(2024) 18:119
Shaikh et al. BMC Chemistry
https://doi.org/10.1186/s13065-024-01196-1

BMC Chemistry

RESEARCH

Open Access

Synthesis, biological evaluation,and molecular docking of novel
1,3,4‑substituted‑thiadiazole derivatives
as potential anticancer agent
Samin A. Shaikh1,3*, Satish N. Wakchaure2,6*, Shivaji R. Labhade3, Raju R. Kale3, Rajasekhar R. Alavala4,Santosh S. Chobe5, Kamlesh S. Jain1, Hrishikesh S. Labhade3 and Dipak D. Bhanushali1

Abstract
In an attempt to develop potent anti-cancer agents, a new 1,3,4-substituted-thiadiazole derivatives (8b-g), startingfrom 4-substituted-thiazol-2-chloroacetamides (4b-g), were synthesized and evaluated for their cytotoxic effects
on multiple human cancer cell lines, including the hepatocellular carcinoma (HEPG-2), human lung carcinoma (A549),
human breast carcinoma (MCF-7) and pseudo-normal human embryonic liver (L02) cancer cell lines by an MTT assay.
Among all synthesized compounds, compound 8d showed the potent anti-cancer activities with ­GI50 values of 2.98,
2.85 and 2.53 μM against MCF-7, A549 and HepG-2 cell lines respectively as compared to standard drug Doxorubicin.
Furthermore, molecular modelling studies have spotlighted the anchoring role of 1,3,4-substituted-thiadiazole moiety
in bonding and hydrophobic interaction with the key amino acid residues. Therefore, these results can provide promising starting points for further development of best anti-cancer agents.
Keywords Anticancer agents, Thiazole-Thiadiazole compounds, Antiproliferative activity, Synthesis optimization,
Molecular docking*Correspondence:
Samin A. Shaikhsaminshaikh80@gmail.com
Satish N. Wakchauresatish_nclpune@yahoo.co.in
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​
mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Shaikh et al. BMC Chemistry(2024) 18:119

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Graphical Abstract

Introduction
Cancer is presently one of the main causes of deathworldwide. The International Agency for Research on
Cancer (IARC) estimated that globally, 1 in 5 people
develop cancer during their lifetime, and 1 in 8 men
and 1 in 11 women die from the disease suggesting that
more than 50 million people are living within five yearsof a past cancer diagnosis [1]. In past decades, anticancer research on the design of effective oncology drugs
for the application in chemotherapy has improved
treatments which led to remarkable results and many
drugs have been approved [2, 3]. However, many of theapproved drugs still being characterized by high systemic toxicity mainly due to the lack of tumor selectivity and present pharmacokinetic drawbacks, including
low water solubility, that negatively affect the drug circulation time and bioavailability which limit its clinical application [4]. The above stated disadvantages of
conventional anticancer drugs are the reason why thedevelopment of new anticancer drug or improvement inpresent anticancer drug is still demanding. Therefore,
the newer generations of CDK9 inhibitors are alreadyraising as an anticancer therapy and present ongoing
research in this direction are helping to develop better,more selective inhibitors.
HO

Thus, the best suitable inhibition strategies are required
to be able to distinguish between normal rapidly proliferating cells like the T cells and cancer cells. Thiazole andthiadiazole possess unique properties that make them
useful scaffolds in medicinal chemistry [5–10], few examples are shown in Fig. 1. As bioisosteres of pyrimidines,
their derivatives can potentially interact with DNA andRNA and can readily cross cell membranes and interact
with biological compounds in unique ways. For example,
Filanesib (Array-520, Fig. 1) is anti-cancer drug used specifically for multiple myeloma. The best strategy showed
when it was combined with bortezomib and dexamethasone had a favorable safety profile. The resistance toDabrafenib (Fig. 1) and other BRAF inhibitors could be
reduced when the Dabrafenib was combined with the
MEK inhibitor Trametinib [11]. Recently Noblejas-López
et al. had verified that CDK9 PROTAC THAL-SNS-032
showed potent and efficient anti-tumor properties [12].
However, no clinical trials of THAL-SNS-032 (Fig. 1, see
structure SNS-032) have been initiated till date.
Literature data revealed that many research groups
used these scaffolds as a basic core for the developmentof new molecules for anticancer activity. In this study, we
have followed the same strategy and proposed to synthesize novel derivatives (8b-g) having two basic scaffolds,

N
N

N

N
O
NH
Cl

NH

S
N

F

O

S
F

H
N

F

O
N

N

S

O

N
N

N

S
O

NH2

O
S
N

NH

NH

F

O

N N
S

F

Dasatinib

Dabrafenib

SNS-032

Fig. 1 Some examples of thiazole and diathiazole scaffold-based (marked in pink circle) anti-cancer drugs

ARRY-520

NH2

Shaikh et al. BMC Chemistry(2024) 18:119

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on one side is thiazole ring and on another side is 5-aminodiathiazole moiety that could mimic Dabrafenib’s side
chain (2-aminopyrimidine) to some extent (Fig. 2). Additionally, hydrophobic side chains on these scaffolds could
offer the hydrophobic sphere in a molecule which could
contribute to enhance the CDK9 selectivity.

