Design, synthesis and evaluation of indole-2-carboxamides with pan anti-mycobacterial activity

Article abstract of DOI:10.1016/j.bmc.2017.05.015

Current treatment regimens for non-tuberculous mycobacteria (NTM) and tuberculosis (TB) generally require long duration of therapy with multiple drugs, some of which are broad spectrum antibiotics. Despite some advances in antimicrobial compounds, there remains a need in therapy for antibiotics with specific mycobacterial targets. It has been shown that MmpL3 is an essential transporter required for the translocation of mycolic acids to the mycobacterial cell envelope. Here, we synthesized a series of indole-2-carboxamides that inhibit MmpL3 and have potent pan-activity against mycobacterial species. The compounds were tested against several fast and slow-growing Mycobacterium species, including M. abscessus, M. massiliense, M. bolletii, M. chelonae, M. tuberculosis, M. avium, M. xenopi and M. smegmatis. The target of these indole-based compounds makes them selective for mycobacteria, while showing no clinically relevant bactericidal activity against S. aureus or P. aeruginosa. These compounds were tested against THP-1, a human-cell line, and showed minimal in vitro cytotoxicity and good selectivity indices. The data shown and discussed suggest that lead indole-2-carboxamides are strong contenders for further preclinical testing as NTM therapeutics.

Full text of DOI:10.1016/j.bmc.2017.05.015

Accepted Manuscript
Design, Synthesis and Evaluation of Indole-2-carboxamides with Pan Anti-my-
cobacterial Activity
Nicholas D. Franz, Juan Manuel Belardinelli, Michael A. Kaminski, Louis C.
Dunn, Vinicius Calado Nogueira de Moura, Michael A. Blaha, Dan D. Truong,
Wei Li, Mary Jackson, E. Jeffrey North
PII:
S0968-0896(17)30499-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.05.015
BMC 13733
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 March 2017
4 May 2017
6 May 2017
Please cite this article as: Franz, N.D., Belardinelli, J.M., Kaminski, M.A., Dunn, L.C., de Moura, V.C.N., Blaha,
M.A., Truong, D.D., Li, W., Jackson, M., Jeffrey North, E., Design, Synthesis and Evaluation of Indole-2-
carboxamides with Pan Anti-mycobacterial Activity, Bioorganic & Medicinal Chemistry (2017), doi: http://
dx.doi.org/10.1016/j.bmc.2017.05.015
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Design, Synthesis and Evaluation of Indole-2-carboxamides
with Pan Anti-mycobacterial Activity
Nicholas D. Franz1, Juan Manuel Belardinelli2, Michael A. Kaminski1, Louis C. Dunn1, Vinicius Calado
Nogueira de Moura2, Michael A. Blaha1, Dan D. Truong1, Wei Li 2, Mary Jackson2, E. Jeffrey North1*
1Department of Pharmacy Sciences, Creighton University, 2500 California Plaza, Omaha, NE 68178, USA
2Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology,
Colorado State University, Fort Collins, CO 80523, USA
*Corresponding author:
E. Jeffrey North
Department of Pharmacy Sciences
School of Pharmacy & Health Professions
Creighton University
2500 California Plaza
Hixon-Lied Sciences Building, Room 149
Omaha, NE 68178
E-mail: jeffreynorth@creighton.edu
Voice: (402) 280-2871
Fax: (402) 280-1883
Keywords: Non-tuberculous mycobacteria; NTM; indole-2-carboxamide; Mycobacterium;
Mycobacterium tuberculosis; Mycobacterium abscessus

Abstract
Current treatment regimens for non-tuberculous mycobacteria (NTM) and tuberculosis (TB) generally
require long duration of therapy with multiple drugs, some of which are broad spectrum antibiotics.
Despite some advances in antimicrobial compounds, there remains a need in therapy for antibiotics with
specific mycobacterial targets. It has been shown that MmpL3 is an essential transporter required for
the translocation of mycolic acids to the mycobacterial cell envelope. Here, we synthesized a series of
indole-2-carboxamides that inhibit MmpL3 and have potent pan-activity against mycobacterial species.
The compounds were tested against several fast and slow-growing Mycobacterium species, including M.
abscessus, M. massiliense, M. bolletii, M. chelonae, M. tuberculosis, M. avium, M. xenopi and M.
smegmatis. The target of these indole compounds makes them selective for mycobacteria, while
showing no clinically relevant bactericidal activity against S. aureus or P. aeruginosa. These compounds
were tested against THP-1, a human-cell line, and showed minimal in vitro cytotoxicity and good
selectivity indices. The data shown and discussed suggest that lead indole-2-carboxamides are strong
contenders for further preclinical testing as NTM therapeutics.

1.1 - Introduction
Non-tuberculous mycobacteria (NTM) are ubiquitous environmental pathogens that can cause a
wide variety of infections, such as: progressive pulmonary disease, skin and soft tissue infections,
lymphadenitis, and disseminated disease.1, 2 The most common human pathogens are the species M.
avium complex (MAC) and M. kansasii, however other clinically relevant species include the M.
abscessus complex (MABSC, including the subspecies M. abscessus, M. massiliense and M. bolletii)
and M. fortuitum.3 NTM commonly cause pulmonary disease in older, immunocompromised patients
who have underlying lung disease. The precise frequency of NTM disease is unknown because reporting
for NTM is not mandatory in the United States and many other countries.4, 5 Furthermore, current
incidence and prevalence data are likely underestimated due to the indolent nature of NTM pulmonary
disease, the most common form of NTM infections. A growing number of studies suggest that the
number of NTM infections and mortality rates continue to increase.4, 6 Growing prevalence of NTM
isolates between 1997 (9.1/100,000) and 2003 (14.1/100,000) has been observed in Ontario, Canada.7
More recently, Taiwan has reported increasing incidence of NTM disease, 10.2 cases per 100,000
persons in 2008 compared to 2.7 cases per 100,000 persons in 2000.8, 9 In addition, estimates from 1999
to 2010 suggest the number of immediate NTM-related deaths in the United States rose significantly
and are expected to increase over the next few years, given the increasing median age in North
America.10 Although NTM are opportunistic pathogens, they represent a matter of significant concern
to health practitioners because of increasing incidence and mortality rates. As the number of patients
with immune-compromising conditions continues to grow, both opportunistic and primary cases of NTM
infection present a significant challenge for current antibiotic therapy.
In contrast to the insidious nature of NTM infections, M. tuberculosis is the cause of highly
transmittable disease and infects about one-third of the world’s population.11 In 2015, tuberculosis (TB)
became the leading infectious disease killer in the world and causes illness in approximately 9.6 million
people, killing 1.5 million each year.11 The global incidence of multi-drug-resistant tuberculosis (MDR-
TB) remains unchanged at 3.3%, causing growing concern for healthcare professionals.11 MDR-TB is
classified as resistant to both rifampicin and isoniazid, two critical antibiotics used in standard
treatment. This form of TB goes largely undetected, as the estimated number of cases of MDR-TB in
2014 was 480,000 people and the number of these patients who were started on appropriate treatment
was only about 111,000.11 Current treatment against mycobacterial infections are inadequate and this
may be due, at least in part, to the absence of timely diagnostics and a paucity of narrow spectrum
antimycobacterial antibiotics. The current treatment of NTM recommended by the American Thoracic

