2,4-Dinitroimidazole: Microwave Assisted Synthesis and Use in Synthesis of 2,3-Dihydro-6-nitroimidazo[2,1-b]oxazole Analogues with Antimycobacterial Activity

Article abstract of DOI:10.1002/jhet.5570410108

2,4-Dinitroimidazole (2,4-DNI), an important starting material for nitroimidazooxazole and nitroimidazooxazine types of antitubercular agents was synthesized by rearrangement of 1,4-dinitroimidazole (1,4-DNI) under microwave irradiation. Various new nitro

Full text of DOI:10.1002/jhet.5570410108

Jan-Feb 2004
1
2,4-Dinitroimidazole: Microwave Assisted Synthesis and Use in
Synthesis of 2,3-Dihydro-6-nitroimidazo[2,1-b]oxazole Analogues
with Antimycobacterial Activity
Kankan Bhaumik,b and K. G. Akamanchia*
a. Department of Pharmaceutical Sciences & Technology, Mumbai University Institute of Chemical
Technology, Matunga, Mumbai-400 019. India.
b. Chembiotek Research International Pvt. Ltd., Block BN, Sector V, Salt Lake, Kolkata- 700 091, India
E-mail: kgap@rediffmail.com.
Fax: +91-22-24145614
2,4-Dinitroimidazole (2,4-DNI), an important starting material for nitroimidazooxazole and nitroimidazoox-
azine types of antitubercular agents was synthesized by rearrangement of 1,4-dinitroimidazole (1,4-DNI) under
microwave irradiation. Various new nitroimidazooxazoles analogues were prepared using 2,4-DNI and were
tested preliminarily against Mycobacterium tuberculosis, H Rv strain. Some were found to be active.
37
J. Heterocyclic Chem., 41, 1 (2004).
Introduction.
Crystalline 2,4-dinitroimidazole (2,4-DNI) [1a-d] is an
explosive, high-energy material. It is much less sensitive
than RDX and HMX, and is about 15-20% more energetic
than TATB [2]. It has both good thermal stability and
impact insensitivity [1a]. It can be synthesized by reflux-
ing 1,4-DNI in chlorobenzene [1d]. However, reaction is
slow and requires more than 24 h (~50 hours) to give a
yield of 80%. It can also be prepared in 63% yield by nitra-
tion of 2-nitroimidazole [3]. However, 2-nitroimidazole is
not readily available. A process to convert 1,4-DNI into
2,4-DNI by direct melting is reported in a patent [1a], how-
e v e r, we were unable to reproduce this molten state
rearrangement. The use of microwave is finding wide
application in acceleration of many reactions [4a,4b]. We
considered the possibility of acceleration of conversion of
1,4-DNI to 2,4-DNI under microwave irradiation and
observed that the reaction was very fast, and within 2 min
more than 70% conversion was observed. The proceedure
gave reproducible results and is convenient to employ on a
routine basis.
Apart from the high energetic value, 2,4-DNI is a very
good radiosensitizing agent and also a valuable starting
material for the synthesis of many potent chemotherapeu-
tic agents [3,5a,5b]. Nitroimidazooxazoles [6] and
nitroimidazooxazines [7] have also being well investigated
for their antitubercular activity. CGI-17341 (I) and PA-824
(II) (Figure 1) are two of such agents that have exhibited
potent activities against both active and latent tubercle
bacilli. It is important to note that nitroimidazooxazoles
and nitroimidazooxazines do not generate any cross-resis-
tance [7] with antitubercular drugs such as INH, rifampin,
streptomycin, ciprofloxacin, etc. Amongst a series of
nitroimidazooxazoles possessing antimycobacterial activ-
ity, CGI-17341 (I) is the lead compound. Our preliminary
efforts are directed towards the synthesis of newer poten-
tial analogues of bicyclic nitroimidazooxazole pharma-
cophore using 2,4-DNI as a starting material.
Figure 1. The chemical structures of CGI-17341(I), PA-824 (II), Dup-
721(III) and Linezolid (IV).
