Phosphonate Analogues of Cyclopropavir Phosphates and Their E-isomers. Synthesis and Antiviral Activity

Abstract

Z- and E-phosphonate analogues 12 and 13 derived from cyclopropavir and the corresponding cyclic phosphonates 14 and 15 were synthesized and their antiviral activity was investigated. The 2,2-bis(hydroxymethylmethylenecyclopropane acetate (17) was transformed to tetrahydropyranyl acetate 18. Deacetylation gave intermediate 19 which was converted to bromide 20. Alkylation with diisopropyl methylphosphonate afforded after protecting group exchange (21 to 22) acetylated phosphonate intermediate 22. Addition of bromine gave the dibromo derivative 16 which was used in the alkylation-elimination procedure with 2-amino-6-chloropurine to give Z- and E-isomers 23 and 24. Hydrolytic dechlorination coupled with removal of all protecting groups gave the guanine phosphonates 12 and 13. Cyclization afforded the cyclic phosphonates 14 and 15. Z-Phosphonate 12 was a potent and non-cytotoxic inhibitor of human and murine cytomegalovirus (HCMV and MCMV) with EC₅₀ 2.2-2.7 and 0.13 μM, respectively. It was also an effective agent against Epstein-Barr virus (EBV, EC₅₀ 3.1 μM). The cyclic phosphonate 14 inhibited HCMV (EC₅₀ 2.4-11.5 μM) and MCMV (EC₅₀ 0.4 μM) but it was ineffective against EBV. Both phosphonates 12 and 14 were as active against two HCMV Towne strains with mutations in UL97 as they were against wild-type HCMV thereby circumventing resistance due to such mutations. Z-Phosphonate 12 was a moderate inhibitor of replication of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) but it was a potent agent against varicella zoster virus (VZV, EC₅₀ 2.9 μM). The cyclic phosphonate 14 lacked significant potency against these viruses. E-isomers 13 and 15 were devoid of antiviral activity.

1. Introduction

Methylenecyclopropane analogues of nucleosides are established antiviral agents, particularly effective against herpesviruses such as cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpes virus 6 and 8 (HHV-6 and HHV-8).¹ The most potent are the Z-isomers of purine nucleosides 1 and 2 although the individual E-isomers 3 and 4 (Chart 1) are active against EBV, especially in the series of fluorinated analogues.^2,3 The Z-guanine analogue 2b, cyclopropavir, is under preclinical development as a potential drug against human cytomegalovirus (HCMV) infections.^4-6 It is accepted that methylenecyclopropane analogues follow the intracellular activation process (monophosphate - diphosphate - triphosphate) generally established for nucleosides and their analogues. In several cases, metabolically stable mimics of nucleoside phosphates, phosphonates, have yielded antiviral agents.^7,8 These include phosphonate derivatives^9-12 of antiherpetic drugs acyclovir (Zovirax), ganciclovir (Cytovene) 5a, 5b. and the cyclic phosphonate 6. By contrast, phosphonates of methylenecyclopropanes 7 and 8 did not exhibit antiviral potency¹³ with a single exception of compound 7b (n = 1) which was a moderate inhibitor of replication of varicella zoster virus (VZV).

Chart 1.

Whereas the cyclopropavir phosphate (9) is an effective prodrug of the parent compound 2b, the pattern of antiviral activity of the cyclic phosphate 10 is different.¹⁴ Although it had limited potency against HCMV in Towne strain of the virus, it was effective against AD169 strain with efficacy comparable to the cyclic phosphate of ganciclovir 11. Compound 10 also exhibited potent activity against hepatitis B virus (HBV). It was therefore of interest to synthesize phosphonate analogues 12 - 15 and investigate their antiviral activity.

2. Results and discussion

2.1. Synthesis

Alkylation-elimination method which had been successfully exploited¹³ for synthesis of phosphonates 7 and 8 formed also the basis of our approach to analogues 12 - 15. The suitably protected reagent 16 for alkylation-elimination was prepared as follows (Scheme 1). The previously described¹⁵ monoacetate 17 was converted to tetrahydropyranyl (THP) derivative 18 in 82% yield using 3,4-dihydro-2H-pyran in CH₂Cl₂ under acid catalysis. Ammonolysis afforded intermediate 19 (98%). The latter was converted to bromide 20 in 76% yield by CBr₃-triphenylphosphine reagent. This reagent is not compatible¹⁶ with acid-labile groups like THP but inclusion of triethylamine in the reaction mixture successfully removed this obstacle. This modification may significantly expand use of the reagent. Reaction of 20 with lithium salt of diisopropyl methylphosphonate in THF gave the phosphonate intermediate 21 (81%). The presence of THP group was considered a potential liability for the bromination step and, therefore, the THP was replaced with acetyl in a single step¹⁷ using acetyl chloride in CH₂Cl₂ to give acetate 22 in 92% yield. Addition of bromine using pyridinium perbromide in CH₂Cl₂ was uneventful to provide dibromo derivative 16 as a mixture of cis,trans isomers (92%).

Scheme 1.

Reagents and conditions: (a) 3,4-Dihydro-2H-pyran, MeSO₃H, CH₂Cl₂; (b) NH₃, MeOH, Δ; (c) CBr₄, Ph₃P, NEt₃, CH₂Cl₂; (d) CH₃P(O)(i-PrO)₂, BuLi, THF; (e) AcCl, CH₂Cl₂; (f) Pyridine.HBr₃, CH₂Cl₂; (g) 1. B-H, Cs₂CO₃, DMF, Δ. 2. Chromatography; (h) 80% HCO₂H, Δ; (i) 1. Me₃SiBr, DMF. 2. NH₄OH. 3. Chromatography; (j) N,N′-Dicyclohexyl-4-mopholinecarboxamidine, DCC, pyridine. 2. NH₄OH. 3. Dowex 50 (H⁽⁺⁾).

