Anti-mycobacterial activity of a bis-sulfonamide

Article abstract of DOI:10.1016/j.bmcl.2006.11.079

A bis-arylsulfonamide, 7, has been identified that exhibits growth inhibition of Mycobacterium smegmatis at less than 25 μg/mL, but has no such activity against Escherichia coli or Staphylococcus aureus. A closely related bis-arylsulfonamide (8) was much

Full text of DOI:10.1016/j.bmcl.2006.11.079

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1355–1357
Anti-mycobacterial activity of a bis-sulfonamide
Brendan L. Wilkinson,a Laurent F. Bornaghi,a Anthony D. Wright,b
Todd A. Houstonc,* and Sally-Ann Poulsena,*
aEskitis Institute for Cell and Molecular Therapies, Griffith University, Nathan, Qld 4111, Australia
bAustralian Institute for Marine Science, Townsville, Qld 4810, Australia
cInstitute for Glycomics, Griffith University, Gold Coast, Qld 9726, Australia
Received 27 October 2006; revised 24 November 2006; accepted 30 November 2006
Available online 3 December 2006
Abstract—A bis-arylsulfonamide, 7, has been identified that exhibits growth inhibition of Mycobacterium smegmatis at less than
25 lg/mL, but has no such activity against Escherichia coli or Staphylococcus aureus. A closely related bis-arylsulfonamide (8)
was much less active, but was the only other compound among 54 arylsulfonamides tested with detectable growth inhibition of
M. smegmatis.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Since the introduction of prontosil over 70 years ago,
sulfa drugs have been widely used to treat a broad
spectrum of infectious microorganisms. The bioactive
component of prontosil, sulfanilamide (1), inhibits
6-hydroxymethyl-7,8-dihydropteroate synthase (DHPS)
selectively limiting folate synthesis in prokaryotes and
lower eukaryotes thus disrupting the integrity of their
DNA synthesis.1 The evolution of drug resistance in
infectious microorganisms underpins an immense and
ongoing need for new therapies to treat infectious dis-
eases. The identification of new and novel chemical enti-
ties with activity against cells infected with the
microorganism is a crucial component of this anti-infec-
tive drug development pathway.2 Although sulfona-
mides are not specifically incorporated into current
tuberculosis treatment, many sulfur-,3,4 sulfonyl-,4–6
and sulfonamide-containing7,8 compounds are active
against mycobacteria. This prompted us to screen our
growing library of triazole-based sulfonamides (e.g., 2)
for anti-mycobacterial activity. Herein, we report identi-
fication of a novel bis-sulfonamide with anti-microbial
activity against Mycobacterium smegmatis that is rela-
tively inert toward common bacterial and fungal strains.
The syntheses of the bis-sulfonamides 7 and 8 are shown
in Scheme 1. The propargyl derivatives 4 and 5 were syn-
thesized from 4-carboxybenzene sulfonamide (3) by
esterification (EDC/HOBT, 38%) and amide coupling
(HBTU/HOBT, 82%), respectively.9 Compounds 7 and
8 were prepared by reaction of 4 and 5 with the azido
arylsulfonamide 6 using the ‘click-tailing’ approach de-
scribed previously for the construction of a diverse
library of glycosyl triazole-linked arylsulfonamides.9
Using Cu(I) catalysis the ester-linked bis-sulfonamide 7
(42%) and amide-linked analog 8 (48%) were each ob-
tained cleanly from 6.9–11 Yields were less than ideal,
owing to poor product solubility during purification.
As both 4 and 7 are potentially labile to cellular esteras-
es, compounds 2 and 3 were included in the biological
screening as control compounds to identify if these
esters were behaving as prodrugs.
Keywords: Sulfonamide; Mycobacterium; Mycobacterium smegmatis;
Triazole.
Along with a commercial sample of sulfanilamide 1 and
4-carboxybenzene sulfonamide 3, synthetic compounds
*
Corresponding authors. Tel.: +61 7 3735 7825; fax: +61 7 3735
7656; e-mail: s.poulsen@griffith.edu.au
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.11.079

