Facile synthesis of novel bis-derivatives of 2,5-diamino-thiadiazole/N,N′-thiocarbohydrazide and their biological perspective

Article abstract of DOI:10.1007/s11164-018-3554-2

A series of new bis derivatives of 2,5-thiadiazole and N,N′-thiocarbohydrazide along with their transition metal complexes were synthesized by green methods (sonochemical and microwave), i.e., energy and time efficient and operationally more simple than existing conventional methods. All the synthesized compounds were fully characterized by routine analytical techniques and subjected to a preliminary biological screening. These novel characterized compounds were screened for antimicrobial activities and showed promising results. A few potent compounds were also tested for antitumor activities against MDA-MB231 (human breast carcinoma cell line) and compound 5 has emerged as a potential antitumor compound followed by compound 2, 10 and 9, with IC₅₀ values of 29.48, 32.25, 32.34 and 34.34?μg/ml, respectively. To understand the binding interactions of these compounds, in silico studies were performed using the AUTODOCK 4.2 suite and found to be supportive of the experimental results.

Full text of DOI:10.1007/s11164-018-3554-2

Research on Chemical Intermediates
https://doi.org/10.1007/s11164-018-3554-2(0123456789(). -, vo Vl
)
(
0
1
2
3
4
5
6
7
8
9
(
)
.
-
,
v
o
l
V
)
Facile synthesis of novel bis-derivatives
0
of 2,5-diamino-thiadiazole/N,N -thiocarbohydrazide
and their biological perspective
Priyanka Bhatt1 • K. Sreekanth1 • Anjali Jha1
Received: 14 May 2018 / Accepted: 9 August 2018
Ó Springer Nature B.V. 2018
Abstract
A series of new bis derivatives of 2,5-thiadiazole and N,N -thiocarbohydrazide
0
along with their transition metal complexes were synthesized by green methods
(
sonochemical and microwave), i.e., energy and time efficient and operationally
more simple than existing conventional methods. All the synthesized compounds
were fully characterized by routine analytical techniques and subjected to a pre-
liminary biological screening. These novel characterized compounds were screened
for antimicrobial activities and showed promising results. A few potent compounds
were also tested for antitumor activities against MDA-MB231 (human breast car-
cinoma cell line) and compound 5 has emerged as a potential antitumor compound
followed by compound 2, 10 and 9, with IC50 values of 29.48, 32.25, 32.34 and
34.34 lg/ml, respectively. To understand the binding interactions of these com-
pounds, in silico studies were performed using the AUTODOCK 4.2 suite and found
to be supportive of the experimental results.
Keywords Ultrasonication/microwave assisted synthesis Á 2,5-diamino-thiadiazole Á
0
N,N -thiocarbohydrazide Á Transition metal complexes Á Molecular
docking Á Antimicrobial/anticancer studies
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11164-
018-3554-2) contains supplementary material, which is available to authorized users.
&
Anjali Jha
anjali.jhamanish@gitam.edu
1
Department of Chemistry, GITAM Institute of Science (GIS), GITAM (Deemed to be
University), Rushikonda, Visakhapatnam 530045, India
123

P. Bhatt et al.
Introduction
In spite of much research and intensive efforts, the challenges raised by the emergence
of new, mutant and resistant forms of pathogenic microorganisms are still at an
alarming state in the world. Considering the devastating nature of many diseases, an
effective and immediate solution seems to be the supply and diversification of the
current small molecular [1] arsenal. Most of these small molecules are easily
accessible through ordinary laboratory procedures, in contrast to their natural
counterparts with complex structures. Unfortunately, most of the conventional
synthetic methods rely on less efficient energysources, which result in excessive use of
solvents/reagents, low yield, longer reaction times and higher costs of the process.
Several modifications have been reported in the literature to tackle these problems,
such as solvent-free reactions, microwave irradiation [2], ultrasound-assisted
procedures [3], reactions in ionic liquid [4], multicomponent reactions [5], etc. Some
practical advantages of these green methodologies involve large reductions in reaction
time and enhancements in the conversion, selectivity and yield of a reaction. Use of
ultrasonic (US) radiation to accelerate chemical reactions, or sonochemistry, is one
such outstanding and greener technique [6]. In this method, reactions proceed through
the formation and then subsequent devastation of cavitation bubbles which act as
micro-reactors, creating a localized hot spot which leads to chemical reactions
occurring in a liquid medium, while in microwave-assisted organic synthesis, the
dipole moments of molecules are important in order to proceed with the chemical
reactions [7]. An instantaneous localized superheating of a substance takes place in
this process, leading to the formation of products without any heating of vessels.
The thiadiazole analogues have been reported as an integral part of several
pharmacologically essential scaffolds [8], together with good fluorescent properties
[9, 10] responsible for innumerable purposes from cell imaging to automated DNA
sequencing. Isatin and its derivatives exhibit diverse biological activities, i.e.,
anticonvulsant, antineoplastic, antimicrobial, antiviral and anticancer properties
[11, 12]. Chalcones, having two benzene rings joined by a three-carbon a, b-
unsaturated carbonyl system, are the key precursors of many biologically important
compounds and show an array of pharmacological activities [13, 14]. Similarly,
acridones, a rigid hetero tricyclic compound with nitrogen at the 10th position and a
keto group at the 9th position, forms the structure motif of a myriad of natural products
and displays a wide range of biological effects [15]. In the past few decades, many
macro-hetero-multi-donor ligands have been explored for their medicinal and
biological activities [16, 17]. The macrocycle ligands with various donor atoms/centers
have exciting possibilities toward the construction of novel supramolecular architec-
tures. These large-sized structures may be important for our understanding of highly
specific and important functions of a cell, so these molecular systems are now a focal
theme of many of research endeavors covering the area of organic and coordination
chemistry [18]. Additionally, the emergence of Cisplatin as a promising anticancer drug
(
first metal-based drug) in the twentieth century has inspired several researchers to
explore the presence of other metal ions in various drugs. The photochemical and
various oxidation states of metal ions make them active in treating diverse diseases [19].
1
23

