Synthesis of Disulfide-Bridged N-Phenyl-N′-(alkyl/aryl/heteroaryl)urea Derivatives and Evaluation of Their Antimicrobial Activities

Article abstract of DOI:10.1002/cbdv.201900461

The discoveryof new antimicrobial agents is extremely needed to overcome multidrug-resistant bacterial and tuberculosis infections. In the present study, eight novel substituted urea derivatives (10a–10h) containing disulfide bond were designed, synthesized and screened for their in vitro antimicrobial activities on standard strains of Gram-positive and Gram-negative bacteria as well as on Mycobacterium tuberculosis. According to the obtained results, antibacterial effects of the compounds were found to be considerably better than their antimycobacterial activities along with their weak cytotoxic effects. Molecular docking studies were performed to gain insights into the antibacterial activity mechanism of the synthesized compounds. The interactions and the orientation of compound 10a (1,1′-((disulfanediylbis(methylene))bis(2,1-phenylene))bis(3-phenylurea)) were found to be highly similar to the original ligand within the binding pocket E. faecalis β-ketoacyl acyl carrier protein synthase III (FabH). Finally, a theoretical study was established to predict the physicochemical properties of the compounds.

Full text of DOI:10.1002/cbdv.201900461

Title: Synthesis of Disulfide-Bridged N-Phenyl-N’-(Alkyl/Aryl/
Heteroaryl)Urea Derivatives and Evaluation of Their Antimicrobial
Activities
Authors: Şengül Dilem Doğan, Sümeyye Buran, Miyase Gözde
Gündüz, Ceren Özkul, Vagolu Siva Krishna, and Dharmarajan
Sriram
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To be cited as: Chem. Biodiversity 10.1002/cbdv.201900461
Link to VoR: http://dx.doi.org/10.1002/cbdv.201900461

10.1002/cbdv.201900461
Chemistry & Biodiversity
Synthesis of Disulfide-Bridged N-Phenyl-N’-(Alkyl/Aryl/Heteroaryl)Urea Derivatives
and Evaluation of Their Antimicrobial Activities
Şengül Dilem Doğana, Sümeyye Burana, Miyase Gözde Gündüzb*, Ceren Özkulc,
Vagolu Siva Krishnad, Dharmarajan Sriramd
aDepartment of Basic Sciences, Faculty of Pharmacy, Erciyes University, Kayseri, Turkey
bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Hacettepe University,
Sıhhiye, 06100 Ankara, Turkey, E-mail address:miyasegunduz@yahoo.com
cDepartment of Pharmaceutical Microbiology, Faculty of Pharmacy, Hacettepe University,
Sıhhiye, 06100 Ankara, Turkey
dDepartment of Pharmacy, Birla Institute of Technology and Science-Pilani, Hyderabad, India
The recognition of new antimicrobial agents is extremely needed to overcome multidrug-
resistant bacterial and tuberculosis infections. In the present study, eight novel substituted
urea derivatives containing disulfide bond (10a-h) were designed, synthesized and screened
for their in vitro antimicrobial activities on standard strains of Gram-positive and Gram-
negative bacteria as well as on Mycobacterium tuberculosis. According to the obtained results,
antibacterial effects of the compounds were found to be considerably better than their
antimycobacterial activities along with their weak cytotoxic effects. Molecular docking
studies were performed to gain insights into the antibacterial activity mechanism of the
synthesized compounds. The interactions and the orientation of compound 10a were found to
be highly similar to the original ligand within the binding pocket E. faecalis β-ketoacyl acyl
carrier protein synthase III (FabH). Finally, a theoretical study was established to predict the
physicochemical properties of the compounds.