Alkylation-elimination of 2-amino-6-chloropurine with 16 (Cs₂CO₃, DMF, 75 °C, 20 h) furnished Z- and E-isomers 23 and 24 which were separated by column chromatography on silica gel in 31 and 30% yield, respectively. Hydrolytic dechlorination of the Z-isomer 23 by 80% formic acid afforded after chromatographic separation a mixture of acetyl and formyl esters 25a + 25b in the ratio of 4 : 1 and 89% yield. A smaller amount (4%) of deacylated phosphonate 25c was also obtained. In this case, conversion to guanine moiety was accompanied by a partial deacetylation followed by formylation. Formylation of hydroxy groups in the course of this procedure was observed before.¹⁹ Dealkylation of 25a + 25b with trimethylsilyl bromide in DMF followed by ammonolysis, chromatography on DEAE Sephadex in NH₄HCO₃ buffer and then Dowex 1 (HCO₂⁽⁻⁾) in formic acid gave phosphonate 12 in 81% yield as a free acid. Cyclization was performed using a protocol previously employed for the corresponding cyclic phosphate¹⁴10 with N,N′-dicyclohexyl-4-morpholinecarboxamidine and N,N′-dicyclohexylcarbodiimide (DCC) in pyridine. Cyclic phosphonate 14 was obtained as a free acid with the aid of Dowex 50 (H⁽⁺⁾) in 85% yield. In a similar fashion, hydrolytic dechlorination of the E-isomer 24 furnished a mixture of acetyl and formyl esters 26a + 26b in the ratio of 4 : 1 and 88% yield as well as deacetylated product 26c (5%). Dealkylation, ammonolysis and work-up employed for the Z-isomer 12 gave the E-phosphonate 13 (80%). Cyclization as described above then afforded the cyclic E-phosphonate 15 in 82% yield.

2.2. The Z- and E-isomeric Assignment

The UV spectra of intermediates 23, 24 (l_max 311 nm) and final products 12, 13 (l_max 267-268 nm) have indicated that the phosphonylated side-chain is attached in the 9 position of the purine base as found also for phosphonates¹³ 7 and 8. As far as the Z/E isomerism is concerned, a general trend¹³ that Z-isomers are less polar (moving faster on silica gel) than E-isomers was also observed with compounds 23 and 24. In addition, the C_3′ chemical shifts of the Z-isomers are more shielded than those of E-isomers (Table 1). This was also found in analogues 7 and 8 and their derivatives.¹³ However, the reversed pattern of the C_4′ chemical shifts found in analogues 7 and 8 was not followed possibly because of a quarternary character of C_4′. Final confirmation of the Z- and E-assignment came from the NOE experiments with compounds 23 and 24 (Table 2). In the Z-isomer 23, the NOE enhancements were observed between the cis-related atoms such as the H₈ of the heterocyclic base in an anti-like conformation and H_4′′ and H_5′ and between the H_1′ and H_3′. As expected, the strongest interactions in the E-isomer 22 were observed between the H₈ and H_3′ as well as H_1′ and H_4′′, H_6′, H_5′. A weaker NOE enhancements were found between the H_1′ and CH₃ of the acetyl and isopropyl groups.

Table 1.

Comparison of the C_3′ ¹³C NMR chemical shifts of isomeric methylenecyclopropane phosphonates

Compounda	Isomer	C3′ (ppm)	Compounda,b	Isomer	C3′ (ppm)
23	Z	12.3	7c,d,e	Z	8.8
24	E	17.0	8c,d,e	E	11.2
25c	Z	11.9	7be	Z	9.1
26c	E	15.9	8be	E	11.4
12	Z	11.8	7b	Z	8.1f
13	E	16.0	8b	E	10.6f

^aCD₃SOCD₃ as solvent unless stated otherwise. For numbering of atoms see Table 2.

^bThe values are from ref. 13.

^cCDCl₃.

^dB = 2-Amino-6-chloropurine.

^eDiisopropyl ester, n = 2.

^fSodium salt, D₂O, n = 2.

Table 2.

NOE data of the Z- and E-isomers 23 and 24 (500 MHz, CDCl₃)

Compound	Hirr	d	□obs	□	.□□□
23	H8	8.10	CH3 of Ac	1.97	1.50, 2.66a
	H8	8.10	H5′	2.20	1.81
	H8	8.10	H4″	3.83	0.84
	H5′	2.20	H8	8.10	3.85, 4.73a
	H4″	3.83	H8	8.10	0.85, 0.95a
	H3′	1.46	H1′	7.26	2.43, 4.24a
	H3′	1.39	H1′	7.26	1.47, 2.89a
	H1′	7.26	H3′	1.46	0.55
	H1′	7.26	H3′	1.39	0.51
	H1′	7.26	H3′	1.39 + 1.46	0.64
24	H8	8.17	H3′	1.66	1.54
	H8	8.17	H3′	1.58	1.37
	H3′	1.66	H8	8.17	2.16, 3.82a
	H3′	1.58	H8	8.17	2.28, 3.16a
	H1′	7.46	H4″	4.16	0.04
	H1′	7.46	H4″	4.03	0.28
	H1′	7.46	H6′, H5′	1.84	1.10
	H1′	7.46	CH3 of Ac	2.09	0.17
	H1′	7.46	CH3 of i-Pr	1.30	0.21
	CH3 of Ac	2.09	H1′	7.46	0.13

^aTwo different scans.

2.3. Antiviral Activity

All phosphonates were tested against the following viruses: Human cytomegalovirus (HCMV), murine cytomegalovirus (MCMV), herpes simplex virus types 1 and 2 (HSV-1, HSV-2), varicella zoster virus (VZV), Epstein-Barr virus (EBV), hepatitis B virus (HBV) and hepatitis C virus (HCV). As mentioned at the outset, the first series of phosphonates of methylenecyclopropanes 7 and 8 designed as analogues of acyclovir phosphonates 5a lacked any significant antiviral activity.¹³ It is therefore surprising (and rewarding) that among analogues 12 - 15 new potent antivirals were found. Thus, the Z-isomeric phosphonate 12 and the corresponding cyclic derivative 14 which can be regarded as mimics of ganciclovir phosphonate 5b and cyclic phosphonate 6, are potent and non-cytotoxic inhibitors of replication of HCMV comparable to ganciclovir (Table 3). Analogues 12 and 14 were virtually equipotent against the Towne strain of HCMV with EC₅₀'s 2.2 and 2.4 μM, respectively. Against the AD169 strain, phosphonate 12 was somewhat more effective (EC₅₀ 2.7 μM) than the cyclic derivative 14 (EC₅₀ 11.6 μM). In comparison with the cyclic phosphate¹⁴ 10, cyclic phosphonate 14 was significantly more effective in Towne strain and somewhat less potent in AD169 strain. Both phosphonates 12 and 14 inhibited murine cytomegalovirus (MCMV) with EC₅₀ 0.13 and 0.4 μM, respectively, surpassing the cyclic phosphate¹⁴ 10. Comparison with cyclopropavir⁴ (2b) reveals reduced potency of the phosphonates 12 and 14 against HCMV in vitro but against MCMV the efficacy was about the same (Table 3). A distinct advantage of phosphonates 12 and 14 is circumventing the first step of activation (phosphorylation) of cyclopropavir (2b) most likely catalyzed by HCMV UL97 phosphotransferase.⁵ Therefore, analogues 12 and 14 may overcome resistance of cyclopropavir (2b) caused by mutations in this enzyme.^19,20 To test this hypothesis, cyclopropavir (2b) and its phosphonates 12 and 14 were assayed in two strains of Towne HCMV with mutations in UL97 that are known to produce resistance to cyclopropavir.^19,20 Strain E8 has two point mutations¹⁹ in UL97 whereas strain 2696r has most of UL97 sequence deleted.²⁰ In both strains, cyclopropavir (2b) was 13 to 47 times less active than in wild-type Towne strain (Table 4). In contrast, phosphonates 12 and 14 were as active against both mutant strains as they were against wild-type Towne strain of HCMV. We conclude, therefore, that 12 and 14 do circumvent the necessity of UL97 to phosphorylate cyclopropavir (2b) to produce their activity.

