The novel economical synthesis and antimicrobial activity of a trithiocarbonate derivative

Article abstract of DOI:10.1016/j.bioorg.2019.103157

The compound diethyl 2,2′-(thiocarbonyl-bis(sulfanediyl))-diacetate 4 belongs to the trithiocarbonate class containing a trithiocarbonate function group flanked by ethyl acetate. In this procedure, a novel economic synthesis route to obtain compound 4 is described. This compound proved to possess broad-spectrum antimicrobial activity both in vitro and in vivo, and could be used as a lead compound. It is worth mentioning that this compound has been patented [No. US 9,988,348 B1; date of patent: June 5, 2018].

Full text of DOI:10.1016/j.bioorg.2019.103157

Journal Pre-Proof
The Novel Economical Synthesis and Antimicrobial Activity of a Trithiocar-
bonate Derivative
Yahia N. Mabkhot, Jamal M.A. Khaled, Naiyf S.H.A. Alharbi, Mujeeb A.S.
Sultan, Salim S. Al-Showiman, Hazem A. Ghabbour, Abdulrhman Alsayari,
Abdullatif Bin Muhsinah, H. Algarni
PII:
S0045-2068(19)30551-6
DOI:
https://doi.org/10.1016/j.bioorg.2019.103157
YBIOO 103157
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Accepted Date:
9 April 2019
24 July 2019
Please cite this article as: Y.N. Mabkhot, J.M.A. Khaled, N.S.H. Alharbi, M.A.S. Sultan, S.S. Al-Showiman, H.A.
Ghabbour, A. Alsayari, A. Bin Muhsinah, H. Algarni, The Novel Economical Synthesis and Antimicrobial Activity
of a Trithiocarbonate Derivative, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103157
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JOURNAL PRE-PROOF
The Novel Economical Synthesis and Antimicrobial Activity
of a Trithiocarbonate Derivative
Yahia N. Mabkhot1*, Jamal M. A. Khaled2, Naiyf S. H. A. Alharbi2, Mujeeb A.
S. Sultan3, Salim S. Al-Showiman4, Hazem A. Ghabbour5, Abdulrhman
Alsayari6, Abdullatif Bin Muhsinah6 and H. Algarni 7,8
1Department of Pharmaceutical Chemistry, College of Pharmacy, King Khalid University, Abha,
Saudi Arabia; E-mail: ygaber@kku.edu.sa
2Department of Botany and Microbiology, College of Science, King Saud University, Saudi Arabia; E-
mail: gkhaled@ksu.edu.sa; nalharbi1@KSU.EDU.SA
3Department of Chemistry, Science College, Riyadh, 11451, Kingdom of Saudi Arabia E-mail:
Alhosami1983@yahoo.co.uk
4 Department of Chemistry, Science College, King Saud University, P. O Box 2455 E-mail:
showiman@KSU.EDU.SA
5 Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura
35516, Egypt. E-mail: ghabbourh@yahoo.com
6 Department of Pharmacognosy, College of Pharmacy, King Khalid University, Abha, Saudi Arabia;
E-mail: alsayari@kku.edu.sa; ajmohsnah@kku.edu.sa
7Department of Physics, Faculty of Sciences, King Khalid University, P. O. Box 9004, Abha,
Saudi Arabia. halgarni@kku.edu.sa
8Research Centre for Advanced Materials Science (RCAMS), King Khalid University, Abha,
61413131413, P. O. Box 9004, Saudi Arabia. halgarni@kku.edu.sa
______________________________________________________________________________
Abstract
The compound diethyl 2,2'-(thiocarbonyl-bis(sulfanediyl))-diacetate 4 belongs to the
trithiocarbonate class containing a trithiocarbonate function group flanked by ethyl
acetate. In this procedure, a novel economic synthesis route to obtain compound 4 is
described. This compound proved to possess broad-spectrum antimicrobial activity
both in vitro and in vivo, and could be used as a lead compound. It is worth mentioning
that this compound has been patented [No. US 9,988,348 B1; date of patent: June 5,
2018]
Keywords: Antimicrobial activity, diethyl 2,2' (thiocarbonyl bis (sulfanediyl))
diacetate; synthesis, trithiocarbonate.
_____________________________________________________________________
1. Introduction
The trithiocarbonates have been investigated for years, with various synthetic routes
having been employed for their synthesis [1]. They are simply and efficiently
incorporated into many organic compounds and pharmaceutical therapeutic agents. In
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organic synthesis, for instance, they have been used in surface and colloidal
nanotechnology [2], as reagents for platinum group metals flotation [3], as an
electrolyte additive in lithium batteries [1], for monodisperse surface modification [4]
and for polymer formation [5]. As pharmaceutical therapeutic agents, they represent a
promising class. As leads for novel biologically active compounds, they act as
biological toxicants [6], anti-radiation agents in mice [7], antitumor agents [8,9],
inhibitors for carbonic anhydrase, and anti-glaucoma effects in vivo [10]. Their activity
as an inhibitor for carbonic anhydrase (CA) is seen through the formation of a mono-
coordination bond of its sulfur atoms with the Zn(II) ion from the CA enzyme active
site, as proved by X-ray crystal structure [11-13].
The development of antimicrobial agents never stops, but there is no doubt that the
major microbial pathogens are increasingly developing resistance to the current
antimicrobial drugs. Broad-spectrum antimicrobial agents must kill or inhibit specific
targets in pathogenic microorganisms. These targets exist in most human microbial
pathogens as part of an essential pathway. Absent from animal cells, not inhibited by
broadly applied antimicrobial drugs, easy to evaluate in vitro and in vivo, highly specific
for the pathogenic microorganisms, non-toxic for animals or humans, not result in the
quick gaining of resistance [14,15].
