Thiophene containing trisubstituted methanes [TRSMs] as identified lead against Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.ejmech.2015.03.036

Triarylmethanes (TRAMs) and thiophene containing trisubstituted methanes (TRSMs) have been reported by us, having potential against Mycobacterium tuberculosis and Mycobacterium fortuitum strains, respectively. Further, extension through synthesis and biol

Full text of DOI:10.1016/j.ejmech.2015.03.036

Accepted Manuscript
Thiophene containing Trisubstituted Methanes [TRSMs] as identified lead against
Mycobacterium tuberculosis
Priyanka Singh, Sudipta Kumar Manna, Amit Kumar Jana, Tiash Saha, Pankaj
Mishra, Saurav Bera, Maloy Kumar Parai, Srinivas Lavanya Kumar M, Sankalan
Mondal, Priyanka Trivedi, Vinita Chaturvedi, Shyam Singh, Sudhir Sinha, Gautam
Panda
PII:
S0223-5234(15)00203-2
10.1016/j.ejmech.2015.03.036
EJMECH 7781
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 February 2015
Revised Date: 16 March 2015
Accepted Date: 17 March 2015
Please cite this article as: P. Singh, S.K. Manna, A.K. Jana, T. Saha, P. Mishra, S. Bera, M.K. Parai,
S.L. Kumar M, S. Mondal, P. Trivedi, V. Chaturvedi, S. Singh, S. Sinha, G. Panda, Thiophene containing
Trisubstituted Methanes [TRSMs] as identified lead against Mycobacterium tuberculosis, European
Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.03.036.
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Thiophene containing Trisubstituted Methanes [TRSMs] as
identified lead against Mycobacterium tuberculosis
Leave this area blank for abstract info.
P Singh, S K Manna, A K Jana, T Saha, P Mishra, S Bera, M K Parai, S L Kumar M., S Mondal, P Trivedi, V Chaturvedi, S
Singh, S Sinha and G Panda
Lead S006-830 exhibited MIC; 1.33 mg/L, non-toxic against Vero C-1008 cell with SI>10, ex vivo efficacy
equivalent to first line TB drugs in mouse and human bone marrow derived macrophages.

ACCEPTED MANUSCRIPT
Thiophene containing Trisubstituted Methanes [TRSMs] as identified lead against Mycobacterium
tuberculosis
a
a
a
a
a
a
Priyanka Singh, Sudipta Kumar Manna, Amit Kumar Jana, Tiash Saha, Pankaj Mishra , Saurav Bera,
a
a
a
b
Maloy Kumar Parai, Srinivas Lavanya Kumar M. , Sankalan Mondal, Priyanka Trivedi, Vinita
Chaturvedi, Shyam Singh, Sudhir Sinha * and Gautam Panda *
b
b
b
a
a
b
Medicinal and Process Chemistry Division, Biochemistry Division; CSIR-Central Drug Research
Institute, B.S. 10/1, Jankipuram Extension, Sitapur Road, Lucknow-226031, UP, India.
Fax: (+) 91-522-2771941, E-mail: gautam.panda@gmail.com; gautam_panda@cdri.res.in
Fax: (+) 91-522-2771941, E-mail: sinha.sudhir@gmail.com, sudhir_sinha@cdri.res.in
Keywords: Trisubstituted Methanes, Mycobacterium tuberculosis H37R , Antimycobacterial agents
V
1

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Abstract
Triarylmethanes (TRAMs) and thiophene containing trisubstituted methanes (TRSMs) have been reported
by us, having potential against M. tuberculosis and M. fortuitum strains, respectively. Further, extension
through synthesis and biological evaluation of novel TRSMs resulted into an identified lead 36 (S006-
8
1
30) [(diisopropyl-(2-{4-[(4-methoxy-phenyl)- thiophen-2-yl-methyl]-phenoxy}-ethyl)-amine) with MIC;
.33 mg/L, non-toxic against Vero C-1008 cell line with selectivity index >10, ex vivo efficacy equivalent
to first line TB drugs-isoniazid (INH), rifampicin and pyrazinamide in the mouse and human bone
7
marrow derived macrophages tuberculosis model, respectively and CFU count of 2.2 x 10
7
(
(
approximately 15 fold lesser than untreated mice (31 x 10 ) with comparable efficacies to ethambutol
7
7
EMB) (1.27 x 10 ) and PZA (1.9 x 10 ). Further, S006-830 also showed potent bactericidal activity
against multi-drug resistant and single-drug resistant clinical isolates of M. tuberculosis.
. Introduction
1
Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tb) is not merely a disease rather it is an
epidemic which is prevalence in modern era of mankind and is considered as the world’s most deadliest
1,2
communicable disease by World Health Organization (WHO). Worldwide according to WHO report
2
014, about 9 million people were infected with TB in the year 2013 and an approximate of 1.4 million
1
,2
died in a single year of 2011 and the scenario is being worsen by multidrug-resistant (MDR-TB) and
extensively drug-resistant (XDR-TB), often emerged from the patient poor compliances to the standard
drugs therapy regimen. Hence, discovery of newer inhibitors of M. tb a global and urgent need. In our on-
3
-6
going campaign against TB, we previously reported triarylmethanes (TRAMs)
and thiophene
7
containing trisubstitued methanes (TRSMs) as novel cores having anti-tubercular activity (Fig. 1.) In
2
012, we reported the potential of few novel TRSMs against acute and persistent infection of M. fortuitum
(
responsible for the opportunist non-tubercular infection in humans) in a murine infection model and
8
identified two promising lead compounds (Fig. 1.). Further, optimization through synthetic and medicinal
chemistry approach led us to present the current work with potential applicability of novel TRSMs as
anti-mycobacterial compounds.
Figure 1. Evolution of triaryl methanes into thiophene containing TRSMs identified potential lead 36
(
S006-830) against M. tuberculosis .
2
2
. Results and Discussion
.1 Chemistry
2

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7, 8
The reaction scheme followed the similar protocol as outlined in our previous report.
We emphasized
on the design, synthesis and anti-tubercular activity of thiophene containing TRSMs with alkylaminoethyl
chains A.
Figure 2. General skeleton of designed molecules
In this work, we emphasized on the design, synthesis and anti-tubercular activity of thiophene containing
TRSMs with alkylaminoethyl chains A (Fig.2.). Initially, the Grignard reagent aryl magnesium bromide
1
a-j were reacted with various commercially available thiophene-2-aldehyde 2 to synthesize carbinol 3
.
Then Friedel-Crafts alkylation of 3 with phenol in the presence of conc. H SO in dry benzene furnished
2
4
4
in satisfactory yields. Alkylated product 4 was refluxed with various dialkylaminoethyl chloride
CO in acetone to obtain compounds 5-36 in good yields,
hydrochloride chains in presence of the K
Scheme 1).
2
3
(
Scheme 1. Synthesis of Trisubstituted Methanes (TRSMs)
0
Reagents and conditions (a) Dry THF, 0 C-rt, 30 min.; (b) Phenol, dry benzene, catalytic H
2
SO
4
, reflux,
1
2 3
h; (c) Alkylaminoethyl hydrochloride, dry acetone, anhyd. K CO , reflux, 6-7 h;
Scheme 2. Synthesis of dithiophene substituted compounds
0
Reagents and conditions (a) Dry THF, 0 C-rt, 30 min.; 1h; (b) Alkylaminoethyl hydrochloride, dry
acetone, anhyd. K CO , reflux, 6-7 h;
2
3
Initially, synthesis of compounds without any substitution on benzene ring were carried out by
nucleophilic addition of phenylmagnesium bromide on 2-thiophene carbaldehyde to provide phenyl
thiophen-2-yl carbinol. After this similar protocol was followed as above to synthesize compounds 5-8
(
Table 1). To explore the effect of substitution on benzene ring, various substitutions were incorporated in
the basic framework like halogen, methoxy and thiomethoxy substituted arylbromide to synthesize the
grignard reagent and then compounds 9-36 (Table 1) were synthesized by procedure stated above.
3

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Similarly, to understand the effect of substitution at 3 position of thiophene, 3-thiophene carboxaldehyde
was also incorporated to provide p-methoxy substituted phenyl thiophen-3-yl carbinol which were used
subsequently to synthesize compounds 37-39 by above mentioned procedure. Later dithiophene
substituted compounds 40-49 were also synthesized by the same procedure.
2
2
. 2. Biological Evaluation
.2.1. In vitro screening of compounds using virulent strain of M. tuberculosis to select ‘hits’:
This assay measures the capability of the test compound to kill or inhibit the multiplication of pathogenic
M. tuberculosis H37Rv and considered as ‘primary screening method’, as by principle compounds
11
7
showing MIC
was evident that the amino alkyl chain (Fig. 1. II) exhibited the most impact on the anti-tubercular activity
MIC less than 12.5 mg/L) and para-substituted chain (R ) over the ring C remains critical for the
≤ 6.25 mg/L can only be considered for further evaluation . From our previous report, it
(
B
activity as the ortho- substituted derivatives demonstrated very poor activity (MIC: para-substituted = <
2.5 mg/L; ortho-substituted = >12.5 mg/L). Surprisingly, both electron-donating group (EDG) and
1
electron withdrawing group (EWG) substituted over the para/meta- position (R ) of ring A exhibited
B
good anti-tubercular activity. In line with the observations from the previous report, we decided to
continue our work by changing the best possible substitutions on the amino alkyl chain R on ring C and
2
observed their anti-tubercular effects. All thirty one compounds were found to be active against M.
tuberculosis H37Rv with MIC ranging from 0.78 to 25.0 mg/L (Table 1). Nine compounds [MIC: 9 =
0
.78 mg/L, 15 = 1.34 mg/L,12 = 1.25 mg/L, 20 = 1.24 mg/L, 32 = 1.28 mg/L, 14 = 1.29 mg/L, 10 = 1.56
mg/L, 26 = 1.57 mg/L, and S006-830 = 1.33 mg/L] demonstrated notable inhibitory capacity against M.
tuberculosis H37R strain. While ring A substituted with para-chloro and N(CH ) as amino alkyl chain 9
v
2 5
showed the most potent inhibition with MIC of 0.78 mg/L, meta-substituted counterpart 14 showed
somewhat diminished activity MIC = 1.29 mg/L. While ortho-fluoro substituted compound 20 with the
similar alkyl chain [N(CH ) ] exhibited diminished inhibition [MIC = 1.24 mg/L] as compared to the
2
5
chloro substituted derivative 9, the meta-fluoro substituted compound 25 showed highly diminished
activity [MIC = 19.8 mg/L]. Towards the observation with EDGs at ring A, somewhat comparable
inhibition has been observed with compound 32 carrying ortho-OCH
MIC = 1.28 mg/L. Additionally, compound 36 (S006-830) with para-OCH and N{CH(CH ) }
3 2 2
3 2 5
with [N(CH ) ] side chain with
3
demonstrated potent inhibition of M. tuberculosis H37R strain with MIC = 1.33 mg/L as comparison to
v
their ortho-OCH substituted counterpart 34 with very low activity (25.0 mg/L). Surprisingly, all 3-
3
substituted compounds (37-39, 44-49) exhibited low activity (25.0 mg/L). Moreover, 2-substituted
dithiophene containing compounds also exhibited moderate activity (12.5 mg/L).
Table 1: Biological Activity of Synthesized TRSMs (5-36)
4