Keywords: Drug design, synthesis; dimerization; antibacterial; urea
Introduction
Bacterial infections, which are caused by mycobacterial and Gram-positive/Gram-negative
pathogens, still remain one of the most severe health-threating problems worldwide.[1]
Infectious diseases take place among five leading causes of life lost together with
cardiovascular diseases and cancer.[2] Tuberculosis (TB), caused by Mycobacterium
tuberculosis, is ranked in the top ten causes of death and the leading cause of an infectious
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disease. The World Health Organization (WHO) estimated that ten million people developed
TB disease in 2017.[3] The rising antimicrobial resistance to the current drugs makes the
treatment of infectious diseases complicated. Therefore, continuous efforts focus on
developing new molecules with diverse structures to control different types of infections
successfully.[4]
Urea functionality is one of the most important pharmacophores integrated into chemistry and
life sciences. Thus, a remarkable amount of studies focus on alkyl/aryl substituted urea
moieties for the rational design of new drug candidates.[5] Various urea-containing molecules
have been reported with diverse biological applications as antibacterial,[6] antitubercular,[7]
antifungal,[8] anti-inflammatory,[9] antiviral,[10] antidepressant,[11] and anticancer agents.[12,13]
Among them, various aryl/alkyl-substituted urea derivatives were reported with their
antimicrobial activities.[14–19] (Figure 1)
Figure 1. Some examples of substituted urea derivatives possessing antimicrobial activities
Numerous disulfide bonds are present in antimicrobial peptides isolated from both plants and
animals.[20,21] These peptides have been shown to possess protective effects against
antibacterial infections in the living hosts.[22] Disulfide bond containing analogs of
vancomycin (Figure 2) and teixobactin,[23,24] well-known antibacterial compounds, have been
reported with improved antibacterial activities. Consequently, combining the pharmacophores
in the same molecule by using a disulfide bond is a rational approach to obtain novel
antimicrobial agents.
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10.1002/cbdv.201900461
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Figure 2. Vancomycin disulfide dimer possessing antibacterial activity
In light of these considerations, we aimed to synthesize disulfide-bridged N-phenyl-N’-
(alkyl/aryl/heteroaryl)urea derivatives (Figure 3) and evaluate their antimicrobial activities on
different Gram-positive and Gram-negative bacteria as well as on Mycobacterium
tuberculosis.
Figure 3. The design strategy of 10a-h
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Results and Discussion
Chemistry
To obtain the target novel disulfide-bridged N-phenyl-N′-(alkyl/aryl/heteroaryl) urea
derivatives, 10a-h, 2,2'-(disulfanediylbis(methylene))dibenzoic acid (5) was employed as the
key compound. Commercially available methyl 2-methylbenzoate (1) was brominated in the
presence of NBS/AIBN at reflux temperature to give the product 2. 2-Bromomethyl
substituted ester 2 treated with thiourea resulted in the formation of thiouronium salt 3. Basic
hydrolysis of thiouronium salt 3 gave carboxylic acid derivative 4 with high yield. The
proposed structure of the obtained product 4 was evaluated by NMR spectral data. After the
synthesis of acid 4, the attempts to synthesize urea compound 8 were failed. Thiophthalide (7)
was formed with the azidination reaction of 4 with ethyl chloroformate in the presence of
triethylamine following by the addition of a solution of NaN3 in water, unexpectedly (Scheme
1). In order to synthesize bisulfide bond containing urea derivative 10a-h, thiol group
oxidative coupling reaction takes place before the azidination reaction. Oxidative coupling
reaction of thiol group in 4 took place with air within one or two weeks in open vessel,
without any heavy metal ion or a base, giving the disulfide containing compound 5 (Scheme
1). The high-resolution mass spectrum data obviously indicated the formation of product 5,
obtained from two moles of 4. The experimental value of compound 5 [M-H]-= 333.0245
(C16H13O4S2) was in an excellent agreement with the theoretical value, [M-H]-=333.0275.
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Scheme 1. Synthetic scheme for the preparation of the key compound, 2,2'-
(disulfanediylbis(methylene))dibenzoic acid (5) .
In order to synthesize target urea derivatives 10a-h, azidination reaction was performed on the
2,2'-(disulfanediylbis(methylene))dibenzoic acid (5). In the azidination reaction, [25,26] acid 5
was initially reacted with ethyl chloroformate in presence of triethylamine, then a solution of
NaN3 in water was added to synthesize azide 9 in 81% yield (Scheme 2). The azide function
was convenient to generate the corresponding isocyanate via Curtius rearrangement.[27,28]
Hence, acyl azide 9 was refluxed in benzene and this gave rise to the formation of the
isocyanate 11. It was not isolated and reacted with various amines to achieve the target urea
derivatives 10a-h (Scheme 2).
The structures of the title compounds 10a-h were evaluated by 1H‐ and 13C‐NMR, IR, HRMS.
In the 1H‐NMR spectra, the singlet signal for the CH2 group of 10a-h was observed at 3.5-4.0
ppm. The signals for -NH groups were observed at 8.0-12.0 ppm in all synthesized urea
derivatives 10a-h.
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10.1002/cbdv.201900461
Chemistry & Biodiversity
Scheme 2. General synthesis of urea derivatives 10a-h.
