Design, synthesis, and biological evaluation of indole-2-carboxamides: A promising class of antituberculosis agents

Article abstract of DOI:10.1021/jm4012774

Indole-2-carboxamides have been identified as a promising class of antituberculosis agents from phenotypic screening against mycobacteria. One of the hits, indole-2-carboxamide analog (1), had low micromolar potency against Mycobacterium tuberculosis (Mtb), high mouse liver microsomal clearance, and low aqueous solubility. Structure-activity relationship studies revealed that attaching alkyl groups to the cyclohexyl ring significantly improved Mtb activity but reduced solubility. Furthermore, chloro, fluoro, or cyano substitutions on the 4- and 6-positions of the indole ring as well as methyl substitution on the cyclohexyl ring significantly improved metabolic stability. 39 and 41, the lead candidates, displayed improved in vitro activity compared to most of the current standard TB drugs. The low aqueous solubility could not be mitigated because of the positive correlation of lipophilicity with Mtb potency. However, both compounds displayed favorable oral pharmacokinetic properties in rodents and demonstrated in vivo efficacy. Thus, indole-2-carboxamides represent a promising new class of antituberculosis agents.

Full text of DOI:10.1021/jm4012774

Subscriber access provided by University Libraries, University of Memphis
Article
Design, synthesis and biological evaluation of indole-2-
carboxamides, a promising class of anti-tuberculosis agents
Ravinder Reddy Kondreddi, Jan Jiricek, Srinivasa P S Rao, Suresh B Lakshminarayana, Luis
R Camacho, Ranga Rao, Maxime Herve, Pablo Bifani, Ngai Ling Ma, Kelli Kuhen, Anne Goh,
Arnab K Chatterjee, Thomas Dick, Thierry T Diagana, Ujjini H. Manjunatha, and Paul W Smith
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm4012774 • Publication Date (Web): 03 Oct 2013
Downloaded from http://pubs.acs.org on October 4, 2013
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Design, synthesis and biological evaluation of
indoleꢀ2ꢀcarboxamides, a promising class of antiꢀ
tuberculosis agents
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
Ravinder Reddy Kondreddi,*† Jan Jiricek,† Srinivasa P S Rao,† Suresh B. Lakshminarayana,†
Luis R. Camacho,† Ranga Rao,† Maxime Herve,† Pablo Bifani,† Ngai Ling Ma,† Kelli Kuhen,‡
Anne Goh,† Arnab K. Chatterjee,‡, § Thomas Dick,†# Thierry T. Diagana,† Ujjini H. Manjunatha †
and Paul W. Smith†
†Novartis Institute for Tropical Diseases, 10 Biopolis Road #05ꢀ01 Chromos Singapore 138670
‡Genomics Institute of the Novartis Research Foundation, 10675 John J. Hopkins Drive, San
Diego, California 92121
KEYWORDS. Indoleꢀ2ꢀcarboxamides, antiꢀtuberculosis agents, structure activity relations,
structure property relations
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 47
1
2
3
4
5
6
7
8
9
ABSTRACT. Indoleꢀ2ꢀcarboxamides have been identified as a promising class of antiꢀ
tuberculosis agents from phenotypic screening against mycobacteria. One of the hits indoleꢀ2ꢀ
carboxamide analog (1) had low ꢁM potency against Mycobacterium tuberculosis (Mtb), high
mouse liver microsomal clearance and low aqueous solubility. Structure activity relationship
studies revealed that attaching alkyl groups to the cyclohexyl ring significantly improved Mtb
activity but reduced solubility. Further, chloro or fluoro or cyano substitutions on the 4ꢀ and 6ꢀ
positions of the indole ring and methyl substitution on the cyclohexyl ring significantly improved
metabolic stability. 39 and 41, the lead candidates displayed improved in vitro activity compared
to most of the current standard TB drugs. The low aqueous solubility could not be mitigated due
to the positive correlation of lipophilicity with Mtb potency. However, both compounds
displayed favorable oral pharmacokinetic properties in rodentsand demonstrated in vivo efficacy.
Thus, indoleꢀ2ꢀcarboxamides represent a promising new class of antiꢀTB agents.
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
INTRODUCTION
Mycobacterium tuberculosis (Mtb), causative agent of tuberculosis (TB) infects one third of
the world population and 5ꢀ10% of those infected will eventually develop active disease in their
lifetime1. In 2011, 8.7 million new TB cases were reported with 1.4 million deaths, including
430,000 HIV positive patients2. Currently, drug susceptible TB is treated using drugs discovered
more than 50 years ago. There are increasing reports on multiꢀdrug resistant (MDR) and
extensively drug resistant (XDR) tuberculosis in recent years2. Some strains of Mtb are
becoming resistant to most of the currently available antiꢀmycobacterial drugs rendering them to
be totally drug resistant (TDR)3. Hence, there is an urgent need to develop novel drugs with new
modes of action in order to fight all the different forms of TB ꢀ MDR, XDR and TDR. Recently,
drug discovery initiatives have yielded a few new interesting compounds that are being evaluated
2
ACS Paragon Plus Environment

Page 3 of 47
Journal of Medicinal Chemistry
1
2
for the treatment of TB namely TMC207 (42)4, OPCꢀ67683 (43)5 and PAꢀ824 (44)6 (Figure S1).
Recently, 42 was approved by U S Food and Drug Administration in 2012 for treatment of
MDR TB patients as a part of new combination therapy when other alternatives have failed7.
In order to find new lead compounds, both academic institutions and pharmaceutical
companies have conducted phenotypic screens on compound libraries and made the hits publicly
available (GSK, SRI, Novartis)8ꢀ10. These efforts will help many academic researchers to select
starting points for further optimization using medicinal chemistry approaches to reach preꢀ
clinical stage. In a similar effort the Novartis inꢀhouse compound library was subjected to whole
cell based screening against M. bovis BCG using ATP content as a surrogate marker for bacterial
growth11. Novel chemical entities identified were further reꢀconfirmed for their activity against
virulent MtbH37Rv and chemically clustered. The hit reconfirmation process resulted in a few
interesting scaffolds for hit to lead optimization. Our criteria used for hit selection were based on
potency, bactericidal activity and low cytotoxicity against mammalian cells. New antiꢀtubercular
drugs need to have high chemical stability and be cost effective for TB treatment due to high
burden disease in underdeveloped and developing countries. Hence, ease of synthesis and
chemical stability were also taken into consideration for choosing the hit molecule. One of the
hits, indoleꢀ2ꢀcarboxamide (1), showed promising antiꢀmycobacterial activity (minimum
inhibitory concentration, MIC = 0.8 ꢁM). Recently, Ballell et al. reported indole 2ꢀ
3
4
5
6
7
8
9
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
carboxamides as a hit in antiꢀmycobacterial phenotypic high throughput screening assay9, 12 and
Onajole et al.13 published preliminary structure activity relationship (SAR) of related indoleꢀ2ꢀ
carboxamides against Mtb. In the present study, we describe SAR, structure property
relationship (SPR), in vivo pharmacokinetic (PK) and mouse efficacy of indoleꢀ2ꢀcarboxamides.
We primarily focused on modifying the indole and cyclohexyl moieties to simultaneously
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 47
1
2
3
4
5
6
7
8
9
improve Mtb activity and optimize PK properties. The molecular target of indoleꢀ2ꢀ
carboxamides has been identified as mmpl3 protein using whole genome sequencing of resistant
mutants and lipid profiling to be described elsewhere14. In this study, we have successfully
identified the advanced lead candidates 39 and 41 with good PK properties and excellent in vivo
efficacy in an acute mouse model of Mtb.
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
CHEMISTRY
Indoleꢀ2ꢀcarboxamides were synthesized in a straightꢀforward way by coupling of substituted
indoleꢀ2ꢀcarboxylic acids with the corresponding aromatic, alkyl or cycloalkyl amines (Scheme
1) in the presence of 2ꢀ(7ꢀazaꢀ1Hꢀbenzotriazoleꢀ1ꢀyl)ꢀ1,1,3,3ꢀtetramethyluronium
hexafluorophosphate (HATU) and N,Nꢀdiisopropylethylamine (DIPEA) in dimethylformamide
(DMF). The starting materials i.e., indoleꢀ2ꢀcarboxylic acids were either commercially available
or prepared by standard methods such as the Fisher indole synthesis or Hemetsberger–Knittel
indole synthesis following published procedures15ꢀ17. The alkylamines used were commercially
available except (1R,2S)ꢀ2ꢀmethylcyclohexanamine that was prepared as previously described18,
19. The tetrahydro indoleꢀ2ꢀcarboxamide 16 was synthesized as shown in scheme 1 by reduction
of 9 with sodium cyano borohydride. 4,6ꢀdimethylꢀNꢀ1Hꢀbenzo[d]imidazoleꢀ2ꢀcarboxamide 17
was obtained in an efficient oneꢀpot synthesis using 3,5ꢀdimethylbenzeneꢀ1,2ꢀdiamine and
sodium 2ꢀ(((1R,2S)ꢀ2ꢀmethylcyclohexyl)amino)ꢀ2ꢀoxoacetate in the presence of POCl3 as
previously reported20. The synthesis of 19 was performed analogously to the procedure described
in the literature21 followed by HATU coupling.
4
ACS Paragon Plus Environment

