Development of 3-phenyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine derivatives as novel Mycobacterium tuberculosis pantothenate synthetase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2013.08.036

Forty 3-phenyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine derivatives were synthesized from piperidin-4-one by five step synthesis and evaluated for Mycobacterium tuberculosis (MTB) pantothenate synthetase (PS) inhibition study, in vitro activities against MTB, cytotoxicity against RAW 264.7 cell line. Among the compounds, 1-benzoyl-N-(4-nitrophenyl)-3-phenyl-6,7-dihydro-1H-pyrazolo[4, 3-c]pyridine-5(4H)-carboxamide (6ac) was found to be the most active compound with IC₅₀ of 21.8 ± 0.8 μM against MTB PS, inhibited MTB with MIC of 26.7 μM and it was non-cytotoxic at 50 μM.

Full text of DOI:10.1016/j.ejmech.2013.08.036

Accepted Manuscript
Development of 3-phenyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine derivatives as
novel Mycobacterium tuberculosis pantothenate synthetase inhibitors
Ganesh Samala, Parthiban Brindha Devi, Radhika Nallangi, Perumal Yogeeswari,
Dharmarajan Sriram
PII:
S0223-5234(13)00553-9
10.1016/j.ejmech.2013.08.036
EJMECH 6383
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 April 2013
Revised Date: 22 August 2013
Accepted Date: 25 August 2013
Please cite this article as: G. Samala, P.B. Devi, R. Nallangi, P. Yogeeswari, D. Sriram, Development of
3-phenyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine derivatives as novel Mycobacterium tuberculosis
pantothenate synthetase inhibitors, European Journal of Medicinal Chemistry (2013), doi: 10.1016/
j.ejmech.2013.08.036.
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ACCEPTED MANUSCRIPT
Development of 3-phenyl-4,5,6,7-tetrahydro-1H-
pyrazolo[4,3-c]pyridine derivatives as novel
Mycobacterium
tuberculosis
Pantothenate
synthetase
inhibitors
Ganesh Samala,ParthibanBrindha Devi, RadhikaNallangi, PerumalYogeeswari*,DharmarajanSriram* Department of
Pharmacy, Birla Institute of Technology & Science- Pilani, Hyderabad Campus, R.R. District-500078, Andhra Pradesh,
India
Compound 1-benzoyl-N-(4-nitrophenyl)-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxamide(3) was
found to be the most active compound with IC50 of 21.8±0.8 µM against MTB PS, inhibited MTB with MIC of 26.7 µM
and it was non-cytotoxic at 50 µM.

ACCEPTED MANUSCRIPT
Development of 3-phenyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine
derivatives as novel Mycobacterium tuberculosis pantothenate synthetase
inhibitors
Ganesh Samala, Parthiban Brindha Devi, Radhika Nallangi,
Perumal Yogeeswari *, Dharmarajan Sriram *
Antitubercular Drug Discovery Laboratory, Department of Pharmacy, Birla Institute of
Technology and Science - Pilani, Hyderabad Campus, Jawahar Nagar, Hyderabad – 500 078,
India.
Abstract
Forty 3-phenyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine derivatives were synthesized from
piperidin-4-one by five step synthesis and evaluated for Mycobacterium tuberculosis (MTB)
pantothenate synthetase (PS) inhibition study, in vitro activities against MTB, cytotoxicity
against RAW 264.7 cell line. Among the compounds, 1-benzoyl-N-(4-nitrophenyl)-3-phenyl-6,7-
dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (6ac) was found to be the most active
compound with IC50 of 21.8±0.8 µM against MTB PS, inhibited MTB with MIC of 26.7 µM and
it was non-cytotoxic at 50 µM.
Key words: Tuberculosis, Pantothenate synthetase, Cytotoxicity
*For Correspondence: Email- pyogee@hyderabad.bits-pilani.ac.in; drdsriram@yahoo.com
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1. Introduction
Tuberculosis (TB) is becoming a global health threat due to the emergence of multi-drug
resistant strains of Mycobacterium tuberculosis (MTB) and the deadly synergy of TB with HIV
[1]. This point to the critical need to develop new drugs against novel targets of MTB.
Pantothenate synthetase (PS; EC 6.3.2.1), encoded by the panC gene, catalyzes the essential
adenosine triphosphate (ATP)–dependent condensation of D-pantoate and ß-alanine to form
pantothenate in bacteria [2]. Pantothenate is a key precursor of coenzyme A and acyl carrier
protein, essential for many intracellular processes including fatty acid metabolism, cell signaling,
synthesis of polyketides and non-ribosomal peptides. Both microorganisms and plants must
synthesize pantothenate, while mammals must obtain it from their diet, and its biosynthetic
pathway is not present. A PanC gene knockout (KO) of PS in MTB results in a highly attenuated
phenotype in immunocompromised SCID mice and in immunocompetent BALB/c mice [3],
whereas the ∆lysA ∆pan CDKO mutant exhibits substantially reduced replication and persistence
[4]. This observation indicates that a functional pantothenate biosynthetic pathway is essential
for persistent growth and virulence of MTB and PS is an appropriate target for developing new
therapeutics to treat TB [5]. Till date some of the MTB PS inhibitors reported were (Fig 1) 5-
tert-butyl-N-pyrazol-4-yl-4,5,6,7-tetrahydrobenzo[d]isoxazole-3-carboxamide derivatives (A)
[6], actinomycin D (IC50 of 250.72 µM) [6], indole derivative (B) [7], nafronyl oxalate [8], and
benzofuran derivatives (C) [9]. In this work, we discovered novel 3-phenyl-4,5,6,7-tetrahydro-
1H-pyrazolo[4,3-c]pyridine derivatives as a novel scaffold as MTB PS inhibitors.
2. Results and discussion
2.1. Design and Chemistry
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In a protein data bank (PDB) crystal structure of a PS from MTB in complex with a reaction
intermediate, pantoyl adenylate was reported (PDB ID: 1N2H) with 2.0 Å resolution. The
crystal structure of the PS protein in complex with the reaction intermediate shows that the
reaction intermediate, pantoyl adenylate, has strong interactions with the active site atoms. This
tight binding is necessary to stabilize this highly reactive compound. It can be expected that a
nonreactive analog of pantoyl adenylate will be a good inhibitor [5]. A nonreactive analog of
pantoyl adenylate is expected to be very specific to PS as well. Because there is no pantothenate
biosynthesis in humans, this analog will be unlikely to inhibit any human enzyme. A high-
throughput virtual screening of our in-house (BITS-Pilani) database was carried out using Glide
XP (extra precision) docking and we identified 1-benzoyl-N-(4-bromophenyl)-3-phenyl-6,7-
dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxamide (Compound 6aa) as an inhibitor in the
pantoyl adenylate site with docking score of -6.92, while the pantoyl adenylate docking score
was -9.8 (Fig 2). Compound 6aa showed a percentage inhibition of 60.6 at 100 µM when tested
for MTB PS inhibitory activity. To investigate the potential of this lead, we synthesized a series
of 3-phenyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine derivatives to screen against MTB PS
enzyme activity for establishing structure-activity relationship (SAR), and to evaluate
antimycobacterial activity and cytotoxicity.
The target molecules were synthesized in five steps (Scheme 1), starting with commercially
available, less expensive, 4-piperidone hydrochloride salt (1). Initially the amine was protected
by t-butyloxycarbonyl (Boc) protecting group to get 4-N-Boc-piperidone (2), and then it was
subjected to stark-enamine reaction conditions using morpholine, p-toluenesulfonic acid
(catalytic) and benzoyl chloride to produce 1, 3-dicarbonyl intermediate; which is then treated in
situ with hydrazine hydrate to get Pyrazole ring (3) according to procedure reported by X. M. Ye
et al [10]. Compound 3 was then deprotected using trifluoroacetic acid to get compound 4. Under
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mild conditions the more nucleophilic amine of aliphatic ring was reacted selectively with
various substituted isocyanates, isothiocyanates and arylsulfonyl halides using DIPEA as base
and DMF as solvent at room temperature to yield corresponding urea, thiourea and
sulphonamides (5 and 8). The free amino group of pyrazole ring was treated with
benzoyl/cyclohexanecarbonyl chloride using DIPEA as base to get target compounds.
2.2 Pantothenate synthetase enzyme inhibition studies
Synthesized compounds were assayed for MTB PS inhibition study, that couples the AMP
produced in the condensation of β-alanine and pantoate with the reduction of NADH to NAD+
through myokinase, pyruvate kinase and lactate dehydrogenase [2]. The NAD+ produced can be
monitored spectrophotometrically at 340 nm. Initial screening at 100µM, compounds showed
inhibition ranged from 9.0-95.7%. Seventeen compounds showed >50% inhibition against MTB
PS and compounds which showed % inhibition of >60 were further selected for IC50 calculation.