Facile synthesis of novel bis-derivatives…
In light of the above observations and in continuation with our research interest
in the development of green chemical techniques for biologically active new,
potent, and less toxic compounds, we here report an efficient synthesis of 2,5-
0
diamino-thiadiazole and N,N -thiocarbohydrazide derivatives employing ultrasonic,
microwave irradiation and also conventional methods. We have also studied in vitro
and in silico antimicrobial activities against Gram-positive and Gram-negative
bacteria, together with a fungus, along with the anticancer activities of the
characterized compounds in order to understand the synergistic effects of various
pharmacophores present in these novel molecules.
Experimental
Materials and methods
Reagents such as semicarbazide hydrochloride, potassium thiocynate, thiocarbohy-
Ò
drazide and isatin were purchased from Across and used as received. Bithiourea, 2,
5
-diamino-1,3,4-thiadiazole [20], chalcone [29] and acridone [30] were synthesized
by the following reported methods. The solid IR spectra and electronic spectra of
the ligands and the complexes were recorded in KBr and DMSO on Perkin Elmer
FTIR and Perkin Elmer-Spectrum-Lambda-25 spectrophotometers, respectively, at
GITAM, Visakhapatnam, India. ESR spectra were acquired at room temperature
(
RT) and liquid nitrogen temperature (LNT) on a Varian E 112 instrument using
1
13
DPPH as standard at SAIF, IIT Chennai, India. H and C NMR spectra were
obtained using the Bruker 400 MHz NMR facility at IISc, Bangalore, India.
Chemical shift values are reported in parts per million (ppm) using TMS as an
internal standard and DMSO-d /CDCl as NMR solvents. Ultrasonication (US)
6
3
experiments were carried out using a probe sonicator (12/20-mm probe) with an
ultrasonic processor (250 W) operating at a fixed frequency of 50 Hz with a speed
of 238 rpm. Microwave reactions were performed using a Flexiwave-Milestone
Microwave Synthesis Platform instrument (model No. VA1601178FLEX49050).
Synthesis
Bithiourea was synthesized according to the reported literature procedure [31].
Semicarbazide hydrochloride and potassium thiocyanate were mixed thoroughly
and dissolved in water while refluxing. In an alternative method, ultrasonication for
about 30 min and microwave irradiation for 20 min were found to be adequate to
obtain similar outcomes. The final product was recrystallized from boiling water
(
Scheme 1).
Scheme 1 Synthetic procedure of 1,3,4-thiadiazole-2,5-diamine
123

P. Bhatt et al.
Preparation of 1,3,4-thiadiazole-2,5-diamine
,5-diamino-1,3,4-thiadiazole was prepared according to previously reported
2
methods. The use of ultrasonic/microwave radiation was also found to be effective
for this step. The isolated thiadiazole product was characterized by spectral means
and found to be structurally consistence with the expected structure.
0
Preparation of 2,5-thiadiazolo and N,N -thiocarbohydrazide bis-Isatin (L , L )/
1
4
chalcone (L , L )/acridone(L , L )
2
5
3
6
The synthetic approaches implemented to acquire the target compounds are outlined
in Schemes 2 and 3. The bis isatin/chalcone/acridone derivatives of 2,5-thiadiazolo
0
and N,N -thiocarbohydrazide were prepared by conventional methods as well as by
ultrasonication/microwave radiation. An ethanolic solution (10 ml) of isatin/
chalcone/acridone (0.02 mol) was added dropwise to a solution of 1,3,4-thiadia-
0
zole-2,5-diamine/N,N -thiocarbohydrazide, (0.01 mol, 10 mL ethanol) in separate
reactions. The reaction mixture was refluxed/ultrasonicated/microwaved with a few
drops of glacial acetic acid. The progress of the reaction was monitored by TLC.
The obtained reaction mixture from all the methods was allowed to cool to room
temperature and kept in a refrigerator overnight. The separated solids were
recrystallized and characterized.
1 3
Scheme 2 Synthetic pathway for compound L –L ; a conventional, b ultrasonication, c microwave
irradiation
1
23

Facile synthesis of novel bis-derivatives…
S
S
L6
L5
HN
N
NH
N
HN
N
NH
N
NH
HN
S
a/b/c
a/b/c
O
O
HN
NH2
NH
NH2
N
H
O
O
a/b/c
N
H
S
HN
N
NH
N
N
H
O
O
N
H
L4
4 6
Scheme 3 Synthetic pathway for compound L –L ; a conventional, b ultrasonication, c microwave
irradiation
0 0
3Z,3 Z)-3,3 -((1,3,4-thiadiazole-2,5-diyl)bis(azanylylidene))bis(indolin-2-one)(L-1) (1)
(
-
1
IR (KBr, cm ): 3250 (NH), 3140 (Ar–H), 1731 (C=O), 1620 (C=N); 1514 (C=C);
1
13
HNMR (400 MHz DMSO-d ): d7.9 (s, 2H, NH), 7.5–6.8 (m, 8H, Ar–H); CNMR
6
(
100 MHz, DMSO-d ): d184.35 (C=O), 166.43 (C=N), 159.32, 150.69 (thiazole-C),
6
1
38.34, 137.7, 131.09, 128.67, 122.7, 117.7, 112.1 (Ar–C); MS (m/z, %): 374.38
?
(
M) . Anal. Calcd. for C H N O S (%); C, 57.75; H, 2.69; N, 22.45; Found: C,
18 10 6 2
5
7.82; H, 2.73; N, 22.51.
2
5
(
N Z,N Z)-N2,N5-bis((E)-1,3-diphenylallylidene)-1,3,4-thiadiazole-2,5-diamine(L-2) (2)
-
IR (KBr, cm ): 3062 (Ar–H), 1661, 1616 (C=N); 1575, 1495, 1216; HNMR
1
1
(
400 MHz, CDCl ): d8.03–8.09 (d, 4H, CH=CH); 7.83–7.41 (m, 20H, Ar–H);
3
1
3
CNMR (100 MHz, CDCl ): d144.83, 138.23 (thiazole-C); 132.75, 130.52, 128.95,
3
?
28.49, 122.14 (Ar–C); MS (m/z, %): 496 (M) ; Anal. Calcd. C H N S (%); C,
1
3
2 24 4
7
7.41; H, 4.83; N, 11.29; Found: C, 77.49; H, 4.77; N, 11.35.
123

