U.S. patent application number 09/777013 was filed with the patent office on 2002-06-06 for peptido-mimetic compounds containing rgd sequence useful as integrin inhibitors.
Invention is credited to Giannini, Giuseppe, Scolastico, Carlo.
Application Number | 20020068695 09/777013 |
Document ID | / |
Family ID | 26331621 |
Filed Date | 2002-06-06 |
United States Patent
Application |
20020068695 |
Kind Code |
A1 |
Scolastico, Carlo ; et
al. |
June 6, 2002 |
Peptido-mimetic compounds containing RGD sequence useful as
integrin inhibitors
Abstract
The present invention discloses compounds of formula (I) 1
wherein n is the number 0, 1 or 2. There are also disclosed
processes for the preparation of said compounds, together with
methods for treating pathologies related to an altered
.alpha..sub.v.beta..sub.3 integrin-mediated cell attachment, in
particular wherein the inhibition of angiogenesis is desired, for
example in tumors, also associated with metastasis.
Inventors: |
Scolastico, Carlo; (Milan,
IT) ; Giannini, Giuseppe; (Pomezia, IT) |
Correspondence
Address: |
NIXON & VANDERHYE P.C.
8th Floor
1100 North Glebe Road
Arlington
VA
22201-4714
US
|
Family ID: |
26331621 |
Appl. No.: |
09/777013 |
Filed: |
February 6, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09777013 |
Feb 6, 2001 |
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09366198 |
Aug 4, 1999 |
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6235877 |
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Current U.S.
Class: |
530/330 ;
514/13.3; 514/15.4; 514/16.9; 514/19.1; 514/19.8; 514/21.1 |
Current CPC
Class: |
C07C 67/31 20130101;
C07C 67/31 20130101; C07C 227/32 20130101; C07C 227/32 20130101;
A61K 38/00 20130101; C07C 69/675 20130101; C07K 7/64 20130101; C07K
5/06139 20130101; C07C 229/22 20130101 |
Class at
Publication: |
514/9 ;
530/330 |
International
Class: |
C07K 005/08; C07K
005/12; A61K 038/06 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 16, 1998 |
IT |
MI98A002477 |
Claims
1. Compounds of formula (I) 8wherein n is the number 0, 1 or 2, Arg
is the amino acid l-arginine, gly is the amino acid glycine and asp
is the amino acid l-aspartic acid and the pharmaceutically
acceptable salts thereof, their racemates, single enantiomers and
diastereoisomers.
2. A compound of claim 1 which is 9
3. A compound of claim 1, which is 10
4. A compound of claim 1, which is 11
5. A compound of claim 1, which is 12
6. A compound of claim 1, which is 13
7. A compound of claim 1, which is 14
8. A compound of claim 1, which is 15
9. A compound of claim 1, which is 16
10. A process for the preparation of the compounds of claim 1
comprising the following steps: a) Horner-Emmons olefination of a
compound of formula (II) 17wherein R is a lower alkyl residue;
R.sub.1 is a suitable nitrogen protecting group, to give a compound
of formula (III); 18wherein R.sub.3 is a suitable nitrogen
protecting group, R.sub.4 is a lower alkyl residue; b)
hydrogenation of said compound of formula (III) and cyclisation;
and, if desired c) separation of the stereoisomeric mixture; d)
building of the RGD cyclic sequence; and, if desired e) separation
of the stereoisomeric mixture.
11. A process for the stereoselective synthesis of the compounds of
claim 1, comprising the following steps: a) Horner-Emmons
olefination of a compound of formula (II) 19wherein R is a lower
alkyl residue; R.sub.1 is a suitable nitrogen protecting group, to
give a compound of formula (III); 20wherein R.sub.3 is a suitable
nitrogen protecting group, R4 is a lower alkyl residue; b)
hydrogenation of said compound of formula (III) by chiral
phosphine-Rh catalysed hydrogenation and cyclisation; and, if
desired c) separation of the stereoisomeric mixture; d) building of
the RGD cyclic sequence; and, if desired e) separation of the
stereoisomeric mixture.
12. Pharmaceutical composition comprising a therapeutically or
preventive effective dose of at least a compound of claim 1 in
admixture with pharmaceutically acceptable vehicles and/or
excipients.
13. A method for selectively inhibiting {acute over
(.alpha.)}.sub.v{circumflex over (.alpha.)}.sub.3 integrin-mediated
cell attachment to an RGD-containing ligand, comprising contacting
said ligand with an effective amount of a compound of claim 1.
14. A method for treating a subject suffering from a pathology
related to an altered .alpha..sub.v.beta..sub.3 integrin-mediated
cell attachment comprising administering to said subject a compound
of claim 1.
15. A method according to claim 14, wherein said pathology is
retinopathy.
16. A method according to claim 14, wherein said pathology is acute
renal failure.
17. A method according to claim 14, wherein said pathology is
osteoporosis.
18. A method for treating a subject suffering from altered
angiogenesis, comprising administering to said subject a compound
of claim 1.
19. A method for the treatment of tumors in a subject comprising
administering to said subject a compound of claim 1.
20. A method according to claim 19, wherein said tumor, is
associated with metastasis.
Description
[0001] The present invention relates to cyclic peptido-mimetic
compounds, in particular to cyclic peptido-mimetic compounds having
azabicycloalkane structure and containing the RGD (Arg-Gly-Asp)
sequence. Said compounds have inhibiting action on
.alpha..sub.v.beta..sub.3 -receptor of the integrin family. The
compounds of the present invention are endowed with antiangiogenic
properties, hence are useful as medicaments, preferably for the
treatment of tumors.
BACKGROUND OF THE INVENTION
[0002] The first molecule with antiangiogenic activity was
discovered in 1975 by Henry Brem and Judah Folkman in cartilaginous
tissues.
[0003] In the 80s it was found that interferon (.alpha./.beta.) is
effective in inhibiting tumor angiogenesis.
[0004] In 1998, it was widely published, also in the media, that
angiostatin and endostatin discovered by J.Folkman at Harvard
Medicinal School and Boston Children's Hospital were giving very
encouraging results in tumor treatment.
[0005] To-date, about 30 molecules are tested in clinical trials
(Phase I-III).
[0006] Of these 30 molecules, only two drugs, of which one is an
antibody, are in clinical trials for their activity in inhibiting
endothelial specific integrins.
[0007] It is calculated that only in the USA, about 9 million
patients could benefit from an antiangiogenic therapy.
[0008] Recently, FDA has approved clinical trials for the
combination of IL-10 with Thalidomide and Methoxyestradiol.
[0009] Angiogenesis is intended as the formation of new capillary
blood vessels. This natural phenomenon is involved both in
physiological processes, as reproduction, and in pathological
occurrences, as wound healing, arthritis and tumor
vascularization.
[0010] A number of growth factors have been identified as capable
of promoting angiogenesis, through direct induction of
proliferation and/or chemiotaxis of endothelial cells. Other
factors, instead, act indirectly, by stimulating other cell types
(mast cells, macrophages), which, on their turn, produce angiogenic
factors. The presence of growth factors, such as bFGF and VEGF,
near a resting capillary net, suggested that angiogenesis might be
the outcome of an unbalance between pro-and anti-angiogenic
factors.
[0011] In the last years, it was reported that tumor growth and
metastasis formation is strictly dependent on the development of
new vessels capable of vascularizing the tumor mass.
[0012] Antiangiogenic tumor therapy is strongly desired by
physicians for the following reasons:
[0013] specificity: tumor neovascularization is the target;
[0014] bioavailability: the antiangiogenic agent is targeted toward
endothelial cells, easily reached without the well-known problems
of chemotherapy, which is directed on the tumor cell;
[0015] chemoresistance: this is the most striking advantage, in
fact, endothelial cells are genetically stable and it is quite
difficult to observe drug resistance;
[0016] angiogenic blockade avoids metastatic cells to diffuse
through blood circulation;
[0017] apoptosis: blocking angiogenesis makes tumor cell suffer
from oxygen and nutrition lack, thus inducing apoptosis;
[0018] antiangiogenic therapy does not give rise to side effects
typical of chemotherapy.
[0019] The endogenous pro-angiogenic factors to date known are
acid/basic Fibroblast Growth factor (a/bFGF) and Vascular
Endothelial Growth Factor (VEGF), and its subtype B and C,
Angiogenin, Endothelial Growth Factor (EGF), Platelet
derived-Endothelial Cell Growth Factor (PD-ECGF), Transforming
Growth Factor-.alpha. (TGF-.alpha.), Transforming Growth
Factor-.beta. (TGF-.beta.), Tumor Necrosis Factor-.alpha.
(TNF-.alpha.).
[0020] Retinoids are tested as potential antiangiogenic agents.
[0021] Some PK-C inhibitors, such as Calphostin-C, phorbol esters
and Staurosporin, can block angiogenesis, either partially or
totally.
[0022] Integrins are a class of receptors involved in the mechanism
of cell adhesion and alterations in the function of these receptors
are responsible in the occurrence of a number of pathologic
manifestations, for example embryogenic development, blood
coagulation, osteoporosis, acute renal failure, retinopathy,
cancer, in particular metastasis. Among the molecular targets
involved in angiogenesis, .alpha..sub.v.beta..sub.3 integrins play
an important role in adhesion, motility, growth and differentiation
of endothelial cells. .alpha..sub.v.beta..sub.3 integrins bind the
RGD sequence (Arg-Gly-Asp), which constitutes the recognition
domain of different proteins, such as laminin, fibronectin and
vitronectin. The RGD sequence represent the minimal amino acid
domain, in several extra-cellular matrix proteins, which has been
demonstrated to be the binding site of the transmembrane integrins
proteins family (G. Bazzoni, E. Dejana and M. G. Lampugnani. 1999,
Current Opinion in Cell Biology; (11) pp. 573-581). Indeed
replacement of just one single amino acid of this short sequence
result in loss of binding activity to integrins (F. E. Ali, R.
Calvo, T. Romoff, I. Samanen, A. Nichols. 1990, Peptides:
Chemistry. Structure and Biology (Eds: J. E. Rivier, G. R.
Marshall) ESCOM Science Leiden (Netherland) pp. 94-96). In the last
years has been demonstrated that RGD peptide isolated from phage
peptides library or biochemically synthesised, were able to compete
with extracellular matrix proteins to bind integrins (R. Haubner,
D. Fisinger and H. Kessler. 1997, Angew. Chem. Int. Ed. Engl. ;
(36) pp. 1374-1389).
[0023] The role of RGD sequence is described, for example, in Grant
et al., J. Cell Physiology, 1992, Saiki et al., Jpn. J. Cancer Res.
81; 668-75. Carron et al, 1998, Cancer Res. 1; 58(9):1930-5
disclosed an RGD-containing tripeptide, named SC-68448, capable of
inhibiting the binding between .alpha..sub.v.beta..sub.3 integrin
with vitronectin (IC.sub.50=1 nM). Other works (Sheu et al., 1197,
BBA; 1336(3):445-54-Buckle at al., 1999, Nature 397:534-9) showed
that RGD peptides can diffuse through the cell membrane and bind to
the protein caspase-3, inducing apoptosis.
[0024] Therefore, RGD sequence is the basis for developing
antagonists of the different integrins. To date, the reasons for
which in many cases a high selectivity for certain integrins is
observed is not quite clear, although a different conformation of
the RGD sequence can be taken as an explanation. Recent data
demonstrated that this sequence is often inserted into a type
II-.beta.-turn between two .beta.-sheets extending from the core of
the protein.
[0025] Thus the problem to provide substances having high
selectivity toward integrins has not been fully satisfied yet.
[0026] There is a structural constraint to this research, namely,
the RGD sequence must be kept unaltered, since it is well known
that any modification to this sequence implies a loss of
activity.
[0027] To find the correct structure that can block the molecule in
a precise reverse-turn conformation, inducing a .beta.-turn
geometry, is very critical.
[0028] It is well known that the
.alpha..sub.v.beta..sub.3-receptor, a member of the integrin
family, is implicated in angiogenesis and in human tumor
metastasis.
[0029] Metastasis of several tumor cell lines as well as
tumor-induced angiogenesis can be inhibited by antibodies or small,
synthetic peptides acting as ligands for these receptors
(Friedlander et al.: Science 1995, 270, 1500-1502.
[0030] In order to have an inhibiting property, all the peptides
must contain the Arg-Gly-Asp (RGD) sequence. Notwithstanding this
RGD sequence, a high substrate specificity is present, due to
different conformations of the RGD sequence in different matrix
proteins (Ruoshlati et al. Science 1987, 238, 491-497). This
flexibility of particular RGD portion is an obstacle to the
determination of the bioactive conformation to be used in the
widespread structure-activity drug design.
[0031] A solution was provided by Haubner et al. (J. Am. Chem. Soc.
1996, 118, 7881-7891) by inserting the RGD sequence in cyclic,
rigid peptide structure. Spatial screening led to the highly active
first-generation peptide c(RGDfV) (cyclic Arg-Gly-Asp-D-Phe-Val;
WO97/06791), which shows a .beta.II'/.gamma.-turn arrangement. A
reduction of the flexibility is a technical goal to be achieved in
order to obtain antagonists of integrins. Due to the width of the
integrin family and to the number of different physiological
activities of said integrins, it is highly desired to obtain active
agents having highly selective inhibiting action.
[0032] A solution proposed in the art was to introduce in the
peptido-mimetic structure a rigid building block (turn
mimetics).
[0033] Despite different tentatives and a number of structures
proposed, Haubner et al. (J. Am. Chem. Soc. 1996, 118, 7881-7891),
identified an RGD"spiro" structure capable of providing the desired
.beta.II'/.gamma.-turn arrangement. Actually, four different
structures are enabled in this work: an (S)-proline derivative, an
(R)-proline derivative, a thiazabicyclo structure and a
diaza-spiro-bicyclic structure. Non-homogeneous results were
obtained. The spiro structure was the only one able to adopt a
.beta.II'/.gamma.-turn conformation, but lacks of biological
activity. The (R)-proline is very active, but less selective. The
(S)-proline is active and selective. The thiazabicyclo-structure is
active, but has the disadvantage to be less selective.
[0034] WO91/15515 discloses cyclic peptides, also containing the
RGD sequence, useful for treating thrombosis, through the selective
inhibition of the platelet aggregation receptor GPIIb/IIa.
[0035] WO92/17492 discloses cyclic peptides, also containing the
RGD sequence, useful for treating thrombosis, through the selective
inhibition of the platelet aggregation receptor GPIIb/IIa. These
peptides contain also a positively charged nitrogen containing
exocyclic moiety stably bonded to the cyclic peptide through a
carbonyl. No beta-turns are contained in these structures.
[0036] WO94/29349 discloses a long peptide containing a
-Cys-S-S-Cys- cyclic portion for the treatment of a venous or
arterial thrombotic condition. This trifunctional peptide combines
both catalytic and anion binding exosite inhibition of thrombin
with GP IIb/IIIa receptor inhibition.
[0037] Other peptides active in treating thrombosis are disclosed
in WO95/00544.
[0038] WO97/06791 discloses the use of c(RGDfV) as selective
inhibitor of .alpha..sub.v/.gamma..sub.5 and useful as inhibitor of
angiogenesis
[0039] WO97/08203 discloses circular RGD-containing peptides, which
comprise the motif (/P)DD(G/L)(W/L)(W/L/M).
[0040] U.S. Pat. No. 5,767,071 and U.S. Pat. No. 5,780,426 disclose
non-RGD amino acid cyclic peptides binding
.alpha..sub.v/.gamma..sub.3 integrin receptor.
[0041] U.S. Pat. No. 5,766,591 discloses RGD-peptides for
inhibiting .alpha..sub.v/.gamma..sub.3 receptor and useful as
antiangiogenesis agents. No beta turn portions are taught.
[0042] WO98/56407 and WO98/56408 disclose fibronectin antagonists
as therapeutic agents and broad-spectrum enhancers of antibiotic
therapy. Said fibronectin antagonists bind to a
.alpha..sub.5.beta..sub.1 integrin to the purpose to prevent
intracellular invasion by microbial pathogens. Some of these
inhibitors are linear or cyclic peptides containing the RGD
structure or antibodies. Integrin antagonists are specifically
disclosed for their selectivity against .alpha..sub.5.beta..sub.1
integrin. The best of them proved to be
(S)-2-[2,4,6-trimethylphenyl)
sulfonyl]amino-3-[[7benzyloxycarbonyl-8-(2-pyridinylaminomethyl)-1-oxa-2,-
7-diazaspiro- [4,4] -non-2-en-3-yl]carbonylamino]propionic
acid.
[0043] U.S. Pat. No. 5,773,412 discloses a method for altering
.alpha..sub.v.beta..sub.3 integrin receptor-mediated binding of a
cell to a matrix, said cell being an endothelial or smooth muscle
cell, by contacting said cell with a RGD-containing cyclic peptide.
Also disclosed there is a method for inhibiting angiogenesis by
using this cyclic peptide. The cyclic peptide disclosed in U.S.
Pat. No. 5,773,412 contains at least 6 amino acids and the RGD
sequence is flanked, on the D-side, by a first amino acid which can
provide a hydrogen bond interaction with an integrin receptor (Asn,
Ser or Thr) and a second amino acid, that has the characteristics
of hydrophobicity or conformational constraint (Tic, i.e.
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, Pro, Phe or Ile).
A selection of these peptides are taught as useful for altering the
binding of osteoclasts to a matrix such as bone or for selectively
altering integrin receptor binding. It has now been found that
cyclic pseudopeptides having an RGD mimetic structure characterized
by an azabicycloalkane structure are endowed with selective
inhibition of .alpha.v.beta.3 integrin-mediated cell attachment.
This activity makes them useful as therapeutical agents, in
particular for treating pathologies due to an altered angiogenesis,
for example tumors.
[0044] A further object of the present invention is a method for
treating a subject, whether human or animal, suffering of a tumor,
by inducing an inhibition of angiogenesis, in particular for
inhibiting or reducing or blocking metastatic proliferation, with
the administration of a therapeutic or preventive dose of at least
a compound of formula (I). Also objects of the present invention
are: a method for selectively inhibiting .alpha..sub.v.beta..sub.3
integrin-mediated cell attachment to an RGD-containing ligand,
comprising contacting said ligand with an effective amount of a
compound of formula (I); a method for treating a subject suffering
from a pathology related to an altered .alpha..sub.v.beta..sub.3
integrin-mediated cell attachment comprising administering to said
subject a compound of formula (I); said pathologies being for
example retinopathy, acute renal failure, osteoporosis.
[0045] From the industrial application point of view, the present
invention also comprises pharmaceutical compositions comprising an
effective dose of at least a compound of formula (I) in admixture
with pharmaceutically acceptable vehicles and/or excipients.
[0046] The present invention shall be disclosed in detail in the
foregoing also by means of examples and figures, wherein, in the
figures:
[0047] FIG. 1 represents, in an exemplary way, the general
synthesis of the lactams;
[0048] FIG. 2 represents a preferred embodiment of the synthesis of
6,5-fused "cis" lactams;
[0049] FIG. 3 represents a preferred embodiment of stereoselective
hydrogenation with chiral phosphine-Rh catalyst;
[0050] FIG. 4 represents a preferred embodiment of the synthesis of
7,5-fused "cis" lactams;
[0051] FIG. 5 represents a preferred embodiment of the synthesis of
5,5-fused "cis" lactams;
[0052] FIG. 6 represents another preferred embodiment of the
synthesis of 5,5-fused "cis" lactams;
[0053] FIG. 7 represents a preferred embodiment of the synthesis of
6,5-fused "trans" lactams;
[0054] FIG. 8 represents a preferred embodiment of the synthesis of
7,5-fused "trans" lactams.
[0055] FIG. 9 represents a preferred embodiment of bicyclic lactam
templates Fmoc-protected;
[0056] FIG. 10 represents a preferred embodiment of linear
pseudopeptides tBu-, Pmc-protected;
[0057] FIG. 11 represents a preferred embodiment of protected
cyclic pseudopeptides;
[0058] FIG. 12 represents a preferred embodiment of RGD cyclic
pseudopeptides;
DETAILED DESCRIPTION OF THE INVENTION
[0059] In its broadest aspects, the present invention relates to
compounds of the above formula (I).
[0060] The compounds of formula (I) are peptido-mimetics containing
an RGD sequence. Said compounds can be seen as formed by an
azabicycloalkane scaffold and an RGD sequence.
[0061] For sake of clarity, in formula (I), there is a variable
part, given by the different values of n, and a fixed part, given
by the RGD sequence. When n is 0, the scaffold is referred to as
5,5 azabicycloalkane, when n is 1, the scaffold is referred to as
6,5 azabicycloalkane and when n is 2, the scaffold is referred to
as 7,5 azabicycloalkane. The bonds written in formula (I) as a wavy
line represents a stereo bond, which can be either above the plane
of the page (thick bond) either below the plane of the page (thin
bond). The compounds of formula (I) can exist in different
stereoisomers, according to the orientation of the wavy bond.
[0062] A first class of preferred compounds of formula (I) are 7,5
azabicycloalkane, in particular those having trans configuration as
to the positions 7 and 10 and (R) configuration as to the carbon
atom at position 3.
[0063] A second class of preferred compounds of formula (I) are 6,5
azabicycloalkane, in particular those having trans configuration as
to the positions 6 and 9 and (S) configuration as to the carbon
atom at position 3.
[0064] A particularly preferred compound is the one of the
following formula (also named as ST 1646). 2
[0065] In the following table there are represented the preferred
compounds of formula (I): 3
[0066] Within the boundaries of the present invention, there is
disclosed a process for the preparation of the compounds of formula
(I), comprising the following steps:
[0067] a) Horner-Emmons olefination of a compound of formula (II)
4
[0068] wherein
[0069] R is a lower all residue;
[0070] R.sub.1 is a suitable nitrogen protecting group, to give a
compound of formula (III); 5
[0071] wherein R.sub.3 is a suitable nitrogen protecting group,
R.sub.4 is a lower alkyl residue;
[0072] b) hydrogenation of said compound of formula (III) and
cyclisation; and, if desired
[0073] c) separation of the stereoisomeric mixture;
[0074] d) building of the RGD cyclic sequence, and if desired
[0075] e) separation of the stereoisomeric mixture.
