(Z)- and (E)-2-(1,2-Dihydroxyethyl)methylenecyclopropane Analogues of 2’-Deoxyadenosine and 2’-Deoxyguanosine. Synthesis of All Stereoisomers, Absolute Configuration and Antiviral Activity

Abstract

Chiral Z and E-stereoisomers of (1,2-dihydroxyethyl)methylenecyclopropane analogues of 2’-deoxyadenosine and 2’-deoxyguanosine were synthesized and their antiviral activity investigated. (S)-Methylenecyclopropylcarbinol (16) was converted in seven steps to reagents 26 and 27 which were used for alkylation-elimination of adenine and 2-amino-6-chloropurine to get ultimately analogues 12a, 12b, 13a, 13b, 14a, 14b, 15a and 15b. The enantiomeric series ent-12a, ent-12b, ent-13a, ent-13b, ent-14a, ent-14b, ent-15a and ent-15b was obtained by similar procedures starting from (R)-methylenecyclopropylcarbinol (ent-16). The Z-isomer ent-12b was an inhibitor of two strains of human cytomegalovirus (HCMV) with EC₅₀ 6.8 and 7.5 µM and murine cytomegalovirus (MCMV) with EC₅₀ 11.3 µM. It was less active against HCMV with mutated gene UL97. It inhibited Epstein-Barr virus (EBV) with EC₅₀ 8 µM. The E-isomers ent-15a, ent-13a and 15b were less effective. All adenine analogues with the exception of the Z-isomers ent-12a and ent-14a were moderate substrates for adenosine deaminase.

Introduction

Methylenecyclopropane analogues of nucleosides are established antiviral agents¹ effective against β-herpesviruses (cytomegalovirus, CMV; human herpes virus 6, HHV-6) and γ-herpesviruses (Epstein-Barr virus, EBV; human herpes virus 8, HHV-8). In the first generation analogues which have only a single hydroxymethyl group, the Z-isomers 1 (Chart 1) are most effective whereas the E-isomers 2 are active only exceptionally. In the second generation group, the antiviral activity is more narrow but the trend was similar. The Z-isomers 3 were effective against CMV and EBV whereas the anti-EBV effect was found also in some of the E-isomers 4. In this group of methylenecyclopropanes containing two geminal hydroxymethyl functions, the most potent analogue, cyclopropavir (3b), is now in preclinical development as a potential drug against human cytomegalovirus (HCMV) infections.^2–5 Vicinal cis-bis-hydroxymethyl analogues 5a and 5b were ineffective as antiviral agents.⁶ Adenine trans analogue 6 was also synthesized but antiviral activity has not been reported.⁷ Addition of a third hydroxymethyl group (Z- and E-isomers 7 and 8) led to a loss of antiviral activity.⁸ Analogues 1 and 2 can be regarded as analogues of antiherpetic drug acyclovir (9) whereas bis-hydroxymethyl methylenecyclopropanes 3 and 4, and particularly cyclopropavir (3b), are related to ganciclovir (10), a drug used against HCMV.

The analogy between C-O-C grouping of acyclovir (9) or ganciclovir (10) and methylenecyclopropane moiety was crucial in designing analogues 1 – 4. It was therefore of interest whether this type of relationship can be extended to other antiviral nucleoside analogues. We determined that 9-[(2,3-dihydroxy-1-propoxy)methyl]guanine (11) would be a good starting point for such a design. Structurally, it is a positional isomer of ganciclovir (10) with a strong potency against herpes simplex virus 1 and 2 (HSV-1, HSV-2) and HCMV.^9,10 The S-enantiomer of 11 is more potent than R-enantiomer. A design of methylenecyclopropane analogues based on compound 11 is stereochemically more complex. Thus, from a single heterocyclic base, eight stereoisomers can be derived given the two chiral centers present and taking Z and E isomerism into account (Chart 2, formula 12 – 15 and ent-12 - ent-15). All 16 analogues comprising two bases, adenine and guanine, were synthesized and tested for antiviral activity. It is important to note that vertical relationships in Chart 2 (columns) are enantiomeric whereas horizontal (rows) correspond to diastereoisomers or Z,E-isomers.

Synthesis

Enantiomeric (methylenecyclopropyl)methanols^11–13 16 and ent-16 served as convenient starting materials for synthesis of all stereoisomeric analogues reported herein. Importantly, the originally assigned^11,12 absolute configurations of 16 and ent-16 were later reversed.¹³ In the 4’S series of analogues 12 through 15, (S)-(+)-(methylenecyclopropyl)-methanol (16) was first oxidized to the respective aldehyde 17 using oxalyl chloride and DMSO reagent¹² (Scheme 1). The crude aldehyde 17 was transformed to the diastereoisomeric cyanohydrin 18 using NaCN under phase transfer conditions¹⁴ in 54% yield. Hydrolysis gave the corresponding acid 19 which, in turn, were converted to benzyl esters 20 and 21 by the action of benzyl bromide, NBu₄I and K₂CO₃ in DMF. Both diastereoisomers were readily separated by column chromatography on silica gel to give the 1S, 2S-stereoisomer 20 (35%) and 1S, 2R-stereoisomer 21 (38%). Compounds 20 and 21 were reduced with diisobutylaluminum hydride (DIBALH) in hexanes to give diols 22 and 23 in 74 and 79% yield, respectively. Acetylation afforded diacetates 24 and 25 (84 and 86%). Bromination of 24 using pyridinium tribromide in CH₂Cl₂ furnished a mixture of the Z/E-isomers 26 (86%) whereas 25 gave dibromides 27 in 76% yield.

Scheme 1.

The 4’S,5’S series of analogues 12 and 13 were obtained using dibromide 26. Alkylation-elimination protocol with adenine and 26 (K₂CO₃ in DMF at 100–105 °C) gave an inseparable mixture of the Z- and E-isomeric acetates 28a in 56% yield. In a similar fashion, 2-amino-6-chloropurine and 26 afforded intermediates 28b (57% yield). Deacetylation of 28a with K₂CO₃ in methanol furnished, after chromatographic separation, the Z-isomer 12a (36%) and E-isomer 13a (49%). Deprotection of 28b was performed using NH₃ in methanol to give the Z- and E-isomers 12c and 13c in 41 and 48% yield, respectively. Hydrolysis of 12c and 13c in 80% formic acid at 80 °C followed by treatment with NH₃ in methanol gave guanine Z- and E-isomers 12b and 13b (83 and 84%).

In the 4’S,5’R series 14 and 15, reaction of adenine with dibromide 27 was performed under the conditions similar to those used for dibromide 26 to give the Z/E isomers 29a (58%). 2-Amino-6-chloropurine and 27 gave the isomeric mixture 29b (55% yield). Deacetylation of 29a gave the Z- and E-isomer 14a and 15a in 35 and 48% yield, respectively, whereas 29b afforded the Z- and E-isomers 14c and 15c (34 and 45%). Hydrolytic dechlorination furnished guanine analogues 14b and 15b (86 and 83%).

Synthesis of the enantiomeric series ent-12 through ent-15 started from (R)-(−)-(methylenecyclopropyl)methanol (ent-16) and followed the procedures outlined in Scheme 1 (ent-16 through ent-29). The enantiomeric enhancement (ee) of adenine stereoisomers 12a through 15a and ent-12a through ent-15a determined by chiral HPLC was >95% (Table 1).

Table 1.

Chiral HPLC of adenine stereoisomers^a 12a - 15a and ent-12a - ent-15a

Stereoisomer	RT(min)b	ee(%)c	Stereoisomer	RT(min)b	ee(%)c
12a	10.62	95.3	ent-12a	8.10	100.0
13a	11.94	96.5	ent-13a	8.0	100.0
14a	11.69	99.0	ent-14a	7.21	100.0
15a	7.80	96.4	ent-15a	6.37	100.0

^aChiralpak AD column, 25 × 0.46 cm, methanol, 1.0 mL/min, detection at 277 nm.

^bRT, retention time.

^cee, enantiomeric enhancement.

Assignment of Absolute Configuration and Z/E Isomerism

The absolute configuration of diastereoisomers 12 – 15 and ent-12 – ent-15 were assigned as follows. The configurations at the cyclopropane carbon atom C-4’ of analogues were guaranteed by enantiomeric starting materials 16 and ent-16 (see carbon C-1 in structure 20 and 21). To establish configuration at the neighboring exocyclic carbon C-5’ (C-2 of of 20 and 21) both esters were transformed to conformationally locked oxabicyclohexanes 30 and 31 (Scheme 2). Stereoisomer 20 was first converted to cis,trans-dibromide 32 which was, in turn, cyclized to oxabicyclohexane 30 (35%). Similarly, compound 21 afforded isomeric derivative 31 via dibromide 33 in 32% yield. In both cases, only the cis isomers of 32 and 33 can undergo cyclization. A similar reaction of unprotected racemic methylenecyclopropyl alcohols with iodine followed by cyclization was described.¹⁵ Structure and configuration of both stereoisomers 30 and 31 followed from NMR spectroscopy including nuclear Overhauser effect (NOE) experiments. A strong NOE enhancement (2.79 and 3.07%) was found in compound 31 between the cis-configured H-1 and H-2 whereas none was present in isomer 30 where these hydrogens are trans. This established that absolute configuration is 1S,2R in 30 and 1R,2R in 31, respectively. Consequently, the configurations of esters 20 and 21 are 1S,2S and 1S,2R. The enantiomers ent-20 and ent-21 have then opposite configurations. Thus, the absolute configurations of all four key intermediates 20, 21, ent-20 and ent-21 were secured.

Scheme 2.

Dibromides 26, 27, ent-26 and ent-27 obtained from these intermediates (Scheme 1) were used for synthesis of all stereoisomeric analogues. Consequently, two pairs of adenine Z-and E-enantiomers 12a, ent-12a and 13a, ent-13a were readily identified and their relationship with 14a, 15a and ent-14a, ent-15a must be diastereoisomeric. Similar conclusions apply for the respective guanine analogues.

As observed wih other methylenecyclopropane analogues,¹⁶ the cis(Z)-isomers were less polar moving faster when chromatographed on silica gel than trans(E)-isomers. Final isomeric assignment was confirmed by NMR spectroscopy (Table 2). The chemical shifts of H_1’ and C_3’ followed the trend observed before^2,16 H_1’(E) > H_1’(Z) and C_3’(E) > C_3’(Z). Nevertheless, the “usual” pattern of chemical shifts of H₈ and 5’-OH /H₈(Z) > H₈(E) and 5’-OH(Z) > 5’-OH(E)/held only for the 4’S,5’S series 12a, 13a, 12b and 13b. In the 4’S,5’R series 14a, 15a, 14b and 15b, no significant differences in these chemical shifts were found for the Z- or E-isomers. This indicates a lack of deshielding of H₈ of the Z-isomers 14a and 14b by the 5’-OH. At any rate, these differences may help in distinguishing both stereoisomeric series of analogues. No significant change in the 6’-OH chemical shifts was observed in any of these analogues. The C_4’ chemical shifts C_4’(Z) > C_4’(E) then followed the trend seen in other methylenecyclopropane analogues.¹⁷ Needless to say, all these patterns were also reflected in the corresponding enantiomeric series 4’R,5’R and 4’R,5’S.

