Design and synthesis of a series of pyrido[2,3-d]pyrimidine derivatives as CCR4 antagonists

Article abstract of DOI:10.3390/molecules17089961

A series of pyrido[2,3-d]pyrimidine derivatives were designed and synthesized based on known CC chemokine receptor 4 (CCR4) antagonists. The activities of all the newly synthesized compounds were evaluated using a chemotaxis inhibition assay. Compound 6b

Full text of DOI:10.3390/molecules17089961

Molecules 2012, 17, 9961-9970; doi:10.3390/molecules17089961
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Design and Synthesis of a Series of Pyrido[2,3-d]pyrimidine
Derivatives as CCR4 Antagonists
Hongwei Gong 1,2,3, Hui Qi 4, Wei Sun 1, Yang Zhang 4, Dan Jiang 2, Junhai Xiao 2,*,
Xiaohong Yang 1,*, Ying Wang 4 and Song Li 2
1
School of Pharmaceutical Sciences, Jilin University, Changchun 130021, China;
E-Mails: gonghw@jlu.edu.cn (H.G.); wsun@jlu.edu.cn (W.S.)
2
Laboratory of Computer-Aided Drug Design & Discovery, Beijing Institute of Pharmacology &
Toxicology, Beijing 100850, China; E-Mails: jiangdan@263.net.cn (D.J.); lis@nic.bmi.ac.cn (S.L.)
3
School of Public Health, Jilin University, Changchun 130021, China
4
Department of Immunology, School of Basic Medical Sciences, Key Laboratory of Medical
Immunology, Ministry of Health, Peking University Health Science Center, Beijing 100191, China;
E-Mails: qh20021983@163.com (H.Q.); byzhangyang08@163.com (Y.Z.); yw@bjmu.edu.cn (Y.W.)
* Authors to whom correspondence should be addressed; E-Mails: xiaojunhai@139.com (J.X.);
xiaohongyang88@126.com (X.Y.); Tel.: +86-10-6693-0673-712 (J.X.); +86-431-8561-9660 (X.Y.).
Received: 26 June 2012; in revised form: 6 August 2012 / Accepted: 13 August 2012 /
Published: 20 August 2012
Abstract: A series of pyrido[2,3-d]pyrimidine derivatives were designed and synthesized
based on known CC chemokine receptor 4 (CCR4) antagonists. The activities of all the
newly synthesized compounds were evaluated using a chemotaxis inhibition assay.
Compound 6b was proven to be a potent CCR4 antagonist that can block cell chemotaxis
induced by macrophage-derived chemokine (MDC), thymus and activation regulated
chemokine (TARC), and CKLF1, the natural ligands of CCR4. In addition, compound 6b
is more effective than budesonide in the murine rhinitis model. The intravenous injection
LD50 of compound 6b is 175 mg/kg and the oral LD50 is greater than 2,000 mg/kg.
Keywords: CC chemokine receptor 4 (CCR4) antagonists; CKLF1; TARC; MDC;
inflammatory disease

