Design, synthesis, and pharmacological evaluation of fluorinated azoles as anti-tubercular agents

Article abstract of DOI:10.1002/ardp.201700294

Design, synthesis, and biological screening of 2,2-dimethyl-2,3-dihydrobenzofuran tethered 1,3,4-oxadiazole derivatives as anti-tubercular agents were described. The synthesis of the target compounds was conducted by a series of reaction schemes. All the synthesized compounds were characterized by IR, ¹H NMR, ¹³C NMR, and mass spectrometry. The therapeutic potential of the synthesized compounds was confirmed by molecular docking studies. Among the synthesized compounds, 12a, 12c, 12d, 12e, 12g, and 12j were found to be more active against non-replicating than against replicating cultures of Mycobacterium tuberculosis H37Ra ex vivo and in vitro. These compounds exhibit minimum inhibitory concentration (MIC) values in the range of 2.31–23.91 μg/mL. The cytotoxicity study was conducted against the cell lines THP-1, A549 and PANC-1, and the compounds were observed to be non-toxic to host cells. Molecular docking was conducted with InhA (FabI/ENR) and suggested the antimycobacterial potential of the synthesized compounds. The investigation presented here was found to be adventitious for the development of new therapeutic agents against Mycobacterium infection.

Full text of DOI:10.1002/ardp.201700294

Received: 21 September 2017
DOI: 10.1002/ardp.201700294
Revised: 26 November 2017
Accepted: 30 November 2017
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FULL PAPER
Design, synthesis, and pharmacological evaluation of
fluorinated azoles as anti-tubercular agents
Somnath Gholap1
Dhiman Sarkar2
Macchindra Tambe1
Laxman Nawale2
Manoj Damale4
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Jaiprakash Sangshetti3
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1 Postgraduate Department of Chemistry and
Research Centre, Padmashri Vikhe Patil
College, Pravaranagar, A/P-Loni kd, Tal.-
Rahata, Ahmadnagar, India
Abstract
Design, synthesis, and biological screening of 2,2-dimethyl-2,3-dihydrobenzofuran
tethered 1,3,4-oxadiazole derivatives as anti-tubercular agents were described. The
synthesis of the target compounds was conducted by a series of reaction schemes. All
the synthesized compounds were characterized by IR, 1H NMR, 13C NMR, and mass
spectrometry. The therapeutic potential of the synthesized compounds was confirmed
by molecular docking studies. Among the synthesized compounds, 12a, 12c, 12d, 12e,
12g, and 12j were found to be more active against non-replicating than against
replicating cultures of Mycobacterium tuberculosis H37Ra ex vivo and in vitro. These
compounds exhibit minimum inhibitory concentration (MIC) values in the range of
2.31–23.91 μg/mL. The cytotoxicity study was conducted against the cell lines THP-1,
A549 and PANC-1, and the compounds were observed to be non-toxic to host cells.
Molecular docking was conducted with InhA (FabI/ENR) and suggested the
antimycobacterial potential of the synthesized compounds. The investigation
presented here was found to be adventitious for the development of new therapeutic
agents against Mycobacterium infection.
2 Division of Organic Chemistry, Combichem-
Bioresource Centre, National Chemical
Laboratory, Pune, India
3 Y. B. Chavan College of Pharmacy,
Aurangabad, India
4 Bhagwan College of Pharmacy, Aurangabad,
India
Correspondence
Dr. Somnath Gholap, Postgraduate
Department of Chemistry and Research
Centre, Padmashri Vikhe Patil College,
Pravaranagar, A/P-Loni kd, Tal.-Rahata, Dist.-
Ahmadnagar, Pin-413713 (MS), India.
Email: ssgholap2002@gmail.com
K E Y W O R D S
1,3,4-oxadiazole, 2,2-dimethyl-2,3-dihydrobenzofuran, anti-tubercular agents, cytotoxicity,
molecular docking
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1
INTRODUCTION
to 1 year to cure the infection completely with complex medication
course.[8,9] The treatment of multi drug resistant tuberculosis suffered
Tuberculosis is a leading infectious bacterial disease caused by
Mycobacterium tuberculosis.[1,2] At present, approximately 30
Mycobacterium genus are responsible to cause human diseases among
the bacterial genus.[3] In particular, human immune deficiency virus
(HIV) infected persons are more susceptible to Mycobacterium
infections.[4] Nowadays, multi-drug resistant (MDR) tuberculosis is
more common,[5] as well as continuing problem of the extremely drug
resistant (XDR) or totally drug resistant (TDR) forms of tuberculosis.[6,7]
The available first line anti-TB chemotherapy required nearly 6 months
by toxic, poorly tolerated, and less effective medication for long-
time.[10] Hence, there is urgent need to develop effective drug
candidate against M. tuberculosis.
The 1,3,4-oxadiazole derivatives are well known nitrogen hetero-
cycles possessing broad spectrum of bioactivities and found to be good
bioisosters of amide and ester functionalities.[11] The structural
features of the oxadiazole core displayed interesting H-bond acceptor
properties, which improves lipophilicity profile and adequate ADME
properties.[12] Some of the 1,3,4-oxadiazole derivatives such as A, B, C,
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Arch Pharm Chem Life Sci. 2018;e1700294.
https://doi.org/10.1002/ardp.201700294
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
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and D was reported as good anti-TB activity with MIC 0.78 to
1.6 µg/mL, respectively[13] and zibotentan (E) was found to be potent
anticancer agent[14] (Figure 1).
acid (2) was synthesized by N-acetylation of 2-hydroxy-4-amino-
benzoic acid (1). The N-acetylated derivative on subsequent
nitration gaves 4-acetamido-2-hydroxy-5-nitrobenzoic acid (3).
The esterification of 4-acetamido-2-hydroxy-5-nitrobenzoic acid
followed by deprotection of N-acetyl was achieved in a one-pot
reaction with methanol and catalytic amount of sulfuric acid
furnished to methyl 4-amino-2-hydroxy-5-nitrobenzoate (4). The
alkylation of compound 4 with 3-chloro-2-methyl propene in N,N-
dimethyl formamide at 70°C gave methyl 2-(2-methyl allyloxy)-4-
amino-5-nitrobenzoate (5). The Claisen rearrangement of interme-
Moreover, 1,3,4-oxadiazole derivatives exhibit diverse biological
profile such as anti-HIV,[15] antimalarial,[16] analgesic,[17] antiinflam-
matory,[18] anticonvulsant,[19] and as lipid peroxidase inhibitors.[20]
Interestingly, the constrained analogue of isopropyl phenyl ether such
as 2,2-dimethyl-2,3-dihydrobenzofuran has been found in zatosetron
(F) as 5-HT3 antagonist[21] and derivative G as serotonin 2C agonist[22]
(Figure 2). In the view of the biocompatibility and biological potential of
1,3,4-oxadiazole as well as 2,2-dimethyl-2,3-dihydrobenzofuran
core, herein, we have reported the design, synthesis, molecular
docking, and biological screening of some new 1,3,4-oxadiazole based
2,3-dihydrobenzofuran derivatives (Scheme 1). The targeted com-
pounds were designed in such a way that the 1,3,4-oxadiazole and 2,2-
dimethyl-2,3-dihydrobenzofuran core will be conserved together with
incorporation of the groups as H-bond acceptors and H-bond donors
at particular sites[23,24] (Figure 3).
