Dipeptidyl peptidase IV dependent water-soluble prodrugs of highly lipophilic bicyclic nucleoside analogues

ARTICLE

pubs.acs.org/jmc

Dipeptidyl Peptidase IV Dependent Water-Soluble Prodrugs

of Highly Lipophilic Bicyclic Nucleoside Analogues

Alberto Diez-Torrubia,^†Jan Balzarini,^‡Graciela Andrei,^‡Robert Snoeck,^‡Ingrid De Meester,^§

María-Josꢀe Camarasa,^†and Sonsoles Velꢀazquez*^,†

^†Instituto de Química Mꢀedica (C.S.I.C.), Juan de la Cierva 3, E-28006 Madrid, Spain

^‡Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium

^§Laboratory of Medical Biochemistry, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium

S Supporting Information

b

ABSTRACT: We present the ﬁrst report of the application of

the dipeptidyl peptidase IV (DPPIV/CD26) based prodrug

approach to hydroxy-containing drug derivatives. In particular,

we applied this strategy to the highly lipophilic antiviral drug

family of bicyclic furanopyrimidine nucleoside analogues

(BCNA) in order to improve their physicochemical and phar-

macokinetic properties. Our stability data demonstrated that

the prodrugs eﬃciently release the parent BCNA drug upon

selective conversion by puriﬁed DPPIV/CD26 and by soluble DPPIV/CD26 present in bovine, murine, and human serum.

Vildagliptin, a speciﬁc inhibitor of DPPIV/CD26, was able to completely block the hydrolysis of the prodrugs in the presence of

puriﬁed DPPIV/CD26 human, murine, and bovine serum. Several novel prodrugs showed remarkable increases in water solubility

(up to more than 3 orders of magnitude) compared to the poorly soluble parent drug. We also demonstrated a markedly enhanced

oral bioavailability of the prodrugs versus the parent drug in mice.

’ INTRODUCTION

through an amide bond, which is speciﬁcally cleaved by the

endogenous DPPIV/CD26 activity (Figure 1).

Dipeptidyl-peptidase IV (DPPIV/CD26) belongs to a unique

class of membrane-associated peptidases and is identical to the

lymphocyte surface glycoprotein CD26.¹It is endowed with an

interesting (dipeptidyl) peptidase catalytic activity, and it has

high hydrolytic selectivity for natural peptides carrying a proline,

or to a lesser extent an alanine, at the penultimate position of the

N-terminus end. Instead, it tolerates a much wider range of

amino acid residues at the N-terminal end. A free, nonsubstituted

amino group on the ultimate (amino terminal) amino acid

position is one of the prerequisites for substrate recognition by

the enzyme.^2-4The enzyme is not only expressed on a variety of

leukocyte cell subsets but also on several types of epithelial,

endothelial, and ﬁbroblast cells. A soluble form of the enzyme is

detected in plasma and cerebrospinal ﬂuid at low amounts.^5,6

In 2005, it was shown, for the ﬁrst time, that a synthetic small

molecule [GPG-NH₂(glycyl-prolyl-glycynamide)] can be con-

verted to an antiviral drug through the speciﬁc action of DPPIV/

CD26.⁷This was the ﬁrst demonstration that a diﬀerentiation/

activation leukocytic enzyme marker acts as a highly speciﬁc and

obligatory activator of a synthetic anti(retro)viral prodrug that is

otherwise inactive as such. On the basis of this study, we pre-

viously reported^8,9a novel type of prodrug approach that could

be applied to improve the solubility and formulation properties

of therapeutic agents. In our approach a di- (or oligo)peptide

moiety was linked to a free amino group of a non-peptide drug

For proof-of-concept of this novel prodrug technology, we

focused on the lipophilic TSAO derivatives^10-12that potently

inhibit HIV-1 replication. This lentivirus infects lymphocytes that

abundantly express DPPIV/CD26 in their membrane. Inparticular,

the N-3 aminopropyl TSAO-T derivative was chosen as model

compound because its primary amine functionality would allow the

formation of an amide bond between the prodrug peptide moiety

and the TSAO molecule. A variety of dipeptidyl and tetrapeptidyl

amide prodrugs of NAP-TSAO-T derivatives deprotected at the

peptide N-terminus were synthesized and studied. Our data revea-

led that puriﬁed DPPIV/CD26 could speciﬁcally recognize these

prodrugs as eﬃcient substrates to be converted to the parent com-

pound. Interestingly, it was possible to modify the enzymatic and

serum hydrolysis rate (half-life) of the prodrug conjugates by

changing the nature and the length of the peptide promoiety.^8,9

We recently extended the applicability of this prodrug ap-

proach to a variety of amine-containing drugs.¹³Our studies

demonstrated that XaaPro dipeptides linked to a free amino

group present on an aromatic ring [6-aminoquinoline (6-AQ)]

or on a sugar entity (doxorubicin) behave as prodrugs that eﬃ-

ciently release the parent drug upon conversion by puriﬁed

DPPIV/CD26 and soluble DPPIV/CD26 present in bovine

Received: December 22, 2010

Published: February 18, 2011

r

2011 American Chemical Society

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Journal of Medicinal Chemistry

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Figure 1. [(Xaa-Pro)_n]-[drug] conjugates cleavable by DPPIV/CD26.

and human serum. When the amino group is present on a hetero-

cyclic pyrimidine or purine ring (i.e., cytarabine or vidarabine),

the dipeptide derivatives are chemically unstable, whereas the

tetrapeptide derivatives were much more stable in solution and

eﬃciently converted to the parent drug by the action of DPPIV/

CD26. Thus, our studies revealed that a wide variety of drug

types containing a free amino moiety on an aromatic, carbohy-

drate, or heterocyclic pyrimidine or purine ring could be deriva-

tized with di- or tetrapeptides to become eﬃcient substrates for

the dipeptidyl peptidase activity of DPPIV/CD26.¹³Vildagliptin,

a speciﬁc inhibitor of DPPIV/CD26, was able to completely

block the hydrolysis of the prodrugs in the presence of puriﬁed

CD26 but also in human and bovine serum. Furthermore, we

could demonstrate that the 6-AQ and vidarabine peptide pro-

drugs exhibited a considerably higher water solubility than the

parent compounds.¹³

On the basis of these promising results with amino-containing

drugs, we now successfully expand for the ﬁrst time our prodrug

strategy to hydroxy-containing drug derivatives. In amine-con-

taining drugs, the peptide promoiety is attached directly to the

amino group of the drug through an amide bond that is

speciﬁcally cleaved by DPPIV/CD26 (Figure 1). In hydroxy-

containing drugs, however, this is not possible, and for the design

of these novel prodrugs a heterobifunctional connector is

required between the peptide promoiety and the hydroxyl group

of the drug. As connector in the proposed conjugates (general

formula I, Figure 2), an amino acid was chosen to conjugate via

its amine function to the acid end of the dipeptide and via its acid

function to the OH group of the drug through a metabolically

labile ester linkage. Thus, the release of the parent drug from the

designed novel prodrugs would involve a two-step mechanism:

(1) DPPIV/CD26-mediated hydrolysis to release an amino acid

ester of the drug and (2) subsequent chemical or enzymatic

cleavage of the ester bond of this intermediate resulting in the

formation of the free parent drug and the connector.

Figure 2. Design of DPPIV/CD26-cleavable prodrugs of the BCNA

compound 1 of general formula I.

both water solubility and oral bioavailability.^17,18This com-

pound is currently subject of phase II clinical trials.

In the present study, we report the application of the DPPIV/

CD26-based prodrug approach to the prototype BCNA drug 1 in

order to improve its physicochemical and pharmacokinetic pro-

perties. The design and synthesis of a broad variety of water-

soluble prodrugs of 1 (Figure 2) and evaluation of the pharma-

cokinetic properties of the prodrugs including chemical and

enzymatic stability (cleavage rates), water solubility, and oral

pharmacokinetics have been investigated. The results of our

studies on the eﬀect of the site of esteriﬁcation, the number of

peptide promoieties, and the inﬂuence of the nature of the

peptide promoiety on the rate of hydrolysis and pharmacoki-

netics of these novel prodrugs are now reported. Several of the

synthesized prodrugs combine high aqueous solubility and

stability with an eﬃcient and selective conversion of the prodrug

to the biologically active parent compound in the presence of

DPPIV/CD26. Moreover, plasma levels of 1 after oral adminis-

tration of selected prodrugs to mice proved markedly higher than

their plasma levels obtained after oral administration of the

parent drug. Therefore, the data obtained for the synthesized

prodrugs of the highly insoluble and poor bioavailable BCNA

analogue 1 may form the basis and rationale for the synthesis of

similar prodrug types of molecules that suﬀer from poor solubi-

lity and/or unfavorable pharmokinetic (i.e., poor bioavailability)

properties.

As hydroxy-containing drugs, in particular, we wanted to apply

this strategy to bicyclic furanopyrimidine nucleoside analogues

(BCNAs) which represent a family of highly lipophilic antivirals,

which display high inhibitory potency and unusual selectivity

against varicella zoster virus (VZV).^14-16The most potent anti-

VZV prototype compound is the p-pentylphenyl BCNA ana-

logue Cf1743¹⁵(1) (Figure 2), which exhibits activity against a

broad range of VZV isolates at low nanomolar concentrations

and little or no detectable toxicity at micromolar concentrations.

However, the clinical potential of this highly lipophilic com-

pound is mainly limited by its low oral bioavailability. Moreover,

this compound has a poor aqueous solubility. Formulation-

based approaches were successful in improving the solubility

of 1 (Cf1743) but did not have a signiﬁcant impact on oral

pharmacokinetics.¹⁷To overcome this problem, a prodrug

approach was successfully designed. The most promising pro-

drug that emerged from these studies proved to be the HCl salt of

the 5⁰-valyl ester, designated as FV-100, which was endowed with a

promising stability proﬁle and a signiﬁcant enhancement of

’ RESULTS AND DISCUSSION

Chemistry. Our initial efforts focused on the preparation of

the model thymidine prodrug 6 (Scheme 1) to explore the

viability of the prodrug approach in hydroxy-containing drugs. In

this model system, the dipeptide ValPro sequence (efficiently

recognized by DPPIV/CD26 in natural peptides^2,3and in

previous [XaaPro]-[drug] conjugates^8,13) is attached to the

hydroxyl group of the 5⁰-position of the nucleoside through L-

valine as the connector. ^L-Valine was first chosen as connector, as

it has been successfully used as a promoiety to enhance its

intestinal absorption by the h-PEPT1 transporter (e.g., valacy-

clovir and valgancyclovir).^19-21The synthesis of the proposed

conjugate involved a four-step procedure: (i) regioselective

acylation of the 5⁰-OH position of the nucleoside with conve-

niently protected R-Val-OH, (ii) deprotection of the amino

group of the amino acid ester intermediate of the nucleoside,

(iii) coupling of the 5⁰-Val-thymidine intermediate with the

commercially available dipeptide R-Val-Pro-OH (formation of

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Journal of Medicinal Chemistry

ARTICLE

a N-protected tripeptide conjugate), and finally (iv) N-deprotec-

tion of the tripeptide ester of the nucleoside.

5⁰-valyl ester intermediate 3 was isolated in 86% yield after

puriﬁcation by short ﬂash column chromatography. Catalytic

hydrogenation of 3 in the presence of 10% Pd/C in methanol

aﬀorded the N-deprotected valyl ester nucleoside derivative 4

in 93% yield. Coupling of 4 with Z-Val-Pro-OH in the presence

of BOP and TEA yielded the N-protected tripeptide ester

conjugate 5 in moderate yield (44%). Removal of the benzyl

groups by catalytic hydrogenation of 5 in the presence of 10%

Pd/C in methanol aﬀorded the desired model prodrug 6 in

good yields (90%).

Initial attempts of coupling of thymidine (2) with Z-Val-OH

using conventional coupling method (DCC/DMAP) gave a

mixture of 3⁰-, 5⁰-, and 3⁰, 5⁰-di-O-valyl ester thymidine deriva-

tives (data not shown). The regioselective coupling of amino

acids on the 5⁰position of a nucleoside via Mitsunobu conden-

sation^22,23was next explored. Thus, condensation of thymidine

(2) with Z-Val-OH in the presence of Ph₃P and DIAD in dry

THF at room temperature overnight under argon aﬀorded exclu-

sively the 5⁰-valyl nucleoside 3 in 49% yield. However, repeated

chromatographic puriﬁcations were required to separate the

desired material from the byproduct triphenylphosphine oxide

and the reduced azodicarboxylate. Improved procedures for

the Mitsunobu coupling reaction that facilitate puriﬁcation of the

desired products have been previously described.^24-26Among

these, the combined use of polymer-supported triphenylpho-

sphine (PS-PPh₃) and the acid-labile di-tert-butyl azodicarbox-

ylate (DBAD) allows the destruction/removal of reagent

byproduct through simple ﬁltration and evaporation after an

acid workup.²⁶By use of this combination of reagents, thymidine

was coupled with Z-Val-OH in dry THF at room temperature

overnight. The crude mixture was then treated with 4 M HCl in

dioxane for 1 h, ﬁltered, and washed with aqueous HCl. The

Next, the regioselective synthesis of the 5⁰-Val-Pro-Val pro-

drug 13 (Scheme 2) was carried out in a way similar to that

described for the model thymidine conjugate 6. Thus, reaction of

the parent drug 1, which was synthesized by the previously

described method,¹⁷with Z-Val-OH under the above Mitsunobu

mentioned conditions (PS-PPh₃and DBAD) gave the 5⁰-valyl

ester intermediate 7 in 66% yield. It is noted that acidic workup

with aqueous HCl could not be used in this case because such

acidic conditions led to partial base cleavage. Catalytic hydro-

genation of 7 (H₂, Pd/C) followed by coupling Z-Val-Pro-OH

under standard conditions (BOP and TEA) gave a mixture of the

desired tripeptide ester conjugate 8 in low yield (27%) together

with the unexpected acyclic derivative 9 (32% yield). Ring-

opening of the furan ring and subsequent reduction of the double

bond at the C-5 substituent under the catalytic hydrogenation

conditions could explain formation of compound 9. Scarce ring-

opening reactions of furanopyrimidine nucleoside derivatives

with nucleophiles have been previously described.^27-29The

absence of ﬂuorescence in the unexpected product 9 clearly

indicated that the product did not contain a bicyclic base moiety.

