Hit to lead optimization of a series of N-[4-(1,3-benzothiazol-2-yl)phenyl]acetamides as monoacylglycerol lipase inhibitors with potential anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2016.05.038

A total of thirty five new N-[4-(1,3-benzothiazol-2-yl)phenyl]acetamide derivatives were synthesized and structures of all the compounds were confirmed on the basis of elemental analysis and collective use of IR, ¹H NMR, ¹³C NMR and

Full text of DOI:10.1016/j.ejmech.2016.05.038

Accepted Manuscript
Hit to lead optimization of a series of N-[4-(1,3-benzothiazol-2-yl)phenyl]acetamides
as monoacylglycerol lipase inhibitors with potential anticancer activity
Obaid Afzal, Md Sayeed Akhtar, Suresh Kumar, Md Rahmat Ali, Manu Jaggi,
Sandhya Bawa
PII:
S0223-5234(16)30436-6
10.1016/j.ejmech.2016.05.038
EJMECH 8630
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 February 2016
Revised Date: 27 April 2016
Accepted Date: 19 May 2016
Please cite this article as: O. Afzal, M.S. Akhtar, S. Kumar, M.R. Ali, M. Jaggi, S. Bawa, Hit to lead
optimization of a series of N-[4-(1,3-benzothiazol-2-yl)phenyl]acetamides as monoacylglycerol lipase
inhibitors with potential anticancer activity, European Journal of Medicinal Chemistry (2016), doi:
10.1016/j.ejmech.2016.05.038.
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ACCEPTED MANUSCRIPT

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Hit to lead optimization of a series of N-[4-(1,3-benzothiazol-2-yl)phenyl]acetamides as
monoacylglycerol lipase inhibitors with potential anticancer activity
Obaid Afzal1, Md Sayeed Akhtar2, Suresh Kumar1, Md Rahmat Ali1, Manu Jaggi3, Sandhya
Bawa1*
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard University, New Delhi-110062,
India.
2 Department of Pharmacology, Faculty of Pharmacy, Hamdard University, New Delhi-110062, India.
3 Dabur Research Foundation, Ghaziabad, Uttar Pradesh, India.
* Corresponding author:
Sandhya Bawa (Ph.D)
Associate Professor
Department of Pharmaceutical Chemistry
Faculty of Pharmacy
Jamia Hamdard (Hamdard University)
New Delhi-110062
Tel.: +91 11 26059688(5644).
E-mail: drsbawa@rediffmail.com
sbawa@jamiahamdard.ac.in

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Abstract
A total of thirty five new N-[4-(1,3-benzothiazol-2-yl)phenyl]acetamide derivatives were
synthesized and structures of all the compounds were confirmed on the basis of elemental
1
13
analysis and collective use of IR, H-NMR, C-NMR and mass spectral data. Compounds
were tested for their ability to inhibit human monoacylglycerol lipase (hMAGL) enzyme.
Eight compounds 4, 19-21, 24-26, and 34 reduced the hMAGL activity less than 50 % at 100
nM concentrations. The halogen substituted aniline derivatives 20, 21 and 24-26 were found
to be most active among all the synthesized compounds having IC50 value in the range of
6.5–9 nM. Twenty five compounds were selected by NCI, USA for one dose anticancer
screening. Compound 21 (NSC: 780167) and 24 (NSC: 780168) fulfilled prearranged
doorstep growth inhibition criteria and further selected for NCI full panel five dose assay at
10-fold dilutions of five different concentrations (0.01, 0.1, 1, 10 and 100 ꢀM). Both the
compounds 21 and 24 were found to be most active against MCF7 and MDA-MB-468 breast
cancer cell lines. The GI50 value of 32.5 nM (MCF7) and 23.8 nM (MDA-MB-468) was
observed for compound 21. Compound 24 showed GI50 values of 37.1 nM against MCF7
breast cancer cell line and 25.1 nM against MDA-MB-468 breast cancer cell line.
Keywords
1,3-Benzothiazole; In-vitro anticancer activity; Monoacylglycerol lipase; NCI 60 cell line.

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1. Introduction
Human monoacylglycerol lipase (MAGL) is a serine hydrolase enzyme that belongs to the
super family of α/β-hydrolases. It is a 33-kDa protein consisting of 303 amino acid residues.
It hydrolyzes one of the major endocannabinoid 2-AG (2-arachidonoylglycerol), an
endogenous full agonist at G-protein coupled cannabinoid receptors CB1 and CB2, into free
fatty acids (FFA) and glycerol through a classical Ser122, Asp239 and His269 catalytic triad
[1-4].
The regulatory importance of MAGL in cancer has gained recently. Cancer cells adjust their
metabolism on the way to a lipolytic pathway by the over expression of MAGL, to produce
indispensable lipid blocks for their accelerated metabolism and proliferation and to
synthesize various pro-tumorigenic lipid mediators [5,6]. It has been reported that MAGL
regulates a fatty acid network in cancer and is highly aggravated in aggressive cancer cells,
including melanoma, breast cancer, ovarian cancer, colorectal carcinoma, and prostate
cancer. Pharmacological inhibition or genetic knockdown studies showed that MAGL
correlates with high proliferation, pathogenesis and in vivo tumour growth [7-9]. The cancer
promoting activity of MAGL is not due to deactivation of its usual substrate
monoacylglycerol (2-AG), but due to an elevated free fatty acids (FFA) level, which
suggested a new metabolic function of this enzyme [10]. MAGL activity also leads to an
increase in specific FFA-derived oncogenic signalling lipids, such as phosphatidic acid (PA),
lysophosphatidic acid (LPA), sphingosine-1-phosphate (S1P) and prostaglandin E2 (PGE2).
These local lipid mediators induce cell proliferation, survival, and migration/invasion, thus
favouring tumor growth, angiogenesis, and metastasis in cancer [11]. The exciting role of
elevated endocannabinoid tone in the anticancer effects of MAGL inhibition cannot be
excluded, in view of the widespread pharmacological indication of the cancer-inhibiting
properties of cannabinoid (CB) agonists. The dual advantage of MAGL inhibition could be
beneficial in the treatment of cancer. In view of the fact that MAGL is not required for cell
survival, but instead promotes progression to a more aggressive phenotype, inhibitors of
MAGL may have a better safety profile and offer advantages over existing treatments for
cancer.
In recent years, novel MAGL inhibitors have been identified that includes LY2183240 [12-
14], URB602 [15], CAY10499 [16, 17], SPB01403 [18], JZL184 [19], KML29 [20], ML30
[21] and JJKK-048 [22]. Recently, the 3D-structure of the human MAGL protein was
elucidated by X-ray diffraction method (PDB ID: 3HJU, Resolution: 2.2 Å) [23].
Simultaneously, the crystal structure of the MAGL in bound with the inhibitor SAR629 (a

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triazolo carboxamide) was also reported [24]. Another X-ray crystal structure of the inhibitor
(ZYH) bound form of human MAGL was determined with the use of molecular replacement
method to a resolution of 1.35 Å (PDB ID: 3PE6) [25]. The structure of the above mentioned
representative MAGL inhibitors, along with their IC50 values are presented in Fig. 1.
Our group has recently identified a novel human MAGL inhibitor, ZINC24092691 (N-[4-
(1,3-benzothiazol-2-yl)phenyl]-2-(benzotriazol-1-yl)acetamide; Fig. 1) by docking based
virtual screening of ZINC database, with an IC50 value of 10 nM [26]. In addition, the
anticancer potential of benzothiazole derivatives especially 2-(4-aminophenyl)-benzothiazole
(CJM 126), 2-(4-Amino-3methylphenyl)benzothiazole (DF 203), 2-(3,4-Dimethoxyphenyl)-
5-fluorobenzothiazole (GW 610) and related compounds are well documented in literature
[27-30]. The anticancer potential of MAGL inhibitors as well as of benzothiazole derivatives
prompted us for further hit to lead optimization of the identified MAGL inhibitor and
screening for their potential anticancer activity.
2. Result and discussion
ZINC24092691 (Compound 4), was identified as a potential hit, after docking-based virtual
screening and in vitro MAGL inhibition assay, having an IC50 value of 10 nM [26]. It was
chosen for further hit to lead optimization. The syntheses of ZINC24092691 (4) and its
analogues (5-38) were carried out as per Fig. 2 (Scheme 1).
2.1. Chemistry
2-(4-Nitrophenyl)benzo[d]thiazole (1) was synthesized by direct condensation of 2-
aminobenzenethiol and 4-nitrobenzoyl chloride. Usually the reaction is exothermic, but
complete conversion and higher yield was achieved by refluxing the reactants in pyridine.
Reduction of the nitro compound (1) with stannous chloride in absolute ethanol under
nitrogen atmosphere furnished amine (2). Compounds 1 and 2 were synthesized as per the
reported procedure with slight modification [27]. The 2-(4-aminophenyl)benzo[d]thiazole (2)
was further reacted with chloroacetyl chloride in the presence of powdered anhydrous
potassium carbonate as base in anhydrous dichloromethane to yield penultimate intermediate
N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-chloroacetamide (3). The final secondary amine
derivatives (4-16) were synthesized by stirring and heating equimolar quantities of N-(4-
(benzo[d]thiazol-2-yl)phenyl)-2-chloroacetamide (3) and appropriate secondary amines in
anhydrous N,N-dimethylformamide containing powdered anhydrous potassium carbonate.
However, primary amine derivatives (17-38) were obtained by refluxing equimolar quantities
of N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-chloroacetamide (3) and appropriate primary amines
in absolute ethanol containing catalytic amount of triethylamine. The structures of all the

