Identification of Phenoxyalkylbenzimidazoles with Antitubercular Activity

Article abstract of DOI:10.1021/acs.jmedchem.5b00546

We conducted an evaluation of the phenoxyalkylbenzimidazole series based on the exemplar 2-ethyl-1-(3-phenoxypropyl)-1H-benzo[d]imidazole for its antitubercular activity. Four segments of the molecule were examined systematically to define a structure-activity relationship with respect to biological activity. Compounds had submicromolar activity against Mycobacterium tuberculosis; the most potent compound had a minimum inhibitory concentration (MIC) of 52 nM and was not cytotoxic against eukaryotic cells (selectivity index = 523). Compounds were selective for M. tuberculosis over other bacterial species, including the closely related Mycobacterium smegmatis. Compounds had a bacteriostatic effect against aerobically grown, replicating M. tuberculosis, but were bactericidal against nonreplicating bacteria. Representative compounds had moderate to high permeability in MDCK cells, but were rapidly metabolized in rodents and human liver microsomes, suggesting the possibility of rapid in vivo hepatic clearance mediated by oxidative metabolism. These results indicate that the readily synthesized phenoxyalkylbenzimidazoles are a promising class of potent and selective antitubercular agents, if the metabolic liability can be solved.

Full text of DOI:10.1021/acs.jmedchem.5b00546

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Article
Identification of phenoxyalkylbenzimidazoles with anti-tubercular activity
N Susantha Chandrasekera, Torey Alling, Mai Ann Bailey, Megan Files, Julie V
Early, Juliane Ollinger, Yulia Ovechkina, Thierry Masquelin, Prashant V Desai,
Jeffrey W Cramer, Philip A Hipskind, Joshua O. Odingo, and Tanya Parish
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 21 Aug 2015
Downloaded from http://pubs.acs.org on August 21, 2015
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Identification of phenoxyalkylbenzimidazoles with
antiꢀtubercular activity.
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a
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N. Susantha Chandrasekera , Torey Alling , Mai A. Bailey , Megan Files , Julie V. Early ,
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Juliane Ollinger , Yulia Ovechkina , Thierry Masquelin , Prashant V. Desai , Jeffrey W
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Cramer , Philip A. Hipskind , Joshua O. Odingo , Tanya Parish
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Infectious Disease Research Institute, 1616 Eastlake Ave E, Seattle, WA 98102.
Lilly Research Laboratories, Indianapolis, IN 46285, USA.
b
KEYWORDS: antiꢀtubercular, drug discovery, structureꢀactivity relationship, bactericidal
activity, high throughput screening
ABSTRACT: We conducted an evaluation of the phenoxyalkylbenzimidazole series based on
the exemplar 2ꢀethylꢀ1ꢀ(3ꢀphenoxypropyl)ꢀ1Hꢀbenzo[d]imidazole for its antiꢀtubercular activity.
Four segments of the molecule were examined systematically to define a structureꢀactivity
relationship with respect to biological activity. Compounds had subꢀmicromolar activity against
Mycobacterium tuberculosis; the most potent compound had a minimum inhibitory concentration
(MIC) of 52 nM and was not cytotoxic against eukaryotic cells (selectivity index = 523).
Compounds were selective for M. tuberculosis over other bacterial species, including the closely
related Mycobacterium smegmatis. Compounds had a bacteriostatic effect against aerobicallyꢀ
grown, replicating M. tuberculosis, but were bactericidal against nonꢀreplicating bacteria.
Representative compounds had moderate to high permeability in MDCK cells, but were rapidly
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metabolized in rodents and human liver microsomes suggesting the possibility of rapid in vivo
hepatic clearance mediated by oxidative metabolism. These results indicate that the readilyꢀ
synthesized phenoxyalkylbenzimidazoles are a promising class of potent and selective antiꢀ
tubercular agents, if the metabolic liability could be solved.
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INTRODUCTION
Tuberculosis (TB), caused by Mycobacterium tuberculosis, is the second leading cause of death
from an infectious disease and is a major global health problem. In 2013, according to the World
Health Organization (WHO), 9 million new cases and 1.5 million deaths from the disease were
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reported, including 360 000 deaths among HIVꢀpositive people . In addition, oneꢀthird of the
world’s population is infected with latent TB, 10% of which is expected to develop active TB at
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some point in their lives . Hence there is a desperate need for new TB drugs.
The benzimidazole core is a wellꢀknown privileged structure in medicinal chemistry as it is a
versatile heterocycle, possessing a wide spectrum of biological activities including antiꢀ
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bacterial , antiꢀparasitic , antiꢀfungal, and antiꢀviral activities . The benzimidazole scaffold
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has also shown antiꢀmycobacterial activity in vitro . Ojima et al reported trisubstituted
benzimidazoles with activity against M. tuberculosis and demonstrated that these are FtsZ
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inhibitors. Gong et al also reported benzimidazoleꢀbased compounds with activity against M.
tuberculosis. The benzimidazoles reported in these studies represent a variety of pharmacophores
with most having extensive variation at the Cꢀ2 position for modulation of activity. The SAR of
the series we report here has a particularly strict requirement for Cꢀ2 position with an ethyl group
as the favored substituent. This could point to a novel mechanism of action or target distinct
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from those already reported in the literature. Therefore it is an important pharmacophore for the
discovery of new drugs.
We conducted an exploratory study to understand the structureꢀactivity relationship of a
benzimidazole series regarding antiꢀtubercular activity. We defined key features required for
activity and determined that, importantly, the compounds lack significant cytotoxicity, are
specific for M. tuberculosis, and are bactericidal against nonꢀreplicating bacteria, features
which are desirable in a new therapeutic for tuberculosis.
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RESULTS AND DISCUSSION
Confirmation of anti-tubercular activity
We were interested in the phenoxyalkylbenzimidazole (PAB) class of compounds since
phenoxyalkylimidazoles and derivatives were previously identified as having activity against
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M. tuberculosis . In this previous study 27/88 analogs tested had some activity, with the
benzimidazole compound, 2ꢀethylꢀ1ꢀ(3ꢀphenoxypropyl)ꢀ1Hꢀbenzo[d]imidazole being the most
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active . We were interested in using this compound (Figure 1; 5) as a starting point to explore
the potential of the PAB class of compounds as a lead series for tuberculosis treatment.
We first wanted to confirm that the reference benzimidazole compound was active. We
synthesized 2ꢀethylꢀ1ꢀ(3ꢀphenoxypropyl)ꢀ1Hꢀbenzo[d]imidazole (5) and tested it for activity. We
determined the minimum inhibitory concentration (MIC) against M. tuberculosis H37Rv
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London Pride) . We found the MIC of the synthesized compound (5) to be very similar to that
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previously reported ꢀ 5.2 µM (Table 1) as compared to 3.4 µM .
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Structure-activity relationship (SAR) studies on 2-ethyl-1-(3-phenoxypropyl)-1H-
benzo[d]imidazole (5)
We wanted to explore the structureꢀactivity relationship of the PAB series to determine its
potential for progression as a drug candidate. Therefore, we designed, synthesized and tested a
large number of analogs in this process. PABs were synthesized according to Scheme1.
Condensation of the 1,2ꢀdiaminobenzene with the appropriate carboxylic acid yielded the core
benzimidazole intermediate (1) upon heating. Subsequent alkylation of 1 to generate the product
was achieved by either of two methods. Either the benzimidazole (1) was deprotonated with
sodium hydride and reacted with phenoxyalkyl bromide or it was reacted with a molar equivalent
of dibromobutane and the resulting Nꢀ(bromoalkyl)benzimidazole treated with the appropriate
phenol. Similarly, anilines were used in place of phenol to generate the corresponding aniline
derivatives.
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All compounds were tested for activity against M. tuberculosis in aerobic culture to determine
the minimum inhibitory concentration (MIC). All active compounds were tested against
eukaryotic cells to determine the cytotoxic concentration (TC ). From these data we calculated
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the selectivity index (SI = MIC/TC ).
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Four segments of the reference molecule 2ꢀethylꢀ1ꢀ(3ꢀphenoxypropyl)ꢀ1Hꢀbenzo[d]imidazole (5,
Fig 1) were examined systematically to define the structureꢀactivity relationships influencing
potency. First we probed the consequence of alkyl chain length variation on antiꢀtubercular
activity. Two sets of analogs were prepared bearing a 2ꢀ, 3ꢀ or 4ꢀcarbon linker with either a
methyl group or an ethyl group as the substituent at the Cꢀ2 position of the benzimidazole
moiety. The 4ꢀcarbon linked analog (6) was the most potent (MIC = 1.1 µM) and also had good
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selectivity (SI = 19). The 2ꢀcarbon linked analog and analogs with branched spacer were not
active (MIC > 20 µM) (Table 1). Interestingly, none of the analogs with methyl group as the Cꢀ2
substituent (7 – 10) were active (MIC >20 µM), suggesting the importance of ethyl group as the
preferred substituent at Cꢀ2 position (Table 1).
