353
J . Med. Chem. 1993,36, 353-362
Syntheses and Biological Evaluations of 3’-Deoxy-3’-C-Branched-Chain-Substituted Nucleosides Tai-Shun Lin,’ Ju-Liang Zhu, Ginger E. Dutachman, Yung-Chi Cheng, and William H. Prusoff Department of Pharmacology and Comprehensive Cancer Center, Yale University School of Medicine, New Haven, Connecticut 06510 Received July 24, 1992
Various 3’-deoxy-3’-C-(hydroxymethyl)-,3’-deoxy-3’-C-(fluoromethyl)-, 3‘-deoxy-3’-C-(azidomethy1)-, and 3’-deoxy-3’-C-(aminomethyl)-substituted nucleosides (total 12 compounds) have been synthesized and evaluated against L1210, P388, S-180,and CCRF-CEM cells and HSV-1, HSV-2, and HIV-1 in culture. Only 3’-deoxy-3’-C-(hydroxymethyl)thymidine(36) was found to show significant anticancer activity against L1210, P388,S-180, and CCRF-CEM cells with ED50 values of 50,5,10, and 1NM,respectively. None of these compounds demonstrated significant antiviral activity against HSV-1, HSV-2, or HIV-1. These compounds were also evaluated against thymidine kinases derived from HSV-I (strain KOS), HSV-2 (strain 333),and mammalian (K562) cells. The thymidine kinase (HSV-1 strain KOS) was inhibited significantly by both 3’-deoxy-3’-C(hydroxymethy1)- and 3’-deoxy-3’-C-(fluoromethyl)thymidine. Branched-chain sugar nucleosides, such as 9- [3-deoxy3-C-(2-hydroxyethyl)-~-~-ribofuranosyll adenine and related compounds, were first synthesized by Rosenthal et al.1*2Later, Acton et aL3 reported the synthesis of 3’4(hydroxymethyl)-2’,3’-dideoxythioguanosine and ita a-isomer. Both the a- and j3-nucleosides were equally inhibitory to the growth of WI-L2 human lymphoblastoid cells. Recently, Pudlo and Townsend4described a novel approach toward the synthesis of 3‘-deoxy- and 2‘,3‘dideoxy-3’-hydroxymethylribofuranosides. Subsequentreported the syntheses and antiviral ly, Bamford et evaluation of a series of 3’-deoxy-3’-C-(fluoromethyl)-,3’deoxy-3’-C-(difluoromethy1)-,and 3’-deoxy-3’-C-(hydroxymethyl)-arabino-pentofuranosylnucleosides and other related compounds. Sterzyckiet al.’ described the synthesis and antiviral evaluation of various 3’-branched nucleoside analogues; and Svansson et al.* reported the synthesis of 2’,3‘-dideoxy-3’-C-(hydroxymethyl)nucleosides as potential inhibitors of HIV. Tseng et aL9 also described the synthesisof 3’-C-(hydroxymethyl)-2‘,3‘-dideoxyadenosine, which possessed an activity profile similar to oxetanocin A against HIV in ATH 8 cells. This report describes the syntheses and biological evaluation of the 3’-deoxy-3’-C-(azidomethyl)and 3’deoxy-3’-C-(fluoromethyl)homologues of 3’-azido-3’-deoxythymidine (AZT) and 3’-fluoro-3’-deoxythymidine(3’FddT), which are among the most potent anti-human immunodeficiency virus type 1 (HIV-1) and other related compounds. al.536
Chemistry Various 3’-deoxy-3‘-C-branched-chain-substituted nucleoside analogues have been synthesized by coupling the silylated base with the appropriate 3-deoxy-3-branchedchain-substituted sugar derivatives. The syntheses of the key intermediates, 3-deoxy-3-(fluoromethyl)-,3-deoxy-3(azidomethyb, and 3-deoxy-3-(hydroxymethyl)ribofuranosy1 acetates and their related nucleoside analogues are described as follows. The 3-deoxy-3-fluoromethyl sugar derivative 7 was synthesized as depicted in Scheme I. The 3-deoxy-3hydroxymethyl derivative 1 was reacted with (diethylamido)sulfur trifluoride (DAST)13 to yield 3-deoxy-3-
L
BOX CFaCOOH
P
4
9*P phcHzo
OH
Ae,O/Pyridino
PhCHZo
F
1
B