Table 2.

Selected Chemical Shifts (δ) of Adenine and Guanine Analogues 12a - 15a and 12b - 15b

Compound (Isomer)	H1’	H8	5’-OH	C3’	C4’
12a (Z)	7.38	8.96	5.23	6.7	20.8
13a (E)	7.49	8.46	4.83	8.7	19.0
14a (Z)	7.36	8.46	4.91	5.8	21.2
15a (E)	7.42	8.47	4.90	9.6	19.8
12b (Z)	7.10	8.56	5.10	6.6	20.7
13b (E)	7.24	8.02	4.78	8.5	18.8
14b (Z)	7.09	8.01	4.85	5.7	20.9
15b (E)	7.17	8.04	4.85	9.5	19.8

The assignment of the Z- and E-isomers was unequivocally confirmed by the NOE results with guanine analogues 12b, 13b, 14b and 15b (Table 3 and Table 4). As expected,¹⁶ the interactions of the cis-related protons with the H₈ of purine in an anti-like conformation were crucial for assignment. In the Z-isomers 12b and 14b, strong effects were seen between the H₈ and 5’-OH, H_6’(H_5’) and H_4’ complemented by NOE enhancements between the H_1’ and H_3’. By contrast, interactions between the H₈ and H_3’ as well as between H_1’ and H_4’ were typical for the E-isomers 12b and 15b. In case of 15b, the long-range effects between the H_1’ and H_5’(H_6’) and even 6’-OH assisted possibly by the rigidity of methylenecyclopropane scaffold were also observed.

Table 3.

NOE data of the Z- and E-isomers 12b and 13b (DMSO-d₆, 300 MHz)

Compound	Hirr	δ	Hobs	δ	%NOE
12b	H8	8.56	5’-OH	5.10	2.07
	H8	8.56	H6’(H5’)	3.39–3.46	4.18
	H8	8.56	H6’ (H5’)	3.10	1.33
	H8	8.56	H4’	1.90	3.61
	5’-OH’	5.10	H8	8.56	3.29
	H6’(H5’)	3.39–3.46	H8	8.56	0.72
	H6’(H5’)	3.10	H8’	8.56	1.38
	H4’	1.90	H8	8.56	3.50
	H3’	1.42	H1’	7.10	1.59
13b	H8	8.02	H3’	1.36–1.40	0.56
	H8	8.02	H3’	1.61	1.01
	H4’	1.79–1.84	H1’	7.24	0.88
	H3’	1.36–1.42	H8	8.02	1.47
	H3’	1.61	H8	8.02	2.77
	H1’	7.24	H4’	1.79–1.84	1.67

Table 4.

NOE data of the Z- and E-isomers 14b and 15b (DMSO-d₆, 300 MHz)

Compound	Hirr	δ	Hobs	δ	%NOE
14b	H8	8.01	5’-OH	4.84	0.17
	H8	8.01	6’-OH	4.64	0.18
	H8	8.01	H6’(H5’)	3.51	2.33
	H8	8.01	H4’	2.05–2.09	1.62
	5’-OH’	4.84	H8	8.01	0.71
	6’-OH	4.64	H8	8.01	4.33
	H5’(H6’)	3.51	H8	8.01	5.05
	H5’(H6’)	3.25–3.31	H8	8.01	5.84
	H4’	2.05–2.09	H8	8.01	3.45
	H3’	1.30–1.38	H1’	7.09	1.41
	H1’	7.09	H3’	1.30–1.38	1.65
15b	H8	8.04	H3’	1.64	1.07
	H8	8.04	H3’	1.35–1.40	0.82
	6’-OH	4.63	H1’	7.17	2.05
	H5’(H6’)	3..37–3.46	H1’	7.17	1.27
	H5’(H6’)	2.97–3.03	H1’	7.17	2.62
	H4’	1.72–1.77	H1’	7.17	0.74
	H3’	1.64	H8	8.04	1.48
	H3’	1.30–1.38	H8	8.04	2.30

The final confirmation of absolute configuration and Z,E-isomeric structure came from X-ray diffraction of a single crystal of diacetate 34 derived from adenine analogue ent-12a (Figure 1). The X-ray confirmed the Z-configuration of 34 as well as the anti-like conformation of the purine base. Given the fact that diacetate 34 does not contain any heavy atom and molybdenum tube was used in the experiment, determination of only relative configuration at the C-4’ and C-5’ (C-9 and C-10 in Figure 1) was possible. Nevertheless, as indicated above configuration at the C-4’ is R by virtue of the starting (R)-(−)-(methylenecyclopropyl)methanol (ent-16) used for the synthesis of analogue ent-12a and diacetate 34. The absolute configuration at carbon C-5’ is then R confirming thus the ¹H NMR spectroscopic assignment based on intermediates 20, 21 and respective enantiomers ent-20, ent-21 (see above). This established not only the 4’R,5’R configuration and structure of the E-isomer ent-13a but also of the opposite 4’S,5’S enantiomers 12a and 13a. Stereoisomers 14a, 15a and ent-14a, ent-15a must then have the 4’S,5’R and 4’R,5’S configuration, respectively.

Figure 1. Crystal structure of (4’R,5’R)-diacetate 34.

ORTEP-3 view of diacetate 34 molecule showing 50% probability displacement ellipsoids.

Biological Activity. A. Antiviral Effects

Analogues 12a - 15a. 12b - 15b and enantiomers ent-12a - ent-15a, ent-12b - ent-15b were tested for antiviral activity against the following viruses in vitro: herpes simplex virus 1 and 2 (HSV-1, HSV-2), varicella zoster virus (VZV), human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), hepatitis B and C virus (HBV, HCV). The results for HCMV and EBV are summarized in Table 5. Activity against HCMV was found only for the 4’R,5’R guanine Z-isomer ent-12b. It was effective in plaque reduction assay against Towne and AD169 strain of HCMV with EC₅₀ 6.8 and 7.5 µM, respectively. It was also active in murine cytomegalovirus (MCMV) assay (EC₅₀ 11.5 µM). It is not cytotoxic and its efficacy is somewhat lower than that of ganciclovir. The fact that the new active anti-HCMV agent ent-12b is the Z-isomer is not suprising because the Z-guanine analogues 1b and 3b were highly effective.¹ None of the E-isomers of the present analogues were effective against HCMV which is in line with the previous results with other methylenecyclopropane analogues¹ such as 2 and 4. More intriguing is a lack of HCMV potency of adenine analogues 12a - 15a and ent-12a -ent-15a because analogues 1a and 3a were effective against HCMV.¹ The very narrow range of potent anti-HCMV activity limited to a single compound ent-12b with unique 4’R,5’R stereochemistry from a total of eight Z-isomeric analogues is a hallmark of this series. The rigidity of methylenecyclopropane system coupled with two centers of chirality is probably responsible for this marked stereoselectivity. Interestingly, an opposite trend, decreased enantioselectivity, was observed in less rigid dihydroxyanalogue 11 with only a single center of asymmetry where both enantiomers were active against HCMV.⁹

Table 5.

Inhibition of HCMV and EBV Replication by Stereoisomeric 1,2-(Dihydroxyethyl)methylenecyclopropane Analogues of Nucleosides

		EC50/CC50 (µM)
		HCMV/HFF
Compound	Configuration	Townea,b	AD169c,d	EBV/Akatae
12a	Z,4’S,5’S	>100/>100	300/>300	>20/50.5
13a	E,4’S,5’S	>100/>100	>60/>300	>20/57
14a	Z,4’S,5’R	>100/>100	>60/>300	43/55
15a	E,4’S,5’R	>100/>100	>300/>300	50.4/50.6
12b	Z,4’S,5’S	>100/>100	>300/>300	43/54
13b	E,4’S,5’S	>100/>100	>60/>300	>20/67
14b	Z,4’S,5’R	>100/>100	>300/>300	>20/56
15b	E,4’S,5’R	>100/>100	>300/>300	18/61
ent-12a	Z,4’R,5’R	>100/>100	>60/194	52.6/61.5
ent-13a	E,4’R,5’R	>100/>100	>60/264	17/73
ent-14a	Z,4’R,5’S	>100/>100	>60/206	60/93.5
ent-15a	E,4’R,5’S	>100/>100	>60/194	9.8/72.7
ent-12b	Z,4’R,5’R	6.8/>100f	7.5/299a,g	8/78
ent-13b	Z,4’R,5’R	>100/>100	>60/298	40/75
ent-14b	Z,4’R,5’S	>100/>300	>300/>300	>20/62.6
ent-15b	E,4’R,5’S	>100/>300	>300/>300	58.7/86.6
3b	-	0.46/>100h	0.49/>100h	0.22//46i
Control		1.2–2.4/>100j	0.04–1.5/>100j	3.3/>100k

^aPlaque reduction assay in HFF cells‥

^bVisual cytotoxicity in uninfected HFF’s used in plaque reduction assays.

^cCytopathic effect (CPE) inhibition assay.

^dCytotoxicity by neutral red uptake.

^eDNA hybridization assay.

^fAverage of three experiments.

^gEC₅₀ 11.3 µM by plaque reduction assay in MCMV/ MEF, EC₅₀ of ganciclovir was 5.5 µM.

^hRef. ².

ⁱRef. ²⁸.

^jGanciclovir.

^kAcyclovir.

Analogue ent-12b was also the most effective against EBV in Akata cell culture with EC₅₀ 8 µM (Table 5). It is somewhat less potent than acyclovir (9). The other less potent analogues included the E-isomers ent-15a, ent-13a and 15b with EC₅₀ 9.8 – 18 µM indicating that the 4’ R,5’ R stereochemistry and Z/E isomerism are less important for anti-EBV than an anti-HCMV effect. It should be noted that effectivity of the E-isomers of methylenecyclopropane analogues against EBV was observed before in several instances.^1,18 Interestingly, the pattern of anti-HCMV and anti-EBV effects of analogue ent-12b parallel those of cyclopropavir 3b although the former is a weaker antiviral. Compound ent-12b also exhibited a moderate effect against HSV-1 (EC₅₀/CC₅₀ 50/>100 µM) in BSC-1 cells (ELISA) but it was inactive against HSV-1 and HSV-2 in human foreskin fibroblast (HFF) culture. It was also somewhat effective in VZV/HFF assay (EC₅₀/CC₅₀ 47/284 µM). None of the analogues was active against hepatitis B or C virus. It should be noted that somewhat similar cyclopropane analogue 35 (n = 0, B = Gua) described in the patent literature¹⁹ as a mixture of diastereoisomers was without significant activity against HSV-1. The 1’S,2’R,4’S stereoisomers 35 (n = 0 or 1, B = Thy, Ura, Ade or Gua) were also devoid of antiviral activity.^20,21 Nevertheless, it should be emphasized that HCMV and EBV assays with 35 have not been reported.