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Following up on the good activity of compound 6, we investigated the effects of modulating the
electronics of the phenyl ring region. Compounds bearing either electronꢀreleasing groups or
electronꢀdonating groups on the phenyl ring were synthesized. Both weak electronꢀreleasing
groups (such as methyl) which inductively donate electrons to the phenyl ring, and strong
electronꢀdonating groups such as methoxy were investigated; ortho, meta and para-methylated
phenyl analogs were synthesized and evaluated. All these compounds (12ꢀ14) displayed a slight
increase in potency over 6 (Table 2). The pꢀmethyl substituted analog (14, 0.15 µM) was the
most potent and showed the highest selectivity (SI = 93). The addition of the bulkier isopropyl
group at ortho positon (16, 20 µM) was detrimental to the antiꢀtubercular activity, relative to
methyl group (12, 0.32 µM) suggesting that a stericallyꢀhindered group at the ortho position of
the phenyl ring has a negative impact on activity. An analog with a strong electronꢀdonating
group such as the methoxy group (20, 1.2 µM) had comparable activity to 6. Replacement of the
phenyl ether in 6 by benzyl ether as in 19 (3.6 µM) resulted in a threeꢀfold decrease in potency
(Table 2).
We synthesized a set of compounds incorporating strong electronꢀwithdrawing substituents such
as chloroꢀ, nitroꢀ and cyanoꢀgroups in the phenoxy region (21ꢀ31) (Table 2). These analogs
were, in general, less potent compared to 6. The monoꢀ and diꢀhalogenated compounds had
similar activity to 6 except the dichloro compound (24, 20 µM) which was 20ꢀfold less active.
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The strongly electronꢀwithdrawing cyano analog (28, 16 µM) and nitro analog (31, 7.9 µM) were
much less potent relative to 6.
We next investigated the influence of substituents on the benzo portion of the benzimidazole
core (Table 3). The weak electronꢀdonating methyl group at Cꢀ6 (34) and the weak electronꢀ
withdrawing chloride group at Cꢀ5 (33) resulted in the best potency and selectivity. The
introduction of Nꢀatoms (38, 39), had a negative impact on activity (MIC > 20 µM). However,
the aza group in combination with either a chloro (35, 6.1 µM) or a methyl (40, 1.5 µM)
substitution resulted in active compounds. The introduction of strong electronꢀwithdrawing
group such as cyano (41, 42) was detrimental to the antiꢀtubercular activity. A methoxy
substitution at Cꢀ6 (36, 0.9 µM) was favorable compared to Cꢀ5 (37) showing activity similar to
that of compound 6.
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Next, we optimized the Cꢀ2 substitution on compound 6 (Table 4). Compounds were
synthesized with a wide variety of Cꢀ2 substituents including electronꢀdonating groups, electronꢀ
withdrawing groups, heterocyclic groups (with both saturated and unsaturated identities) and a
sterically hindered group (tertꢀbutyl). Interestingly, none of the compounds showed activity
comparable to compound 6. Three of the compounds, namely phenyl (43, 16 µM), ethanolꢀ1ꢀylꢀ
(45, 20 µM and 46, 11 µM) and acetamido (47, 11 µM) analogs, had a 10 ꢀ15ꢀfold lower activity
compared to compound 6.
We then probed the effect of replacement of the linker oxygen on activity (Table 5). The amine
analog (54, 0.47 µM) improved the selectivity index by a factor of 4.7 whereas a thioether analog
(53, 1.4 µM) had no effect on activity and selectivity.
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From these initial SAR studies based on 6, we determined that the key substituents for activity
enhancement are: a methyl group at the Cꢀ6 position, an ethyl group at the Cꢀ2 position of the
benzimidazole, nitrogen as the hetero atom in the alkyl chain, and a methyl group at the para
position of the phenyl ring. We used this knowledge to synthesize another set of analogs in an
attempt to improve potency and selectivity further. This resulted in analogs with subꢀmicromolar
potency (Table 6). The most potent compound (58) had a MIC of 0.11 µM and a SI of 109.
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We also applied the same SAR lessons to improving the potency of compound 5 (Table 6).
Again we obtained compounds with subꢀmicromolar activity and were able to improve potency
100ꢀfold, as well selectivity by 55ꢀfold. The substituted aniline analog 62 was the most potent
with an MIC of 0.052 µM and a SI of 523.
Finally we explored the effect of replacing the phenyl ring with other heterocycles (Table 7).
Replacement with the 3ꢀpyridyl (64), 4ꢀpyridyl (65), 8ꢀquinolinyl (66) or methyl (69) groups
were all detrimental to the antiꢀtubercular activity (MIC > 20 µM). However, surprisingly a
combination of chlorophenyloxadiazole and a thiother linker as in compound 68 was potent
(MIC = 0.1 µM). Replacements of the benzimidazole moeity with an indole (71–73) or an
indazole (70) resulted in loss of activity (Table 8).
In silico and in vitro ADME profile
ADME was evaluated using predictive in silico models (data not shown). Based on these results,
a representative set of compounds was tested in key in vitro assays including solubility,
permeability and liver microsomal metabolic turnover (Table 9). Passive permeability across
MDCK cells was moderate to high for the four compounds tested. Thermodynamic equilibrium
solubility at pH 2 was >0.5 mg/mL for all compounds. This was in line with the expected
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ionization of the basic benzimidazole group with pKa close to 6. However, at pH 6 and 7.4, the
solubility was significantly lower, suggesting a potential impact on absorption, especially
through the intestine. The compounds were rapidly metabolized with >90% lost in 30 min in
rodents and human liver microsomes. These results suggested the possibility of rapid in vivo
hepatic clearance mediated by oxidative metabolism. The relatively poor solubility at pH 6 and
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7.4 as well as rapid microsomal turnover were not unexpected given relatively high lipophilicity
with cLogP > 4 for most compounds.
The PAB series is bactericidal against non-replicating M. tuberculosis
We determined the microbiological profile of selected compounds from this series. We
determined whether compounds were bactericidal or bacteriostatic under two conditions ꢀ
aerobic growth (replicating conditions) and under nutrient starvation (nonꢀreplicating
conditions).
We selected compounds, 5, 54, 59, 62, and 68 based on potency and diversity and tested these
for bactericidal activity in aerobic culture (Figure 2). M. tuberculosis was exposed to a range of
compound concentrations (from 1ꢀ10X MIC) for 21 days and viable bacteria counted. None of
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the compounds was bactericidal (defined as >3 log kill in 21 days) against M. tuberculosis
under replicating conditions. An MBC (minimum bactericidal concentration) was not obtained
for any compound. Only compound 5 showed any killing activity, with 2 logs of kill over 21
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days. According to microbiological definition , all five compounds are bacteriostatic, since the
MBC/MIC was >4. Compounds 5 and 68 were also tested for activity against nonꢀreplicating
bacteria generated by nutrient starvation (Figure 3). In contrast to aerobic culture, compounds
were clearly bactericidal against nonꢀreplicating bacteria, resulting in >3 logs kill over 21 days.
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Even at 1x MIC, both compounds had sterilizing activity of more than 3 logs under these
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conditions. The bactericidal activity was timeꢀdependent i.e. the same rate of kill was noted at
all concentrations.
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PAB series activity is specific for M. tuberculosis
To determine the spectrum of activity, we measured MICs on solid medium against a
representative Gram negative species (Escherichia coli), a representative Gram positive species
(Staphylococcus aureus) and a nonꢀpathogenic mycobacterial species (Mycobacterium
smegmatis). Compounds 6, 53, and 68 were inactive against all three species, with MIC > 100
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µM on solid medium (Table 10), but were active against M. tuberculosis with MIC of 2.5ꢀ10
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µM on solid medium. Thus the growth inhibitory activity of the PAB series is specific to M
tuberculosis.
CONCLUSION
We conducted a systematic exploration of the phenoxyalkylbenzimidazole (PAB) series for its
activity against M. tuberculosis. The compounds in this series show good activity and selectivity.
The methyl group in the Cꢀ6 position of the benzimidazole and para position of the phenyl ring,
ethyl group at the Cꢀ2 position of the benzimidazole core, 3 or 4 carbon atom linker, nitrogen as
the hetero atom on the alkyl chain and benzimidazole as the core moiety are key determinants of
the activity of the compounds in this series.
Interestingly, this series is bacteriostatic under replicating conditions but bactericidal under nonꢀ
replicating conditions. The increased activity against nonꢀreplicating bacteria is of particular
interest, since current therapeutic agents largely target replicating organisms. Thus development
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of the PAB series has the potential to provide new agents which could shorten antibiotic therapy
and treat latent infections. Based on these properties, we propose the PAB is a promising series
for further exploration, if the physicochemical and ADME properties can be improved.
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EXPERIMENTAL SECTION
Determination of minimum inhibitory concentration (MIC)
MIC were determined against M. tuberculosis H37Rv (London Pride), a laboratoryꢀpassaged
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derivative of H37Rv (ATCC 25618) which has been sequenced . MICs were run as described
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; briefly MICs were determined in Middlebrook 7H9 medium containing 10% OADC (oleic
acid, albumin, dextrose, catalase) supplement (Becton Dickinson) and 0.05% w/v Tween 80
7H9ꢀTwꢀOADC) under aerobic conditions. Compounds were prepared as 10ꢀpoint twoꢀfold
(
serial dilutions in DMSO with a starting concentration of 20 ꢁM. The final concentration of
DMSO in the assay was 2%. Bacterial growth was measured by OD590 after 5 days of incubation
at 37°C. Growth inhibition curves were plotted and fitted using the Gompertz model. The MIC
was defined as the minimum concentration at which growth was completely inhibited and was
calculated from the inflection point of the fitted curve to the lower asymptote (zero growth).
