Synthesis, absolute stereochemistry and molecular design of the new antifungal and antibacterial antibiotic produced by Streptomyces sp.201

Article abstract of DOI:10.1016/j.bmcl.2004.04.025

The absolute stereochemistry of the new antifungal and antibacterial antibiotic produced by Streptomyces sp.201 has been established by achieving the total synthesis of the product. A series of analogues have also been synthesized by changing the side chain and their bioactivity assessed against different microbial strains. Among them, 1e (R=C₈H₁₇) was found to be the most potent with MIC of 8μg/mL against Mycobacterium tuberculosis, 12μg/mL against Escherichia coli and 16μg/mL against Bacillus subtilis 6μg/mL against Proteus vulgaris. This was followed by 1b (R=C₅H₁₁) with MIC of 10-20μg/mL range and 1d (R=C ₇H₁₅) with MIC of 14-24g/mL, whereas 1a (R=C ₄H₉) and 1f (R=C₁₈H₃₅) were found to be completely inactive. Besides, 1c (R=C₆H₁₃) showed certain extent of antibacterial activity in the range of 24-50μg/mL. Mycobacterium tuberculosis was very sensitive to 1e (R=C₈H ₁₇) with MIC of 8μg/mL. Antifungal activity of analogues 1d (R=C₇H₁₅) and 1e, (R=C₈H₁₇) against Fusarium oxysporum and Rhizoctonia solani were found promising with MFCs in the 15-18μg/mL range.

Full text of DOI:10.1016/j.bmcl.2004.04.025

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3571–3574
Synthesis, absolute stereochemistry and molecular design
of the new antifungal and antibacterial antibiotic
produced by Streptomyces sp.201
J. Boruwa,a B. Kalita,a N. C. Barua,a,* J. C. Borah,a
S. Mazumder,b D. Thakur,b D. K. Gogoib and T. C. Borab
aNatural Products Chemistry Division, Regional Research Laboratory (CSIR), Jorhat 785006, Assam, India
bBiochemistry Division, Regional Research Laboratory (CSIR), Jorhat 785006, Assam, India
Received 13 February 2004; revised 6 April 2004; accepted 8 April 2004
Abstract—The absolute stereochemistry of the new antifungal and antibacterial antibiotic produced by Streptomyces sp.201 has
been established by achieving the total synthesis of the product. A series of analogues have also been synthesized by changing the
side chain and their bioactivity assessed against different microbial strains. Among them, 1e (R ¼ C8H17) was found to be the most
potent with MIC of 8 lg/mL against Mycobacterium tuberculosis, 12 lg/mL against Escherichia coli and 16 lg/mL against Bacillus
subtilis 6 lg/mL against Proteus vulgaris. This was followed by 1b (R ¼ C5H11) with MIC of 10–20 lg/mL range and 1d (R ¼ C7H15)
with MIC of 14–24 g/mL, whereas 1a (R ¼ C4H9) and 1f (R ¼ C18H35) were found to be completely inactive. Besides, 1c (R ¼ C6H13)
showed certain extent of antibacterial activity in the range of 24–50 lg/mL. Mycobacterium tuberculosis was very sensitive to 1e
(R ¼ C8H17) with MIC of 8 lg/mL. Antifungal activity of analogues 1d (R ¼ C7H15) and 1e, (R ¼ C8H17) against Fusarium oxy-
sporum and Rhizoctonia solani were found promising with MFCs in the 15–18 lg/mL range.
Ó 2004 Elsevier Ltd. All rights reserved.
O
1. Introduction
O
NHNH2
OR
In our continued search for novel microbial metabolites
having agricultural and pharmaceutical potential a
number of Streptomyces strains were screened. This has
resulted in characterization of several antibiotic-pro-
ducing strains isolated from untapped mega biodiversity
hot zone of Indo-Burma belt. We have previously re-
ported the isolation and structure elucidation of a broad
spectrum and novel antibiotic 2-methylheptyl isonicoti-
nate 1 (R ¼ 2-methylheptyl), a natural analogue of anti-
TB drug isoniazid.1–5 In view of its promising in vitro
activity against Mycobacterium tuberculosis H37rv, the
compound has been investigated in details. In this article
we have reported the total synthesis of both the isomers
of the molecule in the chirally pure form as well as the
outcome of our studies on minimum structural
requirement for optimum activity.
N
N
1 R = 2-methylheptyl
Isoniazid
2. Materials and methods
2.1. Test pathogens
Fusarium semitectum Berkeley and Ravenel and Rhiz-
octonia solani Kuehn were obtained through the cour-
tesy of ICRISAT, Hyderabad, India. Pathogenic
bacteria such as M. tuberculosis MTCC 300, E. coli
DSMZ 1103 and P. vulgaris ATCC 6380, B. subtilis
ATCC 11774 were obtained from MTCC and Gene
Bank, IMTECH, Chandigarh, India.
2.2. Antimicrobial agents
Keywords: 2-Methylheptylisonicotinate; Streptomyces sp.; Synthesis;
Stereochemistry; MIC.
Stock solution of antibiotic 1 and their derivatives 1a–f,
(R)-(þ)-1 and (S)-(ꢀ)-1 were prepared in dimethyl
* Corresponding author. Tel.: +91-0376-2370055; fax: +91-0376-237-
0011; e-mail: ncbarua2000@yahoo.co.in
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.04.025

