Triazolothiadizepinylquinolines as potential MetAP-2 and NMT inhibitors: Microwave-assisted synthesis, pharmacological evaluation and molecular docking studies

Article abstract of DOI:10.1016/j.molstruc.2019.127445

The enzymes MetAP-2 and NMT play a crucial role in the process of myristoylation of oncoproteins which is deregulated in many types of cancers. Execution of both these enzymes is considered as strategy for the intervention of various cancers and relative fungal infections, and hence the discovery of novel MetAP-2 and NMT inhibitors necessitate their high relevancy. In this investigation, we have synthesized a series of novel seven-membered triazolothiadiazepinyl quinolines 10(a–m) distinctively under microwave irradiation technique and identified as selective MetAP-2 and NMT inhibitors. Amongst the functionalized derivatives when evaluated for the in vitro antifungal assay, compounds 10b, 10c, 10e and 10f were considered promising due to notable inhibitory effects (MIC = 0.2 mg/mL) on Aspergillus fumigatus. Screening of the anticancer activity against NCI-60 Human tumor cell lines portrayed that conjugates 10b, 10c, 10e and 10f were found to be moderately effective against the Renal Cancer cell line UO-31. The data acquired from biological studies was further validated by molecular docking studies and pharmacokinetic evaluation.

Full text of DOI:10.1016/j.molstruc.2019.127445

Journal Pre-proof
Triazolothiadizepinylquinolines as potential MetAP-2 and NMT inhibitors: Microwave-
assisted synthesis, pharmacological evaluation and molecular docking studies
Saba Kauser J. Shaikh, Ravindra R. Kamble, Praveen K. Bayannavar, Shilpa M.
Somagond, Shrinivas D. Joshi
PII:
S0022-2860(19)31554-6
DOI:
https://doi.org/10.1016/j.molstruc.2019.127445
MOLSTR 127445
Reference:
To appear in: Journal of Molecular Structure
Received Date: 1 October 2019
Revised Date: 1 November 2019
Accepted Date: 18 November 2019
Please cite this article as: S.K.J. Shaikh, R.R. Kamble, P.K. Bayannavar, S.M. Somagond, S.D. Joshi,
Triazolothiadizepinylquinolines as potential MetAP-2 and NMT inhibitors: Microwave-assisted synthesis,
pharmacological evaluation and molecular docking studies, Journal of Molecular Structure (2019), doi:
https://doi.org/10.1016/j.molstruc.2019.127445.
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GRAPHICAL ABSTRACT
Triazolothiadizepinylquinolines as potential MetAP-2 and NMT inhibitors:
Microwave-assisted synthesis, pharmacological evaluation and molecular
docking studies
a
a,
a
Saba Kauser J. Shaikh , Ravindra R. Kamble *, Praveen K. Bayannavar ,
Shilpa M. Somagonda, Shrinivas D. Joshi b

Triazolothiadizepinylquinolines as potential MetAP-2 and NMT inhibitors:
Microwave-assisted synthesis, pharmacological evaluation and molecular
docking studies
a
a,
a
Saba Kauser J. Shaikh , Ravindra R. Kamble *, Praveen K. Bayannavar ,
Shilpa M. Somagonda, Shrinivas D. Joshi b
a
Department of Studies in Chemistry, Karnatak University Dharwad, Pavate Nagar,
Dharwad-580 003, Karnataka, India
b Novel Drug Design and Discovery Laboratory, Department of Pharmaceutical Chemistry,
S.E.T.’s College of Pharmacy, Sangolli Rayanna Nagar, Dharwad-580 002, Karnataka, India
ABSTRACT
The enzymes MetAP-2 and NMT play a crucial role in the process of myristoylation of
oncoproteins which is deregulated in many types of cancers. Execution of both these enzymes
is considered as strategy for the intervention of various cancers and relative fungal infections,
and hence the discovery of novel MetAP-2 and NMT inhibitors necessitate their high
relevancy. In this investigation, we have synthesized a series of novel seven-membered
triazolothiadiazepinyl quinolines 10(a-m) distinctively under microwave irradiation
technique and identified as selective MetAP-2 and NMT inhibitors. Amongst the
functionalized derivatives when evaluated for the in vitro antifungal assay, compounds 10b,
10c, 10e and 10f were considered promising due to notable inhibitory effects (MIC = 0.2
mg/mL) on Aspergillus fumigatus. Screening of the anticancer activity against NCI-60
Human tumor cell lines portrayed that conjugates 10b, 10c, 10e and 10f were found to be
moderately effective against the Renal Cancer cell line UO-31. The data acquired from
biological studies was further validated by molecular docking studies and pharmacokinetic
evaluation.
Keywords: Triazolothiadiazepinylquinoline; Microwave-assisted synthesis; Antifungal assay;
Anticancer activity; Molecular docking; N-Myristoyl transferase; Methionine aminopeptidase
type-2 (MetAP-2); Pharmacokinetic parameters.
*Corresponding author at: Department of Studies in Chemistry, Karnatak University,
Pavate nagar, Dharwad-580003, Karnataka, India.
E-mail address: kamchem9@gmail.com (R. R. Kamble)
1

1. Introduction
Angiogenesis is the formation of new blood vessels and the process is controlled by
vascular-related pathologies. Angiogenesis is identified as a crucial process because it assists
in the growth of cancers because solid tumors need supply of blood if they have to grow
beyond some size. Inhibition of angiogenesis is considered to be one of the remarkable
pathways of inhibiting the tumor growth [1]. Methionine aminopeptidase (MetAP-2) has been
identified as an important enzyme because it plays a crucial role in angiogenesis and in
progressions of solid tumour cancers, cell proliferation and different tumor growth [2].
MetAPs found in all organisms are proteases that catalyze the hydrolytic removal of N-
terminal methionine residues from nascent proteins, involved in protein maturation. It is
essential prior to the N-terminal modifications such as acetylation and myristoylation.
MetAP-2 is recognized as the cellular target of angiostatic agents and the significant growth
inhibition was observed in cells sensitive to MetAP-2 inhibition, have attracted more
attention towards the same [3].
N-terminal myristoylation on the other hand is an irreversible co-translational protein
modification which is catalyzed by N-myristoyltransferase (NMT). It is also a modification
carried out by NMT after MetAP-2 enzyme removes methionine from the amino terminal
of the protein as depicted in the Figure 1 [4].
Poly A tail
CH3
S
CO
NH
H3C
AA2-AA3.......
NH
CO
NH2
Methionine
Glycine
MetAP2
CH3
Poly A tail
NH
protein with
N-terminal glycine
AA2-AA3.......
NH2
CO
myristoyl-CoA
NMT
Poly A tail
CH3
NH
myristoyl
AA2-AA3.......
N
CO
myristolylated protein
Figure 1. Representation of protein N-myristoylation
2

In the year 2004, Selvakumar et al examined the first study demonstrating the levels of
expression of MetAP-2, NMT, in human colon cancer cell lines (HCCLs) [5]. Fumagillin and
its congeners are identified originally as an antibacteriophagic and amebicidal agent [6,7]
isolated from microbial organism Aspergillus fumigatus in 1949. Fumagillin has also been
found to bind selectively and covalently to MetAP-2 and block its aminopeptidase activity by
cleaving the N-terminal methionine from the proteins, a required process for the exposure of
N-terminal glycine residues of NMT protein substrates [8].
Diazepines and their analogues are classified as agonists at gamma-aminobutyric acid-
A (GABA-A) receptors and possess amnesic, anxiolytic, anticonvulsant, hypnotic, sedative
and skeletal muscle relaxant properties [9]. Also these are commonly used for their
antibacterial, antiviral, anti-protozoan and also have been designed as potential lead for
anticancer and antiviral agents [10]. 1,3,4-Thiadiazepine also classified under the diazepines
is notably a remarkable heterocycle due to the presence of the -N=C-S group and is found to
be a better therapeutic agent [11]. Thiadiazepines have been reported to exhibit potent anti-
microbial activities I, II [12], analgesic III, IV, V [13], cytotoxic activities VI [14] as shown
in the Figure 2. The 1,3,4-Thiadiazepine skeleton also possess anti-HIV and antidepressant
properties [15,16]. They also act as intermediates for the preparations of substituted
caprolactams useful for treatment of HIV disease [17] and excellent charge generating agents
[18]. The rationale behind designing and investigating the sulfur-based poly-fused
heterocyclic analogues is the presence of sulfur in biological systems as well as its increasing
interest as a regulatory agent [19].
The purpose of the present study is to synthesize, evaluate the synthesized compounds
pharmacologically, computationally and further identifying them as potent MetAP-2 and
NMT inhibitors. Amongst the compounds screened, we observed that compounds 10b, 10c,
10e and 10f as the inhibitors of MetAP-2 and NMT despite being structurally divergent from
Fumagillin.
3