Cytotoxicity against eukaroytic cells
Cytotoxicity was determined against the African green monkey kidney cell line (Vero: ATCC
CCLꢀ81). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM), High Glucose,
GlutaMAX, 10% FBS, and 1x of penicillinꢀstreptomycin solution (100 units/mL of penicillin,
100 µg/mL of streptomycin). Compounds were prepared as 10ꢀpoint threeꢀfold serial dilutions in
DMSO with a starting concentration of 50 ꢁM. CellTiterꢀGlo® Reagent (Promega) was added to
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6ꢀwell plates after 2 days of incubation at 37°C, 5% CO and relative luminescent units (RLU)
2
measured. Inhibition curves were fitted using the Levenberg–Marquardt algorithm. Toxic
concentration (TC ) was defined as the concentration of compound that gave 50% inhibition of
5
0
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growth.
Bactericidal activity
For replicating conditions, a late log phase culture of M. tuberculosis was adjusted to OD590=0.1
in 7H9ꢀTwꢀOADC and 50 µL used to inoculate 5 mL of 7H9ꢀTwꢀOADC containing compounds
o
(final DMSO concentration of 2%). Cultures were incubated standing at 37 C and serial dilutions
plated to determine colony forming units (CFUs) on Middlebrook 7H10 agar plus 10 % v/v
OADC supplement. Plates were incubated for 4 weeks before colonies were counted.
For nonꢀreplicating conditions, late log phase bacterial cultures were grown in 7H9ꢀTwꢀOADC,
harvested, resuspended in PBSꢀTy (PBS + 0.05% w/v Tyloxapol) to an OD590=0.1, and incubated
o
at 37 C for 14 d prior to addition of compound. Compounds were added at indicated
o
concentrations (final DMSO concentration of 2%). Cultures were incubated standing at 37 C and
serial dilutions plated to determine colony forming units (CFUs) on Middlebrook 7H10 agar plus
10 % v/v OADC supplement. Plates were incubated for 6 weeks before colonies were counted.
Spectrum of activity
21
MICs were determined on solid medium using the serial proportion method . LB agar was used
2
for E. coli BL21 and S. aureus RN4220; 7H10ꢀOADC was used for M. smegmatis mc 155 and
o
M. tuberculosis. Plates were incubated at 37 C for 1 day for E. coli, 2 days for S. aureus, 5ꢀ7
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days for M. smegmatis and 21ꢀ28 days for M. tuberculosis. MIC was defined as the concentration
of compound which yielded less than 1% CFUs.
ADME
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MDCK Permeability ꢀ Test compound (10 µM) transport was measured across MDCK cell line
in presence of a Pꢀgp inhibitor in the absorptive direction and expressed as the percent transport
over the incubation period. Benchmark compounds, atenolol and dexamethasone, were used to
define three levels of transport to classify the test compounds as having low, medium, or high
permeability. To determine microsomal turnover, test compounds were incubated (2µM) with
liver microsomes in the presence of NADPH and loss of parent molecule was measured by
LC/MS after 30 min. High throughput thermodynamic equilibrium solubility was measured
utilizing 10 mM DMSO solutions. The DMSO was removed by drying before buffers at various
22
pH were added. Fraction unbound in plasma was measured as described . Briefly, test
compound and mouse plasma were mixed together and placed into a dialysis block with plasma
mixture on one side and buffer on the other. After 4.5 h incubation, samples taken from both
sides. The fraction unbound was calculated by dividing the LC/MS/MS area of the buffer side by
the LC/MS/MS area of the protein side.
Compound synthesis
General Methods
1
H and NMR spectral data were recorded in CDCl or Acetoneꢀd6 on a 300 MHz Bruker NMR
3
spectrometer. Column chromatography was conducted on Revelaris flash chromatography
system. Reactions were monitored using thinꢀlayer chromatography (TLC) on silica gel plates.
HPLC techniques and high resolution mass spectrometry were used to determine the purity of
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compounds. Purity of all final products was >95% as determined by HPLC analysis conducted
on an Agilent 1100 series LC system (Agilent ChemStation Rev.A.10.02; PhenomenexꢀLunaꢀ
C18, 4.8 mm × 150 mm, 5 ꢁm, 1.0 mL/min, UV 254nm, room temperature) with MeCN/H O
2
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49
50
51
52
53
54
55
56
57
58
59
60
(
0.05% TFA or HCOOH buffer) gradient elution. HPLCꢀHRMS was performed on a Gilson 321
HPLC with detection performed by a Gilson 170 DAD and a Finnigan AQA mass spectrometer
operating in electrospray ionization mode using a Phenomenex Gemini C18 150x4.6mm column.
General procedure for synthesis of 1H-benzo[d]imidazole intermediates (1)
A mixture of diamine (1 eq) and acid (1 eq) was heated to the boiling point of the acid for 4 h.
The reaction mixture was poured into a beaker containing ice water and neutralized with 2M
NaOH. The resulting precipitate was filtered and dried under the vacuum. All of these
intermediates were purchased from commercial sources except 2ꢀethylꢀ6ꢀmethylꢀ1Hꢀ
imidazo[4,5ꢀc]pyridine (1a)
General procedure for synthesis of compounds 2–10, 43–47, 51, 52, 53 and 70-71
To a solution of benzimidazole in anhydrous dimethylformamide was added 3 eq of sodium
hydride and stirred for 0.5 h. Phenoxyalkylbromide (2 eq) was then added and stirred at R. T.
until the disappearance of starting material monitored by TLC. The reaction was quenched with
methanol. The organic layer was washed with water and dried with anhydrous sodium sulfate
then concentrated in vacuo. The resulting crude mixture was purified by column
chromatography.
General Procedure for synthesis of 1-(2-bromoalkyl)-1H-benzo[d]imidazole intermediates
(11)
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To a solution of benzimidazole in acetone was added 5 eq of sodium hydroxide, dibrimoalkane
o
and a catalytic amount of sodium iodide. The reaction mixture was heated to 50 C and stirred
overnight. The acetone was then evaporated. The crude mixture was dissolved in ethyl acetate
and washed with water. The organics were dried with anhydrous sodium sulfate and concentrated
in vacuo. The crude reaction mixture was purified by column chromatography.
0
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General procedure for synthesis of compounds 12–42, 59–61, 63–69, 71 and 72
To a solution of benzimidazole in dimethylformamide were added 5 eq of potassium carbonate
and 2 eq of the phenol (or thiophenol). The reaction was stirred overnight at room temperature or
until all of the starting material disappeared. The reaction mixture was washed with water and
extracted with ethyl acetate. The organics were dried with anhydrous sodium sulfate and
concentrated in vacuo. The crude mixture was purified by column chromatography.
General procedure for synthesis of compounds 54–58, 62 and 73
To a solution of benzimidazole in dimethylformamide were added 5 eq of potassium carbonate
o
and 2 eq of the aniline. The reaction was stirred at 50 C overnight (or until disappearance of the
starting material). The reaction mixture was washed with water and extracted with ethyl acetate.
The organics were dried with anhydrous sodium sulfate and concentrated in vacuo. The crude
mixture was purified by column chromatography.
2-ethyl-6-methyl-1H-imidazo[4,5-c]pyridine (1a)
1
Yield 1a: (0.7 g, 56%). H NMR (300 MHz, CDCl ): δ 1.50 (t, J = 7.6 Hz, 3H), 2.73 (s, 3H),
3
3.06 (q, J = 7.5 Hz, 2H), 7.09 (d, J = 8.1 Hz, 1H), 7.92 (d, J = 8.0 Hz, 1H), 12.63 (s, 1H).
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(
98.1% purity).
2-ethyl-1-(2-phenoxyethyl)-1H-benzo[d]imidazole (2)
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Yield 2: (0.32 g, 89%). H NMR (300 MHz, CDCl ): δ 1.49 (t, J = 7.5 Hz, 3H), 2.94 (q, J = 7.5
3
Hz, 2H), 4.13 (t, J = 5.3 Hz, 2H), 4.36 (t, J = 5.3 Hz, 2H), 6.60 – 7.04 (m, 3H), 7.09 – 7.51 (m,
5H), 7.70 – 7.86 (m, 1H).
+
LCMS – ESI (M+H) : 267.2 (99.3% purity). HRMS (ESI): calcd for C H N O, 267.1497;
1
7
19
2
found, 267.1493.