[0076] A process for the stereoselective synthesis of the compounds
of formula (I), comprises the following steps:
[0077] a) Horner-Emmons olefination of a compound of formula (II)
6
[0078] wherein
[0079] R is a lower alkyl residue;
[0080] R.sub.1 is a suitable nitrogen protecting group, to give a
compound of formula (III); 7
[0081] wherein R.sub.3 is a suitable nitrogen protecting group,
R.sub.4 is a lower alkyl residue;
[0082] b) hydrogenation of said compound of formula (III) by chiral
phosphine-Rh catalysed hydrogenation and cyclisation; and, if
desired
[0083] c) separation of the stereoisomeric mixture;
[0084] d) building of the RGD cyclic sequence and if desired
[0085] e) separation of the stereoisomeric mixture.
[0086] As lower alkyl residue it is normally understood a
C.sub.1-C.sub.4 alkyl, for example, methyl, ethyl, propyl, butyl
and all the possible isomers, but also higher alkyls are suitable,
provided their compatibility with reaction conditions. As suitable
nitrogen protecting groups, the skilled person is able to select,
according the general common knowledge, the suitable protecting
group, as it will appear from the following examples, but also in
the available technical literature and commercial catalogues.
[0087] Also disclosed are pharmaceutical compositions comprising a
therapeutically or preventive effective dose of at least a compound
of formula (I) in admixture with pharmaceutically acceptable
vehicles and/or excipients.
[0088] In its broadest aspect, the present invention advantageously
teaches a method for selectively inhibiting
.alpha..sub.v.beta..sub.3 integrin-mediated cell attachment to an
RGD-containing ligand, comprising contacting said ligand with an
effective amount of a compound of formula (I), a method for
treating a subject suffering from altered angiogenesis, comprising
administering to said subject a compound of formula (I), a method
for the treatment of tumors in a subject comprising administering
to said subject a compound of formula (I), optionally in
combination with other active ingredients, in particular other
antitumour agents.
[0089] The present invention shall be described in detail also by
means of examples and figures, wherein,
[0090] Best Mode for Carrying out the Invention
[0091] The synthesis of so-called peptido-mimetics molecules has
been a very active and productive field of research in drug design
(J. Gante, Angew. Chem., Int. Ed. Engl. 1994, 33, 1699.-G. L.
Olson, et al.: J. Med. Chem. 1993, 36, 3039.-D. C. Horwell,
Bioorg.Med. Chem. Lett. 1993, 3, 797.-A. Giannis et al.: Angew.
Chem., Int. Ed. Engl. 1993, 32, 1244.-B. A. Morgan: Annu. Rep. Med.
Chem. 1989, 24, 243). The expectation is that these molecules will
have the same biological effects as natural peptides, but at the
same time, will be metabolically more stable. Of particular
interest has been the replacement of reverse-turn dipeptide motifs
with constrained molecules that reproduce their conformational
features (ibid; M. Kahn, Ed., Peptide Secondary Structure Mimetics.
Tetrahedron Symposia-in-Print No. 50 1993, 49, 3433-3689 and
references therein). This goal has been frequently achieved using
the azaoxobicyclo[X.Y.0]alkane skeleton and/or heteroatom
analogues. This has created a demand for efficient synthetic
approaches toward such molecules, and many methods have been
introduced and recently reviewed (S. Hanessian et al: Tetrahedron
1997, 38, 12789-12854). One particularly effective and versatile
route has been developed by Lubell et al. and employed for the
preparation of enantiopure indolizidinone-type 6,5-fused bicyclic
lactams (H. -G. Lombartet al.: J. Org. Chem. 1996, 61,
9437-9446.-F. Polyak et al.: J. Org. Chem. 1998, 63, 5937-5949 and
references therein for the syntheses of azabicycloalkane amino
acids--F. Gosselin et al.: J. Org. Chem. 1998, 63, 7463-7471).
Several procedures are also available for the synthesis of
7,5-fused bicyclic lactams, the majority of which require
relatively long synthetic sequences. On the contrary, there is not
many published protocol that allow the synthesis of 5,5-fused
bicyclic lactams.
[0092] According to the present invention, the beta-turn portion of
the cyclic peptide consists in an azabicycloalkane amino acid
scaffold, selected from a 5,5-, 6,5- or 7,5-fused bicyclic lactams.
Several 6,5-and 7,5-fused 1-aza-2-oxabicyclo[X.3.0]alkane amino
acids have been synthesised, using radical (L. Colombo et al.:
Tetrahedron Lett. 1995, 36, 625-628.-L. Colombo et al.: Gazz. Chim.
It. 1996, 126, 543-554) or ionic reactions (L. Colombo et al.
Tetrahedron 1998, 54, 5325-5336). These structures can be regarded
as conformationally restricted substitutes for Ala-Pro and Phe-Pro
dipeptide units, and, if their conformations meet certain criteria,
they can be used to replace the central (i+1 and i+2) residues of
.beta.-turns.
[0093] The present invention provides an improved reaction
sequence, amenable to large scale preparation, and allowing the
synthesis of different bicyclic lactams from common intermediates,
as described in the appended FIG. 1.
[0094] Starting from 5-allyl/formyl prolines 13-18, a Z-selective
Horner-Emmons olefination followed by double bond reduction has
been used to build the second ring. The starting aldehydes have
been stereoselectively synthesised by modifications of known
procedures (vide infra). Stereorandom double bond reduction can be
performed using H.sub.2/Pd to yield, after cyclisation, mixtures of
easily separable epimers. Stereoselective hydrogenation is studied
for the synthesis of 6,5-fused lactams, and achieved with d.e. 80%
using Rh-chiral phosphine catalysts. Structural diversity, in terms
of ring size and stereochemistry of the azabicycloalkane fragment,
is provided by the new strategy, and access to the less common
5,5-fused bicyclic scaffold is also secured.
[0095] Examples of bicyclic dipeptide derivatives 1-12 are shown in
FIG. 2.
[0096] Synthesis of the Fused Bicyclic Lactams 1-12
[0097] The synthesis of lactams 1-12 follows the common steps
reported in FIG. 1. Starting from the cis or trans 5-alkyl proline
aldehydes 13-18, a Horner-Emmons olefination with the potassium
enolate of (.+-.)-Z-.alpha.-phosphonoglycine trimethyl ester (U.
Schmidt, A. Lieberknecht, J. Wild, Synthesis 1984, 53-60) sets up
the necessary carbon chain. Following protecting group manipulation
(vide infra), reduction of the enamino acrylic acids and treatment
with condensing agents gives the lactams of both the "cis" and
"trans" series in good yields.
[0098] In all cases where stereoisomeric mixtures of lactams are
formed, they can be easily separated by flash chromatography, and
their configuration can be assigned with n.O.e. experiments.
[0099] The synthetic scheme is best illustrated by the synthesis of
the 6,5-fused "cis"-lactams 2a and 8a (FIG. 3). The necessary cis
aldehyde 14 is obtained from the known cis 5-allyl-proline
derivative 25 (M. V. Chiesa, L. Manzoni, C. Scolastico, Synlett
1996, 441-443) and reacted with the commercially available
phosphonate 26 (U. Schmidt, A. Lieberknecht, J. Wild, Synthesis
1984, 53-60) to give 20 in 98% yield and 7:1 Z:E ratio.
[0100] Hydrogenation of 20 occurs initially at the enamino Cbz
group, and thus results in a complex mixture of products. To
circumvent this problem, the substrate is treated with Boc.sub.2O
to give 27 (98%). Reduction of 27 with H.sub.2/Pd(OH).sub.2
followed by reflux in MeOH gives a 1:1 mixture of 8a and 2a, which
are easily separated by flash-chromatography. From 14 the whole
sequence requires only two chromatographic separations
(purification of 20 and separation of 8a from 2a) and can easily be
carried out in multigram scale.
[0101] The stereoselective preparation of the two epimers 8a and 2a
(FIG. 3) is carried out using chiral phosphine-Rh catalysed
hydrogenation of the enamino acid 28.
[0102] Chiral phosphine-Rh catalyst is well-known to represent a
powerful and well-established way of access to naturally and
non-naturally occurring amino acids and the catalytic asymmetric
hydrogenation of dehydropeptides is the logical extension of this
methodology to the preparation of biologically active chiral oligo-
and polypeptides.
[0103] In asymmetric catalytic hydrogenations using chiral
phosphine-Rh catalysts (Z) olefins usually gives the highest
stereolsomeric purity of the products, but the most stringent
requirement for the substrate remains the presence of an acetamido
or an equivalent group on the double bond. (K. E. Koenig in
Asymmetric Synthesis, J. D. Morrison Editor, Vol 5, Academic Press
Inc. 1985, 71) The amide-type carbonyl is needed in order to allow
two-point co-ordination of the substrate to the metal, which
increases the sterical demand as it has been fully elucidated
experimentally. (J.Halpern, ibidem, 41) For applications to the
synthesis of peptides protecting groups other than the acetamido,
like Boc or Cbz should be used, thus permitting differential
deprotection. However, very few examples of asymmetric catalytic
hydrogenation are known in which these protecting groups are found
on the enamino nitrogen: (B. Basu, S. K. Chattopadhyay, A. Ritzen,
T. Frejd, Tetrahedron Asymmetry, 1997, 8, 1841) (S. D. Debenham, J.
D. Debenham, M. J. Burk, E. J. Toone, J.Am.Chem.Soc. 1997, 119,
9897) more frequently Boc or Cbz protecting groups are present in
different position of dehydropeptides being hydrogenated at the
N-terminus. (A. Hammadi et al. Tetrahedron Lett. 1998, 39, 2955-I.
Ojima, Pure & Appl. Chem. 1984, 56, 99). For the catalytic
asymmetric hydrogenation of 28 [Rh(Phosphine)(COD)]CLO.sub.4
catalysts is used. The catalysts were prepared by displacing one
cyclooctadiene ligand of [Rh(COD).sub.2]CLO.sub.4 with the
appropriate phosphine. The ligands investigated are (R)-Prophos 29
and (+) or (-) BitianP 30 and 31. BitianP is a chiral atropisomeric
chelating phosphine belonging to a new class of ligands based on
biheteroaromatic framework, which gives very high e.e. % in the
asymmetric hydrogenation of olefins and ketones. (E. Cesarotti et
al. J.Chem.Soc.Chem.Comm. 1995, 685-Cesarotti et al. J.Org.Chem.
1996,61,6244).
[0104] The results of asymmetric hydrogenation are reported in the
Table 1. The conversion is always quantitative but the highest
stereodifferentiation is obtained with [Rh/(-)-BitianP] (entry 3).
The results suggest that the newly created stereocentre is mainly
determined by the catalyst, which overruns the effect of the
stereocentre on the substrates (entry 2 and 3). The results also
indicate that the Boc protecting group on the enamino nitrogen
fulfils the requirements and allows the olefin to chelate to the
catalyst.
1TABLE 1 Asymmetric hydrogenation of 28 Entry Catalyst 32/33 d.e. %
1 Rh-29 86/14 72 2 Rh-30 13/87 74 3 Rh-31 90/10 80
[0105] Reactions were carried out at R.T. for 24 h under 10 atm of
H.sub.2.
[0106] Treatment of crude 32 and 33 with CH.sub.2N.sub.2, followed
by hydrogenation and cyclisation under the usual conditions
(H.sub.2/Pd-C followed by reflux in MeOH) allows a stereoselective
route to lactams 8a and 2a.
[0107] All the remaining lactams 1-12 can be synthesised following
essentially the same sequence described above. Thus, the 7,5-fused
lactams 3a and 9a (FIG. 4) can be made starting from the cis
aldehyde 15, easily prepared from the cis 5-allyl proline 25. (M.
V. Chiesa, L. Manzoni, C. Scolastico, Synlett 1996, 441-443)
Horner-Emmons reaction of 15 with 26 gives a 6:1 Z:E mixture of
enamino acrylates. After N-protection they are reduced with
H.sub.2/Pd-C. The thermic cyclisation of methyl ester 34 can be
carried out in a suitable solvent, for example xylene. Better
results are obtained upon ester hydrolysis followed by EDC/HOBT
promoted lactam formation to give 3a and 9a, which are easily
separable by flash chromatography (51% overall yield from 25).
[0108] The starting material for the synthesis of the 5,5-fused
"cis" lactams (FIG. 5) is alcohol 36. Oxidation and Horner-Emmons
reaction with 26 followed by N-Boc protection gives 37 as a 5:1 Z:E
mixture in 57% yield. Hydrogenation of 37 (H.sub.2/Pd(OH).sub.2)
results in a complex mixture of products, from which the 1,2
diamino ester 38 is anyway isolated in 40% yield. The formation of
38 may result from initial N-debenzylation of 37 followed by
intramolecular Michael addition to the enamino ester double bond
and hydrogenolysis of the resulting aziridine. The problem can be
partly circumvented by performing the hydrogenation starting from
the acid 39. Treatment of 39 with H.sub.2/Pd-C followed by reflux
in MeOH gives an easily separable 1:1 mixture of 1a and 7a in 40%
yield.
[0109] An alternative synthesis of these lactams is also provided
starting from the trifluoroacetamido aldehyde 13 (FIG. 6). Aldehyde
13 is synthesised from 36 with a series of 5 high-yielding steps.
Horner-Emmons and nitrogen protection gives 40 (46% over 7 steps),
which could be directly reduced to give a 1:1 mixture of the fully
protected ester 41 (77%). Removal of the trifluoroacetamido
protecting group (NaBH.sub.4 in MeOH, 84%) followed by treatment in
refluxing xylene gives the lactams 1a and 7a in 78% yield.
[0110] The same synthetic schemes are equally adopted for the
synthesis of the "trans" lactam series.
[0111] Starting material for the 6,5-fused "trans" lactams 5a and
11a is the trans-substituted proline 17 (FIG. 7). Aldehyde 17 is
best obtained from ester 43, which is made in one step from
N-Cbz-5-hydroxy proline tert-Butyl ester as 4:1 trans:cis mixture,
following a published procedure. (I. Collado et al., Tetrahedron
Lett., 1994, 43, 8037) The Horner-Emmons reaction with the
potassium enolate of 26 proceeds with 98% yield. Treatment with
Boc2O and cis/trans isomers separation, followed by unselective
H.sub.2/Pd-C hydrogenation of the crude and treatment in refluxing
MeOH gives a 1:1 mixture of easily separated 5a and 11a.
[0112] Finally, synthesis of the 7,5-fused "trans" lactams 6a and
12a is achieved starting from the "trans' allyl proline 45 (FIG.
8). (M. V. Chiesa et al. Synlett 1996, 441-443) Hydroboration and
Swern oxidation (80% over 2 steps) gives the aldehyde 18, which
reacted with 26 to give, after nitrogen protection, 46 as a 6:1 Z:E
mixture. The usual sequence (NaOH; H.sub.2/Pd-C) allowed the
isolation of 6a and 12a in 40% overall yield.
[0113] As far as the synthesis of the cyclic RGD portion, synthetic
methods are well known in the art. It is convenient to use the
solid phase synthesis approach, although other methods could be
used.
[0114] The classical solid-phase synthesis is preferred.
[0115] The solid-phase synthesis is carried out as outlined in
C.Gennari et al. Eur.J.Org. Chem. 1999, 379-388.
[0116] The protected amino acid is condensed on a suitable resin,
for example a Wang-Merrifield resin. Protecting groups are known in
this art. 9-fluorenylmethoxycarbonyl (FMOC) is preferred.
[0117] After having activated the resin, N-FMOC-Gly is attached to
the Wang-Merrifield resin by means of a suitable condensing agent,
preferably diisopropylcarbodiimide (DIC) /1-hydroxybenzotiazole
(HOBt)/4-dimethylaminopyridine (DMAP) (J.Org. Chem, 1996, 61,
6735-6738.
[0118] Subsequently, N-FMOC-Arg(Pmc)OH is attached, followed by the
bicyclic N-FMOC-lactam (IIIa) or (IIIb) and finally N-FMOC-Asp
(tBu) OH.
[0119] In a still preferred embodiment of the present invention,
the solid phase synthesis of cyclic peptides containing the RGD
sequence bonded to the bicyclic lactam was performed with
9-flourenylmethoxy carbonyl (Fmoc) strategy. Thus the N-Boc
protecting group had to be exchanged by Fmoc group in the bicyclic
lactam. The synthesis was performed using Merrifield solid phase
peptide synthesis with SASRIN (Super Acid Sensitive Resin) applying
Fmoc strategy. Asp was protected at the carboxy group in the side
chain as t-butylester and Arg was protected at the guanidino group
as Pmc (2,2,5,7,8 PentamethyI chroman-6 sulphonyl). Linear
polipeptides were assembled leaving the glycine residue at the
C-terminus to prevent racemization and steric hindrance during the
cyclization step. The Fmoc group was cleaved with 20% piperidine in
DMF. The Fmoc-protected aminoacid and bicyclic lactams were coupled
with HOAT (Azahydroxy Benzotriazole) in the presence of DIC
(Diisopropylcarbodiimid- e) or with HOAT/HATU {Azahydroxy
Benzotriazole)/{O-(7-Azabenzotriazol-1-yl-
)-N,N,N',N'-tetramethyluronioesafluorophosphate} using collidine as
base. Peptides were cleaved from SASRIN solid support by 1% TFA in
DCM and subsequent neutralisation of TFA with Py. This procedure
leads to peptides with intact side chain protective groups. Final
cyclization was performed in the same conditions i.e. HOAT/HATU and
final deprotection was done with trifluoroacetic acid in the
presence of scavengers to avoid side alkylations.
[0120] The compounds of the present invention are endowed with
interesting physiological properties, which make them useful as
medicaments. In particular, the compounds of formula (I) herein
disclosed are selective antagonists of .alpha..sub.v.beta..sub.3
integrins. This antagonist activity provides the use of said
compounds for the preparation of medicaments useful in inhibiting
the action of .alpha..sub.v.beta..sub.3 integrins. In particular,
said medicaments will be used in the treatment of tumors, namely in
inhibiting tumor growth and/or angiogenesis or metastasis.
[0121] Receptor Binding Assay
[0122] By way of example, the tests were performed on the preferred
compound ST 1646 (see claim 9) and for comparison purposes, the
highly active compound of the prior art, namely c(RGDfV), i.e.
cyclo (Arg-Gly-Asp-D-Phe-Val), in the attached report named as the
"KESSLER" peptide, disclosed in WO 9706791was used. Both ST 1646
and "KESSLER" are also named "RGD".
[0123] Materials and Methods.
[0124] The receptor binding assay was performed as described by
Orlando and Cheresh (Arginine-Glycine-Aspartic Acid Binding Leading
to Molecular Stabilization between Integrin
.alpha..sub.v.beta..sub.3 and Its Ligand. J. Biol. Chem. 266:
19543-19550, 1991). .alpha..sub.v.beta..sub.3 was diluted at 500
ng/ml in coating buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 2 mM
CaCl.sub.2, 1 mM MgCl.sub.2, 1 mM MnCl.sub.2) and an aliquot of 100
.mu.l/well was added to a 96-well microtiter plate and incubated
overnight at 4.degree. C. The plate was washed once with
blocking/binding buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 2 mM
CaCl.sub.2, 1 mM MgCl.sub.2, 1 mM MnCl.sub.2, 1% bovine serum
albumin), and incubated an additional 2 h at room temperature. The
plate was rinsed twice with the same buffer and incubated with
radiolabelled ligand at the indicated concentrations. For
competition binding, unlabelled competitor and competing peptides
were included at the concentration described. After additional
three washing, counts were solubilized with boiling 2N NaOH and
subjected to y-counting.
[0125] Cell Culture
[0126] Bovine microvascular endothelial cells (BMEC) were
maintained in DMEM supplemented with 20% foetal calf serum, 50
units/ml heparin, 50 .mu.g/ml bovine brain extract, 100 units/ml
gentamycin.
[0127] BMEC were cultured on 1% gelatine-coated culture flasks and
employed in experiments between passage 6-12.
[0128] Human prostate carcinoma cells (PC3) were purchased from
American Type Collection Culture (ATCC) and maintained in RPMI
supplemented with 10% foetal calf serum, 10 mM L-glutamine, 1%
sodium piruvate and 100 units/ml gentamicin.
[0129] Murine lung carcinoma cells (M109) were purchased from
American Type Collection Culture (ATCC) and maintained in RPMI
supplemented with 10% foetal calf serum, 10 mM L-glutamine and 100
units/ml gentamicin.
[0130] Cells were passaged and used for the experiments before
reaching confluence.
[0131] Adhesion Test
[0132] Ninety-six-well plates (Falcon) were coated with either
fibronectin or vitronectin (both at 5 g/ml in phosphate buffered
saline) overnight at 4.degree. C. Cells were detached using EDTA (1
mM)/trypsine (0,25%) and resuspended in own medium described above.
Approximately 40.000 cells/100 1 were applied for each well and
allowed to adhere for 60 min at 37.degree. C. in presence of
different amounts of RGD peptides. For all experiments the
non-adherent cells were removed with PBS and the remaining cells
were fixed with 4% paraformaldehyde for 10 min.
[0133] Cells were stained with 1% toluidine blue for 10 min and
rinsed with water.
[0134] Stained cells were solubilized with 1% SDS and quantified on
a microtiter plate reader at 600 nm.
[0135] Experiments described were performed in quadruplicate and
repeated a minimum of three times.
[0136] Results were presented as mean and standard deviation.
[0137] Results
[0138] Binding Assay
[0139] Both purified and membrane-bound integrin
.alpha..sub.v.beta..sub.3 bind to the disintegrin echistatin with
high affinity, which can be competed efficiently by linear and
cyclic RGD peptides (C. C. Kumar, Huiming-nie, C. P. Rogers, M.
Malkowski, E. Maxwell, J. J. Catino and L. Armstrong. 1997, The
Journal of Pharmacology and Experimental Therapeutics; (283) pp
843-853). Therefore to assess the affinity of these peptides for
this integrin we used an experimental protocol of competition with
the [.sup.125]I-echistatin as described in materials and
methods.
[0140] Our results are showing that ST1646 (the compound of claim
9) is the more effective peptide to shift echistatin from its
interaction with the .alpha..sub.v.beta..sub.3 integrin. Indeed
affinity of the RGD peptide ST1646 reported in table 2 as IC.sub.50
of the binding concentration was almost 20 time higher than the
Kessler cyclic peptides used as reference peptide. Therefore these
data are providing clear evidence that the structural constrain of
the RGD sequence introduced by the ST 1646 result unexpectedly in
an affinity for the .alpha..sub.v.beta..sub.3 integrin notably
higher than Kessler peptide.