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2
.2.2. Cytotoxicity assessment through in vitro toxicity assay and ex vivo activity in the Mouse and
Human Bone Marrow Derived Macrophages (M/HBMDM) tuberculosis model: Initially, in vitro
cytotoxicity of active compounds (having MIC 6.25 mg/L) was measured using Vero cells (Vero C-
008 cell line) and if found non-toxic, were then taken further for their testing on the ex vivo assay against
≤
1
mouse or human bone marrow derived macrophages (M/HBMDM) developed tuberculosis model. The
basis for the selection of this model lies in the fact that the macrophage model mimics growth
environment of natural infection and demonstrates the ability of the candidate molecule to penetrate host
cell membrane, phagocytic vacuole and bacilli residing within the vacuole, and hence give the advantage
to reach the desired drug targets. In addition, it also serves as a model for hypoxia induced latent TB
infection, since tissue concentration of oxygen is considerably lower than that in the ambient air. Many
latency marker proteins of the pathogen are known to get expressed during its intracellular regime. From
VeroC-1008 cell line studies, four compounds viz. 9, 10, 26 and 36 (S006-830) could be evidenced to be
non-toxic demonstrating SI of greater than 10 and taken further for their assessment in the MBMDM
model. All four compounds (at 4 × MIC) killed greater than 90% of the intracellular M. tuberculosis , as
judged by counting the viable bacilli (colony forming units or CFUs) in lysates of the drug-treated or
untreated infected macrophages. As assessed from the statistical data, the ex vivo efficacy of 9, 10, 26 and
S006-830 were comparable to that of the first line TB drugs-INH, RFM and PZA (Fig. 2) in the
MBMDM model. While two compounds 26 and S006-830 showed a comparable ex vivo activity to that of
the standard drug INH (INH) in the HBMDM model, which were experimentally infected with M.
tuberculosis H37Rv(Fig. 3, 4).
Figure 3. Percent reduction in intracellular CFU of M. tuberculosis H37R
derived macrophages by 9, 10, 26 and S006-830. Isoniazid (INH), rifampicin (RFM) and pyrazinamide
PZA) were used as standard drugs.
V
in mouse bone marrow
(
5

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Figure 4. Compounds 10 and S006-830 were also able to kill intracellular M. tuberculosis H37Rv in
human monocyte derived macrophages in a manner similar to the standard drug INH (corresponding
mouse macrophage data are shown for comparison).
2
.2.3 In vivo assay against M. tuberculosis H37R infected Mouse model
V
When infected with a high number of M. tuberculosis CFUs by the i.v. route, the mouse harbors a
bacillary population that is similar in number and in metabolic state to that present in the lung cavity of
human TB. Thus, the mouse model is able to reproduce bacteriologic conditions close to those present in
the natural human disease and provide information on compound activity that can be extrapolated to
humans.The most frequently used is the outbred ‘Swiss’ mouse. The differences in the immune status
among outbred Swiss mice also exist among humans, and a drug or regimen that is active against the
mycobacteria in Swiss mouse is likely to be active in humans. The strain of choice for tubercle bacilli in
v
the mouse model is the H37R strain of M. tuberculosis, which maintains virulence through regular
passages in the mouse. Efficacy of a compound is monitored by survival/mortality rate, evaluation of
body weight, extent of gross lesions, and the enumeration of the CFUs in organs. Usually the CFU counts
are performed in the spleen or lungs or both. Since spleen and lung display similar overall results, it is
unnecessary to enumerate the CFU in both organs. In general, effective dosages of compounds (in mg/kg
body weight) in the laboratory animals are larger than those in humans. One possible reason for this is
that drug metabolism correlates better with body surface area than body weight. The ratio of body surface
area to body weight decreases sharply with increasing body weight. Thus, for most drugs the equipotent
dosage is 12 times larger in the mouse than in humans. This assay has been performed on Outbred Swiss
mice (obtained from CDRI-LAD) infected with M. tuberculosis H37R strain. All four in vitro and ex vivo
v
active compounds 9, 10, 26 and S006-830 were evaluated for their in vivo efficacy in the mouse model of
7
TB. Swiss mice were infected intravenously with M. tuberculosis H37R (10 CFU/ mouse). The
V
experimental groups received daily oral doses of test compounds (100 mg/kg body weight, for 30 days)
dissolved in corn oil. Drug-treated control groups received INH (25 mg/kg body weight) or EMB (10
mg/kg body weight) or PZA (100 mg/kg body weight) which were dissolved in water. Untreated control
groups received only the vehicles (corn oil or water). Mice were observed up to day-40 for determination
of percentage survivors and mean survival time (MST). Maximum survivors (42% in treated and 0% in
untreated) were seen in the S006-830 treated groups, followed by 10 and 26 treated groups. No survivors
were left in 9 treated mice. The highest enhancement in MST (7 days, p< 0.05) was also seen with S006-
8
30. All dead animals were autopsied and their lungs were found full of typical TB lesions. ‘Impression
smears’ of the cut surface of lungs were microscopically examined after staining with Ziehl-Neelsen stain
6

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and found full of Acid Fast Bacilli (AFB) confirming that the deaths were due to M. tuberculosis
infection (Fig. 5b).
Three of the surviving mice from the group treated with lead compound S006-830 was sacrificed on day
3
0 for determination of CFU in the lungs. Fig.5a shows results of treatment with S006-830 and the anti-
TB drugs-ethambutol (EMB) and pyrazinamide (PZA) in terms of viable bacilli (CFU) in the lungs of
7
mice. S006-830 treated mice showed CFU count of 2.2 x 10 (mean of 3 animals) which was
7
approximately 15 fold lesser than that of the untreated mice (31 x 10 ), and comparable with the efficacies
7
7
of EMB (1.27 x 10 ) and PZA (1.9 x 10 ) (Fig. 6). Based on the result of this and prior studies, compound
S006-830 has been chosen as the lead molecule and taken forward for the further assessment.
Figure 5. Results of in vivo protection experiments in mice infected (i.v.) with M. tuberculosis H37Rv.
A) S006-830 treated mice (100 mg/kg body wt, by oral gavage, 6 d/w, x 4w) showed appx.15 fold
reduction in CFU in lungs, which was equivalent to in vivo efficacy of standard drugs ethambutol (EMB)
and pyrazinamide (PZA) B) represents the Z-N staining of lung homogenate untreated and treated with
S006-830.
Figure 6. CFU in the lungs of mice treated with the S006-830 and standard drugs ethambutol (EMB) and
pyrazinamide (PZA). UNTR stands for untreated.
2
.2.4. Minimum Bactericidal Activity (MBC) determination of the lead compound 36 (S006-830)
through in vitro and ex vivo studies and potency assessment on multi-drug and single-drug resistant
M. tuberculosis
These experiments were aimed at determining the capacity of test compounds to kill the bacilli, in
addition to inhibiting their multiplication. The assays were thus designed to record killing of the seed
culture (inoculum) and in principle, if a compound in in vitro study reduces the CFU of the inoculum; it
can be considered as bactericidal and corresponding concentration as its MBC. If a compound do not kill
the inoculum but only prevents its multiplication (i.e., reduction in day-14 CFU), it can be considered as
bacteriostatic (as per method described in the experimental section). For ex vivo, if a compound reduces
the day-0 CFU (as per method described in the experimental section) of macrophages infected with M.
tuberculosis it can be considered as bactericidal. If it reduces only the day-5 CFU of such macrophages, it
15
can be considered as bacteriostatic . The lead compound 36 (S006-830) produced 100% killing (zero
7

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v
CFU) in vitro and 75% killing (1200 CFU) ex vivo of the inoculum of M. tuberculosis H37R . The
bactericidal potency was comparable with standard drug RFM (Fig. 7). S006-830 also showed potent
bactericidal activity against multi-drug resistant (i.e., resistant to RFM and INH) and single-drug resistant
(
to RFM) clinical isolates of M. tuberculosis (Fig. 8).
Figure 7. in vitro and ex vivo bactericidal property of 36 (S006-830) in comparison with rifampicin
RFM).
(
Figure 8. Activity of 36 (S006-830) against sensitive (H37R ), single- and multi- drug resistant M.
V
tuberculosis.
Further, in order to develop S-006-830 as identified lead, further pharmacokinetics (PK) studies were
conducted at Pharmacokinetics & Metabolism Division of our institute CSIR-CDRI using newly
developed and validated methods on HPLC and LC-MS/MS for quantitative estimation in rat plasma and
demonstrated good PK properties with fast intestinal absorption, peak plasma concentration appeared
8

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after one hour post oral dose, optimum elimination half-life of 9-13 hours, plasma protein binding (PPB)
of ~60%, mean residence time of around 18-20 hours and favourable bioavailability in the range of 45-
5
0%. As evident that the plasma protein binding was not too high, which in turn leads to unbound
fraction of S006-830 to favour tissue redistribution or clearance from the body and remains for longer
9,10
time in the body. Further, S006-830 was found stable in rat plasma under various operating conditions
.
S006-830 is racemic and after separation through chiral column chromatography, we are separately
evaluating the anti-TB activity of both enantiomers and results will be communicated in future.
3
. Conclusion
While it was evident from our earlier report that TRSMs can have significant potential against the M.
tuberculosis, this series led us to identify a highly potent and viable lead S006-830. Collectively, from
7,8
earlier reported compounds and the current work, a preliminary structure activity relationship could be
drawn and directed to the subsequent consideration: (a) presence of benzene ring with amino alkyl chains
is important for potent anti-tubercular activity; (b) TRSMs without any substituent on benzene ring (5-8)
showed moderate anti-tubercular activity; (c) Among TRSMs with electron withdrawing group, para-
substituted chloro (9, 10) containing compounds showed promising in vitro activity than their meta
analogues 11-16. On the other hand, TRSMs with electron donating group i.e. methoxy and thiomethoxy
at para position of benzene ring 26, 36 showed good anti-tubercular activities than ortho isomers 27-35.
Anti-tubercular activity of four TRSMs, which showed MIC of <6.25 mg/L against Mycobacterium
tuberculosis H37R and lacked cytotoxicity towards mammalian cells showed >90 % inhibition in
v
multiplication of M. tuberculosis within mouse and human macrophages (ex vivo), which was
comparable to ex vivo efficacy of standard TB drugs- isoniazid, rifampicin and pyrazinamide. The lead
molecule S006-830 brought approximately 15 fold reduction in the viable bacilli (CFUs) in lungs of the
infected mice, which was comparable to the in vivo efficacy of ethambutol and pyrazinamide. S006-830
with noticeable microbiological profile and encouraging PK properties can be considered as potential lead
against M. tuberculosis.
4
4
4
. Experimental section
.1 Chemistry
.1.1 General Methods
All reagents and solvents were purchased from commercial sources and used without further purification.
Organic solvents were dried by standard methods. Analytical TLC was performed using 2.5×5 cm
9

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aluminium plates coated with a 0.25 mm thickness of silica gel (60F-254), visualization was
accomplished with iodine and under UV lamp. The spots on TLC were also visualized by warming ceric
0
sulphate (2% CeSO in 2N H SO ) sprayed plates in hot plate or in oven at about 100 C. Column
4
2
4
1
chromatography was performed using silica gel (60-120 and 100-200 mesh). H NMR spectra were
recorded on 200 and 300 MHz spectrometer in CDCl (all signals are reported in ppm with the internal
3
0
13
chloroform signal at 7.26 ppm as standard) at 25 C. C NMR spectra were recorded on 50 and 75 MHz
3
spectrometer in CDCl (all signals are reported in ppm with the internal chloroform signal at 77.00 ppm
0
as standard) at 25 C. In a few cases tetramethylsilane (TMS) at 0.00 ppm was used as the reference
1
standard. Coupling constants (J values) are given in hertz (Hz). H NMR splitting patterns are designated
as singlet (s), doublet (d), double doublet (dd), triplet (t), quartet (q) or multiplet (m). IR spectra were
-1
recorded using a FTIR spectrophotometer in cm .
4
.1.1.1. General procedure for the preparation of heteroarylcarbinols (3) through Grignard
reaction
To a suspension of Mg (1.50 g, 61.7 mmol) in dry THF (40 mL) was added drop wise a solution of 4-
bromoanisole (6.52 mL, 52.1 mmol) in dry THF (40 mL). After stirring the mixture for 30 min. a solution
of thiophenylcarbaldehyde (4.21 mL, 45 mmol) in dry THF (30 mL) was added drop wise and the
resulting solution was allowed to stir for further 30 min. After quenching by adding a saturated solution
of NH Cl (20 mL), the reaction mixture was extracted with ethyl acetate (100 mL), washed with water
4
(
100 mL), brine (2x50 mL) and then dried over Na
2 4
SO . The organic layer was dried under reduced
pressure. The crude product was purified by column chromatography.
4
.1.1.2. General procedure for the preparation of thiophene containing trisubstituted methanes
through Friedel Crafts alkylation reaction:
0
Phenol, benzene, 1-2 drops of sulphuric acid along with the carbinol 3 was refluxed at 60-75 C for 2-3
hrs. Completion of reaction was confirmed by checking TLC by which compound 4 was obtained. After
completion, reaction was quenched with water, reaction mixture was extracted with ethyl acetate (100
mL), washed with water and brine and dried over Na
and purified by column chromatography.
2 4
SO . Solvent was removed under reduced pressure
4
.1.1.3. Diethyl-{2-[4-(phenyl-thiophen-2-yl-methyl)-phenoxy]-ethyl}-amine (5):
Compound 4 (0.2 g, 0.8 mmol) refluxed with K CO (0.2 g, 1.9 mmol), 1-(2-chloroethyl)-diethyl amine
2
3
1
hydrochloride (0.2 g, 0.7 mmol) in acetone furnished 5 (0.2 g, 72.8%) as brown viscous oil. H NMR
300 MHz, CDCl3): 7.18-7.15 (m, 8H), 6.91-6.78 (m, 3H), 6.64-6.63 (m, 1H), 5.59 (s, 1H), 4.02 (t, J =
.22, 2H), 2.87 (t, J = 6.2, 2H), 2.65 (q, J = 7.1, 4H), 1.08 (t, J = 7.0, 6H); C NMR (CDCl , 50 MHz):
(
δ
13
6
δ
3
10

