Potential O-acyl-substituted (-)-epicatechin gallate prodrugs as inhibitors of DMBA/TPA-induced squamous cell carcinoma of skin in swiss albino mice

Article abstract of DOI:10.1002/cbdv.201000219

(-)-Epicatechin-3-gallate (1) is one of the principal catechins of green tea and exhibits cancer-preventive activities in various animal models. However, this compound is unstable in neutral or alkaline medium and, therefore, has a poor bioavailability. To improve its stability, O-acyl derivatives of 1 were prepared by isolating the partially purified tea catechin fraction from green tea extract and treating it with a variety of acylating agents. The resulting derivatives, compounds 2-6, were screened for their antitumor potential against 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced squamous cell carcinogenesis of skin in mice. The results showed that the antitumor activity decreased with the increase in size of the chain length of the acyl groups, i.e., from compound 2, derivative with an Ac group, to compound 6, possessing a valeryl group. Moreover, the C₄ derivative with a branched acyl chain, 5, had a lower activity than the linear C₄ derivative 4. This reduction in the inhibitory activity may be due to the steric hindrance by the two Me groups. Moreover, significant increases in the protein levels analyzed by ELISA of c-Jun, p65, and p53 were observed in the skin of DMBA/TPA treated mice, whereas mice treated with 2 and DMBA/TPA had a similar expression of these transcription factors than the control mice. The prodrug potential of the O-acyl derivatives 2-6 showed that they were adequately stable to be absorbed intact from the intestine, more stable at gastric pH, and suitable for oral administration. Copyright

Full text of DOI:10.1002/cbdv.201000219

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
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Potential O-Acyl-Substituted (ꢀ)-Epicatechin Gallate Prodrugs as Inhibitors
of DMBA/TPA-Induced Squamous Cell Carcinoma of Skin in Swiss Albino
Mice
by Sandeep Vyasa), Benu Manona), Tej Vir Singhb), Pritam Dev Sharmaa), and Manu Sharma*c)
a) University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh-160014, India
b) Department of Chemistry, Panjab University, Chandigarh-160014, India
c) Department of Pharmacy, Jaypee University Information Technology, Waknaghat-173215, Distt. Solan
(HP), India (phone: þ91-1792-245219; fax: þ91-1792-245362; e-mail: lantadene@hotmail.com)
(ꢀ)-Epicatechin-3-gallate (1) is one of the principal catechins of green tea and exhibits cancer-
preventive activities in various animal models. However, this compound is unstable in neutral or alkaline
medium and, therefore, has a poor bioavailability. To improve its stability, O-acyl derivatives of 1 were
prepared by isolating the partially purified tea catechin fraction from green tea extract and treating it
with a variety of acylating agents. The resulting derivatives, compounds 2–6, were screened for their
antitumor potential against 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-
acetate (TPA)-induced squamous cell carcinogenesis of skin in mice. The results showed that the
antitumor activity decreased with the increase in size of the chain length of the acyl groups, i.e., from
compound 2, derivative with an Ac group, to compound 6, possessing a valeryl group. Moreover, the C4
derivative with a branched acyl chain, 5, had a lower activity than the linear C4 derivative 4. This
reduction in the inhibitory activity may be due to the steric hindrance by the two Me groups. Moreover,
significant increases in the protein levels analyzed by ELISA of c-Jun, p65, and p53 were observed in the
skin of DMBA/TPA treated mice, whereas mice treated with 2 and DMBA/TPA had a similar expression
of these transcription factors than the control mice. The prodrug potential of the O-acyl derivatives 2–6
showed that they were adequately stable to be absorbed intact from the intestine, more stable at gastric
pH, and suitable for oral administration.
Introduction. – Green tea derived from the leaves of Camellia sinensis has been
under extensive investigations for its health benefits, and there is evidence that various
catechins (polyphenolic compounds) are responsible for its biological activities. (ꢀ)-
Epicatechin-3-gallate ((ꢀ)-ECG, 1; Fig. 1), one of the principal catechins of green tea,
has shown cancer preventive activities in various animal models [1–5]. Compound 1 is
hydrophilic and unstable in neutral and alkaline media and its bioavailability is poor
[6]. Glucuronidation, methylation, sulfation, and microbial degradation in the colon
are major barriers for its bioavailability [7][8]. These factors limit further development
of catechins as therapeutic agents. To overcome the aforementioned drawbacks, the
prodrug concept can be used, which involves the development of chemically modified
derivatives of the pharmacologically active agent that must undergo transformation in
vivo to release the active drug. Such chemical alterations of the physicochemical
properties impart the stability and consequently improve the bioavailability and
thereby increase the developability and usefulness of a potential molecule. In most
cases, prodrugs are simple chemical derivatives that require only one to two chemical or
ꢀ 2011 Verlag Helvetica Chimica Acta AG, Zꢁrich

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Fig. 1. Chemical structure of (ꢀ)-epicatechin-3-gallate (1)
enzymatic (e.g., by esterases and phosphatases) transformation steps to yield the active
parent drug. In some cases, a prodrug may consist of two pharmacologically active
drugs that are coupled together in a single molecule so that each drug acts as a
promoiety for the other. Currently, 5–7% of the drugs approved worldwide can be
classified as prodrugs, and ca. 15% of all new drugs approved in 2001 and 2002 were
prodrugs [9]. Esters are the most common prodrugs used, and it is estimated that ca.
49% of all marketed prodrugs are activated by enzymatic hydrolysis. Ester prodrugs are
accomplished by masking of the polar groups, resulting in enhanced lipophilicity and
making OH groups unavailable for phase II biotransformation or oxidative degrada-
tion [10].
A number of studies have been carried out for the preparation and evaluation of
acylated derivatives as potential catechin prodrugs with improved stability, bioavail-
ability, and pharmacological activities [11–26]. Most of the investigators have carried
out their studies with commercially available tea catechins as starting material for the
synthesis of O-acyl derivatives. Previously, we have reported the semisynthetic
synthesis and antitumor evaluation of O-acyl derivatives of (ꢀ)-epigallocatechin-3-
gallate [15]. In the present study, we report the semisynthetic preparation of O-acyl
derivatives of (ꢀ)-epicatechin-3-gallate ((ꢀ)-ECG), 2–6, from the partially purified
catechin fraction and their inhibitory effect on 7,12-dimethylbenz[a]anthracene
(DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) induced squamous cell carci-
noma of skin in Swiss albino mice. The effect of the most active derivative, i.e., the O-
acetyl derivative 2, on the expression of various transcriptional factors (AP-1, NF-kB,
and p53) was also investigated, and the physicochemical properties of compounds 2–6
were evaluated to assess their prodrug potential.
Result and Discussion. – Chemistry. Five potential O-acyl-substituted (ꢀ)-ECG
prodrugs, 2–6, were synthesized by treatment of the partially purified catechin fraction
of green tea leaves with various acylating agents, i.e., Ac2O, propionic anhydride,
butyric anhydride, isobutyric anhydride, and valeroyl chloride (Scheme). The dry green
tea leaves were extracted with hot H2O. The extract was treated with an aqueous AlCl3
solution to precipitate the tea catechins as Alꢀcatechin complexes. The complexes were
decomposed with H2SO4, and the liberated catechins were extracted with AcOEt. The
removal of the solvent under reduced pressure gave a partially purified catechin
fraction as brown colored semisolid material, which was washed with small amounts of
CHCl3 to remove any traces of caffeine.

