Potential bacterial biofilm, MRSA, and DHFR inhibitors based on new morpholine-linked chromene-thiazole hybrids: One-pot synthesis and in silico study

Article abstract of DOI:10.1016/j.molstruc.2021.131476

We report herein the utility of the morpholine-linked benzaldehyde as a key building block in the preparation of two series of new chromene-thiazoles with potential bioactivities. In this regard, a one-pot protocol was developed involving the reaction of the prior aldehyde, 2-cyanothioacetamide and α-bromoketones in dioxane in the presence of triethylamine to give the target hybrids. Thiazole hybrids 9d and 9e, attached to two chromene units at thiazole-C2 and C4, and linked to methyl or methoxy groups, respectively, showed the best antibacterial activity. The previous hybrids exhibited MIC/MBC values of 3.9/7.8 and 1.7/3.5 μM, respectively, against S. aureus, S. mutans and E. coli strains. In addition, the same hybrids gave MIC values of 7.7–32.0 μM and MBC values of 15.5–64.1, μM against MRSA ATCC:33,591 and ATCC:43,300 strains. Furthermore, 9d and 9e gave the best bacterial biofilm inhibitory activity against S. aureus, S. mutans and E. coli strains with IC₅₀ values ranging from 3.9 to 4.6 μM. Also, 9e and 9d displayed superior dihydrofolate reductase enzyme inhibitory activity with IC₅₀ values of 0.122 and 0.131 μM, respectively, compared to the standard sulfadiazine (IC₅₀ value of 0.138 μM). Molecular docking study was used to determine the binding energies of some new hybrids with the previous enzyme. Moreover, physicochemical, pharmacokinetic properties, and drug-likeness of some new hybrids were calculated using SwissADME.

Full text of DOI:10.1016/j.molstruc.2021.131476

Journal of Molecular Structure 1248 (2022) 131476
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Potential bacterial biofilm, MRSA, and DHFR inhibitors based on new
morpholine-linked chromene-thiazole hybrids: One-pot synthesis and
in silico study
Sherif M.H. Sanada,∗, Ahmed E.M. Mekkya, Tamer T. El-Idreesya,b
a Chemistry Department, Faculty of Science, Cairo University, Giza 12613, Egypt
b Department of Chemistry, School of Sciences and Engineering, The American University in Cairo, New Cairo 11835, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
We report herein the utility of the morpholine-linked benzaldehyde as a key building block in the
preparation of two series of new chromene-thiazoles with potential bioactivities. In this regard, a one-
pot protocol was developed involving the reaction of the prior aldehyde, 2-cyanothioacetamide and α-
bromoketones in dioxane in the presence of triethylamine to give the target hybrids. Thiazole hybrids 9d
and 9e, attached to two chromene units at thiazole-C2 and C4, and linked to methyl or methoxy groups,
respectively, showed the best antibacterial activity. The previous hybrids exhibited MIC/MBC values of
3.9/7.8 and 1.7/3.5 μM, respectively, against S. aureus, S. mutans and E. coli strains. In addition, the same
hybrids gave MIC values of 7.7–32.0 μM and MBC values of 15.5–64.1, μM against MRSA ATCC:33,591 and
ATCC:43,300 strains. Furthermore, 9d and 9e gave the best bacterial biofilm inhibitory activity against
S. aureus, S. mutans and E. coli strains with IC50 values ranging from 3.9 to 4.6 μM. Also, 9e and 9d dis-
played superior dihydrofolate reductase enzyme inhibitory activity with IC50 values of 0.122 and 0.131 μM,
respectively, compared to the standard sulfadiazine (IC50 value of 0.138 μM). Molecular docking study
was used to determine the binding energies of some new hybrids with the previous enzyme. Moreover,
physicochemical, pharmacokinetic properties, and drug-likeness of some new hybrids were calculated us-
ing SwissADME.
Received 23 July 2021
Revised 3 September 2021
Accepted 8 September 2021
Available online 10 September 2021
Keywords:
Bacterial biofilm inhibitors
DHFR inhibitors
Morpholine-thiazole hybrids
MRSA inhibitors
SwissADME prediction
© 2021 Elsevier B.V. All rights reserved.
1. Introduction
Various morpholine derivatives can be isolated from naturally oc-
curring sources [13] These include Polygonapholine extracted from
The ability of the dihydrofolate reductase (DHFR) enzyme as a
therapeutic target in the treatment of infections has been demon-
strated since the middle of the last century [1]. Using NADPH,
DHFR catalyzes the reduction of dihydrofolate to tetrahydrofolate
and is involved in the synthesis of cell proliferation raw material,
both in prokaryotic and eukaryotic cells [2]. DHFR inhibitors are
widely used in the treatment of fungal, bacterial and mycobacte-
rial infections and in the fight against malaria and other proto-
zoal infections as well as fight against cancer [3–6]. These include
pyrimethamine and proguanil as antimalarial drugs [7]; trimetho-
prim, a widely used sulfonamide-associated antibacterial medica-
tion such as sulfamethoxazole [8,9]; and methotrexate, the first
discovered anticancer agent that act by inhibition of DHFR [10].
Morpholine is commonly used in industry as well as in the
synthesis of various biologically active organic derivatives [11,12].
the rhizome methanol extract of Polygonatum altelobatum and used
as a tonic drink in Taiwan [14], as well as Lidamycin isolated
from Streptomyces globisporus and used as an antitumor antibi-
otic [15]. Numerous publications reported the synthesis of hete-
rocyclic derivatives incorporating morpholine units [16,17]. This is
attributed to the wide spectrum of medicinal applications of these
derivatives including antidepressant [18,19], appetite suppressant
[20], antitumor [21], antioxidant activities [22,23]. They also act
selective inhibitors of acetylcholinesterase [24,25], monoamine ox-
idase A and B [25], cyclooxygenase-1 and 2 [25], and glucosidase
enzymes [26]. In addition, morpholine units are frequently selected
as key synthons for different biologically important derivatives in-
cluding the preparation of enantiomerically pure α-amino acids, β-
amino alcohols, and peptides [27–29].
Thiazole derivatives have shown remarkable antimicrobial [30],
anti-inflammatory, analgesic [31], anticancer [32], antihypertensive
[33], anti-HIV [34], anti-hypoxic [35], and anti-asthmatic activities
[36]. Moreover, chromene hybrids act as antimicrobial [37], anti-
inflammatory, analgesic [38], antioxidant [39], and antifungal [40],
∗
Corresponding author.
E-mail address: sherif_hamed1980@yahoo.com (S.M.H. Sanad).
https://doi.org/10.1016/j.molstruc.2021.131476
0022-2860/© 2021 Elsevier B.V. All rights reserved.

