U.S. patent application number 12/491839 was filed with the patent office on 2009-10-22 for hemiasterlin analogs.
This patent application is currently assigned to THE UNIVERSITY OF BRITISH COLUMBIA. Invention is credited to Raymond ANDERSEN, John Coleman, James Nieman, Edward Piers, Michel Roberge.
Application Number | 20090264487 12/491839 |
Document ID | / |
Family ID | 4161925 |
Filed Date | 2009-10-22 |
United States Patent
Application |
20090264487 |
Kind Code |
A1 |
ANDERSEN; Raymond ; et
al. |
October 22, 2009 |
HEMIASTERLIN ANALOGS
Abstract
This invention provides analogs of hemiasterlin, methods of
synthesis of the analogs and use of the analogs as a cytotoxic
anti-mitotic agents.
Inventors: |
ANDERSEN; Raymond;
(Vancouver, CA) ; Piers; Edward; (Vancouver,
CA) ; Nieman; James; (Kalamazoo, MI) ;
Coleman; John; (Halifax, CA) ; Roberge; Michel;
(Vancouver, CA) |
Correspondence
Address: |
ARENT FOX LLP
1050 CONNECTICUT AVENUE, N.W., SUITE 400
WASHINGTON
DC
20036
US
|
Assignee: |
THE UNIVERSITY OF BRITISH
COLUMBIA
Vancouver
CA
|
Family ID: |
4161925 |
Appl. No.: |
12/491839 |
Filed: |
June 25, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09581511 |
Oct 6, 2000 |
7579323 |
|
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PCT/CA1998/001184 |
Dec 18, 1998 |
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12491839 |
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Current U.S.
Class: |
514/376 ;
435/375; 514/533; 514/563; 548/229; 560/41; 562/442 |
Current CPC
Class: |
C07K 5/0205 20130101;
A61P 35/00 20180101; C07C 271/22 20130101; A61K 38/00 20130101;
Y02P 20/55 20151101 |
Class at
Publication: |
514/376 ;
435/375; 514/533; 514/563; 548/229; 560/41; 562/442 |
International
Class: |
A61K 31/421 20060101
A61K031/421; C12N 5/00 20060101 C12N005/00; A61K 31/216 20060101
A61K031/216; A61K 31/195 20060101 A61K031/195; C07D 263/04 20060101
C07D263/04; C07C 229/34 20060101 C07C229/34; A61P 35/00 20060101
A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 19, 1997 |
CA |
2225325 |
Claims
1. A compound or pharmaceutically acceptable salt thereof, having
the formula: ##STR00036## wherein: R.sub.1 and R.sub.2 are
independently selected from the group consisting of: H, R, and
ArR--, and where at least one of R.sub.1 and R.sub.2 is R the other
is not ArR--; R.sub.1 and R.sub.2 together may optionally be a
three to seven member non-aromatic ring; R.sub.3 and R.sub.4 are
independently selected from the group consisting of: H, R, and
ArR--, and where at least one of R.sub.3 and R.sub.4 is R the other
is not ArR, R.sub.3 and R.sub.4 together may optionally be a three
to seven member non-aromatic ring; R.sub.5 is selected from the
group consisting of: H, R, ArR--, and Ar; R.sub.6 is selected from
the group consisting of: H, R, and ArR--; R.sub.7 and R.sub.8 are
independently selected from the group consisting of: H, R, and
ArR--; and R.sub.9 is: ##STR00037## and wherein, R is defined as a
saturated or unsaturated moiety having a linear, branched, or
non-aromatic cyclic skeleton containing one to ten carbon atoms,
zero to four nitrogen atoms, zero to four oxygen atoms, and zero to
four sulfur atoms, and the carbon atoms are optionally substituted
with: .dbd.O, .dbd.S, --OH, --OR.sub.10, --O.sub.2CR.sub.10, --SH,
--SR.sub.10, --SOCR.sub.10, --NH.sub.2, --NHR.sub.10,
--N(R.sub.10).sub.2, --NHCOR.sub.10, --NR.sub.10COR.sub.10, --I,
--Br, --Cl, --F, --CN, --CO.sub.2H, --CO.sub.2R.sub.10, --CHO,
--COR.sub.10, --CONH.sub.2, --CONHR.sub.10, --CON(R.sub.10).sub.2,
--COSH, --COSR.sub.10, --NO.sub.2, --SO.sub.3H, --SOR.sub.10,
--SO.sub.2R.sub.10, wherein R.sub.10 is a linear, branched or
cyclic, one to ten carbon saturated or unsaturated alkyl group, X
is defined as a moiety selected from the group consisting of: --OH,
--OR, .dbd.O, .dbd.S, --O.sub.2CR, --SH, --SR, --SOCR, --NH.sub.2,
--NHR, --N(R).sub.2, --NHCOR, --NRCOR, --I, --Br, --Cl, --F, --CN,
--CO.sub.2H, --CO.sub.2R, --CHO, --COR, --CONH.sub.2, --CONHR,
--CON(R).sub.2, --COSH, --COSR, --NO.sub.2, --SO.sub.3H, --SOR, and
--SO.sub.2R; Ar is an aromatic ring selected from the group
consisting of: phenyl, naphthyl, anthracyl, phenanthryl, furyl,
pyrrolyl, thiophenyl, benzofuryl, benzothiophenyl, quinolinyl,
isoquinolyl, imidazolyl, thiazolyl, oxazolyl, and pyridinyl,
optionally substituted with R or X; Y is a linear, unsaturated, one
to six carbon alkyl group, optionally substituted with R, ArR--, or
X; and Z is defined as a moiety selected from the group consisting
of: --NHR; and --N(R).sub.2.
2. The compound of claim 1 wherein Ar is phenyl, naphthyl,
anthracyl, or pyrrolyl.
3. The compound of claim 2 wherein R.sub.5 is phenyl, naphthyl,
anthracyl, or pyrrolyl.
4. The compound of claim 1 wherein R.sub.3 and R.sub.4 are
independently selected from the group consisting of: methyl, ethyl,
n-propyl, and n-butyl; or R.sub.3 and R.sub.4 together are joined
to form a moiety selected from the group consisting of:
cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
5. The compound of claim 1 wherein R.sub.1 and R.sub.2 are
independently selected from the group consisting of: H, methyl,
ethyl, propyl, n-butyl and acetyl; or R.sub.1 and R.sub.2 together
are joined to form a moiety selected from the group consisting of:
cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
6. The compound of claim 1 wherein R.sub.1 and R.sub.2 are
independently: H, methyl, or acetyl.
7. The compound of claim 1 wherein R.sub.1 is H and R.sub.2 is
methyl.
8. The compound of claim 1 wherein R.sub.7 is a three to six carbon
alkyl group.
9. The compound of claim 1 wherein R.sub.6 and R.sub.8 are
independently: H or methyl.
10. The compound of claim 1 wherein R.sub.6 is H, R.sub.7 is
--C(CH).sub.3).sub.3 and R.sub.8 methyl.
11. The compound of claim 1 wherein R.sub.3 and R.sub.4 are each
R.
12. The compound of claim 1 wherein R.sub.3 and R.sub.4 are each
methyl.
13. The compound of claim 1 wherein R.sub.5 is phenyl.
14. A method of inhibiting mitosis of a tumor cell comprising
contacting the tumor cell with an effective amount of a compound
according to claim 1.
15. A method of treating colon cancer comprising administering to a
patient in need thereof an anti-mitotic effective amount of a
compound according to claim 1.
16. A method of treating breast cancer comprising administering to
a patient in need thereof an anti-mitotic effective amount of a
compound according to claim 1.