ACCEPTED MANUSCRIPT
1
1
55.8, 147.9, 143.7, 137.7, 130.1, 129.8, 128.7(2C), 128.4(2C), 128.3(2C), 126.7, 126.6, 126.3, 124.6,
-1
14.5(2C), 65.6, 51.2, 48.8, 47.7, 11.2 ; IR (Neat): 3019, 2928, 2855, 2362, 1612, 1509, 1217, 761 cm ;
+
MS (ESI): m/z 366 (M+H) ; Anal. Calcd for C H NOS: C, 75.57; H, 7.45; N, 3.83. Found: C, 75.61; H,
23
27
7
.52; N, 3.72.
4
.1.1.4. 1-{2-[4-(Phenyl-thiophen-2-yl-methyl)-phenoxy]-ethyl}-piperidine (6):
CO (259.42 mg, 1.87 mmol), 1-(2-chloroethyl)-
Compound 4 (200 mg, 0.75 mmol) refluxed with K
2
3
piperidine hydrochloride (138.23 mg, 0.75 mmol) in acetone furnished 6 (203.8 mg, 71.9%) as brown
1
viscous oil. H NMR (300 MHz, CDCl ):
δ 7.28-7.15 (m, 6H), 7.09-7.06 (m, 2H), 6.90-6.87 (m, 1H),
3
6
4
1
5
3
.81-6.78 (m, 2H), 6.64-6.63 (m, 1H), 5.58 (s, 1H), 4.07 (t, J = 6.0, 2H), 2.76 (t, J = 6.0, 2H), 2.52 (t, J =
1
3
.9, 4H), 1.65-1.58 (m, 4H), 1.48-1.42 (m, 2H); ); C NMR (CDCl
3
, 50 MHz): δ 157.2 , 156.6 , 148.5 ,
36.1 , 132.8, 129.8(2C), 129.6(2C), 127.7 , 126.3, 126.0 , 123.9 , 120.4, 114.1 , 110.5, 96.1 , 65.5 , 57.8,
-1
5.5 , 54.9, 43.8 , 25.6, 24.0 ; IR (Neat): 3020, 2936, 2362, 1610, 1508, 1217, 761 cm ; MS (ESI): m/z
+
78 (M+H) ; Anal. Calcd for C H NOS: C, 76.35; H, 7.21; N, 3.71. Found: C, 76.40; H, 7.15; N, 3.69.
24
27
4
.1.1.5. 1-{2-[4-(Phenyl-thiophen-2-yl-methyl)-phenoxy]-ethyl}-azepane (7):
Compound 4 (200 mg, 0.75 mmol) refluxed with K CO (259.42 mg, 1.87 mmol), 1-(2-Chloro-ethyl)-
2
3
azepane hydrochloride (148.58 mg, 0.75 mmol) in acetone furnished 7 (220 mg, 75%) as brown viscous
1
oil. H NMR (300 MHz, CDCl ):
δ
7.21-7.06 (m, 8H), 6.91-6.78 (m, 3H), 6.64-6.63 (m, 1H), 5.59 (s, 1H),
3
1
3
4
.07 (t, J = 6.0, 2H), 2.97 (t, J = 6.1, 2H), 2.81 (t, J = 5.6, 4H), 1.69-1.61 (m, 8H); C NMR (CDCl , 50
3
MHz):
δ
157.5, 148.4, 144.1, 136.2, 129.8(2C), 128.7(2C), 128.4(2C), 126.6, 126.5, 126.2, 124.4,
1
7
7
14.4(2C), 65.7, 57.8, 54.9(2C), 51.3, 25.7(3C), 24.1; IR (Neat): 3019, 2938, 2362, 1610, 1509, 1218,
-1
+
63 cm ; MS (ESI): m/z 392 (M+H) ; Anal. Calcd for C25H29NOS: C, 76.68; H, 7.46; N, 3.58. Found: C,
6.72; H, 7.39; N, 3.61.
4
.1.1.6. 4-{2-[4-(Phenyl-thiophen-2-yl-methyl)-phenoxy]-ethyl}morpholine (8):
CO (259.42 mg, 1.87 mmol), 4-(2-Chloro-ethyl)-
Compound 4 (200 mg, 0.75 mmol) refluxed with K
2
3
morpholine hydrochloride (258.45 mg, 0.75 mmol) in acetone furnished 8 (205.1 mg, 72%) as brown
1
viscous oil. H NMR (300 MHz, CDCl ):
δ 7.29-7.15 (m, 6H), 7.10-7.06 (m, 2H), 6.90-6.78 (m, 3H),
3
6
.64-6.63 (m, 1H), 5.59 (s, 1H), 4.06 (t, J = 5.7, 2H), 3.70 (t, J = 4.4, 4H), 2.77 (t, J = 5.6, 2H), 2.55 (t, J
13
=
4.6, 4H); C NMR (CDCl , 50 MHz): δ 157.4, 148.3, 144.0, 136.3, 129.8, 128.7, 128.7(2C), 128.5(2C),
3
1
2
28.4, 126.6, 126.5, 126.2, 124.4, 114.4(2C), 66.7, 65.5, 57.6, 54.0(2C), 51.3; IR (Neat): 3018, 2927,
-1
+
+
+
862, 1609, 1509, 1217, 753 cm ; MS (ESI): m/z 378 (M-H) , 379 (M ) , 380 (M+H) ; Anal. Calcd for
C H NO S: C, 72.79; H, 6.64; N, 3.69. Found: C, 72.82; H, 6.70; N, 3.55.
2
3
25
2
11

ACCEPTED MANUSCRIPT
4
.1.1.7. 1-(2-{4-[(4-Chloro-phenyl)-thiophen-2-yl-methyl]-phenoxy}-ethyl)-piperidine (9)
1
Compound 4 p-Cl substituted at Ar (0.20 g, 0.667 mmol) refluxed with K CO (0.280 g, 2.00 mmol), 1-
2
3
(
2-Chloroethyl)-piperidine hydrochloride (0.135 g, 0.734 mmol) in acetone furnished 9 (0.205 g, 75%) as
1
wine red viscous liquid. H NMR (200 MHz, CDCl ):
δ 7.15-6.94 (m, 7H), 6.82-6.52 (m, 4H), 5.47 (s,
3
1
3
1
5
1
H), 3.99 (t, J = 4.0, 2H), 2.67 (t, J = 4.0, 2H), 2.45-2.41 (m, 4H), 1.57-1.37 (m, 6H); C NMR (CDCl
3
,
0 MHz): 158.1, 148.1, 143.0, 135.8, 132.8, 130.4(2C), 130.0(2C), 128.9(2C), 126.9, 126.6, 125.0,
δ
-1
14.8(2C), 77.4, 66.2, 58.3, 55.4, 51.0, 30.1, 26.3, 24.6; IR (Neat) 3438, 2990, 1712, 1623, 775, 664 cm ;
+
MS (ESI): m/z 412 (M+H) ; Anal. Calcd for C H ClNOS: C, 69.97; H, 6.36; N, 3.40. Found: C, 70.04;
24
26
H, 6.34; N, 3.46.
4
.1.1.8. (2-{4-[(4-Chloro-phenyl)-thiophen-2-yl-methyl]-phenoxy}-ethyl)-diisopropyl-amine (10):
1
Compound 4 p-Cl substituted at Ar (0.20 g, 0.67 mmol) refluxed K CO (0.28 g, 2.00 mmol), (2-Chloro-
2
3
ethyl)-diisopropyl-amine hydrochloride (0.15 g, 0.74 mmol) in acetone furnished 10 as yellow viscous oil
1
(
0.21 g, 73%). H NMR (200 MHz, CDCl
3
):
δ
7.29-7.19 (m, 4H), 7.14-7.05 (m, 3H), 6.94-6.64 (m, 4H),
13
5
.58 (s, 1H), 3.87 (t, J = 7.4, 2H), 3.07-3.00 (m, 2H), 2.80 (t, J = 7.4, 2H), 1.03 (d, J = 6.5, 12H); C
NMR (CDCl , 50 MHz):
δ 156.6, 146.5, 141.4, 134.0, 131.1, 128.8(2C), 128.4(2C), 127.2(2C),
3
1
1
3
25.3(2C), 124.9, 123.3, 113.1(2C), 67.9, 49.4, 48.2, 43.1, 19.5(4C) ; IR (Neat): 3312, 2967, 1602, 1446,
-1
+
353, 1023, 759 cm ; MS (ESI): m/z 428 (M+H) ; Anal. Calcd for C H ClNOS: C, 70.15; H, 7.06; N,
25
30
.27. Found: C, 70.13; H, 7.13; N, 3.31.
4
.1.1.9. 2-(4-((3-chlorophenyl)(thiophen-2-yl)methyl)phenoxy)-N,N-dimethylethanamine (11):
1
Yield, 72%; H NMR (300 MHz, CDCl )
δ 7.19-7.18 (m 4H), 7.09-7.07 (m, 3H), 6.93-6.90 (m, 1H),
3
6
6
1
.86-6.84 (m, 2H), 6.66-6.65 (m, 1H), 5.57 (s, 1H), 4.04 (t, J = 5.7, 2H), 2.72 (t, J = 5.7, 2H), 2.33 (s,
13
H); C NMR (75 MHz, CDCl
3
): δ 157.6, 147.3, 146.0, 135.2, 134.1, 129.6, 129.5(2C), 128.7, 126.9,
26.7, 126.5, 126.3, 124.6, 114.3(2C), 65.7, 58.1, 50.8, 45.8(2C); IR (Neat) 3471, 2980, 1748, 1643, 768
-
1
+
cm ; MS (ESI) m/z 372 (M+H) ; Anal. Calcd for C H ClNOS: C, 67.82; H, 5.96; Cl, 9.53; N, 3.77.
21
22
Found: C, 67.77; H, 5.92; N, 3.71.
4
.1.1.10. 2-(4-((3-chlorophenyl)(thiophen-2-yl)methyl)phenoxy)-N,N-diethylethanamine (12):
1
Yield, 75%; H NMR (300 MHz, CDCl )
δ 7.21-7.18 (m, 4H), 7.09-7.07 (m, 3H), 6.94-6.91 (m, 1H),
3
6
7
.85-6.82 (m, 2H), 6.67-6.66 (m, 1H), 5.58 (s, 1H), 4.02 (t, J = 6.4, 2H), 2.86 (t, J = 6.4, 2H), 2.63 (q, J =
1
3
.1, 4H), 1.06 (t, J = 7.1, 6H); C NMR (50 MHz, CDCl3):
δ 157.4, 146.1, 135.5, 134.2, 129.8,
12