Results and discussion
To confirm the anticancer properties for our new compounds (8b-g), first we have referred our previously
reported compounds (4a-g) [13], and interestingly, thedocking studies of them confirmed their potential affinity to selective CDK9/Cyclin T1 (1BLQ), inhibitors

R

R

R

N
R

N

S

R

N

S

First we have synthesized 4-substituted-thiazol-2-chloroacetamide compounds (4a-m) starting from aromatic
ketones and ethyl acetoacetates derivatives in threesteps using synthetic methods reported in our previous research articles (Scheme 1) [13] and used them forthe synthesis of 1,3,4-substituted-diathiazole derivatives(8a-g). The synthetic protocols for (4a-m) and (8a-g)
are given in the Additional file 1 (see Additional file 1 forgeneral experimental procedure).

H
S

O

Cl

Synthetic chemistry

Heterocyclic linker

N

H

O

Hydrophobic moiety toentrance binding side

R

S

NH2

N N

Extension of heterocycle with amine gr.
(8a-g)(4a-g)(Fig. 4a and Table 2). Based on this results; we proposed
the design and synthesis of 1,3,4-substituted-thiadiazole
compounds (8a-g) as shown in Fig. 2. Although, Gang
Yan et al. has reported the compound 8a, however its
anticancer activity is not yet fully explored [14]. The
structures of the newly synthesized compounds were elucidated by FT-IR, 1H NMR, 13C NMR, and LC − MS analysis methods. These compounds (8a-g) were screened
for in vitro anticancer activity against hepatocellular carcinoma cell lines (HEPG-2), human lung carcinoma cellline (A549) and human breast carcinoma cell (MCF-7)
and pseudo-normal human embryonic live cell line (L02)by MTT assay. In addition, molecular docking studies
were applied to investigate the anti-proliferative effects ofthese novel compounds.

Fig. 2 Structural modifications of 4-substituted-thiazol-2-chloroac
etamides (4a-g) clubbed with 5-amino-1,3,4-thiadiazole-2-thiol (7)moietyAromatic substituents

O

O

a

Ar

Br

80-90%

Ar

+

S
H2N

R1

b
NH2

R2

74-90%

2a: Ar= -Ph
2b: Ar= -4-Br-Ph
2c: Ar= -4-Cl-Ph
2d: Ar= -4-OMe-Ph
2e: Ar= -4-Me-Ph
2f: Ar= -4-NO2-Ph
2g: Ar= -4-F-Ph
2h: Ar= -3-NO2-Ph
2i: Ar= -3-Br-Ph
2j: Ar= -3-OMe-Ph
2k: Ar =

O

O

NH2

N

R1

c
R2

80-98%

S

H
N

N
S

3a: R1 = -H, R2 = -H(80%)
3b: R1 = -H, R2 = -Br (82%)
3c: R1 = -H, R2 = -Cl (85%)
3d: R1 = -H, R2 = -OMe(90%)
3e: R1 = -H, R2 = -Me (87%)
3f: R1 = -H, R2 = -NO2 (75%)
3g: R1 = -H, R2 = -F (76%)
3h: R1 = -NO2, R2 = -H (78%)
3i: R1 = -Br, R2 = -H (80%)
3j: R1 = -OMe, R2 = -H (82%)

4a: R1 = -H, R2 = -H (89%)
4b: R1 = -H, R2 = -Br (94%)
4c: R1 = -H, R2 = -Cl (98%)
4d: R1 = -H, R2 = -OMe (80%)
4e: R1 = -H, R2 = -Me (83%)
4f: R1 = -H, R2 = -NO2 (85%)
4g: R1 = -H, R2 = -F (81%)
4h: R1 = -NO2, R2 = -H (83%)
4i: R1 = -Br, R2 = -H (82%)
4j: R1 = -OMe, R2 = -H (81%)

3k: Ar =4k: Ar =

O

O

(83%)

O

O

Cl
O

(90%)

Aliphatic substituents
O

Y

X O

O

2l: X= -H, Y= -Cl
2m: X= -Cl, Y= -H

+

S
H2N

b
NH2

75-85%

R1
R2

N
S

NH2

c
80-82%

3l: R1= -Me, R2= -COOEt (85%)
3m:R1= -COOEt, R2= -H (75%)

R1

N

R2

S

NH
O

Cl

4l: R1= -Me, R2= -COOEt (82%)
4m: R1= -COOEt, R2= -H (80%)

Scheme 1 Synthesis of 4-substituted-thiazol-2-chloroacetamide compounds (4a-m); Reagents and conditions: a. Bromination: B­ r2/AcOH, 0–5 °C,
3 h, (for 2k: Bromination: B­ r2/CHCl3, 55 °C, 4 h); b Hantzsch reaction: thiourea, EtOH, reflux, 8h; c Amidation: CAC, TEA, DMF, 0–5 °C, 0.5–2.5 hShaikh et al. BMC Chemistry(2024) 18:119

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observed (Scheme 2, 8b, 8c, 8f and 8 g: 91–94%). Thismay be due to the negative resonance effect of -NO2
group and strong electro-negative atoms such as -Br, -Cl,and -F which made electrophilic substitution reaction
better for the 4-substituted-thiazol-2-chloroacetamide
compounds 4a to 4 g.