Page 5 of 47
Journal of Medicinal Chemistry
1
2
3
4
RESULTS AND DISCUSSION
5
6
7
8
9
A promising hit from cell based screening, 1, had displayed attractive features, sub ꢁM
potency, mycobactericidal activity and easy synthetic feasibility. However, 1 has low aq
solubility (<4 ꢁM at pH 6.8), high lipophilicity (cLogP = 4.48) and high in vitro clearance in
mouse liver microsomes (CLint = 693 ꢁL/min/mg). We hypothesized that the electron rich 4,6ꢀ
dimethyl indole and cyclohexyl group of 1 are metabolically susceptible (Figure 1) leading to
high turnover. Therefore we reasoned that blocking or modifying these positions should help to
improve metabolic stability. Additionally, attaching polar groups to lower lipophilicity should
improve aqueous solubility. Medicinal chemistry efforts focused on modifying the indole ring
and cyclohexyl group are shown in tables 1ꢀ3. All compounds synthesized were analyzed in a
whole cell assay for in vitro MIC against Mtb H37Rv11 and screened for cytotoxicity against
THPꢀ1 and HepG2 mammalian cell lines. A few selected compounds were subjected to in vitro
metabolic stability and solubility assays to understand SAR and SPR. The lead compounds 39
and 41 were further profiled in vivo to assess the PK properties and in vivo efficacy in a mouse
model of TB infection.
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
SAR to improve potency and aqueous solubility. Our initial efforts to reduce lipophilicity of
1 by introducing a basic nitrogen in the cyclohexane ring (2, cLogP = 2.5) resulted in significant
increase in aqueous solubility but Mtb activity was lost by >25 fold (Table 1). A basic amine is
not tolerated in the amide side chain of the indolcarboxamide and loss of Mtb activity could be
attributed to reduced lipophilicity and/or presence of ionisable group. In order to understand the
importance of the hydrophobic interaction of the cyclohexyl group, different alkyl and cycloalkyl
groups were explored on the amide. Replacing the cyclohexane ring of 1 with an isopropyl group
(3) dramatically decreased its activity >25 fold, whereas increasing lipophilicity from secꢀ
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 47
1
2
3
4
5
6
7
8
9
butylamide (4, cLogP = 3.81) to cycloheptylamide (8, cLogP = 5.03) or (1R,2S)ꢀ2ꢀ
methylcyclohexylamide (9, cLogP = 4.99) enhanced its activity against Mtb by 100 fold (Table
1). Further a positive correlation was observed between cLogP and MIC50. Hit 1 and potent
compound 9 dispersed along the same LiPE22 line (Figure 2). The SAR of alkyl and cycloalkyl
groups on the amide revealed that hydrophobic groups are important for Mtb activity and
suggested a critical hydrophobic interaction with the molecular target. In addition, improved
cellular activity may also be linked to an improved cell penetration of lipophilic analogues into
highly waxy mycobacterial cell wall. Additional evidence of a lipophilic binding pocket was
provided by the loss of Mtb activity with the introduction of other polar groups 10 (cLogP =
2.65), 11 (cLogP = 2.07) and 12 (cLogP = 3.28).
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
The replacement of the cyclohexyl group of 1 with a phenyl group (13) reduced potency
approximately 20 fold. This is probably due to less lipophilic interaction of flat phenyl group
with the threeꢀdimensional active site of the potential molecular target as compared to the
cyclohexyl ring. Our initial medicinal chemistry efforts on the cyclohexyl group led to the
identification of a highly potent compound 9 (MIC = 79 nM) with 4 fold better activity against
Mtb as compared to the standard TB drug isoniazid (MIC = 330 nM). We then investigated the
indole ring and amide bonds to understand the pharmacophore.
SAR of indole and amide groups. The SAR on indole NH, amide NH and indole ring was
investigated to understand the pharmacophore and the importance of both NHs for Mtb activity.
The methylation of amide NH to NMe (14) reduced Mtb activity by more than 20 fold as
compared to 1 (Table 2). Likewise, Nꢀmethyl indole derivative (15) was also significantly less
potent against Mtb (>285 fold) compared to the corresponding analog 9. The indoline (16) also
displayed reduced Mtb activity (16 fold lower than 9), suggesting that indole ring planarity is
6
ACS Paragon Plus Environment

Page 7 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
important for the potency. Introducing nitrogen atoms into the indole ring (17, 18 and 19)
reduced lipophilicity, but also resulted in significant reduction of Mtb activity. Improvement of
aqueous solubility was observed for low liphophilic compound 19. The SAR on indole ring and
amide functionalities suggested that both NH and indole ring are important for Mtb activity.
Thus, we identified the indoleꢀ2ꢀcarboxamides as a pharmacophore for potency against Mtb in
the series. SAR on cyclohexyl group and indole core led to a highly potent compound 9 (MIC =
79 nM). However, it has a high in vitro mouse microsomal clearance (CLint = 433 ꢁL/min/mg)
and poor aqueous solubility (<4 ꢁM) (Table 3). Therefore the SAR on 9 was focused on the
improvement of the metabolic stability.
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
SAR to mitigate metabolic stability. In silico analysis of metabolic soft spots on 9 using
commercial available software tool StarDrop23 predicted both 4ꢀ and 6ꢀmethyl groups on the
indole as potential sites for high metabolic clearance (Figure 1). In order to improve the
metabolic stability of 9, we modified the indole methyl groups by incorporating stable
chloro/fluoro/cyano groups. Replacement of both indole methyl groups with chloro (20)
significantly improved in vitro metabolic stability by 5 fold (Table 3) while retaining excellent
potency (MIC = 0.053 ꢁM). Repositioning of the methyl group on cyclohexyl in 20 further
improved metabolic stability by 2 fold in 21, while maintaining similar Mtb activity and
removed the chirality. However the same modification with fluoro group (22 and 23) didnot
improve metabolic stability. Other substitutions on indole with 6ꢀchloroꢀ4ꢀfluoro (24), 4ꢀchloroꢀ
6ꢀfluoro (25) and dicyano (26) have improved metabolic stability similar to 20 with slightly
reduced activity against Mtb. The high microsomal clearance of 9 was mitigated with
metabolically stable 21 while retaining similar Mtb activity. The improvement of in vitro
metabolic stability was also reflected in good in vivo PK properties (Table 6 and Figure 3). The
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 47
1
2
3
4
5
6
7
8
9
metabolically stable compound 21 is a mixture of cis and trans isomers (38:62). The pure cis
(27) and trans (28) isomers displayed similar potency. However, a significant improvement in
metabolic stability (>10 fold) was observed with the trans isomer (28). The replacement of both
methyls on indole with other groups displayed the solubility similar to 9. As predicted, metabolic
soft spots were blocked with metabolically stable chloro groups on the indole ring. The SAR on
cyclohexyl and indole ring resulted in the identification of lead compounds 27 and 28 based on
in vitro potency and metabolic stability. The lead compound 28 was further optimized to
improve Mtb activity by altering the chloro position on the indole ring.
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
SAR towards the optimum position of the chloro substituents on indole ring. Further
investigation was focused on optimization of chloro substituents at different positions on the
indole ring (Table 4). In the monochloroindole series, both 30 and 32 had similar Mtb activity
compared to 21 with >10 fold reduced metabolic stability. The decreased potency of other two
monochloroindoleꢀ2ꢀcarboxamides 31 and 33 indicated that chloro substitution on the 7ꢀposition
and 5ꢀposition of the indole are less favorable for Mtb activity respectively. However, 5ꢀchloro
substitution as in 31 displayed improved metabolic stability over 4ꢀ and 6ꢀpositions. Overall
SAR of mono chloro indoleꢀ2ꢀcarboxamides indicated that 4ꢀ and 6ꢀpositions are preferred for
activity and 5ꢀposition is vulnerable for metabolic clearance. In fact monochloro indoleꢀ2ꢀ
carboxamides are either less active or less metabolically stable compared to 21. Therefore we
moved on to investigate the effect of dichloro substitution on the indole ring.
Out of the six dichloroindoleꢀ2ꢀcarboxamide analogues, 35 showed significantly less activity,
whilst 37 and 38 exhibited moderately reduced activity on Mtb. The chloro substitution on the 7ꢀ
position of the indole alone or in combination with other positions is less favorable for Mtb
activity. The other two compounds 34 and 36 had a comparable potency with 21. Even though
8
ACS Paragon Plus Environment

Page 9 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
monoꢀchloro analogues on the 4ꢀ or 6ꢀposition are metabolically less stable, dichloro
substitutions at the 4,5ꢀ or 5,6ꢀpositions improved metabolic stability. The metabolic stability of
21 was superior to the 4,5ꢀ or 5,6ꢀdichloro indoles. Overall 4,6ꢀdichloro substitution on indole is
found to be optimal for activity and metabolic stability.
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
Improving the potency of the lead compound. Additional optimization of lead compound 28
was focused on the improvement of potency against Mtb by exploring the hydrophobic space on
the 4ꢀposition of the cyclohexane ring (Table 5). The introduction of gemꢀdimethyl group (39)
increased cLogP (from 5.56 to 6.08) and also resulted in a 10 fold boost in potency as compared
to 28. The introduction of the gemꢀdimethyl group removed cis and trans isomerism. The
difluoro analog 40 did not show any additional improvement in metabolic stability and has
reduced potency by > 20 fold. The loss of Mtb activity of 40 was likely due to reduced cLogP.
Likewise, the difluoro substitution on the indole ring (41) retained Mtb activity, however,
metabolic stability was reduced by >5 fold. A similar trend in metabolic stability was observed
between 4,6ꢀdifluoro (29) and 4,6ꢀdichloro (28) derivatives. Although in vitro metabolic stability
of 41 is less than 39, this difference was not reflected in vivo where both compounds showed
similar clearance in mouse and good exposure (Table 6).
Lead candidates 39 and 41 were selected for further profiling as they displayed excellent
potency against Mtb and improved metabolic stability. Overall, optimization of potency and
metabolic stability of the hit to the lead candidates also increased lipophilicity. The aqueous
solubility of these lead candidates remained poor. The high lipophilicity of lead compounds 39
and 41 thus reduced “druglikeness”24. However, cLogPs of both indoleꢀ2ꢀcarboxamide lead
compounds are still lower than the recently approved MDR TB drug 427 (cLogP = 7.25) and
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 47
1
2
similar to 4325 (cLogP = 5.2) which is in phase II clinical trial. Indolcarboxamides 39 and 41
showed potent activity against all tested MDR TB clinical isolates to be described elsewhere14.
In vivo PK and efficacy. The in vivo pharmacokinetics of the potent analogues 21, 39 and 41
were evaluated in mice by oral (p.o.) and intravenous (i.v.) routes at doses of 25 and 5 mg/kg
respectively. Like other high lipophilic compounds26, a special delivery microemulsion
preconcentrate (MEPC) formulation was used to address the solubility limitation of 39 and 41
for oral dosing. The lead compounds 21, 39 and 41 displayed favorable PK properties with low
total systemic clearance and moderate to high volume of distribution with elimination halfꢀlives
ranging from 4.5 to 7 hours (Figure 3 and Table 6). Overall, these compounds achieved good
oral exposure in systemic circulation that resulted in good oral bioavailability (48ꢀ53%).
Based on the potent in vitro activity and promising in vivo PK profile of 39 and 41, we
conducted in vivo efficacy studies in an acute mouse model of Mtb infection11 along with the
moderately potent analogue 28 (MIC 10 fold lower than 39). Treatment was initiated 7 days post
Mtb infection in Balb/c mice. The compounds were orally administered at 25 and 75 mg/kg once
daily for 28 days. The lead candidate 39 showed a 2.6 and 4.8 log lung colony forming unit
(CFU) reductions at 25 and 75 mg/kg respectively compared to the untreated control (Figure 4).
In addition, the other lead candidate 41 displayed 2.4 and 4.6 log lung CFU reduction at 25 and
75 mg/kg respectively. However less potent analogue 28 achieved less than a log CFU reduction
compared to the untreated control. A significantly better efficacy was observed with both lead
candidates 39 and 41 at 75 mg/kg over the firstꢀline TB drug ethambutol (100 mg/kg).
3
4
5
6
7
8
9
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
In summary, we have identified promising indoleꢀ2ꢀcarboxamides from whole cell phenotypic
highꢀthroughput screening and examined their SAR and SPR. The SAR clearly indicated that i)
cyclohexyl group is interacting with a hydrophobic site of the potential molecular target, ii) a
10
ACS Paragon Plus Environment

