOH in CH2C2 affording 177.6 mg (0.246 mmol, 30%) of product. APCI-HRMS e/z calc. for C.sub.36H.sub.43N.sub.5O.sub.11: 721.2959, found 722.3027 [M+H].

Example 190—6-((bis(pyridin-2-ylmethyl)amino)methyl)-N-ethyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)nicotinamide

(363) ##STR00212##

(364) The ester (255 mg, 0.73 mmol) was dissolved in 5 mL THF and LiOH monohydrate (154 mg, 3.67 mmol) was added with the aid of 2 mL water. The mixture was stirred for 2 hours at room temperature before 2.94 mL 1M HCl (2.94 mmol) was added to neutralize the reaction. The mixture was then concentrated under reduced pressure to give a white powder which was dissolved in 5 mL DMF. EDC hydrochloride (140 mg, 0.73 mmol), n-ethyl-d-glucamine (153 mg, 0.73 mmol) and HOAT (99 mg, 0.73 mmol) were added before NMM (161 μL, 1.47 mmol) was added. The mixture was heated to 50° C. and left for 18 hours before it was cooled to room temperature and concentrated under reduced pressure. Purification of the product was achieved by way of dry column vacuum chromatography on C18 bondesil material, using a stepwise elution from 20% to 50% methanol in water affording 0.135 g (0.22 mmol, 30%) of product.

(365) .sup.1H NMR (400 MHz, MeOD) δ 8.55 (d, J=23.8 Hz, 1H), 8.44 (d, J=4.5 Hz, 2H), 7.85 (dt, J=15.6, 7.4 Hz, 3H), 7.70 (dd, J=19.7, 8.4 Hz, 3H), 7.33-7.23 (m, 2H), 4.22-3.94 (m, 1H), 3.94-3.85 (m, 6H), 3.84-3.33 (m, 9H), 1.27, 1.12 (2×t, J=6.7 Hz, 1H). .sup.13C NMR (101 MHz, MeOD) δ 171.72, 171.57, 161.58, 160.98, 159.93, 149.55, 148.36, 147.32, 138.71, 137.60, 136.47, 133.00, 132.74, 124.98, 124.41, 124.06, 123.92, 74.09, 73.37, 73.00, 72.62, 71.75, 71.44, 64.73, 61.35, 61.12, 60.85, 52.65, 46.66, 41.62, 14.08, 12.66. APCI-HRMS e/z calc. for C.sub.27H.sub.35N.sub.5O.sub.6: 525.2587, found 526.2660 [M+H].

Example 191—Methyl 4-((bis(pyridin-2-ylmethyl)amino)methyl)benzoate

(366) ##STR00213##

(367) Dipicolylamine (1.84 mL, 10 mmol) was dissolved in 150 mL THF and mixed with methyl 2-bromomethyl benzoate (2.29 mg, 10 mmol) and DIPEA (2.7 mL, 16.0 mmol). The mixture was stirred at room temperature for 24 hours before it was filtered through a plug of celite. The filtrate was concentrated under reduced pressure, re-dissolved in 50 mL Et.sub.2O and filtered through a plug of celite. The filtrate was concentrated under reduced pressure to give 3.683 grams (>99%) of material with trace solvent impurities.

Example 192—4-((bis(pyridin-2-ylmethyl)amino)methyl)-N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)benzamide

(368) ##STR00214##

(369) The methyl 4-((bis(pyridin-2-ylmethyl)amino)methyl)benzoate (0.560 g, 1.61 mmol, 1.0 eq.), prepared in the previous example, was dissolved in a mixture of 10 mL THF and 10 mL H2O at room temperature. To this solution was added LiOH.H2O (0.203 g, 4.84 mmol, 3 eq.) and the reaction progress monitored by TLC on alumina using 5% MeOH in CH2C12. After 16 h, the crude reaction mixture was concentrated under reduced pressure and the residue dissolved in 5 mL dest. H2O. The pH of the basic solution was adjusted to 4 with 2M HCl and the mixture concentrated under reduced pressure. The obtained 4-((bis(pyridin-2-ylmethyl)amino)methyl)benzoic acid was used for the next reaction without further purification. The 4-((bis(pyridin-2-ylmethyl)amino)methyl)benzoic acid obtained in the previous reaction (0.537 g, 1.61 mmol, 1.0 eq.) was dissolved in 20 mL dry DMF at room temperature. N-Methyl-D-Glucamine (0.471 g, 2.41 mmol, 1.5 eq.), EDCl (0.462 g, 2.41 mmol, 1.5 eq.), HOAt (0.328 g, 2.41 mmol, 1.5 eq.) and NMM (0.266 mL, 2.41 mmol, 1.5 eq.) were then added. The mixture was heated to 50° C. for 16 h with stirring and then concentrated under reduced pressure. Purification of the product was achieved by way of dry column vacuum chromatography on C18 bondesil material, using a stepwise elution from 10% to 90% methanol in water affording 0.108 g (0.212 mmol, 13%) of product. .sup.1H NMR (400 MHz, MeOD) δ 8.43 (d, J=4.5 Hz, 2H), 7.81 (t, J=7.3 Hz, 2H), 7.68 (d, J=7.8 Hz, 2H), 7.46 (ddd, J=29.4, 16.9, 8.5 Hz, 4H), 7.33-7.19 (m, 2H), 4.21-3.91 (m, 1H), 3.86-3.42 (m, 13H), 3.13, 3.05 (2×s, 3H). .sup.13C NMR (101 MHz, MeOD) δ 174.87, 174.42, 174.29, 167.61, 160.36, 151.72, 149.45, 143.89, 142.08, 141.70, 141.53, 140.45, 138.73, 136.78, 136.42, 131.67, 130.11, 129.94, 128.94, 128.59, 128.40, 128.07, 127.37, 124.78, 124.64, 123.85, 121.06, 74.06, 73.72, 73.61, 73.33, 73.02, 72.92, 72.59, 71.98, 71.62, 71.43, 70.99, 64.76, 62.10, 60.88, 60.72, 60.20, 59.49, 59.36, 55.83, 55.38, 54.38, 52.33, 40.07, 34.01. APCI-HRMS e/z calc. for C.sub.27H.sub.35N.sub.4O.sub.6: 510.2478, found 511.2551 [M+H].

(370) M. N. Discovery of a Cyclic Boronic Acid β-Lactamase Inhibitor (RPX7009) with Utility vs Class A Serine Carbapenemases Hecker et al. J. Med. Chem., 2015, 58, 3682-3692.

Example 193—tert-butyl 3-((3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)methyl)benzoate

(371) ##STR00215##

(372) tert-butyl 3-(bromomethyl)benzoate (1000 mg, 3.69 mmol) was mixed with KOAc (542 mg, 5.53 mmol) and Pd(dppf)Cl2 DCM complex (139 mg, 0.17 mmol) and suspended in 20 mL dioxane. The mixture was purged with argon three times, heated to 90° C. and stirred overnight. The mixture was then cooled to room temperature, concentrated under reduced pressure, dissolved in a 1:1 mixture of heptane:DCM (15 mL) and filtered. The solids were washed with DCM (3×15 mL) and the combined organic filtrate was concentrated under reduced pressure. The crude material was purified by column chromatography on SiO2 using 2-5% EtOAc in heptane giving 899 mg (66%) of a clear oil. .sup.1H NMR (300 MHz, Chloroform-d) δ 7.81 (td, J=1.8, 0.6 Hz, 1H), 7.76 (dt, J=7.5, 1.6 Hz, 1H), 7.36 (ddd, J=7.7, 2.0, 1.4 Hz, 1H), 7.30 (dd, J=7.6, 0.6 Hz, 1H), 4.28 (dd, J=8.7, 2.0 Hz, 1H), 2.38 (s, 2H), 2.31 (ddt, J=14.4, 8.9, 2.4 Hz, 1H), 2.19 (dtd, J=10.9, 6.0, 2.2 Hz, 1H), 2.04 (dd, J=6.0, 5.0 Hz, 1H), 1.93-1.76 (m, 2H), 1.38 (s, 3H), 1.27 (s, 3H), 1.07 (d, J=10.9 Hz, 1H), 0.83 (s, 3H). NMR in according to literature (J. Med. Chem. 2010, 53, 7852)

