Comparative Analysis of Acyclovir Esters Stability in Solutions: The Influence of the Substituent Structure, Kinetics, and Steric Effects

Article abstract of DOI:10.1002/kin.20943

Reversed-phase high-performance liquid chromatography has been applied to the determination of acyclovir (ACV) esters such as acetate, isobutyrate, pivalate, ethoxycarbonate, and nicotinate. All analyses were carried out at laboratory temperature using a column LiChrospher RP-18 (250 × 4 mm, 5 μm) and a proper mobile phase consisting of acetonitrile and phosphate buffer (pH 6 or 6.7) or acetonitrile and potassium dihydrogen phosphate, and acetic acid. The methods were validated by the determination of the following parameters: selectivity, precision, accuracy, and linearity. Kinetic studies on the hydrolysis were investigated in solutions at 310 K over the pH range 0.42-1.38. The pH-profiles indicated specific acid-catalyzed and spontaneous water-catalyzed degradation. The stability of the studied ACV esters were determined not only by steric factors. In the case of ethoxycarbonyl ester of ACV, the hydrolysis was a two-step reaction.

Full text of DOI:10.1002/kin.20943

Comparative Analysis of
Acyclovir Esters Stability in
Solutions: The Influence of
the Substituent Structure,
Kinetics, and Steric Effects
MONIKA A. LESNIEWSKA,1 MICHAŁ GOLA,1 ZBIGNIEW DUTKIEWICZ,2 IZABELA MUSZALSKA1
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Poznan University of Medical Sciences, Grunwaldzka 6,
60–780, Poznan, Poland
2Department of Chemical Technology of Drugs, Faculty of Pharmacy, Poznan University of Medical Sciences, Grunwaldzka 6,
60–780, Poznan, Poland
Received 22 January 2015; revised 7 July 2015; accepted 8 August 2015
DOI 10.1002/kin.20943
Published online 22 September 2015 in Wiley Online Library (wileyonlinelibrary.com).
ABSTRACT: Reversed-phase high-performance liquid chromatography has been applied to the
determination of acyclovir (ACV) esters such as acetate, isobutyrate, pivalate, ethoxycarbonate,
and nicotinate. All analyses were carried out at laboratory temperature using a column LiChro-
spher RP-18 (250 × 4 mm, 5 µm) and a proper mobile phase consisting of acetonitrile and
phosphate buffer (pH 6 or 6.7) or acetonitrile and potassium dihydrogen phosphate, and acetic
acid. The methods were validated by the determination of the following parameters: selectivity,
precision, accuracy, and linearity. Kinetic studies on the hydrolysis were investigated in solu-
tions at 310 K over the pH range 0.42–1.38. The pH-profiles indicated specific acid-catalyzed
and spontaneous water-catalyzed degradation. The stability of the studied ACV esters were
determined not only by stꢀeCric factors. In the case of ethoxycarbonyl ester of ACV, the hydrolysis
was a two-step reaction.
2015 Wiley Periodicals, Inc. Int J Chem Kinet 47: 724–733, 2015
time. In the 1960s, trifluridine and vidarabine were ap-
INTRODUCTION
plied to therapeutic use, i.e. drugs that are DNA poly-
merase inhibitors [1]. However, both of them are char-
acterized by low selectivity and high toxicity. A new
generation of an antiviral drug initiated by acyclovir
(ACV), a guanosine analogue with marked selectivity
in regard to the virus and the safe use, was a break-
through. At a further stage, ganciclovir and pencyclovir
as well as ester modifications of ACV (valacyclovir)
and pencyclovir (famcyclovir) were developed and
Owing to the prevalence of herpes viruses in the hu-
man population, and the fact that the infection is of-
ten asymptomatic, dedicated research and development
work on effective drugs have been in progress for a long
Correspondence to: Izabela Muszalska; e-mail: imuszals@
ump.edu.pl.
ꢀC
2015 Wiley Periodicals, Inc.

COMPARATIVE ANALYSIS OF ACYCLOVIR ESTERS STABILITY IN SOLUTIONS
725
introduced into therapeutics [1]. The relatively low tox-
icity of ACV results from the mechanism of the action
that requires its phosphorylation to a monophosphate.
In this reaction, viral thymidine kinase is involved.
