COMPOUNDS FOR THE MODULATION OF CYCLOPHILINS FUNCTION

Abstract

The present invention relates to compounds of formula (I) or a pharmaceutically acceptable salt thereof useful as inhibitors of Cyclophilins and modulators of cyclophilin-like proteins. The invention also relates to uses of said compounds in the treatment of various disorders. Formula (I), wherein: R.sup.1 and R.sup.2 are each independently is selected from the group consisting of —R, -haloalkyl, -hydroxyalkyl, —OR, —C(O)R, —CO2R, —C(O)N(R)2, —NRC(O)R, and —N(R)2; wherein R.sup.2 could also be a sulphide; each R is independently hydrogen, C.sub.1-6 aliphatic, C.sub.3-10 aryl, a 3-8 membered saturated or partially unsaturated carbocyclic ring, a 3-7 membered heterocylic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, sulphur, or a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms selected from nitrogen, oxygen, or sulphur; R.sup.3 is Formula (Ia) selected from the group consisting of —OEt, and wherein R.sup.5 and R.sup.6 are independently selected from the group consisting of H, halide, methoxy, thiomethyl, morpholine and trifluoromethyl; R.sup.4 is selected from the group consisting of C.sub.1-6-alkyl-, and R.sup.4.1—CH.sub.2— wherein, R.sup.4.1 is C.sub.3-6-cycloalkyl-, a 5-6 membered heterocyclic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, sulphur; optionally substituted with one R.sup.4.1.1 wherein, R.sup.4.1.1 is selected from —H, C.sub.1-4-alkyl, optionally substituted with one substituent selected from H.sub.2N(O)C— or EtO(O)C—; X is carbon or nitrogen; A is selected from the group consisting of 6 membered unsaturated ring, with 1-3 nitrogen atoms, which is optionally substituted by —NH.sub.2, and Formula (Ib), wherein ring B is a fused 5-10 membered saturated or partially unsaturated heterocyclic mono-bicyclic ring having 1-3 heteroatoms selected from nitrogen, oxygen or sulphur, which is optionally substituted by —OH; and m is 1 or 2; and n is 1 or 2.

##STR00001##

Claims

1. A compound of formula I, ##STR00073## or a pharmaceutically acceptable salt thereof, wherein: R.sup.1 and R.sup.2 are each independently selected from the group consisting of —R-haloalkyl, -hydroxyalkyl, —OR, —C(O)R, —CO2R, —C(O)N(R)2, —NRC(O)R, and —N(R)2; wherein R.sup.2 may also be a sulphide; each R is independently hydrogen, C.sub.1-6 aliphatic, C.sub.3-10 aryl, a 3-8 membered saturated or partially unsaturated carbocyclic ring, a 3-7 heterocyclic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, sulphur, or a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulphur; R.sup.3 is selected rom the group consisting of —OEt, and ##STR00074## wherein R.sup.5 and R.sup.6 are independently selected from the group consisting H, halide, methoxy, thiomethyl, morpholine and trifluoromethyl; R.sup.4 is selected from the group consisting of C.sub.1-6-alkyl-, and R.sup.4.1-—CH.sub.2— wherein, R.sup.4.1 is C.sub.3-6-cycloalkyl-, a 5-6 membered heterocyclic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, sulphur, optionally substituted with one R.sup.4.1.1 wherein, R.sup.4.1.1 is selected from —H, C.sub.1-4-alkyl, optionally substituted with one substituent selected from H.sub.2N(O)C— or EtO(O)C—; X is carbon or nitrogen; A is selected from the group consisting of 6 membered unsaturated ring, with 1-3 nitro atoms, which is optionally substituted by —NH.sub.2, and ##STR00075## wherein ring B is a fused 5-10 membered saturated or partially unsaturated heterocyclic mono-bicyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, sulphur, which is optionally substituted by —OH; and m is 1 or 2; and n is 1 or 2.

2. The compound or the pharmaceutical salt hereof according to claim 1, wherein R.sup.3 is —OEt.

3. The compound or the pharmaceutical salt thereof according to claim 1, wherein R.sup.3 is ##STR00076##

4. The compound or the pharmaceutical salt thereof according to claim 3, wherein R.sup.6 is H and R.sup.5 is selected from the group consisting of —Br, —Cl, —OH, —OMe, —SMe, —CF.sub.3, -morpholine and —H.

5. The compound or the pharmaceutical salt thereof according to claim 1, wherein R.sup.1 and R.sup.2 are —H, and X is nitrogen.

6. The compound or the pharmaceutical salt thereof according to claim 1, wherein A is selected from the group consisting of ##STR00077##

7. The compound according to claim 1, of formula (II) ##STR00078## or the pharmaceutical salt thereof, wherein R.sup.3 and R.sup.4 are as defined above; Y is carbon or nitrogen and Z is carbon or nitrogen.

8. The compound or the pharmaceutical salt thereof according to claim 7 wherein Y and/or Z are carbon atoms.

9. The compound according to claim 2 selected from the group consisting of ##STR00079## ##STR00080## and the pharmaceutical salt thereof.

10. The compound according to claim 4 selected from the group consisting of ##STR00081## and the pharmaceutical salt thereof.

11. The compound according to claim 4 selected from the group consisting of ##STR00082## ##STR00083## and the pharmaceutical salt thereof.

12. A pharmaceutical composition comprising the compound or the pharmaceutical salt thereof of claim 1.

13. A method of treatment in medicine in a patient, preferably in the treatment of a cyclophilin-mediated disease or disorder or for modulation of cyclophilin or cyclophilin-like protein induced signalling in cells wherein such signalling modulation is beneficial for the treatment of a disease or disorder comprising administering to the patient the compound or the pharmaceutical salt thereof of claim 1.

14. The method of treatment according to claim 13 wherein the disease or disorder is selected from the group consisting of fibrosis of the kidney, liver, lung or pancreas, cardiac failure, viral infections, inflammation, cancer, Alzheimer's disease, Parkinson's disease, Amyotrophic Lateral Sclerosis (ALS), Dementia, Multiple Sclerosis, and Huntington's disease.

15. The method of treat treatment according to claim 13 wherein the disease or disorder is treatable using immunotherapy.

Description

BRIEF DESCRIPTION OF THE FIGURE

[0083] FIG. 1 shows (A) a depiction of a type-I binding mode for prior-art compounds (B) a depiction of a type-II binding mode for novel tri-vector compounds disclosed in this invention. (C) co-crystal structure of CypA with a prior-art compound in a type-I binding mode. (D) co-crystal structure of CypA with tri-vector compound 12 that adopts a type-I binding mode. (E) co-crystal structure of CypA with tri-vector compound 19 that adopts a type-II binding mode. (F) co-crystal structure of CypA with tri-vector compound 4 that adopts a type-II binding mode.

EXAMPLES

Synthesis of Selected Compounds

[0084] Compounds have been prepared by the synthetic procedures shown in Scheme 1.

##STR00014##

[0085] The first key intermediate of the synthesis, namely the substituted amino derivative, can be prepared from either p-nitrobenzaldehyde using a reductive amination procedure, or from a p-nitrophenyl derivative by a nucleophilic substitution reaction. The urea moiety is obtained by reaction with ethyl isocyanatoacetate. After hydrolysis of the ester, the corresponding carboxylic acid undergoes an amide coupling reaction with the haloarylpirrolidine derivative. Reduction of the nitro group led to the synthesis of the desired final products. Where intermediates were not commercially available, they were prepared according to reported procedures. Details concerning the preparations methods are provided below.

[0086] Chemicals, Materials, and Methods

[0087] Abbreviations used in the description of the examples that follow are: Acetonitrile (MeCN); ammonium chloride (NH.sub.4Cl); BnBr (benzyl bromide); carbonyldiimidazole (CDI); cesium carbonate (Cs.sub.2CO.sub.3); cyclohexane (Cy); chloroform (CHCl.sub.3); deuterated dimethylsulfoxyde (DMSO-d6); dichloromethane (DCM); dimethylsulfoxyde (DMSO); N,N-diisopropylethylamine (DIPEA); dimethylformamide (DMF); di-tert-butyldicarbonate (Boc.sub.2O); 4-(dimethylamino)-pyridine (DMAP); ethylene glycol monomethyl ether (EGME); ethanol (EtOH); electrospray ionization (ESI); ethyl acetate (EtOAc); hydrochloric acid (HCl); mass spectrometry (MS); microwave (MW); sulfuric acid (H.sub.2SO.sub.4); iodomethane (MeI); N,N-dimethylformamide (DMF); lithium hydroxide (LiOH); magnesium sulphate (MgSO.sub.4); methanol (MeOH); nuclear magnetic resonance (NMR); room temperature (RT); palladium acetate (Pd(OAc).sub.2); potassium carbonate (K.sub.2CO.sub.3); sodium bicarbonate (NaHCO.sub.3); sodium borohydride (NaBH.sub.4); tetrabutylammonium iodide (TBAI); triethylsilane (TES); tetrahydrofurane (THF); thin layer chromatography (TLC); triethylamine (Et.sub.3N or TEA) and trifluoroacetic acid (TFA).

[0088] Automated column chromatography purifications were conducted using Biotage Isolera One apparatus with prepacked silica gel columns of different sizes (10 and 25 g).

[0089] Mixtures of increasing polarity of cyclohexane and ethyl acetate or dichloromethane and methanol were used as eluents.

[0090] Microwave heating was performed using Biotage Initiator instrument.

[0091] Nuclear magnetic resonance (NMR) spectra were recorded at ambient temperature (298 K, unless otherwise stated) on a Bruker AVA400, AVA500 or AVA600 spectrometer running at 400, 500, or 601 MHz (.sup.1H spectra) or 101, 126, 151 Hz (.sup.13C spectra, respectively). Chemical shifts (δ values) are reported in parts-per-million (ppm) relative to tetramethylsilane (.sup.1H and .sup.13C spectra; δTMS=0) and are calibrated to the residual solvent peak.

[0092] Mass spectra were obtained by electrospray ionization (ESI) on a Bruker microTOF II mass spectrometer. Mass-to-charge ratios (m/z) of all parent (molecular) ions ([M]+/) and their intensities are reported, followed by (major) fragment or adduct ions and their intensities. LC-MS analyses were run on a Bruker microTOF II system equipped with an electrospray ionization interface and a photodiode array detector. PDA range was 210-400 nm.

[0093] Electrospray ionization was applied in positive modes.

[0094] UPLC mobile phases were (A) H.sub.2O/Formic Acid (99.9:0.1), and (B) MeCN/Formic acid (99.9:0.1). Analyses were performed with the method reported below. Gradient: 5-100% B over 10 min. Flow rate: 200 μL/min. Temperature: 30° C. Column: Phenomenex Kinetex C18 (5 μm, 2.1 mm×50 mm). Melting points (mp) were determined on a Gallenkamp Electrothermal Melting Point apparatus.

[0095] General Procedure 1 for the Synthesis of the N-Substituted p-Nitrobenzylamine Derivatives (Steps a and b, Scheme 1)

[0096] Method A (Reductive Amination)

[0097] To a solution of p-nitrobenzaldehyde (1 eq.) in dry ethanol was added the amine derivatives (1.1 eq.) under a nitrogen atmosphere. The solution was stirred at room temperature for 16 hours and then refrigerated to 0° C., and NaBH.sub.4 (2 eq.) was added portionwise until disappearance of the intermediate imine (TLC analysis, approximately 8 hours). The reaction mixture was concentrated in vacuo and the residue dissolved in DCM. The organic phase was washed sequentially with a saturated solution of NaHCO.sub.3, water and brine, dried over MgSO.sub.4, filtered, and concentrated in vacuo to yield the title compounds as pale-yellow oils, which were used without further purification.

[0098] Method B (Nucleophilic Substitution).

[0099] A mixture of 4-nitrobenzyl bromide or 4-nitrobenzylamine hydrochloride (1 eq.), alkyl amines or alkyl halides (1 eq.) and K.sub.2CO.sub.3 (2 eq.) in acetonitrile was stirred at 70° C. for 6 hours. The suspension was filtered and the residue washed with acetone several times. The filtrates were concentrated under reduced pressure to give the desired intermediates as oily products, which were pure enough for the next step.

[0100] General Procedure 2 for the Synthesis of Urea Derivatives (Step c, Scheme 1)

[0101] Ethyl isocyanatoacetate (1 eq.) was dissolved in DCM. p-nitrophenylamino intermediates (1 eq.) were added and the reaction mixture was stirred at room temperature overnight. After this time, water was added to the mixture and the organic phase was collected. The water phase was back-extracted with DCM several times and the combined organic phases were dried over MgSO.sub.4, filtered, and evaporated under reduced pressure, yielding the products as an oil.

[0102] General Procedure 3 for the Synthesis of the Amide Derivative (Step e, Scheme 1)

[0103] Ethyl ester derivatives (1 eq.) were dissolved in a 1:1 mixture of THF/MeOH and treated with LiOH monohydrate (5 eq.) dissolved in water (Water/THF/MeOH ratio: 1:2:2). The reaction was kept under stirring at RT for 2 hours, then the reaction mixture was concentrated, diluted with water and washed with DCM. The aqueous phase was treated with 1 N aqueous HCl until acidic pH and extracted several times with EtOAc. The combined ethyl acetate organic phases were dried over MgSO.sub.4, filtered, and evaporated to afford the desired products, which were used directly in the next step.

[0104] 2-(2-Bromophenyl)-pyrrolidine (1 eq.), carboxylic acid derivatives (1.1 eq.) and HATU (1.4 eq.) were dissolved in DMF. DIPEA (1.5 eq.) was added and the reaction mixture was stirred at room temperature for 18 hours. After this time, the reaction mixture was diluted with EtOAc and washed with water and brine. The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. The crude product was purified by flash column chromatography eluting with MeOH in DCM. The desired compounds were obtained as an oily product.

[0105] General Procedure 4 for the Synthesis of the Final Aniline Derivatives (Step d, Scheme 1)

[0106] p-nitrophenylurea intermediates (1 eq.) were placed in a round-bottomed flask and ethanol was added. Fe powder (3 eq.) was added and the reaction taken to reflux temperature (90° C.) at which time water (EtOH/water ratio: 10:2) was added through the top of the condenser together with calcium chloride (1 eq.). After 4 hours the reaction was allowed to cool and filtered through Celite. The reaction solvent was removed under reduced pressure by rotary evaporator, yielding a crude which was dissolved in DCM and washed with water and the organic phase was collected, dried over magnesium sulfate, filtered and concentrated, yielding the crude as a yellow oil. The resultant crude was purified by column chromatography on silica gel (DCM/MeOH 9:1). The organic fractions were collected and evaporated, yielding the desired compounds as an oily product.

Synthesis of the Intermediates

Ethyl[(4-nitrophenyl)methyl]amine

[0107] ##STR00015##

[0108] The title compound was synthesized applying the general procedure 1 method A using 4-nitrobenzaldehyde (500 mg, 3.31 mmol), dry ethanol (10 mL), ethylamine (2 M solution in THF, 1.80 mL, 3.64 mmol) and NaBH.sub.4 (250 mg, 6.62 mmol). Yellow oil 555 mg (93%). LC-MS: Rt 2.8 min; m/z 181 [M+H].sup.+. .sup.1H NMR (601 MHz, DMSO-d6) δ 8.19-8.16 (m, 2H), 7.63-7.59 (m, 2H), 3.81 (s, 2H), 2.51 (q, J=7.0 Hz, 2H), 2.21 (bs, 1H), 1.03 (t, J=7.1 Hz, 3H).

