Synthesis of novel 3-cyclohexylpropanoic acid-derived nitrogen heterocyclic compounds and their evaluation for tuberculostatic activity

Article abstract of DOI:10.1016/j.bmc.2011.11.020

A series of novel 3-cyclohexylpropanoic acid derivatives and 3-cyclohexylpropanoic acid-derived nitrogen heterocyclic compounds (1-8) have been synthesized and evaluated for tuberculostatic activity. Compounds 1a, 1c, 1e and 1f bearing benzimidazole or benzimidazole-like systems showed the most potent tuberculostatic activity against Mycobacterium tuberculosis strains with MIC values ranging from 1.5 to 12.5 μg/mL. More importantly 1a (6-chloro-2-(2-cyclohexylethyl)-4-nitro-1H-benzo[d]imidazole) and 1f (2-(2-cyclohexylethyl)-1H-imidazo[4,5-b]phenazine) appeared selective for M. tuberculosis as compared with eukaryotic cells (human fibroblasts), and other antimicrobial strains. These compounds may thus represent a novel, selective class of antitubercular agents. Additionally compound 1a stimulated type I collagen output by fibroblasts, in vitro.

Full text of DOI:10.1016/j.bmc.2011.11.020

Bioorganic & Medicinal Chemistry 20 (2012) 137–144
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of novel 3-cyclohexylpropanoic acid-derived nitrogen heterocyclic
compounds and their evaluation for tuberculostatic activity
Katarzyna Gobis a, , Henryk Foks , Krzysztof Bojanowski , Ewa Augustynowicz-Kopec ,
⇑
a
b
c
´
Agnieszka Napiórkowska c
a Department of Organic Chemistry, Medical University of Gdan´sk, 107 Gen. Hallera Ave., 80-416 Gdan´sk, Poland
b Sunny BioDiscovery, 722 East Main Str., Santa Paula, CA 93060, USA
c Department of Microbiology, Institute of Tuberculosis and Pulmonary Diseases, 26 Płocka Str., 01-138 Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 3-cyclohexylpropanoic acid derivatives and 3-cyclohexylpropanoic acid-derived nitro-
gen heterocyclic compounds (1–8) have been synthesized and evaluated for tuberculostatic activity.
Compounds 1a, 1c, 1e and 1f bearing benzimidazole or benzimidazole-like systems showed the most
potent tuberculostatic activity against Mycobacterium tuberculosis strains with MIC values ranging from
Received 19 September 2011
Revised 8 November 2011
Accepted 11 November 2011
Available online 20 November 2011
1.5 to 12.5 lg/mL. More importantly 1a (6-chloro-2-(2-cyclohexylethyl)-4-nitro-1H-benzo[d]imidazole)
and 1f (2-(2-cyclohexylethyl)-1H-imidazo[4,5-b]phenazine) appeared selective for M. tuberculosis as
compared with eukaryotic cells (human fibroblasts), and other antimicrobial strains. These compounds
may thus represent a novel, selective class of antitubercular agents. Additionally compound 1a stimu-
lated type I collagen output by fibroblasts, in vitro.
Keywords:
3-Cyclohexylpropanoic acid
Benzimidazole
Synthesis
Ó 2011 Elsevier Ltd. All rights reserved.
Tuberculostatic activity
Antibacterial activity
Cytotoxic activity
Type I collagen output
1. Introduction
tics, as well as antibiotics such as rifampicin (RMP), rapidly induce
multidrug resistance (MDR) and cause serious side effects, like
Tuberculosis caused by Mycobacterium tuberculosis takes the
leading place in the reports of incidence and mortality among pop-
ulations of developed countries.1 The infection can affect the respi-
ratory system, central nervous system, lymphatic system,
genitourinary system, bones, and even skin.2 In 1990, there were
over 6 million new cases, and in 2007 this figure rose to 9.2 mil-
lion.3 Furthermore, new multidrug-resistant tuberculosis strains
(MDR-TB) are appearing at an alarming speed. The cure of multi-
drug-resistant tuberculosis requires a longer treatment period,
and the success rate is only 52% in newly diagnosed patients, and
29% among previously treated patients.4
Tuberculosis is one of the opportunistic infections in AIDS pa-
tients. For this reason, it represents a serious threat for this group
of individuals. Immune deficiency increases vulnerability inci-
dence to tuberculosis up to 50%.
At the same time there is only a small number of effective anti-
tuberculous chemotherapeutics. Among them isoniazid (INH) and
pyrazinamide (PZA) belong to the most commonly administrated
drugs (Fig. 1). Unfortunately, the most effective chemotherapeu-
hepatotoxicity, neurotoxicity, acute pancreatitis and hypersensi-
tivity reactions.5,6 Therefore, the search for new antituberculous
drugs active against resistant strains of M. tuberculosis should be
one of the priority tasks of medicinal chemistry.
Most of the administrated drugs belong to the group of nitrogen
heterocyclic compounds. That is why potential antituberculous
drugs are searched for in this chemical group. Many pyridine and
pyrazine derivatives obtained in the course of these studies exhib-
ited high antituberculous activity, for example 4-(5-pentadecyl-
1,3,4-oxadiazol-2-yl)pyridine containing 1,3,4-oxadiazole ring
fused with pyridine system.7 Other examples of potent compounds
are pyrazinamidine-, pyridinamidine-, pyrazinecarbohydrazide-
and pyridinecarbohydrazide-derived hydrazinecarbodithioic acid
esters and amides described earlier by us.8,9 After numerous syn-
theses of pyridine and pyrazine derivatives, our interest turned
in the direction of other nitrogen heterocyclic systems including
benzimidazole.
The compounds of the benzimidazole structure described previ-
ously, just like their biological effects, are characterized by great
variety. An example might be 2-benzylbenzimidazole, for which
the SciFinder Database provides over three hundred reports con-
cerning its biological activity. The same 2-benzylbenzimidazole
and its derivatives have effects on the cardiovascular system10
⇑
Corresponding author. Tel.: +48 58 349 31 44; fax: +48 58 349 31 45.
E-mail address: kgobis@gumed.edu.pl (K. Gobis).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.020

138
K. Gobis et al. / Bioorg. Med. Chem. 20 (2012) 137–144
Figure 1. Structures of some tuberculostatic drugs and benzimidazoles of tuberculostatic activity.
and cholesterol level.11 They exhibit anti-inflammatory and anal-
gesic,12 antineoplastic,13 anticonvulsant, antidiabetic14 and also
anthelmintic activity.15 In addition, benzimidazoles are gonadotro-
pin-releasing hormone (GnRH) receptor antagonists.16 Their anti-
viral17 and antimicrobial18 activities have been also demonstrated.
However, there are only few reports on the antituberculous
activity of benzimidazoles.19 Significant tuberculostatic activity of
2-phenylalkyl- and 2-cyclohexylalkylbenzimidazoles (Fig. 1) has
been presented by us.20 The designated minimum concentration
inhibiting the growth of M. tuberculosis strains (MIC) in vitro was
at the level appropriate for applied chemotherapeutics.20,21 We
previously found that tested compounds were more active against
resistant than sensitive strains and the presence of cyclohexylethyl
substituent at position C-2 of the benzimidazole system was
important for their activity.
thiosemicarbazide structure. Those derivatives cyclized to 4-
substituted 1,2,4-triazole-5-thiones 4a–d in a 10% solution of lye.
