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| United States Patent |
6,043,272
|
|
Brugnara
, et al.
|
March 28, 2000
|
Substituted diphenyl indanone, indane and indole compounds and analogues
thereof useful for the treatment of prevention of diseases
characterized by abnormal cell proliferation
Abstract
The present invention provides substituted 3,3-diphenyl indanone, indane
and indole compounds, as well as analogues thereof, which are specific,
potent and safe inhibitors of mammalian cell proliferation. The compounds
can be used to inhibit mammalian cell proliferation in situ as a
therapeutic approach towards the treatment or prevention of diseases
characterized by abnormal cell proliferation, such as cancer.
| Inventors:
|
Brugnara; Carlo (Newton Highlands, MA);
Halperin; Jose (Brookline, MA);
Fluckiger; Rudolf (Brookline, MA);
Bellott, Jr.; Emile M. (Beverly, MA);
Lombardy; Richard John (Littleton, MA);
Clifford; John J. (Arlington, MA);
Gao; Ying-Duo (North Edison, NJ);
Haidar; Reem M. (Woburn, MA);
Kelleher; Eugene W. (Somerville, MA);
Moussa; Adel M. (Burlington, MA);
Sachdeva; Yesh P. (Concord, MA);
Sun; Minghua (Libertyville, IL);
Taft; Heather N. (Littleton, MA)
|
| Assignee:
|
Ion Pharmaceuticals, Inc. (New York, NY);
President and Fellows of Harvard College (Cambridge, MA);
Children's Medical Center Corporation (Boston, MA)
|
| Appl. No.:
|
975391 |
| Filed:
|
November 20, 1997 |
| Intern'l Class: |
A61K 031/335; C07C 255/00; C07C 069/76; C07C 069/52 |
| Field of Search: |
514/467,475,544,546,617,640,717
549/430,453,550
558/388
560/56,57,221
564/180,265
568/659,808,809
570/183
|
References Cited
U.S. Patent Documents
| 3546165 | Dec., 1970 | Morgan | 260/47.
|
| Foreign Patent Documents |
| 0 636 608 A1 | Feb., 1995 | EP.
| |
Other References
Chem. Abstr., vol. 109, No. 19, Nov. 7, 1998 (Columbus, OH, USA) p. 696,
col. 2, the abstract No. 170175x, Nishio, T. et al. "A novel route to
indolines by photochemical desulfurization of indoline-2-thiones." J.
Chem. Soc. Commun. 1988, (9), 572-3 (Eng).
Chem. Abstr., vol. 105, No. 23, Dec. 8, 1986 (Columbus, OH, USA) p. 531,
col. 2, the abstract No. 208158b, Barton, D.H.R. et al. "Pentavalent
organobismuth reagents. Part 3. Phenylation of enols and of enolate and
other anions." J. Chem. Soc., Perkin Trans. 1, 1985, (12), 2667-75 (Eng).
Chem. Abstr., vol. 94, No. 1, Jan. 5, 1981 (Columbus, OH, USA) p. 368, col.
1, the abstract no. 3984s Bergman, J. et al. "Synthesis and studies of
tris-indolobenzenes and related compounds." Tetrahedron 1980, 36 (10),
1445-50 (Eng).
Chem. Abstr., vol. 81, No. 13, Sep. 30, 1974 (Columbus, OH, USA) p. 448,
col. 1, the abstract No. 77766j, Isobe, M. et al. "3H-indole. I. General
synthetic route of 3,3-disubstituted 3H-indoles via the corresponding
oxindoles and the indolines." Yakugaku Zasshi 1974, 94(3), 343-50 (Japan).
Dakkouri et al., Chem. Abstract 126:305266, 1997.
Enokida et al., Chem. Abstract 126:39836, 1996.
Barili et al., Chem. Abstract 107:58774, 1987.
Manning et al., Chem. Abstract 94:102538, 1981.
Starnes, Jr., Chem. Abstract 69:43555, 1968.
Koelsch, Chem. Abstract 55:9360, 1961.
Gagnon, Beilstein Reg. No. 4924895, 1929.
|
Primary Examiner: Shah; Mukund J.
Assistant Examiner: Rao; Deepak R.
Attorney, Agent or Firm: Fish & Richardson P.C.
Claims
What is claimed is:
1. A compound having the structural formula:
##STR4##
or a pharmaceutically acceptable salt or hydrate thereof, wherein:
The bond - - - designates a single or double bond;
m is 0, 1, 2, 3 or 4;
each n is independently 0, 1, 2, 3, 4 or 5;
Y is absent, (C.sub.1 -C.sub.6) alkyl, (C.sub.1 -C.sub.6) alkenyl or
(C.sub.1 -C.sub.6) alkynyl;
R.sub.1 is H, --OR, --SR, --O--C(O)R, --S--C(O)R, --O--C(S)R, --S--C(S)R,
or when taken together with R.sub.2 is a .dbd.O, .dbd.S, .dbd.N--OR, a 3-8
membered heterocycloalkyl or a substituted 3-8 membered heterocycloalkyl;
R.sub.2 is absent or --H;
R.sub.3 is absent or --H;
R.sub.4 is --H, --OR', --SR', --NR'.sub.2, --CN, --NO.sub.2, (C.sub.3
-C.sub.8) cycloalkyl, 3-8 membered heterocycloalkyl, --C(O)R', --C(S)R',
--C(O)OR', --C(S)OR', --C(O)SR', --C(S)SR', C(O)NR'.sub.2 or
--C(S)NR'.sub.2 ;
each R.sub.5, R.sub.6 and R.sub.7 is independently selected from the group
consisting of halogen, --R', --OR', --SR', --NR'.sub.2, --ONR'.sub.2,
--SNR'.sub.2, --NO.sub.2, --CN, --C(O)R', --C(S)R', --C(O)OR', --C(O)SR',
--C(S)OR', --CS(S)R', --C(O)NR'.sub.2, --C(S)NR'.sub.2, --C(O)NR'(OR'),
--C(S)NR'(OR'); --C(O)NR'(SR'), --C(S)NR'(SR'), --CH(CN).sub.2,
--CH[C(O)R'].sub.2, --CH[C(S)R'].sub.2, --CH[C(O)OR'].sub.2,
--CH[C(S)OR'].sub.2, --CH[C(O)SR'].sub.2 and --CH[C(S)SR'].sub.2 ; with
the following exceptions:
when - - - is single bond, R.sub.1 and R.sub.2 when taken together are
.dbd.O, Y is absent, R.sub.3 and R4 are H, m=0, n=1; then (a) if R.sub.5
is absent. then R.sub.6 is not Br (para), or OMe (para) or OH (para); (b)
if R.sub.5 is OH (para) then R.sub.6 is not --NMe.sub.2 at the para
position; or
when - - - is single bond, both R.sub.1 and R.sub.2 are H, Y is absent,
R.sub.3 and R4 both are H, m=0; (a) then n is not 0; and (b) if n is 1,
then both R.sub.5 and R.sub.6 are not --NH.sub.2 at para positions; (c) if
n is 1, then both R.sub.5 and R.sub.6 are not --OH at the para position;
or
when - - - is double bond, Y, R.sub.2 and R.sub.3 are absent, R.sub.1 and
R.sub.4 are H, m=0; (a)then n is not 0; (b) if n is 1, R.sub.5 is absent,
then R.sub.6 is not --OMe (para), or Br (para), or --CN (para); or
when - - - is single bond, R.sub.1 and R.sub.2 taken together are .dbd.O, Y
is absent, R.sub.3 is Me, R.sub.4 is 1, m=0, n=1, then both R.sub.5 and
R.sub.6 are not --OH at the para position; or
when - - - is single bond, R.sub.1 and R.sub.2 taken together are .dbd.O, Y
is absent, R.sub.3 is --C(O)OEt, R.sub.4 is H, m=0, n=1, R.sub.5 is
absent, then R.sub.6 is not --OH at the para position; or
when - - - is single bond, R.sub.1 is --OH, R.sub.2 is H, Y is absent,
R.sub.3 and R.sub.4 are H, m=0, n=1, R.sub.5 is absent, then R.sub.6 is
not --Br at the para position; or
when - - - is single bond, R.sub.1 and R.sub.2 taken together are
.dbd.N--OH.HCl, Y is absent, R.sub.3 and R.sub.4 are H, m=0, then n is not
0; and
each R is independently selected from the group consisting of --H, (C.sub.1
-C.sub.6) alkyl, (C.sub.1 -C.sub.6) alkenyl, (C.sub.1 -C.sub.6) alkynyl,
(C.sub.5 -C.sub.20) aryl, substituted (C.sub.5 -C.sub.20) aryl, (C.sub.6
-C.sub.26) alkaryl and substituted (C.sub.6 -C.sub.26) alkaryl;
the heterocycloalkyl substituents are each independently selected from the
group consisting of --CN, --NO.sub.2, --NR'.sub.2, --OR', --C(O)NR'.sub.2,
--C(S)NR'.sub.2, --C(O)OR', --C(S)OR', --C(O)SR', --C(S)SR' and
trihalomethyl;
the aryl substituents are each independently selected from the group
consisting of halogen, --C(O)R', --C(S)R', --C(O)OR', --C(S)OR',
--C(O)SR', --C(S)SR', --C(O)NR'.sub.2, --C(S)NR'.sub.2 and trihalomethyl;
each R' is independently selected from the group consisting of --H,
(C.sub.1 -C.sub.6) alkyl, (C.sub.1 -C.sub.6) alkenyl and (C.sub.1
-C.sub.6) alkynyl.
2. The compound of claim 1, wherein said compound is selected from the
group consisting of
##STR5##
3. A pharmaceutical composition comprising an effective amount of a
compound and a pharmaceutically acceptable excipient, carrier or diluent,
said compound having the structural formula (I): or a pharmaceutically
acceptable salt or hydrates thereof, wherein:
--- designates a single or double bond;
m is 0, 1, 2, 3 or 4;
Y is absent, (C.sub.1 -C.sub.6) alkyl, (C.sub.1 -C.sub.6) alkenyl or
(C.sub.1 -C.sub.6) alkynyl;
R.sub.1 is H, --OR, --SR,, --O--C(O)R, --S--C(O)R, --O--C(S)R, --S--C(S)R,
or when taken together with R.sub.2 is .dbd.O, .dbd.S, .dbd.N--OR, a 3-8
membered heterocycloalkyl or a substituted 3-8 membered heterocycloalkyl;
R.sub.2 is absent or --H;
R.sub.3 is absent or --H;
R.sub.4 is --H, --OR', --SR', --NR'.sub.2, --CN, --NO.sub.2, (C.sub.3
-C.sub.8) cycloalkyl, 3-8 membered heterocycloalkyl, --C(O)R', --C(S)R',
--C(O)OR', --C(S)OR', --C(O)SR', --C(S)SR', --C(O)NR'.sub.2 or
--C(S)NR'.sub.2 ;
each R.sub.5, R.sub.6 and R.sub.7 is independently selected from the group
consisting of halogen, --R', --OR', --SR', --NR'.sub.2, --ONR'.sub.2,
--SNR'.sub.2, --NO.sub.2, --CN, --C(O)R', --C(S)R', --C(O)OR', --C(O)SR',
--C(S)OR', --CS(S)R', --C(O)NR'.sub.2, --C(S)NR'.sub.2, --C(O)NR'(OR'),
--C(S)NR'(OR'); --C(O)NR'(SR'), --C(S)NR'(SR'), --CH(CN).sub.2,
--CH[C(O)R'].sub.2, --CH[C(S)R'].sub.2, --CH[C(O)OR'].sub.2,
--CH[C(S)OR'].sub.2, --CH[C(O)SR'].sub.2 and --CH[C(S)SR'].sub.2 ; with
the following exceptions:
when - - - is single bond, R.sub.1 and R.sub.2 when taken together are
.dbd.O, Y is absent, R.sub.3 and R.sub.4 are H, m=0, n=1; then (a) if
R.sub.5 is absent, then R.sub.6 is not Br (para), or OMe (para) or OH
(para); (b) if R.sub.5 is OH (para) then R.sub.6 is not --NMe.sub.2 at the
para position; or
when - - - is single bond, both R.sub.1 and R.sub.2 are H, Y is absent,
R.sub.3 and R.sub.4 both are H, m=0; (a) then n is not 0; and (b) if n is
1, then both R.sub.5 and R.sub.6 are not --NH.sub.2 at para positions; (c)
if n is 1, then both R.sub.5 and R.sub.6 are not --OH at the para
position; or
when - - - is double bond, Y, R.sub.2 and R.sub.3 are absent, R.sub.1 and
R.sub.4 are H, m=0; (a)then n is not 0; (b) if n is 1, R.sub.5 is absent,
then R.sub.6 are is --OMe (para), or Br (para), or --CN (para); or
when - - - is single bond, RI and R.sub.2 taken together are .dbd.O, Y is
absent, R.sub.3 is Me, R.sub.4 is H, m=0, n=1, then both R.sub.5 and
R.sub.6 are not --OH at the para position; or
when - - - is single bond, R.sub.1 and R.sub.2 taken together are .dbd.O, Y
is absent, R.sub.3 is --C(O)OEt, R.sub.4 is H, m=0, n=1, R.sub.5 is
absent, then R.sub.6 is not --OH at the para position; or
when - - - is single bond, R.sub.1 is --OH, R.sub.2 is H, Y is absent,
R.sub.3 and R.sub.4 are H, m=0, n=1, R.sub.5 is absent, then R.sub.6 is
not --Br at the para position; or
when - - - is single bond, R.sub.1 and R.sub.2 taken together are
.dbd.N--OH.HCl, Y is absent, R.sub.3 and R4 are H, m=0, then n is not 0;
and
each R is independently selected from the group consisting of --H, (C.sub.1
-C.sub.6) alkyl, (C.sub.1 -C.sub.6) alkenyl, (C.sub.1 -C.sub.6) alkynyl,
(C.sub.5 -C.sub.20) aryl, substituted (C.sub.5 -C.sub.20) aryl, (C.sub.6
-C.sub.26) alkaryl and substituted (C.sub.6 -C.sub.26) alkaryl;
the heterocycloalkyl substituents are each independently selected from the
group consisting of --CN, --NO.sub.2, --NR'.sub.2, --OR', --C(O)NR'.sub.2,
--C(S)NR'.sub.2, --C(O)OR', --C(S)OR', --C(O)SR', --C(S)SR' and
trihalomethyl;
the aryl and alkaryl substituents are each independently selected from the
group consisting of halogen, --C(O)R', --C(S)R', --C(O)OR', --C(S)OR',
--C(O)SR', --C(S)SR', --C(O)NR'.sub.2, --C(S)NR'.sub.2 and trihalomethyl;
each R' is independently selected from the group consisting of --H,
(C.sub.1 -C.sub.6) alkyl, (C.sub.1 -C.sub.6) alkenyl and (C.sub.1
-C.sub.6) alkynyl.