Antibacterial activity evaluation
The in vitro antibacterial activities of the title compounds were evaluated against three Gram-
positive and two Gram-negative bacteria by measuring their minimum inhibitory
concentration (MIC) values. Gentamicin and piperacillin-tazobactam were used as positive
controls during the experiments. Table 1 reports the antibacterial activities of the designed
compounds against tested bacteria.
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Table 1. The minimum inhibitory concentration (µg/mL) of 10a-h against tested bacteria
Gram-positive bacteria Gram-negative bacteria
S. aureus
E. faecalis
MRSA
E. coli
P. aeruginosa
Compound
ATCC 29213 ATCC 29212 ATCC 43300 ATCC 25922 ATCC 27853
16
256
128
16
2
16
8
32
256
128
32
16
256
256
32
256
128
256
128
128
128
128
128
1
10a
10b
10c
2
10d
64
8
64
128
128
128
128
1
10e
32
4
32
10f
64
4
64
10g
128
1
4
64
10h
16
32
Gentamicin
Piperacillin-
tazobactam
0.5
1
NA
0.5
0.5
NA: Not applicable.
The title compounds possessed antimicrobial activity against all tested microorganisms with
MIC values ranging between 2 and 256 µg/mL. According to obtained results, the tested
compounds have high antimicrobial activity against E. faecalis which is a Gram-positive
bacterium present in the gastrointestinal tract and may cause enteric infections (MIC values
ranging between 2 and 16 µg/mL). All tested compounds have higher or equal MIC values
when compared to reference compound gentamicin against E. faecalis. The obtained results
clearly revealed that the test compounds (10a-h) had higher antimicrobial activity against
Gram-positive bacteria. Furthermore, three of the compounds (10a, 10d, and 10f) had an
activity on methicillin-resistant S. aureus (MRSA) with a MIC of 32 µg/mL as determined for
gentamicin.
When the obtained results are figured out in terms of chemical structure, the substitution of
the phenylurea moiety with either aryl or alkyl group did not mediate a significant change in
the antibacterial activity of the compounds. Introducing a substituent into the 4- position of
the phenyl ring in 10a did not enhance the activity, nevertheless, compound 10d carrying
chlorine atom possessed equipotent activity to 10a. Changing one of the phenyl rings in 10a
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10.1002/cbdv.201900461
Chemistry & Biodiversity
with other aromatic rings (pyridine and pyrimidine) did not cause an improvement in the
preferential activity, suggesting that this modification is not critical.
Antitubercular activity evaluation
The synthesized compounds were evaluated for their in vitro antitubercular activity against M.
tuberculosis H37Rv using the Microplate Alamar Blue Assay (MABA) method. Isoniazid,
rifampin, and ethambutol were employed as reference compounds. The antimycobacterial
activities of the compounds are provided as MIC values in Table 2.
Table 2. The antitubercular activity of compound 10a-h
Compound
10a
MIC (μg/mL)
>25
>25
10b
25
10c
>25
10d
>25
10e
>25
10f
>25
10g
>25
10h
0.05
0.1
Isoniazid
Rifampicin
Ethambutol
1.56
Among the tested compounds, only 10c exhibited moderate activity with MIC value of 25
μg/mL. The rest of the compounds were found to have MIC values higher than 25 μg/mL.
These data indicated that the compounds were not effective inhibitors of M. tuberculosis.
In vitro cytotoxicity screening
As the discovery of new antimicrobial agents with low cytotoxicity is of utmost importance,
the compounds were also screened for their cytotoxic effects on RAW 264.7 cells at 50
μg/mL concentration using MTT assay. The percentages of inhibition data for all compounds
are summarized in Table 3.
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Table 3. Cytotoxicity of the synthesized compounds
Compound
% Inhibition at 50
(µg/mL) against RAW
cells
16.25
9.86
10a
10b
10c
10d
10e
10f
21.74
22.08
13.90
24.61
22.81
19.42
10g
10h
According to the obtained values, all compounds were found to be non-toxic with <50%
inhibition.