S.M.H. Sanad, A.E.M. Mekky and T.T. El-Idreesy
Journal of Molecular Structure 1248 (2022) 131476
anti-hepatitis C virus [41], antitumor activities [42], and promis-
ing acetylcholinesterase inhibitors [43,44]. Fig. 1 represents some
important morpholine-based drugs [45–47], in addition to some
promising DHFR inhibitors incorporating a thiazole or chromene
unit [48–50]. Based on the aforementioned findings and in con-
nection with our efforts to prepare potential antibacterial agents
as well as DHFR inhibitors [51–59], we designed herein facile syn-
thetic routes for the preparation of novel thiazole-chromene hy-
brids incorporating a morpholine unit.
137.2, 145.8, 150.9, 157.1, 196.2; MS m/z (%): 303 (M+, 59.2); Anal.
for C15H17 N3O2S: C, 59.39; H, 5.65; N, 13.85; found: C, 59.21; H,
5.47; N, 13.94%.
2.2.2. Synthesis of thiazole-chromene hybrids 7 and 9
2.2.2.1. Method ‘A’ (synthesis of hybrid 7a).
A mixture of 2-
imino-2H-chromene-3-carbothioamide 5 (5 mmol) and 2–bromo-
1-phenylethan-1-one 6a (5 mmol) in dioxane (15 mL) in the pres-
ence of four drops of piperidine was boiled at reflux for 3 h to
afford a sole product as detected by TLC analysis. The reaction was
cooled, filtrated, washed with cold ethanol and the reaction prod-
uct was recrystallized from dioxane / ethanol mixture.
A pharmacophore can be defined by determining complemen-
tarities between a ligand and its corresponding binding site [60].
A pharmacophore’s most important elements can range from a
group of atoms to a portion of the molecule’s volume. Hydrophobic
and/or aromatic rings, as well as hydrogen bond acceptors and/or
donors, are key features of a pharmacophore [61]. Sulfadiazine,
a reference DHFR inhibitor, contains a sulfonamide group and a
pyrimidine-N atom, both of which act as hydrogen bond accep-
tors to the DHFR binding sites [60]. Our designed new hybrids
have the same pharmacophoric elements as sulfadiazine due to the
chromene-CO group(s) and morpholine-O atom. Fig. 2 compares
the pharmacophoric elements of the reference sulfadiazine with 9d
as an example of the designed hybrids.
2.2.2.2. Method ‘B’ (one-pot protocol).
A
ternary mixture of
2-hydroxybenzaldehyde 3 (5 mmol), 2-cyanothioacetamide
4
(5 mmol) and the appropriate of α-bromoketones 6a 6e or 8a
8e (5 mmol) in dioxane (15 mL) in the presence of triethylamine
(0.4 mL) was boiled at reflux for 6–8 h to give a sole product in
each case as detected by TLC analyses. The reaction was cooled,
filtrated, washed with cold ethanol and the reaction product was
recrystallized from the proper solvent.
In this study, the pharmacological activities of the new
morpholine-linked thiazole-chromene hybrids as potential bacte-
rial biofilm, MRSA and DHFR inhibitors were tested. Furthermore,
the SwissADME program was used to calculate the physicochemi-
cal, pharmacokinetic, and medicinal chemistry properties of some
new hybrids, as well as their drug-likeness.
2.2.3. 6-(Morpholinomethyl)−3-(4-phenylthiazol-2-yl)−2H-
chromen-2-one (7a)
Pale yellow powders (dioxane / ethanol mixture, 70% using
method ‘A’; 85% using method ‘B’); m.p. 210 °C; IR (υ cm−1): 1716
(CO); 1H NMR (DMSO–d6): δ 2.40 (t, J = 4.8 Hz, 4H, morpholine-
NCH2), 3.60 (t, J = 4.8 Hz, 4H, morpholine–OCH2), 3.65 (s, 2H,
CH2), 7.22–7.25 (m, 2H, H7 and H8), 7.32–7.38 (m, 3H, Ar-H’s), 7.66
(s, 1H, H5), 7.85 (d, J = 8.4 Hz, 2H, Ar-H’s), 7.93 (s, 1H, thiazole-H),
8.34 (s, 1H, H4); 13C NMR (DMSO–d6): δ 53.5, 60.6, 66.3, 113.2,
117.2, 118.1, 122.4, 124.2, 126.8, 128.5, 129.0, 131.5, 132.0, 132.4,
144.3, 147.2, 151.3, 160.4, 164.6; MS m/z (%): 404 (M+, 47.3); Anal.
for C23H20N2O3S: C, 68.30; H, 4.98; N, 6.93; found: C, 68.42; H,
5.11; N, 7.05%.
2. Experimental
2.1. Materials
All solvents were acquired from commercial sources and used
as received unless otherwise stated. All other chemicals were ac-
quired from Merck or Aldrich and used without further purifi-
cation. The melting points were measured on a Stuart melting
point apparatus and are uncorrected. IR spectra were recorded on
a Smart iTR, which is an ultra-high-performance, versatile Attenu-
ated Total Reflectance (ATR) sampling accessory on the Nicolet iS10
FT-IR spectrometer. NMR spectra were recorded on Bruker Avance
III 400 MHz spectrophotometer (400 MHz for 1H and 100 MHz for
13C) using TMS as an internal standard and DMSO–d6 as solvent
and chemical shifts were expressed as δ ppm units. Mass spec-
tra were recorded on a GC–MS-QP1000EX spectrometer using inlet
type at 70 eV. Elemental analyses were carried out on a EuroVector
instrument C, H, N, S analyzer EA3000 Series. All spectral analyses
as well as the biological screening were conducted in the laborato-
ries of Cairo University.
2.2.4. 3-(4-(4-Chlorophenyl)thiazol-2-yl)−6-(morpholinomethyl)−2H-
chromen-2-one (7b)
Pale yellow powders (dioxane, 77%); m.p. 234–236 °C; IR (υ
cm−1): 1717 (CO); 1H NMR (DMSO–d6): δ 2.43 (t, J = 4.8 Hz, 4H,
morpholine-NCH2), 3.58 (t, J = 4.8 Hz, 4H, morpholine–OCH2), 3.63
(s, 2H, CH2), 7.24–7.27 (m, 2H, H7 and H8), 7.45 (d, J = 8.4 Hz,
2H, Ar-H’s), 7.62 (s, 1H, H5), 7.89 (d, J = 8.4 Hz, 2H, Ar-H’s),
7.96 (s, 1H, thiazole-H), 8.38 (s, 1H, H4); 13C NMR (DMSO–d6):
δ 53.6, 60.8, 66.4, 113.4, 117.0, 118.2, 122.6, 124.4, 127.5, 128.7,
131.4, 132.3, 132.7, 135.6, 144.2, 146.6, 150.8, 159.8, 164.8; Anal. for
C23H19 ClN2O3S (438.9): C, 62.94; H, 4.36; N, 6.38; found: C, 63.10;
H, 4.45; N, 6.27%.
2.2.5. 3-(4-(4-Bromophenyl)thiazol-2-yl)−6-(morpholinomethyl)−2H-
chromen-2-one (7c)
2.2. The procedures and spectral data
Pale yellow powders (dioxane, 79%); m.p. 246 °C; IR (υ cm−1):
2.2.1. Synthesis of
1718 (CO); 1H NMR (DMSO–d6): δ 2.40 (t,
J
=
4.8 Hz, 4H,
2-imino-6-(morpholinomethyl)−2H-chromene-3-carbothioamide (5)
morpholine-NCH2), 3.57 (t, J = 4.8 Hz, 4H, morpholine–OCH2), 3.63
(s, 2H, CH2), 7.20 (d, J = 8.4 Hz, 1H, H7), 7.28 (d, J = 8.4 Hz,
1H, H8), 7.49 (d, J = 8.4 Hz, 2H, Ar-H’s), 7.64 (s, 1H, H5), 7.80 (d,
J = 8.4 Hz, 2H, Ar-H’s), 8.00 (s, 1H, thiazole-H), 8.33 (s, 1H, H4);
13C NMR (DMSO–d6): δ 53.5, 60.6, 66.5, 113.3, 117.2, 117.9, 121.9,
122.8, 124.7, 126.5, 131.3, 131.6, 131.8, 132.0, 144.3, 145.6, 150.4,
159.3, 164.7; Anal. for C23H19 BrN2O3S (483.3): C, 57.15; H, 3.96; N,
5.80; found: C, 57.02; H, 4.13; N, 5.96%.
A
mixture of 2-hydroxybenzaldehyde 3 (5 mmol) and 2-
cyanothioacetamide 4 (5 mmol) in ethanol (15 mL) in the presence
of four drops of piperidine was boiled at reflux for 3 h to give a
sole product as detected by TLC analysis. The reaction was cooled,
filtrated, washed with cold ethanol and the reaction product was
recrystallized from dioxane / ethanol mixture as pale yellow pow-
ders (78%); m.p. 188 °C; IR (υ cm−1): 3404, 3316, 3170 (NH, NH2);
1H NMR (DMSO–d6): δ 2.44 (t, J = 4.8 Hz, 4H, morpholine-NCH2),
3.61 (t, J = 4.8 Hz, 4H, morpholine–OCH2), 3.66 (s, 2H, CH2), 7.18
(d, J = 8.4 Hz, 1H, H8), 7.30 (d, J = 8.4 Hz, 1H, H7), 7.63 (s, 1H,
H5), 8.24 (s, 1H, H4), 8.78, 9.00 (s, 2H, NH2), 10.13 (s, 1H, NH); 13C
NMR (DMSO–d6): δ 53.8, 60.9, 66.5, 117.0, 121.1, 122.1, 127.6, 131.3,
2.2.6. 6-(Morpholinomethyl)−3-(4-(p-tolyl)thiazol-2-yl)−
2H-chromen-2-one (7d)
Pale yellow powders (dioxane / ethanol mixture, 84%); m.p.
208–210 °C; IR (υ cm−1): 1716 (CO); 1H NMR (DMSO–d6): δ 2.36
2

