Preparation and Biological Evaluation of Indole, Benzimidazole, and

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Preparation and Biological Evaluation of Indole, Benzimidazole, and Thienopyrrole Piperazine Carboxamides: Potent Human Histamine H4 Antagonists Jennifer D. Venable,* Hui Cai, Wenying Chai, Curt A. Dvorak, Cheryl A. Grice, Jill A. Jablonowski, Chandra R. Shah, Annette K. Kwok, Kiev S. Ly, Barbara Pio, Jianmei Wei, Pragnya J. Desai, Wen Jiang, Steven Nguyen, Ping Ling, Sandy J. Wilson, Paul J. Dunford, Robin L. Thurmond, Timothy W. Lovenberg, Lars Karlsson, Nicholas I. Carruthers, and James P. Edwards Johnson & Johnson Pharmaceutical Research and Development, L.L.C., 3210 Merryfield Row, San Diego, California 92121 Received March 7, 2005

Three series of H4 receptor ligands, derived from indoly-2-yl-(4-methyl-piperazin-1-yl)methanones, have been synthesized and their structure-activity relationships evaluated for activity at the H4 receptor in competitive binding and functional assays. In all cases, substitution of small lipophilic groups in the 4 and 5-positions led to increased activity in a [3H]histamine radiolabeled ligand competitive binding assay. In vitro metabolism and initial pharmacokinetic studies were performed on selected compounds leading to the identification of indole 8 and benzimidazole 40 as potent H4 antagonists with the potential for further development. In addition, both 8 and 40 demonstrated efficacy in in vitro mast cell and eosinophil chemotaxis assays. Introduction Until recently, the pharmacological effects of the endogenous chemical mediator histamine were attributed to its interaction with three known G-protein coupled receptors H1, H2 and H3.1 The human histamine H1 receptor is expressed widely throughout the body and is involved in numerous functions including smooth muscle and endothelial cell contraction causing increases in vascular permeability.2,3 These effects account for the role of the H1 receptor in allergic and inflammatory responses and consequently for the application of H1 receptor antagonists for the treatment of allergies. Although the H2 receptor also participates in vasodilation, due to its expression in vascular smooth muscle, the major physiological role of this receptor is in the gastrointestinal tract where histamine stimulates acid secretion.4 Antagonists of this receptor are widely used for treatment of ulcerative conditions. The histamine H3 receptor behaves as a presynaptic autoreceptor on histamine neurons and as a heteroreceptor on nonhistamine neurons controlling the release of histamine and other neurotransmitters in the central nervous system.5,6 While the therapeutic importance has not been clinically defined, it has been implicated in various conditions involving deficits in arousal, vigilance, and cognition. The fourth human histamine receptor was disclosed in 2000 as a 390 amino acid GPCR with a 3743% sequence homology to the human H3 receptor.7-12 This high degree of similarity is reflected by the affinity of well-known H3 ligands thioperamide (1) and (R)-Rmethylhistamine (2) (Figure 1) for the H4 receptor.13 While the H3 receptor is found in the peripheral and central nervous systems, the H4 receptor is primarily expressed in eosinophils, mast cells, dendritic cells, and other leukocytes, implying that the H4 receptor may * To whom correspondence should be addressed. Phone: +1-858320-3365. Fax: +1-858-784-3116. [email protected].

Figure 1. Dual H3/H4 Ligands Thioperamide and (R)-RMethylhistamine and H4 Selective Antagonist 3.

play a key role in inflammation and regulation of the immune system.2,9,14,15 The H4 receptor is known to mediate chemotaxis of eosinophils and mast cells14-16,35,36 and is also implicated in histamine-induced release of interleukin-16 from CD-8+ T cells.2 Additionally, a selective H4 receptor antagonist demonstrated antiinflammatory activity in a mouse peritonitis model.16 A screen of our corporate compound collection in a [3H]histamine radiolabeled ligand competitive binding assay resulted in the identification of piperazine 3, which displayed a Ki ) 38 nM at the H4 receptor (Figure 1) with 240-fold selectivity over the H3 receptor. Analogues of indole 3 were synthesized and found to exhibit excellent affinity (4-150 nM) for the H4 receptor with 100-1500-fold selectivity over the H3 receptor. Further profiling in vitro and in vivo studies revealed a promising metabolic and pharmacokinetic profile for this series

10.1021/jm0502081 CCC: $30.25 © 2005 American Chemical Society Published on Web 11/23/2005

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of indolylpiperazines. Using these data, modifications were made to the heterocyclic nucleus of the pharmacophore with the intent of maintaining activity at the H4 receptor while exploring what effects this had on the metabolic and pharmacokinetic parameters. Therefore, each ring of the fused heterocycle was alternately replaced with an appropriate bioisostere, resulting in the development of the thienopyrroles and benzimidazole carboxamides as two additional series of potential H4 receptor ligands.

Venable et al. Table 1. Binding Affinity and Functional Data for Indole-Substituted Analogues

Results and Discussion Initial examination of the high-throughput screening hit, indolylpiperazine 3, revealed the N-methylpiperazine moiety as the optimal amine terminus, with all other modifications yielding decreased affinity for the H4 receptor, as previously disclosed.13 Thus, attention turned toward the indole core and substituent effects on the arene ring. As a result, a variety of substituted indolyl N-methylpiperazine-carboxamides (4-24, Table 1) were constructed, the synthesis of which was described in the previous report.13 The indole carboxamide analogues were first examined in a recombinant human histamine H4 competitive binding assay using [3H]histamine as the radioligand (Table 1). For compounds with Ki values of less than 75 nM, the functional antagonism was measured by Schild analysis of a forskolin-stimulated cAMP-mediated reporter gene assay in SK-N-MC cells and reported as a pA2 value. The parent N-methylpiperazine carboxamide 4 was essentially equipotent (Ki ) 17 nM) to the high-throughput screening hit 3 (Ki ) 38 nM) and displayed encouraging results in the functional assay with a pA2 ) 7.4. A considerable amount of information could be obtained by examining substitution at various positions around the indole nucleus. Placement of a bromine atom in the 4-position gave 5, which was equipotent to the unsubstituted indole 4 in the binding assay, whereas bromine substitution at C(6) (6) resulted in decreased affinity for the receptor. However, the 5-bromoindole 7 displayed increased affinity for the receptor (Ki ) 8 nM) and showed a marked improvement in the functional assay (pA2 ) 7.8). With this information in hand, we surveyed a variety of substituents at the indole 5-position. The 5-chloro and 5-fluoro compounds 8 and 9 were both found to be potent ligands for the H4 receptor, with indole 8 having excellent efficacy in the functional assay with a pA2 ) 8.1. Nonhalo substituents at C(5) (1012) reduced affinity for the receptor 10-100 fold compared to 4. Small electron-donating groups were also tolerated at this position, with the 5-hydroxy compound 13 and 5-aminoindole 14 retaining potent H4 activity (Ki ) 23 nM and 15 nM, respectively). Introduction of similar substituents at the 7-position (15-18) also showed improved activity in the binding and functional assays when compared with the parent 4. In general, the 5-position appears to have the greatest effect on in vitro potency. Interestingly, once the requisite 5-position is occupied, additional substituents in the 4 or 7-positions (19-20 and 21-24 respectively) are tolerated without a loss in H4 binding affinity. With the structure-activity relationships around the indole core evaluated, bioisosteric replacement18,19 of the indole arene ring with thiophene afforded a series of

a Displacement of [3H]histamine from the recombinant histamine H4 receptor. Ki values are the geometric mean ( SEM of three or more independent determinations and calculated according to Cheng and Prusoff.17 b Antagonism of histamine inhibition of forskolin-stimulated cAMP-mediated reporter gene activity in SK-N-MC cells expressing the human histamine H4 receptor.

