Note: Descriptions are shown in the official language in which they were submitted.
CA 02193703 2004-10-13
1
PROTEIN KINASE C INHIBITORS
Protein kinase C (PKC) consists of a family of
closely related enzymes that function as serine/threonine
kinases. Protein kinase C plays an important role in.cell-
cell signaling, gene expression, and in the control of cell
differentiation and growth. At present, there are currently
at least ten known isozymes of PKC that differ in their
tissue distribution. enzymatic specificity, and regulation.
Nishizuka Y. ,gnu. Rev. $~.ochem. ,~"~: 31-44 (1989; Nishizuka
Y. Science ~,: 607-614 (2992?.
Protein kinase C isozymes are single polypeptide
chains ranging from 592 to ?37 amino acids in length. The
isozymes contain a regulatory domain and a catalytic domain
connected by a linker peptide. The regulatory and catalytic
domains can be further subdivided into constant and variable
regions. The catalytic domain of protein kinase C is very
similar to that seen in other protein kinases while the
regulatory domain is unique to the PKC isozymes. The PKC
isozymes demonstrate between 40-80% homology at the amino
acid level among the group, however, the homology of a single
isozyme between different species is generally greater than
9?%.
Protein kinase C is a membrane-associated enzyme
that is allosterically regulated by a number of factors.
including membrane phospholipids, calcium, and certain
membrane lipids such as diacylglycerols that are liberated in
response to the activities of phospholipases. Bell, R.M. and
Burns, D.3., J. ~i,5~1. Chem. ?~6: 4661-4664 11991): Nishizuka,
Y. Science ~8_: 607-614 (1992?. The protein kinase C
isozymes, alpha, beta-1, beta-2 and gamma, require membrane,
phospholipid, calcium and diacylglycerol/phorbol esters for
full activation. The delta, epsilon, eta, and theta forms of
PKC are calcium-independent in their mode of activation. The
zeta and lambda forms of PKC are. independent of both calcium
and diacylglyceroi and are believed to require only membrane
phospholipid for their activation.
2~93°~~3
R'O 95135294 ' ; PCT1US95/07791
Only one or two of the protein kinase C isozymes
may be involved in a given disease state. For example, the
elevated blood glucose levels found in diabetes lead to an
isozyme-specific elevation of the beta-2 isozyme in vascular
tissues. Inoguchi et al., Proc. Natl. Acad. Sci. USA ~Q:
11059-11065 (1992). A diabetes-linked elevation of the beta
isozyme in human platelets has been correlated with their
altered response to agonists. Bastyr III, E.J. and Lu, J.
D~ah ~ ~, (Suppl 1) 97A (1993). The human vitamin D
receptor has been shown to be selectively phosphorylated by
protein kinase C beta. This phosphorylation has been linked
to alterations in the functioning of the receptor. Hsieh et
al., Proc. Natl. Acad. Sci. LTSA ,~$: 9315-9319 (1991); Hsieh
et al., s7. Biol. Chem. 2~: 15118-15126 (I993). In addition,
recent work has shown that the beta-2 isozyme is responsible
for erythroleukemia cell proliferation while the alpha
isozyme is involved in megakaryocyte differentiation in these
same cells. Murray et al., J. Biol. Chem. ~: 15847-15853
(1993).
The ubiquitous nature of the protein kinase C
isozymes and their important roles in physiology provide
incentives to produce highly isozyme selective PKC
inhibitors. Given the evidence demonstrating linkage of
certain isozymes to disease states, it is reasonable to
assume that inhibitory compounds that are selective to one or
two protein kinase C isozymes relative to the other PKC
isozymes are superior therapeutic agents. Such compounds
should demonstrate greater efficacy and lower toxicity by
virtue of their specificity.
The art recognizes various classes of compounds as
protein kinase C inhibitors. Some of these compounds are
also known to demonstrate specificity to protein kinase C.
However, very little is known regarding isozyme selectivity.
Studies of the PKC-selective compound, 3-[1-(3-
dimethylaminopropyl)-indol-3-yl]-4-(1H-indol-3-yl)-1H-
pyrrole-2,5-dione, suggest a slight selectivity for the
calcium dependent isozymes, but find no isozyme selectivity
~O 95!35294
PCTIUS95/07791
- 3 -
between alpha, beta-I, beta-2, and gamma. Toullec et al.,
'-°-~ 2~~: 15771-15781 (1991). Martiny-Baron, et al.,
B=~~~ 2.b.$: 9194-9197 (1993), tested the same
compound and found slight selectivity for isozymes, alpha and
beta versus delta, epsilon, and zeta. Martiny-Baron observed
no differences in the selectivity between alpha and beta-1
isozymes. Wilkinson, et al., Biocue: 335-337
(1993), failed to observe any high degree of isozyme
selectivity and suggest only slight selectivity for the alpha
isozyme and equal inhibition of beta, gamma, and epsilon for
several species of bis-indolemaleimides. Therefore, despite
years of research, there remains a need for therapeutically
effective isozyme-selective inhibitors.
This invention provides compounds that are highly
isozyme selective. The compounds selectively inhibit protein
kinase C beta-1 and beta-2 isozymes. Accordingly, the -
present invention also provides a method of selectively
inhibiting protein kinase C isozymes beta-1 and beta-2. As
isozyme selective inhibitors of beta-1 and beta-2, the
compounds are therapeutically useful in treating conditions
associated with diabetes mellitus and its complications, as
well as other disease states associated with an elevation of
the beta-1 and beta-2 isozymes.
This invention provides compounds., which
selectively inhibit protein kinase C beta-1 and beta-2 -
isozyme, of the Formula I:
R (I)
wherein:
R1 is of the Formula II:
W0 95135294 PCT/US95I07791
r ø -
X
O
Rq
NHP1. ( II ) ;
R2 is hydrogen, alkyl, acyl; alkoxyallcyl,
hydroxyalkyl, aminoalkyl, monoalkylaminoalkyl,
dialkylaminoalkyl, acylaminoalkyl, N3-alkyl, or an amino acid
of the Formula (III):
0
R4
NHP1 (III);
R3 is H or CH3;
Rø is an amino acid side chain;
X is -(CHZ)n-NH-, -(CH2)n-0-, phenylene-NH-,
phenylene-O-, or a bond;
P1 is H, alkyl, or an-amino protecting group; and
n is l, 2 or 3.
