New spiro-oxindole constructed with pyrrolidine/thioxothiazolidin-4-one derivatives: Regioselective synthesis, X-ray crystal structures, Hirshfeld surface analysis, DFT, docking and antimicrobial studies

Article abstract of DOI:10.1016/j.molstruc.2017.09.086

In this work, polycyclic heterocycles containing spirooxindole, pyrrolidine, and thioxothiazolidin-4-one rings have been synthesized via the regioselective 1,3-dipolar cycloaddition of azomethine ylide, which is generated in situ by the condensation of th

Full text of DOI:10.1016/j.molstruc.2017.09.086

Accepted Manuscript
New spiro-oxindole constructed with pyrrolidine/thioxothiazolidin-4-one derivatives:
Regioselective synthesis, X-ray crystal structures, Hirshfeld surface analysis, DFT,
docking and antimicrobial studies
Assem Barakat, Saied M. Soliman, Abdullah Mohammed Al-Majid, M. Ali, Mohammad
Shahidul Islam, Yaseen A.M.M. Elshaier, Hazem A. Ghabbour
PII:
S0022-2860(17)31284-X
10.1016/j.molstruc.2017.09.086
MOLSTR 24335
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 14 June 2017
Revised Date: 21 August 2017
Accepted Date: 22 September 2017
Please cite this article as: A. Barakat, S.M. Soliman, A.M. Al-Majid, M. Ali, M.S. Islam, Y.A.M.M.
Elshaier, H.A. Ghabbour, New spiro-oxindole constructed with pyrrolidine/thioxothiazolidin-4-one
derivatives: Regioselective synthesis, X-ray crystal structures, Hirshfeld surface analysis, DFT, docking
and antimicrobial studies, Journal of Molecular Structure (2017), doi: 10.1016/j.molstruc.2017.09.086.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
New spiro-oxindole constructed with
pyrrolidine/thioxothiazolidin-4-one derivatives:
Regioselective synthesis, X-ray crystal structures,
Hirshfeld surface analysis, DFT, docking and
antimicrobial studies
Assem Barakata,c,*, Saied M. Solimanb,c, Abdullah Mohammed Al-Majida, M. Alia,
Mohammad Shahidul Islama, Yaseen A. M. M. Elshaierd, and Hazem A. Ghabboure,f
a
Department of Chemistry, College of Science, King Saud University, P.O. Box 2455,
Riyadh 11451, Saudi Arabia.
b Department of Chemistry, College of Science & Arts, King Abdulaziz University, P.O. Box
344 Rabigh 21911, Saudi Arabia.
3c
Department of Chemistry, Faculty of Science, Alexandria University, P.O. Box 426,
Ibrahimia, Alexandria 21321, Egypt.
d
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Al-Azhar
University, Assuit 71524, Egypt.
e Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.
O. Box 2457, Riyadh 11451, Saudi Arabia.
f
Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura,
Mansoura 35516, Egypt.
* Correspondence: ambarakat@ksu.edu.sa; Tel.: +966-114-675-901; Fax:
+966-114-675-992.

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Abstract: In this work, polycyclic heterocycles containing spirooxindole, pyrrolidine, and
thioxothiazolidin-4-one rings have been synthesized via the regioselective 1,3-dipolar
cycloaddition of azomethine ylide, which is generated in situ by the condensation of the
dicarbonyl compound isatin and the secondary amino acid (L-proline), with
5-arylidine-2-thioxothiazolidin-4-one as the dipolarophile. The structure of the synthesized
compounds 4a and 4b, was determined by using X-ray single crystal diffraction, and also,
Hirshfeld surface analysis were reported. Their geometric parameters were calculated using
density functional theory at the B3LYP/6-311G (d,p) level of theory. Both compounds
showed antimicrobial and antifungal activity better than selected standards (ampicillin and
gentamicin in case of antibacterial activity and Amphotricin A and fluconazole in case of
antifungal activity). Molecular docking study of the synthesized compounds indicated that
phenyl group plays an important role in determination of compound interaction inside the
receptors.
Keywords: spirooxindole; thioxothiazolidin-4-one; Hirshfeld surface analysis; DFT;
anti-microbial activity
1. Introduction
The spirocyclic ring structure is a unique feature of a number of natural and synthetic
products that possess interesting biological activities. The potential use of spiro derivatives
in medicinal chemistry has been well recognized owing to their antimycobacterial,
antitumor, antiproliferative, and anti-tuberculosis activities [1−15]. Similarly, the
4-thiazolidinone moiety has been utilized for the synthesis of a variety of useful heterocyclic
products including drugs [16,17], dyes, and intermediates such as thioflavin T., thiazole
yellow, and thidiazuron [18]. Furthermore, 4-thiazolidinone derivatives have been used as