analogous thienopyrrole carboxamides. The desired thienopyrrole system has two possible regioisomers: one isomer positions the sulfur and nitrogen on the same ring fusion carbon atom (head-to-head), alternatively sulfur and nitrogen may be placed on different carbons (head-to-tail). The head-to-head thienopyrroles were synthesized via Hemetsberger reaction of thiophene 27, derived from the condensation of thiophene-3-carboxaldehyde and ethyl azidoacetate (Scheme 1).20 Ester 28 could be functionalized further by treatment with Nchlorosuccinamide (NCS) to give the 5-chloro compound 29, yielding a similar substitution pattern to the indoles already described. After cyclization, the esters were hydrolyzed and coupled with N-methylpiperazine to give the desired amide products 30 and 31. The head-to-tail thienopyrroles were synthesized utilizing the same

Human Histamine H4 Antagonists

Scheme 1a


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Table 2. Activity of Thienopyrroles

a Reagents and conditions: (i) EtONa, EtOH, 0 °C; (ii) xylenes, 145 °C; (iii) NCS (1 equiv), AcOH, CHCl3, rt; (iv) LiOH, THF/ MeOH/H2O, 65 °C; (v) (COCl)2, cat. DMF, CH2Cl2, rt; or EDCI, CH2Cl2; (vi) N-methylpiperazine, CH2Cl2, rt.

Scheme 2a

a Displacement of [3H]histamine from the recombinant histamine H4 receptor. Ki values are the geometric mean ( SEM of three or more independent determinations and calculated according to Cheng and Prusoff.17 b Antagonism of histamine inhibition of forskolin-stimulated. cAMP-mediated reporter gene activity in SK-N-MC cells expressing the human histamine H4 receptor.

a

Reagents and conditions: (i) EtONa, EtOH, 0 °C; (ii) xylenes, 145 °C; (iii) LiOH, THF/MeOH/H2O, 65 °C; (iv) (COCl)2, cat. DMF, CH2Cl2, rt; (v) N-methylpiperazine, CH2Cl2, rt.

methodology starting with variously substituted thiophene-2-carboxaldehydes (Scheme 2).21,22 As postulated, replacement of the benzene portion of indole with thiophene did indeed yield potent antagonists at the human histamine H4 receptor (Table 2), although the unsubstituted compounds 30 and 32 displayed a 3-4-fold decrease in affinity in comparison with the parent indole 3. In the indole series, the most consistently active compounds were obtained from 5-substituted indoles (e.g. 8, Ki ) 4 nM, pA2 ) 8.1). A similar trend was found in both regioisomers of the thienopyrrole analogues; when either R4 or R5 of the thiophene moiety was substituted, activity at the H4 receptor was increased as demonstrated by compound 31 in the head-to-head series (R5 ) Cl) and compound 33 (R4 ) Cl) in the head-to-tail series with Kis of 25 and 40 nM, respectively. Substitution of R5 in the head to tail series also led to the H4 ligand 34 (Ki ) 21 nM). These effects appeared to be additive in this series with di-substituted thienopyrrole 35 having a 10-fold increase in affinity for the H4 receptor (Ki ) 3 nM) over either monosubstituted analogue 33 or 34. However this did not translate to a marked difference in the functional assay. Replacement of the pyrrole ring of the indole carboxamides for imidazole was also examined. This approach

has also been taken by Terzioglu et al.29 There are relatively few literature methods for constructing benzimidazole 2-carboxylic esters, acids and amides.23-29 One synthesis involves oxidation of 2-hydroxymethyl benzimidazole to the carboxylic acid30 followed by standard amide coupling. Unfortunately, many of the benzimidazole-2-carboxylic acids were found to be unstable, readily decarboxylating upon standing and under coupling conditions, giving only moderate to low yields of the desired compounds. Following the work of Holan,31 an alternative two-step route was developed avoiding this labile intermediate. Commercially available phenylenediamines were treated with methyl 2,2,2-trichloroacetimidate in glacial acetic acid to afford 2-trichloromethylbenzimidazoles,29,32 followed by ammination/ hydrolysis directly to the desired amides with N-methylpiperazine and excess sodium hydrogencarbonate in wet acetonitrile (Scheme 3). The benzimidazoles synthesized by this method were tested for affinity at the H4 receptor (Table 3). For simplicity, the numbering system in Table 3 follows that of the indole series. However, when comparing data for the benzimidazoles to that of the indole series, one must keep in mind that C(4) of the benzimidazole is equivalent to C(7) due to tautomerization of the benzimidazole moiety. Likewise, C(5) and C(6) are also interchangeable. The parent benzimidazole 36 was found to be comparable in activity to the parent N-methylpiperazinylindole 4. Contrary to the indole and thienopyrrole series, introduction of substituents in the 5-position (39-42) did not greatly improve affinity for the receptor.

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Scheme 3a

Venable et al. Table 4. In Vitro Metabolism and Phamacokinetic Data

a Reagents and conditions: (i) Methyl 2,2,2-trichloroacetimidate, AcOH, rt; (ii) LiOH, EtOH; N-methylpiperazine, NaHCO3, AcOH/ H2O, rt.

Table 3. Activity for Benzimidazole-2-carboxamides

a Displacement of [3H]histamine from the recombinant histamine H4 receptor. Ki values are the geometric mean ( SEM of three or more independent determinations and calculated according to Cheng and Prusoff.17 bAntagonism of histamine inhibition of forskolin-stimulated cAMP-mediated reporter gene activity in SK-N-MC cells expressing the human histamine H4 receptor.

This core was also less tolerant of substitution in general, as demonstrated by the reduced affinity of the 5-methylbenzimidazole 41 (Ki ) 528 nM). Di-substituted 5-fluoro-4-methylbenzimidazole 43 maintained similar activity to the corresponding indole analogue (20). The 4,6-disubstituted compound 45 maintained good affinity for the receptor but showed diminished activity in the functional assay. Interestingly, the 4,6-dichlorobenzimidazole 46 was 20-fold less active than the corresponding 5,7-substituted indole 22. In Vitro Metabolism and In Vivo Pharmacokinetics. On the basis of these results, the 5-chloroindole 8 (JNJ 7777120), and its direct analogues from the

thienopyrrole and benzimidazole systems, thienopyrrole 31 and benzimidazole 4033 (JNJ 10191584), were selected for further profiling. All three compounds were found to be potent ligands for the H4 receptor (Ki ) 4 nM, 25 nM, and 26 nM, respectively) and showed excellent functional antagonism in H4 receptor transfected cells with pA2’s ranging from 7.7 to 8.1. In vitro metabolism studies in both rat and human liver microsomes and S9 fraction were performed with each heterocyclic nucleus for comparison with the observed in vivo pharmacokinetic data (Table 4). The indolylpiperazine 8 showed a moderate rate of metabolism in both human and rodent with a t1/2 ) 28 min in human liver microsomes and t1/2 ) 64 min in rodent S9 fraction. This compares with an in-vivo half-life of 2h when dosed orally in rats at 10 mg/kg with a Cmax ) 1.8 µM. The oral bioavailability of the compound was confirmed by its activity in the zymosan induced mouse peritonitis model.16 From the in vitro metabolism results, it appeared that the chlorosubstituted head-to-head (31) thienopyrrole was metabolized similarly to the indole 8. Unfortunately, in vivo pharmacokinetic studies of 31 in rodents unexpectedly showed the compound had no oral bioavailability at 10 mg/kg and an extremely short intravenous half-life (t1/2 ) 6 min at 2 mg/kg). Replacement of the pyrrole ring of indole 8 with imidazole to give the benzimidazole 40 did show increased stability