Examples 194 and 195—Examples of Peptide-Based Analogs for Experimental Comparison with Non-Peptidic Compounds in the Present Invention

Example 194—peptide-based analog—Methyl (2-(4-((bis(pyridin-2-ylmethyl)amino)methyl)1H-1,2,3-triazol-1-yl)acetyl)-D-alanyl-D-alaninate

(373) ##STR00216##

(374) The compound was prepared as described in WO 2015/049546. Copper acetate (200 mg, 1.0 mmol, 1.0 eq.) in 2.5 mL H2O and sodium-(+)ascorbate (396 mg, 2.0 mmol, 2.0 eq.) in 2.5 mL H20 were added simultaneously to a stirring solution of the alkyne (237 mg, 1.0 mmol, 1.0 eq.) in 2.5 mL tBuOH. The azide prepared in Example 32 (257 mg, 1.0 mmol, 1.0 eq) was then added and the solution was stirred at room temperature for 16 hours. EDTA (293 mg, 1.0 mmol, 1.0 eq) was then added to the stirring solution and left for 60 minutes before the mixture was diluted with 50 mL H.sub.20 and the pH of the mixture was adjusted to >10 with 1M NaOH. The slurry was then extracted with 2×50 mL dichloromethane. The combined organic phases were dried over K.sub.2C03 and concentrated under reduced pressure to give a dark red oil. The crude products were purified using column chromatography by eluting a neutral Al.sub.2O.sub.3 column with 0-5% methanol in dichloromethane to give 134 mg of the title compound as a pale orange oil (27%). 1H NMR (400 MHz, DMSO-d6) δ 8.59 (d, J=7.5 Hz, 1H), 8.49 (d, J=4.7 Hz, 2H), 8.43 (d, J=7.2 Hz, 1H), 8.04 (s, 1H), 7.77 (t, J=7.7 Hz, 2H), 7.57 (d, J=7.8 Hz, 2H), 7.29-7.21 (m, 2H), 5.13 (s, 2H), 4.42-4.21 (m, 2H), 3.77-3.70 (m, 6H), 3.62 (s, 3H), 1.31-1.21. (m, 6H). 13C NMR (101 MHz, DMSO) δ 172.8, 171.8, 165.0, 159.0, 148.8, 143.1, 136.5, 125.3, 122.5, 122.1, 58.7, 51.8, 51.4, 48.0, 47.9, 47.5, 31.3, 18.3, 16.8. HRMS e/z calculated for C24H30Ns04: 494.2390, found 495.2463 (M+H).

Example 195—Peptide-Based Analog—N-(4-(2-((R)-2-((R)-2-aminopropanamido)propanamido)ethyl)phenyl)-6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinamide hydrochloride

Example 195a—Tert-butyl (R)-1-((R)-1-(4-(6-((bis(pyridin-2-ylmethyl)amino) methyl)nicotinamido)phenethylamino)-1-oxopropan-2-ylamino)-1-oxopropan-2-yl carbamate

(375) ##STR00217##

(376) N-(4-(2-aminoethyl)phenyl)-6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinamide from Example 167b (218 mg, 0.48 mmol, 1.0 eq.) was dissolved in 3 mL dry DMF, cooled to 0° C. in an ice bath. To this solution was added Boc-D-Ala-D-Ala-OH (132 mg, 0.506 mmol, 1.05 eq.), HATU (192 mg, 0.506 mmol, 1.05 eq.) and NMM (116 μL, 1.056 mmol, 2.2 eq.) and the solution stirred at 0° C. for 30 min, then at room temperature for 16 h. The mixture was concentrated under reduced pressure and the product purified by dry column vacuum chromatography on C18 material, using a stepwise elution from 10% to 90% methanol in water affording 217.7 mg (0.313 mmol, 65%) of the product as a pale yellow oil. .sup.1H NMR (600 MHz, MeOD) δ 8.96 (d, J=1.7 Hz, 1H), 8.44 (d, J=4.3 Hz, 2H), 8.25 (dd, J=8.2, 2.2 Hz, 1H), 7.78 (ddd, J=10.3, 6.1, 2.2 Hz, 3H), 7.67 (d, J=7.9 Hz, 2H), 7.61 (d, J=8.4 Hz, 2H), 7.27 (ddd, J=7.3, 5.1, 0.8 Hz, 2H), 7.20 (d, J=8.5 Hz, 2H), 4.30 (q, J=7.0 Hz, 1H), 4.03 (q, J=7.0 Hz, 1H), 3.92 (s, 2H), 3.88 (s, 4H), 3.48-3.41 (m, 1H), 3.38-3.32 (m, 1H), 2.78 (t, J=7.0 Hz, 2H), 1.44 (s, 9H), 1.30 (dd, J=13.2, 7.0 Hz, 6H). .sup.13C NMR (151 MHz, MeOD) δ 175.63, 174.74, 166.17, 163.43, 159.86, 157.98, 149.57, 148.92, 138.68, 137.89, 137.50, 136.92, 130.97, 130.23, 124.92, 124.24, 123.90, 122.28, 80.73, 61.15, 60.88, 51.95, 50.36, 41.96, 35.88, 28.73, 18.21, 18.02. APCI-HRMS e/z calc. for C.sub.3H.sub.46N.sub.8O.sub.5: 694.3591, found: 695.3659 [M+H].

Example 195b—N-(4-(2-((R)-2-((R)-2-aminopropanamido)propanamido)ethyl)phenyl)-6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinamide hydrochloride

(377) ##STR00218##

(378) (R)-1-((R)-1-(4-(6-((bis(pyridin-2-ylmethyl)amino)methyl)nicotinamido)phenethylamino)-1-oxopropan-2-ylamino)-1-oxopropan-2-ylcarbamate from example 195a (176 mg, 0.253 mmol, 1.0 eq.) was dissolved in 5 mL CH.sub.2Cl.sub.2, cooled to 0° C. in an ice bath. To this solution was added TFA (0.97 mL, 12.65 mmol, 50 eq.) and the mixture was stirred at room temperature until full conversion monitored by .sup.1H-NMR. The mixture was concentrated under reduced pressure, the residue dissolved in a mixture of EtOAc/dest. H.sub.2O/sat. aq. K.sub.2CO.sub.3 (30 mL/10 mL/30 mL) and transferred into a separation funnel. The organic phase was separated, the aq. phase extracted with EtOAc (2 times 30 mL) and the combined organics dried over K.sub.2CO.sub.3, filtered and concentrated under reduced pressure affording a white foamy solid. This was dissolved in 2 mL CH.sub.2C2 and 2 M HCl in diethyl ether was added in excess resulting in a white precipitate. The mixture was stored in the fridge for 4 h, filtered with suction and the solid washed with diethyl ether, turning into a yellowish oil. This oil was dissolved in warm H.sub.2O, collected into a flask and concentrated under reduced pressure to afford 59.4 mg (0.094 mmol, 37%) of product as a yellowish oil. .sup.1H NMR (400 MHz, D.sub.2O) δ 9.14 (s, 1H), 8.78 (d, J=5.8 Hz, 2H), 8.66 (dd, J=8.2, 1.9 Hz, 1H), 8.55 (t, J=7.9 Hz, 2H), 8.10 (t, J=13.5 Hz, 2H), 8.00 (dd, J=11.3, 6.3 Hz, 3H), 7.52 (d, J=7.7 Hz, 2H), 7.33 (d, J=7.2 Hz, 2H), 4.47 (s, 4H), 4.38 (s, J=7.6 Hz, 2H), 4.24 (q, J=7.0 Hz, 1H), 4.09 (q, J=7.0 Hz, 1H), 3.66-3.54 (m, 1H), 3.48-3.33 (m, 1H), 2.96-2.76 (m, 2H), 1.51 (d, J=7.0 Hz, 3H), 1.29 (d, J=7.1 Hz, 3H). .sup.13C NMR (101 MHz, D.sub.2O) δ 174.42, 170.44, 164.00, 156.34, 151.62, 147.44, 144.52, 142.00, 141.71, 137.18, 134.69, 131.72, 129.77, 129.72, 127.46, 126.65, 126.30, 122.54, 57.78, 56.36, 49.96, 48.91, 40.27, 34.08, 16.75, 16.52. APCI-HRMS e/z calc. for C.sub.33H.sub.38N.sub.8O.sub.3 (free amine): 594.3067, found: 595.3135 [M+H].