Society (ATS) and Infectious Diseases Society of America (IDSA) include regimens of multiple antibiotics,
potentially including macrolides, aminoglycosides, fluoroquinolones, oxazolidinones, tigecycline,
carbapenems, cephalosporins, sulfonamides, ethambutol, and rifampicin.3 Many of these
recommendations are made on the basis of preliminary in vitro efficacy studies due to the lack of a
standardized animal model.3 Depending on the organism and site of infection, two to four of these
agents are often used in combination for a duration of at least 12 months after the first negative
culture.3 The use of multiple antibiotics for prolonged periods of time is challenging due to common
complications such as drug interactions and noncompliance.3
Problems surrounding current anti-TB therapy are similar to that of NTM. The treatment of
drug-susceptible TB continues to revolve around the same four-antibiotic regimen (isoniazid, rifampicin,
ethambutol, and pyrazinamide) introduced more than 40 years ago.11 Although the treatment of TB has
been extensively studied, therapeutic regimens are still somewhat lacking in that: 1) they require the
use of multiple antibiotics and 2) the duration of therapy is long. These are both factors contributing to
non-compliance and treatment failure, which can lead to the emergence of MDR-TB and extensively
drug-resistant tuberculosis (XDR-TB). The treatment of MDR-TB/XDR-TB is more difficult and requires
multiple broad-spectrum antibiotics, which exposes patients to complications like Clostridium difficile
infections and other resistant microorganisms.12 Clinical practice guidelines for the treatment of drug-
resistant TB are currently under development by the ATS and IDSA.13 Depending on the susceptibilities
of particular drug-resistant TB strains, second-line anti-TB drugs are typically used for a duration of 2
years and treatment success rates range from 30 to 80%, underscoring the need for newer anti-
mycobacterial drugs and treatment regimens that maximize efficacy and shorten duration of
treatment.14-21
Current drugs that are in the pharmaceutical pipeline for mycobacterial infections are largely
being used against M. tuberculosis and not specifically against NTM. Despite the number of new drugs
being studied in pre-clinical and clinical trials for TB, they are being studied in combination drug
regimens with other broad-spectrum antibiotics. Furthermore, these drugs, including DC-159a,22 SQ-
641,23 CPZEN-45,24 BTZ043,25 bedaquiline,26 and pretomanid27, lack bactericidal activity against many
NTM species. The development of narrow-spectrum anti-mycobacterial drugs could revolutionize the
treatment of both TB and NTM.
Indole-2-carboxamides (IC) have been reported as novel antitubercular agents with activity
against drug-resistant strains and in in vivo efficacy mouse models.28-32 IC are bioisosteric isomers of
published urea based M. tuberculosis inhibitors and have similar structure activity relationships.33-35

Herein, we report the expansion of i) the chemical space for the published antitubercular IC compounds
and ii) the spectrum of antimycobacterial activity. In addition, lead compounds have demonstrated a
safe pharmacological profile and are inhibitors of the mycolic acid biosynthetic pathway in both TB and
NTM strains. Specifically, they inhibit the translocation of trehalose-monomycolate (TMM) to the outer
membrane, suggestive of Mycobacteria membrane protein large 3 (MmpL3) inhibition.

2.1 - Results and Discussion
2.2 - Chemistry. Indole-2-carboxamide (IC) compounds were generated using published
methods and is shown in scheme 1.30 Briefly, the reaction of the arylhydrazine with ethyl pyruvate in
the presence of p-toluenesulfonic acid (pTsOH) afforded ethyl indole-2-carboxylate. After NaOH-
mediated saponification of the ester, commercially available amines were coupled to indole-2-carboxylic
acid using standard coupling conditions.
Scheme 1. Synthetic pathway for the production of IC compounds. Reagents and conditions: (a) Ethyl
pyruvate, pTsOH, EtOH, reflux, 6 hr; (b) NaOH, EtOH, reflux, 3 hr; (c) amine, DCC, DMAP, CH2Cl2, r.t.,
overnight.
2.3 - Anti-Mycobacterial Activity. Two IC series were evaluated for their anti-mycobacterial
activity against various mycobacterial pathogens, including M. abscessus, M. massiliense, M. bolletii, M.
chelonae, M. tuberculosis, and M. smegmatis. The first series is an unsubstituted indole (R = H,
compounds 5-19) and the second was as 4,6-dimethyl indole (R = CH3, compounds 20-34). A variety of
commercially available bulkyl aliphatic and aromatic head groups were incorporated into final
compounds that are consistent with published SAR for previously evaluated urea-based compounds and
IC.28, 30, 32-35
Table 1 shows the minimum inhibitory concentration (MIC) values for the synthesized
unsubstituted IC compounds. Overall, these compounds have limited pan-antimycobacterial activity as
demonstrated by inhibition from compounds 5 and 16. Excluding M. smegmatis, compounds 5 and 16
show the best antimycobacterial activity with sub-μg/mL MIC values and compound 16 obtaining 2-4-
fold increased activity over 5. An adamantyl substitution in compound 5 confers activity, however the 3-
ethyl adamantyl (6) or adamantyl with an ethyl or methyl linker (7, 8) is not tolerated. Interestingly,
changing the connection point from the 1-position (5) to the 2-position (9) on adamantine abolished
NTM activity, however retained potent activity against M. tuberculosis. The methyl-linked adamantyl
substitution on compound 8 elicits greater activity against NTM than the larger ethyl-linked compound
7, suggesting an upper limit for linker bulk. Saturated cycloalkyl rings that are 8 carbons or smaller (10,
11, 12, and 13) abolish all NTM activity. However, adding methyl components to smaller aliphatic rings

can increase activity as seen with the isopinocampheyl substituted (16), which attains the highest pan-
activity of the series.
The antitubercular SAR is much looser, as the constraints on for the bulky aliphatic group are
less restrictive. A cyclooctyl (10) substituent maintains antimicrobial activity against M. tuberculosis
with an MIC of 0.39 μg/mL, despite being absent for NTM species. However, smaller rings like
cycloheptyl, cyclohexyl, and cyclopentyl (11, 12, and 13) are not tolerated for any species. There remains
modest anti-TB activity with the 3-ethyl adamantyl (6) with an MIC of 1.25 μg/mL.
Table 1. Anti-NTM activity of unsubstituted indole-2-carboxamides. MIC values are in µg/mL.
O
R
N
H
HN
5 - 19
M.
abscessus
M.
M.
M.
M.
smeg.
M. tb
massiliense bolletii chelonae
#
R
H37 Rv
(mc2-
6206)
ATCC
19977
DCI
#21
CIP10
8297
CRM001
9
ATCC
14472
ATCC 35752
mc2155
5
6
0.25 0.25 0.5
>32 >32 >32
0.25
>32
0.25
>32
0.25
0.2
1.56
>32
1.25
>20
7
8
16
2
16
2
8
2
8
16
2
2
1
>5
>5
>20
>20
>32
9
>32 >32 >32
>32 >32 >32
32
32
32
32
>32
>32
0.39
0.39
>25
25
10
11
12
13
14
15
>32 >32 >32
>32 >32 >32
>32 >32 >32
>32 >32 >32
>32
>32
>32
>32
16
>32
>32
>32
>32
16
>32
>32
>32
>32
16
>5
>10
>10
>10
5
>20
>25
>25
>25
12.5
>32 >32
16