S
H2N

N
H

+

NH2

5

CS2

6
a.
R2

R2

R1

N N
H2N

7

N
NH

S
O

S

b.
Cl

R1

SH

Anti‑proliferative activity

N
NH

S
O

N

S
S

N
NH2

4a: R1= -H, R2= - H
4b: R1= -H, R2= - Br
4c: R1= -H, R2= - Cl
4d: R1= -H, R2= - OMe
4e: R1= -H, R2= - Me
4f: R1= -H, R2= - NO2
4g: R1= -H, R2= - F

8a: R1= -H, R2= - H (84%)
8b: R1= -H, R2= - Br (94%)
8c: R1= -H, R2= - Cl (91%)
8d: R1= -H, R2= - OMe (78%)
8e: R1= -H, R2= - Me (81%)
8f: R1= -H, R2= - NO2 (91%)
8g: R1= -H, R2= - F (94%)

Scheme 2 Synthesis of substituted-thiadiazole derivatives (8a-g);
Reagents and conditions: a. ­NaHCO3, EtOH, reflux, 5 h, 90%; b. ­K2CO3,
THF, rt, 0.5–1 h, 78–94%

The intermediates 2-aminothiazoles derivatives (3am) were prepared by reaction of aromatic ketones and­Br2 in acetic acid or chloroform followed by Hantzsch
reaction of direct condensation of intermediates (2a-m)
with thiourea in a good to excellent yields. Further amidation reaction using -CAC​ (chloro acetyl chloride) in
DMF furnished 4-substituted-thiazol-2-chloroacetamide
derivatives (4a-m) in excellent yield (Scheme 1). We havefollowed reported procedure and optimized reaction
conditions to obtain these compounds in better yield [13,15–19].
Our main focus was to synthesize 1,3,4-substitutedthiadiazoles derivatives (8a-g). The synthetic strategy
has been developed by clubbing of the 4-substitutedthiazol-2-chloroacetamide compounds (4a-g) with
5-amino-1,3,4-thiadiazole-2-thiol (7) via thial linkage
to furnish thiadiazole derivatives (8a-g). The optimized
reaction condition for (8a-g) is depicted in Scheme 2. The
excellent yield of these derivatives (8a-g) was achievedby substitution reaction of compounds (4a-g) with
5-amino-1,3,4-thiadiazole-2-thiol (7) in THF at room
temperature (Scheme 2, 78–24%). Surprisingly, the substitution reaction worked well for any substituent present
on thiazole ring such as -Ph, -4-Br-Ph,-4-Cl-Ph, -4-OMePh, -4-Me-Ph, -4-NO2-Ph and -4-F-Ph (Scheme 2, 8a-g).
The 8d was synthesized with less yield (8d:78%), thismay be due to the strong positive resonance effect of-OMe group (­R2) present on the phenyl ring. Especially, when -4-Br-Ph, -4- Cl-Ph, -NO2-Ph and -4-F-Ph
groups were present on thiazole ring then highest yields

Several structural alterations in compounds (8a-g) in
relation to the substitution at C4 of the thiazole moiety
were made to examine whether the nature of the substituent would affect selective toxicity for hepatocellular carcinoma cell lines (HEPG-2), human lung carcinoma cellline (A549) and human breast carcinoma cell line (MCF7) and pseudo-normal human embryonic liver cell line
(L02) by MTT assay. The doxorubicin is used as a standard drug; it is a reasonable standard cytotoxic agent thatis a well-known chemotherapy medication. Interestingly,
the compounds 4c, 4j, 8b, 8d and 8f showed the bestanti-proliferative activity with I­ C50 values in the range of
1.82 to 4.07 µM against the MCF-7 cell line. They werealso equally potent against A549 and HepG-2 cell lines­(GI50 values in the range of 2.61 to 18.43 and 2.38 to
5.86 µM respectively) as compared to the doxorubicin­(GI50 = 3.26 and 3.87 µM respectively, Table 1).
As hypothesized, the compound 4a has unsubstituted
phenyl ring and exhibited poor growth anticancer inhibition properties with ­GI50 values of 23.76, 18.74, 12.86
and 21.63 µM in all cancer cell lines (Table 1). Substitution at the -para position of phenyl ring of 4a by halogen-bromo (4b) resulted moderate growth inhibition properties against MCF-7 cell line (Table 1, ­GI50 of 18.25 µM)
while it showed potent growth inhibition against A549
and HepG-2 cell lines with ­GI50 values of.
2.97 and 3.62 µM respectively as compared to thestandard doxorubicin (Table 1). Here, the activity was
improved, this may be due to the -para bromo substituted phenyl ring. Introduction of another substituent by–chloro in –para position of phenyl ring of 4a brought
the greatest improvement in the activity of the resulting
compound 4b against MCF-7, A549 and HepG-2 celllines ­(GI50 value of 2.63, 3.86 and 3.92 µM respectively)
and indicating it was superior in potency against MCF-7
cell line compared to the doxorubicin (Table 1). Thispotency trend could be explained by the -para substituted halogen could have a potential role of drug–target
binding affinity in above three cell lines.
In the next stage of biological activity, more compounds with substituted phenyl groups (electron-donating and electron-withdrawing) installed on the C4- andC5-positions of phenyl ring were prepared including
substituents -OMe, -Me, -NO2 and -F (4d, 4e, 4f, 4 g,
4 h and 4i) diminished the potency against all four cell

