Enhancing the intestinal absorption of molecules containing the polar guanidino functionality: A double-targeted prodrug approach

Article abstract of DOI:10.1021/jm9011559

A prodrug strategy was applied to guanidino-containing analogues to increase oral absorption via hPEPT1 and hVACVase. L-Valine, L-isoleucine, and L-phenylalanine esters of [3-(hydroxymethyl)-phenyl]guanidine (3-HPG) were synthesized and evaluated for transport and activation. In HeLa/ hPEPT1 cells, Val-3-HPG and Ile-3-HPG exhibited high affinity to hPEPT1 (IC₅₀: 0.65 and 0.63 mM, respectively), and all three L-amino acid esters showed higher uptake (2.6- to 9-fold) than the parent compound 3-HPG. Val-3-HPG and Ile-3-HPG demonstrated remarkable Caco-2 permeability enhancement, and Val-3-HPG exhibited comparable permeability to valacyclovir. In rat perfusion studies, Val-3-HPG and Ile-3-HPG permeabilities were significantly higher than 3-HPG and exceeded/matched the high-permeability standard metoprolol, respectively. All the L-amino acid 3-HPG esters were effectively activated in HeLa and Caco-2 cell homogenates and were found to be good substrates of hVACVase (k _cat/K_m in mM^-1 · s^-1: Val-3-HPG, 3370; Ile-3-HPG, 1580; Phe-3-HPG, 1660). In conclusion, a prodrug strategy is effective at increasing the intestinal permeability of polar guanidino analogues via targeting hPEPT1 for transport and hVACVase for activation.

Full text of DOI:10.1021/jm9011559

624 J. Med. Chem. 2010, 53, 624–632
DOI: 10.1021/jm9011559
Enhancing the Intestinal Absorption of Molecules Containing the Polar Guanidino Functionality:
A Double-Targeted Prodrug Approach
Jing Sun,† Arik Dahan,‡ and Gordon L. Amidon*,†
†College of Pharmacy, University of Michigan, 428 Church Street, Ann Arbor, Michigan 48109-1065 and ‡Department of Clinical Pharmacology,
School of Pharmacy, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel
Received August 3, 2009
A prodrug strategy was applied to guanidino-containing analogues to increase oral absorption via
hPEPT1 and hVACVase. L-Valine, L-isoleucine, and L-phenylalanine esters of [3-(hydroxymethyl)-
phenyl]guanidine (3-HPG) were synthesized and evaluated for transport and activation. In HeLa/
hPEPT1 cells, Val-3-HPG and Ile-3-HPG exhibited high affinity to hPEPT1 (IC50: 0.65 and 0.63 mM,
respectively), and all three L-amino acid esters showed higher uptake (2.6- to 9-fold) than the parent
compound 3-HPG. Val-3-HPG and Ile-3-HPG demonstrated remarkable Caco-2 permeability enhan-
cement, and Val-3-HPG exhibited comparable permeability to valacyclovir. In rat perfusion studies,
Val-3-HPG and Ile-3-HPG permeabilities were significantly higher than 3-HPG and exceeded/matched
the high-permeability standard metoprolol, respectively. All the L-amino acid 3-HPG esters were
effectively activated in HeLa and Caco-2 cell homogenates and were found to be good substrates of
hVACVase (kcat/Km in mM-1 -1: Val-3-HPG, 3370; Ile-3-HPG, 1580; Phe-3-HPG, 1660). In conclu-
s
3
sion, a prodrug strategy is effective at increasing the intestinal permeability of polar guanidino
analogues via targeting hPEPT1 for transport and hVACVase for activation.
Introduction
valganciclovir, the valine ester prodrugs of poorly absorbed
antiviral drugs acyclovir and ganciclovir, respectively, exhibit
more than 50% oral bioavailability by hPEPT1-mediated
membrane transport and extensive activation to their corre-
sponding parent drugs.5-8 The rapid activation of valacyclo-
vir and valganciclovir is due to the cooperation of chemical
hydrolysis and multiple activating enzymes. However, some
particular esterase(s), such as a specific amino acid ester
prodrug-activating enzyme we identified recently, human
valacyclovirase (hVACVase), may be primarily responsible
for this activation process.9 Encouraged by the success of
valacyclovir and valganciclovir, the amino acid ester pro-
drug strategy has also been applied to other poorly absorbed
drugs, especially nucleosides10 such as zidovudine,11
floxuridine,12-15 gemcitabine,16 levovirin,17 and cytarabine.18
Results suggested that the membrane permeability was in-
creased greatly by targeting hPEPT1, and many of the
prodrugs could be activated by hVACVase.19
Frequently required for the high potency of drugs and drug
candidates, the guanidino functionality is also an obstacle for
oral absorption due to polarity and positive charge. Unless
paracellular or active transport mechanism exists, such as the
cases of metformin20,21 and famotidine,22 guanidino-contain-
ing drugs are expected to have very limited oral absorption.
Forexample, the influenza neuraminidaseinhibitorzanamivir
and the thrombin inhibitor argatroban (Scheme 1) are dosed
by inhalation23 and intravenous administration,24 respec-
tively, due to low oral bioavailability. When oral route of
administration is desired, guanidino group has to be replaced
in many cases although activity may also be compromised. To
retain the potency as well as increase oral bioavailability, the
Prodrugs are bioreversible derivatives of drug molecules
designed to overcome pharmaceutical, pharmacokinetic, or
pharmacodynamic barriers such as low oral absorption, lack
of site specificity, insufficient chemical stability, poor solubi-
lity, toxicity, etc.1 To improve oral absorption, a classic
prodrug approach can be adopted to enhance drug lipophili-
city and passive diffusion. In recent years, the understanding
of membrane transporters has promoted a novel targeted
prodrug approach utilizing carrier-mediated transport to
increase intestinal permeability.2 Once the goal is achieved,
prodrugs must be converted to the active parent drug to exert
therapeutic effect. This activation process is not necessary to
be specific; however, a good understanding of the possible
activating enzymes will help the rational design of successful
prodrugs.
