Design, synthesis, cytotoxic evaluation and molecular docking studies of novel thiazolyl α-aminophosphonates

Article abstract of DOI:10.1007/s11164-020-04321-6

Abstract: A new class of thiazolyl α-aminophosphonate derivatives was synthesized by one-pot Kabachnik–Fields reaction of ethyl 2-(3-formyl-4-isobutoxyphenyl)-4-methylthiazole-5-carboxylate with various aryl amines and diethyl phosphite under solvent-free conditions using β-cyclodextrin supported sulfonic acid (β-CD-SO₃H) as an efficient, reusable and heterogeneous solid acid catalyst. The products were obtained in good to excellent yields at shorter reaction time. All the title compounds were screened for cytotoxic activity against human breast cancer (MCF-7 and MDA-MB-231), prostate cancer (DU-145) liver cancer (HepG2) and HeLa cancer cell lines using sulfarodamine-B (SRB assay). Compounds (8b, –4OMe), (8h, –4NO₂) and (8j, –2I, –4CF₃) showed better anticancer activity when compared with standard drug Adriamycin. Further in-silico target hunting reveals the anticancer activity of the designed compounds by inhibiting DNA topoisomerase II. Graphical abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s11164-020-04321-6

Research on Chemical Intermediates
https://doi.org/10.1007/s11164-020-04321-6
Design, synthesis, cytotoxic evaluation and molecular
docking studies of novel thiazolyl α‑aminophosphonates
Mohan Gundluru, et al. [full author details at the end of the article]
Received: 12 September 2020 / Accepted: 31 October 2020
©
Springer Nature B.V. 2020
Abstract
A new class of thiazolyl α-aminophosphonate derivatives was synthesized by one-
pot Kabachnik–Fields reaction of ethyl 2-(3-formyl-4-isobutoxyphenyl)-4-methylth-
iazole-5-carboxylate with various aryl amines and diethyl phosphite under solvent-
free conditions using β-cyclodextrin supported sulfonic acid (β-CD-SO H) as an
3
eꢀcient, reusable and heterogeneous solid acid catalyst. The products were obtained
in good to excellent yields at shorter reaction time. All the title compounds were
screened for cytotoxic activity against human breast cancer (MCF-7 and MDA-
MB-231), prostate cancer (DU-145) liver cancer (HepG2) and HeLa cancer cell
lines using sulfarodamine-B (SRB assay). Compounds (8b, –4OMe), (8h, –4NO )
2
and (8j, –2I, –4CF ) showed better anticancer activity when compared with standard
3
drug Adriamycin. Further in-silico target hunting reveals the anticancer activity of
the designed compounds by inhibiting DNA topoisomerase II.
Graphical abstract
Keywords Thiazolyl α-aminobisphosphonates · β-CD-SO H · Cytotoxic activity ·
3
Molecular docking studies · DNA topoisomerase II inhibition
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s1116
4
-020-04321-6) contains supplementary material, which is available to authorized users.
Vol.:(012
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M. Gundluru et al.
Introduction
The structural diversity and biocidal importance of phosphorylated heterocycles
have made them attractive scaꢁolds for the synthesis of pharmacological agents
in medicinal chemistry [1–3]. In recent years, some of the α-aminophosphonate
derivatives containing heterocycle moieties have been synthesized which have
shown interesting biological activities. It has been shown that the existence of
heterocyclic motifs in the structure of the α-aminophosphonate molecules signiꢂ-
cantly enhanced their bioactivities [4–7].
α-Aminophosphonates (APs) are an important class of organophosphorus com-
pounds because of their applications in various ꢂelds like catalysis [8] and materials
chemistry [9, 10]. Due to the structural similarity of α-aminophosphonates to that of
natural α-amino carboxylic acids and tetrahedral conꢂguration at phosphorus [11],
they can act as enzyme inhibitors [12], antibiotics [13] herbicides [14], insecticides
[
15], plant growth regulators [16], antitumor [17], anti-inꢃammatory [18], antimi-
crobials [19], antioxidants [20], antivirals [21] and antitubercular agents [22]. Fur-
thermore, the introduction of aminophosphonate group to a pharmacophore is capa-
ble of enhancement of the anticancer activity and many of them have demonstrated
potent inhibitory activities against various human cancers including human cervical
cancer (HeLa), human breast adenocarcinoma (SK-BR-3) [23] human chronic mye-
loid leukemia (K 562) and human colon carcinoma cells (Colo 205) [24].
In the ꢂeld of medicinal chemistry, thiazole is an important class of hetero-
cyclic compound which is known to be present in many commercially available
products with a wide range of pharmacological activities [25]. Because of its low
toxicity, thiazole moiety is a key pharmacophore for the synthesis of several bio-
logical molecules like vitamin B1 (thiamine) which helps in the regular function-
ing of the nervous system by its role in the synthesis of acetylcholine [26]. Fur-
thermore, thiazole derivatives play a key role in the ꢂeld of medicinal chemistry
and normally present in the structure of various natural products and bioactive
compounds including antimicrobial [27], anticonvulsant [28], anti-inꢃammatory
[
29], antiviral [30], insecticidal [31], antioxidant [32], antihypertensive [33], anti-
cancer [34], analgesic [35], anti-HIV [36], anti-ꢂlarial [37], antimalarial [38],
anti-leishmanial [39] and enzyme inhibition activities [40]. Thiazole is present in
natural products as a subunit in numerous terrestrial and marine compounds with
diverse bioactivities that signiꢂes its key role in drug discovery [41, 42]. Apart
from biological activities, thiazoles also have applications in liquid crystals, poly-
mers, ꢃuorescent dyes and photo-nucleases and so on [43–45].
Molecular docking studies were carried out to understand their possible tar-
get-based interactions that led to the anticancer activity of α-aminophosphonates
against human Topo IIa. DNA helix and tubulin polymerization inhibitory
activity of α-aminophosphonates showed promising antitumor activity. These
results encouraged us to design and synthesis novel derivatives of thiazolyl
α-aminophosphonates and evaluated their anticancer activity. The design strategy
is to club the aminophosphonate and thiazole moieties and generate novel thia-
zolyl α-aminophosphonates (Fig. 1) [17, 46–49].
1
3

Design, synthesis, cytotoxic evaluation and molecular docking…
R
F
O
OEt
P
O
N
S
P
OEt
OEt
EtO
NH
S
NH
HN
N
R
R
O
P
O
P
OR'
N
S
OEt
R
O
R'O
R'O
N
S
NH
P
OEt
HN
OEt
N
S
OEt
N
O
R
EtO
Ph OEt
O
P
O
S
P
O
OEt
N
NH
Cl
Designed hybrid (8a-8j)
HN
N
OR'
S
F
O
O
OEt
OEt
EtO
EtO
H
N
H
N
P
P
N
N
R
S
S
R
Cl
Fig. 1 Strategy adopted to design novel thiazolyl α-aminophosphonate derivatives
Encouraged by the aforesaid ꢂndings and in continuation of our research in the
development of new methodologies for the construction of bio-potent heterocyclic
α-aminophosphonates, it was thought worthwhile to synthesize some novel thiazolyl
α-aminophosphonate derivatives and test their antitumor activities in vitro against
several selected tumor cell lines in addition to a study of their structure–activity
relationship (SAR) in order to develop new potentially bioactive synthetic drugs.
Besides this, molecular docking studies were also carried out against X-ray crystal
structure of human type IIA DNA topoisomerase and colchicine binding site at α/β-
tubuline interface by using AutoDock 4.2.
Results and discussion
Chemistry
The synthesis of thiazolyl α-aminophosphonate derivatives (8a–8j) has been
accomplished by a two-step process. Initially, the cyclization of 4-hydroxy-
thiobenzamide (1) with 2-bromoacetoacetic acid ethyl ester (2) in reꢃux-
ing ethanol provides 2-(4-hydroxyphenyl)-4-methylthiazole-5-carboxylic acid
ethyl ester (3), which is formylated by reaction with hexamethylenetetramine
(
HMTA) and triꢃuoroacetic acid (TFA) in hot HOAc/water to aꢁord 2-(3-formyl-
4
-hydroxyphenyl)-4-methylthiazole-5-carboxylic acid ethyl ester (4) [50]. Alkyla-
tion of (4) with isobutyl bromide, K CO and KI in DMF gives ethyl 2-(3-formyl-
2
3
4
-isobutoxyphenyl)-4-methylthiazole-5-carboxylate (5) as starting compound
1
3