Hydrazinecarbodithioic acid esters 5–7 were synthesized in the
reaction of 3-cyclohexylpropanoic acid hydrazide with carbon
disulfide and suitable halides (methyl iodide, 1,2-dibromoethane)
in a methanol solution of triethylamine (TEA). In the reaction with
1,2-dibromoethane a two-fold molar excess of triethylamine was
used to obtain 1,3-dithiolane 6. In the case of methyl diester 7, a
two-fold molar excess of both triethylamine and methyl iodide
was needed. That methyl diester was used to obtain 5,6,7,8-tetra-
hydro-1,2,4-triazolopyrimidine 8 in the reaction with 1,3-diami-
no-2,2-dimethylpropane. The formation of this kind of
compounds has been already reported by our research team.23
Promising results of biological studies encouraged us to under-
take the synthesis of other benzimidazole type compounds 1b–f.
Imidazopyridines 1b and 1d were obtained by the heating of 3-
cyclohexylpropanoic acid and appropriate diamine in a diglyme
(di(2-dimethoxyethyl) ether) solution (method B). Due to the li-
quid form of 2-(2-cyclohexylethyl)-3H-imidazo[4,5-c]pyridine the
compound 1d was synthesized as dihydrochloride. Syntheses of
compounds 1c, 1e and 1f were carried out according to the method
described above for benzimidazole 1a.
These findings prompted us to extend our studies on the devel-
opment of novel tuberculostatic agents, here we disclose the syn-
thesis of novel 3-cyclohexylpropanoic acid derivatives.
2. Results and discussion
2.1. Chemistry
All the newly synthesized compounds were characterized by IR
and 1H NMR spectra as well as the elemental analysis listed in the
experimental section. The spectral analyses were in accordance
with the assigned structures. The spectral data of compound 6
indicated two multiplets for SCH2 groups at 3.45 and 3.64 ppm in
the 1H NMR spectrum, which suggested the magnetic inequiva-
lence of both groups. A similar phenomenon was observed in the
case of the compound 7 with two SCH3 groups observed in the
1H NMR spectrum as two singlets at 2.46 and 2.51 ppm. Magnetic
inequivalence seen in the 1H NMR spectrum of compound 6 was
confirmed by the 13C NMR spectrum performed for that derivative.
Carbon atoms of SCH2 groups gave no signal together, but did apart
at 37.8 and 39.4 ppm.
We synthesized structures in which the cyclohexylethyl group
is connected to the benzimidazole system or systems of benzimid-
azole type structure, as well as other heterocyclic rings. Derivatives
of 3-cyclohexylpropanoic acid hydrazide were also obtained:
mono- and diester of hydrazinecarbodithioic acid and appropriate
hydrazinecarbothioamides. The synthesized compounds have been
screened for their tuberculostatic, antibacterial and cytotoxic
activities. Finally, target structures were planned in order to inves-
tigate the preliminary structure–activity relationship, with the
emphasis on the crucial role of the benzimidazole system.
The synthetic route of the studies was outlined in Scheme 1.
Syntheses of the target 3-cyclohexylpropanoic acid derivatives
1–8 were achieved in 36–98% yields by procedures described be-
low. The starting compound for a series of syntheses was the
commercially available 3-cyclohexylpropanoic acid. 6-Chloro-2-
cyclohexylethyl-4-nitrobenzimidazole 1a was obtained by the
heating of 3-cyclohexylpropanoic acid with 5-chloro-3-nitroben-
zene-1,2-diamine according to the method described by Algul
et al.22 and based on the use of polyphosphoric acid (PPA) as a
solvent with strong acidic properties (method A).
Compounds 2–8 were synthesized from 3-cyclohexylpropanoic
acid hydrazide, which was obtained from starting acid via methyl
ester in the reaction with hydrazine hydrate. Then, upon treatment
of carbon disulfide in an alkaline water–ethanol solution, hydra-
zide gave 1,3,4-oxadiazole-2-thione derivative 2a. The same sub-
strate in the reaction with ammonium thiocyanate without
solvent underwent cyclization to 1,2,4-triazole-5-thione 2b. The
reaction of hydrazide with appropriate isothiocyanates (methyl,
allyl, cyclohexyl, 4-chlorophenyl) yielded compounds 3a–d of
2.2. Biological activity
All of the obtained 3-cyclohexylpropanoic acid derivatives 1–8
were evaluated for their in vitro tuberculostatic activity against
the M. tuberculosis H37Rv strain and two ‘wild’ strains isolated from
tuberculosis patients: one (Spec. 210) resistant to p-aminosalicylic
acid (PAS), isonicotinic acid hydrazide (INH), etambutol (ETB) and
rifampicin (RMP) and the another (Spec. 192) fully sensitive to the
administrated tuberculostatics. The MIC values were determined
as the minimum concentration inhibiting the growth of tested
tuberculous strains in relation to the probe with no tested com-
pound. INH, PZA and RMP were used as reference drugs. Com-
pounds were also tested for their antibacterial activity against
Propionibacterium acnes (ATCC11827) and Brevibacterium linens
(ATCC9174). The most active compounds of the benzimidazole
structure 1a–f which exhibited the highest tuberculostatic activity

K. Gobis et al. / Bioorg. Med. Chem. 20 (2012) 137–144
139
Scheme 1. Synthesis of novel 3-cyclohexylpropanoic acid derivatives 1–8. Reagents and conditions: (i) method A (compounds 1a, 1c, 1e, 1f): diamine, PPA, 180–200 °C;
NaHCO3/H2O; method B (compounds 1b, 1d): diamine, dyglime, reflux; (ii) hydrazine hydrate, MeOH, reflux; (iii) for 2a: KOH/H2O, CS2, EtOH, reflux; for 2b: NH4SCN, 170–
180 °C; NaOH/H2O, reflux; (iv) R4NCS, MeOH, reflux; (v) NaOH/H2O, reflux, conc. HCl; (vi) TEA, CS2, MeI, EtOH, room trmperature; (vii) TEA (2 equiv), CS2, 1,2-dibromoethane,
EtOH, room temperature; (viii) TEA (2 equiv), CS2, MeI (2 equiv), EtOH, room temperature; (ix) 1,3-diamino-2,2-dimethylpropane (2 equiv), reflux.
were then tested for effect on the proliferation of neonatal human
dermal fibroblasts (ATCC PCS-201–010). MAP (magnesium ascor-
byl phosphate) and bFGF (basic fibroblast growth factor) were used
as the positive control. Non-cytotoxic compounds 1a, 1d, 1f were
additionally tested for effect on type I collagen output in human
dermal fibroblast populations by ELISA. Type I collagen is the pre-
dominant form of collagen in the body and its decrease is associ-
ated with the ageing of many organs, such as skin and tendons.
The ELISA test used in the studies detects stimulation of soluble
type I collagen by human dermal fibroblast populations, due either
to the stimulation of fibroblast proliferation, stimulation of colla-
gen production or the inhibition of metaloproteinases, which di-
gest collagen.
low tuberculostatic activity against all strain types. Three of the
obtained compounds 1b, 1d and 2a exhibited moderate tuberculo-
static activity with MIC values in range 12.5–50 lg/mL. Their activ-
ities against sensitive and resistant strain were also comparable.
