9-{[3-Fluoro-2-(hydroxymethyl)cyclopropylidene]methyl}adenines and guanines. Synthesis and Antiviral Activity of All Stereoisomers¹

Abstract

All stereoisomers of adenine and guanine methylene-3-fluoromethylenecyclopropane analogues of nucleosides 9a, 9b, 10a, 10b, 11a, 11b, 12a and 12b were synthesized and their antiviral activities were evaluated. A highly convergent approach permitted the synthesis of all these analogues using a single intermediate 15. Reaction of aldehyde 13 with fluorotrichloromethane and tri-n-butylphosphine gave fluoroalkenes 14a + 14b (83:17). Addition of carbene derived from ethyl diazoacetate gave cyclopropane 15 as the major product. Reduction (19), bromination (20) and phenylselenenylation (21) followed by Se-oxidation and β-elimination gave cis-methylenecyclopropane 22. Addition of bromine provided the reagent 23 for alkylation-elimination. Reaction of 23 with adenine led to isomeric mixture 25a + 26a which after deprotection afforded analogues 9a and 10a. The 2-amino-6-chloropurine furnished 25e + 26e and after deblocking (9e and 10e) and hydrolysis gave targets 9b and 10b. Intermediate 15 provided after debenzylation (27), 2-nitrophenylselenenylation (28), reduction (29), benzylation (30) and oxidation-elimination trans-methylenecyclopropane 31. Addition of bromine gave reagent 32. Further transformations followed the sequence outlined for analogues 9a, 9b, 10a and 10b. Analogue 9b was effective against HCMV (Towne) with EC₅₀ 2.9 μM. The trans-isomer 10b inhibited AD169 strain of HCMV (EC₅₀ 15 μM) and MCMV (EC₅₀ 2.5 μM). Compound 12a was effective against EBV (EC₅₀ <0.03 μM). Analogue 9a inhibited VZV (EC₅₀ 5.9 μM) and HIV-1 (EC₅₀ 5.2 μM). Analogues 9a, 10a and 11a are moderate substrates for adenosine deaminase. The structure-activity relationships will be discussed in context with other methylenecyclopropane analogues.

Methylenecyclopropane analogues of nucleosides are antiviral agents effective especially against human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV).^2,3 The antiviral potency of the first generation series resides mostly in purine Z(cis)-isomers 1 (Chart 1) whereas the E(trans) isomers 2 and pyrimidine analogues are active only exceptionally. The second generation Z(cis) isomers 3 have a more narrow antiviral effect^4–6 but the guanine analogue cyclopropavir (3, B = Gua) is effective in vivo⁷ and it is currently being developed as a potential drug against HCMV infections. As in the first generation series, the E(trans) isomers 4 lack anti-HCMV activity but some EBV potency has been noted.^4,6

Chart 1.

Frequently, fluoro analogues of biologically active compounds have yielded effective agents in many areas of biology and biochemistry.^8.9 For these reasons, we focused our attention on methylenecyclopropane analogues of nucleosides fluorinated in the cyclopropane moiety. In the previous work, we reported on 3,3-difluoromethylenecyclopropane analogues¹⁰ 5 and 6 and, more recently, 2-fluoro substituted compounds¹¹ 7 and 8. Although activity of compounds 5 and 6 was limited to a moderate potency¹⁰ of the E(cis)-isomer 5a against HCMV, several Z(cis)- and E(trans)-isomers of the series 7 and 8 were effective¹¹ against HCMV and EBV. Also, the methylene-3,3-difluorocyclopropanes 5 and 6 have limited stability¹⁰ which may have affected the biological activity but monofluoro compounds 7 and 8 are stable.¹¹ It was then of interest to investigate the isomeric methylene-3-fluorocyclopropane analogues 9, 10, 11 and 12.

Synthesis

At the outset, it was clear that a convergent approach utilizing a single intermediate for synthesis of all anticipated analogues 9, 10, 11 and 12 would be most convenient. Such a key compound should comprise the fluorocyclopropane ring carrying two different but modifiable substituents at the remaining cyclopropane carbon atoms. The different fluorocyclopropane stereochemistry necessary for cis-fluoro analogues 9 and 10 vs. trans-isomers 11 and 12 could then be generated by manipulation of the cyclopropane substituents.

The synthesis of such a key intermediate, compound 15, is described in Scheme 1. Benzyloxyacetaldehyde 13 was converted to an isomeric mixture of fluoroalkenes 14a + 14b (Z/E = 83:17) in 68% yield by a modified Wittig reaction using fluorotrichloromethane and tri-n-butylphosphine in dichloromethane followed by alkaline hydrolysis.¹² Addition of carbene¹³ derived from ethyl diazoacetate catalyzed by copper(II) acetylacetonate in dichloromethane gave a mixture of four cyclopropane stereoisomers 15, 16, 17 and 18 (65% conversion) and two unidentified fluorine containing components. The major stereoisomer 15 was formed by addition of carbene from a less hindered side of the double bond of the cis-isomer 14a. It was readily obtained by chromatography on a silica gel column in 42% yield. The other three stereoisomers 16, 17 and 18 were obtained as an unresolvable mixture identified by ¹⁹F NMR spectroscopy. Reduction of 15 with diisobutylaluminum hydride in tetrahydrofuran afforded hydroxymethylcyclopropane 19 (95%). Bromination using bromine - triphenylphosphine complex¹⁴ in dichloromethane gave crude bromomethylcyclopropane 20 (91%) which was, in turn, converted to phenylselenenylcyclopropane 21 in 94% yield using sodium phenylselenide generated in situ from diphenyl diselenide.⁵ Oxidation with 30% hydrogen peroxide was followed by β-elimination catalyzed by diisopropylethylamine in toluene¹⁰ at 80–85 °C to give methylenecyclopropane 22 (68%). Addition of bromine via pyridinium tribromide in dichloromethane afforded dibromide 23 (83%) obtained as a single stereoisomer of 95% isomeric purity. Alkylation-elimination protocol with adenine (K₂CO₃, DMF, 100–105 °C, 48 h) led to the alkylated product 24 and isomeric mixture of methylenecyclopropanes 25a + 26a. The elimination procedure was repeated with 24 to give additional 25a + 26a (total yield 46%). Finally, debenzylation with boron trichloride in dichloromethane at −78 °C furnished, after chromatographic separation, analogues 9a and 10a in 47% yield each. Alkylation-elimination with 2-amino-6-chloropurine and 23 under the conditions described for adenine isomers 25a + 26a gave isomeric mixture 25e + 26e (56%). Debenzylation gave the E- and Z-isomers 9e and 10e in 47% yield each. Hydrolysis with 80% formic acid afforded guanine analogues 9b and 10b (both in 95% yield).

Scheme 1.

The key intermediate 15 served also as a staring material for synthesis of the isomeric series 11 and 12 (Scheme 2). The O-debenzylation of 15 with boron trichloride in dichloromethane at −78 °C furnished hydroxyester 27 (83%). Reaction with 2-nitrophenyl selenocyanate and tri-n-butylphosphine in tetrahydrofuran (THF) using the procedure described¹⁰ for difluoro analogues 5 and 6 gave 2-nitrophenylselenenyl derivative 28 in 92% yield. Reduction with diisobutylaluminum hydride in THF afforded hydroxymethylcyclopropane 29 (95%). Benzylation with benzyl bromide using sodium hydride in THF led to intermediate 30 (71%). Oxidation with hydrogen peroxide in THF followed by β-elimination (see also Scheme 1, 21 → 22) provided methylenecyclopropane 31 (trans-isomer of 22 from Scheme 1) in 73% yield. Addition of bromine furnished dibromocyclopropane 32 (83%) obtained, in contrast to isomeric derivative 23, as a mixture of cis- and trans-isomers. Further transformations followed those described in Scheme 1 but the alkylation and elimination steps were separated. Alkylation of adenine (K₂CO₃, DMF, 25 – 40 °C) with 32 gave alkylated product 33a in 86% yield. β-Elimination was effected with K₂CO₃ in DMF at 100 °C to furnish E- and Z-isomers 34a and 35a which were separated by chromatography in 27 and 18% yield, respectively. The O-debenzylation afforded adenine analogues 11a and 12a (74 – 79%). The reaction sequence with 2-amino-6-chloropurine proceeded in a similar fashion: 32 → 33e (87%) → 34e and 35e (29 and 33%) → 11e and 12e (74% each). Hydrolysis then provided guanine analogues 11b and 12b in 73 – 76% yield.

Scheme 2.

Isomeric Structure of Analogues 9, 10, 11 and 12

A preliminary isomeric assignment of cis-and trans-alkene isomers 9 vs. 10 and 11 vs. 12 was made on the basis of chromatographic mobility that followed the pattern observed previously³ for other methylenecyclopropane analogues. The isomers 9 and 11 with a cis-configured base are faster moving than the respective trans-isomers 10 and 12. Although this “rule” may be of value for distinguishing cis- and trans-isomers at the alkene bond, it has little relevance for determining the cis- and trans-configuration at the cyclopropane moiety in 9 vs. 11 and 10 vs. 12. All these isomeric structures were readily established from ¹H and ¹⁹F NMR spectra (Table 1). The chemical shifts of the cis-isomers of the purine H₈, alkene H_1′ and OH of 9a and 11a are all located downfield from the respective trans-isomers 10a and 12a. A similar deshielding pattern was observed previously¹⁰ for the corresponding 3,3-difluoromethylene analogues 5a and 6a. The ³J_F,H coupling constants were then instrumental for isomeric assignment of the substituents in the cyclopropane moiety. The fluorine signals of isomers 9a and 10a with trans situated proton and fluorine appear as doublets with ³J_F,H <1 Hz whereas compounds 11a and 12a having cis-configured protons exhibit ³J_F,H 12.2 and 10.9 Hz, respectively. This is in accord with the general pattern in fluorocyclopropanes¹⁵ where the ³J_F,H-cis are much larger than ³J_F,H-trans. As expected, cis and trans located geminal fluorine atoms of difluoro analogues 5a and 6a also follow these trends.¹⁰ Similar relationships were observed for analogues containing bases other than adenine, compounds 9b, 10b, 9e and 10e. The ³J_F,H‘s of a similar magnitude, <1 and 13.9 Hz, were also observed for cis- and trans-methylenecyclopropanes 22 and 31 lacking the heterocyclic bases.

Table 1.

Chemical shifts (δ) and coupling constants ³J_F,H of the relevant ¹H NMR signals of fluorinated methylenecyclopropanes 5a, 6a, 9a, 10a, 11a and 12a

Compounda	H1′	H8	OH	3JF,H (Hz)
5a	8.19	8.68	5.39	<1, 7.5
6a	7.64	8.16	5.24	<1, 6.3
9a	7.89	8.72	5.2	<1
10a	7.55	8.35	5.03	<1
11a	7.93	8.71	5.17	12.2
12a	7.57	8.34	5.00	10.9

^aDMSO-d₆ as solvent. Values for 5a and 6a were taken from ref.10

The NOE experiments with analogues 9a, 10a, 11a and 12a confirmed these assignments (Table 2). As expected, in compounds 9a and 11a with a cis-configured adenine, the NOE enhancements were observed between the H₈ and H_4′ and OH and H₈. In trans isomers 10a and 12a, an interaction between the H₈ and H_3′ was observed. The NOE enhancements between the cis-configured cyclopropane protons H_3′ and H_4′ were noted for analogues 9a and 10a but they were absent in 11a and 12a where this relationship is trans.

