1,5-Disubstituted benzimidazoles that direct cardiomyocyte differentiation from mouse embryonic stem cells

Accepted Manuscript 1,5-Disubstituted benzimidazoles that direct cardiomyocyte differentiation from mouse embryonic stem cells Karl J. Okolotowicz, Paul Bushway, Marion Lanier, Cynthia Gilley, Mark Mercola, John R. Cashman PII: DOI: Reference:

S0968-0896(15)00657-4 http://dx.doi.org/10.1016/j.bmc.2015.07.073 BMC 12502

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

9 June 2015 23 July 2015 30 July 2015

Please cite this article as: Okolotowicz, K.J., Bushway, P., Lanier, M., Gilley, C., Mercola, M., Cashman, J.R., 1,5Disubstituted benzimidazoles that direct cardiomyocyte differentiation from mouse embryonic stem cells, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.07.073

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Graphical Abstract

1,5-Disubstituted benzimidazoles that direct cardiomyocyte differentiation from mouse embryonic stem cells

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Karl J. Okolotowicza, ‡ , Paul Bushwayb, ‡, Marion Laniera, Cynthia Gilleya, Mark Mercolab, John R. Cashmana a) Human BioMolecular Research Institute, 5310 Eastgate Mall, San Diego, CA 92121; b) The Sanford Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037 and Department of Bioengineering, University of California, San Diego, 9500 Gilman Drive MC 0695 La Jolla, CA 92093-0695

Bioorganic & Medicinal Chemistry j o ur n al h o m e p a g e : w w w . e l s e v i e r . c o m

1,5-Disubstituted benzimidazoles that direct cardiomyocyte differentiation from mouse embryonic stem cells Karl J. Okolotowicza,∗,‡, Paul Bushwayb,‡, Marion Laniera, Cynthia Gilleya, Mark Mercolab and John R. Cashmana a

Human BioMolecular Research Institute, 5310 Eastgate Mall, San Diego, CA 92121, USA The Sanford Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037 and Department of Bioengineering, University of California, San Diego, 9500 Gilman Drive MC 0695 La Jolla, CA 92093-0695, USA

b

AR T IC LE IN F O

A B S TR A C T

Article history: Received Received in revised form Accepted Available online

Cardiomyopathy is the leading cause of death worldwide. Despite progress in medical treatments, heart transplantation is one of the only current options for those with infarcted heart muscle. Stem cell differentiation technology may afford cell-based therapeutics that may lead to the generation of new, healthy heart muscle cells from undifferentiated stem cells. Our approach is to use small molecules to stimulate stem cell differentiation. Herein, we describe a novel class of 1,5-disubstituted benzimidazoles that induce differentiation of stem cells into cardiac cells. We report on the evaluation in vitro for cardiomyocyte differentiation and describe structure activity relationship results that led to molecules with drug-like properties. The results of this study show the promise of small molecules to direct stem cell lineage commitment, to probe signaling pathways and to develop com pounds for the stimulation of stem cells to repair damaged heart tissue.

Keywords: Cardiomyogenesis Embryonic stem cells Benzimidazoles Cardiomyocytes High Content Screen

——— ∗ Corresponding author. Tel.: +1-858-458-9305; fax: +1-858-458-9311; e-mail: [email protected] ‡

Authors contributed to the manuscript equally.

2009 Elsevier Ltd. All rights reserved.

1. Introduction More than 5 million Americans are estimated to suffer from heart failure1. Costs due to hospitalization, therapies and loss of productivity are estimated to be more than $37.2 billion/year1. Current therapies for heart disease do little to regenerate damaged heart muscle. The adult human heart has a limited regenerative capability but regeneration is too low to compensate for loss due to ischemic or non-ischemic heart disease2-5. Administration of a small molecule stem cell (SC) differentiation agent to stimulate the generation of heart cells may prove useful for heart repair6. For example, we have reported on the ability of a class of 1,5-

Figure 1. Screening “hit”, compound 1. dihydropyridines that inhibit TGF-β signaling and a class of novel Wnt inhibitors to stimulate cardiomyogenesis (CM)7-10. In addition, other groups have reported on small molecule inducers of cardiomyogenesis11-13. Herein, we describe potent and selective 1,5-disubstituted benzimidazoles that represent a new class of small molecules that instruct stem cell differentiation to

cardiomyocytes. A commercially available library of 13,360 chemically diverse compounds 14 was screened in a high content cell-based assay employing mouse embryonic stem cells (mESCs) to identify small molecules that stimulated SC differentiation. The cellbased screen identified four chemically distinct “hits”. Of these “hits,” two compounds were robust and reproducible inducers of cardiomyogenesis. One of the “hits” was reported previously8, 9. Herein, we focus on the second of these “hits”, (i.e., 1-phenyl-5(4-methylphthalazinyl)aminobenzimidazole, 1) (Figure 1). The “hit” from the screen (i.e., 1) had poor drug-like properties including large predicted lipophilicity and an unfavorable PSA value (i.e., predicted cLogP = 5.44, PSA = 52.35 Å2)15, unfavorable water solubility (i.e., less than 1 mg/ml) that mitigated against 1 as a drug candidate. Accordingly, elaboration of a more potent and drug-like candidate from the “hit” to improve upon pharmaceutical properties of “hit” 1 was a goal of this work. As shown in Figure 1, molecular dissection of 1 showed that the molecule could be divided into three regions including: a benzimidazole core, (region A), aryl substitution at the 1-position of the benzimidazole core (i.e., region B), and a phthalazine group at the 5-position of the benzimidazole core (i.e., region C). Structure-activity relationship (SAR) studies of derivatives of 1 using Stem Cell Dynamic Medicinal Chemistry8 afforded a series of substituted benzimidazoles that stimulated differentiation of cardiomyocytes from mESCs.

Scheme 3. Synthesis of compounds 68-74 via HartwigBuchwald coupling. Reagents and conditions: (a) R1PhNH2, THF, 25 °C, 15 h; (b) sodium dithionite, Ethanol/H2O, 70 °C, 1 h; (c) trimethylorthoformate, cat. p-toluenesulfonic acid, 110 °C, 1 h; (d) R6NH2, Pd(OAc)2, Cs2CO3, rac-BINAP in toluene or Xantphos in dioxane, 100 °C, 15 h.

Scheme 1. Synthesis of benzimidazoles 1, 30-48, and 75. Reagents and conditions: (a) R1NH2, THF, r.t., 15 h. (b) H2, Pd/C, ethyl acetate, 15 h; (c) formic acid 110 °C, 1 h; (d) R2Cl, iPrOH, 150 °C, 1 h; (e) R3 CHO, borane-pyridine complex, ethanol, 25 °C, overnight; (f) R4COCl, TEA, DCM, 25 °C, overnight.

Scheme 4. Synthesis of indole 78. Reagents and conditions: (a) PhB(OH)2, Cu(OAc) 2, DIPEA, DCM, 25 °C, 15 h; (b) sodium dithionite, Ethanol/H2O, 70 °C, 1 h; (c) 1-chloro-4methylphthalazine, iPrOH, 90 °C, 2 h.

Scheme 2.

Synthesis of 7-N-benzimidazole 65. Reagents

Scheme 5. Synthesis of 2-aminobenzimidazoles 82 and 83 and benzotriazole 85. Reagents and conditions: (a) phosgene iminium chloride, TEA, DCM, 25 °C, 15 h; (b) cyanogen

2. Results and Discussion 2.1. Chemistry Benzimidazoles 1, 30-48, and 75 were prepared in a sequence of four steps from commercially available 2,4-dinitro-haloarene 2 (Scheme 1). Using a modified procedure from the literature16-18, one equivalent of a desired amine, R1NH2, was combined with 2 at room temperature in tetrahydrofuran to give substituted anilines 3-15. Anilines 3-15 were hydrogenated via palladium on carbon to afford the diamino-aniline intermediate that was used without further purification or characterization. Cyclization to the

benzimidazole core 16-29 was accomplished using formic acid in hydrochloric acid19 at 110 °C. The substituent at the 5-amino group was installed either via N-arylation (1, 30-48, 75), reductive amination (49-58) or acylation (59, 60) of intermediates 16-29 (Scheme 1). Benzimidazole 65 incorporated a pyridine in the benzimidazole core and was prepared following similar protocols starting with commercially available 2,4-dinitrohaloarene 61 (Scheme 2). The desired amine was combined with 61 at room temperature in tetrahydrofuran to give intermediate 62. Compound 62 was reduced using sodium dithionite in aqueous ethanol to give the triamine 6320. Cyclization to the benzimidazole core 64 was accomplished using formic acid in

Figure 2 (A) 13,360 compounds were tested at two concentrations (1 and 5 µg/mL). Mouse ESCs in growth media without Leukemia Inhibitory Factor (LIF) were applied to 384 well microtiter plates as a monolayer on day 0 and compounds were administered on days 2 and 4. On days 6 and 8 the cells were fed growth media. Differentiated cell cultures were fixed on day 10 with paraformaldehyde. (B) Nine field image mosaics from representative wells treated with DMSO (vehicle control; top row) or the benzimidazole 1 (bottom row). Intense fluorescence of eGFP expressed from the αMHC promoter was apparent in the cells treated with 1. Only sparse puncta were apparent in the DMSO-treated cells (center column). Nuclear DAPI staining (left column) indicated the relative density of the culture was similar in the 1- and DMSO-treated conditions. Non-specific red (nsRED) images indicated a measure of broad-spectrum auto fluorescence and were used for subtraction of fluorescent artifacts. There was no significant increase in this non-specific fluorescent signal in 1-treated cells compared to DMSO-treated cells (right column). hydrochloric acid19 at 110 °C. Finally, 1-chloro-4-methylphthalazine was condensed with 64 to give 65. The use of cross coupling reactions were used for benzimidazoles that incorporated various 5-amino-pyrimidine (69), -pyrazine (70), -pyridine (71, 72) or -aryl substituents (73, 74, 68), starting with 5-bromobenzimidazoles 67a or 67b (Scheme 3). Compounds 67a and 67b were obtained in three steps starting from 4-bromo-1-fluoro-2-nitrobenzene, 66. Nucleophilic aromatic substitution of 66 with a substituted aniline gave a nitroaniline intermediate that was reduced with sodium dithionite and cyclized to give benzimidazoles 67a and 67b. Aryl and heteroaryl groups (i.e., R7) in compounds 68-74 were installed in good yield using Hartwig-Buchwald crosscoupling conditions21. In addition to benzimidazoles, we investigated the synthesis and testing of substituted indoles (Scheme 4). The synthesis of substituted indole 78 began with commercially available 5nitroindole 76 that was coupled with phenylboronic acid under Hartwig-Buchwald conditions22, 23 to afford 1-phenyl-indole 77. Compound 78 was prepared in two steps, first reduction of the nitro group of 77 using sodium dithionite, followed by nucleophilic aromatic substitution with 1-chloro-4methylphthalazine. Other modifications to the benzimidazole core included modifications to the 2-position. This included substitution or replacement of a benzimidazole moiety with a benzotriazole group (Scheme 5). To prepare 2-aminobenzimidazoles, compound 79 was treated with either phosgene iminium chloride to give the 2-dimethylamino-benzimidazole 80 or cyanogen bromide to give the 2-amino-benzimidazole 8124, 25. Hydrogenation of 80 or 81 followed by introduction of the methylphthalazine ring system as above afforded 82 and 83. The benzotriazole analog 84 was obtained when 79 was treated with isoamyl nitrite26. Hydrogenation of 84 followed by introduction of the methylphthalazine ring system gave 85. 2.2. Biology The high content screen used herein was based on an assay previously described12. mESCs were stably transfected with an enhanced green fluorescent protein (eGFP) construct regulated by a myosin heavy-chain alpha gene promoter (i.e., αMHC-eGFP)12. Early stage mouse cardiomyocytes express high amounts of this myosin subunit necessary for normal function of the cardiac contractile apparatus. Thus, αMHC (Myh6) expressed in

mesoderm-derived cardiac muscle from day 10-differentiated mESC cultures was used as a biomarker of differentiated cardiomyocytes (i.e., see Supporting Figure S1 for a cell fate map). Other biological markers along the cardiomyocyte differentiation pathway were examined. These included; Bra and GSC for the mesendoderm linage; Cer1 and Sox17 for the endoderm linage; and Cdx2 and MesP1 for the mesoderm linage6, 27, 28 . For the initial screen, compounds were administered to mESCs in fortified media during days 2-6 (see Methods). After day 6, differentiated cells were reseeded and expanded in normal media (Figure 2a). Fold-change in cardiomyogenesis was determined on day 10 by quantification of αMHC-eGFP fluorescence levels in cell culture. Along with the quantification of αMHC-eGFP fluorescence, additional biological markers were examined with compound treatment from days 1 – 1029. After the initial primary screen, the time window of compound administration was restricted to days 2-4 for the SAR studies. This time window corresponded to the time point where “hit” 1 was most potent. Treatment of mESCs with functionally potent benzimidazoles resulted in widespread αMHC-driven eGFP expression within the cell culture well (Figure 2b). An automated image capture and custom image quantification algorithm was employed in high-content screens to score lineage bias as an indicator of eGFP expression30 (see Methods). Immunohistochemistry for αActinin, another marker of the cardiac contractile apparatus, co-localized with αMHC-eGFP expression. This verified that eGFP expression was associated with cardiomyocyte formation. Differentiation of mESCs treated with 1 or vehicle control were monitored for 12 days of development. On day 12, the cultures were fixed in paraformaldehyde and immunostained with mouse monoclonal anti-αActinin followed by a cocktail of 2-(4-amidinophenyl)-1H -indole-6-carboxamidine (DAPI) and anti-mouse Alexa568 (Figure 3a). DAPI, eGFP, and Alexa568 images were collected and quantified by image analysis. After administration of 6 µM compound 1 to the cell cultures at days 2 and 4, αMHC-eGFP and αActinin Alexa568 showed a 20-fold increase over vehicle control (integrated intensity of αMHC-eGFP and αActininAlexa568 fluorescence in control samples were 4.2% and 5.0%, respectively, of that of 1-treated samples (Figure 3b). A few αActinin foci appeared to lack eGFP, possibly caused by leaching out of eGFP during cell permeabilization for