The imrention further provides a method of
selectively inhibiting protein kinase C beta-1 and beta-2
isozyme, which comprises administering to a mammal in need of
such treatment a pharmaceutically effective amount of a
compound of the Formula I. As selective inhibitors, the
invention further provides a method for treating diabetes
mellitus, which comprises administering to a mammal in need
of such treatment a pharmaceutically effective amount of a
compound of the Formula I. The invention also provides
~O 95!35294 PCTIUS95/07791
- 5 -
pharmaceutical formulations comprising a compound of the
Formula I associated with one or more pharmaceutically
acceptable excipients, carriers, or diluents.
As noted above, the invention provides compounds of
the Formula I which selectively inhibit isozymes of protein
kinase C.
The preferred compounds are compounds of the
Formula Ia:
Ra (Ia)
wherein: R1 is
0
Rq
~P~ ~ and
R2 is amino alkyl, rnonoalkylamino alkyl, or
dialkylamino alkyl.
Particularly preferred compounds are those wherein
Rq is H, CH3, CH2CH(CH3)2, or
and P1 is t-butoxycarbonyl (BOC) or benzyloxycarbonyl (CBZ).
As used herein, the term "alkyl", alone or in
combinations, means a straight or branched-chain alkyl group
containing from one to seven, preferably one to four, carbon
R'O 95135294 PCTIUS95107791
z
,.. a 't6 '~. ~' '
atoms such as methyl, ethyl, propyl, isopropyl, butyl, sec-
butyl, t-butyl and pentyl. The term "C1-4 alkyl" is an alkyl
limited to one to four carbon atoms. '
The term "alkoxy", alone or in combinations, is an
alkyl covalently bonded to the parent moiety by a -0- '
linkage. Examples of alkoxy groups are methoxy, ethoxy,
propoxy, isopropoxy, butoxy and t-butoxy. An alkoxyalkyl is,
for example, CH3(CH2)z-O-(CH2)z- wherein z is from one to
seven or preferably one to four.
The acyl moiety, alone or in combination, is
derived from an alkanoic acid containing a maximum of seven,
preferably a maximum of four, carbon atoms (e. g. acetyl,
propionyl or butyryl) or from an aromatic carboxylic acid
(e. g. benzoyl). An acylamino is, for example, CH3(C=O)NH-
I5 (acetylamino). Likewise, an acylaminoalkyl is
CH3(C=0)NH(CH2)z- wherein z is from one to seven or
preferably one to four.
The term "amino acid side chain" represents the
variable region of the naturally occurring amino acids, which
are of the formulas:
CH3 CH3
/CH- /CH-CH2- CH3~H2 ~H_
CH3-, ~3 , CH3 , CH3 ,
(Ala) (Val) (Leu) (Ile)
H2
H2C~ C\ ~ C-CHz-
H2C~ ~ ~ ~ CH2- ~ ~ /ICH
H N
, , H
,
(Pro) (Phe) (Trp)
CH3-S-CHZ-CH2-, H-, HO-CH2, HzN-CH2-CHZ-CH2-CHZ-
CA 02193703 2004-10-13
7
tMet) iGly) tSer) (Lys)
off
1
~, _ ~..~. j ~-~Hz
xo ~ ~ cH2 -
o~
FiS--CH2~, ,
tThr) (Cys) (Tyr) tAsn)
HC ~ -~H2- HEN
H2N--C NH-CH2-CHz-CH=- N ~ / N'H ~ C' CH2 - CH2 .
NH , ' H , O
(Arg) (His) (Gln)
Ho' xo\
C-CH2 - ~ C-CH2 ~ CH2
. Or ~
(Asp) (Glu)
The term "amino protecting group" as used herein refers
to substituents conunonly employed to block or protect the
amino functionality. Preferred amino-protecting groups are
t-butoxycarbonyl and benzyloxycarbonyl. Other amino
protecting groups are found in-J. W. Barton, Protective
Groups in Oraani~ C',~hemistrv, J.G.W. McOmie, Ed., Plenum
Press, New York, N.Y., 1973, Chapter 2, and T. W. Greene,
o 'v r s 'n r a 'c , John Wiley and Sons,
New York, N.Y., 1981, Chapter 7.
The related term "protected amino" defines an
amino group substituted with an amino protecting group as
previously discussed.
The term "pharmaceutically effective amount", as
used herein, represents an amount of a compound of the
X9.93703
WO 95135294 '~ ''. ' " PCT/U595/07791
.:..iL Y 1..
- -
invention that is capable of selectively inhibiting PKC
isozyme activity in mammals. The particular dose of the
compound administered according to this invention will, of
course, be determined by a physician under the particular
circumstances surrounding the case, including the compound
administered, the route of administration, the particular
condition being treated, and similar considerations. The
compounds can be administered by a variety of routes
including the oral, rectal, transdermal, subcutaneous,
topical, intravenous, intramuscular or intranasal routes.
The term "treating," as used herein, describes the
management and care of a patient for the purpose of combating
the disease, condition, or disorder and includes the
administration of a compound of present invention to prevent
the onset of the symptoms or complications, alleviating the
symptoms or complications, or eliminating the disease,
condition, or disorder.