diate compound
5 was conducted with anhydrous AlCl3 in
dichloromethane to afforded methyl-4-amino-2,3-dihydro-2,2-di-
methyl-5-nitrobenzofuran-7-carboxylate (6). Sandmayer reaction of
compound 6 was furnished compound methyl-2,3-dihydro-4-iodo-
2,2-dimethyl-5-nitrobenzofuran-7-carboxylate 7, which was further
hydrolyzed using LiOH to afforded 2,3-dihydro-4-iodo-2,2-di-
methyl-5-nitrobenzofuran-7-carboxylic acid (8) followed by treat-
ment with oxalyl chloride and 4-trifluoromethyl phenyl hydrazide to
give
4-iodo-5-nitro-2,2-dimethyl-N-[4-(trifluoromethyl)benzoyl]-
2,3-dihydrobenzofuran-7-carbohydrazide (9). The N-benzoyl hydra-
zide (9) on cyclization with POCl3 afforded 2-[4-(trifluoromethyl)
phenyl-5-(2,3-dihydro-4-iodo-2,2-dimethyl-5-nitrobenzofuran-7-
yl]-1,3,4 oxadiazole (10). The compound 10 on reduction with Fe
followed by the reaction of pivolyl chloride in THF at room
temperature afforded N-(7-(5-(4-(trifluoromethyl)phenyl)-1,3,4-ox-
adiazol-2-yl)-2,3-dihydro-4-iodo-2,2-dimethyl benzofuran-5-yl)piv-
alamide (11). The compound 11 on Sonogashira coupling[32–37] with
terminal alkynes using PdCl2(PPh3)2 at 80°C furnished target
compounds 12a–j in a good to excellent yield (70–85%) (Scheme
1, Figure 4, Table 1). The purification of all synthesized compounds
was conducted by column chromatography using methanol/
dichloromethane (4%) as eluent system. As representative IR
spectrum of 12a showed absorption at 3426 cm−1 due to the
presence of amide (-NH stretching), absorption at 2211 cm−1 due to
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2
RESULTS AND DISCUSSION
In continuation to our ongoing research on the synthesis of biologically
active oxadiazole derivatives,[25–31] herein, we have reported the
synthesis of novel structurally diverse 1,3,4-oxadiazole possessing 2,2-
dimethyl-2,3-dihydrobenzofuran core (12a–j) (Table 1) and evaluated
their biological potential. The structure of the synthesized compounds
was confirmed by their characterization data obtained by IR, NMR, and
mass spectral analysis study.
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2.1 Chemistry
The target compounds 12a–j were synthesized as depicted in
Scheme 1. The starting compound 4-acetamido-2-hydroxy benzoic
FIGURE 1 Potentially active 1,3,4-oxadiazole derivatives

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FIGURE 2 Bioactive 2,2-dimethyl-2,3-dihydrobenzofuran derivatives
−1
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�
�
C
C, at 1678 cm due to C O stretching of amide group, peak at
2.2 Biological activity
1494 cm−1 due to C─F streching, absorption at 1324 cm−1 due to
C
N. The NMR spectrum of 12a, 1H NMR (300 MHz, DMSO-d6): δ
2.2.1 Anti-tubercular activity studies
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�
1.28 (s, 9H), 1.58 (s, 6H), 3.28 (s, 2H), 7.48 (d, J = 7.6 Hz, 3H), 7.55 (d,
J = 7.6 Hz, 2H), 7.92 (s, 1H), 8.03 (d, J = 7.8 Hz, 2H), 8.30 (d,
J = 7.8 Hz, 2H), 9.15 (s, 1H, exchangeable with D2O); 13C NMR
(125.7 MHz, CDCl3): δ 27.8, 28.5, 40.2, 42.9, 88.4, 89.6, 90.7, 106.1,
117.6, 120.8, 126.1, 127.3, 127.5, 127.6, 128.7, 128.9, 129.7,
130.2, 131.7, 131.8, 132.0, 132.2, 135.9, 153.6, 162.9, 176.2. The
mass spectrum of 12a (M.F. C32O3N3F3H28, M.W. = 559.57) showed
molecular ion peak at m/z = 560.03 (M+H)+.
The synthesized compounds 12a–j were screened for their ex vivo/in
vitro antitubercular activity against M. tuberculosis H37Ra and M.
bovis BCG with concentrations of 30, 10, and 3 μg/mL using XRMA
(H37Ra) and nitrate reductase model (BCG) as per protocols
reported in literature.[38–40] The percentage inhibition at above
concentrations was calculated and collected in Table S1 (Supporting
Information). The compounds 12c, 12g, and 12j exhibited higher
SCHEME 1 Synthesis of 1,3,4-oxadiazole derivatives. Reagents and conditions: (a) Ac2O, EtOH, reflux, 3 h, 88%. (b) NaNO3, TFA, 0°C, 3 h,
70%. (c) Methanol, H2SO4, reflux, 24 h, 97%. (d) 3-Chloro-2-methyl propene, K2CO3, DMF, 70°C, 5 h, 80%. (e) Anhy. AlCl3, CH2Cl2, −78 to
25°C, 24 h, 83%. (f) Conc. HCl, NaNO2, KI, 0°C, 3 h, 90%. (g) LiOH, THF, H2O, 2 h, 93.90%. (h) CH2Cl2, oxalyl chloride, 25°C, 2 h, 85%. (i) 4-
Trifluoromethyl phenyl hydrazide, TEA, THF, 0°C, (ii) POCl3, reflux, 10 h, 74.1%. (j) Fe, MeOH, conc. HCl, 1 h, 80%. (k) Pivolyl chloride, TEA,
THF, 25°C, 76.8%. (l) Acetylene, PdCl2(PPh3)2, TBAF, DMSO, 80°C, 3 h, 70–85%

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2.2.2 Cytotoxicity studies
The cytotoxicity study of the synthesized compounds 12c, 12g, 12j,
12a, 12d, and 12e was conducted as per reported procedures[40] using
various concentrations as 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, and
0.7813 µg/mL against three human cancer cell lines (THP-1, A549, and
PANC-1)[40] (Table 3). In order to interpret the cytotoxicity results, the
synthesized compounds and indication of their specific inhibition
against MTB, the compounds 12c, 12g, and 12j were evaluated for the
determination of GI50/GI90 values. It has been found that all of the
compounds showed very low cytotoxicity at GI50 <90; >100 μM
against THP-1 and >147 μM against A549 and PANC-1 cells. The
selectivity of 12a, 12c, 12d, 12e, 12g, and 12j toward human cell lines
against MTB and BCG is described in terms of the selectivity index
(Table 4).
FIGURE 3 Molecular structure of designed compound
anti-mycobacterial potency (inhibiting >90% of mycobacterial
growth at 30 μg/mL with rifampicin as reference drug). In general,
the newly synthesized compounds showed excellent selectivity
toward M. tuberculosis H37Ra and M. bovis BCG strains. The anti-
tubercular data obtained showed growth inhibition MTB and BCG
can be imparted by the introduction of a hydroxyprop-1-ynyl group
at 4-position of 12j. Most of the compounds were more active
against non-replicating than replicating cultures of M. tuberculosis
H37Ra by ex vivo as well as by in vitro. The percentage inhibition at
above concentrations was calculated and collected in Table S1.
Three of the compounds viz. 12c, 12g, and 12j showed MIC in the
range of 2.31–23.91 μg/mL proving their anti-tubercular potential
(Table 2).
The selectivity index (SI) reflects the concentration of the
compound at which it is active against mycobacteria M. bovis BCG
but is not toxic toward host cells. The higher selectivity index value, the
compound can be used as a therapeutic agent. The synthesized
compound “12j” showed a very high SI index, which is actually good
inhibitor of M. tuberculosis and M. bovis BCG. Although the selectivity
index of rifampicin is very high, it is important to consider the
significance of this study with respect to the developing resistance
TABLE 1 Synthesis of 1,3,4-oxadiazole bearing 2,2-dimethyl-2,3-dihydrobenzofuran derivatives (12a–j)
Entry
R
Compound (12)
12a
Time (h)
Yield (%)a,b,c
M.P. (°C)
1.
2.5
85
188–189
2.
3.
4.
5.
12b
12c
12d
12e
2.0
2.5
2.3
4.0
82
80
82
75
171–172
172–173
176–177
190–191
6.
7.
12f
5.5
6.3
78
70
198–199
212–213
12g
8.
12h
12i
12j
4.5
4.5
4.0
75
70
75
126–127
132–133
182–183
9.
