Dipeptidomimetic ketomethylene isosteres as pro-moieties for drug transport via the human intestinal di-/tripeptide transporter hPEPT1: Design, synthesis, stability, and biological investigations

Article abstract of DOI:10.1021/jm040780c

Five dipeptidomimetic-based model prodrugs containing ketomethylene amide bond replacements were synthesized from readily available α,β- unsaturated γ-ketoesters. The model drug (BnOH) was attached to the C-terminus or to one of the side chain positions of the dipeptidomimetic. The stability, the affinity for the di-/tripeptide transporter hPEPT1, and the transepithelial transport properties of the model prodrugs were investigated. ValΨ[COCH₂]-Asp(OBn) was the compound with highest chemical stability in buffers at pH 6.0 and 7.4, with half-lives of 190 and 43 h, respectively. All five compounds showed high affinity for hPEPT1 (K_i values < 1 mM), and PheΨ[COCH₂]Asp(OBn) and ValΨ[COCH ₂]Asp(OBn) had the highest affinities with K_i values of 68 and 19 μM, respectively. An hPEPT1-mediated transport component was demonstrated for the transepithelial transport of three compounds, a finding that was corroborated by hPEPT1-mediated intracellular uptake. The results indicate that the stabilized Phe-Asp and Val-Asp derivatives are promising pro-moieties in a prodrug approach targeting hPEPT1.

Full text of DOI:10.1021/jm040780c

J . Med. Chem. 2004, 47, 4755-4765
4755
Dip ep tid om im etic Ketom eth ylen e Isoster es a s P r o-m oieties for Dr u g Tr a n sp or t
via th e Hu m a n In testin a l Di-/Tr ip ep tid e Tr a n sp or ter h P EP T1: Design ,
Syn th esis, Sta bility, a n d Biologica l In vestiga tion s
J on Våbenø,† Carsten Uhd Nielsen,‡ Truls Ingebrigtsen,†, Tore Lejon, Bente Steffansen,‡ and
Kristina Luthman*,†,§
Department of Medicinal Chemistry, Institute of Pharmacy, University of Tromsø, N-9037 Tromsø, Norway;
Department of Pharmaceutics, The Danish University of Pharmaceutical Sciences, Universitetsparken 2,
DK-2100 Copenhagen, Denmark; Department of Chemistry, University of Tromsø, N-9037 Tromsø, Norway; and
Department of Chemistry, Medicinal Chemistry, Go¨teborg University, SE-412 96 Go¨teborg, Sweden
Received J anuary 26, 2004
Five dipeptidomimetic-based model prodrugs containing ketomethylene amide bond replace-
ments were synthesized from readily available R,â-unsaturated γ-ketoesters. The model drug
(BnOH) was attached to the C-terminus or to one of the side chain positions of the
dipeptidomimetic. The stability, the affinity for the di-/tripeptide transporter hPEPT1, and
the transepithelial transport properties of the model prodrugs were investigated. ValΨ[COCH2]-
Asp(OBn) was the compound with highest chemical stability in buffers at pH 6.0 and 7.4, with
half-lives of 190 and 43 h, respectively. All five compounds showed high affinity for hPEPT1
(Ki values < 1 mM), and PheΨ[COCH2]Asp(OBn) and ValΨ[COCH2]Asp(OBn) had the highest
affinities with Ki values of 68 and 19 µM, respectively. An hPEPT1-mediated transport
component was demonstrated for the transepithelial transport of three compounds, a finding
that was corroborated by hPEPT1-mediated intracellular uptake. The results indicate that
the stabilized Phe-Asp and Val-Asp derivatives are promising pro-moieties in a prodrug
approach targeting hPEPT1.
In tr od u ction
In recent years the human di/tri-peptide transporter
hPEPT11,2 has been extensively studied3 due to its
ability to not only transport di- and tripeptides but also
peptidomimetic drugs, e.g. â-lactam antibiotics4 and
angiotensin converting enzyme (ACE) inhibitors,5 across
the intestinal epithelium. Additionally, a number of
prodrugs based on amino acid or dipeptide pro-moieties
have been shown to be substrates for hPEPT1, including
valacyclovir (the L-valyl ester of acyclovir),6 Gly-Val-
acyclovir,7 and the stabilized model prodrug D-Asp-
(OBn)-Ala.8 Thus, a drug/prodrug delivery approach
F igu r e 1. Structures of the synthesized dipeptidomimetic
model prodrugs.
targeting hPEPT1 is promising, and an extensive search
for suitable pro-moieties has been initiated.9-11
pounds are derivatives of PheΨ[COCH2]Gly (1), ValΨ-
[COCH2]Gly (2), PheΨ[COCH2]Asp (3), ValΨ[COCH2]-
Asp (4) and AspΨ[COCH2]Gly (5), to which the model
drug benzyl alcohol (BnOH) was linked via an ester
bond to either the C-terminus or to carboxylic acid
groups in the amino acid side chain positions. In the
present study we report the design and synthesis, and
studies of the chemical and biological stabilities (buffer
stability in the absence and presence of Caco-2 cells,
respectively), the affinities for hPEPT1 and the trans-
port properties, of these dipeptidomimetic model pro-
drugs.
Design . Our previous study on Phe-Gly dipeptido-
mimetics showed that the Phe-Gly ketomethylene iso-
stere 26 had affinities for hPEPT1 and rPEPT2 similar
to the natural dipeptide Phe-Gly.14 An hPEPT1-medi-
ated transport component was also demonstrated, which
was in contrast to what was observed for hydroxyl
containing amide bond isosteres.14 Therefore, the ke-
hPEPT1 has been termed a low affinity/high capacity
transporter12 since di- and tripeptides, its natural
substrates, typically have Ki values in a narrow range
of 0.1-3.0 mM.13 We have earlier published hPEPT1
affinity and transport data on a series of five Phe-Gly
dipeptidomimetics which showed that the ketomethyl-
ene isostere had a Ki value for hPEPT1 of 0.40 mM (in
this context considered as high affinity) and was trans-
ported into and across Caco-2 cells in an hPEPT1-
dependent manner.14 These results encouraged us to
further investigate the use of pro-moieties containing
the ketomethylene isostere. Hence, the five model
prodrugs 1-5 (Figure 1) were synthesized. The com-
* To whom correspondence should be addressed. Phone: +46-31-
772-2894, fax: +46-31-772-3840, e-mail: luthman@chem.gu.se.
† Department of Medicinal Chemistry, University of Tromsø.
‡ The Danish University of Pharmaceutical Sciences.
Department of Chemistry, University of Tromsø.
§ Go¨teborg University.
10.1021/jm040780c CCC: $27.50 © 2004 American Chemical Society
Published on Web 08/10/2004

4756 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19
Våbenø et al.
Sch em e 1
tomethylene amide bond replacement was considered
a good starting point for a peptidomimetic prodrug
approach targeting hPEPT1. BnOH was chosen as the
model drug due to the inherent synthetic advantages,
its ease of detection by UV and our previous experience
with this type of model prodrugs.8,9,15-20
To investigate which position is best suited for drug
attachment, we intended to link the model drug (BnOH)
to all available positions that a natural dipeptide offers
for an alcohol containing compound; the C-terminus (1
and 2), the R2- (3 and 4), and the R1-side chain positions
(5) (Figure 1). We have earlier made thorough investi-
gations of using the R1-position as attachment point for
different (model) drugs11,15,20 and wanted to extend these
studies to also involve attachments to the R2- and
C-terminal positions. Our previous studies have shown
that the D-Asp(OcHx)-Ala model prodrug has affinity
for hPEPT1,15 and that the benzyl alcohol model pro-
drugs D-Glu(OBn)-Ala, D-Asp(OBn)-Ala and D-Ser(Bn)-
Ala (ether linkage) are all substrates for hPEPT1.8,9
Interestingly, the corresponding Asp(OBn)-Sar model
compound seemed to be an inhibitor.8 High hPEPT1-
affinities have also been found for Glu-Sar ester pro-
drugs of acyclovir and the model compound thymine
(ester linkage via a 1-(2-hydroxyethyl) spacer) whereas
the corresponding D-Glu-Ala derivatives showed poor
affinities.11 However, based on intracellular accumula-
tion studies, Glu[acyclovir]-Sar appears to be an inhibi-
tor of hPEPT1.21
In the present study the model drug was linked to
the different pro-moieties via an ester bond since this
covalent linkage is generally more stable at slightly
acidic pH than at slightly basic pH.22 Therefore, a higher
aqueous (chemical) stability of the prodrug is expected
in the intestinal lumen than after passage across the
brush border membrane. At the same time the linkage
is bioreversible since it is liable to enzyme-catalyzed
hydrolysis (i.e. esterases) once in the brush border
membrane or the systemic circulation.
ing results from the biological investigations of the Phe-
Gly ketomethylene isostere,14 this derivative was a
natural starting point for the pro-moiety design. But
indications from the literature on the beneficial effects
on affinity of an N-terminal valine residue6,23,24 led us
to extend the series of compounds from the phenylala-
nine-based compounds (1 and 3) to also include the
valine analogues (2 and 4). This would enable a direct
comparison of effects due to different R1 side chains not
taking direct part in the attachment of the model drug,
providing further structural information about the
hPEPT1 binding site.
