Application of the dipeptidyl peptidase IV (DPPIV/CD26) based prodrug approach to different amine-containing drugs

Article abstract of DOI:10.1021/jm901590f

Here we explore the applicability of the dipeptidyl peptidase IV (DPPIV/CD26) based prodrug approach to a variety of amine-containing drugs. Efficient procedures have been developed for the synthesis of dipeptide and tetrapeptide amide prodrugs including

Full text of DOI:10.1021/jm901590f

J. Med. Chem. 2010, 53, 559–572 559
DOI: 10.1021/jm901590f
Application of the Dipeptidyl Peptidase IV (DPPIV/CD26) Based Prodrug Approach to Different
Amine-Containing Drugs
†
Alberto Diez-Torrubia,† Carlos Garcı
ꢀ
and Sonsoles Velazquez*
´
a-Aparicio,† Silvia Cabrera,† Ingrid De Meester,‡ Jan Balzarini,§ Marı
´
ꢀ
a-Jose Camarasa,
,†
†
‡
Instituto de Quımica Medica (C.S.I.C.), Juan de la Cierva 3, E-28006 Madrid, Spain, Laboratory of Medical Biochemistry, University of
´
Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium, and Rega Institute for Medical Research, Katholieke Universiteit Leuven,
ꢀ
§
Minderbroedersstraat 10, B-3000 Leuven, Belgium
Received June 17, 2009
Here we explore the applicability of the dipeptidyl peptidase IV (DPPIV/CD26) based prodrug
approach to a variety of amine-containing drugs. Efficient procedures have been developed for the
synthesis of dipeptide and tetrapeptide amide prodrugs including N-acylation protocols of the exocyclic
amino function of cytidine and adenosine nucleosides. Our studies demonstrated that XaaPro
dipeptides linked to a free amino group present on an aromatic ring or on a sugar entity are prodrugs
that efficiently release the parent drug upon conversion by purified DPPIV/CD26 as well as soluble
DPPIV/CD26 in bovine and human serum. Vildagliptin, a specific inhibitor of DPPIV/CD26, was able
to completely block the hydrolysis of the prodrugs in the presence of purified CD26 but also in human
and bovine serum. When the amino group is present on a pyrimidine or purine ring, the dipeptide
derivatives are chemically unstable, whereas the tetrapeptide derivatives (i.e., ValProValPro or
ValAlaValPro) were much more stable in solution and efficiently converted to the parent drug by the
action of DPPIV/CD26. This DPPIV/CD26-directed prodrug technology can be useful to increase
solubility of the parent drug molecules and/or to allow better formulation properties.
Introduction
the enzyme.2-4 DPPIV/CD26 truncates several bioactive
peptides of medical importance.2-4 It is expressed not only
on a variety of leukocyte cell subsets but also on several types
of epithelial, endothelial, and fibroblast cells. A soluble form
of the enzyme is detected in plasma and cerebrospinal fluid at
low amounts.5
In 2005, it was demonstrated for the first time that a
synthetic small molecule (GPG-NH2) can be converted to
an antiviral drug through the specific action of DPPIV/
CD26.6 This was the first demonstration that a differentia-
tion/activation leukocytic marker acts as a highly specific and
obligatory activator of a synthetic anti(retro)viral prodrug
thatis otherwise inactive as such. On the basis of this study, we
recently reported7,8 a novel type of prodrug approach that
could be applied to mediate the solubility, formulation, and
potentially also the bioavailability of therapeutic agents. In
our approach a di- (or oligo)peptide moiety was linked to a
free amino group of a nonpeptidic drug through an amide
bond that is specifically cleaved by the endogenous DPPIV/
CD26 enzyme (Figure 1).7,8
Many prodrug technologies have already been developed
depending on the nature of the drug that has to be released.9-11
Coupling of peptides or amino acids as carriers of a thera-
peutic agent has already been pursued in the past. Examples
of amino acid coupling to drugs are valacyclovir and valgan-
cyclovir, the valyl ester prodrugs of the antiherpetic acyclo-
vir and ganciclovir, respectively,12-14 and most recently
also the valine derivative of the highly specific antivaricella
zoster virus bicyclic nucleoside analogue (BCNA) drug
Cf1743 (designated FV-100).15 The markedly increased oral
The lymphocyte surface glycoprotein CD26,a originally
described as a T-cell activation/differentiation marker,1 be-
longs to a unique class of membrane-associated peptidases. It
is characterized by an array of diverse functional properties,
and it is identical to dipeptidyl peptidase IV (DPPIV).2-4
DPPIV/CD26isamemberof the prolyloligopeptidasefamily,
a group of atypical serine proteases able to hydrolyze the
prolyl bond. It is endowed with an interesting (dipeptidyl)
peptidase catalytic activity, and it has high selectivity for
peptides with a proline or to a lesser extent an alanine at the
penultimate position of the N-terminus of a variety of natural
peptides. Instead, it tolerates a much wider range of amino
acid residues at the N-terminal end. A free, nonsubstituted
amino group on the ultimate (amino terminal) amino acid
position is oneof the prerequisites for substrate recognition by
*To whom correspondence should be addressed. Phone: (þ34)
912587458. Fax: (þ34) 91 5644853. E-mail: iqmsv29@iqm.csic.es.
a Abbreviations: 6-AQ, 6-aminoquinoline; ara-A, 9-β-D-arabinofura-
nosyladenosine; ara-C, 1-β-D-arabinofuranosylcytosine; BCNA, bicyclic
nucleoside analogue; BOP, 1-benzotriazolyloxy-tris-dimethylaminopho-
sphonium hexafluorophosphate; BS, bovine serum; CCTLC, centrifugal
circular thin layer chromatography; CD26, cluster of differentiation 26;
DIC, diisopropylcarbodiimide; DKP, diketopiperazine; Dox, doxorubi-
cin; DPPIV, dipeptidyl peptidase IV; HATU, O-(7-azabenzotriazo-
lyl)tetramethyluronium hexafluorophosphate; HCTU [bis(dimethyl-
amino)methylene]-5-chloro-1H-benzotriazolium 3-oxide hexafluoropho-
sphate; hPEPT1, human intestinal oligopeptide transporter 1; HIV-1,
human immunodeficiency virus type 1; HOBt, 1-hydroxybenzotriazole;
HS, human serum; NAP, N-3-aminopropyl; SPE, solid phase extraction;
TFE, 2,2,2-trifluoroethanol; TSAO, (tert-butyldimethylsilyl-β-D-ribo-
furanosyl)-30-spiro-400-amino-100,200-oxathiole-200,200-dioxide.
r
2009 American Chemical Society
Published on Web 12/10/2009
pubs.acs.org/jmc