ACCEPTED MANUSCRIPT
1
29.6(2C), 128.8(2C), 126.9, 126.8, 126.6, 126.4, 124.7, 114.4(2C), 65.7, 51.5, 50.9, 47.6(2C), 11.1(2C);
-1
+
IR (Neat) 3433, 2985, 1747, 1611, 779 cm ; MS (ESI) m/z 400 (M+H) ; Anal. Calcd for C23H26ClNOS:
C, 69.07; H, 6.55; N, 3.50. Found: C, 69.13; H, 6.64; N, 3.55.
4
.1.1.11. 1-(2-(4-((3-chlorophenyl)(thiophen-2-yl)methyl)phenoxy)ethyl) pyrrolidine (13):
1
Yield, 68%; H NMR (300 MHz, CDCl
3
)
δ
7.19-7.18 (m, 4H), 7.10-7.07 (m, 3H), 6.93-6.90 (m, 1H),
6
.86-6.83 (m, 2H), 6.66-6.65 (m, 1H), 5.57 (s, 1H), 4.12 (t, J = 5.8, 2H), 2.94 (t, J = 5.8, 2H), 2.69 (s,
13
4
H), 1.82 (s, 4H); C NMR (50 MHz, CDCl3):
δ 157.5, 147.2, 146.0, 135.3, 134.1, 129.6, 129.5,
1
28.7(2C), 126.8, 126.7, 126.5, 126.2, 124.6, 114.4(2C), 66.5, 54.8, 54.5(2C), 50.8, 23.3(2C); IR (Neat)
-1
+
3
451, 2962, 1745, 1611, 871 cm ; MS (ESI) m/z 398 (M+H) ; Anal. Calcd for C H ClNOS: C, 69.42;
23 24
H, 6.08; N, 3.52. Found: C, 69.33; H, 6.04; N, 3.55.
4
.1.1.12. 1 - (2-(4-((3-chlorophenyl)(thiophen-2-yl)methyl)phenoxy)ethyl) piperidine (14):
1
Yield, 79%; H NMR (300 MHz, CDCl )
δ
7.19-7.17 (m, 4H), 7.09-7.06 (m, 3H), 6.93-6.90 (m, 1H),
3
6
.85-6.82 (m, 2H), 6.67-6.66 (m, 1H), 5.57 (s, 1H), 4.08 (t, J = 6.6, 2H), 2.77 ( t, J = 5.9, 2H), 2.53-2.50
13
(
m, 4H), 1.64-1.57 (m, 4H), 1.47-1.43 (m, 2H); C NMR (75 MHz, CDCl
3
):
δ 157.5, 147.2, 146.0, 135.1,
1
34.0, 129.6, 129.4, 128.6, 126.8(2C), 126.7, 126.5, 126.2, 124.6, 114.3(2C), 65.6, 57.7, 54.8(2C), 50.8,
+
2
5.6(2C), 23.9; MS (ESI) m/z 412 (M+H) ; Anal. Calcd for C H ClNOS: C, 69.97; H, 6.36; N, 3.40.
24 26
Found: C, 69.89; H, 6.34; N, 3.45.
4
.1.1.13. N-(2-(4-((3-chlorophenyl)(thiophen-2-yl)methyl)phenoxy)ethyl)-N-isopropylpropan-2-amine
15):
Yield, 63%; H NMR (300 MHz, CDCl )
(
1
δ
7.19-7.18 (m, 4H), 7.09-7.06 (m, 3H), 6.92-6.89 (m, 1H),
3
6
7
1
.84-6.81 (m, 2H), 6.66 (d, J = 3.3, 1H), 5.57 (s, 1H), 3.87 (t, J = 7.4, 2H), 3.07-2.98 (m, 2H), 2.80 (t, J =
1
3
.5, 2H), 1.02 (d, J = 6.5, 12H); C NMR (75 MHz, CDCl
3
): δ 157.4, 147.4, 146.1, 135.0, 134.2, 129.6,
29.5, 128.8, 126.9(2C), 126.8, 126.6, 126.3, 124.6, 114.3(2C), 69.2, 50.9, 49.6(2C), 44.4, 20.8(4C); IR
-1
+
(
Neat) 3432, 2990, 1742, 1631 cm ; MS (ESI) m/z 428 (M+H) ; Anal. Calcd for C H ClNOS: C, 70.15;
25 30
H, 7.06; N, 3.27. Found: C, 69.99; H, 7.14; N, 3.30.
4
.1.1.14. 4-(2-(4-((3-chlorophenyl)(thiophen-2-yl)methyl)phenoxy)ethyl) morpholine (16):
1
Yield, 68%; H NMR (300 MHz, CDCl )
δ
7.20-7.18 (m, 4H), 7.10-7.08 (m, 3H), 6.94-6.92 (m, 1H),
3
6
4
.85-6.83 (m, 2H), 6.67-6.66 (m, 1H), 5.58 (s, 1H), 4.09 (t, J = 4.5, 2H), 3.72 (t, J = 3.5, 4H), 2.79 (t, J =
13
.4, 2H), 2.57 (d, J = 3.0, 4H); C NMR (75 MHz, CDCl ):
δ 157.6, 147.3, 146.1, 135.4, 134.2, 129.7,
3
13

ACCEPTED MANUSCRIPT
1
29.5, 128.8, 126.9(2C), 126.8, 126.6, 126.3, 124.7, 114.5(2C), 66.9(2C), 65.7, 57.6, 54.0(2C), 50.9; IR
-1
+
(
2
Neat) 3435, 2980, 1732, 1630 cm . MS (ESI) m/z 414 (M+H) ; Anal. Calcd for C23H24ClNO S: C,
6
6.73; H, 5.84; N, 3.38. Found: C, 66.79; H, 5.88; N, 3.31.
4
.1.1.15. 2-(4-((2-fluorophenyl)(thiophen-2-yl)methyl)phenoxy)-N,N-dimethylethanamine (17)
1
Isolated as light brownish oily liquid (yield 71%). H NMR (300 MHz, CDCl
3
):
δ
7.25-7.22 (m, 1H),
7
.21-7.12 (m, 1H), 7.09-7.02 (m, 5H), 6.94-6.91 (m, 1H), 6.87-6.67 (m, 2H), 6.66 (d, J = 2.5, 1H), 5.93
13
(
s, 1H), 4.05 (t, J = 5.7, 2H), 2.73 (t, J = 5.7, 2H), 2.33 (s, 6H). C NMR (50 MHz, CDCl ):
δ
162.7,
3
1
5
57.6, 146.9, 134.8, 130.1, 129.6, 128.5, 126.6(2C), 126.3, 124.5, 124.0, 115.5, 115.1, 114.4(2C), 65.8,
-1
8.2, 45.8(2C), 43.7; IR (Neat): 3432, 2904, 1611, 1445, 1249, 1176, 1023, 823, 778, 661 cm ; MS
+
(
ESI): m/z 356 (M+H) , Anal. Calcd for C H FNOS: C, 70.96; H, 6.24; N, 3.94. Found: C, 70.91; H,
21
22
6
4
.23; N, 3.97.
.2.2.16 N, N-diethyl-2-(4-((2-fluorophenyl)(thiophen-2-yl)methyl)phenoxy) ethanamine (18):
1
Isolated as light blackish oily liquid (yield 64%). H NMR (300 MHz, CDCl
3
):
δ
7.24-7.12 (m, 2H), 7.09-
7
2
1
6
.01 (m, 5H), 6.93-6.90 (m, 1H), 6.84-6.81 (m, 2H), 6.66 (d, J = 3.4, 1H), 5.93 (s, 1H), 4.04 (t, J = 6.3,
13
H), 2.88 (t, J = 6.3, 2H), 2.65 (q, J = 7.1, 4H), 1.07 (d, J = 7.1, 6H). C NMR (75 MHz, CDCl ):
δ
3
62.7, 157.6, 146.9, 134.7, 130.1, 129.6, 128.4, 128.3, 126.5, 126.2(2C), 124.5, 123.9, 115.5, 114.3(2C),
6.2, 51.7, 47.7(2C), 43.7, 11.6(2C); IR (Neat): 3433, 2904, 1617, 1445, 1254, 1176, 1033, 823, 779, 662
-
1
+
cm ; MS (ESI): m/z 384 (M+H) , Anal. Calcd for C23H26FNOS: C, 72.03; H, 6.83; N, 3.65. Found: C,
7
2.09; H, 6.80; N, 3.67.
4
.1.1.17. 1-(2-(4-((2-fluorophenyl)(thiophen-2-yl)methyl)phenoxy)ethyl) pyrrolidine (19):
1
Isolated as brownish oily liquid (yield 68%). H NMR (300 MHz, CDCl ):
δ
7.25-7.12 (m, 2H), 7.09-6.99
3
(
m, 5H), 6.94-6.91 (m, 1H), 6.86-6.83 (m, 2H), 6.66 (d, J = 0.9, 1H), 5.93 (s, 1H), 4.09 (t, J = 5.9, 2H),
1
3
2
1
5
.90 (t, J = 5.9, 2H), 2.63 (s, 4H), 1.80 (d, J = 3.8, 4H). C NMR (50 MHz, CDCl ):
δ
157.7, 146.9,
3
39.2, 134.7, 130.1, 129.6, 128.5, 128.3, 126.5(2C), 126.3, 124.5, 124.0, 123.9, 115.5, 114.4(2C), 66.8,
-1
4.6(2C), 43.7, 23.4(2C); IR (Neat): 3433, 2909, 1617, 1445, 1235, 1176, 1043, 823, 779, 660 cm ; MS
+
(
ESI): m/z 382 (M+H) , Anal. Calcd for C23
H
24FNOS: C, 72.41; H, 6.34; N, 3.67. Found: C, 72.42; H,
6
.39; N, 3.71.
4
.1.1.18. 1-(2-(4-((2-fluorophenyl)(thiophen-2-yl)methyl)phenoxy)ethyl) piperidine (20):
14

ACCEPTED MANUSCRIPT
1
Isolated as light brownish oily liquid (yield 75%). H NMR (300 MHz, CDCl
3
):
δ
7.24-7.12 (m, 2H),
7
6
.09-6.98 (m, 5H), 6.93-6.90 (m, 1H), 6.84-6.81 (m, 2H), 6.66 (d, J = 3.4, 1H), 5.92 (s, 1H), 4.09 (t, J =
1
3
.0, 2H), 2.78 (t, J = 6.0, 2H), 2.52 (t, J = 4.6, 4H), 1.64-1.57 (m, 4H), 1.47-1.40 (m, 2H); C NMR (75
159.1, 157.6, 146.9, 134.8, 131.3, 129.7(2C), 128.5, 128.4, 126.6, 124.5, 115.4,
14.4(2C), 65.6, 57.8, 54.9(2C), 43.8, 43.7, 37.9, 25.7(2C), 24.0 ; IR (Neat): 3423, 2914, 1617, 1425,
MHz, CDCl ):
δ
3
1
1
7
-1
+
254, 1176, 1031, 823, 774, 661 cm ; MS (ESI): m/z 396 (M+H) , Anal. Calcd for C24H26FNOS: C,
2.88; H, 6.63; N, 3.54. Found: C, 72.83; H, 6.57; N, 3.59.
4
.1.1.19. N-(2-(4-((2-fluorophenyl)(thiophen-2-yl)methyl)phenoxy)ethyl)-N-isopropylpropan-2-amine
(
21):
1
Isolated as light brownish oily liquid (yield 72%). H NMR (300 MHz, CDCl
3
):
δ 7.22-7.11 (m, 2H),
7
7
1
6
.08-6.97 (m, 5H), 6.92-6.89 (m, 1H), 6.83-6.81 (m, 2H), 6.66 (d, J = 2.5, 1H), 5.92 (s, 1H), 3.86 (t, J =
13
.3, 2H), 3.07-2.98 (m, 2H), 2.80 (t, J = 7.3, 2H), 1.02 (d, J = 6.5, 12H). C NMR (75 MHz, CDCl ):
δ
3
62.7, 157.8, 147.0, 134.4, 130.1, 129.6, 128.4, 126.5, 126.2(2C), 124.4, 123.9, 115.5, 115.0, 114.2(2C),
9.1, 49.6(2C), 44.4, 43.7, 20.8(4C); IR (Neat): 3432, 2924, 1621, 1458, 1249, 1166, 1033, 833, 768, 663
-
1
+
cm ; MS (ESI): m/z 412 (M+H) , Anal. Calcd for C25H30FNOS: C, 72.96; H, 7.35; N, 3.40. Found: C,
7
2.92; H, 7.27; N, 3.46.
4
.1.1.20. 4-(2-(4-((2-fluorophenyl)(thiophen-2-yl)methyl)phenoxy)ethyl) morpholine (22):
1
Isolated as brownish oily liquid (yield 71%). H NMR (300 MHz, CDCl ):
δ
7.24-7.21 (m, 1H), 7.19-7.12
3
(
m, 1H), 7.09-6.98 (m, 5H), 6.93-6.90 (m, 1H), 6.84-6.81 (m, 2H), 6.66 (d, J = 3.3, 1H), 5.92 (s, 1H),
1
3
4
1
6
.09 (s, 2H), 3.72 (t, J = 4.5, 4H), 2.80 (s, 2H), 2.60 (d, J = 4.5, 4H); C NMR (75 MHz, CDCl
3
): δ 161.5,
59.1, 146.6, 135.0, 129.7, 128.5(2C), 128.4(2C), 126.5(2C), 124.6(2C), 115.2, 114.4(2C), 66.8(2C),
-1
5.6, 57.6, 54.0(2C), 37.9; IR (Neat): 3453, 2904, 1617, 1445, 1224, 1176, 1033, 823, 770, 662 cm ; MS
+
(
ESI): m/z 398 (M+H) , Anal. Calcd for C H FNO S: C, 69.49; H, 6.09; N, 3.52. Found: C, 69.51; H,
2
3
24
2
6
.13; N, 3.48.
4
.1.1.21.2-(4-((3-fluorophenyl)(thiophen-2-yl)methyl)phenoxy)-N,N-dimethylethanamine (23):
1
Colourless oil; yield, 78%; H NMR (300 MHz, CDCl )
δ 7.20-7.17 (m, 3H), 7.00-6.86 (m, 7H), 6.65 (s,
3
1
3
1
1
1
1
7
3
H), 6.07 (s, 1H), 4.02-3.96 (m, 2H), 2.55 (t, J = 5.7, 2H), 2.23 (s, 6H); C NMR (75 MHz, CDCl ): δ
57.6 , 147.3 , 146.0 , 135.2 , 129.5 , 128.7(2C) , 126.9, 126.7, 126.5, 125.9, 125.4, 125.0, 124.6 ,
14.3(2C) , 65.7 , 58.1 , 50.8 , 45.8(2C) ; IR (Neat): 3015, 2871, 2121, 1580, 1434, 1251, 1013, 729 cm-
+
.
; MS (ESI): m/z 356 [M+H] ; Anal. Calcd for C H FNOS: C, 70.96; H, 6.24; N, 3.94. Found: C,
21
22
0.90; H, 6.30; N, 3.90.
15

