Dipeptidyl Peptidase IV-Activated Prodrugs of Anti-Varicella Zoster Virus Bicyclic Nucleoside Analogues Containing Different Self-Cleavage Spacer Systems

Article abstract of DOI:10.1002/cmdc.201200295

A new type of double prodrug of the antiviral family of bicyclic nucleoside analogues (BCNA) bearing cyclization self-cleavage spacers between the Val-Pro dipeptide sequence as well as the parent compound were synthesized and evaluated with regard to activation by the DPPIV/CD26 enzyme and for their stability in human and bovine serum. In buffer solution, carbamate and ester prodrugs were found to be chemically stable. Most prodrugs containing a dipeptidyl linker efficiently converted into the BCNA parent drug. In contrast, the Val-Pro alkyldiamino prodrugs converted predominantly into their alkyldiamino prodrug intermediates in the presence of CD26 and human serum. A marked increase in water solubility was observed for all prodrugs. In contrast to the parent compound, a tetrapeptide prodrug containing the Val-Val dipeptide as a self-cleavage spacer released substantial amounts of the BCNA parent drug at the basolateral side of Caco-2 cell cultures and exhibited 15- to 20-fold increased bioavailability in mice relative to the poorly bioavailable parent compound.

Full text of DOI:10.1002/cmdc.201200295

MED
DOI: 10.1002/cmdc.201200295
Dipeptidyl Peptidase IV-Activated Prodrugs of Anti-
Varicella Zoster Virus Bicyclic Nucleoside Analogues
Containing Different Self-Cleavage Spacer Systems
Alberto Diez-Torrubia,[a] Silvia Cabrera,[a] Ingrid De Meester,[b] Marꢀa-Josꢁ Camarasa,[a]
Jan Balzarini,[c] and Sonsoles Velꢂzquez*[a]
Dedicated to Prof. Josꢀ Luis Garcꢁa Ruano on the occasion of his 65th birthday
A new type of double prodrug of the antiviral family of bicyclic
nucleoside analogues (BCNA) bearing cyclization self-cleavage
spacers between the Val-Pro dipeptide sequence as well as the
parent compound were synthesized and evaluated with regard
to activation by the DPPIV/CD26 enzyme and for their stability
in human and bovine serum. In buffer solution, carbamate and
ester prodrugs were found to be chemically stable. Most pro-
drugs containing a dipeptidyl linker efficiently converted into
the BCNA parent drug. In contrast, the Val-Pro alkyldiamino
prodrugs converted predominantly into their alkyldiamino pro-
drug intermediates in the presence of CD26 and human
serum. A marked increase in water solubility was observed for
all prodrugs. In contrast to the parent compound, a tetrapep-
tide prodrug containing the Val-Val dipeptide as a self-cleavage
spacer released substantial amounts of the BCNA parent drug
at the basolateral side of Caco-2 cell cultures and exhibited 15-
to 20-fold increased bioavailability in mice relative to the
poorly bioavailable parent compound.
Introduction
Dipeptidyl peptidase IV (DPPIV, also termed CD26) is a serine
peptidase that is part of the prolyl oligopeptidase superfamily,
which removes X-Pro (and to a lesser extent X-Ala) dipeptides
from the N-terminus of natural peptides, including many che-
mokines, neuropeptides, and peptide hormones.[1–4] This
enzyme occurs in two forms, an extracellular membrane-
bound peptidase (highly expressed on endothelial cells, epi-
thelial cells, and lymphocytes) and a soluble form that is found
in plasma.[5,6]
alkyl chains (N-3 aminopropyl derivative of the anti-HIV TSAO
compounds)[9,10] as well as on heteroaromatic (6-aminoquino-
line), carbohydrate (doxorubicin), heterocyclic pyrimidine (ara-
C and TSAO-m5C), and purine rings (ara-A).[11] In these pro-
drugs, the compounds/drugs and the peptide moiety are di-
rectly coupled via an amide bond which is specifically cleaved
by DPPIV/CD26 (Figure 1). Our data revealed that purified
DPPIV/CD26 could recognize these prodrugs as substrates and
efficiently release the parent compound. Interestingly, it was
possible to modulate the enzymatic and serum hydrolysis rates
(half-lives) of the prodrug conjugates by changing the nature
and the length of the peptide promoiety.[8,12]
We also recently reported[13] the application of this prodrug
approach to hydroxy-containing drugs, in particular, the fura-
nopyrimidine bicyclic nucleoside analogues (a prototype of
which is designated Cf1743 (1), Figure 2), which represent
a promising new family of antivirals with extreme potency and
specificity for varicella zoster virus (VZV) infections but have
In 2005, it was first demonstrated that a synthetic small mol-
ecule [GPG-NH2, (glycylprolylglycinamide)] can be converted
into an antiviral drug (glycinamide) through the specific action
of DPPIV/CD26.[7] Based on these results, we previously report-
ed[8] a novel type of DPPIV/CD26-directed prodrug technology
that can be applied to improve the solubility and/or bioavaila-
bility of therapeutic agents. With our technology, the prodrugs
are conjugates of a therapeutic drug and a di- or oligopeptide
moiety that is cleavable by the DPPIV/CD26 activity. This ap-
proach was first successfully applied to a broad variety of
amine-containing drugs having a free amino group on their
[a] Dr. A. Diez-Torrubia, Dr. S. Cabrera, Prof. M.-J. Camarasa, Dr. S. Velꢂzquez
Instituto de Quꢁmica Mꢀdica (CSIC)
Juan de la Cierva 3, 28006 Madrid (Spain)
E-mail: sonsoles@iqm.csic.es
[b] Dr. I. De Meester
Laboratory of Medical Biochemistry
University of Antwerp, Universiteitsplein 1, 2610 Antwerp (Belgium)
[c] Prof. J. Balzarini
Rega Institute for Medical Research
Katholieke Universiteit (KU) Leuven
Minderbroedersstraat 10, 3000 Leuven (Belgium)
Figure 1. Application of the DPPIV/CD26-based prodrug approach to amine-
containing drugs.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201200295.
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Figure 3. General approaches for the design of DPPIV/CD26-activated pro-
drugs of antiviral Cf1743 bearing cyclization connectors as self-cleavage
spacers. Structure of target prodrugs 2a and b and 3a–c.
Figure 2. Two-step activation of DPPIV/CD26-based prodrugs of hydroxy-
containing drugs.
unfavorable pharmacokinetic properties.[14,15] Thus, our pur-
pose was twofold: to study for the first time the applicability
of this strategy to hydroxy-containing drugs, and to improve
the poor aqueous solubility and low oral bioavailability of
these highly lipophilic antiviral drugs. In hydroxy-containing
drugs, the peptide sequence cannot be linked directly to the
hydroxy group through an ester bond because the DPPIV/
CD26 enzyme specifically recognizes amide bonds. Thus, heter-
obifunctional spacers such as amino acids were designed for
attachment to the dipeptide through an amide bond (cleava-
ble by DPPIV/CD26) and for attachment to the OH group of
the drug through a metabolically labile ester linkage (Figure 2).
Liberation of the parent drug from these tripartite prodrugs
should therefore take place through a two-step hydrolysis se-
quence, first involving DPPIV/CD26-mediated enzymatic cleav-
age of the terminal dipeptide, followed by chemical or enzy-
matic (esterase-catalyzed) hydrolysis of the ester bond
(Figure 2). Our data proved that the prodrugs efficiently release
the parent drug upon selective conversion by purified DPPIV/
CD26, as well as by soluble DPPIV/CD26 present in bovine and
human serum. DPPIV/CD26 was solely responsible for the effi-
cient hydrolysis of the dipeptide moiety from the prodrugs as
the DPPIV/CD26-specific inhibitor vildagliptin was able to com-
pletely block the enzymatic conversion in human, bovine and
murine serum. Several prodrugs showed remarkable increases
in water solubility and markedly enhanced oral bioavailability
in mice relative to the parent free compound.[13a]
approach A). After DPPIV/CD26 hydrolysis the spacer should
spontaneously cyclize to give a cyclic urea derivative, thereby
releasing free Cf1743.
Second, peptide ester prodrugs 3a–c containing a dipeptide
cyclization spacer (XaaYaa) between the dipeptide and CF1743
were prepared (Figure 3, approach B). These tetrapeptide pro-
drugs were designed to release the parent compound through
an initial enzymatic conversion step (DPPIV/CD26), followed by
spontaneous cyclization of the dipeptide conjugate intermedi-
ate via diketopiperazine (DKP).[16a,18–20] It is well documented in
the literature that dipeptide-based ester prodrugs containing
a C-terminal proline residue readily cyclize to the DKP due to
the geminal-dimethyl effect.[21] The rate of drug release also
depends on the size of the side chains of the dipeptide carrier,
that is, the bulkier the amino acids that are incorporated in the
dipeptide backbone, the slower the drug release.[18,22] Based on
these reports, we incorporated prodrugs 3a–c, bearing dipep-
tides (XaaYaa) with a C-terminal proline residue and/or (b-
branched) amino acids of different molecular size, as cycliza-
tion spacers in order to diversify the half-lives of the prodrugs.