Among the five compounds (6aa, 6ac, 6ag, 9aa, and 9ac) tested, showed IC50 ranged from
21.8±0.8 to 82.1±2.3 µM. Compound 6ac (1-benzoyl-N-(4-nitrophenyl)-3-phenyl-6,7-dihydro-
1H-pyrazolo[4,3-c]pyridine-5(4H)-carboxamide) was found to be the most potent compound
with IC50 of 21.8 ± 0.8µM (Fig 3) and it was found to be four times more potent than our initial
compound 6aa. With respect to structure activity relationship, we have prepared twenty benzoyl
and twenty cyclohexanecarbonyl derivatives at 1st position of 4,5,6,7-tetrahydro-1H-
pyrazolo[4,3-c]pyridine scaffold (R2). Benzoyl group substituted compounds (6aa-6ai, 7aa-7af
and 9aa-9ae) showed better activity than cyclohexanecarbonyl derivatives (6ba-6bi, 7ba-7bf and
9ba-9be) against MTB PS. We have also done modification at 5th position by preparing urea
(6aa-6ai, 6ba-6bi), thiourea (7aa-7af, 7ba-7bf) and sulfonamides (9aa-9ae, 9ba-9be). In
general the order of activity as follows urea (6aa-6ai) > sulfonamides (9aa-9ae) > thiourea (7aa-
7af). Among the urea derivatives (6aa-6ai), we have prepared (sub) phenyl, naphthyl, benzyl and
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alkyl derivatives. Electron withdrawing substituents in the phenyl ring (6aa-6ac) favors activity
than electron donating substituents (6ae, 6af).
2.3 In-vitro MTB screening
All the new 3-phenyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine derivatives were screened
for their in vitro anti-tubercular activity against Mycobacterium tuberculosis H37Rv
(ATCC27294) using an agar dilution method using drug concentration from 50 µg/mL to
0.78µg/mL in duplicates. The minimum inhibitory concentration (MIC) was determined for each
compound. The MIC is defined as the minimum concentration of compound required to
completely inhibit the bacterial growth. Isoniazid and Ethambutol were used as a reference
compound for comparison. The MIC values of the synthesized compounds along with the
standard drug for comparison were reported in Table 1. Among the forty compounds screened
seventeen compounds showed activity against MTB with MIC ≤ 60 µM. Two compounds (6ac,
and 9ab) inhibited MTB with MIC of < 30 µM. Compound 9ab ((5-(4-nitrophenylsulfonyl)-3-
phenyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)(phenyl)methanone) was found to be
the most active compound in vitro with MIC of 25.58 ìM against log-phase culture of MTB.
Structural changes at N-1 position do not affect activity appreciably, whereas sulfonamides
derivatives at N-5 position showed better activity in general.
2.4. In-vitro cytotoxicity study
All the compounds were also tested for in-vitro cytotoxicity against RAW 264.7 cells at 50 µM
concentration by (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Percentage inhibition of cells was reported in Table 1. The most promising anti-TB compound
6ac was devoid of cytotoxicity at 50µM [0% inhibition].
3. Conclusion
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We identified 1-benzoyl-N-(4-bromophenyl)-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-
5(4H)-carboxamide (Compound 6aa) as an inhibitor in the pantoyl adenylate site of MTB PS by
virtual screening. To study the SAR we synthesized forty compounds and compound 6ac was
found to be the most potent compound with IC50 of 21.8±0.8 µM against MTB PS. Compound
6ac also inhibit the growth of MTB with MIC of 26.7 µM and non-cytotoxic to RAW 264.7 cells
till 50 µM.
4. Experimental
4.1. Chemistry
4.1.1 Preparation of tert-butyl 4-oxopiperidine-1-carboxylate (2)
Et3N (26.45 mL, 185.17 mmol) was added drop wise to a stirred solution of compound 1 (10.0 g,
74.07 mmol) in CH2Cl2 (100 mL) and MeOH (10 mL) at 0 °C, then (Boc)2O (19.37 mL, 88.88
mmol) was added drop wise to the reaction mixture at same temperature and allowed to stir at
room temperature for 16 h. The reaction mixture was diluted with H2O (100 mL) and extracted
with CH2Cl2 (3 X 200 mL). The separated organic layer was concentrated under reduced
pressure and the crude residue was washed with hexanes to get compound 2 (11.5 g, 78%) as an
off-white solid. ESI-MS found 200 [M+H]+and carried to next step.
4.1.2 Preparation of tert-butyl 3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-
carboxylate (3)
In a 100 mL round-bottom flask equipped with a Dean-stark trap, a reflux condenser and an
internal thermocouple, Compound 2 (1.0 g, 4.63 mmol), toluene (10 mL), morpholine (0.42 mL,
4.63 mmol), and p-toluenesulfonic acid (catalytic) were added sequentially. The reaction solution
was refluxed under N2 for 16 h. The solvent was evaporated and the crude was dissolved in
CH2Cl2 (20 mL) and then triethylamine (1.07 mL, 7.53 mmol) was added at 0 °C, under N2, a
solution of benzoyl chloride (0.58mL, 5.02 mmol) in CH2Cl2 (10 mL) was added over 10 min,
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the ice bath was then removed and the reaction solution was stirred at room temperature for 4 h.
All the volatile solvents were removed in vacuo and the residue was dissolved in ethanol, at 0
°C, and hydrazine hydrate (0.25mL, 7.53 mmol) was then added over 5 min (exothermic
reaction), the reaction solution was stirred at r.t. for 16h and the reaction mixture was
concentrated under reduced pressure and the crude residue was purified by column
chromatography to get compound 3 (1.34 g, 90%) as an off-white solid. ESI-MS found 300
[M+H]+. 1H NMR (300 MHz, CDCl3) δ 9.45 (s, 1H), 7.5–7.2 (m, 5H), 4.80 (s, 2H), 3.90 (t, J =
13
7.8 Hz, 2H), 2.81 (t, J = 7.8 Hz, 2H), 1.40 (s, 9H); C NMR (75 MHz, CDCl3) δ 163.5, 147.6,
143.5, 136.6(2C), 132.7, 129,3(2C), 127.3, 117.5, 81.7, 43.9, 36.9, 31.5(3C), 27.7; Anal. calcd
for C17H21N3O2: C, 68.20; H, 7.07; N, 14.04% Found: C, 68.24; H, 7.09; N, 14.13%
4.1.3 Preparation of 3-phenyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine (4)
CF3COOH (0.65 mL, 8.36 mmol) was added drop wise to a stirred solution of compound 3 (0.6
g, 1.67 mmol) in CH2Cl2 (10 mL) at 0 °C, and allowed to stir at room temperature for 4 h. The
reaction mixture was concentrated under reduced pressure and the crude residue was washed
with hexanes and diethyl ether to get compound 4 (0.45 g, 91%) as an off-white solid. ESI-MS
showed 200 [M+H]+. 1H NMR (300 MHz, DMSO-d6): 7.38–7.10 (m, 5H), 4.22 (s, 2H), 3.45 (t, J
= 7.2 Hz, 2H), 2.61 (t, J = 7.8 Hz, 2H).
4.1.4 Preparation of 5
Substituted arylisocyanate/arylisothiocyanate (1.20 equiv.), was added to the stirred solution of
Compound 4 (1.0 equiv) and Et3N (2.5 equiv) in DMF at 0 °C under N2 atm, and allowed to stir
at room temperature for 6 h. The reaction mixture was diluted with EtoAc and washed with
Brine solution, the separated organic layer was concentrated under reduced pressure and the
crude residue was purified by column chromatography (EtOAc/Hexanes) to get compounds 5.
4.1.5 Preparation of 8
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Substituted arylsulphonyl halide (1.20 equiv), was added to the stirred solution of Compound 4
(1.0 equiv) and Et3N (2.5 equiv) in DMF at 0 °C under N2 atm, and allowed to stir at room
temperature for 6 h. The reaction mixture was diluted with EtOAc and washed with Water, the
separated organic layer was concentrated under reduced pressure and the crude residue was
purified by column chromatography (EtOAc/Hexanes) to get compounds 8.
4.1.6a. Preparation of 6aa-6ai, 7aa-7af, and 9aa-9ae:
Benzoylchloride (1.20 equiv), was added to the stirred solution of Compound 5/Compound 8
(1.00 equiv) and DIPEA (2.20 equiv) in DMF at 0 °C under N2 atm, and allowed to stir at room
temperature for 12 h. The reaction mixture was quenched with water, extracted with EtOAc and
washed the EtOAc layer with Brine solution, the separated organic layer was concentrated under
reduced pressure and the crude residue was purified by column chromatography
(EtOAc/Hexanes as eluent) to get compounds 6aa-6ai, 7aa-7af, and 9aa-9ae.
4.1.6b. Preparation of 6ba-6bi, 7ba-7bf, and 9ba-9be:
Cyclohexanecarbonylchloride (1.20 equiv), was added to the stirred solution of Compound
5/Compound 8 (1.00 equiv) and DIPEA (2.20 equiv) in DMF at 0 °C under N2 atm, and allowed
to stir at room temperature for 12 h. The reaction mixture was quenched with water, extracted
with EtOAc and washed the EtOAc layer with Brine solution, the separated organic layer was
concentrated under reduced pressure and the crude residue was purified by column
chromatography (EtOAc/Hexanes as eluent) to get compounds 6ba-6bi, 7ba-7bf, and 9ba-9be.
