U.S. patent application number 11/883880 was filed with the patent office on 2008-06-12 for peptidomimetic compounds and preparation of biologically active derivatives.
Invention is credited to Daniela Arosio, Leonardo Manzoni, Carlo Scolastico.
Application Number | 20080139461 11/883880 |
Document ID | / |
Family ID | 36570882 |
Filed Date | 2008-06-12 |
United States Patent
Application |
20080139461 |
Kind Code |
A1 |
Scolastico; Carlo ; et
al. |
June 12, 2008 |
Peptidomimetic Compounds and Preparation of Biologically Active
Derivatives
Abstract
The invention relates to novel cyclic peptidomimetic compounds
containing the sequence RGD for the preparation of appropriately
functionalised antagonists of .alpha.v.beta.3 and .alpha.v.beta.5
integrins, and intended, for example, for the treatment of altered
angiogenic phenomena or for the preparation of diagnostically
useful compounds.
Inventors: |
Scolastico; Carlo; (Milan,
IT) ; Manzoni; Leonardo; (Milan, IT) ; Arosio;
Daniela; (Milan, IT) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Family ID: |
36570882 |
Appl. No.: |
11/883880 |
Filed: |
March 3, 2006 |
PCT Filed: |
March 3, 2006 |
PCT NO: |
PCT/IB06/00460 |
371 Date: |
October 18, 2007 |
Current U.S.
Class: |
514/13.3 ;
514/19.1; 514/19.8; 514/21.1; 514/212.07; 514/299; 530/317;
540/523; 546/183 |
Current CPC
Class: |
C07K 5/06139 20130101;
A61P 35/04 20180101; A61P 43/00 20180101; A61P 19/10 20180101; A61P
35/00 20180101; C07D 471/04 20130101; A61P 9/10 20180101; A61P 9/00
20180101; A61P 13/12 20180101; C07K 7/64 20130101; C07D 401/06
20130101 |
Class at
Publication: |
514/9 ; 546/183;
540/523; 530/317; 514/299; 514/212.07 |
International
Class: |
A61K 38/06 20060101
A61K038/06; C07D 221/04 20060101 C07D221/04; C07D 487/04 20060101
C07D487/04; A61K 31/55 20060101 A61K031/55; A61P 35/00 20060101
A61P035/00; A61K 31/435 20060101 A61K031/435; C07K 5/02 20060101
C07K005/02 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 3, 2005 |
IT |
MI2005A000328 |
Claims
1. Compounds of general formula (I): ##STR00025## wherein: n has
the value of 1 or 2, R.sub.1 is H, (C.sub.1-C.sub.4) alkyl or a
protective group; R.sub.2 is H or a protective group; X is N.sub.3,
--NH--R.sub.3, --N(R.sub.3).sub.2, --NAlkR.sub.3,
--NH--CO--R.sub.3, --NH--CS--R.sub.3, --NH--CO--NHR.sub.3,
--NH--CS--NHR.sub.3, or ##STR00026## wherein: Alk=(C.sub.1-C.sub.4)
linear or branched alkyl R.sub.3.dbd.H, a protective group, a
biologically active molecule; their salts, racemic mixtures,
individual enantiomers, individual diastereoisomers and mixtures
thereof in whatever proportion.
2. Compounds according to claim 1, of general formulae (Ia) and
(Ib) ##STR00027## wherein n, R.sub.4, R.sub.5 and X are defined
according to claim 1 and the wedge-shaped and dashed bonds indicate
that the substituents are positioned above and below the plane
respectively.
3. A process for the preparation of the compounds of formulae (I),
(Ia) and (Ib) as defined in claim 1, characterized by the following
steps of schemes 1 or 2: a. hydrogenation of the isoxazolidine of
compounds 1-4; b. protection of the amine group with a protective
group; c. transformation of the free hydroxyl group into an azide
to give the compounds of formulae 6, 9, 12, 15; d. reduction of the
azide group into an amine by means of the Staudinger reaction or by
means of hydrogenation to give the compounds of formulae (I), (Ia)
or (Ib) wherein X is NH.sub.2; e. optional transformation of said
compounds into other compounds of formulae (I), (Ia) or (Ib) and/or
into a salt thereof; f. or from compounds 6, 9, 12, 15, 1,3-dipolar
reaction (click chemistry) with a biologically interesting molecule
(sugar, fluorescein, biotin) linked to a suitable linker and g.
possible transformation of said compounds into different compounds
having formula (I), (Ia) or (Ib) and/or into their salts.
4. Compounds of general formula (II): ##STR00028## wherein: n has
the value of 1 or 2, R.sub.4 and R.sub.5 together constitute the
sequence Asp-Gly-Arg, X is N.sub.3, --NH--R.sub.6,
--N(R.sub.6).sub.2, --NAlkR.sub.6, --NH--CO--R.sub.6,
--NH--CS--R.sub.6, --NH--CO--NHR.sub.6, --NH--CS--NHR.sub.6, or
##STR00029## wherein Alk=(C.sub.1-C.sub.4) linear or branched alkyl
R.sub.6.dbd.H, a protective group, a biologically active molecule,
therein comprising a sugar; their salts, racemic mixtures,
individual enantiomers, individual diastereoisomers and mixtures
thereof in whatever proportion.
5. Compounds according to claim 1, of general formulae (Ia) and
(IIb): ##STR00030## wherein n, R.sub.4, R.sub.5 and X are as
defined in claim 1 and the wedge-shaped and dashed bonds indicate
that the substituents are positioned above and below the plane
respectively.
6. Compound according to claim 4, having the following structure:
##STR00031##
7. Compounds according to claim 4, wherein X is NH.sub.2.
8. Compounds according to claim 4, wherein X is a substituted or
unsubstituted amide.
9. Compounds according to claim 4, wherein X is a substituted or
unsubstituted urea or thiourea.
10. Compounds according to claim 4, wherein X is a substituted or
unsubstituted triazole system.
11. Compounds according to claim 4, or their pharmaceutically
acceptable salts thereof, wherein R.sub.6 is a drug.
12. Compounds according to claim 11, wherein said drug is a
cytotoxic and/or antitumour drug.
13. Compounds of claim 4, for their use as drugs.
14. Compounds of claim 1, for the preparation of antagonist drugs
towards .alpha.v.beta.3 and .alpha.v.beta.5 integrins.
15. Compounds of claim 1, for the preparation of drugs with
antiangiogenic activity.
16. Compounds according to claim 1, for the preparation of drugs
intended for the treatment and/or the prophylaxis of altered
angiogenic processes, metastasised tumour processes, retinopathies,
acute renal damage and osteoporosis.
17-21. (canceled)
22. Compounds according to claim 4, as drug carriers.
23. A pharmaceutical composition containing at least one compound
of formulae (II), (IIa) or (IIb), or their pharmaceutically
acceptable salts thereof, according to claim 4, as active
ingredient, optionally in combination with one or more
pharmaceutically acceptable carriers or excipients.
24. A process for the preparation of the compounds of formulae
(II), (Ia) and (IIb) as defined in claim 4, characterized by the
following steps of schemes 3 to 5: transformation of the hydroxyl
group into the corresponding azide in accordance with a procedure
known to those skilled in the art (through the Mitsunobu reaction,
or mesylation and subsequent nucleophilic substitution with sodium
azide) to give compounds 21, 23, 25, 27; subsequent reduction by
means of catalytic hydrogenation or the Staudinger reaction;
transformation into the corresponding amides by means of a coupling
reaction; optional conjugation with molecules of biological
interest; subsequent deprotection of the aminoacid side chain
protective groups to give the compounds of formulae 22, 24, 26, 28;
or from compounds 21, 23, 25, 27 1,3-dipolar reaction (click
chemistry) with a biologically interesting molecule (sugar,
fluorescein, biotin) linked to a suitable clinker; and subsequent
elimination of protective groups of the amino acids side chains.
Description
[0001] The present invention relates to novel cyclic peptidomimetic
compounds having an azabicycloalkane structure, and the preparation
of antagonists of integrins .alpha.v.beta.3 and .alpha.v.beta.5,
useful for example, in the treatment of altered angiogenetic
phenomena. The invention also concerns a process for the
transformation of the functional groups of said cyclic
peptidomimetic compounds and the biologically active derivatives
thereof.
BACKGROUND OF THE INVENTION
[0002] A great number of physiological processes involve
biologically active peptides, through their interactions with
receptors and enzymes. However, peptides are not to be considered
ideal drugs, given their poor metabolic stability, rapid excretion
and low selectivity for specific receptors. A valid alternative
involves the design of peptide analogues which are capable of
mimicking the action of the natural peptide at the receptor level
(peptidomimetic) [(a) Kahn, M. (Editor). Peptide Secondary
Structure Mimetics. Tetrahedron Symposia-in-Print No. 50 1993, 49,
3433-3689. (b) Gante, J. Angew. Chem. Int. Ed. Engl. 1994, 33,
1699-1720. (c) Olson, G. L.; Bolin, D. R.; Bonner, M. P.; Bos, M.;
Cook, C. M.; Fry, D. C.; Graves, B. J.; Hatada, M.; Hill, D. E.;
Kahn, M.; Madison, V. S.; Rusiecki, V. K.; Sarabu, R.; Sepinwall,
J.; Vincent, G. P.; Voss, M. E. J. Med. Chem. 1993, 36, 3039-3049.
(d) Kitagawa, O.; Velde, D. V.; Dutta, D.; Morton, M.; Takusagawa,
F.; Aube, J. J. Am. Chem. Soc. 1995, 117, 5169-5178. (e) Giannis,
A.; Kolter, T. Angew. Chem.; Int. Ed. Engl. 1993, 32, 1244. (f)
Aube, J. Tetrahedron Symposia-in-Print No. 50, 2000, 56,
9725-9842].
[0003] During our research into peptide secondary structure
mimetics, certain 6,5- and 7,5-azabicycloalkane aminoacids have
been synthesised [(a) Colombo, L.; Di Giacomo, M.; Scolastico, C.;
Manzoni, L.; Belvisi, L.; Molteni, V. Tetrahedron Lett. 1995, 36,
625; (b) Colombo, L.; Di Giacomo, M.; Belvisi, L.; Manzoni, L.;
Scolastico, C. Gazz. Chim. It. 1996, 126, 543; (c) Colombo, L.; Di
Giacomo, M.; Brusotti, G.; Sardone, N.; Angiolini, M.; Belvisi, L.;
Maffioli, S.; Manzoni, L.; Scolastico, C. Tetrahedron 1998, 54,
5325-5336; (d) Angiolini, M.; Araneo, S.; Belvisi, L.; Cesarotti,
E.; Checchia, A.; Crippa, L.; Manzoni, L.; Scolastico, C. Eur. J.
Org. Chem. 2060, 2571-2581; (e) Manzoni, L.; Colombo, M.; May, E.;
Scolastico, C. Tetrahedron 2001, 57, 249; (f) Belvisi, L.; Colombo,
L.; Colombo, M.; Di Giacomo, M.; Manzoni, L.; Vodopivec, B.;
Scolastico, C. Tetrahedron 2001, 57, 6463; (g) EP 1 077 218; (h)
Colombo, L.; Di Giacomo, M.; Vinci, V.; Colombo, M.; Manzoni, L.;
Scolastico, C. Tetrahedron, 2003, 59, 4501-4513; (i) Manzoni, L.;
Colombo, M.; Scolastico, C. Tetrahedron Lett. 2004, 45, 2623-2625;
(l) Belvisi, L.; Colombo, L.; Manzoni, L.; Potenza, D.; Scolastico,
C. Synlett, 2004, 1449-1471.
[0004] These structures may be considered as conformationally
constrained analogues of the Ala-Pro and Phe-Pro dipeptide units.
[(a) Belvisi, L.; Bernardi, A.; Manzoni, L.; Potenza, D.;
Scolastico, C. Eur. J. Org. Chem. 2000, 2563-2569; (b) Gennari, C.;
Mielgo, A.; Potenza, D.; Scolastico, C.; Piarulli, U.; Manzoni, L.
Eur. J. Org. Chem. 1999, 379].
[0005] The functionalisation of such molecules with heteroalkyl
substituents is an aim of great interest, since the side chains may
increase the affinity of the peptide for the receptor by
interacting with the hydrophobic or hydrophilic sites of the
receptor itself. A further advantage of such systems is the
possibility of binding to different pharmacophoric groups and hence
the possibility of creating a library, with the member components
of which having different biological properties and activities.
During our research into peptide secondary structure mimetics,
certain 6,5- and 7,5-azabicycloalkane aminoacids have been
synthesised which have been functionalised with heteroalkyl
appendages [(a) Artale, E.; Banfi, G.; Belvisi, L.; Colombo, L.;
Colombo, M.; Manzoni, L.; Scolastico, C. Tetrahedron, 2003, 59,
6241-6250; (b) Bracci, A.; Manzoni, L.; Scolastico, C. Synthesis
2003, 2363-2367; (c) Bravin, F. M.; Busnelli, G.; Colombo, M.;
Gatti, F.; Manzoni, L.; Scolastico, C. Synthesis, 2004, 353; (d)
Manzoni, L.; Belvisi, L.; Colombo, M.; Di Carlo, E.; Forni, A.;
Scolastico, C. Tetrahedron Lett. 2004, 45, 6311-6315].
[0006] Furthermore, analogously to what occurs for non-substituted
conformationally constrained dipeptide mimetics [Belvisi, L.;
Bernardi, A.; Checchia, A.; Manzoni, L.; Potenza, D.; Scolastico,
C.; Castorina, M.; Cupelli, A.; Giannini, G.; Carminati, P.;
Pisano, C. Org. Lett. 2001, 3, 1001, C. Scolastico, L. Manzoni, G.
Giannini. Brit. UK Pat. Appl. 2004. GB 2395480] such heteroalkyl
substituted lactams may be incorporated into cyclic
pseudo-peptides, containing RGD sequence.
[0007] Such molecules may be selectively targeted to those tissues
over-expressing certain receptors (e.g. epithelial cells involved
in vascular growth), so as to be able to be used to inhibit
angiogenesis and selectively control the release of any drugs
optionally bound to the substituent groups on the lactam ring
[Arap, W.; Pasqualini, R.; Ruoslahti, E. Science, 1998, 279,
377].
[0008] The low number of "scaffolds" reported in the literature
necessitates the design and synthesis of novel conformationally
constrained dipeptide mimetics, functionalised with
hetero-substituted side chains for interaction with various
receptors.
DESCRIPTION OF THE INVENTION
[0009] It has now been found that certain conformationally
constrained azabicyclic[X.Y.0]alkanes satisfy the characteristics
required for the application of this kind of technology.
Particularly, it has been found that compounds containing the
conformationally constrained homoSer-Pro dipeptide unit structure
are useful as drugs, particularly as drugs with antagonistic action
towards the .alpha.v.beta.3 and .alpha.v.beta.5 integrins.
[0010] Thus, according to one of the aspects thereof, the invention
concerns compounds of general formula (I)
##STR00001##
[0011] where:
[0012] n has the value of 1 or 2,
[0013] R.sub.1 is H, (C.sub.1-C.sub.4) linear or branched alkyl or
a protective group;
[0014] R.sub.2 is H or a protective group;
[0015] X is N.sub.3, --NH--R.sub.3, --N(R.sub.3).sub.2,
--NAlkR.sub.3, --NH--CO--R.sub.3, --NH--CS--R.sub.3,
[0016] --NH--CO--NHR.sub.3,
[0017] --NH--CS--NHR.sub.3, or
##STR00002##
[0018] where
[0019] Alk=(C.sub.1-C.sub.4) linear or branched alkyl
[0020] R.sub.3.dbd.H, a protective group, a biologically active
molecule;
[0021] their salts, racemic mixtures, individual enantiomers,
individual diastereoisomers and mixtures thereof in whatever
proportion.