(Cyclopropylmethyl)[(4-nitrophenyl)methyl]amine

[0109] ##STR00016##

[0110] The title compound was synthesized applying the general procedure 1 method A using 4-nitrobenzaldehyde (620 mg, 4.10 mmol), dry ethanol (10 mL), cyclopropylmethylamine (0.40 mL, 4.51 mmol) and NaBH.sub.4 (310 mg, 8.21 mmol). Yellow oil 800 mg (95%). LC-MS: Rt 2.9 min; m/z 207 [M+H].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.20-8.15 (m, 2H), 7.64-7.60 (m, 2H), 3.84 (s, 2H), 2.37 (d, J=6.7 Hz, 2H), 2.33 (s, 1H), 0.93-0.84 (m, 1H), 0.41-0.37 (m, 2H), 0.10-0.06 (m, 2H).

tert-butyl N-{[(1R,9S,10S)-10-hydroxy-12-oxa-8-azatricyclo[7.3.1.0.SUP.2,7.]trideca-2,4,6-trien-4-yl]methyl}carbamate

[0111] ##STR00017##

[0112] A mixture of 2-deoxy-D-ribose (314 mg, 2.34 mmol), 4-[(N-Boc)aminomethyl]aniline (520 mg, 2.34 mmol) and Montmorillonite (1.2 g) were stirred at RT in MeCN (10 mL) for three days. The mixture was filtered through a celite pad and washed with methanol. The filtrate was concentrated under reduced pressure. The crude was purified by flash column chromatography on silica gel (hexane:EtOAc). 273 mg (36%). .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 6.82 (bs, 1H), 6.56 (d, J=8.2 Hz, 1H), 6.47 (d, J=8.2 Hz, 1H), 6.29 (s, 1H), 6.08 (s, 1H), 4.94 (d, J=3.9 Hz, 1H), 4.51 (s, 2H), 4.33 (t, J=5.1 Hz, 1H), 3.59 (dtd, J=10.8, 5.6, 3.1 Hz, 1H), 3.49-3.34 (m, 5H), 3.28 (bs, 1H), 1.39 (s, 9H)

(1R,9S,10S)-4-[(ethylamino)methyl]-12-oxa-8-azatricyclo[7.3.1.0.SUP.2,7.]trideca-2,4,6-trien-10-ol

[0113] ##STR00018##

[0114] To a solution of tert-butyl N-{[(1R,9S,10S)-10-hydroxy-12-oxa-8-azatricyclo[7.3.1.0.sup.2,7]trideca-2,4,6-trien-4-yl]methyl}carbamate (520 mg, 1.62 mmol) in DCM was added 1 mL of 4N HCl in dioxane. The reaction was stirred for 1 h, then concentrated to give the intermediate which was used in the next step without purification. This crude material was dissolved in ethanol (10 mL) and acetaldehyde (0.1 mL, 1.9 mmol) added and stirred at room temperature for 12 h. NaBH.sub.4 (144 mg, 3.8 mmol) was added and the reaction stirred for 6 h. The solvent was dissolved in water and the residue dissolved in water and acidified to pH 12 with 1 N NaOH. The mixture was extracted with ethyl acetate and concentrated to give the title compound as a yellow oil 199 mg (42%). This material was telescoped into the next reaction.

({[(4-nitrophenyl)methyl]amino}methyl)-1H-1,2,3-triazol-1-yl]acetate

[0115] ##STR00019##

[0116] To a solution of 2-{[(4-nitrophenyl)methyl]amino}acetonitrile (600 mg, 3.15 mmol), ethylazidoactetate (448 mg, 3.47 mmol) and CuSO.sub.4 (201 mg, 1.26 mmol) was added sodium ascorbate (1.25 g, 6.31 mmol) and the mixture stirred at RT for 16 h. The reaction was quenched with crushed ice and extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over anhydrous MgSO.sub.4, filtered, and concentrated under reduced pressure to afford the desired compound. Brown oil 900 mg (89%). LC-MS: Rt 2.6 min; m/z 320 [M+H].sup.+. .sup.1H NMR (601 MHz, DMSO-d.sub.6) δ 8.23-8.17 (m, 2H), 8.00 (s, 1H), 7.69 (d, J=8.8 Hz, 1H), 7.64 (d, J=8.5 Hz, 1H), 5.41-5.34 (m, 2H), 4.22-4.11 (m, 4H), 4.11 (s, 1H), 3.74-3.69 (m 2H), 1.19 (t, J=7.1 Hz, 3H)

2-{[(4-nitrophenyl)methyl]amino}acetonitrile

[0117] ##STR00020##

[0118] The title compound was synthesized applying the general procedure 1 method B using 4-nitrobenzylamine hydrochloride (700 mg, 3.71 mmol), chloroacetonitrile (0.23 mL, 3.71 mmol) and K.sub.2CO.sub.3 (1026 mg, 7.42 mmol) in acetonitrile (10 mL). Brown oil 705 mg (99%). LC-MS: Rt 2.4 min; m/z 192 [M+H].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.24-8.15 (m, 2H), 7.66-7.58 (m, 2H), 3.90 (d, J=6.1 Hz, 2H), 3.65 (d, J=7.2 Hz, 2H), 3.29-3.23 (m, 1H).

[(4-nitrophenyl)methyl](prop-2-yn-1-yl)amine

[0119] ##STR00021##

[0120] The title compound was synthesized applying the general procedure 1 method B using 4-nitrobenzyl bromide (700 mg, 3.24 mmol), propargylamine (0.21 mL, 3.24 mmol) and K.sub.2CO.sub.3 (896 mg, 6.48 mmol) in acetonitrile (10 mL). Brown oil 603 mg (98%). LC-MS: Rt 1.2 min; m/z 191 [M+H].sup.+. .sup.1H NMR (601 MHz, DMSO-d.sub.6) δ 8.20-8.17 (m, 2H), 7.64-7.59 (m, 2H), 3.88 (s, 2H), 3.81 (s, 1H), 3.31 (d, J=2.3 Hz, 2H), 3.11 (t, J=2.4 Hz, 1H).

[(4-nitrophenyl)methyl](propyl)amine

[0121] ##STR00022##

[0122] The title compound was synthesized applying the general procedure 1 method B using 4-nitrobenzyl bromide (700 mg, 3.24 mmol), 1-propylamine (0.27 mL, 3.24 mmol) and K.sub.2CO.sub.3 (896 mg, 6.48 mmol) in acetonitrile (10 mL). Brown oil 600 mg (95%). LC-MS: Rt 1.2 min; m/z 195 [M+H].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.20-8.14 (m, 2H), 7.64-7.58 (m, 2H), 3.80 (s, 2H), 2.44 (t, J=7.4 Hz, 2H), 2.23 (s, 1H), 1.43 (sxt, J=7.4 Hz, 2H), 0.87 (t, J=7.4 Hz, 3H).

(carbamoylmethyl)(diazyn-1-ium-1-yl)azanide

[0123] ##STR00023##

[0124] 2-bromoacetamide (600 mg, 4.35 mmol) was dissolved in 10 mL of DMSO. Sodium azide (622 mg, 9.57 mmol) was added and the mixture stirred overnight at rt. The reaction was diluted with water and extracted with ethyl acetate. The combined organic phases were dried over anhydrous MgSO.sub.4, filtered, and the solvent removed under reduced pressure to give the title compound as a colorless oil 650 mg (90%).

[0125] .sup.1H NMR (601 MHz, DMSO-d.sub.6) δ 7.52 (s, 1H), 7.25 (s, 1H), 3.78 (s, 2H).

2-[4-({[(4-nitrophenyl)methyl]amino}methyl)-1H-1,2,3-triazol-1-yl]acetamide

[0126] ##STR00024##

[0127] A mixture of (carbamoylmethyl)(diazyn-1-ium-1-yl)azanide (347 mg, 3.47 mmol), [(4-nitrophenyl)methyl](prop-2-yn-1-yl)amine (600 mg, 3.15 mmol), CuSO.sub.4 (503 mg, 3.15 mmol) and sodium ascorbate (1249 mg, 6.31 mmol) were dissolved in EtOH/water (20 mL, 1:1) and stirred at rt for 16 h. The reaction mixture was quenched with crushed ice and extracted with ethyl acetate. The combined organic fractions were washed with brine and dried over anhydrous MgSO.sub.4, filtered and the solvent removed in vacuo. The crude oil was purified by column chromatography on silica gel (DCM/MeOH). Pale yellow solid 250 mg (27%). LC-MS: Rt 1.0 min; m/z 291 [M-Na].sup.+. .sup.1H NMR (601 MHz, DMSO-d.sub.6) δ 8.20 (d, J=8.3 Hz, 2H), 7.92 (s, 1H), 7.69 (s, 1H), 7.64 (d, J=8.3 Hz, 2H), 7.36 (s, 1H), 5.04 (s, 2H), 3.97-3.68 (d, J=66.3 Hz, 4H).

Ethyl 2[({[1-(carbamoylmethyl)-1H-1,2,3-triazol-4-yl]methyl}[(4-nitrophenyl)methyl]carbamoyl)amino]acetate

[0128] ##STR00025##

[0129] The title compound was synthesized applying the general procedure 2 using 2-[4-({[(4-nitrophenyl)methyl]amino}methyl)-1H-1,2,3-triazol-1-yl]acetamide (250 mg, 0.86 mmol), Ethyl isocyanatoacetate (0.1 mL, 0.86 mmol) in 10 mL of DMF. Yellow oil 800 mg (97%). LC-MS: Rt 5.2 min; m/z 420 [M+H].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.19 (d, J=8.8 Hz, 2H), 7.93 (s, 1H), 7.70 (s, 1H), 7.48 (d, J=8.8 Hz, 2H), 7.38 (s, 2H), 5.05 (s, 2H), 4.61 (s, 2H), 4.48 (s, 2H), 4.09 (q, J=7.1 Hz, 2H), 3.79 (d, J=5.9 Hz, 2H), 1.19 (t, J=7.1 Hz, 3H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 171.45, 171.38, 167.68, 158.22, 157.94, 144.35, 128.99 (20), 125.29, 123.58 (20), 61.93, 60.65, 50.82, 48.76, 42.96, 14.45.

tert-butyl N-{2-[2-(2-chlorophenyl)pyrrolidin-1-yl]-2-oxoethyl}carbamate

[0130] ##STR00026##

[0131] Pivaloyl chloride (0.2 mL, 1.54 mmol) was added to a solution of Boc-glycine (270 mg, 1.54 mmol) and Et.sub.3N (0.5 mL, 3.47 mmol) in DCM (5 ml) at 0° C. The reaction was stirred for 1 h, where after Et.sub.3N (0.5 mL, 3.47 mmol) and 2-(2-Chlorophenyl)-pyrrolidine (308 mg, 1.70 mmol) were added in succession. The reaction was allowed to warm to room temperature and react overnight. The reaction mixture was washed with aqueous 0.5 M citric acid, sat. NaCl and sat. NaHCO.sub.3. The DCM phase was dried with anhydrous MgSO.sub.4 and evaporated. 500 mg, 96%. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.42 (td, J=6.0, 5.5, 2.9 Hz, 2H), 7.31-7.19 (m, 2H), 7.19-7.10 (m, 1H), 6.77 (t, J=5.8 Hz, 1H), 3.83 (qt, J=12.3, 5.9 Hz, 2H), 3.75-3.49 (m, 2H), 2.31-2.19 (m, 1H), 2.01-1.63 (m, 4H), 1.21 (s, 9H).

3-{2-[2-(2-chlorophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-[(4-nitrophenyl)methyl]-1-(prop-2-yn-1-yl)urea

[0132] ##STR00027##

[0133] tert-butyl N-{2-[2-(2-chlorophenyl)pyrrolidin-1-yl]-2-oxoethyl}carbamate (460 mg, 1.36 mmol) was placed in a round-bottom flask followed by dry DCM (10 mL). 2-Chloropyridine (0.39 mL, 4.07 mmol) was added, followed by trifluoromethanesulfonic anhydride (0.34 mL, 2.04 mmol), and the reaction mixture was stirred for 50 min at room temperature. Then Et.sub.3N (0.4 mL, 2.85 mmol) and 2-{[(4-nitrophenyl)methyl]amino}acetonitrile (516 mg, 2.72 mmol) were added, and the reaction mixture was stirred at room temperature for 20 h. The crude product was purified using column chromatography (30%-50% EtOAc in hexane) to afford the desired product of adequate purity for the next step.

3-{2-[2-(2-chlorophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-[(1-methyl-1H-1,2,3-triazol-4-yl)methyl]-1-[(4-nitrophenyl)methyl]urea

[0134] ##STR00028##

[0135] To a solution of Iodomethane (0.1 mL, 1.6 mmol) in H.sub.2O/DMF 1:4 (10 mL), NaN.sub.3 (99 mg, 1.5 mmol), Na.sub.2CO.sub.3 (440 mg, 4.1 mmol), ascorbic acid (157 mg, 0.9 mmol), CuSO.sub.4*5H.sub.2O (148 mg, 0.6 mmol) and 3-{2-[2-(2-chlorophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-[(4-nitrophenyl)methyl]-1-(prop-2-yn-1-yl)urea (450 mg, 1.0 mmol) were added. The reaction mixture was stirred at rt overnight, diluted with saturated NH.sub.4Cl, treated with solid EDTA and extracted with EtOAc. The combined organic extracts were washed with H.sub.2O, dried over anhydrous MgSO.sub.4, and concentrated under reduced pressure. The resulting residue was further purified by column chromatography on silica gel (Eluent: Chloroform/MeOH), as orange oil (196 mg, 39% yield). LC-MS: Rt 5.9 min; m/z 512 [M+H].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.16 (d, J=8.7 Hz, 2H), 7.96 (d, J=3.4 Hz, 1H), 7.63-7.45 (m, 2H), 7.46-7.41 (m, 1H), 7.40-7.30 (m, 1H), 7.30-7.12 (m, 2H), 6.77 (dt, J=39.7, 5.4 Hz, 1H), 5.41-5.22 (m, 1H), 4.58 (s, 1H), 4.06-3.98 (m, 2H), 3.92-3.84 (m, 1H), 3.68-3.58 (m, 2H), 2.90 (s, 3H), 2.35-2.21 (m, 1H), 2.00-1.66 (m, 3H).

tert-butyl N-[(4R)-4-(2-bromophenyl)-4-(methanesulfonyloxy)butyl]carbamate

[0136] ##STR00029##

[0137] Bromoiodobenzene (0.5 mL, 3.88 mmol) was added to iPrMgCl.LiCl (3 mL, 3.89 mmol) cooled to −15° C. and stirred for 30 minutes. Tert-butyl-2-oxopyrrolidine-1-carboxylate (0.68 mL, 3.97 mmol), was added and the reaction stirred at 0° C. for 2 h then left overnight at room temperature. NH.sub.4Cl (sat, 20 mL) was added and the mixture extracted with ethyl acetate. The combined organic layers were dried over MgSO.sub.4, filtered, and the solvent removed in vacuo. This crude was dissolved in THF (12 mL) and added dropwise to (R)-2-methyl-CBS-oxaborolidine (206 mg, 0.75 mmol) in BH.sub.3-DMS (2.48 mL, 2.48 mmol). The reaction was stirred for 3 h at RT, quenched with 1M HCl, and extracted with ethyl acetate. The combined organic layers were dried over MgSO.sub.4, filtered, and the solvent removed in vacuo to an oil (781 mg, 91%). The oil was dissolved in DCM (9 mL) and MsCl (0.51 mL, 6.57 mmol) and NEt.sub.3 (1.22 mL, 8.76 mmol) added. The mixture was stirred at RT overnight. The mixture was washed with NaHCO.sub.3, water and brine and dried over MgSO.sub.4, filtered and the solvent removed in vacuo affording an oil which was used directly in the next step.

