Preliminary structure - Activity relationships and biological evaluation of novel antitubercular indolecarboxamide derivatives against drug-susceptible and drug-resistant Mycobacterium tuberculosis strains

Article abstract of DOI:10.1021/jm4003878

Tuberculosis (TB) remains one of the leading causes of mortality and morbidity worldwide, with approximately one-third of the world's population infected with latent TB. This is further aggravated by HIV coinfection and the emergence of multidrug- and extensively drug-resistant (MDR and XDR, respectively) TB; hence the quest for highly effective antitubercular drugs with novel modes of action is imperative. We report herein the discovery of an indole-2-carboxamide analogue, 3, as a highly potent antitubercular agent, and the subsequent chemical modifications aimed at establishing a preliminary body of structure-activity relationships (SARs). These efforts led to the identification of three molecules (12-14) possessing an exceptional activity in the low nanomolar range against actively replicating Mycobacterium tuberculosis, with minimum inhibitory concentration (MIC) values lower than those of the most prominent antitubercular agents currently in use. These compounds were also devoid of apparent toxicity to Vero cells. Importantly, compound 12 was found to be active against the tested XDR-TB strains and orally active in the serum inhibition titration assay.

Full text of DOI:10.1021/jm4003878

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Article
Preliminary Structure–Activity Relationships and Biological Evaluation
of Novel Antitubercular Indolecarboxamide Derivatives Against
Drug-Susceptible and Drug-Resistant M. tuberculosis Strains
Oluseye K Onajole, Marco Pieroni, Suresh K Tipparaju, Shichun Lun, Jozef Stec, Gang Chen,
Hendra Gunosewoyo, Haidan Guo, Nicole C Ammerman, William R. Bishai, and Alan P. Kozikowski
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm4003878 • Publication Date (Web): 23 Apr 2013
Downloaded from http://pubs.acs.org on April 28, 2013
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Preliminary Structure–Activity Relationships and
Biological Evaluation of Novel Antitubercular
Indolecarboxamide Derivatives Against Drugꢀ
Susceptible and DrugꢀResistant M. tuberculosis
Strains
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Oluseye K. Onajole,† Marco Pieroni,†,^ Suresh K. Tipparaju,†,§, Shichun Lun,║ Jozef Stec†,┴
Gang Chen,† Hendra Gunosewoyo,† Haidan Guo,║ Nicole C. Ammerman,║,▲ William R.
Bishai║,▲,¶* and Alan P. Kozikowski.†*
†Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of
Illinois at Chicago, 833 South Wood Street, Chicago, Illinois, 60612, United States
║Center for Tuberculosis Research, Department of Medicine, Division of Infectious Disease,
Johns Hopkins School of Medicine, Baltimore, Maryland, 21231ꢀ1044, United States
▲KwaZuluꢀNatal Research Institute for Tuberculosis and HIV, Nelson R. Mandela School of
Medicine, 719 Umbilo Road, Durban, 4001, KwaZuluꢀNatal, South Africa
¶Howard Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase, Maryland, 20815ꢀ
6789, United States
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ABSTRACT
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Tuberculosis (TB) remains one of the leading causes of mortality and morbidity worldwide with
approximately one third of the world’s population infected with latent TB. This is further
aggravated by HIV coꢀinfection and the emergence of multidrugꢀ and extensively drugꢀresistant
(MDR and XDR, respectively) TB; hence the quest for highly effective antitubercular drugs with
novel modes of action is imperative. We report herein the discovery of an indoleꢀ2ꢀcarboxamide
analogue, 3, as a highly potent antitubercular agent, and the subsequent chemical modifications
aimed at establishing a preliminary body of structure–activity relationships (SARs). These efforts
led to the identification of three molecules (12–14) possessing an exceptional activity in the low
nanomolar range against actively replicating Mycobacterium tuberculosis, with minimum
inhibitory concentration (MIC) values lower than those of the most prominent antitubercular
agents currently in use. These compounds were also devoid of apparent toxicity to Vero cells.
Importantly, compound 12 was found to be active against the tested XDRꢀTB strains and orally
active in the serum inhibition titration assay.
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INTRODUCTION
TB is a highly contagious and insidious disease caused by Mycobacterium tuberculosis (M.
tuberculosis) and is currently the second leading cause of mortality due to infectious disease
worldwide.1 Despite its known etiology, TB still remains one of the most threatening health
problems globally, claiming 1.4 million lives in 2011.2 Firstꢀline TB chemotherapy options have
not changed in nearly five decades with the most recent drug rifampin having been introduced in
early 1960s.3 The current treatment regimen for drug susceptible M. tuberculosis requires the
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daily administration of firstꢀline drugs which include ethambutol, isoniazid, pyrazinamide and
rifampin for two months followed by an additional four months daily administration of isoniazid
and rifampin.4 However, several factors such as inconsistent treatment, patient nonꢀcompliance,
and the nonꢀavailability of drugs during this period have contributed to the failure to achieve a
cure, making the emergence of resistance an event practically unavoidable in manyꢀresourceꢀ
limited countries.5
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Drug resistantꢀTB can be classified into two categories. MDRꢀTB is caused by infection with
M. tuberculosis resistant to isoniazid and rifampin, and requires 18−24 months of combination
therapy with secondꢀline drugs which are generally more toxic, expensive and difficult to
administer. XDRꢀTB is caused by M. tuberculosis resistant to isoniazid, rifampin, at least one
fluoroquinolone and one of the injectable antitubercular drugs such as amikacin, kanamycin or
capreomycin. The success rate for the treatment of XDRꢀTB is very low depending on the extent
of drug resistance and whether or not the immune system of the patient is compromised.
Consequently in the development of novel antitubercular chemotherapeutics, the lack of crossꢀ
resistance with existing drugs remains pivotal, along with good compound potency and little or
no toxicity.6
At present, there are several drugs being evaluated in clinical trials for the treatment of TB.7ꢀ9
Some of these represent novel chemical entities, such as TMC207 (1)10 and PA824 (2)11,
whereas others are either approved drugs being repurposed for TB or they belong to known
chemical series.12ꢀ14 The United States Food and Drug Administration (FDA) recently approved
TMC207 (bedaquiline, trade name Sirturo®)15 for the treatment of MDRꢀTB (including XDRꢀ
TB) in adults, when no other treatment options are effective. This is the first antitubercular drug
to be approved by the FDA in over four decades. Unfortunately, like other known secondꢀline
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antitubercular drugs, it has been reported to possess unwanted side effects, in particular its ability
to cause QT (the time interval measured from the beginning of the QRS complex to the end of
the T wave in an electrocardiogram) prolongation could potentially lead to abnormal heart
rhythms.15 A singular FDA approval in 40 years coupled with the staggering volume of mortality
from TB underscore the impetus for the discovery and development of new TB
chemotherapeutics.
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In our continuous efforts to discover novel antitubercular scaffolds,16ꢀ22 a phenotypic screening
of a carefully selected library of 6800 compounds was carried out.23 The overall physicochemical
properties of this library fall within Lipinski’s rules of 5, and thus are leadꢀlike. The phenotypic
screening of this library against replicating M. tuberculosis led to the identification of an indoleꢀ
2ꢀcarboxamide scaffold (Figure 1) as one of the hit series.22 The indoleꢀ2ꢀcarboxamide, 3, was
found to possess potent antiꢀTB activity (MIC = 0.93 ꢀM) with low toxicity against Vero cells
(half maximal inhibitory concentration [IC50] > 200 ꢀM). Studies on the identification of the
molecular target for this class of indoleꢀ2ꢀcarboxamides using genome sequencing suggest that
they may inhibit M. tuberculosis growth by acting on the MmpL3 protein, which belongs to the
resistance, nodulation and cell division (RND) family of membrane transporters.24 Herein we
describe the synthesis, the in vitro biological evaluation, and bioavailability study of a number of
indolecarboxamide analogues rationally designed so as to define a preliminary body of SAR as
potential antitubercular agents.