To provide “handles” for drug attachment in the R1-
and R2-positions, a carboxylic acid functionality had to
be introduced in the pro-moiety. Since the basic idea
for this kind of prodrug is to mimic a natural dipeptide,
Glu and Asp side chains were the two alternatives. As
mentioned above, promising pro-moieties based on
stabilized dipeptides (containing D-amino acids or N-
methylated amide bonds) with both Glu and Asp in the
R1-position have earlier been investigated by our re-
search groups,9,20 but no unambiguous conclusion of
which of these that is the most advantageous for binding
and transport has yet been reached. We have earlier
reported that model prodrugs based on stabilized dipep-
tides with Asp(OBn) side chains in the R1-position were
less stable than the corresponding Glu(OBn) deriva-
tives,19 but as this was a result of intramolecular
cyclization due to attack from the amide NH-group, this
effect is not likely to apply to ketomethylene isosteres.
Due to the increased synthetic complexity of peptido-
mimetics (compared to amino acid coupling), the Asp
side chain (CH2COOH) was chosen in this study mainly
because of the synthetic feasibility of such derivatives
(see Chemistry section).
Resu lts a n d Discu ssion
Ch em istr y. To obtain compounds with L-stereochem-
istry of the side chains, the synthetic strategy had to
be designed accordingly, enabling all five compounds to
be synthesized in a stereoselective manner. The syn-
thetic strategy for 1-4 is outlined in Scheme 1. The
syntheses of the key intermediates 8,25 12,25 and 1426
have been described earlier. Selective double bond
reduction of 8 and 12 in the presence of the benzyl ester
was achieved using Wilkinson’s catalyst under H2-
atmosphere27,28 to give 9 and 13, respectively. To
introduce the L-Asp side chain in the R2-position com-
pounds 16 and 19 were synthesized via a diastereose-
lective Michael addition of the anion of allyl benzyl
malonate to the unsaturated tert-butyl esters 14 and
17, respectively, followed by deallylation and decar-
boxylation. The Michael reaction has earlier been
investigated by Deziel et al.25 who reported the highest
Considering our previous synthetic experience with
Phe-Gly peptidomimetics, and especially the encourag-

Dipeptidomimetic Ketomethylene Isosteres
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19 4757
Sch em e 2
The suspected racemization was confirmed after cata-
lytic hydrogenation of 25, which gave racemic 24.
However, due to the low chemical stability of the final
racemic product 5 (see below), no further attempts were
made to synthesize the pure L-enantiomer.
Selective N-Boc deprotection of 9 and 13 using HCl-
(g) in EtOAc gave the hydrochloride salts of 1 and 2,
while selective deprotection of the N-Boc and C-terminal
tert-butyl ester of 16, 19 and 25, in the presence of the
benzyl ester, was achieved with TFA in CH2Cl2, afford-
ing the trifluoroacetate salts of 3-5 (Schemes 1 and 2).
Pure 3 and 4 were obtained by semipreparative reversed
phase HPLC.
Ch em ica l a n d Biologica l Sta bility of 1-5. It is
evident that the stability of the prodrug is crucial for
hPEPT1-mediated drug transport, as the prodrug must
be sufficiently stable to survive the gastrointestinal
environment, and at the same time be sufficiently labile
to release the drug once it reaches the systemic circula-
tion. Another important aspect of stability studies is to
verify that the compounds are stable enough to make
sound interpretations of data from in vitro assays, such
as the Caco-2 cell assay used in this study. Hence, the
chemical and biological stabilities of 1-5 were assessed
by stability studies in buffer in the absence and presence
of Caco-2 cells, respectively.
diastereoselectivity in the addition of the anion of allyl
tert-butyl malonate to unsaturated γ-keto benzyl esters
using NaH as base and THF as solvent at -78 °C. The
Michael additions to 14 and 17 were therefore carried
out analogously and were shown to be both regio- and
stereoselective. The diastereomeric ratios were >80:20
A. Ch em ica l Sta bility Stu d ies. The chemical sta-
bilities of the model prodrugs 1-5 were investigated in
both apical (pH 6.0) and basolateral (pH 7.4) transport
buffers at 37 °C (Table 1). It was observed that com-
pounds 1-5 all as expected were more stable at pH 6.0
than at 7.4 (maximum p ) 0.0056). This effect was most
pronounced for compounds 1 and 2, being 11-12 times
more stable at pH 6.0 than at 7.4, whereas 3-5 were
3-5 times more stable at the lower pH. When compar-
ing the Phe- and Val-derivatives, compounds 2 and 4
were 2-3 times more stable than their corresponding
Phe-derivatives 1 and 3 at either pH value (maximum
p ) 0.007). In terms of absolute values, 4 was the most
stable compound at either pH value, whereas 5 was the
most unstable compound at either pH value. It was also
observed that the two Val-derivatives (2 and 4) were
the most stable compounds at pH 6.0, while the two R2-
benzylated compounds (3 and 4) were the most stable
at pH 7.4.
1
for both compounds, assessed by H NMR spectroscopy
of the diastereomeric mixture of 16 and 19 and their
(3R)-epimers and by analytical HPLC of crude 3 and 4
after deprotection.
The synthetic strategy for 5 is outlined in Scheme 2.
Initial attempts to make the phosphonates from Boc-
Glu(OBn)-OMe or Boc-Glu-OMe, analogously to the
synthesis of 7, were unsuccessful. For Boc-Glu(OBn)-
OMe this could be due to an unselective attack of the
phosphonate anion on the two ester functionalities. No
reaction was observed for Boc-Glu-OMe, probably due
to electrostatic repulsion from the deprotonated car-
boxylic acid.
B. Biologica l Sta bility Stu d ies. To assess the
influence of enzyme-catalyzed hydrolysis of the com-
pounds, stability studies in buffer in the presence of
Caco-2 cells were carried out by investigating the
amount of intact compound remaining on the apical side
of the Caco-2 cell monolayers after the 2 h transport
experiments (see below) for all compounds (Table 2).
When comparing these results with the expected amount
remaining in buffer (pH 6) after 2 h, calculated from
the kobs values obtained from the chemical stability
studies, a significant decrease was observed for 1 and
2. However, for the Asp(OBn) derivatives 3-5 there was
no significant difference in the degradation in the
absence or presence of Caco-2 cells. These results
suggest the involvement of esterases in the degradation
of 1 and 2;31 thus, it seemed as the C-terminal esters
were more prone to enzymatic hydrolysis than the Asp-
(OBn) esters in the R1- or R2-positions. Overall, the
results in Tables 1 and 2 indicated a sufficient stability
Instead, an alternative approach to the R1-benzylated
Asp-derivative was undertaken. Reaction of the phos-
phonate anion on the N-Boc protected lactone 20,
synthesized from the commercially available (S)-R-
amino-γ-butyrolactone, provided the phosphonate 21,
which was converted to the unsaturated ketoester 22
using the standard Horner-Wadsworth-Emmons reac-
tion. Subsequent catalytic hydrogenation using H2/Pd
in EtOAc gave the saturated analogue 23. The primary
alcohol was then oxidized with PDC in DMF29 to give
the desired carboxylic acid (aspartate side chain) 24,
which was finally benzylated using Cs2CO3 and benzyl
bromide30 to afford the diester 25. This benzylation
procedure is reported to be a mild method for esterifi-
cation of amino acids and peptides,30 but surprisingly
the optical activity of 25 was lost during this reaction.

4758 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19
Våbenø et al.
Ta ble 1. Estimated Rate Constants and Half-Lives ((SD) for Chemical Degradation of Compounds 1-5 in Transport Buffers (pH 6.0
and 7.4) at 37 °C, Studied for Several Half-Lives or over a 48-h Period for Slower Degradations (n ) 3)a
pH 6.0b
pH 7.4b
compound
kobs (min-1
)
t1/2 (min)
kobs (min-1
)
t1/2 (min)
1
2
3
4
5
2.5 (( 0.1) × 10-4
8.4 (( 1.8) × 10-5
1.8 (( 0.5) × 10-4
6.1 (( 0.3) × 10-5
3.5 (( 0.2) × 10-3
2803 ( 131
8529 ( 1943
4085 ( 928
11381 ( 560
200 ( 9
2.7 (( 0.2) × 10-3
9.7 (( 1.3) × 10-4
5.9 (( 0.4) × 10-4
2.7 (( 0.1) × 10-4
255 ( 20
721 ( 96
1171 ( 81
2591 ( 78
67 ( 2
1.04 (( 0.03) × 10-2
a
b
Assessed by HPLC, see Experimental Section. For buffer composition, see Experimental Section.