Regarding the mechanism of antiviral action of active 1,2-dihydroxyethyl analogues, we hypothesize that the phosphorylation cascade generally applicable for activation of nucleosides (including methylenecyclopropane analogues¹) phosphate ➔ diphosphate ➔ triphosphate also is operative here. Because none of the compounds is active against HSV-1 or HSV-2, viral thymidine kinase likely seems unable to effect the first phosphorylation in contrast to ganciclovir analogue 11 where the hydroxymethyl group is phosphorylated by this enzyme.¹⁰ There is evidence that methylenecyclopropanes 1b, 2b and 3b are phosphorylated by the action^3,22–25 of HCMV-encoded phosphotransferase pUL97 and this appears to be the case with ent-12b as well. Comparison of the activity of ent-12b to that of cyclopropavir (3b) and ganciclovir (10) in two HCMV strains with mutated UL97 genes showed that all three compounds were significantly less active against HCMV with UL97 mutations than to wild-type virus (Table 6). Together with the known substrate specificity²⁶ of pUL97 for ganciclovir (10), we take this as a strong evidence that this enzyme phosphorylates ent-12b.

Table 6.

Activity of ent-12b against Drug Resistant HCMV

Compound	Towneb	EC50 (µM)a Virus Strain 2696rc	E8d
ent-12b	8	>100	72
cyclopropavir (3b)	0.6	28	8
ganciclovir (10)	2	42	22

^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.

^dVirus with two point mutations introduced into gene UL97.

B. Adenosine Deaminase (ADA)

Adenine analogues 12a - 15a and ent-12a - ent-15a were tested for substrate activity toward ADA (Table 7). All active analogues are only moderate substrates for the enzyme. As observed in the previous cases of methylenecyclopropane analogues, the E-isomers are more reactive than the Z-isomers. This effect does not depend much on the stereochemistry of the side-chain. Thus, all E-isomers were deaminated from 80–100% within 24–48 h whereas the Z-analogues ent-12a and ent-14a were resistant to deamination.

Table 7.

Deamination of Adenine 1,2-(Dihydroxyethyl)methylenecyclopropane Analogues of Nucleosides by Adenosine Deaminase^a

Compound	Isomer	Configuration	%Deamination
			24 h	48 h
12a	Z	4’S,5’S	45	80
13a	E	4’S,5’S	85	100
14a	Z	4’S,5’R	65	80
15a	E	4’S,5’R	100	100
ent-12a	Z	4’R,4’R	0	0
ent-13a	E	4’R,4’R	100	100
ent-14a	Z	4’R,5,S	0	0
ent-15a	E	4’R,5’S	65	85

^aFor details see Experimental.

Experimental Section

General Methods

The UV spectra were measured in ethanol and NMR spectra were determined in CD₃SOCD₃ at 400 MHz (¹H) and 100 MHz (¹³C) unless stated otherwise. Mass spectra were determined in electron-impact (EI-MS) or electrospray ionization (ESI-MS, methanol - AcONa) mode. Optical rotations were measured on JASCO Digital Polarimeter DIP-370. The (S)-(+)- and (R)-(−)-(methylenecyclopropyl)methanols (16) and (ent-16) were prepared as described^11–13 from (S)-(+)- and (R)-(−)-epichlorohydrin. Enantiomeric epichlorohydrins were obtained as reported.²⁷ They were converted to benzyl (S)- and (R)-glycidyl ethers²⁷ using a procedure for racemic compounds²⁸ and enantiomeric enhancements (ee) were determined by chiral HPLC (Chiralcel AD column was used instead of Chjralcel OD²⁷). The ees of benzyl (S)- and (R)-glycidyl ether were 96 and 99%, respectively. A characterization of enantiomeric compounds by UV (where applicable), ¹H, ¹³C NMR and MS is reported for only one set of enantiomers. The data of the opposite series was essentially identical. The prefixes ”ent-“ before formula numbers indicate opposite enantiomers. Optical rotation was determined for all enantiomeric compounds. All biologically tested analogues were ≥95% pure as indicated by C,H,N analyses. The optical purity of all adenine analogues was at least 95% as shown by chiral HPLC (Table 1).

(−)-(R,S)-2-Hydroxy-2-[(S)-2-methylenecyclopropyl]acetonitrile (18)

To a solution of oxalyl chloride (7.5 mL, 85.0 mmol) in CH₂Cl₂ (100 mL) at −60 ⁰C was added DMSO (12.5 mL, 175.0 mmol) in CH₂Cl₂ (25 mL) with stirring at -60 °C followed, after 45 min, by a dropwise addition of (S)-(+)-(methylenecyclopropyl)methanol (16, 4.2g, 50.0 mmol) in CH₂Cl₂ (25 mL). The stirring was continued for 45 min whereupon triethylamine (35 mL, 0.25 mol) was added and the reaction mixture containing crude aldehyde 17 was allowed to warm to room temperature. 1 M HCl saturated with NaCl (275 mL) was added and the aqueous layer was extracted with CH₂Cl₂ (4 × 50 mL). The organic layers were combined and saturated solution of NH₄Cl (30 mL) was added with stirring and external ice-cooling followed by NaCN (3.67 g, 75 mmol). The stirring was continued for 30 min, the layers were separated and the aqueous layer was extracted with CH₂Cl₂ (6 × 25 mL). The combined organic phase was dried over MgSO₄. The solvent was evaporated and the crude product was chromatographed on a silica gel column using hexanes - diethyl ether (10 : 1 to 1 : 1) to give nitrile 18 as a colorless oil (2.96 g, 54.3%). [α]_D²⁶ −11.8° (c 1.0, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ 1.17–1.27 (m, 1H), 1.43–1.52 (m, 1H), 1.93–2.01 (m, 1H, cyclopropane), 3.90 (bs, 1H, OH), 4.10, 4.29 (2d, J = 8.4, 7.2 Hz, 1H, CHO), 5.53–5.55 (m, 1H), 5.61–5.65 (m, 1H, =CH2). ¹³C NMR (75 MHz) 7.5, 9.2, 18.9, 19.2 (cyclopropane), 63.4, 63.7 (CHO), 107.00, 107.04 (=CH₂), 118.7, 119.1 (=C), 128.6, 129.5 (CN). EI-HRMS calcd for C₆H₇NO (M) 109.0528, found 109.0529.

(+)-(R,S)-2-Hydroxy-2-[(R)-2-methylenecyclopropyl]acetonitrile (ent-18)

The procedure described above was performed with (R)-(−)-(methylenecyclopropyl)methanol (ent-16, 4.8 g, 57 mmol) to give nitrile ent-18 via aldehyde ent-17 (3.55 g, 57%), [α]_D²³ 11.4° (c 1.58, CHCl₃).

(−)-Benzyl (S)-2-Hydroxy-2-[(S)-2-methylenecyclopropyl)]acetate (20) and (−)-Benzyl (R)-2-Hydroxy-2-[(S)-2-methylenecyclopropyl)]acetate (21)

A solution of nitrile 18 (2.76 g, 25.32 mmol) in CH₂Cl₂ (100 mL) was stirred with 37% HCl (25 mL) at room temperature for 2 h. The volatiles were evaporated at 18 torr and room temperature and a solution of the residue in ether was passed through a 5-cm silica gel pad. The pad was washed with hexanes - Et₂O (1 : 1, 180 mL) and the filtrate was concentrated to give acid 19 (2.91 g, 90%) which was directly used in the next step.

Benzyl bromide (8.4mL, 70.3 mmol) was added dropwise with stirring to acid 19 (2.8 g, 21.9 mmol) in DMF (33 mL), anhydrous K₂CO₃ (5.07 g, 36.7 mmol) and NBu₄I (2.60 g, 7.03 mmol) at room temperature. The stirring was continued for 3.5 h, the solvent was evaporated and the residue was partitioned between water (60 mL) and Et₂O (4 × 60 mL). The organic phase was washed with 10% HCl (25 mL), 5% NaHCO₃ (25 mL) and brine (20 mL). After drying (MgSO)₄, the solvent was evaporated and the crude product was chromatographed on a silica gel column in hexanes - Et₂O (10 : 1 to 5 : 1) to give the (S, S)-isomer 20 (1.67 g, 35%) followed by (S,R)-isomer 21 (1.81 g, 38%).

(S,S)-Isomer 20

[α]_D²⁶ −7.3° (c 1.0, CHCl₃). ¹H NMR (CDCl₃) δ 1.28–1.34 (m, 2H), 1.82–1.85 (m, 1H, cyclopropane), 2.73 (s, 1H, OH), 4.04 (d, 1H, J = 5.6 Hz, CHO), 5.23 (AB, 2H, J_AB = 12 Hz, CH₂ of Bn), 5.46 (d, 2H, J = 2.4 Hz, =CH₂), 7.37 (m, 5H, Ph). ¹³C NMR 6.8, 18.8 (cyclopropane), 67.5 (CHO), 71.0 (CH₂ of Bn), 105.7 (=CH), 128.5, 128.7, 128.9, 130.9, 135.5 (=C, Ph), 174.3 (C=O). ESI-MS 241.3 (M + Na, 100.0). Anal. Calcd for C₁₃H₁₄O₃×0.2 H₂O.

(S,R)-Isomer 21

[α]_D²⁵ −45.3° (c, 1.02, CHCl₃). ¹H NMR (CDCl₃) δ 1.19 (m, 1H), 1.37 (tt, 1H, J = 8.8, 2.4 Hz), 1.72–1.79 (m, 1H, cyclopropane), 2.93 (s, 1H, OH), 3.80 (d, 1H, J = 7.2 Hz, CHO), 5.20 (AB, 2H, J_AB = 12 Hz, CH₂ of benzyl), 5.40, (s, 1H), 5.43 (d, 1H, J = 1.6 Hz, =CH₂), 7.37 (m, 5H, Ph). ¹³C NMR 8.0, 19.4 (cyclopropane), 67.8 (CHO), 72.6 (CH₂ of Bn), 106.0 (=CH₂), 128.6, 128.8, 128.9, 130.9, 135.4 (=C, Ph), 174.2 (C=O). ESI-MS 241.3 (M + Na, 100.0). Anal. (C₁₃H₁₄O₃x0.3 H₂O) C, H.