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Figure 1. Structure of TMC207 (1), PAꢀ824 (2) and hit compound 3
Replacement of the
indole ring
Substitution by hydrophilic
or lipophilic atoms
O
N
HN
H
Alkylation of
nitrogen atoms
Replacement by substituted
or non-substituted aryl and
cycloaliphatic groups
Figure 2. Strategies for modification of hit compound 3
CHEMISTRY
The hit compound 3 obtained from high throughput screening (HTS) was resynthesized to
confirm the activity along with 40 novel derivatives (4-44) employing an efficient amide
coupling protocol (Scheme 1 and 2). Briefly, following a Fischer indole synthesis protocol, 3,5ꢀ
dimethylphenylhydrazine hydrochloride (45) was reacted with ethyl pyruvate under acidic
conditions to afford the disubstituted indoleꢀ2ꢀcarboxylate 46, and subsequent basic hydrolysis
afforded the corresponding acid 47. Nꢀmethylation of 46 followed by basic hydrolysis gave the
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carboxylic acid 48. The carboxylic acids were subsequently reacted with their corresponding
amine in the presence of 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide hydrochloride
(EDC.HCl) and hydroxybenzotriazole (HOBt) as coupling agents and triethylamine as a base to
obtain compounds 3−18 (Scheme 1). 5ꢀChlorobenzofuranꢀ2ꢀcarboxylic acid (53)25 and 4,6ꢀ
dimethylbenzofuranꢀ2ꢀcarboxylic acid (54)26 were obtained following a modified literature
protocol (Scheme 1).27,28 Compound 54 was reacted with the appropriate amines under standard
amide coupling conditions to obtain compounds 19−23. Following similar conditions,
compounds 24 and 25 were obtained by reacting carboxylic acid 53 with the appropriate amines
(Scheme 1).
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The unsubstituted and monosubstituted carboxylic acids (55−59) were reacted with their
corresponding amines to afford compounds 26−31, 33 and 34 while compound 32 was obtained
from its methoxy precursor 30 using boron tribromide (Scheme 2). 3,5ꢀ
Bis(trifluoromethyl)phenylhydrazine hydrochloride (60) was reacted with ethyl pyruvate under
microwave irradiation to obtain its hydrazone intermediate 61, which was further subjected to
acidic conditions to obtain the cyclized indole (62). Basic hydrolysis then afforded the desired
carboxylic acid 63. Compound 63 was reacted with cycloheptylamine or cyclooctylamine to
provide the amides 35 and 36. 3,5ꢀDimethylbenzeneꢀ1,2ꢀdiamine (64) was reacted with methylꢀ
2,2,2ꢀtrichloroacetimidate to afford its trichloromethyl intermediate 65, followed by basic
hydrolysis to give the corresponding acid 66. Decarboxylation of indole 47 with copper powder
in quinoline gave the desired intermediate 67, which was subsequently reacted with
trichloroacetyl chloride to give the trichloromethyl intermediate 68. Subsequent basic hydrolysis
afforded 4,6ꢀdimethylꢀ1Hꢀindoleꢀ3ꢀcarboxylic acid (69). Compounds 66 and 69 were reacted
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with their corresponding amines under standard amide coupling conditions to obtain compounds
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37−44 (Scheme 2).
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Scheme 1a
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aReagents and conditions: (a) ethyl pyruvate, pꢀTsOH, toluene, reflux, overnight; (b) LiOH,
EtOH, reflux, 3 h; (c) CH3I, NaH, DMF, rt; (d) EDC.HCl, HOBt, corresponding amines, Et3N,
CH2Cl2, rt, 12–16 h; (e) ethyl bromoacetate, K2CO3, 140 °C, 6 h; (f) (i) EtOH, C2H5ONa, rt to 70
°C, 1 h (ii) 3N NaOH, 70 °C, 20 min (iii) 1N HCl, rt; (g) THF/EtOH (1:1), 3N NaOH, rt, 3−4 h.
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Scheme 2a
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aReagents and conditions: (a) EDC.HCl, HOBt, corresponding amines, Et3N, CH2Cl2, rt, 12–16
h; (b) BBr3, CH2Cl2, ꢀ78 °C to rt, 3 h; (c) ethyl pyruvate, EtOH, MW, 150 °C, 10 min; (d)
polyphosphoric acid, toluene, reflux, 3 h; (e) LiOH, EtOH, reflux, 3 h; (f) EDC.HCl, HOBt,
corresponding amines, Nꢀmethylmorpholine, DMF, rt, 12−16 h; (g) acetic acid, methylꢀ2,2,2ꢀ
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trichloroacetimidate, rt, 16 h; (h) THF, 1N NaOH, rt, 3−4 h; (i) Cu powder, quinoline, 240 °C, 3
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h; (j) trichloroacetyl chloride, C5H5N, CH2Cl2, 3 h; (k) 2N KOH, THF, rt, 3 h.
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RESULTS AND DISCUSSION
The general strategy for the chemical modifications of the hit compound 3 aimed at improving
the antitubercular activity is summarized in Figure 2. The SAR investigation was focused on the
role of different substituents on the amide nitrogen, the indole nitrogen, replacement of the
indole core with other heterocycles, substitution at the 3ꢀposition of the indole ring, and on the
benzene ring of the indole group (Figure 2). Target compounds 3−44 were screened in vitro
against the M. tuberculosis strain H37Rv to obtain the MIC values using the microplate Alamar
Blue assay (MABA) while cytotoxicity was assessed for selected compounds using Vero cells.
The initial round of SAR modifications was directed towards substitutions for the cyclohexyl
group while maintaining the 4,6ꢀdimethylꢀ1Hꢀindoleꢀ2ꢀcarboxamide scaffold. First, we
substituted the cyclohexyl group for an unsubstituted benzene ring to ascertain the possibility of
π–π stacking interactions with the biological target as well as the impact of the planarity of the
aromatic ring on activity. Compounds 4 (MIC = 3.8 ꢀM) and 5 (MIC = 1.7 ꢀM) were
approximately 4ꢀfold and 2ꢀfold less active than the hit compound 3 (MIC = 0.93 ꢀM),
respectively, while the pyridine analog 6 (MIC = 240 ꢀM) showed a dramatic loss in activity.
These results suggest that the presence of an aromatic ring on the amide nitrogen is not
favorable. The nitrogenꢀcontaining cycloaliphatic groups (7, MIC = 449 ꢀM; 8, MIC = 204 ꢀM,
and 9, MIC = 428 ꢀM) were subsequently tested for their antitubercular activity. However, none
of these analogs were found to be active, therefore implying that hydrophilic substituents are
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generally not favorable for biological activity. This trend also includes the pyridyl analog 6,
which was 63ꢀfold less active compared to the phenyl derivative 4.
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Based on these data, the next iteration was performed to examine cycloaliphatic rings. The
introduction of a small ring, such as cyclopropyl, led to a complete loss of activity (10, MIC =
561 ꢀM) whereas a bulkier substituent, such as the cycloheptyl, yielded a highly active
compound (11, MIC = 0.055 ꢀM). Of particular note, compound 11 is approximately 5ꢀfold
more potent than INH (MIC = 0.29 ꢁM), the standard firstꢀline drug used against actively
replicating M. tuberculosis, and it is comparable in activity to the antitubercular agents now on
the market or in clinical trials, namely TMC207 (1) (MIC = 0.05 ꢁM)10 and PA824 (2) (MIC =
0.04–0.70 ꢁM).11 Once it was established that a bulky cycloaliphatic ring on the amide nitrogen
plays an important role for activity, the cyclooctyl and the adamantyl rings were introduced
(analogs 12–14). These modifications led to the identification of the most active compounds in
the series (12–14), with MIC values of 0.013 ꢀM (12) and 0.012 ꢀM (13 and 14), respectively
(Table 1), which are approximately 3ꢀ to 4ꢀfold lower than those published for TMC207 (1)10
and PA824 (2).11 Furthermore, the addition of a methylene spacer between the amide nitrogen
and the cycloaliphatic ring was tolerated (compound 15, MIC = 0.88 ꢀM). Although the increase
in activity was accompanied by a rise in the lipophilicity [the ClogD (pH = 7.4) values calculated
using the ACD/Percepta software are as follow: 3 = 4.36; 11 = 4.78; 12 = 5.32; 13 = 5.03; 14 =
4.96], it must be considered that TMC207 (1) (Sirturo®), recently approved by the FDA for
treatment of MDRꢀTB, has a CLogD value of 5.32. These findings lead us to hypothesize that a
rather large hydrophobic pocket may be present in the target binding site within proximity of the
amide moiety, which is able to accommodate bulky groups such as the cyclooctyl and adamantyl
groups. More importantly, compounds 11−14 exhibited high selectivity indices (SI) ranging from
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>3,600 for 11 and 12 to >16,600 for 13 and 14 when tested against Vero cells, indicating that
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these compounds lack apparent cyctoxicity in this system.
