Synthesis and anti-mycobacterial activity of 4-(4-phenyl-1H-1,2,3-triazol-1-yl)salicylhydrazones: revitalizing an old drug

Article abstract of DOI:10.1007/s12272-016-0882-x

The antitubercular drug; para-aminosalicylic acid (PAS) was used as the core scaffold for the design of a series of 1H-1,2,3-triazolylsalicylhydrazones upon coupling with triazole and arylhydrazone moietis to furnish a single molecular architecture. The o

Full text of DOI:10.1007/s12272-016-0882-x

Arch. Pharm. Res.
DOI 10.1007/s12272-016-0882-x
RESEARCH ARTICLE
Synthesis and anti-mycobacterial activity of 4-(4-phenyl-1H-1,2,3-
triazol-1-yl)salicylhydrazones: revitalizing an old drug
Hajjaj H. M. Abdu-Allah1
Bahaa G. M. Youssif1,2 Mostafa H. Abdelrahman3
•
•
•
Mohammed K. Abdel-Hamid4 Rudraraju Srilakshmi Reshma Perumal Yogeeswari
Tarek Aboul-Fadl4 Dharmarajan Sriram5
5
5
•
•
•
•
Received: 13 August 2016 / Accepted: 22 December 2016
Ó The Pharmaceutical Society of Korea 2016
Abstract The antitubercular drug; para-aminosalicylic
acid (PAS) was used as the core scaffold for the design of a
series of 1H-1,2,3-triazolylsalicylhydrazones upon cou-
pling with triazole and arylhydrazone moietis to furnish a
single molecular architecture. The obtained derivatives
were screened against Mycobacterium tuberculosis H37Rv
revealing good to high activity for the active compounds
(MIC values of 0.39–1.5 lg/mL) compared to the marketed
drugs isoniazid, rifampicin and ethambutol. Moreover, the
most active analogue N-(1-(4-chlorobenzyl)-2-oxoindolin-
3-ylidene)-2-hydroxy-4-(4-phenyl-1H-1,2,3-triazol-1-yl)-
benzohydrazide (20) was found to be ten-fold more potent
than PAS and equipotent to rifampicin (MIC 0.39 lg/mL),
while exhibiting low cytotoxicity with a selectivity index
of [128. In addition, this compound was shown to be
active against persistent forms of mycobacteria comparable
to standard drugs in nutrient starvation model. Accord-
ingly, we introduce compound 20 as a valuable lead for
further development. A 3D-QSAR study was also con-
ducted to help in explaining the observed activity and to
serve
as
a
tool
for
further
development.
Keywords Antitubercular Á p-Aminosalicylic Á Dormant
TB Á Triazole Á Isatin Á Hydazide and modeling
Introduction
Tuberculosis (TB), one of the most refractory and deadly
infectious diseases worldwide, is a chronic bacterial
infection caused by Mycobacterium tuberculosis (MTB).
According to the WHO, nearly one third of the population
is infected with tuberculosis that kills nearly 1.4 million
people each year (WHO 2015). Most infections in human
result in asymptomatic, latent infection and about one in
ten latent infections eventually progresses to active disease
(WHO 2015). The situation is more complicated by the
emergence of multidrug resistant (MDR-TB) and exten-
sively drug resistant (XDR-TB) strains. Therefore, the
discovery of new drugs with different modes of action and
toxicity profiles is urgently needed for use in the global
effort to combat and eradicate the disease (Zumla et al.
2013). A common approach in drug design is the use of so-
called privileged structures, which refers to substructures
or scaffolds that appear frequently in drugs, natural prod-
ucts, or bioactive molecules (Evans et al. 1988; Verma
et al. 2013). One of those privileged frameworks is the
aminosalicylate moiety which has been shown in various
drug classes that exhibit a broad range of applications such
Electronic supplementary material The online version of this
article (doi:10.1007/s12272-016-0882-x) contains supplementary
material, which is available to authorized users.
& Hajjaj H. M. Abdu-Allah
hagag.abdallah@pharm.au.edu.eg
& Dharmarajan Sriram
dsriram@hyderabad.bits-pilani.ac.in
1
Pharmaceutical Organic Chemistry Department, Faculty of
Pharmacy, Assiut University, Assiut 71526, Egypt
2
Department of Pharmaceutical Chemistry, College of
Pharmacy, Aljouf University, Aljouf, Sakaka 42421, Saudi
Arabia
3
Department of Organic Chemistry, Faculty of Pharmacy, Al-
Azhar University, Assiut 71524, Egypt
4
Medicinal Chemistry Department, Faculty of Pharmacy,
Assiut University, Assiut 71526, Egypt
5
Medicinal Chemistry and Antimycobacterial Research
Laboratory, Pharmacy Group, Birla Institute of Technology
& Science-Pilani, Hyderabad Campus, Jawahar Nagar,
Hyderabad, Telangana 500078, India
123

H. H. M. Abdu-Allah et al.
as anticancer, antioxidant, antibacterial, and neuroprotec-
tive activity (Abdu-Allah et al. 2016a). In particular, para-
aminosalicylic acid (PAS) which entered the clinical
market as antitubercular agent in 1946 as the second
exclusive drug after streptomycin (Minato et al. 2015).
While PAS was initially a first-line treatment TB drug, the
introduction of more potent antitubercular agents relegated
PAS to the second line of treatment of drug-resistant My-
cobacterium tuberculosis infections. The reported molec-
ular and biochemical mechanisms involve PAS-mediated
disruption of iron acquisition and corrupting one-carbon
metabolism through inhibition of the formation of reduced
folate species (Minato et al. 2015). Due to the short serum
half-life of PAS, large oral doses (10–12 g per day) are
required which result in a wide range of adverse effects,
such as gastrointestinal symptoms, hypersensitivity reac-
tions, and renal failure (Humma 1996; Peloquin et al.
1999). While it has been more than ten years since the
synthesis of the last reported PAS antitubercular (Ren-
garajan et al. 2004; Patole et al. 2006), it would be nec-
essary to revisit this molecule aiming for the production of
new analogues that may be effective against existing PAS-
resistant strains. On the other hand, different families of
compounds have been reported with antitubercular activi-
Triazole pharmacophore
Variable substituents
N
N
R
N
C
O
N
H
N
Ar
OH
PAS pharmacophore
Fig. 1 Design strategy for the synthesis of triazolylsalicylhydrazones
(4–21)
was prepared according to the reported method (Gano et al.
