Design and discovery of a novel dipeptidyl-peptidase IV (CD26)-based prodrug approach

Article abstract of DOI:10.1021/jm0606490

Here we describe a novel type of enzyme-based prodrug approach in which a dipeptide moiety is linked to a nonpeptidic therapeutic drug through an amide bond which is specifically cleaved by the dipeptidyl-peptidase IV (DPP IV/CD26) enzyme activity present in plasma and on the surface of certain cells. DPP IV has high substrate selectivity for peptides with a proline (or an alanine) at the penultimate amino acid position at the N-terminus but tolerates a wide range of natural amino acids at the amino terminal end. A variety of dipeptidyl amide prodrugs of anti-HIV TSAO molecules were synthesized and evaluated for their ability to act as substrates for the enzyme. Our data revealed that DPP IV/CD26 can efficiently recognize such prodrugs as substrates, releasing the parent compound. Moreover, it is possible to modify the half-life and the lipophilicity of the prodrugs by changing the nature of the dipeptide. All conjugates have shown marked in vitro antiviral activities irrespective the the nature of the terminal and/or the penultimate amino acid moiety.

Full text of DOI:10.1021/jm0606490

J. Med. Chem. 2006, 49, 5339-5351
5339
Design and Discovery of a Novel Dipeptidyl-peptidase IV (CD26)-Based Prodrug Approach
Carlos Garc´ıa-Aparicio,† Mar´ıa-Cruz Bonache,† Ingrid De Meester,§ Ana San-Fe´lix,† Jan Balzarini,*,‡ Mar´ıa-Jose´ Camarasa,† and
Sonsoles Vela´zquez*,†
Instituto de Qu´ımica Me´dica (C.S.I.C.), Juan de la CierVa 3, E-28006 Madrid, Spain, Rega Institute for Medical Research, K. U. LeuVen,
Minderbroedersstraat 10, B-3000 LeuVen, Belgium, and Laboratory of Medical Biochemistry, UniVersity of Antwerp, 2610 Wilrijk, Belgium
ReceiVed June 1, 2006
Here we describe a novel type of enzyme-based prodrug approach in which a dipeptide moiety is linked to
a nonpeptidic therapeutic drug through an amide bond which is specifically cleaved by the dipeptidyl-
peptidase IV (DPP IV/CD26) enzyme activity present in plasma and on the surface of certain cells. DPP IV
has high substrate selectivity for peptides with a proline (or an alanine) at the penultimate amino acid position
at the N-terminus but tolerates a wide range of natural amino acids at the amino terminal end. A variety of
dipeptidyl amide prodrugs of anti-HIV TSAO molecules were synthesized and evaluated for their ability to
act as substrates for the enzyme. Our data revealed that DPP IV/CD26 can efficiently recognize such prodrugs
as substrates, releasing the parent compound. Moreover, it is possible to modify the half-life and the
lipophilicity of the prodrugs by changing the nature of the dipeptide. All conjugates have shown marked in
vitro antiviral activities irrespective the the nature of the terminal and/or the penultimate amino acid moiety.
Introduction
ing a proline at the penultimate position of the N-terminus, as
a carrier, wherein the conjugate [(Xaa-Pro)n]-[drug] (I, Figure
1) is specifically cleavable by the endogenous dipeptidyl-
peptidase IV enzyme (DPP IV/CD26) present on the surface of
certain cells or in plasma.
The lymphocyte surface glycoprotein CD26, originally de-
scribed as a T-cell activation/differentiation marker,1 belongs
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 (DPP IV).2-4 DPP IV
is a member of the prolyl oligopeptidase family, a group of
atypical serine proteinases able to hydrolyze a prolyl peptide
bond. CD26 is strongly expressed on a variety of leukocyte cell
subsets (e.g. T cells, B cells, natural killer cells, and macro-
phages), but also on several types of epithelial, endothelial, and
fibroblast cells. A soluble form of this enzyme also exists and
is detected in plasma and cerebrospinal fluid at lower amounts.2-4
CD26 is endowed with an interesting (dipeptidyl)peptidase
catalytic activity, and it has high selectivity for peptides with a
proline or alanine at the penultimate position of the N-terminus
of a variety of natural peptides but tolerates a wide range of
amino acyl residues at the ultimate N-terminal end. A free amino
group on the ultimate amino acid is a prerequisite for substrate
activity by the enzyme. DPP IV truncates several bioactive
peptides of medical importance.2-4
In 2005, it was demonstrated for the first time that this
enzyme plays a direct regulatory and indispensable role in the
eventual antiretroviral activity of small synthetic molecules such
as the antiretroviral prodrug GlyProGly-NH2 (inactive) releasing
glycinamide (Gly-NH2) (active).5 This is the first demonstration
that a differentiation/activation leukocytic marker, abundantly
present on activated T-lymphocytic cells, acts as a highly
specific and obligatory activator of a synthetic anti(retro)viral
prodrug that is otherwise inactive as such.
Many prodrug technologies have already been developed
depending on the kind of drug that has to be converted.6,7
Coupling of peptides or amino acids as carriers to a therapeutic
agent has already been pursued in the past. Examples of amino
acid coupling to drugs are valacyclovir and valgancyclovir, the
valyl ester prodrugs of the antiherpetic acyclovir and ganciclovir
drugs, respectively.8-10 The 3-5-fold increased oral bioavail-
ability of the prodrugs have been shown to be caused by their
affinity for the human peptide transporter hPEPT-1 located in
the membrane of the upper small intestine epithelial cells.11 The
prior art for ameliorating solubility and bioavailability reveals
however only amino acid prodrugs (only one amino acid
coupled) of small organic molecules whereby the amino acid
is usually coupled through ester bonds, allowing easy back
conversion to the free therapeutic agent by esterases. However,
the prodrugs have low stability at physiological pH and their
delivery is often not optimal.8a
Our novel dipeptide-prodrug approach would allow us to
ameliorate the solubility and/or bioavailability of therapeutic
agents in a potentially more successful manner since coupling
of a di- (or oligo)peptide moieties (instead of one single amino
acid) to a therapeutic agent is performed. Moreover, the
therapeutic drug is linked to the peptide through a more stable
amide bond (instead of an ester bond) which is specifically
cleaved by DPP IV/CD26. The presence of a proline near the
N-terminus may also serve as efficient structural protection
against nonspecific proteolytic degradation.12
These findings prompted us to explore an entirely novel
enzyme-based prodrug approach that provides conjugates of
therapeutic agents with a di- (or oligo) peptide moiety, contain-
Design. The novel prodrug approach should be used on drugs
that contain a free amino group that can be directly coupled
with the carboxyl group of amino acids via an amide bond. Since
human immunodeficiency virus (HIV), the ethiological agent
of AIDS, mainly infects lymphocytes or macrophages that
abundantly express CD26/DPP IV enzyme in their membrane
we focused on anti-HIV compounds, in particular on TSAO
derivatives. These compounds belong to an unique family of
* To whom correspondence should be addressed. Telephone: (+34)-
915622900. Fax: (+34)-91 5644853. E-mail: iqmsv29@iqm.csic.es.
(J.B.) Telephone: (+32)-16-337367. Fax: (+32)-16-337340. E-mail:
jan.balzarini@rega.kuleuven.be.
† Instituto de Qu´ımica Me´dica (C.S.I.C.).
‡ Rega Institute for Medical Research.
§ University of Antwerp.
10.1021/jm0606490 CCC: $33.50 © 2006 American Chemical Society
Published on Web 08/01/2006

5340 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17
Garc´ıa-Aparicio et al.
derivative Z-Xaa-Yaa-OH followed by deprotection of the amino
group of the intermediate N-protected conjugates by catalytic
hydrogenation. The presence of tert-butyldimethylsilyl groups
(TBDMS) at positions 2′ and 5′, sensitive to basic and acid
media, respectively, but essential for the antiviral activity of
TSAO compounds, implies the selection of a benzyloxycarbonyl
protecting strategy due to the smooth deprotection reaction
reaction conditions compatible with TBDMS groups. The
precursor NAP-TSAO-T (2) was obtained as described previ-
ously.17
Figure 1. [Xaa-Pro]-[drug] conjugates of general formula I cleavable
by CD26.
First, we focused on the synthesis of the model prodrug 5
(Scheme 1) bearing the Val-Pro dipeptide sequence, easily
recognized by the enzyme DPPIV/CD26 in natural peptides, as
the promoiety. Thus, coupling of compound 2 with the com-
mercially available dipeptide Z-Val-Pro-OH (3a) in CH2Cl2, in
the presence of BOP and TEA, gave the corresponding fully
protected dipeptide 4a in 54% yield after purification by flash
column chromatography. Catalytic hydrogenation of 4a in the
presence of 10% Pd/C in methanol afforded the deprotected
model prodrug 5 in 83% yield. No deprotection of the TBDMS
groups was observed.
Figure 2. Structures of TSAO-T (1), NAP-TSAO-T (2), and [Xaa-
Yaa]-[NAP-TSAO-T] conjugates of general formula II.
The biological results (see below) obtained with the model
prodrug 5 encouraged us to prepare a novel series of conjugates
[Xaa-Pro]-[NAP-TSAO-T] (compounds 6-13, Scheme 1)
where the proline of the model conjugate 5 was maintained and
the valine was replaced by a broad range of different natural
amino acids (Xaa) such as hydrophobic (alanine, phenylalanine,
tyrosine), basic (lysine), neutral (glycine, asparagine) or acid
(aspartic acid, glutamic acid) residues.
nonnucleoside reverse transcriptase inhibitors discovered in our
laboratories. The prototype compound is the thymine derivative
designated as TSAO-T (1, Figure 2).13-15 A lot of experience
has been gained over the past years with regard to the chemical
synthesis and the efficient introduction of modifications in its
structure. Therefore, the TSAO-T molecule was chosen as a
prototype compound for proof of concept. Due to the low
reactivity of the 4′′-amino group of the spirosultone moiety of
TSAO derivatives,16 the N-3 aminopropyl TSAO-T derivative
(NAP-TSAO-T, 2, Figure 2) has been chosen as a model
compound because its primary amine functionality would allow
the formation of an amide bond between the peptide and the
TSAO molecule. Also, it is known, in the whole series of N-3
substituted TSAO derivatives, that attachment of the (CH2)3-
NH2 substituent to the N-3 atom of the thymine of the prototype
TSAO-T preserves the antiviral activity of the compound.17 It
has been shown that the N-3-substituted moiety of the TSAO
derivatives run parallel to the subunit interface and is exposed
to the solvent. This explains why sometimes large substituents
at N-3 of TSAO-T, such as an AZT molecule linked to N-3 by
a methylene spacer, do not compromise the anti-HIV (and anti-
RT) activity of those compounds.17,18
We designed and synthesized a variety of dipeptidyl amide
prodrugs of TSAO molecules deprotected at the peptide
N-terminus (II, Figure 2) and evaluated their ability to act as
efficient substrates for the enzyme DPPIV/CD26. The aim for
the design of the dipeptide-TSAO conjugates is to explore
whether (1) DPP IV/CD26 is able to efficiently recognize
dipeptide sequences when linked through an amide bond to a
molecule (i.e. TSAO) different from a natural peptide, and if
so, whether (2) it is possible to modify the enzymatic and serum
hydrolysis rate (half-life) of the conjugates changing the nature
(Xaa, Yaa) of the dipeptide promoieties, and (3) to study the
influence of the nature of the dipeptide moiety on the overall
lipophilicity of the prodrugs. In addition, we have also evaluated
the TSAO-dipeptide prodrugs against HIV-1 RT in a cell-free
enzyme assay and against HIV-1 replication in cell culture.
The synthesis of compounds 6-13 (Series 1, Scheme 1) was
carried out in a similar way to that described for the model
prodrug 5 starting from the appropriate dipeptide derivative
Z-Xaa-Yaa-OH (3b-i). Noncommercially available dipeptides
Z-Tyr(OBzl)-Pro-OH (3d),19 Z-Lys(Z)-Pro-OH (3e),20 Z-Gly-
Pro-OH (3f),21 Z-Asn-Pro-OH (3g),22 and Z-Asp(OBzl)-Pro-
OH (3i),23 were synthesized as previously described. Novel
dipeptide intermediate Z-Glu(OBzl)-Pro-OH (3h) (Scheme 1)
was prepared using standard coupling/deprotection techniques
as follows. Thus, coupling of Z-Glu(Bzl)-OH in CH2Cl2 in the
presence of BOP and TEA gave the corresponding fully
protected dipeptide Z-Asp(OBzl)-Pro-OH (21) (Scheme 1) in
63% yield after purification by flash column chromatography.
Acid hydrolysis of compound 21 in a TFA solution yielded the
C-deprotected derivative 3h in 87% yield. Next, the appropriate
dipeptide derivatives Z-Xaa-Yaa-OH (3b-i) were reacted with
NAP-TSAO-T (2) in the presence of BOP and TEA to give the
protected intermediate conjugates 4b-i in moderate to good
yields (40-89%). Removal of the benzyl groups by catalytic
hydrogenation in the presence of 10% Pd/C in CH3OH afforded
the desired final deprotected conjugates 6-13 in good yields
(66-97%).
To further explore the substrate activity requirements for
DPPIV/CD26 a second series of conjugates [Val-Yaa]-[NAP-
TSAO-T] (Series 2, Scheme 1) was also prepared. In these
conjugates the valine of the model conjugate 5 is maintained
and the proline is changed by an alanine (14), also recognized
although less efficiently by the enzyme in natural peptide
substrates, or a glycine, leucine, or phenylalanine (15-17) as
negative control compounds. Also, unnatural amino acids such
as D-proline (18) or modified prolines [trans-4-hydroxy-L-proline
(Hyp, 19, 20) and 2,3-trans-3,4-cis-3,4-dihydroxy-L-proline
(DHP, 29, Scheme 2)] whose substrate specificity is unknown
in natural peptides, were introduced. Conjugates 14-20 (Scheme
1) were prepared by coupling of the appropriate C-deprotected
Results and Discussion
Chemistry. For the synthesis of the target conjugates we
designed a two-step procedure that consisted of the coupling
of NAP-TSAO-T 2 with the appropriate C-deprotected dipeptide

