Synthesis, Antimicrobial Activity and Molecular Docking Study of Novel α-(Diphenylphosphoryl)- and α-(Diphenylphosphorothioyl)cycloalkanone Oximes

Article abstract of DOI:10.1002/cbdv.202000217

A series of novel α-(diphenylphosphoryl)- and α-(diphenylphosphorothioyl)cycloalkanone oximes have been synthesized in search for novel bioactive molecules. Their structures were characterized by various spectroscopic methods including IR, NMR (¹H, ³¹P, ¹³C), mass spectrometry and single crystal X-ray diffraction. The newly synthesized phosphorus-containing oximes were screened for their in vitro antimicrobial activity against Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), Gram-negative bacteria (Escherichia coli and Salmonella typhimurium) and fungal strains (Candida albicans and Candida glabrata). The biological assays showed that all the studied compounds exhibited high antibacterial and antifungal activities at only 0.1–2.1 μg/mL. In silico molecular docking studies in FabH enzyme active site were performed in order to predict the possible interaction modes and binding energies of the drug candidates at the molecular level.

Full text of DOI:10.1002/cbdv.202000217

Title: Synthesis, antimicrobial activity and molecular
docking study of novel α-(diphenylphosphoryl)- and α-
(diphenylphosphorothioyl)cycloalkanone oximes
Authors: Nejib Jebli, Selma Hamimed, Kristof Van Hecke, Jean-
François Cavalier, and Soufiane Touil
This manuscript has been accepted after peer review and appears as an
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To be cited as: Chem. Biodiversity 10.1002/cbdv.202000217
Link to VoR: https://doi.org/10.1002/cbdv.202000217

10.1002/cbdv.202000217
Chemistry & Biodiversity
Synthesis, antimicrobial activity and molecular docking study of novel α-
(diphenylphosphoryl)- and α-(diphenylphosphorothioyl)cycloalkanone
oximes
Nejib Jeblia*, Selma Hamimedb, Kristof Van Heckec, Jean-François Cavalierd, Soufiane Touila
aUniversity of Carthage, Faculty of Sciences of Bizerte, Laboratory of Hetero-Organic Compounds and
Nanostructured Materials (LR18ES11), CP 7021-Jarzouna, Tunisia.
bUniversity of Carthage, Faculty of Sciences of Bizerte, Laboratory of Biochemistry and Molecular Biology,
7021 Jarzouna, Tunisia.
cXStruct, Department of Inorganic and Physical Chemistry, Krijgslaan 281-S3, CP 9000-Ghent, Belgium.
dAix-Marseille Univ, CNRS, LISM, IMM FR, CP 3479-Marseille, France.
*Corresponding author: Dr. Nejib Jebli
Office: +21626516906
E-mail: nejib.jebli@yahoo.com
Abstract:
A
series
of
novel
α-(diphenylphosphoryl)-
and
α-
(diphenylphosphorothioyl)cycloalkanone oximes have been synthesized in search for novel
bioactive molecules. Their structures were characterized by various spectroscopic methods
including IR, NMR (1H, 31P, 13C), mass spectrometry and single crystal X-ray diffraction. The
newly synthesized phosphorus-containing oximes were screened for their in vitro antimicrobial
activity against Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), Gram-
negative bacteria (Escherichia coli and Salmonella typhimurium) and fungal strains (Candida
albicans and Candida glabrata). The biological assays showed that all the studied compounds
(2a-f) exhibited high antibacterial and antifungal activities at only 0.1-2.1 μg/mL. In silico
molecular docking studies in FabH enzyme active site were performed in order to predict the
possible interaction modes and binding energies of the drug candidates at the molecular level.
Keywords: oximes; phosphine oxides; phosphine sulfides; phosphonyloximes; antibacterial
activity; antifungal activity; molecular docking.
Introduction
The emergence of multidrug-resistant strains of bacteria and fungi has brought an urgent
need for the discovery of novel antimicrobial agents with other modes of action.[1-3] One
possible and useful strategy to attain new classes of drug molecules is to develop synthetic
antimicrobials whose chemical structures do not occur in nature, and would thus be
evolutionarily foreign to microbes.
