Synthesis of novel hetero ring fused pyridine derivatives; Their anticancer activity, CoMFA and CoMSIA studies

Article abstract of DOI:10.1016/j.bmcl.2018.04.031

A series of novel furo[2,3-b]pyridine-2-carboxamide 4a–h/pyrido[3′,2′:4,5]furo[3,2-d] pyrimidin-4(3H)-one derivatives 5a–p were prepared from pyridin 2(1H) one 1 via selective O-alkylation with α-bromoethylester followed by cyclization, then reaction with

Full text of DOI:10.1016/j.bmcl.2018.04.031

Accepted Manuscript
Synthesis of novel hetero ring fused pyridine derivatives; their anticancer ac-
tivity, CoMFA and CoMSIA studies
G. Santhosh Kumar, Y. Poornachandra, Shravan Kumar Gunda, K. Ratnakar
Reddy, Jaheer Mohmed, Kamal Shaik, C. Ganesh Kumar, B. Narsaiah
PII:
S0960-894X(18)30343-3
https://doi.org/10.1016/j.bmcl.2018.04.031
BMCL 25778
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 February 2018
8 April 2018
11 April 2018
Please cite this article as: Santhosh Kumar, G., Poornachandra, Y., Kumar Gunda, S., Ratnakar Reddy, K., Mohmed,
J., Shaik, K., Ganesh Kumar, C., Narsaiah, B., Synthesis of novel hetero ring fused pyridine derivatives; their
anticancer activity, CoMFA and CoMSIA studies, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://
doi.org/10.1016/j.bmcl.2018.04.031
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Leave this area blank for abstract info.
Synthesis of novel hetero ring fused pyridine derivatives; their anticancer activity, CoMFA and
CoMSIA studies
G. Santhosh Kumar, Y. Poornachandra, Shravan Kumar Gunda, K. Ratnakar Reddy, Jaheer Mohmed, Kamal
Shaik, C. Ganesh Kumar and B. Narsaiah*
Alignment
IC50 (µM) = 5.2 ± 0.18
CoMFA steric contour map
Steric Contribution = 73.6%
2

Bioorganic & Medicinal Chemistry Letters
Synthesis of novel hetero ring fused pyridine derivatives; their anticancer
activity, CoMFA and CoMSIA studies
G. Santhosh Kumar a, Y. Poornachandra b, Shravan Kumar Gunda c, K. Ratnakar Reddy a,
Jaheer Mohmed c, Kamal Shaik d, C. Ganesh Kumar b, B. Narsaiah a, 
aFluoro Organic Division, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad-500007, India.
bMedicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad-500007, India.
cBioinformatics Division, PGRRCDE, Osmania University, Hyderabad–500007, India;
dDepartment of Chemical Engineering, Visvesvaraya National Institute of Technology, Nagpur–440010, India.
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A
series of novel furo[2,3-b]pyridine-2-carboxamide 4a-h/pyrido[3',2':4,5]furo[3,2-d]
pyrimidin-4(3H)-one derivatives 5a-p were prepared from pyridin 2(1H) one 1 via selective O-
alkylation with α-bromoethylester followed by cyclization, then reaction with different aliphatic
primary amines to obtain 4 and further reaction with triethyl orthoacetate/triethyl orthoformate.
Also prepared novel furo[2,3-b]pyridine-2-carbohydrazide Schiff’s bases 7a-h and pyrido
[3',2':4,5]furo[3,2-d]pyrimidin-4(3H)-one derivatives 8a-h starting from furo[2,3-b]pyridine
carboxylate derivatives 3 by reaction with hydrazine hydrate to form 6 and reaction with diverse
substituted aldehydes and cyclization. Products 4a-h, 5a-p, 7a-h and 8a-h were screened against
four human cancer cell lines (HeLa, COLO205, Hep G2 and MCF 7) and one normal cell line
(HEK 293). Compounds 4e, 4f, 4g, 5h, 7c, 7d, 7e and 7f showed significant anticancer activity
against all the cell lines at micro molar concentration and found to be non-toxic to normal cell
line. Studies for HeLa, COLO205 and MCF-7 using CoMFA and CoMSIA. Models from 3D-
QSAR provided a strong basis for future rational design of more active and selective HeLa,
COLO205 and MCF-7 cell line inhibitors.
Keywords:
Carbohydrazide Schiff’s bases
Pyrido[3',2':4,5]furo[3,2-d]pyrimidin-4(3H)-one,
anti-cancer activity,
3D QSAR, CoMFA and CoMSIA Studies
2009 Elsevier Ltd. All rights reserved.
Present statistics indicate that the cancer disease is second
common cause of death after heart disease and continuously
growing as worldwide killer.1 Although chemotherapy is the
backbone for cancer treatment, the use of chemotherapeutics is
often limited due to undesirable side effects. Therefore,
identification of new agents as targets for the treatment of
cancer is very important. Some of the furopyridine and pyrimidi
none derivatives (5,6 fused ring systems) found to have
biological and chemotherapeutic importance. Furopyridines are
structural analogues to indoles, pyrrolopyridines which are
frequently employed as core scaffolds and play a significant
role in promoting activity.2,3 Furo[2,3-b]pyridine skeleton is
rarely found in naturally occurring alkaloids;4 however, it
showed activity against HIV,5 CNS disorders,6 skin diseases7
and hyperglycemia.8 Furo[2,3-b]pyridine derivatives also
demonstrated in vitro activity for tubulin polymerization,9 Lck10
and Akt11 kinase inhibitors. A specific drug Cicletanine, L-754,
394 based on furopyridine, shows an antihypertensive with
vasorelaxant, diuretic property12,13 and potent HIV protease
inhibitor. Fluorouracil and Tegafur based on pyrimidinone
skeleton used as anticancer agents are outlined in Figure 1.
Figure 1. Bio-active compounds based on furopyridine and
pyrimidinone scaffolds.
———
 Corresponding author. Tel.: +91-40-27193185
fax: +91-40-27160387; E-mail: narsaiahbanda84@gmail.com.
1

