Antimycobacterial activity of chiral aminoalcohols with camphane scaffold

Article abstract of DOI:10.1016/j.ejmech.2014.05.007

A series of aminoalcohols were synthesized by reaction of aminolysis of camphor derived oxiranes with chosen amines. The compounds were evaluated for their in vitro activity against Mycobacterium tuberculosis H37Rv. Ten of the new structures show much higher activity than the classical anti-TB drug ethambutol. Some of the most active compounds were tested against MDR strain 43, and four of them demonstrated excellent activities with MICs 0.27-0.72 μM. The cytotoxicity of representative exerting antimycobacterial activity compounds was assessed. Quantitative structure-activity relationship (QSAR) model is derived to estimate the contribution of each structural fragment to the activity. The camphane-based aminoalcohols are promising lead compounds for further development of novel antimycobacterial agents.

Full text of DOI:10.1016/j.ejmech.2014.05.007

European Journal of Medicinal Chemistry 81 (2014) 150e157
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Antimycobacterial activity of chiral aminoalcohols with camphane
scaffold
Zhanina Petkova a, Violeta Valcheva b, Georgi Momekov c, Petar Petrov d,
,
a,
*
Vladimir Dimitrova, Irini Doytchinova c, Georgi Stavrakovc , Malinka Stoyanova
*
a Institute of Organic Chemistry with Center of Phytochemistry, Bulgarian Academy of Sciences, Acad. Bonchev 9, Sofia 1113, Bulgaria
b Institute of Microbiology, Bulgarian Academy of Sciences, Akad. Bonchev 26, Sofia 1113, Bulgaria
c Faculty of Pharmacy, Medical University of Sofia, Str. Dunav 2, Sofia 1000, Bulgaria
d Faculty of Chemistry and Pharmacy, Sofia University St. Kliment Ohridsky, James Bourchier Blvd. 1, Sofia 1164, Bulgaria
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of aminoalcohols were synthesized by reaction of aminolysis of camphor derived oxiranes with
chosen amines. The compounds were evaluated for their in vitro activity against Mycobacterium tuber-
culosis H37Rv. Ten of the new structures show much higher activity than the classical anti-TB drug
ethambutol. Some of the most active compounds were tested against MDR strain 43, and four of them
Received 31 January 2014
Received in revised form
12 March 2014
Accepted 1 May 2014
Available online 4 May 2014
demonstrated excellent activities with MICs 0.27e0.72 mM. The cytotoxicity of representative exerting
antimycobacterial activity compounds was assessed. Quantitative structureeactivity relationship (QSAR)
model is derived to estimate the contribution of each structural fragment to the activity. The camphane-
based aminoalcohols are promising lead compounds for further development of novel antimycobacterial
agents.
Keywords:
Camphor
Mycobacterium tuberculosis
MDR TB
Ó 2014 Elsevier Masson SAS. All rights reserved.
Cytotoxicity
QSAR
1. Introduction
part because of its very low toxicity and relatively few side-effects.
Based on structureeactivity relationship (SAR) studies it appeared
Mycobacterium tuberculosis (MTB) infects latently one-third of
the world’s population causing approximately 9 million cases of
active disease each year [1]. The WHO-recommended anti-TB
therapy involves four drugs: isoniazid (INH), rifampicin (RIF), pyr-
azinamide (PZA) and ethambutol (EMB), but the emergence of
multi drug-resistant bacteria (MDR TB) against which the first-line
drugs have become ineffective, requires treatment for up to two
years with more toxic, less active and more expensive drugs. The
latter usually involve any first-line drugs to which the strain is still
susceptible and alternative or second-line drugs [2]. The long cur-
rent drug regimen, the emergence of drug resistant strains and HIV
co-infection necessitate the urgent development of new and
effective anti-TB drugs.
that crucial for its activity is the distance between the two nitro-
gens, the presence of -aminoalcohol motifs, and the small side
b
chains [4]. Lately, the occurrence of a ‘better ethambutol’ has been
systematically investigated through virtual screening or a combi-
natorial approach [5,6]. Several 1,2-diamines, such as SQ 109 (Fig. 1.
II), displaying improved antimycobacterial potencies and prom-
ising pharmacokinetic properties have thus been reported [7]. It is
very likely that the highly lipophilic adamantane structure was
integral for the antitubercular activity.
Camphor and its derivatives are of particular importance among
the numerous monoterpenoids. Camphor is a readily available and
inexpensive chiral source for the synthesis of a variety of struc-
turally diverse compounds [8,9]. Inspired by the b-aminoalcohol
The synthesis and activity of EMB (Fig. 1. I) was first reported by
Wilkinson and coworkers [3]. Despite the relatively modest MIC of
10 mM, EMB is a useful addition to tuberculosis chemotherapy, in
fragment in the molecule of EMB and the analogy of the camphane
scaffold as a compact lipophilic moiety to the adamantyl fragment
in SQ 109, we dedicated our efforts towards the development of
camphor derived structures. Recently, we have accomplished a
practical synthesis of new b-amido-alcohols and amido-diols on
the base of 3-exo-aminoisoborneol (Fig. 1. III) and isobornylamine
(Fig. 1. IV) [10,11]. Some of the compounds show 25 times higher
antimycobacterial activity than EMB.
* Corresponding authors.
E-mail addresses:
(G. Stavrakov), mstoyanova@orgchm.bas.bg (M. Stoyanova).
gstavrakov@hotmail.com,
gstavrakov@pharmfac.net
http://dx.doi.org/10.1016/j.ejmech.2014.05.007
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

Z. Petkova et al. / European Journal of Medicinal Chemistry 81 (2014) 150e157
151
diisopropyl, piperidine, methylpiperidine, morpholine and fluoro
or chloro substituted N-phenylpiperazine moieties. As evident from
the calculated log P values, most of the compounds are lipophilic
and thus have low water solubility (Table 2). However, the com-
pounds possess amino groups, which allow their transformation
into corresponding water soluble ammonium salts. The combina-
tion of these two features conditions good cell permeability and
facilitates preparation of pharmaceutical forms for various
applications.
The diisopropylamine and piperidine derived aminoalcohols
with R-configuration (3a and 5a) exhibited high activity against
M. tuberculosis H37Rv with MICs of 0.37 and 0.38 mM. The isomers
with S-configuration 3b and 5b gave low activity. Interestingly,
both isomers derived from 4-methyl-piperidine 6a,b demonstrated
excellent activity with MIC values of 0.36 and 0.72 mM, respectively.
Fig. 1. Structures of ethambutol (I), SQ 109 (II), 3-exo-aminoisoborneol based amido-
alcohols (III), isobornylamine based amido-alcohols (IV), camphor derived amino-
alcohols (V).
On the other hand, introducing of N-Me-piperazine 7a,b and N-
phenyl-piperazine 8a,b fragments in the structure of the amino-
alcohols resulted in activity lower than that of EMB. Various
substituted piperazines (N-o-F-phenyl 9a,b, N-p-F-phenyl 10a,b, N-
p-Cl-phenyl 11a,b) were also used as pharmacophores in combi-
nation with camphane moiety. The o-F-phenyl- and p-F-phenyl-
piperazine derivatives 9a,b, and 10a,b exhibited extremely high
Encouraged by these observations, we expanded the approach
to the synthesis of aminoalcohols possessing a camphane skeleton
(Fig. 1. V). Here, we report the synthesis, antimycobacterial activity
and cytotoxicity of 21 compounds and derive a QSAR model to
estimate the contribution of each structural fragment to the ac-
tivity. Thus, we continue the exploration of camphane based
structures as a novel class of anti-TB compounds.
activity for both isomers: MICs of 0.28
phenyl-piperazine, the more active isomer was the one with S-
configuration 11b, MIC 0.27 M. The R-isomer 11a displayed po-
tency comparable to EMB. The morpholine derived aminoalcohol
with R-configuration 12a showed excellent MIC of 0.37 M, while
mM. In the case of p-Cl-
m
2. Results and discussion
m
2.1. Chemistry
the S-isomer 12b had low activity. The thiomorpholine derivatives
13a,b were not active.