ACV is not a substrate for the host’s cellular-type ki-
nase [2,3]. For the improvement of pharmacokinetic
properties of drugs containing ACV, different prepa-
rations with modified release to allow a reduction in
the frequency of drug administration, are being devel-
oped [4,5]. There is also research work being carried
out to apply mucoadhesive microspheres composed
of ethyl cellulose and Carbapol resulting in a signifi-
cant prolongation of drug residence in the upper and
middle sections of the digestive system in compari-
son with conventional microspheres [6]. In the case
of ophthalmic drugs, the potential application of mi-
crospheres prepared by the spray drying method and
nanocapsules made by the ionotropic gelation method
(a combination of chitosan and tripolyphosphate) is
being considered [7].
a thiazole ring does not have such an effect [16,17].
For the purpose of use after oral and nasal mucosa
administration, the stability of aliphatic (ACV) esters
(n-butyrate, pivalate, valerate, and hexanoate) has been
investigated. These esters are characterized by higher
lipophilicity and reduced solubility in water in com-
parison with ACV [18]. Their stability has been in-
vestigated in plasma. It has been proven that the pi-
valoyl ester is more resistant to the impact of plasma
esterase, and only the hexane ester has shown satis-
factory properties relating to penetration through the
nasal mucosa [18].
However, in the literature, there are no comprehen-
sive studies on the stability of ACV esters in water
solutions and the evaluation of the impact of hydrogen
ions on the observed hydrolysis reaction. The main
aim of this study is to determine kinetic parameters
of the hydrolysis of ACV esters (acetate, isobutyrate,
pivalate, etoxycarbonate, and nicotinate; Fig. 1) under
conditions of constant ionic strength (0.50 mol L−1
)
Another way to improve the effectiveness of the
therapy with ACV is the chemical modification of
the molecule toward the formation of an ester within
the pseudosugar group. The only ACV ester approved
for trading is valacyclovir (combined with L-valine),
and it is rapidly converted into ACV. Its bioavailability
is about 54%, and it is several times higher than for
ACV [8]. This is a drug of high stability in an acidic
environment, but it is metabolized intensively in the
intestines and liver [8–10]. ACV esters have also been
synthesized with other amino acids, such as glycine,
alanine, glutamic acid, serine, and isoleucine [8,11].
Glycine and alanine esters proved to be too unstable at
physiological pH [8,11]. In the intestinal lumen and in
homogenizates of Caco-2 and liver cells, the ester of
γ -glutaminic acid has been the most stable, whereas
the stability of L-serine ester has turned out to be sim-
ilar to valacyclovir [11]. Furthermore, L-serine and L-
isoleucine esters have been recognized as promising
ACV precursors because they have revealed the best
absorption from the intestinal lumen [11]. For dipep-
tide derivatives, it has been demonstrated that direct
binding with valine significantly stabilizes the ester
binding that is influenced by steric properties of this
amino acid [12]. The bivaline derivative is also char-
acterized by higher solubility in water in comparison
with ACV, and better bioavailability as well [12–14].
The low stability of amino acid esters in water so-
lutions does not allow a drug being developed to be
administered as an injection. Therefore, ACV esters
with the aminomethylbenzoyl group have been ana-
lyzed to prove that the incorporation of an aromatic
ring between the amino group and the carboxyl group
stabilizes the molecule [15], whereas the insertion of
and at a temperature of 310 K (37°C). Additionally,
to determine catalytic rate constants by a kinetic equa-
tion describing the observed reaction and analysis of
the correlation between calculated parameters with the
steric factors.
EXPERIMENTAL
ACV was received from JELFA S.A. (Jelenia Go´ra,
Poland). Esters of ACV (Fig. 1): acetyl (Ac-ACV),
iso-butyryl (iBut-ACV), pivaloyl (Piv-ACV), ethoxy-
carbonyl (Etc-ACV), and nicotinoyl (Nic-ACV) were
synthesized in a laboratory, for the purpose of the
present study [19]. Acetonitrile for HPLC (isocratic
basis) and methyl 4-hydroxybenzoate (99%) were ob-
tained from POCH S.A. (Gliwice, Poland) but sul-
fathiazole (98.0%) and sulfamerazine (99.0%) were
obtained from Sigma-Aldrich (St. Louis, MO). All
other chemicals of the highest purity were commer-
cially available.