4. The pharmaceutical composition of claim 3, wherein in the compound of
structural formula (I):
##STR6##
--- is a single or double bond; m is 0 or 1;
each n is independently 0 or 1;
Y is absent, (C.sub.1 -C.sub.3) alkyl, (C.sub.1 -C.sub.3) alkenyl or
(C.sub.1 -C.sub.3) alkynyl;
R.sub.1 is --H, --OR, --O--C(O)R, or when taken together with R.sub.2 is
.dbd.O, --NR.sub.2, .dbd.N--OR, a 3-5 membered oxirane or 3-5 membered
substituted oxirane;
R.sub.2 is absent or --H;
R.sub.3 is absent or --H;
R.sub.4 is --H, --OR, --NR.sub.2, --CN, --C(O)OR, --C(O)NR.sub.2 or 5-6
membered dioxoycycloalkyl;
each R.sub.5, R.sub.6 and R.sub.7 is independently selected from the group
consisting of --R', --F, --Cl or --Br; with the following exceptions:
when - - - is single bond, R.sub.1 and R.sub.2 when taken together are
.dbd.O, Y is absent, R.sub.3 and R.sub.4 are H, m=0, n=1; and if R.sub.5
is absent, then R.sub.6 is not Br at the para position; or
when - - - is single bond, both R.sub.1 and R.sub.2 are H, Y is absent,
R.sub.3 and R.sub.4 both are H, m=0; then n is not 0; or
when - - - is double bond, Y, R.sub.2 and R.sub.3 are absent, R.sub.1 and
R.sub.4 are H, m=0; then n is not 0; (b) if n is 1, R.sub.5 is absent,
then R.sub.6 is not --Br at the para position; or
when - - - is single bond, R.sub.1 is --OH, R.sub.2 is H, Y is absent,
R.sub.3 and R.sub.4 are H, m=0, n=1, R.sub.5 is absent, then R.sub.6 is
not --Br at the para position;
each R is independently selected from the group consisting of --H, (C.sub.1
-C.sub.3) alkyl, (C.sub.1 -C.sub.3) alkenyl, (C.sub.1 -C.sub.3) alkynyl,
(C.sub.5 -C.sub.10) aryl, substituted (C.sub.5 -C.sub.10) aryl, (C.sub.6
-C.sub.13) alkaryl, substituted (C.sub.6 -C.sub.13) alkaryl;
the oxirane substituent is --CN, --NO.sub.2, --NR'.sub.2, --OR' and
trihalomethyl;
the aryl and alkaryl substituents are each independently selected from the
group consisting of --F, --Cl, --Br, --CN, --NO.sub.2, --NR'.sub.2,
--C(O)R', --C(O)OR' and trihalomethyl;
R' is --H, (C.sub.1 -C.sub.3) alkyl, (C.sub.1 -C.sub.3) alkenyl or (C.sub.1
-C.sub.3) alkynyl.
5. The pharmaceutical composition of claim 4, wherein said compound is
selected from the group consisting of
##STR7##
6. A method of inhibiting mammalian cell proliferation, said method
comprising the step of contacting a mammalian cell in situ with an
effective amount of a compound having the structural formula: or a
pharmaceutically acceptable salt or hydrate thereof, wherein:
--- designates a single or double bond;
m is 0, 1, 2, 3 or 4;
Y is H, --OR, --SR, --O--C(O)R, --S--C(O)R, --O--C(S)R, --S--C(S)R, or when
taken together with R.sub.2 is .dbd.O, .dbd.S, .dbd.N--OR, a 3-8 membered
heterocycloalkyl or a substituted 3--8 membered heterocycloalkyl;
R.sub.2 is absent or --H;
R.sub.3 is absent or --H;
R.sub.4 is --H, --OR', --SR', --NR'.sub.2, --CN, --NO.sub.2, (C.sub.3
-C.sub.8) cycloalkyl, 3-8 membered heterocycloalkyl, --C(O)R', --C(S)R',
--C(O)OR', --C(S)OR', --C(O)SR', --C(S)SR', --C(O)NR'.sub.2 or
--C(S)NR'.sub.2 ;
each R.sub.5, R.sub.6 and R.sub.7 is indepedently selected from the group
consisting of -halogen, --R', --OR', --SR', --NR'.sub.2, --ONR'.sub.2,
--SNR'.sub.2, --NO.sub.2, --CN, --C(O)R', --C(S)R', --C(O)OR', --C(O)SR',
--C(S)OR', --CS(S)R', --C(O)NR'.sub.2, --C(S)NR'.sub.2, --C(O)NR'(OR'),
--C(S)NR'(OR'); --C(O)NR'(SR'), --C(S)NR'(SR'), --CH(CN).sub.2,
--CH[C(O)R'].sub.2, --CH[C(S)R'].sub.2, --CH[C(O)OR'].sub.2,
--CH[C(S)OR'].sub.2, --CH[C(O)SR'].sub.2 and --CH[C(S)SR'].sub.2 ; with
the following exceptions:
when - - - is single bond, RI and R.sub.2 when taken together are .dbd.O, Y
is absent, R.sub.3 and R.sub.4 are H, m=0, n=1; then (a) if R.sub.5 is
absent, then R.sub.6 is not Br (para), or OMe (para) or OH (para); (b) if
R.sub.5 is OH (para) then R.sub.6 is not --NMe.sub.2 at the para position;
or
when - - - is single bond, both R.sub.1 and R.sub.2 are H, Y is absent,
R.sub.3 and R.sub.4 both are H, m=0; (a) then n is not 0; and (b) if n is
1, then both R.sub.5 and R.sub.6 are not --NH.sub.2 at para positions; (c)
if n is 1, then both R.sub.5 and R.sub.6 are not --OH at the para
position; or
when - - - is double bond, Y, R.sub.2 and R.sub.3 are absent, R.sub.1 and
R.sub.4 are H, m=0; (a) then n is not 0; (b) if n is 1, R.sub.5 is absent,
then R.sub.6 are is --OMe (para), or Br (para), or --CN (para); or
when - - - is single bond, R.sub.1 and R.sub.2 taken together are .dbd.O, Y
is absent, R.sub.3 is Me, R.sub.4 is H, m=0, n=1, then both R.sub.5 and
R.sub.6 are not --OH at the para position; or
when - - - is single bond, R.sub.1 and R.sub.2 taken together are .dbd.O, Y
is absent, R.sub.3 is --C(O)OEt, R.sub.4 is H, m=0, n=1, R.sub.5 is
absent, then R.sub.6 is not --OH at the para position; or
when - - - is single bond, R.sub.1 is --OH, R.sub.2 is H, Y is absent,
R.sub.3 and R.sub.4 are H, m=0, n=1, R.sub.5 is absent, then R.sub.6 is
not --Br at the para position; or
when - - - is single bond, R.sub.1 and R.sub.2 taken together are
.dbd.N--OH.HCl, Y is absent, R.sub.3 and R.sub.4 are H, m=0, then n is not
0; and
each R is independently selected from the group consisting of --H, (C.sub.1
-C.sub.6) alkyl, (C.sub.1 -C.sub.6) alkenyl, (C.sub.1 -C.sub.6) alkynyl,
(C.sub.5 -C.sub.20) aryl, substituted (C.sub.5 -C.sub.20) aryl, (C.sub.6
-C.sub.26) alkaryl and substituted (C.sub.6 -C.sub.26) alkaryl;
the heterocycloalkyl substituents are each independently selected from the
group consisting of --CN, --NO.sub.2, --NR'.sub.2, --OR', --C(O)NR'.sub.2,
--C(S)NR'.sub.2, --C(O)OR', --C(S)OR', --C(O)SR', --C(S)SR' and
trihalomethyl;
the aryl and alkaryl substituents are each independently selected from the
group consisting of halogen, --C(O)R', --C(S)R', --C(O)OR', --C(S)OR',
--C(O)SR', --C(S)SR', --C(O)NR'.sub.2, --C(S)NR'.sub.2 and trihalomethyl;
each R' is independently selected from the group consisting of --H,
(C.sub.1 -C.sub.6) alkyl, (C.sub.1 -C.sub.6) alkenyl and (C.sub.1
-C.sub.6) alkynyl.
7. T he method of claim 6, wherein in the compound of structural formula
(1):
--- is a single or double bond;
m is 0 or 1;
Y is absent, (C.sub.1 -C.sub.3) alkyl, (C.sub.1 -C.sub.3) alkenyl or
(C.sub.1 -C.sub.3) alkynyl;
R.sub.1 is --H, --OR, --O--C(O)R, or when taken together with R.sub.2 is
.dbd.O, --NR.sub.2, .dbd.N--OR, 3-5 membered oxirane or 3-5 membered
substituted oxirane;
R.sub.2 is absent or --H;
R.sub.3 is absent or --H;
R.sub.4 is --H, --OR, --NR.sub.2, --CN, --C(O)OR, --C(O)NR.sub.2, or 5-6
membered dioxoycycloalkyl;
each R.sub.5, R.sub.6 and R.sub.7 is independently selected from the group
consisting of --R', --F, --Cl or --Br; with the following exceptions:
when - - - is single bond, R.sub.1 and R.sub.2 when taken together are
.dbd.O, Y is absent, R.sub.3 and R.sub.4 are H, m=0, n=1; and if R.sub.5
is absent, then R.sub.6 is not Br at the para position; or
when - - - is single bond, both R.sub.1 and R.sub.2 are H, Y is absent,
R.sub.3 and R.sub.4 both are H, m=0; then n is not 0; or
when - - - is double bond, Y, R.sub.2 and R.sub.3 are absent, R.sub.1 and
R.sub.4 are H, m=0; then n is not 0; (b) if n is 1, R.sub.5 is absent,
then R.sub.6 is not --Br at the para position; or
when - - - is single bond, R.sub.1 is OH, R.sub.2 is H, Y is absent,
R.sub.3 and R.sub.4 are H, m=0, n=1, R.sub.5 is absent, then R.sub.6 is
not --Br at the para position;
each R is independently selected from the group consisting of --H, (C.sub.1
-C.sub.3) alkyl, (C.sub.1 -C.sub.3) alkenyl, (C.sub.1 -C.sub.3) alkynyl,
(C.sub.5 -C.sub.10) aryl, substituted (C.sub.5 -C.sub.10) aryl, (C.sub.6
-C.sub.13) alkaryl, substituted C.sub.6 -C.sub.13) alkaryl;
the oxirane substituent is --CN, --NO.sub.2, --NR'.sub.2, --OR' and
trihalomethyl;
the aryl and alkaryl substituents are each independently selected from the
group consisting of --F, --Cl, --Br, --CN, --NO.sub.2, --NR'.sub.2,
--C(O)R', --C(O)OR' and trihalomethyl;
R' is --H, (C.sub.1 -C.sub.3) alkenyl (C.sub.1 -C.sub.3) alkenyl or
(C.sub.1 -C.sub.3) alkynyl.
8. The method of claim 7, wherein said compound is selected from the group
consisting of
##STR8##
9. The method of claim 6, wherein said mammalian cell is an endothelial
cell, a fibrotic cell or a vascular smooth muscle cell.
10. A method of treating or preventing a disorder characterized by abnormal
cell proliferation, said method comprising the step of administering to a
subject in need thereof a therapeutically effective mount of a
pharmaceutical composition according to claim 3.
