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Doubly Homologated Dihalovinyl and Acetylene Analogues of Adenosine: Synthesis, Interaction with S-Adenosyl-L-homocysteine Hydrolase, and Antiviral and Cytostatic Effects Stanislaw F. Wnuk,*,† Carlos A. Valdez,† Jahanzeb Khan,† Priscilla Moutinho,† Morris J. Robins,‡ Xiaoda Yang,§ Ronald T. Borchardt,§ Jan Balzarini,# and Erik De Clercq# Department of Chemistry, Florida International University, Miami, Florida 33199, Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602-5700, Departments of Biochemistry and Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045, and Rega Institute for Medical Research, Katholieke Universiteit of Leuven, Leuven, Belgium Received September 23, 1999
Treatment of the 6-aldehyde derived by Moffatt oxidation of 3-O-benzoyl-1,2-O-isopropylideneR-D-ribo-hexofuranose (2c) with the dibromo- or bromofluoromethylene Wittig reagents generated in situ with tetrabromomethane or tribromofluoromethane, triphenylphosphine, and zinc gave the dihalomethyleneheptofuranose analogues 3b and 3d, respectively. Acetolysis, coupling with adenine, and deprotection gave 9-(7,7-dibromo-5,6,7-trideoxy-β-D-ribo-hept-6enofuranosyl)adenine (5a) or its bromofluoro analogue 5b. Treatment of 5a with excess butyllithium provided the acetylenic derivative 9-(5,6,7-trideoxy-β-D-ribo-hept-6-ynofuranosyl)adenine (6). The doubly homologated vinyl halides 5a and 5b and acetylenic 6 adenine nucleosides were designed as putative substrates of the “hydrolytic activity” of S-adenosyl-Lhomocysteine (AdoHcy) hydrolase. Incubation of AdoHcy hydrolase with 5a, 5b, and 6 resulted in time- and concentration-dependent inactivation of the enzyme (Ki: 8.5 ( 0.5, 17 ( 2, and 8.6 ( 0.5 µM, respectively), as well as partial reduction of enzyme-bound NAD+ to E-NADH. However, no products of the “hydrolytic activity” were observed indicating these compounds are type I mechanism-based inhibitors. The compounds displayed minimal antiviral and cytostatic activity, except for 6, against vaccinia virus and vesicular stomatitis virus (IC50: 15 and 7 µM, respectively). These viruses typically fall within the activity spectrum of AdoHcy hydrolase inhibitors. Introduction The enzyme S-adenosyl-L-homocysteine (AdoHcy) hydrolase (EC 3.3.1.1) effects hydrolytic cleavage of AdoHcy to adenosine (Ado) and L-homocysteine (Hcy).1 The cellular levels of AdoHcy and Hcy are critical because AdoHcy is a potent feedback inhibitor of crucial transmethylation enzymes,1,2 and elevated plasma levels of Hcy in humans have been shown to be a risk factor in coronary artery disease.3 A number of inhibitors which function as substrates for the “3′-oxidative activity” of AdoHcy hydrolase and convert the enzyme from its active form (NAD+) to its inactive form (NADH, type I inhibition) have been prepared.1 Inhibitors which function as substrates for the “5′/6′-hydrolytic activity” were also synthesized, which included vinyl fluoride4 A, homovinyl halides5 B, and oxime derivatives of adenosine 5′-aldehyde,6 but not their 3′-deoxy analogues.6a,7 Moreover, the propensity of homoadenosine 6′-aldehyde (formed during processing of B by the enzyme) to depurinate decreases the efficiency of enzyme inactivation by B.5b * To whom correspondence should be addressed at Florida International University. Tel: 305-348-6195. Fax: 305-348-3772. E-mail:
[email protected]. † Florida International University. ‡ Brigham Young University. § University of Kansas. # Katholieke Universiteit of Leuven.