Table 3.

Inhibition of human and murine cytomegalovirus (HCMV and MCMV) and Epstein-Barr virus (EBV) replication by methylenecyclopropane phosphonates

	EC50/CC50 (μM)
	HCMV/HFF
Compound	Townea,b	AD169c,d	MCMV/MEFa	EBV/Akatae
2b	0.46/>100f	0.49/>380a,f	0.27/>380f	0.22/46g
10	20/>100h	6.0/>301a,h	7.2/>301h	0.96/150h,i
12	2.2/>100	2.7/>300a	0.13/100	3.1/>100
13	>100/>100	>300/>300	-	>100/>100
14	2.4/>100	11.6/>300a	0.4/>100	>100/>100
15	>100/>100	>300/>300	-	>100/>100
Control	2.9/>100j	0.09/>100a,j	2.6/>100j	8.4/>100k

^aPlaque reduction assay in HFF cultures.

^bVisual cytotoxicity of HFF's in cells unaffected by virus.

^cCytopathic effect (CPE) assay.

^dCytotoxicity by neutral red uptake.

^eDNA hybridization assay.

^fRef ⁴.

^gRef. ²¹.

^hRef. ¹⁴.

ⁱDaudi cells, viral capsid antigen (VCA) assay. The EC₅₀/CC₅₀ in H-1 cells (DNA hybridization assay) was >20/>100 μM, cytotoxicity was determined in CEM cells.

^jGanciclovir.

^kAcyclovir.

Table 4.

Activity of Phosphonates 12 and 14 against Drug Resistant HCMV

Compound	Towneb	EC50 (μM)a Virus Strain 2696rc	E8d
12	3.5	3	3.2
14	4	3	4
cyclopropavir (3b)	0.6	28	8

^aData from a plaque reduction assay with four drug concentrations in duplicate.

^bWild-type virus from which isolates 2696r and E8 were obtained.

^cVirus isolated for resistance to cyclopropavir (3b) that has a truncated UL97 gene.²⁰

^dVirus with two point mutations¹⁹ introduced into gene UL97.

The cyclic phosphonate 14 may be either either a prodrug of 12 or have an entirely different mechanism of action. It is interesting that against EBV in Akata cells, only the Z-isomeric phosphonate 12 was effective (EC₅₀ 3.1 μM) whereas the corresponding cyclic phosphonate 14 was inactive. This precludes that the latter analogue can function as a prodrug of phosphonate 12 under the conditions of this assay. Interestingly, the cyclic phosphate 10 was active against EBV although the assays used were different.¹⁴

Against α-herpes viruses, the most potent activity was found for analogue 12 in varicella zoster virus (VZV), EC₅₀ 2.9 μM whereas cyclic phosphonate 14 and cyclopropavir⁴ (2b) were ineffective (Table 5). Interestingly, phosphonate 7b (n = 1) was also effective against VZV but to a much lesser extent¹³ (EC₅₀ 24 μM) than 12. Similar to other methylenecyclopropane analogues,¹ only moderate activity was found against herpes simplex virus types 1 and 2 (HSV-1 and HSV-2). Phosphonate 12 was the most effective against HSV-1 in a plaque assay in BSC-1 cells (EC₅₀ 15 μM) but it was less potent against HSV-1 and HSV-2 in a cytopathic effect (CPE) inhibition assay in HFF cultures. Regardless, we hypothesize that the activity of 12 against the α-herpes viruses is the result of delivering this monophosphate analogue into virus-infected cells thereby circumventing the necessity of an initial phosphorylation step by a viral-specified kinase. This would explain the activity of 12 against the α-herpes viruses compared to the inactivity of 2b.

Table 5.

Inhibition of herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2) and varicella zoster virus (VZV) replication by methylenecyclopropane phosphonates

	EC50/CC50 (μM)
Compound.	HSV-1/BSC-1a	HSV-1/HFFb,c	HSV-2/HFFb,d	VZV/HFFb,d
2b	>100/>100e	>380/>380e	>380e	>380e
10	20/>100f	>301/>301f	242f	>301f
12	15/>100	59.4/>300	76.5	2.9g
13	100/>100	>300/>300	>300	191
14	40/>100	>300/>300	>300	>300
15	100/>100	>300/>300	>300	>300
Acyclovir	0.3	1.3/>300	1.2	4.4g

^aELISA in BSC-1 cells was used for compounds 2b and 12; other compounds were assayed by plaque reduction in BSC-1 cells. Cytotoxicity was determined in replicating KB cells.

^bCytopathic effect (CPE) assay.

^cCytotoxicity by neutral red uptake.

^dFor cytotoxicity see HSV-1.

^eRef. ⁴.

^fRef. ¹⁴.

^gPlaque reduction assay.

All analogues including the cyclic phosphonate 14 were inactive against HBV and HCV. By contrast, the cyclic phosphate 10 is an effective anti-HBV agent.¹⁴ The E-isomers 13 and 15 were devoid of potency against all tested viruses.

3. Conclusion

Phosphonates 12, 13, 14 and 15 were synthesized and they were evaluated for antiviral activity. The Z-phosphonates 12 and 14 were effective inhibitors of replication of HCMV and MCMV in HFF and MEF culture. Compounds 12 and 14 also inhibited two Towne strains of HCMV with mutations in UL97. Phosphonate 12 was effective against EBV in Akata cells and VZV in HFF culture whereas cyclic phosphonate 14 was inactive. Analogue 12 was a moderate inhibitor of HSV-1 and HSV-2. The E-isomers 13 and 15 were devoid of antiviral activity.

4. Experimental

4.1. General Methods

The UV spectra were measured in ethanol and NMR spectra were determined on Varian instruments at 300, 400 or 500 MHz (¹H), 75 or 100 MHz (¹³C) and 121 or 161 MHz (³¹P) in CDCl₃ unless stated otherwise. Mass spectra were determined in electrospray ionization (ESI-MS) mode using methanol - sodium acetate or by negative ESI-MS.