M. Gundluru et al.
Scheme 1 Synthesis of ethyl 2-(3-formyl-4-isobutoxyphenyl)-4-methylthiazole-5-carboxylate (5)
(
Scheme 1) [51]. The structure elucidation of compound (5) was based on the full
1 13
sets of IR, H NMR, C NMR and mass spectral data.
At the outset, an improved one-pot, three-component Kabachnik–Fields reac-
tion of the starting compound ethyl 2-(3-formyl-4-isobutoxyphenyl)-4-methylth-
iazole-5-carboxylate (5) with diversely substituted aromatic amines (6a–6j) and
diethyl phosphite (7) was taken as a typical reaction and run it in the presence of
β‐CD‐SO H under solvent-free conditions for about 30 min at 50 °C. The desired
3
products (8a–8j) were obtained in good to excellent (90–96%) yields (Scheme 2).
To accomplish optimum reaction conditions for the synthesis of thiazolyl
α-aminophosphonates (8a–8j), the model reaction was carried out by taking
ethyl 2-(3-formyl-4-isobutoxyphenyl)-4-methylthiazole-5-carboxylate (5), aniline
(
6a) and diethyl phosphite (7) as reactants under catalyst and solvent-free condi-
tions at r.t. Even after heating the reaction mixture for about 24 h, the desired
product was not obtained (Table 1, entry 1). Subsequently, the reaction was con-
ducted in the presence of various catalysts like FeCl , AlCl , LaCl , ZnCl , NiCl ,
3
3
3
2
2
CuCl , CuBr , BF .SiO , Fe O , TiO and β‐CD‐SO H in the absence of solvent
2
2
3
2
3
4
2
3
(
Table 1, Entries 2–12). Remarkably, the model reaction showed excellent prod-
uct yields (95%) within 30 min reaction time in the presence of β‐CD‐SO H as
3
catalyst under solvent-free conditions at 50 °C (Table 1, entry 12). Later, the sol-
vent eꢁect on the reaction was also studied by running the reaction with various
protic and non-protic solvents like methanol, ethanol, THF, DCM, DMF and tolu-
ene (Table 1, Entry 13–18) and also in solvent-free conditions. To our delight,
high product (8a) yield was obtained in solvent-free conditions with β‐CD‐SO H
3
as catalyst.
Scheme 2 Synthesis of thiazolyl α-aminophosphonate derivatives (8a–8j)
1
3

Design, synthesis, cytotoxic evaluation and molecular docking…
Table 1 Optimization of the model reaction under varied catalyst conditions
Entry
Catalyst (7.5 mol%)
Solvent
Temperature
°C)
Time (h)
Yield (%)b
(
1
2
3
4
5
6
7
8
9
Catalyst free
FeCl3
AlCl3
LaCl3
ZnCl2
NiCl2
CuCl2
CuBr2
–
–
–
–
–
–
–
–
–
–
–
–
r.t
24
10
10
9
10
7
9
12
8
8
6
0.5
6
5
nrc
55
48
54
56
60
58
60
62
56
68
96
90
90
78
65
60
80
80
80
80
80
80
80
80
80
80
80
50
50
50
50
50
50
50
BF .SiO
3
2
10
11
12
13
14
15
16
17
18
Fe O
3
4
TiO2
β‐CD‐SO H
3
β‐CD‐SO H
MeOH
EtOH
THF
DCM
DMF
Toluene
3
β‐CD‐SO H
3
β‐CD‐SO H
6
3
β‐CD‐SO H
10
10
4
3
β‐CD‐SO H
3
β‐CD‐SO H
3
a
Reaction conditions: Ethyl 2-(3-formyl-4-isobutoxyphenyl)-4-methylthiazole-5-carboxylate (5; 1 mmol),
aniline (6a; 1 mmol) and diethyl phosphite (7; 1 mmol), in the presence of various catalysts and solvents
along with solvent-free conditions
Isolated yields
No reaction
b
c
After establishment of the optimal reaction conditions, we investigated the
scope of the reaction by condensing ethyl 2-(3-formyl-4-isobutoxyphenyl)-4-meth-
ylthiazole-5-carboxylate (5) with various commercially available substituted aryl
amines having diꢁerent electronically activating or deactivating substituents (6a–6j)
and diethyl phosphite (7) to obtain corresponding thiazolyl α-aminophosphonates
(
(
8a–8j). The details of physical data like yield and melting points are illustrated in
Table 2).
1
3

M. Gundluru et al.
Table 2 Synthesis of thiazolyl α-aminophosphonates (8a–8j) under neat conditions using β-CD-SO H
3
as catalyst via Scheme 2
a
S. no. Structures of
aryl amines
Structures of products (8a–8j)
Time (min) Yield (%) Melting point (°C)
(
6a–6j)
1
30
30
96
96
235–237
255–257
2
3
4
30
30
95
93
286–288
242–244
5
30
92
247–249
1
3

Design, synthesis, cytotoxic evaluation and molecular docking…
Table 2 (continued)
a
S. no. Structures of
aryl amines
Structures of products (8a–8j)
Time (min) Yield (%) Melting point (°C)
(
6a–6j)
6
30
90
275–277
7
8
30
30
91
92
290–292
288–290
9
1
30
30
94
92
276–278
294–296
0
Reaction conditions: Ethyl 2-(3-formyl-4-isobutoxyphenyl)-4-methylthiazole-5-carboxylate (5; 1 mmol),
various aryl amines (6a–6j; 1 mmol) and diethyl phosphite (7; 1 mmol) in the presence of β-CD-SO H
3
(
7.5 mol%) as catalyst, under solvent-free conditions at 50 ºC for 30 min
Isolated yields (96–90%)
a
1
3

M. Gundluru et al.
Formation of α-aminophosphonates (8a–8j) in the presence of β‐CD‐SO H as
3
catalyst involves nucleophilic amine addition to the electrophilic carbonyl car-
bon of the aldehyde with simultaneous elimination of water from it. The β‐CD‐
SO H (a) as catalyst initially protonates the carbonyl oxygen of the aldehyde (b)
3
and renders the carbon more electrophilic and this factor facilitates the amine
NH (c) nucleophilic attack on the carbonyl carbon to form the imine intermedi-
2
ate (e). Nucleophile [P] of phosphite (f) attacks the electrophilic C of the imine
intermediate (e) leading to the formation of C–P bond. Abstraction of the proton
from phosphonate (f) by sulfonate anion of the catalyst (g) makes the P of the
phosphonate (f) more nucleophile and accelerates P–C bond formation leading to
α-aminophosphonates (h) (Scheme 3).
The title compounds (8a–8j) were characterized by physical and spec-
tral (IR, NMR and Mass) data. They showed strong IR absorption bands in the
−
1
−1
−1
region of 3296–3260 cm , 1710–1690, 1258–1228 cm , 1026–1012 cm and
−
1
7
60–730 cm for –NH, –C=O, –P=O, –P–O–C and P–C aliphatic stretching
1
frequencies, respectively. In H NMR spectra, the chemical shifts in the region
of 8.50–6.10 ppm are due to aromatic protons and the doublet at 5.50–5.10 ppm
corresponds to HC–P proton. The singlet at δ 5.00–4.82 ppm conꢂrmed the
presence of –NH proton. The multiplet in the region of 4.40–3.60 ppm is due
to the –O–CH –CH protons. The singlet in the region of 2.80–2.60 ppm and a
2
3
multiplet in the region 2.30–2.10 ppm are due to the –CH and –CH protons,
3
respectively. Two triplets in the region of 1.40–1.20 ppm conꢂrmed the pres-
ence of –O–CH –CH protons. The doublet and triplet in the range of 1.14–1.12
2
3
1
3
and 1.10–1.00 ppm conꢂrmed the presence of –CH protons. In C NMR spec-
3
tra, the chemical shifts in the region of 184.50–110.00 ppm are assigned to car-
bons of aromatic ring; the signals in the region of 64.30–58.00, 52.00–46.00,
2
9.00–26.00, 19.60–19.20, 16.70–16.10 and 14.60–14.10 ppm conꢂrmed the
Scheme 3 Plausible mechanism for the catalytic activity of β-CD-SO H in the formation of
3
α-aminophosphonates (8a–8j)
1
3

