Synthesis, cytotoxicity and hDHFR inhibition studies of 2H-pyrido[1,2-a]pyrimidin-2-ones

Article abstract of DOI:10.1039/c3md00013c

Synthesis of the titled scaffolds was achieved by the condensation of Baylis-Hillman acetates with 2-aminopyridines under solvent-free conditions. Resulting compounds were evaluated for anticancer activity against five different cancer cell lines. Compoun

Full text of DOI:10.1039/c3md00013c

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Synthesis, Cytotoxicity and hDHFR inhibition studies of 2Hꢀ
Pyrido[1,2ꢀa]pyrimidinꢀ2ꢀones
a
a **
b
b
Sreevani Rapolu , Manjula Alla , Roopa Jones Ganji , Venkateshwarlu Saddanapu ,
Chandan Kishor b, Vittal Rao Bommena a,* Anthony Addlagatta b,***
aCrop Protection Chemicals Division, Indian Institute of Chemical Technology, Tarnaka,
Hyderabad, 500607. Eꢀmail: manjula@iict.res.in; Tel: 091 040 27191441; Fax: 091 040
27193382
bCentre for Chemical Biology, Indian Institute of Chemical Technology, Tarnaka, Hyderabad,
500607. Eꢀmail: anthony@iict.res.in;
Synthesis of the titled scaffolds was achieved by the condensation of BaylisꢀHillman acetates
with 2ꢀaminopyridines under solvent free conditions. Resulting compounds were evaluated for
anticancer activity against five different cancer cell lines. Compounds 3cꢀ3g displayed lowꢀ
micromolar inhibition with IC50 values ranging from 0.86ꢀ0.94µM and 3b, 3h, 3i and 3j between
8.6ꢀ9.8µM against neuroblastoma (SKꢀnꢀSH) cancer cell line. 3b, 3i and 3j inhibited the breast
cancer cell (MCFꢀ7) proliferation at 10µM. hDHFR inhibitory studies revealed 3i, 3j are with
IC50 values 2.7 and 3.1µM while 3o at 8.7µM. Molecular docking studies established the mode
of binding of these compounds into methotrexate binding pocket of hDHFR. Structure activity
relationship study indicates a clear preference for some substitutions while others were deterrent.
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Introduction
Investigation of new chemical entities for the treatment of cancer is an emerging area in
pharmaceutical research. Cancer is a disease posing serious threat to human health all over the
world today. Anticancer drugs currently in clinical use throughout the world are associated with
various side effects like gastrointestinal toxicity, constipation, lowering of blood pressure, heart
failure and hair loss.1 Major obstacle for successful treatment of cancer is the multidrug
resistance mediated by Pꢀglycoprotein (Pgp) or multidrug–resistanceꢀassociated protein (MRP).
Inhibition of Pgp and MRP transport is important for high efficacy of anticancer drugs.2 Due to
these drawbacks; there is an immense need for development of more efficacious, selective and
safe chemical agents for the treatment of cancer.
The compatibility of small chemical entities with biological systems is the prime reason
for the design and development of drugs based on small organic molecules such as nucleoside
analogues. A number of pyrimidine antagonists such as 5ꢀfluorouracil, gemcitabin, capecitabine
and floxuridine are currently in use as anticancer drugs.3 In this context, pyridopyrimidines are
an important class of azaꢀbridgehead fused heterocyclic compounds with extremely rich
biological activities due to their structural similarities with nucleobases. Fused pyrimidines are
found to exhibit antiviral,4 antibacterial,5 antiꢀHIV,6 antiꢀplatelet and antithrombotic7
antinociceptive activities.8 They are extensively used in treatment of neurodegenerative disorders
10
like Parkinson’s disease,9 antiꢀanxiety disorders
and depression.11 Their applications are
especially attractive due to inhibition of multidrug resistance. Fused pyrimidine antifolates act as
antitumor agents thus making folate metabolism as an attractive target for cancer chemotherapy
12 and are also proven as selective MRP1 inhibitors. Wide range of pharmacological applications
of pyridopyrimidinone is demonstrated aptly by the drugs already in the market namely
2

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13
14
antiasthematic agent pemirolast
basmastine.15
the tranquillizer pirenperone
and the antiallergic
The role of pyrimidines as MRP1 inhibitors and antifolates, as well as the diverse
biological profile of fused pyrimidines prompted us to develop novel 2Hꢀpyrido[1,2ꢀa]pyrimidinꢀ
2ꢀones as a class of potential anticancer agents. In an earlier study, we have developed
imidazo[1,2ꢀa]pyrazines derivatives as cytotoxic compounds.16 Herein, we report the synthesis of
2Hꢀpyrido[1,2ꢀa]pyrimidinꢀ2ꢀone derivatives, their cytotoxicity against cervical, breast, hepatic,
lung and neuronal cancer cell lines. As pyrimidinone analogues exhibit anticancer activity by
inhibiting dihydrofolate reductase (DHFR) enzyme, we have also tested the inhibition against
hDHFR. Molecular modeling studies were performed to understand the molecular basis for the
DHFR inhibition.