Fused pyrimidines have a long and distinguished history
extending from the days of their discovery as important
constituents of nucleic acids to their current use in the
chemotherapy. Hydrazones have been demonstrated to possess
antimicrobial, anticonvulsant, analgesic, anti-inflammatory,
anti-platelet, anti-tubercular and anti-tumor activities.
Hydrazide derivatives are not only intermediates, coupling
products can be synthesized by using the active hydrogen
component of –CONHN=CH– azo-methine group.14 Similarly,
anticancer activity against four human cancer cell lines.
Compounds 4e, 4f, 4g, 5h, 7c, 7d, 7l and 7o which showed
promising anticancer activity have been identified.
The 3-cyano-4-trifluoromethyl-6-substituted pyridine 2(1H) one
1 was reacted with 2-bromoethyl acetate under basic conditions
and obtained selectively 2-O-ethylacetoxy-3-cyano-4-trifluoro
methyl-6-substituted pyridine derivatives 2. Compounds 2 were
cyclized in DMF using potassium carbonate as base and
obtained furo[2,3-b]pyridine derivatives 3. The reaction
sequence includes selective O-alkylation then abstraction of
proton by base from an active methylene followed by
cyclization onto nitrile carbon. This type of cyclization is also
known as Thorpe-Ziegler cyclization. Compounds 3 were
reacted with different substituted aliphatic primary amines to
thieno[2,3-b]quinoline-2-carboxamide
derivatives
are
considered as anticancer agents.15 Further, it was found that the
fluorine16 or trifluoromethyl17,18 group at a strategic position of
an organic molecule dramatically alters the properties of
molecule in terms of lipid solubility, oxidative thermal stability
thereby enhances the transport mechanism and bio-efficacy.
However, no reports are available on synthesis of fluorinated
furo [2,3-b] pyridine derivatives except our reports.19,20 In
continuation of our efforts, we designed and synthesized a
series of novel trifluoromethyl substituted furo [2,3-b] pyridine-
2-carbohydrazide Schiff’s bases and pyrido [3',2':4,5] furo [3,2-
d] pyrimidin-4(3H)-one derivatives and screened them for
result in carboxamide derivatives
4 and were further
independently reacted with triethyl orthoacetate and triethyl
orthoformate to obtain pyrido furo pyrimidinone derivatives 5a-
p. The sequence of reactions outlined in Scheme 1.
Scheme 1
Reagents and conditions (i) EtOH, Piperidine, reflux, 4-5 h (ii) K2CO3, NaI, Acetone, reflux, 6h (iii) DMF, K2CO3, 110 ºC, 6h. (iv) Neat, Δ, 6h (v) Acetic acid,
140 ºC, 3 h.
2