1-(2-(4-chlorophenoxy)ethyl)-2-ethyl-1H-benzo[d]imidazole (3)
1
Yield 3: (0.27 g, 70%). H NMR (300 MHz, CDCl ): δ 1.49 (t, J = 7.5 Hz, 3H), 2.94 (q, J = 7.4
3
Hz, 2H), 4.12 (t, J = 5.4 Hz, 2H), 4.39 (t, J = 4.6 Hz, 2H), 6.63 (d, J = 8.9 Hz, 2H), 7.05 – 7.18
+
(
m, 2H), 7.18 – 7.34 (m, 3H), 7.70 – 7.81 (m, 1H).LCMS – ESI (M+H) : 301.10 (98.8% purity).
HRMS (ESI): calcd for C H N OCl, 301.1108; found, 301.1107.
1
7
18
2
1-((2,3-dihydrobenzo[b][1,4]dioxin-2-yl)methyl)-2-ethyl-1H-benzo[d]imidazole (4)
1
Yield 4: (0.065 g, 16%). H NMR (300 MHz, CDCl ): δ 1.41 – 1.56 (m, 3H), 2.81 – 2.98 (m,
3
2H), 3.89 – 4.02 (m, 1H), 4.15 – 4.26 (m, 1H), 4.29 – 4.41 (m, 2H), 4.49 – 4.62 (m, 1H), 6.75 –
+
6
.99 (m, 4H), 7.14 – 7.38 (m, 3H), 7.67 – 7.88 (m, 1H).. LCMS – ESI (M+H) : 295.10 (99%
purity). HRMS (ESI): calcd for C H N O , 295.1447; found, 295.1447.
1
8
19
2
2
2-ethyl-1-(3-phenoxypropyl)-1H-benzo[d]imidazole (5)
1
Yield 5 (1): (0.32 g, 83%). H NMR (300 MHz, CDCl ): δ 1.41 (3H, CH , t, J = 7.5 Hz); 2.24
3
3
(m, 2H); 2.98 (2H, q, J = 7.5 Hz); 3.86 (2H, t, J = 5.3 Hz); 4.30 (2H, t, J = 6.7 Hz); 6.84 – 7.75
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1
2
2
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2
2
2
2
2
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4
4
4
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+
(
m, 9H). LCMS – ESI (M+H) : 281.2 (99.5% purity). HRMS (ESI): calcd for C H N O,
18 21 2
2
81.1654; found, 281.1651.
2
-ethyl-1-(4-phenoxybutyl)-1H-benzo[d]imidazole (6)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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1
2
3
4
5
6
7
8
9
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
Yield 6: (0.40 g, 99%). H NMR (300 MHz, CDCl ): δ 1.45 (3H, CH , t, J = 7.4 Hz); 1.66ꢀ1.84
3
3
(m, 2H); 1.92 (m, 2H); 2.83 (2H, q, J = 2. 7.5 Hz); 3.88 (2H, t, J = 6.7 Hz); 4.06 (2H, t, J = 7.5
1
3
Hz);; 6.81 – 6.94 (m, 3H); 7.17 – 7.28 (m, 5H); 7.72 – 7.77 (m, 1H). C NMR (CDCl ): δ 11.92,
3
2
0.85, 26.67, 26.90, 43.32, 67.10, 109.16, 114.41, 119.28, 120.90, 121.72, 121.99, 129.53,
+
135.10, 142.71, 155.91, 158.72. LCMS – ESI (M+H) : 295.2 (99.4% purity). HRMS (ESI): calcd
for C H N O, 295.1810; found, 295.1806.
1
9
23
2
2
-methyl-1-(2-phenoxyethyl)-1H-benzo[d]imidazole (7)
1
Yield 7: (0.31 g, 77%). H NMR (300 MHz, CDCl ): δ 2.68 (3H, CH , s); 4.23 (2H, t, J = 5.1
3
3
Hz); 4.47 (2H, t, J = 5.4 Hz); 6.72 – 6.84 (m, 2H); 6.86 – 7.01 (m, 1H); 7.19 – 7.26 (m, 4H); 7.31
+
– 7.36 (m, 1H); 7.66 – 7.72 (m, 1H). LCMS – ESI (M+H) : 253.2 (99% purity). HRMS (ESI):
calcd for C H N O, 253.1341; found, 253.1340.
1
6
17
2
1
-(2-(4-chlorophenoxy)ethyl)-2-methyl-1H-benzo[d]imidazole (8)
1
Yield 8: (0.37 g, 85%). H NMR (300 MHz, CDCl ): δ 2.62 (3H, CH , s); 4.10 (2H, t, J = 5.1
3
3
Hz); 4.34 (2H, t, J = 5.7 Hz); 6.59 – 7.09 (m, 2H); 7.08 – 7.13 (m, 2H); 7.19 – 7.23 (m, 3H); 7.66
+
–
7.71 (m, 1H). LCMS – ESI (M+H) : 287.10 (99.1% purity). HRMS (ESI): calcd for
C H N OCl, 287.0951; found, 287.0951.
16
16
2
2
-methyl-1-(3-phenoxypropyl)-1H-benzo[d]imidazole (9)
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1
Yield 9: (0.36 g, 89%). H NMR (300 MHz, CDCl ): δ 2.15ꢀ2.19 (m, 2H); 2.50 (3H, CH , s);
3
3
3
.76 ꢀ 3.83 (m, 2H); 4.21 ꢀ 4.26 (m, 2H); 6.70 – 7.06 (m, 3H); 7.06 – 7.41 (m, 5H); 7.56 – 7.81
+
(
m, 1H). LCMS – ESI (M+H) : 267.2 (97.1% purity). HRMS (ESI): calcd for C H N O,
1
7
19
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
67.1497; found, 267.1488.
2-methyl-1-(4-phenoxybutyl)-1H-benzo[d]imidazole (10)
1
Yield 10: (0.36 g, 84%). H NMR (300 MHz, CDCl ): δ 1.79 ꢀ 1.86 (m, 2H); 1.94 – 2.04 (m,
3
2
H); 2.60 (3H, CH , s); 3.95 (2H, t, J = 5.9 Hz); 4.17 (2H, t, J = 7.9 Hz); 6.84 – 7.69 (m, 9H);
3
+
6.85 – 6.96 (m, 3H); 7.19 – 7.32 (m, 5H); 7.67 – 7.72 (m, 1H). LCMS – ESI (M+H) : 281.2
(98.3% purity). HRMS (ESI): calcd for C H N O, 281.1654; found, 281.1645.
18 21 2
1
-(4-bromobutyl)-2-ethyl-1H-benzo[d]imidazole (11a)
1
Yield 11a: (1.4 g, 49%). H NMR (300 MHz, CDCl ): δ 1.48 (t, J = 7.7 Hz, 3H), 1.77 – 2.11 (m,
3
4
H), 2.87 (q, J = 7.6 Hz, 2H), 3.36 (t, J = 6.0 Hz, 2H), 4.08 (t, J = 7.0 Hz, 2H), 7.10 – 7.33 (m,
+
3H), 7.59 – 7.86 (m, 1H). LCMS – ESI (M+H) : 281.1 (97.2% purity).
1
-(4-bromobutyl)-2-ethyl-6-methyl-1H-benzo[d]imidazole (11b)
1
Yield 11b: (1.39 g, 47%). H NMR (300 MHz, CDCl ): δ 1.49 (t, J = 7.5 Hz, 3H), 1.86 – 2.08
3
(m, 4H), 2.50 (br. s, 3H), 2.89 (q, J = 7.5 Hz, 2H), 3.43 (t, J = 6.0 Hz, 2H), 3.96 – 4.28 (m, 2H),
+
6
.97 – 7.24 (m, 2H), 7.46 – 7.70 (m, 1H).. LCMS – ESI (M+H) : 295.1 (98.2% purity).
1-(3-bromopropyl)-2-ethyl-6-methyl-1H-benzo[d]imidazole (11c)
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1
1
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1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
Yield 11c: (1.1 g, 63%). H NMR (300 MHz, CDCl ): δ 1.29 – 1.59 (m, 3H), 2.13 – 2.39 (m,
3
2
H), 2.37 – 2.54 (br. s, 3H), 2.77 – 3.04 (m, 2H), 3.17 – 3.51 (m, 2H), 3.94 – 4.45 (m, 2H), 6.81
7.26 (m, 2H), 7.42 – 7.67 (m, 1H).
–
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
.
LCMS – ESI (M+H) : 281.1 (99.2% purity).
2-ethyl-1-(4-(o-tolyloxy)butyl)-1H-benzo[d]imidazole (12)
1
Yield 12: (0.044 g, 79%). H NMR (300 MHz, CDCl ): δ 1.51 (t, J = 7.0 Hz, 3H), 1.82 – 1.97
3
(
m, 2H), 1.97 – 2.14 (m, 2H), 2.24 (s, 3H), 2.93 (q, J = 6.4, 7.0 Hz, 2H), 4.00 (t, J = 5.3 Hz, 2H),
4.21 (t, J = 7.4 Hz, 2H), 6.74 – 6.84 (m, 1H), 6.84 – 6.94 (m, 1H), 7.10 – 7.38 (m, 5H), 7.71 –
+
7
.82 (m, 1H).LCMS – ESI (M+H) : 309.2 (99.4% purity). HRMS (ESI): calcd for C H N O,
2
0
25
2
3
09.1967; found, 309.1954
2
-ethyl-1-(4-(m-tolyloxy)butyl)-1H-benzo[d]imidazole (13)
1
Yield 13: (0.04 g, 72%). H NMR (300 MHz, CDCl ): δ 1.45 (t, 3H, CH , J = 7.0 Hz); 1.82 (m,
3
3
2H); 1.97 (m, 2H); 2.31 (s, 3H, CH ); 2.84 (2H, q, J = 6.3, 7.1 Hz); 3.95 (2H, t, J = 5.3 Hz); 4.17
3
(
2H, t, J = 6.7 Hz); 6.81 – 6.84 (m, 2H), 7.10 – 7.38 (m, 5H), 7.71 – 7.75 (m, 1H).. LCMS – ESI
+
(
M+H) : 309.2 (99.2% purity). HRMS (ESI): calcd for C H N O, 309.1967; found, 309.1958.