Molecular Docking
In order to investigate the antibacterial activity mechanism of the compounds, compound 10a
was selected for molecular docking studies. 10a was found to be more active on Gram-
positive bacteria, especially on E. faecalis with MIC value of 2 µg/mL. E. faecalis is one of
the major causes of hospital-acquired infections, thus the development of new antibacterials
against this bacteria is of utmost importance due to the increased antimicrobial drug
resistance.[29] The bacterial fatty acid biosynthesis (fab) pathway is essential for cell growth
and viability, thus stands as an attractive target to obtain antibacterial activity. β-Ketoacyl
acyl carrier protein synthase III (KAS III) encoded by the fabH gene initiates fatty acid
synthesis in bacteria and is one of the key enzymes to overcome the antibiotic resistance
problem.[30] Particularly, molecules containing disulfide bonds were reported to inhibit
FabH,[31] therefore this situation prompted us to dock compound 10a into the active site of E.
faecalis FabH with the PDB code 3IL5.
Common interactions of superimposed co-crystallized ligand and the top-ranked docking pose
of 10a within the binding pocket of the enzyme are provided in Figure 4. It is clear that the
original ligand and 10a have a similar orientation in the active site and we surmise that
compound 10a is likely to show its antibacterial effect by inhibiting the mentioned enzyme
considering the high number of common interactions with the enzyme.
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Figure 4. Common interactions of the co-crystallized ligand (green) with compound 10a
(red) in the binding pocket of E. faecalis FabH. Color-coded pharmacophore features:
hydrogen bond acceptor (red vector), hydrophobic interaction (yellow sphere)
More closely focusing on the interactions of 10a; the binding conformation of 10a in the
active pocket of FabH, the pharmacophore features in 3D and 2D representations are provided
in Figure 5. β-Ketoacyl acyl carrier protein synthase III (KAS III) catalyzes the condensation
reaction between acyl-CoA and malonyl-ACP during fatty acid biosynthesis.[30] The active
site of FabH consists of the catalytic triad formed by the residues Cys117, His250 and Asn280
in which Cys117 is involved in acetyl transfer, while His250 and Asn280 play an active role
in decarboxylation.[32] Thus, it is important to note that 10a interacts with this catalytic triad
via forming three hydrogen bonds. The proposed binding mode of 10a involves several
hydrophobic contacts as well.
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Figure 5. Predicted binding mode of 10a in the active pocket of 3IL5. 3D- (A) and 2D- (B)
representations of the pharmacophores. Hydrophobic and aromatic interactions are
represented as yellow spheres and blue circle. Hydrogen bond acceptor and donor interactions
are represented as red and green arrows, respectively. The residues forming the catalytic triad
are represented as ball and stick models.
In silico prediction of physicochemical properties of 10a-h
Molecular property prediction is considered to be a useful tool to identify chemical leads in
the drug discovery process.[33] Here, we carried out some computational calculations, using
Molinspiration online software,[34] to obtain the physicochemical properties 10a-h. (Table 4).
Table 4. Calculated physicochemical properties data of 10a-h
Compound LogP[a] M.W.[b] HBA[c] HBD[d] TPSA[e]
nROTB[f]
6.84
6.76
6.96
8.18
7.26
7.33
5.04
4.04
514.68
604.67
574.73
583.57
526.77
502.75
516.65
518.63
6
12
8
4
4
4
4
4
4
4
4
82.25
173.19
100.72
82.25
9
11
11
9
10a
10b
10c
10d
10e
10f
6
6
82.25
9
6
82.25
15
9
8
108.03
133.82
10g
10h
10
9
[a]LogP: logarithm of n-octanol-water partition coefficient
[b]M.W: molecular weight
[c]HBA: number of hydrogen bond acceptors
[d]HBD: number of hydrogen bond donors
[e]TPSA: topological polar surface area
[f]NROTB: number of rotatable bonds
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Lipinski’s rule of five describes the main molecular descriptors which are used to predict the
cell permeability and oral bioavailability of the new drug candidate molecules.[35] Any drug
candidate must adhere to at least three of the following rules: M.W≤500 Da, logP≤5, HBA≤10,
HBD≤5. In addition to these parameters, we additionally calculated NROTB and TPSA which
are also considered to be important descriptors of oral bioavailability of drugs.[36] The TPSA
values for the most known drug molecules are below 140–150 Å2.[37] According to the
obtained data, most of the compounds showed a slight violation regarding their molecular
weights or logP values. Only 10b has additional violation due to its number of hydrogen bond
acceptors. In the present study, we combined repeating pharmacophores with disulfide bonds,
so it is expected that the compounds slightly exceed the accepted values of molecular weight.
Additionally, only 34.4 % of marketed antibacterial drugs have logP value in 0-5 range.[38]
Altogether, it can be concluded that compound 10a-h exhibit reasonable physicochemical
properties as antibacterial drug candidates.