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1. Introduction
CC chemokine receptor 4 (CCR4) is a pivotal factor in the development of allergic inflammations,
such as asthma, dermatitis, and rhinitis [1]. It consists of a seven-transmembrane G-protein-coupled
receptor that is selectively expressed on Th2 cell membranes. CCR4 has three natural ligands:
(1) thymus and activation regulated chemokine (TARC/CCL17); (2) macrophage-derived chemokine
(MDC/CCL22); and (3) chemokine-like factor 1 (CKLF1) [2–4]. Through the chemotaxis of the three
ligands of CCR4, the Th2 cells are attracted to the sites of allergic inflammation. The number of MDC,
TARC, and CCR4-expressing T cells is increased in asthmatic lungs and airways. In murine asthmatic
models, the CCR4 blocking antibody attenuates airway eosinophilia and goblet cell hyperplasia and
diminishes IgE synthesis and bronchial hyperreactivity [5]. Like the CCR4 antibody, the special Ab
against TARC and MDC can also reduce airway eosinophilia and hyperresponsiveness in asthmatic
mice elicited by OVA [6,7]. Furthermore, CCR4 knockout mice treated with Mycobacterium bovis
Bacille–Calmette–Guerin experience a two-week delay in bacterial clearance and diminished late-stage
inflammation [8]. Therefore, CCR4 and its three ligands (TARC, MDC, and CKLF1) play important
roles in allergic inflammations, and CCR4 antagonists have a huge potential in the therapeutics of the
allergic diseases. In addition, interrupting the interaction between CCR4 and its ligands is a potential
therapeutic route for autoimmune diseases.
CKLF1 is the third natural ligand of CCR4. Although it bears no significant similarity to TARC and
MDC, there are some same pivotal amino acids beside the conserved CC motif [4]. Mice with
overexpressed CKLF1 have significant pathological changes that are similar to those of asthma, such
as peribronchial leukocyte infiltration, tracheal epithelial shedding, and collagen deposition in lungs.
Obvious pathological changes also appeared in the lungs of the CKLF1 transgenic mice, whereas no
such change was observed in other organs [9]. Interestingly, the CKLF1 C-terminal peptides C19 can
inhibit chemotaxis induced by both CKLF1 and TARC. In the asthmatic mouse model, C19 can reduce
airway eosinophilia, lung inflammation, and airway hyperresponsiveness. However, the CKLF1
C-terminal peptide C27 has the same functional activity as that of CKLF1 [10].
As the studies on CCR4 deepen, an increasing number of highly active small molecular
CCR4 antagonist classes have been discovered [11–20]. All the CCR4 antagonists are inhibitors of
TARC and MDC. Our research aimed to develop more potent CCR4 antagonists that can inhibit
the emigration of CCR4-expresing cells induced by MDC, TARC, and CKLF1, so a series of
pyrido[2,3-d]pyrimidine derivatives were designed and synthesized, and the activities of all the newly
synthesized compounds were evaluated using a chemotaxis inhibition assay.
2. Results and Discussion
2.1. Chemistry
Compound BMS-397 (Figure 1) is the most potent CCR4 antagonist for TARC and MDC among all
the antagonists [11]. By researching the structure-activity realationship of BMS-397, we presumed that
the section A of BMS-397 has large contribution to the activity. This led us to modify this site by
introducing different lengths of carbochains and carbocycles, including heteroatoms. Following the
design, 6b, 7a–d and 8 have been synthesized, and the synthetic routes are illustrated in Scheme 1.

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According to well-established literature procedures, the thermal cyclization of commercially
available 2-aminonicotinic acid and urea produced 2, which was further chlorinated with phosphorus
oxychloride to produce 3 [21]. Intermediate 3 was sequentially nucleophilically substituted with
2,4-dichlorobenzylamine and piperazine to produce 5. Then, 5 was condensed with (R)-1-(tert-butoxy-
carbonyl)piperidine-2-carboxylic acid or (R)-4-(tert-butoxycarbonyl)thiomorpholine-3-carboxylic acid
to produce the corresponding amides. Thereafter, Boc deprotection with trifluoroacetic acid and
dichloromethane was performed to produce the desired compounds 6a and 6b, respectively. Finally,
compounds 7a to 7d were synthesized by the condensation reaction of 5 with different carboxylic
acids (R2COOH), and compound 8 was then obtained by the nucleophilic substitution of 5 and
bromopropane. All of the synthesized compounds were characterized using 1H-NMR and MS analyses.
2.2. Chemotaxis Inhibition Assay
The activities of all the synthesized compounds were evaluated using a CCR4-MDC/TARC/C27
chemotaxis inhibition assay (Table 1). Considering that the functional activity of CKLF1-C27 is
similar to that of CKLF1, we performed a chemotaxis inhibition assay induced by C27 [10,22].
Compound 6a (BMS-397) was used as the calibration or comparison standard for the results in all the
assays, because it is one of the most potent CCR4 antagonists [11]. In Table 1, compound 6b
demonstrate proximate chemotaxis inhibition activities for MDC/TARC and better chemotaxis
inhibition activities for C27 compared with compound BMS-397.
According to biological evaluation results (Table 1), the introduction of carbonyl groups interacting
with piperazine increases inhibitory potency, because 7d exhibits a higher activity than 8. The changes
from 7a, 7b, to 7c, 7d demonstrate that the CCR4-MDC/TARC chemotaxis inhibitory potency is
enhanced by increasing the volume of the groups interacting with carbonyl. Furthermore, the cyclic
groups with hydrogen bond donors exhibit the best contribution to the activity, because both 6a and 6b
are more effective than 7a. However, for the CCR4-C27 chemotaxis inhibition, the structure–activity
relationship follows an opposite rule (i.e., 7a with a larger substitute exhibits a lower inhibitory
activity than 7b, 7c and 7d for CCR4-C27).
Figure 1. The structure of BMS-397.