Page 11 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
strong relationship between Mtb activity and cLogP, iii) optimally positioned chloro and fluoro
substituents on indole and gemꢀdimethyl functionality on the cyclohexyl ring improved
metabolic stability, iv) hydrophilic groups improved aqueous solubility but lowers Mtb activity,
v) indole planarity is important for activity and vi) 4,6ꢀdichloro substitution is optimal for Mtb
activity and metabolic stability. The lead candidates 39 and 41 displayed promising
microbiological and pharmacological properties and also demonstrated potent oral efficacy in an
acute TB mouse model. They also showed adequate safety profile with no cytotoxicity,
mutagenicity, cardiotoxicity and phototoxicity, which will be described in detail elsewhere14.
The data presented in this study suggest that 39 and 41 are promising lead candidates with a
potential for MDR and XDR TB treatment, thus warranting further preꢀclinical evaluation.
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
EXPERIMENTAL
Chemistry. Reagents and solvents were purchased from Aldrich, Acros, or other commercial
sources and used without further purification. Thin layer chromatography (TLC) was performed
on precoated silica gel 60 F254 plates from Merck. Compounds were visualized under UV light,
ninhydrin, or phosphomolybdic acid (PMA) stain. NMR spectra were obtained on a Varian 400
MHz Mercury plus NMR or Bruker 400 MHz Ultrashield spectrometer using CDCl3 and DMSOꢀ
d6 as solvents. Compound purity was determined by method 1: LC/MS using an Agilent
UHPLC 1290 coupled with API 3200; Acquity UPLC BEH C18 column, 1.7ꢁm, 2.1x50 mm;
gradient of 98:2 H2O (0.1% formic acid):CH3CN to 2:98 H2O (0.1% formic acid):CH3CN for 2
minutes run time with 1.0 mL/min flow rate / method 2: Waters Acquity UPLC coupled with
MS waters ZQ 2000; Acquity UPLC BEH C18 column, 1.7ꢁm, 2.1x50mm; gradient of 98:2 H2O
(0.1% formic acid):CH3CN to 2:98 H2O (0.1% formic acid):CH3CN for 2 minutes run time with
1.0 mL/min flow rate / method 3: Waters Quattro Micro UPLCꢀLCMS equipped with a Acquity
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 47
1
2
3
4
5
6
7
8
9
BEH C18 column, 1.7ꢁm, 2.1x50mm using a gradient of 90:10 H2O (0.025% trifluoro acetic
acid):CH3CN (0.025% trifluoro acetic acid) to 10:90 H2O (0.025% trifluoro acetic acid):CH3CN
(0.025% trifluoro acetic acid) for 5 minutes run time with 0.4 mL/min flow rate and HPLC
purity using method 4: Waters Acquity UPLC equipped with a Acquity UPLC HSS T3 column,
1.8ꢁm, 2.1x50mm; gradient of 95:5 H2O (0.1% formic acid):CH3CN to 2:98 H2O (0.1% formic
acid):CH3CN for 2 minutes run time with 1.0 mL/min flow rate / method 5: Waters UPLC
equipped with a Acquity, BEH C18 column, 1.7ꢁm, 2.1x100mm using a gradient (4 minutes) of
70:30 H2O (0.025% trifluoro acetic acid):CH3CN (0.025% trifluoro acetic acid) to 20:80 H2O
(0.025% trifluoro acetic acid):CH3CN (0.025% trifluoro acetic acid) for 6 minutes run time with
0.3 mL/min flow rate. The purity of all compounds reported were > 95% measured at 214/254
nM. The cis and trans isomers ratio was measured by HPLC or NMR.
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
Minimum inhibitory concentration (MIC) determination. M. tuberculosis H37Rv (ATCC
27294) strain maintained in Middlebrook 7H9 broth medium supplemented with 0.05 % Tween
80 and 10 % ADS supplement was used for MIC determination. Compounds dissolved in 90 %
DMSO were three fold serialꢀdiluted in duplicates in 384ꢀwell clear plates. A volume of 50 ꢂL of
early log phase culture of Mtb diluted to OD600 0.02 was added to each well, and the assay plates
were incubated at 37 °C for 5 days. Absorbance was recorded using a Spectramax M2
spectrophotometer, and MIC50, a concentration required to inhibit growth by 50% was
determined by plotting nonꢀlinear curves using GraphPad Prism 5 software. Isoniazid was used
as internal control in all our assays, it showed an MIC50 of 0.33 ꢁM.
Solubility. Solubility was measured using high through put equilibrium solubility (HTꢀEq sol)
assay using a novel miniaturized shakeꢀflask approach and streamlined HPLC analysis. Briefly,
transfer DMSO sample stock solution from source plate to MPV (miniꢀprep filer vials,
12
ACS Paragon Plus Environment

Page 13 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Whatman) chamber and subjected to centrifugal evaporator to remove DMSO. Add aqueous
buffer to MPV chamber and insert MPV plunger till the membrane on the bottom of the plunger
touches the surface of the solution in the chamber. Incubate the assembled MPV on orbital
shaker for 24h and push the plunger to the bottom of the chamber followed by shaking for
another 30 min and filtrate was analyzed using HPLC27.
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
Mouse, Rat and Human in vitro metabolic stability. The metabolic stability of drug
candidates was determined in mouse/rat/human liver microsomes using compound depletion
approach and quantified by LC/MS/MS. The assay measured the rate and extent of metabolism
of chemical compounds by measuring the compounds in vitro halfꢀlife (T1/2) and hepatic
extraction ratios (ER) and predicted metabolic clearance in all species28, 29
.
In vivo PK. All animal experiments were approved by the Institutional Animal Care and Use
Committee (IACUC) of Novartis Institute for Tropical Diseases. Mice were allowed for
acclimatization before initiation of pharmacokinetic (PK) experiments. Food and water were
given ad libitum. Compounds were formulated at a concentration of 2.5 mg/mL for a dose of 25
mg/kg given orally (p.o.) and at 1 mg/mL concentration for a dose of 5 mg/kg given
intravenously (i.v.). 21 was formulated using 20% 1,2ꢀpropandiol, 10% solutol HS15 and top up
with pH 4.6 acetate buffer for both i.v and oral administration. 39 and 41 were formulated with
50% MEPC (containing 30% Capmul MCM, 50% Cremophor RH40, 20% Ethanol) diluted with
50% of 50 mM pH 6.8 phosphate buffer for p.o. administration and with 20% 1,2ꢀPropylene
Glycol, 10% Cremophor EL, 70% of 50 mM acetate buffer pH 4.65 for i.v. administration. Blood
samples were collected at 0.08, 0.25, 0.75, 1.5, 3, 8, 16 and 24 hours following oral dosing. The
sampling schedule was identical following i.v. dosing except the first time point was at 0.02 h.
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 47
1
2
3
4
5
6
Groups of three mice were used for each time point. Blood was centrifuged at 13,000 rpm for 7
min at 4 ºC, plasma harvested and stored at ꢀ20 ºC until analysis.
7
8
9
Extraction and LCMS analysis. Plasma samples were extracted with
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
acetonitrile:methanol:acetic acid (90:9.8:0.2) containing warfarin as internal standard, using a 8.8
to 1 extractant to plasma ratio. Analyte quantitation was performed by LC/MS/MS. Liquid
chromatography was performed using an Agilent 1200 HPLC system (Santa Clara, CA) coupled
with a API4000 triple quadruple mass spectrometer (Applied Biosystems, Foster City, CA) using
the Agilent Zorbax Phenyl (3.5ꢁm, 4.6x75 mm) column at 45 ºC. Instrument control and data
acquisition were performed using Applied Biosystems software Analyst, version 1.4.2. The
mobile phases used were (A) 0.2% of acetic acid in water (99.8:0.2, v/v) and (B) 0.1% of acetic
acid in methanol (99.9:0.1, v/v), using a gradient with flow rate of 1.0 mL/min and run time of 5
min. Compound detection on the mass spectrometer was performed in electrospray positive
ionization mode for 41 and negative mode for 21, 39 and using multiple reaction monitoring
(MRM) for specificity (transitions: 21 323.2 / 183.8, 39 337.2 / 184.0 and 41 307.4 / 197.1)
together with their optimized MS parameters. The lower limit of quantification ranged between 1
and 3 ng/mL.
Pharmacokinetic analysis. The mean value from the three animals at each time point was
plotted against time to give plasma concentrationꢀtime profile. Pharmacokinetic parameters were
determined using Watson LIMS, version 7.2 (Thermo Electron Corporation, PA, U.S.), by nonꢀ
compartmental analysis. The oral bioavailability (F) was calculated as the ratio between the area
under the curve (AUC) following oral administration and the AUC following intravenous
administration corrected for dose (F = AUCp.o. x dosei.v. / AUCi.v. x dosep.o.).
14
ACS Paragon Plus Environment

