2-methyl-3-chloro-9-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidine hydrochloride: Crystal structure and interaction with DNA

Article abstract of DOI:10.1007/s10870-012-0383-9

The title compound, C₉H₈ClN₂O₂ ⁺·Cl^-, is a hydrochloric acid salt of 2-methyl-3-chloro-9- hydroxy-4H-pyrido[1,2-a]pyrimidine-4-one, which is an important intermediate for the synthesis of biologically active heterocyclic compounds. The synthesized compound was characterized by ¹H NMR and X-ray crystallography. The structure was solved in monoclinic, space group P2₁/n with a = 11.8295 (12), b = 6.2214 (6), c = 13.8133 (15) A?, β = 97.7860 (10) , V = 1007.23 (18) A?³, Z = 4, and with R _int = 0.077. The cation of the title compound, as shown by the single-crystal structure determination, has two conjugated aromatic rings that are almost coplanar with a dihedral angle of 0.230. The crystal packing is stabilized by intermolecular N-H···Cl, O- H···Cl hydrogen bonds, which link the molecules into centrosymmetric dimers, and by weak π-π stacking interactions (average distance 3.352 A?). The interaction of native calf thymus DNA (ctDNA) with the compound at physiological pH was monitored by UV-Vis spectroscopy and viscosimetric techniques. It was found that the compound might interact with ctDNA by a groove mode of binding via hydrogen bonds.

Full text of DOI:10.1007/s10870-012-0383-9

J Chem Crystallogr (2013) 43:70–75
DOI 10.1007/s10870-012-0383-9
ORIGINAL PAPER
2-Methyl-3-chloro-9-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidine
Hydrochloride: Crystal Structure and Interaction with DNA
•
Huaihong Zhang Rong Huang Zhaosheng Cai
•
•
•
Chunxiang Xu Baiwang Sun
Received: 22 April 2012 / Accepted: 6 November 2012 / Published online: 27 November 2012
Ó Springer Science+Business Media New York 2012
Abstract The title compound, C9H8ClN2O2 ꢀCl-, is a
hydrochloric acid salt of 2-methyl-3-chloro-9- hydroxy-
4H-pyrido[1,2-a]pyrimidine-4-one, which is an important
intermediate for the synthesis of biologically active het-
erocyclic compounds. The synthesized compound was
Keywords Pyrido[1,2-a]pyrimidine ꢀ Crystal structure ꢀ
?
DNA ꢀ Interaction
Introduction
1
characterized by H NMR and X-ray crystallography. The
structure was solved in monoclinic, space group P21/n with
˚
a = 11.8295 (12), b = 6.2214 (6), c = 13.8133 (15) A,
Aza-bridgehead fused heterocyclic compounds are attrac-
tive and such compounds have always been the main focus
of interest in the search for new pharmaceutical candidates.
Many pyrimidine and pyridopyrimidine derivatives show
interesting pharmaceutical properties, such as: antiviral,
antibacterial, anti-HIV, antiallergic, and antitumoral
activities. For example, pyrimidine ring containing sul-
fonamides constitute one of the oldest groups of antibac-
terial agents. Bicyclic pyrimidines and their derivatives,
especially those having substituents on the pyrimidine ring,
are common drug intermediates and possess significant
biological activities [1–9].
°
3
˚
b = 97.7860 (10) , V = 1007.23 (18) A , Z = 4, and with
Rint = 0.077. The cation of the title compound, as shown
by the single-crystal structure determination, has two
conjugated aromatic rings that are almost coplanar with a
dihedral angle of 0.230°. The crystal packing is stabilized
by intermolecular N–HꢀꢀꢀCl, O–HꢀꢀꢀCl hydrogen bonds,
which link the molecules into centrosymmetric dimers, and
by weak p–p stacking interactions (average distance
˚
3.352 A). The interaction of native calf thymus DNA
(ctDNA) with the compound at physiological pH was
monitored by UV–Vis spectroscopy and viscosimetric
techniques. It was found that the compound might interact
with ctDNA by a groove mode of binding via hydrogen
bonds.
