Coumarin tethered cyclic imides as efficacious glucose uptake agents and investigation of hit candidate to probe its binding mechanism with human serum albumin

Article abstract of DOI:10.1016/j.bioorg.2019.103212

A series of novel coumarin-cyclic imide conjugates (1a–1j) were designed and synthesized to evaluate their glucose uptake activity by insulin resistant liver hepatocyte carcinoma (HepG2) cells through 2-NBDG uptake assay. Compounds (1a–1j) were characterised using various analytical methods such as ¹H NMR, ¹³C NMR, IR, GC–MS, elemental and single-crystal X-ray diffraction techniques. Compounds (1a–1j) exhibited 85.21 – 65.80% of glucose uptake and showed low level of cytotoxicity towards human embryonic kidney cells (HEK-293) indicating good selectivity and safety profile. Compound 1f was identified as a hit candidate exhibiting 85.21% of glucose uptake which was comparable with standard antidiabetic drug Metformin (93.25% glucose uptake). Solution stability study under physiological pH conditions ≈ (3.4 – 8.7), indicates that compound 1f is sufficiently stable at varied pH conditions and thereby compatible with bio-physiological environments. Interaction of 1f with human serum albumin (HSA) were also studied which quantifies that compound 1f binds with HSA efficiently through facile binding reaction in solution. Fluorescence, UV–vis spectrophotometry and molecular modeling methodologies were employed for studying the interaction mechanism of compound 1f with protein.

Full text of DOI:10.1016/j.bioorg.2019.103212

Journal Pre-proofs
Coumarin Tethered Cyclic Imides as Efficacious Glucose Uptake Agents and
Investigation of Hit candidate to probe its binding mechanism with Human Se-
rum Albumin
Dinesh S. Reddy, Manasa Kongot, Vishal Singh, Neha Maurya, Rajan Patel,
Nitin Kumar Singhal, Fernando Avecilla, Amit Kumar
PII:
S0045-2068(19)30916-2
DOI:
https://doi.org/10.1016/j.bioorg.2019.103212
YBIOO 103212
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
7 June 2019
6 August 2019
20 August 2019
Please cite this article as: D.S. Reddy, M. Kongot, V. Singh, N. Maurya, R. Patel, N. Kumar Singhal, F. Avecilla,
A. Kumar, Coumarin Tethered Cyclic Imides as Efficacious Glucose Uptake Agents and Investigation of Hit
candidate to probe its binding mechanism with Human Serum Albumin, Bioorganic Chemistry (2019), doi: https://
doi.org/10.1016/j.bioorg.2019.103212
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Coumarin Tethered Cyclic Imides as Efficacious Glucose Uptake Agents and Investigation
of Hit candidate to probe its binding mechanism with Human Serum Albumin
Dinesh S. Reddya, Manasa Kongota, Vishal Singhb, Neha Mauryac, Rajan Patelc, Nitin Kumar
Singhalb, Fernando Avecillad, Amit Kumar a,*
aCentre for Nano and Material Sciences, Jain University, Jain Global Campus, Jakkasandra
Post,
Bangalore 562112, India
bNational Agri Food Biotechnology Institute, Mohali 140306, India
cBiophysical Chemistry Laboratory, Centre for Interdisciplinary Research in Basic Sciences,
Jamia Millia Islamia (A Central University), New Delhi 110025, India
dGrupo Xenomar, Centro de Investigacións Científicas Avanzadas (CICA), Departamento de
Química, Facultade de Ciencias, Universidade da Coruña, Campus de A Coruña, 15071 A
Coruña, Spain
*To whom correspondence should be addressed
Dr. Amit Kumar (amit.kumar@jainuniversity.ac.in) Phone: +91-80-27577251, Fax: +91-80-2757 7211
ABSTRACT
A series of novel coumarin-cyclic imide conjugates (1a–1j) were designed and synthesized to
evaluate their glucose uptake activity by insulin resistant liver hepatocyte carcinoma (HepG2)
cells through 2-NBDG uptake assay. Compounds (1a–1j) were characterised using various
analytical methods such as 1H-NMR, 13C-NMR, IR, GC-MS, elemental and single-crystal X-ray
diffraction techniques. Compounds (1a–1j) exhibited 85.21 – 65.80% of glucose uptake and
showed low level of cytotoxicity towards human embryonic kidney cells (HEK-293) indicating
good selectivity and safety profile. Compound 1f was identified as a hit candidate exhibiting
85.21% of glucose uptake which was comparable with standard antidiabetic drug Metformin
(93.25% glucose uptake). Solution stability study under physiological pH conditions ≈ (3.4 –
8.7), indicates that compound 1f is sufficiently stable at varied pH conditions and thereby
compatible with bio-physiological environments. Interaction of 1f with human serum albumin
(HSA) were also studied which quantifies that compound 1f binds with HSA efficiently through
facile binding reaction in solution. Fluorescence, UV–vis spectrophotometry and molecular
modeling methodologies were employed for studying the interaction mechanism of compound 1f
with protein.
1

Keywords: Coumarins; Cyclic imides; Glucose uptake; Human serum albumin (HSA); Solution
stability studies.
1. Introduction
Diabetes affects a large section of the world population with an estimation of 1.6 million deaths
worldwide in 2015 alone. As per World Health Organization (WHO), the number of diabetic
patients have increased from 0.18 billion in 1980 to 0.42 billion in 2014, indicating its alarming
prevalence in the coming decades [1]. Among the different categories of diabetes, type-2
diabetes affects the larger population, accounting for 90% of all diabetic cases. This condition
involves the loss of insulin sensitivity by the body cells, thereby causing hyperglycemia [2].
Today most of the antidiabetic drugs generally include insulin secreting agents such as
Sulfonylureas, Glinides, Thiazolidinediones and Biguanides which perform via the mechanism of
α-glucosidase inhibition [3]. Recently, dipeptidyl peptidase IV (DPP-IV) inhibitors which work
by inhibiting incretin catabolism and thus increasing insulin secretion, have also emerged as
promising antidiabetic agents to treat type-2 diabetes [4]. To name a few, Sitagliptin, Saxagliptin,
Vidagliptin, Linagliptin, Omarigliptin and Alogliptin are among the different classes of DPP-IV
inhibitors [3-5]. Although DPP-IV inhibitors are effective in diabetic treatment, majority of such
drugs have certain side effects such as weight gain, edema, fractures, lactic acidosis,
gastrointestinal intolerance, hypoglycaemia and cardiovascular diseases [3, 6-8]. Thereby, it is a
challenging task for the researchers to develop an effective and safe anti-diabetic drugs in order
to minimize the diabetic complications from the patient’s body.
Coumarin scaffolds are well established structural motifs found mainly in plants and in some
microorganisms [9]. They have found to exhibit a broad range of bioactivities and thereby, have
been evolved as favourable lead targets for medicinal uses [9, 10]. Further, coumarin has a
unique character which allows its derivatives to interact easily with various enzymes through
weak bond interactions and thereby have greater scope as medicinal drugs [11]. Among the
various biological activities, antidiabetic activity of coumarin is well known from ancient times,
e.g. Marmesin, a coumarin derivative has been used against diabetes in Indian Ayurvedic
medicine [12-14]. It is known that coumarins (Figure 1) exhibit their antidiabetic effects by
various mechanisms [15] such as antioxidative [15b], antiinflammatory action [15c], modulation
of pancreatic function [15d], improvement of abnormal insulin signalling [15e], α-glucosidase
inhibition [15a, 15f], and PTP1B inhibition [15a]. However, despite its unique and widespread
2