(2S)-2-(2-bromophenyl)pyrrolidine

[0138] ##STR00030##

[0139] tert-butyl N-[(4R)-4-(2-bromophenyl)-4-(methanesulfonyloxy)butyl]carbamate (739 mg, 1.75 mmol), was dissolved in DCM/TFA (12 mL, 5:1) and stirred at RT for 2 h. The mixture was concentrated in vacuo and the residue dissolved in 1 M NaOH (7 mL) in MeOH (6 mL) and stirred for 2 days at RT. The reaction was concentrated, neutralized with 1M HCl and extracted with ethyl acetate. The combined organic layers were dried over MgSO.sub.4, filtered, and the solvent removed in vacuo. Yellow solid (395 mg, quant.).sup.1H NMR (601 MHz, DMSO-d.sub.6) δ 7.70-7.65 (m, 1H), 7.57 (ddd, J=7.8, 5.4, 1.5 Hz, 1H), 7.50-7.42 (m, 1H), 7.42-7.33 (m, 1H), 3.94 (tt, J=7.4, 2.1 Hz, 1H), 3.32 (bs, 1H), 3.28-3.10 (m, 1H), 2.96-2.87 (m, 1H), 2.40-2.31 (m, 2H), 2.03-1.90 (m, 1H), 1.88-1.71 (m, 1H).

Ethyl 2-({ethyl[(4-nitrophenyl)methyl]carbamoyl}amino)acetate

[0140] ##STR00031##

[0141] The title compound was synthesized applying the general procedure 2 using Ethyl[(4-nitrophenyl)methyl]amine (480 mg, 2.66 mmol), Ethyl isocyanatoacetate (0.3 mL, 2.66 mmol) in 10 mL of DCM. Yellow oil 800 mg (97%). LC-MS: Rt 5.6 min; m/z 332 [M-Na].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.17-8.23 (m, 2H), 7.53-7.48 (m, 2H), 6.95 (t, J=5.9 Hz, 1H), 4.57 (s, 2H), 4.10 (q, J=6.9 Hz, 2H), 3.77 (d, J=6.0 Hz, 2H), 3.24 (q, J=7.0 Hz, 2H), 1.20 (t, J=7.1 Hz, 3H), 1.04 (t, J=7.0 Hz, 3H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 171.48, 157.88, 148.24, 146.95, 128.62 (2C), 123.90 (2C), 60.60, 48.95, 42.86, 41.49, 14.59, 13.81.

Ethyl 2-{[(cyclopropylmethyl)[(4-nitrophenyl)methyl]carbamoyl]amino}acetate

[0142] ##STR00032##

[0143] The title compound was synthesized applying the general procedure 2 using (Cyclopropylmethyl)[(4-nitrophenyl)methyl]amine (380 mg, 1.84 mmol) and Ethyl isocyanatoacetate (0.2 mL, 1.84 mmol) in 10 mL of DCM. Yellow oil 500 mg (81%). LC-MS: Rt 5.8 min; m/z 358 [M-Na].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.23-8.13 (m, 2H), 7.54-7.46 (m, 2H), 6.98 (t, J=5.8 Hz, 1H), 4.67 (s, 2H), 4.14-4.07 (m, 2H), 3.76 (d, J=5.9 Hz, 2H), 3.13 (d, J=6.8 Hz, 2H), 1.24-1.14 (m, 3H), 0.95 (ddtd, J=11.7, 8.0, 6.8, 4.9 Hz, 1H), 0.40-0.31 (m, 2H), 0.21-0.12 (m, 2H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 171.45, 158.05, 148.24, 146.86, 128.47 (2C), 123.80 (2C), 60.88, 51.05, 49.59, 42.78, 14.58, 10.66, 3.81 (2C).

Ethyl 2-{[(cyanomethyl)[(4-nitrophenyl)methyl]carbamoyl]amino}acetate

[0144] ##STR00033##

[0145] The title compound was synthesized applying the general procedure 2 using 2-{[(4-nitrophenyl)methyl]amino}acetonitrile (600 mg, 3.14 mmol) and Ethyl isocyanatoacetate (0.35 mL, 3.14 mmol) in 10 mL of DCM. Brown oil 900 mg (90%). LC-MS: Rt 5.5 min; m/z 321 [M+H].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.26-8.21 (m, 2H), 7.58-7.54 (m, 2H), 7.51-7.48 (m, 1H), 4.72 (s, 2H), 4.38 (s, 2H), 4.13-4.09 (m, 2H), 3.83-3.79 (m, 2H), 1.22-1.19 (m, 3H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 170.95, 157.32, 147.30, 145.77, 128.84 (2C), 123.99 (2C), 67.48, 60.79, 50.49, 36.21, 25.60, 14.57.

Ethyl 2-({ethyl[(4-nitrophenyl)methyl]carbamoyl}amino)acetate

[0146] ##STR00034##

[0147] The title compound was synthesized applying the general procedure 3 part 1 using Ethyl 2-({ethyl[(4-nitrophenyl)methyl]carbamoyl}amino)acetate (340 mg, 1.1 mmol) and LiOH (185 mg, 4.4 mmol) in THF/methanol (12 mL, 1:1). Yellow solid 230 mg (74%). .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 12.38 (s, 1H), 8.20 (s, 2H), 7.51 (d, J=8.7 Hz, 2H), 6.84 (t, J=5.9 Hz, 1H), 4.56 (d, J=6.1 Hz, 2H), 3.71 (t, J=6.2 Hz, 2H), 3.23 (q, J=7.1 Hz, 2H), 1.03 (t, J=7.0 Hz, 3H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 172.96, 157.96, 148.34, 146.94, 128.69 (2C), 123.89 (2C), 60.22, 48.96, 21.23, 13.84.

3-{2-[2-(2-bromophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-ethyl-1-[(4-nitrophenyl)methyl]urea

[0148] ##STR00035##

[0149] The title compound was synthesized applying the general procedure 3 part 1 using Ethyl 2-({ethyl[(4-nitrophenyl)methyl]carbamoyl}amino)acetate (224 mg, 0.8 mmol), 2-(2-bromo)-pyrrolidine (150 mg, 0.66 mmol), HATU (328 mg, 0.86 mmol) and DIPEA (0.2 mL, 1.00 mmol), in DMF (15 mL). The product was purified by column chromatography (EtOAc/hexane) colourless liquid 100 mg (31%). LC-MS: Rt 6.2 min; m/z 491 [M+H].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ .sup.1H NMR (500 MHz, Chloroform-d) δ 8.14 (dd, J=8.8, 1.9 Hz, 2H), 7.55 (ddd, J=14.6, 7.9, 1.3 Hz, 1H), 7.42-7.37 (m, 2H), 7.30-7.19 (m, 1H), 7.12 (dtd, J=28.5, 7.7, 1.7 Hz, 1H), 6.99 (ddd, J=39.6, 7.7, 1.7 Hz, 1H), 5.48-5.41 (m, 1H), 4.59 (q, J=16.5 Hz, 1H), 4.55 (s, 1H), 4.15 (d, J=4.0 Hz, 1H), 4.11 (q, J=7.1 Hz, 1H), 3.89-3.69 (m, 1H), 3.62 (ddd, J=10.0, 8.7, 7.2 Hz, 1H), 3.35 (dd, J=17.5, 3.5 Hz, 1H), 3.31-3.19 (m, 2H), 2.44 (dddd, J=12.7, 11.2, 8.0, 6.9 Hz, 1H), 2.34 (dddd, J=12.6, 10.4, 8.2, 7.0 Hz, 1H), 2.02-1.95 (m, 1H), 1.95-1.87 (m, 1H), 1.25 (t, J=7.1 Hz, 1H), 1.19-1.09 (m, 2H).

3-{2-[2-(2-bromophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-(cyclopropylmethyl)-1-[(4-nitrophenyl)methyl]urea

[0150] ##STR00036##

[0151] The title compound was synthesized applying the general procedure 3 Ethyl 2-{[(cyclopropylmethyl)[(4-nitrophenyl)methyl]carbamoyl]amino}acetate (350 mg, 1.04 mmol) and LiOH (219 mg, 5.22 mmol), in THF/water (8 mL, 1:1) followed by HATU (235 mg, 0.62 mmol), and 2-(2-bromophenyl)-pyrrolidine (100 mg, 0.44 mmol) and DIPEA (0.12 mL, 0.66 mmol). The crude product was purified by flash column chromatography eluting DCM/MeOH to yield the product as a brown oil 110 mg, (48%). .sup.1H NMR (600 MHz, Chloroform-d) δ 7.95 (apt, J=8.8 Hz, 2H), 7.38 (dddd, J=49.7, 23.1, 7.9, 1.5 Hz, 1H), 7.26 (d, J=8.7 Hz, 2H), 7.18-6.96 (m, 1H), 6.95-6.76 (m, 1H), 4.54 (q, J=7.1 Hz, 2H), 4.02-3.97 (m, H), 3.96-3.89 (m, 2H), 3.71-3.63 (m, 2H), 3.60-3.43 (m, 2H), 3.06-2.92 (m, 2H), 2.36-2.11 (m, 1H), 1.86-1.57 (m, 2H), 1.08 (t, J=7.1 Hz, 3H), 0.86-0.71 (m, 2H), 0.30 (ddd, J=20.5, 8.1, 1.2 Hz, 1H).

3-{2-[(2S)-2-(2-bromophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-[(1-methyl-1H-1,2,3-triazol-4-yl)methyl]-1-[(4-nitrophenyl)methyl]urea

[0152] ##STR00037##

[0153] The title compound was synthesised according to the general procedure 3 using Ethyl 2-({[(1-methyl-1H-1,2,3-triazol-4-yl)methyl][(4-nitrophenyl)methyl]carbamoyl}amino)acetate (752 mg, 2.00 mmol), LiOH (419 mg, 9.99 mmol) in THF/MeOH (8 mL, 1:1). The acid was then coupled with (2S)-2-(2-bromophenyl)pyrrolidine (150 mg, 0.66 mmol), using HATU (353 mg, 0.93 mmol) and DIPEA (0.17 mL, 1.0 mmol). Brown oil (111 mg, 30%). LC-MS: Rt 1.1 min; m/z 556 [M+H].sup.+.

2-{[(cyanomethyl)[(4-nitrophenyl)methyl]carbamoyl]amino}acetic acid

[0154] ##STR00038##

[0155] The title compound was synthesized applying the general procedure 3 using Ethyl 2-{[(cyanomethyl)[(4-nitrophenyl)methyl]carbamoyl]amino}acetate (450 mg, 1.4 mmol) and LiOH (295 mg, 7.02 mmol) in THF/methanol (12 mL, 1:1). Yellow solid 170 mg (41%). .sup.1H NMR (601 MHz, DMSO-d.sub.6) δ 12.90 (s, 1H), 8.33 (d, J=8.8 Hz, 2H), 8.22-8.09 (m, 2H), 7.40 (t, J=6.2 Hz, 1H), 3.64 (d, J=6.2 Hz, 2H), 3.55 (s, 2H), 3.18 (s, 2H).

Ethyl 2-({[(4-nitrophenyl)methyl](prop-2-yn-1-yl)carbamoyl}amino)acetate

[0156] ##STR00039##

[0157] The title compound was synthesized applying the general procedure 2 using [(4-nitrophenyl)methyl](prop-2-yn-1-yl)amine (480 mg, 2.52 mmol) and Ethyl isocyanatoacetate (0.28 mL, 2.5 mmol) in 10 mL of DCM. Red oil 800 mg (99%). LC-MS: Rt 5.4 min; m/z 320 [M+H].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.21 (m, 2H), 7.57-7.51 (m, 2H), 7.20 (t, J=5.8 Hz, 1H), 4.66 (s, 2H), 4.17-4.06 (m, 4H), 3.77 (d, J=5.8 Hz, 2H), 3.18 (t, J=2.4 Hz, 1H), 1.24-1.16 (m, 3H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 171.17, 157.54, 147.09, 146.81, 128.78 (2C), 123.87 (2C), 80.35, 75.24, 60.67, 49.29, 42.90, 36.34, 14.58.

Ethyl 2-({[(4-nitrophenyl)methyl](propyl)carbamoyl}amino)acetate

[0158] ##STR00040##

[0159] The title compound was synthesized applying the general procedure 2 using [(4-nitrophenyl)methyl](propyl)amine (600 mg, 3.09 mmol) and Ethyl isocyanatoacetate (0.35 mL, 3.09 mmol) in 10 mL of DCM. Yellow oil 990 mg (99%). LC-MS: Rt 5.8 min; m/z 346 [M-Na].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.24-8.16 (m, 2H), 7.52-7.46 (m, 2H), 6.94 (t, J=5.8 Hz, 1H), 4.58 (s, 2H), 4.09 (q, J=7.1 Hz, 2H), 3.76 (d, J=5.8 Hz, 2H), 3.17-3.09 (m, 2H), 1.49 (sxt, J=7.3 Hz, 2H), 1.20 (t, J=7.2 Hz, 3H), 0.81 (t, J=7.4 Hz, 3H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 171.45, 158.10, 148.25, 146.93, 128.57 (2C), 123.87 (2C), 60.62, 49.45, 48.54, 42.89, 21.46, 14.59, 11.41.

Ethyl 2-({[(4-nitrophenyl)methyl][(1H-1,2,3,4-tetrazol-5-yl)methyl]carbamoyl}amino)acetate

[0160] ##STR00041##

[0161] To a 100 mL round-bottomed flask equipped with a stir bar was added Ethyl 2-{[(cyanomethyl)[(4-nitrophenyl)methyl]carbamoyl]amino}acetate (700 mg, 2.19 mmol), 12 mL of DMF, sodium azide (256 mg, 3.93 mmol), and ammonium chloride (222 mg, 4.15 mmol). The reaction vessel was stirred at 90° C. overnight (18 h). The reaction was cooled to RT and diluted with 50 mL of 1 M HCl (aq), then extracted with ethyl acetate (3×20 mL). The organic phase was collected, dried over magnesium sulfate and filtered, then evaporated to give the desired product. Brown oil 571 mg (72%). LC-MS: Rt 5.4 min; m/z 364 [M+H].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 16.23 (s, 1H), 8.24-8.19 (m, 2H), 7.56-7.51 (m, 2H), 7.31 (t, J=5.7 Hz, 1H), 4.75 (s, 2H), 4.72 (s, 2H), 4.09 (q, J=7.1 Hz, 2H), 3.77 (d, J=5.7 Hz, 2H), 1.18 (t, J=7.1 Hz, 3H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 171.11, 163.73, 162.75, 157.83, 146.94, 128.65 (20), 124.17 (2C), 61.16, 50.07, 42.50, 36.25, 15.03.

