Discovery of antimycobacterial spiro-piperidin-4-ones: An atom economic, stereoselective synthesis, and biological intervention

Article abstract of DOI:10.1021/jm800545k

An atom economic and stereoselective synthesis of several spiro-piperidin-4-ones through 1,3-dipolar cycloaddition of azomethine ylides generated in situ from isatin and α-amino acids viz. proline, phenylglycine, and sarcosine to a series of 1-methyl-3,5-bis[(E)- arylmethylidene]tetrahydro-4(1H)-pyridinones is described. These compounds were evaluated for their in vitro and in vivo activity against Mycobacterium tuberculosis H37Rv (MTB), multidrug resistant Mycobacterium tuberculosis (MDR-TB), and Mycobacterium smegmatis (MC²). Compound 4-(4-fluorophenyl)-5-phenylpyrrolo(spiro[2.3″]oxindole)spiro[3.3′] -1′-methyl-5′-(4-fluorophenylmethylidene)piperidin-4′-one (4e) was found to be the most active in vitro with a MIC value of 0.07 μM against MTB and was 5.1 and 67.2 times more potent than isoniazid and ciprofloxacin, respectively. In vivo, compound 4e decreased the bacterial load in lung and spleen tissues with 1.30 and 3.73-log 10 protections respectively and was considered to be promising in reducing bacterial count in lung and spleen tissues.

Full text of DOI:10.1021/jm800545k

J. Med. Chem. 2008, 51, 5731–5735
5731
Discovery of Antimycobacterial Spiro-piperidin-4-ones: An Atom Economic, Stereoselective
Synthesis, and Biological Intervention
Raju Ranjith Kumar,† Subbu Perumal,*,† Palaniappan Senthilkumar,‡ Perumal Yogeeswari,‡ and Dharmarajan Sriram‡
Department of Organic Chemistry, School of Chemistry, Madurai Kamaraj UniVersity, Madurai 625021, Tamil Nadu, India, Medicinal
Chemistry Research Laboratory, Pharmacy Group, Birla Institute of Technology and Science, Pilani 333031, Rajasthan, India
ReceiVed May 12, 2008
An atom economic and stereoselective synthesis of several spiro-piperidin-4-ones through 1,3-dipolar
cycloaddition of azomethine ylides generated in situ from isatin and R-amino acids viz. proline, phenylglycine,
and sarcosine to a series of 1-methyl-3,5-bis[(E)-arylmethylidene]tetrahydro-4(1H)-pyridinones is described.
These compounds were evaluated for their in vitro and in vivo activity against Mycobacterium tuberculosis
H37Rv (MTB), multidrug resistant Mycobacterium tuberculosis (MDR-TB), and Mycobacterium smegmatis
(MC2). Compound 4-(4-fluorophenyl)-5-phenylpyrrolo(spiro[2.3′′]oxindole)spiro[3.3′]-1′-methyl-5′-(4-fluo-
rophenylmethylidene)piperidin-4′-one (4e) was found to be the most active in vitro with a MIC value of
0.07 µM against MTB and was 5.1 and 67.2 times more potent than isoniazid and ciprofloxacin, respectively.
In vivo, compound 4e decreased the bacterial load in lung and spleen tissues with 1.30 and 3.73-log 10
protections respectively and was considered to be promising in reducing bacterial count in lung and spleen
tissues.
Introduction
of new agents and the lack of pharmaceutical industry
research in this area.7 Hence, the discovery of fast-acting
effective new drugs to effectively combat TB, including
multidrug resistant tuberculosis, is imperative.
