PRO-SURVIVAL COMPOUNDS

Abstract

Disclosed herein are a class of compounds useful in cell culture, in particular, the in vitro culture of stem cells. The compounds have been found to promote the survival and/or maintenance of stem cells in (or during) culture and/or throughout passage.

Claims

1. A compound according to formula (I): ##STR00033## wherein: R.sup.1 is H, aryl, substituted aryl, alkyl, CH(CH.sub.3)R.sup.5, or CH.sub.2R.sup.5; R.sup.5 is aryl, substituted aryl, heteroaryl, substituted heteroaryl or heterocyclic; X is NZ, O, CH.sub.2 or S, or alternatively X may not be present; Z is H or alkyl; R.sup.2 and R.sup.3 are each independently selected from H, alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, or alternatively R.sup.2 and R.sup.3 together form an aromatic or heteroaromatic ring, optionally comprising one or more substituents; A is CH.sub.2, CH.sub.2CH.sub.2 or CH(CH.sub.3); Y is C(═O)NZ′, C(═O)O, SO.sub.2NH, NHC(═O), C(═O)NHC(═O), NH, or 0; Z′ is H, alkyl, alkoxyalkyl, or wherein Z′ and R.sup.4 together form a 5- or 6-membered ring heterocyclic group with at least one N atom; n is 0, 1, 2 or 3; W is H or alkyl, wherein when n is greater than 1, each W is independently selected from H or alkyl; R.sup.4 is H, alkyl, cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, OR.sup.6, heterocyclic, alkylamino, C≡N or C(═O)R.sup.7; R.sup.6 is H, alkyl, haloalkyl, aryl, alkoxyalkyl; and R.sup.7 is hydroxy, alkoxy, amino or alkylamino.

2. A compound according to formula (II), (III) or (IV): ##STR00034## wherein R.sup.1, X, A, Y, W, n and R.sup.4 are as defined for formula (I); ##STR00035## wherein R.sup.1, R.sup.2, R.sup.3 and X are as defined for formula (I); X′ is a hydrogen bond acceptor site; n′ is 1 or 2; the dotted lines indicate that the hydrogen bond acceptor site is incorporated into a heteroaryl or heterocyclic ring, or alternatively the dotted lines are not present and the hydrogen bond acceptor site is present on an acyclic side chain, in either case the cyclic or acyclic side chain optionally comprises one or more alkyl substituents; and wherein one or more of the starred carbon atoms (*) is substituted with a heteroatom; ##STR00036## wherein R.sup.1, R.sup.2, R.sup.3 and X and are as defined for formula (I); and X″ is a heteroaryl, substituted heteroaryl or an alkoxyalkyl group.

3. A compound having the following structure: ##STR00037##

4. A composition comprising one or more compounds according to claim 1.

5.-12. (canceled)

13. A composition comprising one or more compounds according to claim 2.

14. A composition comprising one or more compounds according to claim 3.

15. A cell culture medium or cell culture media supplement comprising one or more compounds according to claim 1.

16. A cell culture medium or cell culture media supplement comprising one or more compounds according to claim 2.

17. A cell culture medium or cell culture media supplement comprising one or more compounds according to claim 3.

18. A method of maintaining a cell or cells in culture, said method comprising contacting the cell or cells with one or more compounds according to claim 1.

19. A method of maintaining a cell or cells in culture, said method comprising contacting the cell or cells with one or more compounds according to claim 2.

20. A method of maintaining a cell or cells in culture, said method comprising contacting the cell or cells with one or more compounds according to claim 3.

21. The method of claim 18, wherein the cell or cells is a stem cell or population of stem cells.

22. The method of claim 19, wherein the cell or cells is a stem cell or population of stem cells.

23. The method of claim 20, wherein the cell or cells is a stem cell or population of stem cells.

24. A kit for maintaining stem cells in culture and/or for use in a method of maintaining stem cells in culture as described herein, said kit comprising a compound according to claim 1

25. The kit of claim 24, wherein the kit further comprises one or more components selected from the group consisting of: (a) receptacles for the culture and/or maintenance of stem cells, embryoid bodies and/or cells; (b) tools and/or implements for adding supplements to media; and (c) instructions for use.

26. A kit for maintaining stem cells in culture and/or for use in a method of maintaining stem cells in culture as described herein, said kit comprising a compound according to claim 2.

27. The kit of claim 26, wherein the kit further comprises one or more components selected from the group consisting of: (a) receptacles for the culture and/or maintenance of stem cells, embryoid bodies and/or cells; (b) tools and/or implements for adding supplements to media; and (c) instructions for use.

28. A kit for maintaining stem cells in culture and/or for use in a method of maintaining stem cells in culture as described herein, said kit comprising a compound according to claim 3.

29. The kit of claim 26, wherein the kit further comprises one or more components selected from the group consisting of: (a) receptacles for the culture and/or maintenance of stem cells, embryoid bodies and/or cells; (b) tools and/or implements for adding supplements to media; and (c) instructions for use.

Description

DETAILED DESCRIPTION

[0157] Note: compound SC332 (also referred to as “T16”) is 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(5-methylisoxazol-3-yl)acetamide and its structure is illustrated in the following Examples section).

[0158] The present invention will now be described by way of example only, and with reference to the following Figures, wherein:

[0159] FIG. 1 shows the percentage survival (24 hour post passage) of cell lines treated with either 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(5-methylisoxazol-3-yl)acetamide (SC332), a representative compound of the present invention, or Y27632, or left untreated.

[0160] FIG. 2 shows hPSC survival after long term exposure to pro-survival compounds. SC332 or Y27632 was used to enzymatically passage the hESC line H1 for 30 consecutive passages. Survival was assayed 24 hrs post passage. Data up to passage 20 represents mean survival ±SEM, n=3. Data after passage 20 represents cell survival, n=1. The horizontal red line represents the average survival of untreated cells ±SEM, n=3.

[0161] FIG. 3 shows the cytogenetic stability of cells treated with compound SC332. hPSC (H1 hESC) treated with SC332 or Y27632 for 30 consecutive passages were independently tested for cytogenetic stability (Yorkhill hospital, NHS Greater Glasgow and Clyde). Cells remained karyotypically normal (46, XY) until at least P30.

[0162] FIG. 4 shows flow cytometric analysis of hPSC surface markers. HPSC (H1 hESC) were enzymatically passaged using SC332 or Y27632 for 30 consecutive passages. Cells were harvested at the point of passage and analysed via flow cytometry for the pluripotency marker SSEA4 and differentiation marker SSEA1. The data shows that both SC332 and Y27632 treated cells maintained consistently high expression of SSEA4 over 30 consecutive passages, with negligible expression of SSEA1 being observed. Data shown as mean percentage of positive cells ±SEM (n=3) up to and including passage 20. Passages 25 and 30 represent values from an n=1.

[0163] FIG. 5 shows passive differentiation of hPSC after treatment with compound SC332. HPSC treated for 30 passages with SC332 or Y27632 were passively differentiated for 2 weeks in suspension culture, followed by 2 weeks adherent culture, fixed and stained for markers of the three germ layers. Control cells that had been mechanically passaged and unexposed to any survival compound were also differentiated and stained in the same way. PAX6 is stained in the left hand column, AFP in the centre column and SMA in the right hand column. All cells were co-stained with DAPI. Scale bar represents 100 μm.

[0164] FIG. 6 shows the effect of compound SC332 on the activity of Rho-associated protein kinase 2 (ROCK2), evaluated at 10 μM and 30 μM concentrations using a radioactive filter-binding assay at the Dundee International Centre for Kinase Profiling. A 30 μM concentration of compound SC332 did not inhibit either ROCK2 or the closely related kinase, Serine/threonine-protein kinase N2 (PRK2).

[0165] FIG. 7 shows analysis of levels of phosphorylated myosin light chain (pMLC) after treatment and dissociation of hPSC. Protein samples were taken from iPSC (cell line NMF-iPS6) that were treated with SC332, Y27632 or had no treatment (control) before being dissociated. Samples were harvested 15 mins, 30 mins, 45 mins, 1 hr, 2 hr and 4 hr post dissociation. Western blot analysis was performed. (A) shows representative immunoblots for pMLC and α-tubulin. (B) shows expression of pMLC relative to α-tubulin analysed via densitometry. Both SC332 treated and untreated cells had significantly higher levels of pMLC 15 mins post dissociation when compared to Y27632 treated cells (P=<0.001 and P=<0.01 respectively). SC332 treated cells had significantly higher levels of pMLC when compared to Y27632 treated cells after 30 mins (P=<0.05), 45 mins (P=<0.05) and 1 hr (P=<0.001). Data shown as mean±SEM, n=3.