Ethyl 2-({[(1-methyl-1H-1,2,3-triazol-4-yl)methyl][(4-nitrophenyl)methyl]carbamoyl}amino)acetate

[0162] ##STR00042##

[0163] To a solution of Iodomethane (0.2 mL, 433 mg, 3.05 mmol) in H.sub.2O/DMF 1:4 (15 mL), NaN.sub.3 (229 mg, 3.52 mmol), Na.sub.2CO.sub.3 (996 mg, 9.39 mmol), ascorbic acid (331 mg, 1.88 mmol), CuSO.sub.4*5H.sub.2O (235 mg, 0.95 mmol) and Ethyl 2-({[(4-nitrophenyl)methyl](prop-2-yn-1-yl)carbamoyl}amino)acetate (750 mg, 2.35 mmol) were added. The reaction mixture was stirred at RT overnight, diluted with a saturated solution of NH.sub.4Cl (20 mL), treated with solid EDTA (1.0 g) and extracted with EtOAc (2×20 mL). The combined organic extracts were washed with H.sub.2O (20 mL), dried over anhydrous MgSO.sub.4, filtered, and concentrated under reduced pressure. The resulting residue was washed with hexane and dried under vacuum, to give the desired compound. Orange oil 860 mg (97%). LC-MS: Rt 5.4 min; m/z 377 [M+H].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.22-8.17 (m, 2H), 7.91 (s, 1H), 7.52-7.48 (m, 2H), 7.20 (t, J=5.8 Hz, 1H), 4.59 (s, 2H), 4.45 (s, 2H), 4.11 (q, J=7.1 Hz, 2H), 4.01 (s, 3H), 3.79 (d, J=5.8 Hz, 2H), 1.20 (t, J=7.1 Hz, 3H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 171.34, 162.76, 157.95, 147.02, 144.22, 128.71 (20), 123.90 (20), 60.67, 49.18, 42.95, 41.68, 36.68, 31.24, 14.57.

Ethyl 2-({[(2-methyl-2H-1,2,3,4-tetrazol-5-yl)methyl][(4-nitrophenyl)methyl]carbamoyl}amino)acetate

[0164] ##STR00043##

[0165] The tetrazole derivative Ethyl 2-({[(4-nitrophenyl)methyl][(1H-1,2,3,4-tetrazol-5-yl)methyl]carbamoyl}amino)acetate (700 mg, 1.93 mmol) was dissolved in a solution of triethylamine (0.4 mL, 2.70 mmol) and acetonitrile (10 mL). The solution was heated to reflux temperature (90° C.), followed by the dropwise addition of Iodomethane (0.2 mL, 2.60 mmol). Upon completion, the solution was allowed to cool and stirred at room temperature for three days, then evaporated to dryness. The crude liquid containing a mixture of the 2- and 1-regioisomers was purified using silica gel chromatography (elution with DCM/MeOH) to give the desired 2-regioisomer. Brown oil 170 mg (23%). LC-MS: Rt 5.4 min; m/z 378 [M+H].sup.+. .sup.1H NMR (601 MHz, DMSO-d.sub.6) δ 8.21-8.19 (m, 2H), 7.53-7.49 (m, 2H), 7.25 (t, J=5.8 Hz, 1H), 4.72 (s, 2H), 4.68 (s, 2H), 4.31 (s, 3H), 4.09 (q, J=7.1 Hz, 2H), 3.77 (d, J=5.7 Hz, 2H), 1.19 (t, J=7.1 Hz, 3H). .sup.13C NMR (151 MHz, DMSO-d.sub.6) δ 171.30, 163.73, 162.76, 157.86, 147.23, 128.50 (20), 123.91 (20), 60.69, 49.70, 42.91, 36.25, 31.24, 14.56.

Ethyl 2-[({[1-(2-ethoxy-2-oxoethyl)-1H-1,2,3-triazol-4-yl]methyl}[(4-nitrophenyl)methyl]carbamoyl)amino]acetate

[0166] ##STR00044##

[0167] The title compound was synthesized applying the general procedure 2 using ({[(4-nitrophenyl)methyl]amino}methyl)-1H-1,2,3-triazol-1-yl]acetate (350 mg, 1.1 mmol) and ethyl isocyanatoacetate (0.12 ml, 1.1 mmol) in 10 mL of DCM. Yellow oil 248 mg (50%). LC-MS: Rt 5.7 min; m/z 449 [M+H].sup.+. .sup.1H NMR (601 MHz, DMSO-d.sub.6) δ 8.19-8.17 (m, 2H), 7.97 (s, 1H), 7.96 (s, 1H), 7.48 (d, J=8.8 Hz, 2H), 5.36 (s, 2H), 4.61 (s, 2H), 4.50 (s, 2H), 4.18 (q, J=7.1 Hz, 2H), 4.09 (q, J=7.2 Hz, 2H), 3.77 (d, J=6.0 Hz, 2H), 1.23 (t, J=7.2 Hz, 3H), 1.19 (t, J=7.1 Hz, 3H)

Ethyl (2S)-2-({ethyl[(4-nitrophenyl)methyl]carbamoyl}amino)-4-(methylsulfanyl)butanoate

[0168] ##STR00045##

[0169] CDI (200 mg, 1.23 mg) was dissolved in DCM (10 mL). L-Methionine ethyl ester hydrochloride (263 mg, 1.23 mmol) and DIPEA (0.4 mL, 2.3 mmol) were added and stirred for 6 h at RT. Ethyl[(4-nitrophenyl)methyl]amine (200 mg, 1.11 mmol) in DCM (2 mL) was added and the mixture stirred at RT overnight. The solvent was removed in vacuo and the residue purified by column chromatography eluting DCM:MeOH. Yellow oil 400 mg (94%). LC-MS: Rt 6.0 min; m/z 384 [M+H].sup.+. .sup.1H NMR (601 MHz, DMSO-d.sub.6) δ 8.20 (d, J=8.8 Hz, 2H), 7.51-7.47 (m, 2H), 6.70 (d, J=7.9 Hz, 1H), 4.59 (s, 2H), 4.27 (td, J=7.9, 6.9 Hz, 1H), 4.10 (q, J=10.8 Hz, 1H), 4.09 (q, J=7.1 Hz, 1H), 3.27 (q, J=7.0 Hz, 2H), 2.51 (dt, J=3.6, 1.8 Hz, 2H), 2.04 (s, 3H), 1.99-1.92 (m, 2H), 1.19 (t, J=7.1 Hz, 3H), 1.03 (t, J=7.0 Hz, 3H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 173.53, 157.74, 148.30, 128.61 (2C), 123.90 (2C), 60.75, 55.38, 53.39, 49.00, 41.51, 30.78, 30.55, 15.09, 14.58, 13.87.

3-{2-[2-(2-bromophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-[(4-nitrophenyl)methyl]-1-(prop-2-yn-1-yl)urea

[0170] ##STR00046##

[0171] The title compound was synthesized applying the general procedure 3 using Ethyl 2-({[(4-nitrophenyl)methyl](prop-2-yn-1-yl)carbamoyl}amino)acetate (2100 mg, 6.58 mmol) and LiOH monohydrate (1380 mg, 32.88 mmol) in 16 mL of MeOH/THF 1:1, then 4 mL of water. The so-obtained carboxylic acid (1368 mg, 4.70 mmol) was then reacted with 2-(2-Bromophenyl)-pyrrolidine (905 mg, 4.00 mmol) HATU (2130 mg, 5.60 mmol) and DIPEA (1.05 mL, 6.00 mmol) in DMF (20 mL). Brown oil 1499 mg (46% over two steps). LC-MS: Rt 6.1 min; m/z 499 [M+H].sup.+. .sup.1H NMR (601 MHz, DMSO-d.sub.6) δ 8.20-8.15 (m, 2H), 7.55-7.50 (m, 2H), 7.32-7.23 (m, 2H), 7.20-7.10 (m, 2H), 6.77 (t, J=5.3 Hz, 1H), 4.65 (s, 2H), 4.08 (d, J=2.5 Hz, 2H), 3.97 (d, J=5.4 Hz, 2H), 3.94-3.87 (m, 1H), 3.66-3.58 (m, 2H), 3.17 (t, J=2.4 Hz, 1H), 2.30-2.23 (m, 1H), 1.98-1.90 (m, 1H), 1.84-1.66 (m, 2H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 168.20, 157.50, 147.04, 142.38, 133.02, 128.99, 128.84, 128.81 (2C), 127.93, 127.26, 123.86 (2C), 121.83, 80.39, 75.27, 60.74, 49.45, 46.75, 43.49, 36.44, 32.29, 23.34.

3-{2-[2-(2-bromophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-[(4-nitrophenyl)methyl]-1-(prop-2-yn-1-yl)urea

[0172] ##STR00047##

[0173] 2-(2-Bromophenyl)-pyrrolidine (100 mg, 0.44 mmol, 1 equiv), 2-{[(cyanomethyl)[(4-nitrophenyl)methyl]carbamoyl]amino}acetic acid (142 mg, 0.49 mmol, 1.1 equiv) and HATU (235 mg, 0.62 mmol, 1.4 equiv) were dissolved in DMF (10 mL). DIPEA (0.12 mL, 0.66 mmol, 1.5 equiv) was added and the reaction mixture was stirred at room temperature overnight. After this time, the reaction mixture was diluted with EtOAc (40 mL) and washed with water (40 mL) and brine (40 mL). The organic layer was dried over MgSO.sub.4, filtered and concentrated in vacuo. (65 mg, 29%). Reaction was telescope to the final step.

3-{2-[2-(2-bromophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-[(1-methyl-1H-1,2,3-triazol-4-yl)methyl]-1-[(4-nitrophenyl)methyl]urea

[0174] ##STR00048##

[0175] The title compound was synthesized applying the general procedure 3 using Ethyl 2-({[(1-methyl-1H-1,2,3-triazol-4-yl)methyl][(4-nitrophenyl)methyl]carbamoyl}amino)acetate (600 mg, 1.59 mmol) and LiOH monohydrate (334 mg, 7.97 mmol) in 8 mL of MeOH/THF 1:1, then 2 mL of water. The so-obtained carboxylic acid (254 mg, 0.73 mmol) was then reacted with 2-(2-Bromophenyl)-pyrrolidine (150 mg, 0.66 mmol), HATU (353 mg, 0.93 mmol) and DIPEA (0.17 mL, 1.00 mmol) in DMF (10 mL). Brown oil 311 mg (35% over two steps). LC-MS: Rt 5.9 min, m/z 556 [M+H].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.18-8.14 (m, 2H), 7.91 (s, 1H), 7.51-7.46 (m, 2H), 7.31-7.23 (m, 2H), 7.21-7.12 (m, 2H), 6.81 (t, J=5.4 Hz, 1H), 4.58 (s, 2H), 4.43 (s, 2H), 3.99 (d, J=5.4 Hz, 2H), 3.96 (s, 3H), 3.93-3.86 (m, 1H), 3.61 (qd, J=11.1, 10.3, 5.4 Hz, 2H), 232-2.27 (m, 2H), 1.98-1.66 (m, 2H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 169.44, 156.94, 147.65, 138.41, 137.91, 136.57, 132.88, 128.83, 128.04 (2C), 127.62, 125.34, 124.66, 124.42 (2C), 121.84, 64.96, 52.05, 46.78, 43.43, 42.85, 37.72, 32.46, 24.61.

3-{2-[2-(2-bromophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-[(2-methyl-2H-1,2,3,4-tetrazol-5-yl)methyl]-1-[(4-nitrophenyl)methyl]urea

[0176] ##STR00049##

[0177] The title compound was synthesized applying the general procedure 3, using Ethyl 2-({[(2-methyl-2H-1,2,3,4-tetrazol-5-yl)methyl][(4-nitrophenyl)methyl]carbamoyl}amino)acetate (602 mg, 1.60 mmol), and LiOH monohydrate (335 mg, 7.98 mmol) in 8 mL of MeOH/THF 1:1, then 2 mL of water. The so-obtained carboxylic acid (255 mg, 0.73 mmol) was then reacted with 2-(2-Bromophenyl)-pyrrolidine (150 mg, 0.66 mmol) HATU (353 mg, 0.93 mmol) and DIPEA (0.17 mL, 1.00 mmol) in DMF (10 mL). Brown oil 221 mg (25% over two steps). LC-MS: Rt 5.9 min; m/z 558 [M+H].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.29-8.22 (m, 2H), 7.63-7.53 (m, 2H), 7.48-7.42 (m, 2H), 7.08-7.03 (m, 2H), 6.27 (s, 1H), 4.65 (s, 2H), 4.54 (s, 2H), 4.09 (s, 3H), 3.97 (d, J=5.2 Hz, 2H), 3.93-3.86 (m, 1H), 3.61-3.58 (m, 2H), 2.30-2.23 (m, 1H), 1.98-1.90 (m, 1H), 1.84-1.66 (m, 2H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 168.82, 157.22, 147.35, 140.39, 135.46, 132.78, 130.86, 128.82 (20), 127.98, 127.44, 125.00, 124.14 (20), 121.84, 70.11, 56.39, 48.11, 45.09, 43.17, 37.08, 32.37, 23.97.

Synthesis of Final Products

Ethyl 2-({[(4-aminophenyl)methyl](ethyl)carbamoyl}amino)acetate hydrochloride (12)

[0178] ##STR00050##

[0179] The title compound was synthesized according to the general procedure 4, starting from Fe powder (235 mg, 4.21 mmol), calcium chloride (156 mg, 1.40 mmol), Ethyl 2-({ethyl[(4-nitrophenyl)methyl]carbamoyl}amino)acetate (434 mg, 1.40 mmol), in Ethanol/Water (12 mL, 10:2). The resultant oil was dissolved in a small amount of ethanol, to which 1.25 M HCl in ethanol was added. Evaporation of the solvent produced the title compound as yellow solid 145 mg (33%). Mp: 88-91° C. LC-MS: Rt=1.3 min, m/z 302 [M+Na].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 10.22 (bs, 2H), 7.38-7.31 (m, 4H), 6.91 (s, 1H), 4.45 (s, 2H), 4.09 (q, J=7.2 Hz, 2H), 3.76 (s, 2H), 3.19 (q, J=7.0 Hz, 2H), 1.20 (t, J=7.0 Hz, 3H), 1.01 (t, J=6.8 Hz, 3H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 171.62, 157.82, 139.80, 130.95, 128.81 (2C), 123.50 (2C), 60.57, 48.61, 42.86, 41.06, 14.60, 13.75. ESI+(m/z): [M+Na].sup.+ calculated for C.sub.14H.sub.21N.sub.3O.sub.3Na 302.1475; found 302.1460 [M+Na].sup.+. LC-MS purity: 96%

Ethyl 2-({[(4-aminophenyl)methyl](cyanomethyl)carbamoyl}amino)acetate hydrochloride (13)

[0180] ##STR00051##

[0181] The title compound was synthesized according to the general procedure 4, starting from Fe powder (124 mg, 2.22 mmol), calcium chloride (82 mg, 0.74 mmol), Ethyl 2-{[(cyanomethyl)[(4-nitrophenyl)methyl]carbamoyl]amino}acetate (237 mg, 0.74 mmol), in Ethanol/Water (8 mL, 6:2). The resultant oil was dissolved in a small amount of ethanol, to which 1.25 M HCl in ethanol was added. Evaporation of the solvent produced the title compound as an orange solid 600 mg (25%). Mp: 91-95° C. LC-MS: Rt=1.3 min, m/z 314 [M+Na].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 9.73 (bs, 2H), 7.32 (d, J=8.3 Hz, 2H), 7.25 (d, J=8.0 Hz, 2H), 6.97 (t, J=7.9 Hz, 1H), 4.52 (s, 2H), 4.22 (s, 2H), 4.17-4.13 (m, 2H), 4.03 (d, J=7.9 Hz, 2H), 1.23-1.20 (m, 3H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 172.19, 170.08, 167.90, 156.09, 129.28 (4C), 122.40, 61.78, 43.56, 33.40, 25.86, 14.51. ESI+(m/z): [M+Na].sup.+ calculated for C.sub.14H.sub.19N.sub.4O.sub.3Na 314.1349; found 314.1360 [M+Na].sup.+. LC-MS purity: 95%.