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insecticides [19,20] and herbicides [21] owing to their low toxicity toward human beings and
excellent biological activity. The thiazolidinone moiety is also associated with a broad
spectrum of biological activities including antidiabetic [22], antifungal, antibacterial,
anti-inflammatory, anticonvulsant, hypnotic, anti-tuberculosis, antihistaminic, antiviral,
cardiovascular, anthelmintic, and anticancer properties [23,24]. A number of
4-thiazolidinone derivatives have been investigated for their inhibitory effect on the
oxidation of β-hydroxybutyrate substrate by rat brain homogenates and in the tricarboxylic
acid cycle during respiratory activity [25]. Therefore, development of new spiro-
heterocycles having oxindole, pyrrolidine, or thiazolidinone rings is worthwhile from the
perspective of medicinal chemistry.
Recently, several methods have been reported in literature for the synthesis of oxindole
derivatives with spiro heterocycles [26,27]. Generally, isatin and its derivatives have been
employed as the starting materials for 1,3-dipolar cycloaddition reactions that yield the
spirooxindole core owing to the facile preparation of the corresponding azomethine ylides
in the presence of α-amino acids [28−33]. Ponnala et al. have reported an example of
1,3-dipolar cycloaddition reactions with olefins that proceed regio- and stereoselectively to
furnish the novel dispiropyrrolidine scaffold [34]. Liu et al. have developed an efficient
three-component
reaction
of
5-arylidene-4-thioxo-1,3-thiazolidine-2-one
or
5-arylidene-1,3-thiazolidine-2,4-dione, sarcosine, and isatin for the synthesis of
dispiropyrrolidine derivatives in ethanol under ultrasound irradiation [35]. Another example
reported by Prasad and coworkers is a facile method for the syntheses of
pyrrolizidine-substituted
benzo[h]quinoline,
quinoline,
dispiropyrrolidine,
and

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thiapyrrolizidine compounds in good yields via multicomponent 1,3-dipolar cycloaddition
reactions of azomethine ylide [36]. However, to the best of our knowledge, there are no
reports on the synthesis of this regioisomers and diastereoisomers of dispiro compounds
containing rhodanine.
In this work, the synthesis and X-ray crystal structures of diastereomerically pure
spiro-oxindole, pyrrolidine, and thiazolidinone rings have been reported for the first time.
Moreover, the anti-microbial activities and molecular docking study as well was addressed
on this work.
2. Materials and Methods
2.1. General remarks
“All chemicals were purchased from Aldrich, Sigma-Aldrich, and Fluka and used
without further purification, unless otherwise stated. All melting points were measured on a
Gallenkamp melting point apparatus in open glass capillaries and are uncorrected. IR spectra
were measured as KBr pellets on a Nicolet 6700 FT-IR spectrophotometer. NMR spectra
1
were recorded on a Varian Mercury Jeol-400 NMR spectrometer. H (400 MHz) and
13C-NMR (100 MHz) were measured in deuterated dimethyl sulfoxide (DMSO-d6).
Chemical shifts (δ) are in ppm, and J-coupling constants are given in Hz. Mass spectra were
recorded on a Jeol JMS-600 H instrument. Elemental analysis was carried out on an Elmer
2400 Elemental Analyzer in CHN mode. For X-ray diffraction analysis, data were collected
on a Bruker APEX-II D8 Venture diffractometer with an area detector”.

