Unequivocal synthesis of (Z)-alkene and (E)-fluoroalkene dipeptide isosteres to probe structural requirements of the peptide transporter PEPT1

Article abstract of DOI:10.1021/ol052781k

Described is a novel synthetic route for dipeptide isosteres containing (Z)-alkene and (E)-fluoroalkene units as cis-amide bond equivalents via organocopper-mediated reduction of γ-acetoxy- or γ,γ-difluoro- α,β-unsaturated-δ-lactams. The synthesized isost

Full text of DOI:10.1021/ol052781k

ORGANIC
LETTERS
2006
Vol. 8, No. 4
613-616
Unequivocal Synthesis of (Z)-Alkene and
(E)-Fluoroalkene Dipeptide Isosteres To
Probe Structural Requirements of the
Peptide Transporter PEPT1
Ayumu Niida,† Kenji Tomita,† Makiko Mizumoto,† Hiroaki Tanigaki,†
Tomohiro Terada,‡ Shinya Oishi,† Akira Otaka,†,§ Ken-ichi Inui,‡ and
Nobutaka Fujii*,†
Graduate School of Pharmaceutical Sciences, Kyoto UniVersity, Sakyo-ku, Kyoto
606-8501, Japan, Department of Pharmacy, Kyoto UniVersity Hospital, Sakyo-ku,
Kyoto 606-8507, Japan, and Graduate School of Pharmaceutical Sciences, The
UniVersity of Tokushima, Tokushima 770-8505, Japan
nfujii@pharm.kyoto-u.ac.jp
Received November 17, 2005
ABSTRACT
Described is a novel synthetic route for dipeptide isosteres containing (Z)-alkene and (E)-fluoroalkene units as cis-amide bond equivalents via
organocopper-mediated reduction of -acetoxy- or -difluoro- -unsaturated- -lactams. The synthesized isosteres were evaluated in terms
γ
γ
,
γ
r,
â
δ
of their affinities for the peptide transporter PEPT1. trans-Amide isosteres tended to possess higher affinities for PEPT1 as compared to the
corresponding cis-amide bond equivalents.
In postgenomic drug discovery research, the rapid elucidation
of structural requirements of the ligands for newly identified
drug targets (e.g., GPCRs, enzymes, transporters, etc.) is
strongly needed in the arena of medicinal chemistry.1 Many
protein drug targets interact with proteinic or peptidic ligands.
Therefore, development of peptidomimetic small molecules
is important for investigating criteria for the mutual molecular
recognition.2 Alkene-type dipeptide isosteres represent po-
tential amide bond mimetics (Figure 1).3 Fluoroalkene
dipeptide isosteres were designed as electrostatically favor-
able mimetics as compared to simple alkene isosteres.4 These
isosteres have structural similarities with the parent peptides
(2) (a) Burgess, K. Acc. Chem. Res. 2001, 34, 826. (b) Bursavich M.
G.; Rich, D. H. J. Med. Chem. 2002, 45, 541. (c) Hruby, V. J. J. Med
Chem. 2003, 46, 4215.
(3) (a) Oishi, S.; Kamano, T.; Niida, A.; Odagaki, Y.; Hamanaka, N.;
Yamamoto, M.; Ajito, K.; Tamamura, H.; Otaka, A.; Fujii, N. J. Org. Chem.
2002, 67, 6162. (b) Wipf, P.; Xiao, J. Org. Lett. 2005, 7, 103. (c) Xiao, J.;
Weisblum, B.; Wipf, P. J. Am. Chem. Soc. 2005, 127, 5742.
(4) (a) Abraham, R. J.; Ellison, S. L. R.; Schonholzer, P.; Thomas, W.