Ethyl 2-({[(4-aminophenyl)methyl][(1H-1,2,3,4-tetrazol-5-yl)methyl]carbamoyl}amino)acetate hydrochloride (14)

[0182] ##STR00052##

[0183] The title compound was synthesized according to the general procedure 4, starting from Fe powder (263 mg, 4.71 mmol), calcium chloride (174 mg, 1.57 mmol), Ethyl 2-({[(4-nitrophenyl)methyl][(1H-1,2,3,4-tetrazol-5-yl)methyl]carbamoyl}amino)acetate (570 mg, 1.57 mmol), in Ethanol/Water (12 mL, 10:2). The resultant oil was dissolved in a small amount of ethanol, to which 1.25 M HCl in ethanol was added. Evaporation of the solvent produced the title compound as an orange solid 145 mg (25%). Mp: 146-149° C. LC-MS: Rt=1.4 min, m/z 334 [M+H].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 10.05 (bs, 2H), 7.37-7.26 (m, 5H), 4.66 (s, 2H), 4.56 (s, 2H), 4.07 (q, J=6.7 Hz, 2H), 3.76 (d, J=5.2 Hz, 2H), 1.05 (t, J=6.8 Hz, 3H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 171.18, 157.88, 147.54, 128.93 (4C), 122.94 (2C), 60.70, 56.49, 49.70, 42.91, 14.61. ESI+(m/z): [M+H].sup.+ calculated for C.sub.14H.sub.19N.sub.7O.sub.3 333.1543; found 333.1550 [M+H].sup.+. LC-MS purity: 100%.

Ethyl (2S)-2-({[(4-aminophenyl)methyl](ethyl)carbamoyl}amino)-4-(methylsulfanyl)butanoate hydrochloride (15)

[0184] ##STR00053##

[0185] The title compound was synthesized according to the general procedure 4, starting from Fe powder (124 mg, 2.22 mmol), calcium chloride (82 mg, 0.74 mmol), Ethyl 2-{[(cyanomethyl)[(4-nitrophenyl)methyl]carbamoyl]amino}acetate (237 mg, 0.74 mmol), in Ethanol/Water (8 mL, 6:2). The resultant oil was dissolved in a small amount of ethanol, to which 1.25 M HCl in ethanol was added. Evaporation of the solvent produced the title compound as an orange solid 145 mg (42%). LC-MS: Rt=5.2 min, m/z 376 [M+Na].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 10.38 (bs, 2H), 7.49-7.25 (m, 4H), 6.73-6.56 (m, 1H), 5.38 (bs, 2H), 4.81-4.67 (m, 1H), 4.47 (s, 2H), 4.44-4.32 (m, 2H), 4.31-4.19 (m, 2H), 4.16-3.98 (m, 2H), 3.29-3.06 (m, 2H) 2.04 (s, 2H), 2.01-1.90 (m, 1H), 1.19 (t, J=7.1 Hz, 3H), 1.00 (t, J=7.0 Hz, 3H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 206.95, 173.6, 158.00, 157.74, 139.97, 130.79, 128.85 (2C), 123.64 (2C), 60.71, 53.38, 31.17, 30.79, 30.58, 15.11, 14.60, 13.80. LC-MS purity: 100%.

Ethyl 2-({[(4-aminophenyl)methyl](propyl)carbamoyl}amino)acetate (16)

[0186] ##STR00054##

[0187] The title compound was synthesized according to the general procedure 4, starting from Fe powder (503 mg, 9.00 mmol), calcium chloride (333 mg, 3.00 mmol), Ethyl 2-({[(4-nitrophenyl)methyl](propyl)carbamoyl}amino)acetate (970 mg, 3.00 mmol), in Ethanol/Water (24 mL, 20:4). Yellow oil 550 mg (62%). LC-MS: Rt=4.7 min, m/z 316 [M+Na].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 6.92-6.89 (m, 2H), 6.72 (t, J=5.6 Hz, 1H), 6.52-6.49 (m, 2H), 4.24 (s, 2H), 4.09 (q, J=7.1 Hz, 2H), 3.76 (d, J=5.8 Hz, 2H), 3.03-2.97 (m, 2H), 1.49 (h, J=7.3 Hz, 2H), 1.20 (t, J=7.2 Hz, 3H), 0.81 (t, J=7.4 Hz, 3H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 171.66, 157.97, 147.90, 128.65 (2C), 126.10, 114.38 (2C), 60.77, 49.02, 47.26, 42.76, 21.13, 14.80, 11.54. ESI+(m/z): [M+Na].sup.+ calculated for C.sub.15H.sub.23N.sub.3O.sub.3Na 316.1631; found 316.1614 [M+Na].sup.+. LC-MS purity: 99%

Ethyl 2-({[(4-aminophenyl)methyl](cyclopropylmethyl)carbamoyl}amino)acetate hydrochloride (17)

[0188] ##STR00055##

[0189] The title compound was synthesized according to the general procedure 4, starting from Fe powder (75 mg, 1.34 mmol), calcium chloride (50 mg, 0.45 mmol), Ethyl 2-{[(cyclopropylmethyl)[(4-nitrophenyl)methyl]carbamoyl]amino}acetate (150 mg, 0.45 mmol), in Ethanol/Water (8 mL, 6:2). The resultant oil was dissolved in a small amount of ethanol, to which 1.25 M HCl in ethanol was added. Evaporation of the solvent produced the title compound as an orange solid 150 mg (98%). Mp: 101-105° C. LC-MS: Rt=4.8 min, m/z 328 [M+Na].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 10.32 (bs, 2H), 7.37-7.31 (m, 4H), 6.94 (s, 1H), 4.56 (s, 2H), 4.09 (q, J=7.1 Hz, 2H), 3.75 (s, 2H), 3.07 (d, J=6.8 Hz, 2H), 2.88 (dd, J=6.7, 1.4 Hz, 1H), 1.20 (t, J=6.7 Hz, 3H), 0.39-0.35 (m, 2H), 0.18-0.10 (m, 2H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 171.52, 158.08, 139.79, 128.76, 128.70 (2C), 123.54 (2C), 60.58, 50.51, 49.12, 42.91, 14.60, 10.55, 3.75, 3.44. ESI+(m/z): [M+Na].sup.+ calculated for C.sub.16H.sub.23N.sub.3O.sub.3Na 328.1631; found 328.1650 [M+Na].sup.+. LC-MS purity: 99%.

Ethyl 2-({[(4-aminophenyl)methyl][(1-methyl-1H-1,2,3-triazol-4-yl)methyl]carbamoyl}amino)acetate hydrochloride (18)

[0190] ##STR00056##

[0191] The title compound was synthesized according to the general procedure 4, starting from Fe powder (85 mg, 1.51 mmol), calcium chloride (56 mg, 0.50 mmol), Ethyl 2-({[(1-methyl-1H-1,2,3-triazol-4-yl)methyl][(4-nitrophenyl)methyl]carbamoyl}amino)acetate (190 mg, 0.50 mmol), in Ethanol/Water (8 mL, 6:2). The resultant oil was dissolved in a small amount of ethanol, to which 1.25 M HCl in ethanol was added. Evaporation of the solvent produced the title compound as an orange solid 155 mg (80%). Mp: 144-147° C. LC-MS: Rt=1.2 min, m/z 369 [M+Na].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 10.29 (bs, 2H), 7.87 (s, 1H), 7.41-7.30 (m, 4H), 7.18 (s, 1H), 4.47 (s, 2H), 4.39 (s, 2H), 4.13-4.07 (m, 2H), 4.01 (s, 3H), 3.78 (s, 2H), 1.23-1.17 (m, 3H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 171.41, 157.94, 144.36, 138.82, 130.99, 129.01 (2C), 124.60, 123.62 (2C), 60.64, 56.48, 48.74, 42.94, 36.70, 14.60. ESI+(m/z): [M+Na].sup.+ calculated for C.sub.16H.sub.22N.sub.6O.sub.3Na 369.1645; found 369.1650 [M+Na].sup.+. LC-MS purity: 95%.

Ethyl 2-({[(4-aminophenyl)methyl][(2-methyl-2H-1,2,3,4-tetrazol-5-yl)methyl]carbamoyl}amino)acetate hydrochloride (19)

[0192] ##STR00057##

[0193] The title compound was synthesized according to the general procedure 4, starting from Fe powder (67 mg, 1.19 mmol), calcium chloride (44 mg, 0.40 mmol), Ethyl 2-({[(2-methyl-2H-1,2,3,4-tetrazol-5-yl)methyl][(4-nitrophenyl)methyl]carbamoyl}amino)acetate (150 mg, 0.40 mmol), in Ethanol/Water (8 mL, 6:2). The resultant oil was dissolved in a small amount of ethanol, to which 1.25 M HCl in ethanol was added. Evaporation of the solvent produced the title compound as an orange solid 50 mg (33%). Mp: 128-131° C. LC-MS: Rt=2.9 min, m/z 370 [M+Na].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.87 (bs, 2H), 7.37-7.31 (m, 2H), 7.31-7.25 (m, 2H), 7.23 (t, J=5.5 Hz, 1H), 4.64 (s, 2H), 4.53 (s, 2H), 4.32 (s, 3H), 4.09 (q, J=7.1 Hz, 2H), 3.77 (d, J=5.5 Hz, 2H), 1.20 (t, J=7.1 Hz, 3H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 169.32, 158.73, 156.94, 144.54, 128.55 (2C), 126.17 (2C), 117.15, 61.61, 52.05, 43.44, 41.86, 32.67, 14.60. ESI+(m/z): [M+Na].sup.+ calculated for C.sub.15H.sub.21N.sub.7O.sub.3Na 370.1598; found 370.1600 [M+Na].sup.+. LC-MS purity: 95%.

({[(4-aminophenyl)methyl]({[1-(2-ethoxyprop-2-en-1-yl)-1H-1,2,3-triazol-4-yl]methyl})carbamoyl}amino)acetate hydrochloride (20)

[0194] ##STR00058##

[0195] The title compound was synthesized according to general procedure 4, starting from Fe powder (92 mg, 1.64 mmol), calcium chloride (61 mg, 0.55 mmol), ethyl 2-[({[1-(2-ethoxy-2-oxoethyl)-1H-1,2,3-triazol-4-yl]methyl}[(4-nitrophenyl)methyl]carbamoyl)amino]acetate (245 mg, 0.55 mmol), in Ethanol/Water (8 mL, 3:1). The resultant oil was dissolved in a small amount of ethanol, to which 1.25 M HCl in ethanol was added. Evaporation of the solvent produced the title compound as an orange solid. 98 mg (39%). LC-MS: Rt=4.7 min, m/z 419 [M+H].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 10.30 (s, 2H), 7.99 (s, 1H), 7.41-7.27 (m, 4H), 7.21 (s, 1H), 5.38 (s, 2H), 4.86 (bs, 2H), 4.48 (s, 2H), 4.42 (s, 2H), 4.19 (q, J=7.2 Hz, 2H), 4.10 (q, J=7.1 Hz, 2H), 3.82-3.75 (m, 2H), 1.23 (t, J=7.1 Hz, 3H), 1.19 (t, J=7.2 Hz, 3H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 171.4, 167.7, 157.9, 144.3, 129.0 (2C), 125.1, 123.6 (2C), 61.9, 60.6, 50.8, 48.8, 43.0, 41.9, 41.2, 35.5, 14.6, 14.4. LC-MS purity: 80%.

Ethyl 2-({[(4-aminophenyl)methyl][1-(carbamoylmethyl)-1H-1,2,3-triazol-4-yl]carbamoyl}amino)acetate (21)

[0196] ##STR00059##

[0197] The title compound was synthesized according to the general procedure 4, starting from Fe powder (60 mg, 1.07 mmol), calcium chloride (40 mg, 0.36 mmol), Ethyl 2-[({[1-(carbamoylmethyl)-1H-1,2,3-triazol-4-yl]methyl}[(4-nitrophenyl)methyl]carbamoyl)amino]acetate (150 mg, 0.36 mmol), in Ethanol/Water (8 mL, 6:2). The resultant oil was dissolved in a small amount of ethanol, to which 1.25 M HCl in ethanol was added. Evaporation of the solvent produced the title compound as an orange solid which was recrystalised from diethylether 60 mg (39%). LC-MS: Rt=9.0 min, m/z 413 [M+Na].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 10.17 (bs, 1H), 8.94-8.62 (m, 2H), 8.34-8.16 (m, 2H), 8.00-7.66 (m, 1H), 7.44-7.20 (m, 1H), 5.04 (s, 2H), 4.40-4.22 (m, 1H), 4.03 (s, 2H), 3.88-3.60 (m, 2H), 3.53-2.99 (m, 4H), 2.80 (s, 2H), 1.27-1.11 (m, 3H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 167.67, 158.05, 144.33, 129.00 (2C), 125.28, 123.29 (2C), 61.70, 60.67, 50.75, 48.69, 42.77, 41.22, 35.49, 31.64, 19.45.

Ethyl 2-({[(4-aminophenyl)methyl](prop-2-yn-1-yl)carbamoyl}amino)acetate (22)

[0198] ##STR00060##

[0199] The title compound was synthesized according to the general procedure 4, starting from Fe powder (487 mg, 8.73 mmol), calcium chloride (323 mg, 2.91 mmol), Ethyl 2-({[(4-nitrophenyl)methyl](prop-2-yn-1-yl)carbamoyl}amino)acetate (929 mg, 2.91 mmol), in Ethanol/Water (24 mL, 20:4). Yellow oil 350 mg (42%). LC-MS: Rt=4.3 min, m/z 312 [M+Na].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.40-7.37 (m, 2H), 7.36-7.33 (m, 2H), 7.19 (t, J=6.0 Hz, 1H), 4.54 (s, 2H), 4.10 (q, J=7.1 Hz, 1H), 4.02 (d, J=2.5 Hz, 2H), 3.77 (d, J=5.9 Hz, 2H), 3.18 (t, J=2.4 Hz, 1H), 1.20 (t, J=7.1 Hz, 3H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 171.26, 157.55, 138.24, 131.25, 128.90 (2C), 123.59 (2C), 80.43, 75.02, 60.65, 48.91, 42.90, 35.79, 14.60. ESI+(m/z): [M+Na].sup.+ calculated for C.sub.15H.sub.19N.sub.3O.sub.3Na 312.1318; found 312.1316 [M+Na].sup.+. LC-MS purity:

Ethyl 2-{[ethyl({[(1R,9S,10S)-10-hydroxy-12-oxa-8-azatricyclo[7.3.1.0.SUP.2,7.]trideca-2,4,6-trien-4-yl]methyl})carbamoyl]amino}acetate hydrochloride (23)

[0200] ##STR00061##

[0201] The title compound was synthesized according to the general procedure 2, starting from (1R,9S,10S)-4-[(ethylamino)methyl]-12-oxa-8-azatricyclo[7.3.1.0.sup.2,7]trideca-2,4,6-trien-10-ol (187 mg, 0.75 mmol), and Ethyl isocyanatoacetate (97 mg, 0.75 mmol) in DCM. The crude product was purified by column chromatography (DCM/MeOH). The product of which was dissolved in EtOH and HCl 1M in ethanol added. The volatiles were removed and the solid dissolved in water and washed with DCM before freeze drying to a dark solid 50 mg (16%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.10 (bs, 1H), .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 9.11 (d, J=8.6 Hz, 1H), 8.38-8.02 (m, 1H), 7.77-7.55 (m, 1H), 7.18-6.91 (m, 1H), 6.68-6.46 (m, 1H), 6.27 (s, 2H), 5.23 (d, J=33.4 Hz, 2H), 4.64-4.40 (m, 2H), 4.29-3.97 (m, 4H), 3.83-3.76 (m, 3H), 3.67-3.59 (m, 4H), 3.11-3.05 (m, 4H), 2.99-2.89 (m, 4H).

