Synthesis of novel morpholine, thiomorpholine and N-substituted piperazine coupled 2-(thiophen-2-yl)dihydroquinolines as potent inhibitors of Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.ejmech.2018.12.043

A series of novel morpholine, thiomorpholine and N-substituted piperazine coupled 2-(thiophen-2-yl)dihydroquinolines 7a–p was designed and synthesized from 2-acetyl thiophene in six step reaction sequence involving modified Bohlmann-Rahtz and Vilsmeier-Haack-Arnold reactions as key transformations. 2-(Thiophen-2-yl)dihydroquinoline was formylated and subsequently chlorinated using DMF-POCl₃. The resulting aldehyde was reduced to give an alcohol and then converted to bromide using PBr_3. Further coupling of bromide with morpholine, thiomorpholine and N-substituted piperazines resulted in the desired quinolines 7a-p in very good yields. All the new derivatives 7a–p were characterized by their NMR and mass spectral analysis. In vitro screening of new compounds for antimycobacterial activity against Mycobacterium tuberculosis H37Rv (MTB), resulted in two derivatives 7f and 7p as most potent antitubercular agents (MIC:1.56 μg/mL) with lower cytotoxicity profiles.

Full text of DOI:10.1016/j.ejmech.2018.12.043

Accepted Manuscript
Synthesis of novel morpholine, thiomorpholine and N-substituted piperazine coupled
2-(thiophen-2-yl)dihydroquinolines as potent inhibitors of Mycobacterium tuberculosis
Sandeep Kumar Marvadi, Vagolu Siva Krishna, Dharmarajan Sriram, Srinivas
Kantevari
PII:
S0223-5234(18)31080-8
DOI:
https://doi.org/10.1016/j.ejmech.2018.12.043
EJMECH 10976
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 April 2018
Revised Date: 4 December 2018
Accepted Date: 17 December 2018
Please cite this article as: S.K. Marvadi, V.S. Krishna, D. Sriram, S. Kantevari, Synthesis of novel
morpholine, thiomorpholine and N-substituted piperazine coupled 2-(thiophen-2-yl)dihydroquinolines as
potent inhibitors of Mycobacterium tuberculosis, European Journal of Medicinal Chemistry (2019), doi:
https://doi.org/10.1016/j.ejmech.2018.12.043.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Synthesis of novel morpholine, thiomorpholine and N-substituted piperazine coupled 2-
(thiophen-2-yl)dihydroquinolines as potent inhibitors of Mycobacterium tuberculosis
*
Sandeep Kumar Marvadi, Vagolu siva krishna, Dharmarajan Sriram, Srinivas Kantevari

ACCEPTED MANUSCRIPT
Synthesis of novel morpholine, thiomorpholine and N-substituted
piperazine coupled 2-(thiophen-2-yl)dihydroquinolines as potent
inhibitors of Mycobacterium tuberculosis
a
b
b
*a,c
Sandeep Kumar Marvadi, Vagolu Siva Krishna, Dharmarajan Sriram, Srinivas Kantevari,
a
Fluro & Agrochemicals Division, CSIR- Indian Institute of Chemical Technology, Hyderabad-
00007, Telangana, INDIA.
5
b
Medicinal Chemistry and Antimycobacterial Research Laboratory, Pharmacy Group, Birla
Institute of Technology & Science-Pilani, Hyderabad Campus, Jawahar Nagar, Hyderabad-
5
00078, Telangana, India.
c
Academy of Scientific and innovative Research, CSIR- Indian Institute of Chemical Technology,
Hyderabad-500 007, Telangana, India.
Abstract
A series of novel morpholine, thiomorpholine and N-substituted piperazine coupled 2-(thiophen-
2
-yl)dihydroquinolines 7a–p was designed and synthesized from 2-acetyl thiophene in six step
reaction sequence involving modified Bohlmann-Rahtz and Vilsmeier-Haack-Arnold reactions
as key transformations. 2-(Thiophen-2-yl)dihydroquinoline was formylated and subsequently
chlorinated using DMF-POCl . The resulting aldehyde was reduced to give an alcohol and then
3
converted to bromide using PBr3. Further coupling of bromide with morpholine, thiomorpholine
and N-substituted piperazines resulted in the desired quinolines 7a-p in very good yields. All the
new derivatives 7a–p were characterized by their NMR and mass spectral analysis. In vitro
screening of new compounds for antimycobacterial activity against Mycobacterium tuberculosis
H37Rv (MTB), resulted in two derivatives 7f and 7p as most potent antitubercular agents
(MIC:1.56 µg/mL) with lower cytotoxicity profiles.
Keywords:
Thiophene; Piperazine; Quinoline; Mycobacterium tuberculosis; Vilsmeier-Haack-Arnold
reaction

ACCEPTED MANUSCRIPT
1
. Introduction
Tuberculosis (TB) is one of the deadliest airborne infectious disease caused mainly by
pathogen Mycobacterium tuberculosis (Mtb) [1,2]. According to Global tuberculosis report 2018
of the World Health Organization (WHO), an estimated 1.3 million deaths and approximately
1
0.4 million people were affected with tuberculosis in 2017 [3]. Today TB is responsible for a
greater number of deaths than HIV and ranked as the leading killer in infectious diseases [3-8].
Further TB threat has acquired a new dimension with the emergence of both multidrug-resistant
TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) [9-13]. In 2017, globally, 558,000
people developed TB that was resistant to first line anti TB drug rifampicin, and of these 82%
had MDR & XDR -TB [3,14-16].
After 40 years of long wait, approval of Bedaquiline (Fig. 1), a quinoline based drug for
treating multi drug resistant TB [17-19], have gained momentum to investigate various quinoline
derivatives as antimycobacterial agents [20-25]. Previously we reported [26-32] that 2-substiuted
dihydroquinolones exhibit potential antitubercular activity against H37RV. Also recent reports
suggested that the groups like morpholine, piperazine helps improving pharmacological
properties [33,34]. Our continued interest [35,36] in developing new antimycobacterial agents
led to hybridization of 2-(thiophenyl)dihydroquinolines with morpholine, thiomorpholine, or N-
substituted piperazines in one molecular platform to generate a new scaffold for
biological evaluation. We herein report an efficient synthesis and evaluation of novel
dihydroquinoline derivatives 7a-p in excellent yields. Screening all sixteen new compounds for
in vitro activity against Mycobacterium tuberculosis H37Rv resulted in two compounds 7f and
7
p (MIC: 1.56 µg/mL) as most potent antitubercular agents.
Figure 1 >
Broadly, the designed scaffold (Fig. 2) has two fragments. The first fragment is 2-
thiophen-2-yl)dihydroquinoline pharmacophore having antitubercular activity[36]. The second
<
(
fragment is morpholine, thiomorpholine and piperazine pharmacophores present in clinical
antitubercular drugs II-VIII (Figure.1). Combining these two fragments into single molecular
frame resulted newer scaffold as depicted in figure 2. Alkyl, aryl, pyridinyl variants appended on
piperazine unit could provide lipophilicity control in the proposed scaffold.
<
Figure 2 >
2
. Results and Discussion

