K H W A J A ET AI..
8-Substituted Derivatives of Adenosine 3’,5’-Cyclic Phosphate Require an Unsubstituted 2’-Hydroxyl Group in the Ribo Configuration for Biological Activity’ Tasneem A. Khwaja,t Kay H. Boswell, Roland K. Robins, and Jon P. Miller*
ABSTRACT: Derivatives of adenosine 3’,5’-cyclic phosphate
(CAMP) with modifications in both the 2‘ and the 8 positions were synthesized and their enzymic activities as activators of CAMP-dependent protein kinase and as substrates for and inhibitors of cAMP phosphodiesterases were determined. Three types of derivatives were investigated: 8-substituted derivatives of 02’-Bt-cAMP, 8-substituted derivatives of 9-/3-D-arabinofuranosyladenine 3’,5’-cyclic phosphate (ara-CAMP), and 8-substituted derivatives of 8,2’anhydro-9-/3-D-arabinofuranosyladenine3’,5’-cyclic phosphate (8,2’-anhydro-cAMP). The 8-substituted 02’-BtcAMP derivatives were synthesized by acylation of the preformed 8-substituted cAMP (8-HS-cAMP, 8-MeS-cAMP, and 8-PhCH2S-CAMP). 8-Br-02‘-tosyl-cAMP was used as an intermediate for the preparation of 8,2’-anhydro-cAMP derivatives (8-HO-, 8-SH-, 8-H2N-, and 8-H3 C H N derivatives of 8,2’-anhydro-cAMP). 8-Substituted ara-CAMP derivatives were obtained by ring opening of 8-HO-8,2’-anhydro-CAMP with H + / H 2 0 , NH3/MeOH, or MeONa/ MeOH (to yield the 8-HO-, 8-H2N-, and 8-MeO-ara-
w e have been interested in the effects of modification of the purine, carbohydrate, and phosphate moieties of 3’3’cyclic nucleotides on their interaction with various enzymes of cyclic nucleotide metabolism (for review, see Simon et al., 1973; Meyer and Miller, 1974). We have previously shown that many 8-substituted analogs of CAMP‘ activated bovine brain CAMP-dependent protein kinase more efficiently than did cAMP (Muneyama et al., 1971; Miller et al., 1973a). Any 2‘ substitution of cAMP was not tolerated by this enzyme and a free 2’-hydroxyl group in the rib0 configuration was essential for its activation (Miller et al., 1973b). In contrast, 8-substituted derivatives of cAMP were very poor substrates for rabbit kidney phosphodiesterase (Muneyama et al., 1971; Miller et al., 1973a), whereas the 2‘ derivatives were hydrolyzed at substantial rates by this enzyme (Miller et al., 1973b). We have now begun to investigate the effects of multiple substitutions of cAMP on its biological activity. Some of these studies on purine 5’-thio-5’-deoxynucleoside3’3’-cyclic phosphorothioates have suggested that the interaction of cAMP with the above two enzymes involved binding of both the 6-amino group and the 5’ oxygen in a synergistic
cAMP derivatives). All of these doubly modified derivatives of cAMP were less than one-hundredth as active as cAMP at activating protein kinase and did not serve as substrates for the phosphodiesterase. These data show that the general inactivity of 2’ derivatives of cAMP with the kinase was not overcome by addition of an 8-substituent, even though many 8-substituted derivatives of cAMP activate the kinase more efficiently than does cAMP itself. In addition they show that while 2’-modifications were tolerated by the phosphodiesterase, addition of an 8-substituent countermanded the allowable 2’-modification. The 8-substituted derivatives of 02’-Bt-cAMP were found in general to be slightly better inhibitors of phosphodiesterase than the parent compounds containing no 02’-Bt substitution. As a group, the 8-substituted ara-CAMP derivatives were poorer inhibitors of phosphodiesterase than 8-substituted cAMP derivatives while the 8,2‘-anhydro-CAMP derivatives were much poorer inhibitors than the 8-substituted ara-CAMP derivatives.
