Synthesis and exploration of 2-morpholino-4-phenylthiazol-5-yl acrylamide derivatives for their effects against carbonic anhydrase I, II, IX and XII isoforms as a non-sulfonamide class of inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.115090

Novel series of 2-morpholino-4-phenylthiazol-5-yl acrylamide derivatives (8a–s) have been synthesized and explored as a non-sulfonamide class of carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. The newly synthesized molecules were evaluated for their CA in

Full text of DOI:10.1016/j.bmc.2019.115090

Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and exploration of 2-morpholino-4-phenylthiazol-5-yl acrylamide
derivatives for their effects against carbonic anhydrase I, II, IX and XII
isoforms as a non-sulfonamide class of inhibitors
Baijayantimala Swaina, Chander Singh Digwala, Andrea Angelib, Mallika Alvalaa, Priti Singha,
⁎
⁎
Claudiu T. Supuranb, , Mohammed Arifuddina,
a Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Balanagar, Hyderabad 500037, India
b Università degli Studi di Firenze, Neurofarba Dept., Sezione di Scienze Farmaceutiche e Nutraceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy
A R T I C L E I N F O
A B S T R A C T
Keywords:
Novel series of 2-morpholino-4-phenylthiazol-5-yl acrylamide derivatives (8a–s) have been synthesized and
explored as a non-sulfonamide class of carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. The newly synthesized
molecules were evaluated for their CA inhibitory potency against four isoforms: the cytosolic isozyme hCA I, II as
well as trans-membrane tumor associated isoform hCA IX and hCA XII taking acetazolamide (AAZ) as standard
drug. The results revealed that most of the compounds showed good activity against hCA II, IX, and XII whereas
none of them were active against hCA I (Ki > 100 μM). It is observed that the physiologically most important
cytosolic isoform hCA II was inhibited by these molecules in the range of Ki 9.3–77.7 μM. It is also found the both
the transmembrane isoforms hCA IX and XII were also inhibited with Kis ranging between 54.7–96.7 μM and
4.6–8.8 μM, respectively. The binding modes of the active compounds within the catalytic pockets of hCA II, IX
and XII were evaluated by docking studies. This new non-sulfonamide class of selective inhibitors of hCA II, IX
and XII over the hCA I isoform may be used for further understanding the physiological roles of some of these
isoforms in various pathologies.
Carbonic anhydrase
Nonsulfonamide
Acrylamide
Isoform selective inhibitor
Glaucoma
Cancer
1. Introduction
morpholine, phenyl acrylamide etc., are frequently used for the
synthesis of various heterocyclic compounds with wide ranges of
Carbonic anhydrases (CAs, EC 4.2.1.1) are the essential enzymes
that effectively catalyze the reversible hydration of CO2 to bicarbonate
and proton, an important reaction for many physiological processes.1
This Zn(II) containing metalloenzyme (α-CA) has sixteen different iso-
forms (I–XV, VA and VB) in mammals (Fig. 1), which may promote
pathological situations under their malfunctioning and/or altered ex-
pression.2 Therefore, it is important to develop an effective approach
that can selectively inhibit isoforms involved in different diseases. Al-
though, there are several commercially available CAIs, such as acet-
azolamide, ethoxzolamide, dorzolamide, well known for their antic-
onvulsant, anti-glaucoma, and anti-infective activities,3 none of them
shows selectivity for a specific isoenzyme, therefore causing side ef-
fects. Nowadays, the major concern of the researchers working in this
field is to develop new concept for designing new families of com-
pounds that are capable to block specifically the process catalyzed by
one or two isoforms of these enzymes.
pharmacological activities. As an important class of heterocyclic com-
pounds, the thiazoles are associated with almost all the biological and
pharmacological activities such as antimicrobial (Sulfathiazole)
(Fig. 2A), antiretroviral (Ritonavir) (Fig. 2B), antifungal (Abafungin)
(Fig. 2C), and antineoplastic (Tiazofurin) (Fig. 2D) with recent appli-
cations for the treatment of allergies, hypertention, inflammation,
schizophrenia, bacterial, HIV infections, hypnotics, and more recently
for the treatment of pain, as fibrinogen receptor antagonists with an-
tithrombotic activity and as new inhibitors of bacterial DNA gyrase B.4
The morpholine linked thiazole moiety was found to be relevant in
the structure of inhibitors of the DNA binding domain of the androgen
receptor (Fig. 2E),5 PI3K inhibitors (Fig. 2F, G)6 and as antifungal
agents (Fig. 2H).7 On the hand acrylamide derivatives were explored by
various researchers for numerous biological activities such as antic-
ancer (Fig. 2I),8 antiparasite (Fig. 2J),9 antioxidant (Fig. 2K),10 and also
as a lipid lowering agent (Fig. 2L).11
In this perspective, privileged scaffolds such as thiazole,
Coming to CA inhibition, sulfonamides were the most investigated
⁎ Corresponding authors.
E-mail addresses: claudiu.supuran@unifi.it (C.T. Supuran), arif.niperhyd@gov.in (M. Arifuddin).
https://doi.org/10.1016/j.bmc.2019.115090
Received 13 June 2019; Received in revised form 27 August 2019; Accepted 4 September 2019
0968-0896/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:BaijayantimalaSwain,etal.,Bioorganic&MedicinalChemistry,https://doi.org/10.1016/j.bmc.2019.115090

B. Swain, et al.
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acrylic acid (7a–d) derivatives which were finally subjected to acid-
amine coupling in the presence of EDCI-HOBt as coupling reagent to
afford the target compound (8a–s) in good yield. It selectively yields E-
isomer and were found to be geometrically pure with trans configura-
tion (J = 13–16 Hz) from 1H NMR (i.e. in compound 8a the unsaturated
proton at 6.20 showing a doublet with J = 14.9 Hz). The newly syn-
thesized compounds (8a–s) are shown in the Table 1.
