The tandem chain extension aldol reaction used for synthesis of ketomethylene tripeptidomimetics targeting hPEPT1

Article abstract of DOI:10.1016/j.bmcl.2011.05.108

The rationale for targeting the human di-/tripeptide transporter hPEPT1 for oral drug delivery has been well established by several drug and prodrug cases. The aim of this study was to synthesize novel ketomethylene modified tripeptidomimetics and to investigate their binding affinity for hPEPT1. Three related tripeptidomimetics of the structure H-Phe-ψ[COCH₂]- Ser(Bz)-X_aa-OH were synthesized applying the tandem chain extension aldol reaction, where amino acid derived β-keto imides were stereoselectively converted to α-substituted γ-keto imides. In addition, three corresponding tripeptides, composed of amide bonds, were synthesized for comparison of binding affinities. The six investigated compounds were all defined as high affinity ligands (K_i-values <0.5 mM) for hPEPT1 by measuring the concentration dependent inhibition of apical [ ¹⁴C]Gly-Sar uptake in Caco-2 cells. Consequently, the ketomethylene replacement for the natural amide bond and α-side chain modifications appears to offer a promising strategy to modify tripeptidic structures while maintaining a high affinity for hPEPT1.

Full text of DOI:10.1016/j.bmcl.2011.05.108

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4597–4601
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The tandem chain extension aldol reaction used for synthesis of ketomethylene
tripeptidomimetics targeting hPEPT1
Karina Thorn a, , Carsten Uhd Nielsen b, Palle Jakobsen c, Bente Steffansen b, Charles K. Zercher d,
⇑
Mikael Begtrup e
a Protein Chemistry, Biogen Idec Hemophilia, 9 Fourth Avenue, Waltham, MA 02451, USA
b Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark
c Insulin Chemistry, Novo Nordisk A/S, Måløv Byvej 200, 2760 Måløv, Denmark
d Department of Chemistry, University of New Hampshire, 23 Academic Way, Durham, NH 03824, USA
e Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
The rationale for targeting the human di-/tripeptide transporter hPEPT1 for oral drug delivery has been
well established by several drug and prodrug cases. The aim of this study was to synthesize novel ketom-
ethylene modified tripeptidomimetics and to investigate their binding affinity for hPEPT1. Three related
Received 3 April 2011
Revised 26 May 2011
Accepted 26 May 2011
Available online 6 June 2011
tripeptidomimetics of the structure H-Phe-
dem chain extension aldol reaction, where amino acid derived b-keto imides were stereoselectively con-
verted to -substituted -keto imides. In addition, three corresponding tripeptides, composed of amide
w[COCH2]-Ser(Bz)-Xaa-OH were synthesized applying the tan-
a
c
Keywords:
hPEPT1
Ketomethylene
Peptides
Peptidomimetics
TCEA reaction
bonds, were synthesized for comparison of binding affinities. The six investigated compounds were all
defined as high affinity ligands (Ki-values <0.5 mM) for hPEPT1 by measuring the concentration depen-
dent inhibition of apical [14C]Gly-Sar uptake in Caco-2 cells. Consequently, the ketomethylene replace-
ment for the natural amide bond and
a-side chain modifications appears to offer a promising strategy
to modify tripeptidic structures while maintaining a high affinity for hPEPT1.
Ó 2011 Elsevier Ltd. All rights reserved.
