Synthesis and characterization of novel dipeptide ester prodrugs of acyclovir.

Article abstract of DOI:10.1016/S1386-1425(03)00007-6

Four dipeptide (Gly-Gly, Gly-Val, Val-Val, Val-Gly) ester prodrugs of 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir, ACV) were synthesized. LC/MS was used to characterize the new prodrugs. Both 1H NMR and 13C NMR spectra of the four prodrugs of ACV were measured and assigned based on spectral comparison with compounds of similar structures.

Full text of DOI:10.1016/S1386-1425(03)00007-6

Spectrochimica Acta Part A 59 (2003) 2033ꢀ2039
/
www.elsevier.com/locate/saa
Synthesis and characterization of novel dipeptide ester
prodrugs of acyclovir
Yasser E. Nashed, Ashim K. Mitra *
Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri ꢀ
/
Kansas City, 5005 Rockhill Road, Kansas City,
MO 64110-2499, USA
Received 17 September 2002; received in revised form 10 December 2002; accepted 13 December 2002
Abstract
Four dipeptide (Glyꢀ
/
Gly, Glyꢀ
/
Val, Valꢀ
/
Val, ValꢀGly) ester prodrugs of 9-[(2-hydroxyethoxy)methyl]guanine
/
(acyclovir, ACV) were synthesized. LC/MS was used to characterize the new prodrugs. Both H NMR and 13C NMR
spectra of the four prodrugs of ACV were measured and assigned based on spectral comparison with compounds of
similar structures.
1
# 2003 Elsevier Science B.V. All rights reserved.
Keywords: Acyclovir; Dipeptide; Prodrug; Esterfication; 1H NMR; 13C NMR; LC/MS
1. Introduction
far from being optimal. The drug cannot be given
as aqueous eye drops or by intramuscular injection
because of its limited solubility in water. Pre-
viously a number of ACV prodrugs were devel-
oped with the aim of improving the delivery
characteristics. Different approaches have been
implemented to serve these aims. Colla et al. [8]
and Maudgal et al. [9] have added various amino
acid moieties to ACV to form amino acid esters
prodrugs, which were potentially useful in the
preparation of eye drops formulation. Beauchamp
et al. [10] have used similar approach and prepared
several amino acid esters prodrugs as potential
prodrugs for oral administration. Shao et al. and
Yang et al. [11,12] have reported the synthesis,
nasal and ocular absorption, and metabolism of
A direct synthesis of acyclovir (ACV) (9-[(2-
hydroxyethoxy)methyl]guanine) from guanonsine
via exchange of glycosyl substituents is the most
efficient method reported so far [1,2]. Boryski [3,4]
and Boryski and coworkers [5,6] have studied the
mechanism of conversion of guanosine to ACV in
details. Schaeffer et al. [7] first reported ACV as a
highly specific inhibitor of herpes virus prolifera-
tion. Because of its poor aqueous solubility and
lipophilicity, the intravenous and oral delivery is
* Corresponding author. Tel.: ꢁ
816-235-5190.
E-mail address: mitraa@umkc.edu (A.K. Mitra).
/
1-816-235-1615; fax: ꢁ1-
/
1386-1425/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved.
doi:10.1016/S1386-1425(03)00007-6

2034
Y.E. Nashed, A.K. Mitra / Spectrochimica Acta Part A 59 (2003) 2033ꢀ2039
/
various aliphatic acid ester prodrugs of ACV.
Bundgaard et al. [13] evaluated a series of N-
substituted 3- or 4-(amino-methyl) benzoate esters
of ACV as solution-stable, biolabile prodrug form.
Recently, Gao and Mitra [14] have synthesized
several series of N2-acyl-ACV, a-, b-, g-amino acid
esters and dicarboxylic acid esters of ACV to
investigate their effectiveness in enhancing mem-
brane transport.