2
0
25
2
2
-ethyl-1-(4-(p-tolyloxy)butyl)-1H-benzo[d]imidazole (14)
1
Yield 14: (0.042 g, 76%). H NMR (300 MHz, CDCl ): δ 1.45 (3H, CH , t, J = 7.4 Hz); 1.82 ꢀ
3
3
1
.91 (m, 2H,); 2.00 ꢀ 2.02 (m, 2H); 2.27 (s, 3H, CH ); 2.90 (2H, q, J = 6.2, 7.5 Hz); 3.94 (2H, t, J
3
=
5.3 Hz); 4.17 (2H, t, J = 6.7 Hz); 6.78 – 7.01 (m, 2H); 7.12ꢀ7.51 (m, 4H); 7.67 – 7.74 (m, 2H).
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+
LCMS – ESI (M+H) : 309.2 (97.2% purity). HRMS (ESI): calcd for C H N O, 309.1967;
20
25
2
found, 309.1962.
1
-(4-(3,4-dimethylphenoxy)butyl)-2-ethyl-1H-benzo[d]imidazole (15)
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16
17
18
19
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23
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25
26
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Yield 15: (0.055 g, 95%). H NMR (300 MHz, CDCl ): δ 1.47 (t, J = 7.4 Hz, 3H), 1.73 – 1.91
3
(m, 2H), 1.92 – 2.09 (m, 2H), 2.18 (s, 3H), 2.22 (s, 3H), 2.90 (q, J = 7.3 Hz, 2H), 3.94 (t, J = 5.1
Hz, 2H), 4.19 (t, J = 6.0 Hz, 2H), 6.54 – 6.81 (m, 2H), 7.02 (d, J = 8.1 Hz, 1H), 7.12 – 7.41 (m,
+
3
H), 7.74 (s, 1H).. LCMS – ESI (M+H) : 323.2 (99% purity). HRMS (ESI): calcd for
C H N O, 323.2123; found, 323.2125.
21
27
2
2
-ethyl-1-(4-(2-isopropylphenoxy)butyl)-1H-benzo[d]imidazole (16)
1
Yield 16: (0.037 g, 61%). H NMR (300 MHz, CDCl ): δ 1.11 – 1.26 (m, 6H), 1.37 – 1.56 (m,
3
3
H), 1.77 – 1.94 (m, 2H), 1.94 – 2.15 (m, 2H), 2.80 – 3.00 (m, 2H), 3.19 – 3.38 (m, 1H), 3.90 –
4
.05 (m, 2H), 4.10 – 4.27 (m, 2H), 6.72 – 6.85 (m, 1H), 6.85 – 7.00 (m, 1H), 7.06 – 7.36 (m,
+
5H), 7.68 – 7.80 (m, 1H).. LCMS – ESI (M+H) : 337.3 (99.5% purity). HRMS (ESI): calcd for
C H N O, 337.2280; found, 337.2271.
22
29
2
4
-(4-(4-(2-ethyl-1H-benzo[d]imidazol-1-yl)butoxy)-2,5-dimethylbenzyl)morpholine (17)
1
Yield 17: (0.105 g, 92%). H NMR (300 MHz, CDCl ): δ 1.48 (t, J = 7.5 Hz, 3H), 1.78 – 1.91
3
(
m, 2H), 1.98 – 2.08 (m, 2H), 2.15 (s, 3H), 2.32 (s, 3H), 2.40 (t, J = 4.6 Hz, 4H), 2.90 (q, J = 6.0
Hz, 2H), 3.35 (s, 2H), 3.67 (t, J = 4.6 Hz, 4H), 3.96 (t, J = 5.8 Hz, 2H), 4.19 (t, J = 7.4 Hz, 2H),
6
.58 (s, 1H), 6.98 (s, 1H), 7.18 – 7.25 (m, 2H), 7.27 – 7.34 (m, 1H), 7.69 – 7.79 (m, 1H).. LCMS
+
–
ESI (M+H) : 422.3 (98.7% purity). HRMS (ESI): calcd for C H N O , 422.2808; found,
2
6
36
3
2
422.2800.
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Journal of Medicinal Chemistry
Page 20 of 57
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-(4-(4-(2-ethyl-1H-benzo[d]imidazol-1-yl)butoxy)phenyl)morpholine (18)
1
Yield 18: (0.055 g, 80%). H NMR (300 MHz, CDCl ): δ 1.48 (3H, CH , t, J = 7.5 Hz); 1.79 ꢀ
3
3
1
.88 (m, 2H); 2.01 ꢀ 2.04(m, 2H); 2.84 (2H, q, J = 6.0, 6.4 Hz); 3.13 (4H, t, J= 4.8); 3.83 (4H, t,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
J = 4.8 Hz); 3.96 (2H, t, J = 6.0 Hz); 4.16 (2H, t, J = 7.5 Hz); 6.26 – 6.65 (m, 3H); 7.04 – 7.41
1
3
13
(m, 4H); 7.71 – 7.75 (m, 1H). C NMR (CDCl ): δ C NMR (75 MHz, CDCl ) δ 11.90, 11.92,
3
3
20.83, 26.70, 26.90, 43.30, 49.23, 66.87, 67.13, 76.68, 77.10, 77.53, 102.67, 105.15, 108.66,
109.20, 119.23, 121.74, 122.00, 129.93, 135.06, 142.61, 152.73, 155.90, 159.80.. LCMS – ESI
+
(M+H) : 380.2 (99.2% purity). HRMS (ESI): calcd for C H N O , 380.2338; found, 380.2334.
2
3
30
3
2
1-(4-(benzyloxy)butyl)-2-ethyl-1H-benzo[d]imidazole (19)
1
Yield 19: (0.010 g, 23%). H NMR (300 MHz, CDCl ): δ 1.45 (3H, CH , t, J = 7.5 Hz); 1.66 (m,
3
3
2
H); 1.90 (m, 2H); 2.41 (m, 4H); 2.89 (m, 2H); 3.50 (m, 2H); 4.10 (m, 2H); 4.44 (s, 2H, CH2);
+
7.20 – 7.73 (m, 9H). LCMS – ESI (M+H) : 309.2 (97.9% purity). HRMS (ESI): calcd for
C H N O, 309.1967; found, 309.1959.
20
25
2
2
-ethyl-1-(4-(4-methoxyphenoxy)butyl)-1H-benzo[d]imidazole (20)
1
Yield 20: (0.023 g, 50%). H NMR (300 MHz, CDCl ): δ 1.50 (t, J = 7.5 Hz, 3H), 1.77 – 1.91
3
(m, 2H), 1.94 – 2.12 (m, 2H), 2.93 (q, J = 7.5 Hz, 2H), 3.79 (s, 3H), 3.96 (t, J = 6.0 Hz, 2H),
4
7
3
.22 (t, J = 7.4 Hz, 2H), 6.72 – 6.93 (m, 4H), 7.17 – 7.27 (m, 2H), 7.30 – 7.38 (m, 1H), 7.67 –
+
.91 (m, 1H). LCMS – ESI (M+H) : 325.0 (98.2% purity). HRMS (ESI): calcd for C H N O ,
20 25
2
2
25.1916; found, 325.1913.
1
-(4-(3-chlorophenoxy)butyl)-2-ethyl-1H-benzo[d]imidazole (21)
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Page 21 of 57
Journal of Medicinal Chemistry
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Yield 21: (0.043 g, 73%). H NMR (300 MHz, CDCl ): δ 1.45 (3H, CH , t, J = 7.4 Hz); 1.85 (m,
3
3
2
H); 2.02 (m, 2H); 2.88 (2H, q, J = 7.4 Hz); 3.95 (2H, t, J = 5.3 Hz); 4.18 (2H, t, J = 6.7 Hz);
+
6
.77 – 6.90 (m, 2H); 7.10 – 7.25 (m, 5H); 7.75 ꢀ 7.79 (m, 1H). LCMS – ESI (M+H) : 329.2
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
99.2% purity). HRMS (ESI): calcd for C H N OCl, 329.1421; found, 329.1424.