2001). Melting points were determined on an electro
thermal melting point apparatus [Stuart Scientific, model
SMP3, England, UK], and were uncorrected. Pre-coated
silica gel plates (kieselgel 0.25 mm, 60G F254, Merck,
Germany) were used for TLC monitoring of reactions. The
spots were detected at 254 nm wavelength using ultraviolet
lamp (Spectroline, model CM-10, USA). IR spectra (KBr
discs) were recorded on a shimadzu IR-470 spectrometer
(Shimadzu, Kyoto, Japan) at Faculty of Pharmacy, Assiut
University, Assiut, Egypt. NMR Spectra were taken using a
Varian Unity INOVA 400 MHz spectrometer for proton
and carbon at 400 and 100 MHz, respectively, University
of Aberdeen, Aberdeen, UK. High resolution mass spec-
trometric data were obtained using the EPSRC mass
spectrometry center in Swansea (UK) on a LTQ Orbitrap
XL instrument.
ˇ´
´
ties such as hydrazones (Vavrıkova et al. 2011, Pavan et al.
2010) and isatins (Aboul-Fadl et al. 2010, 2011, Sriram
et al. 2006). Investigation of SAR of isatin derivatives has
revealed that 5-halogenation and N-alkylation have resul-
ted in marked increase in antitubercular activity (Aboul-
Fadl et al. 2010, 2011, Sriram et al. 2006). Moreover, tri-
azoles have recently received much attention as central
core in the synthesis of antitubercular agents (Emmadi
et al. 2015; Suman et al. 2015; Menendez et al. 2012;
Shanmugavelan et al. 2011; Jorda˜o et al. 2011). In continue
of our interest in the development of antitubercular agents
(Aboul-Fadl et al. 2010, 2011, Sriram et al. 2006) and
synthesis and biological activities of aminosalicylate
derivatives (Abdu-Allah et al. 2005, 2016b; Abdel-Alim
et al. 2005), the present work describes the synthesis and
biological evaluation of a series of 1H-1,2,3-triazolylsali-
cylhydrazones starting from PAS as an attempt to its
revitalization as antitubercular drug. The designed com-
pounds 3–21 combine the core fragments of PAS, triazole
and hydrazones in a single molecular architecture with the
aim of improving the spectrum, potency and safety
(Fig. 1).
Synthesis of ethyl 2-hydroxy-4-(4-phenyl-1H-1,2,3-
triazol-1-yl)-benzoate (2)
To a solution of compound 1 (4.14 g, 20.0 mmol) in a
mixture of THF/H2O [2:1 (v/v), 30 mL], phenyl acetylene
(2.44 g, 24.0 mmol), CuSO4.5H2O (0.24 g, 1.0 mmol) and
sodium ascorbate (0.40 g, 2.0 mmol) were added. The
mixture was stirred at room temperature for 20 h. the
residue was extracted with CHCl3 (3 9 15 mL), the
organic phase was washed with water, dried over anhy-
drous Na2SO4 and concentrated. The crude product was
crystallized from ethanol to afford pure compound as a
white solid in 86% yield. m.p. 162–163 °C; IR (KBr):
3390, 3125, 3034, 2914, 1660, 1594, 1503, 1466, 1406,
1231, 874, 771, 688 cm-1; 1H NMR (DMSO-d6): d (ppm):
10.91 (s, 1H); 9.40 (s, 1H); 7.95 (t, 2H, J = 9.5 Hz);
7.96–7.23 (m, 6H); 4.50–4.24 (q, 2H, J = 8.2 Hz); 1.34 (t,
3H, J = 8 Hz,); 13C NMR (DMSO-d6): d (ppm): 167.86;
160.92; 147.44; 141.09; 131.87; 131.54; 129.83; 128.86;
128.26; 125.24; 119.47; 112.93; 110.24; 107.60; 61.48;
13.85.
Materials and methods
All chemicals and solvents were obtained from commercial
suppliers and used without further purification. The starting
material ethyl 4-azido-2-hydroxybenzoate, compound 1
123

Synthesis and anti-mycobacterial activity…
Synthesis of 2-hydroxy-4-(4-phenyl-1H-1,2,3-triazol-
1-yl)-benzohydrazide (3)
3H, J = 20.7, 5.5 Hz); 7.50 (t, 2H, J = 7.7 Hz); 7.41 (dt,
2H, J = 15.0, 7.6 Hz); 13C NMR (DMSO-d6): d (ppm):
163.85; 159.87; 147.51; 140.01; 136.50; 132.84; 131.02;
130.71; 130.00; 129.01; 128.38; 126.39; 125.40; 122.19;
119.59; 116.21; 110.04; 107.87; HRESI-MS m/z calcd for
C22H16BrN5O2 [M?H]? 462.0560 Found 462.0550.
To a solution of ethyl 2-hydroxy-4-(4-phenyl-1H-1,2,3-
triazol-1-yl)benzoate (2) (3.09 g, 10.0 mmol) in absolute
ethanol (40 mL), hydrazine hydrate 99% (0.75 g,
15.0 mmol) was added. The reaction mixture was refluxed
for 6 h, and then cooled. The precipitated product was
filtered, washed with cold ethanol, dried, and crystallized
from ethanol as white crystals in 85% yield. m.p.
295–297 °C; IR (KBr): 3335, 3278, 1620, 1583, 1525,
N1-(3-Chlorobenzylidene)-2-hydroxy-4-(4-phenyl-1H-
1,2,3-triazol-1-yl)-benzohydrazide (6)
Yield 71%, m.p. 301–302 °C; IR (KBr): 3327, 3275, 3129,
1661, 1616, 1590, 1549, 1517, 1411, 1347, 1238, 1155,
;
1076, 779, 686 cm-1 1H NMR (DMSO-d6): d (ppm):
1
1448, 1335, 1252, 933, 847, 748 cm-1; H NMR (DMSO-
d6): d (ppm): 10.33 (s, 1H); 9.35 (s, 1H); 8.05 (d, 1H,
J = 8.5 Hz,); 7.98–7.90 (m, 2H); 7.56–7.44 (m, 4H);
7.41–7.34 (m, 1H); 13C NMR (DMSO-d6): d (ppm):
160.71; 147.44; 139.72; 130.05; 129.06; 128.36; 125.38;
119.57; 114.59; 109.73; 107.86; HRESI-MS m/z calcd for
C15H13N5O2 [M?H]? 296.1142 Found 296.1134.
12.33 (s, 1H); 12.02 (s, 1H); 9.37 (s, 1H); 8.45 (s, 1H); 8.11
(d, 1H, J = 8.6 Hz); 7.95 (d, 2H, J = 7.6 Hz); 7.79 (s,
1H); 7.71 (t, 1H, J = 4.4 Hz); 7.65–7.56 (m, 2H);
7.56–7.45 (m, 5H); 7.38 (t, 1H, J = 7.4 Hz); 13C NMR
(DMSO-d6): d (ppm): 163.88; 159.92; 147.51; 147.26;
140.02; 136.27; 133.69; 130.74; 130.68; 130.01; 129.96;
129.00; 128.37; 126.50; 125.97; 125.40; 119.57; 116.14;
110.02; 107.78; HRESI-MS m/z calcd for C22H16ClN5O2
[M?H]? 418.1065 Found 418.1051.