DNA is a major target for drugs and some toxic chem-
icals. Small molecules normally interact with DNA via
noncovalent interaction modes, for instance, (i) intercalat-
ing between stacked base pairs, (ii) noncovalent groove
binding, or (iii) electrostatic interaction with the negatively
charged nucleic acid sugar–phosphate structure [10].
Research into the binding mechanism of some small mol-
ecules with DNA has been regarded as one of the key
topics during the past few decades [11–14]. It is of interest
to understand the action mechanism of some antitumor and
antivirus drugs in order to design new and more efficient
DNA targeted drugs to deal with genetic diseases.
H. Zhang ꢀ R. Huang ꢀ B. Sun (&)
College of Chemistry and Chemical Engineering,
Southeast University, Nanjing 211189, China
e-mail: zhuaih99@yahoo.com.cn
H. Zhang ꢀ Z. Cai
Previously, we performed X-ray diffraction and spec-
troscopic studies of the pyrido[1,2-a] pyrimidine-4-one
analogue [15]. Herein, we report the synthesis and crys-
tal structural of the 2-methyl-3-chloro-9-hydroxy-4H-pyr-
ido[1,2-a]pyrimidine-4-one hydrochloride (MCHPP). The
School of Chemistry and Biology, Yancheng Institute
of Technology, Yancheng 224051, China
C. Xu
State Key Lab of Bioelectronics, Southeast University,
Nanjing 211189, China
123

J Chem Crystallogr (2013) 43:70–75
71
interaction of MCHPP with the native calf thymus DNA
(ctDNA) using in vitro conditions was also investigated.
We believe that this work will provide some useful infor-
mation for the evaluation of the bioactivity of 2-methyl-3-
chloro-9-hydroxy-4H-pyrido[1,2-a]pyrimidine-4-one.
[MCHPP]/[ctDNA] ratio in Tris buffers, where g0 is the
viscosity of the ctDNA solution alone.
Synthesis of 2-methyl-3-chloro-9-hydroxy-
4H-pyrido[1,2-a]pyrimidine-4-one (1)
A suspension of 0.88 g (0.005 mol) of the 2-methyl-9-
hydroxy-4-pyrido[1,2-a]pyrimidine-4-one in 20 mL of
68 % hydrochloric acid was heated in a steam bath until
complete dissolved, 6 mL of 20 % H2O2 was added, and
the heating was continued for 1 h. After cooling, the pre-
cipitate was filtered off and recrystallized from a 7:1 eth-
anol–water mixture, 0.5 g of the title compound was
Experimental
Chemicals and Reagents
The ctDNA was purchased from Sigma. The stock solution
of ctDNA was prepared by dissolving ctDNA in 0.01 M of
Tris buffer at pH = 7.2 (0.01 M of tris(hydroxy-
methyl)aminomethane [Tris] with NaCl concentration at
0.01 M)) and dialyzed exhaustively against the same buffer
for 24 h. The ctDNA solution gave a UV 260/280 nm
absorbance ratio of [1.8, indicating that DNA was suffi-
ciently free of protein. The ctDNA concentration of the
stock solution was determined by UV spectrophotometry in
properly diluted samples, using a molar absorption coeffi-
cient (e = 6,600 M-1 cm-1) at 260 nm. The stock solution
was stored at 4 °C and was used within 5 days. An indi-
vidual stock solution for each compound containing
5 9 10-5 mol L-1 MCHPP, was prepared by dissolving an
appropriate amount of the individual MCHPP in Tris buf-
fer. A fresh working solution was prepared daily by
diluting the stock solution with Tris buffer and used for
different studies. Other used chemicals were of analytical
reagent grade and were used as received. 2-Methyl-9-
hydroxyl-pyrido[1,2-a]pyrimidine-4-one was synthesized
as previously described [16].
1
obtained for a yield of 48 %. H NMR (DMSO-d6, ppm):
14.46 (1H, phenolic), 8.43 (1H, para to nitrogen of pyri-
dine), 7.20–6.06 (2H, aromatic), 1.79 (3H, methyl). Ele-
ment analysis calcd (%) for C9H7ClN2O2: C 51.31, H 3.34,
N 13.31; Found: C 61.13, H 3.59, N 13.72. IR (KBr,
cm-1):3530 (–OH stretching); 3057 (aromatic); 2984 (C–H
stretching of methyl); 1735 (C=O stretching); 1605 (C=N
stretching); 1510, 1471 (aromatic); 1278 (O–H deforma-
tion); 783 (C–Cl). MS (m/e): 209.6 [M?].
