Design, synthesis and anti-tumoractivity of novel amidine derivatives of doxifluridine

Article abstract of DOI:10.1016/j.cclet.2009.10.013

A series of novel amidine derivatives of doxifluridine were synthesized using acid amide as the starting material, and their anti-tumor activity was evaluated in A549 cells. Compounds 10 and 11 demonstrated were more potent than 5-Fu, which was used as a positive control. Compound 10, which were found to be the most potent one with IC₅₀ of 3.2 μmol/L, was 16 times more potent than 5-Fu with IC₅₀ of 52 μM to the A549 cells. A new route was designed to synthesize 5′-deoxy-5-fluorocytidine. All compounds were characterized by ¹H NMR, MS and X-ray spectras in detail.

Full text of DOI:10.1016/j.cclet.2009.10.013

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Chinese Chemical Letters 21 (2010) 163–166
www.elsevier.com/locate/cclet
Design, synthesis and anti-tumoractivity of novel amidine
derivatives of doxifluridine
*
Ji Lu Feng, Hai Ling Sun, Dong Ping Geng, Ke Li
Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical University, Shanghai 200433, China
Received 18 June 2009
Abstract
A series of novel amidine derivatives of doxifluridine were synthesized using acid amide as the starting material, and their anti-
tumor activity was evaluated in A549 cells. Compounds 10 and 11 demonstrated were more potent than 5-Fu, which was used as a
positive control. Compound 10, which were found to be the most potent one with IC50 of 3.2 mmol/L, was 16 times more potent than
5-Fu with IC50 of 52 mM to the A549 cells. A new route was designed to synthesize 50-deoxy-5-fluorocytidine. All compounds were
1
characterized by H NMR, MS and X-ray spectras in detail.
# 2009 K. Li. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Doxifluridine; Amidine; Synthesis; Anti-tumor; Crystal structure
1. Introduction
Nucleoside analogues have been used as antiviral and anticancer agents for several decades [1–3]. Among various
nucleoside analogues, uridine and cytidine nucleoside analogues have shown significant antiviral and antineoplastic
activity [4–6]. Our interest in nucleoside and nucleotide analogues is to prepare novel amidine derivatives of
doxifluridine. Derivatives of doxifluridine are of great importance because of their remarkable anti-tumor and anti-
virus activity [7]. Amidines have been investigated for their capacity to protect deoxyadenosine, deoxycytidine, 5-
methyldeoxycytidine, cytidine, and deoxyguanosine and improve their bioavailability [8]. New formamidine-3TC
(3TC, 2,3-dideoxy-3-thiacytidine) analogues have been synthesized and should not be considered as classical
prodrugs reported by Anastasi et al. [9]. Therefore, we designed and synthesized a series of novel amidine derivatives
of doxifluridine and their anti-tumor activity was evaluated in A549 cells [10]. Most of compounds showed good
inhibition against A549 cells in vitro. A new route was designed to synthesize 50-deoxy-5-fluorocytidine and its yield
was higher than reported [11]. The crystal of compound 7 was grown in alcohol, and its structure determined by X-ray
diffraction and the result indicated that the configuration of N(7) C(8) was in an (E)-form.
The preparation of target compounds 6–11 was illustrated in Scheme 1. We designed a novel synthetic of 50-deoxy-
5-fluorocytidine 3. Firstly, compound 1 was treated with acetic anhydride to protect its 20,30-dihydroxy group, then
mixed with phosphorus pentasulfide under refluxing in anhydrous pyridine for 10 h to form product 2 [12] in 90%
* Corresponding author.
E-mail address: proflike@sina.com (K. Li).
1001-8417/$ – see front matter # 2009 K. Li. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
doi:10.1016/j.cclet.2009.10.013