16
0.12 0.5 0.06 0.06
0.12
<0.06
0.05
0.78
17
18
>32 >32 >32
>32 >32 >32
>32
>32
>32
>32
>32
>32
>10
>10
>25
>25
19
>32 >32 >32
>32
>32
>32
>5
>20
The second series evaluated for pan-antimycobacterial activity was the 4,6-dimethyl indole
substitution and contributes to greater potency against the mycobacterial panel than with the
unsubstituted indole compounds as demonstrated in table 2. The most potent pan-activity is seen with
cyclooctyl (25) and cycloheptyl (26) head groups with MIC values ranging from 0.0039 to 0.625 μg/mL,
depending on the species of Mycobacterium. The 4-methylcyclohexyl substituted compounds (29 and
30) also display significant anti-NTM and anti-TB activity with the exception of M. smegmatis. The pure
trans isomer (29) and cis/trans mixture (30) achieved the same MIC values showing no preference for
stereochemical orientation at the 4-position. The dimethyl indole series allow for smaller aliphatic
substituents, presumably through the maintaining of lipophilicity from the dimethyl groups. Aliphatic
rings smaller than cyclooctyl confer anti-mycobacterial activity that was not present in the unsubstituted
indoles 11, 12, 13 and 14. Substituents the size of the cyclooctyl (25) or cycloheptyl (26) have activity
against M. abscessus, M. massiliense, M. bolletii, M. chelonae, M. smegmatis, and M. tuberculosis but
begin to lose potency as the ring size decreases, until any clinically relevant activity is lost at a size of a
cyclopentyl (28). Compounds with substitutions larger than adamantyl (20, 21, 22, 23) lose all MABSC
NTM activity. Comparable to the unsubstituted indoles of table 1, activity against M. tuberculosis in the
dimethyl series is less restrictive to substituent size than that of NTM species. The majority of these
compounds (20, 21, 22, 23, 24, 25, 26, 27, 29, 30, and 31) exhibit anti-tuberculous activity with MIC
values ranging from 0.00195 to 0.625 μg/mL.
Table 2. Anti-NTM activity of substituted 4, 6-dimethylindole-2-carboxamides. MIC values are in µg/mL.
M.
M.
M.
smeg.
M. abscessus
M. massiliense
M. tb
bolletii chelonae
#
Structure
ATCC
CIP1082
CRM0019
97
ATCC
14472
ATCC
35752
H37 Rv
(mc2-6206)
DCI #21
19977
mc2155

1.25-
2.50
20
21
>32
>32
32
>32
>32
>32
>32
>32
>32
>32
>32
0.0195
0.039
>32
>20
>20
22
23
16
>32
8
8
16
16
0.31
0.16
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>20
>20
24
25
>32
0.04
0.313-
0.625
0.063 0.0078 0.031 0.0078 0.0039
0.063
0.0195
26
27
28
29
30
≤0.06 ≤0.06 ≤0.06 ≤0.06
≤0.06
0.25
2
≤0.06
0.25
2
0.08
0.625
2.5
0.625
2.5
0.5
4
0.25
4
0.25
8
0.25
8
5
≤0.06 ≤0.06 ≤0.06 ≤0.06
≤0.06 ≤0.06 ≤0.06 ≤0.06
≤0.06
≤0.06
≤0.06
≤0.06
0.31
0.31
>20
>20
1.25-
2.50
31
0.125 0.125 0.125 0.125
0.125
0.125
0.0195
32
33
>32
>32
>32
>32
>32
>32
>32
>32
32
>32
>32
>5
5
2.50-
5.00
>32
>20
34
>32
>32
>32
>32
>32
>32
>5
>20
Regarding the unsubstituted ICs, only two compounds demonstrate sub-µg/mL pan-activity
potencies compared to six total dimethyl ICs. Furthermore, preference for bulky aliphatic rings is shown,
since potency is lost with decreasing size in both series, albeit more drastically in the unsubstituted
series. It is clearly seen that the 4, 6-dimethylated indole is preferred due to the acquisition of previously
absent anti-mycobacterial activity (25, 26-29), which is most likely due to the boost in lipophilicity from
the two methyl groups. Supporting this observation is the attainment of anti-NTM activity for smaller
saturated cycloalkyl substitutions (table 2, compounds 25, 26, 27, 29, and 30) when the indole ring is
dimethylated. The anti-NTM activity seen with adamantyl (table 1, 5) in the unsubstituted ICs is
abolished when the indole is 4,6-dimethylated (table 2, 20), however, this is not the case for the

isopinocampheyl substitution in either series (table 1, 16 and table 2, 31). It seems when this minimum
liphophilic threshold is not met, such as with the aliphatic rings smaller than an 8-carbon ring size in the
unsubstituted series (table 1, 10, 11, 12, and 13), the compounds suffer a loss of activity. Lastly, no
mycobacterial activity is observed in either series for uncyclized straight alkyl chains (table 1,
compounds 17, 18; table 2, compounds 32, 33).
To further evaluate the spectrum of anti-mycobacterial activity of the IC compounds, we also
tested these compounds against MAC strains including M. avium and M. xenopi (table 3). Compounds
that were not effective anti-mycobacterial agents were not tested. Overall, current IC compounds are
more active against M. xenopi over M. avium. The best potential candidate for pan-activity against
mycobacteria in the cyclooctyl substituted 4,6-dimethyl-indole (25), which also showed the most potent
activity in this panel with MIC values ranging from 0.25-1 μg/mL. Compounds 26 and 31 also showed
good activity against M. avium in addition to M. xenopi with MIC values between 2-4 μg/mL.
Table 3. Anti-NTM activity of substituted 3,5-dimethylindole-2-carboxamides. MIC values are in µg/mL.
M. avium
#
M. xenopi WT
Intracellulare
1956
104
5
6
7
8
8
>32
>32
>32
>32
>32
>32
8
8
>32
32
0.5-1.0
1.0-2.0
8
>32
>32
>32
>32
8
>32
>32
>32
>32
>32
1
4
2
9
10
15
16
20
21
22
23
24
25
26
27
28
29
30
31
32
33
2
16
0.5-1.0
>32
>32
>32
>32
>32
>32
>32
0.25-0.05
2
>32
1
0.5-1.0
0.25
0.06-0.12
1.0-2.0
8-16
0.25-1
0.5-1
0.25-1
>32
4
>32
>32
>32
>32
2
>32
>32
>32
>32
2
>32
>32
>32
>32
>32

2.4 - Indolecarboxamides are inactive against S. aureus and P. aeruginosa. Lead compounds in
this IC series appear to be selective for mycobacteria, as IC compounds 5, 16, 25, 26, 30 and 31 were
inactive against P. aeruginosa and S. aureus (table 4). All compounds tested showed no bacterial growth
inhibition up to 160 μg/mL except for 16, which inhibited S. aureus growth at 40 μg/mL.
Table 4. MIC values against P. aeruginosa and S. aureus
MIC (µg/mL)
Compound #
P. aeruginosa PAO-1
S. aureus ATCC25923
5
>160
>160
>160
>160
>160
>160
>160
40
>160
>160
>160
>160
16
25
26
30
31
2.5 - Indolecarboxamides are potent bactericidal agents. Minimum bactericidal concentration
(MBC) values were determined for IC compounds 16, 25, 26 and 30. Our results show that for these
tested IC compounds, MBC90 values were highly potent and similar to determined MIC values. MBC90
values for IC 16, 25, 26 and 30 are 0.06, 0.015, 0.015 and 0.03 μg/mL, respectively.
2.6 - In vitro toxicity characterization. The cytotoxicity of compounds 5, 16, 25, and 31 was
tested against THP-1 cells, a human cell line (Table 5). With the exception of Compound 16 (TD50 of 11.3
µg/mL), the ICs were found not to be cytotoxic up to 50 µM. Across several Mycobacterium species,
these compounds have exceptional selectivity indices, ranging from 22.6 to at least 3,800. Compound
25, the most potent of the scaffold, shows the most promise against NTM, with selectivity indices of
>1,910 for M. abscessus, M. massiliense, and M. bolleti, while compound 31 holds the most selectivity
against M. tuberculosis.
Table 5. Cytotoxicity profiles of selected indole-carboxamides against THP-1 cells.
Selectivity Indices (TD50/MIC)
M. abscessus M. massiliense
CRM0019
M. chelonae M. tb
H37Rv
Compound TD50 (μg/mL)
M. bolletii
>58
92
>3820
>130
DCI #21
ATCC14472
5
>14.7
11.3
>58
22.6
>58
188
>58
>73
226
16
25
31
>188
>236
>130
>14.9
>16.2
>1910
>130
>1910
>130
>764
>830
2.7 - Indolecarboxamides inhibit the transfer of mycolic acids to their cell envelope acceptors in
M. abscessus complex strains. IC compounds, including compound 5, have been reported to target the

M. tuberculosis trehalose monomycolate (TMM) transporter, MmpL3, resulting in an inhibition of the
transfer of mycolic acids to their cell envelope acceptors, arabinogalactan and trehalose dimycolates
(TDM).29, 31, 36 To determine whether the NTM inhibitors identified herein displayed a similar activity on
M. abscessus complex strains, cultures of M. massiliense strain 1513 treated with different
concentrations of compounds 5, 16, 25 and 31 were metabolically labeled with [1,2-14C]acetate and
their lipid and cell wall-bound mycolic acid contents analyzed by TLC. Inhibitor treatments resulted in a
concentration-dependent build-up of TMM in the cells that accompanied a decrease in mycolic acid
transfer onto cell wall arabinogalactan and TDM (figure 1). Inhibitor concentration-dependence was less
marked in the case of compound 5. Thus, similar to the situation in M. tuberculosis, the IC under study
herein do not inhibit mycolic acid biosynthesis per se but rather the transfer of these products to their
cell envelope acceptors. It is likely that the killing of M. abscessus complex strains that ensues results
from the inhibition of the formation of their outer membrane.

