Insertion of 2-carboxysuccinate and tricarballylic acid fragments into cyclic-pseudopeptides: New antagonists for the human tachykinin NK-2 receptor

Article abstract of DOI:10.1016/S0960-894X(01)00841-1

A series of cylic pseudopeptides were the synthesized containing the sequence -Trp-Phe-(D)PheΨCH₂NH-, the terminal ends of which were bound to 2-carboxy succinate or enantiomerically enriched tricarballylic acid to give the final cyclic structu

Full text of DOI:10.1016/S0960-894X(01)00841-1

Bioorganic & Medicinal Chemistry Letters 12 (2002) 693–696
Insertion of 2-Carboxysuccinate and Tricarballylic Acid
Fragments into Cyclic-Pseudopeptides: New Antagonists
for the Human Tachykinin NK-2 Receptor
Nicholas J. S. Harmat,* Danilo Giannotti, Rossano Nannicini, Enzo Perrotta,
Marco Criscuoli, Riccardo Patacchini, Anna-Rita Renzetti, Sandro Giuliani,
Maria Altamura and Carlo A. Maggi
Menarini Ricerche S.p.A. Laboratori di Firenze, Via Rismondo 12/A, 50131 Firenze, Italy
Received 4 October 2001; revised 22 November 2001; accepted 10 December 2001
Abstract—A series of cyclic pseudopeptides were synthesized containingthe sequence -Trp-Phe-( d)-PheÉCH2NH-, the terminal
ends of which were bound to 2-carboxy succinate or enantiomerically enriched tricarballylic acid to give the final cyclic structures.
These two molecules and their subsequent derivatives were screened for h-NK2 receptor bindingand functional antagonist activity
on the rabbit urinary bladder. # 2002 Published by Elsevier Science Ltd.
The mammalian tachykinins comprise a group of small
peptides, referred to as substance P (SP), neurokinin A
(NKA) and neurokinin B (NKB) that are located in the
central and peripheral nervous systems. They exert a
variety of biological responses, such as smooth muscle
contraction and relaxation, pain transmission, vasodi-
lation and neurogenic inflammation by interacting with
the receptor types NK-1, NK-2 and NK-3.1,2 NKA
induces a biological response by binding to the NK-2
receptor. Our studies have focused on the human NK-2
receptor (h-NK2) which has been identified as a target
for the discovery of new drugs for the treatment of
inflammatory diseases in the respiratory, gastro-
intestinal and genitourinary tracts.3ꢀ5
With these premises in mind we decided to prepare a
series of analogues of MEN 11558 in which the succi-
nate fragment is replaced by 2-carboxy succinic acid or
enantiomerically enriched tricarballylic acid (TCA),
giving, respectively, analogue structures 2 and 3, which
were each converted to the amide derivatives 8 and 12.
In the course of our work on the discovery of new small
molecules as antagonists for the h-NK2 receptor we
recently communicated the SAR on a series of cyclic
pseudopeptides which gave the lead MEN 11558, 1 with
a potent in vitro h-NK2 receptor bindingactivity and
functional activity.6 The aim of our work presented here
taking 1 as a lead, was to replace the succinoyl moiety
with other functionalities without changing the struc-
tural characteristics of the monocyclic compounds
essential for h-NK2 antagonist activity and yet have
compounds with improved potency.
Chemistry
The syntheses of both these orthogonally protected
2-carboxy succinic acid7 and enantiomerically enriched
TCA8 buildingblocks have been described in earlier
communications. It was therefore envisaged to insert
the protected 2-carboxy succinic acid and TCA moi-
eties in a regioselective manner into the orginal tripep-
tide fragment common to 1, openingthe possibility for
*Correspondingauthor. Fax: +39-055-5680-419; e-mail: chimfarm@
menarini.ricerche.it
0960-894X/02/$ - see front matter # 2002 Published by Elsevier Science Ltd.
PII: S0960-894X(01)00841-1