2.2. Carbonic anhydrase inhibition
The inhibition data against four CA isoenzymes, hCA I, II (cytosolic
isoforms involved in glaucoma and other pathogenic conditions) and
the transmembrane tumor-associated hCA IX, and XII (anticancer drug
target) with the newly synthesized molecules 8a–s along with the
standard drug AAZ, are shown in Table 2. The screening was done by a
stopped flow CO2 assay.20
Fig. 1. The subcellular localization of catalytically active human CA iso-
enzymes.
class of CA inhibitors (CAIs) that bind to the metal ion from the enzyme
active site by displacing the metal-bound hydroxide ion/water which is
situated at the bottom of a (15 Å) deep active site pocket and co-
ordinated by three amino acid residues, His94, His96 and His119 in a
tetrahedral geometry (Fig. 3A).12,13 On the other hand, the carboxylates
were found to be the most complicated family of inhibitors, showing a
variety of inhibition mechanism such as monodentate coordination of
carboxylate to the metal ion, or anchoring to the zinc coordinated
water, or binding at the entrance or even outside the active site cavity
(Fig. 3).14–17
The following structure activity relationship can be figured out from
the inhibition data of Table 2:
All the synthesized compounds (8a–s) were very weak or ineffective
•
inhibitors for isoform hCA I, which is a slow cytosolic and off-target
isozyme for anti-epileptics.
The anti-glaucoma drug target hCA II was inhibited by five com-
•
pounds (8k, 8m, 8n, 8o and 8r) in moderate to low micro molar
range (Ki = 9.3–77.7 μM) whereas other compounds were in-
effective. It is assumed that these compounds i.e. 8k (Ki = 39.2 μM),
8m (Ki = 9.3 μM), 8n (Ki = 77.7 μM), 8o (Ki = 50.4 μM), 8r
(Ki = 67.8 μM) were showing inhibition might be due to the pre-
sence of 4-NO2, 4-Cl as R1 group and 4-OMe, 4-Br and 2-NH2 as R2
group.
Recently, one of us presented17 a comparison study of carbon versus
sulfur based zinc binding groups where the carbon based compounds
showed an interesting inhibition profile against all the mammalian
isoforms of carbonic anhydrase CA I–XV, although with a lower efficacy
compared to the sulfonamides (Fig. 4A). It has also been explained that
the inhibition depends on: the pKa of the ZBG, its geometry (tetra-
hedral, i.e. sulfur-based, versus trigonal, i.e. carbon-based ZBGs), or-
ientation of the organic scaffold induced by the nature of the ZBG
where the carboxamide has shown very good inhibition potency against
CA isoforms.18 Di Fiore et al. showed that the hydroxamate have in-
hibited all 12 CA isoforms with inhibition constants in the range of
0.94–179 μM and are less effective with compared to the sulfonamide
but exhibiting a comparable activity with reference to that of the N-
substituted sulfonamides (Fig. 4B).19 Also Carradori et al. has described
the amide derivatives of Probenecid as selective inhibitors of carbonic
anhydrase IX and XII (Fig. 4C).20 Based on these literatures findings on
carbonic anhydrase and the diverse activity associated with thiazole
our endeavor to discover novel CA inhibitors and hence this particular
scaffold non-sulfonamide based 2-morpholinothiazolophenylacryla-
mide has been synthesized and evaluated for CA inhibitions.
hCA IX, the tumor associated transmembrane isoform was inhibited
•
in low micro molar range by some of the synthesized molecules and
the rest of the molecules were ineffective. The molecules (8m, 8n,
8o and 8r) which contain -NO2 and -Cl substitution at R1 position
were showing inhibition with Ki ranging 54.7–96.7 μM.
In the case of tumor associated isoform hCA XII the compounds 8k,
•
8m and 8o exhibited moderate to weak inhibition properties with Ki
in the range 4.6–8.8 μM.
Thus in brief the results summarised from the screening data against
•
the four isoforms: The molecules have showed good inhibition when
R1 substituted with -NO2 and -Cl as compared to 4-OMe and -H.
Hence the electron withdrawing groups were facilitating the good
inhibition. It was observed that the compounds with the substitution
on R2 as 4-OMe, 4-Br and 2-NH2 exhibiting better inhibitory potency
when compared to the di- and tri -OMe derivatives. It was also no-
ticed that the compounds have shown inhibitory potency selectivity
towards hCA II, IX, XII over hCA I.
2. Results and discussion
2.1. Chemistry
2.3. Docking studies
The current work was designed to selectively inhibit hCA I, II, IX,
XII isoforms, with derivatives which do not incorporate a sulfonamide
moiety. The designed molecule 2-morpholino-4-phenylthiazol-5-yl
cinnamamide (8a–s) was synthesized according to the general synthetic
plan depicted in Scheme 1. Acyl chloride (1a–d) was refluxed with
KSCN in dry CH3CN, cooled to room temperature and followed by ad-
dition of a solution of morpholine to give N-(morpholine-4-carbo-
nothioyl)benzamide (2a–d). Compound 2a–d was treated with TEA
followed by ethyl bromoacetate at room temperature in ethanol to af-
ford the thiazole ester derivative (3a–d). The compound 3a–d on re-
duction with lithium aluminum hydride produces the corresponding
thiazole alcohol (4a–d) which in turn was subjected to Swern oxidation
to provide compound 5a–d. Furthermore, compound 5a–d was sub-
jected to Wittig reaction to give the corresponding acryl ester 6a–d. In
the next step the compound 6 was saponified to give the corresponding
Molecular docking calculations were performed to understand the
interaction of synthesized analogues at active site pocket of CA iso-
forms. The docking scores of 8a–s against various isoforms of CA were
shown in Table 3. All the synthesized compounds shown lower docking
scores in comparison to standard co-crystals of hCA II, IX, XII, but some
of them were showing interactions with amino acid (AA) residues
within the binding site. All the docking scores and interacting AA are
displayed in Table 4.
With respect to SAR, it has been observed that the compounds with
electron donating groups are less interactive than electron withdrawing
groups. The compounds 8k, 8m, 8n, 8o, 8r which are showing good
inhibition against several hCA isoforms demonstrated strong interac-
tion. These results are supporting the in-vitro enzymatic assay results.
Furthermore, these new class of compounds also not belongs to any
non-classical CA inhibitors. It is anticipated that these new class of
2

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Fig. 2. Thiazoles, 2-morpholinothiazole and acrylamides derivatives used for the treatment of various diseases.