Dipeptidyl-peptidase IV-Based Prodrug Approach
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17 5341
Scheme 1. Synthesis of [Xaa-Yaa]-[NAP-TSAO-T] Conjugates 5-20a
a Reagents: (i) BOP, TEA, CH2Cl2; (ii) 2 N NaOH or TFA; (iii) H2, Pd(C), CH3OH.
Scheme 2. Synthesis of [Val-DHP]-[NAP-TSAO-T] Conjugate 29a
a Reagents: (i) Z-Val-OH, BOP, TEA, CH2Cl2; (ii) OsO4, NMO, 1,4-dioxane/H2O; (iii) 2 N NaOH, CH3OH; (iv) NAP-TSAO-T (2), BOP, TEA, CH2Cl2;
(v) H2, Pd(C), CH3OH.
dipeptide derivatives Z-Xaa-Yaa-OH 3j-p with NAP-TSAO-T
(2) in the presence of BOP/TEA to afford the Z-conjugates 4j-p
in moderate to good yields (37-89%) that were subsequently
hydrogenated (H2, Pd/C, CH3OH) to give the final deprotected
prodrugs 14-20.
Conjugate 29, bearing a 2,3-trans-3,4-cis-3,4-dihydroxy-L-
proline (DHP) amino acid residue, was prepared from the
commercially available 3,4-dehydroproline amino acid as shown
in Scheme 2. Coupling of H-3,4-dehydro-Pro-OCH3‚HCl with
Z-Val-OH gave the N-protected dipeptide ester derivative 25

5342 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17
Garc´ıa-Aparicio et al.
Scheme 3. Synthesis of [Val-Pro-Val]-[NAP-TSAO-T]
Conjugates 33
Biological Studies
Anti-HIV Activity of Dipeptidyl Prodrugs of NAP-TSAO-
T. Stock solutions of the compounds were made in DMSO prior
to dilution in cell culture medium and initiation of the antiviral
experiment. The [Xaa-Pro]-[NAP-TSAO] derivatives 5 to 13
and the [Val-Yaa]-[NAP-TSAO-T] derivatives 14 to 20, 29,
31, and 33 were evaluated for their antiviral activity against
HIV-1 and HIV-2 in CEM and MT-4 cell cultures (Table 1).
The parent NAP-TSAO-T derivative 2 was active at an EC50
of 0.14 µM in CEM and 0.52 µM in MT-4 cell cultures against
HIV-1, but not active against HIV-2. As a rule, none of the
prodrug compounds were inhibitory to HIV-2 in CEM and MT-4
cell cultures. In contrast, the dipeptidyl NAP-TSAO prodrugs
containing a penultimate Pro as the amino acid (5 to 13)
invariably inhibited HIV-1-induced syncytium formation. The
prodrugs were virtually as active as the parent compound 2.
Taking all [Xaa-Pro]-[NAP-TSAO-T] prodrugs together (5 to
13), the EC50s against HIV-1 ranged between 0.10 µM and 1.1
µM in CEM, and between 0.14 µM and 1.2 µM in MT-4 cell
cultures. Also the cellular toxicity of compounds 5 to 13 was
invariably in the same order of magnitude against MT-4 and
CEM cells (CC50s ranging between 10 µM and g125 µM).
Similar data were obtained for the dipeptide prodrugs in which
the NH2-terminal Val was kept, but the penultimate proline had
been replaced by a variety of natural (14-17), but also other,
less common or synthetic amino acids including the Val D-Pro
(18), Val Hyp(Bzl) (19), Val Hyp (20), and Val DHP (29)
derivatives. Thus, as a rule, [Xaa-Yaa]-[NAP-TSAO-T] deriva-
tives have shown marked in vitro antiviral activities irrespective
the nature of the terminal and/or the penultimate amino acid
moiety. Interestingly, the [Val-Pro-Val]-[NAP-TSAO-T] de-
rivative 33 proved also at least as inhibitory to HIV as the
[Val]-[NAP-TSAO-T] derivative 31 and the parent compound
2 (Table 1).
Inhibitory Activity of Dipeptidyl Prodrugs of NAP-
TSAO-T on Recombinant Purified HIV-1 Reverse Tran-
scriptase. The compounds were also examined on their
inhibitory activity against HIV-1 RT. The parent NAP-TSAO-T
derivative 2 inhibited the reaction at an IC50 of 8.1 µM. All
dipeptidyl prodrug derivatives inhibited the RT reaction at a
similar extent (Table 1).
Inhibitory Effect of [Val-Pro]-[NAP-TSAO-T] (5) against
CD26-Catalyzed Conversion of GlyPro-p-nitroanilide (pNA)
to GlyPro and pNA. The enzymatic activity of purified CD26
has been measured by conversion of the artificial substrate
GlyPro-pNA to GlyPro and pNA. During the hydrolysis
reaction, yellow-colored pNA is released from the uncolored
GlyPro-pNA. The formation of pNA can be monitored by
spectrophotometric absorption measurements at 400 nm. Com-
pound 5 dose-dependently inhibited the hydrolysis of 1 mg/
mL (3.4 mM) GlyPro-pNA in the presence of 1.5 mUnits of
purified CD26. At 100 µM 5, the reaction was inhibited by
∼70% (Figure 3). Also, 20 µM 5 could still inhibit the reaction
by ∼50%. The corresponding Z-blocked analogue 4a was
completely inactive in this assay. The dose-dependent inhibition
of the CD26-catalyzed conversion of GlyPro-pNA to GlyPro
and pNA by compound 5 is obviously due to competition of 5
with the artificial substrate for the enzyme.
Reagents: (i) Z-Val-OH, BOP, TEA, CH2Cl2; (ii) H2, Pd(C), CH3OH;
(iii) Z-Val-Pro-OH, BOP, TEA, CH2Cl2.
in 68% yield. The stereoselective osmium tetroxide oxidation
of compound 25 with N-methyl-morpholine-N-oxide (NMO)24
afforded the 2,3-trans-3,4-cis-3,4-dihydroxy-L-proline dipeptide
ester derivative 26 as the major diasteromeric derivative in good
yield (82%). Cleavage of the methyl ester group under standard
saponification conditions yielded the desired C-deprotected
dipeptide derivative 27 in 89% yield. Compound 27 and NAP-
TSAO-T (2) were similarly coupled in the presence of BOP/
TEA to give Z-conjugate 28 in 40% yield which was subse-
quently hydrogenated in the presence of 10% Pd/C in CH3OH
to give the final desired conjugate 29 in good yields (87%).
The structure of intermediate conjugates Z-[Xaa-Yaa]-[NAP-
TSAO-T] (4a-p, 28) and final deprotected conjugates H-[Xaa-
Yaa]-[NAP-TSAO-T] (5-20 and 29) was determined by
analytical and spectroscopic data. Racemization was not ob-
served by HPLC or NMR except in the Val-Ala (4j, 14), Val-
Leu (4l, 16) and Val-Phe (4m, 17) conjugates where an
epimerization of the C-terminal amino acid residue by 15%,
30%, and 24%, respectively, was observed. These results are
in agreement with previous studies on racemization during
coupling reactions that showed that Phe or Leu are the most
susceptible residues to a-inversion.25
Finally, we prepared the valyl-prolyl-valyl derivative 33
(Scheme 3) in which the dipeptide model sequence Val-Pro was
linked to the primary amino group of the valyl-NAP-TSAO-T
intermediate 31 in order to investigate whether this compound
would be also an efficient substrate of the DPP IV/CD26
enzyme. Coupling of 2 with Z-Val-OH in the presence of BOP
and TEA gave the protected valyl TSAO derivative 30 in 72%
yield. Subsequent standard catalytic hydrogenation of 30 (H2,
Pd/C) yielded the corresponding deprotected derivative 31
(72%). Compound 31 was finally coupled with the commercially
available Z-Val-Pro-OH in the presence of BOP and TEA to
give the Z-conjugate 32 in 70% yield and hydrogenated (H2,
Pd/C) to afford the deprotected tripeptide conjugate 33 (59%).
Conversion of [Val-Pro]-[NAP-TSAO-T] (5) to NAP-
TSAO-T (2) and Val-Pro by Purified CD26 and Human and
Bovine Serum. When 50 µM 5 was exposed to purified CD26,
conversion to compound 2 took place as a function of incubation
time (Table 2). After 1 h, 37% conversion was observed, and
after 4 and 24 h, ∼60% of prodrug derivative was hydrolyzed.