Figure 1. Effect of indolecarboxamides on the transfer of mycolic acids to their cell envelope acceptors.
M. massiliense strain 1513 were either untreated or treated with compound 5 (4 µg/ml [4 x MIC] or 10
µg/ml [10 x MIC]), 16 (2 µg/ml [4 x MIC] or 5 µg/ml [10 x MIC]), 31 (2 µg/ml [4 x MIC] or 5 µg/ml [10 x
MIC]) or 25 (0.5 µg/ml [4 x MIC] or 1.25 µg/ml [10 x MIC]) and labeled with [1,2-14C]acetate as described
under the experimental section. Panel (A): Analyses of lipids from untreated and treated cells. The same
volume of [14C]-labeled lipids from bacterial cells either treated or untreated were analyzed by TLC in
the solvent system [CHCl3:CH3OH:H2O, 20:4:0.5] and revealed by phosphorimaging. TMM/TDM ratios,
expressed relative to the value measured in the untreated control (arbitrarily set to 1), are presented as
histograms. PE, phosphatidylethanolamine; CL, cardiolipin; GPLs, glycopeptidolipids; FM, free mycolates.
Panel (B): Analyses of cell wall-bound mycolic acid methyl esters (MAMEs) prepared from the same
untreated and treated cells as in (A). TLC plates were developed thrice in the solvent system [n-
hexane:ethyl acetate, 95:5] and revealed by phosphorimaging. The amount of radioactivity incorporated
in MAMEs was semi-quantified using a PhosphorImager and the results, expressed as a percentage of
the value measured in the untreated control, are presented as histograms.

3.1 - Conclusions
We have expanded the spectrum of activity of the IC class of antituberculars to include a panel
of NTM species, including the most prevalent NTM strains MABSC and MAC. Against MABSC and M.
tuberculosis, the IC class was found to be subdivided into three categories; 1) pan-active against the M.
abscessus complex and M. tuberculosis; 2) pan-inactive against the M. abscessus complex and M.
tuberculosis; 3) solely active against M. tuberculosis. M. tuberculosis was generally more susceptible to
the IC class over the M. abscessus complex. Eight IC compounds showed sub-μg/mL potency against the
M. abscessus complex. In addition, IC compounds were generally inactive against M. avium, however
compound 25 also showed sub-µg/mL potency against M. avium. As seen in previous reports and our
current data, this IC series has a safe pharmacological profile and are nontoxic against a human cell line
with large selectivity indexes. The mechanism of action for NTM inhibition against M. massiliense
suggests similar macromolecular activity to antitubercular MmpL3 inhibition, which is consistent with
mycolic acid transport inhibition. The mechanism of action seems to be specific to mycobacteria as lead
IC compounds were generally inactive against gram-positive and gram–negative bacteria. Recently,
compound 25 was shown to achieve in vivo efficacy in TB infected mouse models, demonstrating its
excellent pharmacokinetics/pharmacodynamics and is especially intriguing for evaluation in NTM mouse
models.29 Altogether, compounds 5, 16, 25 and 31 are promising lead candidates for treatment of
MABSC and MAC strains and future preclinical studies are warranted.

4.1 - Experimental
4.2 - Materials and instrumentation. All chemicals and solvents were purchased from commercial
sources. The chemical reactions were tracked by TLC using Biotage Silica Gel 60F254 plates and spots
were visualized by UV lamp or I2 condensation. Compounds were purified using a Biotage SP silica gel
1
column on a Biotage Isolera One. H and 13C NMR were recorded on a 400 MHz Bruker NMR and
chemical shifts were reported relative to solvent peak. Analytical RP-HPLC was determined on a Waters
Acquity UPLC system equipped with an Acquity BEH C18 column (1.7 µm), flow rate of 0.5 mL/min and a
gradient of solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid): 0-
0.25 min 97% A; 0.25-3.0 min 3-100% B (linear gradient); 3.0-4.5 min 100% B; 4.5-4.75 min 0-97% A
(linear gradient); 4.75-5.0 min 97% A. UV absorbance at 254 nm was used as the detection method. All
compounds were found to have >95% purity with the described analytical methods.
4.3 - Methyl 4,6-H-indole-2-carboxylate (2). Compound 2 was synthesized according to the published
protocol of Onajole et al.30 1.75 mg (27.7%) of tan powder; 1H NMR (DMSO-d6) δ = 1.32 (3H, t, J = 8 Hz),
2.34 (3H, s), 2.43 (3H, s), 4.30 (2H, q, J = 8 Hz), 6.69 (1H, s), 7.03 (1H, s), 7.10 (1H, s), 11.67 (1H, s); 13C
NMR (DMSO-d6) δ = 14.31, 18.24, 21.52, 60.17, 106.41, 109.53, 122.17, 124.96, 126.04, 130.73, 134.21,
137.69, 161.35; ESI-MS: [M+H]+ calculated for C13H16NO2: 218.1, found: 218.1.
4.4 - 4,6-H-indole-2-carboxylate (4). Compound 4 was synthesized according to the published protocol
of Onajole et al.30 438 mg (>99%) of white powder; 1H NMR (DMSO-d6) δ = 2.34 (3H, s), 2.42 (3H, s), 6.68
(1H, s), 7.02 (1H, s), 7.04 (1H, s), 11.54, (1H, s), 12.74, (1H, s); 13C NMR (DMSO-d6) δ = 18.26, 21.51,
106.03, 109.49, 121.96, 125.07, 127.10, 130.56, 133.79, 137.53, 162.83: [M+H]+ calculated for
C11H10NO2: 188.1, found: 187.8.
4.5 - General synthetic method for unsubstituted indole-2-caboxamide series. Indole-2-carboxamide
(1.5 eq), DCC (1.2 eq), DMAP (0.1 eq) and amine (1.0 eq) were all dissolved in anhydrous CH2Cl2 (10
mL/1.0 mmol amine) and stirred at room temperature for 24 hours. The evolved ppt was filtered and
the filtrate was removed under reduced pressure. The residue was purified by normal phase flash
column chromatography using a hexane to ethyl acetate gradient.
4.5.1 - N-(1-adamantyl)-1H-indole-2-carboxamide (5). 197 mg (33.5%) of white powder; 1H NMR
(CDCl3) δ = 1.75 (6H, br), 2.18 (9H, br), 5.92 (1H, s), 6.76 (1H, s), 7.12 (1H, t, J = 8 Hz), 7.25 (1H, t, J = 8
Hz), 7.50 (1H, d, J = 8 Hz), 7.61 (1H, s, 8 Hz) 9.99 (1H, s); 13C NMR (CDCl3) δ = 29.52, 36.36, 41.86, 52.50,