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Table 1. Antitubercular activity of compounds 3−18 against the M. tuberculosis strain H37Rv.
b
b
MICa
(ꢀM)
IC50
MICa
(ꢀM)
IC50
Compd
3
R
Compd
12
R
(ꢀM)
(ꢀM)
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>200
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0.012
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>200
>200
NTc
NT
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>200
>200
>200
NT
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448
204
428
450
N
NT
>499
450
NT
NT
NT
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NT INHd
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>200
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aThe lowest concentration of drug leading to at least a 90% reduction of bacterial growth
signal by the microplate Alamar Blue assay (MABA). MIC values are reported as an average of
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d
three individual measurements; cytotoxicity against Vero cells; NT = not tested; INH =
Isoniazid.
The importance of hydrogen bond donors on the indoleamide core for potent antitubercular
activity was also investigated. First, the influence of the hydrogen atom on the amide motif was
explored; this led to the synthesis of compounds 16 and 17, which were bereft of antiꢀTB
activities (MIC values of > 400 ꢀM, Table 1). Secondly, Nꢀmethyl indole analogue 18 (MIC =
450 ꢀM) was approximately 500ꢀfold less potent than the corresponding analogue 3 (MIC = 0.93
ꢀM), and replacement of the indole moiety with a benzofuran (19−21 vs 11−13) resulted in a
dramatic attenuation of activity, ranging from ~250 up to 2,000ꢀfold difference (Table 2). The
other synthesized benzofuran analogues 22−24 had MIC values of 113, 59 and 26 ꢀM
respectively, while analogue 25 (MIC = >388 ꢀM) was found to be > 1,000ꢀfold less potent than
the indole counterpart 3129 (MIC = 0.38 ꢀM). These data highlight the importance of the two NH
groups at the indole and amide moieties for potent antiꢀTB activity.
Subsequent SAR investigation was focused on the effects of indole substituents on antiꢀTB
activity (Table 2). Interestingly, 4,6ꢀdidesmethyl indole analogues (26 and 27) were found to be
devoid of activity (MIC = ~500 ꢀM) while replacement of the methyl groups with fluoroꢀ (28
and 29) or trifluoromethylꢀ (35 and 36) substituents resulted in a 3ꢀ to 17ꢀ fold drop in activity
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compared to their counterparts 11 and 12. Introduction of a methoxy group at Cꢀ6 (30, MIC =
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0.77 ꢀM) gave a 2ꢀfold higher activity than a chlorine atom at Cꢀ5 (31, MIC
= 0.38 ꢀM),
7
8
9
whereas the more polar 6ꢀhydroxy substituent (32, MIC = 12.9 ꢀM), resulted in 17ꢀfold
reduction of activity compared to 30. Thus far, the 4,6ꢀdimethyl substituents on the indole ring
resulted in compounds with the most potent antiꢀTB activity in this series.
10
11
12
13
14
15
16
17
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44
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60
Aiming to improve the water solubility by introduction of ionizable nitrogen groups, the 5ꢀazaꢀ
6ꢀmethoxyindoles 33 and 34 were synthesized whereby the benzene ring of the indole nucleus
was replaced with a pyridine ring. Unfortunately, these modifications resulted in a 510ꢀfold and a
125ꢀfold decrease in activity, respectively, compared to the MIC values of compounds 12 and
13. Further attempts to replace the indole core included the replacement with benzo[d]imidazole
(37−40). However, these compounds did not show improved antitubercular activity compared to
their corresponding indoleꢀ2ꢀcarboxamide counterparts 11−14. The positioning of the
carboxamide on the indole ring was also explored (Scheme 2). Shifting the carboxamide moiety
to position 3 of the indole ring (41−44, Scheme 2) resulted in a drastic drop in the activity (MIC
values of ~ 200 ꢀM), supporting the importance of the carboxamide group at position 2 of the
indole ring. Thus, these data point to the indoleꢀ2ꢀcarboxamide core as the superior scaffold for
potent and nonꢀtoxic antiꢀTB leads.
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Table 2. Antitubercular activity of compounds 19−40 against M. tuberculosis strain H37Rv.
5
6
7
8
9
10
11
12
13
14
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17
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19
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MICa
(ꢀM)
MICa
(ꢀM)
Compd
X
R
Compd
X
R
4,6ꢀ
dimethyl
56
27
6ꢀOCH3
0.77
0.38
13
19
30
4,6ꢀ
dimethyl
5ꢀCl
20
21
22
23
24
25
26
27
31
32
33
34
35
36
37
38
4,6ꢀ
dimethyl
3.1
6ꢀOH
4,6ꢀ
dimethyl
113
59
ꢀ
ꢀ
6.6
4,6ꢀ
dimethyl
1.5
4,6ꢀ
bis(CF3)
5ꢀCl
5ꢀCl
H
26
0.64
0.04
>224
1.7
4,6ꢀ
bis(CF3)
≥388
>528
477
ꢀ
ꢀ
H
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4,6ꢀ
difluoro
0.86
0.10
ꢀ
ꢀ
0.39
1.5
28
29
39
40
4,6ꢀ
difluoro
10
11
12
13
14
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19
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aThe lowest concentration of drug leading to at least a 90% reduction of bacterial growth
signal by microplate Alamar Blue assay (MABA). MIC values are reported as an average of
three individual measurements.
Table 3. Antitubercular activity of compound 3, 11 and 12 against susceptible, MDR and XDR
strains of M. tuberculosis.
V4207
(DS)a
TF274
R506
KZN494
(MDR)c
V2475
Compd
(XDR)b
(XDR)b
(MDR)c
MICd (ꢀM)
0.93
0.11
0.026
0.46
0.46
3.7
0.93ꢀ1.9
0.11
3
0.055
0.055
0.11
eNT
11
0.026
0.0067
eNT
12
aDrug susceptible strain of M. tuberculosis; bextensively drug resistant strain of M.
c
d
tuberculosis; multiꢀdrug resistant strain of M. tuberculosis; the lowest concentration of drug
leading to at least a 90% reduction of bacterial growth signal by microplate Alamar Blue assay
e
(MABA); reported MIC values are an average of three individual measurements; NT = not
tested.
Selected compounds 3, 11 and 12 were tested for their ability to inhibit the growth of the
acquired clinical MDRꢀTB (KZN494 and V2475) and XDRꢀTB (TF274 and R506) strains from
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KwaZuluꢀNatal, South Africa (Table 3).30 To our delight, these indoleꢀ2ꢀcarboxamides
maintained similar excellent activities against the susceptible M. tuberculosis strain H37Rv in all
the tested drugꢀresistant strains.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
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41
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44
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52
53
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60
Figure 3. Serum inhibition titration result for compound 12.
nNH = number of hydrogen bond donors; nON = number of hydrogen bond acceptors; MW =
Molecular Weight; TPSA = Topological polar surface area; nRot. bond = number of rotatable
bonds calculated using the molinspiration online service (www.molinspiration.com); ClogD was
calculated using the ACD/lab Percepta software; BALB/c mice were orally gavaged with two
doses (100 and 300 mg/kg) of compound 12, with blood collected at different time points and
serum separated 60 min later. Growth inhibition of serially diluted serum on H37Rv was
determined using the Alamar Blue assay; Vehicle, 0.5 % CMC (carboxylmethyl cellulose); INH,
isoniazid at 10 mg/kg (positive control).
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The most potent indoleꢀ2ꢀcarboxamides 11–14 were evaluated in the serum inhibition titration
assay.31 Briefly, each compound was administered at 100 and 300 mg/kg to BALB/c mice by
oral gavage using carboxymethyl cellulose as vehicle, after which blood samples were collected
at 15, 30 and 60 minutes. The sera were separated and prepared in 2ꢀfold dilutions and incubated
with a bacterial suspension for 7 days. Bacterial growth was measured using MABA. As
illustrated in Figure 3, the cyclooctyl derivative 12 was present in the serum at potent
concentrations up to a 1:128 dilution following a 300 mg/kg oral dose at the 30 minute time
point. In contrast, the cycloheptyl derivative 11 and both adamantyl derivatives 13 and 14 were
not active (data not shown) in this assay which may be a consequence of any of a number of
factors including poor absorption, tight serum protein binding, rapid metabolism, etc.32 The
attractive results achieved with compound 12 suggest that it or one of its analogs may be worth
developing further in order to identify potential antiꢀTB chemotherapeutics.