For the past few years, oxime derivatives have attracted the increasing interest of
researchers due to their wide-range of useful applications as antimicrobial,[4-7] antiparasitic,[8,9]
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10.1002/cbdv.202000217
Chemistry & Biodiversity
antiviral,[10] antihistaminic,[11] and anti-HIV[12] agents. Some examples of bioactive oxime
derivatives containing commercial drugs are chromanone and nafimidone oximes which have
demonstrated good performances as antibacterial agents,[13,14] and oxiconazole, an antifungal
medication used to treat skin infections, such as athlete's foot, jock itch and ringworm[15,16]
(Figure 1).
On the other hand, organophosphorus derivatives are a valuable class of compounds in
medicinal chemistry that possess interesting biological effects and different binding affinities
to diverse microbiological targets,[17-19] and the introduction of a phosphoryl or thiophosphoryl
group to the oxime unit could enhance the antimicrobial activity of such compounds, in
accordance with the active substructure combination principle.[20,21] Indeed, some
commercialized antibiotic drugs, such as fosfomycin,[22] tedizolid phosphate[23] and
phosphopyricin (Figure 1),[24] contain in their structures a phosphonic acid, phosphate or
diphenylphosphine moiety. Furthermore, a number of antibiotic and antimalarial drugs used in
clinical medicine, such as fosmidomycin[25] and FR-900098[26] (Figure 1), possess in their core
chemical structure a phosphorus moiety as well as an hydroxamic acid functionality which is
a structural analog of the oxime pharmacophore.[27]
Figure 1. Structures of some antimicrobial agents bearing an oxime or phosphoryl group
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10.1002/cbdv.202000217
Chemistry & Biodiversity
In view of the above, and in the continuation of our research program concerning the
preparation of novel phosphorus-containing compounds with possible biological properties,[28-
31]
we now report the synthesis of unprecedented α-(diphenylphosphoryl)- and α-
(diphenylphosphorothioyl)cycloalkanone oximes bearing both the phosphoryl
/
phosphorothioyl and oxime pharmacophores. The antimicrobial and antifungal properties of
these compounds were studied in vitro, and the results obtained were corroborated by in silico
molecular docking studies.
Results and discussion
Chemistry
The starting α-(diphenylphosphoryl)- and α-(diphenylphosphorothioyl)cycloalkanones 1
were easily prepared from the reaction of a cyclic enamine with P-chlorodiphenylphosphine in
the presence of triethylamine, followed by oxidation or sulfurization and hydrolytic work-up,
according to the reported procedure.[32-34] Reaction of compounds 1 with hydroxylamine
hydrochloride, conducted in refluxing ethanol for 24 h in the presence of an equimolar quantity
of potassium hydroxide, led to the desired α-(diphenylphosphoryl)- and α-
(diphenylphosphorothioyl)cycloalkanone oximes 2. The isolated yield of the reaction ranges
from 81 to 89% (Table 1).
The structures of phosphorus-containing oximes 2 were established from their IR, NMR
(1H, 31P, 13C), and mass spectral data, which indicate that they are obtained as a mixture of two
inseparable Z and E isomers (Scheme 1). Their relative proportions were estimated from the
31P NMR spectra where a singlet for each isomer is present (Table 1). The Z and E
13
configurations were assigned on the basis of the C chemical shifts of carbons in α position
with respect to the C=N double bond. Indeed, according to some literature data regarding the
stereochemistry of oximes,[35-37] the carbon adjacent to the C=N double bond resonates at
higher field when it is in the syn position relative to the oxime OH group. It should be
mentioned that our attempts to separate the Z and E isomers by column chromatography or by
fractional crystallization, have failed.
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10.1002/cbdv.202000217
Chemistry & Biodiversity
Table 1. Synthesis of α-(diphenylphosphoryl)- and α-(diphenylphosphorothioyl)-
cycloalkanone oximes 2
Entry
1
Cycloalkanones 1
Oximes 2
Z-2 (%)[b]
E-2 (%)[b]
73
27
2
55; 35[c]
10
3
4
91
42
9
58
5
6
21
86
68; 11[d]
14
[a] Isolated yield. [b] Determined from the 31P NMR spectra. [c] Two Z-diastereoisomers are present
due to asymmetric carbons. [d] Two E-diastereoisomers are present due to asymmetric carbons.