The pharmacophore, bicyclic nitroimidazooxazole that
is present in CGI-17341 (I) has close similarity with oxa-
zolidinones like Dup-721 [8] and (III) Linezolid [9] (IV)
(Figure 1) that are potent anti-bacterial agents against
gram-positive bacteria. This encouraged us to synthesize
newer analogues for anti-tubercular activity. Also random
derivatization, and application of simple physicochemical
principles across nitroimidazooxazole pharmacophore of
CGI-17341 (I) may improve the understanding of structure
activity relationships.
Chemistry.
An improved procedure for synthesis of 2,4-DNI 2 start-
ing from 1,4-DNI 1 was developed using microwave irra-
diation. 2,4-DNI was efficiently prepared in a short time.
All nitroimidazooxazole analogues were synthesized start-
ing from 4-nitroimidazole [10] (Scheme 1). Reaction of
2,4-DNI with racemic epichlorohydrin generated a mix-
ture containing three compounds [11]. The desired com-
pound 3 was isolated by a solvent treatment and character-
ized. The aryl sulfide analogues 4 were prepared by react-
ing compound 3 with the corresponding arylthiophenols.

2
K. Bhaumik, and K. G. Akamanchi
Vol. 41
The sulfides 4 thus obtained were oxidized into sulfone
derivatives using hydrogen peroxide in the presence of a
catalytic amount of sodium tungstate as a reagent system
to get a set of sulfones 5. All the sulfide (4a-d) and sulfone
(5a-d) analogues (Figure 2) were tested against the
Mycobacterium tuberculosis H37RV strain.
and power level of irradiation. The best results are tabu-
lated in Table 1.
Table 1
MW Assisted Rearrangement of 1,4-DNI to 2,4-DNI
Solvent
Concentration Power Level Time of Exposure Yields
(%) [a]
(%)
(min)
(%) [b]
PhCl
PhCN
PhCN:PhCl
(3:7)
10
10
10
90
50
90
10
2
5
51
79
71
[a] 10% was found to be an optimum concentration; at higher concentra-
tion colored impurities developed; [b] yields are the isolated yields after
silica gel column chromatography.
4
5
4a,
4b,
4c,
4d,
5a Ar = 2-thienyl;
5b, Ar = phenyl;
5c, Ar = 4-chlorophenyl;
5d, Ar = 4-methylphenyl
To have a ready comparison side by side with the con-
ventional heating, a set of parallel experiments were per-
formed. In all cases the reaction required longer times.
The results are given in Table 2.
Figure 2. Chemical structures of racemic sulfides and sulfones synthesized
and tested against the M. tuberculosis H Rv strain.
37
Scheme 1
Table 2
Comparative Results of Conventional Heating, MW assisted, and Molten
state Rearrangement of 1,4-DNI to 2,4-DNI
Series
No.
Solvent
Temp.
(°C)
Time
(h/min)
Yields
(%)
Reference
1
2
3
PhCl
PhCN
PhCN:PhCl
(3:7)
125-130
125-130
125-130
50 h
12 h
12 h
80
51
48
1d
--
--
4
5
PhCN
---
MW
95-98
(Molten State)
2 min 79
25 min No reaction
-
1a
To demonstrate the microwave activity of 1,4-DNI in
PhCN a series of parallel experiments were run and the
temperatures were recorded at the end of 1 min of expo-
sure (by external thermometer) at 50% power level. Figure
3 clearly shows that 1,4-DNI is microwave active, since in
the initial one minute temperature rises with increase in the
concentration of 1,4-DNI.
Reagents, conditions and isolated yields: (i) AcOH, Fuming nitric acid (Sp. Gr.
1.52) and Ac O, 67.60%. (ii) MW/solvent, 79 % (iii) Epichlorohydrin/ EtOH,
2
reflux, 24 h, 22% (iv) ArSH, K CO , DMF, rt, 22-35% (v) 30% H O ,
2
3
2 2
Na WO .2H O, Ethyl acetate: water (5:1), rt, 55-65%.