Figure 2. Inhibition of mast cell (A) and eosinophil (B) chemotaxis by 8 (b)and 40 (O). The results shown are the average of at least two experiments and the error bars represent the S. E. M.

in both human and rodent liver microsomes (3-4 fold) and had added stability in rodent S9 fraction. However, this improvement in vitro did not translate to a longer half-life in vivo in rodent. Oral administration of benzimidazole 40 at 10 mg/kg in rats gave a profile similar to that of 8 with a Cmax ) 1.8 µM, but with a shorter half-life (1 h). In Vitro Mast Cell and Eosinophil Chemotaxis. As previously mentioned, the H4 receptor is primarily expressed on hematopoietic cells such as eosinophils and mast cells.15,34,35 Both types of effector cells are important during an inflammatory response and have been implicated in the pathology of several diseases including asthma and rheumatoid arthritis. In recent publications, we described the in vitro histamine-induced chemotaxis of mouse bone-marrow mast cells and human eosinophils.14-16 Using specific histamine receptor antagonists, as well as mast cells derived from H4 receptor deficient mice, this chemotaxis was determined to be an H4-mediated effect. The induction of eosinophil chemotaxis by histamine is also mediated by the H4 receptor.35,36 We have previously shown that 8 can inhibit both eosinophil and mast cell chemotaxis with IC50 values of 86 nM and 40 nM, respectively.16,35 In the present study a second H4 antagonist, 40, was shown to be efficacious in both assays, inhibiting chemotaxis of eosinophils and mast cells with IC50 values of 530 nM and 138 nM, respectively (Figure 2). The relative potency of the indole 8 and benzimidazole 40 in the chemotaxis assays is consistent with the differences in their affinity for the H4 receptor. Conclusion After determining the indolyl piperazine 3 was a potent human histamine H4 receptor antagonist, a medicinal chemistry effort based on this series led to the synthesis of the 5-chloroindole 8 (Ki ) 4 nM). Alternately replacing each ring of the indole moiety with an appropriate bioisostere yielded two additional series of potent H4 antagonists, the thienopyrrole and benz-


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imidazole carboxamides. Specifically, the activity of the indoles was matched in the head-to-tail series of the thienopyrroles by substitution with small lipophilic groups (e.g. 35, Ki ) 3 nM), whereas analogues from the benzimidazole series were generally found to be 5-10-fold less potent than the corresponding indole carboxamide (e.g. 40, Ki ) 26 nM). Comparison of the chloro-substituted indole 8, thienopyrrole 31, and benzimidazole 40 in in vitro metabolism studies demonstrated the effect of the heterocyclic core on in vitro stability. The benzimidazole 40 showed an increased stability over the indole 8 and thienopyrrole 31, with half-lives 2-4-fold longer in human liver microsomes and 1.5-2-fold longer in rodent S9 fraction; however, the same was not observed in vivo. The indole and benzimidazole displayed similar bioavailability with Cmax ) 1.8 µM in both cases, however the benzimidazole had a 2-fold shorter half-life. The thienopyrrole 31 had no oral bioavailability in-vivo in rat. The efficacy of the two orally bioavailable compounds, indole 8 and benzimidazole 40, were examined in both the human eosinophil and mouse bone-marrow mast cell chemotaxis assays. These compounds were able to block chemotaxis of these inflammatory cells in vitro with IC50s from 40 to 530 nM. Additionally, compounds 8 (JNJ 7777120) and 40 (JNJ 10191584) have been shown to be selective for the H4 receptor over the H3 receptor (Ki ) 5125 ( 1087)16 and 14053 ( 4878 nM, respectively) and displayed no cross-reactivity in a CEREP panel of 50 other receptors, enzymes, transporters, and ion-channels. We hope to utilize the compounds developed from all three series of potent human histamine H4 antagonists to further discern the role of the newest histamine receptor in inflammatory pathways. Experimental Section General Experimental Information. 1H NMR spectra were recorded on Bruker DPX-400 or DPX-500 NMR spectrometers in the specified deuterated solvent. Column chromatography was performed employing an Isco SG100 automated flash chromatography system using prepacked silica gel columns purchased from either Isco, Inc. or Biotage, Inc. Low resolution mass spectra (MS) were determined on an Agilent HP1100/MSD. High-resolution mass spectra (HRMS) were determined on a Bruker MicroTOF with Eksigent capillary HPLC express-100 using a Zorbax 300SB-C18 (3.5 micrometer, 50 × 0.3 mm), 100 nL injection, 100% ACN (with 0.1% formic acid) for 3 min at 10 µL/min. Elemental analyses were performed by Desert Analytics, Tucson, AZ, or Numega Laboratories, San Diego, CA, and are within (0.4% of the calculated values unless otherwise stated. IR spectra were obtained on a Thermo-Nicolet Avatar 360. Reagents were purchased commercially and used without purification unless otherwise stated. Abbreviations used below include CDI (1,1′carbonyldiimidazole), DMF (N,N-dimethylformamide), EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), HATU (O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate), HOAT (1-hydroxy-7-azabenzotriazole), HOBT (1-hydroxybenzotriazole hydrate), TFA (trifluoroacetic acid), and THF (tetrahydrofuran). The synthesis of compounds 3-9, 12-19, and 21-23 has been previously described.13 (5-Methyl-1H-indol-2-yl)-(4-methyl-piperazin-1-yl)-methanone (10). A mixture of 5-methylindole-2-carboxylic acid (1.0 g, 5.7 mmol) and EDCI (1.6 g, 8.3 mmol) in CH2Cl2 (10 mL) was treated with 1-methylpiperazine (0.63 mL, 5.7 mmol) and stirred at room temperature for 18 h. The reaction mixture was quenched with saturated NaHCO3 solution and extracted with CH2Cl2 and dried over MgSO4, filtered, and concentrated

