Synthesis and evaluation of 4′,5′-dihydrospiro[piperidine-4,7′-thieno[2,3-c]pyran] analogues against both active and dormant Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.bmc.2017.12.044

Need for new drugs to fight against tuberculosis (TB) is increasing day by day. In the present work we have taken a spiro compound (GSK 2200150A) reported by GSK as a lead and we modified the structure of the lead to study the antitubercular activity. For structure activity profiling twenty-one molecules have been synthesized, characterized and evaluated for their antimycobacterial potency against both active and dormant TB. Compound 06, 1-((4-methoxyphenyl)sulfonyl)-4′,5′-dihydrospiro[piperidine-4,7′-thieno[2,3-c]pyran] was found to be the most potent compound (MIC: 8.23 μM) in active TB and was less effective than the lead but more potent than standard first line drug ethambutol. It was also found to be more efficacious than Isoniazid and Rifampicin and equipotent as Moxifloxacin against dormant Mycobacterium tuberculosis (MTB). Compound 06 also showed good inhibitory potential against over expressed latent MTB enzyme lysine ε-amino transferase with an IC₅₀ of 1.04 ± 0.32 μM. This compound is a good candidate for drug development owing to potential against both active and dormant stages of MTB.

Full text of DOI:10.1016/j.bmc.2017.12.044

Accepted Manuscript
Synthesis and Evaluation of 4',5'-Dihydrospiro[piperidine-4,7'-thieno[2,3-
c]pyran] Analogues Against Both Active and Dormant Mycobacterium tuber-
culosis
Kiran Kumar Alluri, Rudraraju Srilakshmi Reshma, Raghuram Suraparaju,
Suryanarayana Gottapu, Dharmarajan Sriram
PII:
S0968-0896(17)32239-3
https://doi.org/10.1016/j.bmc.2017.12.044
BMC 14149
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
16 November 2017
27 December 2017
28 December 2017
Please cite this article as: Alluri, K.K., Reshma, R.S., Suraparaju, R., Gottapu, S., Sriram, D., Synthesis and
Evaluation of 4',5'-Dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran] Analogues Against Both Active and Dormant
Mycobacterium tuberculosis, Bioorganic & Medicinal Chemistry (2017), doi: https://doi.org/10.1016/j.bmc.
2
017.12.044
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Synthesis and Evaluation of 4',5'-Dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]
Analogues Against Both Active and Dormant Mycobacterium tuberculosis
a
a
b
Kiran Kumar Alluri , Rudraraju Srilakshmi Reshma , Raghuram Suraparaju , Suryanarayana
b
a
Gottapu , Dharmarajan Sriram , *
a
b
Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad Campus,
Hyderabad-500078, India.
GSN Life Sciences Pvt Ltd, Kukatpally, Hyderabad-500072, India.
Corresponding Author*
1

D. Sriram, Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad Campus,
Jawahar nagar, Hyderabad - 500078. INDIA. Telephone: +91-40663030506; Fax: +91-4066303998;
Email: dsriram@hyderabad.bits-pilani.ac.in
ABSTRACT:
Need for new drugs to fight against tuberculosis (TB) is increasing day by day. In the present
work we have taken a spiro compound (GSK 2200150A) reported by GSK as a lead and we
modified the structure of the lead to study the antitubercular activity. For structure activity
profiling twenty-one molecules have been synthesized, characterized and evaluated for their
antimycobacterial potency against both active and dormant TB. Compound 06, 1-((4-
methoxyphenyl)sulfonyl)-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran] was found to be
the most potent compound (MIC : 8.23µM) in active TB and was less effective than the lead but
more potent than standard first line drug ethambutol. It was also found to be more efficacious
than Isoniazid and Rifampicin and equipotent as Moxifloxacin against dormant Mycobacterium
tuberculosis(MTB). Compound 06 also showed good inhibitory potential against over expressed
latent MTB enzyme lysine ε-amino transferase with an IC of 1.04 ± 0.32 µM. This compound
5
0
is a good candidate for drug development owing to potential against both active and dormant
stages of MTB.
Key words: Tuberculosis, Mycobacterium tuberculosis, Spiro compounds, Dormant TB
2

1
. Introduction
Tuberculosis (TB) is caused by Mycobacterium tuberculosis (MTB) that have evolved both as a
1
symbiont and pathogen more than 150 million years ago. The co-evolution of MTB with
humans for such long periods has resulted in extensive evolutionary adaptation, which facilitated
2
MTB survival inside the lungs and established it as a potent pathogen. It earned apt sobriquets
such as The Robber of youth, Captain of these men of death, The Grave yard Cough and King’s
th
3
evil during 19 century due to massive deaths caused by the disease. MTB can remain in lungs
in asymptomatic latent form for a very long time, which can get activated at any opportune time.
Stringent & lipid rich cell envelope, slow metabolic rate and precise regulation of pathogenic
4
factors make it a dreadful pathogen. WHO reports showed that one third of the world population
is suffering with latent TB, which has ~10 % lifetime chance to convert into active TB. In 2015,
there are 10.4 million new cases reported across the world. 4,80,000 people developed multi drug
resistant TB. Despite the availability of treatment regimens TB still remains among top 10 causes
5
of death worldwide. Manmade amplification of disease led to emergence of MDR and XDR
forms of TB, which are resistant to most of first line and second line drugs available. The
treatment options for resistant and dormant forms of TB are limited and costly, and causing more
side effects. Newer drugs need to be developed which can lower duration of therapy and become
6
more efficacious against resistant and dormant forms. Various reasons are behind the lack of
new medicines for the treatment of TB and other neglected diseases over the last 40 years, but
most of them are generally related to a lack of critical mass in terms of funding, R&D capacity,
7
and commercial interest. Recently GlaxoSmithKilne screened around 2 million compounds
phenotypically against MTB and identified and reported a total of 177 compounds with MIC <
9
5
8
1
0 µM and therapeutic index of (HepG2IC /MTB H37Rv MIC) >50. They also reported seven
50
chemical families with strong antitubercular actives as potential lead compounds. Among the
3

reported leads; a spiro compound GSK2200150A with a cluster size of seven compounds
showed MTB MIC of 0.38µM. We have taken this spiro compound as a lead compound to
synthesize twenty-one compounds and evaluated biological evaluation against active and
dormant H37Rv strain of MTB.
2
. Results and discussion
2
.1. Designing and synthesis
In this work we have taken GSK2200150A as the starting point to design a new series by shifting
of sulfur atom and also studied the effect of N1 alkyl, sulfonyl and acyl derivatives (Fig 1) to
investigate the structure activity relationship (SAR) of the lead compound. To develop series of
molecules 4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran] core was retained as such and
the amine was reacted with aromatic sulfonyl chlorides, acids and halides. Electron donating and
withdrawing substituents are introduced on the aromatic ring in all 3 classes of sulfonamides,
amides and N-alkyl derivatives to study SAR.
Designed molecules were synthesized using the protocol shown in the Fig. 2. 2-(thiophen-3-
yl)acetic acid (01) was reduced with lithium aluminium hydride in THF to afford 2-(thiophen-3-
yl)ethanol (02) which was reacted with N-boc protected 4-piperidone in a solvent such as
dichloromethane in the presence of a suitable acid such as trifluroacetic acid gives 4',5'-
dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-ium (03) as trifluoroacetate salt which was
treated with various arylsulfonyl chlorides, aryl carbonyl chlorides and aryl alkyl chlorides in
presence of base such as triethyl amine in appropriate solvent such as DMF and dichlromethane
at room temperature to give corresponding crude sulfonamides, amides and alkyl derivatives,
which were triturated with methyl tert butyl ether to obtain as off-white to brown solids (04-24)
9
-11
(Table 1).
The purity of the synthesized compounds was checked by LC-MS and the
4