pharmacological properties, extensive efforts were not made in designing coumarin-based
antidiabetic drugs. This prompted researchers to exploit antidiabetic potential of this class of
compounds and thus synthetic coumarin analogs have been recently developed and reported to
display significant antidiabetic effects [16-18].
Figure 1. Marmesin, an Indian Ayurvedic medicine used for diabetic treatment and structures of
some coumarin analogs exhibiting anti-diabetic affects by various mechanisms.
Cyclic imides are another important class of compounds with unique pharmacokineticproperties
[19] and are known to possess diverse medicinal activities such as antibacterial [20], antifungal
[21], antitumor [22], antitubercular [23] and analgesic activities [20]. Recently, 4,4-
dimethylpiperidine-2,6-dione derived compounds were explored as effective antihypertensive
agents revealing the therapeutic potential of cyclic imide scaffold [24]. In particular, cyclic
imides can also be found in several antidiabetic drugs such as Trelagliptin [25], Alogliptin [26]
and Linagliptin [27] which are known for treating type-2 diabetes. Another favourable cyclic
imide inhibitor Fidarestat [28] is under clinical trials and has shown to inhibit the functional
developments of diabetic neuropathy[29] and also halt the growth in sorbitol pathway flux in
diabetic patients [30]. In some case, compound having cyclic imide ring have shown distinct
antidiabetic activity than the standard drug Glibenclamide [31]. Structures of some antidiabetic
3

drugs (Trelagliptin, Alogliptin, Linagliptin) and agent (Fidarestat) with cyclic imide ring are
represented in Figure 2.
Figure 2. Structures of some antidiabetic drugs and agents. Most of the compounds contain
cyclic imide ring (drawn in blue).
Considering all the aforementioned pharmacological significance of cyclic imides particularly in
diabetic treatment enthralled our curiosity in exploring these versatile motifs as conjugates with
bio-active coumarin analogs, so that the synergetic effect of such combination may help in
enhancing the overall activity of the compound. Apart from designing the desired compounds, it
was also essential to see that the compounds fall well within the Lipinski rule of five (RO5) [32].
Reports have suggested that candidate drugs that conform to the RO5 tend to have lower attrition
rates during clinical trials and hence have an increased chance of reaching the market [32, 33].
Hence, keeping these factors in view the architecture of the compounds was designed (Figure 3).
In the present study, we have designed, synthesized and characterized ten structural analogues of
coumarin-cyclic imide conjugates. All the compounds were investigated for in-vitro glucose
uptake activity potency and their safety profile on healthy cells. The solution state stability
behavior over a wide range of pH conditions were studied to predict their nature under
4

physiological environment. The most active compound was further studied for its interaction
with human serum albumin protein (HSA).
Figure 3. Designed structural analogs which follow the Lipinski rule of five.
2. Results and discussion
2.1. Chemistry
In order to synthesize the requisite coumarin-cyclic imide conjugates (1a–1j), SN2 substitution
reaction was employed (Scheme 1). The starting material, 4-bromomethyl coumarin derivatives
[34] (a–j) were synthesized through Pechmann cyclization of phenols (iii) with ethyl 4-
bromoacetoacetate (ii) using H2SO4 as cyclizing agent. The obtained 4-bromomethyl coumarin
derivatives (a–j) on treating with 4,4-dimethylpiperidine-2,6-dione (iv) in presence of anhydrous
K2CO3 afforded coumarin cyclic-imide derivatives (1a-1j) with 73–83% yields. The structures of
the new compounds were confirmed by 1H NMR, 13C NMR, mass, IR and elemental analysis.
Further, through single crystal X-ray diffraction, structures of compounds 1a-1f, 1h and 1i were
elucidated.
In the case of compound 1a (R = 6-CH3), in the 1H-NMR spectrum, a sharp singlet was observed
in the up-field region at δ 1.14 ppm which corresponds to the di-methyl protons of the
glutarimide ring. The two methylene protons at ortho position of glutarimide ring were observed
as singlet at δ 2.62 ppm. The methyl proton at C-6 position of the coumarin was observed as
singlet at δ 2.39 ppm. The methylene proton (–CH2) linking the coumarin with glutarimide ring
was observed as singlet at δ 5.10 ppm. Two singlets were observed at δ 5.90 ppm and δ 7.42 ppm
corresponding to the C2-H and C5-H of the coumarin moiety respectively. The remaining
aromatic protons of the coumarin unit were observed in the range δ 7.20-7.34 ppm. 13C NMR
data also support the structure of 1a, wherein the two carbonyl carbons of the glutarimide ring
were observed at δ 166.75 ppm and the carbonyl carbon of the coumarin moiety was observed at
δ 155.85 ppm. The two methyl carbons of the glutarimide ring were observed at δ 23.12 and δ
5

23.22 ppm respectively. The methylene carbon of the glutarimide ring was observed at δ 34.21
ppm, whereas the methylene carbon connecting coumarin to glutarimide ring was observed at δ
41.38 ppm. The carbon at C2 position of the coumarin ring was observed at δ 106.82 ppm. The
remaining aromatic carbons showed signals in the range δ 112.25-146.91 ppm and are in good
agreement with the predicted values. The molecular ion peak at 313 [M]+ also confirmed the
structure of 1a. Spectral data of all other compounds are also in good agreement with their
assigned structures (See Supporting Information page no. S2 - S11).
Scheme 1 Schematic representation to synthesize coumarin-cyclic imide derivatives (1a-1j) a,b,c
b
a% yield of the purified products; Log P: Calculated by http://www.molinspiration.com;
cpKa: Calculated by potentiometric titration method; N.D: Not determined.
2.2. X-ray crystal structure determination
Bruker Kappa Apex CCD diffractometer were used to collect 3-D X-ray crystal information for
compounds 1a-1f, 1h and 1i by using the - scan method. Reflections were measured using a
hemisphere of data, each covering 0.3º in  and collected from frames. A total of 53993 for 1a,
29522 for 1b, 37632 for 1c, 30537 for 1d, 34083 for 1e, 31606 for 1f, 35538 for 1h and 144652
6

for 1i, reflections measured were corrected for absorption by multi-scan methods and for Lorentz
and polarization effects by symmetry equivalent reflections. Of the total, 3197 for 1a, 2856 for
1b, 2970 for 1c, 2878 for 1d, 3042 for 1e, 2962 for 1f, 3008 for 1h and 12051 for 1i, independent
reflections surpassed the significance level (F/F) > 4.0. Upon data collection, a multi-
scan absorption correction (SADABS) [35] was induced in each and every scan, and the
structure was solved and refined by direct methods and full matrix least-squares on F2 data
respectively using SHELX program suite [36]. All hydrogen atoms were included in calculated
positions and refined through the riding mode for all structures, except for 1a, which was located
in a different Fourier map and left to refine freely. Refinements were carried by allocation for
thermal anisotropy of non-hydrogen atoms. ORTEP diagrams for compounds 1a-1f, 1h and 1i
are shown in Figure 4. Further details of the crystal structure refinement for the compounds are
given in Table 1. Selected bond distances and angles are given in Supporting Information (see
page no. S25).
Table 1 Crystal Data and Structure Refinement for compounds 1a, 1b, 1c, 1d, 1e, 1f, 1h and 1i.
1a
1b
1c
1d
C18 H19 N O4
313.34
100(2)
C18 H19 N O4
313.34
100(2)
C17 H16 Cl N O4 C17 H16 Cl N O4
Formula
Formula weight
T, K
333.76
333.76
100(2)
100(2)
Wavelength, Å
Crystal system
Space group
a/Å
b/Å
0.71073
Monoclinic
C2/c
18.7857(13)
11.4459(9)
14.6202(11)
90
0.71073
Monoclinic
P21/n
15.5791(7)
5.7144(3)
19.1011(9)
90
0.71073
Monoclinic
C2/c
18.6800(6)
11.3537(3)
14.6679(4)
90
0.71073
Monoclinic
P21/c
7.3640(2)
15.5756(6)
13.4200(5)
90
c/Å
/º
β/º
γ/º
V/Å3
Z
94.156(4)
90
3135.4(4)
8
111.790(2)
90
1578.98(13)
4
94.0540(10)
90
3103.09(15)
8
90.678(2)
90
1539.15(9)
4
F000
1328
664
1392
696
Dcalc/g cm-3
/mm-1
/ (º)
Rint
1.328
0.094
1.318
0.093
1.429
0.266
1.440
0.268
2.17 to 27.18
0.0293
2.82 to 26.40
0.0333
2.10 to 26.42
0.0257
2.00 to 26.41
0.0395
7