ACCEPTED MANUSCRIPT
4
.1.1.22. N, N-diethyl-2-(4-((3-fluorophenyl)(thiophen-2-yl)methyl)phenoxy) ethanamine (24):
1
Colourless oil; yield, 75%; H NMR (300 MHz, CDCl )
δ 7.14-7.10 (m, 3H), 7.02-6.99 (m, 2H), 6.86-
3
6
0
1
2
.59 (m, 5H), 6.59 (s, 1H), 5.52 (s, 1H), 3.94 (t, J = 6.3, 2H), 2.78 (t, J = 6.3, 2H), 2.58-2.51 (m, 4H),
13
.98 (t, J = 2.3, 6H); C NMR (75 MHz, CDCl ):
δ
164.5, 157.7, 147.5, 135.3, 129.7, 126.6, 126.3(2C),
3
24.6(2C), 115.8, 115.5, 114.4(2C), 113.6, 113.3, 66.4, 51.7, 50.9, 47.8(2C), 11.8(2C); IR (neat): 3076,
-1
+
811, 2190, 1581, 1446, 1242, 1039, 777 cm ; MS (ESI): m/z 384 (M+H) ; Anal. Calcd for C23H26FNOS:
C, 72.03; H, 6.83; N, 3.65. Found: C, 72.06; H, 6.89; N, 3.71.
4
.1.1.23. 1-(2-(4-((3-fluorophenyl)(thiophen-2-yl)methyl)phenoxy)ethyl) piperidine (25):
1
Colourless oil; yield, 77%; H NMR (300 MHz, CDCl
3
)
δ
7.11-7.06 (m, 3H), 6.92-6.56 (m, 7H), 6.55 (s,
13
1
H), 5.98 (s, 1H), 3.97-3.92 (m, 2H), 2.53 (t, J = 5.7, 2H), 2.28 (t, J = 4.3, 4H), 1.45-1.17 (m, 6H); C
NMR (75 MHz, CDCl3):
δ 164.0, 155.8, 147.0, 139.2, 131.9, 129.5, 128.2, 126.5(2C), 126.4(2C),
1
1
24.3(2C), 115.9, 115.7, 114.6(2C), 66.5, 57.8(2C), 44.8, 25.8(3C); IR (Neat): 3042, 2862, 2115, 1587,
-1
+
491, 1211, 1047, 769 cm ; MS (ESI): m/z 396 (M+H) ; Anal. Calcd for C24H26FNOS: C, 72.88; H, 6.63;
N, 3.54. Found: C, 72.82; H, 6.69; N, 3.50.
.1.1.24. N-isopropyl-N-(2-(4-((4-(methylthio)phenyl)(thiophen-2-yl)methyl)phenoxy)ethyl)propan-2-
amine (26):
Compound 4 p-SMe substituted at Ar (0.10 g, 0.35 mmol) refluxed with K CO (0.135 g, 0.96 mmol),
4
1
2
3
(
2-Chloro-ethyl)-diethyl-amine hydrochloride (0.07 g, 0.35 mmol) in acetone furnished 26 as a light
1
yellow viscous oil (0.105 g, 74%); H NMR (200 MHz, CDCl
3
):
δ
7.18-6.99 (m, 7H), 6.86-6.85 (m, 1H),
6
.74 (d, J = 8.1, 2H), 6.60 (d, J = 3.6, 1H), 5.49 (s, 1H), 3.80 (t, J = 7.4, 2H), 2.99-2.93 (m, 2H), 2.73 (t, J
13
=
7.4, 2H), 2.38 (s, 3H), 0.96 (d, J = 6.4, 12H); C NMR (75 MHz, CDCl ): δ 158.1, 148.7, 141.6, 136.9,
3
1
2
36.1, 130.1(2C), 129.6(2C), 127.0(2C), 126.6(2C), 124.8, 114.7(2C), 69.5, 51.2, 50.1(2C), 44.9,
-1
+
1.2(4C), 16.3; IR (Neat): 2964, 1608, 1505, 1248 cm ; MS (ESI): m/z 440 (M+H) , Anal. Calcd for
C H NOS : C, 71.02; H, 7.57; N, 3.19. Found: C, 71.04; H, 7.63; N, 3.24.
2
6
33
2
4
.1.1.25.
Dimethyl-(2-{4-[(2-methylsulfanyl-phenyl)-thiophen-2-yl-methyl]-phenoxy}-ethyl)-amine
(
27):
1
Compound 4 o-SMe substituted at Ar (200 mg, 0.64 mmol) refluxed with K
2
CO
3
(221.17 mg, 1.6 mmol),
1
7
6
-(2-chloroethyl)-dimethyl amine hydrochloride (92.20 mg, 0.64 mmol) in acetone furnished 27 (174 mg,
1
0.8%) as brown viscous oil; H NMR (300 MHz, CDCl ):
δ 7.22-7.14 (m, 3H), 7.05-7.01 (m, 4H), 6.89-
3
.78 (m, 3H), 6.59-6.57 (m, 1H), 6.07 (s, 1H), 4.02 (t, J = 5.8, 2H), 2.70 (t, J = 5.7, 2H), 2.36 (s, 3H),
16

ACCEPTED MANUSCRIPT
1
3
2
,
.32 (s, 6H); C NMR (75 MHz, CDCl
3
): δ 157.7, 156.7, 136.1, 129.8(3C) , 127.8 , 126.4 , 126 , 124(2C)
120(2C) , 114(2C), 110 , 65.8 , 58.3 , 55.6 , 45.8(2C) , 43.8; IR (Neat): 3017, 2926, 2361, 2338, 1609,
-
1
+
1
6
508, 1465, 1217, 1040, 762 cm ; MS (ESI): m/z 384 (M+H) ; Anal. Calcd for C H NOS : C, 68.89; H,
22 25 2
.57; N, 3.65. Found: C, 68.95; H, 6.62; N, 4.09.
4
.1.1.26. Diethyl-(2-{4-[(2-methylsulfanyl-phenyl)-thiophen-2-yl-methyl]-phenoxy}-ethyl)-amine (28):
1
Compound 4 o-SMe substituted at Ar (200 mg, 0.64 mmol)refluxed with K
2
CO
3
(221.17 mg, 1.6 mmol),
1
7
6
-(2-chloroethyl)-diethyl amine hydrochloride (110.16 mg, 0.64 mmol)in acetone furnished 28 (191.7 mg,
1
2.7%) as brown viscous oil; H NMR (300 MHz, CDCl ,):
δ 7.21-7.14 (m, 4H), 7.08-6.99 (m, 4H),
3
.89-6.76 (m, 2H), 6.59-6.58 (m, 1H), 6.06 (s, 1H), 4.01 (t, J = 6.3, 2H), 2.86 (t, J = 6.2, 2H), 2.67-2.60
13
(
m, 4H), 2.36 (s, 3H), 1.07 (t, J = 7.0, 6H); C NMR (75 MHz, CDCl
3
):
δ 157.4 , 137.5 , 135.4 ,
1
1
30.1(2C) , 129.1(2C) , 127.3 , 126.6(3C) , 125.1(2C) , 124.5 , 114.3(2C) , 65.8 , 51.5 , 47.7 , 47.6 , 42.3 ,
-1
+
6.5 , 11.3(2C); IR (Neat): 2967, 2358, 1617, 1222, 1040, 767 cm ; MS (ESI): m/z 412 (M+H) , Anal.
Calcd for C H NOS : C, 70.03; H, 7.10; N, 3.40. Found: C, 70.10; H, 7.19; N, 3.30.
24
29
2
4
.1.1.27. (2-{4-[(2-Methoxy-phenyl)-thiophen-2-yl-methyl]-phenoxy}-ethyl)-dimethyl-amine (29):
1
Compound 4 o-OMe substituted at Ar (400 mg, 1.349 mmol) refluxed with K CO (466.32 mg, 3.374
2
3
mmol), 1-(2-chloroethyl)-dimethyl amine hydrochloride (194.32mg, 1.349 mmol) in acetone furnished 29
1
(
350 mg, 70.5%) as brown viscous oil; H NMR (300 MHz, CDCl ):
δ
7.21-6.99 (m, 5H), 6.89-6.78 (m,
3
5
H), 6.61-6.60 (m, 1H), 6.00 (s, 1H), 4.02 (t, J = 5.8, 2H), 3.74 (s, 3H), 2.71 (t, J = 5.7, 2H), 2.32 (s, 6H).
13
C NMR (75 MHz, CDCl
3
): δ 157.3, 156.7, 136.1, 129.8(3C), 127.8, 126.4(2C), 126.0(2C), 124.0,
1
1
20.4, 114.2(2C), 110.7, 65.5, 58.1, 55.4, 45.6(2C), 43.8; IR (Neat): 3011, 2933, 26 21, 1608, 1509, 1463,
-1
+
+
+
243, 1218, 1032, 758 cm ; MS (ESI): m/z 368 (M+H) , 295 (C H O S) , 72 (C H N) ; Anal. Calcd
18
15
2
4
10
for C H NO S: C, 71.90; H, 6.86; N, 3.81. Found: C, 71.92; H, 6.83; N, 3.85.
22
25
2
4
.1.1.28. Diethyl-(2-{4-[(2-methoxy-phenyl)-thiophen-2-yl-methyl]-phenoxy}-ethyl)-amine (30):
1
As described above, compound 4 o-OMe substituted at Ar (400 mg, 1.349 mmol)refluxed with K CO
2
3
(
466.32 mg, 3.37 mmol), 1-(2-chloroethyl)-diethyl amine hydrochloride (232.16 mg, 1.349 mmol) in
1
acetone furnished 30 (360 mg, 67.5%) as brown viscous oil; H NMR (300 MHz, CDCl
3
):
δ 7.24-7.02 (m,
5
6
1
4
H), 6.91-6.79 (m, 5H), 6.63-6.62 (m, 1H), 6.03 (s, 1H), 4.02 (t, J = 6.3, 2H), 3.73 (s, 3H), 2.85 (t, J =
13
.3, 2H), 2.66-2.59 (m, 4H), 1.05 (t, J = 7.1, 6H); C NMR (300 MHz, CDCl ):
δ
157.3, 156.6, 148.6,
3
35.9, 132.8, 129.7(2C), 129.5, 127.7, 126.3, 125.9, 123.9, 120.3, 114.1(2C), 110.6, 66.3, 55.5, 51.7,
-1
7.7(2C), 43.8, 11.7(2C); IR (Neat): 3017, 2971, 2935, 2836, 2362, 1607, 1509, 1244, 1217, 764 cm ;
17