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3.2. General procedure for the synthesis of 4a,b (GP1)
A stirred mixture of isatin 2 (74 mg, 0.5 mmol), L-proline 3 (57.5 mg, 0.5 mmol), and
5-benzylidene-2-thioxothiazolidin-4-one 1a,b (0.5 mmol) in MeOH (10 ml) was heated
under reflux conditions for the specified period of time. After completion of the reaction was
indicated by TLC, the solvent was evaporated under reduced pressure and the residue was
purified by column chromatography using petroleum ether–ethyl acetate (3:1) as the eluent.
(1'R,7a'R)-2''-Thioxo-2'-(p-tolyl)-5',6',7',7a'-tetrahydro-2'H-dispiro[indoline-3,3'-pyrrolizin
e-1',5''-thiazolidine]-2,4''-dione, 4a
IR (KBr, ν, cm−1): 3246, 1722, 1618, 1595, 1515, 1471; 1H NMR (400 MHz, DMSO-d6) δ:
11.80 (bs, 1H, NH), 9.86 (bs, 1H, NH), 7.84−7.66 (m, 8H, Ar-H), 5.49−5.41 (d, J = 9.3 Hz,
1H) , 4.10 (m, 2H), 2.91 (t, 2H), 2.28 (m, 2H), 2.10 (s, 3H), 1.12 (m, 2H); 13C NMR (100
MHz, DMSO-d6) δ: 26.9, 30.6, 47.3, 51.5, 54.9, 63,6, 71.5, 73.7, 107.4, 111.9, 113.3,
122.2, 129.7, 131.5, 136.4, 144.2, 162.0, 170.2, 174.2, 178.1; HRMS (m/z) calculated for
[M+1]+ C23H21N3O2S2 435.11, found: 436.11. Elemental Analysis: C, 63.42; H, 4.86; N,
9.65; S, 14.72; found C, 63.45; H, 4.85; N, 9.70; S, 14.71.
(1'R,7a'R)-2'-Phenyl-2''-thioxo-5',6',7',7a'-tetrahydro-2'H-dispiro[indoline-3,3'-pyrrolizine-
1',5''-thiazolidine]-2,4''-dione, 4b
1
IR (KBr, ν, cm−1): 3340, 1710, 1610, 1466; H NMR (400 MHz, DMSO-d6) δ: 11.81 (bs,
1H, NH), 9.91 (bs, 2H, NH), 7.98−7.70 (m, 8H, Ar-H), 5.55−5.51 (d, J = 9.3 Hz, 1H) , 4.12
(m, 2H), 2.95 (t, 2H), 2.29 (m, 2H), 1.17 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ: 26.5,
48.0, 53.8, 56, 67.8, 72.6, 79.6, 83.4, 100.5, 108.3, 111.0, 122.4, 129.8, 131.4, 139.3, 144.0,

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162.3, 170.0, 174.1, 178.0; HRMS (m/z) calculated for C22H19N3O2S2 [M+1]+: 421.09,
found: 422.09; Elemental Analysis: C, 62.69; H, 4.54; N, 9.97; S, 15.21; found: C, 62.71; H,
4.55; N, 10.00; S, 15.22.
2.3 Computational details
The X-ray structures of the studied compounds were optimized by using the Gaussian 09
program [37,38]. For this task, the B3LYP method and 6-311G(d,p) basis sets were used for
all atoms. Frequency calculations gave no imaginary frequencies, indicating that the
optimized structure is an energy minimum as there were no negative modes.
2.4. Hirshfeld surface analysis
Hirshfeld surfaces (HSs) and 2D fingerprint plots (FPs) were generated by using the
Crystal Explorer 3.1 software [39]. The crystallographic information files (CIF) obtained
from the X-ray single crystal measurements were used for performing the HS analysis. The
HSs and FPs were constructed based on the electron distribution calculated as the sum of
spherical atom electron densities [40−47].
2.4. Agar Diffusion Well Method to Determine the Antimicrobial Activity
2.4.1. Antibacterial Activity of Compound 4a,b
“Antibacterial activities were investigated by using agar well diffusion method, against
the Staphylococcus pneumonia (RCMB 010010) and Bacillus subtilis (RCMB 010067) (as
Gram-positive bacteria) and Pseudomonas aeruginosa (RCMB 010043) and Escherichia
coli (RCMB 0100052) (as Gram-negative bacteria). The solution of 10 mg/mL of compound
in DMSO was prepared for testing against bacteria. Centrifuged pellets of bacteria from 24 h

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old culture containing approximately 104–106 CFU (colony forming unit) per mL were spread
on the surface of nutrient agar (type tone 1%, yeast extract 0.5%, NaCl 0.5%, agar, and 1000
mL of distilled water, pH 7.0) which was autoclaved under 121 °C for at least 20 min. Wells
were created in medium with the help of sterile metallic bores and then cooled down to 45 °C.
The activity was determined by measuring the diameter of the inhibition zone
(in mm). A volume of 100 ꢀL of the tested sample (10 mg/mL) was loaded into the wells of
the plates. A solution of the compound was prepared in DMSO, while DMSO was also loaded
as control. The plates were kept for incubation at 37 °C for 24 h and then the plates were
examined for the formation of zones of inhibition. Each inhibition zone was measured three
times by caliper to get an average value. The test was performed three times for each bacterium
and the average was taken. Ampicillin and Gentamicin were used as antibacterial standard
drugs” [48].
2.4.2. Antifungal Activity of Compound 4a,b
Tested sample was screened in vitro for its antifungal activity against various fungi,
namely, Aspergillus fumigatus (RCMB 002568), Syncephalastrumracemosum (RCMB 016001)
Geotricumcandidum (RCMB 05097) and Candida albicans (RCMB 05036). The antifungal
activity was performed by agar well diffusion method [48].
“Fungal strains were grown in 5 mL Sabouraud dextrose broth (glucose/peptone; 40/10) for
3–4 days to obtain 105 CFU/mL cells. The fungal culture (0.1 mL) was spread uniformly on the
Sabouraud dextrose agar plates by sterilized triangular folded glass rod. Plates were left for
5–10 min so the culture was properly adsorbed on the surface. Then small wells 4 mm × 2
mm were cut into the plates with the help of well cutter and the bottoms of the wells were
sealed with 0.8% soft agar to prevent the flow of test sample at the bottom of the well. 100 ꢀL
of the tested sample (10 mg/mL) was loaded into the wells of the plates. Compound 1
dissolved in DMSO, while pure DMSO was also used as control. The plates were kept for
incubation at 30 °C for 3–4 days and then examined for the formation of zones of inhibition. The