Fig. 3. CA inhibition mechanisms: A. Zinc-binders; B. Compounds which anchor to the metal ion coordinated water/hydroxide ion; C. Occlusion of the active site
entrance. D. Out of the active site binding. Adapted from Ref. 3b.
compounds might be interacting with a CA via different mechanism
which is beyond the present scope of work. Some docking poses of
compounds synthesized in this work, when bound to the active site of
isoforms hCA II, IX and XII are shown in Fig. 5. It should be mentioned
that the suggested binding modes of Fig. 5 were not yet validated by
means of crystallographic studies.
3

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Fig. 4. Carbon and sulfur based CAIs investigated in Ref. 17.
3. Conclusion
and reactions involving air- or moisture-sensitive compounds were
performed under a nitrogen atmosphere using dried glassware and
syringe techniques to transfer solutions. Melting points (mp) were
measured on Stuart digital melting-point apparatus/SMP-30 in open
capillary tubes and uncorrected. Analytical thin-layer chromatography
(TLC) was carried out on Merck silica gel 60 F-254 aluminum plates.
Column chromatography purifications were performed on silica gel
(60–120 mesh) as the stationary phase and ethyl acetate /n-hexane
(3:7) were used as eluents. Nuclear magnetic resonance (1H NMR and
13C NMR) spectra were recorded using an Advanced Brucker NMR
spectrometer at 500 MHz, 125 MHz in DMSO‑d6. Chemical shifts (δ)
values reported in parts per million (ppm) relative to tetramethylsilane
(TMS) as an internal reference, and the coupling constants (J) expressed
in hertz (Hz). Splitting patterns are symbolized as follows: s, singlet; d,
doublet; t, triplet; m, multiplet; dd, doublet of doublet; q, quartet.
HRMS were conformed with Agilent QTOF mass spectrometer 6540
series instrument and were carried out in the ESI techniques at 70 eV.
In conclusion, we have synthesized 2-morpholinothiazole phenyla-
crylamide derivatives (8a–s) and screened against the various CA iso-
forms. Among them compound 8k, 8m, 8n, 8o and 8r are shown no-
table inhibitory activity against three isoforms hCA II, IX and XII with Ki
in the range of 4.6–96.7 μM. Since these compounds are not belonging
to neither classical nor non-classical CAIs functionality, thus it is an-
ticipated that further optimization and exploration of such kind of
novel scaffolds will lead to structurally diverse ZBGs and play important
role in the design and development of new isoform selective CAIs with
better inhibition potency and less side effects. Hence it may be con-
cluded that the further modification of this newly designed 2-mor-
pholinothiazole phenylacrylamide derivatives (8a–s) will emerges as
isoform selective CAIs.
4. Experimental
4.1. General
4.1.1. General procedure for the synthesis of N-(morpholine-4-
carbonothioyl)benzamide derivatives (2a–d)
All the reagents implemented here in the present work were pur-
chased from commercial suppliers and used without further purifica-
tion. All the solvents were purified and dried using standard methods
prior to use. All reactions were performed under atmospheric pressure
A stirred solution of acyl chloride 1 (10 mmol) and KSCN (12 mol)
in dry CH3CN (150 mL) was refluxed for 3 h. After the mixture was
cooled to room temperature, a solution of the morpholine (12 mol) in
CH3CN (40 mL) was added drop wise (temperature maintained below
Scheme 1. Reagents and conditions: (i) KSCN, CH3CN, reflux, 3 h; (ii) Morpholine, CH3CN, rt, 1–3 h; (iii) Ethyl bromoacetate, TEA, EtOH, rt (iv) LiAlH4, dry THF,
0 °C (v) MnO2, DCM, 0 °C (vi) Ph3PCHCOOEt, benzene, reflux (vii) EtOH: 10% NaOH (1:1), 50 °C (viii) EDCI, HOBt, TEA, DMF, rt.
4

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Table 1
Table 3
Structure of compounds 8a–s.
Docking scores for the compounds 8a–s against hCA I, II, IX, XII.
S. No
Compound
hCA II
hCA IX
hCA XII
1
8a
8b
8c
8d
8e
8f
−4.322
−3.794
−3.762
−3.217
−4.492
−4.028
−4.384
−3.277
−3.82
−4.865
−4.906
−1.605
−1.032
−4.408
−1.728
−1.104
−1.544
−0.975
−4.431
−4.911
−4.765
−5.075
−4.802
−3.405
−3.935
−3.418
−1.838
−4.79
−4.394
−4.86
2
2
−4.219
−2.549
−4.43
4
5
6
−3.384
−3.832
−3.76
7
8g
8h
8i
8
9
−2.916
−4.515
−3.762
−3.233
−4.513
−4.013
−3.403
−3.733
−3.658
−3.502
−4.482
10
11
12
13
14
15
16
17
18
19
8j
−4.251
−4.045
−3.591
−3.945
−5.359
−4.187
−4.709
−2.859
−2.062
−5.174
Compound
R1
R2
Isolated yield
8k
8l
8a
8b
8c
8d
8e
8f
-H
-4-OMe
-3,4-OMe
-3,4,5-OMe
-4-Br
80%
82%
86%
42%
66%
83%
83%
88%
44%
69%
78%
80%
36%
56%
80%
82%
85%
38%
64%
8m
8n
8o
8p
8q
8r
8s
-H
-H
-H
-H
-2-NH2
-OMe
-OMe
-OMe
-OMe
-OMe
-NO2
-NO2
-NO2
-NO2
-Cl
-4-OMe
-3,4-OMe
-3,4,5-OMe
-4-Br
8g
8h
8i
8j
-2-NH2
recrystallized from EtOH/H2O to provide white solid of thiazole ester
8k
8l
-4-OMe
-3,4-OMe
-4-Br
derivatives 3a–d.