Commercially available 10-camphorsulfonyl chloride 1 was
selected as the key (þ)-camphor-derived starting compound. Its
reaction with diazomethane and triethylamine followed by elimi-
nation of sulphur dioxide afforded 7,7-dimethyl-1-vinyl-nor-
bornan-2-on in 77% yield [12]. Subsequent epoxidation using 3-
chloroperbenzoic acid led to diastereoisomeric mixtures of oxir-
anes 2a and 2b (Scheme 1) [13].
Both oxiranes 2a and 2b were isolated as pure diastereoisomers,
after chromatographic separation, in 52% and 33% yield respec-
tively [13]. In our previous work we determined the configuration
of the newly formed stereogenic centre by advanced NMR experi-
ments and X-ray crystallography [13]. The two products 2a and 2b
were then converted into aminoalcohols by aminolysis with excess
of secondary amines at 50 ꢀC in acetonitrile in the presence of
LiClO4 (Scheme 1). The synthesis of compounds 3a,be6a,b was
reported in our previous work considering their use as chiral li-
gands for asymmetric Zn-mediated catalysis [13]. The initial anti-
mycobacterial activity testing of these compounds directed us
towards the creation of a small library of camphane based amino-
alcohols and evaluation of their biological properties. The amino-
lytic cleavage of the oxirane ring with various amines containing
groups with pharmacophore properties afforded 14 new com-
pounds 7a,be13a,b. Consequently, 21 compounds were syn-
thesised and tested for the antimycobacterial activity reported
herein.
The aminoalcohols were obtained in very good to excellent
yields as summarized in Table 1. All compounds were characterized
by NMR spectroscopy, MS data, [a]
20 and elemental analysis.
D
2.2. Antimycobacterial activity
All synthesized camphane-based aminoalcohols were evaluated
for their in vitro activity against M. tuberculosis H37Rv using the
method of Canetti (Table 2). Ten of these compounds have shown
excellent activity, between 10 and 27 times higher than that of EMB,
used as reference (Table 2). The most active compounds possess
Scheme 1. Synthesis of aminoalcohols 3a and 3b: (a) Et3N, CH2N2, 95 ꢀC, 77%; (b)
MCPBA, CH2Cl2, 53% 2a and 32% 2b; (c) HNR2, LiClO4, up to 99%.

152
Z. Petkova et al. / European Journal of Medicinal Chemistry 81 (2014) 150e157
Table 1
Structures of the synthesized aminoalcohols.
Compound
Starting oxirane 2a
Yield
82
Compound
Starting oxirane 2b
Yield
71
3a
3b
4a
5a
93
83
e
e
e
5b
94
6a
7a
98
85
6b
7b
86
79
8a
9a
99
80
8b
9b
99
91
10a
11a
99
73
10b
11b
98
76
12a
13a
91
72
12b
13b
84
68
It is worth noting that the stereogenic center at C10 does not
influence the activity. Hence, the proper choice of amine containing
pharmacophores appears to be crucial.
Remarkable is that 4-methyl-piperidine 6b, p-F-phenylpiperazine
10a,b, and p-Cl-phenylpiperazine 11b derivatives gave excellent
activity with MICs 0.27e0.72 mM. Piperidine 5a and morpholine
Some of the most active compounds were tested for anti-
mycobacterial activity towards MDR strain 43 of M. tuberculosis.
12a demonstrated low activity: MICs of 18.91 and 18.70 mM,
respectively.

Z. Petkova et al. / European Journal of Medicinal Chemistry 81 (2014) 150e157
153
Table 2
2.4. Quantitative structureeantimycobacterial activity
relationships (QSAR)
Data for antimycobacterial activity, cytotoxicity and lipophilicity of synthesized
compounds.
No
MTB
MIC (
MDR MTB
Cytotoxicity
IC50
M)d
Log Pe
The antimycobacterial activity of the studied compounds to-
wards the reference strain of M. tuberculosis H37RV was presented
in p-units (pMIC). According to these values, the compounds are
almost equally distributed into two classes: class 1 e high active
compounds (pMIC > 5) and class 2 e low active compounds
(pMIC < 5). Eleven (3a, 5a, 6a, 9a-12a, 6b, 9b, 10b, 11b) have
m
M)a
MIC (
m
M)b,c
(m
3a
3b
4a
5a
5b
6a
6b
7a
7b
8a
8b
9a
0.37
10.66
19.89
0.38
18.84
0.72
NT
NT
NT
18.91
NT
NT
0.72
NT
NT
NT
NT
NT
NT
0.55
0.55
NT
0.27
>18.70
NT
166.3
NT
NT
143.2
NT
158.4
169.3
NT
2.86
2.86
1.92
2.49
2.49
2.98
2.98
0.55
0.55
2.96
2.96
3.33
3.33
3.05
3.05
3.69
3.69
0.83
0.83
1.82
1.82
0.06g
MIC < 10
mM and enter class 1; ten compounds (4a, 7a, 8a, 13a, 3b,
0.36
5b, 7b, 8b, 12b, 13b) have antimycobacterial activity above 10
mM
17.83
17.83
14.60
14.60
0.28
0.28
0.28
0.28
7.96
0.27
0.37
18.70
17.64
17.64
7.22
and enter class 2. The chemical structure of the studied compounds
was divided into two parts: a camphane scaffold which is common
for all derivatives and an N-containing substituent. The N-con-
taining substituents were described by binary coded descriptors.
Twelve descriptors were used: NME (N-dimethyl), PYR (pyrroli-
dine), PPD (piperidine), MET-PPD (methyl-piperidine), PPZ (piper-
azine), PHE-PPZ (phenyl-piperazine), oF-PHE-PPZ (o-flouro-phenyl-
piperazine), pF-PHE-PPZ (p-flouro-phenyl-piperazine), pCL-PHE-
PPZ (p-chloro-phenyl-piperazine), MOR (morpholine), THI-MOR
(thiomorpholine) and (R)-OH (hydroxyl group at carbon with (R)-
configuration). If a particular substituent presents in the structure,
the corresponding element in the binary string takes 1; otherwise,
it takes 0. The coded structures are shown in Table 3.
Discriminantanalysisbypartial leastsquares (DA-PLS)was applied
to the dataset as implemented in SIMCA 13.0 [14]. The first principal
component (PC) accounts for 79% of the variance in the set; thesecond
PC adds only 1% and it was omitted. The score and loadings plots are
given in Fig. 2. The sets of highly and low active compounds are well
distinguished on the score plot (Fig. 2a). The loadings plot (Fig. 2b)
points the descriptors contributing positively to the class 1 activity.
They are MET-PPD, oF-PHE-PPZ, pF-PHE-PPZ, pCL-PHE-PPZ and (R)-OH.
The contributions of substituents to class 1 activity are given
below:
NT
NT
NT
128.6
119.8
82.2
65.1
8.6
9.2
>200
NT
9b
10a
10b
11a
11b
12a
12b
13a
13b
EMB$2HClf
NT
NT
NT
NT
NT
NT
a
Antimycobacterial activity towards reference strain of Mycobacterium tubercu-
M).