Human peptide transporter 1 (hPEPT1a), the oligopeptide
transporter expressed in the apical brush border membrane of
the small intestine, mediates the transport of di- and tripep-
tides and a variety of peptidomimetics across the intestinal
wall.3,4 The broad substrate specificity of hPEPT1 makes it an
attractive target for prodrug design. By conjugating to an
amino acid promoiety, a low permeability drug can be
recognized by hPEPT1 transporter and obtain significantly
higher oral absorption. As examples, valacyclovir and
*To whom correspondence should be addressed. Phone: 734-764-
2226. Fax: 734-764-6282. E-mail: glamidon@umich.edu.
a Abbreviations: hPEPT1, human peptide transporter 1; hVACVase,
human valacyclovirase; 3-HPG, [3-(hydroxymethyl)phenyl]guanidine;
Boc, tert-butyloxycarbonyl; OCT, organic cation transporter; TEA,
tetraethylammonium.
r
pubs.acs.org/jmc
Published on Web 12/03/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 625
Scheme 1. Structures of Guanidino-Containing Drugs Zana-
mivir and Argatroban
Scheme 2. Structures of 3-HPG and Its L-Valine, L-Isoleucine,
L-Phenylalanine, and D-Valine Esters
classic prodrug strategy can be used to mask the charge and
facilitate passive diffusion, following activation to the parent
drug.25-28 However, this strategy has met with only limited
success, partly due to the insufficient membrane transport and
complicated activation. Moreover, if the drug is zwitterionic
such as zanamivir and argatroban, all the charges need to be
shielded, which further complicates both the synthesis and the
in vivo activation. On the other hand, oral bioavailability can
be increased by a targeted prodrug strategy using mucosal
transporters which may tolerate charges, such as hPEPT1, as
prodrug carrier. It is well-known that guanidino group occurs
in arginine-containing small peptides, which are natural sub-
strates of hPEPT1 transporter. Although hPEPT1 appears to
prefer neutral side chains instead of the charged ones,3,4,29,30
many arginine-containing small peptides still have at least
medium affinity to hPEPT1 transporter and show evidence of
transport.29-32 As for the amino acid ester prodrug strategy,
the questions may arise are: (1) whether the guanidino func-
tionality on the parent drug structure affects recognition and
transport by hPEPT1, and (2) if the guanidino functionality
plays a negative role, whether it canbecompensated by a good
amino acid promoiety so the prodrug will be transported. If
the guanidino group can be tolerated by hPEPT1, the amino
acid ester prodrug strategy may be applied to guanidino-
containing drugsaslong as anesterbondcanbemadethrough
a hydroxyl group in the drug structure or a linker.
Another challenge for prodrug design is the activation
process; the prodrug form should be retained prior to absorp-
tion and be quickly metabolized to the parent compound
following absorption. The classic prodrug strategy suffered
from the unpredictable activation of the shielded guanidino
functionality. The targeted prodrug strategy, however, cir-
cumvents this problem by leaving the guanidino group intact
and at the same time introducing a well-studied and relatively
predictable amino acid ester bond. We have found that one of
the primary amino acid ester prodrug-activating enzymes is
hVACVase,9 a serine hydrolase containing a catalytic triad
S122-H255-D227. The very specific preference for amino
acid ester substrates is attributed to the critical residue D123
forming electrostatic interaction with the R-amino group of
substrate. hVACVase contains a large leaving group accom-
modating groove, which accommodates various leaving
groups including nucleoside analogues acyclovir, ganciclovir,
floxuridine, gemcitabine, zidovudine, and BDCRB as well as
simple alcohols such as methanol, ethanol, and benzyl alco-
hol.9,19,33 In addition, the crystal structure of hVACVase
revealed negative electrostatic potential in the groove, which
may prefer a positively charged leaving group. Therefore,
hVACVase may be a good target for guanidino-containing
prodrugs in order to control the activation process and secure
the quick release of parent drug in vivo.
The purpose of this study was to apply a double-targeted
(hPEPT1 and hVACVase) prodrug strategy for guanidino-
containing molecules. We have designed and synthesized
L-valine, L-isoleucine, and L-phenylalanine esters of a model
parent compound, [3-(hydroxymethyl)phenyl]guanidine
(3-HPG, Scheme 2). These promoieties were chosen based
on affinity to hPEPT1 transporter and stability profiles
learned from our previous studies of amino acid nucleoside
prodrugs. D-Valine ester of 3-HPG was synthesized and used
as a control to help determine the transport mechanism. The
different model compoundswere tested for hPEPT1-mediated
uptake and transport across HeLa/hPEPT1 and Caco-2 cells,
intestinal permeability in the rats, and activation by hVAC-
Vase. This setup allowed us to evaluate the feasibility of the
suggested strategy to improve oral absorption of low perme-
able guanidino-containing drugs.
Results
Synthesis of 3-HPG and Its Valine, Isoleucine, and Phenyl-
alanine Esters. To synthesize 3-HPG, 3-amino benzyl alcohol
was treated with 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-
thiopseudourea to convert the amino group to Boc-pro-
tected guanidino group. Followed by cleavage of the Boc
group, 3-HPG was obtained as TFA salt (Scheme 3).
Amino acid esters were synthesized by coupling intermedi-
ate 2 and Boc-protected amino acids followed by deprotec-
tion (Scheme 3).