8m
8n
8o
8p
8q
8r
8s
-2-NH2
4.1.3. Synthesis of (2-morpholino-4-phenylthiazol-5-yl)methanol (4a–d)
To a suspension of Lithium aluminum hydride (2 equiv.) in THF, a
solution of compound 3a–d (1 equiv.) in THF (13 mL) was added
dropwise at 0 °C. The reaction mixture was slowly warmed up to rt and
was stirred for 30 min. It was then cooled to 0 °C. Water, aq. NaOH
(1 N), and water were added successively. The mixture was stirred at rt
for 15 min, then filtered through Celite. The filtrate was extracted with
DCM and the combined DCM layer was concentrated to provide crude
compound (4a–d) which was used without further purification.
-4-OMe
-3,4-OMe
-3,4,5-OMe
-4-Br
-Cl
-Cl
-Cl
-Cl
-2-NH2
Table 2
Inhibitory potency KI (µM) data for compounds 8a–s against hCA I, II, IX, XII.
Compounds
hCA I
hCAII
hCA IX
hCAXII
4.1.4. Synthesis of 2-morpholino-4-phenylthiazole-5-carbaldehyde (5a–d)
To a suspension of MnO2 (30 equiv) in DCM maintained at 0 °C, a
solution of compound 4a–d in DCM (13 mL) was added dropwise
through dropping funnel over period of 1 h. After completion of the
reaction, the reaction mixture was filtered through Celite and celite.
The filtrate was concentrated to provide crude compounds 5a–d which
were purified through column chromatography (30% EA:Hexane).
8a
8b
8c
8d
8e
8f
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
0.250
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
39.2
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
54.7
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
4.6
8g
8h
8i
8j
4.1.5. Synthesis of ethyl (E)-3-(2-morpholino-4-phenylthiazol-5-yl)acrylate
(6a–d)
8k
8l
> 100
9.3
> 100
8.8
To a solution of compound 5a–d (1 equiv.) in benzene (30 mL) was
added ethyl (triphenylphosphoranylidene)acetate (2 equiv.) at room
temperature. The reaction mixture was then refluxed overnight and
then concentrated. The residue was purified by column chromato-
graphy to give compounds 6a–d.
8m
8n
8o
8p
8q
8r
77.7
96.7
> 100
7.5
50.4
94.9
> 100
> 100
67.8
> 100
> 100
96.5
> 100
> 100
> 100
> 100
0.006
8s
> 100
0.012
> 100
0.026
AAZ
4.1.6. Synthesis of (E)-3-(2-morpholino-4-phenylthiazol-5-yl)acrylic acid
(7a–d)
*Mean from 3 different assays, by a stopped flow technique (errors were in the
range of 5–10% of the reported values), KI = inhibitor constant in μM.
A solution of compound 6a–d (1 equiv.) in 10% aq. NaOH:EtOH
(5 mL) was heated at 50 °C until the complete consumption of starting
material. Water (20 mL) was added and the reaction mixture was
acidified using 1 N HCl to provide compound 7a–d as solid. The solid
was collected after filtration, air dried and used without further pur-
ification.
50 °C). The mixture was stirred for 30 min and poured into an ice/water
mixture (500 mL) with stirring. The precipitate was collected and re-
crystalized from EtOH/H2O to provide white solid product 2.
4.1.2. General procedure for the synthesis of thiazole esters (3a–d)
To a stirred solution of compound 2 (10 mmol, 1 equiv.) in ethanol
(100 mL), was added TEA (12 mmol, 1.2 equiv.) followed by addition of
ethyl bromoacetate (12 mmol, 1.2 equiv.) at room temperature. The
reaction mixture was stirred overnight and then poured into an ice/
water mixture (500 mL) with stirring. The precipitate was collected and
4.1.7. Synthesis
of
(E)-3-(2-morpholino-4-phenylthiazol-5-yl)-N-
phenylacrylamide (8a–s)
To a solution of 7a–d (1 equiv.) and aniline (1.2 equiv.) in DMF
(5 mL) was added EDCI (1.2 equiv.) and HOBt (0.1 equiv.). The mixture
was stirred at room temperature until complete consumption of starting
materials. The reaction mixture was then added to H2O and extracted
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Table 4
Docking interactions with important amino acids for the compounds 8a–s against hCA I, II, IX, XII.
Compound
Substitution
H bond interaction
R1
R2
hCA II
hCA IX
hCA XII
8a
8b
8c
8d
8e
8f
H
OMe
TRP 5, π-π HIS94
GLN 92
π-π HIE 66, TRP 4
H
Di-OMe
Tri-OMe
Br
ASN 62
x
π-π HIE 66, TRP 4
H
ASN 62
x
ASN 64
H
π-π TRP 5
HIE 64, GLN 92, π-π HIS94
π-π LYS 69
H
NH2
THR 199, THR 200, π-π HIS94
VAL 19
ASN 71, LYS 69, π-cat LYS 69
OMe
OMe
OMe
OMe
OMe
NO2
NO2
NO2
NO2
Cl
OMe
GLN 94, π-π HIS94
x
x
8g
8h
8i
Di-OMe
Tri-OMe
Br
GLN 94, π-π HIS94
GLN 94, π-π HIS94
LYS 69
x
x
LYS 69
x
x
ASN 64, π-π HIS91
ASN 64, HIE 66, TRP 4, π-π HIE 66
8j
NH2
GLU 69, π-π HIS94
THR 199
8k
8l
OMe
π-π HIS64, HIS 94
THR 199, GLN 92, π-π HIS64, HIS 94, Salt bridge Zn
TRP 4
Di-OMe
Br
TRP 5
x
x
8m
8n
8o
8p
8q
8r
8s
TRP 5
GLN 92, THR 199, TRP 5, Salt brdg Zn
LYS 69, TRP 4
NH2
ASN 67, GLN 92, π-π HIS94, Salt brdg Zn
GLN 92, TRP 5
TRP 4, π-cat LYS 69
OMe
GLN 92, π-π HIS94
x
ASN 64
Cl
Di-OMe
Tri-OMe
Br
TRP 5, π-π HIS94, PHE 131
GLN 92, π-π HIS94
x
Cl
x
π-π TRP 5
LYS 69, π-cat LYS 69
Cl
x
HIE 64, GLN 92, π-π HIS64, HIS94, Salt brdg. Zn
THR 199, THR 200, π-π HIE 64, TRP 5
x
Cl
NH2
HIS 64, π-π HIS94
THR 88, π-cat LYS 69
X = no interaction.