Crystal size/ mm3
0.50 x 0.48 x
0.45
0.34 x 0.33 x
0.19
1.055
0.49 x 0.49 x
0.39
1.033
0.48 x 0.41 x
0.33
1.036
Goodness-of-fit on F2 1.050
R1[I>2(I)] a
0.0337
0.0345
0.0285
0.0367
wR2 (all data) b
0.0943
0.0935
0.0781
0.1011
Largest differences
peak and hole (eÅ-3)
0.332 and
-0.212
0.278 and
-0.215
0.328 and
-0.258
0.631 and
-0.280
1e
C19 H21 N O4
Formula weight 327.37t
1f
1h
1i
Formula
C19 H21 N O4
327.37
100(2)
C17 H16 Br N O4 C21 H19 N O4
378.22
100(2)
349.37
100(2)
T, K
100(2)
Wavelength, Å
Crystal system
Space group
a/Å
b/Å
0.71073
Triclinic
P-1
0.71073
Monoclinic
P21/n
17.0983(17)
5.6965(6)
17.4465(17)
90
0.71073
Monoclinic
C2/c
18.9597(6)
11.3867(4)
14.6964(5)
90
0.71073
Triclinic
P-1
7.1642(3)
9.2495(3)
12.7646(5)
106.592(2)
100.370(2)
91.893(2)
794.18(5)
2
12.4115(7)
14.1571(8)
19.8887(11)
85.607(3)
82.596(3)
81.261(3)
3419.5(3)
8
c/Å
/º
β/º
γ/º
V/Å3
110.092(5)
90
1595.9(3)
4
94.5580(10)
90
3162.75(18)
8
Z
F000
348
696
1536
1472
Dcalc/g cm-3
/mm-1
/ (º)
Rint
Crystal size/
mm3
1.369
0.096
1.363
0.096
1.589
2.619
1.357
0.094
2.90 to 26.40
0.0213
0.49 x 0.45 x
0.42
2.06 to 26.41
0.0226
0.47 x 0.45 x
0.44
2.16 to 26.41
0.0260
0.46 x 0.41 x
0.33
1.03 to 26.52
0.0415
0.46 x 0.41 x
0.39
Goodness-of-fit 1.049
on F2
1.083
1.077
1.060
R1[I>2(I)] a
0.0334
0.0336
0.1040
0.303 and
-0.239
0.0209
0.0504
0.389 and
-0.440
0.0366
0.1012
0.243 and
-0.273
wR2 (all data) b
Largest
0.0881
0.313 and
-0.218
difference peak
and hole (eÅ-3)
8

1a
1e
1b
1f
1c
1d
1i
1h
Figure 4. ORTEP diagram for compounds 1a, 1b, 1c, 1d, 1e, 1f, 1h and 1i. All non-hydrogen
atoms are represented by their 50% probability ellipsoids. Hydrogen atoms have been omitted for
clarity purpose.
2.3. Antidiabetic activity studies
The coumarin-cyclic imide conjugates (1a-1j) were initially tested for their glucose uptake
activity at 50 nM, 100 nM, 200 nM and 500 nM concentrations (Table 2). Different doses of
compounds were given to the cells along with insulin resistant media for 24 h prior to the 2-
NBDG uptake. Insulin resistant HepG2 cells treated with the test compounds showed increase in
2-NBDG (fluorescent glucose) uptake and the results are presented in Figure 5. Compound 1f
and 1b exhibited distinct activity with 85.21% and 80.89% of glucose uptake at 50 nM
concentration respectively, which is comparable with standard antidiabetic drug Metformin
(93.25 % of glucose uptake). The second line of activity were observed by compounds 1e, 1h
and 1a with 74.44%, 73.64% and 73.14 % of glucose uptake respectively at 50 nM
concentration. Compounds 1c, 1d, 1g, 1i and 1j showed moderate activity in the range of 65.80 -
72.08 % at 50 nM concentration. It was noted that even at higher doses (100 to 500 nM), there
was no significant change in the NBDG uptake (Table 2); which suggest that the compounds
were active at minimal dose concentration. From structural point of view, it was noted that 7,8
di-CH3 substituted coumarin (1f) was found to be highly active with exceptional glucose uptake
of 85.21%. The next line of activity was observed by –CH3 substituent at C-7 position (1b) with
80.89% followed by 5,7 di-CH3 substituent (1e) with 74.44% of glucose uptake at 50 nM
concentration. The -Br substituent at C-6 position (1h) and
–CH3 substituent at C-6 position
9

(1a) obtained comparable activity with 73.64 and 73.14% of glucose uptake respectively. The
compound with –Cl substituent at C-6 (1c) and C-7 (1d) position showed 70.17% and 68.21% of
glucose uptake respectively. The -OCH3 substituted at C-7 position (1g) of the coumarin ring
showed 72.08% of glucose uptake. Whereas 5,6 Benzo (1i) and 7,8 Benzo (1j) substituted
coumarin derivatives showed 65.80% and 71.00% of glucose uptake respectively. Overall it was
observed that –CH3 substituted compounds were found to be more favourable candidates for
enhancing the activity.
Table 2 Glucose uptake by insulin resistant liver hepatocyte carcinoma (HepG2) cells.
Compound % of glucose uptake by HepG2 cells at 50-500 nM concentration
50 nM
100 nM
79.26%
85.31%
84.28%
74.98%
79.05%
200 nM
81.56%
88.38%
85.41%
87.36%
80.00%
500 nM
83.17%
90.86%
90.62%
88.38%
81.57%
73.14%
80.89%
70.17%
68.21%
74.44%
1a
1b
1c
1d
1e
85.21%
72.08%
87.68%
85.56%
89.67%
86.45%
91.76%
87.47%
1f
1g
73.64%
65.80%
84.69%
67.37%
78.11%
85.37%
70.58%
83.96%
87.04%
78.57%
87.16%
1h
1i
71.00%
93.25%
1j
N.D
N.D
N.D
Metformin
N.D – Not Done
10

Figure 5. Antidiabetic activity for compounds (1a-1j), assessed by 2-NBDG uptake assay which
gave the % of glucose uptake by insulin resistant HepG2 cells at 50, 100, 200 and 500 nM
concentration of compounds.
2.4. Correlation between X-ray crystal structure and antidiabetic activity
A notable observation was illustrated from X-ray crystal studies that compounds 1f and 1b which
exhibited excellent glucose uptake of 85.21 and 80.89% respectively have shown C=O-π
interactions (Figure 6). The X-ray crystal packing diagram for 1f and 1b is given in supporting
information (See page no. S23, Figure S20). To visualize these aspects, atropoisomers images
for 1f and 1b have been depicted in supporting information (See page no. S24, Figure S21).
Compounds 1a, 1c, 1d, 1e, 1h and 1i which demonstrated moderate glucose uptake have shown
π-π interactions between coumarin rings (See page no. S25, Figure S22 in supporting
information). The atropoisomers images for compounds 1a, 1c, 1d, 1e, 1h and 1i are depicted in
supporting information (See page no. S25, Figure S23). The enhanced activities exhibited by 1f
and 1b can be related with the dihedral angles between the plane that cuts in half the cyclic
imides and the plane defined for the coumarin ring atoms by rotation around the C8-C9 link.
This theoretical evidence clearly indicates that there is structural correlation between the
structural angle (dihedral angle) and antidiabetic activity of the compound. The obtained results
may serve in future to predict the bio-activity of the compounds through single crystal X-ray
studies.
11