Ta ble 2. Remaining Amount (%) of 1-5 after 120 min at 37
°C, pH 6, in the Absence and Presence of Caco-2 Cells
compound
buffera
Caco-2b
1
2
3
4
5
97
99
98
99
66
2.2 ( 2.2
48 ( 1.3
100c
100c
73.3 ( 7.8
a
Amount (%) remaining intact after 120 min in apical transport
b
buffer (pH 6.0), calculated from kobs (see Table 1). Observed
amount (%) remaining intact on the apical side of Caco-2 cell
monolayers after the transport assay. c No detectable appearance
of BnOH.
for all five compounds in the affinity studies (incubation
time 5 min) but revealed that a careful examination of
the data from the transepithelial transport assay (time
course 120 min) was necessary for 1, 2, and 5.
F igu r e 2. Inhibition of [14C]-Gly-Sar uptake by 1 (0), 2 (9),
3 (O), 4 (b), and 5 (4) in Caco-2 cells. The lines describe the
best fit to the experimentally obtained points using eq 1. Error
bars represent SE.
The bioreversible ester bond is a widely used linkage
in prodrugs, especially in the lipophilic prodrug ap-
proach, benefiting from the difference in pH between
the small intestine and the blood. This strategy is also
applicable when designing a prodrug for hPEPT1, as
the stability of all five compounds was higher at pH 6.0
than at 7.4. However, the results revealed large differ-
ences in chemical stability between the different com-
pounds, indicating that some pro-moieties are better
suited than others in this respect. It should be men-
tioned that the stability of an ester prodrug is highly
dependent also on the structural properties of the
attached drug20 and is therefore likely to be different
when a drug other than the model drug used in this
study is attached.
Affin ity a n d Tr a n sp or t Stu d ies of 1-5 in Ca co-2
Cell Mon ola yer s. The synthesized derivatives 1-5
were tested for their affinity for hPEPT1 based on
inhibition of [14C]-Gly-Sar uptake using the Caco-2 cell
assay as described earlier.14 The transepithelial trans-
port of 1-5 across Caco-2 monolayers, including uptake
(intracellular accumulation), was also investigated. In
these studies Gly-Sar was used as the reference com-
pound, and mannitol as the marker of paracellular
transport.
A. Affin ity for h P EP T1. The affinities of 1-5 for
hPEPT1 in confluent mature Caco-2 cell monolayers
were determined by their ability to inhibit the transport
of [14C]-Gly-Sar, a standard substrate for hPEPT1. The
results (Figure 2, Table 3) demonstrated that in the
context of hPEPT1 all compounds had high affinities
(Ki < 1 mM), and that PheΨ[COCH2]Asp(OBn) (3) and
ValΨ[COCH2]Asp(OBn) (4) had the highest affinities,
with Ki values of 68 and 19 µM, respectively. The
differences in the Ki values for the Val-derivatives (2
and 4) and their corresponding Phe-derivatives (1 and
3, respectively) are statistically significant (Table 3).
Ta ble 3. IC50 and Ki Values of 1-5 for hPEPT1 in Caco-2
Cells
compound
IC50 (µM)
Ki (µM)
1
2
3
4
5
915 ( 37
134 ( 13
69 ( 6
905
133
68
19
417
19 ( 2
422 ( 33
Using Student’s t-test a significant difference in affinity between
1 and 2 was found (p < 0.0001, n ) 3) and similarly a significant
difference between 3 and 4 (p ) 0.0013, n ) 3).
B. Tr a n sep ith elia l Tr a n sp or t a n d Up ta k e Stu d -
ies. As affinity studies only give information about
binding to hPEPT1, transepithelial transport studies
were subsequently carried out to investigate the overall
transport across the Caco-2 cell monolayer. Additionally,
the uptake, i.e., the amount of compound accumulated
intracellularly after completion of the transport studies
(2 h), was assessed to investigate the actual cellular
accumulation mediated by hPEPT1. To demonstrate
active transport, these experiments were carried out in
the absence and presence (20-fold excess) of the hPEPT1
competitive inhibitor Gly-Pro.
The results from the transport experiments showed
that 3-5 were transported across the Caco-2 cell mono-
layer (Figure 3). A significant reduction (p < 0.05, n )
3) in the flux of 3-5 was observed in the presence of
Gly-Pro, indicating an hPEPT1-mediated transport
component in the overall transepithelial transport of
these three compounds. The same was observed for the
reference compound Gly-Sar, confirming the involve-
ment of hPEPT1. As there was no significant difference
in the flux of the paracellular marker mannitol in the
presence and absence of Gly-Pro (Papp values of 8.7 (
0.6 × 10-7 and 9.4 ( 0.2 × 10-7 cm × s-1, respectively,
n ) 3, p > 0.05), the changes in the flux of 3-5 were
not likely to be an effect of decreased paracellular

Dipeptidomimetic Ketomethylene Isosteres
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19 4759
F igu r e 4. Intracellular uptake of 1 mM compound in Caco-2
cells in the absence (gray) and in the presence (black) of 20
mM Gly-Pro after 2 h. (A) Gly-Sar, 3 and 4; (B) 5 and 5
including BnOH. NM: not measurable. Error bars represent
SE. * p < 0.05 and *** p < 0.001.
F igu r e 3. Transepithelial flux (J ) of 1 mM compound across
Caco-2 cell monolayers in the absence (gray) and in the
presence (black) of 20 mM Gly-Pro. (A) Gly-Sar, 3 and 4; (B)
5 and 5 including BnOH. NM: not measurable. Error bars
represent SE. ** p < 0.01.
presence of 20 mM Gly-Pro intact 2 could not be
detected, giving a weak indication that 2 in fact is a
substrate for hPEPT1.
transport. Also the uptake of 3-5 (Figure 4) was
significantly different in the presence and absence of
Gly-Pro, corroborating transcellular transport mediated
by hPEPT1. A significant release of BnOH during the
time course of the experiments (2 h) was observed for
5, which could be expected from the chemical stability
studies. The transepithelial transport of BnOH was
relatively fast with a Papp value of 1.40 ( 0.04 × 10-4
cm × s-1. This indicates that the apically released
BnOH is transported across the Caco-2 cells, thereby
contributing to the flux calculated for 5 including BnOH,
as illustrated in Figure 3B. The flux for 5 is thus the
difference in total transport of 5 and BnOH in the
absence and presence of Gly-Pro (approximately 0.7
nmol × min-1 × cm-2). For 3 and 4 no release of BnOH
was observed, suggesting that not only the chemical
stabilities of these two compounds, but also their
biological stabilities, could be sufficient to warrant the
use of these as pro-moieties in a prodrug approach
targeting hPEPT1. Although the chemical stability of
1 and 2 was sufficient, the biological stability studies,
i.e., in the presence of Caco-2 cells, showed that 1 and
2 were degraded during the course of the experiment.
Thus, it was impossible to determine accurate flux
values for 1 and 2, although for 2 intact compound was
observed basolaterally in the first sample (t ) 40 min,
results not shown). But as no compound was left at the
end of the experiment (t ) 120 min), a significant
basolateral esterase activity was also evident. In the
Str u ctu r e-Affin ity Rela tion sh ip s for h P EP T1.
As the 3D structure of hPEPT1 not yet has been
established, only indirect evidence for the transporter
topology can be obtained, based on epitope tagging32 and
site-directed mutagenesis studies.33-37 Thus, ligand
based design is currently the only way to extract
structure-affinity information for the transporter. The
affinities of a large number of natural peptides have
been determined, whereas the effects of modification of
the side chains of natural peptides have been investi-
gated to a lesser degree. Still, an interaction of the R2
side chain with a proposed hydrophobic pocket has been
suggested,38 and studies have also indicated that sub-
strates may interact with a hydrophobic pocket in the
R1-position.20 A reasonable amount of data has been
accumulated on the importance of the different func-
tional groups in the peptide backbone, such as charged
N- and C-termini and the carbonyl functionality of the
N-terminal amino acid. Over the years several template
models for hPEPT1 have been proposed, including a

4760 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19
Våbenø et al.
general model for both di- and tripeptides38 based on
molecular modeling of a diverse set of substrates/
inhibitors. Recently a more detailed model for dipeptides
and amino acid aryl amides based on CoMFA and
CoMSIA was published.39
beneficial effect on affinity compared to Phe. There are
two possible explanations for this observation: the
isopropyl group contributes to the stabilization of the
bioactive conformation, or the binding site of hPEPT1
has a preference for the isopropyl group, indicating an
especially favorable interaction with residues in the
binding site of hPEPT1. It is also seen that modifications
in the R2-position are better tolerated, and even seem
advantageous, compared to the R1-position and the
C-terminus.
Str u ctu r e-Tr an spor t Relation sh ips for h P EP T1.