P. Bhatt et al.
2
5
(
N ,N )-di(acridin-9(10H)-ylidene)-1,3,4-thiadiazole-2,5-diamine(L-3) (3)
-
IR (KBr, cm ): 3260 (NH), 3120 (Ar–H), 1614 (C=N), 1550, 1512 (C=C); HNMR
1
1
1
3
(
400 MHz, DMSO-d ): d8.16 (s, 2H, NH),7.9–7.4 (m, 16H, Ar–H); CNMR
6
(
100 MHz, DMSO-d ):d 144.0, 137.53 (thiazole-C), 133.17, 130.65, 128.4, 122.03,
6
?
Ar–C); MS (m/z, %): 470 (M) ; Anal. Calcd. for C H N S (%); C, 71.48; H,
(
2
8 18 6
3
.82; N, 17.87; Found: C, 71.52; H, 3.89; N, 17.77.
0
N,N -Thiocarbohydrazone bis (isatin) (L-4) (4) (Conventional synthesis was reported
for L-4 = Ref 18)
-
IR (KBr, cm ): 3345 (NH), 1690 (C=O), 1619 (C=N), 1555, 1512 (C=C); HNMR
1
1
(
400 MHz, DMSO-d ): d11.0 (s, 2H, NH), 7.8(s, 2H, NH), 7.7–6.9 (m, 8H, Ar–H);
6
1
3
CNMR (100 MHz, DMSO-d ): d186.35 (C=O), 136.7, 132.09, 127.67, 120.7,
6
?
18.7, 112.1 (Ar–C); MS (m/z, %): 368 (M) ; Anal. Calcd. C H N O S (%) C,
5.73; H, 4.37; N, 22.95, Found: C, 55.69; H, 4.40; N, 22.87.
1
5
1
7 16 6 2
0
N,N -Thiocarbohydrazone bis (chalcone (L-5) (5)
-
IR (KBr, cm ): 3350 (NH), 3015 (Ar–H), 1612 (C=N), 1517 (C=C); HNMR
1
1
(
400 MHz, CDCl ): d10.2 (s, 2H, NH), 8.3–8.1 (s, 4H, CH=CH); 7.8–7.31 (m, 20H,
3
1
3
Ar–H); CNMR (100 MHz, CDCl ): d 134.75, 130.52, 127.95, 125.49, 122.14
(Ar–C), 110–115 (CH=CH); MS (m/z, %): 486 (M) ; Anal. Calcd. C H N S (%)
31 26 4
C, 74.17; H, 5.39; N, 11.61; Found: C, 74.22; H, 5.44; N, 11.72.
3
?
0
N,N -Thiocarbohydrazone bis (acridone) (L-6) (6)
-
1
1
IR (KBr, cm ): 3316 (NH), 3108 (Ar–H), 1615, 1514 (C=N); HNMR (400 MHz,
1
3
DMSO-d ) d 10.6 (s, 2H, NH),8.2 (s, 2H, NH), 7.9–7.4 (m, 16H, Ar–H); CNMR
6
(
100 MHz, DMSO-d ): d134.7, 131.65, 128.4, 125.03, (Ar–C); MS (m/z, %):
60.55 (M) ; Anal. Calcd. C H N S (%) C, 70.43; H, 4.14; N, 17.42; Found: C,
27 20 6
6
?
4
7
0.51; H, 4.22; N, 17.49.
General procedure for the preparation of metal complexes
The Ni and Cu complexes of the above novel ligands (L –L ) were prepared
6
1
following the reported literature method [32]. Alternatively, the possible utility of
ultrasonic/microwave radiation was also explored, and it was found that only a few
minutes of ultrasonication/microwave were enough for similar results. The
complexes were characterized by standard analytical methods, i.e., IR, UV–visible,
and ESR (for Cu complexes only).
1
23

Facile synthesis of novel bis-derivatives…
L –Ni/[NiL Cl Á(H O) ](7)
1
1
2
2
2
-
1
IR (KBr, cm ): 3244 (NH), 3108(Ar–H), 1710 (C=O), 1608 (C=N), 1514 (C=C);
Anal. Calcd. for NiC H O N SCl :C, 55.75; H, 2.77; N, 22.45; Ni, 11.71; Cl,
1
8
10
2
6
2
1
4.09; Found: C, 55.70; H, 2.82; N, 22.41; Ni, 11.85; Cl, 14.25.
L –Cu/[Cu L Cl Á(H O) ] (8)
1
1
2
2
2
-
1
IR (KBr, cm ): 3240 (NH), 3100 (Ar–H), 1718 (C=O), 1610 (C=N), 1514 (C=C);
Anal. Calcd. for CuC H O N SCl : C, 55.75; H, 2.75; N, 22.45; Cu, 12.58; Cl,
1
8
10
2
6
2
1
3.90; Found: C, 55.70; H, 2.79; N, 22.42; Cu, 12.45; Cl, 13.85.
L –Ni/[NiL Cl Á(H O) ](9)
2
2
2
2
2
-
1
IR (KBr, cm ): 3055 (Ar–H), 1661, 1610 (C=N); 1575, 1495, 1216, Anal. Calcd.
for NiC H O N SCl : C, 77.39; H, 4.23; N, 11.28; Ni, 8.91; Cl, 10.72; Found: C,
3
2
28
2
4
2
7
7.43; H, 4.28; N, 11.22; Ni, 8.82; Cl, 10.79.
L –Cu/[CuL Cl Á(H O) ] (10)
2
2
2
2
2
-
1
IR (KBr, cm ): 3050 (Ar–H), 1661, 1616 (C=N); 1575, 1495, 1216; Anal. Calcd.
for CuC H O N SCl : C, 77.39; H, 4.20; N, 11.28; Cu, 9.60; Cl, 10.65; Found: C,
3
2
28
2
4
2
7
7.44; H, 4.26; N, 11.32; Cu, 9.55; Cl, 10.75.
L –Ni/[NiL Cl Á(H O) ] (11)
3
3
2
2
2
-
1
IR (KBr, cm ): 3245 (NH), 3102 (Ar–H), 1610 (C=N), 1550, 1512 (C=C); Anal.
Calcd. for NiC H O N SCl : C, 71.47; H, 3.40; N, 17.86; Ni, 9.28; Cl, 11.16;
Found: C, 71.43; H, 3.45; N, 17.92; Ni, 9.38; Cl, 11.22.
2
8
22
2
6
2
L –Cu/[CuL Cl Á(H O) ] (12)
3
3
2
2
2
-
1
IR (KBr. cm ): 3242 (NH), 3110 (Ar–H), 1604 (C=N), 1550, 1512 (C=C); Anal.
Calcd. for CuC H O N SCl : C, 71.47; H, 3.43; N, 17.86; Cu, 9.99; Cl, 11.08;
Found: C, 71.52; H, 3.38; N, 17.81; Cu, 9.85; Cl, 11.14.
2
8
22
2
6
2
L –Ni/[NiL Cl Á(H O) ] (13)
4
4
2
2
2
-
1
IR (KBr, cm ): 3345 (NH), 1675 (C=O), 1609 (C=N), 1555, 1512 (C=C); Anal.
Calcd. for NiC H O N SCl : C, 55.42; H, 4.01; N, 22.81; Ni, 11.84; Cl, 14.25;
Found: C, 55.47; H, 4.08; N, 22.85; Ni, 11.75; Cl, 14.35.
1
7
20
4
6
2
123