2TABLE 2 Competition binding of RGD to Integrin
.alpha..sub.v.beta..sub.3 Receptor RGD IC.sub.50 .+-. SD (nM) Ki
.+-. SD (nM) KESSLER RGD 36.9 .+-. 6.4 34.06 .+-. 5.9 ST 1646 2.2
.+-. 0.32 2.03 .+-. 0.29
[0141] Effect of RGD compounds on the binding of [125I] Echistatin
to .alpha..sub.v.beta..sub.3 integrin.
[0142] IC.sub.50, the concentration of compounds required for 50%
inhibition of echistatin binding, were estimated graphically by
program Allfit. The Ki of the competing ligands were calculated
according to the Cheng and Prusoff equation.
[0143] Values are the mean.+-.standard deviation of triplicate
determinations.
[0144] Saturation binding isotherms of .sup.125I-echistatin binding
to .alpha..sub.v.beta..sub.3 receptor were determined in a
solid-phase receptor binding assay as described in materials and
method. Integrin .alpha..sub.v.beta..sub.3 was coated and incubated
with various concentrations (0.05-10 nM) of 125I-echistatin. Non
specific binding was evaluated by carrying out the binding assay in
the presence of an excess of cold echistatin and was subtracted
from the total binding to calculate specific binding.
[0145] In competition binding .sup.1251-echistatin was added to the
wells to a final concentration of 0.05 nM in binding buffer in the
presence of competing ligand. Cold unlabelled echistatin and
peptides dissolved in binding buffer at concentrations ranging
between 10-4 M to 10.sup.-9.
[0146] Endothelial Cells Adhesion Assay
[0147] Since transmembrane .alpha..beta. integrins family are
involved in adhesion of endothelial cells to extracellular matrix
proteins we assayed adhesion inhibition of bovine microvascular
endothelial cells (BMEC) to both vitronectin and fibronectin when
these cells were treated with different concentration of our cyclic
RGD.
[0148] According to the binding experiment the cyclic RGD peptide
ST1646 was the more effective in inhibiting adhesion than the other
peptide tested. Since vitronectin is a more specific ligand of
.alpha..sub.v.beta..sub.3 integrin than fibronectin we observed
that the RGD tested were able to more efficiently inhibit adhesion
of BMEC cells on vitronectin than on fibronectin coated plates
(Compare Table 3 with Table 4). Comparing adhesion inhibition, we
observed that the cyclic RGD ST1646 was about 10 time more
effective than the Kessler peptide inhibiting adhesion of BMEC
cells to both fibronectin and vitronectin (see Table 5).
[0149] To asses the ability of ST1646 peptide to compete with
vitronectin in adhesion assay also on other cells type, we
performed this experiment using microvascular endothelial cells
(HMEC), human prostate carcinoma cells (PC3) and murine lung
carcinoma cells (M109). Table 6 (a, b and c) show a good activity
of the ST1646 peptide in inhibiting adhesion of all cells type.
Indeed the reported adhesion inhibition of the ST1646 on HMEC, PC3
and M109 cells have shown higher percentage than the Kessler RGD
peptide.
[0150] Putting together these data we have, therefore, showed high
activity of the RGD cyclic peptide ST 1646 on several cellular type
coherently with binding affinity experiment previously
described.
3TABLE 3 Adhesion inhibition of BMEC to Vitronectin RGD %
inhibition t-test versus control KESSLER 96 P < 0.0001 ST 1646
99 P < 0.0001
[0151] The percentages of adhesion inhibition refer to 100 M
concentration of each peptide of and it's calculated by the
following formula (control-sample/control.times.100) where control
was RGD untreated sample. Each percentage is the mean of 4
independent samples treated with the same peptide. The t-test has
been calculated, using the Mann Witney non parametric test, by the
instat program.
4TABLE 4 Adhesion inhibition of BMEC to Fibronectin RGD %
inhibition t-test versus control KESSLER 30 P < 0.0001 ST 1646
60 P < 0.0001
[0152] The percentages of adhesion inhibition refer to 100 .mu.M
concentration of each peptide of and it's calculated by the
following formula (control-sample/control.times.100) where control
was RGD untreated sample. Each percentage is the mean of 4
independent samples treated with the same peptide. The t-test has
been calculated, using the Mann Witney non parametric test, by the
instat program.
5TABLE 5 IC50 of adhesion inhibition of BMEC IC.sub.50 (.mu.M) RGD
Fibronectin Vitronectin KESSLER >100 7.8 .+-. 1.2 ST 1646 44
.+-. 4 0.8 .+-. 0.06
[0153] Several concentrations (in quadruplicate) of the indicate
RGD peptides ranging between 100 to 0.6 .mu.M has been tested in
adhesion experiment as described in materials and methods. The
IC.sub.50 which represent the RGD peptide concentration able to
inhibit 50% of the adhesion of BMEC to the indicate substrate, has
been calculate by the linear regression analysis using the Allfit
program. The IC.sub.50 for each RGD has been reported together with
the standard deviation.
6TABLE 6(a) Adhesion assay on Vitronectin HMEC CELLS % IC.sub.50
t-test versus RGD Inhibition (.mu.M) control KESSLER 39 4.23 .+-.
0.31 P < 0.01 ST 1646 58 1.27 .+-. 0.375 P < 0.0005
[0154] Serial concentrations (in quadruplicate) of indicated RGD
peptide over a wide range (0.01-100 .mu.M has been tested in
adhesion test, on vitronectin, as described in material and
methods.
[0155] The IC.sub.50 represents the average value of 3 experiments
and indicates that RGD peptide concentration able to inhibit the
50% of cell adhesion.
[0156] The percentages of adhesion inhibition refer to 1.5 .mu.M
concentration of each peptide and were calculated by the following
formula (control-sample/control.times.100) where control was RGD
untreated sample.
7TABLE 6(b) Adhesion assay on Vitronectin PC3 CELLS % IC.sub.50
t-test versus RGD Inhibition (.mu.M) control KESSLER 69 2.5 .+-.
0.2 P < 0.0001 ST 1646 96 0.3 .+-. 0.08 P < 0.0001
[0157] Serial concentrations (in quadruplicate) of indicated RGD
peptide over a wide range (0.01-100 .mu.M has been tested in
adhesion test, on vitronectin, as described in material and
methods.
[0158] The IC.sub.50 represents the average value of 3 experiments
and indicates that RGD peptide concentration able to inhibit the
50% of cell adhesion.
[0159] The percentages of adhesion inhibition refer to 1.5 .mu.M
concentration of each peptide and were calculated by the following
formula (control-sample/control.times.100) where control was RGD
untreated sample.
8TABLE 6(c) Adhesion assay on Vitronectin M109 CELLS % IC.sub.50
t-test versus RGD Inhibition (.mu.M) control KESSLER 70 0.46 .+-.
0.5 P < 0.0001 ST 1646 99 0.048 .+-. 0.06 P < 0.0001
[0160] Serial concentrations (in quadruplicate) of indicated RGD
peptide over a wide range (0.01-100 .mu.M has been tested in
adhesion test, on vitronectin, as described in material and
methods.
[0161] The IC.sub.50 represents the average value of 3 experiments
and indicates that RGD peptide concentration able to inhibit the
50% of cell adhesion.
[0162] The percentages of adhesion inhibition refer to 1.5 .mu.M
concentration of each peptide and were calculated by the following
formula (control-sample/control.times.100) where control was RGD
untreated sample.
[0163] The t-test has been calculated using the Mann Witney non
parametric test, by the instat program. In the top left side of the
two panels it's shown the cell type the adhesion experiment it's
referred to.
[0164] Antitumor And Antimetastatic Activity Of St 1646 Vs. Kessler
Peptide On M109 Lung Carcinoma-Bearing Balb/C Mice
[0165] Balb/c mice were injected i.m. with M109 lung carcinoma
cells (3.times.10.sup.5 cells/mouse) into the hind leg muscle. One
day after tumor injection, mice were treated with ST 1646 (300
.mu.g/mouse=15 mg/kg) or Kessler peptide (200 .mu.ug/mouse=10
mg/kg) according to a qdx9 treatment schedule (every day for 9
administration, i.p. route).
[0166] Tumors were excised at day 10.sup.th after tumor implant.
Mice were sacrificed at day 16.sup.th from tumor implant and lungs
were removed. The number of lung metastases has been evaluated on
tumor-excised mice (3 mice/group) using a dissecting
microscope.
[0167] TVI % (tumor volume inhibition)=100-[(mean tumor weight of
treated group/mean tumor weight of control group).times.100].
Calculated on day 16.sup.th after tumor implant (just before mice
sacrifice) on nonoperated mice.
[0168] The results obtained, reported in table 7, shown that ST1646
is more effective than Kessler peptide in reducing both the number
of the metastasis and the volume of the tumor.
9TABLE 7 Antitumor and antimetastatic activity of ST 1646 vs.
Kessler peptide on M109 lung carcinoma-bearing Balb/c mice. Mean
no. of Group Schedule metastases TVI % Untreated / 34 / Kessler 200
.mu.g/mouse qdx9 23 / (10 mg/kg) ST 1646 300 .mu.g/mouse qdx9 20 3
(15 mg/kg)
[0169] Angiogenesis Inhibition On Cam Assay with St1646
Cyclopeptide
[0170] Angiogenesis on CAM (chicken embryo chorioallantoic
membrane) assay has been quantified by counting the number of
vessels interfacing the implanted gelatin sponge on each embryos
and calculating the average for each single experimental point (6-8
eggs for peptide concentration). A single treatment means that the
embryo received the peptide, at the concentration indicated in the
table, only one times at the beginning of the experiment while in
the repeated treatment the peptide has been added to the embryo
every day for three days. In some experiments we have refereed our
sample to control where angiogenesis occurred spontaneously on the
chorioallantoic membrane during embryo development (Table 8). In
others experiment (Table 9) instead we have used control where
angiogenesis has been stimulated by bFGF (400 ng/embryo).
10TABLE 8 Angiogenesis inhibition occurred spontaneously on the
chorioallantoic membrane Inhibition Standard Deviation Treatment
(%) (%) Control 0 ST1646 -70 .+-.27 (100 .mu.g single treatment)
ST1646 -27 .+-.8 (20 .mu.g repeated treatment)
[0171] Inhibition (%)=[(mean vessels treated group-mean vessels
control group)/control group].times.100
11TABLE 9 Angiogenesis inhibition on the chorioallantoic membrane
where angiogenesis has been stimulated by bFGF. Inhibiton Standard
Deviation Treatment (%) (%) Control bFGF (400 ng) 0 ST1646 -56
.+-.18 (100 .mu.g single treatment) ST1646 -84 .+-.30 (100 .mu.g
repeated treatment)
[0172] Inhibition (%)=[(mean vessels treated group-mean vessels
control group)/control group].times.100
[0173] The results obtained provide a clear evidence that the
structural constrain of the RGD sequence introduced by the ST 1646
result unexpectedly in an affinity for the
.alpha..sub.v.beta..sub.3 integrin notably higher than Kessler
peptide. Paralleled to these results in in vitro competition
binding assay, ST 1646 assesses its activity in inhibiting the
binding of several cell types to fibronectin and vitronectin
proteins [table 3-4-5-6(a. b and c)]. According to the binding
assay experiments (Table 3), cellular inhibition assay show that ST
1646 is at least 10 folds more active than Kessler peptide.
Moreover, ST 1646 is extremely specific in inhibiting cellular
binding to vitronectin. This is an additional evidence, in which ST
1646 shows a good selectivity towards cellular
.alpha..sub.v.beta..sub.3 integrin implicated in binding to
vitronectin substrates. In in vivo experiments the results obtained
shown that ST 1646 inhibits the growth of M109 lung metastasis
(table 7). In addition, ST 1646 strongly inhibits angiogenesis both
in FGF-induced and spontaneous angionenesis (table 8 and 9
respectively). This results show that ST 1646 is a very effective
antitumoral and antiangiogenic compound.
[0174] The compounds of the present invention have azabicycloalkane
structure and contain the RGD (Arg-Gly-Asp) sequence are selective
inhibitors of .alpha.v.beta.3 receptor, and they are useful agents
for treating pathologies due to an altered activation of the
.alpha.v.beta.3 receptor. It is well known that the activation of
.alpha.v.beta.3 receptor is linked to several pathological
processes.
[0175] As above mentioned, the experimental results above reported
shown that compounds according to the invention are/have: selective
inhibitor of .alpha.v.beta.3 receptor; inhibitors of the adhesion
of cell lines to fibronectin; antitumoral activity (reduction of
the number of the metastasis); antiangiogenetic activity.
[0176] As far as the industrial aspects of the present invention
are concerned, the compounds of formula (I) shall be suitably
formulated in pharmaceutical compositions. Said compositions will
comprise at least one compound of formula (I) in admixture with
pharmaceutically acceptable vehicles and/or excipients. According
to the therapeutic necessity, the bioavailability of the selected
compound, its physico-chemical characteristics, the pharmaceutical
compositions according to the present invention will be
administered by enteral or parenteral route. Enteral pharmaceutical
compositions may be both in the liquid or solid from, for example
tablets, capsules, pills, powders, sachets, freeze dried powders to
be readily dissolved or in any other way soluble powders,
solutions, suspensions, emulsions. Parenteral formulation will be
in injectable form, as solutions, suspensions, emulsions or in
powdery form to be dissolved immediately before use. Other
administration routes are also provided for example intranasal,
transdermal or subcutaneous implant. Special pharmaceutical
compositions can also be provided. For example controlled release
formulations or particular vehicles, for example liposomes.
[0177] The preparation of the pharmaceutical compositions according
to the present invention is absolutely within the general knowledge
of the person skilled in this art.
[0178] The dosage will be established according to the type of the
pathology to be treated, its severity, and the conditions of the
patient (weight, age, and sex).
[0179] The following examples further illustrate the invention.
[0180] Examples 1-12 may be read easier by making reference to
FIGS. 1-8.
[0181] General: .sup.1H and .sup.13C NMR spectra were recorded in
CDCl.sub.3 or C.sub.6D.sub.6 as indicated, at 200 (or 300) and 50.3
MHz, respectively. The chemical shift values are given in ppm and
the coupling constants in to Hz. Optical rotation data were
obtained on Perkin-Elmer model 241 polarimeter. Thin-layer
chromatography (TLC) is carried out using Merck precoated silica
gel F-254 plates. Flash chromatography is carried out with Merck
Silica Gel 60, 200-400 mesh. Solvents were dried with standard
procedure, and reactions requiring anhydrous conditions were
performed under a nitrogen atmosphere. Final product solutions were
dried over Na.sub.2SO.sub.4, filtered and evaporated under reduced
pressure on a Buchi rotary evaporator.
EXAMPLE 1
[0182] Preparation of Enamides via Horner-Emmons Reaction.
[0183] General Procedure A:
[0184] To a stirred solution of tBuOK (7.36 mmol) in 40 ml of dry
CH.sub.2Cl.sub.2 under nitrogen atmosphere, at -78.degree. C., was
added a solution of Z-.alpha.-phosphonoglycine trimethyl ester 26
(7.36 mmol) in 5.0 ml of dry CH.sub.2Cl.sub.2. The solution was
stirred for 30 min at this temperature and then a solution of
aldehyde (6.13 mmol) in dry CH.sub.2Cl.sub.2 (25 ml) was added.
After 5 hours the solution was neutralised with a phosphate buffer.
The aqueous phase was extracted with CH.sub.2Cl.sub.2, dried over
Na.sub.2SO.sub.4 and the solvent evaporated under reduced pressure.
The crude was purified by flash chromatography (hexane/ethyl
acetate), affording the enamide in a Z:E diastereoisomeric
mixture.
[0185] Preparation of N-Boc-protected Enamide.
[0186] General Procedure B:
[0187] A solution of enamide (11.0 mmol), (Boc).sub.2O (22.0 mmol)
and a catalytic quantity of DMAP in 40 ml of dry THF, was stirred
for 30 min. under nitrogen. The solution was then quenched with 40
ml of water and extracted with ethyl acetate. The organic phase was
dried over Na.sub.2SO.sub.4 and the solvent evaporated under
reduced pressure. The crude was purified by flash chromatography
(hexane/ethyl acetate), yielding the Boc-protected enamide.
[0188] Preparation of Alcohol via Hydroboration.
[0189] General Procedure C:
[0190] To a solution of allyl proline (2.34 mmol) in dry THF (4.2
ml) was added a 0.5 M solution of 9-BBN in THF (1.26 mmol). The
reaction was stirred for 12 h. and then cooled at 0.degree. C. and,
water (0.6 ml), a 3 N solution of NaOH (0.5 ml) and H.sub.2O.sub.2
30% (0.44 ml) were added. The reaction was stirred for 1 h. at room
temperature and then refluxed for other 2 h. The aqueous phase was
extracted with AcOEt, the collected organic phases were dried over
Na.sub.2SO.sub.4, filtered and evaporated under reduced pressure,
the crude was purified by flash chromatography (hexane/ethyl
acetate), yielding the alcohol as yellow oil.
[0191] Preparation of Aldehyde via Swern Oxidation.
[0192] General Procedure D:
[0193] To a stirred solution of oxalyl chloride (16.9 mmol) in 35
ml of CH.sub.2Cl.sub.2, cooled at -60.degree. C., were added DMSO
(23.1 mmol), alcohol (5.66 mmol) dissolved in 21 ml of
CH.sub.2Cl.sub.2, TEA (28.2 mmol). The reaction was warmed at room
temperature. After one hour the reaction was washed with 50 ml of
water and the aqueous phase was extracted with CH.sub.2Cl.sub.2.
The collected organic layers were dried over Na.sub.2SO.sub.4. The
solvent was evaporated under reduced pressure and the crude
purified by flash chromatography (hexane/ethyl acetate), yielding
the aldehyde.
EXAMPLE 2
[0194] Aldehyde (14):
[0195] A stirred solution of 25 (6.0 g, 17.4 mmol) in 84 ml of
CH.sub.2Cl.sub.2 was cooled at -60.degree. C. and bubbled with
O.sub.3 (flow rate=1/hour). After 1.5 hours the reaction was
allowed to warm to room temperature and bubbled with N.sub.2 in
order to eliminate the excess of O.sub.3. The solution was then
cooled at 0.degree. C. with an ice bath and Me.sub.2S (101.8 mmol,
38 ml) was added. After 5 days of stirring at room temperature the
solvent was evaporated under reduced pressure and the crude was
purified by flash chromatography (hexane/ethyl acetate, 8:2),
yielding 4.53 g of 14 (75%) as yellow oil.
-[.alpha.]D.sup.22=-22.03 (c=1.27, CHCl.sub.3). --.sup.1H NMR (200
MHz, CDCl.sub.3), (signals were splitted for amidic isomerism):
.delta. =1.4-1.5 [2 s, 9 H, C(CH.sub.3).sub.3], 1.6-2.4 (m, 4 H,
CH.sub.2--CH.sub.2), 2.4-3.2 (2 m, 2 H, CH.sub.2CHO), 4.3-4.5 (m, 2
H, CH.sub.2--CH--N, N--CH--COOtBu), 5.15 (s, 2 H, CH.sub.2Ph), 7.30
(m, 5 H, aromatic), 9.8 (2 s, 1 H, CHO). --.sup.13C NMR (50.3 MHz,
CDCl.sub.3) (signals were splitted for amidic isomerism):
.delta.=200.8, 171.7, 154.0, 136.2, 128.3, 128.0, 127.8, 127.6,
81.4, 67.0, 66.9, 60.8, 60.3, 54.0, 53.2, 49.0, 48.3, 31.0, 30.2,
29.5, 28.9, 28.0, 27.7.-FAB.sup.+MS: calcd. for
C.sub.19H.sub.25NO.sub.5 347.4, found 348.
EXAMPLE 3
[0196] Enamide (20):
[0197] The general procedure A was followed using 14 and the crude
was purified by flash chromatography (hexane/ethyl acetate, 65:35),
affording 20 (98%) in a 7:1 Z:E ratio as colourless oils. Z-isomer:
-[.alpha.]D.sup.22=+38.78 (c=1.26, CHCl.sub.3). --.sup.1H NMR (200
MHz, CDCl.sub.3) (signals were splitted for amidic isomerism):
.delta. =1.3-1.5 [2 s, 9 H, C(CH.sub.3).sub.3], 1.5-2.3 (m, 4 H,
CH.sub.2--CH.sub.2), 2.4-2.7 (2 m, 2 H, .dbd.CH--CH.sub.2), 3.7 (2
s, 3 H, COOCH.sub.3), 4,2 (2 m, 2 H, --CH.sub.2--CH--N,
N--CH--COOtBu), 5.10 (m, 4 H, CH.sub.2Ph), 6.15 (m, 1 H, .dbd.CH),
7.30 (m, 10 H, aromatic). --.sup.13C NMR (50.3 MHz, CDCl.sub.3)
(signals were splitted for amidic isomerism): .delta.=172.4, 164.9,
154.5, 136.2, 132.5, 128.3, 128.2, 127.8, 127.7, 127.6, 81.8, 67.2,
66.9, 60.8, 60.3, 57.9, 57.2, 52.1, 33.8, 33.2, 30.7, 29.8, 29.5,
29.0, 28.0, 27.7, 27.6.--FAB.sup.+MS: calcd. for
C.sub.30H.sub.36N.sub.2O.sub.8552.6, found 553.--E-isomer:
-[.alpha.]D.sup.22=-4.08 (c=1.17, CHCl.sub.3). --.sup.1H NMR (200
MHz, CDCl.sub.3) (signals were splitted for amidic isomerism):
.delta. =1.25-1.50 [3 s, 9 H, C(CH.sub.3).sub.3], 1.5-2.3 (m, 4 H,
CH.sub.2--CH.sub.2), 2.8-3.3 (2 m, 2 H, .dbd.CH--CH.sub.2), 3.8 (2
s, 3 H, COOCH.sub.3), 4,1 (m, 1 H, --CH.sub.2--CH--N), 4.25 (m, 1
H, N--CH--COOtBu), 5.15 (2 s, 4 H, CH.sub.2Ph), 6.30 (m, 1 H,
.dbd.CH), 7.30 (m, 10 H, aromatic). --.sup.13C NMR (50.3 MHz,
CDCl.sub.3) (signals were splitted for amidic isomerism):
.delta.=171.8, 164.4, 154.1, 153.6, 136.4, 135.9, 128.7, 128.4,
128.2, 128.1, 128.0, 127.8, 127.7, 127.6, 126.5, 125.9, 81.2, 80.9,
66.7, 61.0, 60.6, 60.2, 58.8, 58.1, 52.2, 32.7, 32.0, 31.8, 29.9,
29.5, 29.2, 28.8, 27.8, 27.7, 22.5, 14.0.