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10.1002/cbdv.202000217
Chemistry & Biodiversity
Scheme 1. Z and E isomers for oximes 2a-f
X-ray crystallography
Unambiguous structure elucidation of the α-(diphenylphosphoryl)cyclohexanone oxime
2a was achieved through single-crystal X-ray diffraction analysis. The crystallographic data
and structural refinement parameters are summarized in Table 2. Single crystals of 2a were
obtained by slow evaporation at room temperature of a methanol/hexane/ethyl acetate solution.
Oxime 2a crystallized in the monoclinic space group P21/n, with one molecule in the
asymmetric unit (Figure 2a).
The molecule exhibits a regular spatial configuration with habitual distances and angles
(Table S1). The dihedral angle between the two phenyl groups on the phosphorus atom is
104.29°, showing a quasi-orthogonal arrangement of the two phenyl rings due to steric reasons.
The cyclohexanone oxime ring is in the characteristic chair conformation and its
geometrical parameters are reported in Table S1 indicating that the C-C and C-N bond lengths
as well as the C-C-C and C-C-N angles are in conformity with those observed in similar
molecules.[38] The conformation of the cyclohexanone oxime ring can be described in terms of
Cremer and Pople puckering coordinates,[39] by evaluating the parameters Q (total puckering
amplitude), q2, q3,  and . Their calculated values for the C1-C2-C3-C4-C5-C6 ring are: Q =
0.5370 Å, q2 = 0.0811Å, q3 = 0.5308Å,  = 176.34° and  = 8.69°, indicating that the
cyclohexanone oxime ring has been distorted from the standard chair conformation by the
oxime moiety.
The oxime function is predominantly observed in the Z configuration. However, a slight
disorder was observed for this functionality, adopting an E configuration. The disorder was
properly refined in two parts with final occupancy factors of 0.73 and 0.27, for the Z and E
configurations, respectively (Figure 2b). This was in perfect agreement with the NMR results,
which showed the same ratio (73/27) for the Z and E isomers, respectively (Table 1).
The crystal packing indicates that the molecules are interconnected via C=N-O-H…O=P
intermolecular hydrogen bonds to form chains along the a-axis (Figure S1).
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10.1002/cbdv.202000217
Chemistry & Biodiversity
Table 2. Crystallographic data and structure refinement parameters for compound 2a.
Empirical formula
C18H20NO2P
313.32
100
1.54184
Monoclinic
P21/n
7.4395(3)
22.9744(9)
9.4000(4)
90
Formula weight [g mol-1]
Temperature (K)
Wavelength
Crystal system
Space group
a [Å]
b [Å]
c [Å]
α (°)
β (°)
γ (°)
V (Å3)
96.447(4)
90
1596.47(11)
4
Z
ρcalc [g cm-3]
1.304
Absorption coefficient μ [mm-1] 1.577
F(000)
664.0
Crystal size (mm3)
0.218 × 0.111 × 0.091
7.696 to 147.254
9, 28,11
θ range [°]
h, k, l ranges
Reflections collected
Independent reflections [Rint]
Completeness to θ
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2σ(I)]
R indices (all data)
Largest diff. peak/hole [e Å−3]
15096
3181 (0.0494)
98.7%
Full-matrix least squares on F2
3181/10/208
1.045
R1 = 0.0611, wR2 = 0.1700
R1 = 0.0738, wR2 = 0.1853
0.52/-0.40
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10.1002/cbdv.202000217
Chemistry & Biodiversity
Figure 2. (a) X-ray molecular structure of compound 2a. (b) X-ray molecular structure of 2a
showing both Z and E (in yellow) isomers. Thermal displacements ellipsoids are drawn at the
30% probability level.
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Antimicrobial activity
The antimicrobial activity of the synthesized compounds (2a-f) was assessed against Gram-
negative bacteria (Escherichia coli JW 1772, Salmonella typhimurium ATCC 14028), Gram-
positive bacteria (Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633), and
fungal strains (Candida albicans ATCC 28367, Candida glabrata ATCC 15126) using the
broth microdilution method according to the guidelines provided by the National Committee
for Clinical Laboratory Standards.[40,41] Stock solutions of tested compounds (2a-f) (0.1 to 60
µg/mL final concentration) were incubated with an aliquot of each microorganism (0.5
McFarland turbidity standard scale) overnight at 37 °C and 29 °C for bacteria and fungi,
respectively. Ciprofloxacin and fluconazole were used as standard antibacterial and antifungal
drugs, respectively.
