Antimycobacterial activity: A facile three-component [3+2]-cycloaddition for the regioselective synthesis of highly functionalised dispiropyrrolidines

Article abstract of DOI:10.1016/j.bmcl.2012.06.047

A series of twelve dispiropyrrolidines were synthesized using [3+2]-cycloaddition reactions. The synthesized compounds were screened for their antimycobacterial activity against M. tuberculosis H₃₇Rv and INH resistant M. tuberculosis strains using agar dilution method, four of them showed good activity with MIC of less than 1 μM. Compound 4′-[5-(4-fluorophenyl)pyridin-3-yl]-1′-methyldispiro[indan-2, 2′ pyrrolidine-3′,2″-indan]-1,3,1″-trione (4b) was found to be the most active with MIC of 0.1215 and 5.121 μM, respectively.

Full text of DOI:10.1016/j.bmcl.2012.06.047

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4930–4933
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antimycobacterial activity: A facile three-component [3+2]-cycloaddition
for the regioselective synthesis of highly functionalised dispiropyrrolidines
Ang Chee Wei a, Mohamed Ashraf Ali a,d,e, , Yeong Keng Yoon a, Rusli Ismail a, Tan Soo Choon a,
⇑
Raju Suresh Kumar b, Natarajan Arumugam b, Abdulrahman I. Almansour b, Hasnah Osman c
a Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Minden 11800, Penang, Malaysia
b Department of Chemistry, College of Science, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabia
c School of Chemical Sciences, Universiti Sains Malaysia, Minden 11800, Penang, Malaysia
d New Drug Discovery Research, Department of Medicinal Chemistry, Alwar Pharmacy College, Alwar, Rajasthan 301030, India
e New Drug Discovery Research, Department of Medicinal Chemistry, Sunrise University, Alwar, Rajasthan 301030, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of twelve dispiropyrrolidines were synthesized using [3+2]-cycloaddition reactions. The synthe-
sized compounds were screened for their antimycobacterial activity against M. tuberculosis H37Rv and
INH resistant M. tuberculosis strains using agar dilution method, four of them showed good activity with
Received 25 April 2012
Revised 5 June 2012
Accepted 15 June 2012
Available online 21 June 2012
MIC of less than
pyrrolidine-30,200-indan]-1,3,100-trione (4b) was found to be the most active with MIC of 0.1215 and
5.121 M, respectively.
1 l
M. Compound 40-[5-(4-fluorophenyl)pyridin-3-yl]-10-methyldispiro[indan-2,20
l
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
Antimycobacterial activity
Intermolecular 1,3-dipolar cycloaddition
Azomethineylide
Indanone
Dispiropyrrolidines
Tuberculosis (TB) remains a global health problem and the sec-
ond leading cause of death from an infectious disease worldwide
after AIDS. In 2010, there were 8.8 million incident cases of TB
and 1.2–1.5 million deaths.1 In recent years, the resurgence in TB
is further fuelled by the emergence of HIV and resistance of
Mycobacterium tuberculosis strains. The overall incidence of TB in
HIV-positive patients is 50 times more than the rate in HIV-nega-
tive individuals.2 Furthermore, the rise of multi drug resistant
(MDR), extensively drug resistant (XDR) and extremely drug resis-
tant (XXDR) TB strains3 as a consequence of lengthy treatment,
makes patient compliance more difficult. No new classes of drugs
have been developed for the past 40 years. The last drug with a
new mechanism of action approved (rifampicin) was discovered
in 1963.4 Therefore there is an urgent need for development of
new drug leads to combat this chronic infectious disease.
construct heterocyclic units in a single reaction without the need
to purify the intermediates. Previous studies have shown that
chalcone and chalcone-like compounds exert various biological
activities including antimycobacterial,8 anti-inflammatory,9
anti-cancer10 and anti-bacterial.11 In continuation of our research
on the synthesis of biologically active heterocycles and in view of
the importance of indanone derivatives in medicinal chemistry,
we focused our attention in synthesizing dispiro substituted inda-
none derivatives, there seems to be no report on the biological
activity of indanone substituted spiro compounds.
Herein we report the synthesis of indanone substituted dispiro-
pyrrolidines through [3+2]-cycloaddition reaction of azomethine
ylide generated in situ from ninhydrin and sarcosine to arylmethy-
lideneindanones. The synthesized compounds were screened for
their antimycobacterial activity and the results are presented in
this paper.
Heterocycles with five and six-membered rings have drawn
considerable attention due to their highly pronounced biological
activities.5 Especially spiro compounds are known for their anti-
mycobacterial properties and many of them have shown compara-
ble or even better activities than some of the fist-line TB drugs.6,7
1,3-Dipolar cycloaddition is proved to be an efficient method to
Substituted aryl-10-methyldispiro[indan-2,20 pyrrolidine-30,200-
indan]-1,3,100-trione analogues 4a–l described in this study is
shown in Table 1, and a reaction sequence for the preparation is
outlined in Scheme 1. In the initial step, 2-[(E)-1-arylmethyli-
dene]-1-indanones were synthesized following the literature
method12 by condensing indanone with appropriate aromatic
aldehydes in dilute ethanolic sodium hydroxide solution at room
temperature, followed by synthesis of dispiropyrrolidines via
[3+2]-cycloaddition of azomethine ylides generated in situ from
⇑
Corresponding author.
E-mail address: asraf80med@yahoo.com (M.A. Ali).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.047