17. A method of treating lung cancer comprising administering to a
patient in need thereof an anti-mitotic effective amount of a
compound according to claim 1.
Description
FIELD OF INVENTION
[0001] This invention relates to biologically active compounds and
compositions, their use and derivation.
BACKGROUND
[0002] As described in Talpir, R. et al. (1994) Tetrahedron Lett.
35:4453-6 and in international patent application PCT/GB96/00942
published Oct. 24, 1996 under number WO96/33211, the compound
hemiasterlin may be obtained from marine sponges or synthesized. As
set forth in PCT/GB96/00942, hemiasterlin and the synthetic analogs
described therein are cytotoxic and anti-mitotic.
[0003] Compounds that differ from hemiasterlin in the region of the
indole moiety of hemiasterlin are novel. It has now been found that
analogs of hemiasterlin wherein the indole moiety of hemiasterlin
has been deleted or replaced demonstrate potent anti-mitotic and
cytotoxic activity.
SUMMARY OF INVENTION
[0004] This invention provides a compound or pharmaceutically
acceptable salt thereof, having the formula
##STR00001##
wherein,
[0005] R.sub.1 and R.sub.2 are independently selected from the
group consisting of: H, R, and ArR--, and where at least one of
R.sub.1 and R.sub.2 is R and neither are ArR--, R.sub.1 and R.sub.2
together may optionally be a three to seven membered ring;
[0006] R.sub.3 and R.sub.4 are independently selected from the
group consisting of: H, R, ArR--, and where at least one of R.sub.3
and R.sub.4 is R and neither are ArR-- or Ar, R.sub.3 and R.sub.4
together may optionally be a three to seven membered ring;
[0007] R.sub.5 is selected from the group consisting of: H, R,
ArR--, and Ar;
[0008] R.sub.6 is selected from the group consisting of: H, R, and
ArR--;
[0009] R.sub.7 and R.sub.8 are independently selected from the
group consisting of: H, R, and ArR--; and
[0010] R.sub.9 is:
##STR00002##
[0011] and wherein,
[0012] R is defined as a saturated or unsaturated moiety having a
linear, branched, or cyclic skeleton containing one to ten carbon
atoms, zero to four nitrogen atoms, zero to four oxygen atoms, and
zero to four sulfur atoms, and the carbon atoms are optionally
substituted with: .dbd.O, .dbd.S, OH, --OR.sub.10,
--O.sub.2CR.sub.10, --SH, --SR.sup.10, --SOCR.sub.10, --NH.sub.2,
--NHR.sub.10, --N(R.sup.10).sub.2, --NHCOR.sub.10,
--NR.sub.10COR.sub.10, --I, Br, --Cl, --F, --CN, --CO.sub.2H,
--CO.sub.2R.sub.10, --CHO, --COR.sub.10, --CONH.sub.2,
--CONHR.sub.10, --CON(R.sub.10).sub.2, --COSH, --COSR.sub.10,
--NO.sub.2, --SO.sub.3H, --SOR.sub.10, --SO.sub.2R.sub.10, wherein
R.sub.10 is a linear, branched or cyclic, one to ten carbon
saturated or unsaturated alkyl group;
[0013] X is defined as a moiety selected from the group consisting
of: --OH, --OR, .dbd.O, .dbd.S, --O.sub.2CR, --SH, --SR, --SOCR,
--NH.sub.2, --NHR, --N(R).sub.2, --NHCOR, --NRCOR, --I, --Br, --Cl,
--F, --CN, --CO.sub.2H, --CO.sub.2R, --CHO, --COR, --CONH.sub.2,
--CONHR, --CON(R).sub.2, --COSH, --COSR, --NO.sub.2, --SO.sub.3H,
--SOR, and --SO.sub.2R;
[0014] Ar is defined as an aromatic ring selected from the group
consisting of: phenyl, naphthyl, anthracyl, phenanthryl, furyl,
pyrrolyl, thiophenyl, benzofuryl, benzothiophenyl, quinolyl,
isoquinolyl, imidazolyl, thiazolyl, oxazolyl, and pyridyl,
optionally substituted with R or X;
[0015] Y is defined as a moiety selected from the group consisting
of: a linear, saturated or unsaturated, one to six carbon alkyl
group, optionally substituted with R, ArR--, or X; and,
[0016] Z is defined as a moiety selected from the group consisting
of: --OH, --OR; --SH; --SR; --NH.sub.2; --NHR; --N(R).sub.2;
--NHCH(R.sub.11)COOH; and --NRCH(R.sub.11)COOH, wherein R.sup.11,
is a moiety having the formula: R, or
--(CH.sub.2).sub.nNR.sub.12R.sub.13, wherein n=1-4 and R.sub.12 and
R.sub.13 are independently selected from the group consisting of:
H; R; and --C(NH)(NH.sub.2).
[0017] This invention also provide methods of preparing the
aforementioned compound of formula I, and precursors thereof, as
described herein.
[0018] This invention also provides the use of the aforementioned
compound of formula I, or a pharmaceutically acceptable salt
thereof, for:
[0019] (a) manufacture of a medicament;
[0020] (b) in a method whereby cells, including tumor cells, which
are susceptible to the cytotoxic effects of the compound are
treated with the compound or a pharmaceutically acceptable salt
thereof; and
[0021] (c) in a method whereby cells are treated with the compound
or a pharmaceutically acceptable salt thereof, to bring about
mitotic arrest in the cells, or the production of abnormal mitotic
spindles in the cells.
DESCRIPTION OF FIGURES
[0022] FIG. 1 is a schematic showing a preferred scheme for
synthesis of a compound of this invention.
[0023] FIG. 2 is a schematic showing a preferred scheme for
synthesis of the amino acid used in the coupling reaction shown in
FIG. 1.
[0024] FIG. 3 is a schematic showing steps in the synthesis of a
compound of this invention as described in the examples herein.
[0025] FIG. 4 is a schematic showing steps in the synthesis of the
dipeptide shown in FIG. 3, as described in the examples herein.
[0026] FIGS. 5A and 5B are graphs comparing the cytoxicity of
hemiasterlin to SPA-110, as described in the examples herein.
[0027] FIG. 6 is a graph comparing the anti-mitotic activity of
SPA-110 (.diamond-solid.) to hemiasterlin (.quadrature.), as
described in the examples herein.
DETAILED DESCRIPTION OF THE INVENTION
[0028] Except where otherwise stated, the recitation of a compound
herein covers all possible salts of the compound, and denotes all
possible isomers possible within the structural formula given for
such compound, including geometrical and optical isomers. Unless
otherwise stated, materials described herein comprising a compound
for which isomers exist, are to be regarded as covering individual
isomers, and, mixtures of isomers including racemic mixtures.
[0029] In the compound of formula I set out above, bonds drawn in
wavy line are from carbon atoms which may be optical centers.
Preferably, the following absolute configurations predominate:
##STR00003##
[0030] Except where otherwise stated, any moiety referred to herein
which is described as "alkyl" will preferably be straight chain or,
branched when possible, and will preferably have up to eight, more
preferably up to six and even more preferably up to four carbon
atoms. Except where otherwise stated optionally substituted alkyl
groups are preferably unsubstituted. Methyl is the most preferred
alkyl group.
[0031] In this specification, reference is made to alkyl moieties
being saturated or unsaturated, thereby including within the
definition of the moiety, alkene and alkyne groups (whether
internal, terminal or part of a ring).