101.18, 112.14, 120.42, 121.69, 124.04, 127.68, 131.93, 136.41, 160.93; ESI-MS: [M+H]+ calculated for
C19H23N2O: 295.2, found: 295.0.
4.5.2 - N-(3-ethyl-1-adamantyl)-1H-indole-2-carboxamide (6). 189 mg (29.3%) of white powder; 1H
NMR (CDCl3) δ = 0.83 (3H, t, J = 8 Hz), 1.21 (2H, q, J = 8 Hz), 1.44-1.52 (4H, m), 1.60-1.73 (2H, m), 1.88
(2H, s), 2.07-2.15 (4H, m), 2.21 (2H, s), 5.92 (1H, s), 6.75 (1H, s), 7.12 (1H, t, J = 8 Hz), 7.26 (1H, t, J = 8
Hz), 7.50 (1H, d, J = 8 Hz), 7.61 (1H, d, J = 8 Hz), 9.94 (1H, s); 13C NMR (CDCl3) δ = 7.05, 29.70, 34.61,
35.85, 35.95, 40.76, 41.46, 46.08, 53.37, 101.14, 112.06, 120.45, 121.70, 124.08, 127.68, 131.86, 136.27,
160.86; ESI-MS: [M+H]+ calculated for C21H27N2O: 323.2, found: 323.2.
4.5.3 - N-(1-(1-adamantyl)ethyl)-1H-indole-2-carboxamide (7). 198 mg (30.7%) of white powder; 1H
NMR (CDCl3) δ = 1.17 (3H, d, J = 4 Hz), 1.58-1.74 (12H, m), 2.01 (3H, s), 4.03 (1H, t, J = 8 Hz), 6.13 (1H, d, J
= 8 Hz), 6.86 (1H, s), 7.12 (1H, t, J = 8 Hz), 7.26 (1H, t, J = 8 Hz), 7.45 (1H, d, J = 8 Hz), 7.62 (1H, d, J = 8
Hz), 10.26 (1H, s); 13C NMR (CDCl3) δ = 14.66, 28.29, 36.19, 37.01, 38.49, 53.18, 101.15, 111.95, 120.63,
121.74, 124.34, 127.63, 131.10, 136.18, 161.03; ESI-MS: [M+H]+ calculated for C21H27N2O: 323.4, found:
323.3.
4.5.4 - N-(1-(1-adamantyl)methyl)-1H-indole-2-carboxamide (8). 266 mg (43.1%) of white powder; 1H
NMR (CDCl3) δ = 1.17 (3H, d, J = 4 Hz), 1.58-1.74 (13H, m), 4.03 (1H, quin, J = 8 Hz), 6.13 (1H, d, J = 4 Hz),
6.86 (1H, s), 7.12 (1H, t, J = 8 Hz), 7.26 (1H, t, J = 8 Hz), 7.45 (1H, d, J = 8 Hz), 7.62 (1H, d, J = 8 Hz), 10.26
(1H, s); 13C NMR (CDCl3) δ = 28.19, 34.13, 36.88, 40.27, 50.98, 101.32, 111.90, 120.67, 121.80, 124.42,
129.51, 130.90, 136.12, 161.68; ESI-MS: [M+H]+ calculated for C20H25N2O: 309.4, found: 309.3
4.5.5 - N-(2-adamantyl)-1H-indole-2-carboxamide (9). 202 mg (34.3%) of white powder; 1H NMR
(CDCl3) δ = 1.72 (1H, s), 1.75 (1H, s), 1.80 (1H, s), 1.88 (1H, s), 1.92 (8H, s), 2.09 (2H, s), 4.29 (1H, d, J = 8
Hz), 6.50 (1H, d, J = 8 Hz), 6.86 (1H, s), 7.14 (1H, t, J = 8 Hz), 7.28 (1H, t, J = 8 Hz), 7.44 (1H, d, J = 8 Hz),
7.64 (1H, d, J = 8 Hz), 9.54 (1H, s); 13C NMR (CDCl3) δ = 27.23, 32.04, 37.13, 27.50, 53.54, 101.33, 111.99,
120.62, 121.78, 124.34, 127.69, 131.23, 136.26, 160.76; ESI-MS: [M+H]+ calculated for C19H23N2O: 295.4,
found: 295.3.
4.5.6 - N-cyclooctyl-1H-indole-2-carboxamide (10). 233 mg (43.1%) of white powder; 1H NMR (CDCl3) δ
= 1.62-1.72 (13H, m), 1.96-2.01 (2H, m), 4.25 (1H, sep, J = 4 Hz), 6.10 (1H, d, J = 8 Hz), 6.80 (1H, s), 7.14
(1H, t, J = 4 Hz), 7.28 (1H, t, J = 4 Hz), 7.44 (1H, d, J = 8 Hz), 7.63 (1H, d, J = 8 Hz), 9.49 (1H, s); 13C NMR
(CDCl3) δ = 23.73, 27.19, 32.46, 49.71, 101.33, 111.96, 120.59, 121.80, 124.31, 127.69, 131.18, 136.21,
160.4; ESI-MS: [M+H]+ calculated for C17H24N2O: 271.4, found: 271.4.

4.5.7 - N-cycloheptyl-1H-indole-2-carboxamide (11). 231 mg (45.1%) of white powder; 1H NMR (CDCl3)
δ = 1.57-1.69 (8H, m), 1.71-1.94 (2H, m), 2.04-2.08 (2H, m), 4.19 (1H, quin, , J = 4 Hz), 6.09 (1H, d, J = 8
Hz), 6.80 (1H, s), 7.14 (1H, t, J = 4 Hz), 7.28 (1H, t, J = 8 Hz), 7.43 (1H, d, J = 8 Hz), 7.63 (1H, d, J = 8 Hz),
9.41 (1H, s); 13C NMR (CDCl3) δ = 24.14, 23.04, 31.05, 50.78, 101.36, 111.71, 111.94, 120.61, 121.82,
124.34, 127.69, 131.15, 136.19, 160.46; ESI-MS: [M+H]+ calculated for C16H21N2O: 257.3, found: 257.2.
4.5.8 - N-cyclohexyl-1H-indole-2-carboxamide (12). 233 mg (48.1%) of white powder; 1H NMR (CDCl3) δ
= 1.11 (1H, quin, J = 8 Hz), 1.18-1.47 (5H, m), 1.66 (1H, s), 1.76-1.95 (4H, m), 2.05 (1H, d, J = 8 Hz), 6.00
(1H, d, J = 8 Hz), 7.14 (1H, t, J = 8 Hz), 7.28 (1H, t, J = 8 Hz), 7.43 (1H, d, J = 8 Hz), 7.63 (1H, d, J = 8 Hz),
9.29 (1H, s); 13C NMR (CDCl3) δ = 24.60, 24.92, 25.54, 32.44, 33.32, 48.54, 101.39, 106.48, 111.89,
120.63, 121.85, 124.38, 127.71, 136.13, 160.64; ESI-MS: [M+H]+ calculated for C15H19N2O: 243.3,
found: 243.3.
4.5.9 - N-cyclopentyl-1H-indole-2-carboxamide (13). 277 mg (60.7%) of white powder; 1H NMR (CDCl3)
δ = 1.54 (2H, quin, J = 4 Hz), 1.66-1.77 (4H, m), 2.11 (2H, sex, J = 4 Hz), 4.43 (1H, sex, J = 4 Hz), 6.09 (1H,
d, J = 4 Hz), 6.80 (1H, s), 7.13 (1H, t, J = 8 Hz), 7.28 (1H, t, J = 8 Hz), 7.43 (1H, d, J = 8 Hz), 7.63 (1H, d, J = 8
Hz), 9.41 (1H, s); 13C NMR (CDCl3) δ = 23.81, 26.16, 33.32, 51.49, 101.47, 111.94, 120.62, 121.85, 124.38,
127.70, 131.01, 136.19, 161.22; ESI-MS: [M+H]+ calculated for C14H17N2O: 229.3, found: 229.2.
4.5.10 - N-(4-methylcyclohexyl)-1H-indole-2-carboxamide pure trans isomer (14). 277 mg (54.0%) of
white powder; 1H NMR (CDCl3) δ = 1.04-1.15 (4H, m), 1.33 (3H, q, J = 8 Hz), 1.56-1.67 (3H, m), 1.87 (5H,
d, J = 4 Hz), 3.67-3.74 (1H, m), 4.39 (1H, s), 6.47 (1H, s), 7.02 (1H, s), 7.10 (1H, t, J = 8 Hz), 7.62 (1H, d, J =
8 Hz), 9.31 (1H, s); 13C NMR (CDCl3) δ = 24.66, 25.37, 25.99, 32.43, 50.21, 106.53, 111.72, 120.70, 122.37,
129.50, 136.14, 154.03; ESI-MS: [M+H]+ calculated for C16H21N2O: 257.3, found: 257.3.
4.5.11 - N-(4-methylcyclohexyl-1H-indole-2-carboxamide cis/trans mix (15). 280 mg (54.6%) of white
powder; 1H NMR (CDCl3) δ = 0.97 (3H, d, J = 4 Hz), 1.23-1.29 (2H, m), 1.64-1.85 (7H, m), 4.27 (1H, s), 6.27
(1H, d, J = 8 Hz), 6.85 (1H, s), 7.14 (1H, t, J = 8 Hz), 7.28 (1H, t, J = 8 Hz), 7.44 (1H, d, J = 8 Hz), 7.64 (1H, d,
J = 8 Hz), 9.77 (1H, s); 13C NMR (CDCl3) δ = 21.13, 26.15, 29.41, 30.09, 30.48, 33.86, 45.80, 101.42,
112.05, 120.58, 121.77, 124.31, 127.66, 136.36, 160.96; ESI-MS: [M+H]+ calculated for C16H21N2O: 257.3,
found: 256.9.
4.5.12 - N-((1R,2R,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-yl)-1H-indole-2-carboxamide (16). 378
mg (63.8%) of white powder; 1H NMR (CDCl3) δ = 0.94 (1H, d, J = 8 Hz), 1.13 (3H, s), 1.19 (3H, d, J = 8 Hz),
1.27 (3H, s), 1.66-1.71 (1H, m), 1.88-1.98 (2H, m), 2.00-2.04 (1H, m), 2.45-2.51 (1H, m), 2.70-2.78 (1H,