OCF3
N
N
N
N
N
N
N
S
N
S
N
N
OCH3
N
HN
O
Cl
II
I
R
N
N
N
Ar
S
N
S
N
8
R'
N
H
H
N
R
N
N
N
O
O
R
O
VI
III: R = Phenyl, R' = H, Ar = Phenyl
IV: R = o-Cresyloxymethyl, R' = CH3, Ar = p-Anisyl
V: R = p-Cresyloxymethyl, R' = OCH3, Ar = p-Anisyl
Figure 2. Examples of biological significant 1,3,4-thiadiazepines derivatives
2. Results and discussion
2.1. Synthesis
Synthesis of one of the precursor 4-amino-5-(pyridine-2'-thiol)-4H-1,2,4-triazole-3-
thiol 5 involves a multistep synthesis which was achieved by the initial esterification of 2-
mercaptopyridine-3-carboxylic acid 1 with methanol. Further, acid hydrazide 3 was formed
from 2-mercaptopyridine-3-methylcarboxylate 2 by reacting with hydrazine hydrate in
presence of absolute alcohol. Later, potassium dithiocarbazinate 4 was formed reacting acid
hydrazide 3 with carbon disulfide in presence of KOH at room temperature. The cyclo-
condensation of 4 with hydrazine hydrate in water resulted in 4-amino-5-(pyridine-2'-thiol)-
4H-1,2,4-triazole-3-thiol 5 as shown in Scheme 1. The synthesis of 3-(bromomethyl)-2-
chlorosubstituted quinolines 8a-m has been synthesized by known methods [20a] wherein 2-
chloro-6-substituted-quinoline-3-carbaldehyde 6a-m was prepared by Vilsmeier-Haack
reaction. The substituted carbaldehyde 6a-m was further reduced using NaBH4 to the
corresponding alcohol 7a-m. This later gave 3-(bromomethyl)-2-chloro-6-substituted
quinoline 8a-m when treated with phosphorous tribromide in DCM.
4

O
COOH
SH
COOCH3
SH
MeOH
NH2NH2
NH NH2
Conc. H2SO4
Abs. EtOH
N
SH
N
N
3
1
2
Abs. EtOH
CS2/KOH
N
N
O
SH
N
NH2NH2
H2O
NH NH
NH2
S
N
SH
N
SH
S
K
4
5
Scheme 1. Synthesis of precursor 4-amino-5-(pyridine-2'-thiol)-4H-1,2,4-triazole-3-thiol (5)
In order to obtain the desired tricyclic fused thiadiazepine derivatives, 4-amino-5-
(pyridine-2'-thiol)-4H-1,2,4-triazole-3-thiol 5 and 3-(bromomethyl)-2-chloro-substituted
quinolines 8a-m as precursors were treated under mild basic condition in presence of freshly
distilled dimethylformamide solution at room temperature. The reaction was carried and
completed under conventional method at room temperature for 4-6 h but the yields recorded
were relatively low, with intermediate formation and product formed consumed more time.
Looking into the relatively less yields, microwave irradiation method was employed with
equimolar ratios of 5 and 8a-m which were taken in a glass vial and irradiated by 120W
microwave radiation at 393.15K (120˚C) for 5-10 min. The yields increased and the time to
complete the reaction was less compared to the conventional norm to give 3-(2'-
mercaptopyridine)12H-dihydro-(alkyl)quinolino[4,3-e][1,2,4]-triazolo-[3,4-b][1,3,4]-
thiadiazepine 10a-m as shown in Scheme 2.
The reaction proceeded with the route that bromomethyl group on the quinoline was
substituted by the sulfur atom towards nucleophilic displacement which was evidenced by the
earlier reported methods [21]. Later the chlorine atom at the second position of the quinoline
undergoes cyclocondensation resulting in the desired novel tricyclic heterocycles 10a-m
without any traces of the by-products. In conventional method, it can be deduced that the
nucleophilic displacement by sulfur atom viz., also evidenced by the intermediate 9a-m which
was established by previous published reports [22]. Further, compounds 9a-m was stirred at
room temperature so as to get the final derivatives, resulting in no product formation. It was
learnt that the cyclocondensation requires high temperatures such that the C-Cl bond of the
quinoline cleaves to undergo cyclization to obtain the final compounds 10a-m [22].
5

Apparently, microwave irradiation method gave the final compounds 10a-m in flourishing
yields without any ado in the reaction pathway with minimal time.
R4
O
R4
NaBH4
MeOH
R3
R2
R3
R2
H
OH
N
Cl
RT
N
Cl
R1
R1
7a-m
6a-m
PBr3, DCM
0°C
H
R4
H
R4
N
N
MWI, 120W,
110-120°C,5-10 min
R3
R2
S
R3
R2
SH
Br
N
N
DMF, K2CO3
N
N
NH2
N
N
N
Cl
N
SH
H
R1
HS
R1
8a-m
Conventional
rt, 4-6 h
5
DMF, K2CO3
Reflux
N
DMF, K2CO3
10a-m
N
N
R4
R3
R2
N
S
N
HS
NH2
N
Cl
R1
9a-m
a) R1 = H, R2 = H, R3 = H, R4 = H; b) R1 = H, R2 = H, R3 = CH3 , R4 = H; c) R1 = H, R2 = H, R3 = OCH3 , R4 = H;
d) R1 = H, R2 = H, R3 = Cl, R4 = H; e) R1 = H, R2 = H, R3 = F, R4 = H ; f) R1 = H, R2 = H, R3 = Br, R4 = H;
g) R1 = H, R2 = CH3 , R3 = H, R4 = H; h) R1 = H, R2 = OCH3 , R3 = H, R4 = H; i) R1 = H, R2 = Cl, R3 = H, R4 = H;
j) R1 = CH3 , R2 = H, R3 = H, R4 = H; k) R1 = OCH3 , R2 = H, R3 = H, R4 = H; l) R1 = CH3 , R2 = H, R3 = H, R4 = CH3 ;
m) R1 = H, R2 = CH3 , R3 = H, R4 = CH3
Scheme 2. Cyclocondensation for the formation of 3-(2'-mercaptopyridine)-12H-dihydro-
(alkyl)quinolino[4,3-e][1,2,4]-triazolo-[3,4-b][1,3,4]-thiadiazepine 10a-m
The precursor 5 was confirmed by IR and GC-MS. The IR spectrum showed a
medium band at 3058 cm-1 which corresponds to the C-H stretching and band at 2770 cm-1
corresponds to S-H stretching. A band at 1333 cm-1 corresponds to C-N stretching and
adsorption band appears around 3280 cm-1 due to the N-H stretching of the triazol-thiol ring
(Spectrum no. 1, Supplementary Information). The mass spectral analysis has shown a
molecular ion peak at 225 which corresponds to the molecular mass of compound 5
(Spectrum no. 2, Supplementary Information). The 1H NMR of the isolated intermediate 9b
showed a singlet at 4.82 ppm attributed to thiomethylene (CH2S) group. A broad singlet at
5.14 ppm due to the NH2 group as well as signals appearing around 8.17-7.30 ppm
corresponds to the aromatic protons clearly confirms the depicted structure (Spectrum no. 3,
Supplementary Information). The 13C NMR spectrum demonstrates eighteen signals
corresponding carbons of the desired compound 9b (Spectrum no. 4, Supplementary
6