S.M.H. Sanad, A.E.M. Mekky and T.T. El-Idreesy
Journal of Molecular Structure 1248 (2022) 131476
(s, 3H, p-CH3), 2.43 (t, J = 4.8 Hz, 4H, morpholine-NCH2), 3.59 (t,
J = 4.8 Hz, 4H, morpholine–OCH2), 3.65 (s, 2H, CH2), 7.18–7.21 (m,
2H, H7 and H8), 7.30 (d, J = 8.4 Hz, 2H, Ar-H’s), 7.61 (s, 1H, H5),
7.74 (d, J = 8.4 Hz, 2H, Ar-H’s), 7.96 (s, 1H, thiazole-H), 8.41 (s,
1H, H4); 13C NMR (DMSO–d6): δ 20.5, 53.4, 60.5, 66.6, 113.2, 116.8,
118.2, 121.7, 124.4, 127.2, 128.8, 131.2, 131.4, 131.9, 139.3, 144.8,
146.3, 150.8, 159.8, 164.5; MS m/z (%): 418 (M+, 52.8); Anal. for
C24H22N2O3S: C, 68.88; H, 5.30; N, 6.69; found: C, 69.04; H, 5.45;
N, 6.78%.
2.2.11. 6-Methyl-3-(2-(6-(morpholinomethyl)−2-oxo-2H-chromen-3-
yl)thiazol-4-yl)−2H-chromen-2-one (9d)
Beige powders (DMF / ethanol mixture, 72%); m.p. 262–264 °C;
IR (υ cm−1): 1730, 1694 (CO); 1H NMR (DMSO–d6): δ 2.37 (s, 3H,
CH3), 2.42 (t, J = 4.8 Hz, 4H, morpholine-NCH2), 3.58 (t, J = 4.8 Hz,
4H, morpholine–OCH2), 3.63 (s, 2H, CH2), 7.15–7.18 (m, 2H, H7 and
H7΄), 7.23–7.26 (m, 2H, H8 and H8΄), 7.55 (s, 1H, H5΄), 7.60 (s, 1H,
H5), 7.90 (s, 1H, thiazole-H), 8.24 (s, 1H, H4), 8.40 (s, 1H, H4΄);
13C NMR (DMSO–d6): δ 20.8 (CH3), 53.5, 60.5, 66.4, 115.0, 116.0,
116.2, 117.2, 117.4, 118.4, 118.7, 126.7, 127.7, 130.2, 133.5, 133.8,
134.3, 134.7, 142.0, 147.7, 151.8, 152.7, 163.5, 164.1, 165.4; Anal. for
C27H22N2O5S (486.5): C, 66.65; H, 4.56; N, 5.76; found: C, 66.72;
H, 4.67; N, 5.53%.
2.2.7. 3-(4-(4-Methoxyphenyl)thiazol-2-yl)−6-
(morpholinomethyl)−2H-chromen-2-one (7e)
Pale yellow powders (dioxane, 80%); m.p. 230 °C; IR (υ cm−1):
1717 (CO); 1H NMR (DMSO–d6): δ 2.41 (t,
J
=
4.8 Hz, 4H,
2.2.12. 6-Methoxy-3-(2-(6-(morpholinomethyl)−2-oxo-2H-chromen-
3-yl)thiazol-4-yl)−2H-chromen-2-one (9e)
morpholine-NCH2), 3.57 (t, J = 4.8 Hz, 4H, morpholine–OCH2), 3.61
(s, 2H, CH2), 3.83 (s, 3H, p–OCH3), 6.95 (d, J = 8.4 Hz, 2H, Ar-H’s),
7.20 (d, J = 8.4 Hz, 1H, H7), 7.27 (d, J = 8.4 Hz, 1H, H8), 7.64 (s, 1H,
H5), 7.79 (d, J = 8.4 Hz, 2H, Ar-H’s), 7.99 (s, 1H, thiazole-H), 8.36 (s,
1H, H4); 13C NMR (DMSO–d6): δ 53.6, 55.3, 60.7, 66.8, 112.9, 114.1,
116.9, 118.3, 122.0, 124.2, 127.2, 129.5, 131.4, 132.3, 144.5, 146.1,
150.7, 159.7, 160.1, 164.6; Anal. for C24H22N2O4S (434.5): C, 66.34;
H, 5.10; N, 6.45; found: C, 66.17; H, 4.98; N, 6.37%.
Beige powders (DMF / ethanol mixture, 70%); m.p. 270–272 °C;
IR (υ cm−1): 1730, 1698 (CO); 1H NMR (DMSO–d6): δ 2.42 (t,
J
=
4.8 Hz, 4H, morpholine-NCH2), 3.57 (t,
J
=
4.8 Hz, 4H,
morpholine–OCH2), 3.62 (s, 2H, CH2), 3.80 (s, 3H, OCH3), 7.16 (d,
J = 8.4 Hz, 1H, H7), 7.21–7.26 (m, 3H, H5΄, H7΄ and H8), 7.45 (d,
J = 8.4 Hz, 1H, H8΄), 7.59 (s, 1H, H5), 7.93 (s, 1H, thiazole-H), 8.19
(s, 1H, H4), 8.28 (s, 1H, H4΄); 13C NMR (DMSO–d6): δ 53.4, 55.8,
60.6, 66.2, 113.7, 115.3, 116.4, 116.8, 117.3, 117.5, 117.9, 118.3, 118.6,
127.4, 130.3, 133.0, 133.6, 142.7, 147.3, 149.9, 151.4, 154.7, 163.4,
163.9, 165.2; MS m/z (%): 502 (M+, 36.4); Anal. for C27H22N2O6S:
C, 64.53; H, 4.41; N, 5.57; found: C, 64.38; H, 4.55; N, 5.74%.
2.2.8. 6-(Morpholinomethyl)−3-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-
yl)−2H-chromen-2-one (9a)
Beige powders (dioxane, 77%); m.p. 260–262 °C; IR (υ cm−1):
1728, 1696 (CO); 1H NMR (DMSO–d6): δ 2.42 (t, J = 4.8 Hz, 4H,
morpholine-NCH2), 3.59 (t, J = 4.8 Hz, 4H, morpholine–OCH2), 3.63
(s, 2H, CH2), 7.19–7.22 (m, 2H, H7 and H8), 7.36–7.42 (m, 2H, H6΄
and H8΄), 7.56–7.60 (m, 2H, H5 and H7΄), 7.82 (d, J = 8.8 Hz, 1H,
H5΄), 7.90 (s, 1H, thiazole-H), 8.35 (s, 1H, H4), 8.48 (s, 1H, H4΄); 13C
NMR (DMSO–d6): δ 53.5, 60.6, 66.6, 114.2, 115.3, 116.6, 117.6, 117.8,
118.3, 123.4, 125.7, 128.5, 128.8, 129.6, 130.2, 134.3, 134.6, 144.8,
148.7, 150.2, 151.3, 162.1, 163.2, 165.3; MS m/z (%): 472 (M+, 40.5);
Anal. for C26H20N2O5S: C, 66.09; H, 4.27; N, 5.93; found: C, 65.87;
H, 4.34; N, 6.08%.
2.3. The in vitro antibacterial screening
2.3.1. Minimum inhibitory concentration (MIC) determination
The inhibitory activities were estimated against each of Staphy-
lococcus aureus (ATCC:6538), Streptococcus mutans, (ATCC:25,175),
Enterococcus faecalis (ATCC:29,212), Escherichia coli (ATCC:9637),
Pseudomonas aeruginosa (ATCC:27,953) and Klebsiella pneumonia
(ATCC:10,031)] as well as MRSA (ATCC:33,591 and ATCC:43,300)
bacterial strains [62]. MIC values were determined using micro-
broth serial dilution method [63] in a sterile 96-well microtiter
plate after overnight incubation of tested bacteria at 37 °C. This as-
say was performed in triplicates for consistency in accordance with
guidelines provided by CLSI (2012) [64]. Ciprofloxacin (100 μg sus-
ceptibility disk) was used as a standard drug. The concentration of
the tested hybrids as well as ciprofloxacin used in the study ranged
from 250 to 0.9 μg/mL. The sterile Muller-Hinton broth (MHB) was
enriched with 2% NaCl before the tested antimicrobial agents were
inserted into the well at concentration gradient in a serial dilu-
tion. Then the diluted bacterial suspension at final inoculum of
106 CFU/mL was added. The tested compound in MHB was used
as negative control to ensure medium sterility, while the inocu-
lum in MHB served as positive control to ensure the adequacy of
the broth for bacterial growth. To facilitate the observation of the
growth of bacteria in each well, 20 μL of 2,3,5-triphenyltetrazolium
chloride (TTC) at 2 mg/mL was added into each well.
2.2.9. 6-Chloro-3-(2-(6-(morpholinomethyl)−2-oxo-2H-chromen-3-
yl)thiazol-4-yl)−2H-chromen-2-one (9b)
Beige powders (DMF / ethanol mixture, 74%); m.p. 274 °C; IR (υ
cm−1): 1729, 1697 (CO); 1H NMR (DMSO–d6): δ 2.40 (t, J = 4.8 Hz,
4H, morpholine-NCH2), 3.60 (t, J = 4.8 Hz, 4H, morpholine–OCH2),
3.66 (s, 2H, CH2), 7.18 (d, J = 8.4 Hz, 1H, H7), 7.25 (d, J = 8.4 Hz,
1H, H8), 7.36 (d, J = 8.8 Hz, 1H, H8΄), 7.62 (s, 1H, H5), 7.74–7.76
(m, 2H, H5΄ and H7΄), 7.94 (s, 1H, thiazole-H), 8.28 (s, 1H, H4), 8.34
(s, 1H, H4΄); 13C NMR (DMSO–d6): δ 53.3, 60.4, 66.5, 114.2, 117.2,
117.7, 118.0, 118.2, 118.4, 123.2, 128.0, 129.2, 129.4, 130.4, 133.2,
133.9, 134.3, 144.4, 148.9, 151.8, 152.4, 163.0, 163.4, 165.4; Anal. for
C26H19 ClN2O5S (506.9): C, 61.60; H, 3.78; N, 5.53; found: C, 61.73;
H, 3.64; N, 5.68%.
2.2.10. 6-Bromo-3-(2-(6-(morpholinomethyl)−2-oxo-2H-chromen-3-
yl)thiazol-4-yl)−2H-chromen-2-one (9c)
2.3.2. Minimum bactericidal concentration (MBC)
Beige powders (DMF / ethanol mixture, 78%); m.p. 270–272 °C;
IR (υ cm−1): 1724, 1695 (CO); 1H NMR (DMSO–d6): δ 2.43 (t,
The tested thiazoles were screened against each of Staphylococ-
cus aureus (ATCC:6538), Streptococcus mutans (ATCC:25,175) and E.
coli (ATCC:9637) as well as MRSA (ATCC:33,591 and ATCC:43,300)
bacterial strains to estimate their MBC values [65]. Each of the
tested strains was cultured in sterile broth medium for 24 h at
37 °C. The assay was performed in 2 mL microcentrifuge tubes
with concentrations ranging from 250 to 0.9 μg/mL of the tested
derivatives. To each concentration of the tested derivatives, 0.1 mL
of the cultured bacterial strain was added and then, allowed to in-
cubate for 24 h at 37 °C. 10 μL sample was collected post incuba-
tion and seeded onto the agar plates and left to incubate for 24 h
J
=
4.8 Hz, 4H, morpholine-NCH2), 3.58 (t,
J
=
4.8 Hz, 4H,
morpholine–OCH2), 3.63 (s, 2H, CH2), 7.15 (d, J = 8.8 Hz, 1H, H8΄),
7.22 (d, J = 8.4 Hz, 1H, H7), 7.28 (d, J = 8.4 Hz, 1H, H8), 7.60–
7.62 (m, 2H, H5 and H7΄), 7.90 (s, 1H, thiazole-H), 7.95 (s, 1H, H5΄),
8.27 (s, 1H, H4), 8.32 (s, 1H, H4΄); 13C NMR (DMSO–d6): δ 53.4,
60.6, 66.6, 114.3, 117.5, 117.8, 118.0, 118.3, 118.6, 118.8, 123.5, 129.3,
130.2, 130.6, 133.6, 134.4, 135.8, 144.2, 149.2, 151.4, 153.0, 162.8,
163.3, 165.6; Anal. for C26H19 BrN2O5S (551.4): C, 56.63; H, 3.47; N,
5.08; found: C, 56.49; H, 3.57; N, 4.93%.
3