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J. Boruwa et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3571–3574
sulfoxide. Further dilutions were made in phosphate
buffer, pH 7.4 and with culture broth as diluent. The
antimicrobial agents were tested in the final concentra-
tion range of 1–600 lg/mL.
lowest concentration at which there was 100% inhibition
of growth compared to antibiotic free control.
2.3. Medium
3. Results and discussion
M. tuberculosis was grown in M-33 medium (g/L): Yeast
extract 2.5, Tryptone 5.0, Glucose 1.0, pH 7.0. B. sub-
tilis, E. coli and P. vulgaris were grown in Nutrient broth
medium (Hi-Media Laboratories, Mumbai, India).
F. semitectum and R. solani were grown in M2 agar
medium (g/L): Yeast extract 5.00, NaCl 10.00, KH2PO4
0.10, MgSO4Æ7H2O 0.05, dextrose 10.00, agar 15.0,
pH 7.1.
It has been already established that 2-methylheptyliso-
nicotinate 1, is a natural analogue of potent anti-TB
drug isoniazid (INH).1–5 The mode of action of INH
also have been firmly established. New insights into the
mechanism of INH have been provided by the applica-
tion of genetic tools.12 However, another interesting
observation is that INH is not reported to have anti-
fungal properties. In contrast, compound 1 has shown
promising activity2 against fungi viz Fusarium semitec-
tum and Rhizoctonia solani. As the structural difference
between 1 and INH is only in the side chain; hence, the
side chain might play a vital role in exhibiting activity in
certain fungi. In order to study the effect of the chain
length, and to establish minimum structural requirement
for optimum bioactivity, a series of analogues of 1
(R ¼ 2-methylheptyl) were synthesized by changing the
chain length, and these analogues were tested against
several bacterial and fungal test pathogens. The results
have been presented in Table 1. It is evident from Table
1 that the length of the chain has indeed got a major
effect on bioactivity. The analogue with a carbon chain
length of 8, 1e (R ¼ C8H17) showed maximum activity
with MICs in the 8 lg/mL against M. tuberculosis, 12 lg/
mL against E. coli, 16 lg/mL against B. subtilis and 6 lg/
mL against P. vulgaris. This was followed by 1b
(R ¼ C5H11) a 5-carbon chain with MICs in the 10–
20 lg/mL range and 1d (R ¼ C7H15) with MICs in the
15–24 lg/mL. Interestingly, 1a (R ¼ C4H9) and 1f
(R ¼ C18H37) were found to be completely inactive;
although 1c (R ¼ C6H13) showed certain extent of anti-
bacterial activity in the 24–50 lg/mL range. The anti-
fungal activity of 1e (R ¼ C8H17), 1b (R ¼ C5H11) and 1d
(R ¼ C7H15) showed consistency in their antifungal
potency with MFCs in the 10–50 lg/mL, whereas 1a
(R ¼ C4H9) and 1f (R ¼ C18H37) were found to be inac-
tive with MFCs in the 500–600 lg/mL range. The fact
that analogue 1e (R ¼ C8H17) having MFCs value very
close to that of the natural 1 (R ¼ 2-methylheptyl)
indicates that the methyl group at position 2 of the
hydrocarbon chain does not have a marked effect on
the antimicrobial activity of 1. On the other hand it is
2.4. Antifungal and antibacterial susceptibility tests
Broth minimum inhibitory concentration (MIC) and
minimum fungicidal concentration (MFC) were deter-
mined in the culture tube containing 4.5 mL of either
nutrient broth or M-33 broth or molten M2 agar. Final
volume was adjusted to 5 mL after addition of culture
media and the required antibiotic solution. Spore sus-
pensions were prepared in Ringer’s solution and
adjusted to a final inoculum size of 3 ꢁ 105 colony
forming units (CFU)/mL. After inoculation the culture
tubes were shaked well and then incubated at 35 °C for
24 h for pathogenic bacteria, 48 h for fungi and 120 h for
M. tuberculosis, respectively. Incubated broth showed
visible turbidity in both control and the tubes with
sublethal doses of antibiotic.
2.5. Determination of MIC and MFC
MICs of bacterial test pathogens were determined after
a definite period of incubation by removing 10 lL of the
contents from each tube showing no visible growth and
spreading them onto agar plates of specific medium. The
plates were incubated at 35 °C and growth (if any) was
carefully examined under microscope. Minimum fungi-
cidal concentration (MFCs) were determined in molten
M2 agar following the same procedure and incubated
for 48 h. MFC of an antibiotic was defined as the lowest
concentration at which 95% of the inoculum was killed.
MICs of the bioactive molecules were defined as the
Table 1. Antimicrobial activity of 2-methylheptylisonicotinate and its derivatives
Acid esters tested
MIC (lg/mL)
MFC (lg/mL)
Rhizoctonia solani
Bacillus subtilis
Escherichia coli
Mycobacterium
tuberculosis
Proteus vulgaris
Fusarium
semitectum
1a, R ¼ C4H9
1b, R ¼ C5H11
1c, R ¼ C6H13
1d, R ¼ C7H15
1e, R ¼ C8H17
1f, R ¼ C18H37
(R)-(þ)-1
600
20
500
20
35
16
12
56
30
40
280
10
24
16
8
500
14
36
16
6
500
10
600
14
50
24
44
15
44
15
16
16
18
600
16
320
10
14
560
6
500
24
600
24
(S)-(ꢀ)-1
20
14
30
32