The tuberculostatic activity of benzimidazole 1 and compounds
of the benzimidazole type structure 1c, 1e and 1f was much better
than the other 3-cyclohexylpropanoic acid derivatives. Among this
‘benzimidazole’ series, the compound 1a containing the benzene
ring in benzimidazole system and two substituents—a chlorine
atom in the C-6 position and a nitro group in the C-4 position—
exhibited the weakest tuberculostatic activity with MIC values of
12.5 lg/mL against all tested strains. Compound 1c containing a
pyridine ring instead of benzene in the benzimidazole system
The bioactive data were summarized in Tables 1–3.
and a bromine atom in the C-6 position showed good tuberculo-
static activity with MIC values of 6.2 lg/mL. Noticeably, com-
2.2.1. Tuberculostatic activity
pounds 1e and 1f with larger condensed systems instead of
The results of tuberculostatic activity indicated that most of the
synthesized compounds exhibited moderate to low activity against
M. tuberculosis strains in vitro. As seen in Table 1, compounds 2b–8
exhibited comparable tuberculostatic activity against sensitive
(Spec. 192) and resistant (Spec. 210) strains, as well as the standard
benzimidazole exhibited excellent tuberculostatic activity with
MIC values at 1.5–3.1 lg/mL. In the 1e case, we also observed no
difference in activity against sensitive and resistant strains.
All of the tested compounds showed tuberculostatic activity
lower than INH and RMP used as reference drugs with MIC values
H37Rv strain. MIC values ranging from 50 to100
l
g/mL indicated
at 0.5–1.1 lg/mL and 1.2–2.5 lg/mL respectively. However, they

140
K. Gobis et al. / Bioorg. Med. Chem. 20 (2012) 137–144
Table 1
presence of benzimidazole or other benzimidazole type system
in their molecules. The structures containing other nitrogen het-
erocyclic rings (1,2,4-triazole-5-thione, 1,3,4-oxadiazole-2-thi-
one) or hydrazinecarbothioyl group fused with cyclohexylethyl
moiety did not exhibit enhanced antituberculous activity in
vitro.
In vitro tuberculostatic and antibacterial activities of compounds 1–8a,b,c
Compds
MIC [lg/mL]
M. tuberculosis
P. acnes
B. linens
H37Rv
Spec. 192
Spec. 210
1a
1b
1c
1d
1e
1f
2a
2b
3a
3b
3c
3d
4a
4b
4c
4d
5
12.5
25
6.5
25
1.5
3.1
25
50
50
50
50
50
50
50
50
50
50
50
50
50
0.5
25
1.2
12.5
50
6.2
25
1.5
1.5
25
12.5
50
6.2
25
1.5
3.1
12.5
25
50
50
50
50
50
50
50
50
50
50
50
50
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
—
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
—
2.2.2. Antibacterial activity against anaerobic strains
Interestingly, all of the synthesized compounds exhibited poor
antibacterial activity against the anaerobic bacteria P. acnes and
B. linens, (Table 1), which indicates that their mechanism of action
may involve interference with aerobic functions of M. tuberculosis.
Indeed, this microorganism unusually has a high oxygen require-
ment for growth.
50
100
100
100
50
100
50
100
100
50
100
50
2.2.3. Cytotoxic activity
The results of cytotoxicity tests have indicated that compounds
1a–f represent a whole range of cytotoxic activity between 10
lg/
mL and 100 g/mL, from no effect (1d), to mildly cytostatic (1a,
l
1f), to 100% kill (1c) (Table 2). It appears that compounds 1a and 1f
have the best therapeutic potential, because of their high tuberculo-
static activity and no (or low) cytotoxicity to tested eukaryotic cells.
6
7
8
INH
PZA
RMP
100
0.5
25
1.1
>400
2.5
—
—
—
—
2.2.4. Effect of 1a on type I collagen output
Besides having substantial anti-tuberculotic activity and no
cytotoxic effect on fibroblasts, the compound 1a stimulated type I
1.2
a
Minimum inhibitory concentrations for bacterial strains were determined by
two-fold serial dilution method for microdilution plates and for mycobacterial
strains by two-fold classical test-tube method of successive dilution.
collagen output in a statistically-significant manner at 100 lg/mL
(Table 3). This stimulation was not due to the increase of cell
number, as measured by the MTT assay. Furthermore, it appears
to be selective to type I collagen, as measured by the sulforhoda-
mine B total insoluble protein stain. No other tested compounds
and concentrations stimulated type I collagen output. Benzimid-
azole 1a seems to be the most interesting among three tested
compounds and further investigations of its mechanism of collagen
I stimulation by itself and its derivatives is greatly warranted.
b
INH isoniazid; PZA pyrazinamide; RMP rifampicin.
M. tuberculosis H37Rv, Spec. 192, Spec. 210, P. acnes (ATCC 11827), B. linens
c
(ATCC 9174).
Table 2
Effect of compounds 1a-f on the proliferation of human fibroblasts at compounds
concentration 10 and 100
l
g/mL.a,b,c
Compds
Inhibition (%]
3. Conclusions
10
lg/mL
100 lg/mL
1a
1b
1c
1d
1e
1f
0
0
17
0
16
0
18
41
100
0
74
11
In conclusion, a series of novel 3-cyclohexylpropanoic acid
derivatives were successfully synthesized. All these new com-
pounds were confirmed by IR and 1H NMR spectra as well as ele-
mental analysis. Their tuberculostatic activity was evaluated
against M. tuberculosis H37Rv, Spec. 192 and Spec. 210 strains, using
the two-fold serial dilution MIC method. The results showed that
most of the synthesized derivatives exhibited moderate tuberculo-
static activity; however compounds 1a, 1c, 1e and 1f bearing benz-
imidazole or benzimidazole-like systems exhibited potent
tuberculostatic activity with MIC values ranging from 1.5 to
MAP 100
bFGF 15 ng/mL
lg/mL
138
160
a
b
c
Effect expressed as % of non-treated control.
MAP and bFGF were used as the positive controls.
Neonatal human dermal fibroblasts (ATCC PCS-201-010).
12.5 lg/mL. More importantly, active compounds 1a and 1f exhib-
ited no cytotoxic effect on the proliferation of neonatal human der-
mal fibroblasts in vitro. Compound 1a also stimulated type I
collagen output. These findings demonstrated that 2-cyclohexyl-
ethylbenzimidazoles are of biological significance, with the poten-
tial to become a new member of tuberculostatic agents.
Table 3
Stimulation of type I collagen output by compound 1a.a
Compound
Collagen concentration (ng/mL]
Stimulation (%)
Control
1a
11.3
18.8
100
166
MAP
bFGF
141.9
23.1
1255
204
4. Experimental
4.1. Chemistry
a
Stimulation of type I collagen output as compared with positive controls (bFGF
and MAP).
All materials and solvents were of analytical reagent grade.