The term "isozyme selective" means the preferential
inhibition of protein kinase C beta-1 or beta-2 isozymes over
protein kinase C isozymes, alpha, gamma, delta, epsilon,
zeta, and eta. In general, the compounds demonstrate a
minimum of a eight fold differential, preferably a ten fold
differential, in fhe dosage required to inhibit PKC beta-1 or
beta-2 isozymes and the dosage required for equal inhibition
of the alpha protein kinase C isozyme as measured in the PKC
assay. The compounds demonstrate this differential across
the range of inhibition and are exemplified at the IC50,
i_e., a 50~ inhibition. Accordingly, the invention provides
a method for selectively inhibiting the beta-1 or beta-2
protein kinase C '5a ozyme. A related phrase is "selectively
inhibiting prote;n k;nase C beta-1 and beta-2 isozymes,"
which refers to isozyme selective inhibition. Thus, because
one needs a substantially higher concentration of compound to
inhibit the other,protein kinase C isozymes (e.g., Example 1
discloses 50~ inhibition at a concentration of 0.031 ~tmol/L
for the beta-2 isozyme while the IC50 with respect to the
alpha protein kinase C isozyme is 1.4 Eimol/L) a
CA 02193703 2004-10-13
.. g
pharmaceutically effective dosage of the compound inhibits
beta-1 and beta-2 protein kinase C isozymes with lower
toxicity by virtue of their minimal inhibition of the other
isozymes. Surprisingly, the bis-indolyl maleimide containing
two amino acids (i.e., R1 is of the Formula II and.R2. is of
the Formula III) are yellow in appearance. This is a marked
advantage because a yellow compound does not interfere with
testing or monitoring of the urine of patients taking the
drug.
The preparation of bis-indolylmaleimides of the
Formula:
R~, (IV)
wherein R1'is hydrogen, and R2' is hydrogen, alkyl,
alkoxyalkyl, hydroxyalkyl, aminoalkyl, monoalkylaminoalkyl,
dialkylaminoalkyl, or acylaminoalkyl, is disclosed in U.S.
Patent 5,057,614 and known in the art as evidenced by EPO 397
060 (1990) and Bit et al., J. Met em. ,~: 21-29 (1993).
The compounds of Formula I. are prepared by
reacting a compound of Formula IV with an activated amino
acid of the Formula V:
0
Pz~ HR4C
(V)
wherein P2 is an amino protecting group; and'R4 is an amino
acid side chain.
W095l35294 ~ ~ ~ , ~,, ,,, ' PCTlU595107791
~- 10 -
The acylation of Compound IV with the activated
amino acid (Compound V) is carried out in acetoniLrile in the
presence of 18-crown-6 and fluorine anion under basic
conditions. The compounds containing an amine (e. g. Example
2) are prepared preferably with pyridine as the base for
solubility purposes. The preferred base when the solubility
of the substrate is not important is diisopropylethylamine.
The reaction conditions and other acceptable solvents are
known in the art and described in Rlausner, et al., J. Chem.
Soc. Perkin I: 507-631 (1977); and Nakagawa, et al., J.J. Am.
Chem. Soc., ~: 3709-3710 (1983).
The compounds of Formula V are commercially
available or can be prepared by known methodology, e.g.,
Wolman Y et al, J Chem. Soc. C: 689 (1967); Bodanszky M et
IS aI, J. Amer. Chem. Soc., 81: 2504 (1959); Sakakibara S et
al, g~,l. Chem. Soc. Jun., 37: 1231 (1964)
When preparing the compounds of Formula I, wherein
X is -(CH2)n-NH-, -(CHZ)n-O-, phenylene-NH-, or phenylene-O-,
the linking moiety, X, is appendaged to the bis-
indolylmaleimide-prior to the coupling. Thus, for example,
xs
N
~C
P1HN ~ H2)n
when R1 is o , R1'is aminoalkyl. Likewise,
xa
P HN~~C
II HZ)n
when R1 is o , R1' is hydroxyalkyl. The
coupling reaction when X is -(CHZ)n-O- or phenylene-O- may be
alternatively carried out with dicyclohexylcarbodiimide and
4-dimethylaminopyridine under standard coupling conditions
known in the art.
By virtue of their acidic moieties, the compounds
of Formula I include the pharmaceutically acceptable base
addition salts thereof. Such salts include those derived
from inorganic bases such as ammonium and alkali and alkaline
earth metal hydroxides, carbonates, bicarbonates, and the
'~I ~3?03
~O 95135294 PCT/US95107791
- 11 -
like, as well as salts derived from basic organic amines such
as aliphatic and aromatic amines, aliphatic diamines, hydroxy
alkamines, and the like. Such bases useful in preparing the
salts of this invention thus include ammonium hydroxide,
potassium carbonate, sodium bicarbonate, calcium hydroxide,
methylamine, diethylamine, ethylenediamine, cyclohexylamine,
ethanolamine and the like.
Because of the basic moiety, the compounds of
Formula I can also exist as pharmaceutically acceptable acid
addition salts. Acids commonly employed to form such salts
include inorganic acids such as hydrochloric, hydrobromic,
hydroiodic, sulfuric and phosphoric acid, as well as organic
acids such as para-toluenesulfonic, methanesulfonic, oxalic,
para- bromophenylsulfonic, carbonic, succinic, citric,
benzoic, acetic acid, and related inorganic and organic -
acids. Such pharmaceutically acceptable salts thus include -
sulfate, pyrosulfate, bisulfate, sulfite, bisulfate,
phosphate, monohydrogenphosphate, dihydrogenphosphate,
metaphosphate, pyrophosphate, chloride, bromide, iodide,
acetate, propionate, decanoate, caprylate, acrylate, formate,
isobutyrate, caproate, heptanoate, propiolate, oxalate,
malonate, succinate, suberate, sebacate, fumarate, maleate,
2-butyn-1,4-dioate, 3-hexyn-2, 5-dioate, benzoate,
chlorobenzoate, hydroxybenzoate, methoxybenzoate, phthalate,
xylenesulfonate, phenylacetate, phenylpropionate,
phenylbutyrate, citrate, lactate, hippurate, (3-
hydroxybutyrate, glycollate, maleate, tartrate,
methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, -
naphthalene-2-sulfonate, mandelate and the like salts.
In addition to pharmaceutically-acceptable salts,
other.salts are included in the invention. They may serve as
intermediates in the purification of compounds or in the
preparation of other salts, or are useful for the
identification, characterization or purification.
The pharmaceutically acceptable salts of compounds
of Formula I can also exist as various solvates, such as with
water, methanol, ethanol, dimethylformamide, ethyl acetate
'193703
W095135294 . k~ ~, ' ' fCTIUS'J5~07791
,- 12 -
and the like. Mixtures of such solvates can also be
prepared. The source of such solvate can be from the solvent
of crystallization, inherent in the solvent of preparation or
crystallization,-or adventitious to such solvent. Such
solvates are within the scope of-the present invention.
