Synthesis and in vitro antimycobacterial activity of compounds derived from (R)- and (S)-2-amino-1-butanol - The crucial role of the configuration

Article abstract of DOI:10.1016/j.ejmech.2011.11.035

The synthesis of 47 structurally diverse compounds incorporating the (R)-2-amino-1-butanol motif has been realized. Ten of these compounds were found to exhibit in vitro specific activity against Mycobacterium tuberculosis H37Rv in a MIC range of 0.65 μM-14.03 μM. Five of the most active compounds 11, 22, 23, 31 and 42 (5.7-11.1 fold more active than ethambutol) can be outlined with very low cytotoxicity towards human embryonal kidney non-tumour cells (SI ranging from 91.2 to 375.4). For the purpose of comparison the (S)-enantiomers of these most active compounds have been synthesized and evaluated towards M. tuberculosis H₃₇Rv showing no activity even at 20-32 fold higher concentrations.

Full text of DOI:10.1016/j.ejmech.2011.11.035

European Journal of Medicinal Chemistry 48 (2012) 45e56
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and in vitro antimycobacterial activity of compounds derived from
(R)- and (S)-2-amino-1-butanol e The crucial role of the configuration
,
b
b
c
Georgi M. Dobrikov a , Violeta Valcheva , Margarita Stoilova-Disheva , Georgi Momekov ,
*
Pavleta Tzvetkova a, Angel Chimov a, 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 Department of Pharmacology, Pharmacotherapy and Toxicology, Faculty of Pharmacy, Medical University e Sofia, 2 Dunav Str., Sofia 1000, 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 47 structurally diverse compounds incorporating the (R)-2-amino-1-butanol motif has
been realized. Ten of these compounds were found to exhibit in vitro specific activity against Myco-
bacterium tuberculosis H37Rv in a MIC range of 0.65 mMe14.03 mM. Five of the most active compounds 11,
22, 23, 31 and 42 (5.7e11.1 fold more active than ethambutol) can be outlined with very low cytotoxicity
towards human embryonal kidney non-tumour cells (SI ranging from 91.2 to 375.4). For the purpose of
comparison the (S)-enantiomers of these most active compounds have been synthesized and evaluated
towards M. tuberculosis H37Rv showing no activity even at 20e32 fold higher concentrations.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Received 17 August 2011
Received in revised form
13 November 2011
Accepted 16 November 2011
Available online 28 November 2011
Keywords:
Antimycobacterial
Ethambutol analogues
Enantiomers
Antibacterial
2-Amino-1-butanol
1. Introduction
World Health Organization (WHO) for the treatment of TB [6]. The
problems with current TB treatment are complex and include:
Tuberculosis (TB) is one of the most wasteful diseases primarily
due to several decades of neglect, HIV infection, immigration and
globalization [1]. Approximatelyone-third of the world’s population
has been infected with the causative organism Mycobacterium
tuberculosis (MTB), eight million people become sick with TB and
globally it accounts for approximately two million deaths, per year.
The synergy of TB/HIV infections [2] and the emergence of multi
drug resistance (MDR-TB) and extensively drug resistance tubercu-
losis (XDR-TB) pose a threatening challenge to chemotherapy of
tuberculosis with significant problems and complications [3e5].
One fifth of all deaths of adults in developing countries are due toTB
and the problem is particularly re-emerging in many industrialized
countries mostly because of the free movement of people in the
globalized world. In affected regions, the disease is recognized as
serious hindrance to economic and social development.
a prolonged standard course regimen of six months, which often
result in patient noncompliance; emergency of XDR-TB strains; lack
of effective drugs against the latent state. One approach to decrease
treatment time is improvement of potency of currently used anti-
tuberculosis drugs [7] and discovery of new active compounds
[8e10].
The simple diamine EMB (Scheme 1) was synthesized by reacting
1,2-dihaloethane with (S)-2-amino-1-butanol [11,12]. An alternative
synthetic method was also described [13]. The EMB is primarily
a bacteriostatic anti-tuberculosis agent with not fully known
mechanism of action. It targets the arabinosyl transferases respon-
sible for arabinogalactan biosynthesis, a key component of the
unique mycobacterial cell-wall [14e16]. Despite of modest anti-
mycobacterial activity and due to its synergy with other drugs, and
lower toxicity, EMB is used in combination with more potent front-
line antimycobacterial agents. Early SAR study indicates that the
distance between the two nitrogens, the presence of two hydroxy
groups, and the small side chains in the molecule are key pharma-
cophore elements [17]. The configuration of the molecule is deci-
sivelyimportantfortheactivity, sinceEMB(withS,S-configuration)is
approx. 200e500 fold more potent than its (R,R)-enantiomer [17].
The leading drugs isoniazid (INH), rifampin (RIF), pyrazinamide
(PZA), and ethambutol (EMB) are currently recommended by the
* Corresponding authors. Tel.: þ359 2 9606152; fax: þ359 2 8700225.
E-mail addresses: gmdob@orgchm.bas.bg (G.M. Dobrikov), vdim@orgchm.bas.bg
(V. Dimitrov).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.11.035

46
G.M. Dobrikov et al. / European Journal of Medicinal Chemistry 48 (2012) 45e56
Scheme 1.