4.1.7. 1-Benzoyl-N-(4-bromophenyl)-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-
1
carboxamide (6aa): Yield: 78%, m.p. 167–169 °C; MS(ESI) m/z 501 [M+H]+; H NMR (400
MHz, CDCl3) δ 8.40 (s, 1H), 7.89 (d, J = 7.6 Hz, 2H), 7.50–7.25 (m, 12H), 4.70 (s, 2H), 3.82 (t,
13
J = 8.0 Hz, 2H), 3.10 (t, J = 7.6 Hz, 2H); C NMR (100 MHz, CDCl3) δ 169.2, 159.4, 145.6,
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144.6(2C), 134.9, 134.3, 133.7, 132.2, 131.4, 129.2, 128.3(2C), 127.1, 126.6(2C), 125.0, 124.2,
124.0, 123.4(2C), 122.7, 118.9, 54.7, 48.4, 29.6. Anal. calcd for C26H21BrN4O2: C, 62.28; H,
4.22; N, 11.17% Found C, 62.31; H, 4.26; N, 11.21%
4.1.8. 1-Benzoyl-N-(4-chlorophenyl)-3-phenyl-6, 7-dihydro-1H-pyrazolo [4,3-c]pyridine-
1
5(4H)-carboxamide (6ab): Yield: 85%, m.p. 160–162 °C; MS(ESI) m/z 457 [M+H]+; H NMR
(400 MHz, DMSO-d6) δ 8.90 (s, 1H), 7.90 (d, J = 8.0 Hz, 2H), 7.78 (d, J = 8.4 Hz, 2H), 7.60–
13
7.24 (m, 10H), 4.75 (s, 2H), 3.85 (t, J = 8.0 Hz, 2H), 3.21 (t, J = 8.0 Hz, 2H); C NMR (100
MHz, DMSO-d6) δ 171.2, 160.0, 145.2, 144.9, 141.3, 135.4(2C), 132.6, 131.9, 130.6, 130.1,
129.6(2C), 128.4, 128.5, 127.9, 127.4, 126.4, 126.0, 125.3(2C), 123.9, 119.6, 54.9, 48.6, 29.9.
Anal. calcd for C26H21ClN4O2: C, 68.34; H, 4.63; N, 12.26% Found C, 68.36; H, 4.69; N,
12.34%
4.1.9. 1-Benzoyl-N-(4-nitrophenyl)-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-
1
carboxamide (6ac): Yield: 75%, m.p. 172–175 °C; MS(ESI) m/z 468 [M+H]+; H NMR (400
MHz, DMSO-d6) δ 9.45 (s, 1H), 8.16 (d, J = 12.0 Hz, 2H), 8.03 (d, J = 10.0 Hz, 2H), 7.74–7.46
13
(m, 10H), 4.82 (s, 2H), 3.87 (t, J = 6.8 Hz, 2H), 3.26 (t, J = 7.2 Hz, 2H); C NMR (75 MHz,
DMSO-d6) δ 167.2, 154.4, 150.2, 147.2, 142.6, 141.1, 132.8, 132.3, 131.4, 131.1, 129.2(2C),
129.0(2C), 128.0(2C), 127.1(2C), 124.6(2C), 118.6(2C), 116.6, 41.3, 40.8, 25.2. Anal. calcd for
C26H21N5O4: C, 66.80; H, 4.53; N, 14.98% Found C, 66.88; H, 4.58; N, 14.91%
4.1.10. N-(4-Acetylphenyl)-1-benzoyl-3-phenyl-6, 7-dihydro-1H-pyrazolo [4, 3-c] pyridine-
1
5(4H)-carboxamide (6ad): Yield: 64%, m.p. 166–168 °C; MS(ESI) m/z 465 [M+H]+; H NMR
(400 MHz, CDCl3) δ 8.48 (bs, 1H), 7.63 (d, J = 6.8 Hz, 2H), 7.42–7.15 (m, 12H), 4.28 (s, 2H),
13
3.45 (t, J = 7.6 Hz, 2H), 3.05 (t, J = 7.6 Hz, 2H), 2.46 (s, 3H); C NMR (100 MHz, CDCl3) δ
187.6, 172.4, 164.4, 143.6, 142.6, 141.9, 136.7, 136.4, 135.7, 134.2(2C), 132.9, 129.1(2C),
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128.6, 128.3, 127.6(2C), 127.1, 126.9, 126.4, 124.7, 122.1, 120.3, 56.9, 45.7, 29.1, 25.9: Anal.
calcd for C28H24N4O3: C, 72.40; H, 5.21; N, 12.06% Found C, 72.45, H, 5.24, N, 12.09%
4.1.11.
1-Benzoyl-3-phenyl-N-p-tolyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5
(4H)-
1
carboxamide (6ae): Yield: 66%, m.p. 163–165 °C; MS(ESI) m/z 437 [M+H]+; H NMR (400
MHz, CDCl3) δ 8.45 (bs, 1H), 7.89–7.21 (m, 14H) ,4.92 (s, 2H), 3.45 (t, J = 8.0 Hz, 2H), 3.15 (t,
J = 7.6 Hz, 2H), 2.40 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 165.6, 162.4, 139.8, 139.7, 138.6,
137.6, 137.8, 136.8, 134.8, 134.6(2C), 133.8, 133.0, 131.8, 130.8, 127.8, 126.7(2C), 125.1,
124.8(2C), 124.1, 119.9, 56.8, 44.7, 31.7, 25.2; Anal. calcd for C27H24N4O2: C, 74.29; H, 5.54;
N, 12.84% Found C, 74.34; H, 5.59; N, 12.88%.
4.1.12. 1-Benzoyl-N-(4-ethoxyphenyl)-3-phenyl-6, 7-dihydro-1H-pyrazolo [4, 3-c] pyridine-
1
5(4H)-carboxamide(6af): Yield: 68%, m.p. 155–158 °C; MS(ESI) m/z 467 [M+H]+; H NMR
(400 MHz, CDCl3) δ 7.65–7.30 (m, 15H), 4.70 (s, 2H), 4.21–4.18 (m, 2H), 3.45 (t, J = 8.0 Hz,
2H), 2.95 (t, J = 7.2 Hz, 2H), 1.42 (t, J = 7.6 Hz, 3H); 13C NMR (100 MHz, CDCl3) 167.4,
158.3, 153.9, 144.6, 144.2, 142.6, 137.9, 136.6, 135.6, 133.4, 132.6(2C), 130.3(2C), 131.1,
128.9, 128.2, 126.7, 125.6, 124.3(2C), 121.9, 116.3, 66.3, 56.3, 47.3, 27.8, 16.9. Anal. calcd for
C28H26N4O3: C, 72.09; H, 5.62; N, 12.01% Found C, 72.12; H, 5.66; N, 12.18%
4.1.13.
1-Benzoyl-N-(naphthalen-1-yl)-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-
1
5(4H)-carboxamide (6ag): Yield: 85%, m.p. 185–187 °C; MS(ESI) m/z 473 [M+H]+; H NMR
(300 MHz, DMSO-d6) δ 8.92 (s, 1H), 8.06 (t, J = 7.5 Hz, 2H), 7.93–7.39 (m, 15H), 4.78 (s, 2H),
3.95 (t, J = 7.6 Hz, 2H), 3.12 (t, J = 8.0 Hz, 2H); 13C NMR (75 MHz, DMSO-d6) δ 167.3, 156.3,
150.2, 142.8, 135.3, 133.7, 132.8, 132.4, 131.5, 131.1, 129.6, 129.1(2C), 128.9(2C), 128.0(2C),
127.9(2C), 127.0(2C), 125.8, 125.5, 125.2, 123.5, 123.3, 117.0, 41.3, 40.6, 25.3. Anal. Calcd. for
C30H24N4O2: C, 76.25; H, 5.12; N, 11.86 Found C, 76.29; H, 5.16; N, 11.90%
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4.1.14.
1-Benzoyl-N-benzyl-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-
1
carboxamide (6ah): Yield: 64%, m.p. 176–178 °C; MS(ESI) m/z 437 [M+H]+; H NMR (400
MHz, CDCl3) δ 8.45 (t, 1H), 7.65–7.18 (m, 15H), 4.92 (s, 2H), 4.65 (d, J = 6.8 Hz, 2H), 3.75 (t,
13
J = 8.0 Hz, 2H), 2.96 (t, J = 7.6 Hz, 2H); C NMR (100 MHz, CDCl3) δ 164.4, 159.5, 138.5,
138.0, 137.4, 136.3, 135.9, 133.8, 133.0, 132.3(2C), 131.8, 129.0, 127.5, 126.4, 123.8,
122.4(2C), 121.6, 121.0(2C), 120.6, 120.0, 51.8, 43.7, 39.3, 22.5. Anal. calcd for C27H24N4O2: C,
74.29; H, 5.54; N, 12.84% Found C, 74.36; H, 5.52; N, 12.92%.
