Regioselective synthesis of acyclovir and its various prodrugs

Article abstract of DOI:10.1081/SCC-100104050

High-yield regioselective synthesis of 9-[(2-hydroxyethoxy)methyl]guanine (Acyclovir 1, Scheme 1) was achieved from guanine via trisilylated guanine. N²-acylacyclovir 9a-9b were prepared from N², O-diacylacyclovir (4, 8b-8d) using regioselective deacylation procedure. N²-Acylacyclovir 11 and 13 were prepared via protection of primary hydroxyl groups. Three amino acid esters of acyclovir were synthesized as water-soluble prodrugs, which form protonated cations in pH 7.4 phosphate buffer. Two water-soluble ester prodrugs with free carboxylic acids, which form anionic species in pH 7.4 phosphate buffer, were also synthesized.

Full text of DOI:10.1081/SCC-100104050

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REGIOSELECTIVE SYNTHESIS
OF ACYCLOVIR AND ITS
VARIOUS PRODRUGS
Hongwu Gao a & Ashim K. Mitra b
a Division of Pharmaceutical Science, School of
Pharmacy, University of Missouri-Kansas City,
5005 Rockhill Road, Kansas City, MO, 64100-2499,
U.S.A.
b Division of Pharmaceutical Science, School of
Pharmacy, University of Missouri-Kansas City,
5005 Rockhill Road, Kansas City, MO, 64100-2499,
U.S.A.
Version of record first published: 09 Nov 2006.
To cite this article: Hongwu Gao & Ashim K. Mitra (2001): REGIOSELECTIVE
SYNTHESIS OF ACYCLOVIR AND ITS VARIOUS PRODRUGS, Synthetic Communications:
An International Journal for Rapid Communication of Synthetic Organic Chemistry,
31:9, 1399-1419
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SYNTHETIC COMMUNICATIONS, 31(9), 1399–1419 (2001)
REGIOSELECTIVE SYNTHESIS OF
ACYCLOVIR AND ITS VARIOUS
PRODRUGS
Hongwu Gao and Ashim K. Mitra*
Division of Pharmaceutical Science, School of Pharmacy,
University of Missouri-Kansas City, 5005 Rockhill Road,
Kansas City, MO64100-2499
ABSTRACT
High-yield regioselective synthesis of 9-[(2-hydroxy-
ethoxy)methyl]guanine (Acyclovir 1, Scheme 1) was achieved
from guanine via trisilylated guanine. N2-acylacyclovir 9a–9b
were prepared from N2, O-diacylacyclovir (4, 8b–8d) using
regioselective deacylation procedure. N2- Acylacyclovir 11
and 13 were prepared via protection of primary hydroxyl
groups. Three amino acid esters of acyclovir were synthesized
as water-soluble prodrugs, which form protonated cations in
pH 7.4 phosphate buffer. Two water-soluble ester prodrugs
with free carboxylic acids, which form anionic species in pH
7.4 phosphate buffer, were also synthesized.
INTRODUCTION
9-[(2-Hydroxyethoxy)methyl]guanine (Acyclovir 1, Scheme 1) is a
widely used therapeutic agent for the treatment of herpes virus infection.1
The oral and intravenous delivery of acyclovir is far from optimal.
* Corresponding author.
1399
Copyright & 2001 by Marcel Dekker, Inc.
www.dekker.com

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GAO AND MITRA
Previously, a number of acyclovir prodrugs were developed with the aim of
improving the in delivery characteristics. Krenitsky et al. developed 6-deox-
yacyclovir (desciclovir) which was shown to be readily absorbed (about
75%) after oral administration.2,3 Colla et al. have prepared various a-
amino acid esters that are highly water-soluble and potentially useful in
the preparation of eyedrop formulations4,5 Beauchamp et al. synthesized
18 amino acid esters of acyclovir as potential prodrugs for oral administra-
tion.6 Bundgaard et al. evaluated a series of N-substituted 3- or 4-(amino-
methyl)benzoate esters of acyclovir as solution-stable, biolabile prodrug
from.7 Previously, we reported the synthesis, nasal and ocular absorption,
and metabolism of acyclovir acetate, propitate, butyrate, pivalate, valerate,
and hexanoate prodrugs.8,9 In this study, acyclovir was regioselectively
synthesized from guanine via trisilylated guanine. Several series of N2-acyl-
acyclovir, a-, b-, g-amino acid esters and dicarcoxylic acid esters of acyco-
lovir were synthesized to investigate their effectiveness in enhancing
membrane transport. The intermediates as well as the final product struc-
tures were confirmed by their NMR chemical shifts, MS and HPLC. NMR
spectral data of several series of novel acyclovir prodrugs are reported in this
article. The NMR assignments of the carbon signals are based on spectral
comparison with compounds of similar structures.
Scheme 1. Reagents and conditions: i. acetic anhydride, HOAc, DMAP, 160ꢀC, 8 h,
3: 88%; ii. P-TsOH, 1,3-dioxolane, Ac2O, HOAc; then, toluene, relfux, 1 h, 4: 18%;
5: 2.3%; 4 and 5: 52%; iii. 40% aqueous CH3NH2, 1: 93%.