Hydrolysis in Buffer and Cell Homogenates. Hydrolysis
studies of 3-HPG and its L-amino acid esters were conducted
in buffer and cell homogenates and the estimated half-lives
(t1/2) obtained from linear regression of pseudo-first-order
plots are shown in Table 1. The half-life of the reference
amino acid ester prodrug, valacyclovir, is also listed for
comparison. In all buffers and cell homogenates, the parent
compound 3-HPG exhibited good stability, which makes it
an excellent model compound for subsequent studies. All the
L-amino acid esters were also stable in pH 6 uptake buffer
and relatively stable in pH 7.4 phosphate buffer. In both
HeLa and Caco-2 cell homogenates, the estimated half-lives
of L-amino acid esters were much shorter than that in pH 7.4
buffer, suggesting predominant contribution of enzymatic
hydrolysis.
hVACVase-Mediated Hydrolysis. The estimated Michaelis-
Menten kinetic parameters of 3-HPG esters as well as
valacyclovir are listed in Table 2. All L-amino acid esters
of 3-HPG showed high affinity to hVACVase, with Km
values ranging from 9 to 207 μM. Interestingly, the kcat
values followed the same order as Km values (Phe-3-HPG

626 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Sun et al.
Scheme 3. Synthesis of 3-HPG and Its Valine, Isoleucine, Phenylalanine, and D-Valine Esters
Table 1. Estimated Half-Lives in pH 6 Uptake Buffer, pH 7.4 Phos-
phate Buffer, HeLa Cell Homogenates, and Caco-2 Cell Homogenates
(Mean ( SEM; n = 3)
Table 3. [3H]Gly-Sar Uptake Inhibition in HeLa/hPEPT1 Cells (Mean
( SEM; Valacyclovir, n = 6; others n = 4)
IC50 (mM)
half-lives (min)
3-HPG
Val-3-HPG (5a)
Val-OBz
>3
HeLa cell Caco-2 cell
pH 6 buffer pH 7.4 buffer homogenates homogenates
0.65 ( 0.04
0.39 ( 0.04
0.63 ( 0.04
1.05 ( 0.13
2.33 ( 0.45
>3
Ile-3-HPG (5b)
Ile-OBz
3-HPG
stablea
stablea
stablea
stableb
stablea
stablea
stablea
20.3 ( 0.7
137.2 ( 3.6
,5
stablea
4.8 ( 0.2
59.6 ( 2.3
,1
Val-3-HPG (5a)
Ile-3-HPG (5b)
Phe-3-HPG (5c)
valacyclovir
493.6 ( 10.4
704.8 ( 9.7
261.8 ( 5.0
Phe-3-HPG (5c)
Phe-OBz
valacyclovir
829.4 ( 25.0 142.2 ( 1.2
31.3 ( 0.8
1.42 ( 0.18
a No area decrease detected in HPLC after 2 h. b Less than 5%
decrease after 2 h.
Table 4. Direct Uptake and Stability in HeLa/hPEPT1 and Normal
HeLa Cells (Mean ( SEM; n = 3)
Table 2. Michaelis-Menten Kinetic Parameters for hVACVase-
Mediated Hydrolysis (Mean ( SEM; n = 3)
HeLa/hPEPT1 cells
HeLa cells
uptake
uptake
Km
(μM)
Vmax
(nmol/min/μg)
kcat
(s-1
kcat/Km
(mM-1 s-1
(nmol/mg stabilitya (nmol/mg stabilitya hPEPT1/
)
)
3
in 60 min)
(%)
in 60 min)
(%)
control
Val-3-HPG (5a)
Ile-3-HPG (5b)
46 ( 5
9.0 ( 1.2
321 ( 9
30 ( 1
718 ( 16
120 ( 2
154 ( 4
14 ( 1 1580 ( 260
345 ( 8
58 ( 1
3370 ( 460
3-HPG
5.5 ( 0.6
3.8 ( 0.1
<2
<2
1.4 ( 0.2
>25
>13
Val-3-HPG (5a) 50.2 ( 1.6 96.9 ( 0.0
Ile-3-HPG (5b) 27.7 ( 1.4 98.7 ( 0.0
Phe-3-HPG (5c) 14.8 ( 1.7 83.8 ( 0.3 1.7 ( 0.2 87.2 ( 0.9 8.8 ( 2.0
valacyclovir 5.5 ( 0.3 96.8 ( 0.1 <0.2 99.2 ( 0.0 >25
98.0 ( 0.1
99.0 ( 0.1
Phe-3-HPG (5c) 207 ( 16
valacyclovir 68 ( 4
1660 ( 160
850 ( 66
> Val-3-HPG > Ile-3-HPG), so the specificity constant,
kcat/Km, did not differ as much as Km or kcat value itself.
When compared with valacyclovir, Val-3-HPG exhi-
bited higher, but not significantly different affinity. All the
3-HPG esters showed higher specificity constant than val-
acyclovir, clearly indicating that they are good substrates of
hVACVase.
a Percentage of ester form in donor solution at 60 min.
although it was not as good as valacyclovir. Comparing with
the corresponding benzyl esters, all the 3-HPG esters showed
similar or higher affinity to hPEPT1.
Direct Uptake. The results of direct uptake study in HeLa/
hPEPT1 cells and normal HeLa cells are summarized in Table
4. The parent compound 3-HPG, its valine and isoleucine
esters and valacyclovir were very stable in donor solution
(>95% remaining after 1 h). Phe-3-HPG was also relatively
stable (>80% remaining after 1 h). Valacyclovir, a known
substrate of hPEPT1 transporter, showed more than 25-fold
higher uptake in HeLa/hPEPT1 cells compared to that in
normal HeLa cells. The 3-HPG esters displayed 8- to more
than 25-fold increased uptake in HeLa/hPEPT1 cells com-
pared with in normal HeLa cells, whereas 3-HPG only showed
little enhancement. In HeLa/hPEPT1 cells, the uptake of the
amino acid esters was 2.6- to 9-fold higher than 3-HPG.