Scheme 2
Reagents and conditions: (vi) N2H4.H2O, EtOH, 80 ºC, 4-6h. (vii) R′CHO, EtOH, cat. Piperidine, 80 ºC, 2h (viii) cat. Acetic acid, 120 ºC, 3h.
Compounds 3 were reacted with hydrazine hydrate to result
carbohydrazide derivatives 6 which were further reacted with
diverse substituted aromatic aldehydes to obtain furo[2,3-
b]pyridine-2-carbohydrazide Schiff’s bases 7. Compounds 7
were reacted with TEOF (triethyl orthoformate) in the presence
of catalytic amount of acetic acid and obtained the final
products of furo pyrido pyrimidinone derivatives 8a-h. The
sequence of reactions outlined in Scheme 2.
Table 2. In vitro cytotoxicity of compounds 5a-p.
Compd.
IC50 values (in µM)
HeLa
COLO205
HepG2
MCF7
HEK293
5a
5b
5c
5d
5e
5f
5g
5h
5i
---
---
---
---
---
---
---
---
---
---
---
---
---
---
220.9 ± 0.41 ---
---
---
---
---
---
---
---
82.4 ± 0.52 121 ± 0.52
85.5 ± 0.36 90 ± 0.48
---
---
---
116.8±0.38 52 ± 0.32
22.8 ± 0.22 96 ± 0.28
---
---
---
62.7 ± 0.38 ---
---
---
31.5 ± 0.42 68 ± 0.34
--- 124 ± 0.22
---
---
---
Anticancer activity & structure-activity relationship
Compounds 4a-h and 5a-p were screened against four human
cancer cell lines such as HeLa (cervical cancer, CCL-2),
COLO-205 (colon cancer, CCL-222), HepG2 (liver cancer, HB-
8065) and MCF7 (breast cancer, HTB-22) using MTT assay.21
Among all the compounds screened, compounds 4e, 4f, 4g, and
5h showed promising activity at micro molar concentration. The
structure-activity relationship studies revealed that the
carboxamide derivatives 4a-h showed more promising activity
as compared to pyrimidinone derivatives 5a-p. It was attributed
to the presence of amide and primary amine group in
compounds 4a-h and was considered as polar groups. Among
all the compounds screened in 4a-h series, compounds 4e-g
showed promising activity. Similarly, in 5a-p series, the
presence of thiophenyl group in 6th position was crucial for
activity and compound 5h exhibited the promising activity. The
activity data is shown in Table 1 & 2.
18.2 ± 0.12 18.7 ± 0.28 17.1 ± 0.20
---
---
---
---
---
---
---
---
---
---
---
---
5j
5k
5l
50.2 ± 0.42 42.5 ± 0.36 49.8 ± 0.23
5m
5n
5o
5p
---
---
---
---
---
---
---
---
28.5 ± 0.18 18.9 ± 0.18 22.8 ± 0.42
--- --- ---
5-Fluorouracil (Std control)
1.8 ± 0.09 1.9 ± 0.11 1.7 ± 0.08
1.8 ± 0.07 19.6 ± 0.18
---indicates IC50 value > 220 µg/mL; Cell lines used: HeLa - Cervical cancer (CCL-2);
COLO 205- Colon cancer (CCL-222); HepG2- Liver cancer (HB-8065); MCF7 - Breast
cancer (HTB-22); HEK-293 –Human Embryonic Kidney cells (CRL-1573).
Compounds 7a-h and 8a-h were also screened against four
human cancer cell lines such as HeLa, COLO-205, HepG2 and
MCF7. Compounds 7a-h are uncyclized Schiff’s base products,
while compounds 8a-h were cyclized pyrimidinone products.
The structure verses activity revealed that the presence of –NH2
on furyl and amide linkage (–NHCO) are crucial for activity.
Among all the compounds screened, compounds 7a-h showed
promising activity as compared to compounds 8a-h. Compounds
7c, 7d, 7e and 7f were found to exhibit high activity. Among all
the compounds 7c, 7d, 7e and 7f were found to be more potent
with IC50 values of < 10 µg/mL on all the tested cancer cell
lines. The activity data to this regard is tabulated in Table 3 & 4.
Compounds 7c and 7d were considered as lead molecules for
further optimization. The introduction of thiophenyl group at 6th
position of furopyridine and chloro, bromo substituents at 4th
position of phenyl ring enhanced the activity to a greater extent
as compared to other compounds. However, the same
substituents for compounds 8c and 8d could not show activity
Table 1. In vitro cytotoxicity of compounds 4a-h.
Compd.
IC50 values (in µM)
COLO205 HepG2 MCF7
HeLa
HEK293
4a
40.2 ± 0.22
36.8 ± 0.26 41.2 ± 0.28 37.4 ± 0.18 62 ± 0.26
29.2 ± 0.24 42.8 ± 0.14 54.0 ± 0.22 70 ± 0.44
38.9 ± 0.23 37.4 ± 0.27 35.6 ± 0.32 81 ± 0.51
51.1 ±0.41 32.4 ± 0.32 59.8 ± 0.28 85 ± 0.22
17.8 ± 0.24 12.3 ± 0.32 18.0 ± 0.18 75 ± 0.35
18.7 ± 0.28 12.1 ± 0.20 21.8 ± 0.22 62 ± 0.19
15.3 ± 0.31 17.2 ± 0.17 20.4 ± 0.20 64 ± 0.22
18.4 ± 0.29 20.8 ± 0.15 22.9 ± 0.19 51 ± 0.16
4b 36.2 ± 0.21
4c 36.1 ± 0.32
4d 25.8 ± 0.28
15.6 ± 0.25
14.2 ± 0.12
4g 18.9 ± 0.18
4h 21.8 ± 0.09
4e
4f
5-Fluorouracil (Std control)
1.8 ± 0.09 1.9 ± 0.11
1.7 ± 0.08
1.8 ± 0.07 19.6 ± 0.18
3