10.
aIsolated yields of the products after purification.
bYields of the products in final step.
cAll synthesized compounds were characterized by IR, 1H-NMR, 13C-NMR and mass spectral data.

GHOLAP ET AL.
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FIGURE 4 Structures of synthesized compounds
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among microorganisms against available antibiotics. According to a
study of Hartkoorn et al.,[41] on the drug susceptibility of TB,
antimycobacterial activity was considered to be specific when the
selectivity index was >10. In the current report, compound 12j
exhibited highest selectivity index of >10, indicating its potential as an
antitubercular agent, and thus it should be investigated further.
2.3 Molecular docking study
Furthermore, the molecular docking study supports the InhA
inhibition, binding mode, and identification of the structural features
of the active molecules from series of synthesized compounds. As InhA
(FabI/ENR) is an enoyl-ACP reductase as one of the key enzymes of M.
tuberculosis implicated in the biosynthesis of cell wall constituents such
as mycolic acids, the docking study was conducted with the
mycobacterial enoylreductase (InhA) (PDB code 4TZK).[42–44]* [*The
complex X-ray crystal structures of enoyl-ACP reductase with
inhibitors pyrrolidine carboxamide (PDC) for Mycobacterium was
retrieved from the RCSB protein data bank (http://www.rcsb.org/
pdb) and used for the docking study. The molecular docking study has
been performed with Tripos SYBYL X 2. 2.1 program.[45]
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2.2.3 Antibacterial screening study
The anti-tubercular screening results strengthen the fact that the
synthesized compounds will exhibit antimicrobial activity. Therefore,
the antibacterial screening of the synthesized compounds was
conducted against four bacteria strains (Gram-negative strains: E.
coli, S. aureus; Gram-positive strains: P. aeruginosa and B. subtilis).[40]
The compounds 12a, 12c, 12d, 12e, 12g, and 12j showed higher
In the previous studies of synthesis and biological evaluation of
1,3,4-oxadiazole, derivative has been reported for inhibition of
mycobacterial enoylreductase (InhA) which made a firm basis for
selection of this enzyme as the potential target. The molecular docking
specificity toward MTB as these compounds showed nil anti-bacterial
[40]
activity up to 100 μg/mL. (Table 5)
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TABLE 2 Experimentally determined anti-mycobacterial activity (ex
vivo/in vitro) of the key compounds
molecular interactions study between active site amino acid residue
and components of the molecule was carried out to understand the
thermodynamic stability of different 1,3,4-oxadiazole bearing 2,2-
dimethyl-2,3-dihydrobenzofuran derivatives which also provide infor-
mation about binding modes observed within the active site cavity. The
theoretical values of the molecular docking study are presented in
Table 6.
IC50 and IC90 values (µM) of compounds with
SD (±) values
M. tuberculosis
H37Ra (ATCC
25177)
M. tuberculosis
H37Ra (ATCC
25177)
M. bovis BCG
(ATCC 35743)
Ex vivo
(dormant)
In vitro
(dormant)
In vitro
(dormant)
To represent the details of docking score following terms is used
as Total score as Total Docking Score, Crash Score as degree of
inappropriate penetration by the ligand into the protein and of
interpenetration between ligand atoms that are separated by rotatable
bonds of compounds and Polar Score gives an idea about the
contribution of the polar non-hydrogen bonding interactions to the
total score is shown in Table 6. The value of Total Score (inhibition
constant-logKi) provides an indication of how potent an inhibitor is.
Higher the value for the total score (inhibition constant-logKi), more is
the potency of inhibitor.[50] The high value of total score indicates
potency of highest value 12j which indicate that it is most active among
the six docked molecules. The compound binds and inhibits with a total
docking score of 7.3242, Crash Score of −3.1839 which indicate
degree of inappropriate penetration by the ligand into the protein and
of interpenetration between ligand atoms that are separated by
rotatable bonds and Polar Score of 1.0449 contributions of the polar
non-hydrogen bonding interactions to the total score.
Compound
12a
IC90
IC90
IC90
>53.67
>53.67
>53.67
12c
40.24 ± 1.05
>57.25
43.39 ± 0.29
>57.25
24.46 ± 1.28
>57.25
12d
12e
>47.77
>47.77
>47.77
12g
19.22 ± 0.6
4.50 ± 0.69
0.972 ± 0.04
21.22 ± 1.11
5.67 ± 0.89
0.911 ± 0.01
19.73 ± 1.55
5.40 ± 0.92
1.009 ± 0.02
12j
Rifampicin
SD (±), standard deviation.
study will act as a key indicator to understand the basis of molecular
inhibition of enoyl-ACP reductase activity.[46–49] It was also helped in
correlation of in vivo and in vitro activity of the newly synthesized
compounds.
The detailed analysis of the binding interactions and binding
pose of 12j showed that it is stabilized within the active site of InhA
through an extensive network of favorable non-covalent interactions
such as π interaction, π–π T-shaped interaction, alkyl interaction,
and π-alkyl interactions. As a part of non-covalent interactions,
fluorine atom of tri-fluoromethyl interacts with amino acids such as
Asp42 and Agr43 forms H–F interactions with distance of 2.81, 3.14,
and 275 Å, respectively. π–π T-shaped interactions Phe41 with aryl
ring of distance 4.67 Å. The hydrophobic amino acid Ile16 forms π
and π-alkyl interactions with aryl, oxadiazole, and methyl groups of
The computational (theoretical) prediction data from the molecu-
lar docking study was found to be replicated in the results of
experimental anti-tubercular activity. All the compounds 12a, 12b,
12c, 12d, 12e, 12f, and 12j were successfully docked into the active
site of target enzyme mycobacterial enoylreductase (InhA/FabI/ENR)
and it has been observed that they have varying degrees of affinity to
the active site residues. Majority of the amino acids present in the
active site cavity such as glycine, alanine, leucine, isoleucine, proline,
phenylalanine, tyrosine, methonine, arginine, lysine, aspartic acid, and
glutamic acid. Depending on the physicochemical properties, these are
non-polar or hydrophobic, polar charged, non-charged, negatively
charged, and positively charged amino acid residues. The detailed
benzofuryl ring with
a distance of 4.66, 4.33, and 4.89 Å,
respectively. The amino acid Ala198 forms π and π-alkyl intera-
ctions with dihydrobenzofuryl group and methyl substituent of
TABLE 3 Cytotoxicity of selected compounds in three human cancer cell lines after 48 h of exposure
Cytotoxic profile of key compounds against human cancer cell lines with SD (±) values
A549
PANC-1
THP-1
Compound ID
GI50
GI90
GI50
GI90
GI50
GI90
12a
>178.89
>178.89
>181.49
>190.84
>159.24
>151.98
>194.93
0.083 ± 0.09
>178.89
>181.49
>190.84
>159.24
>151.98
147.54 ± 0.97
0.150 ± 0.17
>178.89
>181.49
>190.84
>159.24
>151.98
>194.93
6.693 ± 0.15
>178.89
>178.89
>181.49
>190.84
>159.24
>151.98
>194.93
0.088 ± 0.2
12c
>181.49
101.00 ± 1.09
>190.84
12d
>190.84
12e
>159.24
>159.24
12g
>151.98
112.67 ± 1.03
169.90 ± 0.76
0.006 ± 0.00014
12j
193.37 ± 0.87
0.004 ± 0.00012
cPaclitaxel
SD (±), standard deviation.