1356
B. L. Wilkinson et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1355–1357
4–16 lg/mL) and had only modest growth inhibition
of the yeast strain, C. albicans. M. smegmatis is used
as a surrogate host to perform virulence studies of
Mycobacterium tuberculosis.13,14 None of these sulfon-
amides displayed dose-dependent growth inhibition of
any of the three bacterial strains tested: E. coli, S.
aureus, and V. harveyi. In this regard, the selective
anti-microbial activity displayed by the bis-arylsulf-
onamide 7 is remarkable as previous work on 35 sul-
fanilamides showed comparable inhibitory effects
against both E. coli and M. smegmatis.15 Among the
54 arylsulfonamides screened in this study, bis-aryl-
sulfonamide 7 was the only compound with significant
activity against M. smegmatis at concentrations of less
than 100 lg/mL.
In vivo ester hydrolysis of 7 would give rise to 2 and 3,
neither of which had any activity against C. albicans,
and much less activity against M. smegmatis (ꢀ20%
growth inhibition at 100 lg/mL). The increased activity
of the ester 7 versus the amide 8 may be due to greater
flexibility of the linker in the former. Calculations of
the logP values for the ester (logP = 0.52) indicate that
it is more hydrophobic than the amide (logP =
ꢁ0.654).16 This would undoubtedly improve membrane
passage of 7 through the waxy mycobacterial exterior
thus increasing its bioavailability relative to the amide.
Scheme 1. Synthesis of bis-sulfonamides.
2, 4–8 were tested for anti-microbial activity against five
microorganisms: M. smegmatis, Candida albicans, Esch-
erichia coli, Staphylococcus aureus, and Vibrio harveyi.
Each compound was assessed at five doses (100, 25,
6.25, 1.56, and 0.39 lg/mL) and scored for % growth
inhibition of the microorganisms (average of two-inde-
pendent determinations).12 Bioassay results are present-
ed in Table 1.
Compound 7 may be exploiting a unique feature in the
active site periphery of mycobacterial DHPS or it may
be acting on a novel cellular target unique to mycobac-
teria. Sulfonamides display increasingly well-understood
pharmacological effects most notably the potent inhibi-
tion of the carbonic anhydrase catalyzed hydration of
carbon dioxide to give bicarbonate and a proton:
CO2 þ H2O $ HCO3ꢁ þ Hþ.17,18 Recently it has been
shown that both M. smegmatis and C. albicans encode
for carbonic anhydrases and that this enzyme is critical
for the growth of these pathogens.19,20 Structure–activi-
ty work to identify the role of the two arylsulfonamide
moieties is underway. These investigations will be
reported in due course.
The results in Table 1 demonstrate that only the bis-
sulfonamides 7 and 8 were able to inhibit the growth
of M. smegmatis at concentrations of 100 lg/mL or
less. The ester 7 had activity comparable to some cur-
rent antitubercular agents (aminoglycosides such as
kanamycin, a Class B antitubercular drug, are active
against most mycobacterial strains in the range of
Table 1. Minimum inhibitory concentration (MIC50) values in lg/mL
versus various microorganisms
Compound
Mycobacterium
smegmatis
Candida
albicans
Bacterial
strains
Acknowledgments
1
>100
>100
>100
>100
>100
NA
<25a
ꢀ100
—
NA
NA
NA
NA
NA
NA
ꢀ100b
NA
—
NA
NA
NA
NA
NA
NA
NA
NA
500
—
The authors thank the Australian Research Council
(Grant No. F00103312) and Griffith University for
research funding, and J. Doyle and A. Muirhead,
Australian Institute for Marine Science, for undertaking
anti-microbial assays.
2
3
4
5
6
7
8
Ampicillinc
Amphotericin
Streptomycin
References and notes
—
500
25
—
—
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Bacterial strains tested: Escherichia coli, Staphylococcus aureus, Vibrio
harveyi; NA, not active.
a 68% growth inhibition at 25 lg/mL (92% at 100 lg/mL).
b 36% growth inhibition at 25 lg/mL (49% at 100 lg/mL).
c The three controls, ampicillin, amphotericin, and streptomycin, were
all used at dose levels that guaranteed 100% mortality of the test
organism.

