Synthesis and structure-activity relationship of tyrosinase inhibiting novel bi-heterocyclic acetamides: Mechanistic insights through enzyme inhibition, kinetics and computational studies

Article abstract of DOI:10.1016/j.bioorg.2019.01.036

The present research was designed for the selective synthesis of novel bi-heterocyclic acetamides, 9a-n, and their tyrosinase inhibition to overwhelm the problem of melanogenesis. The structures of newly synthesized compounds were confirmed by spectral techniques such as ¹H NMR, ¹³C NMR, and EI-MS along with elemental analysis. The inhibitory effects of these bi-heterocyclic acetamides (9a-n) were evaluated against tyrosinase and all these molecules were recognized as potent inhibitors relative to the standard used. The Kinetics mechanism was analyzed by Lineweaver-Burk plots which explored that compound, 9h, inhibited tyrosinase competitively by forming an enzyme-inhibitor complex. The inhibition constants K_i calculated from Dixon plots for this compound was 0.0027 μM. The computational study was coherent with the experimental records and these ligands exhibited good binding energy values (kcal/mol). The hemolytic analysis revealed their mild cytotoxicity towards red blood cell membranes and hence, these molecules can be pondered as nontoxic medicinal scaffolds for skin pigmentation and related disorders.

Full text of DOI:10.1016/j.bioorg.2019.01.036

Accepted Manuscript
Synthesis and Structure-activity relationship of Tyrosinase Inhibiting novel bi-
heterocyclic acetamides: Mechanistic Insights through Enzyme Inhibition, Ki-
netics and Computational Studies
Abdul Rehman Sadiq Butt, Muhammad Athar Abbasi, Aziz-ur-Rehman,
Sabahat Zahra Siddiqui, Hussain Raza, Mubashir Hassan, Syed Adnan Ali Shah,
Muhammad Shahid, Sung-Yum Seo
PII:
S0045-2068(18)30957-X
https://doi.org/10.1016/j.bioorg.2019.01.036
YBIOO 2740
DOI:
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 September 2018
7 January 2019
21 January 2019
Please cite this article as: A. Rehman Sadiq Butt, M. Athar Abbasi, Aziz-ur-Rehman, S. Zahra Siddiqui, H. Raza,
M. Hassan, S. Adnan Ali Shah, M. Shahid, S-Y. Seo, Synthesis and Structure-activity relationship of Tyrosinase
Inhibiting novel bi-heterocyclic acetamides: Mechanistic Insights through Enzyme Inhibition, Kinetics and
Computational Studies, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.01.036
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Synthesis and Structure-activity relationship of Tyrosinase Inhibiting novel bi-heterocyclic
acetamides: Mechanistic Insights through Enzyme Inhibition, Kinetics and Computational
Studies
Abdul Rehman Sadiq Butt1, Muhammad Athar Abbasi1,2,*, Aziz-ur-Rehman1, Sabahat Zahra
Siddiqui1, Hussain Raza2, Mubashir Hassan2, Syed Adnan Ali Shah3,
Muhammad Shahid4 and Sung-Yum Seo2,*
1Department of Chemistry, Government College University, Lahore-54000, Pakistan.
2College of Natural Sciences, Department of Biological Science, Kongju National University,
Gongju, 32588, South Korea.
3Faculty of Pharmacy and Atta-ur-Rahman Institute for Natural Products Discovery (AuRIns),
Level 9, FF3, Universiti Teknologi MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam,
Selangor Darul Ehsan, Malaysia.
4Department of Biochemistry, University of Agriculture, Faisalabad-38040, Pakistan.
*Corresponding Authors: Dr. Muhammad Athar Abbasi, E-mail: abbasi@gcu.edu.pk Tel:
(+92)-42-111000010 Ext. 266 and Prof. Dr. Sung-Yum Seo, E-mail: dnalove@kongju.ac.kr
Tel: (+82)-416-8508503.
1

ABSTRACT
The present research was designed for the selective synthesis of novel bi-heterocyclic
acetamides, 9a-n, and their tyrosinase inhibition to overwhelm the problem of melanogenesis.
The structures of newly synthesized compounds were confirmed by spectral techniques such as
13
1H-NMR, C-NMR, and EI-MS along with elemental analysis. The inhibitory effects of these
bi-heterocyclic acetamides (9a-n) were evaluated against tyrosinase and all these molecules were
recognized as potent inhibitors relative to the standard used. The Kinetics mechanism was
analyzed by Lineweaver-Burk plots which explored that compound, 9h, inhibited tyrosinase
competitively by forming an enzyme-inhibitor complex. The inhibition constants Ki calculated
from Dixon plots for this compound was 0.0027 µM. The computational study was coherent with
the experimental records and these ligands exhibited good binding energy values (kcal/mol). The
hemolytic analysis revealed their mild cytotoxicity towards red blood cell membranes and hence,
these molecules can be pondered as nontoxic medicinal scaffolds for skin pigmentation and
related disorders.
Keywords: Thiazole, Triazole, Acetamides, Tyrosinase, Melanogenesis, Hemolytic, Binding
energy.
2

1. Introduction
Heterocyclic compounds are prevalent in various fields of life sciences. These
compounds carry out several significant tasks in technology, nature and medicine. Heterocyclic
hybrids have become most promising molecules due to its numerous potential as novel drug
candidates in modern drug discovery. It is a challenging and critical issue for the pharmaceutical
industry and academic researchers to develop new drugs and pharmaceuticals [1,2]. Heterocyclic
compounds have wide range of medicinal properties which inspired Chemists to synthesize their
new hybrids and screen its pharnacophoric properties. Heterocyclic compounds containing
nitrogen, especially azoles, and sulfur have attained constant interest in organic synthesis
because they are pharmacologically active and important pharmaceutically [3-5].
Thiazoles are familiar aromatic five member ring heterocyclic compound containing both
sulfur and nitrogen atom in the ring and encompassing extensive biological activities and their
utility as medicaments is very much established. They are fundamental part of all the available
penicillins which have revolutionized the therapy of bacterial diseases [6]. A literature survey
revealed that derivatives of thiazoles have numerous biological activities such as antimicrobial
[7,8], anticancer [9-12], antifungal [13], anti-inflammatory [14], anti-hypertension [15],
antitubercular [16], antioxidant [17], analgesic [18], and anthelmintic [19] activities.
Triazoles are five membered heterocyclic compounds containing two carbons and three
nitrogen atoms. These molecules have maintained a unique position in medicinal and organic
chemistry due to their numerous biological activities. The literature survey shows that
derivatives of triazole contain extensive range of pharmacological activities such as anticancer
[20], antimicrobial [21,22], antiviral [23], anti-inflammatory, local anaesthetic [24], antimalarial
[25], analgesic [26], antineoplastic [27], antiproliferative [28], and anticonvulsant [29]. Many of
the triazole based derivatives are now available as medicines [30].
Tyrosinase, also known as polyphenol oxidase enzyme, is widely distributed in nature
including higher plants, fungi, bacteria and animals [31]. It is responsible for browning of
vegetables, beverages, fruits and melanogenesis in animals. It catalyzes the hydroxylation of
monophenols to ortho-diphenols and subsequently oxidizes the products to corresponding ortho-
quinones by using molecular oxygen [32]. Champignon mushroom, Agaricus bisporus is a
source of tyrosinase. Tyrosinase is extracted from it and is well suited as a model for the studies
3