Figure 6. Compounds having C=O–π interactions exhibited significant antidiabetic activity.
2.5. Toxicity study against healthy cells
Compounds were evaluated against Human Embryonic Kidney cells (HEK293) to check the
safety profile. Their toxicity levels were evaluated through MTT assay. The results (Figure 7)
indicated that none of the compounds showed any significant toxicity against HEK293 cells at
50, 100, 500 and 1000 nM concentration, suggesting great potential for their in-vivo use as
antidiabetic agents. It was found that, when the cells were treated with compounds 1a-1h and 1j,
the % survival of HEK293 cells were well above 85% at 50 nM, above 80% at 500 nM and
above 65% at 1000 nM concentration. This clearly indicates that the compounds were well
within the toxicity limits and thereby exhibited good safety profile. An exception was compound
1i which showed moderate cytotoxicity with 84, 67 and 59% survival of HEK293 cells at 50, 500
and 1000 nM concentration respectively. It is noteworthy to mention that compound 1f and 1b
which showed significant glucose uptake also revealed remarkable safety profile with 94 and
68% survival of HEK293 cells at 1000 nM concentration respectively, signifying that they have
a high prospective for in-vivo use as antidiabetic agents.
12

Figure 7. Cytotoxicity study of compounds (1a-1j) at 50 to 1000 nM concentration against
Human
Embryonic Kidney cells (HEK293) by MTT assay.
Further, to evaluate the drug like properties of the compounds, physicochemical studies were
performed (http://www.molinspiration.com) to calculate important pharmacological properties
like log P, polar surface area, number of hydrogen bond donors and acceptors. All the
compounds were found to follow Lipinski rule of five (RO5) [32] according to which, molecular
weights were below 500 Da, lipophilicity expressed as a logP was less than 5 (Scheme 1); the
number of hydrogen bond donors as well as acceptors were also less than 5. The results clearly
indicates that none of the compounds violate the rules and they fall well within the range as
stated by the RO5 to qualify as a drug candidate [32, 33].
2.6. Solution stability studies
The physical and chemical properties of bio-active compounds such as hydrophobicity and tissue
penetrability are significantly dependent on their pKa value. To determine the pKa of the newly
synthesized coumarin-cyclic imide conjugates, potentiometric titration method was employed.
Further, UV-Visible spectroscopic technique was employed at varied pH ≈ (3.4 – 8.7) conditions
[37] to check the stability of active compounds. Initially, pH of active compounds 1a, 1b, 1e, 1f,
1g, 1h and 1j (10-4 M each) were in the range 6.9 to 7.6 and this pH was steadily brought down
to acidic (pH ~3) by titration with HCl (25 mM). After attaining pH ~3, the acidic solution was
13

further titrated with KOH (25 mM) till pH reached basic (pH ~ 9). Thereby, through this process,
pH variation trend was prudently studied and a graphical plot of volume of KOH added vs pH
was drawn. From the obtained equivalence point, pKa values were calculated (See Figure S1 to
S7 in Supporting Information). The pKa values of the tested compounds were found to be in
between 4.3 to 6.4 (Scheme 1), which suggest that the newly synthesized compounds have
capability to cross the biological membranes [38].
In order to evaluate the solution stability of active compounds (1a, 1b, 1e, 1f, 1g, 1h and
1j), UV-Visible absorption analysis was done in the pH range of 3.4 to 8.7. For example, in case
of compound 1f initial pH = 7, was brought to acidic pH by slowly titrating with HCl (25 mM)
which resulted in the increased intensity of the absorption band at 330 nm. The addition of HCl
was continued until pH reached 3.4 (pH = 7.0 to 3.4, given in Figure 8 (A)). The solution
reaction was found to be reversed from pH 3.4 by slowly titrating with KOH (25 mM) (pH = 3.4
to 8.3, given in Figure 8 (B)). From Figure 8 (B), it could be seen that absorbance bands
retained their peak positions during the experiments and no additional bands were detected
which clarifies that compound 1f is stable at varied pH conditions.
Similarly, for other active compounds (1a, 1b, 1e, 1g, 1h and 1j) stability studies were
established in the pH range 3.5 – 8.7 (See Figure S8 to S19 in Supporting Information).
(B)
(A)
Figure 8. UV-Visible titration study of compound 1f (10–4 M) with HCl (25 mM) (A) and KOH
(25 mM) (B).
2.7. HSA interaction study
Assessment of the bioavailability of a prospective drug is of utmost importance and in this
direction; plasma protein binding study plays a highly decisive role. It is one of the crucial steps
14

involved before screening the potential therapeutic agent in-vivo [39]. One such important
plasma protein is human serum albumin (HSA) which is not only abundantly present in plasma
(60% of total plasma protein), but also is a transporter of metabolic compounds and drug
molecules in body. It can act as a reserve for drug molecules and allow for passive targeting due
to its preferential uptake in diseased tissues and cells [40]. The study of human serum albumin
binding will help for further prediction of the pharmacokinetics of a prospective drugs.
Interaction of the most active compound 1f with HSA was studied using a combination of
fluorescence, UV-Visible spectroscopic techniques and in-silico modeling study.
2.7.1. Steady-state fluorescence quenching studies
HSA has intrinsic fluorophore residues such as tryptophan and tyrosine which mainly emits at
around 340 nm when excited at about 280 nm [41]. This emission is sensitive to the environment
around the protein and hence can provide information about the binding of compound with HSA.
The emission spectra of HSA (5μM) at various concentrations of 1f (0.83–8.19 μM) in pH 7.4
phosphate buffer were recorded at three different temperatures, viz. 298 K, 303 K and 308 K. At
all the temperatures, the emission maxima which were observed at 337 nm got steadily quenched
upon successive addition of 1f (See Figure 9A).
(A)
(B)
Figure 9. (A) Intrinsinc fluorescence quenching study of HSA at 298 K with the titration of
compound 1f at ten different concentrations; (B) Stern-Volmer plot obtained from the quenching
study at three temperatures from which quenching constants were calculated
To evaluate the type of quenching of fluorophore HSA by the quencher 1f, Stern-Volmer plot
(F0/F vs [Q]) was plotted using equation 1 [42].
15

푭
ퟎ
= ퟏ + 풌풒흉ퟎ[푸] = ퟏ + 푲푺푽[푸] (1)
푭
where F0 and F are the emission intensities of pure HSA and HSA added with different
concentrations of 1f respectively. kq is the rate constant of the biomolecular quenching reaction
and Ksv, the Stern-Volmer quenching constant. 흉ퟎ is the average lifetime of molecules of HSA
protein (≈ 5x10-9 s) [41] and [Q] is the concentration of quencher compound 1f. Figure 9B gives
the Stern-Volmer plot at different temperatures. All the SV plots gave almost linear graphs with
R2 in the range of 0.998-0.999. The values of KSV and kq (Table 3) show that the quenching
constants increase with increasing temperature thereby suggesting dynamic or collisional
quenching [42].
(A)
(B)
Figure 10. (A) Double log graph to calculate the binding sites and binding constants at different
temperatures; (B) van’t Hoff’s plot to find out thermodynamic parameters ΔH, ΔS and ΔG.
To find the number of binding site(s) in the protein for the compound 1f, a double log graph of
log [(F0–F)/F] vs log [Q] (Figure 10A) was drawn and using equation 2, the value of binding
sites (n) and binding constant (Ka) was calculated at different temperatures.
ퟎ
풍풐품 (푭 −푭) = 풍풐품푲풂 + 풏풍풐품[푸]
(2)
푭
The values obtained are given in table 4 which suggest that there is one binding site in the
protein for 1f and that the binding between compound and HSA is strong.
The thermodynamics involved in the binding reaction of 1f and HSA was evaluated by drawing
the van’t Hoff’s plot (Figure 10B) from which the thermodynamic parameters, change in
enthalpy (ΔH) and change in entropy (ΔS) were calculated using the van’t Hoff’s equation 3.
16