[0022] According to another aspect thereof, object of the invention
are compounds of formulae (Ia) and (Ib)
##STR00003##
[0023] where n, R.sub.1, R.sub.2 and X are as defined above and the
wedge-shaped and dashed bonds indicate that the substituents are
positioned above and below the plane respectively.
[0024] According to the present invention, the term
"(C.sub.1-C.sub.4) alkyl" designates a linear or branched,
saturated or unsaturated alkyl substituent comprising from 1 to 4
carbon atoms such as for example methyl, ethyl, propyl, isopropyl,
butyl, tert-butyl. However, it is possible to use alkyl
substituents containing a higher number of carbon atoms providing
they are compatible with the reaction conditions of the present
invention.
[0025] According to the present invention, the expression
"protective group" designates a protective group adapted to
preserving the function to which it is bound, specifically the
amino function or carboxyl function. Appropriate protective groups
include for example benzyl, benzyloxycarbonyl, alkyl or benzyl
esters, or other substituents commonly used for the protection of
such functions, which are well known to those skilled in the art,
for example those described in conventional manuals such as T. W.
Green, Protective Groups in Organic Synthesis (Wiley, N.Y.
1981).
[0026] In the present description, by "biologically active
molecule" is meant any molecule that may be used as a drug, or also
as a "targeting" molecule, a diagnostically useful molecule, or
even a sugar etc.
[0027] Said biologically active molecule may be bound to compound
of formula (I), either directly or through an appropriate spacer
allowing or promoting binding, and optionally release into the site
of action.
[0028] The salts of the compounds of formulae (I), (Ia) and (Ib)
according to the present invention comprise both those with mineral
or organic acids allowing the expedient separation or
crystallisation of the compounds of the invention, and those
forming physiologically and pharmaceutically acceptable salts, such
as for example hydrochloride, hydrobromide, sulphate, hydrogen
sulphate, dihydrogen sulphate, maleate, fumarate,
2-naphthalenesulphonate, para-toluenesulphonate, oxalate etc.
[0029] Salts of the compounds of formulae (I), (Ia) and (Ib)
according to the present invention also further include
physiologically and pharmaceutically acceptable quaternary ammonium
salts.
[0030] Said salts are prepared according to the well known
techniques for the person skilled in the art.
[0031] When there is a free carboxyl group (R.sub.2.dbd.H) present,
the salts of the compounds of the invention also comprise salts
with organic or mineral bases, such as for example alkaline metal
or alkaline earth metal salts, such as sodium salts, potassium or
calcium salts, or with an amine such as trometamol (tromethamine),
or salts of arginine, lysine or any other physiologically and
pharmaceutically acceptable amine.
[0032] According to one preferred embodiment, object of the
invention are compounds of formulae (I), (Ia) and (Ib) wherein n is
1, R.sub.1 is benzyloxycarbonyl, R.sub.2 is tert-butyl, X is
N.sub.3 or NH.sub.2.
[0033] According to another preferred embodiment, object of the
invention are compounds of formulae (I), (Ia) and (Ib) wherein n is
2, R.sub.1 is benzyloxycarbonyl, R.sub.2 is tert-butyl, R.sub.3 is
N.sub.3 or NH.sub.2.
[0034] A process for the synthesis of such compounds is described
in detail over the course of the present description, making
reference to the synthetic schemes reported in the enclosed
figures.
[0035] According to the present invention, compounds of formulae
(I), (Ia) and (Ib) may be prepared according to the processes
described hereinafter.
[0036] Particularly, compounds of general formulae (Ia) 6,5-trans-
and (Ib) 6,5-cis-fused, may be prepared according to a synthetic
process outlined in Scheme 1 (wherein n=1), comprising the
following stages: [0037] a) hydrogenation of the isoxazolidine of
compound 1 or of compound 2, for example with H.sub.2, Pd/C in
MeOH; [0038] b) protection of the amine group with a suitable
protective group, such as for example Cbz, Boc, etc.; [0039] c)
transformation of the free hydroxyl group into an azide through the
Mitsunobu reaction, or by means of any other known method (for
example, transformation into mesylate and subsequent nucleophilic
substitution with sodium azide), to give compounds of formulae 6,
9; [0040] d) reduction of the azide group into an amino group
through the Staudinger reaction, or by means of hydrogenation to
give compounds 7 and 10.
[0041] The compounds of general formulae (Ia) 7,5-trans and (Ib)
7,5-cis fused, may be prepared according to a synthetic process
outlined in Scheme 2 (wherein n=2), comprising the following
stages: [0042] a) hydrogenation of the isoxazolidine of compound 3
or of compound 4, for example with H.sub.2, Pd/C in MeOH; [0043] b)
protection of the amine group with a suitable protective group,
such as for example Cbz, Boc, etc.; [0044] c) transformation of the
hydroxyl group into an azide through the Mitsunobu reaction, or by
means of any other known method (transformation into mesylate and
subsequent nucleophilic substitution with sodium azide), to give
compounds of formulae 12, 15; [0045] d) reduction of the azide
group into an amino group through the Staudinger reaction, or by
means of hydrogenation to give compounds 13, 16.
[0046] To the resulting lactams, following the introduction of the
azide group 6, 9, 12 and 15, may be bound the desired biologically
active molecules or compounds of biological importance such as
sugars, through appropriate reactions known to those skilled in the
art (Click chemistry).
[0047] Or moreover, to the amide or thioamide groups (compounds
wherein X NH--CO--R.sub.3 or NH--CS--R.sub.3) derived from lactams
7, 10, 13 and 16 may be bound appropriate substituents or known
spacers, to give for example ureas or thioureas (i.e. to form
compounds wherein X.dbd.--NH--CO--NHR.sub.3, --NH--CS--NHR.sub.3),
prepared by reacting with isocyanates or isothiocyanates according
to procedures known to the person skilled in the art.
[0048] Practical examples are reported in the experimental section
of the present description.
[0049] The tricyclic starting compounds 1-4 (n=1 and n=2, FIG. 1)
may be prepared according to the procedures described in Scheme
A--Preparation of the starting products.
[0050] The compounds of the present invention of formulae (I), (Ia)
and (Ib) may be used as conformationally constrained scaffolds with
the possibility to replicate the geometry of the backbone and of
the side chains of a peptide residue within the active site, and be
used for the preparation of biologically active compounds.
[0051] The need to conjugate drugs or other molecules of biological
interest to systems of this kind is linked to the possibility for
providing molecules having functional groups forming biologically
stable bonds, for example containing nitrogen, present for example
as an amino or amide group, azide or triazole.
[0052] Schemes 6-9 relates to conjugation examples through click
chemistry between sugars, fluorescein and biotin and compounds of
general formula (I). Scheme 8 refers to the preparation of one
utilized linker, taken as an example.
[0053] In pursuit of such aims, it has now been found that certain
of the novel compounds of general formula (II), reported
hereinafter, satisfy the required characteristics for the
application of this type of technology. Particularly, it has been
found that compounds having general formula (II) are useful as
drugs, particularly as drugs having antagonistic action against the
.alpha.v.beta.3 and .alpha.v.beta.5 integrins, particularly those
compounds deriving from the transformation of the compounds of
formulae (I), (Ia) and (Ib) as set out above, wherein the peptide
sequence RGD has been introduced.
[0054] Thus, in accordance with another aspect thereof, object of
the invention are novel compounds of formula (II)
##STR00004##
[0055] wherein:
[0056] n has the value of 1 or 2,
[0057] R.sub.4 and R.sub.5 together constitute the sequence
Asp-Gly-Arg,
[0058] X is N.sub.3, --NH--R.sub.6, --N(R.sub.6).sub.2,
--NAlkR.sub.6, --NH--CO--R.sub.6, --NH--CS--R.sub.6,
[0059] --NH--CO--NHR.sub.6,
[0060] --NH--CS--NHR.sub.6, or
##STR00005##
[0061] wherein
[0062] Alk=(C.sub.1-C.sub.4) linear or branched alkyl
[0063] R.sub.6.dbd.H, a protective group, a biologically active
molecule;
[0064] their salts, racemic mixtures, individual enantiomers,
individual diastereoisomers and mixtures thereof in whatever
proportion.
[0065] According to one preferred aspect, the invention concerns
compounds of formulae (IIa) and (IIb)
##STR00006##
[0066] wherein n, R.sub.4, R.sub.5 and X are as defined above and
the wedge-shaped and dashed bonds indicate that the substituents
are positioned above and below the plane respectively.
[0067] The peptide sequence Asp-Gly-Arg is advantageously bound to
compounds (II), (IIa) and (IIb) in such a manner whereby the
carboxyl group is attached to the aminoacid arginine, and the amino
group is attached to aspartic acid.
[0068] The details provided above for the variable substituents
(alkyl, etc.) and the salts of the compounds of formula (I) are
also applicable to the compounds of formulae (II), (IIa) and
(IIb).
[0069] Thus, just as for the compounds of formula (I), X may
represent an amino residue, an amide residue, a urea, a thiourea,
said residues advantageously being substituted.
[0070] The Asp-Gly-Arg chain may be introduced by adapting the
compounds of formulae (I), (Ia) and (Ib) described above, according
to a process comprising the following stages: [0071] when R.sub.2
is a protective group, chemoselective deprotection reaction of the
carboxyl group of compound of general formula (I) and condensation
with the appropriately protected Arg-Gly dipeptide; [0072]
reduction of the oxazolidine by means of catalytic hydrogenation;
[0073] transformation of the methyl ester of glycine into the
benzyl ester through a transesterification reaction, followed by
the simultaneous removal of the protective group from the glycine
and the amino group from the aspartic acid by catalytic
hydrogenation; [0074] condensation agent mediated intramolecular
cyclisation and subsequent deprotection of the amino acid side
chain protective groups.
[0075] The functional group protection and deprotection reactions
may be carried out in accordance with known techniques.
[0076] Compounds (IIa) and (IIb) may hence be obtained, according
to a process comprising the following stages (schemes 4-5): [0077]
transformation of the hydroxyl group of compounds 17, 18, 19, 20
into the corresponding azides according to known procedures, for
example through the Mitsunobu reaction, or mesylation and
subsequent nucleophilic substitution with sodium azide, to give
compounds 21, 23, 25, 27; [0078] subsequent reduction by means of
catalytic hydrogenation or Staudinger reaction [0079]
transformation into the corresponding amides by means of coupling
reactions using known reactions. [0080] Optional conjugation with
molecules of biological interest by means of known reactions;
[0081] subsequent deprotection of the aminoacid side chain
protective groups to give the compounds of formulae 22, 24, 26,
28.
[0082] To the resulting compounds, following the introduction of
the azide group 21, 23, 25 and 27, may be bound the desired
biologically active molecules or compounds of biological importance
such as for example sugars, through appropriate reactions known to
those skilled in the art (Click chemistry).
[0083] Or even, to the amine groups of the previously prepared
compounds may be bound appropriate substituents or known spacers,
to give for example ureas or thioureas (i.e. to form compounds
wherein X.dbd.--NH--CO--NHR.sub.3, --NH--CS--NHR.sub.3, prepared by
reaction with isocyanates or isothiocyanates in accordance with
known procedures.
[0084] Examples and details of such reactions are provided in the
experimental section of the present description.
[0085] According to one preferred embodiment, object of the
invention are compounds of formulae (II), (IIa), and (IIb) wherein
n is 1 and X is azide, amine, valeroyl amide and triazole,
functionalized with a sugar, fluorescein, biotin and aliphatic
chain.
[0086] According to one preferred embodiment, object of the
invention are compounds of formulae (II), (IIa), and (IIb) wherein
n is 2 and X is azide, amine, valeroyl amide and triazole,
functionalized with a sugar, fluorescein, biotin and aliphatic
chain.
[0087] The functional group protection and deprotection reactions
may be carried out in accordance with known techniques, such as
those described in the experimental section of the present
description.
[0088] The scheme for the preparation of valeroyl amides starting
from compounds of general formula (I) is reported in schemes 4 and
5.
[0089] The preparation of compounds 6,5- and 7,5-cis is reported in
scheme 4.
[0090] The preparation of compounds 6,5- and 7,5-trans is reported
in scheme 5.
[0091] The preparation of functionalised triazoles starting from
azides of compound of general formula (I) is reported in schemes
10-12.
[0092] Details of this kind of preparation for different, even if
structurally analogous compounds, are provided in the Italian
patent application N.sup.o MI2003A 002102 filed on Oct. 30, 2003,
which will be available to the public at the publication date of
the present application.
[0093] The compounds of formulae (II), (IIa) and (IIb) possess
interesting pharmacological properties, particularly an
antagonistic effect towards the .alpha.v.beta.3 and .alpha.v.beta.5
integrins, and display interesting antiangiogenic activities.
[0094] A further object of the present invention is hence the use
of the compounds of general formulae (II), (IIa) and (IIb) for the
preparation of drugs and/or compounds for diagnostic use,
particularly useful for their antagonistic action towards the
.alpha.v.beta.3 and .alpha.v.beta.5 integrins.
[0095] More particularly, the invention concerns the use of
compounds of general formulae (II), (IIa) and (IIb) for the
preparation of drugs useful for the treatment of both altered
angiogenic phenomena, and for those that may be encountered in
metastasising tumour processes, retinopathies, acute renal damage
and osteoporosis.
[0096] Biological tests for the evaluation of the activities of the
compounds of general formulae (II), (IIa) and (IIb) towards the
.alpha.v.beta.3 and .alpha.v.beta.5 integrins have been performed
using known tests also described in the literature [C. C. Kumar, H.
Nie, C. P. Rogers, M. Malkowski, E. Maxwell, J. J. Catino, L.
Armstrong, J. Pharmacol. Exp. Ther. 1997, 283, 843] for example
such as the one reported in patent application EP 1 077 218.
[0097] In such tests, certain compounds representative of the
invention have shown interesting biological and pharmacological
activities.
[0098] Regarding their use as drugs according to the invention, the
compounds of general formulae (II), (IIa) and (IIb), their
pharmaceutically acceptable salts, racemic mixtures, individual
enantiomers, individual diastereoisomers and mixtures thereof in
any proportion, are advantageously formulated into pharmaceutical
compositions according to conventional techniques, well known to
those skilled in the art.
[0099] Thus, according to another aspect thereof, the invention
concerns pharmaceutical compositions containing, as active
ingredient, at least one compound of general formulae (II), (IIa)
or (IIb), the pharmaceutically acceptable salts, racemic mixtures,
individual enantiomers, individual diastereoisomers and mixtures
thereof in whatever proportion, in combination with one or more
possible pharmaceutically acceptable carriers or excipients.
[0100] The compositions of the invention may be prepared according
to conventional techniques well known to those skilled in
pharmaceutical techniques.
[0101] In order to obtain the desired prophylactic or therapeutic
effect, the dose of active ingredient is advantageously
administered in the form of a unit dose, one or more times daily.
The daily dosages are obviously selected by the health professional
prescribing the drug depending on the biologically active molecule
introduced.
[0102] As already mentioned, the compounds of formulae (II), (IIa)
and (IIb) may be conjugated to various drugs, for example with a
cytotoxic type drug, active towards the tumour pathology, or with
appropriate ligands for diagnostic use.
[0103] In fact, the functionalised side chain (--X substituent) of
the compounds of the invention have been selected to be exploited
as a site for the introduction of pharmacologically significant
groups, in order to enhance protein-protein or protein-receptor
interactions.
[0104] Thus, for example, in the compounds of formulae (II), (IIa)
and (IIb) the nitrogen residue contained in X is available for easy
conjugation to a drug or a diagnostically useful compound.
[0105] According to another aspect thereof, the invention concerns
the use of compounds of formulae (II), (IIa) and (IIb) as mediators
for the transport and release of drugs.
[0106] Thus, the drug conjugated to the compounds of formulae (II),
(IIa) or (IIb) is then transported to the desired site of action in
order to fulfil its pharmacological activity.
[0107] Conjugates of the compounds of formulae (I), (IIa) or (IIb)
as described above, with cytotoxic and antitumour drugs, constitute
an advantageous aspect of the present invention.
