Gold catalyzed double condensation reaction: Synthesis, antimicrobial and cytotoxicity of spirooxindole derivatives

Article abstract of DOI:10.1016/j.bmcl.2016.07.036

Microwave assisted synthesis of spirooxindoles via tandem double condensation between isatins and 4-hydroxycoumarin under gold catalysis is reported. The reaction is practical to perform, since the products can be isolated by simple filtration without requiring tedious column chromatography. The scope of this chemistry is exemplified by preparing structurally diverse spirooxindoles (22 examples) in excellent yields. Antimicrobial evaluation of the synthesized compounds revealed that three compounds (3a, 3f and 3s) exhibited significant MIC values in comparison to the standard drugs. Molecular docking studies of these compounds with AmpC-β-lactamase receptor revealed that 3a exhibited minimum binding energy (?117.819?kcal/mol) indicating its strong affinity towards amino acid residues via strong hydrogen bond interaction. All compounds were also evaluated for their in vitro cytotoxicity against COLO320 cancer cells. Biological assay and molecular docking studies demonstrated that 3g is the most active compound in terms of its low IC₅₀value (50.0?μM) and least free energy of binding (?8.99?kcal/mol) towards CHK1 receptor, respectively.

Full text of DOI:10.1016/j.bmcl.2016.07.036

Accepted Manuscript
Gold catalyzed double condensation reaction: Synthesis, antimicrobial and cy-
totoxicity of spirooxindole derivatives
K. Parthasarathy, Chandrasekar Praveen, J.C. Jeyaveeran, A.A.M. Prince
PII:
S0960-894X(16)30749-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.07.036
BMCL 24080
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
23 May 2016
7 July 2016
18 July 2016
Please cite this article as: Parthasarathy, K., Praveen, C., Jeyaveeran, J.C., Prince, A.A.M., Gold catalyzed double
condensation reaction: Synthesis, antimicrobial and cytotoxicity of spirooxindole derivatives, Bioorganic &
Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.07.036
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Gold catalyzed double condensation reaction: Synthesis, antimicrobial and cytotoxicity
of spirooxindole derivatives
K. Parthasarathy a, Chandrasekar Praveen b,*, J. C. Jeyaveeran c, A. A. M. Prince c
a Department of Phytochemistry, Central Council for Research in Siddha, Arumbakkam,
Chennai-600106, Tamil Nadu, India
b Functional Materials Division, CSIR-Central Electrochemical Research Institute Karaikudi-
630003, Tamil Nadu, India
c Department of Chemistry, Ramakrishna Mission Vivekananda College, Mylapore, Chennai-
600004, Tamil Nadu, India
ABSTRACT:
Microwave assisted synthesis of spirooxindoles via tandem double condensation between
isatins and 4-hydroxycoumarin under gold catalysis is reported. The reaction is practical to
perform, since the products can be isolated by simple filtration without requiring tedious
column chromatography. The scope of this chemistry is exemplified by preparing structurally
diverse spirooxindoles (22 examples) in excellent yields. Antimicrobial evaluation of the
synthesized compounds revealed that three compounds (3a, 3f and 3s) exhibited significant
MIC values in comparison to the standard drugs. Molecular docking studies of these
compounds with AmpC-β-lactamase receptor revealed that 3a exhibited minimum binding
energy (-117.819 kcal/moL) indicating its strong affinity towards amino acid residues via
strong hydrogen bond interaction. All compounds were also evaluated for their in vitro
cytotoxicity against COLO320 cancer cells. Biological assay and molecular docking studies
demonstrated that 3g is the most active compound in terms of its low IC50 value (50.0 μM)
and least free energy of binding (-8.99 kcal/mol) towards CHK1 receptor, respectively.
Keywords:
Microwave chemistry, Gold catalysis, Spirooxindoles, Biological activity, Molecular docking
*Corresponding author; email: chandrasekar.praveen@gmail.com
1