Thin-layer chromatography was performed on Merck silica gel
60F254 plates and visualized with UV. The results of elemental anal-
yses (% C, H, N) for all of obtained compounds were in agreement
with calculated values within 0.3% range. 1H NMR spectra and
13C NMR spectra in CDCl3 or DMSO-d6 were recorded on Varian
Unity Plus (500 MHz) and Varian Gemini (200 MHz) instruments.
exhibited higher activity than PZA with MIC values at 25 to
>400 lg/mL, commonly used as second line chemotherapeutic.
Importantly, as opposed to clinically used drugs, these compounds
did not have a decreased activity towards resistant strain 210.
The obtained results indicate that tuberculostatic activity of
3-cyclohexylpropanoic acid derivatives was associated with the

K. Gobis et al. / Bioorg. Med. Chem. 20 (2012) 137–144
141
IR Spectra (KBr) were determined as KBr pellets of the solids on a
Satellite FT-IR spectrophotometer. Melting points were determined
on a BOETIUS apparatus and were uncorrected.
(
m
C–H), 1621, 1535 (d N–H), 1425, 1394 (
m
C@C), 1266 (d C–H), 930
(c
C–H), 685 cmꢁ1 1H NMR (200 MHz, DMSO-d6): d 0.92, (t, 2H,
;
CH2, J = 11 Hz), 1.11–1.21 (m, 4H, 2CH2), 1.51–1.75 (m, 7H, 6H
3CH2 and 1H CH), 2.83 (t, 2H, CH2, J = 7.5 Hz), 8.13 (d, 1H, pyridine,
J = 1.8 Hz), 8.31 (d, 1H, pyridine, J = 1.8 Hz), 11.08 (br s, 1H, NH)
ppm. Anal. Calcd for C14H18BrN3 (308.22): C, 54.56; H, 5.89; N,
13.63. Found: C, 54.62; H, 5.89; N, 13.63.
4.1.1. Synthesis of 3-cyclohexylpropanoic acid hydrazide
A
stirred solution of 3-cyclohexylpropanoic acid (10 mL,
64 mmol) and 3 mL of thionyl chloride (137 mmol) in 30 mL of
methanol was refluxed for 3 h. Then methanol was evaporated, res-
idue was neutralized with a saturated solution of sodium hydrogen
carbonate and extracted with dichloromethane (2 ꢀ 40 mL). After
drying, the solvent was evaporated, and 40 mL of methanol and
10 mL of 98% hydrazine hydrate were added to the oily residue.
The mixture was refluxed for 3 h. Then methanol was evaporated,
and the residue was treated twice with 30 mL of benzene, which
in each case was evaporated. The oily residue crystallized when
cooled on ice. The step of recrystallization from methanol yielded
10 g (90%) of hydrazide, which was obtained as colourless crystals
melting at 92 °C, in agreement with literature data.24
4.1.2.4. 2-(2-Cyclohexylethyl)-3H-imidazo[4,5-c]pyridine dihy-
drochloride (1d).
In the case of compound 1d obtained by
method B, the water solution was extracted with dichloromethane
(3 ꢀ 8 mL). Organic fractions were collected, dried with MgSO4 and
evaporated. The crude product was diluted with 10 mL of metha-
nol and treated with 10 mL of dry diethyl ether saturated with
hydrochloride. The precipitate was filtered, dried and recrystalized.
Compound 1d in dihydrochloride form was obtained as white solid
(after crystallization from the methanol/ethyl ether mixture 1:1) in
36% yield (1 g), mp 148–150 °C. IR (KBr): 3024, 2920, 2849 (
2572 ( C@C), 1405 (d C–H), 1226, 801
+N–H), 1639, 1497, 1473 (
(c
C–H), 626 cmꢁ1; 1H NMR for free amine (500 MHz, DMSO-d6): d
1.02–1.08 (m, 2H CH2) 1.19–1.44 (m, 4H, 2CH2), 1.69–1.90 (m, 7H,
6H 3CH2 and 1H CH), 3.24 (t, 2H, CH2, J = 8.0 Hz), 8.27 (d, 1H, pyr-
idine, J = 6.3 Hz), 8.72 (d, 1H, pyridine, J = 6.3 Hz), 9.40 (s, 1H, pyr-
idine), 11.18 (br s, 1H, NH) ppm. Anal. Calcd for C14H19N3 ꢀ 2HCl
(302.24): C, 55.63; H, 7.00; N, 13.90. Found: C, 55.68; H, 7.01; N,
13.87.
m C–H),
m
m
4.1.2. General procedure for the synthesis of arenoimidazoles
(1a–f)
Method A: 3-Cyclohexylpropanoic acid (15 mmol), appropriate
diamine (10 mmol) and PPA (5 mL) were heated and stirred at
180–200 °C for 5 h, then the mixture was allowed to cool to ambi-
ent temperature and poured into cold water (50 mL). The mixture
was neutralized with a saturated solution of sodium hydrogen car-
bonate and the resulting precipitate was filtered off, washed sev-
eral times with water and recrystallized from a methanol/water
mixture (1:1) with the addition of activated carbon.
4.1.2.5.
(1e). This compound was obtained by method A as beige solid
in 87% yield (2.42 g), mp 157–158 °C. IR (KBr) 2923, 2851 ( C–
H), 1564 (d N–H), 1449, 1384 ( C@C), 1087, 1004, 809, 754 (
C–H), 523 cmꢁ1 1H NMR (200 MHz, DMSO-d6): d 0.96 (t, 2H,
CH2, J = 12 Hz), 1.13–1.44 (m, 4H, 2CH2), 1.62–1.80 (m, 7H, 6H
CH2 and 1H CH), 2.93 (t, 2H, CH2, J = 7.8 Hz), 7.43 (t, 1H, ArH,
J = 7.2 Hz), 7.57 (t, 1H, ArH, J = 7.1 Hz), 7.96 (d, 1H, ArH,
J = 8.1 Hz), 8.36 (d, 1H, ArH, J = 7.9 Hz), 10.46 (br s, 1H, NH) ppm.
Anal. Calcd for C19H22N2 (278.39): C, 81.97; H, 7.97; N, 10.06.
Found: C, 82.11; H, 7.99; N, 10.04.
2-(2-Cyclohexylethyl)-1H-naphto[2,3-d]imidazole
Method B: 3-Cyclohexylpropanoic acid (2 mL, 12 mmol) was
m
added to
a
stirred mixture of appropriate diaminopyridine
m
c
(1.09 g, 10 mmol) and 2 mL of diglyme. The mixture was refluxed
for 3 h. After cooling, 20 g of ice was added. The precipitate was fil-
tered and purified. The hot methanol solution of crude product
with the addition of activated carbon was filtered and cooled. Then
the product was precipitated by the water addition, filtered off,
dried and recrystallized again.
