Synthesis and biological evaluation of new enantiomerically pure azole derivatives as inhibitors of Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.bmcl.2009.02.101

A series of novel enantiomerically pure azole derivatives was synthesized. The new compounds, bearing both an imidazole as well as a triazole moiety, were evaluated as antimycobacterial agents. One of them proved to have activity against Mycobaterium tube

Full text of DOI:10.1016/j.bmcl.2009.02.101

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2203–2205
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of new enantiomerically pure azole
derivatives as inhibitors of Mycobacterium tuberculosis
Daniele Castagnolo a, Marco Radi a, Filippo Dessì a, Fabrizio Manetti a, Manuela Saddi b, Rita Meleddu b,
Alessandro De Logu b, Maurizio Botta a,c,
*
a Università degli Studi di Siena, Dipartimento Farmaco Chimico Tecnologico, via A. De Gasperi 2, 53100 Siena, Italy
b Sezione di Microbiologia Medica, Dipartimento di Scienze e Tecnologie Biomediche, Università di Cagliari, Viale Sant’Ignazio 38, 09123 Cagliari, Italy
c Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, BioLife Science Bldg., Suite 333,
1900 N 12th Street, Philadelphia, PA 19122, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel enantiomerically pure azole derivatives was synthesized. The new compounds, bearing
both an imidazole as well as a triazole moiety, were evaluated as antimycobacterial agents. One of them
proved to have activity against Mycobaterium tuberculosis comparable to those of the classical antibacte-
rial/antifungal drugs Econazole and Clotrimazole.
Received 5 February 2009
Revised 24 February 2009
Accepted 25 February 2009
Available online 28 February 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Azole
Click chemistry
Tuberculosis
Antifungal agents
Tuberculosis (TB) is a common and deadly infectious disease
caused by mycobacteria, mainly Mycobacterium tuberculosis. Over
one-third of the world’s population has been exposed to the TB
bacterium, and new infections occur at a rate of one per second.1
In 2005, mortality and morbidity statistics included 14.6 million
chronic active TB cases, 8.9 million new cases, and 1.6 million
deaths, mostly in developing countries.2 In addition, the rising
number of people who are contracting TB because of their immune
systems compromised by immunosuppressive drugs or substance
abuse or HIV/AIDS, is a serious threat to TB control and prevention.
Moreover the emergence of multi-drug resistance TB (MDR-TB),
defined as resistance to at least isoniazid and rifampin, and the
extensively drug resistant (XDR-TB) strains make the discovery
and the development of new drugs a priority.3 In the last few years,
the determination of the genome sequence of Mycobacterium
tuberculosis4,5 (MTB) provided a much needed boost for research
into new drug targets against this pathogen. In particular, recent
promising data suggest that targeting the lipid metabolism path-
ways of MTB may provide an excellent route to attenuating or kill-
ing the bacterium. The genome of MTB encodes for a relatively
large number of cytochrome P450 enzymes. These data indicate
important physiological roles for these enzymes which, given that
the substrate preference of the majority of P450s is for hydropho-
bic molecules, most are likely to be involved in lipid metabolism.
McLean and co-workers cloned and expressed MTB CYP51 and
CYP121, two types of P450 from MTB.6 They demonstrated that
CYP51 and CYP121 bind azole antifungal drugs tightly and that
azole compounds are potent inhibitor of cell growth of Mycobacte-
rium bovis and Mycobacterium smegmatis, two mycobacterial spe-
cies which closely resemble MTB. Azole drugs proved to have
also antitubercular activities in mice.7 Finally, recent studies fur-
nished the crystal structure of MTB cytochrome P450 CYP121 in
complex with the fluconazole.8 It has been found that MTB-
CYP121 binds commercially available azole drugs as clotrimazole
or econazole and that it may be the true target for the antimyco-
bacterial activity of the azoles in vivo as suggested by gene-knock-
out studies.9,10
In this context, as a continuation of our previous works on the
discovery of new antimicrobial agents,11,12 we decided to focus
our attention on the synthesis and preliminary biological evalua-
tion against MTB of novel azole analogues with polycyclic structure
A which resemble the classical antifungal/antibacterial azole drugs
(Fig. 1). Being the target compounds chiral, we were also attracted
by the possibility of synthesizing these derivatives in enantiomeri-
cally pure form in order to evaluate the biological profile of the sin-
gle enantiomers.
* Corresponding author. Present address: Università degli studi di siena, Dipar-
timento Farmaco Chimico Tecnologico, via A. De Gasperi 2, 53100 Siena, Italy.