[0166] FIG. 8 shows the long term survival produced using human induced pluripotent stem cells (hiPSC). Cells (NMF-iPS6) were treated with either SC332 or Y27632 and passaged enzymatically for 30 consecutive passages. Data represents survival from a single experiment (n=1). The red line represents the average survival of untreated cells ±SEM, n=3.

[0167] FIG. 9: Kinase profile of SC332 at 10 μM and 30 μM. Kinase inhibition on 121 kinases was assayed using a radioactive filter-binding assay at the Dundee International Centre for Kinase Profiling.

EXAMPLES SECTION

[0168] Chemistry

[0169] As used in the following example reaction schemes, R.sup.1, X, R.sup.2 and R.sup.3 simply represent variable groups and have been used for illustrative purposes throughout each reaction scheme. The appropriate selection of these groups is dependent upon the targeted compound and will be apparent to the skilled person. They may not necessarily be defined as described in the foregoing.

##STR00009##

[0170] Compounds of formula (I) in which A is CH.sub.2 and Y is simultaneously C(═O)NZ′ may be prepared as illustrated in Scheme 2, wherein a 2-substituted benzimidazole (1; X═O, S, NH) is alkylated with a chloroacetamide derivative (2) under basic conditions, typically by addition of sodium hydride to a solution of the benzimidazole (1) in anhydrous N,N-dimethylformamide solvent followed by addition of the chloroacetamide (2). The chloroacetamide (2) may be prepared by reaction of a suitable amine (Scheme 2, R.sup.2—NH—R.sup.3) with chloroacetyl chloride in the presence of a base (typically triethylamine) in an aprotic anhydrous solvent such as dichloromethane. Bromo- or iodoacetamide analogs of chloroacetamide 2 may alternatively be used. The benzimidazole precursor (1) may be commercially available or prepared by general methods known and widely used in the art such as: (A) N-alkylation of 2-aminobenzimidazole with an alcohol catalysed by dichloro(pentamethylcyclopentadienyl)iridium(III) dimer (F. Li et al., Eur. J. Org. Chem., 2012, 5085-5092); (B) reductive amination of 2-aminobenzimidazole, typically through condensation with an aldehyde or ketone and treatment of the intermediate imine with a reducing agent such as sodium triacetoxyborohydride in an appropriate solvent; (C) reaction of 2-(methylsulfonyl)-1H-benzo[d]imidazole with a sulfur, oxygen or nitrogen nucleophile (P. Lan et al., Tetrahedron Lett., 2008, 49, 1910-1914).

##STR00010##

[0171] Compounds of formula (I) in which A is CH.sub.2 and Y is simultaneously C(═O)O may be prepared as illustrated in Scheme 3, wherein a 2-substituted benzimidazole (1; X═O, S, NH) is alkylated with a chloroacetate ester (4) under basic conditions, typically by addition of sodium hydride to a solution of the benzimidazole (1) in anhydrous N,N-dimethylformamide solvent followed by addition of the chloroacetate ester (4). The chloroacetate (4) may be commercially available (e.g. R.sup.2=methyl, ethyl, tert-butyl) or prepared by reaction of a suitable alcohol (Scheme 3, R.sup.2—OH) with chloroacetyl chloride in the presence of a base (typically triethylamine) in an aprotic anhydrous solvent such as dichloromethane. The benzimidazole precursor (1) may be commercially available or prepared as presented in Route 1. Acid-catalysed or base-mediated solvolysis of esters 5 may afford access to the corresponding carboxylic acids 6. Alternatively an acid-catalysed elimination reaction on 5 (R.sup.2=tert-butyl), typically accomplished by treatment with trifluoroacetic acid or a mixture of trifluoroacetic acid in dichloromethane, may afford carboxylic acids 6.

##STR00011##

[0172] Compounds of formula (I) in which A is CH.sub.2CH.sub.2 and Y is simultaneously 0 may be prepared as illustrated in Scheme 4, wherein a 2-substituted benzimidazole (1; X═O, S, NH) is alkylated with a bromoethyl ether (4) under basic conditions, typically by addition of sodium hydride to a solution of the benzimidazole (1) in anhydrous N,N-dimethylformamide solvent followed by addition of the bromoethyl ether (4). Chloro- or iodoethyl ether analogs of 4 may be used or the corresponding toluenesulfonates or trifluoromethanesulfonates. The benzimidazole precursor (1) may be commercially available or prepared as presented in Route 1.

##STR00012##

[0173] Compounds of formula (I) in which A is CH.sub.2 and Y is simultaneously C(═O)NZ′ and X is simultaneously N-alkyl may be prepared as illustrated in Scheme 5, wherein a benzimidazolylacetate derivative (5; X═NH) is alkylated under basic conditions, typically by addition of sodium hydride to a solution of 5 in anhydrous N,N-dimethylformamide solvent followed by addition of an alkyl halide (Scheme 5 R.sup.3-Hal). Conversion of esters 9 into acids 10 may be accomplished by acid-catalysed or base-mediated solvolysis of the ester or alternatively by an acid-catalysed elimination reaction on 9 (R.sup.2=tert-butyl), typically through treatment with trifluoroacetic acid or a mixture of trifluoroacetic acid in dichloromethane. Acids 10 may be converted into their para-nitrophenol ester derivatives (11), for example by treatment with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and para-nitrophenol in dichloromethane. Reaction of esters 11 with a suitable primary amine pre-treated with sodium hydride in N,N-dimethylformamide may then afford amides 12. Those skilled in the art will appreciate that numerous alternative reagents and conditions may be applied to activate carboxylic acids such as 11 for coupling to amines.

##STR00013##

[0174] Compounds of formula (I) in which A is CH.sub.2 and Y is simultaneously C(═O)NZ′ may also be prepared as illustrated in Scheme 6, wherein 2-aminobenzimidazole is alkylated with a chloroacetamide derivative (13) under basic conditions, typically by addition of sodium hydride to a solution of 2-aminobenzimidazole in anhydrous N,N-dimethylformamide solvent followed by 13. The chloroacetamide (13) may be prepared by reaction of a suitable amine (Scheme 6, R.sup.1—NH—R.sup.2) with chloroacetyl chloride in the presence of a base (typically triethylamine) in an aprotic anhydrous solvent such as dichloromethane. Bromo- or iodoacetamide analogs of chloroacetamide 13 may alternatively be used. Substituted benzimidazoles 14 may then be converted into targets 15 by reaction with suitable alcohols (R.sup.3OH) catalysed by dichloro(pentamethylcyclopentadienyl)iridium(III) dimer.

##STR00014##

[0175] Compounds of formula (I) in which A is CH.sub.2 and Y is simultaneously C(═O)NZ′ may additionally be prepared as illustrated in Scheme 7, wherein 2-chlorobenzimidazole is alkylated with a chloroacetamide derivative (13) under basic conditions, typically by addition of sodium hydride to a solution of 2-chlorobenzimidazole in anhydrous N,N-dimethylformamide solvent followed by 13. As previously, bromo- or iodoacetamide analogs of chloroacetamide 13 may alternatively be used. Substituted benzimidazoles 16 may then be converted into targets 15 by reaction with suitable amines (R.sup.3—NH.sub.2), for example by heating in an appropriate solvent (e.g. 1,4-dioxane or n-butanol) in a sealed pressure vessel (if required) and sometimes in the presence of a suitable additive such as potassium dihydrogen phosphate or a silver salt.

##STR00015##

[0176] A variant on the preceding route to compounds of formula (I) in which A is CH.sub.2 and Y is simultaneously C(═O)NZ′ is illustrated in Scheme 8, wherein 2-chlorobenzimidazole is alkylated with 4-nitrophenyl 2-bromoacetate (17), for example by addition of sodium hydride to a solution of 2-chlorobenzimidazole in anhydrous N,N-dimethylformamide solvent followed by 17. For this purpose bromoacetate 17 may first be prepared by reaction of bromoacetyl bromide with para-nitrophenol. Reaction of intermediate esters 18 with a suitable primary amine (R.sup.3—NH.sub.2) pre-treated with sodium hydride in N,N-dimethylformamide may then afford amides 19. The latter may then be converted into targets 20 by a reaction with a suitable amine (R.sup.4—NH.sub.2), for example by heating in an appropriate solvent (e.g. 1,4-dioxane or n-butanol) in a sealed pressure vessel (if required) and sometimes in the presence of a suitable additive such as potassium dihydrogen phosphate or a silver salt.

