Enantiopure antituberculosis candidates synthesized from (-)-fenchone

Article abstract of DOI:10.1016/j.ejmech.2014.03.025

The synthesis of new enantiopure N-acyl compounds derived from (-)-fenchone has been performed. The evaluation of their in vitro activity against Mycobacterium tuberculosis H₃₇Rv showed for most of them moderate activity. The structures bearing

Full text of DOI:10.1016/j.ejmech.2014.03.025

European Journal of Medicinal Chemistry 77 (2014) 243e247
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Enantiopure antituberculosis candidates synthesized from
(ꢀ)-fenchone
,
b
a
c
Georgi M. Dobrikov a , Violeta Valcheva , Yana Nikolova , Iva Ugrinova ,
*
Evdokia Pasheva c, Vladimir Dimitrova
,
*
a Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Bl. 9, Acad. G. Bonchev Str., Sofia 1113, Bulgaria
b Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Bl. 26, Acad. G. Bonchev Str., Sofia 1113, Bulgaria
c Institute of Molecular Biology “Roumen Tsanev”, Bulgarian Academy of Sciences, Bl. 21, Acad. G. Bonchev Str., Sofia 1113, Bulgaria
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of new enantiopure N-acyl compounds derived from (ꢀ)-fenchone has been performed.
The evaluation of their in vitro activity against Mycobacterium tuberculosis H37Rv showed for most of
them moderate activity. The structures bearing sulfonamide functionality have comparable activity to
ethambutol and possess low cytotoxicity.
Received 10 January 2014
Received in revised form
6 March 2014
Accepted 8 March 2014
Available online 12 March 2014
Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Fenchone
Chiral
Amides
Antimycobacterial
Tuberculosis
1. Introduction
researchers for several years starting with the discovery of SQ109
[5]. The recent studies of Onajole et al. [2,6] confirm the importance
Despite the availability of highly efficacious treatment for dec-
ades, tuberculosis (TB) remains a major global health challenge. In
1993, the World Health Organization (WHO) declared TB a global
public health emergency, at a time when an estimated 7e8 million
cases and 1.3e1.6 million deaths occurred each year. In 2010 there
were an estimated 8.5e9.2 million cases and 1.2e1.5 million deaths
(including deaths from TB among HIV-positive people). TB is the
second leading cause of death from an infectious disease world-
wide (after HIV, which caused an estimated 1.8 million deaths in
2008) [1].
An urgent need for highly potent, more effective drugs with
fewer or no side effects and shorter treatment periods to combat
the increasing TB pandemic is therefore apparent. Potential anti-TB
drug candidates such as diarylquinolones (TMC207), nitro-
imidazoles (OPC67683 and PA824), pyrroles (LL3858), diamines [2]
are in different stages of clinical trials [3,4]. The incorporation of
lipophilic polycyclic aliphatic compounds into drug-structures with
promising anti-TB applications has enjoyed much attention from
of further investigations of polycyclic compounds as potent anti-TB
agents.
In the course of our research for novel anti-tubercular com-
pounds, we recently demonstrated the role of chirality and wide
spectrum of substituted acyl groups, attached to nitrogen atom of
(R)-2-aminobutanol [7,8]. The present study aims at investigating
of new subclass diastereomerically pure bicyclic N-substituted
compounds with fenchane skeletons and the possibility of further
enhancing/improving their anti-TB activity. Based on this, 15 novel
amidoalcohols and 2 other N-carbonyl derivatives bearing the lip-
ophilic fenchane skeleton were synthesized and screened for
activity against drug sensitive (H37Rv) strain of tuberculosis.
2. Results and discussion
2.1. Chemistry
The starting aminoalcohol 3 (Scheme 1) was synthesized by
using enantiomerically pure (ꢀ)-fenchone as a natural source of
chirality. Desired amides 13e21, 25e27 and 31e33 were synthe-
sized through three main methods of N-acylation of 3, as shown
below (Schemes 2e4). It was found that the N-acylation by using
* Corresponding authors.
E-mail address: gmdob@orgchm.bas.bg (G.M. Dobrikov).
http://dx.doi.org/10.1016/j.ejmech.2014.03.025
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

244
G.M. Dobrikov et al. / European Journal of Medicinal Chemistry 77 (2014) 243e247
acylchlorides or activated acids (TBTU) is more efficient than the
aminolysis of esters applied previously [7]. Compounds 36e37
were synthesized by other methods (Scheme 5). All of the syn-
thesized compounds inherit the configuration of 3 and were iso-
lated in high purity after column chromatography or crystallization.