2
4
2
Results and Discussion.
All microwave reactions were carried out by simply
irradiating a solution of 1,4-DNI in a suitable solvent by
using household microwave oven (IFB make) modified
with refluxing system; and a power of 750 watts with max-
imum frequency of 2450 MHz. The reaction was standard-
ized with respect to solvent, concentration, reaction time,
Figure 3

2,4-Dinitroimidazole: Microwave Assisted Synthesis
Jan-Feb 2004
3
Table 1
The studies showed that conversion of 1,4-DNI
increases as the concentration increases up to 30% with no
significant increase in conversion thereafter (Figure 4).
However, formation of colored impurities and decomposi-
tion were observed at 20% concentration and above.
Activity Test Tesults Against Mycobacterium tuberculosis H Rv Strain
37
Compound No
(Figure 2)
In vitro activity
(MIC, µg/ml) [a,b,c]
3
200
4a
4b
4c
4d
5a
5b
5c
5d
-
-
-
200
200
-
200
-
[a] All results were recorded in form of minimum inhibitory concentra-
tion (MIC) defined as the minimum drug concentrations to inhibit the
growth of M. tuberculosis strain H Rv; [b] all compounds are soluble in
37
DMSO and range of concentration used 200 µg/ml- 2 µg/ml (blank cell
indicate in this concentration range compound is inactive); [c] MIC of
CGI-17341 (I) is 0.06 µg/ml and PA-824 (II) is 0.015 µg/ml.
Figure 4
recorded on 60 MHz JEOL NMR. Mass spectra were recorded
on a Finnigan Mat 1020B Mass Spectrometer and using EI at 70
eV for ionization. The Microwave reactions were carried out
using an IFB make household microwave oven modified with
refluxing system, and a power of 750 watts with a maximum fre-
quency of 2450 MHz. Unless otherwise specified, reagents used
for the synthesis were obtained commercially.
Therefore, 10% was chosen as an optimum concentra-
tion for subsequent experiments. The reactions were
repeated several times and results were highly repro-
ducible. No explosions were experienced. Nevertheless
care must be taken for potential explosion hazards. In all
the reactions performed, formation of 4-nitroimidazole
was observed. The rearrangement was also attempted by
microwave irradiation of a neat pure crystalline sample of
1,4-DNI and also 1,4-DNI on silica gel. In the former case
an explosion took place (CAUTION!) and in the latter
decomposition occurred where charred material was
obtained with small amounts of 2,4-DNI.
The various synthesized nitroimidazooxazoles were
tested preliminarily against H37Rv strain. Minimum
Inhibitory Concentrations (MICs) were considerably low,
however, some of the compounds showed activity at 200
µg/ml (Table 3). There was no clear structural relationship
of the synthesized analogues with respect to observed
activity. These newly tested analogues may be of interest
to medicinal chemists, for further structural variation and
to get some insight into structural requirements.
1,4-Dinitroimidazole (1,4-DNI) 1.
A stirred solution of 4-nitroimidazole (10.00 g, 88.5 mmol) in
glacial acetic acid (20 mL) was cooled to 0 °C by external cool-
ing in an ice-bath. Freshly prepared fuming nitric acid (6.5 mL)
(Sp.gr 1.52) was added slowly followed by addition of acetic
anhydride (18 mL). Contents of the flask were stirred at this tem-
perature till complete dissolution of solid takes place and solution
becomes golden yellow in colour (about 2 h). The mixture was
stirred at room temperature overnight. Reaction was quenched by
pouring the contents into ice-cold water (125 mL) with vigorous
stirring and the solid precipitated was collected by filtration and
washed with ice-cold water (about 100 mL). This solid was fur-
ther purified by recrystallization from benzene to give 1,4-DNI as
white fine needles (9.45 g, 67.60%). m.p. 92 °C (Lit. [1a], 92 °C);
1
H-NMR (CDCl ) δ 8.7 (s, 1H, H-5), 8.5 (s, 1H, H-2).