Broad-spectrum antimicrobials have several advantages in initial empirical therapy,
particularly against serious microbial infections. In a severe microbial disease, a therapy
must be administered before the identification of the pathogenic microbe, because the
time of diagnosis is a powerful predictor of mortality [16,17]. The patents of novel
antifungal drugs include azole compounds (new imidazole, fuconazole analogs, azole
with 2,6-di-tert-butyl-4-methylphenol), enzyme inhibitors (chitin inhibitors, β-1,3-D-
glucan synthases inhibitors), peptides (fusion peptides), phenylalkane nitriles (esters of
2-phenylalkane nitriles), boron compounds, pyridine derivatives, cyclic guanidines and
quinolinium compounds, and arylalkyl (azole derivatives in the structure of oxime
ester) [18-20]. In the present research, we report on a simple and economical synthesis
of diethyl 2,2' (thiocarbonyl bis (sulfanediyl)) diacetate 4 and investigate its biological
activity as a novel broad-spectrum antimicrobial agent in vitro and in vivo.
2. Results and discussion
2.1. Chemistry
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Trithiocarbonate 4 has been synthesized through a novel, simple and economical route
from commercially available materials. Although compound 4 is available from the
Sigma Company, it is very expensive; 5 mg costs $126.50 EUR. In comparison, the
production of 5 mg costs $0.125 EUR, according to our synthetic route. Furthermore,
Sigma noted on its website that compound 4 is rare and unique. However, that is not
the case. The reaction of ethyl cyanoacetate 1, carbon disulfide (CS2) 2 and ethyl 2-
chloroacetate 3 was used to obtain the trithiocarbonate 4, as illustrated in Scheme 1.
O
O
S
C
NC
Cl
OC2H5
OC2H5
S
3
1
2
DMF
K2CO3
S
C2H5O
OC2H5
S
S
O
O
4
Scheme 1. Synthesis of trithiocarbonate 4
This reaction was carried out in the presence of anhydrous K2CO3 as a base in DMF at
room temperature. An unsuccessful attempt was implemented to obtain trithiocarbonate
by reacting CS2 2 and ethyl 2-chloroacetate 3 in the absence of ethyl cyanoacetate 1. A
plausible mechanism was then proposed to rationalize the formation of trithiocarbonate
4, as depicted in Scheme 2.
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S
C
S
O
O
C
O
C
C
Cl
EtO
2
CN
S
S
C
S
O
C
DMF
CN
SK
3
EtO
EtO
CN
K2CO3
EtO
C
OEt
EtO
C
S
KS
1
O
O
2
O
C
O
CN
CN
S
C
S
S
C
C
EtO
C
EtO
S
EtO
OEt
EtO
C
OEt
S
S
S
S
O
O
O
O
4
Scheme 2. Mechanism proposed for the formation of trithiocarbonate 4
The structure of the synthesized compound 4 was revealed by using IR, 1H-NMR, 13C-
NMR, HRMS and X-ray single crystallography. The IR spectrum of this compound
displayed the characteristic absorption of the carbonyl and thiocarbonyl groups at υ
1
1732 and 1155 cm-1, respectively. The H-NMR and 13C-NMR spectra showed the
symmetrical pattern of compound 4. The 1H-NMR spectrum exhibited a triplet signal
at δ 1.23 ppm, with coupling constants J = 7 Hz integrated for the protons assigned for
the methyl group (-OCH2CH3), and a quartet signal at δ 4.16 ppm with coupling
constants J = 7 Hz integrated for the protons assigned for the methylene group of the
ester functional group (-OCH2CH3). The signal of protons assigned for the other
13
methylene group (-S-CH2CO-) appeared as a singlet at δ 4.14 ppm. In the C-NMR
spectrum, the peaks which appeared at δ 14.0, 38.9 and 62.1 ppm were attributed to (-
OCH2CH3), (-S-CH2CO-) and (-OCH2CH3), respectively. The carbonyl group peak
appeared at δ 167.0 ppm, while the peak of thiocarbonyl downshifted and appeared at
δ 220.2 ppm.
The x-ray single diffraction analysis clearly elucidated the molecular structure of
compound 4. The crystallographic data and refinement information for this compound
are summarized in Table 1. The selected bond lengths and bond angles are listed in
Table 2. The asymmetric unit contains one independent molecule, as shown in Figure
1. All the bond lengths and angles are within normal ranges [21]. Regarding the crystal
packing, shown in Figure 2, the molecules are linked via three non-classical
intermolecular hydrogen bonds (Table 3).
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Table 1. Experimental data
Crystal data
C9H14O4S3
282.38
Chemical formula
Mr
Crystal system, space group
Temperature (K)
a, b, c (Å)
Monoclinic, P21/c
293
15.1354 (7), 4.8723 (2), 18.6434 (8)
β (°)
91.866 (3)
1374.11 (10)
4
V (Å3)
Z
Radiation type
µ (mm−1)
Cu Kα
4.93
Crystal size (mm)
Data collection
Diffractometer
0.37 × 0.19 × 0.12
Bruker APEX-II D8 venture
diffractometer
Absorption correction
Multi-scan
SADABS Bruker 2014
0.266, 0.584
Tmin, Tmax
No. of measured, independent and observed
[I > 2σ(I)] reflections
Rint
12063, 2196, 1348
0.060
Refinement
R[F2 > 2σ( F2)], wR( F2), S
No. of reflections
No. of parameters
No. of restraints
0.077, 0.243, 1.03
2196
147
2
H-atom treatment
H atoms treated by a mixture of
independent and constrained refinement
0.47, −0.36
Δρmax, Δρmin (e Å−3)
Table 2. Selected geometric parameters (Å, °)
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S1—C4
1.788 (5)
O1—C3
O2—C3
O3—C7
O4—C7
O4—C8
1.308 (9)
1.198 (8)
1.213 (8)
1.313 (9)
1.486 (11)
S1—C5
1.729 (5)
1.617 (4)
1.740 (5)
1.784 (5)
1.450 (12)
103.6 (2)
103.1 (2)
116.6 (7)
118.5 (6)
117.2 (10)
124.4 (6)
108.9 (5)
126.6 (6)
113.2 (4)
S2—C5
S3—C5
S3—C6
O1—C2
C4—S1—C5
C5—S3—C6
C2—O1—C3
C7—O4—C8
O1—C2—C1
O1—C3—O2
O1—C3—C4
O2—C3—C4
S1—C4—C3
S1—C5—S2
S1—C5—S3
S2—C5—S3
S3—C6—C7
O3—C7—O4
O3—C7—C6
O4—C7—C6
O4—C8—C9
126.5 (3)
108.1 (2)
125.5 (3)
114.4 (4)
124.8 (6)
124.6 (6)
110.6 (5)
111.7 (9)
Table 3. Hydrogen-bond geometry (Å, °)
D—H···A
D—H
0.9900
0.9900
0.9900
H···A
2.4600
2.5800
2.6000
D···A
D—H···A
145.00
C4—H4A···O2i
C6—H6A···O3ii
C6—H6A···O3iii
3.319 (7)
3.353 (7)
3.260 (8)
135.00
124.00
Symmetry codes: (i) x, y−1, z; (ii) x, y+1, z; (iii) −x+1, y+1/2, −z+3/2.