[0032] In a compound of formula I, the following substituents
alone, or in combination, are preferred: [0033] (a) R.sub.1 and
R.sub.2 independently: H, methyl, ethyl, propyl, n-butyl, acetyl;
or, where R.sub.1 and R.sub.2 are joined: cyclopropyl, cyclobutyl,
cyclopentyl or cyclohexyl; more preferably R.sub.1 and R.sub.2 are
independently: H or CH.sub.3; most preferably R.sub.1 is H and
R.sub.2 is CH.sub.3; [0034] (b) preferably no more than one of
R.sub.3 and R.sub.4 is H; more preferably, R.sub.3 and R.sub.4 are
independently: methyl, ethyl, n-propyl or n-butyl, or, where
R.sub.3 and R.sub.4 are joined: .beta.-cyclopropyl,
.beta.-cyclobutyl, .beta.-cyclopentyl or .beta.-cyclohexyl; most
preferably R.sub.3 and R.sub.4 are each methyl; [0035] (c) R.sub.5:
Ar in the definition of R.sub.5 is preferably phenyl, naphthyl,
anthracyl or pyrrolyl; preferably R.sub.5 is phenyl, methyl or H;
most preferably R.sup.5 is phenyl or methyl; [0036] (d) R.sub.6 and
R.sub.8 independently: H or methyl, more preferably R.sub.6 is H
and R.sub.8 is methyl; [0037] (e) R.sub.7: a three to six carbon,
branched alkyl group; more preferably R.sub.7 is
--C(CH.sub.3).sub.3; and [0038] (f) in R.sub.9, Z is preferably OH,
--OR.sub.14 (wherein R.sub.14 is a linear or branched one to six
carbon alkyl group, --NHCH(R).sub.11)COOH or
--NCH.sub.3CH(R.sub.11)COOH wherein R.sub.11, is R, or
--(CH.sub.2).sub.nNHC(NH)(NH.sub.2); or R.sub.9 is preferably
--C--C.dbd.C(R.sub.16)
[0038] ##STR00004## [0039] wherein R.sub.15 is methyl, ethyl,
n-propyl, isopropyl, tert-butyl, iso-butyl, or sec-butyl and
R.sub.16 is H, methyl, ethyl, propyl, iso-propyl, n-butyl,
iso-butyl or sec-butyl; more preferably Z is OH and R.sub.9, as a
whole is:
##STR00005##
[0039] Where R.sub.9 has a chiral center in moiety Y, the following
absolute configuration is preferred, with reference to an example
where the chiral center has a methyl substituent:
##STR00006##
[0040] Compounds of formula I may be prepared by coupling moieties
A, B and C as represented below using standard procedures,
including procedures for coupling amino acids through a peptide
bond.
##STR00007##
[0041] A coupling agent, for example PyBroP, is suitably used in
the reaction. The reaction suitably comprises connecting amino acid
moieties in the presence of the coupling agent, a base such as
4-dimethylaminopyridine and an organic solvent such as methylene
chloride. Standard reaction quenching and purification procedures
known in the art produce the coupled compound.
[0042] Preparation of moieties B and C as described above may be
carried out using procedures and starting materials known in the
art, for example by following the methods described in the
aforementioned PCT/GB96/00942. The methods as set out in the
examples herein may be employed, with suitable modification to
materials and reagents, according to particular substituents of
moieties A, B and C.
[0043] One aspect of this invention is a method for preparing a
compound of formula I in which a compound of the formula:
##STR00008##
is coupled with a compound of the formula:
##STR00009##
[0044] A compound of formula III may be prepared by known methods
(such as described in PCT/GB96/00942) and by the method described
in the examples herein.
[0045] A further aspect of this invention is a method for preparing
a compound of the formula II described above in which a compound of
the formula:
##STR00010##
is treated with a base such as dilute sodium hydroxide in a solvent
such as methanol for enough time to allow removal of OR; followed
by acidification to about pH 3. Preferably R in formula IV is a
simple alkyl chain, such as CH.sub.3, and a protecting agent such
as tert-butoxycarbonyl(Boc) may be used to protect the amine group;
i.e. R.sub.1 or R.sub.2 is replaced by Boc. The Boc group is
suitably removed by a reaction such as TFA/CH.sub.2Cl.sub.2 for
about 1 hour at ambient temperatures. An appropriate isolation
protocol produces the TFA salt. Subsequently another group (eg.
R.sub.1 or R.sub.2) could be introduced on the nitrogen by standard
techniques known to any person in the art, which produces compound
of formula IV.
[0046] A further aspect of this invention is a method for preparing
a compound of formula IV described above in which a compound of the
formula:
##STR00011##
is coupled with a compound of the formula:
##STR00012##
[0047] Preparation of compound V can be accomplished by many
procedures known to person skilled in the art. One such example is
described below in FIG. 2. Compounds of formula VI may be prepared
by methods known to persons of skill in the art.
[0048] A preferred method according to this invention for preparing
a compound of formula I, is to prepare a dipeptide comprising
moieties B and C and couple the dipeptide to moiety A. In this
method, a compound of the following formula, wherein Q and T
together are a combination of any two of the substituents: R.sub.1,
R.sub.2 and a protecting group:
##STR00013##
is coupled with a compound of the formula:
##STR00014##
[0049] A compound of formula VIII may be prepared by coupling a
compound of formula III as described above, with a compound of the
formula:
##STR00015##
[0050] Compounds of formula IX may be prepared by methods known to
persons of skill in the art.
[0051] A further aspect of this invention is a method to prepare a
compound of formula VII as described above in which a compound of
the formula:
##STR00016##
is treated with a base followed by an azide compound. The azide
derivative so produced is reduced to form an amine which is then
treated with groups selected from: R.sub.1, R.sub.1 and Boc in the
presence of a base such as sodium hydride.
[0052] FIG. 1 sets out a preferred scheme for preparation of a
compound of formula I involving the coupling of amino acid moiety A
with a dipeptide comprising moieties B and C. In the embodiment
shown in the figure, the substituents of the dipeptide are those of
hemiasterlin. The Boc protected dipeptide portrayed in the figure
may be obtained by the methods set out in the examples herein. On
the A moiety, Boc may replace R.sub.1 rather than R.sub.2 or both
R.sub.1 and R.sub.2 may be present on the A moiety prior to
coupling.
[0053] FIG. 2 sets out a preferred scheme for preparation of moiety
A as used in the scheme shown in FIG. 1. R.sub.2 may be added in
place of Boc and Boc may replace R.sub.1.
[0054] Compounds of formula I are biologically active. The
invention includes the use of a compound of formula I. Compounds of
formula I may have pesticidal, for example insecticidal activity.
Preferably, however, the use is in the pharmaceutical field for
medical or veterinarial applications.
[0055] The compounds described herein have utility as cytotoxic
agents, particularly against tumor cells and may have utility as
anti-bacterial or anti-viral agents. Therefore, this invention
includes a pharmaceutical composition comprising an effective
amount of a compound of formula I, in association with a
carrier.
[0056] This invention further provides the use of a compound of
formula I for the manufacture of a medicament for use in the
treatment of cancer or a tumor in a mammal.
[0057] In using a compound of formula I for medical or veterinarial
applications, the compound is preferably administered in a
pharmaceutical composition comprising also a pharmaceutically
acceptable carrier, and optionally, one or more other biologically
active ingredients. Such compositions may be in any form used for
administering pharmaceuticals, for example any form suitable for
oral, topical, vaginal, intravenous, subcutaneous, parenteral,
rectal and inhalatory application. The compositions may be provided
in discrete dose units. The carriers may be particulate, with the
compositions being, for example, tablets or powders, or liquid,
with the compositions being, for example, oral syrups or injectable
liquids, or aerosol, for inhalatory application.
[0058] For oral administration an excipient and/or binder may be
present. Examples are sucrose, kaolin, glycerin, starch dextrins,
sodium alginate, carbosymethylcellulose and ethyl cellulose.