19 22 2
1-(4-(2-chlorophenoxy)butyl)-2-ethyl-1H-benzo[d]imidazole (22)
1
Yield 22: (0.04 g, 64%). H NMR (300 MHz, CDCl ): δ 1.49 (t, J = 7.5 Hz, 3H), 1.82 – 2.00 (m,
3
2
H), 2.00 – 2.19 (m, 2H), 2.93 (q, J = 7.5 Hz, 2H), 4.06 (t, J = 5.7 Hz, 2H), 4.26 (t, J = 7.3 Hz,
2H), 6.81 – 7.02 (m, 2H), 7.17 – 7.31 (m, 3H), 7.31 – 7.45 (m, 2H), 7.70 – 7.87 (m, 1H).. LCMS
+
–
3
ESI (M+H) : 329.1 (99% purity). HRMS (ESI): calcd for C H N OCl, 329.1421; found,
1
9
22
2
29.1413.
1
-(4-(4-chlorophenoxy)butyl)-2-ethyl-1H-benzo[d]imidazole (23)
1
Yield 23: (0.036 g, 61%). H NMR (300 MHz, CDCl ): δ 1.45 (3H, CH , t, J = 7.4 Hz); 1.85 (m,
3
3
2H); 2.02 (m, 2H); 2.88 (2H, q, J = 7.4 Hz); 3.95 (2H, t, J = 5.3 Hz); 4.18 (2H, t, J = 6.7 Hz);
+
6
.77 – 6.90 (2m, 1H); 7.17 – 7.35 (m, 5H); 7.73 – 7.75 (m, 1H). LCMS – ESI (M+H) : 329.2
(97.5% purity). HRMS (ESI): calcd for C H N OCl, 329.1421; found, 329.1411.
19 22 2
1-(4-(2, 6-dichlorophenoxy)butyl)-2-ethyl-1H-benzo[d]imidazole (24)
1
Yield 24: (0.057 g, 87%). H NMR (300 MHz, CDCl ): δ 1.45 (3H, CH , t, J = 7.4 Hz); 1.90 (m,
3
3
2
H); 2.10 (m, 2H); 2.94 (2H, q, J = 7.4 Hz); 4.03 (2H, t, J = 5.3 Hz); 4.24 (2H, t, J = 6.7 Hz);
+
6
.98 – 7.02 (m, 3H); 7.45 – 7.75 (m, 4H). LCMS – ESI (M+H) : 363.2 (98.2% purity). HRMS
(ESI): calcd for C H N OCl , 363.1031; found, 363.1015.
19 21 2 2
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Journal of Medicinal Chemistry
Page 22 of 57
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-(4-(2,4-dichlorophenoxy)butyl)-2-ethyl-1H-benzo[d]imidazole (25)
1
Yield 25: (0.042 g, 64%). H NMR (300 MHz, CDCl ): δ 1.47 (3H, CH , t, J = 7.5 Hz); 1.88 ꢀ
3
3
1
.90 (m, 2H); 1.99 ꢀ 2.05 (m, 2H); 2.90 (2H, q, J = 7.5 Hz); 3.99 (2H, t, J = 5.3 Hz); 4.22 (2H, t,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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5
6
7
8
9
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
J = 6.7 Hz); 6.78 ꢀ 7.79 (m, 1H); 7.02 – 7.48 (m, 5H); 7.64 – 7.88 (m, 1H). LCMS – ESI
+
(M+H) : 363.1 (99% purity). HRMS (ESI): calcd for C H N OCl , 363.1031; found, 363.1028.
1
9
21
2
2
1
-(4-(4-chloro-3-fluorophenoxy)butyl)-2-ethyl-1H-benzo[d]imidazole (26)
1
Yield 26: (0.053 g, 85%). H NMR (300 MHz, CDCl ): δ 1.48 (t, J = 6.2 Hz, 3H), 1.73 – 1.90
3
(m, 2H), 1.90 – 2.09 (m, 2H), 2.89 (q, J = 7.8 Hz, 2H), 3.82 – 4.00 (m, 2H), 4.05 – 4.28 (m, 2H),
+
6
.49 – 6.73 (m, 2H), 7.13 – 7.38 (m, 4H), 7.66 – 7.82 (m, 1H). LCMS – ESI (M+H) : 347.1
(98.7% purity). HRMS (ESI): calcd for C H N OFCl, 347.1326; found, 347.1316.
19 21 2
1-(4-(4-chloro-2-fluorophenoxy)butyl)-2-ethyl-1H-benzo[d]imidazole (27)
1
Yield 27: (0.04 g, 64%). H NMR (300 MHz, CDCl ): δ 1.45 (3H, CH , t, J = 7.5 Hz); 1.85ꢀ1.93
3
3
(m, 2H); 2.02 ꢀ 2.04 (m, 2H); 2.88 (2H, q, J = 7.5 Hz); 3.98 (2H, t, J = 5.3 Hz); 4.21 (2H, t, J =
6
.7 Hz); 6.59 – 6.98 (m, 2H); 6.97 – 7.20 (m, 2H); 7.21 – 7.52 (m, 2H); 7.72 – 7.77 (m, 1H).
+
LCMS – ESI (M+H) : 347.1 (98.2% purity). HRMS (ESI): calcd for C H N OClF, 347.1326;
1
9
21
2
found, 347.1325.
4
-(4-(2-ethyl-1H-benzo[d]imidazol-1-yl)butoxy)benzonitrile (28)
1
Yield 28: (0.046 g, 80%). H NMR (300 MHz, CDCl ): δ 1.36 – 1.58 (m, 3H), 1.76 – 1.92 (m,
3
2H), 1.94 – 2.11 (m, 2H), 2.79 – 2.99 (m, 2H), 3.89 – 4.08 (m, 2H), 4.09 – 4.29 (m, 2H), 6.79 –
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Page 23 of 57
Journal of Medicinal Chemistry
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.98 (m, 2H), 7.13 – 7.34 (m, 3H), 7.44 – 7.63 (m, 2H), 7.65 – 7.83 (m, 1H). LCMS – ESI
+
(M+H) : 320.2 (99.6% purity). HRMS (ESI): calcd for C H N O, 320.1763; found, 320.1754.
20 22
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-(4-(3-chloro-4-methylphenoxy)butyl)-2-ethyl-1H-benzo[d]imidazole (29)
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11
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13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
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36
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46
47
48
49
50
51
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57
58
59
60
1
Yield 29: (0.051 g, 83%). H NMR (300 MHz, CDCl ): δ 1.45 (3H, CH , t, J = 7.5 Hz); 1.82 ꢀ
3
3
1.85 (m, 2H); 2.00 – 2.02 (m, 2H); 2.27 (3H, CH , s); 2.90 (2H, q, J = 7.5 Hz); 3.92 (2H, t, J =
3
5
.3 Hz); 4.17 (2H, t, J = 6.7 Hz); 6.78 – 7.24 (m, 4H), 7.44 – 7.63 (m, 2H), 7.65 – 7.74 (m, 1H)..
+
LCMS – ESI (M+H) : 343.2 (99.1% purity). HRMS (ESI): calcd for C H N OCl, 343.1577;
2
0
24
2
found, 343.1572.
1
-(4-(4-chloro-2-methylphenoxy)butyl)-2-ethyl-1H-benzo[d]imidazole (30)
1
Yield 30: (0.038 g, 62%). H NMR (300 MHz, CDCl ): δ 1.45 (3H, CH , t, J = 7.5 Hz); 1.85 –
3
3
1
.87 (m, 2H); 2.02 – 2.04 (m, 2H); 2.15 (3H, CH , s); 2.84 (2H, q, J = 7.5 Hz); 3.93 (2H, t, J =
3
5
.3 Hz); 4.19 (2H, t, J = 6.7 Hz); 6.62 – 6.68 (m, 1H); 7.06 – 7.09 (m, 2H); 7.17 – 7.40 (m, 3H);
+
7.63 – 7.84 (m, 1H). LCMS – ESI (M+H) : 343.2 (99.4% purity). HRMS (ESI): calcd for
C H N OCl, 343.1577; found, 343.1566.
20
24
2
1
-(4-(4-chloro-5-methyl-2-nitrophenoxy)butyl)-2-ethyl-1H-benzo[d]imidazole (31)
1
Yield 31: (0.057 g, 82%). H NMR (300 MHz, CDCl ): δ 1.35 – 1.62 (m, 3H), 1.72 – 2.22 (m,
3
4
H), 2.39 (s, 3H), 2.79 – 3.09 (m, 2H), 3.92 – 4.39 (m, 4H), 6.84 (s, 1H), 7.09 – 7.45 (m, 3H),
+
7
.64 – 7.84 (m, 1H), 7.82 – 7.99 (m, 1H).LCMS – ESI (M+H) : 388.1 (95.1% purity). HRMS
(
ESI): calcd for C H N O Cl, 388.1428; found, 388.1411.
20 23 3 3
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Journal of Medicinal Chemistry
Page 24 of 57
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5
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5
5
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6
6
-chloro-2-ethyl-1-(4-phenoxybutyl)-1H-benzo[d]imidazole (32)
1
Yield 32: (0.023 g, 13%). H NMR (300 MHz, CDCl ): δ 1.46 (t, J = 7.3 Hz, 3H), 1.75 – 1.92
3
(m, 2H), 1.93 – 2.10 (m, 2H), 2.88 (q, J = 7.4 Hz, 2H), 3.97 (t, J = 5.8 Hz, 2H), 4.16 (t, J = 7.3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
Hz, 2H), 6.82 – 7.06 (m, 3H), 7.14 – 7.40 (m, 4H), 7.70 (s, 1H).. LCMS – ESI (M+H) : 329.2
(97.2% purity). HRMS (ESI): calcd for C H N OCl, 329.1421; found, 329.1418.