on melanogenesis. Mushroom tyrosinase is the only commercially available enzyme and
therefore almost all the tyrosinase inhibitory studies are conducted on it [33].
Human skin is frequently exposed to ultraviolet radiation, which is an induction factor of
reactive oxygen species. Formation of pathological skin pigmentation or direct DNA damage is
the cause of excessive reactive oxygen species and leads to induce skin injury. Antioxidant
defense system helps oxidative stress [34-36]. Prevention of damage due to ultraviolet radiation
to skin pigmentation is also done by another mechanism. These ultraviolet radiations are being
absorbed by Melanin thus protecting skin cells from them [37]. Therefore, for human health,
normal skin pigmentation is very essential [38]. Tyrosinase is a copper containing enzyme and is
essential in the melanin biosynthesis and therefore, responsible for pigmentation of skin and hair
in mammals [39-41]. Apart from the production of melanin, tyrosinase has many other functions
including detoxification of host plant defensive phenols for symbiotic bacteria [42-43] and
synthesis of amino acid based antibiotics [44].
Although some azole derivatives have been recently reported as tyrosinase inhibitors [45-
48], yet there is a need to discover some unique and safe inhibitors of this enzyme to devastate
the problems of melanogenesis. Therefore, in continuation of our previous studies on bioactivity
of related bi-heterocyclic bi-amides [49], the present investigation was designed to seek some
novel 2-aminothiazole-phenyltriazole hybrid molecules bearing N-(aralkyl/aryl)acetamides as
tyrosinase inhibitors.
2. Results and discussions
2.1. Chemistry
The targeted bi-heterocyclic N-substituted acetamides were synthesized in several steps as
delineated in scheme 1a and scheme 1b and the varying groups have been listed in Table 1. The
synthesis was planned by refluxing ethyl 2-(2-amino-1,3-thiazol-4-yl)acetate (1) in methanol and
hydrazine hydrate to convert it into 2-(2-amino-1,3-thiazol-4-yl)acetohydrazide (2). The
hydrazide, 2, was taken in methanol and refluxed with phenyl isothiocyante (3) to transform it
into a solid intermediary compound 2-[2-(2-amino-1,3-thiazol-4-yl)acetyl]-N-phenyl-1-
hydrazinecarbothioamide (4) which was cyclized further to a nucleophilic molecule, 5-[(2-
amino-1,3-thiazol-4-yl)methyl]-4-phenyl-4H-1,2,4-triazole-3-thiol (5). In a complementary set of
4

reactions, various electrophiles were synthesized by the reactions of un/substituted
anilines/benzyl amine/phenethyl amine (6a-n) with 2-bromoacetyl bromide (7) in an aqueous
medium,
resulting
in
the
formation
of
2-bromo-N-(un/substituted-
phenyl/benzyl/phenethyl)acetamides (8a-n). To attain the peak success in this synthesis, the
nucleophile 5 was dissolved in DMF and a pinch of LiH was added into it. The mixture was
stirred for about 30 minutes for activating its mercapto position, and then in this final step,
various electrophiles, 8a-n, were coupled in equimolar amounts with the activated 5 to yield the
targeted bi-heterocyclic molecules, 2-({5-[(2-amino-1,3-thiazol-4-yl)methyl]-4-phenyl-4H-1,2,4-
triazol-3-yl}sulfanyl)-N-(aralkyl/aryl)acetamides (9a-n).
S
O
CH3
NH
C
NH
N
NH2
Ph
N
B
N
N
A
N
O
O
H
H
O
N
C
S
H2N
H2N
4
S
S
3
1
2
H2N
S
C
3"
3"
5"
5"
R1
H
9a-l
R1
1"
3""
1"
N
C
O
Br
O
N
N
1""
2"'
4'
5""
S
4'
SH
C
1"'
5'
1'
N
6
6
5'
3'
3'
N
8a-l
D
N
R2
N
3
4
3
1'
R2
4
2'
2'
H
N
2
N
2
5
N
5
1
1
H2N
H2N
S
S
5
3"
5"
O
C
1"
H
N
Br
9m
O
C
O
Br
N
N
4'
7""
H
2"'
S
C
1"'
6
5'
1'
3'
N
N
H
8m
8n
3
4
3""
2'
1""
N
2
N
5
5""
1
D
D
H2N
S
3"
5"
1"
O
3""
1""
N
8""
5""
4'
2"'
S
C
1"'
6
5'
1'
N
3'
N
N
3
4
5
2'
7""
H
2
N
1
H2N
9n
S
Scheme 1a. Outline for the synthesis of 2-({5-[(2-amino-1,3-thiazol-4-yl)methyl]-4-phenyl-4H-
1,2,4-triazol-3-yl}sulfanyl)-N-(aralkyl/aryl)acetamides. Reagents Conditions: (A)
&
MeOH/N2H4·H2O/refluxing for 2 hrs. (B) MeOH/3/Refluxing for 1 hr. (C) The ppt. of 4
dissolved in 10% NaOH/filtration/acidification of filtrate in cold state to get ppt. of 5. (D)
DMF/LiH/stirring for 10-12 hrs.
5

R1
R1
H
N
NH2
O
C
C
O
Br
E
+
+
Br
Br
R2
R2
8a-l
7
6a-l
O
O
C
C
Br
NH2
N
E
E
Br
Br
H
Br
Br
6m
6n
8m
7
H
N
O
NH2
C
O
Br
C
+
8n
7
Scheme 1b. Synthesis of various electrophiles involved in scheme 1a. Reagents & Conditions:
(E) Aq. Na2CO3 soln./pH 9-10/vigorous manual shaking at RT for 2-3 hrs.
Table 1. Different groups (–R1 & –R2) in scheme 1a and 1b.
Compd.
-R1
-R2
Compd.
-R1
2-CH3
-R2
-H
-H
6-CH3
-H
2-CH3 3-CH3
2-CH3 4-CH3
2-CH3 5-CH3
2-CH3 6-CH3
3-CH3 4-CH3
3-CH3 5-CH3
6a, 8a, 9a
6b, 8b, 9b
6c, 8c, 9c
6d, 8d, 9d
6e, 8e, 9e
6f, 8f, 9f
6g, 8g, 9g
6h, 8h, 9h
6i, 8i, 9i
6j, 8j, 9j
6k, 8k, 9k
6l, 8l, 9l
2-CH2-CH3
2-CH2-CH3
4-CH2-CH3
4-O-CH2-CH3 -H
-H -H
The morphology of the synthesized compounds was well justified as well as their
structures were corroborated with aid of spectral data obtained by the detailed spectroscopic
studies. Structural analysis of one compound is discussed hereby for the benefit of readers. The
molecule 9j was synthesized as bright yellow amorphous solid having melting point of 100-101
oC. Its molecular formula, C22H22N6OS2, was recognized by CHN analysis data and molecular
mass was justified by the molecular ion peak in its EI-MS at m/z 450. The mass fragmentation
pattern also supported to erect the said molecular formula. Moreover, counting the number of
1
protons in its H-NMR spectrum and the carbon resonances in its 13C-NMR spectrum, also
augmented this assignment. IR spectrum was used to recognize various functionalities in the
molecule. The characteristic peaks appeared at  3358 (N-H stretching), 3048 (C-H stretch of
aromatic ring), 2922 (-CH2- str.), 1642 (C=O stretching), 1578 (C=C stretch of aromatic ring),
1
1521 (C=N stretch), 1167 (C-N-C bond stretch), and 625 cm-1 (C-S stretch). In its H-NMR
6

spectrum, the phenyl ring linked with nitrogen (4′) of 1,2,4-triazole heterocyclic ring was
identified by two prominent multiplets in the aromatic region at δ 7.52-7.50 (m, 3H, H-3'', H-4''
& H-5'') and 7.37-7.35 (m, 2H, H-2'' & H-6''). A 4-ethylphenyl moiety attached with nitrogen of
acetamidic unit was clearly identified by A2B2 spin system in aromatic region, represented by
two ortho-coupled broad doublets at δ 7.45 (br.d, J = 8.5 Hz, 2H, H-2'''' & H-6''''), and δ 7.14
(br.d, J = 7.14 Hz, 2H, H-3'''' & H-5''''), along with characteristic quartet and a triplet for ethyl
group at δ 2.55 (q, J = 7.6, 2H, CH3-CH2-4′′′′) and 1.15 (t, J = 7.6, 3H, CH3-CH2-4′′′′). The 2-
amino-1,3-thiazol-4-yl moiety was characterized by two singlets at δ 6.84 (br.s, 2H, H2N-2), and
δ 5.85 (s, 1H, H-5), while a singlet δ 3.77 (s, 2H, CH2-6) was assignable to a methylene group
linking the two heterocycles in the molecule. The acetamidic unit in the molecule was unified by
1
two peculiar singlets at δ 10.24 (s, 1H,-CO-NH-1'''') and δ 4.10 (s, 2H, CH2-2′′′). The H-NMR
spectrum of this molecule has been shown in Fig. S1(a). Its expanded aromatic region has been
shown in Fig. S1(b) while its expanded aliphatic region has been presented in Fig. S1(c). The
13C-NMR spectrum (Fig. S2) was also used to substantiate all the assignments. Overall, eighteen
carbon resonances were observed due to some symmetrical carbons in the molecule. The 2-
amino-1,3-thiazol-4-yl moiety was signified by two quaternary signals at δ 168.20 (C-2), and
145.60 (C-4), besides a methine signal at δ 102.18 (C-5). Likewise, the other heterocycle, i.e.
(1,2,4-triazol-5-yl)sulfanyl was also rationalized by two quaternary signals at δ 153.39 (C-5') and
149.71 (C-3') while a methylene joining the two heterocycles (4-position of the former
heterocycle with 5′-position of the latter heterocycle) was obvious at δ 27.48 (C-6). The 4-
ethylphenyl moiety was apparent with two quaternary signals at δ 138.89 (C-1′′′′), and 136.43
(C-4′′′′) along with two symmetrical methine duplets at δ 127.92 (C-3′′′′ & C-5′′′′) and δ 119.12
(C-2′′′′ & C-6′′′′). The 4-ethyl group was easily characterized by δ 27.55 (CH3-CH2-4′′′′) and δ
15.61 (CH3-CH2-4′′′′). The central acetamidic unit was corroborated by a downfield carbonyl
signal at δ 165.27 (C-1′′′) and a methylene signal at δ 35.93 (C-2'''). The phenyl ring attached to
the nitrogen (4′) atom of 1,2,4-triazole ring was recognized by a quaternary signal at δ 132.90
(C-1'') and three methine resonances at δ 129.78 (C-4''), 129.55 (C-3'' & C-5'') and 127.14 (C-2''
& C-6''). Various connectivities in the carbon skeleton were thoroughly ascertained by its HMBC
spectrum. This spectrum, along with important correlations, is shown in Fig. S3. So, on the basis
of aforementioned accumulative evidences, the structure of 9j was confirmed and it was named
as
2-({5-[(2-amino-1,3-thiazol-4-yl)methyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)-N-(4-
7