P. Bhatt et al.
L –Cu/[CuL Cl Á(H O) ] (14)
4
4
2
2
2
-
1
IR (KBr, cm ): 3345 (NH), 1672 (C=O), 1611 (C=N), 1555, 1510 (C=C); Anal.
Calcd. for CuC H O N SCl :C, 55.42; H, 3.30; N, 22.81; Cu, 10.58; Cl, 11.74;
Found: C, 55.45; H, 3.35; N, 22.85; Cu, 10.62; Cl, 11.79.
1
7
20
4
6
2
L –Ni/[NiL Cl Á(H O) ] (15)
5
5
2
2
2
-
1
IR (KBr, cm ): 3320 (NH), 3015 (Ar–H), 1610 (C=N), 1517 (C=C); Anal. Calcd.
for NiC H O N SCl : C, 76.51; H, 4.60; N, 11.51; Ni, 9.05; Cl, 10.89; Found: C,
3
1
30
2
4
2
7
6.55; H, 4.65; N, 11.57; Ni, 9.15; Cl, 10.95.
L –Cu/[CuL Cl Á(H O) ](16)
5
5
2
2
2
-
1
IR (KBr, cm ): 3335 (NH), 3015 (Ar–H), 1608 (C=N), 1515 (C=C); Anal. Calcd.
for CuC H O N SCl : C, 76.51; H, 4.54; N, 11.51; Cu, 9.74; Cl, 10.81; Found: C,
3
1
30
2
4
2
7
6.56; H, 4.59; N, 11.48; Cu, 9.65; Cl, 10.87.
L –Ni/[NiL Cl Á(H O) ] (17)
6
6
2
2
2
-
1
IR (KBr, cm ): 3306 (NH), 3078 (Ar–H), 1605, 1514 (C=N); Anal. Calcd. for
NiC H O N SCl : C, 70.41; H, 3.82; N, 18.25; Ni, 9.41; Cl, 11.32; Found: C,
2
7
24
2
6
2
7
0.46; H, 3.85; N, 18.29; Ni, 9.47; Cl, 11.38.
L –Cu/[CuL Cl Á(H O) ] (18)
6
6
2
2
2
-
1
IR (KBr, cm ): 3302 (NH), 3088 (Ar–H), 1602, 1514 (C=N); Anal. Calcd. for
CuC H O N SCl : C, 70.41; H, 3.80; N, 18.25; Cu, 10.15; Cl, 11.26; Found: C,
2
7
24
2
6
2
7
0.47; H, 3.77; N, 18.29; Cu, 10.25; Cl, 11.31.
Antimicrobial assay
The synthesized compounds (1–18) were evaluated for their antimicrobial activity
against Gram-negative bacteria EC—Escherichia coli (MTCC 1687), PA—Pseu-
domonas aeruginosa (MTCC 1688) and Gram-positive bacteria BS—Bacillus
subtilis (MTCC 441), SA—Staphylococcus aureus (MTCC 96) and a fungus CA—
Candida albicans (MTCC 227) using a well diffusion method. The antimicrobial
activities of DMSO solution of compounds were determined by the well diffusion
method. The whole procedure was adopted from our previous work [33].
Ciprofloxacin and ketaconazole were used as antibacterial and antifungal reference
drugs, respectively. The zone of inhibition (mm) was determined at 10 lg/ml for
each compound in triplicate experiments; the mean values were calculated and are
listed in Table 3.
1
23

Facile synthesis of novel bis-derivatives…
Antitumor assay
The breast adenocarcinoma cells (MDA-MB231) were placed in a 96-well plates
(
4
1 9 10 cells/well) and incubated with L-15 medium containing different
concentrations of the compounds for 24 and 48 h at 37 °C. After that, 20 ll of
MTT [34] reagent was added (0.5 mg/ml) to the medium and incubated for the next
3
h. Formazen crystals were dissolved in DMSO and their absorbance was
measured using an ELISA microplate reader at 570 nm. Finally, IC50 (concentration
required to reduce 50% viability) values were calculated by the method of
regression analysis.
Docking methodology
Molecular docking is a computerized technique which serves to verify the binding
affinity and interaction sites of ligands within the binding pocket of target proteins.
For our docking study, the crystal structure of a 24-kDa fragment of Escherichia
coli topoisomerase II DNA gyrase (PDB ID: 1KZN) in complex with antimicrobial
agent clorobiocin (a natural coumarin) and BRCT domain of BRCA1 (a protein
kinas of Homo sapiens) in complex with a minimal recognition tetrapeptide with a
free carboxy C-terminus (PDB ID: 3K0K) were obtained from the Protein Data
Bank. Both the proteins play a crucial role in the maintenance of genomic integrity
and cellular proliferation. The molecular docking study was performed using the
AUTODOCK 4.2 suite, automated docking tools that use a Lamarckian genetic
algorithm. Protein structures were cleaned by removing the crystallographic water,
bound substrates and co-factors. Polar hydrogen atoms were added and Gasteiger
charges were computed in ADT. 2D structures of all the ligands were drawn using
Chem Draw ultra 12 and converted to 3D. Finally, all the structures were energy
minimized and geometrically optimized using an AM1 force field.
˚
In the docking process, the grid points were fixed at a distance of 0.375 A with a
˚
grid box size of 40 9 40 9 40 A (number of points in x, y and z axes for both
proteins). For the final docking run, default parameters were used (numbers of final
conformations: 10, population size: 150, maximum number of energy evaluations:
6
.5 9 10 , and maximum number of generations: 27,000). Results were evaluated
2
according to the lowest binding energy criteria. All docked poses were inspected in
detail for their binding interactions with protein using UCSF Chimera.
In silico study of molecular properties and drug-likeness
For a compound to be labeled as ‘‘lead/drug-like’’, its molecular properties such as
lipophilicity, molecular weight (MW), number of rotatable bonds, number of
hydrogen bond donors and number of hydrogen bond acceptors should be within an
acceptable range. To determine this range, Lipinski’s rule of five [35] is an
important guideline. All the above-mentioned properties for synthesized compounds
were computationally estimated using the Molinspiration Cheminformatics tool and
are listed in Tables S1 and S2 (supporting information). The degree of absorption is
123