Apparatus and Chromatographic
Conditions
The high-performance liquid chromatography (HPLC)
with a system consisting of a Rheodyne 7120, 20 µL
fixed-loop injector, an LC 3-UV detector (Pye Uni-
cam, England), an L-6000A pump (Merck-Hitachi,
Germany), and an A/C transmitter with Chromed soft-
ware (Medson, Poland), was used for the determination
of tested substances and their decomposition product
– ACV. As a stationary phase, a LiChrospher RP-18
column (250 × 4 mm, 5 µm; Merck, Germany) was
International Journal of Chemical Kinetics DOI 10.1002/kin.20943

726
LESNIEWSKA ET AL.
Figure 1 Chemical structure of ACV esters.
Table I The Composition of the Mobile Phase and Solutions of Internal Standards for the Determination of Various
Esters
Retention Time (min)
Internal Standard
Solution (µg mL−1
Internal
Compound
Ac-ACV
Mobile Phase Composition
)
ACV Compound Standard
ACN and 20 mmol L−1 phosphate buffer
pH 6 (25:75)
Sulfathiazole (120)*
1.99
2.48
3.28
3.70
7.15
iBut-ACV ACN and phosphate buffer 20 mmol
L−1 pH 6.7 (30:70)
Piv-ACV
Methyl 4-hydroxybenzoate (90)* 1.95
4.05
4.55
Etc-ACV
KH2PO4 0.5 mol L−1 – ACN –
CH3COOH (99,9%) – H2O
(40:200:5:755)
Sulfamerazine (240)*
2.09
6.20
Nic-ACV
3.50
*In a mixture of ACN and phosphate buffer 0.2 mol L−1 pH 6 (1:1).
used. The flow rate of the mobile phases (Table I) was
1.0 mL min−1. The UV detection was carried out at
254 nm.
The pH values of the buffer solutions were mea-
sured on a HI 110 pH-Meter (HANNA Instruments,
Romania).
used as a component of the mobile phase was a mixture
of K2HPO4 (17 mmol L−1) and KH2PO4 (3 mmol L−1),
and the phosphate buffer pH 6.7 was the mixture of
KH2PO4 (11 mmol L−1) and Na2HPO4 (9 mmol L−1),
respectively.
After hydrolysis in acidic medium, the solu-
tions of tested substances at a concentration of ca.
180 µg mL−1 in mixtures with internal standards and
the phosphate buffer pH 6 (0.2 mol L−1) (1:1:1) were
chromatographed using a column LiChrospher RP-
18 (250 × 4 mm, 5 µm) and a proper mobile phase
(Table I). Simultaneously, the retention times were
Validation of the HPLC Method
All analyses was carried out at laboratory temperature
using a reversed phase technique (RP-HPLC) on the
analytical column RP-18. The phosphate buffer pH 6
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COMPARATIVE ANALYSIS OF ACYCLOVIR ESTERS STABILITY IN SOLUTIONS
727
Table II The Quantitative Parameters and Statistical Data for Determination of ACV Esters by the Proposed HPLC
Method: Calibration Curves
Range
(µg mL−1
Correlation
Coefficient (r)
LOD
(µg mL−1
LOQ
(µg mL−1
)
Compound
)
n
Slope (b)
)
Ac-ACV
iBut-ACV
Piv-ACV
Etc-ACV
Nic-ACV
5.0–120.0
5.0–150.0
10.0–150.0
10.0–200.0
10.0–150.0
11
10
9
11
9
0.0130 0.0005
0.0100 0.0003
0.0097 0.0004
0.0040 0.0001
0.0068 0.0002
0.9985
0.9994
0.9984
0.9989
0.9994
4.19
3.20
5.75
5.57
3.62
12.70
9.70
17.42
16.87
10.70
determined for ACV (50 µg mL−1 in the mobile phase)
and nicotinic acid (50 µg mL−1 in the mobile phase)
as the ester degradation products.
The linearity was assessed in the range from ca. 5 to
ca. 200 µg mL−1 of the esters (Table II) in the appro-
priate mobile phase (Table I). For each sample, the test
was performed three times. A statistical evaluation of
the above relationships was described by the equation
y = bx (the intercepts where statistically insignificant).
The limits of detection (LOD) and quantitation (LOQ)
were determined by using the following formula:
was added and the contents of the tested compounds
were determined by the HPLC method. The final con-
centration of tested substances in the mixture applied
to the column was ca. 60 µg mL−1
.