ACCEPTED MANUSCRIPT
+
2
MS (ESI): m/z 396 (M+H) ; Anal. Calcd for C24H29NO S: C, 72.87; H, 7.39; N, 3.54. Found: C, 72.95; H,
7
.36; N, 3.58.
4
.1.1.29. 1-(2-{4-[(2-Methoxy-phenyl)-thiophen-2-yl-methyl]-phenoxy}-ethyl)-pyrrolidine (31):
1
Compound 4 o-OMe substituted at Ar (500 mg, 1.687 mmol) refluxed with K CO (582.83 mg, 4.217
2
3
mmol), 1-(2-chloroethyl)-pyrrolidine hydrochloride (286.92 mg, 1.687 mmol) in acetone furnished 31
1
(
478.6mg, 72.1%) as brown viscous oil; H NMR (300 MHz, CDCl
3
):
δ
7.24-6.98 (m, 5H), 6.88-6.77 (m,
5
4
1
H), 6.60-6.59 (m, 1H), 5.99 (s, 1H), 4.09 (t, J = 5.9, 2H), 3.73 (s, 3H), 2.92 (t, J = 5.9, 2H), 2.68 (bs,
13
H), 1.84-1.80 (m, 4H); C NMR (75 MHz, CDCl3):
δ 157.0, 156.5, 148.4, 136.1, 132.6, 129.7(2C),
29.5, 127.7, 126.3, 125.9, 123.9, 120.3, 114.1(2C), 110.4, 66.1, 55.4, 54.7, 54.5(2C), 43.8, 23.4(2C); IR
-1
+
(
Neat): 3017, 2964, 2601, 1607, 1509, 1224, 1217, 769 cm ; MS (ESI): m/z 394 (M+H) ; Anal. Calcd for
C H NO S: C, 73.25; H, 6.92; N, 3.56. Found: C, 73.18; H, 6.95; N, 3.54.
2
4
27
2
4
.1.1.30. 1-(2-{4-[(2-Methoxy-phenyl)-thiophen-2-yl-methyl]-phenoxy}-ethyl)-piperidine (32):
1
Compound 4 o-OMe substituted at Ar (500 mg, 1.687 mmol) refluxed with K
2
CO
3
(582.83 mg,
4
.217mmol), 1-(2-chloroethyl)-piperidine hydrochloride (310.59 mg, 1.687 mmol) in acetone furnished
1
3
2 (630 mg, 91.62%) as brown viscous oil; H NMR (300 MHz, CDCl ):
δ
7.11-6.99 (m, 5H), 6.88-6.76
3
(
m, 5H), 6.61-6.59 (m, 1H), 5.99 (s, 1H), 4.06 (t, J = 6.0, 2H), 3.73 (s, 3H), 2.76 (t, J = 5.9, 2H), 2.52 (t, J
13
=
5.2, 4H), 1.61 (q, J = 5.6, 4H), 1.47-1.42 (m, 2H); C NMR (75 MHz, CDCl ): δ 157.2, 156.6, 148.5,
3
1
5
36.0, 132.8, 129.8, 129.6, 127.7, 126.3, 126.0, 123.9, 120.4, 114.2(2C), 110.5, 96.2 65.5, 57.8, 55.5,
-1
4.9(2C), 43.8, 25.6(2C), 24.1; IR (Neat): 3020, 2962, 260, 1610, 1217, 1091, 929, 761 cm ; MS (ESI):
+
m/z 408 (M+H) ; Anal. Calcd for C H NO S: C, 73.67; H, 7.17; N, 3.44. Found: C, 73.76; H, 7.21; N,
25
29
2
3
.34.
4
.1.1.31. 1-(2-{4-[(2-Methoxy-phenyl)-thiophen-2-yl-methyl]-phenoxy}-ethyl)-azepane (33):
1
Compound 4 o-OMe substituted at Ar (200 mg, 0.674 mmol) refluxed with K CO (233.16 mg, 1.68
2
3
mmol), 1-(2-Chloro-ethyl)-azepane hydrochloride (265.47 mg, 0.674 mmol) in acetone furnished 33
1
(
197mg, 69.2%) as. brown viscous oil; H NMR (300 MHz, CDCl ):
δ
7.19-6.98 (m, 5H), 6.87-6.75 (m,
3
5
4
1
2
H), 6.60-6.58 (m, 1H), 5.99 (s, 1H), 4.04 (t, J = 5.8, 2H), 3.73 (s, 3H), 2.95 (t, J = 5.5, 2H), 2.79 (t, J =
1
3
.2, 4H), 1.67 (bs, 4H), 1.60 (bs, 4H); C NMR (75 MHz, CDCl
3
):
δ
157.2, 156.6, 148.5, 136.1, 132.8,
29.8(2C), 129.6, 127.7, 126.3, 125.9, 123.9, 120.4, 114.2(2C), 110.5, 65.8, 56.5, 55.7, 55.4(2C), 43.8,
-1
7.3(2C), 27.0(2C); IR (Neat): 3017, 2937, 1609, 1509, 1242, 1217, 767 cm ; MS (ESI): m/z 422
18

ACCEPTED MANUSCRIPT
+
+
(
8 2
M+H) ; 126 (C H16N) ; Calcd for C26H31NO S: C, 74.07; H, 7.41; N, 3.32. Found: C, 74.02; H, 7.39; N,
3
.33.
4
.1.1.32. Diisopropyl-(2-{4-[(2-methoxy-phenyl)-thiophen-2-yl-methyl]-phenoxy}-ethyl)-amine (34):
1
Compound 4 o-OMe substituted at Ar (500mg, 1.687 mmol) refluxed with K CO (582.83 mg, 4.217
2
3
mmol, (2-Chloro-ethyl)-diisopropyl-amine hydrochloride (337.65 mg, 1.687 mmol) in acetone furnished
1
3
4 (520 mg, 72.7%) as yellow viscous oil; H NMR (300 MHz, CDCl
3
):
δ
7.17-6.98 (m, 5H), 6.88-6.78
(
m, 5H), 6.60-6.59 (m, 1H), 5.99 (s, 1H), 3.87 (bs, 2H), 3.74 (s, 3H), 3.06 (bs, 2H), 2.82 (t, J = 6.7, 2H),
1
3
1
1
.05 (d, J = 6.2, 12H); ); C NMR (75 MHz, CDCl ):
δ
157.7, 156.8, 135.9, 133.0, 129.9(2C), 129.7(2C),
3
27.9, 126.5, 126.1, 124.1, 120.5, 114.2(2C), 110.8, 69.3, 55.6, 49.8(2C), 44.6, 44.0, 21.0(4C) ; IR
-1
+
(
Neat): 3018, 2968, 2930, 2361, 1605, 1508, 1217, 764 cm ; MS (ESI): m/z 424 (M+H) ; Anal.
C H NO S: C, 73.72; H, 7.85; N, 3.31. Found: C, 73.81; H, 7.90; N, 3.25.
2
6
33
2
4
.1.1.33. 4-(2-{4-[(2-Methoxy-phenyl)-thiophen-2-yl-methyl]-phenoxy}-ethyl)-morpholine (35):
1
Compound 4 o-OMe substituted at Ar (400mg, 1.349 mmol), K
2
CO
3
(466.32mg, 3.37mmol), 4-(2-
Chloro-ethyl)-morpholine hydrochloride (251.02g, 1.349 mmol) furnished 35 (510mg, 92.2%) as brown
1
viscous oil. H NMR (200 MHz, CDCl ):
δ 7.23-6.98 (m, 5H), 6.88-6.75 (m, 5H), 6.60-6.59 (m, 1H),
3
5
4
1
2
7
.99 (s, 1H), 4.05 (t, J = 5.7, 2H), 3.73 (s, 3H), 3.69 (t, J = 4.5, 4H), 2.75 (t, J = 5.7, 2H), 2.53 (t, J = 4.6,
13
H); C NMR (75 MHz, CDCl ):
δ
157.2, 156.6, 148.4, 136.1, 132.7, 129.8(2C), 129.6, 127.8, 126.3,
3
26.0, 123.9, 120.4, 114.1(2C), 110.5, 66.8(2C), 65.6, 57.7, 55.4, 54.3(2C), 43.8; IR (Neat): 3012, 2936,
-1
+
3
863, 1605, 1508, 1243, 1220, 767 cm . MS (ESI): m/z 410 (M+H) ; Anal. Calcd for C24H27NO S: C,
0.39; H, 6.65; N, 3.42. Found: C, 70.43; H, 6.72; N, 3.43.
4
.1.1.34. Diisopropyl-(2-{4-[(4-methoxy-phenyl)-thiophen-2-yl-methyl]-phenoxy}-ethyl)-amine (36):
1
Compound 4 p-OMe substituted at Ar (0.10 g, 0.33 mmol), K
2
CO
3
(0.14 g, 1.01 mmol), (2-Chloro-ethyl)-
diisopropyl-amine hydrochloride (0.07g, 0.37 mmol) furnished 36 as yellow viscous oil (0.11 g, 75%).
H NMR (300 MHz, CDCl3): 7.17 (d, J = 4.5, 1H), 7.12-7.07 (m, 4H), 6.92-6.89 (m, 1H), 6.84-6.80
m, 4H), 6.67-6.64 (d, J = 3.0, 1H), 5.56 (s, 1H), 3.86 (t, J = 7.3, 2H), 3.76 (s, 3H), 3.07-2.98 (m, 2H),
1
δ
(
2
1
.80 (t, J = 7.5, 2H), 1.03 (d, J = 6.3, 12H); 13C NMR (75 MHz, CDCl3): δ 158.2, 157.5, 148.9, 136.3,
36.2, 129.6(4C), 126.4, 125.9, 124.2, 114.2(2C), 113.6(2C), 68.8, 55.1(2C), 50.5, 49.9, 44.5, 20.6(4C);
+
IR (Neat): 2964, 1654, 1611, 1509 cm-1; MS (ESI): m/z 424 (M+H) ; Anal. Calcd for C H NO S: C,
2
6
33
2
7
3.72; H, 7.85; N, 3.31. Found: C, 73.79; H, 7.79; N, 3.29.
.1.1.35. 1-(2-(4-((4-methoxyphenyl)(thiophen-3-yl)methyl)phenoxy)ethyl)piperidine (37):
4
19