Colouring and/or flavouring agents may be present. A coating shell
may be employed. For rectal administration oleaginous bases may be
employed, for example lanolin or cocoa butter. For an injectable
formulation buffers, stabilizers and isotonic agents may be
included.
[0059] The dosage of a compound of formula I may depend upon the
weight and physical condition of the patient; on the severity and
longevity of the illness; and on the particular form of the active
ingredient, the manner of administration and the composition
employed. A daily dose of about 0.0001 to about 100 mg/kg of body
weight taken singly or in separate doses of up to 6 times a day, or
by a continuous infusion, embraces the effective amounts most
typically required. A preferred range is about 0.001 to about 50
mg/kg of body weight, per day, most preferably about 0.01 to about
30 mg/kg of body weight, per day.
[0060] It is to be understood that use of a compound of formula I
in chemotherapy can involve such a compound being bound to an
agent, for example a monoclonal or polyclonal antibody, a protein
or a liposome, which assists the delivery of the said compound to
tumor cells.
[0061] This invention also includes the use of a compound of
formula I as an antimitotic agent. Such use may be in procedures
that require blocking cells in mitosis, such as the preparation of
mitotic spreads for karyotype analysis.
[0062] The compounds of this invention can also be used to probe
microtubule function in mitotic cells.
EXAMPLES
[0063] The following examples provide a detailed description of
preferred methods of synthesis of a preferred compound of this
invention, SPA-110. Also described are precursor compounds and
characterization of various compounds of this invention. FIGS. 3
and 4 schematically portray the synthesis of the a SPA-110 salt
according to the examples. Reference numerals in the examples
correspond to labelling of compounds in FIGS. 3 and 4 and labelling
of compounds depicted in the examples.
3-methyl-3-phenylbutanoic acid (2)
##STR00017##
[0065] 3-Methyl-2-butenoic acid (1, 5.10 g, 50.9 mmol) and
AlCl.sub.3 (20.4 g, 153 mmol) were placed in a one-neck
round-bottomed flask. Benzene (50 mL) was added, which produced
vigorous bubbling. Upon completion of the bubbling, a capped
condenser (i.e. closed system) was attached, the reaction mixture
was stirred and placed in an oil bath at 65.degree. C. The pressure
in the system was occasionally released. The progress of the
reaction was followed by following the loss of starting material by
GC. If the reation was not complete within 1 h, a small quantity of
AlCl.sub.3 was added and stirring was continued. To the solution
was added diethyl ether and the mixture was cooled to 0.degree. C.
Slowly conc. HCl and some water were added until all the solid
dissolved and the pH was less than 2. The aqueous layer was
extracted with diethyl ether three times. The organic layer was
concentrated to 150 mL and then was extracted with a saturated
sodium hydrogen carbonate solution six times. The combined aqueous
layer were acidified with conc. HCl until the pH was less than 2.
The acidic aqueous layer was extracted with diethyl ether three
times and the accumulated organic layer was dried with magnesium
sulfate. The solution was filtered and the diethyl ether was
removed in vacuo producing a white solid (8.51 g, 47.7 mmol) in 94%
yield, which did not need further purification. mp 55-56.degree. C.
.sup.1H-NMR (400 MHz, CDCl.sub.3) 10.45 (bs, 1H, CO.sub.2H), 7.38
(d, 2H, J=7.2 Hz, H-11 and H-7), 7.32 (t, 2H, J=7.2 Hz, H-10 and
H-8), 7.21 (t, 2H, J=7.2 Hz, H-9), 2.65 (s, 2H, H-2), 1.47 (s, 6H,
H-5 and H-4); Mass spectrum (EI) 178 (23, M.sup.+), 119 (100,
[C.sub.9H.sub.11].sup.+). For pioneering work to form 2 see: F. J.
Eijkman (1908) Chem. Kentr. II, p. 110; or A. Hoffman (1929) J. Am.
Chem. Soc. 51:2542.
(4S)-3-(3-methyl-3-phenyl-1-oxobutyl)-4-isopropyl-2-oxazolidinone
(3)
##STR00018##
[0067] 3-Methyl-3-phenylbutanoic acid (2, 1.00 g, 5.61 mmol) was
dissolved in 70 mL of THF and cooled to -78.degree. C.
Triethylamine (1.17 mL, 8.42 mmol) and trimethylacetyl chloride
(0.760 mL, 6.17 mmol) were added to the reaction flask producing a
white solid. The resulting mixture was warmed to 0.degree. C. for 1
h and then cooled back down to -78.degree. C. In a second flask
butyllithium (6.84 mL, 1.6 M in hexanes, 10.9 mmol) was added
dropwise with vigorous stirring to a solution of
(4S)-(-)-4-isopropyl-2-oxazolidinone (1.45 g, 11.2 mmol) at
-78.degree. C. in THF (60 mL) producing a white precipitate. The
resulting suspension of the lithiated oxazolidinone was added via
cannula to the reaction flask. Stirring was continued for 2 h,
water was added and the reaction mixture was warmed to room
temperature, whereupon it was extracted three times with diethyl
ether. The combined organic extracts were dried over magnesium
sulfate, and concentrated in vacuo. The product was purified by
radial chromatography (4 mm plate, 3:7 diethyl ether-pet. ether)
affording compound 3 as a clear, colourless oil in 84% yield (1.37
g, 4.74 mmol). .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.38 (d, 2H, J=7.3
Hz, H-19 and H-15) 7.28 (t, 2H, J=7.3 Hz, H-18 and H-16), 7.16 (t,
1H, J=7.3 Hz, H-17), 4.22-4.18 (m, 1H, H-4), 4.05 (dd, 1H, J=9.0
and 2.8 Hz, 1H-5), 4.00 (t, 1H, J=9.0 Hz, 1H-5), 3.38-3.30 (m, 2H,
H-10), 2.16-2.12 (m, 1H, H-6), 1.48 (s, 3H, H-13 or H-12), 1.47 (s,
3H, H-13 or H-12), 0.79 (d, 3H, J=7.1 Hz, H-8 or H-7), 0.71 (d, 3H,
J=6.9 Hz, H-8 or H-7); Mass spectrum (EI) 289 (8, M..sup.+), 119
(100, [C.sub.9H.sub.11].sup.+). Optical rotation obtained was
[.alpha.].sub.D.sup.25+69.5 (c 1.16, CHCl.sub.3). Compound 3 was
prepared according to D. A. Evans et al. (1988) Tetrahedron
44:5525.