Design, synthesis, cytotoxic evaluation and molecular docking…
presence of –OCH –CH , HC–P, –CH–, (CH ) , –OCH –CH and –CH carbons.
2
3
3 2
2
3
3
3
1
The P NMR chemical shifts of the title compounds appeared in the range of
2
2.00 to 24.80 ppm.
Pharmacology
In vitro anticancer activity
The synthesized thiazolyl α-aminophosphonates (8a–8j) were tested against human
breast (MCF-7 and MDA-MB-231), prostate cancer (DU-145) liver cancer (HepG2)
and HeLa cancer cell lines sulforhodamine-B (SRB) cytotoxic assay [52] to investi-
gate the eꢁectiveness of the in vitro cell cytotoxic properties (Table 3). All the data
were expressed as IC values. The obtained data revealed that the compound 8h
5
0
with nitro substitution exhibited highest cell growth inhibitory eꢁects on MCF-7,
MDA-MB-231, DU-145 HepG2 and HeLa cell lines with IC values when com-
5
0
pared to that of IC of the Adriamycin standard used. Compound 8b with 4-OMe
5
0
and compound 8j having 2I, 4-CF has exhibited excellent growth inhibitory eꢁects
3
on all the cell lines. While compounds 8d, 8c, 8a and 8g showed moderate cytotoxic
activity remaining compounds 8e, 8f and 8i could not show eꢁective cytotoxic activ-
ity on all the ꢂve cell lines (Fig. 2).
Molecular docking studies against DNA topoisomerase II
All the synthesized compounds were docked with X-ray crystal structure of
tubulin inhibitor (PDB ID: 3E22), and human type IIA DNA topoisomerase
(
[
PDB: 1ZXM), using AutoDock, protocol reported in earlier communications
53–59]. Human type IIA DNA topoisomerase tremendously expressed cancer
Table 3 In vitro anticancer activity of thiazolyl α-aminophosphonate derivatives (8a–8j)
Compound
MCF-7
MDA-MB-231
DU-145
HepG2
HeLa
IC50 (μg/mL± S.D.)a
8
8
8
8
8
8
8
8
8
8
a
b
c
d
e
f
18.32± 0.019
12.54± 0.027
17.23± 0.016
17.16± 0.042
22.38± 0.051
24.74± 0.024
19.65± 0.028
12.30± 0.018
27.98± 0.016
14.13± 0.016
16.18± 0.017
10.03± 0.026
8.18± 0.018
10.08± 0.016
9.25± 0.008
16.68± 0.014
18.02± 0.016
10.17± 0.001
6.30± 0.002
22.12± 0.008
8.54± 0.008
8.64± 0.006
5.25± 0.028
3.02± 0.018
4.63± 0.016
4.61± 0.028
10.91± 0.014
13.96± 0.028
5.32± 0.026
2.13± 0.018
15.65± 0.024
3.34± 0.028
3.82± 0.024
30.51± 0.003
22.01± 0.008
28.35± 0.006
28.23± 0.001
45.34± 0.004
46.02± 0.006
30.63± 0.006
20.89± 0.008
50.49± 0.005
26.14± 0.001
26.68± 0.002
13.97± 0.014
10.68± 0.019
13.88± 0.016
13.34± 0.012
28.95± 0.015
32.06± 0.016
14.17± 0.012
9.60± 0.016
38.83± 0.028
12.30± 0.018
12.65± 0.018
g
h
i
j
Adriamycin
aIC values are expressed as mean± SD of three experiments
5
0
1
3

M. Gundluru et al.
60
50
40
30
20
10
0
MCF-7
MDA-MB-231
DU-145
HepG2
HeLa
Tested Compounds
Fig. 2 Anticancer activities of tested compounds (quantiꢂed version of Table 3)
proliferating cells and plays an important function in cellular processes such as
replication, transcription, chromatin assembly. In order to understand the possible
mechanism of action, molecular docking studies were carried out against X-ray
crystal structure of Topo IIa (DNA gyrase, PDB: 1ZXM) [60]. All the synthe-
sized compounds completely occupied the active site residue (Leu89, ALA93,
Arg98, Ala92, Cys104, Lys96, Trp119, Tyr214 and Val90) of human Topo IIa
and shown excellent fee energy of binding from − 7.08 to − 8.69 kcal/mole with
good inhibitory constant up to ki = 0.42 µM (Table 4). The active compound 8b
the methoxy oxygen interacts with terminal amino hydrogen of Lys157 (bond
distance 2.10 Å), with free binding energy − 7.55 kcal/mole, while in the com-
pound 8h, both the oxygen of nitro group establish two hydrogen bonds one with
terminal amine hydrogen of Lys168 (bond distance 2.08 Å), another with amine
Table 4 Molecular docking
Compound
DNA gyrase
K i(µM)
Tubulin protein
studies against active site of
DNA gyrase and tubulin protein
(Kcal/mol)
Ki (µM)
(Kcal/mol)
8
8
8
8
8
8
8
8
8
8
a
b
c
d
e
f
6.44
2.92
0.59
3.01
1.64
6.48
0.72
1.23
1.93
0.42
−7.08
−7.55
−8.49
−7.53
−7.89
−7.08
−8.38
−8.06
−7.79
−8.69
13.67
4.95
2.04
4.89
5.07
14.84
14.61
4.61
8.83
4.20
−6.64
−7.24
−7.76
−7.25
−7.22
−6.59
−6.60
−7.28
−6.89
−7.34
g
h
i
j
1
3

Design, synthesis, cytotoxic evaluation and molecular docking…
Fig. 3 Binding mode analysis and interaction of compounds in the active site of human type IIA DNA
topoisomerase (PDB: 1ZXM)
hydrogen of Ala167 (bond distance 1.97 Å), with free binding energy of binding
−
7.28kacl/mole (Fig. 3).
Molecular docking with colchicine binding site
Tubulin is recognized as an important target for anticancer drug development. All
the synthesized compounds interacted with both α, β interface of tubulin [61–63]
in the colchicine binding pocket, i.e., αAsn101, αSer178, αVal181, αThr179,
βLys352, βThr353, βLeu248, βGln247 and exhibit signiꢂcant fee energy of binding
from −7.24 to −7.88 kcal/mole with good inhibitory constant up to Ki=1.67 µM
(
Table 4). Compound 8h oxygen of nitro group established a hydrogen bond with
amine hydrogen of Cys352, i.e., bond distance 1.78 Å with free binding energy of
binding-7.28kacl/mole, while compound 8j, the sulfur of thiazole ring established
one hydrogen bond with carbonyl oxygen of Thr353, i.e., bond distance of 2.95 Å,
with free binding energy of binding-7.34kacl/mole (Fig. 4).
The reason behind the cytotoxic activity of compounds (8b, 8h and 8j) may be
due to the additional hydrogen bonding with the active site residue in the target,
and we are not trying to correlate our observation with the experimental results as
other modes of action are possible for this type of chemical scaꢁold. This study has
1
3