Its structure was unequivocally assigned on the basis of its

spectroscopic and analytical data. The ¹H NMR spectrum for 9

showed the disappearance of the characteristic signals of the

furanopyrimidine moiety (two singlets at δ 7.18 and 8.71

ppm assigned to H-4 and H-5) and the presence of two new

singlets (one broad singlet at 10.08 ppm and a narrow one at 7.20

ppm corresponding to the NH-3 and H-6) and new multiplets

between 2.5 and 2.8 ppm corresponding to the new -CH₂CH₂-

system at the C-5 substituent.

Scheme 1. Synthesis of 5⁰-Val-Pro-Val Thymidine Model

Prodrug 6^a

An alternative Fmoc strategy for the synthesis of the target

prodrug 13 is depicted in Scheme 2. Thus, coupling of 1 with

Fmoc-Val-OH under the above-mentioned optimized Mitsuno-

bu conditions followed by reaction with piperidine in DMF

^aReagents: (i) (a) Z-Val-OH, DBAD, PS-PPh₃, THF; (b) 4 M HCl in

dioxane; (ii) H₂, Pd/C, HCl (cat.), CH₃OH; (iii) Z-Val-Pro-OH, BOP,

TEA, CH₂Cl₂.

Scheme 2. Synthesis of 5⁰-Val-Pro-Val Prodrug 13^a

^aReagents: (i) Z-Val-OH or Fmoc-Val-OH, PS-PPh₃, DBAD, THF; (ii) (a) H₂, Pd(C), HCl (cat.), CH₃OH; (b) Z-Val-Pro-OH, BOP, TEA, CH₂Cl₂;

(iii) (a) piperidine 10%, DMF; (b) Fmoc-Val-Pro-OH, BOP, TEA, CH₂Cl₂; (iv) piperidine 10%, DMF.

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Scheme 3. Synthesis of 3⁰,5⁰-Val-Pro-Val and 3⁰-Val-Pro-Val

Prodrugs 17 and 22^a

Scheme 4. Synthesis of 5⁰-Val-Pro-Xaa Prodrugs 30, 32, and

34 Modiﬁed at the Connector Xaa Residue^a

^aReagents: (i) Fmoc-Val-OH, DCC, DMAP, DMF; (ii) piperidine 10%,

DMF; (iii) Fmoc-Val-Pro-OH, BOP, TEA, CH₂Cl₂; (iv) TBDMSCl,

Py; (v) Fmoc-Val-OH, DCC, DMAP, DMF; (vi) Fmoc-Val-Pro-OH,

BOP, TEA, CH₂Cl₂; (vii) (a) HCl 0.1 N, MeOH; (b) piperidine

10%, DMF.

^aReagents: (i) Fmoc-Val-Pro-OH, BOP, TEA, CH₂Cl₂; (ii) TFA,

CH₂Cl₂; (iii) Fmoc-Xaa-OH, PS-PPh₃, DBAD, THF; (iv) piperidine

10%, DMF; (v) Fmoc-Val-Pro-OH, BOP, TEA, CH₂Cl₂; (vi) Fmoc-Val-

Pro-Ala-OH (24), PS-PPh₃, DBAD, THF.

for 5 min gave the 5⁰-valyl nucleoside derivative (11)¹⁷in good

yields after chromatographic puriﬁcation. Intermediate 11 was

then reacted with Fmoc-Val-Pro-OH in the presence of BOP and

TEA to give the N-protected tripeptide ester conjugate 12 in 74%

yield. Final removal of the Fmoc group by brief treatment of 12

with piperidine aﬀorded the desired ﬁnal deprotected prodrug 13

in 89% yield. The biological results (see below) obtained with the

prodrug 13 encouraged us to prepare novel series of Cf1743

prodrugs.

A novel series of prodrugs (compounds 30, 32, and 34,

Scheme 4) was next prepared where the valine used as connector

was replaced by diﬀerent natural amino acids (Xaa) with hydro-

phobic (Ala, Leu) or aromatic residues (Phe). The synthesis of

prodrugs 30 and 32 (Scheme 4) was carried out following a four-

step procedure similar to that described for the synthesis of

prodrug 13. Thus, condensation of 1 with the appropriate Fmoc-

Xaa-OH under the above-mentioned Mitsunobu conditions (PS-

PPh₃and DBAD) provided the protected amino acid ester

intermediates 25 and 27 in 53% and 56% yield, respectively,

that were subsequently reacted with piperidine to yield the

unprotected amino acid ester intermediates 26 and 28 in

excellent yields. Coupling of intermediates 26 and 28 with

Fmoc-Val-Pro-OH in the presence of BOP and TEA followed

by removal of the Fmoc group with piperidine aﬀorded the

desired ﬁnal deprotected conjugates 30 and 32 in good yields.

For the synthesis of the corresponding conjugate 34, condensa-

To evaluate the eﬀect of the site of esteriﬁcation on the rate of

hydrolysis and pharmacokinetic properties of the prodrugs, the

synthesis of the 3⁰,5⁰-diester and 3⁰-monoester prodrugs 17 and

22 was next investigated (Scheme 3). Reaction of 1 with an

excess of Fmoc-Val-OH (3 equiv) under standard coupling

conditions (DCC/DMAP) in dry DMF aﬀorded the protected

3⁰,5⁰-di-O-valyl ester intermediate 14 in 70% yield after chroma-

tographic puriﬁcation. The Fmoc groups were deprotected by

treatment with piperidine to give the N-deprotected valyl inter-

mediate 15 in excellent yield which was ﬁnally converted into the

target 3⁰,5⁰-di-O-tripeptide ester derivative 17 (Scheme 3) in

good yield by coupling with Fmoc-Val-Pro-OH in the presence

of BOP/TEA followed by N-deprotection with piperidine. The

synthesis of the 3⁰-monoester prodrug 22 was carried out in a way

similar to that described for the corresponding 5⁰-monoester

prodrug 13 starting from the 5⁰-silyl protected derivative 18

(Scheme 3). Thus, reaction of 1 with TBDMSCl in anhydrous

pyridine at room temperature for 48 h provided the 5⁰-silyl

protected nucleoside 18 in 63% yield after ﬂash column chro-

matography. Condensation of 18 with Fmoc-Val-OH in the

presence of DCC/DMAP followed by treatment with piperidine

yielded the N-deprotected 3⁰-valyl ester intermediate 20 in good

yield. Coupling of 20 with Fmoc-Val-Pro-OH under standard

conditions (BOP/TEA) provided the tripeptide ester prodrug

21 in 80% yield. Finally, treatment of intermediate 21 with 0.1 N

HCl in MeOH (5⁰-silyl deprotection) followed by reaction with

piperidine (Fmoc removal) gave the fully deprotected 3⁰-tripep-

tide target conjugate 22 in good yields (Scheme 3).

tion of 1 with Fmoc-Ala-OH H₂O was ﬁrst attempted using

3

similar Mitsunobu conditions, but only starting material was

recovered. As an alternative, 1 was directly coupled with Fmoc-

Val-Pro-Ala-OH 24 (previously prepared using standard cou-

pling/deprotection protocols) under the optimized Mitsunobu

conditions (PS-PPh₃and DBAD). Although longer reaction

times were needed in this case (6 days), the desired protected

tripeptide prodrug 33 could be obtained in 58% yield using this

strategy. Further treatment of 33 with piperidine aﬀorded the

fully deprotected tripeptide ester derivative 34 in 74% yield.

As will be discussed below, it appears that the chemical stability of

the ester bond in the prodrugs is greatly inﬂuenced by the nature of

the amino acid used as connector. The highest chemical stability,

together with an adequate activation proﬁle of the prodrug bearing a

Val residue as the connector (compound 13) compared to the

prodrugs 30, 32, and 34 (bearing a Leu, Phe, and Ala residue,

respectively), prompted us to maintain the Val residue as connector

for the subsequent modiﬁcations of prodrugs of Cf1743.

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Scheme 5. Synthesis of 5⁰-Yaa-Pro-Val Prodrugs 42, 44, and

46 Modiﬁed at the N-Terminal Residue of the Peptide

Promoiety^a

45 in the presence of a catalytic amount of Pd(PPh₃)₄and

PhSiH₃as hydride donor (acting as allyl group scavenger) in dry

CH₂Cl₂at room temperature for 1 h, followed by brief treatment

with piperidine, aﬀorded the desired deprotected conjugate 46 in

83% yield.

The target prodrugs were characterized by ¹H and ¹³C NMR,

mass spectrometry, and microanalysis, all data conﬁrming the

structure and purity of the novel compounds.

Aqueous Solubility and Chemical Stability. Aqueous drug

solubility has long been recognized as a key molecular property in

pharmaceutical science and is important for efficient drug

delivery, transport, and biodistribution. The aqueous solubilities

of the DPPIV-dependent prodrugs of compound 1 were mea-

sured at 25 °C (Table 1). Most of the compounds were highly

stable in PBS buffer at pH 7.4 (>95% intact prodrug after 24 h at

room temperature, Table 1), and thermodynamic solubilities

have been measured after 24 h. However, the prodrugs 30, 32,

and 34 in which the valine residue connector was replaced by

other amino acids (Leu, Phe, or Ala, respectively) showed

markedly lower chemical stability (52-74% intact prodrug after

24 h in PBS) (Table 1). For this reason, their aqueous solubility

was measured after 4 h to minimize the error due to instability of

these derivatives. As shown in Table 1, all novel prodrugs showed

dramatic increases in water solubility compared to the sparingly

soluble parent drug solubility. Values ranged from 1.1 to 77 mg/

mL, which correspond to a 64- to 4.294-fold higher water solu-

bility than that of the parent drug 1. No close correlation was

observed between the water solubility values and the structure of

the prodrugs or the ClogP values. In general, prodrugs 42, 44,

and 46 containing polar N-terminal residues (capable of hydro-

gen bonding with water molecules) such as Asn, Lys, and Asp,

respectively, ranked among the most water-soluble compounds

(2.186-fold, 3.061-fold, and 838-fold solubility increases, re-

spectively). However, the 3⁰,5⁰-diester prodrug 17 bearing twice

the less polar ValProVal peptide promoiety showed the highest

increase in solubility compared with the parent drug (4.294-fold

increase) (Table 1).

Biological Studies. Anti-VZV Activity of Peptide Prodrugs in

HEL Cell Cultures. The variety of peptide prodrugs has been

evaluated for their anti-VZV activity in HEL cell cultures. The

parent compound 1 inhibited VZV-induced cytopathicity at an

EC₅₀of 0.003 μM (both YS and OKA strains) (Table 2). The 5⁰-

Val-Pro-Xaa prodrugs 30, 32, and 34 inhibited VZV within the

same order of magnitude as the parent drug (EC₅₀: 0.003-0.013

μM). Also, the 5⁰-Yaa-Pro-Val prodrugs 42, 44, and 46 showed

substantial antiviral activity as well as the 5⁰-, 3⁰-, and 3⁰,5⁰-Val-

Pro-Val prodrugs (13, 17, and 22). These findings indicate that

the peptide prodrugs of compound 1 are invariably efficiently

converted to the parent drug in the virus-infected cell cultures

(Table 2) over the course of the antiviral assays (∼5 days).

Enzymatic Stability Studies of Peptide Prodrugs. In the first

series of experiments, the 5⁰-, 3⁰-, and 3⁰,5⁰-Val-Pro-Val prodrugs

(compounds 13, 22, and 17) were exposed to purified DPPIV/

CD26, 10% human serum (HS), or 10% bovine serum (BS) for

different time periods (up to 24 h). Exposure to purified DPPIV/

CD26 revealed hydrolysis of the prodrugs to the corresponding

valine derivatives, followed by further conversion to the parent

compound. The latter observations point to a certain instability

of the valine (5⁰-, 3⁰-, or 3⁰,5⁰-) derivative of compound 1

(spontaneous conversion to parent compound). Exposure of

these compounds to HS or BS revealed the appearance of the

same type of metabolites but often a more extensive conversion

^aRegents: (i) H-Pro-O^tBu, BOP, TEA, CH₂Cl₂; (ii) TFA, CH₂Cl₂; (iii)

Fmoc-Yaa-Pro-OH, BOP, TEA, CH₂Cl₂; (iv) piperidine 10%, DMF; (v)

(a) Pd(PPh₃)₄, PhSiH₃, CH₂Cl₂; (b) piperidine 10%, DMF.

Finally, a novel series of prodrugs (compounds 42, 44, and 46,

Scheme 5) was prepared to diversify the half-life and to study the

physicochemical and pharmacokinetic properties of these pro-

drugs. In these conjugates the Val linker was maintained but the

N-terminal Val residue was changed by polar residues such as Asn

(compound 42), Lys (compound 44), and Asp (compound 46).