ACYCLOVIR
1401
RESULTS AND DISCUSSION
Syntheses
Syntheis of Acyclovir
The previous synthetic methods of acyclovir encompass routes that
proceed through alkylation of 2,6-dichloropurine,1 2-chloro-6-iodopurine,10
2-amino-6-chloropurine,11 or guanine.12–14 The 6-amino or 6-chloro moiety
of 2-amino-6-substituted purines was hydrolyzed with adenosine deaminase
to yield acyclovir.11 Acyclovir was also prepared from 2-amino-9-[(2-
hydroxyethoxy)methyl]-9H-purine (6-deoxyacyclovir) by oxidation with
xanthine oxidase.2 A five-step synthesis of acyclovir from 7-formamido-
5-methylthiofurazano [3,4-d]pyrimidine was reported by Kelley and
Schaffer.15 9-Regioselective alkylations were accomplished by condensation
of trimethylsilyl derivative of 2-bromo-6-(4-nitrophenylethoxy)purine16 or
2-N-acetyl-6-O-diphenylcarbamoylguanine17 with (2-acetoxyethoxy)methyl
bromide. A new synthesis of 9-substituted guanines from 1-alkyl-5-[(thio-
carbamoyl)-amino]imidazole-4-carboxamides by ring closure under alkaline
conditions was developed by Alhede et al.18
In this article, we describe a modified Matsumoto’s method13 for
the synthesis of acyclovir from guanine (Scheme 1). N2,9-Diacetyl guanine
was first prepared according to the procedure of Robins and Hatfield.19
1H NMR spectra of 3 did not show N2,7-isomer. 2-Oxa-1,4-butanediol
diacetate was prepared from acetic anhydride and 1,3-dioxolane with
p-toluenesulfonic acid as the catalyst. Without further purification, the
product was refluxed with diacetylguanine 3 in toluene for one hour.
Chromatographic separations resulted in N2-acetyl-9-(2-acetoxyethoxy-
methyl)guanine (4) and N2-acetyl-7-(2-acetoxyethoxymethyl)guanine (5) in
18% and 2.3% yields, respectively. The major fraction was the mixture of
regioisomers 4 and 5. Treatment of 4 with 40% aqueous methyl amine
gave rise to 1 in 93% yield, which indicates that overall yield of 1 from
guanine is 15%.
Our next attempt to synthesize acycolovir by Zou’s procedure17 was
unsuccessful because the diphenylcarbamoyl proctective group used was
unstable during the reaction. Finally, acyclovir was successfully synthesized
by a process reported by Scholemer et al.19 (Scheme 2). Guanine (2) was first
protected as trisilyl derivative by reacting with a silylating agent,
hexamethyldisilazane (HMDS), at reflux temperature in the presence of
trifluoromethansulfonic acid as a catalyst. The protected compound 6 was
treated with 1,3-dioxolane to generate 9-(2-trimethylsilylethoxymethly)gua-
nine (7) in 93% yield. NMR spectra of 7 did not show the presence of
7-isomer. Subsequent hydrolysis of trimethylsilyl group was accomplished

1402
GAO AND MITRA
Scheme 2. Reagents and conditions: i. HMDS, CF3SO3H, 135ꢀ–140ꢀC, 32 h, 6; ii.
ꢀ
Acetone, H2O, rt, 0.5 h, 7: 93%; iii. 2% HOAc/H2O , 80C, 2 h, 1: 85%.
by heating compound 7 in 2% aqueous acetic acid at 80ꢀC for 2h. The
overall yield was 79%.
Synthesis of N2-Amide Prodrugs of Acyclovir
Synthesis of N2, O-diacylacyclovir was successfully carried out with
acid anhydride in refluxing pyridine in the presence of 4-dimethylamino-
pyridine (DMAP) as a catalyst (Scheme 3). The use of a strong base (8M
solution of sodium methoxide in methanol) for selective removal of the
O-acetyl in the presence of the N-acetyl in 4 was reported by Stimac and
Kobe in 1991.20 We successfully repeated the procedure to synthesize com-
pound 4 and applied the same protocol to prepare compounds 9b–9d. To
avoid potential difficulty in selective hydrolysis of O-acyl groups, the
N2-acyl derivatives of acyclovir were also synthesized by selective protection
of primary hydroxyl group of acyclovir. The first method employed direct
silylation of acyclovir in DMF with tert-butyldimethylsilyl chloride in the
presence of imidazole (Scheme 4). However, the protection group was found
to be unstable during refluxing with valeric anhydride in pyridine. The N2,
O-divalerylacyclovir (10c) was separated in 11% yield. Treatment of result-
ing product 10b with tetrabutylammonium fluoride (TBAF) in DMF gave
the N2-valerylacyclovir (11) in 73% yield. The second method was also
accomplished by directed condensation of acyclovir (1) with levulinic acid
Scheme 3. Reagents and conditions: i. acid anhydride, DMAP, pyridine, reflux, 1 h,
4: 75%, 8b: 60%, 8c, 70%, 8d, 80%; ii. 8M NaOMe, MeOH, rt, 16 min, 9a: 67%, 9b,
90%, 9c, 80%, 9d, 87%.

ACYCLOVIR
1403
using the coupling agent dicyclohexylcarbodiimide (DCC) and DMAP as a
catalyst (Scheme 4). The amino functional group in 12a, following reaction
with trimethyl acetic anhydride to yield the desired ester prodrug 12b, and
the levulinyl group at the 40-position, were removed with hydrazine mono-
hydrate to give 13 in 92% yield.
Scheme 4. Reagents and conditions: i. TBDMSCI, imidazole, DMF, rt, 24 h, 10a:
79%; ii. Valeric anhydride, DMAP, pyridine, reflux, 1 h, 10b: 55%, 10c: 11%; iii.
TBAF, THF, DMF, 1 h, 11: 73%; iv. Levulinic acid, DCC, DMAP, DMF, rt, 4 h
12a: 71%; v. trimethylacetic anhydride, DMAP, pyridine, reflux, 1.5 h, 12b: 73%; vi.
NH2NH2H2O, HOAc/pyridine, rt, 10 min, 13: 92%.
Synthesis of water-soluble prodrugs of acyclovir
The amino acid esters (16a-16b) were prepared in two steps according
to the method of Beauchamp et al. (Scheme 5). A solution of acyclovir in
dimethylformamide (DMF) was treated with the N-carbobenzyloxy-
protected amino acid using the coupling agent DCC and DMAP as a cat-
alyst. The resulting N-protected ester derivatives (15a,15b) were purified by
column chromatography on silica gel and subsequently subjected to cataly-
tic hydrogenation in the presence of HCl to generate the target esters as the
hydrochloride salts. Because of intramolecular catalysis by the neighboring
amino group, these two a-amion acid esters were highly unstable in aqueous
solution (pH 3–6). To avoid this problem, the b- and g- amino acid esters
(15c, 15d) were also synthesized by the same method. However, subsequent
catalytic hydrogenation of compound 15c or 15d gave a mixture of several
by-products. Replacement of methyl ester with benzyl protection group
resulted in two more b- or g-amino acid esters (15e, 15f). However, the