Antimycobacterial activity towards MDR strain 43 of Mycobacterium tubercu-
losis, MIC ( M).
losis H37Rv, MIC (
m
b
m
c
NT e not tested.
d
e
In vitro cytotoxicity towards human embryonal kidney cell line 293T, IC50 (mM).
Log P, octanolewater partitioning coefficient, was calculated using ACDLabs/
ChemSketch 12.01.
f
EMB$2HCl e ethambutol dihydrochloride (reference compound).
Log P and water solubility of EMB$2HCl are known in the literature: N.R. Budha,
g
R.E. Lee and B. Meibohm, Curr. Med. Chem. 15 (2008) 809.
DAðclass 1Þ ¼ 1:024 ꢁ 0:015 NME ꢁ 0:220 PYR ꢁ 0:015 PPD þ 0:290 MET ꢁ PPD ꢁ 0:319 PPZ ꢁ 0:319 PHE ꢁ PPZ þ 0:290
oF ꢁ PHE ꢁ PPZ þ 0:290 pF ꢁ PHE ꢁ PPZ þ 0:290 pCL ꢁ PHE ꢁ PPZ ꢁ 0:015 MOR ꢁ 0:319 THI ꢁ MOR þ 0:222
ðRÞ ꢁ OH n ¼ 21 r2 ¼ 0:790 q2 ¼ 0:331at threshold ¼ 0:5 sensitivity ¼ 1:000 specificity ¼ 1:000 accuracy ¼ 1:000
2.3. Cytotoxicity
According to this model, the substituents methyl-piperidine
(MET-PPD), o-fluoro-phenyl-piperazine (oF-PHE-PPZ), p-fluoro-
phenyl-piperazine (pF-PHE-PPZ), o-chloro-phenyl-piperazine (oCL-
PHE-PPZ) and hydroxyl group at carbon with (R)-configuration ((R)-
OH) increase the antimycobacterial activity of the studied com-
pounds. The remaining substituents decrease the activity. At
threshold 0.5, the model classifies the studied compounds with
100% accuracy e compounds with predicted values above 0.5 were
classified as class 1, those with values below 0.5 e as class 2.
In order to examine the selectivity of the antiproliferative ef-
fects, the cytotoxic activity of representative compounds, exerting
antimycobacterial activity, was assessed against the human
embryonal kidney cell line 293T, after 72 h exposure. Evident from
the IC50 values summarized in Table 2, the compounds were
generally of low-to-moderate cytotoxicity against the human cells;
with the exception of 11a and 11b. Disappointing is the result for
compound 11b, which demonstrated high activity both against the
reference strain and the MDR strain of M. tuberculosis. Compounds
3. Conclusion
10a and 10b have moderate cytotoxicity with IC50 82.2 and 65.1
respectively. Compounds 3a, 5a, 6a,b and 9a,b induced 50% inhi-
bition of cellular viability at concentrations exceeding 100 M.
Compound 12a did not induce 50% inhibition of cellular viability
within the tested range of concentrations (12.5e200 M), hence its
cytotoxicity was exceeding 200 M.
Structure 6b exhibits both high antimycobacterial activity
against the reference and the MDR strains, and low cytotoxicity,
which makes it a promising lead compound for antimycobacterial
agents.
mM
In summary, we are presenting a small library of compounds
synthesized by aminolytic cleavage of camphor derived oxiranes
with a set of amines. The compounds were screened for their
antimycobacterial activity against M. tuberculosis H37Rv. Some of
the new compounds show 25 times higher activity than the clas-
sical anti-TB drug ethambutol. The activity shifts from micromolar
to nanomolar inhibitory concentrations depending on the nitrogen
containing fragments. Quantitative structureeactivity relationship
(QSAR) model was derived to estimate the contribution of each
m
m
m

154
Z. Petkova et al. / European Journal of Medicinal Chemistry 81 (2014) 150e157
Table 3
Antimycobacterial activity (pMIC) and binary coded structures of the studied compounds.
ID
pMIC
Class
NMEa
PYRb
PPDc
MET-PPDd
PPZe
PHE-PPZf
oF-PHE-PPZg
pF-PHE-PPZh
pCl-PHE-PPZi
MORj
THI-MORk
(R)eOHl
3a
4a
5a
6a
7a
8a
9a
10a
11a
12a
13a
3b
5b
6b
7b
8b
6.449
4.701
6.420
6.143
4.749
4.836
6.553
6.553
5.089
6.432
4.754
4.972
4.725
6.444
4.749
4.836
6.553
6.553
6.569
4.728
4.754
1
2
1
1
2
2
1
1
1
1
2
2
2
1
2
2
1
1
1
2
2
1
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
9b
10b
11b
12b
13b
a
N-dimethyl.
Pyrrolidine.
Piperidine.
Methyl-piperidine.
Piperazine.
b
c
d
e
f
Phenyl-piperazine.
g
h
i
o-Flouro-phenyl-piperazine.
p-Flouro-phenyl-piperazine.
o-Chloro-phenyl-piperazine.
Morpholine.
Thiomorpholine.
Hydroxyl group at carbon with (R)-configuration.
j
k
l
structural fragment to the activity. Four of the compounds
demonstrate excellent potency against MDR strain 43. Noteworthy,
one of them proved to exert low, and two of them moderate
cytotoxic activity against a human embryonal kidney cell line 293T.
On this basis, the camphane-based aminoalcohols appear to be
promising lead compounds for development of antimycobacterial
agents.
analyses were performed in Faculty of Chemistry and Pharmacy,
University of Sofia, using Vario ELIII CHNS(O). Dimethyl sulfoxide
(DMSO) for testing of bioactivities was commercial (spectroscopic
grade) and was used without distillation.
4.1.1. General procedure for the preparation of aminoalcohols 3a,be
13a,b
To a stirred solution of oxirane 2a or 2b (0.55 mmol) in 5 ml
CH3CN was added LiClO4 (0.55 mmol) at room temperature and the
mixture was stirred until the salt was completely dissolved. Sec-
ondary amine (1.7 mmol, 3 eq) was added to the solution and the
mixture was stirred at 50 ꢀC until the starting oxirane was
consumed (TLC). After aqueous quench, the mixture was acidified
with 2N HCl (pH 3e4) and extracted twice with ether to remove
traces of starting oxirane. The aqueous layer was basified with 10%
aqueous Na2CO3 and extracted with CH2Cl2 (4 ꢂ 15 ml). The com-
bined organic layers were dried over anhydrous Na2SO4, filtered
and concentrated under reduced pressure. The residue was purified
by flash column chromatography on silica gel (eluent: ether or
hexane/ether ¼ 2:3).
