Schiff base conjugates of p-aminosalicylic acid as antimycobacterial agents

Article abstract of DOI:10.1016/j.bmcl.2005.12.035

Schiff base conjugates of p-aminosalicylic acid have been synthesized and characterized by elemental analysis, IR and ¹H NMR spectroscopy and cyclic voltammetry. Compounds containing hydroxyl-rich side chains show enhanced antimycobacterial act

Full text of DOI:10.1016/j.bmcl.2005.12.035

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1514–1517
Schiff base conjugates of p-aminosalicylic acid
as antimycobacterial agents
Jayendra Patole,a Dipti Shingnapurkar,a Subhash Padhyea,* and Colin Ratledgeb
aDepartment of Chemistry, University of Pune, Pune 411 007, India
bDepartment of Biological Sciences, University of Hull, Hull HU6 7RX, UK
Received 3 June 2005; revised 28 November 2005; accepted 12 December 2005
Available online 18 January 2006
Abstract—Schiff base conjugates of p-aminosalicylic acid have been synthesized and characterized by elemental analysis, IR and 1H
NMR spectroscopy and cyclic voltammetry. Compounds containing hydroxyl-rich side chains show enhanced antimycobacterial
activity against Mycobacterium smegmatis and Mycobacterium bovis BCG. Higher ClogP values and superior radical scavenging
activities are thought to be the contributing factors for their enhanced antimycobacterial activities.
Ó 2005 Elsevier Ltd. All rights reserved.
Tuberculosis (TB) is one of the most refractory and
deadly infectious killer diseases worldwide caused by
the pathogen Mycobacterium tuberculosis which is
responsible for infecting nine million people globally
with three million deaths every year according to World
Health Organization (WHO) report.1 Over the years, the
organism has acquired resistance to nearly all first-line
antitubercular drugs such as isoniazid, rifamcin, ethi-
onamide, etc., posing serious problems in treating the
infection clinically.2 The problem of clinical treatment
has become more acute especially in immuno-compro-
mised AIDS patients where the rise in TB incidence
and consequent deaths over the past two decades has
escalated by more than 12%. There is thus an urgent
need to evolve new antitubercular drugs.
accommodated by the cells as they up-regulate the syn-
thesis of various iron-containing proteins. Salicylic acid,
which can also be found extracellularly in cultures of
mycobacteria grown with a deficiency of iron, is a pre-
cursor of mycobactin and carboxymycobactin that are
essential for growth of M. tuberculosis in macrophages.4
Salicylate, however, is too weak a chelator of iron for it
to act as a siderophore.5 Some time ago Brown & Ratl-
edge6 suggested that p-aminosalicylic acid (PAS), which
is a second-line antitubercular drug, acts as an antisali-
cylate compound by interfering with the metabolism of
salicylic acid, possibly by preventing its conversion to
carboxymycobactin and mycobactin.3
More recent evidence7 has shown that when Mycobacte-
rium smegmatis was treated with PAS, the concentration
of salicylate increased by 5- to 6-fold and that of myco-
bactin decreased by about 70–80% in keeping with these
proposals. In addition, a salicylate-requiring auxotroph
had a considerably heightened sensitivity to PAS. How-
ever, other evidence,8 which carefully eschewed mention
of all contrary evidence, has suggested that PAS may be
acting as an anti-folate compound as PAS-resistant mu-
tants of Mycobacterium bovis show diminished activity
of thymidylate synthase. Whatever is the site of action
of PAS, it is easy to synthesize and is an inexpensive
drug for the treatment of tuberculosis. As it is now many
years since various analogues of PAS have been synthe-
sized9 it would now seem desirable to revisit this mole-
cule and produce further modifications of it in order
to find novel chemotherapeutic agents that may possibly
be effective against existing PAS-resistant strains.
Mycobacteria have a specific need to acquire iron for
their metabolism, as described by Ratledge.3 This task
is accomplished by the synthesis of specialized com-
pounds known as siderophores,which, in the mycobacte-
ria, are carboxymycobactin in the pathogens and
exochelins in saprophytic bacteria.3 Mycobactin, which
is found exclusively intracellularly in the envelope of
mycobacteria, acts as an intracellular storage compound
for iron and allows the subsequent slow release of iron
into the cytoplasm of the cells at a rate, which can be
Keywords: para-Amino salicyclic acid; Mycobacterium bovis; Antimy-
cobacterial agents.
*
Corresponding author. Tel.: +91 20 569 1728; fax: +91 20 5691728;
e-mail: sbpadhye@chem.unipune.ernet.in
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.12.035