(+)-Benzyl (R)-2-Hydroxy-2-[(R)-2-methylenecyclopropyl)]acetate (ent-20) and (+)-Benzyl (S)-2-Hydroxy-2-[(R)-2-methylenecyclopropyl)]acetate (ent-21)

The protocol described above was repeated with nitrile ent-18 (3.10 g, 28 mmol) to give esters ent-20 (1.71 g, 28%) and ent-21 (2.52 g, 41%) via acid ent-19 as an intermediate. ent-20: [α]_D²⁶ 6.3° (c 1.04, CHCl₃). ent-21: [α]_D²⁶ 44.9° (c 1.02, CHCl₃).

(−)-(S)-1-[(S)-2-Methylenecyclopropyl]ethane-1,2-diol (22)

DIBALH in hexanes (1M, 24 mL, 24 mmol) was added to a solution of the (S, S)-ester 20 (1.50 g, 6.9 mmol) in THF (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%, 35 mL). After stirring for 2 h, it was extracted with Et₂O (15 × 30 mL). The organic phase was washed successively with saturated NaHCO₃ (2 × 30 mL) and brine (2 × 30 mL). After drying (MgSO₄), the solvents were evaporated and the crude product was chromatographed on a silica gel column in hexanes – Et₂O (2 : 1 to 1 : 3) to give diol 22 (580 mg, 74%) as a colorless oil, [α]_D²⁴ −3.5° (c1.03, CHCl₃). ¹H NMR (CDCl₃) δ 0.97–1.01 (m, 1H), 1.27 (tt, 1H, J = 8.8, 1.6 Hz), 1.57–1.62 (m, 1H, cyclopropane), 3.10 (bs, 2H, OH), 3.32 (dt, 1H, J = 7.6, 3.2 Hz), 3.56 (dd, 1H, J = 11.4, 8.2 Hz), 3.73 (dd, 1H, J = 11.2, 3.2 Hz, CHO, CH₂O), 5.44, 5.53 (2m, 2H, =CH₂). ¹³C NMR 7.3, 17.9 (cyclopropane), 66.4 (CH₂O), 74.9 (CHO), 104.7 (=CH), 132.6 (=CH₂). EI-HRMS calcd for C₆H₈O (M - H₂O): 96.0575, found: 96.0572. Anal. (C₆H₁₀O₂x0.2 H₂O) C, H.

(+)-(R)-1-[(R)-2-Methylenecyclopropyl]ethane-1,2-diol (ent-22)

The procedure described above was performed with (R,R)-ester ent-20 (1.65 g, 7.6 mmol) to furnish diol ent-22 (630 mg, 73%). [α]_D²⁵ 2.7° (c 1.05, CHCl₃).

(+)-(R)-1-[(S)-2-Methylenecyclopropyl]ethane-1,2-diol (23)

The reduction of (R,S)-ester 21 (1.6 g, 7.34 mmol) followed the above procedure to give diol 23 (660 mg, 79%) as a hite solid, mp 69–71 °C, [α]_D²⁷ 25.4° (c 1.0, CHCl₃). ¹H NMR (CDCl₃) δ 1.06–1.10 (m, 1H), 1.34 (tt, 1H, J = 8.8, 1.6 Hz), 1.54–1.60 (m, 1H, cyclopropane), 3.01, 3.10 (2bs, 2H, OH), 3.21 (dt, 1H, 8.0, J = 2.7 Hz), 3.59 (dd, 1H, J = 10.8, 7.2 Hz), 3.69 (poorly resolved dd, 1H, J = 10.4 Hz, CHO, CH₂O), 5.40 (d, 1H, J = 2.4 Hz), 5.42 (d, 1H, J = 1.6 Hz, =CH₂). ¹³C NMR 8.3, 18.5 (cyclopropane), 66.7 (CH₂O), 75.2 (CHO), 104.8 (=CH), 132.0 (=CH₂). EI-HRMS calcd for C₆H₈O M - H₂O): 96.0575, found: 96.0580. Anal. (C₆H₁₀O₂x0.45 H₂O) C, H.

(−)-(S)-1-[(R)-2-Methylenecyclopropyl]ethane-1,2-diol (ent-23)

The reduction of (S,R)-ester ent-21 (2.0 g, 9.17 mmol) followed the above procedure to give diol ent-23 (805 mg, 77%), mp 73–74 °C, [α]_D²⁶ −25.0° (c 1.05, CHCl₃).

(+)-(S)-1-[(S)-2-Methylenecyclopropyl]ethane-1,2-diyl Diacetate (24)

Acetic anhydride (3 mL) was added dropwise to a stirred solution of diol 22 (520 mg, 4.56 mmol) in pyridine (1.5 mL) at room temperature. The stirring was continued for 10 h, the reaction was quenched with water, and product was extracted with ice cold Et₂O (100 mL). The combined organic phase was washed successively with saturated aqueous CuSO₄, 5% HCl (4 × 25 mL), aqueous NaHCO₃ (3 × 20 mL) and brine (3 × 20 mL). It was dried (MgSO₄), the solvent was evaporated and the residue was chromatographed on a silica gel column (hexanes - Et₂O, 50 : 1 to 10 : 1) to give diacetate 24 (760 mg, 84%) as a colorless oil, [α]_D²⁴ 80.9° (c 0.97, CHCl₃). ¹H NMR (CDCl₃) δ 1.06–1.09 (m, 1H), 1.37 (tt, 1H, J = 9.2, 1.6 Hz), 1.71 (m, 1H, cyclopropane), 2.05, 2.07 (2s, 6H, CH₃), 4.09 (dd, 1H, J = 12.0, 7.2 Hz), 4.34 (dd, 1H, J = 12.4, 2.8 Hz,), 4.71 (dt, 1H, J = 8.8, 3.2 Hz, CHO, CH₂O), 5.42, 5.44 (2s, 2H, =CH₂). proton). ¹³C NMR 7.9, 15.8 (cyclopropane), 21.0, 21.3 (CH₃), 65.0 (CH₂O), 73.8 (CHO), 105.2 (=CH), 131.2 (=C), 170.5, 171.0 (C=O). ESI-MS 221 (M + Na, 100.0). EI-HRMS calcd for C₁₀H₁₅O₄: 199.0970, found: 199.0966. Anal. (C₁₀H1₄O₄) C, H.

(−)-(R)-1-[(R)-2-Methylenecyclopropyl]ethane-1,2-diyl Diacetate (ent-24)

The protocol described above was followed with diol ent-22 (600 mg, 5.26 mmol) to give diacetate ent-24 (840 mg, 81%), [α]_D²⁷ -78.1o (c 1.0, CHCl₃).

(+)-(R)-1-[(S)-2-Methylenecyclopropyl]ethane-1,2-diyl Diacetate (25)

The procedure described above was performed with diol 23 (610 mg, 5.35 mmol) to give diacetate 25 (910 mg, 86%) as a colorless oil, [α]_D²⁶ 31.6° (c 1.0, CHCl₃). ¹H NMR (CDCl₃) δ 1.10–1.14 (m, 1H), 1.28 (tt, 1H, J =8.8, 2.4 Hz), 1.58–1.63 (m, 1H, cyclopropane), 1.99, 2.00 (2s, 6H, CH₃), 4.08–4.19 (m, 2H), 4.53–4.68 (m, 1H, CH₂O, CHO), 5.40 (m, 2H, =CH₂). ¹³C NMR 8.8, 16.5 cyclopropane), 20.9, 21.2 (CH₃), 65.4 (CH₂O), 73.6 (CHO), 105.6 (=CH), 131.0 (=C), 170.6, 170.7 (C=O). ESI-MS 221 (M + Na, 100.0). Anal. (C₁₀H₁₄O₄) C, H.

(−)-(S)-1-[(R)-2-Methylenecyclopropyl]ethane-1,2-diyl Diacetate (ent-25)

The protocol described above was followed with diol ent-23 (720 mg, 6.32 mmol) to give diacetate ent-25 (1.04 g, 81%), [α]_D²⁷ −33.9° (c 0.98, CHCl₃).

(S)-1-[(1R,2S)-2-Bromo-2-(bromomethyl)cyclopropyl]ethane-1,2-diyl Diacetate + (S)-1-[(1R,2R)-2-Bromo-2-(bromomethyl)cyclopropyl]ethane-1,2-diyl Diacetate (26)

Pyridinium tribromide (1.64 g, 5.15 mmol) was added with stirring to a solution of diacetate 24 (680 mg, 3.43 mmol) in CH₂Cl₂ (18 mL) at −20 °C. The reaction mixture was allowed to warm to room temperature. After 12 h, it was diluted with AcOEt (100 mL) and the resultant solution was washed sequentially with saturated Na₂S₂O₃ (2 × 25 mL), NaHCO₃ (2 × 20 mL) and water (20 mL). The organic phase was dried over MgSO₄ and the solvents were evaporated. The crude product was chromatographed on a silica gel column in hexanes - Et₂O (20 : 1 to 5 : 1) to afford cis,trans-dibromide 26 (1.06 g, 86%) as a colorless oil, [α]_D²⁵ 46.5° (c 1.1, CHCl₃). ¹H NMR (CDCl₃) δ 1.00 (t, J = 6.4 Hz), 1.22–1.31 (m), 1.34–1.43 (m), 1.60–1.64 (m, 3H, cyclopropane), 2.06, 2.07, 2.09, 2.10 (4s, 6H, CH₃), 3.64–3.80 (m, 2H, CH₂Br), 4.03–4.14 (m), 4.35–4.46 (m, 2H), 4.68–4.73 (m), 4.81–4.85 m, 1H, CH₂O, CHO). ¹³C NMR 20.9, 21.0, 21.2, 21.3, 22.3, 22.6, 27.1, 32.0, 35.5, 38.0, 40.2, 43.4, 64.6, 64.7, 70.8, 74.7, 170.1, 170.4, 170.8, 170.9. ESI-MS (MeOH +AcOK) 395, 397, 399 (M + K, 48.8, 100.0, 55.6).

(R)-1-[(1S,2R)-2-Bromo-2-(bromomethyl)cyclopropyl]ethane-1,2-diyl Diacetate + (R)-1-[(1S,2S)-2-Bromo-2-(bromomethyl)cyclopropyl]ethane-1,2-diyl Diacetate (ent-26)

The reaction was performed as described above with diacetate ent-24 (740 mg, 3.74 mmol) to give cis,trans-dibromide ent-26 (1.14 g, 85%), [α]_D²⁵ −52.8° (c 1.05, CHCl₃).