1404
GAO AND MITRA
following deprotection product of 15e, 16e, was found to be sparingly water-
soluble. The hydrolysis of 15f was not performed. For the synthesis of
acyclovir mono-esters of di-carboxylic acids, mono-benzyl protected
carboxylic acids were first coupled with acyclovir using the coupling agent
DCC and DMAP as a catalyst, and subsequently subjected to catalytic
hydrogenation to produce the target esters with free carboxylic acid end
groups. The reagent mono-benzyl glutarate (15h) was not commercially
available and prepared from glutaric anhydride and benzyl alcohol
(Scheme 5).
Scheme 5. Reagents and conditions: i. acid 14a–14h, DCC, DMAP, DMF, rt, 24 h,
then recharge acid, DMAP, DCC, rt 24 h, 15a: 86%, 15b: 58%, 15e: 63%, 15f: 55%,
15g: 67%, 15h: 37%, ii. H2, 10% Pd/C, ethanol/water 16a: 97%, 16b: 100%, 16e:
61%, 16g: 95%, 16h, 72%.
NMR Spectroscopy
1H NMR spectral assignments of compounds 4, 8b–8d, 10b, 12b;
9a–9d, 11, 13; and 15a–15h are listed in Tables 1a, 1b, and 1c, respectively.
13C NMR spectral assignments of compounds 4, 8b–8d, 10b, 12b; 9a–9d, 11,
13; and 15a–15h are listed in Tables 2a, 2b, 2c, respectively. Table 3 shows
the substituent effects of various carbonyl groups on the parent compound 1
(acyclovir), calculated from the shielding data listed in Tables 1a–c and
2a–c. Their structures are illustrated in Schemes 3–5. All NMR assignments
are based on comparison with compounds of similar structure.
As shown in Table 1c, conversion of OH to OR (aliphatic esters) leads
1
to deshielded H NMR chemical shifts of 0.64–0.70 ppm for the protons of
carbon (CH2OCO), to which these groups are attached. The immediately
adjacent protons (CH2O) are also deshielded by 0.18–0.23 ppm. In compar-
ison to acyclovir, other protons produced no change in chemical shift.

ACYCLOVIR
1405

1406
GAO AND MITRA

ACYCLOVIR
1407

1408
GAO AND MITRA
Table 2a. 13C NMR Data of Acyclovir and Its Diacyl Derivatives
Carbon
1
4
8b
8c
8d
10b
12b
2
4
5
6
153.80
151.40
116.37
156.80
137.80
148.82
148.09
120.14
154.84
140.05
148.94
148.28
120.21
154.99
140.21
148.86
148.16
120.21
154.86
140.52
148.98
148.47
120.41
155.03
140.40
148.86
148.20
120.33
154.92
140.21
148.86
148.71
120.25
154.96
140.13
8
NCH2O72.00
CH2OCO
CH2O70.38
RCOO
72.39
59.89
66.65
72.51
62.65
66.80
72.39
62.69
66.65
8.70
26.71
72.59
62.46
66.80
13.28
17.86
35.17
13.35
17.86
37.77
72.78
62.65
70.37
18.71
33.23
72.39
61.87
66.53
62.81
20.50
À5.34
27.44
29.50
37.26
26.24
40.06
25.66
RCONH
23.72
8.90
26.98
18.94
34.90
13.66
21.66
26.67
33.46
RCOO
170.21
173.55
173.63
177.16
172.62
176.30
176.07
180.42
172.23
206.74
181.43
RCONH
176.46
Table 2b. 13C NMR Data of Acylcovir and Its Amide Derivatives
Carbon
1
9a
9b
9c
9d
11
13
2
4
5
6
153.80
151.40
116.37
156.80
137.80
148.78
148.01
120.06
154.84
140.21
148.90
148.16
120.09
154.95
139.97
148.86
148.12
120.10
154.92
140.13
148.82
148.28
120.10
154.92
140.01
148.86
148.24
119.98
154.92
140.36
148.82
148.63
120.18
154.99
140.17
8
NCH2O72.00
CH2OH
CH2O70.38
RCONH
72.55
59.89
70.45
72.62
59.82
70.52
72.63
59.93
70.53
8.73
29.34
72.63
59.93
70.49
13.39
17.89
37.77
72.51
60.01
70.41
72.59
59.78
70.45
59.93
23.68
18.83
34.74
13.59
21.50
26.47
35.63
176.38
26.24
40.02
RCONH
173.51
177.00
176.27
175.94
181.39
As summarized in Table 1a, derivatization of OH to OR and H2N- to
RHN- led to the same chemical shift change on 9-side chain. Additionally,
both H-1 and H-8 deshielded by 0.68–1.28 ppm and 0.33–0.38 ppm,
respectively. The 2-amino group diappeared from the spectra with the loss