We herein describe the synthesis, water solubility, and chem-
ical and enzymatic stability studies of these novel prodrugs of
antiviral compound Cf1743 (1). In addition, permeability/trans-
port across colon carcinoma Caco-2 cell layers with selected
prodrugs and oral bioavailability in vivo in one selected tetra-
peptide prodrug were also studied.
Based on these encouraging results, we decided to explore
self-cleavage spacers in the design of novel two-step activation Results and Discussion
prodrugs of the antiviral Cf1743 (Figure 3), attached to the 5’-
hydroxy group of the nucleoside through a carbamate or ester
Synthesis
linkage. They were designed to undergo enzymatic activation
via DPPIV/CD26, followed by spontaneous release of the
parent drug. In this way, ideally, and contrary to what occurred
when amino acids were used as spacers in our previous stud-
ies,[13] final generation of the free drug may not be dependent
on enzymatic hydrolysis (esterase-catalyzed) of the ester bond.
Cyclization self-cleavage spacers were selected.[16] We first
turned our attention to the prodrugs of Cf1743 (2a and b), in-
corporating ethylene- and propylenediamine spacers[17] linked
to the dipeptide moiety via an amide bond and to the 5’-hy-
droxy group of the nucleoside via a carbamate bond (Figure 3,
The target prodrugs were prepared by coupling of the 5’-hy-
droxy group of the antiviral nucleoside and the spacer end of
the appropriate dipeptide-spacer moiety. Val-Pro was selected
as the dipeptide for the prodrugs because it is efficiently rec-
ognized by DPPIV/CD26, as shown in previous studies.[8,12,13]
We first focused on the synthesis of carbamate prodrugs 2a
and b, bearing diamine self-cleavage spacers. The required di-
peptide-spacer amines 5 and 7 (Scheme 1) were prepared in
80% and 95% yield by standard coupling of commercially
available Fmoc-Val-Pro-OH with N-Boc-ethylenediamine or N-
Boc-propylenediamine in the presence of dicyclohexylcarbodii-
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spacer (compound 7), followed by TBDMS deprotection, afford-
ed final target prodrug 2b in good yield.
Tetrapeptide prodrugs 3a–c, bearing a variety of dipeptide
cyclization spacers, were next prepared by coupling of appro-
priate tetrapeptide sequences to parent nucleoside
1 (Scheme 2). The tetrapeptide pro-moieties 16a–c were pre-
Scheme 2. Synthesis of tetrapeptide ester prodrugs 3a–c. Reagents and con-
ditions: a) Fmoc-Val-OH, HCTU, DIEA, DMF, RT; b) piperidine (20%), DMF;
c) Fmoc-Pro-OH, HCTU, DIEA, DMF, RT; d) CH2Cl2/TFA (95:5), RT; e) Fmoc-Val-
Pro-Val-Xaa-OH (16a–c), PPh3, DBAD, DMF, 808C, MW; f) piperidine (5%),
DMF.
Scheme 1. Synthesis of carbamate prodrugs 2a and b from 3’-protected nu-
cleoside 8. Reagents and conditions: a) NH2-(CH2)n-NHBoc, BOP, Et3N, CH2Cl2,
RT; b) HCl (1.25 m), MeOH, RT; c) TBDMSCl, imidazole, DMAP, DMF, RT;
d) 1. CDI, Et3N, DMF, RT; 2. H2O; e) compound 5 or 7, 408C; f) TBAF (1.1 m),
THF, RT.
pared in good yields by solid-phase synthesis using an Fmoc
strategy on a 2-chlorotrityl polystyrene resin as previously de-
scribed (see Supporting Information).[12] Regioselective cou-
pling of these peptides on the 5’ position of the parent nu-
cleoside via Mitsunobu condensation was first investigated in
a similar way to that described previously for the 5’-O-acylation
of 1 with amino acids.[13] Disappointingly, reaction of 1 with
Fmoc-Val-Pro-Val-Pro-OH (16a) in the presence of PPh3 and di-
tert-butylazodicarboxylate (DBAD) in dry DMF (under argon at-
mosphere) at room temperature for several days failed to give
5’-tetrapeptide prodrug 17a. However, treatment of 1 with
pro-moiety 16a under the aforementioned Mitsunobu condi-
tions (PPh3 and DBAD) under microwave irradiation (MW,
808C) for 8 h afforded desired tetrapeptide prodrug 17a in
45% yield after chromatographic purification.
mide (DCC) and triethylamine (TEA), followed by acid hydroly-
sis of protected dipeptide intermediates 4 and 6 in a 1.25 m
HCl solution. To introduce the carbamate linkage between the
nucleoside and the dipeptide-spacer moiety, we needed to se-
lectively activate the 5’-hydroxy group of the antiviral nucleo-
side. Initial attempts to form 5’-carbamate prodrug 2a were
carried out by in situ activation of unprotected nucleoside 1,
followed by reaction of the activated intermediate with dipep-
tide-spacer amine 5. However, activation of nucleoside 1,
either with 4-nitrophenyl chloroformate and TEA or with car-
bonyldiimidazole (CDI) and pyridine in dry DMF, followed by
reaction of the carbonate intermediates with amine 5 at room
temperature afforded mixtures of the 5’- and 3’-carbamates in
low yields along with starting material as the major com-
pound. We next investigated an alternative approach for the
synthesis of 5’-carbamate 2a using the 3’-tert-butyldimethylsil-
The syntheses of compounds 17b and c were carried out in
a similar manner, starting from the appropriate tetrapeptide
derivatives (16b and c) (Scheme 2). Removal of the Fmoc
group by brief treatment of 17a–c with piperidine afforded
the target-deprotected prodrugs 3a–c in good to excellent
yl (TBDMS)-protected nucleoside
9 as starting material
(Scheme 1). The synthesis of protected nucleoside 9 was car-
ried out in high yield by treatment of the fully TBDMS protect-
ed nucleoside 8 with 1.25 m HCl solution in methanol. Activa-
tion of the nucleoside was performed by treatment of 9 with
an excess (5 equiv) of CDI and TEA at room temperature. After
quenching the excess reagent through addition of water, the
activated intermediate 10 was reacted directly with peptide-
spacer amine 5 at 408C for 24 h to afford the 3’-TBDMS-pro-
tected carbamate 11 in good yield (51% over the two steps).
Under these conditions, simultaneous deprotection of the
Fmoc group was observed, likely due to the imidazole generat-
ed in the reaction media. The silyl protecting group was readily
removed by reaction of 11 with 1.1 m TBAF solution in THF at
room temperature to give the desired fully deprotected pro-
drug 2a in 63% yield. Similarly, treatment of activated nucleo-
side 10 with the corresponding peptide–propylenediamine
1
yields (76–97%). The target prodrugs were characterized by H
and 13C NMR, mass spectrometry, and microanalysis; all data
confirmed the structure and purity of the novel compounds.
Water solubility studies
In addition, the water solubility of the [Val-Pro]-[self-cleavage
spacer]-[Cf1743] prodrugs 2a, 2b, and 3a–c were determined
and compared with that of the poorly soluble parent com-
pound (Table 1). All prodrugs dramatically improved the water
solubility. Values ranged from 7.810 to 32.469 mgmLÀ1, corre-
sponding to 434- to 1804-fold higher water solubility in com-
parison with the poorly soluble parent drug Cf1743
(0.018 mgmLÀ1). These results further support the applicability
of the DPPIV/CD26 prodrug approach for increasing the water
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Table 1. Experimental aqueous solubility and other physicochemical pa-
rameters of [Val-Pro]-[self-cleavage spacer]-[Cf1743] prodrugs 2a, 2b, 3a–
c, and parent compound 1.
[d]
Compd
Aqueous solubility
logS ClogP[c] tR
mp
[8C]
[a]
[mgmLÀ1
]
Increase[b]
[min]
Cf1743 (1)
0.018Æ0.004
32.469Æ0.303
29.838Æ0.167
10.855Æ0.229
7.810Æ0.329
9.089Æ0.394
–
À4.35
À1.32
À1.37
À1.86
À2.01
À1.93
2.48
2.6
–
3.27
3.60
3.09
35.7 204–207
2a
2b
3a
3b
3c
1804
1658
603
434
505
31.6
–
84–86
–
35.8 108–110
37.6 113–115
34.3 119–121
[a] Thermodynamic water solubility measured at 258C after 24 h; values
are typically means of three measurements. [b] Fold increase in water sol-
ubility relative to parent drug 1. [c] Calculated logP values obtained with
the ALOGPS 2.1 program (http://www.vcclab.org/lab/alogps/). [d] HPLC
retention times determined with a reversed-phase column [Lichro Card
(LiChrospher 60 RP-select B; 5 mm; Merck)] (4.6 mmꢄ250 mm).
solubility of hydrophobic drugs. In general, the aqueous solu-
bility of small molecules is influenced by their hydrophobicity
(logP).[23] Thus, a classical strategy for improving aqueous solu-
bility has been the introduction of hydrophilic groups into
molecules. However, this approach is far from being universally
effective. It has also been proposed that disruption of crystal
packing could be an alternative method for improving aque-
ous solubility.[24] In an attempt to explain the improved solubili-
ty of prodrugs 2a, 2b, and 3a–c, the effect of changes in hy-
drophobicity as well as modification of crystal packing of pro-
drugs versus parent drug was examined next. To clarify the
contributions of the different mechanisms, physicochemical
parameters including ClogP, retention time on reversed-phase
HPLC (related to hydrophobicity), and melting point values (re-
lated to crystal packing) were evaluated (Table 1).