Synthesis of 2-methyl-3-chloro-9-hydroxy-
4-pyrido[1,2-a]pyrimidin-4-one Hydrochloride (2)
A weighed sample (2 9 10-3 mol) of compound 1 was
dissolved in ethanol (10 mL), and then added a quantitative
ZnCl2. The pH of the solution was brought to 2 using HCl
with a concentration of 1.0 mol/L. After 2 h stirring at
room temperature, the solution was filtrated and then put in
a peaceful environment. Several days later, the title salt,
2-methyl-3-chloro-9-hydroxy-4-pyrido[1,2-a]- pyrimidin-
4-one hydrochloride (MCHPP), needle-shaped crystals
precipitated. The isolated crystals were filtered off, washed
with ethanol, and dried under vacuum.
Apparatus and Methods
IR spectra were recorded in the range 400–4,000 cm-1 on
Perkin-Elmer Spectrum One FT-IR spectrometer using a
KBr pellet. Elemental analyses (C, H and N) were per-
formed on a Perkin Elmer 2400II CHN elemental analyzer.
1H NMR spectra were measured on a Bruker ARX300
spectrometer with DMSO as solvent. The UV–Vis spectra
for MCHPP interactions with DNA were obtained using a
Unico (UV 2100) spectrophotometer. Solutions of ctDNA
and MCHPP were scanned using a 0.5-cm (1 mL) quartz
cell. The spectra were recorded by progressive addition of
pure MCHPP to the ctDNA solution. A SCHOT AVS450
viscometer in a constant water bath, set at 25 °C, was used
to measure viscosity. Flow time was measured with a
digital stopwatch and the mean values of three replicated
measurements were used to evaluate the viscosity (g) of the
Crystallographic Data Collection and Refinement
Intensity data for the MCHPP hydrochloride was collected
at 298 (2) K on a Siemens SMART CCD diffractometer with
˚
graphite monochromatic Mo Ka radiation (k = 0.7107 A).
Data reductions and absorption corrections were performed
with the SAINT [17] and SADABS software packages [18],
respectively. The structures were solved by direct methods
using SHELXS-97 [19, 20] and were refined by full matrix
least-squares methods using SHELXL-97 [21]. Anisotropic
displacement parameters were refined for all non-hydrogen
atoms. All C-bound H-atoms were placed in idealized
locations and were refined using a riding model, with C–
˚
Uiso(H) = 1.2Ueq(C). The H-atoms
H = 0.93 A and
attached to nitrogen were located in difference Fourier
˚
map and refined with the restraints N–H = 0.86 A and
1=3
samples. The data were reported as ðg=g0Þ versus the
123

72
J Chem Crystallogr (2013) 43:70–75
Table 1 Crystal data and structure refinement information for
MCHPP
Molecular formula
Formula weight
T (K)
[C9H8ClN2O2]?ꢀCl-
247.07
298 (2) K
0.71073
Monoclinic
P21/n
˚
k (A)
Crystal system
Space group
˚
a (A)
11.8295 (12)
6.2214 (6)
13.8133 (15)
97.7860 (10)
1,007.23 (18)
4
˚
b (A)
˚
c (A)
b (°)
3
˚
V (A )
Z
l (mm-1
)
0.623
F(0 0 0)
504
Scheme 1 The synthetic route of the compound (1) and its
hydrochloride salt (2)
Total reflections
Unique reflections
Goodness-of-fit on F2
Rint
4,647
1,770
1.064
9-hydroxy-4H-pyrido[1,2-a]pyrimidine-4-one. The struc-
ture of the title compound was confirmed by mass spec-
1
trum, elemental analyses, and H NMR. The salt of the
0.077
R1 [I C 2r(I)]
wR2 (all data)
0.0458
0.1535
compound (1) was obtained by reacting with HCl in
presence of the ZnCl2. The structure of the salt was
determined by single-crystal X-ray diffraction.