EXAMPLE 4
[0198] Enamide (27):
[0199] The general procedure B was followed using 20 and the
resulting crude was purified by flash chromatography (hexane/ethyl
acetate, 7:3), yielding 27 (98%) as yellow oil. -Z-isomer:
-[a]D.sup.22=.+-.16.95 (c=1.86, CHCl.sub.3). --.sup.1H NMR (200
MHz, CDCl.sub.3) (signals were splitted for amidic isomerism):
.delta. =1.3-1.5 [2 s, 18 H, C(CH.sub.3).sub.3], 1.6-2.2 (m, 4 H,
CH.sub.2--CH.sub.2), 2.3-2.8 (2 m, 2 H, .dbd.CH--CH.sub.2), 3.7 (s,
3 H, COOCH.sub.3), 4,1-4.2 (2 m, 2 H, .dbd.CH--CH.sub.2--CH--N,
N--CH--COOtBu), 5.15 (m, 4 H, CH.sub.2Ph), 6.95 (dd, J=8.5, J=6.4
Hz, 1 H, .dbd.CH), 7.30 (m, 10 H, aromatic). --.sup.13C NMR (50.3
MHz, CDCl.sub.3) (signals were splitted for amidic isomerism):
.delta.=171.4, 163.8, 154.6, 154.3, 152.1, 150.4, 139.0, 138.8,
136.2, 135.1, 129.7, 128.3, 128.2, 128.1, 127.8, 127.6, 83.3, 81.2,
77.1, 68.2, 66.8, 60.9, 60.4, 57.5, 56.7, 52.1, 32.8, 32.1, 29.9,
29.1, 28.8, 27.7.-E-isomer: -[.alpha.]D.sup.22=+7.34 (c=1.33,
CHCl.sub.3). --.sup.1H NMR (200 MHz, CDCl.sub.3) (signals were
splitted for amidic isomerism): .delta. =1.3-1.5 [2 s, 18 H,
C(CH.sub.3).sub.3], 1.6-2.2 (m, 4 H, CH.sub.2--CH.sub.2), 3.0-3.3
(m, 2 H, .dbd.CH--CH.sub.2), 3.75 (2 s, 3 H, COOCH.sub.3), 4,1-4.2
(2 m, 2 H, .dbd.CH--CH.sub.2--CH--N, N--CH--COOtBu, 5.1-5.2 (m, 4
H, CH.sub.2Ph), 6.3 (m, 1 H, .dbd.C), 7.30 (m, 10 H, aromatic).
--.sup.13C NMR (50.3 MHz, CDCl.sub.3) (signals were splitted for
amidic isomerism): .delta.=171.6, 163.8, 154.5, 154.3, 152.1,
150.4, 142.8, 142.5, 136.3, 135.2, 128.7, 128.3, 128.2, 128.1,
127.9, 127.8, 127.6, 83.2, 81.1, 68.2, 66.8, 61.1, 60.6, 58.1,
57.4, 51.7, 32.7, 32.0, 29.5, 29.4, 28.9, 28.7, 27.7.
EXAMPLE 5
[0200] 6,5-Fused Bicyclic Lactam (2a, 8a):
[0201] A solution of 0.320 g of 27 (0.49 mmol) and a catalytic
quantity of Pd/C 10% in 5 ml of MeOH was stirred under H.sub.2 for
one night. The catalyst was then filtered through celite and the
filtration bed was washed with MeOH. The solvent was evaporated
under reduced pressure, the residue was dissolved in MeOH and
refluxed for 48 h. The solvent was removed and the two
diastereoisomers formed were separated by flash chromatography
(hexane/ethyl acetate, 7:3), yielding 0.122 g of 8a and 2a (70%) in
a 1.4:1 diastereoisomeric ratio as white foam.
-[.alpha.]D.sup.22=-10.70 (c=1.29, CHCl.sub.3). --.sup.1H NMR (200
MHz, CDCl.sub.3): .delta. =1.43-1.45 [2 s, 18 H,
C(CH.sub.3).sub.3], 1.5-2.5 (m, 8 H, CH.sub.2--CH.sub.2,
BocN--CH--CH.sub.2--CH.sub.2), 3.69 [m, 1 H, CH--N], 4.1 (m, 1 H,
CH--NBoc), 4.38 (dd, J=7.7 Hz, J=1.8 Hz, 1 H, N--CH--COOtBu), 5.59
(d, J=5.4 Hz, 1 H, NH). --.sup.13C NMR (50.3 MHz, CDCl.sub.3):
.delta.=170.7, 165.8, 155.8, 147.1, 81.4, 79.3, 59.0, 56.2, 49.9,
32.0, 29.5, 29.1, 28.2, 27.8, 27.0, 26.5.-FAB.sup.+MS: calcd. for
C.sub.18H.sub.32N.sub.2O.sub.5 354.46, found 354. -8a
-[.alpha.]D.sup.22=-45.07 (c=1.69, CHCl.sub.3). --.sup.1H NMR (200
MHz, CDCl.sub.3): .alpha. =1.44-1.46 [2 s, 18 H,
C(CH.sub.3).sub.3], 1.55-2.2 (m, 7H, CH.sub.2--CH.sub.2,
BocN--CH--CH--CH.sub.2), 2.5 (m, 1H, BocN--CH--CHH), 3.75 [tt,
J=11.2 Hz, J=4.2 Hz, 1 H, CH--N], 3.90 (m, 1 H, CH--NBoc), 4.32 (d,
J=9.2 Hz, 1 H, N--CH--COOtBu), 5.59 (broad, 1 H, NH). --.sup.13C
NMR (50.3 MHz, CDCl.sub.3): .delta. =170.6, 167.9, 155.7, 81.2,
79.4, 77.5, 60.4, 59.0, 52.2, 31.4, 28.5, 28.3, 28.2, 27.8,
27.6.-FAB.sup.+MS: calcd. for C.sub.18H.sub.32N.sub.2O.sub.5
354.46, found 354.
[0202] Acid (28):
[0203] To a solution of 27 (0.640 g, 0.980 mmol) in 4.9 ml of MeOH
was added 4.9 ml of 1N NaOH (4.9 mmol). After 18 hours of stirring
at room temperature the solvent was evaporated under reduced
pressure. The solid residue was dissolved in 5 ml of water and 2N
HCl was added until pH 3, then the aqueous solution was extracted
with CH.sub.2Cl.sub.2. The organic phase was dried with
Na.sub.2SO.sub.4, the solvent evaporated under reduced pressure and
the crude was purified by flash chromatography
(CH.sub.2Cl.sub.2/MeOH, 95:5), yielding 0.420 g of 28 (85%) as a
white solid.
[0204] Z isomer: -[.alpha.]D.sup.22=-57.01 (c=1.99, CHCl.sub.3).
--.sup.1H NMR (200 MHz, CDCl.sub.3) (signals were splitted for
amidic isomerism): .delta. =1.30-1.50 [2 s, 18 H,
C(CH.sub.3).sub.3], 1.7-2.7 (m, 6 H, CH.sub.2--CH.sub.2,
.dbd.CH--CH.sub.2), 4.2-4.3 (m, 2 H, .dbd.CH--CH.sub.2--CH--N,
N--CH--COOtBu), 5.1 (m, 2 H, CH.sub.2Ph), 6.6 (m, 1 H, .dbd.CH),
7.30 (m, 6 H, aromatic, NHBoc). --.sup.13C NMR (50.3 MHz,
CDCl.sub.3) (signals were splitted for amidic isomerism): .delta.
=171.5, 168.3, 154.8, 154.5, 140.6, 136.4, 136.1, 133.9, 133.5,
128.3, 128.2, 128.1, 127.8, 127.4, 126.9, 81.3, 80.9, 67.1, 66.9,
65.0, 66.9, 65.0, 57.5, 56.8, 33.4, 32.4, 29.5, 28.5, 28.5, 28.0,
27.8, 27.7, 27.4.
[0205] E isomer: -[a]D.sup.22=-41.63 (c=1.87, CHCl.sub.3).
--.sup.1H NMR (200 MHz, CDCl.sub.3) (signals were splitted for
amidic isomerism): .delta. =1.35-1.50 [3 s, 18 H,
C(CH.sub.3).sub.3], 1.7-2.4 (m, 4 H, CH.sub.2--CH.sub.2), 2.7-3.2
(m, 2 H, .dbd.CH--CH.sub.2), 4,2-4.3 (m, 2 H,
.dbd.CH--CH.sub.2--CH--N, N--CH--COOtBu), 5.1 (m, 2 H, CH.sub.2Ph),
6.7-6.9 (m, 2 H, .dbd.CH, NHBoc), 7.30 (m, 5 H, aromatic).
--.sup.13C NMR (50.3 MHz, CDCl.sub.3) (signals were splitted for
amidic isomerism): .delta. =171.7, 167.2, 154.9, 154.5, 154.3,
136.5, 136.2, 128.3, 128.2, 127.7, 127.5, 126.9, 126.3, 126.1,
81.2, 80.4, 66.9, 65.0, 60.7, 60.4, 58.3, 57.7, 32.9, 32.0, 29.5,
28.4, 28.1, 27.8, 27.7, 27.4, 27.1, 14.0.
[0206] Acid (32, 33):
[0207] To the [Rh-(-)-BitianP] catalyst prepared as described in
the literature was added 28 (0.16 mmol) and MeOH (30 ml), the
resulting solution was stirred for 30 min. A 200 ml stainless-steel
autoclave equipped with a magnetic stirrer and a thermostatic bath
was pressurised with hydrogen and vented three times. The solution
was transferred into the autoclave with a syringe and the autoclave
was pressurised at 10 KPa with hydrogen. The solution was stirred
for 24 h. at 30.degree. C. The hydrogen pressure was released, the
solvent evaporated. The crude was submitted to the next reaction
without further purification.
[0208] 6,5-fused Bicyclic Lactam (2a):
[0209] To a solution of 32 and 33 as diastereomeric mixture in MeOH
(1.5 ml) was added a solution of CH.sub.2N.sub.2 in Et.sub.2O until
the TLC showed that the reaction was complete. The solution was
evaporated and the crude was dissolved in MeOH (2 ml) and a
catalytic quantity of Pd/C was added, the mixture was stirred under
H.sub.2 for 12 h. The catalyst was then filtered through celite pad
and washed with MeOH. The solvent was evaporated under reduced
pressure and the crude, as a white foam, was refluxed in MeOH for
48 h. The solvent was evaporated under reduced pressure and the
crude was purified by flash chromatography (hexane/ethyl acetate
7:3) affording 2a (85%) as a white solid.
EXAMPLE 6
[0210] 6,5-fused Bicyclic Lactam (8a):
[0211] This bicyclic lactam was achieved with the same synthetic
sequence followed for the lactam 2a using for the asymmetric
hydrogenation the [Rh-(+)-BitianP] catalyst.
[0212] Aldehyde (15):
[0213] The general procedure C was followed using 25 and the
resulting residue was purified by flash chromatography
(hexane/ethyl acetate, 7:3), yielding the alcohol (95%) as yellow
oil. --.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. =1.4 [s, 9 H,
C(CH.sub.3).sub.3], 1.6-2.4 (m, 8 s H, CH.sub.2--CH.sub.2), 3.5-3.8
(2 m, 2 H, CH.sub.2OH), 4.1 (m, 1 H, CH.sub.2--CH--N), 4.25 (m, 1
H, N--CH--COOtBu), 5.15 (s, 2 H, CH.sub.2Ph), 7.30 (m, 5 H,
aromatic).
[0214] The general procedure D was followed using the previous
alcohol and the resulting crude residue was purified by flash
chromatography (hexane/ethyl acetate, 7:3), yielding 15 (89%) as an
oil. --.sup.1H NMR (200 MHz, CDCl.sub.3), (signals were splitted
for amidic isomerism): .delta. =1.4-1.5 [2 s, 9 H,
C(CH.sub.3).sub.3], 1.6-2.8 (m, 4 H, CH.sub.2--CH.sub.2), 4.05 (m,
1 H, CH.sub.2--CH--N), 4.25 (m, 1 H, N--CH--COOtBu), 5.15 (s, 2 H,
CH.sub.2Ph), 7.30 (m, 5 H, aromatic), 9.6-9.8 (2 s, 1 H, CHO).
[0215] Aminoester (34):
[0216] The general procedure A was followed using 15 and the
resulting residue was purified by flash chromatography yielding the
enamide (95%) as yellow oil. The compound previously synthesised
was submitted to the general procedure B and the resulting residue
was purified by flash chromatography yielding the N-Boc protected
compound (95%) as white solid. A solution of this compound (0.96
mmol) in MeOH (1 mL) and a catalytic quantity of Pd/C were stirred
under hydrogen atmosphere for 12 h. The catalyst was then filtered
through a celite pad. The solvent was evaporated under reduced
pressure yielding 0.320 g of 34 (83%) as a white solid (mixture of
two diastereoisomers). --.sup.1H NMR (200 MHz, CDCl.sub.3): .delta.
=1.47, 1.48 [2 s, 18 H, C(CH.sub.3).sub.3], 1.40-2.1 (m, 10 H,
CH.sub.2--CH.sub.2, BocN--CH--CHH--CH.sub.2), 3.00 (m, 1 H, CH--N),
3.6 (m, 1 H, N--CH--COOtBu), 4.3 (m, 1 H, CH--NBoc), 5.0(db, 1H,
NH).
[0217] Amino Acid (35):
[0218] To a solution of 34 (0.288 g, 0.720 mmol) in MeOH was added
1N NaOH, after 1.5 h. the solution was acidified until pH 3 with IN
HCl, then the solution was evaporated. The crude was submitted to
the next reaction without further purification.
EXAMPLE 7
[0219] 7,5-fused Bicyclic Lactams (3a, 9a):
[0220] To a solution of the crude 35 (0.720 mmol) in
CH.sub.2Cl.sub.2 (80 ml) was added in the order: Et.sub.3N (0.720
mmol, 0.220 ml), HOBt (0.166 g, 1.22 mmol) and a catalytic quantity
of DMAP. After 15 min was added EDC (0.180 g, 0.937 mmol) and the
solution was stirred for 24 h. To the solution was added H.sub.2O
(40 ml), the aqueous phase was extracted with CH.sub.2Cl.sub.2 and
the collected organic layers were dried with Na.sub.2SO.sub.4
filtered and evaporated under reduced pressure affording 0.191 g of
3a and 9a in a 1:1 diastereoisomeric ratio and 72% of yield over 2
steps.
[0221] (3a). --.sup.1H NMR (200 MHz, CDCl.sub.3): .delta. =1.41,
1.42 [2 s, 18 H, C(CH.sub.3).sub.3], 1.5-2.5 (m, 10 H,
CH.sub.2--CH.sub.2), 3.80 (m, 1 H, CH--N), 4.2 (m, 1 H, CH--NBoc),
4.51 (dd, J=4.8 Hz, 1 H, N--CH--COOtBu), 5.54 (db, 1 H, NH). -(9a).
--.sup.1H NMR (200 MHz, CDCl.sub.3): .delta. =1.42, 1.43 [2 s, 18
H, C(CH.sub.3).sub.3], 1.50-2.2 (m, 10H, CH.sub.2--CH.sub.2), 3.8
[m, 1 H, CH--N], 4.25 (dd, J=4.6 Hz, J=9.6 Hz, 1 H, CH--NBoc), 4.42
(dd, J=2.3 Hz, J 7.2 Hz, 1 H, N--CH--COOtBu), 5.30 (bs, 1 H,
NH).
[0222] Enamide (37): The general procedure D was followed using 36
and the crude was purified by flash chromatography (hexane/ethyl
acetate, 7:3), yielding the aldehyde (81%) as an oil. --.sup.1H NMR
(200 MHz, CDCl.sub.3), (signals were splitted for amidic
isomerism): .delta. =1.48 [s, 9 H, C(CH.sub.3).sub.3], 1.8-2.2 (m,
4 H, CH.sub.2--CH.sub.2), 3.21 (m, 1 H, CH.sub.2--CH--N), 3.45 (m,
1 H, N--CH--COOtBu), 3.70 (d, J=12 Hz, 1 H, HCHPh), 4.10 (d, J=12
Hz, 1 H, HCHPh), 7.30 (m, 5 H, aromatic), 9.12 (d, 1 H, CHO).
[0223] The general procedure A was followed using the previous
aldehyde and the crude was purified by flash chromatography
(hexane/ethyl acetate, 65:35), affording the enamide (98%) in a 9:1
Z:E ratio as colourless oils. Z-isomer --.sup.1H NMR (200 MHz,
CDCl.sub.3) .delta. =1.31 [s, 9 H, C(CH.sub.3).sub.3], 1.7-2.2 (m,
4 H, CH.sub.2--CH.sub.2), 3.3 (m, 1 H, N--CH--COOtBu) 3.5 (s, 1 H,
CH.sub.2--CH--N), 3.66 (d, J=13.2 Hz, HCHPh) 3.73 (s, 1 H,
COOCH.sub.3), 3.79 (d 1 H, HCEPh), 5.11 (d, J=12.5 Hz, 1 H,
OHCHPh), 5.15 (d, J=12.5 Hz, 1 H, OHCHPh), 6.07 (d, J=7.4 Hz, 1 H,
.dbd.CH), 7.10-7.6 (m, 10 H, aromatic), 8.15 (sb, 1 H, --NH).
--.sup.13C NMR (50.3 MHz, CDCl.sub.3): .delta. =173.7, 165.1,
154.1, 137.4, 136.1, 129.5, 128.5, 128.3, 128.0, 127.8, 127.7,
127.1, 80.5, 66.9, 65.3, 62.3, 57.5, 52.0, 30.1, 28.9, 27.7.
[0224] The general procedure B was followed using the enamide
previous synthesised. The crude was purified by flash
chromatography (hexane/ethyl acetate, 7:3) yielding 37 (98%) as a
white solid. --.sup.1H NMR (200 MHz, CDCl.sub.3) (signals were
splitted for amidic isomerism): .delta. =1.3-1.5 [2 s, 18 H,
C(CH.sub.3).sub.3], 1.6-2.2 (m, 4 H, CH.sub.2--CH.sub.2), 3.1 (m, 1
H, N--CH--COOtBu), 3.5 (m, 1 H, CH.sub.2--CH--N), 3.7 (s, 1 H,
COOCH.sub.3), 3.7 (d, J=12 Hz, 1 H, HCHPh), 3.9 (d, J=12 Hz, 1 H,
HCHPh), 5.20 (d, J=12 Hz, 1 H, HCHPh), 7.0 (d, J=8.6 Hz, 1 H,
.dbd.CH), 7.1-7.4 (m, 10 H, aromatic).
[0225] Amino acid (39): To a solution of 37 (0.424 g, 0.713 mmol)
in MeOH (4 ml) was added IN NaOH (4 mmol, 4 ml) and stirred for 1.5
h. The solution was acidified until pH 3 with 1N HCl, then the
solution was evaporated. The crude was submitted to the next
reaction without further purification. --.sup.1H NMR (200 MHz,
CDCl.sub.3) (signals were splitted for amidic isomerism): .delta.
=1.35, 1.5 [2 s, 18 H, C(CH.sub.3).sub.3], 1.7-2.3 (m, 4 H,
CH.sub.2--CH.sub.2), 3.3 (m, 1 H, N--CH--COOtBu), 3.65 (m, 1 H,
CH.sub.2--CH--N), 3.7 (d, J=12.8 Hz, 1 H, HCHPh), 3.9 (d, J=12.8
Hz, 1 H, HCHPh), 6.5 (d, J=7.6 Hz, 1 H, .dbd.CH, 7.1-7.4 (m, 10 H,
aromatic), 9.00 (bs, 1 H, --COOH).
EXAMPLE 8
[0226] 5,5-fused Bicyclic Lactams (1a, 7a):
[0227] A solution of 39 (0.713 mmol) and a catalytic quantity of
Pd(OH).sub.2/C 20% in 1 ml of MeOH (7 ml) was stirred under
hydrogen atmosphere for 12 h. The catalyst was then filtered
through a celite pad and the solvent was evaporated under reduced
procedure. The crude was dissolved in MeOH and refluxed for 48 h.
The solvent was evaporated under reduced pressure and the crude was
purified by flash chromatography (hexane/ethyl acetate 6:4)
affording 0,097 g of 1a and 7a as a white solid in 40% of yield
(over 2 steps) and 1:1 diastereomeric ratio. 1a:
-[.alpha.]D.sup.22=-4.80 (c=1.20, CHCl.sub.3). --.sup.1H NMR (200
MHz, CDCl.sub.3): .delta. =1.50, 1.51 [2 s, 18 H,
C(CH.sub.3).sub.3], 1.6-2.4 (m, 5 H, CH.sub.2--CH.sub.2,
BocN--CH--CHH), 2.95 (m, 1 H, BocN--CH--CHH), 3.85 [m, 1 H,
(CH--N], 4.15 (d, J=8.8 Hz, 1 H, N--CH--COOtBu), 4.60 (m 1 H,
CH--NBoc), 5.25 (broad, 1 H, NH). --.sup.13C NMR (50.3 MHz,
CDCl.sub.3) (signals were splitted for amidic isomerism): .delta.
=171.7, 169.7, 155.6, 81.8, 79.5, 58.8, 56.5, 56.0, 55.8, 39.5,
33.4, 29.5, 28.2, 27.8.-FAB.sup.+MS: calcd. for
C.sub.17H.sub.28N.sub.2O.- sup.5 340.41, found 341. -2a:
[.alpha.]D.sup.22=-4.80 (c=1.20, CHCl.sub.3). --.sup.1H NMR (200
MHz, CDCl.sub.3): .delta. =1.45 [2 s, 18 H, C(CH.sub.3).sub.3],
1.5-2.5 (m, 6 H, CH.sub.2--CH.sub.2, BocN--CH--CH.sub.2), 4.05 (d,
J=8.8 Hz, 1 H, N--CH--COOtBu), 4.12 (m, 1 H, CH--N), 4.25 (m, 1 H,
CH--NBoc), 5.05 (broad, 1 H, NH). --.sup.13C NMR (50.3 MHz,
CDCl.sub.3) (signals were splitted for amidic isomerism): .delta.
=170.9, 169.8, 155.2, 82.2, 81.8, 79.9, 77.1, 61.2, 58.8, 57.6,
56.0, 55.8, 20 34.4, 33.8, 33.4, 29.9, 29.5, 29.2, 28.5, 28.1,
27.7.-FAB.sup.+MS: calcd. for C.sub.17H.sub.28N.sub.2O.sup.5
340.41, found 341.