A. Tetrahedron 1986, 42, 2101. (b) Allmendinger, T.; Furet, P.; Hunger-
bu¨hler, E.; Tetrahedron Lett. 1990, 31, 7297. (c) Allmendinger, T.; Furet,
P.; Hungerbu¨hler, E.; Tetrahedron Lett. 1990, 31, 7301. (d) Otaka, A.;
Watanabe, J.; Yukimasa, A.; Sasaki, Y.; Watanabe, H.; Kinoshita, T.; Oishi,
S.; Tamamura, H.; Fujii, N. J. Org. Chem. 2004, 69, 1634. (e) V. d. Veken,
P.; Senten, K.; Kerte`sz, I.; D. Meester, I.; Lambeir, A.-M.; Maes, M.-B.;
Scharpe`, S.; Haemers, A.; Augustyns, K. J. Med. Chem. 2005, 48, 1768.
(f) Nakamura, Y.; Okada, M.; Sato, A.; Horikawa, H.; Koura, M.; Saito,
A.; Taguchi, T. Tetrahedron 2005, 61, 5741.
† Graduate School of Pharmaceutical Sciences, Kyoto University.
‡ Kyoto University Hospital.
§ The University of Tokushima.
(1) (a) Drews, J. Science 2000, 287, 1960. (b) Klabunde, T.; Hessler, G.
ChemBioChem 2002, 3, 928. (c) Tyndall, J. D. A.; Pfeiffer, B.; Abbenante,
G., Fairlie, D. P. Chem. ReV. 2005, 105, 793.
10.1021/ol052781k CCC: $33.50
© 2006 American Chemical Society
Published on Web 01/24/2006

Scheme 1. Synthetic Route for (Z)-Alkene and
(E)-Fluoroalkene Dipeptide Isosteres
Figure 1. Cis/trans equilibrium of peptide bond and the corre-
sponding alkene- or fluoroalkene isosteres.
via 3,6-dihydropyridin-2-ones, in which the â,γ-(Z)-alkene
unit was constructed by Grubbs’ RCM after condensation
of chiral allylamines with chiral vinyl acetic acids.8 The
present method provides a new entity for the synthesis of
(Z)-alkene isosteres in a divergent fashion. That is comple-
mentary to their method as well as our alternative method
based on organocoper-mediated anti-SN2′ reaction.9 It is
noteworthy that to our knowledge, this is the first unequivocal
synthesis of (E)-fluoroalkene dipeptide isosteres.
Substrates for the organocopper-mediated reduction were
synthesized by the sequence of reactions shown in Scheme
2. Synthesis of acetate 6 started from a known phenylalanine
derivative 1.9 Conversion of the N-protecting group of 1 to
N-Ns (Ns ) 2-nitrobenzenesulfonyl)10 followed by O-
protection with a TBS group gave N-Ns amide derivative 2.
Treatment of 2 with DMB (2,4-dimethoxybenzyl) alcohol
under Mitsunobu conditions afforded the N-DMB sulfon-
amide 3. After removal of the N-Ns group of 3, acylation of
the resulting secondary amine followed by O-TBS depro-
tection gave the acrylamide derivative 4. RCM reaction of
4 with Grubbs’ ruthenium catalyst11 proceeded smoothly at
room temperature to yield the γ-hydroxy-R,â-unsaturated
δ-lactam 5. Lactam 5 was converted to acetate 6 by Ac2O
treatment in the presence of pyridine.
and resist enzymatic degradation. Peptide bonds exist in cis/
trans equilibrium, while alkene isosteres serve as defined
trans-amide or cis-amide equivalents, which do not isomerize
to each other. Cis/trans isomerization of peptide bonds
(especially Xaa-Pro sequences) in several bioactive peptides
tends to play an important role in their conformations and
biological activities.5 Therefore, alkene and fluoroalkene
isosteres might be promising tools for conformational
analysis of bioactive peptides and proteins.6 We have been
engaged in the development of synthetic methodologies for
(E)-alkene or (Z)-fluoroalkene dipeptide isosteres as trans-
amide bond equivalents utilizing organocopper reagents or
SmI2. However, the lack of efficient synthetic methodologies
for the preparation of (Z)-alkene or (E)-fluoroalkene dipeptide
isosteres as cis-amide bond equivalents has limited an
extensive application of alkene and fluoroalkene isosteres
in the analysis of amide bond geometries in bioactive
peptides and proteins. In this paper, we describe a new
synthetic approach for the preparation of (Z)-alkene or (E)-
fluoroalkene dipeptide isosteres. We also include the ap-
plication of these isosteres to probe structural requirements
of the peptide transporter PEPT1.