-3
˚
Max/min electron density (e A
)
0.515/-0.551
Uiso = 1.2Ueq(N). The crystallographic details and selected
˚
Crystal Structure of MCHPP
bond lengths (A) and torsion angles (°) are provided in
Tables 1 and 2. Images were created with the Diamond
Single-crystal X-ray analysis reveals that the title salt
crystallizes in the monoclinic space group P21/n. The
asymmetric unit of the crystal is illustrated in Fig. 1.
program [22].
Results and Discussion
Syntheses of MCHPP and its Hydrochloride
The MCHPP was prepared according to Scheme 1. At the
first step of the synthesis, 2-methyl-9- hydroxy-4H-pyr-
ido[1,2-a]pyrimidine-4-one reacted with HCl in presence
of the H2O2 under heated to provide the 2-methyl-3-chloro-
˚
Table 2 Selected bond lengths (A) and torsion angles (°) for
MCHPP
Bond lengths
Cl2–C2
1.725 (3)
1.352 (4)
1.358 (4)
1.353 (4)
N2–C8
N2–C3
O1–C3
O2–C5
1.383 (4)
1.469 (4)
1.211 (4)
1.339 (4)
N1–C4
N1–C1
N2–C4
Torsion angles
C3–N2–C4–C5
C8–N2–C4–N1
C3–N2–C4–N1
-179.5 (3)
-180.0 (3)
-0.7 (5)
C1–C2–C3–O1
C9–C1–C2–Cl2
N2–C4–C5–O2
-176.1(4)
-2.5 (5)
179.2 (3)
Fig. 1 View of the molecule of MCHPP, showing the atom-labelling
scheme. Displacement ellipsoids are drawn at the 30 % probability
level. H atoms are represented by circles of arbitrary size
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J Chem Crystallogr (2013) 43:70–75
73
Excepting methyl hydrogens, the cation of the salt is
almost co-planar as evidenced by the two conjugated rings
with a dihedral angle of only 0.230° and the C8–N2–C4–
N1, Cl2–C2–C3–O1, and C9–C1–C2–C3 torsion angles of
-180.0 (3), 3.5 (5), and 177.1 (4)°, respectively. The Cl2–
ribbon via the face-to-face stacking interactions between
conjugated rings of pyrido[1,2-a]pyrimidine heterocyclic
˚
moiety (average distance 3.352 A) (Fig. 3). Furthermore,
two neighbouring ribbons interact via the weak C3–O1ꢀꢀꢀp
interactions into two-dimension sheets. All hydrogen bond
parameters are listed in Table 3.
˚
C2 bond distance is 1.725(3) A, which is similar to the
found in the crystal structure of chlorophene analogue, i.e.
˚
1.745(5) A [23]. The C–N bond lengths of the pyrido[1,2-
a]pyrimidine heterocyclic moiety are in the range of 1.352
˚
(4)–1.469 (4) A. The N2–C3 bond is longer than the
Study of the MCHPP Interaction with DNA
remaining four C–N bonds within pyrido[1,2-a]pyrimidine
heterocyclic moiety (Table 2). The elongation of the N2–
C3 bond is presumed by the electron withdraw effect of the
chlorine substituent that disturbs the delocalisation of the p
electron of the double bond O1–C3 over these three atoms
(O1, C3 and N2). The O1–C3 bond with a distance of 1.211
Effect of the MCHPP on UV Spectra of ctDNA
The absorption band at around 260 nm of DNA arises
because of the p–p* transition of DNA bases. Changes in
absorbance and wavelength shifts of this characteristic
band reflect the corresponding structural changes of the
DNA, including alteration of stacking pattern, loss of the
hydrogen bonds between complementary strands, covalent
binding of DNA bases, intercalation between aromatic
rings of molecules, etc. [24, 25]. Previous investigations
have shown that hypochromism results from the contrac-
tion of DNA in the helix axis and from the changes in the
conformation of DNA, whereas hyperchromism results
from the disassociation, or melting, of the DNA double
helix strands from each other [26]. The results obtained in
Fig. 4 showed that with increasing amounts of MCHPP, a
slight bathochromic shift (*3 nm) of the band of DNA
centered at the 263 nm was observed, with a significant
˚
˚
(4) A is shorter than the O2–C5 bond (1.339 (4) A), which
is due to the double bond in O1–C3.