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1
compounds were confirmed on the basis of elemental analysis and collective use of IR, H-
13
NMR, C-NMR and mass spectral data. The elemental analysis of the final compounds was
found to be within ± 0.4 % limit.
The IR spectrum of the prototype compound 4 (ZINC24092691) showed stretching bands at
1
3379 and 1695 cm-1 due to characteristic N-H and C=O groups respectively. In H NMR
spectrum, the singlet appearing at δ 10.97 showed the presence of D2O exchangeable amide
N-H proton. All the twelve aromatic protons of benzothiazole, benzotriazole and phenyl ring
appeared resonating between δ 8.09-7.43. A singlet appearing at δ 5.76 was assigned to two
CH2 protons. The mass spectrum of this prototype compound 4 showed the molecular ion
peak i.e. m/z (M+) at 385.10, which confirmed its successful synthesis.
In general, IR spectra of the compounds 5-16, showed characteristic stretching bands at
around 3338 and 1668 cm-1 for N-H and C=O groups of acetamide respectively. In 1H NMR
spectra of these compounds, one singlet appeared at around δ 10.49 which indicated the
presence of N-H proton and it disappeared by addition of D2O as confirmation for this group.
The other characteristic singlet for two CH2 protons of acetamide appeared at around δ 4.51.
Among compounds 17-38, the spectral discussion of unsubstituted aniline containing
derivative 19 is provided hereafter. The IR spectrum of compound 19 showed stretching
bands at 3094, 1592 and 1516 cm-1, due to Ar C-H, C=N and C=C groups respectively. The
bands at 3422 and 3310 cm-1 showed characteristic N-H stretching. While, the band at 1657
1
cm-1 showed characteristic C=O stretching. In H NMR spectrum, the singlet appearing at δ
10.33 showed the presence of amide N-H proton. The multiplet resonating between δ 8.13-
8.01 indicated four aromatic protons, H-4 & H-7 of benzothiazole and 2ꞌ & 6ꞌ of the phenyl
ring. The doublet appeared resonating between δ 7.84-7.81 (J = 9 Hz) showed two aromatic
protons 3ꞌ and 5ꞌ of phenyl ring. The triplet appearing at δ 7.52 (J = 7.5 Hz) and δ 7.43 (J =
7.5 Hz) indicated two protons, H-5 and H-6 of benzothiazole ring respectively. The triplet
resonating at δ 7.10 (J = 7.5 Hz) indicated three protons 3ꞌꞌ, 4ꞌꞌ and 5ꞌꞌ of the distal phenyl
ring. The doublet appeared resonating between δ 6.63-6.60 (J = 9 Hz) showed two aromatic
protons 2ꞌꞌ and 6ꞌꞌ of distal phenyl ring. A broad singlet at δ 6.01 (D2O exchangeable)
indicated one phenyl N-H proton. A singlet appearing at δ 3.93 was assigned to two CH2
protons. The mass spectrum of the compound 19 showed the molecular ion peak i.e. m/z (M+)
at 359.13, which is in agreement with the structure of this compound.
2.2. In vitro human MAGL enzyme inhibition assay
The in vitro human MAGL inhibition assay was carried out by the method reported by
Muccioli et al. [17]. Out of the thirty compounds tested, most of them were found to inhibit

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MAGL. Eight compounds 4, 19-21, 24-26, and 34 reduced the MAGL activity less than 50 %
at 100 nM concentrations. Compounds 4-6, 14-21, 24-26 and 34 showed IC50 value in
nanomolar range, while compounds 11-13, 29, 31, 33 and 35-38 showed IC50 value in
micromolar range. Compounds 7-10 and 27 had much weaker inhibitory effect against
hMAGL upto 100 ꢀM concentrations. The halogen substituted aniline derivatives 20, 21 and
24-26 were found to be most active among all the synthesized compounds having IC50 value
in the range of 6.5 – 9 nM. The results for CAY10499 (IC50 = 424 nM) were comparable to
that reported previously (IC50 = 350 nM) [17].
The structure activity relationship (SAR) in relation to hMAGL inhibition revealed that
replacement of benzotriazole moiety in compound 4 with benzimidazole or indole ring,
decreases activity, but IC50 remains within nanomolar range. Five membered heterocyclic
derivatives like imidazole, triazole, pyrazole or isoxazole and six member saturated
heterocyclic derivatives having piperidine, morpholine and piperazine are more potent than
the derivatives having bulky groups like isatin, diphenylamine, carbazole or
tetrahydrocarbazole. Substitution with cyclohexylamine and benzylamine also decreased
activity, but not to greater extent. Substitution with halogen substituted anilines increased
activity and the order is 3-Cl, 4-F > 2,6-Cl = 4-F > 4-Cl = 4-Br (IC50 = 6.5 - 9 nM). Nitro
group at 4th position completely abolished the activity, while 4-CH3, 4-OCH3 and 4-OH
substituted aniline products showed IC50 in ꢀM range. Replacement with unsubstituted
aniline and 4-SO2NH2 aniline showed almost similar potency. 4-Cl Phenyl thiazole or 4-Cl
phenyl oxadiazole substituted products showed similar potency with an IC50 of 47 and 48 ꢀM
respectively.
2.3. Anticancer screening
Compounds were submitted to National Cancer Institute (NCI) USA, for anticancer
screening.
2.3.1. One dose assay
All the synthesized compounds were submitted to NCI, USA, out of which twenty five
compounds were selected for one dose anticancer screening. Single dose results for each
tested compounds were reported as a mean graph of the percent growth (% G) of the treated
cells and deliberated by comparing with untreated control cells as percentage of growth
inhibition (% GI) over all the tested cell lines [31, 32] and are presented in Table
(Supplementary materials). Compound 21 (NSC: 780167) and 24 (NSC: 780168) were the
most potent anticancer compounds detected in one dose anticancer screening by NCI and
both of them showed potent anticancer activity towards all the cancer cell lines. 4-Cl

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substituted derivative (21) showed GI50 value in range of 23.8 nM – 13 ꢀM, while 2,6-Cl
derivative (24) showed GI50 in the range of 25.1 nM – 13.80 ꢀM against all the 60 cell lines.
Unsubstituted aniline derivative (19), sulphonamide derivative (34) and N-methyl piperazine
derivative (16) also showed potent anticancer activity, but were not selected for further study.
Remaining of the selected derivatives, irrespective of their potency towards MAGL
inhibition, showed potent anticancer activity towards MDA-MB-468 breast cancer cell line.
2.3.2. Five dose assay
The compounds that reduced the growth of the cell lines to 30 % or less (negative number
indicate kills) were considered in vitro active [33]. Compound 21 (NSC: 780167) and 24
(NSC: 780168) fulfilled prearranged doorstep growth inhibition criteria and were further
selected for NCI full panel five dose assay at 10-fold dilutions of five different concentrations
(0.01, 0.1, 1, 10 and 100 ꢀM). The results of tested compounds were given by three response
parameters (GI50, TGI and LC50) for each cell line from log concentration verses % growth
inhibition curves on nine cancer disease. The in vitro anticancer five dose assay (0.01, 0.1, 1,
10, and 100 ꢀM) results of compounds 21 and 24 against NCI 60 cell lines, in terms of
response parameters like GI50, TGI & LC50 (in ꢀM) is presented in Table-2. Compound 21
and 24 exhibited excellent anticancer activity against all the tested cell lines with MID GI50
values 2.08 and 1.99 ꢀM, beneath responsive range and outstanding activity. Compound 21
showed an overall GI50 value ranging from 23.8 nM – 13 ꢀM and TGI value ranging from
353 nM – 61.80 ꢀM. While, compound 24 exhibited an overall GI50 value ranging from 25.1
nM – 15.30 ꢀM and TGI value ranging from 137 nM – 69.30 ꢀM. With regard to the
sensitivity against individual cell lines both the compounds 21 and 24 were found to be most
active against MCF7 and MDA-MB-468 breast cancer cell lines. The GI50 value of 32.5 nM
(MCF7) and 23.8 nM (MDA-MB-468) was observed for compound 21. Compound 24
showed GI50 values of 37.1 nM against MCF7 breast cancer cell line and 25.1 nM against
MDA-MB-468 breast cancer cell line. The obtained results from Developmental Therapeutics
Program, NCI, for compounds 21 and 24 were compared with the standard drug Fluorouracil
in terms of potency by three response parameter (in ꢀM) and presented in Table-3. In terms
of MID GI50 value, both the compounds were found to be comparable with 5-FU, while in
terms of MID TGI and MID LC50, both showed greater potency.
Dose response curves for all cancer cell lines in the NCI 60 panel exposed to synthetic
compounds 21 (NSC: 780167) and 24 (NSC: 780168) were colour coded by tissues of origin:
red, leukemia cell line; blue, lung cancer; green, colon cancer; gray, central nervous system
cancer; coral, melanoma; purple, ovarian cancer; gold, renal cancer; turquoise, prostate

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cancer; pink, breast cancer (Fig. 3a & 3b). The dose response curve was created by plotting
the % G against log10 of corresponding sample concentration (molar) for each cell line.
Horizontal lines have been provided at the % G values of +50, 0 and -50. The concentrations
of tested compounds corresponding to points where the curves cross these lines are GI50, TGI
and LC50, respectively [34, 35].
3. Summary and Conclusion
The synthesis of ZINC24092691 (4) analogues (5-38) were carried out successfully as per the
scheme outlined in Fig. 2 (Scheme 1). A total of thirty selected compounds were tested for
their ability to inhibit human MAGL. The halogen substituted aniline derivatives 20, 21 and
24-26 were found to be most active among all the synthesized compounds having IC50 value
in the range of 6.5–9 nM. All the synthesized compounds were submitted to NCI, US, out of
which twenty five compounds were selected for one dose anticancer screening. Overall one
dose assay results showed that majority of the compounds were active against MCF7 and
MDA-MB-468 breast cancer cell lines. Compound 21 (NSC: 780167) and 24 (NSC: 780168)
were the most potent anticancer compounds detected in one dose anticancer screening. They
fulfilled prearranged doorstep growth inhibition criteria and further selected for NCI full
panel five dose assay. With regard to the sensitivity against individual cell lines both the
compounds 21 and 24 were found to be most active against MCF7 and MDA-MB-468 breast
cancer cell lines. The GI50 value of 32.5 nM (MCF7) and 23.8 nM (MDA-MB-468) was
observed for compound 21. Compound 24 showed GI50 values of 37.1 nM against MCF7
breast cancer cell line and 25.1 nM against MDA-MB-468 breast cancer cell line. The results
obtained from Developmental Therapeutics Program, NCI, for compounds 21 and 24 were
compared with the standard drug Fluorouracil. In terms of MID GI50 value, both the
compounds were found to be comparable with 5-FU, while in terms of MID TGI and MID
LC50, both showed greater potency. In conclusion, the overall study confirmed that
pharmacological inhibition of MAGL function may confer significant therapeutic benefits
and a better safety profile over existing treatments for cancer.
4. Experimental
4.1. Chemistry
Required reagents and chemicals used for the experimental work were purchased from Sigma
Aldrich (India), E. Merck (India) Ltd., S. D. Fine (India) Chemicals Ltd. and Qualigens
(India). Progress of reaction and purity of the compounds were assessed by TLC. Iodine
chamber and UV lamps were used for visualization of TLC spots. The silica gel G (E. Merck
India Ltd.) was used as stationary phase for TLC. The mobile phase solvent system used was

