A facile three-component [3+2]-cycloaddition for the regioselective synthesis of highly functionalised dispiropyrrolidines acting as antimycobacterial agents

Article abstract of DOI:10.1016/j.bmcl.2012.12.069

A series of fourteen dispiropyrrolidines were synthesized using [3+2]-cycloaddition reactions and were screened for their antimycobacterial activity against Mycobacterium tuberculosis H₃₇Rv in HTS (High Throughput Screen). Most of the compounds showed moderate to good activity with MIC of less than 20 μM. Compound 4′-(4-bromophenyl)-1′- methyldispiro[acenaphthylene-1,2′-pyrrolidine-3′,2″-indane]-2, 1″(1H)-dione (4c) was found to be the most active with MIC of 12.50 μM.

Full text of DOI:10.1016/j.bmcl.2012.12.069

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1383–1386
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A facile three-component [3+2]-cycloaddition for the regioselective
synthesis of highly functionalised dispiropyrrolidines acting as
antimycobacterial agents
a
a
a
Ang Chee Wei a, Mohamed Ashraf Ali a,b,c, , Yeong Keng Yoon , Rusli Ismail , Tan Soo Choon ,
⇑
Raju Suresh Kumar d
a Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Minden 11800, Penang, Malaysia
b New Drug Discovery Research, Department of Medicinal Chemistry, Alwar Pharmacy College, Alwar, Rajasthan 301030, India
c New Drug Discovery Research, Department of Medicinal Chemistry, Sunrise University, Alwar, Rajasthan 301030, India
d Department of Chemistry, College of Science, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of fourteen dispiropyrrolidines were synthesized using [3+2]-cycloaddition reactions and were
Received 17 October 2012
Revised 15 November 2012
Accepted 22 December 2012
Available online 4 January 2013
screened for their antimycobacterial activity against Mycobacterium tuberculosis
(High Throughput Screen). Most of the compounds showed moderate to good activity with MIC of less
than 20
M. Compound 40-(4-bromophenyl)-10-methyldispiro[acenaphthylene-1,20-pyrrolidine-30,200-
indane]-2,100(1H)-dione (4c) was found to be the most active with MIC of 12.50
M.
H37Rv in HTS
l
l
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Antimycobacterial activity
[3+2]-Cycloaddition
Azomethine ylide
Intermolecular
Dispiroheterocycle
Over the last century, tuberculosis (TB) has killed more than
100 million people and this has continued relatively unchanged
over the last 50 years, despite the development of effective antitu-
bercular drugs. TB, disproportionally affecting the world’s poorest
populations, remains one of the biggest public health problems
in the 21st century.1 TB continues to kill young and middle-aged
adults faster than any other disease apart from AIDS. In 2010, the
number of people who fell ill with TB is 8.8 million cases, including
1.1 million cases among people with HIV. There was an estimated
prevalence of 650,000 cases of multidrug-resistant TB (MDR-TB),
and in 2008 it was estimated there were 150,000 MDR-TB deaths
annually.2 Today, TB remains one of the leading infectious disease
killers around the world. Emerging drug-resistant strains such as
multidrug-resistant tuberculosis (MDR-TB) and extensively drug-
resistant tuberculosis (XDR-TB) are presenting a new challenge in
the ever changing battle to control and prevent TB.3 Many pub-
lished reports4,5 on the resistance of the current available first line
drugs against the virulence strains of MTB incite the researchers to
develop new anti TB agents on the priority basis.
Heterocyclic compounds with five and six-membered rings
have drawn considerable attention with their highly pronounced
biological activities.6 Spiro compounds especially are known for
their antimycobacterial properties and some of them have shown
comparable or even better activities than the fist-line TB drugs.7–9
Hence our group has synthesized and evaluated a series of novel
spiro-heterocyles using 1,3-dipolar cycloaddition to obtain novel
and more potent antitubercular agents. 1,3-Dipolar cycloaddition
is proved to be an efficient method to construct heterocyclic units
in a single step without the need to purify the intermediates.
Previous studies have shown that chalcone and chalcone-like
compounds exert various biological activities including antituber-
cular,10 anti-inflammatory,11 antioxidant,12 anticancer13 and anti-
microbial.14 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 indanone derivatives.
In our previous study15 we have reported the synthesis of highly
functionalised dispiropyrrolidines by [3+2]-cycloaddition reaction
of sarcosine and ninhydrin as a source of azomethine ylide with
indanone dipolarophiles. As part of the ongoing research program,
herein we wish report the synthesis of spiropyrrolidines by the
same cycloaddition reaction with different azomethine ylides
⇑
Corresponding author.
E-mail address: asraf80med@yahoo.com (M.A. Ali).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.069

