Solid-phase combinatorial synthesis of peptide-biphenyl hybrids as calpain inhibitors

Article abstract of DOI:10.1021/ol048216j

(Chemical Equation Presented) The combinatorial parallel synthesis of peptide-biphenyl hybrids on solid support using state of the art of peptide synthesis is reported. Key steps were the N to C addition of an amino moiety, hydrolysis of the methyl ester,

Full text of DOI:10.1021/ol048216j

ORGANIC
LETTERS
2004
Vol. 6, No. 22
4089-4092
Solid-Phase Combinatorial Synthesis of
Peptide Biphenyl Hybrids as Calpain
Inhibitors†,‡
−
|
Ana Montero,§, Fernando Albericio,*, Miriam Royo,*, and Bernardo Herrado´n*,§
Instituto de Qu´ımica Orga´nica General, C.S.I.C., Juan de la CierVa 3, 28006-Madrid,
Spain, and Combinatorial Chemistry Unit and Barcelona Biomedical Research
Institute, Barcelona Science Park, UniVersity of Barcelona, 08028 Barcelona, Spain
herradon@iqog.csic.es; mroyo@pcb.ub.es; albericio@pcb.ub.es
Received September 3, 2004
ABSTRACT
The combinatorial parallel synthesis of peptide-biphenyl hybrids on solid support using state of the art of peptide synthesis is reported. Key
steps were the N to C addition of an amino moiety, hydrolysis of the methyl ester, and the absence of cross-linked compounds when the
2,2′-diamino-1,1′-biphenyl was incorporated. When tested for activity as calpain inhibitors, some of the compounds exhibited IC50 values in the
nanomolar range.
Peptide-biphenyl hybrids A (Figure 1) are compounds in
which peptides or single amino acids are bound to the 2-
and 2′-positions of a biphenyl ring system.1 Both peptides
and biaryl compounds have been used as chiral catalysts,2
as building blocks for the design of technological materials,3
† Taken in part from the Ph.D. Thesis of A.M. (Universidad Auto´noma,
Madrid, 2004).
Figure 1. Generic structure of peptide-biphenyl hybrid A.
‡ Abbreviations: CDI, carbonyldiimidazole; DIEA, N,N-diisopropyl-
ethylamine; DIPCDI, N,N′-diisopropylcarbodiimide; DMF, N,N-dimethyl-
formamide; HOAt, 1-hydroxy-7-azabenzotriazole; HOBt, 1-hydroxyben-
zotriazole; MBHA, 4-methylbenzhydrylamine; PS, polystyrene; PyBOP,
(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate; TEA,
triethylamine; TFA, trifluoroacetic acid; THF, tetrahydrofuran. Amino acid
symbols denote L-configuration.
as biologically active compounds, and in other applications.4
The combination of both structural fragments in a single
molecule provides compounds with synergistic properties.1,5
§ C.S.I.C.
Combinatorial Chemistry Unit, Barcelona Science Park, University of
Barcelona.
(3) (a) Voyer, N. Top. Curr. Chem. 1997, 184, 1. (b) Hartley, C. S.;
Lazar, C.; Wand, M. D.; Lemieux, R. P. J. Am. Chem. Soc. 2002, 124,
13513.
| Barcelona Biomedical Research Institute, Barcelona Science Park,
University of Barcelona.
(4) (a) Hajduk, P. J.; Bures, M.; Praestgaard, J.; Fesik, S. W. J. Med.
Chem. 2000, 43, 3443. (b) Kutzki, O.; Park, H. S.; Ernst, J. T.; Orner, B.
P.; Yin, H.; Hamilton, A. D. J. Am. Chem. Soc., 2002, 124, 11838. (c)
Wang, W.; Cai, M.; Xiong, C.; Zhang, J.; Trivedi, D.; Hruby, V. J.
Tetrahedron 2002, 58, 7365. (d) Peukert, S.; Brendel, J.; Pirard, B.;
Bru¨ggemann, A.; Below, P.; Kleemann, H.-W.; Hemmerle, H.; Schmidt,
W. J. Med. Chem. 2003, 46, 486. (e) Sheppard, G. S.; Kawai, M.; Craig,
R. A.; Davidson, D. J.; Majest, S. M.; Bell, R. L.; Henkin, J. Biorg. Med.
Chem. Lett. 2004, 14, 865.
(1) (a) Brandmeier, V.; Feigel, M.; Bremer, M. Angew. Chem., Int. Ed.
Engl. 1989, 28, 486. (b) Weigand, C.; Feigel, M.; Landgrafe, C. Chem.
Commun. 1998, 679. (c) Mann, E.; Montero, A.; Maestro, M. A.; Herrado´n,
B. HelV. Chim. Acta 2002, 85, 3624. (d) Amine, M.; Atmani, Z.; El Hallaoui,
A. A.; Giorgi, M.; Pierrot, M.; Re´glier, M. Biorg. Med. Chem. Lett. 2002,
12, 57.
(2) (a) Jarvo, E. R.; Miller, S. J. Tetrahedron 2002, 58, 2481. (b)
McCarthy, M.; Guiry, P. J. Tetrahedron 2001, 57, 3809.
10.1021/ol048216j CCC: $27.50
© 2004 American Chemical Society
Published on Web 09/29/2004