It is noted that the selected dipeptide sequences YaaPro were also

recognized by DPPIV/CD26 in natural peptides^2,3and in

previous [YaaPro]-[drug] conjugates⁸with diﬀerent conversion

rates to the parent compounds. The synthesis of compounds 42,

44, and 46 (Scheme 5) was carried out in a way similar to that

described for prodrug 13 starting from the appropriate dipeptide

derivatives Fmoc-Yaa-Pro-OH and the 5⁰-valyl nucleoside deri-

vative 11. Noncommercially available dipeptides Fmoc-Asn-Pro-

OH (36), Fmoc-Asp-(OAll)-Pro-OH (38), and Fmoc-Lys-

(Fmoc)-Pro-OH (40) were prepared using standard peptide

coupling/deprotection procedures. Next, the appropriate dipep-

tide derivatives Fmoc-Yaa-Pro-OH (36, 38, and 40) were reacted

with the 5⁰-valyl nucleoside derivative 11 in the presence of BOP

and TEA to give the protected intermediate conjugates 41, 43,

and 45 in good yields (72-82%). Removal of the Fmoc groups in

the presence of piperidine in DMF aﬀorded the desired ﬁnal

deprotected prodrugs 42 and 44 in good yields. For the synthesis

of the corresponding conjugate 46, simultaneous deprotection of

the aspartic allyl ester group and the base labile Fmoc was ﬁrst

attempted by using a mild palladium(0)-catalyzed allyl transfer to

various amines (such as morpholine or pyrrolidine) as the accep-

ting nucleophile.^30,31However, treatment of protected prodrug

45 in the presence of catalytic amounts of Pd(PPh₃)₄and mor-

pholine in dry THF under an argon atmosphere at room temp-

erature for 1 h provided low yields of the desired fully depro-

tected conjugate 46 because of ring-opening of the furan ring of

the bicyclic heterocyclic base of the nucleoside as determined by

HPLC-MS analysis. Among the wide variety of allyl group

scavengers that have been previously described, phenyltrihydro-

silane (PhSiH₃) was next selected as a neutral allyl group

scavenger.³²Thus, palladium catalyzed deprotection of prodrug

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Table 1. Aqueous Solubility and Chemical Stability of 5⁰-Yaa-Pro-Xaa Prodrugs and Parent Compound 1

Xaa

site of

aqueous solubility

(mg/mL)^b

fold increase

of solubility^c

chemical stability

(% remaining at 24 h)^d

compd

Yaa

(connector)

esteriﬁcation

ClogP ^a

1 (parent compound)

2.48

2

0.018

1.2

77

13

17

22

30^e

32^e

34^e

42

44

46

Val

Asn

Lys

Asp

Val

Leu

Phe

Ala

Val

5⁰

3⁰, 5⁰

3⁰

5⁰

68

4294

290

>95

69

1.77

1.97

2.12

2.53

1.63

1.06

0.41

1.3

5.2

14

766

1.1

6.1

39

64

52

337

74

2186

3061

838

>95

55

15

^aClogP values were obtained by using the ALOGPS 2.1 program (www.vcclab.org/lab/alogps/). ^bThermodynamic water solubilities measure at 25 °C

after 24 h. Values are typically the mean of two measurements, with variations of <20%. ^cFold increase in water solubility relative to the parent drug 1.

^dChemical stability in PBS buﬀer at pH 7.4 as determined by HPLC after 24 h at room temperature. ^eAqueous solubility was determined after 4 h instead

of 24 h because of the lower chemical stability of these prodrugs.

Table 2. Anti-VZV Activity of Peptide Prodrugs in HEL Cell Cultures

EC₅₀^a(μM)

compd

VZV (YS)

VZV (OKA)

CC₅₀^b(μM) (HEL)

MCC ^c(μM) (HEL)

1: parent compound

0.004

0.154

0.260

0.016

0.006

0.010

0.003

0.085

0.022

0.073

0.003

0.089

0.127

0.018

0.009

0.013

0.006

0.042

0.018

0.032

g100

>100

50

>20

>2

13: 5⁰-Val-Pro-Val prodrug

17: 3⁰,5⁰-di-Val-Pro-Val prodrug

22: 3⁰-Val-Pro-Val prodrug

30: 5⁰-Val-Pro-Leu prodrug

32: 5⁰-Val-Pro-Phe prodrug

34: 5⁰-Val-Pro-Ala prodrug

42: 5⁰-Asn-Pro-Val prodrug

44: 5⁰-Lys-Pro-Val prodrug

46: 5⁰-Asp-Pro-Val prodrug

>5

17

60

31

>0.5

>5

40

44

56

29

>5

52

>5

^a50% eﬀective concentration or compound concentration required to inhibit VZV-induced cytopathicity by 50%. ^b50% cytostatic concentration or

compound concentration required to inhibit HEL cell proliferation by 50%. ^cMinimal cytotoxic concentration or compound concentration required to

aﬀord a microscopical alteration of HEL cell morphology.

to parent compound 1 (presumably due to the presence of

hydrolytic enzymes in the sera) (Figure 3a). It is noted that in the

HPLC chromatograms of especially 3⁰,5-di-Val-Pro-Val prodrug

17 (Figure 3a), one more metabolite eluting slightly later than

the prodrug and parent compound 1 appears in function of time.

It is rather minor in quantity and may represent one or more

intermediates that may eventually appear upon conversion of the

prodrug to the parent compound. In the second series of

experiments, three 5⁰-Yaa-Pro-Val prodrug derivatives (42, 44,

and 46) were investigated (Figure 3b). Although all of them were

converted to the 5⁰-valyl intermediate and eventually to the

parent compound 1, the 5⁰-Asp-Pro-Val 46 was clearly less

efficiently hydrolyzed by DPPIV/CD26, an observation that

was most evident in the HS and BS samples. This higher stability

is in agreement with literature data and our earlier observations

that [Asp-Pro]-based prodrugs are less good substrates for CD26

than [Val-Pro]- or [Lys-Pro]-based prodrugs.^2,8In a third series

of experiments, three 5⁰-Val-Pro-Xaa prodrugs (30, 32, and 34)

were investigated (Figure 3c). These prodrugs, although less

stable in PBS than the other compounds mentioned above, were

more efficiently converted to the parent drug than the 5⁰-Yaa-

Pro-Val prodrug series.

Hydrolysis of Peptide Prodrugs of Compound 1 in the

Presence of a Specific Inhibitor of DPPIV/CD26. Compounds

6 and 13 (5⁰-Val-Pro-Val thymidine and compound 1 prodrugs)

have been subjected to conversion studies upon exposure to

purified DPPIV/CD26 or human, bovine, and murine (only for

13) serum (20% in PBS) in the presence of varying concentrations

of vildagliptin, a selective and potent DPPIV/CD26 inhibitor

(Figure 4, panels a and b). Human serum was used to enable

comparison of the DPPIV/CD26-hydrolytic activity with the other

sera included in our study and thus showed the relevance of the

obtained data in animal sera to the human situation. Bovine serum

was used to enable correlation with the activity of the compounds in

cell culture, since the cell culture medium used for the antiviral

experiments contained 10% bovine serum. Murine serum was used

to enable correlation with the In Vivo (mice) data and to ascertain

that the data obtained in murine serum (known to contain an

abundant amount of proteases) were comparable to those obtained

in the other (especially human) sera. The tripeptide prodrugs were

efficiently and time-dependently converted to the 5⁰-Val intermedi-

ates, respectively. Interestingly, vildagliptin dose-dependently in-

hibited the hydrolysis of both prodrugs 6 and 13. In fact, at 2.5 μM

inhibitor, the DPPIV/CD26-catalyzed hydrolysis of 13 was virtually

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Figure 3. Continued

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Figure 3. (a) Conversion of 5⁰-, 3⁰-, and 3⁰,5⁰-Val-Pro-Val prodrugs (13, 22, and 17) to the corresponding valyl intermediates and parent drug 1 in the

presence of puriﬁed DPP IV/CD26, bovine, and human serum (20%). The open bars (0) in (a) represent unidentiﬁed metabolite(s). (b) Conversion of

5⁰-Yaa-Pro-Val prodrugs (42, 44, and 46) to 5⁰-Val intermediate and parent drug in the presence of puriﬁed DPP IV/CD26, bovine, and human serum

(20%). (c) Conversion of 5⁰-Val-Pro-Xaa prodrugs (30, 32, and 34) to 5⁰-Xaa intermediates and parent drug in the presence of puriﬁed DPP IV/CD26,

bovine and human serum (20%).

completely blocked, not only in the presence of purified enzyme but

also in the presence of (20%) human, bovine, and murine serum.

These findings point to a highly specific conversion of these types of

prodrugs by DPPIV/CD26. It is noted that the murine serum

expressed a somewhat higher DPPIV/CD26 activity (∼2-fold after

15 min compared to human serum). This higher activity may be

related to the ∼2.5- to 5-fold higher concentration of vildagliptin

required to afford a similar inhibitory potency of CD26-catalyzed

prodrug conversion in murine versus human serum (Figure 4a).

Recently, several classes of compounds (the so-called gliptins,

including vildagliptin) have been developed as novel oral glu-

cose-lowering agents to be used to treat patients with type 2

diabetes mellitus. These drugs can be safety administered to such

patients without signiﬁcant side eﬀects.^33-35Interestingly, poor

if any drug-drug interactions have been observed for the

gliptins. Although caution should be given to combine DPPIV/

CD26 inhibitors with DPPIV/CD26-dependent prodrugs (this

combination may potentially delay prodrug cleavage to the

parent drug and/or may negatively eﬀect its impact on glucose

levels), the DPPIV/CD26-dependent prodrug approach may be

applicable to a much broader range of diseases to eventually

deliver eﬀective drugs with unfavorable PK properties when used

as its nonderivatized parental drug as such.

The hydrolytic release of the Val-Pro dipeptide in CaCo-2 cell

cultures from the 5⁰-Val-Pro-Val prodrug 13 and the eﬀect of

vildagliptin on this DPPIV/CD26-driven prodrug conversion

has also been studied. CaCo-2 cells are human colon carcinoma

cells that are generally used to study the potential oral bioavail-

ability of candidate drug compounds. It is well-known that colon

cells express high amounts of DPPIV/CD26 but also the pepT1

receptor that recognizes valine and valine-based compounds for

their internalization at the apical side and subsequent release of

valine and excretion of the parent (valine-lacking) drug at the

basolateral side. We found prodrug 13 very eﬃciently hydrolyzed

(80% of prodrug disappeared within 15 min of incubation), and

the DPPIV/CD26-driven hydrolysis was eﬃciently inhibited by

vildagliptin applied at 25 μM. Longer incubation times of the

CaCo-2 cells with prodrug and vildagliptin decreased the in-

hibitory eﬃciency of the DPPIV/CD26 inhibitor and resulted in

a time-dependent hydrolysis of the intact drug presumably due to

the overwhelming amounts of DPPIV/CD26 present on the

CaCo-2 cells (Figure 5).

Release of Compound 1 at the Baselateral Side of Prodrug

13-Exposed CaCo-2 Cell Cultures. CaCo-2 cells have also been

seeded in 24-well microtiter plates and grown until confluency.

Then prodrug 13 was added to the cell cultures (apical side) and

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Journal of Medicinal Chemistry

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Figure 4. (a) Hydrolysis of prodrug 13 in the presence of a speciﬁc inhibitor of DPP IV/CD26. (b) Hydrolysis of thymidine prodrug 6 in the presence of

a speciﬁc inhibitor of DPP IV/CD26.

the appearance of prodrug and parent compound 1 at the

basolateral side of these cell cultures was quantified. Interest-

ingly, whereas administration of 100 μM parent compound 1 to

the CaCo-2 cell cultures resulted in a limited appearance of this

drug at the baselateral side, the cells more efficiently released 1

from prodrug 13 in function of time. No significant amounts of

prodrug were found to appear at the basolateral side, pointing (i)

to the potential oral bioavailability of the prodrug and (ii) to an

efficient hydrolysis and processing/transport of the (pro)drug by

the CaCo-2 cell cultures.

In Vivo Experiments in Mice (with Selected Peptide Prodrugs).

Several peptide prodrugs of compound 1 have been examined for

their oral bioavailability compared with the parent compound 1 in

mice. Plasma levels of 1 were determined at different time points

after oral gavage of the peptide drugs. In none of the experiments,

intact prodrug could be detected in the plasma of the exposed mice.

As evident from Figure 6, parent compound 1 has a low oral

bioavailability. However, the 5⁰-Val-Pro-Val prodrug 13 showed a

markedly increased oral bioavailability. Its bioavailability (average of

three independent experiments) was higher than if the 5⁰-Val

intermediate 11 was administered to the mice (Figure 6). When

the 5⁰-Val-Pro-Val, 3⁰,5⁰-di-Val-Pro-Val, and 3⁰-Val-Pro-Val deriva-

tives 13, 17, and 22 were compared, oral bioavailability of all

prodrugs was markedly increased to a comparable extent (Table 3).

Also, the 5⁰-Val-Pro-Leu and the 5⁰-Lys-Pro-Val prodrugs 32 and 40

showed a comparable increased oral bioavailability (Table 3).

’ CONCLUSIONS

We successfully developed water-soluble prodrugs of the

highly lipophilic BCNA compound 1. In some cases, the water

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Table 3. Fold-Increased Oral Bioavailability of the Tripep-

tide Prodrugs versus Parent Compound 1

fold-increased plasma

levels of parent compound 1

within 0 and 180 min after

compd

oral gavage of the prodrug^a

13: 5⁰-Val-Pro-Val prodrug

22: 3⁰-Val-Pro-Val prodrug

17: 3⁰, 5⁰-di-Val-Pro-Val prodrug

44: 5⁰-Lys-Pro-Val prodrug

30: 5⁰-Val-Pro-Leu prodrug

1: parent compound

13

7

12

15

11

^aSeven time points (15, 30, 45, 60, 90, 120, 180 min) were taken into

consideration for each compound.

Figure 5. Appearance of parent compound 1 at the basolateral side of

CaCo-2 cell cultures upon incubation of the cells by compound 1 or the

5⁰-Val-Pro-Val 13 prodrug at the apical side of the CaCo-2 cell cultures.

with Me₄Si as internal standard. Analytical thin-layer chromatography

(TLC) was performed on silica gel 60 F₂₅₄(Merck). Separations on

silica gel were performed by flash column chromatography with silica gel

60 (230-400 mesh) (Merck) or preparative centrifugal circular thin

layer chromatography (CCTLC) on a Chromatotron (Kieselgel 60

PF₂₅₄(Merck), layer thickness of 1 mm, flow rate of 5 mL/min). Liquid

chromatography was performed using a force flow (flash chromato-

graphy) Horizon HPFG system (Biotage) with Flash 25 or 40 silica gel

cartridges. The dipeptide derivatives Cbz-Val-Pro-OH and Fmoc-Val-

Pro-OH were purchased from Bachem Feinchemikalien. Compound

1 was synthesized as previously described.¹⁷The purity of novel com-

pounds was determined to be >95% by elemental analysis.