Preparation of the 4-isopropyl-2-oxazolidione 4
##STR00019##
[0069] Oxazolidinone 3 (472 mg, 1.63 mmol), dried under high vacuum
for 0.5 h, was dissolved in THF and cooled to -78.degree. C. (10
mL). Freshly titrated potassium bis(trimethylsilyl)amide (15.6 mL,
0.115 M in THF, 1.79 mmol) was added and the resulting solution was
stirred at -78.degree. C. for 1 h. A solution of
2,4,6-triisopropylbenzenesulfonyl azide (625 mg, 2.04 mmol) in THF
(5 mL) at -78.degree. C. was added via cannula and after 2 min. the
orange coloured reaction mixture was treated with glacial acetic
acid (0.429 mL, 7.50 mmol), warmed to 40.degree. C. in a water bath
and stirred for a further hour. To the light yellow mixture was
added brine (35 mL), water (35 mL) and the aqueous phase was
extracted three times with 80 mL diethyl ether. The combined
organic extracts were washed with a saturated sodium hydrogen
carbonate solution (20 mL), dried with magnesium sulfate and
concentrated in vacuo. The product was purified by radial
chromatography (4 mm plate, 3:7 diethyl ether-pet. ether, sample
was loaded with diethyl ether) affording azide 5 as a colourless
oil (482 mg, 1.46 mmol) in 89% yield. .sup.1H-NMR (400 MHz,
CDCl.sub.3) 7.39 (d, 2H, J=7.2 Hz, H-19 and H-15), 7.31 (t, 2H,
J=7.2 Hz, H-18 and H-16), 7.23 (t, 1H, J=7.2 Hz, H-17), 5.64 (s,
1H, H-10), 3.95 (dd, 1H, J=8.7 and 2.2 Hz, 1H-5), 3.89-3.85 (m, 1H,
H-4), 3.56 (t, 1H, J=8.7 Hz, 1H-5), 2.31-2.26 (m, 1H, H-6), 1.54,
1.52 (s, 3H, H-13 and H-12), 0.83 (d, 3H, J=7.0 Hz, H-8 or H-7),
0.79 (d, 3H, J=6.9 Hz, H-8 or H-7); Mass spectrum (DCI, NH.sub.3)
349 (45, [M+NH.sub.5].sup.+), 348 (100, [M+NH.sub.4].sup.+), 331
(12, [M+H].sup.+), 303 (57, [M-N.sub.2].sup.+), 119 (94,
[C.sub.9H.sub.11].sup.+). Optical rotation obtained was
[.alpha.].sub.D.sup.25+121.5 (c 1.1, CHCl.sub.3). Compound 4 was
prepared according to the methodology developed by D. A. Evans et
al. (1990) J. Am. Chem. Soc. 112:4011.
2,4,6-Triisopropylbenzenesulfonyl azide was prepared by the method
of O. C. Dermer et al. (1955) J. Am. Chem. Soc. 77:70.
Preparation of the 4-isopropyl-2-oxazolidinone 5
##STR00020##
[0071] Azide 4 (418 mg, 1.26 mmol), 10% palladium on charcoal (280
mg), and di-tert-butyl dicarbonate (608 mg, 2.78 mmol) were placed
in a 100 mL flask. Ethyl acetate (37 mL) was added and the
resulting black suspension was stirred at room temperature. The
mixture was flushed with argon, then with hydrogen and was stirred
under a hydrogen balloon overnight (.about.14 h). The reaction
mixture was filtered through silica gel and the collected material
was washed with ethyl acetate. The combined filtrate was
concentrated in vacuo and the crude mixture was purified by flash
column chromatography (3:7 diethyl ether-pet. ether) to afford
compound 5, a viscous colourless oil, in 78% yield (400 mg, 0.989
mmol). .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.40 (d, 2H, J=7.4 Hz,
H-19 and H-15), 7.29 (t, 2H, J=7.4 Hz, H-18 and H-16), 7.21 (t, 1H,
J=7.4 Hz, H-17), 6.12 (d, 1H, J=9.9 Hz, H-10), 5.11 (bs, 1H, N--H),
3.89 (d, 1H, J=8.4 and 1.9 Hz, H-5), 3.82-3.79 (m, 1H, H-4), 3.45
(t, 1H, J=8.4 Hz, H-5), 2.26-2.22 (m, 1H, H-6), 1.41 (s, 9H, H-24,
H-23 and H-22), 0.80 (d, 3H, J=7.0 Hz, H-8 or H-7), 0.76 (d, 3H,
J=6.9 Hz, H-8 or H-7); Mass spectrum (DCI, CH.sub.4/NH.sub.3 mix)
405 (1, [M+H].sup.+), 349 (7, [M-C.sub.4H.sub.9].sup.+), 230 (100,
[C.sub.9H.sub.14N.sub.2O.sub.5].sup.+). Optical rotation obtained
was [.alpha.].sub.D.sup.24+118.4 (c 0.935, CHCl.sub.3). Compound 5
was prepared according to the methodology developed by D. A. Evans
et al. (1990) [supra].
Methyl
(2S)-2-(tert-butyloxycarbonyl)amino-3-methyl-3-phenylbutanoate
(6)
##STR00021##
[0073] Oxazolidinone 5 (245 mg, 0.65 mmol) was dissolved in a
mixture of 7.1 mL THF and 1.8 mL water. This solution was cooled to
0.degree. C. and hydrogen peroxide (0.618 mL, 30% aqueous, 5.45
mmol) and lithium hydroxide (1.82 mL, 1.0 M, 1.82 mmol) were added.
The resulting mixture was stirred at room temperature overnight
(.about.15 h). The excess peroxide was quenched by addition of
sodium hydrogen sulfite (7.1 mL, 1.5 M, 10.7 mmol) and stirring was
continued for 1 h. The aqueous phase was acidified with 1.0 M
citric acid and the mixture was extracted three times with ethyl
acetate. The combined ethyl acetate extracts were dried over
magnesium sulfate and concentrated in vacuo. To the remaining crude
material was added a solution of diazomethane in diethyl ether
until the solution stayed yellow. After bubbling argon through the
solution for 15 min., the remaining volatile components were
removed in vacuo to afford crude compound 6. Purification of ester
6 was accomplished by radial chromatography (2 mm plate, 37 diethyl
ether-pet. ether, sample was loaded with CHCl.sub.3), producing a
clear colourless oil (171 mg, 0.555) in 92% yield. .sup.1H-NMR (400
MHz, CDCl.sub.3) 7.33-7.27 (m, 4H, H-16, H-15, H-13, H-12), 7.20
(t, 1H, J=6.7 Hz, H-14), 4.99 (bd, 1H, J=8.8 Hz, H-2), 4.50 (bd,
1H, J=8.8 Hz, N--H), 3.48 (s, 3H, H-17), 1.41, 1.38 (s, 3H, H-5 and
H-4), 1.37 (s, 9H, H-10, H-9, and H-8); Mass spectrum (EI) 307
(0.1, M..sup.+), 234 (2, [M-Ot-Bu].sup.+), 119 (100,
[C.sub.9H.sub.11].sup.+). Optical rotation obtained was
[.alpha.].sub.D.sup.25+35.2 (c 2.98, CHCl.sub.3). Compound 5 was
prepared according to the methodology developed by D. A. Evans et
al. (1990) [supra].
(2S)--N-tert-butoxycarbonyl-N-methyl-3-methyl-3-phenylbutanoic acid
(7)
##STR00022##
[0075] To a vigorously stirred solution of ester 6 (43.4 mg, 0.141
mmol) in 2 mL dry DMF were added sodium hydride (10.2 mg, 4.24
mmol) followed by methyl iodide (0.088 mL, 1.41 mmol) and the
resulting grey suspension was stirred overnight (.about.20 h) at
room temperature. The excess sodium hydride was quenched by
cautious addition of water and the mixture was acidified by
dropwise addition of 1.0 M citric acid. The acidic mixture was
extracted three times with ethyl acetate, the combined organic
layer extracted three times with brine, dried over magnesium
sulfate and concentrated in vacuo. The resulting light orange oil
was dissolved in 4 mL methanol in a 25 mL flask. To the solution
was added 1.0 mL of water, followed by 1.13 mL of 1.0 M lithium
hydroxide. The reaction mixture was heated at 60.degree. C.