4.2. 2-Acetoxymethyl-2-(tetrahydropyranyloxy)methyl-1-methylenecyclopropane (18)

To a solution of monoacetate¹⁷ 17 (5.0 g, 32.04 mmol) and 3,4-dihydro-2H-pyran (7.31 mL, 80.1 mmol) in CH₂Cl₂ (50 mL) was added methanesulfonic acid (0.03 mL, 0.46 mmol) in CH₂Cl₂ (2 mL) dropwise with stirring at 0 °C. The stirring was continued for 6 h. The reaction was quenched with triethylamine (0.07 mL, 0.50 mmol), the solvent was evaporated, the residue was dissolved in ethyl acetate (60 mL). The organic phase was washed with saturated aqueous NaHCO₃ (3 × 20 mL) and brine (3 × 20 mL) whereupon it was dried (MgSO₄) and the solvent was evaporated. The crude product was chromatographed on a silica gel column using ethyl acetate-hexane (0.5 : 10) to furnish compound 18 (6.3 g, 82%) as a sirup. ¹H NMR (400 MHz) δ 5.52, 5.48 (2t, J = 2-3 Hz, 1H), 5.41 (s, 1H, CH₂=), 4.63, 4.61 (2t, J = 3-4 Hz, 1H, CHO of THP), 4.17-4.05 (m, 2H, CH₂OAc), 3.81 (m, 1H), 3.68 (t, J = 9.6 Hz, 1H, CH₂OTHP), 3.46 (m, 1H), 3.39 (dd, J = 10.0, 3.4 Hz. 1H, CH₂O of THP), 2.05 (s, 3H, CH₃), 1.8-1.5 (cluster of m, 6H, 3×CH₂ of THP), 1.29-1.26 (m, 2H, H₃). ¹³C NMR (100 MHz) 171.3 (C=O), 135.5, 135.1 (C=), 105.11, 105.05 (CH₂=), 98.6, 98.2 (CHO of THP), 69.2, 68.9, 66.51, 66.48, 62.3, 61.9 (CH₂O), 30.8, 30.7, 25.68, 25.65, 19.6, 19.3 (3×CH₂ of THP), 23.9, 23.8 (C₂), 21.2 (CH₃), 14.1, 13.9 (C₃). ESI-MS 241 (7.4, M + H), 263 (100.0, M + Na). Anal. Calcd for C₁₃H₂₀O₄: C, 64.98; H, 8.39. Found: C, 65.08; H, 8.35.

4.3. 2-Hydroxymethyl-2-(tetrahydropyranyloxy)methyl-1-methylenecyclopropane (19)

A solution of compound 18 (5.7 g, 23.74 mmol) in NH₃/MeOH (30%, 100 mL) was stirred at 0 °C for 30 min and at room temperature for 16 h. The volatile components were evaporated and the product 19 obtained as a sirup (4.6 g, 98%) was used directly in the next step. For analysis, a sample of 19 was chromatographed on a silica gel column using ethyl acetate - hexane (1 : 5). ¹H NMR (400 MHz) δ 5.50, 5.48 (2t, J = 2.4 Hz, 1H), 5.40 (poorly resolved t, 1H, CH₂=), 4.63 (2 overlapped t, 1H, CHO of THP), 3.94-3.36 (cluster of m, 6H, CH₂O), 2.69, 2.61 (2t, J = 6 Hz, 1H, OH), 1.84-1.52 (cluster of m, 6H, 3×CH of THP), 1.31-1.20 (m, 2H, H₃). ¹³C NMR (100 MHz) 135.9, 135.5 (C=), 104.5, 104.4 (CH₂=), 99.22, 99.15 (CHO of THP), 72.2, 71.9, 67.29, 67.27, 62.81, 62.7 (CH₂O), 30.83, 30.78, 25.5, 19.9, 19.8 (3×CH₂ of THP), 26.6, 26.5 (C₂), 14.1, 14.0 (C₃). ESI-MS 199 (5.0, M + H), 221 (100.0, M + Na). Anal. Calcd for C₁₁H₁₈O₃: C, 66.64; H, 9.15. Found: C, 66.34, H, 9.38.

4.4. 2-Bromomethyl-2-(tetrahydropyranyloxy)methyl-1-methylenecyclopropane (20)

Triphenylphosphine (25.15 g, 95.9 mmol) in CH₂Cl₂ (25 mL) was added to a solution of CBr₄ (31.8 g, 95.9 mmol) in CH₂Cl₂ (75 mL) at −5 °C with stirring which continued for another 10 min. Triethylamine (16.04 mL, 0.12 mol) was added, followed by a dropwise addition of compound³ 19 (3.8 g, 19.18 mmol) in CH₂Cl₂ (15 mL) over a period of 10 min. Reaction was complete in 30 min. The reaction mixture was diluted with hexane (130 mL), the insoluble portion was filtered and it was washed with hexane (50 mL). The filtrate was concentrated and the crude product was chromatographed on a silica gel column using ethyl acetate - hexane (0.2 : 10) to furnish compound 20 (3.79 g, 76%) as a sirup. ¹H NMR (400 MHz) δ 5.56, 5.54 (2t, J = 2.8-3.0 Hz, 1H), 5.37 (poorly resolved d, 1H, CH₂=), 4.66, 4.64 (partially overlapped 2t, J = 3.6 Hz, 1H, CHO of THP), 3.89-3.47 (cluster of m, 6H, CH₂Br, CH₂O ), 1.89-1.49 (m, 6H, 3×CH₂ of THP), 1.43-1.42 (m, 1H), 1.31-1.28 (2 poorly resolved t, 1H, H₃).

¹³C NMR (100 MHz) 137.6, 137.5 (C=), 104.83, 104.79 (CH₂=), 98.9, 98.5 (CHO of THP), 69.1, 68.9, 62.5, 62.1 (CH₂O), 39.1 (CH₂Br), 30.8, 30.7, 25.67, 25.65, 19.7, 19.4 (3×CH₂ of THP), 26.1, 26.0 (C₂), 17.2, 17.1 (C₃). ESI-MS 283, 285 (98.8, 100.0, M + Na). Anal. Calcd for C₁₁H₁₇BrO₂×0.05 H₂SiO₃: C, 49.84; H, 6.50. Found: C, 49.90; H, 6.47.