M. Gundluru et al.
Fig. 4 Binding mode analysis and interaction of compounds in the active site of colchicine binding site at
tubulin protein (PDB: 3E22)
clearly shown that the compounds are well accommodated in the active site of DNA
topoisomerase II and colchicine binding site in tubulin protein, though activity can
be ascertained only through in vitro enzyme-based assay methodology.
Structure activity relation (SAR) studies
The SAR studies related to the in vitro cytotoxicity of the title compounds (8a–8j)
against MCF-7, MDA-MB-231, DU-145, HepG2 and HeLa cancer cell lines revealed
that though the basic skeletal structure of title compounds remains same among all the
compounds, diverse groups on the phenyl ring substituted at the α-carbon of the phos-
phonates shows varying percent of inhibition among all the cancer cell lines. Further-
more, the same phosphonate is not continuously active on the cancer cell lines. This
shows that not only the basic skeleton structure of compounds, but also its substitu-
ent groups are the deciding factor for the activity of a certain compound. Amongst all,
the compound 8h (R=4–NO ) exerts extremely higher percentage of inhibition among
2
all the ꢂve cancer cell lines than the standard Adriamycin. After that, compounds 8b
(
R=4–OMe) and 8j (R=2I, 4-CF ) exhibit higher percentage of inhibition on ꢂve cell
3
lines, respectively. The results of the docking studies are also in support with the cyto-
toxic studies having good binding energies. Thus, these compounds form a colony of
1
3

Design, synthesis, cytotoxic evaluation and molecular docking…
novel cytotoxic lead molecules and by further ꢂne tuning their structure with respect to
bioactivity, they may oꢁer a new generation of potential and safe cytotoxic compounds.
Conclusion
In summary, we have developed an eꢀcient and eco-friendly protocol for the synthesis
of thiazolyl α-aminophosphonate derivatives through the β‐CD‐SO H catalyzed reac-
3
tion of an aldehyde with various aryl amines and diethyl phosphite. This new method
is endowed with green reaction conditions such as low cost, use of non-toxic catalyst,
solvent-free medium, easy work-up process and good yields. The title compounds were
screened for anticancer activity against human breast (MCF-7 and MDA-MB-231),
prostate cancer (DU-145) liver (HepG2) and HeLa cancer cell lines with sulfaroda-
mine-B (SRB) assay. The compounds 8b, 8g and 8j showed better anticancer activity
when compared with the standard drug Adriamycin. In-silico target hunting reveals the
anticancer activity of the designed compounds by inhibiting DNA topoisomerase II and
tubulin polymerase inhibition. The design strategy provides a base for evaluating this
class of molecules for their anticancer activity and also be utilized to rationale design
and synthesis of a new library of compounds for anticancer activity.
Experimental
Analysis and instruments
All the required chemicals were purchased from Sigma-Aldrich and the solvents from
Merck and were used without further puriꢂcation. The completion and purity of the
reactions were monitored by TLC, performed on silica gel aluminum 60 F-254 thin
layer plates procured from Merck, and visualization on TLC was achieved by UV light
and iodine indicator. Melting points of the compounds were determined on Guna dig-
ital melting point apparatus using open capillary tubes and are uncorrected. Infrared
spectra were recorded on FT-IR Bruker ALPHA Interferometer and wave numbers
−1
are given in cm . NMR spectra were recorded on a Bruker instrument operating at
1
13
31
4
00 MHz for H, 100 MHz for C and 161.9 MHz for P in CDCl . TMS was used
3
as an internal standard. Assignments of the signals are based on the chemical shifts
and intensity patterns. Chemical shift (δ) and coupling constant (J) were expressed in
ppm and Hertz, respectively. Mass spectra were recorded on a LC–MS/MS-TOF API
QSTAR PULSAR spectrometer; samples were introduced by the infusion method
using the electrospray ionization technique (ESI).
General procedure for synthesis of the starting compound ethyl
2
‑(3‑formyl‑4‑isobutoxyphenyl)‑4‑methylthiazole‑5‑carboxylate (5)
Initially, 4-hydroxythiobenzamide (1) 2-bromoacetoacetic acid ethyl ester (2) and
0 ml of ethanol were taken in a round-bottomed ꢃask and run the reaction under
1
1
3

M. Gundluru et al.
reꢃuction conditions for about 2 h to get 2-(4-hydroxyphenyl)-4-methylthiazole-
5
-carboxylic acid ethyl ester (3), which is formylated by reaction with hexamethyl-
enetetramine (HMTA) and triꢃuoroacetic acid (TFA) in hot HOAc/water for about
2
.5 h to aꢁord 2-(3-formyl-4-hydroxyphenyl)-4-methylthiazole-5-carboxylic acid
ethyl ester (4). Finally, alkylation of (4) with isobutyl bromide, in the presence of
K CO and KI in DMF, gives ethyl 2-(3-formyl-4-isobutoxyphenyl)-4-methylthia-
2
3
zole-5-carboxylate (5) as starting compound.
Ethyl 2‑(3‑formyl‑4‑isobutoxyphenyl)‑4‑methylthiazole‑5‑carboxylate (5)
−
1
White solid; Yield: 90%, M.p. 253–255 °C; FT-IR (neat, cm ): ν 2979 (–C–H),
1
1
1
704 (ester -C=O), 1696 (aldehydic –C=O). H-NMR (400 MHz, CDCl , ppm): δ
3
0.52 (s, 1H, H–C=O), 8.35 (s, 1H, Ar–H), 8.19 (d, J=8.0 Hz, 1H, Ar–H), 7.04 (d,
J=8.0 Hz, 1H, Ar–H) 4.36–4.31 (m, 2H –OCH ), 3.91 (d, J=8.0 Hz, 2H, –OCH ),
2
2
2
1
1
7
.75 (s, 3H, –CH ), 2.22–2.16 (m 1H, –CH), 1.37 (t, J =8.0 Hz, J =8.0 Hz 3H),
3
1
2
1
3
.06 (d, J=8.0 Hz, 6H, –CH ). C-NMR (100 MHz, CDCl , ppm): δ 188.88,
3
3
68.56, 163.29, 162.32, 161.12, 133.87, 127.13, 125.94, 125.08, 121.65, 113.18,
+
5.27, 61.32, 28.36, 19.25, 17.57, 14.41. HRMS (ESI) calcd. for C H NO S
1
8
21
4
+
[
M+H] : 348.1225 and found 348.1221.
General procedure for synthesis of thiazolyl α‑aminophosphonates (8a–8j)
Ethyl 2-(3-formyl-4-isobutoxyphenyl)-4-methylthiazole-5-carboxylate (5) (1 mol),
various heteroaryl amines (6a–6j) (1 mol), diethyl phosphite (7) (1.5 mmol) and
7
.5 mol% of β‐CD‐SO H were taken in a round-bottomed ꢃask and the reaction
3
was run under neat conditions at for about 30 min. The progress of the reaction was
monitored by TLC (3:2; n-hexane/ethyl acetate) for every 10 min. After completion
of reaction, the mixture was dissolved in 10 mL of DCM and ꢂltered to remove the
catalyst as residue. The organic layer was washed with water (2×5 mL) and the
water layer was discarded. The combined organic mixture was washed with brine
solution (5 mL), dried over anhydrous Na SO and concentrated under reduced pres-
2
4
sure. The solids obtained were washed with cold water, air-dried and recrystallized
from ethanol to get the pure compounds.
Ethyl 2‑(3‑((diethoxyphosphoryl)(phenylamino)
methyl)‑4‑isobutoxyphenyl)‑4‑methyl thiazole‑5‑carboxylate (8a)
−
1
White solid; Yield: 96%, M.p. 235–237 °C. FT-IR (neat, cm ) ν 3290 (–NH) 2967
1
(
–C–H), 1702 (–C=O), 1246 (–P=O), 1019 (–P–O–C), 749 (–P–C–). H-NMR
(
400 MHz, CDCl , ppm): δ 8.04 (s, 1H, Ar–H), 7.85 (d, J=8.0 Hz, 1H, Ar–H),
3
7
.09 (t, J =8.0 Hz, J =8.0 Hz 2H, Ar–H), 6.91(d, J=8.0 Hz, 1H, Ar–H), 6.67 (d,
1
2
J=4.0 Hz, 2H, Ar–H), 6.63 (d, J=8.0 Hz, 2H, Ar–H), 5.41 (d, J=24.0 Hz, 1H,
P–CH), 4.93 (s, 1H, –NH), 4.34–4.28 (dd, J=8.0 Hz, 2H, –OCH ), 4.18–3.67
2
(
m, 6H –OCH ), 2.72 (s, 3H, –CH ), 2.25–2.17 (m 1H, –CH), 1.35 (t, J =8.0 Hz,
2
3
1
J =4.0 Hz 3H), 1.29 (t, J =8.0 Hz, J =4.0 Hz 3H, –OCH ), 1.12 (d, J=8.0 Hz,
2
1
2
3
1
3