A. C. Wei et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4930–4933
4931
Table 1
Comparison of the reaction time, yields for thermal and microwave-assisted [3+2]-cycloaddition reactions and antimycobacterial activity of pyrrolidine derivatives 4a–l
5'
Ar
CH3
O
O
4'
3'
1'
2'
N
1"
3
3"
1
O
Compd
Ar
Conventional method
(MeOH, reflux)
Microwave
irradiation (100 °C,
Rf
(MIC)
MTBa
lM
(MIC)
MTBb
l
M
Cytotoxicity (
ml)
lg/
valuec
100 W)
Time (h) Yield
(%)
Yield
(%)
Time
(min)
Yield
(%)
4a
4b
4c
4d
4e
4f
4g
4h
4i
Phenyl
4
3.5
2
65
68
69
67
64
67
65
68
63
60
65
68
2
3
2
2.5
3
2
2
2
3
92
94
96
95
89
91
92
93
87
85
92
93
0.85
0.83
0.81
0.83
0.79
0.84
0.83
0.81
0.83
0.79
0.84
0.88
6.219
26.219
5.121
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
5-[(4-Fluorophenyl)-pyridine
4-Chlorophenyl
4-Bromophenyl
4-Carboxy phenyl
3-Nitrophenyl
4-Trifluoromethylphenyl-
4-Trifluoromethoxyphenyl-
4-Methoxyphenyl
Benzo[d][1,3]dioxole
4-Dimethylaminophenyl
1-(4-(Morpholine-1-
yl)phenyl)
0.1215
1.195
1.372
0.295
0.810
0.638
0.214
>6.25
2.415
3.319
1.215
24.195
21.372
18.295
16.810
13.638
12.214
>6.25
32.415
43.319
27.215
3
2.5
4.5
3
3
2.5
4
4j
4k
4l
3.5
2
2
3
3
Isoniazid
—
—
—
—
—
—
0.73
11.23
>62.5
a
b
c
Mycobacterium tuberculosis H37Rv.
INH resistant Mycobacterium tuberculosis.
Pet.ether: ethylacetate.
O
Ar-CHO
EtOH
NaOH
3-4hrs
O Ar
O
O
O
H
N
O
N
CH3
MeOH
O
O
+
+
O
H3C
OH
Reflux
Ar
2-4.5hrs
1
3
2
4a-l
Scheme 1. Protocol for synthesis of titled compound.
the decarboxylative condensation of ninhydrin and sarcosine with
appropriate dipolarophiles, 2-[(E)-1-arylmethylidene]-1-indanon-
es (1) in 60–68% yield after recrystalization with pet ether: ethyl
acetate (4:1).
The structure of the newly synthesized dispiropyrrolidines were
elucidated using NMR, CHN, mass spectrometry and X-ray analysis.
All the analytical and spectral data showed that the synthesized
compounds were in full agreement with the proposed structures.
In the 1H NMR spectrum, the signals of the respective protons of
the synthesized compounds were verified on the basis of their
chemical shifts, multiplicities and coupling constants. 1H NMR
spectrum of 4b showed a singlet at d 2.47 ppm corresponding to
N-CH3 group; a triplet at 4.95 ppm with J = 9.0 Hz is due to H-40.
The triplet at 4.03 ppm (J = 9.0 Hz) and the doublet at 3.01 ppm
(J = 17.7 Hz) is due one proton each of 50-CH2 and 300-CH2, respec-
tively while the other protons of 50-CH2 and 300-CH2 appears as
multiplet at 3.67–3.77 ppm. The aromatic protons appear around
7.12–8.55 ppm. In 13C NMR spectrum, the N-CH3 signal appear at
35.9 ppm, aliphatic ring carbons appear at 47.1, 56.0 ppm while
the peaks at 68.4 and 81.6 ppm is due to the spiro carbons. In
the p-fluoropheny ring, the carbon directly attached to fluorine
appears as a doublet at 163.0 ppm with J = 247.5 Hz; ortho- and
meta- carbons appears as doublets at 116.1 and 128.7 ppm with
The reactions were initially performed by heating an equimolar
mixture of the starting materials under reflux in methanol and
were subsequently investigated also under microwave irradiation
in a focused microwave synthesizer, as this technique has evolved
as a valuable alternative to conventional heating for the introduc-
tion of energy into reactions and has clear benefits in many chem-
ical transformations, including cycloadditions, in terms of rate
accelerations and yield enhancements. As in the case of the ther-
mal reactions, an equimolar mixture of the starting materials 1–3
in methanol was subjected to microwave irradiation13 for 6–
8 min until completion of the reaction (TLC) and the pyrrolidine
derivatives purified by crystallisation. The results obtained for both
methods are summarized in (Table 1), and clearly show that micro-
wave irradiation led to an enhancement in the yield of the product
(85–96%) over the conventional thermal method (60–68%).