Dipeptidyl-peptidase IV-Based Prodrug Approach
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17 5343
Table 1. Antiviral, Cytostatic, and Anti-RT Activity of Dipeptidyl Prodrugs of NAP-TSAO-T (2)
EC50 (HIV-1) (µM)a
CC50 (µM)b
IC50 (µM)c
compd
amino acids at N-3 position
MT-4
CEM
MT-4
CEM
HIV-1 RT (polyrC.dG)
2
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
29
31
33
0.52 ( 0.31
0.30 ( 0.12
0.30 ( 0.27
0.20 ( 0.12
0.14 ( 0.03
0.14 ( 0.09
0.13 ( 0.08
0.42 ( 0.18
0.10 ( 0.03
0.15 ( 0.04
0.16 ( 0.06
0.20 ( 0.10
0.37 ( 0.24
0.26 ( 0.01
1.1 ( 0.64
0.08 ( 0.01
0.05 ( 0.01
0.19 ( 0.11
0.09 ( 0.01
0.09 ( 0.01
0.09 ( 0.01
0.40 ( 0.0
14 ( 7.8
12 ( 8.2
10 ( 0.64
19 ( 4.2
14 ( 6.6
10 ( 0.28
47 ( 7.4
51 ( 12
8.1 ( 4.6
6.6 ( 4.2
Val-Pro
Ala-Pro
Phe-Pro
Tyr-Pro
Lys-Pro
Gly-Pro
Asn-Pro
Glu-Pro
Asp-Pro
Val-Ala
Val-Gly
Val-Leu
Val-Phe
Val-DPro
Val-Hyp(Bzl)
Val-Hyp
Val-DHP
Val
11 ( 0.99
20 ( 0.17
19 ( 0.14
11 ( 13
8.1 ( 5.1
4.7 ( 0.5
13 ( 6.0
4.0 ( 0.2
87 ( 54
0.39 ( 0.11
0.59 ( 0.08
0.49 ( 0.04
1.2 ( 1.0
0.23 ( 0.03
0.14 ( 0.02
0.21 ( 0.06
0.26 ( 0.09
0.49 ( 0.25
0.27 ( 0.12
0.41 ( 0.09
0.77 ( 0.02
0.30 ( 0.06
0.22 ( 0.07
13 ( 0.57
22 ( 6.1
64 ( 19
25 ( 3.0
30 ( 8.8
69 ( 23
g125
7.2 ( 4.3
5.4 ( 1.2
3.8 ( 0.2
4.6 ( 0.1
6.8 ( 3.0
22 ( 6.0
28 ( 1.0
16 ( 10
22 ( 2.6
9.9 ( 6.3
13 ( 6.5
14 ( 5.3
22 ( 0.78
2.0 ( 0.12
10 ( 0.14
46 ( 0.85
8.2 ( 3.3
11 ( 9.4
11 ( 0.71
10 ( 0.19
11 ( 0.78
10 ( 1.4
11 ( 0.78
2.1 ) 0.26
15 ( 1.6
51 ( 3.0
9.6 ( 0.38
11 ( 0.78
0.35 ( 0.21
0.06 ( 0.03
0.07 ( 0.01
4.8 ( 0.3
6.7 ( 2.7
4.4 ( 0.3
Val-Pro-Val
a 50% effective concentration required to inhibit HIV-induced cytopathicity by 50%. b 50% cytostatic concentration required to inhibit MT-4 or CEM cell
proliferation by 50%. c Inhibitory concentration ) 50% of the compound concentration required to inhibit recombinant HIV-1 RT activity by 50%.
Figure 3. Inhibitory effect of different concentrations of [Val-Pro]-
[NAP-TSAO-T] 5 against CD26-catalyzed conversion of GlyPro-pNA
to GlyPro and pNA at 5, 10, or 15 min of reaction.
Figure 4. Inhibitory effect of different concentrations of the dipeptide
Val-Pro against CD26-catalyzed conversion of GlyPro-pNA to GlyPro
and pNA at 5, 10, or 15 min of reaction.
Table 2. Conversion of Dipeptidyl Prodrugs of NAP-TSAO-T (2) by
Purified CD26 (1.5 mUnits) after 1, 4, or 24 h of Incubation
catalyzed GlyPro-pNA conversion, it dose-dependently inhibited
the reaction (Figure 4). At 40 µM, the release of pNA was
prevented by ∼50%.
percent of conversion to parent compound 2
1 h
4 h
24 h
2 (NAP-TSAO-T)
4a
5
6
7
Interestingly, conjugate 4a containing a benzyloxycarbonyl
group (Z) at the terminal amino group of valine completely
lacked substrate activity for CD26 (Table 2). Even after 24 h
of incubation, no traces of NAP-TSAO-T (2) could be observed.
Thus, a free amino group on the NH2-terminal amino acid is a
prerequisite for substrate activity by CD26.
0
37
0
62
81
37
66
85
20
27
48
7.9
6.5
0
0
61
88
58
79
99
58
73
92
30
35
0
8
9
43
5
10
11
12
13
14
15
16
17
18
19
20
29
Human serum (HS) and bovine serum (BS) also hydrolyzed
compound 5 to ProVal and compound 2. Indeed, when diluted
to 5, 2.5, 1, or 0.5% in PBS, both HS and BS converted 5 to 2
in a serum concentration-dependent manner (Figure 5). A human
serum concentration as low as 2.5% in PBS hydrolyzed 5 up to
30% after 3 h and up to 50% after 6 h. BS had a slower
conversion rate than HS. After 6 h, 2 was formed from 5 by
almost 20%, and 50% of 5 had converted to 2 after overnight
(24 h) incubation (Figure 5).
0
0
0
0
0
0
0
2.2
0
0
0
4.2
0
4.1
0
18
0
43
0
Inhibition of CD26-Catalyzed Conversion of Compound
5 to Compound 2 by CD26 Inhibitors. Both Diprotin A and
IlePyr are known inhibitors of CD26.26-28 When 5 was added
to the reaction mixture containing purified CD26, 2.5% HS or
2.5% BS in PBS and 500 µM or 50 µM IlePyr or 1000 µM or
100 µM Diprotin A, the conversion of 5 to 2 after 5 h was
markedly inhibited. In fact, at the highest inhibitor concentration,
Higher (i.e. 4-fold) CD26 concentrations did not further increase
the amount of conversion (data not shown). The fact that the
reaction did not further proceed after a 4 h exposure time nor
in the presence of higher CD26 enzyme concentrations is most
likely due to feedback inhibition of the reaction by the hydrolysis
product ValPro. Indeed, when ValPro was exposed to CD26-

5344 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17
Garc´ıa-Aparicio et al.
Figure 7. Conversion of [Val-Pro-Val]-[NAP-TSAO-T] 33 to [NAP-
TSAO-T] 5 in the presence of purified CD26 in function of time.
potency of inhibition may depend on the nature of the released
dipeptide.
In the second series of compounds, the penultimate proline
was replaced by another natural amino acid and the first (NH2-
terminal) amino acid consisted of a Val (compounds 14 to 20
and 29). None of the compounds, except for the ValAla NAP-
TSAO-T derivative 14, showed significant substrate activity
against CD26. Interestingly, the Val D-pro derivative 18 showed
measurable but rather marginal substrate activity. Instead, Val-
Hydroxyproline (ValHyp) was markedly converted to the parent
2, albeit at a lower efficiency than the corresponding ValPro
derivative. The Val-dihydroxyproline (ValDHP) derivative 29
was devoid of any CD26 substrate activity.
Figure 5. Conversion of [Val-Pro]-[NAP-TSAO-T] 5 to the parent
compound [NAP-TSAO-T] 2 by human and bovine serum.
The ValProVal tripeptide derivative of 2 (compound 33) has
also been synthesized and investigated for substrate activity
against purified CD26. Interestingly, the tripeptide prodrug was
efficiently converted by CD26 to the Val derivative 31, which
itself is not a substrate for CD26. When the substrate activity
of 33 was compared with compound 5 in the presence of 5.7
mUnits CD26, it proved to be slightly less efficiently converted
to its hydrolysis product than compound 5 (Figure 7).
Modulation of the Lipophilicity of the Dipeptidyl Deriva-
tives of NAP-TSAO-T. The cLogP values for the different
dipeptidyl derivatives of NAP-TSAO-T have been calculated
and compared with the parent compound 2 (Supporting Infor-
mation). We also correlated the cLogP data with the retention
time of the prodrug derivatives on a reverse phase HPLC
column. There was a good correlation between both values. We
found a correlation coefficient of r ) 0.73 between cLogP and
tR (compounds 6, 12, and 19 were excluded because their tR
values were obtained through a different HPLC gradient system).
Interestingly, when considering the Xaa Pro prodrugs 5 to 13,
the lipophilicity of the prodrugs could be markedly modulated
depending the nature of the NH2-terminal amino acids. Indeed,
the AspPro and GluPro derivatives (cLogP: -0.27 and -0.69)
had a 1000-fold lower lipophilicity than parent drug 2 (cLogP:
2.69). Several other dipeptidyl prodrugs showed a cLogP value
between these extremes. However, lipophilicity could be also
increased by at least 1 order of magnitude by the PhePro prodrug
(cLopP: 3.88). Also, among the ValYaa prodrugs (i.e. 14 to
20 and 29), the lipophilicity could be markedly modulated
depending the nature of the penultimate amino acid. Thus, the
lipophilicity of the parent drug can be easily optimized
depending on the eventual needs and nature of the drug.
Figure 6. Effect of the specific CD26 inhibitors diprotin A and IlePyr
on the conversion of [Val-Pro]-[NAP-TSAO-T] 5 to [NAP-TSAO-T]
2.
the reaction was completely blocked in the presence of CD26,
HS, or BS. At the lowest inhibitor concentration, ∼70% of the
reaction was inhibited for CD26, and >80% for HS and BS
(Figure 6). These observations point to CD26 as the main and
predominant enzyme responsible in HS and BS to remove the
dipeptide part from the prodrug.
Conversion of Dipeptidyl Prodrugs of Compound 2 by
Purified CD26. A variety of dipeptides were introduced on the
parent compound 2. In a first series of compounds, the second
amino acid consisted of a proline (compounds 5 to 13). All
prodrugs proved to be a substrate for CD26 (Table 2).
Compounds 6 (AlaPro) and 9 (LysPro) ranked among the most
efficient substrates whereas 10 (GlyPro) and 13 (AspPro) were
among the least efficient substrates of CD26. In none of the
cases except for compound 9, conversion was complete after
24 h. Obviously, as earlier demonstrated for ValPro, the released
dipeptide may feedback inhibit the enzymatic reaction, and the
Discussion
The dipeptidyl-peptidase IV occurs as a membrane-bound
enzyme present on several cell types, but also as a soluble form

Dipeptidyl-peptidase IV-Based Prodrug Approach
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17 5345
present in plasma.2 It predominantly cleaves the dipeptidyl part
from the amino terminal side of peptides when the penultimate
amino acid is preferentially a proline.2 However, peptides with
penultimate alanine are also known to be a substrate for CD26
albeit at a lower efficiency as when a proline is present.2,3 In
fact, the structure (amino acid)-activity relationship for CD26/
DPP IV has also been demonstrated among the XaaPro-
prodrugs of NAP-TSAO-T. The stringent selectivity conferred
by the penultimate amino acid, but also the freedom to have a
broad variety of natural amino acids at the amino terminal end
of the peptide, let us to apply these principles in a novel type
of prodrug approach in which a dipeptide is linked to the free
amino-terminal end of a nonpeptidic drug. Our data revealed
that CD26 can indeed recognize these prodrugs as efficient
substrates to be converted to the parent compound.
nature of the different amino acids at the amino terminal end,
as well as the presence of a proline, a hydroxyproline or an
alanine as the second amino acid of the prodrug part clearly
determines the half-life of the prodrug in the plasma. This will
allow modulation of the release of the parent compound as a
function of the particular needs. Third, if required, the lipophi-
licity of the prodrug can be profoundly modulated by the choice
of the first amino acid (i.e. aspartic acid or glutamic acid on
the one extreme versus phenylalanine on the other extreme).
Fourth, it is also obvious that the nature of the amino-terminal
amino acid will also modulate the solubility of the parent drug
and may allow more efficient or optimized formulation of the
particular parent compound. The fact that CD26 is abundantly
present in plasma in its soluble form and on several cell types
will guarantee that the prodrug will be eventually converted to
the parent drug.
In fact, the known substrate specificity of CD26 to cleave
natural peptides29 also proved valid when a synthetic compound
such as TSAO-T had been linked to the dipeptide: Xaa-Pro is
recognized as well as Xaa-Ala; hydrophobic aliphatic residues
are favored at the amino terminal position, and negatively
charged Xaa such as Asp is among the least favored amino acids,
whereas the positively charged Xaa Lys is usually a well-
accepted substrate for CD26. Also, CD26 is strongly stereospe-
cific. The scissile and Xaa-Pro bonds must be in trans
configuration.29 Thus, the substrate activity and selectivity of
dipeptidyl derivatives of synthetic compounds to be cleaved by
CD26 can be more or less predicted as based on the known
SAR of CD26 for its natural peptidic substrates.
Experimental Section
Microanalyses were obtained with a Heraeus CHN-O-RAPID
instrument. Electrospray mass spectra were measured on a qua-
dropole mass spectrometer equipped with an electrospray source
(Hewlett-Packard, LC/MS HP 1100). 1H NMR spectra were
recorded with a Varian XL-300 and a Bruker AM-200 spectrometer
operating at 300 and at 200 MHz 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 chromatog-
raphy (CCTLC) on a Chromatotron (Kiesegel 60 PF254 gipshaltig
(Merck), layer thickness (1 mm), flow rate (5 mL/min). The
dipeptide derivatives Z-Val-Pro-OH, Z-Ala-Pro-OH, Z-Phe-Pro-
OH and Z-Val-Ala-OH were purchased from Bachem Feinchemi-
kalien. Z-Tyr(OBzl)-Pro-OH,19 Z-Lys(Z)-Pro-OH,20 Z-Gly-Pro-
OH,21 Z-Asn-Pro-OH,22 Z-Asp(OBzl)-Pro-OH,23 Z-Val-Gly-OH,30
Z-Val-Leu-OH,31 and Z-Val-Phe-OH32 were synthesized as previ-
ously described.
Synthesis of Novel Dipeptide Derivatives Z-Xaa-Yaa-OH
(3h,n,o,p). General Coupling Procedure for the Synthesis of
Z-Xaa-Yaa-OR. To a solution of the corresponding Z-Xaa-OH (1
equiv) in CH2Cl2 (2-3 mL) were successively added (benzotriazol-
1-yl-oxy)-tris(dimethylamino)phosphonium hexafluorophosphate
(BOP) (1.2 equiv), the corresponding C-protected amino acid
H-Yaa-OR‚HCl (1.2 equiv), and TEA (2.2 equiv), and the mixture
was stirred at room temperature for 15 h. After removal of the
solvent in vacuo, 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 flash
column chromatography. The chromatography eluent, yield of the
isolated products, analytical and spectroscopic data are indicated
below for each compound.
The dipeptidyl derivatives of NAP-TSAO generally showed
a pronounced anti-HIV activity in cell culture irrespective the
nature of the amino acids in the dipeptide part of the molecule.
These findings are not unexpected since these TSAO derivatives
invariably inhibited HIV RT in cell-free assays, due to the
flexibility that is allowed on the N-3 position of the thymine
ring to introduce bulky substituents at this position.14,15 It is
therefore not clear whether the anti-HIV activity observed in
the cell culture assays is due to the presence of the prodrug or
to the released parent NAP-TSAO-T compound. However, when
107 CEM cells were suspended in PBS and exposed for 3 h at
37 °C to compound 5, up to 63.5% of the prodrug has been
converted to the parent NAP-TSAO-T compound in the PBS
supernatant within this short time period, and 62% of the parent
compound has been found intracellularly after 3 h. This
conversion had been obviously due to the CD26 present in the
CEM cell membrane. Given the fact that there is also CD26
present on the culture medium of HIV-infected CEM cell
cultures, it is reasonable to assume that the eventual antiviral
activity recorded at day 4 postinfection is most likely due to
the parent compound released from the prodrug.
Z-Glu(Bzl)-Pro-OtBu (21). According to the general procedure,
Z-Glu(OBzl)-OH (0.55 g, 1.48 mmol) was reacted with H-Pro-Ot-
Bu‚HCl (0.21 g, 1.24 mmol). The residue was purified by flash
column chromatography (hexane:ethyl acetate, 7:3) to give 0.40 g
(63%) of 21 as a white foam. MS (ESI+): m/z 483.3 (M + 1)+.
Anal. for C29H36N2O7: C, H, N.
Z-Val-D-Pro-OCH3 (22). Via the general coupling procedure,
Z-Val-OH (0.47 g, 1.90 mmol) was treated with H-D-Pro-OMe‚
HCl (0.20 g, 1.58 mmol). The residue was purified by flash column
chromatography (hexane:ethyl acetate, 2:1) to give 0.43 g (75%)
of 22 as a white foam. MS (ESI+): m/z 363.3 (M + 1)+. Anal. for
C19H26N2O5: C, H, N.
In this study, the major aims were to demonstrate that
dipeptidyl (particularly Xaa-Pro) prodrugs of synthetic small
molecules can be cleaved to the parent compound by the
selective action of CD26. This has now been clearly demon-
strated with purified enzyme, as well as human and bovine
serum. Although we have also shown preservation of antiviral
activity of the prodrugs in cell culture, these data do not reveal
whether the prodrugs are efficiently converted to the parent drug
since the prodrugs also proved inhibitory to the TSAO target
HIV-1 reverse transcriptase.
In conclusion, the advantages of this approach are multiple.
First, upon conversion of the prodrug to its parent compound,
a natural product (a dipeptide) is released. Therefore, the prodrug
would not be expected to create additional side effects after
cleavage to the parental drug and the prodrug part. Second, the
Z-Val-Hyp(Bzl)-OCH3 (23). Z-Val-OH (0.30 g, 1.21 mmol) was
reacted with H-Hyp(Bzl)-OMe‚HCl (0.39 g, 1.46 mmol) according
to the general coupling procedure. The final residue was purified
by flash column chromatography (hexane:ethyl acetate, 1:1) to give