Design, synthesis, cytotoxic evaluation and molecular docking…
1
3
6
H, –CH ) 1.05 (t, J =8.0 Hz, J =8.0 Hz 3H, –CH ). C-NMR (100 MHz,
3 1 2 3
CDCl , ppm): δ 169.68, 162.45, 161.02, 159.00 (d, J =6.0 Hz), 153.75, 146.27
3
P–C
(
d, J =14.0 Hz), 129.25, 127.83 (d, J =2.0 Hz), 127.21 (d, J =5.0 Hz),
P–C P–C P–C
1
J
25.95, 120.98, 113.57 (d, J =5.0 Hz), 111.39, 75.06, 63.26 (dd, J
=2.0 Hz,
1P–C
P–C
=2.0 Hz), 61.17, 55.75, 48.08 (d, J =153.0 Hz), 28.52, 19.44 (d,
2
P–C
P–C
P–C
3
1
J
=3.0 Hz), 17.54, 16.53 (d, J =5.0 Hz), 16.24 (d, J =5.0 Hz), 14.41. P-
P–C P–C
+
NMR (162 MHz, CDCl , ppm): δ 23.5. HRMS (ESI) calcd. for C H N O PS
3
28 37
2
6
+
[
M+H] : 561.2143 and found 561.2140.
Ethyl 2‑(3‑((diethoxyphosphoryl)((4‑methoxyphenyl)amino)
methyl)‑4‑isobutoxyphenyl)‑4‑methylthiazole‑5‑carboxylate (8b)
−
1
Brown solid; Yield: 96%, M.p. 255–257 °C. FT-IR (neat, cm ) ν 3288 (–NH)
1
2
928 (–C–H), 1703 (–C=O), 1249 (–P=O), 1025 (-P-O-C), 751 (–P–C–). H-NMR
(
400 MHz, CDCl , ppm): δ 8.03 (s, 1H, Ar–H), 7.85 (d, J=8.0 Hz, 1H, Ar–H),
3
6
.90 (d, J=8.0 Hz, 1H, Ar–H), 6.67 (d, J=8.0 Hz, 2H, Ar–H), 6.58 (d, J=8.0 Hz,
H, Ar–H), 5.33 (d, J=28.0 Hz, 1H, P–CH), 4.93 (s, 1H, –NH), 4.33–4.28 (dd,
2
J=8.0 Hz, 2H, –OCH ), 4.18–3.69 (m, 6H –OCH ), 3.66 (s, 3H, –OCH ), 2.72
2
2
3
(
s, 3H, –CH ), 2.24–2.18 (m 1H, –CH), 1.35 (t, J =8.0 Hz, J =4.0 Hz 3H), 1.29
3
1
2
(
t, J =8.0 Hz, J =4.0 Hz 3H, –OCH ), 1.12 (d, J=8.0 Hz, 6H, –CH ) 1.05 (t,
1
2
3
3
1
3
J =8.0 Hz, J =8.0 Hz 3H, –CH ). C-NMR (100 MHz, CDCl , ppm): δ 169.71,
1
2
3
3
1
62.44, 160.99, 159.07 (d, J =6.0 Hz), 152.74, 140.33 (d, J =15.0 Hz), 127.75
P–C P–C
(
d, J =2.0 Hz), 127.23 (d, J =5.0 Hz), 120.95, 114.87 (d, J =5.0 Hz),
P–C P–C P–C
1
J
11.38, 75.04, 63.20 (dd, J
=15.0 Hz, J =14.0 Hz), 61.17, 55.75, 48.95 (d,
2P–C
1
P–C
=152.0 Hz), 28.51, 19.44 (d, J =3.0 Hz), 17.53, 16.54 (d, J =6.0 Hz),
P–C
P–C
P–C
3
1
1
6.26 (d, J =6.0 Hz), 14.41. P-NMR (162 MHz, CDCl , ppm): δ 23.68. HRMS
P–C 3
+
+
(
ESI) calcd. for C H N O PS [M+H] : 591.2249 and found 591.2246.
29 39 2 7
Ethyl 2‑(3‑((diethoxyphosphoryl)((4‑(phenylthio)phenyl)amino) methyl)‑4‑isobutoxy
phenyl) ‑4‑methylthiazole‑5‑carboxylate (8c)
−
1
White solid; Yield: 96%, M.p. 286–288 °C. FT-IR (neat, cm ) ν 3275 (–NH) 2967
1
(
(
(
–C–H), 1699 (–C=O), 1245 (–P=O), 1023 (–P–O–C), 764 (–P–C–). H-NMR
400 MHz, CDCl , ppm): δ 7.88 (d, J=8.0 Hz, 1H, Ar–H), 7.75 (s, 1H, Ar–H), 7.49
3
d, J=8.0 Hz, 1H, Ar–H), 7.18–7.06 (m, 6H Ar–H) 6.91 (d, J=8.0 Hz, 1H, Ar–H)
6
1
.69 (t, J =8.0 Hz, J =8.0 Hz 1H, Ar–H) 6.54 (d, J=8.0 Hz, 1H, Ar–H), 6.14 (t,
1 2
J =8.0 Hz, J =8.0 Hz 1H, Ar–H), 5.43–5.35 (d, J=32.0 Hz, 1H, –P–C–H), 4.96
2
(
s, 1H, –NH), 4.38–4.33 (dd, J=8.0 Hz, 2H, –OCH ), 4.02–3.68 (m, 6H –OCH ),
2
2
2
3
–
J
.73 (s, 3H, –CH ), 2.26–2.19 (m 1H, –CH), 1.40 (t, J =8.0 Hz, J =8.0 Hz
3
1
2
H), 1.18–1.12 (m, 9H, –OCH –CH ), 1.05 (t, J =8.0 Hz, J =8.0 Hz, 3H,
3
,
3
1
2
1
3
OCH ) ppm; C-NMR (100 MHz, CDCl ): δ 169.65, 162.52, 161.05, 158.89 (d,
3
3
=6.0 Hz), 157.96, 147.83 (d, J =14.0 Hz), 137.76, 136.78, 131.30, 129.06,
P–C
P–C
1
1
6
27.78 (d, J =4.0 Hz), 127.07 (d, J =8.0 Hz), 125.60, 120.97, 118.21, 115.64,
P–C
P–C
P–C
11.36 (d, J =10.0 Hz), 75.07, 63.23 (dd, J
=15.0 Hz, J
=14.0 Hz),
1P–C
2P–C
1.18, 48.11 (d, J =152.0 Hz), 28.50, 19.46 (d, J =2.0 Hz), 17.59, 16.47
P–C
P–C
3
1
(
d, J =6.0 Hz), 16.26 (d, J =6.0 Hz), 14.48. P-NMR (162 MHz, CDCl ):
P–C
P–C
3
1
3