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Scheme. Semisynthetic Synthesis of the O-Acyl Derivatives of (ꢀ)-Epicatechin-3-gallate, 2–6
For the synthesis of the O-acyl derivatives, the partially purified catechin fraction
was treated with an acylating agent in the presence of pyridine (Scheme). The acylated
material was subjected to tandem preparative-scale elution chromatography (SGC I
and SGC II) [27] and preparative TLC (PTLC), to obtain the O-acyl derivatives of
(ꢀ)-ECG present in green tea. The structures of these compounds were determined by
physical data and spectral studies. The relative Rf values show that the polarity of the O-
acyl derivatives decreased with the increase in size of the chain length of the acyl
groups, i.e., from compound 2, derivative with an Ac group, to compound 6, possessing
a valeroyl group. The O-acyl derivatives of (ꢀ)-ECG were obtained as semisolids,
except the O-isobutyryl derivative 5, which was obtained as white flakes with a m.p. of
178–1808. The optical rotations ([a]2D5 ) of the derivatives were ꢀ27.0, ꢀ31.81, ꢀ20.0,
ꢀ27.8, and ꢀ30.7 (CHCl3), respectively. The UV spectra showed the characteristic
bands of (acyloxy)phenyl rings (260–270 nm) and a band in the 216–228-nm region for
the substituted gallate moiety. The IR spectra showed bands at ca. 1673–1428 cmꢀ1
,
indicative for the presence of aromatic rings, whereas the bands in the range of 1764–
1775 cmꢀ1 were characteristic for the presence of acyl functional groups. The bands in
1
the range of 1723–1727 cmꢀ1 were characteristic for the BzO group. In the H-NMR
spectra, for all O-acyl derivatives of (ꢀ)-ECG, 2–6, a broad singlet appeared in the
range of d(H) 4.97–5.19 for HꢀC(2) and a multiplet in the range of d(H) 5.37–5.62 for
HꢀC(3). Other signals appeared in the range of d(H) 2.91–3.07 (dd, J¼4.2–4.6, 17.6–
18.1) for HaꢀC(4), 2.81–2.98 (dd, J¼1.5–2.5, 17.5–18.0) for HbꢀC(4), 6.65–6.73 (d,
J¼2.2) for HꢀC(6), and 6.49–6.59 (d, J¼2.2) for HꢀC(8). The signals for the ring B

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H-atoms appeared at d(H) 7.30–7.33 (d, J¼2.0–2.1) for HꢀC(2’), 7.15–7.20 (d, J¼
7.45–7.62) for HꢀC(5’), and 7.26–7.28 (d, J¼1.94–2.00) for HꢀC(6’). The signals for
the ring D H-atoms appeared at d(H) 7.60–7.61 as singlet for HꢀC(2’’) and HꢀC(6’’).
The signals of the alkyl H-atoms of the acyl chains appeared in the range of d(H) 2.22–
2.28 as overlapping singlets for the seven MeCO of the O-acetyl derivative of (ꢀ)-ECG,
2, 1.21–1.28 for the seven MeCH2CO and 2.49–2.62 for the seven MeCH2CO of O-
propionoyl derivative 3, 0.73–1.44 for the seven MeCH2CH2CO, 1.7–2.1 for the seven
MeCH2CH2CO, and 2.4–2.5 for the seven MeCH2CH2CO of O-butyryl derivative 4,
1.25–1.34 for the seven Me2CHCO and 2.68–2.83 for the seven Me2CHCO of O-
isobutyryl derivative 5, and 0.91–0.95 for the seven MeCH2CH2CH2CO, 1.35–1.39 for
the seven MeCH2CH2CH2CO, 1.63–1.71 for the seven MeCH2CH2CH2CO, and 2.41–
2.50 for the seven MeCH2CH2CH2CO of O-valeroyl derivative 6. In the 13C-NMR
spectra, the signals of the CO C-atoms of the acyl groups appeared in the range of d(C)
176.5–163.1 and the signals for the C-atoms adjacent to the CO group (O¼CꢀC) at
d(C) 21.0–20.1, 29.6–27.1, 36.2–35.5, 34.2–33.8, and 33.7–33.2 for compounds 2–6,
respectively. The mass spectra showed the molecular-ion peaks ([MþNH4]þ ) at m/z
754.16, 852.71, 950.38, 950.38, and 1048.49 for compounds 2–6, respectively. The purity
of the compounds was verified by HPLC (Fig. 2).
Antitumor Activity of the O-Acyl Derivatives of (ꢀ)-Epicatechin-3-gallate, 2–6.
Cancer is a multistep process involving the sequential phases of initiation, promotion,
and progression. The mouse skin carcinogenesis model is a well-characterized model
for studying the genetic and biological changes associated with the chemical initiation
of the lesions and their subsequent transition to squamous cell carcinoma. The various
O-acyl derivatives of (ꢀ)-ECG, 2–6, were screened for their tumor inhibitory potential
against 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-ace-
tate (TPA)-induced skin carcinogenesis in Swiss albino mice. The administration of 2–
6 at the maximal tolerated dose of 50 mg/kg body weight showed a significant delay in
the onset and an overall reduction of papillomas as well as a better survival rate in
comparison to the treatment with DMBA/TPA alone, suggesting an antitumor activity
of the (ꢀ)-ECG derivatives. The onset of papillomas was observed in the fifth week in
the DMBA/TPA treated mice and found to be 18%. There was a gradual rise in the
incidence of tumors that reached 100% during the 14th week (Fig. 3). The incidence of
DMBA/TPA-induced papillomas was delayed for 4 weeks by 2–4 and by 2 weeks for 5
and 6. Moreover, as shown in Fig. 3, compounds 2–6 showed a significant decrease in
the incidence of cancer (34, 39, 44, 48, and 58% vs. 100%, resp.) at the end of 19 weeks,
as compared with DMBA/TPA alone (P<0.001, P<0.01). The antitumor profile of
these compounds showed that there was a significant decrease in the activity with the
increase in size of the chain length of the acyl groups (P<0.01). Compound 2 showed a
better antitumor activity than all other derivatives (P<0.001), and compound 6 was
the least active compound among them.
The survival rate of the mice decreased significantly in the DMBA/TPA-treated
mice, as compared with the vehicle-treated group (30 vs. 80%). The survival rate of the
animals treated with the (ꢀ)-ECG derivatives was significantly higher in comparison to
the DMBA/TPA-treated group (Fig. 4). The group treated with the acetyl derivative 2
showed a survival rate of 70%, whereas those treated with 3–6 showed a survival rate
of 60% (P<0.05).

