Inhibition of the β-carbonic anhydrases from Mycobacterium tuberculosis with C-cinnamoyl glycosides: Identification of the first inhibitor with anti-mycobacterial activity

Article abstract of DOI:10.1016/j.bmcl.2012.11.085

A small series of C-cinnamoyl glycoside containing the phenol moiety was tested for the inhibition of the three Mycobacterium tuberculosis β-carbonic anhydrases (CAs, EC 4.2.1.1) with activities in the low micromolar range detected. The compounds were also tested for the inhibition of growth of M. tuberculosis H₃₇Rv strain, leading to the identification of (E)-1-(2′,3′,4′,6′-tetra-O-acetyl-β-d- glucopyranosyl)-4-(3-hydroxyphenyl)but-3-en-2-one (1) as the first carbonic anhydrase inhibitor with anti-tubercular activity.

Full text of DOI:10.1016/j.bmcl.2012.11.085

Bioorganic & Medicinal Chemistry Letters 23 (2013) 740–743
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibition of the b-carbonic anhydrases from Mycobacterium tuberculosis
with C-cinnamoyl glycosides: Identification of the first inhibitor with
anti-mycobacterial activity
María V. Buchieri b, Leonardo E. Riafrecha a, Oscar M. Rodríguez a, Daniela Vullo c, Héctor R. Morbidoni b,
,
⇑
a,
Claudiu T. Supuran c, , Pedro A. Colinas
⇑
⇑
a LADECOR, Departamento de Química, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, 47 y 115, 1900 La Plata, Argentina
b Cátedra de Microbiología, Virología y Parasitología, Facultad de Ciencias Médicas, Universidad Nacional de Rosario, Santa Fe 3100, 2000 Rosario, Argentina
c Universitá degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino, Firenze, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A small series of C-cinnamoyl glycoside containing the phenol moiety was tested for the inhibition of the
three Mycobacterium tuberculosis b-carbonic anhydrases (CAs, EC 4.2.1.1) with activities in the low micro-
molar range detected. The compounds were also tested for the inhibition of growth of M. tuberculosis
Received 2 November 2012
Revised 15 November 2012
Accepted 20 November 2012
Available online 1 December 2012
H37Rv strain, leading to the identification of (E)-1-(20,30,40,60-tetra-O-acetyl-b-
D-glucopyranosyl)-4-(3-
hydroxyphenyl)but-3-en-2-one (1) as the first carbonic anhydrase inhibitor with anti-tubercular activity.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
C-Glycosides
Mycobacterium tuberculosis
Carbonic anhydrase
Anti-tubercular activity
Infection with Mycobacterium tuberculosis and related mycobac-
teria affects a large fraction of the world population, with an esti-
mated 9.4 million new cases and 1.7 million deaths annually.1
Furthermore, tuberculosis (TB) infection including both
multidrug-resistant tuberculosis (MDR-TB) and drug-resistant
tuberculosis (XDR-TB) is a leading cause of death worldwide. The
development of novel anti-TB drugs that do not possess cross-
resistance with current anti-mycobacterial drugs and have mini-
mal toxicity, is urgently needed due to the increasing incidence
of MDR-TB, XDR-TB, and TB–HIV co-infection. In the last 10 years
the research on M. tuberculosis has made much progress with the
genome unrevealed2 and the discovery of possible new drug tar-
gets which may lead to the development of compounds possessing
a novel mechanism of action and thus resolve the drug resistance
problem mentioned above. Among the new such proteins identi-
fied after the M. tuberculosis genome was published, there are also
three b-carbonic anhydrases (CAs, EC 4.2.1.1) which have been
cloned, purified and characterized by Jones’ and Supuran’s
groups.3,4
CAs are a superfamily of zinc metalloenzymes that catalyze the
reversible hydration of carbon dioxide to give bicarbonate and a
proton.5 Whereas the
a
-CA family, mainly present in mammals
has been thoroughly investigated from the drug design viewpoint,6
only recently CAs belonging to the b- and -CA families, which are
widespread in bacteria and fungi (the b-CAs) and Archaea (the b-
and -CAs), respectively, started to be considered for such a pur-
c
c
pose. Two b-CAs from M. tuberculosis were successfully cloned
and crystallized by Jones’ group: Rv1284, also denominated mtCA
1, and Rv3588c, also designated as mtCA 2.3 Supuran’s group then
identified, cloned and characterized the third such enzyme, en-
coded by the gene Rv3273, and denominated mtCA 3.4 The precise
physiological function of the three b-CAs from this pathogen is not
known for the moment. Rv1284 is generally believed to be essen-
tial for survival of Mycobacterium and was found to be highly
up-regulated under starvation conditions. In contrast, there is no
evidence suggesting that Rv3273 is essential for mycobacterial
growth.7,8 Several sulfonamides and sulfamates inhibitors of the
three mycobacterial carbonic anhydrases has been reported.9 Such
studies shows mtCAs to be druggable targets for developing anti-
mycobacterial agents with a diverse mechanism of action com-
pared to the clinically used drugs for which many strains led to
the development of MDR-TB and XDR-TB. However the in vivo
proof of concept study showing that inhibition of these enzymes
leads to mycobacterial death or growth inhibition is still missing.9
⇑
Corresponding authors. Tel.: +54 341 5689618 (H.R.M.); tel.: +39 055 457 3005;
fax: +39 055 457 3385 (C.T.S.); tel.: +54 221 4243104; fax: +54 221 4226947
(P.A.C.).