11. The method of claim 10, wherein in the compound of structural formula
(I): --- is a single or double bond;
m is 0 or 1;
each n is independently 0 or 1;
Y is absent, (C.sub.1 -C.sub.3) alkyl, (C.sub.1 -C.sub.3) alkenyl or
(C.sub.1 -C.sub.3) alkynyl;
R.sub.1 is --H, --OR, --O--C(O)R, or when taken together with R.sub.2 is
.dbd.O, --NR.sub.2, .dbd.N--OR, or a 3-5 membered oxirane or 3-5 membered
substituted oxirane;
R.sub.2 is absent or --H;
R.sub.3 is absent or --H;
R.sub.4 is --H, --OR, --NR.sub.2, --CN, --C(O)OR, --C(O)NR.sub.2 or 5-6
membered dioxoycycloalkyl;
each R.sub.5, R.sub.6 and R.sub.7 is independently selected from the group
consisting of --R', --F, --Cl or --Br; with the following exceptions:
when - - - is single bond, R.sub.1 and R.sub.2 when taken together are
.dbd.O, Y is absent, R.sub.3 and R.sub.4 are H, m=0, n=1; and if R.sub.5
is absent, then R.sub.6 is not Br at the para position; or
when - - - is single bond, both R.sub.1, and R.sub.2 are H, Y is absent,
R.sub.3 and R.sub.4 both are H, m=0; then n is not 0; or
when - - - is double bond, Y, R.sub.2 and R.sub.3 are absent, R.sub.1 and
R.sub.4 are H, m=0; then n is not 0; (b) if n is 1, R.sub.5 is absent,
then R.sub.6 is not --Br at the para position; or
when - - - is single bond, R.sub.1 is --OH, R.sub.2 is H, Y is absent,
R.sub.3 and R.sub.4 are H, m=0, n=1, R.sub.5 is absent, then R.sub.6 is
not --Br at the para position;
each R is independently selected from the group consisting of --H, (C.sub.1
-C.sub.3) alkyl, (C.sub.1 -C.sub.3) alkenyl, (C.sub.1 -C.sub.3) alkynyl,
(C.sub.5 -C.sub.10) aryl, substituted (C.sub.5 -C.sub.10) aryl, (C.sub.6
-C.sub.13) alkaryl, substituted (C.sub.6 -C.sub.13) alkaryl;
the oxirane substituent is --CN, --NO.sub.2, --NR'.sub.2, --OR' and
trihalomethyl;
the aryl and alkaryl substituents are each independently selected from the
group consisting of --F, --Cl, --Br, --CN, --NO.sub.2, --NR'.sub.2,
--C(O)R', --C(O)OR' and trihalomethyl;
R' is --H, (C.sub.1 -C.sub.3) alkyl, (C.sub.1 -C.sub.3) alkenyl or (C.sub.1
-C.sub.3) alkynyl.
12. The method of claim 11, wherein said compound is selected from the
group consisting of
##STR9##
13. The method of claim 10, wherein said disease characterized by abnormal
cell proliferation is cancer, a blood vessel proliferative disorder, a
fibrotic disorder or an arteriosclerotic condition.
14. The method of claim 13, wherein said administration of said compound is
per oral, parenteral or intravenous.
15. The method of claim 10, wherein said disease characterized by abnormal
cell proliferation is a dermatological disease or Kaposi's sarcoma and
said administration is transdermal.
16. The method of claim 15, wherein said dermatological disease is selected
from the group consisting of keloids, hypertonic scars, seborrheic
dermatosis, papilloma virus infection, eczema and actinic keratosis.
Description
1. FIELD OF THE INVENTION
The present invention relates to aromatic organic compounds which are
specific, potent and safe inhibitors of the Ca.sup.2+ -activated potassium
channel (Gardos channel) of erythrocytes and/or of mammalian cell
proliferation. The compounds can be used to reduce sickle erythrocyte
dehydration and/or delay the occurrence of erythrocyte sickling or
deformation in situ as a therapeutic approach towards the treatment or
prevention of sickle cell disease. The compounds can also be used to
inhibit mammalian cell proliferation in situ as a therapeutic approach
towards the treatment or prevention of diseases-characterized by abnormal
cell proliferation.
2. BACKGROUND OF THE INVENTION
Sickle cell disease has been recognized within West Africa for several
centuries. Sickle cell anemia and the existence of sickle hemoglobin (Hb
S) was the first genetic disease to be understood at the molecular level.
It is recognized today as the morphological and clinical result of a
glycine to valine substitution at the No. 6 position of the beta globin
chain (Ingram, 1956, Nature 178:792-794. The origin of the amino acid
change and of the disease state is the consequence of a single nucleotide
substitution (Marotta et al., 1977, J. Biol. Chem. 252:5040-5053). The
major source of morbidity and mortality of patients suffering from sickle
cell disease is vascular occlusion , caused by the sickled cells, which
causes repeated episodes of pain in both acute and chronic form and also
causes ongoing organ damage with the passage of time. It has long been
recognized and accepted that the deformation and distortion of sickle cell
erythrocytes upon complete deoxygenation is caused by polymerization and
intracellular gelation of sickle hemoglobin, hemoglobin S (Hb S). The
phenomenon is well reviewed and discussed by Eaton and Hofrichter, 1987,
Blood 70:1245. The intracellular gelatin and polymerization of Hb S can
occur at any time during erythrocyte's journey through the vasculature.
Thus, erythrocytes in patients with sickle cell disease containing no
polymerized hemoglobin S may pass through the microcirculation and return
to the lungs without sickling, may sickle in the veins or may sickle in
the capillaries.
The probability of each of these events is determined by the delay time for
intracellular gelation relative to the appropriate capillary transit time
(Eaton et al., 1976, Blood 47:621). In turn, the delay time is dependent
upon the oxygenation state of the hemoglobin, with deoxygenation
shortening the delay time. Thus, if it is thermodynamically impossible for
intracellular gelation to take place, or if the delay time at venous
oxygen pressures is longer than about 15 seconds, cell sickling will not
occur. Alternatively, if the delay time is between about 1 and 15 seconds,
the red cell will likely sickle in the veins. However, if the delay time
is less than about 1 second, red cells will sickle within the capillaries.
For red cells that sickle within the capillaries, a number of possible
consequent events exist, ranging from no effect on transit time, to
transient occlusion of the capillary, to a more permanent blockage that
may ultimately result in ischemia or infarction of the surrounding cells,
and in destruction of the red cell.
It has long been recognized that the cytoplasm of the normal erythrocyte
comprises approximately 70% water. Water crosses a normal erythrocyte
membrane in milliseconds; however, the loss of cell water causes an
exponential increase in cytoplasmic viscosity as the mean cell hemoglobin
concentration (MCHC) rises above about 32 g/dl. Since cytoplasmic
viscosity is a major determinate of erythrocyte deformability and
sickling, the dehydration of the erythrocyte has substantial rheological
and pathological consequences. Thus, the physiological mechanisms that
maintain the water content of normal erythrocytes and the pathological
conditions that cause loss of water from erythrocytes in the blood
circulation are critically important. Not surprisingly, regulation of
erythrocyte dehydration has been recognized as an important therapeutic
approach towards the treatment of sickle cell disease. Since cell water
will follow any osmotic change in the intracellular concentration of ions,
the maintenance of the red cell's potassium concentration is of particular
importance (Stuart and Ellory, 1988, Brit J. Haematol. 69:1-4).
Many attempts and approaches to therapeutically treating dehydrated sickle
cells (and thus decreasing polymerization of hemoglobin S by lowering the
osmolality of plasma) have been tried with limited success, including the
following approaches: intravenous infusion of distilled water (Gye et al.,
1973, Am. J. Med. Sci. 266:267-277); administration of the antidiuretic
hormone vasopressin together with a high fluid intake and salt restriction
(Rosa et al., 1980, M. Eng. J. Med. 303:1138-1143; Charache and Walker,
1981, Blood 58:892-896); the use of monensin to increase the cation
content of the sickle cell (Clark et al., 1982, J. Clin. Invest.
70:1074-1080; Fahim and Pressman, 1981, Life Sciences 29:1959-1966);
intravenous administration of cetiedil citrate (Benjamin et al., 1986,
Blood 67:1442-1447; Berkowitz and Orringer, 1984, Am. J. Hematol.
17:217-223; Stuart et al., 1987, J. Clin. Pathol. 40:1182-1186); and the
use of oxpentifylline (Stuart et al., 1987, J. Clin. Pathol.
40:1182-1186).
Another approach towards therapeutically treating dehydrated sickle cells
involves the administration of imidazole, nitroimidazole and triazole
antimycotic agents such as Clotrimazole (U.S. Pat. No. 5,273,992 to
Brugnara et al.). Clotrimazole, an imidazole-containing antimycotic agent,
has been shown to be a specific, potent inhibitor of the Gardos channel of
normal and sickle erythrocytes, and prevents Ca.sup.2+ -dependent
dehydration of sickle cells both in vitro and in vivo (Brugnara et al.,
1993, J. Clin. Invest. 92:520-526; De Franceschi et al., 1994, J. Clin.
Invest. 93:1670-1676). When combined with a compound which stabilizes the
oxyconformation of Hb S, Clotrimazole induces an additive reduction in the
clogging rate of a micropore filter and may attenuate the formation of
irreversibly sickled cells (Stuart et al., 1994, J. Haematol. 86:820-823).
Other compounds that contain a heteroaryl imidazole-like moiety believed
to be useful in reducing sickle erythrocyte dehydration via Gardos channel
inhibition include miconazole, econazole, butoconazole, oxiconazole and
sulconazole. Each of these compounds is a known antimycotic. Other
imidazole-containing compounds have been found to be incapable of
inhibiting the Gardos channel and preventing loss of potassium.
As can be seen from the above discussion, reducing sickle erythrocyte
dehydration via blockade of the Gardos channel is a powerful therapeutic
approach towards the treatment and/or prevention of sickle cell disease.
Compounds capable of inhibiting the Gardos channel as a means of reducing
sickle cell dehydration are highly desirable, and are therefore an object
of the present invention.
Cell proliferation is a normal part of mammalian existence, necessary for
life itself. However, cell proliferation is not always desirable, and has
recently been shown to be the root of many life-threatening diseases such
as cancer, certain skin disorders, inflammatory diseases, fibrotic
conditions and arteriosclerotic conditions.
Cell proliferation is critically dependent on the regulated movement of
ions across various cellular compartments, and is associated with the
synthesis of DNA. Binding of specific polypeptide growth factors to
specific receptors in growth-arrested cells triggers an array of early
ionic signals that are critical in the cascade of mitogenic events
eventually leading to DNA synthesis (Rozengurt, 1986, Science
234:161-164). These include (1) a rapid increase in cystolic Ca.sup.2+,
mostly due to rapid release of Ca.sup.2+ from intracellular stores; (2)
capacitative Ca.sup.2+ influx in response to opening of ligand-bound and
hyperpolarization-sensitive Ca.sup.2+ channels in the plasma membrane
that contribute further to increased intracellular Ca.sup.2+
concentration (Tsien and Tsien, 1990, Annu. Rev. Cell Biol. 6:715-760;
Peppelenbosch et al., 1991, J. Biol. Chem. 266:19938-19944); and (3)
activation of Ca2+-dependent K.sup.+ channels in the plasma membrane with
increased K.sup.+ conductance and membrane hyperpolarization (Magni et
al., 1991, J. Biol. Chem. 261:9321-9327). These mitogen-induced early
ionic changes, considered critical events in the signal transduction
pathways, are powerful therapeutic targets for inhibition of cell
proliferation in normal and malignant cells.
One therapeutic approach towards the treatment of diseases characterized by
unwanted or abnormal cell proliferation via alteration of the ionic fluxes
associated with early mitogenic signals involves the administration of
Clotrimazole. As discussed above, Clotrimazole has been shown to inhibit
the Ca.sup.2+ -activated potassium channel of erythrocytes. In addition,
Clotrimazole inhibits voltage- and ligand-stimulated Ca.sup.2+, influx
mechanisms in nucleated cells (Villalobos et al., 1992, FASEB J.
6:2742-2747; Montero et al., 1991, Biochem. J. 277:73-79) and inhibits
cell proliferation both in vitro and in vivo (Benzaquen et al., 1995,
Nature Medicine 1:534-540). Recently, Clotrimazole and other
imidazole-containing antimycotic agents capable of inhibiting Ca.sup.2
+-activated potassium channels have been shown to be useful in the
treatment of arteriosclerosis (U.S. Pat. No. 5,358,959 to Halperin et
al.), as well as other disorders characterized by unwanted or abnormal
cell proliferation.
As can be seen from the above discussion, inhibiting mammalian cell
proliferation via alteration of ionic fluxes associated with early
mitogenic signals is a powerful therapeutic approach towards the treatment
and/or prevention of diseases characterized by unwanted or abnormal cell
proliferation. Compounds capable of inhibiting mammalian cell
proliferation are highly desirable, and are therefore also an object of
the present invention.
3. SUMMARY OF THE INVENTION
These and other objects are provided by the present invention, which in one
aspect provides a class of organic compounds which are potent, selective
and safe inhibitors of the Ca.sup.2+ -activated potassium channel (Gardos
channel) of erythrocytes, particularly sickle erythrocytes, and/or of
mammalian cell proliferation. The compounds are generally substituted
3,3-diphenyl indanone, indane or (3-H) indole compounds, or analogues
thereof. In one illustrative embodiment, the compounds capable of
inhibiting the Gardos channel and/or mammalian cell proliferation
according to the invention are compounds having the structural formula:
##STR1##
or pharmaceutically acceptable salts or hydrates thereof, wherein: m is 0,
1, 2, 3 or 4;
each n is independently 0, 1, 2, 3, 4 or 5;
X is C or N;
Y is absent, (C.sub.1 -C.sub.6) alkyl, (C.sub.1 -C.sub.6) alkenyl or
(C.sub.1 -C.) alkynyl;
R.sub.1 is absent, --OR, --SR, .dbd.O, .dbd.S, .dbd.N--OR, --O--C(O)R,
--S--C(O)R, --O--C(S)R, --S--C(S)R, or when taken together with R.sub.2 is
a 3-8 membered heterocycloalkyl or a substituted 3-8 membered
heterocycloalkyl;
R.sub.2 is absent or --H;
R.sub.3 is absent or --H;
R.sub.4 is --H, --OR', --SR', --NR'.sub.2, --CN, --NO.sub.2, (C.sub.3
-C.sub.8) cycloalkyl, 3-8 membered heterocycloalkyl, --C(O)R', --C(S)R',
--C(O)OR', --C(S)OR', --C(O)SR', --C(S)SR', --C(O)NR'.sub.2 or
--C(S)NR'.sub.2 ;
each R.sub.5, R.sub.6 and R.sub.7 is independently selected from the group
consisting of -halogen, --R', --OR', --SR', --NR'.sub.2, --ONR'.sub.2,
--SNR'.sub.2, --NO.sub.2, --CN, --C(O)R', --C(S)R', --C(O)OR', --C(O)SR',
--C(S)OR', --CS(S)R', --C(O)NR'.sub.2, --C(S)NR'.sub.2, --C(O)NR'(OR'),
--C(S)NR'(OR'); --C(O)NR'(SR'), --C(S)NR'(SR'), --CH(CN).sub.2,
--CH[C(O)R'].sub.2, --CH[C(S)R'].sub.2, --CH[C(O)OR'].sub.2,
--CH[C(S)OR'].sub.2, --CH[C(O)SR'].sub.2 and --CH[C(S)SR'].sub.2 ;
each R is independently selected from the group consisting of --H, (C.sub.1
-C.sub.6) alkyl, (C.sub.1 -C.sub.6) alkenyl, (C.sub.1 -C.sub.6) alkynyl,
(C.sub.5 -C.sub.20) aryl, substituted (C.sub.5 -C.sub.20) aryl, (C.sub.6
-C.sub.26) alkaryl and substituted (C.sub.6 -C.sub.26) alkaryl;
the heterocycloalkyl substituents are each independently selected from the
group consisting of --CN, --NO2, --NR'.sub.2, --OR', --C(O)NR'.sub.2,
--C(S)NR'.sub.2, --C(O)OR', --C(S)OR', --C(O)SR', --C(S)SR' and
trihalomethyl;
the aryl and alkaryl substituents are each independently selected from the
group consisting of halogen, --C(O)R', --C(S)R', --C(O)OR', --C(S)OR',
--C(O)SR', --C(S)SR', --C(O)NR'.sub.2, --C(S)NR'.sub.2 and trihalomethyl;
each R' is independently selected from the group consisting of --H,
(C.sub.1 -C.sub.6) alkyl, (C.sub.1 -C.sub.6) alkenyl and (C.sub.1
-C.sub.6) alkynyl; and
--- designates a single or double bond.