1-[(4-aminophenyl)methyl]-3-{2-[2-(phenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-ethylurea (9)

[0202] ##STR00062##

[0203] 3-{2-[2-(2-bromophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-ethyl-1-[(4-nitrophenyl)methyl]urea (100 mg, 0.2 mmol) and ammonium formate (90 mg, 1.4 mmol) were dissolved in THF/MeOH (5 mL, 1:4). Pd/C 10% (22 mg, 0.2 mmol) was added and the mixture stirred at RT overnight. The solution was filtered on a celite pad and concentrated to yield an oil. This was dissolved in DCM and washed with water. The organic phase was collected dried over MgSO.sub.4, filtered and concentrated yielding a yellow solid. 58 mg, (75%). .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.38 (t, J=7.6 Hz, 1H), 7.28 (ddd, J=7.8, 5.9, 1.6 Hz, 1H), 7.25-7.21 (m, 2H), 7.20-7.11 (m, 1H), 6.90 (dd, J=9.6, 8.3 Hz, 1H), 6.48 (d, J=8.1 Hz, 2H), 6.18 (dq, J=49.3, 5.2 Hz, 1H), 5.17-5.03 (m, 1H), 4.93 (s, 1H), 4.30-4.09 (m, 2H), 4.02-3.84 (m, 1H), 3.78 (ddd, J=9.9, 8.1, 3.3 Hz, 1H), 3.68-3.51 (m, 1H), 3.15-3.03 (m, 1H), 2.41-2.30 (m, 1H), 2.21 (dddd, J=12.2, 10.8, 8.1, 6.8 Hz, 1H), 1.91 (dtt, J=14.1, 7.2, 3.7 Hz, 1H), 1.86-1.76 (m, 2H), 1.72 (ddt, J=12.2, 6.1, 2.9 Hz, 1H), 1.14 (td, J=7.1, 2.1 Hz, 1H), 0.95 (dt, J=12.5, 7.0 Hz, 3H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 169.06, 157.69, 128.83 (2C), 128.78 (2C), 128.49 (2C), 125.99 (2C), 114.26, 60.55, 48.70, 47.35, 46.35, 43.37, 40.52, 36.54, 34.06, 23.61, 21.60, 13.56.

1-[(4-aminophenyl)methyl]-3-{2-[2-(2-bromophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-ethylurea hydrochloride (10)

[0204] ##STR00063##

[0205] The title compound was synthesized according to general procedure 4, starting from Fe powder (34 mg, 0.61 mmol), calcium chloride (23 mg, 0.2 mmol), 3-{2-[2-(2-bromophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-ethyl-1-[(4-nitrophenyl)methyl]urea (245 mg, 0.55 mmol), in Ethanol/Water (8 mL, 3:1). The resultant oil was dissolved in a small amount of ethanol, to which 1.25 M HCl in ethanol was added. Evaporation of the solvent produced the title compound as an orange solid. 40 mg (39%). LC-MS: Rt=5.5 min, m/z 459 [M+H].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 10.30 (s, 1H) 7.68 (d, J=7.8 Hz, 2H), 7.60 (d, J=7.8 Hz, 2H), 7.50-7.34 (m, 1H), 7.34-7.25 (m, 1H), 7.25-7.11 (m, 1H), 7.04 (d, J=8.0 Hz, 1H), 6.38 (s, 1H), 5.23-5.14 (m, 2H), 4.52 (d, J=23.1 Hz, 2H), 4.35 (d, J=22.5 Hz, 2H), 4.03-3.87 (m, 2H), 3.68-3.54 (m, 2H), 3.18-3.10 (m, 1H), 2.26 (dtd, J=11.5, 7.7, 3.6 Hz, 1H), 2.01-1.91 (m, 1H), 1.90-1.77 (m, 1H), 1.74-1.66 (m, 1H), 1.06-0.91 (m, 4H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 168.69, 157.78, 142.44, 133.48, 133.03, 128.92, 127.95, 127.29, 125.58, 121.85, 60.73, 47.70, 46.74, 43.35, 34.56, 32.28, 23.36, 13.70.

1-[(4-aminophenyl)methyl]-3-{2-[2-(2-bromophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-(cyclopropylmethyl)urea (11)

[0206] ##STR00064##

[0207] The title compound was synthesized according to general procedure 4, starting from Fe powder (34 mg, 0.61 mmol), calcium chloride (23 mg, 0.2 mmol), 3-{2-[2-(2-bromophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-(cyclopropylmethyl)-1-[(4-nitrophenyl)methyl]urea (105 mg, 0.0.2 mmol), in Ethanol/Water (8 mL, 3:1). The resultant oil was dissolved in a small amount of ethanol, to which 1.25 M HCl in ethanol was added. Evaporation of the solvent produced the title compound as an orange solid. 60 mg (56%). LC-MS: Rt=5.5 min, m/z 487 [M+2H].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 9.54 (s, 1H) 8.23-8.12 (m, 1H), 8.05 (ddd, J=8.4, 5.3, 2.9 Hz, 1H), 7.67 (d, J=8.0 Hz, 2H), 7.60 (d, J=7.9 Hz, 2H), 5.92 (s, 1H), 5.20 (t, J=7.1 Hz, 2H), 4.69-4.49 (m, 2H), 4.00-3.78 (m, 4H), 3.73 (ddd, J=11.7, 8.1, 3.1 Hz, 1H), 3.61 (dtd, J=27.2, 10.5, 10.1, 7.4 Hz, 2H), 3.08 (dq, J=29.9, 6.5 Hz, 3H), 2.84 (dd, J=41.7, 6.7 Hz, 2H), 2.32-2.23 (m, 1H), 1.94 (ddq, J=14.1, 7.0, 3.5 Hz, 2H), 1.86-1.63 (m, 1H), 1.02-0.90 (m, 1H), 0.44-0.27 (m, 2H).

1-[(4-aminophenyl)methyl]-3-{2-[2-(2-bromophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-(prop-2-yn-1-yl)urea hydrochloride (1)

[0208] ##STR00065##

[0209] The title compound was synthesized according to the general procedure 4, starting from Fe powder (502 mg, 8.99 mmol), calcium chloride (333 mg, 3.00 mmol), 3-{2-[2-(2-bromophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-[(4-nitrophenyl)methyl]-1-(prop-2-yn-1-yl)urea (1496 mg, 3.00 mmol), in Ethanol/Water (24 mL, 20:4). The resultant oil was dissolved in a small amount of ethanol, to which 1.25 M HCl in ethanol was added. Evaporation of the solvent produced the title compound as an orange solid 600 mg (40%). Mp: 136-140° C. LC-MS: Rt=5.3 min, m/z 469 [M+H].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 10.28 (s, 2H), 7.60 (ddd, J=7.9, 2.6, 1.2 Hz, 1H), 7.37 (dd, J=8.6, 2.1 Hz, 2H), 7.35-7.24 (m, 3H), 7.16 (dddd, J=12.6, 9.4, 7.6, 1.7 Hz, 2H), 6.71 (s, 1H), 5.22-5.16 (m, 1H), 4.53 (s, 2H), 4.02 (d, J=2.4 Hz, 2H), 4.00-3.94 (m, 2H), 3.90 (tdd, J=7.7, 4.6, 2.3 Hz, 1H), 3.60 (dtd, J=16.7, 9.6, 7.7 Hz, 1H), 3.16 (dt, J=7.3, 2.4 Hz, 1H), 2.26 (tdd, J=11.8, 9.7, 6.8 Hz, 1H), 1.95 (tq, J=10.4, 3.6 Hz, 1H), 1.80-1.73 (m, 1H), 1.68 (ddt, J=13.6, 10.2, 6.2 Hz, 1H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 168.25, 157.49, 142.39, 138.47, 133.47, 133.02, 131.11, 129.81, 129.02 (2C), 127.95, 127.27, 123.60 (2C), 80.47, 75.02, 60.72, 56.48, 49.00, 43.47, 35.97, 32.28, 24.61. ESI+(m/z): [M+H].sup.+ calculated for C.sub.23H.sub.25.sup.79BrN.sub.4O.sub.2468.1161; found 468.1193 [M+H].sup.+; calculated for C.sub.23H.sub.25.sup.81BrN.sub.4O.sub.2 (97.3%) 470.1140; found 470.1147 [M+H].sup.+. LC-MS purity: 100%.

3-{2-[2-(2-bromophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-(cyanomethyl)-1-[(4-nitrophenyl)methyl]urea (2)

[0210] ##STR00066##

[0211] The title compound was synthesized according to the general procedure 4, starting from Fe powder (20 mg, 0.36 mmol), calcium chloride (13 mg, 0.12 mmol), 3-{2-[2-(2-bromophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-[(4-nitrophenyl)methyl]-1-(prop-2-yn-1-yl)urea (60 mg, 0.12 mmol), in Ethanol/Water (8 mL, 6:2). The resultant oil was dissolved in a small amount of ethanol, to which 1.25 M HCl in ethanol was added. Evaporation of the solvent produced the title compound as an orange solid which was recrystallized from acetone 50 mg (82%). .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 10.06 (s, 1H), 7.67 (dd, J=7.9, 1.2 Hz, 1H), 7.60 (dd, J=7.9, 1.3 Hz, 2H), 7.34 (d, J=8.4 Hz, 2H), 7.21-7.11 (m, 2H), 6.79 (d, J=8.5 Hz, 2H), 5.28-5.19 (m, 2H), 3.90-3.79 (m, 2H), 3.59 (td, J=9.7, 7.0 Hz, 2H), 2.33-2.18 (m, 2H), 2.02-1.58 (m, 5H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 206.95, 168.29, 158.37, 142.34, 129.05, 128.64, 127.98, 127.26, 123.29, 121.82, 121.58, 60.72, 60.27, 47.68, 46.62, 42.82, 40.52, 34.45, 32.34, 31.17, 23.30, 21.23.

1-[(4-aminophenyl)methyl]-3-{2-[2-(2-bromophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-[(1-methyl-1H-1,2,3-triazol-4-yl)methyl]urea hydrochloride (3)

[0212] ##STR00067##

[0213] The title compound was synthesized according to the general procedure 4, starting from Fe powder (92 mg, 1.64 mmol), calcium chloride (61 mg, 0.55 mmol), 3-{2-[2-(2-bromophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-[(1-methyl-1H-1,2,3-triazol-4-yl) methyl]-1-[(4-nitrophenyl)methyl]urea (305 mg, 0.55 mmol), in Ethanol/Water (8 mL, 6:2). The resultant oil was dissolved in a small amount of ethanol, to which 1.25 M HCl in ethanol was added. Evaporation of the solvent produced the title compound as an orange solid 300 mg (97%). Mp: 201-205° C. LC-MS: Rt=5.3 min, m/z 527 [M+2H].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 10.33 (bs, 2H), 7.89 (s, 1H), 7.62-7.59 (m, 1H), 7.36-7.32 (m, 3H), 7.32-7.23 (m, 2H), 7.21-7.13 (m, 3H), 5.21 (dd, J=8.1, 2.4 Hz, 1H), 4.46 (s, 2H), 4.38 (s, 2H), 4.01-3.95 (m, 5H), 3.92-3.88 (m, 1H), 3.61 (qd, J=10.0, 7.1 Hz, 1H), 2.26 (tt, J=12.1, 8.2 Hz, 1H), 1.95 (dtt, J=11.0, 7.9, 4.0 Hz, 1H), 1.90-1.73 (m, 1H), 1.70 (ddd, J=12.6, 6.2, 3.0 Hz, 1H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 168.46, 157.93, 144.41, 142.40, 139.01, 133.47, 133.02, 130.86, 129.34 (2C), 127.95, 127.39, 124.66, 123.67 (2C), 121.84, 60.73, 56.48, 46.76, 43.41, 38.72, 36.80, 32.29, 23.34. ESI+(m/z): [M+H].sup.+ calculated for C.sub.24H.sub.28.sup.79BrN.sub.7O.sub.2 (100.0%) 525.1488; found 525.1473 [M+H].sup.+; calculated for C.sub.24H.sub.28.sup.81BrN.sub.7O.sub.2 (97.3%) 527.1467; found 527.1478 [M+H].sup.+ LC-MS purity: 99%

1-[(4-aminophenyl)methyl]-3-{2-[2-(2-bromophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-[(2-methyl-2H-1,2,3,4-tetrazol-5-yl)methyl]urea hydrochloride (4)

[0214] ##STR00068##

[0215] The title compound was synthesized according to the general procedure 4, starting from Fe powder (64 mg, 1.15 mmol), calcium chloride (43 mg, 0.39 mmol), 3-{2-[2-(2-bromophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-[(2-methyl-2H-1,2,3,4-tetrazol-5-yl)methyl]-1-[(4-nitrophenyl)methyl]urea (215 mg, 0.39 mmol), in Ethanol/Water (8 mL, 6:2). The resultant oil was dissolved in a small amount of ethanol, to which 1.25 M HCl in ethanol was added. Evaporation of the solvent produced the title compound as an orange solid 120 mg (55%). Mp: 212-214° C. LC-MS: Rt=1.0 min, m/z 549 [M+Na].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 10.27 (bs, 2H), 7.62-7.58 (m, 1H), 7.36-7.24 (m, 5H), 7.16-7.10 (m, 3H), 5.19 (dt, J=8.3, 2.5 Hz, 1H), 4.46 (s, 2H), 4.38 (s, 2H), 4.30 (s, 3H), 4.01-3.95 (m, 2H), 3.85 (td, J=11.6, 10.4, 7.1 Hz, 1H), 3.59 (dtd, J=12.6, 6.9, 2.8 Hz, 1H), 2.26 (dtt, J=10.4, 8.0, 5.1 Hz, 1H), 1.94 (ddt, J=11.7, 7.0, 3.6 Hz, 1H), 1.79 (dtd, J=8.2, 5.4, 4.7, 2.6 Hz, 1H), 1.69 (ddt, J=11.8, 5.7, 2.6 Hz, 1H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 168.26, 162.76, 142.32, 133.03, 129.80, 129.03 (2C), 129.00, 128.59, 128.56, 127.97, 127.24, 123.45 (2C), 121.81, 60.71, 52.07, 46.67, 43.08, 36.25, 32.33, 31.25, 23.37. ESI+(m/z): [M+H].sup.+ calculated for C.sub.23H.sub.27.sup.79BrN.sub.8O.sub.2 (100.0%) 526.1440; found 526.1470 [M+H].sup.+; calculated for C.sub.23H.sub.27.sup.81BrN.sub.8O.sub.2 (97.3%) 528.1420; found 528.1428 [M+H].sup.+ LC-MS purity: 97%.