Figure 2. Molecular packing of compound 4 viewed as hydrogen bonds drawn as
dashed lines along the b axis.
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2.2. Biological activity tests
Antimicrobial agents are used in the treatment and prevention of microbial infections.
These agents (drugs) have been produced by both biological and chemical methods.
Many antimicrobial agents, such as sulfonamids, oxazolidinones and quinolones, are
produced via chemical synthesis [22]. The production of a broad-spectrum
antimicrobial agent is extremely significant when countering the threat of pathogenic
microorganisms, particularly antibiotic resistant microorganisms. The production of
broad-spectrum antibacterial and antifungal agents through chemical synthesis was the
principle objective of this work.
The biological activity of compound 4 as an antimicrobial and anticancer agent was
first evaluated. The results indicated that the compound showed no biological activity
as an anticancer against the MCF-7 human breast cancer cell line. The data in Table 4
did confirm that compound 4 showed biological activity as an antibacterial and
antifungal agent against all pathogenic microorganisms that were used in this research.
Both the water- and DMSO-soluble forms indicated efficacy against the
microorganisms.
All tested fungi (C. albicans, Cr. neoformans, A. niger, P. chrysogenum, Fusairum sp.)
except C. parapsilosus were significantly (P <0.05) susceptible to the compound.
Regarding the A. fumigatus mold, the inhibition shown by the compound was
significantly (P <0.05) stronger than that of the standard antifungal drugs
Cycloheximide, Canesten and Caspofungin. The A. fumigatus used in this work is one
of the strains isolated from humans who suffered from aspergillosis disease in the city
of Riyadh, KSA, as seen in our previous study [23]. The A. fumigatus mold, which lives
both outdoors and indoors, is considered a real risk for individuals with
immunodeficiency or lung illnesses [24]. Compound 4 also showed high biological
activity as an antifungal agent against Cr. neoformans, with the inhibition zone and
minimal inhibitory concentration reaching 30 mm and 0.03125 mg/ml, respectively.
The Cr. neoformans strain used in this study confirmed that Caspofungin has limited
activity against this fungi [25]. This pathogenic encapsulated yeast causes several
diseases called cryptococcosis [26]. The results in Table 4 refer to the efficiency of
compound 4 to inhibit both pathogenic yeasts and molds, particularly its effectiveness
in inhibiting Cycloheximide, Canesten and Caspofungin resistant C. parasilosus. Both
the gram-positive and -negative bacteria which were used in this research were
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inhibited by compound 4 (Table 4). Additionally, the biological activity against S.
mutans (plays a major role in tooth decay) and N. meningitides (can cause meningitis)
was also very good.
Regarding in vitro uses, the present work concluded that compound 4 exhibits
biological activity as a novel broad-spectrum antimicrobial agent.
To confirm the results obtained from the in vitro study, an animal experiment was also
implemented. In this stage, the role of the water-soluble compound 4 was evaluated to
treat mice infected with C. albicans (candidemia). The animal experiments add
abundant evidence to support the use of Compound 4. In the following results, Co1,
Co2, FluCal, ComCal and Comp are given to several groups of mice, treated with sterile
normal saline, infected with C. albicans, treated with Diflucan (fluconazole) (Pfizer
Inc. New York, N.Y, U.S.A), treated with compound 4 or injected only with the
chemical, respectively. In Table 5, a percentage change refers to the change in weight
of the treated groups compared to the control group (Co1). At the end of the 15th day,
the data showed that the percentage change of groups treated with fluconazole and
compound 4 were convergent changes, whereas the percentage change of the group
infected with C. albicans (Co2) was the highest. Food consumption (g/day/mice) and
water consumption (ml/day/mice) were arranged as noted in Tables 6 and 7. Both
fluconazaole and compound 4 had nearly identical effects on food and water
consumption. The investigated compound 4 had no negative side effects on food and
water consumption compared to the control group (Tables 6, 7). Table 8 shows the
daily mortality rate (%) in albino male mice infected with C. albicans and treated with
fluconazole or compound 4. The C. albicans caused dramatic increases in daily
mortality compared to the control group. The results demonstrated that compound 4, at
a loading dose of 800 mg, followed by 400 mg/d for 2 weeks, reduced the mortality (%)
caused by C. albicans. The results confirmed that compound 4 has very similar effects
to fluconazole (Table 8). The mortality in the group treated only with compound 4 was
also very similar to the control group.
The effectiveness of the previous treatments on the serum marker enzymes of the mice
is shown in Table 9. The activity of the serum marker enzymes (SGOT, SGPT, alkaline
phosphatase, GGT and bilirubin) significantly (p<0.001) increased in the group
infected with C. albicans (negative control) when compared with the control group.