5⁰-O-[N-(Benzyloxycarbonyl)valyl]thymidine (3). Under an

argon atmosphere, to a solution of thymidine 2 (150 mg, 0.62 mmol) in

anhydrous THF (6 mL), Cbz-Val-OH (311 mg, 1.24 mmol), polymer-

bound triphenylphosphine (PS-PPh₃) (413 mg, 1.24 mmol), and di-tert-

butyl azodicarboxylate (DBAD) (285 mg, 1.24 mmol) were added. The

reaction mixture was stirred at room temperature for 15 h. After this time,

4 M HCl solution in dioxane (4 mL) was added, and the mixture was stirred

for 1 h. The resin was filtered off, washed with ethyl acetate, and the filtrate

was evaporated to dryness under reduced pressure. The residue was

dissolved in ethyl acetate (20 mL) and washed with 4 M HCl (3 ꢀ 20

mL). The organic layer was dried (Na₂SO₄), filtered, and evaporated to

dryness. The final residue was purified by flash chromatography (CH₂Cl₂/

MeOH, 10:1) to give 255 mg of 3 (86%) as a white foam. MS (ESI^þ): m/z

476.3 (M þ 1^þ). Anal. for C₂₃H₂₉N₃O₈: C, H, N.

Figure 6. Administration of 5⁰-Val-Pro-Val (13), 5⁰-Val intermediate

(11), and parent drug to mice by oral gavage.

solubility could be boosted by more than 3 orders of magnitude

when compared to the parent drug. DPPIV/CD26 was solely

responsible for the eﬃcient hydrolysis of the dipeptide moiety

from the prodrugs, since the DPPIV/CD26-speciﬁc inhibitor

vidagliptin could completely block the enzymatic conversion in

human, bovine, and murine serum. We also demonstrated a

markedly enhanced oral bioavailability of the prodrugs versus the

parent drug in mice that were receiving the prodrugs by oral

gavage. The DPPIV/CD26-based prodrug approach may there-

fore prove useful to increase solubility and oral bioavailability of

lipophilic drugs with poor systemic absorption characteristics

exempliﬁed by the BCNA compound 1. Since compound 1

served as a prototype drug in our studies to demonstrate the

power and usefulness of this novel DPPIV/CD26-based prodrug

concept, the method may now be extended to other unrelated

drugs on the condition that they contain a free hydroxyl moiety.

5⁰-O-(Valyl)thymidine (4). To a solution of compound 3 (75 mg,

0.158 mmol) in ethyl acetate (3 mL), Pd(OH₂) (23 mg, 30% w/w) was

added. The reaction mixture was hydrogenated at 20 psi at room

temperature for 2 h. The reaction mixture was filtered, and the filtrate

was evaporated to dryness under reduced pressure. The residue was

dissolved in water and liophilized to give 50 mg of 4 (93%) as a yellow

foam. MS (ESI^þ): m/z 342.1 (M þ 1^þ), 364.1 (M þ Na^þ), 683.2 (2M

þ 1^þ). Anal. for C₁₅H₂₃N₃O₆: C, H, N.

5⁰-O-[N-(Benzyloxycarbonyl)valylprolylvalyl]thymidine

(5). To a solution of 4 (125 mg, 0.32 mmol) in CH₂Cl₂(3 mL), Cbz-

Val-Pro-OH (132 mg, 0.38 mmol), (benzotriazol-1-yl-oxy)tris(dimeth-

ylamino)phosphonium hexafluorophosphate (BOP) (168 mg, 0.38

mmol), and TEA (0.048 mL, 0.38 mmol) were successively added.

The reaction mixture was stirred at room temperature for 15 h. The

solvent was removed under reduced pressure, and the residue was

dissolved in ethyl acetate (20 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 CCTLC on

the Chromatotron (CH₂Cl₂/MeOH, 10:1) to give 97 mg of protected

’ EXPERIMENTAL SECTION

Chemical Procedures. Microanalyses were obtained with a Heraeus

CHN-O-RAPID instrument and the analytical results were within

0.4% of the theoretical values. Electrospray mass spectra were measured

on a quadropole mass spectrometer equipped with an electrospray

source (Hewlett-Packard, LC/MS HP 1100). Spectra were recorded

with a Varian Inova-300 or Varian Inova-400 spectrometers operating at

300 or at 400 MHz for ¹H NMR and at 75 or at 100 MHz for ¹³C NMR

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conjugate 5 (44%) as a yellow foam. MS (ESI^þ): m/z 672.3 (M þ 1^þ).

Anal. for C₃₃H₄₅N₅O₁₀: C, H, N.

Val₁, CdO, Val₂, CdO, Pro). MS (ESI^þ): m/z 832.3 (M þ 1^þ), 854.3

(M þ Na^þ), 1685.6 (2M þ Na^þ). Anal. for C₄₅H₆₁N₅O₁₀: C, H, N.

From the slowest moving fractions, an amount of 45 mg (27%) of

compound 8 as a yellow foam was isolated. ¹H NMR (400 MHz,

acetone-d₆): δ 0.84-0.95 (m, 15H, 2γ-CH₃, Val₁and Val₂, CH₃),

1.23-1.35 (m, 4H, CH₂), 1.62 (m, 2H, CH₂), 1.92-2.17 (m, 6H, β-

CH, Val₁and Val₂, β-CH₂, Pro, γ-CH₂, Pro), 2,34 (m, 1H, H-2⁰a),

2.58-2.66 (m, 3H, H-2⁰b, CH₂), 3.65-3.82 (m, 2H, δ-CH₂, Pro),

4.23-4.52 (m, 6H, 2H-5⁰, H-4⁰, H-3⁰, R-CH, Val₁, OH), 4.57 (m, 1H, R-

CH, Val₂), 4.68 (m, 1H, R-CH, Pro), 5.04 and 5.08 (AB system, 2H,

CH₂, Cbz, J = 12.6 Hz), 6.18 (d, 1H, NH, Val₁, J = 8.2 Hz), 6.27 (t, 1H,

H-1⁰, J = 6.0 Hz), 7.18 (s, 1H, H-5), 7.30-7.37 (m, 7H, Ar, Cbz), 7.23

(d, 1H, NH, Val₂, J = 7.5 Hz), 7.79 (AA⁰BB⁰system, 2H, Ar, J = 8.2 Hz),

8.71 (s, 1H, H-4). ¹³C NMR (100 MHz, acetone-d₆): δ 13.6 (CH₃),

17.6, 18.1, 18.7, 19.1 (4C-γ, Val₁and Val₂), 22.5 (CH₂), 25.1 (C-γ, Pro),

28.5, 30.8, 31.0, 31.2 (3CH₂, C-β, Pro), 31.5, 35.6 (2C-β, Val₁and Val₂),

41.8 (C-2⁰), 47.7 (C-δ, Pro), 58.1, 58.6 (C-R, Val₂, C-R, Pro), 59.9 (C-

R, Val₁), 64.1 (C-5⁰), 66.1 (CH₂, Cbz), 70.5 (C-3⁰), 85.3 (C-4⁰), 88.5

(C-1⁰), 99.0 (C-5), 107.5 (C-4a), 125.0, 126.8, 127.9, 128.0, 128.1 128.6

(4C-Ar, Cbz, C-Hb, ipso-C), 129.2 (C-Ha), 137.1 (C-4), 144.6 (para-

C), 154.4, 154.8 (C-6, CdO, Cbz), 156.5 (C-2), 171.5, 171.7, 172.0,

172.2 (CdO, Val₁, CdO, Val₂, CdO, Pro, C-7a). MS (ESI^þ): m/z

828.3 (M þ 1^þ), 850.3 (M þ Na^þ), 1677.6 (2M þ Na^þ). Anal. for

C₄₅H₅₇N₅O₁₀: C, H, N.

3-[2⁰-Deoxy-5⁰-O-[N-(fluorenylmethoxycarbonyl)valyl]-β-

D-ribofuranosyl]-6-(p-pentylphenyl)-2,3-dihydrofuro[2,3-d]-

pyrimidin-2-one (10). According to the procedure described for

compound 7, a solution of compound 1 (100 mg, 0.25 mmol) in an-

hydrous THF (3 mL), under an argon atmosphere, was reacted with

Fmoc-Val-OH (170 mg, 0.5 mmol) in the presence of PS-PPh₃(184 mg,

0.55 mmol) and DBAD (118 mg, 0.5 mmol). The final residue was puri-

fied by CCTLC on the Chromatotron (CH₂Cl₂/MeOH, 40:1) to give

110 mg of compound 10 (61%) as a white foam. MS (ESI^þ): m/z 720.2

(Mþ1^þ), 742.2 (MþNa^þ), 1439.0 (2M þ 1^þ). Anal. for C₄₂H₄₅N₃O₈:

C, H, N.

5⁰-O-(Valylprolylvalyl)thymidine (6). According to the depro-

tection procedure described for compound 4, protected conjugate 5 was

hydrogenated to give 41 mg of 6 (90%) as a yellow foam. ¹H NMR (300

MHz, acetone-d₆): δ 0.83-0.97 (m, 12H, 2γ-CH₃, Val₁and Val₂), 1.84

(s, 3H, CH₃-5), 1.87-2.12 (m, 5H, β-CH, Val₁, β-CH₂, Pro, γ-CH₂,

Pro), 2.21-2.32 (m, 3H, H-2⁰, β-CH, Val₂), 3.47-3.77 (m, 2H, δ-CH₂,

Pro), 3.94 (d, 1H, R-CH, Val₁, J = 8.3 Hz), 4.07-4.38 (m, 4H, H-5⁰,

H-4⁰, R-CH, Val₂), 4.48 (m, 1H, H-3⁰), 4.57 (m, 1H, R-CH, Pro), 6.29

(t, 1H, H-1⁰, J = 6.7 Hz), 7.47 (s, 1H, H-6). ¹³C NMR (100 MHz,

acetone-d₆): δ 11.9 (C-C₅), 17.7, 18.6, 18.8, 19.3 (4C-γ, Val₁and

Val₂), 25.1 (C-γ, Pro), 27.5 (C-β, Pro), 31.1, 31.7 (2C-β, Val₁and Val₂),

39.4 (C-2⁰), 47.2 (C-δ, Pro), 58.0 (C-R, Val₂), 60.1 (C-R, Pro), 64.5 (C-

R, Val₁), 70.5 (C-3⁰), 71.4 (C-5⁰), 84.4, 85.2 (C-1⁰, C-4⁰), 110.5 (C-5),

136.0 (C-6), 150.6 (C-2), 163.5 (C-4), 171.4, 171.8, 171.9 (CdO, Val₁,

CdO, Val₂, CdO, Pro). MS (ESI^þ): m/z 538.3 (M þ 1^þ). Anal. for

C₂₅H₃₉N₅O₈: C, H, N.

3-[5⁰-O-[N-(Benzyloxycarbonyl)valyl]-2⁰-deoxy-β-D-ribo-

furanosyl]-6-(p-pentylphenyl)-2,3-dihydrofuro[2,3-d]pyri-

midin-2-one (7). Following the procedure described for thymidine

derivative 3, compound 1 (100 mg, 0.25 mmol) was reacted with Z-Val-

OH (125 mg, 0.5 mmol), PS-PPh₃(183 mg, 0.55 mmol) and DBAD

(116 mg, 0.5 mmol). The resin was filtered off, washed with ethyl

acetate, and the filtrate was evaporated to dryness under reduced

pressure. The residue was dissolved in ethyl acetate (20 mL) and washed

with brine (1 ꢀ 20 mL). The organic layer was dried (Na₂SO₄), filtered,

and evaporated to dryness. The final residue was purified by flash

chromatography (CH₂Cl₂/MeOH, 20:1) to give 105 mg of the valyl

derivative 7 (66%) as a white foam. MS (ESI^þ): m/z 632.3 (M þ 1^þ).

Anal. for C₃₅H₄₁N₃O₈: C, H, N.

3-[5⁰-O-[N-(Benzyloxycarbonyl)valylprolylvalyl]-2⁰-deoxy-

β-^D-ribofuranosyl]-6-(p-pentylphenyl)-2,3-dihydrofuro[2,3-

d]pyrimidin-2-one and 5⁰-O-[N-(Benzyloxycarbonyl)valyl-

prolylvalyl]-2⁰-deoxy-5-[2-(p-pentylphenyl)ethyl]uridine (8

and 9). A solution of 7 (125 mg, 0.2 mmol) in ethyl acetate (3 mL)

was hydrogenated at 20 psi at room temperature in the presence of

Pd(OH₂) (38 mg, 30% w/w) for 2 h. After this time, TLC showed the

disappearance of starting material and the formation of a mixture of two

compounds. The catalyst was filtered off, and the filtrate was evaporated

to dryness under reduced pressure. A solution of the residue in CH₂Cl₂

(3 mL) was treated with Cbz-Val-Pro-OH (84 mg, 0.24 mmol), BOP

(106 mg, 0.24 mmol), and TEA (0.033 mL, 0.24 mmol) for 15 h

following the coupling procedure described for thymidine derivative 5.