In another aspect, the present invention provides pharmaceutical
compositions comprising one or more compounds according to the invention
in admixture with a pharmaceutically acceptable carrier, excipient or
diluent. Such a preparation can be administered in the methods of the
invention.
In still another aspect, the invention provides a method for reducing
sickle erythrocyte dehydration and/or delaying the occurrence of
erythrocyte sickling or deformation in situ. The method involves
contacting a sickle erythrocyte in situ with an amount of at least one
compound according to the invention, or a pharmaceutical composition
thereof, effective to reduce sickle erythrocyte dehydration and/or delay
the occurrence of erythrocyte sickling or deformation. In a preferred
embodiment, the sickle cell dehydration is reduced and erythrocyte
deformation is delayed in a sickle erythrocyte that is within the
microcirculation vasculature of a subject, thereby preventing or reducing
the vaso-occlusion and consequent adverse effects that are commonly caused
by sickled cells.
In still another aspect, the invention provides a method for the treatment
and/or prevention of sickle cell disease in a subject, such as a human.
The method involves administering a prophylactically or therapeutically
effective amount of at least one compound according to the invention, or a
pharmaceutical composition thereof, to a patient suffering from sickle
cell disease. The patient may be suffering from either acute sickle crisis
or chronic sickle cell episodes.
In yet another aspect, the invention provides a method for inhibiting
mammalian cell proliferation in situ. The method involves contacting a
mammalian cell in situ with an amount of at least one compound according
to the invention, or a pharmaceutical composition thereof, effective to
inhibit cell proliferation. The compound or composition may act either
cytostatically, cytotoxically or a by a combination of both mechanisms to
inhibit proliferation. Mammalian cells in this manner include vascular
smooth muscle cells, fibroblasts, endothelial cells, various types of
pre-cancer cells and various types of cancer cells.
In still another aspect, the invention provides a method for treating
and/or preventing unwanted or abnormal cell proliferation in a subject,
such as a human. In the method, at least one compound according to the
invention, or a pharmaceutical composition thereof, is administered to a
subject in need of such treatment in an amount effective to inhibit the
unwanted or abnormal mammalian cell proliferation. The compound and/or
composition may be applied locally to the proliferating cells, or may be
administered to the subject systemically. Preferably, the compound and/or
composition is administered to a subject that has a disorder characterized
by unwanted or abnormal cell proliferation. Such disorders include, but
are not limited to, cancer, epithelial precancerous lesions, non-cancerous
angiogenic conditions or arteriosclerosis.
In a final aspect, the invention provides a method for the treatment and/or
prevention of diseases that are characterized by unwanted and/or abnormal
mammalian cell proliferation. The method involves administering a
prophylactically or therapeutically effective amount of at least one
compound according to the invention, or a pharmaceutical composition
thereof, to a subject in need of such treatment. Diseases that are
characterized by abnormal mammalian cell proliferation which can be
treated or prevented by way of the methods of the invention include, but
are not limited to, cancer, blood vessel proliferative disorders, fibrotic
disorders and arteriosclerotic conditions.
3.1 Definitions
As used herein, the following terms shall have the following meanings:
"Alkyl:" refers to a saturated branched, straight chain or cyclic
hydrocarbon radical. Typical alkyl groups include, but are not limited to,
methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl,
isopentyl, hexyl, and the like. In preferred embodiments, the alkyl groups
are (C.sub.1 -C.sub.6) alkyl, with (C.sub.1 -C.sub.3) being particularly
preferred.
"Substituted Alkyl:" refers to an alkyl radical wherein one or more
hydrogen atoms are each independently replaced with other substituents.
"Heterocycloalkyl:" refers to a saturated cyclic hydrocarbon radical
wherein one or more of the carbon atoms are replaced with another atom
such as Si, Ge, N, O, S or P. Typical heterocycloalkyl groups include, but
are not limited to, morpholino, thiolino, piperadyl, pynolidinyl,
piperazyl, pyrazolidyl and the like. Preferably, the heterocycloalkyl
group contains 3-8 atoms. In a particularly preferred embodiment the
heteroatoms are oxygen, and the heterocycloalkyl group is a 3-8 membered
oxirane, preferably 2,3-oxirane or a 5-8 membered dioxycycloalkyl,
preferably 1,3-dioxolanyl.
"Substituted Heterocycloalkvl:" refers to a heterocycloalkyl group wherein
one or more hydrogen atoms are each independently replaced with other
substituents.
"Alkenyl:" refers to an unsaturated branched, straight chain or cyclic
hydrocarbon radical having at least one carbon-carbon double bond. The
radical may be in either the cis or trans conformation about the double
bond(s). Typical alkenyl groups include, but are not limited to, ethenyl,
propenyl, isopropenyl, butenyl, isobutenyl, tert-butenyl, pentenyl,
hexenyl and the like. In preferred embodiments, the alkenyl group is
(C.sub.1 -C.sub.6) alkenyl, with (C.sub.1 -C.sub.3) being particularly
preferred.
"Substituted Alkenyl:" refers to an alkenyl radical wherein one or more
hydrogen atoms are each independently replaced with other substituents.
"Alkynyl:" refers to an unsaturated branched, straight chain or cyclic
hydrocarbon radical having at least one carbon-carbon triple bond. Typical
alkynyl groups include, but are not limited to, ethynyl, propynyl,
butynyl, isobutynyl, pentynyl, hexynyl and the like. In preferred
embodiments, the alkynyl group is (C.sub.1 -C.sub.6) alkynyl, with
(C.sub.1 -C.sub.3) being particularly preferred.
"Substituted Alkynyl:" refers to an alkynyl radical wherein one or more
hydrogen atoms are each independently replaced with other substituents.
"Alkoxy:" refers to an --OR radical, where R is alkyl, alkenyl or alkynyl,
as defined above.
"Alksulfanvl:" refers to an --mSR radical, where R is alkyl, alkenyl or
alkynyl, as defined above.
"Aryl:" refers to an unsaturated cyclic hydrocarbon radical having a
conjugated .pi. electron system. Typical aryl groups include, but are not
limited to, penta-2,4-diene, phenyl, naphthyl, anthracyl, azulenyl,
indacenyl, and the like. In preferred embodiments, the aryl group is
(C.sub.5 -C.sub.20) aryl, with (C.sub.5 -C.sub.10) being particularly
preferred.
"Substituted Aryl:" refers to an aryl radical wherein one or more hydrogen
atoms are each independently replaced with other substituents.
"Alkaryl:" refers to a straight-chain alkyl, alkenyl or alkynyl group
wherein one of the hydrogen atoms bonded to a terminal carbon is replaced
with an aryl moiety. Typical alkaryl groups include, but are not limited
to, benzyl, benzylidene, benzylidyne, benzenobenzyl, naphthenobenzyl and
the like. In preferred embodiments, the alkaryl group is (C.sub.6
-C.sub.26) alkaryl, i.e., the alkyl, alkenyl or alkynyl moiety of the
alkaryl group is (C.sub.1 -C.sub.6) and the aryl moiety is (C.sub.5
-C.sub.20). In particularly preferred embodiments, the alkaryl group is
(C.sub.6 -C.sub.13) alkaryl, i.e., the alkyl, alkenyl or alkynyl moiety of
the alkaryl group is (C.sub.1 -C.sub.3) and the aryl moiety is (C.sub.5
-C.sub.10).
"Substituted Alkaryl:" refers to an alkaryl radical wherein one or more
hydrogen atoms on the aryl moiety of the alkaryl group are each
independently replaced with other substituents.
"In Situ:" refers to and includes the terms "in vivo," "ex vivo," and "in
vitro" as these terms are commonly recognized and understood by persons
ordinarily skilled in the art. Moreover, the phrase "in situ" is employed
herein in its broadest connotative and denotative contexts to identify an
entity, cell or tissue as found or in place, without regard to its source
or origin, its condition or status or its duration or longevity at that
location or position.
4. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a general reaction scheme for synthesizing certain compounds
according to the invention; and
FIG. 2 is a general reaction scheme for synthesizing certain compounds
according to the invention.
5. DETAILED DESCRIPTION OF THE INVENTION
As discussed in the Background section, blockade of sickle dehydration via
inhibition of the Gardos channel is a powerful therapeutic approach
towards the treatment and/or prevention of sickle cell disease. In vitro
studies have shown that Clotrimazole, an imidazole-containing antimycotic
agent, blocks Ca.sup.2+ -activated K.sup.+ transport and cell dehydration
in sickle erythrocytes (Brugnara et al., 1993, J. Clin. Invest.
92:520-526). Studies in a transgenic mouse model for sickle cell disease
(SAD mouse, Trudel et al., 1991, EMBO J. 11:3157-3165) show that oral
administration of Clotrimazole leads to inhibition of the red cell Gardos
channel, increased red cell K.sup.+ content, a decreased mean cell
hemoglobin concentration (MCHC) and decreased cell density (De Franceschi
et al., 1994, J. Clin. Invest. 93:1670-1676). Moreover, therapy with oral
Clotrimazole induces inhibition of the Gardos channel and reduces
erythrocyte dehydration in patients with sickle cell disease (Brugnara et
al., 1996, J. Clin. Invest. 97:1227-1234). Other antimycotic agents which
inhibit the Gardos channel in vitro include miconazole, econazole,
butoconazole, oxiconazole and sulconazole (U.S. Pat. No. 5,273,992 to
Brugnara et al.). All of these compounds contain an imidazole-like ring,
i.e., a heteroaryl ring containing two or more nitrogens.
Also as discussed in the Background section, the modulation of early ionic
mitogenic signals and inhibition of cell proliferation are powerful
therapeutic approaches towards the treatment and/or prevention of
disorders characterized by abnormal cell proliferation. It has been shown
that Clotrimazole, in addition to inhibiting the Gardos channel of
erythrocytes, also modulates ionic mitogenic signals and inhibits cell
proliferation both in vitro and in vivo.
For example, Clotrimazole inhibits the rate of cell proliferation of normal
and cancer cell lines in a reversible and dose-dependent manner in vitro
(Benzaquen et al., 1995 Nature Medicine 1:534-540). Clotrimazole also
depletes the intracellular Ca.sup.2+ stores and prevents the rise in
cystolic Ca.sup.2+ that normally follows mitogenic stimulation. Moreover,
in mice with severe combined immunodeficiency disease (SCID) and
inoculated with MM-RU human melanoma cells, daily administration of
Clotrimazole resulted in a significant reduction in the number of lung
metastases observed (Benzaquen et al., supra).
It has now been discovered that substituted 3,3-diphenyl indanone, indane
and (3-H) indole compounds, as well as analogues of these classes of
compounds, also inhibit the Gardos channel of erythrocytes and/or
mammalian cell proliferation. Thus, in one aspect, the present invention
provides a new class of organic compounds that are capable of inhibiting
the Ca.sup.2+ -activated potassium channel (Gardos channel) of
erythrocytes, particularly sickle erythrocytes and/or of inhibiting
mammalian cell proliferation, particularly mitogen-induced cell
proliferation.
The activities of these compounds are quite surprising.
Significantly, the compounds of the invention do not contain an imidazole
or imidazole-like moiety. The imidazole or imidazole-like moiety is
well-recognized as the essential functionality underlying the antimycotic
and other biological activities of Clotrimazole and the other
above-mentioned antimycotic agents. Thus, the substituted 3,3-diphenyl
indanone, indane or (3-H) indole compounds and analogues of the invention
provide an entirely new class of compounds capable of effecting inhibition
of the Gardos channel and/or mammalian cell proliferation.
In another aspect, the invention provides a method of reducing sickle cell
dehydration and/or delaying the occurrence of erythrocyte sickling in situ
as a therapeutic approach towards the treatment of sickle cell disease. In
its broadest sense, the method involves only a single step--the
administration of at least one pharmacologically active compound of the
invention, or a composition thereof, to a sickle erythrocyte in situ in an
amount effective to reduce dehydration and/or delay the occurrence of cell
sickling or deformation.