Fig. 5. (I) Docking pose of compound 8m with hCA II (II) docking pose of compound 8m with hCA IX (III) docking pose of compound 8n with hCA IX (IV) docking
pose of compound 8m with hCA XII. The pink arrows in the figure indicate hydrogen bonding interactions. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
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with CH2Cl2 (3×). The combined organic phases were washed with
H2O, dried over NaSO4, and concentrated in vacuo. The crude products
were purified using silica gel chromatography in EA:Hexane gradients.
130.81 (s), 120.89 (s), 119.30 (s), 118.00 (s), 114.45 (d, J = 10.0 Hz),
65.84 (s), 55.71 (d, J = 14.7 Hz), 48.38 (s). HRMS (ESI) m/z: [M+H]+
calculated for C24H26N3O4S+ 452.1639, found 452.1652.
4.1.8. Characterization of compounds 8a–s
4.1.8.7. (E)-N-(3,4-Dimethoxyphenyl)-3-(4-(4-methoxyphenyl)-2-
4.1.8.1. (E)-N-(4-Methoxyphenyl)-3-(2-morpholino-4-phenylthiazol-5-yl)
acrylamide (8a). Light yellow solid, yield: 80%, mp: 302–304 °C; 1H
NMR (500 MHz, DMSO‑d6) δ 9.95 (s, 1H), 7.65–7.44 (m, 8H), 6.90 (t,
J = 14.5 Hz, 2H), 6.20 (d, J = 14.9 Hz, 1H), 3.78–3.73 (m, 4H), 3.72 (s,
3H), 3.58–3.51 (m, 4H). 13C NMR (125 MHz, DMSO‑d6) δ 168.86,
163.53, 155.66, 155.26, 134.80, 133.07, 131.15, 129.45, 129.21,
129.02, 120.92, 119.86, 119.06, 114.41, 65.83, 55.64, 48.39. HRMS
(ESI) m/z: [M+H]+ calculated for C23H24N3O3S+ 422.1533, found
422.1534.
morpholinothiazol-5-yl) acrylamide (8g). Light yellow solid, yield: 83%,
mp: 302–303 °C; 1H NMR (500 MHz, DMSO‑d6) δ 9.94 (s, 1H), 7.62 (d,
J = 14.9 Hz, 1H), 7.52 (d, J = 8.6 Hz, 2H), 7.44 (s, 1H), 7.09 (dd,
J = 12.9, 9.0 Hz, 3H), 6.89 (d, J = 8.7 Hz, 1H), 6.16 (d, J = 14.9 Hz,
1H), 3.83 (s, 3H), 3.77–3.71 (m, 10H), 3.58–3.51 (m, 4H). 13C NMR
(125 MHz, DMSO‑d6)
δ 168.73, 163.72, 160.18, 155.26, 149.11,
133.66, 131.53, 130.80, 127.26, 119.23, 117.99, 114.49, 112.81,
65.84, 56.29, 55.84, 55.76, 48.39. HRMS (ESI) m/z: [M+H]+
calculated for C25H28N3O5S+ 482.1744, found 482.1753.
4.1.8.2. (E)-N-(3,4-Dimethoxyphenyl)-3-(2-morpholino-4-phenylthiazol-
5-yl)acrylamide (8b). Light yellow solid, yield: 82%, mp: 285–287 °C;
1H NMR (500 MHz, DMSO‑d6) δ 9.96 (s, 1H), 7.64–7.45 (m, 7H), 7.43
(d, J = 2.2 Hz, 1H), 7.10 (dd, J = 8.7, 2.3 Hz, 1H), 6.89 (d, J = 8.8 Hz,
1H), 6.20 (d, J = 14.9 Hz, 1H), 3.77–3.74 (m, 4H), 3.72 (d, J = 4.0 Hz,
6H), 3.57–3.53 (m, 4H). 13C NMR (125 MHz, DMSO‑d6) δ 168.89,
163.58, 155.34, 149.03, 145.26, 134.75, 133.55, 131.21, 129.44,
129.23, 129.04, 119.75, 119.01, 112.66, 111.30, 104.58, 65.83,
56.21, 55.78, 48.38. HRMS (ESI) m/z: [M+Na]+ calculated for
4.1.8.8. (E)-3-(4-(4-Methoxyphenyl)-2-morpholinothiazol-5-yl)-N-(3,4,5
trimethoxyphenyl) acrylamide (8h). Light yellow solid, yield: 88%, mp:
327–329 °C; 1H NMR (500 MHz, DMSO‑d6) δ 10.02 (s, 1H), 7.64 (d,
J = 14.9 Hz, 1H), 7.51 (t, J = 12.3 Hz, 2H), 7.12–7.02 (m, 4H), 6.15 (d,
J = 14.9 Hz, 1H), 3.83 (d, J = 5.6 Hz, 3H), 3.76 (d, J = 20.0 Hz, 10H),
3.63 (d, J = 7.2 Hz, 3H), 3.54 (dd, J = 12.2, 7.6 Hz, 4H). 13C NMR
(125 MHz, DMSO‑d6)
δ 168.81, 163.99, 160.21, 155.53, 153.22,
136.05, 131.91, 130.81, 127.20, 118.92, 117.90, 114.50, 97.28,
65.84, 60.58, 56.17, 55.77, 48.39. HRMS (ESI) m/z: [M+H]+
calculated for C26H30N3O6S+ 512.1850, found 512.1896.
C
24H25N3NaO4S 474.1664, found 474.1669.
4.1.8.3. (E)-3-(2-Morpholino-4-phenylthiazol-5-yl)-N-(3,4,5-
trimethoxyphenyl)acrylamide (8c). Light yellow solid, yield: 86%, mp:
297–299 °C; 1H NMR (500 MHz, DMSO‑d6) δ 10.03 (s, 1H), 7.64 (d,
J = 14.9 Hz, 1H), 7.59 (d, J = 1.5 Hz, 1H), 7.57 (s, 1H), 7.55–7.45 (m,
3H), 7.04 (s, 2H), 6.19 (d, J = 14.9 Hz, 1H), 3.78–3.72 (m, 10H), 3.62
(s, 3H), 3.58–3.53 (m, 4H). 13C NMR (125 MHz, DMSO‑d6) δ 168.95,
163.87, 155.58, 153.22, 135.99, 134.74, 133.94, 131.59, 129.44,
129.24, 129.03, 119.49, 118.95, 97.29, 65.83, 60.58, 56.17, 48.41.