Figure 3. αMHC-eGFP cardiomyocyte expression from mESCs treated with 1 was confirmed by fluorescence immunohistochemical staining of cardiomyocyte-specific αActinin. (A) DAPI staining (left; gray) shows the relative confluence of the culture at day 10. Widespread αMHC-eGFP expression (center; green) was apparent after treatment with compound 1. αActinin immunostaining (right; red) co-localized with αMHC-eGFP fluorescence. (B) Quantification of αMHCeGFP and αActinin–Alexa568 showed similar trends in response to DMSO vehicle and compound 1 with a 10-fold and 20-fold increase (at 3 µM and 6 µM concentration, respectively) in integrated intensity of eGFP signal over DMSO vehicle control. (C) An overlay of αMHC-eGFP (green) and αActinin (red) fluorescence showed co-localized expression of both cardiomyocyte markers in yellow. immunostaining (Figure 3a,c). For this reason, αMHC-eGFP fluorescent intensity likely represented a conservative estimate of cardiomyocyte conversion. As further evidence that the αMHCeGFP foci contained functional cardiomyocytes, the cells were imaged live at day 10 and the image showed functionally active and coherent contractile cell beating, a property unique to cardiomyocytes (See Supporting Movie). Taken together, the increase in αMHC-eGFP expression in mESC cultures by day 10 indicated that compound 1 significantly enhanced cardiomyocyte differentiation 2.3. SAR discussion Although “hit” 1 was moderately potent in the cardiomyogenesis assay, 1 had several drawbacks as a drug-like molecule: compound 1 had poor water solubility and was lipophilic with a ClogP value of 5.44 and a tPSA value of 52.4. The synthesis of analogs of 1 focused on ways to improve the overall potency and water solubility of the molecule. To identify the regions of the molecule that contributed to potency, 1 was divided into three regions for modification: region A, the benzimidazole core; region B, the benzimidazole 1-subsituent; region C, the benzimidazole 5-substituent (Figure 1). A structure-activity relationship (SAR) of analogs of 1 was developed from results of testing and synthesis of 400 compounds. Herein, we describe the biological results of a select subset of these synthetic compounds. The results focused on the modification of the aryl groups with either electron-withdrawing groups or electron-donating groups as well as modulation of lipophilicity of benzimidazoles. The effect of modification at the 1-position of benzimidazole 1 (Region B, Figure 1) was investigated for potency on mESC differentiation to cardiomyocytes. Compared to unsubstituted 1, substitution of electron-donating groups on the aryl ring at the 1position of benzimidazole 1 increased cardiomyogenesis (Table 1). In contrast, substitution of electron-withdrawing groups led to compounds with diminished potency for cardiomyogenesis. For example, substitution with alkoxyphenyl groups (i.e., 30, 31 or 35), increased cardiomyogenesis by 4.3-, 18.6- and 10.0-fold, respectively. Hydroxyphenyl-substituted benzimidazole 37

increased cardiomyogenesis by 2.1-fold. By comparison, electron-withdrawing substituted benzimidazoles were generally less potent. For example, trifluoromethylphenyl-substituted benzimidazoles 33 and 36 afforded a 0.5 and 1.6 fold-change in cardiomyogenesis. Alkyl-substituted benzimidazoles such as isopropylphenyl (i.e., 34) or n-propylphenyl (i.e., 32) afforded a 4.4- and a 0.7-fold-change in cardiomyogenesis, respectively. These results suggested that addition of aryl-substituted alkyl

Table 1. Effect of R2 modification of region B of 1 on cardiomyogenesis in mESCs.

a

R2

CM Fold-changea

1

Ph

9.6 ± 1.1

30

2-OMe Ph

4.3 ± 0.8

31

3-OMe Ph

18.6 ± 7.2

32

4-nPr Ph

0.7 ± 0.3

33

3-CF3 Ph

0.5 ± 0.1

34

2-iPr Ph

4.4 ± 0.8

35

4-OMe Ph

10.0 ± 1.9

36

4-CF3 Ph

1.6 ± 0.5

37

2-OH

2.1 ± 0.4

38

Me

1.6 ± 0.6

39

Cyclopropyl

0.9 ± 0.3

40

Cyclohexyl

3.3 ± 0.8

41

2-Py

2.2 ± 0.4

42

4-THP

1.6 ± 0.3

43

4-Me piperidine

4.4 ± 0.8

Fold-change in cardiomyogenesis compared to vehicle (i.e., DMSO)treated cells.

groups at the 1-position of benzimidazoles could afford moderately potent cardiomyogenesis agents. However, these lipophilic substituents did not improve the solubility of 1 and were less potent than benzimidazoles with methoxy substituents at the 2- and 3-position of the aryl ring. Accordingly, addition of lipophilic substituents to the 1-position of benzimidazole was not explored further. Replacing the aryl group in region B with a nonaromatic moiety (i.e., cyclohexyl-substituted 40) increased cardiomyogenesis 3.3-fold. In contrast, 1-substitution of the benzimidazole with smaller alkyl groups such as methyl (i.e., 38) or cyclopropyl (i.e., 39) gave only a 1.6-fold and 0.9-fold change in cardiomyogenesis, respectively. No overall improvement in cardiomyogenesis was observed by introduction of increased polarity into region B by the addition of heteroatoms (e.g., 2pyridine 41, 4-tetrahydropyran-substituted, 42 or N-methyl 4piperidine, 43). Compounds 41, 42 and 43 increased cardiomyogenesis 2.2-, 1.6-, and 4.4-fold, respectively. These results showed that an aryl group with electron-donating substituents at the 2- or 3-position of Region B was necessary to facilitate an improvement in cardiomyogenesis potency. In contrast, addition of polarity to the ring either by the addition of pyridine or piperidine rings decreased cardiomyogenesis. Overall, the results indicated 2- and 3-methoxyaryl groups at region B improved cardiomyogenesis and were used at the 1position in subsequent studies. The effect of modification of the benzimidazole at the 5position (Region C, Figure 1) on compound potency for mESC differentiation was investigated by synthesizing and testing more than a hundred analogs. A representative number of benzimidazoles containing substituents at the 5-position were listed in Table 2 to illustrate the SAR. Modification of the phthalazine moiety was thus explored to probe the effects of heteroatoms and other ring systems in this region on mESC differentiation potency. Table 2 shows the results of the effects of substituents at region C of benzimidazole 1 on cardiomyogenesis in mESCs. “Hit” 1 contained a phthalazine ring tethered through a 5amino group to the benzimidazole core (i.e., region C, Figure 1). Modification of the phthalazine at the 4-position was explored by synthesis and testing of compounds 44 – 48. The results showed that small substituents including hydrogen (i.e., 44), chloro (i.e., 45) or methoxy (i.e., 46) increased cardiomyogenesis 13.7-, 6.7and 7.1-fold, respectively. Larger substituents (e.g., benzyl, 47 or phenyl, 48) did not improve cardiomyogenesis, because, compared to 1, they only increased cardiomyogenesis 2.0- and 1.0-fold, respectively. Replacing the phthalazine ring (region C, Figure 1) by a naphthalene ring (i.e., 68), afforded only a 3.1fold-increase in cardiomyogenesis. We conclude that at least one of the two nitrogen atoms in the phthalazine ring was essential for significant potency. Replacement of the phthalazine moiety with a 4-methylpyridazinyl group (i.e., 75) increased cardiomyogenesis 7.9-fold. This showed that smaller ring systems in region C of benzimidazole 1 could afford significant potency. To decrease the lipophilicity and improve the overall drug-like properties of benzimidazole 1, additional modification of region C was undertaken. Compared to 1 (cLogP = 5.44) the absence of the fused phenyl ring in region C from the phthalazine ring in 75 decreased the overall lipophilicity by more than one cLogP unit (4.42). Several additional nitrogen-containing monocyclic analogs (i.e., 69-72) as well as the simple phenyl analog (i.e., 73) were prepared and tested to investigate the necessity and/or position of the nitrogen atom in region C of 1. Introduction of the phenyl moiety (i.e., 73) in region C slightly decreased cardiomyogenesis (i.e., 0.7 fold-change). This result suggested that one or both of the nitrogen atoms in region C might be

Table 2. Effect of R3 modification of region C of 1 on cardiomyogenesis in mESCs R 2a

R 3b

CM Foldchangea

16

H

H

3.0 ± 0.4

44

3-OMe

Phthalazin-1-yl

13.7 ± 4.1

45

3-OMe

4-Cl-Phthalazin-1-yl

6.7 ± 1.5

46

3-OMe

4-MeO-Phthalazin-1-yl

7.1 ± 2.2

47

3-OMe

4-Bn-Phthalazin-1-yl

2.0 ± 0.9

48

3-OMe

4-Ph-Phthalazin-1-yl

1.0 ± 0.8

49

3-OMe

Ph-CH2CH2

1.4 ± 0.2

50

3-OMe

2-MeO-Ph-CH2

1.7 ± 0.3

51

H

2-Furan-CH2

2.2 ± 0.4

52

H

N-Me-Pyrrol-2-ylCH2

1.6 ± 0.3

53

H

(2-Cl-Pyrid-3-yl)CH2

1.4 ± 0.3

54

H

5-Br-Thien-2-yl CH2

3.1 ± 1.0

55

H

Thien-2-yl-CH2

0.75 ± 0.2

56

H

(2-Ph-Pyrid-5-yl)CH2

3.0 ± 0.5

57

H

(2-CF3-Pyrid-5-yl)CH2

2.9 ± 0.7

58

3-OMe

(N-Bn-piperidin-4yl)CH2

4.3 ± 0.8

59

3-OMe

2-Py-CO

1.7 ± 0.3

60

H

Ac

1.2 ± 0.2

68

2-OMe

2-Naphthyl

3.1 ± 0.6

69

2-OMe

2-Pyrimidinyl

1.1 ± 0.2

70

H

2-Pyrazinyl

2.4 ± 0.5

71

H

3-Pyridyl

7.8 ± 0.8

72

2-OMe

2-Pyridyl

1.5 ± 0.2

73

2-OMe

Ph

0.7 ± 0.1

74

2-OMe

3-MeO-Ph

3.8 ± 0.7

75

3-Me

4-methyl Pyridazinyl

7.9 ± 0.4

a Fold-change in cardiomyogenesis compared to vehicle (i.e., DMSO)treated cells. b R3, substituent on the 5-amino group of the benzimidazole core. c Fold-change over vehicle (i.e., DMSO)-treated cells.