It is recognized that various stereoisomeric forms
of the compounds of Formula I may exist; for example, R1
introduces a chiral carbon atom. The compounds are normally
prepared as racemates and can conveniently be used as such,
but individual enantiomers can be isolated or synthesized by
conventional techniques if so desired. Such racemates and
individual enantiomers and mixture thereof form part of the
present invention.
The following examples are provided merely to
further illustrate the invention. The scope of the invention
is not construed as merely consisting of the following
examples. In the following eXamples and preparations;-
melting point, nuclear magnetic resonance spectra, mass
spectra, high pressure liquid chromatography, N,N-
dimethylformamide, palladium on charcoal, diisobutylaluminum
hydride, acetonitrile, and tetrahydrofuran are abbreviated
M_Pt., NMR, MS, HPLC, DMF, Pd/C, DIBAL, ACN and THF,
respectively. The terms "NMR"- and "MS" indicate that the
spectrum was consistent with the desired structure.
Example 1
3-[1-(3-azidopropyl)-3-indolyl]-4-[(1-N-tBOC)-glycine-3
indolyl]-1H-pyrrol-2,5-dione
3-[1-(3-azidopropyl)-3-indolyl]-4-[3-indolyl]-1H-
pyrrol-Z,5-dione -(0.2D g, 0.49 mol., 1 eq.) was transferred
to an oven dried 50 mL round bottom flask equipped with a
stir bar, septum, and N2 balloon. The red solid was
dissolved in about 25 mL of anhydrous acetonitrile (delivered
via canula) and stirring started. To the red solution was
added (N-tBOC)glycine, p-nitrophenyl ester (0.22 g, 0.74
21 ~3 X03
~O 95135294 PCTlUS95107791
- 13 -
mmol, 1.5 eq), 18-crown-6 (0.13 g, 0.49 mmol, 1 eq), and N,N-
diisopropylethylamine (0.08 g, 0.61 mmol, 1.25 eq, 0.11 mL),'
and potassium fluoride (0.06 g, 0.98 mmol, 2 eq) with
vigorous stirring. This red solution was allowed to stir
under a N2 atmosphere for 5 days.
TLC (50$ EtOAc in hexanes) of the reaction
indicated total loss of starting materials. The reaction was
diluted with 100 mL of EtOAc, transferred to a separatory
funnel, washed with water, and brine. The organic layer was
dried over MgS04. The solvent was removed in vacuo to obtain
a red oil. This oil was purified by silica flash
chromatography using 37.5 EtOAc in hexanes as the mobile
phase to give 0.2055 g of an orange solid.
NMR, MS
Elemental Analysis:
Theory: C 63.48 N 17.27 H 5.15
Found: C 63.53 N 16.28 H 5.64
Examn~A
3-[1-(3-N,N'-dimethylaminopropyl)-3-indolyl]-4-[(1-N-tBOC)
glycine-3-indolyl]-1H-pyrrol-2,5-dione
3-[1-(3-N,N'-dimethylaminopropyl)-3-indolyl]-4-[3-
indolyl]-1H-pyrrol-2,5-dione (0.05 g, 0.12 mmol, 1 eq) was
transferred to a 100 mL round bottom flask equipped with a stir
bar, septum, and N2 balloon. Anhydrous acetonitrile (70 mL) was
delivered via canula followed by pyridine (0.05 g, 0.62 mmol, 5
eq, 0.05 mL) via syringe. The resulting orange suspension was
heated to 50°C for 60 minutes to help solubilize the compound.
After 60 minutes, (N-tBOC)glycine, p-nitrophenyl ester (0.07 g,
0.24 mmol, 2 eq), 18-crown-6 (0.06 g, 0.24 mmol, 2 eq), and dry
potassium fluoride (0.03 g, 0.48 mmol, 4 eq) were added as solids
with vigorous stirring. The resulting yellow/orange solution was
allowed to stir at room temperature under an atmosphere of N2 for
48 hours. MS.
WO 95135294 _ N .- ;':. : PCT1US95/07791
- 14 -
Proton NMR (300 MHz in CDC13): 1.48 (s, 9H, t-butyl grp
of BOC), 2.4 (2H, NCH2CH2CH2N(CH3)2), 2.95 (6H, -N{CH3)z), 3.25
(2H, CH2N(CH3)2), 3.7 (2H, C(=0)CH2NHBOC), 4.55 (2H,
NCHaCH2CH2N(CH3)2), 5.18 (1H, NHBOC), 6.57-7.4 (10H, aromatics),
8.2 (d, J=9 Hz, 1H, C2' proton of acylated indole), 8.6 (IH,
maleimide NH).
Example 3
3,4-Bis[(1-N-tBOC)-phenylalanine-3-indolyl]-1H-pyrrol-2,5
dione
0
0
NH AN
3,4-Bis[3-indolyll-1H-pyrrol-2,5-dione {0.165 g,
0.5 mmol, 2 eq) was transferred to an oven dried 50 mL round
bottom flask equipped with stir bar, septum, and N2 balloon.
Anhydrous acetonitrile (25 mL) was added via canula and
stirring startedto produce a red solution. (N-
tBOC)pheny7.alanine, p-nitrophenyl ester (0.67 g, I.75 mmol,
3,.5 eq), 18-crown-6 (0.13 g, 0.5 mmol, 2 eq), and
diisopropylethylamine (0.16 g, I.25 mmol, 2.5 eq, 0.22 mL)
were then added to the solution with vigorous stirring. When
everything had dissolved, anhydrous potassium fluoride (0.12
g, 2 mmol, 4 eq) was added as a solid. The solution was
allowed to stir under nitrogen at room temperature overnight.
After 16 hours of stirring, TLC (50~ EtOAc in
hexanes) showed loss of starting material. The yellow
solution was transferred to a separatory funnel with -150 mL
of EtOAc. This solution was washed with water and brine.
~.~ ~3 ~~~ . i
~O 95135294 PCT/US95107791
- 15 -
The organic layer was collected and then dried over Mg504.