4.5. 2-[(2-Diisopropylphosphono)ethyl]-2-(tetrahydropyranyloxymethyl-1-methylenecyclopropane (21)

1-Butyllithium (1.6 M in hexanes, 12.1 mL, 19.29 mmol) was added to a solution of diisopropyl methylphosphonate (3.35 mL, 18.15 mmol) in THF (25 mL) at −78 °C with stirring which was continued for 30 min. Compound 20 (2.95 g, 11.34 mmol) in THF (15 mL) was then slowly added over a period of 10 min and the reaction mixture was stirred for 2 h at −78 °C. The reaction mixture was then allowed to warm to room temperature and, after 30 min, it was quenched with saturated aqueous NH₄Cl (15 mL). Ethyl acetate (80 mL) was added, the organic phase was washed with brine (3 × 30 mL), saturated aqueous NaHCO₃ (3 × 30 mL) and brine (3 × 30 mL). After drying (MgSO₄), the solvent was evaporated and the crude product was chromatographed on a silica gel column using ethyl acetate - hexane (2 : 1) to furnish phosphonate 21 (3.3 g, 81%) as a sirup. ¹H NMR δ 5.44, 5.41 (poorly resolved 2t, 1H), 5.35 (s, 1H, CH₂=), 4.70-4.57 (m, 3H, CH of i-PrO, CHO of THP), 3.82 (m, 1H), 3.47 (m, 1H, CH₂O of THP), 3.66, 3.22 and 3.59, 3.32 (2AB's, J = 10.4 and 10.6 Hz, 2H, CH₂OTHP), 1.93-1.49 (cluster of m, 10H, CH₂ of THP, H₄ and H₅), 1.30-1.28 (2 poorly resolved d, 12H, CH₃), 1.17- 1.04 (m, 2H, H₃). ¹³C NMR 138.3, 137.7 (C=), 104.0, 103.8 (CH₂=), 98.6, 98.1 (CHO of THP), 71.3, 70.7 (CH₂O of THP), 70.0 (d, J = 6.7 Hz, CH of i-PrO), 62.4, 62.0 (CH₂OTHP), 30.8, 30.7, 25.68, 25.65, 19.7, 19.4 (CH₂ of THP), 26.84, 26.80 (2 overlapped d, J = 4.5 Hz, C₄), 24.58, 24.54 (2d, J = 140.2 Hz, C₅), 24.57, 24.36 (C₂), 24.3 (d, J = 3.8 Hz, CH₃), 15.1, 14.7 (C₃). ³¹P NMR (161 MHz) 31.24. ESI-MS 361 (6.3, M + H), 283 (100.0, M + Na). Anal. Calcd for C₁₈H₃₃O₅P: C, 59.98; H, 9.23. Found: C, 60.24, H, 9.44.

4.6. 2-(Acetoxymethyl)-2-[2-(diisopropylphosphono)ethyl]-1-methylenecyclopropane (22)

Acetyl chloride (8.9 mL, 0.13 mol) was added to a solution of phosphonate 21 (3.0 g, 8.33 mmol) in CH₂Cl₂ (50 mL). The reaction mixture was stirred at room temperature for 6 h, it was concentrated to a half of its original volume and the reaction was quenched with saturated aqueous NaHCO₃ (30 mL). Ethyl acetate (50 mL) was then added and the organic layer was washed with saturated aqueous NaHCO₃ (3 × 25 mL) and brine (3 × 25 mL). After drying (MgSO₄), the solvents were evaporated and the crude product was chromatographed on a silica gel column using ethyl acetate - hexane (1 : 1) to give acetate 22 (2.44 g, 92%) as a sirup. ¹H NMR (400 MHz) δ 5.47, 5.40 (2 poorly resolved t, 2H, CH₂=), 4.67 (m, 2H, CH of i-PrO), 3.97 (s, 2H, CH₂OAc), 2.06 (s, 3H, CH₃ of Ac), 1.92-1.62 (cluster of m, 4H, H₄ and H₅), 1.30 (d, J = 2.4 Hz), 1.29 (d, 1.6 Hz, 12H, CH₃ of i-PrO), 1.20, 1.11 (poorly resolved t of AB, J_AB = 10 and 9.2 Hz, 2H, H₃). ¹³C NMR (100 MHz) 171.2 (C=O), 136.7 (C=), 104.8 (CH₂=), 70.1 (d, J = 6.7 Hz, CHO of i-PrO), 68.0 (CH₂OAc), 26.6 (d, J = 4.5 Hz, H₄), 24.4 (d, J = 140.9 Hz, H₅), 24.2 (d, J = 4.4 Hz, CH₃ of i-PrO), 23.5 (d, J = 21.5 Hz, C₂), 21.1 (CH₃ of Ac), 14.9 (C₃). ³¹P NMR (161 MHz) 30.57. ESI-MS 319 (13.3, M + H), 341 (100.0, M + Na). Anal. Calcd for C₁₅H₂₇O₅P: C, 56.59; H, 8.55. Found: C, 56.56, H, 8.64.

4.7. (cis,trans)-2-(Acetoxymethyl)-2-[2-(diisopropylphosphono)ethyl]-1-bromo-1-bromomethylcyclopropane (16)

Pyridinium hydrobromide perbromide (3.32 g, 10.37 mmol) was added in three portions to a solution of acetate 22 (2.2 g, 6.92 mmol) in CH₂Cl₂ (50 mL) at-20 °C. Reaction mixture was stirred for 1 h whereupon it was diluted with diethyl ether (50 mL). The precipitate was filtered off and it was washed with diethyl ether (25 mL). The organic phase was washed with saturated aqueous Na₂S₂O₃ (3 × 25 mL), water (3 × 25 mL) and brine (3 × 25 mL). The solvents were evaporated to give dibromophosphonate 16 (3.03 g, 92%) as a sirup. ¹H NMR (400 MHz) δ 4.69 (m, 2H, CH of i-PrO), 4.43-3.68 (4 overlapped AB's, 4H, CH₂Br, CH₂OAc), 2.10, 2.08 (2s, 3H, CH₃ of Ac), 2.27-2.19, 2.04-1.67 (cluster of m, 4H, H₄ and H₅), 1.24-1.36 (cluster of d, CH₃ of i-PrO), 1.12 (d, J = 7.6 Hz, part of H₃ obscured by CH₃, total 14H). ³¹P NMR (161 MHz) 29.55, 29.44. ESI-MS 477, 479, 481 (M + H, 9.6, 16.7, 7.4), 499, 501, 503 (M + Na, 47.5, 100.0, 49.4), Anal. Calcd for C₁₅H₂₇Br₂O₅P: C, 37.68; H, 5.69. Found: C, 37.72, H, 5.73.

4.8. (Z)- and (E)-2-Amino-6-chloro-9-{[2-(2-diisopropylphosphonoethyl)-2-(acetoxymethyl)cyclopropylidene]methyl}purine (23 and 24)

A mixture of dibromophosphonate 16 (2.7 g, 5.67 mmol), 2-amino-6-choropurine (0.96 g, 5.67 mmol) and Cs₂CO₃ (9.24 g, 28.36 mmol) in DMF (30 mL) was stirred at room temperature for 5 h and at 75 °C for 20 h. After cooling, the insoluble portion was filtered off and it was washed with DMF (10 mL). The solvent was evaporated in vacuo and the crude product was chromatographed on a silica gel, using methanol - CH₂Cl₂ (0.3 : 10) to obtain the Z-isomer 23 (850 mg, 31%) followed by E-isomer 24 (820 mg, 30%).