The final residue was purified by CCTLC on the Chromatotron

(CH₂Cl₂/MeOH, 20:1). The fastest moving fractions afforded 53 mg

of the unexpected acyclic derivative 9 (32%) as a yellow foam. ¹H NMR

(400 MHz, acetone-d₆): δ 0.85-1.00 (m, 15H, 2γ-CH₃, Val₁and Val₂,

CH₃), 1.27-1.39 (m, 4H, CH₂), 1.57 (m, 2H, CH₂), 1.89-2.14 (m, 7H,

2β-CH, Val₁and Val₂, β-CH₂, Pro, γ-CH₂, Pro, H-2⁰a), 2,20 (m, 1H,

H-2⁰b), 2.53-2.65 (m, 4H, CH₂, CH₂-Ph), 2.76 (m, 2H, CH₂-5), 3.61-

3.83 (m, 2H, δ-CH₂, Pro), 4.02- 4.38 (m, 6H, 2H-5⁰, H-4⁰, H-3⁰, 2R-CH,

Val₁and Val₂, OH), 4.56 (m, 1H, R-CH, Pro), 5.03 and 5.07 (AB

system, 2H, CH₂, Cbz, J = 12.5 Hz), 6.23 (t, 1H, H-1⁰, J = 6.2 Hz), 6.32

(d, 1H, NH, Val₁, J = 8.8 Hz), 7.09-7.35 (m, 9H, Ar, Ar, Cbz), 7.20 (s,

3-(2⁰-Deoxy-5⁰-O-valyl-β-D-ribofuranosyl)-6-(p-pentyl-

phenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one (11). To a

solution of 10 (143 mg, 0.2 mmol) in DMF (4 mL) piperidine (0.2

mL, 2.0 mmol) was added. The mixture was stirred at room temperature

for 5 min and evaporated to dryness under reduced pressure. The

residue was purified by CCTLC on the Chromatotron (CH₂Cl₂/

1

MeOH, 20:1) to give 88 mg of 11 (89%) as a yellow foam. H NMR

(300 MHz, acetone-d₆): δ 0.88-0.95 (m, 9H, γ-CH₃, Val, CH₃), 1.31-

1.36 (m, 4H, CH₂), 1.59-1.67 (m, 2H, CH₂), 1.84 (m, 1H, β-CH, Val),

2.25 (m, 1H, H-2⁰a), 2.55-2.72 (m, 3H, CH₂, H-2⁰b), 3.94 (d, 1H, R-

CH, Val, J = 6.8 Hz), 4.26- 4.59 (m, 5H, 2H-5⁰, H-4⁰, H-3⁰, OH), 6.39 (t,

1H, H-1⁰, J = 6.4 Hz), 7.02 (s, 1H, H-5), 7.34 and 7.73 (AA⁰BB⁰system,

2H, Ar, J = 8.5 Hz), 8.74 (s, 1H, H-4). ¹³C NMR (100 MHz, acetone-

d₆): δ 13.9 (CH₃), 17.3, 19.2 (2C-γ, Val), 22.8 (CH₂), 30.2, 31.4, 32.6

(3CH₂), 35.9 (C-β, Val), 41.9 (C-2⁰), 60.6 (C-R, Val), 64.0 (C-5⁰), 70.2

(C-3⁰), 85.9 (C-4⁰), 88.5 (C-1⁰), 98.6 (C-5), 107.9 (C-4a), 125.2 (C-

Hb), 126.9 (ipso-C), 129.6 (C-Ha), 137.4 (C-4), 145.2 (para-C), 154.7

(C-6), 155.3 (C-2), 171.8 (C-7a), 175.7 (CdO, Val). MS (ESI^þ): m/z

498.3 (M þ 1^þ), 995.5 (2M þ 1^þ). Anal. for C C₂₇H₃₅N₃O₆: C, H, N.

3-[2⁰-Deoxy-5⁰-O-[N-(fluorenylmethoxycarbonyl)valyl-

prolylvalyl]-β-^D-ribofuranosy]-6-(p-pentylphenyl)-2,3-di-

hydrofuro[2,3-d]pyrimidin-2-one (12). A solution of compound

11 (95 mg, 0.19 mmol) in CH₂Cl₂(5 mL) was reacted with Fmoc-Val-

Pro-OH (100 mg, 0.23 mmol), BOP (101 mg, 0.23 mmol), and TEA

(0.032 mL, 0.23 mmol) according to the coupling procedure described

for compound 5. The final residue was purified by CCTLC on the

Chromatotron (CH₂Cl₂/MeOH, 25:1) to give 130 mg of 12 (74%) as a

white foam. MS (ESI^þ): m/z 916.3 (M þ 1^þ), 938.0 (M þ Na^þ). Anal.

for C₅₂H₆₁N₅O₁₀: C, H, N.

1H, H-6), 7.64 (d, 1H, NH, Val₂, J = 8.2 Hz), 10.08 (bs, 1H, NH-3). ¹³

C

NMR (100 MHz, acetone-d₆): δ 13.7 (CH₃), 17.7, 17.8, 18.8, 19.1 (4C-γ,

Val₁and Val₂), 22.5 (CH₂), 25.1 (C-γ, Pro), 28.2, 28.6, 29.8, 31.0, 31.5,

31.6 (3CH₂, C-β, Pro, C-C-₅, C-ipso-C), 34.3, 35.4 (2C-β, Val₁and

Val₂), 39.4 (C-2⁰), 47.7 (C-δ, Pro), 58.0, 58.1 (C-R, Val₂, C-R, Pro),

59.9 (C-R, Val₁), 64.6 (C-5⁰), 66.1 (CH₂, Z), 71.5 (C-3⁰), 84.4 (C-4⁰),

85.3 (C-1⁰), 113.8 (C-5), 127.9, 128.0, 128.5, 128.6, 128.7, 128.9 (4C-

Ar, Cbz, C-Ha, C-Hb), 136.6 (C-6), 138.9, 140.4 (ipso-C, para-C),

150.5 (C-2), 156.5 (CdO, Z), 163.2 (C-4), 171.4, 171.6, 171.7 (CdO,

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3-[2⁰-Deoxy-5⁰-O-(valyl-prolyl-valyl)-β-D-ribofuranosyl]-

6-(p-penthylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-

one (13). Following the deprotection procedure described for 11, a

solution of compound 12 (120 mg, 0.13 mmol) in DMF (3 mL) was

treated with piperidine (0.15 mL, 1.5 mmol). The final residue was

purified by CCTLC on the Chromatotron (ethyl acetate/MeOH, 10:1)

to give 81 mg of 13 (89%) as a white solid after trituration with Et₂O.

NMR (300 MHz, acetone-d₆): δ 0.88-1.02 [m, 27H, 4γ-CH₃, (Val₁,

Val₂)-3⁰, (Val₁, Val₂)-5⁰, CH₃], 1.29-1.40 (m, 4H, 2CH₂), 1.65 (m, 2H,

CH₂), 1.72-2.57 [m, 14H, 2H-2⁰, 4β-CH, (Val₁, Val₂)-3⁰, (Val₁, Val₂)-

5⁰, 2β-CH₂, Pro-3⁰, Pro-5⁰, 2γ-CH₂, Pro-3⁰, Pro-5⁰], 2.67 (t, 2H, CH₂, J =

7.7 Hz), 3.60-4.58 (m, 13H, 2H-5⁰, H-4⁰, 4R-CH, (Val₁, Val₂)-3⁰, (Val₁,

Val₂)-5⁰, 2R-CH Pro-3⁰, Pro-5⁰, 2δ-CH₂, Pro-3⁰, Pro-5⁰), 5.45 (m,

1H, H-3⁰), 6.35 (m, 1H, H-1⁰), 7.20 (s, 1H, H-5), 7.34 and 7.70 (AA⁰BB⁰

system, 2H, Ar, J = 8.1 Hz), 7.75-7.89 (m, 2H, 2NH Val₂-3⁰, Val₂-5⁰), 8.68

(s, 1H, H-4). MS (ESI^þ): m/z 989.5 (M þ 1^þ). Anal. for C₅₂H₇₆N₈O₁₁:

C, H, N.

3-[5⁰-O-(tert-Butyldimethylsilyl)-2⁰-deoxy-β-D-ribofura-

nosyl]-6-(p-pentylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-

2-one (18). To a solution of compound 1 (200 mg, 0.5 mmol) in

anhydrous pyridine (4 mL), TBDMSCl (302 mg, 2.0 mmol) was added.

The reaction mixture was stirred at room temperature under an argon

atmosphere for 48 h. The solvent was removed under reduced pressure.

The residue was purified by flash column chromatography on silica gel

(CH₂Cl₂/MeOH, 40:1) to give 160 mg of 18 (63%) as a white foam. MS

(ESI^þ): m/z 513.3 (M þ 1^þ), 535.3.3 (M þ Na^þ), 1025.5 (2M þ 1^þ).

Anal. for C₂₈H₄₀N₂O₅Si: C, H, N.

1

Mp: 76-79 °C. H NMR (300 MHz, acetone-d₆): δ 0.78-0.97 (m,

15H, 2γ-CH₃, Val₁and Val₂, CH₃), 1.17-1.41 (m, 4H, CH₂), 1.65 (m,

2H, CH₂), 1.83-2.35 (m, 7H, 2β-CH, Val₁and Val₂, β-CH₂, Pro, γ-

CH₂, Pro, H-2⁰a), 2.59-2.70 (m, 3H, H-2⁰b, CH₂), 3.51-3.78 (m, 2H,

δ-CH₂, Pro), 3.93 (d, 1H, R-CH, Val₁, J = 8.1 Hz), 4.32-4.58 (m, 7H,

2H-5⁰, H-4⁰, H-3⁰, R-CH, Val₂, R-CH, Pro, OH), 6.31 (t, 1H, H-1⁰, J =

5.8 Hz), 7.25 (s, 1H, H-5), 7.33 (AA⁰BB⁰system, 2H, Ar, J = 8.5 Hz),

7.75-7.86 (m, 3H, Ar, NH, Val₂), 8.71 (s, 1H, H-4). ¹³C NMR (75

MHz, acetone-d₆): δ 13.9 (CH₃), 18.4, 19.0, 19.1, 19.6 (4C-γ, Val₁and

Val₂), 22.8 (CH₂), 25.4 (C-γ, Pro), 28.2, 31.0, 31.4, 31.8 (3CH₂, C-β,

Pro), 32.5 (C-β, Val₂), 42.0 (C-2⁰), 47.6 (C-δ, Pro), 58.8 (C-R, Val₂),

60.1, 60.4 (C-R, Val₁, C-R, Pro), 64.4 (C-5⁰), 70.8 (C-3⁰), 85.6 (C-4⁰),

88.8 (C-1⁰), 99.4 (C-5), 108.2 (C-4a), 125.3 (C-Hb), 127.1 (ipso-C),

129.5 (C-Ha), 137.5 (C-4), 144.9 (para-C), 154.8 (C-6), 155.1 (C-2),

168.9, 172.0, 172.3, 172.7 (CdO, Val₁, CdO, Val₂, CdO, Pro, C-7a).

MS (ESI^þ): m/z 694.3 (M þ 1^þ), 716.5 (M þ Na^þ), 1387.7 (2M þ

1^þ), 1409.6 (2M þ Na^þ). Anal. for C₃₇H₅₁N₅O₇: C, H, N.

3-[5⁰-O-(tert-Butyldimethylsilyl)-2⁰-deoxy-3⁰-O-[N-(fluorenil-

methoxycarbonyl)valyl]-β-^D-ribofuranosyl]-6-(p-pentyl-

phenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one (19). To a

solution of 18 (170 mg, 0.33 mmol) in DMF (3 mL), DMAP (12 mg,

0.1 mmol), Fmoc-Val-OH (225 mg, 0.66 mmol) and DCC (137 mg,

0.66 mmol) were successively added at 0 °C. The reaction mixture was

stirred at room temperature for 2 h. The white solid was filtered off,

washed with DMF, and the solvent was evaporated to dryness. The

residue was dissolved in ethyl acetate (20 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 on silica gel (CH₂Cl₂/MeOH,

60:1) to give 230 mg of 19 (83%) as a white foam. MS (ESI^þ): m/z

834.3 (M þ 1^þ). Anal. for C₄₈H₅₉N₃O₈Si: C, H, N.

3-[2⁰-Deoxy-3⁰,5⁰-bis-O-[N-(fluorenilmethoxycarbonyl)-

valyl]-β-^D-ribofuranosyl]-6-(p-pentylphenyl)-2,3-dihydro-

furo[2,3-d]pyrimidin-2-one (14). To a solution of compound 1

(188 mg, 0.47 mmol) in DMF (4 mL) at 0 °C, DMAP (17 mg, 0.14

mmol), Fmoc-Val-OH (481 mg, 1.42 mmol), and DCC (292 mg, 1.42

mmol) were successively added. The reaction mixture was stirred at

room temperature for 15 h. The white solid was filtered off, washed with

DMF, and the solvent was evaporated to dryness. The filtrate was

dissolved in ethyl acetate (20 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 on silica gel (hexane/ethyl acetate, 3:1) to

give 343 mg of 14 (70%) as a white foam. MS (ESI^þ): m/z 1041.3 (M þ

1^þ). Anal. for C₆₂H₆₄N₄O₁₁: C, H, N.

3-(2⁰-Deoxy-3⁰,5⁰-bis-O-valyl-β-D-ribofuranosyl)-6-(p-pen-

tylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one (15). Following

the deprotection procedure described for compound 11, a solution of

14 (330 mg, 0.32 mmol) in DMF (12 mL) was treated with piperidine

(0.6 mL, 6.0 mmol). The final residue was purified by CCTLC on the

Chromatotron (CH₂Cl₂/MeOH, 20:1) to give 181 mg of 15 (95%) as

a yellow foam. MS (ESI^þ): m/z 597.3 (M þ 1^þ), 1193.7 (2M þ 1^þ).

Anal. for C₃₂H₄₄N₄O₇: C, H, N.