The solvent was removed to give a bright yellow/orange
residue. This residue was purified by silica flash
chromatography using 37.5 EtOAc in hexanes as the mobile
phase to give a bright yellow film after the solvent was
removed. HPLC of this solid still showed some impurities so
the product was purified using the gel permeation hydrophobic
columns with CHC13 as the mobile phase to give 229 mg of a
bright yellow solid. NMR, MS (FD (in MeOH): MW-821.94; m/z
822 (MH+),
Rx m~~ 1 d~
3-[1-(3-acetamidepropyl)-3-indolyl]-4-[(1-N-tBOC)-glycine-3
indolyl]-1H-pyrrol-2,5-dione
H
H
TT
N mnam N NHBOC
3
H
3-[1-(3-azidopropyl)-3-indolyl]-4-[(1-N-tBOC)-
glycine-3-indolyl]-1H-pyrrol-2,5-dione (80 mg, 0.14 mmol, 1
eq) was dissolved in ethyl acetate in a 100 mL round bottom
flask equipped with a stir bar and 14/22 adapter. Lindlar~s
catalyst (catalytic, 8 mg) and acetic anhydride {40 mg, 0_38
mmol, 3 eq) were added with vigorous stirring to the red
solution. A hydrogen balloon was placed on top of the flask.
The flask was then evacuated and filled with hydrogen 3 times
to remove dissolved oxygen from the solution. The reaction
was allowed to stir at room temperature under the hydrogen
atmosphere overnight.
After stirring overnight, TLC (50~ EtOAc in
hexanes) of the reaction indicated loss of the starting
CA 02193703 2004-10-13
- 16 -
material. The reaction was filtered through a pad of celite
to remove the hydrogenation catalyst. The red solution was
transferred to a separatory funnel and diluted with '100 mL
of EtOAc. The organic layer was washed with water and brine
and then collected and dried over MgS04. The solvent was
removed to give an~orange residue. This residue was
purified using size exclusion gel per~ation columns using
chloroform as the mobile phase to give 52.4 mg of an orange
residue. MS (FD (iwMeOH): MW-583.65; m/z 584 (MH+),426
(M+-amino acid).
Example 5
3-[1-(3-azidopropyl)-3-indolyl]-4-[(1-N-tBOC)-alanine-3-
indolyl]-1H-pyrrol-2,5-dione
H F3
f
wt
Ns
The azide (0.36 g, 0.88 mmol, 1 eq) was transferred to
a.dried 100 mL round bottom flask equipped with stir bar,
septum, and N2 balloon. Anhydrous acetonitrile was
transferred to the flask via canula and stirring started.
18-Crown-6 (0.23 g, 0.88 mmol, 1 eq) and (N-HOC)alanine, p-
nitrophenyl ester (0.48 g, 1.54 mmol, 1.75 eq) were added as
solids and diisopropylethylamine (0.14 g, 1.10 mmol, 1.25 .
eq) was added via syringe to the vigorously stirred red
solution. The red solution was allowed to stir under N2
overnight.
After stirring for 16 hours, TLC (37.5% EtOAC in
heganes) showed the loss of the starting material. The
reaction was diluted with EtOAc and transferred to a
* Trade-mark
095135294 2~~370~ PCT/US95107791
separatory funnel and washed with water and brine. The
organic layer was collected, dried over MgS04, and the
solvent removed. The resulting orange oil was purified
using a silica get flash column using 37.5% EtOAc in hexanes
as the mobile phase to obtain 0.42 g (82% yield) of a
red/orange solid.
(300 MHz in CDC13): 1.42 (d, J=6 Hz, 3H, methyl grp of
alanine side chain), 1.5 (s, 9H, t-butyl group of BOC
protecting group), 2.0 (m, 2H, NCH2CH2CH2N3), 3.2 (m, 2H,
NCH2CH2CH2N3), 4.2 (m, 2H, NCH2CH2CH2N3), 5.13 (m, 1H, a
proton of alanine), 6.8-8.4 (m, 10H, aromatics).
Example 6
3-[1-(3-N,N'-dimethylaminopropyl)-3-indolyl]-4-[3-indolyl]-
1H-pyrrol-2,5-dione
H
3-[1-(3-N,N'-dimethylaminopropyl)-3-indolyl]-4-[3-
indolyl]-1H-pyrrol-2,5-dione (0.11 g, 0.27 mmol, 1 eg) was
transferred to a dry 100 mL round bottom flask equipped with
stir bar, septum, and N2 balloon. Anhydrous acetonitrile
(25 m7L) was delivered via canula and stirring started. To
the red/orange suspension was added pyridine (0.11 g, 1.35
mmol, 5 eq) via syringe. 18-Crown-6 (0.14 g, 0.54 mmol, 2
eq) and (N-BOC)leucine p-nitrophenylester (0.19 g, 0.54
mmol, 2 eq) were then added as solids to the suspension.
Dry KF (0.06 g, 1.08 mmol, 4 eq) was then quickly added as a
SUBSTITUTE SN~~T ,rr,Uy 2G
WO 95135294 ~ ',' ~~' ' PCTJU595/07791
_ 1s -
solid with vigorous stirring. Vigorous stirring was
continued under N2 at 50°C using a water bath. (After about
3 hours, the suspension became a red/orange solution.)
After 3 days, TLC (10~ MeOH in acetone) showed loss of
starting material. The reaction was diluted with EtOAc and
washed-with water and then brine. The organic layer was
collected, dried over MgS04, and the solvent removed to give
a red/orange oil. This oil was purified by flash
chromatography (silica gel) using 10% MeOH in acetone as the
mobile phase to give 0.11 g of a reddish solid. MS.
Proton NMR (300 MHz in d6-DMSO): 0.70 and 0.76 (d, 3H
each, J=6.6 Hz, the -CH3 groups of the leucine side chain),
0.83 (t, J=7.2 Hz, 1H, CH of leucine side chain), 1.17 (s,
6H, N(CH3)2), 1.39 (s, 9H, BOC group), 1.59 (m, 2H, beta H
on leucine side chain), 1.84 (m, 2H, NCH2CH2CH2N), 2.17 (m,
2H, NCH2CH2CH2N(CH3)2), 4.28 (m, 2H, NCH2CH2CH2N(CH3)2).