due to non-availability of –NH2 group and uncyclized amide
linkage (–NHCO). Remaining compounds were not active upto
the concentration of 60 µg/mL. The activity data is shown in
Table 3.
biological activities with chemical structures, and also used to
predict the biological activity of non-synthesized compounds,
which are structurally related to training sets. The present
investigation reports the first application of 3D-QSAR to study
of furo [2,3-b] pyridine and pyrido [3',2':4,5] furo [3,2-d]
pyrimidin-4(3H)-one derivatives as potent anticancer agents. We
studied twenty one compounds for HeLa and COLO205 cell
lines, twenty six compounds for MCF-7 cell line inhibitors as
anticancer agents using CoMFA (comparative molecular field
analysis)22 and CoMSIA (comparative molecular similarity
indices analysis).23 Models obtained from 3D-QSAR studies
provided a strong basis for future rational design of more active
and selective HeLa, MCF-7 and COLO205 cell line inhibitors.
Table 3. In vitro cytotoxicity of compounds 7a-h & 8a-h.
Compd
IC50 Values in (µM)
HeLa
COLO205
HepG2
MCF7
HEK293
7a
7b
7c
7d
7e
7f
7g
7h
8a
8b
8c
8d
8e
8f
11.8±0.21 9.7±0.11
24.4±0.11 21.2±0.32
9.9±0.22
19.8±0.23 18.7±0.19
8.1±0.46
6.1±0.26
13.1±0.31 14.2±0.48
8.9±0.44 10.2±0.11
12.1±0.14 11.9±0.09
19.9±0.13 18.7±0.22
---
---
12.1±0.21
72±0.36
78±0.21
83±0.22
86±0.28
70±0.16
69±0.22
81±0.52
64±0.35
90±0.41
---
102±0.34
---
---
---
---
8.0±0.19
6.4±0.32
7.8±0.48
5.9±0.34
7.4±0.52
5.2±0.18
13.4±0.33 12.2±0.28
11.8±0.51 9.1±0.21
4.1±0.15
11.1±0.46
23.9±0.41 21.2±0.05
42.3±0.13 ---
42.3±0.23 ---
---
Results and discussion
CoMFA and CoMSIA
29.7±0.28
42.1±0.22
---
---
---
---
---
---
---
---
51.2±0.31 ---
---
---
---
43.6±0.4
---
CoMFA and CoMSIA methods were applied to derive 3D-
QSAR models for furo [2,3-b] pyridine and pyrido [3',2':4,5]
furo [3,2-d] pyrimidin-4(3H)-one derivatives as potential
anticancer inhibitors. The statistical results of CoMFA and
CoMSIA analysis are summarized in Table 4. Best predictions
were obtained with CoMFA standard model involving cross-
validated coefficient (q2) = 0.820, 0.876 and 0.853 for HeLa,
MCF-7 and COLO205 cell lines, respectively, correlation
coefficient (r2)= 0.960, 0.959 and 0.978 for HeLa, MCF-7 and
COLO205, respectively. Standard Error of Estimate (SEE)
=0.084, 0.081 and 0.056 for HeLa, MCF-7 and COLO205,
respectively. Cross Validation (cv) = 0.818, 0.867 and 0.841 and
Fischer statistic (F-value) = 47.511, 75.303 and 87.829 for
HeLa, MCF-7 and COLO205, respectively with number of
components as 5 and column filtering 2.0 kcal/mol. In CoMSIA,
the standard model predictions obtained are q2 = 0.812, 0.824
and 0.885, r2 = 0.946, 0.959 and 0.980, SEE =0.102, 0.084 and
0.056, CV = 0.804, 0.814 and 0.889 and F-value = 26.373,
59.015 and 72.306 respectively for HeLa, MCF-7 and COLO205
respectively, with number of components as 6 and column
filtering 1.0 kcal/mol. Except for COLO205, CoMFA results
show a higher predictive ability for furo [2,3-b] pyridine and
pyrido [3',2':4,5] furo[3,2-d] pyrimidin-4(3H)-one against
anticancer on comparison with the CoMSIA results.
---
---
---
---
---
---
8g
8h
56.8±0.25
---
---
5-Fluorouracil (Std control)
1.8±0.09 1.9±0.11
1.7±0.08
1.8±0.07
19.6 ± 0.18
---indicates IC50 value > 220 µg/mL; Cell lines used: HeLa - Cervical
cancer (CCL-2); COLO 205- Colon cancer (CCL-222); HepG2- Liver
cancer (HB-8065); MCF7 - Breast cancer (HTB-22); HEK-293 –Human
Embryonic Kidney cells (CRL-1573).
All the above compounds were also screened against human
normal cell line (HEK-293, Human Embryonic Kidney cells,
CRL-1573) and they were found to be not cytotoxic upto the
concentration of 51 µg/mL to 124 µg/mL as compared to 5-
Fluorouracil (19.6 µg/mL).
QSAR Introduction
In general, the 3D-QSAR techniques are valuable methods of
ligand-based drug design by correlating physicochemical
properties from a set of related compounds to their known
molecular property or molecular activity values. Validation of
QSAR models plays the vital role in defining the applicability of
the QSAR model for the prediction of designed molecules.
QSAR model is mostly used to correlate properties, i.e.
Table 4. The statistical results of CoMFA and CoMSIA analysis
MCF-7
HeLa
COLO205
CoMFA
0.820
CoMSIA
0.812
CoMFA
0.876
CoMSIA
0.824
CoMFA
0.853
CoMSIA
0.885
q2
r2
0.960
0.084
0.946
0.959
0.959
0.978
0.980
0.102
0.081
0.084
0.056
0.056
SEE
47.511
0.818
5
26.373
0.804
6
75.303
0.867
5
59.015
0.814
6
87.829
0.841
5
72.306
0.889
6
F-Value
CV
N
Bootstrap
Mean
0.055
0.983
Std.dev Mean
Stddev Mean
Std.dev Mean
Stddev Mean
Std.dev Mean
Stddev
0.026
0.004
0.051
0.015
0.052
0.984
0.059
0.023
0.064
0.973
0.042
0.012
0.067
0.973
0.045
0.010
0.034
0.991
0.026
0.005
0.030
0.994
SEE
r2
Field Contribution (%)
66.7
23.1
21.5
34.0
16.3
5.1
69.9
32.8
26.9
07.8
17.1
15.4
73.6
21.7
19.2
30.8
23.0
05.3
Steric
33.3
30.1
26.4
Electrostatic
Hydrophobic
Donor
-
-
-
-
-
-
-
-
-
Acceptor
4

For COLO205, CoMSIA results are better predictive ability for
these compounds. Bootstrapping method is used to evaluate the
robustness and the statistical confidence of the QSAR model. It
involves simulating a large number of datasets which are of the
The table 4 shows the results of relative contributions for
CoMFA and CoMSIA methods. For CoMFA, 66.7%, 69.9%
and 73.6% field contribution was observed for steric, and
33.3%, 30.1% and 26.4% field contribution was observed for
electrostatic, respectively, for HeLa, MCF-7 and COLO205 cell
lines. For CoMSIA, the observed contributions for steric,
electrostatic, hydrophobic, donor and acceptor properties are
given in Table 4, respectively. Electrostatic property is the main
contributor in CoMSIA analysis. The affinities between
same size as original and are produced by randomly selecting
samples from the original set. Biological activities, predicted and
residual values of both training set and test set CoMFA and
CoMSIA are shown in Table 4.
experimental and calculated values of the training set and the
test set of CoMFA and CoMSIA models derived from non-
cross-validated analysis of HeLa; MCF-7; COLO205 cell lines
are plotted in Figure 3(a), 3(b); 3(c), 3(d); 3(e) 3(f),
respectively. CoMSIA and the best CoMFA models were used
to predict the inhibitory activities of the compounds in the test
set.
CoMFA
CoMSIA
(b)
(a)
(d)
(c)
(f)
(e)
Figure 3. Calculated pIC50 versus experimental pIC50 values for the molecules of the training set (Blue color) and test set (red color) obtained
by PLS analysis using CoMFA and CoMSIA models for (a), (b); (c), (d); (e) (f)
5