Table 2.

The NOE enhancements of relevant ¹H NMR signals of 3-fluoromethylenecyclopropanes 9a, 10a, 11a and 12a

Compounda	Hiir	δ	Hobs	δ	NOE (%)
9a	H8	8.72	H4′	2.48	2.84
	H4′	2.48	H8	8.72	2.20
	OH	5.21	H8	8.72	1.98
	H4′	2.48	H3′	5.32, 5.49a	1.87, 2.40
10a	H8	8.35	H3′	5.53, 5.70a	2.47, 2.44
	H3′	5.53, 5.70a	H8	8.35	5.23, 3.67
	H4′	2.37	H3′	5.53, 5.70a	3.33, 3.76
	H3′	5.53, 5.70a	H4′	2.37	13.05, 12.57
11a	H8	8.71	H4′	2.60	1.54
	H4′	2.60	H8	8.71	3.36
	H8	8.71	OH	5.17	0.30
	OH	5.17	H8	8.71	2.58
12a	H8	8.34	H3′	5.28, 5.45a	1.08, 0.68
	H3′	5.28, 5.45a	H8	8.34	4.44, 2.33

^aDoublets of the H_3′ signals of 9a and 10a were treated separately as two singlets. Likewise, the doublets of doublets of 11a and 12a were treated as two doublets.

Biological Activity

Antiviral Activity

Analogues 9a, 9b, 10a, 10b, 11a. 11b, 12a and 12b were tested against the following viruses: human cytomegalovirus (HCMV), herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Epstein-Barr virus (EBV), varicella zoster virus (VZV), human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV). The results are summarized in Tables 3 and 4. Analogue 9b was active against HCMV/HFF (EC₅₀/CC₅₀ 2.9/>100 μM) in Towne strain of the virus (Table 3) but it had little effect against the AD169 strain. Conversely, trans-isomer 10b was somewhat effective against HCMV/HFF in the AD169 strain (EC₅₀/CC₅₀ 15/266 μM) and inactive against Towne strain. It was active with 2.5/>100 μM against the murine virus MCMV/MEF. Analogues 11a, 11b, 12a and 12b were without significant effect.

Table 3.

Inhibition of HCMV and HSV-1 replication by 3^′-fluoromethylenecyclopropane analogues of nucleosides

EC50/CC50 (μM)
Compound	HCMV/HFF		HSV-1/BSC-1a
	Towneb,c	AD169d,e
9a	31/>100	44.3/>100	80/>100
10a	>100/>100	>100/>100	>100/>100
9b	2.9/>100	>12/>300	70/>100
10b	>100/>100	15/266b,f	20/>100
11a	>100/>100	45/>300	20/>100
12a	>100/>100	285/>300	50/>100
11b	>100/>100	>60/255	>100/>100
12b	>100/>100	>60/183	>100/>100
Control	1.8/>100g	0.15/>100g	3.5/>100h

^aELISA. Cytotoxicity was determined in KB cells. All listed compounds were inactive against HSV-1 or HSV-2 in Vero (EC₅₀/CC₅₀ >50/>50 μM)^b and HFF cells (EC₅₀/CC₅₀ >100/>100 μM)^d culture.

^bPlaque reduction assay.

^cVisual cytotoxicity.

^dCytopathic effect (CPE) inhibition assay.

^eCytotoxicity by neutral red uptake.

^fAgainst MCMV/MEF^b the EC₅₀/CC₅₀ was 2.5/>100 μM. Because of a lack of significant potency against HCMV/AD169, other compounds listed in Table 1 were not tested.

^gGanciclovir.

^hAcyclovir.

Table 4.

Inhibition of EBV, VZV, HIV-1 and HBV replication by 3′-fluoromethylene-cyclopropane analogues of nucleosides

	EC50/CC50 (μM)
Compound	EBV
	Daudia	H-1b,c	VZV HFFd,e	HIV-1LAI MT-2d	HBV2.2.15b,c
9a	>50/>50	>20/>100	5.9f	5.2/>10	>20
10a	>50/>50	>20/>100	54.6f	>10/>10	>20
9b	>100/>100	13/74	>100f	>10/>10	>20
10b	>100/>100	>20/>100	68.3f	>10/>10	>20
11a	>100/>100	>10/9	33.6f	>10/>10	>10
12a	<0.03/>100	>10/>100	>60	>10/>10	>10
11b	>100/>100	>30/>100	>100	>10/>10	>30
12b	91.6/>100	>30/>100	>60	>10/>10	>30
Control	0.33/>100g	5h	0.03g	0.02/>10i	0.02/>100j

^aViral capsid antigen (VCA) ELISA.

^bDNA hybridization assay.

^cCytotoxicity was determined in CEM cells.

^dCytopathic effect (CPE) assay.

^eOnly the EC₅₀ values are listed, for CC₅₀‘s see HCMV(AD169)/HFF in Table 1.

^fPlaque reduction assay.

^gAcyclovir.

^hGanciclovir.

ⁱAZT.

^jLamivudine.

Moderate antiviral effects were detected by ELISA against HSV-1/BSC-1. The most potent compounds 10b and 11a had EC₅₀/CC₅₀ 20/>100 μM. Against EBV/Daudi (VCA-ELISA), the trans-isomer 12a was the most effective analogue with EC₅₀/CC₅₀ <0.03/>100 μM but it was devoid of potency in H-1 cells as determined by DNA hybridization assay (Table 4).

Under the latter conditions, guanine analogue 9b was moderately active with EC₅₀/CC₅₀ 13/74 μM. It was inactive in Daudi cells. The adenine analogue 9a was the most potent compound against VZV/HFF with EC₅₀/CC₅₀ 5.9/>100 μM and it also inhibited HIV-1/MT-2 (EC₅₀/CC₅₀ 5.2/>10 μM). The tested compounds were inactive against HBV in 2.2.15 cells and they were non-cytotoxic with the exception of analogue 11a with CC₅₀ 9 μM in CEM cells.

The analogues described herein conclude the first generation of methylenecyclopropanes 1 and 2 with a single fluorine atom in the cyclopropane moiety. Therefore, some generalizations regarding the structure-activity relationships of antiviral activity in this whole series of compounds can be made. The antiviral activity of the purine methylenecyclopropane analogues with a cis configuration of the nucleobase follows approximately the order 1 > 7 > 9 > 11 > 5. Introduction of the fluorine appears to narrow the antiviral effects.^10,11 Two geminal fluorines (5a, 5b, 6a and 6b) decrease the chemical stability of the analogues.¹⁰ Compounds with a trans-configured nucleobase 10 and 12 were mostly devoid of significant antiviral activity. Strong potency, sometimes in submicromolar range, was detected against EBV/Daudi by VCA-ELISA but it was not always reproduced in DNA hybridization assays. Analogues 8a, 8b, 8c (ref.¹¹) and 12a serve as examples. It is likely that the mechanism of action of active analogues of this series follows the conversion to the corresponding triphosphates which then inhibit the relevant DNA polymerase or reverse transcriptase. This was documented¹⁶ for non-fluorinated analogues 1 and 2.

Adenosine Deaminase (ADA)

Adenine fluoroanalogues 9a, 10a, 11a and 12a were investigated as substrates for adenosine deaminase from calf intestine. Whereas compounds 9a, 10a and 11a are moderate substrates which were about 50% deaminated after 24 h, analogue 12a was resistant to deamination up to 48 h.

Experimental Section

General Methods

The UV spectra were measured in ethanol and NMR spectra were determined at 300 or 400 MHz (¹H), 75 or 100 MHz (¹³C) and 376 MHz (¹⁹F). For ¹⁹F NMR CFCl₃ was used as a reference. For atom numbering of 3-fluoromethylenecyclopropane analogues see Table 2, formula 9a. Mass spectra were determined in electron-impact (EI-MS) or electrospray ionization (ESI-MS, methanol - NaCl) mode. Benzyloxyacetaldehyde¹⁷ (13) and 2-nitrophenyl selenocycanate¹⁸ were prepared as described. For the nomenclature of compounds with a double cis-trans isomerism in the cyclopropane ring an r,c,t system¹⁹ was adopted. The substituents were denoted as follows: r (reference), c (cis) and t (trans). Adenosine deaminase from calf intestine, X6B8577, 19.8 units/mg solid, was the product of Worthington, Lakewood, New Jersey.

(Z,E)-1-Benzyloxymethyl-2-fluoroethene (14a + 14b)

Tributylphosphine (205 mL, 0.825 mmol) was added dropwise with stirring at 0 °C to a mixture of CH₂Cl₂ (270 mL) and CFCl₃ (25.7 mL, 0.275 mol). The stirring was continued for 1 h at 0 °C and then 3 h at rt. Benzyloxyacetaldehyde¹⁷ (13, 33.3 g, 0.22 mol) in CH₂Cl₂ (20 mL) was added at 0 °C with stirring which was continued for 16 h at rt. Sodium hydroxide solution (10%, 330 mL) was then added and the stirring was continued for 24 h. The reaction mixture was cooled to 0 °C and the pH was adjusted to 5.0 by a careful addition of HCl. The organic phase was separated, it was washed with 5% HCl (5%, 2 × 150 mL) and dried over MgSO₄. The solvents were removed in vacuo, the residue was dissolved in hexanes and the solution was filtered through a silica gel pad. After removal of hexanes, the crude product was chromatographed on a silica gel column in hexanes to hexanes - Et₂O (40:1) to give Z- and E-isomers 14a and 14b (25.2 g, yield 68%, 83% 14a and 17% 14b as determined by ¹⁹F NMR) as a colorless oil which was of sufficient purity to be used in the next step. ¹H NMR (CDCl₃) d 3.98 (dt, J = 7.2, 1.6 Hz, 14b), 4.22 (dt, 2H, J = 7.2, 2.0 Hz, 14a, CH₂O), 4.54 (s, 14b), 4.56 (s, 2H, CH₂Ph, 14a), 5.09 (ddt, J = 42.0, 7.2, 4.8 Hz, 14a), 5.60 (ddt, 1H, J = 18.4, 11.2, 7.2 Hz, 1H, 14b, CH=), 6.67 (ddd, J = 84.4, 4.8, 1.6 Hz, 14a), 6.75 (ddd, 1H, J = 83.6, 11.2, 1.6 Hz, 14b, CHF=), 7.36 (m), 7.41 (m, 5H, Ph). ¹³C NMR 61.9 (d, J = 6.7 Hz, 14a), 64.8 (d, J = 14.2 Hz), 14b, CH₂O), 72.2 (14b), 72.5 (14a, CH₂Ph), 108.5 (d, J = 3.7 Hz, 14a), 108.9 (d, J = 9.8 Hz, 14b, CH=), 128.0 (14a), 128.1 (14b), 128.2 (14a, 14b), 128.7 (14a), 128.8 (14b), 138.2 (14b,) 138.4 (14a, Ph), 149.9 (d, J = 261.9 Hz, 14a), 152.1 (d, J = 261.2 Hz, 14b, CHF=). ¹⁹F NMR −126.07 (dd, J = 83.9, 44.4 Hz, 14a), −125.54 (ddt, J = 83.9, 18.1, 3.0 Hz, 14b). EI-MS 166 (M, 3.0), 165 (M − H, 4.2), 91 (PhCH₂, 100.0). EI-HRMS calcd for C₁₀H₁₁OF 166.0794, found 166.0791.