4.1.15.
1-Benzoyl-N-isopropyl-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-
1
carboxamide (ai): Yield: 60%, m.p. 144–146 °C; MS(ESI) m/z 389 [M+H]+; H NMR (400
MHz, CDCl3) δ 7.86–7.58 (m, 10H), 4.42 (s, 2H), 4.23 (m, 1H), 3.65 (t, J = 7.2 Hz, 2H), 3.06 (t,
13
J = 7.6 Hz, 2H); 1.38 (d, J = 9.6 Hz, 6H); C NMR (100 MHz, CDCl3) δ 161.4, 158.3, 142.7,
142.2, 141.7, 140.1, 133.5, 133.1, 131.7, 130.9(2C), 130.3, 130.0, 127.5, 126.9, 126.6, 125.1,
51.3, 43.9, 40.5, 29.7, 21.6(2C). Anal. calcd for C23H24N4O2: C, 71.11; H, 6.23; N, 14.42%
Found C, 71.21; H, 6.24; N, 14.52%.
4.1.16.
1-Benzoyl-N-(4-chlorophenyl)-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-
1
5(4H)-carbothioamide (7aa): Yield: 75%, m.p. 176–178 °C; MS(ESI) m/z 473 [M+H]+; H
NMR (400 MHz, CDCl3) δ 9.28 (bs, 1H), 8.05–7.40 (m, 14H), 4.95 (s, 2H), 3.82 (t, J = 7.6 Hz,
13
2H), 3.12 (t, J = 7.6 Hz, 2H); C NMR (100 MHz, CDCl3) δ 179.3, 163.9, 144.3, 144.0, 143.5,
141.8, 138.9, 137.8, 136.5, 135.9, 135.5, 134.9, 134.2, 133.9, 132.9, 129.6(2C), 129.1, 128.6,
127.6(2C), 126.7, 125.8, 59.6, 49.9, 27.3. Anal. calcd for C26H21ClN4OS: C, 66.02; H, 4.48; N,
11.85% Found C, 66.12; H, 4.54; N, 11.89%.
4.1.17. 1-Benzoyl-N-(4-fluorophenyl)-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-
carbothioamide (7ab): Yield: 81%, m.p. 176–177 °C; MS(ESI) m/z 473 [M+H]+; 1H NMR (400
MHz, CDCl3) δ 8.28 (s, 1H), 7.92 (d, J = 8.0 Hz, 2H), 7.80 (d, J = 8.4 Hz, 2H), 7.62–7.35 (m,
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ACCEPTED MANUSCRIPT
10H), 4.85 (s, 2H), 3.92 (t, J = 7.2 Hz, 2H), 3.06 (t, J = 7.6 Hz, 2H); 13C NMR (100 MHz,
CDCl3) δ 162.7, 159.6, 153.7, 144.3, 144.1, 143.7, 142.5, 139.9, 138.6, 135.9, 134.4, 133.8,
132.9, 132.3, 131.9, 130.7(2C), 129.6, 129.0, 128.5(2C), 126.3, 124.8, 61.6, 52.7, 26.9. Anal.
calcd for C26H21FN4OS: C, 68.40; H, 4.64; N, 12.27% Found C, 68.54; H, 4.68; N,12.31%
4.1.18
1-Benzoyl-3-phenyl-N-p-tolyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-
carbothioamide (7ac): Yield: 69%, m.p. 189–190 °C; MS(ESI) m/z 453 [M+H]+; 1H NMR (400
MHz, CDCl3) δ 7.85–7.21 (m, 13H), 7.18 (d, J = 7.6 Hz, 2H), 4.85 (s, 2H), 3.58 (t, J = 7.6 Hz,
13
2H), 2.95 (t, J = 7.6 Hz, 2H), 2.52 (s, 3H); C NMR (100 MHz, CDCl3) δ 176.4, 165.7, 138.9,
137.4, 137.0, 136.5, 136.0, 134.9, 134.3, 134.1(2C), 131.8, 130.0, 129.8, 127.4, 127.0,
125.9(2C), 123.8(2C), 123.4, 122.1, 118.8, 58.9, 46.8, 29.5, 22.8. Anal. calcd for C27H24N4OS:
C, 71.65; H, 5.35; N, 12.38% Found C, 71.73; H, 5.40; N, 12.45%.
4.1.19.
1-Benzoyl-N-(4-methoxyphenyl)-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-
1
5(4H)-carbothioamide (7ad): Yield: 73%, m.p. 169–172 °C; MS(ESI) m/z 469 [M+H]+; H
NMR (400 MHz, CDCl3) δ 9.08 (s, 1H), 7.72–6.98 (m, 14H), 4.75 (s, 2H), 4.05 (s, 3H) 3.82 (t, J
= 7.6 Hz, 2H), 2.89 (t, J = 7.2 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 172.6, 163.7, 156.8,
142.6, 142.2, 141.6, 141.0, 139.2, 138.4, 137.8, 133.9, 132.6, 129.3(2C), 129.1, 128.6, 127.0,
126.5, 124.9, 122.9(2C), 120.9, 119.4, 60.9, 52.7, 44.1, 23.4. Anal. calcd for C27H24N4O2S: C,
69.21; H, 5.16; N, 11.96% Found C, 69.24; H, 5.19; N, 12.04%.
4.1.20.
1-Benzoyl-N-benzyl-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-
carbothioamide (7ae): Yield: 61%, m.p. 180–181 °C; MS(ESI) m/z 453 [M+H]+; 1H NMR (400
MHz, CDCl3) δ 9.15 (t, J = 7.6 Hz, 1H), 7.69–7.28 (m, 15H), 4.89 (s, 2H), 4.45 (d, J = 6.8 Hz,
13
2H), 3.85 (t, J = 7.6 Hz, 2H), 2.88 (t, J = 7.6 Hz, 2H); C NMR (100 MHz, CDCl3) δ 169.4,
155.5, 139.5, 136.9, 135.4, 134.9, 134.0, 133.8, 133.2, 132.8(2C), 1320, 128.9, 127.8, 127.3,
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ACCEPTED MANUSCRIPT
125.9, 125.4(2C), 124.4, 121.8(2C), 120.8, 119.5, 55.8, 43.5, 39.9, 20.9. Anal. calcd for
C27H24N4OS: C, 71.65; H, 5.35; N, 12.38% Found C, 71.69; H, 5.39; N, 12.41%
4.1.21.
N-Allyl-1-benzoyl-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-
carbothioamide (7af): Yield: 84%, m.p. 184–186 °C; MS(ESI) m/z 403 [M+H]+; 1H NMR (400
MHz, CDCl3) δ 7.99–7.16 (m, 11H), 6.02 (m, 1H), 5.23 (m, 2H), 4.79 (s, 2H), 4.38 (m, 2H),
13
3.96 (t, J = 8.0 Hz, 2H), 2.95 (t, J = 7.6 Hz, 2H); C NMR (100 MHz, CDCl3) δ 174.5, 165.7,
140.7, 140.2, 133.5, 132.9, 131.9, 130.5(2C), 130.3, 129.5, 127.5, 126.5(2C), 126.2, 125.6,
124.5, 119.1, 114.3, 51.3, 45.9, 43.2, 21.6. Anal. calcd for C23H22N4OS: C, 68.63; H, 5.51; N,
13.92% Found C, 68.69; H, 5.56; N, 13.99%.
4.1.22. (5-(4-fluorophenylsulfonyl)-3-phenyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-
1
yl)(phenyl)methanone (9aa): Yield: 71%, m.p. 193–195 °C; MS(ESI) m/z 462 [M+H]+; H
NMR (400 MHz, DMSO-d6) δ 8.02–7.91 (m, 4H), 7.7–7.42 (m, 10H), 4.45 (s, 2H), 3.55 (t, J =
7.6 Hz, 2H), 3.20 (t, J = 7.6 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 167.8, 167.1, 163.7, 150.8,
141.7, 133.5, 133.0, 132.3, 131.7(2C), 130.3, 130.2, 129.3, 129.0(2C), 128.0(2C), 127.2(2C),
116.8, 116.5, 115.0, 43.1, 42.9, 25.5. Anal. calcd for C25H20FN3O3S: C, 65.06; H, 4.37; N, 9.10%
Found C, 65.12; H, 4.41; N, 9.16%
4.1.23.