Example 196—HPLC Purity of Example 26

(379) Isocratic HPLC analysis of the product from Example 26 with 91% MeOH/9% 0.5 M HCOOH as eluent detected at 254 nm showed >99.5% purity of the compound as shown in FIG. 1.

Example 197—General Protocol for Microbroth Dilution Method for Evaluating Antibacterial Activity of Compounds or Synergistic Effect of Compound-Antibiotic Combination

(380) Preparation of Bacteria

(381) Day 1:

(382) Plate bacterial strain(s) on appropriate media: Gram-negative bacteria with ESBLs or carbapenemases: green agar plates with 100 mg/L ampicillin Gram-negative bacteria without β-lactamases: green agar plates Gram-positive bacteria (Staphylococci and Enterococci): blood agar plates Incubate o.n. at 37° C.
Day 2:
Prepare the Bacterial Inoculum: Prepare a 0.5 McFarland suspension of bacteria in 0.85% NaCl. (Should be used within 15 min of preparation). Dilute the 0.5 McFarland suspension 1:100 into MH broth.
Check the inoculum by diluting the prepared bacterial suspension 1:100 (10 μl bacterial suspension+990 μl 0.85% NaCl). Plate 10 μl of the dilution on MH agar plates (×2). Incubate o.n. at 37° C., count the colonies and calculate the final CFU/ml inoculum in the plate by multiplying the average number of colonies with 10000 and divide by 2. The final inoculum should be between 3-7×10.sup.5 CFU/ml. Add 50 μl of the prepared bacterial suspension to each well in the microtiter plate except negative growth control.
Preparation of Compounds/Antibiotics Calculate the desired concentration-range and volume of the compounds/antibiotics in the assay. For antibiotics this will depend on the MIC to meropenem of the bacterial strains to be tested. Dilute the stock solution in MH broth. Make subsequent 2-fold dilutions in MH broth of the desired concentrations if a concentration range is to be tested (remember the extra dilution factor in the assay plate). Always include extra volume for pipetting. Take into consideration stock solutions that are made in buffers that have an effect on bacterial growth (e.g. DMSO).
Assay: Determining the MIC of compounds/antibiotics alone: Add 25 μl of each concentration of compound/antibiotic to row 2-11 (highest concentration in row 2) Add 25 μl MH broth to row 2-11 Add 50 μl MH broth to row 12 (positive control) Add 100 μl MH broth to row 1 Add 50 μl bacterial suspension to row 2-12. Determining the MIC of antibiotics+compounds: Add 25 μl of each concentration of antibiotic to row 2-11 (highest concentration in row 2) Add 25 μl of compound to row 2-11 Add 50 μl MH broth to row 12 (positive control) Add 100 μl MH broth to row 1 Add 50 μl bacterial suspension to row 2-12. Incubate the plate for 20 hrs at 37° C. and determine the MIC in the presence and absence of inhibitor.

(383) This experimental design was used for the results in Tables 2-6 below. In Table 2, inhibitor concentration dependency on the MIC of meropenem was studied.

(384) TABLE-US-00002 TABLE 2 Study of inhibitor concentration dependency on the MIC of meropenem Ref. no K66-45 K34-7 Species K. pneumoniae P. aeruginosa MBL NDM-1 VIM-2 Conc. (μM) 125 62.5 50 31.25 15.63 125 62.5 50 31.25 15.63 Inhibitor no inhibitor 32-64 32-64 Example 9 ≤0.5 0.125 0.125 2 ≤0.5 1 16 32 Example 12 0.25 0.125 4 1 1 4 Example 14 0.125 0.125 16 2 2 32 Example 23 0.125 0.125 0.125 0.125 1 1 1 32 Example 24 0.125 0.125 16 1 2 32 Example 25 0.125 0.125 0.125 8 2 1 1 32 Example 26 0.125 0.125 8 1 1 Example 27 0.125 0.125 8 64 1 1 1 32 Example 84 0.5 2 8 16 2 8 16 32 Example 85 0.5 2 8 16 2 8 32 32 Example 87 0.125 0.125 16 2 0.5 32 Example 91 0.125 0.125 8 1 2 16

(385) The results of a study of the minimum inhibitory concentration (MIC) of meropenem against a broad spectrum of clinical isolates of MBL-positive and MBL-negative gram negative bacteria on the MIC of meropenem is given in Table 3. In the presence of inhibitor, the MIC values for meropenem are dramatically lowered, while the effect on bacteria lacking MBL is absent. As shown in Table 4, all examples show the same dramatic effect on the MIC values of meropenem in two strains of clinically isolated multiresistant strains of P. aeruginosa harbouring VIM-2 and K. pneumoniae harbouring NDM-1, except for Examples 101, 157, 180, 182, 185 and 187.

(386) TABLE-US-00003 TABLE 3 Study of inhibitor effect on a broad spectrum of MBL-positive and MBL-negative gram negative bacteria on the MIC of meropenem Presence MBL-negative of MBL: MBL-positive Gram-negatives Gram-negatives Ref. no: K34-7 50692172 K66-45 A3-81 K71-77 50639799 50808021 50732159 Species: P. aeruginosa P. aeruginosa K. pneumoniae K. pneumoniae E. coli E. coli P. aeruginosa K. pneumoniae MBL: VIM-2 NDM-1 NDM-1 VIM-1 NDM-1 VIM-1 No MBL No MBL Meropenem 32-64 32-128 32-64 64-256 1-8 8-32 128-256 32-64 activity without inhibitor: Inhibitor concen- Example tration Meropenem activity (mg/L) in presence of inhibitor TPEN 63 mg/L 1 ≤0.5 ≤0.5 ≤0.5 ≤0.064 ≤0.064 64 ≤0.064 Captopril 63 mg/L 32 32-64 64 64-128 2-4 16 Example 9 63 mg/L ≤0.5 ≤0.5 Example 22a 63 mg/L 1 0.25 Example 27 63 mg/L 1 0.125 Example 23 25 mg/L 1 1 0.125 0.125 ≤0.03 ≤0.03 >64 32 Example 25 25 mg/L 2 2 0.125 0.125 ≤0.03 ≤0.03 >64 64 Example 84 25 mg/L 8 4 Example 85 25 mg/L 8 4 Example 91 25 mg/L 1 0.125 Example 83 25 mg/L 16 8 Example 24 25 mg/L 1 0.125 Example 12 25 mg/L 1 0.25 Example 14 25 mg/L 2 0.125 Example 26 25 mg/L 1 4 0.125 0.125 ≤0.03 0.06 >64 32 Example 95 25 mg/L 16 4 Example 194 25 mg/L 1 32 0.125 64 0.5 2 >64 32 Example 104 25 mg/L 8 4 Example 98 25 mg/L 8-32 4 Example 99 25 mg/L 1 0.25 1 0.06 0.125 >64 32 Example 100 25 mg/L 1 0.125 0.5 0.06 0.06 >64 32