In recent years diverse derivatives of (S)-2-amino-1-butanol and
1,2-ethylenediamine have been synthesized and evaluated for their
antimycobacterial activities and mechanisms of action [18e22]. In
general, most of the compounds containing (S)-2-amino-1-butanol
motif are showing similar but not significantly higher activity than
EMB. It is important to note that the utilization of (R)-2-amino-1-
butanol motif for the synthesis of anti TB drug candidates has
been obviously neglected. There are only isolated examples of such
derivatives showing results that are not encouraging enough with
respect to antimycobacterial activity [18,21,23], but possessing
promising antifungal activity [24].
Herein, we report the synthesis of small library of structurally
diverse compounds incorporating (R)-2-amino-1-butanol motif
and the evaluation of their in vitro antimycobacterial, antibacterial
and antifungal activities. For selected structures the (S)-configured
analogues have also been synthesized and evaluated.
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of compounds 10e17
A series of N-monoacylated compounds 10e17 was synthesized
and good to excellent yields were achieved using standard condi-
tions for acylation of 1 (0 ꢀC and Et3N in dry DCM) with acid
chlorides 2e9 (Scheme 2). All compounds were obtained in
excellent purity (>99%) after column chromatography or crystalli-
zation. The synthesis of 13e15 and 17 has been published earlier,
with no data about the purity whatsoever [25e29]. In the case of 11,
the (S)-enantiomer has been obtained through rather complicated
procedure in enantiomerically enriched form (75e97 % ee) [25].
2.1.2. Synthesis of compounds 21e23
The amide derivatives 21e23 were synthesized using simple
solvent-free aminolysis of esters 18e20 with 1 by heating at
90e100 ꢀC(Scheme3). Compounds21e23wereisolatedinverygood
yields and purities (by column chromatography or crystallization).
2.1.3. Synthesis of compounds 31e37
The synthesis of compounds 31e37 (Scheme 4) was performed
by applying standard procedures described in literature [30e36] e
heating of 1 with 24e30, and using either solvent-free conditions
or different solvents (e.g. Et2O, THF, EtOH etc.). Compounds 31, 32
and 35 have been obtained previously [30e32,36], however, the
data published were insufficient in respect of purity and of
Scheme 2. Synthesis of compounds 10e17.

G.M. Dobrikov et al. / European Journal of Medicinal Chemistry 48 (2012) 45e56
47
Scheme 3. Synthesis of compounds 21e23.
properties. It is interesting to note that the reaction of 1 with 26
under those particular conditions led to ester 33. Compound 34 was
obtained from 1 and 27 as a result of migration of the double bond.
Similar rearrangement reaction has been previously observed [37].
2.1.4. Synthesis of compounds 41e43 and 47e49
It is known that some ureas and thioureas are effective drugs
against a range of MDR strains of M. tuberculosis [10,38e41].
Therefore it is worthy to evaluate the activity of similar structures
incorporating the (R)-2-aminobutanol moiety. The synthesis of
compounds 41e43 was performed by mixing 1 and isothiocyanate
38 and isocyanates 39, 40 respectively, in THF as a solvent (Scheme
5). Compounds 41e43 were obtained in very high yields and
excellent purities. The formation of 41 [42e44] and 43 [45] was
described elsewhere in connection with different studies, not being
related to the present investigations.
Scheme 4. Synthesis of compounds 31e37.
The heterocycle 47 was synthesized in two steps. In the first step
the salt of 1 was formed and isolated after mixing of 1 and
homophthalic acid (44) in THF. The salt was then heated without
solvent for 5 h to form 47 in good yields. Structures similar to 47
have shown anti-inflammatory and analgesic activity [46].
The quinazolinone derivative 48 was prepared in acceptable
yield, in boiling toluene by mixing 1, anthranilic acid (45) and
triethyl orthoformate, in presence of catalytic amounts of p-tolue-
nesulfonic acid (PTSA). In the present case, the published solvent-
free procedure for synthesis of quinazolinones [47] was not
successful. Some 4(3H)-quinazolinones have shown antitubercular
[48] and antibacterial [49] activity.
Compound 49 was interesting to synthesize due to the expected
lipophilicity and hydrolytic stability caused by the steric hindrance
of the camphene skeleton. The reaction was carried out by
condensation of (þ)-camphor (46) and 1 in the presence of catalytic
amount of anhydrous ZnCl2, according to published procedure [50].
Imine 49 was obtained in excellent purity by column chromatog-
raphy and crystallization.
2.1.5. Synthesis of compounds 52e54, 59e62 and 65e67
Three series of N-substituted aminoalcohols have been
synthesized by using (R)-2-amino-1-butanol (1) as central chiral
unit, in reaction with aryl- and a-aryl-alkyl halogenides (Scheme 6).
Compounds 59e62 were prepared in good yields, under stan-
dard conditions, by using excess of K2CO3 in refluxing acetonitrile.