Our synthetic routes for the preparation of (Z)-alkene and
(E)-fluoroalkene isosteres are depicted in Scheme 1. We
envisioned key synthetic intermediates B would be synthe-
sized by organocopper-mediated reduction of lactam A with
predominant formation of â,γ-(Z)-alkenes or (E)-fluoroalk-
enes as cis-amide equivalents. This strategy could be
expanded into consecutive one-pot reduction/R-alkylation
methodologies for the synthesis of structurally diverse
R-alkylated (Z)-alkene and (E)-fluoroalkene dipeptide iso-
steres.7 First, we synthesized γ-acetoxy- or γ,γ-difluoro-R,â-
unsaturated lactams and examined the organocopper-medi-
ated reduction of these substrates to confirm whether this
approach was applicable to the synthesis of cis-amide bond
isosteres.
γ,γ-Difluoro-R,â-unsaturated δ-lactam 12 was synthesized
from the â-amino ester 10, which was prepared from
phenylacetaldehyde 7 and the chiral amine 8 via rhodium
catalized diastereoselective Reformatsky-Honda reaction.4d,12
After DIBAL-H treatment of 10, (Z)-selective Horner-
Wadsworth-Emmons reaction13 of the resulting aldehyde
gave (Z)-enoate 11 in 72% yield with a concomitant
formation of small amount of (E)-isomer (4%). After
deprotection of the Boc and t-Bu groups of 11 using 4 M
HCl in dioxane, cyclization with EDC gave the desired
lactam 12.
(8) Boucard, V.; S.-Dorizon, H.; Guibe´, F. Tetrahedron 2002, 58, 7275.
(9) Niida, A.; Oishi, S.; Sasaki, Y.; Mizumoto, M.; Tamamura, H.; Fujii,
N.; Otaka, A. Tetrahedron Lett. 2005, 46, 4183.
(10) Fukuyama, T.; Jow, C.-K.; Cheung, M. Tetrahedron Lett. 1995, 36,
6373.
Guibe´ et al. reported a similar but inherently different
convergent approach to the synthesis of (Z)-alkene isosteres
(11) Scholl, M.; Ding, S.; Lee, C. W.; Grubbs, R. H. Org. Lett. 1999, 1,
953.
(12) Honda, T.; Wakabayashi, H.; Kanai, K. Chem. Pharm. Bull. 2002,
50, 307.
(5) Dugave, C.; Demange, L. Chem. ReV. 2003, 103, 2475.
(6) Wang, X. J.; Xu, B.; Mullins, A. B.; Neiler, F. K.; Etzkorn, F. A. J.
Am. Chem. Soc. 2004, 126, 15533.
(7) Otaka, A.; Watanabe, H.; Yukimasa, A.; Oishi, S.; Tamamura, H.;
Fujii, N. Tetrahedron Lett. 2001, 42, 5443, and references cited thererin.
(13) Ando, K.; Oishi, T.; Hirama, M.; Ohno, H.; Ibuka, T. J. Org. Chem.
2000, 65, 4745.
614
Org. Lett., Vol. 8, No. 4, 2006

3LiBr also gave the desired reduction product 15 in excellent
yield (92%).
Scheme 2. Synthesis of Requisite Substrates for
Organocopper-Mediated Reduction
Next, we carried out the hydrolysis of the amide bond in
lactams 14 and 15 to accomplish the synthesis of the cis-
amide bond isosteres. Lactams 14 and 15 were converted to
lactim ethers using Me3O‚BF4. Hydrolysis of the lactim
ethers15 under acidic conditions followed by HPLC purifica-
tion using 0.1% TFA aqueous MeCN gave Phe-Gly type (Z)-
alkene dipeptide isostere (Phe-ψ[(Z)-CHdCH]-Gly 16) and
(E)-fluoroalkene dipeptide isostere (Phe-ψ[(E)-CFdCH]-Gly
17),16 respectively as TFA salts.