Synthesis of compounds (4–21)
To a suspension 2-hydroxy-4-(4-phenyl-1H-1,2,3-triazol-1-
yl)benzohydrazide (3) (0.29 g, 1.0 mmol) in ethanol
(10 mL) and the appropriate aryl carbonyl compound
(1.0 mmol), 2 drops of glacial acetic acid were added, then
the reaction mixture was heated under reflux for 6 h. The
reaction mixture was cooled and the precipitated product
was filtered, washed with cold ether and recrystallized from
aqueous DMF to give yellow solid product.
N1-(4-Fluorobenzylidene)-2-hydroxy-4-(4-phenyl-1H-
1,2,3-triazol-1-yl)-benzohydrazide (7)
Yield 47%, m.p. [300 °C; IR (KBr): 3420, 3267, 3086,
1624, 1559, 1508, 1407, 1236, 1152, 1040, 925, 833, 788,
1
764, 691 cm-1; H NMR (DMSO-d6): d (ppm): 12.39 (s,
1H); 11.97 (s, 1H); 9.38 (d, 1H, J = 3.6 Hz); 8.47 (d, 1H,
J = 3.5 Hz); 8.12 (dd, 1H, J = 8.6, 3.6 Hz); 7.95 (dd, 2H,
J = 7.7, 3.6 Hz); 7.81 (ddd, 2H, J = 8.8, 5.5, 3.0 Hz);
7.65–7.53 (m, 2H); 7.49 (td, 2H, J = 7.6, 3.5 Hz); 7.38 (td,
1H, J = 7.5, 3.6 Hz); 7.30 (td, 2H, J = 8.7, 3.5 Hz); 13C
NMR (DMSO-d6): d (ppm): 164.52; 163.83; 162.05;
160.14; 147.87; 147.50; 139.98; 130.67; 130.56; 130.02
129.54; 129.46; 129.01; 128.37; 125.39; 119.58; 116.07;
115.85; 109.92; 107.84; HRESI-MS m/z calcd for
C22H16FN5O2 [M?H]? 402.1361 Found 402.1350.
N1-(4-(Dimethylamino)benzylidene)-2-hydroxy-4-(phenyl-
1H-1,2,3-triazol-1-yl)- benzohydrazide (4)
Yield 57%, m.p 293–294 °C; IR (KBr): 3424, 3287, 3134,
2914, 1651, 1614, 1593, 1533, 1350, 1314, 1234 1188, 845,
1
766 cm-1; H NMR (DMSO-d6): d (ppm): 12.67 (s, 1H);
11.73 (s, 1H); 9.38 (s, 1H); 8.33 (s, 1H); 8.14 (d, 1H,
J = 8.4 Hz); 7.96 (d, 2H, J = 7.7 Hz); 7.54 (dt, 6H,
J = 30.2, 7.6 Hz); 7.39 (t, 1H, J = 7.5 Hz); 6.75 (d, 2H,
J = 8.4 Hz); 2.97 (s, 6H); 13C NMR (DMSO-d6): d (ppm):
163.63; 160.52; 151.71; 150.13; 147.49; 139.86; 130.06;
130.03; 129.02; 128.75; 128.38; 125.39; 121.10; 119.75;
115.65; 111.74; 109.84; 107.83; 39.73; HRESI-MS m/z
calcd for C24H22N6O2 [M?H]? 427.1877 Found 427.1863.
N1-(4-Chlorobenzylidene)-2-hydroxy-4-(4-phenyl-1H-
1,2,3-triazol-1-yl)-benzohydrazide (8)
Yield 86%, m.p. 300–301 °C; IR (KBr): 3423, 3274, 3064,
1620, 1548, 1515, 1490, 1405, 1237, 1090, 923, 823, 763,
N1-(3-Bromobenzylidene)-2-hydroxy-4-(4-phenyl-1H-
1
690, 627 cm-1; H NMR (DMSO-d6): d (ppm): 12.04 (s,
1,2,3-triazol-1-yl)-benzohydrazide (5)
1H); 11.95 (s, 1H); 9.14 (d, 1H, J = 3.6 Hz); 8.50 (d, 1H,
J = 3.5 Hz); 7.96 (br. d, 1H); 7.81 (dd, 2H, J = 7.5,
3.4 Hz); 7.52–7.43 (m, 9H); 13C NMR (DMSO-d6): d
(ppm): 164.52; 163.83; 162.05; 160.14; 147.9; 147.50;
139.98; 130.30; 130.56; 130.02 129.40; 129.46; 129.01;
128.8; 120.4; 119.58; 116.07; 115.85; 109.92; 107.84;
Yield 71%, m.p. [300 °C; IR (KBr): 3450, 3255, 3035,
1
1639, 1617, 1551, 1515, 1253, 1042, 755, 795 cm-1; H
NMR (DMSO-d6): d (ppm): 12.31 (s, 1H); 12.03 (s, 1H);
9.38 (s, 1H); 8.44 (s, 1H); 8.11 (d, 1H, J = 8.6 Hz); 7.95
(d, 3H, J = 7.7 Hz); 7.75 (d, 1H, J = 7.7 Hz); 7.60 (dt,
123

H. H. M. Abdu-Allah et al.
HRESI-MS m/z calcd for C22H16ClN5O2 [M?H]?
(s, 6H); 13C NMR (DMSO-d6): d (ppm): 160.23; 149.39;
148.91; 148.03; 147.51; 139.95; 130.39; 130.02; 129.02;
128.39; 125.40; 123.75; 119.58; 109.94; 108.51; 105.26;
101.65; 65.00; HRESI-MS m/z calcd for C24H22N5O4
[M?H]? 444.1666 found 444.1649.
418.1071 Found 418.1000.
2-Hydroxy-N1-(2-hydroxybenzylidene)-4-(4-phenyl-1H-
1,2,3-triazol-1-yl)-benzohydrazide (9)
Yield 65%, m.p. [300 °C; IR (KBr): 3440, 3260, 3064,
1636, 1620, 1546, 1516, 1466, 1406, 1361, 1237, 1037,
863, 752, 690 cm-1; 1H NMR (DMSO-d6): d (ppm): 12.15
(s, 1H); 11.16 (s, 1H); 9.38 (s, 1H); 8.71 (s, 1H); 8.13 (d,
1H, J = 8.2 Hz); 7.95 (d, 2H, J = 7.4 Hz); 7.70–7.26 (m,
9H); 6.94 (t, 2H, J = 6.8 Hz); 13C NMR (DMSO-d6): d
(ppm): 157.47; 147.48; 131.70; 129.96; 129.00; 128.38;
125.36; 119.60; 119.40; 116.43; 110.06; HRESI-MS m/z
calcd for C22H17N5O3 [M?H]? 400.1404 Found 400.1395.