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Scheme 1. Synthesis of 6a and 6b, 7a–d, and 8.
Table 1. Chemotaxis inhibition assay results for compounds (Inh. % at 1 μM).
Compound
BMS-397
6b
7a
7b
7c
7d
8
CCR4-MDC
60.3 ± 3.5 61.4 ± 1.0 52.2 ± 4.3 40.0 ± 4.9 32.0 ± 0.3 29.7 ± 3.7 23.3 ± 6.7
CCR4-TARC 59.2 ± 4.0 57.3 ± 4.7 48.8 ± 5.4 47.7 ± 7.2 48.7 ± 6.3 48.1 ± 6.6 34.8 ± 0.6
CCR4-C27
CCR3-CCL11
CCR5-CCL5
CXCR1-IL-8
CXCR4-SDF-1
9.6 ± 3.5
21.4 ± 1.5 16.8 ± 5.2 35.7 ± 7.0 34.7 ± 0.7 37.1 ± 1.8 −1.5 ± 8.8
2.9 ± 8.9
−7.4 ± 41.4
2.9 ± 5.1
−4.8 ± 3.2
a Values are means of three independent experiments.

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Compound 6b demonstrates high chemotaxis inhibition activities for CCR4. In order to study the
receptor specificity of compound 6b, we evaluated its chemotaxis inhibition activities for the related
chemokine receptors, CCR3, CCR5, CXCR1 and CXCR4 (Table 1). As shown in Table 1, the poor
chemotaxis inhibition activities proved that compound 6b has high selectivity when tested against the
related chemokine receptors (CCR3, CCR5, CXCR1 and CXCR4).
2.3. Effects of 6b Administration on Symptoms of Murine Allergic Rhinitis
In the murine rhinitis model (sensitized with ovalbumin), budesonide (an efficient glucocorticoid)
was used as the calibration or comparison standard to assess the relative efficacy of the compound.
Five parameters was used to assess the effects of compounds administration on symptoms of murine
allergic rhinitis: (1) the number of sneezing in ten minutes; (2) the number of rubbing nose in ten
minutes; (3) the IL-4 level in the bronchoalveolar lavage fluid; (4) the IgE level of serum; (5) the
number of eosinophils in noses and pulmonary tissues [23]. The efficacy of 1.28 mg/Kg of budesonide
in the five parameters was achieved by only 10 μg/Kg of compound 6b (data not published).
2.4. Determination of Acute Toxicity
The acute toxicity of compound 6b was determined with up-and-down procedure. The intravenous
injection LD50 of compound 6b in female Kunming mice is 175 mg/kg and the oral LD50 is greater
than 2,000 mg/kg. The results indicate that compound 6b has low bioavailability and the security is
poor. Considering the administration dose is only 10 μg/Kg, the therapeutic window is very wide.
3. Experimental
3.1. Chemistry
3.1.1. Materials and Reagents
Melting points were determined using a YRT-3 melting point detector and were uncorrected. The
NMR spectra were recorded using a Bruker ARX 400 spectrometer (Karlsruhe, Germany). The mass
spectra were determined using an Agilent 5875(EI) spectrometer (Palo Alto, CA, USA). All solvents
and reagents were purchased commercially and used without further purification.
3.1.2. Chemical Synthesis
Pyrido[2,3-d]pyrimidine-2,4-diol (2). Compound 2 was synthesized according to a well-established
1
literature procedure [21]. Yield 54%. H-NMR (DMSO-d6) δ ppm: 11.69 (1H, s), 11.48 (1H, s), 8.61
(1H, m), 8.27 (1H, m), 7.26 (1H, m).
2,4-Dichloropyrido[2,3-d]pyrimidine (3). Compound 3 was synthesized according to a
1
well-established literature procedure [21]. Yield 85%. H-NMR (CDCl3) δ ppm: 9.34 (1H, m), 8.66
(1H, m), 7.76 (1H, m); EI-MS (m/z): 199.0 [M]+.