560 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Diez-Torrubia et al.
bioavailability of these prodrugs has been shown to be caused
through their recognition by the human peptide transporter
hPEPT-1 located in the membrane of the upper small intestine
epithelial cells.16 The prior art for ameliorating solubility and
bioavailability predominantly reveals amino acid prodrugs
(only one amino acid coupled) of small organic molecules
whereby the amino acid is usually linked through ester bonds,
allowing easy back-conversion to the free therapeutic parental
agent by esterases. However, such prodrugs have low stability
at physiological pH and their delivery is often not optimal.12
The peptide-prodrug approach would allow us to amelio-
rate the solubility and formulation of therapeutic agents in a
conceptually entirely different and potentially more successful
manner, since coupling of di- (or oligo)peptide moieties
(instead of one single amino acid) to a therapeutic agent is
performed through a more stable amide bond (instead of an
ester bond) which is specifically cleaved by DPPIV/CD26
(Figure 1). The presence of a proline near the N-terminus
serves as structural protection against nonspecific proteolytic
degradation (many exopeptidases do not recognize such
sequences),17 which restricts the use of peptides as promoi-
eties. Moreover, the fact that DPPIV/CD26 is present in
plasma in its soluble form and in the membrane of several
cell types guarantees that the prodrug will eventually be
converted to the parent drug. It is also worth noticing that
upon conversion of the prodrug to the parent compound, a
natural product (di- or oligopeptide) is released that is not
expected to generate any undesired side effects.
For proof of the concept of our novel prodrug technology,
we initially focused on the lipophilic TSAO compounds18-20
that inhibit HIV-1 replication. This retrovirus infects lympho-
cytes that abundantly express DPPIV/CD26 in their mem-
brane. In particular, the N-3-aminopropyl TSAO-Tderivative
(NAP-TSAO-T, Figure 2) was chosen as model compound
because its primary amine functionality would allow the
formation of an amide bond between the peptide and the
TSAO molecule. A variety of dipeptide and tetrapeptide
amide prodrugs of NAP-TSAO-T molecules of general for-
mulas I and II deprotected at the peptide N-terminus
(Figure 2) were synthesized and studied. Our data7,8 revealed
that purified DPPIV/CD26 could specifically recognize these
prodrugs as efficient substrates to be converted to the parent
compound. Tetrapeptides of formula II were cleaved in two
successive dipeptidylpeptidase-directed steps to release the
parent drug. Soluble DPPIV/CD26 activity present in human
and bovine serum was the predominant and often sole enzyme
responsible for removing the dipeptide part of the prodrugs in
these biological media. Our studies indicated that it was
possible to modify the enzymatic and serum hydrolysis rate
(half-life) of the prodrug conjugates by changing the nature
and length of the peptide promoiety. Also, the nature of the
peptide promoiety markedly influenced the overall lipophili-
city of the prodrugs, resulting in some cases in a substantial
higher water solubility than the parent compound.8
Whereas in the previous studies the peptidic sequence in the
[peptide]-[NAP-TSAO-T] conjugates was linked to a primary
amino group on an aliphatic alkyl chain, we now extend the
applicability of this prodrug strategy to a variety of amine-
containing drugs of different nature (Figure 3). As an amine-
containing aromatic compound, the fluorescent 6-aminoqui-
noline (6-AQ) bearing a primary amino group bound to the
aromatic ring was selected.21 Antracyclineantibioticscontain-
ing an amino sugar such as doxorubicin (Dox), the most
commonly prescribed intercalating agent for the treatment of
cancer, were next chosen.22 Finally, in order to study whether
the strategy was feasible in primary amino groups bound to
ring heterocyclic systems, the anti-HIV-1 TSAO-cytosine
derivative (TSAO-m5C)23 and the anticancer drug cytarabine
(ara-C)24 were selected as model pyrimidine nucleosides and
the antiviral drug vidarabine (ara-A)25 as model purine
nucleoside (Figure 3). As the promoiety, we focused on the
dipeptide ValPro sequence that is efficiently recognized by
DPPIV/CD26 in natural peptides2,3 andinthe [peptide]-[NAP-
TSAO-T] conjugates.7 The synthesis and stability of the corre-
sponding prodrugs (general formula III, Figure 3), their ability
to act as efficient substrates for DPP-IV/CD26, and their
human and bovine serum hydrolysis profiles and solubility in
aqueous solutions are herein described.
Results and Discussion
Chemistry. The target 6-aminoquinoline (6-AQ) dipeptide
conjugate 4a (Scheme 1) was prepared following our pre-
viously described two-step procedure7 that consisted of the
coupling of 4a with the commercially available dipeptide
Z-Val-Pro-OH followed by catalytic hydrogenation of the
N-protected conjugate intermediate. Thus, coupling of 6-AQ
with Z-Val-Pro-OH in CH2Cl2 in the presence of (benzo-
triazol-1-yloxy)-tris-(dimethylamino)phosphonium hexafluoro-
phosphate (BOP) and TEA gave the corresponding protected
Figure 1. [(Xaa-Pro)n]-[drug] conjugates cleavable by DPPIV/
CD26.
Figure 2. Structures of [Xaa-Pro]-[NAP-TSAO-T] and [Xaa-Pro-Xaa1-Pro]-[NAP-TSAO-T] conjugates of general formulas I and II.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 561
Scheme 1. Synthesis of [Xaa-Pro]-[6-AQ] Conjugates 4a,ba
a Reagents: (i) H-Pro-OMe HCl, BOP, TEA, CH2Cl2, room temp; (ii)
3
2 N NaOH; (iii) Cbz-Val-Pro-OH (2a) or Boc-Asp(OtBu)-Pro-OH (2b),
BOP, TEA, CH2Cl2, room temp; (iv) H2, Pd (C), CH3OH, room temp;
(v) TFA, CH2Cl2, room temp.
Scheme 2. Synthesis of [Val-Pro]-[Dox] Conjugate 7a
Figure 3. Structures of target [Val-Pro]-[drug] conjugates of general
formula III.
dipeptide conjugate 3a (Scheme 1) in 65% yield, after puri-
fication by flash column chromatography. Catalytic hydro-
genation of 3a in the presence of 10% Pd/C in methanol
afforded the N-deprotected prodrug 4a in 48% yield to-
gether with the partially reduced tetrahydroquinoline
derivative 5 (Scheme 1) as minor compound (12% yield).
Examples of catalytic hydrogenation of quinolines at the
pyridine ring generating tetrahydroquinoline derivatives in
the presence of H2, Pd/C have been described.26,27
a Reagents: (i) Fmoc-Val-Pro-OH, HATU, DIEA, DMSO, room
temp; (ii) piperidine 50%, DMF, room temp.
The conjugate 4b bearing the Asp-Pro dipeptide sequence,
which showed a much lower conversion rate to the parent
compound by CD26 in previous studies with [Xaa-Yaa]-[NAP-
TSAO-T] conjugates,7 was next prepared (Scheme 1). The
synthesis of compound 4b was carried out in a way similar to
that described above following a tert-butoxycarbonyl (BOC)
strategy in order to avoid the partial catalytic hydrogenation
of the quinoline system in the final deprotection step. The
noncommercially available dipeptide Boc-[Asp(OtBu)-Pro]-
OH (2b) was prepared using standard coupling/deprotection
techniques (Scheme 1). Coupling of Boc-[Asp(OtBu)-Pro]-
OH (2b) with 6-aminoquinoline in the presence of BOP and
TEA gave the protected intermediate 3b in 45%. Acid
hydrolysis of compound 1b in TFA/CH2Cl2 solution yielded
exclusively the fully deprotected conjugate 4b in 83% yield.
The base-labile 9-fluorenylmethoxycarbonyl (Fmoc) pro-
tecting group was used in the preparation of the doxorubicin
(Dox) dipeptide conjugate 7 (Scheme 2). Thus, commercially
available Fmoc-Val-Pro-OH was coupled with Dox in
DMSO in the presence of hexafluorophosphate salt of the
O-(7-azabenzotriazolyl)tetramethyluronium hexafluorophos-
phate (HATU) as the coupling agent28 and DIEA as
base to afford the protected conjugate 6 in 45% yield. The
Fmoc protecting group was removed by treatment of 6 with
50% piperidine in DMF. The final deprotected conjugate 7
was obtained in 50% yield after purification by reverse phase
chromatrography using solid-phase extraction (SPE) car-
tridges.
Next, we focused on the synthesis of pyrimidine nucleoside
prodrugs bearing the model Val-Pro dipeptide sequence as
promoiety (Scheme 3). A benzyloxycarbonyl (Cbz)-protect-
ing strategy was selected for the synthesis of dipeptide
prodrugs of TSAO-m5C. Thus, TSAO-m5C was reacted with
the commercially available dipeptide Cbz-Val-Pro-OH in
CH2Cl2, in the presence of BOP and TEA at room tempera-
ture, to give the corresponding protected dipeptide conjugate
8a in 53% yield (Scheme 3). Because of the low nucleophi-
licity of the cytosine amino group, an extra amount of
coupling reagent and base was required to drive the reaction
to completion.
Peptide bond formation at the N4-position of ara-C
proved to be more complicated by the lower solubility of
the compound due to the presence of three free hydroxyl
groups. Peptide prodrugs of ara-C at the N4-position have
already been described to overcome the drawbacks for the
clinical use of ara-C such as toxicity, low plasma levels, and
rapid enzymatic degradation.29-32 In most of the examples
protection of the glycoside moiety of the nucleosides was
required. Interestingly, a mild procedure for the synthesis of
peptidyl-ara-C derivatives, which does not require protection of

562 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Diez-Torrubia et al.
Scheme 3. Attempted Synthesis of Dipeptide Conjugates of Cytidine Nucleosides TSAO-m5C or ara-Ca
a Reagents: (i) Cbz-Val-Pro-OH, BOP, TEA, CH2Cl2, room temp; (ii) R-Val-Pro-OH or Cbz-Val-Ala-OH, HOBt, DIC, TEA, DMF, room temp;
(iii) H2, Pd (C) 10%, CH3OH; (iv) HCl, AcOEt, room temp.
ara-C, has also been reported.31 Following this protocol,
ara-C was reacted with dipeptide Cbz-Val-Pro-OH in DMF
using 1,3-diisopropylcarbodiimide (DIC) as coupling agent
and N-hydroxybenzotriazole (HOBt) as additive to give the
Cbz-protected conjugate intermediate 8b in 27% yield.
Catalytic hydrogenation of protected conjugates 8a and 8b
in the presence of 10% Pd/C in methanol showed that parent
nucleosides TSAO-m5C and ara-C were spontaneously
released from the N-deprotected conjugates with concomi-
tant formation of the known diketopiperazine (DKP) such as
compound 1033 (Scheme 3). It is well-documented in the
literature34-37 that dipeptide esters including Val-Pro-de-
rived dipeptide inhibitors of DPP IV38 readily cyclize to a
DKP derivative (to a much lesser extent in dipeptide amides
owing to their lower electrophilic reactivity). It is noted,
however, that the spontaneous intramolecular cyclization of
the Val-Pro dipeptide promoiety had not been observed,
neither in the N-deprotected dipeptide conjugates of 6-AQ
and Dox 4a,b and 7 herein described nor in the previously
described NAP-TSAO-T dipeptide conjugates that proved
to be chemically stable.7 A much lower electrophilicity of the
N-4 amide in the cytidine conjugates could explain why the
cytidine drugs are better leaving groups than 6-AQ, Dox, or
NAP-TSAO-T. In order to enhance the chemical stability of
the dipeptide prodrugs of ara-C and to suppress spontaneous
release of the parent drug, several approaches were at-
tempted. Boc was first selected as protecting group, instead
of a Cbz-protecting strategy, because it could be removed
under acidic conditions, affording the corresponding salts
that may prevent spontaneous cyclization.30 Thus, treatment
of ara-C with the commercially available Boc-Val-Pro-OH
under the above-mentioned coupling conditions gave the
intermediate conjugate 9b in 28% yield. Conjugate 9b was
deprotected with HCl/ethyl acetate. However, the desired
hydrochloride salt of the dipeptide-ara-C conjugate was not
chemically stable and only ara-C and DKP 1033 were isolated
after chromatographic purification. Since it is known that a
proline at the penultimate position significantly facilitates
the rate of DKP formation owing to the greater propensity to
adopt a cis-amide conformation,36 the Val-Pro dipeptide was
next replaced by the Val-Ala sequence (which is also recog-
nized by DPP IV/CD26 as previously described).7 Thus,
commercially available Cbz-Val-Ala-OH was coupled with
ara-C in DMF in the presence of DIC and HOBt to give the
intermediate conjugate 11b (Scheme 3) in 26% yield. How-
ever, in the catalytic hydrogenation of 11b only ara-C and the
corresponding DKP derivative 1239 were isolated. Our find-
ings indicate that the Val-Pro and Val-Ala dipeptide
sequences cannot be used as useful promoieties in cytidine
nucleoside drugs because of an extremely high tendency for
cyclization. On the other hand, there is one example reported
in the literature where other dipeptide sequences with non-
proteinogenic amino acids (such as R,R-disubstituted amino
acids) have been successfully employed as carriers in pro-
drugs of ara-C.30 Therefore, our results point to a key role of
the nature of the dipeptide on the rate of release of the parent
drug via DKP formation.
To further investigate whether the DPP IV/CD26 prodrug
strategy could be applied to cytidine nucleoside drugs,
tetrapeptide prodrugs of TSAO-m5C and ara-C were