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2
.1 Chemistry
Initiating the synthesis, (Scheme 1) β-enaminone 1 required was prepared by the
condensation of 2-acetylthiophene with N,N-dimethylformamide dimethyl acetal (DMF-DMA)
refluxing in xylene[37]. Enaminone 1 was reacted with cyclohexane-1,3-dione and ammonium
acetate in the presence of CeCl .7H O-NaI at reflux in propan-2-ol to obtain the compound 2 in
3
2
very good yield [36-38]. Reaction of 2 was subjected to Vilsmeier-Haack-Arnold reaction
o
(
POCl dimethylformamide in CHCl at 60 C for 4 h) to afford compound 3 in 84% yield. 3
3
,
3
1
13
were fully characterized by H and C NMR, and mass (ESI-MS and HR-MS) spectral data.
Scheme 1 >
The aldehyde 3 was reduced with sodium borohydride in methanol to give alcohol 4 in 88%
yield. The alcohol 4 was converted to the respective bromide 5 in 82% yield using PBr in
<
3
diethyl ether at RT for 1 h. Further coupling of 5 with various amines 6a-p (morpholine,
thiomorpholine, N-substituted piperazines) in the presence of K CO at RT in acetone resulted
2
3
7
a-p in very good yields (72-94%). All of the compounds 7a-p were purified through silica gel
column chromatography (HPLC purity >95%) and were fully characterized by IR, was fully
1
13
characterized by H and C NMR, and mass (ESI-MS and HR-MS) spectral analysis. For
1
example, H NMR spectra of 7a showed aromatic protons at δ 7.81 - 7.10, morpholine protons at
δ 3.74 – 3.69 (4H) as multiplet (N-attached) and δ 2.54 – 2.47 (4H) as multiplet (O-attached),
2
methylene protons (-CH ) at δ 3.35 (2H) as singlet, triplet signals appeared for dihydroquinoline
1
3
protons at δ 3.04 ( J = 8.4 Hz, 2H), 2.66 (t, J = 8.4 Hz, 2H). C NMR spectrum showed
methylene carbon at δ 53.6, N-attached carbons in morpholine at δ 60.5 and O-attached carbons
in morpholine at δ 66.9. HRMS analysis of 7a displayed a molecular ion peak at m/z 347.09794
+
[
M+H] suggesting the molecular formula of C H ON ClS. Additionally, the IR spectra for the
1
8
20
2
-1
target compounds 7a-p exhibited characteristic absorption bands at 1637-1688 cm , 2958-2802
-1
-1
cm and 1010 - 1245 cm which corresponded to C=O, C-H and C-N respectively.
Table 1 >
<
2
2
.2 Pharmacological evaluation
.2.1 Antimycobacterial activity
All the newly synthesized dihydroquinoline derivatives 7a–p was screened for in vitro
antimycobacterial activity against M. Tuberculosis H37Rv strain by micro plate alamar blue
assay method [39]. Resazurin, used as an oxidation-reduction (REDOX) indicator in alamar

ACCEPTED MANUSCRIPT
blue® assay undergoes colorimetric change in response to the proliferation of mycobacterium
tuberculosis and was measured quantitatively. The minimum inhibitory concentration (MIC) is
defined as the minimum concentration of compound required to completely inhibit the bacterial
growth. The MIC values (µg/mL) of 7a-p along with isoniazid, Rifampicin, ciprofloxacin and
ethambutol as standard drugs for comparison are presented in Table 1. All sixteen products 7a-p
screened have shown in vitro inhibitory activity against Mtb with MIC ranging from 1.56-25.0
µg/mL. With respect to parent compound 2 (MIC: 12.5 µg/mL) six derivatives 7a, 7d, 7f, 7k, 7m
and 7p are more potent with lower MICs. When compared to first line anti-TB drugs, all the
compounds are less potent than Isoniazid (0.1 µg/mL), and Rifampicin (0.2 µg/mL). Two
compounds 7f & 7p (MIC: 1.56 µg/mL) are more potent than ethambutol and equipotent to
another standard drug ciprofloxacin (MIC: 1.56 µg/mL). Compound 7d (MIC: 3.125 µg/mL) is
equipotent to ethambutol. Among other new analogues, three compounds 7a, 7k and 7m
exhibited MIC 6.25 µg/ mL, and two compounds 7e, 7g exhibited MIC 12.5 µg/mL, and eight
compounds 7b, 7c, 7h, 7i, 7j, 7l, 7n, 7o exhibited MIC 25.0 µg/mL
.
Structure-activity relationship (SAR) of new compounds 7a-p indicated interesting in
vitro antimycobacterial activity patterns against M. tuberculosis H37Rv (MTB). To note that,
thiomorpholine analog 7b is less potent than parent 2-(thiophen-2-yl)dihydro quinoline 2 (MIC:
1
2.5 µg/mL) whereas morpholine analog 7a exhibited better potency than both 2 & 7b. The best
active compounds are N-substituted piperazine analogs. Substation on piperazine played crucial
role in the inhibition of MTB. For example, phenyl ring bearing 2-methoxy and 2-fluoro groups
7
k & 7m showed moderate activity (MIC: 6.25 µg/mL) and 1-cyclopropane carbonyl analog 7d
displayed good MTB inhibition (MIC: 3.125 µg/mL). Further replacement of phenyl unit with
pyridine and benzyl groups resulted most potent analogs 7p & 7f with best MTB inhibition
activity and low cytotoxicity.
Lipophilicity (CLogP) is one of the important physicochemical properties analyzed in
antitubercular drug discovery to predict drug-like characteristics. In recent years, computational
methods are being used in calculating lipophilicity measurements since experimental methods
are cost intensive and time consuming. In general, Chemdraw® or quantitative structure–activity
databases are used as state-of-the-art software for calculating cLogP values [41].These
calculations are straightforward and provide the ClogP for a large number of molecules. Most of

ACCEPTED MANUSCRIPT
the first line antitubercular drugs show low lipophilicity which includes isoniazid (cLogP =
−
−
0.67), ethambutol (cLogP = 0.12), pyrazinamide (cLogP = −0.68), and cycloserine (cLogP =
1.2). However, recent precedents suggested that highly lipophilic compounds should not be
discarded in screening programs for antituberculosis drugs. We employed ChemDraw
Professional 17.1 to calculate CLogP values for 2-(thiophene-2-yl)dihydroquinolines 7a–p to
assess their lipophilic characteristics. The data (Table 1) suggested that compounds described in
this study has cLogP values in a range from 2.92 to 7.09. Compound 7d (MIC: 3.125 µg/mL) has
CLogP 4.20 which is in the limits of Lipinski rule. It is interesting to note that the ClogP values
of the active molecules 7f and 7p are 6.32 and 5.08 respectively. The success of Bedaquiline and
Delaminid as examples with lipophilicity (cLogP values of 7.3 and 5.6, respectively) [41, 42]
prompted us to consider active molecules 7f and 7p as hit analogues for further in vivo and
mechanistic evaluations. This information on structure/lipophilicity–antitubercular activity
relationship could help further in generating drug like lead analogs.
2
.2.2 In vitro cytotoxicity
In pre-clinical drug discovery, potential candidates are usually tested against mammalian
cell lines such HEK-293T cells in order to assess any cytotoxic effects the compound could exert
on the body’s own cells. The safety profile of antitubercular active dihydroquinoline derivatives
with MIC ≤6.25 µg/mL was assessed by testing in vitro cytotoxicity against Human Embryonic
Kidney (HEK-293T) cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(
MTT) assay. Reduction of a yellow tetrazolium salt (MTT) gives direct measure to cellular
metabolic activity as a proxy for cell viability. The promising antitubercular compounds 7a, 7d,
f, 7k, 7m and 7p exhibited 34.04%, 28.94%, 19.86%, 36.72%, 32.58% and 20.06% inhibition,
7
respectively, at a 50 µg/mL concentration (Figure 3). The results indicated that most potent
analogs 7f and 7p are also less toxic compared to other derivatives of this scaffold.
<
Figure 3 >
3
. Conclusion
In conclusion, we have designed and synthesized a series of novel morpholine,
thiomorpholine -and N-substituted piperazine coupled 2-(thiophene-2-yl)dihydroquinolines 7a–p
in excellent yields. All the new analogues 7a–p was fully characterized by their NMR and mass
spectral data and were evaluated for their in vitro antimycobacterial activity against
Mycobacterium tuberculosis H37Rv (Mtb). Two compounds 7f and 7p (MIC: 1.56 µg/mL) were