Information). The mass spectrum also showed the molecular ion peak at m/z 414 (M+)
corresponding to the molecular mass of intermediate 9b (Spectrum no. 5, Supplementary
Information).
All the previously unknown compounds 10a-m were characterized by IR, 1H, 13C NMR
and GC-MS analysis. The absorption band appears around 3354-3328 cm-1 and medium
intense band ranging around 3069-3031 cm-1 correspond to N-H and C-H stretching,
1
respectively. In case of H NMR, CH2S hydrogen of thiadiazepine appeared as a singlet
around 4.76-4.73 ppm. The signals appeared around 8.62-7.07 ppm was attributed to the
aromatic protons in all the compounds. In the case of 13C NMR spectral analyses, the signals
ranging from 34.97-34.13 ppm are assigned to the carbon of the methylene group viz.,
attached to the sulfur atom. The mass spectral analyses of all the compounds 10a-m have
shown the molecular ion peak corresponding to their molecular mass.
2.2. Pharmacology
2.2.1. In vitro antifungal activity
Learning the fact that quinoline-based scaffolds [20] tend to show outstanding
antifungal activities, the seven-membered derivatives were evaluated against fungi of clinical
importance, such as Candida albicans, Aspergillus fumigatus, Aspergillus flavus and
Aspergillus niger. Till date the majority of invasive fungal infections (IFIs), known to the
mankind are due to Candida and Aspergillus species. IFI is significantly observed in
immunocompromised patients, including those having AIDS and malignant diseases because
they are at a higher risk of developing an infection. Both the species Candida and
Aspergillus, are classified as an etiological agent of many opportunistic infections in
immunocompromised hosts [23]. Candida albicans and Aspergillus fumigatus are
humongously found in the environment and major cause of any infection followed by
Aspergillus flavus, Aspergillus niger. As a result, there is an emerging need for the constant
development of antifungals.
As the synthesized compounds 10a-m contain substitutions at 6th, 7th and 8th position of
quinoline and to properly analyze the observed antifungal activity in terms of MIC values, the
compounds were categorized into three groups: (i) group I contains the non-substituted and 6-
substituted quinolines; (ii) group II represents the 7-substituted quinolines and (iii) group III
comprises of 8-substituted quinolines as well as disubstituted (5,8- and 5,7-) quinolines. The
Zone of Inhibition (ZOI) and Minimum Inhibitory Concentrations (MIC) were summarized in
Table 1. Antifungal activities with MIC values correspond to the minimum concentration of
7

a compound necessary to inhibit the fungi growth was observed in the range from 0.20 to 25
mg/mL for all the synthesized derivatives.
All the synthesized compounds 10a-m inhibited growth of C. albicans, A. fumigatus, A.
flavus and A. niger at different concentration when compared to Fluconazole as a standard.
However, A. fumigatus was found to be more sensitive to all the tested compounds with MIC
value lower than 3.12 mg/mL than rest of pathogens. Group I compounds such as 10b, 10c,
10e and 10f showed maximum inhibition against A. fumigatus with MIC value of 0.20
mg/mL, whereas 10a (non-substituted) and 10d showed MIC value of 0.80 mg/mL. Further
the compounds of group I were also found to be sensitive towards fungal strains C. albicans,
A. flavus and A. niger with MIC values ranging from (25-0.40) mg/mL.
While considering group II, compounds 10g, 10h and 10i inhibited all the pathogens
under test with MIC values ranging from (25-0.80) mg/mL and amongst them compounds
10g, 10h and 10i displayed good inhibition against A. fumigatus. The MIC value of 0.80
mg/mL was recorded for 10g and 10i, whereas 10h exhibited minimum inhibitory effects of
1.60 mg/mL against A. fumigatus. Also, compounds under group III exhibited remarkable
activity against all test strains with MIC value from (3.12-0.40) mg/mL. Indeed compounds
10b (-CH3), 10c (-OCH3), 10e (-F) and 10f (-Br) comprising of mono-substitution at 6th
position of quinoline have shown activity increased by 4-folds comparatively. Whereas,
antifungal activity of compounds of group II and III (with substitution at 7th and 8th position)
were found to be less compared to group I. Disubstituted compounds 10l and 10m exhibited
good activity, but in particular against A. fumigatus displayed MIC value of 1.60 mg/mL with
negligible difference in activity when compared to mono-substituted derivatives implying
prominent inhibition.
8

Table 1. In vitro antifungal activities of 10a-m expressed in ZOI (mm) and MIC (mg/ml).
A. fumigatus
MIC (mg/mL)
0.80
A. flavus
A. niger
C. albicans
Category
ZOI (mm)
8
ZOI (mm)
10
ZOI (mm)
8
ZOI (mm)
10
Entry No.
MIC (mg/mL)
MIC (mg/mL)
0.40
MIC (mg/mL)
1.60
3.12
10a
10b
10c
10d
10e
10f
12
10
12
10
10
13
12
20
8
0.40
6.25
3.25
12.5
25
12
10
10
10
13
12
12
10
10
10
13
12
26
0.20
0.20
0.80
0.20
0.20
0.80
1.60
0.80
0.80
0.80
1.60
1.60
30
10
10
20
8
3.12
0.80
0.80
0.40
1.60
1.60
25
10
10
10
8
1.60
12.5
1.60
3.12
12.5
3.12
0.80
3.12
0.80
3.12
1.60
3.12
30
I
10
8
8
1.60
3.12
6.25
0.80
1.60
1.60
0.40
30
10
8
10g
10h
10i
II
10
10
10
10
8
1.60
3.12
3.12
0.40
0.80
30
10
10
8
10j
10
10
8
10k
10l
III
8
8
10
26
10m
Std*
24
26
* Standard: Fluconazole
9

2.2.2. Docking Studies against N-myristoyl transferase
To investigate the detailed intermolecular interactions between the ligand and the target
protein, Surflex-Dock program was used. Three-dimensional structure information on the
target protein was taken from the PDB entry 4CAW. Processing of the protein included the
deletion of the ligand and the solvent molecules as well as the addition of hydrogen atoms.
All the compounds were docked into the active site of N-myristoyl transferase (NMT) as
shown in Figure S24 (Supplementary Information). The predicted binding energies of the
compounds are listed in Table 2.
As depicted in the Figure 3(a) and (b), the compound 10b makes two hydrogen
bonding interactions at the active site of N-myristoyl transferase, among them a bonding
interaction from nitrogen atom of triazole ring with hydrogen of SER378 (-N----H-SER378,
1.96 Å), and remaining one more interaction from the nitrogen atom of pyridine ring with
hydrogen of ASN434 (-N----H-ASN434, 2.22 Å). The binding interaction of compound 10b
is depicted in Figure S25 (Supplementary Information).
(a)
(b)
Figure 3. Docked view of compound 10b at the active site of N-myristoyl transferase (PDB
ID: 4CAW)
As depicted in the Figure 4 (a) and (b), the compound 10f makes three hydrogen
bonding interactions at the active site of N-myristoyl transferase, among them two bonding
interactions from 1st nitrogen atom of triazole ring and 2nd nitrogen atom of triazole ring with
hydrogen atoms of SER378 (-N----H-SER378, 2.55 Å; 2.02 Å, respectively), and the
remaining one more interaction from the nitrogen atom of pyridine ring with hydrogen atom
of ASN434 (-N----H-ASN434, 2.09 Å). The binding interaction of compound 10f is depicted
in Figure S26 (Supplementary Information).
10

(a)
(b)
Figure 4. Docked view of compound 10f at the active site of N-myristoyl transferase (PDB
ID: 4CAW)
The binding interaction of myristoyl CoA and a pyrazole sulphonamide ligand with the
active sites of N-myristoyl transferase shows three bonding interactions and the docked view
of the same has been depicted in Figure S27 (Supplementary information). To comparatively
evaluate the binding interactions of the compounds under investigation, fluconazole was also
docked into the active site of the enzyme. The binding interaction of fluconazole with N-
myristoyl transferase shows two bonding interactions and the docked view of the same have
been depicted in Figure S28 (Supplementary information). All the compounds showed
consensus score in the range 22.38- 16.77, indicating the summary of all forces of interaction
between ligands and the enzyme.
The compounds 10a-m also showed similar kind of interaction with amino acid residue
(SER378) as that of myristoyl CoA with pyrazole sulphonamide ligand and fluconazole. This
indicates that compounds 10a-m preferentially bind to N-myristoyl transferase in comparison
to the myristoyl CoA with pyrazole sulphonamide ligand and fluconazole as shown in Table
2. The interactions of hydrophobic and hydrophilic amino acid residues that are surrounded to
compounds 10b and 10f are shown in the Figure S29 (Supplementary information).
11