(5-(4-nitrophenylsulfonyl)-3-phenyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-
1
yl)(phenyl)methanone (9ab): Yield: 64%, m.p. 187–189 °C; MS(ESI) m/z 489 [M+H]+; H
NMR (400 MHz, CDCl3) δ 7.93 (d, J = 9.2 Hz, 2H), 7.78 (d, J = 8.4 Hz, 2H), 7.72–7.39 (m,
10H), 4.86 (s, 2H), 3.95 (t, J = 8.0 Hz, 2H), 3.28 (t, J = 8.0 Hz, 2H); 13C NMR (100 MHz,
CDCl3) δ 172.3, 144.8, 143.6, 143.1(2C), 139.8, 133.5, 132.2, 131.7, 130.6, 129.5, 129.2,
128.7(2C), 127.6, 127.0, 125.8, 124.9, 122.8(2C), 120.4, 120.8, 55.9, 42.8, 23.4. Anal. calcd for
C25H20N4O5S: C, 61.47; H, 4.13; N, 11.47% Found C, 61.54; H, 4.19; N, 11.54%.
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4.1.24.
1-(4-(1-Benzoyl-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridin-5(4H)-
ylsulfonyl)phenyl)ethanone (9ac): Yield: 72%, m.p. 176–179 °C; MS(ESI) m/z 486 [M+H]+; 1H
NMR (400 MHz, CDCl3) δ 7.83–6.96 (m, 14H), 4.68 (s, 2H), 3.65 (t, J = 7.6 Hz, 2H), 3.05 (t, J
13
= 8.0 Hz, 2H), 2.52 (s, 3H); C NMR (100 MHz, CDCl3) δ 189.9, 168.5, 143.6, 142.9, 142.2,
139.9, 138.4, 137.9, 136.7, 134.7, 133.9, 132.6(2C), 131.6, 129.6, 127.6(2C), 126.6, 125.2,
124.6, 124.0, 123.1, 122.3, 57.9, 46.3, 29.5, 22.5. Anal. calcd for C27H23N3O4S: C, 66.79; H,
4.77; N, 8.65% Found C, 66.84; H, 4.79; N, 8.69%
4.1.25. Phenyl(3-phenyl-5-tosyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)methanone
(9ad): Yield: 81%, m.p. 120–123 °C; MS(ESI) m/z 458 [M+H]+; 1H NMR (400 MHz, CDCl3) δ
7.99–7.21 (m, 14H), 4.62 (s, 2H), 3.65 (t, J = 7.6 Hz, 2H), 3.09 (t, J = 8.0 Hz, 2H), 2.42 (s, 3H);
13C NMR (100 MHz, CDCl3) δ 174.4, 137.9, 137.4, 136.8, 136.5, 136.0, 133.9, 133.1,
131.4(2C), 129.8, 129.2, 128.8, 127.5, 126.7, 125.5(2C), 124.6(2C), 122.5, 121.5, 119.9, 59.5,
48.8, 31.5, 23.4. Anal. calcd for C26H23N3O3S: C, 68.25; H, 5.07; N, 9.18% Found C, 68.30; H,
5.11; N, 9.21%.
4.1.26.
Phenyl(3-phenyl-5-(thiophen-2-ylsulfonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-
1
c]pyridin-1-yl)methanone (9ae): Yield: 75%, m.p. 221–223 °C; MS(ESI) m/z 450 [M+H]+; H
NMR (400 MHz, CDCl3) δ 7.79–7.01 (m, 13H), 4.22 (s, 2H), 3.25 (t, J = 7.6 Hz, 2H), 2.86 (t, J
13
= 7.6 Hz, 2H); C NMR (100 MHz, CDCl3) δ 172.2, 146.9, 145.3, 139.3, 138.7, 137.4, 136.7,
131.8, 131.5, 130.5, 128.4, 126.6, 125.7, 124.3, 122.8, 121.9, 120.4, 119.6, 119.2, 118.6, 56.7,
45.9, 23.6. Anal. Calcd. for C23H19N3O3S2: C, 61.45; H, 4.26; N, 9.35% Found C, 61.50; H, 4.30;
N, 9.40%.
4.1.27. N-(4-Bromophenyl)-1-(cyclohexanecarbonyl)-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-
c]pyridine-5(4H)-carboxamide (6ba): Yield: 84%, m.p. 173–176 °C; MS(ESI) m/z 507 [M+H]+;
1H NMR (400 MHz, CDCl3) δ 7.89 (d, J = 9.6 Hz, 2H), 7.76 (d, J = 9.6 Hz, 2H), 7.62–7.48 (m,
14

ACCEPTED MANUSCRIPT
13
6H), 4.84 (s, 2 H), 3.88 (t, J = 7.6 Hz, 2H), 3.52 (t, J = 8.0 Hz, 2H), 2.36–1.25 (m, 11H): C
NMR (100 MHz, CDCl3) δ 171.5, 165.6, 144.7, 142.9, 132.3, 131.3, 128.9(2C), 127.5,
126.4(2C), 122.5(2C), 121.9(2C), 120.2, 119.4, 60.1, 49.4, 28.6(2C), 25.8(2C), 23.8, 23.4, 22.5.
Anal. Calcd. for C26H27BrN4O: C, 61.54; H, 5.36; N, 11.04% Found C, 61.60; H, 5.40; N,
11.12%.
4.1.28. N-(4-Chlorophenyl)-1-(cyclohexanecarbonyl)-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-
c]pyridine-5(4H)-carboxamide (6bb): Yield: 92%, m.p. 160–162 °C; MS(ESI) m/z 463 [M+H]+;
1H NMR (400 MHz, CDCl3) δ 7.98 (d, J = 10.4 Hz, 2H), 7.86 (d, J = 9.6 Hz, 2H), 7.68–7.46 (m,
5H), 6.52 (s, 1H) 4.74 (s, 2H), 3.82 (t, J = 7.6 Hz, 2H), 3.42 (t, J = 8.0 Hz, 2H), 2.25–1.25 (m,
11H): 13C NMR (100 MHz, CDCl3) δ 169.6, 151.7, 145.6, 143.7, 131.6, 130.8, 129.4(2C), 125.7,
121.6(2C), 121.0(2C), 119.9(2C), 119.2, 117.9, 61.5, 48.6, 29.3(2C), 26.2(2C), 24.9, 24.4, 23.4.
Anal. calcd for C26H27ClN4O2: C, 67.45; H, 5.88; N, 12.10% Found C, 67.54; H, 5.93; N,
12.23%.
4.1.29.
1-(Cyclohexanecarbonyl)-N-(4-nitrophenyl)-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-
c]pyridine-5(4H)-carboxamide (6bc): Yield: 62%, m.p. 157–159 °C; MS(ESI) m/z 474 [M+H]+;
1H NMR (400 MHz, CDCl3) δ 7.98–7.42 (m, 10H), 4.84 (s, 2H), 3.95 (t, J = 7.2 Hz, 2H), 3.08 (t,
J = 7.6 Hz, 2H), 2.30 (s, 1H), 2.05–1.25 (m, 10H): 13C NMR (100 MHz, CDCl3) δ 179.4, 166.6,
154.7, 153.7, 143.6, 136.8, 132.5(2C), 124.8, 123.7(2C), 122.6(2C), 121.2(2C), 119.2, 117.9,
59.6, 38.8, 26.3(2C), 25.8, 26.9(2C), 23.9, 22.1. Anal. calcd for C26H27N5O4: C, 65.95; H, 5.75;
N, 14.79% Found C, 65.99; H, 5.83; N, 14.88%.
4.1.30. N-(4-Acetylphenyl)-1-(cyclohexanecarbonyl)-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-
c]pyridine-5(4H)-carboxamide (6bd): Yield: 78%, m.p. 169–171 °C; MS(ESI) m/z 471 [M+H]+;
1H NMR (400 MHz, CDCl3) δ 7.91 (d, J = 9.6 Hz, 2H), 7.72 (d, J = 9.2 Hz, 2H), 7.56–7.44 (m,
5H), 6.75 (s, 1H), 4.79 (s, 2H), 3.82 (t, J = 7.6 Hz, 2H), 3.32 (t, J = 7.6 Hz, 2H), 2.55 (s, 3H),
15

ACCEPTED MANUSCRIPT
13
2.16–1.21 (m, 11H): C NMR (100 MHz, CDCl3) δ 169.3, 164.3, 154.3, 148.6, 145.6, 136.5,
133.5(2C), 128.4, 125.7(2C), 125.4, 124.8(2C), 122.7(2C), 121.6, 120.4, 58.5, 46.5, 37.6, 35.2,
28.4, 26.5(2C), 24.8, 24.3, 21.6. Anal. calcd for C28H30N4O3: C, 71.47; H, 6.43; N, 11.91%
Found C, 71.52; H, 6.48; N, 11.85%.
4.1.31. 1-(Cyclohexanecarbonyl)-3-phenyl-N-p-tolyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-
1
5(4H)-carboxamide (6be): Yield: 88%, m.p. 171–172 °C; MS(ESI) m/z 443 [M+H]+; H NMR
(400 MHz, CDCl3) δ 7.89 (d, J = 10.8 Hz, 2H), 7.72 (d, J = 9.6 Hz, 2H), 7.78–7.52 (m, 5H), 6.78
(s, 1H) 4.59 (s, 2H), 3.82 (t, J = 7.6 Hz, 2H), 3.18 (t, J = 8.0 Hz, 2H), 2.39 (s, 3H), 2.26 (s, 1H),
13
2.05–1.25 (m, 10H); C NMR (100 MHz, CDCl3) δ 182.6, 161.3, 155.9, 151.6, 141.5, 139.5,
133.7(2C), 125.6, 124.6(2C), 124.4, 122.6, 121.9(2C), 118.5, 118.0, 58.9, 39.5, 34.9, 27.6(2C),
28.8, 26.5(2C), 24.3, 23.4. Anal. calcd for C27H30N4O2: C, 73.28; H, 6.83; N, 12.66% Found C,
73.32; H, 6.92; N, 12.69%.