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under reduced pressure to yield the crude product as a pale yellow solid. The crude product was purified via silica gel chromatography (0-100% acetone/CH2Cl2) to give the title compound (1.16 g, 79%). 1H NMR (400 MHz, CDCl3): δ 9.04 (br s, 1H), 7.39 (d, J ) 0.7 Hz, 1H), 7.29 (d, J ) 8.4 Hz, 1H), 7.09 (dd, J ) 8.4, 1.4 Hz, 1H), 6.67 (m, 1H), 3.92 (br s, 4H), 2.47 (t, J ) 5.1 Hz, 4H), 2.42 (s, 3H), 2.32 (s, 3H); Anal. (C15H19N3O) C, H, N. (5-Trifluoromethyl-1H-indol-2-yl)-(4-methyl-piperazin1-yl)methanone (11). The title compound was prepared according to the procedure described for compound 10. 1H NMR (400 MHz, CDCl3): δ 10.94 (s, 1H), 7.93 (s, 1H), 7.48 (dd, J ) 13.1 Hz, 9.0 Hz, 2H), 6.84 (d, J ) 1.6 Hz, 1H), 4.00 (br s, 4H), 2.53 (t, J ) 4.9 Hz, 4H), 2.36 (s, 3H); MS (electrospray): mass calculated for C15H16F3N3O, 311.12; m/z found, 312.1 [M+ + H], Anal. (C15H16F3N3O); C, H, N. (5-Fluoro-4-methyl-1H-indol-2-yl)-(4-methyl-piperazin1-yl)-methanone (20). The title compound was prepared according to the procedure described for compound 10. 1H NMR (400 MHz, CDCl3): δ 9.98 (br s, 1H), 7.18 (dd, J ) 4.89 Hz, 3.9 Hz, 1H), 6.99 (t, J ) 9.8 Hz, 1H), 6.74 (d, J ) 2.2 Hz, 1H), 3.99 (br s, 4H), 3.51 (t, J ) 5.3 Hz, 4H), 2.44 (s, 3H), 2.36 (s, 3H); MS (electrospray): exact mass calculated for C15H18FN3O, 275.14; m/z found, 276.2 [M+ + H], Anal. (C15H18FN3O); C, H, N. (7-Chloro-5-methyl-1H-indol-2-yl)-(4-methyl-piperazin1-yl)-methanone (24). The title compound was prepared according to the procedure described for compound 10.1H NMR (400 MHz, CDCl3): δ 9.71 (br s, 1H), 7.20 (s, 1H), 7.30 (s, 1H), 7.11 (s, 1H), 6.71 (d, J ) 2.3 Hz, 1H), 3.95 (br s, 4H), 2.50 (t, J ) 4.80 Hz, 4H), 2.42 (s, 6H), 2.35 (s, 3H); MS (electrospray): mass calculated for C15H18ClN3O, 291.11; m/z found, 292.2 [M+ + H], Anal. (C15H18ClN3O) C, H, N. General Procedures for the Preparation of Compounds 30-35. General Procedure A: Annulation of Aldehyde with Ethyl Azidoacetate. A solution of the aldehyde (1 equiv) and ethyl azidoacetate (4 equiv) was added dropwise to a solution of NaOEt (4 equiv) in ethanol (0.15 M) at 0 °C. The reaction mixture was stirred at 0 °C for 1 h and room temperature for an additional 1 h. The reaction mixture was then poured into sat. NH4Cl and extracted with ether. The combined organics were dried (Na2SO4) and concentrated in vacuo. The residue was purified by silica gel column chromatography to provide the desired acrylate. A solution of the resultant acrylate in xylene (0.2 M) was heated at 145 °C for 10-60 min and then allowed to cool to room temperature. The xylene solution was either cooled further to induce product crystallization or directly subjected to silica gel column chromatography to obtain the desired annulation product. General Procedure B: Ester Hydrolysis. A solution (0.2 M) of the ethyl ester (1 equiv, from General Procedure A) and LiOH (5 equiv) in THF/MeOH/H2O (3:1:1) was heated at 65 °C overnight, cooled to room temperature, acidified with 2 N HCl, and extracted with EtOAc. The organic layer was separated, dried over Na2SO4, and concentrated to give the desired crude acid which was taken to the next step without further purification. General Procedure C: Amide Formation Using 1-(3Dimethylaminopropyl)-3-ethylcarbodimide Hydrochloride (EDCI). A mixture of acid (1 equiv, from General Procedure B), amine (1.5 equiv), and EDCI (2.0 equiv) in CH2Cl2 (0.2 M) was stirred at room-temperature overnight and then partitioned between CH2Cl2 and sat. NaHCO3. The organic layer was separated, washed with H2O, dried over Na2SO4, and concentrated. The crude product was further purified with silica gel column chromatography. General Procedure D: Amide Formation via Acyl Chloride Intermediate. A mixture of acid (1 equiv, from General Procedure B) in CH2Cl2 (0.5 M) was treated at 0 °C with oxalyl chloride (1.2 equiv) followed by 1-2 drops of DMF. The reaction mixture was stirred at 0 °C for 30 min then slowly warmed to room temperature and stirred for an additional 1 h. All volatiles were removed to provide the crude acyl chloride. The resultant acyl chloride was treated with amine (5.0 equiv)

in CH2Cl2 (0.2 M) and allowed to stir at room temperature for 3 h. The reaction mixture was partitioned between CH2Cl2 and sat. NaHCO3. The organic layer was separated, washed with H2O, dried over Na2SO4, and concentrated. The crude product was further purified with silica gel column chromatography. 6H-Thieno[2,3-b]pyrrole-5-carboxylic Acid Ethyl Ester (28). Thiophene-3-carbaldehyde (2.24 g, 20 mmol) was annulated according to General Procedure A to provide 6H-Thieno[2,3-b]pyrrole-5-carboxylic acid ethyl ester (1.2 g, 31%) as a white solid. TLC (silica, 20% EtOAc/hexanes): Rf ) 0.50. 1H NMR (CDCl3, 400 MHz): δ 10.30 (br s, 1H), 7.10 (d, J ) 1.9 Hz, 1H), 6.96 (d, J ) 5.4 Hz, 1H), 6.87 (d, J ) 5.4 Hz, 1H), 4.39 (q, J ) 7.1 Hz, 2H), 1.38 (t, J ) 7.1 Hz, 3H). (4-Methyl-piperazin-1-yl)-(6H-thieno[2,3-b]pyrrol-5yl)-methanone (30). 6H-Thieno[2,3-b]pyrrole-5-carboxylic acid ethyl ester (835 mg, 4.3 mmol) was hydrolyzed according to General Procedure B to provide the crude acid as a paleyellow solid. 1H NMR (CD3OD, 400 MHz): 7.02 (s, 1H), 6.96 (s, 1H), 6.95 (s, 1H). The carboxylic acid (60 mg, 0.35 mmol) was coupled with N-methylpiperazine according to General Procedure C to provide the title compound (44 mg, 50%) as a light yellow solid. TLC (silica, 10% MeOH/CH2Cl2): Rf ) 0.4. MS (electrospray): mass calculated for C12H15N3OS, 249.34; m/z found, 250.1 [M+ + H]. 1H NMR (CD3OD, 400 MHz, TFA salt): 6.97 (s, 1H), 6.96 (s, 1H), 6.85 (s, 1H), 4.20-3.10 (m, 8H), 2.96 (s, 3H). Anal. (C12H15N3OS) C, H, N, S. 2-Chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic Acid Ethyl Ester (29). A solution of compound 28 (580 mg, 3.0 mmol) in acetic acid (6 mL) and CHCl3 (6 mL) was treated with three portions of N-chlorosuccinimide (total 415 mg, 3.15 mmol) at 0 °C over 2 h. The reaction mixture was slowly warmed to room temperature and stirred overnight. CHCl3 was then removed, and the residue was basified with 4 N NaOH and extracted with EtOAc. The combined organics were washed with sat. NaHCO3, dried over Na2SO4, and concentrated. Column chromatography (silica, 5-10% EtOAc/hexanes) gave 600 mg (88%) of 2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid ethyl ester as a white solid. TLC (silica, 20% EtOAc/hexanes): Rf ) 0.5. 1H NMR (CDCl , 400 MHz): δ 10.50 (br s, 1H), 6.97 (d, J ) 3 2.0 Hz, 1H), 6.85 (s, 1H), 4.39 (q, J ) 7.2 Hz, 2H), 1.35 (t, J ) 7.2 Hz, 3H). (2-Chloro-6H-thieno[2,3-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone (31). Compound 29 (102 mg, 0.45 mmol) was hydrolyzed (General Procedure B) and coupled with N-methylpiperazine (procedure D) to provide the title compound (102 mg, 80% for two steps) as an off-white solid. TLC (silica, 10% MeOH/CH2Cl2): Rf ) 0.4. HRMS (electrospray): calculated for C12H14ClN3OS, 284.0619; m/z found, 284.0611 [M+ + H]. 1H NMR (CDCl3, 400 MHz): δ 10.50 (br s, 1H), 6.87 (s, 1H), 6.61 (d, J ) 1.8 Hz, 1H), 3.92 (t, J ) 5.1 Hz, 4H), 2.50 (t, J ) 5.1 Hz, 4H), 2.35 (s, 3H). Anal. (C12H14ClN3OS‚C4H4O4) calcd C, 48.06; H, 4.54; N, 10.51, S, 8.02. Found C, 47.58. H, 4.07; N, 10.95; S, 8.50. (4-Methyl-piperazin-1-yl)-(4H-thieno[3,2-b]pyrrol-5yl)-methanone (32). A solution of thiophene-2-carbaldehyde (1.10 mL, 11.7 mmol) was annulated according to General Procedure A to provide 4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester as a yellowish solid. 1H NMR (400 MHz, CDCl3) δ 9.06 (br s, 1H), 7.33 (d, J ) 5.3 Hz, 1H), 7.14 (m, 1H), 6.96 (m, 1H), 4.37 (q, J ) 7.3 Hz, 2H), 1.39 (t, J ) 7.3 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 161.3, 140.9, 129.2, 126.9, 124.6, 110.9, 107.3, 60.4, 14.2. The ethyl ester (98.5 mg, 0.50 mmol) was then hydrolyzed according to General Procedure B and coupled with N-methylpiperazine (General Procedure C) to provide the title compound in 51% yield. 1H NMR (400 MHz, CDCl3) δ 9.26 (br s, 1H), 7.26 (m, 1H), 6.97 (m, 1H), 6.74 (m, 1H), 3.99 (m, 4H), 2.59 (m, 4H), 2.43 (br, 3H). MS (electrospray): mass calculated for C12H15N3OS, 249.34; m/z found, 250.1 [M+ + H]. Anal. (C12H15N3OS) C, H, N, S. (2-Chloro-4H-thieno[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone (33). 5-Chloro-thiophene-2-carbaldehyde (2.92 g, 20 mmol) was annulated according to General Procedure A to provide the annulated product (2.8 g, 61%) as a white solid. TLC (silica, 20% EtOAc/hexanes): Rf ) 0.48.