3. Conclusion
In this study, we presented the synthesis of novel compounds containing disulfide bridged
repeating urea pharmacophores. The obtained compounds were screened for their
antimicrobial activities on various Gram-positive/Gram-negative bacteria and on
Mycobacterium tuberculosis. Some compounds turned out to be promising antibacterial
agents with a good safety profile in this series. The docking results identified FabH as the
possible target enzyme for the compounds to possess their antibacterial activities. Altogether,
our data show that combining two same pharmacophores with a disulfide bond may serve as a
good approach to achieve the antibacterial activity.
Experimental Section
Material and methods
All chemicals were purchased from commercial sources and used as received. The reaction
mixtures were monitored by thin layer chromatography (TLC) using glass-backed TLC plates.
Melting points were determined using open glass capillaries and were uncorrected. Infrared
(IR) spectra were obtained via ATR diamond in the range 4000–600 cm−1. 1H (400 MHz) and
13C NMR spectra (100 MHz) were obtained on a Bruker AM 400 spectrometer in DMSO-d6
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or CDCl3 solution. Coupling constants, J, are reported in hertz. MS spectra were carried out
on an LC/MS High-Resolution Time of Flight (TOF) Agilent 1200/6530 instrument at the
Atatürk University-East Anatolian High Technology Research and Application Center
(DAYTAM).
Chemistry
Synthesis of the methyl-2-(bromomethyl)benzoate (2)[39]
Methyl 2-methylbenzoate (1) (4 g, 26.4 mmol), N-bromosuccinimide (NBS) (4.74 g, 26.40
mmol) and azaisobutryonitrile (AIBN) (100 mg, 0.60 mmol) were refluxed in 80 mL
chloroform for 24 h. After the reaction was completed, the solid succinimide was filtered, and
the organic phase was evaporated to achieve product 2. This product was used directly for the
next step without further purification. Yellow oil. (Yield 95%).
Synthesis of tiouronium salt 3[40]
Thiourea was added (0,11 mol, 8.4 g) was added to the solution of methyl 2-
(bromomethyl)benzoate (2) (8.7 mmol, 2.0 g) in acetone (30 mL). The resulting mixture was
refluxed for 10 h., then cooled and filtered to remove the solid. The filtrate was evaporated to
half volume. The desired solid tiouronium salt was filtered. The obtained product was used
directly for the next step without further purification. White solid. Yield: 45%. Mp: 171-
1
173 °C. H NMR (400 MHz, DMSO) δ 9.12 (s,4H), 7.96 (d, J = 7.8 Hz, 1H), 7.67-7.56 (m,
2H), 7.51 (t, J = 7.5 Hz, 1H), 4.74 (s, 2H), 3.86 (s,3H). 13C NMR (100 MHz, DMSO) δ 169.7,
167.1, 136.9, 133.5, 131.7, 131.6, 129.2, 129.1, 52.9, 33.7.
Synthesis of 2,2'-(disulfanediylbis(methylene))dibenzoic acid (5) [40]
A solution of 2M KOH (20 mL) was added to a solution of 3 (4.73g, 0,01 mol) in 20 mL of
MeOH and the reaction was refluxed for 1h. After the reaction was completed, MeOH was
removed using a rotary evaporator, and the residue was acidified with 1N HCl. The obtained
mixture was extracted with Et2O (3X25 mL), organic layers were collected, dried over
1
MgSO4 and evaporated. White solid. Yield 72%. Mp: 117-119 °C. H NMR (400 MHz,
DMSO) δ 13.06 (s, 1H), 7.84 (d, J = 7.7 Hz, 1H), 7.50 (t, J = 7.4 Hz, 1H), 7.43 (d, J = 7.6 Hz,
1H), 7.34 (t, J = 7.5 Hz, 1H), 4.03 (d, J = 8.3 Hz, 2H), 2.75 (t, J = 8.2 Hz, 1H). 13C NMR (100
MHz, DMSO) δ 168.7, 139.3, 132.2, 132.2, 131.4, 130.1, 128.1, 41.0. After one or two weeks
on an open vessel, the colour of synthesized compound 5 became yellow. Mp: 158-160 °C.
HRMS (EI): [M-H]–, found 333.0275. C28H26N4NaO2S2 requires 333.0255.