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 563
Scheme 4. Solid-Phase Synthesis of Tetrapeptides 17-19a
Scheme 6. Synthesis of the Tetrapeptide Conjugate of ara-A
29a
a Reagents: (i) Fmoc-Val-OH, HCTU, DIEA, DMF; (ii) piperidine
20%, DMF; (iii) Fmoc-Pro-OH or Fmoc-Ala-OH, HCTU, DIEA,
DMF; (iv) R-Val-OH, HCTU, DIEA, DMF; (v) AcOH, TFE, DCM.
Scheme 5. Synthesis of Tetrapeptide Conjugates of Cytidine
Nucleosides 22a,c and 23ca
a Reagents: (i) Ac2O, py, room temp; (ii) TBSCl, imidazole, DMF,
room temp; (iii) Fmoc-Val-Pro-OH, BOP, TEA, CH2Cl2, room temp;
(iv) piperidine 20%, DMF, room temp, 5 min; (v) Cbz-Val-Pro-Val-Pro-
OH (17), BOP, TEA, CH2Cl2, 70 °C; (vi) (a) TBAF, THF, room temp;
(b) CaCO3, Dowex 50Wx8, CH3OH, room temp; (vii) H2, Pd (C) 10%,
CH3OH, room temp.
Fmoc N-protecting group to afford tripeptide derivatives 15
and 16. The N-terminal residue Cbz-Val-OH or Fmoc-Val-
OH was finally incorporated under similar conditions. Final
tetrapeptides 17-19 were obtained by cleavage from the
resin with AcOH/2,2,2-trifluoroethanol (TFE). The tetra-
peptides were isolated in excellent yields (92-96%) and at a
high purity (>95%) and were used without further purifica-
tion in coupling with nucleosides. This solid-phase strategy
afforded the tetrapeptides in better yields and enhanced
efficacy than the previous solution methodology.8
a Reagents: (i) R-Val-Pro-Val-Pro-OH (17), BOP, TEA, CH2Cl2,
room temp; (ii) Fmoc-Val-Xaa-Val-Pro-OH (18 or 19), BOP, TEA,
DMF, room temp or 45 °C; (iii) H2, Pd (C) 10%, CH3OH ; (iv) TBAF,
THF, room temp.
designed (Scheme 5). On the basis of our previous studies8
showing that tetrapeptide derivatives of NAP-TSAO were
efficiently converted to the parent compound in two steps by
DPPIV/CD26, the tetrapeptide sequences Val-Pro-Val-Pro
and Val-Ala-Val-Pro (with different conversion rates to the
parent compound) were selected.8
The synthesis of the target tetrapeptide conjugates 22a,c
and 23c (Scheme 5) utilized a combination of solid-phase and
solution chemistry. The tetrapeptide promoieties 17-19
were prepared by solid-phase synthesis using a Fmoc strat-
egy on a 2-chlorotritylpolystyrene resin (Scheme 4). This
solid support does not favor the on-resin formation of
diketopiperazines because of steric hindrance of the bulky
2-chlorotrityl. Resin, preloaded with the first amino acid
(H-Pro), was coupled to Fmoc-Val-OH in the presence of
1-[bis(dimethylamino)methylene]-5-chloro-1H-benzotriazo-
lium 3-oxide hexafluorophosphate (HCTU)/DIEA to give
intermediate dipeptide 14. Fmoc-Pro-OH or Fmoc-Ala-OH
was then similarly incorporated after deprotection of the
Coupling of tetrapeptide 17 with TSAO-m5C in the pre-
sence of excess amounts of BOP and TEA gave the corre-
sponding protected intermediate conjugate 21a in moderate
yield (41%) (Scheme 5). Removal of the benzyl group by
catalytic hydrogenation in the presence of 10% Pd/C in
CH3OH afforded the final desired deprotected conjugate
of TSAO-m5C 22a, which proved to be chemically stable, in
excellent yield (95%). Initial attempts to couple tetrapeptides
18 and 19 with unprotected ara-C in DMF in the presence of
DIC and HOBt (under similar conditions described above
for the dipeptide conjugates 8b, 9b, and 11b) afforded the
desired tetrapeptide conjugates 21b and 23b in very low
yields (6% and 9%, respectively). However, when silyl
protected ara-C (20b)40 was condensed with tetrapeptide 18
or 19 in the presence of excess amounts of BOP and TEA, a
much higher yield of the fully protected conjugates 21b and
23b was obtained (56% and 43%, respectively) (Scheme 5).
Removal of both the tert-butyldimethylsilyl (TBS) and
Fmoc groups of compounds 21b and 23b was carried out by

564 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Diez-Torrubia et al.
Figure 4. (a) Conversion of [ValPro]-[6-AQ] (4a) to parent 6-AQ in the presence of purified CD26, bovine and human serum (20%), and PBS.
(b) Conversion of [AspPro]-[6-AQ] (4b) to parent 6-AQ in the presence of purified CD26, bovine and human serum (20%), and PBS.
treatment with tetrabutylammonium fluoride (TBAF) to
give the target fully deprotected conjugates 22c and 23c
(Scheme 5) in good yields (60% and 63%, respectively).
Finally, the synthesis of purine nucleoside prodrugs of the
antiviral vidarabine (ara-A) bearing di- and tetrapeptide
sequences as promoieties was performed (Scheme 6). The
preparation of the fully blocked dipeptide derivative 26
(Scheme 6) was carried out in moderate yields (56%) by
coupling of the acetyl protected nucleoside 2441 with Fmoc-
Val-Pro-OH in the presence of excess amounts of BOP and
TEA at room temperature. A higher reaction temperature
(90 °C) was required for an efficient coupling of ara-A with
tetrapeptide sequences. After evaluation of a range of
N-terminal protecting groups including Fmoc, Boc, or
Cbz, the final candidate for protection of the peptide pro-
moiety was the Cbz group that could be easily removed
under mild conditions.42 Thus, treatment of tetrapeptide 17
with the conveniently protected nucleoside 2543 in the pre-
sence of excess amounts of BOP and TEA at 90 °C gave the
fully protected intermediate conjugate 27 in moderate yield
(59%) (Scheme 6).
Similar to that observed with cytidine dipeptide prodrugs,
N-deprotection of dipeptide conjugate 26 with 20% piper-
idine showed that parent nucleoside ara-A was sponta-
neously released with concomitant formation of the known
DKP 1033 whereas the tetrapeptide prodrug was much more
stable. The Cbz-tetrapeptide conjugate derivative 27 was
first subjected to TBAF-promoted TBS removal in THF.
A recently described simple and efficient workup to remove
excess TBAF and TBAF-derived materials,44 which avoids
the conventional aqueous-phase extraction protocol
(precluded in our water-soluble compound), was used. This
method involves addition of a commercially available sulfo-
nic acid resin and calcium carbonate, followed by filtration