(387) TABLE-US-00004 TABLE 4 Study of inhibitor effect on two MBL-positive multiresistant clinical isolates of gram negative bacteria on the MIC of meropenem. In all cases, the concentration of the inhibitor was 25 mg/L Ref. no K34-7 K66-45 Species P. aeruginosa K. pneumoniae MBL VIM-2 NDM-1 MIC values (mg/L) of meropenem without inhibitor 32-64 32-64 MIC values (mg/L) of meropenem in Example presence of inhibitor Example 101 16 8    Example 24 1-4  0.25  Example 24  2 0.125 Example 195  1 0.125 Example 195  1 0.125 Example 195  2 0.125 Example 26  1 0.125 Example 26  1 0.25  Example 25  1 0.125 Example 25  1 0.125 Example 105  2 0.125 Example 147  1 0.25-0.125     Example 69  1 0.25-0.125     Example 147  2 0.125 Example 157 16 8    Example 160 2 0.125 Example 163b 1 0.125 Example 163b 1 0.125 Example 180 32 8    Example 147 1 0.25  TPA 1 0.125 Example 182 32 16    Example 169 1 0.125 Example 185 32 64    Example 187 32 32   

Summary of Example 197

(388) In Examples 101 and 157, the chelating moiety comprises the structural elements DPA-triazol-p-phenyl-Linker (2B), a structural element showing less activity as an adjuvant at 25 mg/L compared to structural elements based on TPA and tripyridyl-ethylene-diamine in many of the other examples comprising the structural elements in the scheme 23(A). However, compounds comprising the chelator moiety shown in scheme 23(B) achieve full adjuvant effect in concentrations >50 mg/L. Introduction of a thiophene group in the chelator as in Examples 180 and 182 (scheme 23(C)), or removal of one or two nitrogen atoms in the pyridine rings in the chelator as in Examples 185 and 187 (C) completely abolishes the adjuvant effect at an inhibitor concentration of 25 mg/L. In conclusion, scheme 23 shows structural chelator elements leading to superior adjuvant effects. These chelators may give abolishment of antibacterial resistance with carbapenems at concentrations between 8 and 15 mg/L.

(389) ##STR00219## ##STR00220##

Example 198—Effect of Examples 26 and 195 on Different Strains of Gram-Negative Bacteria Harboring an MBL

(390) The protocol given in Example 197 was used to compare a non-peptidic compound represented by Example 26 with a peptidic compound represented by Example 195, having the same chelator in both examples (TPA), in a large number of different strains producing MBLs. The results show that the peptidic and the non-peptidic compounds show a similar dramatic adjuvant effect together with meropenem, restoring the effect of the antibacterial drug.

(391) TABLE-US-00005 TABLE 5 Effect of Examples 26 and 195 on different strains of Gram-negative bacteria harboring an MBL MIC MIC MIC meropenem + meropenem + meropenem 25 mg/L 25 mg/L alone Example 26 Example 195 Species [mg/L] [mg/L] [mg/L] MBL C. freundii 4 0.06 0.125 NDM-1 P. stuartii 8 0.06 0.125 NDM-1 P. mirabilis 32 0.125 0.125 NDM-1 M. morganii 8 1 1 OXA48+ NDM-1 K. pneumoniae 64 <0.03 <0.03 NDM-7 E. coli 64 0.25 0.25 NDM-5 K. pneumoniae 64 0.125 0.125 NDM-1 E. coli 0.5 <0.03 <0.03 VIM-1 K. pneumoniae 4 0.06 0.125 VIM K. pneumoniae 64 <0.03 <0.03 NDM-1 K. pneumoniae 64 <0.03 <0.03 NDM-1 E. coli 64 0.06 0.06 NDM-5 K. pneumoniae 64 0.06 0.06 NDM E. coli 32 0.125 0.25 NDM E. coli 32 0.125 0.125 NDM P. stuartii 64 0.25 0.25 NDM E. coli 64 0.125 0.25 NDM K. pneumoniae 64 0.06 0.06 NDM K. pneumoniae 64 0.06 0.06 NDM E. coli 32 0.125 0.125 NDM C. freundii 16 0.06 0.06 NDM E. coli 4 <0.03 <0.03 NDM E. coli 32 0.25 0.25 NDM K. pneumoniae 8 0.06 0.06 NDM E. coli 8 0.06 0.06 NDM E. cloacae 8 <0.03 <0.03 VIM E. coli 32 0.06 0.125 NDM K. pneumoniae 32 0.125 0.125 NDM-1 K. pneumoniae 32 0.06 0.06 NDM-1 E. coli 4 <0.03 <0.03 NDM-5

Example 199—the Effect of Example 26 on Resistant Strains of Clinical Isolates of K. pneumoniae K66-45 and P. aeruginosa K34-7 with Different Carbapenems

(392) Table 6 shows the results when the protocol as given in Example 197 was used to test the synergistic effect of Example 26 on a population of growing clinical isolates of K. pneumoniae K.sub.66-45 and P. aeruginosa K.sub.34-7 harbouring VIM-2 and NDM-1, resistant to carbapenems in general. The two carbapenems chosen was meropemen and doripenem, Three different batches of Example 26 in a concentration of 25 mg/L was used in the study. The results are given in Table 5. A MIC value of 32-64 represents resistance to the carbapenem. The results show that all three batches of Example 26 counteracts resistance in the bacterial strains and potentiates carbapenems with a factor 32-256 times. The results also shows that Example 26 can be resynthesized reproductively.

(393) TABLE-US-00006 TABLE 6 The effect of Example 26 on resistant strains of clinical isolates of K. pneumoniae K66-45 and P. aeruginosa K34-7 harbouring VIM-2 and NDM-1 MIC of carbapenem MIC value MIC Value in table in mg/L Ref. no in K34-7 in K66-45 Compound: Species P. aeruginosa K. pneumoniae Batches Example 26. MBL VIM-2 NDM-1 Batch 1 + MEM MEM 1 0.125 Batch 2 + MEM MEM 1 0.125 Batch 3 + MEM MEM 1 0.25  Meropenem alone MEM 32-64 32-64 Batch 1 + DOR DOR 1 0.25  Batch 2 + DOR DOR 1 0.125 Batch 3 + DOR DOR 1 0.125 Doripenem alone DOR 64 >64    

Example 200—Checkerboard Study of Example 26 and Meropenem

(394) The following protocol was used to study the synergy of Example 26 with meropenem against meropenem-resistant K. pneumonia and E. coli. A checkerboard MIC assay was using the broth dilution method according to CLSI guidelines. A sample of Example 26 was dissolved in sterile saline (0,9%) to 16 mg/ml and further diluted in Mueller Hinton BBL II-broth (MH-broth) to 1.6 mg/ml. A stock solution of 80 mg/ml concentration of meropenem (Mylan, 51B0424/08-2017) was prepared by dissolving 1 g meropenem in 12.50 ml sterile saline (0,9%). Dilutions to 4 mg/ml was performed by adding+4.75 ml sterile saline to 0.25 ml of the stock solution, and dilutions to 512 μg/ml was performed by adding 10.9 ml MH-broth to the 4 mg/ml solution. Inoculums of bacterial strains as described in Table 4 were prepared by suspending fresh overnight colonies from 5% horse blood agar were to a turbidity of 0.5 McFarland and further diluted to 1×10.sup.6 CFU/ml in MH-broth. Aliquots of 50 μl of MH-broth were added to each well of polystyrene microtiter plates. Thereafter, the 512 μg/ml meropenem solution was added to column 1 and titrated 2-fold throughout column 11. Solutions of 50 μl pre-titrated Example 26 were added to all wells and finally 100 μl diluted bacterial suspension was added to the wells. The plates were incubated at 35° C., 16-20 hours.