To establish the antimicrobial activity of the synthesized compounds on the bacterial and
fungal growth, their respective minimum inhibitory concentration (MIC) and minimum
bactericidal concentration (MBC) [i.e., the minimum fungicidal concentration (MFC) for C
albicans and C. glabatra] were determined by optical density (OD600) reading and the agar
plating method, respectively (see Supplementary Information for detailed protocols).[42]
The critical MIC, MBC (and MFC) values of the tested compounds against each pathogenic
strain are summarized in Table 3 together with the activity of the reference molecules
ciprofloxacin and fluconazole.
The inhibition growth kinetics against E. coli, S. typhimurium, S. aureus, and B. subtilis
containing different concentrations of the synthesized compounds 2a-f (Figure S2) point out a
significant inhibition of all bacterial strains. Indeed, the results show that all the synthesized
compounds (2a-f) displayed good antibacterial activity, better than the standard drug
ciprofloxacin, against all bacterial strains, with MIC values ranging from 0.7 to 1.8 μg/mL. The
five-membered ring compounds 2c and 2f exhibited the best antibacterial activity with MIC
values 3 to 8-fold more potent than those of the standard drug ciprofloxacin. Interestingly, the
tested compounds (2a-f) were found to be more effective against Gram-positive bacteria (MIC
= 0.7-1.2 μg/mL) than Gram-negative ones (MIC = 1.2-1.8 μg/mL), which could be due to the
difference in bacterial membrane composition.[43]
Furthermore, a significant bactericidal activity (99.9%) was noticed for all the studied
compounds (2a-f) with MBC values ranging from 2.0 to 2.1 μg/mL. Five-membered ring
compounds 2c and 2f were confirmed to be the most potent agents, with 2c showing the
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10.1002/cbdv.202000217
Chemistry & Biodiversity
strongest MBC activity against S. typhimurium and S. aureus, and 2f giving the highest MBC
activity against E. coli and B. subtilis.
Importantly, the ratio MBC/MIC was found in the range of 1.1-2.9 (Table 4), indicating
that all the synthesized compounds (2a-f) were bactericidal in nature and not bacteriostatic.[44-
47]
Table 3. MIC, MBC and MFC (µg/mL) of compounds 2a-f and standard drugs against selected
human pathogens.
Gram-negative bacteria
Gram-positive bacteria
Fungi
Compound
E.C. JW
1772
S.T. ATCC
14028
S.A. ATCC
25923
B.S. ATCC
6633
C.A. ATCC
C.G. ATCC
15126
28367
MFC
0.1
MIC MBC
MIC MBC
MIC MBC
MIC
MBC
MFC
1.8
2.116
1.6
2.030
1.0
2.086
1.0
2.132
0.1
2a
1.8
1.6
2.094
2.115
2.106
2.103
2.059
62.5
1.8
1.2
1.8
1.5
1.3
5.8
2.135
2.006
2.032
2.016
2.022
31.2
0.9
0.7
1.2
1.0
0.8
5.8
2.070
2.056
2.089
2.114
2.082
31.2
0.9
0.8
0.9
1.0
0.7
1.9
2.083
2.090
2.080
2.083
2.065
15.6
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
2b
2c
2d
2e
1.8
1.8
1.6
2f
10.9
Ciprofloxacin
Fluconazole
-
-
64.0
64.0
-
-
-
-
-
-
-
-
E.C.: Escherichia coli; S.T.: Salmonella typhimurium S.A.: Staphylococcus aureus; B.S.: Bacillus subtilis;
C.A.: Candida albicans; C.G.: Candida glabrata.
Table 4. Ratio MBC/MIC of compounds 2a-f.
Compounds
2a
2b
2c
2d
2e
2f
Strains
1.17
1.26
1.16
1.18
1.32
1.67
1.17
1.12
1.16
1.34
1.28
1.55
Escherichia coli
Salmonella typhimurium
Staphylococcus aureus
Bacillus subtilis
2.08
2.13
2.30
2.31
2.93
2.61
1.74
2.31
2.11
2.08
2.60
2.95
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The antifungal activity of the synthesized compounds was assessed through the inhibition
of Candida albicans and Candida glabrata as fungal strains. As shown in Table 3, all the tested
compounds (2a-f) also displayed good fungicidal activity, better than the standard drug
fluconazole, with an excellent MFC value of 0.1 µg/mL.