These physicochemical parameters and log(solubility) (logS)
were plotted as shown in Figure 4. In all cases, logS was more
strongly correlated with melting point values (Figure 4a) than
with hydrophobicity (ClogP) and retention time parameters
(Figure 4b,c). Thus, the increase in aqueous solubility does not
seem to be caused by a decrease in hydrophobicity. Several re-
ports have examined the relationship between solubility and
crystal packing or melting point values.[24] Despite the limited
number of prodrug analogues examined in our study, it
became clear that our results represent another example in
which disruption of crystal packing could play an important
role in improved aqueous solubility.
Figure 4. Relationships between experimentally observed solubility values
and physicochemical data of prodrugs 2a, 3a–c, and parent compound 1:
a) melting point; b) ClogP; c) HPLC retention time (tR).
into an intermediate that lacked the terminal Val-Pro moiety.
After 4 h, virtually complete conversion into this intermediate
was observed (figures S1 and S2, panels B and C). Interestingly,
in bovine serum, the prodrugs not only converted into the al-
kyldiamino-Cf1743 intermediate, but a time-dependent release
of the parent Cf1743 drug was also observed (figures S1 and
S2, panel D). In fact, after 24 h, almost 40% Cf1743 was formed
from the 2a prodrug, and >95% Cf1743 was derived from the
2b prodrug. This means that the spacer was released (either
spontaneously or enzymatically) from the 2a and 2b prodrugs
in bovine serum, but not in human serum. When the 2b pro-
drug was exposed to bovine serum in the presence of vilda-
gliptin, a potent inhibitor of CD26 activity, no intermediate was
released from the prodrug, nor could the parent drug (Cf1743)
be detected (data not shown). These observations revealed
that the eventual release of Cf1743 from the prodrug can only
be performed after the dipeptide moiety has been released
from the prodrug. Thus, the enzyme responsible for the even-
Biological evaluation
The Cf1743 prodrugs were examined for their stability in PBS
(pH 7.4), their susceptibility to degradation by CD26 enzymatic
activity, and their conversion into parent compound in human
and bovine serum (20% in PBS). Two prodrugs of Cf1743 were
investigated in which the terminal Val-Pro dipeptide was linked
to the parent molecule through an ethylenediamino (2a) or
propylenediamino (2b) spacer (figures S1 and S2, Supporting
Information). Both prodrugs were fully stable in PBS. Exposure
to CD26 or human serum revealed time-dependent conversion
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tual release of Cf1743 must specifically act on the alkyldiamino
Cf1743 conjugate. Similar observations were observed with
bovine serum that was extensively dialyzed in PBS to remove
any small molecules from the enzyme preparation. This points
to the lack of necessity of participation of additional small or-
ganic molecule substrates such as amino acids or sugars in the
enzymatic conversion reaction.
The Val-Pro-Val-Pro-Cf1743 tetrapeptide prodrug (3a) proved
stable in PBS (Figure 5a) but was highly susceptible to CD26-
catalyzed hydrolysis of the parent compound (Figure 5b). Con-
version of the tetrapeptide to the parent compound proceed-
ed very efficiently, and no intermediary Val-Pro dipeptide pro-
drug was observed during the conversion process to parent
drug Cf1743. Also, human and bovine serum efficiently re-
leased the parent compound without detectable release of the
dipeptide intermediate (Figure 5c,d).
Next, Val-Pro-Val-Val-Cf1743 (3b) was studied for stability in
PBS and CD26 susceptibility against the purified enzyme and
in serum. While very stable in PBS (only ~6% conversion into
the parent drug after 24 h) (Figure 6a), the tetrapeptide pro-
drug was rapidly converted into Val-Val-Cf1743 and Cf1743
(60% and 13%, respectively, after 1 h) in the presence of
CD26. After 24 h, all of the prodrug was converted into a 40:60
mixture of Val-Val-Cf1743 and Cf1743 (Figure 6b). Human and
bovine serum (Figure 6c,d) showed a similar conversion profile.
After 24 h, appearance of Cf1743 in the serum was still inferior
to formation of the Val-Val-Cf1743 intermediate. When similar
incubations with Val-Val-Cf1743 were performed, the dipeptide
prodrug was quite stable in PBS as well as in the CD26 assay
and in serum incubations (Figure S3). Only after 24 h incuba-
tion was the Val-Val-Cf1743 dipeptide prodrug further convert-
ed into Cf1743, to a limited extent.
Upon investigation of the tetrapeptide Val-Pro-Val-Ala-
Cf1743 (3c), the formation of small amounts of Cf1743 in PBS
was observed as a function of time (figure S4, panel A, Sup-
porting Information). However, CD26 efficiently converted the
tetrapeptide prodrug to Cf1743. Only trace amounts of the in-
termediary dipeptide Val-Ala-Cf1743 could be observed (fig-
ure S4, panel B). Most likely, CD26 also cleaved the Val-Ala di-
peptide from the dipeptide prodrug intermediate to release
Cf1743. The Val-Ala dipeptide has indeed been shown to act as
a substrate recognition motif for CD26.[22] Both human and
bovine serum also efficiently and time-dependently converted
the tetrapeptide prodrug to Cf1743 (figure S4, panels C and D).
To determine the potentially increased oral bioavailability of
the prodrugs compared with the poorly oral bioavailable
parent drug Cf1743, uptake and release of the tetrapeptide
prodrug Val-Pro-Val-Val-Cf1743 (3b) and its parent Cf1743 were
investigated in confluent Caco-2 monolayer cell cultures. In
this assay model, the drugs were exposed at the apical side of
the cell culture monolayer, and release of the parent drug (or
prodrug/intermediates) at the basolateral side of the cell cul-
Figure 5. Stability of [Val-Pro][Val-Pro]-Cf1743 (3a; &) in a) PBS and its con-
version into parent drug Cf1743 (&) in the presence of b) purified CD26,
c) human serum (HS), and d) bovine serum (BS).
amounts of the dipeptide intermediate (Val-Val-Cf1743) were
also observed to be released at the basolateral side of the
Caco-2 cell cultures, but these prodrug levels were invariably
well below the Cf1743 amounts.
tures was determined as
a function of exposure time
(Figure 7). Whereas Cf1743 was poorly transported through the
Caco-2 monolayers, exposure of equimolar amounts of the tet-
rapeptide prodrug 3b resulted in a pronounced appearance of
Cf1743 in the basolateral compartment. Also, measurable
These data indicate a markedly increased potential for oral
bioavailability of the tetrapeptide prodrug versus parent drug
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Figure 7. Appearance of Cf1743 (&) and [Val-Val]-Cf1743 (&) at the basolat-
eral side of monolayer Caco-2 cell cultures exposed to a) Cf1743 or b) [Val-
Pro][Val-Val]-Cf1743.
Figure 8. Oral bioavailability of equimolar drug concentrations (25 mgmLÀ1
À1
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Cf1743 ( ) or 50 mgmL [Val-Pro][Val-Val]-Cf1743 (3b; )) when adminis-
tered to mice by oral gavage.
Figure 6. Stability of [Val-Pro][Val-Val]-Cf1743 (3b; &) in a) PBS and its con-
version into parent drug Cf1743 (&) and intermediates [Val]-Cf1743 (&) and
[Val-Val]-Cf1743 (&) in the presence of b) purified CD26, c) human serum
(HS), and d) bovine serum (BS).
to 20-fold more orally bioavailable than the parent drug. In
this respect, the oral bioavailability of the Val-Pro-Val-Val-
Cf1743 prodrug was superior (ꢀ2-fold) to the bioavailability
previously observed for Val-Pro-Val-Cf1743.[13a] It should also be
noted that only Cf1743 was detected in the murine plasma
and not any tetrapeptide prodrug or dipeptide (Val-Val) inter-
mediate (Figure 8).