Shaikh et al. BMC Chemistry(2024) 18:119

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Table 1 Structures and in vitro biological activities of compounds (4a-m) and (8a-f)
R2

R1

R2

N

O

(4a-k)

Cl

R1

N
NH

S
O

NH

S

R2

R1

Ar

N
N

S

Cl

H
S

O

(4l-m)(8a-f)S
N N

NH2

Growth inhibition, G­ I50 (μM)b

Substituent(% inhibition
at 100 µM)b

Compoundb

R1

R2

MCF-7

A549

HepG-2

L02

4a

−H

−H

23.76 ± 0.24

18.74 ± 0.41

12.86 ± 0.25

21.63 ± 0.43

4b

−H

− Br

18.25 ± 0.39

2.97 ± 0.19

3.62 ± 0.14

17.82 ± 0.51

4c

−H

− Cl

2.63 ± 0.14

3.86 ± 0.22

3.92 ± 0.17

18.45 ± 0.48

4d

−H

− OMe

26.54 ± 0.32

15.41 ± 0.23

8.37 ± 0.23

12.76 ± 0.22

4e

−H

− Me

31.84 ± 0.46

13.67 ± 0.34

7.28 ± 0.16

15.86 ± 0.24

4f

−H

− ­NO229.43 ± 0.32

19.25 ± 0.32

18.92 ± 0.29

28.16 ± 0.27

4g

−H

−F

36.68 ± 0.41

17.54 ± 0.26

22.65 ± 0.46

16.26 ± 0.21

4h

− ­NO2−H

38.15 ± 0.27

28.62 ± 0.47

25.81 ± 0.23

18.43 ± 0.34

4i

− Br

−H

33.74 ± 0.32

31.36 ± 0.36

15.96 ± 0.43

17.64 ± 0.38

4j

− OME

−H

1.82 ± 0.21

2.61 ± 0.12

2.38 ± 0.24

17.29 ± 0.23

26.35 ± 0.28

25.28 ± 0.39

14.26 ± 0.28

22.97 ± 0.35

4k
Ar = −

O

O

4l

Me

COOEt

37.26 ± 0.37

32.85 ± 0.46

29.83 ± 0.34

26.04 ± 0.47

4m

COOEt

H

12.38 ± 0.42

23.68 ± 0.43

4.35 ± 0.16

16.34 ± 0.28

8a

−H

−H

34.87 ± 0.31

36.23 ± 0.39

26.75 ± 0.28

22.56 ± 0.42

8b

−H

− Br

4.07 ± 0.19

18.43 ± 0.46

5.86 ± 0.24

15.76 ± 0.28

8c

−H

− Cl

20.68 ± 0.36

7.86 ± 0.24

4.12 ± 0.18

18.25 ± 0.22

8d

−H

− OMe

2.98 ± 0.15

2.85 ± 0.18

2.53 ± 0.10

21.79 ± 0.38

8e

−H

− Me

27.99 ± 0.34

29.76 ± 0.37

18.28 ± 0.34

23.76 ± 0.42

8f

−H

− ­NO2

3.71 ± 0.26

4.64 ± 0.23

3.46 ± 0.13

20.65 ± 0.37

doxorubicin––

4.15 ± 0.18

3.26 ± 0.24

3.87 ± 0.16

82.15 ± 1.26a

The cytotoxicity was detected using the MTT assay; bThe data are expressed as the mean ± SD of three independent experiments

lines, (Table 1). Surprisingly, compound 4j has -methoxy
substituent at -meta position of phenyl ring exhibited
highest potent growth anticancer inhibition properties
against MCF-7, A549 and HepG-2 cell lines (Table 1, ­GI50
of 1.82, 2.61 and 2.38 µM respectively) as compared tothe doxorubicin (­ GI50 of 4.15, 3.26 and 3.87 µM respectively), this is mainly because of electrons donating effectof -methoxy group at –meta position could constructs πelectrons interactions and contributing positively for theanti-proliferative activity (Table 1).
To learn the impact of the substituted-diathiazole
compounds to potency, we synthesized compounds(8a-g) and screened for the anticancer activity. The
substitution reaction on 4a by 5-amino-1,3,4-thiadiazole-2-thiol moiety resulted compound 8a in moderate reduction of antiproliferative activity as compared
to 4a (Table 1). However, for –para substituted chloro