3-[2⁰-Deoxy-3⁰,5⁰-bis-O-[N-(fluorenilmethoxycarbonyl)-

valylprolylvalyl]-β-^D-ribofuranosyl]-6-(p-pentylphenyl)-2,3-

dihydrofuro[2,3-d]pyrimidin-2-one (16). Following the cou-

pling procedure described for compound 5, a solution of 15 (113 mg,

0.19 mmol) in CH₂Cl₂(5 mL) was reacted with Fmoc-Val-Pro-OH

(198 mg, 0.45 mmol), BOP (201 mg, 0.45 mmol), and TEA (0.063 mL,

0.45 mmol) at room temperature for 15 h. The final residue was purified

by CCTLC on the Chromatotron (CH₂Cl₂/MeOH, 40:1) to give 184

mg of 16 (68%) as a yellow oil. MS (ESI^þ): m/z 1434.4 (M þ 1^þ). Anal.

for C₈₂H₉₆N₈O₁₅: C, H, N.

3-(2⁰-Deoxy-3⁰,5⁰-bis-O-valylprolylvalyl-β-D-ribofuranosyl)-

6-(p-pentylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one

(17). Following the deprotection procedure described for compound

11, a solution of 16 (164 mg, 0.11 mmol) in DMF (12 mL) was reacted

with piperidine (0.25 mL, 2.5 mmol). The final residue was purified by

CCTLC on the Chromatotron (CH₂Cl₂/MeOH, 10:1) to give 98 mg of

17 (87%) as a white solid after trituration with Et₂O. Mp: 85-89 °C. ¹H

3-[5⁰-O-(tert-Butyldimethylsilyl)-2⁰-deoxy-3⁰-O-valyl-β-D-ribo-

furanosyl]-6-(p-pentylphenyl)-2,3-dihydrofuro[2,3-d]pyri-

midin-2-one (20). Following the deprotection procedure described

for 11, compound 19 (179 mg, 0.22 mmol) was reacted with piperidine

(0.25 mL, 2.5 mmol). The final residue was purified by CCTLC on the

Chromatotron (CH₂Cl₂/MeOH, 20:1) to give 122 mg of 20 (93%) as a

yellow foam. MS (ESI^þ): m/z 612.4 (M þ 1^þ), 634.3 (M þ Na^þ),

1224.7 (2M þ 1^þ). Anal. for C₃₃H₄₉N₃O₆Si: C, H, N.

3-[5⁰-O-(tert-Butyldimethylsilyl)-2⁰-deoxy-3⁰-O-[N-(fluorenil-

methoxycarbonyl)valylprolylvalyl]-β-^D-ribofuranosyl]-6-

(p-pentylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one

(21). Following the coupling procedure described for compound 5, a

solution of 20 (81 mg, 0.13 mmol) in CH₂Cl₂(2 mL) was treated with

Fmoc-Val-Pro-OH (69 mg, 0.16 mmol), BOP (70 mg, 0.16 mmol), and

TEA (0.022 mL, 0.16 mmol). The final residue was purified by CCTLC

on the Chromatotron (CH₂Cl₂/MeOH, 50:1) to give 109 mg of 21

(80%) as a white foam. MS (ESI^þ): m/z 1030.3 (M þ 1^þ), 1052.6 (M þ

Na^þ). Anal. for C₅₈H₇₅N₅O₁₀Si: C, H, N.

3-[2⁰-Deoxy-3⁰-O-(valylprolylvalyl)-β-D-ribofuranosyl]-

6-(p-pentylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one

(22). Compound 21 (109 mg, 0.11 mmol) was dissolved in MeOH

(2 mL), and a solution of 0.1 N HCl in MeOH (1.1 mL, 0.11 mmol) was

added. The reaction mixture was stirred at room temperature for 2 h, and

the solvent was evaporated to dryness. The residue was dissolved in

CH₂Cl₂(40 mL) and washed with a 10% aqueous NaHCO₃(3 ꢀ

20 mL). The organic layer was dried, filtered, and evaporated to dryness.

The residue was dissolved in DMF (2 mL). Piperidine (0.10 mL,

1.0 mmol) was added, and the mixture was stirred at room temperature

for 5 min. The solvent was removed under reduced pressure and the

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Journal of Medicinal Chemistry

ARTICLE

residue was purified by CCTLC on the Chromatotron (CH₂Cl₂/

MeOH, 15:1) to give 50 mg of 22 (62%) as a white solid after trituration

with Et₂O. Mp: 71-72 °C. ¹H NMR (300 MHz, acetone-d₆): δ 0.83-

1.02 (m, 15H, 2γ-CH₃, Val₁, Val₂, CH₃), 1.29-1.39 (m, 4H, 2CH₂),

1.65 (m, 2H, CH₂), 1.88-2.48 (m, 8H, 2β-CH, Val₁,Val₂, β-CH₂, Pro,

γ-CH₂, Pro, 2H-2⁰), 2.66 (t, 2H, CH₂, J = 7.5 Hz), 3.53-3.76 (m, 3H, R-

CH, Val₁, δ-CH₂, Pro), 3.93-4.55 (m, 6H, 2H-5⁰, H-4⁰, R-CH Val₂, R-

CH, Pro, OH), 5.43 (m, 1H, H-3⁰), 6.37 (m, 1H, H-1⁰), 7.05 (s, 1H,

H-5), 7.34 and 7.74 (AA⁰BB⁰system, 2H, Ar, J = 8.5 Hz), 7.69 (d, 1H,

NH, Val₂, J = 7.9 Hz), 8.86 (s, 1H, H-4). ¹³C NMR (100 MHz, acetone-

d₆): δ 14.0 (CH₃), 17.3, 18.3, 18.9, 19.7 (4C-γ, Val₁y Val₂), 22.0 (CH₂),

24.5 (C-γ, Pro), 28.9, 29.8, 30.4, 30.8, 31.6 (3CH₂, C-β, Pro, C-β, Val₂),

34.9 (C-β, Val₁), 38.6 (C-2⁰), 46.7 (C-δ, Pro), 57.3 (C-R, Val₂), 58.7 (C-

R, Val₁), 61.0 (C-R, Pro), 62.4 (C-5⁰), 74.4 (C-3⁰), 83.7 (C-4⁰), 87.7 (C-

1⁰), 98.7 (C-5), 107.3 (C-4a), 124.6 (C-Hb), 125.8 (ipso-C), 129.1 (C-

Ha), 137.7 (C-4), 144.2 (para-C), 153.8, 154.1 (C-6, C-2), 171.2, 172.0,

172.5, 173.7 (C-7a, CdO, Val₁and Val₂, CdO, Pro). MS (ESI^þ): m/z

916.3 (M þ 1^þ), 938.3 (M þ Na^þ). Anal. for C₃₇H₅₁N₅O₈: C, H, N.

3-[2⁰-Deoxy-5⁰-O-[N-(fluorenylmethoxycarbonyl)leucyl]-

β-^D-ribofuranosyl]-6-(p-pentylphenyl)-2,3-dihydrofuro[2,3-

d]pyrimidin-2-one (25). Following the coupling procedure de-

scribed for compound 7, compound 1 (186 mg, 0.46 mmol) was reacted

with Fmoc-Leu-OH (330 mg, 0.93 mmol), DBAD (215 mg, 0.93 mmol),

and PS-PPh₃(341 mg, 0.93 mmol) in anhydrous THF (3 mL). The final

residue was purified by CCTLC on the Chromatotron (CH₂Cl₂/MeOH,

40:1) to give 180 mg of 25 (53%) as a white foam. MS (ESI^þ): m/z

734.3 (M þ 1^þ), 1467.4 (2M þ 1^þ). Anal. for C₄₃H₄₇N₃O₈: C, H, N.

3-(2⁰-Deoxy-5⁰-O-leucyl-β-D-ribofuranosyl)-6-(p-pentyl-

phenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one (26). Following

the deprotection procedure described for compound 11, a solution of

25 (175 mg, 0.24 mmol) in DMF (5 mL) was treated with piperidine

(0.25 mL, 2.5 mmol). The final residue was purified by CCTLC on the

Chromatotron (CH₂Cl₂/MeOH, 15:1) to give 110 mg of 26 (90%) as a

yellow foam. MS (ESI^þ): m/z 512.3 (M þ 1^þ), 1023.5 (2M þ 1^þ).

Anal. for C₂₈H₃₇N₃O₆: C, H, N.

126.7 (C-Hb, ipso-C), 129.2 (C-Ha), 137.1 (C-4), 144.5 (para-C),

154.4, 154.8 (C-6, C-2), 171.4, 172.0, 172.4, 172.7 (CdO, Val, CdO,

Leu, CdO, Pro, C-7a). MS (ESI^þ): m/z 708.3 (M þ 1)^þ, 730.3 (M þ

Na)^þ, 1416.8 (2M þ 1)^þ. Anal. for C₃₈H₅₃N₅O₈: C, H, N.

3-[2⁰-Deoxy-5⁰-O-[N-(fluorenylmethoxycarbonyl)phenyl-

alanyl]-β-^D-ribofuranosyl]-6-(p-pentylphenyl)-2,3-dihydro-

furo[2,3-d]pyrimidin-2-one (27). Following the coupling proce-

dure described for compound 7, a solution of compound 1 (250 mg,

0.628 mmol) in anhydrous THF (6 mL) was reacted with Fmoc-Phe-

OH (487 mg, 1.256 mmol), DBAD (295 mg, 1.256 mmol), and PS-PPh₃

(461 mg, 1.382 mmol). The final residue was purified by flash column

chromatography on silica gel (CH₂Cl₂/MeOH, 22:1) to give 265 mg of

27 (56%) as an amorphous solid. MS (ESI^þ): m/z 768.3 (M þ 1^þ),

770.2 (M þ Na^þ). Anal. for C₄₆H₄₅N₃O₈: C, H, N.

3-(2⁰-Deoxy-5⁰-O-phenylalanyl-β-D-ribofuranosyl)-6-(p-pen-

tylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one (28). Following

the deprotection procedure described for compound 11, a solution of

27 (237 mg, 0.309 mmol) in DMF (8 mL) was treated with piperidine

(0.4 mL, 4.0 mmol). The final residue was purified by CCTLC on the

Chromatotron (CH₂Cl₂/MeOH, 15:1) to give 143 mg of 28 (85%) as a

white foam. MS (ESI^þ): m/z 546.3 (M þ 1^þ), 568.3 (M þ Na^þ),

1091.5 (2M þ 1^þ). Anal. for C₃₁H₃₅N₃O₆: C, H, N.

3-[2⁰-Deoxy-5⁰-O-[N-(fluorenylmethoxycarbonyl)valyl-

prolylphenylalanyl]-β-^D-ribofuranosy]-6-(p-penthylphenyl)-

2,3-dihydrofuro[2,3-d]pyrimidin-2-one (31). Following the

coupling procedure described for compound 5, a solution of 28 (90 mg,

0.165 mmol) in CH₂Cl₂(2 mL) was reacted with Fmoc-Val-Pro-OH

(86 mg, 0.198 mmol), BOP (88 mg, 0.198 mmol), and TEA (0.028 mL,

0.198 mmol). The final residue was purified by CCTLC on the

Chromatotron (CH₂Cl₂/MeOH, 35:1) to give 126 mg of 31 (79%) as a

white foam. MS (ESI^þ): m/z 964.3 (M þ 1^þ). Anal. for C₅₆H₆₁-

N₅O₁₀: C, H, N.

3-[2⁰-Deoxy-5⁰-O-(valylprolylphenylalanyl)-β-D-ribofu-

ranosyl]-6-(p-penthylphenyl)-2,3-dihydrofuro[2,3-d]pyri-

midin-2-one (32). Following the deprotection procedure described

for compound 11, a solution of 31 (115 mg, 0.119 mmol) in DMF (3 mL)

was reacted with piperidine (0.15 mL, 1.5 mmol). The final residue was

purified by CCTLC on the Chromatotron (CH₂Cl₂/MeOH, 20:1) to

give 79 mg of 32 (90%) as a white solid after trituration with Et₂O. Mp:

3-[2⁰-Deoxy-5⁰-O-[N-(fluorenylmethoxycarbonyl)valyl-

prolylleucyl]-β-^D-ribofuranosy]-6-(p-penthylphenyl)-2,3-

dihydrofuro[2,3-d]pyrimidin-2-one (29). Following the coupling

procedure described for compound 5, a solution of 29 (66 mg, 0.13 mmol)

in CH₂Cl₂(2 mL) was reacted with Fmoc-Val-Pro-OH (67 mg, 0.15 mmol),

BOP (68 mg, 0.15 mmol), and TEA (0.021 mL, 0.15 mmol). The final

residue was purified by CCTLC on the Chromatotron (CH₂Cl₂/MeOH,

30:1) to give 104 mg of 41 (87%) as a white foam. MS (ESI^þ): m/z 930.3

(M þ 1^þ), 952.3 (M þ Na^þ). Anal. for C₅₃H₆₃N₅O₁₀: C, H, N.