The pH values of the hydrochloric acid at the tem-
perature of the study were calculated from the activity
coefficient data [20].
Statistical Calculation
Statistical analyses of the results were performed us-
ing a spreadsheet of MS Excel program. The errors
of the values were estimated and presented as a con-
fidence interval for a level of significance α = 0.05,
standard deviation (SD), and coefficient of variation
(RSD,%). Repeatability of results for series of assays
was analyzed by the F-Snedecor test for comparing of
variance and the t-student test for comparing of mean
values. The correlation analyses were performed using
the test of significance of the correlation coefficient (r).
The assessment of significance of the regression coef-
ficients was included in the regression analysis with
the significance level α = 0.05.
κ · SDa
LOD(LOQ) =
(1)
b
where к is 3.3 for LOD and 10 for LOQ, SDa is the
standard deviation of the intercept, and b is the slope.
The precision, accuracy, and reproducibility of the
method was assessed by determining the content of
tested substance in solution in the mobile phase, at
two different concentrations (µg mL−1; Ac-ACV: 25.0,
90.0; iBut-ACV and Piv-ACV: 25.0, 125.0; Etc-ACV:
25.0, 175.0; Nic-ACV: 40.0, 125.0). The study was
performed in two days (interday, intraday), and the test
was repeated six times for each series of solutions.
RESULTS AND DISCUSSION
Kinetic Procedures
HPLC Determination: Validation
of the Method
Hydrochloric acid solutions (0.05–0.50 mol L−1) were
prepared while maintaining a constant ionic strength
(µ 0.50 mol L−1) by adding varying amounts of the
sodium chloride solution (2 mol L−1). The solutions
(9.5 mL) were equilibrated at the temperature of the
study (310 K) prior to initiation of the reaction. The
reaction was initiated by adding 0.5 mL of the ester so-
lution in the same hydrochloric acid (ca. 3.6 mg mL−1).
At specified time points, a 0.2-mL of the reaction mix-
ture was transferred into 5 mL tubes and neutralized
by adding 0.2 mL of NaOH solution with a concentra-
tion equal to the concentration of the acid. Then, the
mixture was cooled in ice water to stop the reaction. To
each mixture, 0.2 mL of the internal standard solution
Separately prepared conditions (Table I) were used to
develop the RP-HPLC method for the determination of
all five esters. According to the selectivity, the peaks
position of the compounds: the signals of substrates,
expected degradation products, as well as the inter-
nal standards were separately observed (Table I). In
the case of Ac-, iBut-, Piv-, and Etc-ACV, the ACV
molecule and an aliphatic acid, which does not have
UV absorption, were formed by hydrolysis. The re-
tention time of nicotinic acid (a hydrolysis product of
ester Nic-ACV) was compatible with the elution time
of ACV (Table I). For the linearity study of the de-
termination of esters, solutions of each compounds in
International Journal of Chemical Kinetics DOI 10.1002/kin.20943

728
LESNIEWSKA ET AL.
Table III The Precision and Accuracy of the Proposed HPLC Methods for the Determination of ACV Esters in Solution
(n = 6)
Amount Found (mg) (Recovery, %)
Declared Content
Repeatability
RSDr%
Compound
Ac-ACV
(µg mL−1
)
Interday
Intraday
25.0
90.0
25.0
125.0
25.0
125.0
25.0
175.0
40.0
125.0
23.2 0.2 (92.8 0.8)
88.6 1.1 (98.5 1.3)
23.8 0.4 (95.4 1.7)
131.6 1.6 (105.3 1.3)
23.3 0.4 (93.1 1.5)
117.3 0.7 (93.8 0.5)
24.2 0.3 (96.6 1.2)
176.2 0.6 (100.7 0.4)
39.7 0.7 (99.2 1.8)
124.6 0.6 (99.7 0.5)
24.0 0.3 (96.1 1.1)
85.8 0.4 (95.3 0.4)
25.2 0.3 (101.0 1.1)
129.4 1.1 (103.5 0.9)
23.0 0.3 (92.2 1.0)
117.6 1.2 (94.0 0.9)
23.8 0.4 (95.3 1.5)
177.6 1.0 (101.5 0.6)
42.0 0.5 (104.9 1.1)
137.3 1.4 (109.8 1.1)
0.37
0.24
0.37
0.28
0.36
0.31
0.35
0.18
0.37
0.31
iBut-ACV
Piv-ACV
Etc-ACV
Nic-ACV
appropriate mobile phases in the range of 5 to about
200 µg mL−1 were prepared (Table II). The relation-
ships of the observed signals analyzed as a function
of the concentration of esters had the linear character
described by the equation y = bx. The values of correla-
tion coefficients ࣙ0.9984 indicated a strong correlation
of the examined relationships. Calculated parameters
of the equations were used to determine the limits of
detection (LOD: 3.2–5.8 µg mL−1) and quantification
(LOQ: 9.7–16.9 µg mL−1) of the tested compounds
(Table II). Comparison of the regression coefficients
(the slope) can sort the HPLC method in terms of sen-
sitivity. Thus, sensitivity for the determination of the
tested compounds was decreased in the order: Ac-ACV
> iBut-ACV > Piv-ACV > Nic-ACV > Etc-ACV
(Table II). To assess the precision, accuracy, and re-
peatability of the methods, the esters were studied in
two solutions, in significantly different concentrations.