ethylphenyl)acetamide. A similar protocol was exercised for the structural characterization of
other synthesized molecules (Fig. S4-S17).
2.2. Urease inhibition and structure-activity relationship
The newly synthesized bi-heterocyclic acetamides (9a-n) were evaluated for their
inhibitory potentials against tyrosinase enzyme and their results are tabulated in Table 2. All
these compounds exhibited very potent inhibitory activities against this enzyme, as evident from
their lower IC50 (M) values, relative to standard, kojic acid, having IC50 value of 16.8320 ±
1.1600 M. Though the rendered activity is accumulative of a whole molecule, yet a limited
structure-activity relationship (SAR) was recognized by examining the effect of different aryl
entity on the inhibitory potential, as it was the only varying part and all other parts were intact in
all molecules. The general structural parts of the studied acetamides are featured in Fig. 1.
Phenyl
Part
Aralkyl/Aryl
2-Aminthiazole
Part
Part
O
C
N
0-2
R1
S
N
N
H
N
N
H2N
S
Acetamide
Part
R2
Triazole
Part
Fig. 1. General structural features of compounds 9a-n.
8

Table 2. Tyrosinase inhibitory and hemolytic activity of bi-heterocyclic acetamides, 9a-n.
Tyrosinase activity Hemolysis (%)
Compounds
9a
Aryl part
IC50 ± SEM (µM)
(Mean ± SEM)
H3C
CH3
19.62 ± 0.04
2.0181 ± 0.1629
H3C
17.98 ± 0.02
16.89 ± 0.03
0.0547 ± 0.0211
0.0589 ± 0.0011
9b
9c
CH3
H3C
CH3
H3C
4.11 ± 0.02
13.33 ± 0.03
19.22 ± 0.02
0.0513 ± 0.0035
0.1133 ± 0.0038
0.2465 ± 0.0019
9d
9e
9f
H3C
CH3
CH3
CH3
CH3
H3C
0.0156 ± 0.0021
0.0652 ± 0.0044
9g
9h
17.22 ± 0.04
16.77 ± 0.04
H3C
CH2
H3C
CH2
0.1943 ± 0.0063
9i
21.4 ± 0.05
H3C
9

CH2 CH3
0.1224 ± 0.0023
0.1726 ± 0.0149
0.1330 ± 0.0129
1.0108 ± 0.0614
0.0161 ± 0.0014
9j
9k
9l
20.22 ± 0.05
13.77 ± 0.02
4.21 ± 0.01
18.42 ± 0.02
18.01 ± 0.03
O
CH2 CH3
H2C
9m
9n
H2C
H2C
Kojic acid
16.8320 ± 1.1600
-
-
89.11 ± 0.01
Triton X
SEM= Standard error of the mean; values are expressed in mean ± SEM.
PBS Hemolysis = 2.93 ± 0.01%.
Among the following four di-methylated regio-isomers, compound 9a in which the two
methyl groups were present on adjacent positions (2,3-dimethylphenyl), and probably due to
steric crowding it exhibited least inhibitory activity (IC50 = 2.0181 ± 0.1629 µM) as compared to
other isomers. Even it was even least active in the whole series, yet it was much better inhibitor
than standard, kojic acid (IC50 = 16.8320 ± 1.1600 µM). The other analogues 9b, 9c and 9d
possessed very resembling inhibitory potential with IC50 values of 0.0547 ± 0.0211, 0.0589 ±
0.0011 and 0.0513 ± 0.0035 µM respectively, indicating that separation of the methyl groups
probably augmented the suitable interactions with the active site of the enzyme (Fig. 2).
10

CH3
9a
9b
H3C
H3C
CH3
O
C
O
C
N
N
S
S
N
N
N
H
N
H
N
N
N
N
H2N
H2N
S
S
IC50 = 0.0547 ± 0.0211M
IC50 = 2.0181 ± 0.1629 M
9c
9d
H3C
H3C
O
O
C
N
N
S
S
C
N
N
CH3
H2N
N
H
N
H
N
N
N
N
CH3
H2N
S
S
IC50 = 0.0513 ± 0.0035M
IC50 = 0.0589 ± 0.0011 M
Fig. 2. Structure-activity relationship of compounds 9a, 9b, 9c, and 9d.
Contrary, when the inhibitory potential of other two isomers was compared it was
observed that 9e with adjacent methyl groups (3,4-dimethylphenyl), had a better activity (IC50 =
0.1133 ± 0.0038 µM), relative to 9f (IC50 = 0.2465 ± 0.0019 µM), probably due to the reason that
the hereby adjacent methyl groups in 9e were away from the point of attachment of aromatic
ring, thus the same steric factor was diminished to some extent and this molecule became
comparatively more suitable for inhibition of tyrsosinase enzyme (Fig. 3).
CH3
CH3
9e
9f
CH3
O
C
O
C
N
N
S
S
N
N
CH3
N
N
H
H
N
N
N
N
H2N
H2N
S
S
IC50 = 0.2465 ± 0.0019 M
IC50 = 0.1133 ± 0.0038 M
Fig. 3. Structure-activity relationship of 9e and 9f.
When the inhibitory potential of ortho-substituted molecules was compared, it was
revealed that 9g with a smaller methyl group was more prone to the inhibition of tyrosinase (IC50
11

= 0.0156 ± 0.0021µM). The presence of little bulky ethyl group at ortho-position retarded the
activity of 9h (IC50 = 0.0156 ± 0.0021µM) and a further decrease was observed in 9i (IC50 =
0.1943 ± 0.0063µM) in which both ortho-positions were occupied, one with a smaller methyl
group and other with a little bulky ethyl group (Fig. 4). It means that the presence of only smaller
group/s at ortho-position can favour the inhibitory activity in these molecules and indeed 9g was
the most active compound in the synthetic series.
CH3
9g
9h
H3C
O
C
O
C
N
N
S
S
N
N
N
H
N
H
N
N
N
N
H2N
H2N
S
S
IC50 = 0.0652 ± 0.0044M
IC50 = 0.0156 ± 0.0021 M
CH3
9i
O
C
N
S
N
N
H
N
N
CH3
H2N
S
IC50 = 0.1943 ± 0.0063M
Fig. 4. Structure-activity relationship of 9g, 9h and 9i.
In between the para-substituted molecules, 9j with 4-ethyl group in aryl part exhibited
slightly better inhibitory potential (IC50 = 0.1224 ± 0.0023µM) as compared to 9k (IC50 = 0.1726
± 0.0149 µM) in which 4-ethoxy group was present (Fig. 5). It means the presence of a slightly
bulky polar group was not a suitable entity to impart a superb inhibitory activity to a molecule.
12