M. Gundluru et al.
+
+
δ 22.28. HRMS (ESI) calcd. for C H N O PS [M+H] : 669.2177 and found
3
4
41
2
6
2
6
69.2174.
Ethyl 2‑(3‑((diethoxyphosphoryl)((4‑ꢀuorophenyl)amino)
methyl)‑4‑isobutoxyphenyl)‑4‑methylthiazole‑5‑carboxylate (8d)
−
1
White solid; Yield: 94%, M.p. 242–244 °C. FT-IR (neat, cm ) ν 3279 (–NH), 2965
1
(
(
(
(
(
–C–H), 1700 (–C=O), 1248 (–P=O), 1026 (–P–O–C), 764 (–P–C–). H-NMR
400 MHz, CDCl , ppm): δ 8.02 (s, 1H, Ar–H), 7.86 (d, J=8.0 Hz, 1H, Ar–H), 6.91
3
d, J=8.0 Hz, 1H, Ar–H), 6.79 (t, J =8.0 Hz, J =8.0 Hz, 2H, Ar–H), 6.56–6.53
1
2
m, 2H, Ar–H), 5.32 (d, J=28.0 Hz, 1H, P–C-H), 4.80 (s, 1H, –NH), 4.36–3.64
m, 8H –OCH ), 2.72 (s, 3H, –CH ), 2.24–2.15 (m 1H, –CH), 1.36 (t, J =8.0 Hz,
2
3
1
J =8.0 Hz, 3H), 1.30 (t, J =8.0 Hz, J =8.0 Hz 3H, –OCH ), 1.11 (d, J=4.0 Hz,
2
1
2
3
1
3
6
H, –CH ) 1.06 (t, J =8.0 Hz, J =4.0 Hz 3H, –CH ). C-NMR (100 MHz, CDCl ,
3 1 2 3 3
ppm): δ 169.58, 162.44, 161.05, 159.03, 155.19, 133.89, 127.87, 127.13, 126.03,
1
1
2
J
25.62, 125.07, 122.57, 121.03, 116.08, 115.61, 114.50 (d, J =7.0 Hz), 113.19,
P–C
11.45, 75.07, 63.26 (dd, J
=3.0 Hz, J
=3.0 Hz), 48.45 (d, J =204.0 Hz),
2P–C P–C
1
P–C
8.50, 19.44 (d, J =3.0 Hz), 19.25, 17.56, 16.54 (d, J =6.0 Hz), 16.26 (d,
P–C
P–C
3
1
+
=7.0 Hz), 14.41. P-NMR (162 MHz, CDCl , ppm): δ 23.42. HRMS (ESI)
P–C
3
+
calcd. for C H FN O6PS [M+H] : 579.2049 and found 579.2046.
2
8
36
2
Ethyl 2‑(3‑(((3‑chlorophenyl)amino)(diethoxyphosphoryl)
methyl)‑4‑isobutoxyphenyl)‑4‑methylthiazole‑5‑carboxylate (8e)
−
1
White solid; Yield: 92%, M.p. 247–249 °C. FT-IR (neat, cm ) ν 3284 (–NH) 2966
1
(
(
(
–C–H), 1703 (–C=O), 1245 (–P=O), 1020 (–P–O–C), 755 (–P–C–). H-NMR
400 MHz, CDCl , ppm): δ 8.12 (s, 1H, Ar–H), 7.99 (d, J=8.0 Hz, 1H, Ar–H), 7.77
3
d, J=8.0 Hz, 2H, Ar–H), 7.34 (s, 1H, Ar–H), 7.25 (t, J =12.0 Hz, J =16.0 Hz 1H,
1
2
Ar–H) 7.20 (s, 1H, Ar–H), 6.90 (d, J=16.0 Hz, 1H, Ar–H), 5.31 (d, J=32.0 Hz,
1
H, P–C-H), 4.83 (s, 1H, –NH), 4.23–3.50 (m, 8H –OCH ), 2.61 (s, 3H, –CH ),
2 3
1
3
2
.24–2.09 (m, 1H, –CH), 1.27–1.04 (m, 15H, –OCH –CH ). C-NMR (100 MHz,
3
,
3
CDCl , ppm): δ 169.32, 162.13, 160.69, 158.64, 154.05, 145.81 (d, J =19.0 Hz),,
3
P–C
1
1
J
29.81, 128.89 (d, J =26.0 Hz), 127.37, 126.52, 126.01, 124.74, 123.42, 121.34,
P–C
11.68 75.23, 64.07 (d, J =10.0 Hz), 63.87 (d, J =10.0 Hz), 58.26, 47.10 (d,
P–C
P–C
=210.0 Hz), 28.35, 19.34 (d, J =10.0 Hz), 16.41 (d, J =7.0 Hz), 16.16 (d,
P–C
P–C
P–C
P–C
3
1
+
J
=8.0 Hz), 14.30. P-NMR (162 MHz, CDCl , ppm): δ 21.59. HRMS (ESI)
3
+
calcd. for C H ClN O6PS [M+2H] : 596.1691 and found 596.1688.
2
8
36
2
Ethyl 2‑(3‑(((4‑bromophenyl)amino)(diethoxyphosphoryl)
methyl)‑4‑isobutoxyphenyl)‑4‑methylthiazole‑5‑carboxylate (8f)
−
1
White solid; Yield: 90%, M.p. 275–277 °C. FT-IR (neat, cm ) ν 3286 (–NH) 2964
1
(
(
(
(
–C–H), 1701 (–C=O), 1248 (–P=O), 1025 (–P–O–C), 756 (–P–C–). H-NMR
400 MHz, CDCl , ppm): δ 8.00 (s, 1H, Ar–H), 7.84 (d, J=8.0 Hz, 1H, Ar–H), 6.90
3
d, J=8.0 Hz, 1H, Ar–H), 6.76 (t, J =8.0 Hz, J =8.0 Hz, 2H, Ar–H), 6.58–6.50
1
2
m, 2H, Ar–H), 5.34 (d, J=32.0 Hz, 1H, P–C-H), 4.86 (s, 1H, –NH), 4.34–3.62
1
3