While not intending to be bound by any particular theory, it is believed
that administration of the active compounds described herein in
appropriate amounts to sickle erythrocytes in situ causes nearly complete
inhibition of the Gardos channel of sickle cells, thereby reducing the
dehydration of sickle cells and/or delaying the occurrence of cell
sickling or deformation. In a preferred embodiment, the dehydration of a
sickle cell is reduced and/or the occurrence of sickling is delayed in a
sickle cell that is within the microcirculation vasculature of the
subject, thereby reducing or eliminating the vaso-occlusion that is
commonly caused by sickled cells.
Based in part on the surmised importance of the Gardos channel as a
therapeutic target in the treatment of sickle cell disease, the invention
is also directed to methods of treating or preventing sickle cell disease.
In the method, an effective amount of one or more compounds according to
the invention, or a pharmaceutical composition thereof, is administered to
a patient suffering from sickle cell disease. The methods may be used to
treat sickle cell disease prophylactically to decrease intracellular Hb S
concentration and/or polymerization, and thus diminish the time and
duration of red cell sickling and vaso-occlusion in the blood circulation.
The methods may also be used therapeutically in patients with acute sickle
cell crisis, and in patients suffering chronic sickle cell episodes to
control both the frequency and duration of the crises.
The compounds of the invention are also potent, specific inhibitors of
mammalian cell proliferation. Thus, in another aspect, the invention
provides methods of inhibiting mammalian cell proliferation as a
therapeutic approach towards the treatment or prevention of diseases
characterized by unwanted or abnormal cell proliferation. In its broadest
sense, the method involves only a single step--the administration of an
effective amount of at least one pharmacologically active compound
according to the invention to a mammalian cell in situ. The compound may
act cytostatically, cytotoxically, or by a combination of both mechanisms
to inhibit cell proliferation. Mammalian cells treatable in this manner
include vascular smooth muscle cells, fibroblasts, endothelial cells,
various pre-cancer cells and various cancer cells. In a preferred
embodiment, cell proliferation is inhibited in a subject suffering from a
disorder that is characterized by unwanted or abnormal cell proliferation.
Such diseases are described more fully below.
Based in part on the surmised role of mammalian cell proliferation in
certain diseases, the invention is also directed to methods of treating or
preventing diseases characterized by abnormal cell proliferation. In the
method, an effective amount of at least one compound according to the
invention, or a pharmaceutical composition thereof, is administered to a
patient suffering from a disorder that is characterized by abnormal cell
proliferation. While not intending to be bound by any particular theory,
it is believed that administration of an appropriate amount of a compound
according to the invention to a subject inhibits cell proliferation by
altering the ionic fluxes associated with early mitogenic signals. Such
alteration of ionic fluxes is thought to be due to the ability of the
compounds of the invention to inhibit potassium channels of cells,
particularly Ca.sup.2+ -activated potassium channels. The method can be
used prophylactically to prevent unwanted or abnormal cell proliferation,
or may be used therapeutically to reduce or arrest proliferation of
abnormally proliferating cells. The compound, or a pharmaceutical
formulation thereof, can be applied locally to proliferating cells to
arrest or inhibit proliferation at a desired time, or may be administered
to a subject systemically to arrest or inhibit cell proliferation.
Diseases which are characterized by abnormal cell proliferation that can be
treated or prevented by means of the present invention include blood
vessel proliferative disorders, fibrotic disorders, arteriosclerotic
disorders and various cancers.
Blood vessel proliferative disorders refer to angiogenic and vasculogenic
disorders generally resulting in abnormal proliferation of blood vessels.
The formation and spreading of blood vessels, or vasculogenesis and
angiogenesis, respectively, play important roles in a variety of
physiological processes such as embryonic development, corpus luteum
formation, wound healing and organ regeneration. They also play a pivotal
role in cancer development. Other examples of blood vessel proliferative
disorders include arthritis, where new capillary blood vessels invade the
joint and destroy cartilage and ocular diseases such as diabetic
retinopathy, where new capillaries in the retina invade the vitreous,
bleed and cause blindness and neovascular glaucoma.
Another example of abnormal neovascularization is that associated with
solid tumors. It is now established that unrestricted growth of tumors is
dependent upon angiogenesis and that induction of angiogenesis by
liberation of angiogenic factors can be an important step in
carcinogenesis. For example, basic fibroblast growth factor (bFGF) is
liberated by several cancer cells and plays a crucial role in cancer
angiogenesis. The demonstration that certain animal tumors regress when
angiogenesis is inhibited has provided the most compelling evidence for
the role of angiogenesis in tumor growth. Other cancers that are
associated with neovascularization include hemangioendotheliomas,
hemangiomas and Kaposi's sarcoma.
Proliferation of endothelial and vascular smooth muscle cells is the main
feature of neovascularization. The invention is useful in inhibiting such
proliferation, and therefore in inhibiting or arresting altogether the
progression of the angiogenic condition which depends in whole or in part
upon such neovascularization. The invention is particularly useful when
the condition has an additional element of endothelial or vascular smooth
muscle cell proliferation that is not necessarily associated with
neovascularization. For example, psoriasis may additionally involve
endothelial cell proliferation that is independent of the endothelial cell
proliferation associated with neovascularization. Likewise, a solid tumor
which requires neovascularization for continued growth may also be a tumor
of endothelial or vascular smooth muscle cells. In this case, growth of
the tumor cells themselves, as well as the neovascularization, is
inhibited by the compounds described herein.
The invention is also useful for the treatment of fibrotic disorders such
as fibrosis and other medical complications of fibrosis which result in
whole or in part from the proliferation of fibroblasts. Medical conditions
involving fibrosis (other than atherosclerosis, discussed below) include
undesirable tissue adhesion resulting from surgery or injury.
Other cell proliferative disorders which can be treated by means of the
invention include arteriosclerotic conditions. Arteriosclerosis is a term
used to describe a thickening and hardening of the arterial wall. An
arteriosclerotic condition as used herein means classical atherosclerosis,
accelerated atherosclerosis, atherosclerotic lesions and any other
arteriosclerotic conditions characterized by undesirable endothelial
and/or vascular smooth muscle cell proliferation, including vascular
complications of diabetes.
Proliferation of vascular smooth muscle cells is a main pathological
feature in classical atherosclerosis. It is believed that liberation of
growth factors from endothelial cells stimulates the proliferation of
subintimal smooth muscle which, in turn, reduces the caliber and finally
obstructs the artery. The invention is useful in inhibiting such
proliferation, and therefore in delaying the onset of, inhibiting the
progression of, or even halting the progression of such proliferation and
the associated atherosclerotic condition.
Proliferation of vascular smooth muscle cells produces accelerated
atherosclerosis, which is the main reason for failure of heart transplants
that are not rejected. This proliferation is also believed to be mediated
by growth factors, and can ultimately result in obstruction of the
coronary arteries. The invention is useful in inhibiting such obstruction
and reducing the risk of, or even preventing, such failures.
Vascular injury can also result in endothelial and vascular smooth muscle
cell proliferation. The injury can be caused by any number of traumatic
events or interventions, including vascular surgery and balloon
angioplasty. Restenosis is the main complication of successful balloon
angioplasty of the coronary arteries. It is believed to be caused by the
release of growth factors as a result of mechanical injury to the
endothelial cells lining the coronary arteries. Thus, by inhibiting
unwanted endothelial and smooth muscle cell proliferation, the compounds
described herein can be used to delay, or even avoid, the onset of
restenosis.
Other atherosclerotic conditions which can be treated or prevented by means
of the present invention include diseases of the arterial walls that
involve proliferation of endothelial and/or vascular smooth muscle cells,
such as complications of diabetes, diabetic glomerulosclerosis and
diabetic retinopathy.
The compounds described herein are also potent antineoplastic agents and
are therefore useful in treating or preventing various types of neoplastic
diseases. Neoplastic diseases which can be treated by means of the present
invention include, but are not limited to, biliary tract cancer; brain
cancer, including glioblastomas and medulloblastomas; breast cancer;
cervical cancer; choriocarcinoma; colon cancer; endometrial cancer;
esophageal cancer; gastric cancer; hematological neoplasms, including
acute and chronic lymphocytic and myelogenous leukemia, multiple myeloma,
AIDS associated leukemias and adult T-cell leukemia lymphoma;
intraepithelial neoplasms, including Bowen's disease and Paget's disease;
liver cancer; lung cancer; lymphomas, including Hodgkin's disease and
lymphocytic lymphomas; neuroblastomas; oral cancer, including squamous
cell carcinoma; ovarian cancer, including those arising from epithelial
cells, stromal cells, germ cells and mesenchymal cells; pancreas cancer;
prostate cancer; rectal cancer; sarcomas, including leiomyosarcoma,
rhabdomyosarcoma, liposarcoma, fibrosarcoma and osteosarcoma; skin cancer,
including melanoma, Kaposi's sarcoma, basocellular cancer and squamous
cell cancer; testicular cancer, including germinal tumors (seminoma,
non-seminoma (teratomas, choriocarcinomas)), stromal tumors and germ cell
tumors; thyroid cancer, including thyroid adenocarcinoma and medullar
carcinoma; and renal cancer including adenocarcinoma and Wilms tumor.
The compounds of the invention are useful with hormone dependent and also
with nonhormone dependent cancers. They also are useful with prostate and
breast cancers. They further are useful with multidrug resistant strains
of cancer.
In addition to the particular disorders enumerated above, the invention is
also useful in treating or preventing dermatological diseases including
keloids, hypertrophic scars, seborrheic dermatosis, papilloma virus
infection (e.g., producing verruca vulgaris, verruca plantaris, verruca
plan, condylomata, etc.), eczema and epithelial precancerous lesions such
as actinic keratosis; other inflammatory diseases including proliferative
glomerulonephritis; lupus erythematosus; scleroderma; temporal arthritis;
thromboangiitis obliterans; mucocutaneous lymph node syndrome; and other
pathologies mediated by growth factors including uterine leiomyomas.
The compounds and methods of the invention provide myriad advantages over
agents and methods commonly used to treat sickle cell disease and/or cell
proliferative disorders. The compounds and methods of the invention also
provide myriad advantages over the treatment of sickle cell disease and/or
cell proliferative disorders with Clotrimazole or other antimycotic
agents. Most significantly, the compounds of the invention have reduced
toxicity as compared with Clotrimazole and other antimycotic agents, and
therefore provide consequential therapeutic benefits in clinical settings.
For example, for clotrimazole, it is well-known that the imidazole moiety
is responsible for inhibiting a wide range of cytochrome P-450 isozyme
catalyzed reactions, which constitutes their main toxicological effects
(Pappas and Franklin, 1993, Toxicology 80:27-35; Matsuura et al., 1991,
Biochemical Pharmacology 41:1949-1956). The compounds of the invention do
not contain an imidazole or imidazole-like moiety and therefore may not
share Clotrimazole's known toxicities.
5.1 The Compounds
The compounds which are capable of inhibiting the Gardos channel and/or
mammalian cell proliferation according to the invention are generally
substituted 3,3-diphenyl indanone, indane and (3-H) indole compounds, as
well as analogues of these classes of compounds wherein the atoms at ring
positions 1 and 2 are connected via a double bond.
In one illustrative embodiment, the compounds capable of inhibiting the
Gardos channel and/or mammalian cell proliferation according to the
invention are compounds having the structural formula:
##STR2##
or pharmaceutically acceptable salts or hydrates thereof, wherein: m is 0,
1, 2, 3 or 4;
each n is independently 0, 1, 2, 3, 4 or 5;
X is C or N;
Y is absent, (C.sub.1 -C.sub.6) alkyl, (C.sub.1 -C.sub.6) alkenyl or
(C.sub.1 -C.sub.6) alkynyl;
R.sub.1 is absent, --OR, --SR, .dbd.O, .dbd.S, .dbd.N--OR, --O--C(O)R,
--S--C(O)R, --O--C(S)R, --S--C(S)R, or when taken together with R.sub.2 is
a 3-8 membered heterocycloalkyl or a substituted 3-8 membered
heterocycloalkyl;
R.sub.2 is absent or --H;
R.sub.3 is absent or --H;
R.sub.4 is --H, --OR', --SR', --NR'.sub.2, --CN, --NO.sub.2,
(C.sub.3-C.sub.8) cycloalkyl, 3-8 membered heterocycloalkyl, --C(O)R',
--C(S)R', --C(O)OR', --C(S)OR', --C(O)SR', --C(S)SR', --C(O)NR'.sub.2 or
--C(S)NR'.sub.2 ;
each R.sub.5, R.sub.6 and R.sub.7 is independently selected from the group
consisting of -halogen, --R', --OR', --SR', --NR'.sub.2, --ONR'.sub.2,
--SNR'.sub.2, --NO.sub.2, --CN, --C(O)R', --C(S)R', --C(O)OR', --C(O)SR',
--C(S)OR', --CS(S)R', --C(O)NR'.sub.2, --C(S)NR'.sub.2, --C(O)NR'(OR'),
--C(S)NR'(OR'); --C(O)NR'(SR'), --C(S)NR'(SR'), --CH(CN).sub.2,
--CH[C(O)R'].sub.2, --CH[C(S)R'].sub.2, --CH[C(O)OR'].sub.2,
--CH[C(S)OR'].sub.2, --CH[C(O)SR'].sub.2 and --CH[C(S)SR'].sub.2 ;
each R is independently selected from the group consisting of --H, (C.sub.1
-C6) alkyl, (C.sub.1 -C.sub.6) alkenyl, (C.sub.1 -C.sub.6) alkynyl,
(C.sub.5 -C.sub.20) aryl, substituted (C.sub.5 -C.sub.20) aryl, (C.sub.6
-C.sub.26) alkaryl and substituted (C.sub.1 -C.sub.26) alkaryl;
the heterocycloalkyl substituents are each independently selected from the
group consisting of --CN, --NO.sub.2, --NR'.sub.2, --OR', --C(O)NR'.sub.2,
--C(S)NR'.sub.2, --C(O)OR', --C(S)OR', --C(O)SR', --C(S)SR' and
trihalomethyl;
the aryl and alkaryl substituents are each independently selected from the
group consisting of halogen, --C(O)R', --C(S)R', --C(O)OR', --C(S)OR',
--C(O)SR', --C(S)SR', --C(O)NR'.sub.2, --C(S)NR'.sub.2 and trihalomethyl;
each R' is independently selected from the group consisting of --H,
(C.sub.1 -C.sub.6) alkyl, (C.sub.1 -C.sub.6) alkenyl and (C.sub.1
-C.sub.6) alkynyl; and
--- designates a single or double bond.