(395) Results:

(396) The results of the checker boards are shown in table 7 as MICs for the single compound and in combination between the two. In addition to the different MBLs, the isolates were producers of a large number of non-MBL β-lactamases. Table 7 shows a strong synergistic effect of EXAMPLE 26 in combination with meropenem for all of the MDR isolates tested. For some of the strains, the amplification of the effect of meropenem was more than a 1000 times.

(397) TABLE-US-00007 TABLE 7 Checkerboard results in the study of Example 26 with meropenem with a variety of resistant Gram-negative strains harboring MBL MIC alone MIC in Isolate β-lactamase/ (mg/L) combination (mg/L) category carbapenemase Other β-lactamases isolate id Example 26 Meropenem Example 26 Meropenem NDM NDM-1 CTX-M-15; CMY-6; OXA-1 50676002 >400 32 12.5 ≤0.0625 E. coli NDM-1 CTX-M-15; CMY-16(v); OXA10; OXA-1; 50739822 >400 32 12.5 ≤0.0625 TEM1 NDM-1 CMY-6; DHA-1-like; OXA-1; TEM-1C AMA817 >400 32 12.5 ≤0.0625 NDM-5 CTX-M14 AMA1563 >400 64 25 ≤0.0625 VIM VIM-4 CTX-M-15; CTX-M-9; OXA-1 AMA323 >400 8 12.5 ≤0.0625 E. coli VIM-1 nd 50921466 >400 2 12.5 ≤0.0625 NDM-1 NDM-1 SHV-11; CTX-M-15; OXA-1; OXA-9; TEM-1 K66-45 >400 64 12.5 ≤0.0625 K. pneumonia NDM-1 SHV-11; CTX-M-15; OXA-1; CMY-6 50627996 >400 64 12.5 0.125 NDM-1 SHV-1; CTX-M-15; OXA-1; OXA-9; TEM-1 50752501 >400 64 25 ≤0.0625 NDM-1 SHV11(v); OXA-1; TEM-1 50690310 >400 16 12.5 ≤0.0625 NDM-1 SHV-11; CTX-M-15; OXA-9; TEM-1 50825040 >400 64 12.5 ≤0.0625 NDM-1 CMY-6; SHV-11 50877064 >400 64 12.5 ≤0.0625 VIM VIM-1 SHV-12 K45-67 >400 32 12.5 ≤0.0625 K. pneumonia VIM-1 SHV-12; TEM-1 K46-62 >400 32 12.5 ≤0.0625 VIM ND 50923744 >400 2 12.5 ≤0.0625

Example 201—General Protocol and Results for In Vitro Toxicity of Compounds in Human hepG2 Cells

(398) Materials and methods:

(399) Cells

(400) The human hepatoblastoma cell line HepG2 (HB-8065, ATCC, Manassas, Va., USA) was cultured in DMEM-Glutamax™ (5.5 mM glucose) supplemented with 10% foetal bovine serum, streptomycin (100 μg/ml) and penicillin (100 units/ml) at 37° C. in 5% CO.sub.2. For viability assays, cells were seeded in white 96-well Nunclon plates at a density of 20,000 cells/well and left overnight to adhere before experiments were conducted.

(401) Cell Viability Assay

(402) Zn chelators dissolved in DMSO were added to white 96-well plates containing 20000 HepG2 cells/well at concentrations ranging from 1 to 1×10.sup.−6 mM (max DMSO concentration never exceeded 1%) and incubated for 24 hours at 37° C. After 24 hours, AlamarBlue® cell viability reagent (Thermo Fisher, Carlsbad, USA) was added (10% final concentration) and incubated for 4 hours at 37° C. This dye is a red-ox indicator yielding a fluorescent signal proportional to viable cells in each well that was measured in a fluorescence plate reader at ex550 nm/em603 nm (Clariostar, BMGlabtech, Germany) (O'Brien et al., 2000). Data from 8 replicates were fitted by non-linear regression to estimate IC50 values using GraphPad Prism (GraphPad Software Inc, USA).

REFERENCES

(403) O'Brien, J., Wilson, I., Orton, T., Pognan, F., 2000. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem 267, 5421-5426.
Results:

(404) The numerical calculations of the IC50 values for the in vitro in hepG2 cells for compounds of the invention are given in Table 8. The curves in FIGS. 2 and 3 show the log 10 concentration of Example No. as a function of relative fluorescence units (RFU) in suspensions of hepG2 cells. None of the compounds gave a RFU<10.sup.3 in the cell-free medium alone.

(405) TABLE-US-00008 TABLE 8 Compound IC50 R square TPEN 7.1 0.9372 Example 12 16.5 0.9363 Example 14 12.4 0.9911 Example 25 113.3 0.7048 Example 26 >1000 0.2191, calculation and visual inspection Example 84 >1000 0.3462, calculation and visual inspection Example 91 165.2 0.9321 Example 96 >1000 0.8415, calculation and visual inspection Example 97 130.6 0.885 Example 98 259.1 0.8232 Example 99 217.3 0.7271 Example 100 >100 0.7154 Example 101 ~220 Visual inspection Example 103 67.61 0.89 Example 104 224.5 0.884 Example 105 ~210 Visual inspection Example 107 >500 0.4354, calculation and visual inspection Example 108 >1000 0.5954, calculation and visual inspection Example 109 127.7 0.839 Example 147 ~10 Visual inspection Example 163b ~600 Visual inspection Example 180 ~80 Visual inspection Example 186 ~215 Visual inspection Example 195 >1000 0.8331, calculation and visual inspection

Example 202—General Protocol and Results for In Vitro Human Red Blood Cell Toxicity

(406) Human blood samples were incubated at 37° C. with six concentrations of the compounds given in Table 1 in the range 1-500 μM concentrations. The blood samples were centrifuged to remove whole cells, and the supernatants were analyzed spectrophotometrically. None of the compounds induced hemolysis below 500 μM concentration.

Example 203—General Protocol and Results for In Vitro Identification of Bacterial Ambler Class

(407) 1. Isolate a carbapenen-resistant bacterial strain of interest and spread it on an agar plate. 2. Prepare the following disks: a. Disk A with meropenem alone. b. Disk B with meropenem+Example 26. c. Disk C with imipenem alone. d. Disk D with imipenem+Example 115. e. Disk E with temocillin. 3. Add the five disks at five different places in the agar plate or in four different plates. 4. Incubate overnight at 37° C., 5. Read all zone diameters of the lobes. 6. The response as read by zone diameters may be used to discriminate between producers of MBL, KPC and OXA as described by Teethaisong et al in Journal of Applied Microbiology (2016), 121, 408-414, according to Table 9.

(408) TABLE-US-00009 TABLE 9 Disk Result A − − − B − + − C − − − D − + + E − − Suggested Pathogen without MBL, MBL-based KPC-based conclusion KPC but indicates OXA-48 pathogen pathogen Symbols: +: increased zone. −: minimally changed zone.

Example 204—Time-Kill Studies

(409) Materials:

(410) Mueller-Hinton broth (MHB) was warmed from fridge temperature cold. Materials used in the study wsa LB agar, preferably square plates, sterile PBS, 96-well plates, 30 mL Media bottles, inoculating loops, tips for multichannel, sterile micro-centrifuge tubes (1.5 mL), 40 mg/mL freshly prepared meropenem (MEM) solution in DMSO appearing as clear/pale yellow solutions. A sample of Example 26 was dissolved in DMSO.