m), 4.55-4.63 (1H, m), 6.11 (1H, d, J = 8 Hz), 6.86 (1H, d, J = 4 Hz), 7.13 (1H, t, J = 8 Hz), 7.27 (1H, t, J = 8
Hz), 7.47 (1H, d, J = 8 Hz), 7.63 (1H, d, J = 8 Hz), 9.95 (1H, s); 13C NMR (CDCl3) δ = 20.83, 23.38, 28.05,
35.48, 38.50, 41.64, 46.49, 47.85, 48.21, 101.43, 112.12, 120.53, 121.77, 124.25, 127.65, 131.07, 136.45,
161.25; ESI-MS: [M+H]+ calculated for C19H25N2O: 297.4, found: 297.3.
4.5.13 - N-heptyl-1H-indole-2-carboxamide (17). 354 mg (68.5%) of white powder; 1H NMR (CDCl3) δ =
0.89 (3H, t, J = 8 Hz), 1.28-1.41 (8H, m), 1.65 (2H, quin, J = 8 Hz), 3.49 (2H, q, J = 8 Hz), 6.19 (1H, s), 6.82
(1H, s), 7.14 (1H, t, J = 8 Hz), 7.28 (1H, t, J = 8 Hz), 7.43 (1H, d, J = 8 Hz), 7.63 (1H, d, J = 8 Hz), 9.56 (1H, s);
13C NMR (CDCl3) δ = 14.06, 22.60, 26.95, 28.99, 29.80, 31.76, 39.79, 101.51, 112.00, 120.61, 121.83,
124.38, 127.67, 130.91, 136.26, 161.63; ESI-MS: [M+H]+ calculated for C16H23N2O: 259.4, found: 259.1.
4.5.14 - N-octyl-1H-indole-2-carboxamide (18). 267 mg (49.0%) of white powder; 1H NMR (CDCl3) δ =
0.88 (3H, t, J = 8 Hz), 1.28- 1.39 (10H, m), 1.64 (2H, quin, J = 8 Hz), 3.48 (2H, q, J = 8 Hz), 6.17 (1H, s), 6.81
(1H, s), 7.14 (1H, t, J = 4 Hz), 7.28 (1H, t, J = 8 Hz), 7.43 (1H, d, J = 8 Hz), 7.63 (1H, d, J = 8 Hz), 9.46 (1H, s);
13C NMR (CDCl3) δ = 14.09, 22.65, 26.99, 29.22, 29.79, 31.80, 39.79, 101.49, 111.97, 120.64, 121.84,
124.40, 127.88, 130.91, 136.22, 161.58; ESI-MS: [M+H]+ calculated for C17H25N2O: 273.3, found: 273.3.
4.5.15 - N-(3-chlorobenzyl)-1H-indole-2-carboxamide (19). 454 mg (79.7%) of white powder; 1H NMR
(CDCl3) δ = 2.97 (2H, s), 4.61 (2H, s), 6.99 (1H, s), 7.12 (1H, t, J = 4 Hz), 7.24-7.31 (4H, m), 7.35 (1H, s),
7.42 (1H, d, J = 12 Hz), 7.63 (1H, d, J = 8 Hz); 13C NMR (CDCl3) δ = 42.90, 48.92, 49.35, 49.57, 49.78,
103.21, 112.12, 120.60, 121.97, 124.61, 125.82, 127.71, 130.05, 134.59, 140.41, 162.27; ESI-MS: [M+H]+
calculated for C16H14ClN2O: 285.7, found: 285.0.
4.5.16 - N-1-adamantyl-4,6-dimethyl-1H-indole-2-carboxamide (20). 54 mg (31.0%) of white powder;
1H NMR (CDCl3) δ = 1.75 (6H, s), 2.16 (9H, s), 2.42 (3H, s), 2.51 (3H, s), 5.85 (1H, s), 6.71 (1H, s), 6.77 (1H,
s), 7.08 (1H, s), 9.23 (1H, s); 13C NMR (CDCl3) δ = 18.62, 21.62, 29.52, 36.36, 41.90, 52.39, 99.67, 109.18,
122.67, 130.70, 130.86, 134.42, 136.39, 160.90; ESI-MS: [M+H]+ calculated for C21H27N2O: 323.4, found:
323.3.
4.5.17 - N-(3-ethyl-1-adamantyl)-4,6-dimethyl-1H-indole-2-carboxamide (21). 61 mg (32.2%) of white
powder; 1H NMR (CDCl3) δ = 0.81 (3H, t, J = 8 Hz), 1.20 (3H, q, J = 8 Hz), 1.46 (4H, s), 1.56 (1H, s), 1.84
(1H, s), 2.06 (4H, q, J = 12 Hz), 2.20 (3H, s), 2.42 (3H, s), 2.51 (3H, s), 5.85 (1H, s), 6.70 (1H, s), 6.77 (1H, s),
7.05 (1H, s), 9.02 (1H, s); 13C NMR (CDCl3) δ = 7.05, 18.61, 21.62, 35.86, 40.79, 41.52, 46.23, 53.28, 99.68,
109.11, 122.72, 130.39, 134.49, 136.31, 160.89; ESI-MS: [M+H]+ calculated for C23H31N2O: 351.5, found:
351.7.