4932
A. C. Wei et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4930–4933
116.1,
4.95, t, J =9.0 Hz
47.1
sively one stereoisomer despite of the presence of three
stereocentres.
J=21.3 Hz
163.0,
128.7,
J=247.5 Hz
J=8.8 Hz
4. 03, t, J=9.0 Hz
3. 67-3.77, m
56.0
The synthesized compounds 4a–l were tested for their antimy-
cobacterial activity in vitro against Mycobacterium tuberculosis
(MTB-H37Rv) and INHR-MTB using agar dilution method15,16 for
the determination of minimum inhibitory concentration (MIC).
The MIC was defined as the minimum concentration of compound
required to inhibit 90% of bacterial growth and MIC’s of the
compounds were reported in (Tables 1) with standard drug INH
for comparison. Among the twelve compounds synthesized, four
compounds were found to be most active with minimum inhibitory
F
N
133.4,
J=3.8 Hz
2.47, s
H
CH3
35.9
O
O
N
3.01, d, J =17.7 Hz
3. 67-3.77, m
31.4
203.0
68.4
200.4
81.6
198.4
7.12-8.55, m
aromatic
O
Figure 1. Selected 1H and 13C NMR chemical shifts of 4b.
concentration of less than
1 lM. Among them compound
40-[5-(4-fluorophenyl)pyridin-3-yl]-10-methyldispiro[indan-2,20-
pyrrolidine-30,200-indan]-1,3,100-trione (4b) was the most active
agent and even equally active as INH against MTB and more active
against INHR-MTB strains.
Among the twelve newly synthesized compounds, compound
4b produced highest efficacy and exhibited >90% inhibition
against MTB at a concentration of 0.1215
MTB 5.121 M, followed by 4h, 4g, and 4f showing moderate
inhibitory activity at 0.214, 0.638, 0.810 M, against INHR-MTB
12.214,13.368 and 16.810 M and, respectively. The pyridine
lM and against INHR-
l
l
l
4-fluoro phenyl group substitution (4b) derivatives displayed rela-
tively higher inhibitory activity in general. However the electron
rich group such as 4-trifluorometoxy, 4-trifluoromethyl and 3-ni-
tro substituted analogue compounds produced moderate inhibi-
tory activity against MTB Mycobacterium tuberculosis. On the
other hand the analogue methylene dioxy group (4j), dimethyl-
amino phenyl (4k), morpholino phenyl (4i), 4-bromophenyl (4d),
4-carboxyphenyl (4e) and 4-methoxy phenyl (4i) showed rela-
tively moderate to low antitubercular activity. These reports
clearly showed that the increases of the presence of 4-fluoro
substitution causes remarkable improvement in antimycobacterial
activity.
All the compounds were tested for cytotoxicity (IC50) in VERO
cells at concentrations of 62.5 lg/mL or 10 times. After 72 h of
exposure, viability was assessed on the basis of cellular conversion
of MTT into a formazan product using the Promega Cell Titer 96