J. Boruwa et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3571–3574
OH
3573
OH
OR
OH
O2N
ii
i
+
(+)-
3
2
(R)-(+)-3
(S )-(-)-3, R=Ac
(S )-(-)-4, R=H
iii
O
O
OH
O
Cl
N
N
N
(R)-(+)-3
iv
(R)-(+)-1
O
O
O
OH
Cl
N
iv
(S )-(-)-1
(S )-(-)-4
Scheme 1. Reagents and conditions: (i) n-Bu3SnH, AIBN, dry benzene, 80 °C, 6 h; (ii) Candida rogusa lipase, vinyl acetate/hexane, 12 h; (iii) K2CO3/
MeOH, 1 h; (iv) Amberlyst A-21, dry CH3CN, MW (40%, 5 min).
further observed that the MICs and MFCs of 1e
(R ¼ C8H17) are better than that of the natural molecule,
which indicates that the length of the chain plays a
major role on the antimicrobial activity of these types of
molecules. Another important observation is that the R-
isomer of 1 is slightly more active than the corre-
sponding S-isomer indicating that the methyl group in
position 2 of the hydrocarbon chain has got some effect
on the bioactivity.
resembled both in bioactivity and sign of optical rota-
tion [a] with that of natural 2-methylheptyl isonicoti-
nate. Hence the absolute stereochemistry of the methyl
group of natural 2-methylheptyl isoniconate has been
assigned as ‘R’. The synthetic steps are depicted in
Scheme 1.
The alarming spread of tuberculosis especially in
developing and underdeveloped countries due to HIV
infection has focused the attention on the need to
understand the pathogenesis of this disease. In July 2003
report, the WHO has called TB, and not AIDS, India’s
biggest health concern.9–11 The report says that over
4.5 million people in India suffer from TB annually, the
highest number of cases reported in the world. It is also
reported that the last two decades witnessed the fre-
quencies and types of life threatening fungal infections
that have increased dramatically among the immuno-
compromised patients.13–15 Moreover, secondary fungal
infections that appear towards the later part of medi-
cation, among the TB patients, aggravate the situation.6
Hence the need for novel anti-TB drug with antifungal
activity is highly felt. In view of this isoniazid analogue 1
isolated by us and the analogue 1e R ¼ C8H17 having
both antifungal and antibacterial activity reported here
hold lot of promise towards developing new drug to
fight this menace.
4. Total synthesis of the individual isomers of
2-methylheptyl isonicotinate (R)-(+)-1 and (S)-())-1
The racemic alcohol 2-methylheptanol was prepared
starting from 2-bromoheptane. Treatment of 2-bromo-
heptane with NaNO2 in dimethylformamide yielded the
2-nitroheptane in 60% yield as a gum, which was sub-
jected to formylation with 30% aqueous formaldehyde
solution in the presence of K2CO3 at rt to give nitro
alcohol 2 in 72% yield. Denitration of 2 was effected by
refluxing a solution of 2 in dry benzene with n-tri-
butyltinhydride in the presence of azobisisobutyronitrile
to give (ꢂ)-3 in 75% yield.7 The individual R- and S-
isomers of the resulting 2-methyl-1-heptanol, (ꢂ)-3 were
resolved by lipase catalyzed transesterification with
lipase from Candida rogusa giving (R)-(þ)-3 in 49% yield
20
D
having ½aꢃ þ13.8 (c 0.8, CHCl3) with ee 96% and the
20
20
D
acetate (S)-(ꢀ)-3 in 46% yield,8 with ½aꢃ ꢀ12.4 (c 0.8,
Acknowledgements
CHCl3) and ee 97%. The (S)-(ꢀ)-4, with ½aꢃ ꢀ11.3 (c
D
The authors thank Director R. R. L. Jorhat for his
initiatives and providing facilities for this work. This
study was a partly funded by CSIR SRF Grants, New
Delhi, India.
0.7, CHCl3) and ee 95% was generated by hydrolyzing
acetate (S)-(ꢀ)-3 with K2CO3 in methanol in an inert
atmosphere.8 Esterification of isonicotinoyl chloride
hydrochloride with (R)-(þ)-3 yielded (R)-(þ)-116 in 52%
20
yield having ½aꢃ þ8.3 (c 0.6, CHCl3) with ee 94% and
D
esterification of isonicotinoyl chloride hydrochloride
References and notes
with alcohol (S)-(ꢀ)-4 gave (S)-(ꢀ)-116 in 40% yield,
20
½aꢃ ꢀ9.5 (c 0.8, CHCl3) and ee 95%. Both (R)-(þ)-1 and
1. Bordoloi, G.; Kumari, B.; Guha, A.; Bordoloi, M. J.; Roy,
M. K.; Bora, T. C. Biosci. Biotechnol. Biochem. 2001, 65,
1856–1858.
D
gave (S)-(ꢀ)-1 were separately subjected for bioassay
against different test organisms. Compound (R)-(þ)-1