[0228] Aldehyde (13):
[0229] To a stirred solution of 36 (1.5 g, 5.14 mmol) in 39 ml of
dry CH.sub.2Cl.sub.2 under nitrogen were added in the order:
TBDMSC1 (0.931 g, 6.17 mmol), TEA (6.17 mmol, 0.94 ml) and DMAP
(0.063 g, 0.51 mmol). After 12 h. the solvent was evaporated under
reduced pressure and the crude purified by flash chromatography
(hexane/ethyl acetate, 9:1), yielding 1.910 g of compound (94%) as
a colourless oil. -[.alpha.].sub.D.sup.22=-3.61 (c=2.52,
CHCl.sub.3). --.sup.1H NMR (200 MHz, CDCl.sub.3): .delta. =-0.5 [s,
6 H,CH.sub.3Si], 0.85 [s, 9 H, (CH.sub.3).sub.3C--Si], 1.4 [s, 9 H,
C(CH.sub.3).sub.3], 1.5-2.1 (m, 4 H, CH.sub.2--CH.sub.2), 2.9 (m, 1
H, SiO--CH.sub.2--CH--N), 3.3-3.4 (m, 3 H, N--CH--COOtBu,
SiO--CH.sub.2), 3.9 (s, 2 H, CH.sub.2Ph), 7.3 (m, 5 H, aromatic).
--.sup.13C NMR (50.3 MHz, CDCl.sub.3): .delta. =173.6, 139.3,
129.1, 127.9, 126.7, 19.9, 67.5, 66.8, 65.8, 58.8, 28.4, 28.0,
27.8, 25.8, 18.1,-3.6.
[0230] A solution of the silyl protected alcohol (1.850 g, 4.55
mmol) and Pd(OH).sub.2/C 20% (0.250 g, 0.45 mmol) in 45 ml of MeOH
was stirred under hydrogen atmosphere for 4 hours. Then the
catalyst was filtered through celite pad and washed with MeOH, the
solvent was evaporated under reduced pressure, yielding 1.34 g of
hydrogenated compound (94%) as colourless oil.
-[.alpha.]D.sup.22=-5.80 (c=1.99, CHCl.sub.3). --.sup.1H NMR (200
MHz, CDCl.sub.3): .delta. =0.4 (s, 6 H,CH.sub.3Si), 0.92 [s, 9 H,
(CH.sub.3).sub.3C--Si], 1.49 [s, 9 H, C(CH.sub.3).sub.3], 1.5-2.1
(m, 4 H, CH.sub.2--CH.sub.2), 2.35 (broad, 1 H, NH), 3.2 (m, 1 H,
SiO--CH.sub.2--CH--N), 3.65 (m, 3 H, N--CH--COOtBu,
SiO--CH.sub.2).
[0231] To a stirred solution of the previous compound (1.2 g, 3.79
mmol) in 38 ml of CH.sub.2Cl.sub.2 were added pyridine (11.39 mmol,
0.92 ml) and (CF.sub.3CO).sub.2O (8.35 mmol, 1.16 ml). After 1.5
hours the solvent was evaporated under reduced pressure and the
crude purified by flash chromatography (hexane/ethyl acetate, 9:1),
yielding 1.4 g of the N-protected pyrrolidine (89%) as colourless
oil. -[.alpha.]D.sup.22=-8.62 (c=2.11, CHCl.sub.3). --.sup.1H NMR
(200 MHz, CDCl.sub.3): .delta. =0.4 (s, 6 H,CH.sub.3Si), 0.9 [s, 9
H, (CH.sub.3).sub.3C--Si], 1.47 [s, 9 H, C(CH.sub.3).sub.3],
1.7-2.4 (m, 4 H, CH.sub.2--CH.sub.2), 3.5 (m, 1 H, SiO--CHH), 3.75
(dd, J=10.6 Hz, J=4.2 Hz, 1 H, SiO--CHH), 4.2 (m, 1 H,
SiO--CH.sub.2--CH--N), 4.35 (t, J=8.5 Hz 1 H, N--CH--COOtBu).
[0232] To a stirred solution of N-protected pyrrolidine (1.2 g,
2.91 mmol) in 29 ml of THF, cooled at -40.degree. C., was added a
1M solution of TBAF in THF (3.20 mmol, 3.2 ml). Then the solution
was allowed to warm at room temp. After 2.5 hours was added 30 ml
of brine and the resulting mixture was extracted with ethyl
acetate. The organic phase was dried with Na.sub.2SO.sub.4 and the
solvent evaporated under reduced pressure. The crude was purified
by flash chromatography (hexane/ethyl acetate, 6:4), yielding 0.850
g of O-deprotected compound (98%) as colourless oil.
-[.alpha.]D.sup.22=-6.40 (c=1.45, CHCl.sub.3). --.sup.1H NMR (200
MHz, CDCl.sub.3): .delta. =1.5 [s, 9 H, C(CH.sub.3).sub.3], 2.0-2.4
(m, 4 H, CH.sub.2--CH.sub.2), 3.4-3.7 (m, 2 H, HO--CH.sub.2),
4.2-4.6 (m, 3 H, N--CH--COOtBu, HO--CH.sub.2--CH--N).
[0233] The general procedure D was followed using the alcohol and
the residue was purified by flash chromatography (hexane/ethyl
acetate, 6:4), yielding the aldehyde (93%) as white solid.
-[.alpha.]D.sup.22=+22.48 (c=1.53, CHCl.sub.3). --.sup.1H NMR (200
MHz, CDCl.sub.3): .delta. =1.5 [s, 9 H, C(CH.sub.3).sub.3], 1.8-2.5
(m, 4 H, CH.sub.2--CH.sub.2), 4.5-4.7 (m, 2 H, CHO--CH--N,
N--CH--COOtBu), 9.7 (s, 1 H, CHO).
[0234] Enamide (40):
[0235] The general procedure A was followed using 13 and the crude
residue was purified by flash chromatography affording the enamide
(68%) as colourless oil (diastereoisomeric ratio Z:E=1:1).
--.sup.1H NMR (200 MHz, CDCl.sub.3) (signals were splitted for
amidic isomerism and were referred to the mixture of two
diastereoisomers): .delta. =1.5 [s, 9 H, C(CH.sub.3).sub.3],
1.6-2.45 (m, 4 H, CH.sub.2--CH.sub.2), 3.75 (s, 3 H, COOCH.sub.3),
4.6 (m, 1 H, N--CH--COOtBu), 4.8 (dd, J=18 Hz, J=10 Hz, 1 H,
.dbd.CH--CH--N), 5.12 (s, 2 H, CH.sub.2Ph), 6.3, 6.8 (2d, J=10 Hz,
1 H, .dbd.CH of Z-isomer, E-isomer), 7.35 (m, 5 H, aromatic).
[0236] The general procedure B was followed using the enamide and
the crude was purified by flash chromatography affording 40 with a
95% of yield as colourless oil. --.sup.1H NMR (200 MHz,
C.sub.6D.sub.6) (signals were splitted for amidic isomerism and
were referred to the mixture of two diastereoisomers): .delta.
=1.3, 1.5 [2 s, 18 H, C(CH.sub.3).sub.3], 1.6-2.35 (m, 4 H,
CH.sub.2--CH.sub.2), 3.7 (s, 3 H, COOCH.sub.3), 4.6-4.8 (m, 2 H,
N--CH--COOtBu, .dbd.CH--CH--N), 5.25 (m, 2 H, CH.sub.2Ph), 7.0 (m,
1 H, .dbd.CH), 7.35 (m, 5 H, aromatic). --.sup.13C NMR (50.3 MHz,
C.sub.6D.sub.6) (signals were splitted for amidic isomerism and
were referred to the mixture of two diastereoisomers): .delta.
=169.1, 163.9, 141.2, 136.1, 129.9, 128.4, 128.2, 127.4, 119.4,
113.7, 83.6, 82.5, 82.0, 68.8, 68.5, 68.2, 62.5, 60.9, 60.8, 58.5,
57.6, 56.8, 53.2, 51.9, 51.7, 51.6, 33.7, 31.8, 30.2, 27.7, 27.5,
26.9.
[0237] Aminoester (41)
[0238] A Z/E mixture of 40 (0.609 g, 1.01 mmol) and Pd(OH).sub.2/C
20% (0.054 g) in 10 ml of MeOH was stirred under hydrogen
atmosphere for 18 h. The catalyst was filtered through a celite pad
and washed with MeOH. The solvent was evaporated under reduced
pressure and the crude purified by flash chromatography
(toluene/Et.sub.2O, 85:15), yielding 0.365 g of 40 (77%) as yellow
oil. --.sup.1H NMR (200 to MHz, CDCl.sub.3) (signals were splitted
for amidic isomerism and were referred to the mixture of two
diastereoisomers): .delta. =1.45 [s, 18 H, C(CH.sub.3).sub.3],
1.6-2.7 (m, 6 H, CH.sub.2--CH.sub.2, BocN--CH--CH.sub.2), 3.75 (2
s, 3 H, COOCH.sub.3), 4.25-4.4 (2 m, 2 H, BocN--CH,
BocN--CH--CH.sub.2--CH), 4.55 (m, 1 H, N--CH--COOtBu), 5.30 (d,
J=8.5 Hz, 1 H, NH). --.sup.13C NMR (50.3 MHz, CDCl.sub.3) (signals
were splitted for amidic isomerism and were referred to the mixture
of two diastereoisomers): .delta. =172.4, 170.0, 155.8, 128.9,
128.0, 82.7, 82.0, 79.7, 61.4, 60.6, 58.0, 56.5, 52.2, 51.5, 37.7,
36.4, 35.5, 30.2, 29.7, 29.0, 28.4, 28.1, 27.6, 25.5. -FAB.sup.+MS:
calcd. for C.sub.20H.sub.31F.sub.3N.sub.2O.sub.7 468.47, found
468.
[0239] Amino acid (42)
[0240] A solution of 41 (0.184 g, 0.393 mmol) and NaBH.sub.4
(0.0298 g, 0.781 mmol) in 8 ml of MeOH was stirred for 1 hour at
room temperature. The solution was concentrated and 10 ml of water
was added. The aqueous solution was extracted with ethyl acetate,
the collected organic phases were dried on Na.sub.2SO.sub.4 and the
solvent evaporated under reduced pressure. The two diastereoisomers
formed in the previous reactions were separated at this step by
flash chromatography (ethyl acetate/hexane, 6:4), achieving 0.123 g
of 42 (R) and 42 (S) (84%) in a 2.6:1 diastereoisomeric ratio as
colourless oil. -42 (R): --.sup.1H NMR (200 MHz, C.sub.6D.sub.6)
(signals were splitted for amidic isomerism): .delta. =1.30, 1.45
[2 s, 18 H, C(CH.sub.3).sub.3], 1.5-1.9 (m, 6 H,
CH.sub.2--CH.sub.2, BocN--CH--CH.sub.2), 2.85 (m, 1 H,
BocN--CH--CH.sub.2--CH), 3.2-3.4 (m, 4 H, COOCH.sub.3,
N--CH--COOtBu), 4.65 (m, 1 H, BocN--CH), 6.6 (broad, 1 H, NHBoc).
--.sup.13C NMR (50.3 MHz, C.sub.6D.sub.6) (signals were splitted
for amidic isomerism): .delta. =174.1, 173.2, 155.8, 81.4, 81.3,
79.5, 60.6, 60.4, 56.5, 56.3, 52.5, 52.0, 37.7, 31.9, 30.0, 29.8,
28.2, 28.0, 27.9.-FAB.sup.+MS: calcd. for
C.sub.18H.sub.32N.sub.2O.sub.6 372.46, found 373. -42 (S):
--.sup.1H NMR (200 MHz, C.sub.6D.sub.6) (signals were splitted for
amidic isomerism): .delta. =1.30, 1.50 [2 s, 18 H,
C(CH.sub.3).sub.3], 1.50-1.80 (m, 6 H, CH.sub.2--CH.sub.2,
BocN--CH--CH.sub.2), 2.8 (m, 1 H, BocN--CH--CH.sub.2--CH), 3.3 (s,
3 H, COOCH.sub.3), 3.4 (dd, J=9.1 Hz, J=5.9 Hz, 1 H,
N--CH--COOtBu), 4.45 (m, 1 H, BocN--CH), 5.3 (broad, 1 H, NHBoc).
--.sup.13C NMR (50.3 MHz, C.sub.6D.sub.6) (signals were splitted
for amidic isomerism): .delta.=171.7, 171.5, 164.2, 164.0, 154.7,
154.3, 153.5, 136.6, 136.4, 135.8, 128.4, 128.3, 128.2, 128.1,
127.7, 126.2, 125.9, 125.8, 81.0, 87.1, 66.8, 66.6, 60.8, 60.4,
58.2, 57.5, 52.3, 52.2, 32.8, 31.9, 28.5, 28.1, 27.8, 27.7, 27.4,
27.1.-FAB.sup.+MS: calcd. for C.sub.18H.sub.32N.sub.2O.sub.6
372.46, found 373.
EXAMPLE 9
[0241] 5,5-Fused Bicyclic Lactam [1a]:
[0242] A stirred solution of 42 (S) (0.028 g, 0.075 mmol) in 1.5 ml
of p-xylene was warmed at 130.degree. C. for 24 hours. The solvent
was then evaporated under reduced pressure and the crude purified
by flash chromatography (hexane/ethyl acetate, 7:3), yielding 19 mg
of 1a (74%) as a white foam. -.alpha..sub.D.sup.22=-4.80 (c=1.20,
CHCl.sub.3). --.sup.1H NMR (200 MHz, CDCl.sub.3): .delta. =1.50,
1.51 [2 s, 18 H, C(CH.sub.3).sub.3], 1.6-2.4 (m, 5 H,
CH.sub.2--CH.sub.2, BocN--CH--CHH), 2.95 (m, 1 H, BocN--CH--CHH),
3.85 [m, 1 H, (CH--N], 4.15 (d, J=8.8 Hz, 1 H, N--CH--COOtBu), 4.60
(m 1 H, CH--NBoc), 5.25 (broad, 1 H, NH). --.sup.13C NMR (50.3 MHz,
CDCl.sub.3) (signals were splitted for amidic isomerism): .delta.
=171.7, 169.7, 155.6, 81.8, 79.5, 58.8, 56.5, 56.0, 55.8, 39.5,
33.4, 29.5, 28.2, 27.8. -FAB.sup.+MS: calcd. for
C.sub.17H.sub.28N.sub.2O.sub.5 340.41, found 341.
EXAMPLE 10
[0243] 5,5-Fused bicyclic lactam [7a]:
[0244] The compound [7a] was achieved from compound 42 (R), by
using the same procedure described for the synthesis of compound
1a, with a 65% of yield as white foam. -[.alpha.]D.sup.22=-4.80
(c=1.20, CHCl.sub.3). --.sup.1H NMR (200 MHz, CDCl.sub.3): .delta.
=1.45 [2 s, 18 H, C(CH.sub.3).sub.3], 1.5-2.5 (m, 6 H,
CH.sub.2--CH.sub.2, BocN--CH--CH.sub.2), 4.05 (d, J=8.8 Hz, 1 H,
N--CH--COOtBu), 4.12 (m, 1 H, CH--N), 4.25 (m, 1 H, CH--NBoc), 5.05
(broad, 1 H, NH). --.sup.13C NMR (50.3 MHz, CDCl.sub.3) (signals
were splitted for amidic isomerism): .delta. =170.9, 169.8, 155.2,
82.2, 81.8, 79.9, 77.1, 61.2, 58.8, 57.6, 56.0, 55.8, 34.4, 33.8,
33.4, 29.9, 29.5, 29.2, 28.5, 28.1, 27.7.-FAB.sup.+MS: calcd. for
C.sub.17H.sub.28N.sub.2O.sub.5 340.41, found 341.
[0245] Ester (43)
[0246] To a stirred suspension of KH (0.777 g, 19.4 mmol) in
anhydrous DMF (80 ml) the triethyl phosphonoacetate (19.4 mmol, 3.9
ml) was added. The mixture was stirred at room temperature for 1 h
and then a solution of hemiaminal (5.2 g, 16.2 mmol) in DMF (80 ml)
was added. The reaction was stirred overnight at room temperature,
quenched with satured aqueous NH.sub.4Cl solution and extracted
with AcOEt. The combined organic extract were dried over
Na2SO.sub.4 and the solvent was evaporated to dryness and purified
by flash chromatography yielding 4.8 g of 43 (75%) in a 4:1
trans:cis diastereoisomeric ratio. --.sup.1H NMR (200 MHz,
CDCl.sub.3) (signals are splitted for amidic isomerism): .delta.
=1.2-1.35 (m, 3 H, CH.sub.3CH.sub.2O), 1.35, 1.40, 1.45, 1.50 [4 s,
9 H, C(CH.sub.3).sub.3], 1.60-2.60 (m, 5 H, CH.sub.2--CH.sub.2,
CHCO.sub.2Et), 2.70-3.1 (2 dd, J.sub.1=4 Hz, J.sub.2=15 Hz, 1 H,
CHCO.sub.2Et, trans isomer), 3.2-3.5 (2 dd, J.sub.1=4 Hz,
J.sub.2=15 Hz, 1 H, CHCO.sub.2Et, cis isomer), 4.13 (dq,
J.sub.1=J.sub.2=7 Hz , 2 H, CH.sub.3CH.sub.2O) 4.27 (m, 1 H,
CHCO.sub.2tBu), 4.45 (m, 1 H, CH.sub.2--CH--N), 5.15-5.35 (m, 2 H,
CH.sub.2Ph), 7.3-7.4 (m, 5 H, aromatic). --.sup.13C NMR (50.3 MHz,
CDCl.sub.3) (signals are splitted for amidic isomerism): 5=171.4,
171.3, 171.1, 171.0, 154.4, 154.1, 153.8, 136.5, 136.3, 128.3,
128.2, 127.7, 127.6, 81.2, 66.9, 66.8, 60.8, 60.5, 60.3, 60.2,
55.5, 55.2, 54.5, 39.1, 38.0, 30.4, 29.7, 28.9, 28.7, 28.2, 28.0,
27.8, 27.7, 27.1, 14.1.-FAB.sup.+MS: calcd. for
C.sub.21H.sub.29NO.sub.6 391.2, found 392.
[0247] Aldehyde (14, 17):
[0248] To a stirred solution of 43 (1.205 g, 3.08 mmol) in dry
diethylether (31 mL) at -10.degree. C., LiBH.sub.4 2M in THF (1.5
mL, 3.08 mmol) was added. After 24 h a saturated solution of
NaHCO.sub.3 (40 ml) was added and the resulting mixture was
extracted with AcOEt. The organic phase was dried over
Na.sub.2SO.sub.4 and evaporated to dryness. The crude product was
purified by flash chromatography (hexane/ethyl acetate 1:1),
yielding 1.01 g of alcohol (94%) as a yellow oil. -Trans-isomer:
[.alpha.]D.sup.22=-32.3 (c=1.02, CHCl.sub.3). -.sup.1H NMR (200
MHz, CDCl.sub.3): .delta. =1.35 [s, 9 H, C(CH.sub.3).sub.3],
1.5-2.4 (m, 6 H, CH.sub.2--CH.sub.2, CH.sub.2--CH.sub.2--O),
3.5-3.7 (m, 2 H, CH.sub.2OH), 3.82 (bs, 1 H, OH), 4.22 (dd, J=7.5,
J.about.0, 1 H, CHCO.sub.2tBu), 4.38 (m, 1 H, CH.sub.2--CH--N),
5.15 (m, 2 H, CH2Ph), 7.32 (s, 5 H, aromatic). --.sup.13C NMR (50.3
MHz, CDCl.sub.3) (signals were splitted for amidic isomerism):
.delta. =171.4, 156.1, 136.0, 128.4, 128.3, 127.9, 127.8, 127.7,
81.2, 81.1, 67.2, 67.0, 60.4, 59.9, 59.0, 55.2, 55.1, 38.6, 37.7,
28.9, 28.7, 27.8, 27.7.-Cis-isomer: [.alpha.]D.sup.22=-54.0
(c=1.51, CHCl.sub.3). --.sup.1H NMR (200 MHz, CDCl.sub.3): .delta.
=1.33 [s, 9 H, C(CH.sub.3).sub.3], 1.4-1.24 (m, 6 H,
CH.sub.2--CH.sub.2, CH.sub.2--CH.sub.2--O), 3.6-3.9 (m, 2 H,
CH.sub.2OH), 4.08 (dd, J=9.5, J=4, 1 H, OH), 4.25 (dd, J=J 8.5, 1
H, CHCO.sub.2tBu), 4.40 (m, 1 H, CH.sub.2--CH--N), 5.15 (m, 2 H,
CH.sub.2Ph), 7.35 (s, 5 H, aromatic). --.sup.13C NMR (50.3 MHz,
CDCl.sub.3): .delta. =27.7, 28.9, 30.4, 37.4, 55.4, 58.8, 60.5,
67.4, 81.3, 127.7, 127.9, 128.3, 136.1, 155.9, 171.8.
[0249] A solution of the alcohol (0.304 g, 0.87 mmol) in dry
CH.sub.2Cl.sub.2 (2.5 mL) was added to a suspension of Dess-Martin
periodinane (0.408 g, 1.13 mmol) in dry CH.sub.2Cl.sub.2 (2.5 mL)
at room temperature. After 1 h Et.sub.2O and NaOH 1N were added
till clear solution. The aqueous phase was extracted twice with
Et.sub.2O; the collected organic layers were washed with H.sub.2O,
dried with Na.sub.2SO.sub.4, and evaporated to dryness. The crude
product was purified by flash chromatography (hexane/ethyl acetate
7:3) affording 0.277 g of 17 (92%). --Trans-isomer:
[.alpha.]D.sup.22=-48.65 (c=1.01, CHCl.sub.3). --.sup.1H NMR (200
MHz, CDCl.sub.3) (signals were splitted for amidic isomerism):
.delta. =1.35-1.45 [2 s, 9 H, C(CH.sub.3).sub.3], 1.6-2.6 (m, 4 H,
CH.sub.2--CH.sub.2), 2.8-3.1 (2 m, 2 H, CH.sub.2CHO), 4.3 (m, 1 H,
CHO--CH.sub.2--CH--N), 4.6 (m, 1 H, N--CH--COOR), 5.15 (m, 2 H,
CH.sub.2Ph), 7.30 (m, 5 H, aromatic), 9.1, 9.3 (2 m, 1H, CHO).