Ethyl 2-[4-({[(4-aminophenyl)methyl]({2-[2-(2-bromophenyl)pyrrolidin-1-yl]-2-oxoethyl}carbamoyl)amino}methyl)-1H-1,2,3-triazol-1-yl]acetate hydrochloride (5)

[0216] ##STR00069##

[0217] A mixture of 1-[(4-aminophenyl)methyl]-3-{2-[2-(2-bromophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-(prop-2-yn-1-yl)urea hydrochloride (13) (250 mg, 0.5 mmol, 1 equiv), ethyl azidoacetate (25% solution in Ethanol, 0.4 mL, 0.6 mmol, 1.2 equiv), CuSO.sub.4 (123 mg, 0.5 mmol, 1 equiv) and sodium ascorbate (196 mg, 1.0 mmol, 2 equiv) in EtOH/H.sub.2O (20 mL, 1:1) was stirred at rt for 16 h. The reaction mixture was quenched with crushed ice and extracted with ethyl acetate (10 mL×3). The organic extracts were washed with brine solution (20 mL) and dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure to afford the desired compound. The resultant brown oil was dissolved in a small amount of ethanol, to which 1.25 M HCl in ethanol was added. Evaporation of the solvent produced the title compound as an orange solid 30 mg (10%). Mp: 186-190° C. LC-MS: Rt=5.3 min, m/z 598 [M+H].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.97 (t, J=9.6 Hz, 1H), 7.67 (dt, J=8.0, 1.5 Hz, 1H), 7.36-7.22 (m, 4H), 7.22-7.11 (m, 4H), 5.30-5.16 (m, 1H), 4.58-4.48 (m, 3H), 4.39 (d, J=11.2 Hz, 2H), 4.11-4.01 (m, 3H), 3.98 (d, J=3.0 Hz, 2H), 3.67-3.54 (m, 2H), 1.95 (ddt, J=12.5, 6.4, 3.1 Hz, 1H), 1.90-1.65 (m, 3H), 1.15 (t, J=7.1 Hz, 3H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 205.02, 163.93, 141.94, 140.54, 138.41, 138.20, 133.03, 132.88, 129.02 (2C), 128.83, 127.97, 127.62, 127.28, 123.57 (2C), 121.84, 60.76, 60.29, 48.95, 47.72, 46.76, 43.52, 42.80, 32.29, 23.36, 14.45. ESI+(m/z): [M+H].sup.+ calculated for C.sub.27H.sub.32.sup.79BrN.sub.7O.sub.4 (100.0%) 597.1699; found 597.1670 [M+H].sup.+; calculated for C.sub.27H.sub.32.sup.31BrN.sub.7O.sub.4 (97.3%) 599.1679; found 599.1668 [M+H].sup.+ LC-MS purity: 97%.

Ethyl 3-[4-({[(4-aminophenyl)methyl]({2-[2-(2-bromophenyl)pyrrolidin-1-yl]-2-oxoethyl}carbamoyl)amino}methyl)-1H-1,2,3-triazol-1-yl]propanoatehydrochloride (6)

[0218] ##STR00070##

[0219] A mixture of 1-[(4-aminophenyl)methyl]-3-{2-[2-(2-bromophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-(prop-2-yn-1-yl)urea hydrochloride (200 mg, 0.4 mmol), ethylazidopropionate (62 mg, 0.43 mmol), CuSO.sub.4 (99 mg, 0.4 mmol) and sodium ascorbate (157 mg, 0.8 mmol) were stirred in EtOH/water (10 mL, 1:1) at RT for 16 h. Crushed ice was added and the mixture extracted with ethyl acetate. The combined organic extracts were dried over MgSO.sub.4, filtered and the solvent removed in vacuo to a dark oil which was purified by flash column chromatography. Brown solid 51 mg, (20%) LC-MS: Rt=5.3 min, m/z 614 [M+2H].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 10.10 (s, 1H), 8.02-7.95 (m, 1H), 7.68 (dt, J=7.9, 1.5 Hz, 1H), 7.60 (ddd, J=7.9, 4.1, 1.3 Hz, 1H), 7.49-7.37 (m, 1H), 7.36-7.24 (m, 5H), 7.22-7.07 (m, 3H), 6.78-6.61 (m, 1H), 5.29-5.17 (m, 1H), 4.58-4.50 (m, 2H), 4.48-4.36 (m, 2H), 4.06 (q, J=7.1 Hz, 2H), 3.67-3.53 (m, 2H), 2.98-2.85 (m, 3H), 2.32-2.21 (m, 1H), 2.00-1.58 (m, 4H), 1.20-1.08 (m, 4H). .sup.13C NMR (126 MHz, DMSO-ds) δ 170.70, 168.26, 157.93, 144.27, 142.42, 133.04, 129.05 (2C), 127.95, 127.29 (2C), 124.03, 123.37, 121.85, 60.77, 60.71, 48.92, 47.72, 46.76, 45.66, 43.50, 41.34, 35.41, 34.43, 32.29, 23.35, 21.28, 14.46.

1-[(4-aminophenyl)methyl]-3-{2-[(2S)-2-(2-bromophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-[(1-methyl-1H-1,2,3-triazol-4- yl)methyl]urea hydrochloride (7)

[0220] ##STR00071##

[0221] The title compound was synthesized according to the general procedure 4, starting from Fe powder (32 mg, 0.6 mmol), calcium chloride (21 mg, 0.20 mmol), 3-{2-[(2S)-2-(2-bromophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-[(1-methyl-1H-1,2,3-triazol-4-yl)methyl]-1-[(4-nitrophenyl)methyl]urea (105 mg, 0.19 mmol), in Ethanol/Water (8 mL, 6:2). The resultant oil was dissolved in a small amount of ethanol, to which 1.25 M HCl in ethanol was added. Evaporation of the solvent produced the title compound as an orange solid 50 mg (47%) αD=+1.24. LC-MS: Rt=5.3 min, m/z 526 [M+H].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 10.12 (s, 1H), 8.44 (s, 1H), 8.01-7.78 (m, 1H), 7.81-7.66 (m, 1H), 7.66-7.52 (m, 1H), 7.54-7.47 (m, 1H), 7.45-7.37 (m, 2H), 7.36-7.24 (m, 2H), 6.79 (d, J=8.5 Hz, 2H), 5.38-4.65 (m, 2H), 4.61-4.12 (m, 2H), 4.45 (s, 3H), 4.11-3.90 (m, 3H), 3.79-3.48 (m, 2H), 2.06-1.60 (m, 2H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 157.95, 144.45, 142.42, 136.10, 134.44, 133.04, 131.77, 129.08 (2C), 128.64, 127.96 (2C), 127.30, 124.64, 123.38, 63.45, 60.74, 56.48, 48.96, 36.70, 32.29, 23.35, 21.21.

1-[(4-aminophenyl)methyl]-3-{2-[2-(2-chlorophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-[(1-methyl-1H-1,2,3-triazol-4-yl)methyl]urea hydrochloride (8)

[0222] ##STR00072##

[0223] The title compound was synthesized according to general procedure 4, starting from Fe powder (90 mg, 1.61 mmol), calcium chloride (66 mg, 0.6 mmol), 3-{2-[2-(2-chlorophenyl)pyrrolidin-1-yl]-2-oxoethyl}-1-[(1-methyl-1H-1,2,3-triazol-4-yl) methyl]-1-[(4-nitrophenyl)methyl]urea (192 mg, 0.38 mmol), in Ethanol/Water (10 mL, 3:1). The resultant oil was dissolved in a small amount of ethanol, to which 1.25 M HCl in ethanol was added. Evaporation of the solvent produced the title compound as an orange solid. 30 mg (15%). LC-MS: Rt=5.2 min, m/z 482 [M+2H].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 10.38 (s, 1H), 7.99-7.83 (m, 2H), 7.54-7.37 (m, 3H), 7.31-7.22 (m, 2H), 7.22-7.12 (m, 1H), 5.38-5.20 (m, 1H), 5.17-4.80 (s, 5H), 4.65-4.30 (m, 3H), 4.05-3.92 (m, 3H), 3.80-3.52 (m, 2H), 2.37-2.16 (m, 2H), 1.99-1.61 (m, 3H). .sup.13C NMR (126 MHz, DMSO-d.sub.6) δ 168.50, 158.00, 144.47, 140.92, 131.39, 129.80 (2C), 129.03 (2C), 127.43, 127.19, 125.63, 124.75, 123.67, 58.53, 57.98, 48.96, 46.64, 43.52, 37.02, 36.68, 34.41, 32.14, 23.47.

[0224] Protein Expression and Purification

[0225] The plasmids of HisCypA, HisCypB and HisCypD were provided by the Edinburgh Protein Production Facility (EPPF). Protein expression and purification protocols were slightly modified from Wear et al. for their usage in ITC and X-ray studies (Wear 2017).

[0226] Recombinant Cyclophilins (Cyps) were produced in C41 BL21(DH3) E. coli cell lines (Lucigen, Middleton, Wis., USA). Briefly, 1 μL of the stock plasmids were added to the competent cells, left on ice for about half an hour and after a two-minute heat shock at 42° C. were incubated by shaking (250 rpm) in SOC media (500 μL) at 37° C. for 45 minutes. They were left overnight to colonize in agar plates (100 μL/plate) containing carbenicillin (100 μg mol.sup.−1). After this a single colony was picked and grown for six hours in LB media. Subsequently, 20% v/v glycerol was added and these glycerol stocks were used in future reference.

[0227] A 100 mL pre-culture was left overnight in LB media using a glycerol stock and carbenicillin as antibiotics (100 μg mol.sup.−1). The cultures were centrifuged for five minutes at 1,500 g and new 500 mL cultures were made by transferring the cell pellets, adding carbenicillin and incubating by shaking (250 rpm) until OD.sub.600 0.6-0.8 at 37° C. and then induced at 30° C. with 0.5 mM IPTG for four hours. Finally, the cultures were pelleted by centrifugation at 8,000 g for 20 minutes at 4° C. prior to cell lysis.

[0228] All purifications were performed on ÄKTA Pure (GE Healthcare) equipment at 4° C. Prior to purification cell pellets were lysed using protease inhibitors (Roche) in loading buffer (20 mM phosphate, 300 mM NaCl, 20 mM imidazole, pH 7.4) by a double passage on a Constant Systems Cell Disruptor (1.1 kW TS Benchtop) at 22 kpsi followed by one-hour centrifugation at 4° C. (55,000 g). A two-step purification protocol was used in all cases, i.e. Immobilized Metal Ion Affinity Chromatography (IMAC) and Size Exclusion Chromatography (SEC) using the HiTrap IMAC FF 5 mL and the HiLoad Superdex 75 μg 16/60 columns, respectively. The buffer used in the SEC purification step was similar to the ITC buffer and for the IMAC elution 20 mM phosphate, 300 mM NaCl, 500 mM imidazole, pH 7.4. Protein His-Tag was cleaved for further use of the protein in ITC and X-ray studies, whereas for the SPR experiments the protein was uncleaved. Proteins were desalted to cleavage buffer (100 mM Tris, 100 mM NaCl, pH 7.5) using a HiPrep 26/10 desalting column prior to the addition of TEV protease (200 ng TEV/40 μg protein). Samples were left incubating at 30° C. for about four hours and the cleaved His-tag was removed by IMAC. At the end of each purification the purity of the fractions was tested by using precast gels (Biorad®) in Tris/Glycine/SDS, pH 8.3 buffer. The molecular weights of HisCypA and free CypA are 20.893 and 18.069 kDa, respectively. Protein concentration was determined by measuring the absorbance at 280 nm and the extinction coefficients 14440 and 8480 M.sup.−1 cm′, respectively.

[0229] Isothermal Titration Calorimetry (ITC)

[0230] All ITC experiments for the ester series compounds were carried out at 25° C. on a MicroCal Auto iTC200 (GE Healthcare) instrument. The buffer used in the titrations of the compounds belonging to the ester series was 50 mM phosphate buffer, pH 6.5 and the concentration of DMSO was 2% v/v for all the compounds, unless otherwise stated in Table 51. Final compound solutions were heated to 65° C. and/or sonicated prior to the experiment. Each experiment consisted of an initial injection of 0.4 μL followed by nineteen 2 μL injections and in most of cases for these compounds, the “continue injections” protocol was used leaving the cell intact and performing a second series of titrations using the above protocol to achieve saturation. Control experiments were performed, where each compound was titrated into buffer and when small amount of heat was detected due to heat of dilution, it was subtracted when processing the data using a linear fit method. In all cases the first injection was omitted from the data processing. All data were analysed using the MicroCal PEAQ-ITC Analysis software. Because some compounds have affinity that lie in the low-mid micromolar range and the c value (Wiseman constant) is very small, a fixed stoichiometry to 1 was applied during the non-linear regression of the raw data for fitting the data (Tellinghuisen, J., Isothermal titration calorimetry at very low c. Analytical biochemistry, 373 (2), 395-7, 2008). Control runs on literature compounds were run as a control at the end of each experiment to verify that CypA remained active during the duration of the experiment.

[0231] Compound belonging to the arryl-pyrrolidine series, were tested using a reverse titration method or a reverse titration using a competition-based method as previously described with CsA (Low-Affinity Binding Determined by Titration calorimetry Using a High-Affinity Coupling Ligand: A Thermodynamic Study of Ligand Binding to Protein Tyrosine Phosphatase. Zhang Y, Zhang Z. Analytical biochemistry, 261, 139-148, 1998). The buffer used for these titrations was: PBS, 0.05% v/v P20 surfactant, 50 μM EDTA in presence of 2% v/v EtOH and the pH was set to 7.4. For the reverse competitive titrations Cyclophilin A (60 μM) was titrated into 4 μM CsA in presence of 10 μM compound in cell using a 15-injection protocol. A control experiment comprising of a titration of 60 μM CsA into 4 μM CsA using the same instrument parameters was performed.

[0232] Surface Plasmon Resonance (SPR)

[0233] SPR measurements were performed on a BIAcore T200 instrument (GE Healthcare). Ni.sup.2+-nitrilotriacetic acid (NTA) sensor chips, 1-ethyl-3-(3-diaminopropyl) carbodiimide hydrochloride (EDC) and Nhydroxysuccinimide (NHS) were purchased from GE Healthcare.

[0234] Pure His-cyclophilins were immobilized and covalently stabilized on the NTA sensor chip according to the protocol described in Thermo-kinetic analysis space expansion for cyclophilin-ligand interactions—identification of a new nonpeptide inhibitor using Biacore T200. Wear, M. A.; Nowicki, M. W.; Blackburn, E. A.; McNae, I. W.; Walkinshaw, M. D., FEBS open bio 7 (4), 533-549 (2017), using 200 nM concentrations of each protein, in Running Buffer (PBS, pH 7.4; 0.05% surfactant P20, 2% v/v ethanol; 50 μM EDTA), at 30 μl min.sup.−1 with 60 second contact times on the activated NTA surfaces. This gave signals of 1,921 RU for His-CypA, 1932 RU for His-CypB and 1,397 RU for His-CypD. Specific surface protein activity was assayed by passing saturating amounts of CsA (2 μM) in Running Buffer over these surfaces; values of 94.1%, 95.5% and 95.6% activity were obtained for His-CypA, -B and -D, respectively.

[0235] Single cycle kinetic titration binding experiments were performed using SPR in triplicate at 25° C. 3-fold dilution concentration series of CsA, ranging from 2.45 nM to 200 nM, in Running Buffer (PBS, pH 7.4, 50 μM mM EDTA; 0.05% v/v surfactant P20; 2% v/v ethanol), were injected over the sensor surface, at 100 μl.Math.min.sup.−1 with a 90 s contact time and a 90 s dissociation time. The sensor surface was regenerated between experiments by dissociating any formed complex in running buffer for at least 1,200 seconds. The apparent on-rate (k+) and off-rate (k−) constants and the equilibrium dissociation constant (K.sub.d) were calculated from reference corrected sensorgrams by global fitting of a 1:1 binding model, including a mass transport term, using analysis software (v. 2.02, GE Healthcare) provided with the BIAcore T200 instrument.