Tuberculosis (TB) caused by Mycobacterium tuberculosis
bacteria (MTB)a is one of the most prevalent diseases that is
responsible for the deaths of about one billion people during
the last two centuries.1TB remains a serious public health
problem in India, accounting for nearly one-third of the global
burden, and it has been estimated that 3.5 million of the
population are infected with TB.1,2 Currently, the recom-
mended standard chemotherapeutic regimen for TB treatment
is prescribed under DOTS. The chemotherapeutic regimen
consists of an initial 2-month phase of treatment with
isoniazid, rifampicin, pyrazinamide, and ethambutol followed
by a continuation phase of treatment lasting 4 months with
isoniazid and rifampicin.3,4 Poor patient compliance can
promote the emergence of drug resistance, and this is
particularly true in TB chemotherapy.3,4 The emergence of
strains resistant to either of these drugs causes major concern,
as it leaves only drugs that are far less effective, have more
toxic side effects, and result in higher death rates, especially
among HIV-infected persons. Serial selection of drug resis-
tance, thus, is the predominant mechanism for the develop-
ment of MDR strains; the patients with MDR strains
constitute a pool of chronic infections, which propagate
primary MDR resistance. In addition to accumulation of
mutations in the individual drug target genes, the permeability
barrier imposed by the MTB cell wall can also contribute to
the development of low-level drug resistance.5,6 In the last
50 years, only a few drugs have been approved by the Food
and Drug Administration (FDA) to treat TB, which reflects
the inherent difficulties in the discovery and clinical testing
1,3-Dipolar cycloaddition of azomethine ylides to exocyclic
olefins constitutes a versatile protocol for the construction of
poly functionalized spiro-heterocycles viz. pyrrolidines8 and
pyrrolizines,9 which widely occur in natural products and
biologically active compounds. In general, spiro compounds10
and nitrogen heterocycles such as pyridines,11 pyrroles,12 and
pyrazolines13 display good antimycobacterial activities. Re-
cently, we have reported an atom economic synthesis and
evaluation of antimycobacterial activities of (i) spiro pyrido-
pyrrolizines and pyrrolidines14 and (ii) 4H-pyrano[3,2-c]pyridine
derivatives,15 which inhibited in vitro MTB and MDR-TB. In
the course of screening to discover new compounds that could
be useful for the chemotherapy of tuberculosis, we identified
spiro-piperidin-4-one derivatives 3-5, which inhibited in vitro
and in vivo MTB and MDR-TB. We present the preliminary
results on the synthesis and the antimycobacterial activities of
the first representative series of this family.
Chemistry
The 1,3-dipolar cycloaddition of azomethine ylides generated
in situ from the reaction of isatin with (i) proline, (ii) phenyl-
glycine, and (iii) sarcosine, to 1a-m afforded spiro-[2.3′′]oxin-
dole-spiro-[3.3′]-5′-arylmethylidene-1′-methyltetrahydro-4′(1H)-
pyridinone-4-aryl-hexahydro-1H-pyrrolizines (3a-m), 4-aryl-
5-phenylpyrrolo(spiro[2.3′′]-oxindole)-spiro[3.3′]-5′-arylmethylidene-
1′-methylpiperidin-4′-ones (4a-m), and 1-methyl-4-arylpyrrolo-
(spiro[2.3′′]-oxindole)-spiro[3.3′]-5′-arylmethylidene-1′-
methylpiperidin-4′-ones (5a-m) in excellent yields, respectively
(Scheme 1). All the reactions proceed (i) chemoselectively, as
the cycloaddition occurs on only one CdC of 1 furnishing
exclusively the mono spiro-cycloadducts 3-5, ascribable to the
steric hindrance exerted by these cycloadducts for the second
cycloaddition, (ii) regioselectively, as the electron-rich carbon
of the dipole adds to the ꢀ carbon of 1, and (iii) stereoselectively,
* To whom correspondence should be addressed. Phone/Fax: +91-452-
2459845. Email: subbu.perum@gmail.com.
†
Department of Organic Chemistry, School of Chemistry, Madurai
Kamaraj University.
‡
Medicinal Chemistry Research Laboratory, Birla Institute of Technology
and Science.
a Abbreviations: MTB, Mycobacterium tuberculosis; MDR-TB, multidrug
resistant tuberculosis; MC2, Mycobacterium smegmatis; MIC, minimum
inhibitory concentration, DOTS, directly observed treatment short-course.
10.1021/jm800545k CCC: $40.75
2008 American Chemical Society
Published on Web 08/21/2008

5732 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Scheme 1. Synthesis of Spiro-piperidin-4-ones 3-5
Kumar et al.