1
structures were identified by spectral data. In the nuclear magnetic resonance spectra ( H NMR),
the signals of the respective protons of the prepared spiro derivatives were verified on the basis
of their chemical shifts, multiplicities, and coupling constants.
Figure 1: Modifications in lead molecule GSK2200150A by shifting of sulfur atom
5

Figure 2: Synthetic strategy employed for synthesis of final compounds 04-24.
2
.2. Biological evaluation
All the synthesized compounds (04-24) showed activity against replicative MTB with MIC
ranging from 8.23 to 83.49 µM(Table 1). Five compounds (05, 06, 13, 16 and 22) showed MIC
values less than 10 µM and were more potent than standard first line antitubercular drug
Ethambutol.
Compound
1-((4-methoxyphenyl)sulfonyl)-4',5'-dihydrospiro[piperidine-4,7'-
thieno[2,3-c]pyran] (6) was found to be the most potent among the synthesized derivatives with
MIC of 8.23 µM and was less active than GSK lead.
6

Table 1: Structures and biological activity results of synthesized compounds
COMPD
X
R
MTB MIC Cytotoxicity
% inhib) at
(
(
µM)
50 µg/ml
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
2
2
2
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
SO
SO
SO
SO
SO
SO
SO
2
2
2
2
2
2
2
H
71.53
8.59
NT
4-CH
22.16
12.32
NT
3
4-OCH
4-F
8.23
3
34.01
65.11
16.21
29.87
79.76
19.08
9.09
4-Cl
NT
2,4-Di F
2,4-Di Cl
H
31.08
NT
CO
CO
CO
CO
CO
CO
CO
NT
4-CH
23.16
14.59
18.98
NT
3
4-OCH
4-F
3
18.85
71.86
8.94
4-Cl
2,4-Di F
2,4-Di Cl
H
34.06
NT
32.69
83.49
79.75
23.14
19.69
9.35
CH
CH
CH
CH
CH
NT
2
4-CH
NT
2
2
2
2
3
4-OCH
4-F
NT
3
21.67
26.54
4-Cl
2
2
3
4
CH
2
2
2,4-Di F
37.26
33.93
NT
NT
CH
2,4-Di Cl
7

INH
0.66
9.84
2.16
0.38
NT
NT
NT
NT
Ethambutol
Ofloxacin
GSK2200150A
NT indicates not tested
With regard to structure activity relationship, we have prepared total of twenty-one compounds;
seven each with sulfonyl, carbonyl, and methylene bridge (X) between spiro nucleus and aryl
ring. In general, sulfonyl and carbonyl bridge showed better activity than methylene bridge. In
general substituents in the aryl ring enhances activity. Among the sulfonyl bridged molecules
(04-10); aryl ring substituents (R) alter the activity considerably. Electron donating substituents
like methyl (05) and methoxy (06) groups showed very potent activity (MIC < 10 µM) than
electron withdrawing substituents like chloro and fluoro groups. When compared to mono chloro
th
or mono fluoro substituent at 4 position of aryl ring; di-substitution with same group at 2,4
position enhances activity two times. Similar kind of trend is observed carbonyl bridged
compounds (11-17) also; whereas in methylene bridged compounds mono fluoro (21) and mono
chloro (22) substituted compounds showed good activity.
Further we have screened selected compounds (each one from sulfonyl, carbonyl and methylene
bridged) against nutrient starved dormant TB model. Most antibiotics target biosynthetic
processes that bacteria need to increase their biomass. It is not surprising that such antibiotics are
more effective against replicating than non-replicating bacteria. However, a major need in global
health is to eradicate persistent or non-replicating subpopulations of bacteria such as MTB.
Worldwide, an estimated 1 person in 3 is infected with MTB; in about 9 of every 10 infected,
5
MTB persist in a largely non-replicating (“dormant”) state throughout the lifetime of the host.
8

MTB organisms subjected to stress conditions goes into dormancy phase. Three types of stress
were applied hypoxia, nutrient starvation and acidic environment. J.C. Betts et al. earlier
established an in vitro model, in which nutrient starvation caused M. tuberculosis to arrest
growth, minimized aerobic metabolism and became resistant to existing antitubercular drugs
1
3
while maintaining viability. This model shows the changes associated with dormancy such as
loss of Ziehl-Neelsen staining, low and continuous type of respiration, resistance to antibiotics.
Genes which are found to be up regulated are involved in antibiotic production and resistance,
insertion sequence elements, repeated sequences and phage, nucleotide biosynthesis and
metabolism, putative enzymes and regulatory function. Nutrient starvation may therefore mimic
some of the features of M. tuberculosis during the persistent state. In this model the MTB culture
is starved in PBS for 6 weeks then treated with inhibitor at a concentration of 10 µg/ml for 7
days.
7
6
5
4
3
2
1
0
l
I
r
H
F
I
t
06
16
22
X
N
I
R
C
MO
Compounds
Figure 3: Biological activities of the active compounds against M. tuberculosis in the nutrient
starvation model. Bacterial count estimation (Mean ± S.D., n = 3) for control and treated groups
9