manner to the enzymes (Shuman et al., 1973). We here report the synthesis of cAMP analogs with modifications in both the 02‘and 8 positions. Three different types of derivatives were investigated: (1) 8-substituted 8,2’-anhydrocAMP derivatives, (2) 8-substituted ara-CAMP derivatives, and (3) 8-substituted 02’-Bt-cAMP derivatives. The in vitro enzymic properties of these derivatives were investigated by studying their ability to serve as substrates for and inhibitors of cyclic nucleotide phosphodiesterases and to activate a CAMP-dependent protein kinase. Experimental Section
Synthetic, 8-Substituted cAMP analogs were synthesized as previously described (Muneyama et al., 1971). 8-Br02‘-tosyl-cAMP (2) (Khwaja et al., 1972) was used as a versatile intermediate for the preparation of 8,2’-anhydrocAMP derivatives (11-13) by reaction with an appropriate nucleophile: NaSH, ”3, or CH3NH2 (Scheme I). 8-Substituted ara-CAMP derivatives (15, 16, and 18) were obtained by ring opening 8,2’-anhydro-9-/3-D-arabinofuranosyl-8-hydroxyadenine 3’,5’-cyclic phosphate (10, 8-HO8,2’-anhydro-cAMP) (Khwaja et al., 1972; Mian et al., 1974) with methanolic ammonia, dilute acid, or methanolic sodium methylate (Scheme 11). 9-/3-~-ArabinofuranosyladFrom the ICN Pharmaceuticals, Inc., Nucleic Acid Research Inenine 3’,5’-cyclic phosphate (14, ara-CAMP) and 8-thio-9stitute, Irvine, California 92664. Received April 14, 1975. * Present address: Advanced Therapeutics Program, LAC/USC /3-D-arabinofuranosyladenine3’,5’-cyclic phosphate (17, 8Cancer Center, Los Angeles, California 90033. were synthesized as previously described ’ Abbreviations used are: CAMP, adenosine 3’,5’-cyclic phosphate; HS-ara-CAMP) (Khwaja et al., 1972; Mian et al., 1974). The ‘ H nuclear ara-CAMP, 9-/3-D-arabinofuranosyladenine 3’,5’-cyclic phosphate; 8,2’-anhydro-cAMP, 8,2’-anhydro-9-/3-D-arabinofuranosyladenine magnetic resonance (‘H N M R ) spectra of these arabinose 3’,5’-cyclic phosphate. cyclic phosphates exhibit characteristic doublets (6 6-7) for
4238
BIOCHEMISTRY. VOL.
14, NO. 19, 1975
BIOLOGICAL ACTIVITY OF CAMP
Scheme I1
Scheme I
Tf ?”*
”, I
O=P-
0
I
OH
(5/GH;,CHIOH 18
I
”*
I
OH 11
0
I
H
I
100,000
>100,000
12 13
>100,000 >100,000
>100,000 15,000
20 21 22
CUM)
Rabbit Lung
Beef Heart
5.6
5.6
130 32 12
130 70 13
a The assay for the inhibition of cAMP hydrolysis was performed as described in Biochemical Methods. Results are reported as I,, values, where I,, is the concentration of compound that produces 50% of the uninhibited rate. b The form of the substituent for the 8,2‘-anhydro derivatives is given in parentheses.