1384
A. C. Wei et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1383–1386
O
O
Ar
O
Ar-CHO
NaOH, EtOH
3-4 hrs
CH3
N
Ar
O
MeOH
1
O
O
Reflux
2-4.5 hrs
H
N
O
+
H3C
OH
4a-n
3
2
Scheme 1. Protocol for synthesis of titled compound.
generated in situ by decarboxylative condensation of acenaphtho-
quinone and sarcosine (Scheme 1). The synthesized compounds
were screened for antimycobacterial activity and the results are
presented in this paper.
2-[(E)-1-Arylmethylidene]-1-indanones (1) were synthesized
according to the literature method.16 An equimolar mixture of
the starting materials were refluxed in methanol until completion
of the reaction (as evident from TLC) and the pyrrolidine deriva-
tives were purified by crystallization.
The purity of the newly synthesized dispiropyrrolidines was
checked 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 4c showed a singlet at d 2.22 ppm corresponding to
N–CH3 group; a triplet at 4.43 ppm with J = 9.0 Hz is due to H-40.
The triplets at 3.74 ppm (J = 9.0 Hz) and 4.16 ppm (J = 9.3 Hz) are
due to protons of 50-CH2 while the doublets at 2.63 ppm
(J = 17.7 Hz) and 2.91 ppm (J = 17.7 Hz) were due to protons of
300-CH2. The aromatic protons appear around 6.80–8.01 ppm. In
13C NMR spectrum, the N–CH3 signal appears at 35.6 ppm, ali-
phatic ring carbons appear at 35.0, 50.5 and 59.9 ppm while the
peaks at 67.6 and 82.0 ppm are due to the spiro carbons. The
C@O of acenaphthenequinone ring appears at 207.1 ppm while
the C@O of indanone appears at 208.4 ppm. A selected 1H and
13C chemical shift of 4c is shown in Figure 1. The product 4c was
further confirmed by mass spectrometry with the molecular ion
peak at m/z 509 [M+]. The regio- and stereochemical outcome of
the cycloaddition reaction was ascertained by single crystal X-ray
analysis18 of 4c (Fig. 2). This molecular structure is stabilized by
intermolecular and intramolecular C–Hꢀ ꢀ ꢀO hydrogen bonds.
The mechanism for the formation of the novel pyrrolidine deriv-
ative is proposed and summarized in Scheme 2. The reaction of
acenaphthenequinone and sarcosine affords the azomethine ylide
(5), which adds to C@C bond of the dipolarophile from the bottom
to form the corresponding cycloadduct. The reaction proceeds with
complete regioselectivity affording regioisomer (4) and also with
Figure 2. ORTEP diagram of 4c.
complete stereoselectvity furnishing only one stereoisomer of the
cycloadduct despite of the presence of three stereocentres.
All the newly synthesized compounds 4a–n were screened for
their in vitro antimycobacterial activity against Mycobacterium
tuberculosis H37RV (MTB-H37Rv) in a HTS (High Throughput Screen)
using an assay adapted from the microdilution alamar Blue (AB)
broth assay reported by Collins and Franzblau17 and additionally
used an alternative method for end-point detection was assessed
using the Promega reagent BacTiter-Glo™ Microbial Cell Viability
(BTG). The BTG assay is a quantitative ATP assay for bacteria using
luciferase production as an end-point detection point. Six standard
drugs were used to test together with the synthesized compound
as references for the assay. Data was analyzed using the IDBS
Activity Base software and the dose response result was analyzed
using a four parameter logistic fit to the data (Excel Fit equation
205) with the maximum and minimum locked at 100 and 0. From
these curves, EC90 and EC50 values were calculated. The MIC was
defined as the minimum concentration of compound required to
Br
4.43, t, J = 9.0 Hz
50.5
3.74, t, J = 9.0 Hz
4.16, t, J = 9.3 Hz
59.9
208.4
H
O
2.22, s
35.6
CH3
67.6
N
O
207.1
82.0
2.63, d, J = 17.7 Hz
6.80-8.10, m
aromatic
2.91, d, J = 17.7 Hz
35.0
Figure 1. Selected 1H and 13C NMR chemical shifts of 4c.