4. Experimental
4.1. Chemistry
Reagents were of commercial grade and used without further
purification. Thin layer chromatography (TLC) was performed on
aluminium sheets pre-coated with Merck Kieselgel 60 F254
0.25 mm (Merck). Flash column chromatography was carried out
using Silica Gel 60 230e400 mesh (Merck). Commercially available
solvents for reactions, TLC and column chromatography were used
after distillation (and were dried when needed). Melting points of
the compounds were determined using “Electrothermal” MEL-
TEMP apparatus (uncorrected). Optical rotation ([a] ) were
20
D
measured on PerkineElmer 241 polarimeter. The NMR spectra
were recorded on a Bruker Avance IIþ 600 spectrometer (600 for 1H
MHz, 150 MHz for 13C NMR) and Bruker Avance DRX 250 spec-
trometer (250 for 1H MHz; 62 for 13C MHz) with TMS as internal
4.1.1.1. (1R,4R)-1-((R)-1-hydroxy-2-(4-methylpiperazin-1-yl)ethyl)-
7,7-dimethylbicyclo[2.2.1]heptan-2-one 7a. Yield: 85%; colourless
20
crystals; mp 110e112 ꢀC; [
a
]
¼ ꢁ12.24 (c ¼ 1, CHCl3); 1H NMR
D
(250 MHz, CDCl3)
d
¼ 1.02 (s, 3H, 8-H), 1.15 (s, 3H, 9-H), 1.25e1.40
standards for chemical shifts (
d
, ppm). 1H and 13C NMR data are
(m, 2H, 5-Hendo, 6-Hendo), 1.78 (d, 1H, 3-Hendo, J ¼ 18.3 Hz), 1.95e2.21
(m, 3H, 4-H, 6-Hexo, 5-Hexo), 2.29 (dd, 1H, 11-Hsin, J ¼ 12.1, 10.9 Hz),
2.27 (s, 3H, 16-H), 2.31e2.51 (m, 7H, 3-Hexo, 12-H, 13-H, 14-H, 15-H),
2.70e2.78 (m, 2H, 13-H, 14-H), 2.92 (dd, 1H, 11-Hanti, J ¼ 12.2,
3.1 Hz), 3.94 (dd, 1H, 10-H, J ¼ 10.9, 3.1 Hz); 13C NMR (62 MHz,
reported as follows: chemical shift, multiplicity (s ¼ singlet,
d ¼ doublet, t ¼ triplet, q ¼ quartet, br ¼ broad, m ¼ multiplet),
coupling constants (Hz), integration, identification. The assignment
of the 1H and 13C NMR spectra was made on the basis of DEPT, COSY,
HSQC, HMBC and NOESY experiments. Mass spectra were recorded
on Hewlet Packard MS 5973 (30 eV) spectrometer. Elemental
CDCl3):
d
¼ 20.52 (C-8), 21.92 (C-9), 21.94 (C-6), 26.84 (C-5), 43.69
(C-3), 44.18 (C-4), 46.04 (C-16), 48.32 (C-7), 55.33 (C-13, C-12, C-14,

Z. Petkova et al. / European Journal of Medicinal Chemistry 81 (2014) 150e157
155
15-H), 2.87e2.95 (m, 2H, 12-H, 15-H), 3.04 (dd, 1H, 11-Hanti, J ¼ 12.2,
3.00 Hz), 3.10e3.17 (m, 4H,13-H,14-H), 4.01 (dd,1H,10-H, J ¼ 10.09,
3.00 Hz), 6.88e7.09 (m, 4H, Ph); 13C NMR (62 MHz, CDCl3):
d
¼ 20.57
(C-8), 21.95 (C-9), 22.01 (C-6), 26.87 (C-5), 43.71 (C-3), 44.20 (C-4),
48.37 (C-7), 50.69 (C-13), 50.74 (C-14), 53.11 (C-15, C-12), 60.28 (C-
11), 62.68 (C-1), 64.02 (C-10), 116.14 (d, 1C, C-20, J ¼ 20.7 Hz),118.92
(d,1C, C-18, J ¼ 3.00 Hz),122.45 (d,1C, C-17, J ¼ 7.9 Hz),124.43 (d,1C,
C-19, J ¼ 3.6 Hz), 140.04 (d, 1C, C-16, J ¼ 8.5 Hz), 155.72 (d, 1C, C-21,
JCeF ¼ 245.7 Hz), 217.89 (C-2); MS (þESI) m/z (rel. int.): 383 (100,
MþNa), 361 (91, Mþ1); Anal. Calcd for C21H29FN2O2: C 69.97; H 8.11;
N 7.77; Found: C 69.82; H 8.24; N 7.78.
4.1.1.4. (1R,4R)-1-((R)-2-(4-(4-fluorophenyl)piperazin-1-yl)-1-
hydroxyethyl)-7,7-dimethylbicyclo[2.2.1]heptan-2-one
10a.
¼ ꢁ11.48
20
Yield: 99%; colourless crystals; mp 104e107 ꢀC; [
a
]
D
(c ¼ 1.02, CHCl3); 1H NMR (600 MHz, CDCl3)
d
¼ 1.04 (s, 3H, 8-H),
1.17 (s, 3H, 9-H), 1.34e1.40 (m, 2H, 6-Hendo, 5-Hendo), 1.81 (d, 1H, 3-
H
endo, J ¼ 18.4 Hz), 1.99e2.07 (m, 2H, 4-H, 6-Hexo), 2.17e2.23 (m, 1H,
5-Hexo), 2.35e2.40 (m, 2H, 3-Hexo, 11-Hsin), 2.57e2.61 (m, 2H, 12-H,
15-H), 2.87e2.91 (m, 2H, 12-H, 15-H), 3.00 (dd, 1H, 11-Hanti, J ¼ 12.2,
3.00 Hz), 3.08e3.15 (m, 4H, 13-H, 14-H), 4.00 (dd, 1H, 10-H,
J ¼ 10.92, 3.00 Hz), 6.86e6.89 (m, 2H, Ph), 6.94e6.98 (m, 2H, Ph);
13C NMR (150 MHz, CDCl3):
d
¼ 20.55 (C-8), 21.93 (C-6), 21.97 (C-9),
26.88 (C-5), 43.72 (C-3), 44.16 (C-4), 48.39 (C-7), 50.34 (C-13, C-14),
53.03 (C-12, C-15), 60.24 (C-11), 62.70 (C-1), 64.02 (C-10), 115.53 (d,
2C, C-18, C-20, J ¼ 21.7 Hz), 117.80 (d, 2C, C-17, C-21, J ¼ 8.5 Hz),
147.89 (d, 1C, C-16, J ¼ 2.4 Hz), 156.30 (d, 1C, C-19, JCeF ¼ 235.4 Hz),
218.15 (C-2); MS (þESI) m/z (rel. int.): 383 (100, MþNa), 361 (90,
Mþ1); Anal. Calcd for C21H29FN2O2: C 69.97; H 8.11; N 7.77; Found:
C 70.21; H 7.87; N 7.80.
Fig. 2. The score plot (a). The first PC accounts for 79% of the variance in the dataset.
The highly active compounds with MIC < 10 M (class 1) are given as black circles. The
low active compounds with MIC > 10 M (class 2) are given as grey circles. The loading
4.1.1.5. (1R,4R)-1-((R)-2-(4-(4-chlorophenyl)piperazin-1-yl)-1-
m
m
hydroxyethyl)-7,7-dimethylbicyclo[2.2.1]heptan-2-one
11a.
¼ ꢁ10.84
20
Yield: 73%; colourless crystals; mp 140e143 ꢀC; [
a
]
plot (b). X and Y descriptors are given as grey and black circles, respectively.
D
(c ¼ 1.02, CHCl3); 1H NMR (600 MHz, CDCl3)
d
¼ 1.04 (s, 3H, 8-H),
1.17 (s, 3H, 9-H), 1.33e1.40 (m, 2H, 5-Hendo, 6-Hendo), 1.81 (d, 1H, 3-
C-15), 60.10 (C-11), 62.67 (C-1), 63.90 (C-10), 217.82 (C-2); MS
(þESI) m/z (rel. int.): 281 (100, Mþ1); Anal. Calcd for C16H28N2O2: C
68.53; H 10.06; N 9.99; Found: C 68.59; H 10.21; N 9.97.