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TABLE 4 Selectivity index (SI) of selected key compounds on three human cell lines against M. tuberculosis H37Ra (ex vivo/in vitro) and M. bovis
BCG (in vitro)
SI on A549
Ex vivo
SI on PANC-1
Ex vivo
SI on HeLa
Ex vivo
In vitro
In vitro
In vitro
In vitro
In vitro
In vitro
H37Ra
H37Ra
BCG
H37Ra
H37Ra
BCG
H37Ra
H37Ra
BCG
Entry
12a
SI against dormant stage of M. tuberculosis H37Ra and M. bovis BCG
3
3
3
3
0
3
3
3
3
12c
5
4
7
5
0
7
3
2
4
12d
3
3
3
3
0
3
3
3
3
12e
3
3
3
3
0
3
3
3
3
12g
8
7
8
8
1
8
6
5
6
12j
43
125
34
133
36
120
33
125
11
167
27
120
38
125
30
133
31
120
Rifampicin
dihydrobenzofuryl group with atomic distance of 3.27 and 4.33 Å,
respectively. The hydrophobic amino acids such as Ile21 and polar
charged amino acid form π-alkyl interactions with acetyl methyl
hydrogen of distance of 4.87, 3.99, and 4.59 Å, respectively, as
shown in Figure 5.
molecular weight (<500), partition coefficient LogP (<5), number of
hydrogen bond donor (<10), number of hydrogen bond acceptor
(<5), number of rotatable bonds (<10), total polar surface area
(<75 Å2). The % ABS: percentage absorption also predicated using
formula ABS = 109 − (0.345 × TPSA). To increase the rate of drug
likeness predication Veber suggested some variations like molecular
weight can be considered above 500 Da, total polar surface area
(<150 Å2), partition coefficient LogP (<5.6). All the compounds
showed significant values for the various parameters analyzed and
showed good drug-like characteristics based on Lipinski's rule of five
and its variants that characterized that these agents as likely orally
active.[52] The values of % ABS, polar surface area (PSA), n-HBA, and
n-HBD for synthesized compounds 12a, 12c, 12d, 12e, 12g, and 12j
indicated good oral bioavailability as depicted in Table 7. The in silco
assessment of all the synthetic compounds showed that they have
very good pharmacokinetic properties which was reflected in their
physicochemical values and which ultimately contributing for
pharmacological properties of these molecules.
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2.4 The physicochemical and pharmacokinetic
parameters prediction studies
The physicochemical and pharmacokinetic parameters prediction
was conducted by calculation and analysis of various physical
descriptors and pharmaceutical relevant properties for ADMET
prediction by using FAFDrugs2 and data is summarized in Table 7.
The drug likeness was predicted by analyzing absorption, distribu-
tion, metabolism, and elimination (ADME) parameters based on
Lipinski's rule of five and its variants.[51] This approach has been
widely used as a filter for substances that would likely be further
developed for drug design programs who has assumptions as
TABLE 5 Antibacterial activity against E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis
In vitro anti-bacterial activity of selected key compounds
E. coli
P. aeruginosa
S. aureus
B. subtilis
Gram-negative
Gram-positive
Entry
12a
IC90 (µM)
177.17 ± 2.53
>181.49
IC90 (µM)
175.56 ± 6.96
>181.49
IC90 (µM)
151.06 ± 5.24
>181.49
IC90 (µM)
166.78 ± 6.36
>181.49
12c
12d
188.40 ± 4.66
>159.24
186.68 ± 4.95
>159.24
185.76 ± 3.46
>159.24
184.85 ± 2.30
>159.24
12e
12g
>151.98
>151.98
142.26 ± 4.46
>194.93
136.44 ± 4.57
>194.93
12j
>194.93
>194.93
Ampicillin
Kanamycin
4.18 ± 0.72
3.34 ± 1.62
12.48 ± 1.88
1.01 ± 0.50
2.86 ± 0.98
>61.92
29.54 ± 1.25
2.79 ± 0.19
SD (±), standard deviation.

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TABLE 6 Experimentally determined anti-mycobacterial activity (ex vivo/in vitro) of the key compounds and molecular docking details
IC50 and IC90 values (µM) of compounds with CI (±) values
Molecular docking score
M. tuberculosis H37Ra
(ATCC 25177)
M. tuberculosis H37Ra
(ATCC 25177)
M. bovis BCG
(ATCC 35743)
In vitro (dormant)
Ex vivo (dormant)
In vitro (dormant)
Entry
12a
IC90
IC90
IC90
Total score (−logKi)
4.1411
Crash score
−5.1433
−1.674
Polar score
0.3551
0.5103
0.0028
0.4167
0.0001
1.0449
NA
>53.67
>53.67
>53.67
12c
40.24 ± 1.05
>57.25
43.39 ± 0.29
>57.25
24.46 ± 1.28
>57.25
4.7562
12d
7.2417
−4.581
12e
>47.77
>47.77
>47.77
4.5721
−2.0919
−4.5584
−3.1839
NA
12g
19.22 ± 0.6
4.50 ± 0.69
0.972 ± 0.04
21.22 ± 1.11
5.67 ± 0.89
0.911 ± 0.01
19.73 ± 1.55
5.40 ± 0.92
1.009 ± 0.02
5.3567
12j
7.3242
Rifampicin
NA
Total score, total docking score; Crash, degree of inappropriate penetration by the ligand into the protein and of interpenetration between ligand atoms that
are separated by rotable bonds; Polar, contribution of the polar non-hydrogen bonding interactions to the total score.
|
2.5 The biotransformation prediction
oxidation, and epoxidation), and eliminations (e.g., dealkylation, ester,
and amide hydrolysis) are engaged.[55] In addition, the atom neighboring
the added oxygen is highlighted as a reaction center. The most active
anti-tubercularcompounds12j (Figure 5) and 12c (Figure 6) are analyzed
for metabolic product prediction. The plot of MetaPrint2D-React shows
possible site of metabolism of individual atom neither indicated by
Normalized Occurrence Ratio (NOR) values. The higher NOR value
indicates site frequently undergoes through particular metabolism
phase I metabolic reactions.[56,57] The NOR ratio for 12j was observed as
Red 0.66 ≤ NOR ≤ 1.00, Orange 0.33 ≤ NOR < 0.66, Green 0.15 ≤
NOR < 0.33, White (no color) 0.00 ≤ NOR < 0.15, and Gray little/no
data. The NOR ratio for 12c was observed as Red 0.66 ≤ NOR ≤ 1.00,
Yellow 0.33 ≤ NOR < 0.66, Green 0.15 ≤ NOR < 0.33, and White (no
color) 0.00 ≤ NOR < 0.15. The NOR values and atoms marks for 12j
indicate that >C(CH3)2 group of dihydrobenzofuran and −CF3 of phenyl
ring mostly undergoes through the hydroxylation reactions, whereas, in
The biotransformation prediction was performed by MetaPrint2D-
React, a metabolic product predictor server developed by Unilever
Cambridge, Center for Molecular Science Informatics, University of
Cambridge, UK. It is online tool that predicts fate of xenobiotics
metabolism through data-mining and statistical analysis of known
metabolic transformations reported in scientific literature.[53–54] The
MetaPrint2D data is generated through processing of the trans-
formations found in the Symyx Metabolite database previously known
as MDL, for each transformation, the differences between the
structures of the reactant. In order to make predictions of on the
drug molecule, each atom environment is calculated and Symyx
Metabolite database mining is done to search similar environment.
As based on the interest of the study, only Phase I additions (defined
as the addition of a single oxygen atom; covering hydroxylation,
FIGURE 5 Binding pose and molecular interactions of 12j into the active site of InhA

GHOLAP ET AL.
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|
TABLE 7 Pharmacokinetic parameters important for agents to have good oral bioavailability of synthesized compounds
ID
MW
LogP
PSA
% ABS
n-RotB
HBD
HBA
Toxicity
12a
12c
12d
12e
12g
12j
559.57823
551.59929
523.54613
627.5762
657.67817
513.5082
7.5933
7.7352
6.955
77.25
77.25
77.25
77.25
103.55
97.48
81.6535
81.6535
81.6535
81.6535
72.3433
74.49208
5
5
5
6
5
5
1
1
1
1
1
2
5
5
5
5
7
6
Non toxic
Non toxic
Non toxic
Non toxic
Non toxic
Non toxic
8.6121
8.3371
5.5373
MW, molecular weight; LogP, logarithm of partition coefficient of compound between n-octanol and water; PSA, polar surface area; n-RotBond, number of
rotatable bonds; HBA, hydrogen bond acceptors; and HBD, hydrogen bond donor.
|
12d dimethyl group of dihydrobenzofuran undergoes hydroxylation
and −CF3 also undergoes the hydroxylation reaction. Above
observations indicated that some atoms in skeleton of the target
compounds were less prone to metabolic deactivation marked by
grey color (Figures 7 and 8).