5346 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17
Garc´ıa-Aparicio et al.
0.51 g (90%) of 23 as a white foam. MS (ESI+): m/z 469.3 (M +
1)+. Anal. for C26H32N2O6: C, H, N.
Chromatotron (dichloromethane-methanol, 40:1) to give 0.12 g
1
(82%) of compound 26 as a syrup. H NMR (200 MHz, CDCl3):
δ 0.95 (d, 3H, γ-CH3a, Val, J ) 6.8 Hz), 1.01 (d, 3H, γ-CH3b,
Val, J ) 6.8 Hz), 2.01 (m, 1H, b-CH, Val), 3.70 (m, 6H, OCH3,
δ-CH2, γ-CH, Hyp), 4.28 (m, 2H, R-CH, Val, Hyp), 4.41 (d, 1H,
â-CH, Hyp, J ) 5.3 Hz), 5.07 (s, 2H, CH2, Z), 5.60 (d, 1H, NH,
Val, J ) 8.6 Hz), 7.34 (m, 5H, Ar, Z). MS (ESI+): m/z 395.2 (M
+ 1)+. Anal. for C19H26N2O7: C, H, N.
Z-Val-Hyp-OCH3 (24). Following the general procedure, Z-Val-
OH (0.33 g, 1.33 mmol) was reacted with H-Hyp-OCH3‚HCl (0.16
g, 1.11 mmol). The final residue was purified by flash column
chromatography (hexane:ethyl acetate, 1:2) to give 0.22 g (45%)
of 24 as a white foam. MS (ESI+): m/z 379.2 (M + 1)+. Anal. for
C19H26N2O6: C, H, N.
General C-Deprotection Procedure for the Synthesis of
Z-Xaa-Yaa-OH. Method A (saponification). A solution of the
corresponding Z-Xaa-Yaa-OCH3 (1 equiv) in CH3OH (7-9 mL)
was treated with a 2 N NaOH solution (1.5 equiv). The mixture
was stirred at room-temperature overnight, and the solvent was
evaporated to dryness. The residue was dissolved in water (15 mL)
and acidified with a 1 N HCl solution (pH ) 3), and the product
was extracted with ethyl acetate (3 × 15 mL). The organic layer
was washed with brine (3 × 20 mL), dried (Na2SO4), filtered, and
evaporated to dryness. The final residue was dissolved in water
and liophilized.
Method B (acid hydrolysis). A solution of the corresponding
Z-Xaa-Yaa-OtBu (1 equiv) in CH2Cl2 (4-5 mL) was reacted with
TFA (1.5 equiv), and the mixture was stirred at room temperature
for 3 h. The solvent was removed in vacuo, and the residue was
coevaporated several times with CH2Cl2. The final residue was
dissolved in water and liophilized.
The deprotection method, yield of the isolated products, analytical
and spectroscopic data are indicated below for each compound.
Z-Glu(Bzl)-Pro-OH (3h). Following the general C-deprotection
procedure (Method B), compound 21 (0.40 g, 1.48 mmol) was
reacted with TFA to give 0.31 g (87%) of 3h as a colorless syrup.
MS (ESI+): m/z 483.3 (M + 1)+. Anal. for C25H28N2O7: C, H, N.
Z-Val-D-Pro-OH (3n). According to the general deprotection
procedure (Method A), Z-Val-D-Pro-OCH3 (22) (0.37 g, 0.95 mmol)
was treated with a 2 N NaOH solution to give 0.31 g, (86%) of 3n
as a white foam. MS (ESI+): m/z 349.1 (M + 1)+. Anal. for
C18H24N2O5: C, H, N.
Z-Val-(3R,4S)-3,4-dihydroxy-Pro-OH (27). Following the gen-
eral deprotection procedure (Method A), compound 26 (0.12 g, 0.31
mmol) was treated with a 2 N NaOH solution to yield 0.10 g (89%)
1
of dipeptide derivative 27 as a white foam. H NMR (300 MHz,
DMSO-d6): δ 0.95 (d, 3H, γ-CH3a, Val, J ) 6.8 Hz), 1.01 (d, 3H,
γ-CH3b, Val, J ) 6.8 Hz), 2.01 (m, 1H, â-CH, Val), 3.59 (dd, 1H,
δ-CH2a, Hyp, J ) 5.0 Hz, J ) 10.0 Hz), 3.72 (dd, 1H, δ-CH2b,
Hyp, J ) 5.4 Hz, J ) 10.0 Hz), 4.02 (m, 4H, R-CH, Val, R-CH,
â-CH, γ-CH, Hyp), 5.01 (dd, 2H, CH2, Z, J ) 12.6, J ) 17.6 Hz),
5.14 (bs, 1H, OH), 5.43 (bs, 1H, OH), 7.43 (m, 5H, Ar, Z), 7.43
(d, 1H, NH, Val, J ) 8.4 Hz). MS (ESI+): m/z 381.2 (M + 1)+.
Anal. for C18H24N2O7: C, H, N.
General Coupling Procedure for the Synthesis of Compounds
4a-p, 28, 30, and 32. A solution of the corresponding C-
deprotected dipeptide (1.5 equiv) in CH2Cl2 (2 mL) was succes-
sively treated with BOP (1.5 equiv), the N-3-aminopropyl TSAO
derivative (NAP-TSAO-T) 2 (1 equiv), and triethylamine (2.5
equiv). The reaction mixture was stirred at room temperature for
24 h, and the solvent was evaporated to dryness. The residue was
dissolved in ethyl acetate 26(50 mL), 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 flash column chromatography or by CCTLC on
the Chromatotron. The chromatography eluent, yield of the isolated
products, and analytical and spectroscopic data are indicated below
for each compound.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[Na(benzyloxycarbonyl)-valyl-prolylamino]propyl]thym-
ine]-3′-spiro-5′′-[4′′-amino-1′′,2′′-oxathiole-2′′,2′′-dioxide] (4a).
According to the general coupling procedure, NAP-TSAO-T 2 (99
mg, 0.15 mmol) was reacted with Z-Val-Pro-OH 3a (64 mg, 0.18
mmol). The final residue was purified by CCTLC on the Chroma-
totron (dichloromethane:methanol, 70:1) to give 81 mg (54%) of
Z-Val-Hyp(Bzl)-OH (3o). Z-Val-Hyp(Bzl)-OCH3 (23) was
deprotected with a 2 N NaOH solution via the general procedure
(Method A) to yield 0.34 g, (75%) of 3o as an amorphous white
solid. MS (ESI+): m/z 455.3 (M + 1)+. Anal. for C25H30N2O6: C,
H, N.
Z-Val-Hyp-OH (3p). Z-Val-Hyp-OCH3 (24) (0.20 g, 0.54 mmol)
was deprotected with a 2 N NaOH solution according to the general
procedure (Method A) to yield 0.20 g, (98%) of 3p as a white foam.
MS (ESI+): m/z 365.2 (M + 1)+. Anal. for C18H24N2O6: C, H, N.
Z-Val-3,4-dehydro-Pro-OCH3 (25). Z-Val-OH (0.31 g, 1.26
mmol) was reacted with H-3,4-dehydro-Pro-OCH3‚HCl (0.24 g,
1.51 mmol) according to the general procedure. The final residue
was purified by flash column chromatography (hexane:ethyl acetate,
1
4a as a white foam. H NMR (300 MHz, CDCl3) d 0.92 (d, 3H,
γ-CH3a, Val, J ) 6.7 Hz), 0.96 (d, 3H, γ-CH3b, Val, J ) 6.7 Hz),
1.76 (m, 2H, -CH2-), 2.00 (m, 8H, CH3-5, â-CH, Val, â-CH2,
γ-CH2, Pro), 3.14 (m, 2H, CH2NHCO), 3.58 (m, 2H, δ-CH2, Pro),
3.95 (m, 4H, 2H-5′, N3-CH2), 4.29 (m, 1H, H-4’), 4.32 (dd, 1H,
R-CH, Val, J ) 6.1 Hz, J ) 9.0 Hz), 4.49 (m, 2H, H-2′, R-CH,
Pro), 5.05 (s, 2H, CH2, Z), 5.45 (d, 1H, NH, Val, J ) 9.2 Hz),
5.55 (s, 1H, H-3′′), 5.58 (bs, 2H, NH2-4′′), 5.89 (d, 1H, H-1′, J )
8.0 Hz), 7.03 (m, 1H, NHCO), 7.15 (s, 1H, H-6), 7.32 (m, 5H, Ar,
Z). MS (ESI+): m/z 977.2 (M + 1)+. Anal. for C45H72N6O12SSi2:
C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-
3-N-[3-N′-[Na(benzyloxycarbonyl)-alanyl-prolylamino]propyl]-
thymine]-3′-spiro-5′′-[4′′-amino-1′′,2′′-oxathiole-2′′,2′′-dioxide]
(4b). Following the general coupling procedure, nucleoside 2 (100
mg, 0.15 mmol) was reacted with Z-Ala-Pro-OH 3b (59 mg, 0.18
mmol). The residue was purified by CCTLC on the Chromatotron
(dichloromethane:methanol, 70:1) to give 125 mg (85%) of 4b as
a white foam. MS (ESI+): m/z 949.3 (M + 1)+. Anal. for
C43H68N6O12SSi2: C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[Na(benzyloxycarbonyl)-phenyalanyl-prolylamino]pro-
pyl]thymine]-3′-spiro-5′′-[4′′-amino-1′′,2′′-oxathiole-2′′,2′′-diox-
ide] (4c). Nucleoside 2 (100 mg, 0.15 mmol) was reacted with
Z-Phe-Pro-OH 3c (74 mg, 0.18 mmol) according to the general
coupling procedure. The residue was purified by flash column
chromatography (dichloromethane:methanol, 70:1) to give 141 mg
(89%) of 4c as a white foam. MS (ESI+): m/z 1026.2 (M + 1)+.
Anal. for C49H72N6O12SSi2: C, H, N, S.
1
1:1) to give 0.22 g (45%) of 25 as a white foam. H NMR (300
MHz, CDCl3): δ 0.96 (d, 3H, γ-CH3a, Val, J ) 6.8 Hz), 1.05 (d,
3H, γ-CH3b, Val, J ) 6.8 Hz), 2.01 (m, 1H, â-CH, Val), 3.70 (s,
3H, OCH3), 4.27 (m, 1H, R-CH, Val), 4.43 (dd, 1H, δ-CH2a, Pro,
J ) 2.8 Hz, J ) 14.5 Hz), 4.60 (dd, 1H, δ-CH2b, Pro, J ) 2.2 Hz,
J ) 14.5 Hz), 5.05 (s, 2H, CH2, Z), 5.18 (m, 1H, R-CH, Pro), 5.42
(d, 1H, NH, Val, J ) 9.3 Hz), 5.81 (m, 1H, γ-CH, Pro), 5.96 (m,
1H, â-CH, Pro), 7.31 (m, 5H, Ar, Z). MS (ESI+): m/z 361.2 (M +
1)+. Anal. for C19H24N2O5: C, H, N.
Z-Val-(3R,4S)-3,4-dihydroxy-Pro-OCH3 (26). A solution of
compound 25 (0.13 g, 0,27 mmol) was placed in a mixture of 1,4-
dioxane (3 mL) and water (1 mL) to which was added N-
methylmorpholine oxide (NMO, 50% aqueous solution, 93.3 mL,
0.45 mmol) followed by a tert-butyl alcohol solution of OsO4 (2.5%
w/w, 61.88 µL, 0.01 mmol). The reaction was stirred under an argon
atmosphere for 2 days, and then it was quenched with a 10%
solution of Na2S2O3 (2 mL). The reaction was concentrated to
remove THF, and the aqueous fraction was extracted with ethyl
acetate (3 × 20 mL). The combined organic fractions were washed
with 10% aqueous NaHCO3 (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