important for cardiogenic potency. Compared to 1, introduction of a 3-pyridyl substituent (i.e., 71) increased cardiomyogenesis by 7.8-fold. The 2-pyridinyl analog (i.e., 70) was less potent with only a 2.4-fold increase in cardiomyogenesis. These data suggested that either a basic interaction or a hydrogen-bond interaction might be important in region C for optimal cardiomyogenesis potency in benzimidazole 1. For modifications in region C, the results from this series (i.e., 44-75) showed the necessity of a nitrogen atom at the 3-position of the substituent. To investigate a possible role of a linker group (i.e., a bridge group between region A and region C of 1) in compound potency, several analogs were prepared and tested. For example, a methylene linker for compounds 49 – 58; a carboxy group for 59 and 60; or no moiety, compound 16 was investigated. Compounds 49-53, 55, 59 and 60 did not possess increased cardiomyogenesis potency. In contrast, compounds 54, 56, 57

and 58 that were based on thien-2-yl or pyrid-5-yl groups at the nitrogen on C-5 (Table 2) increased cardiomyogenesis by 3.1-, 3.0-, 2.9- and 4.0-fold, respectively. The results suggested that certain heteroaromatics linked through a methylene spacer at the 5-position in region C of the benzimidazole afforded compounds that increased cardiomyogenesis. Results listed in Table 3 showed the effect of introduction of additional nitrogen atoms in region A on cardiomyogenesis potency. Replacement of carbon or nitrogen atoms at the 2, 3 and 7-positions of region A of benzimidazole 1 (i.e., compounds 65, 78, 82, 83 and 85) afforded compounds with modest increases in cardiomyogenesis, 2.5-, 2.0-, 3.7-, 2.1- and 3.1-fold, respectively. Compared to compound 65, 2-amino-benzimidazoles 82 and 83 gave small increases in potency 3.7- and 2.1-fold, respectively. Replacing N-3 of region A with a carbon atom (i.e., 78) gave slightly decreased potency (i.e., 2.0-fold). Compared to 1, replacing C-2 of region A with a nitrogen atom to elaborate a triazole (i.e., 85) gave slightly increased potency (i.e., 3.1-fold). However, compared to 1, none of these compounds substituted in region A significantly improved the overall cardiogenic potency.

Table 3. Effect of modification of region A of 1 on cardiomyogenesis in mESCs.

2,4-dinitrofluorobenzene (Matrix Scientific, Columbia, SC). 5nitro indole (Aldrich, St. Louis, MO), 2-chloro-3,5dinitropyridine (Alfa Aesar, Ward Hill, MA) and 5-bromo-2fluoronitrobenzene (TCI America, Portland, OR) were commercially available and used without further purification. Microwave reactions were conducted using a Biotage Initiator microwave synthesizer (Biotage, Uppsala, Sweden). Reaction products were purified, when necessary, by chromatography on silica gel (Silicycle, Quebec City, Quebec) (40-63 µm) with the solvent system indicated. Nuclear magnetic resonance (NMR) data were recorded on a Varian Mercury 300 MHz Spectrometer (Agilent, Santa Clara, CA) using TMS as an internal standard and CDCl3 as a solvent except where indicated. Electrospray ionization (ESI) mass spectral data was obtained using a Hitachi M-8000 (Hitachi, Dallas, TX). Final test compounds had a purity greater than 95% on the basis LCMS analysis using UV-vis detection at 254 nM and 220 nM. Compounds were prepared as the hydrochloride salt unless otherwise noted. Hydrochloride salts were prepared by dissolution of the benzimidazole in a minimum amount of dichloromethane and addition of excess 2 M HCl in ether. The solvent was evaporated and benzimidazole hydrochlorides were used directly for evaluation. Aqueous solubilities were determined by preparing a DMSO stock solution of the desired compound at 10 mM then diluting the stock solution in distilled water to the desired final concentration or until turbidity was observed. 4 . 1. 1. 2, 4- Di n it ro- N- ph en yl a ni li ne ( 3)

R2

R8

X1

X2

X3

CM Foldchangea

65

2-MeO

H

N

C

N

2.5 ± 0.5

78

H

H

C

C

C

2.0 ± 0.3

82

H

N(Me)2

N

C

C

3.7 ± 0.4

83

H

NH2

N

C

C

2.1 ± 0.3

85

H

H

N

N

C

3.1 ± 0.5

a

Fold-change in cardiomyogenesis compared to vehicle (i.e., DMSO)treated cells.

3. Conclusions In summary, 1,5-disubstituted benzimidazole 1, discovered from a high content screen, markedly stimulated cardiomyocyte differentiation in mESC cultures. In addition to αMHC activation, 1 enhanced additional gene biomarkers for mESC differentiation to cardiomyocytes 29. Medicinal chemistry and SAR development of 1 from over 400 benzimidazole analogs yielded 31, 44 and 75. These compounds showed increased potency relative to 1 and other published small molecule inducers11. In addition, 31, 44 and 75 trended toward improved drug-like properties, compared to 1. Thus, in the 1,5-disubstituted benzimidazole series examined herein, 1, 31, 44 and 75 constituted compounds that are useful and reliable molecular tools to direct cardiomyogenesis in mESCs. 4. Experimental 4.1. Chemistry General. Reagents, starting materials, and solvents were purchased in the highest purity available from commercial suppliers and used as received. 1-Chloro-4-methylphthalazine, 1chloro-4-phenylphthalazine (Enamine LLC, Kiev, Ukraine), 1chloro-phthalazine and 1-benzyl-4-chloro-phthalazine (Frontier Scientific, Newark, DE), 1-chloro-4-methoxy-phthalazine and

To a flask containing 25 mL of THF was added 2 g (10.7 mmol) 2,4-dinitrofluorobenzene and 1.1 g (11.8 mmol) of aniline and stirred at 25 °C. After stirring 15 h the solvent was concentrated in vacuo to give a crude solid that was recrystallized from ethanol to afford a yellow solid (98% yield). 1H NMR δ 7.17 (d, J = 9.6 Hz, 1H), 7.31 (m, 2H), 7.39 (m, 1H), 7.51 (m, 2H), 8.17 (ddd, J = 9.6, 2.5, 0.6 Hz, 1H), 9.19 (d, J = 2.5 Hz, 1H), 9.99 (bs, 1H). 4 . 1. 2. N 1 -P he n yl b en zen e- 1 , 2, 4- t ri ami n e. To a flask containing 2 g (16.9 mmol) of 3 in 75 mL of ethyl acetate, 200 mg of Pd/C was added. The flask was purged 3 times by vacuum and each time refilled with an atmosphere of H2. H2 was introduced to the flask and the mixture was stirred at 25 °C until the mixture showed no more starting material as determined by TLC (hexane/ethyl acetate, 90:10, v/v). The reaction mixture was filtered through celite and rinsed with ethyl acetate. The solvent was evaporated and the crude product was used without further purification. 4 . 1. 3. 1-P h e nyl- 1H - b en zo[ d] i mi d azol - 5- a mi ne (16) To a flask containing 1.5 g (7.5 mmol) N1-phenylbenzene-1,2,4triamine was added 25 mL of 4N HCl and 1 mL of formic acid and heated under reflux until no starting material was detected as determined by TLC. The reaction was then cooled to 0 °C and basicified with solid NaOH until a pH 11 was obtained. The aqueous fraction was extracted 3 times with ethyl acetate (100 mL). The organic phases were combined, dried over sodium sulfate and concentrated. The crude material was purified via column chromatography (silica gel, DCM with a gradient from 2 to 5% methanol) to give a red brown solid (80% yield). 1H NMR δ 3.73 (bs, 2H), 6.74 (dd, J = 2.0, 8.5 Hz, 1H), 7.14 (d, J = 2.0 Hz, 1H), 7.33 (dd, J = 8.5, 0.6 Hz, 1H), 7.38 – 7.56 (m, 5H), 8.00 (s, 1H).

4 . 1. 4. N -( 4-M et h yl p ht h al a zi n- 1- yl )- 1- ph en yl - 1Hb e n zo[ d ] i mi d azol - 5- a mi ne • HC l (1) To a microwave vial was added 16 mg (0.07 mmol) of 1-phenyl1H-benzo[d]imidazol-5-amine, 27 mg (0.15 mmol) of 1-chloro4-methylphthalazine in 0.5 mL of isopropyl alcohol. The mixture was heated to 150 °C for 1 h. The mixture was concentrated to dryness and ethyl acetate (1 mL) was added to precipitate the product. The hydrochloride salt of 1 was isolated by filtration and washing with cold ethyl acetate to give 17 mg (70% yield) as a tan solid. 1H NMR (CD3OD) δ 2.87 (s, 3H), 7.59 (dd, J = 8.5, 2.2 Hz, 2H), 7.68 – 7.70 (m, 4H), 7.80 (d, J = 8.5 Hz, 1H), 8.06 (d, J = 2.2 Hz, 1H), 8.23 – 8.27 (m, 2H), 8.38 – 8.42 (m, 1H), 8.60 (s, 1H), 8.74 – 8.77 (m, 1H). LRMS (ESI) m/z calcd 351.15; found 352.28(M+1)+.

( d, J = 6. 9 H z, 3H ), 1. 22 ( d, J = 6. 6 H z, 3 H), 2. 5 2 – 2 . 6 5 ( m, 1H ), 2. 8 8 (s, 3H), 7. 30 – 7. 40 ( m, 2 H), 7 . 4 4 – 7. 4 9 ( m, 1H), 7 . 55 ( d, 8. 5 H z, 1 H) , 7. 6 1 – 7 . 6 9 ( m, 2H ), 8. 0 9 (s, 1H), 8. 21 – 8. 29 ( m, 2 H), 8 . 3 7 – 8. 4 2 ( m, 1 H), 8. 5 3 (s, 1 H), 8. 74 – 8. 77 (m, 1 H ). LR MS ( ES I) m/ z ca l cd 3 94 . 19 ; fou nd 3 95 . 4( M+ 1) + . 4 . 1. 10. 5 - ( 4-M et h yl p ht h al a zi nyl - 1- a mi no) -1-( 4 me t hoxyp h en yl )b e n zi mi da zol e • H Cl (3 5).

Compounds 6-24 were prepared using procedures similar to those described for the synthesis of 1.

T he h yd r ochl or i d e s alt of 3 5 ( 6 5 % yi e l d) was ob t ai n e d a s a t a n s ol i d s ta rti ng fr om 4 me t h ox ya n i li ne. 1 H NMR (C D 3 O D ) δ 2 . 8 8 ( s, 3 H), 3 . 9 1 (s, 3 H ), 7. 2 1 (d, J = 8 . 8 H z, 2 H), 7 . 60 ( d, J = 8 . 8 H z, 3H ), 7. 7 4 ( d, J = 8 . 8 H z, 1 H ), 8. 09 ( s, 1 H), 8 . 2 5 – 8. 2 8 ( m, 2 H), 8. 4 0 – 8. 43 ( m, 1H ), 8. 67 (s , 1 H ), 8. 7 5 – 8. 7 8 ( m, 1H). LR MS ( E SI) m/ z cal cd 3 81 . 4 3; fou nd 38 2. 13( M+ 1) + .

4 . 1. 5. 5- ( 4-Me t hyl ph t hal azi n yl - 1 - ami n o) - 1-( 2me t hoxyp h e nyl ) be n zi mi da zol e • H Cl ( 30).

4 . 1. 11. 5 - ( 4-M et h yl p ht h al a zi nyl - 1- a mi no) -1-( 4 t ri fl u or ome t hyl ph en yl )b e n zi mi da zol e • H Cl ( 36 ).

T he h yd r ochl or i d e s alt of 3 0 ( 60 % yi e l d) was ob t ai n e d a s a t a n s ol i d s ta rti n g fr om 2 me t h ox ya n i li ne. 1 H NMR (C D 3 O D ) δ 2. 8 4 ( s, 3 H), 3 . 8 8 ( s, 3 H), 7. 1 9 – 7. 25 ( m, 2H ), 7. 30 ( d, J = 8.8 H z, 1 H ), 7. 3 5 ( d, J = 8 . 3 H z, 1 H), 7. 54 – 7 .58 (m, 2 H ), 7. 70 – 7. 7 4 ( m, 1H), 8. 0 0 – 8 . 03 ( m, 2 H ), 8. 16 – 8. 1 9 ( m, 1 H), 8. 2 3 ( s, 1H ), 8. 32 (s, 1 H), 8. 5 2 – 8 . 5 5 ( m, 1H ). LR M S ( E SI) m/ z cal cd 38 1. 4 3; fou nd 3 8 2. 5( M+ 1) + .