Spirooxindoles have received paramount importance, because of their prevalence as
substructure in numerous bioactive agents and natural products.1 On the other hand, 4-
hydroxycoumarin is a structural component present in various pharmaceuticals/clinical drugs
such as warfarin, phenprocoumon, acenocoumarol, tioclomarol and brodifacoum (Figure 1).2
Inspired by their pharmacological properties and synthetic feasibility, it was thought
worthwhile to fuse both these scaffolds as a single molecular entity in view of enhanced
biological activity. Towards this end, we intended a simple and straightforward synthesis of
hybrid-spirooxindoles via bis-condensation between isatin and 4-hydroxycoumarin. Only
limited reports were documented for this type of transformations though with narrow
substrate scope (N-unsubstituted). For example, a substituent at the isatinyl nitrogen was not
reported in the previously reported p-TSA and I2 catalyzed protocols.3 In another finding, the
solvent-induced synthesis of similar compounds under dry conditions was disclosed albeit
with less product selectivity.4 In order to circumvent these limitations coupled with our
research endeavors in homogeneous gold catalysis,5 we sought to investigate whether it
would be possible to accomplish the aforementioned transformation under gold catalysis. To
this end, we herein report a practical and efficient gold(III)-catalyzed double condensation
reaction between isatin and 4-hydroxycoumarin under microwave condition. The resulting
spirooxindole[pyrano-bis-2H-l-benzopyrans] were evaluated for their antimicrobial and
cytotoxic properties. Additionally, molecular docking of most active compounds with
appropriate receptors was performed and all these results were disclosed in this paper.
Figure 1. Pharmaceuticals containing of 4-hydroxycoumarin scaffold.
Based on our previous experience with gold catalysis,5 we initially screened few Au(I) and
Au(III) catalysts for the prototype reaction between isatin (1.0 mmoL) and 4-
o
hydroxycoumarin (2.0 mmoL) at 80 C (Scheme 1, Table 1). In this effort, we found that
2

AuCl, Ph3PAuCl and AuCl3 afforded poor yield of product 3a in both dichloroethane and
ethanol (entries 1-5). Among the triflimide complexes screened Ph3PAuNTf2 gave slightly
better yield over t-Bu3PAuNTf2 (entries 6 and 7). The use of alkali gold salts such as KAuCl4
and NaAuCl4.2H2O enhanced the yield of 3a to 57% and 59% respectively (entry 8 and 9).
Since, NaAuCl4.2H2O is relatively cheaper than KAuCl4, our further investigation focused
only on the use of the sodium salt. Reducing the catalytic amount of NaAuCl4.2H2O from 5
to 2 mol% resulted in no appreciable change in yield (entry 10) and thus evaluated for
subsequent screening. A drastically reduced yield was observed, when the reaction was
performed at room temperature suggesting the necessity of elevated temperature in this
transformation (entry 11). Through this observation, we rationalized that the reaction rate
could be escalated by microwave irradiation instead of conventional heating. To our delight,
o
microwave irradiation of model reaction at 25 C afforded 79% yield of 3a (entry 12). A
slight increase of temperature to 40 oC resulted in an excellent yield of 92% (entry 13). When
the solvent was changed from ethanol to dichloroethane more or less same yield of 3a (90%)
was obtained (entry 14). A blank reaction led to no product formation confirmed the role of
NaAuCl4.2H2O as catalytic species (entry 15). In view of the fact that, ethanol has a loss
tangent (tan δ value) of 0.941 in microwave promoted reactions and dielectric constant (ε') of
24.6; we preferred it over dichloroethane for further studies.6 From the screening data, it is
o
quite obvious that 2 mol% of NaAuCl4.2H2O in ethanol at 40 C under an irradiation of 200
W was effective for the desired transformation (entry 13). Hence, this reaction condition was
employed for a wide range of substrates toward the preparation of structurally diverse
spirooxindoles.
Scheme 1. Proposed synthesis of target 3a.
Table 1 Screening of Lewis acidsa
Entry
Catalyst
AuCl
AuCl
Ph3PAuCl
AuCl3
mol Solvent Temp Time Yield
%
5
(oC) (min) (%)b
1
2
3
4
(CH2Cl)2
EtOH
80
80
80
80
60
60
60
60
25
26
22
15
5
5
EtOH
5
(CH2Cl)2
3