[0177] General Chemistry Experimental

[0178] Tetrahydrofuran (THF), diethyl ether (Et.sub.2O), dichloromethane (DCM) and toluene (PhMe) were dried by passage through commercial columns in an in-house solvent purification system. Methanol (MeOH), ethyl acetate (EtOAc), chloroform, acetone, hexane and light petroleum were used as supplied from Fisher. ‘Light petroleum’ refers to the fraction boiling between 40° C. and 60° C. Anhydrous N,N-dimethylformamide (DMF) was purchased from Aldrich and used as supplied from Sure/Seal™ bottles. Analytical thin layer chromatography (TLC) was carried out using aluminium backed plates coated with Merck Kieselgel 60 GF254 (Art. 05554). Developed plates were visualized under ultra-violet light (254 nm) and/or alkaline potassium permanganate dip. Preparative chromatography was performed using flash silica (60 Å; 35-70 μM) from Fisher in glass columns or over Strata® SI-1 (70 Å; 55 μM) silica Giga™ Tube cartridges from Phenomenex.

[0179] IR spectra were recorded on a Thermo Scientific iD5 Diamond ATR/Nicolet iS5 FT-IR spectrometer with samples as neat solids or liquids. Mass spectra were obtained under electrospray ionisation (ESI) conditions through the Edinburgh University Mass Spectrometry Service. .sup.1H NMR spectra were recorded at 300 and 400 MHz on Bruker AVIII-300 and AVIII-400 spectrometers; .sup.13C NMR spectra were recorded at 75 and 101 MHz on the same instruments. Chemical shifts are recorded in parts per million (8 in ppm) and are referenced against solvent signals (δ.sub.C 77.16 for chloroform, δ.sub.C 39.52 for methyl sulfoxide) for .sup.13C spectra and solvent residual resonances (δ.sub.H 7.26 for chloroform, δ.sub.H 3.31 for methanol, δ.sub.H 2.50 methyl sulfoxide) for .sup.1H spectra. Chemical shift values and are accurate to ±0.01 ppm and ±0.1 ppm in .sup.1H and .sup.13C spectra respectively. J values are given in Hz. Multiplicity designations used are: s, d, t, q, sept and m for singlet, doublet, triplet, quartet, septet and multiplet respectively; broadened signals are denoted br. In .sup.13C NMR spectra, signals corresponding to CH, CH.sub.2, or CH.sub.3 groups are assigned from DEPT. Elemental analyses were carried out by the analytical service of the at Heriot-Watt University using an Exeter CE-440 Elemental Analyser.

Example 1

2-(2-(Benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(2-methoxyethyl)acetamide (SC050) was prepared according to Route 1

[0180] Step 1. N-Benzyl-1H-benzo[d]imidazol-2-amine was prepared following a published procedure (F. Li et al., Eur. J. Org. Chem., 2012, 5085-5092). Thus, a flame-dried, heavy-walled, sealable flask fitted with a magnetic stir bar was charged under argon with 2-aminobenzimidazole (1.00 g, 7.51 mmol), dichloro(pentamethylcyclopentadienyl)iridium(III) dimer (12.0 mg, 15.1 μmol), cesium carbonate (245 mg, 752 μmol) and benzyl alcohol (3.11 mL, 30.1 mmol). The flask was sealed and the mixture heated at 130° C. with stirring. After 24 h the mixture was cooled, diluted with EtOAc (60 mL) and washed with brine (3×20 mL). The organic phase was dried (MgSO.sub.4), filtered and evaporated to afford a residual green oil that was subjected to silica gel chromatography (gradient elution with 90% v/v hexane/EtOAc to 100% EtOAc). Fractions containing the target material (R.sub.f 0.32, 10% v/v MeOH/DCM) were combined and evaporated to afford N-benzyl-1H-benzo[d]imidazol-2-amine (1.52 g, 6.79 mmol; 90%) as a light brown powder: δ.sub.H (300 MHz, CD.sub.3OD) 7.38-7.42 (2H, m), 7.30-7.36 (2H, m), 7.17-7.28 (3H, m), 6.94-7.00 (2H, m), 4.59 (2H, s).

[0181] Step 2. To an ice-cooled solution of 2-methoxyethylamine (623 mg, 8.29 mmol) and triethylamine (1.50 mL, 10.8 mmol) in anhydrous DCM (50 mL) under argon was added dropwise chloroacetyl chloride (795 μL, 10.0 mmol). After 4 h the mixture was flushed through a Strata® SI-1 silica cartridge (20 g Giga™ Tube), eluting with DCM. Fractions containing the target material (R.sub.f 0.65, 100% EtOAc) were combined and evaporated to afford 2-chloro-N-(2-methoxyethyl)acetamide (1.14 g, 7.50 mmol; 90%) as a brown oil: δ.sub.H (300 MHz, CDCl.sub.3): 6.92 (1H, br s), 4.02 (2H, s), 3.44-3.49 (4H, m), 3.34 (3H, s).

[0182] Step 3. To an ice-cooled solution of N-benzyl-1H-benzo[d]imidazol-2-amine (224 mg, 1.00 mmol) in anhydrous DMF (5 mL) under argon was added NaH (60% w/w dispersion in mineral oil; 44.0 mg, 1.10 mmol). After 20 min a solution of 2-chloro-N-(2-methoxyethyl)acetamide (194 mg, 1.28 mmol) in DMF (5 mL) was added. The mixture was stirred and allowed to come ambient temperature over the course of 1 h. TLC analysis (100% EtOAc) indicated complete consumption of the benzimidazole substrate (R.sub.f 0.30) and formation of a product (R.sub.f 0.37). The reaction mixture was evaporated to dryness (70 CC, 12 mbar) and the resulting residue diluted with EtOAc (50 mL). This solution was then washed with brine (3×20 mL), dried (Na.sub.2SO.sub.4), filtered and evaporated to afford a solid residue. Recrystallization of the solid residue from hot CHCl.sub.3/hexane afforded 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(2-methoxyethyl)acetamide (285 mg, 842 μmol; 84%) as a colourless powder: δ.sub.H (300 MHz, CDCl.sub.3) 7.46-7.49 (1H, ddd, J 7.8, 1.0 and 0.6), 7.36-7.40 (2H, m), 7.23-7.35 (3H, m), 7.12-7.17 (1H, m), 7.06 (1H, td, J 7.4 and 1.2), 7.01 (1H, ddd, J 7.8, 1.6 and 0.6), 6.51 (1H, br s), 5.65 (1H, br t, J 5.5), 4.67 (2H, d, J 5.3), 4.47 (2H, s), 3.27-3.33 (4H, m), 3.19 (3H, s).

Example 2

2-(2-(Benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(2-hydroxyethyl)acetamide (SC705) was prepared according to Route 1

[0183] Step 1. A flame-dried flask fitted with a magnetic stir bar was charged under argon with anhydrous THF (15 mL) and NaH (60% w/w dispersion in mineral oil; 110 mg, 2.75 mmol). To the resulting suspension was added ethanolamine (76 μL, 1.3 mmol) followed after 10 min by tert-butyldimethylsilyl chloride (192 mg, 1.27 mmol). After stirring at ambient temperature for 1 h chloroacetyl chloride (100 μL, 1.26 mmol) was added and stirring continued for a further 1.5 h to afford ‘Mixture A’ as a colourless liquid containing a fine solid suspension. In parallel with the preparation of Mixture A, a separate flask was charged with N-benzyl-1H-benzo[d]imidazol-2-amine (283 mg, 1.27 mmol), anhydrous THF (35 mL) and NaH (60% w/w dispersion in mineral oil; 60.0 mg, 1.50 mmol), generating a homogenous yellow solution as ‘Mixture B’ after stirring for 30 min. Mixture A was then cannulated into Mixture B and stirring continued. After 24 h the combined mixture was filtered through Celite® and the filtrate evaporated to dryness. The resulting residue was subjected to chromatography over a Strata® SI-1 silica cartridge (20 g Giga™ Tube) using gradient elution: 100% light petroleum >25% v/v DCM/light petroleum >75% v/v DCM/light petroleum >100% DCM >10% v/v EtOAc/DCM >20% v/v EtOAc/DCM >30% v/v EtOAc/DCM >40% v/v EtOAc/DCM. Fractions containing the product, which eluted in 30-40% v/v EtOAc/DCM, were combined and evaporated to afford 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acetamide (274 mg, 625 μmol; 50%) as a colourless powder: δ.sub.H (300 MHz, CDCl.sub.3) 7.52 (1H, br d, J 7.7), 7.27-7.42 (5H, m), 7.16 (1H, td, J 7.4 and 1.5), 7.08 (1H, td, J 7.4 and 1.1), 7.03 (1H, br dd, J 7.8 and 1.0), 6.11 (1H, br t, J 4.7), 5.04 (1H, br t, J 5.3), 4.73 (2H, d, J 5.5), 4.51 (2H, s), 3.57 (2H, t, J 5.3), 3.31 (2H, q, J 5.3), 0.75 (9H, s), −0.08 (6H, s).