Our previous research on the inhibition of calpain by
peptide-biphenyl hybrids yielded several nanomolar inhibitors
(Figure 2).5a To establish structure-activity relationships for
is incorporated, the chain reverses sense. Optimization of
the solid-phase process was carried out for type XXX (4)
and type XXXX (13) compounds. The synthesis is illustrated
in Scheme 1.
Rink-MBHA-PS-resin (the loading of intial MBHA-PS
resin was 0.7 mmol/g) was transformed into the solid-phase
linked dipeptide 5 by standard methodology involving the
appropriate Fmoc-removal [piperidine-DMF (1:4)] and cou-
pling (DIPCDI/HOBt, 3 equiv each) to the corresponding
N-Fmoc-amino acid (Val and Phe, sequentially, 3 equiv).
All of the reactions involving resin-bound amines were
monitored by Kaiser’s test,8 and when necessary, the coupling
was repeated until a negative test result was obtained. The
first biphenyl fragment was introduced by the reaction of 5
with diphenic anhydride in the presence of TEA to give the
acid 6. The free acid of 6 was activated with PyBOP/HOAt
and then coupled with H-L-Phe-OMe to give 7. The reactions
involving resin-bound carboxylic acids were monitored by
the malachite green test.9 At this stage an aliquot of 7 was
treated with TFA to give 8, which had a purity of over 95%
by HPLC-MS.10 The next step, the hydrolysis of the methyl
ester with LiOH in THF to afford the acid 9, required
extensive optimization.11 Thus, the amount of LiOH, reaction
temperature, and reaction time were all investigated in order
to achieve a satisfactory result. This process was readily
followed by HPLC/MS analysis of samples obtained after
TFA cleavage of aliquots of 9. Complete conversion and a
purity of >95% of the acid 10 were obtained when the
reaction was carried out with 25 equiv of LiOH, at 50 °C
for 17 h.11 The penultimate step of the synthesis was
activation of the acid functionality of 9 with CDI (10 equiv)
followed by addition of the rather poor nucleophile 2,2′-
diamino-1,1′-biphenyl (5 equiv) to give the monoacylated
product 11. No diacylated cross-linked product was detected
under these conditions. This absence can be attributed to the
“pseudo-dilution phenomenon”12 associated with the solid
phase, as well as to the poor nucleophilicity of the amino
group of compound 11. Finally, TFA cleavage from the resin
yields in the target molecule 4 (as the trifluoroacetate salt).
Compound 4 was analyzed by HPLC-MS.10 It is interesting
to note that at room temperature, the chromatogram of the
peptide-biphenyl 4 shows two close peaks at 9.61 and 9.73
min that coalesce (9.60 min) when the temperature is
increased to 40 °C (Figure 4, Supporting Information). These
two peaks may be attributed to the two possible atropisomers
Figure 2. Structures and IC50 values of previously reported peptide-
biphenyl hybrid calpain inhibitors 1-3 as well as the structure of
target 4.
peptide-biphenyl hybrids, a large variety of peptide-biphenyl
hybrids is needed, and a solid-phase combinatorial synthetic
strategy is desired.6 Our target library includes compounds
of the types XXX and XXXX (Figure 3), which exhibit
structural similarity to the lead molecule 3. The target
compounds possess two common biphenyl fragments and
either three or four amino acid residues that are used to
generate molecular diversity.
The synthesis of peptide-biphenyl hybrids was previously
carried out in solution using an efficient coupling reaction
between a nucleophile (either an amino acid or a peptide
derivative) and an electrophile [either conveniently activated
2,2′-biphenyl-1,1′-dicarboxylic acid or dibenzo[c,e]oxepine-
5,7-dione (diphenic anhydride)]. The aforementioned strategy
is not readily applicable to the solid phase, as it does not
facilitate linkage to a solid support. Alternatively, a standard
solid-phase peptide strategy that involves linkage of the first
amino acid to the solid support through the C-terminal
carboxylic acid and elongation of the chain through the
N-terminus was probed.7 In this case, however, when the
biphenyl residue, which contains two carboxylic moieties,
1
of compound 4 that have been also detected by H NMR.1c
The synthesis is very efficient, providing 4 in an overall yield
13
of 75% for the 11 step sequence.
The last coupling for the preparation of type XXXX
involves the reaction of the poor nucleophile aromatic amine
(5) (a) Montero, A.; Mann, E.; Chana, A.; Herrado´n, B. Chem. BiodiVer.
2004, 1, 442. (b) Montero, A.; Alonso, M.; Benito, E.; Chana, A.; Mann,
E.; Navas, J. M.; Herrado´n, B. Bioorg. Med. Chem. Lett. 2004, 14, 2753.
(c) Herrado´n, B.; Benito, E.; Chana, A.; Mann, E.; Montero, A. Spanish
Patent Application 200301125; PCT Application ES2004/070034.
(6) Nicolaou, K. C.; Hanko, R.; Hartwig, W., Eds. Handbook of
Combinatorial Chemistry; Wiley-VCH Verlag GmbH & Co. KGaA:
Weinheim, Germany, 2002
(8) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.
Biochem. 1970, 34, 594.
(9) Attardi, M. E.; Portu, G.; Taddei, M. Tetrahedron Lett. 2000, 41,
7391.
(10) HPLC analyses are included as Supporting Information.
(11) Cantel, S.; Desgranges, S.; Martinez, J.; Fehrentz, J.-A. J. Peptide
Sci. 2004, 10, 326.
(12) Mazur, S.; Jayalekshmy, P. J. Am. Chem. Soc. 1979, 101, 677.
(13) Starting from 50 mg of resin (0.035 mmol of reacting amine)
afforded 25.6 mg of 4 (93% purity).
(7) Lloyd-Williams, P.; Albericio, F.; Giralt, E. Chemical Approaches
to the Synthesis of Peptides and Proteins; CRC Press: Boca Raton, FL,
1997.
4090
Org. Lett., Vol. 6, No. 22, 2004