2. Materials and techniques
2.1. NMR spectroscopy
The 1H and 13C NMR were recorded on a
Bruker AC 250-modified Tecmag DSPect Fourier
transform NMR spectrometer at 250 and 63 MHz,
respectively, for 0.25ꢀ0.5 M DMSO-d6 solutions
/
at 23 8C. Chemical shifts are expressed in parts
per million relative to tetramethylsilane as the
internal standard. For the 13C NMR spectra, the
spectral width was 20 000 Hz and the number of
data points was 16 000, generating a 2.5 Hz per
point digital resolution. The flip angle was 4 ms
(908) and the acquisition time was 0.409 s with a
The amino acid ester prodrugs of ACV have low
stability at the physiological pH (7.4) and their
delivery is still not optimal. The overall objectives
of this study are to synthesize prodrugs of ACV
possessing stability, solubility and membrane
transport at the physiological pH enabling optimal
delivery. The oligopeptide transporter (PEPT1) is
an essential mammalian protein that actively
transports small peptides (active transport) [15].
PEPT1 is found on the wall of the small intestine
of mammals [16,17] and also on the cornea and
internal eye structures [18]. Based on these studies,
we have chosen the approach of synthesizing
dipeptide ester prodrugs of ACV. In addition,
dipeptide prodrugs can exist in several more
possible conformers than the amino acids pro-
drugs, which may lead to more stable forms. Also
the possibility of intramolecular hydrogen bonding
between the carbonyl group of the ester link and
the free amino group of the dipeptide to form
more stable prodrug, is higher in the case of
dipeptide esters than in the case of the amino
acid prodrugs. Both geometrical isomerism and
hydrogen bonding might enhance the stability of
the prodrugs. In some cases increasing the solubi-
lity using the prodrug strategy would help in drug
delivery by concentration gradient (passive trans-
port) and by transporter mediated delivery (facili-
tated transport). Such compounds would probably
have high potential for oral administration and
also eye drops formulation. For the first time, four
pulse delay of 3 s; 500ꢀ/1000 scans were accumu-
lated for each spectrum.
2.2. Liquid chromatography/mass spectrometry
LC/MS was carried out by coupling an high-
performance liquid chromatographic (HPLC) sys-
tem (Spectra System) to a Finnigan aQa single
quadruple mass spectrometer using an electro-
spray interface held at 350 8C. The electrospray
needle was maintained at 3.00 kV with decluster-
ing potential set at 30 V. Ultrapure nitrogen was
used as nebulizer gas. The mass spectrometer was
operated in the positive ion mode. The flow rate
was 1.0 ml/min, and a 20-min isocratic of 20%
CH3CN with 0.1 trifluoroacetic acid (TFA) and a
C8 column of 10-cm length were used. The
compounds were dissolved in CH3OH (1 mg/ml)
and 10 ml was injected directly for the LC/MS
analysis.
2.3. Materials
dipeptide (Glyꢀ
/
Gly, Valꢀ
/
Gly, Glyꢀ
/
Val, ValꢀVal)
/
All of the chemicals (Bocꢀ
/
L
ꢀ
/
Val, BocꢀGly,
/
ester prodrugs of ACV have been synthesized and
characterized to investigate its aqueous solubility,
stability, antiviral activity and its membrane
delivery. During the synthesis, the intermediate
guanosine, 1,3-dioxolane, p-toluenesulfonic acid
monohydrate, TFA, dimethylformamide and
acidic anhydride) were purchased from Sigma-
Aldrich (St. Louis, MO). HPLC grade acetonitrile
and methanol were obtained from Fisher Scientific
Company (St. Louis, MO).
1
and product structures were confirmed by H and
13C NMR, TLC and LC/MS analysis.