4.56 (br, 1H, NHBOC), 4.77 (m, 1H, alpha proton of leucine
side chain), 6.6-8.2 (m, 10H, aromatic protons), 11.2 (s, 1
H, maleimide NH).
Example 7
3-[1-(3-N,N'-dimethylaminopropyl)-3-indolyl]-4-[3-indolyl]
1H-pyrrol-2,5-dione hydrochloride
3-[1-(3-N,N'-dimethylaminopropyl)-3-indolyl]-4-[3-
indolyl]-IH-pyrrol-2,5-dione hydrochloride (0.25 g, 0.56
SUB~TiU"~~~,'-_.r.,::~=~
j'l'O 95135294 PCT/US95107791
- 19 -
mmol, 1 eq) was suspended in 25 mL of anhydrous acetonitrile
in a dry 100 mL round bottom flask equipped with stir bar,
septum, and N2 balloon. The 18-crown-6 (0.30 g, 1.12 mmol,
2 eq) and the (N-BOC)phenylalanine, p-nitrophenyl ester
(0.43 g, 1.12 mmol, 2 eq) were added as solids followed by
pyridine (0.27 g, 3.36 mmol, 6 eq) via syringe. Anhydrous
RF was added as a solid to the vigorously stirred
suspension. The temperature of the flask was raised to 55°C
with a water bath and the suspension was allowed to stir
overnight under a nitrogen atmosphere at 55°C (after about 3
hours the suspension turned into a solution).
After 36 hours of stirring at 55°C, the reaction was
diluted with 300 mh of ethyl acetate and transferred to a
separatory funnel and washed with water and then brine. The
organic layer was collected, dried over MgS04, and the
solvent removed on the rotary evaporator. The orange oil
was purified by flash chromatography on silica gel using 10%
MeOH in acetone as the mobile phase. Removal of the solvent
gave 0.13 g (39~) of an orange solid. MS.
Carbon NMR (75 MHz in CDC13): 26.4, 28.3, 28.5, 29.3,
31.7, 38.8, 44.2, 44.3, 53.8, 54.2, 55.5, 80.3, 105.4,
110.0, 113.7, 116.5, 121.0, 121.8, 122.0, 122.8, 123.8,
124.0, 125.6, 125.7, 126.2, 126.5, 127.1, 128.0, 128.3,
128.4, 129.5, 133.0, 133.3, 135.2, 135.6, 136.3, 170.2,
171.6, 172.0
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PCTIUS95107791
W O 95!35294
- 20 -
Example 8
3-[1-(3-N,N'-dimethylaminopropyl)-3-indolyl]-4-[3-indolyl]- ,
1H-pyrrol-2,5-dione hydrochloride
H H
H3~ ' HCl
N
H3C
3-[1-(3-N,N'-dimethylaminopropyl)-3-indolyl]-4-[3-
indolyl]-1H-pyrrol-2,5-dione hydrochloride (0.25 g, 0.56
mmol, 1 eq) was transferred to a dry 100 mL round bottom
flask equipped with stir bar, septum, and N2 balloon.
Anhydrous acetonitrile (25 mL) was delivered to the round
bottom via canula followed by pyridine (0.27 g, 3.36 mmol, 6
eq) via syringe. 18-Crown-6 (0.30 g, 1.12 mmol, 2 eq), (N-
CBZ)phenylalanine p-nitrophenylester (0.47 g, 1.12 mmol, 2
eq), and KF (0.13 g, 2.24 mmol, 4 eq) were added as solids
to the vigorously stirred suspension. The suspension was
heated to 50°C with a water bath. (The suspension turned
into a red/orange solution after about 3 hours of stirring.)
The solution was allowed to stir at 50°C for 4 days.
After 4 days, TLC (10% MeOH in acetone) showed no
starting material. The reaction was diluted with EtOAc and
transferred to a separatory funnel. It was washed with
water and brine. The organic layer was collected, dried
over MgS04, and the solvent removed to give an orange oil.
This oil was purified by silica gel flash chromatography
using 10% MeOH in acetone as the mobile phase. The
resulting orange solid still contained some impurities and
i ~T4~m.~~m_, ~,~.
~0 95135294 PCT/U595/07791
- 21
was purified using gel filtration chromatography using CHC13
as the mobile phase. MS.
Carbon NMR (75 MHz in CDC13): 24.7, 43.4, 43.7, 45.8,
55.2, 66.8, 110.1, 113.3, 133.4, 116.4, 121.2, 121.7, 122.0,
122.8, 123.9, 125.7, 126.6, 127.0, 127.6, 127.7, 127.8,
127.9, 128.0, 128.1, 128.3, 128.4, 128.6, 129.6, 129.8,
134.4, 134.6, 135.0, 136.0, 154.9, 172.0
As previously noted, the compounds of the present
invention are potent, beta-1 and beta-2 isozyme selective
PRC inhibitors. As such, they are useful in the treatment
of conditions associated with diabetes mellitus and its
complications, as well as other disease states associated
with an elevation of the beta-1 and beta-2 isozymes.
Protein kinase C beta-1 and beta-2 has been linked
to diabetes. Inoguchi et al., Proc. Natl. Acad. Sci USA
89: 11059-11065 (1992). Excessive activity of protein
kinase C has been linked to insulin signaling defects and
therefore to the insulin resistance seen in Type II
diabetes. Rarasik, A. et al., J. Biol. Chem. 265: 10226-
10231 (1990); Chen, R.S. et al., Trans. Assoc. Am.
Physicians ~p4: 206-212 (1991); Chin, J.E. et al., J. Biol. _ _
Chem. 268: 6338-6347 (1993). Further, studies have
demonstrated a marked increase in protein kinase C activity
in tissues known to be susceptible to diabetic complications
when exposed to hyperglycemic conditions. Lee, T.-S. et
al., J. Clin. Invest. 83: 90-94 (1989); Lee, T.-S. et al.,
Proc. Natl. Acad. Sci USA 86: 5141-5145 (1989); Craven,
P.A. and DeRubertis, F.R. J. Clin. Invest. 83: 1667-1675
(1989); Wolf, B.A. et al., J. Clin. Invest. 87: 31-38
(1991); Tesfamariam, B, et al., J. Clin. Invest. 87: 1643-
1648 (1991); Bastyr III, E.J. and Lu, J., Diabetes 42:
(Suppl 1) 97A (1993).