5
6
7
8
9
10
11
12
13
14
AuCl3
5
5
5
5
5
2
2
2
2
2
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
(CH2Cl)2
80
80
80
80
80
80
25
25
40
40
60
60
60
60
60
60
120
60
15
15
18
39
28
57
59
Ph3PAuNTf2
t-Bu3PAuNTf2
KAuCl4
NaAuCl4.2H2O
NaAuCl4.2H2O
NaAuCl4.2H2O
NaAuCl4.2H2O
NaAuCl4.2H2O
NaAuCl4.2H2O
58
10
79c
92c
90c
a All reactions were performed using 1.0 mmoL of isatin and 2.0 mmoL of 4-hydroxycoumarin.
b Isolated yield after filteration.
c Reaction was performed under microwave irradiation with a power level of 200 W
Initially, we prepared a series of N-substituted isatins required for our study by several
closely related N-alkylation procedures in synthetically good yields.7 In order to fully
examine the scope of our chemistry, various isatins and 4-hydroxycoumarin were tested
under our optimized conditions (Scheme 2, Table 2). The reaction appears to be general and
works well regardless of the electronic nature of the peripheral substituent (R and R1) on the
isatin moiety and afforded good to excellent yield of products 3a-3u (Table 2). This
uniformity in yields irrespective of the electronic nature of the substituent can be explained
by looking at the increased electrophilicity at the C3-carbonyl of isatin via Au(III) activation,
thus facilitating the nucleophilic addition of 4-hydroxycoumarin. The protocol is amenable to
substrates containing peripheral substituent at all positions (R and R1), whereas a substitution
at the isatinyl nitrogen was not reported in other catalytic protocols.3 One of the significant
advantages of the current methodology is its scope towards substrate possessing C-C multiple
bonds (3q and 3r), since it is well known that, Au(III)-catalyst activates unsaturated bonds
via typical π-activation chemistry.8
Scheme 2. Synthesis of targeted spirooxindoles 3a-3u.
Table 2 Synthesis of 3a-3u under optimized conditions
Entry
1
R
H
R1
H
Producta
Yield (%)b
92
3a
4

2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
H
H
H
H
H
H
H
H
H
4-Cl
5-Me
5-F
5-Cl
5-Br
5-NO2
6-Cl
7-F
7-Cl
H
96
93
92
89
88
86
91
95
90
95
95
94
95
91
90
93
91
94
92
89
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
Me
Et
H
H
H
H
H
H
H
H
n-Pr
n-Bu
Pentyl
Hexyl
Allyl
Propargyl
Bn
2-F-Bn
Me
H
5-F
3t
3u
a All products were characterized by IR, 1H NMR 13C NMR & mass.
b Isolated yield after filtration.
The utility of this chemistry was further manifested by its applicability to afford product with
propargyl tether 3r without observing any allene-isomerization.9 Interestingly, our Au(III)-
catalyzed spirooxindole formation strategy is not only limited to mono-systems but also can
be extended to bis-system. As illustrated in Scheme 3, we have been able to apply this
chemistry to the synthesis of complex bis-isatinyl system. Thus, treatment of 1,1'-(propane-
1,3-diyl)bis-isatin 1v (1.0 mmoL) with 4-hydroxycoumarin (4.0 mmoL) under our standard
conditions generated the corresponding bis-spiro skeleton 3v in 81% yield.
Scheme 3. Synthesis of bis-spiroxindole system 3v.
5

In another study, we subjected N-methylisatin (1.0 mmoL) with 4-hydroxy-6-methyl-2H-
pyran-2-one (2.0 mmoL) under our standard conditions in view of obtaining the bis-
condensation product 3w (Scheme 4). In contrast to our expectation, the product 4w was
obtained as the sole product in low yield. This observation was in sharp agreement with
results obtained using Rh(cod)2BF4 as catalyst by Stephenson et al.10 Nevertheless, we were
able to achieve excellent diastereoselectivity in this transformation (dr = 95:05 vs 5:1).
Scheme 4. Au(III)-catalyzed formation of compound 2b.
1
13
The structure of all products was confirmed by spectral data (FTIR, H NMR, C NMR and
MS) and elemental analyses. As a representative example, the IR spectrum of compound 3a
showed a broad peak at 3411 cm-1 and a sharp peak at 1708 cm-1, which corresponds
respectively to the –NH and carbonyl functionalities of the oxindole core. Sharp stretching
bands at 1732 and 1662 cm-1 suggested the presence of carbonyl group characteristic of the δ-
1
lactone ring. In H NMR spectrum, twelve protons resonated at aryl region (δH 6.84-8.48
ppm) and a broad singlet at δH 10.85 ppm corresponds to -NH proton (D2O exchangeable) of
13
the oxindole moiety. The C NMR spectrum of 3a recorded in DMSO-d6 showed seventeen
carbons. A less intense peak at δc 46.5 ppm indicates the presence of a quaternary carbon in
the aliphatic region (spiro carbon). Two peaks at δc 157.9 and 176.4 ppm corresponds to the
carbonyl carbon of the coumarin and oxindole, respectively. This observation was supported
by DEPT-135 and 2D chemical shift correlation experiments (see supplementary data).
Finally, HRMS data showed a peak at m/z(obs.)=458.0641 corresponds to [M+Na]+, where M
is the molecular mass of 3a is in well agreement with the expected m/z(expd.)=458.0635. All
these experimental data unambiguously confirmed the structure of compound 3a.
Based on the observed results, a tentative mechanism was proposed (Scheme 5) for the
formation of representative compound 3a. According to which, 4-hydroxycoumarin 2
6