[0236] Kinetic titration binding experiments were performed in triplicate at 25° C. 2-fold dilution concentration series of the compounds, ranging from 0.0195 μM to 20 μM, in Running Buffer (PBS, pH 7.4, 50 μM mM EDTA; 0.05% v/v surfactant P20; 2% v/v ethanol), were injected over the sensor surface, at 100 μl.Math.min.sup.−1 with a 15 s contact time and a 600 s dissociation time. The sensor surface was regenerated between experiments by dissociating any formed complex in running buffer for at least a further 600 seconds. The apparent on-rate (k+) and off-rate (k−) constants and the equilibrium dissociation constant (K.sub.d) were calculated from reference corrected sensorgrams by global fitting of a 1:1 binding model, including a mass transport term, using analysis software (v. 2.02, GE Healthcare) provided with the BIAcore T200 instrument.

X-Ray Diffraction Experiments

[0237] Purified and his-tag-cleaved CypA was buffer-exchanged into PBS and concentrated to ˜29 mg ml.sup.−1. For crystallisation 1 μL of protein was mixed with an equal volume of the well solution, consisting of 100 mM Tris-HCl pH8.0 and 20-22% v/v PEG 8000, and crystal formation came about after equilibration overnight in 6° C. by vapour diffusion using the hanging drop method over 1 mL of the same well solution. Apo CypA crystals were soaked overnight into different ligand solution consisting of 100 mM Tris-HCl pH 8.0, 35% w/v PEG 8000, 5% v/v Glycerol, 5% v/v DMSO and 5 mM ligand, before flash frozen into liquid nitrogen. X-ray data were collected at the Diamond synchrotron-radiation facility in Oxford-shire, England at 100K. Structures were solved by molecular replacement using DIMPLE from the CCP4i suite. Modelled structures were visualised and manually adjusted as needed using Coot10 and further refined using REFMAC5 from CCP4i.

[0238] Cell Assays

[0239] Dulbecco's Modified Eagle's Medium (DMEM, with high glucose, sodium bicarbonate and L-glutamine), myo-inositol and folic acid were purchased from Sigma-Aldrich, Alpha MEM without ribonucleosides and deoxyribonucleosides, fetal bovine serum (FBS), heat-inactivated FBS heat-inactivated horse serum, β-mercaptoethanol and Ready-To-Use Geltrex were purchased from Life Technologies, and recombinant human IL-2 was purchased from Peprotech. Draq 7 was purchased from New England Biolabs and NucView 488 was purchased from Biotium. 384-well microclear tissue culture-treated plates for microscopy were purchased from Greiner Bio-One. Cyclophilin A antibody (rat polyclonal), cyclophilin B antibody (rabbit monoclonal) and GAPDH antibody (rabbit monoclonal) were purchased from New England Biolabs. Cyclophilin D antibody (mouse monoclonal) and mammalian protein extraction reagent (M-PER) was purchased from Thermo-Fisher Scientific. Complete EDTA-free protease inhibitor and phosSTOP phosphatase inhibitor were purchased from Roche. IRDye 800CW goat anti-rabbit, IRDye 800CW goat anti-mouse antibodies and IRDye 680RD were purchased from Li-Cor BioSciences. 4-15% mini protean TGX stain-free gels, 10× tris/glycine/SDS PAGE buffer and Transblot Turbo Midi nitrocellulose transfer packs were purchased from Bio-Rad Laboratories.

[0240] The tumorigenic, breast, epithelial adenocarcinoma cell lines MDA-MB-231_NLG, MDA-MB-231_NLR, MDA-MB-468_NLR, SKBR3-NLR, MCF7_NLR and the normal, lung fibroblast cell line, IMR90 were cultured as adherent monolayers in DMEM with 10% volume FBS in an atmosphere with 5% CO.sub.2 and 95% humidity and were routinely sub-cultured upon reaching 80-90% confluence. The natural killer cell line NK92 was cultured in suspension in alpha MEM medium with 12.5% volume heat inactivated fetal bovine serum, 12.5% volume heat-inactivated horse serum, 0.02 mM folic acid, 0.1 mM 3-mercaptoethanol, 0.2 mM myo-inositol and 200 U/mL IL-2. The latter medium with 2000 U/mL IL-2 was used for NK92 cell killing assays. MDA-MB-231 NLG and MDA-MB-468_NLR cells are a variant of MDA-MB-231 cells, expressing nuclear-restricted green fluorescent protein or nuclear-restricted mKate2 fluorescent protein respectively while MDA-MB-468_NLR, SKBR3_NLR and MCF7_NLR are, respectively, variants of MDA-MB-468, SKBR3 and MCF7 cells expressing nuclear-restricted mKate2 fluorescent protein; the cell lines with nuclear-restricted fluorescent protein were produced by stable transduction of cells with NucLight Green lentivirus or Nuclight Red lentivirus (Essen Bioscience), following the manufacturer's protocol.

[0241] To determine the direct effect of the compounds on cell proliferation, cells were seeded at a density of 500 cells per well in cell culture medium in 384-well cell culture plates and allowed to adhere overnight (about 16 hours), incubated in a humidified atmosphere with 5% CO.sub.2. Subsequently, cell culture medium was refreshed, supplemented with Draq 7 (3 μM final concentration) and test compound at the indicated concentrations with three wells being treated for each condition tested. Cells were then returned to the cell culture incubator and imaged with a 10× objective every 3 hours for 120 hours using an IncuCyte ZOOM microscope from Essen Bioscience.

[0242] Using the IncuCyte ZOOM software, custom image analysis procedures were developed and applied for each cell line to determine cell confluency, cell number and number of dead cells over the time course of the experiment. Phase contrast was used to determine relative area of each image occupied by cells (confluency), while green nuclear counts were used to determine number of MDA-MB-231_NLG cells and red nuclear counts were used to determine number of dead (Draq7-positive) cells.

[0243] Cell viability was determined relative to vehicle-treated (0.1% DMSO) controls using the GI.sub.50 method established by the National Cancer Institute with GI.sub.50 values (concentration of compound causing 50% growth inhibition) being determined by fitting non-linear regression curves to the data and extrapolating the required values using GraphPad Prism 6. Statistical analyses in cell viability assays to compare the effect of compound treatment to treatment with vehicle were performed using GraphPad Prism 6 (2-way ANOVA with Bonferroni correction post hoc).

[0244] To determine and quantify the expression of cyclophilins in the cell lines, cells were lysed with ice-cold MPER supplemented with protease and phosphatase inhibitor cocktails. Clarified lysates were resolved on 4-15% Tris-glycine gels by SDS-PAGE and total protein transferred to nitrocellulose membrane. Membranes were blocked with Li-Cor Buffer, probed with appropriate primary antibodies overnight, followed by washing and probing with appropriate fluorescence-conjugated secondary antibodies. Membranes were imaged and fluorescence intensity on the membranes recorded using the Li-Cor Odyssey CLx imager.

[0245] To determine the effect of the compounds on immune cell killing of target cancer cells, cancer cells (MDA-MB-231_NLR, MDA-MB-468_NLR, SKBR3_NLR or MCF7_NLR) were seeded at a density of 250 cells per well in NK92 cell culture medium with 2000 U/mL IL2, supplemented with 2.5 μM NucView 488 in GelTrex-coated 384-well cell culture plates. NK92 cells were then added at a density of 1000 cells per well and 10 μM test compound was added. Cells were imaged with a 10× objective every 3 hours for 120 hours using an IncuCyte ZOOM microscope from Essen Bioscience.

[0246] Using the IncuCyte ZOOM software, custom image analysis procedures were developed and applied for each cell line to determine cell number (red nuclei) and number of apoptotic cells (NucView 488-positive nuclei with co-localized red and green fluorescent signal) over the time course of the experiment. Apoptotic fraction of the population (number of nuclei with co-localized red and green fluorescence divided by total number of red nuclei in the population) and fold increase in number of red nuclei were determined at various time points.

[0247] Cyps Binding

[0248] FIG. 1A summarises the mode of binding of certain prior-art urea small molecule Cyp inhibitors. In the so-called ‘type-I binding mode’ the compounds may interact with the Abu and Pro pockets of the Cyps surfaces. In the so called novel ‘type-II binding mode’ depicted in FIG. 1B the compounds may interact with the Abu, Pro and 3 o'clock pockets of the Cyps surfaces. A type-II binding mode compound may be obtained by replacement of the shaded H atom in a type-I binding mode compound with a suitably chosen R group to create tri-vector Cyclophilin inhibitor derivatives. This modification is not obvious given the prior-art because the vector between the nitrogen atom bonded to the shaded H atom and the shaded H atom points away from the 3 o'clock. Thus conversion from a type-I binding mode to a type-II binding mode involves a 180 degree flip of the urea moiety to position the R group towards the 3 o'clock pocket. Only certain non-obvious R groups are able to accommodate this urea flip. FIG. 1C depicts a crystal structure of CypA in complex with a prior-art compound (R═H) showing that the compound does not access the 3 o'clock pocket. FIG. 1D depicts the crystal structure of CypA in complex with a novel tri-vector derivative (R=Et) that does not adopt a type-II binding mode. FIG. 1E and FIG. 1F depict crystal structures of CypA in complex with novel tri-vector derivatives (R=tetrazolyl-methyl) that adopt a type-II binding mode enabling interactions between their R groups and the 3 o'clock pocket.

[0249] Table 1 shows that tri-vector derivatives show a range of binding constants measured by isothermal titration calorimetry experiments (ITC) consistent with CypA inhibition with K.sub.d values ranging from high micromolar to mid-nanomolar.

[0250] Table 1 also shows that selected tri-vector derivatives show a range of binding constants measured by surface plasmon resonance (SPR) experiments consistent with CypA, CypB and CypD inhibition, with K.sub.d values ranging from low micromolar to mid-nanomolar. In the case of CypD, some tri-vector derivatives show a potency that approaches that of the drug Cyclosporine A. For comparison the most potent literature compounds (Fragment-based discovery of a new family of non-peptidic small-molecule cyclophilin inhibitors with potent antiviral activities. Ahmed-Belkacem A, Colliandre L, Ahnou N, Nevers Q, Gelin M, Bessin Y, Brillet R, Cala O, Douguet D, Bourguet W, Krimm I, Pawlotsky J M1, Guichou J F. Nature Communications 7: 12777, 2016) are reported to have IC.sub.50 values of ca. 0.2 micromolar against CypD. Thus some of the present tri-vector derivatives are more potent than the previous generation of small molecule Cyp inhibitors. The data in Table 1 also shows that some of the tri-vector derivatives show up to 25-fold selectivity for CypA, B or D, which is over 10 times greater than what is measured for the drug Cyclosporine A or other small molecule inhibitors

[0251] Table 1 shows that some of the tri-vector derivatives show superior growth inhibition 50 (GI50) values over the drug Cyclosporine A in growth inhibition experiments against a triple-negative breast cancer cell line that has previously been reported to be sensitive to Cyclosporine A (Prolyl isomerase cyclophilin A regulation of Janus-activated kinase 2 and the progression of human breast cancer. Zheng J, Koblinski J E, Dutson L V, Feeney Y B, Clevenger C V. Cancer Res. 68(19):7769-78, 2008). Table 1 shows that the tri-vector derivatives inhibit growth without causing cell death in the MDA-MB-231 cancer cell line and the non-cancerous control cell line IMR-90, whereas Cyclosporine A causes cell death in both cases. Western Blotting experiments confirmed that both cell lines express CypA, CypB and CypD.

[0252] Table 2 shows that the tri-vector derivatives are not toxic to NK92 cells whereas CsA shows pronounced toxicity, and that the combination of tri-vector derivatives with NK92 cells is more effective at reducing proliferation of MDA-MB-231 cancer cell lines than the combination of CsA with NK92 cells. This shows that immunotherapies based on combination of novel tri-vector cyclophilin ligands with NK cells may be more effective than immunotherapies based on combination of CsA with NK cells.

[0253] Table 1 summarises binding and inhibition experiments carried out on tri-vector derivative. Table 2 summarises results from additional cell assay experiments carried out on compounds 1 and 4.

[0254] Altogether the data in Table 1, Table 2 and FIG. 1 confirm the usefulness of tri-vector Cyclophilin ligands as potent and selective Cyclophilin inhibitors with reduced toxicity over other scaffolds such as the drug Cyclosporine A.

TABLE-US-00001 TABLE 1 Summary of binding and inhibition activity. All figures are in micromolar. CsA: Cyclosporine A. Toxicity Gl50 MDA- Toxicity CypA CypA CypA CypB CypD MDA- MB- IMR- Kd X-ray Kd Kd Kd MB-231 231@10 90@10 Compound (ITC) cocrystal (SPR) (SPR) (SPR) cells μM μM CsA 0.011 ± 0.001 Y 0.024 ± 0.006 0.010 ± 0.003 0.019 ± 0.005 6.2 ± 0.8 Y Y  1 2 ± 1 Y 3.2 ± 1.3 1.4 ± 0.4 0.2 ± 0.1 5.1 ± 0.9 N N  2 ~1 Y 1.7 ± 0.8 0.9 ± 0.3 0.06 ± 0.03 ND N N  3 ~1 Y 1.7 ± 1.0 0.3 ± 0.2 2.9 ± 1.2 NT NT NT  4 0.8 ± 0.1 Y 0.6 ± 0.4 0.2 ± 0.1 0.07 ± 0.03 2.8 ± 0.9 N N  5 ~3 N 4.3 ± 1.0 1.9 ± 0.9 1.0 ± 0.4 ND NT NT  6 0.30 ± 0.08 N NT NT NT NT NT NT  7 3.0 ± 0.5 Y 4.5 ± 1.0 0.7 ± 0.4 4.7 ± 2.9 NT NT NT  8 1.1 ± 0.4 N NT NT NT NT NT NT  9 35 ± 5 Y NT NT NT NT NT NT 10 ~25 N NT NT NT NT NT NT 11 ~10 Y NT NT NT NT NT NT 12 200 ± 5  Y NT NT NT NT NT NT 13 990 ± 20  Y NT NT NT NT NT NT 14 139 ± 4  N NT NT NT NT NT NT 15 35 ± 5  Y NT NT NT NT NT NT 16 266 ± 3  Y NT NT NT NT NT NT 17 ND Y NT NT NT NT NT NT 18 135 ± 6  Y NT NT NT NT NT NT 19 184 ± 13  Y NT NT NT NT NT NT 20 >600 Y NT NT NT NT NT NT 21 ND Y NT NT NT NT NT NT 22 >1000 Y NT NT NT NT NT NT 23 700 ± 235 N NT NT NT NT NT NT NT Not tested ND Not determined Y observed in cocrystal or evidence of dose dependent cytotoxicity N not observered in cocrystal or evidence of dose dependent cytotoxicity

TABLE-US-00002 TABLE 2 Summary of anti-proliferative activity Apoptotic Fold increase fraction of the MDA-MB-231 MDA-MB-231 cells in presence population in of NK92 cells Fold increase in NK92 the presence of after 72 h @ 10 cells after 72 h @ 10 NK92 cells after Compound μM μM 72 h @ 10 μM DMSO 2.6 ± 0.4 4.1 ± 0.3 0.56 ± 0.03 CsA 3.0 ± 0.6 2.5 ± 0.3 0.38 ± 0.01 1 2.0 ± 0.3 4.3 ± 0.7 0.64 ± 0.03 4 1.0 ± 0.4 4.7 ± 0.5 0.58 ± 0.03