3
Microwave Preparation of 2,4-Dinitroimidazole (2,4-DNI) 2.
Caution!: We never experienced any explosion in any of our
experiments and while handling 1,4-DNI and 2,4-DNI. However,
we would like to caution that these compounds are potential
explosives [1a], and care must be taken while carrying out these
reactions.
1,4-DNI (1.0 g, 6.33 mmol) was taken in benzonitrile (10 mL)
and irradiated in a microwave oven at 50% power level for 2 min.
The reaction mixture was cooled to room temperature and the
solid precipitated was collected by filtration to give crude 2,4-
DNI. The mother liquor was treated with hexane (25 mL) to
recover a second crop of 2,4-DNI. The crude 2,4-DNI was puri-
fied by silica gel (60-120 mesh) chromatography using ethyl
acetate:hexane (8:2) to give crystalline 2,4-DNI (0.79 g, 79%).
2,4-DNI can also be purified by recrystallization from a mixture
of acetonitrile and chloroform. m.p. 264-267 °C (Lit. [1c], 264-
Conclusion.
An improved and a convenient method based on
microwave irradiation has been developed for the synthe-
sis of 2,4-DNI. Using 2,4-DNI various sulfide and sulfone
analogues of nitroimidazooxazoles were prepared. All the
newly synthesized analogues were screened for antimy-
cobacterial activity against H37Rv strain. Some of these
analogues showed moderate antitubercular activity.
EXPERIMENTAL
Melting points are uncorrected. Infrared (IR) spectra were
1
recorded on BUCK Scientific IR Spectrometer and H NMR

4
K. Bhaumik, and K. G. Akamanchi
Vol. 41
Table 2
Physical Constant and Spectral Data
No.
3
Yield
(%)
Melting Point
(°C)/State
(recryst solvent)
NMR and MS
22
30
170-171(White solid)
(ethanol)
d 8.03 (s, 1H, H-5); 5.60 (m, 1H, H-2); 4.58-4.05 (m, 4H, H-3 & CH Cl).
2
MS (EI): m/z 205/203 (M , Cl/ M , Cl), 189/187, 170/168, 159/157, 147/145,
131/129, 118, 102, 93, 83, 75, 67, 56, 41(base peak). Anal. Calcd. for C H N O Cl: C,
+
37
+ 35
6
6 3 3
35.38; H, 2.93; N, 20.60 ;Cl, 17.44. Found: C, 35.32; H, 2.91; N, 20.00; Cl, 17.49.
δ 8.03 (s, 1H, H-5), 7.18 (m, 1H, thiophene), 7.11-6.98 (m, 2H, thiophene), 5.44-5.24
4a
184-185(White solid)
(benzene)
+
(m, 1H, H-2), 4.38-4.01(m, 2H, H-3), 2.39 (m, 1H, CH S). MS (EI): m/z 283 (M ),
2
255, 237, 227, 206, 149, 129, 115(base peak), 91, 71, 57, 41. Anal. Calcd. for
C
14.72; S, 22.55.
H N O S : C, 42.25; H, 3.52; N, 14.79; S, 22.54. Found: C, 42.11; H, 3.51; N,
10 10 3 3 2
4b
4c
35
30
174-175 (light yellow solid)
(benzene)
δ 8.03(s, 1H, H-5), 7.29 (m, 5H, aromatic), 5.63-5.23(m, 1H, H-2), 4.56-4.04 (m, 2H,
+
H-3), 2.43 (m, 2H, CH S). MS(EI): m/z 277(M ), 203, 149, 135, 123, 109, 93, 77, 67,
2
41(base peak). Anal. Calcd. for C
H N O S: C, 51.99; H, 3.97; N, 15.16; S, 11.55.
12 11 3 3
Found: C, 51.85; H, 3.93; N, 15.11; S, 11.59.