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 565
and evaporation. Thus, treatment of 27 with excess of TBAF
in THF, followed by this workup method, afforded the
desilylated conjugate 28 in 76% yield after filtration, eva-
poration, and chromatography purification of the crude
residue. Finally, removal of the Cbz group by catalytic
hydrogenation in the presence of 10% Pd/C in CH3OH
afforded the desired fully deprotected conjugate 29 in 85%
yield (Scheme 6).
In addition, water solubility studies were carried out with
the 6-aminoquinoline (6-AQ) dipeptide prodrugs 4a and 4b
and the vidarabine tetrapeptide prodrug 29 and compared to
that of the parent compounds. The solubility of [ValPro]-[6-
AQ] 4a and the most hydrophilic [AspPro]-[6-AQ] prodrug
4b in aqueous solution were 33.18 and 47.82 mg/mL, respec-
tively, while the solubility of 6-AQ was 7.01 mg/mL. Thus, it
is possible to modify the water solubility of the conjugates by
changing the nature of the dipeptide promoiety. Further-
more, the vidarabine Val-Pro-Val-Pro tetrapeptide prodrug
29 improved the water solubility (34.74 mg/mL) more than
60-fold in comparison with the parent compound (0.526 mg/
mL). Thus, the solubility of peptide-based prodrugs in-
creased between 5- and more than 60-fold compared with
their parent compounds. This result suggests that the
DPPIV/CD26 prodrug approach could be useful for increas-
ing the water solubility of poorly soluble parent drug mole-
cules.
Biological Studies. Among the aromatic dipeptide pro-
drugs, the [ValPro]-[6-AQ] (4a) and the [AspPro]-[6-AQ]
(4b) prodrugs have been evaluated for their conversion to
the parent 6-AQ compound by purified CD26 and in the
presence of 20% bovine serum (BS) and 20% human
serum (HS) in PBS (Figure 4). In the absence of enzyme,
50 μM [ValPro]-[6-AQ] (4a) (Figure 4a) and [AspPro]-[6-
AQ] (4b) (Figure 4b) solutions in PBS were fairly stable
for 4 h and slightly (spontaneously) converted to the
parent compound 6-AQ after 24
h
(∼10-20%)
(Figures 4). Instead, both prodrugs were efficiently con-
verted to 6-AQ by purified CD26 and by 20% bovine and
human serum. Complete conversion was noticed for
[ValPro]-[6-AQ] within 1 h of incubation, whereas
[AspPro]-[6-AQ] needed at least 4 h (in BS and HS) for
optimal conversion. It is noted that [ValPro]-[6-AQ] could
be fully converted to the parent compound, whereas the
maximal conversion of [AspPro]-[6-AQ] to its parent
compound was around 70%, even after 24 h. This is
obviously due to the feedback inhibitory activity of the
released dipeptide AspPro against CD26, an observation
that has also earlier been made for the [AspPro]-[NAP-
TSAO-T] prodrug.7 The higher efficiency (rate) of con-
version for [ValPro]-[6-AQ] (4a) than [AspPro]-[6-AQ]
(4b) is also in agreement with earlier findings7 and with
the known cleavage specificities of CD26 against natural
peptides.2,3 The presence of a positively charged (i.e., Lys)
or lipophilic (i.e., Val) amino acid as N-terminal amino
acid is preferred over a negatively charged (i.e., Asp)
amino acid for hydrolytic cleavage after the penultimate
proline by CD26.
Figure 5. Conversion of [ValPro]-[Dox] (7) to parent doxorubicin
in the presence of purified CD26, bovine and human serum (20%),
and PBS.
Two peptide prodrug derivatives were prepared using the
amine function of cytosine as the entity to link the peptide
moiety. [ValProValPro]-[TSAO-m5C] (22a) was not very
stable in PBS. However, it proved to be a substrate for
CD26 (∼50% conversion after 2 h) and was also efficiently
converted to the parent compound in 20% HS (∼20% con-
version after 2 h) and 20% BS (∼40% conversion after 2 h).
The dipeptide intermediate has not been detected during the
TSAO-m5C release process (data not shown). The tetrapep-
tide derivatives of ara-C, in which the ValProValPro (22c) and
ValAlaValPro (23c) tetrapeptides were linked to the amine at
the cytosine heterocyclic ring, proved to be much more stable
in PBS (Figure 6). Indeed, upon incubation during the first 4 h
inPBS, release of hardlyany parent compound(a few percent)
could be detected. After 24 h, 18-24% of the tetrapeptide
prodrug was converted to parental ara-C. However, in the
presence of purified CD26, HS, or BS, the ara-C prodrug 22c
The ValPro derivative of doxorubicin (7) in which the
dipeptide is linked to the molecule through the sugar amide
moiety proved to be a good substrate for CD26 (Figure 5).
While fully stable in PBS for at least 24 h, CD26 was able to
substantially convert the prodrug to its parent compound
within 1 h of incubation. Also BS and HS time-dependently
and efficiently hydrolyzed the prodrug (Figure 5).

566 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Diez-Torrubia et al.
Figure 6. (a) Conversion of [ValProValPro]-[ara-C] (22c) to parent ara-C in the presence of purified CD26, bovine and human serum (20%),
and PBS. (b) Conversion of [ValAlaValPro]-[ara-C] (23c) to parent ara-C in the presence of purified CD26, bovine and human serum (20%),
and PBS.
was converted to ara-C by 80-90% within 4 h (Figure 6a). No
ara-C dipeptide intermediate could be detected by HPLC at
any time point investigated, an observation that is in agree-
ment with the observed high instability of the ara-C dipeptide
prodrug. Therefore, although it cannot be excluded that the
intermediate ara-C dipeptide prodrug is a substrate for CD26
and can also be catalytically (very rapidly) cleaved a second
time, wemay assume thatonce the ara-C tetrapeptideprodrug
has been enzymatically converted to the ara-C dipeptide
prodrug by the action of CD26, this intermediate would be
instantly converted to ara-C by spontaneous nonenzymatic
hydrolysis. Similar data were observed for the [ValAla-
ValPro]-[ara-C] prodrug 23c (Figure 6b).
compound was fully stable in PBS for up to 24 h, the
tetrapeptide ara-A derivative converted to ara-A by CD26,
as well as by 20% HS and BS (Figure 7). No dipeptide ara-A
could be detected during the conversion process by CD26,
presumably because of the noted instability of this inter-
mediate metabolite (as also observed for the ara-C dipeptide
prodrug (see above)). The tetrapeptide ara-A compound 29
seemed to be less efficiently converted to the parent drug
than the corresponding ara-C prodrug 22c (Figure 6a). It is
mentioned that no formation of ara-Hx (deamination pro-
duct of ara-A) was observed in the CD26-catalyzed reaction
or under the 20% HS and 20% BS experimental conditions.
Since the disappearance of the prodrug in the samples
was also measured (instead of appearance of parental
compound), potential deamination of ara-A to ara-Hx
would not influence the conversion kinetic measurements.
The stability in PBS and susceptibility of the ValProVal-
Pro tetrapeptide derivative of ara-A 29 upon exposure
to CD26 or 20% serum was investigated. Whereas this

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 567
exposed to CD26 in the presence of a variety of the vilda-
gliptin inhibitor concentrations, a dose-dependent inhibition
of prodrug conversion was observed. At 2 μM, the inhibitor
was still able to fully prevent prodrug conversion to ara-A.
At a concentration as low as 0.05 μM vildagliptin, the CD26-
catalyzed reaction was inhibited by ∼50% (Figure 8). This
vildagliptin concentration closely corresponded to the 50%
inhibitory concentration of this drug reported against DPP-4
activity in vitro (IC50 = 62 nM).47
The doxorubicin and ara-C prodrugs have been evaluated
for their inhibitory activity against the proliferation of
murine leukemia L1210, human lymphocyte CEM, and
cervix carcinoma HeLa cells (Table 1). The prodrugs vir-
tually kept their cytostatic activity compared with the par-
ental compounds doxorubin and ara-C. This observation
points to an efficient conversion of the prodrug derivatives to
the parent drugs by CD26 activity present in the cell cultures
and is in agreement with the kinetic conversion data. Also,
the TSAO-m5C prodrug kept its anti-HIV-1 activity in CEM
cell cultures when compared to TSAO-m5C (EC50 = 0.07 μM),
and the [ProValProVal]-[ara-A] prodrug was virtually
equally antivirally active against herpes simplex virus type
1 (HSV-1) and vaccinia virus (VV) in HEL cell cultures as the
parent ara-A (Table 2). However, in the presence of 20 μM
vildagliptin, the antiviral activity was unaltered for ara-A but
5- to 10-fold decreased for the prodrug of ara-A. These
findings indicate that the CD26 inhibitor could prevent
conversion of [ValProValPro]-[ara-A] to ara-A by at least
80-90% under the experimental conditions (addition of one
single administration of the inhibitor at the time of initiation
of the virus infection).
The kinetic parameters (Km, Vmax) were also determined
for [ValPro]-[6-aminoquinoline], [AspPro]-[6-aminoquino-
line], [ValPro]-[doxorubicine], and [ValProValPro]-[ara-A]
against purified CD26 (Table 3). Whereas the Km values were
rather similar for the three ValPro prodrugs (irrespective the
nature of the parent drug linked to the ValPro-moiety (Km =
25-56 μM)), [AspPro]-[6-aminoquinoline] was endowed
with a much higher Km value (524 μM). Instead, the Vmax
values differ much more depending the nature of the parent
drug: the highest Vmax values were observed for the 6-
aminoquinoline prodrugs and the lowest Vmax values (7-fold
lower) for the [ValProValPro]-[ara-A] prodrug (Table 3).
The overall hydrolyzing capacity of CD26 (ratio Vmax/Km)
differed up to ∼10-fold for the four prodrugs investigated.
The Km values of the prodrugs were somewhat higher than
reported for a variety of natural substrates including SDF-
1R, IP-10, RANTES, and LD78β (1-4 μM).48 However, the
Km for eotoxin was 25 μM,48 a value comparable to that
found for [ValPro]-[doxorubicine] and [ValPro]-[6-
aminoquinoline] (Table 3).
Figure 7. Conversion of [ValProValPro]-[ara-A] (29) to parent ara-
A in the presence of purified CD26, bovine and human serum
(20%), and PBS.
To ascertain that the observed conversions of the prodrugs
to their parental compounds were solely due to the action of
CD26 but not to other enzymatic activities, the conversion of
the [ValPro]-[6-aminoquinoline], [AspPro]-[6-aminoquino-
line], [ValPro]-[doxorubicine], and [ValProValPro]-[ara-A]
by CD26, 20% HS, and 20% BS was studied in the presence
of vildagliptin, a specific inhibitor of CD26.45-47 At 20 μM
vildagliptin, no signs of hydrolysis of any of the prodrugs was
observed, neither in the presence of purified CD26 nor in the
presence of 20% HS or 20% BS. These findings clearly
indicate that the observed conversions of the prodrugs to
their parent compounds were exclusively due to the action of
CD26. When the ValProValPro prodrug of ara-A was
In conclusion, we could earlier demonstrate that purified
CD26 (as well as human and bovine serum) could efficiently
recognize dipeptide derivatives where the dipeptide is linked
to a nonpeptidic drug through an aminoalkyl side chain. We
now could extend this observation to drugs at which a free
amino group is located at an aromatic ring (i.e., 6-amino-
quinoline) or an amino sugar (i.e., doxorubicine). Whereas
linkage of a dipeptide to an amino group at a heterocyclic
ring such as in cytosine and adenine is highly unstable,
tetrapeptide derivatives of ara-C and ara-A instead afford
fairly stable prodrugs that are efficiently recognized by CD26
and well-cleaved in bovine and human serum. Thus, our
studies revealed that a wide variety of drug types containing a