conducted by using the MPN (most probable number) assay. All compounds gave significant
inhibition of growth of M. tuberculosis in this model as compared to the control (p < 0.0001, two
way ANOVA using GraphPad Prism Software).
In nutrient starvation model compound 06 and 16 show 2.8 and 2.7 log fold reduction in bacterial
count equipotent to standard drug Moxifloxacin and more potent than first line drugs Isoniazid
(1.7 fold) and Rifampicin (2.0 fold). Compound 22 shows 2.4 log reduction which is potent than
standard drugs Isoniazid and Rifampicin (Fig 3). These results suggest that Compound 06 is not
only able to combat active stages but also dormant forms of MTB which is helpful in lowering
the duration of therapy.
To get insight into mechanism of action of inhibitors the most active compounds in nutrient
starvation were evaluated for inhibitory potential against lysine Ɛ-amino transferase enzyme. The
reason behind selection of this enzyme is it is overexpressed 40 times in nutrient starvation
model and has pivotal role in persistence and antibiotic tolerance. LAT is a PLP dependent type
II aminotransferase enzyme which catalyzes reversible transamination from lysine to α-
ketoglutaric acid resulting in piperidine-6-carboxylic acid and glutamate. The most active
compound in nutrient starvation compound 06 shows correlating IC50 of 1.04 ± 0.32 µM.
Compound 16 and 22 exhibits IC values of 3.48 ± 0.74 µM and 6.83 ± 0.54µM.
5
0
Docking studies of these compounds in lysine and ketoglutarate active sites of LAT (PDB code:
CJHand 2CJD) revealed that compounds06, 16and 22 are targeting lysine binding site (Fig 4).
2
Compound 06 has retained crucial interactions with active site by four hydrogen bonds.
Dihydropyran is involved in hydrogen bonding with Lys 300, the phenyl group mediates
hydrogen bond with Phe 167, sulfonamide is involved in interaction with Ala 129 and methoxy
1
0

group participates in interaction with Asp 271 in compound 06. Amide derivative 16 with
difluoro substitution has only one interaction with Phe 167 but fits into active site. In case of N-
alkyl derivative 22 there are 2 hydrogen interactions in active site. The dihydropyran ring
participates in hydrogen bonding with Lys 300 and phenyl ring is involved in interaction with
Phe 167.
1
1

Figure 4: Interaction profile of Compounds 06, 16 and 22 with lysine binding site of LAT
The safety profile of all the synthesized compounds was evaluated by testing there in vitro
cytotoxicity in mouse macrophage cell line (RAW 264.7) cells at 50 µg/mL concentration using
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Since MTB reside
inside the macrophage, the monocyte macrophage cell lines (RAW 264.7) were mostly used for
tuberculosis research; in order to check whether the screened compounds were not toxic towards
macrophages but toxic to the bacteria. Compound 06 showed 12.32 % inhibition which suggests
that compound does not have cytotoxicity at highest concentration tested.
3
. Conclusion
As there is growing need for new antimycobacterial agents we attempted to derivatizespiro lead
reported in the literature by shifting of sulfur atom position in the spiro nucleus. Among series of
twenty-one
compounds,
compound
06,
1-((4-methoxyphenyl)sulfonyl)-4',5'-
dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran] was found to be effective than standard drug
ethambutol in terms of MIC. It was also found to be more efficacious than Isoniazid and
Rifampicin and equipotent as Moxifloxacin against dormant stages of Mtb. Compound 06also
shows good inhibitory potential against latent target LAT with an IC of 1.04 ± 0.32 µM. As it
5
0
has minimal cytotoxicity at higher concentration it can be taken for further studies for
development of new drugs in area of TB.
4
4
4
. Experimental Section
.1. Chemistry
.1.1. General
1
2

Reagents were purchased from commercial sources and were used as received. Proton nuclear
magnetic resonance spectra were obtained on a Bruker AVANCE 400 spectrometer at 400 MHz
with tetramethylsilane used as an internal reference. Carbon nuclear magnetic resonance spectra
were obtained on a Bruker AVANCE 300 spectrometer at 100 MHz with the solvent peak used
as the reference. Thin-layer chromatography (TLC) was performed using Whatman No.4500-101
(Diamond No MK6F silica-gel 60 A˚) plates. Visualization of TLC plates was performed using
UV light (254 nm).
4
.1.2. 2-(Thiophen-3-yl)ethanol (02): To a 0 °C cooled stirred suspension of lithium aluminium
hydride (8.0 g, 0.21 mol) in THF (100 mL) was added 3-Thiopheneacetic acid 01(20 g, 0.14
mol) in THF (100 mL) drop wise over 1 h. Reaction mixture was warmed to room temperature
and stirred for 6 h. Reaction progress was checked by TLC for completion and then quenched
with 10% NaOH solution. The mixture was filtered over celite, washed with ethylacetate.
Aqueous layer was extracted with ethylacetate and the combined organic layer was washed with
1
brine. Dried over sodium sulfate and concentrated to yield 2 as a yellow liquid (14.2 g, 78%) H
NMR (MeOD, 400 MHz): δ 5.92- 5.87 (m, 1H), 5.69- 5.66 (m, 1H), 5.57 (s, 1H) 2.90 (d, J =
1
3
1
1
1
4
4.0 Hz, 1H), 2.38 (d, J = 14 Hz, 1H), 1.55- 1.42 (m, 2H). C NMR (DMSO-d 100 MHz):
6,
45.53, 129.13, 126.54, 121.45, 64.46, 38.85; ESI-MS m/z: (Calcd for C H OS: 128.03); Found:
6
8
+
28.9 [(M +H] .
.1.3. 4',5'-Dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-ium trifluoroacetate (03): To
a solution of 02 (50 g, 0.39 mol) in dichloromethane (500 mL) was added N-tert-butoxycarbonyl-
-piperidone (85.4 g, 0.43 mol) and stirred for 10 min. at room temperature. Reaction mixture
4
was cooled to 0-5 °C then trifluoroacetic acid (133 g, 1.17 mol) was added drop wise over 30
min. Reaction mixture was warmed to room temperature and stirred for 16 h. Reaction progress
1
3

was monitored by TLC. When TLC showed complete consumption of starting material, reaction
mixture was concentrated under reduced pressure to yield crude compound and it was triturated
with methyl tert-butyl ether, filtered and washed to obtain 4',5'-dihydrospiro[piperidine-4,7'-
1
thieno[2,3-c]pyran]-1-ium trifluoroacetate03 as beige solid (115 g, 91%). H NMR (CDCl 400
3
,
MHz): δ 9.73 (br s, 1H), 9.24 (br s, 1H), 7.20 (d, J = 4.8 Hz 1H), 6.79 (d, J = 5.2 Hz, 1H), 3.90 –
1
3
3
.93 (m, 2H), 3.26 – 3.36 (m, 4H), 2.70 – 2.73 (m, 2H), 2.12 – 2.27 (m, 4H); C NMR (DMSO-
d 100 MHz): 203.03, 158.89, 139.53, 133.54, 127.13, 123.54, 70.54, 58.98, 42.21, 38.85, 37.30,
6
,
3
4.20, 25.76; ESI-MS m/z: (Calcd for C H F NO S: 323.08); Found: 210.1 [(M –CF COOH)
13 16 3 3 3
+
+
H] .
4
.1.4. General procedure for the synthesis of sulfonamides (04-10): To a solution of 4',5'-
dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-ium trifluoroacetate (03) (200 mg, 0.62
mmol) in DMF (5 mL) was added triethylamine (187 mg, 1.86 mmol) followed by benzene
sulfonylchloride (0.685 mmol) at ambient temperature. The reaction mixture was stirred for 5 h
at room temperature. When TLC showed complete consumption of starting material, the reaction
mixture was poured on ice-cold water under stirring and then filtered, washed with water, dried
under vacuum at 60 °C for 2 h. The crude product was triturated with tert-butylmethyl ether to
obtain pure corresponding substituted 1-(phenylsulfonyl)-4',5'-dihydrospiro[piperidine-4,7'-
thieno[2,3-c]pyran] (04-10) in good yield.
4
.1.4.1. 1-(Phenylsulfonyl)-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran] (04): The
compound was synthesized according to the general procedure using 4',5'-
dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-ium trifluoroacetate (03) (200 mg, 0.62
mmol) and benzenesulfonyl chloride (120 mg, 0.685 mmol) to obtain 04 as off white solid (190
1
mg, 88%). H NMR (CDCl 400 MHz): δ 7.81 – 7.78 (m, 2 H), 7.64 – 7.52 (m, 3H), 7.14 (d, J =
3,
1
4