2-Hydroxy-4-(4-phenyl-1H-1,2,3-triazol-1-yl)-N1-
(pyridine-2-ylmethylene)-benzohydrazide (13)
Yield 78%, m.p. 287–288 °C; IR (KBr): 3435, 3263, 3137,
3052, 1635, 1618, 1552, 1467, 1237, 1040, 940, 778, 742,
1
629 cm-1; H NMR (DMSO-d6): d (ppm): 12.12 (s, 1H);
9.38 (s, 1H); 8.63 (d, 1H, J = 4.6 Hz); 8.50 (s, 1H); 8.20-
7.85 (m, 7H); 7.66–7.34 (m, 9H); 13C NMR (DMSO-d6): d
(ppm): 164.14; 160.02; 152.99; 149.57; 149.08; 147.52;
140.05; 136.89; 130.63; 130.02 128.36; 125.41; 124.59;
120.17; 119.58; 116.23; 109.99; 107.79; HRESI-MS m/z
calcd for C21H16N6O2 [M?H]? 385.1408 Found 385.1396.
N1-(2,6-Dichlorobenzylidene)-2-hydroxy-4-(4-phenyl-1H-
1,2,3-triazol-1-yl)-benzohydrazide (10)
Yield 97%, m.p. 279–280 °C; IR (KBr): 3421, 3282, 3128,
3066, 1678, 1616, 1579, 1546, 1413, 1237, 1158, 1066,
931, 765, 688 cm-1; 1H NMR (DMSO-d6): d (ppm): 12.21
(s, 1H); 9.40 (s, 1H); 8.70 (s, 1H); 8.13 (d, 1H,
J = 8.5 Hz); 7.96 (d, 2H, J = 7.8 Hz); 7.65–737 (m,
10H); 13C NMR (DMSO-d6): d (ppm): 164.15; 160.06;
147.52; 144.39; 140.10; 134.01; 131.38; 130.67; 130.35;
130.01; 129.13; 129.02; 128.39; 125.40; 119.63; 116.10;
110.04; 109.55; 107.83; HRESI-MS m/z calcd for C22H15
Cl2N5O2 [M?H]? 452.0676 Found 452.0662.
2-Hydroxy-4-(4-phenyl-1H-1,2,3-triazol-1-yl)-N1-((z)-3-
phenylallylidene)-benzohydrazide (14)
Yield 83%, m.p. 284–285 °C; IR (KBr): 3438, 3280, 3058,
1625, 1609, 1581, 1518, 1245, 1142, 1077, 969, 915, 759,
690 cm-1; 1H NMR (DMSO-d6): d (ppm): 12.43 (s, 1H); 11.85
(s, 1H); 9.39 (s, 1H); 8.27 (d, 1H, J = 6.3 Hz); 8.11 (d, 1H,
J = 8.6 Hz); 7.96 (d, 2H, J = 7.6 Hz); 7.71–7.30 (m, 13H);
7.10 (d, 2H, J = 5.3 Hz); 13C NMR (DMSO-d6): d (ppm):
163.81; 160.15; 151.09; 147.50; 140.02; 139.96; 135.78;
130.48; 130.01; 129.02; 128.85; 128.39; 127.22; 125.40;
125.34; 119.61; 116.00; 109.96; 107.80; HRESI-MS m/z calcd
for C24H20N5O2 [M?H]? 410.1612 Found 410.1602.
N1-(Benzo[d][1,3]dioxol-5-ylmethylene)-2-hydroxy-4-(4-
phenyl-1H-1,2,3-triazol-1-yl)-benzohydrazide (11)
1
Yield 87%, H NMR (DMSO-d6): d (ppm): 12.44 (s, 1H);
N1-(1-(2-Fluorophenyl)ethylidene)-2-hydroxy-4-(4-phenyl-
11.87 (s, 1H); 9.38 (s, 1H); 8.12 (s, 1H); 7.95 (d, 2H,
J = 7.2 Hz); 7.64–7.55 (m, 2H); 7.50 (t, 3H, J = 7.4 Hz);
7.39 (t, 1H); 7.33 (s, 1H); 7.21 (d, 1H, J = 7.6 Hz); 7.00
(d, 1H, J = 7.4 Hz); 6.1 (s, 2H); 13C NMR (DMSO-d6): d
(ppm): 160.23; 149.39; 148.91; 148.03; 147.51; 139.95;
130.39; 130.02; 129.02; 128.39; 125.40; 123.75; 119.58;
109.94; 108.51; 105.26; 101.65; HRESI-MS m/z calcd for
C23H17N5O4 [M?H]? 428.1353 Found 428.1340.
1H-1,2,3-triazol-1-yl)-benzohydrazide (15)
Yield 75%, m.p. 281–282 °C; IR (KBr): 3439, 3318, 3137,
3075, 1674, 1614, 1519, 1257, 915, 753 cm-1; H NMR
1
(DMSO-d6): d (ppm): 11.37 (s, 1H); 9.37 (s, 1H); 9.30 (s,
1H); 8.19 (dd, 2H, J = 19.9, 8.5 Hz); 7.95 (dd, 3H,
J = 12.6, 7.6 Hz); 7.72–7.23 (m, 16H); 2.32 (s, 3H); 13C
NMR (DMSO-d6): d (ppm): 161.25; 158.77; 157.53, 156.69;
150.62; 147.53; 147.48; 139.73; 132.94; 132.59; 130.03;
129.99; 129.91; 129.00; 128.37; 125.62; 125.42; 125.39;
124.49; 124.46; 119.60; 119.55; 118.03; 116.26; 116.04;
110.69; 107.65; 107.45; 24.31; 17.21; 17.16; HRESI-MS m/z
calcd for C23H18FN5O2 [M?H]? 416.1517 Found 416.1503.
N1-(2,5-Dimethoxybenzylidene)-2-hydroxy-4-(4-phenyl-
1H-1,2,3-triazol-1-yl)-benzohydrazide (12)
Yield 90%, m.p. 277–278 °C; IR (KBr): 3459, 3265, 3080,
2943, 2835, 1662, 1610, 1548, 1516, 1495, 1466, 1263,
1
1263, 1041, 938, 764, 709 cm-1; H NMR (DMSO-d6): d
2-Hydroxy-N1-(2-oxoindolin-3-ylidene)-4-(4-phenyl-1H-
(ppm): 12.04 (s, 1H); 11.23 (s, 1H); 9.38 (s, 1H); 8.12 (s,
1H); 7.95 (d, 2H, J = 7.2 Hz); 7.64–7.55 (m, 2H); 7.50 (t,
3H, J = 7.4 Hz); 7.39 (t, 1H,); 7.33 (s, 1H); 7.21 (d, 1H,
J = 7.6 Hz); 7.00 (d, 1H, J = 7.4 Hz); 6.1 (br.s, 2H); 3.85
1,2,3-triazol-1-yl)-benzohydrazide (16)
Yield 80%, m.p. [300 °C; IR (KBr): 3439, 3296, 3133,
1723, 1680, 1612, 1504, 1482, 1465, 1401, 1212, 1156,
123

Synthesis and anti-mycobacterial activity…
1
1152, 1052, 757 cm-1; H NMR (DMSO-d6): d (ppm):
NMR (DMSO-d6): d (ppm): 12.60 (s, 1H); 9.36 (s, 1H); 8.24
(d, 1H, J = 8.6 Hz); 7.96 (d, 2H, J = 7.6 Hz); 7.69–7.61 (m,
2H); 7.56 (d, 1H, J = 8.7 Hz); 7.49 (t, 2H, J = 7.6 Hz);
7.45–7.32 (m, 6H); 7.14 (t, 1H, J = 7.6 Hz); 7.04 (d, 1H,
J = 7.9 Hz); 5.00 (s,2H); 13C NMR (DMSO-d6): d (ppm):
159.86, 157.65; 147.56; 142.51; 140.37; 134.98; 132.26;
131.30; 129.97; 129.32; 128.99; 128.71; 128.38; 125.43;
123.18; 120.76; 119.95; 119.60; 110.11; 107.42; HRESI-MS
m/z calcd for C30H21ClN6O3 [M?H]? 549.1436 Found
549.1414.