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J.L. Feng et al. / Chinese Chemical Letters 21 (2010) 163–166
Scheme 1. Reagents and conditions: (a) anhydrous pyridine, (CH3CO)2O, 0 8C, 20 h; (b) P2S5, anhydrous pyridine, 110 8C 10 h; (c) NH3ꢂH2O
(20%), CH3OH, 0 8C, 5 h; (d) NH3, CH3OH, 60 8C, 10 h; (e) methyl sulfate, 90 8C, 4 h; (f) CH3ONa, petroleun ether, 0 8C, 30 min; (g) CH3OH, r.t.,
10 h. (6)
R
R N(CH3)2; (7) R N(CH2CH3)2; (8) R N(CH2CH2CH2CH3)2; (9) R N(CH(CH3)2)2; (10) R NCH2CH2CH2CH2; (11)
NCH2CH2CH2CH2CH2.
yield. After removing acetyl group at 20,30-position of compound 2 by ammonia water, treatment with dry ammonia
gas under 60 8C for 10 h in methyl alcohol gave compound 3 in 75% yield.
Acetal compounds 5 were prepared from secondary amine according to the method described by Yu et al. [10].
Treatment of secondary amine with acetic anhydride gave compounds 4. Followed, freshly prepared sodium
methoxide and compounds 4 was strongly stirring in petroleun ether (bp: 30–60 8C) at 0 8C for 30 min resulted in
compounds 5 in 70–80% yield. The prepared acetal compounds 5 were moisture and oxygen sensitive, so it is better to
utilize these compounds to prepare amidine immediately. Subsequently, compound 5 and 50-deoxy-5-fluorocytidine 3
were stirring together in anhydrous methyl alcohol at room temperature for 10 h obtained compounds 6–11, which
were easily separated by silica gel chromatography and characterized by NMR spectra [13].
In order to determine the structure of target compounds, we obtained a monocrystal of compound 7, as shown in
Fig. 1, and its CCDC No. was 724668. The colorless single crystal was obtained in ethanol at 4 8C. The crystal
structure belong to orthorhombic, crystal size 0.12 mm ꢀ 0.10 mm ꢀ 0.08 mm, space group P21/c with a = 6.071(3),
3
3
˚
˚
b = 12.877(7), c = 11.168(6) A, a = b = g = 908, Z = 2, V = 865.0(8) A , Dc = 1.314 Mg/m , F(0 0 0) = 364,
m = 0.103 mmꢁ1, the final R = 0.0485 and Rw = 0.1240 for 3904 observed reflections (I > 2s(I)). The torsion angle
of C(4)–N(7)–C(8)–N(9) is 165.1(3)8 indicated that the configuration of N(7) C(8) was in an (E)-form.
The anti-tumor activities of synthesized compounds 6–11 were tested against A549 cells in vitro by the standard
MTT method with 5-Fu as a positive control and the results were summarized in Table 1. As shown in Table 1, all
synthesized compounds, except for compounds 6 and 8, had good anti-tumor activities. These compounds
demonstrated potent anti-tumor activity with IC50 values of 3.00–500 mmol/L. Compounds 10 and 11 exhibited more
potent anti-tumor activity than 5-Fu with IC50 values of 3.2 and 7.2 mmol/L, respectively. The most active one,
compound 10, was almost 16 times more with IC50 values of 3.2 mmol/L to A549 cells. The SAR research showed that
compounds 10 and 11 with piperidine and pyrrolidine group had noticeable anti-tumor activities. However,
compounds 6, 7 and 8 with one, two and four carbon atoms of straight-chain group and compound 9 with branched-

J.L. Feng et al. / Chinese Chemical Letters 21 (2010) 163–166
165
Fig. 1. Crystal structure of compound 7.
Table 1
Anti-tumor evaluation of all synthesized compounds.
Compounds
R
A549/IC50 (mmol/L)
6
N(CH3)2
>600
476
>600
339
3.2
7
N(CH2CH3)2
8
N(CH2CH2CH2CH3)2
N(CH(CH3)2)2
9
10
11
5-Fu
NCH2CH2CH2CH2
NCH2CH2CH2CH2CH2
7.2
52
‘‘IC50’’ is 50% inhibitory concentration.
chain group have lower potent than 5-Fu. Given the potency of the compounds against A549, further structural
modification aiming at discovering more potent compounds is under investigation in our laboratory.
In summary, a series of novel amidine derivatives of doxifluridine were successfully synthesized and the single
crystal X-ray diffraction depicted that the structure was the one which we wanted to obtain. Furthermore,
investigations on anti-tumor effect in vitro showed exciting results. This suggested that this kind of novel amidine
derivatives of doxifluridine are worthy of further investigation to discover new anti-tumor agents.
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[8] L.J. Mcbride, R. Kierzek, S.L. Beaucage, et al. J. Am. Chem. Soc. 108 (1986) 2040.
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[13] Selective data for the new compounds. Compound 6: white solid, yield 36.6%; m.p.: 173–174 8C; ESI-MS (m/z, %): 315.2 ([MH+]); 1H NMR
(300 Hz, CDCl3): d 1.29 (d, 3H, J = 13 Hz, CH3), 2.25 (s, 3H, CH3), 3.09 (s, 3H, NCH3), 3.14 (s, 3H, NCH3), 3.68 (dd, 1H, J = 11 Hz, H-20),
3.86 (m, 1H, H-40), 4.02 (dd, 1H, J = 9 Hz), 5.71 (d, 1H, J = 6 Hz, H-10), 7.74 (d, 1H, J = 6 Hz, H-6). Compound 7: white solid, yield 38.2%;
m.p.: 170–171 8C; ESI-MS (m/z, %): 343.3 ([MH+]); 1H NMR (300 Hz, CDCl3): d 1.19 (t, 3H, J = 14 Hz, CH2CH3), 1.26 (t, 3H, J = 14 Hz,
CH2CH3), 1.37 (d, 3H, J = 13 Hz, CH3), 2.38 (s, 3H, CH3), 3.44 (m, 2H, CH2CH3), 3.57 (m, 2H, CH2CH3), 3.92 (dd, 1H, J = 11 Hz, H-20), 4.17
(dd, 1H, J = 9 Hz, H-30), 4.36 (m, 1H, H-40). Compound 8: white solid, yield 28.4%; m.p.: 126–127 8C; ESI-MS (m/z, %): 399.1 ([MH+]); 1H