The tests were repeated the next day for parameter es-
timation and validation on an interday and an intraday.
The values of the coefficients of variation less than
1.9% indicated an adequate precision for the research
methods. The coefficients of variation for repeatability
did not exceed 0.4% (Table III). The accuracy of the de-
veloped methods was evaluated on the basis of recovery
in the range from 93% to 96% for Ac-ACV and from
99% to 110% for Nic-ACV (Table III). The results of
statistical analyses have confirmed that the developed
chromatographic conditions provide precise, accurate,
and reproducible analyses of the esters studied in
solutions.
(0.42–1.38) at a temperature of 310 K, which was ac-
cepted as suitable for testing at a temperature approach-
ing body temperature (Table IV). All solutions were
applied to a constant value of the ionic strength (µ =
0.50 mol L−1) by the addition of an appropriate volume
of sodium chloride solution (2.0 mol L−1). The pKa of
acyclovir (pKa1 = 2.27, pKa2 = 9.25) [21] concluded
that esters of ACV exist in the protonated (BH+) and
neutral (B) forms in this pH range, and these forms
were hydrolyzed. Therefore, in the acid medium the
neutral forms are between 1.4% in pH solutions 0.42
and 11.4% in pH solutions 1.38. For the formation
of the ionized forms (BH+), the amine groups at the
C2 position of ACV esters have been protonated. In the
acidic medium, the observed hydrolysis reactions were
the pseudo–first-order reactions (Fig. 2a) described by
the following equation:
ln Pt = ln P0 − kobs · t
(2)
where Pt and P0 are the ratio of the peak areas
of the substrate to the internal standard peak deter-
mined for the respective time t and t0, kobs is the ob-
served rate constant, and t is the time of the reaction
(Table IV).
The values of the observed rate constant (kobs) cor-
responding to kpH values (acid–base catalysis) were
used to determine the catalytic rate constants, describ-
ing the catalytic effect of hydrogen ions (kH+) and the
spontaneous hydrolysis by water (kH O) on the hydrol-
2
ysis of esters (Table IV). The overall reaction of esters
hydrolysis in pH range 0.42–1.38 can be defined by the
following equation:
Observed and Catalytic Rate Constants
Kinetic studies of the decomposition of esters was car-
ried out in hydrochloric acid (0.05–0.50 mol L−1) pH
kpH = kH+ · aH+ · f1 + kH O · f1
(3)
2
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COMPARATIVE ANALYSIS OF ACYCLOVIR ESTERS STABILITY IN SOLUTIONS
729
Table IV The Kinetic Parameters (kobs) of Hydrolysis of ACV Esters in the Hydrochloric Acid Medium
(310 K, µ = 0.50 mol L−1
Compound
)
cHCl (mol L−1
)
pH
kobs ꢀkobs (s−1
)
–r
n
Parameters of kpH Equation (3)
Ac-ACV
0.05
0.10
0.20
0.30
0.40
0.50
1.38
1.10
0.81
0.65
0.52
0.42
(1.30 0.12) × 10−5