12.44 (s, 1H); 11.17 (s, 1H); 9.36 (s, 1H); 8.22 (d, 1H,
J = 8.7 Hz); 8.00–7.92 (m, 2H); 7.68 (d, 1H, J = 2.1 Hz);
7.63–7.32 (m, 2H); 7.09 (td, 1H, J = 7.6, 1.0 Hz); 6.93 (dt,
1H, J = 7.7, 0.9 Hz); 2.54 (s, 3H); HRESI-MS m/z calcd
for C23H16N6O3 [M?H]? 425.1357 Found 425.1345.
2-Hydroxy-N1-(2-oxo-5-(trifluoromethoxy)indolin-3-
ylidene)-4-(4-phenyl-1H-1,2,3-triazol-1-yl)-
benzohydrazide (17)
Yield 72%, m.p. [300 °C; IR (KBr): 3430, 3140, 3064,
1671, 1613, 1485, 1403, 1277, 1248, 1205, 1138, 1055,
929, 827, 675 cm-1; 1H NMR (DMSO-d6): d (ppm): 11.35
(s, 1H); 9.36 (s, 1H); 8.23 (d, 1H, J = 8.6 Hz); 8.00-7.92
(m, 2H); 7.68 (d, J = 2.1 Hz, 1H); 7.56 (dd, 1H, J = 8.7,
2.1 Hz); 7.52–7.45 (m, 3H); 7.43–7.33 (m, 2H); 7.01 (d,
1H, J = 8.5 Hz); HRESI-MS m/z calcd for C24H15F3 N6O4
[M?H]? 509.1180 Found 509.1169.
2-Hydroxy-N1-(1-(3-methoxybenzyl)-2-oxoindolin-3-
ylidene)-4-(4-phenyl-1H-1,2,3-triazol-1-yl)-
benzohydrazide (21)
Yield 73%, m.p. 286–287 °C; IR (KBr): 3402, 3214, 3128,
3067, 1702, 1670, 1614, 1489, 1466, 1357, 1241, 1143,
1
1018, 765 cm-1; H NMR (DMSO-d6): d (ppm): 9.36 (s,
1H); 8.25 (d, 1H, J = 8.6 Hz); 8.00-7.90 (m, 3H); 7.69-
7.62 (m, 2H); 7.53 (dt, 4H, J = 24.2, 8.0 Hz); 7.43–7.33
(m, 2H); 7.26 (t, 1H, J = 7.9 Hz); 7.14 (t, 1H,
J = 7.5 Hz); 7.05 (d, 1H, J = 7.9 Hz); 7.01–6.90 (m, 2H);
6.86 (dd, 1H, J = 8.3, 2.5 Hz); 4.96 (d, 2H, J = 6.5 Hz);
3.72 (d, 3H, J = 2.3 Hz,); 13C NMR (DMSO-d6): d
(ppm):160.58; 159.87; 159.51; 157.65; 147.58; 142.73;
140.39; 137.48; 133.62; 130.03; 129.98; 129.93; 129.14;
129.03; 128.40; 125.44; 125.38; 123.15; 120.74; 119.89;
119.63; 119.38; 117.42; 113.47; 112.73; 110.81; 110.25;
109.79; 107.84; 107.45; 55.09; HRESI-MS m/z calcd for
C31H24N6O4 [M?H]? 545.1932 Found 545.1919.
Sodium 3-(2-(2-hydroxy-4-(4-phenyl-1H-1,2,3-triazol-1-
yl)benzoyl)hydrazono)-2-oxo- indoline-5-sulfonate (18)
Yield 57%, m.p. [300 °C; IR (KBr): 3399, 3139, 3088,
1717, 1685, 1617, 1537, 1510, 1212, 1190, 1152, 1029,
1
726 cm-1; H NMR (DMSO-d6): d (ppm): 12.03 (s, 1H);
10.60 (s, 1H); 8.97 (d, 2H, J = 7.1 Hz); 7.94 (s, 1H); 7.86
(d, 1H, J = 8.6 Hz); 7.58 (d, 3H, J = 7.8 Hz); 7.42–7.35
(m, 2H); 7.16 (dt, 6H, J = 34.0, 8.3 Hz,); 7.01 (d, 2H,
J = 7.3 Hz); 6.56 (d, 1H, J = 8.1 Hz); 13C NMR (DMSO-
d6): d (ppm): 165.27; 147.57; 147.49; 142.48; 140.42;
138.65; 133.21; 130.40; 130.04; 129.06; 128.42; 125.45;
125.41; 121.59; 119.63; 114.58; 110.60; 110.19; 109.88;
108.15; 107.85; 107.66; HRESI-MS m/z calcd for
C23H15N6NaO6S [M?H]? 527.0744 Found 528.07649.
Biological screening
Anti-mycobacterial activity: In-vitro MABA assay
N1-(5-Chloro-2-oxoindolin-3-ylidene)-2-hydroxy-4-(4-
The anti-mycobacterial activities of compounds 4–21 were
evaluated against M. tuberculosis H37Rv using Microplate
Alamar Blue assay (MABA) (Franzblau et al. 1998).