δ 8.03 (s, 1H, H-5), 7.79-7.31(m, 4H, aromatic), 5.54-5.29(m, 1H, H-2), 4.38-4.00
168-169 (Yellowish solid)
(benzene)
+
37
+ 35
(m, 2H, H-3), 2.38(m, 2H, CH S). MS (EI): m/z 313/311(M , Cl/ M , Cl), 239/237,
2
200, 183, 169, 159/157(base peak), 145/143,108, 93, 67, 41. Anal. Calcd. for
C
H N O SCl: C, 46.22; H, 3.21; N, 13.48; S, 10.27; Cl, 11.39. Found: C, 46.20; H,
12 10 3 3
3.11; N, 13.42; S, , 10.30; Cl 11.30
4d
32.50
165-166(White solid)
(benzene)
δ 8.03 (s, 1H, H-5), 7.18 (m, 1H, thiophene), 7.11-6.98 (m, 2H, thiophene), 5.44-5.24
+
(m, 1H, H-2), 4.38-4.01(m, 2H, H-3), 2.39(m, 1H, CH S). MS (EI): m/z 283 (M ),
2
255, 237, 227, 206, 149, 129, 115(base peak), 91, 71, 57, 41. Anal. Calcd. for
C
H N O S: C, 53.61; H, 4.47; N, 14.43; S, 11.00. Found: C, 53.58; H, 4.46; N,
13 13 3 3
14.41; S, 11.02.
MS(EI): m/z 315 (M ), 287, 149. Anal. Calcd. for C H N O S : C, 37.97; H, 3.16; N,
+
5a
5b
5c
61
70
160-161 (White solid)
(benzene)
212-214(Yellowish solid)
(benzene)
10
9 3 5 2
13.29; S, 20.25. Found: C, 37.91; H, 3.06; N, 13.30; S, 20.50.
+
MS(EI): m/z 309 (M ), 235, 41. Anal. Calcd. for C H N O S: C, 46.60; H, 3.56; N,
12 11 3 5
13.59; S, 10.36. Found: C, 46.52; H, 3.55; N, 13.57; S, 10.38.
+
37
35
55.50
206-208(White solid)
(benzene)
MS(EI): m/z 345/343 (M , Cl,/ Cl),271/269,149,41. Anal. Calcd. for
N O SCl: C, 41.92; H, 2.91; N, 12.22; S, 9.32; Cl, 10.33. Found: C, 41.88; H,
C
H
12 10
3 5
2.90; N, 12.21; S, 9.33; Cl 10.38
+
5d
65
174-175(White solid)
(benzene)
Mass Spectra (EI): m/z 323(M ), 278,246,149. Anal. Calcd. for C
H
N O S: C,
3 5
13 13
48.29; H, 4.02; N, 13.00; S, 9.91. Found: C, 48.20; H, 3.98; N, 13.02; S, 9.99.
1
°C); H-NMR (CDCl + DMSO-d ): δ 9.6 (s, 1H (exchanges
2 - ( 4 - M e t h y l t h i o p h e n o x y ) m e t h y l - 2 , 3 - d i h y d r o - 6 - n i t r o i m i-
dazo[2,1-b]oxazole (4d).
3
267
with D O), NH), 8.60 (s, 1H, H-5).
6
2
Anal. Calcd. for C H N O : C, 22.72; H, 1.27; N, 35.44.
3
Found: C, 22.72; H, 1.25; N, 35.41.
2 4 4
Compound 3 (100 mg, 0.49 mmol) and 4-methylthiophenol
(61 mg, 0.49 mmol) were taken in dry DMF (2 mL) with potas-
sium carbonate (68 mg, 0.49 mmol). The reaction mixture was
then stirred at room temperature for 10 min. It was quenched with
copious volume of water (ca. 30 mL). The solid separated out
was collected by filtration, washed with dilute sodium hydroxide
solution and purified by crystallization from boiling benzene to
give 4d as white solid (50 mg, 32.65%). m.p.,165-166 °C, IR
2-Chloromethyl-2,3-dihydro-6-nitroimidazo[2,1-b]oxazole (3).