Table 5. Experimental and predicted activities (pIC50) of the
training set molecules for furo[2,3-b]pyridine and
pyrido[3',2':4,5]furo[3,2-d]pyrimidin-4(3H)-one derivatives as
HeLa Cell line inhibitors.
Table 8. Experimental and predicted activities (pIC50) of the
test set molecules for furo[2,3-b]pyridine and pyrido [3',2':4,5]
furo[3,2-d]pyrimidin-4(3H)-one derivatives as MCF-7 Cell line
inhibitors
HeLa - Training Set
MCF-7 - Test Set
C.No
pIC50
CoMFA
CoMSIA
C.No
pIC50
CoMFA
CoMSIA
Predicted Residual Predicted Residual
Predicted Residual Predicted Residual
4a
4b
4c
4d
4g
4h
5i
7a
7b
7c
7d
7e
7f
4.34
4.44
4.44
4.59
4.72
4.66
4.30
4.93
5.04
5.10
5.19
4.87
4.93
5.39
4.37
4.37
4.48
4.49
4.71
4.50
4.48
4.62
4.27
4.97
5.00
5.33
5.23
4.95
4.98
4.99
4.21
4.41
-0.05
-0.05
-0.27
0.09
0.24
0.04
0.03
-0.04
0.04
-0.23
-0.24
-0.08
-0.05
0.35
0.16
-0.04
4.38
4.47
4.71
4.54
4.47
4.65
4.36
4.95
4.93
5.28
5.35
4.95
4.99
4.99
4.36
4.38
0.05
-0.03
-0.27
0.05
0.25
0.01
-0.06
-0.02
0.11
-0.18
-0.16
-0.08
-0.06
0.35
0.01
-0.01
4d
5l
7a
4.22
4.20
4.92
4.95
4.55
4.77
5.18
5.25
-0.33
-0.57
-0.26
-0.30
4.48
4.92
5.25
5.31
-0.26
-0.72
-0.33
-0.18
7b
Table 9. Experimental and predicted activities (pIC50) of the
training set molecules for furo [2,3-b] pyridine and pyrido
[3',2':4,5] furo [3,2-d] pyrimidin-4(3H)-one derivatives as
COLO205 Cell line inhibitors
COLO205 - Training Set
CoMFA
7g
8a
8b
C.No
pIC50
CoMSIA
Predicted Residual
Predicted Residual
4a
4b
4c
4d
4e
4f
4g
4h
5l
5a
7c
7d
7e
7f
4.43
4.54
4.41
4.29
4.75
4.73
4.81
4.73
4.37
5.01
5.10
5.23
4.91
5.04
4.95
4.29
4.52
4.50
4.60
4.42
4.64
4.77
4.66
4.70
4.33
5.13
5.21
5.15
4.79
4.91
4.89
4.49
-0.09
0.04
-0.19
-0.13
0.11
-0.04
0.15
0.03
0.04
-0.12
-0.11
0.08
0.12
0.13
0.06
-0.20
4.52
4.46
4.62
4.45
4.68
4.77
4.64
4.69
4.30
5.20
5.19
5.17
4.80
4.89
4.89
4.47
-0.09
0.08
-0.21
-0.16
0.07
-0.04
0.17
0.04
0.07
-0.19
-0.09
0.06
0.11
0.15
0.06
-0.18
Table 6. Experimental and predicted activities (pIC50) of the
test set molecules for furo[2,3-b]pyridine and pyrido [3',2':4,5]
furo[3,2-d]pyrimidin-4(3H)-one derivatives as HeLa Cell line
inhibitors
HeLa - Test Set
C.No
pIC50
CoMFA
CoMSIA
Predicted Residual Predicted Residual
4e
4f
5h
5o
7h
4.81
4.85
4.74
4.54
4.62
4.41
4.49
4.22
4.28
5.23
0.40
0.36
0.52
0.26
-0.61
4.39
4.47
4.36
4.35
5.27
0.42
0.38
0.38
0.19
-0.65
7g
8c
Table 7. Experimental and predicted activities (pIC50) of the
training set molecules for furo[2,3-b]pyridine and pyrido
[3',2':4,5] furo[3,2-d]pyrimidin-4(3H)-one derivatives as MCF-
7 Cell line inhibitors
Table 10. Experimental and predicted activities (pIC50) of the
test set molecules for furo [2,3-b] pyridine and pyrido [3',2':4,5]
furo[3,2-d]pyrimidin-4(3H)-one derivatives as COLO205 Cell
line inhibitors
MCF-7 - Training Set
C.No pIC50
CoMFA
CoMSIA
COLO205 - Test Set
Predicted Residual Predicted Residual
C.No
pIC50
CoMFA
CoMSIA
4a
4b
4c
4e
4f
4.43
4.27
4.45
4.74
4.66
4.69
4.64
4.08
4.07
3.93
4.64
4.50
4.93
5.13
5.28
4.85
4.99
4.92
4.73
4.53
4.37
4.24
4.47
4.40
4.37
4.71
4.64
4.73
4.78
4.00
4.01
4.25
4.61
4.27
4.81
5.22
5.15
4.82
4.83
4.81
4.89
4.56
4.54
4.15
-0.04
-0.13
0.08
0.03
0.02
-0.04
-0.14
0.08
0.06
-0.32
0.03
0.23
0.12
-0.09
0.13
0.03
0.16
0.11
-0.16
-0.03
-0.17
0.09
4.40
4.39
4.29
4.76
4.71
4.61
4.66
3.94
4.13
4.26
4.59
4.31
4.96
5.22
5.10
4.97
4.88
4.80
4.94
4.42
4.59
3.98
0.03
-0.12
0.16
-0.02
-0.05
0.08
-0.02
0.14
-0.06
-0.33
0.05
0.19
-0.03
-0.09
0.18
-0.12
0.11
0.12
-0.21
0.11
-0.22
0.26
Predicted Residual
Predicted Residual
5h
5o
7b
7h
8g
4.73
4.72
4.99
4.67
4.36
4.05
4.36
5.02
4.93
4.05
0.68
0.36
-0.03
-0.26
0.31
4.12
4.39
5.25
4.98
4.06
0.61
0.33
-0.26
-0.31
0.30
4g
4h
5b
5c
5g
5h
5o
5p
7c
7d
7e
7f
Contour analysis
CoMFA and CoMSIA steric contour plots
For HeLa, the CoMFA model was used to generate the three
dimensional contour maps to represent the quantitative structure
activity result. A single green polyhedron present above Br at
4th position of C6H4. It indicates that the position contains more
steric hindrance. The compound shows more yellow contours at
thien-2-yl and 4-BrC6H4 positions, which indicates that steric
hindrance is less. In CoMSIA a very large green contour present
above the C6H4 ring, which increases the steric hindrance and a
large yellow contour present at C6H4. Adding bulk groups at
this ring decreases the biological activity. In MCF-7 CoMFA, a
medium and a small green contours present around C6H4 and
7g
7h
8b
8c
8g
6