Bacterial inoculi of Mycobacterium tuberculosis H37Rv
were prepared from fresh LJ medium and re-suspended in
Middlebrook 7H9 broth supplemented with 10% OADC
(HiMedia Laboratories). This culture was grown at 37 °C
until the turbidity matched with McFarland no.1 turbidity
standard. The culture was diluted to 1:25 with media and
100 lL was used as an inoculum. Each drug stock solution
was thawed and diluted in Middlebrook 7H9-OADC at
four-fold the final highest concentration tested. Serial two
fold dilutions of each drug were prepared directly in a
sterile 96-well microtiter plate using 100lL Middlebrook
7H9-OADC. Each plate also contained wells containing
MTB cultures without addition of the tested compounds
and wells with only sterile medium as controls. Sterile
phenyl-1H-1,2,3-triazol-1-yl)-benzohydrazide (19)
Yield 75%, m.p. [300 °C; IR (KBr): 3450, 3140, 3057, 1721,
1683, 1616, 1511, 1465, 1243, 1147, 1023, 817, 758 cm-1; 1H
NMR (DMSO-d6): d (ppm): 11.27 (s, 1H); 9.34 (d, 1H,
J = 7.5 Hz); 8.21 (d, 1H, J = 8.6 Hz); 7.95 (d, 2H,
J = 7.4 Hz); 7.69–7.63 (m, 1H); 7.58–7.45 (m, 4H); 7.38 (t,
2H, J = 7.6 Hz); 6.92 (d, 1H, J = 8.4 Hz); HRESI-MS m/z
calcd for C23H15ClN6O3 [M?H]? 459.0967 Found 459.0954.
N1-(1-(4-Chlorobenzyl)-2-oxoindolin-3-ylidene)-2-
hydroxy-4-(4-phenyl-1H-1,2,3-triazol-1-yl)-
benzohydrazide (20)
Yield 77%, m.p. 277–279 °C; 3482, 3140, 3069, 1701, 1672,
1613, 1490, 1467, 1356, 1222, 1142, 1016, 763 cm-1; H
1
123

H. H. M. Abdu-Allah et al.
water was added to all outer wells to avoid evaporation
during incubation. The plate was covered, sealed in plastic
bags and incubated at 37 °C. MIC values were determined
using MABA assay (Franzblau et al. 1998). After 5 days of
incubation, 50 lL of 1:1 mixture of alamar blue solution
(Sigma Aldrich) and sterile 10% tween 80 (Nice chemi-
cals) were added to each well and the plate was re-incu-
bated overnight (reading on 7th day). A change in color
from blue (oxidized state) to pink (reduced) indicated
growth of bacteria and the minimum inhibitory concen-
tration (MIC) was defined as the lowest concentration of
the compound that prevented this change in color.
centrifuged at 1200 rcf for about 3 min and the super-
natants were removed, subsequently to each well 200 lL of
DMSO was added. The absorbance was measured at a
wavelength of 490 nm on Victor-3-microplate reader
against the blank. Three replicate wells were done for each
concentration of drug to minimize the error rate. The
cytotoxicity of each compound was expressed as % inhi-
bition. The selective activities of the compounds were
determined by calculating the selectivity index (SI). SI was
calculated by dividing 50 lg/mL by MIC. As Cytotoxicity
is less than 50% at 50 lg/mL. All the values are mentioned
by ‘‘[’’. Selectivity index (SI) = 50 lg/mL/MIC lg/mL.
Nutrient starvation model
Pharmacophore modeling
Compounds showing promising MIC (B1.5 lg/mL) were
evaluated for their efficiency against dormant forms of
bacteria by nutrient starvation model. MTB dormant cul-
ture was prepared using nutrient starvation method (Betts
et al. 2002; De Man 1983). In nutrient starvation model,
culture of MTB H37Rv grown in Middlebrook 7H9 med-
ium supplemented with OADC (nutrient rich medium) was
pelleted and washed twice with PBS (Phosphate Buffer
Saline, obtained from HiMedia Laboratories). The pellet
was suspended in PBS in sealed bottles and was incubated
at 37 °C for 6 weeks. These cultures were treated with
standard drugs like isoniazid, rifampicin and moxifloxacin
along with the synthesized compounds for 7 days at a
concentration of 10 lg/mL. The treated cell suspensions
were diluted 10-fold up to 10—6 using Middlebrook 7H9
medium supplemented with OADC and 100 ll of each
dilution were plated in 48 well plates in triplicates along
with 900 ll of Middlebrook 7H9 medium (HiMedia Lab-
oratories) supplemented with OADC (HiMedia Laborato-
ries). The plates were incubated at 37 °C for 3–4 weeks the
wells with visible bacterial growth were counted as posi-
tive. The bacterial count was determined by using standard
statistical methods using MPN assay.
The pharmacophore model was generated using the
HypoGen algorithm of Catalyst software as implemented
in Discovery Studio 4.1 package (Debnath 2002). Com-
pounds 3–21 were built de novo with standard options
within the 2D/3D editor sketcher of the program. Confor-
mational analysis was performed using the ‘‘BEST’’ option
with a maximum of 250 conformers per molecule and a
20 kcal/mol energy threshold above the calculated global
minimum. The inactivity spread, uncertainty, and spacing
parameters were set to 1.5, 2, and 1.8, respectively, as
proposed by Accelrys for training sets with narrower
activity span than usual. The hydrogen-bond-acceptor
(HBA) and -donor (HBD), hydrophobic (HYD), and aro-
matic ring (AR) chemical features were considered for
hypothesis generation and no excluded volumes. Hypoth-
esis selection was done by a cost analysis procedure (rep-
resented in bit units) based on three terms: weight cost,
error cost (penalizes the deviation between the estimated
activities of the training set and their experimentally
determined values); and configuration cost (penalizes the
complexity of the hypothesis, should not exceed a maxi-
mum value of 18). The error cost contributes the most in
determining the overall cost of a hypothesis. In addition,
the costs of the ideal hypothesis, the simplest possible
hypothesis that fits the data with minimal cost (fixed cost),
and the null hypothesis in which the error cost is high (null
cost) are computed by the HypoGen module. Statistically
significant hypotheses possess total costs close to the fixed
cost and far away from the null cost values.
Evaluation of cytotoxicity
The cytotoxic activity was measured in vitro against
Human Embryonic Kidney (HEK-293T) cells at 50 lg/mL
concentration using 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay (Sykes et al.
´
2012; Bezivin et al. 2003). The cells were cultured in a flat
bottomed 96-well plate till a density of 5 9 105 cells was
reached and the test compounds were respectively added to
each well. While the incubation was permitted at 37 °C,
with 5% CO2 and 95% O2 atmosphere for 48 h before the
cytotoxicity assessments. Just before 4 h of the end of the
incubation, 10 lL of MTT reagent (10 mg mL-1) was
added per well. Four hours later, the 96-well plate was
Results and discussion
Chemistry
The synthesis of the target compounds (3–21) was achieved
starting from PAS (Scheme 1) by the following sequence
of reactions: PAS was esterified, diazotized and treated
123

Synthesis and anti-mycobacterial activity…
Scheme 1 Synthesis of the target compounds 3–21
with sodium azide (Sandmyer reaction) to give ethyl
4-azidosalicylate 1 (Gano et al. 2001). This sequence of
synthesizing 1 is critical for producing a solid easy to
purify, while direct azidation of PAS produces a semisolid
that was difficult to manipulate. Reacting 1 with pheny-
lacetylene, using click chemistry, in presence of sodium
ascorbate and copper sulfate in a mixture of THF/H2O
afforded a new intermediate ethyl 2-hydroxy-4-(4-phenyl-
1H-1,2,3-triazol-1-yl)benzoate; compound 2. The structure
and C = O functions respectively, while it showed no
1
signals derived from ethyl moiety in H NMR spectrum.