The above organocopper-mediated reduction is applicable
to consecutive one-pot R-alkylation (Scheme 4). After
Scheme 4. Synthesis of R-Substituted (Z)-Alkene and
(E)-Fluoroalkene Dipeptide Isosteres Utilizing
Organocopper-Mediated Reduction-Alkylation
Next we examined the organocopper-mediated reduction
of lactams 6 and 12 (Scheme 3). The reaction of acetate 6
with Me3CuLi2‚LiI‚3LiBr14 proceeded smoothly at -78 °C
to yield the â,γ-unsaturated lactam 13 in a good yield (88%).
The DMB group of lactam 13 was easily removed using
TFA. Treatment of difluorolactam 12 with Me3CuLi2‚LiI‚
reduction of lactam 6 with Me3CuLi2‚LiI‚3LiBr, the resulting
metal enolate was trapped by Bn-Br to yield the trans-R-
substituted diketopiperazine mimetic 18 as a main product.
After deprotection of the DMB group using TFA, the
resulting lactam 19 was subjected to ring-opening followed
by N-Boc protection to yield Boc-L-Phe-ψ[(Z)-CHdCH]-D-
Phe-OMe 20 in 40% yield with a small amount of R-epimer-
ized product (13%). Boc-D-Phe-ψ[(E)-CFdCH]-L-Phe-OMe
23 was also synthesized from lactam 21 by a procedure
Scheme 3. Synthesis of Phe-Gly Type (Z)-Alkene- and
(E)-Fluoroalkene Dipeptide Isosteres via
Organocopper-Mediated Reduction of Lactams 6 and 12
(14) Single electron transfer (SET) mechanism has been proposed as one
of the plausible mechanisms of organocopper-mediated reduction. The
electron-transfer potency of Me3CuLi2 was proved to be higher than that
of the corresponding Gilman-type reagent such as Me2CuLi. See: Chounan,
Y.; Horino, H.; Ibuka, T.; Yamamoto, Y. Bull. Chem. Soc. Jpn. 1997, 70,
1953.
(15) Scho¨llkopf, U.; Hartwig, W.; Pospischil, K.-H.; Kehne, H. Synthesis
1981, 966.
(16) Coupling constants of 17 and 23 (3JHF ) 20.7 and 20.5 Hz,
respectively) are consistent with those of R-fluorovinyl groups possessing
a (E)-configuration (3JHFtrans ) 18-22 Hz). See ref 4b.
Org. Lett., Vol. 8, No. 4, 2006
615

similar to that for the synthesis of isostere 20.17 Precise
stereocontrol and introduction of other functional groups at
the R-position are under investigation.
Table 1. Ki Values of Phe-Gly and Various Isosteres Based on
Inhibition of [3H]Gly-Sar Uptake by PEPT1 in Caco-2 Cell
Next, we investigated whether the di/tri-peptide transporter,
PEPT1 recognized synthetic Phe-Gly type isosteres as
substrates. PEPT1 is a membrane protein which has 12
transmembrane domains and mediates intestinal uptake of
not only di-/tripeptides but also several drugs structually
related to small peptides such as â-lactam antibiotics.18
Structure-activity relationship studies of various substrates
for PEPT1 have been carried out in order to apply this
transporter to develop orally bio-available drugs. However
precise recognition mechanisms have not been elucidated.
We envisioned that alkene dipeptide isosteres would be
useful tools for analysis of recognition mechanisms of PEPT1
because of their structural similarity to parent dipeptides. We
also expected that the potency of dipeptide isosteres as amide
bond mimetics could be evaluated by use of the PEPT1
dipeptide transport system.
compd
X
Ki (mM)
Phe-Gly
16
17
24
25
-CO-NH-
0.205
>10.0
>10.0
0.853
1.34
-ψ[(Z)-CHdCH]-
-ψ[(E)-CFdCH]-
-ψ[(E)-CHdCH]-
-ψ[(Z)-CFdCH]-
-ψ[CH2-CH2]-
-ψ[CF2-CH2]-
26
27
2.17
1.67
observed (24 vs 25). Further investigation is required for
verification of the effect of fluorine as a carbonyl oxygen
mimic.