4.1.32. 1-(Cyclohexanecarbonyl)-N-(4-methoxyphenyl)-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-
c]pyridine-5(4H)-carboxamide (6bf): Yield: 76%, m.p. 147–149 °C; MS(ESI) m/z 459 [M+H]+;
1H NMR (400 MHz, CDCl3) δ 7.96 (d, J = 11.2 Hz, 2H), 7.76 (d, J = 9.6 Hz, 2H), 7.75–7.49 (m,
5H), 6.75 (s, 1H) 4.76 (s, 2H), 3.95 (t, J = 8.0 Hz, 2H), 3.90 (s, 3H), 3.38 (t, J = 6.8 Hz, 2H),
13
2.05–1.25 (m, 11H); C NMR (100 MHz, CDCl3) δ 184.5, 160.3, 156.3, 152.2, 146.1, 141.5,
135.6(2C), 129.4, 128.6(2C), 125.4, 124.6, 123.2(2C), 119.5, 117.9, 57.6, 49.9, 38.4, 34.0, 29.4,
27.9(2C), 25.9, 24.3, 23.4. Anal. calcd for C27H30N4O3: C, 70.72; H, 6.59; N, 12.22% Found C,
70.76; H, 6.66; N, 12.25%.
4.1.33. 1-(Cyclohexanecarbonyl)-N-(4-ethoxyphenyl)-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-
c]pyridine-5(4H)-carboxamide (6bg): Yield: 64%, m.p. 177–178 °C; MS(ESI) m/z 473 [M+H]+;
1H NMR (400 MHz, CDCl3) δ 8.05–7.56 (m, 9H), 4.69 (s, 2H), 4.11–4.03 (m, 2H), 3.68 (t, J =
8.4 Hz, 2H), 3.12 (t, J = 8.0 Hz, 2H), 2.41 (s, 1H), 2.45–1.25 (m, 10H), 1.39 (t, J = 8.0 Hz, 3H):
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ACCEPTED MANUSCRIPT
13C NMR (100 MHz, CDCl3) δ 176.9, 164.7, 155.4, 149.6, 147.5, 140.1, 134.2(2C), 125.2,
124.7(2C), 123.6, 123.0, 121.4(2C), 118.2, 115.7, 60.5, 54.9, 48.9, 39.6, 33.8, 27.7, 26.3(2C),
24.9(2C), 21.9. Anal. calcd for C28H32N4O3: C, 71.16; H, 6.83; N, 11.86% Found C, 71.21; H,
6.91; N, 11.92%.
4.1.34. 1-(Cyclohexanecarbonyl)-N-(naphthalen-1-yl)-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-
c]pyridine-5(4H)-carboxamide (6bh): Yield: 74%, m.p. 184–186 °C; MS(ESI) m/z 478 [M+H]+;
1H NMR (400 MHz, CDCl3) δ 8.05–7.15 (m, 13H), 4.66 (s, 2H), 3.72 (t, J = 7.6 Hz, 2H), 3.03 (t,
J = 8.0 Hz, 2H), 2.19–1.30 (m, 11H): 13C NMR (100 MHz, CDCl3) δ 171.8, 166.6, 145.6, 141.7,
138.7, 136.6, 135.2(2C), 134.3(2C), 131.6, 126.3, 126.0, 125.8, 124.6(2C), 123.4, 122.7(2C),
120.6, 119.5, 60.6, 47.2, 29.4(2C), 28.6(2C), 26.3, 24.6, 24.1. Anal. calcd for C30H30N4O2: C,
75.29; H, 6.32; N, 11.71% Found C, 75.32; H, 6.29; N, 11.82%.
4.1.35. N-Benzyl-1-(cyclohexanecarbonyl)-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-
1
5(4H)-carboxamide (6bi): Yield: 69%, m.p. 176–178 °C; MS(ESI) m/z 443 [M+H]+; H NMR
(400 MHz, CDCl3) δ 7.98–7.28 (m, 10H), 6.78 (bs, 1H), 4.82 (s, 2H), 4.64 (d, 2H, J = 8.8 Hz),
13
3.66 (t, J = 7.6 Hz, 2H), 3.21 (t, J = 8.0 Hz, 2H), 2.34 (m, 1H), 2.19–1.30 (m, 10H): C NMR
(100 MHz, CDCl3) δ 176.6, 162.9, 149.3, 144.5, 139.5, 138.6, 137.6, 136.4(2C), 124.9, 124.2,
123.6, 121.9, 120.4(2C), 119.6, 118.4, 59.5, 55.4, 34.5, 28.5(2C), 28.4(2C), 27.4, 23.6, 22.5.
Anal. Calcd. for C27H30N4O2: C, 73.28; H, 6.83; N, 12.66% Found C, 73.43; H, 6.92; N, 12.75%.
4.1.36. N-(4-Chlorophenyl)-1-(cyclohexanecarbonyl)-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-
c]pyridine-5(4H)-carbothioamide (7ba): Yield: 67%, m.p. 192–195 °C; MS(ESI) m/z 479
1
[M+H]+; H NMR (400 MHz, CDCl3) δ 9.05 (s, 1H), 8.10 (d, J = 7.2 Hz, 2H), 7.92 (d, J = 7.2
Hz, 2H), 8.12–7.45 (m, 5H), 4.95 (s, 2H), 3.88 (t, J = 7.6 Hz, 2H), 3.02 (t, J = 7.2 Hz, 2H); 2.18–
13
1.28 (m, 11H): C NMR (100 MHz, CDCl3) δ 180.5, 172.6, 152.6, 143.7, 143.4, 141.6, 137.5,
137.2, 136.3, 132.7, 131.9, 128.5, 124.3(2C), 123.7, 119.3, 116.7, 59.2, 54.6, 31.6, 26.6(2C),
17

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26.1(2C), 24.3, 22.5. Anal. calcd for C26H27ClN4OS: C, 65.19; H, 5.68; N, 11.70% Found C,
65.27; H, 5.76; N, 11.79%.
4.1.37. 1-(Cyclohexanecarbonyl)-N-(4-fluorophenyl)-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-
c]pyridine-5(4H)-carbothioamide (7bb): Yield: 62%, m.p. 180–182 °C; MS(ESI) m/z 463
1
[M+H]+; H NMR (400 MHz, CDCl3) δ 8.02 (s, 1H), 7.92 (d, J = 8.4 Hz, 2H), 7.85 (d, J = 8.4
Hz, 2H), 7.62–7.35 (m, 5H), 4.75 (s, 2H), 3.85 (t, J = 7.6 Hz, 2H), 3.06 (t, J = 6.8 Hz, 2H),
2.31(m, 1H), 2.11–1.28 (m, 10H): 13C NMR (100 MHz, CDCl3) δ 184.7, 179.7, 151.8, 145.6,
144.6, 142.2, 140.9, 139.4, 136.4, 133.3, 131.6, 127.3, 124.7(2C), 123.4, 121.3(2C), 59.8, 58.5,
32.5, 27.8(2C), 26.5(2C), 25.6, 23.8. Anal. calcd for C26H27FN4OS: C, 67.51; H, 5.88; F, 4.11;
N, 12.11% Found C, 67.60; H, 5.93; N, 12.22%.
4.1.38.
1-(Cyclohexanecarbonyl)-N-(4-nitrophenyl)-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-
c]pyridine-5(4H)-carbothioamide (7bc): Yield: 74%, m.p. 178–180 °C; MS(ESI) m/z 490
1
[M+H]+; H NMR (400 MHz, CDCl3) δ 7.98–7.42 (m, 10H), 4.84 (s, 2H), 3.95 (t, J = 7.2 Hz,
13
2H), 3.08 (t, J = 7.6 Hz, 2H), 2.30 (s, 1H), 2.05–1.25 (m, 10H): C NMR (100 MHz, CDCl3) δ
181.4, 176.3, 153.4, 142.8, 141.6, 140.4, 139.4, 138.3, 136.5, 135.3, 129.9, 127.5, 123.9(2C),
122.5, 120.3, 119.5, 61.5, 49.6, 32.3, 27.4(2C), 25.7(2C), 25.6, 22.9. Anal. calcd for
C26H27N5O3S: C, 63.78; H, 5.56; N, 14.30%. Found C, 63.83; H, 5.62; N, 14.41%.