4
EXPERIMENTAL
|
4.1 Chemistry
|
4.1.1 General methods
All the reagents used during the study were purchased from Aldrich and
Spectro-Chem. Solvents were dried and redistilled before use. Melting
points were recorded on Digital Electro thermal Melting point apparatus
(VEEGO, VMP-DS) and are uncorrected. Reaction monitoring was
conducted using thin layer chromatography (TLC) using pre-coated silica
gel 60 F254 plates with layer thickness 0.25 nm purchased from Merck
Ltd. TLC plates were visualized under ultraviolet light at 254 nm
wavelength. IR spectra were recorded on KBr discs on Shimazdzu 470 IR
spectrophotometer. 1H-NMR was recorded on Varian-NMR mercury
300 MHz spectrometer in DMSO-d6 using TMS as an internal standard.
Chemical shifts values (δ) are expressed as parts per million (ppm). Mass
spectra were recorded on a Varian MAT 311 A at 70 eV.
|
3
CONCLUSION
In conclusion, a series of structurally diverse 2,2-dimethyl-2,3-
dihydrobenzofuran based 1,3,4-oxadiazole derivatives were success-
fully synthesized and characterized by spectral data. The synthesized
compounds were screened for ex vivo and in vitro anti-tubercular
studies. The tested compounds 12c, 12g, and 12j were found to have
good anti-tubercular activity against M. tuberculosis H37Ra, M. bovis
BCG strains. The compound 12j shows excellent anti-tubercular
potential (MIC < 6 μM) for ex vivo and in vitro anti-tubercular studies.
The experimental, molecular docking, pharmacokinetics/druglike
properties and metabolic pathway prediction values of synthesized
agents provide a potential pharmacophore for further drug develop-
ment process.
IR and NMR spectra as well as the InChI codes of the investigated
compounds together with some biological activity data are provided as
Supporting Information.
FIGURE 6 Binding pose and molecular interactions of 12d into the active site of InhA

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Synthesis of methyl 4-amino-2-hydroxy-5-nitrobenzoate (4)
To a mixture of 4-acetamido-2-hydroxy-5-nitrobenzoic acid (44 g,
0.287 mol) in methanol (440 mL), conc. H2SO4 (176 mL) was slowly
added. Reaction mixture was stirred at 65°C for 24 h. After completion
of reaction (as indicated by TLC), reaction mixture was cooled and
poured on ice. The product precipitated was filtered to afford 4 as
yellow solid (40 g), yield = 97%; mp = 184–186°C; 1H NMR (300 MHz,
DMSO-d6): δ 3.84 (s, 3H, -CH3), 6.36 (s, 1H, Ar-H), 7.96 (s, 2H,
exchangeable with D2O, Ar-NH2), 8.52 (s, 1H, Ar-H), 10.92 (s, 1H,
exchangeable with D2O, Ar-OH); M.F. = C8O5N2H8 (MW = 212.11) m/
z 213.05 (M+H)+.
FIGURE 7 Metabolic deactivation pathways by MetaPrint-2D
React of compound 12j
Synthesis of methyl 2-(2-methyl allyloxy)-4-amino-5-
nitrobenzoate (5)
To a mixture of methyl-4-amino-2-hydroxy-5-nitrobenzoate (4) (15 g,
0.0707 mol) in dry DMF (90 mL), K2CO3 (17.5 g, 0.127 mol) was added.
Reaction mixture was stirred at 80°C for 1 h. To the above mixture, 3-
chloro-2-methyl prop-1-ene (11.4 g, 0.127 mol) was added slowly and
a reaction mixture was further stirred at 80°C for 3 h. After completion
of reaction (by TLC), reaction mixture was cooled and poured on ice.
The product precipitated was filtered and collected to afford 5 as
yellow solid (15 g), yield = 80 %; mp = 148–149°C; 1H NMR (300 MHz,
DMSO-d6): δ 1.79 (s, 3H,-CH3), 3.75 (s, 3H, -OCH3), 4.47 (s, 2H, -NH2),
4.98 (s, 1H), 5.19 (s, 1H), 6.50 (s, 1H, Ar-H), 7.82 (s, 2H, -CH2-), 8.50 (s,
1H, Ar-H); M.F. = C12O5N2H15 (MW = 266.11) m/z 267.10 (M+H)+.
Synthesis of 4-acetamido-2-hydroxy benzoic acid (2)
To a mixture of 4-amino salicylic acid (50 g, 0.32 mol) in ethanol, acetic
anhydride (43.30 mL, 0.42 mol) was added. The reaction mixture was
stirred at 45–50°C for 3 h. After completion of reaction (as indicated
by TLC), reaction mixture was cooled and poured on ice. The product
precipitated which was then filtered and collected to afford 2 as white
solid (56 g), yield = 88%, mp = 216–217°C. 1H NMR (300 MHz, DMSO-
d6): δ 2.06 (s, 3H, CH3), 7.02 (d, J = 6.9 Hz, 1H, Ar-H), 7.33 (s, 1H, Ar-H),
7.68 (d, J = 8.7 Hz, 1H, Ar-H), 10.20 (s, 1H, -NHCO-), 11.40 (s, 1H
exchangeable with D2O, Ar-OH), 13.00 (s, 1H exchangeable with D2O,
-COOH); M.F. = C9O4NH9 (MW = 195.22) m/z 196 (M+H)+.
Synthesis of methyl-4-amino-2,3-dihydro-2,2-dimethyl-5-
nitrobenzofuran-7-carboxylate (6)
Synthesis of 4-acetamido-2-hydroxy-5-nitrobenzoic acid (3)
To a mixture of 4-acetamido-2-hydroxy benzoic acid (56 g, 0.287 mol)
in trifluoroacetic acid (560 mL), sodium nitrate (26.84 g, 315 mol) was
added slowly and reaction mixture was cooled to 0°C. After
completion of reaction (by TLC), reaction mixture was poured on
ice. The yellow solid separated was filtered to afford 3 (45 g),
yield = 70%, mp = 202–204°C. 1H NMR (300 MHz, DMSO-d6): δ 2.16
(s, 3H, -CH3), 7.64 (s, 1H, Ar-H), 8.44 (s, 1H, Ar-H), 10.43 (s, 1H, Ar-
OH); M.F. = C9O6N2H8 (MW = 240.12) m/z 241 (M+H)+.
To a mixture of methyl-2-(2-methylallyloxy)-4-amino-5-nitrobenzoate
(5) (15 g, 0.056 mol) in dry dichloromethane (90 mL), anhydrous AlCl3
(1.5 g, 0.0112 mol) was added slowly at −78°C. Reaction mixture was
further stirred at −78°C for 1 h. Reaction mixture gradually allowed to
stand at room temperature for 24 h. After completion of reaction (as
indicated by TLC), reaction mixture was cooled and poured on ice. The
product precipitated was filtered and recrystallized from ethyl acetate
to give 6 as a yellow solid (12.5 g), yield = 83%; mp = 173–174°C.
1H NMR (300 MHz, DMSO-d6): δ 1.47 (s, 6H), 2.87 (s, 2H), 3.75 (s, 3H),
7.49 (s, 2H, exchangeable with D2O), 8.51 (s, 1H); M.F. = C12O5N2H14
(MW = 266.11), m/z 267 (M+H)+.