undergoes nucleophilic addition at the C3 carbon of the [Au] activated isatin to form
intermediate I. Dehydration of intermediate I results in the formation of intermediate II. The
Lewis acidic [Au] again enters the catalytic cycle by interacting with the carbonyl oxygen of
II and drives the addition of another molecule of 4-hydroxycoumarin 2 leading to III. The
later upon intramolecular cyclization results in intermediate IV and subsequent dehydration
affords the product 3a.
Scheme 5. Plausible mechanism for the formation of spirooxindole 3a.
In continuation of our on-going project on bio-active heterocycles,11 all the prepared
compounds (3a-3u) were tested for their antibacterial activity against nine bacterial strains
(Staphylococcus epidermidis, Salmonella typhimurium, Klebsiella pneumonia, Bacillus
subtilis, Shigella flexneri, Micrococcus luteus, Enterobacter aerogenes, Staphylococcus
aureus and Staphylococcus aureus MRSA) and antifungal activity against two fungal strains
(Candida albicans, Malassesia pachydermatis) using disc diffusion method.12 Analysis of the
screening results revealed that most of the compounds exhibited comparable antibacterialal
and antifungal activities against reference drugs (Table 3).
Table 3 Zone of inhibition (mm) of compounds 3a-3u (1mg/disc)
Bacteria
Fungi
Compou
nds
S.
S.
K.
B.
S.
M.
E.
S.
S.
C.
M.
epidermi typhimuri pneumon subti flexn
lute aeroge
aure aure
albica pachyderm
dis
um
iae
lis
eri
us
nes
us
us
ns
atis
MRS
A
3a
3b
31
19
26
13
28
12
32
17
35
22
36
21
32
16
21
10
36
21
34
19
22
15
7

3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
21
18
26
31
17
22
17
21
27
28
12
14
13
30
13
26
32
25
20
16
12
19
24
15
15
12
14
20
19
11
12
15
22
11
19
25
18
14
18
17
13
20
26
12
17
13
15
21
22
13
13
12
25
15
21
27
21
11
20
21
20
25
31
11
21
16
18
27
29
12
13
16
29
12
25
33
26
19
25
23
22
31
35
13
24
19
18
31
33
11
11
11
34
12
30
36
30
16
30
20
21
27
30
11
19
18
20
28
23
11
12
11
29
12
27
32
27
12
26
18
17
23
29
11
18
15
13
24
23
13
12
15
24
13
22
30
23
21
22
11
10
15
24
12
10
10
12
17
15
12
12
10
16
11
15
23
15
13
14
23
27
31
38
12
23
20
21
33
32
14
13
12
33
16
31
35
30
17
30
23
21
29
36
12
12
14
16
30
29
14
12
12
32
14
33
33
31
18
28
20
19
27
23
13
10
15
13
28
30
12
13
13
29
13
26
22
27
14
26
3t
3u
Control 25
Control: Streptomycin (standard antibacterial agent) and Ketoconazole (standard antifungal agent)
Out of the 22 compounds screened, 9 compounds (3a, 3e, 3f, 3k, 3l, 3p, 3r, 3s and 3t)
emerged as the most active against all antimicrobial strains. It is noteworthy to mention that
these compounds exhibited greater zone of inhibition than the standard drugs. Other
compounds (3b-3d, 3g-3j, 3m-3o, 3q and 3u) showed moderate activities against the tested
strains as indicated by their reduced zone of inhibition values. Based on these encouraging
results, the most active compounds (3a, 3e, 3f, 3k, 3l, 3p, 3r, 3s and 3t) were assessed in
terms of minimum inhibitory concentration (MIC) by standard reference methods (Table 4).13
MIC is defined as the maximum dilution of the test compound that inhibits the growth of the
microorganism. The selected compounds were dissolved in DMSO to prepare a series of
descending concentration (1000 µM, 500 µM, 250 µM, 125 µM, 62.5 µM, 31.25 µM, 15.62
µM and 7.81 µM). These solutions were then diluted to give serial two-fold dilutions and
were added to each medium in 96 well plates. An inoculum of 100 µL from each well was
inoculated. The references, ketoconazole for fungi and streptomycin for bacteria were
included in the assays as positive controls. For fungi, the plates were incubated for 48 to 72 h
at 28 °C and for bacteria the plates were incubated for 24 h at 37 °C. The MIC for fungi was
defined as the lowest concentration showing no visible fungal growth after the incubation
time. 5 µL of tested broth was placed on the sterile MHA plates for bacteria and incubated at
respective temperature. The MIC for bacteria was determined as the lowest concentration of
the compound inhibiting the visual growth of the test cultures on the agar plate. The MIC
values of active compounds against bacteria and fungi are given in Table 4, which revealed
8