[0184] Step 2. To a stirred solution of 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acetamide (274 mg, 625 μmol) in THF (15 mL) was added tetrabutylammonium fluoride (1 M solution in THF containing 5% water; 750 μL, 750 μmol). After 24 h the mixture was evaporated to dryness, affording a viscous yellow oil that was subjected to chromatography over a Strata® SI-1 silica cartridge (20 g Giga™ Tube) using gradient elution: 100% DCM >20% v/v EtOAc/DCM >40% v/v EtOAc/DCM >5% v/v MeOH/DCM. Fractions containing the product, which eluted in 5% v/v MeOH/DCM, were combined and evaporated to afford 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(2-hydroxyethyl)acetamide (175 mg, 539 μmol; 86%) as a colourless powder: δ.sub.H (300 MHz, CD.sub.3OD) 7.39-7.43 (2H, m), 7.27-7.34 (3H, m), 7.20-7.25 (1H, m), 6.97-7.10 (3H, m), 4.70 (2H, s), 4.66 (2H, s), 3.60 (2H, t, J 5.7), 3.34 (2H, t, J 5.7).

Example 3

tert-Butyl 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)acetate (SC343) was prepared according to Route 2

[0185] To an ice-cooled solution of N-benzyl-1H-benzo[d]imidazol-2-amine (224 mg, 1.00 mmol) in anhydrous DMF (5 mL) under argon was added NaH (60% w/w dispersion in mineral oil; 44.0 mg, 1.10 mmol). After 20 min a solution of tert-butyl bromoacetate (218 mg, 1.12 mmol) in DMF (5 mL) was added. The mixture was stirred and allowed to come ambient temperature over the course of 1 h. TLC analysis (100% EtOAc) indicated complete consumption of the benzimidazole substrate (R.sub.f 0.30) and formation of a product (R.sub.f 0.83). The reaction mixture was evaporated to dryness (70° C., 12 mbar) and the resulting residue diluted with EtOAc (50 mL). This solution was then washed with brine (3×20 mL), dried (Na.sub.2SO.sub.4), filtered and evaporated to afford a viscous oil that was subjected to chromatography over a Strata® SI-1 silica cartridge (20 g Giga™ Tube), eluting with 100% DCM followed by 100% EtOAc. Fractions containing the product were combined and evaporated to afford tert-butyl 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)acetate (316 mg, 937 μmol; 94%) as a colourless oil that solidified on standing: δ.sub.H (300 MHz, CDCl.sub.3) 7.53 (1H, br d, J 7.4), 7.42-7.45 (2H, m), 7.27-7.39 (3H, m), 7.11-7.18 (1H, m), 7.06-7.09 (2H, m), 4.81 (1H, br t, J 5.2), 4.74 (2H, d, J 5.2), 4.50 (2H, s), 1.42 (9H, s).

Example 4

2-(2-(Benzylamino)-1H-benzo[d]imidazol-1-yl)acetic acid (SC287) was prepared according to Route 2

[0186] To a stirred solution of tert-butyl 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)acetate (197 mg, 584 μmop in DCM (4 mL) was added trifluoroacetic acid (4 mL). After 4 h the mixture was evaporated to dryness and the resulting solid residue recrystallized from hot EtOAc to afford 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)acetic acid (110 mg, 391 μmol; 67%) as a colourless solid: δ.sub.H (300 MHz, DMSO-d.sub.6) 13.45 (1H, br s), 9.48 (1H, br s), 7.55-7.61 (1H, m), 7.25-7.44 (8H, m), 5.09 (2H, s), 4.71 (2H, d, J 5.8).

Example 5

N-Benzyl-1-(2-(benzyloxy)ethyl)-1H-benzo[d]imidazol-2-amine (SC175) was prepared according to Route 3

[0187] To an ice-cooled solution of N-benzyl-1H-benzo[d]imidazol-2-amine (225 mg, 1.01 mmol) in anhydrous DMF (5 mL) under argon was added NaH (60% w/w dispersion in mineral oil; 44.0 mg, 1.10 mmol). After 20 min a solution of (2-bromoethyl) benzyl ether (240 mg, 1.12 mmol) in DMF (5 mL) was added. The mixture was stirred and allowed to come ambient temperature over the course of 1 h. TLC analysis (100% EtOAc) indicated complete consumption of the benzimidazole substrate (R.sub.f 0.30) and formation of a product (R.sub.f 0.83). The reaction mixture was evaporated to dryness (70° C., 12 mbar) and the resulting residue diluted with EtOAc (50 mL). This solution was then washed with brine (3×20 mL), dried (Na.sub.2SO.sub.4), filtered and evaporated to afford a viscous oil that was subjected to chromatography over a Strata® SI-1 silica cartridge (20 g Giga™ Tube), eluting with 100% DCM followed by 100% EtOAc. Fractions containing the product were combined and evaporated to afford N-benzyl-1-(2-(benzyloxy)ethyl)-1H-benzo[d]imidazol-2-amine (308 mg, 861 μmol; 86%) as a yellow oil: δ.sub.H (300 MHz, CDCl.sub.3) 7.57 (1H, ddd, J 7.7, 1.2 and 0.6), 7.23-7.34 (8H, m), 7.14-7.20 (3H, m), 7.09 (1H, td, J 7.4 and 1.2), 7.04 (1H, ddd, J 7.7, 1.6 and 0.7), 5.69 (1H, br t, J 5.5), 4.63 (2H, d, J 5.6), 4.45 (2H, s), 4.12-4.17 (2H, m), 3.81-3.86 (2H, m).

Example 6

2-(2-(Benzyl(methyl)amino)-1H-benzo[d]imidazol-1-yl)-N-(5-methylisoxazol-3-yl)acetamide (SC881) was prepared according to Route 4

[0188] Step 1. To an ice-cooled solution of tert-butyl 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)acetate (342 mg, 1.01 mmol) and iodomethane (124 μL, 1.99 mmol) in anhydrous DMF (5 mL) under argon was added NaH (60% w/w dispersion in mineral oil; 48.0 mg, 1.20 mmol). The reaction mixture was stirred and allowed to come to ambient temperature over the course of 48 h. The mixture was then evaporated to dryness (60° C., 12 mbar) and the resulting residue directly subjected to chromatography over a Strata® SI-1 silica cartridge (5 g Giga™ Tube), eluting with 100% DCM followed by 0.5% v/v MeOH/DCM. Fractions containing the product were combined and evaporated to afford tert-butyl 2-(2-(benzyl(methyl)amino)-1H-benzo[d]imidazol-1-yl)acetate (241 mg) as a yellow oil in ca. 90% purity by .sup.1H NMR analysis.

[0189] Step 2. The ester from Step 1 was taken up in DCM (6 mL), ice-cooled and treated with trifluoroacetic acid (2 mL). The mixture was then allowed to come to ambient temperature. After stirring for 16 h the mixture was evaporated to dryness, affording a residue that was subjected to chromatography over a Strata® SI-1 silica cartridge (5 g Giga™ Tube) using 1-10% v/v MeOH/DCM solvent gradient elution. Fractions containing the product were combined and evaporated to afford 2-(2-(benzyl(methyl)amino)-1H-benzo[d]imidazol-1-yl)acetic acid (150 mg, 508 μmol; 50% for Steps 1 and 2 combined) as a colourless solid.

[0190] Step 3. The acid from Step 2 (150 mg, 508 μmop was taken up in DCM (10 mL). para-Nitrophenol (88 mg, 630 μmol), N,N-dimethylaminopyridine (12 mg) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (146 mg, 762 μmol) were then added. After stirring for 1.5 h, the reaction mixture was diluted with DCM (30 mL), washed successively with water (2×10 mL) and brine (15 mL), dried (MgSO.sub.4), filtered and evaporated. The resulting waxy solid (220 mg) was subjected to chromatography over a Strata® SI-1 silica cartridge (5 g Giga™ Tube), eluting with 100% DCM followed by 0.5% v/v MeOH/DCM. Fractions containing the product were combined and evaporated to afford 4-nitrophenyl 2-(2-(benzyl(methyl)amino)-1H-benzo[d]imidazol-1-yl)acetate (164 mg) as a 1:1 mixture with para-nitrophenol according to .sup.1H NMR analysis.