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11
12
13
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CONCLUSIONS
A set of indolecarboxamide derivatives 3−44 has been synthesized and evaluated to build a
body of preliminary SAR based on compound 3 obtained from an highꢀthroughput screening
campaign. The SAR of these analogues leads to the following considerations for antiꢀTB
activity: a) at the amide nitrogen, lipophilic and bulky substituents are beneficial for potent antiꢀ
TB activity (12–14), whereas polar substituents (6−9) lead to loss of the activity suggesting the
presence of a hydrophobic pocket in the target binding site; b) carboxamide substitution at
position 2 of the indole ring is essential in retaining the antiꢀTB activity; c) an unsubstituted
indole nitrogen and a monoꢀsubstituted amide nitrogen are required for the antimycobacterial
activity; d) methyl groups at position Cꢀ4 and Cꢀ6 of the indole ring are important in order to
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confer high activity. The most active compounds in the series, 12–14, have MIC values lower
than that of isoniazid, the most active compound used against actively replicating M.
tuberculosis, and those of TMC207 (1)10 and PA824 (2).11 In addition, compounds 3, 11 and 12
showed excellent activity against the susceptible, the MDRꢀ and the XDRꢀTB strains, indicating
that this series does not suffer from crossꢀresistance with firstꢀ and secondꢀline TB drugs. In
particular, compound 12 was found to be orally active in the serum inhibition titration assay.
Overall, the extraordinary potency against drug susceptible as well as drugꢀresistant M.
tuberculosis strains, and the lack of apparent toxicity, establish these indoleꢀ2ꢀcarboxamides
derivatives as promising novel class of compounds in the pursuit of highly effective antiꢀTB
agents.
10
11
12
13
14
15
16
17
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19
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30
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44
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EXPERIMENTAL SECTION
General information. The following carboxylic acids, 1Hꢀindoleꢀ2ꢀcarboxylic acid, 5ꢀchloroꢀ
1Hꢀindoleꢀ2ꢀcarboxylic acid, 6ꢀmethoxyꢀlHꢀindoleꢀ2ꢀcarboxylic acid, were purchased from
SigmaꢀAldrich while 6ꢀmethoxyꢀ1Hꢀpyrrolo[3,2ꢀc]pyridineꢀ2ꢀcarboxylic acid and 4,6ꢀdifluoroꢀ
1Hꢀindoleꢀ2ꢀcarboxylic acid were purchased from ChemꢀImpex and Combiꢀblocks. Anhydrous
1
13
dichloromethane (CH2Cl2) was obtained by distillation over calcium hydride. H NMR and C
NMR spectra were recorded on a Bruker spectrometer at 400 MHz and 100 MHz, respectively,
with TMS as an internal standard. Standard abbreviation indicating multiplicity was used as
follows: s = singlet, d = doublet, dd = doublet of doublets, t = triplet, q = quadruplet, m =
multiplet and br = broad. HRMS experiments were performed on QꢀTOFꢀ2TM (Micromass) and
ITꢀTOF (Shimadzu) instruments. TLC was performed with Merck 60 F254 silica gel plates.
Flash chromatography was performed using CombiFlash® Rf system with RediSep® columns or
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alternatively using Merck silica gel (40–60 mesh). Final compounds were purified by preparative
HPLC unless otherwise stated. The preparative HPLC employed an ACE 5ꢀAQ (21.2 mm × 150
mm) column, with detection at 254 and 280 nm on a Shimadzu SCLꢀ10A VP detector, flow rate
= 17.0 mL/min. Method 1: 50–100% CH3OH/H2O in 30 min; 100% CH3OH in 5 min; 100–50%
CH3OH/H2O in 4 min. Method 2: 25–100% CH3OH/H2O in 30 min; 100% CH3OH in 5 min;
100–25% CH3OH/H2O in 4 min. Method 3: 15ꢀ100% CH3OH/H2O in 30 min; 100% CH3OH in
5 min; 100–15% CH3OH/H2O in 4 min. Both solvents contains 0.05 vol % of trifluoroacetic acid
(TFA). Purities of final compounds were established by analytical HPLC, which was carried out
using the Agilent 1100 HPLC system with a Synergi 4 ꢂm HydroꢀRP 80A column, on a variable
wavelength detector G1314A. Method 1: flow rate = 1.4 mL/min; gradient elution over 20
minutes, from 30% MeOHꢀH2O to 100% MeOH with 0.05% TFA. Method 2: flow rate = 1.4
mL/min; gradient elution over 20 minutes, from 50% MeOHꢀH2O to 70% MeOH with 0.05%
TFA. The purity of all tested compounds was >95% as determined by the method described
above.
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11
12
13
14
15
16
17
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19
20
21
22
23
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41
42
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44
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General procedure for the synthesis of 3–44
To a solution of the appropriate carboxylic acid (1 equiv) in anhydrous dichloromethane
(CH2Cl2) or dimethylformamide (DMF) (4 mL/mmol) at room temperature were added
anhydrous
hydroxybenzotriazole
(HOBt,
1
equiv)
and
1ꢀethylꢀ3ꢀ(3ꢀ
dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl, 1 equiv) under an argon
atmosphere. After stirring for 10 min, the appropriate substituted amine (1 equiv) and
triethylamine or Nꢀmethyl morpholine (1.5 equiv) were added, and the reaction mixture was
stirred at room temperature until disappearance of the starting material (usually 12 to 16 h). After
this time water (2 mL) was added, and the mixture was extracted with EtOAc (3×10 mL), the
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organic layers were separated, washed with brine, dried over anhydrous Na2SO4, filtered, and
concentrated under reduced pressure. The residue was purified by flash chromatography
(EtOAc–hexane 1:4 unless specified differently) to obtain the indoleamides in yields ranging
from 34 to 95%.
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11
12
13
14
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1
N-Cyclohexyl-4,6-dimethyl-1H-indole-2-carboxamide (3). Yield 92% (white powder). H
NMR (400 MHz, CDCl3)
δ 9.32 (br s, 1H), 7.07 (s, 1H), 6.79 (s, 2H), 6.03 (d, J = 7.6 Hz, 1H),
4.07–3.98 (m, 1H), 2.44 (s, 3H), 2.37 (s, 3H), 2.10–2.06 (m, 2H), 1.83–1.78 (m, 2H), 1.68–1.45
13
(m, 2H), 1.31–1.26 (m, 4H); C NMR (100 MHz, CDCl3)
δ 160.9, 136.5, 134.6, 130.9, 129.9,
125.7, 122.7, 109.2, 99.9, 48.5, 33.3, 25.6, 24.9, 21.8, 18.6. HRMS (ESI) calcd for C17H22N2O
([M+H]+) 271.1805; found: 271.1809.
1
N-Phenyl-4,6-dimethyl-1H-indole-2-carboxamide (4). Yield 67% (white powder). H NMR
(400 MHz, CDCl3) δ 9.47 (br s, 1H), 7.89 (br s, 1H), 7.70 (d, J = 8.0 Hz, 2H), 7.42 (t, J = 7.6 Hz,
2H), 7.19 (t, J = 7.6 Hz, 1H), 7.09 (s, 1H), 7.00 (s, 1H), 6.83 (s, 1H), 2.56 (s, 3H), 2.45 (s, 3H);
13C NMR (100 MHz, CDCl3)
δ
159.9, 139.2, 137.2, 133.3, 130.3, 128.7, 125.3, 123.4, 122.0,
120.0, 109.6, 102.7, 99.6, 21.6, 18.5. HRMS (ESI) calcd for C17H16N2O ([M+H]+) 265.1335;
found: 265.1348
N-(3-Fluoro-4-methylphenyl)-4,6-dimethyl-1H-indole-2-carboxamide (5). Yield 89%
(white powder). 1H NMR (400 MHz, d6ꢀDMSO)
δ 11.61 (s, 1H), 10.26 (s, 1H), 7.80 (d, J = 12.4
Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.45 (s, 1H), 7.24 (t, J = 8.8 Hz, 1H), 7.11 (s, 1H), 6.70 (s,
13
1H), 2.50 (s, 3H), 2.21 (s, 3H), 1.98 (s, 3H); C NMR (100 MHz, d6ꢀDMSO)
δ 161.5 (J = 239
Hz), 159.9, 138.6 (J = 11 Hz), 137.3, 133.4, 131.3 (J = 6.3 Hz), 130.3, 130.0, 125.3, 122.0, 118.6
(J = 17.2 Hz), 115.5, 109.6, 106.7 (J = 27 Hz), 102.8, 21.5, 18.4, 13.7 (J = 2.9 Hz). HRMS (ESI)
calcd for C18H17FN2O ([M+H]+) 297.1252; found: 297.1266.