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General procedure for the synthesis of 10a-h
Compounds 10a-h were synthesized according to the literature procedure.[41] Briefly,
triethylamine in THF was added to the solution of acid 5. After stirring for 30 min., the cold
ethyl chloroformate solution in THF was added slowly. Lastly, sodium azide solution in water
was included. The final mixture was extracted with ethyl acetate, washed with saturated
sodium bicarbonate and water, and dried. The obtained acyl azide 9, obtained after removing
ethyl acetate, was reacted with the corresponding amines to give final product 10a-h. The
experimental procedure is provided in detail as supplementary data.
1,1'-((Disulfanediylbis(methylene))bis(2,1-phenylene))bis(3-phenylurea) (10a)
1
White solid, yield: 32%. mp 198-200 °C; IR (ATR) 3250, 2971, 1699, 1634, 1550, 1149. H
NMR (400 MHz, DMSO) δ 9.02 (s, 1H), 7.97 (s, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.45 (d, J =
7.9 Hz, 2H), 7.32-7.20 (m, 3H), 7.14 (d, J = 7.6 Hz, 1H), 6.96 (t, J = 7.4 Hz, 2H), 3.72 (s,
13
2H). C NMR (100 MHz, DMSO) δ 153.1, 140.2, 137.5, 131.7, 129.3, 123.0, 122.6, 122.3,
118.5, 38.5. HRMS (EI): [M+Na]–, found 537.1327. C28H26N4NaO2S2 requires 537.6518.
1,1'-((Disulfanediylbis(methylene))bis(2,1-phenylene))bis(3-(4-nitrophenyl)urea) (10b)
Yellow solid, yield: 24%. mp 215-217 °C; Rf (EtOAc): 0.62; . IR (ATR) 3285, 3031, 1648,
1551, 1477, 1328. 1H NMR (400 MHz, DMSO) δ 9.73 (s, 1H), 8.36 – 8.04 (m, 3H), 7.79 (d, J
= 8.0 Hz, 1H), 7.69 (d, J = 9.0 Hz, 2H), 7.27 (t, J = 7.3 Hz, 1H), 7.17 (d, J = 7.3 Hz, 1H), 7.03
(t, J = 7.3 Hz, 1H), 3.73 (s, 2H).13C NMR (100 MHz, DMSO) δ 152.6, 146.8, 141.4, 136.7,
131.6, 128.5, 128.2, 125.6, 123.9, 122.6, 117.8, 38.4. HRMS (EI): [M+KH]–, found 643.0733.
C28H24KN6O6S2 requires 643.0839.
1,1'-((Disulfanediylbis(methylene))bis(2,1-phenylene))bis(3-(4-methoxyphenyl)urea)
(10c)
Purple solid, yield: 50%. mp 231-233 °C; Rf (EtOAc:Hexane=1:1): 0.27; IR (ATR) 3247,
1
2902, 1635, 1543, 1443, 1225. H NMR (400 MHz, DMSO) δ 8.84 (s, 1H), 7.88 (d, J = 2.3
Hz, 1H), 7.87 (s, 1H), 7.36 (d, J = 9.0 Hz, 2H), 7.23 (t, J = 7.7 Hz, 1H), 7.13 (d, J = 7.5 Hz,
13
1H), 6.95 (t, J = 7.7 Hz, 1H), 6.87 (d, J = 9.0 Hz, 2H), 3.71 (s, 7H). C NMR (100 MHz,
DMSO) δ 154.8, 153.2, 137.7, 133.2, 131.7, 128.5, 126.8, 122.7, 122.3, 120.2, 114.4, 55.5,
38.3. HRMS (EI): [M+H]+, found 575.1759. C30H31N4O4S2 requires 575.1789.
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1,1'-((Disulfanediylbis(methylene))bis(2,1-phenylene))bis(3-(4-chlorophenyl)urea) (10d)
White solid, yield: 50%. mp 239-241 °C; Rf (EtOAc:Hexane=1:1):0.43; IR (ATR) 3271,
2916, 1633, 1584, 1545, 1237. 1H NMR (400 MHz, DMSO) δ 9.15 (s, 1H), 7.99 (s, 1H), 7.82
(d, J = 8.1 Hz, 1H), 7.51 – 7.45 (m, 2H), 7.35 – 7.29 (m, 2H), 7.28 – 7.21 (m, 1H), 7.14 (dd, J
= 7.6, 1.4 Hz, 1H), 6.98 (td, J = 7.5, 1.1 Hz, 1H), 3.71 (s, 3H). 13C NMR (100 MHz, DMSO)
δ 152.9, 139.1, 137.3, 131.7, 129.1, 128.5, 127.4, 125.7, 123.2, 122.8, 120.0, 38.4. HRMS
(EI): [M+K]–, found 621.0355. C28H24Cl2KN4O2S2 requires 621.0259.