4.5.18 - N-(1-(1-adamantyl)ethyl)-4,6-dimethyl-1H-indole-2-carboxamide (22). 55 mg (29.1%) of white
powder; 1H NMR (CDCl3) δ = 1.16 (3H, d, J = 4 Hz), 1.57-1.74 (14H, m), 2.02 (3H, s), 2.42 (3H, s), 2.53 (3H,
s), 3.94-4.02 (1H, m), 5.98 (1H, d, J = 8 Hz), 6.78 (2H, s), 7.06 (1H, s), 9.33 (1H, s); 13C NMR (CDCl3) δ =
14.69, 18.64, 21.83, 28.3036.21, 37.01, 38.50, 53.08, 99.65, 109.17, 122.77, 125.65, 129.85, 130.86,
134.59, 136.47, 161.19; ESI-MS: [M+H]+ calculated for C23H31N2O: 351.5, found: 351.2.
4.5.19 - N-(1-(1-adamantyl)methyl)-4,6-dimethyl-1H-indole-2-carboxamide (23). 62 mg (34.1%) of
white powder; 1H NMR (CDCl3) δ = 1.60 (6H, s), 1.64 (1H, s), 1.67 (2H, s), 1.72 (2H, s), 1.75 (1H, s), 2.01
(3H, s), 2.42 (3H, s), 2.52 (3H, s), 3.19 (2H, d, J = 8 Hz), 6.24 (1H, s), 6.77 (1H, s), 6.81 (1H, s), 7.07 (1H, s),
9.53 (1H, s); 13C NMR (CDCl3) δ = 18.62, 21.83, 28.22, 34.15, 36.90, 40.28, 50.97, 99.89, 109.18, 122.77,
125.67, 129.68, 130.90, 134.66, 136.49, 161.93; ESI-MS: [M+H]+ calculated for C22H29N2O: 337.4, found:
337.3.
4.5.20 - N-2-adamantyl-4,6-dimethyl-1H-indole-2-carboxamide (24). 83 mg (47.7%) of white powder;
1H NMR (CDCl3) δ = 1.72 (1H, s), 1.75 (1H, s), 1.80 (2H, s), 1.93 (8H, s), 2.52 (4H, s), 2.52 (4H, s), 4.34 (1H,
d, J = 8 Hz), 6.51 (1H, d, J = 8 Hz), 6.76 (2H, d, J = 20 Hz), 7.09 (1H, s), 10.01 (1H, s); 13C NMR (CDCl3) δ =
18.58, 18.66, 21.87, 27.28, 32.07, 37.23, 37.53, 53.66, 99.90, 109.37, 109.46, 110.91, 122.65, 123.53,
130.79, 134.42, 136.88, 137.44, 138.86, 157.48, 161.21; ESI-MS: [M+H]+ calculated for C21H27N2O: 323.4,
found: 323.3.
4.5.21 - N-cyclooctyl-4,6-dimethyl-1H-indole-2-carboxamide (25). 56 mg (34.8%) of white powder; 1H
NMR (CDCl3) δ = 1.57-1.75 (14H, m), 1.93-2.01 (2H, m), 2.42 (3H, s), 2.51 (3H, s), 4.21-4.30 (1H, m), 6.09
(1H, d, J = 8 Hz), 6.77 (2H, s), 7.07 (1H, s), 9.44 (1H, s); 13C NMR (CDCl3) δ = 18.62, 21.83, 23.79, 27.19,
32.55, 49.63, 99.92, 109.23, 122.71, 125.71, 130.88, 134.53, 136.55, 160.65; ESI-MS: [M+H]+ calculated
for C19H27N2O: 299.4, found: 299.0.
4.5.22 - N-cycloheptyl-4,6-dimethyl-1H-indole-2-carboxamide (26). 65 mg (42.3%) of white powder; 1H
NMR (CDCl3) δ = 1.56-1.59 (6H, m), 1.61- 1.73 (4H, m), 2.04-2.09 (2H, m), 2.42 (3H, s), 2.51 (3H, s), 4.19
(1H, s), 6.06 (1H, d, J = 8 Hz), 6.77 (2H, s), 7.06 (1H, s), 9.25 (1H, s); 13C NMR (CDCl3) δ = 18.62, 21.84,
24.17, 28.06, 31.03, 35.32, 50.68, 99.90, 109.17, 122.75, 125.72, 130.92, 134.59, 136.46, 160.61; ESI-MS:
[M+H]+ calculated for C18H25N2O: 285.4, found: 285.3.
4.5.23 - N-cyclohexyl-4,6-dimethyl-1H-indole-2-carboxamide (27). 69 mg (47.3%) of white powder; 1H
NMR (CDCl3) δ = 0.83-0.90 (1H, m), 1.21-1.32 (6H, m), 1.56 (2H, s), 1.75-1.80 (2H, m), 1.91 (2H, m), 2.03
(1H, d, J = 16 Hz), 2.43 (2H, s), 2.48 (2H, s), 6.77 (2H, d, J = 4 Hz), 7.05 (1H, s), 9.01 (1H, s); 13C NMR

(CDCl3) δ = 18.62, 24.95, 25.57, 33.38, 34.94, 48.44, 99.94, 109.09, 122.80, 130.98, 134.68; ESI-MS:
[M+H]+ calculated for C17H23N2O: 271.3, found: 271.2.
4.5.24 - N-cyclopentyl-4,6-dimethyl-1H-indole-2-carboxamide (28). 72 mg (52.0%) of white powder; 1H
NMR (CDCl3) δ = 1.55 (2H, quin, J = 8 Hz), 1.63-1.79 (4H, m), 2.08-2.12 (2H, m), 2.42 (3H, s), 2.50 (3H, s),
4.44 (1H, q, J = 8 Hz), 6.11 (1H, d, J = 8 Hz), 6.77-6.79 (2H, m), 7.05 (1H, s), 9.39 (1H, s); 13C NMR (CDCl3) δ
= 18.61, 21.85, 33.32, 51.43, 100.11, 109.23, 122.72, 125.71, 134.60, 136.55, 161.46; ESI-MS: [M+H]+
calculated for C16H21N2O: 257.3, found: 257.1.
4.5.25 - N-4S-methylcyclohexyl-4,6-dimethyl-1H-indole-2-carboxamide (29). 77 mg (50.1%) of white
powder; 1H NMR (CDCl3) δ = 0.92 (2H, d, J = 8 Hz), 1.08-1.27 (6H, m), 1.60 (1H, s), 1.75 (1H, d, J = 16 Hz),
1.92 (2H, d, J = 16 Hz), 2.08 (1H, d, J = 12 Hz), 2,42 (3H, s), 2.51 (3H, s), 5.93 (1H, d, J = 8 Hz), 6.77 (2H, s),
7.05 (1H, s), 9.18 (1H, s); 13C NMR (CDCl3) δ = 18.61, 21.84, 22.19, 32.01, 33.88, 34.93, 48.73, 99.98,
109.14, 122.75, 125.73, 130.94, 134.63, 136.43, 160.92; ESI-MS: [M+H]+ calculated for C18H25N2O: 285.4,
found: 285.3.
4.5.26 - N-4-methylcyclohexyl-4,6-dimethyl-1H-indole-2-carboxamide (30). 80 mg (52.1%) of white
powder; 1H NMR (CDCl3) δ = 0.92 (1H, d, J = 4 Hz), 0.97 (1H, d, J = 8 Hz), 1.11- 1.28 (4H, m), 1.65- 1.80
(6H, m), 2.10 (1H, d, J = 12 Hz), 2.42-2.52 (6H, m), 6.02-6.27 (1H, m), 6.76-6.80 (2H, m), 7.06 (1H, s), 9.63
(1H, d, J = 16 Hz); 13C NMR (CDCl3) δ = 18.62, 18.66, 21.95, 22.20, 29.40, 30.14, 33.92, 45.84, 48.82,
99.97, 100.06, 109.30, 122.70, 125.68, 130.67, 136.66, 161.14; ESI-MS: [M+H]+ calculated for C18H25N2O:
285.3, found: 285.3.
4.5.27 - N-((1R,2R,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-yl)-4,6-dimethyl-1H-indole-2-
carboxamide (31). 60 mg (34.2%) of white powder; 1H NMR (CDCl3) δ = 0.96 (1H, d, J = 8 Hz), 1.14 (3H,
s), 1.19 (3H, d, J = 8 Hz), 1.27 (3H, s), 1.66-1.72 (1H, m), 1.88-1.97 (2H, m), 2.03 (1H, s), 2.42 (3H, s), 2.45-
2.50 (1H, m), 2.52 (3H, s), 2.70-2.77 (1H, m), 2.88 (1H, d, J = 28 Hz), 4.57 (1H, t, J = 16 Hz), 6.06 (1H, d, J =
8 Hz), 6.77 (1H, s), 6.82 (1H, s), 7.10 (1H, s), 9.66 (1H, s); 13C NMR (CDCl3) δ = 18.64, 20.86, 21.83, 23.48,
28.05, 37.42, 38.52, 41.66, 46.54, 47.87, 48.10, 99.98, 10933, 122.70, 125.71, 130.86, 134.52, 136.70,
161.34; ESI-MS: [M+H]+ calculated for C21H29N2O: 325.4, found: 325.1.
4.5.28 - N-heptyl-4,6-dimethyl-1H-indole-2-carboxamide (32). 99 mg (64.0%) of white powder; 1H NMR
(CDCl3) δ = 0.88 (3H, t, J = 4 Hz), 1.28- 1.39 (7H, m), 1.64 (3H, quin, J = 8 Hz), 1.92-1.95 (1H, m), 2.42 (2H,
s), 2.47 (1H, s), 2.50 (3H, s), 3.47 (2H, q, J = 8 Hz), 6.19 (1H, s), 6.77 (2H, d, J = 8 Hz), 7.05 (1H, s), 9.38
(1H, s); 13C NMR (CDCl3) δ = 14.07, 18.81, 21.84, 22.61, 26.98, 29.02, 31.76, 39.75, 100.14, 109.21,