Considering their overall efficacy, the five-membered ring oximes 2c and 2f were found
to be the most potent of all the tested compounds. However, the increase in the size of the ring
and its substitution resulted only in a very slight decrease of the antibacterial activity. Thus,
compounds 2c and 2f can be regarded as a new valuable source to produce novel oxime
derivatives against bacterial and fungal strains. This indicates that the introduction of
significant phosphorus substituants to cyclopentanone oximes was as effective in obtaining of
potent antimicrobial agents and might be a potential source for synthetic products for the
pharmaceutical industry against bacterial and fungal strains.
Molecular docking analysis
Among the molecular targets in pathogenic strains, the β-ketoacyl-acyl carrier protein
synthase III (FabH), which initiates the fatty acid elongation cycles by catalyzing the first
condensation step between acetyl-CoA and malonyl-ACP,[48] has been validated as an
attractive drug target.[49] Moreover, FabH is not only highly conserved at the sequence and
structural levels among key pathogens; but above all, the amino acid residues (i.e., Cys-His-
Asn) forming its catalytic triad are essentially invariant across Gram-positive and Gram-
negative organisms.[50,51]
Given the fact that oxime derivatives have been previously described to inhibit E. coli
FabH,[49] and the promising antibacterial activity of compounds 2c and 2f against Gram-
positive and Gram-negative, in an attempt to correlate such activity with the putative binding
mode of these latter inhibitors in the active site of this enzyme, in silico molecular docking
experiments were conducted as described previously,[52-54] using the reported crystal structure
of E. coli FabH (EcFabH) in complex with Malonyl-CoA in its active site (PDB entry code:
1HNJ).[55]
The best scoring position obtained (i.e., lowest energy complex) showed that compounds
2c and 2f may be located at the entrance of the EcFabH active site with predicted binding
energy values ΔE2c = −7.0 kcal/mol and ΔE2f = −6.6 kcal/mol, respectively. In both cases, 2c
and 2f would be located at the right entrance of the catalytic cleft, at a distance of around 9 Å
from the catalytic Cys112, thus excluding any covalent interaction (Figure 3A).
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10.1002/cbdv.202000217
Chemistry & Biodiversity
Each of the docked 2c-EcFabH and 2f-EcFabH complexes was further subjected to
interactions analysis using Ligplot+ v.1.4.[56] The Ligplot+ diagram schematically depicts the
hydrogen bonds and hydrophobic interactions between the ligand (i.e., oximes 2c or 2f), and
the active site residues of the protein (i.e., EcFabH) during the binding process. In the obtained
binding model, the two bulky phenyl groups of 2c may be involved in π-stacking interactions
with Trp32, Thr37, GLy152, Ile156, Met207, Gly209, Phe213, and Ala246; while the cyclopentanone
oxime would be stabilized by 2 H-bonding with Arg36 and Asn247 residues and interact with
Asn210 and Trp32 residues (Figure 3B). Although similar hydrophobic interactions were
observed with compound 2f, only one H-bonding involving Asn247 residue would be detected
(Figure 3C).
Such binding modes for both 2c and 2f, would suggest that these two compounds might
be able to block (or at least partially) the access of the substrate into the EcFabH protein’s
active site, therefore resulting in the potential enzyme activity inhibition.