1
9
22
2
5-chloro-2-ethyl-1-(4-phenoxybutyl)-1H-benzo[d]imidazole (33)
1
Yield 33: (0.023 g, 13%). H NMR (300 MHz, CDCl ): δ 1.46 (t, J = 7.3 Hz, 3H), 1.75 – 1.92
3
(m, 2H), 1.93 – 2.10 (m, 2H), 2.88 (q, J = 7.4 Hz, 2H), 3.97 (t, J = 5.8 Hz, 2H), 4.16 (t, J = 7.3
13
Hz, 2H), 6.72 – 7.06 (m, 3H), 7.14 – 7.42 (m, 4H), 7.70 (s, 1H).. C NMR (CDCl ): δ 11.73,
3
20.84, 26.57, 26.80, 43.48, 66.99, 109.36, 114.41, 120.04, 120.97, 122.39, 127.80, 129.56,
+
135.72, 141.24, 156.84, 158.65. LCMS – ESI (M+H) : 329.2 (98.2% purity). HRMS (ESI): calcd
for C H N OCl, 329.1421; found, 329.1407.
1
9
22
2
2-ethyl-6-methyl-1-(4-phenoxybutyl)-1H-benzo[d]imidazole (34)
1
Yield 34: (0.125 g, 66%). H NMR (300 MHz, CDCl ): δ 1.45 (3H, CH , t, J = 7.5 Hz); 1.84 –
3
3
186 (m, 2H); 1.99 – 203 (m, 2H); 2.47 (3H, CH , s); 2.88 (2H, q, J = 6.7 Hz); 3.96 (2H, t, J =
3
5.3 Hz); 4.15 (2H, t, J = 7.5 Hz); 6.88 – 7.05 (m, 3H), 7.14 – 7.31 (m, 4H), 7.28 (s, 1H). LCMS –
+
ESI (M+H) : 309.2 (99% purity). HRMS (ESI): calcd for C H N O, 309.1967; found,
2
0
25
2
309.1964.
6-chloro-2-ethyl-3-(4-phenoxybutyl)-3H-imidazo[4,5-b]pyridine (35)
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Page 25 of 57
Journal of Medicinal Chemistry
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Yield 35: (0.015 g, 17%). H NMR (300 MHz, CDCl ): δ 1.48 (t, J = 7.5 Hz, 3H), 1.64 – 1.78
3
(
m, 2H), 1.89 – 2.01 (m, 2H), 2.89 (q, J = 7.5 Hz, 2H), 3.18 (t, J = 6.9 Hz, 2H), 4.15 (t, J = 7.3
Hz, 2H), 6.40 (s, 1H), 6.49 (d, J = 7.4 Hz, 1H), 6.93 (d, J = 7.4 Hz, 1H), 7.19 – 7.26 (m, 2H),
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
7
.26 – 7.32 (m, 1H), 7.70 – 7.78 (m, 1H).. LCMS – ESI (M+H) : 330.1 (98% purity). HRMS
(ESI): calcd for C H N OCl, 330.1373; found, 330.1364.
1
8
21
3
2
-ethyl-6-methoxy-1-(4-phenoxybutyl)-1H-benzo[d]imidazole (36)
1
Yield 36: (0.025 g, 28%). H NMR (300 MHz, CDCl ): δ 1.46 (3H, CH , t, J = 7.5 Hz); 1.85 –
3
3
1.87 (m, 2H); 2.00 – 2.02 (m, 2H); 2.88 (2H, q, J = 2.1 Hz, 7.5 Hz); 3.98 (3H, CH , s); 4.14 (2H,
3
t, J = 5.7 Hz); 4.23 (2H, t, J = 7.2 Hz); 6.86 – 7.06 (m, 3H), 7.11 – 7.21 (m, 4H), 7.31 (s, 1H).
+
LCMS – ESI (M+H) : 325.2 (99.3% purity). HRMS (ESI): calcd for C H N O , 325.1916;
20 25
2
2
found, 325.1916.
2
-ethyl-5-methoxy-1-(4-phenoxybutyl)-1H-benzo[d]imidazole (37)
1
Yield 37: (0.012 g, 13%). H NMR (300 MHz, CDCl ): δ 1.46 (3H, CH , t, J = 7.5 Hz); 1.83 –
3
3
1
.86 (m, 2H); 2.01 – 2.04 (m, 2H); 2.88 (2H, q, J = 2.1 Hz, 7.5 Hz); 3.98 (3H, CH , s); 4.14 (2H,
3
t, J = 6.0 Hz); 4.23 (2H, t, J = 7.2 Hz); 6.86 – 7.06 (m, 3H), 7.11 – 7.20 (m, 4H), 7.31 (s, 1H).
+
LCMS – ESI (M+H) : 325.2 (99.4% purity). HRMS (ESI): calcd for C H N O , 325.1916;
2
0
25
2
2
found, 325.1909.
2
-ethyl-3-(4-phenoxybutyl)-3H-imidazo[4,5-c]pyridine (38)
1
Yield 38: (0.075 g, 38%). H NMR (300 MHz, CDCl ): δ 1.50 (t, J = 7.5 Hz, 3H), 1.77 – 1.97
3
(
m, 2H), 1.95 – 2.18 (m, 2H), 2.94 (q, J = 7.5 Hz, 2H), 4.00 (t, J = 5.9 Hz, 2H), 4.28 (t, J = 7.4
Hz, 2H), 6.81 – 6.91 (m, 2H), 6.91 – 7.00 (m, 1H), 7.20 – 7.34 (m, 2H), 7.64 (d, J = 5.5 Hz, 1H),
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8
.41 (d, J = 5.6 Hz, 1H), 8.76 (s, 1H).. LCMS – ESI (M+H) : 296.0 (99% purity). HRMS (ESI):
calcd for C H N O, 296.1763; found, 296.1760.
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2
-ethyl-1-(4-phenoxybutyl)-1H-imidazo[4,5-c]pyridine (39)
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Yield 39: (0.045 g, 23%). H NMR (300 MHz, CDCl ): δ 1.51 (t, J = 7.5 Hz, 3H), 1.70 – 1.94
3
(m, 2H), 1.92 – 2.15 (m, 2H), 2.94 (q, J = 7.5 Hz, 2H), 4.01 (t, J = 5.8 Hz, 2H), 4.22 (t, J = 7.4
Hz, 2H), 6.82 – 6.92 (m, 2H), 6.92 – 7.02 (m, 1H), 7.22 – 7.37 (m, 3H), 8.38 (d, J = 5.6 Hz, 1H),
+
9
.03 (s, 1H).. LCMS – ESI (M+H) : 296.0 (99.2% purity). HRMS (ESI): calcd for C H N O,
1
8
22
3
296.1763; found, 296.1758.
2
-ethyl-6-methyl-1-(4-phenoxybutyl)-1H-imidazo[4,5-c]pyridine (40)
1
Yield 40: (0.022 g, 28%). H NMR (300 MHz, CDCl ): δ 1.50 (t, J = 7.5 Hz, 3H), 1.69 – 1.95
3
(
m, 2H), 1.96 – 2.18 (m, 2H), 2.65 (s, 3H), 2.94 (q, J = 7.5 Hz, 2H), 4.03 (t, J = 6.1 Hz, 2H),
4
.33 (t, J = 7.4 Hz, 2H), 6.66 – 7.11 (m, 4H), 7.18 – 7.40 (m, 2H), 7.85 (d, J = 8.1 Hz, 1H).
+
. LCMS – ESI (M+H) : 310.0 (99% purity). HRMS (ESI): calcd for C H N O, 310.1919;
19
24
3
found, 310.1922.
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-ethyl-1-(4-phenoxybutyl)-1H-benzo[d]imidazole-6-carbonitrile (41)
1
Yield 41: (0.022 g, 30%). H NMR (300 MHz, CDCl ): δ 1.47 (3H, CH , t, J = 7.5 Hz); 1.86 –
3
3
1
.89 (m, 2H); 2.03 – 2.05 (m, 2H); 2.94 (2H, q, J = 2.1 Hz, 7.5 Hz); 4.04 (2H, t, J = 5.7 Hz);
4
.23 (2H, t, J = 7.2 Hz); 6.86 – 7.02 (m, 3H), 7.22 – 7.37 (m, 2H), 7.48ꢀ7.77 (m, 2H), 8.04 (s,
+
1
H) LCMS – ESI (M+H) : 320.2 (98.2% purity). HRMS (ESI): calcd for C H N O, 320.1763;
20
22
3
found, 320.1764.