휟푺
푹
풍풏푲풂 = − 휟푹푯푻
+
(3)
The Gibb’s free energy change (ΔG) at different temperatures were calculated using the Gibb’s
free energy relation (Equation 4)
휟푮 = 휟푯 − 푻휟푺
(4)
The obtained thermodynamic parameters suggest that the interaction between 1f and HSA is
mainly governed through hydrophobic forces and that the compound 1f is mainly surrounded by
hydrophobic amino acid residues. As it can be seen from table 4, the value of ΔG was observed
to be negative at all the studied temperatures which showed the spontaneity in the interaction
process [43].
Table 3. Stern–Volmer quenching constants (Ksv) and biomolecular quenching rate constant (kq)
of HSA-1f system at three temperatures
Temp (K)
Ksv(L mol-1)
8.72 x 104
9.13 x 104
11.66 x 104
kq (L mol-1 s-1)
1.74 x 1013
1.83 x 1013
2.33 x 1013
R2
SD
298
0.9990
0.9989
0.9988
0.0026
0.0028
0.0032
303
308
Table 4. The binding constants (Ka), binding site (n) and thermodynamic parameters of HSA and
1f interaction at three temperatures
Temp
∆H 0
∆S0
∆G0
Ka (L mol-1) n
R2
SD
(K)
(kJ M-1)
(J M-1 K-1) (kJ M-1)
298
303
308
3.23 x 103
5.13 x 103
7.59 x 103
0.72 0.9986
0.73 0.9949
0.77 0.9979
0.0501
0.0960
0.0635
-20.04
-21.47
285.59
65.06
-22.90
2.7.2. Synchronous and 3D fluorescence studies
Synchronous fluorescence technique can differentiate between the fluorescence profiles of amino
acid residues like tyrosine and tryptophan and gathers information on HSA vicinity and
environment [44]. Synchronous fluorescence was recorded between λem= 290 –500 nm and λexc=
200–350 nm in the wavelength interval of Δλ=15 nm and Δλ=60 nm with respect to tyrosine and
tryptophan amino acids respectively. A higher % of quenching of the tryptophan fluorescence
was observed (Figure 11A and 11B).
17

(A)
(B)
Figure 11. Synchronous fluorescence measurement of HSA fluorophore upon addition of
compound 1f which measures fluorescence given out by (A) tyrosine residue at Δλ=15 nm and
(B) tryptophan residue at Δλ=60 nm.
Three dimensional fluorescence spectroscopy (Figure 12) helps to simultaneously determine the
excitation and emission profile of protein and hence examines the quenching pattern in a more
reliable way [45]. The 3D spectra of HSA (5 µM) upon titration with an optimal concentration of
1f (8.20 µM) were analysed in the wavelength range of λem= 200–650 nm and λexc= 200–360 nm
at a scan rate of 600 nm/min and wavelength increment, Δλ=2 nm.
18

Figure 12. Three dimensional fluorescence spectra and the respective contour diagrams of (A)
pure HSA; (B) HSA titrated with 1f.
Figure 12A and 12B represent the 3D fluorescence spectra and the respective contour diagrams
of pure HSA and HSA-1f adduct respectively. Peak a is a representation of 1st order Rayleigh
scattering (λem = λex) and peak b is a representation of 2nd order Rayleigh scattering (λem = 2λex).
Peak 1 and peak 2 are due to the π→π* transitions of the aromatic amino acids, mainly, Trp
residues of protein and the typical n→π* transition due to the peptide backbone respectively [46].
Both peak 1 and peak 2 got quenched with the peak 1 getting extensively quenched. The
weakening intensity observed for peaks 1 and 2 upon addition of compound 1f (Table 5)
concludes that there occurs a gradual unfolding of HSA peptide chain which causes
conformational changes in HSA.
The synchronous and 3D fluorescence studies, in combination, signify that during the process of
interaction with HSA, compound 1f causes relatively greater quenching of tryptophan than other
19

amino acid residues by inducing gradual changes in the protein’s secondary conformational
structure.
Table 5. Peak 1, peak 2 changes observed from 3 dimensional fluorescence study.
Samples
Peak 1
λex/λem
Peak 2
Intensity
Intensity
Stokes shift λex/λem
Stokes shift
Δλ (nm)
102
(nm)
Δλ(nm)
(nm)
HSA
280/334
95
64
54
225/327
150
20
HSA+1f 280/333
53
225/337
112
2.7.3. UV-Visible and FRET study
The extent of binding between a drug molecule and a biomacromolecule like HSA can be further
investigated using UV–Visible absorption spectroscopy [47]. HSA exhibits absorption maxima at
around 280 nm [48] which is observed as an important factor in analysing the binding between
HSA and the quencher compound. The absorption spectra of HSA (5μM) upon titration with
various concentrations of compound 1f (0.83 – 8.19 μM) in pH 7.4 phosphate buffer at 298 K is
shown in Figure 13A. With increasing concentrations of 1f, the pattern of absorbance showed an
increasing trend. FRET phenomenon was checked to find out the distance of vicinity at which
the quencher acceptor compound 1f lies from the fluorophore donor HSA. Forster resonance
energy transfer (FRET) occurs when there is transfer of electromagnetic energy non-radiatively
from a fluorophore donor to a vicinal quencher acceptor molecule [49]. Figure 13B represents
the spectral overlap between the fluorescence emission of HSA and the absorption spectrum of
1f. Efficiency of energy transfer (E) between 1f and HSA can be found out using the Forster non-
radiative energy transfer theory [48] according to the equation 5.
ퟔ
푹
ퟔ
푬 = ퟏ − 푭
=
(5)
ퟎ
ퟎ
ퟔ
푭
푹 +풓
ퟎ
where r is the vicinity between binding sites of HSA and 1f and R0 is the Förster critical energy
transfer distance for 50% efficiency of energy transfer. R0 is calculated using equation 6.
푹ퟔퟎ = ퟖ. ퟕퟗ × ퟏퟎ−ퟐퟓ푲ퟐ푵−ퟒ휱푱
(6)
where K2 is the factor of spatial orientation for HSA=2/3, N is the average refractive index of the
medium=1.36, ϕ is the HSA fluorescence quantum yield=0.15 [50] and J is the integral of
20

overlap between the emission spectrum of HSA and absorption spectrum of 1f which can be
calculated using equation 7.
∞
ퟎ
ퟒ
푭(흀)흐(흀)흀 풅흀
∫
푱 =
(7)
∞
( )
푭 흀 풅흀
∫
ퟎ
where F(λ) is the corrected emission intensity of HSA over the wavelength range (λ+dλ) to λ and
ε(λ) is the molar extinction coefficient of 1f at λ [51]. The values of r, R0, J and E were found to
be 2.44 nm, 2.82 nm, 1.97x10-14cm3/L/mol and 0.71 respectively. It was found that the values
(0.5 R0 < r <1.5 R0) are in agreement with the conditions laid by FRET theory [51] which
indicates energy transfer probability between HSA to 1f to be high [52]. The UV-Visible and
FRET studies suggest effective collision between 1f and HSA and that the proximity of protein
from 1f is very close which induces favourable transfer of energy between 1f and HSA during
their interaction.
Figure 13. (A) Absorption spectra of HSA with the titration of increasing concentration of
compound 1f; (B) A representation of the overlap between the absorption spectrum of 1f and
fluorescense emission spectrum of HSA.
2.7.4. Competitive binding study with site markers
To evalaute the specific binding pocket of HSA for 1f binding, two known anti-inflammatory
drugs were used as site markers, viz. Indomethacin (IND) and Ibuprofen (IBU). It is known that
the hydrophobic binding pockets of HSA are situated at subdomains IIA and IIIA respectively.
These drugs specifically bind to site I and II located in the subdomains II and III of HSA
respectively[53]. For this study, the intrinsinc fluorescence of HSA, HSA-IND and HSA-IBU
systems were first measured. The effect of the addition of successive concentrations of
21