--.sup.13C NMR (50.3 MHz, CDCl.sub.3) (signals were splitted for
amidic isomerism): .delta. =200.3, 171.4, 154.1, 136.2, 128.4,
128.2 128.0, 127.8, 127.7, 81.3, 67.1, 66.9, 60.5, 60.1, 53.4,
52.5, 49.0, 48.4, 29.5, 28.6, 28.3, 27.8, 27.7, 27.3.
[0250] N-Boc-protected enamide (44): The mixture of aldehydes 14
and 17 was reacted following the general procedure A. The crude
product was purified by flash chromatography (hexane/ethyl acetate
7:3), affording the enamide in 99% yield, as a trans:cis, Z/E
mixture. Trans-Z-isomer : [.alpha.].sub.D.sup.22=-61.84 (c=1.01,
CHCl.sub.3). --.sup.1H NMR (200 MHz, CDCl.sub.3) (signals were
splitted for amidic isomerism): .delta. =1.35-1.50 [2 s, 9 H,
C(CH.sub.3).sub.3], 1.6-2.3 (m, 4 H, CH.sub.2--CH.sub.2), 2.3-2.8
(2 m, 2 H, .dbd.CH--CH.sub.2), 3.75 (s, 3 H, COOCH.sub.3),
4,15-4.25 (2 m, 2 H, --CH.sub.2--CH--N and N--CH--COOtBu), 5.15 (m,
4 H, CH.sub.2Ph), 6.55 (t, J=8.5 Hz, 1 H, .dbd.CH), 7.35 (m, 10 H,
aromatic). --.sup.13C NMR (50.3 MHz, CDCl.sub.3) (signals were
splitted for amidic isomerism): .delta. =171.4, 164.8, 164.6,
154.4, 153.9, 153.7, 136.4, 136.2, 135.9, 135.7, 133.0, 132.0,
128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.6, 126.7,
81.2, 67.3, 67.2, 67.0, 66.8, 60.6, 60.2, 57.6, 56.7, 52.3, 33.5,
32.5, 28.5, 27.7, 27.4.-FAB.sup.+MS: calcd. for
C.sub.30H.sub.36N.sub.2O.sub.8 552.6, found 552.--
[0251] Trans-E-isomer: [.alpha.].sub.D.sup.22=-50.16 (c=1.48,
CHCl.sub.3). --.sup.1H NMR (200 MHz, CDCl.sub.3) (signals were
splitted for amidic isomerism): .delta. =1.35-1.45 [2 s, 9 H,
C(CH.sub.3).sub.3] 1.6-2.4 (m, 4 H, CH.sub.2--CH.sub.2), 2.7-3.1 (2
m, 2 H, .dbd.CH--CH.sub.2), 3.8 (2 s, 3 H, COOCH.sub.3), 4,1-4.3 (2
m, 2 H, --CH.sub.2--CH--N e N--CH--COOtBu), 5.10 (m, 4 H,
CH.sub.2Ph), 6.50 (m, 1 H, .dbd.CH), 7.25 (m, 10 H, aromatic).
--.sup.13C NMR (50.3 MHz, CDCl.sub.3) (signals were splitted for
amidic isomerism): .delta. =171.7, 171.5, 164.2, 164.0, 154.7,
154.3, 153.5, 136.6, 136.4, 135.8, 128.4, 128.3, 128.2, 128.1,
127.7, 126.2, 125.9, 125.8, 81.0, 87.1, 66.8, 66.6, 60.8, 60.4,
58.2, 57.3, 52.2, 32.8, 31.9, 28.5, 28.1, 27.8, 27.7, 27.4,
27.1.
[0252] The mixture of enamides (0.394 g, 0.71 mmol) was reacted
following the general procedure B. Flash chromatography of the
crude product (hexane/ethyl acetate 75:25) afforded 0.287 g (73%)
of pure trans-isomer 23.--Z-isomer: [.alpha.].sub.D.sup.22=-50.98
(c=1.56, CHCl.sub.3). --.sup.1H NMR (200 MHz, CDCl.sub.3) (signals
were splitted for amidic isomerism): .delta. =1.3-1.5 [4 s, 18 H,
C(CH.sub.3).sub.3], 1.7-2.6 (m, 6 H, CH.sub.2--CH.sub.2 and
.dbd.CH--CH.sub.2), 3.7 (s, 3 H, COOCH.sub.3), 4,1-4.3 (m, 2 H,
--CH.sub.2--CH--N and N--CH--COOtBu), 5.15 (m, 4 H, CH.sub.2Ph),
6.8 (m, 1 H, .dbd.CH), 7.30 (m 10 H, aromatic). --.sup.13C NMR
(50.3 MHz, CDCl.sub.3) (signals were splitted for amidic
isomerism): .delta. =171.4, 163.9, 154.6, 154.5, 150.0, 146.2,
138.5, 138.0, 136.2, 129.9, 128.3, 128.2, 128.1, 127.8, 83.4, 81.2,
68.3, 67.0, 66.8, 60.6, 60.2, 56.9, 56.2, 52.2, 32.9, 32.0, 28.3,
27.8, 27.7, 27.3.-FAB.sup.+MS: calcd. for
C.sub.35H.sub.44N.sub.2O.sub.10 652.7, found 652. --
[0253] E-isomer: .sup.1H NMR (200 MHz, CDCl.sub.3): .delta.
=1.3-1.4 [2 s, 18 H, C(CH.sub.3).sub.3], 1.5-2.3 (m, 4 H,
CH.sub.2--CH.sub.2), 3.0 (2 m, 2 H, .dbd.CH--CH.sub.2), 3.65 (2 s,
3 H, COOCH.sub.3), 4,2 (m, 2 H, --CH.sub.2--CH--N and
N--CH--COOtBu), S.FS(m, 4 H, CH.sub.2Ph), 6.1 (2 t, J=8.5 Hz, 1 H,
.dbd.CH), 7.30 (m, 10 H, aromatic). --.sup.13C NMR (50.3 MHz,
CDCl.sub.3): .delta. 171.5, 163.7, 154.6, 154.3, 152.2, 150.4,
142.7, 142.2, 136.3, 135.1, 128.9, 128.3, 128.2, 128.0, 127.8,
127.7, 83.4, 83.3, 81.1, 77.1, 68.3, 66.9, 66.7, 60.7, 60.3, 57.6,
56.8, 51.7, 32.9, 32.0, 28.4, 28.0, 27.7, 27.3, 27.0.
EXAMPLE 11
[0254] 6,5 fused bicyclic lactams (5a, 11a):
[0255] A solution of 44 (0.489 g, 0.75 mmol) and Pd(OH).sub.2/C 20%
(catalytic) in MeOH (7.5 mL) was stirred under H.sub.2 for one
night. The catalyst was filtered off and the mixture was refluxed
for 24 h. The solvent was then removed and the two
diastereoisomeric products were separated by flash chromatography
(hexane/ethyl acetate 6:4), yielding 0.186 g of 5a and 11a (70%) in
a 1.4:1 diastereoisomeric ratio. --5a: .sup.1H NMR (200 MHz,
CDCl.sub.3): .delta. =1.45-1.50 [2 s, 18 H, C(CH.sub.3).sub.3],
1.55-2.60 (m, 8 H, CH.sub.2--CH.sub.2 and
BocN--CH--CH.sub.2--CH.sub.2), 3.68 [tt, J=14.9 Hz and 4.2 Hz, 1 H,
(R).sub.2CH--N], 4.05 (m, 1 H, CH--NBoc), 4.35 (t, J=8.5 Hz, 1H,
N--CH--COOtBu), 5.28 (broad, 1 H, NH). -FAB.sup.+MS: calcd. for
C.sub.18H.sub.32N.sub.2O.sub.5 354.46, found 354. --
[0256] 11a: [.alpha.].sub.D.sup.22=-107.9 (c=1.7, CHCl.sub.3).
--.sup.1H NMR (200 MHz, CDCl.sub.3): .delta. =1.45-1.50 [2 s, 18 H,
C(CH.sub.3).sub.3], 1.75-2.50 (m, 8 H, CH.sub.2--CH.sub.2 and
BocN--CH--CH.sub.2--CH.sub.2), 3.70 [m, 1 H, CH--N], 4.15 (m, 1 H,
CH--NBoc), 4.50 (t, J=7.0 Hz, 1H, N--CH--COOtBu), 5.55 (broad, 1 H,
NH). --13C NMR (50.3 MHz, CDCl.sub.3): .delta. =170.6, 168.5,
155.5, 81.4, 79.3, 59.0, 56.2, 49.9, 32.3, 28.1, 27.8, 26.5,
25.9.-FAB.sup.+MS: calcd. for C.sub.18H.sub.32N.sub.2O.sub.5
354.46, found 354.
[0257] Aldehyde (18):
[0258] The general procedure C was followed using 43 and the crude
residue was purified by flash chromatography affording the alcohol
with a yield of 98%. --.sup.1H NMR (200 MHz, CDCl.sub.3) .delta.
=1.32 [s, 9 H, C(CH.sub.3).sub.3], 1.4-2.4 (m, 8 H,
CH.sub.2--CH.sub.2), 3.5-3.7 (m, 2 H, CH.sub.2OH), 4.1 (m, 1 H,
CH.sub.2--CH--N), 4.24 (m, 1 H, N--CH--COOtBu), 5.05 (s, 2 H,
CH.sub.2Ph), 7.25 (m, 5 H, aromatic).
[0259] The general procedure D was followed using the alcohol and
the crude was purified by flash chromatography (hexane/ethyl
acetate 6:4) affording 18 with a yield of 82% --.sup.1H NMR (200
MHz, CDCl.sub.3), (signals were splitted for amidic isomerism):
.delta. =1.32, 1.45 [2 s, 9 H, C(CH.sub.3).sub.3], 1.5-2.7 (m, 8 H,
CH.sub.2--CH.sub.2), 4.1 (m, 1 H, CH.sub.2--CH--N), 4.25 (m, 1 H,
N--CH--COOR), 5.15 (s, 2 H, CH.sub.2Ph), 7.20-7.40 (m, H,
aromatic), 9.6-9.8 (2 m, 1 H, CHO).
[0260] Enamide (46):
[0261] The general procedure A was followed using 18 and the crude
was purified by flash chromatography (hexane/ethyl acetate 6:4)
affording the enamide with a yield of 90% (diastereomeric ratio
Z/E=7:1) --1H NMR (200 MHz, CDCl.sub.3), (signals were splitted for
amidic isomerism): .delta. =1.32, 1.42 [s, 9 H, C(CH.sub.3).sub.3],
1.5-2.7 (m, 8 H, CH.sub.2--CH.sub.2), 3.71 (s, 1 H, COOCH.sub.3),
4.1 (m, 1 H, CH.sub.2--CH--N), 4.22 (m, 1 H, N--CH--COOtBu),
5.0-5.20 (m, 4 H, CH.sub.2Ph), 6.6 (m, 1 H, .dbd.CH, 7.20-7.45 (m,
10 H, aromatic).
[0262] The general procedure B was followed using the enamide and
the crude residue was purified by flash chromatography yielding 46
(98%). --.sup.1H NMR (200 MHz, CDCl.sub.3), (signals were splitted
for amidic isomerism): .delta. =1.32, 1.42 [2 s, 18 H,
C(CH.sub.3).sub.3], 1.5-2.2 (m, 8 H, CH.sub.2--CH.sub.2), 3.71 (s,
1 H, COOCH.sub.3), 3.9 (m, 1 H, CH.sub.2--CH--N), 4.22 (m, 1 H,
N--CH--COOtBu), 5.0-5.20 (m, 4 H, CH.sub.2Ph), 6.9 (m, 1 H,
.dbd.CH), 7.20-7.45 (m, 10 H, aromatic). --.sup.13C NMR (50.3 MHz,
CDCl3) (signals were splitted for amidic isomerism): .delta.
=141.6, 128.4, 128.2, 128.1, 127.8, 127.7, 68.2, 66.8, 60.5, 58.1,
52.1, 31.3, 29.5, 27.1, 27.3, 24.6.
EXAMPLE 12
[0263] trans-7,5-fused Bicyclic Lactam (6a, 12a):
[0264] To a solution of 46 (0.093 g, 0.141 mmol) in MeOH (2 ml) was
added 1N NaOH (0.705 mmol, 0.705 ml) and stirred for 1.5 h. The
solution was acidified until pH 3 with IN HCl, then the solution
was evaporated. The crude was submitted to the next reaction
without further purification. --.sup.1H NMR (200 MHz, CDCl.sub.3)
(signals were splitted for amidic isomerism): .delta. =1.25, 1.48
[2 s, 18 H, C(CH.sub.3).sub.3], 1.5-2.4 (m, 8 H,
CH.sub.2--CH.sub.2), 4.1 (m, 1 H, CH.sub.2--CH--N), 4.3 (m, 1 H,
N--CH--COOtBu), 5.12 (s, 2 H, CH.sub.2Ph), 6.65 (m, 1 H, .dbd.C),
7.1-7.4 (m, 5 H, aromatic), 9.00 (bs, 1 H, .dbd.COOH).
[0265] A solution of previous compound in xylene was refluxed for
48 h. The solvent was evaporated and the crude was purified by
flash chromatography yielding 6a and 12a with a 40% of yield.
[0266] 6a --.sup.1H NMR (200 MHz, CDCl.sub.3) (signals were
splitted for amidic isomerism): .delta. =1.43, 1.45 [2 s, 18 H,
C(CH.sub.3).sub.3], 1.51-2.40 (m, 10 H, CH.sub.2--CH.sub.2), 3.75
[m, 1 H, CH--N], 4.22 (m, 1 H, CH--NBoc), 4.48 (t, J=17 Hz, 1H,
N--CH--COOtBu), 5.7 (broad, 1 H, NH).
[0267] 12a --.sup.1H NMR (200 MHz, CDCl.sub.3) (signals were
splitted for amidic isomerism): .delta. =1.47, 1.48 [2 s, 18 H,
C(CH.sub.3).sub.3], 1.55-2.50 (m, 8 H, CH.sub.2--CH.sub.2), 4.0 (m,
1 H, CH--N), 4.30 (m, 1 H, CH--NBoc), 4.50 (dd, J=5.4 Hz, J=17 Hz,
1H, N--CH--COOtBu), 6.0 (bd, 1 H, NH).
EXAMPLE 13
[0268] Using the bicyclic lactams prepared according to the
preceding examples, the respective peptidomimetics compounds,
containing the RGD sequence were prepared according to the method
disclosed in Gennari et al.: Eur. J. Org. Chem., 1999, 379-388.
[0269] Examples 14-47 may be read easier by making reference to
FIGS. 9-12.
EXAMPLE 14
[0270] Reagents and solvents: Sasrin resin (200-400 mesh, 1.02
mmol/g) was purchased from Bachem. All the solvents used for the
solid-phase synthesis were of HPLC quality or Analyticai Reagent
grade and were dried over molecular sieves before use. Flash
chromatography: silica gel (Kieselgel 60, 230-400 mesh). TLC:
silica plates (60 F.sub.254, 0.25 mm, Merck). NMR: Bruker AC-200,
AC-300 and Avance-400 (200 MHz, 300 MHz and 400 MHz for .sup.1H,
50.3 MHz, 75.4 MHz and 100.5 MHz for .sup.13C). Optical rotations:
Perkin Elmer 241 polarimeter. Mass spectrometry: VG 7070 EQ-HF and
PE-SCIEX API-100. Elemental analysis: Perkin Elmer 240. All
solid-phase reaction were carried out on a wrist shaker.
[0271] Abbreviations: DCM: dichloromethane, DIC:
N,N'-diisopropylcarbodiim- ide, HOAt 1-hydroxy-7-azabenzotriazole,
HOBt: 1-hydroxybenzotriazole, HATU:
0-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate, TNBS: 2,4,6-trinitrobenzenesulfonic acid.
[0272] TNBS test was performed following this procedure: a few
resin beads were sampled and washed several times with ethanol. The
sample was then placed in a vial and 1 drop of a 10% solution of
DEPEA in DMF and 1 drop of 1% 2,4,6-trinitrobenzenesulfonic acid
(TNBS) in DMF were added. The sample was then observed and colour
changes were noted. The TNBS test is considered to be positive
(presence of free amino groups) when the resin beads turn orange or
red within 1 min and negative (no free amino groups) when the beads
remain colourless.
[0273] General Procedure 1.
[0274] Preparation of N-Fmoc-Temp-OH (Templ-8). To a solution of
the starting N-Boc-Temp-OtBu (0.62 mmol) in dichloromethane (4.8
ml) was added, under N.sub.2, trifluoroacetic acid (4.8 ml) and the
resulting mixture was stirred at room temperature for 1 h. The
solvents were then evaporated under reduced pressure, the crude
residue was dissolved in THF (0.32 ml) and 10% Na.sub.2CO.sub.3
(0.77 ml) was added. After 15 min the solution was cooled to
0.degree. C., a solution of Fmoc-ONSu (95 mg) in THF (1.4 ml) was
added and the resulting mixture was stirred at room temperature for
3 h (TLC CHCl.sub.3/MeOH/AcOH 75:25:5). THF was then evaporated
under reduced pressure, the aqueous phase was washed with AcOEt,
conc. HCl was added to pH 3-4 and the solution extracted with AcOEt
(3.times.5 ml). The combined organic layers were dried with
Na.sub.2SO.sub.4 and evaporated under reduced pressure to afford
the crude product as a white fbam, which was used without further
purification.
EXAMPLE 15
[0275]
(3S,6S,9S)-1-aza-9-carboxy-3-(9'-fluorenylmetohxycarbonylamino)-2-o-
xo-bicyclo [4.3.0] nonane (Temp 1).
[0276] Was prepared in quantitative overall yield following general
procedure 1. .sup.1H NMR (200 MHz, C.sub.6D.sub.6, 3 23.degree. K)
.delta. =1.1-2.0 (m, 8 H, 4 CH.sub.2), 2.9 (m, 1 H, CH--N), 4.12
(dd, J.sub.1=J.sub.2=6.5 Hz, 1 H, CH--CH.sub.2O), 4.20-4.50 (m, 4
H, CH--NHFmoc, CHCO2H, CH.sub.2O) 6.30 (d, J=7 Hz, 1 H, NE),
7.10-7.30 (m, 4 H, aromatic), 7.45-7.65 (m, 4 H, aromatic), 11.2
(bs, 1 H, CO.sub.2H).
[0277] .sup.13C NMR (50.3 MHz, CDCl3) .delta. =26.5, 26.9, 28.8,
31.9, 47.0, 50.2, 56.9, 58.5, 67.1, 119.8, 125.2, 127.1, 127.6,
141.2, 143.7, 143.9, 156.6, 170.3, 173.2, 174.0.
[0278] MS (FAB.sup.+): 421 (M.sup.+1).
EXAMPLE 16
[0279]
(3R,6S,9S)-1-aza-9-carboxy-3-(9'-fluorenylmethoxycarbonylamino)-2-o-
xo-bicyclo [4.3.0]nonane (Temp2).
[0280] Was prepared in quantitative overall yield following general
procedure 1. .sup.1HNMR--(200 MHz, C.sub.6D.sub.6, 323.degree. K)
.delta. =1.1-2.0 (m, 8 H, 4 CH2), 3.0 (m, 1 H, CH--N), 3.9 (m, 1 H,
CH--NHFmoc), 4.12 (dd, J, =J2=6.5 Hz, 1 H, CH--CH.sub.2O),
4.25-4.55 (m, 3 H, CHCO2H, CH2O)), 6.02 (bs, 1 H, Nh), 7.10-7.25
(m, 4 H, aromatic), 7.45-7.60 (m, 4 H, aromatic), 7.70 (bs, 1 H
Co.sub.2H). .sup.13C NMR (50.3 MHz, CDCl3) .delta. =27.7, 27.8,
28.1, 31.3, 47.0, 51.8, 58.6, 60.5, 67.0, 119.8, 125.1, 127.0,
127.6, 141.1, 143.8, 156.5, 170.0, 173.2, 174.3.
[0281] MS (FAB+): 421 (M+1).
EXAMPLE 17
[0282] (3S, 6R,
9S)-1-aza-9-carboxy-3-(9'-fluorenylmethoxycarbonylamino)-2- -oxo
-bicyclo [4.3.0]nonane (Temp3).
[0283] Was prepared in quantitative overall yield following general
procedure 1. .sup.1H-NMR (200 MHz, C.sub.6D.sub.6, 323.degree. K)
632 1.1-2.0 (m, 8 H, 4 CH.sub.2), 3.0-3.2 (m, 1 H, CH--N), 4.20
(dd, J.sub.1=J.sub.2=7 Hz, 1 H, CH--CH.sub.2o), 4.25-4.50 (m, 4 H,
CH--NHFmoc, CHCO.sub.2H, CH.sub.2o), 6.43 (d, J=1=7 Hz, 1 H, Nh),
7.10-7.30 (m, 4 H, aromatic), 7.40-7.80 (m, 4 H, aromatic), 10.80
(bs, 1 H, Co.sub.2h). .sup.13C-NMR (50.3 MHz, CDCl3) .delta. =27.3,
27.4, 28.0, 32.5, 47.1, 51.9, 58.9, 60.2, 67.1, 119.8, 125.2,
127.0, 127.6, 141.2, 143.8, 143.9, 156.8, 169.5, 173.8. MS
(FAB.sup.+): 421 (M+1).
EXAMPLE 18
[0284]
(3R,6R,9S)-1-aza-9-carboxy-3-(9'-fluorenylmethoxycarbonylamino)-2-o-
xo-bicyclo[4.3.0]nonane (Temp4).
[0285] Was prepared in quantitative overall vield following general
procedure 1. .sup.1H-NMR (200 MHz, C.sub.6D.sub.6, 323.degree. K)
.delta. =0.8-1.9 (m, 8 H, 4 CH.sub.2), 3.15 (m, 1 H, CH--N), 4.10
(dd, J.sub.1=J.sub.2=6 Hz, 1 H, CH--CH.sub.2O), 4.30-4.60 (m, 4 H,
CH--NHFmoc, CHCO.sub.2H, CH.sub.20), 6.20 (bs, 1 H, NH), 7.10-7.30
(m, 4 H, aromatic), 7.50-7.60 (m, 4 H aromatic), 9.80 (bs, 1 H,
CO.sub.2H). .sup.13C-NMR (50.3 MHz, CDCl.sub.3) 5=25.3, 26.6, 27.5,
32.1, 46.9, 49.9, 57.7, 58.8, 67.2, 119.8, 125.1, 127.0, 127.6,
141.1, 143.7, 143.8, 156.6, 173.1, 174.2. MS (FAB.sup.+): 421
(M+1).