Cf1743. Indeed, when Balb/C mice were given Val-Pro-Val-Val-
Cf1743 (3b) or Cf1743 by oral gavage at 25 mgkgÀ1, pro-
nounced levels of Cf1743 appeared in the plasma of the tetra-
peptide prodrug-exposed mice (Figure 8). It was estimated
that the tetrapeptide prodrug of Cf1743 (3b) was at least 15-
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20 mL). The aqueous layer was basified with NH3 and was extract-
ed with CH2Cl2 (5ꢄ20 mL). The organic layer was dried (Na2SO4), fil-
tered, and evaporated to dryness. The final residue was purified by
flash chromatography (HPFC) in a Biotage SP1 to give 522 mg of 4
Conclusions
We have demonstrated that prodrugs of the bicyclic nucleo-
side Cf1743 containing diamines or dipeptide cyclization
spacers are efficient substrates for the dipeptidyl-peptidase ac-
tivity of DPPIV/CD26. Prodrugs 3a–c, based on dipeptide cycli-
zation spacers, efficiently released the parent nucleoside upon
hydrolysis by DPPIV/CD26. All novel prodrugs had substantially
increased water solubility, which proved closely and inversely
correlated to decreased melting point values of the (pro)drugs.
A marked increase in transport through Caco-2 monolayers
and oral bioavailability was observed for the Val-Pro-Val-Val tet-
rapeptide prodrug of Cf1743 (3b). Consequently, tetrapeptide
ester prodrugs of Cf1743 represent a new type of efficient
DPPIV/CD26-based prodrugs that could be extended to other
(lipophilic) hydroxy-containing drugs with poor solubility and/
or unfavorable pharmacokinetic (i.e., poor bioavailability) prop-
erties.
1
(80%) as a white foam: H NMR (300 MHz, [D6]acetone): d=0.98 (d,
3H, g-CH3, Val, J=6.6 Hz), 1.02 (d, 3H, g-CH3, Val, J=6.6 Hz), 1.44 (s,
9H, tBu, Boc), 1.82–2.10 (m, 4H, b-CH2, Pro, g-CH2, Pro), 2.18 (m,
1H, b-CH, Val), 3.16 (m, 2H, CH2-N), 3.27 (m, 2H, N-CH2), 3.62–3.87
(m, 2H, d-CH2, Pro), 4.19–4.41 (m, 5H, a-CH, Val, a-CH, Pro, CH2-
Fmoc, CH-Fmoc), 6.09 (m, 1H, NH-Boc), 6.64 (d, 1H, NH, Val, J=
8.8 Hz), 7.32 (t, 2H, H2,7-Fmoc, J=7.5 Hz), 7.41 (m, 3H, H3,6-Fmoc,
NH-CH2), 7.72 (dd, 2H, H1,8-Fmoc, J=4.6, 7.1 Hz), 7.86 ppm (d, 2H,
H4,5-Fmoc, J=7.3 Hz); MS (ESI+): m/z 579.5 [M+H]+, 601.5 [M+
Na]+.
Fmoc-Val-Pro-NH-(CH2)2-NH3+ClÀ (5): A solution of HCl (1.25 m) in
MeOH (3.16 mL, 3.95 mmol) was added to a solution of compound
4 (457 mg, 0.79 mmol) in THF (5 mL). The reaction mixture was
stirred at room temperature for 6 h, and the solvent was evaporat-
ed to dryness to afford 388 mg of 5 (95%, white foam) as a hydro-
chloride salt: 1H NMR (300 MHz, [D6]acetone): d=0.89–94 (m, 6H,
g-CH3, Val), 1.73–1.84 (m, 2H, b-CHa, Pro, g-CHa, Pro), 1.89–2.10 (m,
3H, b-CHb, Pro, g-CHb, Pro, b-CH, Val), 2.83 (m, 2H, CH2-N), 3.31
(m, 2H, N-CH2), 3.54–3.75 (m, 2H, d-CH2, Pro), 4.04 (t, 1H, a-CH, Val,
J=8.4 Hz), 4.17–4.28 (m, 4H, a-CH, Pro, CH2-Fmoc, CH-Fmoc), 7.31
(t, 2H, H2,7-Fmoc, J=7.2 Hz), 7.41 (t, 2H, H3,6-Fmoc, J=7.5 Hz), 7.60
(d, 1H, NH, Val, J=8.7 Hz), 7.73 (m, 2H, H1,8-Fmoc), 7.88 (d, 2H,
H4,5-Fmoc, J=7.5 Hz), 8.15 (bs, 3H, NH3+), 8.31 ppm (t, 1H, CONH,
J=8.1 Hz); 13C NMR (75 MHz, [D6]DMSO): d=18.9, 19.3 (2C-g, Val),
25.0 (C-g, Pro), 30.1 (C-b, Pro), 36.5 (C-b, Val), 38.9 (CH2-N), 39.6 (N-
CH2), 47.0, 47.6 (C-d, Pro, CH-Fmoc), 58.3 (C-a, Val), 60.1 (C-a, Pro),
Experimental Section
Chemical procedures
General: Microanalyses were obtained with a Heraeus CHN-O-
RAPID instrument, and analytical results were within 0.4% of theo-
retical values. Electrospray mass spectra were measured on a quad-
rupole mass spectrometer equipped with an electrospray source
(Hewlett Packard, LC/MS HP 1100). Spectra were recorded with
Varian Inova-300 or Varian Inova-400 spectrometers operating at
66.0 (CH2-Fmoc), 120.4 (C4,5-Fmoc), 126.0 (C1,8-Fmoc), 127.4 (C2,7
-
300 or 400 MHz for H NMR and at 75 or 100 MHz for 13C NMR with
1
Fmoc), 128.0 (C3,6-Fmoc), 141.0, 144.2 (2C-Fmoc), 156.4 (C=O,
Fmoc), 170.7, 172.5 ppm (C=O, Val, C=O, Pro); MS (ESI+): m/z 515.5
[M+H]+.
Me4Si as an 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 cir-
cular thin layer chromatography (CCTLC) on a Chromatotron (Kie-
segel 60 PF254 gipshaltig (Merck), layer thickness 1 mm, flow rate
5 mLminÀ1. Liquid chromatography was performed using a force
flow (flash chromatography) Horizon HPFG System (Biotage) with
Flash 25 or 40 silica gel cartridges.
Fmoc-Val-Pro-NH-(CH2)3-NH-Boc (6): Fmoc-Val-Pro-OH (500 mg,
1.14 mmol) was reacted with Boc-NH-(CH2)3-NH2 (0.200 mL,
1.14 mmol), BOP (608 mg, 1.37 mmol), and Et3N (0.192 mL,
1.37 mmol) in CH2Cl2 (10 mL), according to the coupling procedure
described for compound 4. The final residue was purified by flash
chromatography (HPFC) in a Biotage SP1 to give 595 mg of 6
1
(88%) as a white foam: H NMR (300 MHz, [D6]acetone): d=0.98 (d,
Microwave reactions were performed using the Biotage Initiator
2.0 single-mode cavity instrument from Biotage (Uppsala). Experi-
ments were carried out in sealed microwave process vials using
the standard absorbance level (400 W maximum power). The tem-
perature was measured with an IR sensor on the outside of the re-
action vessel.
3H, g-CH3, Val, J=6.6 Hz), 1.02 (d, 3H, g-CH3, Val, J=6.6 Hz), 1.41 (s,
9H, tBu, Boc), 1.58 (m, 2H, C-CH2-C), 1.84–2.23 (m, 5H, b-CH2, Pro,
g-CH2, Pro, b-CH, Val), 3.04–3.37 (m, 4H, CH2-N, N-CH2), 3.62–3.89
(m, 2H, d-CH2, Pro), 4.18–4.40 (m, 4H, a-CH, Val, CH2-Fmoc, CH-
Fmoc), 4.43 (m, 1H, a-CH, Pro), 6.07 (m, 1H, NH-Boc), 6.64 (d, 1H,
NH, Val, J=8.8 Hz), 7.32 (m, 3H, H2,7-Fmoc, NH-CH2), 7.41 (d, 2H,
H3,6-Fmoc, J=7.5 Hz), 7.73 (dd, 2H, H1,8-Fmoc, J=4.6, 7.1 Hz),
7.85 ppm (d, 2H, H4,5-Fmoc, J=7.5 Hz); MS (ESI+): m/z 593.3 [M+
H]+, 615.1 [M+Na]+.
The dipeptide derivative Fmoc-Val-Pro-OH was purchased from
Bachem Feinchemikalien. H-Pro-2-chlorotrityl resin was purchased
from GL Biochem (Shanghai), H-Val-2-chlorotrityl resin and H-Ala-2-
chlorotrityl resin were purchased from Irish Biotech, and HCTU was
purchased from Novabiochem. Cf1743 (1) was synthesized as pre-
viously described.[25] The purity of novel prodrugs was determined
to be >95% by elemental analysis.