compound 1f on their fluorescence profile was studied. As seen in the Figure 14A, the addition
of 1f to HSA-IND system drastically decreases the fluorescence, with the intenisty much lower
than that observed in HSA-1f system without the IND marker which suggests that there was a
competition between 1f and IND and that site I marker Indomethacin interfered with the binding
of 1f to HSA. On the contrary, when 1f was added to the HSA-IBU system (Figure 14B), the
fluorescence reduced gradually and the intensity recorded were nearly same as that measured in
the HSA-1f system without the marker which indicates that site II marker did not interfere with
the binding of 1f to HSA. The results obtained summarize that site I marker Indomethacin
competes with 1f for binding with HSA thereby suggesting that the compound 1f binds to the
hydrophobic pocket IIA of HSA. The molecular docking/in-silico study also corroborates with
this conclusion.
(A)
(B)
Figure 14. Depiction of HSA binding site competitive study (A) For HSA-IND-1f system and
(B) For HSA-IBU-1f system.
2.7.5 In-silico study
Molecular docking study of compound 1f with HSA was done to investigate the intermolecular
interactions between 1f and various amino acid residues of HSA. HSA structure consists of three
large homologous domains which are numbered as I (residues 1–195), II (196–383) and III (384–
585). All the three domains comprise of subdomains A and B. The ligand binding hydrophobic
pockets of site I and II are respectively situated at sub domains IIA and IIIA [41]. 1f binds with
the subdomain IIA of HSA (Figure 15). Compound 1f was found to be adjacent to the
hydrophobic residues of HSA such as, TRY150, GLU153, SER192, LYS195, GLN196,
22

LYS199, TRP214, LEU219, ARG222, LEU238, HIS242, ARG257, LEU260, ALA261, ILE264,
SER287, HIS288, ILE290, ALA291 and GLU292 (Figure 15). Compound 1f got accommodated
in the cavity adjacent to TRP214 residue and therefore had a good and favourable interactive
profile inside the cavity [54]. Four hydrogen bonds were found between 1f and GLN196,
LYS199, ARG222 and ARG257 residue of HSA. Molecular docking results suggested that the
main force involved in binding of 1f with HSA is hydrophobic interaction, whereas hydrogen
bonds might have a minor contribution in the interaction between 1f and HSA.
(B)
Figure 15. (A) Representation of docked 1f into HSA at the pocket site (B) Docked 1f
(represented
by green coloured sticks) and neighbouring amino acid residues (represented by magenta
coloured
sticks) of HSA.
3. Experimental section
3.1. Materials and Methods
All the chemicals and reagents procured were of analytical grade and were used directly without
purification. The completion of reaction was confirmed using TLC (Thin layer chromatography)
by visualizing the spots under ultraviolet light (254 nm). To run the TLC, ethyl acetate: hexane
(20: 80) mobile phase system was used. Melting point of the compounds were determined on a
Buchi apparatus in open capillary tubes. 1H and 13C NMR spectra were recorded on a Bruker 400
MHz spectrometer using CDCl3 as solvent. All chemical shifts were reported as δ values (ppm).
Mass spectra were recorded using Shimadzu GCMSQP2010S. IR spectra were recorded on a
Nicolet Impact 410 FTIR spectrometer using KBr pellets. The elemental analysis was carried out
23

using Euro Vector E-3000 system. UV-Visible spectral data were recorded on a Shimadzu, UV-
1800 instrument. X-ray diffraction analysis data for compounds 1a, 1b, 1c, 1d, 1e, 1f, 1h and 1i
were recorded on a Bruker SMART Apex CCD diffractometer. SHELXTL program was used to
record complex scattering factors. Potentiometric titration experiments were carried on a pH
meter (HI5000 Series, Hanna).
3.2. Protocol to synthesize coumarin-cyclic imide conjugates (1a−1j)
4,4-Dimethylpiperidine-2,6-dione (1.41 g, 10 mM) was dissolved in dry acetone (10 mL) in the
presence of anhydrous K2CO3 (4.14 g, 30 mM). The reaction mixture was kept for stirring (30
minutes at 50oC) and then substituted 4-bromomethyl coumarin (a-j, 10 mM) was added. The
reaction mixture was kept for stirring at 55–60oC temperature for 10 h. The progress of the
reaction was monitored using TLC. Once the reaction was complete, the solution was gradually
cooled to room temperature and quenched with crushed ice. The solid product obtained was
further recrystallized with ethanol and dried in vacuo. Elemental analysis, NMR (1H and 13C), IR
and GC-MS data for all the compounds corroborate with their formulation.
3.3. Data for compounds (1a-1j)
4,4-dimethyl-1-((6-methyl-2-oxo-2H-chromen-4-yl)methyl)piperidine-2,6-dione (1a)
[C18H19NO4]. 4-(bromomethyl)-6-methyl-2H-chromen-2-one (a): 2.53 g, 10mM; Yield: 2.39 g
(76.35%); colourless crystals; m.p. 285 – 286oC; Anal. calcd. for. C18H19NO4: C, 68.99; H, 6.11;
N, 4.47. Found: C, 68.97; H, 6.13; N, 4.46; ATR-IR (νmax/cm-1): 1716 (C=O of lactone), 1667
(Cyclic C=O ); 1H NMR (400 MHz , CDCl3, δ ppm) 1.14 (s, 6H, -CH3), 2.39 (s, 3H, -CH3), 2.62
(s, 4H, -CH2), 5.10 (s, 2H, -CH2), 5.90 (s, 1H, -CH), 7.20-7.24 (m, 1H, Ar-H), 7.31-7.34(m, 1H,
Ar-H), 7.42 (s, 1H, Ar-H);13C NMR (100 MHz, CDCl3, δ ppm): 16.22, 23.12, 23.22, 24.54,
34.21, 41.38, 106.82, 112.25, 112.75, 118.70, 128.22, 129.26, 144.94, 146.91, 155.85, 166.75;
GC-MS: 313 [M]+.
4,4-dimethyl-1-((7-methyl-2-oxo-2H-chromen-4-yl)methyl)piperidine-2,6-dione (1b)
[C18H19NO4]. 4-(bromomethyl)-7-methyl-2H-chromen-2-one (b): 2.53g, 10mM; Yield: 2.30 g
(73.48%) , colourless crystal; mp 293–294oC; Anal. calcd. for. C18H19NO4: C, 68.99; H, 6.11; N,
4.47. Found: C, 68.96; H, 6.09; N, 4.45; ATR-IR (νmax/cm-1): 1718 (C=O of lactone), 1663
(Cyclic C=O ); 1H NMR (400 MHz , CDCl3, δ ppm) 1.14 (s, 6H, -CH3), 2.43 (s, 3H, -CH3), 2.62
24