Y.E. Nashed, A.K. Mitra / Spectrochimica Acta Part A 59 (2003) 2033ꢀ
/2039
2035
2.3.1. Acyclovir
ACV was synthesized following the method
reported by Shiragami et al. [1,2].
neutralized Glyꢀ
/
ACV was added to Bocꢀ
/
Gly
amino acid anhydride prepared by the same
procedure mentioned above. The reaction was
initiated at 0 8C and gradually the temperature
was raised to room temperature with continuous
stirring. The reaction was complete in 5 h. The
work-up procedures mentioned earlier were fol-
2.3.2. Dipeptide ester prodrugs of acyclovir
Bocꢀ
/
GlyꢀOH (3.11 g, 17.76 mmol) and dicy-
/
clohexyl carbodiimide (DCC) (1.83 g, 8.88 mmol)
were dissolved in dry DMF (20 ml) under a
nitrogen atmosphere. The mixture was continu-
ously stirred for 1 h at 0 8C. A precipitate, N,N?-
dicyclohexylurea, started to separate almost im-
mediately and its amount gradually increased. A
solution of ACV (2.00 g, 8.88 mmol) and 4-(N,N-
dimethylamino) pyridine (DMAP) (0.06 g, 0.65
mmol) in DMF (100 ml) was added dropwise to
the reaction mixture at 0 8C. The reaction was
stirred and allowed to warm up to the room
temperature. After 24 h, the reaction was checked
by TLC (1:9, methanol to dichloromethane) and
LC/MS and it was complete. The urea derivative
was removed by filtration and washed with
dichloromethane. The solvents of the combined
filtrate and washing were partially removed under
reduced pressure. The resulting oil was added
dropwise to cold ethyl ether and immediately
lowed. The product Glyꢀ
pure. The same scheme (Fig. 1) was applied to
synthesize ValꢀGly, GlyꢀVal and ValꢀVal of
ACV (Fig. 2).
Also we have tried silica gel column chromato-
graphy to purify the intermediates and the pro-
ducts. The silica gel optimal separation conditions
/
GlyꢀACV was highly
/
/
/
/
of all of the compounds were in the range of 1:4ꢀ
/
1:9 (methanol to dichloromethane). The products
1
were highly pure (:98%). The LC/MS and H
/
and 13NMR data were listed in Tables 1 and 2 and
Table 3, respectively.
3. Results and discussion
3.1. Synthesis
Bocꢀ
in the mother liquor was kept in the refrigerator
for 5 h. The BocꢀGlyꢀACV was filtered and
/
Glyꢀ
/
ACV was precipitated. The precipitate
ACV was successfully synthesized following the
method reported by Shiragami et al. [1,2]. Synth-
/
/
esis of Glyꢀ
/
Glyꢀ
/
ACV, Valꢀ
/
Glyꢀ
/
ACV, Glyꢀ
/
washed with small amount of ethyl ether. The
solvents of the filtrate were removed under va-
cuum and again the resulting oil was added
dropwise to the cold ethyl ether. This process
was repeated three times and the precipitation by
the cold ethyl ether afforded a highly pure
compound with 92% yield. The product was
characterized by LC/MS and NMR.
ValꢀACV and Valꢀ
/
/
ValꢀACV dipeptide ester
/
prodrugs of ACV involve (i) formation of N-
protected amino acid anhydrides, (ii) coupling of
the N-protected amino acid anhydride with ACV,
(iii) deprotecting the amino group of the amino
acid ester of ACV, (iv) formation of N-protected
amino acid anhydride, (v) coupling of the N-
protected amino acid anhydride with the amino
acid esters of ACV (formation of dipeptide), and
(vii) finally deprotection of the amino group of the
dipeptide ester of ACV (Fig. 1).
A solution of Bocꢀ
/
GlyꢀACV (3.00 g) in a fresh
/
TFA (60 ml) was stirred at 0 8C for 1 h. The excess
acid was removed under vacuum and the oil
residue was mixed with toluene (100 ml) and
rotary evaporated at 50 8C to provide a white
solid material. The product was washed with ethyl
ether and dried under reduced pressure to yield
A method to attach ACV, a terminal hydroxy
containing model drug to glycine and valine amino
acids through ester linkage was developed. Amino
acid esters of ACV Glyꢀ
/
ACV and ValꢀACV were
/
Glyꢀ
material. Elemental analysis indicated that Glyꢀ
ACV has 1 mole of TFA.