(411) Method:

(412) The 96-well plates were prepared using a multi-channel pipette, adding 270 mL PBS to columns 2-9, or the required number of dilutions needed in the experiment. Each Inoculum was prepared by picking a single colony from the fresh plate that was disperged into 1 mL MHB, the loop was agitated to ensure all the colony comes off the loop. The tube was Vortex mixed, and a 100 μL aliquot was taken from the tube into 9.9 mL of MHB into the media bottle. This results in 10 mL with 10.sup.5 CFU/mL. The procedure was repeated for as many samples needed in the study. Aliquots of 5 mL inoculum were transferred to as many media bottles as needed, e.g. 4 mg/L MEM, 4 mg/L MEM+50 μM test substance, 4 mg/L MEM+100 μM test substance. Then 2×60 mL aliquots were taken from each culture and added to Column 1 on the 96 well plate, e.g. culture 1: 50 μl into A1, 50 mL into B1 etc. for time point 0 h. Then 4 mg/mL MEM was added into each bottle, followed by the required amount of test substance. The bottles were mixed gently to avoid formation of bubbles, and incubated in a secondary container at 37° C. at 180 rpm. Finally, serial dilutions were performed using multi-channel pipette taking 30 μL for column 1, adding to column 2, mixing well, repeating from column 2 to 3 with clean pipette tips.

(413) Result:

(414) FIG. 4 shows the results from the time-kill studies of a resistant clinical isolate of Klebsiella pneumoniae harboring NDM-1 with Example 26 and 4 mg/L meropenem. The data shows that with 4 mg/L meropenem alone the CFU starts to increase after about 5 hours and continues to grow beyond the starting CFU count. With the same concentration of meropenem added Example 26, the CFU drops to below the limit of detection (LOD) after 5-10 hours irrespective of the concentration of Example 26, and stays below LOD in the period of the study (30 hrs.).

Example 205—Resistance Frequency as a Function of Chelator Strength Using the Compounds of Examples 25, 26, 194 and 195 According to Scheme 23B

Example 205A

(415) Resistance frequency of K. pneumoniae K66-45 to meropenem co-treatment with examples 25, 26, 194 and 195 as determined by single-step selection on agar. The following protocol was used: Mueller-Hinton agar (M-A) plates containing either 15, 25 or 50 mg/L of Example 26 were supplemented with 1, 2, 4 or 8 mg/L meropenem. 4 mg/L meropenem corresponds to the mean steady-state antibiotic level in sera when normal doses of meropenem is administered to healthy volunteers. The CFU of K66-45 was −109 (Mueller-Hinton broth, no selection). After plating and grown overnight at 37° C., only single colonies, ignoring any touching the edges of the plate, were counted. Analogously with the same setup, 25 mg/L of Example 196 were run the same way with 1, 2, 4 and 8 mg/L meropenem.

(416) Results and Conclusions:

(417) The results are summarized in Table 10. With Example 194, almost-confluent growth with >1000 colonies, too numerous to count/separate, was observed on nearly all plates. With Example 26 comprising a stronger chelator, no colonies were observed at the anticipated clinical concentration 4 mg/L or higher of meropenem. With Example 25 with an even stronger chelator, no colonies were observed at the sub-clinical MEM concentration 2 mg/L.

(418) TABLE-US-00010 TABLE 10 Resistance frequency of K. pneumoniae K66-45 to meropenem co-treatment with compounds of Examples 25, 26, 194 and 195, determined by single-step selection on agar Inhibitor concentration in all cases: 25 mg/L Inhibitor example Example 194 Example 26 Example 195 Example 25 MEM conc. 1 2 4 8  1  2 4 8  1 2 4 8  1 2 4 8 (mg/L) Sensitivity S S I I S S I I S S I I S S I I RF* - no of tntc tntc tntc tntc 74 35 nc nc 163 nc nc nc 64 nc nc nc colonies MEM = meropenem; n.c. = no colonies, S = sensitive, I = intermediate e.g. 2 < I ≥ 8; tntc = too numerous to count, plates containing either confluent growth or >1000 colonies; nd = not determined. MIC of MEM in the absence of Example 26 >32 mg/L. MIC of MEM in the presence of 31.25 μM, 50 μM and 100 μM Examples 25, 26 and 195 = 0.125 mg/L; *RF = resistance frequency

Example 205B—Determination of Resistance Frequency of Klebsiella pneumoniae K66-45 to Example 26-Meropenem Co-Treatment

(419) To investigate the resistance frequency of Example 26 at varying concentrations, the same protocol as in Example 205A was used to investigate the frequency of resistant mutants in a population of growing K. pneumoniae K66-45. Mueller-Hinton agar plates containing either 31.25 or 50 μM Example 26 were supplemented with 1, 2, 4 or 8 mg/L MEM. 10.sup.9 CFU K.sub.66-45 (Mueller-Hinton broth, no selection) was plated and grown overnight, 37° C. Only single colonies, ignoring any touching the edges of the plate, were counted. A concentration of 4 mg/L meropenem corresponds to a clinical steady-state concentration level in sera when normal doses of meropenem used in healthy volunteers, as described by Leroy A, Fillastre J P, Borsa-Lebas F, Etienne I, Humbert G. in Antimicrobial Agents and Chemotherapy 36 (1992), 2794-2798.

(420) Results and conclusions:

(421) The results are summarized in Table 11. There was a concentration dependency on frequency of number of mutants with increasing concentration of Example 26. At the effective concentration of 25 mg/L of Example 26, no colonies were observed at the anticipated clinical concentration 4 mg/L or higher of meropenem. The 50 mg/L concentration of Example 26 is still a fully acceptable concentration in view of the toxicity data in Examples 202, 203 and 212. At this concentration, no colonies were observed at half the clinical concentration of meropenem on 2 mg/L.

(422) TABLE-US-00011 TABLE 11 Resistance frequency of K. pneumoniae K66-45 to the compound of Example 26 - meropenem co-treatment, determined by single-step selection on agar Resistance/mutant frequency (number of colonies) Inhibitor 1 mg/L MEM 2 mg/L MEM 4 mg/L MEM 8 mg/L MEM (mg/L) (S) (S) (I) (I) 15 137 115 4 n.c. 25  74  35 n.c. n.c. 50  22 n.c. n.c. n.c. MEM = meropenem, n.c. = no colonies, S = sensitive, I = intermediate e.g. 2 < I ≥ 8. MIC of MEM absence of Example 26 > 32 mg/L. MIC of MEM in presence of 31.25 μM, 50 μM and 100 μM Example 26 = 0.125 mg/L

Example 206—Protein Binding of Example 26

(423) To determine the protein binding properties of Example 26, the Transil.sup.XL protein binding kit (Sovicell) was used. Enzymes in the kit are immobilized at a solid phase. Aliquots of 500 μM of Example 26 were incubated for 12 min under shaking (1200 rpm) at different concentration of human serum albumin and α.sub.1-acid glycoprotein (AGP) in a ratio of 24:1 (up to 140 μM). Buffer without inhibitor was included as a control. After incubation, the suspensions were centrifuged and diluted 1:10 in 50 mM HEPES buffer pH 7.5 supplemented with 1 μM zinc.sup.2+ and VIM-2 (final concentration 1 nM). Enzyme and standard was allowed to incubate for 10 and 30 min at 25° C. and measured the residual enzyme activity was measured by adding Nitrocefin as a reporter substrate (final concentration of 50 μM). The absorbance was recorded at 482 nm and 25° C. and the residual enzyme activity was calculated.

(424) Results:

(425) FIG. 5 shows the results for the study of protein binding of Example 26. The bars denoted as 0 in protein concentration are the results for inhibition of VIM-2 without presence of proteins.

(426) Conclusion:

(427) The results show that protein binding is not affecting the inhibitory activity of Example 26 on VIM-2 in any of the protein concentrations.