[0108] The present invention will now be described from the
experimental viewpoint by way of purely illustrative, non-limiting
examples.
EXPERIMENTAL SECTION
[0109] General Observations: The .sup.1H- and .sup.13C-NMR spectra
have been recorded in CDCl.sub.3 as indicated, at 200 (or 300, 400)
and 50.3 (or 75.4, 100.6) MHz, respectively. Chemical shift values
are indicated in ppm and the coupling constants in Hz.--Optical
rotatory powers are measured using a Perkin-Elmer model 241
polarimeter.--Thin layer chromatography (TLC) is performed using
Merck F-254 plates. Flash chromatography is performed using
Macherey-Nagel 60, 230-400 mesh silica gel. Solvents are
anhydrified in accordance with standard procedures and reactions
requiring anhydrous conditions are carried out in nitrogen or argon
atmosphere. Solutions containing the final products are anhydrified
using Na.sub.2SO.sub.4, filtered, and concentrated under reduced
pressure using a rotary evaporator.
The .sup.1H-NMR and .sup.13C-NMR spectra have been recorded in the
solvents indicated using a Bruker Avance-400 instrument at 400 MHz
and 100.6 MHz respectively. Chemical shift values are indicated in
ppm and the coupling constants in Hz. Optical rotatory powers are
measured using a Perkin-Elmer model 241 polarimeter. Thin layer
chromatography (TLC) is performed using Merck F-254 plates. Flash
chromatography is performed using Macherey-Nagel 60, 230-400 mesh
silica gel. Solvents are anhydrified in accordance with standard
procedures and reactions requiring anhydrous conditions are carried
out in an argon atmosphere. FAB.sup.+ mass spectrometry has been
performed using a VG 7070 EQ-HF spectrophotometer, ESI.sup.+ mass
spectrometry has been performed using a Bruker Esquire 3000 plus
spectrophotometer.
Functionalisation of the Compounds Deriving from 1,3 Dipolar
Cyclisation
Scheme 1, Scheme 2, Scheme 7 and Scheme 9
Example 1
Protection of the Free Amino Group
[0110] To a solution of product 5 or 14 (0.51 mmol) in anhydrous
DCM (5 ml) under argon atmosphere and at room temp., are added in
the following sequence TEA (184 .mu.l, 1.33 mmol), Cbz-Cl (95
.mu.l, 0.61 mmol) and finally DMAP (15 mg, 0.126 mmol). The
solution is kept under stirring for approx. 18 hours. After this
period of time, it is taken up with DCM (5 ml) and washed with
NH.sub.4Cl (2.times.5 ml). The organic phase, anhydrated over
Na.sub.2SO.sub.4, is taken to dryness and the crude product thus
obtained is purified by flash chromatography (AcOEt/ETP
7:3.fwdarw.8:2) to give the desired product as a white foam
(60%-78%).
Compound 5-Cbz
[0111] Yield: 78%. [.alpha.].sub.D.sup.22=-13.7 (c=1.0,
CHCl.sub.3). .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta. 1.48 (s,
9H, C(CH.sub.3).sub.3), 1.6 (m, 1H, H-5), 1.7 (m, 1H, H-7), 2.06
(m, 1H, H-8), 2.2 (m, 1H, H-8), 2.23 (m, 1H, H-7), 2.3 (m, 1H,
H-5), 2.75 (m, 1H, H-4), 2.92 (bs, 1H, OH), 3.6 (dd, 1H, HCHOH),
3.71 (m, 1H, HCHOH), 3.72 (m, 1H, H-6), 4.38 (t, 1H, H-3), 4.48 (d,
1H, H-9), 5.15 (dd, 2H, CH.sub.2Ph), 6.0 (d, 1H, NHCbz), 7.28-7.42
(m, 5H, aromatic protons). .sup.13C NMR (100.6 MHz, CDCl.sub.3):
.delta. 171.7, 168.1, 156.7, 136.4, 128.5, 128.8, 127.9, 82.4,
66.9, 63.5, 58.9, 55.7, 52.1, 37.9, 32.5, 32.0, 31.6, 29.7, 29.2,
27.9. MS [FAB.sup.+]: 419.3 [M+1].sup.+. Calculated elemental
analysis C.sub.22H.sub.30N.sub.2O.sub.6: C, 63.14; H, 7.23; N,
6.69; observed C, 62.16; H, 7.25; N, 6.67.
Compound 14-Cbz
[0112] Yield: 60%. [.alpha.].sub.D.sup.22=-25.5 (c=1.0,
CHCl.sub.3). .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta. 1.48 (s,
9H, C(CH.sub.3).sub.3), 1.51 (m, 1H, H-6), 1.53 (m, 1H, H-4), 1.75
(m, 1H, H-5), 1.83 (m, 1H, H-6), 1.98 (m, 1H, H-9), 2.0 (m, 1H,
H-5), 2.2 (m, 1H, H-9), 2.25 (m, 1H, H-8), 2.33 (m, 1H, H-8), 3.4
(t, 1H, HCHOH), 3.72 (d, 1H, OH), 3.82 (d, 1H, HCHOH), 4.03 (t, 1H,
H-7), 4.42 (dd, 1H, H-3), 4.5 (d, 1H, H-10), 5.15 (dd, 2H,
CH.sub.2Ph), 6.48 (d, 1H, NHCbz), 7.31-7.43 (m, 5H, aromatic
protons). .sup.13C NMR (100.6 MHz, CDCl.sub.3): .delta. 170.5,
170.3, 158.0, 136.0, 128.6, 128.2, 128.1, 81.6, 67.5, 64.5, 61.0,
58.7, 55.5, 42.5, 33.9, 32.1, 30.9, 29.7, 29.3, 28.0, 27.2. MS
[ESI.sup.+]: 433.3 [M+H].sup.+, 455.3 [M+Na].sup.+, Calculated
elemental analysis C.sub.23H.sub.32N.sub.2O.sub.6: C, 63.87; H,
7.46; N, 6.48; observed C, 63.85; H, 7.47; N, 6.47.
Example 2
Synthesis of Azide-Derivatives
[0113] To a solution of product 5-Cbz or 14-Cbz (0.29 mmol) in
anhydrous DCM (4 ml) under argon atmosphere and at room temp., are
added in the following sequence MsCl (846 .mu.l, 0.59 mmol) and TEA
(165 .mu.l, 1.18 mmol). The solution is kept under stirring for
approx. 45 minutes. After this period of time, it is taken up with
DCM and washed with NH.sub.4Cl. The organic phase, anhydrated over
Na.sub.2SO.sub.4 is taken to dryness and the crude product thus
obtained dissolved in DMF (3.2 ml) and, under argon atmosphere and
at room temp., NaN.sub.3 (154 mg, 2.37 mmol) is added. The reaction
is kept under stirring at 80.degree. C. for approx. 18 hours. After
this period of time, the DMF is evaporated off to dryness, and the
crude product dissolved in CH.sub.2Cl.sub.2 and washed with
H.sub.2O. The organic phase, anhydrated over Na.sub.2SO.sub.4, is
taken to dryness and the crude product thus obtained is purified by
flash chromatography (AcOEt/ETP 7:3) to give the desired product as
a white foam (76%-90%).
Compound 6.
[0114] Yield: 76%. [.alpha.].sub.D.sup.22=+19.0 (c=1.0,
CHCl.sub.3). .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta. 1.48 (s,
9H, C(CH.sub.3).sub.3), 1.61 (m, 1H, H-5), 1.72 (m, 1H, H-7), 2.08
(m, 1H, H-8), 2.14 (m, 1H, H-8), 2.24 (m, 1H, H-7), 2.31 (m, 1H,
H-5), 2.9 (m, 1H, H-4), 3.28 (dd, 1H, J=Hz, HCHN.sub.3), 3.48 (dd,
1H, HCHN.sub.3), 3.7 (m, 1H, H-6), 4.31 (t, 1H, H-3), 4.39 (d, 1H,
H-9), 5.15 (s, 2H, CH.sub.2Ph), 6.0 (bs, 1H, NHCbz), 7.28-7.42 (m,
5H, aromatic protons). .sup.13C NMR (100.6 MHz, CDCl.sub.3):
.delta. 170.4, 166.7, 156.1, 136.3, 128.5, 128.1, 127.9, 81.9,
67.0, 59.0, 55.3, 55.8, 52.6, 35.4, 32.3, 31.8, 29.2, 28.0. MS
[ESI.sup.+]: 444.3 [M+H].sup.+. Calculated elemental analysis
C.sub.22H.sub.29N.sub.5O.sub.5: C, 59.58; H, 6.59, N, 15.79;
observed C, 59.57; H, 6.58; N, 15.81.
Compound 15
[0115] Yield: 90%. [.alpha.].sub.D.sup.22=-13.8 (c=1.0,
CHCl.sub.3). .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta. 1.48 (s,
9H, C(CH.sub.3).sub.3), 1.5 (m, 1H, H-5), 1.68 (m, 1H, H-4), 1.70
(m, 1H, H-6), 1.72 (m, 1H, H8), 1.82 (m, 1H, H-5), 1.98 (m, 1H,
H-9), 2.13 (m, 1H, H-6), 2.21 (m, 1H, H-9), 2.34 (m, 1H, H-8), 3.26
(dd, 1H, J=18.9 Hz, J=12.1 Hz, HCHN.sub.3), 3.65 (dd, 1H,
HCHN.sub.3), 4.09 (t, 1H, J=9.0 Hz, H-7), 4.42 (dd, 1H, H-3), 4.46
(dd, 1H, J=8.7 Hz, J=2.0 Hz, H-10), 5.12 (dd, 2H, J=15.7 Hz, J=12.2
Hz, CH.sub.2Ph), 6.08 (d, 1H, J=7.18 Hz, NHCbz), 7.3-7.42 (m, 5H,
aromatic protons). .sup.13C NMR (100.6 MHz, CDCl.sub.3): .delta.
170.5, 169.7, 156.6, 136.3, 128.5, 128.1, 81.6, 67.1, 60.9, 58.3,
55.9, 53.7, 40.9, 33.3, 31.9, 31.5, 28.0, 27.2. MS [ESI.sup.+]:
448.2 [M+H].sup.+, 480.2 [M+Na].sup.+, Calculated elemental
analysis C.sub.23H.sub.31N.sub.5O.sub.5: C, 60.38; H, 6.83; N,
15.31; observed C, 60.36; H, 6.84; N, 15.32.
Example 3
Reduction of Azide-Derivatives
[0116] To a solution of product 6 or 15 (0.034 mmol) in anhydrous
DCM (350 .mu.l) under argon atmosphere and at room temp., is added
1M Me.sub.3P in toluene (51 .mu.l, 0.051 mmol). After approx. 2
hours, upon completion of the reaction, the reaction is taken up
with DCM (1 ml), and H.sub.2O (1 ml) added, and the mixture is
allowed under stirring for approx. 10 minutes. After this period of
time, the two phases are separated. The organic phase, anhydrated
over Na.sub.2SO.sub.4, is evaporated to dryness. Compound 7.
[0117] Yield: 93%. [.alpha.].sub.D.sup.22=-13.9 (c=1.0,
CHCl.sub.3). .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta. 1.47 (s,
9H, C(CH.sub.3).sub.3), 1.55 (m, 1H, H-5), 1.72 (m, 1H, H-7), 2.07
(m, 1H, H-8), 2.16 (m, 1H, H-8), 2.21 (m, 1H, H-7), 2.30 (m, 1H,
H-5), 2.68 (m, 1H, H-4), 2.72-2.97 (m, 2H, CH.sub.2NH.sub.2), 3.73
(m, 1H, H-6), 4.31 (t, 1H, H-3), 4.41 (d, 1H, H-9), 5.15 (s, 2H,
CH.sub.2Ph), 6.4 (d, 1H, NHCbz), 7.23-7.42 (m, 5H, aromatic
protons). .sup.13C NMR (100.6 MHz, CDCl.sub.3): .delta. 170.8,
168.1, 156.4, 136.6, 128.5, 128.0, 127.9, 81.8, 67.8, 58.9, 55.6,
53.0, 32.5, 32.0, 29.7, 29.3, 28.0. MS [ESI.sup.+]: 418.4
[M+H].sup.+. Calculated elemental analysis
C.sub.22H.sub.31N.sub.3O.sub.5: C, 63.29; H, 7.48; N, 10.06;
observed C, 63.27; H, 7.47; N, 10.08.
Compound 16
[0118] Yield: 76%. [.alpha.].sub.D.sup.22=-14.5 (c=1.0,
CHCl.sub.3). .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta. 1.45 (s,
9H, C(CH.sub.3).sub.3), 1.5 (m, 1H, H-5), 1.53 (m, 1H, H-4), 1.65
(m, 1H, H-6), 1.72 (m, 1H, H8), 1.81 (m, 1H, H-5), 1.98 (m, 1H,
H-9), 2.14 (m, 1H, H-6), 2.21 (m, 1H, H-9), 2.33 (m, 1H, H-8), 2.83
(bs, 2H, CH.sub.2NH.sub.2), 4.08 (t, 1H, J=9.0 Hz, H-7), 4.41 (m,
1H, H-3), 4.47 (d, 1H, J=8.5 Hz, H-10), 5.13 (dd, 2H, J=15.7 Hz,
J=12.2 Hz, CH.sub.2Ph), 6.16 (d, 1H, J=7.0 Hz, NHCbz), 7.28-7.42
(m, 5H, aromatic protons). .sup.13C NMR (100.6 MHz, CDCl.sub.3):
.delta. 170.6, 156.8, 136.2, 128.5, 128.0, 81.5, 67.0, 60.8, 58.3,
56.3, 44.4, 33.7, 32.0, 31.3, 28.0, 27.2. MS [ESI.sup.+]: 432.5
[M+H].sup.+. Calculated elemental analysis
C.sub.23H.sub.33N.sub.3O.sub.5: C, 64.02; H, 7.71; N, 9.74;
observed C, 64.04; H, 7.72; N, 9.73.
Example 4
Conjugation Through "Click Reaction"
[0119] To a solution of azide 6 o 15 (0.1 mmol) and of an
appropriate alkinyl derivative (0.1 mmol) in H.sub.2O/t-BuOH 1:1
(500 .mu.L.) a solution of sodium ascorbate 0.9 M (44 .mu.L, 0.04
mmol, 0.4 mol eq) and a solution of Cu(OAc).sub.2 0.3 M (67 .mu.L,
0.02 mmol, 0.2 mol eq) are added, respectively. The reaction
mixture is kept under stirring at r. t. for about 18 hs. As the
reaction is completed, compounds 31e 33 have been extracted with
CH.sub.2Cl.sub.2 (2 mL.times.3) and the organic phase has been
washed with a NaHCO.sub.3 (5 mL.times.1) saturated solution and
brine (5 mL.times.1). The organic phase, anhydrated over
Na.sub.2SO.sub.4, has been evaporated under reduced pressure. As
concerns compounds 32, 34, 42-45 at the end of the reaction, the
solvent is evaporated under reduced pressure. The crude products
have been purified by flash chromatography on silica gel.
Compound 31
[0120] Column eluant: EtOAc/ETP 9:1
[0121] Yield: 80%. [.alpha.].sub.D.sup.22=-9.2 (c=1.0, CHCl.sub.3).