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test was performed three times for each fungus and the average was taken. Amphotericin
B/Fluconazole were used as standard antifungal drug” [48].
2.5. Docking studies
This was done using OpenEye molecular Modeling software. A virtual library of
Spirooxoindile derivatives were energy minimized using MMFF94 force field, followed by
generation of multi-conformers using OMEGA application. The whole energy minimized
library will be docked along with the prepared PDB ID: 3HAM and PDB ID: 4WMZ using
FRED application to generate a physical property (ꢁG) reflecting the predicted energy
profile of ligand-receptor complex. Vida application can be employed as a visualization
tool to show the potential binding interactions of the ligands to the receptor of interest
[49-51].
3. Results and discussion
3.1. Synthesis
Synthesis of compounds 4a and 4b has been achieved via the 1,3-dipolar cycloaddition
reaction protocol [52] and illustrated in Scheme 1. In our initial endeavor, Knoevenagel
adducts 1a, b were prepared following our previously reported method [23]. Next, the
three-component reaction between 5-arylidene-2-thioxothiazolidin-4-one (1a and 1b), isatin
(2), and L-proline (3) was carried out at 60 °C in refluxed MeOH, for 5−8 h to produce 4a and
4b with two spiro centers in regioselective manner with 85% and 89% yields, respectively.
The structure of the cycloadduct was confirmed by spectroscopic analyses and it was found
that the reaction afforded adducts 4a and 4b as single regioisomers. The structures of the

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1
compounds were deduced by H and 13C NMR, and IR spectroscopies, MS, elemental
analysis, and X-ray crystallography.
Scheme 1. Synthesis of 4a and 4b
Exclusive regioselectivity of the new spiro-oxindole constructed with
pyrrolidine/thioxothiazolidin-4-one derivatives formation was deduced from spectral
characterization. The 1H-NMR spectrum for compounds 4a shown δ 11.81 and 9.86 (bs) for
the secondary amine proton NH, at δ 7.84−7.66 (m) for the aromatic protons, at δ 5.49−5.41
(d) with J = 9.3 Hz for the benzylic proton, and the pyrrolidine ring protons have been
assigned at δ 4.10, 2.91, 2.28, and 1.12 ppm and finally at δ 2.10 (s, 3H) assigned for the
methyl group protons. Also, 13C-NMR spectrum shown agreement with the expected
compound. Moreover, the MS spectrum was also observed at 436.11(m/z) calculated for
[M+1]+ C23H21N3O2S2. IR spectrum, which correlated with the functional groups including
NH, C=C, C=S and C=O. Confirmation of the assignment of the regioisomer was achieved
by X-ray single crystal diffraction technique. The crystallographic perspective drawing
(Figure 1) shows that the structure of 4a in the solid state is in full accord with the

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regioisomer as elucidated in solution by spectral analysis. Similarly, the spectrum for the
second new spiro-oxiindole 4b was in full agreement with expected product.
The proposed mechanism for the synthesis of spiro-oxindolepyrrolidine
thioxothiazolidin-4-one derivatives (4) is described in Scheme 2. The reaction proceeds via
the generation of azomethine ylide (dipolar structure 6, Scheme 2). Azomethine 6 was
obtained by the condensation of isatin (2) with L-proline (3) and subsequent decarboxylation
of the condensation product 5. Dipolarophiles 1a and 1b react with the respective azomethine
ylides (6) in methanol to give the desired products containing spiro-oxindole, -pyrrolidine,
and -thioxothiazolidin-4-one moieties, 4a-b. The regiochemistry of the cycloaddition
reaction can be explained by considering secondary orbital interactions (SOI) of the arylidine
double bond of the dipolarophile 1 with those of the ylide, as shown in Scheme 2. It is evident
that the formation of regioisomer 4 via path A is more favorable, which is not possible in path
B. this could be attributed due to formation of Z isomer of 1a-b which allow the approach of
azomethine from one side only. Hence, only one regioisomer 4 was obtained in the reaction.
Furthermore, the steric factor plays an additional role in the regioselective formation of
products.
Scheme 2: Plausible approaches for the target compound