P. Bhatt et al.
expressed by the percentage of absorption (% ABS) and calculated by the formula
[
36]:
%
ABS ¼ 109 À ð0:345 Â TPSAÞ
Eventually, a drug-likeness score was calculated (using MolSoft software), which
provides a combined picture of all the physicochemical, pharmacokinetics and
pharmacodynamics aspects of a compound.
Results and discussion
Chemistry
The synthesis of intermediates and target compounds (L –L ) was achieved following
1
6
the steps, as shown in Schemes 1, 2 and 3. Forthe synthesis ofcompounds L , L and L ,
1
2
3
precursor bithiourea and its subsequent cyclized product, 2,5-diamino-1,3,4-thiadia-
zole, were prepared through a reported procedure [20]. Mechanistically, synthesis of
cyclic thiadiazole involved protonation of the thiocarbonyl group of bithiourea, thio-
mercapto tautomerism and final ring closure with the removal of H S. For the synthesis
2
of compounds L , L and L , commercial thiocarbohydrazide was directly condensed
4
5
6
with isatin/chalcone/acridone. Despite moderate to good yields, we also surveyed the
possibility of ultrasonic/microwave radiation for all these reactions. Gratifyingly, we
observed a noticeable improvement in yields with shorter reaction times, than with the
conventional methods (Table 1). Other benefits of these green methods include energy
efficiency, operational and preparative simplicity, better reaction profiles, etc.
Spectral analysis
The IR spectra of the 2,5-diaminothiadiazole exhibited peaks at 3368 and
1
-
3
the ligands (L –L ) upon condensation with isatin/chalcone/acridone, but a single
286 cm which were assigned to NH vibrations. Both peaks disappeared in
2
1
3
-
1
peak for the NH group was noticed over a range of 3265–3200 cm for the ligands
L , L –L . The disappearance of the C=O peak in all the ligands except L and L
4
1
3
6
1
0
was also indicative of the reaction between 2,5-diaminothiadiazole/N,N -thiocarbo-
hydrazide with isatin/chalcone/acridone, which was further confirmed by their
respective NMR spectra and by the presence of a characteristic absorption band at
-
1
1
615–1602 cm in the IR of compounds 1–18 for the azomethine linkage [21, 22].
The complexes were prepared by reacting metal (II) chlorides with the ligands
(
L –L , 1:1). The amorphous nature and poor solubility (except in DMF and
1 6
DMSO) of these complexes restricted us to growing a single crystal for single-
crystal X-ray analysis. The 1:1 ligands–metal stoichiometric ratio was confirmed by
elemental analyses. The standard procedure was followed for metal, nitrogen,
carbon, hydrogen and chloride analyses and found to be consistent with the
expected structure. The solid state IR spectra of all the complexes showed a 15- to
-
1
20-cm lowering in the values of C=O and C=N, indicating coordination through
the O and azomethine N atoms [18]. This coordination with metal was also
1
23

Facile synthesis of novel bis-derivatives…
Table 1 Analytical and physicochemical data of the synthesized compounds
Compd. Compound/molecular
no.
Color/M.P. (°C)
US (%
yield)
MW (%
yield)
Conv. (%
yield)
formula
1
2
3
4
5
6
7
8
9
L
1
L
2
L
3
L
4
L
5
L
6
L
1
L
1
L
2
L
2
L
3
L
3
L
4
L
4
L
5
L
5
L
6
L
6
/C18
/C32
/C28
/C17
/C31
/C27
H
H
H
H
H
H
10
24
18
16
26
20
N
N
N
N
N
N
6
4
6
6
4
6
O
S
2
2
S
Brick red/194–195
Pale yellow/182–184
Green/295–296
25 (82)
35 (85)
35 (80)
20 (86)
20 (80)
25 (82)
25 (78)
25 (80)
30 (89)
35 (80)
25 (78)
20 (70)
22 (70)
18 (76)
25 (72)
28 (70)
20 (72)
25 (80)
25 (74)
32 (24)
30 (70)
32 (70)
8 (76)
7 (72)
S
10 (66)
3 (70)
O
S
S
Orange/285–288
Reddish brown/197–198 35 (89)
12 (72)
15 (69)
10 (75)
12 (60)
12 (62)
10 (68)
12 (63)
10 (60)
11 (64)
10 (69)
12 (67)
10 (52)
12 (63)
10 (60)
S
Light Green/255–257
Brown/185–187
Bluish green/198–200
Dark green/\ 300
Green/289–290
40 (83)
35 (82)
20 (76)
25 (70)
20 (79)
30 (75)
30 (72)
25 (78)
30 (81)
30 (76)
35 (77)
35 (72)
35 (70)
–Ni/[Ni L
–Cu/[Cu L
–Ni/[NiL Cl
–Cu/[CuL Cl
–Ni/[NiL Cl
–Cu/[CuL Cl
–Ni/[NiL Cl
–Cu/[CuL Cl
–Ni/[NiL Cl
–Cu/[CuL Cl
–Ni/[NiL Cl
–Cu/[CuL Cl
1
Cl
Cl
Á(H
Á(H
Á(H
.(H
Á(H
Á(H
Á(H
Á(H
Á(H
Á(H
2
Á(H
.(H
O)
O)
O)
O)
O)
O)
O)
O)
O)
O)
2
O)
2
]
1
2
2
O)
2
]
2
2
2
2
]
10
11
12
13
14
15
16
17
18
2
2
2
2
]
3
2
2
2
]
Green/182–183
3
2
2
2
]
Light green/\ 300
Red/200–202
4
2
2
2
]
4
2
2
2
]
]
]
Brown/259–260
Light green/\ 300
Green/275–276
5
2
2
2
]
5
2
2
2
6
2
2
2
]
Green/172–173
6
2
2
2
Dark green/\ 300
US ultrasonication in minutes; MW microwave irradiation in minutes; Conv. conventional in hours
-
1
supported by the presence of new peaks in the regions 550–595 and 470–485 cm
due to the (M–O) and (M–N) vibrations in 7–18 [23].
Electronic spectra of the ligands and Ni(II) and Cu(II) complexes (Table 2, in
DMSO) displayed two bands at * 285 and * 305 nm which were attributable to
Table 2 Electronic spectral (in DMSO solution) and ESR (solid-state) data of the complexes
a
Complex
k max (mm)
g
II
g\
A
II RT(LNT)
7
8
9
352
325
345
320
355
335
362
328
352
340
355
335
426
410
440
420
434
424
436
434
430
418
435
425
670
607
680
607
665
607
677
607
690
607
685
607
960
789
989
778
980
765
982
770
955
785
985
775
–
–
–
2.241 (2.223)
2.123 (2.110)
165 (159)
–
–
–
1
1
1
1
1
1
1
1
1
a
0
1
2
3
4
5
6
7
8
2.221 (2.103)
2.024 (2.003)
160 (152)
–
–
–
2.230 (2.111)
2.112 (2.002)
162 (158)
–
–
–
2.242 (2.223)
2.122 (2.020)
165 (156)
–
–
–
2.245 (2.220)
–
2.141 (2.103)
–
162 (156)
–
2.232 (2.100)
2.241 (2.002)
160 (155)
Compounds are the same as in Table 1
123