(2.40 0.15) × 10−5
(5.06 0.38) × 10−5
(6.32 0.66) × 10−5
(1.07 0.05) × 10−4
(1.28 0.05) × 10−4
0.9908
0.9928
0.9924
0.9904
0.9968
0.9977
13
18
15
11
16
15
kH+ = (3.38 0.51) × 10−4
SD = 1.96 × 10−5
kH O = ns
2
r = 0.9920
n = 6
iBut-ACV
Piv-ACV
Etc-ACV
Nic-ACV
0.05
0.10
0.20
0.30
0.40
0.50
1.38
1.10
0.81
0.65
0.52
0.42
(6.02 0.22) × 10−6
(1.37 0.06) × 10−5
(2.53 0.07) × 10−5
(4.04 0.28) × 10−5
(4.91 0.28) × 10−5
(6.45 0.55) × 10−5
0.9979
0.9966
0.9989
0.9964
0.9976
0.9937
17
18
13
10
10
11
kH+ = (1.72 0.14) × 10−4
SD = 5.56 × 10−6
kH O = ns
2
r = 0.9973
n = 6
0.05
0.10
0.20
0.30
0.40
0.50
1.38
1.10
0.81
0.65
0.52
0.42
(1.07 0.29) × 10−6
(1.92 0.22) × 10−6
(3.33 0.26) × 10−6
(5.39 0.34) × 10−6
(6.60 0.54) × 10−6
(8.57 0.31) × 10−6
0.9728
0.9919
0.9945
0.9975
0.9950
0.9990
7
9
11
9
10
10
kH+ = (2.30 0.18) × 10−5
SD = 6.84 × 10−7
kH O = ns
2
r = 0.9976
n = 6
0.05
0.10
0.20
0.30
0.40
0.50
1.38
1.10
0.81
0.65
0.52
0.42
(2.48 0.33) × 10−7
(4.06 0.44) × 10−7
(5.47 0.64) × 10−7
(7.26 0.62) × 10−7
(9.53 0.93) × 10−7
(1.15 0.10) × 10−6
0.9614
0.9779
0.9703
0.9836
0.9833
0.9877
22
19
22
22
18
16
kH+ = (2.55 0.25) × 10−6
SD = 8.98 × 10−8
kH O = (1.90 0.57) × 10−7
2
SD = 2.07 × 10−8
r = 0.9975
n = 6
0.05
0.10
0.20
0.30
0.40
1.38
1.10
0.81
0.65
0.52
(2.67 0.30) × 10−7
(4.15 0.57) × 10−7
(6.43 0.71) × 10−7
(8.72 0.54) × 10−7
(9.86 0.71) × 10−7
0.9888
0.9839
0.9894
0.9961
0.9902
11
11
11
12
19
kH+ = (2.76 0.73) × 10−6
SD = 2.28 × 10−7
kH O = (2.19 1.35) × 10−7
2
SD = 4.25 × 10−8
r = 0.9899
n = 5
ns, not significant; kH+ (mol−1 L s−1); kH (s−1).
O
2
or, by its kinetic equivalent:
calculated (Eq. (3)) and designated experimentally
confirmed the usefulness of taking account of the value
of pKa1 = 2.27 in Eq. (3) (Fig. 2b) for the analysis of
the relationship log kpH – pH. The analysis of the re-
lationship between kpH/f1 values and the hydrogen ion
activity (aH+) as well as kpH/aH+ and the fraction of
the neutral forms (f2) of Ac-, iBut-, and Piv-ACV es-
kpH = kH+ · aH+ · f1 + kHꢁ + · aH+ · f2
(4)
where aH+ refers to the hydrogen ion activity, f1 and
f2 are fractions of the ionic forms BH+ and B, respec-
tively:
ters showed that catalytic rate constants kH O are not
2
2
statistically significant (kH O = 0, Table IV) as well
2
(aH+
)
as kH+ and kꢁH+ are statistically equal (kH+ = kꢁH+).
Thus, for these esters Eq. (3) and (4) can take the
form of
f1 =
f2 =
(5)
(6)
(aH+)2 + Ka1 · aH+ + Ka1 · Ka2
Ka1 · aH+
(aH+)2 + Ka1 · aH+ + Ka1 · Ka2
kpH = kH+ · aH+(f1 + f2) = kH+ · aH+
(7)
The pKa values of esters calculated by theoreti-
cal methods showed that the substituents have no ef-
fect on the pKa1 values (MarvinSketch structure-based
calculations). The analysis of compliance kpH values
In the case of Etc- and Nic-ACV esters, the values
of kH+ and kꢁH+ are not statistically equal (kH+ ࣔ kꢁH+).