Due to steric reasons, in the case of products 52 and 54 only mono-
substitution of the amino group was achieved. The reactions,
however, were smooth in both cases (Et3N/DCM for 52 and K2CO3/
18-crown-6 for 54). Attempts for N-acylation of 52 provided only
the O-acylated product 53.
The synthesis of compounds 59 [51e56], 60 [57,58] and 52 has
been previously described. The latter two derivatives have been
used for preparation of antitumour agents [59e66].

48
G.M. Dobrikov et al. / European Journal of Medicinal Chemistry 48 (2012) 45e56
Scheme 5. Synthesis of compounds 41e43 and 47e49.
Aminoalcohol 1 reacted under solvent-free conditions with the
are 1D proton and carbon, DEPT, COSY, HSQC, HMBC and NOESY.
Some of the synthesized compounds required specific NMR exper-
iments for their assignment. High resolution versions of HSQC and
Primitive exclusive HSQC (PE HSQC) experiments were recorded in
order to differentiate and assign the highly crowded areas of signals
mostly in the aromatic region. The PE HSQC experiment resembles
a non-decoupled HSQC experiment [78]. The acquired non-decou-
pled signals have higher resolved shape, allow multiplicity differ-
entiation of the signals in the aromatic areas (singlet, doublet,
triplet) and thus provide additional information. Once the proton
and carbon pairs are assigned from the HSQC/PE HSQC experiment
a long range correlation is needed (HMBC experiment). The crowded
areas of the signals for the aromatic protons and carbons require also
a high resolution experiment to be performed. Despite the HSQC
experiments, if the HMBC is performed in smaller spectral width, the
resulting experiment will contain folded signals and these spurious
signals will prevent spectral assignment. These folded signals will be
present merely in the indirect dimension (13C) and one way of
identifying these spurious signals is to record several HMBC exper-
iments with different spectral widths, therefore these signals that
change position will be false and will be differentiated. Another way
to overcome the appearance of the folded signals is to selectively
irradiate merely the spectral width of interest while leaving the
other areas unaffected. Such an experiment where in one dimension
selective pulses are applied is called semi-selective. We have per-
formed semi-selective versions of HMBC with different selective
pulses depending on the spectral width. Different selective Gaussian
pulses were calibrated to cover spectral widths of 10e40 ppm. The
high resolution semi-selective HMBC experiments were used to
assign the quaternary carbon atoms and also to connect the proton/
carbon pairs within one aromatic ring. In some cases TOCSY exper-
iments with different mixing times were applied to provide identi-
fication of separate spin systems.
heteroaryl bromides 37 and 63, producing compounds 65e66. It is
interesting to note that the bromine atom in 66 was not replaced
with second (R)-2-amino-1-butanol unit. Compound 67 was easily
synthesized from 1 and 64 in refluxing EtOH.
2.1.6. Synthesis of compounds 81e93
A set of benzylic type substituted aminoalcohols 81e93 was
synthesized (Scheme 7) by applying very efficient reductive ami-
nation of aldehydes 68e80 with aminoalcohol 1. Instead of using
the commonly applied (and more expensive) reducing agent NaB-
H(OAc)3 [68e70], we used the cheaper NaCNBH3 [67] and achieved
good results.
Some of the isolated products have been previously mentioned
elsewhere e the S-enantiomer of 81 has been described in patents as
catalyst [71,72]; 83 has been a part of study, concerning prevention
of drug-induced cytotoxicity [73]; aminoalcohol 84 and its S-enan-
tiomer were used as ligands for stereoselective reduction of ketones
[74]; the racemate of 86 has been mentioned as intermediate for
preparation of ferrocenylmethylaminoalkoxy silanes [75]; the S-
enantiomers of 87 and 88 have been previously prepared by
reductive amination of aldehydes 74 and 75 respectively, by using
resin supported cyano borohydride [76]; the formation of 90
throughreductive aminationhas beennoted in a study regardingthe
cytotoxicity of pyrene-containing aminoalcohols [77].
Compounds 81e93 have been isolated in excellent purities.
2.1.7. Synthesis of compounds 95e100
The evaluation of antimycobacterial activity of the compounds
derived from (R)-2-amino-1-butanol, provided very promising
results (see Table 2). Therefore, studying the S-enantiomers of the
most active compounds was necessary to compare the activity data.
Thus, the synthesis of compounds 95e100 (Scheme 8) was ach-
ieved by applying the same procedures already described in this
article for the R-analogues. For the experimental details see
Supplementary data.
Compounds 11, 54, 86, 89 and 90 couldn’t be assigned with the
use of the routine NMR experiments and the above specific
experiments were applied. Phosphinic amide 11 contains two dia-
stereotopic phenyl rings which showed different chemical shifts.