4
.8 Hz, 1H), 6.74 (d, J = 5.1 Hz, 1H), 3.78 (t, J = 5.4 Hz, 2H), 3.72 – 3.68 (m, 2H), 2.75 – 2.52
1
3
(
m, 4H), 2.07 – 1.95 (m, 4H) ppm; C NMR (DMSO-d 100 MHz): 141.62, 138.36, 131.54,
6,
1
3
27.13, 125.61, 124.13, 123.64, 122.25, 121.85, 120.39, 69.54, 55.98, 52.23, 43.21, 41.10, 35.85,
+
0.20, 23.74; ESI-MS m/z: (Calcd for C H NO S : 349.08); Found: 349.8 [M + H] .
1
7
19
3 2
4
.1.4.2. 1-Tosyl-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran] (05): The compound
was synthesized according to the general procedure using 4',5'-dihydrospiro[piperidine-4,7'-
thieno[2,3-c]pyran]-1-ium trifluoroacetate (03) (200 mg, 0.62 mmol) and 4-methylbenzene-1-
1
sulfonyl chloride (130 mg, 0.685 mmol) to obtain 05 as light yellow solid (123 mg, 55%). H
NMR (CDCl 400 MHz): δ 7.67 (d, J = 7.8 Hz, 2H), 7.34 (d, J = 7.8 Hz, 2H), 7.13 (d, J = 4.5
3
,
Hz, 1H), 6.73 (d, J = 4.5 Hz, 1H), 3.78 (t, J = 4.8 Hz, 2H), 3.69 – 3.66 (m, 2H), 2.78 – 2.61 (m,
1
3
4
1
2
H), 2.45 (s, 3H), 2.12 – 1.95 (m, 4H) ppm; C NMR (DMSO-d 100 MHz): 141.62, 140.23,
6,
39.53, 138.36, 131.54, 127.13, 122.25, 114.61, 70.54, 55.98, 42.21, 41.10, 36.85, 31.20, 28.54,
+
4.74; ESI-MS m/z: (Calcd for C H NO S : 363.1); Found: 363.9 [M + H] ; HRMS m/z [M +
1
8
21
3 2
+
H] Calcd for C H O NS : 364.1035, found: 364.1036.
1
8
22
3
2
4
.1.4.3.
1-((4-Methoxyphenyl)sulfonyl)-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-
c]pyran] (06): The compound was synthesized according to the general procedure using 4',5'-
dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-ium trifluoroacetate (03) (200 mg, 0.62
mmol) and 4-methoxybenzene-1-sulfonyl chloride (141 mg, 0.685 mmol) to obtain 06 as a light
1
yellow solid (168 mg, 72%). H NMR (DMSO-d 400 MHz): δ 7.70 (d, J = 8.4 Hz, 2H), 7.38 (d,
6,
J = 5.1 Hz, 1H), 7.17 (d, J = 8.4 Hz, 2H), 6.81 (d, J = 4.8 Hz, 1H), 3.87 (s, 3H), 3.71 (t, J = 4.8
Hz, 2H), 3.54 – 3.50 (m, 2H), 2.57 – 2.56 (m, 4H), 2.02 – 1.97 (m, 2H), 1.84 – 1.74 (m, 2H)
13
ppm; C NMR (DMSO-d 100 MHz): 160.88, 141.62, 138.36, 131.54, 127.13, 122.25, 121.85,
6
,
1
14.61, 69.54, 56.98, 53.23, 44.21, 41.10, 36.85, 31.20, 23.74; ESI-MS m/z: (Calcd for
1
5

+
+
C H NO S : 379.09); Found: 379.9 [M + H] ; HRMS m/z [M + H] Calcd for C H O NS :
18
21
4
2
18 22
4
2
3
80.0984, found: 380.0987.
4
.1.4.4. 1-((4-Fluorophenyl)sulfonyl)-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]
(07): The compound was synthesized according to the general procedure using 4',5'-
dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-ium trifluoroacetate (03) (200 mg, 0.62
mmol) and 4-fluorobenzene-1-sulfonyl chloride (133 mg, 0.685 mmol) to obtain 07 as a light
1
brown solid (94 mg, 41%). H NMR (DMSO-d 400 MHz): δ 7.88 – 7.84 (dd, J = 8.4 Hz, 5.4
6
,
Hz, 2H), 7.52 (t, J = 9.0 Hz, 2H), 7.39 (d, J = 5.1 Hz, 1H), 6.81 (d, J = 5.1 Hz, 1H), 3.74 (t, J =
.1 Hz, 2H), 3.58 – 3.54 (m, 2H), 2.58 – 2.54 (m, 4H), 2.08 – 1.98 (m, 2H), 1.84 – 1.75 (m, 2H)
5
13
ppm; C NMR (DMSO-d 100 MHz): 159.89, 141.65, 139.53, 138.36, 131.54, 127.13, 122.25,
6
,
1
3
14.61, 69.54, 57.98, 42.21, 40.10, 38.85, 34.20, 24.76; ESI-MS m/z: (Calcd for C H FNO S :
17 18 3 2
+
67.07); Found: 367.8 [M + H] .
4
.1.4.5. 1-((4-Chlorophenyl)sulfonyl)-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]
(08): The compound was synthesized according to the general procedure using 4',5'-
dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-ium trifluoroacetate (03) (200 mg, 0.62
mmol) and 4-chlorobenzene-1-sulfonyl chloride (144 mg, 0.685 mmol) to obtain 08 as light
1
yellow solid (110 mg, 47%). H NMR (CDCl 400 MHz): δ 7.41 – 7.22 (m, 4H), 7.16 (d, J = 5.1
3
,
Hz, 1 H), 6.76 (d, J = 4.8 Hz, 1H), 3.86 (t, J = 5.4 Hz, 2H), 3.76 – 3.72 (m, 2H), 3.19 – 3.12 (m,
1
3
2
1
2
H), 2.68 (t, J = 5.4 Hz, 2H), 2.07 – 1.95 (m, 4H) ppm; C NMR (DMSO-d 100 MHz): 152.89,
6,
41.65, 139.53, 138.36, 135.23, 131.54, 127.13, 122.25, 72.52, 56.96, 41.21, 40.25, 38.85, 34.20,
+
4.76; ESI-MS m/z: (Calcd for C H ClNO S : 383.04); Found: 383.9 [M+H] .
1
7
18
3 2
1
6