The human intestinal peptide transporter, hPEPT1, facilitates
the transepithelial transport of dipeptides, tripeptides, and a num-
ber of peptidomimetic drugs.1–4 The active transport via hPEPT1
over the apical membrane is mediated by an electrochemical pro-
ton gradient, and results in a co-transport of protons and sub-
strates.5 hPEPT1 is expressed in the luminal membrane of the
small intestine, and is responsible for the natural absorption of di-
gested protein products from the diet. Consequently, the substrate
recognition of hPEPT1 is broad with mM affinities, and it possesses
a high transport capacity. These features make the transporter an
excellent target for exploitation as a transport system for drug
delivery of small peptidomimetics and prodrugs.6,7
The main challenge with employing peptides as prodrugs or as
potential drugs is their low stability after oral administration. Pro-
teolytic enzymes and exopeptidases distributed in the gastrointes-
tinal (GI) tract rapidly decompose the peptides before the
therapeutic action has occurred. Fortunately, the oral bioavailabil-
ity of peptidic drugs can be increased by chemical modifications.8
Enzymatic degradation of peptides can be avoided by incorpora-
a
-side chains or by replacement of amide bonds with non-hydro-
lysable entities. N-Methylated amides, ketomethylene, hydroxy-
ethylene, and thioamides are among the most commonly used
amide bond bioisosteres.9–12
The choice of amide bond bioisostere in this study was based on
known structural requirements necessary for ligands and sub-
strates interacting with hPEPT1. A previous study of a series of
H-Phe-Gly-OH dipeptidomimetics revealed that the ketomethyl-
ene analog and the native dipeptide possessed a similar affinity
and translocation for hPEPT1, whereas the hydroxyethylene and
hydroxyethylidene analogues were poor substrates.13,14 Conse-
quently, the carbonyl part of the amide bond mimetic appears to
be significant for the hPEPT1 interaction.
We previously suggested H-Phe-Ser-Ala-OH as a lead promoiety
targeting hPEPT1 due to high affinity and translocation.15 In this
tripeptide, the serine
a-side chain is utilized to provide an ester
linkage to a therapeutically active substance with low bioavailabil-
ity containing a carboxylic acid functionality. Phenylalanine serves
as a hydrophobic N-terminus and alanine as a small, uncharged
C-terminal amino acid. The aim of this study was to evaluate if
chemical modifications of the lead structure are tolerated in terms
of hPEPT1 binding. We have thus synthesized the ketomethylene
tion of
D-configured amino acids, by introduction of unnatural
⇑
Corresponding author at present address: Protein Chemistry, Biogen Idec
Hemophilia, 9 Fourth Avenue, Waltham, MA 02451, USA.
peptidomimetic H-Phe-
w[COCH2]-Ser(Bz)-Ala-OH (14A) in which
both an amide bond replacement and a side chain modification
E-mail address: karina.thorn@biogenidec.com (K. Thorn).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.108

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K. Thorn et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4597–4601
Ph
have been incorporated. With the purpose of optimizing the con-
struct, two related peptidomimetics 14B–C were synthesized. In
Ph
Ph
b
a
84 %
PMB
PMB
88 %
these the methyl
a
-side chain of alanine was replaced with propyl
N
COOH
N
H
COOH
H2N
COOH
or cyclohexyl. For comparison of binding affinities, the reference
tripeptides 20A–C were synthesized (Fig. 1).
Cbz
1
2
3
Ketomethylene entities are usually obtained by complicated
multi-step synthetic sequences, many of which do not facilitate
stereoselective incorporation of the
Scheme 1. Double protection of phenylalanine. Reagents and conditions: (a)
(i) p-anisaldehyde, 2 M NaOH, rt, 30 min; (ii) NaBH4, 0 °C, 30 min; (i + ii) repeated;
(b) Cbz-Cl, 1 M NaOH, 0 °C, 1 h.
a
-side chain mimic.16,17 We
used the recently introduced tandem chain extension aldol (TCEA)
reaction for preparation of the ketomethylene entity.18 By this ap-
proach b-keto imides were converted in one-pot to
-keto imides by the highly stereoselective zinc-mediated aldol
reaction directed by chiral oxazolidinones.19,20 Utilization of the
TCEA reaction for stereoselective synthesis of -substituted ketom-
a-substituted
O
O
Ph
c
c
b
6
N
a
5
a
O
78 %
H2N
COOH
ethylene isosteres has been reported for proline as the stereodi-
recting element as well.21
7
4
Ph
A retrosynthetic analysis revealed that the H-Phe-
Ser(Bz)-Xaa-OH construct 14A–C can be derived from the
w
[COCH2]-
-keto
c
Scheme 2. Synthesis of the chiral auxiliary. Reagents and conditions: (a) (i)
BF3OEt2, dry THF, 5 min, reflux; (ii) BH3 Me2S, overnight, reflux; (b) Et3N,
(Cl3CO)2CO, DCM, 2 h, <10 °C; (c) (i) n-BuLi, dry THF, 15 min, À40 °C; (ii) AcCl,
1 h, À40 °C.
imide 11, which in turn can be prepared from the b-keto imide 9
using paraformaldehyde in the TCEA reaction (Fig. 1).