Finally, condensation of 3 with different aldehydes, o-flu-
oroacetophenone and isatins in ethanol gave the corre-
1
sponding hydrazones 4–21. IR, H NMR, 13C NMR and
HRMS spectral data are in agreement with the proposed
structures of the synthesized compounds. Hydrazones 4–21
show carbonyl amide stretching at about 1650 cm-1 and
1
N–H bands in 3250–3200 cm-1 region. H NMR spectra
1
of 2 was confirmed by analyzing its H NMR spectrum
displayed additional signals due to the aromatic ring
derived from aldehyde, acetophenone or isatin moieties in
the aromatic region and showed two signals at about d
7.35 ppm and d 10.31 ppm which were attributed to the
N_CH and NH protons, respectively. N = CH carbon of
the compounds in the hydrazone structures were observed
at d 143.5–149.8 ppm in their 13C NMR spectra.
which shows the appearance of a triplet signal at d
1.34 ppm and quartet at 4.50-4.24 ppm corresponding to
C2H5 moiety, singlet 9.40 (-CH triazole) and singlet at
10.91 corresponding to OH group. The key intermediate p-
(2-hydroxy-4-phenyl-1
H-1,2,3-triazol-1-yl)benzoichy-
drazide 3 was prepared by hydrazinolysis of 2 with
hydrazine hydrate. IR spectrum of compound (3) showed
bands at 3335, 3278 and 1620 cm-1 due to OH, NHNH2
The mass spectra of compounds showed [M?1] peaks in
agreement with their molecular formula. The 1H NMR
123

H. H. M. Abdu-Allah et al.
spectra of the derivatives 4–21 revealed only one N–H
signal for each compound, which was attributed to the (E)-
diastereomer, as reported for similar compounds (Jorda˜o
et al. 2011).
Table 1 Anti-mycobacterial activity of compounds 3-21 against M.
tuberculosis H37Rv strain (ATCC-27294), cytotoxicity and selectiv-
ity index (SI)
Compd. no.
Activity MICa
(lg/mL)
Cytotoxicity
HEK 293 at
50 lg/mLa
Selectivity
index
Anti-mycobacterial activity
(% inhibition)
3
12.5
1.56
1.56
12.5
3.12
6.25
12.5
3.12
12.5
1.56
3.12
25
23.45
[4
[32
[32
[4
[15
[8
[4
[15
[4
[32
[15
–
The target compounds (3–21) were evaluated for their
in vitro activity against M. tuberculosis H37Rv by MABA
(Franzblau et al.1998). The MIC values (lg/mL) along
with the standard drugs are presented in Table 1. Com-
pounds 3–21 showed in vitro activity against MTB with
MIC ranging from 0.39 to 25 lg/mL. Eight compounds (4,
5, 7, 10, 12, 13, 19 and 20) exhibited greater potency than
PAS, while derivatives 4, 5, 12 and 20 exhibited higher
potencies than ethambutol, and analogues 7, 10, 12, 13, 19
were equipotent to ethambutol. Interestingly, compound 20
(MIC 0.39 lg/mL) showed equipotent activity to rifampi-
cin. A preliminary structure activity relationship was
deduced based on the obtained biological activity. It was
noted that the type of substituent on aromatic ring of the
benzylidene group has a significant effect on the observed
biological activity. Among the benzylidene derivatives,
compounds 4 (p-N(CH3)2), 5 (m-Br) and 12 (2,5-diMeO)
showed high potency (MIC 1.5 lg/mL). Accordingly, it
seems that inductively electron withdrawing but
mesomerically electron donating substituents on phenyl
group is/are required for high activity. On the other hand,
only two isatins derivatives (19 and 20) showed promising
activity with MIC 3.12 and 0.39 lg/mL, respectively. The
promising potency of 20 is of interest and reveals the
detrimental effect of N-substituent of isatin on activity.
Surprisingly, introducing trifluoromethoxy or sulfonate at
5-position of isatin or N-(m-methoxybenzyl) drastically
decreased the activity which can be accounted for by either
decreasing the solubility (these compounds are poorly
soluble in common solvents including DMSO), or steric
clash at the binding site.
4
28.97
5
34.0
6
30.9
7
27.8
8
30.9
9
34.56
10
22.68
11
31.23
12
25.56
13
36.75
14
Not tested
40.23
15
6.25
12.5
[25
[25
3.12
0.39
[25
5
[8
[4
–
16
27.89
17
Not tested
Not tested
40.78
18
–
19
[15
[128
–
20
42.31
21
Not tested
NDb
PAS
–
Isoniazid
Rifampicin
Ethambutol
a
0.06
0.39
3.13
21.78
[833
[128
[15
18.12
16.90
Values are means of three experiments
b
Not determined (no inhibition at the tested dose)
Cytotoxicity
The in vitro cytotoxicity for analogues with MIC B12.5 lg/
mL was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-
´
diphenyltetrazolium bromide (MTT) assay (Bezivin et al.
2003; Sykes et al. 2012) against Human Embryonic Kidney
(HEK-293T) cells at 50 lg/mL concentration. The percentage
inhibition is reported in Table 1. The ratio between in vitro
cytotoxicity and antimycobacterial activity (MIC in lg/mL)
determines the selectivity index (SI). The most promising
antitubercular compounds 4, 5, 12, 13, 19 and 20 exhibited
selectivity index [10, revealing good safety margin for the
active compounds.
Activity against dormant forms (nutrient starvation
model)
Compounds which showed MIC B1.56 lg/mL (4, 5, 12
and 20), along with the standard drugs isoniazid, rifampicin
and moxifloxacin, were evaluated for their potency against
dormant forms of mycobacterium by nutrient starvation
model at 10 lg/mL concentration (Betts et al. 2002; De
Man 1983). Among the compounds tested compound 20
showed 2.4 log reduction in bacterial count which is
comparable to the activity of the tested standard drugs
(Fig. 2). Meanwhile, compound 4 was found to be equally
efficacious as isoniazid and rifampicin but less potent when
compared to moxifloxacin.
Molecular modeling
HypoGen algorithm as implemented in Discovery Studio
4.1 package (Accelrys, San Diego, CA) was used for the
generation of a three-dimensional (3D) pharmacophore
123

Synthesis and anti-mycobacterial activity…
1.0 108
process starts with the generation of low-energy confor-
mations of each ligand in the training set followed by the
generation of 10 hypotheses based on HypoGen algorithm
(Table 2).