Scheme 1. Solid-Phase Synthesis of Target Molecules 4 (as a Representative Example of Peptide-biphenyl Hybrids of Type XXX)
and 13 (as a Representative Example of Peptide-biphenyl Hybrids of Type XXXX)a
a Reagents and conditions: (a) HCl H-Phe-OMe (3.0 equiv), PyBOP (3.0 equiv), HOAt (3.0 equiv), DIEA (6.0 equiv), DMF. (b) TFA-
CH2Cl2 (95:5). (c) 2 N LiOH (25 equiv), THF-H2O (7:3), 50 °C. (d) (i) symmetric N-Fmoc-amino acid anhydride (3.0 equiv), TEA (3.0
equiv), CH2Cl2; (ii) piperidine-DMF (1:4).
present in 11 and an N-protected amino acid derivative.
Several procedures such as activation with DIPCDI/HOBt
or use of the acid chloride were tested but proved to be
unsatisfactory.14 The best result was obtained using the
symmetrical anhydride in CH2Cl2, providing 12 (Scheme 1).
Finally, after removal of the N-Fmoc protecting group and
cleavage from the resin, the peptide-biphenyl hybrid 13 was
obtained as a trifluoroacetate salt.
The proof of concept of this methodology was undertaken
by the generation of a small combinatorial library of
compounds of the types XXX and XXXX using a parallel
synthesis strategy. In addition to having studied the scope
and limitation of the synthetic methodology, these experi-
ments should test the utility of the semiautomatic system
proposed for these and future libraries.
The peptide-biphenyl hybrids XXX and XXXX (Figure
3) possess three and four amino acids, respectively. Since
previous results from one of our laboratories indicated that
the peptide-biphenyl hybrids that are most active as calpain
Figure 3. Generic structures of the target peptide-biphenyl hybrids
XXX and XXXX with the sites (R1-R4) for generating molecular
diversity highlighted. XXX and XXXX denote sublibraries contain-
ing three and four amino acids, respectively.
(14) While the reaction promoted by DIPCDI/HOBt was very slow, the
acid chloride methods were not always the best methods for incorporating
urethane-protected amino acids, because they led to the formation of the
less reactive oxazolone. See ref 7.
Org. Lett., Vol. 6, No. 22, 2004
4091