A mixture of 2,4-DNI 2, (2.5 g, 5.80 mmol), absolute ethanol
(100 mL) and epichlorohydrin (25 mL) was heated under reflux
for 20 h. Solvent was removed under rotary evaporator and the
residue obtained was treated with hexane (30 mL), and allowed
to settle. The upper hexane layer was decanted and to the remain-
ing thick slurry was added a chloroform:methanol mixture (2:1)
(15 mL) with stirring. The solid that precipitated was collected by
filtration, dried and was recrystallized from boiling ethanol to
give the product 3 (0.70 g, 22%), m.p. 170-171 °C (Lit. [3,5b],
170-171 °C); IR (KBr): 1607, 1516, 1472, 1459, 1335, 1319,
(KBr): 3099, 2910, 1604, 1511, 1460, 1318, 1282, 1119, 1081,
1
987, 800, 701; H-NMR (DMSO-d ): δ 8.03 (s, 1H, H-5), 7.46-
6
7.18 (m, 4H, aromatic), 5.49-5.31 (m, 1H, H-2), 4.56-3.97 (m,
2H, H-3), 2.41 (m, 2H, CH S), 2.12 ( s, 3H, CH ); MS (EI): m/z
2
3
+
291(M ), 279, 245, 217, 167, 149 (base peak), 137, 123, 91, 79,
67, 57.
Anal. Calcd. for C H N O S: C, 53.61; H, 4.47; N, 14.43; S,
1
746; H-NMR (DMSO-d ): δ 8.03 (s, 1H, H-5), 5.60 (m, 1H, H-
+
6
2), 4.58-4.05 (m, 4H, H-3 & CH Cl); MS (EI): m/z 205/203 (M ,
13 13
3 3
11.00. Found: C, 53.58; H, 4.46; N, 14.41; S, 11.02.
Compounds 4a, 4b, 4c were also synthesized by the above
procedure.
2
37
+ 35
Cl/ M , Cl), 189/187, 170/168, 159/157, 147/145, 131/129,
118, 102, 93, 83, 75, 67, 56, 41 (base peak).

2,4-Dinitroimidazole: Microwave Assisted Synthesis
Jan-Feb 2004
5
REFERECES AND NOTES
2-(4-Methylphenylsulfonyl)methyl-2, 3-dihydro-6-nitroimi-
dazo[2,1-b]oxazole (5d).
[1a] R. Damavarapu, K. Jayasuriya, T. Vladimiroff and S.
Iyer, US Patent 5,387,297 (1995); Chem. Abstr., 1 2 2, P239709n
(1995); [b] A. Deerfield, F. L. Beach, in Nitroazoles: The C-Nitro
Derivatives of Five Membered N- and N,O Heterocycles (Ed. J. A.
Boyer) VCH, New York,1986, pp.79; [c] S. Bulusu, R. Damavarapu,
J. R. Autera, R. Behrens, Jr., L. M. Minier, J. Villanueva, K.
Jayasuriya and T. Axenrod, J. Phys. Chem., 99, 5009 (1995); [d] K.
Nagarajan, V. Sudarsanam, T George, S. J. Shenoy, V. V. Iyer A. P.
Kaulgud, Ind. J. Chem., 21(B), 1022 (1982).
[2] RDX (Royal Demolition explosive) is cyclotrimethylen-
etrinitramine, HMX (High melting explosive), is cycloteramethyl-
enetetramine, and TATB is triamino-tribenzene.
[3] K. C. Agrawal, K. B. Bears, R. K. Sehgal, J. N. Brown, P.
E. Rist and W. D. Rupp, J. Med. Chem., 22, 583 (1979).
[4a] A. K. Bose, M. S. Manhas, S. N. Ganguly, A. H. Sharma
and B. K. Banik, Synthesis, 1578 (2002); [b] N. Kuhnert, Angew.