;
4.1.2.1.
imidazole (1a).
pale yellow crystals in 84% yield (2.6 g), mp 190–192 °C. IR (KBr):
3095, 3015, 2925, 2849 ( C–H), 1517 ( NO2), 1462 ( C@C),
NO2), 1279 (d C–H), 898 ( C–H), 671 ( C–Cl) cm
6-Chloro-2-(2-cyclohexylethyl)-4-nitro-1H-benzo[d]
Compound 1a was obtained by method A as
4.1.2.6. 2-(2-Cyclohexylethyl)-1H-imidazo[4,5-b]phenazine
(1f). Compound 1f was obtained by method A as yellow solid
in 90% yield (2.96 g), mp >270 °C (decomp.). IR (KBr) 3085, 2921,
m
m
m
1402, 1339 (
m
c
m
2849 (
m
C–H), 1563, 1535 (d N–H), 1425 (
m C@C), 1310, 1220,
ꢁ1
;
1H NMR (200 MHz, CDCl3): d 0.99 (t, 2H, CH2, J = 12 Hz), 1.11–
752 (
c
C–H) cmꢁ1 1H NMR (200 MHz, CDCl3): 0.87 (t, 2H, CH2,
;
1.41 (m, 4H, 2CH2), 1.64–1.85 (m, 7H, 6H 3CH2 and 1H CH), 3.01
(t, 2H, CH2, J = 7.5 Hz), 7.99 (m, 1H, Ph), 8.10 (m, 1H, Ph), 10.35
(br s, 1H, NH) ppm; 13C NMR (200 MHz, CDCl3): d 26.2, 26.9,
27.3, 33.5, 35.7, 37.9, 119.1, 121.8, 126.8, 127.6, 132.9, 147.3,
160.0 ppm. Anal. Calcd for C15H18ClN3O2 (307.78): C, 58.54; H,
5.89; N, 13.65. Found: C, 58.48; H, 5.88; N, 13.64.
J = 12 Hz), 1.01–1.38 (m, 4H, 2CH2), 1.58–1.70 (m, 5H, 4H 2CH2
and 1H CH), 1.76–1.87 (m, 2H, CH2), 3.02 (t, 2H, CH2, J = 7.8 Hz),
7.71–7.76 (m, 2H, ArH), 8.15–8.20 (m, 2H, ArH), 8.34 (s, 2H, ArH),
10.52 (br s, 1H, NH) ppm; 13C NMR (200 MHz, CDCl3): d 26.5,
26.8, 28.0, 33.4, 35.5, 37.9, 129.5, 130.1, 140.9, 142.8, 165.1 ppm.
Anal. Calcd for C21H22N4 (330.43): C, 76.33; H, 6.71; N, 16.96.
Found: C, 76.42; H, 6.72; N, 16.95.
4.1.2.2.
2-(2-Cyclohexylethyl)-3H-imidazo[4,5-b]pyridine
(1b). Compound 1b was obtained by method B as colourless crys-
tals (after crystallization from toluene) in 94% yield (3 g), mp 139–
4.1.3. Synthesis of 5-(2-cyclohexylethyl)-1,3,4-oxadiazole-2(3H)-
thione (2a)
140 °C. IR (KBr) 3250 (
1569 (d N–H), 1447 (
591 cmꢁ1 1H NMR (200 MHz, CDCl3):
J = 12 Hz), 1.15–1.32 (m, 4H, 2CH2), 1.55–1.74 (m, 7H, 6H 3CH2
and 1H CH), 2.33 (t, 2H, CH2, J = 7.7 Hz), 6.54–6.60 (m, 1H, pyri-
dine), 6.89–6.93 (m, 1H, pyridine), 7.35–7.38 (m, 1H, pyridine),
11.20 (br s, 1H, NH) ppm. Anal. Calcd for C14H19N3 (229.32): C,
73.33; H, 8.35; N, 18.32. Found: C, 73.45; H, 8.33; N, 18.35.
m
m
N–H), 3033, 2922, 2851 (
C@C), 1398 (d C–H), 889, 763 (
0.92 (t, 2H, CH2,
m
C–H), 1643,
3-Cyclohexylpropanoic acid hydrazide (0.85 g, 5 mmol) was
dissolved in a solution of 1 g (17 mmol) of KOH in 3 mL of water
and 20 mL of ethanol. Then 1 mL (17 mmol) of carbon disulfide
was added and the mixture was refluxed for 4 h. After ethanol
evaporation, 10 mL of water was added and the solution was acid-
ified with concentrated hydrochloric acid. The precipitate was fil-
tered off, dried and recrystallized from cyclohexane/petroleum
ether mixture (1:1) giving colourless needles in 70% yield
c
C–H),
;
d
(0.74 g), mp 81–82 °C. IR (KBr): 3100 (
H), 1616, 1527 (d N–H), 1451 (d C–H), 1191 (
C–H), 758, 667 (
N–H) cmꢁ1 1H NMR (200 MHz, CDCl3): d 0.94
(t, 2H, CH2, J = 11 Hz), 1.03–1.39 (m, 4H, 2CH2), 1.56–1.75 (m, 7H,
m
N–H), 2925, 2850 (
m C–
4.1.2.3. 6-Bromo-2-(2-cyclohexylethyl)-3H-imidazol[4,5-b]pyri-
c
C–O), 975, 956 (
c
dine (1c).
Compound 1c was obtained by method A as white
c
;
solid in 82% yield (2.5 g), mp 229–231 °C. IR (KBr) 3089, 2921, 2850

142
K. Gobis et al. / Bioorg. Med. Chem. 20 (2012) 137–144
6 H 3CH2 and 1H CH), 2.72 (t, 2H, CH2, J = 7.7 Hz), 11.60 (br s, 1H,
NH) ppm. Anal. Calcd for C10H16N2OS (212.31): C, 56.57; H, 7.60; N,
13.19. Found: C, 56.62; H, 7.61; N, 13.21.
(311.49): C, 61.69; H, 9.38; N, 13.49. Found: C, 61.73; H, 9.40; N,
13.50.
4.1.5.4.
zinecarbothioamide (3d).
tained as colourless needles in 88% yield, mp 160–161 °C after
crystallization from dioxane. IR (KBr): 3363, 3243 ( N–H), 2923,
2852 ( C–H), 1681 ( C@O), 1524, 1491 ( C@C), 1088, 1015,
830 (
C–H), 725 (C–Cl) cmꢁ1 1H NMR (200 MHz, CDCl3): d 0.83
(t, 2H, CH2, J = 11 Hz), 1.14–1.30 (m, 4H, 2CH2), 1.49–1.69 (m, 7H,
6H 3CH2 and 1H CH), 2.37 (t, 2H, CH2, J = 7.4 Hz), 7.29 (d, 2H, Ph,
J = 8.8 Hz), 7.50 (d, 2H, Ph, J = 8.8 Hz), 9.17 (br s, 1H, NH), 10.15
(br s, 1H, NH), 10.50 (br s, 1H, NH) ppm. Anal. Calcd for
N-(4-Chlorophenyl)-2-(3-cyclohexylpropanoyl)hydra-
4.1.4. Synthesis of 3-(2-cyclohexylethyl)-1H-1,2,4-triazole-
5(4H)-thione (2b)
Compound 3d (1.5 g) was ob-
3-Cyclohexylpropanoic acid hydrazide (1.7 g, 10 mmol) and
ammonium thiocyanate (1.9 g, 25 mmol) were heated in a metal
bath at 170–180 °C for 0.5 h. After cooling to ambient temperature,
25 mL of 10% NaOH aqueous solution was added and the mixture
was refluxed for 2 h. Then the mixture was cooled and acidified
with acetic acid. The precipitate was filtered off and recrystallized
from ethanol giving 1.6 g (75%) of colourless prisms, mp 233–
m
m
m
m
c
;
234 °C. IR (KBr): 3115 (
H), 1480 (d C–H), 1026 (
cmꢁ1 1H NMR (200 MHz, DMSO-d6):
m
N–H), 2922, 2850 (
N–N), 986 ( C–H), 798, 669 (
0.88 (t, 2H, CH2,
m
C–H), 1604 (d N–
C16H22ClN3OS (339.88): C, 56.54; H, 6.52; N, 12.36. Found: C,
56.48; H, 6.53; N, 12.37.
m
c
c
N–H)
;
d
J = 11 Hz), 1.05–1.27 (4H, 2CH2), 1.50–1.74 (m, 7H, 3CH2 and 1H
CH), 2.47–2.55 (m, 2H, CH2), 13.11–13.25 (br s, 2H, 2NH) ppm.