Page 15 of 47
Journal of Medicinal Chemistry
1
2
In vivo efficacy. In vivo acute mouse efficacy studies were carriedꢀout as described earlier11.
Briefly, on day 0 animals were infected by intranasal route with 103 Mtb H37Rv bacilli. One
week postꢀinfection animals treated for one month (7 days a week) with 25 or 75 mg/kg of NCEs
with ethambutol (100 mg/kg) as positive control. At indicated time points lungs were aseptically
removed, homogenized and bacterial load estimated by serial dilution plating 7H11 agar plates.
39 and 41 were formulated using the formulation as described in PK section. 28 was formulated
using 40% MEPC (micro emulsion pre concentrate containing Cremaphor RH40, Labrafil Mꢀ
2125 CS, 1,2ꢀpropylene glycol and ethanol) diluted with 60% of water for p.o. administration.
All procedures involving mice were reviewed and approved by the institutional animal care and
use committee of the Novartis Institute for Tropical Diseases.
3
4
5
6
7
8
9
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
General procedure for library synthesis of amides. To 4,6ꢀdimethyl indoleꢀ2ꢀcarboxylic
acid (0.108 mmol, 500 ꢁL of 0.2 M in anhydrous DMF) was added HATU (0.108 mmol, 250 ꢁL
of 0.4 M in anhydrous DMF) and Et3N (0.378 mmol, 50 ꢁL of 2.0 M in anhydrous DMF) and
stirred for 5 minutes at room temperature. The amine (0.108 mmol in 520 ꢁL anhydrous DMF)
was added to the reaction mixture. The reaction mixture was stirred for two hours at 45°C and
the solvent was evaporated in vacuo and the residue was purified via preparative Shimadzu
HPLC UPLC equipped with a Bischoff C18 column, 10ꢁm, 20x50mm; a gradient of H2O (0.1%
TFA) and Methanol.
The below compounds were prepared as a library using the above general procedure from
corresponding indoleꢀ2ꢀcarboxylic acids and amines
Nꢀisopropylꢀ4,6ꢀdimethylꢀ1Hꢀindoleꢀ2ꢀcarboxamide (3). Yield 69%. 1H NMR (400 MHz,
DMSOꢀd6): δ 11.31 (br. s, 1H), 8.13 (d, J = 7.82 Hz, 1H), 7.12 (d, J = 1.32 Hz, 1H), 7.02 (s, 1H),
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 47
1
2
3
4
5
6
6.66 (s, 1H), 4.18ꢀ4.05 (m, 1H), 2.44 (s, 3H), 2.34 (s, 3H), 1.18 (d, J = 6.60 Hz, 6H). LCMS
(ESI): m/z 231.8 [M + H]+. HPLC purity: >99% at 254 nM.
7
8
9
Nꢀ(secꢀbutyl)ꢀ4,6ꢀdimethylꢀ1Hꢀindoleꢀ2ꢀcarboxamide (4). Yield 69%. 1H NMR (400 MHz,
DMSOꢀd6): δ 11.29 (s, 1H), 8.04 (t, J = 5.8 Hz, 1H), 7.14 (d, J = 1.6 Hz, 1H), 7.02 (s, 1H), 6.66
(s, 1H), 3.98ꢀ3.88 (m, 1H), 2.43 (s, 3H), 2.33 (s, 3H), 1.56ꢀ1.48 (m, 2H), 1.14 (d, J = 6.6 Hz,
3H), 0.88 (t, J = 7.3 Hz, J = 7.4 Hz, 3H). LCMS (ESI): m/z 243.0 [M – H]–. HPLC purity: >
99.0% at 254 nM.
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
Nꢀisobutylꢀ4,6ꢀdimethylꢀ1Hꢀindoleꢀ2ꢀcarboxamide (5). Yield 24%. 1H NMR (400 MHz,
DMSOꢀd6): δ 11.32 (s, 1H), 8.34 (d, J = 8.0 Hz, 1H), 7.12 (d, J = 1.6 Hz, 1H), 7.02 (s, 1H), 6.66
(s, 1H), 3.11ꢀ3.08 (m, 2H), 2.43 (s, 3H), 2.33 (s, 3H), 1.91ꢀ1.78 (m, 1H), 0.90 (d, J = 6.6 Hz,
6H). LCMS (ESI): m/z 243.2 [M – H]–. HPLC purity: 97.7% at 254 nM.
4,6ꢀdimethylꢀNꢀ(pentanꢀ3ꢀyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (6). Yield 64%. 1H NMR (400 MHz,
DMSOꢀd6): δ 11.29 (s, 1H), 7.93 (d, J = 8.8 Hz, 1H), 7.16 (d, J = 1.2 Hz, 1H), 7.02 (s, 1H),
6.66 (s, 1H), 3.84ꢀ3.75 (m, 1H), 2.44 (s, 3H), 2.34 (s, 3H), 1.59ꢀ1.52 (m, 4H), 0.87 (t, J = 7.4 Hz,
6H). LCMS (ESI): m/z 258.8 [M + H]+. HPLC purity: 98.4% at 254 nM.
Nꢀisopentylꢀ4,6ꢀdimethylꢀ1Hꢀindoleꢀ2ꢀcarboxamide (7). Yield 25%. 1H NMR (400 MHz,
DMSOꢀd6): δ 11.31 (s, 1H), 8.29 (t, J = 5.6 Hz, 1H), 7.08 (d, J = 1.6 Hz, 1H), 7.02 (s, 1H), 6.66
(s, 1H), 3.28ꢀ3.26 (m, 2H), 2.43 (s, 3H), 2.33 (s, 3H), 1.69ꢀ1.59 (m, 1H), 1.45ꢀ1.44 (m, 2H), 0.91
(d, J = 6.6 Hz, 6H). LCMS (ESI): m/z 258.8 [M + H]+. HPLC purity: 97.2% at 254 nM.
Nꢀcycloheptylꢀ4,6ꢀdimethylꢀ1Hꢀindoleꢀ2ꢀcarboxamide (8). Yield 31%. 1H NMR (400 MHz,
DMSOꢀd6): δ 11.28 (s, 1H), 8.13 (d, J = 8.0 Hz, 1H), 7.14 (d, J = 1.6 Hz, 1H), 7.02 (s, 1H), 6.66
16
ACS Paragon Plus Environment