GlyꢀACV (3.11 g) was dissolved in DMF and
neutralized by triethylamine (TEA) (2.20 ml). The
/
ACV a slightly hygroscopic white powdery
prepared in three steps (Fig. 1) using a modified
procedure of method B reported by Beauchamp et
al. [10]. One of the simplest methods for esterifica-
tion of the hydroxymethyl ACV is to use the
symmetrical anhydride of the protected amino acid
/
/

2036
Y.E. Nashed, A.K. Mitra / Spectrochimica Acta Part A 59 (2003) 2033ꢀ2039
/
Fig. 1. (i) Bocꢀ
/
AA, DCC, DMF, 0 8C, 1 h, (ii) DMAP, DMF, 24 h, r.t. (iii) TFA, 0 8C, 60 min (iv) BocꢀAA, DCC, DMF, 0 8C, 1 h,
/
(v) TEA, r.t., (vi) adding the N-protected amino acid anhydride (step iv) to the neutralized amino acid ester of ACV (step v), 5 h, r.t.,
(vii) TFA, 0 8C, 60 min.
one equivalent of DCC in DMF. Preformed
symmetrical anhydrides have been used by many
research groups, mainly in Boc chemistry, because
of their excellent reactivity [19,20]. A solution of
ACV (1) and DMAP in DMF was added to the
reaction mixture. To avoid side products which
could be formed as a result of coupling of the
amino acid with the amine group of ACV the
reaction should be initiated at 0 8C. The choice of
amino acid-labile Boc-protecting group was ap-
propriate since no other parallel reactions were
observed through the reaction steps.
Bocꢀ
/
AAꢀACV compounds were purified by
/
precipitation in cold ethyl ether. The precipitation
method afforded highly pure intermediates and
products and no hydrolysis of the ester linkage was
observed. Beauchamp et al. have reported [10]
Fig. 2. Structures of Glyꢀ
esters of ACV and the dipeptide moieties Glyꢀ
GlyꢀVal and ValꢀVal attached to ACV model drug.
/
ACV and Valꢀ
/
ACV amino acid
/
Gly, ValꢀGly,
/
synthesis of 18 amino acid prodrugs. N-methyl-
alanyl ACV and -2-aminobutyrate ACV were the
L-
/
/
L
only two compounds, which were synthesized by
method B using N-Boc-protecting group. It is
obvious that the real challenge in this method is
in the presence of DMAP. Symmetrical anhydrides
are generated under nitrogen atmosphere using
two equivalents of Boc-protected amino acid and

Y.E. Nashed, A.K. Mitra / Spectrochimica Acta Part A 59 (2003) 2033ꢀ
/2039
2037
Table 1
Yields, measured and calculated m/z ratios of the products
Compound
Yield (%)
Measured MS [Mꢁ
/
1]ꢁ
Measured MS [2Mꢁ
/
1]ꢁ
Calculated MS [Mꢁ
1]ꢁ
/
Glyꢀ
ValꢀGlyꢀ
GlyꢀValꢀ
ValꢀValꢀ
/
Glyꢀ
/
ACV
72
74
70
75
339.7
381.9
381.9
423.9
ꢀ
/
340.13
382.18
382.18
424.22
/
/ACV
763.3
763.4
847.4
/
/ACV
/
/
ACV
the crystallization of the product from 5% CH2Cl2/
MeOH. This crystallization condition might result
in the hydrolysis of the ester linkage to generate
dride for about 5 h. The neutralization step was
essential for completing the reaction to form the
N-protected ACV dipeptide esters. The resulting
products (4) were purified by precipitation follow-
ing the same procedure in step 2, filtered, dried
and the Boc group was deprotected (acidolytic
removal) to yield the desired dipeptide ester
prodrugs of ACV (5).
the starting materials. Bocꢀ
/
GlyꢀACV also hydro-
/
lyzed under similar crystallization conditions.