P. Bhatt et al.
the p ? p* and n ? p* transitions of the (–C=N–NH–CO) group in free ligands.
In the spectra of the complexes, these bands were shifted to the lower frequencies,
which further supported the involvement of the imine nitrogen atom and the oxygen
atom in coordination with the metal ions [24]. Interestingly, the Ni(II) complexes
showed three absorption bands in the regions 426–440, 665–690 and 955–990 nm,
possibly due to 3A (F) ? 3T (P), 3A (F) ? 3T (F) and 3A (F) ? 3T
2
g
1g
2g
1g
2g
2g
(
F) types of transitions, respectively, which were in full agreement with the
octahedral geometry for the Ni(II) ion. Similarly, the electronic spectra of the Cu(II)
complexes displayed strong bands in the range of 324–340 nm, which probably
resultant from an n ? p* transition. A charge transfer LMCT band was also noticed
in the range of 415–440 nm [25]. The spectrum also showed a d–d electronic
transition in the 607–620 and 765–790 nm regions of the spectrum, which
originated from the 2Eg (D) ? 2T2g (D) type of electronic promotion. The distorted
octahedral geometry [26] for the Cu(II) is responsible for the broadness of this band.
The analysis of the X-band powder-state ESR spectral data of the Cu(II)
complexes at RT and LNT (Table 2) showed significant covalent characters in the
metal–ligand bonds. The spin Hamiltonian parameters for the copper complexes
were calculated from the spectra at LNT (Fig. S1 of supporting information). The
gII [ g\ values were found to be supportive of the axially elongated octahedral
geometry of the metal complexes [27]. The unpaired electron lies predominantly in
the d
2
2
orbital as indicated from the analysis of the ESR spectra.
x Ày
All the ligands were also characterized by mass spectral studies. The molecular
ion peaks of all the ligands were found to be consistent with the molecular weights
?
of the expected structures with the (M) ion. On the basis of the analytical and
spectral data, the possible structures of all the metal complexes are depicted in
Fig. 1.
Antimicrobial activities
All the synthesized compounds (1–18) were examined for their antibacterial/
antifungal activity using the well diffusion method [28] and compared with the
standard drugs (Table 3). The important observed trends were chal-
cone [ acridone [ isatin in the case of the Gram–positive bacteria, whereas the
chalcone and isatin ligands displayed higher activity than acridone in the Gram-
negative bacterial strains; however, chalcones containing ligands L2 and L5
displayed higher activities in general. The complexes 9 (L –Ni) and 10 (L –Cu)
2
2
showed promising activities with both the positive and negative bacterial strains as
well as against the fungus C. albincus. All the other metal complexes also exhibited
more activities than their respective ligands. Moreover, the thiadiazole containing
ligands L –L showed slightly higher zones of inhibition over the thiocarbohy-
1
3
drazide ligands, L –L . Additionally, for C. albincus, the observed pattern of
4
6
activities was chalcone [ acridone [ Isatin. All these trends were also supported by
docking studies in the case of all the ligands L –L , and found to be consistent with
1
6
the in vitro values. Since metal–ligand structures were not recognizable in the
AUTODOCK software, in silico studies could not be performed for the metal–
1
23

Facile synthesis of novel bis-derivatives…
Fig. 1 Proposed structures of the synthesized complexes
ligand complexes, although in in vitro antimicrobial studies, all the metal complexes
showed relatively better activities than their parental ligands, suggesting that metal–
ligand complexes can play a significant role in developing new lead drug molecules.
Molecular docking studies
Binding energy scores of ligands 1–6 against Escherichia coli gyrase (1KZN) and
Homo sapiens BRCA1 kinase (3K0 K) are listed in Tables 4 and 5, respectively. In
1
KZN docking, four compounds (1, 3–5) displayed binding energies [ 9.0
kcal/mol, which were quite acceptable scores. Docked pose and hydrogen bonding
interaction between compounds (5 and 6) and neighboring residues are shown in
Figs. 2 and 3. Similarly, in BRCA1 protein kinase docking experiments, two
123