that among the nine compounds tested, compound 3a devoid of any substitution showed
activity against Enterobacter aerogenes (31.25 µM) and Staphylococcus aureus MRSA (7.81
µM). Compound 3f possessing bromo substituent at the C5-carbon of the oxindole core
exhibited activity against Staphylococcus epidermidis (31.25 µM) and Staphylococcus aureus
(7.81 µM). Compound 3s possessing N-benzyl substituent showed activity against
Staphylococcus epidermidis (31.25 µM), Staphylococcus aureus (7.81 µM) and Candida
albicans (31.25 µM). Whereas, other compounds 3e, 3k, 3l, 3p, 3r and 3t displayed
insignificant MIC values against all antimicrobial strains.
Table 4 MIC of most potent compounds
Organism
3a
3e 3f
3k 3l
3p 3r 3s
3t
Control
Bacteria
S. epidermidis
S. typhimurium
K. pneumoniae
B. subtilis
250
250
250
125
500 31.25 500 500
500 250 500 250
500 61.25 500 500
500 125
250 500 31.25 500
25
125 250 125
500 125 250
125
250
500
125
500
500
30
6.25
25
6.25
6.25
25
250 31.25 500 500 125
S. flexneri
M. luteus
E. aerogenes
S. aureus
S. aureus MRSA
Fungi
C. albicans
61.25 500 250
250 500 125
31.25 250 125
250 500 7.81 250 125
7.81 500 125
500 250
500 61.25 250 500 125
250 500 250 500 125
500 125 125
250 250 7.81 61.25 6.25
250 500 250
250 500
250
6.25
25
250 500 500
250 500
250 250 31.25 250
Control: Streptomycin (for antibacterial) and Ketoconazole (for antifungal)
In the present study all compounds (3a-3u) were tested for their cytotoxic potential against
colorectal adenocarcinoma cell lines (COLO320) by MTT assay.14 The cytotoxic results were
compared with reference drug cyclophosphamide which showed 90% inhibition at a
concentration of 156 μM. The tested compounds exhibited maximum cytotoxicity against
COLO320 cells at a concentration of 100 to 50 µM. All concentrations used in the
experiment could decrease the cell viability significantly (P<0.05) in a concentration-
dependent manner. Cytotoxicity of each sample was expressed as IC50 value. The IC50 value
is the concentration of test sample that causes 50% inhibition of cell growth averaged from
three replicate experiments (Table 5). Attempts to understand the SAR of the spirooxindoles
started with the substitution at the C5 position, which revealed that replacement of C5-F (3d,
IC50 = > 100 µM) by C5-Cl (3e, IC50 > 55.5 µM) or C5-Br (3f, IC50 = 52.7 µM) did increased
the potency to a great extent. Similar trend was observed, when C5-Br (3f, IC50 = 52.7 µM)
was replaced by C5-NO2 (3g, IC50 = 50.0 µM) and resulted in an escalation of activity. The
increase in activity with respect to different substitution could be attributed to the increase in
size (F < Cl < Br < NO2) of the respective substituent. Halo substitution other than C5
9