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2-Chloro-N-(2,4-dichlorobenzyl)pyrido[2,3-d]pyrimidin-4-amine (4). 2,4-Dichlorobenzylamine (10.03 g,
0.057 mol) was dropped into the mixture of compound 3 (10.36 g, 0.052 mol) and
N,N-diisopropylethylamine (DIEA, 7.35 g, 0.057 mol) in 1,2-dichloroethane (90 mL) under −10 °C.
After stirring for 5 h, the precipitate was filtered to obtain compound 4 (16.35 g, 93%) as a white
1
solid. H-NMR (DMSO-d6) δ ppm: 9.57 (1H, m), 9.03 (1H, m), 8.79 (1H, m), 7.69 (1H, m), 7.64
(1H, m), 7.45 (2H, m), 4.79 (2H, d, J = 5.2 Hz); EI-MS (m/z): 339.2 [M+H]+.
N-(2,4-Dichlorobenzyl)-2-(piperazin-1-yl)pyrido[2,3-d]pyrimidin-4-amine (5).
A
mixture of
compound 4 (16.35 g, 0.048 mol) and piperazine (8.27 g, 0.096 mol) in ethanol (1,200 mL) was
heated to 60 °C and stirred for 15 h. Ethanol was removed under reduced pressure. The residue was
purified through column chromatography (silica gel) eluted with ethyl acetate, methanol, and ammonia
water (v:v:v = 1:1:0.039) to obtain compound 5 (13.86 g, 74%) as a white solid. 1H-NMR (DMSO) δ
ppm: 8.85 (1H, m), 8.66 (1H, m), 8.46 (1H, m), 7.63 (1H, m), 7.38 (2H, m), 7.10 (1H, m), 4.73 (2H, d,
J = 5.2 Hz), 3.63 (4H, s), 2.61 (4H, s); EI-MS (m/z): 389.2 [M+H]+.
(R)-(4-(4-((2,4-dichlorobenzyl)amino)pyrido[2,3-d]pyrimidin-2-yl)piperazin-1-yl) (thiomorpholin-3-yl)
methanone (6b). A mixture of compound 5 (1.28 g, 3.28 mmol), (R)-4-(tert-butoxycarbonyl)
thiomorpholine-3-carboxylic acid (0.81 g, 3.28 mmol), 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (EDCI) (0.94 g, 4.92 mmol), 1-hydroxybenzotriazole (HOBt)
(0.66 g, 4.92 mmol), and DIEA (0.85 g, 6.56 mmol) in tetrahydrofuran (40 mL) was stirred for 2 h
at room temperature. The solvent was removed under reduced pressure. 20 mL of trifluoroacetic
acid and dichloromethane (v:v = 1:4) were added to the residue, and the mixture was stirred for
2 h at room temperature. The solvent was removed under reduced pressure. Then, 20 mL of water
was added to the residue. The pH of the solution was raised to 8 with 1 N sodium hydroxide, and the
solution was extracted using dichloromethane (3 × 20 mL). The combined dichloromethane extracts
were concentrated under reduced pressure. Thereafter, the residue was purified through column
chromatography (silica gel) eluted with ethyl acetate, methanol, and ammonia water (v:v:v = 6:1.5:0.039)
to produce compound 6b (1.23 g, 73%) as a white solid, m.p.: 168–170 °C. 1H-NMR (CDCl3) δ ppm:
8.79 (1H, m), 7.93 (1H, dd, J = 2.0 Hz, J = 1.7 Hz), 7.44 (1H, d, J = 2.0 Hz), 7.34 (1H, d, J = 8.4 Hz),
7.22 (1H, m), 7.05 (1H, m), 6.26 (1H, t), 4.86 (2H, d, J = 5.9 Hz), 4.09–3.44 (10H, brm), 3.14 (1H, m),
2.82 (2H, m), 2.44 (2H, m). EI-MS (m/z): 518.3 [M+H]+. Elemental analysis, calculated for
C23H25Cl2N7OS (518.46): C, 53.28; H, 4.86; N, 18.91. Found: C, 52.99; H, 4.84; N, 18.86.
Cyclohexyl(4-(4-((2,4-dichlorobenzyl)amino)pyrido[2,3-d]pyrimidin-2-yl) piperazin-1-yl) methan-one
(7a). A mixture of compound 5 (0.4 g, 1.03 mmol), cyclohexanecarboxylic acid (0.13 g, 1.03 mmol),
EDCI (0.30 g, 1.55 mmol), HOBt (0.21 g, 1.55 mmol), and DIEA (0.27 g, 2.06 mmol) in
tetrahydrofuran (10 mL) was stirred for 2 h at room temperature. The solvent was removed under
reduced pressure. Then, the residue was purified through column chromatography (silica gel) eluted
with ethyl acetate, methanol, and ammonia water (v:v:v = 15:1:0.078) to obtain compound 7a (0.41 g,
81%) as a white solid, m.p.: 177–179 °C. 1H-NMR (CDCl3) δ ppm: 8.76 (1H, m), 8.05 (1H, d, J = 7.0 Hz),
7.41 (1H, d, J = 2.0 Hz), 7.35 (1H, d, J = 8.1 Hz), 7.21 (1H, m), 7.04 (1H, m), 6.56 (1H, s), 4.86 (2H,
d, J = 5.9 Hz), 3.96 (4H, d, J = 20.8 Hz), 3.65 (2H, s), 3.52 (2H, s), 2.51 (1H, m), 1.83 (5H, m), 1.52