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TEF (Toluene:Ethylaceate:Formic acid) in the ratio of 5:4:1 and 6:3.5:0.5. Final compounds
were purified by column chromatography technique. Silica gel 60-120 mesh LR purchased
from E. Merck India Ltd., was used for column chromatography as stationary phase. The
solvent system used in column chromatography was hexane: ethyl acetate (4:1). The Infra-red
(IR) spectra of the synthesized compounds were recorded in the region of 4000-400 cm-1
range, on a Shimadzu FT-IR spectrometer by making KBr pellets. The proton nuclear
magnetic resonance spectra (1H NMR) were recorded on Bruker (DPX) 300 MHz instrument
in solvent (DMSO-d6 or CDCl3). The chemical shifts are given in δ (ppm) downfield from
tetramethylsilane (TMS) as internal standard. The carbon-13 nuclear magnetic resonance
spectra (13C NMR) were recorded on Bruker (DPX) 125 MHz instrument in solvent DMSO-
d6. UPLC analyses of the compounds were performed on a Waters ACQUITY UPLC system
(Waters Corp., MA, USA). The mobile phase consisted of acetonitrile : 2 mmol ammonium
acetate buffer (90:10, v/v). Synapt-mass-spectrometric detection was carried out on a UPLC–
MS (Q-TOF-ESI) (Waters Corp., USA) with an electrospray ionisation (ESI) technique.
Elemental analysis was performed on the CHNOS-Elemental analyzer (Vario EL III) using
sulphanilic acid as standard and tungsten (VI) oxide as combusting agent. Sample for analysis
was prepared in Tin boats of dimension 6×6×12 mm. Melting points (M.P.) of the
synthesized compounds were determined using open capillary tubes on electrical melting
point apparatus and were uncorrected.
4.1.1. 2-(4-nitrophenyl)benzo[d]thiazole (1)
To a solution of 4-nitrobenzoyl chloride (0.03 mol) in pyridine (20 ml), 2-aminobenzenethiol
(0.03 mol) was added drop wise with stirring. The reaction mixture was then refluxed for 1
hour. The resulting mixture was cooled to room temperature and poured into crushed ice. The
precipitate thus formed was filtered, washed with water and recrystallised from ethanol. The
progress of reaction and purity of compound was checked by TLC using TEF (5:4:1) as
mobile phase. Yellow solid; Yield: 74 %; M.P.: 229-231 ºC; IR (KBr, cm-1): 3070 (Ar C-H),
1
1681 (C=N), 1560 (C=C), 1519 (NO2, asym), 1340 (NO2, asym); H NMR (DMSO-d6, 300
MHz) δ (ppm): 8.41-8.38 (d, 2H, H-3ꞌ & H-5ꞌ, J =9 Hz), 8.36-8.33 (d, 2H, H-2ꞌ & H-6ꞌ, J =9
Hz), 8.22-8.19 (d, 1H, H-4, J = 9 Hz), 8.16-8.13 (d, 1H, H-7, J = 9 Hz), 7.63-7.58 (t, 1H, H-
5, J = 7.5 Hz), 7.56-7.51 (t, 1H, H-6, J = 7.5 Hz); ESI-MS (m/z): 256.13 (M+); Anal. calcd.
for C13H8N2O2S: C, 60.93; H, 3.15; N, 10.93. Found: C, 60.76; H, 3.12; N, 10.97.
4.1.2. 2-(4-aminophenyl)benzo[d]thiazole (2)
A mixture of 2-(4-nitrophenyl)benzo[d]thiazole (1) (0.015 mol) and stannous chloride (0.075
mol) in absolute ethanol (50 ml) was stirred and refluxed under nitrogen for 6 h. After

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evaporation of ethanol, ethyl acetate was added to the residue and the organic layer was
washed with 2M aqueous NaOH (3×100 ml) and water (2×100 ml) and dried with Na2SO4.
The solvent was removed under reduced pressure. The crude product was crystallized from
ethanol to afford 2-(4-aminophenyl)benzo[d]thiazole (2). The progress of reaction and purity
of compound was checked by TLC using TEF (5:4:1) as mobile phase. Pale yellow solid;
Yield: 90 %; M.P.: 155-157 ºC; IR (KBr, cm-1): 3458 (N-H), 3180 (Ar C-H), 1642 (C=N),
1
1602 (C=C); H NMR (CDCl3, 300 MHz) δ (ppm): 8.03-8.00 (d, 1H, H-4, J = 9 Hz), 7.93-
7.90 (d, 2H, H-2ꞌ & H-6ꞌ, J = 9 Hz), 7.88-7.85 (d, 1H, H-7, J = 9 Hz), 7.49-7.44 (t, 1H, H-5, J
= 7.5 Hz), 7.36-7.31 (t, 1H, H-6, J = 7.5 Hz), 6.77-6.74 (d, 2H, H-3ꞌ & H-5ꞌ, J = 9 Hz), 4.02
(bs, 2H, NH2, D2O exchangeable); ESI-MS (m/z): 226.11 (M+); Anal. calcd. for C13H10N2S:
C, 69.00; H, 4.45; N, 12.38. Found: C, 69.23; H, 4.47; N, 12.41.
4.1.3. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-chloroacetamide (3)
To a solution of 2-(4-aminophenyl)benzo[d]thiazole (2) (0.016 mol) in DCM (30 ml),
powdered anhydrous K2CO3 (1 g) was added. Chloroacetyl chloride (0.03 mol) was added
drop wise with constant stirring at 10 ºC. The reaction mixture was stirred further for 2 h.
After evaporation of DCM, water was added to the residue and stirred for 20 min. The
precipitate thus obtained was filtered, washed with water and dried. The crude product was
crystallized from ethanol. The progress of reaction and purity of compound was checked by
TLC using TEF (5:4:1) as mobile phase. White solid; Yield: 92 %; M.P.: 143-145 ºC; IR
(KBr, cm-1): 3327 (N-H), 3094 (Ar C-H), 1670 (C=O), 1595 (C=N), 1523 (C=C), 761 (C-Cl);
1H NMR (DMSO-d6, 300 MHz) δ (ppm): 10.60 (s, 1H, NH, D2O exchangeable), 8.11-8.00
(m, 4H, H-4, H-7, H-2ꞌ & H-6ꞌ), 7.81-7.78 (d, 2H, H-3ꞌ & H-5ꞌ, J = 9 Hz), 7.54-7.49 (t, 1H,
H-5, J = 7.5 Hz), 7.45-7.40 (t, 1H, H-6, J = 7.5 Hz), 4.30 (s, 2H, CH2); 13C NMR (DMSO-d6,
125 MHz) δ (ppm): 167.22 (C-2), 165.52 (C=O), 154.19 (C-9), 141.73 (C-4ꞌ), 134.74 (C-8),
128.70 (C-1ꞌ), 128.51 (C-2ꞌ & C-6ꞌ), 127.00 (C-5), 125.86 (C-6), 123.03 (C-4), 122.84 (C-7),
120.01 (C-3ꞌ & C-5ꞌ), 44.11 (CH2); ESI-MS (m/z): 302.18 (M+), 304.18 (M+ + 2); Anal.
calcd. For C15H11ClN2OS: C, 59.50; H, 3.66; N, 9.25. Found: C, 59.56; H, 3.57; N, 9.18.
4.1.4. General method for the synthesis of secondary amine derivatives of N-(4-(benzo[d]
thiazol-2-yl)phenyl)-2-chloroacetamide (4-16)
Equimolar quantity of N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-chloroacetamide (3) (10 mmol)
and appropriate secondary amine (10 mmol) was stirred in DMF (15 ml) containing
powdered anhydrous K2CO3 (1 g) at 60 ºC for 4 h. The progress of reaction and purity of
compound was checked by TLC using TEF (6:3.5:0.5) as mobile phase. The reaction mixture
was then cooled, poured into cold water and stirred at room temperature for further 30

ACCEPTED MANUSCRIPT
minutes. The precipitate so obtained was filtered, washed with water, dried and then purified
through column chromatography (Hexane/AcOEt 4:1) to afford final compounds (4-16).
4.1.4.1. 2-(1H-benzo[d][1,2,3]triazol-1-yl)-N-(4-(benzo[d]thiazol-2-yl)phenyl)acetamide (4)
White solid; Yield: 72 %; M.P.: 210-212 ºC; IR (KBr, cm-1): 3379 (N-H), 3055 (Ar C-H),
1
1695 (C=O), 1606 (C=N), 1548 (C=C); H NMR (DMSO-d6, 300 MHz) δ (ppm): 10.97 (bs,
1H, NH, D2O exchangeable), 8.09-7.43 (m, 12H, Ar-H), 5.76 (2H, CH2); 13C NMR (DMSO-
d6, 125 MHz) δ (ppm): 167.31 (C-2), 165.32 (C=O), 154.12 (C-9), 145.60 (C-9ꞌꞌ), 144.53 (C-
8ꞌꞌ), 141.69 (C-4ꞌ), 134.83 (C-8), 128.67 (C-2ꞌ & C-6ꞌ), 127.92 (C-1ꞌ), 127.11 (C-5), 125.83
(C-6), 124.43 (C-4ꞌꞌ), 123.15 (C-4), 122.77 (C-7), 120.09 (C-3ꞌ & C-5ꞌ), 119.55 (C-5ꞌꞌ), 118.38
(C-6ꞌꞌ), 111.46 (C-7ꞌꞌ), 51.09 (CH2); ESI-MS (m/z): 385.10 (M+); Anal. calcd. for
C21H15N5OS: C, 65.44; H, 3.92; N, 18.17. Found: C, 65.67; H, 3.91; N, 18.23.
4.1.4.2. 2-(1H-benzo[d]imidazol-1-yl)-N-(4-(benzo[d]thiazol-2-yl)phenyl)acetamide (5)
White solid; Yield: 88 %; M.P.: 204-205 ºC; IR (KBr, cm-1): 3396 (N-H), 3047 (Ar C-H),
1
1701 (C=O), 1597 (C=N), 1533 (C=C); H NMR (DMSO-d6, 300 MHz) δ (ppm): 10.82 (s,
1H, NH, D2O exchangeable), 8.27 (s, 1H, H-2ꞌꞌ), 8.14-8.01 (m, 4H, H-4, H-7, H-2ꞌ & H-6ꞌ),
7.83-7.80 (d, 2H, H-3ꞌ & H-5ꞌ, J = 9.0 Hz), 7.70-7.68 (d, 1H, H-4ꞌꞌ, J = 6.0 Hz), 7.59-7.41 (m,
3H, H-5, H-6 & H-7ꞌꞌ), 7.27-7.22 (t, 2H, H-5ꞌꞌ & H-6ꞌꞌ, J = 7.5 Hz), 5.25 (s, 2H, CH2); ESI-
MS (m/z): 384.18 (M+); Anal. calcd. for C22H16N4OS: C, 68.73; H, 4.19; N, 14.57. Found: C,
68.94; H, 4.18; N, 14.53.
4.1.4.3. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(1H-indol-1-yl)acetamide (6)
White solid; Yield: 82 %; M.P.: 198-200 ºC; IR (KBr, cm-1): 3298 (N-H), 3053 (Ar C-H),
1
1681 (C=O), 1591 (C=N), 1519 (C=C); H NMR (DMSO-d6, 300 MHz) δ (ppm): 10.63 (s,
1H, NH, D2O exchangeable), 8.28-8.06 (m, 4H, H-4, H-7, H-2ꞌ & H-6ꞌ), 7.82-7.79 (d, 2H, H-
3ꞌ & H-5ꞌ, J = 9 Hz), 7.56-7.51 (t, 1H, H-5, J = 7.5 Hz), 7.46-7.41 (t, 1H, H-6, J = 7.5 Hz),
7.28-7.25 (d, 2H, H-4ꞌꞌ & H-7ꞌꞌ, J = 9 Hz), 7.18-7.13 (t, 2H, H-5ꞌꞌ & H-6ꞌꞌ, J = 7.5 Hz), 7.06-
7.03 (d, 1H, H-2ꞌꞌ, J = 9 Hz), 6.52-6.49 (d, 1H, H-3ꞌꞌ, J = 9 Hz), 5.11 (s, 2H, CH2); ESI-MS
(m/z): 383.16 (M+); Anal. calcd. for C23H17N3OS: C, 72.04; H, 4.47; N, 10.96. Found: C,
72.28; H, 4.48; N, 10.98.
4.1.4.4. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(2,3-dioxoindolin-1-yl)acetamide (7)
Pale yellow solid; Yield: 72 %; M.P.: 174-175 ºC; IR (KBr, cm-1): 3317 (N-H), 3062 (Ar C-
1
H), 1737, 1725, 1683, (C=O), 1610 (C=N), 1543 (C=C); H NMR (DMSO-d6, 300 MHz) δ
(ppm): 10.60 (s, 1H, NH, D2O exchangeable), 8.14-8.01 (m, 4H, H-4, H-7, H-2ꞌ & H-6ꞌ),
7.80-7.77 (d, 2H, H-3ꞌ & H-5ꞌ, J = 9 Hz), 7.56-7.51 (t, 1H, H-5, J = 7.5 Hz), 7.46-7.41 (t, 1H,
H-6, J = 7.5 Hz), 7.71-7.69 (d, 1H, H-7ꞌꞌ, J = 6 Hz), 7.64-7.62 (d, 1H, H-4ꞌꞌ, J = 6 Hz), 7.21-