4
.1.4.6.
1-((2,4-Difluorophenyl)sulfonyl)-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-
c]pyran] (09): The compound was synthesized according to the general procedure using 4',5'-
dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-ium trifluoroacetate (03) (200 mg, 0.62
mmol) and 2,4-difluorobenzene-1-sulfonyl chloride (145 mg, 0.685 mmol) to obtain 09as
1
offwhite solid (69 mg, 29%). H NMR (CDCl 400 MHz): δ 7.95 – 7.84 (m, 1H), 7.16 (d, J =
3
,
5
.1 Hz, 1 H), 7.04 – 6.95 (m, 2H), 6.76 (d, J = 4.8 Hz, 1H), 3.84 (t, J = 5.4 Hz, 2H), 3.77 – 3.73
13
(
m, 2H), 2.98 – 2.91 (m, 2H), 2.67 (t, J = 5.4 Hz, 2H), 2.09 – 1.94 (m, 4H) ppm; C NMR
DMSO-d 100 MHz): 160.88, 155.65, 141.62, 139.53, 138.36, 131.54, 127.13, 122.25, 121.85,
(
6
,
1
3
14.61, 69.54, 56.98, 43.21, 41.10, 37.85, 31.20, 24.74; ESI-MS m/z: (Calcd for C H F NO S :
17 17 2 3 2
+
85.06); Found: 385.8[M+H] .
4
.1.4.7.
1-((2,4-Dichlorophenyl)sulfonyl)-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-
c]pyran] (10): The compound was synthesized according to the general procedure using 4',5'-
dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-ium trifluoroacetate(03) (200 mg, 0.62 mmol)
and 2,4-dichlorobenzene sulfonylchloride (0.685 mmol) to obtain 10as off white solid (167 mg,
1
6
5
2
7%). H NMR (CDCl 400 MHz): δ 7.92 (s, 1H), 7.78 (d, J = 4.8 Hz, 1 H), 7.54 – 7.48 (dd, J =
3,
.1 Hz, J = 3.2 Hz, 1 H), 7.14 (d, J = 5.1 Hz, 1 H), 6.71 (d, J = 4.8 Hz, 1H), 3.82 (t, J = 5.4 Hz,
H), 3.71 – 3.68 (m, 2H), 3.15 – 3.10 (m, 2H), 2.64 (t, J = 5.4 Hz, 2H), 2.01 – 1.92 (m, 4H)
13
ppm; C NMR (DMSO-d 100 MHz): 158.89, 142.65, 139.53, 138.36, 135.23, 133.54, 131.54,
6
,
1
29.11, 127.13, 122.25, 70.54, 58.98, 42.21, 40.10, 38.85, 34.20, 25.76; ESI-MS m/z: (Calcd for
+
C H Cl NO S : 417.00); Found: 417.9 [M+H] .
17
17
2
3 2
4
.1.5. General procedure for the synthesis of amide derivatives (11-17): To a solution of
',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-ium trifluoroacetate (03) (200 mg, 0.62
4
mmol) in DMF (5 mL) was added triethylamine (187 mg, 1.86 mmol) followed by various
1
7

benzoyl chlorides at ambient temperature. The reaction mixture was stirred for 5 h at room
temperature. When TLC showed complete consumption of starting material, the reaction mixture
was poured on ice-cold water under stirring and then filtered, washed with water, dried under
vacuum at 60 °C for 1 h. The crude product was triturated with tert-butylmethyl ether to obtain
corresponding
substituted
(4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-
yl)(phenyl)methanone (11-17) in good yield.
4
.1.5.1.
(4',5'-Dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-yl)(phenyl)methanone
(11): The compound was synthesized according to the general procedure using 4',5'-
dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-ium trifluoroacetate (03) (200 mg, 0.62
mmol) and benzoyl chloride (96 mg, 0.685 mmol) to obtain 11as off white solid (146 mg, 76%).
1
H NMR (CDCl 400 MHz): δ 8.17 – 8.09 (m, 2H), 7.48 – 7.17 (m, 3 H), 7.16 (d, J = 5.1 Hz,
3
,
1
2
1
4
H), 6.77 (d, J = 5.1 Hz, 1H), 3.95 – 3.94 (m, 2H), 3.62 – 3.18 (m, 4 H), 2.72 (t, J = 5.2 Hz,
13
H), 2.16 – 1.79 (m, 4H) ppm; C NMR (DMSO-d 100 MHz): 168.36, 142.62, 140.36,
6
,
31.65, 128.25, 125.61, 124.23, 123.64, 122.25, 120.85, 120.39, 69.54, 55.98, 52.23, 43.21,
+
1.10, 35.85, 30.20, 23;ESI-MS m/z: (Calcd for C H NO S: 313.11); Found: 313.9 [M + H] .
1
8
19
2
4
.1.5.2.
(4',5'-Dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-yl)(p-tolyl)methanone
(12): The compound was synthesized according to the general procedure using 4',5'-
dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-ium trifluoroacetate (03) (200 mg, 0.62
mmol) and 4-methylbenzoyl chloride (105 mg, 0.685 mmol) to obtain 12 as light yellow solid
1
(
162 mg, 80%). H NMR (CDCl 400 MHz): δ 7.35 (d, J = 8.1 Hz, 2H), 7.21 (d, J = 8.1 Hz,
3,
1
2
1
H), 7.15 (d, J = 5.1 Hz, 2H), 6.77 (d, J = 5.1 Hz, 1H), 3.96 – 3.92 (m, 2H), 3.67 – 3.30 (m, 2H),
13
.73 – 2.69 (m, 2H), 2.37 (s, 3H), 2.16 – 1.66 (m, 6H) ppm; C NMR (DMSO-d 100 MHz):
6
,
69.64, 141.62, 138.36, 135.62, 131.54, 127.13, 124.13, 123.64, 122.25, 69.54, 55.98, 42.21,
1
8