overnight (.about.14 h), producing a white precipitate. To the
resultant mixture was added saturated sodium hydrogen carbonate
solution and water; the mixture was then extracted with ethyl
acetate. The aqueous layer was acidified with 1.0 M citric acid
until the pH was .about.4. The mixture was extracted three times
with ethyl acetate. The combined organic layers were dried with
magnesium sulfate and concentrated in vacuo. Compound 7 was also
found in the first ethyl acetate extraction so it was also added to
the crude product. Purification of acid 7 was performed by silica
gel column chromatography (1:2 diethyl ether-pet. ether with 1%
acetic acid) resulting in a 49% yield (21.2 mg, 0.0670 mmol) of a
clear colourless oil. .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.41 (d,
1.3H, J=7.6 Hz, H-17 and H-13), 7.37 (d, 1.3H, J=7.6 Hz, H-17 and
H-13), 7.28 (t, 2H, J=7.6 Hz, H-16 and H-14), 7.18 (t, 1H, J 7.2
Hz, H-15), 5.17 (bs, 0.66H, H-2), 4.93 (bs, 0.33H, H-2), 2.75 (s,
1.05H, H-6), 2.62 (s, 1.95H, H-6), 1.55 (s, 3H, H-5 or H-4),
1.49-1.39 (m, 12H, H-5 or H-4 and H-11, H-10 and H-9); Mass
spectrum (EI) 307 (0.1, M..sup.+), 234 (3, [M-Ot-Bu].sup.+), 119
(100, [C.sub.9H.sub.11].sup.+), 57 (78, [C.sub.4H.sub.9].sup.+);
Exact mass calc d for C.sub.17H.sub.25NO.sub.4: 307.1783. Found
(EI): 307.1793.
Preparation of Compound 9
##STR00023##
[0077] The N-Boc-amino ester 8 (71.6 mg, 0.174 mmol) was dissolved
in 1 mL CH.sub.2Cl.sub.2 and 1 mL of TFA was added. The reaction
mixture was stirred at room temperature for 0.5 h. Removal of the
solvent in vacuo, followed by repeated rinsing of the remaining
material with CH.sub.2Cl.sub.2 (3.times.5 mL) and evaporation of
the residual solvent afforded the TFA salt of the amino acid ester
8. In a separate flask, to a solution (or suspension) of the N-Boc
protected amino acid 7 (51.5 mg, 0.167 mmol) in 0.5 mL
CH.sub.2Cl.sub.2, was added DIEA (0.0875 mL, 0503 mmol), DMAP
(0.031 mg, 0.10 mmol) and PyBroP (0.0781 mg, 0.167 mmol). The
solution was stirred for a few minutes and then a solution of the
TFA salt of 8 was added in 1 mL of CH.sub.2Cl.sub.2 via cannula
addition. The reaction mixture was stirred at room temperature for
18 h. To the mixture was added water, CH.sub.2Cl.sub.2 and ten
drops of 10% aqueous HCl. The resulting biphasic solution was
extracted with CH.sub.2Cl.sub.2 (three times with 20 mL). The
organic layer was extracted with saturated aqueous sodium hydrogen
carbonate (10 mL), dried with magnesium sulfate and the solvent was
removed in vacuo. The product was purified by flash chromatography
(silica gel, 1:1 diethyl ether-pet. ether) affording the protected
tripeptide 9 as a clear colourless oil in 27% yield (0.0272 g,
0.0454 mmol). .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.84 (bd, 1H, J=9.5
Hz, N--H), 7.4-7.30 (m, 5H, H-28, H-27, H-25, H-24), 7.21 (bt, 1H,
J=7.2 Hz, H-26), 6.63 (bd, 1H, J=9.6 Hz, H-6), 5.08 (t, 1H, J=9.6
Hz, H-7), 4.83 (d, 1H, J=9.5 Hz, H-13), 4.17 (q, 2H, J=7.1 Hz,
H-2), 3.02 (s, 3H, H-11), 2.15 (s, 0.66H, H-29), 2.02 (s, 2.37H,
H-29), 1.94-1.81 (m, 1H, H-8), 1.88 (s, 3H, H-5), 1.39-1.38 (m, 9H,
H-34, H-33 and H-32), 1.28 (t, 3H, J=7.1 Hz, H-1), 0.98 (s, 9H,
H-17, H-16 and H-15), 0.83, 0.77 (d, 3H, J=6.6 Hz, H-10 and H-9);
PyBroP is described in E. Frerot et al. (1991) Tetrahedron
47:259.
SPA110-trifluoroacetate salt (10)
##STR00024##
[0079] To a solution of the ethyl ester 9 (23.0 mg, 0.0382 mmol) in
1.1 mL MeOH was added 0.30 mL water and 0.31 mL of a 1.0 M aqueous
solution of lithium hydroxide (0.31 mmol). The reaction mixture was
stirred at room temperature overnight (.about.20 h) whereupon it
was acidified by dropwise addition of 1.0 M citric acid and then
extracted three times with ethyl acetate. The combined organic
extracts were dried with magnesium sulfate and concentrated in
vacuo. Under an argon atmosphere, the crude oil was dissolved in 1
mL CH.sub.2Cl.sub.2 and the solution was treated with TFA (1 mL)
and then was stirred at room temperature for 0.5 h. Removal of the
excess solvents in vacuo, followed by rinsing of the remaining
material three times with CH.sub.2Cl.sub.2 (5 mL) and evaporation
of the residual solvent, produced the TFA salt. HPLC purification
of the crude product using a Magnum reverse phase C-18 column
(H.sub.2O(45): MeOH(55) with 0.05% TFA) afforded the tripeptide 10
as a white powder. .sup.1H-NMR (400 MHz, CD.sub.3OD) 7.53 (d, 2H,
J=7.6, H-25 and H-21), 7.44 (t, 2H, J=7.6 Hz, H-24 and H-22), 7.34
(t, 1H, J=7.6 Hz, H-23), 6.76 (d, 1H, J=9.1 Hz, H-4), 5.04 (t, 1H,
J=10.1 Hz, H-5), 4.91, 4.34 (s, 1H, H-17 and H-11), 3.13 (s, 3H,
H-9), 2.49 (H-26), 2.08-1.99 (m, 1H, H-6), 1.90 (s, 3H, H-3), 1.46,
1.37 (s, 3H, H-19 and H-18), 1.05 (s, 9H, H-15, H-14 and H-13),
0.89 (d, 3H, J=6.1 Hz, H-8 or H-7), 0.88 (d, 3H, J=6.5 Hz, H-8 or
H-7); Mass spectrum (EI) 474 (0.1,
[M-CF.sub.3CO.sub.2.sup.-].sup.+), 458 (0.1,
[M-16-CF.sub.3CO.sub.2.sup.-].sup.+), 382 (2), 162 (62), 69 (74),
45 (100).
N.alpha.Boc-N.alpha.-methyl-1-valine N-methoxy-N-methylamide
(12)
##STR00025##
[0081] To a cold (0.degree. C.) solution of N-Boc-N-methylvaline
(11) (5.0 g, 21.6 mmol), N,O-dimethylhydroxylamine hydrochloride
(2.8 g, 28 mmol), and PyBOP.RTM. (11.2 g, 22 mmol) in
CH.sub.2Cl.sub.2 (22 mL) was added DIEA (8.4 mL, 75 mmol). After 1
min., the reaction mixture was warmed to room temperature and
stirring was continued for 1 h. If the pH value of the mixture was
less than 7, the mixture could be treated with a few drops DIEA to
allow the reaction to go to completion. The mixture was poured into
200 mL of diethyl ether and the resultant mixture was washed
successively with 3 N hydrochloric acid (3.times.30 mL), saturated
aqueous sodium hydrogen carbonate solution (3.times.30 mL), and
saturated aqueous sodium chloride (3.times.30 mL). The organic
layer was dried with magnesium sulfate and the solvent was
evaporated, followed by chromatography of the crude product (silica
gel, 1:3 diethyl ether-pet. ether), afforded 12 (4.46 g, 75% yield)
as a colourless oil. .sup.1H-NMR (200 MHz, CDCl.sub.3): 0.84 (d,
J=6.6 Hz, 4H, (CH.sub.3).sub.2), 0.85 (d, J=6.6 Hz, 2H,
(CH.sub.3).sub.2), 1.41 (s, 6H, Boc-(CH.sub.3).sub.3), 1.44 (s, 3H,
Boc-(CH.sub.3).sub.3), 2.15-2.30 (m, 1H, CH), 2.75 (s, 1H,
NCH.sub.3), 2.78 (s, 2H, NCH.sub.3), 3.10 (bs, 3H, NCH.sub.3), 3.64
(s, 1H, OCH.sub.3), 3.68 (s, 2H, OCH.sub.3), 4.66 (d, J=10 Hz,
0.4H, CH), 4.95 (d, J=10 Hz, 0.6H, CH); Exact mass calc'd for
C.sub.13H.sub.27N.sub.2O.sub.4 (M+H).sup.+:275.19708. Found (DCI):
275.19710. Optical rotation obtained was [.alpha.].sub.D.sup.25:
+128.3 (c 2.9, CHCl.sub.3).