position, such as C4 (3b), C6 (3h) and C7 (3i and 3j) completely failed to show appreciable
cytotoxicity as evidenced by their higher IC50 values (> 100 μM). These comparisons clearly
suggested that the presence of C5-substituent is essential in order to realize good inhibitory
potency. It is apparent from the data presented in Table 5, that with the every increase in the
number of –CH2 spacer, the inhibitory potency also increased. This drift was observed in
analogues containing NH (3a, IC50 = 57.2 μM), NMe (3k, IC50 = 54.5 μM), NEt (3l, IC50 =
53.9 μM) and NPr (3m, IC50 = 51.1 μM), which showed the activity increased in the order
NPr > NEt > NMe > NH. Conversely, a completely opposite trend was observed when the
carbon chain goes beyond the three methylene spacer 3m. For example, a slightly reduced
potency was observed for the NBu analogue (3n, IC50 = 51.8 μM) when compared to the NPr
analogue (3m, IC50 = 51.1 μM). Higher congeners such as the N-pentyl (3o, IC50 > 100 μM),
and N-hexyl (3p, IC50 > 100 μM) exhibited a drastically decreased cytotoxicity. Among the
unsaturated systems, compound with a propargyl group (3r, IC50 > 100 μM) demonstrated
significant cytotoxicity against its allyl counterpart (3q, IC50 = 54.2 μM). Assessment of the
aryl groups revealed that compound with 2-fluorobenzyl substituent (3t, IC50 = 51.8 μM)
exhibited an elevated cytotoxicity than those containing the benzyl (3s, IC50 = 53.1 μM). This
slight enhancement in activity is mainly attributed to the differences in size and
electronegativity offered by the fluorine atom. However, when the fluorine atom was
located on the C5-position (3u, IC50 = 55.3 μM), it renders the molecule less active than its
unsubstituted counterpart (3k, IC50 = 54.5 μM). Analysis of the combined results as depicted
in Table 3, indicates that compound with a -NO2 group (3g, IC50 = 50.0 µM) positioned at the
C5 of the oxindole core emerged as the most active. Compound with N-propyl substituent
(3m, IC50 = 51.1 µM) was found to be the second most active compound. Compounds
possessing butyl (3n, IC50 = 51.8 µM) and 2-fluorobenzyl groups (3t, IC50 = 51.8 µM)
respectively showed same but significant inhibitory potencies and emerged as the third most
active among the screening hits.
Table 5 IC50 and FEB values of compounds 3a-3u
Entry
Compounds
IC50 (µM) FEB (kcal/moL)a
1
2
3
4
5
6
7
8
57.2
>100
>100
>100
55.5
52.7
50.0
>100
-8.24
-7.01
-7.88
-7.77
-7.99
-8.77
-8.99
-6.99
3a
3b
3c
3d
3e
3f
3g
3h
10

9
>100
>100
54.5
53.9
51.1
51.8
>100
>100
54.2
>100
53.1
51.8
55.3
-
-7.75
-8.12
-8.44
-8.34
-8.63
-8.87
-7.58
-6.71
-8.55
-7.74
-8.31
-8.44
-8.33
-9.53
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
10
11
12
13
14
15
16
17
18
19
20
21
22
3u
Standardb,c
a FEB = Free Energy of Binding.
b Cyclophosphamide was used as the standard for cytotoxic studies, which
showed 90% inhibition (156 μM).
c Pyrazolo[1,5-a]pyrimidine was used as the standard for docking.
The precise prediction of ligand-protein complexes is of fundamental importance in modern
structure based drug design.15 The ligand matching and the corresponding docking score
could confirm the binding efficiency of each molecule at the binding site. To this end,
selected ligands were docked into AmpC-β-lactamase and Checkpoint kinase 1 (CHK)
receptors to evaluate the antimicrobial and cytotoxic activities, respectively. Docking of
ligands to the receptor was assessed using Moldock scoring function for antimicrobial and
AUTODOCK for cytotoxicity and all the results were discussed in the following sections.
The level of antimicrobial activity was studied computationally by Molegro Virtual Docker
(MVD) by automated docking of the most active compounds 3a, 3f and 3s to the binding site
of AmpC-β-lactamase. The MolDock scoring function used by MVD is derived from the
piecewise linear potential (PLP) scoring functions. The MolDock further improves these
scoring functions with new hydrogen bonding term and new charge schemes. The ligand
matching and the corresponding scoring could confirm the binding efficiency of each
molecule at the binding site which are evaluated and ranked according to Moldock scoring
function.16 Experimentally, X-ray crystal structure of AmpC-β-lactamase from E.coli was
retrieved from Protein Data Bank (PDB) database -1KE4.17 Computationally, Molegro virtual
docker was used to predict the potential binding site on AmpC-β-lactamase. Based on the
cavities detected, the most active compounds (from the biological assay) 3a, 3f and 3s were
docked with the active site of amino acids, where the hydrogen bonding interactions are
shown by dotted lines (Figure 2, 3 and 4). The active site consisted of amino acid residue that
11