H
endo, J ¼ 18.4 Hz), 1.99e2.07 (m, 2H, 4-H, 6-Hexo), 2.17e2.23 (m, 1H,
5-Hexo), 2.37e2.41 (m, 1H, 3-Hexo), 2.39 (dd, 1H, 11-Hsin J ¼ 12.5,
10.9 Hz), 2.58e2.62 (m, 2H,12-H,15-H), 2.89e2.91 (m, 2H,12-H,15-
H), 3.01 (dd, 1H, 11-Hanti, J ¼ 12.2, 2.8 Hz), 3.13e3.20 (m, 4H, 13-H,
14-H), 4.01 (dd, 1H, 10-H, J ¼ 10.9, 2.8 Hz), 6.82e6.85 (m, 2H, Ph),
4.1.1.2. (1R,4R)-1-((R)-1-hydroxy-2-(4-phenylpiperazin-1-yl)ethyl)-
7,7-dimethylbicyclo[2.2.1]heptan-2-one 8a. Yield: 99%; colourless
7.19e7.22 (m, 2H, Ph); 13C NMR (150 MHz, CDCl3):
d
¼ 20.53 (C-8),
20
crystals; mp 97e100 ꢀC; [
a
]
D
¼ ꢁ10.40 (c ¼ 1, CHCl3); 1H NMR
21.90 (C-6), 21.98 (C-9), 26.88 (C-5), 43.72 (C-3), 44.14 (C-4), 48.40
(C-7), 49.27 (C-13, C-14), 52.90 (C-12, C-15), 60.38 (C-11), 62.66 (C-
1), 64.05 (C-10), 117.27, 124.53, 128.97, 149.879 (Ph), 218.16 (C-2);
MS (þESI) m/z (rel. int.): 399 (100, MþNa), 377 (35, Mþ1); Anal.
Calcd for C21H29ClN2O2: C 66.92; H 7.76; N 7.43; Found: C 66.99; H
7.79; N 7.44.
(250 MHz, CDCl3)
d
¼ 0.97 (s, 3H, 8-H), 1.11 (s, 3H, 9-H), 1.23e1.35
(m, 2H, 5-Hendo, 6-Hendo), 1.74 (d, 1H, 3-Hendo, J ¼ 18.3 Hz), 1.90e2.21
(m, 3H, 4-H, 6-Hexo, 5-Hexo), 2.31 (dd, 1H, 11-Hsin, J ¼ 14.8, 13.7 Hz),
2.25e2.36 (m, 1H, 3-Hexo), 2.48e2.56 (m, 2H, 12-H, 15-H), 2.78e
2.86 (m, 2H, 12-H, 15-H), 2.92 (dd, 1H, 11-Hanti, J ¼ 12.2, 3.00 Hz),
3.05e3.20 (m, 4H, 13-H, 14-H), 3.94 (dd, 1H, 10-H, J ¼ 10.86, 2.9 Hz),
6.75e6.87 (m, 3H, Ph), 7.16e7.22 (m, 2H, Ph); 13C NMR (62 MHz,
CDCl3):
d
¼ 20.53 (C-8), 21.90 (C-6), 21.95 (C-9), 26.88 (C-5), 43.72
4.1.1.6. (1R,4R)-1-((R)-1-hydroxy-2-morpholinoethyl)-7,7-
dimethylbicyclo[2.2.1]heptan-2-one 12a. Yield: 91%; colourless
(C-3), 44.20 (C-4), 48.38 (C-7), 49.33 (C-13, C-14), 53.06 (C-12, C-
15), 60.32 (C-11), 62.68 (C-1), 64.04 (C-10), 116.05, 119.75, 129.11,
151.25 (Ph), 217.89 (C-2); MS (þESI) m/z (rel. int.): 365 (100, MþNa),
343 (86, Mþ1); Anal. Calcd for C21H30N2O2: C 73.65; H 8.83; N 8.18;
Found: C 73.58; H 8.91; N 8.18.
20
crystals, mp 55e57 ꢀC; [
a
]
¼ ꢁ16.68 (c ¼ 1.02, CHCl3); 1H NMR
D
(250 MHz, CDCl3)
d
¼ 1.03 (s, 3H, 8-H), 1.17 (s, 3H, 9-H), 1.32e1.42
(m, 2H, 5-Hendo, 6-Hendo), 1.80 (d, 1H, 3-Hendo, J ¼ 18.3 Hz), 1.96e2.35
(m, 4H, 3-Hexo, 4-H, 6-Hexo, 5-Hexo), 2.29 (dd, 1H, 11-Hsin, J ¼ 12.2,
11.00 Hz), 2.39e2.47 (m, 2H, 13-H, 14-H), 2.68e2.76 (m, 2H, 13-H,
14-H), 2.95 (dd, 1H, 11-Hanti, J ¼ 12.2, 2.9 Hz), 3.64e3.77 (m, 4H, 12-
H, 15-H), 3.97 (dd, 1H, 10-H, J ¼ 10.9, 2.9 Hz); 13C NMR (62 MHz,
4.1.1.3. (1R,4R)-1-((R)-2-(4-(2-fluorophenyl)piperazin-1-yl)-1-
hydroxyethyl)-7,7-dimethylbicyclo[2.2.1]heptan-2-one
9a. Yield:
¼ ꢁ11.44 (c ¼ 1.04,
20
80%; colourless crystals; mp 123e126 ꢀC; [
CHCl3); 1H NMR (250 MHz, CDCl3)
¼ 1.04 (s, 3H, 8-H),1.18 (s, 3H, 9-
H), 1.31e1.43 (m, 2H, 6-Hendo, 5-Hendo), 1.81 (d, 1H, 3-Hendo, J ¼ 18.3
Hz), 1.97e2.24 (m, 3H, 4-H, 6-Hexo, 5-Hexo), 2.37 (dd, 1H, 11-Hsin
J ¼ 12.2,11.0 Hz), 2.33e2.43 (m, 1H, 3-Hexo), 2.57e2.65 (m, 2H,12-H,
a
]
CDCl3):
d
¼ 20.51 (C-8), 21.92 (C-6), 21.94 (C-9), 26.85 (C-5), 43.69
D
d
(C-3), 44.16 (C-4), 48.36 (C-7), 53.50 (C-13, C-14), 60.84 (C-11),
62.61 (C-1), 63.80 (C-10), 67.13 (C-12, C-15), 217.90 (C-2); MS (þESI)
m/z (rel. int.): 290 (100, MþNa), 268 (61, Mþ1); Anal. Calcd for
,
C
15H25NO3: C 67.38; H 9.42; N 5.24; Found: C 67.33; H 9.54; N 5.26.

156
Z. Petkova et al. / European Journal of Medicinal Chemistry 81 (2014) 150e157
4.1.1.7. (1R,4R)-1-((R)-1-hydroxy-2-thiomorpholinoethyl)-7,7-
dimethylbicyclo[2.2.1]heptan-2-one 13a. Yield: 72%; colourless
383 (100, MþNa), 361 (74, Mþ1); Anal. Calcd for C21H29FN2O2: C
69.97; H 8.11, N 7.77; Found: C 70.11; H 8.34; N 7.75.