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Fig. 2. HPLC Chromatograms of the a) O-acetyl, b) O-propionyl, c) O-butyryl, d) O-isobutyryl, and
e) O-valeroyl derivatives of (ꢀ)-epicatechin-3-gallate
Fig. 3. Effect of the O-acyl derivatives of (ꢀ)-epicatechin-3-gallate, 2–6, on the incidence of DMBA/TPA-
induced tumors in mice. Significant differences compared to DMBA/TPA treatment alone: P<0.01 (**),
P<0.001 (***).

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Fig. 4. Effect of the O-acyl derivatives of (ꢀ)-epicatechin-3-gallate, 2–6, on the survival rate of DMBA/
TPA treated mice. Significant differences compared to DMBA/TPA treatment alone: P<0.05 (*), P<
0.001 (***).
The average body weight of the mice treated with 3, 4, 6, or DMBA/TPA alone did
not differ from that of the acetone-treated mice throughout the study. However, there
was a slight increase in the average body weight of the mice treated with 2 and a slight
decrease in the average body weight of the 5-treated mice at the end of the study (P<
0.001 and P<0.05, resp.). The histopathological examination of the depilated backs of
the mice revealed normal skin and subcutaneous tissue in the acetone-treated mice,
whereas the DMBA/TPA-treated animals showed well-differentiated squamous cell
carcinoma with formation of keratin pearls. There was a marked infiltration of cancer
cells in the underlying dermis. The skin section of the (ꢀ)-ECG O-acyl derivative-
treated mice showed hyperplastic papillomatous lesions without evidence of infiltration
or cytological atypia.
The results showed that the antitumor activity decreased with the increase of the
chain length of the acyl groups, i.e., from compound 2, substituted with an Ac group, to
compound 6, bearing a valeryl group. Moreover, the compound with a branched C4 acyl
chain (5) had a slightly lower inhibitory activity than compound 4, with a linear C4 acyl
chain. This may be due to the steric hindrance by the two Me groups. Indeed, this might
inhibit the enzyme-catalyzed hydrolytic cleavage of the ester bond of the O-isobutyryl
derivative of (ꢀ)-ECG (5). The pretreatment of the animals with the (ꢀ)-ECG
derivatives showed their ability to interfere with the initiation of the tumors, which is a
relatively rapid process, and the continuous treatment with the (ꢀ)-ECG derivatives
after the application of TPA interferes with the tumor promotion, which is a slow
process [28]. The compounds also increased the survival rate of the animals. This effect
could be associated with the lower papilloma burden as a result of the inhibitory effect
of the compounds on the carcinogenesis. The slight weight gain with compound 2 could
be the result of recovery from the effect of DMBA/TPA or, alternatively, the better
papilloma control.
Effect of Compound 2 on the Expression of Transcription Factors. Several
transcription factors, including the activator protein-1 (AP-1), the nuclear factor
kappa B (NF-kB), and the signal transducers and activators of transcription (STATs),
have been linked to tumor promotion [29][30]. AP-1 is an important nuclear

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
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transcription factor involved in many cellular functions, such as cell proliferation,
death, survival, and differentiation [31]. The transcription factor AP-1 consists of
homodimers and heterodimers composed of basic region leucine zipper protein that
belongs to the Jun (c-Jun, JunB, and JunD), Fos (c-Fos, Fos B, Fra-1, and Fra-2), Jun
dimerization partners (JDPs and JDP2), and the closely related activation tran-
scription factors. AP-1 Proteins have target genes regulating cell proliferation and
death. The Jun proteins control cell life and death through their ability to regulate the
expression and function of cell cycle modulators. A functional role for AP-1
components in the epidermis of the skin has been suggested for differentiation,
carcinogenesis, UV-response, photo-aging, and wound repair [30]. This might be due to
the regulation of AP-1, which can be achieved at different levels, by changes in the
transcription of genes encoding AP-1 subunits, by controlling the stability of the
mRNAs, by posttranslational processing, turnover, and modification by phosphoryla-
tion of AP-1 proteins, and by specific interaction between AP-1 proteins and other
transcription factors and cofactors [32]. There was a significant increase (P<0.05) in
the level of c-Jun protein as analyzed by ELISA in the skin of DMBA/TPA treated mice
(Group II) as compared to the control mice skin of Group I (Table 1). Moreover, there
was a significant decrease (P<0.001) observed in the level of c-Jun in the mice treated
with compound 2 and DMBA/TPA as compared to the DMBA/TPA treated mice.
Table 1. Effect of Compound 2 on the Expression of Transcription Factors in the Skin of DMBA/TPA
Treated Mice Analyzed by ELISA
Transcription factor
Protein expression [mg/ml]a)
Control
DMBA\TPA
2þDMBA\TPA
AP-1 (c-Jun)
NFkB (p65)
P53
0.223ꢁ0.08
0.156ꢁ0.02
0.228ꢁ0.05
0.562ꢁ0.01*b)
0.365ꢁ0.07**b)
0.742ꢁ0.04*b)
0.208ꢁ0.01***c)
0.116ꢁ0.04*c)
0.308ꢁ0.02*c)
a) Values are meansꢁstandard deviations of six independent observations. b) Significant differences
compared to control group (Group I): *, P<0.05; **, P<0.01. c) Significant differences compared to
DMBA/TPA treated mice (Group II): *, P<0.05; ***, P<0.001.
NF-kB has been implicated in carcinogenesis, as it plays a critical role in cell
survival, cell adhesion, inflammation, differentiation, and cell growth. Cancer is a
hyperproliferative disorder that results from tumor initiation and promotion and
ultimately produces tumor metastasis. Several genes involved in cellular transforma-
tion, proliferation, invasion, and angiogenesis are regulated by NF-kB [33]. Oxidant
stress can result in the degradation of cytoplasmic NF-kB inhibitor (IkB) and its
translocation to the nucleus [34]. In the present study, the protein expression of p65, a
subunit of the NF-kB complex, increased significantly in the DMBA/TPA treated mice
(Table 1). This indicates an activation of NF-kB, which was probably due to the
inhibition of IkB protein that resides in the cytoplasm and, hence, increased the level of
p65 expression in the nuclear fraction. The constitutive activation of NF-kB also
appears to play a role in cell proliferation [35]. A significant decrease (P<0.05) was
observed in the level of p65 in the skin nuclear fraction of the mice treated with 2 and