Chem. Int. Ed., 41, 1863 (2002).
To a stirred slurry of compound 4d (50 mg, 0.171 mmol) and
sodium tungstate dihydrate (5 mg, 0.015 mmol) in ethyl
acetate:water (12 mL, 5:1) was added H O (30 %v/v, 5 mL) and
2
2
stirring was continued for 2 h. Then, the reaction was quenched by
addition of saturated sodium bisulfite solution (5 mL) followed by
extraction with ethyl acetate (2 × 25 mL). The organic layer was
dried over sodium sulfate and evaporated under vacuum. The
solid obtained was crystallized from hot benzene to give 5d as
white solid (36mg. 64.88%), m.p. 174-175 °C; MS (EI): m/z
+
323(M ), 278 (base peak), 262, 246, 167, 149, 107, 65, 44.
Anal. Calcd. for C H N O S: C, 48.29; H, 4.02; N, 13.00; S,
13 13
3 5
9.91. Found: C, 48.20; H, 3.98; N, 13.02; S, 9.99.
Compound 5a, 5b, 5c were also synthesized by the above
procedure.
Pharmacology Methods.
Bacterial Strains.
[5a] J. C. Asquith, M. E. Walts, C. E. Patel and G. E. Adams,
Radiat. Res., 60, 108 (1979); [b] R. K. Sehgal, M. W. Webb and K.
C. Agrawal, J. Med. Chem., 24, 601 (1981).
The bacterial strain used in this study was M. tuberculosis
H Rv, which served as the wild type strain of M. tuberculosis.
37
[6] D. R. Ashtekar, R. C. Perira, K. Nagarajan, N
Vishvanathan, A. D. Bhatt and W. Ritter, A n t i m i c rob . Agents.
Chemother., 37, 183 (1993).
[7] C. K. Stover, P. Warrener, D. R. Va n d a v a n t e r, D. R
Sherman, T. M. Arain, M. H. Langherne, S. W. Anderson, J. A.
Towell, Y. Yuan, D. N. Mac Murray; B. N. Kretsmith, C. E. Barry
(II) and W. R. Baker, Nature, 405, 962 (2000).
[8] W. A. Gregory, D. R. Brittelli, C. L. J. Wang, M. A.
Wuonola, R. J. McRipley, D. C. Eustice, V. S. Eberly, A. M. Slee, M.
Forbes and P. T. Bartholmew, J. Med. Chem., 32,1673 (1989).
[9] S. J. Brickner, D. K. Hutchinson, M. R. Barbachyn, P. R.
Manninen, D. A. Ulanowicz, S. A. Garmon, K. C. Grega, S. K.
Hendges, D. S. Toops, C. W. Ford and G. E. Zurenko, J. Med.
Chem., 39, 673 (1996).
Determination of MICs.
The bacterial suspension were prepared by diluting organisms
grown in Middle brook 7H9 broth medium (with ADC supple-
ment) at 37 °C for 10 days with 0.05% Tween 80 in distilled
6
water to give a bacterial concentration of ca. 10 cfu/ml. The bac-
terial suspension 0.1 ml was taken in test tubes (ca. 10 ml vol-
ume) containing 200-2 µg of drug per mL. The drugs were ini-
tially dissolved in DMSO and the required amount was added to
the broth medium (with ADC supplement) containing 0.05%
Tween 80 in distilled water. The MICs of the drugs were deter-
mined 14 days after the start of cultivation at 37 °C in incubator.
The MICs of the drugs were determined as the minimum drug
concentrations for complete inhibition of organism growth.
[10] R. G. Fargher and F. L. Pyman, J. Chem. Soc., 115, 217
(1919).
Acknowledgments.
KB acknowledges financial support by the UGC of India and
Haffkine Institute, Mumbai, India for the antimycobacterial testing.
[11] K. Nagarajan, R. G. Shankar, S. Rajappa, S. J. Shenoy
and R. Costa-Pereira, Eur. J. Med. Chem., 24, 631 (1989).