Fmoc-Val-Pro-NH-(CH2)3-NH3+ClÀ (7): A solution of HCl (1.25 m) in
MeOH (3.70 mL, 4.63 mmol) was added to a solution of Fmoc-Val-
Pro-NH-(CH2)3-NH-Boc 6 (549 mg, 0.93 mmol) in THF (5 mL). The re-
action mixture was stirred at room temperature for 5 h, and the
solvent was evaporated to dryness to afford 480 mg of 7 (98%,
Fmoc-Val-Pro-NH-(CH2)2-NH-Boc (4): A solution of Fmoc-Val-Pro-
OH (500 mg, 1.14 mmol) in CH2Cl2 (10 mL) was reacted with Boc-
NH-(CH2)2-NH2 (0.181 mL, 1.14 mmol), 1-benzotriazolyloxy-tris-dime-
thylamino-phosphonium hexafluorophosphate (BOP) (608 mg,
1.37 mmol) and Et3N (0.192 mL, 1.37 mmol). The reaction mixture
was stirred at room temperature for 15 h, and the solvent was re-
moved under reduced pressure. The residue was dissolved in
EtOAc (30 mL) and washed with 10% aqueous citric acid (3ꢄ
1
white foam) as a hydrochloride salt: H NMR (300 MHz, [D6]DMSO):
d=0.88–94 (m, 6H, g-CH3, Val), 1.62–1.81 (m, 4H, b-CHa, Pro, g-
CHa, Pro, C-CH2-C), 1.91–2.07 (m, 3H, b-CHb, Pro, g-CHb, Pro, b-CH,
Val), 2.78 (m, 2H, CH2-N), 3.15 (m, 2H, N-CH2), 3.57–3.73 (m, 2H, d-
CH2, Pro), 4.05 (t, 1H, a-CH, Val, J=8.1 Hz), 4.19–4.28 (m, 4H, a-CH,
Pro, CH2-Fmoc, CH-Fmoc), 7.30 (m, 2H, H2,7-Fmoc), 7.40 (t, 2H, H3,6
Fmoc, J=7.2 Hz), 7.57 (d, 1H, NH, Val, J=8.4 Hz), 7.74 (m, 2H, H1,8
-
-
ChemMedChem 0000, 00, 1 – 12
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S. Velꢂzquez et al.
MED
Fmoc), 7.86 (d, 2H, H4,5-Fmoc, J=7.2 Hz), 8.17–8.19 ppm (m, 4H,
NH3+, CONH); 13C NMR (75 MHz, [D6]DMSO): d=18.5, 19.0 (2C-g,
Val), 24.5 (C-g, Pro), 27.1 (C-CH2-C), 29.4 (C-b, Pro), 29.7 (C-b, Val),
35.4 (CH2-N), 36.3 (N-CH2), 46.6, 47.0 (C-d, Pro, CH-Fmoc), 57.9 (C-a,
(m, 1H, H-2’b), 2.61 (t, 2H, CH2, J=7.6 Hz), 3.00–3.36 (m, 5H, N-
CH2-CH2-N, a-CH, Val), 3.43–3.61 (m, 2H, d-CH2, Pro), 4.09 (m, 1H,
H-4’), 4.16–4.28 (m, 4H, 2H-5’, H-3’, a-CH, Pro), 6.21 (t, 1H, H-1’, J=
6.4 Hz), 7.19 (s, 1H, H-5), 7.25 (m, 1H, NHCOO), 7.31 and 7.73
(AA’BB’ system, 4H, Ar, J=8.0 Hz), 7.84 (m, 1H, CONH-CH2),
8.51 ppm (s, 1H, H-4); 13C NMR (100 MHz, [D6]DMSO): d=13.9
(CH3), 17.0, 19.6 (2C-g, Val), 21.8 (CH2), 24.5 (C-g, Pro), 29.0, 30.3,
30.8, 31.1 (2CH2, C-b, Pro, C-b, Val), 37.2 (CH2), 34.8 (CH2-N), 38.4 (N-
CH2), 40.7 (C-2’), 46.7 (C-d, Pro), 57.1 (C-a, Val), 59.5 (C-a, Pro), 63.8
(C-5’), 70.3 (C-3’), 85.3 (C-4’), 87.8 (C-1’), 98.7 (C-5), 107.1 (C-4a),
124.5 (C-Hb), 125.8 (ipso-C), 129.0 (C-Ha), 137.5 (C-4), 144.0 (para-
C), 153.7, 153.9 (C-6, NHCOO), 155.9 (C-2), 171.0, 171.1, 171.9 ppm
(C=O, Val, C=O, Pro, C-7a); MS (ESI+): m/z 681.3 [M+H]+, 703.3
[M+Na]+; Anal. calcd for C35H48N6O8: C 61.75, H 7.11, N 12.34;
found: C 61.85, H 7.07, N 12.41.
Val), 59.6 (C-a, Pro), 65.6 (CH2-Fmoc), 120.0 (C4,5-Fmoc), 125.3 (C1,8
-
Fmoc), 126.9 (C2,7-Fmoc), 127.5 (C3,6-Fmoc), 140.6, 143.7 (2C-Fmoc),
156.1 (C=O, Fmoc), 170.1, 171.9 ppm (C=O, Val, C=O, Pro); MS (ESI+
): m/z 530.1 [M+H]+.
3-[3’,5’-di-O-(tert-Butyldimethylsilyl)-2’-deoxy-b-d-ribofuranosyl]-
6-(p-pentylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one
(8):
Imidazole (773 mg, 11.35 mmol), DMAP (116 mg, 0.95 mmol), and
TBDMSCl (1.426 g, 9.46 mmol) were added to a solution of com-
pound 1 (1.506 g, 3.78 mmol) in dry DMF (15 mL). The reaction
mixture was stirred at room temperature for 1 h, and the solvent
was evaporated to dryness. The crude reaction mixture was dis-
solved in CH2Cl2 (30 mL) and washed with NH4Cl (3ꢄ20 mL). The
organic layer was dried (Na2SO4) and removed by filtration, and the
solvent was eliminated under reduced pressure. The final residue
was purified by flash chromatography (CH2Cl2/MeOH, 40:1) to yield
2.24 g of 8 (95%) as a yellow solid: mp: 89–908C (Et2O); MS (ESI+):
m/z 627.5 [M+H]+; Anal. calcd for C34H54N2O5Si2: C 65.13, H 8.68, N
4.47; found: C 65.61, H 8.69, N 4.56.
3-[3’-O-(tert-Butyldimethylsilyl)-2’-deoxy-5’-O-[2-(valylprolylami-
no)propylcarbamoyl]-b-d-ribofuranosyl]-6-(p-pentylphenyl)-2,3-
dihydrofuro[2,3-d]pyrimidin-2-one (12): According to the proce-
dure described for compound 11, a solution of 9 (200 mg,
0.39 mmol) in dry DMF (15 mL) was treated with Et3N (0.272 mL,
1.95 mmol) and CDI (3175 mg, 1.95 mmol). After 5 h, the reaction
was quenched by the addition of several drops of water, then
Fmoc-Val-Pro-NH-(CH2)3-NH3+ClÀ (7; 166 mg, 0.32 mmol) was
added. The reaction mixture was stirred at 408C for 48 h. The final
residue was purified by CCTLC on the Chromatotron (CH2Cl2/
MeOH, 15:1) to afford 192 mg of 12 (61%) as a yellow foam: MS
(ESI+): m/z 809.7 [M+H]+, 831.7 [M+Na]+; Anal. calcd for
C42H64N6O8Si: C 62.35, H 7.97, N 10.39; found: C 62.59, H 7.93, N
10.45.
3-[3’-O-(tert-Butyldimethylsilyl)-2’-deoxy-b-d-ribofuranosyl]-6-(p-
pentylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one (9): A solu-
tion of HCl (1.25 m) in MeOH (2.82 mL, 3.53 mmol) was added to
a solution of 8 (1.84 g, 2.94 mmol) in MeOH (30 mL). The reaction
mixture was stirred at room temperature for 2 h. The solid was fil-
tered and was washed with MeOH to afford 1.20 g of 9 (80%) as
a white solid: mp: 190–1918C (MeOH); MS (ESI+): m/z 513.5 [M+
H]+, 535.5 [M+Na]+; Anal. calcd for C28H40N2O5Si: C 65.59, H 7.86,
N 5.46; found: C 65.67, H 7.83, N 5.49.