ACYCLOVIR
1409
15h
Table 2c. 13C NMR Data of 15a–15h
15b 15c 15d 15e 15f
Carbon
15a
15g
2
4
5
6
8
153.81 153.91 153.87 153.91 153.91 153.91 153.66 153.87
148.69 151.42 151.38 151.42 151.38 151.42 151.34 151.38
106.62 116.45 116.37 116.41 116.41 116.41 116.45 116.41
156.42 156.12 155.77 156.08 155.88 156.16 156.69 156.82
137.65 137.22 137.72 137.68 137.76 137.76 137.50 137.68
NCH2O71.76
CH2OCO
CH2O66.30
CH2
71.81
63.32
66.45
71.77
63.27
66.30
22.67
28.84
30.20
39.90
–
71.77
63.19
66.42
71.77
62.81
66.30
25.81
29.66
71.77
63.27
66.42
71.75
62.84
66.22
25.77
29.66
71.77
63.70
66.45
62.65
42.01
35.63
35.60
40.88
19.76
32.30
32.37
COOMe
CH
CH2Ar
–
50.23
52.17
65.60
51.90
52.91
65.52
–
–
–
–
–
65.51
53.84
65.13
65.62
50.42
65.64
53.06
65.56
66.03
–
65.39
–
65.41
CH2Ar
127.62 127.09 127.67 127.67 127.71 127.75 127.85 127.86
127.72 127.71 128.29 128.29 128.33 128.33 128.02 128.37
128.28 128.33 136.67 136.79 135.66 135.82 128.33
136.83
136.67 136.79
COO
170.06 172.46 169.78 172.00 169.82 171.80 166.30 172.27
171.26 172.38 170.68 172.03
CONH
170.06 156.12 156.82 156.82 156.82 156.86
156.90
–
–
a 6.52 ppm signal and amide proton surfaced between 11.96 ppm and
12.32 ppm.
As shown in Table 1b, hydrolysis of OR to OH results in the loss of the
chemical shift change in 9-side chain. However, due to the presence of
N2-acyl groups, both H-1 and H-8 are still deshielded by 0.65–1.41 ppm
and 0.29–0.36 ppm, respectively.
As shown in Table 2c, transformation of OH to OR leads to deshield-
ing 13C NMR chemical shifts of 2.76–3.81 ppm for the carbons (CH2OR)
directly attached by the substituent (40-position), but the adjacent carbons
(30-position) are shielded by 3.93–4.08 ppm. The rest of the carbons do not
exhibit any chemical shifts.
As depicted in Table 2a, similar transformation of OH to OR and
H2N- to RHN- lead to the same chemical shift change on 9-side chain.
The C-2, C-4, and C-6 in heterocyclic ring positions are shielded by
4.82–4.98 ppm, 2.69–3.31 ppm, and 1.77–1.96 ppm, respectively. On the

1410
GAO AND MITRA

ACYCLOVIR
1411
Figure 1.
other hand, both C-5 and C-8 are deshielded by 3.77–3.96 ppm and
2.25–2.72 ppm, respectively. These deshielding effects can be explained by
resonance structures as shown in Fig 1. The 2-amide substitutions reduce the
shielding at C-5 and C-8. The ester and amide moieties generated two sets of
data. The chemical shifts decreased as the carbon positions are progressively
removed from the carbonyl groups.
As shown in Table 2b, hydrolysis of OR to OH causes the chemical
shift change in 9-side chain to disappear. However, with N2-acyl groups, C-
2, C-4, and C-6 in heterocyclic ring postions are shielded by 4.90–5.02 ppm,
2.77–3.39 ppm, and 1.81–1.96 ppm, respectively. On the other hand, C-5
and C-8 are deshielded by 3.61–3.81 ppm and 2.17–2.56 ppm, respectively.
CONCLUSION
High-yield regioselective synthesis of acyclovir was accomplished in
two steps via 9-(2-trimethylsilylethoxymethyl)guanine (7). N2-Acylacyclovir
prodrugs were prepared using regioselective deacylation procedure or pro-
tection of primary hydroxyl group. Several water-soluble ester derivatives
were synthesized to investigate their effectiveness in enhancing membrane
transport.
EXPERIMENTAL
Column chromatography was carried out using Grade 62 (60–200
mesh) silica gel, and elution was performed by methanol-methylene
chloride (1:9) solvent system. Melting points were determined on a
Fisher-Johns melting point apparatus and uncorrected. 1H NMR and

1412
GAO AND MITRA
13C NMR spectra were measured at 250 MHz or 63 MHz (Bruker) in
Me2SO-d6 as solvent with TMS as the internal standard. ESI mass spectra
were obtained on a Finnigan TSQ-700 triple-quadrupole mass spectro-
meter equipped with an electrospray source. A high-performance liquied
chromatographic (HPLC) method was used to analyze the samples. The
system comprises a model 515 solvent delivery module (WatersTM of
Millipore Corp., Milford, MA), Rheodyne injector, Waters 486 UV-VIS
Tunable absorbance detector measured at 254 nm (4, 8b–8d, 9a–9d,
11, and 13), or fluorescence detector (Hewlett-Packard Co., Wilmington,
DE) set at an excitation wavelength of 285 nm and emission wavelength
of 370 nm (16a,16b and 16g). Double distilled and deionized water
was used in making the solutions of the compounds. For compounds
4, 8b–8d, 9a–9d, 11, and 13, the mobile phase was composed of
0.005 M KH2PO4 (pH 2.8) and methanol. The proportion of methanol
varied between 30% and 68% according to the different compound.
For compounds 16a–16b, and 16g, the mobile phase was composed of
0.0025 M NH4H2PO4 (pH 2.0) and 1:1 methanol:acetonitrile. The propor-
tion of the mixture of methanol and acetonitrile varied between 1% and
10% according to the different compounds. Data acquistion and
chromatographic analysis were carried out using a HP 3395 integrator
(Hewlett-Packard Co., Wilmington, DE).
N2,9-Diacetyl-guanine (3)
3 was prepared by a published method.17 M.p. 288ꢀ–290ꢀC [Aldrich:
37, 789–9, 285ꢀC], yield: 88%. H NMR: 2.22 (s, 3H, CH3CONH); 2.83 (s,
1
3H, CH3CON); 8.44 (s, 1H, H-8); 11.88 (bs, 1H, NH); 12.21 (s, 1H,
CH3CONH). 13C NMR: 23.79 (CH3CONH); 24.61 (CH3CON); 121.42
(C-5); 137.41 (C-8); 148.32 (C-4); 167.96 (CH3CON); 173.66 (CH3CONH).
N2-Acetyl-9-(2-acetoxyethoxymethyl)guanine (4) and
N2-Acetyl-7-(2-acetoxyethoxy methyl)guanine (5)
p-TsOH (1.12 g, 5.9 mmol, 0.17 equiv) was added to an ice-cold mix-
ture of acetic acid (2.56 mL, 44.7 mmol) and acetic anhydride (15.3 mL,
162 mmol, 4.76 equiv). 1,3-Dioxolane (12.2 mL, 174 mmol, 5.12 equiv) was
added dropwise to this mixture with cooling in an ice bath. After addition,
the reaction mixture was allowed to warm to room temperature, and then
diacetylguanine 3 (8 g, 34 mmol) in toluene (42 mL) was added. The reaction
mixture was refluxed for 1 h, then concentrated to dryness. The resulting oil