(R)-1-[(1R,2S)-2-Bromo-2-(bromomethyl)cyclopropyl]ethane-1,2-diyl Diacetate + (R)-1-[(1R,2R)-2-Bromo-2-(bromomethyl)cyclopropyl]ethane-1,2-diyl Diacetate (27)

The protocol described above was performed with diacetate 25 (810 mg, 4.09 mmol) to give the cis,trans-dibromide 27 (1.11 g, 76%) as a colorless oil, [α]_D²⁷ 11.6° (c 1.0, CHCl₃). ¹H NMR (CDCl₃) δ 1.12 (t, J = 7.5 Hz), 1.15–1.22 (m), 1.31–1.36 (m), 1.41–4.53 (m), 1.83–1.90 (m, 3H, cyclopropane), 2.005, 2.009, 2.02 (3s, 6, CH₃), 3.46, 3.74 (AB, J_AB = 11.2 Hz), 3.70, 3.85 (AB, 2H, J_AB = 12.4 Hz, CH₂Br), 4.17–4.25 (m), 4.36–4.40 (2d, J = 3.2, 4.4 Hz, 2H), 4.77–4.82, 4.84–4.89 (2m, 1H, CH₂O, CHO). ¹³C NMR 20.95, 21.00, 21.17, 21.23, 23.4, 23.7, 26.2, 31.8, 35.8, 37.4, 40.9, 44.0, 64.5, 65.6, 69.8, 74.6, 170.3, 170.6, 170.7. ESI-MS (MeOH + AcOK) 395, 397, 399 (M + K, 50.0, 100.0, 56.9).

(R)-1-[(1S,2R)-2-Bromo-2-(bromomethyl)cyclopropyl]ethane-1,2-diyl Diacetate + (S)-1-[(1S,2S)-2-Bromo-2-(bromomethyl)cyclopropyl]ethane-1,2-diyl Diacetate (ent-27)

The reaction was performed as described above with diacetate ent-25 (740 mg, 3.74 mmol) to give cis,trans-dibromide ent-27 (1.14 g, 85%), [α]_D²⁵ −8.9° (c 1.0, CHCl₃).

9-{(Z)-(S)-2-[(S)-1,2-Diacetoxyethyl)cyclopropylidene]methyl}adenine + 9-{(E)-(S)-2-[(S)-1,2-Diacetoxyethyl)cyclopropylidene]methyl}adenine (28a)

A mixture of adenine (200 mg, 1.48 mmol), dibromide 26 (490 mg, 1.37 mmol) and K₂CO₃ (1.13 g, 8.2 mmol) in DMF (7 mL) was stirred under N₂ at 100–105 °C for 7 h. After cooling, the solids were filtered off using a silica gel pad that was washed with DMF (60 mL). The solvent was evaporated in vacuo and the residue was chromatographed on a silica gel column in AcOEt -MeOH (50 : 1 to 20 : 1) to give the Z,E-isomers 28a (254 mg, 56%), mp 141–145 °C, [α]_D²² 65.8° (c 1.06, MeOH). UV λ_max 224 nm (ε 25,700), 262 (ε 12,800), 279 (ε 9,100). ¹H NMR (CDCl₃) δ 1.43–1.47 (m), 1.52–1.56 (m), 1.70, 1.81 (dt, 1H, J = 8.8, 2.4 Hz), 2.00–2.03 (m, partially overlapped with CH₃ at 2.06), 2.25–2.32 (m, 3H, cyclopropane), 1.91, 2.06, 2.09 and 2.11 (4s, 6H, CH₃), 4.15–4.19 (m), 4.41, 4.44 (dt, 2H, J = 12.0, 1.6 Hz, H_6’), 4.64–4.69 (m), 4.77–4.81 (m, 1H, H_5’), 6.32, 6.40 (2bs, 2H, NH₂), 7.49, 7.56 (m, 1H, H_1’), 8.25, 8.36, 8.37, 8.40 (4s, 2H, H₂, H₈). ¹³C NMR 7.6, 9.3, 15.9, 17.6, 20.99, 21.03, 21.3, 64.6, 64.8, 73.0, 74.4, 112.0, 112.1, 112.9, 113.6, 119.3, 119.4, 137.5, 137.9, 148.9, 152.7, 152.9, 155.4, 170.0, 170.4, 170.87, 170.92. ESI-MS 332 (M + H, 44.5), 354 (M + Na, 100.0), 685 (2M + Na, 85.8).

9-{(Z)-(R)-2-[(R)-1,2-Diacetoxyethyl)cyclopropylidene]methyl}adenine + 9-}(E)-(R)-2-[(R)-1,2-Diacetoxyethyl)cyclopropylidene]methyl}adenine (ent-28a)

A mixture of adenine (230 mg, 1.7 mmol), dibromide ent-26 (580 mg, 1.62 mmol) and K₂CO₃ (1.34 g, 9.7 mmol) in DMF (8 mL) was stirred under N₂ for 48 h at room temperature and then for 8 h at 100–105 °C. The work-up followed the protocol described above to give the Z,E-isomers ent-28a (327 mg, 61%), mp 163–165 °C, [α]_D²² −75.5° (c 1.0, MeOH).

9-{(Z)-(S)-2-[(Z)-(R)-1,2-Diacetoxyethyl)cyclopropylidene]methyl}adenine + 9-}(E)-(S)-2-[(R)-1,2-Diacetoxyethyl)cyclopropylidene]methyl}adenine (29a)

The reaction of adenine with dibromide 27 was performed on the same scale as described for compound 28a (reaction time was 12 h at room temperature and 10 h at 100–105 °C) to give the Z,E-isomers 29a (273 mg, 58%), mp 135–139 °C, [α]_D²² 27.8° (c, 1.0, MeOH). UV λ_max 226 nm (ε 26,500), 263 (ε 12,700), 278 (ε 9,400). ¹H NMR (CDCl₃) δ 1.51–1.55 (m), 1.60 (dt, J = 9.2, 2.0 Hz), 1.66–1.70 (m), 1.79 (dt, J = 8.8, 2.4 Hz, H_3’), 2.00, 2.01, 2.10, 2.11 (4s, 6H, CH₃), 2.07–2.10 (m, partly overlapped with 2.10), 2.18–2.23 (m, 1H, H_4’), 4.24, 4.01 and 4.23, 4.03 (2AB, J_AB = 12.2 Hz, 2H, H_6’), 4.72, 5.21 (2s, 1H, H_5’), 6.28, 6.34 (2bs, 2H, NH₂), 7.49, 7.61 (2d, J = 1.6 Hz. 1H, H_1’), 8.23, 8.24, 8.37 (3s, 2H, H₂, H₈). ¹³C NMR 10.62, 10.5, 16.6, 18.3, 20.9, 20.96, 21.02, 64.6, 65.1, 70.1, 73.0, 112.51, 112.54, 113.1, 114.3, 119.3, 119.4, 137.6, 138.8, 148.9, 151.8., 152.2, 154.9, 155.02, 170.1, 170.6, 170.7, 170.8. ESI-MS 332 (M + H, 38.9), 354 (M + Na, 100.0), 685 (2M + Na, 56.5).

9-{(Z)-(R)-2-[(S)-1,2-Diacetoxyethyl)cyclopropylidene]methyl}adenine + 9-{(E)-(R)-2-[(S)-1,2-Diacetoxyethyl)cyclopropylidene]methyl}adenine (ent-29a)

A mixture of adenine (220 mg, 1.63 mmol), dibromide ent-27 (550 mg, 1.54 mmol) and K₂CO₃ (1.28 g, 9.3 mmol) in DMF (8 mL) was stirred under N₂ for 24 h at room temperature and 8 h at 100–105 °C. The work-up followed the above procedure to furnish the Z,E-isomers ent-29a (280 mg, 55%), mp 152–156 °C, [α]_D²² −36.6° (c, 1.0, MeOH).

2-Amino-6-chloro-9-{(Z)-(S)-2-[(S)-1,2-diacetoxyethyl)cyclopropylidene]methyl}purine + 2-Amino-6-chloro-9-{(E)-(S)-2-[(S)-1,2-diacetoxyethyl)cyclopropylidene]methyl}-purine (28b)

The reaction was performed as described for adenine Z,E-isomers 28a with 2-amino-6-choropurine (270 mg, 1.6 mmol), dibromide 26 (550 mg, 1.54 mmol) and K₂CO₃ (1.27 g, 9.2 mmol) in DMF (8 mL). Reaction time 24 h at room temperature and 6 h at 100–105 °C. The crude product was chromatographed in hexanes - AcOEt (1 : 1 to 1 : 2) to give the E, Z-isomers 28b (320 mg, 57%), mp 142–147 °C, [α]_D²⁶ 80.0° (c 1.18, CHCl3). UV λ_max 311 nm (ε 7,800), 228 (ε 29,000). ¹H NMR (CDCl₃) δ 1.43–1.47, 1.51–1.55 (2m), 1.69, 1.81 (2dt, 1H, J = 8.8, 1.6 Hz, H_3’), 2.25–2.31 (m, 1H, H_4’), 1.96, 2.06, 2.09, 2.12 (4s, 6H, CH₃), 4.14–4.20, 4.34–4.44 (2m, 2H, H_6’), 4.71–4.75, 4.78–4.82 (2m, 1H, H_5’), 5.36 (bs, 2H, NH₂), 7.31, 7.38 (2 poorly resolved d, 1H, H_1’), 8.17, 8.29 (2s, 1H, H₈). ¹³C NMR 7.5, 9.2, 16.0, 17.9, 20.9, 21.0, 21.1, 21.2, 64.5, 64.8, 72.9, 74.3, 111.7, 111.8, 113.1, 113.6, 125.1, 125.2, 138.9, 139.5, 151.6, 151.7, 152.5, 159.50, 159.51, 170.3, 170.4, 170.85, 170.89. ESI-MS 366, 368 (M + H, 22.2, 7.4), 388, 390 (M + Na, 100.0, 30.0).

2-Amino-6-chloro-9-{(Z)-(R)-2-[(R)-1,2-diacetoxyethyl)cyclopropylidene]methyl}purine + 2-Amino-6-chloro-9-{(E)-(R)-2-[(R)-1,2-diacetoxyethyl)cyclopropylidene]methyl}-purine (ent-28b)

A reaction of 2-amino-6-chloropurine (350 mg, 2.07 mmol) with dibromide ent-26 (720 mg, 2.0 mmol) and K₂CO₃ was performed as indicated above to give the E,Z-isomers ent-28b (360 mg, 46%), mp 136–140 °C, [α]_D²⁶ −87.5° (c 1.01, CHCl₃).