compound 8b significantly increased anticancer inhibition properties against MCF-7 and HepG-2 cell lines(Table 1, ­GI50 value of 4.07 and 5.86 µM respectively)
as compared to the compound 4b. This highlighted
5-amino-thiadiazole moiety and 4-substituted halogen
of phenyl ring were playing a major role for the good
activity by exploring binding sides to CDK9/Cyclin
T1. In this series, compound 8d represented remarkable potential in MCF-7, A549 and HepG-2 cell lines(Table 1, ­GI50 of 2.98, 2.85, and 2.53 µM respectively)
as compared to doxorubicin. Amazingly, compound
8f has strong electron-withdrawing substituent -paranitro of phenyl ring showed better activity as comparedto 4f in MCF-7, A549 and HepG-2 cell lines with ­GI50
of 3.71, 4.64 and 3.46 µM respectively and still havecomparable activity with doxorubicin (Table 1).

Shaikh et al. BMC Chemistry(2024) 18:119

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Thus, the compounds 8d and 8f have showed promising activity against MCF-7, A549 and HepG-2 cell lines
as compared compounds (4a-i) and doxorubicin. This isindicating the crucial role of 5-amino-thiadiazole moiety
in the potency.
Annexin V‑FITC/PI assay

The early stages of apoptosis were monitored by Annexin
V-FITC (apoptotic cell marker) and PI (necrotic cellmarker) double staining. The staining method was usedaccording to the Annexin FITC/PI staining kit (Invitrogen™, Thermo Fisher Scientific Inc.). Annexin V- FITC/PI assay was performed to inspect the extent of programmed cell death (Fig. 3, apoptosis) by the compounds
4c, 4j and 8f on breast carcinoma cell line (MCF-7).
The cells were treated with increasing concentrations(5–10 μM) for 24 h and analyzed by flow cytometry.
The untreated cells data has shown 88.26% of live
cells, 0.21% death of cells due to necrosis, 0.18% due tothe late apoptosis and 0.07% because of the early apoptosis (Fig. 3). The compound 4c has shown 62.43% and42.19% of live cells at 5 and 10 μM concentration respectively. Whereas the necrosis is prominent cause of thecell growth inhibition at 5 μM and at 10 μM, the late
apoptosis is the major reason for the cell death. Compound 4j has reduced the viable cell count to the 42.81%
at 5 μM and at 10 μM it was 32.80% only. The cell death
was majorly occurred due to the late apoptosis with
34.84% and 40.33% at the tested concentrations. Compound 4j has showed considerable reduction in theviable cell count as compared to 4c. This may be due tothe electron-donating substituent (-methoxy) present on
the phenyl ring at –meta position of 4j which have muchbetter hydrophilic interactions due to availability of electrons. Whereas in compound 4c, the -chloro substituent
present on phenyl ring at –para position leading minimum reduction of cell counts (Fig. 3). Interestingly, compound 8f has shown 59.08% and 46.14% of live cells at
the tested concentrations. It has shown, the cell death of
16.99 and 14.62% at 5 μM due to necrosis and late apoptosis respectively and at 10 μM, the percentage of death
was 12.44% and 37.15%. Thus, this underlined the role ofsubstituted-diathiazole moiety in apoptosis.

Fig. 3 Cell death (apoptosis) analysis of compounds 4c, 4j and 8f
on breast carcinoma cell line (MCF-7). The cells were treatedwith increasing concentrations for 24 h and analyzed by flow
cytometry. First quadrant Q1 represents necrotic cell, second
quadrant Q2 represents late apoptotic, third quadrant Q3 represents
live cells, and forth quadrant Q4 represents early apoptotic cells.
The untreated cells have shown 88.26% of live cells, 0.21% deathof cells due to necrosis, 0.18% due to the late apoptosis and 0.07%
because of the early apoptosis. The experiments were repeated twice
and representative data were presented

Molecular modeling studies

The target prediction was done by using the Swiss Target Prediction and Pass Online tools. Based on the scoresand ratio data from the two servers, potential anti-cancer targets like Cyclin-dependent kinase 9 (CDK9)/
Cyclin T1–3BLQ signal transducer and activator of transcription 3 (STAT3) were selected [20, 21]. In addition,
Sitemap tool of Glide was used for predicting the binding pockets and one of the predicted five was selected

for docking studies based on the score and volume. Glide
tool of the Maestro (Schrödinger Release 2023-2, New
York, USA) was used for the exploring molecular docking
studies. [22–25]
The docking studies of investigated compounds (4a-m)
displayed XP GScores between − 7.777 and − 3.826 kcal/
mol against CDK9/Cyclin T1 (1BLQ), providing theShaikh et al. BMC Chemistry(2024) 18:119