[0191] Step 4. The para-nitrophenol ester from Step 3 (ca. 1:1 mixture with para-nitrophenol; 162 mg, 0.29 mmol) was taken up in anhydrous DMF (2 mL) under argon and ice-cooled. A solution of 3-amino-5-methylisoxazole (44 mg, 0.45 mmol) in DMF (3 mL) that had been pretreated with NaH (60% w/w dispersion in mineral oil; 22 mg, 0.55 mmol) for 25 min was then added dropwise into the para-nitrophenol ester solution. The combined mixture was allowed to come to ambient temperature. After stirring for 16 h the reaction mixture was evaporated to dryness (60° C., 12 mbar) and the resulting residue reconstituted with CHCl.sub.3 (30 mL). This solution was then washed successively with water (2×10 mL) and brine (15 mL), dried (MgSO.sub.4), filtered and evaporated. The crude material (152 mg) thus obtained was subjected to chromatography over a Strata® SI-1 silica cartridge (5 g Giga™ Tube), eluting with 0.5% v/v MeOH/DCM followed by 1% v/v MeOH/DCM. Fractions containing the product were combined and evaporated. The resulting waxy solid residue was triturated with hexane/Et.sub.2O to afford 2-(2-(benzyl(methyl)amino)-1H-benzo[d]imidazol-1-yl)-N-(5-methylisoxazol-3-yl)acetamide (16.1 mg, 43 μmol; 8% over Steps 3 and 4 combined) as a colourless powder: δ.sub.H (300 MHz, CDCl.sub.3) 8.31 (1H, br s), 7.65 (1H, ddd, J 7.7, 1.2 and 0.6), 7.24-7.35 (6H, m), 7.19 (1H, td, J 7.4 and 1.2), 7.14 (1H, ddd, J 7.7, 1.6 and 0.6), 6.70 (1H, br s), 4.80 (2H, s), 4.46 (2H, s), 2.91 (3H, s), 2.40 (3H, d, J 0.9).

Example 7

2-(2-(Isobutylamino)-1H-benzo[d]imidazol-1-yl)-N-(2-methoxyethyl)acetamide (SC769) was prepared according to Route 5

[0192] Step 1. To a stirred solution of 1H-benzo[d]imidazol-2-amine (1.33 g, 10.0 mmol) in anhydrous DMF (20 mL) under argon was added NaH (60% w/w dispersion in mineral oil; 400 mg, 10.0 mmol) in portions at ambient temperature. After 10 min a solution of 2-chloro-N-(2-methoxyethyl)acetamide (preparation vide supra, EXAMPLE 1; 1.52 g, 10.0 mmol) in DMF (8 mL) was added. The mixture was then stirred at ambient temperature for 16 h. The reaction mixture was evaporated to dryness (60° C., 12 mbar) and the resulting residue subjected to chromatography over a Strata® SI-1 silica cartridge (20 g Giga™ Tube), eluting successively with 2%, 5% and 10% v/v MeOH/DCM. Fractions containing the product were combined and evaporated to afford 2-(2-amino-1H-benzo[d]imidazol-1-yl)-N-(2-methoxyethyl)acetamide (SC593, 925 mg, 3.72 mmol; 37%) as a buff powder: δ.sub.H (300 MHz, CDCl.sub.3) 7.46-7.50 (1H, m), 7.16-7.23 (1H, m), 7.08-7.14 (2H, m), 6.24 (1H, br s), 4.90 (2H, br s), 4.61 (2H, s), 3.42-3.53 (4H, m), 3.31 (3H, s).

[0193] Step 2. A flame-dried, heavy-walled, sealable tube fitted with a magnetic stir bar was charged under argon with 2-(2-amino-1H-benzo[d]imidazol-1-yl)-N-(2-methoxyethyl)acetamide (154 mg, 621 μmop, isobutanol (0.916 mL, 9.92 mmol), dichloro(pentamethylcyclopentadienyl)iridium(III) dimer (5 mg, 6 μmol) and cesium carbonate (20 mg, 62 μmol). The tube was sealed and the mixture heated at 130° C. with stirring. After 20 h the mixture was cooled and evaporated to afford a residue that was subjected to chromatography over a Strata® SI-1 silica cartridge (10 g Giga™ Tube), eluting successively with 10%, 20%, 50% and 75% v/v EtOAc/light petroleum followed by EtOAc. Fractions containing the product were combined and evaporated to afford 2-(2-(isobutylamino)-1H-benzo[d]imidazol-1-yl)-N-(2-methoxyethyl)acetamide (SC769) (88 mg, 0.29 mmol; 47%) as a colourless powder: δ.sub.H (300 MHz, CDCl.sub.3) 7.50-7.54 (1H, m), 7.14-7.20 (1H, m), 7.02-7.11 (2H, m), 6.32 (1H, br s), 5.07 (1H, br t, J 5.6), 4.54 (2H, s), 3.36-3.48 (6H, m), 3.27 (3H, s), 2.02 (1H, nonet, J 6.8), 1.04 (6H, d, J 6.7).

Example 8

N-(2-Methoxyethyl)-2-(2-((pyridin-2-ylmethyl)amino)-1H-benzo[d]imidazol-1-yl)acetamide (SC209) was prepared according to Route 6

[0194] Step 1. To a stirred, ice-cooled solution of 2-chloro-1H-benzo[d]imidazole (1.53 g, 10.0 mmol) in anhydrous DMF (20 mL) under argon was added NaH (60% w/w dispersion in mineral oil; 400 mg, 10.0 mmol) in portions. The mixture was removed from the ice bath, stirred at ambient temperature for 20 min and then returned to the ice bath prior to dropwise addition of a solution of 2-chloro-N-(2-methoxyethyl)acetamide (preparation vide supra, EXAMPLE 1; 1.52 g, 10.0 mmol) in DMF (8 mL). The mixture was then stirred at ambient temperature for 64 h. The reaction mixture was evaporated to dryness (60° C., 12 mbar) and the resulting residue triturated with DCM followed by 10% MeOH/DCM, collecting the solvent fraction by filtration. The filtrate was evaporated and the residual material subjected to chromatography over a Strata® SI-1 silica cartridge (10 g Giga™ Tube), eluting with 33% v/v EtOAc/light petroleum followed by 10% v/v MeOH/DCM. Fractions containing the product were combined and evaporated to afford 2-(2-chloro-1H-benzo[d]imidazol-1-yl)-N-(2-methoxyethyl)-acetamide (2.22 g, 8.05 mmol; 80%) as a colourless powder: δ.sub.H (300 MHz, CDCl.sub.3) 7.74-7.78 (1H, m), 7.33-7.36 (3H, m), 5.86 (1H, br s), 4.88 (2H, s), 3.45-3.50 (2H, m), 3.38-3.42 (2H, m), 3.25 (3H, s).

[0195] Step 2. A flame-dried, heavy-walled, sealable tube fitted with a magnetic stir bar was charged under argon with 2-(2-chloro-1H-benzo[d]imidazol-1-yl)-N-(2-methoxyethyl)-acetamide (134 mg, 501 μmol), pyridin-2-ylmethanamine (216 mg, 2.00 mmol) and anhydrous THF (2.5 mL). The tube was sealed and the mixture heated at 120° C. with stirring. After 18 h the mixture was cooled and evaporated to afford a residue that was subjected to chromatography over a Strata® SI-1 silica cartridge (10 g Giga™ Tube), eluting successively with 1%, 2%, 5% and 10% v/v MeOH/DCM. Fractions containing the product were combined and evaporated to afford N-(2-methoxyethyl)-2-(2-((pyridin-2-ylmethyl)amino)-1H-benzo[d]imidazol-1-yl)acetamide (SC209) (115 mg, 339 μmol; 67%) as a yellow powder: δ.sub.H (300 MHz, CDCl.sub.3) 8.55 (1H, ddd, J 4.9, 1.6 and 0.9), 7.67 (1H, td, J 7.7 and 1.8), 7.49 (1H, dt, J 7.6 and 0.9), 7.32 (1H, ˜d, J 7.8), 7.18-7.22 (1H, m), 7.09-7.17 (1H, m), 7.04-7.08 (2H, m), 6.43 (1H, br t, J 4.9), 6.20 (1H, br s), 4.80 (2H, br s), 4.64 (2H, s), 3.40-3.48 (2H, m), 3.32-3.38 (2H, m), 3.15 (3H, s); δ.sub.C (300 MHz, CDCl.sub.3) 167.1 (C), 156.3 (C), 154.4 (C), 149.0 (CH), 142.6 (C), 136.9 (CH), 134.5 (C), 122.6 (CH), 122.3 (CH), 122.1 (CH), 120.4 (CH), 116.9 (CH), 107.4 (CH), 70.8 (CH.sub.2), 58.8 (CH.sub.3), 47.7 (CH.sub.2), 46.4 (CH.sub.2), 39.5 (CH.sub.2).