Recently, geminal (dihalohomovinyl)adenosines C were designed as putative new substrates for the hydrolytic activity of AdoHcy hydrolase.8 Addition of an enzyme-sequestered water molecule across the 5′,6′double bond of bromo(fluoro)homovinyl analogue C (Y ) F), followed by loss of bromide, was proposed to generate the homoAdo 6′-carboxyl fluoride at the active site of AdoHcy hydrolase.8b Nucleophilic attack by proximal amino acid functionalities caused type II (covalent binding) inhibition. The acetylenic analogue D derived from adenosine is a potent inhibitor of AdoHcy hydrolase5a,9a with antiviral5a,9b and cytostatic activity.5a Addition of water across the 5′,6′-triple bond of haloacetylenes10 E followed by tautomerization of the hydroxyvinyl intermediates was postulated to generate similar acyl halides (C6′ hydroxyl attack) and/or R-halomethyl ketones (C5′ hydroxyl attack) at the enzyme active site. The X-ray structure determination of human AdoHcy hydrolase revealed an unusual dual role for a catalytic water molecule at the active site.11
10.1021/jm990486y CCC: $19.00 © 2000 American Chemical Society Published on Web 03/03/2000
Doubly Homologated Adenosine Analogues
Journal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1181
Scheme 1a
Table 1. Inhibition of AdoHcy Hydrolase with 5a, 5b, and 6 % enzyme activity remaininga concn (µM)
5a
5b
6
0.1 1.0 10 50 100
87.5 ( 4.5 48.5 ( 2.4 17.1 ( 1.0 5.0 ( 0.5 4.0 ( 0.5
90.6 ( 5.0 87.4 ( 4.5 37.4 ( 2.0 9.7 ( 1.0 5.8 ( 1.0
98.3 ( 5.0 73.4 ( 3.8 38.9 ( 2.2 14.3 ( 1.1 8.0 ( 1.0
a AdoHcy hydrolase (42 nM) was incubated with 5a, 5b, and 6 in buffer A at 37 °C for 30 min, and the remaining activity was assayed as described in the Experimental Section. Data are the average of duplicate determinations.
a (a) (i) TBDMSCl/imidazole/DMF, (ii) HCl/MeOH/H O; (b) (i) 2 BzCl/pyridine, (ii) HCl/MeOH/H2O; (c) (i) DCC/DMSO/Cl2CHCO2H, (ii) CBr4 (or CBr3F)/PPh3/Zn/CH2Cl2; (d) (i) TBAF/THF, (ii) BzCl/ pyridine; (e) (i) TFA/H2O, (ii) Ac2O/pyridine/DMAP; (f) adenine/ SnCl4/CH3CN; (g) NH3/MeOH; (h) BuLi/THF/-78 °C.
We now describe syntheses of the first “doubly homologated” vinyl halides 5 and acetylenic 6 adenine nucleosides, their interaction with AdoHcy hydrolase, and their antiviral activities. These derivatives (sp3 hybridized C5′) should have greater conformational flexibility at C5′ relative to analogues that have vinylogous (sp2; B or C) or acetylenic (sp; D or E) functions directly attached to the ribose ring. They also provide probes for evaluation of tolerated distances in the enzyme between the binding site for O3′ (presumably involved with oxidation at C3′) and the protein residues which are responsible for the “hydrolytic activity” (already shown to function at C5′and C6′). Chemistry Synthesis of the “doubly homologated” 6′,7′-unsaturated analogues 5 began with ribo-hexofuranose sugar precursors of type 2, because homologation of adenosine to homoadenosine required multistep procedures which gave low overall yields.12 Moreover, homoAdo 6′-aldehyde, which is the obvious intermediate for the synthesis of 7′-halovinyl nucleosides 5 via the Wittig approach, is known to be unstable.5b Commercial 1,2-O-isopropylidene-R-D-glucose (1) was converted to 6-O-TBDMS-1,2O-isopropylidene-R-D-ribo-hexofuranose (2a) in five steps (∼55%).13 Key steps involved regioselective oxidation [(Bu3Sn)2O/Br2]14 of 1 to the 5-ketone, deoxygenation via its tosylhydrazone, and inversion of configuration at C3 via oxidation and reduction. Silylation (O3) of 2a gave the 3,6-di-O-TBDMS derivative, and regioselective removal of the primary TBDMS group with dilute HCl/MeOH afforded the ribo-hexofuranose 2b.13c Benzoylation (O3) of 2a and desilylation gave 2c (81%; Scheme 1). Moffatt oxidation15 of 2b13c and treatment of the crude 6-aldehyde with (dibromomethylene)triphenylphosphorane (CBr4/Ph3P/Zn)16 gave dibromovinylheptofuranose 3a (89%). Compound 3a was converted to its 3-O-benzoyl derivative 3b, because attempted acetolysis of 3a failed to give the 1-O-acetyl anomers [due to instability of the TBDMS group (O3) under acidic conditions]. Wittig-type olefination of 2c with the dibromomethylene reagent also gave 3b (75%). The iso-