The fact that a significant amount of 1 and 2 was
degraded in the apical compartment during the assay,
and probably also intracellularly and basolaterally,
made interpretations of the transport of the intact
compound impossible.
In terms of absolute values compound 3 showed the
highest flux and uptake; however, flux and uptake of 3
were also measurable in the presence of Gly-Pro. Due
to the two phenyl rings in 3, this compound is more
hydrophobic than the two other zwitterionic compounds
4 and 5, suggesting that the flux and uptake which was
not inhibited by Gly-Pro are contributions from a
passive transcellular transport component.
We have recently shown that the replacement of the
enzymatically labile amide bond of Phe-Gly with the
stable ketomethylene isostere (PheΨ[COCH2]Gly, 26)
only reduced the apparent affinity for hPEPT1 2-fold
(Ki values of 0.20 and 0.40 mM, respectively). The
apparent affinity for rPEPT2 was not significantly
changed, demonstrating that 26 is a high affinity ligand
for both PEPT1 and PEPT2.14 From Table 3 it is seen
that the substitution of the R1 benzyl side chain of 26
with the benzylated aspartate side chain of 5 had a
negligible effect on the affinity for hPEPT1 (Ki values
of 0.40 and 0.45 mM, respectively). The importance of
a hydrophobic R1-side chain has earlier been pointed
out,20 and it is likely that the phenyl ring in 5, despite
of the ester functionality, contributes in this respect, in
the same way as the phenylalanine side chain does for
26. This observation is also in agreement with earlier
studies of R1-benzylated stabilized Glu-Xaa and Asp-
Xaa dipeptides showing high affinity for hPEPT1.9,20
Both flux and uptake were similar for compounds 4
and 5, and as no flux was observed in the presence of
Gly-Pro, a purely carrier-mediated transpithelial trans-
port for these two compounds was indicated. For com-
pound 5, a significant amount of the model drug BnOH
was detected in the basolateral compartment, both in
the presence and absence of inhibitor. As no intact
compound could be detected intracellularly or baso-
laterally when Gly-Pro was added, the basolateral
presence of BnOH under these conditions indicates
apical hydrolysis of the parent compound followed by
passive transpithelial transport of BnOH. The increase
in intracellular and basolateral amount of BnOH in
absence of Gly-Pro indicates that both apical, intra-
cellular, and basolateral hydrolysis of the parent com-
pound take place during the transport experiment.
Overall, the transport and uptake experiments showed
that 3-5 are substrates for hPEPT1. The transepithelial
flux of 3-5 is comparable to the flux measured for orally
active â-lactam antibiotics across Caco-2 cells,4 which
further indicate that dipeptidic ketomethylene isosteres
constitute a promising class of compounds for develop-
ment of pro-moieties for optimizing oral bioavailability
by targeting hPEPT1.
The model prodrug 1 corresponds to the C-terminal
benzyl ester of 26, a modification that reduced the
affinity for hPEPT1 approximately 2-fold. This is in
agreement with earlier studies showing that an unsub-
stituted C-terminus is advantageous for binding to the
transporter. It should also be mentioned that the
solubility and/or dissolution rate of 1 in water was low,
a problem that was overcome by addition of maximum
0.5-1.0% DMSO to the test medium. Even if this was
the prodrug with lowest affinity in our series, a Ki value
below 1 mM is still considered interesting.
Compound 2 is in effect the benzyl ester of ValΨ-
[COCH2]Gly, but contrary to 1 this C-terminally benz-
ylated compound has an improved affinity compared to
26 and an approximately 7-fold improvement in affinity
compared to 1. This is likely to be an effect of the
isopropyl side chain of Val in the R1-position, and it has
been shown in several studies that Val in R1 has an
advantageous effect on binding compared to other amino
acids.
The R2-benzylated Xaa-Asp ketomethylene isosteres
3 and 4 are the two compounds with highest affinity in
this series, with Ki values in the lower µM-range. This
indicates a strong favorable effect of the benzylated
aspartate side chain compared to the hydrogen of 26.
The substrate template of Bailey et al.38 suggests that
a hydrophobic R2-group is an advantage, and in the
same way as for 5 the phenyl ring of the benzyl alcohol
contributes in this respect. But as an Asp(OBn) side
chain in R2 has a much bigger impact on the Ki value
than the same substitution in R1, these results indicate
that there are stronger hydrophobic interactions with
residues in the R2 binding site and probably also less
restriction in size. A significant increase in affinity by
substitution of a benzyl group (Phe) with an isopropyl
group (Val) in the R1-position is also observed for 3 and
4, here resulting in an approximate 3-fold improvement
in affinity for the Val-derivative.
Con clu sion s
Chemical and biological investigations of a series of
structurally related benzyl alcohol model prodrugs
based on the dipeptide ketomethylene isostere have
provided important information on the structure-
affinity and structure-transport relationships for
hPEPT1 and also on the stability of the ester prodrug
linkage. The affinity studies showed that introduction
of the isopropyl side chain of Val in the R1-position is
advantageous for binding affinity, and also for stability,
compared to the benzyl side chain of Phe. It has also
been demonstrated that in terms of affinity drug at-
tachment in the R2-position is better tolerated, and even
seem advantageous, compared to the R1-position or the
C-terminus.
Thus, the affinity studies demonstrate that the iso-
propyl side chain of Val in the R1-position has a

Dipeptidomimetic Ketomethylene Isosteres
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19 4761
atmosphere at 50 °C for 20-24 h. The solvent was evaporated
under reduced pressure, and diethyl ether was added to
precipitate the catalyst. The suspension was filtered through
a short silica gel column (diethyl ether), and the filtrate was
concentrated under reduced pressure. The residue was purified
by flash chromatography on basic Al2O3 using diethyl ether
as eluent to afford 13 (112 mg, 56%) as a colorless oil: [R]D
As the degradation rates for 1 and 2 were much
higher than what were observed in the stability studies
in buffer, the involvement of enzymes, i.e., esterases,
was indicated. Regarding the use of the ester linker in
a potential prodrug, the assessment of the influence of
enzymes (i.e. esterases) on the stability of the prodrug
is needed to enable translation of data into an in vivo
situation. The results of this study imply that PheΨ-
[COCH2]Asp and ValΨ[COCH2]Asp are particularly
promising pro-moieties for a prodrug approach aimed
at increasing oral bioavailability via hPEPT1-mediated
transport, and testing of this hypothesis using poorly
absorbable drugs is presently ongoing in our laboratory.
1
+8.0° (c 1.08, CHCl3); H NMR (CDCl3) δ 7.42-7.32 (m, 5H),
5.16 (br s, 1H), 5.14 (s, 2H), 4.36-4.28 (m, 1H), 3.00-2.90 (m,
1H), 2.84-2.59 (m, 3H), 2.30-2.19 (m, 1H), 1.47 (s, 9H), 1.03
(d, 3H, J ) 6.4 Hz), 0.81 (d, 3H, J ) 6.8 Hz); 13C NMR (CDCl3)
δ 207.9, 172.4, 156.0, 135.8, 128.6 (2 C:s), 128.3, 128.2 (2 C:s),
79.7, 66.6, 63.9, 35.4, 30.3, 28.3 (3 C:s), 27.7, 19.9, 16.7.
Ben zyl (5S)-5-[N-(ter t-Bu toxyca r bon yl)a m in o]-4-oxo-
6-p h en yl-h exa n oa te (9). Reduction of 8 (267 mg, 0.56 mmol)
was performed as described above to afford 129 mg of 9 (48%)
as a white solid: Mp 87.5-89.5 °C; [R]D +5.2° (c 1.0, CHCl3);
1H NMR (CDCl3) δ 7.43-7.17 (m, 10H), 5.15 (s, 2H), 5.10 (d,
1H, J ) 7.6 Hz), 4.57 (dd, 1H, 14.2, 7.0 Hz), 3.14 (dd, 1H, 14.2,
6.2 Hz), 2.96 (dd, 1H, 14.2, 7.0 Hz), 2.83-2.76 (m, 2H), 2.70-
2.64 (m, 2H), 1.43 (s, 9H); 13C NMR (CDCl3) δ 207.5, 172.4,
155.3, 136.3, 135.8, 129.3 (2 C:s), 128.7 (2 C:s), 128.6 (2C:s),
128.3, 128.2 (2 C:s), 127.0, 80.0, 66.6, 60.2, 37.5, 35.1, 28.3 (3
C:s), 27.8.