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N. J. S. Harmat et al. / Bioorg. Med. Chem. Lett. 12 (2002) 693–696
subsequent preparation of various derivatives. The
synthesis of the first series of compounds is shown in
Scheme 1 and starts from the doubly protected tripep-
tide fragment 4, which was prepared by analogy to the
methods previously described6 for obtainingthis key
intermediate.
ester derivatives 9, gave the single epimeric adduct 10
with defined stereochemistry of the a-CH group in the
TCA moiety. The N and C terminus of each separate
epimer of 10 was double de-benzylated by hydro-
genolysis and cyclized by condensation to give 11. Final
de-tert-butylation with TFA and derivatization of the
exo-cyclic CH2–carboxyl terminus by condensation with
various functionalized amines gave the final target
compounds 12a and 12b as separate individual stereo-
isomers with a defined configuration with respect to the
C12 ringcarbon atom.
The Z-(carbobenzyloxy) protectingrgoup in
4 was
removed by hydrogenolysis and the mono-deprotected
intermediate then coupled to the orthogonally di-pro-
tected carboxy succinic acid derivative 5,9 giving the
elaborated 2-carboxy succinamide di-ester 6. In the final
steps to the cyclized pseudopeptide products, inter-
mediate 6 was double de-tert-butylated with TFA at the
N and C termini and the resultingzwitterionic com-
pound cyclized with PyBOP. The final carboxyl pro-
tectingrgoup (PNB) was removed by saponification
giving 7a,b as a 5.6:1 mixture of epimers which were
separable by preparative HPLC.10 We then proceeded
to derivatize the same enriched epimeric mixture 7a,b by
couplingthis to functionalized hydrophilic amines such
as N-acetyl glucosamine11 and 4-piperidinyl piper-
idine,12 both of which are known to confer improved
water solubility, an important consideration for pro-
ducingmolecules with potential in vivo activity. The
resultingderivatives 8a–e were single diastereoisomers,
as indicated by HPLC and NMR data with configura-
tion of the chiral C12 ringatom not beingdefinable. In
one case 8e, two epimers were found to be present in a
ratio of 5.5:1 and these were separable by HPLC as in
the case of the parent compound 7.
Results and Discussion
The cyclo-pseudopeptide target molecules for both the
2-carboxysuccinate series (8a–e) and the tricarballylic
acid (TCA) series (12a–e)13 were evaluated for their in
vitro bindingaffinity to the human NK-2 receptor (p Ki
values)14 and were also screened for their functional
activity (pA2 values) on isolated rabbit urinary bladder
(RUB) tissue.15 The results of both these series of new
cyclo-pseudopeptide derivatives were compared against
our in vitro lead compound MEN 11558, 1.
The results for the first series with insertion of 2-car-
boxysuccinic acid in the cyclopeptide framework are
presented in Table 1. For the derivatization of the free
carboxyl group in with various polar functionalised
amines, we chose as a startingpoint the amino sugar
N-acetyl glucosamine. This substituent is present also in
the form of attachment to a pendant carboxyl group in
the complex bicyclic peptide NK2 antagonist nepadu-
tant (MEN 11420) which is presently in clinical trials.12
The attachment of this sugar group to 7 gave the deri-
vative 8a, which was approximately equipotent with 1 in
terms in receptor bindingaffinity and functional activ-
ity. We then searched for simpler hydroxylated or ring
oxygenated amine derivants the structural features of
which could mimic the sugar moiety yet give molecules
of lower molecular weight.
Because of the ambiguity of assignment of the stereo-
chemistry of the 2-carboxy succinate moiety in the final
cyclized products 7 and 8, we turned our attention to
the application of the homologous tricarballylic acid
(TCA) with the synthesis of this second series of com-
pounds described in Scheme 2. Startingfrom the same
tripeptide intermediate 4, N-Boc deprotection of the
Trp residue and subsequent couplingwith enantiomeri-
cally enriched orthogonally di-protected (R) or (S) TCA
Scheme 1. (a) H2, Pd/C MeOH/DMF; (b) ButO2CCH2CH(CO2PNB)CO2H (5), EDC.HCI/HOBt/DMF; (c) TFA/CH2CL2 (15%, v:v); (d) PyBOP/
DIPEA in DMF; (e) K2CO3/i-PrOH/H2O; (f) R0R00N H, EDC.HCI/HOBt/DMF.