1,1'-((Disulfanediylbis(methylene))bis(2,1-phenylene))bis(3-cyclohexylurea) (10e)
White solid, yield: 10%. mp 214-216 °C; Rf (EtOAc:Hexane=1:1):0.42; IR (ATR) 3303,
2917, 1630, 1556, 1451, 1246. 1H NMR (400 MHz, DMSO) δ 7.92 (d, J = 8.2 Hz, 1H), 7.59
(s, 1H), 7.18 (t, J = 7.8 Hz, 1H), 7.06 (d, J = 7.3 Hz, 1H), 6.88 (t, J = 7.4 Hz, 1H), 6.53 (d, J =
7.6 Hz, 1H), 3.64 (s, 2H), 3.53 – 3.39 (m, 1H), 1.81 (d, J = 11.8 Hz, 2H), 1.66 (dd, J = 9.0,
13
4.0 Hz, 2H), 1.53 (dd, J = 8.3, 3.9 Hz, 1H), 1.37 – 1.23 (m, 2H), 1.23 – 1.03 (m, 3H). C
NMR (100 MHz, DMSO) δ 154.9, 138.5, 131.6, 128.3, 125.6, 121.7, 121.2, 48.2, 38.3, 33.4,
25.7, 24.8. HRMS (EI): [M+K]–, found 565.1999. C28H28KN4O2S2 requires 564.2073.
1,1'-((Disulfanediylbis(methylene))bis(2,1-phenylene))bis(3-pentylurea) (10f)
White solid, yield: 11%. mp 171-173 °C; Rf (EtOAc:Hexane=1:1): 0.5; IR (ATR) 3276, 2924,
1630, 1531, 1450, 1230.1H NMR (400 MHz, DMSO) δ 7.88 (d, J = 8.3 Hz, 1H), 7.66 (s, 1H),
7.19 (t, J = 7.7 Hz, 1H), 7.07 (d, J = 7.2 Hz, 1H), 6.90 (t, J = 7.1 Hz, 1H), 6.56 (t, J = 5.2 Hz,
1H), 3.66 (s, 2H), 3.13 – 3.01 (m, 2H), 1.51 – 1.36 (m, 2H), 1.36 – 1.18 (m, 4H), 0.87 (t, J =
6.0 Hz, 3H). 13C NMR (100 MHz, DMSO) δ 155.7, 138.5, 131.5, 128.3, 125.9, 121.9, 121.5,
39.5, 38.4, 29.8, 29.0, 22.3, 14.4. HRMS (EI): [M+K]–, found 541.2010. C26H38KN4O2S2
requires 541.2073.
1,1'-((Disulfanediylbis(methylene))bis(2,1-phenylene))bis(3-(pyridin-2-yl)urea) (10g)
White solid, yield: 18%. mp 261-263 °C; Rf (EtOAc:Hexane=1:1): 0.71. IR (ATR) 3196,
1
2978, 1694, 1579, 1552, 1540. H NMR (400 MHz, DMSO) δ 11.19 (s, 1H), 9.85 (s, 1H),
8.20 (d, J = 4.7 Hz, 1H), 8.05 (d, J = 8.2 Hz, 1H), 7.80 – 7.67 (m, 1H), 7.32 – 7.25 (m, 1H),
7.22 (d, J = 8.1 Hz, 1H), 7.15 (d, J = 7.6 Hz, 1H), 7.01 (td, J = 7.5, 1.0 Hz, 1H), 6.95 (dd, J =
13
7.1, 5.2 Hz, 1H), 3.77 (s, 2H). C NMR (100 MHz, DMSO) δ 153.5, 152.8, 146.5, 139.3,
137.5, 131.6, 128.7, 126.5, 123.1, 122.1, 117.6, 112.4, 39.1. HRMS (EI): [M+H]+, found
517.1454. C26H25N6O2S2 requires 517.1480.
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10.1002/cbdv.201900461
Chemistry & Biodiversity
1,1'-((Disulfanediylbis(methylene))bis(2,1-phenylene))bis(3-(pyrimidin-2-yl)urea) (10h)
White solid, yield: 14%. mp 262-264 °C; Rf (EtOAc:Hexane=1:1): 0.46; IR (ATR) 3138,
1
2975, 1691, 1578, 1446, 1285. H NMR (400 MHz, DMSO) δ 11.46 (s, 1H), 10.32 (s, 1H),
8.59 (d, J = 4.8 Hz, 2H), 8.00 (d, J = 8.0 Hz, 1H), 7.30 (t, J = 7.9 Hz, 1H), 7.15 (d, J = 7.5 Hz,
1H), 7.11 – 6.99 (m, 2H), 3.78 (s, 2H). 13C NMR (100 MHz, DMSO) δ 158.5, 158.26, 152.0,
137.2, 131.6, 128.7, 127.0, 123.6, 122.6, 115.4 (-CH2 signal overlapped with the solvent).