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insights about their binding mode in the enzyme’s binding cavity. Whereas verifying their potential affinity
for the enzyme’s binding cavity as compared to standard drug Flavopiridol as shown in Table 2 (Fig. 4). We
have chosen reference drug Flavopiridol because it is an
Orphan drug used for the treatment of acute myeloid
leukemia as well as the treatment of arthritis. Flavopiridol is a known inhibitor of CDK9 and it was used as astandard inhibitor for comparing the docking results.
STX-0119 is a co- crystalized ligand in STAT3 protein,
it was used for validating the docking protocol and tocompare the docking score and energy with the designed
compounds. As per the data obtained from the experimentation, some of the designed molecules were foundto possess better docking score with the studied protein
molecules. Especially, compound 4 g was showed highest docking score – 7.777 kcal/mol against CDK9/Cyclin
T1–1BLQ (Table 2) and found to interact with the binding site residues Cys106 (2.05, 2.66 Å) with H-bonding
interactions and π-π stacking with Phe103 (Fig. 4a). As
hypothesized, substitution of phenyl derivatives at C4
position of the thiazole ring afforded moderate to potent

and relatively selective CDK9 inhibitors and other side of
5-amino-thiadiazole moiety could offers free -NH2 and-N groups as a key hydrogen bond donor and acceptor tointeract with the backbone carbonyl and the -NH groupsof the binding pockets respectively and possibly exhibited potential anticancer properties. Therefore, in thisstudy we designed and synthesized six new 1,3,4-substituted-thiadiazole compounds (8b-g) and performed thedocking studies for their potential anticancer properties.
[12–14]In the first series of 4a to 4 m compounds, as discussed above compound 4 g has highest GScores(Table 2, − 7.777 kcal/mol), however experimental
data revealed moderate growth in anticancer inhibition properties (Table 1). The compound 4c has also
showed good GScores (Table 2, − 7.183 kcal/mol) andexhibited the strong binding interactions with the acidic
residues of CDK9/Cyclin T1 and also the experimental
data of anti-proliferative activity showed potent anticancer inhibition properties against MCF-7, A549 and
HepG-2 cell lines (Table 1, 4c). Therefore, experimental data are in good agreement with docking data ofTable 2 Docking score and energy of the tested ­compoundsa
Compounds

CDK9/Cyclin
T1–3BLQ
Docking score

STAT3-1BG1
Glide emodelDocking score

Glide emodel

4a− 7.734− 45.358− 4.842− 37.574

4b− 4.532− 45.126− 4.297− 36.739

4c− 7.183− 43.123− 5.225− 39.644

4d− 7.464− 46.86− 5.239− 38.1214e− 6.376− 45.919− 4.523− 35.675

4f− 6.323− 46.953− 5.269− 37.953

4g− 7.777− 44.228− 4.985− 39.019

4h− 7.254− 49.916− 4.784− 42.581

4i− 7.283− 47.625− 4.408− 38.370

4j− 6.075− 51.949− 5.040− 37.8954k− 4.98− 52.264− 3.374− 34.2164l− 3.826− 47.903− 3.686− 30.5404m− 4.508− 49.863− 4.901− 40.403

8a− 6.479− 60.016− 3.662− 49.1638b− 6.133− 64.789− 5.635− 62.694

8c− 7.451− 61.403− 5.886− 62.6468d− 7.049− 65.300− 5.804− 63.3048e− 5.369− 59.562− 4.543− 53.690

8f− 5.702− 70.403− 5.121− 59.8168g− 7.324− 62.143− 5.341− 62.389

STX− 0119− 6.187− 61.656− 4.242− 60.276(CPB)(Flavopiridol)− 6.238− 53.629––aCalculated using Graph Pad Prism program (Graph Pad software Inc, CA)Shaikh et al. BMC Chemistry(2024) 18:119

Page 8 of 11

Fig. 4 Binding modes of compounds 4g, 8b, 8d and 8g (from the top of the page figure a, b, c and d) with CDK9/Cyclin T1 and an important
residues and interaction distances for key residues. CDK9/Cyclin T1 is shown in green color. The figure was prepared using Glide tool of the Maestro(Schrödinger, LLC, New York, USA)

Shaikh et al. BMC Chemistry(2024) 18:119

Page 9 of 11

compound 4c. Remarkably, the compound 4j showed
highest potent growth anticancer inhibition properties
experimentally as compared to all other compounds andthe docking score for 4j against CDK9/Cyclin T1–1BLQ(Table 1, docking score = -6.075 kcal/mol and glide
emodel = -51.949 kcal/mol) were comparable with thedocking score of STX-0119 and CPB as shown in Table 2.
This may be because of 4j has -methoxy substituent
at –meta position of phenyl ring and it was moderately
water soluble and has showed high GI absorption value
with fulfilling Lipinski rule of five. However same isomeric compound 4d which has –methoxy substituent at–para position of phenyl ring and showed good dockings
core (Table 2, − 7.464 kcal/mol) and found to be inactive
practically (Tables 3 and 4). Similarly, for compounds 4e
and 4f which showed acceptable docking score (Table 2,
GScores − 6.376 and − 6.323 and kcal/mol) but experimental data indicating that they showed moderate antiproliferative activity (Table 1, 4e and 4f ). So in this series
it was observed that -para substituted halogen and -meta
substituted strong electron donating groups determined
an improvement in the potency as compared to othercompounds.
More interestingly, when compounds (4a-g) wereextended by introducing amino-diathiazole moiety
resulted highly potent compounds 8b, 8c, 8d, 8f and