Scheme 1. Synthesis of compounds 2 and 3.
Scheme 2. Synthesis of compounds 13e21.

G.M. Dobrikov et al. / European Journal of Medicinal Chemistry 77 (2014) 243e247
245
Scheme 5. Synthesis of compounds 36 and 37.
Scheme 3. Synthesis of compounds 25e27.
2.1.3. Synthesis of compounds 25e27
The amidoalcohols 25e27 were synthesized using simple sol-
vent-free aminolysis of esters 22e24 with 3 by heating at 90e
100 ꢁC (Scheme 3). Compounds 25e27 were isolated in moderate
yields and high purities after column chromatography (or crystal-
lization in case of the low-soluble 27).
2.1.1. Synthesis of compounds 2 and 3
The synthesis of starting aminoalcohol 3 was performed in two
steps through intermediate 2 (Scheme 1), by modification of
described procedure [9]. The addition of in situ prepared LiCH2CN to
(ꢀ)-fenchone was performed at ꢀ85 ꢁC in THF without using of
anhydrous CeCl3. Other key moment was the hydrolysis of the
reaction mixture e the aqueous NH4Cl was added at ꢀ85 ꢁC and the
reaction immediately allowed to r.t. This improved procedure leads
to quantitative yield of 2 as pure diastereoisomer after column
chromatography. Reduction of 2 to aminoalcohol 3 was performed
quantitatively with LiAlH4 in Et2O, according to the literature [9].
The diastereomerical purity and configurations of 2 and 3 were
unambiguously confirmed by 1D and 2D NMR experiments.
2.1.4. Synthesis of compounds 31e33
Compounds 31e33 were prepared through the coupling reac-
tion between 3 and acids 28e30, respectively, in presence of O-
(Benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium
tetra-
fluoroborate (TBTU) as activating agent (Scheme 4). TBTU was
chosen as one of the most effective coupling reagent in peptide
synthesis [11]. Amides 31e33 were isolated in high yields after
column chromatography. In case of anthranilic acid (29) prelimi-
nary protection of the aromatic amino group was not necessary,
due to its low reactivity in comparison to the high nucleophilic
amino group of 3. To the best of our knowledge, there are no data
published concerning acid 30 and its preparation was introduced in
detail in the part named “Supplementary data”.
2.1.2. Synthesis of compounds 13e21
Series of amidoalcohols 13e21 were synthesized in good to
excellent yields using standard conditions for acylation of 3 (0 ꢁC
and Et3N in dry DCM) with acid chlorides 4e12 (Scheme 2). Sul-
fochloride 12 was prepared from cinnamic acid as described [10].
2.1.5. Synthesis of compounds 36e37
Urea 36 was synthesized through reaction between 3 and ethyl
isocyanate (34) at 0 ꢁC in dry DCM (Scheme 5). The pure product
was obtained in quantitative yield after crystallization from hep-
tane/MTBE. The N-carbobenzyloxy (Cbz) protected guanidine
derivative 37 was prepared in quantitative yield from 3 and com-
mercially
available
1,3-bis(benzyloxycarbonyl)-2-methyl-iso-
thiourea (35) according analogous procedure described in
literature [12].
2.2. Biology
There are no data regarding the antimycobacterial and cytotoxic
activity of the synthesized compounds.
2.2.1. In vitro antimycobacterial activity
The synthesized compounds were evaluated for their in vitro
activity against Mycobacterium tuberculosis H37Rv (Table 1; results
are presented in mM) using the method of Canetti (see Section 4.2).
All compounds are in agreement with the formal Lipinski’s rule of
five (except 18 and 37) and were designed in order to investigate
their antimycobacterial activity in comparison with the activity of
ethambutol (EMB), and the starting bicyclic aminoalcohol 3. Most
Scheme 4. Synthesis of compounds 31e33.

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G.M. Dobrikov et al. / European Journal of Medicinal Chemistry 77 (2014) 243e247
Table 1
In vitro screening data for antimycobacterial activity and cytotoxicity of synthesized compounds 3, 13e21, 25e27, 31e33 and 36e37.