ACYCLOVIR
1413
was chromatographed using CH2Cl2-CH3OH (9:1) as an eluting system.
From the early fractions, 5 (0.24 g, 2.3%) was obtained. From the middle
fractions, a mixture of 4 and 5 (5.5 g, 52%) was obtained. From the later
fraction, 4 (1.84 g, 18%) was obtained. Compound 4: m.p. 190ꢀ–192ꢀC
[ref.13 189ꢀ–190ꢀC]. H NMR: Table 1a. 13C NMR: Table 2a. MS(ESI):
1
619[2Mþ1]þ (24), 310[Mþ1]þ (100). Compound 5: m.p. 173ꢀ–175ꢀC [ref.
(13) 184ꢀ–185ꢀC]. 1H NMR: 1.99–2.21 (m, 6H, CH3COO, CH3CONH); 3.80
(bs, 2H, H-30); 4.13 (bs, 2H, H-40); 5.75 (s, 2H, H-10); 8.39 (s, 1H, H-8); 11.67
(s, 1H, NH); 12.20 (s, 1H, CH3CONH). 13C NMR: 20.57 (CH3COO); 23.76
(CH3CONH); 62.88 (C-30); 66.57 (C-40); 75.11 (C-10); 111.29 (C-5); 145.13
(C-8); 147.46 (C-4); 152.63 (C-2); 157.56 (C-6); 170.40 (CH3COO); 173.59
(CH3CONH). MS(ESI): 619 [2Mþ1]þ (4), 310 [Mþ1]þ (100).
9-[(2-Hydroxyethoxy)methyl]guanine (Acyclovir 1)
A mixture of 4 (1.6 g, 5.2 mmol) and 40% methyl amine aqueous sol-
ution (14 mL) was stirred at 23ꢀC for 1 h. The solvent was then removed in
vacuo and the residue was triturated with 15 mL ethanol. The product was
collected by filtration and acyclovir (1.08 g, 93%) was obtained after recrys-
tallization from water. M.p. 250ꢀ–252ꢀC [ref.11 265ꢀ–266ꢀC]. 1H NMR:
Table 1a. 13C NMR: Table 2a.
Regioselective Synthesis of Acyclovir (1)
To a suspension of guanine 2 (100 g, 0.66 mol) in hexamethyldisilazane
(HMDS, 600 mL, 2.84 mol) was added trifluoromethanesulfonic acid (triflic
acid, 4 mL, 0.045 mol, 0.07 equiv) and the mixture was heated to 160ꢀC for
32 h. The reaction medium was cooled to 70ꢀC, 1,3-dioxolane (100 mL,
1.43 mol, 2.17 equiv) was added, and the resulting mixture refluxed for
12 h. Excess HMDS and 1,3-dioxolane were removed in vacuo, and the
resulting mixture was poured into a mixture of acetone (800 mL) and
water (80 mL). The resulting suspension was stirred at room temperature
for 0.5 h, filtered, and the solid material was washed with cold acetone to
give 9-(2-trimethylethoxymethyl)guanine 7 (183 g, 93%). M.p. 200ꢀ–202ꢀC
1H NMR: À0.03 (s, 9H, Si(CH3)3); 3.52 (m, 2H, H-30); 3.58 (m, 2H, H-40);
5.35 (s, 2H, H-10); 6.57 (bs, 2H, NH2); 7.82 (s, 1H, H-8); 10.79 (bs, 1H, NH).
13C NMR: À5.05 (Si(CH3)3); 61.06 (C-40); 69.99 (C-30); 72.04 (C-10); 116.37
(C-5); 137.88 (C-8); 151.38 (C-4); 153.87 (C-2); 156.94 (C-6). MS(ESI):
MS(ESI): 298[Mþ1]þ (100).