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N-(4-Pyridinyl)-4,6-dimethyl-1H-indole-2-carboxamide (6). Yield 77% (white powder). H
5
6
7
NMR (400 MHz, CD3OD)
δ
8.43 (d, J = 5.2 Hz, 2H), 7.88 (d, J = 6.3 Hz, 2H), 7.43 (s, 1H), 7.27
13
8
9
(s, 1H), 6.87 (s, 1H), 2.54 (s, 3H), 2.42 (s, 3H); C NMR (100 MHz, d6ꢀDMSO)
δ 160.6, 150.4,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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34
35
36
37
38
39
40
41
42
43
44
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59
60
145.9, 137.6, 133.9, 130.6, 129.5, 125.2, 122.3, 113.7, 109.7, 103.8, 99.6, 21.6, 18.5. HRMS
(ESI) calcd for C16H15N3O ([M+H]+) 266.1288; found: 266.1295.
N-(1-Methyl-4-piperidinyl)-4,6-dimethyl-1H-indole-2-carboxamide (7). Purified by column
1
chromatography (EtOAc–hexane 1:1). Yield 65% (white powder). H NMR (400 MHz, d6ꢀ
DMSO) δ 11.24 (s, 1H), 8.18 (d, J = 8 Hz, 1H), 7.13 (s, 1H), 7.02 (s, 1H), 6.58 (s, 1H), 3.76–
3.70 (m, 1H), 2.78–2.75 (m, 2H), 2.43 (s, 3H), 2.33 (s, 3H), 2.16 (s, 3H), 2.00–1.91 (m, 2H),
13
1.77 (m, 2H), 1.65ꢀ1.47 (m, 2H); C NMR (100 MHz, d6ꢀDMSO) δ 160.6, 136.7, 132.5, 130.7,
129.9, 125.3, 121.7, 109.5, 101.4, 54.6, 46.1, 31.7, 21.6, 18.5. HRMS (ESI) calcd for C17H23N3O
([M+H]+) 286.1914; found: 286.1908.
N-(1-Isopropyl-4-piperidinyl)-4,6-dimethyl-1H-indole-2-carboxamide (8). Yield 70%
1
(white powder). H NMR (400 MHz, d6ꢀDMSO) δ 11.43 (s, 1H), 8.15 (d, J = 8.0 Hz, 1H), 7.13
(s, 1H), 7.02 (s, 1H), 6.66 (s, 1H), 3.74–3.72 (m, 1H), 2.81–2.78 (m, 2H), 2.70 (m, 1H), 2.43 (s,
3H), 2.33 (s, 3H), 2.17 (t, J = 12.0 Hz, 2H), 1.97–1.81 (m, 2H), 1.56–1.48 (m, 2H), 0.97 (d, J =
6.4 Hz, 6H); 13C NMR (100 MHz, d6ꢀDMSO) δ 160.5, 136.7, 132.5, 130.7, 129.9, 125.3, 121.7,
109.5, 101.3, 53.7, 47.4, 47.0, 32.2, 21.6, 18.5, 18.2. HRMS (ESI) calcd for C19H27N3O
([M+H]+) 314.2227; found: 314.2216.
N-(1-Methyl-4-azepanyl)-4,6-dimethyl-1H-indole-2-carboxamide (9). Yield 83% (white
1
powder). H NMR (400 MHz, d6ꢀDMSO) δ 11.37 (s, 1H), 9.50 (br s, 1H), 8.11 (d, J = 8.0 Hz,
1H), 7.11 (s, 1H), 7.00 (s, 1H), 6.57 (s, 1H), 4.17 (br s, 1H), 3.00–3.70 (m, 4H), 2.82 (s, 3H),
13
2.47 (s, 3H), 2.33 (s, 3H), 2.07–1.68 (m, 6H); C NMR (100 MHz, d6ꢀDMSO) δ 160.4, 136.7,
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6
132.7, 130.4, 130.0, 125.2, 121.8, 109.5, 101.5, 52.5, 48.1, 47.1, 43.8, 32.2, 21.6, 18.5. HRMS
(ESI) calcd for C18H25N3O ([M+H]+) 300.2070; found: 300.2068.
7
8
9
N-Cyclopropyl-4,6-dimethyl-1H-indole-2-carboxamide (10). Yield 95% (white powder). 1H
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
NMR (400 MHz, CDCl3)
δ 9.47 (br s, 1H), 7.07 (s, 1H), 6.79 (s, 2H), 6.43 (s, 1H), 3.11–2.92 (m,
1H), 2.50 (s, 3H), 2.43 (s, 3H), 0.94–0.88 (m, 2H), 0.76–0.67 (m, 2H); 13C NMR (100 MHz,
CDCl3)
δ 162.9, 137.1, 133.0, 130.9, 130.3, 125.7, 122.1, 109.9, 101.7, 23.1, 21.9, 18.8, 6.2.
HRMS (ESI) calcd for C14H16N2O ([M+H]+) 229.1335; found: 229.1342.
1
N-Cycloheptyl-4,6-dimethyl-1H-indole-2-carboxamide (11). Yield 72% (white powder). H
NMR (400 MHz, CD3OD)
δ 7.12 (s, 1H), 7.05 (s, 1H), 6.70 (s, 1H), 4.09–4.04 (m, 1H), 2.48 (s,
3H), 2.38 (s, 3H), 2.00–1.98 (m, 2H), 1.76–1.54 (m, 10H); 13C NMR (100 MHz, CDCl3)
δ
159.8,
135.2, 131.8, 128.5, 127.9, 123.7, 119.8, 106.9, 99.8, 48.9, 32.7, 25.8, 22.2, 18.6, 15.4. HRMS
(ESI) calcd for C18H24N2O ([M+H]+) 285.1889; found: 285.1892.
1
N-Cyclooctyl-4,6-dimethyl-1H-indole-2-carboxamide (12). Yield 83% (white powder). H
NMR (400 MHz, d6ꢁDMSO) δ 11.29 (s, 1H), 8.14 (d, J = 8.0 Hz, 1H), 7.16 (d, J = 1.6 Hz, 1H),
7.02 (s, 1H), 6.66 (s, 1H), 4.06–4.01 (m, 1H), 2.44 (s, 3H), 2.34 (s, 3H), 1.81–1.65 (m, 6H),
13
1.61–1.51 (m, 8H); C NMR (100 MHz, d6ꢁDMSO) δ 158.9, 134.4, 131.1, 127.7, 127.1, 122.6,
119.0, 106.1, 98.9, 46.8, 29.4, 24.0, 22.7, 21.0, 17.8, 14.6. HRMS (ESI) calcd for C19H26N2O
([M+H]+) 299.2117; found: 299.2115.
N-(1-Adamantyl)-4,6-dimethyl-1H-indole-2-carboxamide (13). Yield 65% (white powder).
1H NMR (400 MHz, d6ꢁDMSO) δ 11.21 (s, 1H), 7.14 (s, 1H), 7.00 (s, 1H), 6.65 (s, 1H), 2.42 (s,
13
3H), 2.33 (s, 3H), 2.09 (s, 6H), 2.07 (br s, 3H), 1.67 (s, 6H); C NMR (100 MHz, d6ꢁDMSO) δ
161.1, 136.9, 132.8, 131.8, 130.3, 125.7, 122.0, 109.8, 101.9, 51.9, 41.6, 36.5, 29.3, 21.9, 18.9.
HRMS (ESI) calcd for C21H26N2O ([M+H]+) 323.2117; found: 323.2105.
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N-(2-Adamantyl)-4,6-dimethyl-1H-indole-2-carboxamide
(14).