Synthesis of methyl-2,3-dihydro-4-iodo-2,2-dimethyl-5-
nitrobenzofuran-7-carboxylate (7)
A mixture of methyl-4-amino-2,3-dihydro-2,2-dimethyl-5-nitroben-
zofuran-7-carboxylate (12 g, 0.0451 mol) and conc. HCl (96 mL, 8 vol)
was cooled at 0°C for 30 min. To this reaction mixture, aqueous
solution of sodium nitrite (4.66 g, 0.0676 mol in water 10 mL) was
added slowly using dropping funnel. The temperature of reaction
mixture was maintained as 0°C. The solution of KI (11.22 g, 0.067 mol
in 20 mL water) was added slowly at 0°C during 1 h. After completion
of reaction, the reaction mixture was extracted by EtOAC, washed
with water and separated. The organic layer was dried over anhydrous
sodium sulphate and solvent was removed under reduced pressure
followed by recrystallization using diethyl ether to give 7 as a yellow
FIGURE 8 Metabolic deactivation pathways by MetaPrint-2D
React of compound 12d

GHOLAP ET AL.
11 of 14
|
solid (15.2 g), yield = 89.41%; mp = 138–139°C. 1H NMR (300 MHz,
DMSO-d6): δ 1.51 (s, 6H), 3.10 (s, 2H), 3.81 (s, 3H), 8.24 (s, 1H);
M.F. = C12O5NIH12 (MW = 377.21) m/z 378 (M+H)+.
mixture conc. HCl (10 mL) was added dropwise. After completion
of reaction, the reaction mixture was basified by saturated
NaHCO3 solution, filtered through celite bed and extracted using
dichloromethane. The organic layer was dried over anhydrous
sodium sulphate and filtered. The solvent was removed under
reduced pressure to afford 7-[5-(4-(trifluoromethyl)phenyl]-1,3,
Synthesis of 2,3-dihydro-4-iodo-2,2-dimethyl-5-
nitrobenzofuran-7-carboxylic acid (8)
To a mixture of methyl-2,3-dihydro-4-iodo-2,2-dimethyl-5-nitroben-
zofuran-7-carboxylate (7) (15 g, 0.039 mol) in THF, LiOH (1.83 g,
0.079 mol) was added at 25°C. The reaction mixture was stirred at
25°C for 1 h. After completion of reaction, THF was removed under
reduced pressure and reaction mixture was acidified by dil. HCl, solid
precipitated out was filtered and collected to afford 8 as a yellow solid
(13.5 g); yield = 93.90%; mp = 120–121°C; 1H NMR (DMSO-d6,
300 MHz): δ 1.50 (s, 6H), 3.09 (s, 2H), 8.23 (s, 1H); M.F. = C11O5NIH10
(MW = 363.11) m/z 364 (M+H)+.
4-oxadiazol-2-yl)-2,3-dihydro-4-iodo-2,
2-dimethylbenzofuran-
5-yl)amine (8 g). This was then dissolved in THF (50 mL) at 0°C
and mixed with triethylamine (10 mL). The reaction mixture was
further stirred at 0°C for 20 min, followed by pivolyl chloride
(1.92 mL) and stirred for 2 h. After completion of reaction, the
reaction mixture was extracted by EtOAC, the organic phase dried
over anhydrous sodium sulfate, then the solvent was removed
under reduced pressure to afford product which was recrystallized
by diethyl ether to give 11 as a yellow solid (8.45 g), yield = 76.88%,
mp = 162–163°C; 1H NMR (300 MHz, DMSO-d6): δ 1.27 (s, 9H),
1.56 (s, 6H), 3.10 (s, 2H), 8.02 (d, J = 8.4 Hz, 2H), 8.30 (d, J = 7.8 Hz,
2H), 9.12 (s, 1H), 9.35 (s, 1H); M.F. = C24O3N3IF3H23 (MW = 584)
m/z 585.92 (M+H)+.
Synthesis of 4-iodo-5-nitro-2,2-dimethyl-N-[4-
(trifluoromethyl)-benzoyl]-2,3-dihydrobenzofuran-7-
carbohydrazide (9)
To the reaction mixture of 2,3-dihydro-4-iodo-2,2-dimethyl-5-nitro-
benzofuran-7-carboxylic acid (8) (13.5 g, 0.037 mol) in dry dichloro-
methane (70 mL), oxalyl chloride (11.82 mL, 0.074 mol) was added at
25°C followed by addition of 2–3 drops of DMF. The reaction mixture
was stirred at 25°C for 1–2 h. After completion of reaction, the solvent
was removed under reduced pressure and residue was mixed with
dichloromethane and cooled to 0°C. Triethylamine (8 mL, 0.074 mol)
and 4-trifluoromethyl phenyl hydrazide (7.5 g, 0.037 mol) was added at
0°C. Reaction mixture was stirred at 0°C for 2 h. After completion of
reaction (by TLC), reaction mixture was cooled and poured on ice. Solid
separated was filtered to afford 9 as a brown yellow solid (15 g);
mp = 154–155°C. 1H NMR (300 MHz, DMSO-d6): δ 1.58 (s, 6H), 3.17
(s, 2H), 7.92 (d, J = 8.4 Hz, 2H), 8.12 (d, J = 8.4 Hz, 2H), 8.26 (s, 1H),
10.16 (s, 1H), 10.88 (s, 1H); M.F. = C19O5N3IF3H15 (MW = 548) m/z
549.13 (M+H)+.
|
4.1.2 General procedure for the synthesis of 12a–j
To a solution of N-{7-[5-(4-trifluoromethylphenyl]-1,3,4-oxadiazol-2-
yl}-2,3-dihydro-4-iodo-2,2-dimethyl benzofuran-5-yl)pivalamide (11)
(150 mg, 0.256 mmol) in DMSO, tetrabutyl ammonium fluoride
monohydrate (TBAF) (217 mg, 0.767 mmol), acetylene derivatives
(0.307 mmol)
and
bis-dichlorotriphenylphospine
palladium(II)
PdCl2(PPh3)2 (3.5 mg, 0.0051 mmol) was added. The reaction mixture
was stirred at 80°C for 3–4 h. After completion of reaction (as
indicated by TLC), reaction mixture was quenched in water and
extracted in ethyl acetate. The solvent was removed under reduced
pressure and collected crude products were purified by column
chromatography (4% methanol/dichloromethane).
N-(2,2-Dimethyl-4-(phenylethynyl)-7-(5-(4-(trifluoromethyl)-
phenyl)-1,3,4-oxadiazol-2-yl)-2,3-dihydrobenzofuran-5-yl)-
pivalamide (12a)
Synthesis of 2-[4-(trifluoromethyl)phenyl-5-(2,3dihydro-4-iodo-
2,2-dimethyl-5-nitrobenzofuran-7-yl]-1,3,4-oxadiazole (10)
A mixture of 4-iodo-5-nitro-2,2-dimethyl-N-[4-(trifluoromethyl)ben-
zoyl]-2,3-dihydrobenzofuran-7-carbohydrazide (9) (15 g) and POCl3
(50 mL) was heated under reflux for 10 h. After completion of reaction
POCl3 was distilled out completely, residue was slowly added in ice
cold water, solid precipitated collected to afford compound 10 as a
brown solid, yield = 74%; mp = 187–188°C. 1H NMR (300 MHz,
DMSO-d6): δ 1.60 (s, 6H), 3.20 (s, 2H), 8.01 (d, J = 7.8 Hz, 2H), 8.30
(d, J = 7.8 Hz, 2H), 8.51 (s, 1H); M.F. = C19O4N3IF3H13 (MW = 530.11)
m/z 531 (M+H)+.