Exp er im en ta l Section
Ch em istr y. Gen er a l. Starting materials and reagents were
purchased from Sigma, Fluka, Aldrich, Senn Chemicals, and
Merck KGaA and were used as received. 1H and 13C NMR
spectra were recorded on a Varian Mercury Plus spectrometer
operating at 400 and 100 MHz, respectively. Chemical shifts
are given relative to the solvent signal (δ 7.26 and δ 77.0 for
CDCl3, and δ 3.49 and δ 49.0 for CD3OD, respectively). TLC
was run on silica gel coated aluminum sheets (silica gel 60
ter t-Bu t yl (5S)-5-[N-(ter t-Bu t oxyca r b on yl)a m in o]-6-
m eth yl-4-oxo-(E)-2-h ep ten oa te (17). Compound 17 was
synthesized in 60% yield (321 mg) from phosphonate 11 (527
mg, 1.63 mmol), as described in the literature for the synthesis
of 14.18 Compound 17 was isolated as a light yellow oil: [R]D
F
254, Merck), with visualization by UV detection and/or 5%
phosphomolybdic acid hydrate (PMA) in EtOH, followed by
heating. Flash chromatography was performed on silica gel
(silica gel 60 (0.040-0.063 mm), Merck) under nitrogen
pressure, if not stated otherwise. THF was distilled from Na/
benzophenone ketyl. Other solvents were of analytical or
synthetic grade and were used without further purification.
Melting points were taken with a Bu¨chi Melting Point B-540
apparatus and are uncorrected. Optical activity was measured
using a Perkin-Elmer 241 Polarimeter. Elemental analyses
were done by Mikro Kemi AB, Uppsala, Sweden. In HPLC a
Waters 510 pump, a Waters 2487 Dual λ Absorbance Detector
operating at 254 nm, and a Varian 4400 Integrator were used.
Reversed phase analytical chromatography of crude 3 and 4
was performed on a Symmetry C18, 5 µm, 3.9 × 150 mm
analytical column from Waters (isocratic: 28% CH3CN, 0.1%
TFA; flow rate 1 mL/min). Purification of compounds 3 and 4
by reversed phase preparative chromatography was performed
on a Prep Nova-Pak HR C18 6 µm 60 Å 25 ×100 mm column
from Waters (isocratic: 3: 34% CH3CN, 0.1% TFA, 4: 28%
CH3CN, 0.1% TFA; flow rate 7.0 mL/min). The syntheses of
8,25 11,25 12,25 7,26 14,26 20,40 and allyl benzyl malonate41 have
been described earlier.
Dim et h yl [(3S)-3-[N-(ter t-Bu t oxyca r b on yl)a m in o]-4-
m eth yl-2-oxop en tyl]p h osp h on a te (11). Compound 11 was
synthesized in 84% yield (4.21 g) from 10 (3.02 g, 13.06 mmol)
according to a literature procedure for the synthesis of 7.26
Compound 11 was isolated as a colorless oil. For spectroscopi-
cal data on 11, see ref 25.
Ben zyl (5S)-5-[N-(ter t-Bu toxyca r bon yl)a m in o]-4-oxo-
6-p h en yl-(E)-2-h exen oa te (8). Compound 8 was synthesized
in 58% yield (972 mg) from 7 (1.60 g, 4.31 mmol), according to
a literature procedure for the synthesis of 14,26 using benzyl
glyoxylate instead of tert-butyl glyoxylate. Compound 8 was
isolated as a yellow oil. For spectroscopical data on 8, see ref
25.
1
+63.8° (c 0.93, CHCl3); H NMR (CDCl3) δ 7.13 (d, 1H, J )
16.0 Hz), 6.75 (d, 1H, J ) 16.0 Hz), 5.20 (d, 1H, J ) 8.4 Hz),
4.56 (dd, 1H, J ) 8.4, 4.0 Hz), 2.25-2.10 (m, 1H), 1.53 (s, 9H),
1.47 (s, 9H), 1.05 (d, 3H, J ) 7.2 Hz), 0.81 (d, 3H, J ) 6.8 Hz);
13C NMR (CDCl3) δ 198.6, 164.4, 155.8, 136.0, 134.2, 82.2, 79.9,
63.2, 30.2, 28.3 (3 C:s), 28.0 (3 C:s), 19.9, 16.7.
Allyl Ben zyl Ma lon a te. A solution of benzyl acetate (1.20
g, 8.0 mmol) in dry THF (25 mL) was cooled to -78 °C, and
LiHMDS (1.0M in THF) (8.0 mL, 8.0 mmol) was added
dropwise over a period of 10 min. The reaction mixture was
stirred at -78 °C for 1 h before allylchloroformate (1.49 g, 12.4
mmol) dissolved in THF (10 mL) was added by needle transfer
over 15 min. The mixture was stirred at -78 °C for 2 h. The
reaction was quenched by addition of 10% citric acid (10 mL).
The resulting solution was extracted with diethyl ether (3 ×
50 mL). The combined organic phase was washed with brine,
dried over MgSO4, and concentrated to give a yellow oil. The
residue was purified by flash chromatography [pentane:diethyl
ether 9:1] to afford 1.30 g allyl benzyl malonate (68%) as a
colorless oil. For spectroscopical data on the product, see ref
41.
Ben zyl
(3S,6S)-3-(ter t-Bu t oxyca r b on yl)-6-[N-(ter t-
bu toxyca r bon yl)a m in o]-7-m eth yl-5-oxo-octa n oa te (19)
(Gen er a l P r oced u r e for th e Mich a el Rea ction , Dea lly-
la tion , a n d Deca r boxyla tion ). (a) Sodium hydride (60% in
mineral oil, 0.45 mmol, 18 mg) was added to a solution of allyl
benzyl malonate (234 mg, 1.00 mmol) in THF (10 mL). After
the gas evolution ceased, the reaction mixture was cooled to
-78 °C and 17 (262 mg, 0.80 mmol) dissolved in THF (10 mL)
was added dropwise over a period of 15 min. The mixture was
stirred at -78 °C for 2.5 h when the reaction was quenched
with 10% citric acid (10 mL). Water (30 mL) was added, and
the mixture was extracted with diethyl ether (3 × 30 mL). The
combined organic phase was washed with brine and dried over
MgSO4. The solvent was evaporated under reduced pressure
to afford 419 mg of crude 18 (93%) as a colorless oil that was
used in the next step without further purification or charac-
terization.
(b) A solution of crude 18 (419 mg, 0.75 mmol) in toluene (5
mL) was added during 5 min to a solution of the catalyst Pd-
(PPh3)4 (45 mg, 0.05 equiv) and pyrrolidine (1.38 mmol, 115
µL) in toluene (10 mL). The reaction was stirred at room
temperature for 7 h. The mixture was diluted with EtOAc (30
mL) and washed with 0.1 M HCl, water, and brine. The organic
phase was dried over MgSO4 and concentrated. The crude
Ben zyl (5S)-5-[N-(ter t-Bu toxyca r bon yl)a m in o]-6-m eth -
yl-4-oxo-(E)-2-h ep ten oa te (12). Compound 12 was synthe-
sized in 47% yield (211 mg) from 11 (420 mg, 1.30 mmol),
according to a literature procedure for the synthesis of 14,26
using benzyl glyoxylate instead of tert-butyl glyoxylate. Com-
pound 12 was isolated as a light yellow oil. For spectroscopical
data on 12, see ref 25.
Ben zyl (5S)-5-[N-(ter t-Bu toxyca r bon yl)a m in o]-6-m eth -
yl-4-oxo-h ep ta n oa te (13) (Gen er a l P r oced u r e for th e
Dou ble Bon d Red u ction of r,â-Un sa tu r a ted γ-Keto Ben z-
yl Ester s). Wilkinson’s catalyst (Ph3P)3RhCl (82 mg, 0.1 equiv)
and 12 (211 mg, 0.61 mmol) were dissolved in benzene (20 mL)
under inert conditions. The reaction was stirred under H2

4762 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19
Våbenø et al.
deallylated product was used in the next step without further
purification or characterization.
silica gel [pentane:diethyl ether 1:1] afforded 22 (420 mg, 53%)
as a colorless oil that was used without further characteriza-
tion.
(c) The crude carboxylic acid was dissolved in xylene (15
mL) and decarboxylated on heating at 130 °C for 4 h. The
mixture was concentrated, and the residue was purified by
flash chromatography on silica gel [pentane:diethyl ether 3:1]
to afford 226 mg of 19 and its 3R-epimer (59% from 17) as a
colorless oil: 1H NMR (CDCl3) (major isomer) δ 7.40-7.30 (m,
5H), 5.20-5.10 (m, 3H), 4.25 (dd, 1H, J ) 8.8, 4.0 Hz), 3.26-
3.20 (m, 1H), 3.03 (dd, 1H, J ) 18.4, 6.8 Hz), 2.77-2.68 (m,
2H), 2.59 (dd, 1H, J ) 16.4, 7.2 Hz), 2.24-2.12 (m, 1H), 1.45
(s, 9H), 1.41 (s, 9H), 1.03 (d, 3H, J ) 6.4 Hz), 0.78 (d, 3H, J )
6.8 Hz); 13C NMR (major isomer) (CDCl3) δ 207.3, 172.4, 171.4,
155.9, 135.7, 128.6 (2 C:s), 128.4 (2 C:s), 128.36, 81.2, 79.7,
66.6, 63.8, 41.6, 37.0, 35.6, 30.1, 28.3 (3C:s), 27.9 (3C:s), 20.0,
16.5.
ter t-Bu tyl (5S)-5-[N-(ter t-Bu toxyca r bon yl)a m in o]-7-h y-
d r oxy-4-oxo-h ep ta n oa te (23). Compound 22 (420 mg, 1.28
mmol) and Pd(C) (10%, 47 mg) were placed in a round-
bottomed flask and EtOAc (25 mL) was added before the flask
was fitted with a hydrogen-filled balloon. The mixture was
stirred at room temperature and was monitored by TLC (SiO2,
diethyl ether:pentane, 1:1), and after 3 h no starting material
could be detected. Filtration through Celite and solvent
removal under reduced pressure left an oil that was filtered
through a silica gel column using diethyl ether as eluent to
afford 23 (250 mg, 59%) as a colorless oil that was used in the
next step without further purification or characterization.