Figure 3. (A) In silico molecular docking of cycloalkanone oximes 2c (yellow color) and 2f (cyan color) into the
crystallographic structure of E. coli FabH in a van der Waals surface representation. The Cys112-His244-Asn274 catalytic residues
of EcFabH active site are in magenta stick representation. Hydrophobic residues (alanine, leucine, isoleucine, valine,
tryptophan, tyrosine, phenylalanine, proline, and methionine) are highlighted in white. (B-C) Ligplot+ analyses results: 3D
representation of the schematic binding mode of compounds 2c (B; yellow color) and 2f (C; cyan color) in complex with
EcFabH showing both hydrogen bonds (yellow dashed lines) and hydrophobic interactions. Selected residues of the EcFabH
binding pocket are shown as gray sticks. The stick representation uses the following atom color-code: nitrogen, blue; oxygen,
red; sulfur, yellow; and phosphorus, orange. Structures were drawn with PyMOL Molecular Graphics System (version 1.4,
Schrödinger, LLC) using the PDB file 1HNJ.[55]
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10.1002/cbdv.202000217
Chemistry & Biodiversity
Conclusion
We have described a two-step approach to unprecedented α-(diphenylphosphoryl)- and α-
(diphenylphosphorothioyl)cycloalkanone oximes (2a-f). Their structures were characterized by
various spectroscopic methods including IR, NMR (1H, 31P, 13C), mass spectrometry and single
crystal X-ray diffraction. The synthesized compounds were screened for their in vitro
antibacterial and antifungal activities against several Gram-negative, Gram-positive and fungal
strains. The biological assays showed that all the studied compounds (2a-f) exhibited high
antibacterial and antifungal activities, better than the standard drugs ciprofloxacin and
fluconazole, with MIC, MBC and MFC values in the range of 0.7-1.8, 2.0 and 0.1 µg/mL,
respectively. Five-membered ring compounds 2c and 2f were found to be the most potent
antibacterial agents. We tried to correlate these results with those obtained in the in silico
molecular docking study with the E. coli β-ketoacyl-acyl carrier protein synthase III (EcFabH).
These experiments have brought useful and informative data regarding the potential binding
mode of oximes 2c and 2f inside the EcFabH enzyme's active site, therefore providing some
clues for developing new lead compounds as potent antibacterial agents. Moreover, from the
molecular docking results, one cannot exclude that the antibacterial activity of compounds 2c
and 2f towards Gram-negative and Gram-positive bacteria might result from the inhibition of
FabH protein.
Experimental Section
Chemistry
Commercially available reagents and solvents were used without further purification.
Melting points were determined in open glass capillaries and are uncorrected. The infrared
spectra were recorded in the 400-4000 cm-1 range with a Nicolet IR200 FT-IR spectrometer
using a neat sample at ambient temperature. The number of scans was 32 and the resolution 4
cm-1. NMR spectra were recorded at 400 and 500 MHz (1H), 161 and 202 MHz (31P), 100 and
126 MHz (13C APT) in CDCl3 at a concentration of 50 mg/mL and at 25 °C. Chemical shifts
(δ) are reported in part per million (ppm) relative to the residual solvent peak. The coupling
constants are reported in Hz. The multiplicities of signals are indicated by the following
abbreviations: s: singlet; d: doublet; t: triplet; and m: multiplet. High-resolution-MS spectra
were performed on a JOEL JMSGCmateII mass spectrometer. Column chromatography was
performed on silica gel (70-230 mesh ASTM) using the reported eluent. Thin layer
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10.1002/cbdv.202000217
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chromatography (TLC) was carried out on 5 x 20 cm plates with a layer thickness of 0.25 mm
(Silica gel 60 F254).
Synthesis of cyclic enamines
The starting cyclic enamines were prepared according to the procedure reported by
Stork[57] with slight modification:
A mixture of cyclic ketone (1 mol) and morpholine (1.7 mol) in dry toluene (30 mL) was heated
at reflux, with Dean-Stark separation of water, for 4 h. The solvent was then removed under
vacuum and the residue obtained was distilled under reduced pressure to give pure enamine in
more than 90% yield.
General
procedure
for
the
synthesis
of
α-(diphenylphosphoryl)-
and
α-
(diphenylphosphorothioyl)cycloalkanones 1a-1f
The α-(diphenylphosphoryl)- and α-(diphenylphosphorothioyl)cycloalkanones 1 were
prepared according to the procedure reported by Barkallah et al: [32]
To a well stirred solution of enamine (1 mmol) and triethylamine (1.1 mmol) in dry acetonitrile
(15 mL), maintained under an inert atmosphere (N2) and cooled at 0 °C, P-
chlorodiphenylphosphine (1 mmol) in dry acetonitrile (3 mL) was added dropwise within 15
min. The resulting solution was warmed up to room temperature and stirred for 2 h. The
reaction mixture was then treated with DMSO or sulphur as follows:
- Oxidation: DMSO (1 mmol) was added and the mixture was heated under reflux for 2 h. After
cooling, 2N aqueous HCl solution (30 mL) was added dropwise at 0 °C and stirring was
continued at room temperature for 12 h. The mixture was then extracted with CH2Cl2 (3 × 10
mL). The organic phase was dried over MgSO4 and concentrated under vacuum. The residue
obtained was chromatographed on a silica gel column using n-hexane/ethyl acetate (50:50 %
v/v) as eluent.