Purified
by
reꢀ
5
6
7
8
1
crystallization from EtOHꢀEt2O. Yield 82% (white powder). H NMR (400 MHz, d6ꢀDMSO) δ
11.30 (s, 1H), 7.72 (d, J = 6.8 Hz, 1H), 7.32 (d, J = 1.6 Hz, 1H), 7.02 (s, 1H), 6.66 (s, 1H), 4.09
(d, J = 5.2 Hz, 1H), 2.45 (s, 3H), 2.34 (s, 3H), 2.14 (d, J = 12.4 Hz, 2H), 1.98 (s, 2H), 1.86–1.83
(m, 6H), 1.73 (s, 2H), 1.54 (d, J = 12.0 Hz, 2H); 13C NMR (100 MHz, d6ꢀDMSO) δ 160.8, 136.7,
132.6, 130.5, 130.0, 125.3, 121.6, 109.4, 102.2, 53.6, 37.2, 36.9, 31.4, 31.1, 26.8, 21.5, 18.4.
HRMS (ESI) calcd for C21H26N2O ([M+H]+) 323.2117; found: 323.2113.
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60
N-(Cyclohexylmethyl)-4,6-dimethyl-1H-indole-2-carboxamide (15). Yield 80% (white
powder). 1H NMR (400 MHz, CDCl3)
δ 9.41 (s, 1H), 7.07 (s, 1H), 6.82 (d, J = 8.9 Hz, 2H), 6.26
(m, 1H), 3.36 (t, J = 6.5 Hz, 2H), 2.53 (s, 3H), 2.44 (s, 3H), 1.85–1.62 (m, 6H), 1.30–1.21 (m,
3H), 1.08–1.02 (m, 2H); 13C NMR (100 MHz, CDCl3)
δ
161.5, 136.2, 134.2, 130.5, 129.3, 125.3,
122.4, 108.8, 99.7, 45.4, 37.8, 30.5, 26.0, 25.4, 21.4, 18.2. HRMS (ESI) calcd for C18H24N2O
([M+H]+) 285.1889; found: 285.1967.
N-Cyclohexyl-N,4,6-trimethyl-1H-indole-2-carboxamide (16). Yield 88% (white powder).
1H NMR (400 MHz, d6ꢀDMSO)
δ 11.33 (s, 1H), 7.05 (s, 1H), 6.74ꢀ6.67 (m, 2H), 4.33 (m, 1H),
3.07 (br s, 3H), 2.45 (s, 3H), 2.35 (s, 3H), 1.81ꢀ1.56 (m, 7H), 1.34ꢀ1.31 (m, 2H), 1.18ꢀ1.10 (m,
13
1H); C NMR (100 MHz, d6ꢀDMSO)
δ 162.6, 136.0, 132.5, 129.9, 129.4, 125.1, 121.7, 109.2,
29.6, 25.3, 24.9, 21.5, 18.3. HRMS (ESI) calcd for C18H24N2O ([M+H]+) 285.1961; found:
285.1969.
(4,6-Dimethyl-1H-indol-2-yl)(piperidin-1-yl)methanone (17). Recrystallization from EtOHꢀ
1
Et2O. Yield 93% (white powder). H NMR (400 MHz, d6ꢀDMSO) δ 11.32 (s, 1H), 7.01 (s, 1H),
6.68 (d, J = 1.2 Hz, 1H), 6.66 (s, 1H), 3.71 (br s, 4H), 2.43 (s, 3H), 2.34 (s, 3H), 1.66–1.64 (m,
13
2H), 1.57–1.56 (m, 4H); C NMR (100 MHz, d6ꢀDMSO) δ 162.5, 136.5, 132.8, 130.3, 129.5,
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Journal of Medicinal Chemistry
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125.4, 122.1, 109.6, 102.5, 26.3, 24.6, 21.9, 18.8. HRMS (ESI) calcd for C16H20N2O ([M+H]+)
257.1648; found: 257.1652.
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8
9
N-Cyclohexyl-1,4,6-trimethyl-1H-indole-2-carboxamide (18). Yield 74% (white powder).
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14
15
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19
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60
1H NMR (400 MHz, CDCl3)
δ 7.01 (s, 1H), 6.79 (d, J = 8.0 Hz, 2H), 6.06 (d, J = 7.6 Hz, 1H),
4.02 (s, 3H), 3.99–3.93 (m, 1H), 2.52 (s, 3H), 2.48 (s, 3H), 2.08–2.05 (m, 2H), 1.82–1.66 (m,
3H), 1.51–1.40 (m, 2H), 1.33–1.19 (m, 3H); 13C NMR (100 MHz, CDCl3)
δ
161.5, 138.9, 133.8,
130.9, 130.4, 123.6, 122.2, 107.1, 101.4, 47.9, 32.9, 31.2, 25.2, 24.6, 21.7, 18.1. HRMS (ESI)
calcd for C18H24N2O ([M+H]+) 285.1889; found: 285.1974.
N-Cycloheptyl-4,6-dimethylbenzofuran-2-carboxamide
(19).
Purified
by
flash
chromatography (CH2Cl2, 100%). Yield 83% (offꢀwhite solid). 1H NMR (400 MHz, d6ꢀDMSO) δ
8.39 (d, J = 8.0 Hz, 1H), 7.55 (s, 1H), 7.23 (s, 1H), 6.94 (s, 1H), 3.96 (m, 1H), 2.46 (s, 3H), 2.40
(s, 3H), 1.86–1.82 (m, 2H), 1.65–1.41 (m, 10H); 13C NMR (100 MHz, d6ꢀDMSO) δ 157.1, 154.5,
148.4, 136.6, 131.7, 125.2, 124.7, 109.0, 107.9, 50.1, 34.2, 27.7, 23.9, 21.4, 18.1. HRMS (ESI)
calcd for C18H23NO2 ([M+H]+) 286.1802; found: 286.1813.
N-Cyclooctyl-4,6-dimethylbenzofuran-2-carboxamide
(20).
Purified
by
flash
1
chromatography (CH2Cl2/CH3OH, 9:1). Yield 69% (offꢀwhite solid). H NMR (400 MHz, d6ꢀ
DMSO) δ 8.37 (d, J = 8.1 Hz, 1H), 7.55 (s, 1H), 7.24 (s, 1H), 6.95 (s, 1H), 4.02 (m, 1H), 2.46 (s,
3H), 2.40 (s, 3H), 1.72–1.69 (m, 6H), 1.62–1.50 (m, 8H); 13C NMR (100 MHz, d6ꢀDMSO) δ
157.1, 154.5, 148.4, 136.6, 131.7, 125.2, 124.7, 109.0, 107.9, 48.9, 31.6, 26.8, 25.1, 23.5, 21.4,
18.1. HRMS (ESI) calcd for C19H25NO2 ([M+Na]+) 322.1778; found: 322.1786.
N-(1-Adamantyl)-4,6-dimethylbenzofuran-2-carboxamide (21). Purified by flash
1
chromatography (CH2Cl2/CH3OH, 9:1). Yield 72% (offꢀwhite solid). H NMR (400 MHz, d6ꢀ
DMSO) δ 7.57 (s, 1H), 7.54 (s, 1H), 7.23 (s, 1H), 6.94 (s, 1H), 2.45 (s, 3H), 2.39 (s, 3H), 2.08
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Page 25 of 38
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(br s, 9H), 1.66 (m, 6H); C NMR (100 MHz, d6ꢀDMSO) δ 157.6, 154.4, 148.5, 136.5, 131.7,
125.2, 124.7, 109.0, 107.8, 51.7, 40.8, 36.0, 28.8, 21.4, 18.1. HRMS (ESI) calcd for C21H25NO2
([M+H]+) 324.1958; found: 324.1966.
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N-(bicyclo[2.2.1]-2-heptanyl)-4,6-dimethylbenzofuran-2-carboxamide (22). Purified by
1
flash chromatography (CH2Cl2, 100 %). Yield 82% (offꢀwhite solid). H NMR (400 MHz, d6ꢀ
DMSO) δ 8.24 (d, J = 6.7 Hz, 1H), 7.59 (s, 1H), 7.24 (s, 1H), 6.94 (s, 1H), 3.72 (m, 1H), 2.46 (s,
3H), 2.40 (s, 3H), 2.24 (s, 1H), 2.18 (d, J = 2.4 Hz, 1H), 1.66–1.40 (m, 5H), 1.22–1.09 (m, 3H);
13C NMR (100 MHz, d6ꢀDMSO) δ 157.7, 154.5, 148.2, 136.6, 131.7, 125.2, 124.7, 109.0, 108.0,
52.6, 42.0, 37.9, 35.2, 34.9, 28.0, 26.3, 21.4, 18.1. HRMS (ESI) calcd for C18H21NO2 ([M+H]+)
284.1645; found: 284.1644.