two small contours present around thien-2-yl position which
indicated that bulky groups had favorable steric interactions.
The compound shows two yellow contours one at thien-2-yl and
another at second position of C6H4 ring, it explains bulky
groups had unfavourable steric interactions. In CoMSIA, a
medium green contour above 2nd position of C6H4 ring. While
adding the bulk groups at this position increases the biological
activity and a very large yellow contour present above 2nd
position of the C6H4 ring which indicates decrease in the
activity. In CoMFA, COLO205 the most active compound
shows a large green polyhedron at 4-BrC6H4, and two small
contours around thien-2-yl ring indicating steric favourable
region where biological activity increases by adding bulky
groups. Two medium sized yellow contours present at 2nd
position of C6H4 ring and at thien-2-yl ring where bulky groups
decreases the activity of the compound. In CoMSIA, the most
active compound shows a large green contour present above Br
of 4th position of C6H4 where activity increases, and a large
yellow contour present above 3rd position of C6H4 which
decreases the activity by adding bulk groups. CoMFA and
CoMSIA steric contour plots are shown in figure 4.
CoMFA steric contour maps
CoMSIA steric contour maps
HeLa
HeLa
MCF-7
MCF-7
COLO205
COLO205
Figure 4. CoMFA and CoMSIA steric contour plots
activity. In MCF-7 only blue color contours are present at thien-2-
yl position and near the NH-N position which increases the
biological activity. Whereas in COLO205 a small blue color
contour present at NH-N position and two medium sized red
colored contours present above oxygen of O-NH position and at N
position. Where addition of negative groups enhance the biological
activity. Figure 5 shows contour maps of CoMFA and CoMSIA
electrostatics contour maps.
CoMFA electrostatics contours plots
For HeLa, the electrostatic contours maps of CoMFA are shown in
blue and red colour. Two medium sized blue contours present near
O-NH-N position indicating addition of positive charge groups may
increase the biological activity of a compound. The blue contours
are in favorable region and red contours in disfavorable region.
There is a large red contour present around the O-NH-N, at this
position electron density is estimated to increase the biological
activity. By adding electronegative groups may enhance the
7

CoMFA electrostatic contour maps
CoMSIA electrostatic contour maps
HeLa
HeLa
MCF-7
MCF-7
`
COLO205
COLO205
Figure 5. CoMSIA steric and electrostatic contour maps for highly active compound
white contour present around C6H6 ring for HeLa, a medium sized
white contour present above N-NH position indicates disfavored
conformation for hydrophobic substitution for MCF-7. A medium
sized white contour present above thien-2-yl ring indicating
disfavored region. CoMSIA hydrophobic contour plots are shown
in figure 6.
CoMSIA Hydrophobic contour analysis
CoMSIA hydrophobic contour maps are shown in white and
yellow. A small to medium sized yellow contours present above Br
of the C6H4 ring for both HeLa and COLO205 targets and a large
yellow contour present at N-NH position for MCF-7 indicates
favorable for hydrophobic substitution increase the activity. A large
MCF
HeLa
8