Faint yellow solid (yield = 85%), mp = 188–189°C. IR (KBr, cm−1) ν
�
�
3426 (NH amide), 2969 CH), 2211 (C C), 1678 (C O amide), 1543
�
(aromatic carbon), 1324 (C N), 1170, 1127 (C-O), 1494 (C-F);
1H NMR (300 MHz, DMSO-d6): δ 1.28 (s, 9H), 1.58 (s, 6H), 3.28 (s,
2H), 7.48 (d, J = 7.6 Hz, 3H), 7.55 (d, J = 7.6 Hz, 2H), 7.92 (s, 1H), 8.03 (d,
J = 7.8 Hz, 2H), 8.30 (d, J = 7.8 Hz, 2H), 9.15 (s, 1H, exchangeable with
D2O); 13C NMR (125.7 MHz, CDCl3): δ 27.8, 28.5, 40.2, 42.9, 88.4,
89.6, 90.7, 106.1, 117.6, 120.8, 126.1, 127.3, 127.5, 127.6, 128.7,
128.9, 129.7, 130.2, 131.7, 131.8, 132.0, 132.2, 135.9, 153.6, 162.9,
176.2; M.F. = C32O3N3F3H28 (MW = 559.58) m/z 560.03 (M+H)+.
Synthesis of N-(7-(5-(4-(trifluoromethyl)phenyl)-1,3,4-
oxadiazol-2-yl)-2,3-dihydro-4-iodo-2,2-dimethyl benzofuran-5-
yl)pivalamide (11)
N-(4-(Cyclohexylethynyl)-2,2-dimethyl-7-(5-(4-
(trifluoromethyl)-phenyl)-1,3,4-oxadiazol-2-yl)-2,3-
dihydrobenzofuran-5-yl)-pivalamide (12b)
A mixture of 2-(4-(trifluoromethyl)phenyl-5-(2,3-dihydro-4-iodo-
2,2-dimethyl-5-nitrobenzofuran-7-yl)-1,3,4-oxadiazole (10) (10 g)
and iron powder (2 g) in methanol was stirred at 25–30°C. To this
White solid (yield = 82%) mp = 171–172°C. IR (KBr, cm−1) ν 3382
�
�
(NH amide), 2930 CH), 2220 (C C), 1671 (C O amide), 1519

12 of 14
GHOLAP ET AL.
|
�
(aromatic carbon), 1325 (C N), 1169, 1118 (C-O), 1428 (C-F);
1H
NMR (300 MHz, DMSO-d6): δ 1.26 (s, 9H), 1.32–1.24 (m, 4H),
Methyl-5-chloro-2-((2,2-dimethyl-5-pivalamido-7-(5-(4-
(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)-2,3-
1.48 (m, 2H), 1.54 (s, 6H), 1.68 (d, 2H), 1.84 (d, 2H), 2.71 (m, 1H),
3.12 (s, 2H), 8.01 (s, 1H), 8.01 (d, J = 8.1 Hz, 2H), 8.28 (d, J = 8.1 Hz,
2H), 8.83 (s, 1H, exchangeable with D2O); 13C NMR (125.7 MHz,
CDCl3): δ 22.2, 27.7, 27.8, 28.4, 28.5, 29.8, 30.8, 40.1, 42.8, 81.4,
90.4, 103.4, 105.4, 114.8, 117.1, 124.5, 126.1, 127.5, 127.6, 128.4,
131.3, 132.1, 153.4, 162.9, 176.5; M.F. = C32O3N3F3H34
(MW = 565.26) m/z 566.03 (M+H)+.
dihydrobenzofuran-4-yl)ethynyl)benzoate (12f)
White solid; (yield = 78%) mp = 198–199°C. IR (KBr, cm−1) ν 3347 (NH
�
�
amide), 2965 CH), 2220 (C C), 1660 (C O amide), 1738 (ester), 1554
�
(aromatic carbon), 1324 (C N), 1170, 1128 (C-O), 1435 (C-F);
1H NMR (300 MHz, DMSO-d6): δ 1.28 (s, 9H), 1.58 (s, 6H), 3.33 (s,
3H), 3.89 (s, 3H), 7.72 (s, 1H), 7.89 (s, 1H), 7.99 (d, J = 8.4 Hz, 2H), 8.01
(s, 1H), 8.31 (d, J = 8.4 Hz, 2H), 9.23 (s, 1H, exchangeable with D2O);
13C NMR (125.7 MHz, CDCl3): δ 28.5, 29.8, 40.2, 43.0, 52.9, 89.7, 98.0,
106.7, 113.7, 118.2, 120.7, 122.8, 126.2, 127.4, 127.6, 130.9, 131.9,
132.0, 132.5, 134.6, 134.9, 135.1, 153.6, 165.3, 176.6;
M.F. = C34O5N3ClF3H29 (MW = 652.06) m/z 652.09 (M+).
N-(4-(Cyclopentylethynyl)-2,2-dimethyl-7-(5-(4-
(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)-2,3-
dihydrobenzofuran-5-yl)pivalamide (12c)
White solid; (yield = 80%) mp = 172–173°C. IR (KBr, cm−1) ν 3382
�
�
(NH amide), 2930 CH), 2220 (C C), 1671 (C O amide), 1519
N-(4-((2,2-Dimethyl-4-oxochroman-8-yl)ethynyl)-2,2-dimethyl-
7-(5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)-2,3-
dihydrobenzofuran-5-yl)pivalamide (12g)
(Ar-H), 1325 (C N), 1169, 1118 (C-O), 1428 (C-F); 1H NMR
�
(300 MHz, DMSO-d6): δ 1.23 (s, 9H), 1.54 (s, 6H), 1.54 (m, 4H),
1.92–1.60 (m, 2H), 2.45 (m, 4H), 3.14 (m, 1H), 3.33 (s, 2H), 7.57 (s,
1H), 8.02 (d, J = 8.4 Hz, 2H), 8.29 (d, J = 8.4 Hz, 2H), 9.15 (s, 1H,
exchangeable with D2O); 13C NMR (125.7 MHz, CDCl3): δ 27.8,
28.4, 28.5, 29.8, 35.1, 40.2, 43.0, 81.3, 88.0, 89.7, 106.7, 113.7,
120.7, 122.8, 126.1, 127.4, 130.9, 131.9, 132.5, 134.6, 135.1,
153.6, 165.3, 176.6; M.F. = C31O3N3F3H32 (MW = 551.60) m/z
552.03 (M+H)+.
Yellow solid; (yield = 70%) mp = 212–213°C. IR (KBr, cm−1) ν 3347 (NH
�
�
amide), 2965 CH), 2220 (C C), 1660 (C O, amide), 1738 (ester),
�
1554 (aromatic carbon), 1324 (C N), 1170, 1128 (C-O), 1435 (C-F);
1H NMR (300 MHz, DMSO-d6): δ 1.48–1.30 (s, 21H), 2.70 (s, 2H), 3.3
(s, 2H), 7.33 (d, J = 7.5 Hz, 1H), 7.82 (d, J = 7.8 Hz, 2H), 7.88 (d,
J = 7.5 Hz, 3H), 8.29 (d, J = 7.8 Hz, 3H); 13C NMR (125.7 MHz, CDCl3): δ
27.8, 27.9, 28.5, 28.6, 40.2, 42.9, 43.0, 48.9, 84.9, 88.4, 89.6, 90.7,
96.6, 106.1, 117.6, 120.8, 126.1, 127.3, 127.5, 127.6, 128.7, 128.9,
129.7, 130.2, 131.7, 131.8, 132.0, 132.2, 135.9, 153.5, 162.9, 176.6,
196.6, M.F. = C37O5N3F3H34 (MW = 657.68) m/z 658.17 (M+H)+.
N-(4-(Cyclopropylethynyl)-2,2-dimethyl-7-(5-(4-
(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)-2,3-
dihydrobenzofuran-5-yl)pivalamide (12d)
White solid; (yield = 82%) mp = 176–177°C. IR (KBr, cm−1) ν 3351 (NH
N-(2,2-Dimethyl-4-(pent-1-ynyl)-7-(5-(4-(trifluoromethyl)-
phenyl)-1,3,4-oxadiazol-2-yl)-2,3-dihydrobenzofuran-5-yl)-
pivalamide (12h)
�
�
amide), 2971 CH), 2220 (C C), 1660 (C O amide), 1556 (Ar-H), 1324
1
�
(C N), 1170, 1131 (C-O), 1462 (C-F); H NMR (300 MHz, DMSO-d6):
δ 0.77 (d, 2H), 0.979 (d, 2H), 1.25 (s, 9H), 1.53 (m, 1H), 1.45 (s, 6H), 2.71
(m, 1H), 3.12 (s, 2H), 7.95 (s, 1H), 8.02 (d, J = 8.4 Hz, 2H), 8.28 (d,
J = 8.4 Hz, 2H), 8.86 (s, 1H, exchangeable with D2O); 13C NMR
(125.7 MHz, CDCl3): δ 0.29, 9.2, 9.3, 28.2, 28.3, 40.0, 42.6, 68.9,
89.2, 90.2, 105.1, 106.3, 114.5, 116.9, 125.9, 126.0, 127.2, 127.4,
130.5, 131.9, 132.1, 153.2, 162.8, 176.3; M.F. = C29O3N3F3H28
(MW = 523.55) m/z 524.00 (M+H)+.