ACCEPTED MANUSCRIPT
7.16 (t, 2H, H-5ꞌꞌ & H-6ꞌꞌ, J = 7.5 Hz), 4.65 (s, 2H, CH2); ESI-MS (m/z): 413.11 (M+); Anal.
calcd. for C23H15N3O3S: C, 66.82; H, 3.66; N, 10.16. Found: C, 66.59; H, 3.65; N, 10.19.
4.1.4.5. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(diphenylamino)acetamide (8)
White solid; Yield: 56 %; M.P.: 245-246 ºC; IR (KBr, cm-1): 3294 (N-H), 3036 (Ar C-H),
1
1653 (C=O), 1602 (C=N), 1518 (C=C); H NMR (DMSO-d6, 300 MHz) δ (ppm): 10.65 (s,
1H, NH, D2O exchangeable), 8.19-8.04 (m, 4H, H-4, H-7, H-2ꞌ & H-6ꞌ), 7.82-7.79 (d, 2H, H-
3ꞌ & H-5ꞌ, J = 9 Hz), 7.59-7.54 (t, 1H, H-5, J = 7.5 Hz), 7.50-7.45 (t, 1H, H-6, J = 7.5 Hz),
7.25-7.20 (t, 4H, H-3ꞌꞌ & H-5ꞌꞌ, J = 7.5 Hz), 7.08-7.06 (d, 4H, H-2ꞌꞌ & H-6ꞌꞌ, J = 6.0 Hz), 6.84-
6.79 (t, 2H, H-4ꞌꞌ, J = 7.5 Hz), 4.67 (s, 2H, CH2); ESI-MS (m/z): 435.21 (M+); Anal. calcd.
for C27H21N3OS: C, 74.46; H, 4.86; N, 9.65. Found: C, 74.69; H, 4.85; N, 9.63.
4.1.4.6. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(9H-carbazol-9-yl)acetamide (9)
White solid; Yield: 78 %; M.P.: 183-185 ºC; IR (KBr, cm-1): 3417 (N-H), 3042 (Ar C-H),
1
1653 (C=O), 1600 (C=N), 1516 (C=C); H NMR (DMSO-d6, 300 MHz) δ (ppm): 10.76 (s,
1H, NH, D2O exchangeable), 8.18-8.01 (m, 6H, H-4, H-7, H-2ꞌ, H-6ꞌ & carbazole), 7.83-7.80
(d, 2H, H-3ꞌ & H-5ꞌ, J = 9 Hz), 7.62-7.38 (m, 5H, H-5, H-6 & carbazole), 7.26-7.15 (m, 3H,
carbazole), 5.32 (s, 2H, CH2); ESI-MS (m/z): 433.17 (M+); Anal. calcd. for C27H19N3OS: C,
74.80; H, 4.42; N, 9.69. Found: C, 74.67; H, 4.43; N, 9.71.
4.1.4.7. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(3,4-dihydro-1H-carbazol-9(2H)-yl)acetamide
(10)
White solid; Yield: 80 %; M.P.: 167-169 ºC; IR (KBr, cm-1): 3398 (N-H), 3037 (Ar C-H),
1
1651 (C=O), 1598 (C=N), 1513 (C=C); H NMR (DMSO-d6, 300 MHz) δ (ppm): 10.52 (s,
1H, NH, D2O exchangeable), 8.19-8.01 (m, 6H, H-4, H-7, H-2ꞌ, H-6ꞌ, H-6ꞌꞌ & H-7ꞌꞌ), 7.81-
7.78 (d, 2H, H-3ꞌ & H-5ꞌ, J = 9 Hz), 7.53-7.44 (m, 4H, H-5, H-6, H-5ꞌꞌ & H-8ꞌꞌ), 4.66 (s, 2H,
CH2), 3.66-3.61 (t, 2H, H-1ꞌꞌ, J = 7.5 Hz), 2.75-2.70 (t, 2H, H-4ꞌꞌ, J = 7.5 Hz), 1.83-1.72 (m,
4H, H-2ꞌꞌ & H-3ꞌꞌ); ESI-MS (m/z): 437.18 (M+); Anal. calcd. for C27H23N3OS: C, 74.11; H,
5.30; N, 9.60. Found: C, 73.89; H, 5.31; N, 9.63.
4.1.4.8. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(1H-imidazol-1-yl)acetamide (11)
White solid; Yield: 78 %; M.P.: 217-218 ºC; IR (KBr, cm-1): 3315 (N-H), 3109 (Ar C-H),
1
1703 (C=O), 1604 (C=N), 1544 (C=C); H NMR (DMSO-d6, 300 MHz) δ (ppm): 10.67 (s,
1H, NH, D2O exchangeable), 8.13-8.01 (m, 4H, H-4, H-7, H-2ꞌ & H-6ꞌ), 7.81-7.78 (d, 2H, H-
3ꞌ & H-5ꞌ, J = 9 Hz), 7.76 (s, 1H, H-2ꞌꞌ), 7.56-7.51 (t, 1H, H-5, J = 7.5 Hz), 7.46-7.41 (t, 1H,
H-6, J = 7.5 Hz), 7.19 (s, 1H, H-5ꞌꞌ), 6.92 (s, 1H, H-4ꞌꞌ), 4.97 (s, 2H, CH2); ESI-MS (m/z):
334.11 (M+); Anal. calcd. for C18H14N4OS: C, 64.65; H, 4.22; N, 16.75. Found: C, 64.83; H,
4.23; N, 16.81.

ACCEPTED MANUSCRIPT
4.1.4.9. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(1H-1,2,4-triazol-1-yl)acetamide (12)
White solid; Yield: 65 %; M.P.: 226-228 ºC; IR (KBr, cm-1): 3319 (N-H), 3105 (Ar C-H),
1
1705 (C=O), 1604 (C=N), 1543 (C=C); H NMR (DMSO-d6, 300 MHz) δ (ppm): 10.75 (s,
1H, NH, D2O exchangeable), 8.58 (s, 1H, H-5ꞌꞌ), 8.14-8.02 (m, 5H, H-4, H-7, H-2ꞌ, H-6ꞌ & H-
3ꞌꞌ), 7.81-7.78 (d, 2H, H-3ꞌ & H-5ꞌ, J = 9 Hz), 7.56-7.51 (t, 1H, H-5, J = 7.5 Hz), 7.47-7.42 (t,
1H, H-6, J = 7.5 Hz), 5.21 (s, 2H, CH2); ESI-MS (m/z): 335.10 (M+); Anal. calcd. for
C17H13N5OS: C, 60.88; H, 3.91; N, 20.88. Found: C, 60.67; H, 3.90; N, 20.95.
4.1.4.10. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(3,5-dimethyl-1H-pyrazol-1-yl)acetamide (13)
White solid; Yield: 58 %; M.P.: 254-255 ºC; IR (KBr, cm-1): 3259 (N-H), 3053 (Ar C-H),
1
1653 (C=O), 1602 (C=N), 1550 (C=C); H NMR (DMSO-d6, 300 MHz) δ (ppm): 10.52 (s,
1H, NH, D2O exchangeable), 8.12-8.01 (m, 4H, H-4, H-7, H-2ꞌ & H-6ꞌ), 7.81-7.78 (d, 2H, H-
3ꞌ & H-5ꞌ, J = 9 Hz), 7.56-7.51 (t, 1H, H-5, J = 7.5 Hz), 7.47-7.42 (t, 1H, H-6, J = 7.5 Hz),
5.85 (s, 1H, H-4ꞌꞌ), 4.89 (s, 2H, CH2), 2.23 (s, 3H, CH3), 2.10 (s, 3H, CH3); ESI-MS (m/z):
362.15 (M+); Anal. calcd. for C20H18N4OS: C, 66.28; H, 5.01; N, 15.46. Found: C, 66.01; H,
5.02; N, 15.51.
4.1.4.11. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(piperidin-1-yl)acetamide (14)
White solid; Yield: 65 %; M.P.: 119-120 ºC; IR (KBr, cm-1): 3305 (N-H), 3022 (Ar C-H),
1685 (C=O), 1593 (C=N), 1521 (C=C); 1H NMR (DMSO-d6, 300 MHz) δ (ppm): 9.98 (s, 1H,
NH, D2O exchangeable), 8.12-8.01 (m, 4H, H-4, H-7, H-2ꞌ & H-6ꞌ), 7.87-7.84 (d, 2H, H-3ꞌ &
H-5ꞌ, J = 9 Hz), 7.55-7.50 (t, 1H, H-5, J = 7.5 Hz), 7.46-7.41 (t, 1H, H-6, J = 7.5 Hz), 3.11 (s,
2H, CH2), 2.47-2.45 (m, 4H, H-2ꞌꞌ & H-6ꞌꞌ), 1.57-1.55 (m, 4H, H-3ꞌꞌ & H-5ꞌꞌ), 1.41-1.39 (m,
13
2H, H-4ꞌꞌ); C NMR (DMSO-d6, 125 MHz) δ (ppm): 169.67 (C-2), 167.40 (C=O), 154.19
(C-9), 141.91 (C-4ꞌ), 134.74 (C-8), 128.51 (C-2ꞌ & C-6ꞌ), 128.32 (C-1ꞌ), 127.00 (C-5), 125.68
(C-6), 123.03 (C-4), 122.65 (C-7), 120.01 (C-3ꞌ & C-5ꞌ), 63.18 (CH2), 54.19 (C-2ꞌꞌ & C-6ꞌꞌ),
25.98 (C-3ꞌꞌ & C-5ꞌꞌ), 24.09 (C-4ꞌꞌ); ESI-MS (m/z): 351.17 (M+); Anal. calcd. for
C20H21N3OS: C, 68.35; H, 6.02; N, 11.96. Found: C, 68.19.; H, 6.01; N, 11.99.
4.1.4.12. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-morpholinoacetamide (15)
White solid; Yield: 58 %; M.P.: 156 ºC; IR (KBr, cm-1): 3342 (N-H), 3049 (Ar C-H), 1653
1
(C=O), 1600 (C=N), 1517 (C=C); H NMR (DMSO-d6, 300 MHz) δ (ppm): 10.07 (s, 1H,
NH, D2O exchangeable), 8.12-8.01 (m, 4H, H-4, H-7, H-2ꞌ & H-6ꞌ), 7.87-7.84 (d, 2H, H-3ꞌ &
H-5ꞌ, J = 9 Hz), 7.55-7.50 (t, 1H, H-5, J = 7.5 Hz), 7.45-7.40 (t, 1H, H-6, J = 7.5 Hz), 3.64
(bs, 4H, H-3ꞌꞌ & H-5ꞌꞌ), 3.18 (s, 2H, CH2), 2.51 (bs, 4H, H-2ꞌꞌ & H-6ꞌꞌ); 13C NMR (DMSO-d6,
125 MHz) δ (ppm): 169.10 (C-2), 167.40 (C=O), 154.19 (C-9), 141.91 (C-4ꞌ), 134.74 (C-8),
128.51 (C-2ꞌ & C-6ꞌ), 128.32 (C-1ꞌ), 127.00 (C-5), 125.68 (C-6), 123.03 (C-4), 122.65 (C-7),