1414
GAO AND MITRA
A mixture of 7 (90 g, 0.3 mol), water (1500 mL), and acetic acid
(30 mL, 0.52 mol, 1.75 equiv) was heated to 80ꢀC for 2 h. The resulting
solution was cooled and kept overnight at 5ꢀC. The crystalline solid was
filtered to yield acyclovir 1 (58 g, 85%). M.p. 258ꢀ–260ꢀC ) [ref.11
265ꢀ–266ꢀC].
General Procedure for Synthesis of Diacyl Prodrugs (4, 8b–d)
To a suspension of acyclovir 1 (5 g, 22.2 mmol) in anhydrous pyridine
(50 mL), was added acid anhydride (133 mmol, 6 equiv) and DMAP
(540 mg, 4.42 mmol, 0.2 equiv). The resulting mixture was refluxed for 1 h,
cooled to 23ꢀC, and the reaction was quenched by addition of water. The
solvent was evaporated in vacuo and the resulting oil was chromatographed
to afford 4 and 8b–8d.
4: 75%. M.p. 198ꢀ–200ꢀC [ref.13 189ꢀ–190ꢀC]. 1H NMR: Table 1a. 13
C
NMR: Table 2a. MS(ESI): 619 [2Mþ1]þ (24), 310 [Mþ1]þ (100). The purity
by the HPLC method was 99.9%.
8b: 60%. M.p. 110ꢀ–112ꢀC. 1H NMR: Table 1a. 13C NMR: Table 2a.
MS(ESI): 675 [2Mþ1]þ (18), 338 [Mþ1]þ (100). The purity by the HPLC
method was 99.6%.
8c: 70%. M.p. 158ꢀ–160ꢀC. H NMR: Table 1a. 13C NMR: Table 2a.
1
MS(ESI): 731 [2Mþ1]þ (28), 366 [Mþ1]þ (50). The purity by the HPLC
method was 98.7%.
8d: 80%. M.p. 160ꢀ–162ꢀC. 1H NMR: Table 1a. 13C NMR: Table 2a.
MS(ESI): 731 [2Mþ1]þ (17), 366[Mþ1]þ (100). The purity by the HPLC
method was 98.9%.
General Procedure for the Synthesis of Amide Prodrugs (9a–d)
Compound 4 (3.09 g, 10 mmol) was added to a freshly prepared sol-
ution of sodium (0.46 g, 20 mmol, 2 equiv) in MeOH (25 mL). The solution
was stirred at 23ꢀC for 16 min, then the reaction was quenched by the
addition of 1.4 M aq HCl (15 mL, 21 mmol, 2 equiv). The mixture was
stirred continuously for 10 min at 23ꢀC and the solvent was evaporated in
vacuo. The resulting oil was chromatographed to afford 9a–9d.
9a: 67%. M.p. 220ꢀ–222ꢀC [ref.20 214ꢀ–215ꢀC]. H NMR: Table 1b.
1
13C NMR: Table 2b. MS(ESI): 535 [2Mþ1]þ (17), 268 [Mþ1]þ (18). The
purity by the HPLC method was >99.0%.

ACYCLOVIR
1415
9b: 90%. M.p. 133ꢀ–135ꢀC. 1H NMR: Table 1b. 13C NMR: Table 2b.
MS(ESI): 563 [2Mþ1]þ (4), 282 [Mþ1]þ (100). The purity by the HPLC
method was >99.0%.
9c: 80%. M.p. 108ꢀ–110ꢀC. 1H NMR: Table 1b. 13C NMR: Table 2b.
MS(ESI): 591 [2Mþ1]þ,32 296 [Mþ1]þ.32 The purity by the HPLC method
was >99.0%.
9d: 87%. M.p. 188ꢀ–190ꢀC. 1H NMR: Table 1b. 13C NMR: Table 2b.
MS(ESI): 591 [2Mþ1]þ (28), 296 [Mþ1]þ (100). The purity by the HPLC
method was >99.0%.
9-[(2-tert-Butyldimethylsilyloxy-ethoxy)methyl]guanine (10a)
A solution of 1 (5.0 g, 22 mmol), imidazole (6.0 g, 88 mmol, 4 equiv),
and tert-butyldimethylsilyl chloride (8 g, 53 mmol, 2.4 equiv) in DMF
(200 mL) was stirred at 23ꢀC for 5 h. The reaction was quenched by the
addition of water (5 mL) and solvent was evaporated in vacuo. The residue
was chromatographed to afford 10a (5.98 g, 79%). M.p. 198ꢀ–200ꢀC
1H NMR: 0.00 (bs, 6H, Si(CH3)2); 0.82 (bs, 9H, SiC(CH3)3); 3.54 (bs, 2H,
H-30); 3.64 (bs, 2H, H-40); 5.38 (s, 2H, H-10); 6.57 (bs, 2H, NH2); 7.84 (s, 1H,
H-8); 10.75 (bs, 1H, NH). 13C NMR: 5.43 (Si(CH3)2); 25.66 (C(CH3)3); 61.80
(C-40); 70.10 (C-30); 72.04 (C-10); 116.41 (C-5); 137.72 (C-8); 151.35 (C-4);
153.83 (C-2); 156.82 (C-6). MS(ESI): 679 [2Mþ]þ (16), 340 [Mþ1]þ (100).
2-Valerylamino-9-[(2-tert-butyldimethylsilyloxy-ethoxy)methyl]-1,9-
dihydro-6H-purin-6-one (10b)
A mixture of 10a (2.5 g, 7.4 mmol), valeric anhydride (8.75 mL,
44.3 mmol, 6 equiv), and DMAP (190 mg, 1.55 mmol, 0.2 equiv) in pyridine
(25 mL) was refluxed at 120ꢀC for 1 h. After cooling, water (5 mL) was
added and the solvent was removed in vacuo. The resulting oil was chro-
matographed to afford 10b (1.7 g, 55%). M.p. 170ꢀ–172ꢀC. H NMR: Tab.
1
1a. 13C NMR: Table 2a. MS(ESI): 847 [2Mþ1]þ (8), 424 [Mþ1]þ (100). 10c
(0.31 g, 11%). M.p. 140ꢀ–142ꢀC [ref.19 143ꢀ–145ꢀC].
2-Valerylamino-9-[(2-hydroxyethoxy)methyl]-1,9-dihydro-6H-purin-6-
one (11)
TBAF (1.0M solution in THF, 10.6 mL, 10.6 mmol, 3 equiv) was
added to a solution of 10b (1.5 g, 3.55 mmol) in DMF (20 mL). The reaction
mixture was stirred at 23ꢀC for 1 h. Water (5 mL) was added and the solvent