ACCEPTED MANUSCRIPT
found to be most effective inhibiting Mtb compared to other evaluated compounds and has low
cytotoxicity. Even though both 7f & 7p show higher ClogP values (6.32 and 5.08 respectively),
the success of Bedaquiline and Delaminid (cLogP values of 7.3 and 5.6, respectively) as drugs
treating TB prompted us to consider 7f and 7p as hit analogues for further in vivo and
mechanistic evaluations. With new anti-TB agents desperately needed, we believe that the
analogues reported in this work will help global efforts for identification of potential lead
antimycobacterial agents for further development.
4
. Experimental Section
Melting points were measured by CINTEX programmable melting point apparatus and
1
13
are uncorrected. H and C NMR spectra of samples in CDCl were recorded on AVANCE- 300
3
MHz, 400 MHz and 500 MHz spectrometers. Chemical shifts (δ) are reported relative to TMS (δ
=
0.0) as the internal standard. Spin multiplicities are described as s (singlet), brs (broad singlet),
d (doublet), t (triplet), q (quartet), or m (multiplet). Coupling constants are reported in hertz (Hz).
Mass spectra were recorded in ESI spectrometers. Analytical HPLC was performed with an
Shimadzu LC-20AD series system using waters acquity UPLC BEC 250 mm, C18, 5 µg column
-1
0
(
2
solvent: ACN:H O; gradient: 1 ml/min ; T = 25 C). All high resolution mass spectra were
recorded on QSTAR XL hybrid ms/ms system (Applied Bio systems/MDS sciex, foster city,
USA), equipped with an ESI source (IICT, Hyderabad). IR was recorded on Thermo Nicolet
nexus-670 spectrometer with reference to KBr. TLC was performed on Merck 60 F-254 silica
gel plates. The chemicals used in this work were obtained from commercial channels and were
used without purification
.1. Synthesis of 5-chloro-2-(thiophen-2-yl)-7,8-dihydroquinoline-6-carbaldehyde (3)
To a solution of N,N-dimethyl formamide (6 mmol) in anhydrous CHCl (15 mL) was cooled to
.
4
3
0
°C. phosphorous oxychloride (4 mmol) was added dropwise over a period of 10 min. The
resulting white suspension was warmed to room temperature and stirred for 30 min and a
solution of dihydro-6H-quinolin-5-one 2 (1 mmol) in chloroform (15 mL) was added dropwise
and mixture was refluxed for 4 h. After completion of the reaction, as monitored by TLC, the
reaction mixture was poured in ice water, solid sodium hydrogen carbonate was carefully added
3
to neutralize the acids and extracted with CHCl .The organic layer was washed with brine, dried
(
Na SO ), and concentrated in vacuo. The crude product was purified on a silica gel column
2
4
eluting with hexane/ethyl acetate to give 3 as yellow color solid. Yield: 84%; mp: 176-178 °C;

ACCEPTED MANUSCRIPT
1
H NMR (500 MHz, CDCl
3
) δ 10.37 (s, 1H, CHO), 8.06 (d, J = 8.2 Hz, 1H, Ar-H), 7.68 (dd, J =
3
.8, 0.9 Hz, 1H, Ar-H), 7.61 (d, J = 8.2 Hz, 1H, Ar-H), 7.46 (dd, J = 5.0, 0.9 Hz, 1H, Ar-H), 7.14
1
3
(
dd, J = 5.0, 3.8 Hz, 1H, Ar-H), 3.08 (t, J = 7.7 Hz, 2H, CH ), 2.78 (t, J = 7.7 Hz, 2H, CH ); C
2
2
3
NMR (75 MHz, CDCl ) δ 189.9, 158.9, 153.5, 143.8, 143.7, 133.7, 131.5, 129.0, 128.3, 125.9,
1
25.7, 116.9, 29.6, 21.0; IR (KBr) 3449, 3060, 2855, 1660, 1545, 1444, 1262, 1165, 831, 712
-1
+
+
cm ; MS (ESI) m/z 276 [M+H] HR-MS (ESI) Calcd for C14H11ONClS [M+H] : 276.02542,
found: 276.02444.
4
.2. Synthesis of (5-chloro-2-(thiophen-2-yl)-7,8-dihydroquinolin-6-yl)methanol (4)
To a mixture of 5-chloro-2-(thiophen-2-yl)-7,8-dihydroquinoline-6-carbaldehyde 3 (1.0 mmol) in
methanol, sodium borohydride (2 mmol) was added at 0 °C and stirred at RT for 1.5 h (Reaction
was monitored by TLC).after completion, the reaction mixture was evaporated under vacuum,
water and ethyl acetate were added ,and the organic layer was separated washed with brine,
dried over anhydrous sodium sulfate and removal of solvent in vacuo. The crude residue was
purified by column chromatography over silica gel to afford pure product (5-chloro-2-(thiophen-
1
2
-yl)-7,8-dihydroquinolin-6-yl)methanol 4 as colorless solid. Yield: 88%; mp: 114-116 °C; H
NMR (400 MHz, CDCl
3
) δ 7.81 (d, J = 8.1 Hz, 1H, Ar-H), 7.59 (dd, J = 3.7, 0.9 Hz, 1H, Ar-H),
.52 (d, J = 8.1 Hz, 1H, Ar-H), 7.38 (dd, J = 5.0, 0.9 Hz, 1H, Ar-H), 7.11 (dd, J = 5.0, 3.7 Hz,
H, Ar-H), 4.52 (s, 2H, CH ), 3.09 (t, J = 8.3 Hz, 2H, CH ), 2.71 (t, J = 8.3 Hz, 2H, CH ), 1.76
7
1
2
2
2
13
(
bs, 1H, OH); C NMR (75 MHz, CDCl
3
) δ 156.3, 150.6, 144.1, 136.0, 131.9, 128.0, 127.5,
1
1
26.7, 124.7, 124.6, 116.8, 62.7, 30.1, 25.5; IR (KBr) 3336, 2925, 1621, 1557, 1447, 1416, 1229,
-
1
+
046, 816, 699 cm ; MS (ESI) m/z 278 [M+H] ; HR- MS (ESI) Calcd For C14H13ONClS
+
[
M+H] : 278.0403, found: 278.0401
.
4
.3. Synthesis of 6-(bromomethyl)-5-chloro-2-(thiophen-2-yl)-7,8-dihydroquinoline (5)
To a solution of (5-chloro-2-(thiophen-2-yl)-7,8-dihydroquinolin-6-yl)methanol 4 (1.0 mmol ) in
diethyl ether was added PBr (0.5mmol) slowly at 0 °C. The resulting mixture was at RT under
3
stirring for 1 h. The reaction progress was monitored by TLC. After completion, the reaction
mixture was quenched with aq. KBr solution. The product was extracted with EtOAc , the
combined organic layers washed with brine solution and dried over anhydrous Na SO . The
2
4
solvent was removed under reduced pressure and the crude residue was subjected to column
1
chromatography to afford pure 5 as colorless solid. Yield: 82%; mp: 128-130 °C; H NMR (300
3
MHz, CDCl ) δ 7.84 (d, J = 8.2 Hz, 1H, Ar-H), 7.61 (dd, J = 3.8, 0.8 Hz, 1H, Ar-H), 7.53 (d, J =