Design, synthesis, cytotoxic evaluation and molecular docking…
(
m, 8H –OCH ), 2.74 (s, 3H, –CH ), 2.26–2.14 (m 1H, –CH), 1.34 (t, J =8.0 Hz,
2 3 1
J =8.0 Hz, 3H), 1.33 (t, J =8.0 Hz, J =8.0 Hz 3H, –OCH ), 1.10 (d, J=4.0 Hz,
2
1
2
3
1
3
6
H, –CH ) 1.02 (t, J =8.0 Hz, J =4.0 Hz 3H, –CH ). C-NMR (100 MHz, CDCl ,
3 1 2 3 3
ppm): δ 169.52, 162.45, 161.02, 159.01, 155.13, 133.80, 127.78, 127.31, 126.04,
1
1
2
J
25.68, 125.00, 122.55, 121.09, 116.02, 115.58, 114.52 (d, J =6.0 Hz), 113.10,
P–C
11.49, 75.02, 63.28 (d d, J
=7.0 Hz, J
=7.0 Hz), 48.48 (d, J =158.0 Hz),
1
P–C
2P–C P–C
8.58, 19.46 (d, J =6.0 Hz), 19.26, 17.62, 16.56 (d, J =6.0 Hz), 16.23 (d,
P–C
P–C
3
1
+
=7.0 Hz), 14.56. P-NMR (162 MHz, CDCl , ppm): δ 22.1. HRMS (ESI)
P–C
3
+
calcd. for C H BrN O6PS [M+2H] : 640.1195 and found 640.1192.
2
8
36
2
Ethyl 2‑(3‑((diethoxyphosphoryl)((2‑iodophenyl)amino)
methyl)‑4‑isobutoxyphenyl)‑4‑methylthiazole‑5‑carboxylate (8g)
−
1
Brown solid; Yield: 91%, M.p. 290–292 °C. FT-IR (neat, cm ) ν 3286 (–NH)
1
2
927 (–C–H), 1702 (–C=O), 1246 (–P=O), 1024 (–P–O–C), 751 (–P–C). H-NMR
(
400 MHz, CDCl , ppm): δ 7.98 (s, 1H, Ar–H), 7.91 (d, J=8.0 Hz, 1H, Ar–H 1H,
3
Ar–H), 7.65 (dd, J =4.0 Hz, J =4.0 Hz 1H, Ar–H), 7.05 (t, J =8.0 Hz, J =8.0 Hz
1
2
1
2
1
H, Ar–H), 6.94 (d, J=8.0 Hz, 1H, Ar–H) 6.43 (t, J =8.0 Hz, J =8.0 Hz 1H,
1
2
Ar–H), 5.44 (d, J=32.0 Hz, 1H, P–C-H), 4.98 (s, 1H, –NH), 4.33 (dd, J =8.0 Hz,
1
J =8.0 Hz, 1H, –OCH ), 4.24–3.78 (m, 6H –OCH ), 2.73 (s, 3H, –CH ), 2.28–2.19
2
2
2
3
(
m 1H, –CH), 1.38 (t, J =8.0 Hz, J =8.0 Hz 3H, –OCH ), 1.33 (t, J =8.0 Hz,
1 2 3 1
J =8.0 Hz 3H, –OCH ), 1.15 (t, J =8.0 Hz, J =8.0 Hz 3H, –OCH ), 1.12 (d,
2
3
1
2
3
3
1
3
J=4.0 Hz, 6H, –CH ). C-NMR (100 MHz, CDCl , ppm): δ 169.53, 162.43, 160.96,
3
1
58.89 (d, J =6.0 Hz),, 145.78 (d, J =14.0 Hz), 139.11, 129.26, 127.80 (d,
P–C P–C
J
=3.0 Hz), 127.06 (d, J =4.0 Hz), 126.02 (d, J =3.0 Hz), 125.25, 121.04,
P–C P–C
P–C
1
4
J
19.83, 111.67,86.17, 75.03, 63.34 (dd, J
=7.0 Hz, J
=7.0 Hz), 61.11,
1
P–C
2P–C
8.95 (d, J =154.0 Hz), 28.43, 21.32, 19.38 (d, J =3.0 Hz), 17.52, 16.54 (d,
P–C
P–C
3
1
=6.0 Hz), 16.29 (d, J =6.0 Hz), 14.36. P-NMR (162 MHz, CDCl , ppm):
P–C
P–C
3
+
+
δ 22.35. HRMS (ESI) calcd. for C H IN O6PS [M+H] : 687.1110 and found
2
8
36
2
6
87.1107.
Ethyl 2‑(3‑((diethoxyphosphoryl)((4‑nitrophenyl)amino)
methyl)‑4‑isobutoxyphenyl)‑4‑methylthiazole‑5‑carboxylate (8h)
−
1
White solid; Yield: 92%, M.p. 288–290 °C. FT-IR (neat, cm ) ν 3288 (–NH) 2920
1
(
–C–H), 1708 (–C=O), 1245 (–P=O), 1024 (–P–O–C), 753 (–P–C–). H-NMR
(
400 MHz, CDCl , ppm): δ 8.04 (s, 1H, Ar–H), 7.87 (d, J=4.0 Hz, 1H, Ar–H),
3
6
.93 (d, J=8.0 Hz, 2H, Ar–H), 6.80 (t, J =16.0 Hz, J =16.0 Hz, 2H, Ar–H), 6.57
1
2
(
t, J =16.0 Hz, J =16.0 Hz, 2H, Ar–H), 5.35 (d, J=24.0 Hz, 1H, P–C-H), 4.82
1 2
(
s, 1H, –NH), 4.40–3.70 (m, 8H –OCH ), 2.73 (s, 3H, –CH ), 2.24–2.18 (m 1H,
2 3
1
3
–
1
1
1
J
CH), 1.43–1.00 (m, 15H OCH & –CH ). C-NMR (100 MHz, CDCl , ppm): δ
3 3 3
69.58, 168.58, 165.78, 163.30, 162.44, 159.03, 157.54, 155.19, 149.94, 138.07,
33.89, 130.46, 127.87, 127.13, 126.03, 125.62, 125.07, 122.52 (d, J =9.0 Hz),
P–C
21.03, 116.08, 115.83, 114.54, 113.19, 75.07, 63.30 (d, J =3.0 Hz), 63.23 (d,
P–C
=3.0 Hz), 48.70 (d, J =153.0 Hz), 28.50, 19.43 (d, J =3.0 Hz), 17.56,
P–C
P–C
P–C
3
1
1
6.54 (d, J =6.0 Hz), 16.25 (d, J =4.0 Hz), 14.41. P-NMR (162 MHz,
P–C P–C
1
3