ACCEPTED MANUSCRIPT
120.20 (C-3ꞌ & C-5ꞌ), 66.58 (C-3ꞌꞌ & C-5ꞌꞌ), 62.61 (CH2), 53.55 (C-2ꞌꞌ & C-6ꞌꞌ); ESI-MS (m/z):
353.15 (M+); Anal. calcd. for C19H19N3O2S: C, 64.57; H, 5.42; N, 11.89. Found: C, 64.80; H,
5.41; N, 11.92.
4.1.4.13. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(4-methylpiperazin-1-yl)acetamide (16)
White solid; Yield: 62 %; M.P.: 151-152 ºC; IR (KBr, cm-1): 3388 (N-H), 3049 (Ar C-H),
1689 (C=O), 1600 (C=N), 1533 (C=C); 1H NMR (DMSO-d6, 300 MHz) δ (ppm): 9.99 (s, 1H,
NH, D2O exchangeable), 8.10-7.98 (m, 4H, H-4, H-7, H-2ꞌ & H-6ꞌ), 7.83-7.80 (d, 2H, H-3ꞌ &
H-5ꞌ, J = 9 Hz), 7.53-7.48 (t, 1H, H-5, J = 7.5 Hz), 7.46-7.41 (t, 1H, H-6, J = 7.5 Hz), 3.13 (s,
13
2H, CH2), 2.35 (bs, 4H, H-2ꞌꞌ & H-6ꞌꞌ) , 2.14 (bs, 4H, H-3ꞌꞌ & H-5ꞌꞌ), 2.06 (s, 3H, CH3); C
NMR (DMSO-d6, 125 MHz) δ (ppm): 169.29 (C-2), 167.40 (C=O), 154.19 (C-9), 141.91 (C-
4ꞌ), 134.74 (C-8), 128.51 (C-2ꞌ & C-6ꞌ), 128.32 (C-1ꞌ), 127.00 (C-5), 125.68 (C-6), 123.03 (C-
4), 122.65 (C-7), 120.20 (C-3ꞌ & C-5ꞌ), 62.23 (CH2), 55.06 (C-3ꞌꞌ & C-5ꞌꞌ), 53.17 (C-2ꞌꞌ & C-
6ꞌꞌ), 46.18 (CH3); ESI-MS (m/z): 366.17 (M+); Anal. calcd. for C20H22N4OS: C, 65.55; H,
6.05; N, 15.29. Found: C, 65.43; H, 6.04; N, 15.33.
4.1.5. General method for the synthesis of primary amine derivatives of N-(4-(benzo[d]
thiazol-2-yl)phenyl)-2-chloroacetamide (17-38)
To a solution of N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-chloroacetamide (3) (10 mmol) in 20
ml of absolute ethanol, a equimolar amount of appropriate primary amines (10 mmol) and 1
ml of triethylamine (TEA) were added. The mixture was refluxed for 8-36 hrs. The progress
of reaction and purity of compound was checked by TLC using TEF (6:3.5:0.5) as mobile
phase. After completion of reaction, the contents of the flask were reduced to half of its
volume and left overnight. The solid mass obtained was filtered, washed with water, dried
and then purified through column chromatography (Hexane/AcOEt 4:1) to afford final
compounds (17-38).
4.1.5.1. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(cyclohexylamino)acetamide (17)
White solid; Yield: 60 %; M.P.: 212-213 ºC; IR (KBr, cm-1): 3338, 3248 (N-H), 3085 (Ar C-
1
H), 1676 (C=O), 1593 (C=N), 1531 (C=C); H NMR (DMSO-d6, 300 MHz) δ (ppm): 10.17
(bs, 1H, CONH, D2O exchangeable), 8.11-8.00 (m, 4H, H-4, H-7, H-2ꞌ & H-6ꞌ), 7.84-7.81 (d,
2H, H-3ꞌ & H-5ꞌ, J = 9 Hz), 7.54-7.49 (t, 1H, H-5, J = 7.5 Hz), 7.44-7.39 (t, 1H, H-6, J = 7.5
Hz), 3.84 (bs, 1H, NH, D2O exchangeable), 3.34 (s, 2H, CH2), 2.40-2.34 (m, 1H, H-1ꞌꞌ,
Cyclohexyl), 1.82-1.52 (m, 5H, Cyclohexyl), 1.25-1.04 (m, 5H, Cyclohexyl); 13C NMR
(DMSO-d6, 125 MHz) δ (ppm): 171.75 (C-2), 167.40 (C=O), 154.19 (C-9), 141.91 (C-4ꞌ),
134.74 (C-8), 128.51 (C-2ꞌ & C-6ꞌ), 128.13 (C-1ꞌ), 127.00 (C-5), 125.68 (C-6), 123.03 (C-4),
122.65 (C-7), 119.82 (C-3ꞌ & C-5ꞌ), 56.76 (C-1ꞌꞌ), 50.71 (CH2), 33.34 (C-2ꞌꞌ & C-6ꞌꞌ), 26.17

ACCEPTED MANUSCRIPT
(C-4ꞌꞌ), 24.85 (C-3ꞌꞌ & C-5ꞌꞌ); ESI-MS (m/z): 365.19 (M+); Anal. calcd. for C21H23N3OS: C,
69.01; H, 6.34; N, 11.50. Found: C, 69.19; H, 6.32; N, 11.47.
4.1.5.2. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(benzylamino)acetamide (18)
White solid; Yield: 66 %; M.P.: 185-187 ºC; IR (KBr, cm-1): 3522, 3298 (N-H), 3035 (Ar C-
1
H), 1680 (C=O), 1602 (C=N), 1523 (C=C); H NMR (DMSO-d6, 300 MHz) δ (ppm): 10.15
(bs, 1H, CONH, D2O exchangeable), 8.10-8.01 (m, 4H, H-4, H-7, H-2ꞌ & H-6ꞌ), 7.84-7.81 (d,
2H, H-3ꞌ & H-5ꞌ, J = 9 Hz), 7.55-7.24 (m, 7H, H-5, H-6, H-2ꞌꞌ, H-3ꞌꞌ, H-4ꞌꞌ, H-5ꞌꞌ & H-6ꞌꞌ),
3.76 (s, 2H, CH2), 3.33 (s, 2H, CH2), 2.82 (bs, 1H, NH, D2O exchangeable); 13C NMR
(DMSO-d6, 125 MHz) δ (ppm): 171.18 (C-2), 167.40 (C=O), 154.19 (C-9), 142.10 (C-1ꞌꞌ),
140.59 (C-4ꞌ), 134.74 (C-8), 128.70 (C-3ꞌꞌ & C-5ꞌꞌ), 128.51 (C-2ꞌ & C-6ꞌ), 128.32 (C-2ꞌꞌ & C-
6ꞌꞌ), 128.13 (C-1ꞌ), 127.38 (C-4ꞌꞌ), 127.19 (C-5), 125.68 (C-6), 123.03 (C-4), 122.65 (C-7),
119.82 (C-3ꞌ & C-5ꞌ), 52.98 (Ph-CH2), 52.41 (CH2); ESI-MS (m/z): 373.15 (M+); Anal. calcd.
for C22H19N3OS: C, 70.75; H, 5.13; N, 11.25. Found: C, 70.83; H, 5.15; N, 11.23.
4.1.5.3. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(phenylamino)acetamide (19)
White solid; Yield: 52 %; M.P.: 160-162 ºC; IR (KBr, cm-1): 3422, 3310 (N-H), 3094 (Ar C-
1
H), 1657 (C=O), 1592 (C=N), 1516 (C=C); H NMR (DMSO-d6, 300 MHz) δ (ppm): 10.33
(bs, 1H, CONH, D2O exchangeable), 8.13-8.01 (m, 4H, H-4, H-7, H-2ꞌ & H-6ꞌ), 7.84-7.81 (d,
2H, H-3ꞌ & H-5ꞌ, J = 9 Hz), 7.55-7.50 (t, 1H, H-5, J = 7.5 Hz), 7.46-7.41 (t, 1H, H-6, J = 7.5
Hz), 7.13-7.08 (t, 3H, H-3ꞌꞌ, H-4ꞌꞌ & H-5ꞌꞌ, J = 7.5 Hz), 6.63-6.60 (d, 2H, H-2ꞌꞌ & H-6ꞌꞌ, J = 9
13
Hz), 6.01 (bs, 1H, NH, D2O exchangeable), 3.93 (s, 2H, CH2); C NMR (DMSO-d6, 125
MHz) δ (ppm): 170.60 (C-2), 167.41 (C=O), 154.12 (C-9), 151.88 (C-1ꞌꞌ), 142.12 (C-4ꞌ),
134.75 (C-8), 128.48 (C-2ꞌ & C-6ꞌ), 128.13 (C-1ꞌ), 127.08 (C-5), 125.74 (C-6), 124.24 (C-4ꞌꞌ),
123.05 (C-4), 122.75 (C-7), 119.95 (C-3ꞌ & C-5ꞌ), 115.07 (C-3ꞌꞌ & C-5ꞌꞌ), 114.13 (C-2ꞌꞌ & C-
6ꞌꞌ), 55.75 (CH2); ESI-MS (m/z): 359.13 (M+); Anal. calcd. for C21H17N3OS: C, 70.17; H,
4.77; N, 11.69. Found: C, 69.98; H, 4.76; N, 11.66.
4.1.5.4. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(4-fluorophenylamino)acetamide (20)
White solid; Yield: 58 %; M.P.: 197-198 ºC; IR (KBr, cm-1): 3351, 3309 (N-H), 3095 (Ar C-
1
H), 1670 (C=O), 1575 (C=N), 1519 (C=C), 1126 (C-F); H NMR (DMSO-d6, 300 MHz) δ
(ppm): 10.34 (bs, 1H, CONH, D2O exchangeable), 8.13-8.00 (m, 4H, H-4, H-7, H-2ꞌ & H-6ꞌ),
7.81-7.78 (d, 2H, H-3ꞌ & H-5ꞌ, J = 9 Hz), 7.55-7.50 (t, 1H, H-5, J = 7.5 Hz), 7.46-7.41 (t, 1H,
H-6, J = 7.5 Hz), 7.15-7.12 (d, 2H, H-3ꞌꞌ & H-5ꞌꞌ, J = 9 Hz), 6.65-6.62 (d, 2H, H-2ꞌꞌ & H-6ꞌꞌ, J
= 9 Hz), 6.24 (bs, 1H, NH, D2O exchangeable), 3.95 (s, 2H, CH2); ESI-MS (m/z): 377.11
(M+); Anal. calcd. for C21H16FN3OS: C, 66.83; H, 4.27; N, 11.13. Found: C, 67.03; H, 4.28;
N, 11.16.