The activity of serum marker enzymes also significantly (p<0.001) increased in the
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group infected with C. albicans and treated with fluconazole, more so than the group
only infected with C. albicans. The results obtained from the group treated with
compound 4 showed significantly (p<0.001) enhancement in activity, with the
exception of SGOT activity. In the group injected only with compound 4, there was
significantly (p<0.01) less SGOT activity than in the control group. Table 10 includes
the concentration of serum lipoproteins (mg/dl) in the tested mice. The data confirmed
that the presence of C. albicans led to a significant (p<0.001) rise in cholesterol,
triglycerides, VLDL and LDL level, whereas it caused a significant (p<0.001) decrease
in the HDL level. These effects of C. albicans significantly (p<0.001) decreased in the
groups treated with compound 4 and fluconazole. The compound 4 group showed a
significant (p<0.001) decrease in cholesterol, triglycerides, VLDL and LDL levels,
along with a non-significant increase in HDL, compared to the control group. The data
in Table 11 demonstrates that C. albicans caused a significant (p<0.001) increase in
glucose and LDH, whereas it caused a significant (p<0.001) decrease in the total protein
level in the serum blood of treated mice. The effects of C. albicans (Table 11)
significantly decreased in the groups treated with compound 4 and fluconazole (p<0.01,
p<0.001 respectively). The glucose and total protein level in serum blood of mice
injected only with the compound 4 significantly decreased (p<0.01) and increased
(p<0.05) respectively, while no significant changes were seen in LDH level.
The MDA, total protein and NP-SH in the liver tissues of the mice were estimated in
the previous treatments (Table 12). Significant (P<0.001) differences were found
between the control group and the negative control group infected with C. albicans.
Compound 4 and fluconazole demonstrated similar results in reducing the biological
effects of C. albicans (Table 12). Table 13 illustrates that all hematologic parameters
in the mice decreased in the group infected with C. albicans, when compared with those
injected with sterile normal saline. The lymphocytes excludes from the previous results
in Table 13 showed significant (p<0.001) increase. The compound 4 and fluconazole
groups noted a decrease in the hematologic changes resulting from C. albicans, with
the exception of monocytes (%).
The data obtained from the in vivo test confirmed that compound 4 significantly reduces
the activity of aspartate aminotransferase (AST), cholesterol, triglycerides, VLDL,
LDL, and glucose, MDA, WBC and lymphocytes in the serum of mice. Additionally,
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it significantly increases other hematologic parameters, the total protein of serum and
the total protein of liver tissue.
Antimicrobial agents can be grouped based on their chemical structures into
aminoglycosides, anasamacrolides, beta-lactams, chloramphenicol and analagues,
lincosamides, macrolides, nucleosides, puromycin, peptides, phenazines, polyenes,
polyethers and tetracyclines [27]. Recent scientific research has focused on the function
and inhibition of fungal β-carbonic anhydrases and bacterial carbonic anhydrases (α-,
β-, and/or γ- carbonic anhydrases familie) as novel antifungal and antibacterial targets
[28,29]. Based on its chemical structure, compound 4 belongs to the trithiocarbonates
class. This class is considered a strong inhibitor of carbonic anhydrases [10,30], which
may explain the effects of compound 4 on the tested microorganisms.
The present results obtained from in vitro and in vivo experiments suggest that
compound 4 may be a novel antibacterial and antifungal drug.
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Table 4. Biological activity of soluble water compound 4 as an antimicrobial agent
against some medical microorganisms.
Compound 4 (4
Inhibition zone diameter (mm) of standard
antimicrobial agents
mg/disk)
Standard
Standard antifungal
agent
antibacterial agent
S. aureus
10.6±0.5
2
14
30
23
N.T
N.T
N.T
F*
S. mutans
E. coli
20±1 D
0.0625
1
35
20
20
40
10
0
N.T
N.T
N.T
N.T
N.T
N.T
11.6±0.5
E
N.
22.6±0.5
C
0.0625
2
21
14
34
30
20
0
N.T
N.T
N.T
N.T
N.T
N.T
meningitides
Sa.
8.6±0.5 F
typhimurium
C. albicans
20±1 D
30±1 B
0.0625
N.T N.T
N.T
N.T
0
26
25
20
0
C.
0.03125 N.T N.T
32
neoformasn
C.
13±1 E
0.05
N.T N.T
N.T
N.T
N.T
N.T
0
0
0
0
parasilosus
P.
30.3±.5
B
0.03125 N.T N.T
0.03125 N.T N.T
0.03125 N.T N.T
23
22
30
30
23
15
chrysoginum
A.
34.6±0.5
A
20
0
fumigatus
Fusarium sp.
30±1 B
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*The mean of inhibition zone diameter (mm) ± Standard division. In the same column, the
means with different letters are significant at P< 0.05.
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Table 5. Daily body weight (gm) of albino male mice infected with C. albicans and
treated with fluconazole and compound 4 for 15 days.
Days
Co1*
Co2
FluCal
Comp.
CompCal.