There are significant intermolecular hydrogen bond
interactions in the crystal of title compound involving O–
˚
HꢀꢀꢀCl (O2–Cl1 = 2.9635 A) uncharged and anionic
˚
chlorine interactions and N–HꢀꢀꢀCl (N1–Cl1 = 3.2055 A)
charge-assisted ones, which makes a discrete dimer
(Fig. 2). The chloride anion is able to act as a proton
acceptor, interacting with the partially charged -NH group.
The results presented herein can form the basis for pre-
dicting hydrogen bonds between protonated multi-hetero-
rings and chlorine anions. The dimers are extended into a
Fig. 2 A view of the 1D ribbon in the compound along b-axis. All do not take part in H-bond (dashed lines) H atoms are omitted for clarity
(Color figure online)
123

74
J Chem Crystallogr (2013) 43:70–75
Fig. 3 The p–p stacking
interactions between the
pyrido[1,2-a]pyrimidine rings of
neighboring cations of the salt
(Color figure online)
˚
Table 3 Hydrogen bonds for MCHPP [A and °]
via traditional intercalating mode but via a groove binding
interaction with MCHPP [27].
D–HꢀꢀꢀA
d(D–H)
d(HꢀꢀꢀA)
d(DꢀꢀꢀA)
\DHA
N1-H1ꢀꢀꢀCl1a
O2-H2ꢀꢀꢀCl1b
a
0.86
0.82
2.37
2.14
3.2055
2.9635
164
177
Viscosity Measurements
x ? 1/2, -y ? 1/2, z-1/2
Optical and photophysical probes offer necessary, but not
sufficient, clues to support a groove binding model. To
further clarify the interaction between the MCHPP and
ctDNA, viscosity measurements were carried out. Viscos-
ity is a hydrodynamic measurements that is sensitive to
length change and is considered the least ambiguous and
the most important test for a binding model in the absence
of crystallographic data [28, 29]. In classical intercalation,
the DNA helix lengthens as base pairs are separated to
accommodate the bound ligand, leading to increased DNA
b
-x ? 3/2, y ? 1/2, -z ? 1/2
hypochromic effect. This indicated that the conformation
of DNA double-helix structure was changed upon MCHPP
addition, which suggests that there must be some interac-
tions between MCHPP and the DNA. As only a slight
bathochromic shift was observed in UV absorption band of
DNA, the conformation changes of DNA structure is not
Fig. 4 UV/Vis spectra of DNA (5.0 9 10-5 M) with increasing
amounts of the MCHPP in Tris buffers (pH = 7.4) with
Ri = [MCHPP]/[ctDNA] 0.0, 0.2, 0.4, 0.6, 0.8, 1.0. The arrow shows
the direction of the absorbance changes upon increasing MCHPP
concentrations
Fig. 5 Effect of the compound on the viscosity of ctDNA (5 9 10-5 M)
with different Ri in Tris–HCl buffers (pH = 7.4) (Ri = [compound]/
[ctDNA] = 0.0, 0.25, 0.5, 0.75 and 1.0)
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J Chem Crystallogr (2013) 43:70–75
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A distinct compound, MCHPP, has been prepared and
characterized. We have performed a prime study on the
title compound structure and its interaction with ctDNA.
The interaction of the native ctDNA with the title com-
pound in vitro was monitored by spectroscopic methods.
The results from the fluorescence and viscosity studies
suggested that title compound interacts with ctDNA
through a groove mode of binding.
Despite the present results indicated that MCHPP might
interact with native DNA, presumably in a groove mode,
intensive investigation and mechanism studies need to be
carried out for a deeper understanding.
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Crystallographic data for the structural analysis has been
deposited with the Cambridge Crystallo- graphic Data
center, CCDC No. 875504. Copies of this information may
be obtained free of charge from The Director, CCDC, 12
Union Road, Cambridge, CB2, IEZ, UK (fax: ?44-1223-
336033; e-mail: deposit@ccdc.cam.ac.uk or http://www.
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Acknowledgments The present work was supported by the
National Natural Science Foundation of China (No. 20671019). The
authors thank the Order Material Central of Southeast University for
supporting this work.
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