The ability of the compounds of the present
invention to selectively inhibit protein kinase C beta-1 and
beta-2 isozyme was determined in the PRC Enzyme assay.
,SUBSTiTUTt .S'~;-~T, t :.;:,:.~ 2G';
WU 95135294 PCT/US95/07791
_ 22
;iPRC Enzyme Assay
PRC enzymes = alpha, beta I, beta II, gamma, delta, epsilon,
eta and zeta.
Assay components in a total volume of 250 uL are
the following:
Vesicles consisting of 120 ug/mL phosphatidylserine (Avanti
Polar Lipids) and sufficient diacylglycerol (Avanti Polar
Lipids) to activate the enzyme to maximum activity in 20 mM
HEPES buffer (Sigma, St. Louis, Missouri), pH 7.5, 940 uM
calcium chloride (Sigma, St. Louis, Missouri) for assaying
the alpha, beta I, beta II and gamma enzyme only, 1 mM EGTA
for all the enzymes, 10 mM magnesium chloride (Sigma, St.
Louis, Missouri) and 30 pM (gamma-32P) ATP (DuPOnt). For
all the enzymes either histone type HL (Worthington) or
myelin basic protein is used as substrate. The assay is
started by addition of protein kinase C enzyme incubated at
30° C for 10 minutes and stopped by adding 0.5 mL of cold
trichloroacetic acid (Amresco) followed by 100 uL of 1 mg/mL
bovine serum albumin (Sigma, St. Louis, Missouri). The
precipitate is collected by vacuum filtration on glass fiber
filters employing a TOMTECT" filtration system and quantified
by counting in a beta scintillation counter.
Using the methodology described, representative
compounds were evaluated and were found to have an IC50
value with respect to the beta-1 and beta-2 isozyme of below
10 Nm. The compounds are isozyme selective, i.e., the
compounds preferentially inhibit protein kinase C beta-I and
beta-2 isozyme over the protein kinase C isozymes, alpha,
gamma, delta, epsilon, zeta, and eta. In general, the
compounds demonstrate a minimum of an eight fold
differential in the dosage required to inhibit PRC beta-I or
beta-2 isozyme and the dosage required for equal inhibition
of the alpha protein kinase C isozyme as measured in this
assay. Therefore, as selective inhibitors of PRC isozyme
beta-1 and beta-2, the compounds are useful in the treatment
of conditions in which PRC beta has demonstrated a role in
SUWTi~UI=S~~~ ;;~.uLL2~~:
~O 95135294 PCTIUS95107791
- 23 -
the pathology, in particular, diabetes mellitus and its
complications.
Table 1 demonstrates the activity of several
representative compounds.
Table 1
ICSn(um1
Ex. a ~1 (32 y b a
1 1.4 0.26 0.031 19 4.6 NA 100 2.6
2 2.3 0.15 0.04 2.6 2.9 6.1 45 0.37
3 83 8 2.6 >100 >I00 NA >100 NA
NA - data not available
The compounds of Formula I are formulated prior to
administration. A pharmaceutical formulation comprises a
compound of the Formula I with one or more pharmaceutically
acceptable excipients, carriers, or diluents.
Pharmaceutical formulations are prepared by known procedures
using well known and readily available ingredients. In
making the compositions, the active ingredient will usually
be mixed with a carrier, or diluted by a carrier, or
enclosed within a carrier, which may be in the form of a
capsule, sachet, paper or other container. When the carrier
serves as a diluent, it may be a solid, semisolid or liquid
material which acts as a vehicle, excipient or medium for
the active ingredient. Thus, the compositions can be in the
form of tablets, pills, powders, lozenges, sachets, cachets,
elixirs, suspensions, emulsions, solutions, syrups, aerosol
(as a solid or in a liquid medium), soft and hard gelatin
capsules, suppositories, sterile injectable solutions and
sterile packaged powders.
Some examples of suitable carriers, excipients,
and diluents include lactose, dextrose, sucrose, sorbitol,
mannitol, starches, gum acacia, calcium phosphate,
alginates, tragacanth, gelatin, calcium silicate
microcrystalline cellulose, polyvinylpyrrolidone, cellulose,
S:JBSTlTI,'C SHr_=GT;~u~G %Gj
2I93~03
W095135294 ~ -: PCT/US95107791
- 24 -
water syrup, methyl cellulose, methyl and
propylhydroxybenzoates, talc, magnesium stearate and mineral
oil. The formulations can additionally include lubricating
agents, wetting agents, emulsifying and suspending agents,
preserving agents, sweetening agents or flavoring agents.
The compositions of the invention may be formulated so as to
provide quick, sustained or delayed release of the active
ingredient after administration to the patient. The
compositions are preferably formulated in a unit dosage
form, each dosage containing from about 5 to about 500 mg,
more usually about 25 to about 300 mg, of the active
ingredient. However, it will be understood that the
therapeutic dosage administered will be determined by the
physician in the light of the relevant circumstances
including the condition to be treated, the choice of
compound to be administered and the chosen route of
administration, and therefore the above dosage ranges are
not intended to limit the scope of the invention in any way.
The term "unit dosage form" refers to physically discrete
units suitable as unitary dosages for human subjects and
other mammals, each unit containing a predetermined quantity
of active material calculated to produce the desired
therapeutic effect, in association with a suitable
pharmaceutical carrier.
In addition to the above formulations, the
compounds of the present invention may be administered
topically. Topical formulations are ointments, creams, and
gels.
Ointments generally are prepared using either (1)
an oleaginous base, i.e., one consisting of fixed oils or
hydrocarbons, such as white petrolatum or mineral oil, or
(2) an absorbent base, i.e., one consisting of an anhydrous
substance or substances that can absorb water, for example
anhydrous lanolin. Customarily, following formation of the
base, whether oleaginous or absorbent, the active ingredient
(compound) is added to an amount affording the desired
concentration.