A. C. Wei et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1383–1386
1385
O
Ar
Ar
O
CH2
CH3
N
H2C
O
N
N
CH3
O
CH3
O
3
+
2
-CO2
4a-n
5
Scheme 2. Mechanism for the regioselective formation of dispiropyrrolidine.
Table 1
Antimycobacterial activity of pyrrolidine derivatives 4a–n
CH3
O
5'
Ar
O
1'
N
4'
3'
2'
1"
3"
1
2
Compd
Ar
EC50
M)
BTG
EC90
M)
BTG
MIC
M)
BTG
Std Dev
EC50
BTG
EC50
M)
AB
EC90
M)
AB
MIC
M)
AB
Std Dev
EC50
AB
Cytotoxicity
(lg/ml)
(
l
(
l
(
l
(
l
(
l
(
l
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
Phenyl
4-Chlorophenyl
4-Bromophenyl
4-Trifluoromethylphenyl-
4-Trifluoromethoxyphenyl-
5-[(4-Fluorophenyl)-pyridine
4-Carboxy phenyl
3-Nitrophenyl
4-Methoxyphenyl
Benzo[d][1,3]dioxole
4-Dimethylaminophenyl
1-(4-(Morpholine-1-yl)phenyl)
2,5-Dimethoxyphenyl
1-(4-(Piperidine-1-yl)phenyl)-
—
—
—
—
—
—
>100.00
6.27
2.20
3.21
8.51
64.46
20.74
10.06
13.43
11.51
9.87
18.85
9.56
17.75
23.55
0.13
>100.00
29.62
10.76
12.29
26.08
>100.00
>100.00
>100.00
62.37
>100.00
55.34
>100.00
>100.00
>100.00
26.38
NA
0.000
>100.00
68.46
15.07
23.43
36.60
>100.00
69.21
77.50
40.90
63.84
55.99
>100.00
67.96
>100.00
24.76
0.19
>100.00
>100.00
>100.00
>100.00
>100.00
>100.00
99.70
>100.00
>100.00
>100.00
>100.00
>100.00
>100.00
>100.00
28.01
NA
NA
NA
NA
NA
NA
100.00
NA
NA
NA
NA
NA
NA
NA
100.00
NA
0.16
0.16
100.00
NA
>9999
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
50.00
12.50
50.00
50.00
NA
100.00
NA
100.00
100.00
100.00
NA
NA
NA
100
0.31
0.04
0.16
100.00
6.25
0.377
0.163
0.258
0.463
27.106
4.062
1.514
1.121
1.478
1.104
1.867
1.065
1.760
1441.00
0.04
31.96
4.54
6.41
5.24
4304.53
5.09
11.80
3.64
5.79
10.18
>9999
14.20
23.72
358.28
0.04
0.40
1458.49
275
Cycloserine
Isoniazid
Rifampin
Amikacin
Pyrimethamine
Ethambutol
0.2
0.03
0.12
46.37
>5.00
0.02
0.14
28.00
0.02
0.07
24.27
1.50
0.00
0.00
1.18
0.02
0.12
25.09
3.45
1.64
3542.14
>200.00
1.60
inhibit 90% of bacterial growth and all the biological results were
reported in Table 1.
All the compounds were also tested for cytotoxicity (IC50) in
VERO cells at concentrations of 62.5 g/mL or 10 times. After
l
Among the fourteen compounds that have been newly synthe-
72 h of exposure, viability was assessed on the basis of cellular
conversion of MTT into a formazan product using the Promega
CellTiter 96 nonradioactive cell proliferation assay. All of the active
sized, compound 4c (40-(4-bromophenyl)-10-methyldispiro[ace-
naphthylene-1,20-pyrrolidine-30,200-indane]-2,100(1H)-dione)
was
M and MIC at
M using BTG. The compound with electron withdrawing
group sustituents, 4d, 4b and 4e showing 50% inhibition at concen-
tration of 3.21, 6.27 and 8.51 M. However substituents with 2,5-
found to be the most active with EC50 of 2.20
12.50
l
compounds were found to be non-toxic up to 62.5 lg/mL. The
l
compounds were bactericidal and the cytotoxic profile was within
an acceptable range. Therefore, a lead optimization programme
was initiated with the goal of achieving potent antitubercular
activity.
These reports clearly showed that the presence of halogen sub-
stitution cause remarkable improvement in antimycobacterial
activity. It is conceivable that derivatives showing antimycobacte-
rial 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 pro-
gress in our laboratory.
l
dimethoxy (4m), 4-dimethylamino (4k), 3-nitro (4h), methylene
dioxy (4j), 4-methoxy (4i), piperidine (4n) and morpholino (4l)
groups showed moderate to low inhibitory with EC50 less than
20 lM. It is to be noted that the halogen substituent in the phenyl
ring causes remarkable improvement in antimycobacterial activity,
especially bromo group. In particular, the compound 40-(4-bromo-
phenyl)-10-methyldispiro[acenaphthylene-1,20-pyrrolidine-30,200-
indane]-2,100(1H)-dione (4c) is endowed with maximum potency in
the titled compounds, being, respectively, 10.70-, 11.0-fold at EC50,
2.45-, 4.30-fold at EC90 and more than 8-fold of MIC than the stan-
dard drugs cycloserine and pyrimethamine. However none of the
compounds screened in the present derivatives are found to be
more potent than other used standard drugs.
Acknowledgments
The authors wish to express their thanks to Pharmacogenetic
and Novel Therapeutic Research, Institute for Research in