Dipeptidyl-peptidase IV-Based Prodrug Approach
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17 5347
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[Na(benzyloxycarbonyl)-O(benzyl)-tyrosyl-prolylamino]-
propyl]thymine]-3′-spiro-5′′-[4′′-amino-1′′,2′′-oxathiole-2′′,2′′-
dioxide] (4d). Nucleoside 2 (75 mg, 0.15 mmol) was reacted with
Z-Tyr(Bzl)-Pro-OH 3d (116 mg, 0.30 mmol) according to the
general coupling procedure. The residue was purified by CCTLC
on the Chromatotron (dichloromethane:methanol, 70:1) to give 80
mg (61%) of 4d as a white foam. MS (ESI+): m/z 1131.6 (M +
1)+. Anal. for C56H78N6O13SSi2: C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-
3-N-[3-N′-[Na,Ne bis(benzyloxycarbonyl)-lysyl-prolylamino]pro-
pyl]thymine]-3′-spiro-5′′-[4′′-amino-1′′,2′′-oxathiole-2′′,2′′-diox-
ide] (4e). Nucleoside 2 (109 mg, 0.15 mmol) was reacted with
Z-Lys(Z)-Pro-OH 3e (148 mg, 0.31 mmol) following the general
coupling procedure. The residue was purified by CCTLC on the
Chromatotron (dichloromethane:methanol, 70:1) to give 76 mg
(40%) of 4e as a white foam. MS (ESI+): m/z 1141.3 (M + 1)+.
Anal. for C54H81N7O14SSi2: C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[Na(benzyloxycarbonyl)-glycyl-prolylamino]propyl]th-
ymine]-3′-spiro-5′′-[4′′-amino-1′′,2′′-oxathiole-2′′,2′′-dioxide] (4f).
Following the general coupling procedure, nucleoside 2 (75 mg,
0.15 mmol) was reacted with Z-Gly-Pro-OH 3f (71 mg, 0.18 mmol).
The residue was purified by CCTLC on the Chromatotron (dichlo-
romethane:methanol, 70:1) to give 57 mg (53%) of 4f as a white
foam. MS (ESI+): m/z 935.3 (M + 1)+. Anal. for C42H66N6O12-
SSi2: C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[Na(benzyloxycarbonyl)-asparaginyl-prolylamino]pro-
pyl]thymine]-3′-spiro-5′′-[4′′-amino-1′′,2′′-oxathiole-2′′,2′′-diox-
ide] (4g). Following the general coupling procedure, nucleoside 2
(100 mg, 0.15 mmol) was reacted with Z-Asn-Pro-OH 3g (84 mg,
0.24 mmol). The residue was purified by flash column chroma-
tography (dichloromethane:methanol, 30:1) to give 76 mg (50%)
of 4g as a white foam. MS (ESI+): m/z 1083.4 (M + 1)+. Anal.
for C51H74N6O14SSi2: C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[Na(benzyloxycarbonyl)-O-benzyl-glutamyl-prolylami-
no]propyl]thymine]-3′-spiro-5′′-[4′′-amino-1′′,2′′-oxathiole-2′′,2′′-
dioxide] (4h). According to the general coupling procedure,
nucleoside 2 (100 mg, 0.15 mmol) was reacted with Z-Glu(Bzl)-
Pro-OH 3h (86 mg, 0.18 mmol). The residue was purified by flash
column chromatography (dichloromethane:methanol, 50:1) to give
110 mg (65%) of 4h as a white foam. MS (ESI+): m/z 1096.2 (M
+ 1)+. Anal. for C52H76N6O14SSi2: C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[Na(benzyloxycarbonyl)-O-benzyl-aspartyl-prolylamino]-
propyl]thymine]-3′-spiro-5′′-[4′′-amino-1′′,2′′-oxathiole-2′′,2′′-
dioxide] (4i). Nucleoside 2 (86 mg, 0.13 mmol) was reacted with
Z-Asp(Bzl)-Pro-OH 3i (73 mg, 0.18 mmol) following the general
coupling procedure. The residue was purified by by flash column
chromatography (dichloromethane:methanol, 70:1) to give 89 mg
(61%) of 4i as a white foam. MS (ESI+): m/z 1083.4 (M + 1)+.
Anal. for C51H74N6O14SSi2: C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[Na(benzyloxycarbonyl)-valyl-alanylamino]propyl]thy-
mine]-3′-spiro-5′′-[4′′-amino-1′′,2′′-oxathiole-2′′,2′′-dioxide] (4j).
Via the general coupling procedure, Z-Val-Ala-OH 3j (132 mg,
0.60 mmol) was treated with NAP-TSAO-T 2 (132 mg, 0.30 mmol).
The residue was purified by CCTLC on the Chromatotron (dichloro-
methane:methanol, 70:1) to give 105 mg (54%) of 4j as a white
foam. MS (ESI+): m/z 951.3 (M + 1)+. Anal. for C43H70N6O12-
SSi2: C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[Na(benzyloxycarbonyl)-valyl-glycylamino]propyl]thy-
mine]-3′-spiro-5′′-[4′′-amino-1′′,2′′-oxathiole-2′′,2′′-dioxide] (4k).
Following the general procedure, Z-Val-Gly-OH 3k (80 mg, 0.26
mmol) was reacted with NAP-TSAO-T 2 (85 mg, 0.13 mmol). The
final residue was purified by CCTLC on the Chromatotron
(dichloromethane:methanol, 70:1) to give 88 mg of 4k (73%) as a
white foam. MS (ESI+): m/z 937.3 (M + 1)+. Anal. for C42H68N6O12-
SSi2: C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[Na(benzyloxycarbonyl)-valyl-leucylamino]propyl]thy-
mine]-3′-spiro-5′′-[4′′-amino-1′′,2′′-oxathiole-2′′,2′′-dioxide] (4l).
Via the general coupling procedure, Z-Val-Leu-OH 3l (102 mg,
0.28 mmol) was treated with NAP-TSAO-T 2 (90 mg, 0.14 mmol).
The residue was purified by flash column chromatography (dichlo-
romethane:methanol, 70:1) to give 61 mg (39%) of 4l as a white
foam. MS (ESI+): m/z 993.5 (M + 1)+. Anal. for C46H76N6O12-
SSi2: C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[Na(benzyloxycarbonyl)-valyl-phenylalanylamino]pro-
pyl]thymine]-3′-spiro-5′′-[4′′-amino-1′′,2′′-oxathiole-2′′,2′′-diox-
ide] (4m). According to the general procedure, Z-Val-Phe-OH 3m
(116 mg, 0.29 mmol) was reacted with NAP-TSAO-T 2 (94 mg,
0.14 mmol). The residue was purified by flash column chroma-
tography (dichloromethane:methanol, 70:1) to give 68 mg (37%)
of 4m as a white foam. MS (ESI+): m/z 1027.3 (M + 1)+. Anal.
for C49H74N6O12SSi2: C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[Na(benzyloxycarbonyl)-valyl-D-prolylamino]propyl]thy-
mine]-3′-spiro-5′′-[4′′-amino-1′′,2′′-oxathiole-2′′,2′′-dioxide] (4n).
Via the general coupling procedure, Z-Val-D-Pro-OH 3n (64 mg,
0.18 mmol) was treated with NAP-TSAO-T 2 (99 mg, 0.15 mmol).
The residue was purified by CCTLC on the Chromatotron (dichloro-
methane:methanol, 70:1) to give 72 mg (48%) of 4n as a white
foam. MS (ESI+): m/z 977.3 (M + 1)+. Anal. for C45H72N6O12-
SSi2: C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[Na(benzyloxycarbonyl)-valyl-(2S,4R)-4-benzyloxypro-
lylamino]propyl]thymine]-3′-spiro-5′′-[4′′-amino-1′′,2′′-oxathiole-
2′′,2′′-dioxide] (4o). Following the general procedure, Z-Val-
Hyp(Bzl)-OH 3o (105 mg, 0.23 mmol) was reacted with NAP-
TSAO-T 2 (75 mg, 0.11 mmol). The final residue was purified by
flash column chromatography (dichloromethane:methanol, 70:1) to
give 112 mg of 4o (89%) as a white foam. MS (ESI+): m/z 1083.6
(M + 1)+.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[Na(benzyloxycarbonyl)-valyl-(2S,4R)-4-O-hydroxypro-
lylaminoprolylamino]propyl]thymine]-3′-spiro-5′′-[4′′-amino-
1′′,2′′-oxathiole-2′′,2′′-dioxide] (4p). Via the general coupling
procedure, Z-Val-Hyp-OH 3p (59 mg, 0.16 mmol) was treated with
NAP-TSAO-T 2 (70 mg, 0.11 mmol). The residue was purified by
flash column chromatography (dichloromethane:methanol, 50:1) to
give 43 mg (40%) of 4p as a white foam.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[(2S,3R,4S)-Na(benzyloxycarbonyl)-3,4-dihydroxypro-
lylamino]propyl]thymine]-3′-spiro-5′′-[4′′-amino-1′′,2′′-oxathiole-
2′′,2′′-dioxide] (28). According to the general procedure, Z-dipeptide
derivative 27 (63 mg, 0.16 mmol) was reacted with NAP-TSAO-T
2 (72 mg, 0.11 mmol). The residue was purified by flash column
chromatography (dichloromethane:methanol, 40:1) to give 44 mg
(40%) of compound 28 as a white foam. MS (ESI+): m/z 1009.5
(M + 1)+. Anal. for C45H72N6O14SSi2: C, H, N, S.
General N-Deprotection Procedure for the Synthesis of
Compounds 5-20, 29, 31, and 33. A solution of the corresponding
protected conjugate intermediate [Z-Xaa-Yaa]-[TSAO-T] (1 equiv)
in methaanol (8 mL) containing Pd/C (10%) (40wt %/wt) was
hydrogenated 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
lyophilized to give the final deprotected conjugates 5-20, 29, 31,
and 33. The yield of the isolated products and spectroscopic and
analytical data are indicated below for each compound.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[valyl-prolylamino]propyl]thymine]-3′-spiro-5′′-[4′′-
amino-1′′,2′′-oxathiole-2′′,2′′-dioxide] (5). According to the general
N-deprotection procedure, conjugate 4a (49 mg, 0.05 mmol) was