0
1
19.20±0.35**
19.9±0.43
19.70±0.47
20.20±0.38
21.00±0.36
20.10±0.31
4.68↑**
21.1±0.31
9.89
19.9±0.37
0.00
19.80±0.35
0.50
20.30±0.36
0.49
21.50±0.26
2.38
20.8±0.38
4.52
21.80±0.24
10.65
22.10±0.23
9.40
22.00±0.25
4.76
2
3
21.7±0.21
13.02
21.7±0.3
9.04
22.30±0.21
13.19
23.60±0.33
16.83
22.3±0.21
6.19
22.2±0.20
15.62
22.22±0.27
11.66
22.40±0.22
13.70
24.20±0.20
19.80
22.90±0.27
9.04
4
22.3±0.21
16.14
24.00±0.26
20.60
23.90±0.31
21.31
24.90±0.23
23.26
23.55±0.37
12.16
5
23.2±0.24
20.83
25.37±0.26
27.51
24.00±0.29
21.82
25.70±0.21
27.22
24.22±0.32
15.34
6
23.9±0.23
24.47
25.12±0.29
26.25
24.22±0.22
22.95
26.20±0.24
29.70
24.66±0.23
17.46
7
24.2±0.24
26.04
26.12±0.29
31.28
24.44±0.24
24.08
26.70±0.26
32.17
24.87±0.29
18.45
8
24.9±0.23
29.68
27.00±0.48
35.67
24.50±0.18
24.36
26.80±0.29
32.67
25.25±0.25
20.23
9
25.00±0.22
30.20
27.42±0.48
37.83
24.87±0.29
26.29
27.00±0.27
33.66
25.37±0.26
20.83
10
11
25.70±0.22
33.85
27.85±0.40
39.98
25.25±0.25
28.17
27.40±0.35
35.64
26.14±0.26
24.48
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26.3±0.27
36.97
27.83±0.40
39.86
25.37±0.26
-28.80
27.66±0.28
36.96
26.28±0.28
25.17
12
13
14
15
26.7±0.38
39.06
29.50±0.42
48.24
26.00±0.30
31.97
27.77±0.27
35.51
27.28±0.28
29.93
27.55±0.29
43.51
29.16±0.47
46.56
26.28±0.28
33.43
28.00±0.37
38.61
28.33±0.49
34.92
28.00±0.40
45.83
30.20±0.73
51.78
26.71±0.28
35.60
28.62±0.41
41.70
28.66±0.33
36.50
* Co1= control group treated with sterile normal saline, Co2= negative control infected with C. albicans,
Flucal= group infected with C. albicans and treated with fluconazole, Comp= group treated with
compound 4 and CompCal= group infected with C. albicans and treated with compound 4. **Mean±SE,
***% change in daily body weight (gm) compared with control group .
Table 6. Food consumption (g/day/mouse) of albino male mice infected with C.
albicans and treated with fluconazole and compound 4.
Days
1
2
3
4
5
6
7
8
9
10
11
12 13 14 15
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* Co1= control group treated with sterile normal saline, Co2= negative control infected with C.
albicans, Flucal= group infected with C. albicans and treated with fluconazole, Comp= group
treated with compound 4 and CompCal= group infected with C. albicans and treated with
compound 4. ** Food consumption (g/day/mice).
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Table 7. Water consumption (ml/day/mouse) of albino male mice infected with C.
albicans and treated with fluconazole and compound 4.
Days
1
2
3
4
5
6
7
8
9
10 11 12
13
14
15
* Co1= control group treated with sterile normal saline, Co2= negative control infected with C. albicans,
Flucal= group infected with C. albicans and treated with fluconazole, Comp= group treated with
compound 4 and CompCal= group infected with C. albicans and treated with compound 4. ** Water
consumption (g/day/mice).
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Table 8. Daily mortality (%) in albino male mice infected with C. albicans and treated
with fluconazole and compound 4.
Days
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15
0**
0
0
0
0
0
0
0
0
0
0
0
0
10 10
0
0
0
0
0
0
0
0
0
0
0
0
10
0
20
0
20 20 20 30 30 30 40 40 40 50
0
0
10 10 20 20 20 20 30 30 30
0
0
0
0
0
0
0
0
10 10 10
0
10
10 10 20 20 20 30 30 30 30 30
* Co1= control group treated with sterile normal saline, Co2= negative control infected with C. albicans,
Flucal= group infected with C. albicans and treated with fluconazole, Comp= group treated with
compound 4 and CompCal= group infected with C. albicans and treated with compound 4. **Mortality
(%).
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Table 9. Activity of serum marker enzymes of albino male mice infected with C.
albicans and treated with fluconazole and compound 4.
107.45
±2.71
27.07
±0.53
4.94
0.54
±0.01
1.67
±
296.88
±4.35
±0.14
204.8
±5.86
***a
125.20
±7.45
***a
414.80
±6.75
***a
9.70
±0.29
***a
39.71
90.59
362.37
9617
209.4
↑
↑
↑
6↑
↑
0.04
***a
0.75
±0.03
***b
143.28
±2.88
*** b
89.07
±3.82
** a
50.48
±3.17
***b
321.28
±6.91
***b
5.60
±0.17
***b
4.40
59.67
22.54
30.03
42.26
55.13
↓
↓
↓
↓
↓
0.57
28.67
5.89
286.62
±4.67 a
3.45
6.04
17.10
11.10
±0.13
±0.017
↓
↓
±1.15 a
↑
↓
↑
a
a
1.15
±
169.33
±2.69
***b
83.50
±2.35
***b
354.66
±5.57
***b
7.18
±0.20
***b
33.30
14.49
17.31
25.94
31.16
↓
↓
↓
↓
↓
0.03
***b
* Co1= control group treated with sterile normal saline, Co2= negative control infected with C. albicans,
Flucal= group infected with C. albicans and treated with fluconazole, Comp= group treated with
compound 4 and CompCal= group infected with C. albicans and treated with compound 4. All values
represent mean ± SEM. ***p<0.001; Student’s- t-test. a As compared with control group. b As compared
with C. albicans group.
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19

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Table 10. Serum lipoproteins of albino male mice infected with C. albicans and treated
with fluconazole and compound 4.
98.17
±
85.3
±
39.0
±
17.0
±
42.0
±
2.1
2.7
0.7
21.1
±
0.5
37.3
±
2.2
154.2
±
212.8
±
186.8
±
116.7
118.8
118.8
45.8
267.1↑
↓
6.0
↑
6.0
↑
0.9
***a
29.8
±
1.21
***a
21.0
±
↑
5.7
***a
83.9
±
***a
134.8
±
***a
105.1
±
41.1
36.6↓
43.7↓
19.6↓
43.7↓
19.6↓
45.5↓
↑
3.6
2.2
0.9
***b
0.4
***b
13.7
±
3.3
***b
23.3
±
***b
78.68
±
***b
68.5
±
41.6
±
6.42
19.85↓
44.35↓
↑
3.2
1.6
0.3
***a
3.0
***a
2.1a
***a
***a
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159.8
±
134.8
±
26.9
±
26.9
±
105.9
±
27.2
24.8↓
27.8↓
27.8↓
31.3↓
↑
3.4
3.8
1.1
**b
0.7
***b
4.6
***b
***b
***b
* Co1= control group treated with sterile normal saline, Co2= negative control infected with C.
albicans, Flucal= group infected with C. albicans and treated with fluconazole, Comp= group
treated with compound 4 and CompCal= group infected with C. albicans and treated with
compound 4. All values represent mean ± SEM. **p<0.01; ***p<0.001; Student’s- t-test. a
As compared with control group. b As compared with C. albicans group.