The amino acid used as starting material needed double protec-
tion of the terminal amine to prevent quenching of the enolate
formed in the TCEA reaction.19–21 We protected the amine group
The final steps of the ketomethylene synthesis are shown in
Scheme 3. The double-protected L-phenylalanine (3) was activated
of L-phenylalanine 1 sequentially with p-methoxybenzyl (PMB)
and benzyloxycarbonyl (Cbz) (Scheme 1). The PMB group was
introduced by reductive amination by treating 1 with p-anisalde-
hyde and sodium borohydride in succession.22 The Cbz group
was inserted under standard conditions using benzyl chlorofor-
mate (Cbz-Cl). The overall yield of the double-protected L-phenyl-
alanine 3 was 74%.
with carbonyldiimidazole to form the intermediate 8. Subse-
quently, 8 was treated with the lithium enolate of 7 which was ob-
tained by deprotonation with lithium diisopropylamide. This gave
the desired b-keto imide 9 in 86% purified yield. Utilizing the one-
pot TCEA reaction, the b-keto imide 9 was converted to the
-substituted c-keto imide 10 in 52% yield. The reaction was ef-
fected by treatment of 9 with the Furukawa reagent, EtZnCH2I, to
give the zinc enolate, which upon treatment in situ with parafor-
a
The 2-substituted oxazolidinone 7, functioning as a chiral aux-
iliary, was prepared from
D
-phenylalanine (4).23–25 Reduction of 4
to the b-amino alcohol 5 was performed by treatment with borane
dimethylsulfide complex and boron trifluoride diethyl etherate.
Subsequent exposure of 5 to triphosgene afforded the oxazolidi-
none 6. Compound 6 was acetylated to give the chiral auxiliary 7
in 78% overall yield (Scheme 2).
maldehyde provided the aldol product 10. The
L-stereochemistry
of the -side chain was controlled by the stereoconfiguration of
a
the chiral auxiliary 7. The facial selectivity in the TCEA reactions
with oxazolidinones was established in earlier studies.18,20 The
product 10 existed as two rotamers in equilibrium with two hemi-
acetals giving rise to complex NMR spectra. The hemiacetal was
particularly characterized by a distinctive 13C NMR signal at
108 ppm and mass determination.
Ketomethylene tripeptidomimetics:
14A R = methyl, X = CH2
Ph
O
R
Subsequently, the serine a-side chain mimic was derivatized by
14B R = propyl, X = CH2
X
O
benzoylation with benzoyl chloride under standard conditions to
give 11 in 94% yield. This esterification pushed the equilibrium in
favor of the desired product 11 due to a higher reactivity of the pri-
mary alcohol group of the open aldol form of 10. The PMB protect-
ing group was removed by treatment with cerium ammonium
nitrate in aqueous acetonitrile.26 Epimerization of 12 during cleav-
age from the chiral auxiliary was suppressed by using the mildest
possible conditions which were found to be 1 equiv lithium
hydroxide in aqueous tetrahydrofuran at À10 °C for 15 min. Alter-
native cleavage of the chiral auxiliary using lithium peroxide27 was
abandoned since competitive Bayer–Villiger oxidation was ob-
served in earlier studies.20 When recycling the recovered auxiliary
7 diastereoselectivity was maintained indicating that the stereo-
center of 7 does not epimerize under the conditions of the reaction.