H3C
CH3
9j
9k
O
O
C
O
C
N
N
S
S
N
N
N
N
H
H
N
N
N
N
H2N
H2N
S
S
IC50 = 0.1726 ± 0.0149M
IC50 = 0.1224 ± 0.0023 M
Fig. 5. Structure-activity relationship of 9j and 9k.
A very interesting trend was observed in the inhibitory activity of molecules with an un-
substituted aryl part. The insertion of one methylene group in between nitrogen atom and phenyl
ring in 9m (IC50 = 1.0108 ± 0.0614µM) decreased its activity relative to 9l (IC50 = 0.1330 ±
0.0129 µM) in which no such methylene was present. However, the presence of two methylenes
in 9m (IC50 = 0.0161 ± 0.0014 µM) enhanced its activity, probably due to more flexibility in the
aryl part of the molecule and it behaved as a promising inhibitor and was indeed the second most
active compound in the series (Fig. 6).
9l
9m
O
C
O
C
N
N
S
S
N
N
N
N
H
H
N
N
N
N
H2N
H2N
S
S
IC50 = 1.0108 ± 0.0614 M
IC50 = 0.1330 ± 0.0129 M
9n
O
C
N
S
N
N
H
N
N
H2N
S
IC50 = 0.0161 ± 0.0014M
Fig. 6. Structure-activity relationship of 9l, 9m and 9n.
13

So, it was envisaged from the structure-activity relationship that in the current synthetic
series, the molecules, either bearing small sized group/s at ortho-position/s in the aryl part like
9g and 9d or a molecule with a flexible aryl part like 9n, generally behaved as highly potent
inhibitors of the tyrosinase enzyme.
2.3. Kinetic analysis
To understand the inhibitory mechanism of synthetic bi-heterocyclic acetamides on
tyrosinase inhibition, kinetic study was performed. Based on our IC50 results, we selected the
most potent compound 9g to determine their inhibition type and inhibition constant. The kinetic
results of the enzyme by the Lineweaver-Burk plot of 1/V versus 1/[S] in the presence of
different inhibitor concentrations gave a series of straight lines, the result of Lineweaver-Burk
plot of compound 9g showed that Vmax remains the same without significantly affecting the
slopes. Km increases with increasing concentration, while Vmax remains the same with an
insignificant difference. This behavior indicated that 9g inhibited the enzyme tyrosinase in a
competitive manner (Fig. 7; A). The second plot (Fig. 7; B) of slope against the concentration of
9g showed EI dissociation constant. Ki was calculated from the inhibitor concentration of 9g
versus the slope and Ki was found to be 0.0027 µM.
14

Fig. 7. Lineweaver–Burk plots for inhibition of tyrosinase in the presence of compound 9g. (A)
Concentrations of 9g were 0.00, 0.0077, and 0.0155 µM, respectively. Substrate L-DOPA
Concentrations were 0.0625, 0.125, 0.25, 0.5, 1 and 2 mM, respectively. (B) The insets
represented the plot of the slope versus inhibitor 9g concentrations to determine inhibition
constant. The lines were drawn using linear least squares fit.
2.4. Hemolytic activity
The synthesized compounds, 9a-n, were also exposed to hemolytic assay to ascertain
their safe utility as therapeutic agents. The cytotoxicity was profiled a percentage results of
hemolysis and the results are shown in Table 2. It was obvious from the results that all
derivatives of this series have modest toxicity towards red blood cell membrane. Maximum
membrane toxicity was shown by the compound 9i (21.4 ± 0.05%) which is much lower than the
positive control, Triton X (89.11 ± 0.01%). The minimum toxicity was rendered by 9d (4.11 ±
0.02%) in the series. In general, it was rational that most of the molecules exhibited very
moderate cytotoxicity and thus can be considered for further applications in drug designing
programs.
2.5. Computational analysis
2.5.1. Bio-chemical properties and Lipinski's rule of five (RO5) validation
The biochemical properties of all the synthesized chemical compounds, 9a-n, were
predicted by using computational servers and tools and validated through Lipinski Rule of five
(RO5) analysis. The RO5 states as compounds must contain <500 (g/mol) molecular mass and
<5 logP values, respectively. Moreover, the compounds should possess no greater than 10
hydrogen bond acceptors (HBA) and 5 hydrogen bond donor (HBD), respectively. The
molecular weight (g/mol), logP, HBA/D all compounds, 9a-n, were not exceeded then standard
value as mentioned in Table 3. The exceed values of HBA and HBD results in poor permeation
[50]. The hydrogen-bonding capacity has been considered as significant parameter for drug
permeability. Our predicted results showed that all the synthesized compounds were present in
standard range and obeyed the RO5 rule. Polar surface area (PSA) is also considered as good
15

descriptor for characterizing the drug absorption, including intestinal absorption, bioavailability
and blood-brain barrier penetration [51]. Our predicted results showed that all compounds, 9a-n,
possess <140 Å2 PSA values which may enhance their absorption efficacy in the body. The
computational drug-likeness prediction is a combination of various molecular properties such as
hydrophobicity, electronic distribution, hydrogen bonding characteristics, molecule size with
flexibility and presence of various pharmacophoric features [52]. Our results showed that
synthetic compounds, 9a-n, showed good drug score values except 9e which showed negative
results. The positive values of synthetic compounds showed good drug-likeness behavior
whereas, compounds with negative values have less drug-likeness probability (Table 3).
Table 3. Biological properties of synthesized compounds.
Drug likeness
Mol.weight No.
No. Mol. MolPSA Mol. Vol
Score
Ligands
(g/mol)
HBA HBD LogP
(A2)
(A3)
0.92
450
450
450
450
450
450
436
450
464
450
446
422
436
450
6
6
6
6
6
6
6
6
6
6
7
6
6
6
3
3
3
3
3
3
3
3
3
3
3
3
3
3
4.23
4.35
4.35
4.23
4.35
4.47
3.95
4.45
4.73
4.57
4.22
3.67
3.64
3.92
79
79
79
78
80
80
79
79
78
80
87
80
81
81
415
414
414
414
414
414
393
412
432
411
422
372
389
408
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
0.24
0.46
1.27
-0.01
0.59
0.77
0.73
1.41
0.85
0.79
0.49
0.48
0.47
* HBA: Hydrogen Bond Acceptor, HBD: Hydrogen Bond Donor
2.5.2. Pharmacokinetic assessment of synthesized compounds
The pharmacokinetics properties such as absorption, distribution, metabolism, excretion
and toxicity (ADMET) of newly synthesized compounds are considered as major hallmark to
predict the lead structures efficacy. The absorption properties like water absorption (log mol/L),
intestinal solubility (% absorbed) and skin permeability (logKp) values predict the therapeutic
potential of newly synthesized compounds. The drug absorption is depends upon routes of
16

administration either orally, intravenously or subcutaneously and chemical compounds target a
specific tissue by using bloodstream pathway. Prior research data showed that ligands having
good absorption value have more chance to cross gut barrier by passive penetration [53]. The
water solubility results justified that compounds, 9a-n showed good absorption and may have
potential to cross gut barriers. Furthermore, all compounds, 9a-n, exhibited good intestinal
solubility results which are comparable to standard value (>30 %abs). The skin permeability
values of all compounds, 9a-n, were also present in the acceptable range compared with standard
value (-2.5 logKp) which predict their drug likeness behavior. The compounds having <30 %
absorption may not considered as good lead like structure. Moreover, Blood Brain Barrier (BBB)
and Central Nervous System (CNS) permeability values of all compounds were also acceptable
with the standard values (>0.3 to <-1 log BB and >-2 to <-3 logPS), respectively. Results showed
that compounds displayed a comparable predicted values with standard and have potential to
cross the barriers and to target the receptor. Moreover, their metabolic behavior was confirmed
by CYP1A2, CYP2C19 and CYP2C9 inhibitor, which are isoform of cytochrome P450. Results
showed the compounds possessed good inhibitory behavior for CYP2C19 and CYP2C9,
however, for CYP1A2 compounds (9h-I, 9k and 9n) were beyond the inhibition behavior. The
excretion and toxicity predicted values were also justified the drug likeness behavior of these
compounds on the basis of total clearance (log ml/min/kg), AMES toxicity, maximum tolerated
dose (MTD) and LD50 values. The non-mutagenic and non-toxic behaviors of synthesized
compounds were observed from AMES toxicity prediction. Compounds showed hepatotoxic
behavior while no one showed skin sensitization effects. The predicted ADMET properties
justified that these novel synthesized compounds may have good lead like potential with mild
hepatotoxic effects (Table 4).
17