- Sulfurization: Ground sulfur (1 mmol) was added and the reaction mixture was stirred at room
temperature until complete dissolution of the sulfur in 2 h. 2N aqueous HCl solution (30 mL)
was then added dropwise at 0 °C and stirring was continued at room temperature for 12 h. The
mixture was then extracted with CH2Cl2 (3 × 10 mL). The organic phase was dried over MgSO4
and concentrated under vacuum. The residue obtained was chromatographed on a silica gel
column using n-hexane/ethyl acetate (50:50 % v/v) as eluent.
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General procedure for the synthesis of α-(diphenylphosphoryl)- and α-
(diphenylphosphorothioyl)cycloalkanone oximes (2a-2f)
To a mixture of hydroxylamine hydrochloride (0.01mol), potassium hydroxide (0.01 mol)
and dry ethanol (20 mL),
a
solution of α-(diphenylphosphoryl)- or α-
(diphenylphosphorothioyl)cycloalkanones 1 (0.01 mol) was added dropwise with stirring, at
25°C, in dry ethanol (10 mL). The reaction mixture was then heated under reflux for 24 h. After
cooling, the solvent was removed under reduced pressure. The residue obtained was diluted
with CHCl3 (30 mL) and washed with water (2×20 mL). The organic phase was dried over
Na2SO4 and concentrated under vacuum. After workup, Column chromatography was
performed when necessary using n-hexane/ethyl acetate (60:40 % v/v) as eluent.
X-ray diffraction data
For the structure of compound C18H20NO2P (2a), X-ray intensity data was collected at 100
K, on a Rigaku Oxford Diffraction Supernova Dual Source (Cu at zero) diffractometer
equipped with an Atlas CCD detector using  scans and CuK ( = 1.577 Å) radiation. The
images were interpreted and integrated with the program CrysAlisPro (Rigaku Oxford
Diffraction).[58] Using Olex2,[59] the structure was solved by direct methods using the
ShelXS[60] structure solution program and refined by full-matrix least-squares on F2 using the
ShelXL[61] program package. Supplementary data related to the X-ray diffraction can be found
at https://www.ccdc.cam.ac.uk. CCDC 1989019 contains the supplementary crystallographic
data
for
this
paper
and
can
be
obtained
free
of
charge
via
www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data
Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: +44-1223-336033; or
deposit@ccdc.cam.ac.uk).
Antimicrobial activity
Microorganisms and growth conditions
Six different strains of pathogenic microorganisms, including Gram-negative bacteria:
Escherichia coli and Salmonella typhimurium, Gram-positive bacteria: Staphylococcus aureus
and Bacillus subtilis, and fungi: Candida albicans and Candida glabrata, were employed to
test the antimicrobial activity of all synthesized compounds. All bacteria were cultured in
Nutrient Broth (BK003HA; Biokar) at 37 °C for 24 h; while the fungi were grown on
Sabouraud Dextrose Broth (AM5088; Microxpress; Tulip Diagnostics) for 48 h at 29 °C.
This article is protected by copyright. All rights reserved.