S.M.H. Sanad, A.E.M. Mekky and T.T. El-Idreesy
Journal of Molecular Structure 1248 (2022) 131476
at 37 °C. All the results were carried out in duplicate and the av-
erage values were determined.
dye. After three hours of incubation at 37 °C, the dye medium
was removed and the microplate was washed twice with 150 μL
PBS to eliminate the unabsorbed neutral red dye contained in the
wells. Then, the cellular morphology of the treated cell lines with
the tested derivatives were detected using Inverted Microscope Le-
ica DMI3000B. Also, the absorbance of acidified ethanol solution
containing extracted neutral red dye was calculated via microplate
reader (BioTek, ELX808) at 540 nm to estimate the optical density
and the cell viability% was determined. All results were performed
2.3.3. Biofilm inhibition assay
The tested thiazoles were screened for their bacterial biofilm
inhibitory activities in sterile 96-well polystyrene microtiter plates
[66]. The assay was performed against each of Staphylococcus au-
reus (ATCC:6538), Streptococcus mutans (ATCC:25,175) and E. coli
(ATCC:9637) strains. The selected strains were cultured in tryptone
soy broth (supplemented with 0.5% glucose) for 24 h. The tested
thiazoles (using concentrations in the range from 0 to 250 μg/mL)
were mixed with 5 × 105 CFU mL−1 concentration of the bacte-
rial suspensions. In each well, 100 μL were distributed and then
allowed to incubate at 37 °C for 24 h under static conditions. To
remove the non-adherent bacteria, the medium was discarded and
then washed with phosphate buffered saline. Each well of the mi-
crotiter plate was stained with 100 μL of 0.1% crystal violet solu-
tion then allowed to incubate for 30 min at rt. Then, the crystal
violet solution was discarded from the plates, washed with dis-
tilled water for 3–4 times and left to dry in air at rt. The crystal
violet stained biofilm was solubilized in 100 μL of ethanol 95% and
the absorbance was measured using spectrophotometer at 540 nm.
Blank wells were used as background check. All results were per-
formed in triplicates. The inhibition data were interpreted from the
dose-response curves and the IC50 values were indicated as mean
SD.
in triplicates. The IC50 values were indicated as mean
SD.
2.5. The in vitro DHFR inhibition assay
The dihydrofolate reductase inhibition assay [68] was per-
formed as per the manual of the DHFR assay kit (Sigma product
code CS0340). All the dilutions were made in assay buffer, pH 7.5.
Stock solutions of the test compounds with different concentra-
tions were prepared in DMSO, and an amount of 20 μL of each
was taken to attain final concentration of 10−8, 10−7, 10−6, 10−5
M
in the respective wells of 96-well plate containing assay buffer.
Methotrexate was diluted to 100-fold dilution with DHFR assay
buffer. 2 μL of tested samples (including methotrexate as positive
control) was added to wells assigned as the test samples, enzyme
control, or inhibitor control. An amount of 0.1 unit of DHFR as sup-
plied in the kit was diluted, and an amount of 2 μL of its 3 × 10−3
unit was added to 798 μL assay buffer, then 98 μL of the diluted
DHFR solution was added to each well of the 96-well plate. Wells
containing 100 μL of assay buffer were used as background control
to check any inhibition of enzyme activity due to solvent. 10 mM
stock solution of NADPH in assay buffer was prepared, mixed by
vortex and kept on ice, then 40 μL of the diluted NADPH solution
was added to wells including the test samples, enzyme control, in-
hibitor control or background control. Avoiding light, wells were
mixed and incubated at room temperature for 10–15 min. 10 mM
Stock solutions of dihydrofolic acid in assay buffer was prepared.
The reactions were started by the addition of 60 μL of dihydro-
folic acid to each well. Absorbance was measured immediately at
340 nm in kinetic mode at room temperature and obtained two
values for the absorbance (A1 and A2) at two-time points (t1 and
t2) in the linear range of the plot. The slope was calculated for all
test inhibitor samples and enzyme control by dividing the net ꢀA
(A1 A2) values with the time ꢀt (t1 t2). Subtract the background
or inhibitor control readings from its paired sample readings.
2.4. Cytotoxicity against eukaryotic cells
2.4.1. Cell line, culture conditions and preparation of compounds
Cytotoxicity was screened against the human breast epithe-
lial MCF-10A, the human breast carcinoma MCF-7, colon cancer
Caco2, and liver hepatocellular carcinoma HEPG2 cell lines, which
were obtained from Cairo University Research Park (CURP), Fac-
ulty of Agriculture. The cell lines were cultivated in Dulbecco’s
Modified Eagle’s Medium (DMEM). All of the growth media were
supplemented with 10% Foetal Bovine Serum (FBS) and antibiotics
(100 U/mL penicillin and 100 mg/mL streptomycin) at 37 °C in a
humidified atmosphere containing 5% CO2. The tested thiazoles as
well as doxorubicin, as a positive control, were dissolved in DMSO
and final concentrations were diluted in culture medium.
2.4.2. Neutral red uptake assay (NRU assay)
The NRU assay relies on the ability of living cells to incorpo-
rate and bind neutral red, a weak cationic dye, in lysosomes [67].
The tested thiazoles were subjected to evaluation of their cyto-
toxicity against MCF-10A, MCF-7, Caco2, and HEPG2 cell lines in
comparison to the reference doxorubicin. Exponentially growing
cells were collected using 0.25% Trypsin-EDTA, then the cell sus-
pension counted using hemocytometer, and cell viability checked
by trypan blue (100% viability). Then, an approximately 1.0 × 105
cell/mL of cells suspension was made by dilution with complete
medium. 200 μL of this suspension, about ≈20,000 cell/well, was
dispensed by multichannel pipette into the inner 60 wells of the
96 well plate and the peripheral wells were filled with PBS. The
plate was allowed to incubate for 24 h to allow cells attachment
to the plate wall before the addition of the tested hybrids. Differ-
ent concentrations of the tested thiazoles (5, 25, 50 and 75 μg/mL)
were made by using DMEM media. Then, 200 μL of treatment me-
dia was dispensed into four replicates for each concentration and
other wells were filled with untreated cells only (as a negative con-
trol) and wells filled with media containing doxorubicin HCL as a
positive control. The 96 well plate allowed to incubate at 37 °C for
48 h. Then, the medium and extracts were discarded and replaced
with 100 μL of neutral red solution (50 mg/mL) and centrifuged
at 1800 rpm for 10 min to eliminate any crystals of precipitated
Slope of enzyme control − Slope of inhibitor sample
% Inhibition =
Slope of enzyme control
X 100
The IC50 values were calculated by plotting a graph between
percentage inhibition and the corresponding concentration of the
compound. All results were performed in triplicates. The IC50 val-
ues were indicated as mean
SD.
2.6. In silico study
2.6.1. Molecular docking
Study of molecular docking was elucidated using Molecular
Operating Environment (MOE) version 2019.01 software (https://
www.chemcomp.com) and it is a rigid molecular docking soft-
ware [69]. Studies of molecular docking have a high significance
for predicting the probable binding modes of the tested active
morpholine-thiazole hybrids against DHFR enzyme from E. coli
(PDB ID: 1RF7). MOE is an interactive molecular graphics software
which could calculate and show feasible docking modes of the tar-
get enzyme and the tested bis(pyrazoles). It requires the tested
compounds and the target enzyme as input in PDB format. The
molecules of water, co-crystallized ligands and other unsupported
4