Table 4. Pharmacokinetic assessment of synthesized compounds, 9a-n.
ADMET Properties
9a
-4.74 -4.71 -4.73 -4.70 -4.67 -4.66 -4.65 -5.02 -5.07 -4.86 -4.47 -4.53 -4.58 -4.92
98.88 98.90 98.88 98.90 98.89 98.87 98.43 98.54 98.98 98.5 100 97.96 95.47 94.65
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
WS
Absorption
Distribution
IS
SP
-2.73 -2.73 -2.73 -2.73 -2.73 -2.73 -2.73 -2.73 -2.73 -2.73 -2.73 -2.73 -2.73 -2.73
-0.82 -0.83 -0.84 -0.83 -0.82 -0.81 -0.85 -0.79 -0.78 -0.83 -1.08 -0.86 -0.83 -0.83
-2.49 -2.49 -2.49 -2.49 -2.52 -2.52 -2.56 -2.57 -2.50 -2.61 -2.86 -2.66 -2.78 -2.83
BBBP
CNSP
CYP1A2
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Metabolism
(inhibitor)
CYP2C19 Yes
CYP2C9
TC
Yes
-0.003 0.043 0.011 0.043 -0.061 -0.076 0.098 0.116 0.006 0.054 0.131 0.144 -0.16 0.18
Excretion
AMES
MTD
ORAT
HT
No
0.72
2.76
Yes
No
No
No
No
No
0.68
2.74
Yes
No
No
0.64
2.75
Yes
No
No
No
No
No
No
No
No
No
0.68 0.68 0.67
2.75 2.76 2.75
0.78 0.84 0.76 0.72 0.80 0.82 0.81 0.79
2.77 2.83 2.80 2.78 2.69 2.77 2.69 2.67
Toxicity
Yes
No
Yes
No
Yes
No
Yes
No
Yes
No
Yes
No
Yes
No
Yes
No
Yes
No
Yes
No
Yes
No
SS
*Abbrevation: WS=Water solubility (log mol/L), IS=intestinal solubility (%abs), SP=Skin permeability (log Kp),
BBBP=blood brain barrier permeability (Log BB), CNSP=CNS permeability (LogPS), TC=Total clearance (log ml/min/kg),
MTD=Max. tolerat. dose ORAT=Oral Rat Acute Toxicity (LD50), HT=Hepatotoxicity, SS=Skin Sensitization.
18

2.5.3. Molecular docking and binding energy analyses
Molecular docking experiment is best approach to study the binding conformation of
ligands within the active region of target proteins [54, 55]. The docked complexes of synthesized
compounds, 9a-n, against mushroom tyrosinase were analyzed on the basis of lowest binding
energy values (kcal/mol) and hydrogen/hydrophobic interaction pattern. Results showed that all
the ligands, 9a-n, exhibited good docking energy values and showed their interaction within
active region of target protein (Table 5). GlideScore is based on ChemScore (fitness function),
but includes a steric-clash term, adds buried polar terms devised by Schrödinger to penalize
electrostatic mismatches. The GScore is calculated as from the equation (i).
GScore = vdW + Coul + Lipo + Hbond + Metal + BuryP + RotB + Site…(i)
vdW = Van der Waals energy, Coul = Coulomb energy, Lipo = Lipophilic, Hbond = Hydrogen-
bonding, Metal = Metal-binding, BuryP = Penalty for buried polar groups, RotB = Penalty for
freezing rotatable bonds and Site = Polar interactions in the active site. Based on in vitro and in
silico docking energy results, 9g was ranked as best ligands which showed good inhibitory
potential against targeted enzyme as compared all other derivatives. Although, the basic nucleus
of all the synthesized compounds were same, therefore most of compounds possess good
efficient energy values and have no big energy fluctuations difference.
Table 5. Docking energy values of 9a-n against mushroom tyrosinase.
Docking Glide Score Docking Glide Score
complexes (kcal/mol) complexes (kcal/mol)
-7.408
-6.689
-5.639
-6.099
-6.172
-6.249
-6.817
-7.740
-5.010
-6.864
-7.945
-6.153
-6.881
-6.313
9a
9b
9c
9d
9e
9f
9h
9i
9j
9k
9l
9m
9n
9g
19

2.5.4. Binding pocket and ligands binding conformations
The binding pocket analysis showed that ligands, 9a-n, were confined in the active region
of target protein. All three docked structures were superimposed to check their binding
configuration in the active region of target protein. Results showed that synthesized compounds
were bound in the binding pocket having similar conformational pattern (Fig. 8).
Fig. 8. Binding pocket conformation of 9a-n against target protein.
2.5.5. Binding analysis
The docked complexes were analyzed on the basis of bonding interactions pattern. Based
on in vitro and docking energy results, the most promising compound (9g) was selected to check
its conformational position inside the active region of target protein (Fig. 9). The 2-methyphenyl
20

ring of 9g formed a couple of hydrophobic interactions at His85 and His259 residues within the
active region of target protein. Both residues are metal bound residues and paly a significance in
protein stability. Similarly, another phenyl ring is attached with His244 and 2-aminothiazole ring
is with His85. The amino group of five thiazol ring showed another significant interaction with
Cys83. The already published computational data results showed good correlation with our
docking results [56, 57]. The 2-dimensional graphical depiction of 9g docking complex is
mentioned in Fig. 10, whereas all other are listed in supplementary data Figs. S18-S30.
Fig. 9. Docking complexes of 9g. The ligand structures 9g is highlighted in purple color while
the interactive residues are depicted in red color. The target protein is justified in different colors
in ribbon format.
21

Fig. 10. Docking complexes of 9g in 2D format.
4. Conclusion
A structurally distinct series of novel molecules, amalgamated with a thiazole, a triazole
and an acetamide moiety, was synthesized and recognized with very superb tyrosinase inhibition.
It was postulated from their SAR studies that molecules particularly bearing small sized methyl
group/s at ortho-position/s in aryl part or a compound with a flexible phenethyl group, generally
inhibited the tyrosinase in an excellent manner. All these molecules also showed mild
cytotoxicity towards red blood cell membranes. Therefore, it was pertinent to conclude that these
bi-heterocyclic acetamides can find their utility as leading medicinal scaffolds for the treatment
of melanogenesis.
22

5. Experimental
5.1. General
All the chemicals, along with analytical grade solvents, were purchased from Sigma
Aldrich, Alfa Aesar (Germany), or Merck through local suppliers. Pre-coated silica gel Al-plates
were used for TLC with ethyl acetate and n-hexane as solvent system. Spots were detected by
UV254. Gallonkamp apparatus was used to detect melting points in capillary tubes. Elemental
analyses were performed on a Foss Heraeus CHN-O-Rapid instrument and were within ± 0.4%
of the theoretical values. IR spectra (ν, cm–1) were recorded by KBr pellet method in the Jasco-
320-A spectrophotometer. EI-MS spectra were measured on a JEOL JMS-600H instrument with
data processing system. 1H-NMR spectra (δ, ppm) were recorded at 600 MHz (13C-NMR spectra,
at 150 MHz) in DMSO-d6 using the Bruker Advance III 600 As- cend spectrometer using BBO
probe.
5.2. Synthesis of 2-(2-amino-1,3-thiazol-4-yl)acetohydrazide (2)
Ethyl 2-(2-amino-1,3-thiazol-4-yl)acetate (0.15 mol.; 1) and 60 mL methanol were taken in
a 500 mL round-bottom flask and hydrazine monohydrate (80%; 20 mL) was added into it. The
reaction mixture was stirred for about 2 hours at room temperature (RT) and as a result 2-(2-
amino-1,3-thiazol-4-yl)acetohydrazide (2) was precipitated out. It was obtained by distilling off
the methanol after absolute conversion of reaction. The obtained precipitates of 2 were washed
with cold n-hexane and air-dried.
5.3. Synthesis of 2-[2-(2-amino-1,3-thiazol-4-yl)acetyl]-N-phenyl-1-hydrazinecarbothio-amide
(4)
2-(2-Amino-1,3-thiazol-4-yl)acetohydrazide (0.13 mol.; 2) was dissolved by heating in
methanol (25 mL) taken in 500 mL round flask and after that phenyl isothiocyanate (0.13 mol.;
3) was added. Reaction mixture was kept on refluxing for an hour. After completion of the
reaction, precipitates of intermediate compound, 2-[2-(2-amino-1,3-thiazol-4-yl)acetyl]-N-
23