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DMBA/TPA as compared to that of DMBA/TPA treated mice (Table 1). This may be
due to the suppression of NF-kB by this compound. Several chemopreventive agents
are inhibitors of NF-kB activation. These inhibitors can block anyone or several steps in
the NF-kB signaling cascade, the translocation of NF-kB into the nucleus, DNA
binding of the dimers, and/or interactions with the basal transcription activation.
The tumor suppressor gene p53 is regarded as a key factor in maintaining the
balance between cell growth and cell death [36][37]. Due to its role in the regulation of
the cell cycle, alterations in p53 are critical events in carcinogenesis [38]. In the present
study, a significant increase in the p53 expression was observed in the skin nuclear
fraction of DMBA/TPA treated mice (Group II) compared to the control group
(Group I). Moreover, there was a significant decrease (P<0.05) observed in the level
of p53 in the skin nuclear fraction of mice treated with 2 and DMBA/TPA as compared
to that of the DMBA/TPA treated mice (Table 1).
Physicochemical Properties and Enzymatic Hydrolysis. In addition to an optimum
solubility and lipophilicity required for a good bioavailability, the prodrug derivatives
should be sufficiently chemically stable and bio-labile to get converted into the parent
drug molecules once absorbed. Therefore, to assess the prodrug potential of the O-acyl
derivatives of (ꢀ)-ECG, 2–6, studies on their physicochemical properties, including
solubility, partition coefficient, and chemical stability, and enzymatic hydrolysis were
carried out.
The gastrointestinal absorption of most drugs depends on the passive transport and
therefore on the combined effects of aqueous solubility and partition coefficients. On
the basis of the available data, drugs having logarithmic octanol/H2O partition
coefficients (log P values)ꢂ2 and aqueous solubilities higher than 10 mg/ml are
assumed to be well absorbed [39–42]. Needless to say that kinetic factors, such as
transport and dissolution rates, are very important in addition to the abovementioned
thermodynamic factors for estimating the absorption. The poor bioavailability of (ꢀ)-
ECG is due to the low lipophilicity and instability, which have limited its development
for clinical use. Esterification of the OH groups gave esters having fair solubilities in
buffer (pH 7.4) and a greater lipophilicity. The solubilities of the O-acyl derivatives of
(ꢀ)-ECG, 2–6, were found to be between 1.05 and 32.23 mg/ml and the log P values
between 2.42 and 6.08 (Table 2). The solubility of the O-acyl-(ꢀ)-ECG derivatives 2–6
decreased from 32.23 to 1.05 mg/ml with the increase in size of the chain length of the
acyl groups. Similarly, the lipophilicity (log P) of derivatives 2–6 increased from 2.42 to
6.08 as the side chain length increased. The solubility decreased while the lipophilicity
increased due to the chain length of the acyl groups. These results indicated that the
potential prodrugs 2–6 might be suitable for oral administration.
Table 2. Lipophilicity (log P) and Solubility of the O-Acyl Derivatives of (ꢀ)-Epicatechin-3-gallate
Compound
log P
Solubility [mg/ml]
2
3
4
5
6
2.42ꢁ0.02
4.84ꢁ0.05
5.08ꢁ0.03
5.40ꢁ0.05
6.08ꢁ0.05
32.23ꢁ0.44
22.61ꢁ0.43
13.86ꢁ0.36
2.88ꢁ0.34
1.05ꢁ0.38

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607
Prodrug derivatives should be sufficiently stable, so that they can be formulated in a
stable dosage form [42]. For a better absorption from the stomach, the drug should be
stable at gastric pH (pH 1–2). To assess these requirements, hydrolysis studies were
carried out in both phosphate buffer at pH 7.4 and HCl buffer at pH 2.0 [43]. The
phosphate buffer at pH 7.4 represented the pH in the intestine. The hydrolysis rate was
monitored by the appearance of the parent compound. The prodrugs followed pseudo-
first order rate kinetics for several half-lives, and the rate constants (k) and half-lives
(t1/2) for the prodrugs in both phosphate and HCl buffer are shown in Table 3. The rates
of hydrolysis at pH 7.4 decreased from 0.0199 to 0.0148 hꢀ1, whereas the rates of
hydrolysis at pH 2.0 increased from 0.0048 to 0.0060 hꢀ1, as the side chain length of the
O-acyl-(ꢀ)-ECG derivatives 2–6 increased. Hence, the t1/2 of the O-acyl derivatives at
pH 7.4 and pH 2.0 increased and decreased, respectively, as the acyl chain length
increased from an Ac to a valeroyl group, as shown in Table 3. These results indicated
that derivatives 2–6 might be adequately stable to be absorbed from the intestine and
that they are more stable at gastric pH, from which they will be absorbed in unionized
form.
Table 3. Hydrolysis Rates and Half-Lives at pH 7.4 and 2.0 of the O-Acyl Derivatives of (ꢀ)-Epicatechin-
3-gallate
Compound
kpH 7.4 [hꢀ1
]
kpH 2.0 [hꢀ1
]
t1/2 (pH 7.4) [h]
t1/2 (pH 2.0) [h]
2
3
4
5
6
0.0199
0.0182
0.0176
0.0162
0.0148
0.0048
0.0050
0.0051
0.0047
0.0060
34.8
38.1
39.8
42.8
46.8
144.4
138.6
135.9
147.5
115.5
The enzymatic cleavage of the O-acyl derivatives of (ꢀ)-ECG, 2–6, was assessed in
80% human plasma. It was observed that these derivatives were readily hydrolyzed
(Table 4). The hydrolysis followed pseudo-first order rate kinetics for several half-lives.
An essential prerequisite for the successful use of a prodrug is its ability to readily
release the parent drug after oral administration [41][42]. The rates of hydrolysis of the
O-acyl-(ꢀ)-ECG derivatives in 80% human plasma were found to be 2.8–2.5 hꢀ1 and
the t1/2 15.4–16.3 min. The slightly higher hydrolysis half-life of the O-isobutyryl
derivative 5 compared to the O-butyryl and O-valeroyl derivatives, 4 and 6,
respectively, may be due to steric hindrance of the Me groups in the isobutyryl
derivative. These observations indicated that compounds 2–6 are readily hydrolyzed in
Table 4. Hydrolysis Rate and Half-live of the O-Acyl Derivatives of (ꢀ)-Epicatechin-3-gallate in 80%
Human Plasma
Compound
kplasma [hꢀ1
]
t1/2 (plasma) [min]
2
3
4
5
6
2.8
2.7
2.6
2.4
2.5
14.6
15.4
15.9
16.7
16.3