propylidene group was removed from 3b (TFA/H2O) and the product was acetylated to give the anomeric acetates 4a (85%; R/β, ∼1:4), which were partially separated by silica gel column chromatography. The β-anomer was obtained after crystallization. Coupling (SnCl4/CH3CN17) of the anomeric mixture of 4a and adenine followed by deacylation gave the crystalline dibromovinyl compound 5a (49%). Treatment of 5a with excess BuLi16,18 effected dehydrobromination to give the acetylenic derivative 6 (65%). Analogously, oxidation of 2b13c and treatment of the crude 6-aldehyde with the bromofluoromethylene Wittig reagent gave the bromofluorovinyl diastereomers 3c [48%; E/Z, ∼45:55; 3JF-H6′ ) 13.2 Hz (Z) and 3JF-H6′ ) 30.9 Hz (E)]. Desilylation of 3c followed by benzoylation gave 3d, which was also prepared directly from 2c (41%; E/Z, ∼45:55). The unreacted 6-aldehydes derived from 2b [1H NMR δ 9.71 (t, J ) 2.4 Hz, 1, H6); 13C NMR δ 200.28 (C6)] and 2c were recovered in moderate yield (20-35%) from column chromatography of the Wittig olefination products. In contrast with the instability of homoAdo 6′-aldehyde,5b these homoribose 6-aldehydes were quite stable. Treatment of 3d with TFA/H2O and acetylation gave 4b (80%; R/β, ∼1:4; E/Z, ∼40:60) which was coupled with adenine. Deacylation of this mixture and RP-HPLC purification gave 9-(7-bromo-5,6,7-trideoxy-7-fluoro-β-D-ribo-hept-6-enofuranosyl)adenine (5b) (31%; E/Z, ∼40:60). Results and Discussion Inactivation of Ado-Hcy Hydrolase. Recombinant human placental AdoHcy hydrolase was inactivated upon incubation with 5a, 5b, or 6 in a concentrationdependent (Table 1) and time-dependent (Figure 1) manner. However, these compounds did not produce complete inactivation of AdoHcy hydrolase even upon prolonged incubation. In the presence of 100 µM AdoHcy, AdoHcy hydrolase could be protected from inactivation by 5a, 5b, and 6 (data not shown). The Ki values of compounds 5a, 5b, and 6 were determined to be 8.5 ( 0.5, 17 ( 2, and 8.6 ( 0.5 µM, respectively. Compounds 5a, 5b, and 6 were designed as putative substrates of the “hydrolytic activity” of AdoHcy hydrolase. Conceptually, addition of the enzyme’s sequestered water11 at the 6′- and/or 7′-positions of 5a or 5b could generate β,β-dihalo alcohols (halohydrins) and/or acyl halide products, respectively; and 6 would give a 6′ketone and/or 7′-aldehyde. Based on our previous studies,4b,5,8 most of the possible products should be released into solution. Some of them might produce covalent modification of the enzyme, but some should be detected by HPLC. However, incubation of AdoHcy
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(IC50: 15 and 7 µM, respectively). Activity against these two viruses in E6SM cells is consistent with what has been found with other AdoHcy hydrolase inhibitors.19 These compounds were also evaluated for their cytostatic activity against a number of tumor cell lines [murine leukemia cells (L1210), murine mammary carcinoma cells (FM3A), and human T-lymphocyte cells (Molt4/C8, CEM)]; no marked inhibitory effects on tumor cell proliferation were observed. Summary and Conclusions
Figure 1. Time-dependent inactivation of AdoHcy hydrolase by 5a (0), 5b (4), and 6 (O). AdoHcy hydrolase (42 nM) was incubated with 10 µM 5a, 5b, and 6 in buffer A for various times. At the indicated times, remaining enzyme activity was determined in the synthetic direction as described in the Experimental Section. Data are the average of duplicate determinations. Table 2. Effects of 5a, 5b, and 6 on NADH Content of AdoHcy Hydrolasea compd NADH content (%)
5a
5b
6
30.2 ( 1.5
49.8 ( 2.5
38.1 ( 2.0
a AdoHcy hydrolase (21 µM, reconstituted NAD+ form) was incubated with 100 µM 5a, 5b, and 6 in buffer A at 37 °C for 2 h, and the NAD+/NADH content was assayed as described in the Experimental Section. Data are the average of duplicate determinations. The NADH content of the control enzyme is
