Summary: Xanthines represent a new, versatile scaffold for combinatorial chemistry. A five-step solid-phase synthesis of xanthine derivatives is described which includes alkylations, a nucleophilic displacement reaction at a heterocycle and a ring closure reaction by condensation of a nitroso function with an activated methylene group. The selected reaction sequence allows the production of a highly diverse small-molecule combinatorial compound librar

Eberle, Alex

Heizmann, Gerhard

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Molecular Diversity, 2 (1996) 171 174 171 ESCOM MOLDIV 055 Xanthines as a scaffold for molecular diversity Gerhard  He izmann and Alex N. Eberle* Laboratory of Endocrinology, Department of Research (ZLF), University Hospital and University Children's" Hospital Basel, CH-4031 Basel, Switzerland Received 6 December 1996 Accepted 6 February 1997 Keywords: Xanthines; Combinatorial chemistry; Diversity; Small molecules Summary Xanthines represent a new, versatile scaffold for combinatorial chemistry. A five-step solid-phase synthesis of xanthine derivatives i described which includes alkylations, a nucleophilic displacement reaction at a heterocycle and a ring closure reaction by condensation of a nitroso function with an activated methylene group. The selected reaction sequence allows the production of a highly diverse small-molecule combinatorial compound library. Introduction Combinatorial compound libraries represent a powerful method to identify new pharmaceutical lead compounds for a given biological target [1]. One of the challenges in generating new kinds of libraries is the development of scaffolds that allow the introduction of a maximum of diversity. Recent investigations howed the successful synthesis on a solid support of several small molecules that are suitable as scaffolds for the generation of molec- ular diversity [2]. The application of solid-phase technol- ogy to the preparation of such new scaffolds for combina- torial chemistry requires the transfer of solution-phase chemistry to conditions that are feasible on the solid support. The reactions hould proceed in resin-swelling solvents and the reaction temperatures should be kept at reasonable values. Xanthines represent a new structurally rigid scaffold for combinatorial chemistry. Certain naturally occurring xanthines (e.g. caffeine, theophylline and theobromine) are well known to exert prominent physiological effects at very low doses, and they have been used as pharmaceuti- cals, narcotics or stimulants for many centuries [3]. To- day, 8-substituted xanthines are studied extensively for clinical application [4], as they inhibit many pharmacolog- ical and physiological effects of adenosine [5]. In order to use the xanthine scaffold for combinatorial chemistry, we have developed a method of solid-phase synthesis of *To whom correspondence should be addressed. 1381-1991/$ 6.00 + 1.00 9 1997 ESCOM Science Publishers B.V. highly substitutable xanthines for the generation of new compound libraries. Results and Discussion The ring closure reaction of 1,3-dialkyl-6-alkylamino-5- nitrosouracils 1 proceeds easily, giving the corresponding xanthine derivatives 2, as long as N1 is blocked by a substituent (Scheme 1) [6]. The application of solid-phase technology to the gener- ation of combinatorial compound libraries necessitates the adaptation of this reaction to solid-phase conditions. As a first step, we coupled bromoacetic acid to the free amino function of a solid support 3, which is modified by the Rink amide linker [7]. We used the Rink linker because of its stability towards a wide variety of reaction condi- tions. 1-Alkyl-4-chlorouracils 5 were then attached to the solid phase by alkylation of the nitrogen at the uracil system to form intermediates 6. The chlorine of the resin- bound uracils 6 can easily be replaced by primary benzyl- amines to form 4-benzylaminouracils 7. The intermediates 7, bound to the solid support, were treated with isopentyl nitrite in the presence of a small amount of acetic acid. The nitroso compounds formed first immediately undergo ring closure to form the xan- thine derivatives 8. This conversion could be followed by UV spectroscopy, since the absorption band at 260 265 nm, characteristic for the uracil-type compounds 7, disap- 172 O O R H RI~N~NO I~N~-... jN ; I H R3 I R2 R2 1 2 Scheme 1. Formation of xanthine derivatives 2 by ring closure of 1,3-dialkyl-6-alkylamino-5-nitrosouracils 1. Note that N1 is blocked. peared and was shifted to 306-310 nm, characteristic for xanthines 8. The progress of all reactions was judged by cleavage of a small amount of the reaction product from the solid phase, followed by analysis by UV spectroscopy and comparison of HPLC characteristics of the product with those of product prepared in solution (if available). Alkylation of these compounds proceeded with excess alkylating agent in the presence of triethylamine to give DMF, NEt3 60 ~ 2 h BrCH2COOH, DMF ii, Diisopropylcarbodiimide R'E 30 rnin o RI~N& J ' O~.,~N .,~CI 6 H2N ~2 DMF 60 ~ 2 h O RI~N& o~"~ N~CI  H 5 o RI~N/JJ ~ O~N~N'~% NH 7 [sopentylnitrite/H + 40 ~ 4 h o R2 LCo NH R3X (X=Halogen), NEt3 1D 60 ~ 2,5 h o R2 NH 9 95% TFA R~ 2 x 15 rain O R2 NH2 10 Fig. 1. Solid-phase synthesis of fully substituted xanthines. 