[3H]Gly-Sar Uptake Inhibition. The IC50 values of 3-HPG,
its valine, isoleucine, and phenylalanine esters to inhibit
[3H]Gly-Sar uptake in HeLa/hPEPT1 cells are listed in Table 3.
The amino acid benzyl esters were used for comparison to
obtain the contribution of guanidino group. Valacyclovir, a
known substrate of hPEPT1, was used as positive control.
The parent compound 3-HPG showed high IC50 value in
[3H]Gly-Sar uptake inhibition study. Valine and isoleucine
esters of 3-HPG, however, exhibited lower IC50 values than
valacyclovir, indicating good affinity to the hPEPT1 trans-
porter. Phe-3-HPG also showed some affinity to hPEPT1,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 627
Figure 1. Apparent apical-to-basolateral permeability coefficient (Papp) across Caco-2 monolayers (mean ( SEM; n = 3; *difference is
statistically significant).
Table 5. Stability in Apical Side during Permeability Study across
Rat Perfusion Studies. The effective permeability (Peff) of
Caco-2 Monolayers (Mean ( SEM; n = 3)
3-HPG, Val-3-HPG, and Ile-3-HPG in rat jejunum perfu-
sion study is shown in Figure 3. Metoprolol was co-perfused
stabilitya (%)
with the test compounds as a reference standard for perme-
ability in close proximity to the low/high permeability class
boundary. It can be seen that Val-3-HPG and Ile-3-HPG
exhibited higher or similar permeability compared to meto-
prolol, respectively, whereas the permeability of 3-HPG was
very low. Hence, the amino acid ester prodrug approach
significantly improved the rat jejunal permeability of
3-HPG, bringing it to high-permeability level.
3-HPG
Val-3-HPG (5a)
Ile-3-HPG (5b)
Phe-3-HPG (5c)
D-Val-3-HPG (5d)
valacyclovir
86.5 ( 0.3
96.6 ( 0.6
3.9 ( 2.0
98.8 ( 0.1
94.7 ( 0.3
a Percentage of ester form in donor solution at 120 min.
Caco-2 Monolayer Permeability Studies. The permeability
of 3-HPG and its amino acid esters across Caco-2 mono-
layers is shown in Figure 1. Val-3-HPG and Ile-3-HPG
exhibited higher permeability than the parent compound
3-HPG, and the permeability of Val-3-HPG was comparable
to valacyclovir. Because both Val-3-HPG and Ile-3-HPG
were stable in the donor solution (Table 5) and only the
parent compound was detected in the receiver solution, most
of the esters appeared to be metabolized in the cells prior to
penetration of the basolateral membrane. Phe-3-HPG did
not show significantly different permeability than 3-HPG.
However, due to its low stability in the donor solution
(Table 5), the permeability may be underestimated. The
stability issues, the lower affinity to hPEPT1 transporter,
and uptake in HeLa/hPEPT1 cells indicated that Phe-3-HPG
was not a favorable candidate for in vivo studies.
In contrast to Val-3-HPG, no transport of D-Val-3-HPG
was detected. Because D-Val-3-HPG and Val-3-HPG are
enantiomers, they should have similar permeability if the
transport mechanism is predominantly passive diffusion.
The much higher permeability of Val-3-HPG suggested
that the transport was carrier-mediated. To identify the
transporter(s) involved in Val-3-HPG transport, we also
investigated the effect of inhibitors on Val-3-HPG perme-
ability, including hPEPT1 inhibitors Gly-Sar and cepha-
lexin, amino acid transporter inhibitors L-valine and
L-arginine, and organic cation transporter (OCT) inhibitor
tetraethylammonium (TEA). As shown in Figure 2, only
hPEPT1 inhibitors reduced Val-3-HPG permeability, sug-
gesting hPEPT1 playing a dominant role in Val-3-HPG
transport.
Discussion and Conclusions
The positively charged guanidino functionality provides
favorable interactions with important drug targets such as
thrombin and influenza neuraminidase. However, the perma-
nent positive charge at all relevant pHs can also cause low
membrane permeability and poor oral bioavailability. To
overcome this drug delivery barrier, we have utilized a tar-
geted prodrug strategy, aiming to exploit hPEPT1 as the
prodrug transport carrier and hVACVase as the predominant
activating enzyme. This double-targeted prodrug approach
may have the advantages of higher transport accompanied by
more predictable activation, leading to favorable properties
for oral delivery.
A primary concern of this research was whether guanidino
functionality can be tolerated and transported by hPEPT1
transporter. In [3H]Gly-Sar uptake inhibition studies, both
the valine and isoleucine esters of 3-HPG showed high affinity
to hPEPT1, while the comparison between benzyl esters and
3-HPG esters did not show significant difference. Therefore,
at least in the framework of this research, there was no
evidence that the guanidino functionality hindered the bind-
ing to hPEPT1 transporter. Theresults indirect uptake, Caco-
2 monolayer permeability, and rat perfusion studies further
support our hypothesis that the amino acid esters of 3-HPG
can be transported by hPEPT1 for improved permeability. In
normal HeLa cells, the uptake of the amino acid esters was
even lower than the parent compound 3-HPG. However, in
HeLa/hPEPT1 cells, the amino acid esters showed 2.6- to
9-fold higher uptake than 3-HPG, which can be attributed to

628 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Sun et al.
Figure 2. Apparent apical-to-basolateral permeability coefficient (Papp) of Val-3-HPG without or with 5 mM inhibitors across Caco-2
monolayers. (mean ( SEM; n = 3; * difference is statistically significant).