4
1.10, 35.85, 30.20, 25.74, 21.63; ESI-MS m/z: (Calcd for C H NO S: 327.13); Found: 327.9
19 21 2
+
[
M + H] .
4
.1.5.3.(4',5'-Dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-yl)(4-
methoxyphenyl)methanone (13): The compound was synthesized according to the general
procedure using 4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-ium trifluoroacetate (03)
(200 mg, 0.62 mmol) and 4-methoxybenzoyl chloride (116 mg, 0.685 mmol) to obtain 13as off
1
white solid (174 mg, 82%). H NMR (CDCl 400 MHz): δ 7.72 (d, J = 8.5 Hz, 2H), 7.14 (d, J =
3
,
5
2
1
5
.1 Hz, 1H), 6.99 (d, J = 8.4 Hz, 2H), 6.74 (d, J = 5.1 Hz, 1H), 3.89 (s, 3H), 3.81 – 3.77 (m,
1
3
H), 3.77 – 3.68 (m, 2H), 2.71 – 2.53 (m, 4H), 2.04 – 2.00 (m, 4H) ppm; C NMR (DMSO-d
6
,
00 MHz): 171.56, 160.88, 140.62, 137.36, 131.54, 128.13, 123.25, 121.85, 115.61, 69.54,
6.98, 53.42, 45.21, 41.10, 36.85, 31.20, 23.74; ESI-MS m/z: (Calcd for C H NO S: 343.12);
19
21
3
+
+
Found: 343.9 [M + H] ; HRMS m/z [M + H] Calcd for C H O NS: 344.1314, found:
19
22
3
3
44.1308.
4
.1.5.4.
(4',5'-Dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-yl)(4-
fluorophenyl)methanone (14): The compound was synthesized according to the general
procedure using 4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-ium trifluoroacetate (03)
(200 mg, 0.62 mmol) and 4-fluorobenzoyl chloride (158 mg, 0.685 mmol) to obtain 14 as off
1
white solid (115 mg, 56%). H NMR (CDCl 400 MHz): δ 7.85 (dd, J = 4.8 Hz, 1.2 Hz, 2H),
3
,
7
5
.35 (dd, J = 4.4 Hz, 1.2 Hz, 2H), 7.15 (d, J = 5.1 Hz, 1H), 6.74 (d, J = 5.1 Hz, 1H), 3.88 (t, J =
13
.4 Hz , 2H), 2.71 – 2.65 (m, 4H), 2.46 – 2.38 (m, 2H), 2.06 – 2.01 (m, 4H) ppm; ; C NMR
(
DMSO-d 100 MHz): 169.54, 157.89, 141.65, 138.53, 138.36, 131.54, 126.13, 122.25, 114.61,
6,
6
9.54, 57.98, 42.21, 40.10, 38.85, 34.20, 24.76; ESI-MS m/z: (Calcd for C H FNO S: 331.10);
18 18 2
+
Found: 331.9 [M + H] .
1
9

4
.1.5.5.
yl)methanone(15): The compound was synthesized according to the general procedure using
',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-ium trifluoroacetate (03) (200 mg, 0.62
(4-Chlorophenyl)(4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-
4
mmol) and 4-chlorobenzoyl chloride (120 mg, 0.685 mmol) to obtain 15 as light yellow solid
1
(
174 mg, 81%). H NMR (CDCl 400 MHz): δ 7.32 – 7.30 (m, 4H), 7.13 (d, J = 5.1 Hz, 1H),
3,
6
2
1
.75 (d, J = 5.1 Hz, 1H), 3.91 (t, J = 5.4 Hz , 2H), 2.73 – 2.67 (m, 4H), 2.49 – 2.40 (m, 2H),
1
3
.01 – 1.93 (m, 4H) ppm; C NMR (DMSO-d 100 MHz): 168.63, 155.89, 142.65, 139.53,
6
,
37.36, 135.23, 131.54, 127.13, 122.25, 72.52, 56.96, 41.21, 40.25, 38.85, 34.20, 24.76; ESI-
+
MS m/z: (Calcd for C H ClNO S: 347.07); Found: 347.9 [M + H] .
18
18
2
4
.1.5.6.
yl)methanone (16): The compound was synthesized according to the general procedure using
',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-ium trifluoroacetate (03) (200 mg, 0.62
(2,4-Difluorophenyl)(4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-
4
mmol) and 2,4-difluorobenzoyl chloride (120 mg, 0.685 mmol) to obtain 16 as a brown solid (85
1
mg, 39%). H NMR (DMSO-d 400 MHz): δ 7.56 (bs, 1H), 7.42 – 7.35 (m, 2H), 7.21 – 7.17 (td,
6
,
J = 2.0 Hz, 8.4 Hz, 1H), 6.85 (d, J = 4.2 Hz, 1H), 4.43 (d, J = 13.2 Hz, 1H), 3.94 – 3.88 (m,
2
1
1
H), 3.43 – 3.26 (m, 2H), 3.10 – 3.04 (m, 1H), 2.89 – 2.87 (m, 1H), 2.66 – 2.63 (m, 2H), 2.09 –
1
3
.71 (m, 3H) ppm; C NMR (DMSO-d 100 MHz): 163.12, 140.37, 133.41, 133.20, 130.39,
6
,
27.08, 123.40, 123.26, 120.79, 112.30, 104.34, 72.33, 58.75, 42.69, 38.01, 37.38, 37.23, 25.91;
+
ESI-MS m/z: (Calcd for C H F NO S: 383.04); Found: 349.9 [M + H] .
18
17
2
2
4
.1.5.7.
yl)methanone (17): The compound was synthesized according to the general procedure using
',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-ium trifluoroacetate (03) (200 mg, 0.62
mmol) and 2,4-dichlorobenzoyl chloride (143 mg, 0.685 mmol) to obtain 17 as off white solid
(2,4-Dichlorophenyl)(4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-
4
2
0

1
(
180 mg, 80%) H NMR (CDCl 400 MHz): δ 7.53 (d, J = 2.1 Hz, 1H), 7.35 – 7.24 (m, 2 H),
3,
7
2
1
4
.20 (d, J = 8.1 Hz, 1H), 6.64 (d, J = 5.1 Hz, 1H), ), 3.80 – 3.76 (m, 2H), 3.74 – 3.63 (m, 2H),
1
3
.68 – 2.49 (m, 4H), 2.09 – 2.02 (m, 4H) ppm; C NMR (DMSO-d 100 MHz): 170.24, 158.89,
6
,
42.65, 139.53, 138.36, 135.23, 133.54, 131.54, 129.11, 127.13, 122.25, 70.54, 58.98, 42.21,
0.10, 38.85, 34.20, 25.76; ESI-MS m/z: (Calcd for C H Cl NO S: 381.04); Found: 381.8 [M +
1
8
17
2
2
+
H] .
4
.1.6. General procedure for the synthesis of N-alkylated derivatives (18-24): To a solution
of 4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-ium trifluoroacetate (03) (200 mg,
.62 mmol) in DMF (5 mL) was added triethylamine (187 mg, 1.86 mmol) followed by various
0
benzyl chlorides at ambient temperature. The reaction mixture was stirred for 16 h at room
temperature. When TLC showed complete consumption of starting material, the reaction mixture
was poured on ice-cold water and was extracted with dichloromethane for 2 times (2 × 15 mL).
The combined organic layers were washed with water (10 mL) and brine (10 mL). The organic
layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The
crude product was purified by column chromatography over 100-200 mesh silica-gel by using 15
%
ethylacetate-n-hexane as eluant to obtain corresponding substituted 1-benzyl-4',5'-
dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran] (18-24) in good yield.
4
.1.6.1. 1-Benzyl-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran] (18): The compound
was synthesized according to the general procedure using 4',5'-dihydrospiro[piperidine-4,7'-
thieno[2,3-c]pyran]-1-ium trifluoroacetate (03) (200 mg, 0.62 mmol) and benzyl chloride (86
mg, 0.685 mmol) to afford the crude product and it was purified by column chromatography over
1
00-200 mesh silica-gel by using 15 % ethylacetate-n-hexane as eluant to obtain 18as off white
1
solid (142 mg, 76%). H NMR (CDCl 400 MHz): δ 8.17 – 8.09 (m, 2H), 7.48 – 7.17 (m, 3 H),
3
,
2
1