Page 17 of 47
Journal of Medicinal Chemistry
1
2
3
4
(s, 1H), 4.05ꢀ3.97 (m, 1H), 2.43 (s, 3H), 2.33 (s, 3H), 1.90ꢀ1.84 (m, 2H), 1.70ꢀ1.40 (m, 10H).
5
6
LCMS (ESI): m/z 283.3 [M – H]–. HPLC purity: >99% at 254 nM.
7
8
9
4,6ꢀdimethylꢀNꢀphenylꢀ1Hꢀindoleꢀ2ꢀcarboxamide (13). Yield 27%. 1H NMR (400 MHz,
DMSOꢀd6): δ 11.54 (s, 1H), 10.12 (s, 1H), 7.81 (d, J = 7.84 Hz, 2H), 7.41 (d, J = 1.6 Hz, 1H),
7.39ꢀ7.33 (m, 2H), 7.12ꢀ7.0 (m, 2H), 6.71 (s, 1H), 2.49 (s, 3H), 2.36 (s, 3H). LCMS (ESI): m/z
265.2 [M + H]+. HPLC purity: >99% at 254 nM.
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
NꢀcyclohexylꢀN,4,6ꢀtrimethylꢀ1Hꢀindoleꢀ2ꢀcarboxamide (14). Yield 42%. 1H NMR (400 MHz,
DMSOꢀd6): δ 11.30 (s, 1H), 7.02 (s, 1H), 6.72 (s,1H), 6.66 (s, 1H), 4.31 (t, J = 11.2 Hz, 1H),
3.07 (s, 3H), 2.43 (s, 3H), 2.34 (s, 3H), 1.82ꢀ1.55 (m, 7H), 1.37ꢀ1.15 (m, 3H). LCMS (ESI): m/z
283.4 [M – H]–. HPLC purity: >99% at 254 nM.
4,6ꢀdichloroꢀNꢀ(4,4ꢀdimethylcyclohexyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (39). To a stirred solution
of 4,6ꢀdichloroꢀ1Hꢀindoleꢀ2ꢀcarboxylic acid (50 mg, 0.217 mmol) in DMF (2 mL) were added
HATU (99.5 mg, 0.26 mmol) and N,Nꢀdiisopropylethylamine (0.188 mL, 1.08 mmol) at room
temperature. The reaction mixture was stirred for 10 min at the same temperature then added 4,4ꢀ
dimethylcyclohexanamine.HCl (39 mg, 0.238 mmol). The resulting reaction mixture was stirred
at room temparature for 16 h. H2O (10 mL) was added to the reaction mixture and extracted with
ethyl acetate (2 x 20 mL). The combined organic layers was washed with brine (1 x 10 mL),
dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude compound
was purified by column chromatography over silica gel (100ꢀ200 mesh) using a solvent gradient
of 30ꢀ40% ethyl acetate in petroleum ether as eluent to afford 30 mg (41%) of 4,6ꢀdichloroꢀNꢀ
(4,4ꢀdimethylcyclohexyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide 39 as a white solid. 1H NMR (400 MHz,
DMSOꢀd6): δ 12.05 (br. s, 1H), 8.43 (d, J = 8.19 Hz, 1H), 7.42 (s, 1H), 7.30 (s, 1H), 7.22 (d, J =
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 47
1
2
3
4
5
6
7
8
9
1.59 Hz, 1H), 3.82ꢀ3.67 (m, 1H), 1.72ꢀ1.62 (m, 2H), 1.61ꢀ1.48 (m, 2H), 1.45ꢀ136 (m, 2H), 1.28
(dt, J = 3.61, 13.17 Hz, 2H), 0.95 (s, 3H), 0.93 (s, 3H). 13C NMR (400 MHz, DMSOꢀd6): δ
159.7, 137.2, 134.3, 128.0, 126.7, 125.3, 119.8, 111.6, 101.0, 48.6, 38.1, 32.6, 29.8, 28.5, 24.6.
LCMS (ESI): m/z 339.5 [M + H]+. HPLC purity: 98.1% at 254 nm. HRMS calcd for
C17H20Cl2N2O [M + H]+ 339.10, found 339.10.
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
The following compounds were synthesized using above procedure from corresponding
substituted indoleꢀ2ꢀcarboxylic acids and amines
Nꢀcyclohexylꢀ4,6ꢀdimethylꢀ1Hꢀindoleꢀ2ꢀcarboxamide (1). Yield 98%. 1H NMR (400 MHz,
DMSOꢀd6): δ 11.30 (s, 1H), 8.10 (d, J = 8.0 Hz, 1H), 7.14 (d, J = 1.6 Hz,1H), 7.02 (s, 1H), 6.66
(s, 1H), 3.78ꢀ3.76 (m, 1H), 2.43 (s, 3H), 2.33 (s, 3H), 1.84ꢀ1.83 (m, 4H),1.76ꢀ1.73 (m, 1H),
1.36ꢀ1.15 (m, 5H).
4,6ꢀdimethylꢀNꢀ(1ꢀmethylpiperidinꢀ4ꢀyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (2). Yield 26.5%. 1H NMR
(400 MHz, DMSOꢀd6): δ 11.31 (s, 1H), 8.14 (d, J = 7.6 Hz, 1H), 7.14 (d, J = 1.2 Hz, 1H), 7.02
(s, 1H), 6.66 (s, 1H), 3.79ꢀ3.69 (m, 1H), 2.78 (d, J = 11.6 Hz, 2H), 2.43 (s, 3H), 2.33 (s, 3H),
2.17 (s, 3H), 1.95 (t, J = 11.6 Hz, 2H), 1.78ꢀ1.76 (m, 2H), 1.63ꢀ1.52 (m, 2H). LCMS (ESI): m/z
284.2 [M – H]–. HPLC purity: 99.0% at 254 nM.
4,6ꢀdimethylꢀNꢀ((1R,2S)ꢀ2ꢀmethylcyclohexyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (9). Yield 44%. 1H
NMR (400 MHz, DMSOꢀd6): δ 11.33 (br. s, 1H), 7.76 (d, J = 8.44 Hz, 1H), 7.29 (d, J = 1.34 Hz,
1H), 7.03 (s, 1H), 6.67 (s, 1H), 4.12 (dt, J = 4.02, 7.98 Hz, 1H), 2.46 (s, 3H), 2.35 (s, 3H), 2.01ꢀ
1.90 (m, 1H), 1.75ꢀ1.63 (m, 2H), 1.61ꢀ1.46 (m, 4H), 1.44ꢀ1.28 (m, 2H), 0.89 (d, J = 6.97 Hz,
3H). 13C NMR (400 MHz, DMSOꢀd6): δ 161.2, 137.1, 133.0, 131.0, 130.4, 125.7, 122.1, 109.8,
18
ACS Paragon Plus Environment

Page 19 of 47
Journal of Medicinal Chemistry
1
2
102.3, 49.9, 33.3, 30.3, 28.9, 23.2, 22.8, 22.0, 18.9, 15.7. LCMS (ESI): m/z 285.28 [M + H]+.
3
4
5
6
HPLC purity: 97.9%.
7
8
9
Nꢀ(2ꢀmethoxyethyl)ꢀ4,6ꢀdimethylꢀ1Hꢀindoleꢀ2ꢀcarboxamide (10). Yield 48%. 1H NMR (400
MHz, DMSOꢀd6): δ 11.35 (s, 1H), 8.41 (t, J = 4.84 Hz, 1H), 7.12 (d, J = 1.6 Hz, 1H), 7.02 (s,
1H), 6.67 (s, 1H), 3.65ꢀ3.41 (m, 4H), 3.29 (s, 3H), 2.43 (s, 3H), 2.33 (s, 3H). LCMS (ESI): m/z
247.6 [M + H]+. HPLC purity: >99% at 254 nM.
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
4,6ꢀdimethylꢀNꢀ(tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (11). Yield 70%. 1H
NMR (400 MHz, DMSOꢀd6): δ 11.34 (s, 1H), 8.22 (d, J = 7.6 Hz, 1H), 7.14 (d, J = 1.2 Hz,1H),
7.02 (s, 1H), 6.66 (s, 1H), 4.05ꢀ3.90 (m, 1H), 3.90ꢀ3.84 (m, 2H), 3.42 (dt, J = 2.0 Hz, 9.6 Hz,
2H), 2.43 (s, 3H), 2.33 (s, 3H), 1.78 (dd, J = 2.28, 13.0 Hz, 2H), 1.59 (dq, J = 4.39, 12.0 Hz, 2H).
LCMS (ESI): m/z 270.9 [M – H]–. HPLC purity: 98.7% at 254 nM.
Nꢀ(2ꢀhydroxycyclohexyl)ꢀ4,6ꢀdimethylꢀ1Hꢀindoleꢀ2ꢀcarboxamide (12). Yield 53%. 1H NMR
(400 MHz, DMSOꢀd6): δ 11.30 (s, 1H), 8.04 (d, J = 8.08 Hz, 1H), 7.13 (s, 1H), 7.02 (s, 1H), 6.6
(s, 1H), 4.65 (d, J = 5.16 Hz, 1H), 3.7ꢀ3.57 (m, 1H), 2.40 (s, 3H), 2.33 (s, 3H), 1.95ꢀ1.81 (m,
2H), 1.69ꢀ1.58 (m, 2H), 1.30ꢀ1.17 (m, 4H). LCMS (ESI): m/z 287.2 [M + H]+. HPLC purity:
>99.0% at 254 nM.
1,4,6ꢀtrimethylꢀNꢀ((1R,2S)ꢀ2ꢀmethylcyclohexyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (15). Yield 50%. 1H
NMR (400 MHz, DMSOꢀd6): δ 7.94 (d, J = 8.35 Hz, 1H), 7.13 (s, 1H), 7.10 (s, 1H), 6.73 (s, 1H),
4.01ꢀ4.14 (m, 1H), 3.89 (s, 3H), 2.46 (s, 3H), 2.39 (s, 3H), 1.98ꢀ1.88 (m, 1H), 1.69ꢀ1.45 (m, 6H),
1.34ꢀ1.32 (m, 2H), 0.89 (d, J = 7.0 Hz, 3H). LCMS (ESI): m/z 299.21 [M + H]+. HPLC purity:
97.4% at 214 nM.
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 47
1
2
3
4
5
6
7
8
9
4,6ꢀDimethylꢀNꢀ((1R,2S)ꢀ2ꢀmethylcyclohexyl)ꢀ1Hꢀpyrrolo[3,2ꢀc]pyridineꢀ2ꢀcarboxamide (18).
Yield 6%. 1H NMR (400 MHz, DMSOꢀd6): δ 11.70 (s, 1H), 7.91 (d, J = 8.24 Hz, 1H), 7.43 (s,
1H), 7.00 (s, 1H), 4.15ꢀ4.07 (m, 1H), 2.61 (s, 3H), 2.45 (s, 3H), 1.99ꢀ1.89 (m, 1H), 1.70ꢀ1.61 (
m, 2H), 1.53ꢀ1.23 (m, 6H), 0.87 (d, J = 6.97 Hz, 3H). LCMS (ESI): 286.18 [M + H]+. HPLC
purity: 96.5% at 254 nm.
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
2,4ꢀDimethylꢀNꢀ((1R,2S)ꢀ2ꢀmethylcyclohexyl)ꢀ7Hꢀpyrrolo[2,3ꢀd]pyrimidineꢀ6ꢀcarboxamide
(19). Yield 13%. 1H NMR (400 MHz, CDCl3): δ 9.39 (br. s, 1H), 6.80 (s, 1H), 6.16 (d, J = 8.24
Hz, 1H), 4.28ꢀ4.26 (m, 1H), 2.76 (s, 3H), 2.74 (s, 3H), 1.94ꢀ2.03 (m, 1H), 1.79ꢀ1.77 (m, 1H),
1.67ꢀ1.65 (m, 3H), 1.52ꢀ1.25 (m, 4H), 0.96 (d, J = 6.97 Hz, 3H). LCMS (ESI): 287.1 [M + H]+.
HPLC purity: 99% at 254 nm.
4,6ꢀdichloroꢀNꢀ((1R,2S)ꢀ2ꢀmethylcyclohexyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (20). Yield 35.4%. 1H
NMR (400 MHz, CDCl3): δ 10.14 (s, 1H), 7.39 (s, 1H), 7.17 (d, J = 1.6 Hz, 1H), 6.89 (d, J = 1.6
Hz, 1H), 6.25 (d, J = 8.88 Hz, 1H), 4.38ꢀ4.28 (m, 1H), 2.08ꢀ2.0 (m, 1H), 1.85ꢀ1.79 (m, 1H),
1.70ꢀ1.60 (m, 3H), 1.58ꢀ1.48 (m, 2H), 1.45ꢀ1.33 (m, 2H), 0.98 (d, J = 6.98 Hz, 3H). LCMS
(ESI): m/z 325.17 [M + H]+. HPLC purity: 97% at 254 nM.
4,6ꢀdichloroꢀNꢀ(4ꢀmethylcyclohexyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (21). Yield 25.5%. Mixture of
cis and trans isomers in 38:62 ratio. 1H NMR (400 MHz, DMSOꢀd6): δ 12.05 (br. s, 1H), 8.43 (d,
J = 8.0 Hz, 0.43H), 8.29 (d, J = 7.46 Hz, 0.58H), 7.42 (s, 1H), 7.38 (s, 0.57H), 7.31 (s, 0.43H),
7.24ꢀ7.20 (m, 1H), 3.99ꢀ3.89 (m, 0.58H), 3.81ꢀ3.68 (m, 0.44H), 1.91ꢀ1.80 (m, 1H), 1.76ꢀ1.64 (m,
2H), 1.63ꢀ1.30 (m, 4H), 1.39ꢀ1.31 (m, 1H), 0.99ꢀ1.10 (m, 1H), 0.96 (d, J = 6.85 Hz, 1.72H), 0.90
(d, J = 6.48 Hz, 1.30H). LCMS (ESI): m/z 325.1 [M + H]+. HPLC purity: >99% at 214 nM.
20
ACS Paragon Plus Environment