Figure 3. Rat jejunal membrane permeability (mean ( SEM; metoprolol, n = 12; others, n = 4; *difference is statistically significant).
hPEPT1-mediated transport. In the same fashion, both valine
and isoleucine esters of 3-HPG exhibited considerably higher
permeability across Caco-2 monolayers than the parent com-
pound. The low permeability of D-Val-3-HPG and the studies
with inhibitors clearly demonstrate that this enhancement can
be attributed to hPEPT1. In rat perfusion studies, the effective
permeability of Val-3-HPG and Ile-3-HPG exceeded or
matched the high-permeability drug metoprolol, indicating
high intestinal permeability. Taken together, these results
demonstrate that it is feasible to target guanidino-containing
compounds to hPEPT1 transporter for enhanced oral absorp-
tion. Up to now, the amino acid ester prodrug strategy has
mainly been limited to nucleoside analogues. The positive
results with guanidino functionality illustrated great promise
to expand this strategy to other polar and charged low-
permeability drugs.
permeability in both cell culture and rat perfusion model,
which is consistent with 50-amino acid ester prodrugs of
floxuridine,12 gemcitabine,16 levovirin,17 and cytarabine.18
The phenylalanine ester exhibited the lowest affinity to
hPEPT1, smallest uptake enhancement, and no significant
improvement in permeability studies. However, the poor
stability in the donor solution of Caco-2 monolayer
(Table 5) inevitably leads to underestimation of the perme-
ability. This is also the case with 50-phenylalanine ester of
levovirin,17 where the low Caco-2 permeability accompanied
low apical stability. Indeed, the 50-phenylalanine ester pro-
drugs of floxuridine and gemcitabine exhibited enhanced
uptake and permeability compared to corresponding parent
drugs. Therefore, phenylalanine should not be excluded as a
promising promoiety for hPEPT1 targeting. However, when
phenylalanine ester prodrugs are designed, attention must be
paid to make sure the prodrug is relatively stable in the brush
border membrane to realize their full potential.
In addition to the guanidino functionality, this study
provided more insights into the influence of amino acid
promoieties on the hPEPT1-mediated transport. Among the
three amino acid esters, valine ester exhibited the highest
When utilizing the prodrug approach to increase oral
absorption, the activation mechanism is often overlooked.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 629
As long as the parent drug can be regenerated, the activation
will be considered successful, and the enzymes responsible for
the prodrug activation may not capture much further atten-
tion. However, the activation process is actually the unique
and one of the most critical steps for a prodrug to exert
therapeutic effect. If the possible activating enzymes are
identified, prodrugs can be designed to target these enzymes,
which greatly increase the chances for the effective production
of the parent drug. Although the amino acid ester prodrug
strategy has been applied to many nucleoside analogues and
improved the oral absorption by targeting hPEPT1, the
activation was not well-studied and was considered nonspe-
cific until the identification of the amino acid prodrug-activa-
ting enzyme hVACVase.9 hVACVase is at least one of the
primary enzymes activating valacyclovir in vivoand iscapable
of activating many other amino acid ester prodrugs of nucleo-
side analogues.19 The large leaving group accommodating
groove of hVACVase makes it an ideal target for prodrug
design. The data presented in this paper demonstrate that a
positively charged leaving group, 3-HPG, can be well tole-
rated by hVACVase, although whether the guanidino func-
tionality helped the binding is still unclear. The high specificity
constant (kcat/Km) of all the 3 L-amino acid esters of 3-HPG is
consistent with their fast activation in Caco-2 cell homo-
genates as well as Caco-2 monolayer permeability studies.
Therefore, hVACVase can be exploited as the target for
activating amino acid ester prodrugs of guanidino-containing
drugs. Atthe same time, the possibility that other enzymes can
also contribute to the activation of amino acid ester prodrugs
cannot be ruled out. We have estimated the hydrolysis rate of
3-HPG estersinCaco-2 cell homogenates withthe kinetic data
in recombinant hVACVase based on the assumption that
hVACVase was the predominant enzyme activating valacy-
clovir. After comparison with the hydrolysis data in Caco-2
homogenates, it turns out that hVACVase was also the
primary enzyme responsible for Val-3-HPG and Ile-3-HPG
hydrolysis, whereas the hydrolysis of Phe-3-HPG may involve
other enzymes.
from Sigma-Aldrich Co. (St. Louis, MO). Cell culture reagents
were obtained from Invitrogen (Carlsbad, CA), and cell culture
supplies were obtained from Corning (Corning, NY) and Falcon
(Lincoln Park, NJ). All chemicals were either analytical or HPLC
grade.
Synthesis. NMR spectra were obtained on a Bruker
AVANCE DRX500 NMR spectrometer. Electronspray ioniza-
tion mass spectra were obtained on a Micromass LCT time-of-
flight mass spectrometer. The purity of all synthesized test
compounds was at least 95% as determined by HPLC.
[3-(Hydroxymethyl)phenyl]guanidine (3-HPG, 3). To a stirred
solution of 3-amino benzyl alcohol (154 mg, 1.25 mmol) in 1 mL
of dry tetrahydrofuran (THF), 1,3-bis(tert-butoxycarbonyl)-2-
methyl-2-thiopseudourea (145 mg, 0.5 mmol) in 1.5 mL of THF
was added dropwise, and then temperature was increased to
50 °C. After 2 h, the reaction was stopped and solvents were
removed. The residue was dissolved in 30 mL of dichloro-
methane and washed twice with 10 mL of brine. The organic
phase was dried over anhydrous MgSO4 and concentrated in
vacuo. The product 2 was purified by column chromatography
(CH2Cl2:MeOH, 60:1) as colorless oil. Yield: 82.4%. Then
119.2 mg of 2 was dissolved in 4 mL of trifluoroacetic acid
(TFA):CH2Cl2 (1:1) and stirred at room temperature for 5 h.