stitution (compounds 20-22) generally did not affect the inhibitory activity of the resulting compounds. The 8-substituted 8,2’-anhydro-cAMP derivatives, with the exception of the 8-HO-8,2’-anhydro-cAMP (lo), were such poor inhibitors that in most cases the 1 5 0 value could not be determined. Both of the cyclic nucleotide preparations were examined for their molecular heterogeneity and substrate specificity. When subjected to gel filtration according to Thompson and Appleman (1971), both the rabbit lung and the beef heart preparations contained primarily what these authors refer to as “Fraction 11” type activity. The preparations used here demonstrated the same K , values for cAMP as did the “Fraction 11” obtained from subjecting these preparations to gel filtration. The K , for cAMP with the rabbit lung phosphodiesterase was 0.32 pLM and with the beef heart phosphodiesterase was 0.085 wA4. 150 values were determined using the “Fraction 11” activity from each preparation for compounds 3, 5, 15, 17, 10, 11, and 20, and were found not to be significantly different from those reported in Table I1 using the preparations described in Biochemical Methods. The detailed properties of these phosphodiesterase preparations will be the subject of a subsequent report. X-Ray data (Travale and Sobell, 1970) indicate that crystals of 8-substituted purine nucleoside exist as syn conformers. Circular dichroism studies (Ikehara et al., 1972) suggest that 8-substituted purine nucleosides retain this syn conformation in aqueous solution. X-Ray analysis of crystals of cAMP (1) revealed two molecules per unit cell, one in anti and the other in the syn conformation (Watenpaugh et al., 1968). ‘ H NMR studies (Schweizer and Robins, 1973) indicate that substitution of cAMP (1) pr ara-CAMP (14) in the 8 position with a bulky group forces the resulting derivatives to exist mainly in the syn form while the parent compounds show a preference for the anti form. In addition, a comparison of the vicinal 13C8-’H1/ coupling con-
stants for CAMP, 8-Br-cAMP, and 8-HO-CAMP (Dea et a]., 1973) can be interpreted to suggest that a solution of cAMP contains some anti conformers. ‘H N M R experiments utilizing the nuclear Overhauser effect were also consistent with this conclusion (Barry et al., 1974). This possibility of cAMP existing in two conformations in aqueous solution raises the questions, whether cAMP will exhibit its biological activity in only the syn or only the anti configuration, and whether an 8-substituted derivative of cAMP when fixed in an anti configuration will also show CAMPlike a ~ t i v i t y8-Substituted .~ 8,2’-anhydro-cAMP derivatives (10-13) which are fixed in an anti-like conformation did not activate bovine brain cAMP dependent protein kinase. However, since these 8-substituted 8,2‘-anhydro-cAMP analogs (10-13) lack a 2’-hydroxyl group in the ribose configuration, which has been shown to be required for protein kinase activation (Miller et al., 1973b) by cAMP and its analogs, the question of the activity of cAMP in the anti conformation in such a system remains open. The 8-substituted 8,2’-anhydro cAMP derivatives probably exist with the C-8 positioned over the C-2’ and remain fixed in that position. The other classes of 8-substituted cAMP derivatives, while being syn-like in conformation, still are significantly less hindered in rotation about the glycosidic bond than the covalently fixed 8-substituted 8,2’anhydro-CAMP derivatives. The torsional angle of the glycosidic bond of cAMP while it is bound to phosphodiesterase may well be such that the C-8 is not positioned over the C-2’. The data, in fact, suggest this. Hampton et al. (1972, Two reports on the conformation of cAMP in solution using lanthanide ions as ‘ H N M R probes (Barry et ai., 1974; Lavallee and Zeltmann, 1974) suggest that cAMP exists in the syn conformer at pH 5.3-5.5 in the presence of lanthanide ions. Without knowing the effects of lanthanide ions on the conformation, we prefer to consider the data from the nonperturbed systems (Schweizer and Robins, 1973; Dea et al., 1973). BIOCHEMISTRY, VOL.