Figure 1. ORTEP diagram of 3a.
as only one diastereomer is obtained exclusively in quantitative
yields, although more than one stereocenter is present in these
cycloadducts 3-5.
Thirty-nine spiro-piperidin-4-ones have been synthesized
in this study by employing cycloadditions. These reactions
were effected by refluxing an equimolar ratio of the corre-
sponding reactants in methanol over a water bath. After
completion of the reactions (TLC), the reaction mixtures were
poured into water to get pure 3-5 as yellow solids. It is
interesting to note that the reaction of 1 with azomethine
ylide generated from isatin and proline is completed within
1-2 min, resulting in quantitative yields of 3, whereas the
reaction with phenylglycine or sarcosine is completed
respectively in 1 h and 30 min. The only byproduct of these
cycloadditions are water and carbon dioxide, and hence the
atom economy is very high (89-90%). The structure of spiro-
Figure 2. ORTEP diagram of 5g.18
responsible for the activity. The MDR-TB clinical isolate was
resistant to isoniazid, rifampicin, ethambutol, and ofloxacin. The
MIC is defined as the minimum concentration of compound
required to completely inhibit the bacterial growth. The MIC’s
of the synthesized compounds along with the standard drugs
for comparison are reported in Table 1.
In the first phase of screening against MTB, all the com-
pounds showed excellent in vitro activity against MTB with
MIC ranging from 0.07-53.30 µM. Eleven compounds (3f, 3h,
3k, 4a, 4c-e, 5d, and 5f-h) inhibited MTB with MIC of less
than 1 µM, and 23 compounds were more potent than standard
fluoroquinolone ciprofloxacin (MIC: 4.71 µM). When compared
to isoniazid (MIC: 0.36 µM), three compounds (3f, 4e, and 5f)
were found to be more active against MTB, and one compound
5h was equipotent as isoniazid. Compound 4e was found to be
the most active in vitro with MIC of 0.07 µM against MTB
and was 5.1 and 67.2 times more potent than isoniazid and
ciprofloxacin, respectively.
1
cycloadducts 3-5 was elucidated with the help of H, 13C
and 2D NMR spectroscopic data and single crystal X-ray
crystallographic studies (Figures 1 and 2).16 The X-ray
structure determination of 3a, which we have noted after the
acceptance of this manuscript, has also been reported by
Kumar et al.16b and the data agree with the data reported by
us except with slight variations. All the spiro compounds
obtained in this work are in racemic form, although two of
the amino acid precursors (proline and phenylglycine)
employed for the generation of the azomethine ylides are
chiral. This is ascribable to the fact that the intermediates
involved in the cycloaddition, viz. the azomethine ylides
generated from the amino acids are achiral.
Subsequently, compounds having MIC less than 3 µM were
evaluated against MDR-TB, and the compounds inhibited MDR-
TB with MIC ranging from 0.08-5.31 µM. All of the 21
compounds screened were found to be more active than isoniazid
(MIC: 45.57 µM) and ciprofloxacin (MIC: 37.73 µM). Eight
compounds (3f, 3h, 3k, 4d, 4e, 5d, 5f, and 5g) inhibited MDR-
TB with MIC of less than 1 µM. Compound 5f was found to
be the most active in vitro with MIC of 0.08 µM against MDR-
TB and was 569.6 and 471.6 times more potent than isoniazid
and ciprofloxacin, respectively. The compounds were also
evaluated against MC2 in which all the compounds inhibited
Biological Results and Discussion
Antimycobacterial Activity. The spiro-piperidin-4-ones 3-5