Anal. Calcd for C10H17N3S (211.33): C, 56.83; H, 8.11; N, 19.88.
Found: C, 56.92; H, 8.13; N, 19.88.
4.1.6. General procedure for the synthesis of 1,2,4-triazole-3-
thiones (4a–d)
Appropriate hydrazinecarbothioamide 3a–d (2 mmol) was re-
fluxed in 20 mL of 10% NaOH aqueous solution for 2 h. The hot
mixture was filtered, cooled and acidified with concentrated
hydrochloric acid to the precipitation of 1,2,4-triazole-3-thione.
The crude product was filtered and recrystalized from ethanol/
water mixture (1:1).
4.1.5. General procedure for the synthesis of
hydrazinecarbothioamides (3a–d)
3-Cyclohexylpropanoic acid hydrazide (5 mmol) was refluxed
in 5 mL of methanol with addition of stechiometric quantity
(5 mmol) of appropriate isothiocyanate (methyl, allyl, cyclohexyl,
4-chlorophenyl). Then the mixture was cooled, precipitate was fil-
tered off and recrystallized from suitable solvent.
4.1.7.1.
4H)-thione (4a).
ourless prisms in 89% yield, mp 171–172 °C. IR (KBr): 3113, 3055
N–H), 2922, 2849 ( C–H), 1576 (d N–H), 1499, 1450 (d C–H),
1343, 1083, 968 ( C–H), 758 (
N–H) cmꢁ1 1H NMR (200 MHz,
3-(2-Cyclohexylethyl)-4-methyl-1H-1,2,4-triazole-5(
Compound 4a (0.4 g) was obtained as col-
(m
m
4.1.5.1. 2-(3-Cyclohexylpropanoyl)-N-methylhydrazinecarboth
c
c
;
ioamide (3a).
Compound 3a (0.92 g) was obtained as white
CDCl3): d 0.96 (t, 2H, CH2, J = 11 Hz), 1.12–1.42 (m, 4H, 2CH2),
1.55–1.76 (m, 7H, 6H 3CH2 and 1H CH), 2.65 (t, 2H, CH2,
J = 8.1 Hz), 3.53 (s, 3H, NCH3), 11.80 (br s, 1H, NH) ppm. Anal. Calcd
for C11H19N3S (225.35): C, 58.63; H, 8.50; N, 18.65. Found: C, 58.69;
H, 8.48; N, 18.67
solid in 77% yield, mp 173–174 °C after crystallization from meth-
anol. IR (KBr): 3285, 3138 (
C@O), 1564 (d N–H), 1447 (d C–H), 1273, 1220 (
N–N), 574 (
N–H) cmꢁ1
m
N–H), 2926, 2850 (
m
m
C–H), 1695 (
C–N), 1043 (
m
m
c
;
1H NMR (200 MHz, DMSO-d6): d 0.87
(t, 2H, CH2, J = 11 Hz), 1.15–1.26 (m, 4H, 2CH2), 1.35–1.46 (m, 2H,
CH2), 1.58–1.74 (m, 5H, 2CH2 and 1H CH), 2.16 (t, 2H, CH2), 2.84
(s, 3H, NCH3), 7.80 (s, 1H, NH), 9.11 (s, 1H, NH), 9.58 (s, 1H, NH)
ppm; 13C NMR (200 MHz, DMSO-d6): d 26.0, 26.4, 31.1, 32.2,
4.1.7.2.
thione (4b).
lid in 98% yield, mp 120–171 °C. IR (KBr): 3098, 3052 (
2923, 2849 ( C–H), 1573 (d N–H), 1501, 1445 (d C–H), 1366,
1273 C–N), 917 C–H), 787
N–H) cmꢁ1 1H NMR
(200 MHz, CDCl3): d 0.88 (t, 2H, CH2, J = 11 Hz), 1.15–1.37 (m, 4H,
2CH2), 1.56–1.74 (m, 7H, 6H 3CH2 and 1H CH), 2.62 (t, 2H, CH2,
J = 7.8 Hz), 4.65 (d, 2H, CH2, J = 5.3 Hz), 5.20 (dd, 2H, CH2,
J1 = 17 Hz, J2 = 11 Hz), 5.79–5.98 (m, 1H, CH), 11.70 (br s, 1H, NH)
ppm. Anal. Calcd for C13H21N3S (251.39): C, 62.11; H, 8.42; N,
16.72. Found: C, 62.18; H, 8.42; N, 16.70.
4-Allyl-3-(2-cyclohexylethyl)-1H-1,2,4-triazole-5(4H)-
Compound 4b (0.49 g) was obtained as white so-
N–H),
m
32.9, 37.0, 172.5, 182.5 ppm. Anal. Calcd for
C
11H21N3OS
m
(243.37): C, 54.29; H, 8.70; N, 17.27. Found: C, 54.13; H, 8.69; N,
17.28.
(m
(c
(
c
;
4.1.5.2. N-Allyl-2-(3-cyclohexylpropanoyl)hydrazinecarbothioa-
mide (3b).
Compound 3b (1.2 g) was obtained as colourless
crystals in 89% yield, mp 132–134 °C after crystallization from
methanol/water mixture (1:1). IR (KBr): 3275, 3182 (
m
m
N–H),
C–N),
2922, 2851 (
m
C–H), 1689 (
m
C@O), 1544 (d N–H), 1196 (
969 ( C–H), 621 (
c
c
N–H) cmꢁ1
;
1H NMR (200 MHz, CDCl3): d
4.1.7.3. 4-Cyclohexyl-3-(2-cyclohexyl)-1H-1,2,4-triazole-5(4H)-
0.91 (t, 2H, CH2, J = 11 Hz), 1.09–1.32 (m, 4H, 2CH2), 1.48–1.72
(m, 7H, 3CH2 and 1H CH), 2.32 (t, 2H, CH2, J = 7.4 Hz), 4.22 (d,
2H, CH2, J = 5 Hz), 5.15–5.28 (m, 2H, CH2), 5.79–5.98 (m, 1H, CH),
8.15 (br s, 1H, NH), 9.00 (br s, 1H, NH), 9.60 (br s, 1H, NH) ppm.
Anal. Calcd for C13H23N3OS (269.41): C, 57.96; H, 8.61; N, 15.60.