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plasma to release the parent drug molecule. The rapid rate of hydrolysis observed in
80% human plasma compared to the slow rate of hydrolysis in absence of plasma under
similar conditions in buffer solution (pH 7.4) was important for these compounds. This
implies that the enzymatic reactivity of 2–6 is independent of their intrinsic ester
reactivity.
Conclusions. – There is enough evidence that (ꢀ)-ECG is a potent catechin present
in green tea and that it is responsible for the antitumor properties of its potential
prodrugs 2–6 after their transformation to release the parent molecule. However, (ꢀ)-
ECG has a poor bioavailability, due to its instability and poor aqueous solubility, and
such compounds are difficult to formulate in therapeutic dosage forms for clinical use.
Many investigators have prepared (ꢀ)-ECG derivatives to overcome these problems
using commercially available (ꢀ)-ECG as the starting material. We have devised a
method for the derivatization of (ꢀ)-ECG from the partially purified catechin fraction
of green tea by the use of acyl chlorides/anhydrides to impart the stability, followed by
isolation of these derivatives. The results of antitumor activity showed that the dose
required in an in vivo cancer model is quite high (50 mg/kg) and further modification
and optimization of the pharmacophore of (ꢀ)-ECG derivatives is required to develop
them as potent clinical antitumor agents.
The authors thank the Indian Council of Medical Research (ICMR), New Delhi, for financial
support.
Experimental Part
General. The acylating agents (Ac2O, propionic anhydride, butyric anhydride, isobutyric anhydride,
and valeroyl chloride) and the solvents (AR or HPLC grade) were purchased from E. Merck/S.D. Fine
Chemicals Ltd., India. Column chromatography (CC): silica gel (SiO2; 60–120 mesh; E. Merck, India).
TLC: precoated G silica-gel plates of appropriate sizes (0.2 mm thickness, plastic base; E. Merck, India).
HPLC: LiChrosphere C18 column (250ꢃ4.6 mm; E. Merck, India), Waters HPLC system equipped with
an automated gradient controller, a 510 pump, a U6K injector, a 481 UV detector, and a 746 data module
(Waters, USA). M.p.: Boetius stage apparatus; uncorrected [33]. Optical rotations: Rudolph Autopol III
automatic polarimeter (Rudolph, USA). UV Spectra: Perkin Elmer LAMBDA-15 spectrophotometer
(Bodenseewerke, Perkin-Elmer & Co. GmbH, Germany). IR Spectra: Perkin Elmer RX-1 spectrometer;
1
˜n in cmꢀ1. H- and 13C-NMR spectra: Bruker AC 30-NMR spectrometer, at 400 and 75 MHz, resp., in
CDCl3; d in ppm rel. to Me4Si, J in Hz. LC/ESI-MS: LC Waters Allianz 2695; mobile phase, ammonium
acetate buffer pH 6.7/MeCN 1:9; software, MassLynx 4.0 (Waters, USA). The spectra were recorded in
positive ion mode between m/z 120 and 1500.
Plant Material. Green tea was collected at the Himachal Pradesh Krishi Vishvavidyalya (HPKV),
accredited institution of the Indian Council of Agriculture Research, Palampur, Himachal Pradesh,
India. A voucher specimen (No. 1453) was deposited with the Herbarium of the University Institute of
Pharmaceutical Sciences, Panjab University, Chandigarh, India.
Extraction and Isolation of the Partially Purified Catechin Fraction. Dry green tea leaves (20 g) were
grounded to coarse powder, transferred to a conical flask, and soaked in H2O (500 ml) at r.t. for 1 h. The
contents were heated at 808 for 1 h. The mixture was cooled to r.t. and filtered. The filtrate was treated
with 15 ml of an aq. AlCl3 soln. (20% (w/v)) and 15 ml of a sat. NaHCO3 soln. to adjust the pH to 5.5–6.5.
The mixture was allowed to stand for 1 h (258). The precipitated material was collected by filtration and
then suspended in H2O (20 ml) and H2SO4 (5 ml, 40% (w/v)). The mixture was extracted with AcOEt
(3ꢃ100 ml), and the org. layer was dried (anh. Na2SO4). The solvent was removed under reduced
pressure. The residue was washed with CHCl3 (3ꢃ50 ml) to yield a partially purified catechin fraction as