Example 9

N-(2-Methoxyethyl)-2-(2-(phenylamino)-1H-benzo[d]imidazol-1-yl)acetamide (SC865) was prepared according to Route 6

[0196] A flame-dried, heavy-walled, sealable tube fitted with a magnetic stir bar was charged under argon with 2-(2-chloro-1H-benzo[d]imidazol-1-yl)-N-(2-methoxyethyl)-acetamide (preparation vide supra, EXAMPLE 8; 134 mg, 501 μmol), aniline (186 mg, 2.00 mmol), KH.sub.2PO.sub.4 (68 mg, 0.52 mmol) and n-butanol (5 mL). The tube was sealed and the mixture heated at 80° C. with stirring. After 60 h the mixture was cooled and evaporated to afford a residue that was subjected to chromatography over a Strata® SI-1 silica cartridge (5 g Giga™ Tube), eluting successively with 33%, 50% and 75% v/v EtOAc/light petroleum followed by 5% v/v MeOH/DCM. Fractions containing the product were combined and evaporated to afford N-(2-methoxyethyl)-2-(2-(phenylamino)-1H-benzo[d]imidazol-1-yl)acetamide (SC865) (133 mg, 409 μmol; 82%) as a colourless powder: δ.sub.H (300 MHz, CDCl.sub.3) 8.27 (1H, br s), 7.48 (2H, d, J 7.2), 7.33 (2H, d, J 7.7), 7.18-7.26 (2H), 7.12 (2H, t, J 7.5), 6.92 (1H, t, J 7.1), 5.05 (2H, s), 3.41-3.50 (4H, m), 3.31 (3H, s).

Example 10

2-(2-(Benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(pyridin-3-yl)acetamide (SC632) was prepared according to Route 7

[0197] Step 1. To a stirred, ice-cooled solution of para-nitrophenol (1.39 g, 10.0 mmol) in anhydrous MeCN (20 mL) under argon was added triethylamine (1.46 mL, 10.5 mmol) followed dropwise by bromoacetyl bromide (915 μL, 10.5 mmol). After 20 min the mixture was removed from the ice bath and stirred at ambient temperature for 1 h. Solvent was then evaporated and the residue directly subjected to chromatography over a Strata® SI-1 silica cartridge (20 g Giga™ Tube), eluting with DCM. Fractions containing the product were combined and evaporated to afford 4-nitrophenyl 2-bromoacetate (2.56 g, 9.85 mmol; 99%) as a colourless powder: δ.sub.H (300 MHz, CDCl.sub.3) 8.28-8.33 (2H, AA′XX′), 7.32-7.37 (2H, AA′XX′), 4.08 (2H, s).

[0198] Step 2. To a stirred solution of 2-chloro-1H-benzo[d]imidazole (153 mg, 1.00 mmol) in anhydrous DMF (5 mL) at ambient temperature under argon was added NaH (60% w/w dispersion in mineral oil; 44 mg, 1.10 mmol). After 1.5 h the mixture was cooled to −40° C. prior to addition of a solution of 4-nitrophenyl 2-bromoacetate (286 mg, 1.10 mmol) in DMF (3 mL). The mixture was allowed to come to ambient temperature and stirred for 16 h. It was then diluted with DCM (25 mL) and washed successively with ice-water (2×100 mL) and brine (2×20 mL). The organic layer was dried (Na.sub.2SO.sub.4) and evaporated to afford a light brown solid (442 mg) that was subsequently triturated with diethyl ether, collecting the solid by filtration and discarding the ethereal phase. The solid was dried in vacuo to afford 4-nitrophenyl 2-(2-chloro-1H-benzo[d]imidazol-1-yl)acetate (221 mg, 666 μmol; 67%) as a buff powder: δ.sub.H (300 MHz, CDCl.sub.3) 8.25-8.30 (2H, AA′XX′), 7.73-7.79 (1H, m), 7.28-7.40 (5H, m), 5.21 (2H, s).

[0199] Step 3. To a solution of 3-aminopyridine (56 mg, 0.59 mmol) in DMF (3 mL) at ambient temperature under argon was added NaH (60% w/w dispersion in mineral oil; 24 mg, 0.60 mmol). After 1 h the mixture was cooled to 0° C. prior to dropwise addition of a solution of 4-nitrophenyl 2-(2-chloro-1H-benzo[d]imidazol-1-yl)acetate (166 mg, 500 μmol) in DMF (3 mL). The mixture was allowed to come to ambient temperature and stirred for 64 h. It was then evaporated to dryness (60° C., 12 mbar) and the resulting residue subjected to chromatography over a Strata® SI-1 silica cartridge (10 g Giga™ Tube), eluting successively with 1%, 2%, 5% and 10% v/v MeOH/DCM. Fractions containing the product were combined and evaporated. The resulting yellow waxy solid (58 mg) was found to contain target material and para-nitrophenol (ca. 1:1) ratio by .sup.1H NMR analysis. The chromatography was therefore repeated over a Strata® SI-1 silica cartridge (5 g Giga™ Tube), eluting successively with 50% v/v EtOAc/light petroleum, EtOAc and 1% v/v MeOH/DCM. Fractions containing the product were again combined and evaporated to afford 2-(2-chloro-1H-benzo[d]imidazol-1-yl)-N-(pyridin-3-yl)acetamide (31 mg; ca. 85-90% mass purity).

[0200] Step 4. The 2-(2-chloro-1H-benzo[d]imidazol-1-yl)-N-(pyridin-3-yl)acetamide (31 mg) prepared in the preceding step was taken up in anhydrous THF (2.5 mL) and transferred under argon into a flame-dried, heavy-walled, sealable tube fitted with a magnetic stir bar. Benzylamine (97 μL, 0.89 mmol) was added. The tube was sealed and the mixture heated at 120° C. with stirring. After 24 h the mixture was cooled and evaporated to afford a residue that was subjected to chromatography over a Strata® SI-1 silica cartridge (5 g Giga™ Tube), eluting successively with 0.5%, 2% and 5% v/v MeOH/DCM. Fractions containing the product were combined and evaporated to afford a residue (34 mg). The residue was triturated with CHCl.sub.3/Et.sub.2O, collecting the solid, to afford 2-(2-(benzylamino)-1H-benzo[c]imidazol-1-yl)-N-(pyridin-3-yl)acetamide (SC632) (17 mg) as a colourless powder: δ.sub.H (300 MHz, CD.sub.3CN) 8.86 (1H, br s), 8.81 (1H, d, J 2.5), 8.35-8.39 (1H, m), 8.08 (1H, dd, J 4.7 and 1.4), 8.05 (1H, br s), 7.31-7.34 (1H, m), 7.10-7.23 (6H, m), 6.94-7.04 (2H, m), 4.88 (2H, s), 4.35 (2H, d, J 6.0). The following compounds were prepared according to the methods outlined above.

By Route 1:

[0201] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(pyridin-2-yl)acetamide (SC938) [0202] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(pyridin-4-yl)acetamide (SC882) [0203] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(o-tolyl)acetamide (SC498) [0204] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(m-tolyl)acetamide (SC386) [0205] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(p-tolyl)acetamide (SC274) [0206] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(5-methylisoxazol-3-yl)acetamide (SC332) [0207] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(3-methylisoxazol-5-yl)acetamide (SC330) [0208] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(2-methoxyphenyl)acetamide (SC162) [0209] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(3-methoxyphenyl)acetamide (SC106) [0210] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(4-methoxyphenyl)acetamide (SC218) [0211] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(2-methoxyethyl)acetamide (SC050) [0212] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(3-methoxypropyl)acetamide (SC399) [0213] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(pyrazin-2-yl)acetamide (SC119) [0214] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(tetrahydrofuran-3-yl)acetamide (SC007) [0215] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(2-isopropoxyethyl)acetamide (SC951) [0216] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(2-ethoxyethyl)acetamide (SC839) [0217] (S)-2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(1-methoxypropan-2-yl)acetamide (SC727) [0218] (R)-2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(1-methoxypropan-2-yl)acetamide (SC321) [0219] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-((tetrahydrofuran-2-yl)methyl)acetamide (SC943) [0220] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(2-(2-methoxyethoxy)ethyl)acetamide (SC831) [0221] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(2-hydroxyethyl)acetamide (SC705) [0222] N-benzyl-2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)acetamide (SC649) [0223] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-cyclopentylacetamide (SC265) [0224] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-butylacetamide (SC977) [0225] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(2-propoxyethyl)acetamide (SC921) [0226] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(pyridin-2-ylmethyl)acetamide (SC153) [0227] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(furan-2-ylmethyl)acetamide (SC537) [0228] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(2-methoxybenzyl)acetamide (SC607) [0229] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(isoxazol-4-yl)acetamide (SC985) [0230] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(3,4-dimethylisoxazol-5-yl)acetamide (SC495) [0231] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(2-methoxyethyl)-N-methylacetamide (SC929) [0232] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(1,3-dimethyl-1H-pyrazol-5-yl)acetamide (SC105) [0233] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(2-(trifluoromethoxy)ethyl)acetamide (SC313) [0234] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-1-morpholinoethan-1-one (SC489) [0235] N-(2-methoxyethyl)-2-(2-methyl-1H-benzo[d]imidazol-1-yl)acetamide (SC666) [0236] 2-(2-(benzylthio)-1H-benzo[d]imidazol-1-yl)-N-(2-methoxyethyl)acetamide (SC697) [0237] N-(2-methoxyethyl)-2-(2-phenethyl-1H-benzo[d]imidazol-1-yl)acetamide (SC352) [0238] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(2-(methylamino)-2-oxoethyl)acetamide (SC257) [0239] methyl (2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)acetyl)glycinate (SC610) [0240] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N,N-bis(2-methoxyethyl)acetamide 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N,N-bis(2-methoxyethyl)acetamide (SC817)2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(cyanomethyl)acetamide (SC933) 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(2-phenoxyethyl)acetamide (SC201) [0241] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(2-methylthiazol-5-yl)acetamide (SC377) [0242] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(5-methyl-1,3,4-oxadiazol-2-yl)acetamide (SC408) [0243] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(1,3,4-thiadiazol-2-yl)acetamide (SC033) [0244] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(5-methyl-1,2,4-oxadiazol-3-yl)acetamide (SC554) [0245] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(3-methyl-1,2,4-oxadiazol-5-yl)acetamide (SC585) [0246] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(2-methyl-2H-tetrazol-5-yl)acetamide (SC761) [0247] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(1-methyl-1H-tetrazol-5-yl)acetamide (SC271)