SUBSI;r.;:Siiut'1;;~~~e u;
~O 95135294 PCTlU595107791
- 25 -
Creams are oil/water emulsions. They consist of
an oil phase (internal phase), comprising typically fixed
oils, hydrocarbons, and the like, such as waxes, petrolatum,
mineral oil, and the like, and an aqueous phase (continuous
phase), comprising water and any water-soluble substances,
such as added salts. The two phases are stabilized by use
of an emulsifying agent, for example, a surface active
agent, such as sodium lauryl sulfate; hydrophilic colloids,
such as acacia colloidal clays, veegum, and the like. Upon
formation of the emulsion, the active ingredient (compound)
customarily is added to an amount to achieve the desired
concentration.
Gels comprise a base selection from an oleaginous
base, water, or an emulsion-suspension base, such as
aforedescribed. To the base is added a gelling agent that -
forms a matrix in the base increasing its viscosity.
Examples of gelling agents are hydroxypropyl cellulose,
acrylic acid polymers, and the like. Customarily, the
active ingredient (compounds) is added to the formulation at
the desired concentration at a point preceding addition of
the gelling agent.
The amount of compound incorporated into a topical
formulation of invention is not critical; the concentration
should only be a range sufficient to permit ready
application of the formulation to the an affected tissue
area in an amount that will deliver the desired amount of
compound. The customary amount of topical formulation to be
applied to an affected tissue will depend upon an affected
tissue size and concentration of compound in the
formulation. Generally, the formulation will be applied to
the an affected tissue in an amount affording from about 1
to about 500 wg compound per cm2 of an affected tissue.
Preferably, the applied amount of compound will range from
about 30 to about 300 ~g/cm2, more preferably, from about 50
to about 200 ~g/cm2, and, most preferably, from about 60 to
about 100 wg/cm2,
r,
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WO 95135294 PCTlUS95107791
- 26 -
The following formulation examples are
illustrative only and are not intended to limit the scope of
the invention in any way.
Formulation 1
Hard gelatin capsules are prepared using the
following ingredients:
Quantity
(mg/capsule)
Active ingredient 250
starch, dried 200
magnesium stearate 10
Total 460 mg
The above ingredients are mined and filled into hard gelatin
capsules in 460 mg quantities.
Formulation 2
A tablet is prepared using the ingredients below:
Quantity
(mg/capsule)
Active ingredient 250
cellulose, microcrystalline 400
silicon dioxide, fumed 10
stearic acid 5
Total 665 mg
The components are blended and compressed to form tablets
each weighing 665 mg.
i[7 y!~=~.s'.
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j'1'0 95135294 PCT/US95107791
- 27
Formulation 3
An aerosol solution is prepared containing the
following components:
Quantity
(mg/capsule)
Active ingredient 0.25
ethanol 29.75
Propellant 22
(chlorodifluoromethane) 70.00
Total 100.00
The active compound is mixed with ethanol. The mixture is
added to a portion of the Propellant 22, cooled to -30°C and
transferred to a filling device. The required amount is then
fed to a stainless steel container and diluted with the
remainder of the propellant. The valve units are then fitted
to the container.
Formulation 4
Tablets each containing 60 mg of active ingredient
are made as follows:
Quantity
(mg/capsule)
Active ingredient 60 mg
starch 45 mg
roicrocrystalline cellulose 35 mg
polyvinylpyrrolidone
(as 10% solution in water) 4 mg
sodium carboxymethyl starch 4.5 mg
magnesium stearate 0.5 mg
talc
1 mg
Total 150 mg
The active ingredient, starch and cellulose are passed
through a No. 45 mesh U.S. sieve and mixed thoroughly. The
solution of polyvinylpyrrolidone is mixed with the resultant
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PCTlUS95107791
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28
powders which are then passed through a No. 14 mesh U.S.
sieve. The granules so produced are dried at 50°C and passed
through a No. 18 mesh U.S. sieve. The sodium carboxymethyl
starch, magnesium stearate and talc, previously passed
through a No. 60 mesh U.S. sieve, are then added to the
granules which, after mixing, are compressed on a tablet
machine to yield tablets each weighing 150 mg.
Formulation 5
Capsules each containing 80 mg of medicament are
made as follows:
Quantity
(mg/capsule)
Active ingredient- 80 mg
starch 59 mg
microcrystalline cellulose 59 mg
magnesium stearate 2 mg
Total 200 mg
The active ingredient, cellulose, starch and magnesium
stearate are blended, passed through a No. 45 mesh U.S.
sieve, and filled into hard gelatin capsules in 200 mg
quantities.
Formulation 6
20. Suppositories each containing 225 mg of active
ingredient may be made as follows:
Quantity
(mg/capsule)
Active ingredient 225 mg
saturated fatty acid glycerides 2,000 mq
Total 2,225 mg
The active ingredient is passed through a No. 60 mesh U.S.
sieve and suspended in the saturated fatty acid glycerides
previously melted using the minimum heat necessary. The
JUB~TI~4'TESY~E R _:~.~~ .
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~O 95!35294 PCTIUS95/07791
- 29 _
mixture is then poured into a suppository mold of nominal 2
g capacity and allowed to cool.
Formulation 7
Suspensions each containing 50 mg of medicament
per 5 mL dose are made as follows:
Quantity
(mg/capsule)
Active ingredient 50 mg
sodium carboxymethyl cellulose 50 mg
sy~p 1.25 mL
benzoic acid solution 0.10 mL
flavor q.v.
color q.v.
purified water to total 5 ~
The medicament is passed through a No. 45 mesh U.S. sieve
and mixed with the sodium carboxymethyl cellulose and syrup
to form a smooth paste. The benzoic acid solution, flavor
and color are diluted with some of the water and added, with
stirring. Sufficient water is then added to produce the
required volume.
Formulation 8
An intravenous formulation may be prepared as
follows:
Quantity
(mg/capsule)
Active ingredient 250 mg
isotonic saline 1000 mg
The solution of the above ingredients is administered
intravenously at a rate of 1 mL ger minute to a subject in
need of treatment.
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