HRMS (ESI) m/z: [M+H]+ calculated for C25H28N3O5S+ 482.1744,
found 482.1758.
4.1.8.9. (E)-N-(4-Bromophenyl)-3-(4-(4-methoxyphenyl)-2-
morpholinothiazol-5-yl) acrylamide (8i). Light yellow solid, yield: 44%,
mp: 341–343 °C; 1H NMR (500 MHz, DMSO‑d6) δ 10.18 (s, 1H), 7.63
(dd, J = 11.7, 10.3 Hz, 3H), 7.49 (dt, J = 27.1, 13.5 Hz, 4H), 7.06 (t,
J = 11.9 Hz, 2H), 6.16 (d, J = 14.9 Hz, 1H), 3.83 (d, J = 5.3 Hz, 3H),
3.79–3.71 (m, 4H), 3.60–3.50 (m, 4H). 13C NMR (125 MHz, DMSO‑d6) δ
168.88, 164.23, 160.26, 155.84, 139.29, 132.40, 132.02, 130.85,
127.18, 121.37, 118.52, 117.83, 115.06, 114.51, 65.83, 55.77, 48.38.
HRMS (ESI) m/z: [M+H]+ calculated for C23H23BrN3O3S+ 500.0638,
found 500.0624.
4.1.8.4. (E)-N-(4-Bromophenyl)-3-(2-morpholino-4-phenylthiazol-5-yl)
acrylamide (8d). Light Yellow solid, yield 42%, mp: 299–301 °C; 1H
NMR (500 MHz, DMSO‑d6) δ 10.20 (s, 1H), 7.65–7.61 (m, 3H),
7.59–7.56 (m, 2H), 7.54–7.47 (m, 5H), 6.20 (d, J = 14.9 Hz, 1H),
3.77–3.73 (m, 4H), 3.58–3.54 (m, 4H). 13C NMR (125 MHz, DMSO‑d6) δ
169.04, 164.12, 155.91, 139.22, 134.69, 132.05, 130.26, 129.48,
129.34, 129.06, 128.81, 121.36, 119.06, 118.84, 115.13, 65.82,
48.38. HRMS (ESI) m/z: [M+H]+ calculated for C22H21BrN3O2S+
470.0532, found 472.0512.
4.1.8.10. (E)-N-(2-Aminophenyl)-3-(4-(4-methoxyphenyl)-2-
morpholinothiazol-5-yl) acrylamide (8j). Light yellow solid, yield: 69%,
mp: 292–294 °C; 1H NMR (500 MHz, DMSO‑d6) δ 9.24 (s, 1H), 7.60 (d,
J = 14.9 Hz, 1H), 7.50 (dd, J = 20.1, 7.7 Hz, 2H), 7.32 (d, J = 7.8 Hz,
1H), 7.07 (d, J = 8.8 Hz, 2H), 6.89 (t, J = 7.4 Hz, 1H), 6.73 (d,
J = 7.9 Hz, 1H), 6.56 (t, J = 7.5 Hz, 1H), 6.27 (d, J = 15.1 Hz, 1H),
4.93 (s, 2H), 3.83 (s, 3H), 3.75 (s, 4H), 3.53 (t, J = 8.2 Hz, 4H). 13C
NMR (125 MHz, DMSO‑d6) δ 168.73, 160.15, 155.19, 130.80, 127.25,
124.86, 117.99, 116.50, 114.48, 65.83, 55.75, 48.34. HRMS (ESI) m/z:
[M+H]+ calculated for C23H25N4O3S+ 437.1642, found 437.1662.
4.1.8.5. (E)-N-(2-Aminophenyl)-3-(2-morpholino-4-phenylthiazol-5-yl)
acrylamide (8e). Light yellow solid, yield: 66%, mp: 280–282 °C; 1H
NMR (500 MHz, DMSO‑d6) δ 9.26 (s, 1H), 7.59–7.56 (m, 3H), 7.52 (dd,
J = 10.0, 4.7 Hz, 2H), 7.47 (dt, J = 5.2, 2.0 Hz, 1H), 7.32 (d,
J = 7.7 Hz, 1H), 6.89 (t, J = 7.4 Hz, 1H), 6.73 (dd, J = 7.9, 1.1 Hz,
1H), 6.57–6.53 (m, 1H), 6.31 (d, J = 15.0 Hz, 1H), 4.91 (s, 2H),
3.77–3.74 (m, 4H), 3.57–3.53 (m, 4H). 13C NMR (125 MHz,
DMSO‑d6) δ 168.87, 163.96, 155.21, 141.87, 134.82, 131.12, 129.44,
129.19, 129.02, 126.03, 124.89, 124.12, 119.84, 119.08, 116.71,
116.43, 65.83, 48.39. HRMS (ESI) m/z: [M+Na]+ calculated for
4.1.8.11. (E)-N-(4-Methoxyphenyl)-3-(2-morpholino-4-(4-nitrophenyl)
thiazol-5-yl) acrylamide (8k). Orange solid, yield: 78%, mp:
305–307 °C; 1H NMR (500 MHz, DMSO‑d6) δ 10.02 (s, 1H), 8.31 (dd,
J = 61.1, 8.5 Hz, 2H), 7.86 (d, J = 8.6 Hz, 2H), 7.71–7.45 (m, 3H), 6.89
(d, J = 8.8 Hz, 2H), 6.30 (d, J = 14.9 Hz, 1H), 3.75 (d, J = 4.4 Hz, 4H),
3.71 (d, J = 19.7 Hz, 3H), 3.55 (t, J = 14.5 Hz, 4H). 13C NMR
(125 MHz, DMSO‑d6)
δ 168.93, 163.21, 155.75, 152.24, 147.66,
140.90, 132.96, 130.52, 130.07, 124.32, 121.62, 121.39, 120.96,
114.45, 65.81, 55.66, 48.42. HRMS (ESI) m/z: [M+H]+ calculated
for C23H23N4O5S+ 467.1384, found 467.1380.