T he h yd r ochl or i d e s alt of 3 6 ( 7 0 % yi e l d) was ob t ai n e d a s a t a n s ol i d s ta rti ng fr om 4 t ri fl u or ome t hyl a nil i ne. 1 H N MR (C D 3 OD ) δ 2. 90 ( s, 3 H), 7. 6 7 ( d, J = 8. 5 H z, 1H), 7. 9 0 ( d, J = 8 .5 H z, 1 H ), 7. 9 4 – 8 . 04 ( m, 4H ), 8. 14 ( s, 1H) , 8. 2 3 – 8 . 2 8 ( m, 2 H), 8. 4 2 – 8 . 44 ( m, 1H ), 8. 7 5 – 8. 81 (m, 1 H ). LR MS ( ES I) m/ z ca l cd 4 19. 1 4; fou nd 4 20 . 0 7( M +1) + .

4 . 1. 6. 5- ( 4-Me t hyl ph t hal azi n yl - 1 - ami n o) - 1-( 3me t hoxyp h e nyl ) be n zi mi da zol e • H Cl ( 31). T he h yd r ochl or i d e s alt of 3 1 ( 75 % yi e l d) was ob t ai n e d as a yel l ow s ol i d st arti ng fr om 3 me t h ox ya n i li ne. LR MS ( ESI) m/ z cal cd 38 1. 43 ; fou nd 38 2. 13( M+ 1) + . 4 . 1. 7. 5- ( 4-Me t hyl ph t hal azi n yl - 1 - ami n o) - 1-( 4p r op yl p he nyl ) be n zi mi da zol e • H Cl (32 ). T he h yd r ochl or i d e s alt of 3 2 ( 50 % yi e l d) was ob t ai n e d as a t a n soli d st arti ng fr om 4 - n p r op yl an ili n e. 1 H NMR (C D 3 O D) δ 1 . 01 (t, J = 7.4 H z, 3 H ), 1. 6 7 – 1. 80 ( m, 2 H), 2. 73 (t, J = 7. 7 Hz, 2 H ), 2. 87 ( s, 3H ), 7. 4 8 ( d, J = 8. 5 H z, 2H), 7. 5 5 – 7 . 6 0 ( m, 3 H ), 7. 75 ( d, J = 8. 8 H z, 1 H), 8. 06 ( d, J = 1 . 7 H z, 1 H), 8. 21 – 8. 28 ( m, 2 H ), 8. 37 – 8. 41 (m, 1 H ), 8. 56 ( s, 1H ), 8. 75 – 8. 8 1 ( m, 1H). LR M S ( ESI) m/ z ca l cd 3 93. 2 0; foun d 39 4. 07 (M+ 1) + . 4 . 1. 8. 5- ( 4-Me t hyl ph t hal azi n yl - 1 - ami n o) - 1-( 3t ri fl u or ome t hyl ph e nyl ) be n zi mi da zol e • H Cl ( 33 ). T he h yd r ochl or i d e s alt of 3 3 ( 65 % yi e l d) was ob t ai n e d a s a t a n s ol i d s ta rti n g fr om 3 t ri fl uor ome t hyl a ni li ne. 1 H N MR (C D 3 OD ) δ 2 . 88 ( s, 3 H), 7. 62 ( dd, J = 1. 9, 8. 8 H z, 2 H), 7. 8 ( d, J = 8 . 8 H z, 1 H) , 7. 9 – 7. 9 1 ( m, 2 H ), 7. 9 9 – 8 . 0 3 (m, 1 H ), 8. 06 (s, 1 H ), 8. 1 ( d, J = 1. 9 H z, 1 H), 8. 2 5 – 8 . 3 0 ( m, 1 H), 8. 3 9 – 8. 43 ( m, 1H ), 8. 7 ( s, 1 H), 8. 75 – 8 . 7 8 ( m, 1H ). LR M S ( ESI) m/ z ca l cd 41 9. 14 ; fou nd 42 0. 05( M+ 1) + . 4 . 1. 9. 5- ( 4-Me t hyl ph t hal azi n yl - 1 - ami n o) - 1-( 2i s opr op yl ph en yl ) b en zi mi d a zol e • H Cl ( 3 4) . T he h yd r ochl or i d e s alt of 3 4 ( 10 % yi e l d) was ob t ai n e d a s an or a nge s ol i d s ta rti n g fr om ( 4 d i met hyl a mi no) a nili ne. 1 H N MR ( CD 3 O D) δ 1 . 18

4 . 1. 12. 5 - ( 4-M et h yl p ht h al a zi nyl - 1- a mi no) -1-( 2 h yd r ox yp he n yl ) be nzi mi da zol e • HC l, 37. T he h yd r ochl or i d e s alt of 3 7 ( 6 0 % yi e l d) was ob t ai n e d a s a t a n s ol i d s ta rti ng fr om 2 me t h ox ya n i li ne. 1 H NMR (C D 3 O D ) δ 3 . 8 0 ( s, 3 H), 6 . 7 1 ( d d, J = 2. 0, 8. 5 H z, 1H ), 7. 0 7 – 7. 12 ( m, 3 H), 7 . 1 4 ( d, J = 2. 0 H z, 1 H), 7. 38 – 7. 4 5 ( m, 2 H), 7. 98 ( s, 1 H ). LR M S (E SI) m/ z ca l cd 3 8 1. 4 3; fou nd 3 82 . 5( M+ 1) + . 4 . 1. 13. 5 - ( 4-M et h yl p ht h al a zi nyl - 1- a mi no) -1me t hyl be nzi mi da zol e • H Cl ( 38). T he h yd r ochl or i d e s alt of 3 8 ( 6 5 % yi e l d) was ob t ai n e d as a t a n s oli d fr om t h e rea cti on of 1 ch l or o-4 -me t h yl p ht h al azi n e a nd comme r ci a ll y a vai la b le 5-a mi no-2 -me t h yl b e nzi mi d azol e. 1 H NMR ( C D 3 O D ) δ 2. 8 3 ( s, 3 H), 3. 96 (s, 3 H), 7. 55 ( d d, J = 8 . 8, 1. 9 H z, 1 H ), 7. 67 ( d, J = 8 . 5 H z, 1 H ) , 7. 95 (d , J = 1. 6 H z, 1H), 8. 1 3 – 8. 1 7 ( m, 1H), 8. 2 5 ( s, 1 H), 8 . 2 7 – 8. 31 ( m, 1H ), 8. 40 – 8. 43 (m, 1H ), 8. 6 3 – 8 . 6 6 ( m, 1 H). LR MS ( ES I) m/ z ca l cd 2 8 9. 1 3; fou nd 2 90 . 0 8( M +1) + . 4 . 1. 14. 5 - ( 4-M et h yl p ht h al a zi nyl - 1- a mi no) -1c ycl opr o p yl be n zi mi da zol e • HC l ( 39 ). T he h yd r ochl or i d e s alt of 3 9 ( 7 0 % yi e l d) was ob t ai n e d as a ye l l ow s ol i d st ar ti ng fr om c ycl op rop yl a mi ne. 1 H NMR ( C D 3 O D) δ 1 . 1 0 – 1. 17 ( m, 2 H ), 1. 21 – 1 . 28 ( m, 2H ), 2. 8 4 (s , 3H), 3 . 5 6 – 3 . 6 1 ( m, 1H), 4. 45 – 4. 5 5 ( m, 1 H), 7. 5 6 ( dd, J = 8 . 5, 1. 6 H z, 1 H ), 7. 87 ( d, J = 8 . 5 H z, 1 H ) , 7. 95 (d , J = 1 . 6 H z, 1 H ), 8. 1 9 – 8. 2 4 ( m, 2 H), 8. 33 – 8. 35 ( m, 1 H ), 8. 36 ( s, 1H), 8. 6 9 – 8. 72 ( m, 1H). LR MS ( E SI) m/ z cal cd 31 5. 15 ; foun d 3 1 6. 2 0( M +1) + . 4 . 1. 15. 5 - ( 4-M et h yl p ht h al a zi nyl - 1- a mi no) -1c ycl ohe x yl b en zi mi d a zol e • H Cl ( 4 0).

T he h yd r ochl or i d e s alt of 4 0 ( 70 % yi e l d) was ob t ai n e d a s a ye l l ow s ol i d st art i ng fr om c ycl oh ex yl ami n e. 1 H N MR (C D 3 O D ) δ 1. 38 – 2 . 22 ( m, 1 0H ), 2. 86 (s, 3H ), 4. 45 – 4 . 55 ( m, 1H ), 7. 58 ( d d, J = 8 . 5, 1. 6 H z, 1H ), 7.8 9 ( d, J = 8. 5 H z, 1 H ), 8 . 0 2 ( d, J = 1. 6 H z, 1H ), 8. 13 – 8 . 17 ( m, 1 H ), 8. 21 – 8. 25 ( m, 1H ), 8. 37 – 8 . 4 0 ( m, 1 H), 8. 6 1 (s, 1 H ), 8 . 7 1 – 8. 74 ( m, 1 H). LR M S ( E SI) m/ z cal cd 35 7 . 20 ; fou nd 35 8. 17( M+ 1) + .

4 . 1. 21. 5 - ( 4-M et h ox yp ht h al a zi n yl - 1- a mi no)- 1-( 3me t hoxyp h en yl ) be n zi mi da zol e • H Cl ( 46 ).

4 . 1. 16. 5 - ( 4-M et h yl p ht h al a zi nyl - 1- a mi no) - 1-( 2 p yr i d yl ) b en zi mi d a zol e • H Cl ( 4 1).

4 . 1. 22. 5 - ( 4-B e nzyl ph t ha l azi nyl - 1 - ami no) - 1-( 3me t hoxyp h en yl )b e n zi mi da zol e • HC l ( 47 ).

T he h yd r ochl or i d e s alt of 4 1 ( 76 % yi e l d) was ob t ai n e d as a yel l ow s ol i d st arti ng fr om 2 a mi n op yr i di n e. 1 H NM R ( C D 3 O D) δ 2. 90 ( s, 3 H), 7 . 4 5 – 7. 49 ( m, 1H), 7. 6 6 ( d, J = 8. 8 H z, 1 H), 8. 02 ( d, J = 8 . 3 H z, 1H ), 8. 1 0 (d, J = 8. 3 H z, 1 H), 8. 15 ( s, 1H ), 8. 2 1 – 8. 32 ( m, 2 H), 8. 4 2 ( d, J = 8. 8 Hz, 2 H ), 8. 66 ( s, 1H ), 9. 09 – 9. 1 4 ( m, 2H). LR M S ( ESI) m/ z ca l cd 3 52. 1 4; foun d 35 3. 22 (M+ 1) + .

T he h yd r ochl or i d e s alt of 4 7 ( 6 5 % yi e l d) was ob t ai n e d as a t an soli d s ta rti ng fr om 1 -chl or o- 4 b e nz yl ph t al a zi n e. 1 H N MR (C D 3 OD ) δ 3 . 93 ( s, 3 H), 7 . 2 6 – 7. 40 ( m, 6H), 7. 6 7( a p par e nt t, 1H ), 7. 9 7 ( bs , 2 H ), 8. 17 – 8. 3 3 ( m, 4 H), 8 .4 0 (s, 1 H) , 8. 47 ( d, J = 8 . 8 H z, 1H ), 8. 8 5 ( d, J = 7. 2 H z, 1H), 9. 7 6 (s, 1 H).

4 . 1. 17. 5 - ( 4-M et h yl p ht h al a zi nyl - 1- a mi no) - 1-( 4 t et r ah yd rop yr an yl ) b en zi mi d a zol e • H Cl ( 4 2) .

T he h yd r ochl or i d e s alt of 4 8 ( 5 8 % yi e l d) was ob t ai n e d as a ye l l ow s ol i d st art ing fr om 1 - chl or o- 4 1 p he n yl p ht ala zi ne. H N MR ( C D 3 O D ) δ 3. 9 3 (s , 3 H ), 7. 14 – 7. 1 8 ( m, 1H), 7. 2 6 – 7 . 29 ( m, 2 H ), 7. 51 – 7. 6 9 ( m, 6 H ), 7. 6 7( a p pa r ent t, 1 H), 7. 8 7 ( d, J = 8 . 8 H z, 1H), 8. 1 7 – 8. 33 ( m, 4H ), 8 . 80 (s, 1 H), 8. 86 ( d, J = 8. 8 H z, 1 H).