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J. Boruwa et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3571–3574
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Bora, T. C. A process for the production of 2-methyl-
heptyl isonicotinate, Indian Patent NF-9/2000.
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Bora, T. C. 2-Methylheptyl isonicotinate as novel antibi-
otic, Indian Patent NF-116/2001.
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53.
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H., Eds.; Chapman and Hall: New York, 1992; pp 349–
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isonicotinate, U.S. Patent 009 NF 2000, 10/027913.
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J. Org. Chem. 1985, 50, 3692–3698.
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16. Selected data: (R)-(þ)-1, oil, ½aꢃ þ8.3 (c 0.6, CHCl3). IR
20
D
(CHCl3): 2956, 2926, 2856, 1732, 1696, 1561, 1380 and
1060 cmꢀ1 1H NMR (300 MHz CDCl3) d ppm: 0.88 (t,
.
J ¼ 6:15 Hz, 3H), 0.93 (d, J ¼ 7 Hz, 3H), 1.25–1.42 (br,
6H), 1.45 (m, 2H), 1.48 (m, 1H), 4.22 (d, J ¼ 6 Hz, 2), 7.51
(dd, J ¼ 4, 6 Hz, 2H), 7.91 (dd, J ¼ 4, 6 Hz, 2H), MS
20
D
CHCl3). IR (CHCl3): 2958, 2930, 2861, 1737, 1690,
(m=z): 258.1 (MþþNa). (S)-(ꢀ)-1, oil, ½aꢃ ꢀ9.5 (c 0.8,
1566, 1377, 1238 and 1050 cmꢀ1 1H NMR (300 MHz
.
CDCl3) d ppm: 0.85 (t, J ¼ 6:5 Hz, 3H), 0.92 (d, J ¼ 7
Hz, 3H), 1.25–1.43 (br, 6H), 1.44 (m, 2H), 1.48 (m,
1H), 4.22 (d, J ¼ 6 Hz, 2H), 7.6 (dd, J ¼ 4, 6 Hz,
2H), 8.0 (dd, J ¼ 4, 6 Hz, 2H), MS (m=z): 258.0 (Mþþ
Na).