1416
GAO AND MITRA
was removed in vacuo. The resulting oil was chromatographed to afford 11
(0.8 g, 73%). M.p. 128ꢀ–130ꢀC. H NMR: Tab. 1b. 13C NMR: Table 2b.
1
MS(ESI): 619 [2Mþ1]þ (28), 310 [Mþ1]þ (44). The purity by the HPLC
method was >99.0%.
9-[(2-levulinyloxy-ethoxy)methyl]guanine (12a)
Compound 1 (12 g, 53.3 mmol) was dissolved in DMF (400 mL) con-
taining DMAP (1.3 g, 10.6 mmol, 0.2 equiv) and mixed with 1,3-dicyclohex-
ylcarbodiimide (DCC, 33 g, 160 mmol, 3 equiv). To this solution, levulinic
acid (22 mL, 215 mmol, 4 equiv) was added. After stirring at 23ꢀC for 4 h,
the reaction was quenched by the addition of water (5 mL), and the white
precipitate was filtered off. The solvent was removed in vacuo, and the
residue was subjected to chromatography to yield 12a (12.3 g, 71%). M.p.
143ꢀ–145ꢀC 1H NMR: 2.11 (s, 3H, COCH3); 2.44 (t, 2H, CH2COCH3); 2.69
(t, 2H, OCOCH2); 3.69 (bs, 2H, H-30); 4.09 (bs, 2H, H-40); 5.40 (s, 2H, H-10);
6.62 (bs, 2H, NH2); 7.88 (s, 1H, H-8); 10.85 (bs, 1H, NH). 13C NMR: 27.44
(COCH3); 29.46 (CH2COCH3); 37.26 (OCOCH2); 62.84 (C-40); 66.45 (C-30);
71.85 (C-10); 116.41 (C-5); 137.92 (C-8); 151.46 (C-4); 153.91 (C-2); 156.94
(C-6). MS(ESI): 647 [2Mþ1]þ (16), 324 [Mþ1]þ (100).
2-Pivaloylamino-9-[(2-levulinyloxy-ethoxy)methyl]-1,9-dihydro-6H-
purin-6-one (12b)
12b was prepared by the same method as 10b. Yield: 73%. M.p. glass.
1H NMR: Table 1a. 13C NMR: Table 2a. MS(ESI): 815 [2Mþ1]þ (12), 408
[Mþ1]þ (100).
2-Pivaloylamino-9-[(2-hydroxyethoxy)methyl]-1,9-dihydro-6H-purin-
6-one (13)
Compound 12b (2.0 g, 4.9 mmol) was dissolved in pyridine (66 mL),
and a solution of hydrazine monohydrate (2.3 mL, 47.4 mmol, 9.7 equiv) in
pyridine-acetic acid (3:2, v/v, 66 mL) was added. After stirring at 23ꢀC for
10 min, the mixture was cooled in an ice bath and quenched by acetone
(50 mL). The solvent was removed and the residue was subjected to chro-
matography to yield 13 (1.4 g, 92%). M.p. 208ꢀ–210ꢀC. 1H NMR: Table 1b.
13C NMR: Table 2b. MS(ESI): 619 [2Mþ1]þ (84), 310 [Mþ1]þ (36). The
purity by the HPLC method was 99.9%.

ACYCLOVIR
1417
Synthesis of Mono-Benzyl Glutarate (14h)
DMAP (4 g, 33 mmol, 0.125 equiv) was added to a ice-cold solution of
glutaric anhydride (30 g, 263 mmol) and benzyl alcohol (27 mL, 261 mmol,
0.99 equiv) in DMF (100 mL). After 1 h, the reaction mixture was allowed to
warm to room temperature, and was stirred at this temperature for an
additional 4 h. The solvent was then removed in vacuo to give glass 14h
(51.4 g, 88%), which was used directly without further purification. 1H
NMR: 1.94 (m, 2H, CH2CH2CH2); 2.39 (bs, 4H, CH2CH2CH2); 5.09 (s,
2H, C6H5CH2); 7.32 (bs, 5H, C6H5CH2); 10.80 (bs, 1H, COOH). MS(ESI):
221 [MÀ1]À (100).
Synthesis of Acyclovir Ester Prodrugs by DCC Coupling
Compounds 15a–15b were synthesized by a published method.6
Compounds 15c–15h were synthesized by the same method as 15a, 15b.
Compounds 16a, 16b were synthesized by a published method.6 Compounds
16e and 16g, 16h were synthesized by a same method as 16a,16b without
using the HCl.
15a: M.p. 133ꢀ–135ꢀC, yield 86%. MS(ESI): 439.16 [MþNa]þ (19),
417.17 [Mþ1]þ (62), 246.08 (100).
15b:M.p. 113ꢀ–115ꢀC, yield 91%, MS(ESI): 622 [Mþ1]þ (4), 256 (21),
242 (100).
15c: M.p. 128ꢀ–130ꢀC, yield 59%, MS(ESI): 512 [MþNaþ1]þ (35),
489[Mþ1]þ (100).
15d: M.p. 125ꢀ–127ꢀC, yield 58%, MS(ESI): 503 [Mþ1]þ (100).
15e: M.p. 138ꢀ–140ꢀC, yield 63%, MS(ESI): 565 [Mþ1]þ (100).
15f: M.p. 115ꢀ–117ꢀC, yield 55%, MS(ESI): 579 [Mþ1]þ (100).
15g: M.p. 148ꢀ–150ꢀC, yield 67%, MS(ESI): 424.16 [MþNa]þ (7),
402.15 [Mþ1]þ (100).
15h: M.p. 168ꢀ–170ꢀC, yield 37%, MS(ESI): 430 [Mþ1]þ (100).
16a: M.p. 153ꢀ–155ꢀC [ref.4 148ꢀ–150ꢀC; ref.6 142ꢀ–180ꢀC], yield 97%.
1H NMR: 3.67(m, 4H, H-30, CH2NHþ3 ); 4.27 (t, 2H, H-40); 5.43 (s, 2H, H-
10); 6.95 (bs, 2H, 2-NH2); 8.25 (s, 1H, H-8); 8.45 (bs, 3H, CH2NH3þ); 11.15
(bs, 1H, NH). 13C NMR: 59.80 (CH2NH3þ); 64.23 (C-30); 66.36 (C-40); 72.29
(C-10); 106.67 (C-5); 137.52 (C-8); 150.90 (C-4); 154.48 (C-2); 155.79 (C-6);
167.51 (COCH2NHþ3 ). MS(ESI): 565 [2Mþ1]þ (90), 283 [Mþ1]þ (100). The
purity by the HPLC method was 99.9%.
16b: Foamy solid [ref.6 142ꢀ–154ꢀC]. yield 100%. H NMR: 1.42 (m,
1
2H, COCH(NH3þ) CH2CH2CH2NH3þ; 1.60 (m, 2H, COCH(NHþ3 )
CH2CH2CH2NHþ3 ); 1.79 (dm, 2H, COCH(NHþ3 )CH2CH2CH2CH2NHþ3 );