E-mail addresses: morbiatny@yahoo.com (H.R. Morbidoni), claudiu.supuran@u-
nifi.it (C.T. Supuran), pcolinas@quimica.unlp.edu.ar (P.A. Colinas).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.085

M. V. Buchieri et al. / Bioorg. Med. Chem. Lett. 23 (2013) 740–743
741
Phenol was reported to act as an inhibitor of the zinc enzyme
carbonic anhydrase.10 Indeed, phenol binds to CA in a diverse man-
ner compared to classical inhibitors of the sulfonamide type, by
interacting with a zinc-bound water molecule through hydrogen
bonding and with no direct interaction between the inhibitor and
the zinc.11 Supuran’s group has recently investigated the interac-
moiety is tethered to a phenol CA pharmacophore through a car-
bon chain.17 The C-glycosides were generally effective mammalian
carbonic anhydrase inhibitors, with inhibition constants in the low
micromolar range.
Very recently galactose-derived aryl enones were shown to be
effective as antimycobacterial agents.18 Likewise alkanoyl glyco-
sides derived from b-C-glycosidic ketones were shown to be inhib-
tion of phenol and some derivatives with
a- and b-carbonic
anhydrases enzymes displaying low micromolar/submicromolar
inhibition.10,12 Thus, by binding in non-classical ways to CAs, phe-
nols as well their derivatives constitute interesting leads to design
novel CA inhibitors with selectivity and/or specificity for some of
the medicinal targets belonging to this enzyme family.
The use of glycomimetics in the design of CA inhibitors has pro-
ven to be a successful approach and now constitutes one of the
most attractive ways to develop new generations of effective and
selective inhibitors.13,14 The stereochemical diversity across the
carbohydrate tails provides the opportunity for interrogation of
subtle differences in active site topology of CA isozymes. On the
last years one of our groups has developed several methodologies
for the synthesis of N-glycosyl sulfonamides by sulfonamidoglyco-
sylation of carbohydrate derivatives. We reported the synthesis of
itors of a
-glucosidase.19
In order to discover novel probes that may help in the investi-
gation and control of tuberculosis through a new mechanism of ac-
tion, we evaluated the M. tuberculosis CAs inhibition and the anti-
tubercular activity of a series of C-cinnamoyl glycosides containing
the phenol moiety.
A set of new C-cinnamoyl glycosides (Fig. 1) was synthesized as
outlined in Scheme 1 and described previously by us.17 C-Cinnam-
oyl glycosides 1–4 have been prepared by aldol condensation of b-
C-glucosyl and b-C-galactosyl ketones with 3-hydroxy or 4-
hydroxybenzaldehyde at room temperature in the presence of pyr-
rolidine as catalyst. The O-acetate protecting groups of the carbo-
hydrate moiety were next removed using triethylamine in
methanol/water to afford the deprotected C-glycosides 5–8.
The inhibitory activity of the C-glycosides 1–8 and phenol
against human CA II and the purified M. tuberculosis b-CAs encoded
by genes Rv1284, Rv3273 and Rv3588c are presented in Table 1.