Table 11. Serum glucose, total protein and LDH of albino male mice infected with C.
albicans and treated with fluconazole and compound 4.
Glucose level
(mg/dl)
Total Protein
(mg/dl)
LDH
Mean±SE
% Change
Mean±SE
% Change
Mean±SE
% Change
113.95
±2.96
7.11
±0.15
158.84
±3.00
204.2
3.55
245.18
79.19↑
29.55↓
15.41↓
18.29↓
50.05↓
58.79↑
7.69↑
54.35↑
24.57↓
2.29↑
±7.58***a
143.85
±0.08***a
5.64
±5.12***a
184.92
±2.47***b
96.38
±0.11***b
7.66
±5.08***b
162.50
±3.62** a
±0.18* a
±1.84 a
166.83
4.91
220.98
38.14↑
9.86↓
±4.32**b
±0.09***b
±3.96**b
* Co1= control group treated with sterile normal saline, Co2= negative control infected with C.
albicans, Flucal= group infected with C. albicans and treated with fluconazole, Comp= group
treated with compound 4 and CompCal= group infected with C. albicans and treated with
21

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compound 4. All values represent mean ± SEM. **p<0.01; ***p<0.001; Student’s- t-test. a
As compared with control group. b As compared with C. albicans group.
Table 12. MDA, Total Protein and NP-SH in liver tissue of albino male mice infected
with C. albicans and treated with fluconazole and compound 4.
MDA
Total Protein
(g/l)
NP-SH
Treatments
(nmol/g)
(nmol/g)
Co1*
Co2
1.24±0.02
115.23±1.80
65.14±2.06***a
92.72±1.54***b
5.70±0.14
6.36±0.25***a
2.33±0.07***b
1.61±0.14***a
4.21±0.18***b
FluCal
Comp.
0.94±0.03***a
4.57±0.25***b
129.04±2.80**a
77.84±2.37**b
6.88±0.22***a
3.56±0.16***b
CompCal.
* Co1= control group treated with sterile normal saline, Co2= negative control infected with C.
albicans, Flucal= group infected with C. albicans and treated with fluconazole, Comp= group
treated with compound 4 and CompCal= group infected with C. albicans and treated with
compound 4. All values represent mean ± SEM. **p<0.01; ***p<0.001; Student’s- t-test. a
As compared with control group. b As compared with C. albicans group.
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Table 13. Hematological studies of albino male mice infected with C. albicans and
treated with fluconazole and compound 4.
Co1*
Co2
FluCal
Comp.
CompCal.
6513.6
8233.2
8795
7540.8
8582.2
±17.7
15.9
±24.0
***a
24.1↓
±83.7
*** b
7.50
26.4↑
±45.9
** a
2.48↑
±87.3
*** b
6.16
↑
4.46
9.26
±0.1
** a
8.14
45.23
68.16
13.72
38.2
±0.19
*** a
±0.23
*** b
11.1
±0.1
↓
↑
↑
↑
±0.17
*** b
9.8
7.62
11.9
12.4
28.6
17.9↓
44.9↓
28.6↓
42.5↓
28.9↓
±0.18
*** b
35.05
±1.5
46.7↑
49.9↑
27.8↑
65.4↑
32.6↑
3.4↑
8.2↑
±0.14
*** b
31.3
±0.16*
** a
↑
±0.18
±0.18 a
23.3
±1.54
*** a
441.2
±24.7
*** a
43.5
42.48
±1.47
45.9
33.8
±0.42
*** b
522.3
±10.1
↑
±1.51 a
*** b
563.8
±17.4
** b
670.7
±15.3
618.0
±15.5
18.3
8.5↑
↑
a
b
*
*
87.31
±1.74
53.7
±2.0
75.84
±3.72
72.04
23.4
±2.5
15.1↑
30.4↑
↑
±2.6*** b
a
b
*** a
21.2
*
*
39.0
±1.0
*** a
26.3
±0.5
*** b
29.9
28.2
23.6
±0.5
↑
±0.97
±0.7*** b
*** a
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19.3
±0.7
*** a
21.0
±0.4
*** a
8.8
32.6
±0.9
** a
23.4
±1.0
27.87
±0.94
24.3
21.1
30.6↓
27.3↓
60.7↑
44.8↓
25.9↑
23.3↑
29.2↑
68.3↑
17.0↑
15.3↑
11.3↓
36.7↑
↑
±0.5*** b
b
*
25.9
±0.5
*** b
6.2
33.3
±1.1
** a
24.1
±0.2
28.88
±0.71
15.0
↑
*** b
7.10
4.8
5.49
19.4
±0.2
*** a
0.96
±0.05
*** a
0.96
±0.1
*** b
1.61
±0.10
*** b
±0.09
** a
±0.07
*** b
1.30
↓
±0.10
2.38
±0.09
*** a
1.74
36.1
±0.02
*** b
↑
±0.04
1.04
7.98
1.06
1.15
10.7
0.88
7.9
11.16
±0.02*
↑
±0.02
↓
±0.02* b
±0.01** a
↑
±0.07 b
↓
a
* Co1= control group treated with sterile normal saline, Co2= negative control infected with C.
albicans, Flucal= group infected with C. albicans and treated with fluconazole, Comp= group
treated with compound 4 and CompCal= group infected with C. albicans and treated with
compound 4. All values represent mean ± SEM. **p<0.01; ***p<0.001; Student’s- t-test. a
As compared with control group. b As compared with C. albicans group.
3. Material and methods
3.1. Chemistry
All chemicals were obtained from commercial sources, including Sigma-Aldrich (St.