7
.16 (d, J = 5.1 Hz, 1H), 6.77 (d, J = 5.1 Hz, 1H), 3.95 – 3.94 (m, 2H), 3.60 (s, 2H), 3.62 – 3.18
1
3
(
m, 4 H), 2.72 (t, J = 5.2 Hz, 2H), 2.16 – 1.79 (m, 4H) ppm; C NMR (DMSO-d 100 MHz):
6,
1
5
2
41.62, 138.36, 131.54, 127.13, 125.61, 124.13, 123.64, 122.25, 121.85, 120.39, 69.54, 55.98,
2.23, 43.21, 41.10, 35.85, 30.20, 23.74.ESI-MS m/z: (Calcd for C H NOS: 299.13); Found:
1
8
21
99.9 [M + H]
4
.1.6.2. 1-(4-Methylbenzyl)-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran] (19): The
compound was synthesized according to the general procedure using 4',5'-
dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-ium trifluoroacetate (03) (200 mg, 0.62
mmol) and 1-(chloromethyl)-4-methylbenzene (96 mg, 0.685 mmol) to obtain the crude product
and it was purified by column chromatography over 100-200 mesh silica-gel by using 12 %
1
ethylacetate-n-hexane as eluant to obtain 19as off white solid (171 mg, 88%). H NMR (CDCl
3
,
4
00 MHz): δ 7.12 (d, J = 5.1 Hz, 1H), 7.08 (d, J = 4.6 Hz, 2H), 7. 01 (d, J = 4.6 Hz, 1H), 6.74
(d, J = 4.8 Hz, 1H), 3.92 (t, J = 5.7 Hz, 2H), 3.64 (s, 2H), 2.80 – 2.68 (m, 4H), 2.32 (s, 3H), 2.63
1
3
–
1
2
2.44 (m, 2H), 2.00 – 1.97 (m, 4H) ppm; C NMR (DMSO-d 100 MHz): 142.62, 139.22,
6,
38.53, 137.36, 131.54, 127.13, 125.25, 121.61, 69.54, 64.46, 54.84, 42.21, 40.10, 35.85, 32.20,
+
7.54, 24.74; ESI-MS m/z: (Calcd for C H NOS: 313.15); Found: 313.9 [M + H] .
1
9
23
4
.1.6.3. 1-(4-Methoxybenzyl)-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran] (20):
The compound was synthesized according to the general procedure using 4',5'-
dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-ium trifluoroacetate (03) (200 mg, 0.62
mmol) and 1-(chloromethyl)-4-methoxybenzene (106 mg, 0.895 mmol) to afford the crude
product and it was purified by column chromatography over 100-200 mesh silica-gel by using 18
1
%
ethylacetate-n-hexane as eluant to obtain 20as off white solid (169 mg, 84%). H NMR
(
CDCl 400 MHz): δ 7.25 (d, J = 6.8 Hz, 2H), 7.13 (d, J = 4.8 Hz, 1H),7.00 (d, J = 7.1 Hz , 2H),
3,
2
2

6
4
1
3
.72 (d, J = 4.8 Hz, 1H), 3.91 (t, , J = 6.9 Hz, 2H), 3.83 (s, 3H), 3.54 (s, 2H), 2.71 – 2.65 (m,
13
H), 2.42 – 2.39 (m, 2H), 2.01 – 1.95 (m, 4H) ppm; C NMR (DMSO-d 100 MHz): 158.67,
6
,
42.62, 137.36, 130.53, 127.13, 123.25, 122.85, 114.61, 69.54, 64.23, 56.98, 53.23, 44.21, 41.10,
+
6.85, 31.20, 23.74; ESI-MS m/z: (Calcd for C H NO S: 329.14); Found: 329.9 [M + H] .
19
23
2
4
.1.6.4. 1-(4-Fluorobenzyl)-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran] (21): The
compound was synthesized according to the general procedure using 4',5'-
dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-ium trifluoroacetate (03) (200 mg, 0.62
mmol) and 1-(chloromethyl)-4-fluorobenzene (99 mg, 0.685 mmol) to afford the crude product
and it was purified by column chromatography over 100-200 mesh silica-gel by using 10 %
1
ethylacetate-n-hexane as eluant to obtain 21 as a light yellow solid (124 mg, 63%). H NMR
(
CDCl 400 MHz): δ 7.35 – 7.30 (dd, J = 8.4 Hz, J = 5.7 Hz, 2H), 7.12 (d, J = 4.8 Hz, 1H),7.00
3,
(t, J = 8.7 Hz , 2H), 6.75 (d, J = 4.8 Hz, 1H), 3.91 (t, , J = 6.9 Hz, 2H), 3.54 (s, 2H), 2.73 – 2.67
13
(
m, 4H), 2.47 – 2.39 (m, 2H), 2.01 – 1.97 (m, 4H) ppm; C NMR (DMSO-d 100 MHz):
6,
1
4
58.71, 142.55, 138.43, 137.36, 132.54, 126.13, 122.25, 114.61, 69.54, 63.54, 56.61, 42.21,
0.10, 36.85, 34.20, 23.76; ESI-MS m/z: (Calcd for C H FNOS: 317.12); Found: 317.9 [M +
1
8
20
+
H] .
4
.1.6.5. 1-(4-Chlorobenzyl)-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran] (22): The
compound was synthesized according to the general procedure using 4',5'-
dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-ium trifluoroacetate (03) (200 mg, 0.62
mmol) and 1-chloro-4-(chloromethyl)benzene (110 mg, 0.685 mmol) to afford the crude product
and it was purified by column chromatography over 100-200 mesh silica-gel by using 10 %
1
ethylacetate-n-hexane as eluant to obtain 22as off white solid (159 mg, 77%). H NMR (CDCl
3
,
4
00 MHz): δ 7.38 – 7.5 (m, 5H), 6.81 (d, J = 5.2 Hz, 1H), 3.84 (t, J = 5.6 Hz, 2H), 3.60 (bs,
2
3