Then solvents were removed and the residue was dissolved in
0.1% TFA, filtered, and lyophilized. The raw product was
further purified by semiprep HPLC to give a colorless oil. Yield:
1
69.1%. H NMR (D2O) δ 7.40 (1H, dd, J = 7.8 Hz, 7.8 Hz,
H-5), 7.29 (1H, d, J=7.8 Hz, H-4 or H-6), 7.22 (1H, s, H-2), 7.17
(1H, d, J=7.8 Hz, H-4 or H-6), 4.56 (2H, s, -CH2OH); ESI-MS:
166.0 (M þ H)þ.
L-Isoleucine [3-[(Aminoiminomethyl)amino]phenyl]methyl Es-
ter (Ile-3-HPG, 5b). First, 33.1 mg of 2 (0.09 mmol), 35.6 mg of
Boc-L-isoleucine (0.154 mmol), and 20.5 mg of 4-(dimethyl-
amino)pyridine were dissolved in 1 mL of dry CH2Cl2 and stirred
in ice bath. Then 30.5 mg of N-(3-dimethylaminopropyl)-N0-
ethylcarbodiimide hydrochloride (EDC HCl, 0.159 mmol) was
3
slowly added to the above solution. The reaction mixture was
stirred overnight at room temperature until reaction was complete.
The solvents were removed and residue was dissolved in 30 mL of
CH2Cl2 and washed with 10% (w/v) citric acid, saturated NaH-
CO3, and brine. The organic phase was dried over anhydrous
MgSO4 and concentrated in vacuo. The mixture was then chro-
matographed on silica gel to obtain 4b as colorless oil. 4b was
treated with 2.2 mL of TFA:CH2Cl2 (1:1.2) under argon for 2 h.
Solvents were removed and residue was dissolved in 0.1% TFA,
filtered, and lyophilized to give 5b as white solid. 5a, 5c, and5d were
synthesized similarly. Yield of two steps: 70.9%. 1H NMR
(methanol-d4) δ 7.53 (1H, dd, J=7.8 Hz, 7.8 Hz, H-5), 7.44 (1H,
d, J=7.8 Hz, H-4 or H-6), 7.38 (1H, s, H-2), 7.33 (1H, d, J=7.8 Hz,
H-4 or H-6), 5.35 (2H, m, -CH2-O-), 4.10 (1H, d, J=3.9 Hz, H-
R), 2.02 (1H, m, H-β), 1.51 (1H, m, CH2-γ), 1.35 (1H, m, CH2-γ),
0.99 (6H, m, CH3-γ, CH3-δ). ESI-MS: 279.1 (M þ H)þ.
Inconclusion, the aminoacid esters ofa guanidino-contain-
ing model compound exhibited high affinity to hPEPT1
transporter, leading to enhanced intestinal permeability ac-
companied by hVACVase-mediated activation to the parent
compound. Hence, it may be feasible to apply the same
strategy to other poorly absorbed guanidino analogues. This
double-targeted approach makes the prodrug design more
rational and less empirical, with the ability to mechanistically
control the two most important aspects governing prodrug
success.
L-Valine [3-[(Aminoiminomethyl)amino]phenyl]methyl Ester
(Val-3-HPG, 5a). Yield of two steps: 39.5%. 1H NMR
(methanol-d4) δ 7.53 (1H, dd, J =7.8 Hz, 7.8 Hz, H-5), 7.45
(1H, d, J=7.8 Hz, H-4 or H-6), 7.38 (1H, s, H-2), 7.33 (1H, d, J=
7.8 Hz, H-4 or H-6), 5.36 (2H, m, -CH2-O-), 4.02 (1H, d, J=
4.5 Hz, H-R), 2.32 (1H, m, H-β), 1.07 (6H, m, H-γ). ESI-MS:
265.1 (M þ H)þ.
Experimental Section
Materials. The tert-butyloxycarbonyl (Boc) protected amino
acids, Boc-L-valine, Boc-L-isoleucine, Boc-L-phenylalanine, and
Boc-D-valine wereobtained from Calbiochem-Novabiochem (San
Diego, CA). Valacyclovir was a gift from GlaxoSmithKline, Inc.
(Research Triangle Park, NC). High-performance liquid chroma-
tography (HPLC) grade acetonitrile was obtained from Fisher
Scientific (St. Louis, MO). L-Isoleucine benzyl ester 4-toluenesul-
fonate salt was obtained from Chem-Impex (Wood Dale, IL).
3-Amino benzyl alcohol, 1,3-bis(tert-butoxycarbonyl)-2-methyl-
2-thiopseudourea, N-(3-dimethylaminopropyl)-N0-ethylcarbodi-
L-Phenylalanine [3-[(Aminoiminomethyl)amino]phenyl]methyl
1
Ester (Phe-3-HPG, 5c). Yield of two steps: 79.7%. H NMR
(methanol-d4) δ 7.49 (1H, dd, J = 7.9 Hz, 7.8 Hz, H-5),
7.20-7.38 (8H, m, aromatic protons), 5.27 (2H, s, -CH2-
O-), 4.41 (1H, t, J = 7.0 Hz, H-R), 3.25 (2H, m, H-β). ESI-
MS: 313.1 (M þ H)þ.