T he h yd r ochl or i d e s alt of 4 2 ( 63 % yi e l d) was ob t ai n e d as a yel l ow s ol i d st arti ng fr om 4 a mi n ot etr a hydr op yr a n. 1 H N MR (C D 3 O D) δ 2 . 1 6 – 2 . 3 4 ( m, 5H ), 2. 88 (s, 3H ), 3. 6 7 – 3. 76 ( m, 2 H), 4 . 1 2 – 4. 20 ( m, 2H), 7. 6 2 ( d, J = 1. 9 H z, 1 H), 7. 95 ( d, J = 8. 8 H z, 1 H), 8. 05 ( d, J = 1. 9 H z, 1 H), 8. 1 2 – 8 . 1 6 ( m, 1 H), 8. 23 – 8 . 26 ( m, 1 H ), 8. 3 7 – 8. 42 (m, 1 H ), 8. 71 ( s, 1H ), 8. 74 – 8. 7 7 ( m, 1H). LR M S ( ESI) m/ z ca l cd 3 59. 1 7; foun d 36 0. 20 (M+ 1) + . 4 . 1. 18. 5 - ( 4-M et h yl p ht h al a zi nyl - 1- a mi no) - 1-( 1 me t hyl pi p eri di n-4 - yl) - 1H- be nzo[ d] i mi da zol - 5 a mi ne • HC l ( 43). T he h yd r ochl or i d e s alt of 4 3 ( 76 % yi e l d) was ob t ai n e d a s a r e d s ol i d st art i ng fr om 1 me t h yl p i peri di n-4 -a mi ne . 1 H N MR ( DMS O -d6 ) δ 2 . 2 0 – 2. 26 ( m, 4 H), 2 .75 ( s, 3 H), 2. 77 (s, 3 H ), 3. 04 – 3. 1 8 ( m, 4 H ), 4. 6 1 – 4. 7 3 ( m, 1 H), 7. 6 5 ( d, J = 8 . 0 H z, 1H ), 7. 74 ( d, J = 9. 6 H z, 1 H) , 7. 9 8 – 8. 00 ( m, 2 H), 8. 07 – 8 .10 ( m, 1H ), 8 .2 3 – 8. 2 8 ( m, 2 H), 8 . 5 9 – 8. 63 ( m, 1 H). LR M S ( E SI) m/ z cal cd 37 2 . 47 ; fou nd 37 3. 5( M +1 ) + . 4 . 1. 19. 5 - (P ht h al a zi nyl - 1- a mi no) - 1-( 3 me t hoxyp h e nyl ) be n zi mi da zol e • H Cl ( 44 ). T he h yd r och l or i d e s al t of 44 ( 6 5%% yi el d) was ob t ai n e d as a yel l ow s ol i d st arti ng fr om 1 ch l or oph t a l azi ne. 1 H N M R (C D 3 O D) δ 3. 9 2 (s , 3 H ), 7. 25 – 7. 4 4 ( m, 4H), 7. 5 9 – 7 . 70 ( m, 2 H ), 7. 90 – 7. 9 9 (m, 2H ), 8. 2 7 – 8. 4 2 ( m, 2H ), 8. 88 – 8. 91 ( m, 1 H) , 9. 32 (s , 1H ), 9. 7 1 ( s, 1H ). LR MS ( ESI) m/ z ca l cd 3 67. 1 4; foun d 36 8. 27 (M+ 1) +. 4 . 1. 20. 5 - ( 4- C hl or oph th al azi n yl - 1- a mi no) -1-( 3me t hoxyp h e nyl ) be n zi mi da zol e • H Cl ( 45 ). T he h yd r ochl or i d e s alt of 4 5 ( 55 % yi e l d) was ob t ai n e d as a yel l ow s ol i d st arti n g fr om 1, 4 d i chl or oph t al a zi ne. 1 H NM R (C D 3 OD ) δ 3. 93 (s , 3 H ), 7. 22 – 7. 25 ( m, 1 H), 7. 35 ( bs, 2 H ), 7. 6 1 – 7. 66 ( m, 1 H), 7. 80 ( d, J = 8. 8 H z, 1 H), 7. 93 ( d, J = 8.8 H z, 1 H ), 8 .24 – 8. 3 0 ( m, 2H) , 8. 3 3 (s, 1 H ), 8. 4 5 (d , J = 7 . 2 H z, 1 H), 8. 81 ( d, J = 7. 2 H z, 1H ), 9. 41 (s , 1 H ). LR MS ( ES I) m/ z ca l cd 4 01. 1 0; fou nd 4 0 2. 1 3( M +1) +.

T he h yd r ochl or i d e s alt of 4 6 ( 6 2 % yi e l d) was ob t ai n e d as a ye l l ow s ol i d st art ing fr om 1 - chl or o- 4 me t h ox yp ht a la zi ne. 1 H N MR ( C D 3 O D) δ 3 . 9 3 (s , 3 H ), 4. 2 4 ( s, 3H ), 7. 1 5 – 7. 17 ( m, 1 H), 7. 2 5 ( bs , 2 H ), 7. 3 2 –7. 66 (m, 5H), 8. 33 –8. 4 5 ( m, 2H), 8. 5 1– 8 . 6 5 ( m, 2 H) .

4 . 1. 23. 5 - ( 4-P h e nyl ph th al azi nyl - 1 -a mi no)- 1-( 3me t hoxyp h en yl )b e n zi mi da zol e • HC l ( 48 ).

4 . 1. 24. 5 - (P he net h yl a mi no) -1-( 3me t hoxyp h en yl )b e n zi mi da zol e • HC l ( 49 ). 5 mg (0 . 03 mmol) of 1 8 ( pr e par e d u si ng t h e met h od d e scri b ed i n S c he me 1) w as sti rr ed wi t h 5. 4 mg ( 0. 0 5 mmol ) of p h en yl a ce t al de hyde an d 15 µl of b ora n e• p yr i di n e compl e x i n 0. 1 mL e t ha n ol for 1 5 h a t 25 ° C in a s eal e d vi al. S ol ve nt w a s eva por at ed a n d t he cr u de rea cti on mi x t ur e pur i fi e d by PT L C ( h e xa ne /et hyl ace t at e, 5 0:5 0, v/ v). 5. 0 mg of 5 ( p he n et h yl ami n o) -1 -( 3-met h ox yp h en yl ) b e nzi mi daz ol e w as obt ai n ed as a fr e e ba s e ( 69% yi e l d). T r eat me nt of t he free bas e ( sol uti on i n DC M) with e x ce s s 1 N H Cl ( 2. 0 M sol u ti on i n et he r) at r oo m t e mp er at ur e, fol l ow ed b y c on cen t r ati on , yi e l de d 49 . 1 H -N M R δ 3 . 8 8 ( s, 3 H), 4. 28 (s, 2H ), 6. 7 2 ( d d, J = 9 , 3 H z, 1H), 7. 1 0-6. 79 ( m, 11 H), 7. 3 6 ( dd, J = 9 H z, 1 H ), 7. 4 4 (t, J = 9 H z, 1 H), 8. 0 6 ( s, 1 H), 8. 63 ( t, J = 6 H z, 1 H) . C omp ou nd s 5 0- 5 8 w e r e pr epa red u si ng a s i mila r p r oce du r e t o t hat d es cr ib ed for t he s ynt h es is of 49. 4 . 1. 25. N -( 2 -Met hoxy be n zyl ) - 1- ( 3me t hoxyp h en yl )- 1H - be nzo[ d] i mi da zol - 5 a mi ne •H Cl ( 50 ). T he h yd r ochl or i d e s alt of 5 0 ( 4 9 % yi e l d) was ob t ai n e d as a ye l l ow oi l fr om 2me t h ox yci n na mal de hyd e a n d 1 8 u s i ng t h e pr oce d ur e d e scri b ed for 2 5 . 1H -NM R δ 3. 8 2 ( s, 3H ), 3. 87 (s , 3 H ), 6. 34 ( b s, 1H), 6. 8 1 -7. 4 8 (m, 13 H). 4 . 1. 26. N -(F u ra n-2- yl me t hyl a mi no) -1- ph e nyl - 1Hb en zi mi da zol e • HCl ( 51 ). T he h yd r ochl or i d e s alt of 5 1 ( 7 5 % yi e l d) was ob t ai n e d as an or a nge oi l from fur a n-2 ca r boxa l d eh yde an d 18 usi n g t h e p r oce d ure d e scri b ed for 2 5 . LR M S ( ESI) m/ z cal cd 2 89 . 1 ; fou nd 29 0. 30( M+ 1) +.

4 . 1. 27. N -( ( 1- Me t hyl- 1H - p yr r ol - 2yl ) met h yl a mi no) - 1- p he nyl - 1H- b e nzi mi da zol e • HC l ( 5 2). T he h yd r ochl or i d e s alt of 5 2 ( 82 % yi e l d) was ob t ai n e d as a n or a n ge oil fr om 1-met h yl -1 H p yr rol e -2-ca r boxa l d eh yde a nd 18 usi n g t he p r oce du r e de s cri b ed for 4 9. LR M S ( ES I) m/ z cal cd 3 0 2. 1 ; foun d 3 0 3. 3 0(M +1 ) +. 4 . 1. 28. N -( ( 2- C hl or op yr i di n - 3- yl) met h yl a mi no) - 1p he nyl - 1 H- b e nzi mi da zol e • HC l ( 53 ). T he h yd r ochl or i d e s alt of 5 3 ( 85 % yi e l d) was ob t ai n e d as a t ra ns par ent oil fr om 2ch l or oni cot i nal d eh yde an d 18 us i n g t he pr oce d ure d e scri b ed for 4 9. LR M S (E SI) m/ z ca l cd 3 34 .0 ; fou nd 33 5. 10( M+ 1) +. 4 . 1. 29. N -( ( 5-B r omoth i ophe n -2 - yl ) me t hyl a mi n o)1 - ph en yl -1H- be nzi mi d azol e• H Cl ( 54). T he h yd r ochl or i d e s alt of 5 4 ( 78 % yi e l d) was ob t ai n e d as a cl ear oi l fr om 5-b r omot h i op he n e-2 ca r boxa l d eh yde a nd 18 usi n g t h e pr oce d ure d e scri b ed for 2 5. LR M S (E SI) m/ z ca l cd 3 8 4.8 ; fou nd 38 6. 00( M+ 1) +. 4 . 1. 30. 1 -P he n yl- N- (t hi op he n- 2 - yl me t hyl ami n o) 1 H- be nzi mi da zol e • H Cl (5 5). T he h yd r ochl or i d e s alt of 5 5 ( 43 % yi e l d) was ob t ai n e d a s a col or l es s oil fr om t hi op he n e-2 ca r ba l deh yde an d 1 8 u si ng t he p r oce du r e d es cri b ed for 49 . LR MS (E SI) m/ z ca l cd 3 0 5. 1 ; fou nd 3 0 6. 3 0( M +1) +. 4 . 1. 31. 1 -P he n yl- N- (( 6- ph en yl p yr i d i n-3 yl ) met h yl a mi no) - 1H- b en zi mi da zol e • HCl ( 56 ). T he h yd r ochl or i d e s alt of 5 6 ( 73 % yi e l d) was ob t ai n e d as a pi n k sol i d fr om 6p h en yl n i cot i n al de h yd e a nd 18 u s i ng t he pr oce d ure d e scri b ed for 4 9. LR M S (E SI) m/ z ca l cd 3 7 5.9 ; fou nd 37 7. 1( M +1 ) +. 4 . 1. 32. 1 -P he n yl- N- (( 6- (t rifl u or omet h yl ) p yr i di n 3 - yl )met hyl )- 1H- b en zo[ d ] i mi d azol - 5- a mi ne •H Cl ( 5 7). T he h yd r ochl or i d e s alt of 5 7 ( 78 % yi e l d) was ob t ai n e d as a w hit e s oli d from 6( tri fl uor ome t hyl ) n i cot i n al d e hyde an d 18 u si ng t he p r oce du r e de s cri b e d for 49 . LR MS ( ES I) m/ z ca l cd 3 6 8. 1 ; foun d 3 6 9. 3 0(M +1 ) +. 4 . 1. 33. N -( ( 1-B e nzyl p i pe r i di n- 4- yl) met h yl )- 1 p he nyl - 1 H- b e nzo[ d ] i mi da zol - 5- a mi ne •H Cl ( 5 8). T he h yd r ochl or i d e s alt of 5 8 (7 5 % yi e l d) was ob t ai n e d a s a n or a nge oi l fr om 1 -be n zyl p i pe ri di ne 4 -car b ox a l de h yd e a nd 1 8 us i n g t he pr oce d ur e d e scri b ed for 49 . LR M S ( ESI) m/ z ca l cd 3 9 5. 2 ; fou nd 39 6. 4( M +1 ) +. 4 . 1. 34. N -( 1 -( 3- M et h ox yphe n yl ) - 1Hb e n zo[ d ] i mi d azol - 5- yl) pic ol i na mi de •H Cl ( 59). T o a 5 m L fl a s k con t ai ni n g 5 0 mg ( 0. 3 5 mmol ) of p i col i n o yl ch l or i d e i n 2 mL D C M was a dde d 8 5 mg ( 0. 3 5 mmol ) of 1 8 an d 0. 1 mL o f t ri et hyl a mi ne. T he mi xt u re wa s st i rre d ove rn i ght at r oom t emp er at ure . T he s ol u ti on w as con cen t r at ed, di l ut e d wit h D CM,