In the compounds of structural formula (I), the bond between the atoms at
ring positions 1 and 2 (designated--) can be either a single or double
bond. It will be recognized by those of skill in the art that when the
bond is a double bond, certain of the substituents must be absent. It will
also be recognized that the identity of X also influences the presence or
absence of certain substituents. Thus, it is to be understood that when X
is N and--is a double bond, R.sub.1, R.sub.2 and R.sub.3 are absent; when
X is C and--is a double bond, R.sub.2 and R.sub.3 are absent. When X is N
and--is a single bond, one of R.sub.1 and R.sub.2 is present and the other
is absent and R.sub.3 is present; when X is C and--is a single bond,
R.sub.1, R.sub.2 and R.sub.3 are each present.
In a preferred embodiment of the invention, the chalcogens in the compounds
of formula (I) are each oxygen.
In another preferred embodiment of the invention, the compounds are those
of structural formula (I) wherein:
m is 0, 1, 2, 3 or 4;
each n is independently 0, 1, 2, 3, 4 or 5;
X is C or N;
Y is absent, (C.sub.1 -C.sub.6) alkyl, (C.sub.1 -C.sub.6) alkenyl or
(C.sub.1 -C.sub.6) alkynyl;
R.sub.1 is absent, --OR, .dbd.O, .dbd.N--OR, --O--C(O)R, or when taken
together with R.sub.2 is a 3-8 membered oxirane or a substituted 3-8
membered oxirane;
R.sub.2 is absent or --H;
R.sub.3 is absent or --H;
R.sub.4 is --H, --OR', --NR'.sub.2, --CN, --NO.sub.2, (C.sub.3 -C.sub.8)
cycloalkyl, 3-8 membered oxiranyl, 5-8 membered dioxycycloalkyl, --C(O)R',
--C(O)OR' or --C(O)NR'.sub.2 ;
each R.sub.5, R.sub.6 and R.sub.7 is independently selected from the group
consisting of -halogen, --R', --OR', --NR'.sub.2, --ONR'.sub.2,
--NO.sub.2, --CN, --C(O)R', --C(O)OR', --C(O)NR'.sub.2, --C(O)NR'(OR'),
--CH(CN).sub.2, --CH[C(O)R'].sub.2 and --CH[C(O)OR'].sub.2 ;
each R is independently selected from the group consisting of --H, (C.sub.1
-C.sub.6) alkyl, (C.sub.1 -C.sub.6) alkenyl, (C.sub.1 -C.sub.6) alkynyl,
(C.sub.5 -C.sub.20) aryl, substituted (C.sub.5 -C.sub.20) aryl, (C.sub.6
-C.sub.26) alkaryl and substituted (C.sub.6 -C.sub.26) alkaryl;
the oxirane substituents are each independently selected from the group
consisting of --CN, --NO.sub.2, --NR'.sub.2, --OR', --C(O)NR'.sub.2,
--C(O)OR' and trihalomethyl;
the aryl and alkaryl substituents are each independently selected from the
group consisting of halogen, --C(O)R', --C(O)OR', --C(O)NR'.sub.2 and
trihalomethyl;
each R' is independently selected from the group consisting of --H,
(C.sub.1 -C.sub.6) alkyl, (C.sub.1 -C.sub.6) alkenyl and (C.sub.1
-C.sub.6) alkynyl; and/or
--- designates a single or double bond.
In another preferred embodiment, the compounds are those of structural
formula (I) wherein:
m is 0 or 1;
each n is independently 0 or 1;
X is C or N;
Y is absent, (C.sub.1 -C.sub.3) alkyl, (C.sub.1 -C.sub.3) alkenyl or
(C.sub.1 -C.sub.3) alkynyl;
R.sub.1 is absent --H, --OR, .dbd.O, --NR.sub.2, .dbd.N--OR, --O--C(O)R, or
when taken together with R.sub.2 is 3-5 membered oxirane or 3-5 membered
substituted oxirane;
R.sub.2 is absent or --H;
R.sub.3 is absent or --H;
R.sub.4 is --H, --OR, --NR.sub.2, --CN, --C(O)OR, --C(O)NR.sub.2 or 5-6
membered dioxoycycloalkyl;
each R.sub.5, R.sub.6 and R.sub.7 is independently selected from the group
consisting of --R', --F, --Cl or --Br;
each R is independently selected from the group consisting of --H, (C.sub.1
-C.sub.3) alkyl, (C.sub.1 -C.sub.3) alkenyl, (C.sub.1 -C.sub.3) alkynyl,
(C.sub.5 -C.sub.10) aryl, substituted (C.sub.5 -C.sub.10) aryl, (C.sub.6
-C.sub.13) alkaryl, substituted C.sub.1 -C.sub.13) alkaryl;
the oxirane substituent is --CN, --NO.sub.2, --NR'.sub.2, --OR' and
trihalomethyl;
the aryl and alkaryl substituents are each independently selected from the
group consisting of --F, --Cl, --Br, --CN, --NO.sub.2, NR'.sub.2,
--C(O)R', --C(O)OR' and trihalomethyl;
R' is --H, (C.sub.1 -C.sub.3) alkyl, (C.sub.1 -C.sub.3) alkenyl or (C.sub.1
-C.sub.3) alkynyl; and/or
--- is a single or double bond.
In still another preferred embodiment, the compounds are those of
structural formula (I) wherein:
m is 0, 1, 2, 3 or 4;
each n is independently 0, 1, 2, 3, 4 or 5;
X is C or N;
Y is absent, (C.sub.1 -C.sub.6) alkyl, (C.sub.1 -C.sub.6) alkenyl or
(C.sub.1 -C.sub.6) alkynyl;
R.sub.1 is absent, --OR, --SR, .dbd.O, .dbd.S, .dbd.N--OR, --O--C(O)R,
--S--C(O)R, --O--C(S)R, --S--C(S)R, or when taken together with R.sub.2 is
a 3-8 membered heterocycloalkyl or a substituted 3-8 membered
heterocycloalkyl;
R.sub.2 is absent or --H;
R.sub.3 is absent or --H;
R.sub.4 is --H, --OR', --SR', --NR'.sub.2, --CN, --NO.sub.2, (C.sub.3
-C.sub.8) cycloalkyl, 3-8 membered heterocycloalkyl, --C(O)R', --C(S)R',
--C(O)OR', --C(S)OR', --C(O)SR', --C(S)SR', --C(O)NR'.sub.2 or
--C(S)NR'.sub.2 ;
each R.sub.5, R.sub.6 and R.sub.7 is independently selected from the group
consisting of -halogen, --R', --OR', --SR', --NR'.sub.2, --ONR'.sub.2,
--SNR'.sub.2, --NO.sub.2, --CN, --C(O)R', --C(S)R', --C(O)OR', --C(O)SR',
--C(S)OR', --CS(S)R', --C(O)NR'.sub.2, --C(S)NR'.sub.2, --C (O) NR'(OR'),
--C(S)NR'(OR'); --C(O)NR'(SR'), --C(S)NR'(SR'), --CH(CN).sub.2,
--CH[C(O)R'].sub.2, --CH[C(S)R'].sub.2, --CH[C(O)OR'].sub.2,
--CH[C(S)OR'].sub.2, --CH[C(O)SR'].sub.2 and --CH[C(S)SR'].sub.2 ;
each R is independently selected from the group consisting of --H, (C.sub.1
-C.sub.6) alkyl, (C.sub.1 -C.sub.6) alkenyl, (C.sub.1 -C.sub.6) alkynyl,
(C.sub.1 -C.sub.20) aryl, substituted (C.sub.5 -C.sub.20) aryl, (C.sub.6
-C.sub.26) alkaryl and substituted (C.sub.6 -C.sub.26) alkaryl;
the heterocycloalkyl substituents are each independently selected from the
group consisting of --CN, --NO, --NR'.sub.2, --OR', --C(O)NR'.sub.2,
--C(S)NR'.sub.2, --C(O)OR', --C(S)OR', --C(O)SR', --C(S)SR' and
trihalomethyl;
the aryl and alkaryl substituents are each independently selected from the
group consisting of halogen, --C(O)R', --C(S)R', --C(O)OR', --C(S)OR',
--C(O)SR', --C(S)SR', --C(O)NR'.sub.2, --C(S)NR'.sub.2 and trihalomethyl;
each R' is independently selected from the group consisting of --H,
(C.sub.1 -C.sub.6) alkyl, (C.sub.1 -C.sub.6) alkenyl and (C.sub.1
-C.sub.5) alkynyl;
--- designates a single or double bond; and
wherein when X is C and R.sub.1 is .dbd.O, .dbd.S or --OR', at least one of
R.sub.5, R.sub.6 or R.sub.7 is other than --R', preferably other than --H,
or I5 Y is present or R.sub.4 is other than --H; and when X is N, --- is a
double bond and R.sub.1, R.sub.2, R.sub.3 and Y are absent, R.sub.4 is
other than --NR'.sub.2, preferably other than --NH.sub.2.
In still another preferred embodiment, the compounds are those of
structural formula (I) wherein:
m is 0, 1, 2, 3 or 4;
each n is independently 0, 1, 2, 3, 4 or 5;
X is C;
Y is absent, (C.sub.1 -C.sub.6) alkyl, (C.sub.1 -C.sub.6) alkenyl or
(C.sub.1 -C.sub.6) alkynyl;
R.sub.1 is absent, --OR, --SR, .dbd.O, .dbd.S, .dbd.N--OR, --O--C(O)R,
--S--C(O)R, --O--C(S)R, --S--C(S)R, or when taken together with R.sub.2 is
a 3-8 membered heterocycloalkyl or a substituted 3-8 membered
heterocycloalkyl;
R.sub.2 is absent or --H;
R.sub.3 is absent or --H;
R.sub.4 is --H, --OR', --SR', --NR'.sub.2, --CN, --NO.sub.2, (C.sub.3
-C.sub.8) cycloalkyl, 3-8 membered heterocycloalkyl, --C(O)R', --C(S)R',
--C(O)OR', --C(S)OR', --C(O)SR', --C(S)SR', --C(O)NR'.sub.2 or
--C(S)NR'.sub.2 ;
each R.sub.5, R.sub.6 and R.sub.7 is independently selected from the group
consisting of --halogen, --R', --OR', --SR', --NR'2 , --ONR'.sub.2,
--SNR'.sub.2, --NO.sub.2, --CN, --C(O)R', --C(S)R', --C(O)OR', --C(O)SR',
--C(S)OR', --CS(S)R', --C(O)NR'.sub.2, --C(S)NR'.sub.2, --C(O)NR'(OR'),
--C(S)NR'(OR'); --C(O)NR'(SR'), --C(S)NR'(SR'), --CH(CN).sub.2,
--CH[C(O)R'].sub.2 '--CH[C(S)R'].sub.2, --CH[C(O)OR'].sub.2
'--CH[C(S)OR'].sub.2, --CH[C(O)SR'].sub.2 and --CH[C(S)SR'].sub.2 ;
each R is independently selected from the group consisting of --H, (C.sub.1
-C.sub.6) alkyl, (C.sub.1 -C.sub.6) alkenyl, (C.sub.1 -C.sub.6) alkynyl,
(C.sub.5 -C.sub.20) aryl, substituted (C.sub.1 -C.sub.20) aryl, (C.sub.6
-C.sub.26) alkaryl and substituted (C.sub.6 -C.sub.26) alkaryl;
the heterocycloalkyl substituents are each independently selected from the
group consisting of --CN, --NO2, --NR'.sub.2, --OR', --C(O)NR'.sub.2,
--C(S)NR'.sub.2, --C(O)OR', --C(S)OR', --C(O)SR', --C(S)SR'and
trihalomethyl;
the aryl and alkaryl substituents are each independently selected from the
group consisting of halogen, --C(O)R', --C(S)R', --C(O)OR', --C(S)OR',
--C(O)SR', --c(S)SR', --C(O)NR'.sub.2, --C(S)NR'.sub.2 and trihalomethyl;
each R' is independently selected from the group consisting of --H,
(C.sub.1 -C.sub.6) alkyl, (C.sub.1 -C.sub.6) alkenyl and (C.sub.1
-C.sub.6) alkynyl;
--- designates a single or double bond; and
wherein when R.sub.1 is .dbd.O or --OH, at least one of R.sub.5, R.sub.6 or
R.sub.7 is other than --R', preferably other than --H, or Y is present or
R.sub.4 is other than --H. In still another preferred embodiment, the
compounds of structural formula (I) are selected from the group of
compounds set forth below:
##STR3##
In still another preferred embodiment, the compounds of structural formula
(I) are selected from the group consisting of Compounds 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20.