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(2H, m), 1.28 (3H, m); EI-MS (m/z): 499.1 [M+H]+. Elemental analysis, calculated for C25H28Cl2N6O
(499.44): C, 60.12; H, 5.65; N, 16.83. Found: C, 59.91; H, 5.62; N, 16.79.
1-(4-(4-((2,4-Dichlorobenzyl)amino)pyrido[2,3-d]pyrimidin-2-yl)piperazin-1-yl)-3-(methylthio)-
propan-one (7b), 1-(4-(4-((2,4-Dichlorobenzyl)amino)pyrido[2,3-d]pyrimidin-2-yl)-piperazin-1-yl)-2-
methylpropan-1-one (7c) and 1-(4-(4-((2,4-dichlorobenzyl)amino)pyrido[2,3-d]-pyrimidin-2-
yl)piperazin-1-yl)propan-1-one (7d). The compounds 7b, 7c and 7d were obtained as white solids
1
through the same method used to obtain compound 7a. Compound 7b, m.p.: 201–203 °C. H-NMR
(CDCl3) δ ppm: 8.75 (1H, m), 8.10 (1H, d, J = 7.6 Hz), 7.41 (1H, d, J = 2.0 Hz), 7.34 (1H, d, J = 8.0 Hz),
7.20 (1H, m), 7.05 (1H, m), 6.71 (1H, s), 4.85 (2H, d, J = 6.4 Hz), 3.91 (4H, brm), 3.67 (2H, m), 3.51
(2H, m), 2.86 (2H, t, J = 7.3 Hz, J = 7.8 Hz), 2.69 (2H, t, J = 7.8 Hz, J = 7.0 Hz), 2.15 (3H, s); EI-MS
(m/z): 495.4 [M+H]+. Elemental analysis, calculated for C22H24Cl2N6OS (491.44): C, 53.77; H, 4.92;
1
N, 17.10. Found: C, 53.69; H, 4.88; N, 17.03. Compound 7c, m.p.: 218–219 °C. H-NMR (CDCl3)
δppm: 8.77 (1H, m), 8.02 (1H, d, J = 7.6 Hz), 7.42 (1H, d, J = 2.0 Hz), 7.35 (1H, d, J = 8.2 Hz), 7.21
(1H, m), 7.05 (1H, m), 6.49 (1H, s), 4.86 (2H, d, J = 5.6 Hz), 3.98 (4H, d, J = 21.6 Hz), 3.66 (2H, m),
3.54 (2H, m), 2.86 (1H, m), 1.17 (6H, d, J = 6.7 Hz); EI-MS (m/z): 459.2[M+H]+. Elemental analysis,
calculated for C22H24Cl2N6O (459.37): C, 57.52; H, 5.27; N, 18.29. Found: C, 57.51; H, 5.26; N, 18.27.
1
Compound 7d, m.p.: 209–210 °C. H-NMR (CDCl3) δ ppm: 8.76 (1H, m), 8.07 (1H, d, J = 7.3 Hz),
7.42 (1H, d, J = 2.2 Hz), 7.35 (1H, d, J = 8.4 Hz), 7.2 (1H, m), 7.05 (1H, m), 6.61 (1H, s), 4.86 (2H, d,
J = 5.9 Hz), 3.97 (4H, d, J = 19.3 Hz), 3.66 (2H, m), 3.50 (2H, m), 2.43 (2H, q), 1.20 (3H, t); EI-MS
(m/z): 444.9 [M+H]+. Elemental analysis, calculated for C21H22Cl2N6O (445.34): C, 56.64; H, 4.98; N,
18.87. Found: C, 56.64; H, 4.99; N, 18.86.
N-(2,4-Dichlorobenzyl)-2-(4-propylpiperazin-1-yl)pyrido[2,3-d] pyrimidin-4-amine (8). A solution of
compound 5 (0.4 g, 1.03 mmol), 1-bromopropane (0.14 g, 1.13 mmol), and DIEA (0.15 g, 1.13 mmol)
in 1-Methyl-2-pyrrolidone (NMP, 10 mL) was stirred for 2 h at room temperature. The solvent
was removed under reduced pressure. The residue was then purified through column chromatography
(silica gel) eluted with ethyl acetate, methanol, and ammonia water (v:v:v = 15:1:0.078) to obtain
compound 8 (0.35 g, 79%) as a white solid, m.p.: 181–182 °C. 1H-NMR (CDCl3) δ ppm: 8.74 (1H, m),
7.90 (1H, d, J = 6.7 Hz), 7.41 (1H, d, J = 2.2 Hz), 7.36 (1H, d, J = 8.1 Hz), 7.21 (1H, m), 6.99 (1H, m),
6.18 (1H, s), 4.86 (2H, d, J = 5.6 Hz), 3.97 (4H, s), 2.48 (4H, s), 2.36 (2H, t, J = 7.6 Hz, J = 7.8 Hz),
1.57 (2H, m), 0.95 (3H, t, J = 7.3 Hz, J = 7.6 Hz); EI-MS (m/z): 431.2 [M+H]+. Elemental analysis,
calculated for C21H24Cl2N6 (431.36): C, 58.47; H, 5.61; N, 18.48. Found: C, 58.25; H, 5.62; N, 18.45.
3.2. Bioassay Methods for Chemotaxis Inhibition
The chemotaxis assay was performed using a 48-well microchemotaxis chamber (Neutroprobe,
Bethesda, MD, USA). C27 (Hybio Engineering Company, Shenzhen, China)/hCCL17/hCCL22
(Peproteche, Rocky Hill, NJ, USA) was diluted using buffer (RPMI 1640, 0.1% BSA) to a
100 ng·mL−1/80 ng·mL−1/10 ng·mL−1 final concentration and placed in the lower wells (27.5 µL/well).
The compounds were diluted with DMSO to achieve a 1 mM concentration and then diluted to a 10 µM
concentration in 0.1% BSA medium. The HEK293 cells that were transfected with pcDI-CCR4 were
suspended in an assay buffer at 1 × 106 cells/mL, incubated with the compounds (1 µM final