Z-isomer 23

Mp 174-176 °C. UV λ_max 311 nm (ε 7,700), 231 (ε 26,700). ¹H NMR (300 MHz, CD₃SOCD₃) δ 8.16 (s, 1H, H₈), 7.28 (s, 1H, H_1′), 7.05 (bs, 2H, NH₂), 4.46 (m, 2H, CH of i-PrO), 4.27, 4.02 (AB, J = 11.7 Hz, 2H, H_4″), 2.05-1.74 (m, 2H, H_6′, overlapped with 1.89 (s, CH₃ of Ac, 3H), 1.68-1.42 (m, H_5′ partially overlapped with split AB, J = 9.2 Hz, 4H, H_3′), 1.19-1.12 (m, 12H, CH of i-PrO). ¹³C NMR (75 MHz) 170.6 (C=O, Ac), 160.8, 153.2, 150.4, 140.9, 123.9 (purine), 120.6 (C_2′), 112.4 (C_1′), 70.0 (d, J = 6 Hz, CH of i-PrO), 68.2 (C_4″), 26.5 (d, J = 22.2 Hz, C_5′), 25.2 (d, J = 2.8 Hz, C_4′), 24.4-24.3 (2 overlapped d, J = 4.0 Hz, CH₃ of i-PrO), 22.8 (d, J = 140.1 Hz, C_6′), 21.0 (CH₃ of Ac), 12.3 (C_3′). ³¹P NMR (121 MHz) 30.57. ESI-MS 486, 488 (M + H, 14.4, 3.8). 508, 510 (M + Na, 100.0, 33.7). Anal. Calcd for C₂₀H₂₉ClN₅O₅P: C, 49.44; H, 6.02; N, 14.41. Found: C. 49.62; H, 6.05; N, 14.33.

E-isomer 24

Mp 86 °C. UV λ_max 311 (ε 7,400), 229 nm (ε 28,600). ¹H NMR (400 MHz, CD₃SOCD₃) δ 8.42 (s, 1H, H₈), 7.43 (s, 1H, H_1′), 7.01 (bs, 2H, NH₂), 4.51 (m, 2H, CH of i-PrO), 4.11, 3.97 (AB, J = 11.2 Hz, 2H, H_4″), 2.03 (s, 3H, CH₃ of Ac), 1.82-1.59 (s + cluster of m, 6H, H_5′ and H_6′ overlapped with H_3′), 1.20 (d, J = 6 Hz, 12H, CH of i-PrO). ¹³C NMR (100 MHz) 171.0 (C=O), 160.8, 153.3, 150.3, 140.2, 123.8 (purine), 120.0 (C_2′), 111.8 (C_1′), 69.9 (d, J = 5.9 Hz), 67.2 (C_4″), 26.6 (d, J = 5 Hz, C_4′), 24.5, 24.4, 24.2 (2 overlapped d, C_5′ and CH₃ of i-PrO), 23.9 (d, J = 138.7 Hz, C_6′), 21.4 (CH₃ of Ac), 17.0 (C_3′). ³¹P (161 MHz) 30.21. ESI-MS 486, 488 (M + H, 19.1, 5.0). 508, 510 (M + Na, 100.0, 19.8, 32.3). Anal. Calcd for C₂₀H₂₉ClN₅O₅P: C, 49.44; H, 6.02; N, 14.41. Found: C, 49.34; H, 5.99; N, 14.38.

4.9. (Z)-9-{[2-(2-diisopropylphosphono)ethyl)-2-(acetoxy/formyloxy/methyl)cyclopropylidene]methyl}guanine (25a + 25b) and (Z)-9-{[2-(2-diisopropylphosphono)ethyl)-2-(hydroxymethyl)cyclopropylidene]methyl}guanine (25c)

A solution of compound 23 (600 mg, 1.24 mmol) in formic acid (80%, 30 mL) was heated at 70 ° for 6 h. Formic acid was evaporated in vacuo, the residue was dissolved in water and the solution was lyophilized. The crude product was chromatographed on a silica gel column using methanol - CH₂Cl₂ (0.5 : 10) to obtain a mixture of acetate and formate 25a + 25b (510 mg, 89 %). The ratio 25a/25b determined from the acetyl and formyl ¹H NMR signals was 4 : 1. Further elution of the column using methanol - CH₂Cl₂ (1 : 5) gave hydroxymethyl phosphonate 25c. Rechromatography in methanol - CH₂Cl₂ (0.5 : 10) afforded 27c (21 mg, 4%).

Z-isomers 25a + 25b

UV λ_max 271, 229 nm. ¹H NMR (300 MHz, CD₃SOCD₃) δ 10.70 (s, 1H, NH), 8.25 (s, 0.25H, CH=O), 7.78, 7.77 (2 overlapped s, 1H, H₈), 7.19, 7.17 (2 overlapped s, 1H, H_1′), 6.56 (s, 2H, NH₂), 4.47-4.45 (m, 2H, CH of i-PrO), 4.30, 3.98 (2 overlapped AB's, J = 11.4 Hz, 2H, H_4″), 2.01, 1.95 (2 overlapped s, 2.25H, CH₃ of Ac), 1.72-1.19 (cluster of m, 6H, H_5′, H_6′, H_3′), 1.19-1.13 (cluster of d, 12H, CH₃ of i-PrO). ¹³C NMR (100 MHz) 170.7 (C=O of Ac), 162.7 (CH=O), 157.3, 154.7, 150.6, 134.7, 118.9, 118.3, 117.1, 112.6, 112.4 (guanine, C_2′, C_1′), 71.0, 70.0 (2 overlapped d, J = 5.2 Hz, CH of i-PrO), 68.1, 67.6 (C_4″), 26.1 (2 overlapped d, J = 21.6 and 22.1 Hz, C_5′), 25.3 (poorly resolved d, C_4′), 24.4, 24.36, 24.6 (2 overlapped d, CH₃ of i-PrO), 23.0 (d, J = 141.9 Hz), 22.9 (d, J = 140.4 Hz, C_6′), 21.1 (CH₃ of Ac), 12.4 (C_3′). ³¹P NMR (121 MHz) 30.29, 30.19. ESI-MS 454 (M + H, 25b, 30.0), 468 (M + H, 25a, 100.0), 476 (M + Na, 25b, 32.3), 490 (M + Na, 25a, 100.0).