The observed NOE proximities of the methyl group and the meth-
ylene proton 3-Hb to the ortho protons of one of the phenyl rings
assigned its signals as 10-H (see Supplementary data). The 1H/13C-
signals of the aromatic rings of compound 54 showed small
chemical shift variation. Their electron environment is different
depending on whether their neighbour is a methyl group or
2.1.8. NMR experiments
The synthesized compounds were studied in detail by NMR
spectroscopy by means of 1H/13C spectra, two dimensional routine
experiments and additional specific techniques. The structural
information obtained is very useful for characterization of similar
compounds in future investigations. The routine experiments used

G.M. Dobrikov et al. / European Journal of Medicinal Chemistry 48 (2012) 45e56
49
Scheme 6. Synthesis of compounds 52e54, 59e62 and 65e67.
a hydroxy group indicating the existence of rotamers. The proton
and carbon signals for compounds 89 and 90 were assigned
following the above procedure: assignment of proton and carbon
pairs, then with the long range correlation fragments were built
and quaternary carbons were assigned. Numbering the pairs of
atoms with similar chemical shifts for 11 and 54 is tentative (see
Supplementary data). Compound 86 contains ferrocenyl moiety
and the high resolution experiments in this case were performed
for this area to resolve chemical shifts. NMR spectra of compounds
95e100 were identical with the spectra of their (R)-enantiomers 11,
13, 22, 23, 31 and 42, respectively.
2.2. Biology
To the best of our knowledge, there are no data published
concerning antimycobacterial, antibacterial and antifungal activity
of the compounds synthesized in this study.

50
G.M. Dobrikov et al. / European Journal of Medicinal Chemistry 48 (2012) 45e56
Scheme 7. Synthesis of compounds 81e93.
2.2.1. In vitro antimycobacterial activity
42 have shown remarkable high activity, which is between 5 and
11 times better than EMB used as reference. For the purpose of
comparison, the most active EMB analogue, the 1,2-diamine SQ
109, is 35 times more potent than EMB [19]. It is interesting to
The synthesized compounds were evaluated for their in vitro
activity against M. tuberculosis H37Rv (Table 1) using the method of
Canetti (see 4.2). Five of the compounds, namely 11, 22, 23, 31 and

G.M. Dobrikov et al. / European Journal of Medicinal Chemistry 48 (2012) 45e56
51
Table 1
In vitro screening data for antimycobacterial activity and cytotoxicity of synthesized compounds.
Entry
Compound
Antimycobacterial activity towards
reference strain of Mycobacterium
In vitro cytotoxicity towards human
Selectivity
index, SIa,b
Log Pc
Solubility in deionized
embryonal kidney cell line 293T,
water at 20 ꢀC (mg/ml)a
tuberculosis H37Rv, MIC (
m
M)
IC50 (m
M)a
1
2
3
4
5
6
7
8
10
11
12
13
14
15
16
17
21
22
23
31
32
33
34
35
36
37
41
42
43
47
48
49
52
53
54
59
60
61
62
65
66
67
81
82
83
84
85
86
87
88
89
90
91
92
93
95
96
97
98
99
100
EMB$2HCle
28.86
0.69
>17.46
9.12
>20.55
>20.55
8.46
>18.37
>25.74
0.65
0.96
0.97
>22.81
23.67
27.29
>29.21
>18.57
>14.36
>22.29
1.25
>19.36
23.23
22.91
>22.39
>15.09
13.39
19.74
10.46
>18.56
>11.73
>12.15
14.03
20.40
>29.90
25.61
>23.89
17.83
21.80
>21.80
17.41
>19.58
17.90
NT
155
NT
211
NT
NT
173
NT
NT
244
257
89
NT
NT
NT
NT
NT
NT
NT
114
NT
NT
NT
NT
NT
114
NT
122
NT
NT
NT
47
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
224.6
NT
23.1
NT
NT
20.5
NT
NT
375.4
267.7
91.8
NT
NT
NT
NT
NT
NT
NT
91.2
NT
NT
NT
NT
NT
8.5
NT
11.7
NT
NT
NT
3.4
NT
NT
NT
NT
NT
NT
NT
NT
NT
0.48ꢁ0.31
NT
<1
NT
<1
NT
NT
1.5
NT
NT
46
1.82ꢁ0.57
0.79ꢁ0.37
1.70ꢁ0.35
2.48ꢁ0.36
2.48ꢁ0.36
1.67ꢁ0.39
1.60ꢁ0.46
ꢂ0.01ꢁ0.37
ꢂ0.27ꢁ0.52
1.59ꢁ0.45
0.49ꢁ0.36
1.81ꢁ0.28
0.07ꢁ0.33
0.66ꢁ0.33
ꢂ0.70ꢁ0.39
1.44ꢁ0.62
2.21ꢁ0.65
1.14ꢁ0.33
ꢂ0.23ꢁ0.35
2.74ꢁ0.40
2.28ꢁ0.75
0.94ꢁ0.29
4.62ꢁ0.47
6.22ꢁ0.53
6.90ꢁ0.40
3.36ꢁ0.56
2.20ꢁ0.38
4.61ꢁ0.42
5.79ꢁ0.55
4.16ꢁ0.38
0.92ꢁ0.98
2.41ꢁ0.62
0.47ꢁ0.57
1.26ꢁ0.35
1.91ꢁ0.36
1.78ꢁ0.48
3.23ꢁ0.34
3.23ꢁ0.34
ed
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
2
1
NT
NT
NT
NT
NT
NT
NT
215
NT
NT
NT
NT
NT
<1
NT
<1
NT
NT
NT
<1
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
100f
3.76ꢁ0.39
4.46ꢁ0.34
3.94ꢁ0.40
4.95ꢁ0.34
0.51ꢁ0.35
1.86ꢁ0.35
1.76ꢁ0.62
1.82ꢁ0.57
1.70ꢁ0.35
ꢂ0.27ꢁ0.52
1.59ꢁ0.45
ꢂ0.23ꢁ0.35
0.49ꢁ0.36
0.06f
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
18.72
16.48
27.74
21.71
20.38
>17.28
>22.80
>16.21
>23.90
>31.21
>24.36
7.22
NT
a
NT e not tested; cytotoxicity, SI and water solubility were tested/calculated/measured only for selected active compounds.