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 1.45 (m, 1H, H-5), 1.48
(s, 9H, C(CH.sub.3).sub.3), 1.66 (m, 1H, H-7), 1.96 (s, 3H,
OCH.sub.3), 1.98 (s, 3H, OCH.sub.3), 2.01 (s, 3H, OCH.sub.3), 2.08
(s, 3H, OCH.sub.3), 2.13 (m, 1H, H-8), 2.15-2.22 (m, 2H, H-8; H-5),
2.23 (m, 1H, H-7), 2.29 (m, 1H, H-4), 3.6-3.73 (m, 2H, H-6; H-5
Glc), 4.06 (dd, 1H, J=2.0 Hz, J=12.0 Hz, H-6 Glc), 4.20 (dd, 1H,
J=4.4 Hz, J=12.0 Hz, H-6 Glc), 4.27 (m, 1H, H-3), 4.35 (m, 1H,
H-9), 4.43 (dd, 1H, J=4.4 Hz, J=13.7 Hz, H--CH--N), 4.60 (d, 1H,
J=8.0 Hz, H-1 Glc), 4.47 (d, 1H, J=12.4 Hz, H--CH--O), 4.84 (d, 1H,
J=12.4 Hz, H--CH--O), 4.93 (m, 1H, H-2 Glc), 5.01 (m, 1H, H-4 Glc),
5.02-5.15 (m, 3H, H-3 Glc, CH.sub.2Ph), 6.0 (m, 1H, NHCbz),
7.22-7.33 (m, 5H, aromatic protons), 7.48 (s, 1H, H triazole).
.sup.13C NMR (100.6 MHz, CDCl.sub.3): .delta. 170.7, 170.4, 169.4,
169.3, 166.5, 156.0, 143.7, 136.2, 128.6, 128.2, 128.1, 124.1,
99.6, 82.3, 72.8, 71.9, 68.4, 67.1, 62.7, 61.9, 58.8, 55.0, 53.0,
51.1, 36.8, 32.5, 30.3, 29.1, 28.0, 20.8, 20.6. MS [ESI.sup.+]:
calc. per C.sub.39H.sub.51N.sub.5O.sub.15: 829.34; observed 852.4
[M+Na].sup.+. Anal. elem. calc. C.sub.39H.sub.51N.sub.5O.sub.15: C,
56.45; H, 6.19; N, 8.44; observed C, 56.46; H, 6.20; N, 8.56.
Compound 33
[0122] Column eluant: EtOAc/ETP 9:1
[0123] Yield: 84%. [.alpha.].sub.D.sup.22=-46.9 (c=1.0,
CHCl.sub.3). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 1.39 (m,
1H, H-6), 1.45 (s, 9H, C(CH.sub.3).sub.3), 1.65 (m, 1H, H-8), 1.69
(m, 1H, H-6), 1.95 (m, 1H, H-9), 1.99 (s, 3H, OCH.sub.3), 2.00 (s,
3H, OCH.sub.3), 2.03 (s, 3H, OCH.sub.3), 2.09 (s, 3H, OCH.sub.3),
2.12-2.25 (m, 2H, H4; H-9), 2.31 (m, 1H, H-8), 3.64 (m, 1H, H-5
Glc), 4.07 (m, 1H, H-7), 4.16 (dd, 1H, J=2.0 Hz, J=9.6 Hz, H-6
Glc), 4.27 (dd, 1H, J=4.8 Hz, J=12.4 Hz, H-6 Glc), 4.31 (m, 1H,
H--CH--N), 4.46 (dd, 1H, J=8.8 Hz, J=2.0 Hz, H-10), 4.51 (m, 1H,
H-3), 4.64-4.72 (m, 2H, H-1 Glc, H--CH--N), 4.79 (d, 1H, J=12.4 Hz,
H--CH--O), 4.92 (d, 1H, J=12.4 Hz, H--CH--O), 5.01 (dd, 1H,
J.apprxeq.0 Hz, J=8 Hz, H-2 Glc), 5.07-5.16 (m, 3H, H-4 Glc,
CH.sub.2Ph), 5.20 (t, 1H, J=9 Hz, H-3 Glc), 6.17 (m, 1H, NHCbz),
7.20-7.31 (m, 5H aromatic protons), 7.40 (s, 1H, H triazole).
.sup.13C NMR (100.6 MHz, CDCl.sub.3): .delta. 170.6, 170.4, 170.2,
169.4, 169.3, 169.1, 168.4, 156.9, 144.0, 136.1, 128.6, 128.2,
128.1, 123.7, 99.9, 81.7, 72.8, 71.9, 71.2, 70.7, 68.4, 67.3, 62.9,
61.9, 60.9, 60.4, 58.2, 56.1, 53.4, 52.0, 41.5, 32.9, 31.9, 30.8,
29.7, 28.0, 27.1, 20.7, 20.6. MS [ESI.sup.+]: Calc. for
C.sub.40H.sub.53N.sub.5O.sub.15: 843.35; observed: 866.2
[M+Na].sup.+, Anal. elem. calc. C.sub.40H.sub.53N.sub.5O.sub.15: C,
56.93; H, 6.33; N, 8.30; observed C, 56.95; H, 6.34; N, 8.32.
Compound 32
[0124] Column eluant: CHCl.sub.3/MeOH 9:1
[0125] Yield: 74%. [.alpha.].sub.D.sup.22=-10.8 (c=1.0,
CHCl.sub.3). .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 1.49 (s,
9H, C(CH.sub.3).sub.3), 1.53 (m, 1H, H-5), 1.66 (m, 1H, H-7), 1.98
(s, 1H, H-5), 2.01 (m, 1H, H-8), 2.13 (m, 1H, H-7), 2.18 (m, 2H,
H-8), 2.98 (m, 1H, H-4), 3.22 (t, 1H, J=8.0 Hz, H-2 Glc), 3.25-3.33
(m, 2H, H-4 Glc, H-5 Glc), 3.35 (m, 1H, H-3 Glc), 3.66-3.74 (m, 2H,
H-6; H-6 Glc), 3.89 (dd, 1H, J=0 Hz, J=12.0 Hz, H-6 Glc), 4.28-4.42
(m, 3H, H-9; H--CH--N, H-1 Glc), 4.50-4.57 (m, 2H, H-3; H--CH--N),
4-77 (d, 1H, J=12.4 Hz, H--CH--O), 4.95 (d, 1H, J=12.4 Hz,
H--CH--O), 5.13 (s, 2H, CH.sub.2Ph), 7.27-7.42 (m, 5H, aromatic
protons), 7.98 (s, 1H, H triazole). .sup.13C NMR (100.6 MHz,
CD.sub.3OD): .delta. 174.4, 167.4, 157.1, 144.4, 136.7, 128.1,
127.7, 127.6, 124.6, 102.2, 81.9, 76.7, 76.6, 73.7, 70.3, 66.7,
61.6, 61.4, 59.0, 57.5, 51.7, 50.9, 38.2, 31.3, 28.9, 28.0, 26.8.
MS [ESI.sup.+]: Calc. per C.sub.31H.sub.43N.sub.5O.sub.11: 661.30;
observed 684.3 [M+Na].sup.+. Anal. elem. calc.
C.sub.31H.sub.43N.sub.5O.sub.11: C, 56.27; H, 6.55; N, 10.58;
observed C, 56.26; H, 6.56; N, 10.59.
Compound 34
[0126] Column eluant: CHCl.sub.3/MeOH 9:1
[0127] Yield: 72%. [.alpha.].sub.D.sup.22=-49.2 (c=1.0,
CHCl.sub.3). .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 1.43 (m,
1H, H-6), 1.44 (s, 9H, C(CH.sub.3).sub.3), 1.58 (m, 2H, 2H-5), 1.75
(m, 1H, H-8), 1.80 (m, 1H, H-6), 1.93 (m, 1H, H-9), 2.18-2.34 (m,
4H, H4; H-8; H-9), 3.21 (dd, 1H, J.about.0 Hz, J=7.8 Hz, H-2 Glc),
3.27-3.33 (m, 2H, H-4 Glc, H-5 Glc), 3.35 (m, 1H, H-3 Glc) 3.68
(dd, 1H, J=5.1 Hz, J=11.9 Hz, H-6 Glc), 3.90 (dd, 1H, J=0 Hz,
J=11.9 Hz, H-6 Glc), 4.16 (m, 1H, H-7), 4.36 (m, 1H, H--CH--N),
4.37 (m, 1H, H-1 Glc), 4.40 (dd, 1H, H-10), 4.56 (m, 1H, H-3), 4.63
(dd, 2H, J=4.3 Hz, J=13.7 Hz, H--CH--N), 4.76 (d, 1H, J=12.6 Hz,
H--CH--O), 4.95 (d, 1H, J=12.5 Hz, H--CH--O), 5.13 (s, 2H,
CH.sub.2Ph), 7.24-7.42 (m, 5H, aromatic protons), 7.95 (s, 1H, H
triazole). .sup.13C NMR (100.6 MHz, CD.sub.3OD): .delta. 171.2,
170.0, 157.2, 144.5, 136.7, 128.1, 127.7, 127.6, 124.5, 102.3,
81.5, 76.7, 76.6, 73.6, 70.2, 66.6, 61.6, 61.4, 61.0, 57.8, 56.0,
51.6, 39.8, 31.5, 31.1, 29.1, 26.8, 26.6. MS [ESI.sup.+]: Calc. per
C.sub.32H.sub.45N.sub.5O.sub.11: 675.31, observed 676.2
[M+H].sup.+, 698.3 [M+Na].sup.+. Anal. elem. calc.
C.sub.32H.sub.45N.sub.5O.sub.1 1: C, 56.88; H, 6.71; N, 10.36;
observed C, 56.86; H, 6.70; N, 10.35.
Compound 42
[0128] Column eluant: CHCl.sub.3/MeOH 9:1
[0129] Yield: 80%. [.alpha.].sub.D.sup.22=-4.1 (c=1.0, MeOH).
.sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 1.28 (m, 2H, CH.sub.2
biotin), 1.31 (s, 9H, C(CH.sub.3).sub.3), 1.39 (m, 1H, H-5), 1.42
(m, 1H, HCH biotin), 1.43-1.52 (m, 3H, H-7, CH.sub.2 biotin),
1.53-1.67 (m, 5H, 2 NCH.sub.2CH.sub.2CH.sub.2O linker, HCH biotin),
1.78-1.87 (m, 2H, H-5; H-8), 1.92-2.07 (m, 4H, H-7; H-8, CH.sub.2
biotin), 2.56 (d, 1H, J=12.8 Hz, SHCH biotin), 2.76 (dd, 1H, J=12.8
Hz, J=4.8 Hz, SHCH biotin), 2.82 (m, 1H, H-4), 3.05 (m, 1H, SHC--
biotin), 3.10 (m, 2H, CH.sub.2N linker), 3.18 (m, 2H, CH.sub.2N
linker), 3.33-3.45 (m, 4H, CH.sub.2O linker), 3.40-3.45 (m, 4H,
CH.sub.2O linker), 3.45-3.505 (m, 4H, CH.sub.2O linker), 3.53 (m,
1H, H-6), 3.98 (s, 2H, COCH.sub.2O linker), 3.94 (s, 2H,
COCH.sub.2O linker), 4.12-4.18 (m, 2H, NCH biotin, H-9), 4.22 (m,
1H, H--CH--N), 4.30-4.41 (m, 5H, H-3; H--CH--N, NCH biotin,
NCH.sub.2C.dbd.), 4.95 (s, 2H, CH.sub.2Ph), 7.10-7.27 (m, 5H,
aromatic protons), 7.74 (s, 1H, H triazole). .sup.13C NMR (100.6
MHz, CD.sub.3OD): .delta. 174.4, 174.3, 170.2, 170.0, 167.2, 164.6,
157.0, 136.6, 128.0, 127.6, 127.4, 81.4, 70.1, 70.0, 69.7, 68.7,
68.5, 66.5, 61.9, 60.1, 58.9, 57.4, 55.6, 51.5, 50.8, 39.6, 38.1,
36.3, 36.2, 35.4, 33.8, 31.3, 29.3, 29.0, 28.9, 28.8, 28.3, 28.0,
27.9, 26.8, 25.4. MS [ESI.sup.+]:
C.sub.49H.sub.74N.sub.10O.sub.13S: 1042.52; observed: 1043.7
[M+H].sup.+, 1065.7 [M+Na].sup.+. Anal. elem. calc.
C.sub.49H.sub.74N.sub.10O.sub.13S: C, 56.41; H, 7.15; N, 13.43;
observed C, 56.43; H, 7.14; N, 13.45.
Compound 44
[0130] Column eluant: CHCl.sub.3/MeOH 9:1
[0131] Yield: 50%. [.alpha.].sub.D.sup.22=-3.83 (c=1.0, MeOH).
.sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 1.21-1.34 (m, 12H,
CH.sub.2 biotin, H-6, C(CH.sub.3).sub.3), 1.36-1.52 (m, 5H, 2H-5;
HCH biotin, CH.sub.2 biotin), 1.53-1.79 (m, 7H, HCH biotin H-6;
H-8, 2 NCH.sub.2CH.sub.2CH.sub.2O linker), 1.77 (m, 1H, H-9),
1.99-2.18 (m, 5H, H-4, CH.sub.2 biotin H-8; H-9), 2.55 (d, 1H,
J=12.8 Hz, SHCH biotin), 2.77 (dd, 1H, J=12.8 Hz, J=4.8 Hz, SHCH
biotin), 3.05 (m, 1H, SHC-- biotin), 3.10 (m, 2H, CH.sub.2N
linker), 3.18 (m, 2H, CH.sub.2N linker), 3.32-3.40 (m, 4H,
CH.sub.2O linker), 3.40-3.45 (m, 4H, CH.sub.2O linker), 3.45-3.50
(m, 4H, CH.sub.2O linker), 3.89 (s, 2H, COCH.sub.2O linker), 3.94
(s, 2H, COCH.sub.2O linker), 3.99 (m, 1H, H-7), 4.14 (m, 1H, NCH
biotin), 4.18 (m, 1H, H--CH--N), 4.23 (m, 1H, H-10), 4.30-4.37 (m,
3H, NCH biotin, NCH.sub.2C.dbd.), 4.39 (m, 1H, H-3), 4.45 (m, 1;
H--CH--N), 4.97 (s, 2H, CH.sub.2Ph), 7.10-7.27 (m, 5H, aromatic
protons), 7.75 (s, 1H, H triazole). .sup.13C NMR (100.6 MHz,
CD.sub.3OD): .delta. 174.4, 171.1, 170.2, 170.0, 169.9, 164.6,
157.0, 136.6, 128.0, 127.6, 127.5, 81.4, 70.0, 69.7, 68.5, 68.4,
66.5, 61.9, 60.9, 60.1, 57.7, 55.8, 55.5, 51.6, 39.6, 39.5, 36.3,
36.2, 35.4, 33.8, 31.5, 31.4, 31.0, 29.2, 29.0, 28.9, 28.3, 28.0,
26.7, 26.5, 25.4. MS [ESI.sup.+]: Calc. per
C.sub.50H.sub.76N.sub.10O.sub.13S: 1056.53, observed 1057.8
[M+H].sup.+, Anal. elem. calc. C.sub.50H.sub.76N.sub.10O.sub.13S:
C, 56.80; H, 7.25; N, 13.25; observed C, 55.81; H, 6.24; N,
13.24.
Compound 43 (Isomers Mixture)
[0132] Column eluant: CHCl.sub.3/MeOH
8:2.fwdarw.CHCl.sub.3/MeOH/H.sub.2O 60:35:5
[0133] Yield: 91%. .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 1.46
(s, 9H, C(CH.sub.3).sub.3), 1.52 (m, 1H, H-5), 1.64 (m, 1H, H-7),
1.74 (m, 2H, NCH.sub.2CH.sub.2CH.sub.2O), 1.82 (m, 1H, H-5),
1.87-2.04 (m, 3H, NCH.sub.2CH.sub.2CH.sub.2O, H-8), 2.12 (m, 1H,
H-7), 2.18 (m, 1H, H-8), 2.93 (m, 1H, H-4), 3.32 (m, 2H, CH.sub.2N
linker), 3.45-3.58 (m, 6H, CH.sub.2N linker, CH.sub.2O linker),
3.59-3.70-3.45 (m, 9H, CH.sub.2O linker, H-6), 4.03 (s, 2H,
COCH.sub.2O linker), 4.09 (s, 2H, COCH.sub.2O linker), 4.29 (m, 2H,
H-9), 4.35 (m, 1H, H--CH--N), 4.41-4.55 (m, 4H, H-3; H--CH--N,
NCH.sub.2C.dbd.), 5.11 (s, 2H, CH.sub.2Ph), 6.63-6.62 (m, 2H,
aromatic protons fluorescein), 6.66-6.80 (m, 4H, aromatic protons,
fluorescein), 7.26-7.42 (m, 5H, aromatic protons Ph), 7.83 (s, 1H,
H triazole), 8.05-8.18 (m, 2H, aromatic protons, fluorescein), 8.45
(m, 1H, aromatic proton, fluorescein). .sup.13C NMR (100.6 MHz,
CD.sub.3OD): .delta. 172.8, 171.5, 168.8, 130.8, 129.5, 129.1,
128.9, 103.7, 83.3, 71.5, 71.4, 71.3, 71.2, 70.3, 70.0, 68.0, 60.4,
59.0, 52.9, 52.2, 39.6, 39.0, 37.6, 35.2, 32.7, 30.4, 30.2, 29.4.