ACCEPTED MANUSCRIPT
8
4
.2 Hz, 1H, Ar-H), 7.40 (dd, J = 4.9, 0.8 Hz, 1H, Ar-H), 7.12 (dd, J = 4.9, 3.8 Hz, 1H, Ar-H),
13
.36 (s, 2H, CH ), 3.12 (t, J = 8.5 Hz, 2H, CH ), 2.73 (t, J = 8.5 Hz, 2H, CH ); C NMR (75
2
2
2
MHz, CDCl ) δ 156.1, 151.1, 144.1, 132.5, 132.3, 128.1, 127.9, 126.4, 124.9, 116.8, 32.9, 30.2,
3
-1
2
7.1; IR (KBr) 3449, 3097, 2926, 1620, 1559, 1443, 1236, 1170, 959, 825, 708 cm .
4
.4. General Procedure for the synthesis of piperazine-dihydroquinoline derivatives (7a-p)
2 3
To a mixture of 6a-p (1.0 mmol) and K CO in acetone ( 10 mL) was added 6-(bromomethyl)-
5
-chloro-2-(thiophen-2-yl)-7,8-dihydroquinoline 5 (1 mmol) at room temperature under stirring
for 12 h (monitored by TLC), After completion the reaction mixture was evaporated and
extracted with ethylacetate and water, the organic layer was separated dried over anhydrous
Na SO and evaporated under vacuum. The resulting crude product was purified by silica gel
2
4.
column chromatography by using EtOAc/hexane as eluent.
4
.4.1. 4-(5-Chloro-2-(thiophen-2-yl)-7,8-dihydroquinolin-6-yl)methyl) morpholine (7a)
1
Colorless solid, yield: 86%; mp: 88-90 °C; H NMR (400 MHz, CDCl ) δ 7.81 (d, J = 8.1 Hz,
3
1
5
2
H, Ar-H), 7.58 (dd, J = 3.6, 0.9 Hz, 1H, Ar-H), 7.51 (d, J = 8.1 Hz, 1H, Ar-H), 7.38 (dd, J =
.0, 0.9 Hz, 1H, Ar-H), 7.10 (dd, J = 5.0, 3.6 Hz, 1H, Ar-H), 3.74-3.69 (m, 4H, 2CH ), 3.35 (s,
2
2 2 2 2
H, CH ), 3.04 (t, J = 8.4 Hz, 2H, CH ), 2.66 (t, J = 8.4 Hz, 2H, CH ), 2.54-2.47 (m, 4H, 2CH );
1
3
C NMR (125 MHz, CDCl ) δ 156.4, 150.6, 144.4, 134.0, 131.9, 128.0, 127.4, 127.0, 126.4,
3
1
1
24.5, 116.7, 66.9, 60.5, 53.6, 30.4, 27.0; IR (KBr) 3451, 3101, 2958, 2849, 2755, 1446, 1280,
-1
+
2
114, 943, 714 cm ; MS (ESI) m/z 347 [M+H] ; HR-MS (ESI) Calcd for C18H20ON ClS
+
[
M+H] : 347.09705, found: 347.09794.
4
.4.2. 4-((5-Chloro-2-(thiophen-2-yl)-7,8-dihydroquinolin-6-yl)methyl)thiomorpholine (7b)
1
Colorless solid, yield: 84%; mp: 132-134 °C; H NMR (400 MHz, CDCl ) δ 7.80 (d, J = 8.1 Hz,
3
1
5
H, Ar-H), 7.58 (dd, J = 3.6, 0.9 Hz, 1H, Ar-H), 7.51 (d, J = 8.1 Hz, 1H, Ar-H), 7.38 (dd, J =
.0, 0.9 Hz, 1H, Ar-H), 7.10 (dd, J = 5.0, 3.6 Hz, 1H, Ar-H), 3.35 (s, 2H, CH ), 3.03 (t, J = 8.4
2
1
3
Hz, 2H, CH ), 2.81-2.59 (m, 10H, 5CH ); C NMR (100 MHz, CDCl ) δ 156.5, 150.5, 144.4,
2
2
3
1
3
34.6, 131.9, 128.0, 127.4, 127.0, 126.2, 124.5, 116.7, 60.7, 54.9, 30.5, 27.9, 26.8; IR (KBr)
-1
448, 3094, 2912, 2803, 1564, 1444, 1415, 1282, 1102, 956, 830, 722 cm ; MS (ESI) m/z 363
+
+
[
M+H] ; HR-MS (ESI) Calcd for C H N ClS [M+H] : 363.07453, found: 363.07509.
18 20 2 2
4
.4.3. tert-Butyl4-((5-chloro-2-(thiophen-2-yl)-7,8-dihydroquinolin-6-yl)methyl)piperazine
carboxylate (7c)