White solid; (yield = 75%) mp = 126–127°C. IR (KBr, cm−1) ν 3322 (NH
�
�
amide), 2964 (CH), 2228 (C C), 1671 (C O amide), 1550 (aromatic
carbon), 1324 (C N), 1168, 1129 (C-O), 1428 (C-F); 1H NMR
�
(300 MHz, DMSO-d6): δ 1.13 (t, 3H), 1.37 (s, 9H), 1.51 (m, 2H), 1.62 (s,
6H), 2.52 (t, 2H), 3.13 (s, 2H), 7.78 (d, J = 7.8 Hz, 2H), 8.19 (s, 1H), 8.30
(d, J = 7.8 Hz, 2H), 8.87 (s, 1H); 13C NMR (125.7 MHz, CDCl3): δ 13.7,
21.8, 22.3, 27.7, 28.4, 40.1, 42.8, 89.3, 90.4, 101.4, 105.3, 114.8,
117.1, 126.2, 127.1, 127.5, 128.4, 130.7, 131.3, 132.0, 153.4, 164.0,
176.5; M.F. = C29O3N3F3H30 (MW = 525.56) m/z 526.69 (M+H)+.
N-(2,2-Dimethyl-7-(5-(4-(trifluoromethyl)phenyl)-1,3,4-
oxadiazol-2-yl)-4-((4-(trifluoromethyl)phenyl)ethynyl)-2,3-
dihydrobenzofuran-5-yl)pivalamide (12e)
N-(4-(Hex-1-ynyl)-2,2-dimethyl-7-(5-(4-(trifluoromethyl)-
phenyl)-1,3,4-oxadiazol-2-yl)-2,3-dihydrobenzofuran-5-yl)-
pivalamide (12i)
Off white solid; (yield = 75%) mp = 190–191°C. IR (KBr, cm−1) ν
�
�
3354 (NH amide), 2965 (CH), 2346 (C C), 1678 (C O amide),
1553 (aromatic carbon), 1325 (C N), 1169, 1128 (C-O), 1460 (C-
White solid; (yield = 70%) mp = 132–133°C. IR (KBr, cm−1) ν 3413 (NH
�
F); 1H NMR (300 MHz, DMSO-d6): δ 1.28 (s, 9H), 1.58 (s, 6H), 3.30
(s, 2H), 7.76 (d, J = 7.8 Hz, 2H), 7.85 (d, J = 7.8 Hz, 2H), 7.90 (s, 1H),
8.01 (d, J = 8.4 Hz, 2H), 8.31 (d, J = 8.4 Hz, 2H), 9.22 (s, 1H,
exchangeable with D2O); 13C NMR (125.7 MHz, CDCl3): δ 27.7,
29.8, 40.1, 42.8, 89.3, 90.4, 103.2, 105.3, 114.8, 117.1, 126.1,
126.2, 127.1, 127.3, 127.5, 127.5, 128.4, 130.7, 131.3, 132.0,
132.0, 153.4, 164.0, 176.5; M.F. = C33O3N3F6H27 (MW = 627.58)
m/z 628.12 (M+H)+.
amide), 2959 (CH), 2217 (C C), 1682 (C O, amide), 1547 (aromatic
�
�
carbon), 1324 (C N), 1163, 1126 (C-O), 1424 (C-F); 1H NMR
�
(300 MHz, DMSO-d6): δ 1.13 (t, 3H), 1.37 (s, 9H), 1.62 (m, 10H), 2.59 (t,
2H), 3.12 (s, 2H), 7.78 (d, J = 7.8 Hz, 2H), 8.18 (s, 1H), 8.30 (d, J = 7.8 Hz,
2H), 8.87 (s, 1H); 13C NMR (125.7 MHz, CDCl3): δ = 13.7, 19.5, 22.2,
27.7, 28.4, 30.9, 40.1, 42.8, 89.4, 90.4, 101.6, 105.4, 114.8, 117.1,
124.5, 126.1, 127.4, 128.4, 130.7, 131.3, 133.3, 153.4, 163.0, 176.5;
M.F. = C30O3N3F3H32 (MW = 539.59) m/z 541.16 (M+H)+.

GHOLAP ET AL.
13 of 14
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N-(4-(3-Hydroxyprop-1-ynyl)-2,2-dimethyl-7-(5-(4-
(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)-2,3-
dihydrobenzofuran-5-yl)pivalamide (12j)
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Off-white solid; (yield = 75%) mp = 182–183°C. IR (KBr, cm−1) ν 3405
�
�
O
(NH amide and OH alcohol), 2971 (CH), 2226 (C C), 1660 (C
�
amide), 1502 (aromatic carbon), 1325 (C N), 1165, 1125 (C-O), 1440
(C-F); 1H NMR (300 MHz, DMSO-d6): δ 1.26 (s, 9H), 1.54 (s, 6H), 3.15
(s, 2H), 4.38 (d, 2H), 5.44 (t, 1H, exchangeable with D2O), 7.62 (s, 1H),
8.03 (d, J = 7.8 Hz, 2H), 8.28 (d, J = 7.8 Hz, 2H), 8.83 (s, 1H,
exchangeable with D2O); 13C NMR (125.7 MHz, CDCl3): δ 27.7,
28.4, 40.1, 42.7, 51.5, 88.1, 89.7, 90.8, 109.6, 117.9, 121.0, 124.8,
126.1, 127.1, 128.6, 130.9, 132.1, 132.3, 153.5, 164.5, 176.9;
M.F. = C27O4N3F3H26 (MW = 513.51) m/z 514.24 (M+H)+.
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11, 119.
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333, 53.
|
4.2 Statistical analysis
All the experiments were performed in triplicates and repeated at least
twice and the data has been presented as mean SD (±). The % inhibition
was calculated with the OriginPro 8 program (Origin Lab, Inc.) and the
associated GI50/IC50/GI90/IC90 values have been noted.
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Simon, M. L. Cohen, J. Med. Chem. 1992, 35, 310.
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2010, 46, 7043.
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Deshmukh, C. R. Chim. 2014, 17, 431.
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ACKNOWLEDGMENT
Authors are thankful to Principal, PVP College, Pravaranagar, for
providing necessary laboratory facilities and National Chemical
Laboratory (NCL), Pune, for providing biological screening facilities.
[29] S. S. Gholap, N. Gunjal, Arabian J. Chem. 2017, 10, S2750.
[30] S. S. Gholap, N. Gunjal, Iranian J. Catal. 2016, 8, 147.
[31] S. R. Ugale, S. S. Gholap, Chem. Pap. 2017, 71 (12), 2435.
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CONFLICT OF INTEREST
The authors declare no conflict of interest.
[34] S. Ursula, B. Frederic, Q. Anna, B. Christian, J. Org. Chem. 2013, 78,
6890.
ORCID
[35] R. Nicholas, Y. Cai-Guang, H. Chauan, Synlett 2006, 8, 1278.
[36] B. Richard, M. Hanniffy, C. Stephes, A. Burke, J. Org. Chem. 2008, 73,
1354.
Somnath Gholap
http://orcid.org/0000-0002-0883-3889
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Additional Supporting Information may be found online in the
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