NMR (CDCl3, 400 MHz): δ 9.10 (br s, 1H), 7.04 (dd, J ) 1.9, 0.7 Hz, 1H), 6.89 (d, J ) 0.7 Hz, 1H), 4.37 (q, J ) 7.2 Hz, 2H), 1.39 (t, J ) 7.2 Hz, 3H). The ethyl ester (230 mg, 1.0 mmol) was hydrolyzed (General Procedure B) and coupled with N-methylpiperazine (General Procedure C) to provide compound 33 (128 mg, 45% for two steps) as an off-white solid. TLC (silica, 10% MeOH/CH2Cl2): Rf ) 0.4. HRMS (electrospray): mass calculated for C12H14ClN3OS, 284.0619; m/z found, 284.0615 [M+ + H]. 1H NMR (CDCl3, 400 MHz): δ 10.1 (br s, 1H), 6.88 (s, 1H), 6.64 (d, J ) 1.4 Hz, 1H), 3.91 (t, J ) 4.4 Hz, 4H), 2.49 (t, J ) 5.1 Hz, 4H), 2.35 (s, 3H). Anal. (C12H14ClN3OS‚C4H4O4) calcd C, 48.06; H, 4.54; N, 10.51, S, 8.02. Found C, 47.53; H, 3.89; N, 11.25; S, 8.60. (4-Methyl-piperazin-1-yl)-(3-methyl-4H-thieno[3,2-b]pyrrol-5-yl)-methanone (34). A solution of 3-methylthiophene (6.76 mL, 70 mmol) in ether (70 mL) was treated with n-butyllithium (2.5 M in hexanes, 28.6 mL, 71.4 mmol) at such a rate that a slight reflux was maintained. The reaction mixture was heated to reflux for 15 min and DMF (7.0 mL, 91 mmol) in ether (30 mL) was added. After stirring for 4 h, the reaction was quenched with addition of sat. NH4Cl (200 mL). The organic layer was separated and washed with brine, H2O, dried over Na2SO4, and concentrated. Column chromatography (silica, 5-10% EtOAc/hexanes) provided a mixture of 4-methylthiophene-2-carbaldehyde and 3-methyl-thiophene-2-carbaldehyde (4.4:1, 8.1 g, 92%) as a light yellow oil. TLC (silica, 10% EtOAc/hexanes): Rf ) 0.55. For 4-methyl-thiophene-2carbaldehyde: 1H NMR (CDCl3, 400 MHz): δ 9.95 (s, 1H), 7.58 (d, J ) 1.2 Hz, 1H), 7.37-7.35 (m, 1H), 2.32 (s, 3H). For 3-methyl-thiophene-2-carbaldehyde: 1H NMR (CDCl3, 400 MHz): 10.02 (s, 1H), 7.64 (d, J ) 4.6 Hz, 1H), 6.97 (d, J ) 4.6 Hz, 1H), 2.58 (s, 3H). The mixture of 4-methyl-thiophene-2carbaldehyde and 3-methyl-thiophene-2-carbaldehyde (2.84 g, 22.5 mmol) was annulated according to General Procedure A to provide the 3-methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester (2.5 g, 65%) as a white solid. TLC (silica, 20% EtOAc/hexanes): Rf ) 0.45. 1H NMR (CDCl3, 400 MHz): δ 9.95 (br s, 1H), 7.12 (d, J ) 1.9 Hz, 1H), 6.90 (d, J ) 1.2 Hz, 1H), 4.39 (q, J ) 7.2 Hz, 2H), 2.35 (s, 3H), 1.39 (t, J ) 7.2 Hz, 3H). The ethyl ester (200 mg, 0.96 mmol) was then hydrolyzed (Procedure B) and coupled with N-methylpiperazine (General Procedure D) to provide compound 34 (197 mg, 78% for two steps) as an off-white solid. TLC (silica, 10% MeOH/CH2Cl2): Rf ) 0.4. HRMS (electrospray): mass calculated for C13H17N3OS, 264.1165; m/z found, 264.1155 [M+ + H]. 1H NMR (CDCl3, 400 MHz): δ 11.10 (br s, 1H), 6.76 (d, J ) 1.2 Hz, 1H), 6.69 (d, J ) 2.0 Hz, 1H), 3.94-3.90 (m, 4H), 2.47 (t, J ) 5.1 Hz, 4H), 2.33 (s, 3H), 2.25 (s, 3H). Anal. (C13H17N3OS‚C4H4O4) calcd C, 53.81; H, 5.58; N, 11.07; S, 8.45. Found C, 49.81; H, 5.50; N, 13.86; S, 7.58. (2-Chloro-3-methyl-4H-thieno[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone (35). 5-Chloro-4-methyl-thiophene-2-carbaldehyde (5.36 g, 33 mmol) was annulated according to Procedure A to provide 2-chloro-3-methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester (4.4 g, 55%) as a white solid. TLC (silica, 20% EtOAc/hexanes): Rf ) 0.45. 1H NMR (CDCl3, 400 MHz): δ 12.30 (br s, 1H), 7.04 (s, J ) 1H), 4.37 (q, J ) 7.2 Hz, 2H), 2.28 (s, 3H), 1.32 (t, J ) 7.2 Hz, 3H). The ethyl ester (243 mg, 1.0 mmol) was hydrolyzed (Procedure B) and coupled with N-methylpiperazine (Procedure D) to provide compound 35 (178 mg, 60% for two steps) as a white solid. TLC (silica, 10% MeOH/CH2Cl2): Rf ) 0.4. MS (electrospray): mass calculated for C13H16ClN3OS, 297.80; m/z found, 298.1 [M+ + H]. 1H NMR (CDCl3, 400 MHz): 11.1 (br s, 1H), 6.62 (d, J ) 1.4 Hz, 1H), 3.92 (m, 4H), 2.48 (t, J ) 5.1 Hz, 4H), 2.33 (s, 3H), 2.18 (s, 3H). Anal. (C13H16ClN3OS‚C4H4O4) C, H, N, S. General Procedure E: Preparaton of 2-Trichloromethyl-1H-benzimidazoles. 2-Trichloromethyl-1H-benzoimidazole. Methyl 2,2,2-trichloroacetimidate (1.63 mL, 9.22 mmol) was added to a solution of phenylenediamine (1.0 g, 9.2 mmol) in acetic acid (30 mL), which was then stirred at room temperature for 1 h. Water was added (20 mL) to the mixture, and the resultant precipitate was collected. The solid