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O
O
N
N
O
-CO2
COOH
+
O
O
O
N
H
N
N
N
H
H
H
2
3
5
6
O
HN
S
O
N
S
O
N
HN
HN
NH
N
S
O
S
6
NH
4
7
+
S
O
O
HN
HN
O
N
S
N
R
HN
O
O
O
NH
Z form: 1a,b
S
HN
4'
S
S
S
7'
3.2. X-ray crystal structure of compounds 4a,b
The crystal structures of compounds 4b and 4b were determined by single-crystal X-ray
diffraction (Figure 1) [53,54]. The crystal and structure refinement data, as well as selected
geometric parameters for compounds 4b and 4b are given in Tables 1, 2, and 4. The
asymmetric unit of 4a and 4b comprised one independent molecule of these compounds and
the solvent molecules methanol and dichloromethane, respectively. In the crystal packing of
4a, its molecules are linked by six hydrogen bonding interactions that result in a
three-dimensional network structure (Figure 2 and Table 3). In case of 4b, the molecules are
also packed together utilizing six intermolecular interactions to form a network structure as
shown in Figure 2 and Table 5.

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4a
4b
Figure 1. ORTEP diagram of the compounds 4a and 4b. Displacement ellipsoids are plotted
at 30% probability level.
4a
4b
Figure 2. Crystal packing showing intermolecular hydrogen bonds as dashed lines.

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Table 1. Crystal data and structure refinement for compounds 4a,b.
Crystal data
4a
4b
Empirical Formula
Formula Weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
2(C23H21N3O2S2)·CH4O
903.18
2(C22H18N3O2S2)·CH2Cl2
928.01
100(2)
(2) 293
Mo Kα radiation, 0.71073
Monoclinic
Cu Kα radiation, 1.54178
Monoclinic
C2/c
C2/c
Unit cell dimensions
a = 9.7855 (5) Å, α = 90.00° a = 17.0062 (10) Å, α =
b = 10.4786 (5) Å, β = 90.00°
92.662 (2)°
b = 13.3291 (9)Å, β =
c = 20.6713 (9) Å, γ = 93.210 (4)°
90.00°
c = 20.3259 (14) Å, γ =
90.00°
Volume (Å3)
2117.31 (17)
4600.2 (5)
4, 1.340
Z, calculated density (mg.m−3) 2, 1.417
Absorption coefficient (mm−1) 0.28
3.36
F(000)
948
1928
Crystal size (mm)
0.40 × 0.29 × 0.14
0.62 × 0.48 × 0.39
4.2° to 69.4°
Theta range for data collection 2.8° to 33.2°
(o)

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Limiting indices
-11 ≤h ≤11, -12 ≤k ≤12, -24 -19 ≤h ≤19, -15 ≤k ≤14, -23
≤l ≤24
≤l ≤22
Reflections collected /unique
50446 / 3658 [R(int) = 24566 / 3372 [R(int) =
0.039]
0.114]
Completeness to theta
Refinement method
25.0° (100 %)
69.4° (100 %)
Full-matrix least-squares on Full-matrix least-squares on
F2
F2
Data/ restraints/ parameters
Goodness of- fit on F2
Final R indices [I > 2σ(I)]
R indices (all data)
3735 / 0 /295
1.08
3665 / 0 /289
1.08
R1 = 0.039, wR2 = 0.053
R1 = 0.114, wR2 = 0.076
R1 = 0.0532, wR2 = 0.136 R1 = 0.0765, wR2 = 0.206
Largest diff. peak and hole (e 0.82 and −0.70
A−3)
1.15 and -0.47
1424967
CCDC reference
1425092
Table 2 Selected geometric parameters (Å, °) of 4a.
S1—C5
S1—C16
S2—C16
O1—C14
O2—C15
1.815 (3)
1.765 (3)
1.668 (3)
1.223 (4)
1.214 (4)
N1—C1
N1—C7
1.521 (4)
1.514 (4)
1.349 (4)
1.408 (4)
1.340 (4)
N2—C14
N2—C13
N3—C16