E-mail address: botta@unisi.it (M. Botta).
Desired compounds were synthesized starting from different
arylpropargylamides 1a–e (Scheme 1) which were obtained in
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.101

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D. Castagnolo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2203–2205
HO
AcO
MeO
OH O
OH OH
O
NHNH2
NH
N
O
N
O
N
OH
O
N
Isoniazid
Rifampin
R
Cl
O
N
Cl
N
N
R'
Cl
N
N
N
Cl
N
N
N
Clotrimazole
Econazole
A
Figure 1. Common drugs used in anti-TB therapy and target compounds A.
R1
the reaction mixtures in good yields. HPLC–MS analysis revealed
that compounds 4a–e are enantiomerically pure and no racemiza-
tion occured during the reaction. The enantiomeric excesses (ee)
and reaction yields are reported in Table 1.
Compounds (R)-4a–e and (S)-4a–e were assayed for their inhib-
itory activity toward M. tuberculosis H37Rv (ATCC27294). The min-
R
R
N
N
N
i.
n = 0, 1
*
*
NHCOCH3
NHCOCH3
1a-e
2a-e
Table 1
ii.
R1
Yields and ee values of azoles 4
R1
Entry
Compounds
R
N
N
N
R
R
R1
n
eea (%)
Yieldb (%)
½ ꢀ
a 2D0
iii.
N
N
n = 0, 1
*
N
1
2
3
4
5
6
7
8
9
(S)-4a
(R)-4a
(S)-4b
(R)-4b
(S)-4c
(R)-4c
(S)-4d
(R)-4d
(S)-4e
(R)-4e
4-F
4-F
4-Br
4-Br
4-F
H
H
H
H
H
H
4-Cl
4-Cl
H
0
0
0
0
1
1
1
1
1
1
95
95
89
89
92
93
95
95
94
95
43
40
45
43
52
53
62
60
55
49
ꢁ9.9 (c 1.0 CHCl2)
+8.3 (c 1.0 CHCl2)
ꢁ10.9 (c 1.0 CHCl2)
+9.2 (c 1.2CH2Cl2)
ꢁ8.7 (c 1.1 CHCl2)
+7.6 (c 1.3CH2Cl2)
ꢁ9.4 (c 1.1 CHCl2)
+9.9 (c 1.0CH2Cl2)
ꢁ9.6 (c 0.9 CHCl2)
+10.1 (c1.0CH2Cl)
n = 0, 1
*
N
NH2
N
4a-e
3a-e
4-F
4-Cl
4-Cl
4-Br
4-Br
Scheme 1. Synthesis of enantiomerically pure azoles. Reagents and conditions: (i)
(For n = 1): NaN3, benzyl chloride, sodium ascorbate/CuSO4, H2O/t-BuOH, MW,
125 °C, 10 min; (For n = 0): PhN3, sodium ascorbate/CuSO4, H2O/t-BuOH, MW,
120 °C, 10 min; (ii) HCl 3 N, 100 °C; (iii) H3PO4, paraformaldehyde, NH4Cl, H2O/
dioxane, MW, 80 °C, 10 min.
10
H
a
Determined by chiral HPLC–MS using an (S,S)-Whelk-O1 column (methanol/
water 95:5, flow rate 0.8 mL/min, UV-254 nm).
b
Isolated yields were reported.
enantiopure form via kinetic enzymatic resolution as described in
our previous works.13 Propargylamides 1a–e were converted into
1,2,3-triazoles 2a–e via a microwave assisted Click reaction in
the presence of Cu/Na Ascorbate to generate in situ the Cu(I) cata-
lyst. Compounds 2a–e were obtained in pure form after simple
aqueous work-up and were used in the next step without any fur-
ther chromatographic purification. Hydrolysis of triazoles 2a–e in
HCl 3 N at reflux afforded the corresponding unstable amines 3a–
e which were immediately used in the next step without further
purification. The synthesis of the imidazole ring from a primary
amine requires generally a multistep procedure and long reaction
times. On the basis of our experience in the microwave-assisted or-
ganic synthesis,13 we decided to circumvent the problem perform-
ing a one-pot synthesis of the imidazole ring under microwave
irradiation.