C
22H22N4NaO2S 429.1361, found 429.1361.
4.1.8.6. (E)-N-(4-Methoxyphenyl)-3-(4-(4-methoxyphenyl)-2-
4.1.8.12. (E)-N-(3,4-Dimethoxyphenyl)-3-(2-morpholino-4-(4-
morpholinothiazol-5-yl) acrylamide (8f). Light yellow solid, yield: 83%,
nitrophenyl)thiazol-5-yl) acrylamide (8l). Yellow solid, yield: 80%, mp:
334–336 °C; 1H NMR (500 MHz, DMSO‑d6) δ 10.03 (s, 1H), 8.39 (t,
J = 14.4 Hz, 2H), 7.86 (d, J = 8.8 Hz, 2H), 7.61 (d, J = 14.9 Hz, 1H),
7.43 (d, J = 2.2 Hz, 1H), 7.11 (dd, J = 8.7, 2.2 Hz, 1H), 6.90 (d,
J = 8.8 Hz, 1H), 6.29 (d, J = 14.9 Hz, 1H), 3.78–3.74 (m, 4H), 3.73
(s, 3H), 3.72 (s, 3H), 3.61–3.54 (m, 4H). 13C NMR (125 MHz, DMSO‑d6)
mp: 295–297 °C; 1H NMR (500 MHz, DMSO‑d6)
δ 9.92 (s, 1H),
7.66–7.46 (m, 5H), 7.10–7.05 (m, 2H), 6.92–6.85 (m, 2H), 6.17 (d,
J = 14.9 Hz, 1H), 3.83 (d, J = 5.3 Hz, 3H), 3.77–3.73 (m, 4H), 3.72 (s,
3H), 3.55 (dd, J = 14.6, 9.6 Hz, 4H). 13C NMR (125 MHz, DMSO‑d6) δ
168.72 (s), 163.64 (s), 160.18 (s), 155.62 (s), 155.20 (s), 131.45 (s),
7

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Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
δ 168.95, 163.27, 152.31, 149.10, 147.66, 145.42, 140.88, 133.46,
130.52, 130.15, 124.33, 121.53, 121.36, 112.77, 111.42, 104.71,
65.81, 56.27, 55.83, 48.43. HRMS (ESI) m/z: [M+H]+ calculated for
504.0143, found 506.0118.
4.1.8.19. (E)-N-(2-Aminophenyl)-3-(4-(4-chlorophenyl)-2-
C
24H25N4O6S+ 497.1489, found 497.1480.
morpholinothiazol-5-yl) acrylamide (8s). Light yellow solid, yield: 64%,
mp: 291–293 °C; 1H NMR (500 MHz, DMSO‑d6) δ 9.28 (s, 1H), 7.57 (d,
J = 18.3 Hz, 5H), 7.32 (d, J = 7.0 Hz, 1H), 6.90 (s, 1H), 6.73 (d,
J = 7.6 Hz, 1H), 6.56 (t, J = 7.2 Hz, 1H), 6.33 (d, J = 14.8 Hz, 1H),
4.92 (s, 2H), 3.75 (s, 4H), 3.55 (s, 4H). 13C NMR (125 MHz, DMSO‑d6) δ
168.88, 163.81, 153.59, 141.88, 133.92, 133.59, 131.12, 130.64,
129.13, 126.07, 124.90, 124.10, 120.36, 119.48, 116.73, 116.45,
65.82, 48.39. HRMS (ESI) m/z: [M+H]+ calculated for
C22H22ClN4O2S+ 441.1147, found 441.1141.
4.1.8.13. (E)-N-(4-Bromophenyl)-3-(2-morpholino-4-(4-nitrophenyl)
thiazol-5-yl) acrylamide (8m). Brick red solid, yield: 36%, mp:
355–357 °C; 1H NMR (500 MHz, DMSO‑d6) δ 10.27 (s, 1H), 8.39–8.34
(m, 2H), 7.88–7.84 (m, 2H), 7.65–7.60 (m, 3H), 7.51–7.48 (m, 2H),
6.29 (d, J = 14.9 Hz, 1H), 3.79–3.73 (m, 4H), 3.61–3.53 (m, 4H). 13C
NMR (125 MHz, DMSO‑d6) δ 169.09, 163.82, 152.83, 147.74, 140.81,
139.11, 132.08, 130.99, 130.57, 124.34, 121.43, 121.14, 120.85,
115.30, 65.80, 48.44. HRMS (ESI) m/z: [M+H]+ calculated for
C
24H25N4O6S+ 497.1489, found 497.1480.
4.2. Carbonic anhydrase inhibition assay
4.1.8.14. (E)-N-(2-Aminophenyl)-3-(2-morpholino-4-(4-nitrophenyl)
thiazol-5-yl)acrylamide (8n). Brick red solid, yield: 56%, mp:
302–304 °C; 1H NMR (500 MHz, DMSO‑d6) δ 9.34 (s, 1H), 8.42–8.33
(m, 2H), 7.86 (d, J = 8.8 Hz, 2H), 7.60 (d, J = 15.0 Hz, 1H), 7.33 (d,
J = 7.8 Hz, 1H), 6.90 (t, J = 7.4 Hz, 1H), 6.74 (d, J = 7.0 Hz, 1H), 6.57
(t, J = 7.5 Hz, 1H), 6.42 (d, J = 14.9 Hz, 1H), 5.00 (s, 2H), 3.80–3.72
(m, 4H), 3.61–3.53 (m, 4H). 13C NMR (125 MHz, DMSO‑d6) δ 168.95,
163.63, 152.21, 147.67, 141.77, 140.93, 130.52, 130.03, 126.15,
124.92, 124.32, 121.61, 121.41, 116.83, 116.52, 65.81, 48.42. HRMS
(ESI) m/z: [M+H]+ calculated for C22H22N5O4S+ 452.1387, found
452.1391.