MS [ESI.sup.+]: Calc per C.sub.60H.sub.70N.sub.8O.sub.17: 1175.49;
observed 1176.5 [M+H].sup.+. Anal. elem. calc.
C.sub.60H.sub.70N.sub.8O.sub.17: C, 61.32; H, 6.00; N, 9.53;
observed C, 61.33; H, 6.01; N, 9.55.
Compound 45
[0134] Column eluant: CHCl.sub.3/MeOH
8:2.fwdarw.CHCl.sub.3/MeOH/H.sub.2O 60:35:5
[0135] Yield: 92%. .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 1.37
(m, 1H, H-6), 1.43 (m, 9H, C(CH.sub.3).sub.3), 1.54 (m, 2H, 2H-5),
1.64-1.84 (m, 5H, H-6; H-8, NCH.sub.2CH.sub.2CH.sub.2O linker),
1.86-1.98 (m, 2H, H-9, NCH.sub.2HCHCH.sub.2O), 2.14-2.30 (m, 3H,
H-4; H-8; H-9), 3.30 (m, 2H, CH.sub.2N linker), 3.43 (m, 2H,
CH.sub.2N linker), 3.45-3.57 (m, 4H, CH.sub.2O linker), 3.58-3.70
(m, 8H, CH.sub.2O linker), 4.03 (s, 2H, COCH.sub.2O linker), 4.08
(s, 2H, COCH.sub.2O linker), 4.11 (m, 1H, H-7), 4.32 (m, 1H,
H--CH--N), 4.39 (m, 1H, H-10), 4.47 (s, 2H, NCH.sub.2C.dbd.), 4.53
(m, 1H, H-3), 4.58 (m, 1; H--CH--N), 5.11 (s, 2H, CH.sub.2Ph),
6.52-6.60 (m, 2H, aromatic protons, fluorescein), 6.66-6.76 (m, 4H,
aromatic protons, fluorescein), 7.22-7.41 (m, 5H, aromatic protons
Ph), 7.81 (s, 1H, H triazole), 8.05-8.20 (m, 2H, aromatic protons,
fluorescein), 8.44 (m, 1H, aromatic proton, fluorescein). .sup.13C
NMR HETCOR (400 MHz, CD.sub.3OD): .delta. 132.9, 129.3, 128.7,
127.8, 127.6, 127.5, 125.8, 125.3, 124.3, 123.5, 113.9, 102.3,
69.9, 69.6, 69.4, 68.9, 68.4, 66.5, 60.8, 57.6, 55.9, 51.5, 39.6,
37.65, 36.2, 33.8, 31.5, 31.4, 31.0, 29.0, 28.9, 26.7, 26.6. MS
[ESI.sup.+]: Calc per C.sub.61H.sub.72N.sub.8O.sub.17: 1189.5;
observed: 1190.2 [M+H].sup.+, 1212.2 [M+Na].sup.+, Anal. elem.
calc. C.sub.61H.sub.72N.sub.8O.sub.17: C, 61.61; H, 6.10; N, 9.42;
observed C, 61.60; H, 6.11; N, 9.43.
Preparation of Amides of the Cyclic Pentapepthde Containing The RGD
sequence (Scheme 4, Scheme 5, Scheme 11 and Scheme 12)
Example 1
Synthesis of Azide-Derivatives
[0136] To a solution of product 17-20 (0.055 mmol) in anhydrous DCM
(700 .mu.l) under argon atmosphere and at room temp., are added in
the following sequence MsCl (8.5 .mu.l, 0.11 mmol) and TEA (30
.mu.l, 0.22 mmol). The solution is kept under stirring for approx.
30 minutes. After this period of time, the solvent is evaporated to
dryness and the crude product filtered over silica gel
(CH.sub.2Cl.sub.2/MeOH 9:1). The crude product thus obtained is
dissolved in DMF (550 .mu.l) and, under argon atmosphere and at
room temp., NaN.sub.3 (36 mg, 0.55 mmol) is added. The reaction is
kept under stirring at 80.degree. C. for approx. 18 hours. After
this period of time, the DMF is evaporated off to dryness, and the
crude product dissolved in CH.sub.2Cl.sub.2 and washed with
H.sub.2O. The organic phase, anhydrated over Na.sub.2SO.sub.4, is
taken to dryness and the crude product thus obtained is purified by
flash chromatography (CH.sub.2Cl.sub.2/iPrOH 9:1.fwdarw.8:2) to
give the desired product as a white foam (30%-75%).
Compound 21
[0137] Yield: 62%. (White solid). [.alpha.].sub.D.sup.22=-6.3
(c=1.0, CHCl.sub.3). .sup.1H NMR (400 MHz, Acetone-D6): .delta.
1.23 (s, 6H, C(CH.sub.3).sub.2 Pmc), 1.38 (s, 9H,
C(CH.sub.3).sub.3), 1.44 (m, 1H, H.beta. Arg), 1.47 (m, 1H, H-7),
1.49 (m, 1H, H-8), 1.51 (m, 1H, H.beta. Arg), 1.60 (m, 1H, H.gamma.
Arg), 1.72 (m, 2H, CH.sub.2CH.sub.2Ar Pmc), 1.74 (m, 1H, H-5), 1.80
(m, 1H, H.gamma. Arg), 2.02 (s, 3H, CH.sub.3 Pmc), 2.1 74 (m, 1H,
H-5), 2.12 (m, 1H, H-4), 2.18 (m, 1H, H-7), 2.3 (m, 1H, H-8), 2.48
(s, 3H, CH.sub.3 Pmc), 2.50 (s, 3H, CH.sub.3 Pmc), 2.54 (m, 2H,
CH.sub.2CH.sub.2Ar Pmc), 2.60 (m, 1H, H.beta. Asp), 2.91 (m, 1H,
H.beta. Asp), 3.17 (m, 1H, H.delta. Arg), 3.19 (m, 1H,
CH.sub.2N.sub.3), 3.23 (m, 1H, H.delta. Arg), 3.48 (m, 1H,
CH.sub.2N.sub.3), 3.6 (m, 1H, H.alpha. Gly), 3.8 (m, 1H, H.alpha.
Gly), 4.0 (m, 1H, H-6), 4.17 (m, 1H, H-9), 4.4 (m, 1H, H-3), 4.63
(m, 1H, H.alpha. Arg), 4.72 (m, 1H, H.alpha. Asp), 6.14 (bs, 1H,
(NH).sub.2C.dbd.NH), 6.37 (bs, 2H, (NH).sub.2C.dbd.NH), 7.32-7.48
(m, 2H, NH Arg, NH bicyclic), 7.8 (bs, 1H, NH Gly), 8.12 (bs, 1H,
NH Asp). .sup.13C NMR HETCOR (400 MHz, Acetone-D6): .delta. 67.5,
54.8, 51.8, 51.4, 50.2, 49.4, 45.8, 40.1, 39.9, 34.7, 32.8, 31.8,
31.5, 30.2, 28.1, 27.2, 26.8, 25.2, 21.3, 18.5, 17.5, 12.1. MS
[FAB.sup.+]: calculated for C.sub.40H.sub.59N.sub.11O.sub.10S:
885.42, observed: 886 [M+H].sup.+. Calculated analysis for
C.sub.40H.sub.59N.sub.11O.sub.10S: C, 54.22; H, 6.71; N, 17.39;
observed C, 54.20; H, 6.72; N, 17.37.
Compound 23
[0138] Yield: 30%. (White solid). [.alpha.].sub.D.sup.22=-65.15
(c=1.0, Acetone). .sup.1H NMR (400 MHz, Acetone-D6): .delta. 1.18
(s, 6H, C(CH.sub.3).sub.2 Pmc), 1.3 (s, 9H, C(CH.sub.3).sub.3),
1.44 (m, 1H, H-8), 1.45 (m, 3H, H-6, H.gamma. Arg), 1.5 (m, 1H,
H.beta. Arg), 1.57 (m, 1H, H.beta. Arg), 1.62 (m, 1H, H-4), 1.65
(m, 1H, H-5), 1.7 (m, 2H, CH.sub.2CH.sub.2Ar Pmc), 1.73 (m, 2H,
H-5, H-9), 1.95 (m, 1H, H-9), 1.96 (s, 3H, CH.sub.3 Pmc), 1.98 (m,
1H, H-6), 2.13 (m, 1H, H-8), 2.46 (s, 3H, CH.sub.3 Pmc), 2.48 (s,
3H, CH.sub.3 Pmc), 2.55 (m, 2H, CH.sub.2CH.sub.2Ar Pmc), 2.7 (m,
1H, H.beta. Asp), 2.78 (m, 1H, H.beta. Asp), 3.07 (m, 1H,
HCHN.sub.3), 3.11 (m, 2H, H.delta. Arg), 3.56 (m, 1H, HCHN.sub.3),
3.63 (m, 1H, H.alpha. Gly), 3.87 (m, 1H, H-7), 3.93 (m, 1H,
H.alpha. Gly), 4.33 (m, 1H, H.alpha. Asp), 4.35 (m, 1H, H-3), 4.42
(m, 1H, H.alpha. Arg), 4.64 (m, 1H, H-10), 6.25 (bs, 1H,
(NH).sub.2C.dbd.NH), 6.36 (bs, 2H, (NH).sub.2C.dbd.NH), 7.46 (m,
1H, NH bicyclic), 7.77-7.9 (m, 2H, NH Gly, NH Arg), 8.05 (bs, 1H,
NH Asp). .sup.13C NMR HETCOR (400 MHz, Acetone-D6): .delta. 61.2,
58.9, 53.5, 53.3, 53.1, 51.4, 43.6, 40.3, 39.6, 36.3, 33.3, 33.1,
31.6, 28.7, 27.3, 26.9, 26.1, 25.8, 25.5, 21.0, 18.0, 16.9, 11.4.
MS [ESI.sup.+]: calculated for C.sub.41H.sub.61N.sub.11O.sub.10S:
899.43, observed: 900.9 [M+H].sup.+. Calculated analysis for
C.sub.41H.sub.61N.sub.11O.sub.10S: C, 54.71; H, 6.83; N, 17.12;
observed C, 54.73; H, 6.82; N, 17.11.
Compound 25
[0139] Yield: 60%. (White solid). [.alpha.].sub.D.sup.22=-65.83
(c=1.15, CHCl.sub.3). .sup.1H NMR (400 MHz, Acetone-D6): .delta.
1.27 (m, 1H, H-5), 1.30 (s, 6H, C(CH.sub.3).sub.2 Pmc), 1.45 (s,
9H, C(CH.sub.3).sub.3), 1.52 (m, 1H, H.beta. Arg), 1.59 (m, 1H,
H-7), 1.60 (m, 1H, H.gamma. Arg), 1.62 (m, 1H, H.beta. Arg), 1.81
(m, 2H, CH.sub.2CH.sub.2Ar Pmc), 1.98 (m, 1H, H.gamma. Arg), 2.0
(m, 1H, H-8), 2.1 (s, 3H, CH.sub.3 Pmc), 2.38 (m, 1H, H-5), 2.42
(m, 1H, H-7), 2.43 (m, 1H, H-8), 2.56 (s, 3H, CH.sub.3 Pmc), 2.58
(s, 3H, CH.sub.3 Pmc), 2.6 (m, 1H.beta. Asp), 2.62 (m, 2H,
CH.sub.2CH.sub.2Ar Pmc), 2.9 (m, 1H, H-4), 2.95 (m, 1H, H.beta.
Asp), 3.19 (m, 1H, H.delta. Arg), 3.25 (m, 1H, H.delta. Arg), 3.37
(m, 2H, CH.sub.2N.sub.3), 3.62 (d, 1H, J=13.3 Hz, H.alpha. Gly),
4.1 (m, 1H, H-6), 4.12 (m, 1H, H.alpha. Gly), 4.25 (m, 2H, H-9),
4.39 (m, 1H, H-3), 4.6 (m, 1H, H.alpha. Arg), 4.67 (m, 1H, H.alpha.
Asp), 6.12 (bs, 1H, (NH).sub.2C.dbd.NH), 6.35 (bs, 2H,
(NH).sub.2C.dbd.NH), 7.32-7.48 (m, 2H, NH Arg, NH bicyclic), 7.82
(bs, 1H, NH Gly), 8.1 (bs, 1H, NH Asp). .sup.13C NMR HETCOR (400
MHz, Acetone-D6): .delta. 62.5, 55.7, 53.4, 51.2, 42.7, 40.6, 40.3,
36.3, 35.5, 34.9, 33.4, 33.3, 30.0, 28.4, 28.3, 27.5, 26.0, 22.1,
20.8, 17.9, 16.7, 13.2, 11.4. MS [FAB.sup.+]: calculated for
C.sub.40H.sub.59N.sub.11O.sub.10S: 885.42, observed: 886
[M+H].sup.+. Calculated analysis for
C.sub.40H.sub.59N.sub.11O.sub.10S: C, 54.22; H, 6.71; N, 17.39;
observed C, 54.21; H, 6.73; N, 17.38.
Compound 27
[0140] Yield: 75%. (White solid). [.alpha.].sub.D.sup.22=-35.74
(c=1.2, CHCl.sub.3). .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta.
1.32 (s, 6H, C(CH.sub.3).sub.2 Pmc), 1.46 (s, 9H,
C(CH.sub.3).sub.3), 1.48 (m, 1H, H-6), 1.5 (m, 1H, H.beta. Arg),
1.60 (m, 1H, H.gamma. Arg), 1.62 (m, 1H, H.beta. Arg), 1.65 (m, 1H,
H-5), 1.7 (m, 1H, H-4), 1.73 (m, 1H, H-8), 1.82 (m, 2H,
CH.sub.2CH.sub.2Ar Pmc), 1.96 (m, 1H, H.gamma. Arg), 2.02 (m, 1H,
H-9), 2.1 (m, 1H, H-5), 2.11 (s, 3H, CH.sub.3 Pmc), 2.21 (m, 1H,
H-8), 2.23 (m, 1H, H-9), 2.45 (m, 1H, H.beta. Asp), 2.56 (s, 3H,
CH.sub.3 Pmc), 2.58 (s, 3H, CH.sub.3 Pmc), 2.64 (m, 2H,
CH.sub.2CH.sub.2Ar Pmc), 2.87 (m, 1H, H.beta. Asp), 3.18 (m, 2H,
H.delta. Arg), 3.22 (m, 1H, HCHN.sub.3), 3.53 (d, 1H, J=13.0 Hz,
H.alpha. Gly), 3.62 (m, 1H, HCHN.sub.3), 4.24 (m, 1H, H-7), 4.26
(m, 1H, H.alpha. Gly), 4.41 (m, 2H, H-10), 4.59 (m, 1H, H.alpha.