The free carboxylic acid obtained after deprotection of 12 was cou-
pled to the C-terminal amino acid. Preactivation of the carboxylic
acid with benzotriazol-1-yl-oxy-tris-dimethylaminophosphonium
+H3N
N
H
14C R = cyclohexyl, X = CH2
Reference tripeptides:
O
O
OBz
20A R = naphthalene, X = NH
20B R = propyl, X = NH
20C R = cyclohexyl, X = NH
Ph
O
R
Pg1
O
OPg3
N
Xc
+
H2N
Pg2
O
OBz
11
Ph
À
PF4 salt (BOP) was followed by treatment overnight with appro-
Pg1
Xc
priate C-terminal protected amino acids to yield products 13A–C.
The protected tripeptidomimetics 13A–C were purified by normal
phase HPLC to remove small amounts (<10% determined by
LC–MS) of the unwanted diastereomer formed by epimerization
during the hydrolysis step using LiOH. Finally, the ketomethylene
tripeptidomimetics 13A–C were deprotected by hydrogenolysis
N
Pg = Protecting group
Xc = Chiral auxiliary
Pg2
O
O
9
Figure 1. Retrosynthetic analysis of the preparation of H-Phe-
aa-OH by the TCEA reaction.
w[COCH2]-Ser(Bz)-
X

K. Thorn et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4597–4601
4599
Cbz
N
Cbz
N
Cbz
N
PMB
Ph
N
PMB
Ph
Xc
a
PMB
Ph
Xc
3
8
c
d
94 %
O
O
b
86 %
O
52 %
O
O
O
HO
O
O
Ph
O
Ph
O
10
9
11
Cbz
Cbz
HN
+H3N
Ph
HN
TFA-
Ph
e
75%
Ph
Ph
Xc
O
O
h
f, g
52%
O
O
O
O
O
63-69%
Ph
O
Ph
O
O
HN
R
O
HN
R
O
12
COOH
14A
14B
14C
:R = methyl
:R = propyl
:R = cyclohexyl
COOPg
13A-C
Pg = Bnor tBu
Scheme 3. Synthesis of ketomethylene tripeptidomimetics H-Phe-
w[COCH2]-Ser(Bz)-Xaa-OH. Reagents and conditions: (a) carbonyldiimidazole, dry THF, 30 min, rt; (b) (i)
i-Pr2NH, n-BuLi, dry THF, 15 min, À5 °C; (ii) compound 7, 1 h, À78 °C; (iii) compound 8, 1 h, À78 °C; (c) (i) Et2Zn, CH2I2, dry DCM, 10 min, 0 °C; (ii) paraformaldehyde,
compound 9, 3 h, rt; (d) pyridine, DMAP, BzCl, DCM, 4.5 h, rt; (e) CAN, 4:1 MeCN H2O, 35 min, rt; (f) LiOHÁH2O, 3:1 THF H2O, 15 min, À10 °C; (g) (i) DIPEA, BOP, DMF, 3 min, rt;
(ii) H-Xaa-OPg, overnight, rt; (h) 10% Pd on C, TFA, MeOH, overnight, rt, H2.
affording the target compounds 14A–C. An ultimate purification of
each compound was carried out by reverse phase HPLC achieving a
higher than 99% purity in each case as determined by LC–MS.
The corresponding tripeptides 20A–C containing only amide
bonds were synthesized as shown in Scheme 4. The dipeptide 6
was composed of Boc-Phe-OH and H-Ser-OBn as a result of peptide
coupling under standard solution phase conditions using 1-ethyl-
3-(3-dimethylaminopropyl)-carbodiimide (EDCI) and 1-hydroxy-
OH
Ph
a, b
H
N
Boc
COOBn
78%
H2N
COOH
N
H
O
15
16
OH
c
83%
benzotriazole (HOBt) as coupling reagents. The serine a-side chain
Ph
Ph
was benzoylated in a similar fashion to that used in the ketometh-
ylene series to give 17 in 83% yield. The benzyl protecting group of
17 was removed quantitatively by hydrogenolysis to afford prod-
uct 18. The three C-terminal amino acids were introduced to give
compound 19A–C applying the peptide coupling protocol de-
scribed above. Finally, the tert-butoxycarbonyl (Boc) and tert-butyl
(tBu) protection groups were removed by exposure of 19A–C to a
2:1 mixture of dichloromethane and trifluoroacetic acid to yield
the desired tripeptides 20A–C with a purity higher than 87%
according to LC–MS.