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°
1
Colorless solid, yield: 82%; mp: 126-128 C; H NMR (400 MHz, CDCl
3
) δ 7.81 (d, J = 8.1 Hz,
1
H, Ar-H), 7.59-7.56 (m, 1H, Ar-H), 7.51 (d, J = 8.1 Hz, 1H, Ar-H), 7.40-7.35 (m, 1H, Ar-H),
7
.12-7.08 (m, 1H, Ar-H), 3.49-3.41 (m, 4H, 2CH ), 3.36 (s, 2H, CH ), 3.04 (t, J = 8.3 Hz, 2H,
2
2
1
3
CH
2
), 2.66 (t, J = 8.3 Hz, 2H, CH
2
), 2.51-2.40 (m, 4H, 2CH
2
3
), 1.46 (s, 9H, 3CH ); C NMR
(
100 MHz, CDCl ) δ 156.4, 154.5, 150.5, 144.3, 133.8, 131.9, 127.9, 127.4, 126.8, 126.5, 124.5,
3
1
1
16.6, 79.5, 60.0, 52.8, 43.3, 30.4, 28.3, 26.9; IR (KBr) 3448, 3004, 2853, 2812, 1688, 1451,
-
1
+
420, 1245, 1164, 1007, 835, 714 cm ; MS (ESI) m/z 446 [M+H] ; HR-MS (ESI) Calcd for
+
C H O N ClS [M+H] : 446.16483, found: 446.16635.
23
29
2
3
4
.4.4. (4-((5-Chloro-2-(thiophen-2-yl)-7,8-dihydroquinolin-6-yl)methyl)piperazinyl)(cyclo
propyl)methanone (7d)
1
Colorless solid, yield: 88%; HPLC purity: 97.17%; mp: 138-140 °C; H NMR (500 MHz,
CDCl ) δ 7.81 (d, J = 8.1 Hz, 1H, Ar-H), 7.58 (dd, J = 3.6, 0.9 Hz, 1H, Ar-H), 7.52 (d, J = 8.1
3
Hz, 1H, Ar-H), 7.38 (dd, J = 5.0, 0.9 Hz, 1H, Ar-H), 7.11 (dd, J = 5.0, 3.6 Hz, 1H, Ar-H), 3.75-
3
.59 (m, 4H, 2CH ), 3.38 (s, 2H, CH ), 3.06 (t, J = 8.3 Hz, 2H, CH ), 2.68 (t, J = 8.3 Hz, 2H,
2
2
2
CH
2
2 2
), 2.59-2.44 (m, 4H, 2CH ), 1.76-1.69 (m, 1H, CH), 1.02-0.96 (m, 2H, CH ), 0.79-0.72 (m,
1
3
2
1
2
H, CH ); C NMR (100 MHz, CDCl ) δ 171.8, 156.4, 150.6, 144.3, 133.9, 131.9, 128.0, 127.4,
2 3
26.9, 126.5, 124.5, 116.6, 60.0, 53.2, 52.9, 45.3, 42.0, 30.4, 27.0, 10.8, 7.3; IR (KBr) 3446,
-
1
+
928, 2806, 1637, 1444, 1234, 1132, 1039, 819, 695 cm ; MS (ESI) m/z 414 [M+H] HR-MS
+
(
ESI) Calcd for C H ON ClS [M+H] : 414.13915 found: 414.14014.
2
2
25
3
4
.4.5. 6-((4-((1,3-Dioxolan-2-yl)methyl)piperazinyl)methyl)-5-chloro-2-(thiophen-2-yl)-7,8-
dihydro quinoline (7e)
Colorless solid, yield: 92%; mp: 114-116 °C; H NMR (400 MHz, CDCl ) δ 7.81 (d, J = 8.1 Hz,
1
3
1
5
3
2
1
3
H, Ar-H), 7.57 (dd, J = 3.6, 0.9 Hz, 1H, Ar-H), 7.51 (d, J = 8.1 Hz, 1H, Ar-H), 7.37 (dd, J =
.0, 0.9 Hz, 1H, Ar-H), 7.10 (dd, J = 5.0, 3.6 Hz, 1H, Ar-H), 5.02 (t, J = 4.4 Hz, 1H, CH), 4.02-
2 2 2 2
.92 (m, 2H, CH ), 3.90-3.81 (m, 2H, CH ), 3.36 (s, 2H, CH ), 3.03 (t, J = 8.5 Hz, 2H, CH ),
1
3
.73-2.46 (m, 12H, 6CH2); C NMR (100 MHz, CDCl ) δ 156.5, 150.4, 144.5, 134.7, 131.9,
3
28.0, 127.3, 127.1, 126.0, 124.4, 116.6, 102.6, 64.7, 61.1, 60.1, 53.9, 50.0, 30.5, 26.9; IR (KBr)
-
1
421, 3097, 2939, 2878, 2809, 1449, 1295, 1139, 1064, 824, 705 cm ; MS (ESI) m/z 432
+
+
[
M+H] ; HR-MS (ESI) Calcd for C H O N ClS [M+H] : 432.14946, found: 432.15070.
22 27 2 3
4
.4.6. 6-((4-Benzylpiperazinyl)methyl)-5-chloro-2-(thiophen-2-yl)-7,8-dihydroquinoline (7f)

ACCEPTED MANUSCRIPT
1
Colorless solid, yield: 94%; HPLC purity: 96.49%; mp: 116-118 °C; H NMR (500 MHz,
CDCl ) δ 7.79 (d, J = 8.2 Hz, 1H, Ar-H), 7.56 (dd, J = 3.6, 0.9 Hz, 1H, Ar-H), 7.50 (d, J = 8.2
3
Hz, 1H, Ar-H), 7.37 (dd, J = 5.0, 0.9 Hz, 1H, Ar-H), 7.34-7.28 (m, 4H, Ar-H), 7.25-7.22 (m, 1H,
Ar-H), 7.09 (dd, J = 5.0, 3.6 Hz, 1H, Ar-H), 3.53 (s, 2H, CH
2 2
), 3.35 (s, 2H, CH ), 3.02 (t, J = 8.3
1
3
Hz, 2H, CH ), 2.64 (t, J = 8.3, Hz, 2H, CH ), 2.59-2.41 (m, 8H, 4CH ); C NMR (125 MHz,
2
2
2
CDCl
3
) δ 156.4, 150.4, 144.4, 137.7, 134.7, 131.8, 129.1, 128.1, 127.9, 127.3, 127.1, 127.0,
1
1
26.0, 124.4, 116.6, 62.8, 60.1, 53.1, 52.9, 30.4, 27.0; IR (KBr) 3450, 3097, 2933, 2802, 1629,
-
1
+
448, 1282, 1140, 1010, 701 cm ; MS (ESI) m/z 436 [M+H] ; HR-MS (ESI) Calcd for
+
C
25
H
27
N
3
ClS [M+H] : 436.15969, found: 436.16087.
4
.4.7.
quinoline (7g)
Colorless solid, yield: 88%; mp: 176-178 °C; H NMR (500 MHz, CDCl ) δ 7.80 (d, J = 8.2 Hz,
5-Chloro-6-((4-(4-chlorobenzyl)piperazinyl)methyl)-2-(thiophen-2-yl)-7,8-dihydro
1
3
1
5
2
2
1
H, Ar-H), 7.57 (dd, J = 3.6, 0.9 Hz, 1H, Ar-H), 7.51 (d, J = 8.2 Hz, 1H, Ar-H), 7.37 (dd, J =
.0, 0.9 Hz, 1H, Ar-H), 7.30-7.22 (m, 4H, Ar-H), 7.10 (dd, J = 5.0, 3.6 Hz, 1H, Ar-H), 3.48 (s,
H, CH ), 3.35 (s, 2H, CH ), 3.03 (t, J = 8.3 Hz, 2H, CH ), 2.64 (t, J = 8.3 Hz, 2H, CH ), 2.58-
2
2
2
2
1
3
2 3
.38 (m, 8H, 4CH ); C NMR (100 MHz, CDCl ) δ 156.4, 150.4, 144.4, 136.5, 134.7, 132.6,
31.8, 130.3, 128.2, 127.9, 127.3, 127.1, 126.0, 124.4, 116.6, 62.0, 60.1, 53.1, 52.9, 30.4, 26.9;
-1
IR (KBr) 3450, 3068, 2926, 2804, 1445, 1330, 1279, 1135, 1005, 830, 690 cm ; MS (ESI) m/z
+
+
4
4
7
26 3 2
70 [M+H] ; HR-MS (ESI) Calcd for C25H N Cl S [M+H] : 470.12096, found: 470.12190.
.4.8. 6-((4-(Benzo[d][1,3]dioxol-5-ylmethyl)piperazinyl)methyl)-5-chloro-2-(thiophen-2-yl)-
,8-dihydroquinoline (7h)
1
Colorless solid, yield: 90%; mp: 112-114 °C; H NMR (400 MHz, CDCl ) δ 7.80 (d, J = 8.1 Hz,
3
1
H, Ar-H), 7.57 (dd, J = 3.6, 0.9 Hz, 1H, Ar-H), 7.51 (d, J = 8.1 Hz, 1H, Ar-H), 7.37 (dd, J =
.0, 0.9 Hz, 1H, Ar-H), 7.10 (dd, J = 5.0, 3.6 Hz, 1H, Ar-H), 6.86 (s, 1H, Ar-H), 6.77-6.71 (m,
H, Ar-H), 5.93 (s, 2H, CH ), 3.44 (s, 2H, CH ), 3.35 (s, 2H, CH ) CH , 3.03 (t, J = 8.5 Hz, 2H,
5
2
2
2
2
2
1
3
CH
2
), 2.64 (t, J = 8.5 Hz, 2H, CH
2
2 3
), 2.60-2.37 (m, 8H, 4CH ); C NMR (125 MHz, CDCl ) δ
1
1
1
56.5, 150.5, 147.5, 146.6, 144.5, 134.6, 131.9, 131.2, 128.0, 127.4, 127.1, 126.1, 124.5, 122.4,
16.7, 109.6, 107.8, 100.8, 62.5, 60.1, 53.0, 52.7, 30.5, 27.0; IR (KBr) 3422, 3111, 2926, 2809,
-
1
+
485, 1448, 1245, 1033, 922, 824, 717 cm ; MS (ESI) m/z 480 [M+H] ; HR-MS (ESI) Calcd for
+
C H O N ClS [M+H] : 480.15205, found: 480.15070.
26
27
2
3
4
.4.9. 5-Chloro-6-((4-phenylpiperazinyl)methyl)-2-(thiophen-2-yl)-7,8-dihydroquinoline (7i)