B. L. Wilkinson et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1355–1357
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also assayed. All assays were performed in duplicate and
incubated overnight at 37 ꢁC.
Procedure for anti-fungal screening. Compounds were
tested against C. albicans. Test cultures were prepared by
inoculating 10 mL of YM broth with a loop of fresh C.
albicans culture. This culture was grown for 18 h at 37 ꢁC
to give a culture with an optical density (OD) of 0.3 at
595 nm, corresponding to a cell density of 5 · 109 cells/mL.
A 100 lL aliquot of this culture was added to 500 mL of
fresh YM broth and well mixed. Assays were carried out
in 96-well microtiter plates in an assay volume of
200 lL—made up of culture (198 lL ꢀ 2 · 105 cells) and
test compound in DMSO (final DMSO concentration of
1% v/v). Controls that contained no test compound
(DMSO 1% v/v) and antibiotic (25 lg/mL amphotericin
B—as a no growth control) were also assayed. All assays
were performed in duplicate and incubated for 48 h at
37 ꢁC.
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Trehan, S. Synthesis 2005, 11, 1801. The general syntheses
for 2, 7, and 8 are as follows: The acetylene (0.25 M) and
azide (0.25 M) were suspended in a 1:1 mixture of tert-
butyl alcohol and distilled H2O. CuSO4Æ5H2O (0.1 equiv)
and sodium ascorbate (0.2 equiv) were added and the
suspension stirred at 40 ꢁC and monitored by TLC
(completion occurred within 2 h). The mixture was evap-
orated and the crude residue purified by chromatography
on silica (100% EtOAc as eluant) to afford analytically
pure material.
Procedure for anti-mycobacterial screening. Compounds
were tested against Mycobacterium smegmatis (ACM916).
Test cultures were prepared by inoculating M. smegmatis
from precultures into approximately 20 mL of Middle-
brook broth in a 50 mL falcon tube. The culture was
vortexed briefly and placed in an incubator at 37 ꢁC, and
120 rpm for 3 days. This culture was then sonicated until
all cells were thoroughly dispersed and then diluted 1:20
into Middlebrook broth to a final volume of 300 mL.
Assays were carried out in 96-well microtiter plates in an
assay volume of 200 lL made up of culture (195 lL) and
test compound in DMSO (final DMSO concentration of
2.5% v/v). Controls that contained no test compound
(DMSO 2.5% v/v) and antibiotic (50 mg/mL streptomy-
cin solution—for a no growth control) were also assayed.
All assays were performed in duplicate. An initial
absorbance reading was taken at 595 nm and the plates
incubated in a humidified incubator at 37 ꢁC for 96 h. At
the end of this period plates were removed form the
incubator and allowed to equilibrate to room tempera-
ture (25 ꢁC).
(1-(4-Sulfamoylphenyl)-1H-1,2,3-triazol-4-yl)methyl-4-
sulfamoylbenzoate (7): 1H NMR (400 MHz, DMSO-d6): d
5.53 (s, 2H, OCH2), 7.50 (br s, 2H, SO2NH2), 7.54 (br s,
2H, SO2NH2), 7.93–8.01 (m, 4H, Ar CH), 8.12–8.16 (m,
4H, Ar CH), 9.06 (s, 1H, triazole CH); 13C {1H} NMR
(100 MHz, DMSO-d6): d 58.90 (OCH2), 121.15 (Ar CH),
124.06 (triazole CH), 126.77 (Ar CH), 128.18 (Ar CH),
130.78 (Ar CH), 123.66 (Ar C), 139.15 (Ar C), 143.90 (Ar
C), 144.67 (triazole C), 148.95 (Ar C), 165.17 (C@O);
ESIMS: m/z [MꢁH]ꢁ 436.2.
4-Sulfamoyl-N-((1-(4-sulfamoylphenyl)-1H-1,2,3-triazol-
1
4-yl)methyl)benzamide (8): H NMR (400 MHz, DMSO-
d6): d 4.62 (d, 3JCH–NH = 5.6 Hz, 2H, NHCH2), 4.76 (br s,
4H, SO2NH2 · 2), 7.89–8.12 (m, 8H, Ar CH), 8.80 (s, 1H,
triazole CH), 9.32 (t, 3JNH–CH = 5.6 Hz, 1H, NH); ESIMS:
m/z [MꢁH]ꢁ 435.3.
For all anti-microbial assays optical densities (ODs) were
recorded at 595 nm at the start and end of the assay
incubation periods. The test compound activity was
calculated as a percent of control according to the
4-(4-(Hydroxymethyl)-1H-1,2,3-triazol-1-yl)benzenesulf-
following equation: Activity
%
of control = 100 ·
1
onamide (2): H NMR (400 MHz, DMSO-d6): d 4.60 (d,
([T0(sample) ꢁ Tend(sample)] ꢁ [T0(neg ctl) ꢁ Tend(neg ctl)])/([T0(pos ctl)
ꢁ
3
3
2H, JCH–OH = 5.2 Hz, 2H, CH2OH), 5.34 (t, JOH–
CH = 5.2 Hz, 1H, OH), 7.48 (br s, 2H, SO2NH2), 7.97–
8.12 (m, 4H, Ar CH), 8.76 (s, 1H, triazole CH); ESIMS: m/
z [MꢁH]ꢁ 253.2.
Tend(pos ctl)] ꢁ [T0(neg ctl) ꢁ Tend(neg ctl)]).
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tested against S. aureus (ATCC 25923), E. coli (ATCC
25922), and V. harveyi (strain C071). Anti-microbial
assays were all conducted in a similar fashion with all
manipulations for S. aureus performed in PYE media
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)
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LB media (BD, Brisbane, Australia). A single colony was
inoculated into 20 mL of sterile growth media and grown
overnight. This culture was then diluted 1000-fold into
fresh media the following day. Assays were carried out in
96-well microtiter plates in an assay volume of 200 lL—
made up of culture (198 lL) and test compound in DMSO
(final DMSO concentration of 1% v/v). Controls that
contained no test compound (DMSO 1% v/v) and antibi-
otic (500 lg/mL ampicillin—as a no growth control) were
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