w a s he d w i t h 2 mL of br i n e, dri ed a nd p u ri fi ed by P T LC ( DC M/ me t ha nol 9/ 1, v/ v) t o a fford 95 mg ( 7 9% yi el d) of a cl ea r l i qui d. 1 H -N MR δ 3 . 89 (s , 3 H ), 6. 95 -7. 18 ( m, 3H), 7. 4 3-7. 5 2 ( m, 2 H), 7. 56 (d , J = 9 H z, 1H ), 7. 8 2 ( d, J = 9 H z, 1 H), 7. 9 3 (t , J = 7 . 8 H z, 1H ), 8. 1 4 ( b s, 1H ), 8. 27 ( bs, 1 H), 8. 34 ( d, J = 8. 1H z, 1 H), 8. 65 ( d, J = 5 H z, 1 H ). 4 . 1. 35. N -( 1 -P he nyl - 1H-b e nzo[ d ] i mi da zol - 5yl ) ac et ami d e (6 0). T o a 5 mL fl a s k con t ai ni n g 5 µ L ( 0. 0 6 mmol) of a cet yl ch l or i de i n 1 mL D C M w as a dd e d 10 mg ( 0. 0 5 mmol ) of 1 8 a n d 5 µL of t r i et hyl a mi ne. T he mi xt u re wa s sti rr ed for 15 h a t 2 5 °C . Th e s ol ut i on w a s con cen t r at ed, di l ut ed wit h D CM, w ash e d wit h 2 mL of b r i n e, dri e d an d pu ri fie d b y PT L C ( D CM/ met ha n ol, 9/ 1, v/ v) t o aff or d 7 mg ( 56 % yi e l d) of a l i gh t bei ge s ol i d. LR MS ( ESI) m/ z cal cd 2 51 . 3 ; foun d 2 52. 2 (M+ 1) +. 4 . 1. 36. N -( 2 -Met hoxy ph e n yl )-3, 5 - di ni t ropyr i di n2 - ami ne (6 2). 2-chl or o-3 , 5 -di ni tr op yr i di n e (9. 8 mmol ), oa n isi di ne ( 9. 8 mmol ) a nd D IEA ( 19 . 6 mmol ) w ere h e ate d at r e fl ux i n 1 00 mL e t ha nol. Aft e r 3 h ou rs , t h e r eacti on w a s comp l et e a n d t he br i ght or a nge p r e ci pit at e w a s filt er e d, w a she d wit h et han ol a nd d ri e d t o gi ve 2. 9 g of a br i ght r e d-or a nge sol i d. 1 H N M R ( 3 00M H z, C DCl 3 ) : 9 . 3 4 ( s, 1 H), 8. 48 (d , J = 6. 3 H z, 1H ), 7. 30 -7. 2 0 ( m, 4H ), 7. 15 -7. 0 0 (m, 3 H ), 3. 99 ( s, 3 H). 4 . 1. 37. N 2 -( 2-M et h ox y phe n yl ) pyr i di n e- 2, 3, 5t ri a mi n e, 63. 1 g of 62 an d 4 g of s od i um d it hi oni t e w er e he at ed a t 7 0 ° C for 1 h i n et ha n ol/ w at er ( 50/ 50 v/ v). The s ol ve n t w a s eva por a t ed an d th e cr ud e pr odu ct w as e x tr act e d w i th et h yl ace t ate t o gi ve 3 40 m g of 6 3 a s a n or a nge s ol i d. Th e comp oun d w a s us e d d ir ectl y w it hout fu rt h er p uri fi ca ti on. 4 . 1. 38. 3 - ( 2-M et h ox yp he n yl ) - 3H-i mi d azo[ 4, 5 b ] pyri d i n- 6 - ami n e ( 64 ). T o a fl a sk con t a i ni n g 33 0 m g ( 1. 4 mmol ) of 6 3 w a s a d de d 5 mL of 4 N H C l an d 0. 2 mL of f or mi c acid a n d he at ed un der r efl u x u nti l n o st arti ng ma t eria l w a s pr es ent as d et ermi n e d b y T LC . U p on c ompl e ti on, t he r ea cti on w as co ol ed t o 0 ° C a nd b a si ci fi e d w it h sol i d N a O H until a p H 11 was r e a ch e d. Th e aq ue ous fr a cti on w as e xtr a cted 3 ti me s wi t h et h yl a ceta te ( 1 00 mL). The or gan ic p ha s e s w er e comb i ne d , dri ed over s od i um s ul fate a n d c on cen t r at ed. Th e cr u de mat er ia l wa s p uri fi ed vi a c ol u mn chr omat ogr a p h y ( s il i ca ge l, et hyl a cet at e wit h a gr a di ent fr om 0 t o 20 % me t ha nol ) to gi ve a br ow n s ol i d ( 8 0% yi el d). . 1 H -NMR δ 3. 81 ( s, 3H), 7. 09 – 7 . 18 ( m, 2H ), 7. 42 -7. 4 7 (m, 1 H), 7 . 5 7 ( d, J = 7 . 7 H z, 1H ), 7. 6 9 ( bs, 2 H ), 8. 3 0 ( d, J = 9 . 1 H z, 1 H), 8. 44 – 8. 47 ( m, 1H), 8. 51 ( d, J = 2.2 H z, 1H ). 4 . 1. 39. N -( 3 -( 2- M et h ox yp he n yl ) - 3H- i mi d azo[ 4, 5b ] pyri d i n- 6 - yl ) -4 - met h yl pht h al a zi n- 1 - ami n e•H Cl ( 6 5). T o a mi cr ow a ve vi al w a s a dd ed 1 0 mg ( 0. 0 4 mmol ) of 1 -ph en yl -1H -b en z o[d ]i mi da zol -5 -ami n e an d 20 mg ( 0. 1 5 mmol) of 1- chl or o-4 -me t hyl ph t h ala zi ne in

0 . 5 mL of i s opr op yl a l coh ol . T he mi xt ure was h e ate d t o 1 50 ° C for 1 h . Th e mi x t ur e was c once nt r at e d t o d ryn es s an d e t h yl a ce t at e ( 1 mL) w a s a dd ed t o pr e ci pit at e t he p r od uct. T he h ydr o ch l or i de salt of 6 5 w a s is ol at ed by fi l tr ati on a n d w a s hi n g w it h col d et h yl ace t at e t o gi ve 1 7 m g ( 7 0% yi e l d) as a t a n s ol i d. LR M S ( ES I) m/ z cal cd 3 8 2. 1 5; fou nd 3 83. 6(M +1 ) +. 4 . 1. 40. 1 - ( 2-M et h ox yphe n yl ) -N -( na ph t hal e n- 2 -yl) 1 H- be nzo[ d] i mi da zol - 5 - ami n e•H Cl ( 6 8). 6 7 a ( 3 0 mg, 0 . 1 mmol ), n a pht hal e n-2 -a mi ne ( 28 mg, 0 . 2 mmol ), p all adi u m( II) ace t at e (~2 mg) , racB IN A P ( ~2 mg), ce si um ca r b on at e (~1 0 mg), a nd 1 . 5 mL of de ga s s ed t ol ue ne we re ad de d t o a 1 gr a m vi a l. Th e vi al w as fl u sh e d wit h ar gon gas , cap p ed w i t h a Te fl on -coa t e d li n er, an d h eat e d over n i gh t a t 1 0 0 ° C. Aft er all ow i n g t h e s ol u ti on t o cool t o r oo m t e mp er at ur e, t he s ol uti on w as filt er e d, con ce n t r at ed , a n d p uri fi ed by fl a s h col umn chr omat ogr ap h y ( D C M w i t h a gr a di ent of 0 -5% me t ha n ol ) t o gi ve 17 . 6 mg ( 4 9% yi el d) of 68 as a r e d oil. LR M S ( ES I) m/z ca l cd 3 65 . 1 5; fou nd 3 66 . 4( M +1 ) + a n d 3 88.0 ( M +N a) +. C omp ou nd s 6 9- 7 8 w er e pr ep ar ed u si n g pr oced ur es s i mil ar t o t h os e de s cri be d for t h e s yn t he s is of 68 . 4 . 1. 41. 1 - ( 2-M et h ox yphe n yl ) -N -( pyr i mi di n- 2- yl)1 H- be nzo[ d] i mi da zol - 5 - ami n e•H Cl ( 6 9). C omp ou nd 6 9 w as pr e par e d usi n g pr oced ur es s i mil ar t o t h os e d e scr ib ed for t he s yn t he si s of 6 8 s ta rti n g fr om 6 7a a n d 2-a mi n o p yr i mi d i ne i n 47% yi e l d. LR M S ( ES I) m/ z cal cd 3 46 . 1 4; fou n d 3 4 7. 4 5( M +1) +. 4 . 1. 42. 1 -P he n yl- N- ( p yr azi n - 2- yl )- 1Hb e n zo[ d ] i mi d azol - 5- a mi ne •H Cl ( 70). C omp ou nd 7 0 w as pr e par e d usi n g pr oced ur es s i mil ar t o t h os e d e scr ib ed for t he s yn t he si s of 6 8 s ta rti n g fr om 6 7 b a nd 2 -a mi n o p yr a zi n e i n 5 9% yi e l d. LR M S ( ES I) m/ z cal cd 2 87 . 3 3; fou n d 2 8 8. 5 3( M +1) + a n d 31 0. 1 3 (M+ N a) + . 4 . 1. 43. 1 -P he n yl- N- ( p yr i di n -3- yl) - 1Hb e n zo[ d ] i mi d azol - 5- a mi ne •H Cl ( 71). C omp ou nd 71 w a s ob t ai ne d i n 23% yi e l d as a cl ea r oi l u si n g p r o ce du r e s si mi la r t o t h ose de s cr i be d for t h e syn t he s is of 6 8 s t art i ng fr om 6 7b an d 3 a mi n op yr i di n e. LR M S ( E S I) m/ z cal cd 28 6 . 34 ; fou nd 28 7. 47 ( M+ 1) +. 4 . 1. 44. 1 - ( 2-M et h ox yphe n yl ) -N -( pyr i di n - 2- yl )- 1Hb e n zo[ d ] i mi d azol - 5- a mi ne •H Cl ( 72). C omp ou nd 7 2 w a s obt a i ned i n 48% yi el d u si ng p r oce du r e s si mila r t o t hos e d es cr i be d for t he s ynt h e si s of 6 8 st a rti n g fr om 67 a a n d 2 a mi n op yr i di n e. LR MS ( E SI) m/ z ca l cd 28 7. 33 ; fou nd 31 7. 73 ( M+ N a) +. 4 . 1. 45. 1 - ( 2-M et h ox yphe n yl ) -N -( ph e nyl )- 1Hb e n zo[ d ] i mi d azol - 5- a mi ne •H Cl ( 73). C omp ou nd 7 3 w a s obt a i ned i n 54% yi el d u si ng p r oce du r e s si mila r t o t hos e d es cr i be d for t he s ynt h e si s of 6 8 s t a rti ng fr om 6 7 a a nd a ni li ne . LR M S (E S I) m/ z cal cd 3 1 5. 3 8; foun d 31 7 . 73 ( M +1) +.