Table 2. Surflex docking scores (kJ/mol) of the compounds 10a-m on N-myristoyl transferase (PDB ID: 4CAW)
Compounds
10a
C Scorea
Crash Scoreb Polar Scorec
D Scored
-469.18
-461.77
-545.50
PMF Scoree
G Scoref
-712.58
-708.02
-856.58
Chem Scoreg
21.67
-2.30
-2.17
-3.64
13.13
12.76
4.18
-222.78
-109.18
10b*
21.84
-272.81
-112.78
10c
19.41
-181.56
-94.70
10d
10e
16.77
19.66
-3.13
-7.44
4.05
4.39
-505.94
-569.92
-234.47
-304.49
-819.20
-868.94
-108.54
-126.39
10f*
10g
21.21
19.62
19.37
18.28
18.86
20.16
22.38
19.37
23.43
38.57
-2.59
-2.05
-3.47
-1.88
-10.41
-2.17
-2.42
-4.72
-3.93
-6.90
11.84
4.93
4.64
4.93
5.35
4.97
12.88
11.46
1.84
4.10
-474.99
-480.87
-496.88
-480.90
-600.53
-501.46
-478.33
-474.59
-515.20
-744.53
-188.93
-233.83
-301.89
-232.19
-305.12
-266.15
-290.25
-279.85
-365.26
-321.93
-755.38
-775.47
-793.90
-758.26
-1050.03
-785.16
-750.27
-787.69
-828.10
-1310.87
-119.21
-104.08
-106.78
-104.13
-120.86
-105.94
-116.39
-124.10
-42.88
10h
10i
10j
10k
10l
10m
Fluconazole
Ligand**
-161.47
12

* Asterisk indicates the compound with highest CScore
** Myristoyl CoA and pyrazole sulphonamide ligand
a CScore (Consensus Score) integrates a number of popular scoring functions for ranking the affinity of ligands
bound to the active site of a receptor and reports the output of total score.
b Crash-score revealing the inappropriate penetration into the binding site. Crash scores close to 0 are favorable.
Negative numbers indicate penetration.
c Polar indicating the contribution of the polar interactions to the total score. The polar score may be useful for
excluding docking results that make no hydrogen bonds.
d D-score for charge and van der Waals interactions between the protein and the ligand.
e PMF-score indicating the Helmholtz free energies of interactions for protein-ligand atom pairs (Potential of
Mean Force, PMF).
f G-score showing hydrogen bonding, complex (ligand-protein), and internal (ligand-ligand) energies.
g Chem-score points for H-bonding, lipophilic contact, and rotational entropy, along with an intercept term.
2.2.3. In vitro anticancer screening at a single high dose concentration (10-5)
All the synthesized compounds 10a-m have been registered under Development
Therapeutics Program (DTP) of National Cancer Institute (NCI), USA and ten (10) of them
were selected by for anticancer screening in a single high dose (10-5 M) concentration against
full 60 human cancer cell lines. The output from the single dose screen is reported as a graph
of mean growth percent of the treated cells and is available for analysis by the COMPARE
program [24]. This allows detecting both growth inhibition values (between 0 and 100) and
cytotoxicity values (less than 0).
The compounds with registration numbers 10a (NSC: D-812121/1), 10b (NSC: D-
812122/1), 10c (NSC: D-812128/1), 10d (NSC: D-812125/1), 10e (NSC: D-812126/1), 10f
(NSC: D-812127/1), 10g (NSC: D-812123/1), 10h (NSC: D-812129/1), 10j (NSC: D-
812124/1), 10m (NSC: D-812130/1) have been screened on all the 60 human cancer cell lines
methodized into nine sub-panels derived from nine different human cancer types: leukemia,
non-small cell lung, cancer, colon, CNS, melanoma, ovarian, renal, prostate and breast cancer
cell lines. Amongst all the 10 compounds, 10b, 10c, 10e and 10f have shown good to
moderate anticancer activity against some cell lines. The percentage growth inhibition (GI %)
of the treated cells at 10-5 M concentration for compound 10b (with 6-tolyl substitution,
Table 3) against Renal Cancer cell line UO-31 (GI % 35.71). Compound 10c (with 6-
13

methoxy substitution, Table 3) against Renal Cancer cell line UO-31 (GI % 22.98). Compound 10e (with 6-fluoro substitution, Table 3) against
CNS cancer cell line SNB-75 (GI % 24.15) and Renal Cancer cell line UO-31 (GI % 17.99). Compound 10f (with 6-bromo substitution, Table
3) against Ovarian Cancer cell line IGROV1 (GI % 33.52) and Renal Cancer cell line UO-31 (GI % 33.88). Subsequently, all the four
compounds have been observed to be potent inhibitors against Renal Cancer UO-31 cell line. The anticancer assay of compounds 10b, 10c, 10e
and 10f are given in Figure S30-S33 (Supplementary Information).
Table 3. Growth inhibition (GI %) in single dose assay (10-5 M) concentration for compound 10b (NSC: D-812122/1), 10c (NSC: D-812128/1),
10e (NSC: D-812126/1) and 10f (NSC: D-812127/1)
Growth Inhibition (GI%)
Panel/Cell line
10b
10c
10e
10f
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
-4.36423
-2.39317
6.95825
8.057758
0.509507
16.66399
-3.25992
-2.04204
-2.17376
-2.64882
-1.96689
-5.89367
1.060425
-5.9901
-6.29652
-1.1685
4.51364
9.6084
3.410641
-2.82145
7.269735
3.086242
3.007055
14.12063
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
0.253275
-1.89899
4.177177
-2.02958
8.475786
1.422126
0.074332
0.834455
-3.07748
4.885924
-8.62947
1.799069
4.012532
-0.77109
-1.18856
-6.54895
-7.70627
5.831781
6.742778
21.9441
6.735636
7.256466
-5.7518
-2.27814
16.22565
6.550636
10.8701
4.755794
7.502436
7.354108
-2.44984
-5.38917
14

NCI-H522
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
16.32633
13.10395
13.22291
19.45808
-10.2473
-6.96855
7.463411
-4.78548
-7.93298
0.311218
-2.21767
-10.5431
-6.04888
4.693211
-12.9491
-1.97249
-3.2342
-15.2225
-3.29968
7.31029
-3.64312
-8.20401
-6.23708
6.522216
-12.2016
-5.94971
14.91311
-2.01896
-6.56187
-2.74045
14.22497
0.794132
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
10.48626
-8.2453
2.65255
9.285093
8.568505
-11.9518
3.368879
-11.5524
5.357517
3.980447
12.55569
-6.29559
1.253602
-4.98941
4.339515
1.906113
24.15288
-7.4708
-12.2016
-5.94971
14.91311
-2.01896
-6.56187
-2.74045
SNB-75
U251
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian Cancer
IGROV1
3.449654
2.344674
6.992669
11.80604
-0.30496
-2.97152
3.648293
-3.3281
-3.57244
0.598851
-2.53203
-0.35815
1.879687
0.09562
0.4343
3.875158
-12.4711
0.959004
-3.40943
1.710061
-1.59395
3.590439
-5.09041
8.338225
7.460168
11.48716
6.62614
1.24726
4.960751
-0.47343
7.808012
-1.64698
17.79054
-2.59949
8.692462
13.0134
17.34889
0.516032
-9.23211
5.381781
-2.96314
9.762811
13.29674
-8.6373
8.53421
33.52549
1.74446
OVCAR-3
OVCAR-4
10.94814
15

OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal Cancer
786-0
0.540403
-0.14464
6.604951
11.19577
9.168406
-3.30405
0.603359
1.769961
4.586821
-4.53389
-3.02793
13.14165
7.880955
-2.55816
2.443624
14.07573
1.293912
-1.02742
2.568467
21.27639
7.192545
-5.29004
35.71891
6.734633
11.72327
2.817682
13.16528
2.171497
5.266687
22.98544
10.3146
7.897646
6.010834
16.21725
1.789203
-10.302
4.253191
6.452962
10.59739
18.74883
6.856853
-7.26562
33.8843
A498
ACHN
CAKI-1
SN12C
TK-10
UO-31
17.99574
Prostate Cancer
PC-3
4.369656
-12.5736
-3.33395
-4.32598
8.47208
-13.6051
5.068147
-7.81167
DU-145
Breast Cancer
MCF7
5.579997
-6.10736
1.334488
5.334221
MDA-MB-231/ATCC
6.579608
3.739711
2.59508
4.836403
-6.53336
-1.91747
6.19185
2.46549
5.588827
3.719795
2.256753
15.88426
-4.7392
20.04835
6.249081
4.800568
20.2163
HS 578T
BT-549
T-47D
9.198604
-10.6383
MDA-MB-468
-10.8225
16

2.2.4. Docking Studies against h-MetAP2
Molecular docking study was performed to support the interaction and preferred
binding mode of the compounds 10a-m with the enzyme. All the inhibitors were docked into
the active site of enzyme as shown in Figure S34 (Supplementary Information). The predicted
binding energies of the compounds are listed in Table 4. Amongst all the compounds 10a-m,
10b, 10c, 10e and 10f displayed highest C-score values with possible interactions and the
details of 10c and 10e have been mentioned below.
As depicted in Figure 5 (a) and (b) compound 10c makes two hydrogen bonding
interactions at the active site of the enzyme (PDB ID: 2EA4), NH hydrogen of thiadiazepino
ring makes hydrogen bonding interaction with nitrogen of HIS231 (NH----N-HIS231, 2.86
Å) and the nitrogen atom of triazole ring makes hydrogen bonding interaction with hydrogen
of HIS382 (N----H-HIS382, 2.24 Å). The binding interaction of compound 10c is depicted in
Figure S35 (Supplementary Information).
(a)
(b)
Figure 5. Docked view of compound 10c at the active site of the enzyme PDB: 2EA4
Also as depicted in Figure 6 (a) and (b), compound 10e makes two hydrogen bonding
interactions at the active site of the enzyme (PDB ID: 2EA4), hydrogen of NH of
thiadiazepino ring makes hydrogen bonding interaction with nitrogen of HIS231 (NH----N-
HIS231, 2.76 Å) and the nitrogen atom of triazole ring makes hydrogen bonding interaction
with hydrogen of HIS382 (N----H-HIS382, 2.23 Å). The binding interaction of compound
10e is depicted in Figure S36 (Supplementary Information).
17

(a)
(b)
Figure 6. Docked view of compound 10e at the active site of the enzyme PDB: 2EA4
The binding interaction of ligand with h-MetAP2 active sites shows four bonding interactions
and the docked view of the same has been depicted in Figure S37 (Supplementary
Information). The compounds have same H-bonding interaction with same amino acid
HIS231 as that of ligand. The interactions of hydrophobic and hydrophilic amino acid
residues that are surrounded to compounds 10c and 10e are shown in the Figure S38
(Supplementary Information).
All the compounds 10a-m under investigation showed the C-score in the range of
26.35-12.38 showing the interaction of the compounds with h-MetAP2. The charge and van
der Waals interactions ranged from -423.09 to -355.98. Whereas, the Helmholtz free energies
of interactions of protein and compounds ranged from -445.86 to -579.30. While the
hydrogen bonding, complex (ligand-protein), and internal (ligand-ligand) energies range from
-667.87 to -961.99. Also the H-bonding, lipophilic contact, and rotational entropy, along with
an intercept term indicated values ranging from -91.97 to -140.21. All these values infer that
the compounds interact with enzyme h-MetAP2 by forming hydrogen bonds in stable
conformations as compared to the ligand.
18

Table 4. Surflex Docking score (kJ/mol) of the compounds for h-MetAP2 complexed with A797859 (PDB ID: 2EA4)
Crash Scoreb
-5.31
Compound
10a
C Scorea
15.98
24.81
26.35
21.88
25.89
24.39
12.38
21.04
14.51
19.45
19.53
23.51
14.64
25.64
Polar Scorec
4.39
D Scored PMF Scoree
G Scoref
-850.77
-848.47
-838.01
-923.80
-862.89
-934.68
-961.99
-871.69
-731.88
-936.01
-667.87
-872.17
-842.05
-755.35
Chem Scoreg
-117.56
-124.16
-124.84
-136.48
-128.90
-138.36
-107.15
-132.69
-106.81
-135.41
-140.21
-131.87
-91.97
-355.98
-385.88
-372.50
-389.77
-363.14
-406.95
-423.09
-412.76
-357.86
-398.47
-381.53
-384.56
-379.06
-488.02
-450.34
-542.30
-557.07
-554.13
-539.84
-548.29
-483.82
-579.30
-445.86
-557.81
-446.89
-567.88
-497.43
-566.36
10b
10c*
-5.81
14.81
11.12
7.94
-3.97
10d
-5.69
10e*
10f
-4.01
11.21
12.00
0.37
-6.86
10g
-8.24
10h
-5.27
7.07
10i
-11.29
-6.77
13.80
6.31
10j
10k
-3.26
15.77
11.00
3.89
10l
-4.60
10m
Ligand**
-5.48
20.20
-9.41
-141.63
* Asterisk indicates the compound with highest C Score
** 2-(2-Aminoethoxy)-3-Ethyl-6-{[(4-Fluorophenyl)sulfonyl]amino}benzoic Acid
19

2.2.5. Structure Activity Relationship (SAR)
The structural requirements for the molecule are explored by structural activity
relationship, where it is relevant for the biological activity to be dependent on its
physicochemical properties. The structure activity relationship (SAR) was proposed looking
into the observations made from the molecular docking, reported biological activities as well
as the pharmacokinetic studies representated in Figure 7.
Thiadiazepine backbone
as a pharmacophore
Quinoline scaffold
behaves as a head group
Binding sites which interact with
the active site of the enzyme pocket
R4
R3
S
N
Modification of substitutents
on quinoline for better
biological activities
N
R2
N
N
N
H
R1
HS
N
Mercaptopyridine
acts a pendent group
Substituent effect is engaging
the biological activities in the
increasing order of R3 > R2 = R1 = H > R4
θ°
HS
= The motif tilts out the plane making
available for hydrophilic interactions
N
Figure 7. SAR of the synthesized compounds
i) Thiadiazepine ring acts as a pharmacophore for its bioactivity forming binding sites which
interacts with the active site of the enzyme pocket under investigation.
ii) The efficacy of quinoline and its scaffolds improves the pharmacological as well as
pharmacokinetic properties displaying the drug-like properties.
iii) The modification of substituents on the quinoline have been able to enhance the biological
activities in the order R3 > R2 = R1 = H > R4, where the maximum bioactivity was observed
for electron withdrawing atoms such as -F, -Br and electron donating groups like -CH3, -
OCH3.
iv) The mercaptopyridine ring attached to the 1,2,4-triazole ring serves as a pendent group.
This ring tilts the plane adjacent to the overall scaffold making available the nitrogen atoms
of the triazole ring for the binding interactions into the active site of the enzyme and also for
the hydrophilic interactions. The computational and structure activity relationship studies
were further validated by in vitro anticancer screening against 60 cell lines and antifungal
studies against four fungal pathogens.
20

2.2.6. In silico pharmacokinetic evaluation
Drug discovery is one of the challenging fields till recent years and this process was
improved based on the high-throughput animal models and also evaluating the toxicity of the
drug candidate. The drug likeliness and pharmacokinetic parameters for the synthesized
compounds 10a-m was obtained by Molinspiration online provider toolkit [25].
Physicochemical properties of compounds 10a-m as molecular weight (MW), number of
hydrogen bond donors (HBD), hydrogen bond acceptors (HBA) and number of rotatable
bonds (rotb) were determined which confirmed Lipinski’s rule of five (RO5) [25]. According
to the results summarized in Table 5, the compounds under investigation are suitable for oral
administration and have a MW < 500, logPo/w < 5, donor HB ≤ 5 and acceptor HB ≤10. The
absorption percentage was measured from TPSA, % Abs =109 - [0.345 × TPSA] [26].
Looking into the known drug candidates and their toxicity by toxic fragments or
chemical structure, a web server Protox estimates rodent oral toxicity [27]. The toxic
quantities are often expressed in LD50 values and the predicted LD50 (lethal dose) for
synthesized compounds 10a-m is equal to 1000 mg/kg as indicated in Table S39
(Supplementary Information). Also all the compounds are classified under class 4 toxicity
category with no toxic fragments.
21