The ketomethylene tripeptidomimetics 14A–C and the control
tripeptides 20A–C were tested for hPEPT1 binding affinity by mea-
suring the concentration dependent inhibition of apical [14C]Gly-
Sar uptake in Caco-2 cells (Table 1). The Ki values ranged between
0.05 and 0.32 mM which means that all of the compounds are de-
fined as high affinity (Ki <0.5 mM) ligands for hPEPT1.
The C-terminal modified tripeptides 20A–C were evaluated
against the lead promoiety H-Phe-Ser-Ala-OH. Compounds 20A
and 20C displayed similar affinity for hPEPT1 compared to
H-Phe-Ser-Ala-OH. For 20B a significantly higher affinity for
hPEPT1 was observed compared to H-Phe-Ser-Ala-OH (p < 0.05,
N = 3). In order to investigate whether larger C-terminal amino
acids would have an effect on hPEPT1 affinity 20A was synthesized.
Compound 20A, containing a naphthalene substituted C-terminal
amino acid, demonstrated that hydrophobic modifications in the
C-terminal part of the tripeptide did not seem to affect hPEPT1
affinity. These results indicate that even though the size of the tri-
peptidomimetic molecule was increased by the derivatized serine
H
H
N
Boc
N
COOH
Boc
COOBn
N
H
N
H
d
O
O
99%
O
O
18
17
O
Ph
O
Ph
e
45-50%
Ph
O
O
R
H
N
f
Boc
96-99%
N
H
N
H
COOPg
O
O
Ph
Ph
19A-C
Pg = Bn or tBu
O
R
H
N
+H3N
TFA-
N
COOH
H
O
O
20A: R = naphthalene
20B: R = propyl
Ph
O
20C: R = cyclohexyl
Scheme 4. Synthesis of reference tripeptides. Reagents and conditions: (a) p-TsOH,
1:1 BnOH CCl4, reflux, Dean–Stark conditions; (b) (i) Boc-Phe-OH, DIPEA, EDCI,
HOBt, DMF, 5 min; (ii) H-Ser-Bn, overnight, rt; (c) pyridine, DMAP, BzCl, DCM, 4.5 h,
rt; (d) 10% Pd on C, MeOH, overnight, rt, H2; (e) (i) DIPEA, EDCI, HOBt, 3 min;
(ii) H-Xaa-OPg, overnight, rt, N2; (f) 2:1 DCM TFA, 6 h, rt.
a-side chain and the altered C-terminal residue, this was not
followed by a reduction of affinity for hPEPT1. This is partly in
agreement with previous observations where hydrophobic modifi-
cations in the R1 or R2 position of dipeptide side chains seem to in-
crease the hPEPT1 affinity.28 In addition, a recent study illustrated
that it is not only the hydrophobic character of the amino acid

4600
K. Thorn et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4597–4601
peptides.29 Thus, further studies are needed to prove unequivocally
Table 1
hPEPT1 affinity dataa
if 14A–C and 20A–C are substrates or inhibitors of hPEPT1. Such
evidence could be acquired by measurements of translocation
using hPEPT1 transfected cell models or oocytes expressing
hPEPT1. Regardless of the translocation properties of 14A–C, these
structures are promising for either the design of substrates or
Product
log Ki log SE
Ki (mM)
H-Phe-Ser-Ala-OH
14A H-Phe-
14B H-Phe-
14C H-Phe-
20A H-Phe-Ser(Bz)-Nal-OH
20B H-Phe-Ser(Bz)-Nva-OH
20C H-Phe-Ser(Bz)-Chg-OH
À0.59 0.09
À0.49 0.05
À0.60 0.09
À1.33 0.09
0.26b
0.32
0.25
0.05
ꢀ0.3c
0.12
0.25
w
[COCH2]Ser(Bz)-Ala-OH
[COCH2]Ser(Bz)-Nva-OH
[COCH2]Ser(Bz)-Chg-OH
w
w
inhibitors. Especially, the 50 lM affinity of 14C could be a starting
point for a chemical effort to find a higher affinity inhibitor which
could be very useful for both in vitro and in vivo studies.