8 g with highest docking score against CDK9/Cyclin
T1–1BLQ inhibitor (Table 2, − 6.133, − 7.451, − 7.049,− 5.702 and − 7.324 kcal/mol respectively). However, theexperimental data revealed that compound 8c showed
moderate anticancer inhibition properties while compounds 8b, 8d and 8f showed potent anticancer inhibition properties (Table 1). The compound 8b has formedhydrophobic interaction with Lys and strong Hydrogenbonding with Leu and Glu amino acids residues (Fig. 4b).
The compound 8d has -methoxy substituent at –para
position of phenyl ring formed of strong Hydrogen bonding with Cys106 (2.82 Å and 2.29 Å) and Arg382(2.97 Å)
as shown in Fig. 4c. Similarly, compound 8f has formedinteractions with Glu107 (2.77 Å), Cys106 (2.24 Å),
Phe103 (2.3 Å), Asp167 (2.19 Å) and Glu66 (2.51 Å) bystrong H-bond and ionic interaction with Lys48 as shownin Fig. 4d. Additionally, all compound 8b, 8d and 8f haveshown good pharmacokinetic properties as predicted by
ADME (Tables 3 and 4).
In the drug likeness prediction and the pharmacokinetic property (ADME) calculations, it was found that
all the compounds have molecular weight well below 500
Daltons and they may not face any difficulty to pass the
biological membranes when given through oral route(Table 3). The same has been supported by the CLogP,
where the compound have shown optimum partition

Table 3 ADME prediction of the designed compounds (4a-m and 8a-g)aCompunds

MW

RB

HBA

HBD

MR

TPSA

CLog P

Log S

ESOL Class

4a252.72

4

2

1

66.66

70.23

2.48

− 3.38Sol4b331.62

4

2

1

74.36

70.23

3.11

− 4.28Mod. Sol4c287.17

4

2

1

71.67

70.23

3.02

− 3.96Sol4d282.75

5

3

1

73.15

79.46

2.47

− 3.43Sol4e266.75

4

2

1

71.62

70.23

2.82

− 3.67Sol

4f

297.72

5

4

1

75.48

116.05

1.65− 3.41Sol

4g

270.71

4

3

1

66.62

70.23

2.8

− 3.53Sol

4h297.72

5

4

1

75.48

116.05

1.62− 3.41Sol

4i

331.62

4

2

1

74.36

70.23

3.11

− 4.28Mod. Sol4j282.75

5

3

1

73.15

79.46

2.5

− 3.43Sol

4k320.75

4

4

1

82.7

100.442.64− 3.82Sol

4l

262.71

6

4

1

62.28

96.53

1.77

− 2.59Sol4m

262.71

7

4

1

61.89

96.53

1.42

− 2.04Sol

8a349.45

6

4

2

90.98

175.572.32− 3.91Sol

8b428.35

6

4

2

98.68

175.57

3.05− 4.81Mod. Sol

8c

383.9

6

4

2

95.99

175.572.94− 4.49Mod. Sol8d

379.48

7

5

2

97.47

184.8

2.33

− 3.96Sol8e363.48

6

4

2

95.94

175.57

2.66

− 4.2

Mod. Sol8f

394.45

7

6

2

99.8

221.391.63− 3.95Sol

8g

367.44

6

5

2

90.93

175.572.72− 4.06

Mod. Sola

MW-Molecular Weight; RB-Number of rotatable bonds; HBA Number of H-Bond acceptors; HBD-Number of H-Bond donors; MR-Molar refractivity; TPSA-Total polar
surface area; CLog P-Consensus Log P

Shaikh et al. BMC Chemistry(2024) 18:119

Page 10 of 11

Table 4 ADME prediction of the designed compounds (4a-m and 8a-g)
Compunds

GI absorption

BBB permeant

Pgp substrate

CYP isoform inhibitorlog Kp (cm/s)