10.1002/cbdv.202000217
Chemistry & Biodiversity
Bioassays
The broth microdilution method was used to determine the antimicrobial activity according
to CLSI guidelines.[40,41] Overnight microorganism’s cultures were diluted with the appropriate
sterile broth to obtain a suspension of about 1.37 × 106 CFU/mL. Stock solutions of tested
compounds (2a-f) were prepared in DMSO by serial two-fold dilution in the previous
appropriate sterile broth to obtain a concentration range from 0.1 to 60 µg/mL. An aliquot of
working suspension (100 µL) was added to a sterile 96-well plate containing (100 µL) of tested
stock solutions. Wells containing DMSO-microorganism inoculum, and those only
microorganism suspensions were served as negative control. The in vitro activities of
ciprofloxacin against bacteria and fluconazole against fungi were evaluated as positive control
with the same serial two-fold dilution as the tested compounds. The microplates were incubated
for 24 h at 37ꢀ°C and 29 °C for bacteria and fungi, respectively. Then, the minimum inhibitory
concentration (MIC) was defined as the lowest concentration of the tested compounds that
completely inhibited bacterial growth via OD600 reading. The minimum bactericidal and
fungicidal concentrations (MBC and MFC) were recorded as the lowest concentration giving
no visible colony on agar plate. These parameters were measured according to Lehtinen et
al.,[42] with some adjustments. We determined the optical density OD600 using a 96-well
microplate reader (VersaMax with SOFTmax). Each broth medium is used as the background,
and the MIC was defined by an optical density equal to 0.2. The agar plate was used to define
the MBC and MFC by counting the colonies (CFU/mL) using the following equation:
N colonies on plate
퐶퐹푈/푚퐿 =
dilution × volume plate
Briefly, at the end of the 24 h-incubation periods, 100 µL of the suspension was plated onto
Nutrient Broth Agar and Sabouraud Dextrose Broth Agar medium. Agar plates were incubated
overnight at the desire temperature described above. Compounds with MIC values close to the
MBC values, were further evaluated through microdilutions, in the concentration range from
2.000 to 2.300 µg/mL, to precisely determine individual MBC value. Each analysis was
performed in triplicate.
Molecular docking experiments
A computational docking and scoring procedure using the Autodock Vina program[62] was
used to generate the putative binding modes of cycloalkanone oximes 2c and 2f into the active
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10.1002/cbdv.202000217
Chemistry & Biodiversity
site of the E. coli β-ketoacyl-acyl carrier protein synthase III (EcFabH) as previously
described.[63,64] The PyMOL Molecular Graphics System (version 1.4, Schrödinger, LLC) was
used as working environment with an in-house version of the AutoDock/Vina PyMOL
plugin.[65] The X-ray crystallographic structure of EcFabI in complex with Malonyl-CoA (PDB
entry code: 1HNJ; 1.46 Å resolution)[55] available at the Protein Data Bank was used as
receptor. Docking runs were performed after removing the ligand (i.e., Malonyl-CoA) from the
enzyme active site. The box size used for the receptor was chosen to fit the whole active site
cleft and to allow non-constructive binding positions. The three-dimensional structures of the
aforementioned compounds were constructed using Chem 3D ultra 11.0 software [Chemical
Structure Drawing Standard; Cambridge Soft corporation, USA (2007)], and their geometry
was refined using the Avogadro open-source molecular builder and visualization tool (version
1.2.0. http://avogadro.cc/)
To check the accuracy of our docking experiment, the N-((3'-(hydroxymethyl)biphenyl-4-
yl)methyl)benzenesulfonamide inhibitor (i.e., Cpd6) for which a 3D structure in complex with
EcFabH is available (PDB entry code: 5BNM)[66] was submitted to in silico molecular docking
into EcFabH active site. Comparison of the generated Cpd6 docking pose with the available
binding geometry observed in the 5BNM crystal structure revealed root mean square deviations
of 0.484 Å for the top ranked pose (predicted binding energy value, ECpd6 = -8.2 kcal/mol).
This re-/cross-docking experiment thus confirms the validity and reliability of our
computational approach.
Acknowledgement
The authors are grateful to the Tunisian Ministry of Higher Education and Scientific Research
for funding.
Author Contribution Statement
N. Jebli synthesized the compounds and analyzed the data. S. Hamimed conducted the
antimicrobial assays. K.V. Hecke performed the X-ray diffraction analysis. J-F. Cavalier
carried out the in silico molecular docking studies. S. Touil designed and supervised the work
and analyzed the data.
Supplementary data
Supplementary data (which contain NMR and mass spectra and other informations regarding
the crystal structure and bioactivity of the title compounds) associated with this article can be
found, in the online version, at http://dx.doi.org.
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10.1002/cbdv.202000217
Chemistry & Biodiversity
Conflict of interest
There are no conflicts to declare.
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Chemistry & Biodiversity
Graphical Abstract
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