568 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Diez-Torrubia et al.
Figure 8. Inhibition of CD26-catalyzed [ValProValPro]-[ara-A] (29) hydrolysis in the presence of different concentrations of vildagliptin.
Table 1. Inhibitory Activity of the ara-C and Its Prodrug against Tumor
Cell Proliferation
Table 2. Antiviral Activity of ara-A and [ValProValPro]-[ara-A] in
HEL Cell Cultures
IC50 a (μM)
EC50 a (μM)
compd
L1210
0.024 ( 0.009 0.024 ( 0.001 0.26 ( 0.01
[ValProValPro]-[ara-C] (22c) 0.10 ( 0.04 0.057 ( 0.016 0.63 ( 0.04
[ValAlaValPro]-[ara-C] (23c) 0.082 0.050 ( 0.005 0.56 ( 0.07
CEM
HeLa
HSV-1
VV
ara-C
compd
as such þvildagliptin as such þvildagliptin
35 ( 15 42 ( 11
[ValProValPro]-[ara-A] 54 ( 7.5 238 ( 20
a EC50 values, or compound concentrations required to reduce the
virus-induced cytopathicity by 50%.
ara-A
8.3 ( 4.0 10 ( 0.7
16 ( 7.5 135 ( 50
a 50% inhibitory concentration, or compound concentration required
to inhibit tumor cell proliferation by 50%.
Table 3. Kinetic Parameters of Prodrugs against Purified CD26
free amino moiety on an aromatic, carbohydrate, or hetero-
cyclic pyrimidine or purine ring can be derivatized with di- or
tetrapeptides to become efficient substrates for the dipepti-
dyl peptidase activity of CD26. Furthermore, the 6-AQ and
vidarabine peptide prodrugs exhibited a considerably higher
water solubility than the parent compounds, a property that
can be beneficial for drug formulation modalities.
kinetic parameters
prodrug
Km (μM) Vmax (nmol/μg/h) Vmax/Km
[ValPro]-[6-aminoquinoline] 25 ( 8.5
[AspPro]-[6-aminoquinoline] 524 ( 207
32 ( 3.2
70 ( 22
15 ( 3.5
10 ( 12
1.3
0.13
0.50
0.18
[ValPro]-[doxorubicin]
[ValProValPro]-[ara-A]
30 ( 8.1
56 ( 19
Experimental Section
in ethyl acetate (50 mL) and washed with 10% aqueous citric
acid (3 ꢀ 20 mL), 10% aqueous NaHCO3 (3 ꢀ 20 mL), water
(3 ꢀ 20 mL), and brine (3 ꢀ 20 mL). The organic layer was dried
(Na2SO4), filtered, and evaporated to dryness. The final residue
was purified by CCTLC on the Chromatotron (hexane/ethyl
acetate, 1:4) to give 3a (0.39 g, 65%) as a yellow foam. MS
(ESIþ): m/z 475.3 (M þ 1þ). Anal. for C27H30N4O4: C, H, N.
6-N-[Nr-(tert-Butoxy)-O4-tert-butylaspartylprolyl]quinoline
(3b). Following the coupling procedure described for compound
3a, 6-aminoquinoline (0.068 g, 0.474 mmol) was reacted with
Boc-Asp(OtBu)-Pro-OH (0.22 g, 0.57 mmol) in the presence of
BOP (0.252 g, 0.57 mmol) and TEA (0.079 mL, 0.57 mmol) in
CH2Cl2 (5 mL). The final residue was purified by CCTLC on the
Chromatotron (CH2Cl2/CH3OH, 40:1) to give 3b (0.105 g, 45%)
as a white foam. MS (ESIþ): m/z 513.3 (M þ 1þ). Anal. for
C27H36N4O6: C, H, N.
Chemical Procedures. Microanalyses were obtained with a
Heraeus CHN-O-RAPID instrument, and the analytical results
were within 0.4% of the theoretical values. Electrospray mass
spectra were measured on a quadropole mass spectrometer
equipped with an electrospray source (Hewlett-Packard, LC/
MS HP 1100). Spectra were recorded with a Varian Inova-300 or
Varian Inova-400 spectrometers operating at 300 or at 400 MHz
for 1H NMR and at 75 or at 100 MHz for 13C NMR with Me4Si
as internal standard. Analytical thin-layer chromatography
(TLC) was performed on silica gel 60 F254 (Merck). Separations
on silica gel were performed by flash column chromatography
with silica gel 60 (230-400 mesh) (Merck) or preparative
centrifugal circular thin layer chromatography (CCTLC) on a
Chromatotron (Kieselgel 60 PF254 gipshaltig (silica gel contain-
ing gypsum) (Merck), layer thickness of 1 mm, flow rate of 5 mL/
min). Preparative reverse phase purification was carried out
using reverse phase SPE cartridges. The dipeptide derivatives
Cbz-Val-Pro-OH, Fmoc-Val-Pro-OH, Boc-Val-Pro-OH, and
Cbz-Val-Ala-OH were purchased from Bachem Feinchemika-
lien. 30,50-TBS-ara-C (20c),39 20,30,50-Ac-ara-A (24),40 and 30-50-
TBS-ara-A (25)42 were synthesized as previously described. H-
Pro-2-chlorotrityl resin and HCTU were purchased from GL
Biochem (Shanghai) Ltd. The purity of novel compounds was
determined to be >95% by elemental analysis.
6-N-(Valylprolyl)quinoline (4a). A solution of Cbz-[Val-
Pro]-[6-AQ] (3a) (0.42 g, 0.88 mmol) in methanol (10 mL)
containing Pd/C (10%) (40 wt %/wt) (0.12 g) was hydrogenated
at 1 atm at room temperature for 2 h. The reaction mixture was
filtered, and the filtrate was evaporated to dryness under
reduced pressure. The residue was purified by CCTLC on the
Chromatotron (CH2Cl2/MeOH, 2:1). From the fastest running
1
fractions, 0.035 g (12%) of 5 was isolated as a yellow oil. H
NMR (300 MHz, acetone-d6): δ 0.83 (d, 3H, γ-CH3a, Val, J =
6.6 Hz), 0.95 (d, 3H, γ-CH3b, Val, J = 6.6 Hz), 1.80-2.30 (m,
7H, H-3, β-CH, Val, β-CH2, γ-CH2, Pro), 2.66 (t, 2H, H-4, J =
6.3 Hz), 3.22 (t, 2H, H-2, J = 5.4 Hz), 3.49-3.78 (m, 2H, δ-CH2,
Pro), 3.96 (d, 1H, R-CH, Val, J = 8.1 Hz), 4.57 (m, 1H, R-CH,
Pro), 6.38 (d, 1H, H-8, J = 8.4 Hz), 7.06 (m, 1H, H-7), 7.14 (s,
1H, H-5), 9.08 (s, 1H, NHCO). MS (ESIþ): m/z 345.1 (M þ 1þ),
6-N-[Nr-(Bencyloxycarbonyl)valylprolyl]quinoline (3a). To a
solution of Cbz-Val-Pro-OH (0.58 g, 1.66 mmol) in CH2Cl2
(3 mL) was successively added BOP (0.73 g, 1.66 mmol), TEA
(0.231 mL, 1.66 mmol), and 6-aminoquinoline (0.20 g, 1.38
mmol). The mixture was stirred at room temperature for 15 h.
After removal of the solvent in vacuo, the residue was dissolved