were screened for their in vitro antimycobacterial activity against
MTB, MDR-TB, and MC2 by agar dilution method for the
determination of MIC in duplicate at pH value of 7.40. The pH
value employed points to the fact that the amino groups in the
compounds cannot be significantly protonated. This, in turn,
suggests that the species with free amine functionality is

DiscoVery of Antimycobacterial Spiro-piperidin-4-ones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5733
Table 1. Minimum Inhibitory Concentrations (µM) of Spiro-piperidin-4-ones 3-5 against Mycobacterial Speciesa
MIC (µg/mL)
MIC (µg/mL)
MIC (µg/mL)
aryl
compd IC50(µM) MTB MDRTB MC2 compd IC50 (µM) MTB MDRTB MC2 compd IC50µM) MTB MDRTB MC2
phenyl
3a
3b
3c
3d
3e
3f
3g
3h
3i
NT
NT
NT
NT
119.05
113.64
NT
113.84
NT
25.56 NT
22.73 NT
12.09 NT
5.70 NT
2.97 2.97
0.35 0.71
12.09 NT
0.71 0.71
5.96 NT
19.94 NT
0.38 0.38
2.65 5.31
53.30 NT
0.36 45.57
4.71 37.73
102.25 4a
90.91 4b
24.18 4c
45.54 4d
23.81 4e
5.69 4f
24.18 4g
2.84 4h
23.81 4i
79.74 4j
3.11 4k
10.61 4l
106.61 4m
45.57
119.05
NT
0.74 1.49
5.27 NT
0.71 1.41
0.67 0.67
0.07 0.16
2.63 5.27
2.82 2.82
1.33 2.67
2.78 NT
2.35 2.35
11.64 NT
5.01 NT
24.75 NT
2.97 5a
42.09 5b
2.82 5c
1.33 5d
0.69 5e
10.52 5f
11.30 5g
1.33 5h
5.58 5i
NT
117.49
NT
119.50
NT
117.48
127.29
119.50
125.25
103.99
131.58
NT
13.49 NT
2.93 NT
6.37 NT
0.75 0.36
6.27 NT
0.17 0.08
0.39 0.39
0.36 1.49
1.56 1.56
1.29 2.59
1.64 3.28
11.10 NT
1.76 3.52
53.99
5.88
50.92
2.98
50.10
1.47
1.59
2.98
6.27
5.21
6.59
22.20
0.88
4-chlorophenyl
4-methylphenyl
4-methoxyphenyl
4-fluorophenyl
2-chlorophenyl
2-methylphenyl
2-methoxyphenyl
3-fluorophenyl
113.02
106.84
111.41
105.22
113.02
106.84
111.41
94.27
NT
2,4-dichlorophenyl 3j
NT
9.43 5j
2-thienyl
1-naphthyl
2-furyl
isoniazid
ciprofloxacin
3k
3l
3m
124.75
106.11
NT
11.64 5k
20.00 5l
49.51 5m
NT
NT
141.08
0.36 45.57 45.57
4.71 37.73 2.35
0.36 45.57 45.57
4.71 37.73 2.35
2.35
a MTB: Mycobacterium tuberculosis; MDR-TB: Multidrug resistant Mycobacterium tuberculosis; MC2: Mycobacterium smegmatis; NT: not tested.
Table 2. In Vivo Activity Data of 4e and Isoniazid against
Mycobacterium tuberculosis ATCC 35801 in Mice
from negative (vehicle treated) controls (Mean culture
forming units (CFU) in lung: 7.31 ( 0.11 and in spleen: 8.93
( 0.20). Compound 4e decreased the bacterial load in lung
and spleen tissues with 1.30 and 3.73-log 10 protections
respectively and was considered to be promising in reducing
bacterial count in lung and spleen tissues. When compared
to isoniazid at the same dose level, 4e was found to be less
active. This could be ascribed to various pharmacokinetic
reasons.
The compounds with MIC less than 3 µM were further
examined for toxicity (IC50) in a mammalian Vero cell line up
to 62.5 µg/mL concentration by serial dilution method. 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 nonradioactive cell proliferation
assay19 and the results are reported in Table 1. Twenty-three
compounds when tested showed IC50 values ranging from
94.27-131.58 µM. The compound 4e was found to be
nontoxic up to 62.5 µg/mL (111.41 µM) and showed
selectivity index (IC50/MIC) of 1634.
compd
lungs (log CFU ( SEM) spleen (log CFU ( SEM)
control
isoniazid (25 mg/kg)
4e (25 mg/kg)
7.31 ( 0.11
5.61 ( 0.13
6.01 ( 0.19
8.93 ( 0.20
4.92 (0.15
5.20 ( 0.13
MC2 with MIC ranging from 0.69-106.61 µM, and 31
compounds were found to be more active than isoniazid (MIC:
45.57 µM).