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
609
amorphous brown solid (4.2 g). The above process was repeated several times to obtain the amount of
partially purified catechin fraction needed for the experiments.
General Procedure for the Synthesis of O-Acyl Derivatives of (ꢀ)-Epicatechin-3-gallate. For the
synthesis of the O-acyl derivatives of (ꢀ)-ECG, the partially purified catechin fraction was treated with
various acylating agents (Ac2O, propionic anhydride, butyric anhydride, isobutyric anhydride, and
valeroyl chloride) in the presence of pyridine. After each reaction, the TLC analysis of the acylated
material showed 4–5 spots. The acylated material was, therefore, subjected to the combined use of CC
(SiO2 (30 g/g of acylated mixture; 75ꢃ3 cm); CHCl3/AcOEt gradient; fraction size, 50 ml) and prep.
TLC to obtain the purified O-acylated (ꢀ)-ECG derivatives. The purity of these O-acyl derivatives was
checked by HPLC and found to be >98%.
(ꢀ)-5,7-O-Diacetyl-3’,4’-O-diacetyl-3’’,4’’,5’’-triacetylepicatechin-3-O-gallate (2). Semisolid (0.35 g,
0.47 mmol, 41.22%). Rf (CHCl3/AcOEt 7:3) 0.45. [a]2D5 ¼ ꢀ27.0 (c¼10.50, CHCl3). UV (MeOH): 266
(4.0), 228 (4.2). IR (KBr): 3498, 2941, 1764, 1726, 1621, 1597, 1496, 1437, 1372, 1190, 1131, 1098, 1049, 892,
790, 701, 669, 592. 1H-NMR: 2.22–2.28 (overlapping s, 7 Me); 2.98 (dd, J¼2.2, 18.0, HbꢀC(4)); 3.07 (dd,
J¼4.6, 18.0, HaꢀC(4)); 5.19 (br. s, HꢀC(2)); 5.62 (m, HꢀC(3)); 6.59 (d, J ¼ 2.2, HꢀC(8)); 6.73 (d, J ¼ 2.2,
HꢀC(6)); 7.20 (d, J ¼ 7.48, HꢀC(5’)); 7.28 (d, J ¼ 1.94, HꢀC(6’)); 7.33 (d, J ¼ 2.0, HꢀC(2’)); 7.61 (2s,
HꢀC(2’’), HꢀC(6’’)). 13C-NMR: 21.0–20.1 (7 Me); 25.9 (C(4)); 66.4 (C(3)); 76.4 (C(2)); 108.0 (C(8));
108.8 (C(6)); 109.5 (C(4a)); 118.9 (C(2’’), C(6’’)); 120.8 (C(2’)); 123.3 (C(5’)); 124.1 (C(6’)); 130.3
(C(1’’)); 135.1 (C(1’)); 141.5 (C(4’’)); 142.7 (C(3’), C(4’)); 143.2 (C(3’’), C(5’’)); 149.7 (C(7)); 149.9
(C(5)); 154.7 (C(8a)); 163.6 (C¼O); 166.4 (C¼O); 167.3 (2 C¼O); 168.1 (2 C¼O); 168.5 (C¼O); 170.2
(C¼O). ESI-MS: 754.16 ([MþNH4]þ ).
(ꢀ)-5,7-O-Dipropionyl-3’,4’-O-dipropionyl-3’’,4’’,5’’-O-tripropionylepicatechin-3-O-gallate (3).
Semisolid (0.55 g, 0.65 mmol, 59.95%). Rf (hexane/AcOEt 7:3) 0.42. [a]2D5 ¼ ꢀ31.81 (c¼11.0, CHCl3).
UV (MeOH): 269 (3.4), 220 (4.2). IR (KBr): 3453, 2968, 1769, 1725, 1624, 1596, 1494, 1440, 1388, 1348,
1219, 1183, 1133, 1089, 918, 874, 837, 771, 681. 1H-NMR: 1.21–1.28 (overlapping s, 7 Me); 2.49–2.62
(overlapping s, 7 CH2); 2.89 (dd, J¼2.1, 17.8, HbꢀC(4)); 2.95 (dd, J¼4.2, 17.7, HaꢀC(4)); 5.10 (br. s,
HꢀC(2)); 5.41 (m, HꢀC(3)); 6.56 (d, J ¼ 2.2, HꢀC(8)); 6.67 (d, J ¼ 2.2 Hz, HꢀC(6)); 6.59 (d, J ¼ 7.62,
HꢀC(5’)); 7.25 (d, J ¼ 1.94, HꢀC(6’)); 7.33 (d, J ¼ 2.0, HꢀC(2’)); 7.52 (2s, HꢀC(2’’), HꢀC(6’’)). 13C-NMR:
9.2–8.7 (7 Me); 29.6–27.1 (7 CH2); 25.9 (C(4)); 66.3 (C(3)); 76.5 (C(2)); 107.9 (C(8)); 108.7 (C(6));
109.4 (C(4a)); 118.6 (C(2’’), C(6’’)); 121.1 (C(2’)); 123.1 (C(5’)); 124.2 (C(6’)); 134.4 (C(1’’)); 135.4
(C(1’)); 141.2 (C(4’’)); 142.4 (C(3’), C(4’)); 143.3 (C(3’’), C(5’’)); 149.6 (C(7)); 149.8 (C(5)); 154.7
(C(8a)); 163.1 (C¼O); 170.2 (C¼O); 171.1 (2 C¼O); 171.9 (2 C¼O); 172.5 (C¼O); 173.9 (C¼O). ESI-
MS: 852.71 ([MþNH4]þ ).
(ꢀ)-5,7-O-Dibutyryl-3’,4’-O-dibutyryl-3’’,4’’,5’’-O-tributyrylepicatechin-3-O-gallate (4). Semisolid
(0.55 g, 0.59 mmol, 65.0%). Rf (hexane/AcOEt 8 :2) 0.47. [a]2D5 ¼ ꢀ20.0 (c¼10.0, CHCl3). UV (MeOH):
268 (4.3), 217 (4.3). IR (KBr): 3443, 2930, 1773, 1725, 1673, 1596, 1495, 1428, 1352, 1319, 1263, 1131, 1081,
1
900, 874, 770, 663. H-NMR: 0.73–1.44 (overlapping s, 7 Me); 1.7–2.1 (overlapping s, 7 CH2); 2.4–2.5
(overlapping s, 7 CH2); 2.81 (dd, J¼1.5, 17.5, HbꢀC(4)); 2.91 (dd, J¼4.3, 17.6, HaꢀC(4)); 4.97 (br. s,
HꢀC(2)); 5.37 (m, HꢀC(3)); 6.53 (d, J ¼ 2.2, HꢀC(8)); 6.65 (d, J ¼ 2.2, HꢀC(6)); 7.17 (d, J ¼ 7.45,
HꢀC(5’)); 7.26 (d, J ¼ 2.0, HꢀC(6’)); 7.31 (d, J ¼ 2.1, HꢀC(2’)); 7.61 (2s, HꢀC(2’’), HꢀC(6’’)). 13C-NMR:
13.7–13.3 (7 CH2); 18.4–18.2 (7 CH2); 36.2–35.5 (7 Me); 26.0 (C(4)); 66.3 (C(3)); 76.5 (C(2)); 108.0
(C(8)); 108.8 (C(6)); 109.5 (C(4a)); 118.7 (C(2’’), C(6’’)); 121.8 (C(2’)); 123.1 (C(5’)); 124.0 (C(6’)); 134.1
(C(1’’)); 135.5 (C(1’)); 141.0 (C(4’’)); 142.0 (C(3’, 4’)); 143.3 (C(3’’), C(5’’)); 149.7 (C(7)); 149.9 (C(5));
154.8 (C(8a)); 163.7 (C¼O); 168.9 (C¼O); 169.8 (2 C¼O); 170.1 (2 C¼O); 170.6 (C¼O); 171.0 (C¼O).
ESI-MS: 950.38 ([MþNH4]þ ).
(ꢀ)-5,7-O-Diisobutyryl-3’,4’-O-diisobutyryl-3’’,4’’,5’’-O-triisobutyrylepicatechin-3-O-gallate (5).
White flakes (0.60 g, 0.64 mmol, 71.5%). M.p. 178–1808. Rf (hexane/AcOEt 8.5 :1.5) 0.40. [a]D25
¼
ꢀ27.8 (c¼10.4, CHCl3). UV (MeOH): 266 (2.4), 216 (3.2). IR (KBr): 3450, 2977, 2938, 1769, 1727, 1627,
1598, 1466, 1434, 1324, 1219, 1131, 1091, 909, 835, 770, 525. 1H-NMR: 1.25–1.34 (overlapping s, 14 Me);
2.68–2.81 (overlapping s, 7 HꢀC); 2.83 (dd, J¼1.5, 17.5, HbꢀC(4)); 2.92 (dd, J¼4.3, 17.6, HaꢀC(4)); 5.11
(br. s, HꢀC(2)); 5.42 (m, HꢀC(3)); 6.50 (d, J ¼ 2.2, HꢀC(8)); 6.67 (d, J ¼ 2.2, HꢀC(6)); 7.16 (d, J ¼ 7.52,
HꢀC(5’)); 7.28 (d, J ¼ 1.97, HꢀC(6’)); 7.32 (d, J ¼ 2.1, HꢀC(2’)); 7.61 (2s, HꢀC(2’’), HꢀC(6’’)). 13C-NMR:
19.0–18.5 (14 Me); 34.2–33.8 (7 CH); 25.9 (C(4)); 66.4 (C(3)); 76.4 (C(2)); 107.9 (C(8)); 108.7 (C(6));