phenyl-1-hydrazinecarbothioamide (4), were obtained which were filtered and dried to proceed
further for cyclization process.
5.4. Synthesis of 5-[(2-amino-1,3-thiazol-4-yl)methyl]-4-phenyl-4H-1,2,4-triazole-3-thiol (5).
The
intermediate
compound,
2-[2-(2-amino-1,3-thiazol-4-yl)acetyl]-N-phenyl-1-
hydrazinecarbothioamide (0.13 mol.; 4), was dissolved in 10% NaOH (100 mL) and the solution
was filtered. The filtrate so obtained was acidified with conc. HCl in cold stated to get the
precipitates of desired cyclized product 5-[(2-amino-1,3-thiazol-4-yl)methyl]-4-phenyl-4H-1,2,4-
o
triazole-3-thiol (5). White crystalline solid; Yield: 92%; m.p. 233-234 C; Mol. Formula:
C12H11N5S2; Mol. Mass.: 289 gmol-1; IR (KBr, ʋ/cm-1): ʋ 3321 (N-H str.), 3040 (C-H str. of
aromatic ring), 2912 (-CH2- str.), 1560 (C=C str. of aromatic ring), 1512 (C=N str.),1145 (C-N-C
bond str.), 609 (C-S str.); 1H-NMR (600 MHz, DMSO-d6, δ/ppm): δ 13.73 (s, 1H, HS-3′), 7.47-
7.46 (m, 3H, H-3′′, H-4′′ & H-5′′), 7.28-7.27 (m, 2H, H-2′′ & H-6′′), 6.88 (s, 2H, H2N-2), 5.85 (s,
1H, H-5), 3.67 (s, 2H, CH2-6); 13C-NMR (150 MHz, DMSO-d6, δ/ppm): δ 168.78 (C-2), 150.05
(C-5′), 150.58 (C-3′), 144.86 (C-4), 134.17 (C-1′′), 129.70 (C-4′′), 129.49 (C-3′′ & C-5′′), 128.63
(C-2′′ & C-6′′), 103.10 (C-5), 28.60 (C-6). Anal. Calc. for C12H11N5S2 (289.38): C, 49.81; H,
3.83; N, 24.20. Found: C, 49.76; H, 3.79; N, 24.17; EI-MS: m/z 289 [M]+, 256 [C12H10N5S]+, 247
[C11H9N3S2]+, 214 [C11H8N3S]+, 138 [C5H4N3S]+, 113 [C4H5N2S]+, 77 [C6H5]+, 71 [C3H3S]+
.
5.5. General Synthesis of 2-({5-[(2-amino-1,3-thiazol-4-yl)methyl]-4-phenyl-4H-1,2,4-triazol-3-
yl}sulfanyl)-N-(aralkyl/aryl)acetamides (9a-n).
5-[(2-Amino-1,3-thiazol-4-yl)methyl]-4-phenyl-4H-1,2,4-triazole-3-thiol (0.2 g, 5) was
dissolved in N,N-dimethyl formamide (DMF, 3 mL) in a 100 mL round bottom flask at room
temperature. One pinch of solid LiH was added to this solution and mixture was stirred for half
an hour for activation. Then, different electrophiles, 2-bromo-N-(un/substituted-
phenyl/benzyl/phenethyl) acetamides (8a-n, one in each reaction), were added in equimolar amount
in each respective reaction and the mixture was stirred for 10-12 hours. The reaction was
monitored by TLC using n-hexane and ethyl acetate solvent system (70:30). A single spot of
product on TLC showed the completion of a reaction. The reaction mixture was quenched with
24

excess ice cold distilled water (50 mL). The respective targeted products, 9a-n, were collected
through filtration, washed with distilled water and dried for further use.
5.5.1. 2-({5-[(2-Amino-1,3-thiazol-4-yl)methyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)-N-(2,3-
dimethylphenyl)acetamide (9a).
o
Light yellow solid; Yield: 90%; m.p. 102-103 C; Mol. Formula: C22H22N6OS2; Mol.
Mass.: 450 gmol-1; IR (KBr, ʋ/cm-1) : 3334 (N-H str.), 3048 (C-H str. of aromatic ring), 2921 (-
CH2- str.), 1665 (C=O str.), 1588 (C=C str. of aromatic ring), 1511 (C=N str.), 1145 (C-N-C
1
bond str.), 612 (C-S str.); H-NMR (600 MHz, DMSO-d6, δ/ppm): 9.74 (s, 1H, -CO-NH-1′′′′),
7.54-7.51 (m, 3H, H-3′′, H-4′′ & H-5′′), 7.38-7.36 (m, 2H, H-2′′ & H-6′′), 7.15 (br.d, J=7.7, 1H,
H-6′′′′), 7.04 (br.t, J=7.6, 1H, H-5′′′′), 7.00 (br.d, J=7.3, 1H, H-4′′′′), 6.86 (br.s, 2H, H2N-2), 5.86
(s, 1H, H-5), 4.12 (s, 2H, CH2-2′′′), 3.79 (s, 2H, CH2-6), 2.23 (s, 3H, CH3-3′′′′), 2.05 (s, 3H, CH3-
2′′′′); 13C-NMR (150 MHz, DMSO-d6, δ/ppm): 168.12 (C-2), 165.69 (C-1′′′), 153.32 (C-5′),
149.72 (C-3′), 145.51 (C-4), 136.85 (C-3′′′′), 135.61 (C-1′′′′), 132.82 (C-1′′), 130.66 (C-2′′′′),
129.71 (C-4′′), 129.48 (C-3′′ & C-5′′), 127.07 (C-2′′ & C-6′′), 126.85 (C-4′′′′), 125.06 (C-5′′′′),
122.88 (C-6′′′′), 102.09 (C-5), 36.12 (C-2′′′), 27.39 (C-6), 20.01 (CH3-3′′′′), 13.77 (CH3-2′′′′).
Anal. Calc. for C22H22N6OS2 (450.58): C, 58.64; H, 4.92; N, 18.65. Found: C, 58.71; H, 4.97; N,
18.72; EI-MS (m/z): 450 [M]+, 330 [C14H12N5OS2]+, 270 [C13H10N4OS]+, 256 [C12H10N5S]+, 148
[C9H10NO]+, 120 [C8H10N]+, 71 [C3H3S]+
.
5.5.2. 2-({5-[(2-Amino-1,3-thiazol-4-yl)methyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)-N-(2,4-
dimethylphenyl)acetamide (9b).
o
Light orange solid; Yield: 89%; m.p. 172-173 C; Mol. Formula: C22H22N6OS2; Mol.
Mass.: 450 gmol-1; IR (KBr, ʋ/cm-1): 3342 (N-H str.), 3052 (C-H str. of aromatic ring), 2918 (-
CH2- str.), 1670 (C=O str.), 1577 (C=C str. of aromatic ring), 1519 (C=N str.), 1152 (C-N-C
1
bond str.), 613 (C-S str.); H-NMR (600 MHz, DMSO-d6, δ/ppm): 9.62 (s, 1H, -CO-NH-1′′′′),
7.53-7.51 (m, 3H, H-3′′, H-4′′ & H-5′′), 7.37-7.36 (m, 2H, H-2′′ & H-6′′), 7.28 (br.d, J=8.1, 1H,
H-6′′′′), 7.01 (br.s, 1H, H-3′′′′), 6.95 (br.d, J=8.1, 1H, H-5′′′′), 6.85 (br.s, 2H, H2N-2), 5.86 (s, 1H,
H-5), 4.11 (s, 2H, CH2-2′′′), 3.78 (s, 2H, CH2-6), 2.23 (s, 3H, CH3-4′′′′), 2.14 (s, 3H, CH3-2′′′′);
25