Ethyl t-2-Benzyloxymethyl-t-3-fluorocyclopropane-r-1-carboxylate (15)

Ethyl diazoacetate (11.5 g, 100 mmol) was added to a refluxing solution of 14a + 14b (16.0 g, 96.38 mmol) and copper acetylacetonate (0.75 g, 2.87 mmol) in CH₂Cl₂ (120 mL) using a syringe pump (0.34 mL/h) with stirring. The stirring was continued for 1 h, solvent was evaporated and the residue was put on a silica gel column which was eluted with hexane - EtOAc (100:0 to 10:1) to give unreacted 14a + 14b (5.6 g, 35%) followed by a mixture of products. Solvents were evaporated, the residue was dissolved in ether (150 mL) and KMnO₄ (15 g) in water (60 mL) was added with external ice-cooling and stirring to remove unsaturated impurities. The stirring was continued for 6 h and excess KMnO₄ was removed by addition of solid Na₂S₂O₃. The mixture was filtered through a short silica gel pad which was eluted with ether. The organic phase was washed successively with saturated NaHCO₃ (2 × 50 mL), water (2 × 50 mL) and it was dried over MgSO₄, The crude product was chromatographed on a silica gel column in hexane - Et₂O, 50:1 to give a faster moving isomer 15 as a colorless oil (6.65 g, 42%). The slower moving fraction was an inseparable mixture consisting of the three remaining isomers 16, 17, 18 and two unidentified fluorine-containing impurities.

Isomer 15

¹H NMR (CDCl₃) δ 1.26 (t, 3H, J = 7.2 Hz, CH₃), 1.91 – 1.99 (m, 2H, H₂, H₁), 3.78 – 3.58 (2 partly overlapped AB’s, 2H, CH₂OBn), 4.08 (2q, 2H, J = 7.2 Hz, CH₂ of Et), 4.52, 4.59 (AB, 2H, J = 11.7 Hz, CH₂Ph), 4.92 (ddd, 1H, J = 63.2, 5.6, 2.4 Hz, H₃), 7.28–7.36 (m, 5H, Ph). ¹³C NMR 14.4 (CH₃), 24.9 (d, J = 12.7 Hz, C₁), 26.3 (d, J = 8.3 Hz, C₂), 61.3 (CH₂ of Et), 65.6 (d, J = 7.5 Hz, CH₂OBn), 73.0 (CH₂Ph), 76.1 (d partly overlapped with CDCl₃, J = 230.5 Hz, C₃), 127.95, 127.98, 128.7, 138.2 (Ph), 170.7 (C=O). ¹⁹F NMR −220.05 (ddd, J = 64.0, 18.1, 7.5 Hz). EI-MS 252 (M, 5.1), 91 (PhCH₂, 100.0). EI-HRMS calcd for C₁₄H₁₇FO₃ 252.1162, found: 252.1164. Anal. C₁₄H₁₇FO₃ (C, H).

Ethyl c-2-Benzyloxymethyl-c-3-fluorocyclopropane-r-1-carboxylate (16)

¹⁹F NMR (CDCl₃) −232.66 (dt, J = 65.5, 9.0 Hz).

Ethyl t-2-Benzyloxymethyl-c-3-fluorocyclopropane-r-1-carboxylate (17)

¹⁹F NMR (CDCl₃) −220.66 (ddd, J = 64.0, 18.1, 7.5 Hz).

Ethyl c-2-Benzyloxymethyl-t-3-fluorocyclopropane-r-1-carboxylate (18)

¹⁹F NMR (CDCl₃) −205.84 (dt, J = 64.0, 18.4 Hz).

t-2-Benzyloxymethyl-t-3-fluorocyclopropyl-r-1-methanol (19)

Diisobutylaluminum hydride (DIBALH) in hexanes (1M, 45.80 mL, 45.80 mmol) was added to a solution of ester 15 (4.62 g, 18.33 mmol) in hexane (40 mL) with stirring at 0 °C during 10 min under N₂. The stirring was continued for 1 h. The reaction was quenched by a dropwise addition of HCl (5%, 50 mL) and then it was extracted with ether (4 × 30 mL). The organic phase was washed successively with saturated NaHCO₃ (2 × 30 mL) and water (2 × 30 mL). The solvents were evaporated and the residue was chromatographed on a silica gel column in hexanes -EtOAc = 10:1 to 5:1 to give compound 19 as a colorless oil (3.66 g, 95%). ¹H NMR (CDCl₃) δ 1.18 (ddd, 1H, J = 13.0, 6.6 Hz, H₂), 1.34 (dddd, 1H, J = 22.0, 13.6, 6.4, 1.6 Hz, H₁), 2.95 (bs, 1H, OH), 3.33 (m, 1H), 3.31–3.46 (m, 1H, CH₂OH), 3.57–3.71(m, 2H, CH₂OBn), 4.50 (ddd, 1H, J = 64.0, 6.4, 2.4 Hz, H₃), 4.44, 4.60 (split AB partly overlapped with H₃, 2H, CH₂Ph), 4.52 (ddd partly overlapped with CH₂Ph, 1H, J = 63.6, 6.0, 2.1 Hz, H₃), 7.28–7.36 (m, 5H, Ph). ¹³C NMR 21.6 (d, J = 10.5 Hz), 25.1 (d, J = 9.7 Hz, C_1′, C₂), 61.8 (CH₂OH), 67.3 (d, J = 8.2 Hz, CH₂OBn), 73.0 (CH₂Ph), 75.1 (d, J = 223.5 Hz, C₃), 128.0, 128.1, 128.7, 138.3 (Ph). ¹⁹F NMR −223.80 (ddd, J = 64.0, 21.5, 4.5 Hz). EI-MS 210 (M, 1.2), 91 (PhCH₂, 100.0). EI-HRMS calcd for C₁₂H₁₅FO₂ 210.1056, found: 210.1057. Anal. C₁₂H₁₅FO₂ (C, H).

t-2-Benzyloxymethyl-t-3-fluoro-r-1-bromomethylcyclopropane (20)

Bromine (2.60 g, 16.24 mmol) was added with stirring to a solution of PPh₃ (4.65 g, 17.71 mmol) in CH₂Cl₂ (20 mL) over 20 min maintaining the temperature below −30 °C. Compound 19 (3.10 g, 14.76 mmol) in CH₂Cl₂ (8 mL) was then added dropwise and the mixture was allowed to warm to rt. It was diluted with hexanes (150 mL) whereupon it was filtered through a silica gel plug (10 g). The plug was washed with hexanes - ethyl acetate (30:1, 150 mL) and the combined filtrates were evaporated to provide compound 20 as a colorless oil containing <10% PPh₃ (3.65 g, 91%). This product was used for preparation of phenylselenenyl derivative 21.

The experiment performed on a 1-mmol scale of 19 gave after chromatography on a silica gel column in hexanes - Et₂O (50:1 to 30:1) compound 20 (250 mg, 92%). ¹H NMR (CDCl₃) δ 1.32 (ddd, J = 12.0, 6.0, 2.0 Hz, 1H, H₂), 1.60 (dddd, J = 21.6, 10.8, 6.4, 2.0 Hz, 1H, H₁), 3.28 (dd, 2H, J = 7.6, 2.0 Hz, CH₂Br), 3.55 (poorly resolved dd, 1H), 3.77 (poorly resolved ddd, 1H, CH₂OBn), 4.54, 4.60 (AB, 2H, J = 12.2 Hz, CH₂Ph), 4.56 (ddd, 1H, J = 63.0, 6.0, 1.6 Hz, H₃), 7.30–7.37 (m, 5H, Ph). ¹³C NMR 25.2 (d, J = 11.2), 26.6 (d, J = 10.5 Hz, C₁, C₂), 32.8 (CH₂Br), 66.6 (d, J = 7.5 Hz, CH₂OBn), 72.9 (CH₂Ph), 77.6 (d, J = 226.8 Hz, C₃), 128.0, 128.1, 128.7, 138.4 (Ph). ¹⁹F NMR −219.63 (ddd, J = 64.0, 20.0, 5.6 Hz). ESI-MS (MeOH - KOAc) 311, 313 (M + K, 92.8 and 100.0). Anal. C₁₂H₁₄BrFO (C, H).

t-2-Benzyloxymethyl-t-3-fluoro-r-1-(phenylselenenylmethyl)cyclopropane (21)

Ph₂Se₂ (1.71 g, 5.50 mmol) was refluxed in ethanol (25 mL) till a clear solution was obtained. After cooling, NaOH (4M, 2.75 mL, 11 mmol) was added followed by NaBH₄ (0.835 g, 11 mmol). The reaction mixture was refluxed for 30 min and then it was cooled again to rt. A solution of compound 20 (3.0 g, 11 mmol) in ethanol (10 mL) was slowly added with stirring. After 3 h, water (125 mL) was added, the mixture was extracted with EtOAc, the organic phase was dried (MgSO₄) and concentrated. The residue was chromatographed on a silica gel column in hexane - EtOAc (30:1) to give compound 21 (3.63 g, 94%) as a colorless oil. ¹H NMR (CDCl₃) δ 1.20 (dd, J = 14.0, 6.4 Hz, H₂), 1.35–1.46 (m, 1H, H₁), 2.75–2.90 (poorly resolved 2 AB’s, 2H, CH₂SePh), 3.49 (poorly resolved dd, 1H), 3.72 (dd, 1H, J = 10.4, 5.6 Hz, CH₂OBn), 4.47 (poorly resolved dd, 1H, J = 63.2, 6.8 Hz, H₃), 4.52, 4.59 (AB, 2H, J = 12.0 Hz, CH₂Ph, partly overlapped with H₃), 7.29–7.34 (m, 4H), 7.37 (bd, 4H), 7.56–7.58 (m, 2H, 2 × Ph). ¹³C NMR 23.3, 25.8 (2d, J =10.5 Hz, C₁, C₂), 28.3 (CH₂SePh), 67.1 (d, J = 8.3 Hz, CH₂OBn), 72.8 (CH₂Ph), 77.8 (d, J = 227.6 Hz, C₃), 127.6, 127.9, 128.0, 128.7, 129.4, 129.6, 133.8, 138.6 (2 × Ph). ¹⁹F NMR −219.63 (ddd, J = 64.0, 21.5, Hz, < 1 Hz). EI-MS 350, 348 (M, 1.90, 0.87), 91 (PhCH₂, 100.0). EI-HRMS calcd for C₁₈H₁₉FO⁸⁰Se for 350.0585, found: 350.0585. Anal. C₁₈H₁₉FOSe (C, H).