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 569
367.1 (M þ Naþ). The slowest moving band afforded 0.14 g
dryness. The residue was dissolved in ethyl acetate (50 mL) and
washed with 10% aqueous citric acid (3 ꢀ 20 mL), 10% aqueous
NaHCO3 (3 ꢀ 20 mL), water (3 ꢀ 20 mL), and brine (3 ꢀ 20 mL).
The organic layer was dried (Na2SO4), filtered, and evaporated
to dryness. The final residue was purified by CCTLC on the
Chromatotron (CH2Cl2/MeOH, 10:1) to give 8b (0.06 g, 27%)
as a white foam. MS (ESIþ): m/z 574.3 (M þ 1þ). Anal. for
C27H35N5O9: C, H, N.
1
(48%) of 6-N-(valylprolyl)quinoline (4a) as a yellow foam. H
NMR (300 MHz, DMSO-d6): δ 0.87 (d, 3H, γ-CH3a, Val, J =
6.6 Hz), 0.95 (d, 3H, γ-CH3b, Val, J = 6.6 Hz), 1.82-2.08 (m,
4H, β-CH, Val, β-CH2, γ-CH2, Pro), 2.21 (m, 1H, β-CH2, Pro),
3.84 (m, 3H, R-CH, Val, δ-CH2, Pro), 4.52 (m, 1H, R-CH, Pro),
7.46 (m, 1H, H-3), 7.78 (d, 1H, H-7, J = 2.2 Hz, J = 9.0 Hz),
7.97 (d, 1H, H-8, J = 9.0 Hz), 8.28 (d, 1H, H-4, J = 7.8 Hz), 8.36
(d, 1H, H-5, J = 2.2 Hz), 8.77 (dd, 1H, H-2, J = 1.7 and 4.1 Hz),
10.39 (bs, 1H, NHCO). MS (ESIþ): m/z 341.2 (M þ 1þ). Anal.
for C19H24N4O2: C, H, N.
4-N-[Nr-(tert-Butoxycarbonyl)valylprolyl]-1-β-arabinofuran-
osylcytosine (9b). Following a similar coupling procedure de-
scribed for compound 8b, Boc-Val-Pro-OH (0.17 g, 0.49 mmol)
was treated with ara-C (0.10 g, 0.41 mmol) to give 9b (0.06 g,
28%) as a white foam. MS (ESIþ): m/z 540.3 (M þ 1þ). Anal. for
C24H37N5O9: C, H, N.
6-N-(Aspartylprolyl)quinoline (4b). To a solution of 3b (0.091 g,
0.18 mmol) in CH2Cl2 (3 mL) was added TFA (0.40 mL), and the
mixture was stirred for 5 h. After removal of the solvent in high
vacuum, the salt formed was liberated with 1 equiv of TEA. The
crude was purified by CCTLC on the Chromatotron (ethyl
acetate/MeOH, 2:1) to give 4b (0.052 g, 83%) as a white foam.
MS (ESIþ): m/z 357.2 (M þ 1þ). Anal. for C18H20N4O4: C, H, N.
N-[Nr-(9-Fluorenylmethoxycarbonyl)valylprolyl]doxorubicine
(6). A solution of Fmoc-Val-Pro-OH (0.07 g, 0.16 mmol) and
doxorubicin hydrochloride (0.10 g, 0.16 mmol) was dissolved in
DMSO (8 mL). DIEA (59.0 μL, 0.34 mmol) was next added, and
the mixture was stirred for 15 min at room temperature
(protected from light). A solution of HATU (0.06 g, 0.18 mmol)
in DMF (2 mL) was added. The reaction mixture was stirred at
room temperature for 15 h, and the solvent was lyophilized. The
residue was dissolved in ethyl acetate (50 mL) and washed with
10% aqueous citric acid (3 ꢀ 20 mL), 10% aqueous NaHCO3
(3 ꢀ 20 mL), water (3 ꢀ 20 mL), and brine (3 ꢀ 20 mL). The
organic layer was dried (Na2SO4), filtered, and evaporated to
dryness. The final residue was dissolved in hot CH2Cl2, filtered,
and evaporated to dryness to give 6 (0.08 g, 45%) as a red foam.
MS (ESIþ): m/z 984.3 (M þ Naþ). Anal. for C52H55N3O15:
C, H, N.
N-(Valylprolyl)doxorubicine (7). The Fmoc-protected conju-
gate 6 (0.06 g, 0.06 mmol) was treated with 50% piperidine in
DMF (4 mL). The reaction mixture was stirred at room tem-
perature for 5 min, and the solvent was evaporated to dryness.
The residue was purified by reverse phase chromatrography
using SPE cartridges (acetonitrile/water, 70:1) to give 7 (0.02 g,
50%) as a red foam. 1H NMR (300 MHz, DMSO-d6): δ 0.81 (m,
6H, 2γ-CH3, Val), 1.11 (d, 3H, H-60, J = 6.4 Hz), 1.43 (m, 1H,
H-2’), 1.74-1.89 (m, 6H, H-20, H-8, β-CH, Val, β-CH2, γ-CH2,
Pro), 2.14 (m, 3H, H-8, β-CH2, Pro), 2.95 (m, 2H, H-10), 3.51
(m, 3H, H-40, δ-CH2, Pro), 3.95 (m, 5H, H-30, OCH3, R-CH,
Val), 4.14 (m, 1H, H-50), 4.35 (m, 1H, R-CH, Pro), 4.56 (bs, 2H,
H-14), 4.78 (d, 1H, OH-40, J = 5.6 Hz), 4.85 (m, 1H, OH-14),
4.93 (bs, 1H, H-7), 5.21 (bs, 1H, H-1’), 5.46 (s, 1H, OH-9), 7.53
(d, 1H, NH-30, J = 8.0 Hz), 7.64 (m, 1H, H-1), 7.91 (m, 2H, H-2,
H-3). MS (ESIþ): m/z 740.2 (M þ 1þ). Anal. for C37H45N3O13:
C, H, N.
4-N-[Nr-(Benzyloxycarbonyl)valylalanyl]-1-β-D-arabinofura-
nosylcytosine (11b). Cbz-Val-Ala-OH (0.17 g, 0.49 mmol) was
treated with ara-C (0.10 g, 0.41 mmol), via a similar coupling
procedure described for compound 8b, to give 11b (0.065 g,
26%) as a white foam. MS (ESIþ): m/z 547.3 (Mþ). Anal. for
C25H33N5O9: C, H, N.
[1-[20,50-Bis-O-(tert-butyldimethylsilyl)-β-D-ribofuranosyl]-4-
N-[Nr-(benzyloxycarbonyl)valylprolylvalylprolyl]cytosine]-30-spiro-
500-[400-amino-100,200-oxathiole-200,200-dioxide] (21a). Following
the coupling procedure described for compound 3a, TSAO-
m5C23 (0.094 g, 0.16 mmol) was reacted with Cbz-Val-Pro-Val-
Pro-OH (0.13 g, 0.24 mmol) in the presence of BOP (0.106 g,
0.24 mmol) and TEA (0.037 mL, 0.24 mmol) in dry CH2Cl2
(4 mL). The final residue was purified by CCTLC on the
Chromatotron (CH2Cl2/MeOH, 50:1) to give 21a (0.07 g,
41%) as a white foam. MS (ESIþ): m/z 1115.4 (M þ 1þ). Anal.
for C52H82N8O13SSi2: C, H, N, S.
30,50-Bis-O-(tert-butyldimethylsilyl)-4-N-[Na-(9-fluorenylmet-
hoxycarbonyl)valylprolylvalylprolyl]-1-β-D-arabinofuranosylcy-
tosine (21b). 30,50-TBS-ara-C (20b)39 (0.10 g, 0.21 mmol) was
treated with Fmoc-Val-Pro-Val-Pro-OH (0.16 g, 0.25 mmol),
via a similar coupling procedure described for compound 3a, to
give 21b (0.13 g, 56%) as a white foam. MS (ESIþ): m/z 1086.6
(Mþ), 1109.3 (M þ Naþ). Anal. for C56H83N7O11Si2: C, H, N.
[1-[20,50-Bis-O-(tert-butyldimethylsilyl)-β-D-ribofuranosyl]-4-
N-[(valylprolylvalylprolyl)cytosine]-30-spiro-500-[400-amino-100,200-
oxathiole-200,200-dioxide] (22a). A solution of the protected tetra-
peptide conjugate 21a (0.045 g, 0.039 mmol) in methanol (6 mL)
containing Pd/C (10%) (40 wt %/wt) (0.021 g) was hydroge-
nated at 25 psi at room temperature for 2 h. The reaction
mixture was filtered, and the filtrate was evaporated to dryness
under reduced pressure. The residue was dissolved in water and
liophilized to give 22a (0.038 g, 95%) as a white foam. MS
(ESIþ): m/z 981.5 (Mþ). Anal. for C44H76N8O11SSi2: C, H, N, S.
4-N-[Valylprolylvalylprolyl]-1-β-D-arabinofuranosylcytosine
(22c). To a solution of Fmoc-[Val-Pro-Val-Pro]-[30,50-TBS-ara-
C] (21b) (0.11 g, 0.10 mmol) in THF (1 mL) was added a 1.1 M
solution of TBAF in THF (0.28 mL, 0.31 mmol). The reaction
mixture was stirred at room temperature for 8 h, and the solvent
was evaporated to dryness. The residue was dissolved in iso-
butanol (50 mL) and washed with brine (3 ꢀ 20 mL). The
organic layer was dried (Na2SO4), filtered, and evaporated to
dryness. The final residue was purified by flash cromatography
(iPrOH:/H2O/NH3, 20:1:0.5) to give 22c (0.039 g, 60%) as a
white foam. 1H NMR (300 MHz, acetone-d6): δ 0.84-0.93 (m,
12H, 4γ-CH3, 2Val), 1.71-2.19 (m, 10H, 2β-CH, 2Val, 2β-CH2,
2Pro, 2γ-CH2, 2Pro), 3.44-3.82 (m, 8H, 2H-50, H-40, R-CH,
Val, 2δ-CH2, 2Pro), 3.92 (m, 1H, H-30), 4.06 (m, 1H, H-2’), 4.32
(m, 1H, R-CH, Val), 4.42 (m, 1H, R-CH, Pro1), 4.51 (m, 1H, R-
CH, Pro2), 6.06 (d, 1H, H-10, J = 3.6 Hz), 7.15 (d, 1H, H-5, J =
7.2 Hz), 7.99 (d, 1H, NH, Val, J = 8.4 Hz), 8.07 (d, 1H, H-6, J =
7.5 Hz), 11.04 (bs, 1H, NH, ara-C). MS (ESIþ): m/z 636.5 (M þ
1þ), 658.5 (M þ Naþ). Anal. for C29H45N7O9: C, H, N.
30,50-Bis-O-(tert-butyldimethylsilyl)-4-N-[Na-(9-fluorenylmet-
hoxycarbonyl)valylalanylvalylprolyl]-1-β-D-arabinofuranosylcy-
tosine (23b). To a solution of Fmoc-Val-Ala-Val-Pro-OH (0.085 g,
[1-[20,50-Bis-O-(tert-butyldimethylsilyl)-β-D-ribofuranosyl]-4-
N-[Nr-(benzyloxycarbonyl)valylprolyl]cytosine]-30-spiro-500-[400-
amino-100,200-oxathiole-200,200-dioxide] (8a). Following a similar
coupling procedure described for compound 3a, TSAO-m5C23
(0.13 g, 0.22 mmol) was reacted with Cbz-Val-Pro-OH (0.11 g,
0.32 mmol) in the presence of BOP (0.14 g, 0.32 mmol) and TEA
(0.066 mL, 0.43 mmol) in dry CH2Cl2 (4 mL) at room tempera-
ture. After 1 h of reaction, an extra amount of BOP (0.14 g,
0.32 mmol) and TEA (0.066 mL, 0.43 mmol) were added to drive
the coupling to completion. The final residue was purified by
CCTLC on the Chromatotron (CH2Cl2/MeOH, 50:1) to give 8a
(0.107 g, 53%) as a white foam. MS (ESIþ): m/z 919.3 (M þ 1þ).
Anal. for C42H66N6O11SSi2: C, H, N, S.
4-N-[Nr-(Benzyloxycarbonyl)valylprolyl]-1-β-D-arabinofura-
nosylcytosine (8b). A solution of Cbz-Val-Pro-OH (0.17 g, 0.49
mmol) (1.2 equiv) in DMF (2 mL) was successively reacted with
HOBt (0.066 g, 0.49 mmol), DIC (0.062 g, 0.49 mmol), and ara-
C (0.10 g, 0.41 mmol) at 0 °C. The reaction mixture was stirred at
room temperature for 15 h, and the solvent was evaporated to