The bioactivities of synthetic Phe-Gly isosteres for PEPT1
were determined by the inhibition of [3H]Gly-Sar uptake in
PEPT1-expressing Caco-2 cell in comparison with trans-
amide type isosteres, 24, 25, and other related compounds
(see the Supporting Information attached). Inhibition con-
stants (Ki) of parent dipeptide Phe-Gly and its isosteres are
shown in Table 1. trans-Amide equivalents 24 and 25
possessed good affinity for PEPT1 corresponding to the
parent dipeptide (Ki: Phe-Gly, 0.205 mM; 24, 0.853 mM;
25, 1.34 mM). It is of note that affinities of the cis-amide
equivalents 16 and 17 for PEPT1 were more than 10 times
weaker than those of trans-isomers. These data suggest that
PEPT1 predominantly recognizes trans-amide conformations
of dipeptides. This is in good accordance with the previous
report by Brandsch et al., in which PEPT1 recognized trans-
conformation of Ala-ψ[CS-N]-Pro.19 Conformationally flex-
ible analogues 26 and 27 retained moderate affinity in
comparison with cis-amide equivalents. Presumably, ana-
logues 26 and 27 could exist as trans-amide-like conformers,
which were favorable for the interaction with PEPT1, due
to their flexibility. Contrary to our expectation, an increase
of affinity by the introduction of fluoroalkene unit was not
In conclusion, we presented a novel unambiguous synthetic
route for the syntheses of (Z)-alkene and (E)-fluoroalkene
dipeptide isosteres as cis-amide bond mimetics via organo-
copper-mediated reduction of γ-acetoxy- or γ,γ-difluoro-R,â-
unsaturated δ-lactams. We also carried out comparative
studies of affinities for peptide transpoter PEPT1 between
the cis-amide mimetics and the corresponding trans-amide
isosteres, and found that peptide transporter PEPT1 pre-
dominantly recognizes trans-amide bond conformations in
dipeptides. Synthetic studies on various R-substituted (E)-
fluoroalkene isosteres and further structure-activity-relation-
ship studies on dipeptide mimetics for PEPT1 are currently
proceeding.
Acknowledgment. We thank Dr. Terrence R. Burke, Jr.,
NCI, NIH, for proofreading this manuscript. This research
was supported in part by 21st Century COE Program
“Knowledge Information Infrastructure for Genome Sci-
ence”, a Grant-in-Aid for Scientific Research from the
Ministry of Education, Culture, Sports, Science and Technol-
ogy, Japan, the Japan Society for the Promotion of Science
(JSPS), and the Japan Health Science Foundation. A.N. is
grateful for Research Fellowships from the JSPS for Young
Scientists.
(17) In 1H NMR experiments, R-protons (position-3) of 3,6-trans isomers
such as 18 or trans-22 appeared upfield from the corrresponding R-protons
of 3,6-cis isomeres. See ref 9.
(18) (a) Våbenø, J.; Lejon, T.; Nielsen, C. U.; Steffansen, B.; Chen, W.;
Ouyang, H.; Borchardt, R. T.; Luthman, K. J. Med. Chem. 2004, 47, 1060.
(b) Våbenø, J.; Nielsen, C. U.; Ingebrigtsen, T.; Lejon, T.; Steffansen, B.;
Luthman, K. J. Med. Chem. 2004, 47, 4755. (c) Terada, T.; Inui, K. Curr.
Drug Metab. 2004, 5, 85. (d) Biegel, A.; Gebauer, S.; Hartrodt, B.; Brandsh,
M.; Neubert, K.; Thondorf, I. J. Med. Chem. 2005, 48, 4410.
(19) Brandsh, M.; Thunecke, F.; Ku¨llertz, G.; Schutkowski, M.; Fischer,
G.; Neubert, K. J. Biol. Chem. 1998, 273, 3861.
Supporting Information Available: Synthesis of com-
pounds 24-27. Experimental procedures and spectral data.
This material is available free of charge via the Internet at
http://pubs.acs.org.
OL052781K
616
Org. Lett., Vol. 8, No. 4, 2006