Results and Discussion
Chemistry
Targeted molecules were accessed by employing an atomꢀeconomy BaylisꢀHillman reaction. The
protocol was found to be versatile for generating diversely substituted 2Hꢀpyrido[1,2ꢀ
a]pyrimidinꢀ2ꢀones library (38 compounds) required for our purpose. In this context, a clean and
green method (i.e., solvent and catalyst free approach) for generation of 3ꢀsubstitutedꢀ2Hꢀ
pyrido[1,2ꢀa]pyrimidinꢀ2ꢀones by addition of various 2ꢀaminopyridines to BaylisꢀHillman
acetates (Scheme 1) has already been developed and reported from our group.17 Employing this
method, compounds 3aꢀr and 3v were synthesized in good yields starting from corresponding
Baylisꢀ Hillman acetates. The BaylisꢀHillman acetates themselves were synthesized following
the standard literature protocols.18
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O
R3
OAc O
1a-n
N
N
R5
R4
R4
R5
R2
R1
R3
a
OEt
+
R2
N
NH2
2a-e
3a-r,v
R1
Scheme 1. Reagents and conditions: a) Neat, rt.
A series of substituted 2Hꢀpyrido[1,2ꢀa]pyrimidinꢀ2ꢀones have been synthesized by varying
substitutions both on phenyl ring and pyridopyrimidinone skeleton. Substitutions such as halo,
alkyl, alkoxy, aryl, hetero aryl, nitro, triflouromethyl and so on, have been incorporated as shown
in Table 1.
To increase the diversity of the library sugar conjugates, 3s and 3t were synthesized starting from
Dꢀglucose as shown in Scheme 2. Dꢀglucose was converted to a diastereomeric mixture of αꢀ
20
methyleneꢀβꢀhydroxy ester (4) by following the literature protocol.19 Acetylation of the ester
(4) gave ethyl 2ꢀ(acetoxy(6ꢀ(benzyloxy)ꢀ2,2ꢀdimethylꢀtetrahydrofuro[3,2ꢀd][1,3]dioxolꢀ5ꢀyl)
methyl)acrylate (5), which on nucleophilic addition with 2ꢀaminopyridines under above
conditions gave the corresponding pyrimidinones 3s and 3t.
O
O
O
OH
H
OAc
H
N
O
O
a
b
O
OEt
OEt
H
N
O
O
O
O
OBn
OBn
O
OBn
O
5
3s-t
R4
4
Scheme 2. Reagents and conditions: a) AcCl, Pyridine, DCM, rt, 4ꢀ5h, 80%; b) 2ꢀamino
pyridines, Neat, rt.
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In contrast to other analogues 3u, was obtained by direct condensation of BaylisꢀHillman adduct
(i.e. by avoiding the acetylating step) with the 2ꢀaminopyridine under solvent free conditions
(Scheme 3).
O
O
HO
S
OH
N
a
OEt
+
N
S
N
2
NH2
6
3u
Scheme 3. Reagents and conditions: a) Neat, rt.
Table 1. Pyrido[1,2ꢀa]pyrimidinꢀ2ꢀones synthesized.
Substituents
R3
R1
R2
R4
R5
Entry
Compound
Time
H
F
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
5 min
12h
6h
1
2
3
4
5
6
7
3a
3b
3c
3d
3e
3f
Cl
Br
Br
Cl
Br
Cl
Br
4h
CH3
OCH3
NO2
14h
15h
11h
3g
(3,5,6ꢀ
trichloropyridinꢀ
2ꢀyloxy)
H
H
Cl
Br
H
H
12h
8h
8
9
3h
3i
(3,5,6ꢀ
trichloropyridin
H
H
ꢀ2ꢀyloxy)
F
Br
H
F
H
H
H
H
H
H
H
H
H
CF3
H
H
CF3
H
H
H
H
CF3
H
H
Br
Cl
H
H
Br
H
H
H
H
H
H
NH2
NH2
NH2
NH2
5 min
5 min
10h
12h
5 min
8h
10h
9h
10h
10
11
12
13
14
15
16
17
18
3j
3k
3l
3m
3n
3o
3p
3q
3r
F
H
H
F
F
Br
benzyloxy)ꢀ2,2ꢀdimethylꢀdihydroꢀ5Hꢀ
furo[3,2ꢀd][1,3]dioxolꢀ5ꢀyl)methyl
benzyloxy)ꢀ2,2ꢀdimethylꢀdihydroꢀ5Hꢀ
furo[3,2ꢀd][1,3]dioxolꢀ5ꢀyl)methyl
hydroxy(thiophenꢀ2ꢀyl)methyl
H
H
H
8h
19
20
3s
3t
Cl
10h
H
H
H
H
9h
5 min
21
22
3u
3v
H
C6H5O
H
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Compounds synthesized were characterized by spectral means as described in the experimental
section. Single crystal Xꢀray diffraction data (Compound 3d, Fig. 1) unequivocally solved the
structural ambiguity. It is interesting to note that compound 3d adopts a similar conformation
both in the crystal structure and in the modelling studies of DHFR active site, this indicates
pyridopyrimidinones are not undergo conformational changes on binding with the active site of
DHFR enzyme.