By Route 2:

[0248] tert-butyl 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)acetate (SC343) [0249] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)acetic acid (SC287)

By Route 3:

[0250] N-benzyl-1-(2-methoxyethyl)-1H-benzo[d]imidazol-2-amine (SC231) [0251] N-benzyl-1-(2-(benzyloxy)ethyl)-1H-benzo[d]imidazol-2-amine (SC175) [0252] N-benzyl-1-(2-(2-methoxyethoxy)ethyl)-1H-benzo[d]imidazol-2-amine (SC873)

By Route 4:

[0253] 2-(2-(benzyl(methyl)amino)-1H-benzo[d]imidazol-1-yl)-N-(5-methylisoxazol-3-yl)acetamide (SC881) [0254] 2-(2-(benzyl(methyl)amino)-1H-benzo[d]imidazol-1-yl)-N-(2-methoxyethyl)acetamide (SC097)

By Route 5:

[0255] 2-(2-amino-1H-benzo[d]imidazol-1-yl)-N-(2-methoxyethyl)acetamide (SC593) [0256] 2-(2-(isobutylamino)-1H-benzo[d]imidazol-1-yl)-N-(2-methoxyethyl)acetamide (SC769)

By Route 6:

[0257] 2-(2-((4-chlorobenzyl)amino)-1H-benzo[d]imidazol-1-yl)-N-(2-methoxyethyl)acetamide (SC719) [0258] N-(2-methoxyethyl)-2-(2-((pyridin-2-ylmethyl)amino)-1H-benzo[d]imidazol-1-yl)acetamide (SC209) [0259] (R)—N-(2-methoxyethyl)-2-(2-((1-phenylethyl)amino)-1H-benzo[d]imidazol-1-yl)acetamide (SC481) [0260] (S)—N-(2-methoxyethyl)-2-(2-((1-phenylethyl)amino)-1H-benzo[d]imidazol-1-yl)acetamide (SC041) [0261] N-(2-methoxyethyl)-2-(2-(phenylamino)-1H-benzo[d]imidazol-1-yl)acetamide (SC865) [0262] N-(2-methoxyethyl)-2-(2-(((tetrahydro-2H-pyran-4-yl)methyl)amino)-1H-benzo[d]imidazol-1-yl)acetamide (SC657) [0263] N-(2-methoxyethyl)-2-(2-((pyridin-3-ylmethyl)amino)-1H-benzo[d]imidazol-1-yl)acetamide (SC520) [0264] 2-(2-((3-chlorobenzyl)amino)-1H-benzo[d]imidazol-1-yl)-N-(2-methoxyethyl)acetamide (SC383) [0265] 2-(2-((2-chlorobenzyl)amino)-1H-benzo[d]imidazol-1-yl)-N-(2-methoxyethyl)acetamide (SC529) [0266] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(2-(dimethylamino)ethyl)acetamide (SC625)

By Route 7:

[0267] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(pyrimidin-5-yl)acetamide (SC425) [0268] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(pyridin-3-yl)acetamide (SC632) [0269] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(pyridazin-3-yl)acetamide (SC809) [0270] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(1-methyl-1H-pyrazol-5-yl)acetamide (SC369) [0271] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(1-methyl-1H-pyrazol-4-yl)acetamide (SC545) [0272] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(1-methyl-1H-pyrazol-3-yl)acetamide (SC576) [0273] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(isoxazol-3-yl)acetamide (SC722) [0274] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(pyridazin-4-yl)acetamide (SC753)

By Other Routes

[0275] (2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)acetyl)glycine (SC433)—By hydrolysis of SC610 [0276] 2-methoxyethyl 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)acetate (SC089)—Route 1, adapted with 2-methoxyethyl 2-chloroacetate [0277] 2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)-N-(2-(dimethylamino)ethyl)acetamide (SC145) Route 1, adapted with 2-chloro-N-(2-methoxyethyl)propanamide [0278] N-(2-(2-(benzylamino)-1H-benzo[d]imidazol-1-yl)ethyl)-2-methoxyacetamide (SC683)—Route 1, adapted to start with alkylation using 2-(2-bromoethyl)isoindoline-1,3-dione followed by hydrazine treatment to liberate the primary amine and N-acylation with methoxyacetyl chloride. [0279] 2-(2-(benzylamino)-1H-imidazol-1-yl)-N-(2-methoxyethyl)acetamide (SC513)—Route 1, adapted to start from 2-(benzylamino)imidazole, which was prepared by reductive amination of the known precusor, 1-trityl-1H-imidazol-2-amine, with benzaldehyde and sodium triacetoxyborohydride followed by detritylation with trifluoroacetic acid.
The structural formula of the above compounds are illustrated below.

##STR00016##

R=

[0280] ##STR00017## ##STR00018## ##STR00019## ##STR00020## ##STR00021## ##STR00022## ##STR00023##

R=

[0281] ##STR00024## ##STR00025## ##STR00026## ##STR00027## ##STR00028## ##STR00029## ##STR00030## ##STR00031## ##STR00032##

Biology

Reagents and Materials

[0282] StemPro® hESC SFM Human Embryonic Stem Cell Culture Medium (Life Technologies)

50 mM 2-Mercaptoethanol (Life Technologies)

[0283] bFGF (R&D)

PBS (−/−) (Life Technologies)

[0284] Fibronectin, Human plasma (Merck Millipore)

TrypLE Select (1×) (Life Technologies)

[0285] H1 and H9 hESC (Thomson et al, 1998)
RC9 (Roslin cells LTD)
iPSC NMF-iPS6 (Sullivan et al, 2010)
6 well culture plates (Corning)
15 ml conical bottomed tubes (Corning)
Serological pipettes (Corning)
Stempro® EZPassage disposable passage tool (Life Technologies)
SSEA4 antibody (BD biosciences)
SSEA1 antibody (BD biosciences)
Smooth muscle actin antibody (Sigma)
Alpha fetoprotein antibody (Abcam)
PAX 6 antibody (Sigma)
α-tubulin antibody (Sigma)
pMLC antibody (Sigma)
Nitrocellulose membranes (Sigma)
4-12% Bis-Tris midi gel (Life Technologies)
MES buffer (Life Technologies)
Transfer buffer (Life Technologies)
Laemmli buffer

Phospho-blocker (Cambridge Bioscience)

[0286] Tris buffered saline with tween (Sigma)
Enhanced Chemiluminescence (ECL) reagent (Thermo Scientific)

Heraeus Multifuge 3 SR (Thermo Scientific)

[0287] FACS Canton II (BD biosciences)

XCell SureLock® Mini-Cell (Life Technologies)

Y27632 (Tocris Bioscience)

[0288] Enzymatic passage of hPSC methodology
1. 24 hrs prior to passage (around 70-80% confluence), supply 1 well of near confluent hPSC with 2 ml StemPro supplemented with 30 μM SC332 (done during usual daily feed).
2. At point of passage, remove media and wash cells 1× in PBS (−/−).
3. Add 1.5 ml of TrypLE Select (1×) to well and return to incubator for 2-5 mins to dissociate to single cells.
4. Add 1.5 ml of StemPro to deactivate TrypLE Select and pipette gently using a 5 ml serological pipette (to dissociate any remaining clumps).
5. Transfer cells to 15 ml conical bottomed tube and centrifuge for 3 mins at 300 g.
6. Aspirate as much supernatant as possible and resuspend cell pellet by gently flicking.
7. Resuspend cells in PBS (−/−) and centrifuge again for 3 mins at 300 g.
8. Aspirate PBS (−/−) and resuspend cells in 1 ml of StemPro. Gently pipette using serological pipette.
9. Perform a cell count and replate hPSC at a density of 5×10.sup.5 cells per well of a pre-coated 6 well plate. Top up to 2 mls total with StemPro supplemented with 30 μM SC332. Wells are pre-coated with 0.3 mg/ml fibronectin (recombinant vitronectin fragment can also be used).
10. Return cells to incubator and supply with fresh StemPro on a daily basis.
11. 24 hrs prior to subsequent passages (around 70-80% confluence), supply 1 well of PSC with 2 ml StemPro supplemented with 30 μM SC332 (done during usual daily feed).
NB SC332 can be used transiently as described above or can be added to culture medium continuously (still at 30 μM).