The present study of selected tripeptidic compounds targeting
À0.91 0.03
À0.61 0.02
Nva, norvaline; Chg, cyclohexylglycine; Nal, naphthylalanine.
hPEPT1 has demonstrated that the tripeptidomimetics H-Phe-
w
a
The Ki-values are measured as the concentration dependent inhibition of 20 lM
[
14C]Gly-Sar apical uptake in Caco-2 cells.
[COCH2]-Ser(Bz)-Xaa-OH maintain high affinity upon replacement
of their N-terminal amide bonds with ketomethylene entities. The
results signify that the amino nitrogen of the natural amide bond
is not vital for hPEPT1 recognition of tripeptidic structures. Conse-
quently, replacement of amide bonds with ketomethylene units is
promising for stabilization against degradation of tripeptides tar-
b
Value obtained by refitting data presented in Thorn et al.15
Value estimated due to limited solubility.
c
substitutions that influence hPEPT1 interaction but also the shape
and orientation of the side chains.29
geting hPEPT1 in the GI tract. Derivatization of the serine
a-side
The ketomethylene analogues 14A and 14B did not have signif-
icantly different affinities for hPEPT1 compared to the lead promo-
iety H-Phe-Ser-Ala-OH, whereas 14C displayed a significantly
higher affinity (p < 0.01, N = 3). Moreover, comparing 14B with
the corresponding tripeptide analogue 20B, a twofold significantly
lower affinity was observed for the ketomethylene product 14B
(p < 0.05, N = 3). Compounds 14C and 20C revealed the opposite
pattern, where the affinity for hPEPT1 was significantly higher
for the ketomethylene analogue (p < 0.001, N = 3). These observa-
tions provide no obvious explanation for the behavior of the fa-
vored ketomethylene analogue based on the knowledge about
ligands interacting with hPEPT1. Perhaps the rotational flexibility
of the ketomethylene part facilitates the adoption of a preferred
conformation of the tripeptidomimetic 14C thereby increasing
chain and incorporation of non-proteinogenic aliphatic amino acids
at the C-terminus were also allowed indicating broad ligand recog-
nition of the tripeptidomimetic core. Furthermore, the efficiency
and application of the stereocontrolled tandem chain extension al-
dol (TCEA) reaction for generation of ketomethylene tripeptidomi-
metics using paraformaldehyde for construction of the
serine mimic was nicely demonstrated.
a-side chain
Supplementary data (Experimental procedures)
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.05.108.
the affinity for hPEPT1. The C-terminal
composed of a cyclohexyl group, which is a sterically large
group compared to the -side chain of 14A or 14B. This disparity
could account for the differences between the ketomethylene
compounds.
a-side chain of 14C is
References and notes
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Ther. Pat. 2005, 15, 153.
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a
All products, 14A–C and 20A–C, displayed high affinity for
hPEPT1 independent on whether the tripeptide skeleton was com-
posed of amide or ketomethylene linkages. In other words, the
nitrogen of the N-terminal amide group of the tripeptidomimetics
did not influence hPEPT1 binding. This finding is supported by a
series of dipeptidomimetics.13,14 The fact that the ketomethylene
compounds maintained the biological affinity for hPEPT1 demon-
strates a promising stabilization strategy towards the environment
in the GI-tract as the ketomethylene linkage is non-hydrolysable.
Furthermore, introduction of the C-terminal non-proteinogenic
amino acids in compound 14B and 14C would presumably in-
crease the stability towards enzymatic degradations caused by
carboxypeptidases.
For the reference tripeptides the stability of similar tripeptides
during the affinity study in Caco-2 cell was investigated in a previ-
ous study. Less than 10% of the tripeptides were decomposed in the
cell assay.30
Recently, a comprehensive investigation of translocation of trip-
eptides via hPEPT1 was published. The study found that 40 out of
55 tripeptides were translocated, 8 interacted with the assay used,
and one (Met-Pro-Pro) was an inhibitor while one (Asp-Ile-Arg)
was not recognized by hPEPT1.31 A series of benzyl esters of
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