N. J. S. Harmat et al. / Bioorg. Med. Chem. Lett. 12 (2002) 693–696
695
Scheme 2. (a) TFA/CH2Cl2 (15% v:v); (b) HO2C CH*2CH(CH2CO2But)CO2Bn, (9), (R) or (S), EDC; HCl/HOBt in DMF; (c) H2, Pd/C MeOH/
DMF; (d) PyBOP/D IPEA in DMF; (e) TFA, 80% in CH2Cl2; (f) 00R0RNH, EDCꢂ HCl/HOBt/DMF.
On this basis, the derivatives 8b–8d were prepared and
from these four products the morpholine derivative 8c
resulted in the best bindingaffinity for the NK-2 recep-
tor and functional activity, both superior to that of 1.
The derivitization of compound 7 with 4-piperidinyl
piperidine gave two isolable epimers 8e and 8f, both
which had markedly superior bindingaffinity and func-
tional activity to 1.
peptide analogues with a derivatizable pendant carboxyl
group. The 2-carboxysuccinate analogues showed more
marked improvements in bindingand functional activ-
ity compared to 1, notably the morpholine derivative 8c
and the basic 4-piperidinyl piperidine epimeric deriva-
tives 8e and 8f. The TCA derivatives overall main-
tained activity compared to the lead 1, with however the
morpholine 12d derivative showingincreased binding
The results for receptor bindingaffinity and functional
activity for the second series with insertion of either
single enantiomer of tricarballylic acid (TCA) into the
cyclopeptide framework, are presented in Table 2.
Table 1. h-NK2 receptor bindingactivity (p Ki) and in vitro functional
activity (pA2) for the 2-carboxy succinate series of derivatives 8a–f
Attachment of the N-acetylglucosamine to the pendant
CH2–carboxyl group of either epimer gave derivatives
12a or 12b which both maintained the receptor binding
and functional activity compared to the lead 1. This first
result also indicated that the activity was independent of
the configuration of the chiral TCA moiety inserted into
the cylopeptide structure. This was confirmed in the
example with the 4-piperidinyl piperidine derivatives 12f
and 12g. Since the (S) enantiomer of TCA was more
readily available we produced further derivatives 12c–
12e both of which maintained a comparable activity
with the lead compound 1, with the exception of the
morpholine derivative 12d which showed pKi and pA2
ꢁ9. However, the improvements in the bindingactivity
are more limited in this second series when compared to
the 2-carboxysuccinate derivatives of Table 1.
Compd
R0R00NH
pKi
pA2 (RUB)
8a
8.6
8.4
8b
8c
8d
8.4
9.9
8.8
8.5
9.2
9.0
In conclusion, we have described the application of
orthogonally protected tricarboxylic acid building
blocks that have been previously reported by us, in the
synthesis of target molecules of interest as potential
NK-2 receptor antagonists. Replacement of the succi-
noyl fragment with either 2-carboxy succinnate or
enantiomerically enriched (R)- or (S)-TCA gave cyclic
8ea
10.3
9.5
8.7
9.1
9.1
8.3
8fa
MEN 11558, 1
–
aTwo epimers formed and separated.