5348 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17
Garc´ıa-Aparicio et al.
1
hydrogenated to give 5 (40 mg, 83%) as a white foam. H NMR
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[glycyl-prolylamino]propyl]thymine]-3′-spiro-′′-[4′′-amino-
1′′,2′′-oxathiole-2′′,2′′-dioxide] (10). Via the general N-deprotection
procedure, conjugate 4f (47 mg, 0.05 mmol) was hydrogenated to
give 10 (47 mg, 97%) as a white foam. 1H NMR (300 MHz,
DMSO-d6): δ 1.59 (m, 2H, -CH2-), 1.88 (m, 8H, CH3-5, â-CH,
Val, â-CH2, γ-CH2, Pro), 3.04 (m, 2H, CH2NHCO), 3.49 (m, 2H,
δ-CH2, Pro), 3.81 (m, 4H, R-CH, Gly, N3-CH2, H-5′a), 3.92 (dd,
1H, H-5′b, J ) 7.5 Hz, J ) 12.4 Hz), 4.22 (m, 1H, H-4′), 4.34 (m,
1H, R-CH, Pro), 4.53 (d, 1H, H-2′, J ) 8.3 Hz), 5.74 (s, 1H, H-3′′),
5.97 (d, 1H, H-1′, J ) 8.3 Hz), 6.96 (bs, 2H, NH2-4′′), 7.62 (s, 1H,
H-6), 7.86 (m, 1H, NHCO). MS (ESI+): m/z 801.3 (M + 1)+. Anal.
for C34H60N6O10SSi2: C, H, N, S.
(300 MHz, DMSO-d6): δ 0.92 (d, 3H, γ-CH3a, Val, J ) 6.7 Hz),
0.96 (d, 3H, γ-CH3b, Val, J ) 6.7 Hz), 1.82 (m, 10H, -CH2-, CH3-
5, â-CH, Val, â-CH2,γ-CH2, Pro), 2.95 (m, 1H, CH2aNHCO), 3.15
(m, 1H, CH2bNHCO), 3.45 (m, 2H, δ-CH2, Pro), 3.82 (m, 4H,
R-CH, Val, N3-CH2, H-5′a), 3.91 (dd, 1H, H-5′b, J ) 7.5 Hz, J )
12.4 Hz), 4.21 (m, 2H, H-4′, R-CH, Pro), 4.75 (d, 1H, H-2′, J )
8.4 Hz), 5.74 (s, 1H, H-3′′), 5.96 (d, 1H, H-1′, J ) 8.4 Hz), 6.97
(bs, 2H, NH2-4′′), 7.62 (s, 1H, H-6), 7.78 (m, 1H, NHCO). MS
(ESI+): m/z 843.2 (M + 1)+. Anal. for C37H66N6O10SSi2: C, H,
N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[alanyl-prolylamino]propyl]thymine]-3′-spiro-5′′-[4′′-
amino-1′′,2′′-oxathiole-2′′,2′′-dioxide] (6). Following the general
N-deprotection procedure, conjugate 4b (76 mg, 0.08 mmol) was
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[asparaginyl-prolylamino]propyl]thymine]-3′-spiro-5′′-
[4′′-amino-1′′,2′′-oxathiole-2′′,2′′-dioxide] (11). According to the
general N-deprotection procedure, conjugate 4g (54 mg, 0.05 mmol)
1
hydrogenated to give 6 (59 mg, 87%) as a white foam. H NMR
(300 MHz, DMSO-d6): δ 1.21 (d, 3H, â-CH2, Ala, J ) 7.0 Hz),
1.58-2.16 (m, 9H, -CH2-, CH3-5, â-CH2, γ-CH2, Pro), 3.10 (m,
2H, CH2NHCO), 3.57 (m, 2H, δ-CH2, Pro), 3.80 (m, 4H, 2H-5′,
N3-CH2), 3.92 (m, 1H, R-CH, Ala), 4.22 (m, 1H, H-4′), 4.32 (m,
1H, R-CH, Pro), 4.54 (d, 1H, H-2′, J ) 8.3 Hz), 5.73 (s, 1H, H-3′′),
5.96 (d, 1H, H-1′, J ) 8.3 Hz), 7.03 (bs, 2H, NH2-4′′), 7.76 (s, 1H,
H-6), 7.91 (m, 1H, NHCO). MS (ESI+): m/z 815.3 (M + 1)+. Anal.
for C35H62N6O10SSi2: C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[phenyalanyl-prolylamino]propyl]thymine]-3′-spiro-5′′-
[4′′-amino-1′′,2′′-oxathiole-2′′,2′′-dioxide] (7). Conjugate 4c (82
mg, 0.08 mmol) was hydrogenated following the general N-
deprotection procedure to give 7 (55 mg, 80%) as a white foam.
1H NMR (300 MHz, DMSO-d6): δ 1.62 (m, 2H, -CH2-), 1.80-
2.08 (m, 8H, CH3-5, â-CH, Val, â-CH2, γ-CH2, Pro), 2.75 (m, 1H,
â-CH2a, Phe), 3.06 (m, 3H, CH2NHCO, â-CH2b, Phe), 3.63 (m,
2H, δ-CH2, Pro), 3.77 (m, 3H, N3-CH2, H-5′a), 3.92 (dd, 1H, H-5′b,
J ) 7.5 Hz, J ) 12.4 Hz), 4.21 (m, 1H, H-4′), 4.29 (m, 1H, R-CH,
Phe), 4.43 (m, 1H, R-CH, Pro), 4.56 (d, 1H, H-2′, J ) 8.3 Hz),
5.75 (s, 1H, H-3′′), 5.97 (d, 1H, H-1′, J ) 8.3 Hz), 6.97 (bs, 2H,
NH2-4′′), 7.26 (m, 5H, Ar, Phe), 7.65 (m, 2H, H-6, NHCO). MS
(ESI+): m/z 891.2 (M + 1)+. Anal. for C41H66N6O10SSi2: C, H,
N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[tyrosyl-prolylamino]propyl]thymine]-3′-spiro-5′′-[4′′-
amino-1′′,2′′-oxathiole-2′′,2′′-dioxide] (8). Conjugate 4d (57 mg,
0.05 mmol) was hydrogenated following the general N-deprotection
procedure to give 8 (34 mg, 66%) as a white foam. 1H NMR (300
MHz, DMSO-d6): δ 1.61 (m, 2H, -CH2-), 1.80-1.92 (m, 8H, CH3-
5, â-CH, Val, â-CH2, γ-CH2, Pro), 2.03 (m, 1H, â-CH2, Pro), 2.65
(m, 1H, â-CH2a, Tyr), 2.94 (dd, 1H, â-CH2b, Tyr, J ) 5.0, J )
13.7 Hz), 3.00 (m, 1H, CH2aNHCO), 3.11 (m, 1H, CH2bNHCO),
3.62 (m, 2H, δ-CH2, Pro), 3.77 (m, 4H, R-CH, Tyr, N3-CH2, H-5′a),
3.93 (dd, 1H, H-5′b, J ) 7.5 Hz, J ) 11.7 Hz), 4.25 (m, 2H, R-CH,
Pro, H-4′), 4.56 (d, 1H, H-2′, J ) 8.3 Hz), 5.74 (s, 1H, H-3′′), 5.97
(d, 1H, H-1′, J ) 8.3 Hz), 6.65 (m, 2H, Ar, Tyr), 6.98 (m, 4H,
NH2-4′′, Ar, Tyr), 7.65 (s, 1H, H-6), 8.08 (m, 1H, NHCO), 9.22
(s, 1H, OH-4, Tyr). MS (ESI+): m/z 907.3 (M + 1)+. Anal. for
C41H66N6O13SSi2: C, H, N, S.
1
was hydrogenated to give 11 (35 mg, 81%) as a white foam. H
NMR (300 MHz, DMSO-d6): δ 1.52-1.98 (m, 9H, CH3-5, -CH2-,
â-CH2, γ-CH2, Pro), 2.24 (m, 1H, â-CH2a, Asn), 2.50 (m, 1H,
â-CH2b, Asn), 3.09 (m, 2H, CH2NHCO), 3.57 (m, 2H, δ-CH2, Pro),
3.83 (m, 4H, 2H-5′, N3-CH2), 4.22 (m, 2H, R-CH, Asn, H-4′), 4.57
(m, 2H, R-CH, Pro, H-2′), 5.74 (s, 1H, H-3′′), 5.97 (d, 1H, H-1′, J
) 7.7 Hz), 6.97 (bs, 3H, CONH2a, Asn, NH2-4′′), 7.65 (m, 2H,
H-6, CONH2b, Asn), 7.78 (m, 1H, NHCO). MS (ESI+): m/z 858.3
(M + 1)+. Anal. for C36H63N7O11SSi2: C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[glutamyl-prolylamino]propyl]thymine]-3′-spiro-5′′-[4′′-
amino-1′′,2′′-oxathiole-2′′,2′′-dioxide] (12). According to the
general N-deprotection procedure, conjugate 4h (55 mg, 0.05 mmol)
1
was hydrogenated to give 12 (28 mg, 72%) as a white foam. H
NMR (300 MHz, DMSO-d6): δ 1.52-2.04 (m, 13H, CH3-5, -CH2-,
â-CH2, γ-CH2, Pro, Glu), 2.93 (m, 1H, CH2aNHCO), 3.07 (m, 1H,
CH2bNHCO), 3.52 (m, 2H, δ-CH2, Pro), 3.79 (m, 3H, N3-CH2,
H-5′a), 3.91 (dd, 1H, H-5′b, J ) 7.5 Hz, J ) 12.4 Hz), 4.21 (m,
2H, R-CH, Glu, H-4′), 4.27 (m, 1H, R-CH, Pro), 4.52 (d, 1H, H-2’,
J ) 8.3 Hz), 5.73 (s, 1H, H-3′′), 5.96 (d, 1H, H-1′, J ) 8.3 Hz),
6.94 (bs, 2H, NH2-4′′), 7.60 (s, 1H, H-6), 7.82 (m, 1H, NHCO).
MS (ESI+): m/z 873.2 (M + 1)+. Anal. for C37H64N6O12SSi2: C,
H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[aspartyl-prolylamino]propyl]thymine]-3′-spiro-5′′-[4′′-
amino-1′′,2′′-oxathiole-2′′,2′′-dioxide] (13). Conjugate 4i (54 mg,
0.05 mmol) was hydrogenated following the general N-deprotection
procedure to give 13 (19 mg, 60%) as a white foam. 1H NMR (300
MHz, DMSO-d6): δ 1.57 (m, 2H, -CH2-), 1.62-1.75 (m, 7H, CH3-
5, â-CH2, γ-CH2, Pro), 2.13 (dd, 1H, â-CH2a, Asp, J ) 9.6 Hz, J
) 15.8 Hz), 2.50 (m, 1H, â-CH2b, Asp), 3.02 (m, 2H, CH2NHCO),
3.62 (m, 2H, δ-CH2, Pro), 3.81 (m, 3H, H-5′a, N3-CH2), 3.94 (m,
2H, R-CH, Asp, H-5′b), 4.24 (m, 2H, R-CH, Pro, H-4′), 4.56 (d,
1H, H-2′, J ) 7.8 Hz), 5.57 (s, 1H, H-3′′), 5.96 (d, 1H, H-1′, J )
7.8 Hz), 6.98 (bs, 2H, NH2-4′′), 7.65 (s, 1H, H-6), 7.88 (m, 1H,
NHCO). MS (ESI+): m/z 859.0 (M + 1)+. Anal. for C36H62N6O12-
SSi2: C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[valyl-alanylamino]propyl]thymine]-3′-spiro-5′′-[4′′-
amino-1′′,2′′-oxathiole-2′′,2′′-dioxide] (14). Conjugate 4j (67 mg,
0.07 mmol) was hydrogenated following the general N-deprotection
procedure to give 14 (45 mg, 75%) as a white foam. 1H NMR (300
MHz, DMSO-d6): δ 0.91 (d, 3H, γ-CH3a, Val, J ) 6.7 Hz), 0.95
(d, 3H, γ-CH3b, Val, J ) 6.7 Hz), 1.19 (d, 3H, â-CH2, Ala, J )
7.1 Hz), 1.92 (m, 4H, CH3-5, â-CH, Val), 3.03 (m, 1H, CH2-
aNHCO), 3.13 (m, 1H, CH2bNHCO), 3.82 (m, 4H, R-CH, Val,
H-5′a, N3-CH2), 3.92 (dd, 1H, H-5′b, J ) 7.1 Hz, J ) 11.7 Hz),
4.22 (t, 1H, H-4′, J ) 2.7 Hz), 4.26 (m, 1H, R-CH, Ala), 4.55 (d,
1H, H-2′, J ) 7.8 Hz), 5.75 (s, 1H, H-3′′), 5.96 (d, 1H, H-1′, J )
7.8 Hz), 6.98 (bs, 2H, NH2-4′′), 7.65 (s, 1H, H-6), 7.98 (m, 1H,
NHCO), 8.07 (m, 1H, NH, Ala). MS (ESI+): m/z 817.3 (M + 1)+.
Anal. for C35H64N6O10SSi2: C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[lysyl-prolylamino]propyl]thymine]-3′-spiro-5′′-[4′′-amino-
1′′,2′′-oxathiole-2′′,2′′-dioxide] (9). Following the general N-depro-
tection procedure, conjugate 4e (57 mg, 0.05 mmol) was
1
hydrogenated to give 9 (42 mg, 93%) as a white foam. H NMR
(300 MHz, DMSO-d6): δ 1.33 (m, 6H, â-CH2, γ-CH2, δ-CH2, Lys),
1.61 (m, 3H, â-CH2a, Pro, -CH2-), 1.82 (m, 2H, γ-CH2, Pro), 1.88
(s, 3H, CH3-5), 2.00 (m, 1H, â-CH2b, Pro), 2.62 (m, 2H, ꢀ-CH2,
Lys), 2.92 (m, 1H, CH2aNHCO), 3.11 (m, 1H, CH2bNHCO), 3.43
(m, 1H, δ-CH2a, Pro), 3.57 (m, 1H, δ-CH2b, Pro), 3.86 (m, 4H,
R-CH, Lys, H-5′a, N3-CH2), 3.93 (dd, 1H, H-5′b, J ) 7.5 Hz, J )
12.4 Hz), 4.25 (m, 2H, R-CH, Pro, H-4′), 4.58 (d, 1H, H-2′, J )
8.2 Hz), 5.72 (s, 1H, H-3′′), 5.96 (d, 1H, H-1′, J ) 8.2 Hz), 6.98
(bs, 2H, NH2-4′′), 7.67 (s, 1H, H-6), 7.78 (m, 1H, NHCO). MS
(ESI+): m/z 872.3 (M + 1)+. Anal. for C38H69N7O10SSi2: C, H,
N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[valyl-glycylamino]propyl]thymine]-3′-spiro-5′′-[4′′-amino-

