Efficient synthesis of new (R)-2-amino-1-butanol derived ureas, thioureas and acylthioureas and in vitro evaluation of their antimycobacterial activity

Article abstract of DOI:10.1016/j.ejmech.2013.02.034

The synthesis of 22 structurally diverse urea, thiourea and acylthiourea derivatives containing the (R)-2-amino-1-butanol motif has been performed. The evaluation of their in vitro activity against Mycobacterium tuberculosis (H ₃₇Rv and strain

Full text of DOI:10.1016/j.ejmech.2013.02.034

European Journal of Medicinal Chemistry 63 (2013) 468e473
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journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Efficient synthesis of new (R)-2-amino-1-butanol derived ureas,
thioureas and acylthioureas and in vitro evaluation of their
antimycobacterial activity
,
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 22 structurally diverse urea, thiourea and acylthiourea derivatives containing the (R)-2-
amino-1-butanol motif has been performed. The evaluation of their in vitro activity against Mycobacte-
rium tuberculosis (H37Rv and strain 43) showed promising results in the case of the acylthiourea de-
Received 19 December 2012
Received in revised form
21 February 2013
Accepted 23 February 2013
Available online 5 March 2013
rivatives (MIC range 0.36e7.46 mM for H37Rv strain).
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
Antimycobacterial
Ethambutol
Chiral
2-Amino-1-butanol
Ureas
Acylthioureas
1. Introduction
standard course regimen of six months, which often result in pa-
tient noncompliance; emergency of extremely drug-resistant
tuberculosis (XDR-TB) strains; lack of effective drugs against the
latent state. One approach to decrease treatment time is improve-
ment of potency of currently used anti-tuberculosis drugs [7],
mainly through discovery of more effective combinations with
newer, more potent and less toxic active compounds [8,9]. There is
a clear trend toward gradually increasing partition of new active
compounds, including derivatives of known anti TB drugs [10] and
natural products [11]. Except of few new chemical entities [12,13]
no other anti MDR-TB drugs with proved novel mechanism of ac-
tion are available in clinical use since last 40 years, but many classes
of new potent compounds [13e15] are currently in different steps
of their anti TB evaluation.
Tuberculosis (TB) is one of the most devastating diseases pri-
marily due to several decades of neglect, HIV infection, immigration
and globalization [1]. Approximately one-third of the world’s pop-
ulation has been infected with the causative organism Mycobacte-
rium tuberculosis (MTB), eight million become sick with TB and
globally it accounts for approximately two million deaths per year.
The spreading of collaborative TB/HIV infections [2] and the ree-
mergence of TB accompanied by an increasing number of drug
resistant and multidrug-resistant (MDR) strains MTB (i.e. resistant to
at least rifampin [RIF] and isoniazid [INH]) has been noted since the
mid-1980s [3e5]. Thus, management of tuberculosis is complicated,
which has become a serious health problem worldwide.
The frontline drugs INH, RIF, pyrazinamide (PZA) and etham-
butol (EMB) are currently recommended by the World Health Or-
ganization (WHO) for the treatment of TB [6]. The problems with
current TB treatment are complex and include: a prolonged
EMB is a simple (S)-2-amino-1-butanol derived 1,2-diamine,
clinically used as primarily bacteriostatic anti-tuberculosis agent
(Scheme 1) with not fully known mechanism of action. It targets the
arabinosyl transferases responsible forarabinogalactan biosynthesis,
a key component of the unique mycobacterial cell-wall [16e18].
Despite modest antimycobacterial activity and due to its synergy
with other drugs and lower toxicity, EMB is used in combinationwith
more potent frontline antimycobacterial agents. The configuration of
* Corresponding authors. Tel.: þ359 2 9606152; fax: þ359 2 8700225.
E-mail address: gmdob@orgchm.bas.bg (G.M. Dobrikov).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.02.034