cis-2-(Benzyloxymethyl)-3-fluoro-1-methylenecyclopropane (22)

Hydrogen peroxide (30%, 11.19 mL, 98.7 mmol) was added dropwise with stirring to a solution of compound 21 (3.49 g, 10 mmol) in THF (30 mL) at −60 °C. The mixture was allowed to warm to rt. After 14 h, water (100 mL) and EtOAc (100 mL) were added, the organic phase was washed with NaHCO₃ (5%) and water, it was dried (MgSO₄) and the solvents were evaporated. The residue was dissolved in toluene (15 mL), diisopropylethylamine (3.50 mL, 20 mmol) was added and the reaction mixture was stirred at 80–85 °C for 2 h. After removal of solvents, the crude product was chromatographed on silica gel in hexanes - EtOAc (30:1) to give compound 22 (1.30 g, 68%) as a colorless oil. ¹H NMR (CDCl₃) δ 2.08–2.17 (m, 1H, H₂), 3.60 (poorly resolved dd, 1H), 3.82 (ddd, 1H, J = 10.7, 5.6, 1.2 Hz, CH₂OBn), 4.56, 4.63 (AB, 2H, J = 12.0 Hz, CH₂Ph), 5.08 (dd, 1H, J = 69.0, 7.5 Hz, H₃), 5.66, 5.90 (2 poorly resolved t, 2H, CH₂=), 7.30–7.39 (2m, 5H, Ph). ¹³C NMR 23.9 (d, J = 15.1 Hz, C₂), 66.5 (d, J = 2.0 Hz, CH₂OBn), 69.3 (d, J = 231.7 Hz, C₃), 72.8 (CH₂Ph), 110.8 (J = 3.0 Hz, CH₂=), 130.9 (C₁), 127.9, 128.0, 128.6, 138.6 (Ph). ¹⁹F NMR −218.27 (dd, J = 68.9, < 1 Hz). EI-HRMS calcd for C₁₂H₁₃FO 192.0950, found: 192.0955. Anal. C₁₂H₁₃FO (C, H).

r-2-Benzyloxymethyl-c or t-1-bromo-c or t-1-bromomethyl)-c-2-fluorocyclopropane (23)

Pyridinium tribromide (3.20 g, 10.24 mmol) was added with stirring to a solution of compound 22 (1.2 g, 6.25 mmol) in CH₂Cl₂ (50 mL) at −78 °C. The reaction mixture was allowed to warm to rt. After 16 h, it was diluted with EtOAc (100 mL) and the resultant solution was washed sequentially with saturated Na₂S₂O₃, NaHCO₃ and water. The organic phase was dried over MgSO₄ and the solvents was evaporated. The crude product was chromatographed on a silica gel column in hexanes - Et₂O (40:1) to afford compound 23 (1.82 g, 83%) as a colorless oil. ¹H NMR (CDCl₃) δ 1.52–1.60 (m, 1H, H₂), 3.6–3.80 (cluster of m, 4H, CH₂Br and CH₂OBn), 4.47 (dd, 1H, J = 64.0, 7.6 Hz, H₃), 4.58 (s, CH₂Ph), 7.30–7.37 (cluster of m, 5H, Ph). ¹³C NMR 29.1 (d, J = 9.7 Hz, C₂), 38.8 (d, J = 9.0 Hz, C₁), 40.5 (d, J = 1.8 Hz, CH₂Br), 66.9 (d, J = 5.9 Hz, CH₂OBn), 73.3 (CH₂Ph), 74.6 (d, J = 235.8 Hz, C₃), 128.04, 128.06, 128.7, 138.1 (Ph). ¹⁹F NMR −213.89 (dd, J = 64.0, 9.0 Hz)²⁰. ESI-MS (MeOH + KOAc) 389, 391, 393 (M + K, 52.7, 100.0, 51.8). Anal. C₁₂H₁₃Br₂FO (C, H).

(Z,E)-9-{[cis-(3-Fluoro-2-benzyloxymethyl)cyclopropylidene]methyl}adenine (25a + 26a) and c or t-9-{[c or t-1-Bromo-c-3-fluoro-r-2-(benzyloxymethyl)cyclopropyl]methyl}adenine (24)

A mixture of adenine (287 mg, 2.2 mmol), compound 23 (704 mg, 2.0 mmol) and K₂CO₃ (1.66 g, 12.0 mmol) in DMF (10 mL) was stirred under N₂ for 4 h at 40 °C and then at 100–105 °C for 45 min. The mixture was rapidly cooled to −78 °C and then it was allowed to warm to rt. The insoluble solid was filtered off using a silica gel pad (5 g) which was washed with DMF (70 mL). The solvent was evaporated in vacuo, the residue was chromatographed on a silica gel column in EtOAc - MeOH (40:1 to 30:1) to give the faster moving E,Z-isomeric mixture 25a + 26a and slower moving intermediate 24 (490 mg, 1.21 mmol). The elimination procedure was repeated with 24 using K₂CO₃ (0.83 g, 6 mmol) and DMF (5.0 mL). The product was chromatographed as described above to give Z,E-mixture 25a + 26a and bromo derivative 24 (120 mg, 14.8%). Both portions of 25a + 26a were combined and they were rechromatographed in EtOAc - MeOH (50:1 to 30:1) to give E,Z-isomers 25a + 26a (300 mg, 46%, E/Z = 1:1).

E,Z-isomers 25a + 26a

Mp 166–170 °C. UV λ_max 237 nm (ε 25,700), 280 (ε 8,500). ¹H NMR (CDCl₃) δ 2.46 (bm, 1H, H_4′), 3.68, 3.85 (2m, 2H, H_5′), 4.47–4.63 (m, 2H, OCH₂Ph), 5.21, 5.37 (2 partially overlapped dd, J = 69.0, 69.6, 6.4 Hz, 1H, H_3′), 6.50, 6.53 (2s, 2H, NH₂), 7.26–7.36 (m, 5H, Ph), 7.59, 7.98 (2bs, 1H, H_1′), 8.38, 8.20, 8.75 (3s, 2H, H₂ and H₈). ¹⁹F NMR −214.83 and −215.88 (2d, J = 70.0, <1Hz). EI-MS 325 (M, 0.36), 91 (100.0). ESI-MS (MeOH) 326 (M + H, 100.0). EI-HRMS calcd for C₁₇H₁₆N₅FO 325.1339, found: 325.1339.

Compound 24

Mp 161–163 °C. UV λ_max 209 nm (ε 21,800), 261 (ε 12,200). ¹H NMR (CDCl₃) δ 1.79–1.87 (m, 1H, H_4′), 3.65–3.75 (m, 2H, H_5′), 4.36–4.52 (overlapped m of CH₂Ph and H_1′, 4H), 4.92 (dd, 1H, J = 64.0, 7.2 Hz, H_3′), 5.85 (s, 2H, NH₂), 7.26–7.36 (m, 5H, Ph), 8.02, 8.32 (2s, 2H, H₂, H₈). ¹³C NMR 26.9 (d, J = 10.5 Hz, C_4′), 39.1 (d, J = 9.8 Hz, C_2′), 51.9 (C_1′), 66.5 (d, J = 5.2 Hz, C_5′), 73.1 (d, J = 234.2 Hz, C_3′), 73.2 (CH₂ of Bn), 119.7 (C₅), 127.9, 128.0, 128.7, 138.0 (Ph), 140.9 (C₈), 150.4 (C₄), 153.4 (C₂), 155.8 (C₆). ¹⁹F NMR −216.85 (ddd, J = 64.0, 9.2 Hz). ESI-MS 406, 408 (M + H, 97.0, 100.0).

(E)-9-{[cis-(3-Fluoro-2-hydroxymethyl)cyclopropylidene]methyl}adenine (9a) and (Z)-9-{[cis-(3-Fluoro-2-hydroxymethyl)cyclopropylidene]methyl}adenine (10a)

Boron trichloride (1M in CH₂Cl₂, 4.44 mL, 4.44 mmol) was added to a solution of the Z,E-isomers 25a + 26a (200 mg, 0.555 mmol) in CH₂Cl₂ (40 mL) at −78 °C under N₂ over 10 min with stirring. The stirring was continued for 5 h at −78°C whereupon the reaction was quenched with methanol (20 mL) and NaHCO₃ (4.0 g, 47.6 mmol). After 20 min, the reaction mixture was allowed to warm to rt and it was stirred for 4 h. The insoluble solid was filtered off using a short silica gel pad (3.5 g) which was washed with CH₂Cl₂ - MeOH (2:1, 60 mL). After removal of solvents, the residue was chromatographed on a silica gel column in hexanes - EtOAc (1.5:1 to 1:1) to give the E- isomer 9a (70 mg, 46.5%) followed by Z-isomer 10a (70 mg, 46.5%).

E-Isomer 9a

Mp 243–245 °C. UV max λ_max 237 nm (ε 24,500), 280 (ε 8,300). ¹H NMR (DMSO-d₆) δ 2.48 (overlapped with DMSO-d₅, H_4′), 3.69 (bs, 2H, H_5′), 5.21 (poorly resolved t, 1H, OH), 5.41 (dd, 1H, J = 70.8 and 6.0 Hz, H_3′), 7.42 (s, 2H, NH₂), 7.89 (bs, 1H, H_1′), 8.20 (s, 1H, H₂), 8.72 (s, 1H, H₈). ¹³C NMR 27.3 (d, J = 14.1 Hz, C_4′), 58.2 (C_5′), 69.1 (d, J = 229.1 Hz, C_3′), 111.7, 116.5 (C_1′, C_2′), 119.2 (C₅), 138.5 (C₈), 149.1 (C₄), 154.0 (C₂), 156.8 (C₆). ¹⁹F NMR − 215.47 (d, J = 70.0 Hz). EI-MS 235 (M, 28.0), 218 (M - OH, 77.9), 205 (M - CH₂O, 24.9), 135 (adenine, 91.7), 136 (adenine + H, 100.0). EI-HRMS calcd for C₁₀H₁₀FN₅O 235.0869, found 235.0865. Anal. C₁₀H₁₀FN₅Ox0.1 H₂O (C, H, N).