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O3—C24
N1—C4
1.426 (8)
1.540 (4)
92.49 (14)
108.2 (2)
107.8 (2)
118.5 (2)
112.0 (2)
113.5 (2)
103.7 (2)
104.7 (2)
106.0 (2)
103.03 (19)
117.7 (2)
116.2 (2)
113.3 (2)
N3—C15
1.370 (4)
C5—S1—C16
C1—N1—C4
C4—N1—C7
C1—N1—C7
C13—N2—C14
C15—N3—C16
N1—C1—C2
N1—C4—C3
N1—C4—C5
S1—C5—C15
S1—C5—C6
S1—C5—C4
N1—C7—C8
N1—C7—C14
N1—C7—C6
N2—C13—C8
N2—C13—C12
O1—C14—C7
N2—C14—C7
O1—C14—N2
O2—C15—N3
O2—C15—C5
N3—C15—C5
S2—C16—N3
S1—C16—N3
S1—C16—S2
108.8 (2)
100.0 (2)
109.8 (2)
127.8 (3)
125.4 (3)
107.8 (2)
126.8 (3)
124.7 (3)
120.0 (3)
115.3 (2)
126.3 (2)
114.8 (2)
118.93 (17)
Table 3. Hydrogen-bond geometry (Å, °) of 4a.
D—H···A
D—H
0.89(4)
0.8800
0.9900
H···A
D···A
D—H···A
161(3)
N2—H1N2···S2i
N3—H3D···N1ii
C1—H1A···O2iii
2.45(4)
1.9800
2.4500
3.306(2)
2.745(3)
3.087(4)
144.00
122.00

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C2—H2A···O2iii
C3—H3A···O1
C9—H9A···S2iii
C24—H24C···N2i
0.9900
0.9900
0.9500
0.9800
2.5800
2.5400
2.7700
2.5000
3.250(4)
3.203(4)
3.705(3)
3.447(6)
125.00
124.00
170.00
162.00
Symmetry codes: (i) −x+2, y+1/2, −z+3/2; (ii) −x+1, y−1/2, −z+3/2; (iii) −x+1, y+1/2, −z+3/2.
Table 4 Selected geometric parameters (Å, °) of 4b.
C11—C23
C12—C23
1.820 (18)
1.564 (19)
1.730 (4)
1.830 (4)
1.645 (4)
1.214 (6)
1.208 (5)
93.51 (18)
107.9 (3)
119.4 (3)
110.5 (3)
112.3 (3)
117.5 (3)
103.1 (4)
N1—C4
N1—C1
1.506 (5)
1.492 (6)
1.482 (5)
1.359 (6)
1.408 (6)
1.348 (5)
1.367 (5)
116.2 (3)
125.9 (4)
107.6 (4)
126.5 (4)
128.6 (4)
108.8 (4)
112.8 (3)
S1—C16
N1—C7
S1—C5
N2—C8
S2—C16
N2—C9
O1—C8
N3—C16
O2—C15
N3—C15
C5—S1—C16
C1—N1—C4
C1—N1—C7
C4—N1—C7
C8—N2—C9
C15—N3—C16
N1—C1—C2
N1—C7—C14
O1—C8—C7
N2—C8—C7
O1—C8—N2
N2—C9—C10
N2—C9—C14
N3—C15—C5

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N1—C4—C3
N1—C4—C5
S1—C5—C6
S1—C5—C15
S1—C5—C4
N1—C7—C6
N1—C7—C8
105.5 (4)
106.0 (3)
117.4 (2)
104.1 (3)
115.9 (3)
101.7 (3)
112.9 (3)
O2—C15—N3
124.0 (4)
123.1 (4)
112.0 (3)
124.9 (3)
123.1 (2)
113.1 (10)
O2—C15—C5
S1—C16—N3
S2—C16—N3
S1—C16—S2
Cl1—C23—Cl2
Table 5. Hydrogen-bond geometry (Å, °) of 4b.
D—H···A
D—H
0.8600
0.8600
0.9700
0.9700
0.9300
0.9300
0.9700
H···A
D···A
D—H···A
173.00
176.00
147.00
129.00
173.00
140.00
153.00
N2—H2C···S2i
N3—H3C···N1ii
C1—H1A···O2iii
C1—H1B···O1
C20—H20A···O2iv
C22—H22A···O1i
C23—H23A···O2v
2.6600
2.0000
2.4600
2.3800
2.5600
2.4800
2.4100
3.512 (3)
2.863 (4)
3.313 (6)
3.087 (5)
3.488 (6)
3.251 (5)
3.307 (17)
Symmetry codes: (i) −x−3/2, −y−3/2, −z+1; (ii) −x−3/2, y−1/2, −z+1/2; (iii) −x−3/2, y+1/2,
−z+1/2; (iv) −x−1, y, −z+1/2; (v) −x−3/2, −y−1/2, −z.