G.M. Dobrikov et al. / European Journal of Medicinal Chemistry 63 (2013) 468e473
469
Scheme 1. Representative examples of compounds possessing antimycobacterial activity.
the molecule is decisively important for the activity, since EMB
is approximately 200e500 fold more potent than its (R,R) enan-
tiomer [19].
obtained in very high yields and excellent purity. The preparation
of 21, 22 [37], and 24 [38,39] has been described previously,
however as racemic mixtures and without evaluation of their
bioactivity.
The urea derivative 30 was synthesized from 1 and 28 (Scheme
3) using a published solvent-free procedure [40]. Compound 31 was
obtained by reacting 1 with 29 under standard acylation conditions
(0 ꢀC and Et3N in dry DCM). The thioureas 34 and 35 were obtained
in high yields in the same manner as ureas 15e27, by mixing 1 and
the isothiocyanates 32 and 33, respectively (Scheme 3). The prep-
aration of 34 has been mentioned earlier [41].
In recent years diverse derivatives of (S)-2-amino-1-butanol and
1,2-ethylenediamine have been synthesized and evaluated in
respect of their antimycobacterial activities and mechanisms of
action [14,20e23]. One of the most active (up to 35 fold more active
in vitro) analogues of EMB of late years is 1,2-diamine SQ109
[14,15,21,24] (Scheme 1). This compound possesses low cytotox-
icity, excellent pharmacokinetic properties and selectivity. It dem-
onstrates synergistic interactions with some “classic” anti TB drugs,
as well. SQ109 and its analogues are currently under the procedures
of their clinical evaluation [24,25].
2.1.2. Synthesis of acylthioureas 41e45
In our recent study [26] the significant role of the chirality has
been demonstrated on the example of a series of (R)-2-amino-1-
butanol derivatives, some being up to 11 times more active than
the S,S-configured EMB. One of the urea derivatives obtained,
namely (R)-1-ethyl-3-(1-hydroxybutan-2-yl)urea (EHBU, Scheme
1), showed very promising activity and low toxicity together with
good water solubility.
The synthesis of compounds 41e45 (Scheme 4) was performed
by recently published procedure [42] by using one-pot reaction of
acylchlorides 36e40 with NH4SCN in the presence of catalytic
amounts of PEG-400, followed by addition of 1 to the reaction
mixture. The yields were moderate, however the application of this
method was easy to perform and convenient in respect of the pu-
rification of the desired products (simple filtration through a pad of
silica provided excellent pure products).
A variety of structurally related thioureas (N-aryl-N0-alkyl and
N,N0-diaryl substituted) have been extensively evaluated against
different strains of MTB [27e32] showing valuable activities, some-
times greatly exceeding EMB. One representative example is isoxyl
(thiocarlide; 4,4-diisoamyloxydiphenylthiourea; Scheme 1), effi-
ciently clinically used drug since 1960 [33,34]. Recently, the subclass
of acylthioureas have been also an object of interest showing
promising anti-TB activity, as summarized by Ananthan et al. [35,36].
Taking into account the above presented results, we were
encouraged to perform the synthesis of new series of ureas, thio-
ureas and acylthioureas incorporating the (R)-2-amino-1-butanol
motif and to evaluate their in vitro antimycobacterial activity.
2.2. Biology
There are no data regarding the antimycobacterial and cytotoxic
activity of the 22 newly synthesized compounds.
2.2.1. In vitro antimycobacterial activity
The synthesized compounds were evaluated for their in vitro
activity against M. tuberculosis H37Rv and MDR strain 43 (Table 1;
results are recalculated in mM) using the method of Canetti (see
Section 4.2). All the compounds synthesized are in agreement with
the formal Lipinski’s rule of five. The first 17 derivatives of (R)-2-
amino-1-butanol e ureas 15e27, 30e31 and thioureas 34e35
(Schemes 2 and 3) were inactive against M. tuberculosis H37Rv
2. Results and discussion
2.1. Chemistry
even at concentrations of 5 mg/ml (100% growth of the bacteria was
observed). The only observed exception was compound 19,
showing activity close to EMB. It is interesting to point out, that
even a small structural change in the molecule EHBU [26] (Scheme
1) induce lack of activity. For example, its inactive near homologues
possess propyl (16), n-butyl (17) and t-butyl (15) groups. Replace-
ment of the carbonyl group with thiocarbonyl (thioureas 34e35)
leads to the same consequences. Similar negative trend was
observed for many derivatives of EMB [7,19].
We have optimized efficient pathways to obtain small libraries
of chiral ureas, thioureas and acylthioureas. All compounds
described have been isolated in pure form and have been charac-
terized by means of NMR, spectroscopy, mass spectrometry and
elemental analysis. Detailed description of the experimental pro-
cedures and the data obtained are available in Supplementary data.
2.1.1. Synthesis of ureas 15e27, 30e31 and thioureas 34e35
The synthesis of compounds 15e27 was performed by mixing 1
and the corresponding isocyanates 2e14, respectively, in THF or
dichloromethane (DCM) as a solvent (Scheme 2). They were
Other series of five new acylthioureas 41e45 (Scheme 4) was
designed to contain important pharmacophore groups (discovered
in our previous study [26]), attached to acylthioureas containing
(R)-2-amino-1-butanol moiety. Compounds 41 and 43e45 showed