HRMS (EI): [M+K]– found 557.0867. C24H22KN8O2S2 requires 557.0944).
Antibacterial activity evaluation
The title compounds were tested against five bacteria including three Gram-positive
(Staphylococcus aureus, Enterococcus faecalis and methicillin-resistant Staphylococcus
aureus) and two Gram-negative microorganisms (Escherichia coli and Pseudomonas
aeruginosa) to evaluate their antibacterial activities. Gentamicin and piperacillin-tazobactam
were used as positive control antibiotics.
Broth microdilution assay reported by the Clinical and Laboratory Standards Institute (CLSI)
was used to determine MIC values. MIC values were identified as the lowest concentration of
the test compounds with no bacterial growth.[42]
Antitubercular activity evaluation
Antitubercular activities of the designed compounds 10a-h were investigated in vitro using
MABA method. The obtained results are reported as MIC values against M. tuberculosis
H37Rv.[43]
In vitro cytotoxicity screening
The in vitro cytotoxicity of the synthesized compounds was assessed by 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay against growth
inhibition of RAW 264.7 cells at 50 μg/mL concentration.[44,45] The values were calculated for
percentage inhibition that enables us to achieve the cytotoxicity data of the test compounds.
The detailed experimental procedures to determine antimicrobial activities and the
cytotoxicity of the compounds are provided as supplementary data.
Molecular docking
The chemical formula of compound 10a was built using ChemDraw Ultra 12.0 and saved as
Simplified Molecule Input Entry System (SMILES) file. The file was transferred to
LigandScout 4.2,[46] the structure was geometrically optimized and energy minimized to the
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10.1002/cbdv.201900461
Chemistry & Biodiversity
3D structure using the MMFF94x force field. The crystal structure of Enterococcus faecalis
FabH with the inhibitor 2-({4-bromo-3-[(diethylamino)sulfonyl]benzoyl}amino)benzoic acid
was obtained from Protein Data Bank with the PDB code 3IL5.[47] 10a was docked into the
active site of the enzyme using AutoDock Vina 1.1, integrated into LigandScout 4.2, with
default parameters. 3D pharmacophores from the cocrystallized ligand of the PDB structure
3IL5 were generated using LigandScout. This pharmacophore model was employed to rank
nine obtained docking poses of 10a. The selection of the most plausible one was based on its
ability to provide the highest amount of chemical interactions defined in the 3D
pharmacophore. Docking results were analyzed using LigandScout 4.2 and Discovery Studio
Visuliazer.[48]
In silico prediction of physicochemical properties of 10a-h
The chemical formulas of compounds 10a-h were built in ChemDraw Ultra 12.0 and saved as
Simplified Molecule Input Entry System (SMILES) file. The calculation of physicochemical
properties was carried out using Molinspiration (https://www.molinspiration.com).
Supplementary Material
The detailed experimental procedures to synthesize the compounds and determine their
1
antimicrobial activities and the cytotoxicity data as well as their H-NMR and 13C-NMR
spectra are provided as supplementary material.
Acknowledgements
ŞDD would like to thank the Scientific and Technological Research Council of Turkey (Grant
No: 115Z261 and the Faculty of Pharmacy at Erciyes University for the financial support. The
author also acknowledges the contribution of the COST (European Cooperation in Science
and Technology) Action CA 15106. MGG would like to thank Prof. Dr. Gerhard Wolber,
Freie Universität Berlin, for providing the license for LigandScout 4.2.
Author Contribution Statement
ŞDD and SB synthesized the compounds and elucidated their structures. CÖ carried out
antibacterial activity evaluation. VSK and DS performed in vitro antitubercular activity and
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10.1002/cbdv.201900461
Chemistry & Biodiversity
cytotoxicity experiments. MGG was responsible for computational studies. ŞDD and MGG
wrote the manuscript.
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Graphical Illustration
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