1386
A. C. Wei et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1383–1386
6. Raj, A. A.; Raghunathan, R.; SrideviKumari, M. R.; Raman, N. Bioorg. Med. Chem.
2003, 11, 407.
7. Karthikeyan, S. V.; Bala, B. D.; Raja, V. P. A.; Perumal, S.; Yogeeswari, P.; Sriram,
D. Bioorg. Med. Chem. Lett. 2010, 20, 350.
8. Ranjith Kumar, R.; Perumal, S.; Senthilkumar, P.; Yogeeswari, P.; Sriram, D. Eur.
J. Med. Chem. 2009, 44, 3821.
9. Kumar, R. R.; Perumal, S.; Senthilkumar, P.; Yogeeswari, P.; Sriram, D. J. Med.
Chem. 2008, 51, 5731.
Molecular Medicine, Universiti Sains Malysia, Pennag and this
work were funded through Research Grant No.RUC (1001/PSK/
8620012), HiCoE research Grant No (311.CIPPM.4401005). This
work was supported by National Institutes of Health and the Na-
tional Institute of Allergy and Infectious Diseases, Contract No.
HHSN272201100012I for antimycobacterial screening.
10. Lin, Y.-M.; Zhou, Y.; Flavin, M. T.; Zhou, L.-M.; Nie, W.; Chen, F.-C. Bioorg. Med.
Chem. 2002, 10, 2795.
Supplementary data
11. Herencia, F.; Ferrándiz, M. L.; Ubeda, A.; Guillén, I.; Dominguez, J. N.; Charris, J.
E.; Lobo, G. M.; Alcaraz, M. J. Free Radical Biol. Med. 2001, 30, 43.
12. Bandgar, B. P.; Patil, S. A.; Totre, J. V.; Korbad, B. L.; Gacche, R. N.; Hote, B. S.;
Jalde, S. S.; Chaven, H. V. Bioorg. Med. Chem. Lett. 2010, 20, 2292.
13. Zhang, H.-J.; Qian, Y.; Zhu, D.-D.; Yang, X.-G.; Zhu, H.-L. Eur. J. Med. Chem. 2011,
46, 4702.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.12.
069.
14. Chauhan, R.; Dwivedi, J.; Siddiqi, A. A.; Kishore, D. Pharm. Chem. J. 2011, 44, 542.
15. Wei, A. C.; Ali, M. A.; Yoon, Y. K.; Ismail, R.; Choon, T. S.; Kumar, R. S.;
Arumugam, N.; Almansour, A. I.; Osman, H. Bioorg. Med. Chem. Lett. 2012, 22,
4930.
16. Ali, M. A.; Samy, J. G.; Manogaran, E.; Sellappan, V.; Hasan, M. Z.; Ahsan, M. J.;
Pandian, S.; ShaharYar, M. Bioorg. Med. Chem. Lett. 2009, 19, 7000.
17. Colins, L.; Franzblau, S. G. Antimicrob. Agents Chemother. 1997, 41, 1004.
18. Wei, A. C.; Ali, M. A.; Choon, T. S.; Arshad, S.; Razak, I. A. Acta Cryst. 2012, E68,
o1340.
References and notes
1. Hand book of anti-tuberculosis agent Tuberculosis 2008, 88, 85.
2. Global tuberculosis control: WHO report 2011, WHO/HTM/TB/2011.16.
3. Ginsberg, A. M. Tuberculosis 2010, 90, 162.
4. Zhao, X.; Yu, H.; Yu, S.; Wang, F.; Sacchettini, J. C.; Magliozzo, R. S. Biochemistry
2006, 45, 4131.
5. Muddassar, M.; Jang, J. W.; Gon, H. S.; Cho, Y. S.; Kim, E. E.; Keum, K. C.; Oh, T.;
Cho, S.-N. T.; Pae, A. N. Bioorg. Med. Chem. 2010, 18, 6914.