ACCEPTED MANUSCRIPT
Compound 1a reacted with 2* to gave corresponding 3* which on refluxing with phenol and two drops of
H
2
SO
4
in benzene provided with 4* which was refluxed with K
2
CO
3
1-(2-chloroethyl)-piperidine
1
hydrochloride in acetone furnished compound 37. H NMR (400 MHz, CDCl ): δ 7.00 (d, J=8Hz, 1H),
3
6
,
.95 (m, 4H), 6.77-6.63 (m , 5H), 6.62 (s,1H), 7.32 (s,1H), 4.32 (t, J=4Hz, 2H), 3.7 (s, 3H), 3.27 (t, J=4Hz
13
2H) , 3.09 (m,4H), 1.88 (m,4H), 1.59 (m,2H) ; C NMR (75 MHz, CDCl3):
δ 158.11 , 155.7 , 145.3 ,
1
5
37.7 , 136.0 , 130.0(2C) , 129.7(2C) , 125.5(2C) , 122.4 , 114.3(2C) , 113.7(2C) , 63.0 , 56.3 , 55.2(2C) ,
4.1 , 50.9 , 23.0(2C) , 21.9.
4
.1.1.36. N, N –diethyl-2-(4-((4-methoxyphenyl)(thiophen-3-yl)methyl)phenoxy)ethanamine (38):
R : 0.4 (20% ethyl acetate in hexane). Isolated as light yellow oily liquid (yield 65%) by elution with 2%
f
1
methanol in dichloromethane from silica gel. H NMR (400 MHz, CDCl
3
): δ 7.19 (t , J= 4Hz,1H ) , 7.0
(
m, 4H ) , 6.97 (m , 5H) , 6.76-6.62 (m, 1H) , 5.32 (s , 1H ) , 4.29 (t , J=4 Hz ,2H) , 3.70 (s , 3H) , 3.43 (t,
J=4Hz, 2H ) , 3.23 (m , 4H) , 1.31 (t, J= 8Hz, 6H ).
4
.1.1.37.
N–isopropyl-N-(2-(4-((4-methoxyphenyl)(thiophen-3-yl)methyl)phenoxy)ethyl)propan-2-
amine (39):
R : 0.43 (20% ethyl acetate in hexane). Isolated as light yellow oily liquid (yield 62%) by elution with
f
1
2
4
,
% methanol in dichloromethane from silica gel. H NMR (300 MHz, CDCl ): δ 7.23 (s , 1H) , 7.03 (m ,
3
H ) , 6.85 (m, 5H) , 6.69 (m , 1H) , 5.39 (s , 1H) , 3.87 (t, J=6Hz, 2H) , 3.76 (s , 3H) , 3.03 (t, J=6Hz, 2H)
2.80 (m , 2H) , 1.04 ( d, J=6Hz, 12H)
4
.1.1.38. 2-(di(thiophen-2-yl)methoxy)-N,N-diethylethanamine(40):
To a solution of 2-thiophene carboxaldehyde, thiophene 2-magnesium bromide (in situ generation of 2-
bromo thiophene in presence of Mg in THF for 1hr) was added and was stirred for another 1hr to furnish
corresponding carbinol. It was then refluxed with diethyl aminoethyl chloride hydrochloride chain in
0
presence of K CO in acetone at 60-75 C to furnish compound 40. Quenched with NH Cl soln, extracted
2
3
4
with ethyl acetate and dried over Na
2
SO
4
. The solvent was removed under reduced pressure. Product was
purified by column chromatography. R
f
: 0.3(2 % methanol in DCM). Isolated as pale yellow, oily liquid,
1
(
(
yield 52%,) , elution with 1.5% methanol in DCM from silica gel. H NMR (400 MHz, CDCl ): δ 7.23
3
13
s, 2H), 6.90 (s, 4H, ), 5.83 (s, 1H), 3.72 (s, 2H,), 3.00-2.90 (m, 6H,), 1.16(d, J= 6.48 Hz, 6H)ppm.
C
NMR (75 MHz, CDCl ): δ 145.1(2C), 126.3(2C), 125.7(2C), 125.6(2C), 75.9 , 66.5, 51.8, 47.6(2C),
3
-
1
1
6
1.1(2C) ppm. IR (Neat cm ): 3442, 2924, 1630, 1218, 772. Anal. calcd. for C H NOS : C 58.39; H,
13 17 2
.41; N, 5.24%; found C, 58.42; H, 6.34; N, 5.15%
20

ACCEPTED MANUSCRIPT
4
.1.1.39. N-(2-(dithiophen-2-ylmethoxy)ethyl)-N-isopropylpropan-2-amine (41):
R : 0.6 (40% ethyl acetate in hexane). Isolated as pale brown, oily liquid, (yield 70%,) by elution with
f
1
0
.5% methanol in DCM from silica gel. H NMR (300 MHz, CDCl
3
): δ 7.21 (d, J= 4.89 Hz, 2H), 6.88 (t,
J= 5.07 Hz ,4H), 5.85 (s, 1H), 3.64 (s, 2H), 3.29 (s, 2H), 2.87(s, 2H), 1.10 (d, J=6.09Hz, 12H)ppm. IR
-1
(
Neat cm ):3440, 2928, 1642, 1218,770. Anal. calcd. for C H NOS : C, 63.11; H, 7.79; N, 4.33 %;
17 25 2
found C, 63.24; H, 7.65; N, 4.41 %.
4
.1.1.40. 2-(di(thiophen-2-yl)methoxy)-N,N-dimethylethanamine(42):
R : 0.35 (2% methanol in DCM ). Isolated as pale brown, oily liquid, (yield 35%,) by elution with 2%
f
1
methanol in DCM from silica gel. H NMR (300 MHz, CDCl
3
): δ 7.25 (d, J= 4.5 Hz, 2H), 6.91 (d, J=
1
3
4
.44 Hz, 4H), 5.85 (s, 1H), 3.87 (t, J= 3.69 Hz, 2H), 3.25 (d, J= 3.75 Hz, 2H), 2.85(s, 6H) ppm. C NMR
(
(
100 MHz, CDCl ): δ 143.7(2C), 126.6(2C), 126.3(2C), 126.1(2C), 76.3, 63.8, 57.3, 44.3(2C) ppm. IR
3
-1
2
Neat cm ): 3435, 2924, 1634, 1217, 770. Anal. calcd. for C15H21NOS : C, 60.98; H, 7.16; N, 4.74 %;
found C, 60.77; H, 7.32; N, 4.71 %.
4
.1.1.41. 1-(2-(di(thiophen-2-yl)methoxy)ethyl)piperidine(43):
R : 0.4 (5% methanol in DCM ). Isolated as pale orange, oily liquid, (yield 33%,) by elution with 1.5%
f
1
methanol in DCM from silica gel. H NMR (400 MHz, CDCl
3
): δ 7.28(dd, J
1
= 1.68, J
2
= 2.88Hz, 2H) ,
6
1
.88 (t, J= 4.64 Hz, 4H ), 5.82 (s, 1H), 3.65 (t, J= 5.88 Hz, 2H), 2.66 (t, J= 5.84 Hz, 2H), 2.51(s, 4H ),
-1
.61-1.55 (m, 4H),1.39 (d, J= 4.96 Hz, 2H)ppm. IR (Neat cm ):3450, 2918, 1620, 1212, 775. (
C H NO). Anal. calcd. for C H NOS : C, 62.50; H, 6.88; N, 4.56%; found C, 62.63; H, 6.78; N,
7
14
16 21
2
4
.56%.
.1.1.42. N-(2-(di(thiophen-3-yl)methoxy)ethyl)-N-isopropylpropan-2-amine (44):
4
R : 0.38 (20% ethyl acetate in hexane). Isolated as yellow oily liquid (yield 64%) by elution with 2%
f
1
methanol in dichloromethane from silica gel. H NMR (400 MHz, CDCl ): δ 7.20-7.17 (m, 4H), 7.01(q,
3
J= 1 Hz,1H), 6.83 (d, J=5.28Hz, 1H), 5.7 (s, 1H), 3.5 (s, 2H), 3.05 (s, 2H), 2.70 (s, 2H), 0.99 (d,
J=5.84Hz, 12H).
4
.1.1.43. 1-(2-(di(thiophen-3-yl)methoxy)ethyl)piperidine (45):
R : 0.20 (30% ethyl acetate in hexane). Isolated as yellow oily liquid (yield 67%) by elution with 5%
f
1
methanol in dichloromethane from silica gel. H NMR (400 MHz, CDCl
3
): δ 7.22-7.19 (m, 4H), 6.99 (q,
21

ACCEPTED MANUSCRIPT
J= 1.6 Hz,1H), 6.86 (d, J=5.28Hz, 1H), 5.76 (s, 1H ), 3.75 (t, J=5.32Hz, 2H), 2.39 (t, J= 5.36Hz, 2H),
2
.78 (s, 4H),1.75-1.69 (m, 4H), 1.52 (s, 2H)
4
.1.1.44. 2-(di(thiophen-3-yl)methoxy)-N,N-diethylethanamine (46):
R : 0.30 (20% ethyl acetate in hexane). Isolated as yellow oily liquid (yield 68%) by elution with 6%
f
1
methanol in dichloromethane from silica gel. H NMR (400 MHz, CDCl ): δ 7.18 (t, J=4.56Hz, 4H),
3
6
2
.99 (q, J= 1.24 Hz, 1H), 6.83 (d, J=5.28Hz, 1H), 5.76 (s, 1H, ), 3.57 (t,J=6.16Hz, 2H), 2.72 (t, J= 6.2Hz,
H), 2.57 (q, J=7.16Hz, 4H),0.99 (t, J= 7.16Hz, 6H)
4
.1.1.45. 2-(di(thiophen-3-yl)methoxy)-N,N-dimethylethanamine (47):
R : 0.30 (20% ethyl acetate in hexane). Isolated as Brown yellow oily liquid (yield 72%) by elution with
f
1
1
4
5
0% methanol in dichloromethane from silica gel. H NMR (400 MHz, CDCl
3
): δ 7.20 (t, J= 3.24 Hz,
H), 7.00 (d, J= 4.72 Hz, 1H), 6.852 (d, J=5.28Hz, 1H), 5.75 (s, 1H, ), 3.63 (t, J=5.52Hz, 2H), 2.69 (t, J=
.52Hz, 2H), 2.35 (s, 6H)
4
.1.1.46. 1-(2-(di(thiophen-3-yl)methoxy)ethyl)pyrrolidine (48):
R
f
: 0.30 (30% ethyl acetate in hexane). Isolated as pale yellow oily liquid (yield 68%) by elution with 4%
1
methanol in dichloromethane from silica gel. H NMR (300 MHz, CDCl ): δ 7.25-7.20 (m, 4H), 7.00 (q,
3
J= 1.48 Hz,1H), 6.86 (d, J=5.28Hz, 1H), 5.77 (s, 1H), 3.82 (q, J=1.92Hz, 2H), 3.14 (t, J= 4.28Hz, 6H),
1
.95 (t, J= 6.6Hz, 4H)
4
.1.1.47. 4-(2-(di(thiophen-3-yl)methoxy)ethyl)morpholine (49):
R
f
: 0.36 (10% ethyl acetate in hexane). Isolated as yellow oily liquid (yield 67%) by elution with 2%
1
methanol in dichloromethane from silica gel. H NMR (400 MHz, CDCl ): δ 7.20 (q, J=1.52Hz, 4H),
3
6
2
.99 (q, J= 1.72 Hz, 1H), 6.86(d, J=5.28Hz,1H), 5.76(s,1H), 3.72(t, J=4.64Hz, 4H ), 3.68 (t, J=5.48Hz,
H), 2.74 (t, J= 5.36Hz, 2H), 2.64 (s, 4H)
4
4
.2. Biological Evaluation
11
V
.2.1. Assay for in vitro activity against M. tuberculosis H37R
The compounds were dissolved in dimethyl sulfoxide (DMSO) to make stocks. Serial dilutions from
stocks were also made in DMSO. To 1.9 ml Middle brook (MB) 7H10 agar supplemented with OADC (in
tubes, held at 45-50° C), 0.1 ml of test compound or DMSO (negative control) or INH (positive control)
was added. The contents were mixed and allowed to solidify (by cooling) as slants. Three-week old
22