Table 5. Pharmacokinetic parameters evaluation of the compounds 10a-m
Entry No.
MWa
miLogPb
TPSAc
n Atoms n ONd n OHNHe
n violation
n rotbf
% ABSg
364.46
378.49
394.49
398.90
382.45
443.36
378.49
394.49
398.90
378.49
394.49
392.51
392.51
3.50
3.93
3.54
4.16
2.67
4.29
3.93
3.54
4.16
3.90
3.51
4.61
4.61
68.53
68.53
77.76
68.53
68.53
68.53
68.53
77.76
68.53
68.53
77.76
68.53
68.53
25
26
27
26
26
26
26
27
26
26
27
27
27
6
6
7
6
6
6
6
7
6
6
7
6
6
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
2
1
1
1
1
2
1
1
2
1
1
85.35
85.35
82.17
85.35
85.35
85.35
85.35
82.17
85.35
85.35
82.17
85.35
85.35
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
a Molecular weight (MW), b Logarithm of partition coefficient between n-octanol and water (miLogP), c Topological polar surface area (TPSA), d Number of hydrogen bond
acceptors (n-ON), e Number of hydrogen bond donors (n-OHNH), f Number of rotatable bonds (n-rotb), g Percentage of absorption (%ABS).
22

3. Conclusions
A new cyclocondensation route was developed for the synthesis of fused seven-
membered bioactive triazolothiadiazepinyl quinolines. The distinctive route was carried out
in mild conditions at room temperature as well as under microwave irradiation technique. The
microwave method was hassle-free, easy and occurred rapidly. However, it is a one-pot
procedure for the preparation of seven-membered ring and the reaction was not interrupted by
any intermediate rather the reaction preceded in a one-attempt.
All the functionalized derivatives were evaluated for the in vitro antifungal assay,
displaying remarkable activities against the test panels of fungal strains compared with
standard drug Fluconazole. The compounds 10b, 10c, 10e and 10f were considered promising
due to notable inhibitory effects on Aspergillus fumigatus with MIC value of 0.20 mg/mL.
While all compounds 10a-m were subjected to docking studies against enzyme N-myristoyl
transferase, compounds 10b and 10f exhibited remarkable C-score values with good
interactions. Furthermore, all the compounds were screened for the anticancer activity against
NCI-60 Human tumor cell line at a single high dose (10-5 M) concentration assay. The
quinoline conjugates 10b, 10c, 10e and 10f were found to be moderately effective against the
Renal Cancer cell line UO-31. Accounting for the anticancer activity, compounds 10a-m
were docked against the active site of the enzyme MetAP-2 revealing that compounds 10c
and 10e displayed good interactions. Hence, it could be inferred that the compounds 10b and
10f are inhibitors of NMT whereas; compounds 10c and 10e are inhibitors of MetAP-2. The
substituents effect on quinoline as well as the interaction of enzymes with the thiadiazepine
and triazole scaffolds was established through SAR study. These results were also validated
by the pharmacokinetic evaluation revealing that physiochemical properties were in good
agreement with the Lipinski’s rule of five. In future, collectively all these inhibitors 10b, 10c,
10e and 10f could demonstrate and develop the levels of expression of MetAP-2 and NMT in
case of Renal Cancer cell line.
4. Experimental
4.1. General procedure for the synthesis of 3-(2'-mercaptopyridine)-12H-dihydro-
(alkyl)quinolino[4,3-e][1,2,4]-triazolo-[3,4-b][1,3,4]-thiadiazepine (10a-m)
Conventional Method
Equimolar ratios of 4-amino-5-(pyridine-2'-thiol)-4H-1,2,4,-triazole-3-thiol 5 (0.010
mol) and 3-(bromomethyl)-2-chloro-substituted quinoline 8a-m (0.010 mol) and anhydrous
potassium carbonate (0.020 mol) in DMF (10.0 ml) was stirred at room temperature. After 4-
23

6 hours of the completion of the reaction viz., monitored by TLC, the reaction mixture was
filtered to remove the K2CO3, washed with small portions of DMF and crushed ice was added
to the filtrate. The resulting precipitate was filtered and washed with ice cold water,
recrystallised with ethyl acetate and dried.
Microwave method
The reaction was performed in a single mode Discover SP (CEM Corporation)
microwave synthesizer with controlled irradiation up to 300 watts having a magnetic stirrer.
An equimolar ratio of compounds 5, 8a-m and anhydrous potassium carbonate were taken in
a sealed glass tube vial (30.0 ml) in DMF (10.0 ml). The sealed vessel with reaction mixture
was pre-stirred for 60 sec at room temperature. Further, the reaction mixture was irradiated
by 120W microwave radiation at 393.15K (120˚C) for 5 min at medium stirring. The reaction
temperatures were monitored with an IR sensor equipped outside of the reactor. Some of the
derivatives consumed more time when compared to others and collectively all the reactions
were completed in 5-10 min when irradiated at 393.15K. After completion of the reaction
(TLC), the reaction mixture was cooled, filtered and quenched in crushed ice. The resulting
precipitate was filtered and washed with ice cold water, recrystallised with ethyl acetate and
dried (Scheme 2).
4.2. Spectral Characterization
4.2.1. 3-(2'-Mercaptopyridine)-12H-dihydro-quinolino[4,3-e][1,2,4]-triazolo-[3,4-b]
[1,3,4]-thiadiazepine (10a)
Yield: 88%, colour: light yellow solid, m.p.: 167-169˚C; IR (νmax, cm-1): 3344 (N-H), 3028
1
(C-H), 2918, 1587, 1477. H NMR (400 MHz, CDCl3, δ ppm): 8.66 (1H, dd, J = 6.4 Hz,
pyridine C6H), 8.38 (1H, s, ArH), 7.85 (1H, dd, J = 8.0 Hz, pyridine C4H), 7.68 (1H, s, ArH),
7.43 (1H, dd, J = 8.0 Hz, pyridine C5H), 7.32-7.24 (3H, m, ArH), 4.64 (2H, s, CH2). 13C
NMR (100 MHz, CDCl3, δ ppm): 160.66, 152.44, 151.78, 150.36, 149.09, 144.76, 138.71,
138.01, 137.45, 132.76, 128.85, 127.32, 126.77, 126.67, 120.98, 118.64, 34.93. Calcd Mass
364.06 MS (m/z): 364 [M+]. Anal. Calcd for C17H12N6S2: C, 56.03; H, 3.32; N, 23.06.
Found: C, 56.11; H, 3.37; N, 23.02.
4.2.2. 3-(2'-Mercaptopyridine)-12H-dihydro-(9-methyl)quinolino[4,3-e][1,2,4]-triazolo-
[3,4-b][1,3,4]-thiadiazepine (10b)
Yield: 90%, colour: light yellow solid, m.p.: 142-144˚C; IR (νmax, cm-1): 3340 (N-H), 3045
1
(C-H), 2922, 1587, 1472. H NMR (400 MHz, DMSO-d6, δ ppm): 8.62 (1H, dd, J = 6.4 Hz,
24