Entry
Compound
Antimycobacterial activity toward reference
strain of Mycobacterium tuberculosis H37Rv, MIC (
Cytotoxicity toward human embryonal
kidney cell line 293T, IC50 (mM)
Selectivity
index, SIa
LogPb
m
M)
1
2
3
4
5
6
7
8
3
13
14
15
16
17
18
19
20
21
25
26
27
31
32
33
15.20
13.92
10.30
15.27
22.15
18.22
13.00
17.62
7.02
42.3
18.9
62.3
23.8
72.5
178.6
21.6
64.8
97.9
335.6
18.8
83.9
106.7
70.3
81.5
62.1
121.2
82.4
2.8
1.4
6.0
1.6
3.3
9.8
1.7
3.7
13.9
54.7
0.9
4.4
9.3
3.6
4.3
4.4
8.1
8.4
e
2.13 ꢂ 0.28
2.80 ꢂ 0.40
2.91 ꢂ 0.42
4.24 ꢂ 0.38
4.76 ꢂ 0.40
3.86 ꢂ 0.42
9.72 ꢂ 0.39
4.36 ꢂ 0.60
3.91 ꢂ 0.44
3.96 ꢂ 0.45
2.79 ꢂ 0.40
3.99 ꢂ 0.46
4.53 ꢂ 0.55
3.42 ꢂ 0.49
3.41 ꢂ 0.43
3.14 ꢂ 0.68
2.31 ꢂ 0.40
6.16 ꢂ 0.65
0.06d
9
10
11
12
13
14
15
16
17
18
19
6.14
19.87
18.92
11.44
19.52
18.96
14.17
14.91
9.85
36
37
EMB$2HClc
7.22
Not tested
a
Selectivity index: SI ¼ IC50/MIC.
b
c
LogP, octanolewater partitioning coefficient, was calculated using ACDLabs/ChemSketch 2012 (www.acdlabs.com).
EMB$2HCl e ethambutol dihydrochloride (reference compound).
d
LogP of EMB$2HCl is known in the literature: N.R. Budha, R.E. Lee and B. Meibohm, Curr. Med. Chem. 15 (2008) 809.
of the amides (15, 17, 19, 25e27 and 36) contain pharmacophore
groups known in our previous studies [7,8]. It is interesting to point
out that the introduction of various substituents at N-atom of 3
doesn’t affect dramatically the activity of the derivatives. As a
whole, the compounds in this study (including 3) showed moderate
but stable level of antimycobacterial activity (in most cases 30e70%
of the activity of reference EMB; 48% for 3). Compounds 20e21 and
37 demonstrated MIC near to EMB. The activity of sulfonamide 20
was expected since recent studies of Malwal et al. [13,14] revealed
the importance of 2,4-dinitrosulfonamide group as in vivo source of
sulfur dioxide that is a key agent for selective radical damaging of
bacterial biomacromolecules. In this context, it is interesting to
3. Conclusions
An efficient synthesis of new enantiopure chiral N-acyl com-
pounds derived from natural (ꢀ)-fenchone has been demonstrated.
The obtained pure structures have been characterized by spectro-
scopic methods and the in vitro biological activity has been eval-
uated against M. tuberculosis H37Rv. The most potent compounds
among this series were 20, 21 and 37 (MIC comparable to EMB).
Almost all of the substances have shown acceptable low to mod-
erate cytotoxicity.
4. Experimental
note that the activity of cinnamic sulfonamide 21 (MIC 6.14 mM) is
comparable with that of 20 although the structures are rather dif-
ferent in respect of the aromatic moieties. Compound 37 is a rare
example of antitubercular agent possessing in its structure the
guanidine motif. This structural motif is common in antibiotics like
streptomycin, capreomycin, viomycin [3,15] and some synthetic
polyamines [16].
4.1. Chemistry
For thin layer chromatography (TLC) aluminum sheets pre-
coated with silica gel 60 F254 (Merck) were used. Flash column
chromatography was carried out using silica gel 60 (0.040e
0.063 mm, 230e400 mesh ASTM, Merck). Commercially available
solvents for reactions, TLC and column chromatography were used
after distillation (and were dried when needed). Melting temper-
atures were determined in capillary tubes on an Electrothermal
MEL-TEMP 1102D-230 VAC apparatus without corrections. The
NMR spectra were recorded on a Bruker Avance IIþ 600 (600.13 for
1H and 150.92 MHz for 13C NMR) spectrometer. In case of CDCl3
TMS was used as internal standard. For other deuterated solvents
1H spectra were calibrated to the residual solvent peaks (DMSO-d6
2.2.2. In vitro cytotoxic activity
The stable level of antimycobacterial activity gave us a good
reason for further more detailed evaluation of the cytotoxic effect
of those compounds and SI (selectivity index) calculation. The
cytotoxic activity of the tested compounds was investigated
against human embryonic kidney cell line (HEK293) using the
MTT dye reduction assay. The corresponding IC50 values (in mM) of
the tested compounds were calculated using nonlinear regression
analysis and summarized in Table 1. As it could be seen the
compounds demonstrated a wide range of cytotoxicity with IC50
between 18.8 and 335.6. Almost all of the substances have shown
acceptable low to moderate cytotoxicity to the cells with only four
exceptions (13, 15, 18 and 25) which showed high cytotoxicity.