1A2

2C19

2C9

2D6

3A4

4a

High

Yes

No

Yes

Yes

No

No

No

− 5.89

4b

High

Yes

No

Yes

Yes

Yes

No

No

− 5.88

4c

High

Yes

No

Yes

Yes

Yes

No

No

− 5.66

4d

High

No

No

Yes

Yes

Yes

No

No

− 6.09

4e

High

Yes

No

Yes

Yes

Yes

No

No

− 5.72

4f

High

No

No

Yes

Yes

No

No

No

− 6.28

4g

High

Yes

No

Yes

Yes

No

No

No

− 5.93

4h

High

No

No

Yes

Yes

No

No

No

− 6.28

4i

High

Yes

No

Yes

Yes

Yes

No

No

− 5.88

4j

High

No

No

Yes

Yes

Yes

No

No

− 6.09

4k

High

No

No

Yes

Yes

Yes

No

No

− 6.31

4l

High

No

No

Yes

No

No

No

No

− 6.45

4m

High

No

No

No

No

No

No

No

− 6.99

8a

Low

No

No

Yes

Yes

Yes

No

Yes

− 6.45

8b

Low

No

No

Yes

Yes

Yes

No

Yes

− 6.44

8c

Low

No

No

Yes

Yes

Yes

No

Yes

− 6.21

8d

Low

No

No

No

Yes

Yes

No

Yes

− 6.66

8e

Low

No

No

Yes

Yes

Yes

No

Yes

− 6.27

8f

Low

No

No

No

Yes

No

No

Yes

− 6.85

8g

Low

No

No

Yes

Yes

Yes

No

Yes

− 6.49coefficient, between 1.42 to 3.11. Whereas, all the compounds were having a minimum of 4 rotatable bonds
required to undergo conformational changes during thebinding to a protein target. The Hydrogen bond acceptors and donors were well within the accepted limits
to meet the Lipinski rule of five. In addition to that thecompounds were found to have high GI absorption and
P-gp non-substrate nature (Table 4) makes them a viablechoice to take them to next stage of exploration.
Thus, by experimental data, compounds 4j, 8b, 8d and
8f were found to be the potent anticancer agent. By theinteraction energy, it was observed that these compounds
have -bromo, -methoxy and -nitro substituent’s at -para
position of phenyl ring of substituted-diathiazole compounds, which strongly assist the molecules to form
potential bonds with the residues of the binding sites. It
was proved that the docking calculations and the experimental data are close in proximity.

Conclusion
A series of novel 1,3,4-substituted-thiadiazole compounds (8b-g) designed and synthesized from 4-substituted-thiazol-2-chloroacetamides (4b-g) and fully
characterized by spectrometric analysis methods(FT − IR, 1H NMR, 13C NMR, and mass). All synthesized compounds (4a-m) and (8a-f) exhibited selective

anti-proliferative activity. Compounds 4j, 8d and 8f demonstrated promising anticancer activity against MCF-7,
A549, HepG-2 and L02 all cancer cell lines. Especiallycompounds 4j and 8d exhibited the most potent anticancer inhibition properties with ­GI50 values of 1.82, 2.61,
2.38 μM and 2.98, 2.85, 2.53 μM against MCF-7, A549
and HepG-2 cell lines respectively as compared to Doxorubicin. Further molecular docking was revealed thebiological activity. Thus, these compounds can provide
promising starting points for further development of bestanti-cancer agents.

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s13065-​024-​01196-1.
Additional file 1. Additional figures and sections.
Acknowledgements
We acknowledge the principal K.T.H.M. College and Kr. V. N. Naik Shikshan
Prasarak Sanstha’s Arts, Commerce and Science College, Department of Chemistry, Savitribai Phule Pune University (SPPU) for providing necessary laboratory
facilities. We appreciate the provision of the required resources by the Director,
SAIF, Chandigarh University for spectral analysis and NCL for providing sample
for biological assay. We are expressing our sincere appreciation to SS Chobe of
MGV College for their strong support and motivation in this research journey.
SN Wakchaure, SA Shaikh, SR Labhade and RR Kale have provided the guidance for research and DD Bhanushali, KS Jain and HS Labhade were performed
the organic synthesis experiments. Thanks to RR Alavala for assistance with

Shaikh et al. BMC Chemistry(2024) 18:119

Page 11 of 11

the Laboratory facility for biological study. We are thankful to Mr. Sandeep
Deshmukh, CMD of Delta Finochem Pvt. Ltd. for motivation, helpful discussion
and providing LC-MS facility.
Author contributions
RR Alavala performed biological studies. SN Wakchaure, SA Shaikh, SS Chobe,
SR Labhade, and RR Kale were constantly guided the projects. DD Bhanushali,
KS Jain and HS Labhade were performed the organic synthesis experiments.
All authors have gone through the final manuscript and approved it. All
authors read and approved the final manuscript.
Funding
No funding has been received from any source.
Availability of data and materials
All data generated or analyzed during this study are included in this publishedarticle [and additionally its supplementary information file includes materials
and methods, general experimental procedure, NMR data, NMR spectra, mass
spectra and IR spectra].

8.

9.
10.
11.
12.

13.

Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Department of Chemistry, Savitribai Phule Pune University, Kr. V. N. Naik
Shikshan Prasarak Sanstha’s Arts, Commerce and Science College, Canada
Corner, Nashik, Maharashtra 422002, India. 2 Department of Synthetic R & D,
Delta Finochem Pvt. Ltd., G. No. 350, Wadivarhe, Igatpuri, Nashik, Maharashtra
422403, India. 3 Department of Chemistry, Savitribai Phule Pune University,
KTHM College, Nashik, Maharashtra 422002, India. 4 SVKM’s NMIMS, Shobhaben
Pratapbhai Patel School of Pharmacy & Technology Management, Vile Parle(W), Mumbai, Maharashtra 400056, India. 5 Department of Chemistry, Savitribai
Phule Pune University, M.G.Vs. L. V. H. Arts, Science and Commerce College,
Panchavati, Nashik, Maharashtra 422003, India. 6 Friedrich Alexander University
Erlangen-Nuremberg (FAU), 91058 Erlangen, Germany.
Received: 15 February 2024 Accepted: 18 April 2024

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