Z-Isomer 25c

Mp 232-234 °C. UV λ_max 273 (ε 12,300), 231 nm (ε 30,100). ¹H NMR (300 MHz, CD₃SOCD₃) δ 10.66 (bs, 1H, NH), 8.21 (s, 1H, H₈), 7.09 (s, 1H, H_1′), 6.57 (s, 2H, NH₂), 5.25 (poorly resolved t, 1H, OH), 4.45 (m, 2H, CH of i-PrO), 3.80, 3.22 (poorly resolved split AB, J = 12.0 Hz, 2H, H_4”), 2.01-1.94 and 1.69-1.32 (2 poorly resolved m, 4H, H_5′, H_6′), 1.31, 1.25 (AB, J = 8.7 Hz, 2H, H_3′), 1.17-1.12 (poorly resolved m, 12H, CH₃ of i-PrO). ¹³C NMR (75 MHz) 157.3, 154.7, 150.4, 134.5, 120.1 (guanine), 117.0 (C_2′), 111.1 (C_1′), 69.9 (d, J = 4.0 Hz, CH of i-PrO), 65.4 (C_4″), 28.7 (d, J =21.1 Hz, C_5′), 24.9 (d, J = 3.8 Hz, C_4′), 24.4 (d, J = 4.0 Hz, CH₃ of i-PrO), 23.1 (d, J = 141.0 Hz), 11.9 (C_3′). ³¹P NMR (121 MHz) 30.57. ESI-MS 426 (M + H, 100.0), 448 (M + Na, 30.9). Anal. Calcd for C₁₈H₂₈N₅O₅P×0.2 H₂O: C, 50.38; H, 6.99; N, 16.33. Found: C, 50.38; H, 6.65; N, 16.12.

4.10. (E)-9-{[2-(2-diisopropylphosphonoethyl)-2-(acetoxy/formyloxy/methyl)cyclopropylidene]methyl}guanine (26a + 26b) and (E)-9-{[2-(2-diisopropylphosphono)ethyl)-2-(hydroxymethyl)cyclopropylidene]methyl}guanine (26c)

The procedure described above for the Z-isomers 25a + 25b and 25c was repeated with the E-isomer 24 (600 mg, 1.24 mmol) to give 26a + 26b (502 mg, 88 %) and 26c (26 mg, 5%). The ratio 26a/26b determined as described above for 25a/25b was 4 : 1.

E-Isomers 26a + 26b

UV λ_max 271, 229 nm. ¹H NMR (300 MHz, CD₃SOCD₃) δ 10.67 (bs, 1H, NH), 8.29 (s, 0.2H, CH=O), 8.03 (s, 1H, H₈), 7.33 (poorly resolved t, 1H, H_1′), 6.53 (s, 2H, NH₂), 4.52 (m, 2H, CH of i-PrO), 4.20, 4.12 and 4.11, 3.97 (2AB, J = 11.6 Hz, 2H, H_4″), 2.04 (s, 2.4H, CH₃ of Ac), 1.9-1.5 (m, 6H, H_5′, H_6′, H_3′), 1.21, 1.20 (2 partly overlapped d, J = 6-6.3 Hz, CH of i-PrO).³¹P NMR (121 MHz, CD₃SOCD₃) 30.21. ESI-MS 454 (M + H of 26b, 11.8), 468 (M + H of 26a, 39.1), 476 (M + Na of 26b, 12.1), 490 (M + Na of 26a, 100.0).

E-Isomer 26c

Mp 154-156 °C. UV λ_max 271 nm (ε 11,100), 228 (ε 27,800). ¹H NMR (300 MHz, CD₃SOCD₃) δ 10.77 (bs, 1H, NH), 8.02 (s, 1H, H₈), 7.27 (s, 1H, H_1′), 6.61 (s, 2H, NH₂), 4.85 (t, J = 5.6 Hz, 1H, OH), 4.52 (m, 2H, CH of i-PrO), 3.37 (H_4‘’, overlapped with H₂O), 1.94-1.58 (m, 4H, H_5′, H_6′), 1.49 (s, 2H, H_3′), 1.22-1.19 (2 poorly resolved d, 12H, CH₃ of i-PrO). ¹³C NMR (75 MHz) 157.4, 154.7, 150.6, 134.4, 120.2 (guanine), 117.0 (C_2′), 111.0 (C_1′), 69.9 (d, J = 7.0 Hz, CH of i-PrO), 65.0 (C_4″), 27.0 (d, J = 20.2 Hz, H_5′), 26.4 (d, J = 4.3 Hz, H_4′), 24.1 (d, J = 139.0 Hz, H_6′), 24.5 (d, J = 3 Hz, CH₃ of i-PrO), 15.9 (C_3′). ³¹P NMR (121 MHz) 30.84. ESI-MS 448 (M + Na, 100.0), 426 (M + H, 90.6). Anal. Calcd for C₁₈H₂₈N₅O₅P×1.2 H₂O: C, 48.34; H, 6.71; N, 15.67. Found: C, 47.98; H, 6.46; N, 15.45.

4.11. (Z)-9-{[2-(Hydroxymethy)l-2-(2-phosphonoethyl)cyclopropylidene]methyl}guanine (12)

Bromotrimethylsilane (3.74 mL, 28.90 mmol) was added dropwise to a solution of phosphonate 25a + 25b (450 mg, 0.97 mmol) in DMF (20 mL) at room temperature with stirring which was continued for 24 h. The solvent was evaporated in vacuo and the residue was dissolved in aqueous NH₄OH (30%, 30 mL). After stirring for 3 h at room temperature, the volatile components were evaporated and the aqueous solution of the crude product was lyophilized. It was chromatographed on DEAE Sephadex (40-120 mesh, HCO₃⁽⁻⁾) column, using a linear gradient of 0 - 0.3 M (500 mL each) NH₄HCO₃. The fractions containing the compound 12 were lyophilized. The product was loaded on Dowex-1 column (X2, 200 mesh, HCO₂⁽⁻⁾) which was eluted first with water (100 mL) followed by formic acid (0.8 M, 800 mL). The fractions containing the compound were lyophilized to give phosphonate 12 (286 mg, 81%) as a white solid, m: >300 °C. UV λ (pH 7) 268 nm (ε 12,800), 230 (ε 29,800). ¹H NMR (400 MHz, D₂O, sodium salt) δ 7.97 (s, 1H, H₈), 7.05 (s, 1H, H_1′), 3.80, 3.55 (AB, J = 12.2 Hz, 2H, H_5″), 2.06, 1.73 (2m, 2H), 1.43, 1.41 (m overlapped with s, 4H, H_5′, H_6′, H_3′. ¹³C NMR (100 MHz, CD₃SOCD₃) 157.3, 154.6, 150.3, 135.0, 120.3, 117.0, 110.8 (guanine, C_2′, C_1′), 65.4 (C_4″), 29.01 (d, J = 20.2 Hz, C_5′), 25.5 (d, J = 8.2 Hz, C_4′), 24.6 (d, J = 136.5 Hz, C_6′ ), 11.8 (C_3′). ³¹P NMR (121 MHz, D₂O, sodium salt) 23.83. Negative ESI-MS 340 (M - H). Anal. Calcd for C₁₂H₁₆N₅O₅P×0.8 H₂O: C, 40.52; H 4.99; N, 19.69. Found: C, 40.50; H, 5.01; N, 19.59.