Page 21 of 47
Journal of Medicinal Chemistry
1
2
4,6ꢀdifluoroꢀNꢀ((1R,2S)ꢀ2ꢀmethylcyclohexyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (22). Yield 36%. 1H
NMR (400 MHz, DMSOꢀd6): δ 11.98 (s, 1H), 7.97 (d, J = 8.25 Hz, 1H), 7.46 (s, 1H), 7.02 (d, J=
8.25 Hz, 1H), 6.89 (t, J = 10.15 Hz, 1H), 4.2ꢀ4.05 (m, 1H), 2.0ꢀ1.9 (m, 1H), 1.7ꢀ1.25 (m, 8H),
0.87 (d, J = 6.98 Hz, 3H). LCMS (ESI): m/z 293.16 [M + H]+. HPLC purity: 97.1% at 214 nM.
3
4
5
6
7
8
9
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
4,6ꢀdifluoroꢀNꢀ(4ꢀmethylcyclohexyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (23). Yield 54%. Mixture of
cis and trans isomers in 39:61 ratio. 1H NMR (400 MHz, DMSOꢀd6): δ 11.98 (br. s, 1H), 8.28 (d,
J = 8.07 Hz, 0.40H), 8.13 (d, J=7.46 Hz, 0.59H), 7.35 (d, J = Hz, 0.61H), 7.26 (s, 0.41H), 7.02
(td, J = 2.38, 9.41 Hz, 1H), 6.92ꢀ6.82 (m, 1H), 3.94 (m, 0.61H), 3.67ꢀ3.80 (m, 0.43H), 1.85 (m,
1H), 1.63ꢀ1.77 (m, 2H), 1.28ꢀ1.62 (m, 5H), 0.99ꢀ1.10 (m, 1H), 0.96 (d, J = 6.85 Hz, 1.84H), 0.90
(d, J = 6.48 Hz, 1.23H). LCMS (ESI): m/z 293.2 [M + H]+. HPLC purity: >99% at 214 nM.
4ꢀchloroꢀ6ꢀfluoroꢀNꢀ(4ꢀmethylcyclohexyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (24). Yield 55%. Mixture
of cis and trans isomers in 40:60 ratio. 1H NMR (400 MHz, DMSOꢀd6): δ 11.96 (s, 1H), 8.36 (d,
J = 8.0 Hz, 0.42H), 8.22 (d, J = 7.4 Hz, 0.61H), 7.35 (s, 0.66H), 7.27 (s, 0.42H), 7.17ꢀ7.10 (m,
2H), 3.94ꢀ3.87 (m, 0.66H), 3.80ꢀ3.67 (m, 0.43H), 1.89ꢀ1.79 (m, 1H), 1.76ꢀ1.63 (m, 2H), 1.61ꢀ
1.26 (m, 5H), 1.08ꢀ0.97 (m, 1H), 0.95 (d, J = 6.84 Hz, 1.87H), 0.89 (d, J = 6.48 Hz, 1.2H).
LCMS (ESI): m/z 307.4 [M – H]–. HPLC purity: >99% 254 nM.
6ꢀchloroꢀ4ꢀfluoroꢀNꢀ(4ꢀmethylcyclohexyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (25). Yield 41.5%.
Mixture of cis and trans isomers in 40:60 ratio. 1H NMR (400 MHz, DMSOꢀd6): δ 12.01 (s,
1H), 8.33 (d, J = 8.0 Hz, 0.4H), 8.18 (d, J = 7.2 Hz, 0.61H), 7.37 (s, 0.61H), 7.32ꢀ7.24 (m, 1.4H),
6.97ꢀ6.92 (m, 1H), 3.94 ꢀ 3.89 (m, 0.62H), 3.80 ꢀ 3.70 (m, 0.39H), 1.86ꢀ1.81 (m, 1H), 1.74ꢀ1.62
(m, 2H), 1.61ꢀ1.28 (m, 5H),1.08ꢀ0.97 (m, 1H), 0.95 (d, J = 6.84 Hz, 1.82H), 0.88 (d, J = 6.48 Hz,
1.18H). LCMS (ESI): m/z 308.9 [M + H]+. HPLC purity: >99% at 254 nM.
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 47
1
2
3
4
5
6
7
8
9
4ꢀcyanoꢀ6ꢀisocyanoꢀNꢀ(4ꢀmethylꢀcyclohexyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (26). Yield 53%.
Mixture of cis and trans isomers in 45:55 ratio. 1H NMR (400 MHz, DMSOꢀd6): δ 12.76 (br. s,
1H), 8.65 (d, J = 7.70 Hz, 0.44H), 8.50 (d, J = 7.09 Hz, 0.55H), 8.16 (s, 1H), 8.11 (s, 1H), 7.64
(s, 0.55H), 7.56 (s, 0.43H), 3.91ꢀ4.03 (m, 0.58H), 3.84ꢀ3.70 (m, 0.46H), 1.92ꢀ1.84 (m, 1H), 1.79ꢀ
1.65 (m, 2H), 1.65ꢀ1.30 (m, 5H), 1.11ꢀ1.00 (m, 1H), 0.97 (d, J = 6.85 Hz, 1.64H), 0.91 (d, J =
6.48 Hz, 1.35H). LCMS (ESI): m/z 307.9 [M + H]+. HPLC purity: 98.9% at 254 nM.
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
4,6ꢀdichloroꢀNꢀ(cisꢀ4ꢀmethylcyclohexyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (27). Yield 50%. 1H NMR
(400 MHz, DMSOꢀd6): δ 12.05 (br. s, 1H), 8.29 (d, J = 7.46 Hz, 1H), 7.43ꢀ7.40 (m, 1H), 7.38 (d,
J = 1.2 Hz, 1H), 7.22 (d, J = 1.59 Hz, 1H), 3.94 (td, J = 3.62, 7.31 Hz, 1H), 1.76ꢀ1.63 (m, 3H),
1.63ꢀ1.49 (m, 4H), 1.49ꢀ1.38 (m, 2H), 0.96 (d, J = 6.85 Hz, 3H).
13C
NMR (400 MHz, DMSOꢀd6): δ 159.5, 136.7, 133.6, 127.5, 126.2, 124.8, 119.3, 111.0, 100.9,
46.5, 29.7, 28.5, 27.9, 19.5. LCMS (ESI): m/z 325.23 [M + H]+ & 327.15 [M + 2H]+. HPLC
purity: 98.05% at 235 nM. HRMS calcd for C16H18Cl2N2O [M – H]– 323.08, found 323.07.
4,6ꢀdichloroꢀNꢀ(transꢀ4ꢀmethylcyclohexyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (28). Yield 66%. 1H
NMR (400 MHz, DMSOꢀd6): δ 12.03 (br. s, 1H), 8.43 (d, J = 8.07 Hz, 1H), 7.42 (dd, J = 0.79,
1.53 Hz, 1H), 7.28ꢀ7.33 (m, 1H), 7.21 (d, J = 1.71 Hz, 1H), 3.81ꢀ3.67 (m, 1H), 1.85 (d, J = 10.03
Hz, 2H), 1.71 (d, J = 11.98 Hz, 2H), 1.36 (dq, J = 3.06, 12.39 Hz, 3H), 0.96ꢀ1.10 (m, 2H), 0.89
(d, J = 6.6 Hz, 3H). 13C NMR (400 MHz, DMSOꢀd6): δ 159.2, 136.7, 133.7, 127.5, 126.2, 124.8,
119.3, 111.0, 100.5, 48.1, 33.6, 32.2, 31.5, 22.1. LCMS (ESI): m/z 325.17 [M + H]+ & 327.15
[M + 2H]+. HPLC purity: 99% at 254 nM. HRMS calcd for C16H18Cl2N2O [M + H]+ 325.08,
found 325.09.
22
ACS Paragon Plus Environment