Selectivity index SI ¼ IC50/MIC.
b
c
d
e
f
Log P, octanol-water partitioning coefficient, was calculated using ACDLabs/ChemSketch 12.01 (www.acdlabs.com).
Log P was not calculated for this compound because of software limitations.
EMB$2HCl e ethambutol dihydrochloride (reference compound).
Log P and water solubility of EMB$2HCl are known in the literature: N.R. Budha, R.E. Lee and B. Meibohm, Curr. Med. Chem. 15 (2008) 809.
note that no analogue structures of 11, 22, 31 and 42 were found,
that possess antimycobacterial activity. Known antimycobacterial
activity of salicylanilide phenolic esters [79,80], shows that the
salicylamide fragment is important for the activity of 23.
Compounds 13, 16, 53, 59 and 65 have shown 50e80% activity
compared to EMB. The cinnamide group of 13 has been incorpo-
rated in diverse structures possessing antimycobacterial activity
[81,82]. The remaining (R)-2-aminobutanol derivatives that were
synthesized have no perceptible activity. All of the compounds
mentioned above (except 53) are in agreement with the formal
Lipinski’s rule of five (Table 1).
The evaluation of the antimycobacterial activity of the S-enan-
tiomers 95e100 has shown absence of activity (100% growth) at
concentrations ca. 2e3 fold higher than MIC of EMB. These results
are in contrast with the high activity observed for the R-configured
enantiomers (11,13, 22, 23, 31 and 42, respectively). This is opposite
to the fact that (S,S)-EMB is approximately 500 fold more active
than (R,R)-EMB [17]. Therefore, the direct comparison of the

52
G.M. Dobrikov et al. / European Journal of Medicinal Chemistry 48 (2012) 45e56
Table 2
Evaluation of in vitro antibacterial and antifungal activity of (R)-2-amino-1-butanol derivatives (50 mg/ml DMSO) against conditioned pathogenic microorganisms.
Compound
Microorganisms
1
2
3
4
5
6
7
8
9
10
11
12
13
10
11
12
13
14
15
16
17
21
22
23
31
32
33
34
35
36
37
41
42
43
47
48
49
52
53
54
59
60
61
62
65
66
67
81
82
83
84
85
86
87
88
89
90
91
92
93
15
18
11
15
NA
16
NA
12
NA
NA
NA
NA
NA
NA
13
11
15
NA
12
NA
NA
NA
NA
NA
NA
12
16
15
NA
12
NA
NA
NA
NA
NA
NA
11
NA
17
15
18
NA
12
NA
NA
NA
NA
NA
NA
11
NA
18
NA
15
NA
NA
NA
NA
NA
NA
13
NA
15
NA
NA
NA
NA
NA
NA
NA
NT
NA
NA
NA
NA
NA
22
NA
11
NA
NA
NA
NA
NA
NA
13
NA
16
NA
13
NA
NA
NA
NA
NA
NA
15
NA
NT
NT
NT
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
11
NA
NA
NA
NA
NA
NA
NA
NA
13
NA
NA
NA
NA
NA
NA
NA
NA
11
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
11 (NT) NA
40 (0.06) 35 (0.06) 40 (0.06) 38 (0.06) 40 (0.0125) 39 (0.06) >40 (0.06) 38 (0.06) 38 (0.3) 29 (1.25) >40 (0.006) >40 (0.006) >40 (0.0125)
21
23
20
24
28
NA
NA
NT
14
NA
NA
18
NA
NA
NA
NA
NA
25
11
NA
11
NA
19
12
14
13
NA
15
14
15
15
21
20
21
22
NA
NA
NA
12
NA
NA
15
NA
NA
NA
NA
17
NT
12
NT
12
NT
18
NA
11
11
NA
17
19
17
17
32
20
21
25
NA
10
NA
NA
NA
NA
NA
NA
NA
NA
NA
12
13
NA
11
NA
NA
NA
NA
13
NA
17
10
10
17
19
15
18
NA
NA
NA
NA
NA
NA
15
NA
NA
NA
13
NA
NA
13
NA
10
NA
NA
22
NT
NA
10
NA
NA
15
NA
12
11
NA
NA
NA
12
NA
NA
15
NA
11
NA
NA
17
NA
NA
NA
12
NA
NA
10
NA
NA
NA
NA
NA
NA
12
NA
NA
NA
NA
12
25
11
NA
15
NA
14
NA
NA
NT
12
NA
NA
13
NA
NA
NA
NA
17
NT
12
10
NA
NA
17
NA
12
NA
NA
NT
16
NA
NA
0
NA
NA
NA
NA
12
NA
NA
NA
16
NA
NA
20
NA
NA
NA
NA
17
NA
NA
NA
15
NA
NA
11
NA
NA
NA
NA
22
NA
NT
NA
NA
35
NA
NA
NA
NA
20
14
14
NA
NA
NA
15
20
11
NA
10
NA
17
NA
11
15
NA
NA
NA
16
NT
11
NA
NA
NA
14
NA
13
10
NA
20
17
15
23
25
21
10
16
10
NA
NA
12
NA
13
NA
14
11
NA
18
17
20
18
NA
NA
NA
NT
17
NA
NA
NA
NA
28
NA
NA
NA
NA
10
NA
NA
16
NA
32
10
14
18
NA
NA
NA
NA
17
NA
12
NA
NA
22
NA
NA
NT
25
NA
NA
NA
NA
30
NA
12
10
NA
16
19
NT
NA
13
17
17
NA
18
19
16
18
NA
10
17
16
19
22
16
28
10
12
18
35
12
40
18
20
20
17
18 (0.3) 19 (1.25) 20 (0.6) 20 (0.6) 19 (0.3)
18 (1.25) 17 (0.6) 16 (1.25) 20 (1.25) 16 (1.25)