2-Amino-6-chloro-9-{(Z)-(S)-2-[(R)-1,2-diacetoxyethyl)cyclopropylidene]methyl}-purine + 2-Amino-6-chloro-9-{(E)-(S)-2-[(R)-1,2-diacetoxyethyl)cyclopropylidene]-methyl}purine (29b)

The protocol described above was followed with 2-amino-6-chloropurine (310 mg, 1.83 mmol), dibromide 27 (620 mg, 1.73 mmol) and K₂CO₃ (1.43 g, 10.4 mmol) at room temperature for 4 h and at 100–105 °C for 7 h. After chromatography in hexanes - AcOEt (1 : 1 to 1 : 3), the E,Z-isomers 29b were obtained (348 mg, 55%). Mp 153–156 °C, [α]_D²⁷ 44.7 ° (c 1.1, CHCl₃). UV λ_max 311 nm (ε 8,000), 228 (ε 35,400). ¹H NMR (CDCl₃) δ 1.50–1.60 (m), 1.67–1.68 (m), 1.78 (t, J = 8.9 Hz, 3H, cyclopropane), 2.06–2.11 (m, partially overlapped with 2.04), 2.35–2.41 (2m, 3H, cyclopropane), 2.00, 2.03, 2.10, 2.12 (4s, 6H, CH₃), 3.96–4.01 (dd, J = 12.0, 6.2 Hz)), 4.24–4.30 (m), 4.73–4.76 (m, 3H, H_6’,H_5’, 5.27, 5.26 (2bs, 2H, NH₂), 7.44, 7.32 (2bs, 1H, J = 1.6 Hz, H_1’), 8.18, 8.19 (2s, 1H, H₈). ¹³C NMR 6.2, 10.4, 16.5, 18.4, 20.9.1, 21.0, 21.2, 64.3, 65.2, 70.3, 72.9, 112.3, 112.6, 114.0, 125.25, 125.27, 138.8, 140.0, 151.8, 152.6, 152.7, 159.57, 159.61, 170.2, 170.6, 170.7, 170.9. ESI-MS 366, 368 (M + H, 26.0, 8.3), 388, 390 (M + Na, 100.0, 32.3).

2-Amino-6-chloro-9-{(Z)-(R)-2-[(S)-1,2-diacetoxyethyl)cyclopropylidene]methyl}-purine + 2-Amino-6-chloro-9-{(E)-(R)-2-[(S)-1,2-diacetoxyethyl)cyclopropylidene]-methyl}purine (ent-29b)

The reaction was performed as described above with dibromide ent-27. Reaction time 24 h at room temperature, 7 h at 100–105 °C, yield 340 mg (53%) of the E,Z-isomers ent-29b, mp 166–169 °C, [α]_D²⁷ −47.0° (c 1.01, CHCl₃).

(+)-9-{(Z)-(S)-2-[(S)-1,2-dihydroxyethyl)cyclopropylidene]methyl}adenine (12a) and (−)-9-{(E)-(S)-2-[(S)-1,2-dihydroxyethyl)cyclopropylidene]methyl}adenine (13a)

A mixture of the Z,E-isomers 28a (240 mg, 0.72 mmol) and K₂CO₃ (200 mg, 1.45 mmol) in methanol (15 mL) was stirred for 45 min at room temperature. The solvent was evaporated and the residue was chromatographed on a silica gel column using AcOEt - CH₂Cl₂ - MeOH (15 : 15 : 1 to 5 : 5 : 1) to give the Z-isomer 12a (64 mg, 36%) followed by E-isomer 13a (88 mg, 49%).

Z-Isomer 12a

Mp 271–272 °C, [α]_D²⁷ 54.6° (c 1.09, DMSO). UV λ_max 278 nm (ε 8,400), 258 (ε 11,900), 229 (ε 25,500). ¹H NMR δ 1.27 (poorly resolved t), 1.47 (t, 2H, J = 8.0 Hz, H_3’), 2.00 (poorly resolved dd, 1H, H_4’), 3.09–3.15 (m, 1H), 3.40–3.51 (m, 2H, H_5’, H_6’), 4.67 (t, 1H, J = 5.0 Hz, 6’-OH), 5.23 (d, J = 4.8 Hz, 5’-OH), 7.31 (bs, 2H, NH₂), 7.38 (s, 1H, H_1’), 8.15 (s, 1H, H₂), 8.96 (s, 1H, H₈). ¹³C NMR 6.7 (C_3’), 20.8 (C_4’), 66.5 (C_6’), 75.1 (C_5’), 110.8, 115.5, 119.0, 137.8, 148.7, 153.6, 156.7 (adenine, C_1’, C_2’). ESI-MS 248 (M + H, 95.9), 270 (M + Na, 100.0). Anal. (C₁₁H₁₃N₅O₂) C, H, N.

E-isomer 13a

Mp 245–247 °C, [α]_D²⁷ −3.6° (c 0.67, DMSO). UV λ_max 278 nm (ε 8,100), 262 (ε 11,100), 228 (ε 24,700). ¹H NMR δ 1.43–1.46 (m), 1.66 (t, 2H, J = 8.8 Hz, H_3’), 1.83–1.88 (m, 1H, H_4’), 3.17–3.22 (m, 1H), 3.42–3.46 (m, 2H, H_5’, H_6’), 4.66 (t, 1H, J = 4.8 Hz, 6’-OH), 4.83 (d, J = 4.0 Hz, 5’-OH), 7.33 (bs, 2H, NH₂), 7.49 (s, 1H, H₁’), 8.16 (s, 1H, H₂), 8.46 (s, 1H, H8). ¹³C NMR 8.7 (C_3’), 19.0 (C_4’), 66.3 C_6’), 73.5 (C_5’), 110.9, 116.0, 119.1, 137.8, 148.9, 153.7, 156.7 (adenine, C_2’, C_1’). ESI- MS 248 (M + H, 100.0), 270 (M + Na, 85.1). Anal. (C₁₁H₁₃N₅O₂) C, H, N.

(−)-9-{(Z)-(R)-2-[(R)-1,2-dihydroxyethyl)cyclopropylidene]methyl}adenine (ent-12a) and (+)-9-{(E)-(R)-2-[(R)-1,2-dihydroxyethyl)cyclopropylidene]methyl}adenine (ent-13a)

The reaction was performed as described above using the Z,E-isomers ent-28a (300 mg, 0.91 mmol) and K₂CO₃ (250 mg, 1.81 mmol) in methanol (18 mL) to give the Z-isomer ent-12a (94 mg, 42%) and E-isomer ent-13a (80 mg, 36%).

Z-Isomer ent-12a

Mp 273–274 °C, [α]_D²⁵ −56.6° (c 1.13, DMSO). Anal. (C₁₁H₁₃N₅O₂) C, H, N.

E-Isomer ent-13a

Mp 251–252 °C, [α]_D²⁶ 5.0° (c 0.71, DMSO). Anal. (C₁₁H₁₃N₅O₂) C, H, N.

(+)-9-{(Z)-(S)-2-[(R)-1,2-dihydroxyethyl)cyclopropylidene]methyl}adenine (14a) and (−)-9-{(E)-(S)-2-[(R)-1,2-dihydroxyethyl)cyclopropylidene]methyl}adenine (15a)

The reaction was performed as in the previous experiments using the Z,E-isomers 29a (260 mg, 0.7 mmol) to give the Z-isomer 14a (68 mg, 35%) and E-isomer 15a (93 mg, 48%).

Z-Isomer 14a

Mp 217–219 °C, [α]_D²⁷ 94.7° (c 0.46, DMSO). UV λ_max 278 nm (ε 8,700), 261 (ε 12,800), 224 (ε 26,900). ¹H NMR δ 1.35–1.43 (m, 2H, H₃’), 2.10–2.15 (m, 1H, H₄’), 3.22–3.32 (m, 2H, H_6’), 3.53–3.58 (m, 1H, H₅’), 4.66 (t, 1H, J = 5.6 Hz, 6’-OH), 4.91 (d, 1H, J = 4.8 Hz, 5’-OH), 7.32 (bs, 2H, NH₂), 7.36 (d, 1H, J = 1.6 Hz, H₁’), 8.16 (s, 1H, H₂), 8.46 (s, 1H, H₈). ¹³C NMR 5.8 (C_3’), 21.2 (C_4’), 66.0 (C_6’), 71.3 (C_5’), 110.3, 116.5, 119.0, 138.6, 148.9, 153.6, 156.7 (adenine, C_2’, C_1’). ESI-MS 248 (M + H, 100.0), 270 (M + Na, 11.2). Anal. (C₁₁H₁₃N₅O₂) C, H, N.

E-isomer 15a

Mp 233–235 °C, [α]_D²⁷ −5.8° (c 0.43, DMSO). UV λ_max 278 nm (ε 8,600), 262 (ε 11,700), 227 (ε 26,400). ¹H NMR δ 1.43–1.47 (m), 1.68 (dt, 2H, J = 8.8, 2.4 Hz, H_3’), 1.78–1.84 (m, 1H, H₄’), 3.04–3.09 (m, 1H), 3.42–3.45 (m, 2H, H_5’’ H_6’), 4.69 (t, 1H, J = 4.8 Hz, 6’-OH), 4.90 (d, 1H, J = 4.8 Hz, 5’-OH), 7.33 (s, 2H, NH₂), 7.42 (s, 1H, H₁’), 8.15 (s, 1H, H2), 8.47 (s, 1H, H₈). ¹³C NMR 9.6 (C_3’), 19.8 (C_4’), 66.4 (C_6’), 74.2 (C₅’), 111.3, 116.0, 119.1, 137.9, 148.9, 153.6, 156.7 (adenine, C₂’, C_1’). ESI-MS 248 (M + H, 100.0), 270 (M + Na, 11.9). Anal. (C₁₁H₁₃N₅O₂) C, H, N.

(−)-9-{(Z)-(S)-2-[(R)-1,2-dihydroxyethyl)cyclopropylidene]methyl}adenine (ent-14a) and (+)-9-{(E)-(S)-2-[(R)-1,2-dihydroxyethyl)cyclopropylidene]methyl}adenine (ent-15a)

The experiment using the Z,E-isomers ent-29a (255 mg, 0.77 mmol) was performed as described above to give the Z-isomer ent-14a (97 mg, 51%) and E-isomer ent-15a (65 mg, 35%).

Z-Isomer ent-14a

Mp 221–223 °C, [α]_D²⁶ −972° (c 0.48, DMSO). Anal. (C₁₁H₁₃N₅O₂) C, H, N.

E-Isomer ent-15a

Mp 230–231 °C, [α]_D²⁸ 7.2° (c 0.53, DMSO). Anal. (C₁₁H₁₃N₅O₂) C, H, N.

(+)-2-Amino-6-chloro-9-{(Z)-(S)-2-[(S)-1,2-dihydroxyethyl)cyclopropylidene]methyl{-purine (12c) and (+)-2-Amino-6-chloro-9-}(E)-(S)-2-[(S)-1,2-dihydroxyethyl)-cyclopropylidene]methyl}purine (13c)

The Z,E-isomers 28b (280 mg, 0.77 mmol) were dissolved in methanol (50 mL), NH₃ in MeOH (20%, 35 mL) was added and the mixture was stirred for 13 h at room temperature. The volatiles were evaporated and the residue was chromatographed on a silica gel column in AcOEt - MeOH (60 : 1 to 20 : 1) to give the Z-isomer 12c (90 mg, 41%) followed by E-isomer 13c (105 mg, 48%).