Page 23 of 47
Journal of Medicinal Chemistry
1
2
4,6ꢀdifluoroꢀNꢀ(transꢀ4ꢀmethylcyclohexyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (29). Yield 52%. 1H
NMR (400 MHz, DMSOꢀd6): δ 11.8 (s, 1H), 8.28 (d, J = 7.91 Hz, 1H), 7.26 (s, 1H), 7.02ꢀ7.0 (m,
1H), 6.89ꢀ6.84 (m, 1H), 3.76ꢀ3.69 (m, 1H), 1.86ꢀ1.83 (m, 2H), 1.72ꢀ1.69 (m, 2H), 1.38ꢀ1.30 (m,
3H), 1.07ꢀ0.98 (m, 2H), 0.89 (d, J = 6.6 Hz, 3H). LCMS (ESI): m/z 293.18 [M + H]+. HPLC
purity: 98.7% at 254 nM.
3
4
5
6
7
8
9
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
4ꢀchloroꢀNꢀ(4ꢀmethylcyclohexyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (30). Yield: 43%. Mixture of cis
and trans isomers in 45:55 ratio. 1H NMR (400 MHz, DMSOꢀd6): δ 12.01 (br. s, 1H), 8.41 (d, J =
8.0 Hz, 0.46H), 8.28 (d, J = 7.46 Hz, 0.54H), 7.40 (dd, J = 3.48, 8.01 Hz, 1H), 7.33 (s, 0.55H),
7.26 (s, 0.45H), 7.20ꢀ7.14 (m, 1H), 7.13ꢀ7.09 (m, 1H), 4.00ꢀ3.90 (m, 0.56H), 3.81ꢀ3.68 (m,
0.48H), 1.86 (d, J = 9.90 Hz, 1H), 1.77ꢀ1.64 (m, 2H), 1.63ꢀ1.29 (m, 5H), 1.10ꢀ1.00 (m, 1H), 0.97
(d, J = 6.85 Hz, 1.68H), 0.90 (d, J = 6.48 Hz, 1.33H). LCMS (ESI): m/z 291.11 [M + H]+. HPLC
purity: >99% at 214 nM.
5ꢀchloroꢀNꢀ(4ꢀmethylcyclohexyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (31). Yield 40%. Mixture of cis
and trans isomers in 45:55 ratio. 1H NMR (400 MHz, DMSOꢀd6): δ 11.74 (d, J = 11.13 Hz, 1H),
8.28 (d, J = 8.04 Hz, 0.45H), 8.14 (d, J = 7.46 Hz, 0.55H), 7.74ꢀ7.64 (m, 1H), 7.43 (dd, J = 3.18,
8.68 Hz, 1H), 7.25ꢀ7.09 (m, 2H), 3.99ꢀ3.88 (m, 0.59H), 3.80ꢀ3.68 (m, 0.48H), 1.85 (d, J = 10.03
Hz, 1H), 1.75ꢀ1.65 (m, 2H), 1.62ꢀ1.29 (m, 5H), 1.1ꢀ0.98 (m, 1H), 0.96 (d, J = 6.8 Hz, 1.76 H),
0.90 (d, J = 6.52 Hz, 1.34H). LCMS (ESI): m/z 291.11 [M + H]+. HPLC purity: >99% at 214
nM.
6ꢀchloroꢀNꢀ(4ꢀmethylcyclohexyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (32). Yield 50%. Mixture of cis
and trans isomers in 25:75 ratio. 1H NMR (400 MHz, DMSOꢀd6): δ 11.69 (br. s, 1H), 8.25 (d, J
= 8.0 Hz, 0.26H), 8.11 (d, J = 7.46 Hz, 0.74H), 7.68ꢀ7.58 (m, 1H), 7.48ꢀ7.38 (m, 1H), 7.25 (s,
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 47
1
2
3
4
5
6
7
8
9
0.78H), 7.17 (s, 0.27H), 7.00ꢀ7.09 (m, 1H), 4.00ꢀ3.88 (m, 0.77H), 3.82ꢀ3.65 (m, 0.28H), 1.90ꢀ
1.63 (m, 3H), 1.63ꢀ1.49 (m, 3H), 1.48ꢀ1.30 (m, 2.4H), 1.10ꢀ0.99 (m, 0.63H), 0.96 (d, J = 6.85
Hz, 2H), 0.90 (d, J = 6.48 Hz, 1H). LCMS (ESI): m/z 291.11 [M + H]+. HPLC purity: >99% at
254 nM.
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
7ꢀchloroꢀNꢀ(4ꢀmethylcyclohexyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (33). Yield 44%. Mixture of cis
and trans isomers in 43:57 ratio. 1H NMR (400 MHz, DMSOꢀd6): δ 11.63 (br. s, 1H), 8.28 (d, J =
7.72 Hz, 1H), 8.16 (d, J = 6.97 Hz, 1H), 7.55ꢀ7.66 (m, 1H), 7.27ꢀ7.32 (m, 1H), 7.22 (s, 0.56H),
7.18 (s, 0.43H), 7.06 (dt, J = 2.63, 7.79 Hz, 1H), 3.91ꢀ4.00 (m, 0.58H), 3.68ꢀ3.80 (m, 0.45H),
1.90 (d, J = 9.90 Hz, 1H), 1.50ꢀ1.79 (m, 5H), 1.29ꢀ1.48 (m, 2H), 0.99ꢀ1.11 (m, 1H), 0.97 (d, J =
6.85 Hz, 1.70H), 0.91 (d, J = 6.48 Hz, 1.33H). LCMS (ESI): 291.11 [M + H]+. HPLC purity:
>99% at 214 nM.
4,5ꢀdichloroꢀNꢀ(4ꢀmethylcyclohexyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (34). Yield 50%. Mixture of
cis and trans isomers in 41:59 ratio. 1H NMR (400 MHz, DMSOꢀd6): δ 12.09 (br. s, 1H), 8.44 (d,
J = 7.92 Hz, 0.42H), 8.30 (d, J=7.46 Hz, 0.61H), 7.45ꢀ7.39 (m, 1H), 7.38ꢀ7.29 (m, 2H), 4.01ꢀ
3.88 (m, 0.64H), 3.81ꢀ3.69 (m, 0.45H), 1.86 (d, J = 10.39 Hz, 1H), 1.78ꢀ1.64 (m, 2H), 1.63ꢀ1.27
(m, 5H), 1.11ꢀ1.00 (m, 1H), 0.97 (d, J = 6.72 Hz, 1.8H), 0.90 (d, J = 6.48 Hz, 1.2H). LCMS
(ESI): m/z 324.98 [M + H]+ & 327.02 [M + 2H]+. HPLC purity: >99% at 254 nM.
4,7ꢀdichloroꢀNꢀ(4ꢀmethylcyclohexyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (35). Yield 41%. Mixture of
cis and trans isomers in 37:63 ratio. 1H NMR (400 MHz, DMSOꢀd6): δ 12.09 (s, 0.5H), 12.01 (s,
0.34H), 7.67ꢀ7.57 (m, 2H), 7.48 (s, 1H), 7.21ꢀ7.17 (m, 1H), 4.03ꢀ4.12 (m, 0.66H), 3.77ꢀ3.73 (m,
0.37H), 1.92ꢀ1.54 (m, 6H), 1.42ꢀ1.25 (m, 2H), 1.08ꢀ1.02 (m, 1H), 0.94 (d, J = 6.32 Hz, 1.97H),
24
ACS Paragon Plus Environment

Page 25 of 47
Journal of Medicinal Chemistry
1
2
0.89 (d, J = 6.54 Hz, 1.2H). LCMS (ESI): 324.9 [M + H]+ & 327.0 [M + 2H]+. HPLC purity:
3
4
5
6
97.0% at 214 nM.
7
8
9
5,6ꢀdichloroꢀNꢀ(4ꢀmethylcyclohexyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (36). Yield 71%. Mixture of
cis and trans isomers in 39:61 ratio. 1H NMR (400 MHz, DMSOꢀd6): δ 11.84 (br. s, 1H), 8.37 (d,
J = 7.99 Hz, 0.41H), 8.23 ( d, J = 7.38 Hz, 0.61H), 7.94 (d, J = 2.4 Hz, 1H), 7.60 (d, J = 2.8 Hz,
1H), 7.24 (s, 0.64H), 7.17 (s, 0.39H), 3.92ꢀ3.91 (m, 0.64H), 3.74ꢀ3.72 (m, 0.41H), 1.89ꢀ1.78 (m,
1H), 1.75ꢀ1.28 (m, 7H), 1.09ꢀ0.98 (m, 1H), 0.95 (d, J = 6.8 Hz, 1.84H), 0.89 (d, J = 6.44 Hz,
1.22H). LCMS (ESI): 325.15 [M + H]+ & 327.10 [M + 2H]+. HPLC purity: 99.0% at 254 nM.
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
5,7ꢀdichloroꢀNꢀ(4ꢀmethylcyclohexyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (37). Yiled 10%. Mixture of
cis and trans isomers in 34:66 ratio. 1H NMR (400 MHz, DMSOꢀd6): δ 11.88 (s, 0.67H), 11.83
(s, 0.35H), 8.32 (d, J = 7.6 Hz, 0.34H), 8.19 (d, J = 6.8 Hz, 0.66H), 7.72 (s, 1H), 7.38 (d, J =
1.32 Hz, 1H), 7.21ꢀ7.16 (m, 1H), 3.95 (m, 0.66H), 3.75ꢀ3.72 (m, 0.34H), 1.9ꢀ1.25 (m, 8H), 1.08ꢀ
1.02 (m, 1H), 0.95 (d, J = 7.2 Hz, 2.01H), 0.89 (d, J = 6.4 Hz, 1.07H). LCMS (ESI): m/z 325.04
[M + H]+. HPLC purity: >99% at 254 nM.
6,7ꢀdichloroꢀNꢀ(4ꢀmethylcyclohexyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (38). Yield 39%. Mixture of
cis and trans isomers in 67:33 ratio. 1H NMR (400 MHz, DMSOꢀd6): δ 11.67 (br. s, 1H), 8.32 (d,
J = 7.82 Hz, 1H), 8.19 (d, J = 7.04 Hz, 1H), 7.67ꢀ7.60 (m, 1H), 7.29ꢀ7.23 (m, 1.33H), 7.21 (s,
0.67H), 4.0ꢀ3.90 (m, 0.35H), 3.80ꢀ3.67 (m, 0.72H), 1.95ꢀ1.85 (m, 1H), 1.78ꢀ1.50 (m, 4H), 1.48ꢀ
1.22 (m, 3H), 1.13ꢀ0.99 (m, 1H), 0.96 (d, J = 6.8 Hz, 0.9H), 0.90 (d, J = 6.48 Hz, 2.16H). LCMS
(ESI): 325.04 [M + H]+ & 327.02 [M + 2H]+. HPLC purity: 98.5% at 254 nM.
4,6ꢀdichloroꢀNꢀ(4,4ꢀdifluorocyclohexyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (40). Yield 39%. 1H NMR
(400 MHz, DMSOꢀd6): δ 12.09 (s, 1H), 8.51 (d, J = 7.50 Hz, 1H), 7.42 (s, 1H), 7.32 (d, J = 1.3
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 47
1
2
3
4
5
6
Hz, 1H), 7. 23 (d, J = 1.3 Hz, 1H), 4.04ꢀ4.02 (m, 1H), 2.07ꢀ1.90 (m, 6H), 1.69ꢀ1.61 (m, 2H).
LCMS (ESI): m/z 347.0 [M + H]+ & 349.0 [M + 2H]+. HPLC purity: 98.8%.
7
8
9
4,6ꢀdifluoroꢀNꢀ(4,4ꢀdimethylcyclohexyl)ꢀ1Hꢀindoleꢀ2ꢀcarboxamide (41). Yield 66.2%. 1H
NMR (400 MHz, DMSOꢀd6): δ 11.96 (br. s, 1H), 8.29 (d, J = 8.07 Hz, 1H), 7.26 (d, J = 0.48 Hz,
1H), 7.02 (dd, J = 1.47, 9.41 Hz, 1H), 6.87 (dt, J = 2.08, 10.39 Hz, 1H), 3.63ꢀ3.81 (m, 1H), 1.73ꢀ
1.62 (m, 2H), 1.60ꢀ1.46 (m, 2H), 1.45ꢀ1.36 (m, 2H), 1.28 (dt, J = 3.73, 13.17 Hz, 2H), 0.95 (s,
3H), 0.92 (s, 3H). LCMS (ESI): m/z 307.11 [M + H]+. HPLC purity: 98.6%. HRMS calcd for
C17H20F2N2O [M + H]+ 307.15, found 307.16.
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
4,6ꢀDimethylꢀNꢀ((1R,2S)ꢀ2ꢀmethylcyclohexyl)indolineꢀ2ꢀcarboxamide (16). To a stirred
solution of 9 (0.10 g, 0.350 mmol) in TFA (8 mL) at 0 °C was added NaCNBH3 (0.308 g, 4.901
mmol) slowly portion wise and stirred for 3 h at room temperature. NaCNBH3 (0.308 g, 4.901
mmol) was added again at 0 °C and continued stirring for 8 h at room temperature. The reaction
mixture was diluted with DCM and washed with aq. sat. NaHCO3. The combined organic layer
was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude
compound was purified by column chromatography over silica gel (100ꢀ200 mesh) using a
solvent gradient of 50% ethyl acetate in petroleum ether followed by prep HPLC to afford 50 mg
( 50% ) of diastereomeric mixture of 4,6ꢀdimethylꢀNꢀ((1R,2S)ꢀ2ꢀmethylcyclohexyl)indolineꢀ2ꢀ
carboxamide 16 as a white solid. 1H NMR (400 MHz, CDCl3): δ 6.49 (s, 1H), 6.45 (s, 1H), 4.45
(br. s, 1H), 4.08 (br. s, 2H), 3.55ꢀ3.45 (m, 1H), 3.0ꢀ2.95 (m, 1H), 2.26 (s, 3H), 2.16 (s, 3H), 1.85
(br. s, 2H), 1.5ꢀ1.19 (m, 6H), 0.89ꢀ0.88 (m, 1H), 0.79 (d, J = 7.0 Hz, 3H). LCMS (ESI): m/z
287.26 [M + H]+. HPLC purity: >99% at 214 nM.
26
ACS Paragon Plus Environment