13C-NMR (150 MHz, DMSO-d6, δ/ppm): 168.21 (C-2), 165.68 (C-1′′′), 153.42 (C-5′), 149.84 (C-
3′), 145.69 (C-4), 134.24 (C-1′′′′), 133.37 (C-2′′′′), 132.89 (C-1′′), 131.16 (C-4′′′′), 130.79 (C-
5′′′′), 129.80 (C-4′′), 129.56 (C-3′′ & C-5′′), 127.15 (C-2′′ & C-6′′), 126.41 (C-3′′′′), 124.36 (C-
6′′′′), 102.18 (C-5), 36.24 (C-2′′′), 27.47 (C-6), 20.42 (CH3-4′′′′), 17.65 (CH3-2′′′′). Anal. Calc. for
C22H22N6OS2 (450.58): C, 58.64; H, 4.92; N, 18.65. Found: C, 58.70; H, 4.94; N, 18.66; EI-MS
(m/z): 450 [M]+, 330 [C14H12N5OS2]+, 270 [C13H10N4OS]+, 256 [C12H10N5S]+, 148 [C9H10NO]+,
120 [C8H10N]+, 71 [C3H3S]+
.
5.5.3. 2-({5-[(2-Amino-1,3-thiazol-4-yl)methyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)-N-(2,5-
dimethylphenyl)acetamide (9c)
o
Dirty yellow solid; Yield: 88%; m.p. 98-99 C; Mol. Formula: C22H22N6OS2; Mol. Mass.:
450 gmol-1; IR (KBr, ʋ/cm-1) : 3349 (N-H str.), 3054 (C-H str. of aromatic ring), 2922 (-CH2-
str.), 1672 (C=O str.), 1561 (C=C str. of aromatic ring), 1517 (C=N str.),1165 (C-N-C bond str.),
623 (C-S str.); 1H-NMR (600 MHz, DMSO-d6, δ/ppm): 9.64 (s, 1H, -CO-NH-1′′′′), 7.53-7.50 (m,
3H, H-3′′, H-4′′ & H-5′′), 7.38-7.36 (m, 2H, H-2′′ & H-6′′), 7.31 (br.s, 1H, H-6′′′′), 7.05 (br.d,
J=7.7, 1H, H-3′′′′), 6.89-6.87 (m, 1H, H-4′′′′), 6.85 (br.s, 2H, H2N-2), 5.86 (s, 1H, H-5), 4.11 (s,
2H, CH2-2′′′), 3.79 (s, 2H, CH2-6), 2.23 (br.s, 3H, CH3-2′′′′), 2.22 (br.s, 3H, CH3-5′′′′); 13C-NMR
(150 MHz, DMSO-d6, δ/ppm): 168.13 (C-2), 165.62 (C-1′′′), 153.48 (C-5′), 149.85 (C-3′),
145.40 (C-4), 135.66 (C-1′′′′), 135.36 (C-5′′′′), 134.86 (C-2′′′′), 132.80 (C-1′′), 129.98 (C-4′′′′),
129.73 (C-4′′), 129.59 (C-3′′′′), 129.49 (C-3′′ & C-5′′), 127.07 (C-2′′ & C-6′′), 124.68 (C-6′′′′),
102.09 (C-5), 36.17 (C-2′′′), 27.40 (C-6), 21.11 (CH3-5′′′′), 16.81 (CH3-2′′′′). Anal. Calc. for
C22H22N6OS2 (450.58): C, 58.64; H, 4.92; N, 18.65. Found: C, 58.76; H, 4.98; N, 18.77; EI-MS
(m/z): 450 [M]+, 330 [C14H12N5OS2]+, 270 [C13H10N4OS]+, 256 [C12H10N5S]+, 148 [C9H10NO]+,
120 [C8H10N]+, 71 [C3H3S]+
.
5.5.4. 2-({5-[(2-Amino-1,3-thiazol-4-yl)methyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)-N-(2,6-
dimethylphenyl)acetamide (9d).
o
Light yellow solid; Yield: 88%; m.p. 166-167 C; Mol. Formula: C22H22N6OS2; Mol.
Mass.: 450 gmol-1; IR (KBr, ʋ/cm-1): 3359 (N-H str.), 3062 (C-H str. of aromatic ring), 2925 (-
26

CH2- str.), 1676 (C=O str.), 1573 (C=C str. of aromatic ring), 1531 (C=N str.),1157 (C-N-C bond
1
str.), 618 (C-S str.); H-NMR (600 MHz, DMSO-d6, δ/ppm): 9.62 (s, 1H, -CO-NH-1′′′′), 7.54-
7.52 (m, 3H, H-3′′, H-4′′ & H-5′′), 7.38-7.37 (m, 2H, H-2′′ & H-6′′), 7.07-7.02 (m, 3H, H-3′′′′, H-
4′′′′ & H-5′′′′), 6.85 (br.s, 2H, H2N-2), 5.85 (s, 1H, H-5), 4.13 (s, 2H, CH2-2′′′), 3.79 (s, 2H, CH2-
13
6), 2.10 (s, 6H, CH3-2′′′′ & CH3-6′′′′); C-NMR (150 MHz, DMSO-d6, δ/ppm): 168.19 (C-2),
165.30 (C-1′′′), 153.33 (C-5′), 149.73 (C-3′), 145.64 (C-4), 135.13 (C-2′′′′ & C-6′′′′), 134.62 (C-
1′′′′), 132.92 (C-1′′), 129.78 (C-4′′), 129.57 (C-3′′ & C-5′′), 127.59 (C-2′′ & C-6′′), 127.14 (C-3′′′′
& C-5′′′′), 126.49 (C-4′′′′), 102.15 (C-5), 35.67 (C-2′′′), 27.46 (C-6), 17.95 (CH3-2′′′′ & CH3-6′′′′).
Anal. Calc. for C22H22N6OS2 (450.58): C, 58.64; H, 4.92; N, 18.65. Found: C, 58.77; H, 4.82; N,
18.69; EI-MS (m/z): 450 [M]+, 330 [C14H12N5OS2]+, 270 [C13H10N4OS]+, 256 [C12H10N5S]+, 148
[C9H10NO]+, 120 [C8H10N]+, 71 [C3H3S]+
.
5.5.5. 2-({5-[(2-Amino-1,3-thiazol-4-yl)methyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)-N-(3,4-
dimethylphenyl)acetamide (9e).
o
Bright yellow solid; Yield: 91%; m.p. 120-121 C; Mol. Formula: C22H22N6OS2; Mol.
Mass.: 450 gmol-1; IR (KBr, ʋ/cm-1): 3338 (N-H str.), 3064 (C-H str. of aromatic ring), 2921 (-
CH2- str.), 1667 (C=O str.), 1563 (C=C str. of aromatic ring), 1527 (C=N str.),1162 (C-N-C bond
1
str.), 632 (C-S str.); H-NMR (600 MHz, DMSO-d6, δ/ppm): 10.16 (s, 1H, -CO-NH-1′′′′), 7.52-
7.50 (m, 3H, H-3′′, H-4′′ & H-5′′), 7.37-7.35 (m, 2H, H-2′′ & H-6′′), 7.32 (br.s, 1H, H-2′′′′), 7.26-
7.25 (m, 1H, H-6′′′′), 7.05 (br.d, J=7.8, 1H,H-5′′′′), 6.87 (br.s, 2H, H2N-2), 5.85 (s, 1H, H-5),
13
4.10 (s, 2H, CH2-2′′′), 3.76 (s, 2H, CH2-6), 2.18 (s, 3H, CH3-4′′′′), 2.14 (s, 3H, CH3-3′′′′); C-
NMR (150 MHz, DMSO-d6, δ/ppm): 168.21 (C-2), 165.18 (C-1′′′), 153.36 (C-5′), 149.72 (C-3′),
145.51 (C-4), 136.47 (C-3′′′′), 136.32 (C-1′′′′), 132.88 (C-1′′), 131.21 (C-4′′′′), 129.77 (C-4′′),
129.54 (C-3′′ & C-5′′), 127.13 (C-2′′ & C-6′′), 120.26 (C-5′′′′), 116.60 (C-2′′′′ & C-6′′′′) 102.18
(C-5), 36.74 (C-2′′′), 27.46 (C-6), 19.58 (CH3-3′′′′) 18.74 (CH3-4′′′′). Anal. Calc. for
C22H22N6OS2 (450.58): C, 58.64; H, 4.92; N, 18.65. Found: C, 58.80; H, 5.02; N, 18.70; EI-MS
(m/z): 450 [M]+, 330 [C14H12N5OS2]+, 270 [C13H10N4OS]+, 256 [C12H10N5S]+, 148 [C9H10NO]+,
120 [C8H10N]+, 71 [C3H3S]+.
27