(s, 4H, -CH2), 5.10 (s, 2H, -CH2), 5.87 (s, 1H, -CH), 7.10-7.13 (m, 2H, Ar-H), 7.52(d, 1H, J =
8.0 Hz); 13C NMR (100 MHz, CDCl3, δ ppm): 21.61, 27.95, 29.30, 38.95, 46.14, 110.71, 115.39,
117.45, 123.28, 125.54, 143.32, 149.85, 153.63, 160.75, 171.55; GC-MS: 313 [M]+.
1-((6-chloro-2-oxo-2H-chromen-4-yl)methyl)-4,4-dimethylpiperidine-2,6-dione (1c)
[C17H16ClNO4]. 4-(bromomethyl)-6-chloro-2H-chromen-2-one (c): 2.73 g, 10mM; Yield: 2.78 g
(83.48%), yellow crystal; mp 312 – 313oC; Anal. calcd. for. C17H16ClNO4: C, 61.18; H, 4.83; N,
4.20. Found: C, 61.16; H, 4.85; N, 4.18; ATR-IR (νmax/cm-1): 1725 (C=O of lactone),
1681(Cyclic C=O );1H NMR (400 MHz , CDCl3, δ ppm) 1.15 (s, 6H, -CH3), 2.63 (s, 4H, -CH2),
5.07 (s, 2H, -CH2), 5.98 (s, 1H, -CH), 7.24-7.29 (m, 1H, Ar-H), 7.47-7.50(m, 1H, Ar-H) 7.63(d,
1H, J = 2.4 Hz); 13C NMR (100 MHz, CDCl3, δ ppm): 27.91, 29.31, 38.80, 46.10, 113.06,
118.68, 118.93, 123.43, 129.87, 131.97, 148.82, 151.96, 159.68, 171.48; GC-MS: 333[M]+.
1-((7-chloro-2-oxo-2H-chromen-4-yl)methyl)-4,4-dimethylpiperidine-2,6-dione (1d)
[C17H16ClNO4]. 4-(bromomethyl)-7-chloro-2H-chromen-2-one (d): 2.73 g, 10mM; Yield: 2.69 g
(80.78%), yellow crystal; mp 318 – 319 oC; Anal. calcd. for. C17H16ClNO4: C, 61.18; H, 4.83; N,
4.20. Found: C, 61.19; H, 4.86; N, 4.22; ATR-IR (νmax/cm-1): 1721 (C=O of lactone), 1673
(Cyclic C=O ); 1H NMR (400 MHz , CDCl3, δ ppm) 1.12 (s, 6H, -CH3), 2.62 (s, 4H, -CH2), 5.06
(s, 2H, -CH2), 5.96 (s, 1H, -CH), 7.24-7.32 (m, 2H, Ar-H), 7.58(d, 1H, J = 8.8 Hz); 13C NMR
(100 MHz, CDCl3, δ ppm): 23.13, 23.20, 24.56, 34.11, 41.30, 107.37, 111.72, 112.76, 119.92,
120.16, 133.21, 144.55, 149.11, 154.85, 166.85; GC-MS: 333[M]+.
1-((5,7-dimethyl-2-oxo-2H-chromen-4-yl)methyl)-4,4-dimethylpiperidine-2,6-dione (1e)
[C19H21NO4]. 4-(bromomethyl)-5,7-dimethyl-2H-chromen-2-one(e): 2.67g, 10mM; Yield: 2.51
g (76.75%), colourless crystal; mp 321 – 323oC; Anal. calcd. for. C19H21NO4: C, 69.71; H, 6.47;
N, 4.28. Found: C, 69.73; H, 6.46; N, 4.26; ATR-IR (νmax/cm-1): 1709 (C=O of lactone), 1670
(Cyclic C=O ); 1H NMR (400 MHz , CDCl3, δ ppm) 1.15 (s, 6H, -CH3), 2.35 (s, 3H, -CH3), 2.63
(s, 4H, -CH2), 2.69 (s, 3H, -CH3), 5.23 (s, 2H, -CH2), 5.71 (s, 1H, -CH), 6.89 (s, 1H, Ar-H),
6.99(s, 1H, Ar-H) ;13C NMR (100 MHz, CDCl3, δ ppm): 21.15, 24.95, 28.03, 29.36, 42.54,
46.08, 109.64, 115.28, 116.29, 130.01, 135.47, 142.33, 152.06, 155.14, 160.48, 171.52; GC-MS:
327 [M]+.
25

1-((7,8-dimethyl-2-oxo-2H-chromen-4-yl)methyl)-4,4-dimethylpiperidine-2,6-dione (1f)
[C19H21NO4]. 4-(bromomethyl)-7,8-dimethyl-2H-chromen-2-one (f): 2.67g, 10mM;Yield: 2.43
g (74.31%), colourless crystal; mp 308 – 309oC; Anal. calcd. for. C19H21NO4: C, 69.71; H, 6.47;
N, 4.28. Found: C, 69.69; H, 6.48; N, 4.30; ATR-IR (νmax/cm-1): 1721 (C=O of lactone), 1664
(Cyclic C=O ); 1H NMR (400 MHz , CDCl3, δ ppm) 1.13 (s, 6H, -CH3), 2.32(s, 3H, -CH3), 2.35
(s, 3H, -CH3), 2.61 (s, 4H, -CH2), 5.09 (s, 2H, -CH2), 5.85 (s, 1H, -CH), 7.08 (d, 1H, J=8.4Hz),
7.36(d, 1H, J=8.4Hz) ; 13C NMR (100 MHz, CDCl3, δ ppm): 11.64, 20.37, 27.97, 29.31, 39.11,
46.10, 110.03, 115.47, 120.26, 124.98, 125.76, 141.86, 150.29, 151.67, 161.03, 171.60; GC-MS:
327 [M]+.
1-((7-methoxy-2-oxo-2H-chromen-4-yl)methyl)-4,4-dimethylpiperidine-2,6-dione (1g)
[C18H19NO5]. 4-(bromomethyl)-7-methoxy-2H-chromen-2-one(g): 2.69g, 10mM; Yield: 2.71 g
(82.37%), white solid; mp 273 – 275oC ; Anal. calcd. for. C18H19NO5: C, 65.64; H, 5.81; N, 4.25.
Found: C, 65.62; H, 5.79; N, 4.24; ATR-IR (νmax/cm-1): 1703 (C=O of lactone), 1669 (Cyclic
C=O );1H NMR (400 MHz , CDCl3, δ ppm) 1.14 (s, 6H, -CH3), 2.63 (s, 3H, -OCH3), 3.85 (s,
4H, -CH2), 5.08 (s, 2H, -CH2), 5.80 (s, 1H, -CH), 6.81-6.87 (m, 2H, Ar-H), 7.55 (d, 1H,
J=8.4Hz);13C NMR (100 MHz, CDCl3, δ ppm): 27.94, 29.30, 38.97, 46.14, 55.76, 101.10,
108.66, 111.35, 112.55, 124.63, 150.03, 155.43, 160.97, 162.84, 171.60; GC-MS: 329 [M]+.
1-((6-bromo-2-oxo-2H-chromen-4-yl)methyl)-4,4-dimethylpiperidine-2,6-dione (1h)
[C17H16BrNO4]. 6-bromo-4-(bromomethyl)-2H-chromen-2-one(h): 3.17g, 10mM; Yield: 2.81 g
(74.33%), light brown crystal; mp 324 – 325oC; Anal. calcd. for. C17H16BrNO4: C, 53.99; H,
4.26; N, 3.70. Found: C, 53.98; H, 4.24; N, 3.68; ATR-IR (νmax/cm-1): 1715 (C=O of lactone),
1666 (Cyclic C=O ); 1H NMR (400 MHz , CDCl3, δ ppm) 1.15 (s, 6H, -CH3), 2.63 (s, 4H, -CH2),
5.07 (s, 2H, -CH2), 5.96 (s, 1H, -CH), 7.21 (d, 1H, J=8.8Hz), 7.61-7.63(m, 1H, Ar-H), 7.78(s,
1H, Ar-H) ;13C NMR (100 MHz, CDCl3, δ ppm): 27.94, 29.36, 38.86, 46.06, 112.82, 117.23,
119.01, 119.39, 126.45, 134.84, 148.78, 152.38, 159.70, 171.56 ; GC-MS: 378 [M]+.
4,4-dimethyl-1-((3-oxo-3H-benzo[f]chromen-1-yl)methyl)piperidine-2,6-dione
[C21H19NO4]. 1-(bromomethyl)-3H-benzo[f]chromen-3-one (i): 2.89g, 10mM; Yield: 2.91 g
(76.98%), brown crystal; mp 343 – 344oC; Anal. calcd. for. C21H19NO4: C, 72.19; H, 5.48; N,
4.01. Found: C, 72.17; H, 5.50; N, 4.02; ATR-IR (νmax/cm-1): 1713 (C=O of lactone), 1668
(1i)
26