ACCEPTED MANUSCRIPT
1
Colorless solid, yield: 82%; mp: 208-210 °C; H NMR (400 MHz, CDCl
3
) δ 7.83 (d, J = 8.1 Hz,
1
5
H, Ar-H), 7.58 (dd, J = 3.6, 0.9 Hz, 1H, Ar-H), 7.52 (d, J = 8.1 Hz, 1H, Ar-H), 7.37 (dd, J =
.0, 0.9 Hz, 1H, Ar-H), 7.29-7.23 (m, 2H, Ar-H), 7.10 (dd, J = 5.0, 3.6 Hz, 1H, Ar-H), 6.96-6.90
), 3.24-3.17 (m, 4H, 2CH ), 3.06 (t, J =
.4 Hz, 2H, CH ), 2.74-2.61 (m, 6H, 3CH ); C NMR (100 MHz, CDCl ) δ 156.4, 151.1, 150.4,
(m, 2H, Ar-H), 6.88-6.82 (m, 1H, Ar-H), 3.41 (s, 2H, CH
2
2
1
3
8
1
4
2
2
3
44.4, 134.5, 131.9, 128.9, 127.9, 127.4, 127.0, 126.1, 124.4, 119.5, 116.6, 115.9, 60.1, 53.1,
-1
9.0, 30.4, 26.9; IR (KBr) 3450, 2939, 2835, 1599, 1501, 1446, 1238, 1137, 1004, 753 cm ; MS
+
+
(
ESI) m/z 422 [M+H] ; HR-MS (ESI) Calcd for C H N ClS [M+H] : 422.14403, found:
24 25 3
4
22.14522.
.4.10. 5-Chloro-2-(thiophen-2-yl)-6-((4-(o-tolyl)piperazinyl)methyl)-7,8-dihydroquinoline
7j)
Colorless solid, yield: 76%; mp: 138-140 °C; H NMR (400 MHz, CDCl ) δ 7.83 (d, J = 8.1 Hz,
4
(
1
3
1
5
H, Ar-H), 7.58 (dd, J = 3.6, 0.9 Hz, 1H, Ar-H), 7.52 (d, J = 8.1 Hz, 1H, Ar-H), 7.38 (dd, J =
.0, 0.9 Hz, 1H, Ar-H), 7.20-7.13 (m, 2H, Ar-H), 7.10 (dd, J = 5.0, 3.6 Hz, 1H, Ar-H), 7.06-7.01
(
m, 1H, Ar-H), 7.00-6.95 (m, 1H, Ar-H), 3.43 (s, 2H, CH ), 3.07 (t, J= 8.3 Hz, 2H, CH ), 2.97-
2
2
1
3
2
1
1
1
.91 (m, 4H, 2CH
2
), 2.74-2.64 (m, 6H, 3CH
2
3 3
), 2.31 (s, 3H, CH ); C NMR (100 MHz, CDCl ) δ
56.6, 151.4, 150.6, 144.5, 132.5, 132.0, 130.9, 128.0, 127.4, 127.1, 126.5, 124.5, 123.0, 118.9,
16.7, 60.2, 53.8, 51.6, 30.5, 27.1, 17.8; IR (KBr) 3447, 3071, 2934, 2820, 1491, 1446, 1222,
-1
+
27 3
131, 927, 825, 721 cm ; MS (ESI) m/z 436 [M+H] ; HR-MS (ESI) Calcd for C25H N ClS
+
[
M+H] : 436.15950, found: 436.16087.
4
.4.11.
5-Chloro-6-((4-(2-methoxyphenyl)piperazinyl)methyl)-2-(thiophen-2-yl)-7,8-di
hydro quinoline (7k)
Colorless solid, yield: 80%; mp: 92-94 °C; H NMR (400 MHz, CDCl ) δ 7.83 (d, J = 8.1 Hz,
1
3
1
5
3
H, Ar-H), 7.58 (dd, J = 3.6, 0.9 Hz, 1H, Ar-H), 7.52 (d, J = 8.1 Hz, 1H, Ar-H), 7.38 (dd, J =
.0, 0.9 Hz, 1H, Ar-H), 7.10 (dd, J = 5.0, 3.6 Hz, 1H, Ar-H), 7.02-6.84 (m, 4H, Ar-H), 3.86 (s,
13
3 2 2 2
H, OCH ), 3.44 (s, 2H, CH ), 3.15-3.03 (m, 6H, 3CH ), 2.76-2.66 (m, 6H, 3CH ); C NMR
(
100 MHz, CDCl ) δ 156.4, 152.0, 150.4, 144.3, 141.1, 134.4, 131.8, 127.9, 127.3, 126.9, 126.2,
3
1
2
24.4, 122.7, 120.8, 118.0, 116.5, 111.0, 60.0, 55.1, 53.3, 50.4, 30.4, 26.9; IR (KBr) 3447, 3062,
-
1
+
931, 2817, 1582, 1497, 1447, 1237, 1021, 745 cm ; MS (ESI) m/z 452 [M+H] ; HR-MS (ESI)
+
Calcd for C H ON ClS [M+H] : 452.15440, found: 452.15579.
2
5
27
3