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3.3. Hirshfeld surface analysis
The Hirshfeld surfaces (HS) mapped over dnorm (A), shape index (B) and curvedness (C)
for compound 4a are shown in Figure 3. The overall fingerprint plots (FPs) and the
decomposed ones for the most significant interactions are given in Figure S1 (Supplementary
data). The decomposed FPs of the studied compounds shed the light on the effect of the
H---H, H---C, S---H, O---H and N---H intermolecular interactions on the molecular
structures of the studied compounds. The contributions of all possible intermolecular
interactions in the crystal of both compounds are illustrated in Figure 4.
According to the crystallographic data obtained for the two compounds 4a and 4b, there
are two main differences in their structures. The first difference is in the nature of the
substituent at the phenyl ring and the second difference is the co-crystallization with
methanol and dichloromethane solvent molecules in 4a and 4b, respectively. It is evident
from Figure 3 that the main intermolecular interactions in both crystals are the same, except
that 4b also shows a significant amount of H---Cl interactions between the protons that are
present inside the surface and the Cl atom of the solvent dichloromethane present in the
crystal. These interactions appear as a sharp spike towards the left of the fingerprint plot
(Figure S1, Supplementary data). The curvedness and shape index plots do not show any
characteristic features of π-π stacking interactions, as evident from the absence of the blue
and red triangles in the shape index as well as the absence of flat areas in the curvedness plot.

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Front View
Back View
Front View
Back View
dnorm
Curvedness
4a
4b
Figure 3. Hirshfeld surfaces of the molecules 4a and 4b.

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Figure 4. Percentage distribution of all possible interactions in the studied crystals.
3.4. DFT studies
The optimized structures of 4a and 4b were matched with their X-ray structures, and the
results are shown in Figure 5. It is clear from these comparison graphs that there is a good
agreement between the calculated and experimental structures. The results shown in Table
S1 (Supplementary data) confirm this conclusion as well. The maximum deviation of the
bond distances is not greater than 0.063 Å (4.2%) and 0.043 Å (2.9%) for 4a and 4b,
respectively. In this regard, the energies of the highest occupied molecular orbital (HOMO)
and the lowest unoccupied molecular orbital (LUMO) were computed using the same level of
theory. These frontier molecular orbitals are important for understanding the electron
transport processes in these compounds. These processes are important parameters for the
biological applications of such molecular systems. A pictorial representation of these orbitals

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is given in Figure 6. It is clear that changing the substituent has little effect on the HOMO and
LUMO energies and geometries. The HOMO level is mainly localized over the
thioxothiazolidin-4-one ring for both molecules while the LUMO level is mainly delocalized
over the conjugated π-system in both compounds. The HOMO-LUMO energy gap of 4.053
eV is almost the same for both compounds.
Figure 5. Structure matching of the calculated and experimental structures of the studied
compounds 4a and 4b.
4a

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HOMO (-5.9743 eV)
LUMO (-1.9211 eV)
4b
HOMO (-5.9991 eV)
LUMO (-1.9464 eV)
Figure 6. Frontier molecular orbitals of 4a and 4b calculated using B3LYP/6-311G(d,p)
method.
3.5. Antimicrobial Evaluation
The synthesized spirooxindole derivatives 4a and 4b were tested for their antibacterial and
antifungal activity at different concentrations in DMSO. Gentamicin and Ampicillin were
used as the positive control drugs for antibacterial. Fluconazole and Amphoterecin-B were
used as the positive control drugs for antifungal tests. The minimum concentration at which
no growth was observed was taken as the minimum inhibitory concentration (MIC) value.
3.5.1. Antimicrobial Activity
Results of the biological activity were illustrated in Table 6; results are expressed as µg/mL
inhibition. The antibacterial activity of synthesized compounds was elucidated against two
gram positive strain and two gram negative strain namely Streptococcus pneumonia, Bacillis
subtilis, Pseudomonas aeruginosa, and Escherichia coli respectively. Antibacterial activity
of compounds 4a, and 4b were compared with the known ampicllin in case of gram positive
bacteria and gentamicin in case of gram negative bacteria. Compounds 4a and 4b were more