Results

[0289] Human pluripotent stem cells (hPSC) were either treated with SC332, Y27632, or left untreated, before being enzymatically passaged. In all experiments untreated cells had an equivalent volume of DMSO added as a carrier control, with cell survival being assayed 24 hrs post passage. HPSC treated with either SC332 or Y27632 had average cell survival that was significantly higher than that observed in untreated controls (P=<0.001) (FIG. 1). In particular, the hESC lines H1, H9 and RC9 and the hiPSC NMF-iPSC6 had cell survival of 72% (±2.9), 73% (±2.8), 69% (±1.9) and 74% (±2.6) respectively when treated with SC332. In the same cell lines, Y27632 resulted in cell survival of 72% (±3.6), 73% (±2.1), 71% (±1.2) and 74% (±4.0) and DMSO alone resulted in survival of 18% (±2.2), 18% (±2.4), 15% (±3.0) and 19% (±2.4) respectively. This result was consistent in both hESC and iPSC. Data shown is the mean survival ±SEM, n=3.

[0290] These results confirm that compound SC332 supports the enzymatic passage of hPSC as effectively as the well-established pro-survival compound Y27632.

[0291] During long term exposure experiments, compound SC332 was shown to support enzymatic passage of hPSC as effectively as Y27632 for at least 30 consecutive passages. In addition to this, long term exposure to compound SC332 had no detrimental effect on the karyotypic stability of hPSC (FIGS. 2 and 3).

[0292] These data show that compound SC332 is a viable alternative to Y27632 as a tissue culture reagent that can be utilised over multiple passages.

[0293] It is important than any reagent used in the general maintenance of hPSC does not negatively impact upon the stem cell identity. In order to confirm this, flow cytometric analysis was performed on cells treated over multiple passages with SC332. FIG. 4 shows the % of SSEA4 (stage-specific embryonic antigen 4) and SSEA1 (stage-specific embryonic antigen 1) positive cells after each subsequent 5 passages. SSEA4 is a cell surface marker present on all undifferentiated hPSC, whereas SSEA1 is only present on differentiated cells. These markers have been routinely used as positive and negative markers of pluripotency in hPSC since their initial isolation (Thomson et al, 1998). As can be seen, consistent enzymatic passage supported by either SC332 or Y27632 has no effect on the expression level of the pluripotency marker SSEA4, with both remaining >90% positive. Furthermore the differentiation marker SSEA1 remains consistently low (expressed on <5% of cells). This shows that SC332 treated cells retain a cell surface marker profile consistent with that expected of hPSC, and comparable to that achieved with Y27632 treatment.

[0294] The differentiation capacity of hPSC after treatment with SC332 was also assessed. HPSC (hESC cell line H1 and NMF-iPS6) that had been enzymatically passaged with either SC332 or Y27632 for 30 consecutive passages were passively differentiated alongside mechanically maintained cells using a mixture of embryoid body based suspension culture and adherent culture. Differentiated cells were subsequently fixed and stained for markers from each of the three germ layers. Long term exposure to either survival compound did not block the differentiation towards mesoderm (smooth muscle actin; SMA), endoderm (alpha fetoprotein; AFP) or ectoderm (paired box protein; PAX6). As expected, untreated mechanically passaged control cells, included as a positive control, were also able to differentiate into each of the three germ layers (FIG. 5).

ROCK Independence

[0295] To reaffirm that SC332 did not inhibit ROCK or the closely related PRK2, the compound was rescreened at the Dundee International Centre for Kinase Profiling at both the standard 10 μM and optimum 30 μM concentrations. This screen utilises a radioactive filter-binding assay (33P-ATP) to assess the effect of compounds on kinase activity. Results confirmed that SC332 had no inhibitory effect on ROCK2 at a concentration of 10 μM (96% activity. SC332 also had very limited effect on the closely related PRK2, showing 76% and 98% activity when used at 10 μM and 30 μM respectively (FIG. 6).

[0296] It has been suggested that the cause of dissociation induced apoptosis of hPSC is the hyperphosphorylation of myosin light chain (MLC) and that the protective activity of Y27632 is to prevent this phosphorylation (Chen et al, 2010; Ohgushi et al, 2010 and Walker et al, 2010), therefore western blot analysis was performed to test whether SC332 shares this biochemical effect. The data shown in FIGS. 7A and B shows that, as expected, the untreated cells had significantly higher levels of pMLC 15 mins post-dissociation and that Y27632 blocked the increase (P=<0.01), however cells treated with SC332 showed high levels of pMLC equivalent to those in untreated cells (P=>0.05). The higher levels of pMLC were maintained in untreated and SC332 treated cells compared to Y27632 treated cells at later time points, with SC332 vs Y27632 being significantly different at 30 mins (P=<0.05), 45 mins (P=<0.05) and 1 hr (P=<0.05). Although untreated cells did not reach significance versus Y27632 treated cells at these subsequent time points, the trend was towards increased levels of pMLC in untreated hPSC.

[0297] These data from untreated and Y27632 treated cells are consistent with those observed by others (Chen et al, 2010; Ohgushi et al, 2010), confirming that in response to dissociation there is a sudden increase in phosphorylated MLC and that treatment with Y27632 is able to prevent this hyperphosphorylation. Importantly though, treatment with SC332 did not inhibit the phosphorylation of MLC strongly suggesting that SC332 does not share the same mechanistic downstream target reported for Y27632. These findings also clearly uncouple the inhibition of MLC hyperphosphorylation and hPSC cell survival, supporting the hypothesis that there is an additional novel pro-survival pathway in these cells that may be elucidated using SC332.

[0298] Summary

[0299] Compounds of the invention have been found to promote survival of enzymatically dissociated hPSC. Kinase assays have confirmed that compounds of the invention do not inhibit ROCK, the reported mechanistic target of Y27632. Furthermore, biochemical analysis has shown that compounds of the invention do not produce a pro-survival effect via inhibition of MLC phosphorylation. Compounds of the invention can be used to support the passage of hPSC for at least 30 consecutive passages whilst retaining expression of pluripotency markers and normal karyotypic stability. Furthermore, cells treated with compounds of the invention retain multi-lineage differentiation capacity. Compounds of the invention are novel stem cell survival compounds with significant potential for commercial exploitation as well offering an alternative means to study the pro-survival pathways involved in dissociation induced apoptosis of hPSC.

REFERENCES

[0300] Watanabe, K., et al., Nat Biotechnol, 2007. 25(6): p. 681-6. [0301] Ohgushi, M., et al., Cell Stem Cell, 2010. 7(2): p. 225-39. [0302] Ohgushi, M. and Y. Sasai, Trends Cell Biol, 2011. 21(5): p. 274-82. [0303] Xu, Y., et al., Proc Natl Acad Sci USA, 2010. 107(18): p. 8129-34. [0304] Andrews, P. D., et al., Biochem J, 2010. 432(1): p. 21-33. [0305] Chen, V. C., et al., Stem Cell Res, 2012. 8(3): p. 388-402. [0306] Amit, M., et al., Stem Cell Rev, 2010. 6(2): p. 248-59. [0307] Singh, H., et al., Stem Cell Res, 2010. 4(3): p. 165-79. [0308] Zweigerdt, R., et al., Nat Protoc, 2011. 6(5): p. 689-700. [0309] Larijani, M. R., et al., Stem Cells Dev, 2011. 20(11): p. 1911-23. [0310] Burton, P., et al., Biochem J, 2010. 432(3): p. 575-84. [0311] Yung, S., et al., Hum Mol Genet, 2011. 20(24): p. 4932-46. [0312] Ken K C, et al, ISSCR 2014, Abstract W2014 [0313] Krawetz, R. J., et al., PLoS One, 2011. 6(11): p. e26484.