(fluoromethyl)-l,2:5,6-di-0-isopropylidene-a-~-allofuranose (2). ‘H NMR spectrum of compound 2 showed that the resonances of 3-CHz-F methylene protons were at 6 4.61-4.74 (J H*,F = 47 Hz) and 6 4.75-4.87 (JH ~ , F= 47 Hz). Selective hydrolysis of compound 2 with HzS04/ MeOH14afforded 3-deoxy-3-(fluoromethyl)-l,2-O-isopropylidene-a-D-allofuranose(3),which was used directly for the next reaction without further purification. Periodate oxidation of compound 3 produced the aldehyde which was immediately reduced with sodium borohydride213to give 3-deoxy-3-(fluoromethyl)-1,2-O-isopropylidene-a-~ribofuranose (4). Benzylation3 of compound 4 gave the 5-0-benzyl derivative 5 which was then hydrolyzed with 80% trifluoroacetic acid, followed by acetylation with acetic anhydride in pyridine to afford the acetate 7. The 3-deoxy-3-azidomethyl sugar derivative 16 was synthesized as shown in Scheme 11. Treatment of 1,20-isopropylidene-a-Dxylofuranose (8) with tert-butyldimethylsilyl chloride in pyridine15 produced the 5-O-(tertbutyldimethylsilyl) derivative 9. Oxidationl6 of compound 9 with chromiumtrioxidelpyridinelaceticanhydride complex (1:2:1, molar ratio) in methylene chlorideyielded the 3-keto derivative 10, which was then converted to the 3-methylene analogue 11 by a Wittig reaction17Jswith
0022-262319311836-0353$04.00/0 0 1993 American Chemical Society
354 Journal of Medicinal Chemistry, 1993, Vol. 36,No.3
Lin et al.
Scheme I1
6%
6% B
+ho
P
lQ
1 M BoronoTTHF
2 N NIOH, 30% H202
Pyridino
HO
methylenetriphenylphosphorane in anhydrous DMSO under nitrogen. Hydroborati~n-oxidation~ of compound 11 afforded the 3-deoxy-3-hydroxymethylderivative 12. Treatment of compound 12 with methanesulfonylchloride in pyridine gave the methanesulfonate 13, which was reacted with lithium azide in DMFl9 to yield the azido analogue 14. DeblockingZ0compound 14 with 50% acetic acid at 100"C for 1h produced compound 15. Acetylation of compound 15 with acetic anhydride/pyridine gave a mixture of a and B anomer8 of 1,2,5-tri-O-acetyl-3-(azidomethyl)-3-deoxy-~-ribofuranose (16). The 3-deoxy-3-hydroxymethylsugar derivative, 1,2-0diacetyl-5-0-benzyl-3-[(benzy1oxy)methyll-3-deoxy-~-ribofuranose (17), was synthesized by hydrolysis of 5-0benzyl-3- [ (benzyloxy)methyll-3-deoxy-l,2-O-isopropylidene-a-~-ribofuranose~.~~ with 80% trifluoroacetic acid, followed by acetylation with acetic anhydride in pyridine.' The syntheses of the 3'-deoxy-3'-C-fluoromethyl and 3'-deoxy-3'-C-hydroxymethyl homologues of 1-8-D-arabinofuranosylthymine (ara-T),l-8-D-ribofuranosylthymine, and 3'-deoxy-3'-fluorothymidine (3'-FddT) are depicted in Scheme 111. Coupling of the 3-deoxy-3fluoromethyl and 3-deoxy-3-hydroxymethylsugar derivatives, compounds 7 and 17, and thymine in the presence of hexamethyldisilazane(HMDS), trimethylchlorosilane (TCS),and potassium nonafluoro-1-butanesulfonate(C4F&03K) by the methodology of Vorbriiggenand Bennua22 yielded the respective nucleosides, compound 18 (84 % yield), and compound 19 (87% yield) and ita a-isomer 20 (6% yield). In the first case,no a-isomerof 18 was isolated. The 'H NMR spectrum showed that the anomeric 1'-H resonance in compound 18 appeared at 6 5.98 as a doublet (51',2' = 3.5 Hz). The 1'-H resonance in compound 19 appeared at 6 6.02 as a doublet (J~',Z! = 3.2 Hz) and the 1'-H resonance in compound 20 appeared at 6 6.80also as a doublet but with a much larger coupling constant ( J ~ J , z # = 9.4 Hz). Treatment of compounds 18 and 19 with methanolic ammonia gave the 2'-deblocked nucleosides 21 and 22. Mesylation22of compounds 21 and 22 afforded the corresponding sulfonates 23 and 24, which were