20
crystals, mp 70e71 ꢀC; [
a
]
D
¼ ꢁ12.89 (c ¼ 0.43, CHCl3); 1H NMR
(250 MHz, CDCl3)
d
¼ 1.01 (s, 3H, 8-H),1.15 (s, 3H, 9-H),1.25e1.43 (m,
4.1.1.11. (1R,4R)-1-((S)-2-(4-(4-fluorophenyl)piperazin-1-yl)-1-
2H, 5-Hendo, 6-Hendo), 1.79 (d, 1H, 3-Hendo, J ¼ 18.3 Hz),1.96e2.20 (m,
3H, 4-H, 5-Hexo, 6-Hexo), 2.20 (dd,1H,11-Hsin, J ¼ 12.4,10.77 Hz), 2.35
(ddd,1H, 3-Hexo, J ¼ 18.3, 4.7, 3.00 Hz), 2.58e2.76 (m, 6H,12-H,15-H),
2.91e3.06 (m, 2H, 13-H, 14-H), 3.03 (dd, 1H, 11-Hanti, J ¼ 12.4,
3.00 Hz), 3.93 (dd, 1H, 10-H, J ¼ 10.8, 3.00 Hz); 13C NMR (62 MHz,
hydroxyethyl)-7,7-dimethylbicyclo[2.2.1]heptan-2-one
10b.
¼ þ24.96
20
Yield: 98%; colourless crystals; mp 134e138 ꢀC; [
a
]
D
(c ¼ 1, CHCl3); 1H NMR (250 MHz, CDCl3)
¼ 1.08 (s, 3H, 9-H),1.14 (s,
d
3H, 8-H), 1.32e1.50 (m, 2H, 6-Hendo, 5-Hendo), 1.82 (m, 1H, 3-Hendo),
1.79e2.01 (m, 3H, 4-H, 6-Hexo, 5-Hexo), 2.27 (dd,1H,11-Hanti, J ¼ 12.2,
3.2 Hz), 2.46 (ddd,1H, 3-Hexo, J ¼ 18.3, 4.3, 3.6 Hz), 2.57e2.65 (m, 2H,
12-H, 15-H), 2.80e2.88 (m, 2H, 12-H, 15-H), 3.07e3.18 (m, 4H, 13-H,
14-H), 3.20 (dd, 1H, 11-Hsin, J ¼ 12.2, 11.0 Hz), 3.88 (dd, 1H, 10-H,
J ¼ 10.88, 3.15 Hz), 6.83e7.01 (m, 4H, Ph); 13C NMR (62 MHz, CDCl3):
CDCl3):
d
¼ 20.46 (C-8), 21.80 (C-6), 21.91 (C-9), 26.86 (C-5), 28.15
(C13, C14), 43.68 (C-3), 44.14 (C-4), 48.38 (C-7), 55.11 (C-12, C-15),
61.12 (C-11), 62.64 (C-1), 63.82 (C-10), 217.84 (C-2); MS (þESI) m/z
(rel. int.): 285 (100, Mþ1), 267 (42, MꢁH2O); Anal. Calcd for
C
15H25NO2S: C 63.56; H 8.89; N 4.94; Found: C 63.71; H 8.96; N 4.94.
d
¼ 20.10 (C-9), 22.30 (C-8), 25.68 (C-6), 26.77 (C-5), 44.05 (C-3),
44.17 (C-4), 48.11 (C-7), 50.31 (C-15), 52.98 (C-12), 59.13 (C-11), 61.77
(C-1), 65.66 (C-10), 115.51 (d, 2C, C-18, C-20, J ¼ 22.1 Hz), 117.81 (d,
2C, C-17, C-21, J ¼ 7.6 Hz),147.90 (d,1C, C-16, J ¼ 2.2 Hz),157.20 (d,1C,
C-19, JCeF ¼ 238.8 Hz), 217.55 (C-2); MS (þESI) m/z (rel. int.): 383
(100, MþNa), 361 (74, Mþ1); Anal. Calcd for C21H29FN2O2: C 69.97; H
8.11; N 7.77; Found: C 69.93; H 8.04; N 7.78.
4.1.1.8. (1R,4R)-1-((S)-1-hydroxy-2-(4-methylpiperazin-1-yl)ethyl)-
7,7-dimethylbicyclo[2.2.1]heptan-2-one 7b. Yield: 79%; colourless
20
crystals; mp 76e79 ꢀC; [
a]
¼ þ23.44 (c ¼ 1.02, CHCl3); 1H NMR
D
(600 MHz, CDCl3)
d
¼ 0.99 (s, 3H, 9-H), 1.06 (s, 3H, 8-H), 1.26e1.38
(m, 2H, 5-Hendo, 6-Hendo,13-H),1.72e1.76 (m,1H, 4-H),1.74 (d,1H, 3-
endo, J ¼ 18.4 Hz), 1.87e1.92 (m, 2H, 6-Hexo, 5-Hexo), 2.12 (dd, 1H,
H
11-Hanti, J ¼ 12.1, 3.2 Hz), 2.22 (s, 3H, 16-H), 2.38 (ddd, 1H, 3-Hexo
,
4.1.1.12. (1R,4R)-1-((S)-2-(4-(4-chlorophenyl)piperazin-1-yl)-1-
J ¼ 18.2, 3.6, 4.3 Hz), 2.18e2.67 (m, 8H, 12-H, 13-H, 14-H, 15-H), 3.09
hydroxyethyl)-7,7-dimethylbicyclo[2.2.1]heptan-2-one
11b.
¼ þ21.99
20
(dd, 1H, 11-Hsin, J ¼ 12.00, 11.3 Hz), 3.75 (dd, 1H, 10-H, J ¼ 11.0,
Yield: 76%; colourless crystals; mp 138e144 ꢀC; [
a
]
D
3.1 Hz); 13C NMR (150 MHz, CDCl3):
d
¼ 20.06 (C-9), 22.30 (C-8),
(c ¼ 1.30, CHCl3); 1H NMR (250 MHz, CDCl3)
d
¼ 1.08 (s, 3H, 9-H),
25.62 (C-6), 26.78 (C-5), 44.07 (C-3), 44.10 (C-4), 46.06 (C-16), 48.11
(C-7), 55.28 (C-13, C-12, C-14, C-15), 59.91 (C-11), 61.68 (C-1), 65.39
(C-10), 217.48 (C-2); MS (þESI) m/z (rel. int.): 281 (100, Mþ1), 303
(19, MþNa); Anal. Calcd for C16H28N2O2: C 68.53; H 10.06; N 9.99;
Found: C 68.61; H 10.29; N 9.90.
1.14 (s, 3H, 8-H), 1.32e1.50 (m, 2H, 5-Hendo, 6-Hendo), 1.76e2.01 (m,
3H, 4-H, 6-Hexo, 5-Hexo), 1.82 (d, 1H, 3-Hendo, J ¼ 18.3 Hz), 2.27 (dd,
1H, 11-Hanti, J ¼ 12.2, 3.1 Hz), 2.46 (ddd, 1H, 3-Hexo, J ¼ 18.3, 4.3,
3.7 Hz), 2.56e2.64 (m, 2H, 12-H, 15-H), 2.79e2.87 (m, 2H, 12-H, 15-
H), 3.10e3.22 (m, 5H, 13-H, 14-H, 11-Hsin), 3.88 (dd, 1H, 10-H,
J ¼ 10.9, 3.1 Hz), 6.80e6.86 (m, 2H, Ph), 7.17e7.23 (m, 2H, Ph); 13
C
4.1.1.9. (1R,4R)-1-((S)-1-hydroxy-2-(4-phenylpiperazin-1-yl)ethyl)-
NMR (62 MHz, CDCl3):
d
¼ 20.10 (C-9), 22.30 (C-8), 25.67 (C-6),
7,7-dimethylbicyclo[2.2.1]heptan-2-one 8b. Yield: 99%; colourless
26.76 (C-5), 44.04 (C-3), 44.17 (C-4), 48.10 (C-7), 49.29 (C-13, C-14),
52.83 (C-12, C-15), 59.17 (C-11), 61.78 (C-1), 65.69 (C-10), 117.23,
124.58, 128.94, 149.84 (Ph), 217.58 (C-2); MS (þESI) m/z (rel. int.):
399 (100, MþNa), 377 (67, Mþ1); Anal. Calcd for C21H29ClN2O2: C
66.92; H 7.76; N 7.43; Found: C 67.11; H 7.72; N 7.44.