N-Boc-N-methyl-1-valinal (13
##STR00026##
[0083] Lithium aluminum hydride (875 mg, 23 mmol) was added to a
solution of N.alpha.-Boc-N.alpha.-methyl-L-valine
N-methoxy-N-methylamide (12) (2.0 g, 7.7 mmol) in dry THF (8 mL)
and the reaction mixture was stirred for 20 min. The mixture was
poured into a stirring solution of potassium hydrogen sulfate (3.14
g, 23 mmol) in water (100 mL). Diethyl ether (75 mL) was added, the
layers separated and the aqueous layer extracted with diethyl ether
(3.times.50 mL). The organic layers were combined, and washed
sequentially with 3 N hydrochloric acid (3.times.30 mL), saturated
aqueous sodium hydrogen carbonate (3.times.30 mL), and saturated
aqueous sodium chloride (3.times.30 mL). The organic layer was
dried with magnesium sulfate and the solvent was evaporated to
yield the crude aldehyde 13 (1.52 g, 92% yield). Aldehyde 13 was
used without further purification. Note: 13 can be stored under
argon for .about.2 weeks, but when stored in organic solvents at
room temperature, undergoes slow decomposition. .sup.1H-NMR (200
MHz, CDCl.sub.3) 0.73 (d, J=6.9 Hz, 3H, CH.sub.3), 0.91 (d, J=6.9
Hz, 3H, CH.sub.3), 1.27 (s, 9H, Boc-(CH.sub.3).sub.3), 2.02-2.15
(m, 1H, CH), 2.63 (s, 2H, NCH.sub.3), 2.72 (s, 1H, NCH.sub.3), 3.44
(d, J=9.5 Hz, 0.5H, CH), 3.86 (d, J=9 Hz, 0.5H, CH), 9.45 (s, 1H,
CH); Exact mass calc'd for C.sub.11H.sub.22NO.sub.3 (M+H).sup.+:
216.15997; Found (DCI): 216.15996; Optical rotation obtained was
[.alpha.].sub.D.sup.25-104.2 (c 5.5, CHCl.sub.3).
Ethyl (2E,4S)--N-Boc-N-methyl-4-amino-2.5-dimethylhex-2-enoate
(14)
##STR00027##
[0085] To a solution of aldehyde 13 (1.75 g, 8.7 mmol) in dry
CH.sub.2Cl.sub.2 (9.0 mL) under an argon atmosphere at room
temperature was added (carbethoxyethylidene)triphenylphosphorane
(4.19 g, 11.3 mmol) and stirring was continued for a 4 h. The
reaction mixture was diluted with water (100 mL) and extracted with
diethyl ether (3.times.100 mL). The combined organic extracts were
washed with saturated aqueous sodium chloride (100 mL), dried with
magnesium sulfate and concentrated in vacuo. The crude oil was
purified by flash chromatography (silica gel, 2:23 diethyl
ether-pet. ether) to afford the required E-2-alkenoate 14 as a
colourless oil (2.13 g, 82% yield). .sup.1H-NMR (200 MHz,
CDCl.sub.3) 0.74 (d, J=6 Hz, 3H, CH.sub.3), 0.79 (d, J=6 Hz, 3H,
CH.sub.3), 1.17 (t, J=7 Hz, 3H, CH.sub.3), 1.34 (s, 9H,
Boc-(CH.sub.3).sub.3), 1.72 (m, 1H, CH), 1.78 (s, 3H, CH.sub.3),
2.60 (bs, 3H, NCH.sub.3), 4.08 (q, J=7 Hz, 2H, CH.sub.2), 4.15-4.20
(m, 0.5H, CH), 4.21-4.32 (m, 0.5H, CH), 6.54 (d, J=8 Hz, 1H, CH);
Exact mass calc'd for C.sub.16H.sub.30NO.sub.4 (M+H).sup.+:
300.21750; Found (DCI): 300.21754. Optical rotation obtained was
[.alpha.].sub.D.sup.25+61.1 (c 9.1, CHCl.sub.3).
General Procedure 1: Trifluoacetic Acid Medicated Cleavage of N-Boc
Groups
[0086] N-Boc-amino acid ester (1.0 equiv.) was treated with
TFA/CH.sub.2Cl.sub.2 (0.1 mmol/1 mL) at room temperature for 0.5 h.
Removal of the solvent in vacuo, followed by repeated rinsing of
the residual material with CH.sub.2Cl.sub.2 (3.times.5 mL) and
evaporation of the remaining traces of solvent afforded the TFA
salt of the amino acid ester in quantitative yield. TFA salts were
used without further purification.
General Procedure 2: Trimethylacetyl Chloride Mediated Peptide
Coupling
[0087] To a cold (-78 C) stirred solution of acid (1.1 equiv.) in
dry THF (1 mL/mmol) under an argon atmosphere was added DIEA (1.5
equiv.) and trimethylacetyl chloride (1.2 equiv.). The resulting
mixture was warmed to 0.degree. C. for 1 h and then re-cooled to
-78 C. DIEA (2.2 equiv.) was added via cannula to the reaction
flask followed by the addition via cannula of the TFA salt of the
amino acid ester (1.0 equiv., prepared by general procedure 1) in
dry THF (0.5 mL/mmol) at -78 C. Stirring was continued for 1 h and
water (40 mL) was added. The mixture was allowed to warm to room
temperature, and extracted with diethyl ether (3.times.50 mL). The
combined organic extracts were washed with saturated aqueous sodium
chloride (50 mL), dried over magnesium sulfate, and concentrated in
vacuo. The crude oil was purified by flash chromatography (silica
gel, diethyl ether-pet. ether) to afford the desired dipeptide as a
colourless oil.
Dipeptide 8
##STR00028##
[0089] Following general procedure 2, dipeptide 8 was prepared with
the following quantities of reagents and solvents:
N-Boc-tert-leucine (15), 156 mg (0.52 mmol); trimethylacetyl
chloride, 64 mL (0.52 mmol); DIEA, 99 mL (0.57 mmol);
N-Boc-MHVV-OEt (14), 110 mg (0.47 mmol); DIEA, 198 mL (1.14 mmol);
THF, 7 mL. Purification of the crude product by flash
chromatography (silica gel, 1:5 diethyl ether-pet. ether) afforded
121 mg of 8 (62% yield). .sup.1H-NMR (200 MHz, CDCl.sub.3) 0.76 (d,
J=6 Hz, 3H, CH.sub.3), 0.80 (d, J=6 Hz, 3H, CH.sub.3), 0.88 (s, 9H,
(CH.sub.3).sub.3), 1.22 (t, J=7 Hz, 3H, CH.sub.3), 1.33 (s, 9H,
Boc-(CH.sub.3).sub.3), 1.79-1.89 (m, 1H, CH), 1.83 (s, 3H,
CH.sub.3), 2.91 (s, 3H, NCH.sub.3), 4.12 (q, J=7 Hz, 2H, CH.sub.2),
4.35 (d, J=10 Hz, 1H, CH), 5.03 (t, J=10 Hz, 1H, CH), 5.14 (d, J=10
Hz, 1H, NH), 6.57 (d, J=8 Hz, 1H, CH); Exact mass calc'd for
C.sub.22H.sub.41N.sub.2O.sub.5 (M+H).sup.+: 413.30154; Found (DCI):
413.30119. Optical rotation obtained was
[.alpha.].sub.D.sup.25-76.9 (c 2.43, CHCl.sub.3).