Z-Isomer 12c

Mp 215–217 °C, [α]_D²⁶ 80.9° (c 0.75, DMF). UV λ_max 311 nm (ε 8,100), 235 (ε 32,800). ¹H NMR δ 1.27 (poorly resolved m), 1.46 (t, 2H, J = 8.4 Hz, H₃’), 2.00 (poorly resolved m, 1H, H_4’), 3.11 (poorly resolved m, 1H), 3.80–3.47 (m, 2H, H_5’, H_6’), 4.70 (t, 1H, J = 4.8 Hz, 6’-OH), 5.14 (d, 1H, J = 4.8, Hz, 5’-OH), 7.01 (s, 2H, NH₂), 7.19 (s, 1H, H₁’), 8.91 (s, 1H, H₈). ¹³C NMR 6.8 (C_3’), 20.7 (C_4’), 66.4 (C_6’), 74.9 (C_5’), 110.3, 116.4, 123.7, 140.9, 150.2, 153.0, 160.7 (purine, C_2’, C_1’). ESI-MS 282, 284 (M + H, 100.0, 31.8), 304, 306 (M + Na, 82.7, 23.8).

E-Isomer 13c

Mp 258–260 °C, [α]_D²⁵ 23.5° (c 0.81, DMF). UV λ_max 311 nm (ε 8,100), 235 (ε 32,800). ¹H NMR δ 1.42–1.45 (m), 1.67 (t, 2H, J = 8.8 Hz, H_3’), 1.83–1.88 (m, 1H, H_4’), 3.19 (poorly resolved m, 1H), 3.43 (poorly resolved m, 2H, H_5’, H_6’), 4.64 (t, 1H, J = 6.0 Hz,6’-OH), 4.81 (d, J = 4.0 Hz, 5’-OH), 7.02 (s, 2H, NH₂), 7.33 (s, 1H, H₁’), 8.43 (s, 1H, H₈). ¹³C NMR 8.8 (C_3’), 19.0 (C_4’), 66.3 (C_6’),, 73.3 (C_5’), 110.5, 117.0, 123.7, 140.2, 150.3, 153.1, 160.7 (purine, C_2’, C_1’). ESI-MS 282, 284 (M + H, 86.5, 25.2), 304, 306 (M + Na, 100.0, 31.4).

(−)-2-Amino-6-chloro-9-{(Z)-(R)-2-[(R)-1,2-dihydroxyethyl)cyclopropylidene]methyl}-purine (ent-12c) and (−)-2-Amino-6-chloro-9-{(E)-(R)-2-[(R)-1,2-dihydroxyethyl)-cyclopropylidene]methyl}purine (ent-13c)

The above procedure starting from the Z,E-isomers ent-28b (340 mg, 0.93 mmol) afforded after chromatography in AcOEt – MeOH (30 : 1 to 20 : 1) the Z-isomer ent-12c (95 mg, 36%) followed by E-isomer ent-13c (130 mg, 50%).

Z-Isomer ent-12c

Mp 210–212 °C, [α]_D²⁸ −87.4° (c 0.85, DMF).

E-Isomer ent-13c

Mp 250–251 °C, [α]_D²⁸ −25.3° (c 0.67, DMF).

(+)-2-Amino-6-chloro-9-{(Z)-(S)-2-[(R)-1,2-dihydroxyethyl)cyclopropylidene]methyl}-purine (14c) and (+)-2-Amino-6-chloro-9-{(E)-(S)-2-[(R)-1,2-dihydroxyethyl)cyclo-propylidene]methyl}purine (15c)

The procedure described above was performed with the Z,E-isomers 29b (320 mg, 0.87 mmol) to give the faster moving Z-isomer 14c (85 mg, 34%) and slower moving E-isomer 15c (110 mg, 45%).

Z-Isomer 14c

Mp 205–207 °C, [α]_D²⁴ 41.3° (c 0.8, DMF). UV λ_max 311 nm (ε 7,900), 234 (ε 31,700). ¹H NMR δ 1.34–1.43 (m, 2H, H_3’), 2.10–2.15 (m, 1H, H_4’), 3.20–3.31 (m, 2H), 3.47–3.50 (m, overlapped with H2O, H_5’, H_6’), 4.63 (t, J = 5.2 Hz, 6’-OH), 4.86 (d, 1H, J = 4.0 Hz, 5’-OH), 7.00 (s, 2H, NH₂), 7.19 (d, 1H, J = 1.6 Hz, H₁’), 8.41 (s, 1H, H8). ¹³C NMR 6.0 (C_3’), 21.3 (C_4’), 66.0 (H_6’), 71.4 (H_5’), 110.9, 117.5, 123.7, 140.9, 150.2, 153.3, 160.7 (purine, C_2’, C_1’). ESI-MS (MeOH + AcOK) 282, 284 (M + H, 16.6, 5.0), 320, 322 (M + K,100.0, 41.4).

E-Isomer 15c

Mp 243–245 °C, [α]_D²⁵ = 51.8 o (c 1.1, DMF). UV λ_max 311 nm (ε 8,000), 234 (ε 32,200). ¹H NMR δ 1.42–1.45 (m), 1.69 (t, J = 8.8 Hz, 2H, H₃’), 1.77–1.82 (m, 1H, H_4’), 3.03 (m, 1H), 3.50 (m, partly overlapped with H₂O, 2H, H_5’, H_6’), 4.67 (poorly resolved t, 1H, 6’-OH), 4.89 (d, J = 4.8 Hz, 5’-OH), 7.02 (s, 2H, NH₂), 7.25 (s, 1H, H₁’), 8.43 (s, 1H, H8). ¹³C NMR 9.7 (C_3’), 20.0 (C_4’), 66.4 (C_6’), 74.2 (C_5’), 111.0, 116.9, 123.7, 140.2, 150.3, 153.1, 160.7 (purine, C_2’, C_1’). ESI-MS 304, 306 (M + Na⁺, 100.0, 32.5), 585, 587 (2M + Na, 57.4, 39.9).

(−)-2-Amino-6-chloro-9-{(Z)-(R)-2-[(S)-1,2-dihydroxyethyl)cyclopropylidene]methyl}-purine (ent-14c) and (−)-2-Amino-6-chloro-9-}(E)-(R)-2-[(S)-1,2-dihydroxyethyl)cyclo-propylidene]methyl}purine (ent-15c)

The experiment described above was performed with the Z,E-enantiomers ent-29b (300 mg, 0.82 mmol) to furnish the Z-isomer ent-14c (94 mg, 41%) and E-isomer ent-15c (106 mg, 46%).

Z-Isomer ent-14c

Mp 199–200 °C, [α]_D²⁷ −39.3° (c 0.75, DMF).

E-Isomer ent-15c

Mp 248–250 °C, [α]_D²⁷ −49.7° (c 1.0, DMF).

(+)-9-{(Z)-{(S)-2-[(S)-1,2-Dihydroxyethyl)cyclopropylidene]methyl}guanine (12b)

A solution of the Z-isomer 12c (85 mg, 0.3 mmol) in 80% formic acid (80%, 8 mL) was heated at 80 oC for 4 h. The volatiles were evaporated in vacuo, the crude product was dissolved in NH₃ in methanol (5%, 40 mL) and the mixture was stirred for 30 min at 0 °C. After removal of solvents, the solid was recrystallized from NH₄OH (28%) to give the guanine Z-isomer 12b (66 mg, 83%), mp >300 ⁰C, [α]_D²⁷ 98.6° (c 0.56, DMSO). UV λ_max 271 nm (ε 10,000), 231 (ε 28,100). ¹H NMR δ 1.22 (m), 1.42 (t, 2H, J = 8.0 Hz, H_3’), 1.94 (m, 1H, H_4’) 3.10 (m, 1H), 3.39–3.46 (m, 2H, partly overlapped with H₂O, H_5’, H_6’), 4.68 (bs, 1H, 6’- OH), 5.10 (bs, 1H, 5’-OH), 6.51 (s, 2H, NH₂), 7.10 (s, 1H, H₁’), 8.56 (s, 1H, H₈), 10.64 (bs, 1H, NH). ¹³C NMR 6.6 (C_3’), 20.7 (C_4’), 66.5 (C_6’), 75.0 (C_5’), 110.8, 115.1, 116.8, 135.4, 150.3, 154.5, 157.4 (guanine, C_2’, C_1’). ESI-MS 264 (M + H, 100.0), 286 (M + Na, 65.7). Anal. (C₁₁H₁₃N₅O₃x0.5 H₂O) C, H, N.

(−)-9-{(Z)-(R)-2-[(R)-1,2-Dihydroxyethyl)cyclopropylidene]methyl}guanine (ent-12b)

The procedure described above was performed with the Z-isomer ent-12c (90 mg, 0.32 mol) to give the title compound ent-12b (73 mg, 87%), mp >300°C, [α]_D²⁷ -101.7° (c 0.52, DMSO). Anal. (C₁₁H₁₃N₅O₃×0.45 H₂O) C, H, N.

(+)-9-{(E)-(S)-2-[(S)-1,2-Dihydroxyethyl)cyclopropylidene]methyl}guanine (13b)

The procedure described above was performed with the E-isomer 13c (90 mg, 0.32 mmol) to give the title compound 13b (71 mg, 84%), mp >300 ⁰C, [α]_D²⁷ 8.8° (c 0.8, DMSO). UV λ_max 271 nm (ε 10,900), 231 (ε 28,200). ¹H NMR δ 1.36–1.40 (m), 1.61 (dt, 2H, J = 8.8, 1.6 Hz, H_3’), 1.79–1.84 (m, 1H, H_4’) 3.17 (m, 1H), 3.40–3.45 (m, partly overlapped with H₂O, 2H, H_5’, H_6’), 4.62 (t, 1H, J = 4.8 Hz, 6’-OH), 4.78 (d, 1H, J = 4.8 Hz, 5’-OH), 6.53 (s, 2H, NH₂), 7.24 (s, 1H, H_1’), 8.02 (s, 1H, H₈), 10.67 (bs, 1H, NH). ¹³C NMR 8.5 (C_3’), 18.8 (C_4’), 66.3 (C_6’), 73.4 (C5_’), 110.8, 115.5, 116.9, 134.4, 150.5, 154.6, 157.4 (guanine, C_2’, C_1’). ESI-MS 264 (M + H, 81.4), 286 (M + Na, 100.0). Anal. (C₁₁H₁₃N₅O₃x0.5 H₂O.

(−)-9-{(E)-(R)-2-[(R)-1,2-Dihydroxyethyl)cyclopropylidene]methyl}guanine (ent-13b)

The procedure described above was performed with the E-isomer ent-13c (110 mg, 0.39 mol) to give the title compound ent-13b (86 mg, 83%), mp >300 °C, [α]_D²⁷ -10.8° (c 0.88, DMSO). Anal. Calcd for C₁₁H₁₃N₅O3×0.45 H₂O) C, H, N.