P. Bhatt et al.
Table 3 Antimicrobial activity
of synthesized compounds (zone
Compounds
Bacteria
EC
Fungus
CA
a
of inhibition in mm at a
PA
BS
SA
concentration of 10 lg/ml)
1
2
3
4
5
6
7
8
9
22
29
25
20
28
24
17
15
30
32
18
13
17
22
19
14
12
10
35
N.A
19
22
19
17
25
21
20
12
25
27
16
14
15
20
17
12
9
15
18
11
15
23
20
22
15
24
19
16
17
15
17
18
20
10
14
28
N.A
20
24
19
23
27
14
19
17
31
29
18
25
21
25
21
24
N.A
12
35
N.A
N.A
19
23
21
15
17
12
10
24
1
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
8
26
24
22
20
21
N.A
14
18
N.A
30
N.A
16
Cephaloflaxin
Ketoconazole
N.A
28
EC Escherichia coli; PA Pseudomonas aeruginosa; BS Bacillus
subtilis; SA Staphylococcus aureus; CA Candida albicans
a
Mean of triplicate experiments in mm, N.A = Not active
compounds, 3 and 6, displayed a higher binding energy ([ 9.0 kcal/mol) than the
rest of the compounds.
In silico estimation on oral bioavailability and drug-likeness
Computationally estimated lead/drug-likeness is now a widely accepted and
frequently used criterion in the drug discovery process. Some important observa-
tions from Tables S1 and S2 (supporting information) were:
1.
2.
3.
While metal complexes in general displayed higher potency in actual assays,
they failed to meet the criteria set for drug-likeness. Compounds 15–18 even
displayed two violations from RO5.
Compounds 3 and 5 displayed the highest drug-likeness scores of 0.08 and 0.50,
respectively, and should be considered as starting points for further optimiza-
tion studies.
Bioactivity score of all the compounds were also computationally calculated.
Compounds 3 and 5 displayed satisfactory bioactivity.
1
23

Facile synthesis of novel bis-derivatives…
Table 4 Molecular docking results of the target molecule with the X-ray crystal structure of the E. coli
24-kDa domain in complex with clorobiocin (PDB-1KZN)
Molecule Docking score Estimated binding
energy (kcal/mol)
H- Amino acids interacted through hydrogen
˚
bond bonding (H-bond length A)
(
kcal/mol)
1
2
3
4
5
6
8.0
6.5
8.2
8.5
8.8
7.9
- 9.11
- 7.51
- 9.54
- 9.03
- 9.18
- 6.95
1
2
1
1
2
3
Gly77 (2.54)
Arg136 (2.85), Asp73 (3.19)
Gly77 (2.74)
Val120 (2.685)
Asn46 (3.23), Arg136 (2.08),
Asn46 (2.41), Val120 (3.09), Ile(2.874)
Table 5 Molecular docking results of the target molecule with the X-ray crystal structure of the BRCA1
BRCT in complex with a minimal recognition tetrapeptide with a free carboxy C-terminus (PDB 3K0K)
Molecule Docking score Estimated binding
energy (kcal/mol)
H- Amino acids interacted through hydrogen
˚
bond bonding (H-bond length A)
(
kcal/mol)
1
2
3
4
5
6.7
6.8
8.2
5.8
7.7
- 7.21
- 8.34
- 9.61
- 7.29
- 9.92
2
2
2
0
3
Leu1701 (3.149), Lys1702 (2.76)
Leu1701 (3.149), Lys1702 (2.84)
Arg1699 (3.01), Arg1699 (3.159)
–
Asn1774 (2.84), Lys1702 (3.06), Leu701
(
3.07)
6
6.7
- 8.71
1
Leu701 (2.70)
Fig. 2 a Docked ligand pose of compound 6 into the binding site pocket of the crystal structure of the
antimicrobial agent Clorobiocin bound to topoisomerase II DNA gyrase (PDB ID:1KZN) in a solid and
ribbon model. b The surface cavity with target compound 6 at the active pocket of the protein
123

P. Bhatt et al.
Fig. 3 a Docked ligand pose of compound 5 into the binding site pocket of the crystal structure of
BRCA1 BRCT in complex with a minimal recognition tetrapeptide with a free carboxy C-terminus (PDB
ID:3K0K) in a solid and ribbon model. b The surface cavity with target compound 5 at the active pocket
of the protein
Table 6 IC50 of tested
Compounds
IC50 (lg/ml)
compounds for antitumor
screening against MDA-MB231
cancer cells
1
2
3
4
5
6
9
37.60
32.25
39.21
63.24
29.48
52.06
34.34
32.34
28.00
1
0
Standard Paclitaxel
Antitumor studies
Antitumor activity data of synthesized compounds are compiled in Table 6, from
which it is obvious that ligand L5 showed higher activity, with an IC50 of
29.48 lg/ml, than the other screened compounds. Compounds L , L –Cu and L –
2 2 2
Ni also showed good to moderate activity with IC50 values of 32.25, 32.34 and
3
4.34 lg/ml, respectively. Similarly, compounds L1 and L6 showed slightly
higher inhibitory concentrations (IC50 values of 37.60 and 63.24 lg/ml r,espec-
tively) (Table 6). These activity trends were also supported by their molecular
docking studies. The acridone-containing ligands and complexes did not show any
satisfactory results, indicating that more structural modifications are required in
these cases.
1
23