These inhibition data were acquired using a stopped flow assay
that monitors the physiological reaction, that is, the CA catalyzed
hidration of CO2.20
a series of
a-D-hex-2-enopyranosyl sulfamides, which was evalu-
ated against CA isozymes; one erythro compound was found to
be a very effective CA IX inhibitor.15 Recently we have prepared no-
vel N-b-glycosyl sulfamides and shown that they selectively target
cancer-associated CAs (IX and XII) with Kis in the low nanomolar
range.16 In the search of non-sulfonamide CAIs belonging to differ-
ent classes of compounds, we have very recently developed the
synthesis of C-cinnamoyl glycosides, where the carbohydrate
A number of structure–activity relationships (SARs) were iden-
tified in this study and are summarized as follows:
OH
OH
OH
AcO
AcO
AcO
OAc
OAc
OAc
OAc
O
O
O
O
OH
OH
OH
OH
AcO
AcO
AcO
AcO
AcO
OAc
OAc
OAc
OAc
O
O
O
O
2
1
4
3
OH
HO
HO
HO
OH
OH
OH
OH
O
O
O
O
HO
HO
HO
HO
HO
OH
OH
OH
OH
O
O
O
O
8
5
7
6
Figure 1. Peracetylated C-glycosides (1–4) and fully deprotected derivatives (5–8).
OH
OHC
O
O
O
(AcO)n
(HO)n
(AcO)n
OH
OH
pyrrolidine
O
O
1-4
Et3N / CH3OH / H2O
O
(HO)n
OH
O
5-8
Scheme 1. Preparation of C-cinnamoyl glycosides 1–8.

742
M. V. Buchieri et al. / Bioorg. Med. Chem. Lett. 23 (2013) 740–743
Table 1
Table 3
Inhibition of hCA II and the b-CAs from Mycobacterium tuberculosis with the C-
Evaluation of antimycobacterial activity on solid medium
cinnamoyl glycosides 1–8 and phenol (as standard)a
Compound
MIC (lg/ml)
Compound
Ki (l
M)b
1
2
7
3.125–6.25
>100
hCA IIc
mt Rv1284
mt Rv3273
mt Rv3588c
100a
1
2
3
4
5
6
7
8
7.0
3.9
7.1
5.5
7.8
8.8
3.1
6.8
5.5
2.1
2.9
3.8
19.0
13.1
15.6
12.0
6.21
4.13
3.25
4.13
79.0
0.64
0.35
0.87
1.15
0.24
0.13
0.51
0.94
ND
a
Pinpoint colonies.
4.5
0.14
0.93
1.16
4.5
This quick method is based on the final turbidity of the micro-
titer well plate and is not quantitative. It is usually taken as a
‘yes/no’ evaluation of activity, although compounds with weak
activities may cause various degrees of inhibition of growth, indi-
cated as (+/ꢀ) in Table 2. A more precise method is the evaluation
of growth inhibition on solid medium that also allows for the
observation of colony morphology and/or size alterations. Thus,
we repeated the tests for the compounds exhibiting the most
promising activity on solid medium containing different concen-
trations of compounds. To this end, we plated small aliquots of
fresh cultures of M. tuberculosis H37Rv containing a low CFU
amount (103) in solid Middlebrook ADS glycerol medium supple-
mented with varying amounts of the selected compounds (1, 2
and 7). The plates were sealed and incubated at 37 °C for 30 days
followed by visual inspection (Table 3). In this case, compound 1
was again the most active, causing a 99.9% reduction in colony
Phenol
64.0
a
All CAs are recombinant enzymes obtained in the authors’ laboratory as
reported earlier.10
b
Errors in the range of 5–10% of the reported value, from three different
determinations.
c
From Ref. 17.
Table 2
Anti-tubercular activity of C-cinnamoyl glycosides
Compound
Concentration
50 g/ml
100
lg/ml
l
625 lg/ml
1
2
3
4
5
6
7
8
MICa (+)b
MIC (+)
No
No
No
(+/ꢀ)
MIC (+)
No
(+/ꢀ)c
(+/ꢀ)
No
No
No
(+/ꢀ)
(+/ꢀ)
No
Nod
No
No
No
No
No
No
No
counts (MIC99.9%) at concentration values as low as 3125–625
ml. Compounds 2 and 7 demonstrated some activity at high con-
centrations (compound MIC99.9% >100 g/ml, compound
MIC99.9% = 100 g/ml) although in the case of 2 an effect on the size
lg/
2
l
7
l
of the colonies growing at sub-inhibitory concentrations was seen.
