P. Hegde et al.
Tuberculosis 129 (2021) 102100
2.1.14. 2-Fluoroisonicotinoic acid hydrazide (11)
(cmꢀ 1) 1562, 1616, 1704.
The title compound was prepared from 2-fluoroisonicotinic acid and
isolated as a white solid in 72% yield. Melting point: 161 ◦C; Rf = 0.71
(95:5 CH2Cl2–MeOH); 1H NMR (DMSO‑d6) δ 8.29 (dd, J = 5.2, 2.2 Hz,
1H), 7.74–7.64 (m, 1H), 7.45 (s, 1H); 13C NMR (DMSO‑d6) δ 165.2,
149.6, 148.4 (d, J = 15.0 Hz), 120.0 (d, J = 4.1 Hz), 107.7, 107.3; HRMS
(EI) calcd for C6H6FN3O [M]+ 155.0489, found 155.0488 (error 1.0
ppm); IR (cmꢀ 1) 1507, 1609, 1668, 2618, 2761, 3159, 3337.
2.1.21. N-(tert-butyloxycarbonyl)piperidine-4-carboxylic acid hydrazide
(18)
The title compound was prepared from methyl N-(tert-butylox-
ycarbonyl)piperidine-4-carboxylate and isolated as a white solid in 87%
yield; 1H NMR (CDCl3) δ 6.81 (d, J = 9.5 Hz, 1H), 4.08 (s, 2H), 3.82 (s,
2H), 2.67 (s, 2H), 2.15 (ddt, J = 11.6, 7.8, 3.9 Hz, 1H), 1.74–1.68 (m,
2H), 1.64–1.54 (m, 2H), 1.38 (s, 9H); 13C NMR (CDCl3) δ 175.2, 154.6,
79.7, 41.6, 28.4; HRMS (ESI) calcd for C11H21N3O3 [M + Na]+
266.1475, found 266.1481 (error 2.4 ppm); IR (cmꢀ 1) 1525, 1629,
1682, 2847, 2933, 2982, 3321.
2.1.15. Furan-2-carboxylic acid hydrazide (12)
The title compound was purchased from Ambeed. Melting point:
79 ◦C; Rf = 0.59 (95:5 CH2Cl2–MeOH); 1H NMR (CDCl3) δ 9.62 (s, 1H),
7.81 (d, J = 1.7 Hz, 1H), 7.08 (d, J = 3.6 Hz, 1H), 6.60 (dd, J = 3.4, 1.7
Hz, 1H), 4.42 (s, 2H); 13C NMR (CDCl3) δ 159.5, 146.6, 144.3, 114.9,
112.1; 1H and 13C NMR matched the reported values [37]; HRMS (EI)
calcd for C5H6N2O2 [M]+ 126.0424, found 126.0420 (error 3.3 ppm); IR
(cmꢀ 1) 1512, 1570, 1592, 1621, 1684, 3024, 3150, 3228, 3312.
2.2. Pathogens and culture conditions for mycobacterial strains
M. abscessus bamboo [39] and M. avium 11 [40] were used as
representative nontuberculous mycobacteria. M. bovis BCG Pasteur
(ATCC 35734) and M. tuberculosis H37Rv (ATCC 27294) were obtained
from the American Type Culture Collection. The other mycobacterial
strains evaluated were M. tuberculosis Euro-American (CDC1551),
M. tuberculosis Beijing (K03b00DS, K07b00DS, K12b00DS, NIH_G1DS,
K04b00DS, K14b00DS, K09b00DS, HN878, K11b00DS, K05b00DS,
0K116), M. tuberculosis Uganda (NIH_G12), M. tuberculosis Haarlem
(NIH_G11R), M. tuberculosis EAI (K5072429), M. tuberculosis West Afri-
can 1 ( NIH_G82, C02), M. africanum, M. tuberculosis Delhi, M. bovis
(AF2122). These other strains were clinical isolates housed in the
tuberculosis research section of the NIAID. Strains were grown in stan-
dard Middlebrook 7H9 broth (BD Difco) supplemented with 0.5% al-
bumin, 0.2% glucose, 0.085% sodium chloride, 0.0003% catalase, 0.2%
glycerol, and 0.05% Tween 80.
2.1.16. 1H-Pyrrole-2-carboxylic acid hydrazide (13)
The title compound was prepared from methyl-1H-pyrrole-2-car-
boxylic acid and isolated as a white solid in 94% yield. Melting point:
227 ◦C; Rf = 0.5 (95:5 CH2Cl2–MeOH); 1H NMR (DMSO‑d6) δ 11.48 (s,
1H), 9.28 (s, 1H), 6.89 (q, J = 2.3 Hz, 1H), 6.81–6.77 (m, 1H), 6.11 (q, J
= 2.7 Hz, 1H), 4.39–4.36 (m, 2H); 13C NMR (DMSO‑d6) δ 161.6, 125.4,
121.6, 109.9, 108.9; HRMS (EI) calcd for C5H7N3O [M]+ 125.0584,
found 125.0585 (error 0.9 ppm); IR (cmꢀ 1): 1561, 1618, 2842, 2927,
3047, 3101, 3202, 3301.