ACCEPTED MANUSCRIPT
4.1.5.5. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(4-chlorophenylamino)acetamide (21)
White solid; Yield: 65 %; M.P.: 226-228 ºC; IR (KBr, cm-1): 3392, 3311 (N-H), 3111 (Ar C-
1
H), 1678 (C=O), 1598 (C=N), 1521 (C=C), 752 (C-Cl); H NMR (DMSO-d6, 300 MHz) δ
(ppm): 10.36 (bs, 1H, CONH, D2O exchangeable), 8.14-8.02 (m, 4H, H-4, H-7, H-2ꞌ & H-6ꞌ),
7.82-7.79 (d, 2H, H-3ꞌ & H-5ꞌ, J = 9 Hz), 7.56-7.51 (t, 1H, H-5, J = 7.5 Hz), 7.47-7.42 (t, 1H,
H-6, J = 7.5 Hz), 7.14-7.11 (d, 2H, H-3ꞌꞌ & H-5ꞌꞌ, J = 9 Hz), 6.63-6.60 (d, 2H, H-2ꞌꞌ & H-6ꞌꞌ, J
= 9 Hz), 5.25 (bs, 1H, NH, D2O exchangeable), 3.93 (s, 2H, CH2); ESI-MS (m/z): 393.09
(M+), 395.09 (M+ + 2); Anal. calcd. for C21H16ClN3OS: C, 64.03; H, 4.09; N, 10.67. Found:
C, 63.93; H, 4.08; N, 10.64.
4.1.5.6. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(3-chlorophenylamino)acetamide (22)
White solid; Yield: 58 %; M.P.: 187-188 ºC; IR (KBr, cm-1): 3415, 3323 (N-H), 3084 (Ar C-
1
H), 1675 (C=O), 1593 (C=N), 1516 (C=C), 756 (C-Cl); H NMR (DMSO-d6, 300 MHz) δ
(ppm): 10.34 (bs, 1H, CONH, D2O exchangeable), 8.18-8.03 (m, 4H, H-4, H-7, H-2ꞌ & H-6ꞌ),
7.83-7.80 (d, 2H, H-3ꞌ & H-5ꞌ, J = 9 Hz), 7.57-7.52 (t, 1H, H-5, J = 7.5 Hz), 7.49-7.44 (t, 1H,
H-6, J = 7.5 Hz), 7.15-7.10 (t, 1H, H-5ꞌꞌ, J = 7.5 Hz), 6.81-6.78 (d, 2H, H-4ꞌꞌ & H-6ꞌꞌ, J = 9
Hz), 6.72 (s, 1H, H-2ꞌꞌ), 5.87 (bs, 1H, NH, D2O exchangeable), 3.97 (s, 2H, CH2);ESI-MS
(m/z): 393.09 (M+), 395.09 (M+ + 2); Anal. calcd. for C21H16ClN3OS: C, 64.03; H, 4.09; N,
10.67. Found: C, 63.98; H, 4.08; N, 10.70.
4.1.5.7. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(2-chlorophenylamino)acetamide (23)
White solid; Yield: 64 %; M.P.: 102-203 ºC; IR (KBr, cm-1): 3432, 3325 (N-H), 3093 (Ar C-
1
H), 1672 (C=O), 1600 (C=N), 1509 (C=C), 751 (C-Cl); H NMR (DMSO-d6, 300 MHz) δ
(ppm): 10.32 (bs, 1H, CONH, D2O exchangeable), 8.11-8.00 (m, 4H, H-4, H-7, H-2ꞌ & H-6ꞌ),
7.81-7.78 (d, 2H, H-3ꞌ & H-5ꞌ, J = 9 Hz), 7.55-7.50 (t, 1H, H-5, J = 7.5 Hz), 7.46-7.43 (t, 1H,
H-6, J = 7.5 Hz), 7.07-6.85 (m, 4H, H-3ꞌꞌ, H-4ꞌꞌ, H-5ꞌꞌ, & H-6ꞌꞌ), 5.76 (bs, 1H, NH, D2O
exchangeable), 3.89 (s, 2H, CH2); ESI-MS (m/z): 393.09 (M+), 395.09 (M+ + 2); Anal. calcd.
for C21H16ClN3OS: C, 64.03; H, 4.09; N, 10.67. Found: C, 64.23; H, 4.10; N, 10.65.
4.1.5.8. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(2,6-dichlorophenylamino)acetamide (24)
White solid; Yield: 57 %; M.P.: 167-169 ºC; IR (KBr, cm-1): 3516, 3319 (N-H), 3049 (Ar C-
1
H), 1676 (C=O), 1597 (C=N), 1519 (C=C), 759 (C-Cl); H NMR (DMSO-d6, 300 MHz) δ
(ppm): 10.38 (bs, 1H, CONH, D2O exchangeable), 8.12-8.01 (m, 4H, H-4, H-7, H-2ꞌ & H-6ꞌ),
7.81-7.78 (d, 2H, H-3ꞌ & H-5ꞌ, J = 9 Hz), 7.56-7.50 (t, 1H, H-5, J = 7.5 Hz), 7.46-7.41 (t, 1H,
H-6, J = 7.5 Hz), 7.17-7.14 (d, 2H, H-3ꞌꞌ & H-5ꞌꞌ, J = 9.0 Hz), 6.81-6.76 (t, 1H, H-4ꞌꞌ, J = 7.5
Hz), 5.51 (bs, 1H, NH, D2O exchangeable), 3.94 (s, 2H, CH2); ESI-MS (m/z): 427.07 (M+),

ACCEPTED MANUSCRIPT
429.07 (M+ + 2); Anal. calcd. for C21H15Cl2N3OS: C, 58.89; H, 3.53; N, 9.81. Found: C,
58.69; H, 3.54; N, 9.78.
4.1.5.9. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(3-chloro-4-fluorophenylamino)acetamide (25)
White solid; Yield: 62 %; M.P.: 238-240 ºC; IR (KBr, cm-1): 3456, 3328 (N-H), 3042 (Ar C-
1
H), 1670 (C=O), 1605 (C=N), 1527 (C=C), 1106 (C-F), 748 (C-Cl); H NMR (DMSO-d6,
300 MHz) δ (ppm): 10.70 (bs, 1H, CONH, D2O exchangeable), 8.10-8.01 (m, 4H, H-4, H-7,
H-2ꞌ & H-6ꞌ), 7.83-7.80 (d, 2H, H-3ꞌ & H-5ꞌ, J = 9 Hz), 7.54-7.49 (t, 1H, H-5, J = 7.5 Hz),
7.44-7.39 (t, 1H, H-6, J = 7.5 Hz), 7.19-7.16 (d, 1H, H-5ꞌꞌ, J = 9.0 Hz), 6.93-6.90 (d, 1H, H-
6ꞌꞌ, J = 9 Hz), 6.78 (s, 1H, H-2ꞌꞌ), 5.33 (bs, 1H, NH, D2O exchangeable), 3.85 (s, 2H, CH2);
ESI-MS (m/z): 411.09 (M+), 413.09 (M+ + 2); Anal. calcd. for C21H15ClFN3OS: C, 61.24; H,
3.67; N, 10.20. Found: C, 61.42; H, 3.66; N, 10.18.
4.1.5.10. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(4-bromophenylamino)acetamide (26)
Light brown solid; Yield: 48 %; M.P.: 180-182 ºC; IR (KBr, cm-1): 3445, 3321 (N-H), 3085
1
(Ar C-H), 1668 (C=O), 1592 (C=N), 1532 (C=C), 658 (C-Br); H NMR (DMSO-d6, 300
MHz) δ (ppm): 10.65 (bs, 1H, CONH, D2O exchangeable), 8.12-8.01 (m, 4H, H-4, H-7, H-2ꞌ
& H-6ꞌ), 7.82-7.79 (d, 2H, H-3ꞌ & H-5ꞌ, J = 9 Hz), 7.55-7.50 (t, 1H, H-5, J = 7.5 Hz), 7.46-
7.41 (t, 1H, H-6, J = 7.5 Hz), 7.14-7.11 (d, 2H, H-3ꞌꞌ & H-5ꞌꞌ, J = 9 Hz), 6.66-6.63 (d, 2H, H-
2ꞌꞌ & H-6ꞌꞌ, J = 9 Hz), 5.23 (bs, 1H, NH, D2O exchangeable), 4.32 (s, 2H, CH2); ESI-MS
(m/z): 437.07 (M+), 439.07 (M+ + 2); Anal. calcd. for C21H16BrN3OS: C, 57.54; H, 3.68; N,
9.59. Found: C, 57.43; H, 3.69; N, 9.61.
4.1.5.11. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(4-nitrophenylamino)acetamide (27)
Yellow solid; Yield: 55 %; M.P.: 206-262 ºC; IR (KBr, cm-1): 3412, 3305 (N-H), 3086 (Ar
C-H), 1667 (C=O), 1592 (C=N), 1522 (C=C), 1495 (NO2); 1H NMR (DMSO-d6, 300 MHz) δ
(ppm): 10.63 (bs, 1H, CONH, D2O exchangeable), 8.18-8.01 (m, 6H, H-4, H-7, H-2ꞌ, H-6ꞌ, H-
3ꞌꞌ & H-5ꞌꞌ), 7.81-7.80 (d, 2H, H-3ꞌ & H-5ꞌ, J = 9 Hz), 7.54-7.49 (t, 1H, H-5, J = 7.5 Hz), 7.45-
7.40 (t, 1H, H-6, J = 7.5 Hz), 6.88-6.85 (d, 2H, H-2ꞌꞌ & H-6ꞌꞌ, J = 9 Hz), 5.25 (bs, 1H, NH,
D2O exchangeable), 4.28 (s, 2H, CH2); ESI-MS (m/z): 404.13 (M+); Anal. calcd. for
C21H16N4O3S: C, 62.36; H, 3.99; N, 13.85. Found: C, 62.51; H, 3.98; N, 13.87.
4.1.5.12. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(2-methylphenylamino)acetamide (28)
White solid; Yield: 60 %; M.P.: 221-223 ºC; IR (KBr, cm-1): 3412, 3324 (N-H), 3073 (Ar C-
1
H), 1678 (C=O), 1587 (C=N), 1526 (C=C); H NMR (DMSO-d6, 300 MHz) δ (ppm): 10.26
(bs, 1H, CONH, D2O exchangeable), 8.13-8.02 (m, 4H, H-4, H-7, H-2ꞌ & H-6ꞌ), 7.82-7.79 (d,
2H, H-3ꞌ & H-5ꞌ, J = 9 Hz), 7.56-7.51 (t, 1H, H-5, J = 7.5 Hz), 7.47-7.420 (t, 1H, H-6, J = 7.5
Hz), 7.16-7.14 (d, 1H, H-3ꞌꞌ, J = 6 Hz), 7.09-7.04 (t, 1H, H-5ꞌꞌ, J = 7.5 Hz), 6.69-6.61 (m, 2H,