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active than standard drugs against all examined bacteria. Compound 4b exhibited better
activity than analogues 4a in case of S. pneumonia, B. subtilis, and E.coli respectively. In
case of activity against P. aeruginosa, compound 4b showed MIC values of 15 while 4a and
gentamicin have MIC values of 16, 19 respectively.
Table 6. Antibacterial activity of compound 4a,b showed in minimal inhibitory
concentrations (MIC)
Gram Positive Bacteria
Gram Negative Bacteria
Streptococcus
pneumoniae
Bacillis
Pseudomonas
aeruginosa
Escherichia
Compound
subtilis
coli
Ampicillin
Gentamicin
25 ± 0.25
19 ± 0.40
23 ± 0.24
33 ± 0.32
28 ± 0.35
30 ± 0.55
19 ± 0.60
16 ± 0.15
15 ± 0.35
19.0 ± 0.33
18.5 ± 0.21
19.0 ± 0.33
Standard
4a
4b
Data are expressed as mean ± SD; Standard = 25 ꢀg/mL.
3.5.2. Antifungal Activity
Based on antibacterial activity of our compounds, our attention was directed to extent the
benefit of these interesting compounds to be broad spectrum antimicrobial especially in
mixed infection disease. In this regard, compounds 4a and 4b were evaluated for their
antifungal activity against Aspergillus fumigates, Syncephalastrum racemosum, Geotricum
candidum, and Candida albicans fungai. The study was done by the diffusion method and
serial dilution method [23].Their activities were compared with the known antifungal agents
Fluconazole and Amphotericin B. Interestingly, both compounds 4a and 4b were more active
than both standard against examined fungai. In a correlation manner, again compound 4b
was most active in this study. Theses correlation data and good activity guided us to
understand the ode of interaction using molecular modeling study.

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Table 7. Antifungal activity of the synthesized compound 4a,b (Zone of inhibition; diameter
in mm).
Fungal Strains
Aspergillus
fumigates
Syncephalastrum
racemosum
Geotricum
candidum
Candida
albicans
Compound
Standard Drug
23 ± 0.54
25 ± 0.11
18 ± 0.46
16 ± 0.50
22.1 ± 0.20
24.1 ± 0.15
19 ± 0.38
14 ± 0.47
29 ± 0.11
30 ± 0.33
27 ± 0.18
24 ± 0.25
26 ± 0.22
28 ± 0.55
27 ± 0.11
25 ± 0.19
I
II
4a
4b
Data are expressed as mean ± SD; standard = 25 ꢀg/mL; I: Amphotericin B; II: Fluconazole.
3.6. Molecular docking
A library of target compounds and standard gentamicin and fluconazole drugs was designed
and energy minimized using MMFF94 force field calculations. Aminoglycoside
Phosphotransferase which was obtained from protein data bank (PDB code: 3HAM) [55]
was selected as the target protein for antibacterial activity of our tested compound.
Lanosterol 14 α-demethylase (CYP51A1) (PDB ID 4WMZ) [56] was chosen as a target protein
for antifungal activity of our compounds
The docking study was prepared for docking using Open Eye® software. This software
package generates consensus scoring which is a filtering processes to obtain virtual binding
affinity, the lower consensus score, the better binding affinity of the ligands towards the
receptor.
3.6.1. Molecular Docking as antibacterial

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The study revealed that both compound 4b and the standard drug gentamicin showed
consensus score of 12 while compound 4a showed consensus score 15. The standard
gentamicin showed hydrogen bonds towards the binding site of ID: 3HAM with ASP:192:A,
ASP:268:A, TYR:272:A (Fig. 7). However, compound 4a showed
a
hydrophobic-hydrophobic interaction towards the binding site (Fig. 8). Furthermore,
Compound 4b showed a hydrogen bonding towards the binding site of ID: 3HAM coming
from thiazolone NH (as donor) and carbonyl (as acceptor) functionalities with LYS:142:A
and ASN: 191: A respectively (Fig. 9). Interestingly, compound 4b (the most active
derivative) occupied the receptor with pose and mode of interaction differs from the analogs
4a. In case of 4b, the carbonyl of thiazolone moiety is oriented close to phenyl arm
(originated from benzaldehyde) while in 4a the substituted phenyl arm (originated from
p-methyl benzaldehyde) sets closely to carbonyl of indolone fragment, (Fig. 10). This
behavior suggests that the presence of P methyl group affect on the geometry of compound
and so the strength and kind of interactions with target receptor.

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Figure 7: Visual representation for gentamicin docked with PDB: 3HAM showing the
hydrophobic-hydrophobic interaction towards the binding site and the dashed green lines
showing the hydrogen bonding

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Figure 8: Visual representation for 4a docked with PDB: 3HAM showing the
hydrophobic-hydrophobic interaction towards the binding site and no detected hydrogen
bonding

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Figure 9: Visual representation for 4b docked with PDB: 3HAM showing the
hydrophobic-hydrophobic interaction towards the binding site and the dashed green lines
showing the hydrogen bonding.
4a
4b