The chemical structural formulae referred to herein may exhibit the
phenomena of tautomerism, conformational isomerism, stereoisomerism or
geometric isomerism. As the structural formulae drawings within this
specification can represent only one of the possible tautomeric,
conformational isomeric, enantiomeric or geometric isomeric forms, it
should be understood that the invention encompasses any tautomeric,
conformational isomeric, enantiomeric or geometric isomeric forms which
exhibit biological or pharmacological activity as described herein.
The compounds of the invention may be in the form of free acids, free bases
or pharmaceutically effective salts thereof. Such salts can be readily
prepared by treating a compound with an appropriate acid. Such acids
include, by way of example and not limitation, inorganic acids such as
hydrohalic acids (hydrochloric, hydrobromic, etc.), sulfuric acid, nitric
acid, phosphoric acid, etc.; and organic acids such as acetic acid,
propanoic acid, 2-hydroxyacetic acid, 2-hydroxypropanoic acid,
2-oxopropanoic acid, propandioic acid, butandioic acid, etc. Conversely,
the salt can be converted into the free base form by treatment with
alkali.
In addition to the above-described compounds and their pharmaceutically
acceptable salts, the invention may employ, where applicable, solvated as
well as unsolvated forms of the compounds (e.g., hydrated forms).
The compounds described herein may be prepared by any processes known to be
applicable to the preparation of chemical compounds. Suitable processes
are well known in the art. Preferred processes are illustrated by the
representative examples. Necessary starting materials may be obtained
commercially or by standard procedures of organic chemistry. Moreover,
many of the compounds are commercially available.
An individual compound's relevant activity and potency as an agent to
affect sickle cell dehydration or deformation and/or mammalian cell
proliferation may be determined using standard techniques. Preferentially,
a compound is subject to a series of screens to determine its
pharmacological activity.
In most cases, the active compounds of the invention exhibit two
pharmacological activities: inhibition of the Gardos channel of
erythrocytes and inhibition of mammalian cell proliferation. However, in
some cases, the compounds of the invention may exhibit only one of these
pharmacological activities. Any compound encompassed by structural formula
(I) which exhibits at least one of these pharmacological activities is
considered to be within the scope of the present invention.
In general, the active compounds of the invention are those which induce at
least about 25% inhibition of the Gardos channel of erythrocytes (measured
at about 10 .mu.M) and/or about 25% inhibition of mammalian cell
proliferation (measured at about 10 .mu.M), as measured using in vitro
assays that are commonly known in the art (see, e.g., Brugnara et al.,
1993, J. Biol. Chem. 268(12):8760-8768; Benzaquen et al., 1995, Nature
Medicine 1:534-540). Alternatively, or in addition, the active compounds
of the invention generally will have an IC.sub.50 (concentration of
compound that yields 50% inhibition) for inhibition of the Gardos channel
of less than about 10 .mu.M and/or an IC.sub.50 for inhibition of cell
proliferation of less than about 10 .mu.M, as measured using in vitro
assays that are commonly known in the art (see, e.g., Brugnara et al.,
1993, J. Biol. Chem. 268(12):8760-8768; Benzaquen et al., 1995, Nature
Medicine 1:534-540).
Representative active compounds according to the invention are Compounds 1
through 20, as illustrated above.
In certain embodiments of the invention, compounds which exhibit only one
pharmacological activity, or a higher degree of one activity, may be
preferred. Thus, when the compound is to be used in methods to treat or
prevent sickle cell disease, or in methods to reduce sickle cell
dehydration and/or delay the occurrence of erythrocyte sickling or
deformation in situ, it is preferred that the compound exhibit at least
about 75% Gardos channel inhibition (measured at about 10 .mu.M) and/or
have an IC.sub.50 of Gardos channel inhibition of less than about 1 .mu.M,
with at least about 90% inhibition and/or an IC.sub.50 of less than about
0.1 .mu.M being particularly preferred.
Exemplary preferred compounds for use in methods related to Gardos channel
inhibition and sickle cell disease include compound numbers 1, 2, 3, 4, 7,
9, 12, 13 and 14.
When the compound is to be used in methods to treat or prevent disorders
characterized by abnormal cell proliferation or in methods to inhibit cell
proliferation in situ, it is preferable that the compound exhibit at least
about 75% inhibition of mitogen-induced cell proliferation (measured at
about 10 .mu.M) and/or have an IC.sub.50 of cell proliferation of less
than about 3.5 .mu.M, with at least about 90% inhibition and/or an
IC.sub.50 of less than about 1 .mu.M being particularly preferred.
Exemplary preferred compounds for use in methods inhibiting mammalian cell
proliferation or for the treatment or prevention of diseases characterized
by abnormal cell proliferation include compound numbers 1, 2, 3, 4, 6, 7,
8, 10, 11, 15, 16, 17, 19 and 20.
5.2 Formulation and Routes of Administration
The compounds described herein, or pharmaceutically acceptable addition
salts or hydrates thereof, can be delivered to a patient using a wide
variety of routes or modes of administration. Suitable routes of
administration include, but are not limited to, inhalation, transdermal,
oral, rectal, transmucosal, intestinal and parenteral administration,
including intramuscular, subcutaneous and intravenous injections.
The compounds described herein, or pharmaceutically acceptable salts and/or
hydrates thereof, may be administered singly, in combination with other
compounds of the invention, and/or in cocktails combined with other
therapeutic agents. Of course, the choice of therapeutic agents that can
be co-administered with the compounds of the invention will depend, in
part, on the condition being treated.
For example, when administered to patients suffering from sickle cell
disease, the compounds of the invention can be administered in cocktails
containing agents used to treat the pain, infection and other symptoms and
side effects commonly associated with sickle cell disease. Such agents
include, e.g., analgesics, antibiotics, etc. The compounds can also be
administered in cocktails containing other agents that are commonly used
to treat sickle cell disease, including butyrate and butyrate derivatives
(Perrine et al., 1993, N. Enql. J. Med. 328(2):81-86); hydroxyurea
(Charache et al., 1995, N. Engl. J. Med. 323(20):1317-1322);
erythropoietin (Goldberg et al, 1990, N. Engl. J. Med. 323(6): 366-372);
and dietary salts such as magnesium (De Franceschi et al., 1996, Blood
88(648a):2580).
When administered to a patient undergoing cancer treatment, the compounds
may be administered in cocktails containing other anti-cancer agents
and/or supplementary potentiating agents. The compounds may also be
administered in cocktails containing agents that treat the side-effects of
radiation therapy, such as anti-emetics, radiation protectants, etc.
Anti-cancer drugs that can be co-administered with the compounds of the
invention include, e.g., Aminoglutethimide; Asparaginase; Bleomycin;
Busulfan; Carboplatin; Carmustine (BCNU); Chlorambucil; Cisplatin
(cis-DDP); Cyclophosphamide; Cytarabine HCl; Dacarbazine; Dactinomycin;
Daunorubicin HCl; Doxorubicin HCl; Estramustine phosphate sodium;
Etoposide (VP-16); Floxuridine; Fluorouracil (5-FU); Flutamide;
Hydroxyurea (hydroxycarbamide); Ifosfamide; Interferon Alfa-2a, Alfa 2b,
Lueprolide acetate (LHRH-releasing factor analogue); Lomustine (CCNU);
Mechlorethamine HCl (nitrogen mustard); Melphalan; Mercaptopurine; Mesna;
Methotrexate (MTX); Mitomycin; Mitotane (o.p'-DDD); Mitoxantrone HCl;
Octreotide; Plicamycin; Procarbazine HCl; Streptozocin; Tamoxifen citrate;
Thioguanine; Thiotepa; Vinblastine sulfate; Vincristine sulfate; Amsacrine
(m-AMSA); Azacitidine; Hexamethylmelamine (HMM); Interleukin 2;
Mitoguazone (methyl-GAG; methyl glyoxal bis-guanylhydrazone; MGBG);
Pentostatin; Semustine (methyl-CCNU); Teniposide (VM-26); paclitaxel and
other taxanes; and Vindesine sulfate.
Supplementary potentiating agents that can be co-administered with the
compounds of the invention include, e.g., Tricyclic anti-depressant drugs
(e.g., imipramine, desipramine, amitriptyline, clomipramine, trimipramine,
doxepin, nortriptyline, protriptyline, amoxapine and maprotiline);
non-tricyclic and anti-depressant drugs (e.g., sertraline, trazodone and
citalopram); Ca.sup.+2 antagonists (e.g., verapamil, nifedipine,
nitrendipine and caroverine); Amphotericin (e.g., Tween 80 and perhexiline
maleate); Triparanol analogues (e.g., tamoxifen); antiarrhythmic drugs
(e.g., quinidine); antihypertensive drugs (e.g., reserpine); Thiol
depleters (e.g., buthionine and sulfoximine); and calcium leucovorin.
The active compound(s) may be administered per se or in the form of a
pharmaceutical composition wherein the active compound(s) is in admixture
with one or more pharmaceutically acceptable carriers, excipients or
diluents. Pharmaceutical compositions for use in accordance with the
present invention may be formulated in conventional manner using one or
more physiologically acceptable carriers comprising excipients and
auxiliaries which facilitate processing of the active compounds into
preparations which can be used pharmaceutically. Proper formulation is
dependent upon the route of administration chosen.
For injection, the agents of the invention may be formulated in aqueous
solutions, preferably in physiologically compatible buffers such as
Hanks's solution, Ringer's solution, or physiological saline buffer. For
transmucosal administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants are generally known
in the art.
For oral administration, the compounds can be formulated readily by
combining the active compound(s) with pharmaceutically acceptable carriers
well known in the art. Such carriers enable the compounds of the invention
to be formulated as tablets, pills, dragees, capsules, liquids, gels,
syrups, slurries, suspensions and the like, for oral ingestion by a
patient to be treated. Pharmaceutical preparations for oral use can be
obtained solid excipient, optionally grinding a resulting mixture, and
processing the mixture of granules, after adding suitable auxiliaries, if
desired, to obtain tablets or dragee cores. Suitable excipients are, in
particular, fillers such as sugars, including lactose, sucrose, mannitol,
or sorbitol; cellulose preparations such as, for example, maize starch,
wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl
cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,
and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may
be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic
acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose,
concentrated sugar solutions may be used, which may optionally contain gum
arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol,
and/or titanium dioxide, lacquer solutions, and suitable organic solvents
or solvent mixtures. Dyestuffs or pigments may be added to the tablets or
dragee coatings for identification or to characterize different
combinations of active compound doses.
Pharmaceutical preparations which can be used orally include push-fit
capsules made of gelatin, as well as soft, sealed capsules made of gelatin
and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can
contain the active ingredients in admixture with filler such as lactose,
binders such as starches, and/or lubricants such as talc or magnesium
stearate and, optionally, stabilizers. In soft capsules, the active
compounds may be dissolved or suspended in suitable liquids, such as fatty
oils, liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration should
be in dosages suitable for such administration.
For buccal administration, the compositions may take the form of tablets or
lozenges formulated in conventional manner.
For administration by inhalation, the compounds for use according to the
present invention are conveniently delivered in the form of an aerosol
spray presentation from pressurized packs or a nebulizer, with the use of
a suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other
suitable gas. In the case of a pressurized aerosol the dosage unit may be
determined by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g. gelatin for use in an inhaler or insufflator may be
formulated containing a powder mix of the compound and a suitable powder
base such as lactose or starch.
The compounds may be formulated for parenteral administration by injection,
e.g., by bolus injection or continuous infusion. Formulations for
injection may be presented in unit dosage form, e.g., in ampoules or in
multi-dose containers, with an added preservative. The compositions may
take such forms as suspensions, solutions or emulsions in oily or aqueous
vehicles, and may contain formulatory agents such as suspending,
stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous
solutions of the active compounds in water-soluble form. Additionally,
suspensions of the active compounds may be prepared as appropriate oily
injection suspensions. Suitable lipophilic solvents or vehicles include
fatty oils such as sesame oil, or synthetic fatty acid esters, such as
ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions
may contain substances which increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally,
the suspension may also contain suitable stabilizers or agents which
increase the solubility of the compounds to allow for the preparation of
highly concentrated solutions.
Alternatively, the active ingredient may be in powder form for constitution
with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal compositions such as
suppositories or retention enemas, e.g., containing conventional
suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds may
also be formulated as a depot preparation. Such long acting formulations
may be administered by implantation or transcutaneous delivery (for
example subcutaneously or intramuscularly), intramuscular injection or a
transdermal patch. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an emulsion in
an acceptable oil) or ion exchange resins, or as sparingly soluble
derivatives, for example, as a sparingly soluble salt.
The pharmaceutical compositions also may comprise suitable solid or gel
phase carriers or excipients. Examples of such carriers or excipients
include but are not limited to calcium carbonate, calcium phosphate,
various sugars, starches, cellulose derivatives, gelatin, and polymers
such as polyethylene glycols.
5.3 Effective Dosages
Pharmaceutical compositions suitable for use with the present invention
include compositions wherein the active ingredient is contained in a
therapeutically effective amount, i.e., in an amount effective to achieve
its intended purpose. Of course, the actual amount effective for a
particular application will depend, inter alia, on the condition being
treated. For example, when administered in methods to reduce sickle cell
dehydration and/or delay the occurrence of erythrocyte sickling or
distortion in situ, such compositions will contain an amount of active
ingredient effective to achieve this result. When administered in methods
to inhibit cell proliferation, such compositions will contain an amount of
active ingredient effective to achieve this result. When administered to
patients suffering from sickle cell disease or disorders characterized by
abnormal cell proliferation, such compositions will contain an amount of
active ingredient effective to, inter alia, prevent the development of or
alleviate the existing symptoms of, or prolong the survival of, the
patient being treated. For use in the treatment of cancer, a
therapeutically effective amount further includes that amount of compound
or composition which arrests or regresses the growth of a tumor.