2.1.17. Piperidine-4-carboxylic acid hydrazide (14)
The title compound was prepared through the Boc-deprotection of N-
(tert-butyloxycarbonyl)piperidine-4-carboxylic acid hydrazide (18) (1
mmol) by stirring in 5 mL of (2:2:1) solution of TFA: THF: water at room
temperature for 20 h. The reaction mixture was dried under vacuum and
washed with hexanes and ethyl acetate forming a white solid precipitate
in 85% yield. Melting point: 139 ◦C; Rf = 0.61 (95:5 CH2Cl2–MeOH); 1H
NMR (DMSO‑d6) δ 10.93 (s, 1H), 8.84 (d, J = 191.4 Hz, 2H), 3.31 (d, J =
12.6 Hz, 2H), 2.93 (d, J = 14.0 Hz, 2H), 2.59 (td, J = 11.1, 5.5 Hz, 1H),
1.88 (dd, J = 14.1, 3.7 Hz, 2H), 1.85–1.71 (m, 2H); 13C NMR (DMSO‑d6)
δ 173.0, 42.7, 37.3, 25.2; HRMS (EI) calcd for C6H13N3O [M]+
143.1053, found 143.1045 (error 5.4 ppm); IR (cmꢀ 1) 1528, 1557,
1591, 1671, 1703, 2528, 2735, 2978, 3205.
2.3. MIC determination for mycobacterial strains
The minimum inhibitory concentration (MIC) defined the concen-
tration at which 90% inhibition of observable bacterial growth was
determined by the microdilution method as previously described with
modifications [39,41]. Briefly, 1
μL of a serial two-fold dilution of
compounds in DMSO, were dispensed into flat bottom 96-well plates
(Corning) using a D300e digital dispenser (Tecan). Test compounds
were dissolved in 100% DMSO to 10 mM. To each well, 200 μL of a
2.1.18. N′-Isopropylisonicotinoic acid hydrazide (15)
mid-log-phase bacterial culture (OD600 = 0.05) was dispensed to result
in final concentration points ranging up to 100 M. Culture plates were
The title compound was purchased from Ambeed. Melting point:
172 ◦C; Rf = 0.58 (95:5 CH2Cl2–MeOH); 1H NMR (CDCl3) δ 8.77–8.61
(m, 1H), 7.61–7.45 (m, 1H), 3.18 (hept, J = 6.3 Hz, 1H), 1.06 (d, J = 6.3
Hz, 3H); 13C NMR (CDCl3) δ 165.4, 150.7, 140.1, 120.7, 51.5, 20.8 (1H
and 13C NMR matched the reported values [37]); HRMS (EI) calcd for
C9H14N3O [M + H]+ 180.1131, found 180.1132 (error 0.1 ppm); IR
(cmꢀ 1) 1544, 1596, 1640, 2877, 2933, 2971, 3235, 3302.
μ
sealed using a Breathe-Easy sealing membrane (Fisher Scientific), put in
a humidified airtight container, and incubated for 3 (M. abscessus), 4
(M. avium) or 5 (M. bovis/M. tuberculosis) days at 37 ◦C on an orbital
shaker at 110 rpm. Turbidity/absorbance was read at 600 nm as a
measure of growth inhibition using a Tecan TM Infinite 200 Pro
microplate reader (Tecan). Percent growth was calculated relative to the
cell density in the untreated wells and inhibition curves were plotted
using Graph Pad Prism 9 software. The MIC, the concentration that re-
duces growth by 90% compared to untreated control, was deduced from
the generated dose-response curves. Isoniazid and clarithromycin were
used as controls. The other mycobacterial strains were grown in Mid-
dlebrook 7H9 medium supplemented with 0.5% BSA fraction V, 0.08%
NaCl, 0.2% glucose, 0.2% glycerol and 0.05% Tween 80 (7H9/ADC/Tw)
to an OD650nm of 0.2. The culture was diluted 1000-fold in 7H9/ADC/Tw
and 50 uL dispensed per well in round-bottom clear 96-well plate
2.1.19. Pyridine-4-amide (16)
The title compound was purchased from Ambeed. Melting point:
156 ◦C; Rf = 0.54 (95:5 CH2Cl2–MeOH); 1H NMR (DMSO‑d6) δ
8.74–8.70 (m, 2H), 8.25 (s, 1H), 7.79–7.75 (m, 2H), 7.73 (s, 1H); 13C
NMR (DMSO‑d6) δ 166.8, 150.7, 150.7, 141.8, 121.8 (1H and 13C NMR
matched the reported values [37]); HRMS (EI) calcd for C6H6N2O [M]+
122.0475, found 122.0472 (error 2.0 ppm); IR (cmꢀ 1) 1550, 1595,
1622, 1655, 2784, 3179, 3366.
(Nunclon) containing 50
μL 7H9/ADC/Tw per well with or without
2.1.20. Isonicotinic acid (17)
2-fold serial dilutions of compound. Plates were sealed in ziplock bags
and incubated at 37 ◦C for 2 weeks. Growth was recorded after 1- and 2-
weeks of growth by monitoring growth using an inverted enlarging
mirror. The MIC was determined as the lowest concentration that
completely inhibited growth. Positive control compounds included
isoniazid and linezolid.
The title compound was purchased from Ambeed. Melting point
>250 ◦C; Rf = 0.34 (95:5 CH2Cl2–MeOH); 1H NMR (CD3OD) δ 8.71–8.56
(m, 2H), 7.93–7.75 (m, 2H); 13C NMR (CD3OD) δ 166.2, 149.6, 139.4,
123.4 (1H and 13C NMR matched the reported values [37]); HRMS (EI)
calcd for C6H5NO2 [M]+ 123.0315, found 123.0312 (error 2.2 ppm); IR
4