Table 2
MIC values for compounds 4a–e
Entry
Compound
Log Pa,b
MIC (l
g mLꢁ1) M. tuberculosis
1
2
3
4
5
6
7
8
9
10
11
12
13
(S)-4a
(R)-4a
(S)-4b
(R)-4b
(S)-4c
(R)-4c
(S)-4d
(R)-4d
4.184
4.184
4.525
4.525
4.329
4.329
5.103
5.103
4.670
4.670
5.399
5.605
0.318
32
32
32
32
64
64
32
32
32
16
20.4
12.5
0.211
(S)-4e
(R)-4e
Clotrimazolec
Econazolec
Isoniazid
Amines 3a–e were dissolved in a 9:1 mixture of water and diox-
ane, and polyphosphoric acid, solid paraformaldehyde, glyoxal
aqueous solution (40%) and NH4Cl saturated solution were added
and the mixture was finally irradiated with microwaves for
6 min at 120 °C. Imidazole derivatives 4a–e were isolated from
Physicochemical properties were predicted using QikProp v2.5.16
Predicted octanol/water partition coefficient; range of recommended val-
a
b
ues(ꢁ2.0)–(+6.5).
c
According to the literature.14

D. Castagnolo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2203–2205
2205
imum inhibitory concentration (l
g mLꢁ1) was determined for each
6. McLean, K. J.; Marshall, K. R.; Richmond, A.; Hunter, I. S.; Fowler, K.; Kieser, T.;
Gurcha, S. S.; Besra, G. S.; Munro, A. W. Microbiology 2002, 148, 2937.
7. Guardiola-Diaz, H.; Foster, L.-A.; Mushrush, D.; Vaz, A. D. N. Biochem.
Pharmacol. 2001, 61, 1463.
8. (a) Ahmad, Z.; Sharma, S.; Khuller, G. K. FEMS Microbiol. Lett. 2006, 261, 181; (b)
Ahmad, Z.; Sharma, S.; Khuller, G. K. FEMS Microbiol. Lett. 2006, 258, 200.
9. Munro, A. W.; McLean, K. J.; Marshall, K. R.; Warman, A. J.; Lewis, G.; Roitel, O.;
Sutcliffe, M. J.; Kemp, C. A.; Modi, S.; Scrutton, N. S.; Leys, D. Biochem. Soc. Trans.
2003, 31, 625.
compound. Clotrimazole and econazole were used a reference
compounds.14 Resulting data are reported in Table 2. Compounds
4a and b showed medium activity toward MTB as well as com-
pounds 4d (entries 1–4 and 7 and 8). In all these cases, both enan-
tiomers presented the same biological profile. On the contrary,
componds 4c resulted to be inactive (entries 5 and 6). Finally the
bromo derivatives 4e showed good MIC values. In particular one
of the two enantiomers, namely (R)-4e (entry 10), proved to have
very promising antimycobacterial activity, showing a biological
profile similar to econazole (entry 12) and better than clotrimazole
(entry 11).15
In conclusion, a new class of azole derivatives has been synthes-
ised. All the compounds were obtained in good yield and in enan-
tiomerically pure form and were tested as new potential
antitubercular agents. One of those compounds, derivative (R)-
4e17,18 showed good activity against MTB. Interestingly, its enan-
tiomer, derivative (S)-4e presented higher MIC values, confirming
the importance of synthesizing enantiomerically pure compounds
due to the different interactions of single enantiomers with chiral
biological systems. Attempts to design and synthesize novel azole
derivatives of 4 to improve the activity against MTB are currently
in progress.
10. Seward, H. E.; Roujeinikova, A.; McLean, K. J.; Munro, A. W.; Leys, D. J. Biol.
Chem. 2006, 281, 39437.
11. Manetti, F.; Magnani, M.; Castagnolo, D.; Passalacqua, L.; Botta, M.; Corelli, F.;
Saddi, M.; Deidda, D.; De Logu, A. Chem. Med. Chem. 2006, 1, 973.
12. Castagnolo, D.; De Logu, A.; Radi, M.; Bechi, B.; Manetti, F.; Magnani, M.;
Supino, S.; Meleddu, R.; Chisu, L.; Botta, M. Bioorg. Med. Chem. 2008, 16, 8587.
13. (a) Castagnolo, D.; Dessı‘, F.; Radi, M.; Botta, M. Tetrahedron: Asymmetry 2007,
18, 1345; (b) Castagnolo, D.; Renzulli, M. L.; Galletti, E.; Corelli, F.; Botta, M.
Tetrahedron: Asymmetry 2005, 16, 2893.
14. Menozzi, G.; Morello, L.; Fossa, P.; Schenone, S.; Ranise, A.; Mosti, L.;
Bondavalli, F.; Loddo, R.; Murgioni, C.; Mascia, V.; La Colla, P.; Tamburini, E.