2
H), 2.60 (t, J = 5.6 Hz, 4H), 2.35 – 2.32 (m, 2H), 1.95 – 1.92 (m, 2H), 1.81 – 1.78 (m, 2H)
13
ppm; C NMR (DMSO-d 100 MHz): 148.89, 142.65, 139.22, 137.35, 134.23, 131.26, 126.13,
6
,
1
22.25, 72.52, 65.21, 55.96, 42.21, 40.25, 37.85, 34.20, 23.76; ESI-MS m/z: (Calcd for
+
+
C H ClNOS: 333.10); Found: 333.9 [M + H] ; HRMS m/z [M + H] Calcd for C H ONClS:
18
20
18 21
3
34.1026, found: 334.1021.
4
.1.6.6. 1-(2,4-Difluorobenzyl)-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran] (23):
The compound was synthesized according to the general procedure using 4',5'-
dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-ium trifluoroacetate (03) (200 mg, 0.62
mmol) and 1-(chloromethyl)-2,4-difluorobenzene (110 mg, 0.685 mmol) to afford the crude
product was purified by column chromatography over 100-200 mesh silica-gel by using 15 %
1
ethylacetate-n-hexane as eluant to obtain 23 as light yellow solid (126 mg, 60%). H NMR
(
CDCl 400 MHz): δ 7.43 – 7.35 (dd, J = 15.3 Hz, J = 6.9 Hz, 1H), 7.12 (d, J = 4.8 Hz, 1H),
3,
6
.88 – 6.76 (m, 2H), 6.75 (d, J = 2.1 Hz, 1H), 3.90 (t, J = 6.9 Hz, 2H), 3.60 (s, 2H), 2.79 – 2.67
13
(
m, 4H), 2.53 – 2.44 (m, 2H), 2.04 – 1.93 (m, 4H) ppm; C NMR (DMSO-d 100 MHz):
6,
1
5
3
58.88, 153.65, 140.62, 139.53, 138.36, 130.54, 128.13, 123.25, 121.85, 114.61, 72.54, 65.21,
9.62, 42.21, 40.10, 36.83, 30.20, 23.74; ESI-MS m/z: (Calcd for C H F NOS: 335.12); Found:
1
8
19 2
+
35.9 [M + H] .
4
.1.6.7. 1-(2,4-Dichlorobenzyl)-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran] (24):
The compound was synthesized according to the general procedure using 4',5'-
dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-ium trifluoroacetate (03) (200 mg, 0.62
mmol) and 2,4-dichloro-1-(chloromethyl)benzene (133 mg, 0.685 mmol) to afford the crude
product and it was purified by column chromatography over 100-200 mesh silica-gel by using
1
1
2 % ethyl acetate-n-hexane as eluant to obtain 24 as a brown solid (145 mg, 64%). H NMR
2
4

(
CDCl 400 MHz): δ 7.50 (d, J = 8.4 Hz, 1H), 7.37 (d, J = 2.1 Hz, 1H), 7.26 – 7.22 (dd, J = 5.4
3,
Hz, J = 3.6 Hz 1H), 7.13 (d, J = 5.1 Hz, 1H), 6.76 (d, J = 4.8 Hz, 1H), 3.92 (t, , J = 5.7 Hz, 2H),
13
3
.64 (s, 2H), 2.80 – 2.68 (m, 4H), 2.63 – 2.44 (m, 2H), 2.00 – 1.97 (m, 4H) ppm; C NMR
(
DMSO-d 100 MHz): 154.89, 141.65, 138.45, 137.12, 135.23, 132.44, 131.54, 128.11, 127.13,
6
,
1
21.25, 70.54, 64.24, 58.98, 42.21, 40.10, 38.85, 34.20, 25.76; ESI-MS m/z: (Calcd for
+
C H Cl NOS: 367.06); Found: 367.9 [M + H] .
18
19
2
4
.2. In vitro active MTB model
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 incubation. The plate was covered, sealed in plastic bags and incubated at
o
3
7 C in normal atmosphere. After 7 days incubation, 50 µl of alamar blue solution was added to
each well, and the plate was re-incubated overnight. A change in colour from blue (oxidized
state) to pink (reduced) indicated the growth of bacteria, and the MIC was defined as the lowest
1
concentration of drug that prevented this change in colour.
4
.3. In vitro dormant MTB model
A culture of M. tuberculosis H37Rv (O.D. of 0.8 - 1.0) grown in Middlebrook 7H9 medium
supplemented with OADC was pelleted and washed twice with PBS. The pellet was re-
2
5

suspended in PBS in sealed bottles and incubated at 37 °C for 6 weeks. Aliquots of these cultures
were then treated with standard drugs like INH, Rif and Moxifloxacin (Mox) and the lead
compounds for 7 days at a concentration of 10 µg/ml. The frequency of persistors was
1
3
enumerated by MPN assay.
4
.4. LAT enzymatic assay
MTB LAT enzymatic assay was performed in 100 μl volume containing 200 mM phosphate
buffer (pH 7.2), 1.5 mM L-lysine, 1.5 mM-ketoglutarate, 15 M pyridoxal 5-phosphate with
MTB LAT for 1hr at 37°C. The compounds were added to the plates with different
concentrations from 50 M to 1 M. Reactions were terminated by adding 10% trichloroacetic
acid in ethanol. The MTB LAT activity was monitored and the end product piperidine-6-
carboxylic acid and glutamate was detected at absorbance of 465 nm and 280 nm. Reactions
were carried out in a heat-controlled Perkin Elmer Victor X3 spectrophotometer. Further the IC50
values were calculated using Graph Pad Prism analysis software. The error values in IC s were
5
0
1
4-15
derived using non-linear regression mode in Graph pad prism software.
.5.MTT assay
4
The synthesized compounds were further examined for its effects on metabolism in mouse
macrophage cell line (RAW 264.7) at 50µg/ml concentration. After 48 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. The compounds are tested at 50
1
6
µg/ml concentration for 48 hrs.
Acknowledgements:
R.S.R. is thankful to Department of Science and Technology, Government of India for the Inspire
2
6

fellowship.DS is thankful to Department of Biotechnology, Government of India for the Tata
innovation fellowship.
Funding: for some of the study we have used funding from Tata Innovation Fellowship of
Department of Biotechnology, India.
References-
1
. Wirth, T.; Hildebrand, F.; Allix-Béguec, C.; Wölbeling, F.; Kubica, T.; Kremer, K.; van
Soolingen, D.; Rüsch-Gerdes S.; Locht, C.; Brisse, S.; Meyer, A. PLoS pathogens2008,
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Biology and Crystallization Communications 2006, 62(6), 572-575.
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2
8

Synthesis
and
Evaluation
of
4',5'-
Dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]
Analogues Against Both Active and Dormant
Mycobacterium tuberculosis
a
a
b
Kiran Kumar Alluri , Rudraraju Srilakshmi Reshma , Raghuram Suraparaju , Suryanarayana
b
a
Gottapu , Dharmarajan Sriram , *
2
9