During the LC/MS measurements, it was also
observed that GlyꢀACV dissolved in methanol,
/
started to hydrolyze within 10 min. After coupling
to the Boc-protected glycine and valine amino
acids, the protecting group was removed by TFA
3.2. Characterization
to give the trifluoroacetate salt of Glyꢀ
ValꢀACV (Fig. 2). The elemental analysis of
ValꢀACV and GlyꢀACV indicated that each of
these prodrugs contains 1 mole of TFA.
Compound (3) (Fig. 1) was neutralized by TEA
and treated with N-protected amino acid anhy-
/
ACV and
L
ꢀ
/
/
Electrospray ionization coupled to a quadruple
mass spectrometer is a powerful analytical tool. It
enables the intact flight of thermally labile mole-
cules. For each of the four compounds, one unique
/
/
peak is observed in the LCꢀESI/MS total ion
/
Table 2
1H NMR data of dipeptide ester prodrugs of ACV in DMSO-d6
1H
ACV
Glyꢀ
/
Glyꢀ
/
ACV
Valꢀ
/
Glyꢀ/ACV
Glyꢀ/Valꢀ/ACV
Valꢀ
/
ValꢀACV
/
1-NH
2-NH2
8-CH
10.72 bs
6.55 bs
7.84 s
10.81 bs
6.65 s
7.82 s
5.35 s
3.59 s
4.14 t
10.89 s
6.69 s
10.83 s
6.64 s
10.89 s
6.64 s
8.15 bs
5.37 s
8.07 bs
5.36 s
8.14 bs
5.34 s
10-CH2
12-CH2
13-CH2
OH
5.36 s
3.47 bs
3.47 bs
4.69 s
3.68 bs
4.15 s
3.67 bs
4.22 t
3.68 m
4.14 m
16-CHR1
17-NH
19-CHR2
20-NH3ꢁ
CH(R1)
CH3(R1)
CH3(R1)
CH(R2)
CH3(R2)
CH3(R2)
TFA
3.92 d
4.04 m
4.13 bs
4.14 m
ꢀ/
ꢀ/
ꢀ/
ꢀ/
3.65 d
8.82 bs
3.89 m
8.89 s
3.67 bs
8.50 d
1.95 m
0.83 d
0.83 d
3.68 m
8.57 d
2.01 m
0.88 d
0.88 d
2.01 m
0.88 d
0.88 d
7.94 s
2.07 m
0.94 d
0.94 d
7.95 s
7.95 s
7.95 s
Glyꢀ
/
Glyꢀ
/
ACV [R1ꢂ
/
R2ꢂ
/
H], Valꢀ
/
Glyꢀ
/
ACV [R1ꢂ
/
H, R2ꢂ
/
(CH3)2CH], Glyꢀ
/
Valꢀ
/
ACV [R1ꢂ
/
(CH3)2CH], R2ꢂ
/
H], Valꢀ
/
Valꢀ
/
ACV [R1ꢂ/R2ꢂ/(CH3)2CH].

2038
Y.E. Nashed, A.K. Mitra / Spectrochimica Acta Part A 59 (2003) 2033ꢀ2039
/
Table 3
13C NMR data of dipeptide ester prodrugs of ACV
13C
ACV
Glyꢀ
/
Glyꢀ
/
ACV
Valꢀ
/
Glyꢀ/ACV
Glyꢀ/Valꢀ/ACV
Valꢀ
/
ValꢀACV
/
2
153.80
151.40
116.37
156.80
137.80
72.00
154.09
151.45
116.45
156.76
137.65
71.86
66.33
63.60
169.43
58.60
166.79
?
154.02
151.20
115.58
156.45
137.71
72.01
66.27
63.44
169.21
57.14
168.34
?
154.10
151.43
116.24
156.80
137.90
72.08
66.53
63.39
171.05
57.37
166.42
?