1

79-81 °C. H NMR (300 MHz, acetone-d₆): δ 0.76-0.92 (m, 9H,

γ-CH₃Val, CH₃), 1.27-1.36 (m, 4H, 2CH₂), 1.58-1.68 (m, 2H, CH₂),

1.74-2.10 (m, 5H, β-CH₂, Pro, γ-CH₂, Pro, H-2⁰a), 2.17-2.29 (m, 1H,

β-CH, Val), 2.47-2.5 (m, 1H, H-2⁰b), 2.64 (t, 1H, CH₂, J = 7.8 Hz),

3.00-3.22 (m, 2H, β-CH₂, Phe), 3.94 (d, 1H, R-CH, Val, J = 8.4 Hz),

4.01 (m, 1H, H-3⁰), 4.25-4.66 (m, 5H, H-5⁰, H-4⁰, R-CH, Pro, R-CH,

Phe), 6.28 (t, 1H, H-1⁰, J = 6.0 Hz), 7.11-7.20 (m, 5H, Ar, Phe), 7.22 (s,

1H, H-5), 7.31 (AA⁰BB⁰system, 2H, Ar, J = 8.1 Hz), 7.82-7-88 (m,

3H, Ar, NH, Phe), 8.69 (s, 1H, H-4). ¹³C NMR (75 MHz, acetone-d₆): δ

15.3 (CH₃), 20.2, 21.0 (2C-γ, Val), 21.3 (CH₂), 26.7 (C-γ, Pro), 29.1,

32.7, 33.1, 33.2 (3CH₂, C-β, Pro), 37.2 (C-β, Val), 39.1, 39.2 (C-β, Phe,

C-β, Pro), 43.4 (C-2⁰), 48.9 (C-δ, Pro), 55.7 (C-R, Phe), 56.7 (C-R

Pro), 61.8 (C-R, Val), 65.9 (C-5⁰), 72.2 (C-3⁰), 86.0 (C-4⁰), 90.2 (C-1⁰),

100.8 (C-5), 109.7 (C-4a), 126.7, 128.3, 128.6, 130.2, 130.8, 130.9,

138.4, 138.8 (5C-Ar, Phe, C-Hb, ipso-C, C-Ha), 146.1 (para-C), 156.1,

156.5 (C-6, C-2), 173.1, 173.4, 173.5, 174.0 (CdO, Val, CdO, Leu,

CdO, Pro, C-7a). MS (ESI^þ): m/z 742.3 (M þ 1^þ), 764.3 (M þ Na^þ),

1484.7 (2M þ 1^þ). Anal. for C₄₁H₅₁N₅O₈: C, H, N.

3-[2⁰-Deoxy-5⁰-O-(valylprolylleucyl)-β-D-ribofuranosyl]-

6-(p-penthylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one

(30). Following the deprotection procedure described for compound

11, to a solution of compound 29 (100 mg, 0.11 mmol) in DMF (2 mL)

piperidine (0.1 mL, 1.0 mmol) was added. The final residue was purified

by CCTLC on the Chromatotron (CH₂Cl₂/MeOH, 15:1) to give 62 mg

of 30 (82%) as a white solid after trituration with Et₂O. Mp: 78-82 °C.

¹H NMR (300 MHz, acetone-d₆): δ 0.77-0.91 (m, 15H, CH₃, γ-CH₃,

Val, δ-CH₃, Leu), 1.28-1.36 (m, 4H, 2CH₂), 1.57-1.79 (m, 5H,

β-CH₂, Leu, γ-CH, Leu, CH₂), 1.83-2.33 (m, 6H, β-CH, Val, β-CH₂,

Pro, γ-CH₂, Pro, H-2⁰a), 2.66 (m, 3H, CH₂, H-2⁰b), 3.52-3.71 (m, 2H,

δ-CH₂, Pro), 3.91 (d, 1H, R-CH, Val, J = 8.4 Hz), 4.33-4.56 (m, 6H,

H-4⁰, H-3⁰, H-5⁰, R-CH, Leu, R-CH, Pro), 6.29 (t, 1H, H-1⁰, J = 6.0 Hz),

7.18 (s, 1H, H-5), 7.33 and 7.84 (AA⁰BB⁰system, 2H, Ar, J = 8.4 Hz),

7.78 (d, 1 H, NH, Leu, J = 7.8 Hz), 8.74 (s, 1H, H-4). ¹³C NMR (75

MHz, acetone-d₆): δ 13.6 (CH₃), 18.6, 19.4 (2C-γ, Val), 21.3, 21.4, 22.5

(2C-δ, Leu, CH₂), 24.7, 25.2 (C-γ, Pro, C-γ, Leu), 28.1, 31.2, 31.5, 31.6

(4CH₂, C-β, Pro), 35.6 (C-β, Val), 40.5 (C-β, Leu), 41.8 (C-2⁰), 47.3

(C-δ, Pro), 51.5 (C-R, Leu), 59.7, 60.6 (C-R Pro, C-R, Val), 64.2 (C-5⁰),

70.5 (C-3⁰), 85.4, 88.6 (C-1⁰, C-4⁰), 99.1 (C-5), 107.8 (C-4a), 125.1,

3-[2⁰-Deoxy-5⁰-O-[N-(fluorenylmethoxycarbonyl)valyl-

prolylalanyl]-β-^D-ribofuranosy]-6-(p-penthylphenyl)-2,3-di-

hydrofuro[2,3-d]pyrimidin-2-one (33). Following the coupling

procedure described for compound 7, a solution of compound 1 (200 mg,

0.502 mmol) in anhydrous THF (5 mL) was reacted with Fmoc-Val-

Pro-Ala-OH (510 mg, 1.0 mmol), DBAD (231 mg, 1.0 mmol), and PS-

PPh₃(368 mg, 1.105 mmol) under Ar at room temperature for 6 days.

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ARTICLE

The final residue was purified by flash column chromatography on silica

gel (CH₂Cl₂/MeOH, 20:1) to give 259 mg of 33 (58%) as a yellow

foam. MS (ESI^þ): m/z 888.3 (M þ 1^þ), 910.3 (M þ Na^þ). Anal. for

C₅₀H₅₇N₅O₁₀: C, H, N.

hydrofuro[2,3-d]pyrimidin-2-one (43). Following the coupling

procedure described for compound 5, a solution of 11 (117 mg, 0.24

mmol) in CH₂Cl₂(4 mL) was reacted with Fmoc-Lys(Fmoc)-Pro-OH

(194 mg, 0.28 mmol), BOP (125 mg, 0.28 mmol), and TEA (0.039 mL,

0.28 mmol). The final residue was purified by CCTLC on the Chro-

matotron (CH₂Cl₂/MeOH, 25:1) to give 197 mg of 43 (72%) as a white

foam. MS (ESI^þ): m/z 1167.8 (M þ 1)^þ, 1189.9 (M þ Na)^þ. Anal. for

C₆₈H₇₄N₆O₁₂: C, H, N

3-[2⁰-Deoxy-5⁰-O-(valylprolylalanyl)-β-D-ribofuranosyl]-

6-(p-penthylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one

(34). Following the deprotection procedure described for compound

11, a solution of 33 (255 mg, 0.287 mmol) in DMF (6 mL) was treated

with piperidine (0.3 mL, 3.0 mmol). The final residue was purified by

CCTLC on the Chromatotron (CH₂Cl₂/MeOH, 10:1) to give 140 mg

of 34 (74%) as a white solid after trituration with Et₂O. Mp: 75-78 °C.

¹H NMR (300 MHz, acetone-d₆): δ 0.78-0.93 (m, 9H, γ-CH₃Val,

CH₃), 1.36 (m, 4H, 2CH₂), 1.38 (d, 3H, β-CH₃Ala, J = 7.1 Hz), 1.65

(m, 2H, CH₂), 1.81-1.99 (m, 4H, β-CH₂, Pro, γ-CH₂, Pro), 2,19-2.34

(m, 2H, β-CH, Val, H-2⁰a), 2.60-2.71 (m, 3H, H-2⁰b, CH₂), 3.47-3.62

(m, 2H, δ-CH₂, Pro), 3.91 (d, 1H, R-CH, Val, J = 8.3 Hz), 4.31-4.56

(m, 6H, H-5⁰, H-4⁰, H-3⁰, R-CH, Ala, R-CH, Pro), 6.28 (t, 1H, H-1⁰, J =

6.1 Hz), 7.19 (s,1H, H-5), 7.34 and 7.86 (AA⁰BB⁰system, 2H, Ar, J = 8.3

Hz), 7.79 (d, 1H, NH, Ala, J = 7.0 Hz), 8.71 (s, 1H, H-4). ¹³C NMR (100

MHz, acetone-d₆): δ 13.7 (CH₃), 16.8(C-β, Ala), 18.6, 19.3 (2C-γ, Val),

22.5 (CH₂), 25.1 (C-γ, Pro), 28.4, 31.1, 31.5, 31.6 (3CH₂, C-β, Pro),

35.6 (C-β, Val), 41.7 (C-2⁰), 47.3, 48.7 (C-δ, Pro, CH Fmoc), 58.0 (C-

R, Ala), 59.6 (C-R, Val), 60.0 (C-R, Pro), 64.2 (C-5⁰), 70.1 (CH₂,

Fmoc), 70.4 (C-3⁰), 85.4 (C-4⁰), 88.6 (C-1⁰), 99.0 (C-5), 107.9 (C-4a),

125.0 (C-Hb), 126.7 (ipso-C), 129.2 (C-Ha), 137.2 (C-4), 144.5 (para-

C), 154.5, 154.8 (C-6, C-2), 171.4, 171.9, 172.4, 172.8 (CdO, Val₁,

CdO, Val₂, CdO, Pro, C-7a). MS (ESI^þ): m/z 666.3 (M þ 1^þ), 688.3

(M þ Na^þ), 1331.6 (2M þ 1^þ). Anal. for C₃₅H₄₇N₅O₈: C, H, N.

3-[2⁰-Deoxy-5⁰-O-[N-(fluorenylmethoxycarbonyl)asparagyl-

prolylvalyl]-β-^D-ribofuranosy]-6-(p-penthylphenyl)-2,3-di-

hydrofuro[2,3-d]pyrimidin-2-one (41). Following the coupling

procedure described for compound 5, a solution of 11 (150 mg,

0.30 mmol) in CH₂Cl₂(3 mL) was reacted with Fmoc-Asn-Pro-OH

(163 mg, 0.36 mmol), BOP (160 mg, 0.36 mmol), and TEA (0.050 mL,

0.36 mmol). The final residue was purified by precipitation in Et₂O

to give 231 mg of 41 (82%) as a white solid. Mp: 93-95 °C. MS (ESI^þ):

m/z 931.5 (M þ 1^þ), 953.5 (M þ Na^þ). Anal. for C₅₁H₅₈N₆O₁₁:

C, H, N.

3-[2⁰-Deoxy-5⁰-O-(lysylprolylvalyl)-β-D-ribofuranosyl]-

6-(p-penthylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one

(44). Following the deprotection procedure described for compound 11,

to a solution of 43 (140 mg, 0.121 mmol) in DMF (6 mL) piperidine (0.3

mL, 3.0 mmol) was added. The final residue was purified by precipitation in

Et₂O togive 82 mg of44 (95%) asayellowsolid. Mp: 68-71 °C. ¹HNMR

(400 MHz, DMSO-d₆): δ 0.840.92 (m, 9H, γ-CH₃, Val, CH₃), 1.14-1.36

(m, 10H, 2CH₂, β-CH₂, Lys, γ-CH₂, Lys, δ-CH₂, Lys), 1.59 (m, 2H,

CH₂), 1.72-2.0. (m, 3H, β-CH, Val, β-CH₂, Pro), 2,22 (m, 1H, H-2⁰a),

2.32-2.45 (m, 3H, γ-CH₂, Pro, H-2⁰b), 2.61 (t, 2H, CH₂, J = 6.0 Hz), 3.15

(m, 2H, ε-CH₂, Lys), 3.41-3.61 (m, 2H, δ-CH₂, Pro), 4.02- 4.38 (m, 6H,

H-5⁰, H-4⁰, H-3⁰, R-CH, Val, R-CH, Lys), 4.51 (m, 1H, R-CH, Pro), 6.19

(t, 1H, H-1⁰, J = 6.0 Hz), 7.08 (s, 1H, H-5), 7.32 and 7.80 (AA⁰BB⁰system,

2H, Ar, J = 8.0 Hz), 8.30 (m, 1H, NH, Val), 8.67 (s, 1H, H-4). ¹³C NMR

(100 MHz, DMSO-d₆): δ 13.9 (CH₃), 18.8, 19.0 (2C-γ, Val), 21.7 (CH₂),

24.5 (C-γ, Pro), 28.6, 28.7, 29.6, 30.4, 30.7, 30.8, 31.3 (3CH₂, C-β, Pro,

C-β, Lys, C-γ, Lys, C-δ, Lys), 34.9 (C-β, Val), 40.8 (C-ε, Lys), 46.5 (C-2⁰),

50.0 (C-δ, Pro), 52.0 (C-R, Lys), 56.8 (C-R, Val), 58.7 (C-R, Pro), 63.9

(C-5⁰), 69.6 (C-3⁰), 84.5 (C-4⁰), 87.9 (C-1⁰), 98.9 (C-5), 107.2 (C-4a),

124.7 (C-Hb), 125.9 (ipso-C), 129.0 (C-Ha), 137.5 (C-4), 144.1 (para-C),

153.7, 153.9 (C-6, C-2), 171.1, 171.6, 172.5, 174.2 (CdO, Val, CdO, Lys,

CdO, Pro, C-7a). MS (ESI^þ): m/z 723.7 (M þ 1^þ), 745.5 (M þ Na^þ).

Anal. for C₃₈H₅₄N₆O₈: C, H, N.

3-[5⁰-O-[[O⁴-Allyl-N-(fluorenylmethoxycarbonyl)aspartyl]-

prolylvalyl]-2⁰-deoxy-β-D-ribofuranosy]-6-(p-penthylphenyl)-

2,3-dihydrofuro[2,3-d]pyrimidin-2-one (45). Following the

coupling procedure described for compound 5, a solution of 11 (170 mg,

0.34 mmol) in CH₂Cl₂(4 mL) was reacted with Fmoc-Asp(OAll)-Pro-

OH (181 mg, 0.34 mmol), BOP (182 mg, 0.41 mmol), and TEA (0.057 mL,

0.41 mmol). The final residue was purified by CCTLC on the Chro-

matotron (CH₂Cl₂/MeOH, 20:1) to give 259 mg of 45 (78%) as a white

foam. MS (ESI^þ): m/z 972.3 (M þ 1^þ), 994.2 (M þ Na^þ), 1943.6

(2M þ 1^þ). Anal. for C₅₄H₆₄N₅O₁₂: C, H, N.