570 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Diez-Torrubia et al.
0.14 mmol) in CH2Cl2 (3 mL) was successively added BOP
(0.062 g, 0.14 mmol), TEA (0.033 mL, 0.23 mmol), and 30,50-
TBS-ara-C (20b) (0.055 g, 0.12 mmol). The mixture was stirred at
45 °C for 15 h. After removal of the solvent in vacuo, the residue
was dissolved in ethyl acetate (15 mL) and washed with 10%
aqueous citric acid (3 ꢀ 10 mL), 10% aqueous NaHCO3 (3 ꢀ
10 mL), water (3 ꢀ 10 mL), and brine (3 ꢀ 10 mL). The organic
layer was dried (Na2SO4), filtered, and evaporated to dryness. The
final residue was purified by CCTLC on the Chromatotron
(CH2Cl2/MeOH, 20:1) to give 23b (0.05 g, 43%) as a white solid.
MS (ESIþ): m/z 1060.9 (M þ 1þ). Anal. for C54H81N7O11Si2:
C, H, N.
4-N-[Valylalanylvalylprolyl]-1-β-D-arabinofuranosylcytosine
(23c). Via a similar deprotection procedure described for com-
pound 22c, Fmoc-[Val-Ala-Val-Pro]-[30,50-TBS-ara-C] (23b)
(0.040 g, 0.039 mmol) was reacted with 1.0 M solution of TBAF
in THF (0.31 mL, 0.31 mmol) for 8 h to afford 23c (0.015 g, 63%)
as a white foam. MS (ESIþ): m/z 610.5 (M þ 1þ). Anal. for
C27H43N7O9: C, H, N.
10 min at room temperature, and then equilibrated overnight at
room temperature. The samples were centrifuged at 14 000 rpm
in an Eppendorf microcentrifuge for 1.5 min at room tempera-
ture. An aliquot of the clear supernatant was removed and
diluted to a concentration within the range of a five-point
standard curve. Water solubility was calculated from each peak
area of the solution by HPLC compared with the sample
dissolved in dimethyl sulfoxide as the standard, the exact
concentration of which is known.
Biological Methods. Compounds and Enzymes. Human solu-
ble CD26 was purified as described.49,50 Human recombinant
CD26, expressed in Sf9 cells, was obtained from Sigma
(St. Louis, MO) (1.4 mg protein/mL, 48 U/mg). Vildagliptin
(a specific CD26 inhibitor)48-50 was kindly provided by I. De
Meester (Antwerp, Belgium). Fetal bovine serum (FBS) was
obtained from Integro (Dieren, The Netherlands), and human
serum was provided by the Blood Bank, Leuven, Belgium.
Conversion of Di- and/or Tetrapeptide Prodrugs to the Corre-
sponding Parent Compound. The test compounds have been
evaluated for their substrate activity against purified CD26,
human serum (HS), and bovine serum (BS) in Eppendorf tubes.
The 400 μL reaction mixtures contained 50 μM test compound
(di- or tetrapeptide prodrugs of 6-aminoquinoline (6-AQ),
doxorubicin, TSAO-m5C, ara-C, and ara-A) in PBS (con-
taining 0.1% DMSO). The reaction was started by the addition
of purified CD26 (1.5 mU) or 20% of HS (in PBS) or BS (in PBS)
at 37 °C. Stability of the prodrugs was performed in PBS in the
absence of enzyme or serum. At different time points (as
indicated in the figures) 100 μL was withdrawn from the reaction
mixture, added to 200 μL of cold methanol, and put on ice for
10 min. Then the mixtures were centrifuged at 13 000 rpm for
5 min at 4 °C and 250 μL supernatant was analyzed by HPLC on
a reverse phase RP-8 column, using the following buffers and
gradients: buffer A, 50 mM NaH2PO4 þ 5 mM heptanesulfonic
acid pH 3.2; buffer B, acetonitrile). Gradient A: 2 min 98% A þ
2% B; 6 min linear gradient to 80% A þ 20% B; 2 min linear
gradient to 75% A þ 25% B; 2 min linear gradient to 65% A þ
35% B; 18 min linear gradient to 50% A þ 50% B; 5 min 50%
A þ 50% B; 5 min linear gradient to 98% A þ 2% B; 5 min
equilibration at 98% A þ 2% B. Gradient B: 2 min 98% A þ 2%
B; 6 min linear gradient to 80% A þ 20% B; 2 min linear
gradient to 75% A þ 25% B; 2 min linear gradient to 65% A þ
35% B; 8 min linear gradient to 50% A þ 50% B; 10 min 50%
A þ 50% B; 10 min linear gradient to 20% A þ 80% B; 5 min 20%
A þ 80% B; 15 min linear gradient to 98% A þ 2% B; 5 min 98%
A þ 2% B. The gradients allowed us to separate the di- and/or
tetrapeptide prodrugs from the corresponding parent drugs.
For the determination of the kinetic parameters (Km, Vmax),
CD26 (human recombinant CD26 derived from Sigma) was
exposed to different concentrations of the test compounds (i.e.,
50, 25, 10, 5, and 2 μM for [ValPro]-[6-aminoquinoline], 1000,
750, 500, 250, 100, and 50 μM for [AspPro]-[6-aminoquinoline],
50, 25, 10, 5, and 2 μM for [ValPro]-[doxorubicine], and 50, 25,
10, 5, and 2 μM for [ValProValPro]-[ara-A]. Hydrolysis
(removal of the prodrug moiety) was measured after 15, 30,
and 45 min as described above. Km and Vmax values were
calculated at each time point, and average values were made
for the data obtained from the three time points. The kinetic
values are the average of at least two to three independent
experiments.
20,30,50-Tris-O-(acetyl)-4-N-[Nr-(9-fluorenylmethoxycarbo-
nyl)valylprolyl]1-β-D-arabinofuranosyladenosine (26). 20,30,50-
AcO-ara-A (24)40 (0.10 g, 0.21 mmol) was treated with Fmoc-
Val-Pro-OH (0.16 g, 0.25 mmol), via a similar coupling proce-
dure described for compound 3a, to give 26 (0.13 g, 56%) as a
white foam. MS (ESIþ): m/z 812.8 (M þ 1þ). Anal. for
C41H45N7O11: C, H, N.
4-N-[Nr-(Benzyloxycarbonyl)valylprolylvalylprolyl]-30,50-bis-
O-(tert-butyldimethylsilyl)-1-β-D-arabinofuranosyladenosine (27).
30,50-TBS-ara-A (25)42 (0.055 g, 0.11 mmol) was treated with
Cbz-Val-Pro-Val-Pro-OH (0.071 g, 0.13 mmol) at 90 °C, via a
similar coupling procedure described for compound 23b, to give
27 (0.066 g, 59%) as a white foam. MS (ESIþ): m/z 1023.0 (M þ
1þ). Anal. for C50H79N9O10Si2: C, H, N.
4-N-[Nr-(Benzyloxycarbonyl)valylprolylvalylprolyl]-1-β-D-ara-
binofuranosyladenosine (28). To Cbz-[Val-Pro-Val-Pro]-[30,50-
TBS-ara-A] (27) (0.040 g, 0.039 mmol) was added a 1.0 M
solution of TBAF in THF (0.31 mL, 0.31 mmol) at room
temperature and under argon atmosphere. The reaction mixture
was stirred for 3 h, and then CaCO3 (0.065 g), DOWEX 50Wx8
(0.194 g), and MeOH (0.5 mL) were added. The resulting
suspension was stirred for 1 h, and the solvent was evaporated
to dryness. The crude was purified by CCTLC on the Chroma-
totron (CH2Cl2/MeOH, 20:1) to give 28 (0.023 g, 76%) as a
white solid. MS (ESIþ): m/z 794.7 (M þ 1þ). Anal. for
C38H51N9O10: C, H, N.
4-N-[Valylprolylvalylprolyl]-1-β-D-arabinofuranosyladenosine
(29). Cbz-[Val-Pro-Val-Pro]-[ara-A] (28) (0.020 g, 0.025 mmol)
was hydrogenated at 1 atm at room temperature for 24 h, via a
similar hydrogenation procedure described for compound 4a, to
1
give 29 (0.014 g, 85%) as a white foam. H NMR (300 MHz,
DMSO-d6): δ 0.82-0.96 (m, 12H, 4γ-CH3, 2Val), 1.53-2.27 (m,
10H, 2β-CH, 2Val, 2β-CH2, 2Pro, 2γ-CH2, 2Pro), 3.16 (d, 2H, δ-
CH2, Pro, J = 5.0 Hz), 3.58-3.83 (m, 5H, H-40, H-50, δ-CH2,
Pro), 4.11-4.24 (m, 3H, H-20, H-30, R-CH, Pro), 4.31-4.53 (m,
2H, 2R-CH, 2Val), 4.85 (bs, 1H, R-CH, Pro), 5.11 (t, 1H, OH-50,
J = 5.4 Hz), 5.58 (d, 1H, OH-30, J = 4.6 Hz), 5.66 (d, 1H, OH-30,
J = 5.3 Hz), 6.37 (d, 1H, H-10, J = 5.0 Hz), 7.92-7.98 (m,1H,
NH, Val), 8.51 (s, 1H, H-8), 8.62 (s, 1H, H-2), 10.87 (bs, 1H, NH,
ara-A). MS (ESIþ): m/z 660.7 (M þ 1þ). Anal. for C30H45N9O8:
C, H, N.
Water-Solubility Studies. Water solubility of the prodrugs
and the parent compounds was determined by HPLC analysis.
HPLC was carried out on a Waters 484 system using Novapack
C18 reverse phase column: flow rate, 1 mL/min; detection, UV
254 nm; gradient solvent system A/B (acetonitrile/water), initial
15% A þ 85% B; 5 min linear gradient to 25% A þ 75% B; 5 min
linear gradient to 35% A þ 65% B; 10 min linear gradient to
45% A þ 55% B; 5 min linear gradient to 60% A þ 40% B; 5 min
linear gradient to 100% A. Excess amount of the prodrug or of
the parent drug was suspended in deionized water, sonicated for
[ValPro]-[6-aminoquinoline], [AspPro]-[6-aminoquinoline],
[ValPro]-[doxorubicine], and [ValProValPro]-[ara-A] (25 μM)
were exposed to CD26, 20% HS, or 20% BS (as described
above) in the presence or absence of different concentrations of
the DPPIV/CD26 inhibitor vildagliptin (ranging between 50
and 0.05 μM). Conversion of the prodrugs to the parent
compounds was evaluated after 15, 30, 45, and 60 min and
analyzed as described above.
Antiviral Assays. Human T lymphocytic CEM cells were
cultured in RPMI-1640 medium (Gibco, Paisley, Scotland)