ACCEPTED MANUSCRIPT
H-4ꞌꞌ & H-6ꞌꞌ), 5.74 (bs, 1H, NH, D2O exchangeable), 3.83 (s, 2H, CH2), 2.09 (s, 3H, CH3);
ESI-MS (m/z): 373.15 (M+); Anal. calcd. for C22H19N3OS: C, 70.75; H, 5.13; N, 11.25.
Found: C, 70.67; H, 5.14; N, 11.23.
4.1.5.13. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(4-methylphenylamino)acetamide (29)
White solid; Yield: 68 %; M.P.: 178-180 ºC; IR (KBr, cm-1): 3408, 3327 (N-H), 3075 (Ar C-
1
H), 1676 (C=O), 1590 (C=N), 1523 (C=C); H NMR (DMSO-d6, 300 MHz) δ (ppm): 10.28
(bs, 1H, CONH, D2O exchangeable), 8.12-8.00 (m, 4H, H-4, H-7, H-2ꞌ & H-6ꞌ), 7.83-7.80 (d,
2H, H-3ꞌ & H-5ꞌ, J = 9 Hz), 7.55-7.50 (t, 1H, H-5, J = 7.5 Hz), 7.45-7.40 (t, 1H, H-6, J = 7.5
Hz), 6.93-6.90 (d, 2H, H-3ꞌꞌ & H-5ꞌꞌ, J = 9 Hz), 6.54-6.52 (d, 2H, H-2ꞌꞌ & H-6ꞌꞌ, J = 6 Hz),
5.81 (bs, 1H, NH, D2O exchangeable), 3.89 (s, 2H, CH2), 2.14 (s, 3H, CH3); ESI-MS (m/z):
373.15 (M+); Anal. calcd. for C22H19N3OS: C, 70.75; H, 5.13; N, 11.25. Found: C, 70.87; H,
5.14; N, 11.26.
4.1.5.14. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(2-methoxyphenylamino)acetamide (30)
White solid; Yield: 58 %; M.P.: 214-216 ºC; IR (KBr, cm-1): 3472, 3328 (N-H), 3056 (Ar C-
1
H), 1682 (C=O), 1603 (C=N), 1535 (C=C); H NMR (DMSO-d6, 300 MHz) δ (ppm): 10.32
(bs, 1H, CONH, D2O exchangeable), 8.13-8.01 (m, 4H, H-4, H-7, H-2ꞌ & H-6ꞌ), 7.82-7.79 (d,
2H, H-3ꞌ & H-5ꞌ, J = 9 Hz), 7.57-7.52 (t, 1H, H-5, J = 7.5 Hz), 7.47-7.42 (t, 1H, H-6, J = 7.5
Hz), 7.04-7.02 (d, 1H, H-3ꞌꞌ or H-6ꞌꞌ, J = 6 Hz), 6.75-6.66 (m, 3H, H-3ꞌꞌ or H-6ꞌꞌ, H-4ꞌꞌ & H-
5ꞌꞌ), 5.61 (bs, 1H, NH, D2O exchangeable), 3.84 (s, 2H, CH2), 3.55 (s, 3H, OCH3); ESI-MS
(m/z): 389.15 (M+); Anal. calcd. for C22H19N3O2S: C, 67.84; H, 4.92; N, 10.79. Found: C,
67.92; H, 4.93; N, 10.77.
4.1.5.15. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(4-methoxyphenylamino)acetamide (31)
White solid; Yield: 64 %; M.P.: 187-188 ºC; IR (KBr, cm-1): 3468, 3330 (N-H), 3055 (Ar C-
1
H), 1685 (C=O), 1600 (C=N), 1539 (C=C); H NMR (DMSO-d6, 300 MHz) δ (ppm): 10.27
(bs, 1H, CONH, D2O exchangeable), 8.12-8.00 (m, 4H, H-4, H-7, H-2ꞌ & H-6ꞌ), 7.83-7.80 (d,
2H, H-3ꞌ & H-5ꞌ, J = 9 Hz), 7.55-7.50 (t, 1H, H-5, J = 7.5 Hz), 7.46-7.41 (t, 1H, H-6, J = 7.5
Hz), 6.76-6.73 (d, 2H, H-3ꞌꞌ & H-5ꞌꞌ or H-2ꞌꞌ & H-6ꞌꞌ, J = 9.0 Hz), 6.59-6.56 (d, 2H, H-2ꞌꞌ & H-
6ꞌꞌ or H-3ꞌꞌ & H-5ꞌꞌ, J = 9.0 Hz), 5.63 (bs, 1H, NH, D2O exchangeable), 3.86 (s, 2H, CH2),
3.60 (s, 3H, OCH3); ESI-MS (m/z): 389.15 (M+); Anal. calcd. for C22H19N3O2S: C, 67.84; H,
4.92; N, 10.79. Found: C, 67.87; H, 4.93; N, 10.81.
4.1.5.16. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(2-hydroxyphenylamino)acetamide (32)
White solid; Yield: 54 %; M.P.: 223-224 ºC; IR (KBr, cm-1): 3445, 3315 (N-H), 3072 (Ar C-
1
H), 1675 (C=O), 1593 (C=N), 1521 (C=C); H NMR (DMSO-d6, 300 MHz) δ (ppm): 10.37
(bs, 1H, CONH, D2O exchangeable), 8.12-8.00 (m, 4H, H-4, H-7, H-2ꞌ & H-6ꞌ), 7.80-7.77 (d,

ACCEPTED MANUSCRIPT
2H, H-3ꞌ & H-5ꞌ, J = 9 Hz), 7.55-7.50 (t, 1H, H-5, J = 7.5 Hz), 7.46-7.41 (t, 1H, H-6, J = 7.5
Hz), 6.86-6.70 (m, 4H, H-3ꞌꞌ, H-4ꞌꞌ, H-5ꞌꞌ & H-6ꞌꞌ), 5.63 (bs, 1H, OH or NH, D2O
exchangeable), 5.38 (bs, 1H, OH or NH, D2O exchangeable), 3.98 (s, 2H, CH2); ESI-MS
(m/z): 375.11 (M+); Anal. calcd. for C21H17N3O2S: C, 67.18; H, 4.56; N, 11.19. Found: C,
67.33; H, 4.55; N, 11.16.
4.1.5.17. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(4-hydroxyphenylamino)acetamide (33)
White solid; Yield: 60 %; M.P.: 219-220 ºC; IR (KBr, cm-1): 3440, 3312 (N-H), 3076 (Ar C-
1
H), 1681 (C=O), 1588 (C=N), 1514 (C=C); H NMR (DMSO-d6, 300 MHz) δ (ppm): 10.38
(bs, 1H, CONH, D2O exchangeable), 8.15-8.02 (m, 4H, H-4, H-7, H-2ꞌ & H-6ꞌ), 7.84-7.81 (d,
2H, H-3ꞌ & H-5ꞌ, J = 9 Hz), 7.57-7.52 (t, 1H, H-5, J = 7.5 Hz), 7.48-7.43 (t, 1H, H-6, J = 7.5
Hz), 6.80-6.78 (d, 2H, H-3ꞌꞌ & H-5ꞌꞌ or H-2ꞌꞌ & H-6ꞌꞌ, J = 6 Hz), 6.72-6.70 (d, 2H, H-2ꞌꞌ & H-
6ꞌꞌ or H-3ꞌꞌ & H-5ꞌꞌ, J = 6 Hz), 5.65 (bs, 1H, OH or NH, D2O exchangeable), 5.37 (bs, 1H, OH
or NH, D2O exchangeable), 3.85 (s, 2H, CH2); ESI-MS (m/z): 375.11 (M+); Anal. calcd. for
C21H17N3O2S: C, 67.18; H, 4.56; N, 11.19. Found: C, 66.95.; H, 4.56; N, 11.22.
4.1.5.18. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(4-sulfamoylphenylamino)acetamide (34)
Pale yellow solid; Yield: 48 %; M.P.: 230-232 ºC; IR (KBr, cm-1): 3410, 3304 (N-H), 3053
1
(Ar C-H), 1676 (C=O), 1595 (C=N), 1510 (C=C); H NMR (DMSO-d6, 300 MHz) δ (ppm):
10.04 (bs, 1H, CONH, D2O exchangeable), 8.12-8.01 (m, 4H, H-4, H-7, H-2ꞌ & H-6ꞌ), 7.82-
7.79 (d, 2H, H-3ꞌ & H-5ꞌ, J = 9.0 Hz), 7.56-7.51 (t, 1H, H-5, J = 7.5 Hz), 7.46-7.41 (t, 1H, H-
6, J = 7.5 Hz), 7.06-7.03 (d, 2H, H-3ꞌꞌ & H-5ꞌꞌ or H-2ꞌꞌ & H-6ꞌꞌ, J = 9.0 Hz), 6.76-6.73 (d, 2H,
H-2ꞌꞌ & H-6ꞌꞌ or H-3ꞌꞌ & H-5ꞌꞌ, J = 9.0 Hz), 5.73 (s, 2H, SO2NH2, D2O exchangeable), 5.59
(bs, 1H, NH, D2O exchangeable), 3.73 (s, 2H, CH2); ESI-MS (m/z): 438.12 (M+); Anal.
calcd. for C21H18N4O3S2: C, 57.52; H, 4.14; N, 12.78. Found: C, 57.74.; H, 4.13; N, 12.76.
4.1.5.19.
N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(4-(4-chlorophenyl)thiazol-2-yl-amino)
acetamide (35)
White solid; Yield: 54 %; M.P.: 205-207 ºC; IR (KBr, cm-1): 3510, 3347 (N-H), 3067 (Ar C-
1
H), 1682 (C=O), 1583 (C=N), 1518 (C=C), 760 (C-Cl); H NMR (DMSO-d6, 300 MHz) δ
(ppm): 10.70 (bs, 1H, CONH, D2O exchangeable), 8.27-7.34 (m, 12H, aromatic), 7.03 (s, 1H,
thiazole), 5.54 (bs, 1H, NH, D2O exchangeable), 4.83 (s, 2H, CH2); ESI-MS (m/z): 476.07
(M+), 478.07 (M+ + 2); Anal. calcd. for C24H17ClN4OS2: C, 60.43; H, 3.59; N, 11.75. Found:
C, 60.61.; H, 3.58; N, 11.71.
4.1.5.20. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl-
amino)acetamide (36)