This was taken as proof of some activity on the mycobacterial
metabolism capable of restricting growth but not large enough to
prevent it. This result is compatible with the fact that there are
three mtCAs, that may be inhibited at different degrees, thus yield-
ing colonies with restricted size.
a
MIC (Minimum Inhibitory Concentration), minimum drug concentration lead-
ing to growth inhibition.
b
(+) indicates complete growth inhibition.
c
(+/ꢀ) indicates partial inhibition.
d
No indicates no inhibition. Growth/no growth was assessed in all cases by
In conclusion, we have investigated the enzyme inhibition pro-
file of a small series of C-cinnamoyl glycosides containing the phe-
nol moiety, against purified M. tuberculosis b-CAs. The C-glycosides
were generally effective CA inhibitors, with inhibition constants in
the low micromolar. Also the glycosides were screened against M.
naked eye observation of turbidity.
(i) Isoform hCA II was inhibited by compounds 1–8 with Kis in
the range 3.1–8.8
of the inhibitory power, similar to that of phenol (Ki of
5.5 M).
lM, showing a flat SAR with less variation
tuberculosis H37Rv and (E)-1-(20,30,40,60-tetra-O-acetyl-b-
D-gluco-
pyranosyl)-4-(3-hydroxyphenyl)but-3-en-2-one (1) was identified
as the first mtCA inhibitor with antimycobacterial activity.
Although the C-glycoside 1 showed no selectivity for the mtCAs
over the ubiquitous isozyme CA II, its physicochemical properties
would lead to a decreased affinity for cytosolic CAs,17 however fur-
ther in vivo studies are necessary to confirm this hypothesis. Thus
1 may be useful as lead compound for the development of novel
chemotherapeutic agents against Mycobacterium tuberculosis.
l
(ii) All investigated C-cinnamoyl glycosides were efficient,
micromolar inhibitors of Rv1284 mtCA with inhibition con-
stants in the range of 0.14–4.5
lM. This inhibition is greater
than that for phenol (Ki of 64.0
lM), this confirms that
attaching carbohydrate moieties to the CA phenol pharma-
cophore could improve its inhibitory activity.
(iii) The Rv3273 mtCA was inhibited very weakly by phenol (Ki of
79.0) while compounds 1–8 exhibited enhanced enzyme
inhibition for this isozyme compared to phenol. Peracetylat-
ed C-glycosides 1–4 exhibited moderate Rv3273 inhibitory
activity with inhibition constants in the range 12.0–
Acknowledgments
This work was financed in part by an EU Grant (Metoxia) to
C.T.S., Grants from FONCyT to H.R.M., UNLP and CONICET to
P.A.C. (Argentina). M.V.B. is a fellow of CONICET. H.R.M. is a career
member of the Science Council of the National University of Rosa-
rio (CIUNR) P.A.C. is member of the Scientific Research Career of
CONICET.
19.0
lM. Deprotected compounds 5–8 were more effective
inhibitors with Kis in the range 4.13–6.21
lM.
(iv) The C-glycosides investigated all showed an efficient low
micromolar or submicromolar inhibitory activity on the
mtCA Rv3588c, with Kis in the range 0.13–1.15 lM.
References and notes
The anti-tubercular activity of compounds 1–8 was investigated
by using fresh cultures of M. tuberculosis H37Rv grown and tested as
previously described.21,22 Tests were carried on by duplicate. Our
preliminary results clearly demonstrated that compounds 1, 2, 6
and 7 displayed growth inhibition activity at concentrations be-
1. Dye, C. Nat. Rev. Microbiol. 2009, 7, 81.
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Feltwell, T.; Gentles, S.; Hamlin, N.; Holroyd, S.; Hornsby, T.; Jagels, K.;
Krogh, A.; McLean, J.; Moule, S.; Murphy, L.; Oliver, K.; Osborne, J.; Quail, M.
tween 50 and 100 lg/ml (Table 2).