With respect to structure-MTB activity relationship, the
results demonstrated that the antimycobacterial activity was
in the order: 5 > 4 > 3. This is evident from the fact that
eight compounds in series 5 (5d, 5f-k, and 5m), five in series
4 (4a, 4c-e, and 4h), and three in series 3 (3f, 3h, and 3k)
were more active against MTB. Among the compounds from
the series 3-5, compounds 3k (MIC: 0.38 µM), 4e (MIC:
0.07 µM), and 5f (MIC: 0.17 µM) was found to be more
active. The influence of substituents at N as well as at carbon
R to the N of the pyrrolidine ring and substituents at different
positions of aryl rings was examined for SAR. Bridging
between N and the adjacent carbon leads to loss of antimy-
cobacterial activity as seen with series 3. Ortho substitution
in the aryl ring enhances activity in both series 3 and 5,
whereas para substitution at the aryl ring facilitates activity
in series 4. Electronegative and monosubstitution favor the
antimycobacterial activity in all series. When the aryl ring
is a sulfur heterocycle, activity is favored as seen in 3k,
whereas the furan ring in 3m and 4m renders these
compounds inactive.
Compound 4e, being the most active in vitro against MTB,
was tested for efficacy against MTB at a dose of 25 mg/kg
(Table 2) in CD-1 mice. The mice were infected intravenously
with Mycobacterium tuberculosis ATCC 35801. Drug treat-
ment by intraperitoneal route began after 10 days of
inoculation of the animal with microorganism and continued
for 10 days. After 35 days postinfection, the spleens and right
lungs were aseptically removed, the number of viable
organisms was determined and compared with the counts
Conclusions
The 1,3-dipolar cycloaddition of azomethine ylide generated
in situ from isatin and R-amino acids to 1-methyl-3,5-bis[(E)-
arylmethylidene]tetrahydro-4(1H)-pyridinones afforded spiro-
piperidin-4-ones 3-5 in quantitative yields. These spiro-
heterocycles displayed good in vitro antimycobacterial activity
against MTB, MDR-TB and MC2. Further studies on the
synthesis and examination of structure-activity relationships
of a wide range of heterocyclic compounds with various
substituents at phenyl ring and N atom of the oxindole moiety
and varying ring size of 1-methyl-4-piperidone are in progress
in our research groups.
Experimental Section
General. The melting points were measured in open capillary
1
tubes and are uncorrected. The H, 13C, and the 2D NMR spectra
were recorded on a Bruker (Avance) 300 MHz NMR instrument

5734 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Kumar et al.
using TMS as internal standard and CDCl3 as solvent. Standard
Bruker software was used throughout. Chemical shifts are given
in parts per million (δ-scale), and the coupling constants are given
in hertz. Silica gel-G plates (Merck) were used for TLC analysis
with a mixture of petroleum ether (60-80 °C) and ethyl acetate as
eluent. Elemental analyses were performed on a Perkin-Elmer 2400
Series II elemental CHNS analyzer.
Acknowledgment. S.P. thanks the Department of Science
and Technology, New Delhi, for funds under major research
project no. SR/S1/OC-70/2006, IRHPA programme for the
purchase of a high resolution NMR spectrometer, and FIST
programme and the University Grants Commission, New Delhi,
for funds under the DRS and ASIST programmes.
Synthesis of 1-Methyl-3,5-bis[(E)-arylmethylidene]tetrahydro-
4(1H)-pyridinones (1). General Procedure. A mixture of 1-methyl-
4-piperidone (0.113 g, 1 mmol), aromatic aldehyde (2 mmol), and
NaOH (1 mL, 30%) in alcohol (25 mL) was stirred for 15 min.
The separated solid was filtered and washed with water (100 mL)
to obtain pure 1 as yellow solid.