Z-Isomer 10a

Mp 230–232 °C. UV λ_max 237 nm (ε 25,200), 280 (ε 8,000). ¹H NMR (DMSO-d₆) δ 2.35 – 2.38 (bm, 1H, H_4′), 3.51 (m, 1H), 3.69 (poorly resolved td, 1H, H_5′), 5.03 (t, 1H, J = 5.2 Hz, OH), 5.61 (dd, 1H, J = 69.0 and 6.6 Hz, H_3′), 7.45 (s, 2H, NH₂), 7.55 (s, 1H, H_1′), 8.20 (s, 1H, H₂), 8.35 (s, 1H, H₈). ¹³C NMR 26.3 (d, J = 14.2 Hz, C_4′), 58.1 (C_5′), 70.0 (d, J = 229.9 Hz, C_3′), 112.1, 114.9 (d, J = 3.2 Hz, C_1′, C_2′), 119.1 (C_5′), 138.0 (C₈), 148.9 (C₄), 154.2 (C₂), 156.8 (C₆).¹⁹F NMR −215.45 (d, J = 70.0 Hz). EI-MS 235 (M, 8.4), 218 (M - OH, 100.0), 135 (adenine, 22.9), 136 (adenine + H, 48.0). EI-HRMS calcd for C₁₀H₁₀FN₅O 235.0869, found: 235.0870. Anal. C₁₀H₁₀FN₅Ox0.1 H₂O (C, H, N).

(E)-2-Amino-6-chloro-9-{[cis-(3-fluoro-2-hydroxymethyl)cyclopropylidene]methyl}purine (9e) and (Z)-2-Amino-6-chloro-9-{[cis-(3-fluoro-2-hydroxymethyl)cyclopropylidene]-methyl}purine (10e)

A mixture of 2-amino-6-chloropurine (170 mg, 1.0 mmol), compound 23 (352 mg, 1.0 mmol) and K₂CO₃ (0.83 g, 6.0 mmol) in DMF (5 mL) was stirred at room temperature for 48 h and then at 100 – 105 °C for 45 min under N₂. The reaction mixture was worked up as described for the isomeric mixture 25a + 26a but the elimination procedure was not repeated. The crude product was chromatographed in EtOAc - hexanes (1:1) to give the (Z,E)- isomeric mixture 25e + 26e (200 mg, 56%). Boron trichloride (1M in CH₂Cl₂, 4.44 mL, 4.44 mmol) was added dropwise to a solution of compound 25e + 26e (200 mg, 0.56 mmol) in CH₂Cl₂ (40 mL) at −78 °C under N₂ over 10 min with stirring which was continued for 5 h. The reaction was quenched with methanol (20 mL) and NaHCO₃ (4 g, 47.6 mmol). After 20 min, the mixture was allowed to warm to room temperature and the stirring was continued for 4 h. The insoluble solid was filtered off using a silica gel pad (3.5g), which washed with CH₂Cl₂ - MeOH (2:1, 60 mL). The solvents were evaporated and the residue was chromatographed in hexanes -EtOAc = 1.5:1 to 1:1 to give the faster moving E-isomer 9e (70 mg, 46.5%) followed by Z-isomer 10e (70 mg, 46.5%).

E-Isomer 9e

Mp 209–211 °C. UV λ_max 239 nm (ε 26,300), 310 (ε 7,400). ¹H NMR (DMSO-d₆) δ 2.44–2.48 (1H, H_4′, overlapped with DMSO-d₅), 3.66 (t, 2H, J = 6.0 Hz, H_5′), 5.15 (t, 1H, J = 5.4 Hz, OH), 5.41 (dd, 1H, J = 70.4, 6.4 Hz, H_3′), 7.10 (s, 2H, NH₂), 7.71 (s, 1H, H_1′), 8.66 (s, 1H, H₈). ¹³C NMR 27.6 (d, J = 13.5 Hz, C_4′), 58.2 (C_5′), 69.1 (d, J = 229.1 Hz, C_3′), 112.5, 116.0 (2d, J = 2 Hz, C_1′, C_2′), 123.8 (C₅), 140.5 (C₈), 150.5 (C₄), 153.4 (C₂), 160.9 (C₆). ¹⁹F NMR −215.58 (d, J = 71.9 Hz). EI-MS 269, 271 (M, 17.6, 6.1), 252, 254 (M - OH, 16.2, 5.6), 169, 171 (2-amino-6-chloropurine, 37.9, 21.4), 170, 172 (2-amino-6-chloropurine + H, 100.0, 31.5). EI-HRMS calcd for C₁₀H₉³⁵ClFN₅O 269.0480, found: 269.0476.

Z-Isomer 10e

Mp 193–195 °C. UV λ_max 239 nm (ε 27,200), 310 (ε 7,200). ¹H NMR (DMSO-d₆) δ 2.35 (1H, poorly resolved dd, H_4′), 3.51 (dt, 1H, J = 11.6, 7.4 Hz, H_5′) and 3.66 (dt, 1H, J = 11.2, 5.6 Hz, H_5′), 5.0 (t, 1H, J = 5.8 Hz, OH), 5.62 (dd, 1H, J = 69.0, 6.6 Hz, H_3′), 7.12 (s, 2H, NH₂), 7.37 (s, 1H, H_1′), 8.31 (s, 1H, H₈). ¹³C NMR 26.4 (d, J = 14.2 Hz, C_4′), 58.0 (C_5′), 70.0 (d, J = 229.8 Hz, C_3′), 113.0, 114.5 (d, J = 3.5 Hz, C_1′, C_2′), 123.8 (C₅), 140.2 (C₈), 150.6 (C₄), 153.2 (C₂), 161.0 (C₆). ¹⁹F NMR −216.25 (d, J = 68.9 Hz). EI-MS 269, 271 (M, 14.9, 5.2), 252, 254 (M - OH, 14.9, 4.7), 169, 171 (2-amino-6-chloropurine, 39.0, 21.1), 170, 172 (2-amino-6-chloropurine + H, 100.0, 32.4). EI-HRMS calcd for C₁₀H₉³⁵ClFN₅O 269.0480, found: 269.0484.

(E)-9-{[cis-(3-fluoro-2-hydroxymethyl)cyclopropylidene]methyl}guanine (9b)

A solution of the E-isomer 9e (90 mg, 0.33 mmol) in 80% HCO₂H (80%, 10 mL) was heated at 80 °C with stirring for 4 h. After cooling, formic acid and water were evaporated in vacuo, the crude product was dissolved in methanol (30 mL), NH₃ (20% in methanol, 10 mL) was added at 0 °C. The reaction mixture was stirred for 4 h at 0 °C. The solvents were removed to give the E-isomer 9b (80 mg, 95%), mp >300 °C. UV λ_max 242 λ_nm (ε 26,300), 273 (ε 10,200). ¹H NMR (DMSO-d₆) δ 2.39 (m, 1H, H_4′), 3.65 (d, 2H, J = 5.6 Hz, H_5′), 5.16 (s, 1H, OH), 5.36 (dd, 1H, J = 71.2, 6.2 Hz, H_3′), 6.62 (s, 2H, NH₂), 7.58 (s, 1H, H_1′), 8.29 (s, 1H, H₈), 10.69 (bs, 1H, NH). ¹³C NMR 27.1 (d, J = 14.2 Hz, C_4′), 58.1 (C_5′), 69.0 (d, J = 229.0 Hz, C_3′), 111.3 (d, J = 3.0 Hz), 116.2 (d, J = 2.2 Hz, C_1′, C_2′), 117.0 (C₅), 134.8 (C₈), 150.8 (C₄), 154.8 (C₂), 157.3 (C₆). ¹⁹F NMR −215.43 (d, J = 71.9 Hz). ESI-MS (MeOH) 252 (M + H, 100.0). Anal. C₁₀H₁₀FN₅O₂ (C, H, N).

(Z)-9-{[cis-3-fluoro-2-(hydroxymethyl)cyclopropylidene]methyl}guanine (10b)

The procedure described above for 9b was performed with the Z-isomer 10e (90 mg, 0.33 mmol) to give compound 10b (80 mg, 95%), mp >300 °C. UV λ_max (EtOH) 242 nm (ε 27,900), 274 (ε 10,800). ¹H NMR (DMSO-d₆) δ 2.31 (m, 1H, H_4′), 3.49 (dd, 1H, J = 10.8, 8.8 Hz), 3.65 (dd, 1H, J =10.4, 5.6 Hz, H_5′), 4.99 (bs, 1H, OH), 5.58 (dd, 1H, J = 68.8, 5.6 Hz, H_3′), 6.67 (s, 2H, NH₂),7.26 (s, 1H, H_1′), 7.91 (s, 1H, H₈), 10.88 (bs, 1H, NH). ¹³C NMR 26.2 (d, J = 15.2 Hz, C_4′), 58.0 (C_5′), 69.0 (d, J = 231.7 Hz, C_3′), 111.8, 114.8 (d, J = 3.0 Hz, C_1′, C_2′), 117.1 (C₅), 134.3 (C₈), 150.6 (C₄), 155.0 (C₂), 157.3 (C₆). ¹⁹F NMR −216.82 (d, J = 68.5 Hz). ESI-MS (MeOH - KOAc) 252 (M + H, 90.0), 290 (M + K, 100.0), 503 (2M + H, 33.0), 541 (2M + K, 50.0). Anal. C₁₀H₁₀FN₅O₂ (C, H, N).

Ethyl t-3-Fluoro-t-2-hydroxymethylcyclopropane-r-1-carboxylate (27)

Boron trichloride (1.0 M in CH₂Cl₂, 40 mL, 40 mmol) was added dropwise with stirring to ester 15 (5.0 g, 19.8 mmol) in CH₂Cl₂ at −78 °C. The reaction mixture was stirred for 1.5 h at −78 °C, 15 min at 0 °C, it was then re-cooled to −78 °C and NaHCO₃ (6.72 g, 80 mmol) was added. The reaction mixture was warmed to room temperature and it was stirred for 4 h. Water (200 mL) was added and the mixture was extracted with CH₂Cl₂ (5 × 50 mL), The organic phase dried over MgSO₄, solvent was evaporated and the crude product was chromatographed on a silica gel column in hexanes: Et₂O = 10:1 to 3:1 to give ester 27 (2.66 g, 83%) as a colorless oil. ¹H NMR (CDCl₃) δ 1.22 (t, 3H, J = 7.4 Hz, CH₃), 1.85 (dd, 1H, J = 14.0, 7.6 Hz, H₂), 1.94 (ddd, 1H, J = 18.4, 6.4, 1.6 Hz, H₁), 2.79 (bs, 1H, OH), 3.84, 3.69 (J_AB = 11.8 Hz), 3.83, 3.71 (J_AB = 11.4 Hz, 2 overlapped AB’s, 2H, CH₂OH), 4.08 (dq, 2H, J = 7.6, 2.4 Hz, CH₂ of Et), 4.88 (ddd, 1H, J = 64.0, 6.4, 1.6 Hz, H₃). ¹³C NMR 14.3 (CH₃ of Et), 24.9 (d, J =12.7 Hz, C₁), 28.6 (d, J = 8.2 Hz, C₁, C₂), 58.6 (d, J = 9.0 Hz, CH₂OH), 61.4 (CH₂ of Et), 76.4 (d, J = 229.8 Hz, C₃), 171.0 (C=O). ¹⁹F NMR −221.34 (ddd, J = 64.4, 18.3, 7.7 Hz). EI-MS 163 (M + H, 0.4), 145 (M -OH, 1.2), 131 (M - CH₂OH, 44.5), 73 (100.0). EI-HRMS calcd for C₇H₁₀FO₂ (M - OH) 145.0665, found 145.0665. Anal. C₇H₁₀FO₂ (C, H).