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N. J. S. Harmat et al. / Bioorg. Med. Chem. Lett. 12 (2002) 693–696
Table 2. h-NK2 receptor bindingactivity (p Ki) and in vitro func-
tional activity (pA2) for the tricarballylic acid series of derivatives
12a,c–f and 12b,g
References and Notes
1. Regoli, D.; Boudon, A.; Fauchere, J. L. Pharmacol. Rev.
1994, 46, 551.
2. Gao, Z.; Peet, N. P. Curr. Med. Chem. 1999, 6, 375.
3. Maggi, C. A.; Manzini, S. Drugs Lung 1994, 507.
4. Von Sprecher, A.; Gerspacher, M.; Anderson, G. P. I.
Drugs 1998, 73.
5. Swain, C. J. Exp. Opin. Ther. Pat. 1999, 9, 571.
6. Giannotti, D.; Perrotta, E.; Di Bugno, C.; Harmat, N. J. S.;
Nannicini, R.; Giolitti, A.; Patacchini, R.; Renzetti, A. R.;
Rotandaro, L.; Giuliani, S.; Altamura, M.; Maggi, C. A. J.
Med. Chem. 2000, 43, 4041.
Compd
R0R00NH
pKi
pA2 (RUB)
7. Perrotta, E.; Raffaelli, B.; Giannotti, D.; Harmat, N. J. S.;
Nannicini, R.; Altamura, M. Synlett 1999, 144.
8. Harmat, N. J. S.; Perrotta, E.; Mangani, S.; Giannotti, D.;
Nannicini, R.; Altamura, M. Tetrahedron Lett. 2000, 41,
1261.
12a
12b
8.1
8.2
8.2
8.1
9. Due to the mobility of the proton of the chiral a-CH in
1,3-dicarboxylic esters such as 5, the orthogonally dipro-
tected ester derivatives of such a structure remain racemic.
However on amidification of one of the 1,3 dicarboxylate
groups, such mobility of the proton in the a-CH group is
lost and such derivatives are found to be stereochemically
stable; see: Evans, D. A.; Brown, D. H.; Johnson, J. S.;
Shaughnessy, E. A. Angew. Chem. Int. Engl. Ed. 1997, 36, 2119.
10. The stereochemistry of the 2-carboxysuccinamide moiety
of each separated epimer was not definable by extensive NMR
studies. Numerous attempts to obtain crystals suitable for X-
ray diffraction as an alternative method to determine the ste-
reochemistry of the chiral centre (C-12 ringatom present in
the 2-carboxy succinamide fragment of 7 were not successful.
11. Hipskind, P. A.; Howbert, J. J.; Bruns, R. F.; Cho, S. Y.;
Crowell, T. A.; Foreman, M. M.; Gehlert, D. R.; Iyengar, S.;
Johnson, K. W.; Krushinski, J. H.; Li, D. L.; Lobb, K. L.;
Mason, N. R.; Muehl, B. B.; Nixon, J. A.; Phebus, L. A.;
Regoli, D.; Simmons, R. M.; Threlkeld, P. G.; Waters, D. C.;
Gitter, B. D. J. Med. Chem. 1996, 39, 736.
12c
12d
12e
12f
8.0
9.2
8.2
8.8
8.2
9.0
8.5
8.6
12g
MEN 11558, 1
8.3
8.7
8.4
8.3
–
affinity and an improvement in in-vitro functional
activity compared to MEN 11558. No apparent varia-
tion in activity with the chirality of the TCA unit was
observed in these analogues of 1. A selection of these
compounds have been taken for further in-depth studies
regarding in vivo activity.
12. Catalioto, R. M.; Criscuoli, M.; Cucchi, P.; Giachetti, A.;
Giannotti, D.; Giuliani, S.; Lecci, A.; Lippi, A.; Patacchini,
R.; Quartara, L.; Renzetti, A. R.; Tramontana, M.; Arc-
amone, F.; Maggi, C. A. Br. J. Pharmacol. 1998, 123, 81.
13. All final compounds were characterised by 1H NMR, m.s.
and had a purity by analytical HPLC of ꢁ95%.
14. Bindingaffinity for the human NK-2 receptor transfected
in CHO cells was determined in competition experiments using
Acknowledgements
[
125I]NKA as the radioligand. The pKi values were calculated
accordingto the procedure described by: Munson, P. J. Anal.
Biochem. 1980, 107, 220.
15. Maggi, C. A.; Astolfi, M.; Giuliani, S.; Goso, C.; Man-
zini, S.; Meini, S.; Patacchini, R.; Pavone, V.; Pedone, C.;
Quartara, L.; Renzetti, A. R.; Giachetti, A. JPET. 1994, 271,
1489.
Thanks are due to Dr. Antonio Triolo and Dr.
Giuseppe Balacco (Chemistry) for spectroscopic
characterisation of compounds and Mr. Marco Guelf
(Pharmacology) for technical assistance in obtaining
the biological data.