Compounds 20e21 and 37 demonstrated MIC near to EMB and
low cytotoxic effect. It is important to note that the most potent
antimycobacterial sample e the cinnamic sulfonamide 21 showed
excellent SI (54.7) and therefore it is of particular interest due to
its highly favorable features low cytotoxicity and significant
antimycobacterial activity which makes it a good candidate for an
efficient therapeutic agent.
d
¼ 2.50). 13C spectra were calibrated in all cases to the residual
solvent peaks (CDCl3
d
¼ 77.00, DMSO-d6
d
¼ 39.52). 31P NMR
spectra was recorded with full proton decoupling and using 85%
H3PO4 as external standard. The calibration of the 31P NMR spectra
was performed through changing of the spectrum reference fre-
quency (specific for the used NMR probe). The following additional
NMR techniques were used for all compounds: DEPT 135, COSY,
HSQC and HMBC. For numbering of the atoms see Supplementary
data. Mass spectra (MS) were recorded on a Thermo Scientific High
Resolution Magnetic Sector MS DFS by chemical ionization (CI) or
electrospray ionization (ESI), and are reported as fragmentation in
m/z with relative intensities (%). Optical rotation [
ments were obtained using a PerkineElmer 241 polarimeter.
20
a
]
measure-
D

G.M. Dobrikov et al. / European Journal of Medicinal Chemistry 77 (2014) 243e247
247
Elemental analyses were performed by the Microanalytical Labo-
ratory for Elemental Analysis of the Institute of Organic Chemistry,
Bulgarian Academy of Sciences. All fine chemicals were commer-
cially available (from SigmaeAldrich, Merck, Fluka, Acros, Alfa
Aesar). Dimethyl sulfoxide (DMSO) for testing of bioactivities was
commercial (spectroscopic grade) and was used without dis-
tillation or purification.
were calculated as percentage of the untreated control. In addition,
IC50 values were derived from the concentration response curves.
Acknowledgments
This study was partially supported by the Bulgarian Science
Fund e project DMU 02-1/2010. The financial support of the Bul-
garian Science Fund for the purchase of Bruker Avance IIþ 600 NMR
spectrometer in the framework of the Program “Promotion of the
Research Potential through Unique Scientific Equipment” e project
UNA-17/2005 is gratefully acknowledged.
4.2. Methodology for evaluation of antimycobacterial activity
The antimycobacterial activity was determined through the
proportional method of Canetti towards reference strain M. tuber-
culosis H37Rv. This method, recommended by the WHO, is the most
commonly used one worldwide for exploration of sensitivity/
resistance of tuberculosis strains towards chemotherapeutics [17e
21]. It allows precise determination of the proportion of resistant
mutants to a certain drug.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.03.025.
A sterile suspension/solution of each tested compound was
added to LöwensteineJensen egg based medium before its coagu-
lation (30 min at 85 ꢁC). Each compound was tested at four con-
References
[1] WHO Report 2011 e Global Tuberculosis Control; Webpage: http://www.
who.int/tb/publications/global_report/2011/gtbr11_full.pdf.
[2] O.K. Onajole, Y. Coovadia, H.G. Kruger, G.E.M. Maguire, M. Pillay, T. Govender,
European Journal of Medicinal Chemistry 54 (2012) 1e9.
[3] R.P. Tripathi, S.S. Bisht, A. Ajay, A. Sharma, M. Misra, M.Pd Gupt, Current
Medicinal Chemistry 19 (2012) 488e517.
[4] L.G. Dover, G.D. Coxon, Journal of Medicinal Chemistry 54 (2011) 6157e6165.
[5] R.E. Lee, M. Protopopova, E. Crooks, R.A. Slayden, M. Terrot, C.E. Barry, Journal
of Combinatorial Chemistry 5 (2003) 172e187.
[6] O.K. Onajole, K. Govender, P. Govender, P.D. van Helden, H.G. Kruger,
G.E.M. Maguire, K. Muthusamy, M. Pillay, I. Wiid, T. Govender, European
Journal of Medicinal Chemistry 44 (2009) 4297e4305.
[7] G.M. Dobrikov, V. Valcheva, M. Stoilova-Disheva, G. Momekov, P. Tzvetkova,
A. Chimov, V. Dimitrov, European Journal of Medicinal Chemistry 48 (2012)
45e56.