Found: C, 57.85; H, 8.60; N, 15.58.
thione (4c).
needles in 84% yield, mp 157–158 °C. IR (KBr): 3125, 3046 (
2923, 2851 ( C–H), 1562 (d N–H), 1502, 1448 (d C–H), 1290 (
N), 983 ( C–H), 739 (
N–H) cmꢁ1 1H NMR (200 MHz, CDCl3): d
0.97 (t, 2H, CH2, J = 11 Hz), 1.10–1.40 (m, 21H, 20H 10CH2 and
1H CH), 2.72 (t, 2H, CH2, J = 7.8 Hz), 4.35–4.65 (m, 1H, CH), 11.80
(br s, 1H, NH) ppm. Anal. Calcd for C16H27N3S (293.47): C, 65.48;
H, 9.27; N, 14.32. Found: C, 65.52; H, 9.29; N, 14.33.
Compound 4c (0.47 g) was obtained as colourless
N–H),
C–
m
m
m
c
c
;
4.1.5.3. N-Cyclohexyl-2-(3-cyclohexylpropanoyl)hydrazinecarb
othioamide (3c).
solid in 71% yield, mp 158–160 °C after crystallization from diox-
ane/water mixture (1:1). IR (KBr): 3295, 3189 ( N–H), 2924,
2852 ( C–H), 1685 ( C@O), 1556 (d N–H), 1450 (d C–H), 1212
C–N), 985 ( C–H), 586 (
N–H) cmꢁ1 1H NMR (200 MHz,
CDCl3): d 0.92 (t, 2H, CH2, J = 11 Hz), 1.15–1.80 (m, 19H, 18H
9CH2 and 1H CH), 1.99–2.05 (m, 2H, CH2), 2.32 (t, 2H, CH2,
J = 7.6 Hz), 3.98–4.28 (m, 1H, CH), 7.00 (br s, 1H, NH), 8.1 (br s,
1H, NH), 9.20 (br s, 1H, NH) ppm. Anal. Calcd for C16H29N3OS
Compound 3c (1.1 g) was obtained as white
4.1.7.4. 4-(4-Chlorophenyl)-3-(2-cyclohexylethyl)-1H-1,2,4-tria-
m
zole-5(4H)-thione (4d).
as white solid in 80% yield, mp 210–212 °C. IR (KBr): 3089 (
2925, 2848 ( C–H), 1570 (d N–H), 1495, 1487 ( C@C), 1331, 1092,
1008, 840 ( C–H), 728 (
C–Cl) cmꢁ1; 1H NMR (500 MHz, CDCl3): d
Compound 4d (0.51 g) was obtained
m
m
m
N–H),
(m
c
c
;
m
m
c
m
0.82 (t, 2H, CH2, J = 12 Hz), 1.07–1.29 (m, 4H, 2CH2), 1.47–1.51 (m,
2H, CH2), 1.59–1.68 (m, 5H, 4H 2CH2 and 1H CH), 2.50 (t, 2H, CH2,
J = 7.8 Hz), 7.32 (d, 2H, Ph, J = 7.8 Hz), 7.57 (d, 2H, Ph, J = 8.3 Hz),

K. Gobis et al. / Bioorg. Med. Chem. 20 (2012) 137–144
143
11.80 (br s, 1H, NH) ppm. Anal. Calcd for C16H20ClN3S (321.87): C,
59.70; H, 6.26; N, 13.06. Found: C, 59.54; H, 6.25; N, 13.04.
(
m
C–N), 1121, 1105 cmꢁ1
;
1H NMR (200 MHz, CDCl3): d 0.95–
0.97 (m, 2H, CH2), 1.11 (s, 6H, 2CH3), 1.16–1.28 (m, 2H, CH2),
1.54–1.79 (m, 7H, 6H 3CH3 and 1H CH), 2.57 (t, 2H, CH2,
J = 8.3 Hz), 3.08 (s, 2H, CH2), 3.43 (s, 2H, CH2), 3.71 (s, 2H, CH2),
5.65 (br s, 1H, NH) ppm; 13C NMR (200 MHz, CDCl3): 22.9, 24.7,
26.7, 33.5, 34.9, 37.7, 51.4, 52.6, 150.2, 153.1 ppm. Anal. Calcd for
4.1.8. Synthesis of methyl 2-(3-cyclohexylpropanoyl)
hydrazinecarbodithioate (5)
3-Cyclohexylpropanoic acid hydrazide (1.7 g, 10 mmol) was
dissolved in 10 mL of ethanol. Then triethylamine (1.4 mL,
10 mmol), carbon disulfide (0.60 mL, 10 mmol) and methyl iodide
(0.62 mL, 10 mmol) were added. The mixture was stirred at room
temperature for 0.5 h. Then the mixture was cooled and precipitate
was filtered off, dried and recrystallized from toluene giving 1.5 g
(58%) of colourless crystals, mp 122–124 °C. IR (KBr) 3171, 3052
C
15H26N4 (262.39): C, 68.66; H, 9.99; N, 21.35. Found: C, 68.78;
H, 9.97; N, 21.33.
4.2. Biological assays
4.2.1. Tuberculostatic assay
(
m
N–H), 2970, 2919, 2849 (
m
C–H), 1688 (
m
C@O), 1546 (d N–H),
The synthesized compounds were examined in vitro for their
tuberculostatic activity against the M. tuberculosis H37Rv strain
and two ‘wild’ strains isolated from tuberculosis patients: one
(Spec. 210) resistant to p-aminosalicylic acid (PAS), isonicotinic
acid hydrazide (INH), etambutol (ETB) and rifampicine (RMP) and
the another (Spec. 192) fully sensitive to the administrated tuber-
culostatics. Investigations were performed by a classical test-tube
method of successive dilution in Youmans’ modification of the
Proskauer and Beck liquid medium containing 10% of bovine ser-
um.25,26 Bacterial suspensions were prepared from 14 days old cul-
tures of slowly growing strains and from 48 h old cultures of
saprophytic strains.27,28 Solutions of compounds in ethylene glycol
were tested. Stock solutions contained 10 mg of compounds in 1
millilitre. Dilutions (in geometric progression) were prepared in
Youmans’ medium. The medium containing no investigated sub-
stances and containing isoniazid (INH), pyrazinamide (PZA) or rif-
ampicin (RMP) as reference drugs were used for comparison.
Incubation was performed at a temperature of 37 °C. The MIC val-
ues were determined as minimum concentration inhibiting the
growth of tested tuberculous strains in relation to the probe with
no tested compound. The influence of the compound on the growth
of bacteria at a certain concentration, 3.1, 6.2, 12.5, 25, 50 and
1220 ( C–N), 1056, 618 (
m
c
N–H) cmꢁ1; 1H NMR (200 MHz, CDCl3):
d 0.87–1.82 (m, 12H 6CH2), 2.35 (t, 2H, CH2, J = 7.5 Hz), 2.63 (s, 3H,
SCH3), 3.15–3.24 (m, 1H, CH), 9.60 (br s, 1H, NH), 10.70 (br s, 1H,
NH) ppm. Anal. Calcd for C11H20N2OS2 (260.42): C, 50.73; H,
7.74; N, 10.76. Found: C, 50.67; H, 7.76; N, 10.76.