Dipeptidyl-peptidase IV-Based Prodrug Approach
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17 5349
1′′,2′′-oxathiole-2′′,2′′-dioxide] (15). Conjugate 4k (47 mg, 0.05
7.30 (m, 5H, Ar, Z), 7.63 (s, 1H, H-6), 7.94 (m, 1H, NHCO). MS
(ESI+): m/z 949.5 (M + 1)+. Anal. for C44H72N6O11SSi2: C, H,
N, S.
mmol) was hydrogenated via the general N-deprotection procedure
1
to give 15 (53 mg, 90%) as a white foam. H NMR (300 MHz,
DMSO-d6): δ 0.81 (d, 3H, γ-CH3a, Val, J ) 6.7 Hz), 0.85 (d, 3H,
γ-CH3b, Val, J ) 6.7 Hz), 1.24 (m, 2H, -CH2-), 1.89 (s, 4H, CH3-
5, â-CH, Val), 3.07 (m, 2H, CH2NHCO), 3.68 (m, 2H, R-CH2,
Gly), 3.82 (m, 4H, R-CH, Val, 2H-5′a, N3-CH2), 3.92 (dd, 1H, 2H-
5′b, J ) 7.1 Hz, J ) 11.7 Hz), 4.21 (t, 1H, H-4′, J ) 2.7 Hz), 4.55
(d, 1H, H-2′, J ) 7.8 Hz), 5.75 (s, 1H, H-3′′), 5.94 (d, 1H, H-1′,
J ) 7.8 Hz), 6.96 (bs, 2H, NH2-4′′), 7.64 (s, 1H, H-6), 7.82 (m,
1H, NHCO), 8.12 (m, 1H, NH, Gly). MS (ESI+): m/z 803.3 (M +
1)+. Anal. for C34H62N6O10SSi2: C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[valyl-leucylamino]propyl]thymine]-3′-spiro-5′′-[4′′-amino-
1′′,2′′-oxathiole-2′′,2′′-dioxide] (16). Following the general N-
deprotection procedure, conjugate 4l (50 mg, 0.05 mmol) was
hydrogenated to give 16 (44 mg, 95%) as a white foam. 1H NMR
(300 MHz, DMSO-d6): δ 0.88 (m, 12H, γ-CH3, Val, δ-CH3, Leu),
1.59 (m, 6H, -CH2-, â-CH2, γ-CH2, Leu), 1.97 (m, 4H, â-CH3,
Val, CH3-5), 3.09 (m, 1H, CH2aNHCO), 3.11 (m, 1H, CH2bNHCO),
3.82 (m, 4H, R-CH, Val, 2H-5′a, N3-CH2), 3.92 (dd, 1H, 2H-5′b,
J ) 7.1 Hz, J ) 11.7 Hz), 4.21 (t, 1H, H-4′, J ) 2.7 Hz), 4.35 (m,
1H, R-CH, Leu), 4.55 (d, 1H, H-2′, J ) 8.5 Hz), 5.74 (s, 1H, H-3′′),
5.95 (d, 1H, H-1′, J ) 8.5 Hz), 6.96 (bs, 2H, NH2-4′′), 7.64 (s, 1H,
H-6), 8.02 (m, 2H, NHCO, NH, Leu). MS (ESI+): m/z 859.0 (M
+ 1)+. Anal. for C38H70N6O10SSi2: C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[valyl-phenylalanylamino]propyl]thymine]-3′-spiro-5′′-
[4′′-amino-1′′,2′′-oxathiole-2′′,2′′-dioxide] (17). Conjugate 4m (51
mg, 0.05 mmol) was hydrogenated following the general N-
deprotection procedure to give 17 (44 mg, 90%) as a white foam.
1H NMR (300 MHz, DMSO-d6): δ 0.66 (d, 3H, γ-CH3a, Val, J )
6.7 Hz), 0.68 (d, 3H, γ-CH3b, Val, J ) 6.7 Hz), 1.60 (m, 2H, -CH2-
), 1.89 (s, 4H, CH3-5, â-CH, Val), 2.82 (m, 1H, CH2aNHCO), 3.00
(m, 3H, CH2bNHCO, â-CH2, Phe), 3.82 (m, 4H, R-CH, Val, 2H-
5′a, N3-CH2), 3.92 (dd, 1H, 2H-5′b, J ) 7.1 Hz, J ) 11.7 Hz),
4.21 (t, 1H, H-4′, J ) 2.7 Hz), 4.55 (m, 2H, H-2′, R-CH, Phe),
5.74 (s, 1H, H-3′′), 5.96 (d, 1H, H-1′, J ) 8.3 Hz), 6.97 (bs, 2H,
NH2-4′′), 7.18 (m, 5H, Ar, Phe), 7.66 (s, 1H, H-6), 8.05 (m, 1H,
NHCO), 8.12 (m, 1H, NH, Phe). MS (ESI+): m/z 893.3 (M + 1)+.
Anal. for C41H68N6O10SSi2: C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[valyl-D-prolylamino]propyl]thymine]-3′-spiro-5′′-[4′′-
amino-1′′,2′′-oxathiole-2′′,2′′-dioxide] (18). Conjugate 4n (68 mg,
0.07 mmol) was hydrogenated according to the general N-
deprotection procedure to give 18 (57 mg, 98%) as a white foam.
1H NMR (300 MHz, DMSO-d6): δ 0.90 (d, 3H, γ-CH3a, Val, J )
6.7 Hz), 0.94 (d, 3H, γ-CH3b, Val, J ) 6.7 Hz), 1.62 (m, 2H, -CH2-
), 1.97 (m, 8H, CH3-5, â-CH, Val, â-CH2,γ-CH2, Pro), 3.04 (m,
2H, CH2NHCO), 3.45 (m, 2H, δ-CH2, Pro), 3.78 (m, 4H, R-CH,
Val, H-5′a, N3-CH2), 3.95 (dd, 1H, H-5′b, J ) 7.1 Hz, J ) 11.7
Hz), 4.20 (t, 1H, H-4′, J ) 2.7 Hz), 4.38 (m, 1H, R-CH, Pro), 4.61
(d, 1H, H-2′, J ) 8.4 Hz), 5.72 (s, 1H, H-3′′), 5.97 (d, 1H, H-1′,
J ) 8.4 Hz), 6.99 (bs, 2H, NH2-4′′), 7.71 (s, 1H, H-6), 7.81 (m,
1H, NHCO). MS (ESI+): m/z 843.3 (M + 1)+. Anal. for
C37H66N6O10SSi2: C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[valyl-(2S,4R)-4-hydroxyprolylamino]propyl]thymine]-
3′-spiro-5′′-[4′′-amino-1′′,2′′-oxathiole-2′′,2′′-dioxide] (20). Fol-
lowing the general N-deprotection procedure, conjugate 4p (50 mg,
0.05 mmol) was hydrogenated to give 20 (26 mg, 58%) as a white
1
foam. H NMR (300 MHz, DMSO-d6): δ 0.86 (m, 6H, γ-CH3,
Val), 1.61 (m, 2H, -CH2-), 1.79-2.14 (m, 6H, CH3-5, â-CH2, Hyp,
â-CH, Val), 2.93 (m, 1H, CH2aNHCO), 3.12 (m, 1H, CH2bNHCO),
3.60 (m, 2H, δ-CH2, Hyp), 3.83 (m, 4H, R-CH, Val, N3-CH2,
H-5′a), 3.91 (dd, 1H, H-5′b, J ) 7.1 Hz, J ) 11.7 Hz), 4.21 (m,
1H, H-4′), 4.26 (m, 2H, R-CH, g-CH, Hyp), 4.56 (d, 1H, H-2′, J )
8.3 Hz), 5.02 (m, 1H, γ-OH), 5.74 (s, 1H, H-3′′), 5.96 (d, 1H, H-1′,
J ) 8.3 Hz), 6.96 (bs, 2H, NH2-4′′), 7.64 (s, 1H, H-6), 7.86 (m,
1H, NHCO). MS (ESI+): m/z 859.5 (M + 1)+. Anal. for
C37H66N6O11SSi2: C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[(2S,3R,4S)-3,4-dihydroxyprolylamino]propyl]thymine]-
3′-spiro-5′′-[4′′-amino-1′′,2′′-oxathiole-2′′,2′′-dioxide] (29). Con-
jugate 28 (30 mg, 0.04 mmol) was hydrogenated according to the
general N-deprotection procedure to give 29 (20 mg, 87%) as a
white foam. 1H NMR (300 MHz, DMSO-d6): δ 0.86 (m, 6H, 2γ-
CH3, Val), 1.64 (m, 2H, -CH2-), 1.90 (m, 4H, CH3-5, â-CH, Val),
3.09 (m, 2H, CH2NHCO), 3.61 (m, 1H, δ-CH2a, DHP), 3.90 (m,
6H, 2H-5′, N3-CH2, δ-CH2b, γ-CH, DHP), 4.05 (m, 3H, R-CH,
Val, DHP, â-CH, DHP), 4.21 (m, 1H, H-4′), 4.60 (d, 1H, H-2′, J
) 8.4 Hz), 5.10 (m, 2H, â-OH, γ-OH), 5.73 (s, 1H, H-3′′), 5.97
(d, 1H, H-1′, J ) 8.4 Hz), 7.00 (bs, 2H, NH2-4′′), 7.70 (s, 1H,
H-6), 7.86 (m, 1H, NHCO). MS (ESI+): m/z 875.5 (M + 1)+. Anal.
for C37H66N6O12SSi2: C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[Na(benzyloxycarbonyl)-valylamino]propyl]thymine]-3′-
spiro-5′′-[4′′-amino-1′′,2′′-oxathiole-2′′,2′′-dioxide] (30). Accord-
ing to the general coupling procedure, Z-Val-OH (80 mg, 0.32
mmol) was reacted with NAP-TSAO-T 2 (138 mg, 0.21 mmol).
The residue was purified by flash column chromatography (dichlo-
romethane:methanol, 70:1) to give 30 (134 mg, 72%) as a white
1
foam. H RMN (300 MHz, CDCl3): δ 0.92 (d, 3H, γ-CH3a, Val,
J ) 6.7 Hz), 0.96 (d, 3H, γ-CH3b, Val, J ) 6.7 Hz), 1.57 (m, 2H,
-CH2-), 1.95 (s, 3H, CH3-5), 2.13 (m, 1H, â-CH, Val), 2.99 (m,
1H, CH2aNHCO), 3.33 (m, 1H, CH2bNHCO), 3.92 (m, 5H, 2H-5′,
N3-CH2, R-CH, Val), 4.30 (t, 1H, H-4′, J ) 2.7 Hz), 4.49 (d, 1H,
H-2′, J ) 7.8 Hz), 5.09 (dd, 2H, CH2, Z, J ) 12.2 Hz, J ) 14.9
Hz), 5.38 (d, 1H, NH, Val, J ) 9.0 Hz), 5.55 (s, 1H, H-3′′), 5.61
(bs, 2H, NH2-4′′), 5.88 (d, 1H, H-1′, J ) 7.8 Hz), 7.18 (s, 1H,
H-6), 7.32 (m, 5H, Ar, Z). MS (ESI+): m/z: 880.3 (M + 1)+. Anal.
for C40H65N5O11SSi2: C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[valylamino]propyl]thymine]-3′-spiro-5′′-[4′′-amino-
1′′,2′′-oxathiole-2′′,2′′-dioxide] (31). According to the general
N-deprotection procedure, compound 30 (123 mg, 0.14 mmol) was
hydrogenated to yield 31 (100 mg, 96%) as a white foam. 1H NMR
(300 MHz, DMSO-d6): δ 0.76 (d, 3H, γ-CH3a, Val, J ) 6.7 Hz),
0.80 (d, 3H, γ-CH3b, Val, J ) 6.7 Hz), 1.62 (m, 2H, -CH2-), 1.86
(m, 4H, CH3-5, â-CH, Val), 3.15 (m, 2H, CH2NHCO), 3.82 (m,
4H, R-CH, Val, N3-CH2, 2H-5′a), 3.91 (dd, 1H, 2H-5′b, J ) 7.5
Hz, J ) 12.4 Hz), 4.22 (t, 1H, H-4′, J ) 2.7 Hz), 4.53 (d, 1H,
H-2′, J ) 7.8 Hz), 5.74 (s, 1H, H-3′′), 5.96 (d, 1H, H-1′, J ) 8.3
Hz), 6.98 (bs, 2H, NH2-4′′), 7.62 (s, 1H, H-6), 7.92 (m, 1H, NHCO).
MS (ESI+): m/z 746.3 (M + 1)+. Anal. for: C32H59N5O9SSi2: C,
H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[valyl-(2S,4R)-4-O-benzyloxyprolylamino]propyl]thy-
mine]-3′-spiro-5′′-[4′′-amino-1′′,2′′-oxathiole-2′′,2′′-dioxide] (19).
According to the general N-deprotection procedure, conjugate 4o
(54 mg, 0.05 mmol) was hydrogenated to give 19 (28 mg, 60%) as
1
a white foam. H NMR (300 MHz, DMSO-d6): δ 0.87 (m, 6H,
γ-CH3, Val), 1.57 (m, 1H, -CH2a-), 1.65 (m, 1H, -CH2b-), 1.93
(m, 5H, â-CH2a, Hyp, CH3-5, â-CH, Val), 2.21 (m, 1H, â-CH2b,
Hyp), 2.97 (m, 1H, CH2aNHCO), 3.12 (m, 1H, CH2bNHCO), 3.61-
(m, 2H, δ-CH2, Hyp), 3.82 (m, 4H, R-CH, Val, N3-CH2, H-5′a),
3.92 (dd, 1H, H-5′b, J ) 7.3 Hz, J ) 11.7 Hz), 4.22 (m, 2H, g-CH,
Hyp, H-4′), 4.31 (m, 1H, R-CH, Hyp), 4.49 (dd, 2H, CH2, Bzl, J
) 11.7 Hz, J ) 17.1 Hz), 4.54 (d, 1H, H-2′, J ) 8.3 Hz), 5.75 (s,
1H, H-3′′), 5.97 (d, 1H, H-1′, J ) 8.3 Hz), 6.95 (bs, 2H, NH2-4′′),
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[Na(benzyloxycarbonyl)-valyl-prolyl-valylamino]propyl]-
thymine]-3′-spiro-5′′-[4′′-amino-1′′,2′′-oxathiole-2′′,2′′-dioxide]
(32). Via the general coupling procedure, Z-Val-Pro-OH (59 mg,
0.17 mmol) was treated with compound 31 (84 mg, 0.11 mmol).
The residue was purified by flash column chromatography (dichlo-
romethane:methanol, 70:1) to give 85 mg (70%) of tripeptide
1
derivative 32 as a white foam. H NMR (300 MHz, CDCl3): δ