was washed with water (2 × 30 mL) and dried under vacuum to afford 1.90 g (88%) of 2-trichloromethyl-1H-benzoimidazole which was used without further purification. MS (electrospray): mass calculated for C8H5Cl3N2, 234.0; m/z found 235.0, [M + H]+. 1H NMR (400 MHz, CDCl3): δ 13.45 (br s, 1H), 7.68 (br m, 2H), 7.35 (m, 2H). 4-Methyl-2-trichloromethyl-1H-benzoimidazole. The reaction was carried out as described in General Procedure E employing 2,3-diaminotoluene. Purification via silica gel chromatography (40% EtOAc/hexanes) afforded 830 mg (34%) of the title compound. MS (electrospray): mass calculated for C9H7Cl3N2, 247.97; m/z found 249.0, [M + H]+. 1H NMR (400 MHz, CDCl3): δ 9.78 (s, 1H), 7.52 (br s, 1H), 7.27 (d, J ) 7.4, 8.1 Hz, 1H), 7.17 (d, J ) 7.4 Hz, 1H), 2.64 (s, 3H). 5-Fluoro-2-trichloromethyl-1H-benzoimidazole. The reaction was carried out as described in General Procedure E using 4-fluoro- 1,2-phenylenediamine (1.0 g, 8.12 mmol) Trituration of the resulting precipitate afforded 1.20 g (60%) of the title compound. MS (electrospray): mass calculated for C8H4Cl3FN2, 251.9; m/z found 253.0, [M + H]+. 1H NMR (400 MHz, CDCl3): δ 7.64 (br s, 1H), 7.31 (br s, 1H), 7.07 (dt, J ) 2.27, 9.1 Hz, 1H). 5-Methyl-2-trichloromethyl-1H-benzoimidazole. The reaction was carried out as described in General Procedure E using 3,4-diaminotoluene Purification via silica gel chromatography (40% EtOAc/hexanes) afforded 980 mg (36%) of the title compound. MS (electrospray): mass calculated for C9H7Cl3N2, 247.97; m/z found 249.0, [M + H]+. 1H NMR (400 MHz, CDCl3): δ 9.77 (br s, 1H), 7.60 (br s, 1H), 7.43 (br s, 1H), 7.19 (dd, J ) 1.3, 8.6 Hz, 1H), 2.50 (s, 3H). 2-Trichloromethyl-5-trifluoromethyl-1H-benzoimidazole. The reaction was carried out as described in General Procedure E using 4-(trifluoromethyl)-1,2-phenylenediamine (1.0 g, 5.68 mmol). Purification via silica gel chromatography (40% EtOAc/hexanes) afforded 930 mg (54%) of the title compound. MS (electrospray): mass calculated for C9H4Cl3F3N2, 301.94; m/z found 303.0, [M + H]+. 1H NMR (400 MHz, CDCl3) rotamers: δ 10.16 (br s, 1H), 8.18 (br s, 0.55H), 7.98 (br d, J ) 8.1 Hz, 0.5H), 7.83 (br s, 0.45H), 7.64 (m, 1.5H). General Procedure F. Preparation of (1H-Benzoimidazol-2-yl)-(4-methyl-piperazin-1-yl)-methanone (36). To a suspension of 2-trichloromethyl-1H-benzoimidazole (100 mg, 0.42 mmol) in acetonitrile/water (3:1 ratio, 4.0 mL) was added N-methylpiperazine (0.93 mL, 0.84 mmol) followed by 4 M K2CO3 (0.30 mL). The reaction was stirred for 24 h before diluting with NaHCO3 (3 mL) and extracting with dichloromethane (3 × 5 mL). The organic extract was dried and evaporated to give a crude product which was purified by chromatography over silica gel (eluent: 4% MeOH/CH2Cl2) to afford 54 mg (52%) of the title compound. MS (electrospray): mass calculated for C13H16N4O, 244.1; m/z found 245.2, [M + H]+. 1H NMR (400 MHz, DMSO): δ 13.20 (s, 1H), 7.74 (d, J ) 7.3 Hz, 2H), 7.53 (d, J ) 8.3 Hz, 2H), 7.30 (m, 2H), 4.43 (m, 2H), 3.71 (t, J ) 5.2 Hz, 2H), 2.41 (m, 4H), 2.22 (s, 3H). 13C NMR (400 MHz, DMSO): 158.1, 145.5, 142.3, 133.2, 124.1, 122.4, 120.1, 112.2, 55.0, 54.4, 46.0, 45.5, 42.3. Anal. (C13H16N4O), C, H, N. (4-Methyl-1H-benzoimidazol-2-yl)-(4-methyl-piperazin1-yl)-methanone (37). The reaction was carried out as described in General Procedure F using 4-methyl-2-trichloromethyl-1H-benzoimidazole (100 mg, 0.40 mmol). Purification afforded 51 mg (50%) of the title compound. MS (electrospray): mass calculated for C14H18N4O, 258.15; m/z found 259.2, [M + H]+. 1H NMR (400 MHz, CDCl3) rotamers: δ 11.53 (br s, 1H), 7.64 (d, J ) 8.3 Hz, 0.5H), 7.32 (d, J ) 8.3 Hz, 0.5H), 7.22 (m, 1H), 7.10 (t, J ) 7.3 Hz, 1H), 4.85 (br m, 1H), 4.78 (br m, 1H), 3.94 (m, 2H), 2.66 (s, 1.5H), 2.57 (m, 4H), 2.50 (s, 1.5H), 2.36 (s, 3H). Anal. (C14H18N4O), C, H, N. (4-Methyl-piperazin-1-yl)-(4-nitro-1H-benzoimidazol2-yl)-methanone. The reaction was carried out as described in General Procedure F employing 4-nitro-2-trichloromethyl1H-benzoimidazole (1.18 g). Filtration and purification by recrystallization from ethyl acetate afforded 800 mg (66%) of the title compound. MS (electrospray): mass calculated for C13H15N5O3, 289.12; m/z found 290.4, [M + H]+. 1H NMR (400