The values of the catalytic rate constants describing
International Journal of Chemical Kinetics DOI 10.1002/kin.20943

730
LESNIEWSKA ET AL.
Figure 3 Correlation between the steric factors (Es) and the
catalytic rate constants kH+ for acid-catalyzed hydrolysis of
ACV esters: acetate, isobutyrate, pivalate, and nicotinate.
•
spontaneous hydrolysis of the protonated form by
water (Etc-ACV and Nic-ACV).
The general scheme of the observed reaction can be
summarized as follows:
BH+ + H+3 O → Products, kH+
B + H+3 O → Products, kHꢁ +
Figure 2 Apparent pseudo–first-order plots for degrada-
tion of acyclovir acetate in hydrochloric acid (pH 0.42–1.38;
µ 0.50 mol L−1; 310 K) (a) and the log k-pH profiles for the
hydrolysis of ACV esters in acid medium pH 0.42–1.38 at
310 K (the circles represent the experimentally determined
values; the lines represent theoretical curves calculated from
the kpH equation (3)) (b).
BH+ + H2O → Products, kH O
2
The Influence of Steric Effects on the
Stability and Mechanism of the Reaction
It is known that structural modifications influence the
stability, physicochemical properties, and biological
activity through polar, steric, or resonance effects. Ac-
cording to the Taft’s analysis, the relative rates of acid-
catalyzed hydrolysis or esterification are determined
only by steric factors, whereas for base-catalyzed reac-
tion both polar and steric factors (Es) are involved [22].
The steric effect can also be described by the v pa-
rameter, which is independent of the kinetic data but
dependent on the van der Waals radius of the sub-
stituent group [23]. Figure 3 shows a good semilog-
arithmic correlation (r = 0.9984) between steric fac-
tors, which differentiate tested esters of ACV and the
the catalytic effect of hydrogen ions on the hydroly-
sis of tested esters (kH+) were calculated from Eq. (3)
with a good correlation of the results (Table IV) since
Eq. (4) involving the form of the neutral molecule (be-
low 11.4%) was the cause of the high error in calcula-
tion of these values.
The log kpH – pH profiles (Fig. 2b) for the hydroly-
sis of ACV esters were constructed from the logarithm
of rate constants kpH and the pH values at 310 K. The
compatibility of the experiment with calculated values
kpH (Eq. (3)) confirms the correctness of the assump-
tions made. Therefore, at 310 K in hydrochloric acid
medium (pH 0.42–1.38) the hydrolysis of the esters is
composed of
catalytic rate constants kH+
describing the catalytic
effect of hydrogen ions on the hydrolysis of these
esters. The steric factors standardized for the CH3
group – Es(CH3) were taken from the literature: methyl
0.00 (for Ac-ACV); iso-propyl –0.47 (for iBut-ACV);
•
hydrolysis of the protonated and neutral forms by
hydrogen ions (all esters) and/or
International Journal of Chemical Kinetics DOI 10.1002/kin.20943

COMPARATIVE ANALYSIS OF ACYCLOVIR ESTERS STABILITY IN SOLUTIONS
731
Scheme 1 Mechanism of acid-catalyzed hydrolysis of ACV esters.
tert-butyl –1.54 (for Piv-ACV); phenyl –2.55 (for Nic-
ACV; 2-pyridinyl and phenyl substituents was consid-
ered as isosteric) [24]. This relationship confirms the
influence of the steric effect on the stability of these
four esters. If the polar effects of substituents are irrel-
evant, Taft’s equation takes the following form:
account the value of kH+. And so, the calculated values
of Es(CH3) were –0.29 (iso-propyl), –1.17 (tert-butyl),
–2.12 (ethoxy), and –2.09 (3-pyridinyl), respectively.
The negative values indicate that these substituents de-
crease the rate of hydrolysis of the examined esters
(show a stabilizing effect) assuming only the influence
of steric effects. It should be noted that if the steric
and also inductive mesomeric effects are present in the
hydrolysis, the calculated values of Es also take into ac-
count the influence of the electronic coupling between
the substituent and the reaction center.
ks
log
= δEs
(8)
k
CH3
where log(ks/kCH ) is the ratio of the rate of the sub-
3
Computational studies suggest that the ester hydrol-
ysis mechanisms of aliphatic and aromatic acids for the
destruction of the ester bond could be different depend-
ing on the accepted model of the reaction [26–28].