Fig. 1 The molecular structure of 3d with the atomꢀnumbering scheme, displacement ellipsoids
are drawn at the 30% probability level and H atoms are shown as small spheres of arbitrary
radius.
Biological evaluation
(A)In vitro anticancer activity
In vitro cytotoxic effects of these compounds were tested against five different human cancer cell
lines i.e., cervical (HeLa), liver (HEP Gꢀ2), breast (MCFꢀ7), neuroblastoma (SKꢀnꢀSH) and lung
(Aꢀ549) cancer cells (Table 2). Majority of the compounds showed good activity against
neuroblastoma cancer cell lines (Fig. 2). MTT assay reveals that compounds 3c, 3d, 3e, 3f and
3g displayed subꢀmicromolar inhibition of the neuronal cancer cells, while compounds 3b, 3h, 3i
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and 3j at about 10µM, which is comparable with the standard doxorubicin. 3b, 3i and 3j also
inhibited the cell proliferation at 10 µM against the breast cancer cell line (MCFꢀ7). Though it is
not clear from the cell based assays as to why compounds other than 3b, 3i and 3j show poor
cytotoxicity against breast cancer cell lines, factors like poor permeability can be one of the
reasons. Current cytotoxic data suggests that a fluorine atom in the R1 position is important to
exhibit activity. The other tolerated group in R1 is hydrogen, and surprisingly a bromine but not
chlorine. Methyl, methoxy or nitro at R1 seems to destabilize the interaction of these molecules
with the target in the cells. Cytotoxicity data clearly indicates that substitution at R3 and R4 are
not critical.
Table 2. Cytotoxic profiles of pyrido[1,2ꢀa]pyrimidinꢀ2ꢀones against selected human cancer cell
lines.
IC50values against cancer cell lines (µM)
Compound
HeLa
>103
HEP Gꢀ2
>103
MCF 7
75.43±1.12
10.36±0.16
>103
SKꢀnꢀSH
95.42±0.99
9.856±0.14
0.94±1.32
0.87±1.37
0.86±1.15
0.94±2.43
0.94±1.75
8.69±1.76
9.24±2.38
8.69±1.22
163±4.92
137±0.75
106±2.17
125±1.77
116±2.46
149±4.39
126±2.17
133±2.31
>103
Aꢀ549
104.48±1.01
98.28±0.21
105.30±1.25
101.96±1.27
>103
3a
>103
>103
3b
3c
>103
>103
931.12±2.34
>103
>103
97.22±2.26
>103
3d
3e
>103
927.80±2.32
927.82±3.43
>103
>103
>103
>103
3f
3g
>103
>103
>103
992±1.28
>103
96.92±1.21
9.77±2.21
9.87±1.12
157±2.92
>103
181±3.30
200±5.87
105±1.42
>103
135±3.41
151±6.99
>103
>103
>103
3h
3i
>103
>103
87.96±1.23
>103
804±1.22
>103
>103
3j
3k
>103
152 ±5.82
>103
>103
>103
3l
3m
>103
>103
139±2.01
112±0.46
>103
>103
>103
3n
3o
131±3.45
>103
98±0.55
>103
3p
3q
>103
>103
>103
>103
>103
>103
3r
3s
>103
>103
199±2.43
182±7.62
248±9.65
>103
>103
>103
>103
3t
3u
3v
166±1.55
>103
>103
166±1.55
243±10.96
1.0±0.11
>103
>103
141±6.29
10.88±0.21
1.12±0.12
0.95±0.26
10.55±1.14
Doxorubicin
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(B) Inhibition of recombinant human DHFR
Literature reports show that, fused pyrimidinones accomplish their activity by inhibiting the
DHFR enzyme and DHFR itself is a good antiꢀcancer target.21ꢀ25 Compounds 3aꢀ3o were tested
for inhibition against this enzyme (hDHFR) as they shown good anticancer activity. Three
compounds displayed subꢀmicromolar inhibition. 3i and 3j inhibited the enzyme with IC50 at 2.7
and 3.1µM while 3o at 8.7µM (Table 3). It is important to note that, compounds 3i and 3j also
display cytotxicity with IC50 lower than 10 µm against MCFꢀ7and SKꢀnꢀSK, while 3o was less
effective. Other compounds that displayed inhibition against the neuronal cell lines at subꢀ
micromolar concentrations have not presented any appreciable inhibition against the purified
DHFR enzyme. Probably an alternative pathway exists in the cancer cells, which accounts for the
inhibition exhibited by these compounds.