M. V. Buchieri et al. / Bioorg. Med. Chem. Lett. 23 (2013) 740–743
743
A.; Rajandream, M. A.; Rogers, J.; Rutter, S.; Seeger, K.; Skelton, J.; Squares,
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the reaction have been used for determining the initial velocity. The
uncatalyzed rates were determined in the same manner and subtracted from
the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in
distilled-deionized water and dilutions up to 0.01 nM were done thereafter
with distilled-deionized water. Inhibitor and enzyme solutions were
preincubated together for 15 min at room temperature prior to assay, in
order to allow for the formation of the E–I complex. The inhibition constants
were obtained by non-linear least-squares methods using PRISM 3, and the
Cheng–Prussoff equation (Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973,
22, 3099) as reported earlier and represent the mean from at least three
different determinations.
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5. Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168.
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21. Bacterial strains and growth media: Mycobacterium tuberculosis strain H37Rv
was grown in Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI, USA)
supplemented with 1/10 v/v of ADS (a solution containing 50 g/L BSA fraction
V, 20 g/L dextrose and 8.1 g/L NaCl), glycerol (1% w/v) herein designated 7H9-
ADS-G for short. Tween 80 was added to prevent clumping (0.05% w/v). When
needed, solid media (Middlebrook 7H11 supplemented with ADS (1/10 v/v)
and glycerol (1% v/v) was used. Cultures were grown at 37 °C under gentle
agitation.
22. Anti-mycobacterial activity assay. Stock solutions for all the tested compounds
were made in DMSO at 10 mg/ml. Working solutions were made by dilution in
the above described 7H9-ADS-G medium at a final concentration of 400
lg/ml.
Antimycobacterial activity was determined by two-fold dilution of the
a
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compounds in Middlebrook 7H9-ADS-G medium. For this purpose 96-well
plates (Falcon, Cat number 3072, BectonDickinson, Lincoln Park, NJ) were used.
The 96-well plates received 100
twofold dilution of the compounds was made directly on the plate. The initial
and final drug concentrations tested were 100 and 0.78 g/ml, respectively.
Four compounds were tested in duplicate in each microtiter plate, Rifampicin
(from 2 to 0.16 g/ml; stock solution prepared as a 10 mg/ml solution in
lL of Middlebrook 7H9 broth and a serial
l
l
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20. An Applied Photophysics stopped-flow instrument has been used for assaying
the CA catalysed CO2 hydration activity as reported by Khalifah (Khalifah, R. G.
J. Biol. Chem. 1971, 246, 2561). Phenol red (at a concentration of 0.02 mM) has
been used as indicator, working at the absorbance maximum of 557 nm, with
20 mM Hepes (pH 7.5) as buffer, and 20 mM Na2SO4 (for maintaining constant
the ionic strength), following the initial rates of the CA-catalyzed CO2
hydration reaction for a period of 10–100 s. The CO2 concentrations ranged
from 1.7 to 17 mM for the determination of the kinetic parameters and
inhibition constants. For each inhibitor at least six traces of the initial 5–10% of
methanol) was used as control drug. Two rows were used for growth control
(medium and inoculum alone) and sterility control (medium alone). The
inoculum was prepared as a 1/25 dilution of a fresh mid-log M. tuberculosis
H
37Rv suspensión (O.D. equivalent to Mc Farland 1.0 scale value) made in
Middlebrook 7H9-ADS-G. A 100-
l
L aliquot (containing approximately 106
Colony Forming Units) was used to inoculate the wells except for the row used
for sterility testing. Plates were sealed with Parafilm and incubated at 37 °C for
7 days. Minimum inhibitory concentration (MIC) was defined as the lowest
drug concentration preventing mycobacterial growth as judged by visual
inspection. Evaluation of activity on solid medium was performed by adding
the chosen amounts of each compound to 3 ml of molten medium, pouring the
mixture on 24 wells microtiter plates, and after hardening, plating aliquots
(ꢁ1 ꢂ 103 CFU) of fresh M. tuberculosis H37Rv cultures onto the surface of the
medium. Incubation proceeded for 30 days at 37 °C before visual inspection.
MIC99.9% was taken as the concentration that caused a 99.9% reduction in cell
counts.