3-[2’-Deoxy-5’-O-[2-(valylprolylamino)propylcarbamoyl]-b-d-ribo-
furanosyl]-6-(p-pentylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-
one (2b): Following the procedure described for compound 2a,
nucleoside 12 (157 mg, 0.19 mmol) was reacted with a solution of
TBAF (1.1 m) in THF (0.266 mL, 0.292 mmol) for 1 h. The final resi-
due was purified by CCTLC on the Chromatotron (CH2Cl2/MeOH,
10:1) to afford 92 mg of 2b (68%) as a yellow foam: 1H NMR
(400 MHz, [D6]DMSO): d=0.86 (t, 3H, CH3, J=7.2 Hz), 0.89–1.00 (m,
6H, g-CH3, Val), 1.26–1.35 (m, 4H, 2CH2), 1.51–1.61 (m, 5H, g-CHa,
Pro, CH2, C-CH2-C), 1.72 (m, 1H, g-CHb, Pro), 1.91 (m, 1H, b-CHa,
Pro), 2.01–2.21 (m, 3H, b-CHb, Pro, b-CH, Val, H-2’a), 2.42 (m, 1H,
H-2’b), 2.62 (t, 2H, CH2, J=7.6 Hz), 2.98–3.06 (m, 4H, N-CH2, CH2-N),
3.03–3.19 (m, 2H, d-CH2, Pro), 3.41 (m, 1H, a-CH, Val), 3.71 (m, 1H,
H-4’), 3.97 (m, 1H, a-CH, Pro), 4.08 (m, 1H, H-3’), 4.16–4.27 (m, 2H,
2H-5’), 6.21 (t, 1H, H-1’, J=6.4 Hz), 7.17 (s, 1H, H-5), 7.20 (t, 1H,
NHCOO, J=7.6 Hz), 7.32 and 7.73 (AA’BB’ system, 4H, Ar, J=
8.0 Hz), 7.97 (t, 1H, CONH-CH2, J=7.0 Hz), 8.02 (bs, 2H, NH2, Val),
8.52 ppm (s, 1H, H-4); MS (ESI+): m/z 695.7 [M+H]+, 718.8 [M+
Na]+; Anal. calcd for C36H50N6O8: C 62.23, H 7.25, N 12.10; found: C
62.07, H 7.27, N 12.18.
3-[3’-O-(tert-Butyldimethylsilyl)-2’-deoxy-5’-O-[2-(valylprolylami-
no)ethylcarbamoyl]-b-d-ribofuranosyl]-6-(p-pentylphenyl)-2,3-
dihydrofuro[2,3-d]pyrimidin-2-one
(11):
Et3N
(0.204 mL,
1.46 mmol) and CDI (245 mg, 1.46 mmol) were added to a solution
of compound 9 (150 mg, 0.29 mmol) in dry DMF (15 mL) under
argon atmosphere. The reaction mixture was stirred at room tem-
perature for 5 h and quenched by the addition of several drops of
water. Then, a solution of Fmoc-Val-Pro-NH-(CH2)2-NH3+ClÀ (5;
166 mg, 0.32 mmol) and Et3N (0.05 mL, 0.35 mmol) in dry DMF
(5 mL) were added. The reaction was stirred at 408C for 24 h. The
solvent was evaporated to dryness, and the residue was dissolved
in EtOAc (30 mL) and washed with 10% aqueous NaHCO3 (3ꢄ
20 mL) and brine (3ꢄ20 mL). The organic layer was dried (Na2SO4)
and removed by filtration, and the solvent was evaporated to dry-
ness. The final residue was purified by CCTLC on the Chromatotron
(CH2Cl2/MeOH, 20:1) to afford 118 mg of 11 (51%) as a yellow
foam: MS (ESI+): m/z 795.9 [M+H]+; Anal. calcd for C41H62N6O8Si: C
61.94, H 7.86, N 10.57; found: C 62.25, H 7.80, N 10.61.
3-[2’-Deoxy-5’-O-[N-(fluorenylmethoxycarbonyl)valylprolylvalyl-
prolyl]-b-d-ribofuranosyl]-6-(p-pentylphenyl)-2,3-dihydrofuro-
[2,3-d]pyrimidin-2-one (17a): A solution of compound 1 (100 mg,
0.25 mmol), Fmoc-Val-Pro-Val-Pro-OH (16a; 199 mg, 0.314 mmol),
PPh3 (99 mg, 0.376 mmol), and DBAD (88 mg, 0.376 mmol) in dry
DMF (3 mL) were placed in a microwave vessel under argon atmos-
phere. The reaction was irradiated at 808C for 8 h. After cooling,
volatiles were evaporated to dryness. The residue was dissolved in
EtOAc (30 mL) and washed with 10% aqueous NaHCO3 (3ꢄ20 mL)
and brine (3ꢄ20 mL). The organic layer was dried (Na2SO4) and fil-
tered, and the solvent was evaporated to dryness. The final residue
was purified by CCTLC on the Chromatotron (CH2Cl2/MeOH, 25:1)
to afford 113 mg of 17a (45%) as a white foam: MS (ESI+): m/z
3-[2’-Deoxy-5’-O-[2-(valylprolylamino)ethylcarbamoyl]-b-d-ribo-
furanosyl]-6-(p-pentylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-
one (2a): A solution of TBAF (1.1 m) in THF (0.180 mL, 0.198 mmol)
was added to a solution of 11 (105 mg, 0.13 mmol) in THF (2 mL).
The reaction mixture was stirred at room temperature for 1 h, and
the solvent was evaporated to dryness. The final residue was puri-
fied by CCTLC on the Chromatotron (CH2Cl2/MeOH, 10:1) to yield
54 mg of 2a (63%) as a yellow solid after trituration with Et2O:
1
mp: 84–868C (Et2O); H NMR (400 MHz, [D6]DMSO): d=0.79 (t, 3H,
CH3, J=7.0 Hz), 0.81–0.88 (m, 6H, g-CH3, Val), 1.22–1.33 (m, 4H,
2CH2), 1.57 (m, 2H, CH2), 1.69–1.78 (m, 3H, g-CH2, Pro, b-CHa, Pro),
1.86–2.00 (m, 2H, b-CHb, Pro, b-CH, Val), 2.18 (m, 1H, H-2’a), 2.43
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DPPIV-Activated Anti-Varicella Zoster Virus Prodrugs
MED
1013.9 [M+H]+, 1035.9 [M+Na]+; Anal. calcd for C57H68N6O11: C
67.57, H 6.76, N 8.29; found: C 68.02, H 6.70, N 8.31.
130.6 (C-Ha), 138.5 (C-4), 145.9 (para-C), 155.7, 156.1 (C-6, C-2),
172.8, 172.9, 173.0, 173.1, 173.3 ppm (C=O, Val1, C=O, Val2, C=O,
Val3, C=O, Pro, C-7a); MS (ESI+): m/z 793.79 [M+H]+; Anal. calcd
for C42H60N6O9: C 63.62, H 7.63, N 10.60; found: C 63.55, H 7.51, N
10.56.
3-[2’-Deoxy-5’-O-(valylprolylvalylprolyl)-b-d-ribofuranosyl]-6-(p-
pentylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one (3a): Piperi-
dine (0.1 mL) was added to
a solution of 17a (255 mg,
0.287 mmol) in DMF (2 mL). The mixture was stirred at room tem-
perature for 5 min and evaporated to dryness under reduced pres-
sure. The residue was purified by CCTLC on the Chromatotron
(CH2Cl2/MeOH, 10:1) to yield 47 mg of 3a (76%) as a white solid
after trituration with Et2O; mp: 108–1108C (Et2O); 1H NMR
(400 MHz, [D6]DMSO): d=0.84–0.94 (m, 15H, 4g-CH3, Val1 and Val2,
CH3), 1.23–1.32 (m, 4H, 2CH2), 1.54–1.62 (m, 2H, CH2), 1.65–2.22 (m,
11H, 2b-CH, Val1 and Val2, 2b-CH2, Pro1 and Pro2, 2g-CH2, Pro1 and
Pro2, H-2’a), 2.43 (m, 1H, H-2’b), 2.61 (t, 2H, CH2, J=7.6 Hz), 3.58–
3.72 (m, 4H, 2d-CH2, Pro1 and Pro2), 4.11 (m, 1H, H-4’), 4.23–4.29
(m, 2H, H-3’, a-CH, Val1), 4.33- 4.46 (m, 5H, 2H-5’, a-CH, Val2, 2a-
CH, Pro1 and Pro2), 5.51 (bs, 2H, NH2), 6.20 (t, 1H, H-1’, J=6.4 Hz),
7.07 (s, 1H, H-5), 7.31 and 7.73 (AA’BB’ system, 4H, Ar, J=8.0 Hz),
8.60 ppm (s, 1H, H-4); 13C NMR (75 MHz, [D6]DMSO): d=13.9 (CH3),
17.2, 18.0, 19.0, 19.1 (4C-g, Val1 and Val2), 22.0 (CH2), 24.7, 24.8 (2C-
g, Pro1 and Pro2), 28.7, 28.9, 30.2, 30.5, 30.8, 30.9 (2CH2, 2C-b, Pro1
and Pro2, 2C-b, Val1 and Val2), 35.0 (CH2), 40.6 (C-2’), 46.9, 47.1 (2C-
d, Pro1 and Pro2), 55.3 (C-a, Val1), 56.4 (C-a, Val2), 58.6, 59.1 (2C-a,
Pro1 and Pro2), 64.1 (C-5’), 69.7 (C-3’), 84.5 (C-4’), 87.8 (C-1’), 98.7 (C-
5), 107.2 (C-4a), 124.7 (C-Hb), 125.8 (ipso-C), 129.0 (C-Ha), 137.7 (C-
4), 144.1 (para-C), 153.7, 153.9 (C-6, C-2), 170.0, 171.1, 171.3, 171.8,
172.0 ppm (C=O, Val1, C=O, Val2, C=O, Pro1, C=O, Pro2, C-7a); MS
(ESI+): m/z 791.7 [M+H]+, 813.7 [M+Na]+; Anal. calcd for
C42H58N6O9: C 63.78, H 7.39, N 10.63; found: C 63.65, H 7.41, N,
10.66.