ACCEPTED MANUSCRIPT
4
.4.12. 1-(4-(4-((5-Chloro-2-(thiophen-2-yl)-7,8-dihydroquinolin-6-yl)methyl)piperazinyl)
phenyl)ethanone (7l)
1
Colorless solid, yield: 84%; mp: 198-200 °C; H NMR (400 MHz, CDCl ) δ 7.93-7.78 (m, 3H,
3
Ar-H), 7.59 (dd, J = 3.6, 0.9 Hz, 1H, Ar-H), 7.53 (d, J = 8.1 Hz, 1H, Ar-H), 7.38 (dd, J = 5.0, 0.9
Hz, 1H, Ar-H), 7.13-7.09 (m, 1H, Ar-H), 6.91-6.82 (m, 2H, Ar-H), 3.44-3.30 (m, 6H, 3CH2),
1
3
3
.06 (t, J = 8.4 Hz, 2H, CH
2 2 3
), 2.74-2.59 (m, 6H, 3CH ), 2.52 (s, 3H, CH ); C NMR (100 MHz,
CDCl ) δ 196.4, 156.4, 154.0, 150.6, 144.4, 134.0, 132.0, 130.38, 130.30, 128.0, 127.5, 126.9,
3
1
1
26.5, 124.5, 116.7, 113.3, 60.0, 52.8, 47.2, 30.5, 27.0, 26.0; IR (KBr) 3446, 3099, 2930, 2837,
-
1
+
668, 1597, 1448, 1229, 1193, 821, 701 cm ; MS (ESI) m/z 464 [M+H] ; HR-MS (ESI) Calcd
+
for C H ON ClS [M+H] : 464.15676, found: 464.15579.
2
6
27
3
4
.4.13. 5-Chloro-6-((4-(2-fluorophenyl)piperazinyl)methyl)-2-(thiophen-2-yl)-7,8-dihydro
quinoline (7m)
Colorless solid, yield: 72%; mp: 186-188 °C; H NMR (400 MHz, CDCl ) δ 7.83 (d, J = 8.1 Hz,
1
3
1
5
2
H, Ar-H), 7.58 (dd, J = 3.6, 0.9 Hz, 1H, Ar-H), 7.52 (d, J = 8.1 Hz, 1H, Ar-H), 7.38 (dd, J =
.0, 0.9 Hz, 1H, Ar-H), 7.10 (dd, J = 5.0, 3.6 Hz, 1H, Ar-H), 7.08-6.88 (m, 4H, Ar-H), 3.44 (s,
1
3
2 2 2 2
H, CH ), 3.17-3.10 (m, 4H, 2CH ), 3.06 (t, J = 8.5 Hz, 2H, CH ), 2.75-2.67 (m, 6H, 3CH ); C
NMR (100 MHz, CDCl ) δ 156.5, 155.6, 150.5, 144.4, 140.0, 134.4, 131.9, 128.0, 127.4, 127.1,
3
1
3
26.3, 124.5, 124.3, 122.3, 118.8, 116.7, 116.0, 60.1, 53.3, 50.4, 30.5, 27.0; IR (KBr) 3449,
-1
+
072, 2824, 1499, 1446, 1237, 1134, 928, 719 cm ; MS (ESI) m/z 440 [M+H] ; HR-MS (ESI)
+
Calcd for C H N ClFS [M+H] : 440.13434, found: 440.13580.
2
4
24
3
4
7
.4.14.
5-Chloro-2-(thiophen-2-yl)-6-((4-(2-(trifluoromethyl)phenyl)piperazinyl)methyl)-
,8-dihydroquinoline (7n)
1
Colorless solid, yield: 76%; mp: 164-166 °C; H NMR (500 MHz, CDCl ) δ 7.83 (d, J = 8.2 Hz,
3
1
2
H, Ar-H), 7.59 (dd, J = 3.6, 0.9 Hz, 1H, Ar-H), 7.53 (d, J = 8.2 Hz, 1H, Ar-H), 7.50-7.45 (m,
H, Ar-H), 7.38 (dd, J = 5.0, 0.9 Hz, 1H, Ar-H), 7.11 (dd, J = 5.0, 3.6 Hz, 1H, Ar-H), 6.94-6.89
2 2 2
(m, 2H, Ar-H), 3.41 (s, 2H, CH ), 3.32-3.25 (m, 4H, 2CH ), 3.06 (t, J = 8.3 Hz, 2H, CH ), 2.72-
1
3
2
1
.63 (m, 6H, 3CH2); C NMR (100 MHz, CDCl ) δ 156.5, 153.2, 150.6, 144.4, 134.3, 132.0,
3
28.0, 127.5, 127.0, 126.33, 126.30, 124.5, 123.3, 116.7, 114.4, 60.1, 52.9, 47.9, 30.5, 27.0; IR
-1
+
(
KBr) 3456, 3059, 2883, 2758, 1568, 1294, 1132, 962, 705 cm ; MS (ESI) m/z 490 [M+H]
+
Calcd for C H N ClF S [M+H] : 490.13136, found: 490.13261.
2
5
24
3
3

ACCEPTED MANUSCRIPT
4
.4.15.
5-Chloro-6-((4-(4-chlorophenyl)piperazinyl)methyl)-2-(thiophen-2-yl)-7,8-dihy
droquinoline (7o)
Colorless solid, yield: 78%; mp: 176-178 °C; H NMR (400 MHz, CDCl ) δ 7.83 (d, J = 8.2 Hz,
1
3
1
5
H, Ar-H), 7.58 (dd, J = 3.6, 1.1 Hz, 1H, Ar-H), 7.53 (d, J = 8.2 Hz, 1H, Ar-H), 7.38 (dd, J =
.1, 1.1 Hz, 1H, Ar-H), 7.22-7.17 (m, 2H, Ar-H), 7.11 (dd, J = 5.1, 3.6 Hz, 1H, Ar-H), 6.86-6.81
2 2 2
(m, 2H, Ar-H), 3.41 (s, 2H, CH ), 3.20-3.14 (m, 4H, 2CH ), 3.06 (t, J = 8.6 Hz, 2H, CH ), 2.72-
1
3
2
1
.63 (m, 6H, 3CH2); C NMR (125 MHz, CDCl ) δ 156.5, 150.6, 149.8, 144.4, 134.4, 132.0,
3
28.8, 128.0, 127.4, 127.1, 126.3, 124.5, 124.3, 117.1, 116.7, 60.1, 53.0, 49.1, 30.5, 27.0; IR
-1
(
KBr) 3447, 3063, 2940, 2821, 1492, 1446, 1229, 1140, 935, 820, 708 cm ; MS (ESI) m/z 456
+
+
[
M+H] Calcd for C H N Cl S [M+H] : 456.10557, found: 456.10625.
24 24 3 2
4
.4.16.
5-Chloro-6-((4-(pyridin-2-yl)piperazinyl)methyl)-2-(thiophen-2-yl)-7,8-dihydro
quinoline (7p)
Colorless solid, yield: 74%; HPLC purity: 96.02%; mp: 102-104 °C; H NMR (400 MHz,
CDCl ) δ 8.21-8.18(m, 1H, Ar-H), 7.83 (d, J = 8.1 Hz, 1H, Ar-H), 7.59 (dd, J = 3.6, 0.9 Hz, 1H,
1
3
Ar-H), 7.53 (d, J = 8.1 Hz, 1H, Ar-H), 7.50-7.45 (m, 1H, Ar-H), 7.38 (dd, J = 5.0, 0.9 Hz, 1H,
Ar-H), 7.11 (dd, J = 5.0, 3.6 Hz, 1H), 6.67-6.59 (m, 2H, Ar-H), 3.61-3.52 (m, 4H), 3.41 (s, 2H,
1
3
CH ), 3.07 (t, J = 8.5 Hz, 2H, CH ), 2.70 (t, J = 8.5 Hz, 2H, CH ), 2.66-2.58 (m, 4H, 2CH ); C
2
2
2
2
NMR (100 MHz, CDCl ) δ 159.4, 156.5, 150.6, 147.8, 144.4, 137.4, 132.0, 128.0, 127.4, 127.1,
3
1
1
4
4
24.5, 116.7, 113.2, 107.0, 60.2, 53.0, 45.1, 30.5, 27.0; IR (KBr) 3448, 3064, 2922, 1634, 1546,
-1
+
+
488, 1256, 1193, 703 cm ; MS (ESI) m/z 423 [M+H] Calcd for C H N ClS [M+H] :
23
24
4
23.13968, found: 423.14047.
.5. Antitubercular evaluation assay
All the compounds were further screened for in vitro antimycobacterial activity against M.
Tuberculosis H37Rv strain by microplate Alamar blue assay method. Briefly, the inoculum was
prepared from fresh LJ medium re-suspended in 7H9-S medium (7H9 broth, 0.1% casitone,
0
.5% glycerol, supplemented oleic acid, albumin, dextrose, and catalase [OADC]), adjusted to a
McFarland tube No. 1, and diluted 1:20; 100 µl was used as inoculum. Each drug stock solution
was thawed and diluted in 7H9-S at four-fold the final highest concentration tested. Serial two-
fold dilutions of each drug were prepared directly in a sterile 96-well microtiter plate using 100
µl 7H9-S. A growth control containing no antibiotic and a sterile control were also prepared on
each plate. Sterile water was added to all perimeter wells to avoid evaporation during the