Ethyl t-2-Fluoro-t-3-(2-nitrophenylselenenyl)cyclopropane-r-1-carboxylate (28)

Tributylphosphine (2.49 g, 14.8 mmol) was added to a mixture of ester 27 (2.0 g, 12.33 mmol) and 2-nitrophenyl selenocyanate¹⁷ (3.36 g, 14.8 mmol) in THF (40 mL) at room temperature with stirring which was continued for 2 h. The solvent was evaporated and the crude product was chromatographed on a silica gel column using hexanes: Et₂O (10:1 to 5:1) to give product 28 (3.93 g, 92%) as a yellow oil. ¹H NMR (CDCl₃) δ 1.25 (t, 3H, J = 7.2 Hz, CH₃), 1.91 (dd, 1H, J = 13.8, 6.6 Hz, H₂), 1.98 (ddd, 1H, J = 18.2, 5.6, 2.4 Hz, H₁), 3.06, 3.11 (J_AB = 12.2 Hz), 3.08, 3.14 (J_AB = 12.0 Hz, 2 overlapped AB, 2H, CH₂Se), 4.12 (2 overlapped q, 2H, J = 7.2 Hz, CH₂ of Et), 4.94 (ddd, 1H, J = 64.4, 6.4, 1.6 Hz, H₃), 7.33 (ddd, 1H, J = 8.0, 5.6, 2.4 Hz), 7.52 (bs, 1H), 7.54 (dd, 1H, J = 8.0, 1.6 Hz), 8.27 (d, 1H, J = 8.4 Hz, 2-NO₂Ph). ¹³C NMR 14.4 (CH₃), 21.4 (d, J = 7.5 Hz, CH₂Se), 25.3 (d, J = 8.2 Hz), 28.0 (d, J = 11.8 Hz), 61.5 (CH₂ of Et), 76.8 (d, J = 231.3 Hz, C₃), 126.0, 126.8, 129.2, 133.0, 134.1 (2-NO₂Ph), 170.3 (C=O). ¹⁹F NMR −220.22 (ddd, J = 64.0, 18.1, 6.0 Hz). EI-MS 347 (M + H, 5.4), 346 (M, 1.4), 125 (100.0). EI-HRMS calcd for C₁₃H₁₄FNO₄⁸⁰Se 347.0072, found: 347.0066.

t-3-Fluoro-t-2-(2-nitrophenylselenenylmethyl)cyclopropane-r-1-methanol (29)

DIBALH in hexane (1M, 21.67 mL, 21.67 mmol) was added to a solution of ester 28 (3.0 g, 8.67 mmol) in hexane (30 mL) at 0 °C during 10 min under N₂. The stirring was continued for 1 h. The reaction was quenched by a dropwise addition of HCl (5%, 50 mL) and then it was extracted with Et₂O (4 × 30 mL). The combined organic phase was washed successively with saturated NaHCO₃ (2 × 30 mL) and water (2 × 30 mL). The solvent was evaporated and the crude product was chromatographed on a silica gel column using hexane: EtOAc (10:1 to 5:1) to give product 29 as a colorless oil (2.49 g, 95%). ¹H NMR (CDCl₃) δ 1.27 (ddd, 1H, J = 13.6, 6.8, 1.6 Hz, H₂), 1.44 (ddd, 1H, J = 21.2, 12.8, 6.4 Hz, H₁), 1.68 (bs, 1H, OH), 3.08, 3.12 (J_AB = 11.6 Hz), 3.06, 3.14 (J_AB = 12.0 Hz, 2 overlapped AB, 2H, CH₂Se), 3.51–3.60 (m, 2H, CH₂O), 4.65 (ddd, 1H, J = 64.0, 6.4, 2.4 Hz, H₃), 7.32 (ddd, 1H, J = 8.0, 5.6, 2.4 Hz), 7.54 (dd, 2H, J = 8.0, 1.6 Hz), 8.29 (d, 1H, J = 8.8 Hz, 2-NO₂Ph). ¹³C NMR 19.8 (d, J = 10.4 Hz, C₁), 23.1 (d, J = 7.5 Hz, CH₂Se), 28.3 (d, J = 9.0 Hz, C₂), 61.8 (CH₂O), 76.0 (d, J = 235.4 Hz, C₃), 125.8, 126.7, 129.4, 134.0 (2- NO₂Ph).¹⁹F NMR −223.87 (ddd, J = 64.0, 21.5, 3.0 Hz). EI-MS 303 (M+ H, 4.9), 186 (100.0). EI-HRMS calcd for C₁₁H₁₂FNO₃⁷⁸Se 302.9974, found 302.9966.

r-1-Benzyloxymethoxymethyl-t-2-fluoro-t-3-(2-nitrophenylselenenylmethyl)cyclopropane (30)

Sodium hydride (50%, 0.63 g, 13.1 mmol) was added to a solution of compound 29 (2.0 g, 6.6 mmol) in THF (30 mL) at 0 °C. The reaction mixture was stirred for 5 h and then 1 h at room temperature. Benzyl bromide (2.57 g, 15 mmol) was added at 0 °C, the reaction mixture was slowly warmed to room temperature and it was stirred for 16 h. The solvent was evaporated and the crude product was chromatographed on a silica gel column in hexane: Et₂O (30:1 to 5:1) to give compound 30 (1.84 g, 71%) as an oil. ¹H NMR (CDCl₃) δ 1.26 (m, 1H, H₂), 1.46 (ddd, 1H, J = 20.9, 6.5, 2.5 Hz, H₁), 3.10 (d, 2H, J = 7.6 Hz, CH₂Se), 3.41 (poorly resolved dd, 1H, J = 6.8, 2.0 Hz, CH₂O), 4.51, 4.48 (AB, 2H, J_AB = 11.8 Hz, CH₂Ph), 4.63 (ddd, 1H, J = 64.0, 6.4, 2.4 Hz, H₃), 7.28–7.36 (m, 6H), 7.48–7.56 (m, 2H), 8.30 (dd, 1H, J = 8.0, 1.6 Hz, Ph + 2-NO₂Ph). ¹³C NMR 20.0 (d, J = 11.3 Hz, C₁), 23.1 (d, J = 6.6 Hz, CH₂Se), 25.9 (d, J = 9.8 Hz, C₂), 68.6 (CH₂O), 72.9 (CH₂Ph), 76.3 (d partly overlapped with CDCl₃, J = 240.0 Hz, C₃), 125.7, 126.7, 127.8, 128.0, 128.7, 129.4, 133.9 (Ph + 2-NO₂Ph). ¹⁹F NMR −223.69 (ddd, J = 65.0, 21.1, 4.5 Hz). EI-MS 395 (M, 0.29), 91 (100.0). EI-HRMS calcd for C₁₈H₁₈FNO₃⁸⁰Se 395.0436, found 395.0434.

trans-3-Benzyloxymethyl-2-fluoromethylenecyclopropane (31)

Hydrogen peroxide (30%, 1.6 mL, 15.66 mmol) was added dropwise to a solution of compound 30 (1.8 g, 4.58 mmol) in THF (20 mL) at 0 °C. The reaction mixture was stirred for 1 h and then 12 h at room temperature whereupon it was partitioned between water (50 mL) and Et₂O (100 mL). The organic phase was washed with water (2 × 50 mL), Na₂S₂O₃ (5%, 2 × 20 mL), NaHCO₃ (5%, 2 × 50 mL), it was dried over MgSO₄ and the solvent was evaporated. A solution of the crude product in toluene (25 mL) was heated at 80–85 °C for 6 h. The solvent was evaporated and the residue was chromatographed on a silica gel column in hexanes: Et₂O (50:1) to give compound 31 (640 mg, 73%) as an oil. ¹H NMR (CDCl₃) δ 2.20–2.22 (m, 1H, H₂), 3.35–3.39 (poorly resolved dd, 1H), 3.37 (poorly resolved dd, 1H), 3.49 (m, 1H, CH₂O), 4.55 (s, 2H, PhCH₂), 4.76 (d, 1H, J =67.2 Hz, H₃), 5.70, 5.92 (2s, 2H, CH₂=), 7.30–7.36 (m, 5H, Ph). ¹³C NMR 25.0 (d, J = 13.0 Hz, C₂), 68.7 (CH₂O), 70.6 (d, J = 230.3 Hz, C₃), 72.8 (PhCH₂), 111.4 (CH₂=), 127.9, 128.0, 128.7, 131.8, 138.2 (C₁ + Ph). ¹⁹F NMR −203.67 (dd, J = 67.4, 13.9 Hz). ESI-MS 215 (M + Na, 42.3), 91 (PhCH₂, 100.0).

r-2-Benzyloxymethyl-c,t-1-bromo-c,t-1-bromomethyl-t-fluorocyclopropane (32)

Pyridinium tribromide (2.0 g, 6.27 mmol) was added to a solution of compound 31 (600 mg, 3.13 mmol) in CH₂Cl₂ (15 mL) at −20 °C with stirring. The reaction mixture was warmed to room temperature and the stirring was continued for 10 h. After removal of the solvent, the crude mixture was chromatographed on a silica gel column in hexanes: Et₂O (50:1 to 20:1) to give product 32 (910 mg, 83%).¹H NMR (CDCl₃) δ 1.73, 2.21 (2m, 1H, H₁), 3.58–3.72 (m, 2H, CH₂Br), 3.76–3.97 (m, 2H, CH₂O), 4.45 (dd, J = 63.8, 4.1 Hz), 4.82 (dd, J = 63.0, 3.6 Hz, 1H, H₃), 4.51, 4.52, 4.59 (3s, 2H, CH₂Ph), 7.33–7.40 (m, 5H, Ph). ¹³C NMR 33.2, 35.8 (2d, J = 11.1, 10.0 Hz, C₂), 38.0–38.1 (2 overlapped d, CH₂Br), 38.5, 40.3 (2d, J = 10.1 Hz, C₁), 64.37, 68.90, 73.2, 73.5 (CH₂O), 76.1, 81.3 (2d, J = 241.7, 243.7 Hz, C₃), 77.0, 77.4, 77.8 (CH₂Ph), 128.1, 128.14, 128.3, 128.8, 128.85, 137.5, 138.0 (Ph). ¹⁹F NMR −201.56 (dd, J = 64.0, 20.0 Hz), −207.76 (dd, J = 62.9, 22.8 Hz). ESI-MS 373, 375, 377 (M + Na, 49.0, 100.0, 48.0).

c,t-9-{[c,t-1-Bromo-t-3-fluoro-r-2-(benzyloxymethyl)cyclopropyl]methyl}adenine (33a)

A mixture of K₂CO₃ (280 mg, 2.22 mmol), adenine (50 mg, 0.37 mmol) and compound 32 (120 mg, 0.34 mmol) was stirred in DMF (2.0 mL) at rt for 8 h and at at 40 °C for 2 h under N₂. The insoluble solid was filtered off and DMF was evaporated in vacuo. The residue was chromatographed on a silica gel column using EtOAc - MeOH (100:0 to 20:1) to give compound 33a (0.12 g, 86%), mp. 112–115 °C. UV λ_max 261 nm (ε 11, 700), 203 (ε 23,400). ¹H NMR (CDCl₃) δ 2.19–2.33 (1H, m, H_4′), 3.46–3.51, 3.57–3.67 (2m, 2H, H_5′), 4.43–5.00 (overlapped m, 5H, CH₂Ph, H_1′, H_3′), 5.84 (s, 2H, NH₂), 7.21–7.34 (m, 5H, Ph), 8.03, 8.06, 8.33 and 8.37 (4s, 1H, H₂, H₈). ¹⁹F NMR −203.35 (ddd, J = 64.0, 19.6, 4.5 Hz), −206.81 (dd, J = 62.5, 23.0 Hz). ESI-MS 406, 408 (M + H, 94.0, 100.0), 428, 430 (M + Na, 24.3, 27.3).