Synthesis of Spiro-[2.3′′]-oxindole-spiro[3.3′]-1′-methyl-5′-(ar-
ylidene)tetrahydro-4′-(1H)-pyridinone-4-arylhexahydro-1H-pyr-
rolizine (3). General Procedure. A mixture of 1 (1 mmol), isatin 2
(0.147 g, 1 mmol), and proline (0.115 g, 1 mmol) was dissolved in
methanol (10 mL) and warmed on a water bath for 1-2 min. After
completion of the reaction as evident from TLC, the mixture was
poured into water (50 mL). The precipitated solid was filtered and
washed with water to obtain pure 3.
Supporting Information Available: Experimental methods for
compounds 3, 3a,b, 3e-m, 4, 4g-j, 5, 5e-l, MIC determination,
cytotoxicity, in vivo studies, This material is available free of charge
via the Internet at http://pubs.acs.org.
References
(1) World Health Organization, Tuberculosis Fact Sheet 2006, http://
www.who.int/tb/publications/2006/tb_factsheet_2006_1_en.pdf.
(2) Granich, R.; Wares, F.; Suvanand, S.; Chauhan, L. S. Tuberculosis
control in India. Lancet Infect. Dis. 2003, 3, 535.
(3) Sharma, D. C. India makes poor tuberculosis progress in 2002. Lancet
Infect. Dis. 2003, 3, 265.
(4) Bass, J. B.; Farer, L. S.; Hopewell, P. C.; O’Brien, R.; Jacobs, R. F.;
Ruben, F.; Snider, D. E.; Thornton, G. Treatment of tuberculosis and
tuberculosis infection in adults and children. American Thoracic
Society and The Centers for Disease Control and Prevention. Am. J.
Respir. Crit. Care Med. 1994, 149, 1359–1374.
(5) Bhowruth, V.; Dover, L. G.; Besra, G. S. Tuberculosis Chemotherapy:
Recent Developments and Future Perspectives. Prog. Med. Chem.
2007, 45, 169–203.
(6) Rattan, A.; Kalia, A.; Ahmad, N. Multidrug-Resistant Mycobacterium
tuberculosis: Molecular Prespectives. Emerg. Infect. Dis. 1998, 4, 195–
209.
(7) O’Brien, R. J.; Nunn, P. P. The Need for New Drugs against
Tuberculosis. Obstacles, Opportunities, and Next Steps. Am. J. Respir.
Crit. Care Med. 2001, 163, 1055–1058.
(8) (a) Watson, A. A.; Fleet, G. W. J.; Asano, N.; Molyneux, R. J.; Nash,
R. J. Polyhydroxylated alkaloids-natural occurrence and therapeutic
applications. Phytochemistry 2001, 56, 265–295. (b) O’Hagan, D.
Pyrrole, pyrrolidine pyridine, piperidine, azepine and tropane alkaloids.
Nat. Prod. Rep. 1997, 14, 637–651. (c) Horri, S.; Fukase, H.; Matsuo,
T.; Kameda, Y.; Asano, N.; Matsui, K. Synthesis and alpha-D-
glucosidase inhibitory activity of N-substituted valiolamine derivatives
as potential oral antidiabetic agents. J. Med. Chem. 1986, 29, 1038–
1046. (d) Spearman, M. A.; Jamieson, J. C.; Wright, J. A. Studies on
the effect of glycoprotein processing inhibitors on fusion of L6
myoblast cell lines. Exp. Cell Res. 1987, 168, 116–126. (e) Karpas,
A.; Fleet, G. W. J.; Dwek, R. A.; Petursson, S.; Mamgoong, S. K.;
Ramsden, N. G.; Jacob, G. S.; Rademacher, T. W. Aminosugar
Derivatives as Potential Anti-Human Immunodeficiency Virus Agents.
Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 9229–9233.