14, NO. 19, 1975
4243
Kf4WAlA
FT
41
1973) have shown that 8,5’-cycloadenosine 5’-phosphate is Meyer, R. B., Jr., and Miller, J. P. (1974), Life Sri. 14, a good substrate for many 5‘-AMP utilizing enzymes while 1019. 8,2’-an hydro-8- hydroxy-9-~-~-arabinofuranosyladenine Meyer, R. B., Jr., Shuman, D. A,, and Robins, R. K. 5’-phosphate and 8,3’-anhydro-8-hydroxy-9-/3-~-arabino- (1973), Tetrahedron Lett., 269. furanosyladenine 5’-phosphate were essentially inactive Meyer, R. B., Jr., Shuman, D. A., Robins, R. K., Bauer, R. with the same enzymes. The compound 8,5’-cyclo-cAMP J., Dimmitt, M. K.. and Simon, L. N . ( 1 972), Bioc,hemistry I I 2704. would indeed provide additional insight into the conformerMian, A. M., Harris, R., Sidwell, R. W., Robins, R. K., and ic form of enzyme-bound CAMP. Khwaja, T. A. (1974), J . Med. Chem. 17, 259. References Miller, J . P., Boswell, K. H., Muneyama, K., Simon, L. N., Barry, C. D., Martin, D. R., Williams, R. J. P.. and Xavier, Robins, R. K . , and Shuman, D. A. (l973a), RiochamisA. V . ( I974), J . Mol. Biol. 84, 491. try 12. 5310. Boswell, K. H., Miller, J . P., Shuman, D. A., Sidwell, R. Miller, J. P., Boswell, K. H., Muneyama, K., Tolman, R. W . , Simon, L. N., and Robins, R. K. (1973), J . Med. L., Scholten, M. B., Robins, R . K . , Simon, L. N., and Shuman, D. A. (1973c), Biochem. Biophys. Res. CbmChem. 16. 1075. niun. 55, 843. Dca, P.,Kreishman, G . P., Schweizer, M. P., Witkowski, J. T., Nunlist, R., and Bramwell, M. (1973), Proc. Inf. Miller, J. P., Shuman, D. A., Scholten, M . B., Dimmitt, M. Conf. Stable Isot. Chem., Biol., Med. 1st. 84. K., Stewart, C. M., Khwaja, T. A . , Robins, R. K., and Simon, L. N. (1973b), Biochemistry 12, 1010. Eckstein. F..Simonson, L. P., and Bar, H.-P. (1974), BioMuneyama, K., Bauer, R. J., Shuman, D. A., Robins, R. chemistry 13. 3806. Giao, M.-B., Cehovic, G., Bayer, M., Gergely, H., and PosK., and Simon, I_. N . ( 1 97 1 ), Biochemistry 10, 2390. Muneyama, K., Shuman, 11. A., Boswell, K. H., Robins, R. ternak, M. T. (1974). C. R. Hebd. Seances Acad. Sci., Srr. I) 279, I 705. K., Simon, L. N., and Miller, J . P. ( 1 974), J . C’arhohydr. Nucle0side.y Nucleotides I , 5 5 . Hampton, A . , Harper, P. J., and Sasaki, T. (1972), Biorheniistr,v I I , 4736. Poqternak, T., Sutherland, E. W., and Henion, W. F. Hampton, A,, and Sasaki, T. (1973), Biochemistry 12, (l962), Biochim. Biophys. Acta 65, 558. 2188. Schweizer, M. P., and Robins, R. K. (1973). in ConformaIkehara, M.. Uesugi, S., and Yoshida. K. (1972), Biochemtion of Biological Molecules and Polymers (Proceedings of Jerusalem Symposium), Israel Academy of Sciences istry I I , 830. Jastorff, B., and Bar, H.-P. (1973), Eur. J . Biochem. 37, and Humanities, Jerusalem, p 329. 497. Shuman, D. A , , Miller, J. P., Scholten, M. R., Simon, L. Jones. G . H., Albrecht, H. P., Damadaran, N . P., and MofN., and Robins, R. K. (1973), Biochemistry 12, 2781. fatt, J. G. (1970),J. A m . Chem. SOC.92, 5510. Simon, L. N., Shuman. D. A., and Robins, R. K . (1973). Khwaja, T.A,, Harris, R., and Robins, R. K. (1972). TetAdi:. C j d i c Nucleotide Res., 225. rahedron Lett., 468 1. Thompson, W. J., and Appleman, M. M. (1971). J . Biol. Chem, 246, 3 145. Kuo, .I. F., and Greengard, P. (1969), Proc. Natl. Acad. Travale, S.S.,and Sobell. M . ( 1 970), J . Mol. Biol. 48, 109. ,Sc.i. U . S . A . 64. 1349. Watenpaugh, K., Dow, J., Jensen, L. H., and Furberg, S. Lavallee, D. K., and Zeltmann, A . H . (1974). J . Anz. (I 968), Science /.59, 206. Chem. Soc. 96, 5 5 5 2 .