Arg), 4.61 (m, 1H, H-3), 4.96 (m, 1H, H.alpha. Asp), 6.32 (bs, 3H,
(NH).sub.2C.dbd.NH), 7.46-7.58 (m, 3H, NH Gly, NH Arg, NH
bicyclic), 7.9 (bs, 1H, NH Asp). .sup.13C NMR HETCOR (400 MHz,
CDCl.sub.3): .delta. 63.4, 58.9, 55.0, 53.8, 51.9, 49.7, 44.3,
40.5, 39.3, 35.2, 33.0, 32.5, 32.6, 31.2, 28.0, 27.8, 26.7, 25.3,
21.3, 18.6, 17.3, 11.9. MS [FAB.sup.+]: calculated for
C.sub.41H.sub.61N.sub.11O.sub.10S: 899.43, observed: 901
[M+H].sup.+. Calculated analysis for
C.sub.41H.sub.61N.sub.11O.sub.10S: C, 54.71; H, 6.83; N, 17.12;
observed C, 54.70; H, 6.83; N, 17.11.
Example 2
Hydrogenation of the Azide Group
[0141] To a solution of product 21, 23, 25 or 27 (0.03 mmol) in
MeOH (1 ml) is added a catalytic quantity of C--Pd 10%. The
suspension is kept stirring under hydrogen atmosphere for approx. 4
hours. After this period of time the reaction mixture is filtered
over a bed of Celite, the collected organic phase is evaporated to
dryness and used in the subsequent reaction without any further
purification.
Example 3
Synthesis of the Amide
[0142] To a solution of product 21-NH.sub.2, 23-NH.sub.2,
25-NH.sub.2 or 27-NH.sub.2 (0.027 mmol) in anhydrous DCM (700
.mu.l) under argon atmosphere and at room temp., are added in the
following sequence: valeroyl chloride (6.5 .mu.l, 0.054 mmol) and
TEA (15 .mu.l, 0.11 mmol). The solution is kept stirring for
approx. 1.5 hours. After this period of time the solvent is
evaporated to dryness and the crude product is purified by flash
chromatography (CH.sub.2Cl.sub.2/iPrOH 85:15) to give the desired
product as a white foam (40%-57%).
Compound 22-PG
[0143] Yield: 40%. (White solid). [.alpha.].sub.D.sup.22=-83.1
(c=0.48, Acetone). .sup.1H NMR (400 MHz, Acetone-D6): .delta. 0.9
(t, 3H, J=7.4 Hz, COCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 1.32 (s, 6H,
C(CH.sub.3).sub.2 Pmc), 1.35 (m, 2H,
COCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 1.45 (s, 9H,
C(CH.sub.3).sub.3), 1.54 (m, 2H, H.gamma. Arg), 1.55 (m, 2H,
COCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 1.6 (m, 1H, H-5), 1.62 (m, 1H,
H.beta. Arg), 1.8 (m, 1H, H-7), 1.85 (m, 2H, CH.sub.2CH.sub.2Ar
Pmc), 1.9 (m, 1H, H.beta. Arg), 1.92 (m, 1H, H-8), 2.08 (m, 1H,
H-5), 2.1 (s, 3H, CH.sub.3 Pmc), 2.12 (m, 2H,
COCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2.19 (m, 1H, H-7), 2.20 (m,
1H, H-8), 2.24 (m, 1H, H-4), 2.57 (s, 3H, CH.sub.3 Pmc), 2.59 (s,
3H, CH.sub.3 Pmc), 2.67 (m, 2H, CH.sub.2CH.sub.2Ar Pmc), 2.78 (m,
1H, H.beta. Asp), 2.82 (m, 1H, HCHNHCO), 2.84 (m, 1H, H.beta. Asp),
3.27 (m, 2H, H.delta. Arg), 3.48 (m, 1H, H.alpha. Gly), 3.57 (m,
1H, HCHNHCO), 3.69 (m, 1H, H-6), 4.14 (m, 1H, H.alpha. Gly), 4.18
(m, 1H, H-9), 4.43 (m, 1H, H.alpha. Arg), 4.62 (m, 1H, H-3), 4.8
(m, 1H, H.alpha. Asp), 6.5 (m, 3H, (N).sub.2C.dbd.NH), 7.08 (m, 1H,
NHCO(CH.sub.2).sub.3CH.sub.3), 7.12 (bs, 1H, NH Arg), 7.88 (m, 1H,
NH Asp), 8.0 (bs, 1H, NH bicyclic), 8.4 (bs, 1H, NH Gly). .sup.13C
NMR HETCOR (400 MHz, Acetone-D6): .delta. 61.9, 60.5, 51.7, 51.3,
50.5, 45.1, 40.5, 40.4, 40.1, 37.7, 36.4, 32.9, 31.7, 30.8, 30.7,
28.2, 28.0, 27.6, 26.4, 25.9, 22.5, 21.6, 18.6, 17.1, 16.6, 11.8.
MS [FAB.sup.+]: calculated for C.sub.45H.sub.69N.sub.9O.sub.11S:
943.48, observed: 944 [M+H].sup.+, 966 [M+Na].sup.+. Calculated
analysis for C.sub.45H.sub.69N.sub.9O.sub.11S: C, 57.25; H, 7.37;
N, 13.35; observed C, 57.23; H, 7.36; N, 13.36.
Compound 24-PG
[0144] Yield: 40%. (White solid). [.alpha.].sub.D.sup.22=-15.9
(c=1.0, Acetone). .sup.1H NMR (400 MHz, Acetone-D6): .delta. 0.9
(t, 3H, J=7.4 Hz, COCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 1.31 (s, 6H,
C(CH.sub.3).sub.2 Pmc), 1.33 (m, 2H,
COCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 1.47 (s, 9H,
C(CH.sub.3).sub.3), 1.49 (m, 1H, H.gamma. Arg), 1.51 (m, 1H,
H.beta. Arg), 1.6 (m, 1H, H.gamma. Arg), 1.62 (m, 2H,
COCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 1.65 (m, 1H, H-6), 1.67 (m,
1H, H-5), 1.69 (m, 1H, H-4), 1.71 (m, 1H, H-8), 1.76 (m, 2H,
CH.sub.2CH.sub.2Ar Pmc), 1.79 (m, 1H, H-6), 1.96 (m, 2H, H-5, H-9),
2.07 (m, 1H, H.beta. Arg), 2.1 (s, 3H, CH.sub.3 Pmc), 2.13 (m, 2H,
COCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2.35 (m, 1H, H-8), 2.38 (m,
1H, H-9), 2.53 (m, 1H, H.beta. Asp), 2.57 (s, 3H, CH.sub.3 Pmc),
2.59 (s, 3H, CH.sub.3 Pmc), 2.68 (m, 2H, CH.sub.2CH.sub.2Ar Pmc),
2.95 (m, 1H, H.beta. Asp), 3.07 (m, 1H, HCHNHCO), 3.21 (m, 2H,
H.delta. Arg), 3.38 (d, 1H, J=14.3 Hz, H.alpha. Gly), 3.64 (m, 1H,
HCHNHCO), 4.25 (m, 1H, H-7), 4.31 (m, 1H, H.alpha. Gly), 4.41 (m,
1H, H-3), 4.46 (m, 1H, H-10), 4.52 (m, 1H, H.alpha. Arg), 4.83 (m,
1H, H.alpha. Asp), 6.32 (bs, 1H, (NH).sub.2C.dbd.NH), 6.5 (bs, 2H,
(NH).sub.2C.dbd.NH), 7.12 (m, 1H, NHCO(CH.sub.2).sub.3CH.sub.3),
7.3 (m, 1H, NH Gly), 7.49 (m, 1H, NH Arg) 7.60 (m, 1H, NH
bicyclic), 8.02 (m, 1H, NH Asp). .sup.13C NMR HETCOR (400 MHz,
Acetone-D6): .delta. 62.0, 59.4, 55.9, 51.8, 50.0, 43.9, 41.1,
40.5, 39.5, 35.7, 35.0, 33.1, 32.7, 32.2, 31.5, 27.8, 27.7, 27.4,
27.1, 26.2, 26.0, 22.3, 21.0, 18.0, 16.8, 13.5, 11.6. MS
[FAB.sup.+]: calculated for C.sub.46H.sub.71N.sub.9O.sub.11S:
957.5, observed: 958 [M+H].sup.+. Calculated analysis for
C.sub.46H.sub.71N.sub.9O.sub.11S: C, 57.66; H, 7.47; N, 13.16;
observed: C, 57.69; H, 7.46; N, 13.15.
Compound 26-PG
[0145] Yield: 54%. (White solid). [.alpha.].sub.D.sup.22=-84.4
(c=0.75, Acetone). .sup.1H NMR (400 MHz, Acetone-D6): .delta. 0.9
(t, 3H, J=7.3 Hz, COCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 1.3 (m, 2H,
COCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 1.32 (s, 6H, C(CH.sub.3).sub.2
Pmc), 1.34 (m, 1H, H-5), 1.45 (s, 9H, C(CH.sub.3).sub.3), 1.5 (m,
1H, H.gamma. Arg, H.beta. Arg), 1.54 (m, 2H,
COCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 1.6 (m, 1H, H.gamma. Arg),
1.63 (m, 1H, H-7), 1.85 (m, 2H, CH.sub.2CH.sub.2Ar Pmc), 2.0 (m,
1H, H-8), 2.08 (m, 1H, H.beta. Arg), 2.1 (s, 3H, CH.sub.3 Pmc),
2.12 (m, 2H, COCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2.4 (m, 1H, H-7),
2.43 (m, 1H, H-8; H-5), 2.56 (m, 1H, H.beta. Asp), 2.57 (s, 3H,
CH.sub.3 Pmc), 2.59 (s, 3H, CH.sub.3 Pmc), 2.68 (m, 2H,
CH.sub.2CH.sub.2Ar Pmc), 2.74 (m, 1H, H-4), 3.01 (dd, 1H, J=16.6
Hz, J=7.0 Hz, H.beta. Asp), 3.12 (m, 1H, HCHNHCO), 3.19 (m, 1H,
HCHNHCO), 3.21 (m, 2H, H.delta. Arg), 3.43 (d, 1H, J=14.2 Hz,
H.alpha. Gly), 4.17 (m, 1H, H-6), 4.2 (m, 1H, H.alpha. Gly), 4.25
(m, 1H, H-9), 4.3 (m, 1H, H-3), 4.55 (m, 1H, H.alpha. Arg), 4.58
(m, 1H, H.alpha. Asp), 6.32 (bs, 1H, (NH).sub.2C.dbd.NH), 6.48 (bs,
2H, (NH).sub.2C.dbd.NH), 6.67 (m, 1H,
NHCO(CH.sub.2).sub.3CH.sub.3), 7.33 (m, 1H, NH bicyclic), 7.42 (m,
1H, NH Arg), 7.58 (m, 1H, NH Gly), 8.032 (m, 1H, NH Asp). .sup.13C
NMR HETCOR (400 MHz, Acetone-D6): .delta. 62.5, 55.7, 53.4, 51.2,
42.7, 40.6, 40.3, 36.3, 35.5, 34.9, 33.4, 33.3, 33.4, 30.0, 28.4,
28.3, 27.5, 26.0, 22.1, 20.8, 17.9, 16.7, 13.2, 11.4. MS
[FAB.sup.+]: calculated for C.sub.45H.sub.69N.sub.9O.sub.11S:
943.48, observed: 944 [M+H].sup.+, 966 [M+Na].sup.+. Calculated
analysis for C.sub.45H.sub.69N.sub.9O.sub.11S: C, 57.25; H, 7.37;
N, 13.35; observed C, 57.26; H, 7.36; N, 13.34.
Compound 28-PG
[0146] Yield: 57%. (White solid). [.alpha.].sub.D.sup.22=-13.78
(c=1.02, Acetone). .sup.1H NMR (400 MHz, Acetone-D6): .delta. 0.92
(t, 3H, J=7.4 Hz, COCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 1.32 (s, 6H,
C(CH.sub.3).sub.2 Pmc), 1.34 (m, 2H,
COCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 1.45 (s, 9H,
C(CH.sub.3).sub.3), 1.48 (m, 1H, H.gamma. Arg), 1.50 (m, 1H,
H.beta. Arg), 1.58 (m, 1H, H.gamma. Arg), 1.6 (m, 2H,
COCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 1.62 (m, 1H, H-6), 1.65 (m,
1H, H-5), 1.67 (m, 1H, H-4), 1.7 (m, 1H, H-8), 1.75 (m, 2H,
CH.sub.2CH.sub.2Ar Pmc), 1.78 (m, 1H, H-6), 1.97 (m, 2H, H-5, H-9),
2.06 (m, 1H, H.beta. Arg), 2.1 (s, 3H, CH.sub.3 Pmc), 2.12 (m, 2H,
COCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2.33 (m, 1H, H-8), 2.38 (m,
1H, H-9), 2.54 (m, 1H, H.beta. Asp), 2.56 (s, 3H, CH.sub.3 Pmc),
2.58 (s, 3H, CH.sub.3 Pmc), 2.67 (m, 2H, CH.sub.2CH.sub.2Ar Pmc),
2.94 (m, 1H, H.beta. Asp), 3.05 (m, 1H, HCHNHCO), 3.2 (m, 2H,
H.delta. Arg), 3.37 (d, 1H, J=14.3 Hz, H.alpha. Gly), 3.65 (m, 1H,
HCHNHCO), 4.24 (m, 1H, H-7), 4.3 (m, 1H, H.alpha. Gly), 4.4 (m, 1H,
H-3), 4.45 (m, 1H, H-10), 4.5 (m, 1H, H.alpha. Arg), 4.85 (m, 1H,
H.alpha. Asp), 6.33 (bs, 1H, (NH).sub.2C.dbd.NH), 6.48 (bs, 2H,
(NH).sub.2C.dbd.NH), 7.13 (m, 1H, NHCO(CH.sub.2).sub.3CH.sub.3),
7.27 (m, 1H, NH Gly), 7.48 (m, 1H, NH Arg) 7.61 (m, 1H, NH
bicyclic), 8.04 (m, 1H, NH Asp). .sup.13C NMR HETCOR (400 MHz,
Acetone-D6): .delta. 63.1, 59.2, 55.7, 51.7, 49.9, 43.6, 40.8,
40.3, 39.6, 35.9, 34.8, 33.0, 32.6, 32.2, 31.4, 27.8, 27.7, 27.5,
27.3, 26.1, 26.0, 22.1, 21.0, 17.9, 16.9, 13.3, 11.4. MS
[FAB.sup.+]: calculated for C.sub.46H.sub.71N.sub.9O.sub.11S:
957.5, observed: 958 [M+H].sup.+. Calculated analysis for
C.sub.46H.sub.71N.sub.9O.sub.11S: C, 57.66; H, 7.47; N, 13.16;
observed: C, 57.67; H, 7.48; N, 13.14.
Example 4
Conjugation Through "Click Reaction"
[0147] To a solution of azide 25 (0.1 mmol) and of an appropriate
alkinyl derivative (0.1 mmol) in H.sub.2O/t-BuOH 1:1 (500 .mu.L.) a
solution of sodium ascorbate 0.9 M (44 .mu.L, 0.04 mmol, 0.4 mol
eq) and a solution of di Cu(OAc).sub.2 0.3 M (67 .mu.L, 0.02 mmol,
0.2 mol eq) are added, respectively. The reaction mixture has been
kept under stirring at r. t. for about 18 hs. At the reaction
completed, the solvent is evaporated under reduced pressure and the
product is isolated by flash chromatography on silica gel.
Compound 46
[0148] Yield: 67%. (White solid). [.alpha.].sub.D.sup.22=-70.5
(c=0.89, MeOH). .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 1.31 (s,
6H, C(CH.sub.3).sub.2 Pmc), 1.44 (m, 1H, H.gamma. Arg), 1.48 (s,
9H, C(CH.sub.3).sub.3), 1.51 (m, 1H, H.beta. Arg), 1.56 (m, 1H,
H.gamma. Arg), 1.6-1.7 (m, 2H, H-5H-7), 1.84 (t, 2H, J=6.8 Hz,
CH.sub.2CH.sub.2Ar Pmc), 1.87 (m, 1H, H-8), 2.06 (m, 1H, H.beta.