5.5.6. 2-({5-[(2-Amino-1,3-thiazol-4-yl)methyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)-N-(3,5-
dimethylphenyl)acetamide (9f).
o
Light yellow solid; Yield: 88%; m.p. 110-111 C; Mol. Formula: C22H22N6OS2; Mol.
Mass.: 450 gmol-1; IR (KBr, ʋ/cm-1): 3364 (N-H str.), 3051 (C-H str. of aromatic ring), 2932 (-
CH2- str.), 1673 (C=O str.), 1574 (C=C str. of aromatic ring), 1531 (C=N str.),1162 (C-N-C bond
1
str.), 614 (C-S str.); H-NMR (600 MHz, DMSO-d6, δ/ppm): 10.17 (s, 1H, -CO-NH-1′′′′), 7.53-
7.50 (m, 3H, H-3′′, H-4′′ & H-5′′), 7.37-7.35 (m, 2H, H-2′′ & H-6′′), 7.17 (s, 2H, H-2′′′′ & H-
6′′′′), 6.86 (br.s, 2H, H2N-2), 6.70 (s, 1H, H-4′′′′), 5.85 (s, 1H, H-5), 4.08 (s, 2H, CH2-2′′′), 3.77
(s, 2H, CH2-6), 2.22 (s, 6H, CH3-3′′′′, CH3-5′′′′); 13C-NMR (150 MHz, DMSO-d6, δ/ppm):
168.13 (C-2), 165.31 (C-1′′′), 153.31 (C-5′), 149.63 (C-3′), 145.52 (C-4), 138.52 (C-1′′′′), 137.68
(C-3′′′′ & C-5′′′′), 132.81 (C-1′′), 129.71 (C-4′′), 129.48 (C-3′′ & C-5′′), 127.06 (C-2′′ & C-6′′),
124.94 (C-4′′′′), 116.79 (C-2′′′′ & C-6′′′′) 102.09 (C-5), 36.67 (C-2′′′), 27.40 (C-6), 20.97 (CH3-
3′′′′, CH3-5′′′′). Anal. Calc. for C22H22N6OS2 (450.58): C, 58.64; H, 4.92; N, 18.65. Found: C,
58.82; H, 4.95; N, 18.84; EI-MS (m/z): 450 [M]+, 330 [C14H12N5OS2]+, 270 [C13H10N4OS]+, 256
[C12H10N5S]+, 148 [C9H10NO]+, 120 [C8H10N]+, 71 [C3H3S]+
.
5.5.7. 2-({5-[(2-Amino-1,3-thiazol-4-yl)methyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)-N-(2-
methyphenyl)acetamide (9g).
o
Light orange solid; Yield: 91%; m.p.: 144-145 C; Mol. Formula: C21H20N6OS2; Mol.
Mass.: 436 gmol-1; IR (KBr, ʋ/cm-1): 3346 (N-H str.), 3067 (C-H str. of aromatic ring), 2928 (-
CH2- str.), 1654 (C=O str.), 1556 (C=C str. of aromatic ring), 1532 (C=N str.),1161 (C-N-C bond
1
str.), 628 (C-S str.); H-NMR (600 MHz, DMSO-d6, δ/ppm): 9.70 (s, 1H, -CO-NH-1′′′′), 7.54-
7.52 (m, 3H, H-3′′, H-4′′ & H-5′′), 7.44 (br.d, J=7.9, 1H, H-6′′′′), 7.38-7.36 (m, 2H, H-2′′ & H-
6′′), 7.22 (br.d, J=7.8, 1H, H-3′′′′), 7.15 (br.t, J=7.8, 1H, H-5′′′′), 7.07 (br.t, J=7.8, 1H, H-4′′′′),
6.84 (br.s, 2H, H2N-2), 5.85 (s, 1H, H-5), 4.12 (s, 2H, CH2-2′′′), 3.79 (s, 2H, CH2-6), 2.13 (s, 3H,
CH3-2′′′′); 13C-NMR (150 MHz, DMSO-d6, δ/ppm): 168.21 (C-2), 165.79 (C-1′′′), 153.40 (C-5′),
149.78 (C-3′), 145.58 (C-4), 135.96 (C-1′′′′), 132.88 (C-1′′), 131.15 (C-2′′′′), 130.29 (C-3′′′′),
129.81 (C-4′′), 129.06 (C-3′′ & C-5′′), 127.15 (C-2′′ & C-6′′), 125.94 (C-5′′′′), 125.23 (C-4′′′′),
124.27 (C-6′′′′) 102.18 (C-5), 36.25 (C-2′′′), 27.47 (C-6), 17.71 (CH3-2′′′′). Anal. Calc. for
28

C21H20N6OS2 (436.55): C, 57.78; H, 4.62; N, 19.25. Found: C, 57.57; H, 4.68; N, 19.38; EI-MS
(m/z): 436 [M]+, 330 [C14H12N5OS2]+, 270 [C13H10N4OS]+, 256 [C12H10N5S]+, 134 [C8H8NO]+,
106 [C7H8N]+, 71 [C3H3S]+
.
5.5.8. 2-({5-[(2-Amino-1,3-thiazol-4-yl)methyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)-N-(2-
ethylphenyl)acetamide (9h).
Brick red solid; Yield: 92%; m.p.: 98-99 oC; Mol. Formula: C22H22N6OS2; Mol. Mass.: 450
gmol-1; IR (KBr, ʋ/cm-1): 3365 (N-H str.), 3045 (C-H str. of aromatic ring), 2915 (-CH2- str.),
1658 (C=O str.), 1582 (C=C str. of aromatic ring), 1519 (C=N str.), 1152 (C-N-C bond str.), 611
(C-S str.); 1H-NMR (600 MHz, DMSO-d6, δ/ppm): 9.69 (s, 1H, -CO-NH-1′′′′), 7.53-7.51 (m, 3H,
H-3′′, H-4′′ & H-5′′), 7.40-7.36 (m, 2H, H-2′′, H-6′′ & H-6′′′′), 7.22 (br.d, J=7.3, 1H, H-3′′′′),
7.17-7.13 (m, 2H, H-4′′′′ & H-5′′′′), 6.85 (br.s, 2H, H2N-2), 5.86 (s, 1H, H-5), 4.13 (s, 2H, CH2-
2′′′), 3.79 (s, 2H, CH2-6), 2.57 (q, J=7.5, 2H, CH3-CH2-2′′′′), 1.09 (t, J=7.5, 3H, CH3-CH2-2′′′′);
13C-NMR (150 MHz, DMSO-d6, δ/ppm): 168.12 (C-2), 165.99 (C-1′′′), 153.35 (C-5′), 149.79 (C-
3′), 145.52 (C-4), 137.24 (C-1′′′′), 135.14 (C-2′′′′), 132.79 (C-1′′), 129.72 (C-4′′), 129.49 (C-3′′ &
C-5′′), 128.45 (C-3′′′′), 127.06 (C-2′′ & C-6′′), 125.82 (C-5′′′′), 125.54 (C-4′′′′), 125.12 (C-6′′′′)
102.08 (C-5), 36.12 (C-2′′′), 27.38 (C-6), 23.58 (CH3-CH2-2′′′′), 14.11 (CH3-CH2-2′′′′). Anal.
Calc. for C22H22N6OS2 (450.58): C, 58.64; H, 4.92; N, 18.65. Found: C, 58.56; H, 4.85; N,
+
+
18.58; EI-MS (m/z): 450 [M]+, 330 [C14H12N5OS2] , 270 [C13H10N4OS] , 256 [C12H10N5S]+,
189 [C9H7N3S] +, 113 [C4H5N2S]+, 91 [C6H5N] +, 77 [C6H5] +, 71 [C3H3S]+
.
5.5.9. 2-({5-[(2-Amino-1,3-thiazol-4-yl)methyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)-N-(2-
ethyl-6-methyphenyl)acetamide (9i).
o
Light yellow solid; Yield: 90%; m.p.: 194-195 C; Mol. Formula: C23H24N6OS2; Mol.
Mass.: 464 gmol-1; IR (KBr, ʋ/cm-1): 3348 (N-H str.), 3059 (C-H str. of aromatic ring), 2926 (-
CH2- str.), 1664 (C=O str.), 1578 (C=C str. of aromatic ring), 1522 (C=N str.), 1144 (C-N-C
1
bond str.), 612 (C-S str.); H-NMR (600 MHz, DMSO-d6, δ/ppm): 9.61 (s, 1H, -CO-NH-1′′′′),
7.53 (m, 3H, H-3′′, H-4′′ & H-5′′), 7.38 (m, 2H, H-2′′ & H-6′′) 7.11 (dis.t, J=7.26, 1H, H-4′′′′),
7.06 (dis.d, J=7.38, 2H, H-3′′′′ & H-5′′′′), 6.85 (br.s, 2H, H2N-2), 5.85 (s, 1H, H-5), 4.14 (s, 2H,
29