(3S)-6-(ter t-Bu toxycar bon yl)-3-[N-(ter t-bu toxycar bon yl)-
a m in o]-4-oxo-h exa n oic Acid (24). Compound 23 (250 mg,
0.75 mmol) was dissolved in freshly distilled DMF (3.5 mL)
and solid pyridinium dichromate (1.41 g, 3.7 mmol) was added
in one portion. The reaction was monitored by TLC (SiO2,
diethyl ether) and after 2.5 h the reaction was quenched by
addition of saturated aqueous NaHCO3 (25 mL). The mixture
was extracted once with pentane and the aqueous phase was
acidified by addition of concentrated HCl. The solution was
extracted with diethyl ether (2 × 20 mL), dried (MgSO4), and
concentrated to afford 24 (161 mg, 62%) as a light yellow oil:
[R]D -10.5° (c 0.82, CHCl3); 1H NMR (CDCl3) δ 5.71 (d, 1H, J
) 8.8 Hz), 4.50-4.48 (m, 1H), 3.05 (dd, 1H, J ) 17.6, 4.4 Hz),
2.94-2.80 (m, 3H), 2.54 (t, 2H, J ) 6.4 Hz), 1.48 (s, 9H), 1.44
(s, 9H); 13C NMR (CDCl3) δ 207.0, 175.8, 172.1, 155.6, 80.9,
80.5, 55.8, 35.5, 33.8, 29.3, 28.3 (3 C:s), 28.0 (3 C:s).
Ben zyl (3S,6S)-3-(ter t-Bu toxyca r bon yl)-6-[N-(ter t-bu -
t oxyca r b on yl)a m in o]-5-oxo-7-p h en yl-h ep t a n oa t e (16).
Crude 15 (453 mg, 90%, colorless oil) was synthesized from
14 (310 mg, 0.83 mmol) as described above for crude 18.
Subsequent deallylation and decarboxylation afforded 228 mg
of 16 and its 3R-epimer (59% from 14) as a light yellow oil:
1H NMR (major isomer) (CDCl3) δ 7.42-7.16 (m, 10 H), 5.15
(app d, 2H), 5.06 (d, 1H, J ) 8.0 Hz), 4.51 (dd, 1H, J ) 14.2,
7.0 Hz), 3.26-3.19 (m, 1H), 3.14 (dd, 1H, J ) 14.0, 6.0 Hz),
3.0-2.88 (m, 2H), 2.78-2.68 (m, 2H), 2.57 (dd, 1H, J ) 16.8,
6.0 Hz), 1.42 (s, 18H); 13C NMR (major isomer) (CDCl3) δ 206.9,
172.4, 171.5, 155.2, 136.3, 135.7, 129.4, 129.4 (2 C:s), 128.7 (2
C:s), 128.6 (2 C:s), 128.4 (2 C:s), 127.0, 81.2, 80.0, 66.5, 60.1,
41.3, 37.3, 37.0, 35.4, 28.3 (3 C:s), 27.9 (3 C:s).
N-(ter t-Bu t oxyca r b on yl)-L-h om oser in e La ct on e (20).
Triethylamine (835 µL, 6 mmol) was added to a suspension of
(S)-R-amino-γ-butyrolactone hydrobromide (1.0 g, 5.5 mmol)
in THF (10 mL) at 0 °C. A solution of di-tert-butyl dicarbonate
(1.32 g, 6 mmol) in THF (5 mL) was added, and the reaction
was stirred for 30 min at 0 °C and then overnight at room
temperature. THF was removed under reduced pressure, and
H2O (25 mL) was added. The aqueous phase was adjusted to
pH ∼5 with 10% aqueous citric acid and extracted with EtOAc
(3 × 30 mL). The organic phase was dried (MgSO4) and the
solvent removed under reduced pressure to give 1.09 g (98%)
of 20 as a yellow solid. For spectroscopical data on 20, see ref
40.
Ben zyl (3R/S)-6-(ter t-Bu t oxyca r b on yl)-3-[N-(ter t-b u -
toxyca r bon yl)a m in o]-4-oxo-h exa n oa te (25). Compound 24
(112 mg, 0.33 mmol) was dissolved in MeOH (2 mL), and H2O
(200 µL) was added. Cs2CO3 (20% aqueous solution) was added
until pH ∼8 (pH paper). After evaporation to dryness under
reduced pressure, DMF (1.2 mL) was added and the solvent
was removed at the pump (water bath kept at 45 °C). DMF
(1.2 mL) was once more added and removed at the pump to
leave a semisolid yellowish material. DMF (1.2 mL) and benzyl
bromide (0.38 mmol, 45 µL) were added, and the solution was
stirred at room temperature for 6 h. Solvents were removed
under reduced pressure and H2O (5 mL) was added. The
aqueous solution was extracted with EtOAc (2 × 5 mL), and
the combined organic extracts were dried (MgSO4) and con-
centrated to afford 25 (102 mg, 72%) as a light yellow oil: 1H
NMR (CDCl3) δ 7.42-7.32 (m, 5H), 5.67 (d, 1H, J ) 8.4 Hz),
5.14 (dd, 2H, J ) 14.4, 12.4 Hz), 4.56-4.52 (m, 1H), 3.04 (dd,
1H, J ) 17.2, 5.2 Hz), 2.94-2.78 (m, 3H), 2.54 (app t, 2H),
1.48 (s, 9H), 1.46 (s, 9H); 13C NMR (CDCl3) δ 207.0, 171.8,
171.3, 155.4, 135.4, 128.6 (2 C:s), 128.4, 128.3 (2 C:s), 80.7,
80.3, 66.9, 56.0, 35.7, 34.0, 29.2, 28.3 (3 C:s), 28.1 (3 C:s).
Ben zyl (5S)-5-Am in o-4-oxo-6-ph en yl-h exan oate (1). Com-
pound 9 (53 mg, 0.13 mmol) was dissolved in EtOAc (5 mL)
saturated with HCl(g). The mixture was stirred at room
temperature for 15 min. Diethyl ether (15 mL) was added, and
the solution was concentrated under reduced pressure. This
procedure was repeated three times. The residue was extracted
with water (2 × 10 mL) and diethyl ether (10 mL). The aqueous
phase was concentrated to afford 41 mg of 1 (92%) as a white
solid: Mp 94-96 °C; [R]D +17.3° (c 0.92, MeOH); 1H NMR
(CD3OD) δ 7.44-7.32 (m, 10H), 5.15 (s, 2H), 4.56-4.48 (m,
1H), 3.39 (dd, 1H, J ) 14.4, 6.0 Hz), 3.03 (dd, 1H, J ) 14.4,
8.0 Hz), 2.95-2.85 (m, 2H), 2.76-2.69 (m, 2H); 13C NMR (CD3-
OD) δ 204.2, 172.4, 136.1, 134.2, 129.1 (2 C:s), 128.9 (2 C:s),
128.2 (2 C:s), 127.9, 127.8 (2 C:s), 127.6, 66.2, 59.6, 35.6, 34.4,
27.2. Anal. (C19H21NO3‚HCl‚0.5 H2O): C, H, N.
Dim et h yl [(3S)-3-[N-(ter t-Bu t oxyca r b on yl)a m in o]-5-
h yd r oxy-2-oxop en tyl]-p h osp h on a te (21). Dimethyl meth-
ylphosphonate (1.6 mL, 15.0 mmol) was dissolved in THF (30
mL), and the solution was cooled to -78 °C under an
atmosphere of nitrogen. Butyllithium (1.6 M in hexane) (9.25
mL, 14.8 mmol) was added in one portion, and the solution
was stirred for 30 min before 20 (1.01 g, 5.0 mmol) dissolved
in THF (25 mL) was added by a double-ended needle. After
stirring for 1 h at -78 °C, 10% citric acid (16 mL) was added,
and the cooling bath was removed. When the solution had
reached room temperature, water (15 mL) was added and the
solution was extracted with diethyl ether (2 × 20 mL). The
combined ethereal extracts were washed with water (20 mL)
and brine (20 mL), dried (MgSO4), and concentrated. Purifica-
tion of the residue by flash chromatography on silica gel using
diethyl ether as eluent afforded 21 (740 mg, 48%) as a colorless
oil that was used in the subsequent reaction without further
purification or characterization.