pyridine C6H), 8.41 (1H, s, ArH), 7.89 (1H, dd, J = 8.0 Hz, pyridine C4H), 7.75 (1H, s, ArH),
7.67 (1H, dd, J = 8.0 Hz, pyridine C5H), 7.29-7.26 (2H, m, ArH), 4.67 (2H, s, CH2), 2.46
(3H, s, CH3). 13C NMR (100 MHz, DMSO-d6, δ ppm): 158.97, 152.31, 151.84, 150.43,
149.04, 144.80, 138.69, 138.03, 137.37, 132.89, 128.99, 127.26, 126.73, 126.60, 120.93,
118.68, 34.97, 21.04. Calcd Mass 387.47 MS (m/z): 387 [M+]. Anal. Calcd for C18H14N6S2:
C, 57.12; H, 3.73; N, 22.21. Found: C, 57.17; H, 3.79; N, 22.25.
4.2.3. 3-(2'-Mercaptopyridine)-12H-dihydro-(9-methoxy)quinolino[4,3-e][1,2,4]-triazolo-
[3,4-b][1,3,4]-thiadiazepine (10c)
Yield: 89%, colour: white solid, m.p.: 172-174˚C; IR (νmax, cm-1): 3352 (N-H), 3069 (C-H),
1
2917, 1588, 1474. H NMR (400 MHz, CDCl3, δ ppm): 8.56 (1H, dd, J = 6.4 Hz, pyridine
C6H), 8.47 (1H, s, ArH ), 8.02 (1H, dd, J = 6.0 Hz, pyridine C4H), 7.89 (1H, dd, J = 9.2 Hz,
ArH), 7.36 (1H, dd, J = 6.0 Hz, pyridine C5H), 7.11-7.08 (2H, m, ArH), 4.73 (2H, s, CH2),
13
3.90 (3H, s, OCH3). C NMR (100 MHz, CDCl3, δ ppm): 164.04, 159.52, 158.40, 150.90,
147.63, 143.26, 138.64, 135.93, 129.56, 128.22, 127.99, 123.66, 123.43, 118.30, 116.99,
105.22, 55.68, 34.28. Calcd Mass 394.47 MS (m/z): 394 [M+]. Anal. Calcd for C18H14N6OS2:
C, 54.81; H, 3.58; N, 21.30. Found: C, 54.76; H, 3.62; N, 21.28.
4.2.4. 3-(2'-Mercaptopyridine)-12H-dihydro-(9-chloro)quinolino[4,3-e][1,2,4]-triazolo-
[3,4-b][1,3,4]-thiadiazepine (10d)
Yield: 87%, colour: chrome yellow solid, m.p.: 174-176 ˚C; IR (νmax, cm-1): 3353 (N-H),
1
3058 (C-H), 2921, 1586, 1475. H NMR (400 MHz, CDCl3, δ ppm): 8.59 (1H, dd, J = 6.4
Hz, pyridine C6H), 8.52 (1H, s, ArH), 8.16 (1H, dd, J = 8.0 Hz, pyridine C4H), 7.94 (1H, dd,
J = 8.8 Hz, ArH), 7.83 (1H, dd, J = 8.8 Hz, pyridine C5H), 7.22-7.13 (2H, m, ArH), 4.75
13
(2H, s, CH2). C NMR (100 MHz, CDCl3, δ ppm): 164.97, 160.15, 151.76, 151.17, 140.45,
138.59, 136.26, 134.32, 130.81, 130.14, 129.96, 129.75, 128.44, 128.06, 121.66, 118.34,
34.79. Calcd Mass 398.02 MS (m/z): 400 [M+2], 398 [M+]. Anal. Calcd for C17H11N6S2Cl:
C, 51.19; H, 2.78; N, 21.07. Found: 51.22; H, 2.81; N, 21.09.
4.2.5. 3-(2'-Mercaptopyridine)-12H-dihydro-(9-fluoro)quinolino[4,3-e][1,2,4]-triazolo-
[3,4-b][1,3,4]-thiadiazepine (10e)
Yield: 84%, colour: yellow solid, m.p.: 148-150 ˚C; IR (νmax, cm-1): 3338 (N-H), 3048 (C-H),
1
2920, 1582, 1474. H NMR (400 MHz, CDCl3, δ ppm): 8.62 (1H, dd, J = 6.4 Hz, pyridine
C6H), 8.50 (1H, s, ArH), 8.14 (1H, dd, J = 8.0 Hz, pyridine C4H), 7.92 (1H, dd, J = 8.0 Hz,
25

ArH), 7.89 (1H, dd, J = 8.8 Hz, pyridine C5H), 7.17-7.07 (2H, m, ArH), 4.76 (2H, s, CH2).
13C NMR (100 MHz, CDCl3, δ ppm): 164.91, 159.54, 151.21, 150.67, 138.67, 138.45,
135.89, 134.28, 130.64, 130.23, 129.87, 129.78, 128.36, 128.09, 121.34, 118.56, 34.67. Calcd
Mass 382.05 MS (m/z): 382 [M+]. Anal. Calcd for C17H11N6S2F: C, 53.39; H, 2.90; N, 21.97.
Found: C, 53.42; H, 2.89; N, 21.94.
4.2.6. 3-(2'-Mercaptopyridine)-12H-dihydro-(9-bromo)quinolino[4,3-e][1,2,4]-triazolo-
[3,4-b][1,3,4]-thiadiazepine (10f)
Yield: 89%, colour: yellow solid, m.p.:177-179˚C; IR (νmax, cm-1): 3336 (N-H), 3054 (C-H),
1
2922, 1585, 1470. H NMR (400 MHz, CDCl3, δ ppm): 8.57 (1H, dd, J = 6.4 Hz, pyridine
C6H), 8.49 (1H, s, ArH), 8.04 (1H, dd, J = 8.0 Hz, pyridine C4H), 7.88 (1H, dd, J = 8.8 Hz,
ArH), 7.79 (1H, dd, J = 8.8 Hz, pyridine C5H), 7.12-7.08 (2H, m, ArH), 4.73 (2H, s, CH2).
13C NMR (100 MHz, CDCl3, δ ppm): 163.91, 159.58, 151.06, 150.97, 138.70, 138.39,
135.96, 134.32, 130.79, 130.04, 129.94, 129.84, 128.16, 128.10, 121.52, 118.29, 34.13. Calcd
Mass 441.34 MS (m/z): 443 [M+2], 441 [M+]. Anal. Calcd for C17H11N6S2Br: C, 46.06; H,
2.50; N, 18.96. Found: C, 46.12; H, 2.45; N, 18.95.
4.2.7. 3-(2'-Mercaptopyridine)-12H-dihydro-(8-methyl)quinolino[4,3-e][1,2,4]-triazolo-[3,4-
b][1,3,4]-thiadiazepine (10g)
Yield: 86%, colour: white solid, m.p.: 166-168˚C; IR (νmax, cm-1): 3354 (N-H), 3054 (C-H),
1
2924, 1589, 1474. H NMR (400 MHz, CDCl3, δ ppm): 8.58 (1H, dd, J = 6.4 Hz, pyridine
C6H), 8.48 (1H, s, ArH ), 8.09 (1H, dd, J = 8.0 Hz, pyridine C4H), 7.63-7.51 (2H, m, ArH),
7.44-7.38 (1H, m, ArH), 7.21-7.08 (1H, m, pyridine C5H), 4.78 (2H, s, CH2), 2.34 (3H, s,
CH3). 13C NMR (100 MHz, CDCl3, δ ppm): 151.93, 143.56, 139.88, 139.34, 138.56, 136.45,
135.87, 131.56, 130.83, 129.77, 127.56, 127.12, 125.56, 125.34, 118.92, 118.29, 34.54,
24.34. Calcd Mass 378.47 MS (m/z): 378 [M+]. Anal. Calcd for C18H14N6S2: C, 57.12; H,
3.73; N, 22.21. Found: C, 57.18; H, 3.76; N, 22.23.
4.2.8.
3-(2'-Mercaptopyridine)-12H-dihydro-(8-methoxy)quinolino[4,3-e][1,2,4]-triazolo-
[3,4-b][1,3,4]-thiadiazepine (10h)
Yield: 88%, colour: off white solid, m.p.: 154-156˚C; IR (νmax, cm-1): 3337 (N-H), 3053 (C-
1
H), 2935, 1585, 1472. H NMR (400 MHz, CDCl3, δ ppm): 8.55 (1H, dd, J = 6.0 Hz,
pyridine C6H), 8.49 (1H, s, ArH ), 8.02 (1H, dd, J = 6.0 Hz, pyridine C4H), 7.88 (1H, dd, J =
9.2 Hz, ArH), 7.36 (1H, dd, J = 6.4 Hz, pyridine C5H), 7.15-7.07 (2H, m, ArH), 4.73 (2H, s,
26