(E)-{[trans-(3-Fluoro-2-benzyloxymethyl)cyclopropylidene]methyl}adenine (34a) and (Z)-{[trans-(3-Fluoro-2-benzyloxymethyl)cyclopropylidene]methyl}adenine (35a)

A mixture of compound 33a (0.40 g, 0.98 mmol) and K₂CO₃ (410 mg, 3 mmol) in DMF (5 mL) was stirred for 55 min at 100 °C. The mixture was cooled to 0 °C, the insoluble portion was filtered off using a silica gel (3.5 g) pad which was washed with DMF (10 mL). The solvent was evaporated and the residue was chromatographed on a silica gel column using hexanes: EtOAc (1:4 to 100% EtOAc) to give the E,Z-isomeric mixture 34a + 35a (50 mg, 16%) followed by starting material 33a (260 mg, 59%). The latter was subjected to another two cycles of elimination and chromatography to give 34a + 35a (105 mg, 33%). The isomeric mixture 34a + 35a (390 mg, 1.2 mmol, E/Z = 1.5:1)) combined from several experiments was chromatographed on silica gel using hexanes - EtOAc = 1:1 to 1:2 to 100% EtOAc to give the faster moving E-isomer 34a (220 mg, 56%) followed by the Z-isomer 35a (150 mg, 38%).

E-Isomer 34a

Mp 173–175 °C. UV λ_max 280 nm (ε 9,500), 238 (ε 26,600). ¹H NMR (CDCl₃) δ 2.62 (m, 1H, H_4′), 3.30 (t, 1H, J = 9.2 Hz), 3.91 (dd, 1H, J = 9.6, 5.6 Hz, H_5′), 4.55 (s, 2H, CH₂Ph), 4.97 (d, 1H, J = 68.8 Hz, H_3′), 6.08 (s, 2H, NH₂), 7.26–7.33 (m, 5H, Ph), 7.99 (s, 1H, H_1′), 8.38, 8.76 (2s, 2H, H₂, H₈). ¹³C NMR (CDCl₃) δ 26.6 (d, J = 13.4 Hz, C_4′), 68.8 (d, J = 234.3 Hz, C_3′), 68.9 (d, J = 3.7 Hz, C_5′), 73.6 (CH₂Ph), 111.4 (d, J = 4.4 Hz), 117.0 (d, J = 3.7 Hz, C_1′, C_2′), 119.6 (C₅), 128.0, 128.3, 128.8, 137.5 (Ph), 138.3 (C₈), 149.3 (C₄), 153.7 (C₂), 155.8 (C₆). ¹⁹F NMR −201.70 (dd, J = 68.9, 10.5 Hz). ESI-MS 326 (M + H, 100.0), 348 (M + Na, 31.6).

Z-Isomer 35a

Mp 159–162 °C. UV λ_max 279 nm (ε 10,000), 237 (ε 29,400). ¹H NMR (CDCl₃) δ 2.58 (m, 1H, H_4′), 3.49 (poorly resolved dd, 1H), 3.60 (m, 1H, H_5′), 4.56 (s, 2H, CH₂Ph), 5.09 (d, 1H, J = 68.0 Hz, H_3′), 6.11 (s, 2H, NH₂), 7.29–7.34 (m, 5H, phenyl), 7.63 (s, 1H, H_1′), 8.19, 8.38 (2s, 2H, H₂, H₈). ¹³C NMR 25.2 (d, J = 12.7 Hz, C_4′), 68.1 (d, J = 3.7 Hz, C_5′), 70.0 (d, J = 229.8 Hz, C_3′), 73.2 (CH₂Ph), 111.9, 116.1 (C_1′, C_2′), 119.6 (C₅), 128.0, 128.2, 128.8, 137.8 (Ph), 138.0 (C₈), 149.0 (C₄), 153.9 (C₂), 155.8 (C₆). ¹⁹F NMR −202.17 (dd, J = 68.3, 10.0 Hz). ESI-MS 326 (M + H, 100.0), 348 M + Na, 48.2).

(E)-{[trans-(3-Fluoro-2-hydroxymethyl)cyclopropylidene]methyl}adenine (11a)

A solution of BCl₃.SMe₂ complex in CH₂Cl₂ (2.0 M, 1.37 mL, 2.74 mmol) was added dropwise to a solution of compound 34a (150 mg, 0.46 mmol) in CH₂Cl₂ (10 mL) at room temperature with stirring which was continued for 5 h. The reaction was quenched by adding NaHCO₃ (4.0 g, 47.6 mmol) and methanol (15 mL) at −78 °C and then it was stirred for 2 h at room temperature. The insoluble solid was filtered off through a silica gel (2.5 g) pad and it was washed with CH₂Cl₂ - MeOH (2:1, 50 mL). The filtrate was concentrated and the residue was chromatographed on a silica gel column to give product 11a (80 mg, 74%), mp 218–220 °C. UV λ_max 280 nm (ε 11,000), 237 (ε 32,600). ¹H NMR (DMSO-d₆) δ 2.60–263 (m, 1H, H_4′), 3.48, 3.67 (2m, H_5′), 5.17 (t, 1H, J = 5.6 Hz, OH), 5.21 (d, 1H, J = 70.4 Hz, H_3′), 7.41 (s, 2H, NH₂), 7.93 (s, 1H, H_1′), 8.19 (s, 1H, H₂), 8.71 (s, 1H, H₈). ¹³C NMR 29.5 (d, J = 11.2 Hz, C_4′), 60.2 (d, J = 3.7 Hz, C_5′), 70.0 (d, J = 229.8 Hz, C_3′), 112.5 (d, J = 2.9 Hz), 116.8 (d, J = 3.7 Hz, C_1′, C_2′), 119.2 (C₅), 138.4 (C₈), 149.1 (C₄), 154.0 (C₂), 156.8 (C₆). ¹⁹F NMR −200.81 (dd, J = 70.2, 12.2). EI-MS 235 (M, 14.9), 218 (M - OH, 100.0), 136 (Ade + H, 48.4), 135 (Ade, 36.0). EI-HRMS calcd for C₁₀H₁₀N₅FO 235.0869, found: 235.0871. Anal. C₁₀H₁₀FN₅O (C, H, N).

(Z)-{[trans-(3-Fluoro-2-hydroxymethyl)cyclopropylidene]methyl}adenine (12a)

The procedure described above for the E-isomer 11a was repeated with the Z-isomer 35a to give compound 12a (85 mg, 79%), mp 239–242 °C. UV λ_max 280 nm (ε 11,300), 237 (ε 31,100). ¹H NMR (DMSO-d₆) δ 2.42–2.45 (m, 1H, H_4′), 3.37 (poorly resolved dd, 1H), 1H), 3.51–3.54 (m, 1H, H_5′), 5.02 (bs, 1H, OH), 5.36 (d, 1H, J = 68.8 Hz, H_3′), 7.44 (s, 2H, NH₂), 7.58 (s, 1H, H_1′), 8.21 (s, 1H, H₂), 8.34 (s, 1H, H₈). ¹³C NMR 27.6 (d, J = 11.1 Hz, C_4′), 60.3 (d, J = 4.4 Hz, C_5′), 70.8 (d, J = 229.8 Hz, C_3′), 112.7 (d, J = 2.2 Hz), 115.7 (d, J = 2.3 Hz, C_1′, C_2′), 119.2 (C₅), 138.1 (C₈), 148.9 (C₄), 154.0 (C₂), 156.7 (C₆). ¹⁹F NMR −201.15 (dd, J = 68.9, 10.9 Hz). EI-MS 235 (M, 17.1), 218 (M - OH, 100.0), 136 (adenine + H, 37.7), 135 (adenine, 28.1). EI-HRMS calcd. for C₁₀H₁₀ N₅FO 235.0869, found: 235.0871. Anal. C₁₀H₁₀FN₅O (C, H, N).

2-Amino-6-chloro-c,t-9-{[c,t-1-bromo-t-3-fluoro-r-2-(benzyloxymethyl)cyclopropyl]-methyl}purine (33e)

A mixture of 2-amino-6-chloropurine (580 mg, 3.43 mmol), compound 32 (1.2 g, 3.41 mmol) in DMF (20 mL) and K₂CO₃ (1.4 g, 10.1 mmol) was stirred for 5 h at 40 °C under N₂. The insoluble solid was filtered off using a short silica gel pad which was washed with DMF (100 mL). The solvent was evaporated in vacuo at room temperature and the residue was chromatographed on a silica gel column in hexanes - EtOAc (3:1 to 2:1) to give compound 33e (1.30 g, 87%), mp 75–79 °C. UV λ_max 311 nm (ε 10,700), 244 (ε 11,100), 223 (ε 34,700). ¹H NMR (CDCl₃) δ 2.14–2.27 (cluster of m, 1H, H_4′), 3.46, 3.65 and 3.90 (3m, 2H, H_5′), 4.42–4.57 (s and m), 4.69–4.91 (m, 5H, H_1′, H_3′, CH₂Ph), 5.16, 5.24 (2s, 2H, NH₂), 7.21–7.37 (2m, 5H, Ph), 7.98, 8.00 (2s, 1H, H₈). 19F NMR −203.69 (dd, J = 64.0, 20.0 Hz), −206.74 (dd, J = 62.5, 24.5 Hz). ESI-MS 440, 442, 444 (M + H, 76.9, 100.0, 24.3), 462, 464, 466 (M + Na, 65.7, 87.6, 18.3).