[0090] Assay for Cytotoxicity
[0091] Cytotoxicity of SPA-110 compared to hemiasterlin as against
p53+ and p53- variants of human breast cancer MCF-7 cells and A549
tumor cells was determined according to the methods described in J.
Immunol. Methods 65:55-63 (1983). Results shown in FIGS. 5A and 5B
show that SPA-110 is more cytotoxic in some instances than the
naturally occurring compound.
[0092] Assay for Antimitotic Activity
[0093] Antimitotic activity is detected by enzyme-linked
immunosorbent assay using a mitosis-specific antibody, TG-3 (from
Albert Einstein College of Yeshiva University, Bronx, N.Y.; and
see: PCT application published Jul. 4, 1996 under WO96/20218).
[0094] MCF-7 mp 53.sup.- cells (expressing a dominant-negative p53
mutation as described in S. Fan, et al. (1955) Cancer Research
55:1649-1654) were cultured as monolayers in DMEM supplemented with
10% fetal bovine serum, 2 mM L-glutamine, 50 units/ml penicillin,
50 .mu.g/ml streptomycin, 1 mM sodium pyruvate, MEM non-essential
amino acids, 1 .mu.g/ml bovine insulin, 1 .mu.g/ml hydrocortisone,
1 ng/ml human epidermal growth factor, and 1 ng/ml .beta.-estradiol
at 37.degree. C. in humidified 5% CO.sub.2. The cells were seeded
at 10,000 cells per well of 96-well polystyrene tissue culture
plates (Falcon) in a volume of 200 .mu.l cell culture medium. The
cells were allowed to grow for 24 hours and compounds were added at
about 1 .mu.g/ml or 10 .mu.g/ml (from 1000-fold stocks in
dimethylsulfoxide) and the cells were incubated for 20 hours.
Nocodazole (Sigma) served as a positive control. After treatment
with the agent to be tested, the cell culture medium was removed
completely and the 96-well tissue culture plates were frozen at
-70.degree. C. for up to 2 hours. The frozen cells were thawed by
addition of 100 .mu.l of ice-cold lysis buffer (0.5 mM
phenylmethylsulfonylfluoride, 1 mM ethylene
glycol-bis(.beta.-aminoethyl ether) N,N,N,N'-tetraacetic acid, pH
7.4,) and lysed by pipeting up-and-down 10 times. The cell lysates
were transferred to 96-well PolySorp ELISA plates (Nunc) and dried
completely by blowing warm air at about 37.degree. C. with a hair
drier positioned about 3 feet above the plates. Protein binding
sites were blocked by adding 200 .mu.l per well of 10 mM Tris HCl
pH 7.4, 150 mM NaCl, 0.1 mM PMSF, 3% (w/v) dried non-fat milk
(Carnation) for 1 hour at room temperature. This was removed and
replaced with 100 .mu.l of the same solution containing 0.1-0.15
.mu.g/ml TG-3 mitosis-specific monoclonal antibody and horseradish
peroxidase-labelled goat anti-mouse IgM (1021-05, Southern
Biotechnology Associates) at a dilution of 1/500. After overnight
incubation at 4.degree. C., the antibody solution was removed and
the wells were rinsed 3 times with 200 .mu.l rinse buffer (10 mM
Tris HCl pH 7.4, 0.02% Tween 20). 100 .mu.l of 120 mM
Na.sub.2HPO.sub.4, 100 mM citric acid, pH 4.0 containing 0.5 mg/ml
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and 0.01%
hydrogen peroxide was added for 1 hour at room temperature and the
plates were read at 405 nm using a BioTek plate reader.
[0095] Results comparing antimitotic activity of hemiasterlin to
SPA-110 are shown in FIG. 6. SPA-110 exhibited considerably greater
antimitotic activity than the naturally occurring compound.
[0096] In Vivo Activity of SPA-110
[0097] The compound SPA-110 was evaluated in vivo using standard
pharmacological test procedures which measure its ability to
inhibit the growth of human tumor xenografts. The human colon
carcinoma LOVO (American Type Culture Collection, Rockville, Md.
#CCL-229) was grown in tissue culture in RPMI supplemented with 10%
FBS. Athymic nu/nu female mice (Charles River, Wilmington, Mass.)
were injected sub-cutaneously in the flank area with
7.0.times.10.sup.6 LOVO cells. When tumors attained a mass of
between 80 and 120 mg, the mice were randomized into treatment
groups (day zero). Animals were treated intravenously once a day on
days 1, 5, and 9 post staging (day zero) with 1 mg/kg/dose of
SPA-110 prepared in 2.5% ethanol in saline or with saline as the
vehicle control. Some animals were treated intraperitonealy once a
day on days 1, 5 and 9 post staging with 1 mg/kg/dose Vincristine
as a positive control. Tumor mass was determined every 7 days
[(length.times.width.sup.2)/2] for 28 days post staging. RTG or
relative tumor growth (Mean tumor mass on days 7, 14, 21 and 28
divided by the mean tumor mass on day zero) was determined for each
treatment group. % T/C was calculated as RTC (treated group) % RTC
(vehicle control group).times.100. Statistical analysis
(Student-t-test) of log relative tumor growth was used to compare
treated verses control group in each experiment. A p-value
(p.ltoreq.0.05), which indicates a statistically significant
reduction in relative tumor growth, was obtained in each case. 5 of
5 animals treated at day 28 with SPA-110 survived. 9 of 10 animals
treated at day 28 with vincristine survived. The results are shown
in the following table.
TABLE-US-00001 % T/C % T/C % T/C % T/C Treatment day 7 day 14 day
21 day 28 Vincristine 34 37 44 47 SPA-110 1 mg /kg 16 18 30 47
[0098] Relative Activity of Compounds of this Invention
[0099] Various analogs of SPA-110 have been synthesized and
characterized for their cytotoxic and anti-mitotic activities. The
high degree of correlation between cytotoxicity and anti-mitotic
capacity indicates that the cytotoxicity of the compounds of this
invention is due to the compounds' anti-mitotic activity. The
following structures are analogs falling within the scope of this
invention depicted in approximately descending order of
cytotoxic/anti-mitotic activity.
TABLE-US-00002 Compound structure (TFA salt) Compound Name Me =
CH.sub.3 SPA110 ##STR00029## SPA115 ##STR00030## SPA123
##STR00031## SPA116 ##STR00032## SPA121 ##STR00033## SPA122
##STR00034## SPA114 ##STR00035##
[0100] As will be apparent to those skilled in the art in the light
of the foregoing disclosure, many alterations and modifications are
possible in the practice of this invention without departing from
the spirit or scope thereof. Accordingly, the scope of the
invention is to be construed in accordance with the substance
defined by the claims herein, which substance includes obvious
chemical equivalents of the compounds and methods set out in the
claims.
* * * * *