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 571
supplemented with 10% fetal bovine serum (FBS) (BioWittaker
Europe, Verviers, Belgium), 2 mM L-glutamine (Gibco), and
0.075 M NaHCO3 (Gibco). HIV-1 (IIIB) was obtained from
Dr. R. C. Gallo and Dr. M. Popovic (at that time at the National
Cancer Institute, NIH, Bethesda, MD).
(6) Balzarini, J.; Andersson, E.; Schols, D.; Proost, P.; Van Damme, J.;
Svennerholm, B.; Horal, P.; Vahlne, A. Obligatory involvement of
CD26/dipeptidyl peptidase IV in the activation of the antiretroviral
tripeptide glycylprolylglycinamide (GPG-NH2). Int. J. Biochem.
Cell Biol. 2004, 36, 1848–1859.
ꢀ
(7) Garcıa-Aparicio, C.; Bonache, M.-C.; De Meester, I.; San-Felix,
´
The CEM cells (∼4.5 ꢀ 105 cells/mL) were suspended in fresh
cell culture medium and infected with HIV (IIIB and ROD) at
100 CCID50 (1 CCID50 being the virus dose infective for 50% of
the cell cultures) per milliliter of cell suspension. Then 100 μL of
the infected cell suspension was transferred to microplate wells,
mixed with 100 μL of appropriate (freshly prepared) dilutions of
the test compounds (i.e., final concentrations of 2000, 400, 80,
16, 3.2, and 0.62 μM), and further incubated at 37 °C. After 4-5
days, giant cell formation was recorded microscopically in the
CEM cell cultures. The 50% effective concentration (EC50)
corresponded to the compound concentrations required to
prevent syncytium formation in the virus-infected cell cultures
by 50%.
The antiherpes simplex virus type 1 [HSV-1 (KOS)] and
vaccinia virus [VV (Lederle)] activity was determined by infect-
ing HEL cell cultures with 100 CCID50/mL in 96-well microtiter
plates in the presence or absence of the prodrug
[ValProValPro]-[ara-A] and 20 μM CD26 inhibitor (vildag-
liptin) (added as one single administration at the initiation of
the virus infection). The antiviral activity was scored at day 3
after infection and based on estimation of the virus-induced
cytopathicity.
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A.-M.; Velazquez, S.; Camarasa, M.-J. Efficient conversion of
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Proliferation Assays. The cytostatic activity against murine
leukemia L1210, human lymphocyte CEM, and cervix carcino-
ma HeLa cells was measured in 200 μL wells of a 96-well
microtiter plate (initial cell number of (5-7.5) ꢀ 104 cells/well).
After 48 (L1210) or 72 (CEM, HeLa) h, the tumor-cell number
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G.; Jones, A. T.; Diez-Torrubia, A.; Camarasa, M.-J.; Velazquez,
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Acknowledgment. We thank Ria Van Berwaer, Lizette van
Berckelaer, Leen Ingels, and Leentje Persoons for excellent
technical assistance. We also thank the Spanish MEC/MI-
CINN (Project SAF2006-12713-C02), the Comunidad de
Madrid (Project BIPEDD-CM ref S-BIO-0214-2006), and
Katholieke Universiteit Leuven (GOA No. 05/19) for finan-
cial support.
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K. Y.; Smith, P. L.; Lee, C.; Oh, D.; Sadee, W.; Amidon, G. L. 5 -Amino
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(18) (a) Balzarini, J.; Perez-Perez, M. J.; San-Felix, A.; Schols, D.;
Perno, C. F.; Vandamme, A. M.; Camarasa, M. J.; De Clercq, E.
20,50-Bis-O-(tert-butyldimethylsilyl)-30-spiro-500-(400-amino-100,200-
oxathiole-200,200-dioxide)pyrimidine (TSAO) nucleoside analogues:
highly selective inhibitors of human immunodeficiency virus type 1
that are targeted at the viral reverse transcriptase. Proc. Natl. Acad.
Supporting Information Available: Elemental analysis data
for novel compounds 1b, 2b, 3a,b, 4a,b, 5-7, 8a,b, 9b, 11b, 18,
19, 21a,b, 22a,c, 23b,c, and 26-29; chemical procedures for the
synthesis of di- and tetrapeptide derivatives 1b, 2b, and 17-19;
1H NMR and 13C NMR chemical shifts assigments of com-
pounds 3a,b, 4b, 6, 8a-c, 11b, 21a,b, 22a, 23b,c, and 26-28. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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