3-[2’-Deoxy-5’-O-[N-(fluorenylmethoxycarbonyl)valylprolylvalyl-
alanyl]-b-d-ribofuranosyl]-6-(p-pentylphenyl)-2,3-dihydrofuro-
[2,3-d]pyrimidin-2-one (17c): Following the procedure described
for compound 17a, a solution of 1 (80 mg, 0.20 mmol), Fmoc-Val-
Pro-Val-Ala-OH 16c (152 mg, 0.25 mmol), PPh3 (79 mg, 0.30 mmol),
and DBAD (70 mg, 0.30 mmol) in dry DMF (3 mL) under argon at-
mosphere was irradiated at 908C for 6 h. Additional amounts of
tetrapeptide (0.12 mmol), PPh3 (0.15 mmol), and DBAD (0.15 mmol)
in DMF (0.5 mL) were then added, and the mixture was irradiated
again at 908C for 6 h. The final residue was purified by CCTLC on
the Chromatotron (CH2Cl2/MeOH, 20:1) to obtain 117 mg of 17c
(59%) as a white foam: MS (ESI+): m/z 988.5 [M+H]+; Anal. calcd
for C55H66N6O11: C 66.92, H 6.74, N 8.51; found: C 67.10, H 6.91, N
8.66.
3-[2’-Deoxy-5’-O-(valylprolylvalylalanyl)-b-d-ribofuranosyl]-6-(p-
pentylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one (3c): A so-
lution of compound 17c (82 mg, 0.083 mmol) in DMF (3 mL) was
reacted with piperidine (0.15 mL), according to the deprotection
method described for compound 17a. The final residue was puri-
fied by CCTLC on the Chromatotron (CH2Cl2/MeOH, 10:1) to afford
61 mg of 3c (97%) as
a
white foam: 1H NMR (400 MHz,
[D6]acetone): 1H NMR (400 MHz, [D6]acetone): d=0.79–1.00 (m,
15H, 4g-CH3, Val1 and Val2, CH3), 1.28–1.44 (m, 7H, 2CH2, b-CH3,
Ala,), 1.60–1.68 (m, 2H, CH2), 1.86–2.00 (m, 5H, b-CH, Val, b-CH2,
Pro, g-CH2, Pro), 2.19–2.31 (m, 2H, b-CH, Val, H-2’a), 2.62–2.67 (m,
3H, H-2’b, CH2), 3.52–3.84 (m, 2H, g-CH, Pro), 3.99 (d, 1H, H-3’, J=
8.5 Hz), 4.29–4.56 (m, 7H, 2a-CH, Val1 and Val2, a-CH, Ala, a-CH,
Pro, 2H-5’, H-4’), 6.29–6.33 (m, 1H, H-1’), 7.18 (s, 1H, H-5), 7.33 and
7.76 (AA’BB’ system, 4H, Ar, J=8.2 Hz), 8.66 ppm (s, 1H, H-4);
13C NMR (100 MHz, [D6]acetone): d=14.7 (CH3), 17.7, 17.9, 18.5,
20.1, (C-b, Ala, 4C-g, Val1 and Val2), 23.5 (CH2), 26.2 (C-g, Pro), 29.1
(C-b, Pro), 32.0, 32.2, 32.5, 32.8 (2CH2, 2C-b, Val1 and Val2), 36.7
(CH2), 42.7 (C-2’), 48.3 (C-d, Pro), 49.6, 58.6, 58.9, 61.5 (4C-a, Ala,
Val1, Val2, Pro), 65.3 (C-5’), 71.7 (C-3’), 86.4 (C-4’), 89.5 (C-1’), 100.0
(C-5), 108.9 (C-4a), 126.0 (C-Hb), 127.7 (ipso-C), 130.3 (C-Ha), 138.2
(C-4), 145.7, (para-C), 155.4, 155.8 (C-6, C-2), 172.4, 172.6, 173.1,
173.6 ppm (C=O, Val1, C=O, Val2, C=O, Ala, C=O, Pro, C-7a); EMAR
(ESI+) for C40H56N6O9 [M+H]+; Calcd: 764.4182, found: 765.4189;
Anal. calcd for C40H56N6O9: C 62.81, H 7.38, N 10.99; found: C 62.95,
H 7.31, N 10.86.
3-[2’-Deoxy-5’-O-[N-(fluorenylmethoxycarbonyl)valylprolylvalyl-
valyl]-b-d-ribofuranosyl]-6-(p-pentylphenyl)-2,3-dihydrofuro[2,3-
d]pyrimidin-2-one (17b): Following the procedure described for
compound 17a, a solution of 1 (100 mg, 0.25 mmol), Fmoc-Val-
Pro-Val-Val-OH (16b; 199 mg, 0.314 mmol), PPh3 (99 mg,
0.376 mmol), and DBAD (88 mg, 0.376 mmol) in dry DMF (3 mL)
under argon atmosphere was irradiated at 908C for 10 h. The final
residue was purified by CCTLC on the Chromatotron (CH2Cl2/
MeOH, 25:1) to obtain 163 mg of 17b (64%) as a white foam: MS
(ESI+): m/z 1016.23 [M+H]+; Anal. calcd for C57H70N6O11: C 67.44, H
6.95, N 8.28; found: C 67.25, H 7.11, N 8.36.
3-[2’-Deoxy-5’-O-(valylprolylvalylvalyl)-b-d-ribofuranosyl]-6-(p-
pentylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one (3b): A so-
lution of compound 17b (143 mg, 0.181 mmol) in DMF (4 mL) was
reacted with piperidine (0.2 mL), according to the deprotection
method described for compound 17a. The final residue was puri-
fied by CCTLC on the Chromatotron (CH2Cl2/MeOH, 10:1) to afford
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 reversed-
phase column (flow rate: 1 mLminÀ1; detection: UV 254 nm; gradi-
ent solvent system A/B (CH3CN/H2O): initial 15% A + 85% B, 5 min
linear gradient to 25% A + 75% B, 5 min linear gradient to 35% A
+ 65% B, 10 min linear gradient to 45% A + 55% B, 5 min linear
gradient to 60% A + 40% B, and 5 min linear gradient to 100%
A). Excess amounts of the prodrug or of the parent drug were sus-
pended in deionized water (pH 5.5), sonicated for 10 min at room
temperature, then equilibrated overnight at room temperature.
The samples were centrifuged at 14000 rpm in an Eppendorf mi-
crocentrifuge for 1.5 min at room temperature. 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 com-
94 mg of 3b (84%) as
a
white foam: 1H NMR (400 MHz,
[D6]acetone): d=0.87–0.99 (m, 18H, 3g-CH3, Val1, Val2 and Val3,
CH3), 1.28–1.35 (m, 4H, 2CH2), 1.62–1.66 (m, 2H, CH2), 1.86–1.97 (m,
3H, g-CH2, Pro, b-CHa, Pro), 2.11–2.35 (m, 5H, 3b-CH, Val1, Val2 and
Val3, b-CHb, Pro, H-2’a), 2.62–2.67 (m, 3H, H-2’b, CH2), 3.50–3.82 (m,
4H, d-CH2, Pro), 3.96 (d, 1H, a-CH, Val1, J=8.6 Hz), 4.33–4.50 (m,
5H, a-CH, Val1, 2H-5’, H-4’, H-3’) 4.52–4.59 (m, 2H, a-CH, Pro, a-CH,
Val), 6.33 (t, 1H, H-1’, J=6.2 Hz), 7.23 (s, 1H, H-5), 7.34 and 7.77
(AA’BB’ system, 4H, Ar, J=8.2 Hz), 8.69 ppm (s, 1H, H-4); 13C NMR
(100 MHz, [D6]DMSO): d=15.0 (CH3), 18.9, 19.6, 19.9, 20.0, 20.4,
20.7 (6C-g, Val1, Val2 and Val3), 23.8 (CH2), 26.5 (C-g, Pro), 29.2 (2C-b,
Pro), 30.7, 30.9, 31.7 (3C-b, Val1, Val2 and Val3), 32.5, 32.8 (2CH2),
37.0 (CH2), 42.9 (C-2’), 48.5 (2C-d, Pro), 59.3, 59.7 (2C-a, Val2 and
Val3), 61.8 (C-a, Pro), 65.6 (C-5’), 72.0 (C-3’), 72.2 (C-a, Val1), 86.6 (C-
4’), 89.8 (C-1’), 100.3 (C-5), 109.1 (C-4a), 126.3 (C-Hb), 128.0 (ipso-C),
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S. Velꢂzquez et al.