Arg), 2.1 (s, 3H, CH.sub.3 Pmc), 2.32-2.46 (m, 3H, H-5; H-7; H-8),
2.55 (s, 3H, CH.sub.3 Pmc), 2.56 (s, 3H, CH.sub.3 Pmc), 2.59 (m,
1H, H.beta. Asp), 2.67 (7, 2H, J=6.8 Hz, CH.sub.2CH.sub.2Ar Pmc),
3.06 (dd, 1H, J=16.6 Hz, J=7.2 Hz, H.beta. Asp), 3.14-3.22 (m, 2H,
H.delta. Arg), 3.26 (m, 1H, H-2 Glc) 3.30-3.34 (m, 3H, H-4; H-4
Glc, H-5 Glc), 3.38 (m, 1H, H-3 Glc), 3.47 (d, 1H, J=13.9 Hz,
H.alpha. Gly), 3.7 (m, 1H, H-6 Glc), 3.91 (d, 1H, J=10.8; H-6 Glc),
4.01 (m, 1H, H-6), 4.16 (dd, 1H, J=6.5 Hz, J=11.0 Hz, H-9), 4.27
(d, 1H, J=14.4 Hz, H.alpha. Gly), 4.3 (m, 1H, HCH--N) 4.35-4.46 (m,
3H, H-3; H-1 Glc, HCH--N), 4.47 (m, 1H, H.alpha. Asp), 4.55 (m, 1H,
H.alpha. Arg), 4.83 (m, 1H, OHCH--), 4.93 (d, 1H, J=12.9 Hz,
OHCH--), 8.02 (s, 1H, H triazole). .sup.13C NMR (100.6 MHz,
CD.sub.3OD): .delta. 174.3 172.4, 172.0, 171.4, 170.7, 168.8,
156.6, 153.3, 144.4, 135.1, 134.7, 133.4, 125.2, 123.5, 118.0,
101.6, 80.8, 76.6, 76.5, 73.6, 70.3, 62.3, 61.4, 55.5, 53.0, 51.5,
51.3, 51.0, 41.7, 40.2, 36.5, 34.5, 33.0, 32.4, 32.2, 30.1, 27.6,
27.0, 25.6, 21.0, 17.5, 16.4, 10.9. MS [ESI.sup.+]: calc. for
C.sub.49H.sub.73N.sub.11O.sub.16S: 1103.5, observed: 1104.5
[M+H].sup.+, 1126.5 [M+Na].sup.+. Anal. calc. for
C.sub.49H.sub.73N.sub.11O.sub.16S: C, 53.3; H, 6.66; N, 13.95;
observed C, 53.28; H, 6.64; N, 13.92.
Compound 48
[0149] Yield: 81%. (White solid). [.alpha.].sub.D.sup.22=-73.3
(c=0.92, Acetone). .sup.1H NMR (400 MHz, Acetone-d6): .delta. 0.98
(t, 3H, J=7.2 Hz, CH.sub.3), 1.35 (m, 1H, H-5), 1.37 (s, 6H,
C(CH.sub.3).sub.2 Pmc), 1.42 (m, 2H, CH.sub.2), 1.5 (s, 9H,
C(CH.sub.3).sub.3), 1.54 (m, 2H, H.beta. Arg, H.gamma. Arg), 1.58
(m, 1H, H.gamma. Arg), 1.65 (m, 1H, H-7), 1.67 (m, 2H, CH.sub.2),
1.9 (t, 2H, J=6.8 Hz, CH.sub.2CH.sub.2Ar Pmc), 2.07 (m, 1H, H-8),
2.12 (m, 1H, H.beta. Arg), 2.15 (s, 3H, CH.sub.3 Pmc), 2.26 (m, 1H,
H-5), 2.40-2.49 (m, 2H, H-7; H-8), 2.62 (s, 3H, CH.sub.3 Pmc), 2.64
(s, 3H, CH.sub.3 Pmc), 2.65-2.76 (m, 5H, CH.sub.2CH.sub.2Ar Pmc,
CH.sub.2, H.beta. Asp), 3.07 (dd, 1H, J=16.8 Hz, J=6.8 Hz, H.beta.
Asp), 3.26 (m, 2H, H.delta. Arg), 3.34 (m, 3H, H-4), 3.53 (d, 1H,
J=14.0 Hz, H.alpha. Gly), 4.11 (m, 1H, HCH--N), 4.22-4.31 (m, 2H,
H-6; H.alpha. Gly), 4.34 (m, 1H, H-9), 4.48 (m, 1H, H-3), 4.52 (m,
1H, HCH--N) 4.62 (m, 1H, H.alpha. Arg), 4.65 (m, 1H, H.alpha. Asp),
6.43 (bs, 1H, NH guanidine), 6.58 (bs, 2H, NH guanidine), 7.42 (d,
1H, J=4.0 Hz, NH scaffold), 7.59 (d, 1H, J=8.8 Hz, NH Arg), 7.66
(d, 1H, J=8.0 Hz, NH Gly), 7.76 (s, 1H, H triazole), 8.50 (d, 1H,
J=7.6 Hz, NH Asp). .sup.13C NMR (100.6 MHz, Acetone-d6): .delta.
174.2 172.2, 171.8, 170.8, 169.4, 157.1, 153.5, 148.1, 135.8,
135.7, 135.2, 123.8, 122.4, 118.5, 80.7, 74.0, 63.2, 56.2, 54.0,
52.2, 52.0, 51.7, 43.5, 41.1, 37.3, 35.5, 33.8, 33.6, 33.2, 32.2,
30.5, 28.0, 26.8, 26.7, 26.6, 25.7, 22.6, 21.7, 18.6, 17.5, 13.9,
12.0. MS [ESI.sup.+]: calc. for C.sub.46H.sub.69N.sub.11O.sub.10S:
967.49, observed: 968.5 [M+H].sup.+. Anal. calc. for
C.sub.46H.sub.69N.sub.11O.sub.10S: C, 57.07; H, 7.18; N, 15.91;
observed C, 57.08; H, 7.20; N, 15.92.
Compound 50
[0150] Yield: 62%. (White solid). [.alpha.].sub.D.sup.22=-26.5
(c=1.06, MeOH). .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 1.3 (s,
6H, C(CH.sub.3).sub.2 Pmc), 1.36-1.52 (m, 14H, H-5; H.gamma. Arg,
CH.sub.2 biotin, C(CH.sub.3).sub.3, H.beta. Arg, H.gamma. Arg),
1.53-1.67 (m, 5H, H-7; H.gamma. Arg, HCH biotin, CH.sub.2 biotin),
1.71 (m, 1H, HCH biotin), 1.72 (m, 2H, NCH.sub.2CH.sub.2CH.sub.2O
linker), 1.78 (m, 2H, NCH.sub.2CH.sub.2CH.sub.2O linker), 1.82 (m,
2H, CH.sub.2CH.sub.2Ar Pmc), 1.87 (m, 1H, H-8), 2.04 (m, 1H,
H.beta. Arg), 2.08 (s, 3H, CH.sub.3 Pmc), 2.18 (m, 2H, CH.sub.2
biotin), 2.27-2.44 (m, 3H, H-5; H-7; H-8), 2.54 (s, 3H, CH.sub.3
Pmc), 2.56 (s, 3H, CH.sub.3 Pmc), 2.57 (m, 1H, H.beta. Asp),
2.63-2.72 (m, 3H, CH.sub.2CH.sub.2Ar Pmc, SHCH biotin), 2.91 (dd,
1H, J=12.7 Hz, J=5.0 Hz, SHCH biotin), 3.04 (dd, 1H, J=16.7 Hz,
J=7.3 Hz, H.sub.13 Asp), 3.12-3.27 (m, 5H, 2H.delta. Arg, H-4,
SHC-- biotin, CH.sub.2N linker), 3.33 (m, 2H, CH.sub.2N linker)
3.41 (d, 1H, J=14.3 Hz, H.alpha. Gly), 3.47-3.55 (m, 4H, CH.sub.2O
linker), 3.56-3.66 (m, 8H, CH.sub.2O linker), 3.98 (m, 1H, H-4),
4.06 (s, 2H, COCH.sub.2O linker), 4.11 (s, 2H, COCH.sub.2O linker),
4.13 (m, 1H, H-9), 4.17-4.32 (m, 3H, HCH--N, H.alpha. Gly, NCH
biotin), 4.33-4.55 (m, 5H, H-3; HCH--N, H.alpha. Arg, H.alpha. Asp,
NCH biotin), 7.40 (m, 1H, NH Asp), 7.80 (s, 1H, H triazole).
.sup.13C NMR (100.6 MHz, Acetone-d6): .delta. 175.6, 173.7, 173.5,
172.7, 172.1, 170.2, 158.0, 154.7, 146.1, 136.5, 136.1, 134.9,
125.5, 124.9, 119.4, 82.2, 74.9, 71.6, 71.5, 71.3, 70.0, 69.9,
63.7, 63.4, 61.6, 57.1, 56.8, 54.4, 52.8, 52.5, 43.3, 41.6, 41.1,
37.9, 37.8, 37.7, 36.9, 35.9, 35.2, 34.4, 33.8, 33.6, 31.5, 30.8,
30.5, 30.4, 29.8, 29.6, 29.0, 28.4, 27.1, 27.0, 26.9, 22.4, 19.0,
17.9, 12.3. MS [ESI.sup.+]: calc. for
C.sub.67H.sub.104N.sub.16O.sub.18S.sub.2: 1484.72, observed: 1485.9
[M+H].sup.+, 743.6 [M+2H].sup.2+. Anal. calc. for
C.sub.67H.sub.104N.sub.16O.sub.18S.sub.2: C, 54.16; H, 7.06; N,
15.08; observed C, 54.14; H, 7.05; N, 15.06.
Example 5
Deprotection of the End-Products Pentapeptides
[0151] To products 22-PG, 24-PG, 26-PG, 28-PG, 46, 48, 50 or 52
(0.015) is added a solution of
CF.sub.3COOH/thioanisole/1,2-ethanedithiol/anisole 90:5:3:2 (1 ml).
Said mixture is kept stirring for approx. 2 hours. After this
period of time, the solvent is evaporated to dryness, the crude
product is dissolved in H.sub.2O and washed with iPr.sub.2O
(twice). The aqueous phases are evaporated to dryness.
Compound 22.
[0152] Yield: 99%. (White solid). [.alpha.].sub.D.sup.22=-67.75
(c=0.72, MeOH). .sup.1H NMR (400 MHz, D.sub.2O): .delta. 0.84 (t,
3H, J=7.4 Hz, NHCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 1.26 (m, 2H,
NHCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 1.42 (m, 1H, H-5), 1.5 (m, 2H,
H.gamma. Arg), 1.53 (m, 2H, NHCH.sub.2CH.sub.2CH.sub.2CH.sub.3),
1.64 (m, 1H, H.beta. Arg), 1.72 (m, 1H, H-7), 1.82 (m, 1H, H.beta.
Arg), 1.95 (m, 1H, H-8), 2.14 (m, 1H, H-5), 2.2 (m, 2H,
NHCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2.23 (m, 1H, H-7), 2.25 (m,
1H, H-8), 2.36 (m, 1H, H-4), 2.79 (m, 2H, H.beta. Asp), 3.07 (m,
1H, HCHNHCO), 3.17 (m, 2H, H.delta. Arg), 3.24 (m, 1H, HCHNHCO),
3.56 (d, 1H, J=14.3 Hz, H.alpha. Gly), 3.66 (t, 1H, J=10.4 Hz,
H-6), 4.04 (d, 1H, J=14.3 Hz, H.alpha. Gly), 4.26 (d, 1H, J=10.1
Hz, H-9), 4.3 (m, 1H, H.alpha. Arg), 4.63 (m, 1H, H.alpha. Asp),
4.74 (m, 1H, H-3). .sup.13C NMR HETCOR (100.6 MHz, D.sub.2O):
.delta. 61.3, 59.9, 51.8, 51.7, 49.7, 44.5, 40.4, 39.8, 38.0, 36.9,
35.6, 30.8, 29.0, 28.9, 27.7, 27.3, 24.3, 21.5, 12.8. MS
[ESI.sup.+]: calculated for C.sub.29H.sub.44N.sub.9O.sub.10:
735.32, observed: 622 [M-TFA].sup.+. Calculated analysis for
C.sub.29H.sub.44N.sub.9O.sub.10: C, 47.34; H, 6.04; N, 14.93;
observed C, 47.35; H, 6.04; N, 14.91.
Compound 24.
[0153] Yield: 98%. (White solid). [.alpha.].sub.D.sup.22=-22.5
(c=1.0, MeOH). .sup.1H NMR (400 MHz, D.sub.2O): .delta. 0.84 (t,
3H, J=7.3 Hz, NHCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 1.25 (m, 2H,
NHCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 1.50 (m, 2H,
NHCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 1.53 (m, 1H, H.gamma. Arg),
1.55 (m, 1H, H-6), 1.6 (m, 2H, H.beta. Arg, H-5), 1.73 (m, 1H,
H-4), 1.83 (m, 1H, H-6), 1.85 (m, 1H, H-8), 1.91 (m, 1H, H.gamma.
Arg), 1.94 (m, 1H, H-5), 2.01 (m, 2H, H.beta. Arg, H-9), 2.22 (m,
2H, NHCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2.26 (m, 1H, H-8), 2.41
(m, 1H, H-8), 2.75 (dd, 1H, J=17.0 Hz, J=6.7 Hz, H.beta. Asp), 3.01
(m, 1H, HCHNHCO), 3.05 (m, 1H, H.beta. Asp), 3.18 (m, 2H, H.delta.
Arg), 3.4 (dd, 1H, J=14.0 Hz, J=3.9 Hz, HCHNHCO), 3.5 (d, 1H,
J=14.5 Hz, H.alpha. Gly), 4.2 (m, 1H, H-7), 4.25 (d, 1H, J=14.5 Hz,
H.alpha. Gly), 4.4 (dd, 1H, J=8.8 Hz, J=4.7 Hz, H-10), 4.45 (dd,
1H, J=10.6 Hz, J=4.2 Hz, H.alpha. Arg), 4.58 (m, 1H, H-3), 4.67
(dd, 1H, J=7.6 Hz, H.alpha. Asp). .sup.13C NMR HETCOR (100.6 MHz,
D.sub.2O): .delta. 62.9, 59.5, 55.0, 52.0, 50.8, 43.2, 41.6, 40.1,
38.3, 36.0, 33.5, 32.0, 31.9, 30.8, 27.7, 27.5, 27.5, 25.7, 21.6,
12.4. MS [ESI.sup.+]: calculated for
C.sub.30H.sub.46F.sub.3N.sub.9O.sub.10: 749.33, observed: 636.7
[M-TFA].sup.+. Calculated analysis for
C.sub.30H.sub.46F.sub.3N.sub.9O.sub.10: C, 48.06; H, 6.18; N,
16.81; observed C, 48.04; H, 6.17; N, 16.82. Compound 26.