Non-radioactive Cell proliferation method. Most of the active com-
Figure 2. ORTEP diagram of 4b.
J = 21.3 and 8.8 Hz, respectively; the carbon directly attached to
the pyridine ring also appears as doublet at 133.4 ppm with
J = 3.8 Hz. The two C@O of ninhydrin ring appears at 198.4 and
200.4 ppm while the C@O of indanone appears at 203.0 ppm. The
molecular ion peak at m/z 503 [M+] confirms the product 4b. A se-
lected 1H and 13C chemical shift of 4b is shown in Figure 1. Finally
the region and stereochemical outcome of the cycloaddition reac-
tion was unambiguously ascertained by single crystal X-ray analy-
sis14of 4b ( Fig. 2). The molecular structure is stabilized by
intermolecular and intramolecular CꢀHꢁ ꢁ ꢁO hydrogen bonds.
The mechanism proposed to rationalise the formation of the no-
vel pyrrolidine derivative is summarized in Scheme 2. The reaction
of ninhydrin and sarcosine affords the azomethine ylide (5), which
adds to C@C bond of the dipolarophile to form the corresponding
cycloadduct. The remarkable product selectivity observed in this
reaction depends on selectivity of sarcosine towards ninhydrin
rather than the potentially competitive indanone ring system.
The reaction proceeded with complete regio- selectivity affording
the cycloadduct 5, and it is pertinent to note that the reaction also
accomplished with complete stereo selectivity, affording exclu-
pounds were found to be non-toxic till 62.5 lg/mL.
In conclusion, we have synthesised successfully a series of inda-
none substituted dispiro derivatives in good yield under micro-
wave irradiation, as this method was found to be synthetically
useful in achieving high yields of the products with reduced reac-
tion time compared to conventional heating. Among the newer
derivatives, compounds 4b exhibited good activity against MTB
and INHR-MTB. It is conceivable that derivatives showing
antimycobacterial activity can be further modified to exhibit better
potency than standard drugs. Further studies to acquire more
information about quantitative structure-activity relationships
(QSAR) are in progress in our laboratory.
O
O
O
Ar
O
O
O
N
CH3
O
Ar
CH2
CH3
O
CH2
O
1
2
O
O
N
N
+
-CO2
OH
CH3
H
N
O
H3C
4a-l
5
3
Scheme 2. Mechanism for the regioselective formation of dispiropyrrolidine.

A. C. Wei et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4930–4933
4933
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The authors wish to express their thanks to Pharmacogenetic
and Novel Therapeutic Research, Institute for Research in Molecu-
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