Synthesis of 4-Aryl-5-phenylpyrrolo(spiro[2.3′′]-oxindole)-spiro-
[3.3′]-1′-methyl-5′-(arylidene)piperidin-4′-ones (4). General Pro-
cedure. A mixture of 1 (1 mmol), isatin 2 (0.147 g, 1 mmol), and
phenylglycine (0.151 g, 1 mmol) in methanol (10 mL) was refluxed
for 1 h. After completion of the reaction as evident from TLC, the
mixture was poured into water (50 mL). The precipitated solid was
filtered and washed with water to obtain pure 4. The yield, melting
point and NMR spectroscopic data of 4a-f and 4k-m agree well
with those reported by our group earlier.17
Synthesis of 1-Methyl-4-arylpyrrolo-(spiro[2.3′′]oxindole)-spiro-
[3.3′]-1′-methyl-5′-(arylidene)piperidin-4′-ones (5). General Pro-
cedure. A mixture of 1 (1 mmol), isatin 2 (0.147 g, 1 mmol), and
sarcosine (0.089 g, 1 mmol) were dissolved in methanol (10 mL)
and refluxed for 30 min. After completion of the reaction as evident
from TLC, the mixture was poured into water (50 mL). The
precipitated solid was filtered and washed with water to obtain pure
5. The physical and spectroscopic data of 5a-d agree well with
those reported in the literature.20
MIC Determination. All compounds were screened for their in
vitro antimycobacterial activity against MTB, MDR-TB, and MC2
in Middlebrook 7H11 agar medium supplemented with OADC by
agar dilution method similar to that recommended by the National
Committee for Clinical Laboratory Standards for the determination
of MIC in duplicate.19 The MDR-TB clinical isolate was obtained
from Tuberculosis Research Center, Chennai, India, and was
resistant to isoniazid, rifampicin, ethambutol, and ofloxacin. The
minimum inhibitory concentration (MIC) is defined as the minimum
concentration of compound required to give complete inhibition
of bacterial growth.
Cytotoxicity. All the compounds were further examined for
toxicity (IC50) in a mammalian Vero cell line up to concentration
of 62.5 µg/mL21 by serial dilution method. 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 nonra-
dioactive cell proliferation assay.
In Vivo Studies. One compound was tested for efficacy against
MTB at a dose of 25 mg/kg in six-week-old female CD-1 mice six
per group. In this model, the mice were infected intravenously
through caudal vein approximately 107 viable Mycobacterium
tuberculosis ATCC 35801. Drug treatment by intraperitoneal route
began after 10 days of inoculation of the animal with microorganism
and continued for 10 days. After 35 days postinfection, the spleens
and right lungs were aseptically removed and ground in a tissue
homogenizer, the number of viable organisms was determined by
serial 10-fold dilutions and subsequent inoculation onto 7H10 agar
plates. Cultures were incubated at 37 °C in ambient air for 4 weeks
prior to counting. Bacterial counts were measured and compared
with the counts from negative controls (vehicle treated) in lung
and in spleen (Table 2).
(9) (a) Liddell, J. R. Pyrrolizidine alkaloids. Nat. Prod. Rep. 1998, 15,
363–370. (b) Michael, J. P. Indolizidine and quinolizidine alkaloids.
Nat. Prod. Rep. 1997, 14, 619–636.
(10) (a) Chande, M. S.; Verma, R. S.; Barve, P. A.; Khanwelkar, R. R.
Facile synthesis of active antitubercular, cytotoxic and antibacterial
agents: a Michael addition approach. Eur. J. Med. Chem. 2005, 40,
1143–1148. (b) Dandia, A.; Sati, M.; Arya, K.; Sharma, R.; Loupy,
A. Facile One-Pot Microwave Induced Solvent-Free Synthesis and
Antifungal, Antitubercular Screening of Spiro[1,5]-Benzothiazepin-
2,3′[3′H]indol-2[1′H]-ones. Chem. Pharm. Bull. 2003, 51, 1137–1141.
(11) Sriram, D.; Yogeeswari, P.; Madhu, K. Synthesis and in vitro
antitubercular activity of some 1-[(4-sub)phenyl]-3-(4-{1-[(pyridine-
4-carbonyl)hydrazono]ethyl}phenyl) thiourea. Bioorg. Med. Chem.
Lett. 2006, 16, 876–878.
(12) Biava, M.; Porretta, G. C.; Poce, G.; Supino, S.; Deidda, D.; Pompei,
R.; Molicotti, P.; Manetti, F.; Botta, M. Antimycobacterial Agents.