20
crystals; mp 103e105 ꢀC; [
a
]
D
¼ þ7.48 (c ¼ 1.03, CHCl3); 1H NMR
(600 MHz, CDCl3)
d
¼ 1.09 (s, 3H, 9-H), 1.14 (s, 3H, 8-H), 1.35e1.47
(m, 2H, 5-Hendo, 6-Hendo), 1.83 (d, 1H, 3-Hendo, J ¼ 18.24 Hz), 1.81e
1.87 (m, 1H, 5-Hexo), 1.95e2.01 (m, 2H, 4-H, 6-Hexo), 2.27 (dd, 1H, 11-
H
anti, J ¼ 12.2, 3.2 Hz), 2.38 (ddd, 1H, 3-Hexo, J ¼ 18.2, 4.6, 3.3 Hz),
2.59e2.63 (m, 2H,12-H, 15-H), 2.83e2.86 (m, 2H,12-H,15-H), 3.17e
3.24 (m, 5H, 11-Hsin, 13-H, 14-H), 3.88 (dd, 1H, 10-H, J ¼ 11.0, 3.2 Hz),
6.87e6.85 (m, 1H, Ph), 6.93e6.94 (m, 2H, Ph), 7.25e7.28 (m, 2H,
4.1.1.13. (1R,4R)-1-((S)-1-hydroxy-2-morpholinoethyl)-7,7-
dimethylbicyclo[2.2.1]heptan-2-one 12b. Yield: 84%; colourless
20
crystals; mp 55e57 ꢀC; [
a
]
D
¼ þ30.68 (c ¼ 1.02, CHCl3); 1H NMR
Ph); 13C NMR (150 MHz, CDCl3):
d
¼ 20.11 (C-9), 22.33 (C-8), 25.67
(600 MHz, CDCl3)
d
¼ 1.07 (s, 3H, 9-H),1.29 (s, 3H, 8-H),1.34e1.45 (m,
(C-6), 26.79 (C-5), 44.08 (C-3), 44.15 (C-4), 48.14 (C-7), 49.31 (C-13,
C-14), 52.97 (C-12, C-15), 59.11 (C-11), 61.76 (C-1), 65.57 (C-10),
116.09, 119.81, 129.14, 151.24 (Ph), 217.62 (C-2); MS (þESI) m/z (rel.
int.): 365 (88, MþNa), 343 (100, Mþ1); Anal. Calcd for C21H30N2O2:
C 73.65; H 8.83; N 8.18; Found: C 73.64; H 8.98; N 8.12.
2H, 5-Hendo, 6-Hendo), 1.82 (d, 1H, 3-Hendo, J ¼ 18.4 Hz), 1.80e1.84 (m,
1H, 6-Hexo), 1.94e2.00 (m, 2H, 4-H, 5-Hexo), 2.22 (dd, 1H, 11-Hanti
,
J ¼ 12.2, 3.1 Hz), 2.43e2.47 (m, 3H, 3-Hexo,13-H,14-H), 2.67e2.69 (m,
2H,13-H,14-H), 3.13 (dd,1H,11-Hsin, J ¼ 12.1,11.1 Hz), 3.69e3.75 (m,
4H, 12-H, 15-H), 3.85 (dd, 1H, 10-H, J ¼ 10.9, 3.1 Hz); 13C NMR
(150 MHz, CDCl3):
d
¼ 20.09 (C-9), 22.30 (C-8), 25.63 (C-6), 26.77 (C-
4.1.1.10. (1R,4R)-1-((S)-2-(4-(2-fluorophenyl)piperazin-1-yl)-1-
5), 44.05 (C-3), 44.13 (C-4), 48.08 (C-7), 53.47 (C-13, C-14), 59.67 (C-
11), 61.71 (C-1), 65.36 (C-10), 67.12 (C12, C15), 217.80 (C-2); MS
(þESI) m/z (rel. int.): 290 (100, MþNa), 268 (27, Mþ1); Anal. Calcd for
hydroxyethyl)-7,7-dimethylbicyclo[2.2.1]heptan-2-one
9b. Yield:
20
91%; colourless crystals; mp 102e104 ꢀC; [
a]
¼ þ22.53 (c ¼ 1.01,
D
CHCl3); 1H NMR (600 MHz, CDCl3)
d
¼ 1.08 (s, 3H, 9-H),1.14 (s, 3H, 8-
C15H25NO3: C 67.38; H 9.42; N 5.24; Found: C 67.44; H 9.71; N 5.17.
H),1.35e1.39 (m,1H, 5-Hendo),1.43e1.48 (m,1H, 6-Hendo),1.83 (d,1H,
3-Hendo, J ¼ 18.2 Hz), 1.82e1.86 (m, 1H, 6-Hexo), 1.96e2.00 (m, 2H, 4-
H, 5-Hexo), 2.28 (dd, 1H, 11-Hanti, J ¼ 12.1, 3.1 Hz), 2.47 (ddd, 1H, 3-
4.1.1.14. (1R,4R)-1-((S)-1-hydroxy-2-thiomorpholinoethyl)-7,7-
dimethylbicyclo[2.2.1]heptan-2-one 13b. Yield: 68%; colourless
¼ þ23.60 (c ¼ 0.99, CHCl3); 1H NMR
20
H
exo, J ¼ 18.2, 4.4, 3.3 Hz), 2.61e2.65 (m, 2H, 12-H, 15-H), 2.85e
crystals; mp 60e63 ꢀC; [
a
]
D
2.89 (m, 2H,12-H,15-H), 3.08e3.15 (m, 4H,13-H,14-H), 3.22 (dd,1H,
11-Hsin, J ¼ 11.76, 11.52 Hz), 3.88 (dd, 1H, 10-H, J ¼ 10.98, 3.1 Hz),
6.92e6.96 (m, 2H, Ph), 7.00e7.07 (m, 2H, Ph); 13C NMR (150 MHz,
(600 MHz, CDCl3)
d
¼ 1.06 (s, 3H, 9-H), 1.13 (s, 3H, 8-H), 1.34e1.44
(m, 2H, 5-Hendo, 6-Hendo), 1.78e1.83 (m, 1H, 6-Hexo), 1.82 (d, 1H, 3-
H
endo, J ¼ 18.4 Hz), 1.95e2.00 (m, 2H, 4-H, 5-Hexo), 2.29 (dd, 1H,
CDCl3):
d
¼ 20.11 (C-9), 22.31 (C-9), 25.62 (C-6), 26.80 (C-5), 44.08
11-Hanti, J ¼ 12.4, 3.2 Hz), 2.44 (ddd, 1H, 3-Hexo, J ¼ 18.4, 4.6, 3.1 Hz),
2.64e2.74 (m, 6H, 12-H, 13-H, 14-H, 15-H), 2.92e2.95 (m, 2H, 13-H,
14-H), 3.09 (dd, 1H, 11-Hsin, J ¼ 12.3, 11.00 Hz), 3.81 (dd, 1H, 10-H,
(C-3), 44.12 (C-4), 48.14 (C-7), 50.70 (C-13, C-14), 53.02 (C-12, C-15),
59.09 (C-11), 61.74 (C-1), 65.49 (C-10), 116.12 (d, 1C, C-20, J ¼ 20.7
Hz), 118.93 (d, 1C, C-18, J ¼ 2.8 Hz), 122.46 (d, 1C, C-17, J ¼ 7.9 Hz),
124.48 (d,1C, C-19, J ¼ 3.4 Hz),140.06 (d,1C, C-16, J ¼ 8.6 Hz),155.74
(d, 1C, C-21, JCeF ¼ 246.5 Hz), 217.61 (C-2); MS (þESI) m/z (rel. int.):
J ¼ 10.9, 3.2 Hz); 13C NMR (150 MHz, CDCl3):
¼ 20.04 (C-9), 22.31
d
(C-8), 25.62 (C-6), 26.77 (C-5), 28.16 (C-12, C-15), 44.07 (C-3), 44.08
(C-4), 48.30 (C-7), 54.99 (C-13, C-14), 59.78 (C-11), 61.73 (C-1),

Z. Petkova et al. / European Journal of Medicinal Chemistry 81 (2014) 150e157
157
65.30 (C-10), 217.86 (C-2); MS (þESI) m/z (rel. int.): 285 (100, Mþ1),
4.4. Discriminant analysis by partial least squares (DA-PLS)
267 (29, MꢁH2O); Anal. Calcd for C15H25NO2S: C 63.56; H 8.89; N
4.94; Found: C 63.62; H 9.08; N 4.90.