17 (0.6) 19 (0.03)
17 (1.25) 17 (1.25)
14
NA
13
18
18 (0.3) 19 (0.3) 12
31 (0.0125) 32 (0.0125) 16 (0.06)
18
13
NA
13
18
16
14
NA
10
16
16 (1.25) 21 (0.06)
20 (1.25)
35
NA
NT
NA
30
14
NA
20
12
NT
14
NA
10
12
30
NA
12
16
13
NA
11
15
NA
16
18
14
NA
13
13
13
NA
14
16
NA
NA
20
40
NA
NT
NA
NA
NA
Streptomycina 35/30/25 30/27/21 30/25/20 35/30/25 30/28/23
(reference)
30/24/20 32/26/20
27/22/19 30/25/20 35/26/23 NT
NT
Gentamycinb 30/27
sulphate
35/29
36/30
32/28
29/26
39/31
35/27
34/28
38/28
31/25
NT
NT
NT
(reference)
Fluconazolec
(reference)
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
35
e
e
e
Itraconazoled NT
(reference)
e
30
Diameter of zone of inhibition are given in mm. Values in brackets are the lowest concentrations (in mg/ml) provoking zone of inhibition >11 mm. Values in brackets are given
only for compounds preserving their activity at concentrations 1.25 mg/ml or less.
NA e not active according first zone inhibition test (at concentration 50 mg/ml); further tests not performed.
NT e zone inhibition test not performed.
aZone inhibition for Streptomycin was measured at three concentrations e 25, 5 and 1 mg/ml.
bZone inhibition for Gentamycin sulphate was measured at concentrations 20 and 4 mg/ml.
cZone inhibition for Fluconazole was measured at concentration 25 mg/ml.
dZone inhibition for Itraconazole was measured at concentration 12.5 mg/ml, but is not completely soluble in DMSO at this concentration.
List of microorganisms: 1 Bacillus subtilis, 2 Bacillus idosus, 3 Bacillus megaterium, 4 Bacillus mycoides, 5 Bacillus cereus, 6 Acinetobacter johnstonii, 7 Staphylococcus aureus, 8
Sarcina lutea, 9 Micrococcus luteus, 10 Escherichia coli, 11 Candida tropicalis, 12 Saccharomyces cerevisiae, 13 Penicillium chrysogenum.
structures presented in this paper with EMB should be handled
with care. Since 95e100 were not interesting as antimycobacterial
agents, their cytotoxicity and other antibacterial activity was not
investigated.
There is no correlation between antimycobacterial activity and
activity against other microorganisms, for the compounds in this
study (see below and compare Tables 1 and 2). This indicates the
specific activity of all potent derivatives of (R)-2-aminobutanol to

G.M. Dobrikov et al. / European Journal of Medicinal Chemistry 48 (2012) 45e56
53
Antifungal activities were examined against the yeast strains
Candida tropicalis ATCC 20336 and Saccharomyces cerevisiae ATCC
9763, and the fungal strain Penicillium chrysogenum CECT 2802.
Fluconazole and itraconazole were used as antifungal reference
compounds.
The most of the compounds tested were not active even at
concentrations 50 mg/ml and consequently are not appropriate for
antimicrobial agents (Table 2). Some of the compounds (11, 33e34,
47, 54, 59, 66, 84e85, 87e89) demonstrated wide spectrum of low-
to-moderate activity at lower concentrations e 25, 10, 5, 2, 1.25 or
1 mg/ml. The data in Table 2 (values in brackets) show that almost
all of the compounds are not active at concentrations 2 mg/ml or
less. Only compounds 33, 88 and 89 were active against all
microorganisms (except E. coli) in significantly lower concentra-
tions. The most potent compound 33 exhibited emphasized activity
against the fungi C. tropicalis, S. cerevisiae and P. chrysogenum even
at concentrations 6, 6 and 12.5
mg/ml, respectively. The intact
maleimido ring of 33 is probably necessary for the observed
activity. On the contrary, the substituted maleimide 34 and the
dihydro analogue 35 showed significantly lower activity and
absence of activity, respectively. Similar trend has been observed in
recent report concerning the antifungal activity of analogous N-
alkyl-aryl substituted maleimide derivatives [84].