J. Patole et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1514–1517
1515
HO
HO
remove trace metals and dispensed as previously de-
scribed.7 When glucose was used, this was sterilized sep-
arately. Medium was supplemented with 0.45 lg
Zn2+ mlÀ1, 0.1 lg Mn2+ mlÀ1 and 40 lg Mg2+mlÀ1 plus
for iron-deficient growth 0.04 lg Fe2+ mlÀ1 and for iron
sufficient growth 2 lg Fe2+ mlÀ1. Cultures were inocu-
lated with 1 ml of 5- to 7-day-old culture grown on
iron-deficient medium and were incubated with shaking
at 37 °C for 6 to 7 days.7
HO
O
N
L1
R =
OCH3
OH
L4
HO
R
HO
HO
OH
OH
L2
L3
L5
L6
N
OH
Scheme 1.
Mycobacterium bovis BCG Glaxo strain was grown as
described above. Culture filtrates were acidified, extract-
ed with chloroform and the extracts were analysed by
HPLC for the presence of salicylic acid by monitoring
the eluate at 237 nm.7 The reason for using iron-defi-
cient growth conditions for testing the compounds was
that it is considered that these are pertinent in vivo con-
ditions that mycobacteria will face when growing in
macrophages.4
In the present work, we describe synthesis and charac-
terization of some new multifunctional conjugates of
PAS (Scheme 1) capable of metal chelation along with
the results on the evaluation of their antimycobacterial
activity against M. smegmatis and M. bovis (BCG).
PAS (Lancaster, UK) was recrystallized before use,
while all aldehydes used in condensation reactions were
purchased from Aldrich and were used as received.
All synthesized Schiff base compounds were character-
ized by elemental analysis, IR and 1H NMR spectrosco-
py (Table 1). The microanalysis data are consistent with
the molecular formula assigned to the compounds. The
formation of the Schiff base derivatives is confirmed by
fingerprinting IR spectra for the appearance of the
Elemental analyses were carried out in the Microanalyt-
ical Laboratory at University of Hull, UK. IR spectra
were recorded in Nujol in the range 4000–450 cmÀ1 on
a Perkin-Elmer 283B FTIR instrument and H NMR
was recorded in DMSO-d6 solvent on the Varian Mercu-
ry Vx 300 instruments.
1
azomethine stretching frequency (C@N) in the range
1607–1616 cmÀ1
and disappearance of the bands
9
around 3000–3400 cmÀ1 due to the symmetric and
asymmetric amino stretches of the para-amino groups.10
The carboxylate carbonyl frequencies in the synthesized
derivatives are observed in the region [1629–
1657 cmÀ1],11 while a broad band located around
3400–3500 cmÀ1 corresponds to the salicylic hydroxyl
group.12 The formation of the Schiff base compounds
All the Schiff base derivatives were synthesized by stir-
ring a methanolic solution of PAS (typically 1.63 g,
0.01 mol) with corresponding aldehydes (typically
1.22 g, 0.01 mol) in 1:1 stoichiometric ratio at room tem-
perature over 24 h. The precipitates obtained were fil-
tered, washed with methanol, and dried in vacuo over
anhydrous CaCl2 and were recrystallized from
methanol.
1
was also confirmed by H NMR spectrum which shows
a proton signal around 8.9 ppm due to –CH@N–
chromophore.13
Mycobacterium smegmatis NCIMB 8548 was grown in
250 ml conical flasks, pre-treated to remove trace met-
als, containing 100 ml medium which consisted of glyc-
erol or glucose, 10 g; KH2PO4, 5 g; L-asparagine, 5 g;
pH to 7.0, or alternative value, with KOH. Medium
was autoclaved with 2% (w/v) aluminium oxide to
The results of the antimycobacterial assays for the com-
pounds L1–L6 against M. smegmatis and M. bovis BCG
are shown in Figures 1 and 2, respectively. M. smegmatis
is fast-growing mycobacteria with a generation time of
3 h compared to 20 h for that of M. tuberculosis and
Table 1. Elemental, IR and 1H NMR spectral data for the compounds L1–L6
Compound
Elemental analysisa
IR spectroscopy (cmÀ1
)
1H NMR (ppm)
d(–CH@N)
8.95
ClogP
% C
% H
% N
5.19
m(OH)
m(C@N)
m(C@O)
L1
L2
L3
L4
L5
L6
65.73
(65.36)
61.87
4.19
(4.28)
3.92
3436
1607
1638
1630
1625
1629
1657
1630
3.42
2.91
2.45
3.40
4.61
2.29
(5.44)
4.81
3420
3350
3446
3435
3413
1616
1616
1610
1608
1618
8.93
8.72
8.93
8.51
8.60
(61.53)
58.73
(4.03)
4.13
(5.12)
4.15
(58.13)
63.01
(3.80)
4.42
(4.84)
4.55
(62.71)
70.24
(4.53)
4.18
(4.87)
4.23
(70.35)
64.07
(64.46)
(4.23)
4.29
(4.13)
(4.56)
10.84
(11.57)
Carboxylate group.
a Values in parentheses are calculated ones.