Example 207—Zinc Restorability of the NDM VIM-2 after In Vitro Incubation with Example 26

(428) PD-10 Desalting Columns comprising Sephadex G-25 size exclusion resin (GE Healthcare Life Sciences) were used to study irreversibility of the interaction between Example 26 and the metallo-β-lactamase VIM-2. The stationary phase retains low-molecular compounds and excludes molecules with higher molecular weight that 10 kD. The eluent used was Chelex-treated HEPES 50 mM buffer, pH 7.5. In experiment 207A, a solution of 50 nM VIM-2 was passed through a column. The enzyme activity towards a standard was the same before and after passage through the column. In experiment 207B, a solution of 50 mg/L of Example 26 was loaded to a PD-10 column and it was shown analytically (spectrophotometrically, HPLC) that Example 26 was completely retained on the column. Thus the eluate only contains either the pure enzyme, or the enzyme that has been mixed with inhibitor pre PD10 column treatment. In experiment 207C, a solution of 50 nM VIM-2 and 140 mg/L Example 26 was incubated on ice for 1 hour. In all experiments, 16 aliquots of 250 μL were taken from the PD-10 column runsout and enzyme activity was analysed. Enzyme activity was measured by adding Nitrocefin as a reporter substrate (final concentration of 50 μM).

(429) Results:

(430) The results are given in FIGS. 6 and 7. It was shown (FIG. 6) that none of the eluate fractions from Example 207B had inhibitory effect on the enzyme. The eluate fractions 5-12 from 207A had full enzyme activity as expected. Of the eluate fractions from Example 207C, fraction 5 showed only 33% residual enzyme activity.

(431) Conclusion: the experiment shows that Example 26 irreversibly inhibits VIM-2.

(432) In Example 207D, aliquots containing 15 nM impaired enzyme from Example 207C were spiked with ZnSO4 in concentrations ranging from 0-100 μM. The results are shown in FIG. 7. Enzyme activity could be restored only to 40%. To investigate if more extreme time-dependent and concentration-dependent conditions could restore enzyme activity, aliquots having final concentrations of 30 nM column-treated enzyme and 1000 μM Zn.sup.2+ on ice and at room temperature over prolonged time. No increase in enzyme activity was observed at any point of time in the experiment.

(433) Conclusion:

(434) The experiment shows that the irreversible inhibition of VIM-2 by Example 26 was not solely a consequence of a reversible removal of Zn.sup.2+ from the enzyme.

Example 208—Zinc Restorability of the NDM VIM-2 after In Vitro Incubation with Example 26 Compared to Incubation with the Standards TPEN and EDTA

(435) The protocol from Example 207 was followed to investigate the effect of increasing concentrations of the literature compound TPEN on purified VIM2 enzyme, and compared to the PAC chelator EDTA at a 100 μM concentration (FIG. 8A). In a parallel experiment, it was investigate how corresponding increasing concentrations of Example 26 affected the VIM-2 enzyme compared to the PAC chelator EDTA at a 100 μM concentration (FIG. 8B).

(436) Conclusion:

(437) The experiments show that VIM-2 was irreversibly inhibited by the zinc-selective 2-pyridyl chelators TPEN and Example 26, and that the inhibition was not solely a consequence of a reversible removal of Zn.sup.2+ from the enzyme. The experiments also show that the typical PAC chelator EDTA did not inhibit VIM-2 irreversibly.

Example 209—In Vitro Interaction of Example 26 with Zinc-Containing Human Enzymes—Comparison to the APC-Chelator EDTA

(438) Protocol:

(439) In order to investigate if representative examples in the present invention are able to interact with human metal containing enzyme, Example 26 was incubated for 30 min at different concentrations ranging from 62.5-500 μM at 25° C. with 0.4 ng/μL enzyme. Enzyme activity was measured by adding Nitrocefin as a reporter substrate (final concentration of 50 μM). The initial reaction velocity was measured at 405 nm. EDTA was included as a representative example of the APC chelator class described above.

(440) Results and Conclusion:

(441) FIG. 9 summarizes the results of the investigation. The data suggest that the compound of Example 26 does not interact with rH glyoxylase II, whereas the APC chelator EDTA does.

Example 210—Solution Stability of a Peptide-Based Inhibitor (Example 195) Compared to a Non-Peptide Inhibitor in the Present Invention (Example 26)

(442) Protocol:

(443) Example 195 and Example 26 was stored for up to 8 months at 2-8° C. and the stability was measured indirectly by performing MIC studies according to the protocol in Example 187 at a final concentration of 25 mg/L of the inhibitors. P. aeruginosa harboring VIM-2 and K. pneumonia bearing NDM-1 was used as two indicator strains.

(444) Results and Conclusions:

(445) The results are summarized in FIG. 10. With Example 195, a loss of activity was observed after 5 months storage, and after 8 months the activity was absent. On the other hand, Example 26 showed no loss of activity even after 8 months storage time. These data indicate that a representative non-peptidic Example (Example 26) from the present invention has a significantly better stability in solution compared to a peptidic inhibitor comprising the same chelator. The peptidic chelator may still be handled as a drug product, e.g. in a lyophilized form.

Example 211—In Vivo Toxicity of Example 26 and Example 195 in Healthy Mice

(446) Protocol:

(447) Female Balb/c mice (4 weeks old, approximately 20 g, Charles River, L'Arbresle, France) acclimatized 4 days in the animal facility before initiation of experiments. Stock solutions with a concentration of 25.6 mg/ml were prepared of the two test compounds, Example 26 and Example 195. Groups of 6 mice were either untreated or treated with 200 μL of Example 26 or Example 197 intraperitoneally once a week with increasing doses each time. Individual weights were followed four days a week. Relative weight was calculated as the ratio between the weight of the day and the weight of the first day. The protocol for experiments in mice was approved by the University of Lyon Animal Ethics Committee. Toxicity experiments were performed by Antineo (www.antineo.fr). After 43 days, the mice in the two test groups had received an accumulated dose of 252 mg/kg.

(448) TABLE-US-00012 TABLE 12 The changes in bodyweight during the experiment Relative weight of mice (% ± SEM) Treatment (mg/kg) Day Untreated mice Example 26 Example 195 4 1 100 ± 0 100 ± 0 100 ± 0 8 8 102 ± 4 101 ± 0  98 ± 5 16 16 109 ± 3 105 ± 0 103 ± 3 32 22 107 ± 3 105 ± 0 103 ± 3 64 29 112 ± 4 106 ± 0 106 ± 6 120 36 114 ± 9 109 ± 0 110 ± 8 No injection, 43 114 ± 9 107 ± 0 110 ± 8 reporting
Results:

(449) The changes in bodyweight of the mice during the experiment are given in Table 12. After 43 days, no mice had died, and no clinical signs of toxicity were observed. The accumulated dose received in the two test groups was 252 mg/kg. A tendency of reduced growth of body weight compared to the control group was observed during the test period. At the highest dose of 128 mg/kg the reduction of growth of body weight was within the standard error of mean (SEM) compared to the untreated group.

Example 212—In Vivo Infection Study of Example 26 in Mice

(450) Purpose of the Study:

(451) The purpose of this study was to investigate the synergy between a typical example of the present invention and a state of art carbapenem in a clinically relevant multiresistant Gram-negative bacterial strain harboring a metallo-β-lactamase in the treatment of live mice infected with the bacterial strain. The chosen compound was Example 26 and the chosen carbapenem was the clinically relevant β-lactam antibiotic meropenem (MEM). The chosen bacterial strain was a clinical isolate of Klebsiella pneumoniae strain 50752501 harbouring NDM-1. The dose of meropenem selected in the present study was 33 mg/kg. This dose was shown to have a minor effect on reducing the bacterial loads but still being a clinically relevant dose for treating susceptible K. pneumoniae strain.