470
G.M. Dobrikov et al. / European Journal of Medicinal Chemistry 63 (2013) 468e473
Scheme 2. Synthesis of compounds 15e27.
activity against M. tuberculosis H37Rv comparable to EMB. Cinnamic
derivative 42 is the most active compound in this study with MIC
0.36 mM. Besides, relatively low cytotoxicity and excellent selec-
tivity index (SI) (119.2) of 42 suggest that this compound is a good
lead structure for further investigations. Encouraging from above
results, we tested 41e45 against MDR strain 43. In this case com-
pounds 41e43 lose their activity (100% growth of bacteria at con-
2.2.2. Cytotoxic activity of the complexes
The findings that the acylthioureas 41e45 exerted good anti-
mycobacterial activity gave us a good reason for further more
detailed evaluation of the cytotoxic effect of those compounds. The
cytotoxic activity of the tested compounds was investigated against
human embryonic kidney cell line (HEK293) using the MTT dye
reduction assay.
centrations 5
45 was only 2 fold less active.
m
M). On the other hand, 44 preserved its activity and
The corresponding IC50 values of the tested compounds were
calculated using nonlinear regression analysis and summarized in

G.M. Dobrikov et al. / European Journal of Medicinal Chemistry 63 (2013) 468e473
471
Scheme 3. Synthesis of compounds 30e31 and 34e35.
Table 1. As it could be seen the compounds demonstrated a wide
range of cytotoxicity with IC50 between 12.8 and 179.6. Perceptible
cytotoxicity to the cells was exhibited by the substances 41e42
while compound 43 showed strong cytotoxic effect. It is note-
worthy that the most potent antimycobacterial sample within this
series, e.g. 42, showed excellent SI (119.2). Therefore it turned out
that compound 42 is of particular interest due to its highly favor-
able features e low cytotoxicity and significant antimycobacterial
activity which makes it a good candidate for an efficient therapeutic
agent.
3. Conclusion
An efficient synthesis of chiral urea, thiourea and acylthiourea
derivatives containing the (R)-2-amino-1-butanol moiety has been
demonstrated. After purification and structure characterization, the
antimycobacterial activity of the compounds was evaluated in vitro
against two MTB strains (H37Rv and strain 43). The most active
compounds among the evaluated series were the acylthiourea de-
rivatives 41e45.
4. Experimental
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) e hexane, heptane,
diethyl ether (Et2O), dichloromethane (DCM), methyl tert-butyl
ether (MTBE), tetrahydrofurane (THF), methanol (MeOH), ethanol
(EtOH), ethylacetate (EtOAc). Melting temperatures were deter-
mined in capillary tubes on an Electrothermal MEL-TEMP
1102D-230 VAC apparatus without corrections. The NMR spectra
were recorded on a Bruker Avance DRX-250 (250.13 for 1H and
62.90 MHz for 13C) and on a Bruker Avance IIþ 600 (600.13 for 1H
and 150.92 MHz and for 13C NMR) spectrometers. In case of CDCl3
TMS was used as internal standard for chemical shifts (d, ppm) and
1H spectra were calibrated to the signal of TMS (
d
¼ 0.0000). For
other deuterated solvents 1H spectra were calibrated to the residual
solvent peaks (DMSO-d6 d ¼ 2.50). 13C spectra were calibrated in all
cases to the residual solvent peaks (CDCl3
d
d
¼ 77.00, DMSO-d6
¼ 39.52). 31P NMR spectra were recorded with full proton
decoupling and using 85% H3PO4 as external standard. The cali-
bration of the 31P NMR spectra was performed through changing of
the spectrum reference frequency (specific for the used NMR
probe). The following additional NMR techniques were used for all
compounds: DEPT 135, COSY, HSQC and HMBC. 1H and 13C NMR
data are reported as follows: chemical shift, multiplicity
(s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, br ¼ broad,
m ¼ multiplet), integration, identification, and coupling constants
(in Hz). 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 negative-ion
electrospray ionization (-ESI), and are reported as fragmentation in
Scheme 4. Synthesis of acylthioureas 41e45.