COLO205
Figure 6. CoMSIA hydrophobic contour maps for highly active compound
CoMSIA hydrogen bond donor contour analysis
The graphical representation of the field contributions of the
hydrogen bond donor is shown in Figure 7. Donor contour maps are
shown in cyan and purple color. Cyan contour maps explains the
position of hydrogen bond donor groups which increases biological
activity, while purple contours are biologically unfavored. For
HeLa, MCF-7, COLO205 the most active compound shows a small
cyan isopleth which enhance the biological activity, and the
presence of three large purple isopleths reduces the biological
activity.
MCF-7
HeLa
COLO205
Figure 7. CoMSIA hydrogen bond donor contour maps for highly active compound
contours. Whereas in COLO205, the most active compound shows
CoMSIA hydrogen bond acceptor contour analysis
Hydrogen bond acceptor contours are represented in magenta and
red in color. Magenta color indicates favorable region and red color
indicates unfavorable region of biological activity. In HeLa, the
most active compound shows a large magenta isopleth only. In
MCF-7, the compound exhibit a large magenta and a small red
a medium sized magenta and a large, a small red contours present
are shown in figure 8.
MCF-7
HeLa
COLO205
Figure 8. CoMSIA hydrogen bond acceptor contour maps for highly active compound
9

Newton, C. L.; Hoffman, J. M.; Darke, P. L.; Zugay-Murphy, J. A.;
Emini, E. A.; Schlief, W. A.; Olsen, D. B.; Stahlhut, M. W.;
Rutkowski, C. A.; Kuo, L. C.; Lin, J. H.; Chen, I. W.; Michelson, S.
R.; Holloway, M. K.; Huff, J. R.; Vacca, J. P. J. Med. Chem. 2000, 43,
3386-3399 (b) Bhupathy, M.; Conlon, D. A.; Wells, K. M.; Nelson, J.
R.; Reider, P. J.; Rossen, K.; Sager, J. W.; Volante, R. P.; Dorsey, B.
D.; Hoffman, J. M.; Joseph, S. A.; McDaniel, S. L. J. J. Heterocycl.
Chem. 1995, 32, 1283-1287 (c) Choi, W. B.; Houpis, I. N.; Churchill,
H. R. O.; Molina, A.; Lynch, J. E.; Volante, R. P.; Reider, P. J.; King,
A. O. Tetrahedron Lett. 1995, 36, 4571-4574 (d) Houpis, I. N.; Choi,
W. B.; Reider, P. J.; Molina, A.; Churchill, H.; Lynch, J.; Volante, R.
P. Tetrahedron Lett. 1994, 35, 9355-9358.
(a) Yonishi, S.; Itani, H.; Sato, Y.; Tsutsumi, H.; Akahane, A. PCT
Int. Appl., 2004, WO 2004089939 A1 (b) Toupence, R. B.;
Debenham, J. S.; Goulet, M. T.; Madsen-Duggan, C. B.; Walsh, T. F.;
Shah, S. K. PCT Int. Appl., 2004, WO 2004012671 A2.
VanSickle, A. P.; Rapoport, H. J. Org. Chem. 1990, 55, 895-901.
Malamas, M. S.; Sredy, J.; Moxham, C.; Katz, A.; Xu, W.; McDevitt,
R.; Adebayo, F. O.; Sawicki, D. R.; Seestaller, L.; Sullivan, D.;
Taylor, J. R. J. Med. Chem. 2000, 43, 1293-1310.
In conclusion,
a series of novel furo [2,3-b] pyridine
carboxamide and pyrido [3',2':4,5] furo [3,2-d] pyrimidinone
derivatives were prepared and evaluated for cytotoxicity against
four human cancer cell lines and one normal (non-tumor) cell
line. Among all the compounds screened, the compounds 4e, 4f,
4g, and 5h showed significant activity against all cell lines at
micro molar concentration. A series of novel furo [2,3-b]
pyridine-2-carbohydrazide Schiff’s base & pyrido [3',2':4,5]
furo [3,2-d] pyrimidin-4(3H)-one derivatives were also
evaluated for anticancer activity. The activity results showed
that Schiff’s base derivatives 7a-h showed promising cytotoxic
activity as compared to pyrido furo pyrimidin-4(3H)-one
derivatives 8a-h. Among the promising ones, compounds 7c,
7d, 7e and 7f exhibited potent anticancer activity against five
human cancer cell lines and all compounds screened against
human normal cell line were found to be non-cytotoxic to
normal cell line, HEK-293. 3D-QSAR CoMFA, CoMSIA
studies have been applied to a set of furo [2,3-b] pyridine
carboxamide and pyrido [3',2':4,5] furo[3,2-d] pyrimidinone
derivatives. Both these CoMFA and CoMSIA models for HeLa,
MFC-7 and COLO205 inhibitors generated have confirmed to
be statistically precise with higher q2 and r2. The information
achieved from CoMFA and CoMSIA models could lead to a
better design of novel selective and higher potent furo[2,3-b]
pyridine carboxamide and pyrido [3',2':4,5] furo [3,2-d]
pyrimidinone derivatives as cancer cell line inhibitors
6.
7.
8.
9.
Flynn, B. L.; Chaplin, J. H.; Paul, D.; Grobelny, D. W.; Kelly, B. B.
PCT Int. Appl., 2008, WO 2008070908 A1.
10. Martin, M. W.; Newcomb, J.; Nunes, J. J.; Bemis, J. E.; McGowan, D.
C.; White, R. D.; Buchanan, J. L.; DiMauro, E. F.; Boucher, C.; Faust,
T.; Hsieh, F.; Huang, X.; Lee, J. H.; Schneider, S.; Turci, S. M.; Zhu,
X. Bioorg. Med. Chem. Lett. 2007, 17, 2299-2304.
11. Wu, Z.; Robinson, R. G.; Fu, S.; Barnett, S. F.; Defeo-Jones, D.;
Jones, R. E.; Kral, A. M.; Huber, H. E.; Kohl, N. E.; Hartman, G. D.;
Bilodeau, M. T. Bioorg. Med. Chem. Lett. 2008, 18, 2211-2214.
12. Chabrier, P. E.; Guinot, P. & Tarrade, T. 1988, 6, 166-179.
13. Bukowski, R. D.; Wagman, P.; De Wan. P & Xue, H. Arch Mal Coeur.
1989, 82, 45-50.
14. Singh, V.; Srivastava, V. K.; Palit, G.; Shanker, K. Drug. Res. 1992,
42, 993-996.
15. Angela, M.; Vedrana, C. C.; Nikolina, R. M.; Milena, V. R.; David,
B.; Euphemia, Y. L.; Johannes. R.; Anita M. Drug Design,
Development and Therapy 2017, 11, 759–769.
Acknowledgements
16. Hertel, L. W.; Kroin, J. S.; Misner, J. W.; Tustin, J. M. J. Org. Chem.,
1988, 53, 2406-2409.
17. Filler, R. Studies in Organic Chemistry. 1993, 48, 362.
18. Chae, J.; Konno, T.; Ishihara, T.; Yamanaka, H. Chem. Lett. 2004, 33,
314-315.
19. Narsaiah, B.; Shivaprasad, A.; Venkataratnam, R. V. J. Fluorine
Chem. 1994, 69, 139-143.
20. Abdel-Rahman, E.; Abdul, E.; Bakhite, Etify, A.; Al-Taifi, Elham. A.
J. Chem. Res. 2005, 2005, 461-468.
21. Mosmann, T. J. Immunol. Methods 1983, 65, 55-63.
22. Cramer, R. D.; Patterson, D. E.; Bunce, J. D. J. Am. Chem. Soc. 1988,
110, 5959-5967.
Authors are thankful to the Council of Scientific and Industrial
Research (CSIR), New Delhi, India for the financial assistance
through a XII five year plan project DITSF code:CSC-0204.
Authors (G. Santhosh Kumar Y. Poornachandra, K. Ratnakar
Reddy) are also thankful to CSIR for providing Senior Research
Fellowship and contingency grant.
23. Klebe, G.; Abraham, U.; Mietzner, T. J. Med. Chem. 1994, 37, 4130-
4146.
References and notes:
24. Gasteiger, J.; Marsili, M. Tetrahedron. 1980, 36, 3219-3228.
25. Roy, K. K.; Dixit, A.; Saxena, A. K. J. Mol. Graph. Model. 2008, 27,
197-208.
26. Cramer III, R. D.; Bunce, J. D.; Patterson, D. E. Quant. Struct. Act.
Relat., 1988, 7, 18-25.
1.
Anand, P.; Kunnumakara, A. B.; Sundaram, C.; Harikumar, K. B.;
Tharakan, S. T.; Lai, O. S.; Sung, B.; Aggarwal, B. B. Pharm. Res.
2008, 25, 2097-2116.
Kawakami, K.; Takahashi, H.; Ohki, H.; Kimura, K.; Miyauchi, S.; R.
Miyauchi, M. Takemura, Chem. Pharm. Bull. 2000, 28, 1667-1672.
Ledoossal, B.; Boazard, D.; Coroneos, E. J. Med. Chem. 1992, 35, 198-
200.
Fokialakis, N.; Magiatis, P.; Aligiannis, N.; Mitaku, S.; Tillequin, F.;
Sévenet, T. Phytochemistry. 2001, 57, 593-596.
(a) Dorsey, B. D.; McDonough, C.; McDaniel, S. L.; Levin, R. B.;
2.
3.
4.
5.
10