ACCEPTED MANUSCRIPT
v
culture of M. tuberculosis H37R was harvested from L-J medium and its suspension (1 mg/ml,
8
equivalent to 10 bacilli approximately) was made in normal saline containing 0.05% Tween-80. 10
ꢀl of
5
1
:10 dilution of this suspension (~10 bacilli) was inoculated into each tube and incubated at 37 ˚C for 4
weeks. The lowest concentration of a compound up to which there was no visible growth of bacilli was its
minimal inhibitory concentration (MIC). Compounds having MIC of 6.25 mg/L were selected as
≤
‘
secondary hits’ for further development.
12
4
.2.2. Assays for in vitro cytotoxicity
In vitro cytotoxicity of active compounds (having MIC
≤ 6.25 mg/L) was measured using Vero cells and
mouse bone-marrow derived macrophages. Vero C-1008 cell line was procured from Laboratory Animals
Division (LAD) of CDRI. For preparation of macrophages, a Swiss mouse (bred in LDA) was euthanized
by exposure to CO and femurs dissected out. The bones were trimmed at each end and marrow flushed
2
out with Dulbecco’s Minimal Essential Medium containing 10% fetal bovine serum (DMEM-FBS) and
antibiotics. The medium was also supplemented with 15% (v/v) L929 fibroblast conditioned medium and
non-essential amino acids. The cell suspension (Vero/macrophage) was plated in 96-well tissue culture
plates (20,000 cells/200
Compounds at different concentrations were added to the wells. A known toxic compound
Staurosporine) was used as a positive control and DMSO was used as negative control. After 24 h of
incubation, 20 L of MTS solution (tetrazolium compound, Owen’s reagent) was added to each well and
incubated for further 2 h (37 ºC, 5% CO ). O. D. was read at 490 nm using a plate reader. A compound
2
ꢀL/well) and incubated overnight (37ºC, 5% CO ) to allow their adherence.
(
ꢀ
2
was considered as potentially toxic if its IC50 (concentration causing 50% loss in cell viability) was
≤ 10
times its MIC for M. tuberculosis H37RV.
13
4
.2.3. Assay for ex vivo activity in the macrophage model of tuberculosis
The effect of selected compounds (MIC 6.25 mg/L and SI 10, i.e., non-toxic) on the survival and
≤
≥
multiplication of M. tuberculosis H37R within mouse bone-marrow derived as well as human blood
v
monocyte derived macrophages was evaluated by light microscopy and counting of colony-forming units
(
CFU). Mouse bone-marrow derived macrophages were prepared as above and the human blood
monocyte derived macrophages were prepared as follows: 10 ml blood from healthy volunteers was
collected in anti-coagulant solution (acid-citrate-dextrose, ACD), centrifuged at 2500 rpm (to remove
plasma) and reconstituted with RPMI 1640 tissue culture medium containing ACD. This suspension was
layered over Ficoll-Isopaque (Sigma Chemical Company, USA) and centrifuged at 600 g for 20 min. The
mononuclear cells on the top of Ficoll layer were collected, washed twice with RPMI medium, and
23

ACCEPTED MANUSCRIPT
6
counted using hemocytometer. The cells were finally suspended at a density of 10 cells/mL in RPMI
containing heat inactivated pooled normal human serum (PNS, 5%).
For evaluation by microscopy, macrophages (human/mouse) were seeded in 8-chambered slides at a
5
density of 5 × 10 cells (in 0.5 ml RPMI-PNS/DMEM-FBS media) per chamber. The cells were incubated
th
at 37 ºC with 5% CO . On 6 day, washed adherent macrophages in each chamber were infected for 3h
2
6
with 0.5 ml suspension containing 2.5 × 10 M. tuberculosis H37R
v
in antibiotic-free medium. Later, the
chambers were thoroughly washed to remove extracellular bacteria and replenished with fresh 0.5 ml
medium containing 4x MIC of standard drugs (INH, RFM and PZA) or test compounds. After further 4
days incubation in the CO incubator, each chamber was gently washed and medium aspirated off. After
2
removing the chambers, infected macrophages on slide were heat-fixed, stained with Ziehl-Neelsen stain,
counter stained with methylene blue, and viewed under oil immersion lens (100×) of a microscope.
6
For evaluation by counting colony forming units (CFU), 5-day old cultures of adherent macrophages (10
6
cells/well, in 24-well plates) were infected for 3h with 5x10 M. tuberculosis H37R in antibiotic-free
v
medium. Later, the wells were washed to remove extracellular bacteria and replenished with fresh 1 ml
antibiotic-free medium containing 4x MIC of standard drugs or test compounds. In order to determine the
number of bacilli phagocytised during the 3 h infection period (0 day count), one well was lysed with
0
.1% saponin (15 min) and lysate (50 ꢀL of 1:100 dilution) plated on MB 7H11 agar (in petri dishes) for
colony counting. Other wells, after further 4 days of incubation, were gently washed, cells lysed and
lysates plated on MB 7H11 agar plates. CFUs were counted after 4 weeks of incubation at 37˚C.
14
4
.2.4. Assay for in vivo activity in the mouse model of tuberculosis
7
Outbred Swiss mice (obtained from CDRI-LAD) were infected with M. tuberculosis H37R
v
(10 CFU/
mouse, i.v.) and divided into groups of 8-10 animals. Each experimental group received daily oral dose of
the test compound (100 mg/kg body weight, for 28 days) dissolved in a suitable vehicle (water/corn oil,
depending on its solubility profile). The drug-treated control groups received INH (25 mg/kg) or EMB
(
10 mg/kg) or PZA (150 mg/kg body weight) for 28 days. The untreated control groups received only the
vehicle. At least 3 mice from each group were sacrificed on day 30 for determination of viable bacilli in
the lungs. Serial dilutions of lung homogenates were placed on MB7H11 agar medium and colonies
(
CFU) were counted after 4 weeks of incubation at 37°C. Remaining mice, if survived, were observed for
further 15 days for determination of mean survival time (MST) and % survivors.
15
4
.2.5. Determination of Minimum Bactericidal Concentration (MBC)
For in vitro MBC determinations, to each culture tube was having 2.5 ml MB7H9 broth, 50 ꢀL inoculum
5
containing 10 M. tuberculosis H37R was added and incubated (37°C) with or without 1× and 4× MIC of
v
test compounds or standard anti-TB drugs. CFU in a tube containing inoculum alone was determined on
24

ACCEPTED MANUSCRIPT
day 0 and in others it was determined on day 14. The same principle was applied to the determination of
ex vivo MBC.
11
4
.2.6. Activity of selected compounds against drug-resistant strains of M. tuberculosis .
The drug sensitive and drug-resistant Indian clinical isolates of M. tuberculosis were obtained from the
DBT Mycobacterium Repository at NJIL-OMD, Agra (Courtesy of Dr VM Katoch, DG-ICMR) and
propagated on L-J medium. The cultures thus obtained were used for testing in vitro sensitivity against
selected active compounds using agar dilution method.
Acknowledgments
We acknowledge the National JALMA Institute of Leprosy & Other Mycobacterial Diseases (NJIL-
OMD), Agra and Dr. VM Katoch, DG-ICMR for providing the drug sensitive and resistant
Mycobacterium tuberculosis H37R strains. Authors also acknowledge Pharmacokinetics & Metabolism
v
Division for conducting the pharmacokinetic evaluation of S006-830. We sincerely feel that addressing
all the questions of the referee resulted in a much thorough presentation of the results and improved the
quality of the paper. This has CDRI communication no xx.
Funding
The study was supported by Council of Scientific and Industrial Research, India (grant number BSC0104
to G. Panda, S. Sinha, Vinita Chaturvedi). CSIR- Senior Research Fellowship to P.S., S.K.M., A.K.J.,
S.B., M.K.P., S.L.K.M., S.M., P.T., S.S., CSIR- project Research Fellowship to T. S., and TATA-CSIR
Open Source Drug Discovery Fellowship (HCP0001P) to P.M.
References
1
2
3
. World Health Organization (WHO). Global tuberculosis report 2014, 4th ed.; WHO Press: Geneva,
Switzerland, 2014 (WHO/HTM/TB/2014.08)
. WHO: Multidrug-resistant tuberculosis (MDR-TB) 2013 Update. World Health Organization.
Retrieved 14 June 2013
. Panda, G.; Shagufta ; Mishra, J. K .; Chaturvedi, V.; Srivastava, A. K.; Srivastava, R.; Srivastava, B.
S. Bioorg. Med. Chem. 2004, 12, 5269.
4
5
6
. Panda, G.; Shagufta; Srivastava, A. K.; Sinha, S. Bioorg. Med. Chem. Lett. 2005, 15, 5222.
. Shagufta; Kumar, A.; Panda, G.; Siddiqi, M. I. J. Mol. Model. 2007, 13, 99.
. Panda, G.; Parai, M. K.; Das, S. K.; Shagufta; Sinha, M.; Chaturvedi, V.; Srivastava, A. K.; Manju,
Y. S.; Gaikwad, A. N.; Sinha, S. Eur. J. Med. Chem. 2007, 42, 410.
25

ACCEPTED MANUSCRIPT
7
8
. Parai, M. K.; Panda, G.; Chaturvedi, V.; Manju, Y. K.; Sinha, S. Bioorg. Med. Chem.Lett. 2008, 18,
2
89.
.
Kashyap, V. K.; Gupta, R. K.; Shrivastava, R.; Srivastava, B. S.; Srivastava, R.; Parai, M. K.; Singh,
P.; Bera, S.; Panda, G. J. Antimicrob. Chemother. 2012, 67, 1188-97.
9
1
. Hidau, M. K.; Singh. Y.; Sahi, S.; Mounika, P.; Singh, S. K. Curr. Pharmaceutl. Anal. 2015, in press.
0. Singh, Y.; Hidau, M. K.; Misra, A.; Kushwaha, H.; Tiwari, A.; Sharma, A.; Singh, Shio.; Drug Test
Anal. in press, 2015.
1
1
1
1
1
1. McClachy, J. K. Lab Med. 1978, 9, 47.
2. Mosmann, T. J. Immunol. Methods. 1983, 65, 55.
3. Orme, I. Antimicrob. Agents. Chemother. 2001, 45, 1943.
4. Canetti, G.; Grumbach, F.; Grosset, J. Am Rev Respir Dis. 1960, 82, 295.
5. Byrne, S. T.; Denkin, S. M.; Gu, P.; Nuermberger, E.; Zhang, Y. J. Med. Microbiol. 2007, 56, 1047.
26

ACCEPTED MANUSCRIPT
a
Compd.
no.
R1
R2
Mol.
CLogP
MIC
Cytotoxic SI
activity,
Weight
(mg/L)
IC50
(
mg/L)
ND
ND
13
b
5
6
7
8
9
H
H
N(C H )
365.53
377.54
391.57
379.52
411.99
428.03
371.92
399.98
397.96
411.99
428.03
413.96
355.57
383.52
381.51
395.53
411.58
397.51
355.47
383.52
393.52
439.68
383.57
411.62
367.50
395.56
393.54
6.36
6.44
6.97
5.11
7.15
7.69
6.02
7.08
6.62
7.15
7.69
5.83
5.45
6.51
6.05
6.58
7.12
5.26
5.45
6.51
6.58
7.18
5.16
6.22
4.82
5.88
5.43
12.47
6.23
3.10
24.98
0.78
1.56
4.65
1.25
2.49
1.29
1.34
5.18
17.8
4.80
19.1
1.24
10.3
19.9
17.8
2.40
19.8
1.57
12.5
25.0
9.19
25.0
25.0
ND
ND
<10
ND
>10
>10
<10
<10
<10
<10
>10
<10
ND
ND
ND
<10
ND
ND
ND
<10
ND
>10
ND
ND
ND
ND
ND
2
5 2
N(CH2)5
H
N(CH2)6
H
N(CH ) O
ND
100
100
10.8
10.1
8.6
2
4
p-Cl
p-Cl
m-Cl
m-Cl
m-Cl
m-Cl
m-Cl
m-Cl
o-F
N(CH2)5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
N{CH(CH ) }
3
2 2
N(CH3)2
N(CH CH )
2
3 2
N(CH2)4
N(CH2)5
8.7
N{CH(CH ) }
2 2
22.0
23.4
ND
ND
ND
3.9
3
N(CH ) O
2
4
N(CH3)2
o-F
N(CH CH )
2
3 2
o-F
N(CH2)4
o-F
N(CH2)5
o-F
N{CH(CH ) }
2 2
ND
ND
ND
5.8
3
o-F
N(CH ) O
2 4
m-F
N(CH3)2
m-F
N(CH CH )
2
3 2
m-F
N(CH2)5
ND
100
ND
ND
ND
ND
ND
p-SCH3
o-SCH3
o-SCH3
o-OCH3
o-OCH3
o-OCH3
N{CH(CH3)2}2
N(CH3)2
N(CH CH )
2
3 2
N(CH3)2
N(CH CH )
2
3 2
N(CH2)4

ACCEPTED MANUSCRIPT
32
33
34
35
36
o-OCH3
o-OCH3
o-OCH3
o-OCH3
p-OCH3
N(CH2)5
N(CH2)6
407.57
421.59
423.61
409.54
423.61
5.96
6.49
6.50
4.63
6.90
1.28
2.64
25.0
10.24
1.33
9.2
12.7
ND
ND
100
<10
<10
ND
ND
>10
N{CH(CH3)2}2
N(CH ) O
2 4
N{CH(CH3)2}2
(
S006-
30)
8