(452) Material and Methods:

(453) Materials

(454) 36 outbred, NMRI female mice, 26-30 gram (Harlan) Apodan® (Cyclophosphamid 1 g) Lot 6F0931 (exp. 06/2019) Meropenem, Mylan 1 g 51B0424 (exp. 06/2019) NZ148, batch CS3071 received 030317 5% Horse Blood Agar plates (SSI: Exp 2017-06-07) 0.9% sterile saline (SSI:) Sterile MilliQ water (SSI.) Nurofen® Junior (20 mg/ml, Novartis) Zoletil mix EDTA coated Eppendorf tubes Vidal glass tubes
Laboratory Animal Facilities and Housing of Mice

(455) The temperature and humidity were registered daily in the animal facilities. The temperature was 22° C.+/−2° C. and can be regulated by heating and cooling. The humidity was 55+/−10%. The air changes per hour were approximately 8-12 times (70-73 times per hours inside cages), and light/dark period was in 12-hours interval of 6 a.m.-6 p.m./6 p.m-6 a.m. The mice had free access to domestic quality drinking water and food (Teklad Global diet 2916C-Envigo) and occasionally peanuts and sunflower seeds (Kge Korn A/S). The mice were housed in Type 3 IVC cages with bedding from Tapvei. Further, the animals were offered Enviro-Dri nesting material and cardboard houses (Bio-serv).

(456) Preparation of Cyclophosphamide

(457) A total of 1 g cyclophosphamide (one ampoule Apondan, 1 g) was dissolved in 50 ml water 20 mg/ml on each day of use. This stock solution was further diluted to 11 mg/ml (16.5 ml of 20 mg/ml+13.5 ml saline) for use on day −4 or to 5.5 mg/ml (8.25 ml of 20 mg/ml+21.75 ml saline) for use on day −1.

(458) Treatment of Mice with Cyclophosphamide

(459) The mice were rendered neutropenic by injecting 0.5 ml cyclophosphamide solution intraperitoneally 4 days (approximately 200 mg/kg) and 1 day (approximately 100 mg/kg) prior to inoculation.

(460) Inoculation of Mice

(461) Fresh overnight colonies from a 5% Horse Blood Agar plate were suspended and diluted in sterile saline to approximately and 10.sup.7 CFU/ml. Mice were inoculated intraperitoneally with 0.5 ml of the suspension. Approximately ½ hr prior to inoculation, mice were treated orally with 45 μl neurophen (20 mg ibuprofen/ml corresponding to approximately 30 mg/kg) as a pain relief.

(462) Treatment of Mice with Meropenem and Example 26

(463) One vial Meropenem, 1 gram, was dissolved in saline to 50 mg/ml and further diluted to 5 mg/ml in saline.

(464) Two vials of Example 26 with 25.7 and 23.7 mg respectively was dissolved in PBS pH 7.4 to 15 mg/ml and pooled. pH was verified to 7.5. The 15 mg/ml stock solution was further diluted to 5 mg/ml and 1.5 mg/ml in PBS pH 7.4.

(465) Mice were treated subcutaneously in the neck region with. 0.2 ml solutions of Example 26 corresponding to 10, 33 and 100 mg/kg. 30 minutes later mice were treated with 33 mg/kg meropenem or vehicle (see Table 13).

(466) Clinical Scoring of Mice

(467) The mice were observed during the study and scored based on their behaviour and clinical signs.

(468) Score: 0 Healthy 1 Minor clinical signs of infection (slower movements, light piloerection in the skin) 2 Moderate signs of infection (lack of curiosity or changed activity, piloerection in the skin, changed body position 3 Severe signs of infection (reduced movements, piloerection in the skin, slightly pinched eyes, tucked up belly, changed body position). 4 Severe signs of infection (stiff movements, piloerection in the skin, pinched eyes, cold, pain). Sacrificed. 5 The mouse does not move, is cold, lying on the side. Sacrificed.
Sampling

(469) Colony counts were determined from blood and peritoneal fluid at 1 and 5 hrs post inoculation. The mice were anaesthetized with Zoletil mix and blood was collected from axillary cutdown in 1.5 ml EDTA coated Eppendorf tubes. The mice were sacrificed immediately after blood sampling and a total of 2 ml sterile saline was injected i.p. and the abdomen gently massaged before it was opened and fluid sampled with a pipette. Each sample was then 10 fold serial diluted in saline and 20-μl spots, ranging from undiluted to a 10.sup.6 dilution, were applied on agar plates in duplicates. In addition, 100 μl undiluted blood from the 4 highest meropenem dosing groups was spread on a separate plate to obtain lowest possible detection limit. All agar plates were incubated 18-22 hrs at 35° C. in ambient air. The overall treatment and sampling schedule is given in Table 13. Colony counts are given in Table 14, and visualized in FIGS. 11 and 12.

(470) TABLE-US-00013 TABLE 13 Treatment and sampling schedule Treatment with Treatment with Sampling Sampling Example 26 T = 1 meropenem T = 1.5 T = 1 T = 5 None None 1-2-3-4  10 mg/kg 33 mg/kg 5-6-7-8  33 mg/kg 9-10-11-12 100 mg/kg 13-14-15-16  10 mg/kg Vehicle 17-18-19-20  33 mg/kg 21-22-23-24 100 mg/kg 25-26-27-28 Vehicle 33 mg/kg 29-30-31-32 Vehicle Vehicle 33-34-35-36
Results:

(471) The actual CFU counts of each inoculum was as intended 7.44 log.sub.10 CFU/ml. Mice were treated with EXAMPLE 26 and meropenem alone or in combination. Colony counts in blood and peritoneal fluid were performed at 1 hour after inoculation corresponding to start of treatment and at 4 hrs after treatment. The CFU counts and the clinical score for each individual mouse are shown in Table 14 and the CFU numbers are also presented in FIGS. 11 and 12.

(472) Treatment with 10-100 mg/kg Example 26 alone did not result in reduction of the CFU levels compared to vehicle treatment in blood and peritoneal fluid. Treatment with meropenem alone resulted in 2.5 and 2.2 log.sub.10 lower CFU levels compared to vehicle treatment in the blood and peritoneal fluid respectively. Combination treatment with Example 26 and meropenem resulted in 3.95-4.38 and 3.57-3.66 log.sub.10 lower CFU levels compared to vehicle treatment in the blood and peritoneal fluid respectively. The CFU levels after combination treatment was significantly lower than the CFU levels after treatment with meropenem alone (p<0.01 in blood and p<0.0001 in peritoneal fluid).

(473) TABLE-US-00014 TABLE 14 Colony counts in blood and peritoneal fluid (PF) from neutropenic mice inoculated intraperitoneally with K.pneumoniae 50752501 Blood Mouse Score at log Pf log id sampling CFU/ml mean CFU/ml mean 1 1 3.98 4.39 6.81 6.77 2 1 5.10 6.72 3 1 3.99 6.80 4 1 4.51 6.74 5 2 1.90 1.61 4.18 3.83 6 2 1.00 3.83 7 2 1.85 3.60 8 2 1.70 3.72 9 1 1.78 1.26 3.88 3.80 10 1 1.78 3.80 11 1 1.00 3.68 12 1 0.50 3.85 13 1 1.30 1.18 3.65 3.74 14 1 0.50 3.74 15 1 1.90 3.92 16 1 1.00 3.65 17 2 6.02 5.88 7.92 7.79 18 2 5.70 7.68 19 2 5.90 7.76 20 2 5.89 7.81 21 1 5.83 5.89 7.68 7.60 22 1 5.54 7.51 23 1 6.78 7.88 24 1 5.40 7.35 25 1 5.83 5.84 8.24 7.93 26 1 6.05 7.89 27 1 5.48 7.63 28 1 6.02 7.98 29 2 3.51 2.98 5.72 5.22 30 2 2.80 4.60 31 2 3.51 5.30 32 2 2.10 5.24 33 2 5.60 5.56 7.40 7.40 34 2 5.90 7.48 35 2 5.18 7.18 36 2 5.57 7.54