Bioorg. Med. Chem. 2004, 12, 5465.
15. M. tuberculosis H37Rv ATCC 27294 was used in this study. It was maintained on
Löwenstein-Jensen (bioMérieux, Marcy l’Étoile, France) agar slants until
needed. MICs were determined by a standard twofold agar dilution method.
Briefly, 1 mL of Middlebrook 7H11 agar (Becton Dickinson BBL, Sparks, MD)
supplemented with 10% oleic acid-albumin-dextrose-catalase enrichment
containing the testing compounds in 24-multiwell plates at concentrations
ranging between 0.0312 and 64 lg/mL was inoculated with 10 lL of a
suspension containing M. tuberculosis H37Rv 1.5 ꢂ 105 cfu/mL grown on
Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI) supplemented with
10% albumin–dextrose–catalase enrichment. Final inoculum was 1.5 ꢂ 103 per
well and was obtained as described previously.11,12 Plates were incubated for
21–28 days and MICs were read as minimal concentrations of compounds
completely inhibiting visible growth of mycobacteria.
Acknowledgements
16. QikProp, version 2.5, Schrödinger, LLC, New York, NY, 2005.
Financial support from the Italian Ministero dell’Istruzione,
dell’Università e della Ricerca (PRIN 2007N7KYCY) is gratefully
acknowledged. CARIPLO (Grant Rif. 2006.0880/10.8485 Bando
2006) is gratefully acknowledged.
17. Synthesis of azoles 4. General procedure.Amine 3 (0.38 mmol) was dissolved into
a solution of H2O/dioxane (3/1 mL) and H3PO4 was added until pH 2. Then solid
paraformaldehyde (15 mg) and glyoxal sol. 40% in water (0.1 mL) were added
and the mixture was stirred at 80 °C for 10 min. NH4Cl saturated solution
(0.5 mL) was added at this temperature and the resulting solution, in a 10-mL
sealed glass vial, was irradiated by microwave for 6 min at 120 °C. The mixture
was then cooled at 0 °C and NaOH was added until pH 12. The alkaline solution
was extracted with AcOEt two times. The combined organic layers were
washed with brine, dried (Na2SO4), filtered and evaporated to give the crude 4.
The crude product was purified by flash chromatography on silica gel, using
AcOEt/MeOH 9:1 as eluant, and obtained as a tan oil.
References and notes
1. World Health Organization (WHO). Tuberculosis Fact sheet No. 104 - Global
and regional incidence. March 2006, Retrieved on 6 October 2006.
2. Raviglione, M. C.; O’Brien, R. J. Tuberculosis. In Harrison’s Principles of Internal
Medicine; Kasper, D. L., Braunwald, E., Fauci, A. S., Hauser, S. L., Longo, D. L.,
Jameson, J. L., Isselbacher, K. J., Eds., 16th ed.; McGraw-Hill Professional, 2004;
pp 953–966.
3. (a) Janin, Y. L. Bioorg. Med. Chem. 2007, 15, 2479; (b) Gutierrez-Lugo, M. T.;
Bewley, C. J. Med. Chem. 2008, 51, 2606.
4. Cole, S. T.; Brosch, R.; Parkhill, J. Nature 1998, 393, 537.
18. Characterization of azole (R)-4e. Yield: 49%. 1H NMR (CDCl3): d 7.48–7.46 (3H,
m, NCHN and Ph), 7.39–7.38 (2H, m, Ph), 7.27–7.24 (3H,m, CCHNBn and Ph),
7.08 (1H, s, NCHCHN), 7.03–7.07 (2H, d, J = 8.21, Ph), 6.91(1H, s, NCHCHN), 6.59
(1H, s, CHN), 5.25 (2H, s, CH2Ph) ppm. 13C NMR (CDCl3): d 146.06, 136.76,
133.70,131.86, 129.48, 128.94, 128.94, 128.75, 128.53, 127.71, 122.51, 122.29,
56.62, 54.09 ppm. MS: 393.04 (M+), 395.04 (M+H)+ 416.04 (M+Na)+. ½a 2D0
ꢀ
+10.1
(c 1.0 CH2Cl2). Anal. Calcd for C19H16BrN5: C, 57.88; H, 4.09; N, 17.76. Found: C,
57.97; H, 4.32; N, 17.87.
5. Camus, J. C.; Pryor, M. J.; Medigue, C.; Cole, S. T. Microbiology 2002, 148, 2967.