[8] G.M. Dobrikov, V. Valcheva, Y. Nikolova, I. Ugrinova, E. Pasheva, V. Dimitrov,
European Journal of Medicinal Chemistry 63 (2013) 468e473.
[9] V. Dimitrov, G. Dobrikov, M. Genov, Tetrahedron: Asymmetry 12 (2001)
1323e1329.
[10] R.J.W. Cremlyn, Journal of the Chemical Society C (1968) 11e16.
[11] E. Valeur, M. Bradley, Chemical Society Reviews 38 (2009) 606e631.
[12] J. Quancard, A. Labonne, Y. Jacquot, G. Chassaing, S. Lavielle, Ph Karoyan,
Journal of Organic Chemistry 69 (2004) 7940e7948.
[13] S.R. Malwal, D. Sriram, P. Yogeeswari, H. Chakrapani, Bioorganic & Medicinal
Chemistry Letters 22 (2012) 3603e3606.
[14] S.R. Malwal, D. Sriram, P. Yogeeswari, V. Badireenath Konkimalla,
H. Chakrapani, Journal of Medicinal Chemistry 55 (2012) 553e557.
[15] Y.L. Janin, Bioorganic & Medicinal Chemistry 15 (2007) 2479e2513.
[16] A. Nefzi, J. Appel, S. Arutyunyan, R.A. Houghten, Bioorganic & Medicinal
Chemistry Letters 19 (2009) 5169e5175.
[17] G. Canetti, N. Rist, J. Grosset, Revue de Tuberculose et de Pneumologie 27
(1963) 217e272.
[18] G. Canetti, S. Froman, J. Grosset, P. Hauduroy, M. Langerova, H.T. Mahler,
G. Meissner, D.A. Mitchison, L. Sula, Mycobacteria: laboratory methods for
testing drug sensitivity and resistance, Bulletin of the World Health Organ-
ization 29 (1963) 565e578.
[19] G. Canetti, W. Fox, A. Khomenko, H.T. Mahler, N.K. Menon, D.A. Mitchinson,
N. Rist, N.A. Smelev, Bulletin de l’Organisation mondiale de la santé 41 (1969)
21e43.
[20] L. Heifets, Conventional methods for antimicrobial susceptibility testing of
Mycobacterium tuberculosis, in: I. Bastian, F. Portaels (Eds.), Multidrug-resist-
ant Tuberculosis, Kluwer Academic Publishers, Dordrecht, The Netherlands,
2000.
[21] CellTiter 96 Non-Radioactive Cell proliferation assay, Technical Bulletin
#TB112, Promega Corporation USA, Revised 12/99.
[22] T. Mosmann, Journal of Immunological Methods 65 (1983) 55e63.
centrations e 5
DMSO). For some compounds, additional tests at concentrations
0.05, 1, 2 and 3 g/mL were performed. Tubes with Löwensteine
mg/mL, 2 mg/mL, 0.2 mg/mL and 0.1 mg/mL (in
m
Jensen medium (5 mL) containing tested compounds and those
without them (controls) were inoculated with a suspension of
M. tuberculosis H37Rv (105 cells/mL) and incubated for 45 days at
37 ꢁC. The ratio between the number of colonies of M. tuberculosis
grown in medium containing compounds and the number of col-
onies in control medium were calculated and expressed as per-
centage of inhibition. The MIC is defined as the minimum
concentration of compound required to inhibit bacterial growth
completely (0% growth). The MIC values are calculated and given as
mM.
4.3. Methodology for evaluation of cytotoxicity
The cell viability was examined as described previously [8].
Briefly a standard MTT-dye reduction based test have been used as
described by Mosmann [22] with some modifications. The method
is based on the biotransformation of the yellow tetrazolium salt
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide) to a violet formazan via the mitochondrial succinate dehy-
drogenase in the viable cells. The exponentially growing cells (HEK
e human embryonic kidney fibroblasts) were seeded in 96-well
flat-bottomed micro-plates (100 m
L/well) at a density of 105 cells
per mL and after 24 h incubation at 37 ꢁC they were exposed to
various concentrations of the tested compounds for 72 h. At least 8
wells were used for each concentration. After the incubation with
the test compounds 10 mL MTT solution (5 mg/mL in PBS) aliquots
were added to each well. The microplates were further incubated
for 4 h at 37 ꢁC and the formazan crystals formed were dissolved
through addition of 100 mL DMSO into each well. The absorbance
was measured on an ELISA plate reader (Bio-Tek Instruments) with
a test wavelength of 570 nm and a reference wavelength of 630 nm
to obtain sample signal (OD570eOD630). The cell survival fractions