Table 3. hDHFR enzyme inhibitory values (IC50 in µM) of the active compounds.
hDHFR inhibition
Compound
(IC50 values)
2.73±0.039
3.10±0.14
3i
3j
3o
8.72±2.7
0.003 ±0.0005
Methotrexate
(C)Molecular docking of the inhibitors against DHFR
Molecular docking experiments were carried out to understand the mode of interaction between
the human DHFR enzyme and its inhibitors (3i, 3j and 3o) discovered in this study (Fig. 3)
Docking was carried out by using AutoDock4 in a predefined methotrexate binding pocket.26ꢀ27
Docking study suggests that pyridopyrimidine moiety of compounds is surrounded by Ile7, Val8,
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Phe31, Phe34, Val115, Tyr121, Val 50, Met52 and NADPH. 3,5,6ꢀtrichloropyridinꢀ2ꢀ
yloxy)benzyl of 3i and 4ꢀfluoroꢀ2ꢀ(trifluoromethyl)benzyl of 3j moves towards methotrexate
binding pocket of DHFR making favorable interactions with enzyme. Amine substitution at R5
position of pyridopyrimidine on compound 3o make series of hydrogen bonds with NADPH
(3.2Ǻ), side chain Tyr121 (2.8Ǻ), main chain of Ile7 (2.8Ǻ) and V115 (2.8Ǻ). It is important to
note that, the compounds 3i and 3j also display cytotxicity with IC50 lower than 10 µm against
MCF7 and SKꢀnꢀSK cell lines in addition to the inhibition of DHFR. Surprisingly, the
substitution pattern on the 3i and 3j are however different. The 3,5,6ꢀtrichloropyridinꢀ2ꢀyloxy at
R2 in 3i and trifluoromethyl group at R3 in 3j suggests that combination of these two groups in
the same molecule should provide a better activity in addition to NH2 at R5 as in 3o. We are in
the process of combining these three groups in the same molecules in addition to variations in
other parts of the skeleton to improve the activity against DHFR. These improved molecules will
be tested against the drug resistant cancer cell lines.
Figure 2. Graphical representation of IC50 values of best inhibitors against SKꢀnꢀSH cancer cell
line.
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Fig. 3 Molecular modeling of DHFR: Ribbon diagram of DHFR is depicted in grey. NADPH is
displayed as light pink sticks. (A): Crystal structure of the human DHFR in complex with
methotrexate (yellow balls and sticks) PDB: 3NZD. This picture gives an overall view of the
enzyme pocket. (B) and (C): Docking of 3i and 3j, respectively. In both these cases, hydrophobic
forces seem to be responsible for the nature of binding. (D): Docking of the 3o in to the enzyme
pocket. The amino group makes series of interactions both with both the enzyme and the
NADPH, while the hydrophobic aromatic region gets sandwiched within the hydrophobic cleft.
Conclusion
In conclusion, a series of 2Hꢀpyrido[1,2ꢀa]pyrimidinꢀ2ꢀones were synthesized under solvent free
conditions and tested for anticancer activity. These compounds displayed variable degree of
cytotoxic activity against selected cancer cell lines. 50% of the compounds of this series are
active against neuroblastoma cancer cell lines (SKꢀnꢀSH) while three compounds showed good
cytotoxic activity against breast cancer cell lines (MCFꢀ7). In addition, three compounds 3i, 3j
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and 3o inhibited human DHFR enzyme at lowꢀmicromolar concentration. Inputs from the human
DHFR inhibition by 3i, 3j and 3o are being utilized further to design specific inhibitors to this
important human enzyme.
Acknowledgements:
We thank Dr. B. Sridhar Crystallography Department for the single crystal Xꢀray data.
SR and CK would like to thank UGC (New Delhi) for the Research Fellowship, AA thanks DST
and DBT, New Delhi for a research grants, VS and RJG acknowledges Senior Research
Fellowship from the CSIR, New Delhi. All authors wish to thank Director, IICT for the support.
Notes and references
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