[0154] Yield: 99%. (White solid). [.alpha.].sub.D.sup.22=-64.9
(c=0.68, MeOH). .sup.1H NMR (400 MHz, D.sub.2O): .delta. 0.83 (t,
3H, J=7.3 Hz, NHCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 1.19 (m, 1H,
H-5), 1.23 (m, 2H, NHCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 1.45 (m,
2H, H.gamma. Arg), 1.48 (m, 2H,
NHCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 1.5 (m, 1H, H.beta. Arg), 1.55
(m, 1H, H-7), 1.60 (m, 1H, H-5), 1.63 (m, 1H, H-7) 1.85 (m, 1H,
H-8), 2.05 (m, 1H, H.beta. Arg), 2.15 (t, 2H, J=7.4 Hz,
NHCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2.42 (m, 2H, CH.sub.2NHCO),
2.45 (m, 1H, H-8), 2.72 (dd, 1H, J=17 Hz, J=7.1 Hz, H.beta. Asp),
2.76 (m, 1H, H-4), 3.06 (dd, 1H, J=17 Hz, J=7.1 Hz, H.beta. Asp),
3.15 (m, 2H, H.delta. Arg), 3.48 (d, 1H, J=14.6 Hz, H.alpha. Gly),
3.98 (m, 1H, H-6), 4.25 (m, 2H, H-9, H.alpha. Gly), 4.36 (t, 1H,
J=7.0 Hz, H-3), 4.5 (t, 1H, J=7.2 Hz, H.alpha. Asp), 4.57 (dd, 1H,
J=10.1 Hz, J=3.4 Hz, H.alpha. Arg). .sup.13C NMR (100.6 MHz,
D.sub.2O): .delta. 177.7, 174.8, 174.5, 174.0, 173.0, 171.6, 170.2,
62.1, 56.0, 52.9, 51.6, 51.3, 42.3, 40.3, 40.1, 35.8, 35.6, 33.2,
32.8, 32.6, 30.1, 27.5, 26.9, 24.3, 21.6, 13.0. MS [ESI.sup.+]:
calculated for C.sub.29H.sub.44F.sub.3N.sub.9O.sub.10: 735.32,
observed: 622 [M-TFA].sup.+. Calculated analysis for
C.sub.29H.sub.44F.sub.3N.sub.9O.sub.10: C, 47.34; H, 6.04; N,
14.93; observed C, 47.33; H, 6.05; N, 14.92.
Compound 28.
[0155] Yield: 99%. (White solid). [.alpha.].sub.D.sup.22=-12.6
(c=1.0, MeOH). .sup.1H NMR (400 MHz, D.sub.2O): .delta. 0.85 (t,
3H, J=7.3 Hz, NHCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 1.25 (m, 2H,
NHCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 1.51 (m, 2H,
NHCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 1.52 (m, 1H, H.gamma. Arg),
1.54 (m, 1H, H-6), 1.62 (m, 2H, H.beta. Arg, H-5), 1.75 (m, 1H,
H-4), 1.82 (m, 1H, H-6), 1.84 (m, 1H, H-8), 1.9 (m, 1H, H.gamma.
Arg), 1.93 (m, 1H, H-5), 2.0 (m, 2H, H.beta. Arg, H-9), 2.2 (m, 2H,
NHCH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2.22 (m, 1H, H-8), 2.4 (m, 1H,
H-8), 2.73 (dd, 1H, J=17.0 Hz, J=6.7 Hz, H.beta. Asp), 3.0 (m, 1H,
HCHNHCO), 3.04 (m, 1H, H.beta. Asp), 3.15 (m, 2H, H.delta. Arg),
3.42 (dd, 1H, J=14.0 Hz, J=3.9 Hz, HCHNHCO), 3.47 (d, 1H, J=14.5
Hz, H.alpha. Gly), 4.17 (m, 1H, H-7), 4.22 (d, 1H, J=14.5 Hz,
H.alpha. Gly), 4.39 (dd, 1H, J=8.8 Hz, J=4.7 Hz, H-10), 4.46 (dd,
1H, J=10.6 Hz, J=4.2 Hz, H.alpha. Arg), 4.56 (m, 1H, H-3), 4.65
(dd, 1H, J=7.6 Hz, H.alpha. Asp). .sup.13C NMR HETCOR (100.6 MHz,
D.sub.2O): .delta. 62.8, 59.3, 55.2, 51.8, 50.6, 43.0, 41.4, 40.0,
38.1, 35.7, 33.2, 31.8, 31.7, 30.5, 27.4, 27.3, 27.0, 25.5, 21.4,
12.8. MS [ESI.sup.+]: calculated for
C.sub.30H.sub.46F.sub.3N.sub.9O.sub.10: 749.33, observed: 636.7
[M-TFA].sup.+. Calculated analysis for
C.sub.30H.sub.46F.sub.3N.sub.9O.sub.10: C, 48.06; H, 6.18; N,
16.81; observed C, 48.05; H, 6.19; N, 16.83.
Compound 47
[0156] Yield: 99%. (White solid). [.alpha.].sub.D.sup.22=-69.0
(c=0.76, MeOH). .sup.1H NMR (400 MHz, D.sub.2O): .delta. 1.33 (m,
1H, H-5), 1.5 (m, 2H, H.gamma. Arg), 1.56 (m, 1H, H.beta. Arg),
1.63 (m, 1H, H-7), 1.82 (m, 1H, H-8), 2.02 (m, 1H, H.beta. Arg),
2.33-2.46 (m, 3H, H-5; H-7; H-8), 2.67 (dd, 1H, J=16.9 Hz, J=7.0
Hz, H.beta. Asp), 3.01 (dd, 1H, J=16.9 Hz, J=7.0 Hz, H.beta. Asp),
3.14 (m, 2H, H.delta. Arg), 3.20-3.29 (m, 2H, H-4; H-2 Glc), 3.33
(m, 1H, H-5 Glc), 3.37-3.45 (m, 2H, H-3 Glc, H-4 Glc), 3.48 (d, 1H,
J=14.7 Hz, H.alpha. Gly), 3.67 (dd, 1H, J=6.1 Hz, J=12.5 Hz, H-6
Glc), 3.85 (dd, 1H, J=1.8 Hz, J=12.5 Hz, H-6 Glc), 4.0 (m, 1H,
H-6), 4.17-4.3 (m, 3H, HCH--N, H.alpha. Arg, H-9), 4.37-4.44 (m,
3H, HCH--N, H.alpha. Asp, H-3), 4.48 (d, 1H, J=8.0 Hz, H-1 Glc),
4.55 (m, 1H, H.alpha. Arg), 4.79 (d, 1H, J=126.0 Hz, OHCH--), 4.93
(d, 1H, J=126.0 Hz, OHCH--), 7.51 (m, 1H, NH Asp), 8.01 (s, 1H, H
triazole). .sup.13C NMR (100.6 MHz, D.sub.2O): .delta. 174.9,
174.8, 174.0, 173.0, 171.6, 169.2, 156.7, 143.6, 126.0, 101.4,
76.0, 75.8, 73.0, 69.7, 62.2, 61.9, 60.8, 58.5, 55.8, 54.3, 52.9,
51.6, 51.5, 51.3, 46.7, 42.4, 40.4, 36.2, 33.3, 32.7, 32.2, 30.0,
27.0, 24.4. MS [ESI.sup.+]: calc. for
C.sub.31H.sub.17N.sub.11O.sub.13: 781.34, observed: 782.5
[M+H].sup.+. Anal. calc. for
C.sub.33H.sub.48F.sub.3N.sub.11O.sub.15: C, 44.25; H, 5.40; N,
17.20; observed C, 44.27; H, 5.41; N, 17.22.
Compound 49
[0157] Yield: 99%. (White solid). [.alpha.].sub.D.sup.22=-68.3
(c=0.77, MeOH). .sup.1H NMR (400 MHz, D.sub.2O): 0.82 (m, 3H,
CH.sub.3), .delta. 1.17-1.33 (m, 3H, H-5, CH.sub.2), 1.42-1.66 (m,
5H, 2H.gamma. Arg, H.beta. Arg, CH.sub.2; H-7), 1.83 (m, 1H, H-8),
2.0 (m, 1H, H.beta. Arg), 2.3-2.46 (m, 3H, H-5; H-7; H-8), 2.66 (m,
1H, CH.sub.2), 2.68 (m, 1H, H.beta. Asp), 2.99 (m, 1H, H.beta.
Asp), 3.13 (m, 2H, H.delta. Arg), 3.24 (m, 1H, H-4), 3.46 (d, 1H,
J=14.7 Hz, H.alpha. Gly), 3.99 (m, 1H, H-6), 4.1-4.29 (m, 3H,
HCH--N, H.alpha. Arg, H-9), 4.31-4.43 (m, 3H, HCH--N, H.alpha. Asp,
H-3), 4.54 (m, 1H, H.alpha. Arg), 7.52 (m, 1H, NH Asp), 7.80 (s,
1H, H triazole). .sup.13C NMR (100.6 MHz, D.sub.2O): .delta. 174.6,
174.0, 173.9, 173.0, 171.6, 170.8, 169.1, 156.7, 148.1, 124.4,
62.2, 55.8, 52.9, 51.8, 51.6, 51.2, 42.4, 40.4, 36.3, 36.1, 33.2,
32.7, 32.2, 30.6, 30.2, 30.0, 27.0, 24.4, 23.7, 21.4, 13.0. MS
[ESI.sup.+]: calc. for C.sub.28H.sub.43N.sub.11O.sub.7: 645.33,
observed: 646.2 [M+H].sup.+. Anal. calc. for
C.sub.30H.sub.44F.sub.3N.sub.11O.sub.9: C, 47.43; H, 5.84; N,
20.28; observed C, 47.42; H, 5.83; N, 20.26.
Compound 51
[0158] Yield: 99%. (White solid). [.alpha.].sub.D.sup.22=-27.3
(c=0.67, MeOH). .sup.1H NMR (400 MHz, D.sub.2O): .delta. 1.25-1.41
(m, 3H, H-5, CH.sub.2 biotin), 1.45-1.67 (m, 8H, 2 CH.sub.2 biotin,
2H.gamma. Arg, H.beta. Arg, H-7), 1.68-1.79 (m, 4H,
NCH.sub.2CH.sub.2CH.sub.2O linker), 1.83 (m, 1H, H-8), 2.02 (m, 1H,
H.beta. Arg), 2.18 (t, 2H, J=7.2 Hz, CH.sub.2 biotin), 2.33-2.46
(m, 3H, H-5, H-7; H-8), 2.64 (dd, 1 Hp J=16.8 Hz, J=6.8 Hz, H.beta.
Asp), 2.69 (dd, 1H, J=13.2 Hz, J=4.8 Hz, SHCH biotin), 3.01 (dd,
1H, J=16.8 Hz, J=7.6 Hz, H.beta. Asp), 3.14 (m, 2H, H.delta. Arg),
3.19 (m, 2H, CH.sub.2N linker), 3.22-3.30 (m, 4H, H-4, SHC--
biotin, CH.sub.2N linker), 3.47 (m, 1H, H.alpha. Gly), 3.48-3.55
(m, 4H, CH.sub.2O linker), 3.56-3.65 (m, 9H, CH.sub.2O linker),
3.99 (m, 1H, H-6), 4.07 (s, 2H, COCH.sub.2O linker), 4.12 (s, 2H,
COCH.sub.2O linker), 4.16 (m, 1H, H-9), 4.20-4.30 (m, 2H, HCH--N,
H.alpha. Gly), 4.33-4.43 (m, 4H, HCH--N, H.alpha. Asp, H-3, NHC
biotin), 4.47 (s, 2H, NCH.sub.2C.dbd.) 4.52-4.58 (m, 2H, H.alpha.
Arg, NHC biotin), 7.49 (m, 1H, NH Asp), 7.87 (s, 1H, H triazole).
.sup.13C NMR (100.6 MHz, D.sub.2O): .delta. 174.6, 174.7, 173.9,
172.9, 171.7, 171.6, 171.5, 169.2, 144.4, 124.5, 70.0, 69.9, 69.6,
69.4, 68.5, 68.4, 62.1, 62.2, 55.7, 55.4, 52.9, 51.6, 51.4, 51.3,
42.3, 40.4, 39.7, 36.3, 36.2, 36.1, 35.5, 33.9, 33.4, 32.7, 32.2,
30.0, 28.3, 27.7, 27.0, 25.2, 24.4. MS [ESI.sup.+]: calc. for
C.sub.49H.sub.78N.sub.16O.sub.15S: 1162.56, observed: 1163.6
[M+H].sup.+, 582.4 [M+2H].sup.2+, Anal. calc. for
C.sub.51H.sub.79F.sub.3N.sub.16O.sub.17S: C, 47.96; H, 6.23; N,
17.54; observed C, 47.97; H, 6.24; N, 17.56.
Compound 53 (Isomers Mixture)
[0159] Yield: 99%. (Yellow solid). .sup.1H NMR (400 MHz, DMSO-d6):
.delta. 1.14 (m, 1H, H-5), 1.3-1.53 (m, 4H, 2H.gamma. Arg, H.beta.
Arg, H-7), 1.66 (m, 2H, NCH.sub.2CH.sub.2CH.sub.2O linker), 1.71
(m, 1H, H-8), 1.79 (m, 2H, NCH.sub.2CH.sub.2CH.sub.2O linker),
1.96-2.08 (m, 2H, H.beta. Arg, H-7), 2.20 (m, 1H, H-5), 2.24 (m,
1H, H-8), 2.52 (m, 1H, H.beta. Asp), 2.94 (m, 1H, H.beta. Asp),
3.03-3.20 (m, 3H, H.delta. Arg, H-4), 3.24 (m, 2H, CH.sub.2N
linker), 3.30 (m, 1H, H.alpha. Gly), 3.34-3.56 (m, 14H, CH.sub.2N
linker, CH.sub.2O linker), 3.92 (s, 2H, COCH.sub.2O linker), 3.93
(m, 1H, HCH--N), 3.96 (s, 2H, COCH.sub.2O linker), 3.99-4.09 (m,
2H, H-6, H.alpha. Gly), 4.16 (m, 1H, H-9), 4.26-4.44 (m, 6H,
HCH--N, H-3, NCH.sub.2C.dbd., H.alpha. Asp, H.alpha. Arg), 651-6.62
(m, 4H, H aromatics fluorescein), 6.7 (m, 2H, H aromatics
fluorescein), 7.27 (m, 1H, NH scaffold), 7.36 (m, 1H, H aromatic
fluorescein), 7.53 (m, 1H, NH Gly), 7.58 (m, 3H, NH guanidine),
7.66 (s, 1H, H aromatic fluorescein), 7.92 (s, 1H, H triazole),
8.02 (m, 1H, NH Asp), 8.07 (m, 1H, H aromatic fluorescein), 8.16
(m, 1H, H aromatic fluorescein), 8.24 (m, 1H, H aromatic
fluorescein), 8.44 (s, 1H, H aromatic fluorescein), 8.53 (m, 1H, NH
Arg), 8.65 (m, 1H, NH linker), 8.80 (m, 1H, NH linker), 9.93 (m,
1H, NH linker), 10.18 (m, 1H, COOH). .sup.13C NMR (100.6 MHz,
DMSO-d6): .delta. 173.2, 172.6, 170.9, 170.8, 170.5, 169.5, 169.4,
168.6, 168.5, 168.3, 168.2, 168.0, 167.8, 164.6, 159.6, 156.5,
151.8, 144.6, 140.8, 136.3, 134.6, 129.2, 129.1, 128.1, 126.4,
124.8, 124.2, 123.6, 123.2, 122.2, 112.6, 109.1, 102.2, 70.2, 70.1,
69.8, 69.7, 69.6, 69.6, 69.4, 68.1, 61.9, 54.8, 52.6, 52.5, 51.1,
50.7, 50.3, 42.6, 36.8, 36.7, 36.1, 35.6, 35.5, 33.8, 33.7, 33.5,
32.8, 32.2, 29.5, 29.2, 29.1, 27.8, 25.3. MS [ESI.sup.+]: calc. for
C.sub.60H.sub.74N.sub.14O.sub.19: 1294.53, observed: 1295.6
[M+H].sup.+, 648.3 [M+2H].sup.2+. Anal. calc. for
C.sub.62H.sub.75F.sub.3N.sub.14O.sub.21: C, 52.84; H, 5.36; N,
13.91; observed C, 52.85; H, 5.35; N, 13.91.
##STR00007## ##STR00008##
##STR00009## ##STR00010##
##STR00011##
##STR00012## ##STR00013##
##STR00014## ##STR00015##
##STR00016##
##STR00017##
##STR00018##
##STR00019##
##STR00020##
##STR00021## ##STR00022##
##STR00023##
##STR00024##
* * * * *