173 TABLE 1 ANALYTICAL DATA OF SYNTHESIZED XANTHINE DERIVATIVES R~N~N,  I R4 R2 No. R~ R 2 R 3 R 4 Yield a (mg) Retention time MW (g/mol) (min) (see text) MS (MH+) b 10a CH 3 ~/NH2 ~ ~  2'-OCH 3 1.94 (10%) 10.87 367.37 " 1 368.1 O 10b ~ ~/ .NH2 ~/ .NH2 4'-CF3 3.63(14%) 13.06 500.44 L j/ -1  -1  502.2 O O 10e CH 3 ~/NH2 H 4'-F 1.80 (19%) 8.75 317.28 " 1 320.4 O 10d CH 3 ~/NHz ~/NH2 4'-F 3.55 (32%) 6.57 374.33 " 1 " 1 377.2 O O 10e CH 3 ~/NH2 H 4'-CH 3 2.34 (25%) 9.45 313.32 c " 1 314.5 O 10f CH 3 /~,~NH2 ,./~NH2 4'-CH 3 3.56(32%) 7.46 371.4 0"37~ O O 10g CH2CH2CH 3 ~/NH2 H 4'-F 2.42 (23%) 10.60 345.33 " 1 344.1 O 10h CH2CH2CH , ~/NH2 ~/NH2 4'-F 3.10(26%) 8.20 402.38 " 1 " 1 403.6 O O 10i CH2CH2CH 3 ~/NH2 H 4'-CH 3 1.31 (13%) 11.23 341.37 " 1 342.4 O 10j CH2CH2CH 3 ~/NH~ ~/NH2 4'-CH 3 1.52 (11%) 8.98 398.42 " 1 " I 400.5 O O a The yield after RP-HPLC purification (based on a resin loading of 0.23 mmol/g) reflects the recovery of pure product. Analysis by RP-HPLC of the crude products (after cleavage from the resin) showed purities indicated by peak areas ranging from 86-98% (X = 300 nm) of 65-89% (X = 220 nm). b Mass spectrometric data were obtained on a MALDI system (Linear Scientific 1700, Reno, CA, U.S.A.). For NMR data see text. the fully substituted xanthines 9. The final products 10 were cleaved from the solid support  by the action of  tr i f luoroacetic acid. The cleavage procedure was as fol- lows. The resin was treated twice with 95% tri f luoroacetic acid and washed with 95% tr i f luoroacetic acid, dichloro- methane and tr i f luoroethanol.  The combined solvents were removed over potass ium hydroxide in an evacuated desiccator, after which the residues were lyophil ized twice f rom dilute acetic acid and directly purif ied by HPLC.  Analyt ical  and preparat ive HPLC was per fo rmed on a Jasco system (Tokyo, Japan). Analyt ical  runs were carr ied out  on a Waters Symmetry  C18 co lumn (3.9 x 150 mm)  using a l inear solvent gradient f rom 5% to 100% solvent B in 20 min (solvent A = 0.1% TFA in water; so lvent  B = 0.1% TFA in acetonitri le/water, 7:3). Preparat ive runs were carr ied out  on a Waters Symmetry  C18 co lumn (19 x 150 mm) with a l inear solvent gradient f rom 25% to 60% solvent B in 22 min. 174 We investigated the applicability of this method by first synthesizing two individual xanthines (10a and 10b). The potential of the synthetic scheme to produce combi- natorial compound libraries was then determined by the synthesis of a group of eight related xanthines (10e-10j). The original amount of resin was continuously divided into smaller portions along with the progressing reaction sequence (see Fig. 1). In this way, eight individual xan- thines were obtained by cleavage from the eight different resin portions. The analytical data of these compounds are summarized in Table 1. The structure of two com- pounds (10e and 10f) was verified by NMR spectroscopy. NMR spectra were recorded on a Bruker 300 MHz in- strument. NMR data of compound 10e: ~H NMR (DMSO-d6, ppm): 7.53 d (2H), 7.36 d (2H) ArH; 4.58 s (2H) CH2CO; 3.30 s (3H) N-CH3; 2.38 s (3H)p-CH 3. NMR data of compound 10f: IH NMR (DMSO-d6, ppm): 7.67 d (2H) CO-NH2; 7.53 d (2H), 7.36 d (2H) ArH; 7.28 d (2H) CO-NH2; 4.94 s (2H) CH2CO; 4.57 s (2H) CH2CO; 3.28 s (3H) N-CH3; 2.38 s (3H)p-CU 3. Conclusions We have demonstrated the successful transfer of sol- ution-phase reactions to a solid-phase synthesis of xan- thine derivatives. The applicability of this synthetic strat- egy for the generation of molecular diversity was tested with the preparation of a small xanthine library consisting of eight individual entities. The method can easily be ex- tended to generate libraries with a large number of com- pounds. A library containing 90 individual compounds was prepared in this way and is currently being tested in various biological assays. Acknowledgements We thank Dr. R. Andreatta, Dr. A. Marzinzik and Mr. H. Miiller, Ciba-Geigy AG, Basel, Switzerland for carrying out the mass spectrometric and NMR analyses. G.H. was supported by the DF fund. References 1 a. Houghten, R.A., Combinatorial libraries. Finding the needle in the haystack, Curr. Biol., 4 (1994) 564-567. b. Eichler, J., Appel, J.R., Blondelle, S.E., Dooley, C.T., Dorner, B., Ostresh, J.M., Perez-Paya, E., Pinilla, C. and Houghten, R.A., Peptide, peptidomimetic, and organic synthetic ombina- torial libraries', Med. Res. Rev., 15 (1995) 481-496. c. Gallop, M.A., Barrett, R.W., Dower, W.J., Fodor, S.EA. and Gordon, E.M., Applications of combinatorial technologies to drug discovery. 1. Background and peptide combinatorial librar- ies, J. Med. Chem., 37 (1994) 1233-1251. d. 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Xanthines as a scaffold for molecular diversity

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Xanthines as a scaffold for molecular diversity

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