M. Gundluru et al.
+
+
CDCl ): δ 23.53. HRMS (ESI) calcd. for C H N O PS [M+H] : 606.1994 and
3
28 36
3
8
found 606.1991.
Ethyl2‑(3‑((diethoxyphosphoryl)((4‑(triꢀuoromethyl)phenyl)amino)
methyl)‑4‑isobutoxyphenyl)‑4‑methylthiazole‑5‑carboxylate (8i)
−
1
Brown solid; Yield: 95%, M.p. 276–278 °C. FT-IR (neat, cm ) ν 3284 (–NH)
1
2
967(–C–H), 1699 (–C=O), 1248 (–P=O), 1023 (–P–O–C), 746 (–P–C–). H-NMR
(
400 MHz, CDCl , ppm): δ 7.96 (s, 1H, Ar–H), 7.82 (d, J=8.0 Hz, 1H, Ar–H),
3
7
.34 (d, J=8.0 Hz, 2H, Ar–H), 6.90 (d, J=8.0 Hz, 1H, Ar–H), 6.56 (d, J=8.0 Hz,
H, Ar–H), 5.26 (d, J=36.0 Hz, 1H, P–CH), 4.89 (s, 1H, –NH), 4.32–4.26 (dd,
2
J=8.0 Hz, 2H, –OCH ), 4.16–3.68 (m, 6H –OCH ), 3.62 (s, 3H, –OCH ), 2.78
2
2
3
(
s, 3H, –CH ), 2.26–2.16 (m 1H, –CH), 1.33 (t, J =8.0 Hz, J =4.0 Hz 3H), 1.27
3
1
2
(
t, J =8.0 Hz, J =4.0 Hz 3H, –OCH ), 1.10 (d, J=8.0 Hz, 6H, –CH ) 1.02 (t,
1
2
3
3
1
3
J =8.0 Hz, J =8.0 Hz 3H, –CH ). C-NMR (100 MHz, CDCl , ppm): δ 169.78,
1
2
3
3
1
62.38, 160.96, 159.06 (d, J =7.0 Hz), 152.72, 140.34 (d, J =17.0 Hz), 127.76
P–C P–C
(
d, J =2.0 Hz), 127.24, 124.46 (d, J =17.0 Hz), 123.19 (d, J =15.0 Hz),
P–C P–C P–C
1
J
20.95, 116.33 (d, J =7.0 Hz), 111.36, 75.10, 63.24 (dd, J
=14.0 Hz,
P–C
1P–C
=14.0 Hz), 61.16, 55.74, 48.98 (d, J =136.0 Hz), 28.50, 19.46 (d,
2
P–C
P–C
P–C
3
1
J
=7.0 Hz), 17.52, 16.58 (d, J =6.0 Hz), 16.28 (d, J =6.0 Hz), 14.61. P-
P–C P–C
+
NMR (162 MHz, CDCl , ppm): δ 22.87. HRMS (ESI) calcd. for C H F N O PS
3
29 36
3
2
6
+
[
M+H] : 629.2017 and found 629.2014.
Ethyl 2‑(3‑((diethoxyphosphoryl)((2‑iodo‑4‑(triꢀuoromethyl)phenyl)amino)
methyl)‑4‑iso butoxyphenyl)‑4‑methylthiazole‑5‑carboxylate (8j)
−
1
White solid; Yield: 94%, M.p. 288–290 °C. FT-IR (neat, cm ) ν 3290 (–NH)
1
2
980 (–C–H), 1702 (–C=O), 1256 (–P=O), 1028 (-P-O-C), 756 (–P–C–). H-
NMR (400 MHz, CDCl , ppm): δ 7.84 (s, 1H, Ar–H), 7.76 (s, 1H, Ar–H), 7.63
3
(
d, J=8.0 Hz, 1H, Ar–H), 7.38 (d, J=8.0 Hz, 1H, Ar–H), 7.12 (d, J=8.0 Hz, 1H,
Ar–H), 6.74 (d, J=8.0 Hz, 1H, Ar–H), 5.24 (d, J=28.0 Hz, 1H, P–C–H), 4.98 (s,
1
3
H, –NH), 4.38 (d, J=8.0 Hz, 1H, –OCH ), 4.18–3.86 (m, 6H –OCH ), 2.50 (s,
2 2
H, –CH ), 2.48–2.30 (m 1H, –CH), 1.36 (t, J =8.0 Hz, J =8.0 Hz 3H), 1.24 (t,
3
1
2
1
3
J =8.0 Hz, J =8.0 Hz 3H, –OCH ), 1.02 (d, J =12.0 Hz, 6H, –CH ). C-NMR
1
2
3
1
3
(
100 MHz, CDCl , ppm): δ 171.36, 165.66, 159.24, 150.51, 149.92, 138.40, 135.96
3
(
d, J =12.0 Hz), 130.44, 129.66 (d, J =12.0 Hz), 126.48, 124.14, 122.18,
P–C
P–C
1
J
17.32, 113.39 (d, J =10.0 Hz), 106.10, 75.94, 74.48, 64.12 (t, J
=11.0 Hz,
1P–C
P–C
3
1
=10.0 Hz), 61.56, 26.89, 19.76, 16.33 (d, J =13.0 Hz), 14.68. P-NMR
2
P–C
P–C
+
(
162 MHz, CDCl , ppm): δ 22.86. HRMS (ESI) calcd. for C H F IN O PS
3
29 35
3
2
6
+
[
M+H] : 755.0984 and found 755.0981.
In vitro anticancer assay
The in vitro cytotoxicity of the title compounds (8a–8j) was tested against pro-
liferation of human breast (MCF-7 and MDA-MB-231), prostate (DU-145) liver
1
3

Design, synthesis, cytotoxic evaluation and molecular docking…
(
HepG2) and HeLa cancer cell lines by performing sulforhodamine-B (SRB)
assay [43]. The cell line of interest was seeded in disinfected ꢃat-bottom 96-well
plate (5000 cells/100 µL) in a medium containing 10% fetal bovine serum and
antibiotics (penicillin and streptomycin). After incubation for about 18–20 h in an
incubator continuously supplied with 5% CO to ensure appropriate adherence of
2
the cells to the surface bottom of the wells, cells were treated with the compounds
or reference standard Adriamycin. To treat cells, working dilutions of concentra-
tion of the compounds were prepared, of which 2 µL aliquot was added to every
well, thereby creating the ꢂnal concentration of compound 0 to 100 µM. Each
compound was tested in triplicate and the cytotoxicity was determined as the
average of that triplicate. DMSO and Adriamycin (as standard control antican-
cer drug) were taken as vehicle and positive controls, respectively. Further, the
cells were allowed to grow for another 48 h in an incubator maintained at 37 °C
with a constant supply of 5% CO . The plates were then air-dried and 100 µL of
2
1
0 mM Tris base was added to each well to solubilize the SRB before reading
the absorbance using Perkin-Elmer Multimode Reader at 510 nm. The amount
of absorbance is directly relative to cell growth and is thus used to calculate the
IC values. In this study for initial screening, ꢂve types of cancer cell lines, i.e.,
5
0
human breast cancer (MCF-7 and MDA-MB-231), prostate cancer (DU-145) liver
cancer (HepG2) and HeLa cancer cell lines were tested for the cytotoxic eꢁect of
the title compounds.
Molecular docking studies
All the synthesized compounds were docked with X-ray crystal structure of tubulin
inhibitor (PDB ID: 3E22) and human type IIA DNA topoisomerase (PDB: 1ZXM),
using AutoDock. Ligand structures were drawn using build panel and prepared using
Ligprep module implemented Maestro-8.4 (Schrodinger LLC). Energy minimization
is carried out using OPLS-2005 force ꢂeld. Structures were saved in. pdb format
and rewritten using open babel for AutoDock compatible atom type. For docking,
grid parameter ꢂle (.gpf) and docking parameter ꢂles (.dpf) were written using MGL
Tools-1.5.6. Receptor grids were generated using 100×100×100 grid points in xyz
with grid spacing of 0.20 Å. Grid box was generated by considering active residues.
Map types were generated using autogrid 4.2. Docking was carried out with the fol-
lowing parameters with number of runs: 50, population size: 150, number of evalu-
ations: 2,500,000 and number of generations: 27,000, using AutoDock 4.2. Analysis
of docking results was done using MGL Tools-1.5.6. Top scoring molecule in the
largest cluster was analyzed for its interaction with the protein.
Acknowledgments The author G. Mohan thanks to Prof. C. Devendranath Reddy, Department of
Chemistry, S.V. University, Tirupati for his helpful discussions and acknowledges DST-PURSE 2nd
Phase Programme facilitated in S.V. University, Tirupati funded by DST, New Delhi, India for provid-
ing instrumentation facility and funding (File No: 17118-UGC-III(3)/DST-PURSE 2nd Phase/2017, Dt:
2
3-08-2018). One of the authors S.H. Yasmin acknowledges DST, New Delhi, India for the ꢂnancial sup-
port under DST-INSPIRE Fellowship Programme (File No. DST/INSPIRE Fellowship/2017/IF170317,
Dated: 24/05/2017).
1
3

M. Gundluru et al.
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Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published
maps and institutional aꢀliations.
Aꢀliations
1
,2
· Vishnu Nayak Badavath3 · Haroon Yasmin Shaik ·
1
Mohan Gundluru
1
4
4
Murali Sudileti · Bakthavatchala Reddy Nemallapudi · Sravya Gundala ·
Grigory V. Zyryanov4 · Suresh Reddy Cirandur1
,5
*
Suresh Reddy Cirandur
csrsvu@gmail.com
1
Department of Chemistry, Sri Venkateswara University, Tirupati, Andhra Pradesh 517 502,
1
3

M. Gundluru et al.
India
2
3
DST-PURSE Centre, Sri Venkateswara University, Tirupati, Andhra Pradesh 517 502, India
Institute for Drug Research, School of Pharmacy, The Hebrew University, 91120 Jerusalem,
Israel
4
5
Department of Organic and Biomolecular Chemistry, Chemical Engineering Institute, Ural
Federal University, 19 Mira Street, Yekaterinburg, Russian Federation 620002
I.Ya, Postovskiy Institute of Organic Synthesis, Ural Division of the Russian Academy
of Sciences, 22 S. Kovalevskoy Street, Yekaterinburg, Russian Federation 620219
1
3