4.1.39. 1-(Cyclohexanecarbonyl)-3-phenyl-N-p-tolyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-
1
5(4H)-carbothioamide (7bd): Yield: 75%, m.p. 170–174 °C; MS(ESI) m/z 459 [M+H]+; H
NMR (400 MHz, DMSO-d6) δ 7.92–7.35 (m, 9H), 7.20 (s, 1H), 4.93 (s, 2H), 3.71 (t, J = 6.8 Hz,
2H), 2.86 (t, J = 7.2 Hz, 2H), 2.49 (s, 3H), 2.24–1.35 (m, 11H): 13C NMR (100 MHz, DMSO-d6)
δ 186.7, 166.7, 148.4, 143.9, 142.4, 139.9, 137.2, 136.9, 134.7, 133.9, 132.3, 132.0, 128.9,
125.7(2C), 123.4, 120.7(2C), 56.2, 45.0, 33.3, 27.7(2C), 25.0(2C), 24.3, 22.7. Anal. calcd for
C27H30N4OS: C, 70.71; H, 6.59; N, 12.22% Found C, 70.78; H, 6.69; N, 12.26%.
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4.1.40. 1-(Cyclohexanecarbonyl)-N-(4-methoxyphenyl)-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-
c]pyridine-5(4H)-carbothioamide (7be): Yield: 69%, m.p. 185–188 °C; MS(ESI) m/z 475
1
[M+H]+; H NMR (400 MHz, CDCl3) δ 9.12 (s, 1H), 7.92–7.38 (m, 9H), 4.85 (s, 2H), 4.18 (s,
13
3H) 3.69 (t, J = 7.6 Hz, 2H), 2.95 (t, J = 8.0 Hz, 2H), 2.35 (m, 1H), 2.05–1.25 (m, 10H): C
NMR (100 MHz, CDCl3) δ 179.2, 163.3, 143.9, 141.4, 139.3, 138.6, 137.4, 136.5, 133.5(2C),
132.7, 131.6, 130.7, 129.6(2C), 124.1(2C), 123.7, 122.7(2C), 62.1, 57.9, 36.1, 31.3, 27.6(2C),
25.9(2C), 24.5, 24.3. Anal. calcd for C27H30N4O2S: C, 68.33; H, 6.37; N, 11.80%. Found C,
68.45; H, 6.46; N, 11.92%.
4.1.41. N-Benzyl-1-(cyclohexanecarbonyl)-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-
1
5(4H)-carbothioamide (7bf): Yield: 61%, m.p. 189–192 °C; MS(ESI) m/z 459 [M+H]+; H
NMR (400 MHz, DMSO-d6) δ 10.15 (bs, 1H), 7.81–7.42 (m, 10H), 4.79 (s, 2H), 4.39 (d, J = 7.2
13
Hz, 2H), 3.99 (t, J = 7.6 Hz, 2H), 3.08 (t, J = 8.0 Hz, 2H), 2.25–1.35 (m, 11H): C NMR (100
MHz, DMSO-d6) δ 176.8, 162.7, 142.9, 140.7, 138.7, 137.2, 135.3, 134.7, 133.9, 132.6, 131.4,
129.7, 124.3(2C), 123.7, 121.4(2C), 58.7, 56.1, 36.6, 34.6, 27.4(2C), 26.1(2C), 25.2, 23.2. Anal.
calcd for C27H30N4OS: C, 70.71; H, 6.59; N, 12.22%. Found C, 71.01; H, 6.68; N, 12.54%.
4.1.42. Cyclohexyl(5-(4-fluorophenylsulfonyl)-3-phenyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-
1
c]pyridin-1-yl)methanone (9ba): Yield: 62%, m.p. 186–188 °C; MS(ESI) m/z 468 [M+H]+; H
NMR (400 MHz, DMSO-d6) δ 8.02–7.38 (m, 9H), 4.56 (s, 2H), 3.65 (t, J = 8.4 Hz, 2H), 3.08 (t,
J = 8.0 Hz, 2H), 2.19–1.16 (m, 11H): 13C NMR (100 MHz, CDCl3) δ 179.8, 148.1, 140.6, 138.8,
137.5, 136.4(2C), 135.8, 134.8, 133.7, 130.7, 126.6, 123.2(2C), 119.5, 118.9, 62.5, 39.2, 34.5,
29.2(2C), 25.7(2C), 24.4, 22.3. Anal. calcd for C25H26FN3O3S: C, 64.22; H, 5.60; N, 8.99%.
Found 64.32; H, 5.67; N, 9.20%.
4.1.43.
Cyclohexyl(5-(4-nitrophenylsulfonyl)-3-phenyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-
1
c]pyridin-1-yl)methanone (9bb): Yield: 81%, m.p.192–193 °C; MS(ESI) m/z 495 [M+H]+; H
19

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NMR (400 MHz, CDCl3) δ 7.93–7.29 (m, 9H), 4.76 (s, 2H), 3.88 (t, J = 7.6 Hz, 2H), 3.31 (t, J =
8.0 Hz, 2H), 2.25–1.30 (m, 11H): 13C NMR (100 MHz, CDCl3) δ 182.6, 142.6, 139.6, 138.4,
136.3, 134.6(2C), 133.5, 132.7, 132.2, 129.6, 127.8, 121.6(2C), 120.7, 119.7, 59.6, 45.2, 33.6,
28.6(2C), 26.9(2C), 25.7, 24.3. Anal. calcd for C25H26N4O5S: C, 60.71; H, 5.30; N, 11.33%.
Found C. 60.83; H, 5.36; N, 11.39%.
4.1.44. 1-(4-(1-(Cyclohexanecarbonyl)-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridin-5(4H)-
ylsulfonyl)phenyl)ethanone (9bc): Yield: 84%, m.p. 206–208 °C; MS(ESI) m/z 492 [M+H]+; 1H
NMR (400 MHz, CDCl3) δ 7.78–6.92 (m, 9H), 4.72 (s, 2H), 3.75 (t, J = 8.0 Hz, 2H), 3.16 (t, J =
7.6 Hz, 2H), 2.49 (s, 3H) 2.21–1.28 (m, 11H): 13C NMR (100 MHz, CDCl3) δ 176.4, 171.5,
148.5, 139.6, 138.3, 137.5, 136.7(2C), 135.7, 134.6, 131.4, 127.5, 126.2, 122.5, 120.1, 119.8,
118.4, 63.4, 51.6, 49.6, 32.7, 28.3(2C), 27.4(2C), 24.8, 21.9. Anal. calcd for C27H29N3O4S: C,
65.97; H, 5.95; N, 8.55%. Found C, 66.02; H, 5.99; N, 8.64%
4.1.45.
Cyclohexyl(3-phenyl-5-tosyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-
1
yl)methanone (9bd): Yield: 69%, m.p. 194–196 °C; MS(ESI) m/z 464 [M+H]+; H NMR (400
MHz, CDCl3) δ 7.86–7.34 (m, 9H), 4.85 (s, 2H), 3.75 (t, J = 8.0 Hz, 2H), 3.12 (t, J = 7.6 Hz,
2H), 2.56 (s, 3H), 2.25–1.32 (m, 11H): 13C NMR (100 MHz, CDCl3) δ 177.9, 146.5, 139.4,
136.9, 133.4, 132.3(2C), 130.3, 129.7(2C), 127.8, 126.3(2C), 122.7(2C), 121.7, 119.5, 64.1,
38.8, 35.7, 30.3(2C), 26.8(2C), 25.8, 23.6. Anal. calcd for C26H29N3O3S: C, 67.36; H, 6.31; N,
9.06%. Found C, 67.43; H, 6.39; N, 9.16%.
4.1.46.
Cyclohexyl(3-phenyl-5-(thiophen-2-ylsulfonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-
1
c]pyridin-1-yl)methanone (9be): Yield: 75%, m.p. 210–214 °C; MS(ESI) m/z 456 [M+H]+; H
NMR (400 MHz, CDCl3) δ 7.69–6.98 (m, 8H), 4.42 (s, 2H), 3.32 (t, J = 7.6 Hz, 2H), 2.98 (t, J =
13
8.0 Hz, 2H), 2.30 (m, 1H), 2.22–1.31 (m, 10H): C NMR (100 MHz, CDCl3) δ 172.6, 145.6,
138.3, 136.7, 131.8, 129.6, 125.7, 124.7, 123.6, 122.6, 121.4, 120.4, 119.2, 118.6, 56.9, 46.9,
20

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31.3(2C), 27.8(2C), 26.7, 24.5, 23.6. Anal. calcd for C23H25N3O3S2: C, 60.63; H, 5.53; N, 9.22%.
Found C, 60.73; H, 5.59; N, 9.31%.