bound to the corresponding amino acids such as ASN346, THR316, ALA318 and SER64.
However, ASN346 was found to be conserved in the active site. The binding energy of ligand
3a with protein active site was found to be -117.819 kcal.mol-1. The best pose for molecule 3s
shows two interactions with lowest energy of -113.067 kcal mol-1 which binds to the active
site amino acids ASN346 and ARG148 respectively. The best poses for molecule 3f shows
three interactions with lowest energy of -107.056 kcal mol-1 which binds to active site amino
acids ASN346, GLU272 and ARG148 respectively. The docking study revealed that
compound 3a exhibited the minimum binding energy which indicates its strong affinity with
the protein and has been stabilized by strong hydrogen bond interaction in the binding pocket.
Figure 2. Putative binding pose of compound 3a in AmpC-β-lactamase (left panel). Docking
of compound 3a in AmpC-β-lactamase, where hydrogen bonding interactions are shown in
dotted lines (right panel).
12

Figure 3. Putative binding pose of compound 3f in AmpC-β-lactamase (left panel). Docking
of compound 3f in AmpC-β-lactamase, where hydrogen bonding interactions are shown in
dotted lines (right panel).
Figure 4. Putative binding pose of compound 3s in AmpC-β-lactamase (left panel). Docking
of compound 3s in AmpC-β-lactamase, where hydrogen bonding interactions are shown in
dotted lines (right panel).
Checkpoint kinase 1 (CHK1) is a protein kinase from the CAMKL kinase family, is a key
element in the DNA damage response pathway and plays a crucial role in the S-G2-phase
checkpoint.18 Each phase of the cell cycle is regulated by several checkpoint mechanisms,
such as DNA damage or spindle assembly checkpoints. First-line therapies for cancer include
radiation and chemotherapy and are designed to induce cell death by damaging the DNA
material of cancerous cells. Because cancer cells utilize the checkpoints to facilitate repair of
their DNA, the inhibition of CHK1 represents an attractive strategy for sensitizing cancer
13

cells to cytotoxic chemotherapeutics. To this end, all compounds were docked into the
relative binding site of CHK1 crystal structures and the optimal conformations of these
compounds were determined. To investigate the potential binding mode of inhibitors, all the
compounds were subjected to molecular docking using the AutoDock 1.5.4 program. As a
reference to other compounds, pyrazolo[1,5-a]pyrimidine was used as a model drug to show
the binding modes of CHK1 inhibitors. From the docking scores, the free energy of binding
(FEB) of all compounds were calculated (Table 5). The overlaid pose of bound and docked
pyrazolo[1,5-a]pyrimidine ligand in the active site of CHK1 is shown in Figure 5. The results
of which revealed that compound 3g as the most active with a calculated binding energy of -
8.99 kcal/mol and the least binding energy was exhibited by compound 3p with a binding
energy of –6.71 kcal/mol (Figure 6). The binding interactions of other potent compounds
exhibiting comparable IC50 and FEB values against the reference drugs such as 3a, 3e, 3f,
3k, 3l, 3m, 3n, 3q, 3s, 3t and 3u were shown in the supporting information.
Figure 5. Overlaid pose of bound and docked co-crystal (left panel). Hydrogen bonding
interactions of reference drug, pyrazolo[1,5-a]pyrimidine with CHK1 receptor.
To elucidate the interaction mechanism, the most potent inhibitors among the dataset, were
selected for more detailed analysis. The ligand-enzyme interaction analysis shows that
Leu137, Glu91, Cys87, Asp148, Ser88, Gly90 and Tyr86 are the important residues present
in the active site. As shown in figure 5, pyrazolo[1,5-a]pyrimidine binds to the kinase
through key H-bond interactions: (i) between the -NH of cyclohexyl ring and the -COO of
Glu91 (−O···HN, 1.90 Å, 160.9°); (ii) between the backbone carbonyl oxygen of Cys87 and
the amino group of pyrimidine ring (−O···HN, 2.46 Å, 129.8°; -O···HN, 3.47 Å, 150.2°); (iii)
14