Louis,MO, USA), and were used as received without further purification, unless
otherwise stated. Melting points were measured on a Gallenkamp melting point
apparatus (Thermo Fisher Scientific, Paisley, UK) in open glass capillaries and are
uncorrected. Infrared spectra (IR) were recorded using the KBr disc technique on a
Perkin Elmer FT-IR spectrophotometer 1000 (PerkinElmer, Waltham, MA, USA). 1H-
24

JOURNAL PRE-PROOF
and13C-NMR spectra were obtained with either a JEOL ECP 600 NMR spectrometer
(Tokyo, Japan) operating at 600 MHz z in deuterated chloroform (CDCl3) or dimethyl
sulfoxide (DMSO-d6). Chemical shifts are expressed in  units and J-coupling constants
are given in Hz. Mass spectra were acquired with the aid of a Shimadzu GCMS-QP
1000 EXmass spectrometer (Tokyo, Japan) at 70 eV. Elemental analysis was carried
out on a Perkin Elmer 2400Elemental Analyzer; CHN mode.
3.1.1. Synthesis of diethyl 2,2'-(thiocarbonylbis(sulfanediyl))diacetate 4
A mixture of ethyl cyanoacetate 1 (0.1 mol) and anhydrous K2CO3 (30 g) in DMF (35
0
mL) was stirred vigorously at 25 C for 10 min, and then CS2 2 (0.2 mol) was added
with stirring for 50 min. The mixture reaction was cooled in an ice bath. Then, ethyl 2-
chloroacetate 3 (0.2 mol) was added with continued stirring for 15 min. The cooling
bath was subsequently removed, and the mixture was stirred for 3 h. The solid product
was precipitated by the addition of H2O collected by filtration, washed with H2O, and
dried. The compounds were recrystallized from EtOH to afford two crystalline
materials:
ethyl
Yield:
3-amino-4-cyano-5-((2-ethoxy-2-oxoethyl)thio)thiophene-2-
60%; m. p. 93° and diethyl 2,2'-
carboxylate,
(thiocarbonylbis(sulfanediyl))diacetate 4. These compounds had the following
characteristics: Yield: 20%; m. p. 40 ºC; IR (KBr, cm–1) ύ = 2978, 2930, 1732, 1475,
1
1367, 1300, 1202, 1155, 1095, 1069; H-NMR (500 MHz, CDCl3) δ = 1.23 (t, J = 7
13
Hz, 3H, -CH3), 4.14 (s, 2H, S-CH2-CO), 4.16 (q, 2H, J = 7 Hz, -O-CH2-); C-NMR
(125 MHz, CDCl3) δ = 14.0, 38.9, 62.1, 167.0, 220.2; MS m/z: 283.0 [M+H]+, 267.1,
239.1, 207.0; Elemental Analysis: C, 38.28; H, 5.00; S, 34.06, Found: C, 38.31; H,
5.12; S, 33.97
3.1.2. Single-Crystal X-ray Diffraction of Compound 4
Compound 4 was obtained as single crystals by slow evaporation from an ethanol
solution of the pure compound at room temperature. Data were collected on a Bruker
APEX-II D8 Venture area diffractometer, equipped with graphite monochromatic Cu
Kα radiation, λ = 1.54178 Å at 293 (2) K. Cell refinement and data reduction were
carried out by a Bruker SAINT. SHELXT [31,32] was used to reveal the structure. The
final refinement was carried out using the full-matrix least-squares technique with
anisotropic thermal data for non-hydrogen atoms on ??. CCDC 1522608 contains the
supplementary crystallographic data for this compound and can be obtained free of charge
25

JOURNAL PRE-PROOF
from
the
Cambridge
Crystallographic
Data
Centre
via
www.ccdc.cam.ac.uk/data_request/cif.
3.2. Biological activity tests
3.2.1. In vitro, antimicrobial activity
To evaluate the antimicrobial activity of compound 4 as a novel anti-bacterial and anti-
fungal agent, the inhibition zone diameter (mm) and the minimal inhibitory
concentration were determined according to literature reports [33,34]. The tests were
carried out on several clinical microorganisms, including gram positive bacteria
(Staphylococcus aureus ATCC and Streptococcus mutans ATCC 35668), gram
negative bacteria (Escherichia coli ATCC 25922, Salmonella typhimurium ATCC
14028 and Neisseria meningitides ATCC 1302), yeasts (Candid albicans ATCC 60193,
Cryptococcus neoformans Wild strain and Candida parapsilosus ATCC 22019), and
molds (Aspergillus fumigatus AUMC 8794, Penicillum chrysogenum AUMC 9476 and
Fusarium sp.).
The clinical microorganisms were cultivated at least three times with suitable culture
mediums and incubating conditions before the susceptibility tests. The concentrations
of microbial inoculation suspensions in sterile sodium chloride solution (0.89 %) were
adjusted to 0.65 at 620 nm by spectrophotometer. In the first stage, 150 mg of
compound 4 was dissolved in 2000 µL of DMSO, then 40 µL of solution was added on
a sterile filter disk (6 mm) to obtain 3 mg per disk. To prepare a water soluble form of
compound 4, 150 mg of compound 5 was treated with sodium ethoxide and dissolved
with 2000 µL of sterile distilled water; then, 40 µL of solution was added to the filter
disk (3 mg/disk). DMSO- and water-soluble forms of compound 4 were investigated
using disk diffusion assay to determine the inhibition zone (mm) resulting from 3 mg
of compound per disk.