D-Valine [3-[(Aminoiminomethyl)amino]phenyl]methyl Ester
(D-Val-3-HPG, 5d). Yield of two steps: 86.6%. 1H NMR
(methanol-d4) δ 7.53 (1H, dd, J=7.8 Hz, 7.8 Hz, H-5), 7.44 (1H,
d, J=7.8 Hz, H-4 or H-6), 7.38 (1H, s, H-2), 7.32 (1H, d, J=7.8 Hz,
H-4 or H-6), 5.35 (2H, m, -CH2-O-), 4.02 (1H, d, J=4.5 Hz,
imide hydrochloride (EDC HCl), 4-(dimethylamino)pyridine
3
(DMAP), trifluoroacetic acid (TFA), L-valine benzyl ester hydro-
chloride, L-phenylalanine benzyl ester hydrochloride, metoprolol,
phenol red, and all other reagents and solvents were purchased

630 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Sun et al.
H-R), 2.32 (1H, m, H-β), 1.06 (6H, m, H-γ). ESI-MS: 265.2
(M þ H)þ.
and 25 mM MES) and incubated with 10 μM Gly-Sar (9.9 μM
Gly-Sar and 0.1 μM [3H]Gly-Sar) and various concentrations
(0.05-5 mM) of test compounds in 0.3 mL uptake buffer for
30 min in an atmosphere of 5% CO2 and 90% relative humidity
at 37 °C. After 30 min, the donor solutions were aspirated and
the cells were washed three times with ice-cold uptake buffer and
solubilized with 0.1% SDS. Aliquots of the suspensions were
used for scintillation counting (Beckman LS 6000SC). IC50
values were determined using nonlinear data fitting
(GraphPad Prism software version 4.01).
Direct Uptake. The carrier-mediated uptake of the test com-
pounds was screened in HeLa/hPEPT1 cells and normal HeLa
cells. Prior to experiment, HeLa cells were grown in 12-well
plates for 5 days and infected with adenovirus containing
hPEPT1 3 days before experiment.34 Cells were washed with
uptake buffer (pH 6.0, 140 mM NaCl, 5.4 mM KCl, 1.8 mM
CaC12, 0.8 mM MgSO4, 5 mM D-glucose, and 25 mM MES) and
incubated with 1 mL/well of fresh uptake buffer in an
atmosphere of 5% CO2 and 90% relative humidity at 37 °C.
After 15 min, uptake buffer was removed and 0.5 mL of freshly
prepared test compound solution (1 mM) in uptake buffer was
added to each well. The cell plate was incubated at 37 °C for
60 min. Then the cells were washed twice with ice-cold
Dulbecco’s phosphate buffered saline and collected in 10 mM
phosphate buffer (pH 7.4) with cell scrapers. The cell suspen-
sion was ultrasonicated in ice bath, and cell lysate was treated
with ice-cold trifluoroacetic acid (final concentration of 7%),
vortexed, and centrifuged for 10 min at 10000 rpm. The super-
natant was then filtered (0.22 μm) and analyzed by HPLC.
Control experiments were performed in normal HeLa cells.
The protein amount of each sample was determined with the
Bio-Rad DC protein assay using bovine serum albumin as
a standard.
Cell Culture. HeLa cells (passage 17-31) and Caco-2 cells
(passage 15-32) from American Type Culture Collection
(Rockvile, MD) were routinely maintained in Dulbecco’s modi-
fied Eagle’s medium (DMEM) containing 10% fetal bovine
serum (FBS), 1% nonessential amino acids, 1 mmol/L sodium
pyruvate, and 1% L-glutamine. Cells were grown in an atmo-
sphere of 5% CO2 and 90% relative humidity at 37 °C.
Hydrolysis in HeLa and Caco-2 Cell Homogenates. HeLa cells
or Caco-2 cells were washed with 0.15 M NaCl solution and then
collected with 10 mM phosphate buffer (pH 7.4). The cell
suspension was ultrasonicated in ice bath to extract enzymes.
The suspension was spun at 10000 rpm for 10 min at 4 °C.
Supernatant was placed in a new tube. The concentration of
enzyme was determined as protein amount with the Bio-Rad
DC assay (Hercules, CA) and adjusted to 500 μg/mL.
Hydrolysis in cell homogenates was determined at 37 °C. The
hydrolysis reaction was initiated by adding 0.75 μL of test
compound solution (200 mM in DMSO) to a reaction tube
containing 749.25 μL of cell homogenates. At various time
points (0, 5, 10, 30, 60, and 120 min), 100 μL of the reaction
mixture was removed and added to a quenching plate containing
100 μL of 10% TFA (in water) and stored in ice. Following the
collection of all samples, quenching plate was filtered (2000 rpm,
4 °C, 10 min). The filtrate was removed and assayed by HPLC.
The apparent first-order degradation rate constants were
determined by plotting the natural logarithm of test compound
remaining as a function of time. All the R2 values were higher
than 0.98. The slope equals to negative rate constant (k). The
degradation half-lives were then estimated by the equation:
t1=2 ¼ 0:693=k
Caco-2 Monolayer Permeability Studies. Caco-2 cells were
grown on 6-well collagen-coated transwell inserts (Corning,
3.0 μm pore size; area 4.67 cm2) and permeability studies were
performed 22-23 days post-seeding. Transepithelial electrical
resistance (TEER) was monitored before and after experiment.
Each well was rinsed with 1.5 mL of MES buffer (pH 6.0,
140 mM NaCl, 5.4 mM KCl, 1.8 mM CaC12, 0.8 mM MgSO4,
5 mM D-glucose, and 25 mM MES) at the apical side and 2.5 mL
of HEPES buffer (pH 7.5, 140 mM NaCl, 5.4 mM KCl, 1.8 mM
CaC12, 0.8 mM MgSO4, 5 mM D-glucose, and 25 mM HEPES)
at the basolateral side. Then 1.5 mL of MES buffer and 2.5 mL
of HEPES buffer were added to the apical side and basolateral
side of each well respectively and the plate was incubated in an
atmosphere of 5% CO2 and 90% relative humidity at 37 °C for
15 min. Experiments were initiated by replacing the apical buffer
with 1.5 mL of 200 μM test compound solution in MES buffer
and the basolateral buffer with 2.5 mL of fresh HEPES buffer
and incubating the plate at 37 °C. 200 μL aliquots of the
basolateral receiver solution were withdrawn at predetermined
intervals and replaced with fresh HEPES buffer. The concentra-
tions were determined by HPLC.