4.12. (E)-9-{[2-(Hydroxymethy)l-2-(2-phosphonoethyl)cyclopropylidene]methyl}guanine (13)

The E-isomer 13 was prepared from a mixture of acetate and formate 26a + 26b (450 mg, 0.98 mmol) as described above for Z-isomer 12 to give phosphonate 13 (279 mg, 80%) as a white solid, mp >300 °C. UV λ_max (pH 7) 267 nm (ε 14,500), 230 (ε 38,800). ¹H NMR (300 MHz, D₂O) δ 8.23 (s, 1H, H₈), 7.22 (s, 1H, H_1′), 3.56, 3.48 (AB, J = 11.7 Hz, 2H, H_4″), 1.84 (m, 1H), 1.68-1.54 (m, 3H, H_5′, H_6′), 11.47 (s, 2H, H_3′). ¹³C NMR (100 MHz, CD₃SOCD₃) 157.4, 154.6, 150.5, 134.3, 120.7, 116.9, 110.6 (guanine, C_2′, C_1′), 65.1 (H_4″), 27.5 (d, J = 20.2 Hz, C_5′), 27.2 (C_4′), 26.2 (d, J = 135.8 Hz, H_6′), 16.0 (C_3′). ³¹P NMR (121 MHz, D₂O) 26.91. Negative ESI-MS 340 (M - H, 100.0). Anal. Calcd for C₁₂H₁₆N₅O₅P×0.9 H₂O: C, 40.32; H, 5.02; N, 19.59. Found: C, 40.35; H, 5.15; N, 19.61.

4.13. Z-Cyclic Phosphonate 14

A mixture of phosphate 12 (150 mg, 0.44 mmol), DCC (727 mg, 3.52 mmol) and N,N′-dicyclohexyl-4-morpholinecarboxamidine (194 mg, 0.66 mmol) in pyridine (15 mL) was refluxed under N₂ with stirring for 12 h. Pyridine was evaporated in vacuo and water (50 mL) was added to the residue. The aqueous layer was extracted with dichloromethane (5 × 25 mL) and it was filtered through a cotton plug. Aqueous NH₃ (30%, 10 mL) was added and the resulting reaction mixture was stirred overnight at room temperature. The volatile components were evaporated and the product was absorbed on Dowex-50 column (WX 2, 200 mesh, H⁽⁺⁾) which was eluted with water (600 mL). The appropriate fractions were concentrated to give cyclic phosphonate 14 (121 mg, 85%) as a white solid, mp >300 °C. UV λ_max (H₂O) 268 nm (ε 12,800), 227 (ε 28,700). ¹H NMR (300 MHz, D₂O) δ 8.54 (s, 1H, H₈), 7.06 (s, 1H, H_1′), 4.10, 3.84 (2t, J = 10.8, 11.9 Hz, 2H, H_4″), 2.00 (m, 1H), 1.83-1.39 (m, 3H, H_5′, H_6′), 1.54, 1.45 (AB, J = 9.3 Hz, 2H, H_3′ partly overlapped with H_5′, H_6′). ¹³C NMR (100 MHz, CD₃SOCD₃) 157.3, 154.6, 150.3, 135.8, 117.4, 117.2, 113.3 (guanine, C_2′, C_1′), 73.2 (d, J = 5.0 Hz, H_4″), 29.8 (d, J = 8.1 Hz, C_4′), 25.4 (d, J = 8.2 Hz, C_5′), 23.6 (d, J = 126.9 Hz, C_6′), 15.7 (C_3′). ³¹P NMR (121 MHz, D₂O) 22.41. Negative ESI-MS 322 (M - H, 100.0). Anal. Calcd for C₁₂H₁₄N₅O₄P× H₂O: C, 42.23; H, 4.73; N, 20.52. Found: C, 42.45; H, 4.83; N, 20.26.

4.14. E-Cyclic Phosphonate 15

The E-isomer 13 (150 mg, 0.44 mmol) was subjected to the same procedure as Z-isomer 12 (see above) to give cyclic phosphonate 15 (116 mg, 82%) as a white solid, mp >300 °C. UV λ_max (H₂O) 268 nm (ε 10,400), 229 (ε 28,500). ¹H NMR (300 MHz, D₂O, sodium salt) δ 7.96 (s, 1H, H₈), 7.22 (s, 1H, H_1′), 3.94, 3.86 (split AB, J = 12.9 Hz, 2H, H_4″), 1.87 (dt, J = 18.9, 6.0 Hz, 2H, H_6′), 1.74-1.63 (m, 2H, H_5′), 1.57, 1.49 (split AB, 2H, H_3′). ¹³C NMR (100 MHz, CD₃SOCD₃) 157.4, 154.7, 150.7, 134.3, 118.6, 116.9, 111.4 (guanine, C_2′, C_1′), 73.3 (d, J = 5.2 Hz), 29.8 (d, J = 7.5 Hz), 24.4 (d, J = 126.9 Hz, C_6′), 22.8 (d, J = 8.2 Hz, C_4′), 16.5 (C_3′). ³¹P NMR (121 MHz, D₂O, sodium salt) 22.52. Negative ESI-MS 322 (M - H, 100.0). Anal. Calcd for C₁₂H₁₄N₅O₄P×H₂O: C, 42.23; H, 4.73; N, 20.52. Found: C, 42.25; H, 4.85; N, 20.17.

4.15. Antiviral Assays

The antiviral assays were performed as described previously.^4,15 The HCMV assays were performed using HFF cell culture with two strains of virus, Towne and AD169, in a plaque reduction or cytopathic effect (CPE) inhibition assay. MCMV was assayed in mouse embryonic fibroblasts (MEF) by plaque reduction. The EBV DNA hybridization assay was run in Akata cells. The HSV-1 assays were performed in BSC-1 cells by ELISA and, together with HSV-2, in HFF cells by CPE inhibition assays. The VZV assays were run in HHF culture using CPE or plaque reduction assays. The results are summarized in Table 3 through 5.