S.M.H. Sanad, A.E.M. Mekky and T.T. El-Idreesy
Journal of Molecular Structure 1248 (2022) 131476
Scheme 1. Synthesis of benzaldehyde derivative 3 with a morpholine unit.
Scheme 2. Synthesis of thiazole-chromene hybrid 7a.
Table 1
elements (e.g., Na, Mg, SO4 etc.,) were removed but the amino-
acid chain was reserved [70]. The ligand’s structure in PDB file for-
mat was created by Gaussian 03 software. The structure of DHFR
enzyme from E. coli was downloaded from the protein data bank
(https://www.rcsb.org/).
Optimizing the yield of chromene-thiazole hybrid 7a.
Entry
∗Solvent
Catalyst
Time (h)
Yield (%)
1
Water
TEA
8
8
8
8
8
8
8
8
7
7
6
6
6
6
6
6
8
Traces
Traces
14
2
Water
Piperidine
TEA
3
Benzene
Benzene
Benzene
Toluene
Toluene
Toluene
Ethanol
Ethanol
DMF
4
DEA
10
2.6.2. ADME prediction
5
Piperidine
TEA
14
Computational studies of new hybrids 9a 9e were performed to
predict molecular properties using the SwissADME online server.
The hybrids were drawn using ChemDraw Professional 16.0.0.82
[72]. Next, all hybrids were inserted into the SwissADME program
to predict all the physicochemical characteristics, lipophilicity, sol-
ubility, pharmacokinetics, drug likeness, and medicinal chemistry
[73]. The program provides predictions of the molecular weight,
logarithm of partition coefficient (log P), number of hydrogen-bond
donors, number of hydrogen-bond acceptors, number of rotatable
bonds, topological polar surface area, and Lipinski’s rule of five of
the new synthesized hybrids. In addition, SwissADME was used to
estimate the BOILED-Egg diagram that works by computing the po-
larity (TPSA) and lipophilicity (WLOGP) of small molecules [74].
Graphical estimations of human gastrointestinal absorption (HIA)
and blood-brain barrier (BBB) penetration of the new hybrids.
6
22
7
DEA
18
8
Piperidine
TEA
20
9
59
10
11
12
13
14
15
16
17
Piperidine
TEA
55
65
DMF
DEA
60
DMF
Piperidine
TEA
62
Dioxane
Dioxane
Dioxane
Dioxane
85
DEA
70
Piperidine
TEA
74
85
∗
All reactions were carried out at reflux.
elucidated by considering its elemental as well as spectral anal-
yses. Its 1H NMR spectrum revealed two triplet signals at δ 2.40
and 3.60 owing to morpholine protons. In addition, it showed four
singlet signals at δ 3.65, 7.66, 7.93 and 8.34 attributed to CH2,
chromene-H5, thiazole-H and chromene-H4 protons, respectively.
Furthermore, it revealed a multiplet signal at δ 7.22–7.85 corre-
sponding to five aromatic, chromene-H7 and chromene-H8 pro-
tons. Its 13C NMR spectrum showed four signals at δ 53.5, 60.6,
66.3 and 164.6 owing to morpholine-C3, CH2, morpholine-C2 and
chromene-CO carbons, respectively. Additionally, it revealed 15 sig-
nals attributed to chromene, thiazole and aromatic carbons (Exper-
imental part).
3. Results and discussion
3.1. Chemistry
In
the
current
study,
the
precursor
2–hydroxy-5-
(morpholinomethyl)benzaldehyde 3 was synthesized and used as
a key synthon for the synthesis of the target thiazole and thiazole-
chromene hybrids with a morpholine unit. Therefore, morpholine
1 was reacted with 5-(chloromethyl)−2-hydroxybenzaldehyde 2
in boiling toluene at reflux in the presence of triethylamine (TEA)
to afford benzaldehyde derivative 3 in a good yield (Scheme 1)
[75,76].
Recently, the multi-component reaction (MCR) has become an
important tool for the synthesis of poly functionalized heterocyclic
hybrids [80]. Some of the benefits of this technique are simplic-
ity, shorter time of reaction, enhanced efficiency and reduced for-
mation of the by-products with the production of diverse "drug-
like" hybrids incorporating heterocyclic units [81,82]. Therefore, we
investigated the synthesis of the target thiazole-chromene hybrid
7a using a one-pot protocol. The reaction was carried out using
the precursors benzaldehyde derivative 3, 2-cyanothioacetamide 4
and 2bromo-1-phenylethan-1-one 6a under different reaction con-
ditions to prepare the target 7a with a maximum yield and mini-
mum reaction time (Scheme 3 and Table 1).
TLC analyses were used to monitor all reactions used in this
study in order to prepare the desired products in a pure state.
At first, we investigated the synthesis of the carbothioamide
derivative 5, linked to chromene-morpholine hybrid, utilizing ben-
zaldehyde derivative 3. Thus, compound
2-cyanothioacetamide 4 in ethanol containing drops of piperi-
dine at reflux for to give the corresponding 2-imino-6-
3 was reacted with
3
h
(morpholinomethyl)−2H-chromene-3-carbothioamide 5 (Scheme 2
and Experimental section) [77,78]. Next, carbothioamide 5 was re-
acted with 2–bromo-1-phenylethan-1-one 6a in dioxane contain-
ing drops of piperidine. TLC analysis revealed that heating the re-
action mixture at reflux for 3 h was sufficient to yield the tar-
get 6-(morpholinomethyl)−3-(4-phenylthiazol-2-yl)−2H-chromen-
2-one 7a as a sole product (Scheme 2) [77,79]. The structure was
Each of piperidine, diethylamine (DEA) and TEA were investi-
gated as basic catalysts for the one-pot synthesis of 7a and in the
presence of different polar or non-polar solvents. TLC analyses re-
vealed that using of water as a polar solvent afforded only traces
of 7a (Table 1 and entries 1 and 2). Using of benzene or toluene as
5