The mechanism proposed for hydrolysis of methyl
stituted reaction compared to the reference reaction
and δ is the sensitivity factor for the reaction to steric
effects. The sensitivity factor in the acid hydrolysis
is 1 [25]. Therefore, the steric factors for a number
of tested compounds can be calculated by taking into
International Journal of Chemical Kinetics DOI 10.1002/kin.20943

732
LESNIEWSKA ET AL.
acetate [26] in acidic conditions assumes the involve-
ment of two water molecules and the formation of a
tetrahedral intermediate. The protonation of carbonyl
oxygen facilitates the addition of one water molecule
at the carbonyl carbon to form a tetrahedral intermedi-
ate, which is stabilized by an additional water molecule
via a hydrogen bond [26]. In this case, the hydrolysis
proceeds in two steps, i.e. the formation of the tetra-
hedral intermediate and its decomposition. A similar
two-step mechanism was proposed for acidic hydrol-
ysis of ethyl acetate and ethyl benzoate studied by a
model cluster consisting of an ester, a H3O+ cation
and 15 water molecules [27]. However, in case of three
explicit water molecules included in the modeled reac-
tion, the hydrolysis proceeds in one step and the tetra-
hedral intermediate does not form in the acid-catalyzed
hydrolysis of ethyl benzoate [28]. Calculated values
of activation energy (ꢀE‡) of the hydrolysis reaction
of methyl acetate, ethyl acetate, and ethyl benzoate
in the proposed models of reaction were 18.3 [26],
19.18 [27], and 25.10 kcal mol−1 [27], respectively.
It is common knowledge that the rates of chemical
reactions are dependent on their activation energies.
The values of the catalytic rate constants of the hy-
drolysis of ACV esters in the present experiments are
approximately two (iBut-), 10 (Piv-), and 100 times
(Etc-, Nic-) lower than the value determined for acetate
ACV (Table IV), which indicates differences in the ac-
tivation energies of their acid hydrolysis. Therefore, in
accordance with [26,27], the stability of these esters is
due to differences in activation energy of the reaction.
This may provide an additional explanation for such a
high stability of nicotinoyl and ethoxycarbonyl esters.
In case of the ethoxycarbonyl ester (carbonate es-
ter), its stability should be explained by the difference
in the mechanism of the reaction in comparison with
the other esters (Scheme 1). That fact may result from
the two-step reaction in which an unstable intermedi-
ate product has been formed. The intermediate peak
was not observed as an additional peak in the chro-
matogram of HPLC, and therefore the kinetic param-
eters of the reaction of its formation and hydrolysis
were not determined. This is a two-step reaction, and
its rate is determined by the first stage of the hydrol-
ysis, which is slower than the second stage. A rapid
preequilibrium protonation step is followed by a slow
bimolecular reaction, the attack of a water molecule
and carbonyl-oxygen fission [29–31]. Subsequently,
the reaction is followed by a fast unimolecular acyl-
oxygen fission of the alkyl hydrogen carbonate [30,32].
The stability studies of the phenolic carbonate esters
demonstrated that the carbonate esters appear to be
less reactive than the corresponding carboxylic acid
ester derivatives [33]. Thus, the mechanism of the
hydrolysis reaction of the Etc-ACV reaction (Scheme
1b) is compliant with the mechanism proposed by
Østergaard and Larsen [33].
CONCLUSIONS
The validated RP-HPLC methods have been developed
for stability studies of ACV esters (Ac-, iBut-, Piv-,
Etc-, Nic-) in acidic medium. The methods are precise,
accurate, linear and simple for this purpose. The devel-
oped chromatographic parameters allow the determi-
nation of ACV esters in the mixture with degradation
products. In the acidic medium (pH 0.42–1.38) at tem-
perature 310 K, the hydrolysis of the protonated form
by hydrogen ions and spontaneous hydrolysis of the
protonated form by water were observed. The stabil-
ity of the ACV esters is determined not only by steric
properties of their chemical structure. The hydrolysis
of the ethoxycarbonyl ester of ACV (Etc-ACV) is a
two-step reaction.
This study was partly supported by the UMP grant
no 502–14–03305411–99674 and 502–01–03319427–
08870.
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