Hydrolysis in pH 6.0 Uptake Buffer and pH 7.4 Phosphate
Buffer. The hydrolysis in buffer was carried out the same way as
above except each reaction well contains 749.25 μL of uptake
buffer (pH 6.0, 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaC12, 0.8
mM MgSO4, 5 mM D-glucose, and 25 mM MES) or 10 mM
phosphate buffer (pH 7.4), and quenching plate contains 100 μL
of 0.1% TFA (in water).
hVACVase-Mediated Hydrolysis. Recombinant hVACVase
was overexpressed and purified from Escherichia coli as
described previously.33 The purified hVACVase was concen-
trated and stored at -80 °C until it was used. The protein
concentration was determined by Bio-Rad DC assay (Hercules,
CA) with bovine serum albumin as a standard. The kinetic
parameters of hVACVase-catalyzed hydrolysis were determined
as follows. Kinetic measurements were carried out in 50 mM
HEPES (pH 7.4) buffer at 37 °C. After preincubation of the
buffer for 5 min, recombinant hVACVase was added, and then
the reaction was initiated by the addition of substrate. Aliquots
were taken at different time points and quenched by adding to
same volume of 10% (v/v) trifluoroacetic acid. Initial velo-
cities were calculated from the linear time course for the product
formation. The kinetic parameters Km and Vmax were deter-
mined by fitting the initial velocity data to the Michaelis-
Menten equation by the nonlinear least-squares regression
analysis in GraphPad Prism software version 4.01. The kcat
value was calculated from Vmax/[enzyme]0 based on the 28.83
kDa molecular mass of hVACVase. Specific activity of valacy-
clovir was routinely monitored to normalize active protein
concentration.
The apparent permeability (Papp; cm/s) across Caco-2 cell
monolayers was calculated using the following equation:
Papp ¼ ð1=C0AÞðdQ=dtÞ
Where C0 is the initial test compound concentration in the donor
solution, A is the surface area of the exposed monolayer, and
dQ/dt is the steady-state appearance rate of the test compound
in the receiver solution.
Rat Perfusion Studies. Male albino Wistar rats (Charles
River, IN) weighing 250-280 g were used for all perfusion
studies. Prior to each experiment, the rats were fasted overnight
(12-18 h) with free access to water. Animals were randomly
assigned to the different experimental groups.
[3H]Gly-Sar Uptake Inhibition. The affinity to hPEPT1 trans-
porter was evaluated by measuring its ability to inhibit the
uptake of [3H]Gly-Sar, a standard hPEPT1 substrate, in
HeLa/hPEPT1 cells. Prior to experiment, HeLa cells were
grown in 12-well plates for 5 days and infected with adenovirus
containing hPEPT1 3 days before experiment.34 Cells were
washed twice with uptake buffer (pH 6.0, 140 mM NaCl,
5.4 mM KCl, 1.8 mM CaC12, 0.8 mM MgSO4, 5 mM D-glucose,
The procedure for the in situ single-pass intestinal perfusion
followed previously published reports.35,36 Briefly, rats were

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 631
anesthetized with an intramuscular injection of 1 mL/kg of
ketamine-xylazine solution (9%:1%, respectively) and placed
on a heated surface maintained at 37 °C (Harvard Apparatus
Inc., Holliston, MA). The abdomen was opened by a midline
incision of 3-4 cm. A jejunal segment of approximately 10 cm
was carefully exposed and cannulated on two ends with flexible
PVC tubing (2.29 mm i.d., inlet tube 40 cm, outlet tube 20 cm,
Fisher Scientific Inc., Pittsburgh, PA). Care was taken to avoid
disturbance of the circulatory system, and the exposed segment
was kept moist with 37 °C normal saline solution (Hospira, Lake
Forest, IL). The isolated segment was rinsed with saline solution
in order to clean out any residual debris.
The perfusion buffer (pH 6.5, 10 mM MES, 135 mM NaCl,
5 mM KCl, 0.1 mg/mL phenol red, 0.4 mg/mL metoprolol, and
0.1 mM test compound) was incubated in a 37 °C water bath. At
the start of the study, perfusion buffer was pumped through the
jejunal segment at a flow rate of 0.2 mL/min (Watson Marlow
Pumps 323S, Watson-Marlow Bredel Inc., Wilmington, MA).
Phenol red was added to the perfusion buffer as a nonabsorb-
able marker for measuring water flux. Metoprolol was co-
perfused as a compound with known permeability that serves
as a marker for the integrity of the experiment and as a reference
standard for permeability in close proximity to the low/high
permeability class boundary.37 The perfusion buffer was first
perfused for 1 h in order to ensure steady state conditions (as
also assessed by the inlet over outlet concentration ratio of
phenol red which approaches 1 at steady state). Following
reaching steady state, samples were taken in 10 min intervals
for 1 h (10, 20, 30, 40, 50, and 60 min). All samples including
perfusion samples at different time points, original drug solu-
tion, and inlet solution taken at the exit of the syringe were
immediately assayed by HPLC. Following the termination of
the experiment, the length of the perfused intestinal segment was
accurately measured.
control group. The difference was termed significant when
p value is smaller than 0.05.
Acknowledgment. We thank Dr. Yasuhiro Tsume for cell
culture trainings and invaluable suggestions. This research
was supported by grant U01AI061457 awarded by the
National Institutes of Health.
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