S.M.H. Sanad, A.E.M. Mekky and T.T. El-Idreesy
Journal of Molecular Structure 1248 (2022) 131476
Scheme 3. One-pot synthesis of thiazole-chromene hybrid 7a.
Scheme 4. One-pot synthesis of thiazole-chromene hybrids 7b 7e.
Scheme 5. One-pot synthesis of thiazole hybrids 9 utilizing α-bromoketones 8 linked to chromene units.
non-polar solvents afforded only 10–22% yields of 7a (Table 1 and
brids were estimated against six different Gram-positive and neg-
ative bacterial strains. For this purpose, the Gram-positive bac-
terial strains of Staphylococcus aureus (ATCC:6538), Streptococcus
mutans, (ATCC:25,175) and Enterococcus faecalis (ATCC:29,212) as
well as the Gram-negative bacterial strains of E. coli (ATCC:9637),
Pseudomonas aeruginosa (ATCC:27,953) and Klebsiella pneumonia
(ATCC:10,031) were selected. To estimate the MIC values for the
new derivatives against the selected bacterial strains, the micro-
broth serial dilution method was used [62,63]. The minimum in-
hibitory concentration (MIC) values were recorded using the ref-
erence ciprofloxacin (MIC values of 2.7 μM against all the tested
bacteria). All findings were listed in Table 2.
entries 3–8). The action of the former basic catalysts was also in-
vestigated in the presence of other polar solvents such as ethanol
and dimethylformamide (DMF) to give 7a in 55–65% yields (Table 1
and entries 9–13). Furthermore, using of dioxane as a solvent was
investigated (Table 1 and entries 14–17). As seen in Table 1, it was
clear that the best yield of 7a was obtained by carrying out the
reaction in dioxane in the presence of TEA as a catalyst at reflux
for 6 h. This last method afforded a pure sample of 7a with high
purity in 85% yield (Table 1 and entry 14).
Using the optimized conditions used for the preparation of 7a,
a new series of chromene-thiazole hybrids 7b 7e was prepared us-
ing the appropriate 1-aryl-2-bromoethan-1-one 6b- instead of 2–
bromo-1-phenylethan-1-one 6a (Scheme 4 and Experimental sec-
tion).
The precursor 2H-chromene-3-carbothioamide 5 showed MIC
values of 25.7 μM against each of S. aureus, S. mutans and E. coli
strains. Nonetheless, compound 5 revealed decreased antibacterial
activities against the other tested strains with MIC values of 206.0
to more than 300 μM. The tested thiazole hybrids showed diverse
antibacterial activities against the tested strains.
Furthermore, we examined the utility of α-bromoketones
with a chromene unit, as versatile synthons for the preparation
of the target chromene-thiazole hybrids. Therefore, the one-pot
reaction of 2–hydroxy-5-(morpholinomethyl)benzaldehyde 3, 2-
cyanothioacetamide 4 and the appropriate 3-(2-bromoacetyl)−2H-
chromen-2-ones 8a 8e in dioxane in the presence of TEA as a cat-
alyst. In all cases, heating the reaction mixture at reflux for 8 h
resulted in the formation of the corresponding thiazole hybrids 9a
9e as sole products as detected by TLC analyses (Scheme 5 and Ex-
perimental section).
In our continuous study to prepare new morpholine-thiazole
hybrids of potential antibacterial efficacies, we described herein
the synthesis of two different series of thiazole hybrids. In each
series, five hybrids were prepared attached to substituents of dif-
ferent electronic properties.
The first series of target hybrids 7a 7e was prepared by attach-
ing a chromene and an arene unit to thiazole-C2 and C4, respec-
tively. The previous series showed, generally, low antibacterial ac-
tivities against all the tested strains. Regarding the S. aureus, S. mu-
tans and E. coli strains, hybrids 7 exhibited MIC values in the range
of 35.9 to 77.1 μM. In addition, the prior hybrids displayed poor
antibacterial activities against the E. faecalis, P. aeruginosa and K.
pneumonia strains with MIC values in the range of 287.6 to more
3.2. Biology
3.2.1. Antibacterial activity: in vitro assay
3.2.1.1. MIC evaluation against standard susceptible ATCC strains. The
in vitro antibacterial activities of the new morpholine-thiazole hy-
6

S.M.H. Sanad, A.E.M. Mekky and T.T. El-Idreesy
Journal of Molecular Structure 1248 (2022) 131476
Fig. 1. Structure of some important morpholine-based drugs I-III and promising DHFR inhibitors IV-VI, as well as the designed hybrids 7 and 9.
Fig. 2. Pharmacophoric elements of the reference sulfadiazine and the designed hybrid 9d [Blue (Acc)/violet (Don), hydrogen bond acceptor/donor; green (Hyd)/orange (Aro),
hydrophobic/aromatic ring].
Table 2
MIC values in μM of new morpholine-thiazole hybrids.
Compound
Compound
MIC in μM
Gram-positive bacterial strains
Gram-negative bacterial strains
S. aureus
S. mutans
E. faecalis
E. coli
P. aeruginosa
K. pneumonia
5
25.7
38.5
71.0
64.5
37.2
35.9
8.2
25.7
77.1
71.0
64.5
37.2
35.9
16.5
30.7
28.2
3.9
206.0
> 300
> 300
> 300
298.6
287.6
66.0
25.7
38.5
71.0
64.5
37.2
35.9
8.2
206.0
> 300
> 300
> 300
298.6
287.6
66.0
> 300
> 300
> 300
> 300
> 300
287.6
66.0
7a
7b
7c
7d
7e
9a
9b
15.3
14.1
3.9
123.2
113.3
16.0
30.7
28.2
3.9
123.1
113.3
16.0
246.4
113.3
32.0
9c
9d
9e
1.7
1.7
15.5
1.7
15.5
15.5
Ciprofloxacin
2.7
2.7
2.7
2.7
2.7
2.7
7