ACCEPTED MANUSCRIPT
o
incubation. The plate was covered, sealed in plastic bags and incubated at 37 C in normal
atmosphere. After 7 days incubation, 30 ml of Alomar blue solution was added to each well, and
the plate was re-incubated overnight. A change in color from blue (oxidized state) to pink
(
reduced) indicated the growth of bacteria, and the MIC was defined as the lowest concentration
of drug that prevented this change in colour.
.6 Evaluation of cytotoxicity
4
Antitubercular active compounds with MIC ≤ 6.25 µg/mL were further examined for toxicity in
a HEK-293T cell line at the concentration of 50 µg/mL. After 72 h of exposure, viability was
assessed on the basis of cellular conversion of MTT into a formazan product using the Promega
Cell Titer 96 non-radioactive cell proliferation assay.
Acknowledgement
The authors thank DBT-Indo-Australia biotechnology fund (BT/Indo-Aus/07/06/2013) for
financial support. SKM is thankful to UGC for senior research fellowship.
References
[1]
R. Ansumana, S. Keitell, G. M. Roberts, F. Ntoumi, E. Petersen, G. Ippolito, A. Zumla,
Int. J. Infect. Dis. 56 (2017) 101.
[
2]
3]
S. Baghdadi, Int. J. Mycobacteriol. (2016) doi: 10.1016/j.ijmyco.2016.10.002.
World Health Organization (WHO), WHO Global Tuberculosis Report 2018, WHO,
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[
Figure/Scheme Captions
Scheme 1. Synthesis of 2-(thiophen-2-yl) dihydroquinoline derivatives 7a–p
Reagents and conditions: (i) DMF-DMA, xylene, reflux, 7 h, 95%; (ii) Cyclohexane-1,3-dione,
o
NH
4
OAc, CeCl
3
.7H
2
O-NaI, propan-2-ol, reflux, 4 h, 86%; (iii) POCl
3
-DMF, CHCl
3
, 60 C, 4 h,

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8
4%; (iv) NaBH
acetone, rt, 12 h, 72–94%.
Table 1. Synthesis and in vitro activity evaluation (against M. tuberculosis H37Rv) of 7a-p.
4 3 2 3
, MeOH, rt, 1.5 h, 88%; (v) PBr , diethyl ether, rt, 1 h, 82%; (vi) 6a–p, K CO ,
Figure 1. Clinical antitubercular agents containing quinoline, piperazine, morpholine, and
thiomorpholine fragments.
Figure 2. Design strategy for new dihydroquinoline derivatives.
Figure 3. Percentage inhibition of HEK-293T cells by new analogs 7a, 7d, 7f, 7k, 7m, 7p at a
concentration of 50 µg/mL.
Reagents and conditions: (i) DMF-DMA, xylene, reflux, 7 h, 95%; (ii) Cyclohexane-1,3-dione,
o
NH
4
OAc, CeCl
3
.7H
2
O-NaI, propan-2-ol, reflux, 4 h, 86%; (iii) POCl
3
-DMF, CHCl
3
, 60 C, 4 h,
8
4%; (iv) NaBH , MeOH, rt, 1.5 h, 88%; (v) PBr , diethyl ether, rt, 1 h, 82%; (vi) 6a–p, K CO ,
4 3 2 3
acetone, rt, 12 h, 72–94%.
Scheme 1. Synthesis of 2-(thiophen-2-yl) dihydroquinoline derivatives 7a–p

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Table 1. Synthesis and in vitro activity evaluation (against M. tuberculosis H37Rv) of 7a-p
a
b
Entry
1
Product
7a
R
Yield(%) CLogP MIC(µg/mL)
86
84
82
4.04
4.77
5.89
6.25
25
2
3
7b
7c
25
4
7d
88
4.20
3.125
5
6
7
8
7e
7f
92
94
88
90
2.92
6.32
7.03
6.29
12.5
1.56
12.5
25
7g
7h
9
7i
82
6.03
25

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1
1
0
1
7j
76
80
6.41
5.93
25
7k
6.25
1
1
2
3
7l
84
72
5.66
6.22
25
7m
6.25
1
1
1
4
5
6
7n
7o
7p
76
78
74
7.09
6.91
5.08
25
25
1.56
Isoniazid
Rifampicin
1
1
1
2
7
8
9
0
---
---
---
---
---
---
---
---
---
---
---
---
0.1
0.2
Ciprofloxacine
1.56
3.13
Ethambutol
a
b
Isolated yield; Calculated using Chemdraw Professional 17.1.

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Figure 1. Clinical antitubercular agents containing quinoline, piperazine, morpholine, and
thiomorpholine fragments.

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Figure 2. Design strategy for new dihydroquinoline derivatives.
50
40
30
20
10
0
3
6.72
3
4.04
32.58
2
8.94
1
9.86
20.06
7
a
7d
7f
7k
7m
7p
Compounds
Figure 3. Percentage inhibition of HEK-293T cells by new analogs 7a, 7d, 7f, 7k, 7m, 7p at a
concentration of 50 µg/mL.

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Research Highlights
1
2
3
. A series of novel 2-thiophenyl dihydroquinoline derivatives were designed and synthesized.
. Morpholine, Thiomorpholine and N-substituted piperazines were coupled to quinoline core.
. 7f & 7p showed most potent in vitro antitubercular activity (MIC: 1.56 µg/ml) & low
cytotoxicity