154.02
151.00
114.62
156.32
137.60
71.89
66.13
63.13
170.95
57.57
168.21
56.89
29.75
17.64
18.41
29.41
17.92
17.23
4
5
6
8
10
12
13
70.38
59.89
15
16
18
19
CH(R1)a
CH3(R1)a
CH3(R1)a
CH(R2)a
CH3(R2)a
CH3(R2)a
30.07
17.80
18.77
29.66
17.45
17.92
The question mark indicates that signal/noise ratio was too low to identify the signals.
a
Glyꢀ
/
Glyꢀ
/
ACV [R1ꢂ
/
R2ꢂ
/
H], Valꢀ
/
Glyꢀ
/
ACV [R1ꢂ
/
H, R2ꢂ
/
(CH3)2CH], Glyꢀ
/
Valꢀ
/
ACV [R1ꢂ
/
(CH3)2CH], R2ꢂ
/
H], Valꢀ
/
Valꢀ
/
ACV [R1ꢂ/R2ꢂ/(CH3)2CH].
current and UV chromatograms. The positive
electrospray mass spectra of GlyꢀGlyꢀACV,
ValꢀGlyꢀACV, GlyꢀValꢀACV and ValꢀValꢀ
ACV eluting at approximately 2.45, 2.45, 2.46
and 2.97 min (figures not presented), respectively,
million. In comparison with ACV, other protons
of the carbons of the ACV moiety of these
dipeptide ester prodrugs of ACV produced almost
no changes in the chemical shift either in 1H NMR
or 13C NMR. The assignments of 13C NMR were
/
/
/
/
/
/
/
/
give the expected Mꢁ
/
1 (Mꢂ/m/z) values of these
straightforward. The highest chemical shifts in 13
C
compounds mainly in monomer and dimer forms
(Table 1).
NMR spectra for each of these compounds were
assigned to the carbon COO (15-C) of the carboxyl
group and the immediate lower chemical shifts
were assigned to the carbon CONH (18-C) of the
amide group (Table 3). Both the highest and
immediate lower chemical shifts indicated the
formation of the ester and peptide bonds, respec-
tively.
Intact cornea and Caco-2 cell permeabilities of
all the ACV dipeptide ester prodrugs were ob-
served to be higher than ACV possibly due to
recognition of the prodrugs by the PEPT1 on the
cornea and Caco-2 cells. Also they exhibited
excellent chemical stability and antiviral activity
against herpes simplex virus thereby rendering
these lead compounds promising drug candidates
against herpes virus infections. More details can be
1
The H and 13C NMR spectral assignments of
the four compounds are listed in Table 2 and Table
3, respectively. As shown in Table 2 the peak at
4.69 ppm for ACV has been assigned to the proton
of the OH group. This peak disappeared upon
conversion of OH to Oꢀ
1). Also this conversion leaded to deshielded H
NMR chemical shifts of 0.67ꢀ0.75 ppm for the
/
AA or Oꢀ
/
AAꢀ
/
AA (Fig.
1
/
protons of carbon CH2OCHO (13-CH2), to which
these groups are attached. Immediate adjacent
protons CH2O (16-CHR1) also deshielded by ꢀ
/
0.4 ppm. These modifications in the 1H NMR
spectra are strong evidence of occurrence of a
substitution reaction and formation of the amino
acid esters of ACV. The proton on carbon 8
produced some shift towards higher parts per
found in Refs. [21ꢀ24].
/

Y.E. Nashed, A.K. Mitra / Spectrochimica Acta Part A 59 (2003) 2033ꢀ
/2039
2039
[11] Z. Shao, G.B. Park, R. Krihnamoorthy, A.K. Mitra,
Pharm. Res. 11 (1994) 237.
Acknowledgements
[12] C. Yang, H. Gao, A.K. Mitra, J. Pharm. Sci. 90 (2001)
615.
This work was supported by NIH grants 2RO1
EY09171-08 and 2RO1 EY10659-07.
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[14] H. Gao, A.K. Mitra, Syn. Commun. 31 (2001) 1399.
[15] D. Meredith, C.A.R. Boyd, J. Membrane Biol. 145 (1995)
1.
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