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J.L. Feng et al. / Chinese Chemical Letters 21 (2010) 163–166
NMR (300 Hz, CDCl3): d 0.95 (t, 3H, J = 14 Hz, CH2CH3), 1.01 (t, 3H, J = 14 Hz, CH2CH3), 1.38 (d, 3H, J = 13 Hz, CH3), 1.62 (m, 8H, 2H,
CH2CH2CH3), 2.42 (S, 3H, CH3), 3.38 (t, 2H, 15 Hz, NCH2), 3.59 (t, 2H, 13 Hz, NCH2), 3.99 (dd, 1H, J = 11 Hz, H-20), 4.21 (dd, J = 9 Hz, H-
30), 4.43 (m, 1H, H-40), 5.70 (d, 1H, J = 6 Hz, H-10), 7.71 (d, 1H, J = 6 Hz, H-6). Compound 9: white solid, yield 31.7%; m.p.: 191.1–191.9 8C;
ESI-MS (m/z, %): 371.2 ([MH+]); 1H NMR (300 Hz, CDCl3): d 1.28 (d, 6H, J = 15 Hz, CH(CH3)2), 1.37 (d, 3H, J = 13 Hz, CH3), 1.47 (d, 6H,
J = 15 Hz, CH(CH3)2), 2.38 (s, 3H, CH3), 3.75 (m, 1H, CH(CH3)2), 3.86 (m, 1H, CH(CH3)2), 3.95 (dd, 1H, J = 11 Hz, H-20), 4.19 (dd, 1H,
J = 9 Hz, H-30) 4.39 (m, 1H, H-40), 5.68 (d, 1H, J = 6 Hz, H-10), 7.64 (d, 1H, J = 6 Hz, H-6). Compound 10: white solid, yield 15.0%; m.p.:
157.6–158.4 8C; ESI-MS (m/z, %): 341.2 ([MH+]); 1H NMR (300 Hz, CDCl3): d 1.37 (d, 3H, J = 13 Hz, 3H), 1.97 (m, 2H, CH2), 2.01 (m, 2H,
CH2), 2.39 (S, 3H, CH3), 3.55 (t, 2H, J = 13 Hz, NCH2), 3.65 (t, 2H, J = 13 Hz, NCH3), 3.94 (dd, 1H, J = 11 Hz, H-20), 4.17 (dd, 1H, J = 9 Hz,
H-30), 4.37 (m, 1H, H-40), 5.69 (d, 1H, J = 6 Hz, H-10), 7.67 (d, 1H, J = 6 Hz, H-6). Compound 11: white solid, yield 29.6%; m.p.: 181.7–
182.4 8C; ESI-MS (m/z, %): 355.2 ([MH+]); 1H NMR (300 Hz, CDCl3): d 1.37 (d, 3H, J = 13 Hz, CH3), 1.69 (m, 6H, CH2CH2CH2), 2.38 (s,
3H, CH3), 3.53 (t, 2H, J = 11 Hz, NCH2), 3.83 (t, 2H, J = 11 Hz, NCH2), 3.93 (dd, 1H, J = 11 Hz, H-20), 4.17 (dd, 1H, J = 9 Hz, H-30), 4.37 (m,
1H, H-40), 5.68 (d, 1H, J = 6 Hz, H-10), 7.66 (d, 1H, J = 6 Hz, H-6).