Figure 4. Structures and IC50 values of the compounds of the library of peptide-biphenyl hybrids. IC50 values were determined using a
previously reported spectrofluorimetric method incorporating calpain I from porcine erythrocytes and fluorescence-labeled casein as a
substrate.5
inhibitors are those with aromatic and hydrophobic amino
acids, the following amino acids were selected: Val and Ile
for position aa1, Phe and Tyr for position aa2, Phe and Ala
for position aa3, and Ala and Phe for position aa4. Peptide-
biphenyl hybrid libraries were prepared according to the
synthetic procedure illustrated in Scheme 1. Figure 4 shows
the structures of the peptide-biphenyl hybrids prepared on a
solid phase.
It is worth mentioning that eight out of the nine compounds
have purities higher than 90%; only compound VYF has a
lower but still acceptable purity of 78%. The overall yields
for the entire sequences (11 steps for compounds of type
XXX and 13 steps for hybrids of type XXXX) ranged from
39 to 75%. Finally, the calpain inhibitory capacity of the
peptide-biphenyl hybrids was determined in the manner
previously reported.5 As expected from our previous experi-
ence, all of the compounds in the library are calpain
inhibitors, although it must be noted that when the biological
activities of the parent compound 3 and the closest analogue
4 are compared, a significant reduction in activity is
observed. This can be attributed to the subtle influence of
the different functionalities at both ends of each molecule.
On the other hand, two of the compounds of the library
(VFFA and VFAA, Figure 4 and Table 1, Supporting
Information) are potent calpain inhibitors, having IC50 values
in the nanomolar range.
In summary, we have shown that peptide-biphenyl hybrids
can be obtained via solid-phase methodology. Key steps of
this protocol were the N to C addition of an amino moiety,
the hydrolysis of the methyl ester, and the total absence of
cross-linked compounds when the 2,2′-diamino-1,1′-biphenyl
was incorporated. The interesting biological activity of these
compounds has inspired an ongoing preparation of a large
number of congeners.
Acknowledgment. This work was partially supported by
MCyT (BQU2003-00089, 2002-02047, and 2001-2270) and
Fundacio´ “la Caixa” (02/162-02). We thank Mercedes Giner
(IQOG, CSIC) for her assistance with the experiments on
calpain inhibition.
Supporting Information Available: Experimental pro-
cedures and copies of HPLC/MS. This material is available
free of charge via the Internet at http://pubs.acs.org.
OL048216J
4092
Org. Lett., Vol. 6, No. 22, 2004