4.2. Biological activity
4.2.1. Protein expression and MTB PS screening
The M. tuberculosis panC gene (Rv3602c) encoding the pantothenate synthetase was cloned and
transformed into BL21 (DE3) cells and the expression of the protein was performed as reported
in literature [2]. For the assay, in a 96-well plate, 60 µL of PS reagent mix containing NADH,
pantoic acid, β-alanine, ATP, phosphoenolpyruvate, MgCl2, myokinase, pyruvate kinase, and
lactate dehydrogenase in buffer was added. Compounds were then added to plates in 1-µL
volumes. The reaction was initiated with the addition of 39 µL of PS, diluted in buffer. The test
plate was immediately transferred to a microplate reader, and absorbance was measured at 340
nm every 12 sec for 120 sec [2]. Percentage inhibition was calculated using following formula,
%Inhibition = 100 X 1- compound rate-background rate
full reaction rate-background rate
4.2.2. In-vitro MTB screening
Two-fold serial dilutions of each test compound/drug were prepared and incorporated into
Middle- brook 7H11 agar medium with oleic acid, albumin, dextrose, and catalase (OADC)
growth supplement to get final concentrations of 50, 25, 12.5, 6.25, 3.13, 1.56, and 0.78 µg/mL.
Inoculum of M. tuberculosis H37Rv ATCC 27294 was prepared from fresh Middlebrook 7H11
agar slants with OADC (Difco) growth supplement adjusted to 1 mg/ mL (wet weight) in Tween
80 (0.05%) saline diluted to 10−2 to give a concentration of ∼107cfu/mL. Five microliters of this
21

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bacterial suspension was spotted onto 7H11 agar tubes containing different concentrations of the
drug as discussed above. The tubes were incubated at 37 °C, and final readings (as MIC in
µg/mL) were determined after 28 days. The MIC is defined as the minimum concentration of
compound required to give complete inhibition of bacterial growth. This method is similar to that
recommended by the National Committee for Clinical Laboratory Standards for the
determination of MIC in triplicate [11].
4.2.3. In vitro cytotoxicity screening
Some compounds were further examined for toxicity in a RAW 264.7 cell line at the
concentration of 50 µM. After 72 h of exposure, viability was assessed on the basis of cellular
conversion of MTT into a formazan product using the Promega Cell Titer 96 non-radioactive cell
proliferation assay [12].
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Acknowledgements
The authors are thankful to Department of Science and Technology (SR/S1/OC-70/2010), New
Delhi, India for their financial assistances, and Ganesh Samala is thankful to Council of
Scientific and Industrial Research (09/1026/0008/2012-EMR-1) for providing Junior Research
Fellowship. We also acknowledge Dr David Eisenberg and Dr Shuishu Wang, Department of
Biochemistry and Molecular biology, USUHS, Maryland for providing MTB PS clone.
References
[1] C. Padmapriyadarsini, G. Narendran, S. Swaminathan, Ind. J. Med. Res. 134 (2011) 850–865.
[2] R. Zheng, J.S. Blanchard, Biochem. 40 (2001) 12904–12912.
[3] V.K. Sambandamurthy, X. Wang, B. Chen, R.G. Russell, S. Derrick, F.M. Collins, S.L.
Morris, W.R. Jr. Jacobs, Nat. Med. 8 (2002) 1171–1174.
[4] V.K. Sambandamurthy, S.C. Derrick, K.V. Jalapathy, B. Chen, R.G. Russell, S.L. Morris,
W.R. Jr. Jacobs, Infect. Immun. 73 (2005) 1196–1203.
[5] S. Wang, D. Eisenberg, Prot. Sci.12 (2003) 1097–1108.
[6] S. Velaparthi, M. Brunsteiner, R. Uddin, B. Wan, S.G. Franzblau, P.A. Petukhov, J. Med.
Chem. 51 (2008) 1999–2002.
[7] Y. Yang, P. Gao, Y. Liu, X. Ji, M. Gan, Y. Guan, X. Hao, Z. Li, C. Xiao, Bioorg. Med.
Chem. Lett. 21 (2011) 3943–3946.
[8] E.L. White, K. Southworth, L. Ross, S. Cooley, R.B. Gill, M.I. Sosa, A. Manouvakhova, L.
Rasmussen, C. Goulding, D. Eisenberg, T.M. Fletcher, J. Biomol. Screen. 12, (2007) 100–
105.
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[9] A.W. Hung, H.L. Silvestre, S. Wen, A. Ciulli, T.L. Blundell, C. Abell, Angew. Chem. 48
(2009) 8452–8456.
[10] X.M. Ye, A.W. Konradi, J. Smith, D.L. Aubele, A.W. Garofalo, J. Marugg, M.L. Neitzel,
C.M. Semko, H.L. Sham, M. Sun, A.P. Truong, J. Wu, H. Zhang, E. Goldbach, J.M. Sauer,
E.F. Brigham, M. Bova, G.S. Basi, Bioorg. Med. Chem. Lett. 20, (2010) 2195–2199.
[11] National Committee for Clinical Laboratory Standards. Antimycobacterial susceptibility
testing for Mycobacterium tuberculosis. Proposed standard M24- T. National Committee for
Clinical Laboratory Standards, Villanova, PA, 1995.
[12] Gundersen, L. L.; Meyer, J. N.; Spilsberg, B. J. Med. Chem. 2002, 45, 1383.
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Figure 1: MTB PS inhibitors [6-9]
Figure 2: Compound (6aa) identified from our database by virtual High Throughput Screening
(vHTS)
Figure 3: Dose response curve for compound 6ac.
Scheme 1: Synthetic protocol of titled compounds
25

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Table 1: Biological activities of synthesized compounds
R2
R2
R2
N
O
O
O
N
N
N
N
N
N
N
N
O
S O
R1
O
NH
R1
S
NH
R1
6aa - 6ai
6ba - 6bi
7aa - 7af
7ba - 7bf
9aa - 9ae
9ba - 9be
Compd
R1
R2
% inhibition at 100
MTB MIC
in µM
Cytotoxicity at 50 µM
(RAW 264.7 cells) %
µM (IC50)
against MTB PS
60.6 (82.1±2.3)
40.2
95.7 (21.8±0.8)
43.7
49.4
47.2
89.8 (38.2±1.7)
46.2
inhibition
42.0
4.9
0
6aa
6ab
6ac
6ad
6ae
6af
6ag
6ah
6ai
4-Bromophenyl
4-Chlorophenyl
4-Nitrophenyl
4-Acetylphenyl
p-Tolyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
99.7
109.43
26.7
>107.6
114.1
>107.1
48.8
12.8
57.1
0
4.7
0.7
10.6
0
0.5
0
0
4-Ethoxyphenyl
1-Naphthyl
Benzyl
Isopropyl
114.1
128.7
105.7
109.5
>110.4
106.7
110.4
>124.22
54.17
25.58
51.49
109.31
55.61
98.54
108.0
52.79
108.1
56.49
109.0
105.8
52.19
54.51
109.0
105.3
53.5
40.9
37.7
43.5
23.9
10.2
52.1
41.7
7aa
7ab
7ac
7ad
7ae
7af
4-Chlorphenyl
4-Fluorophenyl
p-Tolyl
4-Methoxyphenyl
Benzyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
0
6
0
Allyl
9aa
9ab
9ac
9ad
9ae
6ba
6bb
6bc
6bd
6be
6bf
6bg
6bh
6bi
7ba
7bb
7bc
7bd
7be
7bf
9ba
9bb
9bc
9bd
4-Fluorophenyl
4-Nitrophenyl
4-Acetylphenyl
p-Tolyl
Thiophen-2-yl
4-Bromophenyl
4-Chlorphenyl
4-Nitrophenyl
4-Acetylphenyl
p-Tolyl
Phenyl
Phenyl
Phenyl
Phenyl
77.0 (38.6±0.9)
39.7
78.5 (39.1±2.2)
9.0
18.6
5.3
0
6.8
0
4.0
14.9
22.5
15.1
52.5
4.9
4.1
35.7
51.1
58.4
0
Phenyl
48.7
50.5
54.7
50.8
51.4
40.8
38.7
48.5
50.9
49.1
56.3
49.2
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
4-Methoxyphenyl
4-Ethoxyphenyl
1-Naphthyl
Benzyl
4-Chlorphenyl
4-Fluorophenyl
4-Nitrophenyl
p-Tolyl
4-Methoxyphenyl
Benzyl
4-Fluorophenyl
4-Nitrophenyl
4-Acetylphenyl
p-Tolyl
47.3
50.5
50.6
51.4
46.8
50.2
39.2
41.6
51.07
101.7
108.0
53.5
50.5
50.8
27.8
9.5
20.6
1.4
8.3
11.5
0
53.9
26

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9be
Thiophen-2-yl
Isoniazid
Cyclohexyl
52.0
0
0
54.9
0.72
15.28
8.1
-
-
Ethambutol
Figure 1
O
CH3
NH
CH3
N
H3C
O
N
N
H2N
O
O
N
H
OCH3
A
IC50: 90 nM
MTB: 43%inhibition
at 128 uM
B
IC50: 22.44 uM
O
CH3
S
NH
O
O
O
O
OH
N
O
O
O
D
Nafronyl oxalate
MTB PS active at 10 ug/mL
MTB MIC not active at 6.25 ug/mL
C
KD 1.8 uM
27

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Figure 2
28