4 . 1. 46. 1 - ( 2-M et h ox yp he n yl ) -N -( 3me t hoxyp h en yl )- 1H - be nzo[ d] i mi da zol - 5 a mi ne •H Cl ( 74 ). C omp ou nd 7 4 w as ob t a i ne d i n 4 0 % yi e l d u si ng p r oce du r e s si mila r t o t hos e d es cr i be d for t he s ynt h e si s of 6 8 st a rti ng fr om 67 a a n d 3 me t h ox ya n i li ne. LR M S ( ESI) m/ z ca l cd 34 5. 40 ; fou nd 34 6. 60( M+ 1) + an d 36 8. 27 (M+ N a ) +. 4 . 1. 47. N -( 6 -Met hyl pyr i da zi n - 3- yl)- 1- m-t ol yl - 1Hb en zo[ d ]i mi d azol - 5- a mi ne •H Cl ( 75). C omp ou nd 7 5 w as ob t a i ne d i n 3 3 % yi e l d u si ng p r oce du r e s si mila r t o t hos e d es cr i be d for t he s ynt h e si s of 1 s t art i n g fr om 1-m-t ol yl -1 H b e nzo[d ]i mi d azol -5-a mi ne a nd 3 -chl or o- 6 me t h yl p yr i d a zi n e. LR MS ( E SI) m/ z ca l cd 31 5. 15 ; fou nd 31 7. 40( M+ 1) +. 4 . 1. 48. N -P he nyl - 5- n it roi nd ol e ( 7 7). T o a fl a me -d ri ed s e al ed t u b e was ad d ed 5 n it r oi nd ole ( 48 6 mg, 3 mmol ), phe nyl b or oni c aci d ( 9 14 mg, 7 . 5 mmol), a nh yd r ou s copp e r ( II) a cet a te ( 1. 3 6 g, 7. 5 mmol ), dii s opr op yl e t hyl a mi ne ( 1. 3 mL, 7 . 5 mmol ) i n D CM ( 6 mL) . T he mi xt ure w as st i rred a t 2 5 ° C for 4 da ys , con ce nt r at e d i n -vacu o, d il ut ed w it h ch l or ofor m ( 5 0 mL) an d w at er ( 5 0 mL) , a nd fi lt er e d t o r emove pa rt i cul at e s. T he or ga ni c l aye r w a s t he n dri e d ove r s odi u m s u l fat e, c on ce n t r at ed i n -vacu o, a nd pu ri fi e d by fl a s h col umn ch r oma t ogr a ph y ( h ex a ne s/ et hyl a ce t a te, 4:1 , v/ v) t o 7 7 (5 0 2 mg, 70% yi el d ) as a ye l l ow s oli d. 1 H NMR δ 6. 86 ( d, J = 3. 3 H z, 1H ), 7. 4 3-7. 6 ( m, 7 H), 8. 11 ( d d, J = 2. 1, 9. 0 H z, 1 H), 8. 65 ( d, J = 2. 1 H z, 1H ). 4 . 1. 49. N -P he nyl - 5- a mi n oi ndol e. N -Ph en yl -5 -ni tr oi n dol e 77 ( 1 50 mg, 0. 63 mmol ) w a s dis s ol ve d i n et h an ol ( 1 5 mL) . A s ol uti on of s odi u m dit hi onit e ( 3 29 mg, 1. 89 mmol ) i n wa ter (1 mL) w a s a d de d at r oom t e mp er at ur e wi th stir ri ng. T he s ol u ti on w a s t h en h eat e d t o 70 °C u n t il c ompl e te cons u mpt i on of 77 w as ob s er ve d b y T L C ( ~ 2 h our s). Aft e r t h e s ol ut i on w a s co ol e d t o 25 ° C , e t h an ol w as r emove d i n-va cu o an d t he r e s ult i ng cr u de mat eri al w as d il ut e d w it h et hyl a ce t a te , w a s he d wi t h wat er, dri ed ove r s odi u m s ul fat e a nd c once nt r at e d i n-va cu o t o yi el d cr u de N -p he n yl - 5 a mi n oi n dol e ( 62 mg, 4 7%) w hi ch w a s u sed i mme di at el y i n t he fol l ow i n g r ea ct i on w it hou t fu r t he r p ur i fi ca ti on. 1 H NM R δ 3. 5 5 ( bs, 2 H ), 6. 51 ( d d, J = 0. 6 , 3 H z, 1H ), 6. 7 0 ( d d, J = 2. 1, 8. 7 Hz, 1 H ), 7. 0 0 ( d, J = 2. 1 H z, 1H ), 7. 28 -7.5 3 ( m, 7 H). LR MS (E S I) m/ z cal cd 2 08 . 1 0; foun d 20 9. 13 ( M +1) +. 4 . 1. 50. 4 - Met hyl -N -( 1- p he nyl - 1H-i n dol - 5yl ) ph t hal azi n - 1- a mi ne •HC l ( 78 ). T o a mi cr ow a ve vi a l w as ad ded N -P he n yl - 5 a mi n oi n dol e (1 0 mg, 0. 05 mmol) a nd 1 -chl or o- 4 me t h yl p ht h al zi ne (17 mg, 0.09 mmol) in i s opr opa n ol ( 1 mL) . The mi xt ur e w as he at ed at 1 50 ° C for 1 h. Th e r e a cti on mi xtu r e w as con ce n t r at ed t o d r yn e ss an d et h yl a ce t a t e wa s ad d ed to p r e ci pit at e t he hyd r ochl or i d e sa lt of 7 8 as a ye l l ow s ol i d (1 4 mg, 86 % yi e l d). LR M S ( ES I) m/ z cal cd 3 50 . 1 5; fou nd 35 1. 2 (M +1 ) +.

4 . 1. 51. N , N- Di met hyl - 5-ni tr o- 1 - ph en yl - 1Hb e n zo[ d ] i mi d azol - 2- a mi ne ( 8 0). 4 -n i tr o-N 1 -p he n yl b e nze ne -1, 2 -d i ami n e (2 00 mg, 0 . 8 7 mmol) wa s dis sol ved i n di chl or ome t ha ne ( 10 mL) . P hosge ne i mmi ni u m ch l ori d e ( 4 26 mg, 2. 62 mmol ) a nd tr ie thyl a mi ne ( 0. 73 mL, 5 . 2 4 mmol ) w e r e a dd e d t o t he s ol ut i on a nd t h e mi xt ur e was s tir re d at r oom t emp er at ur e ove r ni ght. Th e re a cti on mi xt u re w as car e full y s t op pe d w it h met h an ol, d il ut ed w it h D CM, w a she d wit h w at er, w as hed with b ri n e, dri e d ove r sod i um s ul fat e, a nd con ce n t r at ed . T he cr u de mi xt ur e w as p u ri fi ed b y fl a sh col umn ch r oma t ogr a ph y ( he x an e s wit h a gr a dien t of e t hyl a ce t at e 20 -10 0%) t o yi el d 16 0 mg ( 6 5% yi e l d) of 8 0 . 1 H NM R δ 2. 89 ( s, 6 H), 6. 9 4 ( d, J = 8 . 7 Hz, 1 H ), 7. 42 -7. 6 2 ( m, 5 H ), 7. 95 (d d, J = 2. 4, 8. 4 Hz, 1 H ), 8. 3 7 ( d, J = 2 . 4 H z, 1H ). LR M S ( ES I) m/ z ca l cd 2 82. 1 1; fou nd 28 3 . 6 (M +1 ) +, 30 5. 4 (M +N a ) +. 4 . 1. 52. N 2 , N 2 - Di met hyl - 1- phe n yl - 1Hb e n zo[ d ] i mi d azol e -2, 5- di a mi ne. T o a fl a s k con t a i ni n g 8 0 ( 1 60 mg, 0. 5 7 mmol ) in e t h an ol ( 1 0 mL) w as a dd e d 1 6 mg of 1 0% Pd / C . T he fl a sk w as p ur ge d 3 t i mes b y va cuu m an d e a ch t i me r efi ll ed wit h an at mosp h er e of H 2 . H 2 was i nt r odu ced t o t h e fl a s k a n d t h e mi xt ur e w as st irr ed a t 25 °C for 15 h. Th e mi xt ur e w as t hen filt er ed t hr ough a p a d of cel it e a nd t h e r es ul t i ng s ol u ti on w a s con cen t r at ed t o yi e l d N 2 , N 2 -di met h yl -1 -ph e n yl 1 H -b en zo[d ]i mi da zol e -2, 5-di ami n e ( 1 30 mg, 91 %). LR M S ( ES I) m/ z ca l cd 25 2. 1 4 ; fou n d 2 53 . 2 ( M+ 1)+ , 2 5 4. 4 7 (M +2) +. 2

2

4 . 1. 53. N , N - Di met hyl - N 5-( 4- met h yl pht h al a zi n1 - yl )-1- ph e nyl - 1H- be n zo[ d] i mi d azol e- 2 , 5d i ami ne•H Cl (8 2). T he h yd r ochl or i d e s alt of 8 2 ( 72 % yi e l d) was ob t ai n e d a s a yel l ow s ol i d us i ng t he pr oce d ure d e scri b ed for 1. LR M S ( ESI) m/ z ca l cd 39 4 . 19 ; fou nd 39 5. 00 ( M+ 1) +, 3 96. 0 0 ( M +2 ) +. 4 . 1. 54. 5 - Ni tro- 1 - phe n yl - 1H- be nzo[ d ] i mi da zol - 2 a mi ne ( 81). 4 -N it r o-N 1-p he n yl b en zen e -1, 2 -di ami n e 7 9 ( 20 0 mg, 0 . 8 7 mmol ) wa s di ss ol ve d i n D CM ( 10 mL ). C ya no gen br omi de ( 15 0 mg, 1. 42 mmol) wa s ad d ed t o t h e s ol ut i on an d t he re acti on mi xt ur e wa s st irr ed a t 2 5 ° C for 1 5 h . T LC i nd i cat e d t he rea cti on h ad n ot gone t o compl e ti on. 1 5 0 mg cya n o ge n br omi de w a s ad ded a n d t h e re acti on mi xt ure w a s st irr e d a t r e fl ux for a n ad di t i on al 2 h . Aft e r a ll ow i ng t he s ol ut i on t o c ool t o r oom t emp e ra t ur e a pr e ci pit ate w a s for med . T he p reci pit at e 81 ( 1 40 mg, 63% yi e l d) w a s col le ct ed by fi lt rat i on an d u s ed wit h ou t fu r t her p uri fi ca ti on . 1 H N MR (C DCl 3 + C D 3 O D ) δ 3 . 5 2 ( br s, 2H ), 7. 0 3 ( d, J = 9. 0 H z, 1 H), 7. 43 -7. 46 ( m, 2 H), 7. 6 3-7 . 68 ( m, 3 H), 8. 12 ( dd, J = 2. 4, 9 Hz, 1 H ), 8. 3 2 ( d, J = 1. 8 H z, 1H). LR M S ( ES I) m/z ca l cd 2 54. 0 8; fou nd 25 5 . 33 (M +1 )+ . 4 . 1. 55. 1 -P he n yl- 1H- be nzo[d ] i mi da zol e - 2, 5d i ami ne. 5 -N it r o-1-p he n yl -1 H -be n zo[d]i mi d a zol -2 -ami n e 81 ( 1 40 mg, 0. 5 5 mmol) wa s dis s ol ve d i n 4:1 e t h an ol/ wat er (5 mL). E xce s s s ol i d s odi um d it hi on i te w as ad d ed t o t h e s ol u ti on a nd t he