refluxed with 1 N NaOH/EtOH for 2 hZ3 to yield compounds 27 and 28 via the 2,2'-anhydro intermediates 25 and 26, respectively. Catalytic hydrogenation of compounds 27 and 28 with 10% Pd/C produced the corresponding3'-deoxy-3'-C- (fluoromethyl)and 3'-deoxy3'-C-(hydroxymethyl) arabinonucleosidea29 and 30. Treab menta of compounds 21 and 22 with phenyl chlorothionocarbonateand 4-(dimethy1amino)pyridine in acetonitrile under nitrogen at room temperature gave the 2'-0phenoxythiocarbonyl derivatives 31 and 32. Reduction24 of compounds 31 and 32 with tri-n-butyltin hydride and 2,2'-azobis(2-methylpropionitrile) (AIBN) in toluene at reflux temperature produced the 2'-deoxynucleosides 33 and 34, which were then debenzylated by catalytic hydrogenationwith 10% Pd/C to afford the corresponding 2',3'-dideoxy-3'-C-(fluoromethyl) and 2',3'-dideoxy-3'-C(hydroxymethyl) nucleoside analogues 35 and 36. Catalytic hydrogenation (10% Pd/C) of compounds 21 and 22 yielded the 3'-deoxy-3'-C-(fluoromethyl) and 3'-deoxy3'-C-(hydroxymethyl) ribonucleoside derivatives 37 and 38, respectively. The syntheses of the 3'-deoxy-3'-C-(azidomethyl) and 3'-deoxy3'-C-(aminomethyl) nucleoside homologues of 1-8-arabinofuranosylthymine(ara-T), 1-8-D-ribofuranosylthymine, and 3'-deoxy-3'-azidothyidine (AZT) are described in Scheme IV. Condensation22of the 3-deoxy3-azidomethyl sugar derivative 16 and thymine in the presence of hexamethyldisilazane (HMDS), trimethylchlorosilane (TCS) and potassium nonafluoro-l-butanesulfonate (C4FgS03K) afforded 1-[2,5-0-diacetyl-3-C(azidomethyl)-3-deoxy-8-~-erythro-pentofuranosy1)thymine (39,41% yield) and ita a-isomer (40, 27% yield). The 1'-H resonance of compounds 39 and 40 were a doublet at 6 5.66 (J1:z. = 2.7 Hz) and 6 6.89 (&,r = 9.6 Hz), respectively. Deacetylation of compound 39 with methanolic ammonia gave the 3'-deoxy-3'-C-(azidomethyl) riboside 41. Treatment15.z6of compound 41 with tert-butyldimethylsilyl chloride/silver nitrate/pyridine yielded the 5'-O-tert-butyldimethylailyl-protectad nucleoside 44, which was then converted to the 2'-arabino analogue 47 via 45 and 46, and the 2'-deoxy ana-
Journal of Medicinal Chemistry, 1993, Vol. 36, No.3 366
3'-Deoxy-3'-C-Branched-Chain-Substituted Nucleosides Scheme I11
+
" ' O W
RJ
h c
1NN.W EtOH
RJ
I
1
AIBN, nBuaSnH, Toluenr
10% Pdc,
n,,
Y.OH
RJ
logue 50 via 49, respectively, by the similar methodology as previously described. Reaction15of compound SO with tetra-n-butylammoniumfluoride (n-B@F)/THF affordof ed the 5'-deprotected nucleoside 51. Treatment26127 compounds 41, 47, and 51 with triphenylphoephine in pyridine at room temperature, followed by hydrolysiswith concentrated ammonium hydroxide produced the 3'deoxy-3'-C-(aminomethyl)nucleosideanalogues42,48, and 52, respectively. Compounds 42 and 52 were converted to their corresponding hydrochloride salts and compounds 43 and 53 were isolated as solid products.
Biological Evaluation The synthesized compounds were evaluated in culture for their anticancer activity by growth inhibition studies using three murine cell lines; leukemia L1210, leukemia
RJ
P388, Sarcoma 180, as well as human CCRF-CEM lymphoblastic leukemia cells. Only the 3'-deoxy3'-C-(hydroxymethy1)thymidine (36)showed significant activity against L1210, P388, 5-180,and CCRF-CEM cells with the respective EDM values of 50, 5, 10, and 1 pM. The other compounds were not active against these cell lines at concentrations up to 100 pM. The results are summarized in Table I. Among these 3'-deoxy-3'-branched chain nucleoside analogueg, 1-[2,3-dideoxy-3-C-(fluoromethyl)-19-~erythropentofuranosyllthymine (35) and 1-[2,3-dideoxy-3-C(hydroxymethyl)-8-~-erythro-pentofuranoe(381, were found to demonstrate significant inhibitory effecte against thymidine kinase (TK) derived from herpee simplex type 1(KOS strain) infected HeLa (Bu-26-TK-) cells,28129yielding the corresponding 85.2 9% and 69.19% inhibition at 500 pM concentration. Whereas,at the same
356 Journal of Medicinal Chemistry, 1993, Vol. 36, No.3
Lin et al.