EXAMPLE 19
[0286]
(3S,7S,1OS)-1-aza-10-carboxy-3-(9'-fluorenylmethoxycarbonylamino)-2-
-oxo-bicyclo[5.3.0]decane (TempS).
[0287] Was prepared in quantitative overall yield following general
procedure 1. .sup.1H-NMR (200 MHz, CDCl.sub.3) .delta. =1.2-2.4 (m,
10 H, 5 CH.sub.2), 2.72 (s, 1 H CH--CH.sub.2O), 3.90 (m, 1 H,
CH--N), 4.25 (m, 1 H, CH--CO.sub.2H), 4.4 (m, 2 H, CH.sub.2O), 4.75
(m, 1 H, CH--NHFmoc), 6.35 (d, J =5 Hz, 1 H, NH), 7.3-7.9 (m, 8 H,
aromatic), 9.6 (s, CO.sub.2H). .sup.13C-NMR (50.3 MHz, CDCl3)
.delta. =25.36, 27.15, 27.39, 31.34, 32.97, 34.00, 47.20, 54.75,
59.39, 60.67, 67.08, 119.84, 125.07, 127.02, 127.59, 141.23,
143.84, 143.92, 155.78, 172.04, 174.67. MS (FAB.sup.+): 435
(M+1).
EXAMPLE 20
[0288] (3R;7S,
10S)-1-aza-10-carboxy-3-(9'-fluorenylmethoxycarbonylamino)-- 2-oxo
-bicyclo [5.3.0] decane (Temp6).
[0289] Was prepared in quantitative overall yield following general
procedure 1. .sup.1H-NMR (200 MHz, CDCl.sub.3) .delta. =1.6-2.3 (m,
10 H, 5 CH.sub.2), 2.65 (s, 1 H, CH--CH.sub.2O), 3.98 (m, 1 H,
CH--N), 4.22 (m, 1 H, CH--CO.sub.2H), 4.5 (m, 3 H, CH--NHFmoc,
CH.sub.2O), 6.3 (bs, 1 H, NH), 7.28-7.85 (m, 8 H, aromatic), 9.6
(bs, 1 H, CO.sub.2H). .sup.13C-NMR (50.3 MHz, CDCl.sub.3) .delta.
=22.02, 25.25, 26.63, 32.96, 46.92, 58.54, 60.60, 61.76, 68.74,
119.91, 124.77, 124.82, 127.00, 127.7 1. MS (FAB.sup.+): 435
(M+1).
EXAMPLE 21
[0290]
(3R,7R,10S)-1-aza-10-carboxy-3-(9'-fluorenylmethoxycarbonylamino)-2-
-oxo-bicyclo [5.3.0] decane (Temp7).
[0291] Was prepared in quantitative overall yield following general
procedure 1. .sup.1H-NMR (200 MHz, CDCl.sub.3) 67 =1.70-2.35 (m, 10
H, 5 CH.sub.2), 2.70 (m, 1 H, CH--CH.sub.2O), 4.05 (m, 1 H, CH--N),
4.25 (m, 1 H, CH--NHFmoc), 4.40 (m, 2 H, CH.sub.2O),4.65 (m, 1 H,
CH--CO.sub.2H), 6.15 (bs, 1 H, NH), 7.10-7.80 (8 H, aromatic).
.sup.13C-NMR (50.3 MHz, CDCl.sub.3) .delta. =25.3, 26.9, 29.6,
32.1, 34.0, 47.0, 54.7, 59.4, 60.4, 67.1, 119.8, 119.9, 124.6,
125.1, 127.0, 127.5, 127.6, 131.1. MS (FAB+): 435 (M+1).
EXAMPLE 22
[0292] General Procedure 2.
[0293] Preparation of N-Fmoc-Gly-0-Sasrin Resin.
[0294] In a solid phase reaction vessel, Sasrin resin (500 mg, 0.51
mmol) was suspended in a solution of N-Fmoc-Gly-OH (455 mg, 1.53
mmol), HOBt (206 mg, 1.53 mmol), DIC (0.24 ml, 1.53 mmol) and DMAP
(19 mg, 0.15 mmol) in DMF (10 ml) for 15 h. The solution was
drained and the resin was washed with DMF (3.times.10 ml) and DCM
(3.times.10 ml). The possibly unreacted hydroxy groups present were
capped by treatment with a solution of acetic anhydride (0.096 ml,
1 mmol) and DMAP (57 mg, 0.51 mmol) in DMF (12 ml) for 2 h. The
solution was drained and the resin washed with DMF (3.times.10 ml)
and DCM (3.times.10 ml).
EXAMPLE 23
[0295] General Procedure 3.
[0296] Preparation of N-Fmoc-Arg(Pmc)-Gly-O-Sasrin Resin.
[0297] In a solid phase reaction vessel, N-Fmoc-Gly-O-Sasrin resin
(0.51 mmol) was treated with a 20% piperidine/DMF solution (10 ml,
1.times.3 min, 2.times.17 min). The solution was drained and the
resin was washed with DMF (3.times.10 ml), MeOH (2.times.10 ml) and
DCM (3.times.10 ml). The deprotection was assessed by performing a
TNBS test. N-Fmoc-Arg(Pmc)-OH (1.014 g, 1.53 mmol) and HOAt (208
mg, 1.53 mmol) were dissolved in DCM/DMF 2:1 (10 ml). At 0.degree.
C., DIC (0.24 ml, 1.53 mmol) was added dropwise to this solution.
The resulting mixture was stirred for 10 min at this temperature
and for a further 10 min at room temperature, then added to the
resin. This mixture was shaken at room temperature for 2.5 h. The
solution was drained and the resin washed with DMF (3.times.10 ml)
and DCM (3.times.10 ml). The success of the coupling was assessed
by performing a TNBS test. The unreacted amino groups possibly
present were capped by treatment with a solution of acetylimidazole
(560 mg, 5.1 mmol) in DCM (12 ml) for 2 h. The solution was drained
and the resin washed with DCM (3.times.10 ml).
EXAMPLE 24
[0298] General Procedure 4.
[0299] Preparation of N-Fmoc-Temp-Arg(Pmc)-Gly-O-Sasrin Resin.
[0300] In a solid phase reaction vessel,
N-Fmoc-Arg(Pmc)-Gly-O-Sasrin resin (0.51 mmol) was treated with a
20% piperidine/DMF solution (10 ml, 1.times.3 min, 2.times.17 min).
The solution was drained and the resin was washed with DMF
(3.times.10 ml), MeOH (2.times.10 ml) and DCM (3.times.10 ml). The
deprotection was assessed by performing a TNBS test. The resin was
suspended in a solution of N-Fmoc-Temp-OH (0.54 mmol), HATU (387
mg, 1.02 mmol), HOAt (139 mg, 1.02 mmol) and 2,4,6-collidine (0.135
ml, 1.02 mmol) in DMF/DCM 3:1 (13 ml) for 15 h. The solution was
drained and the resin was washed with DMF (3.times.10 ml), MeOH
(2.times.10 ml) and DCM (3.times.10 ml). The success of the
coupling was assessed by performing a TNBS test. The unreacted
amino groups possibly present were capped by treatment with a
solution of acetylimidazole (560 mg, 5.1 mmol) in DCM (12 ml) for 2
h. The solution was drained and the resin was washed with DCM
(3.times.10 ml).
EXAMPLE 25
[0301] General Procedure 5.
[0302] Preparation of N-Fmoc-Asp(tBu)-Temp-Arg(Pmc)-Gly-O-Sasrin
Resin.
[0303] In a solid phase reaction vessel,
N-Fmoc-Temp-Arg(Pmc)-Gly-O-Sasrin resin (0.51 mmol) was treated
with a 20% piperidine/DMF solution (10 ml, 1.times.3 min,
2.times.17 min). The solution was drained and the resin was washed
with DMF (3.times.10 ml), MeOH (2.times.10 ml) and DCM (3.times.10
Ml). The deprotection was assessed by performing a TN-BS test. The
resin was suspended in a solution of N-Fmoc-Asp(tBu)-OH (840 mg,
2.04 mmol), HATU (776 mg, 2.04 mmol), HOAt (278 mg, 2.04 mmol) and
2,4,6-collidine (0.27 ml, 2.04 mmol) in DMF/DCM 3:1 (13 ml) for 15
h. The solution was drained and the resin was washed with DMF
(3.times.10 ml), MeOH (2.times.10 ml) and DCM (3.times.10 ml). The
success of the coupling was assessed by performing a TNBS test. The
unreacted amino groups possibly present were capped by treatment
with a solution of acetylimidazole (560 mg, 5.1 mmol) in DCM (12
Ml) for 2 h. The solution was drained and the resin was washed with
DCM (3.times.10 ml).
EXAMPLE 26
[0304] General Procedure 6.
[0305] Cleavage of H.sub.2N-Asp(tBu)-Temp-Arg(Pmc)-Gly-OH (1-7)
from the Resin.
[0306] In a solid phase reaction vessel,
N-Fmoc-Asp(tBu)-Temp-Arg(Pmc)-Gly- -O-Sasrin resin (739 mg) was
treated with a 20% piperidine/DMF solution (10 ml, 1.times.3 min,
2.times.17 min). The solution was drained and the resin was washed
with DMF (3.times.10 ml), MeOH (2.times.10 ml) and DCM (3.times.10
ml). The deprotection was assessed by performing a TNBS test. The
resin was treated with 1% TFA/DCM solution (7.4 ml.times.3 min).
The filtrates were immediately neutralized with a 18% pyridine/MeOH
solution (0.89 ml). The fractions containing the product (TLC
DCM/MeOH 8:2) were combined and concentrated under reduced pressure
to yield a residue, which was purified from the pyridinium salts by
size-exclusion chromatography (AMBERLITE XAD-2 resin, H.sub.2O then
MeOH). Evaporation of the combined MeOH fractions containing the
product afforded a yellow residue which was used in the successive
reaction without further purification.
EXAMPLE 27
[0307] H.sub.2N-Asp(tBu)-Templ-Arg(Pmc)-Gly-OH (1). Was prepared
from the corresponding template in 40% overall yield following
general procedures 2-6. .sup.1H NMR (300 MHz, CD.sub.3OD) 6=1.30,
1.32 [2 s, 6 H, (CH.sub.3).sub.2C--O], 1.43 [s, 9 H
(CH.sub.3).sub.3CO], 1.70 (m, 2 H, H--.gamma., Arg), 1.79-1.98 (m,
2 H, H--C.sub.8 Arg), 1.85 (m, 2 H, CH.sub.2CH.sub.2Ar), 1.9 (m, 2
H, H--C.sub.4 Temp), 2.1 (m, 4 H, H--C.sub.5 Temp, H--C.sub.7
Temp), 2.1 (s, 3 H CH.sub.3Ar), 2.2 (m, 2 H, H--C.sub.8 Temp), 2.4
(dd, J=9, 17, 1 H, H--C.sub..beta.Asp), 2.55, 2.57 (2s, 6 H,
CH.sub.3Ar), 2.65 (m, 3 H, H--C.sub..beta.Asp, CH.sub.2CH.sub.2Ar),
3.25 (m, 2 H, H--C.sub..beta.Arg), 3.65 (m, 1 H, H--C.sub.6 Temp),
3.71 (d, J=17, 1 H, H--C, Gly), 3.75-3.95 (m, 1 H, H--C.alpha.
Asp), 3.87 (m, 1 H, H--C.alpha. Gly), 4.25 (m, 1 H, H--C.sub.3
Temp), 4.4 (dd, J=0.8, 1 H, H--C.sub.9 Temp), 4.88 (m, 1 H,
H--C.alpha. Arg). .sup.13C-NMR (50.3 MHz, CD.sub.3OD) .delta.
=12.3, 17.9, 19.0, 22.4, 24.2, 27.0, 28.4, 29.0, 30.7, 33.8, 38.1,
41.4, 42.2, 48.4. 49.2, 50.5, 52.7, 55.2, 55.8, 62.2, 62.3. MS
(FAB.sup.+): 849 (M+1).
EXAMPLE 28
[0308] H.sub.2N-Asp(tBu)-Temp2-Arg(Pmc)-Gly-OH (2).
[0309] Was prepared from the corresponding template in 40% overall
yield following general procedures 2-6. MS (FAB+): 849 (M+1).
EXAMPLE 29
[0310] H.sub.2N-Asp(tBu)-Temp3-Arg(Pmc)-Gly-OH (3). Was prepared
from the corresponding template in 55% overail yield following
general procedures 2-6. .sup.1H-NMR (300 MHz, CD.sub.3OD) .delta.
=1.30 [2 s, 6 H, (CH.sub.3).sub.2C--O], 1.46 [s, 9 H,
(CH.sub.3).sub.3CO], 1.6 (m, 2 H, H--C.sub.7 Temp), 1.70 (m, 2 H,
H--C.gamma. Arg), 1.85 (m, 2 H, CH.sub.2CH.sub.2Ar), 1.95-2.2 (m, 2
H H--C4 Temp), 2.0 (m, 2 H, H--C.beta. Arg), 2.1 (s, 3 H,
CH.sub.3Ar), 2.2 (m, 2 H, H--C.sub.5 Temp), 2.4-2.6 (m, 2 H,
H--C.beta. Asp), 2.55-2.6 (2s, 6 H, CH.sub.3Ar), 2.65 (m, 2 H,
CH.sub.2CH.sub.2Ar), 2.9 (m, 2 H, H--C.beta. Temp), 3.2 (m, 2 H,
H--C.sub.5 Arg), 3.6 (d, J=18, 1 H, H--C.alpha. Gly), 3.80 (m, 1 H,
H--C.sub.6 Temp), 4.0 (m, 1 H, H--C.sub.3 Temp), 4.05 (d, J=18, 1 H
H--C.alpha. Gly), 4.2 (dd, J=6,6, 1 H, H--C.sub.9 Temp), 4.4 (m, 1
H, H--C.alpha. Arg), 4.45 (m, 1 H, H--C.alpha. Asp). .sup.13C-NMR
(50.3 MHz, CD.sub.3OD) .delta. =12.3, 17.9, 19.0, 22.4, 27.0, 28.3,
28.6, 29.6, 30.0, 33.8, 37.0, 41.5, 43.3, 52.7, 54.2, 62.2, 74.9,
83.8, 119.4, 136.5, 169.7, 170.6, 173.9, 174.3. MS (FAB.sup.+): 849
(M+1).
EXAMPLE 30
[0311] H.sub.2N-Asp (tBu)-Temp4-Arg(Pmc)-Gly-OH (4).
[0312] Was prepared from the corresponding template in 30% overall
yield following general procedures 2-6. MS (FAB.sup.+): 850
(M+2).
EXAMPLE 31
[0313] H.sub.2N-Asp (tBu)-Temp5-Arg(Pmc)-Gly-OH (5).
[0314] Was prepared from the corresponding template in 67% overall
yield following general procedures 2-6. MS (FAB.sup.+): 863
(M+1).
EXAMPLE 32
[0315] H.sub.2N-Asp(tBu)-Temp6-Arg(Pmc)-Gly-OH (6).
[0316] Was prepared from the corresponding template in 54% overall
yield following general procedures 2-6. MS (FAB.sup.+): 863
(M+1).
EXAMPLE 33
[0317] H.sub.2N-Asp(tBu)-Temp7-Arg(Pmc)-Gly-OH (7).
[0318] Was prepared from the corresponding template in 63% overall
yield following general procedures 2-6. MS (FAB.sup.+): 863
(M+1).
EXAMPLE 34
[0319] H.sub.2N-Asp(tBu)-Temp8-Arg(Pmc)-Gly-OH (8).
[0320] Was prepared from the corresponding template in 50% overall
yield following general procedures 2-6. MS (FAB.sup.+): 863
(M+1).
EXAMPLE 35
[0321] General Procedure 7.
[0322] Preparation of Cyclo[-Temp-Arg(Pmc)-Gly-Asp(tBu)-] (9-15).
The linear peptide (0. 18 mmol) was dissolved in DMF (45 ml) under
N2. HATU (205 mg, 0.54 mmol), HOAt (73 mg, 0.54 mmol) and
2,4,6-collidine (0.072 ml, 0.54 mmol) were added and the resulting
mixture was stirred for 24 h at room temperature. The solvent was
evaporated under reduced pressure and the residue was dissolved in
AcOEt. The organic phase was washed twice with 5% NaHCO.sub.3,
dried with Na.sub.2SO.sub.4 and evaporated under reduced pressure.
The crude residue was purified by flash chromatography on silica
gel (DCM/MeOH from 95:5 to 9: 1) to afford side-chain protected
cyclopeptide as a yellow foam.
EXAMPLE 36
[0323] Cyclo [-Temp 1-Arg(Pmc)-Gly-Asp(tBu)-] (9).
[0324] Was prepared in 70% yield following general procedure 7.
.sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. =1.25, 1.3 [2 s, 6 H
(CH.sub.3).sub.2C--O], 1.46 [s, 9 H, (CH.sub.3).sub.3CO], 1.5 (m, 2
H, H--Cy Arg), 1.6 (m, 2 H, H--C.sub.4 Temp), 1.8 (m, 4 H,
CH.sub.2CH.sub.2Ar, H--C.sub.5Temp), 2.0 (m, 2 H, H--C.beta. Arg),
2.1 (s, 3 H, CH.sub.3Ar), 2.2 (m, 4 H, H--C.sub.7 Temp, H--C.sub.8
Temp), 2.52, 2.56 (2 s, 6 H, CH.sub.3Ar), 2.6 (m, 3 H,
CH.sub.2CH.sub.2Ar, H--C.beta. Asp), 3.1 (dd, J=5, 17.6, 1 H, 20
H--C.beta. Asp), 3.25 (m, 2 H, H--C.delta. Arg), 3.55 (m, 1 H,
H--C.sub.6 Temp), 3.65 (dd, J=6, 13.6, 1 H, H--C.alpha. Gly), 3.85
(dd, J=4, 13.6, 1 H, H--C.alpha. Gly), 4.22 (dd, J=0, 9, 1 H
H--C.sub.9 Temp), 4.45 (m, 1 H, H--C.sub.3 Temp), 4.54 (m, 1 H,
H--C.alpha. Arg), 4.68 (m, 1 H, H--C.alpha.Asp), 6.3 (s, 3 H,
H--N.epsilon. Arg, HNSO.sub.2, .dbd.NH), 6.8 (d, J=6, 1 H, NHTemp),
7.61 (d, J=9, 1 NHAsp), 7.8 (d, J=8, 1 H, NHArg), 8.9 (bs, 1 H,
NHGly). .sup.13C-NMR (75.4 MHz, CDCl.sub.3) .delta. =12.1, 17.4,
18.5, 21.4, 25.2, 26.2, 26.8, 27.5, 28.0, 29.6, 31.4, 32.2, 32.9,
36.4, 40.2, 46.0, 47.7, 50.3, 51.9, 60.6, 62.1, 73.5, 81.8, 117.8,
123.8, 134.8, 134.9, 135.5, 153.3, 156.5, 168.0, 169.8, 170.2,
170.9, 173.6, 175.0. [.alpha.].sub.D.sup.20=-36.1 (c=1.0,
CHCl.sub.3). MS (FAB.sup.+): 830 (M.sup.+), 853 (M+Na).
EXAMPLE 37
[0325] Cyclo [-Temp3-Arg(Pmc)-Gly-Asp(tBu)-] (10).
[0326] Was prepared in 60% yield following general procedure 7.
.sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. =1.3 [2 s, 6 H,
(CH.sub.3).sub.2C--O], 1.45 [s, 9 H, (CH.sub.3).sub.3CO], 1.5 (m, 4
H, H--C.gamma. Arg, H--C.sub.7 Temp), 1.7 (m, 1 H, H--C.beta. Arg),
1.8 (m, 3 H, CH.sub.2CH.sub.2Ar, H--C.sub.8 Temp), 1.9 (m, 1 H,
H--C.sub.4 Temp), 2.0 (m, 1 H, H--C.beta. Arg), 2.05 (s, 3 H
CH.sub.3Ar), 2.2 (m, 3 H, H--C.sub.4 Temp, H--C.sub.5 Temp), 2.50
(m, 2 H, H--C.sub.8 Asp, H--C.sub.8 Temp), 2.52, 2.56 (2 s, 6 H,
CH.sub.3Ar), 2.6 (m, 2 H, CH.sub.2CH.sub.2Ar), 2.80 (dd, J=8, 16, 1
H, H--C.beta. Asp), 3.25 (m, 2 H, H--C.delta. Arg ), 3.45 (dd, J=5,
16, 1 H, H--C.alpha. Gly), 3.80 (m, 1 H, H--C.sub.6 Temp), 4.10 (m,
1 H, H--C.sub.3 Temp), 4.15 (m, 1 H, H--C.alpha. Gly), 4.35 (dd,
J=10, 10, 1 H, H--C.sub.9 Temp), 4.5 (m, 1 H, H--C.alpha. Arg),
4.70 (m, 1 H, H--C.alpha. Asp), 6.22 (bs, 3 H, H-N" Arg,
HNSO.sub.2, .dbd.NH, 7.1 (d, J=8, 1 H, NH Arg), 7.20 (d, J=8, 1 H,
NHAsp), 7.70 (d, J =8, 1 H, NH Temp), 8.1 (bs, 1 H, NH Gly).
.sup.13C NMR (50.3 MHz, CDCl.sub.3) .delta. =12.0, 17.4, 19.4,
21.3, 25.3, 26.7, 27.4, 27.9, 28.6, 29.6, 32.8, 36.7, 40.4, 45.1,
50.2, 50.7, 51.9, 60.7, 61.6, 73.5, 81.6, 117.8, 123.8, 133.7,
134.7, 135.3, 153.3, 156.3, 168.7, 169.8, 170.7, 171.4, 173.3.
[.alpha.].sub.D.sup.20=-57.1 (c=1.0, CHCl.sub.3). MS (FAB.sup.+):
832 (M+2).
EXAMPLE 38
[0327] Cyclo [-Temp4-Arg(Pmc)-Gly-Asp(tBu)-] (11).
[0328] Was prepared in 40% yield following general procedure 7.
.sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. =1.3 [2 s, 6 H
(CH.sub.3).sub.2C--o] 1.4 (m, 1 H, H--C.sub.5 Temp), 1.45 [s, 9 H
(CH.sub.3).sub.3CO], 1.5-1.6 (m, 4 H, H--C.gamma. Arg, H--C.sub.4
Temp, H--C.sub.8 Temp), 1.8 (m, 2 H, CH.sub.2CH2Ar), 1.97 (m, 1 H,
H--C.sub.8 Temp), 2.0 (m, 4 H, H--C.beta. Arg, H--C.sub.4 Temp,
H--C.sub.5 Temp), 2.15 (s, 3 H, CH.sub.3Ar), 2.17, 2.43 (m, 2 H,
H--C.sub.7 Temp), 2.5 (m, 1 H, H--C.beta. Asp), 2.60 (s, 3 H,
CH.sub.3Ar), 2.60 (m, 2 H, CH.sub.2CH.sub.2Ar), 2.62 (s, 3 H,
CH.sub.3Ar), 2.9 (dd, J=7, 17, 1 H, H--C.beta. Asp), 3.2 (m, 2 H,
H--C.delta. Arg), 3.55 (dd, J=0, 12, 1 H H--C.alpha. Gly), 4.05 (m,
1 H, H--C.sub.9 Temp), 4.1 (m, 1 H, H--C.alpha. Gly), 4.2 (m, 1 H,
H--C.sub.6 Temp), 4.3 (m, 1 H, H--C.sub.3 Temp), 4.6 (m, 1 H,
H--C.alpha. Arg), 4.65 (m, 1 H, H--C.alpha. Asp), 6.2-6.4 (bs, 3 H
H--N.epsilon. Arg, HNSO.sub.2, .dbd.NH), 7.3 (bs, 1 H, NH Temp),
7.45 (bs, 1 H NHArg), 7.90 (bs, 1 H, NHGly), 8.0 (bs, 1 H NH Asp).
.sup.13C-NMR (50.3 MHz, CDCl.sub.3) .delta. =12.0, 17.4, 18.4,
21.3, 22.0, 25.6, 26.2, 26.7, 27.9, 30.1, 32.7, 34.0, 35.0, 50.9,
51.0, 51.8, 56.5, 62.5, 73.5, 81.2, 95.0, 117.8, 123.9, 133.3,
134.7, 135.3, 153.4, 156.2, 170.4, 170.7, 171.9, 172.5, 173.3.
[.alpha.].sub.D.sup.20=-- 71.0 (c=0.7, CHCl.sub.3). MS (FAB.sup.+):
832 (M+2).
EXAMPLE 39
[0329] Cyclo [-Temp5-Arg(Pmc)-Gly-Asp(tBu)-] (12).
[0330] Was prepared in 35% yield following general procedure 7.
.sup.1H-NMR (300 MHz, DMSO-D.sub.6) .delta. =1.25, 1.38 [2 s, 6 H
(CH.sub.3).sub.2CO] 1.31 (m, 2 H, H--C.gamma. Arg), 1.38 [s, 9 H,
(CH.sub.3).sub.3CO], 1.8 (m, 2 H, CH.sub.2CH.sub.2Ar), 2.05 (s, 3
H, CH.sub.3Ar), 2.05 (m, 1 H, H--C.sub.9 Temp), 2,15 (m, 2 H,
H--C.alpha. Arg), 2.35 (dd, J=6.8, 17, 1 H H--C.beta. Asp),
2.50,2,52 (2 s, 6 H, 3 CH.sub.3Ar), 2.5 (m, 1 H, H--C.sub.9 Temp),
2.6 (m, 2 H, CH.sub.2CH.sub.2Ar), 2.8 (dd, J =8.6, 17, 1 H,
H--C.beta. Asp), 3.1 (m, 2 H, H--C.delta. Arg), 3.61 (d, J=9.8, 1
H, H--C.alpha. Gly), 3.98 (d, J=9.8, 1 H, H--C.alpha. Gly), 4.0 (m,
2 H, H--C.alpha. Arg, H--C.sub.7 Temp), 4.31 (m, 2 H, H--C.sub.3
Temp, H--C.sub.10 Temp), 4.55 (m, 1 H, H--C.alpha. Asp), 6.48 (bs,
2 H, H--N.epsilon. Arg, HNSo.sub.2), 6.78 (bs, 1 H, .dbd.NH), 7.68
(d, J=5.1, 1 H, NH Temp), 7.84 (bd, 1 NH Asp), 8.22 (m, 1 H, NH
Arg), 8.5 (bt, 1 H, NH Gly). .sup.13C-NMR (50.3 MHz, DMSO-D.sub.6)
.delta. =11.9, 17.1, 18.2, 26.4, 27.7, 20.8, 25.3, 27.0, 30.6,
32.2, 32.5, 36.3, 38.7, 40.3, 42.4, 49.6, 53.1, 58.8, 62.0, 73.5,
80.3. [.alpha.].sub.D.sup.20=36.7 (c=1, CHCl.sub.3). MS
(FAB.sup.+): 844 (M+).
EXAMPLE 40
[0331] Cyclo[-Temp6-Arg(Pmc)-Gly-Asp(tBu)-] (13). Was prepared in
26% yield following general procedure 7.
[0332] .sup.1H-NMR (300 MHz, DMSO-D.sub.6) .delta. =1.3 [s, 3 H
(CH.sub.3).sub.2C--O], 1.31 (m, 2 H, H--C.gamma. Arg), 1.38 [s, 9
H, (CH.sub.3).sub.3CO], 1.4 [s, 3 H (CH.sub.3).sub.2C--O], 1.8-1.95
(m, 6 H, CH.sub.2CH.sub.2Ar, H--C.sub.9 Temp, H--C.beta. Arg), 2.05
(s, 3 H CH.sub.3Ar), 2.39 (dd, J=4.3, 10.6, 1 H, H--C.beta. Asp),
2.50, 2,52 (2 s, 6 H, 3 CH.sub.3Ar), 2.6 (m, 2 H,
CH.sub.2CH.sub.2Ar), 2.9 (dd, J=4.3, 10.6, 1 H H--C.beta. Asp), 3.1
(m, 2 H H--C.delta. Arg), 3.80 (m, 3 H, H--C.alpha. Gly,
H--C.alpha. Arg), 4.3 (m, 1 H, H--C.sub.3 Temp), 4.35 (m, 1 H,
H--C.sub.10 Temp), 4.48 (m, 1 H, H--C.alpha. Asp), 6.45 (bs, 2 H,
H--N.epsilon. Arg, HNSO.sub.2), 6.60 (bs, 1 H, .dbd.NH), 7.5 (bd, 1
H, NH Asp), 7.51 (bd, 1 H, NHTemp), 8.55 (m, 1 H, NH Arg), 8.75
(bt, 1 H, NH Gly). .sup.13C-NMR (75.4 MHz, CDCl.sub.3) .delta.
=12.1, 14.3, 17.5, 18.6, 21.4, 23.5, 26.8, 28.1, 29.7, 31.7, 32.8,
33.6, 49.7, 60.8, 73.6, 117.9, 124.0, 134.8, 135.5, 153.6, 171.5.
[.alpha.].sub.D.sup.20=-72.9 (c=1, CHCl.sub.3), MS (FAB.sup.+): 844
(M+).
EXAMPLE 41
[0333] Cyclo[-Temp7-Arg(Pmc)-Gly-Asp(tBu)-] (14). Was prepared in
15% yield following general procedure 7.
[0334] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. =1.30 [s, 6 H,
(CH.sub.3).sub.2C--O], 1.41 [s, 9 H, (CH.sub.3).sub.3CO], 1.49 (m,
1 H, H--C.gamma. Arg), 1.50-1.90 (10 H, CH.sub.2CH.sub.2Ar,
H--C.sub.5 Temp, H--C.sub.6 Temp, H--C.sub.8 Temp), 1.51 (m, 2 H,
H--C.sub.4 Temp), 1.60 (m, 1 H, H--C.gamma. Arg), 1.90 (m, 2 H,
H--C.beta. Arg), 1.98 (m, 1 H, H--C.sub.9 Temp), 2.15 (s, 3 H
CH.sub.3Ar), 2.53 (s, 3 H, CH.sub.3Ar), 2.58 (s, 3 H, CH.sub.3Ar),
2.32 (m, 1 H, H--C.sub.9 Temp), 2.51 (m. 1 H, H--C.beta. Asp), 2.85
(m, 1 H, H--C.beta. Asp), 3.20 (m, 2 H, H--C.delta. Arg), 3.51 (bd,
1 H, H--C.alpha., Gly), 4.12 (m, 1 H, H--C.sub.7 Temp), 4.18 (m, 1
H, H--C.alpha. Gly), 4.32 (m, 1 H, H--C.sub.10 Temp), 4.5 (m, 1 H,
H--C.sub.3 Temp), 4.58 (m, 1 H, H--C.alpha. Arg), 4.80 (m, 1 H,
H--C.alpha. Asp), 6.3 (bs, 3 H, H--N.epsilon.Arg, HNSO.sub.2,
.dbd.NH), 7.2 (bd, 1 H, NH Arg), 7.65 (bd, 1 H, NH Temp), 7.80 (bt,
1 H, NH Gly), 7.95 (bd, 1 H, NH Asp).
EXAMPLE 42
[0335] Cyclo[-Temp8-Arg(Pmc)-Gly-Asp(tBu)-] (15). Was prepared in
55% yield following general procedure 7. .sup.1H-NMR (400 MHz,
CDCl.sub.3) .delta. =1.27, 1.31 [2 s, 6 R (CH.sub.3).sub.2C--O],
1.44 [s, 9 H, (CH.sub.3).sub.3CO], 1.50 (m, 3 H, H--C.gamma. Arg,
H--C.sub.6 Temp), 1.60 (m, 2 H, H--C.beta. Arg, H--C.gamma. Arg),
1.70 (m, 2 H, H--C.sub.8 Temp), 1.8 (m, 2 H, CH.sub.2CH.sub.2Ar),
1.85 (m, 1 H, H--C.sub.4 Temp), 1.95 (m, 2 H, H--C.beta. Arg,
H--C.sub.4 Temp), 1.98 (m, 2 H, H--C.sub.5 Temp), 2.11 (s, 3 H,
CH.sub.3Ar), 2.32 (m, 1 H, H--C.sub.9 Temp), 2.56 (s, 3 H
CH.sub.3Ar), 2.57 (dd, J=7.4, 16.7, 1 H, H--C.beta. Asp), 2.58 (s,
3 H CH.sub.3Ar), 2.65 (m, 2 H, CH.sub.2CH.sub.2Ar), 2.87 (dd,
J=7.4, 16.7, 1 H, H--C.beta. Asp), 3.20 (m, 2 H, H--C.sub.3 Arg),
3.54 (bd, 1 H, H--C.alpha. Gly), 4.18 (m, 1 H, H--C.alpha. Gly),
4.22 (m, 1 H, H--C.sub.7 Temp), 4.36 (m, 1 H, H--C.sub.10 Temp),
4.55 (m, 1H, H--C.sub.3 Temp), 4.6 (m, 1 H, H--C.alpha. Arg), 4.83
(m, 1 H, H--C.alpha. Asp), 6.33 (bs, 3 H, H--N.epsilon. Arg,
HNSO.sub.2, .dbd.NH), 7.49 (bd, 1 H, NH Arg), 7.71 (bt, 1 H, NH
Gly), 7.80 (bd, 1 H, NH Temp), 7.95 (bd, 1 H, NH Asp). .sup.13C NMR
(50.3 MHz, CDCl.sub.3) .delta. =12.0, 17.4, 18.4, 26.7, 27.4, 36.4,
21.4, 25.3, 28.5, 29.6, 30.8, 33.0, 34.9, 40.6, 44.3, 49.9, 51.9,
54.1, 59.3, 63.2, 73.5, 81.3, 117.8, 123.9, 133.4, 134.7, 135.3,
153.5, 156.3, 170.3, 170.5, 172.3, 172.6.
[c.alpha.].sub.D.sup.20=-54 (c=0.05, CHCl.sub.3). MS (FAB.sup.+):
844 (M+).
EXAMPLE 43
[0336] General Procedure 8.
[0337] Preparation of Cyclo(-Temp-Arg-Gly-Asp-) (16-22).
[0338] Side-chain protected cyclopeptide (0.1 mmol) was treated
with TFA/thioanisole/1,2-ethanedithiol/anisole 90:53:2 (35 ml) for
2 h. The reaction mixture was evaporated under reduced pressure and
the residue was dissolved in H2O. The aqueous phase was washed
twice with iPr.sub.2O and evaporated under reduced pressure to
afford side-chain deprotected cyclopeptide as a white foam.
Trifluoroacetate ion was substituted with chloride by ion-exchange
chromatography (AMBERLITE IRA-93 resin, chloride form).
EXAMPLE 44
[0339] Cyclo(-Temp1-Arg-Gly-Asp-) (16). Was prepared in
quantitative yield following general procedure 8. .sup.1H-NMR (400
MHz, D.sub.2O) .delta. =1.6-1.75 (m, 2 H, H--C.gamma. Arg),
1.65-1.95 (m, 6 H, H--C.sub.4 Temp, H--C.sub.5 Temp, H--C.sub.7
Temp), 2.2 (m, 2 H, H--C.beta. Arg), 2.3-2.45 (m, 2 H, H--C.sub.8
Temp), 2.73 (dd, J=0, 6, 2 H, H--C.beta. Asp), 3.25-3.50 (m, 2 H,
H--C.delta. Arg), 3.8-3.95 (m, 1 H, H--C.sub.6 Temp), 3.82 (d,
J=13.5, 1 H, H--C.alpha. Gly), 4.25 (d, J=13.5, 1 H, H--C.alpha.
Gly), 4.50 (dd, J=0, 10, 1 H, H--C.sub.9 Temp), 4.55 (dd, J=0, 8, 1
H, H--C.alpha., Arg), 4.58 (m, 1 H, H--C.sub.3 Temp), 4.75 (m, 1 H,
H--C.alpha. Asp). .sup.13C NMR (75.4 MHz, D.sub.2O) 6=25.0, 26.4,
28.2, 29.8, 30.8, 32.2, 39.0, 41.3, 45.8, 48.3, 52.7, 53.1, 61.7,
62.6, 157.7, 170.1, 172.6, 174.0, 175.4, 175.7, 178.4.
[.alpha.].sub.D.sup.20=-52.6 (c=0.88, H.sub.2O). MS (FAB+: 509
(M+1)
EXAMPLE 45
[0340] Cyclo(-Temp3-Arg-Gly-Asp-) (17). Was prepared in
quantitative yield following general procedure 8. .sup.1H-NMR (400
MHz, D.sub.2O) .delta. =1.5 (m, 2 H, H--C.sub.5 Temp), 1.6 (m, 2 H,
H--C.beta. Arg), 1.8 (m, 2 H, H--C.sub.4 Temp), 1.9 (m, 2 H,
H--C.gamma. Arg), 2.2 (m, 2 H, H--C.sub.7 Temp), 2.42-2.52 (m, 2 H,
H--C.sub.8 Temp), 2.7-2.85 (m, 2 H, H--C.beta. Asp), 3.15-3.30 (m,
2 H, H--C.delta. Arg), 3.55 (d, J=14, 1 H, H--C.alpha. Gly),
3.83-3.95 (m, 1 H, H--C.sub.6 Temp), 4.10 (d, J=14, 1 H,
H--C.alpha. Gly), 4.28 (m, 1 H, H--C.sub.3 Temp), 4.37 (dd, J=0, 9,
1 H, H--C.sub.8 Temp), 4.45 (dd, J=5, 10, 1 H, H--C.alpha. Arg),
4.65 (m, 1 H, H--C.alpha. Asp). .sup.13C-NMR (50.3 MHz, D.sub.2O)
.delta. =27.1, 29.3, 30.4, 31.4, 31.8, 35.1, 38.1, 43.3, 47.2,
52.8, 53.5, 54.8, 64.0, 64.5, 159.6, 166.1, 172.5, 174.8, 175.2,
176.4, 176.6, 177.9. [.alpha.].sub.D.sup.20=-94.6 (c=1.32,
H.sub.2O) MS (IS.sup.+): 508 (M+).
EXAMPLE 46
[0341] Cyclo(-Temp4-Arg-Gly-Asp-) (18). Was prepared in
quantitative yield following general procedure 8. This compound was
not stable in aqueous solution over a few days at room temperature.
.sup.1H-NMR (400 MHz, D.sub.2O) .delta. =1.6-1.7 (m, 2 H,
H--C.sub.4 Temp), 1.7 (m, 2 H, H--C.gamma. Arg), 2.0 (m, 2 H,
H--C.sub.7 Temp), 2.2 (m, 2 H, H--C.beta. Arg), 2.4 (m, 2 H,
H--C.sub.5 Temp), 2.6 (m, 2 H, H--C.sub.8 Temp), 2.70 (dd, J=7, 17,
1 H, H--C.beta. Asp), 3.05 (dd, J=7, 17, 1 H, H--C.beta. Asp),
3.15-3.25 (m, 2 H, H--C.sub.8 Arg), 3.52 (d, J=15, 1 H, H--C.alpha.
Gly), 4.05 (m, 1 H, H--C.sub.6 Temp), 4.28 (d, J=15, 1 H,
H--C.alpha. Gly), 4.3 (m, 1 H, H--C.sub.3 Temp), 4.35 (m, 1 H,
H--C.sub.10 Temp), 4.53 (dd, J=7, 7, H--C.alpha. Asp), 4.6 (m, 1H,
H--C.alpha. Arg). [.alpha.].sub.D.sup.20=-63.7 (c=0.95, H.sub.2O).
MS (IS.sup.+): 508 (M+).
EXAMPLE 47
[0342] Cyclo(-Temp5-Arg-GIy-Asp-) (19). Was prepared in
quantitative yield following general procedure 8. .sup.1H-NMR (400
MHz, D.sub.2O) .delta. =1.5-1.8 (m, 2 H, H--C.gamma. Arg), 1.7-2.0
(m, 2 H, H--C.beta. Arg), 2.8 (m, 2 H, H--C.beta. Asp), 3.22 (m, 2
H, H--C.delta. Arg), 4. 0 (m, 2 H, H--C.alpha. Gly, H--C.sub.7
Temp), 4.3 (dd, J=7, 7, 1 H, H--C.alpha. Arg), 4.5-4.6 (m, 1 H,
H--C.sub.3 Temp, H--C.sub.10 Temp), 4.68 (m, 1 H H--C.alpha. Asp).
.sup.13C-NMR (50.3 MHz, D.sub.2O) .delta. =27.2, 29.8, 30.1, 31.0,
33.6, 35.3, 36.2, 39.0, 43.3, 45.7, 53.8, 56.3, 62.8, 65.2, 159.5,
174.3, 174.4, 175.6, 176.4, 178.5. [.alpha.].sub.D.sup.20=-87.4
(c=1.2, H.sub.2O). MS (IS.sup.+): 522 (M+).
EXAMPLE 48
[0343] Cyclo(-Temp6-Arg-Gly-Asp-) (20). Was prepared in
quantitative yield following general procedure 8. .sup.1H-NMR (400
MHz, D.sub.2O) .delta. =1,5-1.8 (m, 2 H, H--C.sub.6 Temp), 1.6 (m,
2 H, H--C.gamma. Arg), 1.75-1.9 (m, 2 H, H--C .beta. Arg), 1.8-1.95
(m, 2 H, H--C.sub.4 Temp), 2.15 (m, 4 H, H--C.sub.8 Temp,
H--C.sub.9 Temp), 2.65-2.8 (m, 2 H, H--C.beta. Asp), 3.2 (m, 2 H,
H--C.delta. Arg), 3.82 (d, J=17, 1 H, H--C.alpha. Gly), 4.05 (d,
J=17, 1 H H--C.alpha. Gly), 4.1 (m, 1 H, H--C.sub.7 Temp), 4.37
(dd, J=0, 7, 1 H, H--C.sub.10 Temp), 4.42 (dd, J=0, 10, 1 H,
H--C.sub.3 Temp), 4.52 (dd, J=5, 10, 1 H, H--C.alpha. Arg), 4.70
(m, 1 H, H--C.alpha. Asp). .sup.13C-NMR (75.4 MHz, D2O) .delta.
=22.3, 25.0, 25.9, 28.7, 30.4, 33.7, 34.2, 37.7, 41.4, 43.2, 51.5,
53.3, 57.5, 59.1, 63.6, 157.6, 171.6, 173.7, 174.2, 175.0, 176.9.
[.alpha.].sub.D.sup.20=-47.9 (c=0.71, H.sub.2O). MS (IS.sup.+): 522
(M+).
EXAMPLE 49
[0344] Cyclo(-Temp8-Arg-Gly-Asp-) (22). Was prepared in
quantitative yield following general procedure 8. .sup.1H-NMR (400
MHz, D.sub.2O) .delta. =1.4 (m, 3 H, H--C.sub.5 Temp, H--C.sub.8
Temp), 1.55-1.7 (m, 2H, H--C.gamma. Arg), 1.8 (m, 4 H, H--C.sub.4
Temp, H--C.sub.8 Temp, H--C.sub.9 Temp), 2.0 (m, 2 H, H--C.beta.
Arg), 2.26 (m, 2 H, H--C.sub.6 Temp), 2.38 (m, 1 H, H--C.sub.9
Temp), 2.68 (dd, J=7, 18, 1 H, H--C.alpha. Asp), 2.98 (dd, J=7,18,
1 H, H--C.alpha. Asp), 3.2 (m, 2 H, H--C.gamma. Arg), 3.5 (d,
J=15,1 H, H--C.alpha. Gly), 4.2 (d, J=15, 1 H, H--C.alpha. Gly),
4.2 (m, 1 H, H--C.sub.7 Temp), 4.38 (m, 1 H, H--C.sub.10 Temp),
4.48 (dd, J=5, 11, 1 H, H--C.alpha. Arg), 4.53 (dd, J=0, 11, 1 H,
H--C.sub.3 Temp), 4.63 (dd, J=7, 7, 2 H, H--C.beta. Asp).
.sup.13C-NMR (75.4 MHz, D.sub.2O) .delta. =27.4, 29.3, 30.2, 30.6,
33.0, 35.1, 36.2, 41.3, 46.1, 53.8, 54.9, 56.8, 62.8, 66.1, 159.5,
174.2, 174.8, 175.9, 176.4, 177.6. [.alpha.]
[0345] D.sup.20=-38.1 (c=1.2, H.sub.2O). MS (IS.sup.+): 522
(M+).
* * * * *