Page 27 of 47
Journal of Medicinal Chemistry
1
2
4,6ꢀdimethylꢀNꢀ((1R,2S)ꢀ2ꢀmethylcyclohexyl)ꢀ1Hꢀbenzo[d]imidazoleꢀ2ꢀcarboxamide (17). 1H
NMR (400 MHz, CDCl3): δ 9.28 (s, 1H), 6.92 (s, 1H), 6.68 (s, 1H), 6.23 (d, J = 8.24 Hz, 1H),
4.35ꢀ4.32 (m, 1H), 2.50 (s, 3H), 2.35 (s, 3H), 2.06 (br. s, 1H), 1.88 (br. s, 1H), 1.66ꢀ1.59 (m,
3H), 1.54ꢀ1.51 (m, 2H), 1.43ꢀ137 (m, 2H), 0.94 (d, J = 6.8 Hz, 3H). LCMS (ESI): 286.18 [M +
H]+. HPLC purity: 99% at 254 nm.
3
4
5
6
7
8
9
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
ASSOCIATED CONTENT
Supporting Information. Experimental details for synthesis of compound 17, a table
describing LCMS and HPLC methods for all compounds and structures of 42, 43 and 44 are
provided. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* Tel: +65ꢀ6722ꢀ2984. Fax: +65ꢀ6722ꢀ2918.
Eꢀmail: ravinder.kondreddi@novartis.com
Present Addresses
§California Institute for Biomedical Research (Calibr)
11119 N. Torrey Pines Rd., Suite 100
La Jolla, CA 92037 USA.
# Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597
ACKNOWLEDGMENT
This work was supported and funded by Novartis Institute for Tropical Diseases and Genomics
Institute of the Novartis Research Foundation. We would like to thank Richard Glynne, Fumiaki
Yokokawa, Kevin Pethe, Gu Feng, Veronique Dartois, Francesca Blasco, David Beer and other
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 47
1
2
3
4
5
6
7
8
9
NITD colleagues from chemistry, pharmacology and TB biology for their support during the
course of this study. We would like to thank Sreehari Babu and Lijun Zhang for formulation
study; Carolyn Shoen and Michael Cynamon for in vivo efficacy study.
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
ABBREVIATIONS
Mtb: Mycobacterium tuberculosis; TB: Tuberculosis; MDR: multiꢀdrug resistant; XDR:
extensively drug resistant; TDR: totally drug resistant; DIPEA: N,Nꢀdiisopropylethylamine;
DMF: dimethylformamide; HATU: 2ꢀ(7ꢀazaꢀ1Hꢀbenzotriazoleꢀ1ꢀyl)ꢀ1,1,3,3ꢀtetramethyluronium
hexafluorophosphate; POCl3: phosphorus oxychloride; MIC: minimum inhibitory concentration;
SAR: structure–activity relationships; LiPE: Lipophilic Efficiency; SPR: structure–property
relationships
REFERENCES
1.
Dye, C.; Williams, B. G. The population dynamics and control of tuberculosis. Science
2010, 328, 856ꢀ861.
2.
Report. World Health Organization (WHO) Global Tuberculosis Report.
http://apps.who.int/iris/bitstream/10665/75938/1/9789241564502_eng.pdf 2012.
3.
tuberculosis in India. Clin Infect Dis 2012, 54, 579ꢀ581.
4. Andries, K.; Verhasselt, P.; Guillemont, J.; Gohlmann, H. W. H.; Neefs, J.ꢀM.; Winkler,
Udwadia, Z. F.; Amale, R. A.; Ajbani, K. K.; Rodrigues, C. Totally drugꢀresistant
H.; Van Gestel, J.; Timmerman, P.; Zhu, M.; Lee, E.; Williams, P.; de Chaffoy, D.; Huitric, E.;
Hoffner, S.; Cambau, E.; TruffotꢀPernot, C.; Lounis, N.; Jarlier, V. A Diarylquinoline Drug
Active on the ATP Synthase of Mycobacterium tuberculosis. Science 2005, 307, 223ꢀ227.
28
ACS Paragon Plus Environment

Page 29 of 47
Journal of Medicinal Chemistry
1
2
3
4
5.
Matsumoto, M.; Hashizume, H.; Tomishige, T.; Kawasaki, M.; Tsubouchi, H.; Sasaki,
5
6
H.; Shimokawa, Y.; Komatsu, M. OPCꢀ67683, a nitroꢀdihydroꢀimidazooxazole derivative with
7
8
9
promising action against tuberculosis in vitro and in mice. PLoS Med 2006, 3, e466.
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
6.
Stover, C. K.; Warrener, P.; VanDevanter, D. R.; Sherman, D. R.; Arain, T. M.;
Langhorne, M. H.; Anderson, S. W.; Towell, J. A.; Yuan, Y.; McMurray, D. N.; Kreiswirth, B.
N.; Barry, C. E.; Baker, W. R. A smallꢀmolecule nitroimidazopyran drug candidate for the
treatment of tuberculosis. Nature 2000, 405, 962ꢀ966.
7.
Cohen, J. Infectious disease. Approval of novel TB drug celebratedꢀꢀwith restraint.
Science 2013, 339, 130.
8.
Ananthan, S.; Faaleolea, E. R.; Goldman, R. C.; Hobrath, J. V.; Kwong, C. D.; Laughon,
B. E.; Maddry, J. A.; Mehta, A.; Rasmussen, L.; Reynolds, R. C.; Secrist, J. A., 3rd; Shindo, N.;
Showe, D. N.; Sosa, M. I.; Suling, W. J.; White, E. L. Highꢀthroughput screening for inhibitors
of Mycobacterium tuberculosis H37Rv. Tuberculosis (Edinb) 2009, 89, 334ꢀ353.
9.
Ballell, L.; Bates, R. H.; Young, R. J.; AlvarezꢀGomez, D.; AlvarezꢀRuiz, E.; Barroso,
V.; Blanco, D.; Crespo, B.; Escribano, J.; Gonzalez, R.; Lozano, S.; Huss, S.; SantosꢀVillarejo,
A.; MartinꢀPlaza, J. J.; Mendoza, A.; RebolloꢀLopez, M. J.; RemuinanꢀBlanco, M.; Lavandera, J.
L.; PerezꢀHerran, E.; GamoꢀBenito, F. J.; GarciaꢀBustos, J. F.; Barros, D.; Castro, J. P.;
Cammack, N. Fueling openꢀsource drug discovery: 177 smallꢀmolecule leads against
tuberculosis. ChemMedChem 2013, 8, 313ꢀ321.
10.
Mak, P. A.; Rao, S. P.; Ping Tan, M.; Lin, X.; Chyba, J.; Tay, J.; Ng, S. H.; Tan, B. H.;
Cherian, J.; Duraiswamy, J.; Bifani, P.; Lim, V.; Lee, B. H.; Ling Ma, N.; Beer, D.; Thayalan, P.;
Kuhen, K.; Chatterjee, A.; Supek, F.; Glynne, R.; Zheng, J.; Boshoff, H. I.; Barry, C. E., 3rd;
Dick, T.; Pethe, K.; Camacho, L. R. A highꢀthroughput screen to identify inhibitors of ATP
29
ACS Paragon Plus Environment