(+)-9-{(Z)-(S)-2-[(R)-1,2-Dihydroxyethyl)cyclopropylidene]methyl}guanine (14b)

The above protocol was performed with the Z-isomer 14c (80 mg, 0.28 mmol) to give the title compound 14b (64 mg, 86%), mp >300 °C, [α]_D²⁷ 62.4° (c 0.53, DMSO). UV λ_max 271 nm (ε 9,700), 230 (ε 22,800). ¹H NMR δ 1.30–1.38 (m, 2H, H_3’), 2.05–2.09 (m, 1H, H_4’), 3.25–3.31 (m, 2H, partially overlapped with H₂O), 3.51 (m, 1H, H_5’, H_6’), 4.64 (s, 1H, 6’-OH), 4.85 (s, 1H, 5’-OH), 6.53 (s, 2H, NH₂), 7.09 (d, 1H, J = 1.6 Hz, H_1’), 8.01 (s, 1H, H₈), 10.71 (bs, 1H, NH). ¹³C NMR 5.7 (C_3’), 20.9 (C_4’), 66.0 (C_6’), 71.4 (C5_’), 111.2, 116.0, 116.9, 135.1, 150.6, 154.6, 157.5 (guanine, C_2’, C_1’). ESI-MS 264 (M + H, 100.0), 286 (M + Na, 39.6). Anal. (C₁₁H₁₃N₅O₃x0.5 H₂O) C, H, N.

(−)-9-{(Z)-(R)-2-[(S)-1,2-Dihydroxyethyl)cyclopropylidene]methyl}guanine (ent-14b)

The procedure described above was performed with the E-isomer ent-13c (85 mg, 0.3 mol) to give the title compound ent-14b (68 mg, 86%), mp >300°C, [α]_D²⁷ −62.3° (c 0.55, DMSO). Anal. (C₁₁H₁₃N₅O₃×0.15 H₂O) C, H, N.

(+)-9-{(E)-(S)-2-[(R)-1,2-Dihydroxyethyl)cyclopropylidene]methyl}guanine (15b)

The procedure described above was performed with the E-isomer 15c (100 mg, 0.35 mmol) to give the title compound 15b (78 mg, 83 %), mp >300°C, [α]_D²⁷ 17.5° (c 0.31, DMSO). UV λ_max 270 nm (ε 9,400), 231 (ε 24,300). ¹H NMR δ 1.35–1.40 (m, 1H), 1.64 (poorly resolved dt, 1H, H_3’), 1.72–1.77 (m, 1H, H_4’), 2.97–3.03 (m, 1H), 3.37–3.46 (m, partly overlapped with H₂O, 2H, H_5’, H_6’), 4.63 (t, 1H, J = 5.2 Hz, 6’-OH), 4.85 (d, 1H, J = 4.0 Hz, 5’-OH), 6.54 (s, 2H, NH₂), 7.17 (s, 1H, H_1’), 8.04 (s, 1H, H₈), 10.64 (s, 1H, NH). ¹³C NMR 9.5 (C_3’), 19.8 (C_4’), 66.4 (C_6’), 74.3 (C5_’), 111.3, 115.5, 116.9, 134.4, 150.5, 154.6, 157.4 (guanine, C_2’, C_1’). ESI-MS 264 (M + H, 47.0), 286 (M + Na, 100.0). Anal. (C₁₁H₁₃N₅O₃×0.5 H₂O) C, H, N.

(−)-9-{(E)-(R)-2-[(S)-1,2-dihydroxyethyl)cyclopropylidene]methyl}guanine (ent-15b)

The procedure described above was performed with the E-isomer ent-15c (95 mg, 0.34 mol) to give the title compound ent-15b (75 mg, 84%), mp >300°C, [α]_D²⁷ -19.4° (c 0.41, DMSO). Anal. (C₁₁H₁₃N₅O₃×0.4 H₂O) C, H, N.

Benzyl (1S,2R,5S)-5-Bromo-3-oxabicyclo[3.1.0]-hexane-2-carboxylate (30)

Pyridinium tribromide (85 mg, 0.27 mmol ) was added with stirring to a solution of (1S,2S)-ester 20 (50 mg, 0.23 mmol) in CH₂Cl₂ (1 mL) at 0 °C. The stirring was continued for 1 h at room temperature to warm to room temperature. The mixture was then filtered through a silica gel pad (1 cm) which was washed with CH₂Cl₂ (10 mL). The solvent was evaporated and the crude dibromide 32 and K₂CO₃ (50 mg, 0.36 mmol) were refluxed in THF (1.5 mL) with stirring for 5 min. After cooling, the mixture was chromatographed on a silica gel column in hexanes - Et₂O (10 : 1) to furnish compound 30 (22 mg, 32%) as a colorless oil. [α]_D²⁵ −17.1° (c 0.70, CHCl₃). ¹H NMR (CDCl₃ δ 1.14 (t, J = 5.6 Hz), 1.38 (dd, 2H, J = 8.0 Hz, H₃), 2.10 (dd, 1H, J = 9.0, 5.0 Hz, H₂), 4.23, 4.10 (AB, 2H, J_AB = 7.8 Hz, H₅), 4.31 (s, 1H, H₁), 5.26, 5.20 (AB, 2H, J_AB = 12.0 Hz, CH₂Ph), 7.37 (m, 5H, Ph). ¹³C NMR 18.4 (C₆), 29.1 (C₁), 30.1 (C₅), 67.2 (C₄), 75.0 (CH₂Ph), 78.1 (C₂), 128.4, 128.7, 128.9, 135.5 (Ph), 171.5 (C=O). ESI-MS 319, 321 (M + Na, 100.0, 100.0), 615, 617, 619 (2M + Na, 38.0, 76.7, 40.0). Anal. (C₁₃H₁₃BrO₃) C, H.

Benzyl (1R,2R,5S)-5-Bromo-3-oxabicyclo[3.1.0]-hexane-2-carboxylate (31)

The above described protocol was followed with (1S,2R)-ester 21 (85 mg, 0.39 mmol) via dibromide 33 to give compound 31 (40 mg, 35%) as a colorless oil. [α]_D²⁶ 48.5° (c 0.86, CHCl₃). ¹H NMR (CDCl₃) δ 1.23 (t, 1H, J = 7.6 Hz), 1.34 (t, 1H, J = 5.8 Hz, H₆, H_6’), 2.19 (ddd, 1H, J = 8.0, 4.4, 1.6 Hz, H₁), 4.24, 3.98 (AB, 2H, J_AB = 8.6 Hz, H₄), 4.67 (d, 1H, J = 3.2 Hz, H₂), 5.20 (d, 2H, J = 1.6 Hz, CH₂Ph), 7.35 (m, 5H, Ph). ¹³C NMR 15.5 (C₆), 28.6 (C₁), 30.7 (C₅), 67.2 (C₄), 75.6 (CH₂Ph), 76.9 (C₂), 128.5, 128.7, 128.9, 135.5 (Ph), 169.4 (C=O). ESI-MS 319, 321 (M + Na, 100.0, 97.4), 615, 617, 619 (2M + Na, 23.7, 48.7, 25.7). Anal. (C₁₃H₁₃BrO₃) C, H.

(Z)-9-{(R)-2-[(R)-1,2-diacetoxyethyl)cyclopropylidene]methyl}adenine (34)

A mixture of the Z-isomer ent-12a (16 mg, 0.065 mmol) and Ac₂O (0.3 mL) in pyridine (1 mL) was stirred for 5 h at room temperature. Solvent was removed at room temperature and 0.04 torr. The crude product was chromatographed on a silica gel column in AcOEt - methanol (50 : 1 to 20 : 1) to give compound 34 (16 mg, 75%). Single crystals for X-ray diffraction were obtained by slow crystallization of a solution in AcOEt. Mp 185–186 °C, [α]_D²⁵ −152.6° (c 1.03, MeOH). UV λ_max 278 nm (ε 8,600), 261 (ε 12,500), 227 (ε 28,200). ¹H NMR (CDCl₃) δ 1.43–1.46 (m), 1.70 (dt, 2H, J = 8.8, 1.6 Hz, H_3’), 1.92, 2.06 (2s, 6H, CH₃), 2.25–2.31 (m, 1H, H_4’), 4.43, 4.17 and 4.42, 4.18 (2AB, 2H, J_AB = 11.6 Hz, H_6’), 4.67 (m, 1H, H_5’), 5.97 (bs, 2H, NH₂), 7.49 (d, 1H, J = 1.6 Hz, H_1’), 8.38 (s, 2H, H₂, H₈). ¹³C NMR 7.6 (C_3’), 17.6 (C_4’), 21.0 (CH₃), 64.6 (C_6’), 74.5 (C_5’), 112.1, 113.1, 119.5, 137.7, 149.1, 153.8, 155.8 (adenine, C_1’, C_2’), 170.0, 170.8 (C=O). ESI-MS: 332 (M + H, 68.9), 354 (M + Na, 100.0), 53.0), 685 (2M + Na, 55). Anal. (C₁₅H₁₇N₅O₄) C, H, N.

Antiviral Assays

Antiviral assays were performed as described previously.^29,30 HCMV assays were run in human foreskin fibroblast (HFF) cell culture with two strains of virus, Towne and AD169, in a plaque reduction or cytopathic effect (CPE) inhibition assay. Virus strains resistant to ganciclovir (10) and cyclopropavir (3b) were isolated from Towne strain by growth in synadenol (1a) (strain E8)²³ or in 3b (isolate 2696r).²⁴ The former strain has two point mutations introduced into gene UL97; the latter has a truncated UL97 gene. The MCMV was assayed in mouse embryonic fibroblast (MEF) cells by plaque reduction. The EBV DNA hybridization assay was run in Akata cells.^30,31

Adenosine Deaminase (ADA) Assay.²⁹

Adenine analogues 12a - 15a and ent-12a - ent-15a (2 µmol) and ADA from calf intestine (1.7 units) were magnetically stirred in 0.05 M Na₂HPO₄ (pH 7.5) in a total volume of 0.6 mL. Aliquots were removed, diluted with buffer, UV spectra were recorded and a decrease of absorbance at 260 nm was followed. The results are summarized in Table 7.

Abbreviations

CMV

cytomegalovirus

HCMV

human CMV

MCMV

murine CMV

UL97

gene for HCMV phosphotransferase

pUL97

phosphotransferase enzyme

EBV

Epstein-Barr virus EBV

HSV-1

herpes simplex virus 1

HSV 2

herpes simplex virus 2

HHV 6

human herpes virus 6

HHV-8

human herpes virus 8

VZV

varicella zoster virus

HFF

human foreskin fibroblasts

MEF

mouse embryonic fibroblasts

CPE

cytopathic effect

ADA

adenosine deaminase

NOE

nuclear Overhauser effect.