S.M.H. Sanad, A.E.M. Mekky and T.T. El-Idreesy
Journal of Molecular Structure 1248 (2022) 131476
Fig. 3. Structure of some important morpholine-based drugs.
Table 3
Table 4
MBC values in μM of some new morpholine-thiazole hybrids
MIC and MBC values in μM of some new morpholine-thiazole hybrids
against ATCC:33,591 and ATCC:43,300 MRSA strains.
against each of S. aureus, S. mutans and E. coli strains.
MBC in μM
MRSA (ATCC:33,591)
MRSA (ATCC:43,300)
Compound
S. aureus
S. mutans
E. coli
Compound
MIC
MBC
MIC
MBC
9a
16.5
30.7
28.2
7.8
16.5
61.4
56.4
7.8
16.5
30.7
28.2
7.8
9a
66.0
123.2
113.3
32.0
15.5
5.2
132.0
246.5
226.6
64.1
33.0
61.5
113.3
16.0
7.7
66.0
123.2
113.3
32.0
15.5
46.2
9b
9b
9c
9c
9d
9d
9e
3.5
3.5
3.5
9e
31.0
Ciprofloxacin
5.4
5.4
5.4
Linezolid
46.2
2.6
Thiazole derivative 9e displayed more powerful efficacy against
all the tested bacterial strains than the reference ciprofloxacin. It
exhibited MBC values of 3.5 μM against each of E. coli, S. mutans
and S. aureus strains. Again, thiazole derivative 9d was the second
best in antibacterial efficacy with MBC values of 7.8 μM against the
prior strains. Other thiazole derivatives 9a 9c displayed low effica-
cies with MBC values in the range of 16.5 to 61.4 μM.
than 300 μM. In conclusion, incorporation of a chromene unit at
thiazole-C2 has low effect on the antibacterial activities of the tar-
get hybrids.
Motivated by the afore-mentioned findings, a second series of
the morpholine-thiazole hybrids 9a 9e with two chromene units at
thiazole-C2 and thiazole-C4 was prepared. Among all tested com-
pounds, in general, the prior series showed enhanced antibacterial
efficacies. Compound 9e, linked to methoxy group, exhibited the
best antibacterial activity against S. aureus, S. mutans and E. coli
strains (MIC value = 1.7 μM) which is exceeding that of the ref-
erence ciprofloxacin (MIC value = 2.7 μM). In addition, compound
9d, linked to methyl group, was the second-best antibacterial agent
with MIC value of 3.9 μM against these three strains. However, hy-
brids 9a 9c show less potency with MIC values ranging between
8.2 and 30.7 μM against the same three strains. On the other hand,
all 9a 9e hybrids exhibited less antibacterial activities against E.
faecalis, P. aeruginosa and K. pneumonia strains with the activities
are slightly better for hybrids 9d and 9e (MIC values in the range
of 15.5 to 32.0 μM), compared to hybrids 9a 9c (MIC values in the
range of 66.0 to 123.1 μM).
In general, all of the tested hybrids’ MBC values are equal to
or twice their MIC values against the same bacterial strains. This
implies that all of the hybrids tested are bactericidal agents.
3.2.1.3. Assessment of antibacterial activity against MRSA strains. In-
spired by the findings of antibacterial assessment against Staphy-
lococcus aureus strain, some of new hybrids were examined as
potential inhibitors of methicillin-resistant Staphylococcus aureus
(MRSA) strains [37]. In this regard, the inhibitory activity of thi-
azole hybrids 9a 9e were examined against two different MRSA
strains (ATCC:33,591 and ATCC:43,300). The results were recorded
using linezolid as a standard drug. Linezolid exhibited MIC/MBC
values of 5.2/46.2 and 2.6/46.2 μM against ATCC:33,591 and
ATCC:43,300 strains, respectively (Table 4).
In conclusion, incorporating a chromene unit at thiazole-C4 sig-
nificantly improves the antibacterial activities of the desired thia-
zole hybrids. Furthermore, the presence of strong electron donat-
ing groups such as methyl and methoxy groups attached to the
The best inhibitory activity was found for the hybrid 9e. It gave
MIC/MBC values of 15.5/31.0 and 7.7/15.5 μM against ATCC:33,591
and ATCC:43,300 MRSA strains, respectively. Thiazole 9d exhib-
ited less inhibitory activity with MIC/MBC values of 32.0/64.1 and
16.0/32.0 μM against ATCC:33,591 and ATCC:43,300 strains, re-
spectively. Other thiazole hybrids 9a 9c displayed the least in-
hibitory activities with MIC and MBC values in the range of 61.5
to 226.6 μM. Generally, the MBC values of the hybrids 9a 9e are
equal to or twice their MIC values against the same strains. This
implies that hybrids 9a 9e are bactericidal agents.
ꢀ
preceding chromene unit at C6 improves the antibacterial activity
against all of the tested hybrids. All of the findings are summarized
in Fig. 3.
3.2.1.2. MBC evaluation against standard susceptible ATCC strains.
Thiazole hybrids 9a 9e displayed the best antibacterial efficacies
against each of E. coli (ATCC:9637), S. mutans (ATCC:25,175) and S.
aureus (ATCC:6538) strains. These hybrids were subjected to the
evaluation of their minimum bactericidal concentration (MBC) val-
ues against the prior strains [65]. All findings were recorded using
ciprofloxacin as the reference drug (MBC values of 5.4 μM against
all the tested strains) (Table 3).
3.2.1.4. Evaluation of anti-biofilm activity. Thiazole hybrids 9a 9e
were chosen for further assessment of their inhibitory activ-
ity against bacterial biofilm [66]. The results were recorded us-
ing the standard ciprofloxacin with IC50 values of 4.3
0.11,
8

S.M.H. Sanad, A.E.M. Mekky and T.T. El-Idreesy
Journal of Molecular Structure 1248 (2022) 131476
Table 5
3.2.4. Molecular modeling of some new thiazoles
Bacterial biofilm inhibition (IC50 in μM
morpholine-thiazole hybrids.
SD) of some new
Out of 20 new thiazoles prepared in this study, we found that
the thiazole-chromene hybrids 9d and 9e exhibited potent DHFR
inhibitory activity. As a result, we decided to use molecular dock-
ing simulations as an in silico study tool to gain a better under-
standing of the structure-activity relationships of these hybrids via
their binding with enzyme. The determined structures of the se-
lected thiazole-chromene hybrids were docked with DHFR enzyme
from E. coli (PDB ID: 1RF7). Computational docking studies were
used to determine the binding energies of the previous interac-
tions [84]. The docking findings thiazole-chromene hybrids 9b, 9d
and 9e as well as the reference sulfadiazine are listed in Table 8.
The in silico study was conducted to predict the capability of
the tested hybrids as potential inhibitors of DHFR enzyme. The
ligand interactions of the reference sulfadiazine with DHFR are
presented in Fig. 4. Docking of sulfadiazine revealed the pres-
ence of strong H-bonding interactions between the two sulfonyl-O
IC50 in μM
SD
S. mutans
Compound
S. aureus
E. coli
9a
4.7
5.2
5.6
4.5
4.0
4.3
0.14
0.17
0.18
0.12
0.10
0.11
4.8
5.1
5.9
4.6
4.2
4.5
0.15
0.16
0.17
0.13
0.12
0.13
4.7
5.0
5.6
4.5
3.9
4.2
0.13
0.16
0.17
0.11
0.11
0.12
9b
9c
9d
9e
Ciprofloxacin
Table 6
The IC50 values (in μM
SD) of some new thiazole hybrids against MCF-
10A, MCF-7, Caco2 and HEPG2 cell lines.
IC50 in μM
Compound
MCF-10A
MCF-7
Caco2
HEPG2
˚
atoms with GLY 15 and GLY 97 residues in DHFR enzyme (3.24 A,
9a
9.4
9.6
9.6
9.2
9.0
9.2
0.23
8.0
8.2
8.4
7.9
7.6
7.9
0.18
0.19
0.13
0.14
0.12
0.16
8.3
8.4
8.5
8.1
7.9
8.0
0.17
0.15
0.17
0.11
0.14
0.19
8.7
8.8
8.9
8.5
8.3
8.4
0.21
˚
−2.0 kcal/mol; 2.82 A, −4.0 kcal/mol) and between pyrimidine-N
9b
0.26
0.28
0.18
0.21
0.25
0.22
0.24
0.19
0.18
0.21
˚
9c
atom with THR 46 (2.96 A, −2.4 kcal/mol).
9d
Experimental study found that morpholine-thiazole hybrids 9d
and 9e, attached to two chromene units at thiazole-C2 and C4, had
the most powerful inhibitory activity against E. coli DHFR enzyme.
Figs. 5 and 6 represented the 2D and 3D ligand interactions of the
prior hybrids with the target enzyme.
9e
Doxorubicin
4.5
0.13 and 4.2
0.12 μM against S. aureus (ATCC:6538), S.
Some of thiazole hybrids 9 exhibited promising DHFR inhibitory
activities. This is consistent with the previously published results
by Alrohily et al. on thiazole-based derivatives [85]. Accounting for
the superior DHFR inhibitory activity of the tested thiazoles 9d and
9e compared to sulfadiazine drug, docking study showed stronger
ligand interactions between thiazoles 9d and 9e with the residues
of amino acids in DHFR enzyme when compared with the refer-
ence sulfadiazine (Fig. 7).
mutans (ATCC:25,175) and E. coli (ATCC:9637) strains, respectively
(Table 5). Thiazole 9e displayed more powerful biofilm inhibitory
activity than ciprofloxacin. It exhibited IC50 values of 4.0
0.10,
4.2 0.12 and 3.9 0.11 μM against S. aureus, S. mutans and E.
coli strains, respectively. Furthermore, other hybrids displayed IC50
values in the range of 4.5 to 5.9 μM.
3.2.2. The in vitro cytotoxicity evaluation
Presence of morpholine unit in hybrids 9d and 9e enhances the
interactions with amino acids residues of the target enzyme [86].
Hence, docking study revealed strong H-bonding interactions be-
tween morpholine-O atom with ARG 98 residue in DHFR enzyme
The neutral red uptake (NRU) assay was performed to assess
the in vitro cytotoxic activities of hybrids 9a 9e [67]. To achieve
this goal, the human breast epithelial MCF-10A, the human breast
carcinoma MCF-7, colon cancer Caco2, and liver hepatocellular car-
cinoma HEPG2 cell lines were chosen to assess the cytotoxicity of
the new hybrids. All the results were compared with the reference
˚
˚
in both 9d (2.55 A, −4.6 kcal/mol) and 9e (2.40 A, −5.2 kcal/mol).
We could observe the dependence of DHFR inhibitory activi-
ties of thiazoles 9 on the presence of chromene units attached
to thiazole-C2 and C4 [87]. Regarding chromene unit attached to
thiazole-C2, docking poses revealed strong H-bonding interactions
doxorubicin having IC50 values of 7.9
0.16, 8.0
0.19, 8.4
0.21
and 9.2 0.25 μM against the cell lines of MCF-7, Caco2, HEPG2
and MCF-10A, respectively (Table 6).
˚
between chromene-CO group with THR 46 residue in 9d (2.71 A,
Thiazole hybrid 9e displayed more potent cytotoxic activities
˚
−2.9 kcal/mol) and 9e (2.56 A, −3.1 kcal/mol). In addition, dock-
than doxorubicin. It exhibited IC50 values of 7.6
0.12, 7.9
0.14,
ing pose of 9e showed hydrophobic contacts (π-H) between the
8.3 0.18 and 9.0 0.21 μM against the cell lines of MCF-7,
˚
chromene unit with residue of HIS 45 (3.39 A, −1.9 kcal/mol). Re-
Caco2, HEPG2 and MCF-10A, respectively. Other hybrids displayed
IC50 values in the range of 7.9 to 9.6 μM against these cell lines
garding a chromene unit attached to thiazole-C4, docking poses
showed favorable H-bonding interactions between chromene-CO
group with amino acid residue in DHFR enzyme as in 9d with
˚
˚
GLY 97 (3.01 A, −2.6 kcal/mol); and in 9e with GLY 15 (2.95 A,
−2.7 kcal/mol).
3.2.3. The in vitro enzyme inhibition of DHFR
DHFR is a ubiquitous enzyme which shows its presence in
protists, plants and animals. In the folate metabolic pathway for
thymidine biosynthesis (precursor for DNA replication), DHFR role
is well known where, with the aid of NADPH, it is responsible for
reducing dihydrofolate to tetrahydrofolate [83]. With its responsi-
bility for generating DNA replication raw materials, DHFR inhibi-
tion forms the basis for the treatment of many infectious diseases
and is the target of antibacterial drugs like trimethoprim [4].
The in vitro inhibitory activities of DHFR enzyme [68] were as-
sessed for hybrids 9a 9e using sulfadiazine as a reference (IC50
The ligand interactions of the reference hybrid 9b with DHFR
are presented in Fig. 8. Docking of 9b revealed the presence
of strong H-bonding interactions between the two chromene-CO
˚
groups with THR 46 and GLY 97 residues in DHFR enzyme (3.13 A,
˚
−1.2 kcal/mol; 3.31 A, −1.6 kcal/mol).
3.2.5. SwissADME predictions of some new hybrids
The data predicted for the physicochemical characteris-
tics, lipophilicity, solubility, pharmacokinetics, drug likeness, and
medicinal chemistry of new hybrids 9a 9e evaluated by Swiss-
ADME (http://www.swissadme.ch) are given in Table S1 (see
Electronic supplemental file) [73]. Hybrids 9a 9e had molecular
weights ranging from 472.51 to 551.41 g/mol, a number of rotat-
able bonds, and H-bond acceptors in the ranges of 4–5 and 7–8, re-
spectively, with no H-bond donors. Furthermore, the previous hy-
value is 0.138
played the same or more powerful DHFR inhibitory activity than
the standard sulfadiazine with IC50 values of 0.131 0.004 and
0.122 0.004 μM, respectively. Furthermore, other hybrids 9a 9c
0.005 μM) (Table 7). Hybrids 9d and 9e dis-
showed lower inhibitory potency with IC50 values in the range of
0.224 to 0.453 μM.
9