1H

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MHz, MeOD): δ 7.37 (d, J ) 8.2 Hz, 1H), 7.27 (t, J ) 7.6 Hz, 1H), 7.37 (d, J ) 7.6 Hz, 1H), 4.12 (m, 2H), 3.82 (m, 2H), 2.45(m, 4H), 2.36 (s, 3H). (4-Amino-1H-benzoimidazol-2-yl)-(4-methyl-piperazin1-yl)-methanone (38). The reaction was carried out as described for compound 14 employing 4-Methyl-piperazin-1yl)-(4-nitro-1H-benzoimidazol-2-yl)-methanone (640 mg, 2.21 mmol) to afford 519 mg (91% yield) of the title compound. MS (electrospray): mass calculated for C13H17N5O, 259.14; m/z found 260.4, [M + H]+. 1H NMR (400 MHz, CDCl3): δ 12.1 (br s, 1H), 7.12 (t, J ) 7.9 Hz, 1H), 7.89 (m, 1H), 7.53 (d, J ) 7.9 Hz, 1H), 4.84 (m, 2H), 4.40 (m, 2H), 3.93 (m, 2H), 2.55(m, 4H), 2.35 (s, 3H). Anal. (C13H17N5O), C, H, N. (5-Fluoro-1H-benzoimidazol-2-yl)-(4-methyl-piperazin1-yl)-methanone (39). The reaction was carried out as described in General Procedure F using 5-fluoro-2-trichloromethyl-1H-benzoimidazole (100 mg, 0.39 mmol). Purification afforded 28 mg (27%) of the title compound. MS (electrospray): mass calculated for C13H15FN4O, 262.12; m/z found 236.2, [M + H]+. 1H NMR (400 MHz, CDCl3) rotamers: δ 11.55 (br s, 1H), 7.74 (br s, 0.5H), 7.46 (br s, 1H), 7.18 (br m, 0.5H), 7.08 (br m, 1H), 4.78 (m, 2H), 3.94 (m, 2H), 2.58 (m, 4H), 2.37 (s, 3H). Anal. (C13H15FN4O) C, H, N, F. (5-Chloro-1H-benzoimidazol-2-yl)-(4-methyl-piperazin1-yl)-methanone (40). The reaction was carried out as described in General Procedure F with commercially available 5-chloro-2-trichloromethyl-1H-benzoimidazole (100 mg, 0.37 mmol). Purification afforded 65 mg (63%) of the title compound. MS (electrospray): mass calculated for C13H15ClN4O, 278.1; m/z found 279.2, [M + H]+. 1H NMR (400 MHz, DMSO): 13.29 (s, 1H), 7.67 (br s, 2H), 7.33 (d, J ) 8.6 Hz, 2H), 4.49 (m, 2H), 3.71 (t, J ) 4.6 Hz, 2H), 2.40 (m, 4H), 2.22 (s, 3H). Anal. (C13H15ClN4O), C, H, N, Cl. (5-Methyl-1H-benzoimidazol-2-yl)-(4-methyl-piperazin1-yl)-methanone (41). The reaction was carried out as described in General Procedure F using 5-methyl-2-trichloromethyl-1H-benzoimidazole (100 mg, 0.40 mmol). Purification afforded 36 mg (35%) of the title compound. MS (electrospray): mass calculated for C14H18N4O, 258.15; m/z found 259.2, [M + H]+. 1H NMR (400 MHz, CDCl3) rotamers: δ 11.24 (br s, 1H), 7.69 (d, J ) 8.3 Hz, 0.6H), 7.60 (br s, 0.4H), 7.39 (d, J ) 8.3 Hz, 0.4H), 7.29 (br s, 0.6H), 7.18 (d, J ) 8.3 Hz, 0.4H) 7.13 (d, J ) 8.3 Hz, 0.6H), 4.79 (br m, 2H), 3.94 (m, 2H), 2.57 (m, 4H), 2.49 (s, 3H), 2.36 (s, 3H). Anal. (C14H18N4O), C, H, N. (4-Methyl-piperazin-1-yl)-(5-trifluoromethyl-1H-benzoimidazol-2-yl)-methanone (42). The reaction was carried out as described in General Procedure F using 2-trichloromethyl-5-trifluoromethyl-1H-benzoimidazole (100 mg, 0.33 mmol). Purification afforded 42 mg (41%) of the title compound. MS (electrospray): mass calculated for C14H15F3N4O, 312.12; m/z found 313.2, [M + H]+. 1H NMR (400 MHz, CDCl3): δ 8.01 (br s, 1H), 7.72 (br m, 1H), 7.58 (dd, J ) 1.3, 8.6 Hz, 1H), 4.78 (m, 2H), 3.95 (m, 2H), 2.59 (m, 4H), 2.37 (s, 3H). Anal. (C14H15F3N4O) C, H, N, F. (5-Fluoro-4-methyl-1H-benzoimidazol-2-yl)-(4-methylpiperazin-1-yl)-methanone (43). (5-Fluoro-4-methyl-2-trichloromethyl-1H-benzoimidazole was prepared as described in General Procedure E using 5-fluoro-4-methyl-1,2-diaminobenzene (500 mg, 3.57 mmol) to afford 900 mg of a crude orange powder. The crude reaction product (100 mg, 0.37 mmol) was used without purification in General Procedure F to afford 43 mg (31% yield) of the title compound. MS (electrospray): mass calculated for C14H17FN4O, 276.14; m/z found 277.8, [M + H]+. 1H NMR (400 MHz, CDCl ) rotamers: δ 11.70 (br s, 1H), 7.57 3 (m, 0.5H), 7.26 (m, 0.5 H), 7.03 (m, 1H), 4.75 (m, 2H), 3.93 (m, 2H), 2.56 (m, 4H), 2.37 (s, 3H). Anal. (C14H17FN4O) C, H, N, F. (4,5-Difluoro-1H-benzoimidazol-2-yl)-(4-methyl-piperazin-1-yl)-methanone (44). (4,5-Difluoro-2-trichloromethyl1H-benzoimidazole was prepared as described in General Procedure E using 4,5-difluoro-1,2-diaminobenzene (200 mg, 1.38 mmol). The crude reaction product was used without purification in General Procedure F to afford 168 mg (44% yield) of the title compound. MS (electrospray): mass calcu-