Novel Diarylpyrrole Derivatives of BM212 Endowed with High
Activity toward Mycobacterium tuberculosis and Low Cytotoxicity.
J. Med. Chem. 2006, 49, 4946–4952.
(13) Shaharyar, M.; Siddiqui, A. A.; Ali, M. A.; Sriram, D.; Yogeeswari,
P. Synthesis and in vitro antimycobacterial activity of N1-nicotinoyl-
3-(4′-hydroxy-3′-methylphenyl)-5-[(sub)phenyl]-2-pyrazolines. Bioorg.
Med. Chem. Lett. 2006, 16, 3947–3949.
(14) Ranjith Kumar, R.; Perumal, S.; Senthilkumar, P.; Yogeeswari, P.;
Sriram, D. A highly atom economic, chemo-, regio- and stereoselective
synthesis, and discovery of spiro-pyrido-pyrrolizines and pyrrolidines
as antimycobacterial agents. Tetrahedron 2008, 64, 2962–2971.
(15) Ranjith Kumar, R.; Perumal, S.; Senthilkumar, P.; Yogeeswari, P.;
Sriram, D. An atom efficient, solvent-free, green synthesis and
antimycobacterial evaluation of 2-amino-6-methyl-4-aryl-8-[(E)-aryl-
methylidene]-5,6,7,8-tetrahydro-4H-pyrano[3,2-c]pyridine-3-carboni-
triles. Bioorg. Med. Chem. Lett. 2007, 17, 6459–6462.

DiscoVery of Antimycobacterial Spiro-piperidin-4-ones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5735
(16) Crystallographic data (excluding structure factors) for 3a in this article
have been deposited with the Cambridge Crystallographic Data Centre
as supplementary publication number CCDC 686497. Copies of the
data can be obtained, free of charge, on application to CCDC, 12 Union
Road, Cambridge CB2 1EZ, UK (fax: +44 (0) 1223 336033 or e-mail:
deposit@ccdc.cam.ac.uk). (b) Kumar, V. V.; Gayathri, D.; Velmuru-
gan, D.; Ravikumar, K.; Sridhar, G. Acta. Cryst. 2007, 63, 2211–
2212.
(17) Ranjith Kumar, R.; Perumal, S. Sacrificial azomethine ylide cycload-
dition controlled chemoselective nitrile oxide cycloaddition to 1-meth-
yl-3,5-bis[(E)-arylmethylidene]tetrahydro-4(1H)-pyridinones: forma-
tion of mono-spiro-isoxazolines. Tetrahedron 2007, 63, 12220–12231.
(18) Crystallographic data (excluding structure factors) for 5g in this article
have been deposited with the Cambridge Crystallographic Data Centre
as supplementary publication number CCDC 686496. Copies of the
data can be obtained, free of charge, on application to CCDC, 12 Union
Road, Cambridge CB2 1EZ, UK (fax: +44 (0) 1223 336033 or e-mail:
deposit@ccdc.cam.ac.uk).H
(19) National Committee for Clinical Laboratory Standards. Antimycobac-
terial susceptibility testing for Mycobacterium tuberculosis; Proposed
Standard M24-T; National Committee for Clinical Laboratory Stan-
dards: Villanova, PA, 1995.
(20) Sridhar, G.; Raghunathan, R. Rapid Access for the Synthesis of 1-N-
Methyl-spiro[2.
3′]oxindole-spiro[3.7′′](3′′-aryl)-5′′-methyl-
3′′,3a′′,4′′,5′′,6′′,7′′-hexahydro-2H-pyrazolo[4,3-c]pyridine-4-aryl-pyr-
rolidines Through Sequential 1,3-Dipolar Cycloaddition and Annulation.
Synth. Commun. 2006, 36, 21–29.
(21) Gundersen, L. L.; Nissen-Meyer, J.; Spilsberg, B. Synthesis and
Antimycobacterial Activity of 6-Arylpurines: The Requirements for
the N-9 Substituent in Active Antimycobacterial Purines. J. Med.
Chem. 2002, 45, 1383–1386.
JM800545K