Scheme I V
+
I
Dlmrthylrmlnopyrldlnr C6H5OC(S)Cl, CH&N
0
0
H N O N
A
y
s)m6H5
N3
44
42
I
I
AIBN, n-BuaSnH, Tolurnr 0
42
HCllMeOH
HCL' H2N
N3-J
N3
1 yNgH 1. ( C d d d P
2. NHdOH
u
u
"9 1
A
or?
concentration,l - B D - a r a b i n o f u r a i n e (am-T)and 3'-deoxy3'-fluorothymidine (3'-FddT), which are known potent inhibitors of thymidine kinase,3O produced 55.1% and 62.4% inhibition of the enzyme, respectively. The findings are summarized in Table 11. None of the compoundsshowed antiviral activityagainst HSV-1 and HSV-2 in culture at a concentration of 100 pM. In addition, the 3'-deoxy-3'-C-(fluoromethyl) deriv-
atives 29,35, and 37 and the 3'-deoxy-3'-C-(azidomethyl) derivatives41,47,and51werealsoevaluatedagainat HIV-1 (HTLV-I11 B) in MT-2 cells and were not active at a concentration up to 100 pM. In cornpariaon to 3'-deoxy3'-fluorothymidine (3'-FddT) and 3'-azido-3'-deoxythythe insertion of a methylene group between midine (AZT), the 3'-carbon and the fluoro or the azido moiety in compounds 35 and 51, respectively, resulted in the total
S-Deoxy-Y- C-Branched-Chain-Substituted Nucleosides Table I. Comparison of the EDw Values of Various 3'-Deoxy-3'-C-Branched-Chain-Substituted Nucleoside Analogues on the Replication of L1210, P388, S-180, and CCRF-CEM Cells in Vitro 0 H N y H 3 OAN
compd
X
Y
R
EDm,O LIM,against cell lines L1210 P388 S-180 CCRF-CEM >lG >lo0 >100 >lo0 >lo0 >lo0 >100 >100 >100 >100 >lo0 >lo0 10 1 50 5 >100 >lo0 >lo0 >lo0 >lo0 >100 >lo0 >100 >100 >lo0 >100 >100 >100 >lo0 >100 >lo0 10 >100 >100 >100 >100 >100 >100 >lo0 >lo0 >100 >loo >lo0 >lo0 >lo0 >100 >lo0
F OH H OH OH H F H H H H H OH H OH H OH H OH OH H 48 OH H H 61 H 63 H H OThe EDw values were estimated from doseresponse curves compiled from at least two independent experiments and represent the drug concentration &M) required to inhibit replication of the respective L1210, P388, S-180, and CCRF-CEM cell lines by 50% after 72-h incubation. 29
30 35 36 37 38 41 43 47
Table 11. Effects of 3'-Deoxy-3'-Branched-Chain Nucleoside Analogues on the Activity of Thymidine Kinases Isolated from Herpes Simplex Type 1 (KOS Strain) and Herpes Simplex Type 2 (333 Strain) Infected HeLa (Bu-25-TK-) Cells, hpectively; and K562 Mammalian C e b activity, % inhibition.0 on thymidine kinaseb conc, HSV-1 HSV-2 mammalian compd pM (KOS) (333) (K562) 10.9 7.1 500 13.8 29 0.7 6.9 500 11.7 30 5.4 4.0 500 85.2 35 3.0 500 59.1 0 36 2.5 500 7.0 4.9 37 0 3.0 38 500 2.6 22.0 5.0 ara-T 500 55.1 60.3 0 3'-FddT 500 62.4 The enzymatic activity is 100% in the absence of the tested analogues. Standard mix contains -90 rM thymidine and 1.1,0.9, or 1.5 units of KOS,333, or K562 enzyme,respectively. The volume of the reation mixture was 0.1 mL.
loss of anti-HIV activity. Compounds 35 and 51 are probably not the substrates for the relevant kinase.