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
109.4 (C(4a)); 118.5 (C(2’’), C(6’’)); 120.4 (C(2’)); 123.6 (C(5’)); 124.2 (C(6’)); 131.0 (C(1’’)); 135.4
(C(1’)); 141.7 (C(4’’)); 142.6 (C(3’), C(4’)); 143.6 (C(3’’), C(5’’)); 149.6 (C(7)); 149.9 (C(5)); 154.8
(C(8a)); 163.4 (C¼O); 172.8 (C¼O); 173.7 (2 C¼O); 174.7 (2 C¼O); 175.2 (C¼O); 176.5 (C¼O). ESI-
MS: 950.38 ([MþNH4]þ ).
(ꢀ)-5,7-O-Divaleroyl-3’,4’-O-divaleroyl-3’’,4’’,5’’-O-trivaleroylepicatechin-3-O-gallate (6). Semisolid
(0.50 g, 0.48 mmol, 59.9%). Rf (hexane/AcOEt 9 :1) 0.43. [a]2D5 ¼ ꢀ30.7 (c¼10.4, CHCl3). UV (MeOH):
266 (3.2), 226 (4.1), 208 (4.2). IR (KBr): 3422, 2940, 2914, 2830, 1775, 1723, 1613, 1597, 1494, 1432, 1377,
1320, 1230, 1124, 1095, 1041, 900, 772, 658. 1H-NMR: 0.91–0.95 (overlapping s, 7 Me); 1.35–1.39
(overlapping s, 7 CH2); 1.63–1.71 (overlapping s, 7 CH2); 2.41–2.50 (overlapping s, 7 CH2); 2.95 (dd, J¼
2.5, 17.7, HbꢀC(4)); 2.98 (dd, J¼4.4, 18.1, HaꢀC(4)); 5.09 (br. s, HꢀC(2)); 5.43 (m, HꢀC(3)); 6.49 (d, J ¼
2.2 Hz, HꢀC(8)); 6.65 (d, J ¼ 2.2, HꢀC(6)); 7.15 (d, J ¼ 7.48, HꢀC(5’)); 7.27 (d, J ¼ 2.0, HꢀC(6’)); 7.30 (d,
J ¼ 2.0, HꢀC(2’)); 7.60 (2s, HꢀC(2’’), HꢀC(6’’)). 13C-NMR: 13.6–13.5 (7 Me); 22.1–22.0 (7 CH2); 26.8–
26.6 (7 CH2); 33.7–33.2 (7 CH2); 26.0 (C(4)); 67.5 (C(3)); 76.4 (C(2)); 107.8 (C(8)); 108.8 (C(6)); 109.2
(C(4a)); 118.4 (C(2’’), C(6’’)); 120.5 (C(2’)); 123.2 (C(5’)); 124.4 (C(6’)); 130.8 (C(1’’)); 142.4 (C(3’),
C(4’)); 135.2 (C(1’)); 141.2 (C(4’’)); 143.2 (C(3’’), C(5’’)); 149.6 (C(7)); 149.8 (C(5)); 154.7 (C(8a)); 163.4
(C¼O); 170.4 (C¼O); 171.8 (C¼O); 172.3 (C¼O); 173.5 (C¼O); 174.8 (C¼O). ESI-MS: 1048.49 ([Mþ
NH4]þ ).
Animals. Female Swiss albino mice (LACCA, six-weeks-old) weighing 18–22 g were obtained from
the Central Animal House of the Panjab University. The animals were kept in the Departmental Animal
House with a controlled temp. of 23ꢁ58, 60ꢁ5% humidity, and a 12 h light/dark cycle. They were fed a
basal diet and H2O. The mice were acclimatized for one week before experimentation. Animal care and
handling was conducted according to the guidelines set by the World Health Organisation (WHO),
Geneva, Switzerland, and the Indian National Science Academy (INSA), New Delhi, India. The protocol
for the antitumor study was approved by the Institutional Animal Ethics Committee.
In vivo Antitumor Activity. Skin tumors were induced in Swiss albino mice (LACCA/female)
according to the method of Azuine and Bhide [43]. Depilatory cream was used to remove the hairs from
the back of the mice. The animals were divided into seven groups and given the following treatments 2 d
after hair removal. The animals (n ¼ 10) of the control group (Group I) were treated with 100 ml vehicle
(acetone). The acetone was topically applied on the depilated back of each mouse twice weekly for 20
weeks. The animals of Group II (n ¼ 10) were topically treated with 7,12-dimethylbenz[a]anthracene
(DMBA; 100 nmol/100 ml acetone) on the depilated back of each mouse twice weekly for 2 weeks,
followed by the topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA; 17 nmol/100 ml
acetone) twice weekly for the next 17 weeks. The animals of groups III–VII (n ¼ 10) were orally treated
with 2–6, resp., suspended in H2O/(methoxycarbonyl)cellulose (5%) at a dose of 50 mg/kg body weight.
The dose of 2–6 was calculated on the basis of the ED50, which was between 50 and 53 mg/kg body
weight. The twice-a-week treatment with 2–6 started one week before the topical application of DMBA
(100 nmol/100 ml acetone) and was continued for the next 19 weeks. The cancer induction and promotion
were achieved as described for the animals of Group II. The body weight, the incidence of skin
papillomas, and the number of surviving animals were recorded weekly. Only those papillomas that
persisted for 2 weeks or more were taken into consideration for the final evaluation of the data.
Histology of Papillomas. The skin papillomas and the normal skin tissue were fixed in Zenkerꢂs
fixative fluid, routinely processed, and embedded in paraffin. Sections (7 mm) were stained with
hematozylin and eosin and examined under a light microscope.
Nuclear Extract Preparation and Protein Estimation by ELISA. The nuclear extract was prepared by
homogenizing the tumor and control skin (10%) in 2 ml of Buffer A (50 mm NaCl, 10 mm HEPES pH 8,
500 mm sucrose, and 1 mm EDTA). After homogenizing, 0.2% Triton-X 100 and 1 mm phenyl-
methylsulfonylfluoride (PMSF) were added. This mixture was then centrifuged at 5,000 rpm for 2 min
under cold conditions. The supernatant (cytoplasmic extract) was removed, and the pellet was re-
suspended in 500 ml of Buffer B (50 mm NaCl, 10 mm HEPES pH 8, 25% glycerol, 1 mm EDTA, and
1 mm PMSF). This mixture was then centrifuged at 5,000 rpm for 3 min under cold conditions. The
supernatant was discarded, and the pellet was resuspended in 50 ml of Buffer C (350 mm NaCl, 10 mm
HEPES pH 8, 25% glycerol, 1 mm EDTA, and 1 mm PMSF), incubated on ice for 30 min with constant
shaking, and then centrifuged at 10,000 rpm for 10 min under cold conditions. The supernatant (nuclear