ter t-Bu tyl (5S)-5-[N-(ter t-Bu toxyca r bon yl)a m in o]-7-h y-
d r oxy-4-oxo-(E)-2-h ep ten oa te (22). Phosphonate 21 (740
mg, 2.4 mmol) and lithium chloride (103 mg, 2.4 mmol) were
dissolved in acetonitrile (20 mL), and the solution was cooled
to 0 °C in an ice bath. Triethylamine (335 µL, 2.4 mmol) was
added, followed by tert-butyl glyoxylate (0.57 g, 4.3 mmol)
dissolved in acetonitrile (16 mL). Stirring was continued for 1
h at 0 °C after which 10% citric acid (50 mL) was added. The
solution was extracted with diethyl ether (2 × 100 mL), and
the combined ethereal extracts were washed with water (50
mL) and brine (50 mL) before drying (MgSO4) and concentra-
tion. Purification of the residue by flash chromatography on
Ben zyl (5S)-5-Am in o-6-m et h yl-4-oxo-h ep t a n oa t e (2).
Compound 13 (74 mg, 0.20 mmol) was deprotected as described
above for 1 to afford 58 mg of 2 (86%) as a white solid: Mp
1
135-140 °C; [R]D +8.6° (c 1.47, MeOH); H NMR (CD3OD) δ
7.40-7.25 (m, 5H), 5.15 (s, 2H), 4.21 (d, 1H, J ) 3.2 Hz), 3.10-
3.00 (m, 1H), 2.94-2.68 (m, 3H), 2.62-2.50 (m, 1H), 1.18 (d,
3H, J ) 6.8 Hz), 0.96 (d, 3H, J ) 7.2 Hz); 13C NMR (CD3OD)

Dipeptidomimetic Ketomethylene Isosteres
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19 4763
δ 204.4, 172.4, 136.1, 128.2 (2 C:s), 127.9, 127.8 (2 C:s), 66.1,
63.6, 34.3, 28.4, 27.1, 18.1, 14.9. Anal. (C15H21NO3‚HCl): C,
H, N.
UV analyses. All quantifications were based on standard
curves measured in the concentration range of 10-125 µM.
All standard curves yielded regression coefficients (r2) better
than 0.99. The detection and quantification limits (DL and QL,
respectively) for 1-5 and BnOH on the L-7450 diode array
detector were calculated from the standard curves using:
QL: 10×(intercept/slope) and DL: 3.3×(intercept/slope). The
DL values were 1.9, 7.4, 3.7, 8.5, 10.2, and 1.3 µM for 1-5
and BnOH, respectively, and the QL values were 5.8, 22.3,
11.2, 25.9, 30.9, and 0.4 µM for 1-5 and BnOH, respectively.
Ch em ica l Sta bility Stu d ies. HPLC-UV was performed
with a system consisting of a Waters pump Model 510, a
Waters variable-wavelength UV detector Lambda-Max model
481 spectrophotometer, and a Merck D-2000 GPC integrator.
For mobile phase systems and retention times, see Supporting
Information.
(2S,5S)-5-Am in o-2-(ben zyloxyca r bon yl-m eth yl)-4-oxo-
6-p h en yl-h exa n oic Acid (3). Compound 16 (104 mg, 0.20
mmol) was dissolved in CH2Cl2 (2 mL) and TFA (2 mL) and
stirred at room temperature for 2 h. The reaction mixture was
concentrated under reduced pressure and the residue extracted
with water (2 × 10 mL) and diethyl ether (2 × 10 mL). The
aqueous phase was concentrated, and the residue was dis-
solved in the mobile phase and purified with reversed phase
preparative HPLC. The combined fractions were evaporated
under reduced pressure to give 50 mg of 3 (53%) as a light
brownish oil. Also 8 mg (8%) of the (2R,5S)-epimer was
isolated: HPLC, major isomer, tR 4.3 min; minor isomer, tR
5.9 min; isomer ratio 90:10.
1
3: [R]D +29.7° (c 1.12, MeOH); H NMR (CD3OD) δ 7.44-
Biologica l Sta bility, Tr a n sp or t a n d Up ta k e Stu d ies.
7.30 (m, 10H), 5.15 (app d, 2H), 4.50-4.40 (m, 1H), 3.46-3.36
(m, 2H), 3.17 (dd, 1H, J ) 18.4, 7.2 Hz), 2.97 (dd, 1H, J )
14.4, 9.2 Hz), 2.85-2.66 (m, 3H); 13C NMR (CD3OD) δ 203.5,
175.0, 171.6, 136.0, 134.3, 129.1 (2 C:s), 128.9 (2 C:s), 128.2 (2
C:s), 127.9 (3 C:s), 127.6, 66.2, 59.5, 40.2, 35.9, 35.6, 34.8. Anal.
(C21H23NO5‚TFA‚H2O): C, H, N.
HPLC-UV was performed with
a Merck/Hitachi system
consisting of an L-6000 pump and an L-7450 diode array
detector. For mobile phase systems and retention times, see
Supporting Information.
Cell Assa ys. A. Ma ter ia ls. Gen er a l. Caco-2 cells were
obtained from the American Type Culture Collection (Manas-
sas, VA). All chemicals for buffer preparations were of labora-
tory grade, obtained from Life Technologies and Sigma.
Transwell plates (12-well) were from Corning Costar Corpora-
tion.
Affin ity Stu d ies. [14C]-Gly-Sar (specific activity: 49.94
mCi/mmol) and [3H]-mannitol (specific activity: 51.50 mCi/
mmol) were obtained from New England Nuclear. Transepi-
thelial electrical resistance (TEER) was measured in tissue
resistance measurement chambers (Endohm) with a voltohm-
meter (EVOM), both from World Precision Instruments. The
shaking plate used for cell culture experiments was a KS 10
DIGI shaker from Edmund Bu¨hler. Radioactivity was deter-
mined in a Packard TriCarb liquid scintillation counter, using
Ultima Gold scintillation fluid from Packcard.
(2S,5S)-5-Am in o-2-(ben zyloxycar bon yl-m eth yl)-6-m eth -
yl-4-oxo-h ep ta n oic Acid (4). Deprotection of 19 (93 mg, 0.20
mmol) as described above for 3 gave 50 mg of 4 (59%) as a
light brownish oil. Also 10 mg (12%) of the (2R,5S)-epimer was
isolated: HPLC, major isomer, tR 2.4 min; minor isomer, tR
2.9 min; isomer ratio 84:16.
1
4: [R]D +43.2° (c 1.14, MeOH); H NMR (CD3OD) δ 7.41-
7.33 (m, 5H), 5.17 (app d, 2H), 4.16-4.10 (m, 1H), 3.40-3.35
(m, 1H), 3.21 (dd, 1H, J ) 18.8, 8.4 Hz), 2.86-2.68 (m, 3H),
2.58-2.48 (m, 1H), 1.17 (d, 3H, J ) 7.2 Hz), 0.95 (d, 3H, J )
6.8 Hz); 13C NMR (CD3OD) δ 203.7, 175.0, 171.6, 136.0, 128.2
(2 C:s), 127.95 (2 C:s), 127.92, 66.2, 63.5, 40.4, 35.8, 34.9, 28.4,
18.2, 14.8. Anal. (C17H23NO5‚TFA): H, N; C: calcd, 52.41;
found, 51.8.
(5R/S)-5-Am in o-6-(ben zyloxyca r bon yl)-h exa n oic Acid
(5). Compound 25 (0.18 mmol, 78.7 mg) was dissolved in CH2-
Cl2 (2 mL) and TFA (2 mL) and stirred at room temperature
for 2 h. The reaction mixture was concentrated under reduced
pressure and the residue extracted with water (2 × 10 mL)
and diethyl ether (2 × 10 mL). The aqueous phase was
concentrated to afford 69 mg of 5 (97%) as a brownish oil: 1H
NMR (CD3OD) δ 7.45-7.35 (m, 5H), 5.24 (s, 2H), 4.50 (dd, 1H,
J ) 7.2, 4.4 Hz), 3.30 (dd, 1H, J ) 18.0, 4.0 Hz), 3.10 (dd, 1H,
J ) 18.2, 7.4 Hz), 2.94-2.76 (m, 2H), 2.67 (app t, 2H); 13C NMR
(CD3OD) δ 202.9, 174.4, 169.5, 135.4, 128.3 (2 C:s), 128.24 (2
C:s), 128.18, 67.2, 55.2, 33.2, 33.0, 27.2. Anal. (C14H17NO5‚0.5
TFA‚H2O): C, H, N.