lated for C13H14F2N4O, 280.11; m/z found 281.2, [M + H]+. 1H NMR (400 MHz, CDCl3) rotamers: δ 11.90 (br s, 1H), 7.32 (br m, 1H), 7.17 (m, 1H), 4.79 (m, 2H), 3.97 (m, 2H), 2.60 (m, 4H), 2.38 (s, 3H). Anal. (C13H14F2N4O) C, H, N, F. (4-Chloro-6-methyl-1H-benzoimidazol-2-yl)-(4-methylpiperazin-1-yl)-methanone (45). 4-Chloro-6-methyl-2-trichloromethyl-1H-benzoimidazole was prepared as described in General Procedure E using 4-chloro-6-methyl-1,2-diaminobenzene (1.0 g, 6.41 mmol). MS (electrospray): mass calculated for C9H6Cl4N2, 281.93; m/z found 283.6, [M + H]+. The crude product was utilized in Procedure F affording 703 mg of the title compound (38% yield). MS (electrospray): mass calculated for C14H17ClN4O, 292.11; m/z found 293.3, [M + H]+. 1H NMR (400 MHz, CDCl3) rotamers: δ 11.20 (br s, 1H), 7.62 (s, 0.5 H), 7.32 (s, 0.5 H), 7.03 (s, 1H), 4.74 (m, 2H), 3.91(m, 2H), 2.62-2.55 (m, 7H), 2.37 (s, 3H). Anal. (C14H17ClN4O) C, H, N, Cl. (4,6-Dichloro-1H-benzoimidazol-2-yl)-(4-methyl-piperazin-1-yl)-methanone (46). 4-Chloro-6-methyl-2-trichloromethyl-1H-benzoimidazole was prepared as described in General Procedure E using 4,6-dichloro-1,2-diaminobenzene (1.0 g, 6.64 mmol). The crude product (200 mg, 0.65 mmol) was utilized in Procedure F affording 59 mg of the title compound (29% yield). MS (electrospray): mass calculated for C14H17Cl2N4O, 313.18; m/z found 315.1, [M + H]+. 1H NMR (400 MHz, CDCl3): 11.7 (br s, 1H), 7.65 (br s, 1 H), 7.36 (s, 1H), 4.74 (m, 2H), 3.91(m, 2H), 2.57 (m, 4H), 2.36 (s, 3H). Anal. (C13H14Cl2N4O) C, H, N, Cl. Biological Materials. SK-N-MC cells were stably transfected with human H4 receptor as previously described.5,9,34 All of the radiolabeled ligands were purchased from PerkinElmer (Boston, MA). Binding Assays. Radioligand binding assays were run as previously described.9,16 Cell pellets from SK-N-MC cells transfected with the human H4 receptor were used. Cell pellets were homogenized in 50 mM TRIS pH 7.5 containing 5 mM EDTA and supernatants from an 800g spin were collected and recentrifuged at 30 000g for 30 min. Pellets were rehomogenized in 50 mM TRIS pH 7.5 containing 5 mM EDTA. For the H4 competition binding studies, human cell membranes were incubated with 10 nM, 40 nM, and 150 nM [3H]histamine (specific activity 23 Ci/mmol), respectively, with or without test compounds for 45 min at 25 °C. Nonspecific binding was defined using 100 µM unlabeled histamine. The Kd values for the human H4 receptor was determined to be 5 nM and the Bmax value was determined to be 1.12 pmol/mg protein. For all studies the Ki values were calculated based on an experimentally determined appropriate Kd values according to Cheng and Prusoff.17 Cell-Based cAMP Assays. Cell-based functional assays were run as previously described.9,16 SK-N-MC cell lines were created that express a reporter gene construct and the human H4 receptor full-coding region. The reporter gene was β-galactosidase under the control of cyclic AMP responsive elements. Cells were plated in 96-well plates the night before the assay. Histamine was used as the agonists for all assays. For the H4 receptor the inhibition of forskolin-stimulated cAMP production was measured. For determination of antagonists activity compounds were added 10 min prior to the addition of agonist, which was added directly to the cell medium. In the case of the H4 receptor assay, forskolin (5 µM final concentration) was added 10 min after the addition of histamine. Cells were returned to the incubator for 6 h at 37 °C. The medium was then aspirated, and the cells were washed with 200 mL of phosphate buffered saline (PBS). Cells were lysed with 25 µL of 0.1× assay buffer (10 mM sodium phosphate, pH 8, 0.2 mM MgSO4, 0.01 mM MnCl2) and incubated at room temperature for 10 min. Cells were then incubated for 10 min with 100 µL of 1× assay buffer containing 0.5% (v/v) Triton and 40 mM β˜ -mercaptoethanol. Color was developed using 25 µL of 1 mg/ mL substrate solution (chlorophenol red β-D-galactopyranoside; Roche Molecular Biochemicals, Indianapolis, IN). Color was quantitated on a microplate reader by measuring the absorbance at 570 nm. The data from each concentration



response curve were fitted to a sigmoidal curve to obtain the maximum response, Hill coefficient and EC50, using Prism (GraphPad Software, San Diego, CA). Dose ratios were calculated from individual concentration response curves of agonists at three antagonist concentrations. An apparent pA2 values were calculated using a Schild plot. Bone-Marrow Mast Cell Culture. Mast cells were differentiated from bone marrow derived from BALB/c mice as previously described.15,16 Briefly bone marrow cells were cultured in RPMI with 10% (v/v) fetal calf serum, 0.1 mM nonessential amino acids, 50 µg/mL penicillin/streptomycin, and 20% (v/v) conditioned medium from WEHI-3 cells (ATCC). After 16 h culture, the nonadherent cells were transferred to a new flask. Four weeks later, the cells were analyzed by flow cytometry for IgE receptor and CD117 (c-kit) expression to confirm the differentiation to mast cells. Greater than 99% of the cells were IgE receptor and CD117 positive. Mast cells of 4-8 weeks of age were used for the experiments. In Vitro Mast Cell Chemotaxis Assay. Mast cell chemotaxis assays were run as previously described.15,16 Transwells (Costar, Cambridge, MA) of a pore size 8 µm were coated with 100 µL of 100 µg/mL bovine fibronectin (Sigma, St. Louis, MO) for 2 h at room temperature. After removal of the fibronectin, 600 µL of RPMI with 0.5% (w/v) bovine serum albumin in the presence of histamine was added to the bottom chamber, and histamine receptor antagonists were added to both chambers. Mast cells (2 × 105/well) were added to the top chamber. The plates were incubated for 3 h at 37 °C. Transwells were removed and the number of cells in the bottom chamber was counted for 1 min using a flow cytometer. Purification of Human Eosinophils. Eosinophils were purified from blood samples collected from healthy volunteers as previously described.35 Briefly, platelet-rich plasma was removed by centrifugation of heparinized whole blood. Polymorphonuclear leukocytes (PMNL), which are enriched with neutrophils and eosinophils, were separated from PBMC by centrifugation at 2000 rpm for 20 min over a discontinuous plasma-Percoll gradient (density1.082 g/mL). Red blood cells in PMNL were removed by hypotonic shock lysis. PMNL were stained with Hematoxylin and Eosin (H&E), and a differential cell count was performed. Eosinophil counts ranged from 2 to 10% of the total PMNL number. Eosinophils were purified from the PMNL by negative selection. PMNL were incubated with a cocktail of anti-CD16, anti-CD3, anti-CD19, and antiCD14 conjugated micro-beads in PBS containing 0.5% BSA and 2 mM EDTA, which selectively bind to neutrophils, T cells, B cells, and monocytes, respectively, in the PMNL suspension. Eosinophils were purified by removing cells bound to microbeads in the AutoMACS system, resulting in eosinophil populations of >97.5% purity according to H&E stain. Purified eosinophils were washed once in buffer (PBS containing 10 mM Ca2+ and Mg2+, 10 mM HEPES, 10 mM glucose, and 0.1% BSA, pH 7.2-7.4) and used immediately for experiments. In Vitro Eosinophil Chemotaxis Assays. Eosinophil chemotaxis assays were carried out as previously described.35 Transwells (Costar, Cambridge, MA) with 5 µm pore size were coated with 100 µL of 100 ng/mL human fibronectin (Sigma) for 2 h at room temperature. After removal of excess fibronectin, 600 µL of RPMI-1640 medium containing 0.5% BSA and different concentrations of histamine (0.01-100 µM) were added to the bottom chamber. Eosinophils (2 × 105/well) were added to the top chamber. Compounds were added to both the top and bottom chambers. The plates were incubated for 2 h at 37 °C, and the number of cells migrated to the bottom chamber was counted for 1 min using flow cytometer. Statistics. Experimental data are presented as mean ( standard deviation (SD) from the number (n) of independent samples. The IC50 or EC50 values were calculated from the concentration-effect curves by nonlinear regression analysis using GraphPad Prism (GraphPad Software Inc., Philadelphia, PA).

analytical expertise. We also thank Drs. Daniel Buzard and Ronald Wolin for the editing of this manuscript.

Acknowledgment. The authors thank Dr. Jiejun Wu, Heather McAllister, and David Tognarelli for their

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Supporting Information Available: Microanalysis data of all final compounds are available free of charge via the Internet at http://pubs.acs.org.

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