N-Hexyl-4,6-dimethylbenzofuran-2-carboxamide (23). Purified by flash chromatography
(CH2Cl2/CH3OH, 9:1). Yield 74% (pale yellow solid). 1H NMR (400 MHz, d6ꢀDMSO) δ 8.57 (t,
J = 5.6 Hz, 1H), 7.51 (s, 1H), 7.23 (s, 1H), 6.95 (s, 1H), 3.24 (m, 2H), 2.46 (s, 3H), 2.40 (s, 3H),
13
1.53–1.48 (m, 2H), 1.27 (br s, 6H), 0.86 (t, J = 6.0 Hz, 3H); C NMR (100 MHz, d6ꢀDMSO) δ
158.1, 154.5, 148.3, 136.7, 131.8, 125.3, 124.7, 109.0, 107.9, 38.6, 31.0, 29.0, 26.1, 22.0, 21.3,
18.1, 13.9. HRMS (ESI) calcd for C17H23NO2 ([M+H]+) 274.1802; found: 274.1805.
5-Chloro-N-cyclooctylbenzofuran-2-carboxamide (24). Purified by flash chromatography
1
(100% CH2Cl2). Yield 79% (offꢀwhite solid). H NMR (400 MHz, d6ꢀDMSO) δ 8.57 (d, J = 7.9
Hz, 1H), 7.86 (d, J = 2.0 Hz, 1H), 7.69 (d, J = 8.8 Hz, 1H), 7.52 (s, 1H), 7.47 (ddd, J = 8.8, 2.1,
0.8 Hz, 1H), 4.04–3.98 (m, 1H), 1.75–1.65 (m, 6H), 1.60–1.45 (m, 8H); 13C NMR (100 MHz, d6ꢀ
DMSO) δ 156.5, 152.6, 150.8, 128.8, 127.9, 126.5, 122.0, 113.4, 108.6, 49.1, 31.6 (2C), 26.7
(2C), 25.1, 23.5 (2C). HRMS (ESI) calcd for C17H20ClNO2 ([M+H]+) 306.1255; found:
306.1252.
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5-Chloro-N-(1-adamantyl)benzofuran-2-carboxamide (25). Purified by reꢀcrystallization
5
6
1
from CH3OH. Yield 84% (offꢀwhite solid). H NMR (400 MHz, d6ꢀDMSO) δ 7.84 (s, 1H), 7.78
7
8
9
(s, 1H), 7.68 (d, J = 8.7 Hz, 1H), 7.51 (s, 1H), 7.45 (d, J = 8.8 Hz, 1H), 2.07 (br s, 9H), 1.65 (m,
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13
6H); C NMR (100 MHz, d6ꢀDMSO) δ 157.0, 152.5, 151.0, 128.8, 127.9, 126.5, 121.9, 113.4,
108.5, 51.9, 40.7, 35.9, 28.8. HRMS (ESI) calcd for C19H20ClNO2 ([M+H]+) 330.1255; found:
330.1264.
1
N-Cyclohexyl-1H-indole-2-carboxamide (26). Yield 95% (white powder). H NMR (400
MHz, d6ꢁDMSO)
δ 11.51 (s, 1H), 8.18 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 7.9 Hz, 1H), 7.43 (d, J =
8.2 Hz, 1H), 7.18–7.14 (m, 2H), 7.02 (t, J = 7.8 Hz, 1H), 3.79 (br s, 1H), 1.85–1.59 (m, 5H),
13
1.38–1.27 (m, 4H), 1.15–1.13 (m, 1H); C NMR (100 MHz, d6ꢁDMSO)
δ 160.2, 136.4, 132.1,
127.1, 123.2, 121.4, 119.7, 112.3, 102.5, 48.0, 32.6, 25.3, 25.0. HRMS (ESI) calcd for
C15H18N2O ([M+H]+) 243.1491; found: 243.1498.
N-(3-Fluoro-4-methylphenyl)-1H-indole-2-carboxamide (27). Yield 83% (white powder).
1H NMR (400 MHz, d6ꢁDMSO)
δ 11.75 (s, 1H), 10.30 (s, 1H), 7.73 (d, J = 12.0 Hz, 1H), 7.67
(d, J = 8.0 Hz, 1H), 7.47 (d, J = 8.0 Hz, 2H), 7.41 (s, 1H), 7.27–7.21 (m, 2H), 7.07 (t, J = 8.0 Hz,
1H), 2.20 (s, 3H); 13C NMR (100 MHz, d6ꢁDMSO)
δ
161.5 (d, J = 239 Hz), 159.8, 138.4 (d, J =
11 Hz), 136.9, 131.5 (d, J = 6 Hz), 131.3, 127.0, 124.0, 121.9, 120.1, 118.9 (d, J = 17 Hz), 115.7
(d, J = 3 Hz), 112.5, 106.9 (d, J = 27 Hz), 104.1, 13.7 (d, J = 3 Hz). HRMS (ESI) calcd for
C16H13FN2O ([M+H]+) 269.1085; found: 269.1087.
1
N-Cycloheptyl-4,6-difluoro-1H-indole-2-carboxamide (28). Yield 68 % (white powder). H
NMR (400 MHz, d6ꢁDMSO)
δ
11.93, 8.33 (d, J = 7.6 Hz, 1H), 7.29 (s, 1H), 7.03 (d, J = 8.8 Hz),
13
6.87 (t, J = 10.4 Hz, 1H), 3.98 (m, 1H), 1.89ꢀ1.85 (m, 2H), 1.65ꢀ1.42 (m, 10H); C NMR (100
MHz, d6ꢁDMSO)
δ
160.2 (d, J = 236 Hz), 159.0, 157.0 (d, J = 246 Hz), 137.5 (t, J = 15.1 Hz),
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133.0, 113.2 (d, J = 22 Hz), 98.2, 95.3 (d, J = 23 Hz), 94.7 (d, J = 26 Hz), 50.1, 34.3, 27.9, 23.8.
5
6
HRMS (ESI) calcd for C16H18F2N2O ([M+H]+) 293.1460; found: 293.1472
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8
9
1
N-Cyclooctyl-4,6-difluoro-1H-indole-2-carboxamide (29). Yield 69 % (white powder). H
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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
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35
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60
NMR (400 MHz, d6ꢁDMSO)
δ
11.93, 8.31 (d, J = 8.0 Hz, 1H), 7.29 (s, 1H), 7.03 (d, J = 9.6 Hz),
13
6.87 (t, J = 10.4 Hz, 1H), 4.04ꢀ4.00 (m, 1H), 1.80ꢀ1.64 (m, 6H), 1.60ꢀ1.50 (m, 8H); C NMR
(100 MHz, d6ꢁDMSO)
δ 160.2 (d, J = 237 Hz), 159.0, 157.0 (d, J = 247 Hz), 137.5 (t, J = 14.9
Hz), 133.1, 113.1 (d, J = 22 Hz), 98.2, 95.3 (d, J = 23 Hz), 94.7 (d, J = 26 Hz), 49.0, 31.5, 26.9,
25.0, 23.4. HRMS (ESI) calcd for C17H20F2N2O ([M+H]+) 307.1617; found: 307.1626.
N-(1-Adamantyl)-6-methoxy-1H-indole-2-carboxamide
(30).
Purified
by
flash
chromatography (EtOAc–hexane 1:3 to 3:2) followed by recrystallization from CH3OH. Yield
77% (pale yellow powder). 1H NMR (400 MHz, d6ꢀDMSO) δ 11.23 (s, 1H), 7.46–7.42 (m, 2H),
7.08 (d, J = 1.8 Hz, 1H), 6.87 (d, J = 1.8 Hz, 1H), 6.67 (dd, J = 8.7, 2.2 Hz, 1H), 3.76 (s, 3H),
2.09–2.06 (m, 9H), 1.67 (br s, 6H); 13C NMR (100 MHz, d6ꢀDMSO) δ 160.5, 156.8, 137.2,
131.5, 122.1, 121.3, 110.7, 103.0, 94.1, 55.0, 51.4, 41.1, 36.1, 28.9. HRMS (ESI) calcd for
C20H24N2O2 ([M+H]+) 325.1911; found: 325.1910.