Experimental Section Melting points were determined with a Thomas-Hoover Unimelt apparatus and are uncorrected. lH NMR spectra were recorded on a Varian EM-390 (90MHz) NMR spectrometer or a Bruker WM-500 (500MHz) spectrometer (for the fiial products 29,30,35,36,37,38,41,43,47,48,51, and 53) with MedSi as the internal reference. Chemical ionization (CI-MS) mass spectra were determined with a Kratos MS80 RFA high-resolution instrument. IR spectra were obtained with a Perkin-Elmer 1420 spectrophotometer. The UV spectra were recorded on a Beckman-25 spectrophotometer. TLC was performed on EM precoated silica gel sheets containing a fluorescent indicator. Elemental analyses were carried out by the Baron Consulting Co., Orange, CT. Where analyses are indicated only by symbols of the elements, the analytical results for those elements were within &0.4% of the theoretical value.
Journal of Medicinal Chemistry, 1993, Vol. 36, No. 3 357
3-Deoxy-3-(fluoromethyl)-lf:6,6-di-O-ieopropylidene-aallofuranome (2). (Diethy1amido)sulfurtrifluoride (DAST, 10.3 g, 64.2 mmol) was dissolved in methylene chloride (150 mL) and pyridine (11mL) at 0 OC under nitrogen. Compound 1 (11.0 g, 40.1 mmol) was added slowly, and the solution was warmed to room temperature. After 4 h, the solvent was removed under vacuum to dryness to give a syrup, which was purified by silica gel column chromatography (eluting with hexane/EtOAc, 101, v/v) to yield 4.2 g (40%)of product as a colorless syrup: lH NMR (CDCb) 6 1.34,1.35,1.38, and 1.53 (4 ~ , 1 H, 2 4 X CHs), 2.36-2.42 (m, 1H, 3-H), 3.72-3.76 (m, 1H, 4-H), 3.92 (dd, 1H, HA,), 4.10 4.61-4.74 (m, 1H, (dd, 1HI~ - H B4.00-4.04 ), (m, 1H, J H ~ , F 47 Hz), F, J H*P 47 Hz), 4.75-4.87 (m, 1HI ~-CHB-F, 4.78 (t, 1 H, 2-H), 5.82 (d, 1 H, 1-HI. Anal. (CisH21FOa) C, H, F. 3-Deoxy-3-(fluoromethyl)-lf-O-isopropylidenaa-~ribofuranose (4). To a solution of compound 2 (4.9 g, 17.7 mmol) in MeOH (150 mL) was added 2 N sulfuric acid (27 mL). After the mixture was stirred at room temperature for 8 h, the reaction mixture was neutralized with solid sodium hydrogen carbonate and extracted with chloroform (3 X 60 mL). The combined chloroform extracts were dried over MgSO4, filtered, and evaporated under diminished pressure to afford a syrup (3). To a solution of syrup 3 in ethanol (150 mL) was added a saturated solution of NaHCO3 (7 mL) followed by sodium metaperiodate solution (4.54 g, 21.2 mmol, in 150 mL of water). After the reaction mixture stirred at room temperature for 4 h, the excess sodium metaperiodate was destroyed by the addition of methylene glycol (7 mL). The resulting aldehyde was immediately reduced with sodium borohydride (1.64 g, 42.4 mmol), and the solution was stirred at room temperature for 18 h. Acetone (4 mL) was added, and the mixture was stirred for an additional 0.5 h. After the solid material was removed by filtration, the filtrate was evaporated to dryness and the residue was chromatographed on a silica gel column (hexane/EtOAc, 1 0 1, v/v) to afford 3.2 g (87%) of product as a syrup: lH NMR (CDCl3) 6 1.33,1.50 (2 s,6 H, 2 X CH3), 2.10 (br, 1H, &OH, D10 exchangeable), 2.25-2.70 (m, 1H, 3-H), 3.45-4.25 (m, 3 H, 4-H, 5-H), 4.30-4.60 (m, 1H, CHAF,J H*,F = 45 Hz), 4.80-5.15 (m, 1 H, C H g , J H ~ = , F45 Hz), 4.70 (t, 1 H, 2-H), 5.70 (d, 1H, 1-H). Anal. (C&$O4) C, H, F. bOBenzyl-b-deoxy-b(fluorom~hyl)-lf-~~p~pylidenea-D-ribofuranose (5). A suspension of 80% sodium hydride/ mineral oil dispersion (0.76 g, 25.2 mmol) in DMF (100 mL) was stirred at 0-5 OC under Nz and treated with a solution of compound 4 (3.47 g, 16.8 mmol) in DMF (10 mL), added in small portions over a period of 10 min. After 30 min at 0-5 OC, benzyl chloride 42.0 mmol) was added and the solution was stirred overnight at room temperature. The mixture was treated with water (35 mL) in portions, with cooling. After 20 min of stirring, the mixture was extracted with EtOAc (3 X 50 mL). The combined organic layers were dried over MgSOl and evaporated (