Molecules 2012, 17
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concentration) for 30 min at 37 °C, and then added to the upper wells (55 µL/well). Between the lower
and upper wells are polyvinylpyrrolidone-free polycarbonate filters (10 µm pores, Neutroprobe) coated
with Rat Tail Collagen Type 1 (Biomedical Technologies). The chamber was incubated for 5 h at 37 °C
with 5% CO2. The filters were removed from the chamber, washed, fixed, and stained using the
Three Step Stain Set (Richard-Allen Scientific Michigan, MI, USA). The migrated cells were counted
in five randomly selected high-power fields (400×) per well. All samples were assayed thrice.
The chemotactic index (CI) is the ratio of the number of cells that migrated to the sample to
the number of cells that migrated to the 0.1% BSA medium. The standard for the significant
chemotaxis is CI > 2. The chemotaxis inhibition percentage was computed using the formula
(1 − CIcompound-pretreated cells/CIDMSO-pretreated cells) × 100%. The chemotaxis assay of compound 6b for
receptors, CCR3, CCR5, CXCR1, CXCR4 was performed using the same method with CCR4
chemotaxis inhibition. The HEK293 cells were transfected with pcDI-CCR3/CCR5/CXCR1/CXCR4,
and the concentration of CCL11/ CCL5/ IL-8/ SDF-1 is 100 ng·mL−1.
4. Conclusions
In summary, we designed and synthesized a series of pyrido[2,3-d]pyrimidine derivatives and
evaluated their in vitro and in vivo activities. Compound 6b was found to be a potent CCR4 antagonist
(in the CCR4-MDC/TARC/C27 chemotaxis inhibition assay). The current study also proved that
compound 6b is even more effective than budesonide in the murine rhinitis model. Although the
intravenous injection LD50 of compound 6b is low (175 mg/kg), the therapeutic window is very wide.
Further studies on the modification and mechanism of these compounds are in progress.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (90813025) and
National Science and Technology Major Project of China (2012ZX09301003).
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Sample Availability: Samples of the compounds 6b, 7a–d, and 8 are available from the authors.
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