472
G.M. Dobrikov et al. / European Journal of Medicinal Chemistry 63 (2013) 468e473
Table 1
In vitro screening data for antimycobacterial activity and cytotoxicity of synthesized compounds 15e27, 30e31, 34e35 and 41e45.
Entry
Compound
Antimycobacterial activity toward
reference strain of Mycobacterium
Antimycobacterial activity toward
In vitro cytotoxicity toward
Selectivity
index, SIb
Log Pc
MDR strain 43 of Mycobacterium
human embryonal kidney
tuberculosis H37Rv, MIC (
m
M)
tuberculosis MIC (
m
M)a
cell line 293T, IC50 (m
M)a
1
2
3
4
5
6
7
8
15
16
17
18
19
20
21
22
23
24
25
26
27
30
31
34
>26.56
>28.71
>26.56
>23.33
4.80
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
9.3
119.2
3.8
14.0
31.3
NT
0.46 ꢂ 0.36
0.30 ꢂ 0.35
0.83 ꢂ 0.35
1.30 ꢂ 0.35
3.04 ꢂ 0.38
2.12 ꢂ 0.39
1.43 ꢂ 0.36
1.01 ꢂ 0.37
1.36 ꢂ 0.38
1.51 ꢂ 0.40
ꢃ0.16 ꢂ 0.49
0.41 ꢂ 0.56
2.80 ꢂ 0.56
ꢃ0.51 ꢂ 0.39
0.05 ꢂ 0.39
ꢃ0.09 ꢂ 0.27
0.44 ꢂ 0.27
1.75 ꢂ 0.60
2.29 ꢂ 0.61
1.73 ꢂ 0.61
1.58 ꢂ 0.62
1.86 ꢂ 0.62
0.06e
>18.77
>21.16
>22.49
>21.16
>24.01
>14.43
>14.76
>11.67
>24.48
>31.21
>30.82
>28.36
7.13
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
35
41
42
43
44
45
NT
NT
>17.83
>17.96
>16.92
7.46
11.49
NT
66.2
42.9
12.8
104.4
179.6
NT
0.36
3.39
7.46
5.74
EMB$2HCld
7.22
a
NT e not tested e for low active compounds against H37Rv strain; cytotoxicity and SI were tested/calculated only for selected active compounds.
Selectivity index: SI ¼ IC50/MIC (H37Rv).
b
c
Log P, octanolewater partitioning coefficient, was calculated using ACDLabs/ChemSketch 12 (www.acdlabs.com).
EMB$2HCl e ethambutol dihydrochloride (reference compound).
d
e
Log P of EMB$2HCl is known in the literature: N.R. Budha, R.E. Lee and B. Meibohm, Curr. Med. Chem. 15 (2008) 809.
20
m/z with relative intensities (%). Optical rotation [
a
]
measure-
percentage 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
D
ments were obtained using a PerkineElmer 241 polarimeter.
Elemental analyses were performed by the Microanalytical Labo-
ratory for Elemental Analysis of the Institute of Organic Chemistry,
Bulgarian Academy of Sciences. All starting chemicals were
commercially available (from SigmaeAldrich, Merck, Fluka, Acros,
Alfa Aesar). Dimethyl sulfoxide (DMSO) for testing of bioactivities
was commercial (spectroscopic grade) and was used without
distillation. (R)-2-Amino-1-butanol (1) was obtained from Alfa-
Aesar, >98 ee (proved as described elsewhere [26]).
as mM.
4.3. Methodology for evaluation of cytotoxicity
The cell viability was assessed using the standard MTT-dye
reduction assay as described by Mosmann [48] 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 bromide) to a violet formazan via the
mitochondrial succinate dehydrogenase in the viable cells. Briefly:
the exponentially growing cells (HEK human embryonic kidney
fibroblasts) were seeded in 96-well flat-bottomed micro-plates
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 and multi-drug resistant strain 43 (resistant to
Rifampin and Isoniazid), recovered from Bulgarian adult HIV-
negative pulmonary TB patient, who was permanent resident of
the country. This method, recommended by the WHO, is the most
commonly used one worldwide for exploration of sensitivity/
resistance of tuberculosis strains towards chemotherapeutics [43e
47]. It allows precise determination of the proportion of resistant
mutants to a certain drug.
(100
m
L/well) at a density of 1 ꢁ 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
550 nm and a reference wavelength of 630 nm to obtain sample
signal (OD570eOD630). The cell survival fractions were calculated
as percentage of the untreated control. In addition, IC50 values were
derived from the concentration response curves.
A sterile suspension/solution of each tested compound was
added to LöwensteineJensen egg based medium before its coag-
ulation (30 min at 85 ꢀC). Each compound was tested at four
concentrations e 5
DMSO). For some compounds, additional tests at concentrations
0.05, 1, 2 and 3 g/ml were performed. Tubes with Löwenstein-
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
Acknowledgements
This study was partially supported by the Bulgarian Science
Fund e projects DO02-231/2008 and DMU02-3/2009. The financial
support of the Bulgarian Science Fund for the purchase of Bruker
Avance IIþ 600 NMR spectrometer in the framework of the Program

G.M. Dobrikov et al. / European Journal of Medicinal Chemistry 63 (2013) 468e473
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