2.2.3. In vitro cytotoxicity
In order to examine the selectivity of the antiproliferative
effects, the cytotoxic activity of representative compounds, exerting
antimycobacterial activity, was assessed against a human embry-
onal kidney non-tumour cell line 293T, after 72 h continuous
exposure. Evident from the IC50 values summarized in Table 1, the
compounds were generally of low-to-moderate cytotoxicity against
the human cells; with few exceptions the compounds induced 50%
inhibition of cellular proliferation and viability at concentrations
greatly exceeding 100
mM. It is noteworthy that the structural
peculiarities affording the highest antimycobacterial activity within
the series e.g. phosphinic amide (11), phthalic diamide (22), sali-
cylamide (23), isoindolinone (31) and urea (42) were also generally
associated with very low antiproliferative/cytotoxic effects against
human cells with selectivity indices ranging from 91.2 to 375.4. The
values of SI for compounds 13, 16, 53, 59 and 65 are significantly
lower, near the acceptable limit values.
Scheme 8. Synthesis of compounds 95e100.
3. Conclusion
the M. tuberculosis, as it is established for the majority of first-line
antimycobacterial agents (including EMB) in clinical use. Further-
more, since all compounds are partial structure analogues of EMB,
it can be assumed that they share similar mode of anti-
mycobacterial action [14e16], although this needs clarification [83].
Therefore, investigating the mechanism of action and the role of
chirality of these compounds would aid further discovery of more
potent analogues.
The synthesis of 47 structurally diverse compounds incorpo-
rating the (R)-2-amino-1-butanol motif has been realized. They are
partial structure analogues (only in respect of the 2-aminobutanol
motif) of the clinically used essential antimycobacterial drug
ethambutol. After purification and unambiguous structure char-
acterization the antibacterial and antimycobacterial activity was
evaluated. Three compounds (33, 88 and 89) showed interesting
antifungal activity. Ten of the compounds showed prominent
antimycobacterial activity with MICs ranging from less than 1 to ca.
2.2.2. Evaluation of in vitro antibacterial and antifungal activity
For the synthesized derivatives of (R)-2-amino-1-butanol
a qualitative evaluation of in vitro antibacterial and antifungal
activities against conditioned pathogenic microorganisms was
performed (Table 2), using agar diffusion test (see 4.3).
Antibacterial activities were examined against the Gram (þ)
strains: Bacillus subtilis ATCC 6633, Bacillus idosus B 241, Bacillus
megaterium NRRL 1353895, Bacillus mycoides DSMZ 274, Bacillus
cereus ATCC 11778, Acinetobacter johnsonii ATCC 17909, Staphylo-
coccus aureus NRRL B 313, Sarcina lutea ATCC 9341, Micrococcus
luteus ATCC 9631, and the Gram (ꢂ) strain Escherichia coli ATCC
8739. Antibiotics streptomycin and gentamicine sulphate were
used as reference compounds.
10 mM. Interestingly, the most potent and promising compounds
from the large series also proved to exert low level of cytotoxic
activity against a human embryonal non-tumour cell line 293T. On
these basis, the (R)-2-amino-1-butanol derivatives bearing phos-
phinic amide (11), phthalic diamide (22), salicylamide (23), iso-
indolinone (31) and urea (42) moieties (possessing 5.7e11.1 fold
higher antimycobacterial activities than EMB) could be regarded as
potential lead compounds for elaboration of antimycobacterial
agents. Cinnamide 13 could be perspective as lead compound, as
well.
The (S)-enantiomers of the 6 compounds mentioned above have
been synthesized as well. They have shown absence of activity

54
G.M. Dobrikov et al. / European Journal of Medicinal Chemistry 48 (2012) 45e56
towards M. tuberculosis H37Rv even at 20e32 fold higher concen-
trations. Therefore, further investigations of the mechanism of
action and the role of chirality of similar compounds would
contribute to discovery of more potent analogues.
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 sensibility/
resistance of tuberculosis strains towards chemotherapeutics
[87e90]. It allows precise determination of the proportion of
resistant mutants to a certain drug.