ACCEPTED MANUSCRIPT
White solid; Yield: 42 %; M.P.: 219-221 ºC; IR (KBr, cm-1): 3451, 3339 (N-H), 3073 (Ar C-
1
H), 1687 (C=O), 1582 (C=N), 1506 (C=C), 763 (C-Cl); H NMR (DMSO-d6, 300 MHz) δ
(ppm): 10.57 (s, 1H, NH, D2O exchangeable), 8.19-8.01 (m, 4H, H-4, H-7, H-2ꞌ & H-6ꞌ),
7.82-7.79 (d, 2H, H-3ꞌ & H-5ꞌ, J = 9 Hz), 7.60-7.57 (d, 2H, H-2ꞌꞌ & H-6ꞌꞌ, J = 9 Hz), 7.51-7.46
(t, 1H, H-5, J = 7.5 Hz), 7.44-7.39 (t, 1H, H-6, J = 7.5 Hz), 7.21-7.18 (d, 2H, H-3ꞌꞌ & H-5ꞌꞌ, J
= 9 Hz), 5.36 (bs, 1H, NH, D2O exchangeable), 4.67 (s, 2H, CH2); ESI-MS (m/z): 461.10
(M+), 463.10 (M+ + 2); Anal. calcd. for C23H16ClN5O2S: C, 59.80; H, 3.49; N, 15.16. Found:
C, 60.01.; H, 3.50; N, 15.10.
4.1.5.21. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(5-methylisoxazol-3-ylamino)acetamide (37)
Pale yellow solid; Yield: 54 %; M.P.: 140-141 ºC; IR (KBr, cm-1): 3486, 3315 (N-H), 3074
1
(Ar C-H), 1694 (C=O), 1602 (C=N), 1518 (C=C); H NMR (DMSO-d6, 300 MHz) δ (ppm):
10.56 (s, 1H, NH, D2O exchangeable), 8.19-8.03 (m, 4H, H-4, H-7, H-2ꞌ & H-6ꞌ), 7.82-7.79
(d, 2H, H-3ꞌ & H-5ꞌ, J = 9 Hz), 7.56-7.51 (t, 1H, H-5, J = 7.5 Hz), 7.47-7.42 (t, 1H, H-6, J =
7.5 Hz), 6.72 (s, 1H, isoxazole), 5.60 (bs, 1H, NH, D2O exchangeable), 4.53 (s, 2H, CH2),
2.39 (s, 3H, CH3); ESI-MS (m/z): 364.13 (M+); Anal. calcd. for C19H16N4O2S: C, 62.62; H,
4.43; N, 15.37. Found: C, 62.46.; H, 4.42; N, 15.42.
4.1.5.22. N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(benzo[d]thiazol-2-yl-amino)acetamide (38)
Light brown solid; Yield: 56 %; M.P.: 245-248 ºC; IR (KBr, cm-1): 3488, 3312 (N-H), 3092
1
(Ar C-H), 1673 (C=O), 1601 (C=N), 1528 (C=C); H NMR (DMSO-d6, 300 MHz) δ (ppm):
11.00 (s, 1H, NH, D2O exchangeable), 8.31-7.30 (m, 12H, aromatic), 5.06 (bs, 1H, NH, D2O
exchangeable), 4.64 (s, 2H, CH2); ESI-MS (m/z): 416.10 (M+); Anal. calcd. for
C22H16N4OS2: C, 63.44; H, 3.87; N, 13.45. Found: C, 63.65.; H, 3.88; N, 13.41.
4.2. In vitro human MAGL enzyme inhibition assay
The in vitro human MAGL enzyme inhibition assay was carried out by Cayman’s MAGL
inhibitor screening assay kit and the method reported by Muccioli et al. [17]. Compounds
were tested for their ability to inhibit MAGL in serial dilutions (1:10) from 100 ꢀM to 1 nM
(six concentrations in triplicate) using dimethyl sulfoxide (DMSO) as solvent. Assay was
carried out in a 96-well plate (180 ꢀl total volumes). Assay buffer 10 mM Tris-HCl, pH 7.2,
containing 1 mM EDTA was used for the assay as well as for the dilution of pure human
MAGL. 4-Nitrophenylacetate was used as substrate, which by the action of MAGL produced
yellow product 4-nitrophenol. The absorbance was measured at 405 nm. The average
absorbance of background well (BW), containing buffer and DMSO was systematically
subtracted from the average absorbance of 100% Initial Activity Wells (IA) and
Test/Standard. The % MAGL activity for the test or standard was calculated by the formula:

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% MAGL activity = Test / IA × 100. IC50 values were calculated from Graph pad prism (ver.
6.01). Results are quoted as % MAGL activity using CAY10499 as standard positive control.
4.3. Anticancer screening
The in vitro anticancer testing was performed by National Cancer Institute (NCI) USA, on 60
different human cancer cell lines, representing melanoma, leukemia and tumors of the ovary,
breast, lung, brain, colon, kidney and prostate. The human tumor cell lines were grown in
RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. Cells were
inoculated into 96 well microtiter plates in 100 µL at densities ranging from 5,000 to 40,000
cells/well depending on the doubling time of individual cell lines. After cell inoculation, the
microtiter plates were incubated at 37 ºC, 5% CO2, 95% air and 100% relative humidity for
24 h, prior to addition of test compounds. After 24 h, two plates of each cell line were fixed
in situ with trichloroacetic acid (TCA), to stand for a measurement of the cell population for
each cell line at the time of sample addition (Tz).
Synthetic compounds for the in vitro anticancer screening were prepared in DMSO:glycerol
9:1, at a concentration of 4 mM for the one dose assay and 40 mM for the 5-dose assay. In
both cases the solution was diluted 1:400, giving a high test concentration of 10 or 100 µM
respectively. For 5-dose assay, the test compounds were solubilised in DMSO:glycerol (9:1)
at 400-fold to obtain the desired final maximum test concentration and stored frozen prior to
use. At the time of compounds addition, an aliquot of frozen concentrate was thawed and
diluted to twice, the desired final maximum test concentration with complete medium
containing 50 ꢀg/mL Gentamicin. Additional four, 10-fold or 1/2 log serial dilutions were
made to provide a total of five drug concentrations plus control. Aliquots of 100 ꢀL of these
different sample dilutions were added to the appropriate microtiter wells already containing
100 ꢀL of medium, resulting in the required final sample concentrations. After the addition of
test compounds, the plates were incubated for an additional 48 h at 37 ºC, 5% CO2, 95% air
and 100% relative humidity. Cells were then fixed in situ by the gentle addition of 50 ꢀL of
cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 min at 4 ºC. The
supernatant was discarded and plates were washed five times with water and air dried.
Sulforhodamine B (SRB) solution (100 ꢀL) at 0.4% (w/v) in 1% acetic acid was added to
each well and plates were incubated for 10 min at room temperature. Bound stain was
subsequently solubilised with 10 mM trizma base (tris buffer) and the absorbance was read
on an automated plate reader at a wavelength of 515 nm [31-33].
The NCI renamed the IC50 value into special concentration parameters (GI50, TGI and LC50).
A plate reader was used to read the optical densities and a microcomputer processed the

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optical densities into the special concentration parameters. % GI was calculated as: [(Ti - Tz)
/ (C - Tz)] × 100 for concentrations for which Ti ≥ Tz; and [(Ti - Tz) / Tz] × 100 for
concentrations for which Ti < Tz. Three dose response parameters (GI50, TGI and LC50) were
calculated for each experimental agent. Growth inhibition of 50% (GI50) calculated from [(Ti
- Tz) / (C - Tz)] × 100 = 50 (drug concentration resulting in a 50% reduction in the net
protein increase), Total growth inhibition (TGI) calculated from Ti = Tz (concentration at
which the total growth inhibition is 100%) and LC50 calculated from [(Ti - Tz) / Tz] × 100= -
50 (concentration of drug resulting in a 50% reduction in the measured protein at the end of
the drug treatment as compared to that at the beginning) indicated net loss of the cells [34,
35].
4.5. Statistical analysis
Statistical analysis was carried out by using Graph pad prism ver. 6.01 (Graph pad software
San Diego, CA). All results are expressed as mean ± standard deviation (S.D). Statistical
comparison of all results were performed using analysis of variance (ANOVA) followed by
Dunnett’s test to observe and identify the significant differences in groups. Differences in
values were considered statistically significant at P<0.05.
Acknowledgments
We are thankful to National Cancer Institute (NCI), Chemotherapeutic Agents Repository
and Fisher Bio Services, USA for in vitro anticancer screening of the compounds. Authors
are also thankful to Department of Pharmaceutical Chemistry, Jamia Hamdard, New Delhi
for providing necessary facilities. The spectral data provided by Central Instrumentation
facility, JNU, New Delhi and CDRI, Lucknow is also acknowledged. One of the authors
(Obaid Afzal) is grateful to the Council of Scientific and Industrial Research (CSIR), New
Delhi, India, for providing financial assistance.

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Fig. 1: Representative MAGL inhibitors along with their IC50 value.
Fig. 2 (Scheme 1): Synthesis of the intermediates and target compounds.
Fig. 3a: Dose response curves (% growth vs. sample concentration at NCI fixed protocol,
µM) for all NCI60 cell line panel after treatment with synthetic compound 21 (NSC: 780167)
at five concentrations (1 log dilutions from 10-4 mol/L to 10-8 mol/L).
Fig. 3b: Dose response curves (% growth vs. sample concentration at NCI fixed protocol,
µM) for all NCI60 cell line panel after treatment with synthetic compound 24 (NSC: 780168)
at five concentrations (1 log dilutions from 10-4 mol/L to 10-8 mol/L).

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Table-1: In vitro hMAGL enzyme inhibition assay of the synthesized compounds (4-38).
Compound
hMAGL IC50
04
10 nM
(ZINC24092691)
117 nM
196 nM
>100 µM
>100 µM
>100 µM
>100 µM
22 µM
17 µM
16 µM
282 nM
780 nM
815 nM
151 nM
242 nM
68 nM
8 nM
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
9 nM
ND
22
ND
23
24
8 nM
25
26
6.5 nM
9 nM
>100 µM
ND
27
28
3 µM
29
ND
30
6.5 µM
ND
31
32
13 µM
74 nM
47 µM
48 µM
24 µM
37 µM
424 nM
33
34
35
36
37
38
CAY10499
ND: Not Determined; CAY10499 used as standard control; IC50 value was calculated from Graph pad prism,
version 6.01.