Research Highlights
 Novel hetero ring fused pyridine derivatives.
 All products screened against four human cancer cell lines and one normal cell line.
Compounds 4e, 4f, 4g, 5h, 7c, 7d, 7e and 7f showed promising anticancer activity.
 All compounds found to be non-toxic to normal cell line.
11

Graphical Abstract
Synthesis of novel hetero ring fused pyridine derivatives; their anticancer activity, CoMFA and
CoMSIA studies
G. Santhosh Kumar, Y. Poornachandra, Shravan Kumar Gunda, K. Ratnakar Reddy, Jaheer Mohmed, Kamal Shaik,
C. Ganesh Kumar and B. Narsaiah*
Alignment
IC50 (µM) = 5.2 ± 0.18
CoMFA steric contour map
Steric Contribution = 73.6%
A series of novel furo[2,3-b]pyridine-2-carboxamide 4a-h/pyrido[3',2':4,5]furo[3,2-d] pyrimidin-4(3H)-one
5a-p, furo [2,3-b] pyridine-2-carbohydrazide Schiff’s base 7a-h and pyrido [3',2':4,5] furo[3,2-d]
pyrimidin-4(3H)-one derivatives 8a-h were prepared. All the final products 4a-h, 5a-p, 7a-h and 8a-h
were screened against four human cancer cell lines (HeLa, COLO205, HepG2 and MCF7) and one normal
cell line (HEK293). Compounds 4e, 4f, 4g, 5h, 7c, 7d, 7e and 7f showed significant anticancer activity
against all the cell lines at micro molar concentration and found to be non-toxic to normal cell line. Further
studied for HeLa, COLO205 and MCF-7 using CoMFA and CoMSIA. Models obtained from 3D-QSAR
studies provided a strong basis for future rational design of more active and selective HeLa, COLO205 and
MCF-7 cell line inhibitors.
12