×
1.0 107
×
1.0 106
×
1.0 105
×
1.0 104
The quality of Hypo1 was tested in terms of fixed cost,
null cost and total cost (17). The total cost for each phar-
macophore model is the summation of the three cost
components; error (E), weight (W), and configuration (C))
multiplied by a coefficient. The fixed cost is the simplest
model that fits the data perfectly, while the null cost rep-
resents the cost of a hypothesis with no features that esti-
mates every activity to be the average activity.
Accordingly, there should be a large difference between the
fixed cost and null cost for a pharmocophore of high pre-
diction power. In addition, the total cost of the model
should be close to the fixed cost. In case of Hypo1, the
difference between the fixed cost (74.1) and the null cost
(119.7) is 45.6 which imply a 75–90% probability for
correlating the experimental and predicted activity data.
Moreover, the total cost for Hypo1 is 82.1 which is close to
the fixed cost (74.1) for the hypothesis calculations.
Analysis of the highest ranked pharmacophore shows a
model of four features; one hydrogen-bond acceptor
(HBA), two aromatic ring features (RA) and a hydrophobic
feature (HB) in addition to a ‘‘shape inclusion’’ boundary
(Fig. 3).
×
1.0 103
×
1.0 102
×
1.0 101
×
1.0 100
×
l
I
4
5
r
H
12
20
N
I
RIF
Ct
MOX
Compounds
Fig. 2 Activity of compounds (4, 5, 12 and 20) against nutrient
starved MTB model. Bacterial count estimation (Mean SD, n = 3)
for control and treated groups conducted by using MPN (most
probable number) assay. The statistical significance (p \ 0.0001)
with respect to control has been analyzed by Two-way ANOVA using
GraphPad Prism Software
model for the 19 (3–21) tested compounds. In the current
study the number of the training set compound (19
derivatives) as well as their biological activity span was
optimal for HypoGen pharmacophore generation. The
Table 2 Characteristics for the 10 models generated by HypoGen
module of catalyst
Mapping of the synthesized active compounds into
Hypo1 (Fig. 4) shows similar alignment with the calculated
pharmacophoric features with the exception of compounds
4 and 20 which show slightly different orientation. For the
majority of the compounds the two aromatic ring features
of Hypo1 are mapped into the triazole ring and its C-4
phenyl substituent. The C-2 hydroxyl group of the middle
phenyl ring fits well into the hydrogen bond acceptor fea-
ture of Hypo1. Finally, the terminal aromatic moiety (either
phenyl or indoline rings) maps into the hydrophobic feature
of Hypo1. Fitting of the synthesized active compounds into
Hypo1 shows similar alignment into the pharmacophore
Model
Total Cost
RMS
Correlation (r)
Hypo1
Hypo2
Hypo3
Hypo4
Hypo5
Hypo6
Hypo7
Hypo8
Hypo9
Hypo10
82.1
85.1
87.1
88.3
88.6
89.4
90.1
90.3
91.5
93.8
1.02
1.13
1.29
1.41
1.51
1.50
1.58
1.59
1.56
1.59
0.89
0.82
0.78
0.77
0.72
0.67
0.67
0.68
0.62
0.64
Fig. 3 Spatial arrangements of
the structural features for the
highest ranked HypoGen model
(Hypo1) showing inter-feature
distances
123

H. H. M. Abdu-Allah et al.
Fig. 4 Active compounds
mapped into Hypo1. Model
features are colour coded
according to Fig. 1
features (Fig. 4) as well as good agreement between pre-
dicted and observed activity (Table 3, R = 0.89).
Table 3 Fit values and experimental and predicted activities of the
compounds (3–21)
The most active compound 20 together with compound
4 are showing different orientation while the triazole and
the hydroxyphenyl ring map nicely into the two aromatic
ring features of Hypo1 (compound 20, Fig. 5). In this case,
the hydrogen bond acceptor feature maps into the N2 of the
hydrazine moiety and the hydrophobic feature fits into the
terminal phenyl ring similarly to other active compounds.
Interestingly, the terminal phenyl ring for these two
molecules (4 and 20) occupy a hypothetical sub-pocket that
is vacant for all other molecules. This may partially explain
the high activity of analogue 20 (MIC = 0.39 lg/
mL).While it was not possible to explain the observed low
activity for compounds 17, 18 and 21 by simple SAR
analysis, mapping of these compounds into Hypo1 shows
unfavorable orientation of the C-5 substituents outside the
shape inclusion feature (Fig. 6). Moreover, calculating the
possible forbidden areas at the receptor side confirmed the
orientation of the trifluromethoxy and the sulfonate groups
(for 17 and 18 respectively) so that they fit into two ‘‘ex-
cluded volumes’’ features (grey maps, Fig. 6). Unfortu-
nately, the model was not able to explain the low activity
for compound 21.
Compd. no.
Fit value
Activity MIC (lg/mL)
Predicted
Experimental
3
6.4
8.4
8.2
7.2
7.8
7.6
7.4
7.7
7.7
8.1
7.9
6.5
7.8
6.9
5.3
5.1
7.5
8.67
6.8
18.586
12.5
1.56
1.56
12.5
3.125
6.25
12.5
3.125
12.5
1.56
3.125
25
4
1.74183
2.96206
7.38495
4.4691
5
6
7
8
4.93569
10.2701
2.10814
6.36325
2.74376
3.648
9
10
11
12
13
14
15
16
17
18
19
20
21
17.8806
3.88851
13.1027
43.7326
31.8313
4.63261
0.931036
16.76334
6.25
12.5
[25
[25
3.125
0.39
[25
Fig. 5 Mapping of the most
active compound (20) into
Hypo1
123

Synthesis and anti-mycobacterial activity…
Fig. 6 Mapping of the inactive
compounds (17 and 18) into
Hypo1
ASA)-4-thiazolinone hybrid derivatives with promising antipro-
liferative activity. Bioorg Med Chem Lett 26:1647–1650
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indoline-2,3-dione (isatin) derivatives and nalidixic acid carbo-
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Conclusion
Reported here is the synthesis and in vitro antituberculous
activities of new 4-phenyl-1H-1,2,3-triazol-1-ylsalicylhy-
drazones. The synthesis was achieved using the inexpensive,
commercially available and clinically used PAS. Among the
derivatives studied, compound 20 showed remarkably high
potency and selectivity index. Moreover, 20 showed 2.4 log
reduction in bacterial count of dormant forms of mycobac-
terium which is comparable with the standard drugs moxi-
floxacin, isoniazid, rifampicin. 3D-QSAR pharmacophore
model was generated and used to evaluate the observed
activity. The results demonstrate the potential utility of
compound 20 as antitubercular lead compound for further
development and mechanistic studies.
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Evaluation of a nutrient starvation model of Mycobacterium
tuberculosis persistence by gene and protein expression profiling.
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Acknowledgement This work was supported by Faculty of Phar-
macy, Assiut University.
Compliance with ethical standards
Conflicts of interest The authors declare no conflict of interest.
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