An SX.18MV-R Applied Photophysics (Oxford, UK) stopped-flow
instrument has been used to assay the catalytic/inhibition of various CA
isozymes.21 Phenol Red (at a concentration of 0.2 mM) has been used as
indicator, working at the absorbance maximum of 557 nm, with 10 mM
Hepes (pH 7.4) as buffer, 0.1 M Na2SO4 or NaClO4 (for maintaining
constant the ionic strength; these anions are not inhibitory in the used
concentration), following the CA-catalyzed CO2 hydration reaction for a
period of 5–10 s. Saturated CO2 solutions in water at 25 °C were used as
substrate. Stock solutions of inhibitors were prepared at a concentration
of 10 μM (in DMSO-water 1:1, v/v) and dilutions up to 0.01 nM done
with the assay buffer mentioned above. At least 7 different inhibitor
concentrations have been used for measuring the inhibition constant.
Inhibitor and enzyme solutions were preincubated together for 10 min
at room temperature prior to assay, in order to allow for the formation
of the E-I complex. Triplicate experiments were done for each inhibitor
concentration, and the values reported throughout the paper are the
mean of such results. The inhibition constants were obtained by non-
linear least-squares methods using the Cheng-Prusoff equation, as re-
ported earlier,22 and represent the mean from at least three different
determinations. All CA isozymes used here were recombinant proteins
obtained as reported earlier by our group.23,24
4.1.8.15. (E)-3-(4-(4-Chlorophenyl)-2-morpholinothiazol-5-yl)-N-(4-
methoxyphenyl) acrylamide (8o). Light yellow solid, yield: 80%, mp:
255–257 °C; 1H NMR (500 MHz, DMSO‑d6) δ 9.96 (s, 1H), 7.62–7.54
(m, 7H), 6.89 (d, J = 9.0 Hz, 2H), 6.22 (d, J = 14.9 Hz, 1H), 3.77–3.71
(m, 7H), 3.58–3.52 (m, 4H). 13C NMR (125 MHz, DMSO‑d6) δ 173.62,
168.17, 160.44, 158.39, 138.69, 138.32, 137.78, 135.97, 135.78,
133.97, 133.79, 125.77, 125.60, 125.14, 124.21, 119.26, 119.11,
70.57, 60.40, 53.14. HRMS (ESI) m/z: [M+H]+ calculated for
C
23H23ClN3O3S+ 456.1143, found 456.1149.
4.1.8.16. (E)-3-(4-(4-Chlorophenyl)-2-morpholinothiazol-5-yl)-N-(3,4-
dimethoxyphenyl) acrylamide (8p). Light yellow solid, yield: 82%, mp:
279–281 °C; 1H NMR (500 MHz, DMSO‑d6) δ 9.98 (s, 1H), 7.63–7.55
(m, 5H), 7.43 (d, J = 2.2 Hz, 1H), 7.11 (dd, J = 8.7, 2.3 Hz, 1H), 6.89
(d, J = 8.8 Hz, 1H), 6.22 (d, J = 14.9 Hz, 1H), 3.76–3.73 (m, 4H), 3.73
(d, J = 4.4 Hz, 6H), 3.57–3.52 (m, 4H). 13C NMR (125 MHz, DMSO‑d6)
δ 168.89, 163.48, 153.71, 149.10, 145.35, 133.94, 133.55, 131.12,
130.74, 129.13, 120.31, 119.44, 112.79, 111.40, 104.73, 65.82, 56.28,
55.83, 48.40. HRMS (ESI) m/z: [M+H]+ calculated for
C24H25ClN3O4S+ 486.1249, found 486.1251.
4.3. Molecular docking studies
The protein preparation wizard was used to prepare of hCA II, IX
and XII downloaded from PDB (hCAII: 1BNM; hCA IX: 3IAI; hCA XII:
4Q0L),25–27 ready for docking i.e. removing waters, adding missing side
chains and energy minimization by OPLS-2005 force field.28 The syn-
thesized compound were sketched and converted to 3D using Ligprep.29
The Glide XP docking algorithm was employed using a grid box volume
of 10x 10x 10 Å at the centre of co-crystal as standard for all isoform
docking.30
4.1.8.17. (E)-3-(4-(4-Chlorophenyl)-2-morpholinothiazol-5-yl)-N-(3,4,5-
trimethoxyphenyl) acrylamide (8q). Light yellow solid, yield: 85%, mp:
285–287 °C; 1H NMR (500 MHz, DMSO‑d6) δ 10.05 (s, 1H), 7.64–7.55
(m, 5H), 7.05 (s, 2H), 6.21 (d, J = 14.9 Hz, 1H), 3.77–3.71 (m, 10H),
3.63 (d, J = 6.9 Hz, 3H), 3.58–3.53 (m, 4H). 13C NMR (125 MHz,
DMSO‑d6) δ 168.96, 163.76, 153.96, 153.23, 135.95, 133.98, 133.51,
131.12, 129.14, 120.01, 119.35, 97.33, 65.82, 60.58, 56.18, 48.42.
HRMS (ESI) m/z: [M+H]+ calculated for C25H27ClN3O5S+ 516.1354,
found 516.1342.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
BS, CSD and PS are thankful to Department of Pharmaceuticals,
Ministry of Chemicals and Fertilizers, Govt. of India, New Delhi, for
awarding the NIPER Ph.D fellowship.
4.1.8.18. (E)-N-(4-Bromophenyl)-3-(4-(4-chlorophenyl)-2-
morpholinothiazol-5-yl) acrylamide (8r). Light yellow solid, yield: 38%,
mp: 273–275 °C; 1H NMR (500 MHz, DMSO‑d6) δ 10.23 (s, 1H),
7.67–7.52 (m, 7H), 7.49 (d, J = 8.9 Hz, 2H), 6.22 (d, J = 14.9 Hz,
1H), 3.80–3.70 (m, 4H), 3.62–3.49 (m, 4H). 13C NMR (125 MHz,
DMSO‑d6) δ 169.03, 164.02, 154.24, 139.20, 134.04, 133.49, 132.04,
131.59, 131.15, 129.15, 121.41, 119.64, 119.27, 115.18, 65.81, 48.41.
HRMS (ESI) m/z: [M+H]+ calculated for C22H20BrClN3O2S+
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmc.2019.115090.
8

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