CH2), 3.90 (3H, s, OCH3). 13C NMR (100 MHz, CDCl3, δ ppm): 164.06, 159.55, 158.38,
150.87, 147.65, 143.28, 138.68, 135.96, 129.51, 128.20, 127.96, 123.65, 123.44, 118.28,
116.99, 105.29, 55.64, 34.33. Calcd Mass 394.47 MS (m/z): 394 [M+]. Anal. Calcd for
C18H14N6OS2: C, 54.81; H, 3.58; N, 21.30. Found: C, 54.84; H, 3.56; N, 21.34.
4.2.9. 3-(2'-Mercaptopyridine)-12H-dihydro-(8-chloro)quinolino[4,3-e][1,2,4]-triazolo-[3,4-
b][1,3,4]-thiadiazepines (10i)
Yield: 85%, colour: yellow solid, m.p.: 168-170˚C; IR (νmax, cm-1): 3345 (N-H), 3066 (C-H),
1
2927, 1587, 1474. H NMR (400 MHz, CDCl3, δ ppm): 8.59 (1H, dd, J = 6.4 Hz, pyridine
C6H), 8.52 (1H, s, ArH), 8.22 (1H, dd, J = 8.0 Hz, pyridine C4H), 7.92 (1H, dd, J = 8.8 Hz,
ArH), 7.80 (1H, dd, J = 8.0 Hz, pyridine C5H), 7.24-7.11 (2H, m, ArH), 4.74 (2H, s, CH2).
13C NMR (100 MHz, CDCl3, δ ppm): 163.89, 159.55, 151.11, 150.77, 138.56, 138.32,
135.78, 134.33, 130.66, 130.02, 129.98, 129.87, 128.21, 128.16, 121.43, 118.09, 34.88. Calcd
Mass 398.02 MS (m/z): 400 [M+2], 398 [M+]. Anal. Calcd for C17H11N6S2Cl: C, 51.19; H,
2.78; N, 21.07. Found: C, 51.24; H, 2.75; N, 21.05.
4.2.10. 3-(2'-Mercaptopyridine)-12H-dihydro-(7-methyl)quinolino[4,3-e][1,2,4]-triazolo-
[3,4-b][1,3,4]-thiadiazepine (10j)
Yield: 92%, colour: white solid, m.p.: 158-160˚C; IR (νmax, cm-1): 3348 (N-H), 3031 (C-H),
1
2922, 1586, 1474. H NMR (400 MHz, CDCl3, δ ppm): 8.56 (1H, dd, J = 6.4 Hz, pyridine
C6H), 8.47 (1H, s, ArH ), 8.04 (1H, dd, J = 8.0 Hz, pyridine C4H), 7.68-7.54 (2H, m, ArH),
7.47-7.42 (1H, m, ArH), 7.10-7.07 (1H, m, pyridine C5H), 4.76 (2H, s, CH2), 2.74 (3H, s,
CH3). 13C NMR (100 MHz, CDCl3, δ ppm): 150.93, 142.61, 139.94, 139.28, 138.58, 136.53,
135.92, 130.90, 130.85, 129.35, 127.32, 127.17, 125.79, 125.72, 118.99, 118.26, 34.38,
17.74. Calcd Mass 378.47 MS (m/z): 378 [M+]. Anal. Calcd for C18H14N6S2: C, 57.12; H,
3.73; N, 22.21. Found: C, 57.16; H, 3.75; N, 22.28.
4.2.11. 3-(2'-Mercaptopyridine)-12H-dihydro-(7-methoxy)quinolino[4,3-e][1,2,4]-triazolo-
[3,4-b][1,3,4]-thiadiazepine (10k)
Yield: 92%, colour: off white solid, m.p.: 177-179˚C; IR (νmax, cm-1): 3346 (N-H), 3054 (C-
1
H), 2920, 1587, 1479. H NMR (400 MHz, CDCl3, δ ppm): 8.58 (1H, dd, J = 6.0 Hz,
pyridine C6H), 8.42 (1H, s, ArH ), 7.99 (1H, dd, J = 6.0 Hz, pyridine C4H), 7.86 (1H, dd, J =
9.2 Hz, ArH), 7.45 (1H, dd, J = 6.4 Hz, pyridine C5H), 7.17-7.06 (2H, m, ArH), 4.74 (2H, s,
CH2), 3.89 (3H, s, OCH3). 13C NMR (100 MHz, CDCl3, δ ppm): 164.14, 159.57, 158.43,
27

150.88, 147.65, 143.28, 138.65, 135.99, 129.59, 128.28, 127.94, 123.64, 123.49, 118.56,
116.78, 105.34, 55.62, 34.58. Calcd Mass 394.47 MS (m/z): 394 [M+]. Anal. Calcd for
C18H14N6OS2: C, 54.81; H, 3.58; N, 21.30. Found: C, 54.79; H, 3.65; N, 21.25.
4.2.12. 3-(2'-Mercaptopyridine)-12H-dihydro-(7,10-dimethyl)quinolino[4,3-e][1,2,4]-
triazolo-[3,4-b][1,3,4]-thiadiazepine (10l)
Yield: 85%, colour: yellow solid, m.p.: 184-186˚C; IR (νmax, cm-1): 3336 (N-H), 3055 (C-H),
1
2918, 1588, 1479. H NMR (400 MHz, CDCl3, δ ppm): 8.52 (1H, dd, J = 6.4 Hz, pyridine
C6H), 8.43 (1H, s, ArH), 8.21 (1H, dd, J = 8.0 Hz, ArH), 7.95 (1H, dd, J = 8.8 Hz, pyridine
C4H), 7.85 (1H, dd, J = 8.8 Hz, pyridine C5H), 7.28-7.14 (1H, m, ArH), 4.67 (2H, s, CH2),
2.28 (6H, s, CH3). 13C NMR (100 MHz, CDCl3, δ ppm): 166.89, 160.35, 151.48, 150.56,
138.89, 138.45, 136.96, 134.78 131.99, 130.54, 129.67, 129.34, 128.06, 126.14, 121.78,
119.89, 34.17, 22.15, 17.87. Calcd Mass 392.09 MS (m/z): 392 [M+]. Anal. Calcd for
C19H16N6S2: C, 58.14; H, 4.11; N, 21.41. Found: 58.17; H, 4.14; N, 21.47.
4.2.13. 3-(2'-Mercaptopyridine)-12H-dihydro-(8,10-dimethyl)quinolino[4,3-e][1,2,4]-
triazolo-[3,4-b][1,3,4]-thiadiazepine (10m)
Yield: 92%, colour: yellow solid, m.p.: 151-153˚C; IR (νmax, cm-1): 3348 (N-H), 3058 (C-H),
1
2927, 1584, 1482. H NMR (400 MHz, CDCl3, δ ppm): 8.61 (1H, dd, J = 6.4 Hz, pyridine
C6H), 8.54 (1H, s, ArH), 8.37 (1H, dd, J = 8.0 Hz, ArH), 7.99 (1H, dd, J = 8.8 Hz, pyridine
C4H), 7.90 (1H, dd, J = 8.8 Hz, pyridine C5H), 7.33-7.11 (3H, m, ArH), 4.69 (2H, s, CH2),
2.27 (6H, s, CH3). 13C NMR (100 MHz, CDCl3, δ ppm): 166.87, 160.45, 151.89, 150.14,
139.43, 138.68, 137.23, 134.56 131.96, 130.54, 129.62, 129.31, 128.026, 126.10, 121.58,
119.92, 34.16, 22.58, 24.17. Calcd Mass 392.09 MS (m/z): 392 [M+]. Anal. Calcd for
C19H16N6S2: C, 58.14; H, 4.11; N, 21.41. Found: 58.19; H, 4.09; N, 21.46.
5. Pharmacological Evaluation
5.1. In vitro antifungal activity
The title compounds were evaluated for their in vitro antifungal activity with Candida
albicans, Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger (cultured) by Disc
Diffusion Method [28] with Sabouraud agar medium. Fluconazole was used as a reference
drug. Each compound was tested at a concentration of 100 ꢀg/mL in DMSO. The zone of
inhibition (mm) was measured at 310.15K (37°C). Briefly, agar (pH = 7.2-7.4) was placed
into Petri dish (100mm size and 4mm depth). The agar plates were then inoculated with broth
28