Discriminant analysis (DA) is a statistical analysis to predict a
categorical dependent variable by one or more continuous or bi-
nary independent variables. Partial least squares (PLS) is a projec-
tion method [22] that can handle matrices with more variables
than observations and with noisy and highly collinear data. PLS
forms new variables, called principal components (PC), as linear
combinations of the initial variables and then uses them to predict
the dependent variable. The DA-PLS used in the present study was
implemented in SIMCA 13.0 [14].
The predictive ability of the derived model was evaluated by
leave-one-out cross-validated coefficient q2. At threshold 0.5,
sensitivity (correctly predicted class 1 compounds/all class 1 com-
pounds), specificity (correctly predicted class 2 compounds/all class
2 compounds) and accuracy (correctly predicted class 1 and class 2
compounds/all compounds) were calculated.
4.2. Antimycobacterial activity
The antimycobacterial activity was determined through the
proportional method of Canetti towards reference strain
M. tuberculosis H37Rv and multi-drug resistant strain 43 (resistant
to Rifampicin and Isoniazid), recovered from Bulgarian adult HIV-
negative pulmonary TB patient, who was a permanent resident of
the country. This method, recommended by the WHO, is the most
commonly used one worldwide for investigation of sensitivity/
resistance of tuberculosis strains towards chemotherapeutics [15e
19]. It allows precise determination of the proportion of resistant
mutants to a certain drug.
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 five con-
centrations e 5 mg/ml, 2 mg/ml, 0.2 mg/ml and 0.1 mg/ml in DMSO.
Tubes with LöwensteineJensen 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 com-
pounds 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
inhibit bacterial growth completely (0% growth). The MIC values
Acknowledgement
Financial support of National Science Fund, Bulgaria (DMU 02/3
e 2009) is gratefully acknowledged.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.05.007.
are calculated and given as mM.
4.3. Cytotoxicity
References
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 foetal calf serum and
[1] World Health Organization, Global Tuberculosis Report 2012, http://www.
who.int/tb/publications/global_report/en/.
[2] E.C. Rivers, R.L. Mancera, Drug Discov. Today 13 (2008) 1090e1098.
[3] R.G. Shepherd, C. Baughn, M.L. Cantrall, B. Goodstein, J.P. Thomas,
R.G. Wilkinson, Ann. N. Y. Acad. Sci. 135 (1966) 686e710.
[4] E.L. Richard, M. Protopopova, E. Crooks, R.A. Slayden, M. Terrot, C.E. Barry III,
J. Comb. Chem. 5 (2003) 172e187.
[5] A. García-García, J. Gálvez, J. De Julián-Ortiz, R. García-Domenech, C. Muñoz,
R. Guna, R.J. Borráz, Biomol. Screen. 10 (2005) 206e214.
[6] R.E. Lee, M. Protopopova, E. Crooks, R.A. Slayden, M. Terrot, C.E. Barry III,
J. Comb. Chem. 5 (2003) 172e187.
[7] L. Jia, J.E. Tomaszewski, C. Hanrahan, L. Coward, P. Noker, G. Gorman,
B. Nikonenko, M. Protopopova, Br. J. Pharmacol. 144 (2005) 80e87.
[8] T. Money, Nat. Prod. Rep. 2 (1985) 253e280.
[9] W. Oppolzer, Pure Appl. Chem. 62 (1990) 1241e1250.
[10] G. Stavrakov, V. Valcheva, I. Philipova, I. Doytchinova, Eur. J. Med. Chem. 70
(2013) 372e379.
2 mM L
-glutamine, at 37 ꢀC in a ‘Heraeus’ incubator with 5% CO2
humidified 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 with some modifications [20,21]. In brief, exponentially
growing cells were seeded in 96-well microplates (100 ml/well) at a
density of 3.5e105 cell/ml and allowed to grow for 24 h prior to
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 con-
centration 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 solution
[11] G. Stavrakov, I. Philipova, V. Valcheva, G. Momekov, Biorg. Med. Chem. Lett. 24
(2014) 165e167.
[12] N. Fischer, G. Opitz, Org. Synth. Coll. vol. 5 (1973) 877.
[13] M.P. Stoyanova, B.L. Shivachev, R.P. Nikolova, V. Dimitrov, Tetrahedron
Asymmetry 24 (2013) 1426e1434.
[14] SIMCA 13.0, Umetrics Ltd., Umeå, Sweden.
[15] G. Canetti, N. Rist, J. Grosset, Rev. Tuberc. Pneumol. 27 (1963) 217e272.
[16] 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, Bull. WHO 29 (1963) 565e578.
[17] G. Canetti, W. Fox, A. Khomenko, H.T. Mahler, N.K. Menon, D.A. Mitchinson,
N. Rist, N.A. Smelev, Bull. Org. Mond. Sante. 41 (1969) 21e43.
[18] L. Heifets, Conventional methods for antimicrobial susceptibility testing of
Mycobacterium tuberculosis, in: I. Bastian, F. Portaels (Eds.), Multidrug-Resistant
Tuberculosis, Kluwer Academic Publishers, Dordrecht, The Netherlands, 2000.
[19] CellTiter 96 Non-Radioactive Cell Proliferation Assay, Technical Bulletin
#TB112, Promega Corporation USA, Revised 12/99.
(10 mg/ml in phosphate-buffered saline) aliquots (100 ml/well) were
added to each well. The plates were further incubated for 4 h at 37 ꢀC
and the MTT-formazan crystals formed were dissolved through
addition of 110 ml of 5% HCOOH in 2-propanol. The MTT-formazan
absorption of the samples was measured by a multimode micro-
plate reader DTX 880 (Beckman Coulter) at 580 nm. Cell survival
fractions were calculated as percentage of the untreated control. The
experimental data were fitted to sigmoidal concentrationeresponse
curves and the corresponding IC50 values (concentrations causing
50% reduction of cellular survival vs. the untreated control) via non-
linear regression (GraphPad Prizm software for PC).
[20] T.J. Mosmann, Immunol. Meth. 65 (1983) 55e63.
[21] S.M. Konstantinov, H. Eibl, M.R. Berger, Br. J. Haematol. 107 (1999) 365e374.
[22] S. Wold, PLS for multivariate Linear Modeling, in: H. van de Waterbeemd (Ed.),
Chemometric Methods in Molecular Design, VCH Verlagsgesellshaft mbH,
Weinheim, 1995.