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pared with the sample dissolved in DMSO as the standard, the
exact concentration of which is known.
0214-2006 and BIPEDD-2-CM ref. S-2010/BMD-2457), and the KU
Leuven (GOA No. 10/014) for financial support.
Keywords: CD26
prodrugs · self-cleavage spacers
· dipeptidyl peptidase IV · peptides ·
Biological methods
Compounds and enzymes: Soluble human CD26 was purified as de-
scribed[26] and kindly provided by I. De Meester and A.-M. Lambeir
(Antwerp, Belgium) or obtained from Sigma–Aldrich (St. Louis, MO,
USA). Fetal bovine serum (FBS) was obtained from Integro (Dieren,
The Netherlands), and human serum was provided by the Blood
Bank, Leuven, Belgium.
[1] D. A. Fox, R. E. Hussey, K. A. Fitzgerald, O. Acuto, C. Poole, L. Palley, J. F.
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1256.
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Conversion of Cf1743 prodrugs to the corresponding parent com-
pound: Test compounds were evaluated for their substrate activity
against purified CD26, human serum (HS), and bovine serum (BS)
in Eppendorf tubes. The 400 mL reaction mixtures contained 50 mm
test compound (prodrugs of Cf1743) in PBS containing 0.1%
DMSO. The reaction was initiated by the addition of purified CD26
(1.5 mU), 20% HS (in PBS), or 20% BS (in PBS) at 378C. At different
time points (as indicated in the figures) 100 mL was withdrawn
from the reaction mixture, added to 200 mL of cold MeOH, and in-
cubated on ice for 10 min. The mixtures were then centrifuged at
13000 rpm for 5 min at 48C, and 250 mL supernatant was analyzed
by HPLC on a reversed-phase RP-8 column with fluorescence de-
tection (excitation l: 340 nm; emission l: 415 nm), using the fol-
lowing buffers and gradients: buffer A: 50 mm NaH2PO4 +5 mm
heptanesulfonic acid, pH 3.2; buffer B: CH3CN; gradient: 90% A
+10% B, 12 min linear gradient to 75% A + 25% B, 13 min linear
gradient to 65% A + 35% B, 8 min linear gradient to 60% A +
40% B, 7 min linear gradient to 50% A + 50% B, 5 min 90% A +
10% B, 5 min equilibration at 90% A + 10% B.
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Vandamme, M. J. Camarasa, E. De Clercq, Proc. Natl. Acad. Sci. USA
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Exposure of Caco-2 cell cultures to Cf1743 and Val-Pro-Val-Val pro-
drug 3b: Human colon carcinoma Caco-2 cells were seeded in the
upper cups of dual-chamber 24-well trays at 1.5ꢄ105 cells per well
in 500 mL of DMEM containing 10% fetal bovine serum. The
bottom cups contained the same medium without cells. After the
cultures in the upper cups became confluent (3 days after seed-
ing), the medium was carefully aspirated and replaced by 800 mL
of DMEM containing either Cf1743 or the 5’-Val-Pro-Val-Val prodrug
at 100 mm. In the bottom wells, the medium was replaced by PBS.
At different time points (i.e., 0, 2, 4, 6, 8, and 24 h), 50 mL superna-
tant was removed from both the upper and bottom cups and
added to 100 mL of cold MeOH. After centrifugation, the drugs and
drug metabolites in the methanolic supernatants were quantified
by HPLC analysis as described above.
[14] a) C. McGuigan, C. J. Yarnold, G. Jones, S. Velꢂzquez, H. Barucki, A. Bran-
cale, G. Andrei, R. Snoeck, E. De Clercq, J. Balzarini, J. Med. Chem. 1999,
42, 4479–4484; b) C. McGuigan, H. Barucki, S. Blewet, A. Carangio, J. T.
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2000, 43, 4993–4997.
[15] a) J. Balzarini, C. McGuigan, Biochim. Biophys. Acta Mol. Basis Dis. 2002,
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149–153.
[16] For reviews of cyclization-activated prodrugs, see: a) P. Gomes, N. Vale,
R. Moreira, Molecules 2007, 12, 2484–2506; b) Prodrugs: Challenges and
Rewards, Part 1–2, (Eds.: V. J. Stella, R. T. Borchardt, M. J. Hageman, R.
Oliyai, H. Maag, J. W. Tilley), Springer, New York, 2007 and references
therein.
[17] For examples of prodrugs bearing these types of diamine cyclization
spacers, see: a) F. M. H. De Groot, L. W. A. Van Berkom, H. W. Scheeren, J.
Med. Chem. 2000, 43, 3093–3102; b) W. S. Saari, J. E. Schwering, P. A.
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[18] Dipeptides have been widely used as drug carriers for cyclization-acti-
vated prodrugs; for example, see: a) C. Santos, M. L. Mateus, A. P.
Santos, R. Moreira, E. Oliveira, P. Gomes, Bioorg. Med. Chem. Lett. 2005,
15, 1595–1598; b) C. Santos, J. Morais, L. Gouveia, E. De Clercq, C. Pan-
necouque, C. U. Nielsen, B. Steffansen, R. Moreira, P. Gomes, ChemMed-
Chem 2008, 3, 970–978.
Administration of compounds to mice by oral gavage: Separate
groups of female Balb/C mice received equimolar doses of Cf1743
(25 mgkgÀ1)and Val-Pro-Val-Val-Cf1743 (50 mgkgÀ1) as a single oral
gavage dose formulated in 0.5% carboxymethylcellulose. The mice
were serially sacrificed at time points ranging from 0.25 to 3 h after
dosing, and plasma samples were taken and analyzed for Cf1743
quantification by HPLC with fluorescence detection. Results are re-
ported as relative peak areas for Cf1743, which assumes that peak
area is directly proportional to drug concentration over the ranges
of the tested different concentrations.
[19] For other examples of dipeptide-based prodrugs, see: a) Y. Tsume, J. H.
Hilfinger, G. L. Amidon, Mol. Pharmaceutics 2008, 5, 717–727; b) R. Jain,
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336, 233–240 and references therein; c) R. S. Talluri, S. K. Samanta, R.
Gaudana, A. K. Mitra, Int. J. Pharm. 2008, 361, 118–124.
[20] Two-step prodrugs have also been proposed, involving an initial enzy-
matic step leading to a dipeptide-based prodrug intermediate; for ex-
ample, see: a) A. Mhaka, S. R. Denmeade, W. Yao, J. T. Isaacs, S. R. Khan,
Bioorg. Med. Chem. Lett. 2002, 12, 2459–2461; b) S. F. Brady, J. M. Paw-
luczyk, P. K. Lumma, D. M. Feng, J. M. Wai, R. Jones, D. DeFeo-Jones,
Acknowledgements
We thank Ria Van Berwaer for excellent technical assistance. We
also thank the Spanish MEC/MICINN (Project SAF2009-13914-
C02), the Comunidad de Madrid (Projects BIPEDD-CM ref. S-BIO-
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DPPIV-Activated Anti-Varicella Zoster Virus Prodrugs
MED
B. K. Wong, C. Miller-Stein, J. H. Lin, A. Oliff, R. M. Freidinger, V. M.
Garsky, J. Med. Chem. 2002, 45, 4706–4715; c) Y. Kohchi, K. Hattori, N.
Oikawa, E. Mizuguchi, Y. Isshiki, K. Aso, K. Yoshinari, H. Shirai, M. Miwa, Y.
Inagaki, M. Ura, K. Ogawa, H. Okabe, H. Ishitsuka, N. Shimma, Bioorg.
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54, 1539–1554 and references therein.
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Andrei, J. Balzarini, J. Antimicrob. Chemother. 2007, 60, 1316–1330.
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ods 1996, 189, 99–105.
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1990–1995; b) R. Keese, M. Meyer, Tetrahedron 1993, 49, 2055–2064;
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44, 2339–2346.
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70, 502–507.
Received: June 13, 2012
Published online on && &&, 0000
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FULL PAPERS
A. Diez-Torrubia, S. Cabrera, I. De Meester,
M.-J. Camarasa, J. Balzarini, S. Velꢂzquez*
Better solubility and oral bioavailabil-
ity! We describe a new type of water-
soluble prodrug of anti-varicella zoster
virus bicyclic nucleosides based on the
cyclization self-cleavage spacers that ef-
ficiently release the parent nucleoside
upon hydrolysis by DPPIV/CD26. A
marked increase in transport through
Caco-2 monolayers and in vivo oral
bioavailability is reported.
&& – &&
Dipeptidyl Peptidase IV-Activated
Prodrugs of Anti-Varicella Zoster Virus
Bicyclic Nucleoside Analogues
Containing Different Self-Cleavage
Spacer Systems
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