r e a cti on mi xt u re w a s he at ed t o 7 0 ° C w i t h st i rri ng for 1 h . Th e r ea cti on mi xt u re w a s di l ut e d with e t h yl a ce t at e a nd wa s he d wit h w at er. T he w ate r l a yer w as e xt ra ct e d s e ver al ti me s w it h et hyl a ce t ate . T he or ga ni c l a ye r s w er e combi n e d, dri e d ove r s odi u m s ul fat e, a nd con ce n t rate d t o yi e l d 1 -ph e nyl 1 H -be n zo [d ]i mi da zol e -2, 5 -di ami n e ( 5 2 mg, 42% yi e l d). LR M S ( ES I) m/ z ca l cd 2 2 4. 1 1; fou nd 22 5 . 07 ( M +1) +, 22 6. 3 3 ( M+ 2) + . 4 . 1. 56. N 5 -( 4- Met hyl ph t hal azi n - 1- yl) - 1- p he nyl 1 H- be nzo[ d] i mi da zol e - 2, 5- di ami ne ( 8 3). T he h yd r ochl or i d e s alt of 8 3 ( 3 9 % yi e l d) was ob t ai n e d a s a ye l l ow s ol i d us i ng t he p r oce d ure d e scri b ed for 1. LR M S ( ES I) m/ z ca l cd 36 6. 16 ; fou nd 36 7. 0 7 ( M+ 1) +, 3 88. 93 ( M +N a) +. 4 . 1. 57. 5 - Ni tro- 1 -p he nyl - 1Hb en zo[ d ][ 1, 2, 3] tr i a zol e ( 84 ). 4 -N it r o-N 1-p he n yl b en zen e -1, 2 -di a mi n e 7 9 (1 2 5 mg, 0 . 5 5 mmol ) w as di ss ol ve d i n chl or of or m ( 15 mL ). Is oam yl ni t rit e ( 1 mL, 7 . 4 mmol ) w as a dd ed t o t he s ol uti on a nd t h e r ea cti on mi xt ur e w as sti rr e d a t r oom t e mpe r at ur e for 1 h. T he s ol uti on w as c once nt r at e d t o gi ve 8 4 ( 1 31 mg, 10 0 % yi e l d) a nd u s ed w it h ou t furt h er pu ri fi cati on . 1 H N MR δ 7 . 57 7 . 7 9 ( m, 5 H ), 7. 8 7 ( d d, J = 0 .6 , 9 H z, 1H), 8. 4 7 (dd , J = 1. 8, 9 H z, 1 H), 9. 09 ( d d, J = 0. 6, 1. 8 H z, 1 H). 4 . 1. 58. 1 -P he nyl- 1H- be nzo[ d ] [ 1, 2, 3 ]t ri a zol - 5a mi ne. T o a fl as k cont ai n i ng 84 ( 13 1 mg, 0. 5 45 mmol) in 8 :1 e t h an ol /t et ra h yd r ofu r a n ( 20 mL) w as ad d ed 13 mg of 1 0% P d/ C. T he fla s k wa s pur ge d 3 t i me s by va cu u m a nd ea ch t i me r efil l ed wi th a n a t mos p here of H 2 . H 2 w as i nt r odu ced t o t h e fl ask a nd t he mi xt u re w a s stir red a t 25 ° C for 1 5 h. T he mi xt ure w a s t he n filt e re d t hr ough a pa d o f cel it e a nd t he r e s ult i ng s ol u ti on w a s con ce n t rat e d t o yi e l d 1 p he n yl -1 H -be n zo [d ][1, 2 , 3]tri a zol -5-a mi ne ( 3 5 mg, 3 1% yi e l d). 1 H N MR δ3. 92 ( br s, 2 H ), 7. 0 ( d d, J = 2 , 8. 7 H z, 1H), 7. 2 8 ( d d, J = 0. 6, 1. 8 H z, 1 H), 7. 45 7 . 6 2 ( m, 5 H) , 7. 74-7 . 78 ( m, 1 H). 4 . 1. 59. 4 - Met hyl -N -( 1- p he nyl - 1Hb en zo[ d ][ 1, 2, 3] tr i a zol - 5 - yl) p ht hal a zi n - 1a mi ne •H Cl ( 85 ). T he h yd r ochl or i d e s alt of 8 5 ( 7 1 % yi e l d) was ob t ai n e d usi n g t h e p r oce du r e d e scri b ed for 1 . LR MS (E S I) m/ z cal cd 3 52 . 1 4; foun d 35 2. 93 ( M +1) +, 37 4. 8 7 ( M+ N a) + . 4.2. Biology General. mESC normal growth media contained high glucose DMEM (prepared by supplementing it with 6% FBS and 5 µM βmercaptoethanol). Additional supplements including sodium pyruvate, non-essential amino acids, glutamine, and pen/strep were added to 1x from 100x stocks (Sigma, St. Louis, MO). Leukemia Inhibitory Factor (LIF) was added at 1 IU/µL (Millipore, Billerica, MA) to maintain mESC pluripotency during scale-up and maintenance. Mouse monoclonal anti-α−Actinin antibody (Millipore) was used at a dilution of 1:200. Donkey anti-mouse Alexa568 (Life Technologies, Carlsbad, CA) was used at a dilution of 1:400. Horse serum and Triton-X100 were used as specified (Sigma).

4 . 2. 1. R ob ot i c s a nd au tomati on The primary screen used a fully integrated Beckman FX fluid handler (Beckman Coulter; San Diego, CA) with a peripheral tip lift, plate hotel, incubation cytomat (ThermoFisher, Pittsburgh, PA) and rail mounted robotic arm. The microtiter plates were imaged at day 10 or day 12 using an IC100 Cell imaging system (Vala Sciences, San Diego, CA) and images were analyzed using Vala Sciences software. The confirmatory and SAR screens for cardiomyogenesis in mESCs used a fully integrated Hamilton STAR robotics system (Hamilton Robotics, Reno, NV) with incubation cytomat and plate hotel (ThermoFisher). The microtiter plates were imaged at day 10 or day 12 using an InCell 1000 (GE Healthcare; Piscataway, NJ) and images were analyzed by a custom built algorithm (see Supporting Information) using Developer Toolbox (GE Healthcare, Pittsburgh, PA). 4 . 2. 2. C omp ou nd ma na ge me nt The primary screen used diluted stocks in 100% DMSO (1 mg/mL daughter plate) dissolved in 20% DMSO in water. The library consisted of 13,360 compounds, the DIVERSet collection (Chembridge; San Diego, CA), at 2 doses of 1 µg/mL and 5 µg/mL. The SAR screen was conducted at 5 concentrations for most of the compounds examined (i.e., 0.78, 1.56, 3.12, 6.25, 12.5 µM). Test compounds were reconstituted in dry DMSO as a 50 mM stock concentration (based on mass of the HCl salt). Fluid transfers were handled with automated liquid handlers with requisite peripheral devices. 4 . 2. 3. mE SC c ult u r e a nd diff e ren ti ati on mESCs were thawed and expanded in feeder free conditions using normal growth media supplemented with LIF. Cells were passaged on poly-D-lysine coated tissue culture plastic 5 times prior to use in the assay. For differentiation experiments, cells were monodispersed and 50 µL were applied to gelatin coated, clear-bottom, black wall, 384-well microtiter plates in normal growth media without LIF. Seeding cell density was held at 92 cells/mm2 or about 1000 cells/well. Cells were grown at 37 °C and 5% CO2. After 2 days in normal growth media, compounds were added to wells by supplying an equivalent volume (50 µL) of normal growth media at 2-fold the final compound concentration. For the primary screen, compounds were refreshed on day 4 by aspirating 50 µL media from each well and replacing with 50 µL of normal growth media fortified with 1x the final compound concentration. Media was replenished with 50 µL of normal growth media on days 6 and 8. For the SAR screen, compounds were not added on day 4, but media was replenished with 50 µL of normal growth media on days 4, 6 and 8. The cell cultures were stopped on day 9 or 10 depending on the maturity of the culture as determined by control experiments. Cell cultures were terminated by adding 25 µL of 4% paraformaldehyde to each well and incubated for 40 minutes. Thereafter, the normal growth media was completely removed by aspiration. Microtiter plates were then washed three times with (1 mM, potassium phosphate, pH 7.4) and then incubated for 30 min with DAPI (Sigma) at 1 µg/mL. The cells were finally washed twice with PBS and then prepared for storage and imaging by adding 40 µL of PBS:Glycerol (1:1 v/v). 4 . 2. 4. I mmu nost a i ni n g mE SC c ul t u re s at da y 10 or da y 12 Cell cultures were terminated by removing media from the plate by aspiration and adding back 25 µL/well 4% paraformaldehyde (Sigma) for 40 minutes at room temperature. Cells were permeablized and blocked for 30 min using 1 x PBS

supplemented with 10% heat inactivated horse serum and 0.2% Triton X-100 (Sigma). Primary antibody (anti-α−Actinin, 15 µL/well) (Millipore) diluted 1:200 was incubated for 60 min, washed three times with 1 x PBS fortified with 0.2% Triton X100. The wells were incubated at 25 °C for 10 min between washes. The secondary antibody (donkey anti-mouse Alexa568) (Life Technologies) was diluted 1:400 in 1 x PBS with 0.2% Triton X-100 and 1 µg/mL DAPI and 15 µL of this solution was added to each well and incubated for 60 min. The cells were washed again 3 times as described above for the primary screen and then each well was stored in 40 µL 0.5x PBS and 50% glycerol prior to imaging. Acknowledgements We are grateful to Jia J. Ding and Tara Wu for helping with the synthesis of some of the benzimidazole compounds. We thank Dr. Marcia Dawson and Dr. Zebin Xia for their intellectual contribution and support throughout the duration of this project. We are grateful to Ying Su for her contribution to the cheminformatics preceding the SAR described in this work. The financial support provided by CIRM (RC1-00132 to Mark Mercola and RS1-00169-1 to John Cashman) and NIH (R37HL059502 and R01HL108176 to Mark Mercola) is gratefully acknowledged. Supplemental Data Synthesis and 1H NMR of compounds 4 – 15 and 17 – 29 and details of the image analysis and supporting figure and movie associated with this article can be found, in the online version, at… References and Notes 1. Roger, V.r.L.; Go, A.S.; Lloyd-Jones, D.M.; Benjamin, E.J.; Berry, J.D.; Borden, W.B.; Bravata, D.M.; Dai, S.; Ford, E.S.; Fox, C.S.; Fullerton, H.J.; Gillespie, C.; Hailpern, S.M.; Heit, J.A.; Howard, V.J.; Kissela, B.M.; Kittner, S.J.; Lackland, D.T.; Lichtman, J.H.; Lisabeth, L.D.; Makuc, D.M.; Marcus, G.M.; Marelli, A.; Matchar, D.B.; Moy, C.S.; Mozaffarian, D.; Mussolino, M.E.; Nichol, G.; Paynter, N.P.; Soliman, E.Z.; Sorlie, P.D.; Sotoodehnia, N.; Turan, T.N.; Virani, S.S.; Wong, N.D.; Woo, D.; Turner, M.B. Circulation 2012, 125, e2-e220. 2. Bergmann, O.; Bhardwaj, R.D.; Bernard, S.; Zdunek, S.; Barnabe-Heider, F.; Walsh, S.; Zupicich, J.; Alkass, K.; Buchholz, B.A.; Druid, H.; Jovinge, S.; Frisen, J. Science 2009, 324, 98-102. 3. Hsieh, P.C.; Segers, V.F.; Davis, M.E.; MacGillivray, C.; Gannon, J.; Molkentin, J.D.; Robbins, J.; Lee, R.T. Nat. Med. 2007, 13, 970-974. 4. Kajstura, J.; Urbanek, K.; Perl, S.; Hosoda, T.; Zheng, H.; Ogorek, B.; Ferreira-Martins, J.; Goichberg, P.; Rondon-Clavo, C.; Sanada, F.; D'Amario, D.; Rota, M.; Del Monte, F.; Orlic, D.; Tisdale, J.; Leri, A.; Anversa, P. Circ. Res. 2010, 107, 305-315. 5. Senyo, S.E.; Steinhauser, M.L.; Pizzimenti, C.L.; Yang, V.K.; Cai, L.; Wang, M.; Wu, T.D.; Guerquin-Kern, J.L.; Lechene, C.P.; Lee, R.T. Nature 2013, 493, 433-436. 6. Willems, E.; Lanier, M.; Forte, E.; Lo, F.; Cashman, J.; Mercola, M. J Cardiovasc. Transl. Res. 2011, 4, 340-350. 7. Lanier, M.; Schade, D.; Willems, E.; Tsuda, M.; Spiering, S.; Kalisiak, J.; Mercola, M.; Cashman, J.R. J. Med. Chem. 2012, 55, 697-708. 8. Schade, D.; Lanier, M.; Willems, E.; Okolotowicz, K.; Bushway, P.; Wahlquist, C.; Gilley, C.; Mercola, M.; Cashman, J.R. J. Med. Chem. 2012, 55, 9946-9957. 9. Willems, E.; Cabral-Teixeira, J.; Schade, D.; Cai, W.; Reeves, P.; Bushway, P.J.; Lanier, M.; Walsh, C.; Kirchhausen, T.; Izpisua Belmonte, J.C.; Cashman, J.; Mercola, M. Cell Stem Cell 2012, 11, 242-252. 10.Willems, E.; Spiering, S.; Davidovics, H.; Lanier, M.; Xia, Z.; Dawson, M.; Cashman, J.; Mercola, M. Circ. Res. 2011, 109, 360-364.

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