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-ethyl-1-(4-phenoxybutyl)-1H-benzo[d]imidazole-5-carbonitrile (42)
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Yield 42: (0.008 g, 11%). H NMR (300 MHz, CDCl ): δ 1.47 (3H, CH , t, J = 7.5 Hz); 1.86 –
3
3
1.89 (m, 2H); 2.03 – 2.04 (m, 2H); 2.94 (2H, q, J = 2.1 Hz, 7.5 Hz); 4.04 (2H, t, J = 5.7 Hz);
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37
38
39
40
41
42
43
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45
46
47
48
49
50
51
52
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54
55
56
57
58
59
60
4
.23 (2H, t, J = 7.2 Hz); 6.86 – 7.02 (m, 3H), 7.22 – 7.37 (m, 2H), 7.47ꢀ7.67 (m, 2H), 7.79 (s,
+
1H).LCMS – ESI (M+H) : 320.2 (98% purity). HRMS (ESI): calcd for C H N O, 320.1763;
20
22
3
found, 320.1769.
1-(4-phenoxybutyl)-2-phenyl-1H-benzo[d]imidazole (43)
1
Yield 43: (0.106 g, 60%). H NMR (300 MHz, CDCl ): δ 1.63 – 1.79 (m, 2H), 1.94 – 2.11 (m,
3
2H), 3.85 (t, J = 6.0 Hz, 2H), 4.32 (t, J = 6.0 Hz, 2H), 6.76 – 6.86 (m, 2H), 6.88 – 7.00 (m, 1H),
7.19 – 7.37 (m, 4H), 7.37 – 7.52 (m, 4H), 7.66 – 7.75 (m, 2H), 7.78 – 7.89 (m, 1H).. LCMS –
+
ESI (M+H) : 343.2 (99.2% purity). HRMS (ESI): calcd for C H N O, 343.1810; found,
2
3
23
2
343.1804.
1-(4-phenoxybutyl)-2-(trifluoromethyl)-1H-benzo[d]imidazole (44)
1
Yield 44: (0.070 g, 78%). H NMR (300 MHz, CDCl ): δ 1.91 – 1.93 (m, 2H); 2.11 ꢀ2.14 (m,
3
2H); 4.00 (2H, t, J = 6.0 Hz); 4.44 (2H, t, J = 7.5 Hz); 6.89 – 7.17 (m, 3H), 7.37 – 7.62 (m, 4H),
+
7.67 – 7.92 (m, 2H). LCMS – ESI (M+H) : 335.0 (99.5% purity). HRMS (ESI): calcd for
C H N OF , 335.1371; found, 335.1361.
18
18
2
3
1-(6-methyl-1-(4-phenoxybutyl)-1H-benzo[d]imidazol-2-yl)ethanol (45)
1
Yield 45: (0.022 g, 24%). H NMR (300 MHz, CDCl ): δ 1.57 (3H, CH , d, J = 6.6 Hz); 1.91 ꢀ
3
3
2.18 (m, 2H); 2.07 (m, 2H); 2.47 (3H, CH , s); 3.98 (2H, t, J = 6.3 Hz); 4.24 – 4.26 (2H, m); 5.09
3
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(
1H, q, J = 6.6 Hz); 6.79 – 7.00 (m, 4H); 7.00 – 7.11 (m, 1H); 7.19 (d, J = 8.2 Hz, 1H); 7.21 –
+
7
.37 (m, 3H). LCMS – ESI (M+H) : 325.3 (97.2% purity). HRMS (ESI): calcd for C H N O ,
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2
2
3
25.1916; found, 325.1911.
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-(1-(4-(3-chloro-4-methylphenoxy)butyl)-6-methyl-1H-benzo[d]imidazol-2-yl)ethanol (46)
1
Yield 46: (0.022 g, 23%). H NMR (300 MHz, CDCl ): δ 1.57 (3H, CH , d, J = 6.6 Hz); 1.97
3
3
(2H, dd, J = 5.9 Hz, 11.7 Hz); 2.07 (2H, dd, J = 5.9 Hz, 11.7 Hz); 2.29 (3H, CH , s); 2.47 (3H,
3
CH , s); 3.99 (2H, t, J = 6.3 Hz); 4.25 (2H, m); 5.09 (1H, q, J = 1.59 – 1.76 (m, 3H), 1.76 – 1.92
3
(m, 2H), 1.91 – 2.16 (m, 2H), 2.29 (s, 3H), 2.47 (d, J = 3.7 Hz, 3H), 3.94 (q, J = 6.4 Hz, 2H),
4
.06 – 4.40 (m, 2H), 5.08 (q, J = 6.6 Hz, 1H), 6.52 – 6.79 (m, 1H), 6.81 – 6.96 (m, 1H), 7.01 –
7
.14 (m, 2H), 7.51 (s, 1H), 7.60 (d, J = 8.1 Hz, 1H). (ESI): calcd for C H N O Cl, 373.1683;
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2
2
found, 373.1685.
N-(1-(5-(3-chloro-4-methylphenyl)pentyl)-1H-benzo[d]imidazol-2-yl)acetamide (47)
1
Yield 47: (0.012 g, 41%). H NMR (300 MHz, CDCl ): δ 1.75 – 1.91 (m, 2H), 1.94 – 2.08 (m,
3
2
H), 2.24 (s, 3H), 2.29 (s, 3H), 4.00 (t, J = 6.1 Hz, 2H), 4.21 (t, J = 7.0 Hz, 2H), 6.62 – 6.77 (m,
1
H), 6.89 (d, J = 2.6 Hz, 1H), 7.10 (d, J = 8.4 Hz, 1H), 7.20 – 7.33 (m, 4H).. LCMS – ESI
+
(M+H) : 371.9 (99.5% purity). HRMS (ESI): calcd for C H NCl, 372.1479; found, 372.1485.
2
5
23
4
-(1-(4-phenoxybutyl)-1H-benzo[d]imidazol-2-yl)morpholine (48)
1
Yield 48: (0.029 g, 49%). H NMR (300 MHz, CDCl ): δ 1.71 – 1.92 (m, 2H), 1.95 – 2.15 (m,
3
2
H), 3.20 – 3.35 (m, 4H), 3.75 – 3.90 (m, 4H), 4.02 (t, J = 5.9 Hz, 2H), 4.13 (t, 2H), 6.89 (d, J =
.1 Hz, 2H), 6.98 (t, J = 7.3 Hz, 1H), 7.12 – 7.24 (m, 2H), 7.25 – 7.36 (m, 3H), 7.54 – 7.72 (m,
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H).. LCMS – ESI (M+H) : 352.3 (96.4% purity). HRMS (ESI): calcd for C H NCl, 352.2025;
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found, 352.2020.
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-(4-(3-chloro-4-methylphenoxy)butyl)-1H-benzo[d]imidazol-2-amine (49)
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11
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Yield 49: (0.07 g, 33%). H NMR (300 MHz, CDCl ): δ 1.77 – 1.91 (m, 2H), 1.94 – 2.08 (m,
3
2H), 2.28 (s, 3H), 4.01 (t, J = 5.7 Hz, 2H), 4.21 (t, J = 7.0 Hz, 2H), 6.62 – 6.77 (m, 1H), 6.89 ꢀ
+
7
.10 (m, 2H), 7.20 – 7.33 (m, 4H).. LCMS – ESI (2M+H O) : 578.1 (98.2% purity).
2
2
-(4-methylpiperazin-1-yl)-1-(4-phenoxybutyl)-1H-benzo[d]imidazole (50)
1
Yield 50: (0.047 g, 76%). H NMR (300 MHz, CDCl ): δ 1.79 – 1.82 (m, 2H); 1.89 ꢀ 2.20 (m,
3
2
H); 2.34 (3H, CH , s); 2.41 – 2.73 (4H, m); 3.21 – 3.41 (4H, m); 4.03 – 4.08 (4H, m); 6.88 –
3
+
6
.97 (m, 3H); 7.08 – 7.42 (m, 5H); 7.50 – 7.72 (m, 1H). LCMS – ESI (M+H) : 365.3 (98.4%
purity). HRMS (ESI): calcd for C H N O, 365.2341; found, 365.2344.
2
2
29
4
2
-chloro-1-(4-phenoxybutyl)-1H-benzo[d]imidazole (51)
1
Yield 51: (0.255 g, 65%). H NMR (300 MHz, CDCl ): δ 1.64 – 1.91 (m, 2H); 1.97 ꢀ 2.15 (m,
3
2
H); 3.97 (2H, t, J = 5.8 Hz); 4.27 (2H, t, J = 6.0 Hz); 6.88 – 6.97 (m, 3H); 7.15 – 7.46 (m, 5H);
+
7
.74 – 7.76 (m, 1H). LCMS – ESI (M+H) : 300.9 (98.1% purity). HRMS (ESI): calcd for
C H N O, 301.1108; found, 301.1103.
22
29
4
1
-(4-phenoxybutyl)-1H-benzo[d]imidazole (52)
1
Yield 52: (0.110 g, 98%). H NMR (300 MHz, CDCl ): δ 1.68 – 1.98 (m, 2H), 1.93 – 2.21 (m,
3
2
H), 3.98 (t, J = 5.9 Hz, 2H), 4.26 (t, J = 7.1 Hz, 2H), 6.78 – 7.04 (m, 3H), 7.21 – 7.38 (m, 3H),
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