S.M.H. Sanad, A.E.M. Mekky and T.T. El-Idreesy
Journal of Molecular Structure 1248 (2022) 131476
Table 7
The IC50 (in μM
SD) of DHFR inhibitory activity of the tested thiazoles.
Compound
IC50
9a
9b
9c
9d
9e
Sulfadiazine
0.224
0.006
0.336
0.009
0.453
0.012
0.131
0.004
0.122
0.004
0.138
0.005
Table 8
Docking results of thiazole-chromene hybrids 9b, 9d and 9e as well as the reference sulfadiazine with Escherichia coli DHFR (PDB ID: 1RF7).
˚
Compound
9b
Ligand moiety
Site
Interaction
Distance (A)
E (kcal/mol)
O 33
O 46
N THR 46 (A)
N GLY 97 (A)
H-acceptor
H-acceptor
3.13
3.31
−1.2
−1.6
O 23
O 33
O 46
NH2 ARG 98 (A)
N THR 46 (A)
N GLY 97 (A)
H-acceptor
H-acceptor
H-acceptor
2.55
2.71
3.01
−4.6
−2.9
−2.6
9d
O 23
O 33
O 46
6-ring
NH2 ARG 98 (A)
N THR 46 (A)
CA GLY 15 (A)
N HIS 45 (A)
H-acceptor
H-acceptor
H-acceptor
Pi-H
2.40
2.56
2.95
3.39
−5.2
−3.1
−2.7
−1.9
9e
O 15
O 16
N 27
CA GLY 15 (A)
N GLY 97 (A)
H-acceptor
H-acceptor
H-acceptor
3.24
2.82
2.96
−2.0
−4.0
−2.4
Sulfadiazine
OG1 THR 46 (A)
Fig. 4. 2D and 3D ligand interactions of sulfadiazine with Escherichia coli DHFR (1RF7).
Fig. 5. A) 2D; and B) 3D ligand interactions of hybrid 9d with Escherichia coli DHFR (1RF7).
10

S.M.H. Sanad, A.E.M. Mekky and T.T. El-Idreesy
Journal of Molecular Structure 1248 (2022) 131476
Fig. 6. A) 2D; and B) 3D ligand interactions of hybrid 9e with Escherichia coli DHFR (1RF7).
Fig. 7. The superposition of sulfadiazine (red, to the front) with either A) 9d (green, to the back); or B) 9e (green, to the back) inside the active site of DHFR (1RF7) structure.
Fig. 8. A) 2D; and B) 3D ligand interactions of hybrid 9b with Escherichia coli DHFR (1RF7).
brids’ topological polar surface area (TPSA) ranged from 114.02 to
solubility log S < 6 (ESOL model) [91], which are favorable for
bioavailable drugs.
2
˚
123.25 A .
The descriptor for lipophilicity is the partition coefficient be-
Lipinski’s rule of five rule states that there is no more than one
violation of the following physicochemical parameters by orally
active drugs: the molecular weight of the tested ligands ≤ 500,
their H-bonding donors ≤ 5, H-bonding acceptors ≤ 10 and log
tween octanol and water (log Po/w) that has a critical importance
for pharmacokinetics drug discovery [88]. The SwissADME pro-
vides access to five predictive models, including XLOGP3 [89] and
WLOGP [90]. The tested hybrids showed XLOGP3 and WLOGP in
the ranges of 3.73–4.42 and 3.99–4.75, respectively. Furthermore,
all tested hybrids, with the exception of 9c, demonstrated water
Po/ ≤ 5 [92]. Only one violation of the above parameters (molec-
w
ular weights > 500) was found in hybrids 9b, 9c, and 9e, while
hybrids 9a and 9d showed no violations. As a result, all tested
11

S.M.H. Sanad, A.E.M. Mekky and T.T. El-Idreesy
Journal of Molecular Structure 1248 (2022) 131476
hybrids were classified as drug-like. However, because of two vi-
olations of lead-likeness (molecular weights > 350 and XLOGP3 >
3.5) [93], all hybrids are not considered lead-like.
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4. Conclusion
A one-pot reaction protocol was adopted to prepare two dif-
ferent series of chromene-thiazole hybrids linked to a morpholine
unit. The protocol involved reacting of the appropriate aldehyde,
2-cyanothioacetamide and α-bromoketones in dioxane in the pres-
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two chromene units at thiazole-C2 and C4, demonstrated good an-
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Declaration of Competing Interest
The authors declare no conflict of interest.
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CRediT authorship contribution statement
Sherif M.H. Sanad: Conceptualization, Methodology, Investi-
gation, Writing – review & editing, Data curation, Supervision.
Ahmed E.M. Mekky: Investigation, Validation, Writing – review
& editing, Project administration, Software, Visualization. Tamer T.
El-Idreesy: Investigation, Resources, Formal analysis, Writing –
original draft.
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doi:10.3390/molecules22081374.
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