(Cyclic C=O ); 1H NMR (400 MHz , CDCl3, δ ppm) 1.16 (s, 6H, -CH3), 2.65 (s, 4H, -CH2), 5.56
(s, 2H, -CH2), 5.97 (s, 1H, -CH), 7.40-7.62 (m, 3H, Ar-H), 7.88-7.96(m, 2H, Ar-H), 8.27(d, 1H,
J=8.4Hz) ;13C NMR (100 MHz, CDCl3, δ ppm): 28.03, 29.42, 43.50, 46.08, 110.41, 113.29,
117.70, 125.28, 125.60, 128.13, 129.36, 129.92, 131.41, 134.11, 152.59, 154.67, 160.30, 171.59;
GC-MS: 378 [M]+.
4,4-dimethyl-1-((2-oxo-2H-benzo[h]chromen-4-yl)methyl)piperidine-2,6-dione (1j)
[C21H19NO4]. 4-(bromomethyl)-2H-benzo[h]chromen-2-one (j): 2.89g, 10mM; Yield: 2.81 g
(74.33%), pale yellow solid; mp 337 – 339oC; Anal. calcd. for. C21H19NO4: C, 72.19; H, 5.48; N,
4.01. Found: C, 72.21; H, 5.49; N, 4.02; ATR-IR (νmax/cm-1): 1719 (C=O of lactone), 1672
(Cyclic C=O ); 1H NMR (400 MHz , CDCl3, δ ppm) 1.17 (s, 6H, -CH3), 2.65 (s, 4H, -CH2), 5.23
(s, 2H, -CH2), 6.03 (s, 1H, -CH), 7.61-7.72 (m, 4H, Ar-H), 7.84-7.87(m, 1H, Ar-H), 8.51-8.53(m,
1H, Ar-H);13C NMR (100 MHz, CDCl3, δ ppm): 28.00, 29.37, 39.48, 46.14, 110.89, 113.13,
119.12, 122.67, 123.14, 124.46, 127.31, 127.66, 128.89, 134.76, 150.85, 160.64, 171.62; GC-
MS: 378 [M]+.
3.4. Biological Assay
3.4.1. Antidiabetic activity
Antidiabetic activity was assessed using 2-NBDG uptake assay. Insulin resistance HepG2
(human liver carcinoma) cells were seeded in 96 well plate with 70% confluency and further
treated with a mixture of fructose, palmitic acid, TNF-alpha, Metformin and test compounds.
After 24 h incubation, the cells were washed with 40µM 2-NBDG and PBS (1 X).
Simultaneously, for every hour 100 nM insulin were added in each of the wells. The cells were
carefully washed again and later lysis buffer was added, after which the fluorescence reading
were taken from each well.
Control refers to cells cultured only in DMEM+10%FBS+1% antibiotic for 24 h prior to the 2-
NBDG uptake analysis. TEST refers to the cells which were grown in insulin resistance media
for 24 h prior to 2-NBDG uptake analysis. Insulin resistance media consists of DMEM + 10%
FBS + 1% antibiotic along with combination of 20 ng/mL TNF-alpha, 0.1 mM of palmitic acid
and 40 mM of fructose. This mixture was used basically to induce insulin resistance in the
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HepG2 cell line. Different doses of different compounds were given to the cells along with
insulin resistance media for 24 h prior to the 2-NBDG uptake.
3.4.2. Cytotoxicity assay
HEK-293 cells (human embryonic kidney cells) were grown in Dulbecco's modified eagle's
medium supplemented with 10% fasting blood sugar and 1% of antibiotic at 370C under 5% of
carbon dioxide. Cells were later harvested using trypsin and seeded in a 96-well cell culture plate
for the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The test
compounds were solubilised in cell culture grade DMSO. Once the cells in 96-well plate were 70
% confluent, the compounds were injected to the cells at 50 nM to 10000 nM concentration
(made in DMEM supplemented with 1% antibiotic and 10% FBS) for 24 h. After 24 h, cells
were washed with PBS and added with 100 µL of fresh media in each well along with 10 µL of
MTT reagent (5 mg/mL) for another 4 h. After 4 h, media was again removed and added 100 µL
cell culture grade DMSO to dissolve the formazan crystals formed by the reduction of MTT by
the live cells. Absorbance of the solution were taken at 570 nm.
3.5. pH titrimetric assay
In water and acetonitrile mixture (8:2) [55], stock solutions for KOH (25 mM), HCl (25 mM)
and test compounds (0.5 mM) were prepared. Potentiometric titration method was employed to
determine the pKa values of compounds. UV-Visible Spectrophotometer was used to study the
absorbance spectra of the test compounds.
3.6. HSA interaction study using spectroscopic and in-silico methods
Standard stock solution of the most active antidiabetic compound 1f (1 mM) was prepared by
dissolution in DMSO. A standard solution of HSA (5 µM) was prepared in pH 7.4 sodium
phosphate buffer and this solution was incubated for 24 h. The absorbance of this solution was
measured at 280 nm to accurately calculate the concentration of HSA standard solution by
knowing the molar absorption coefficient of HSA (ε = 36,500 M-1cm-1) [41]. For the titration
studies, HSA absorbance spectra were recorded on a UV-Visible Spectrophotometer (1800
SHIMADZU) using cuvettes of 1.0 cm path length. HSA fluorescence emission spectra were
recorded using a 150W xenon lamp equipped RF-5301pc SHIMADZU Spectrofluorophotometer.
28

All the fluorescence emission intensities were corrected with the absorbance of compound 1f to
eliminate inner filter effects [42, 53]. Equation 8 was used for this correction.
⁄
ퟐ
푭풄풐풓풓 = 푭풐풃풔풅ퟏퟎ(푨 +푨 ) ퟐ
(8)
ퟏ
where Fcorr and Fobsd are the corrected and observed emission intensities of 1f, respectively; A1
and A2 are sum of absorbances of 1f and HSA at the emission and excitation wavelengths,
respectively.
Molecular docking studies were performed to determine the potential binding environment
between HSA and 1f. The structure of HSA (PDB ID: 1AO6) was acquired from the Protein
Data Bank. The 3D structure of 1f was produced by 3D ChemDraw Ultra 8.0 and MM2 force-
field was applied for energy minimization. Docking studies were carried using the molecular
docking software (AutoDock4.2). AutoDock Tools (ADTs) produce various ligand conformers
using a Lamarckian genetic algorithm (LGA) which is built on adaptive local method search.
Docking was carried out by setting the grid size to 60, 50, 80 along x, y, z axes with a grid
spacing 0.375 A˚. The grid centre was set as 22.556, 35.519, 97.262A˚. The grid map for
different atoms of 1f and HSA was generated by running the AutoGrid. Upon generation of grid
maps, AutoDock was ran and Autodock parameters were found to be as follows: GA population
size: 150; Numbers of generations: 27000; Maximum numbers of energy evaluations: 2.5x106
[53]. A total of 10 runs were performed, and out of all those, minimum energy conformers were
picked for the study based on ranking and scoring.
4. Conclusions
In the present study, we describe the design, synthesis and evaluation of coumarin-cyclic imides
(1a–1j) as possible antidiabetic agents. Among synthesized, compound 1f exhibited distinct
activity with 85.21% of glucose uptake which is comparable with standard drug Metformin
(93.25% glucose uptake). All the compounds (1a–1j) were observed to be least toxic against
Human Embryonic Kidney cells indicating their good safety profile. Single crystals of
compounds 1a, 1b, 1c, 1d, 1e, 1f, 1h and 1i were developed and their crystal parameters were
evaluated. A notable observation from X-ray crystal studies was that compounds 1f and 1b
which exhibited significant glucose uptake activity have shown C=O-π interactions in X-ray
crystal packing. The solution stability studies indicated that the test compounds are sufficiently
stable at varied pH conditions and thereby compatible with bio-physiological environments.
29