The disks were then dried at 250 C under sterile conditions using a safety biological
cabinet. Mueller-Hinton agar (MHA) (Scharlau Microbiogy, Spain) and potato dextrose
agar (PDA) (Scharlau, Spain) were used to perform antibacterial and antifungal tests,
respectively. The media were prepared according to the manufacturer’s guidelines. To
prepare one liter of MHA and one liter of PDA, 21 grams of MHA and 39 grams of
PDA were dissolved in one liter of sterile distilled water, then sterilized at 121 C for
26

JOURNAL PRE-PROOF
15 minutes by autoclave (HL-321, Taiwan). The sterilized media were poured into
sterile plastic petri dishes at 50 ºC. The media were allowed to solidify at room
temperature. Next, 0.1 ml of microbial inoculation suspension was spread on the dried
surface of the medium and kept at 25 0C for 5-10 minutes. Compound 4 and the standard
antimicrobial drug discs (Table 1) were dispensed on the surface of the inoculated
media. The bacteria were incubated at 37±1 ºC for 19-22 hours and the fungi at 25±1
ºC for 48-72 hours. After incubation, the diameters of the inhibition zones (millimetre)
resulting from the biological activity of the compound were recorded.
The minimal inhibitory concentration (MIC) of compound 4 (water-soluble form) was
determined by microdilution assay, using a Mueller-Hinton broth for bacteria and a
potato dextrose broth for fungi. Compound 4 was dissolved in a sterile culture broth (2
mg/mL). Two-fold serial dilutions were carried out to obtain concentrations ranging
from 2 to 0.008 mg/mL. Each dilution of 95 μL was added to 96-well plates, then each
well was inoculated with 5 μL of inoculation microbial suspension (5×108 colony form
unit (CFU)/ml). The plates were incubated at conditions suitable for the tested
microorganism. After incubation, microbial growth was detected in each well by a p-
iodonitrotetrazolium violet reagent (Sigma, USA). Each well received 20 μL of the
reagent (0.5 mg/ml), and was then incubated at 37±1 ºC for 30 min. A violet color
indicated microbial growth in the well. The minimal inhibitory concentration (mg/ml)
of compound 4 was estimated as the lowest concentration that inhibited microbial
growth.
3.2.2. Anticancer test
The biological activity of compound 4 (water soluble form) as an anticancer agent for
human breast cancer was evaluated according to a previous study [35]. Roswell Park
Memorial Institute (RPMI)-1640 medium (Sigma, USA) was prepared according to the
manufacturer’s guidelines and supplemented with fetal bovine serum (10%). To avoid
microbial contamination, streptomycin and penicillin were added to RPMI-1640.
Several concentrations of compound 5, ranging from 4 to 0.008 mg/mL, were tested.
3.2.3. In vivo, antifungal activity
To confirm that the water-soluble form of compound 4 has biological activity as a novel
antimicrobial agent, mice were treated with candidemia and the effectiveness of
27

JOURNAL PRE-PROOF
compound 4 was investigated. The experiment was performed in the Experimental
Animal Care Center (EACC), College of Pharmacy, King Saud University, Riyadh.
Fifty albino male mice weighing nearly 30±2 gram were used. The mice were arranged
in five groups, ten mice in each cage, and given water and feed ad libitum. The negative
control group was infected intravenously with 0.2 ml of C. albicans suspension (106
CFU/mouse), while the control groups received 0.2 ml of sterile normal saline (0.89%
Nacl). The group treated with the standard drug (fluconazole) was infected
intravenously with 0.2 ml of C. albicans suspension (106 CFU/mouse) and treated
(orally) by 12 mg/kg of fluconazole (loading dose) on the first day, followed by 6 mg/ml
for 2 weeks. The group treated with the investigated compound 4 was infected
intravenously with C. albicans at 106 CFU/mouse (0.2 ml) and treated (orally) by 12
mg/kg of compound 4 (loading dose) on the first day, followed by 6 mg/ml for 2 weeks.
To study the biological effects of compound 4, a fifth group was designated. In this
group, the mice were treated intravenously (I.V) with 12 mg/kg of compound 4 on the
first day, followed by 6 mg/ml for 2 weeks. All doses of the standard drug and
compound 4 were given twice daily.
The effectiveness of the treatments on some biological aspects was evaluated. The
biological parameters included daily body weight (gm), mortality (%), food
consumption (g/day/mouse), water consumption (ml/day/mouse), serum glutamic
oxaloacetic transaminase [SGOT(AST) (U/L)], serum glutamic-pyruvic transaminase
[SGPT(ALT)(U/L)], alkaline phosphatase (U/L), gamma-glutamyl transferase (GGT)
(U/L), bilirubin (mg/dl)], cholesterol (mg/dl), triglycerides (mg/dl), high-density
lipoprotein (HDL) (mg/dl), very-low-density lipoprotein (VLDL) (mg/dl), low-density
lipoprotein (LDL) (mg/dl), lactate dehydrogenase (LDH) (U/L), glucose (mg/dl), total
protein (mg/dl), malondialdehyde (MDA) (nmol/g), nonprotein sulfyhydryl (NP-SH)
(nmol/g). Additional hematologic constituents, including white blood cells (WBC)
(cell/mm3), red blood cells (RBC) (106/mm3), hemoglobin (HGB) (g/dL), HCT,
platelets (PLT) (103/mm3), mean corpuscular volume (MCV) (fL), mean corpuscular
hemoglobin (MCH) (pg), mean corpuscular hemoglobin concentration (MCHC) (g/dL),
neutrophils (%), lymphocytes (%), eosinophils (%) and monocytes (%)] were also
evaluated [36-38]. SGOT (AST), SGPT (ALT), ALP, GGT, bilirubin LDH and total
protein were estimated using a Reflotron Plus Analyzer and Roche kits (Roche
Diagnostics GmbH, Mannheim Germany) and United diagnostic kits assay.
28

JOURNAL PRE-PROOF
Hematological tests were performed by Mindray BC-2800VET Auto Hematology
Analyzer.
3.2.4. Experimental design and statistical analysis
The present experiment was designed as a completed random design (CRD). The data
are arranged as mean± standard deviation (SD) or standard error of mean (SEM). The
Statistical Package for the Social Sciences (SPSS 13.0) was used to analyze the mean
of the biological parameters by using the Tukey post-hoc and Student’s- t-test.
ACKNOWLEDGEMENTS
The authors extend their appreciation to the Deanship of Scientific Research at King
Khalid University for its funding of this prolific research group. no R.G. P.
2/23/40/2019.
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