N-(1-Adamantyl)-5-chloro-1H-indole-2-carboxamide
(31).
Purified
by
flash
chromatography (EtOAc–hexane 1:3 to 1:1) followed by reꢀcrystallization from CH3OH. Yield
40% (pale yellow powder). 1H NMR (400 MHz, d6ꢀDMSO) δ 11.63 (s, 1H), 7.65 (br s, 2H), 7.42
(d, J = 8.7 Hz, 1H), 7.17–7.14 (m, 2H), 2.09–2.06 (m, 9H), 1.66 (br s, 6H); 13C NMR (100 MHz,
d6ꢀDMSO) δ 160.1, 134.6, 134.2, 128.1, 124.0, 123.1, 120.4, 113.7, 102.3, 51.7, 41.0, 36.0, 28.9.
HRMS (ESI) calcd for C19H21ClN2O ([M+H]+) 329.1415; found: 329.1399.
N-(1-Adamantyl)-6-hydroxy-1H-indole-2-carboxamide (32). Compound 30 (0.73 mmol)
was dissolved in anhydrous CH2Cl2 (7 mL) and cooled to –78 °C. Subsequently BBr3 (1.0 M
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solution in CH2Cl2, 4.4 mL, 6.0 equiv) was added dropwise and the reaction mixture was
allowed to warm gradually to room temperature within 1 h. Stirring was continued at the same
temperature an additional 3 h. The reaction was quenched with water and extracted with CH2Cl2
(2×50 mL). The combined organic phases were dried over Na2SO4, filtered and concentrated
under reduced pressure. The crude material was purified by column chromatography (EtOAc–
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26
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1
hexane 1:3 to 1:1) followed by preparative HPLC. Yield 52% (white powder). H NMR (400
MHz, d6ꢀDMSO) δ 11.00 (s, 1H), 9.11 (s, 1H), 7.35–7.33 (m, 2H), 7.01 (d, J = 1.5 Hz, 1H), 6.76
13
(d, J = 1.5 Hz, 1H), 6.55 (dd, J = 8.6, 2.1 Hz, 1H), 2.07 (br s, 9H), 1.66 (br s, 6H); C NMR
(100 MHz, d6ꢀDMSO) δ 160.6, 154.6, 137.6, 131.0, 121.9, 120.6, 111.0, 103.1, 96.4, 51.4, 41.1,
36.1, 28.9. HRMS (ESI) calcd for C19H22N2O2 ([M+H]+) 311.1754; found: 311.1767.
N-Cyclooctyl-6-methoxy-1H-pyrrolo[3,2-c]pyridine-2-carboxamide (33). Yield 80% (white
solid). 1H NMR (400 MHz, d6ꢀDMSO) δ 11.67 (s, 1H), 8.39 (br s, 2H), 7.04 (s, 1H), 6.91 (s, 1H),
13
4.09–3.99 (m, 1H), 3.83 (s, 3H), 1.80–1.48 (m, 14H); C NMR (400 MHz, d6ꢀDMSO) δ 159.3,
157.4, 137.3, 135.7, 131.7, 130.7, 100.4, 97.5, 53.6, 49.2, 31.6, 26.9, 25.1, 23.5. HRMS (ESI)
calcd for C17H23N3O2 ([M+H]+) 302.1863; found: 302.1874.
N-(1-Adamantyl)-6-methoxy-1H-pyrrolo[3,2-c]pyridine-2-carboxamide (34). Yield 70%
(white solid).1H NMR (400 MHz, d6ꢁDMSO) δ 11.60 (s, 1H), 8.39 (s, 1H), 7.75 (s, 1H), 7.04 (s,
13
1H), 6.90 (s, 1H), 3.83 (s, 3H), 2.09 (br s, 9H), 1.67 (s, 6H); C NMR (100 MHz, d6ꢀDMSO) δ
159.9, 157.4, 137.9, 135.7, 131.6, 130.6, 100.7, 97.5, 53.6, 51.9, 41.0, 36.1, 28.9. HRMS (ESI)
calcd for C19H23N3O2 ([M+H]+) 326.1863; found: 326.1867.
N-Cycloheptyl-4,6-bis(trifluoromethyl)-1H-indole-2-carboxamide (35). Yield 54 % (white
powder). 1H NMR (400 MHz, d6ꢁDMSO)
δ
12.59 (s, 1H), 8.70 (d, J = 8.0 Hz, 1H), 8.03 (s, 1H),
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7.66 (s, 1H), 7.54 (s, 1H), 4.04 (m, 1H), 1.93ꢀ1.87 (m, 2H), 1.71ꢀ1.55 (m, 10H); C NMR (100
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Page 29 of 38
Journal of Medicinal Chemistry
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MHz, d6ꢁDMSO)
δ
158.5, 137.0, 135.7, 125.6 (d, J = 21 Hz), 125.3, 122.9 (d, J = 21 Hz), 122.8
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(q, J = 32 Hz), 122.0 (q, J = 33 Hz), 114.1, 113.4, 100.4, 50.3, 34.3, 27.9, 23.8. HRMS (ESI)
calcd for C18H18F6N2O ([M+H]+) 393.1396; found: 393.1386.
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N-Cyclooctyl-4,6-bis(trifluoromethyl)-1H-indole-2-carboxamide (36). Yield 61 % (white
powder). 1H NMR (400 MHz, d6ꢁDMSO)
7.66 (s, 1H), 7.55 (s, 1H), 4.07 (m, 1H), 1.82ꢀ1.51 (m, 14H); 13C NMR (100 MHz, d6ꢁDMSO)
δ 12.58 (s, 1H), 8.67 (d, J = 8.0 Hz, 1H), 8.03 (s, 1H),
δ
158.5, 137.0, 135.7, 125.7 (d, J = 20 Hz), 125.3, 123.0 (d, J = 21 Hz), 122.8 (q, J = 32 Hz),
122.0 (q, J = 33 Hz), 114.0, 113.4, 100.4, 49.3, 31.4, 26.8, 25.0, 23.4. HRMS (ESI) calcd for
C19H20F6N2O ([M+H]+) 407.1553; found:407.1562.
N-Cycloheptyl-4,6-dimethyl-1H-benzo[d]imidazole-2-carboxamide (37). Yield 36% (white
1
powder). H NMR (400 MHz, d6ꢁDMSO) δ 8.83 (s, 1H), 7.24 (s, 1H), 7.00 (s, 1H), 4.04–4.00
(m, 1H), 2.55 (s, 3H), 2.40 (s, 3H), 1.91–1.87 (m, 2H), 1.72–1.42 (m, 10H); 13C NMR (100
MHz, d6ꢀDMSO) δ 153.7, 142.3, 136.0, 132.8, 131.4, 127.9, 125.4, 111.3, 51.0, 33.8, 27.9, 23.6,
21.2, 16.7. HRMS (ESI) calcd for C17H23N3O ([M+H]+) 286.1914; found: 286.1921.
N-Cyclooctyl-4,6-dimethyl-1H-benzo[d]imidazole-2-carboxamide (38). Yield 51% (white
1
powder). H NMR (400 MHz, d6ꢁDMSO) δ 9.33 (s, 1H), 7.29 (s, 1H), 7.10 (s, 1H), 4.12–4.04
(m, 1H), 2.58 (s, 3H), 2.42 (s, 3H), 1.81–1.72 (m, 6H), 1.63–1.54 (m, 8H); 13C NMR (100 MHz,
d6ꢀDMSO) δ 154.3, 142.8, 135.4, 133.6, 132.5, 127.4, 125.5, 111.5, 49.9, 31.0, 26.8, 24.9, 23.3,
21.2, 16.7. HRMS (ESI) calcd for C18H25N3O ([M+H]+) 300.2070; found: 300.2080.
N-(1-Adamantyl)-4,6-dimethyl-1H-benzo[d]imidazole-2-carboxamide (39). Yield 40%
1
(white powder). H NMR (400 MHz, d6ꢁDMSO) δ 7.94 (s, 1H), 7.23 (s, 1H), 6.98 (s, 1H), 2.52
(s, 3H), 2.39 (s, 3H), 2.11–2.09 (br s, 9H), 1.68 (s, 6H); 13C NMR (100 MHz, d6ꢀDMSO) δ
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