Sta bility Stu d ies. A. Ch em ica l Sta bility. The buffers
used for stability experiments were HBSS supplemented with
0.05% BSA and 10 mM MES (pH 6.0) (MES-buffer) and 10
mM HEPES (pH 7.4) (HEPES-buffer). The reaction was
initiated by adding 100 µL stock solution to 10 mL preequili-
brated buffer solution to a final concentration of approximately
10-4-10-5 M, and the degradation was studied for several t1/2
or over a 48-h period for slower degradations. During the
experiments, the solutions were kept in a water bath at 37 °C
and at various times, samples were collected and chromato-
graphed immediately. The rates of degradation were deter-
mined using HPLC procedures capable of separating BnOH
from the model prodrugs.
B. Biologica l Sta bility. 10 µL samples were taken from
the apical solution at the start and end (t ) 0 and 120 min,
respectively) of the transport experiments in Caco-2 cell
culture (see below). The samples were transferred to HPLC
vials and frozen for further analysis by HPLC-UV.
HP LC-UV Qu a n tifica tion . Gen er a l. For the stability,
transport and uptake studies quantification was based on high-
performance liquid chromatography (HPLC) and UV-detection
(HPLC-UV). A Waters Spherisorb S5OdS2 reversed-phase
analytical column (5 µm, 250 × 4.6 mm), a flow rate of 1 mL/
min, and UV-detection at 210 nm were used for all HPLC-
Tr a n sp or t a n d Up t a k e St u d ies. Gly-Pro was obtained
from Sigma, and [3H]-mannitol from Amersham. The centri-
fuge was a 1-15K from Sigma, and the sonicator was a
Branson sonifier cell disruptor B15.
B. Cell Cu ltu r e. Caco-2 cells were cultured as previously
described.42 Briefly, cells were seeded in culture flasks and
passaged in Dulbecco’s Modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum, penicillin/strep-
tomycin (100 U/mL and 100 µg/mL, respectively), 1% L-
glutamine, and 1% nonessential amino acids. Caco-2 cells were
seeded onto tissue culture treated 12-well Transwell plates (1.0
cm2, 0.4 µm pore size) at a density of 105 cells × cm-2
.
Monolayers were grown in an atmosphere of 5% CO2-95% O2
at 37 °C. Growth media were replaced every other day. TEER
was measured at room temperature before the experiment
on day 25-27 post seeding giving TEER ) 354 ( 41 Ω ×
cm-2
.
C. Affin ity Stu d ies. The affinity experiments were per-
formed as previously described by Nielsen et al.20 and Våbenø
et al.14 Briefly, the experiment was initiated by adding 0.5 mL
of MES-buffer containing 0.5 µCi of [14C]-Gly-Sar and various
amounts of the respective compounds to the apical side. Due
to the low solubility of 1 in water, 1 was initially dissolved in
DMSO and then further diluted in buffer to give the relevant
concentration used in the affinity studies. The final DMSO
concentration was less than 1.0%, which did not affect the
uptake of [14C]-Gly-Sar. A 1.0 mL amount of HEPES buffer
was added to the basolateral side of each cell monolayer.
During the 5 min incubation, the plates were circularly and
continuously shaken. In all experiments the investigated
compounds were only applied on the apical side. The temper-
ature was maintained at 37 °C. The uptake of 0.5 µCi [14C]-
Gly-Sar was terminated by gentle suction of the uptake
medium followed by three washes of the monolayer with ice-
cold HBSS. Following the washing step the cells were cut from
the Transwell support, placed into scintillation vials and 2 mL
of scintillation fluid was added. The cell-associated radioactiv-

4764 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19
Våbenø et al.
ity was counted via liquid scintillation spectrometry. For these
experiments, the Hill-factor n was in the range of 0.83-1.31,
and Umax was between 0.89 and 0.99.
and the residual concentration of model prodrug at a given
time, t:
D. Tr an sepith elial Tr an spor t acr oss Caco-2 Cell Mon o-
la yer s. Transport of 1-5 and [14C]-Gly-Sar at an apical
concentration of 1 mM was measured using MES-buffer as
apical media and HEPES-buffer as basolateral media. Simi-
larly, the transport of [3H]-mannitol was measured at an apical
concentration of 1 µCi/mL (1.96 × 10-5 M), and BnOH was
measured at apical concentrations of 100, 250, and 500 µM.
For 1, the initial stock solution was made in DMSO, and this
was then diluted to 1 mM giving a final DMSO concentration
of 0.5%. The cell monolayers were rinsed once in prewarmed
HBSS. The cells were then incubated for 15 min with MES-
and HEPES-buffer on the apical and basolateral sides, respec-
tively, on a shaking plate maintained at 37 °C. After this
incubation, the buffers were replaced with the above-men-
tioned concentrations of the compounds in MES-buffer on the
apical side and fresh HEPES-buffer on the basolateral side.
10 µL samples were taken from the apical solution at t ) 0
and 120 min, and 120 µL samples were taken at 20 min
intervals from the basolateral solution and replaced with fresh
buffer (t ) 40, 60, 80, 100, and 120 min; for BnOH: t ) 20,
40, 60, 80, and 100 min). Transport was also measured in the
presence of 20 mM Gly-Pro in the apical solution (except for
BnOH), in the same manner as described above. Samples were
transferred to HPLC vials and frozen for further analysis by
HPLC-UV. Samples containing radioisotopes were treated as
described in section C, Affinity Studies.
E . In t r a cellu la r Up t a k e in Ca co-2 Cell Mon ola yer s.
After completion of the 120 min transport study, the cells were
rinsed in cold HBSS and detached from the Transwell support
using 60 µL of 0.1% Triton X in PBS. Afterward the cell
suspension was frozen. Then it was thawed and 120 µL of
MilliQ water added, and the cell preparation was lysed using
a Branson cell disruptor. The cell suspension was centrifuged
for 15 min at 14 000 rpm, and the clear supernatant was used
for HPLC-UV analysis.
Da ta An a lysis. 1. Tr a n sp or t Kin etics. A. Affin ity.
Affinity for hPEPT1 in Caco-2 cells was determined as inhibi-
tion of [14C]-Gly-Sar uptake in the presence of varying con-
centrations of the respective compound. The degree of inhibi-
tion was fitted to a Michaelis-Menten type equation:
d[model prodrug]
-
) k[model prodrug]t
(4)
dt
kobs was calculated from first-order degradation kinetics. kobs
values were calculated from the linear part of the curve
showing lnAt
was calculated as t1/2 ) ln2/kobs
(A ) area) as a function of time. The half-life
) 0
.
3. Sta tistics An a lysis. Unless stated otherwise, values are
given as means ( SE, and the statistical significance of the
results was determined using two-tailed Student’s t-test using
GraphPad InStat v3.05. p < 0.05 was considered significant.
Affinity, transport, and uptake experiments were performed
in duplicates (N ) 2), in three individual cell passages
(n ) 3). Stability studies were done in triplicates (n ) 3).
Ackn owledgm en t. Financial support for this project
was obtained from the Norwegian Research Council
(128256/420), the Swedish Research Council (621-2001-
1431), the Knut and Alice Wallenberg Foundation
(98.176), and the Danish Medicinal Research Council
via the Center for Drug Design and Transport and via
project grant #22-01-0310. We thank Morten Moe and
Trude Anderssen for excellent assistance with MS-MS
and analytical and preparative HPLC, and Susanne N.
Sørensen, Birgitte Eltong, and Bettina Dinitzen for
doing the cell culturing.
Ap p en d ix
Abbreviations: Boc: tert-butoxycarbonyl; Caco-2: hu-
man colonic adenocarcinoma cell line; CoMFA: com-
parative molecular field analysis; CoMSIA: comparative
molecular similarity index analysis; hPEPT1: human
di-/tripeptide transporter 1; Papp: apparent permeability
coefficient (distance × time-1); rPEPT2: rat di-/tripep-
tide transporter 2; Xaa: any amino acid.
(1 - (U/U0))max × [I]n
Su p p or tin g In for m a tion Ava ila ble: Mobile phase sys-
tems and retention times for HPLC analyses in the stability,
transport, and uptake studies; elemental analysis data for
compounds 1-5. This material is available free of charge via
the Internet at http://pubs.acs.org.
1 - (U/U0) )
(1)
IC50 + [I]n
n
where U ) uptake of [14C]-Gly-Sar, U0 ) uptake of [14C]-Gly-
Sar at zero inhibitor concentration, IC50 ) the concentration
required to inhibit the maximum uptake by 50% (µM), [I] )
compound concentration (µM), and n ) Hill-factor. Ki values
were calculated as described by Cheng and Prusoff43 using a
Km value for Gly-Sar of 1.1 mM, obtained in our laboratory.
B. Tr a n sep ith elia l Tr a n sp or t. The flux values (J ) of the
compounds were calculated by:
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