3-[5⁰-O-(Asparagylprolylvalyl)-2⁰-deoxy-β-D-ribofuranosy]-6-

(p-penthylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one

(42). Following the deprotection procedure described for 11, to a solution

of compound 41 (197 mg, 0.211 mmol) in DMF (5 mL) piperidine

(0.25 mL, 2.5 mmol) was added. The final residue was purified by pre-

cipitation in Et₂O to give 146 mg of 42 (90%) as a white solid. Mp: 97-

99 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 0.83-0.91 (m, 9H, γ-CH₃,

Val, CH₃), 1.24-1.32 (m, 4H, 2CH₂), 1.58 (m, 2H, CH₂), 1.69-1.86

(m, 3H, β-CHa, Pro, γ-CH₂, Pro), 1.99-2.10 (m, 2H, β-CH, Val, β-

CHb, Pro), 2.17-2.46 (m, 4H, β-CH₂, Asn, H-2⁰), 2.61 (t, 2H, CH₂, J =

7.5 Hz), 3.49-3.65 (m, 2H, δ-CH₂, Pro), 4.07 (t, 1H, R-CH, Val, J=7.2Hz),

4.13-4.37 (m, 5H, H-5⁰, H-4⁰, H-3⁰, R-CH, Asn), 4.48 (m, 1H, R-CH,

Pro), 5.59 (m, 1H, OH-3⁰), 6.20 (t, 1H, H-1⁰, J = 6.0 Hz), 6.84 (bs, 1H,

NHa, Asn), 7.16 (s, 1H, H-5), 7.31 and 7.75 (AA⁰BB⁰system, 2H, Ar,

J = 8.1 Hz), 7.43 (bs, 1H, NHb, Asn), 8.25 (d, 1H, NH, Val, J = 7.2 Hz),

8.60 (s, 1H, H-4). ¹³C NMR (100 MHz, DMSO-d₆): δ 14.4 (CH₃),

19.1, 19.4 (C-γ, Val), 22.5 (CH₂), 24.8 (C-γ, Pro), 28.1, 29.4, 301, 30.9

(3CH₂, C-β, Pro), 35.4 (C-β, Val), 41.2 (C-2⁰), 46.7 (C-δ, Pro), 50.2

(C-β, Asn), 58.7 (C-R, Val), 59.4 (C-R, Pro), 64.5 (C-5⁰), 70.3 (C-3⁰),

85.1 (C-4⁰), 88.3 (C-1⁰), 99.3 (C-5), 107.8 (C-4a), 125.1, 126.4 (C-Hb,

ipso-C), 129.6 (C-Ha), 138.1 (C-4), 144.6 (para-C), 154.2, 154.4, 156.4

(C-6, C-2), 171.6, 171.9, 172.7, 173.1, 173.8 (CdO, Val, 2CdO, Asn,

CdO, Pro, C-7a). MS (ESI^þ): m/z 709.7 (M þ 1^þ), 731.5 (M þ Na^þ).

Anal. for C₃₆H₄₈N₆O₉: C, H, N.

3-[5⁰-O-(Aspartylprolylvalyl)-2⁰-deoxy-β-D-ribofuranosy]-

6-(p-penthylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one

(46). To a solution of compound 45 (135 mg, 0.140 mmol) in anhydrous

CH₂Cl₂, Pd(PPh₃)₄(16 mg, 0.014 mmol) and PhSiH₃(34 mL, 0.28 mmol)

were added. The reaction mixture was stirred at room temperature for

1 h, and the solvent was evaporated to dryness under reduced pressure.

The residue was dissolved in DMF (3 mL), and piperidine (0.15 mL,

1.5 mmol) was added. After the mixture was stirred at room temperature

for 5 min, the solvent was evaporated to dryness under reduced pressure.

The final residue was purified by precipitation in ethyl acetate to give 82

1

mg of 46 (83%) as a yellow solid. Mp: 118-122 °C. H NMR (400

MHz, DMSO-d₆): δ 0.84-0.90 (m, 9H, γ-CH₃, Val, CH₃), 1.28 (m, 4H,

2CH₂), 1.58 (m, 2H, CH₂), 1.74-1.88 (m, 3H, γ-CH₂, Pro, β-CHa,

Pro), 2.04-2.10 (m, 2H, β-CH, Val, β-CHb, Pro), 2,16-2.46 (m, 2H,

H-2⁰), 2.61 (t, 2H, CH₂, J = 7.6 Hz), 3.21-3.41 (m, 2H, β-CH₂, Asp),

3.52-3.69 (m, 2H, δ-CH₂, Pro), 3.97-4.05 (m, 2H, H-4⁰, R-CH, Asp),

4.12 (m, 1H, R-CH, Val), 4.23-4.26 (m, 3H, H-5⁰, H-3⁰), 4.48 (m, 1H,

R-CH, Pro), 6.20 (t, 1H, H-1⁰, J = 6.0 Hz), 7.17 (s, 1H, H-5), 7.34 and

7.74 (AA⁰BB⁰system, 2H, Ar, J = 8.0 Hz), 7.00 (d, 1H, NH, Val, J =

7.6 Hz), 8.59 (s, 1H, H-4). ¹³C NMR (100 MHz, DMSO-d₆): δ 13.9

(CH₃), 18.5, 19.0 (2C-γ, Val), 21.9 (CH₂), 24.4 (C-γ, Pro), 29.0, 29.7,

30.4, 30.9 (3CH₂, C-β, Pro), 34.9 (C-β, Val), 37.2 (C-β, Asp), 42.8 (C-

2⁰), 46.6 (C-δ, Pro), 49.3 (C-R, Asp), 58.0 (C-R, Val), 59.1 (C-R, Pro),

3-[2⁰-Deoxy-5⁰-O-[[N,N⁶-di(fluorenylethoxycarbonyl)lysyl]-

prolylvalyl]-β-^D-ribofuranosy]-6-(p-penthylphenyl)-2,3-di-

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Journal of Medicinal Chemistry

ARTICLE

64.0 (C-5⁰), 69.7 (C-3⁰), 84.5 (C-4⁰), 87.7 (C-1⁰), 98.7 (C-5), 107.2 (C-

4a), 124.5, 125.8 (C-Hb, ipso-C), 129.1 (C-Ha), 137.6 (C-4), 144.2

(para-C), 153.8, 154.0 (C-6, C-2), 171.1, 171.3, 171.8, 172.0 (CdO,

Val₁, CdO, Val₂, CdO, Pro, C-7a). MS (ESI^þ): m/z 710.5 (M þ 1^þ),

732.5 (M þ Na^þ), 1420.1 (2M þ 1^þ). Anal. for C₃₆H₄₇N₅O₁₀: C, H, N.

Biological Methods. Compounds and Enzymes. Human solu-

ble CD26 was purified as described and kindly provided by I. De Meester

and A.-M. Lambeir (Antwerp, Belgium) or obtained from Sigma (St.

Louis, MO). Fetal bovine serum (FBS) was obtained from Integro

(Dieren, The Netherlands) and human serum provided by the Blood

Bank, Leuven, Belgium.

Antiviral Activity Assays. The compounds were evaluated against

varicella zoster virus (VZV) strains Oka and YS. The antiviral assay was

based on inhibition of virus-induced cytopathicity (plaque formation in

human embryonic lung (HEL) fibroblasts). Confluent cell cultures in

microtiter 96-well plates were inoculated with 20 plaque forming units

(PFU) of the virus. After a 1-2 h adsorption period, residual virus was

removed, and the cell cultures were incubated in the presence of varying

concentrations of the test compounds. Viral plaque formation was

recorded as soon as it reached completion in the control virus-infected

cell cultures that were not treated with the test compounds. Antiviral

activity was expressed as the EC₅₀or concentration required to reduce

virus-induced cytopathogenicity or viral plaque formation by 50%.

Conversion of Tripeptidyl Prodrugs of Compound 1 to the Corre-

sponding Parent Compound. The test compounds have been evaluated

for their substrate activity against purified CD26, human serum (HS), and

bovine serum (BS) in Eppendorf tubes. The 400 μL reaction mixtures

contained 50 μM test compound (tripeptidyl prodrugs of compound 1) in

PBS (containing 0.1% DMSO). The reaction was started by the addition of

purified CD26 (1.5 mU) or 20% of HS (in PBS) or BS (in PBS) at 37 °C.

At different time points (as indicated in the figures) 100 μL was withdrawn

from the reaction mixture, added to 200 μL of cold methanol, and put on

ice for ∼10 min. Then the mixtures were centrifuged at 13 000 rpm for

5 min at 4 °C and 250 μL supernatant was analyzed by HPLC on a reverse

phase RP-8 column, using following buffers and gradients.

0.5% carboxymethylcellulose. The mice were serially sacrificed at time-

points ranging from 0.25 to 3 h after dosing (3 mice/time-point), and

plasma samples were taken and analyzed for compound 1 concentration

by HPLC with fluorescence detection. Results are reported as relative

peak areas for compound 1, which assumes that peak area is directly

proportional to concentration over the ranges of concentrations.

’ ASSOCIATED CONTENT

S

Supporting Information. Results from elemental anal-

b

ysis data for novel compounds 3-46; chemical procedures for

the synthesis and identiﬁcation of novel tri- and dipeptide

derivatives 23, 24, and 35-40; ¹H NMR and ¹³C NMR chemical

shifts assigments of compounds 3-5, 7, 10, 12, 14-16, 18-21,

25-29, 31, 33, 41, 43, and 45. This material is available free of

charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION

Corresponding Author

*Phone: (þ34)-912587458. Fax: (þ34)-91 5644853. E-mail:

iqmsv29@iqm.csic.es.

’ DEDICATION

This paper is dedicated to the memory of Prof. Rafael Suau who

died on November 11, 2010.

’ ACKNOWLEDGMENT

We thank Ria Van Berwaer, Lizette van Berckelaer, Lies Van

den Heurck, Steven Carmans, and Anita Camps for excellent

technical assistance. We also thank the Spanish MEC/MICINN

(Project SAF2009-13914-C02), the Comunidad de Madrid

(Project BIPEDD-CM ref S-BIO-0214-2006), and the K. U.

Leuven (GOA No. 10/014) for ﬁnancial support. We very much

appreciated the stimulating discussions on the prodrug approach

with Prof. C. McGuigan (Cardiﬀ, U.K.).

Buﬀer A: 50 mM NaH₂PO₄þ 5 mM heptanesulfonic acid, pH 3.2.

Buﬀer B: acetonitrile. Gradient A: 2 min 98% A þ 2% B; 6 min linear

gradient to 80% A þ 20% B; 2 min linear gradient to 75% A þ 25% B;

2 min linear gradient to 65% A þ 35% B; 18 min linear gradient to 50%

A þ 50% B; 5 min 50% A þ 50% B; 5 min linear gradient to 98% A þ 2%

B; 5 min equilibration at 98% A þ 2% B.

’ ABBREVIATIONS USED

Gradient B: 2 min 98% A þ 2% B; 6 min linear gradient to 80% A þ

20% B; 2 min linear gradient to 75% A þ 25% B; 2 min linear gradient

to 65% A þ 35% B; 8 min linear gradient to 50% A þ 50% B; 10 min 50%

A þ 50% B; 10 min linear gradient to 20% A þ 80% B; 5 min 20% A þ

80% B; 15 min linear gradient to 98% A þ 2% B; 5 min 98% A þ 2% B.

The gradients allowed separation of the tripeptidyl compound 1

prodrugs from the corresponding metabolites and parent compound 1.

Exposure of CaCo-2 Cell Cultures to Compound 1 and Its [Val-

Pro]-[Val] Prodrug. Human colon carcinoma CaCo-2 cells were seeded

in the upper cups of dual-chamber 24-well trays at 1.5 ꢀ 10⁵cells/well in

500 μL 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 seeding), the medium was

carefully aspirated and replaced by 800 μL of DMEM containing either

compound 1 or the 5⁰-Val-Pro-Val prodrug 13 at 100 μM. In the bottom

wells, the medium was replaced by PBS. At different time points (i.e., 0,

1, 2, 3, 4, 5, 6, 7, and 24 h), 50 μL supernatant was removed from both

the upper and bottom cup and added to 100 μL of cold methanol. After

centrifugation, the drugs and drug metabolites in the methanolic

supernatants were quantified by HPLC analysis as described above.

Administration of Compounds to Mice by Oral Gavage. Separate

groups of female mice received equimolar doses of compounds 1 (25

mg), 13, 17, 22, 30, and 44 as a single oral gavage dose formulated in

6-AQ, 6-aminoquinoline; BCNA, bicyclic nucleoside analogue;

BOP, 1-benzotriazolyloxy-tris-dimethylaminophosphonium hexa-

ﬂuorophosphate; BS, bovine serum; CCTLC, circular thin layer

chromatography; CD26, cluster of diﬀerentiation 26; DBAD, di-

tert-butyl azodicarboxylate; DCC, N,N⁰-dicyclohexylcarbodii-

mide; DIAD, diisopropyl azodicarboxylate; DMAP, 4-dimethyla-

minopyridine; DPPIV, dipeptidyl peptidase IV; FBS, fetal

bovine serum; GPG-NH₂, glycyl-prolyl-glycynamide; HEL, human

embryonic lung; hPEPT1, human intestinal oligopeptide trans-

porter 1; HS, human serum; NAP, N-3 aminopropyl; PBS, phos-

phate buﬀered saline; PS-PPh₃, polymer-bound triphenylphosphine;

TBDMSCl, tert-butyldimethylsilyl chloride; Thd, thymidine; TSAO,

(tert-butyldimethylsilyl-β-^D-ribofuranosyl)-3⁰-spiro-4⁰⁰-amino-

1⁰⁰,2⁰⁰-oxathiole 2⁰⁰,2⁰⁰-dioxide; VZV, varicella zoster virus

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Article Doi

Dipeptidyl peptidase IV dependent water-soluble prodrugs of highly lipophilic bicyclic nucleoside analogues

DOI: 10.1021/jm101624e

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