4.1.9. Synthesis of 3-cyclohexyl-N0-(1,3-dithiolan-2-
ylidene)propanehydrazide (6)
3-Cyclohexylpropanoic acid hydrazide (1.7 g, 10 mmol) was
dissolved in 10 mL of ethanol. Then triethylamine (2.8 mL,
20 mmol) and carbon disulfide (0.60 mL, 10 mmol) were added
and the mixture was stirred at room temperature for 0.5 h. After
that time 1,2-dibromoethane (0.86 mL, 10 mmol) was added and
the mixture was stirred for another 0.5 h. Then 20 g of ice was
added and the oily product precipitated, which crystallized when
frozen for few hours. The crude solid was filtered off, dried and
recrystallized from cyclohexane/petroleum ether mixture (1:1)
giving 2.5 g (93%) of bright solid, mp 91–92 °C. IR (KBr) 3188,
3017 (
1579, 1534 (d N–H), 1448 (d C–H), 1280 (
C–H) cmꢁ1 1H NMR (200 MHz, CDCl3): d 0.83 (t, 2H, CH2,
m
N–H), 2922, 2850 (
m
C–H), 1679 (
m C@OC@O), 1638,
m
C–N), 1121, 1033, 852
(c
;
J = 10 Hz), 1.12–1.32 (m, 4H, 2CH2), 1.65–1.75 (m, 7H, 6H 3CH2
and 1H CH), 2.55 (t, 2H, SCH2, J = 7.7 Hz), 3.42–3.52 (m, 2H,
SCH2), 3.61–3.69 (m, 2H, SCH2), 7.89 (br s, 1H, NH) ppm; 13C
NMR (200 MHz, CDCl3): d 26.8, 27.1, 30.7, 32.3, 33.6, 35.8, 37.8,
39.4, 154.4, 175.7 ppm. Anal. Calcd for C12H20N2OS2 (272.43): C,
52.90; H, 7.40; N, 10.28. Found: C, 52.81; H, 7.39; N, 10.27.
100 lg/mL, were evaluated.
4.2.2. Anaerobic bacteria assay
Compounds 1–8 immediately before use were dissolved in
100% DMSO at 5 mg/mL and further to 1 mg/mL in 10% DMSO.
Compounds were tested at serial dilutions in bacterial broth start-
ing at 100 lg/mL (final concentration). P. acnes (ATCC11827) was
4.1.10. Synthesis of dimethyl 3-cyclohexylpropanoylcarbonohy
drazonodithioate (7)
grown in thioglycolate nutrient broth (Hardy Diagnostics K29)
for 72 h at 33 °C, then inoculated at the density equivalent to 0.5
McFarland standard and incubated with the compounds for an-
other 72 h in an anaerobic environment. B. linens (ATCC9174) cul-
ture was started from an agar plate, grown in nutrient broth
(Hardy Diagnostics K243) for 24 h at 30 °C, then inoculated at the
density equivalent to 1 MsFarland standard and incubated with
tested compounds for another 24 h. At the end of the incubation
MIC was assessed optically as the lowest concentration of a com-
pound, which caused no bacteria growth.29 This optical assessment
was further confirmed by measuring the absorbance at 655 nm
with the BioRad 3550-UV microplate reader. MIC was defined as
at least 50% inhibition of the control’s (vehicle) OD (optical
density).
3-Cyclohexylpropanoic acid hydrazide (0.8 g, 5 mmol) was dis-
solved in 10 mL of methanol. Then triethylamine (1.4 mL,
10 mmol), carbon disulfide (0.30 mL, 5 mmol) and methyl iodide
(0.62 mL, 10 mmol) were added. The mixture was stirred at room
temperature for 2 h and 40 g of ice was added. The crude product
precipitate was filtered off, dried and recrystallized from petro-
leum ether giving 1.2 g (89%) of white solid, mp 45–46 °C. IR
(KBr) 3166, 3073 (
m
N–H), 2992, 2915, 2849 (
m
C–H), 1664 (
m
C@O), 1450 (d C–H), 1375, 1102, 942 (
c
C–H), 587 (
c
N–H) cm-1;
1H NMR (500 MHz, CDCl3): d 0.89–0.97 (m, 2H, CH2), 1.12–1.32
(m, 4H, 2CH2), 1.57–1.76 (m, 7H, 6H 3CH2 and 1H CH), 2.45 (s,
3H, SCH3), 2.51 (s, 3H, SCH3), 2.64 (t, 2H, CH2, J = 8.3 Hz), 8.91 (br
s, 1H, NH) ppm. Anal. Calcd for C12H22N2OS2 (274.45): C, 52.52;
H, 8.08; N, 10.21. Found: C, 52.46; H, 8.06; N, 10.22.
4.2.3. Cytotoxicity and type I collagen quantification assay
Compounds were dissolved in DMSO at 20 mg/mL and tested
4.1.11. Synthesis of 3-(2-cyclohexylethyl)-6,6-dimethyl-5,6,7,8-
tetrahydro-[1,2,4] triazolo[4,3-a]pyrimidine (8)
at 100
(MAP, 100
l
g/ml and 10
lg/mL. Magnesium ascorbyl phosphate
l
g/mL and basic fibroblast growth factor (bFGF,
Compound 7 (1.7 g, 6.4 mmol) was refluxed with 1,3-diamino-
2,2-dimethylpropane (1.5 mL, 12.7 mmol) for 1.5 h. Then 20 g of
ice was added and product precipitated. The crude product was fil-
tered off, dried and recrystallized from dioxane giving 0.6 g (36%)
15 ng/mL) were used as positive controls. Neonatal human der-
mal fibroblasts (ATCC PCS-201–010) were plated in high glucose
DMEM with 5% cosmic calf serum (Hyclone, Salt Lake City, UT).
Cells were seeded at a density of 3 ꢀ 103 cells/well in 96 well
plates and were exposed to compounds tested for 72 h. The fibro-
blast number and total insoluble proteins were quantified using
of white solid, mp 156–158 °C. IR (KBr) 3241, 3168, 3108 (
m N–
H), 2923, 2849 ( C–H), 1630, 1546 (d N–H), 1447 (d C–H), 1299
m

144
K. Gobis et al. / Bioorg. Med. Chem. 20 (2012) 137–144
the MTT and sulforhodamine B methods as described earlier.30,31
Briefly, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli-
um bromide, Sigma, St. Louis, MO) was added to cell cultures at
the end of the 72 h incubation period and incubation was pursued
for additional 2.5 h. The culture media were then discarded and
the intracellular MTT reduction product formazan was solubilised
in isopropanol. Alternatively, at the end of the experiment cells
were fixed in trichloroacetic acid and stained with protein-
binding dye sulforhodamine B (Sigma). The colorimetric signals
proportional to cell numbers were measured with the BioRad
microplate spectrophotometer 3550-UV at 550 nm (MTT) or
570 nm (sulforhodamine B).
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At the end of the incubation with the tested compounds, cell
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antibodies, followed by streptavidin/avidin-HRP conjugate and
ABTS, according to standard enzyme-linked immunosorbent assay
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This work was supported in part by the National Science Centre,
Cracow, Poland (grant no. 2011/01/B/NZ4/01187).
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