(E)-2-Amino-6-chloro-9-{[trans-(3-fluoro-2-(benzyloxymethyl)cyclopropylidene]methyl}-purine (34e) and (Z)-2-Amino-6-chloro-9-{[trans-(3-fluoro-2-benzyloxymethyl)-cyclopropylidene]methyl}purine (35e)

A mixture of compound 33e (700 mg, 1.59 mmol), K₂CO₃ (660 mg, 4.78 mmol) in DMF (8 mL) was stirred for 45 min at 100 °C. After the work-up (see 33e), the crude product was chromatographed on a silica gel column using hexanes -EtOAc (3:1 to 2:1) the E-isomer 34e (70 mg, 18%) followed by Z-isomer 35e (110 mg, 29%) and unreacted starting material 33e (230 mg, 33%).

E-Isomer 34e

Mp 93–95 °C. UV λ_max 307 nm (ε 9,000), 226 (ε 25,800). ¹H NMR (CDCl₃) δ 2.59–2.61 (m, 1H, H_4′), 3.20 (t, 1H, J = 9.0 Hz, H_5′), 3.87 (dd, 1H, J =10.0, 1.6 Hz, H₅), 4.55 (s, 2H, CH₂Ph), 4.94 (d, J = 68.8 Hz, H_3′), 5.24 (s, 2H, NH₂), 7.26–7.34 (m, 5H, phenyl), 7.81 (s, 1H, H_1′), 8.73 (s, 1H, H₈). ¹³C NMR 26.7 (d, J = 12.7 Hz, C_4′),68.5 (d, J = 3.0 Hz, C_5′), 68.8 (d, J = 235.1 Hz, C_3′), 73.5 (CH₂Ph), 111.7 (d, J = 3.7 Hz), 116.5 (d, J = 2.9 Hz, C_1′, C_2′), 125.5 (C₅), 128.1, 128.3, 128.8, 137.4 (Ph), 139.9 (C₈), 151.8 (C₄), 152.8 (C₂), 159.6 (C₆). ¹⁹F NMR −201.82 (dd, J = 68.5, 12.1 Hz). ESI-MS 360, 362 (M + H, 54,2, 29.2), 382, 384 (M + Na, 100.0, 32.7), 741, 743 (2M + Na, 49.4, 35.7).

Z-Isomer 35e

Mp 99–101 °C. UV λ_max 307 nm (ε 8,500), 226 (ε 24,300). ¹H NMR (CDCl₃) δ 2.54–2.60 m, 1H, H_4′), 3.50–3.60 (m, 2H, H_5′), 4.56 (s, 2H, CH₂Ph), 5.07 (d, J = 68.4 Hz, H_3′), 5.21 (s, 2H, NH₂), 7.30–7.38 (m, 5H, phenyl), 7.45 (s, 1H, H_1′), 8.12 (s, 1H, H₈). ¹³C NMR 25.2 (d, J = 12.7 Hz, C_4′), 68.1 (d, J = 4.5 Hz, C_5′), 69.9 (d, J = 235.7 Hz, C_3′), 73.2 (CH2Ph), 112.2 (d, J = 2.2 Hz), 115.6 (d, J = 2.9 Hz, C_1′, C_2′), 125.5 (C₅), 128.0, 128.2, 128.8, 137.8 (Ph), 139.5 (C₈), 152.0 (C₄), 152.5 (C₂), 159.7 (C₆). ¹⁹F NMR −202.05 (dd, J = 65.5, 12.2 Hz). ESI-MS 360, 362 (M + H, 54.2, 28.6), 382, 384 (M + Na, 100.0, 32.7), 741, 743 (2M + Na, 49.1, 35.7).

(E)-2-Amino-6-chloro-9-{[trans-(3-fluoro-2-hydroxymethyl)cyclopropylidene]methyl-purine (11e)

The procedure described for adenine analogue 11a was followed with the E-isomer 34e (200 mg, 0.56 mmol) to give compound 11e (110 mg, 74%), mp 209–211 °C. UV λ_max 311 nm (ε 8,300), 239 (ε 29,400). ¹H NMR (DMSO-d6) δ 2.60–2.66 (m, 1H, H_4′), 3.42–3.48 (m, 1H), 3.63–3.69 (m, 1H, H_5′′), 5.11 (t partly overlapped with H_3′, 1H, J = 5.6 Hz, OH), 5.20 (d, 1H, J = 71.2 Hz, H_3′), 7.10 (s, 2H, NH₂), 7.74 (s, 1H, H_1′), 8.67 (s, 1H, H₈). ¹³C NMR 29.6 (d, J = 11.9 Hz, C_4′), 60.1 (d, J = 3.8 Hz, C_5′), 70.0 (d, J = 229.1 Hz, C_3′), 113.2 (d, J = 3.7 Hz), 116.3 (d, J = 3.0 Hz, C_1′, C_2′), 123.8 (C₅), 140.5 (C₈), 150.5 (C₄), 153.4 (C₂), 160.9 (C₆). ¹⁹F NMR −201.13 (dd, J = 68.9, 12.4 Hz). ESI-MS 270, 272 (M + H, 100.0, 31.0), 292, 294 (M + Na, 50.0, 15.2).

(Z)-2-Amino-6-chloro-9-{[trans-(3-fluoro-2-hydroxymethylcyclopropylidene]methyl}purine (12e)

The procedure described for compound 11a was performed with the Z-isomer 35e (360 mg, 1.0 mmol) to give 12e (200 mg, 74%), mp 201–203 °C. UV λ_max 311 nm (ε 7,900), 239 (ε 28,700). ¹H NMR (DMSO-d6) δ 2.40–2.46 (m, 1H, H_4′), 3.34–3.40 (overlapped with water), 3.47–3.53 (m, 1H, H_5′), 5.01 (t, 1H, J = 5.8 Hz, OH), 5.36 (d, 1H, J = 67.2 Hz, H_3′), 7.11 (s, 2H, NH₂), 7.40 (s, 1H, H_1′), 8.28 (s, 1H, H₈). ¹³C NMR 27.7 (d, J = 11.2 Hz, C_4′), 60.2 (d, J = 4.4 Hz, C_5′), 70.9 (d, J = 229.8 Hz, C_3′), 113.5 (d, J = 2.2 Hz), 115.3 (d, J = 2.2 Hz, C_1′, C_2′), 123.8 (C₅), 140.1 (C₈), 150.6 (C_4[′), 153.1 (C₂), 161.0 (C₆). ¹⁹F NMR −200.70 (dd, J = 68.9, 12.4 Hz). ESI-MS 270, 272 (M + H, 100.0, 3.6), 292, 294 (M + Na, 50, 15.8).

(E)-9-{[(trans-(3-fluoro-2-hydroxymethyl)cyclopropylidene]methyl}guanine (11b)

The procedure described for compound 9b was followed using the E-isomer 11e (120 mg, 0.45 mmol) to give guanine analogue 11b (82 mg, 73%), mp >300 °C. UV λ_max 273 nm (ε 10,200), 242 (ε 26,300). ¹H NMR (DMSO-d₆) δ 2.48–2.53 (m, 1H, H_4′), 3.61–3.65 (m overlapped with H₂O, H_5′), 5.16 (t overlapped with H_3′, 1H, OH), 5.35 (d, 1H, J = 68.2 Hz, H_3′), 6.59 (s, 2H, NH₂), 7.61 (s, 1H, H_1′), 8.28 (s, 1H, H₈), 10.69 (bs, 1H, NH). ¹³C NMR 29.3 (d, J = 11.2 Hz, C_4′), 60.1 (d, J = 3.7 Hz, C₅), 69.9 (d, J = 229.9 Hz, C_3′), 112.2 (d, J = 3.7 Hz), 116.5 (d, J = 2.9 Hz, C_1′, C_2′), 117.1 (C₅), 134.8 (C₈), 150.8 (C₄), 154.8 (C₂), 157.3 (C₆). 19F NMR −200.84 (dd, J = 70.2, 10.7 Hz). ESI-MS (MeOH - KOAc) 252 (M + H, 100.0), 290 (M + K, 20.9), 503 (2M + H, 16.1), 541 (2M + K, 6.0). Anal. C₁₀H₁₀FN₅O₂ (C, H, N).

(Z)-9-{[(trans-(3-fluoro-2-hydroxymethyl)cyclopropylidene]methyl}guanine (12b)

Procedure described for compound 9b was followed with the Z-isomer 12e (200 mg, 0.74 mmol) to give E-isomer 12b (140 mg, 75.5%), mp >300°C. UV λ_max 274 nm (ε 9,900), 241 nm (ε 26,000). ¹H NMR (DMSO-d₆) δ 2.36–2.40 (m, 1H, H_4′), 3.46–3.53 (m, 1H 4.96 (t, 1H, 5.2 Hz, OH), 5.32 (d, 1H, J = 68.0 Hz, H_3′), 6.88 (s, 2H, NH₂), 7.29 (s, 1H, H_1′), 7.86 (s, 1H, H₈), 10.74 (s, 1H, NH). ¹³C NMR 27.5 (d, J = 11.2 Hz, C_4′), 60.2 (d, J = 4.4 Hz, C_5′), 70.8 (d, J = 229.9 Hz, C_3′), 112.3 (d, J = 1.5 Hz), 115.6 (d, J = 2.3 Hz, C_1′, C_2′), C₅ (117.0), 134.2 (C₈), 150.6 (C₄), 155.2 (C₂), 157.2 (C₆). ¹⁹F NMR −201.23 (dd, J = 67.9, 11.5 Hz). ESI-MS (MeOH + KOAc) 252 (M + H, 100.0), 290 (M + K, 11.9), 503 (2M + H, 11.3), 541 (2M + K, 3.0). Anal. C₁₀H₁₀FN₅O₂ (C, H, N).

Antiviral Assays

The antiviral assays were performed as described previously.¹¹ The HCMV assays were performed with Towne and AD169 strains of the virus in HFF culture by plaque reduction or cytopathic effect (CPE) inhibition assay. The HSV-1 was run in BSC-1 cells by ELISA. In addition, HSV-1 and HSV-2 assays were performed in HFF (CPE inhibition) and Vero cells (plaque reduction). The EBV assays were run in Daudi culture (viral capsid antigen, VCA-ELISA) and in H-1 culture (DNA hybridization). The VZV was assayed in HFF cells (CPE inhibition or plaque reduction), HIV-1 in MT-2 cells (CPE inhibition) and hepatitis B virus (HBV) in 2.2.15 cells. The cytotoxicity assays were performed in HFF, KB or CEM cells. The results are summarized in Table 3 and 4.

Adenosine Deaminase (ADA) Assay.¹¹

Compounds 9a, 10a, 11a and 12a (2.0 – 2.4 μmol) were incubated with ADA (1.1 unit/mL) in 0.05 M Na₂HPO₄ (pH 7.5, 0.47 – 0.54 mL). Aliquots were periodically withdrawn and examined by TLC in CH₂Cl₂ - MeOH (9:1, multiple development, 10a, 11a) and EtOAc - MeOH (10:1, multiple development, 9a, 12a). The extent of deamination of 9a, 10a and 11a was approximately 50% after 24 and 48 h whereas 12a was not deaminated after 48 h.

Supplementary Material

si20060801_120. Supporting Information Available.

Elemental Analyses. This information is available online free of charge via the Internet at http://pubs.acs.org.