Facile synthesis of novel bis-derivatives…
Conclusion
The present work reports a conventional (reflux), US/MW-based protocol for the
0
synthesis of bis-analogs of 2,5-diamino-thiadiazole/N,N -thiocarbohydrazide and
their metal complexes. It was found that the ultrasonication/microwave irradiation-
based method provides better yields in relatively less time. Moreover, these methods
(
US and MW) are more energy efficient, operationally simple and environmentally
benign, and hence are a worthwhile addition to the existing methods. All the
synthesized compounds were well characterized by routine spectral methods. On the
basis of spectral analysis, octahedral geometry was ascertained for the metal
complexes. All 18 compounds were also evaluated for in vitro antimicrobial
activities, and all the ligands (1–6) and a few potential metal complexes with ligands
2
and 5 for in vitro anticancer activities. In general, the complexes exhibited higher
antimicrobial activity than the respective free ligands. Furthermore, the chalcone-
and isatin-containing compounds were found to be more active than acridone;
however, the metal complexes displayed better activity in all cases. Molecular
docking of different inhibitors against topoisomerase II DNA gyrase and of BRCA1
BRCT kinase complex suggested that compounds 3 and 5 are particularly important.
Both these compounds also displayed the highest drug-likenesses with accept-
able bioactivity, and hence can be considered as good starting points for further
optimization. We hope that our findings will be helpful in the design and synthesis
of more effective but safe anticancer/antimicrobial drugs.
Acknowledgements The authors are grateful to the GITAM for providing the instrumental facility and the
University Grants Commission (UGC, Government of India) for providing financial assistance [F. No.
42-239/2013 (SR)]. We also thank the Department of Science and Technology, India under DST-FIST
Program 2014 with Grant No. SR/FST/ETI-373/2016. We also thank Dr P. Lalitha and Dr M. Ramarao,
Department of Microbiology and Department of Biochemistry, GITAM University, respectively, for
performing antimicrobial and anticancer activities. The authors would like to extend their sincere
appreciation to IISc Bangalore, IIT Chennai, for recording the NMR and ESR spectra.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
References
1. S.F.P. Braga, N.C. Fonseca, J.P. Ramos, E.M. De Souza-Fagundes, R.B. De Oliveira, Braz. J. Pharm.
Sci. 52, 299 (2016)
. I. Esirden, M. Tan c¸ , C.T. Supuran, M. Kaya, Bioorg. Med. Chem. Lett. 27, 86 (2017)
. E.M. Hussein, K.S. Khairou, J. Chem. 4, 221 (2014)
. M. Messali, Arab. J. Chem. 7, 63 (2014)
. S. Samai, G.N. Nandi, M.S. Singh, Tetrahedron 68, 1247 (2012)
. H. Kumar, A. Parmar, Ultrason. Sonochem. 15, 129 (2008)
. A.S.-Y. Lee, S.-H.M. Lee, Y.-T. Chang, Res. Chem. Intermed. 43, 3493 (2017)
. G. Revelant, C. Gadais, V. Mathieu, G. Kirsch, S. Hesse, Bioorg. Med. Chem. Lett. 24, 2724 (2014)
. A. Matwijczuk, A. Matwijczuk, D. Karcz, S. Wybraniec, D. Kluczyk, M. Gago s´ , A. Niewiadomy, J.
Phys. Chem. A 119, 10791 (2015)
2
3
4
5
6
7
8
9
123

P. Bhatt et al.
10. A. Matwijczuk, D. Karcz, R. Walkowiak, J. Furso, B. Gładyszewska, S. Wybraniec, A. Niewiadomy,
G.P. Karwasz, M. Gago s´ , J. Phys. Chem. A 121, 1402 (2017)
1. J.F.M. Silva, S.J. Garden, A.C. Pinto, J. Braz. Chem. Soc. 12, 273 (2001)
2. K.L. Vine, L. Matesic, J.M. Locke, M. Ranson, D. Skropeta, Anticancer Agents Med. Chem. 9, 397
1
1
(2009)
1
3. K. Liaras, A. Geronikaki, J. Glamo cˇ lija, A. Dirid, M. Sokovid, Bioorg. Med. Chem. 19, 3135 (2011)
4. R.S. Aliabadi, N.O. Mahmoodi, H. Ghafoori, H. Roohi, V. Pourghasem, Res. Chem. Intermed. 44,
1
2
999 (2018)
5. A. Boumendjel, S. Macalou, A. Ahmed-Belkacem, M. Blanc, A. Di Pietro, Bioorg. Med. Chem. 15,
892 (2007)
1
2
1
1
1
1
2
6. S. Chandra, L.K. Gupta, Spectrochim. Acta A Mol. Biomol. Spectrosc. 60, 1563 (2004)
7. M.N. Aghatabay, Y. Mahmiani, H. Cevik, B. Dulger, Eur. J. Med. Chem. 44, 365 (2009)
8. A.A. Jarrahpour, M. Motamedifar, K. Pakshir, N. Hadi, M. Zarei, Molecules 9, 815 (2004)
9. U. Ndagi, N. Mhlongo, M.E. Soliman, Drug. Des. Devel. Ther. 11, 599 (2017)
0. J.F. Adediji, S.A. Amolegbe, S. Adewuyi, C.A. Akinremi, Y.C. Oyeniran, B.O. Afolayan, Hindawi
Publishing Corporation ISRN Inorganic Chemistry, Article ID 329508, p 7 (2013)
1. A.P. Mishra, H. Purwar, R.K. Jain, Biointerface Res. Appl. Chem. 2, 291 (2012)
2
2
2
2
2
2
2
2
¨
˘
2. N. Sari, N. Pi s¸ kin, H. Og u¨ tc u¨ , N. Kurnaz, Med. Chem. Res. 22, 580 (2013)
3. D. Kumar, A. Syamal, A. Gupta, M. Rani, P.K. Gupta, J. Indian Chem. Soc. 87, 1185 (2010)
4. V.A. Madalina, F.B. Stefania, D. Constantin, L.A. Gabriela, Eur. J. Med. Chem. 45, 2055 (2010)
5. K.C. Patel, D.E. Goldberg, J. Inorg. Nucl. Chem. 34, 637 (1972)
6. B.S.A. Thahira, A.C. Karen, M.T. Ibrahim, R.C. Andrew, M.A. Akbar, Polyhedron 26, 1159 (2007)
7. G.B. Bagihalli, P.G. Avaji, S.A. Patil, P.S. Badami, Eur. J. Med. Chem. 43, 2639 (2008)
8. A. Rahman, M.I. Choudhary, W.J. Thomsen, Bioassay techniques for drug development. Harwood
Academic Publishers, The Netherlands, p 16 (2001)
2
3
3
3
3
3
3
3
9. S. Syam, S.I. Abdelwahab, M.A. Al-Mamary, S. Mohan, Molecules 17, 6179 (2012)
0. A. Girdhar, S. Jain, N. Jain, S. Girdhar, Acta Pol. Pharm. Drug Res. 67, 211 (2010)
1. M.P. Sathisha, V.K. Revankar, K.S.R. Pai, Metal-based drugs, Article ID 362105, p 11 (2008)
2. L. Mishra, A. Jha, Indian J. Chem. 35A, 1001 (1996)
3. A. Jha, Y.L.N. Murthy, U. Sanyal, G. Durga, Med. Chem. Res. 21, 2548 (2012)
4. P. Wang, S.M. Henning, D. Heber, PLoS ONE 5, e10202 (2010)
5. C.A. Lipinski, L. Lombardo, B.W. Dominy, P.J. Feeney, Adv. Drug Deliv. Rev. 46, 3 (2001)
6. Y.H. Zhao, M.H. Abraham, J. Lee, A. Hersey, N.C. Luscombe, G. Beck, B. Sherborne, I. Cooper,
Pharm. Res. 19, 1446 (2002)
1
23