1516
J. Patole et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1514–1517
lipid biosynthesis processes.18 On comparing the
100
80
60
40
20
0
26
ClogP19 values of PAS (0.71) with L5 (4.62), the Schiff
base side chain in the new derivatives clearly contributes
substantially in enhancing the liposolubility of the par-
ent compound thereby facilitating the entry of these
molecules through lipid-enriched bacterial cell wall.20
31
26
27
33
29
L1
L2
L3
L4
L5
L6
Interestingly, the HPLC analysis of the culture filtrates
showed no increased accumulation of salicylic acid (re-
sults not shown), suggesting the probable mechanism
of action of these compounds is not through blocking
the conversion of salicylate into mycobactin or carbox-
ymycobactin. In this connection, there have been prior
reports indicating that the orthohydroxy Schiff bases
of isoniazid had higher antimycobacterial activity than
isoniazid where the enhanced activity was attributed to
radical scavenging activity of the compound.21 It is
likely a similar mechanism may be operating in case of
the compounds described here.
COMPOUNDS (µM)
Figure 1. Antimycobacterial activities of the Schiff base conjugates of
PAS (L1–L6) against Mycobacterium bovisBCG.
120
182
171
311
244
100
80
60
40
20
0
335
389
L1
L2
L3
L4
L5
L6
PAS conjugates containing hydroxyl-rich Schiff base li-
gands have enhanced antimycobacterial activities
against M. smegmatis and M. bovis BCG that may be
useful in developing new and potent antimycobacterial
agents.
COMPOUNDS (µM)
Figure 2. Antimycobacterial activity of the Schiff base conjugates of
PAS (L1–L6) against Mycobacterium smegmatis.
hence is normally employed for the rapid screening
of antitubercular compounds.14 It also has similar
rates of homologous recombination to M. tuberculosis.15
M. smegmatis, unlike M. tuberculosis or BCG, has a
second route of iron uptake that does not involve
salicylic acid3 and, consequently, is considerably less
sensitive to PAS than are the pathogenic mycobacteria.7
M. bovis BCG resembles M. tuberculosis being a slow-
growing pathogen16 and has 99.9% similarity of DNA
sequences with that of M. tuberculosis.17
Acknowledgment
J.P. thank University of Pune (India) for the financial
assistance.
References and notes
1. Bottari, B.; Maccari, M.; Monfort, F.; Ottana, R.;
Rotndo, E.; Vigorita, M. G. Bioorg. Med. Chem. Lett.
2001, 11, 301.
2. Sandbhor, U.; Padhye, S.; Billington, D.; Rathbone, D.;
Franzblau, S.; Anson, C. E.; Powell, A. K. J. Inorg.
Biochem. 2002, 90, 127.
3. Ratledge, C. Tuberculosis 2004, 84, 110.
4. De Voss, J. J.; Rutter, K.; Schroeder, B. G.; Su, H.; Zhu,
Y., ; Barry, C. E., III Proc. Natl. Acad. Sci. U.S.A. 2000,
97, 1252.
5. Chipperfield, J. R.; Ratledge, C. Biometals 2000, 13, 165.