5350 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17
Garc´ıa-Aparicio et al.
0.92 (d, 6H, γ-CH3a, Val, J ) 6.7 Hz), 0.96 (d, 6H, γ-CH3b, Val,
J ) 6.7 Hz), 1.97 (m, 9H, CH3-5, -CH2-, â-CH2, γ-CH2, Val, Pro),
2.23 (m, 1H, â-CH, Pro), 3.00 (m, 1H, CH2aNHCO), 3.29 (m, 1H,
CH2bNHCO), 3.59 (m, 1H, δ-CH2, Pro), 3.72 (m, 1H, δ-CH2, Pro),
3.83 (dd, 1H, H-5’a, J ) 7.5 Hz, J ) 12.4 Hz), 3.95 (m, 3H, H-5′b,
N3-CH2), 4.19 (dd, 1H, R-CH, Val, J ) 5.6 Hz, J ) 8.3 Hz), 4.28
(m, 1H, H-4′), 4.34 (dd, 1H, R-CH, Val, J ) 6.2 Hz, J ) 8.9 Hz),
4.48 (d, 1H, H-2′, J ) 7.8 Hz), 4.66 (m, 1H, R-CH, Pro), 5.06 (dd,
2H, CH2, Z, J ) 12.4 Hz, J ) 15.6 Hz), 5.50 (d, 1H, NH, Val, J
) 8.8 Hz), 5.55 (s, 1H, H-3′′), 5.66 (bs, 2H, NH2-4′′), 5.85 (d, 1H,
H-1′, J ) 7.8 Hz), 6.91 (m, 1H, NHCO), 7.17 (s, 1H, H-6), 7.32
(m, 5H, Ar, Z). MS (ESI+): m/z: 1076.4 (M + 1)+. Anal. for
C50H81N7O13SSi2: C, H, N, S.
of purified CD26 (1.5 mU) or different concentrations of HS (in
PBS) or BS (in PBS) at 37 °C. At different time points (as indicated
in the tables and figures), 100 µL was withdrawn from the reaction
mixture, added to 200 µL methanol, and put on ice for ∼10 min.
Then, the mixture was centrifuged at 13.000 rpm for 5 min at 4
°C, and 250 µL of supernatant was analyzed by HPLC on a reverse
phase RP-8 column, using following gradients:
Gradient A (Buffer A: 50 mM NaH2PO4 + 5 mM heptane-
sulfonic acid pH3.2; buffer B: acetonitrile): 2 min 98% A + 2%
B; 6 min linear gradient to 80% A + 20% B; 2 min linear gradient
to 75% A + 25% B; 2 min linear gradient to 65% A + 35% B; 18
min linear gradient to 50% A + 50% B; 5 min 50% A + 50% B;
5 min linear gradient to 98% A + 2% B; 5 min equilibration at
98% A + 2% B.
Gradient B: 2 min 98% A + 2% B; 6 min linear gradient to
80% A + 20% B; 2 min linear gradient to 75% A + 25% B; 2 min
linear gradient to 65% A + 35% B; 8 min linear gradient to 50%
A + 50% B; 10 min 50% A + 50% B; 10 min linear gradient to
20% A + 80% B; 5 min 20% A + 80% B; 15 min linear gradient
to 98% A + 2% B; 5 min 98% A + 2% B.
Evaluation of the Inhibitory Effect of IlePyr and DiprotinA
on Conversion of Compound 5 to 2 by CD26, Human, and
Bovine Serum. The inhibition assays were performed in eppendorf
tubes. The reaction mixture (RMix) contains 50 µM ValPro-NAP-
TSAO-T (5) and inhibitor (IlePyr 500 or 50 or 0 µM) in PBS. The
reaction is started by addition of 32 µL of CD26 (1.5 mU) or 10
µL of BS or HS (total volume reaction mixture: 400 µL) at 37 °C.
After 5 h, 100 µL of the RMix was withdrawn and added to 200
µL methanol (on ice). The precipitate was then centrifuged at 13.000
rpm during 5 min, and 250 µL of the supernatant was injected on
HPLC (Reverse phase RP-8, Merck) to quantify the reaction
products as described above.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[3-N′-[valyl-prolyl-valylamino]propyl]thymine]-3′-spiro-5′′-
[4′′-amino-1′′,2′′-oxathiole-2′′,2′′-dioxide] (33). Via the general
N-deprotection procedure, 32 (65 mg, 0.06 mmol) was hydrogenated
1
to give 31 mg (59%) of 33 as a white foam. H NMR (300 MHz,
DMSO-d6): δ 0.92 (d, 6H, γ-CH3a, Val, J ) 6.7 Hz), 0.96 (d, 6H,
γ-CH3b, Val, J ) 6.7 Hz), 1.67 (m, 2H, -CH2-), 2.05 (m, 9H, CH3-
5, â-CH2,γ-CH2, Pro, â-CH, Val), 3.00 (m, 1H, CH2aNHCO), 3.04
(m, 2H, CH2NHCO), 3.46 (m, 1H, δ-CH2, Pro), 3.61 (m, 1H,
δ-CH2, Pro), 3.78 (m, 4H, R-CH, Val, N3-CH2, 2H-5′a), 3.91 (dd,
1H, 2H-5′b, J ) 7.5 Hz, J ) 12.4 Hz), 4.02 (m, 1H, R-CH, Val),
4.28 (t, 1H, H-4′, J ) 5.8 Hz), 4.38 (m, 1H, R-CH, Pro), 4.45 (d,
1H, H-2′, J ) 7.8 Hz), 5.72 (s, 1H, H-3′′), 5.96 (d, 1H, H-1′, J )
7.8 Hz), 6.99 (bs, 2H, NH2-4′′), 7.71 (s, 1H, H-6), 7.81 (m, 1H,
NHCO), 8.06 (m, 1H, NH, Val). MS (ESI+): m/z 942.3(M + 1)+.
Anal. for: C42H75N7O11SSi2: C, H, N, S.
Biological Methods. Compounds and Enzymes. GlyPro-pNA
was purchased from Sigma-Aldrich (Bornem, Belgium). CD26 was
purified as described33 and kindly provided by I. De Meester and
A.-M. Lambeir (Antwerp, Belgium). DPP IV/CD26 inhibitor
Diprotin A (Ile-Pro-Ile) was obtained from Bachem. DPP IV/CD26
inhibitor L-isoleucyl pyrrolidine (Ile-Pyr) was synthesized using a
modified procedure (see Supporting Information) to that described
by Christoffers Jens.34 Foetal bovine serum (FBS) was obtained
from Integro (Dieren, The Netherlands) and human serum provided
by the Blood Bank, Leuven, Belgium.
Acknowledgment. We thank Ann Absillis, Lizette van
Berckelaer, and Ria Van Berwaer for excellent technical
assistance. We thank the Spanish MEC (project SAF2003-
07219-C02), and the European Commission [Projects QLK2-
CT-2000-0291 and HPAW-CT-2002-90001 (Rene´ Descartes
Prize-2001) for financial support.
Evaluation of the Inhibitory Effect of Test Compound 5 and
Dipeptide ValPro against Purified CD26 Catalyzed Conversion
of GlyPro pNA to GlyPro and pNA. All enzyme activity assays
were performed in 96-well microtiter plates (Falcon, Becton
Dickinson, Franklin Lakes, NJ). To each well were added 5 µL of
CD26 (at a final concentration of 0.237 milliUnits/200 µL-well),
20 µL of appropriate concentrations of test compound or ValPro
solution in PBS (containing 1% DMSO), and PBS to reach a total
volume of 150 µL. The pH of the reaction mixture was 7.5, which
is virtually identical to the physiological pH of plasma. The reaction
was started by the addition of 50 µL of GlyPro-pNA substrate at
50 µM and carried out at 37 °C. Enzyme activity was examined as
the amount of (yellow-colored) p-nitroaniline (pNA) released from
GlyPro-pNA in function of time. The pNA release was recorded
by the increase of absorption [optical density (OD)] at 400 nm in
a Spectramax microplate spectrometer (Molecular Devices, Sunny-
vale, CA). Under our experimental conditions, the reaction pro-
ceeded linearly for at least 60 min. The 50% inhibitory concen-
trations of the test compounds against CD26 were defined as the
compound concentrations required to inhibit the enzyme (CD26)-
catalyzed hydrolysis of GlyPro-pNA to pNA and GlyPro by 50%.
The OD400 values of blank reaction mixtures (lacking the CD26
enzyme) were subtracted from the obtained OD400 values to
represent the real increase of OD400 value as a measurement of the
enzyme activity.
Supporting Information Available: Results from elemental
analysis for all compounds and 1H NMR chemical shift assignments
of novel dipeptide derivatives 21-24, 3h,n-p and protected
conjugates 4b-p and 30. The chemical procedure for the synthesis
of the DPP IV/CD26 inhibitor IlePyr and a table showing the cLogP
and tR values for dipeptidyl prodrugs of NAP-TSAO-T are also
included. This material is available free of charge via the Internet
at http://pubs.acs.org.
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