4. Experimental
4.1. Chemistry
For thin layer chromatography (TLC) aluminium sheets pre-
coated with silica gel 60 F254 (Merck) were used. Flash column
chromatography was carried out using silica gel 60
(0.040e0.063 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, diethyl ether (Et2O), dichloromethane (DCM), methyl tert-
butyl ether (MTBE), tetrachloroethylene, tetrahydrofurane (THF),
methanol (MeOH), ethanol (EtOH), acetonitrile, xylene. Melting
temperatures were determined in capillary tubes on an Electro-
thermal 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) spectrome-
ters. In case of CDCl3 TMS was used as internal standard for
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 three
concentrations e 5 mg/ml, 2 mg/ml and 0.2 mg/ml (in DMSO). For
compounds showing 0% growth at 0.2 mg/ml, additional test at
concentrations 0.1 mg/ml was performed. Tubes with Löw-
ensteineJensen medium (5 ml) containing tested compounds and
such 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
colonies in control medium were calculated and expressed as
percentage of inhibition. The MIC is defined as the minimum
concentration of compound required to give complete inhibition of
bacterial growth (0% growth). The MIC values are given as mM.
chemical shifts (d
, ppm) and 1H spectra were calibrated to the signal
of TMS (
d
¼ 0.0000). For other solvents 1H spectra were calibrated
4.3. Methodology for evaluation of antibacterial and antifungal
activity
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 ¼ 77.00,
DMSO-d6
d
¼ 39.52). 31P NMR spectra were recorded with full
Antimicrobial and antifungal activities were determined by the
agar diffusion test (or zone inhibition test) according to European
Pharmacopoeia [91]. Test organisms were suspended in melted
nutrient agar and poured into Petri dishes. Holes of 8 mm were cut
proton decoupling and using 85% H3PO4 as external standard. The
calibration 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
in the agar and filled with 50
ml solution (in DMSO) of various
for all compounds: DEPT 135, COSY, HSQC and HMBC. H and 13C
concentrations of the compound. The diameter of clear zone
around the point of application of the compound was measured.
Bacterial strains were grown for 24 h at 37 ꢀC in nutrient agar
(Serva), yeast strains were grown in yeast peptone dextrose agar
(YEPD) and fungi e in potato dextrose agar (PDA). Yeast and fungal
strains were incubated for 72 h at 28 ꢀC.
1
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 electrospray
ionization (ESI), and are reported as fragmentation in m/z with
4.4. Methodology for evaluation of cytotoxicity
relative intensities (%). Optical rotation ½a D20
ꢃ
measurements were
obtained using a PerkineElmer 241 polarimeter. Enantiomeric
purity of selected compounds was determined by using Agilent
1100 HPLC system with diode-array detector (DAD) and Chiralpak
IC chiral column (for conditions see Supplementary data).
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) or are synthesized according literature procedures.
Dimethylsulfoxyde (DMSO) for testing of bioactivities was
commercial (with HPLC grade) and was used without distillation.
(R)-2-Amino-1-butanol (1) was available from Merck (98% chem-
ical purity (GC), enantiomeric purity ee>96%, spec. rotation
The human embryonal kidney cell line 293T cells were obtained
from the German Collection of Microorganisms and Cell Cultures.
Cells were kept in controlled environment e RPMI-1640 medium,
supplemented with 10% heat-inactivated fetal calf serum and 2 mM
L
-glutamine, at 37 ꢀC in a ‘Heraeus’ incubator with 5% CO2 humid-
ified atmosphere.
The cytotoxicity of the newly synthesized compounds was
assessed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide]-dye reduction assay as described
by Mossman [92] with some modifications [93]. In brief, expo-
nentially growing cells were seeded in 96-well microplates (100 ml/
well) at a density of 3.5 ꢄ 105 cell/ml and allowed to grow for 24 h
prior the exposure to the studied compounds. Stock solutions of the
tested compounds were freshly prepared in DMSO and thereafter
were subset to serial dilutions with growth medium in order to
obtain the desired final concentrations. At the final dilutions the
solvent concentration never exceeded 0.5%. Cells were exposed to
the tested agents for 72 h, whereby for each concentration a set of
at least 8 separate wells was used. After the exposure period MTT
½
a 2D0
ꢃ
¼ ꢂ10.5 to ꢂ9.5ꢀ (undiluted)). (S)-2-Amino-1-butanol (94)
was available from Alfa Aesar (98% chemical purity (GC), spec.
rotation ½a 2D0
ꢃ
¼ þ12ꢀ (c ¼ 2 in EtOH)). Intermediate compounds 58
[85] and 80 [86] were not commercially available and were
prepared according described procedures.
4.1.1. Synthetic procedures
solution (10 mg/ml in phosphate-buffered saline) aliquots (100 ml/
The individual procedures for synthesis of the described
compounds are introduced in detail in the part named
“Supplementary data”.
well) were added to each well. The plates were further incubated
for 4 h at 37 ꢀC and the MTT-formazan crystals formed were dis-
solved through addition of 110 ml of 5% HCOOH in 2-propanol. The

G.M. Dobrikov et al. / European Journal of Medicinal Chemistry 48 (2012) 45e56
55
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Acknowledgements
This study was partially supported by the Bulgarian Science
Fund e projects MU0135/2008 and MU0023/2009. The financial
support of the Bulgarian Science Fund for the purchase of Bruker
Avance IIþ 600 NMR spectrometer in the framework of the
Program “Promotion of the Research Potential through Unique
Scientific Equipment”
e Project UNA-17/2005 is gratefully
acknowledged. The multimode detector DTX 880 was purchased
through a grant DCVP02/2 29.12.2009 of the Bulgarian Science
Fund.
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Appendix. Supplementary data
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.11.035. These data include MOL files and
InChiKeys of the most important compounds described in this
article.
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