6. Brown, K. A.; Ratledge, C. Biochim. Biophys. Acta 1975,
385, 207.
In the present study, all the susceptibility testing do not
include the actual MICs, but we have noted the percent
inhibition at a single concentration and the detailed
studies are presently underway. The inhibitory concen-
tration values for almost all of the new compounds as
seen from the preliminary data are considerably lower
than PAS in case of M. smegmatis (MIC > 650 lM) as
well as M. bovis BCG (MIC < 52 lM) indicating conju-
gation of hydroxyl-rich ligands is effective in synergisti-
cally enhancing the antimycobacterial activity of these
compounds. It is difficult to find reliable data in the lit-
erature concerning MICs, because experimental condi-
tions are so different that it could be erroneous to
compare published values.
7. Adilakshmi, T.; Ayling, P. D.; Ratledge, C. J. Bacteriol.
2000, 182, 264.
8. Vergne, A. F.; Walz, A. J.; Miller, M. J. Nat. Prod. Rep.
2000, 17, 99.
9. Rengarajan, J.; Sassetti, C.; Naroditskaya, V.; Sloutsky,
A.; Bloom, B. R.; Rubin, E. J. Mol. Microbiol. 2004, 53,
275.
10. Patole, J.; Dutta, S.; Padhye, S.; Sinn, E. Inorg. Chim.
Acta 2001, 318, 207.
Conjugation of heterocyclic ring gave a slight improve-
ment in the activity of the compounds. This is impor-
tant, as many of the existing first-line antitubercular
drugs possess such structural motif.
11. Afrasiabi, Z.; Sinn, E.; Padhye, S.; Dutta, S.; Padhye, S.;
Newton, C.; Anson, C. E.; Powell, A. K. J. Inorg.
Biochem. 2003, 95, 306.
12. Garg, B. S.; Singh, P. K.; Sharma, J. L. Synth. React.
Inorg. Met.—Org. Chem. 2000, 30, 803.
13. Nawar, N.; Honsy, N. M. Chem. Pharm. Bull. 1999, 47,
944.
Rapid drug resistance developed by M. tuberculosis is
generally due to decreased drug permeability induced
by the bacteria by thickening their cell wall so that only
lipophilic derivatives can penetrate such waxy cell wall.8
The recent genomic data on M. tuberculosis have shown
that about 12% of its genes are involved in cell wall and
14. Sangeetha, N. R.; Pal, S. Bull. Chem. Soc. Jpn. 2000, 73,
357.

J. Patole et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1514–1517
1517
15. Chacon, O.; Feng, Z.; Harris, N. B.; Caceres, E.; Adams,
L. G.; Barletta, R. G. Antimicrob. Agents Chemother.
2002, 46, 47.
18. Colemanm, M. D.; Rathbone, D. L.; Abberley, L.;
Lambert, P. A.; Billington, D. C. Environ. Toxicol.
Pharmacol. 1999, 7, 59.
16. Pavelka, M. S.; Jacobs, W. R. J. Bacteriol. 1999, 181, 4780.
17. Barry, C. E., III; Lee, R. E.; Mdluli, K.; Sampson, A. E.;
Schroeder, B. G.; Slayden, R. A.; Yuan, Y. Prog. Lipid
Res. 1998, 37, 143.
19. ClogP of the synthesized compounds were calculated by
chemsw software, www.chemsw.com.
20. Smith, I. Clin. Microbiol. Rev. 2003, 16, 463.
21. Gao, J.; Richardson, D. R. Neoplasia 2001, 98, 842.