between the nitrogen atom of pyrazole ring and the -NH group of Cys87 residue (−N···HN,
2.37 Å, 157.6°); (iv) the interaction between the nitrogen atom of another pyrazole ring and
the side chain of Asp148 is bridged by a structural molecule W502; (v) pyrazolo[1,5-
a]pyrimidine ring forms arene-cation interaction with Leu137 which further enhances the
binding activity. The docking results were well in consistent with the in vitro CHK1
inhibitory biological activity (IC50). The specific cleft in which the compounds bind in the
active site contains polar residues (Glu85, Tyr86, Cys87, Ser88, Gly89, Gly90, Glu91,
Ser147 and Asp148) and non-polar residues (Ala36, Leu84 and Val23). The compounds 3a,
3f, 3g, 3k, 3l, 3m, 3n, 3q and 3u are well placed inside the active site and demonstrate the
following interactions. (i) The oxygen atom of spirooxindole (figure 7-22) forms H-bond
with –NH group of Cys87 (-O…HN, 2.15 Å). (ii) The –NH group of indole ring forms H-
bond with the backbone –CO of Glu85 (-O...HN, 1.95Å). It is remarkable to note that the
large spirooxindole moiety has sufficient room to get incorporated into the hydrophobic
pocket composed by Ala36, Leu84 and Val23. The docking score was improved, when the R
substituent was hydrogen, methyl, ethyl, propyl and butyl (3a, 3k, 3l, 3m and 3n).
Interestingly, the CHK1 inhibitory activity also improved in the same way. The similar
binding mode and docking score as well as CHK1 inhibitory activity was also observed in
compounds 3f, 3g, 3q and 3u. The pentyl (3o) and hexyl (3p) groups were not able to
accommodate into the active site, hence the docking score and CHK1 inhibitory activity also
reduced. Ligands with bulky substituent like benzyl (3s) and 2-fluorobenzyl (3t) showed
different binding mode on the same active site. The oxygen atom of indole ring forms H-bond
with –NH group of Glu84 (-O…HN, 2.76 Å). The –NH group of indole ring forms H-bond
with the backbone –CO of Glu85 (-O...HN, 2.15 Å). Based on the observations, we conclude
that the network of H-bonds formed between CHK1 and the compounds are crucial for
molecular recognition.
15

Figure 6. Docking pose of most active compound 3g (left panel). Docking pose of least
active compound 3p (right panel).
In summary, we have developed a protocol for the gold(III)-catalyzed bis-condensation
reaction between isatins with 4-hydroxycoumarins. The reaction proceeds under microwave
conditions, extends the substrate class to N-substituted isatins and bis-isatins to provide the
corresponding spirooxindole products with up to 95% yield (22 examples). The developed
methodology shows promising advantages such as high yielding, short reaction time and no
requirement of tedious purification techniques. Antimicrobial screening of spirooxindole
derivatives against a wide panel of antimicrobial strains showed that three compounds (3a, 3f
and 3s) exhibited significant MIC values compared to the reference drugs. Docking with
AmpC-β-lactamase receptor revealed that compound 3a is the most active by exhibiting a
minimum free energy of binding (-117.819 kcal/moL). All compounds were also evaluated
for their cytotoxic potency against COLO320 cancer cells. The results of which indicated that
compound 3g exhibited significant inhibitory potency with IC50 values of 50.0 μM. Molecular
docking with CHK1 receptor also supported the biological results by showing compound 3g
as the most active with a free energy of binding −8.99 kcal/mol. Our future studies will be
directed towards the utilization of these structurally interesting heterocycles in the synthesis
of improved chemical entities (ICE) and new drug discovery research.
Acknowledgements
The work has been financially supported by a fast track grant for young scientists (Grant no:
SERB/F/5666/2015-16) from Science and Engineering Research Board (SERB), India. C. P.
is grateful to Dr. Vijayamohanan K. Pillai, Director, CSIR-CECRI for allowing to publish
16

this work and Dr. D. Jeyakumar, Head, Functional Materials Division, CSIR-CECRI for
providing laboratory and analytical facilities.
Supplementary data
Experimental procedure for synthesis, antimicrobial evaluation, cytotoxicity and molecular
1
docking studies are given in the supporting information. IR, H NMR, 13C NMR, mass
spectra and molecular docking poses were also provided in the same.
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