1418
GAO AND MITRA
2.77 (bs, 2H, COCH(NH3þ)CH2CH2CH2CH2NH3þ); 3.84 (bs, 2H, H-30);
3.95 (bs, 1H, COCH(NH3þ)CH2CH2CH2CH2NHþ3 ); 4.30 (s, 2H, H-40);
5.55 (s, 2H, H-10); 7.39 (bs, 2H, 2-NH2); 8.23 (s, 3H, NHþ3 ); 8.76 (s, 3H,
NHþ3 ); 9.03 (s, 1H, H-8); 11.74 (bs, 1H, NH). 13C NMR: 21.08, 26.05, 29.15
(COCH(NHþ3 )CH2CH2CH2CH2NH3þ); 38.08 (COCH(NHþ3 ) CH2CH2CH2-
NHþ3 ); 51.47 (COCH(NH3þ)CH2CH2CH2CH2NHþ3 ); 64.24 (C-30); 66.76 (C-
40); 72.98 (C-10); 106.67 (C-5); 137.80 (C-8); 150.22 (C-4); 154.41 (C-2);
155.19 (C-6); 169.28 (COCH(NHþ3 )CH2CH2CH2CH2NH3þ); MS(ESI): 354
[Mþ1]þ (14), 270 (22). 186 (45), 169 (100). The purity by the HPLC method
was 99.9%.
16e: M.p. 223ꢀ–225ꢀC, yield 61%. 1H NMR (DMSO-d6þD2Oþ
NaOD): 2.63 (m, 2H, COCH2CH(NH2)COOH); 3.67–3.70 (m, 5H, H-30,
H-40, CO CHCH(NH2)COOH); 5.56 (s, 2H, H-10); 7.94 (s, 1H, H-8). 13C
2
NMR (DMSO-d6þD2OþNaOD): 39.51, 44.63 (COCH2CH(NH2)COOH);
61.25 (C-30); 72.23 (C-40); 73.51 (C-10); 138.73 (C-8); 152.43 (C-4); 162.29
(C-2); 169.00 (C-6); 180.65, 182.78 (COCH2CH(NH2)COOH). MS(ESI):
341 [Mþ1]þ (36).
16g: M.p. 203ꢀ–205ꢀC, yield 72%. 1H NMR: 3.06 (s, 2H,
COCH2COOH); 3.66 (t, 2H, H-30); 4.16 (t, 2H, H-40); 5.37 (s, 2H, H-10);
6.59 (bs, 2H, 2-NH2); 7.84 (s, 1H, H-8); 10.72 (s, 1H, NH). 13C NMR: 41.37
(COCH2COOH); 63.49 (C-30); 66.29 (C-40); 71.79 (C-10); 137.51 (C-8);
151.32 (C-4); 153.87 (C-2); 156.66 (C-6); 166.79, 167.82 (COCH2COOH).
MS(ESI): 312.11 [Mþ1]þ (22), 223.22 (100). The purity by the HPLC
method was 99.9%.
16h: M.p. 200ꢀ–202ꢀC, yield 72%. 1H NMR: 1.70 (pentate, 2H,
CH2CH2CH2COOH); 2.24 (t, 2H, CH2CH2COOH); 2.29 (t, 2H,
CH2CH2CH2COOH); 3.67 (t, 2H, H-30); 4.10(t, 2H, H-40); 5.36 (s, 2H,
H-10); 6.57 (bs, 2H, 2-NH2); 7.83 (s, 1H, H-8); 10.71 (s, 1H, NH). 13C
NMR: 19.76 (CH2CH2CH2COOH); 32.37 (CH2CH2CH2COOH); 32.45
(CH2CH2CH2COOH); 62.61 (C-30); 66.38 (C-40); 71.69 (C-10); 116.37
(C-5); 137.68 (C-8); 151.35 (C-4); 153.83 (C-2); 156.70 (C-6); 172.38
(COCH2CH2CH2COOH); 173.94 (COCH2CH2CH2COOH); MS(ESI): 340
[Mþ1]þ (58), 270 (100).
ACKNOWLEDGMENTS
Supported by NIH grants 2R01 EY09171-05 and 2R01 EY0659-04.
We are grateful to Dr. P. Brown at Hoechst-Marion-Roussell for his help in
running the ESI mass spectra.

ACYCLOVIR
1419
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