122.75, 129.71, 130.93, 134.64, 136.54, 161.79; ESI-MS: [M+H]+ calculated for C18H27N2O: 287.4, found:
287.3.
4.5.29 - N-octyl-4,6-dimethyl-1H-indole-2-carboxamide (33). 101 mg (62.3%) of white powder; 1H NMR
(CDCl3) δ = 0.88 (3H, t, J = 8 Hz), 1.28-1.37 (9H, m), 1.62-1.65 (3H, m), 2.42 (3H, s), 2.50 (3H, s), 3.47 (2H,
q, J = 8 Hz), 6.16 (1H, s), 6.77 (2H, d, J = 4 Hz), 7.05 (1H, s), 9.27 (1H, s); 13C NMR (CDCl3) δ = 14.09, 18.60,
22.65, 27.02, 29.23, 29.85, 31.81, 37.75, 100.13, 109.19, 122.77, 125.73, 130.94, 134.68, 136.52, 161.77;
ESI-MS: [M+H]+ calculated for C19H29N2O: 301.4, found: 301.4.
4.5.30 - N-(3-chlorobenzyl)-4,6-dimethyl-1H-indole-2-carboxamide (34). 103 mg (61.0%) of white
powder; 1H NMR (CDCl3) δ = 1.58 (1H, s), 1.75 (6H, t, J = 12 Hz), 2.16 (8H, s), 2.42 (3H, s), 2.51 (3H, s),
5.85 (1H, s), 6.71 (1H, s), 6.77 (1H, s), 7.08 (1H, s), 9.23 (1H, s); 13C NMR (CDCl3) δ = 18.62, 21.82, 29.52,
36.36, 41.90, 52.39, 99.67, 109.18, 122.67, 130.86, 134,42, 136.39, 160.90; ESI-MS: [M+H]+ calculated
for C18H18ClN2O: 313.7, found: 313.2.
4.6 - Inhibitor susceptibility determinations. MIC values against a number of clinical isolates of
mycobacteria and other bacterial species were determined in 96-well microtiter plates using the
colorimetric resazurin microtiter assay37 and visually scanning for growth. MIC values against M.
tuberculosis H37Rv mc26206 were determined in 7H9-ADC–0.05% tyloxapol medium supplemented with
0.2 % casaminoacids, 48 μg/ml pantothenate and 50 μg/ml L-leucine. MICs against M. smegmatis
mc2155 were determined in 7H9-ADC-0.05% Tween 80. MICs against M. abscessus ATCC 19977, M.
abscessus 21, M. abscessus 103, M. massiliense CIP108297, M. massiliense 1513, M. massiliense CRM-
0019, M. massiliense CRM-0270, M. massiliense 105, M. bolletii ATCC 14472, M. bolletii 88, M. chelonae
ATCC 35752, M. chelonae 28, M. chelonae 49, M. chelonae 69, M. kansasii 662, M. avium 104, M.
intracellulare 1956, M. xenopi 4042, Pseudomonas aeruginosa PAO-1 and Staphylococcus aureus ATCC
25923 were determined in Mueller Hinton II (BD). MBC90 values were determined by subculture from
the MIC wells onto 7H11 agar and defined as the lowest concentration reducing CFU by 90 % relative to
the inoculum.
4.7 - Cytotoxicity determination. The cytotoxicity of compounds was determined by measuring THP-1
cell viability after 3 days in the presence of test compounds. Compounds were prepared as 10-point
serial dilutions in DMSO. The highest concentration of compound tested was 50 µM where compounds
were soluble in DMSO at 10 mM. THP-1 cells were cultured in complete RPMI and differentiated into
macrophage-like cells using 80 nM PMA overnight at 37°C, 5% CO2. Cells were inoculated into assay

plates and cultured for 24h before compound dilutions were added to a final DMSO concentration of
0.5%. Each run included staurosporine as a control. Assay plates were incubated for 3 days at 37°C, 5%
CO2; growth was measured using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) which
uses ATP as an indicator of cell viability. Relative luminescent units (RLU) were measured using a Biotek
Synergy 4 plate reader. The dose response curve was fitted using the Levenberg–Marquardt algorithm.
The IC50 was defined as the compound concentration that resulted in 50% viability.
4.8 - Metabolic labeling and mycolic acid analysis. Cultures of M. massiliense 1513 grown to mid log-
phase in Mueller-Hinton II broth were incubated with different concentrations of inhibitors for 16 hr at
37°C with shaking. 0.5 µCi/ml [1,2-14C]acetic acid (specific activity, 52 Ci/mol, PerkinElmer Inc.) was
added at the same time as the inhibitors. Total lipids and cell wall-bound mycolic acid methyl esters
were prepared from treated and untreated cells and analyzed by TLC as described previously.38 TLCs
were revealed and semi-quantified using a PhosphorImager (Typhoon, GE Healthcare).
5.1 - Acknowledgments. This project was supported by the National Institutes of Health / National
Institute of Allergy and Infectious Diseases research grant AI116525 and the Jack and Lois Wareham
Research Award. The cytotoxicity determination was supported by National Institutes of Health and the
National Institute of Allergy and Infectious Diseases, Contract No. HHSN272201100009I. The content is
solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
The authors would also like to thank the Department of Chemistry at Creighton University for the use of
their NMR and Dr. William Jacobs (Albert Einstein College of Medicine, NY, USA) for the provision of M.
tuberculosis H37Rv mc26206..

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Design, Synthesis and Evaluation of Indole-2-carboxamides
with Pan Anti-mycobacterial Activity
Nicholas D. Franz1, Juan Manuel Belardinelli2, Michael A. Kaminski1, Louis C. Dunn1, Vinicius Calado
Nogueira de Moura2, Michael A. Blaha1, Dan D. Truong1, Wei Li 2, Mary Jackson2, E. Jeffrey North1*
1Department of Pharmacy Sciences, Creighton University, 2500 California Plaza, Omaha, NE 68178, USA
2Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology,
Colorado State University, Fort Collins, CO 80523, USA
Graphical Abstract
M. abscessus
M. Tuberculosis
+
M. massilliense
16
M. bolletii
M. chelonae
M. avium
Non-tuberculous
Mycobacteria
25