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
611
extract) was removed and used for further analyses. The protein concentration in the nuclear extract was
determined using the method described by Bickers and Lowy [44]. ELISAwas carried out to quantify the
concentration of c-Jun, p65, and p53 in the nuclear extract of control skin and tumors. The assay was
standardized by titration of the different concentrations of antigens and antibodies. The wells were
coated with 5 mg of antibodies for c-Jun, p65, and p53 proteins in 100 ml of 0.05m carbonate buffer
(pH 9.6), and the plates were kept overnight at 48 in a moist chamber. The plates were flicked to remove
the unbound antigen/antibody soln., and the wells were blocked with 1% BSA in 0.1m phosphate
buffered saline (PBS pH 7.2) for 1 h at 378. The plates were flicked, and the wells washed thrice with
200 ml of PBS containing 0.05% (v/v) Tween-20. The wells were then incubated with anti-c-Jun (1:250),
anti-p65 (1:1000), anti-p53 (1:300), and primary antibody, resp., diluted in PBS containing 0.05%
Tween-20 and 1% BSA, and kept for 2 h at 378. The plates were washed again, and the wells were
incubated with peroxidase labeled anti-rabbit secondary antibody (Sigma-Aldrich) 1:1000 for c-Jun and
p65 and 1:300 for p53. The wells were incubated with anti-sheep IgG (1:1000; Sigma-Aldrich) for 2 h at
378 and further washed three times as described above. The color was developed by addition of 2,20-
azinodi(3-ethyl)benzothiozolinsulfonic acid reagent, and the absorbance at 405 nm was measured by the
ELISA reader.
HPLC Analysis of the O-Acyl Derivatives of (ꢀ)-Epicatechin-3-gallate. The O-acyl derivatives of
(ꢀ)-ECG, 2–6, were quantified by isocratic reversed-phase (RP) HPLC using a Waters equipment
consisting of two 501 pumps controlled by an automated gradient controller, a Waters 481 UV detector, a
746 data module, a 20 ml Rheodyne loop injection valve, and a RP LiChrosphere C18 column (250ꢃ
4.6 mm; 10 mm particles) eluted with mixtures of MeOH or MeCN and 0.02m phosphate buffer (pH 3.5–
4.5). The composition of the eluent and pH of the buffer were adjusted for each compound in order to
provide an appropriate tR and separation of the O-acyl derivatives of (ꢀ)-ECG. A flow rate of 1 ml/min
was maintained, and the column effluent was monitored at 270 nm. Quantification of the compounds was
carried out by measuring the peak areas or peak heights in relation to those of standard 1
chromatographed under identical conditions.
Solubility Determination. The Solubility of 2–6 was determined by HPLC in 0.05m phosphate buffer
pH 7.4 at 258 using a H2O-bath shaker [45]. An excess amount of each compound was added to buffer
(2.0 ml) in screw-capped test tubes and the suspensions vortexed for 10 min and shaken for 24 h at 258 in
a H2O-bath shaker. The solns. were filtered through membrane filters (0.45 mm) in a warm test tube, and
after appropriate dilution in the same buffer, aliquots of 10 ml were injected. The concentration of each
compound was calculated from the standard plot obtained the same day under similar conditions.
Determinations were performed in triplicate for each compound.
Lipophilicity Determination. The apparent partition coefficients (P) of 2–6 were determined in
octanol/0.05m phosphate buffer pH 7.4 at 258 [35], using the shake-flask method [46]. The phosphate
buffer and octanol were mutually saturated by shaking overnight. The system was left undisturbed for
30 min, and the layers were separated. Saturated solns. of the compounds were prepared in octan-1-ol
(2 ml) and phosphate buffer (5 ml) was added to the solns. in conical flasks. The sealed flasks were kept
shaking in a H2O-bath shaker maintained at 25ꢁ28 for 8 h. The octanol layer was removed and
appropriately diluted. The amount of drug partitioned into the buffer and octanol layers was analyzed by
HPLC by injecting 10 ml of each layer. The concentration was determined by HPLC to afford rapid
evaluation and better reliability. The partition coefficients were calculated with Eqn. 1, and the values
were reported as log P:
Partition coefficient (P)¼AUCoctanol/AUCbuffer ꢃdilution factor
(1)
Chemical Hydrolysis. The chemical stability of the (ꢀ)-ECG derivatives 2–6 in isotonic phosphate
buffer pH 7.4 and in HCl buffer pH 2.0 was studied at 378. The reaction was initiated by adding 20–50 ml
of the stock soln. (2, 1.8 mg/10 ml; 3, 1.3 mg/10 ml; 4, 1.1 mg/10 ml; 5, 12 mg/10 ml; 6, 1.4 mg/10 ml) of
each derivative diluted in MeCN to 2–5 ml of preheated buffer soln. in a screw-capped test tube. The
solns. were kept in a H2O bath at constant temp. and samples were withdrawn at appropriate time
intervals and analyzed by HPLC for the appearance of (ꢀ)-ECG (1). Pseudo first-order rate constants
(kobs) for the hydrolysis of the derivatives were determined from the slopes of the linear plots of the

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
logarithms of residual derivative against time (Eqn. 2). The half-lives (t1/2) were determined from the
pseudo-first-order rate constants using Eqn. 3.
kobs ¼slopeꢃ2.303
t1/2 ¼0.693/kobs
(2)
(3)
Enzymatic Hydrolysis. The enzymatic hydrolysis of the (ꢀ)-ECG derivatives 2–6 was studied in
human plasma (80%) at pH 7.4. The reaction was initiated by adding 20–50 ml of the stock soln. (see
above) of the derivative in MeCN to 2–5 ml of preheated plasma soln. The soln. was kept in a H2O bath
at 378, and samples of 100–250 ml were withdrawn at appropriate time intervals and added to 1–5 ml of
cold MeCN or MeOH to precipitate the plasma proteins. After immediate mixing and centrifugation for
5 min at 7000 rpm, 20 ml of the clear supernatants were analyzed by HPLC for the appearance of
compound 1. The rate constant (kobs) and half-lives (t1/2) were determined as described above (see
Chemical Hydrolysis).
Statistical Analysis. The data were analyzed by analysis of variance (ANOVA), Studentꢂs t-test, and
the c2 test. A statistical value of P<0.05 was considered significant.
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Received July 28, 2010