Determination of an effective amount is well within the capabilities of
those skilled in the art, especially in light of the detailed disclosure
herein.
For any compound described herein the therapeutically effective amount can
be initially determined from cell culture arrays. Target plasma
concentrations will be those concentrations of active compound(s) that are
capable of inducing at least about 25% inhibition of the Gardos channel
and/or at least about 25% inhibition of cell proliferation in cell culture
assays, depending, of course, on the particular desired application.
Target plasma concentrations of active compound(s) that are capable of
inducing at least about 50%, 75%, or even 90% or higher inhibition of the
Gardos channel and/or cell proliferation in cell culture assays are
preferred. The percentage of inhibition of the Gardos channel and/or cell
proliferation in the patient can be monitored to assess the
appropriateness of the plasma drug concentration achieved, and the dosage
can be adjusted upwards or downwards to achieve the desired percentage of
inhibition.
Therapeutically effective amounts for use in humans can also be determined
from animal models. For example, a dose for humans can be formulated to
achieve a circulating concentration that has been found to be effective in
animals. A particularly useful animal model for sickle cell disease is the
SAD mouse model (Trudel et al., 1991, EMBO J. 11:3157-3165). Useful animal
models for diseases characterized by abnormal cell proliferation are
well-known in the art. In particular, the following references provide
suitable animal models for cancer xenografts (Corbett et al., 1996, J.
Exp. Ther. Oncol. 1:95-108; Dykes et al., 1992, Contrib. Oncol. Basel.
Karger 42:1-22), restenosis (Carter et al., 1994, J. Am. Coll. Cardiol.
24(5):1398-1405), atherosclerosis (Zhu et al., 1994, Cardiology
85(6):370-377) and neovascularization (Epstein et al., 1987, Cornea
6(4):250-257). The dosage in humans can be adjusted by monitoring Gardos
channel inhibition and/or inhibition of cell proliferation and adjusting
the dosage upwards or downwards, as described above.
A therapeutically effective dose can also be determined from human data for
compounds which are known to exhibit similar pharmacological activities,
such as Clotrimazole and other antimycotic agents (see, e.g., Brugnara et
al., 1995, JPET 273:266-272; Benzaquen et al., 1995, Nature Medicine
1:534-540; Brugnara et al., 1996, J. Clin. Invest. 97(5):1227-1234). The
applied dose can be adjusted based on the relative bioavailability and
potency of the administered compound as compared with Clotrimazole.
Adjusting the dose to achieve maximal efficacy in humans based on the
methods described above and other methods as are well-known in the art is
well within the capabilities of the ordinarily skilled artisan.
Of course, in the case of local administration, the systemic circulating
concentration of administered compound will not be of particular
importance. In such instances, the compound is administered so as to
achieve a concentration at the local area effective to achieve the
intended result.
For use in the prophylaxis and/or treatment of sickle cell disease,
including both chronic sickle cell episodes and acute sickle cell crisis,
a circulating concentration of administered compound of about 0.001 .mu.M
to 20 .mu.M is considered to be effective, with about 0.1 .mu.M to 5 .mu.M
being preferred.
Patient doses for oral administration of the compounds described herein,
which is the preferred mode of administration for prophylaxis and for
treatment of chronic sickle cell episodes, typically range from about 80
mg/day to 16,000 mg/day, more typically from about 800 mg/day to 8000
mg/day, and most typically from about 800 mg/day to 4000 mg/day. Stated in
terms of patient body weight, typical dosages range from about 1 to 200
mg/kg/day, more typically from about 10 to 100 mg/kg/day, and most
typically from about to 50 mg/kg/day. Stated in terms of patient body
surface areas, typical dosages range from about 40 to 8000 mg/m.sup.2
/day, more typically from about 400 to 4000 mg/m.sup.2 /day, and most
typically from about 400 to 2000 mg/m.sup.2 /day.
For use in the treatment of disorders characterized by abnormal cell
proliferation, including cancer, arteriosclerosis and angiogenic
conditions such as restenosis, a circulating concentration of administered
compound of about 0.001 .mu.M to 20 .mu.M is considered to be effective,
with about 0.1 .mu.M to 5 .mu.M being preferred.
Patient doses for oral administration of the compounds described herein for
the treatment or prevention of cell proliferative disorders typically
range from about 80 mg/day to 16,000 mg/day, more typically from about 800
mg/day to 8000 mg/day, and most typically from about 800 mg/day to 4000
mg/day. Stated in terms of patient body weight, typical dosages range from
about 1 to 200 mg/kg/day, more typically from about 10 to 100 mg/kg/day,
and most typically from about to 50 mg/kg/day. Stated in terms of patient
body surface areas, typical dosages range from about 40 to 8000 mg/m.sup.2
/day, more typically from about 400 to 4000 mg/m.sup.2 /day, and most
typically from about 400 to 2000 mg/m.sup.2 /day.
For other modes of administration, dosage amount and interval can be
adjusted individually to provide plasma levels of the administered
compound effective for the particular clinical indication being treated.
For example, if acute sickle crises are the most dominant clinical
manifestation, a compound according to the invention can be administered
in relatively high concentrations multiple times per day. Alternatively,
if the patient exhibits only periodic sickle cell crises on an infrequent
or periodic or irregular basis, it may be more desirable to administer a
compound of the invention at minimal effective concentrations and to use a
less frequent regimen of administration. This will provide a therapeutic
regimen that is commensurate with the severity of the sickle cell disease
state.
For use in the treatment of tumorigenic cancers, the compounds can be
administered before, during or after surgical removal of the tumor. For
example, the compounds can be administered to the tumor via injection into
the tumor mass prior to surgery in a single or several doses. The tumor,
or as much as possible of the tumor, may then be removed surgically.
Further dosages of the drug at the tumor site can be applied post removal.
Alternatively, surgical removal of as much as possible of the tumor can
precede administration of the compounds at the tumor site.
Combined with the teachings provided herein, by choosing among the various
active compounds and weighing factors such as potency, relative
bioavailability, patient body weight, severity of adverse side-effects and
preferred mode of administration, an effective prophylactic or therapeutic
treatment regimen can be planned which does not cause substantial toxicity
and yet is entirely effective to treat the clinical symptoms demonstrated
by the particular patient. Of course, many factors are important in
determining a therapeutic regimen suitable for a particular indication or
patient. Severe indications such as cancer may warrant administration of
higher dosages as compared with less severe indications such as sickle
cell disease.
5.4 Toxicity
The ratio between toxicity and therapeutic effect for a particular compound
is its therapeutic index and can be expressed as the ratio between
LD.sub.50 (the amount of compound lethal in 50% of the population) and
ED., (the amount of compound effective in 50% of the population).
Compounds which exhibit high therapeutic indices are preferred.
Therapeutic index data obtained from cell culture assays and/or animal
studies can be used in formulating a range of dosages for use in humans.
The dosage of such compounds preferably lies within a range of plasma
concentrations that include the EDS, with little or no toxicity. The
dosage may vary within this range depending upon the dosage form employed
and the route of administration utilized. The exact formulation, route of
administration and dosage can be chosen by the individual physician in
view of the patient's condition. (See e.g. Fingl et al., 1975, In: The
Pharmacological Basis of Therapeutics, Ch. 1 p1).
The invention having been described, the following examples are intended to
illustrate, not limit, the invention.
6. EXAMPLE
Compound Syntheses
This Example demonstrates general methods for synthesizing the compounds of
the invention, as well as preferred methods of synthesizing certain
exemplary compounds of the invention. In all of the reaction schemes
described herein, suitable starting materials are either commercially
available or readily obtainable using standard techniques of organic
synthesis. Where necessary, suitable groups and schemes for protecting the
various funtionalities are well-known in the art, and can be found, for
example, in Kocienski, Protecting Groups, Georg Thieme Verlag, New York,
1994 and Greene & Wuts, Protective Groups in Organic Chemistry, John Wiley
& Sons, New York, 1991.
In FIGS. 1 and 2, the various substituents are defined as for structure
(I), supra.
6.1 Synthesis of Substituted 3,3-Diphenyl Indanones
Referring to FIG. 1, substituted 3,3-diphenyl indanone compounds are
synthesized as follows: substituted triphenylpropionic acid 100 (0.25-0.50
M in sulfuric acid) is stirred at room temperature for 1 hour and then
poured into an equal volume of cold water. The aqueous mixture is
extracted with an equal volume of ethyl acetate and the organics dried
over sodium sulfate. Evaporation gives the desired substituted
3,3-diphenyl indanone compound 102 in about 60-75% yield.
6.2 Synthesis of Substituted 1-Hydroxy-3,3-Diphenyl Indane Compounds
Referring to FIG. 1, substituted 1-hydroxy-3,3-diphenyl indane compounds
are synthesized as follows: a solution of substituted 3,3-diphenylindanone
102 (0.25 M in tetrahydrofuran) is added dropwise to 0.25 volume of a 1.0
M solution of lithium aluminum hydride in tetrahydrofuran at 0-5.degree.
C. The mixture is warmed to reflux and refluxed for 2.5 h, cooled to
0-5.degree. C. and an equal volume of 1 M HCl added slowly. The mixture is
then extracted three times with an equal volume of ethyl acetate. The
combined organic extracts are washed with a saturated aqueous solution of
sodium bicarbonate and dried over sodium sulfate. Evaporation gives the
desired substituted 1-hydroxy-3,3-diphenyl indane compound 104 in about
45-90% yield.
6.3 Synthesis of Substituted 1-N-oxime-3,3-Diphenyl Indanes
Referring to FIG. 1, substituted 1-N-oxime-3,3-diphenyl indane compounds
are synthesized as follows: substituted 3,3-diphenylindanone 102 (1
equivalent) is combined with 5 equivalents of hydroxylamine hydrochloride
and equivalents of sodium acetate and dissolved in methanol. The solution
is stirred at room temperature for 16 h and then an equal volume of water
is added. The mixture is extracted three times with an equal volume of
ethyl acetate and the combined organic extracts are dried over sodium
sulfate. Evaporation gives the desired substituted 1-N-oxime-3,3-diphenyl
indane compound 106 (as a mixture of cis and trans isomers) in about
90-98% yield.
6.4 Synthesis of Substituted 2-Alkyl-3,3-Diphenyl Indanones
Referring to FIG. 1, substituted 2-alkyl-3,3-diphenyl indanone compounds
are synthesized as follows: substituted 3,3-diphenyl indanone 102 (1
equivalent) is dissolved in tetrahydrofuran (0.4-1.0 M) and 1.2
equivalents of potassium hydride is added. The mixture is stirred at room
temperature until the gas evolution subsides and then the bromoalkane (1.2
equivalents) is added. The mixture is stirred at room temperature and
monitored by TLC. The reaction is quenched with water and the mixture
extracted with ethyl acetate. The desired substituted 2-alkyl-3,3-diphenyl
indanone compound 108 is isolated by silica gel chromatography in about
50-75% yield.
6.5 Synthesis of Substituted i-Alkoxy-3,3-Diphenyl Indanes
Referring to FIG. 1, substituted l-alkoxy-3,3-diphenyl indane compounds are
synthesized as follows: substituted 1-hydroxy-3,3-diphenylindanone 104 (1
equivalent) is combined with 2 equivalents of sodium hydride in
N,N-dimethylformamide and stirred at room temperature until the gas
evolution subsides. The haloalkane (2 equivalents) is added and stirred at
room temperature for 16-20 hours. An equal volume of water is added and
the mixture extracted four times with twice the volume of ethyl acetate.
The combined organic extracts are dried over sodium sulfate and the
solvent removed in vacuo. The desired substituted 1-alkoxy-3,3-diphenyl
indane compound 110 is isolated by vacuum distillation.
6.6 Synthesis of Substituted 3,3-Diphenyl-3H-Indoles
Referring to FIG. 2, substituted 3,3-diphenyl-3H-indole compounds are
synthesized as follows: substituted phenyl hydrazine 120 is combined with
an equimolar amount of substituted 1,1-diphenyl-2-ketone 122 in phosphoric
acid. This mixture is stirred at 100-120.degree. C. until the reaction is
complete as determined by TLC. The reaction is cooled to 60-70.degree. C.
and diluted with twice the volume of water while stirring. After cooling
to room temperature, the mixture is filtered, washed with water, and the
crude solid substituted 3,3-diphenyl indole compound 124 is purified by
column chromatography or crystallization.
6.7 Synthesis of Substituted 3,3-Diphenyl-3H-Indolines
Referring to FIG. 2, substituted 3,3-diphenyl-3H-indoline compounds are
synthesized as follows: substituted 3,3-diphenyl indole compound 124 is
reduced with sodium borohydride or sodium cyanoborohydride in a suitable
solvent to yield the substituted 3,3-diphenyl-3H-indoline compound 126.
6.8 Synthesis of Substituted N-Substituted-3,3-Diphenyl Indolines
Referring to FIG. 2, substituted N-substituted-3,3-diphenyl indoline
compounds are synthesized as follows: substituted 3,3-diphenyl indoline
126 (1 equivalent) is combined with an alkyl halide (1 equivalent) and
potassium carbonate (3-4 equivalents) in acetonitrile. The mixture is
stirred at reflux until the reaction is complete as determined by TLC.
Water and ethyl acetate are added and the mixture is extracted with ethyl
acetate. Evaporation of the combined ethyl acetate extracts gives the
crude substituted N-substituted-3,3-diphenyl indoline compound 128, which
is purified by column chromatography.
6.9 Synthesis of 3,3-Diphenylindanone (Compound 2)
3,3-Diphenylindanone (Compound 2) was synthesized as follows:
Triphenylpropionic acid (12 g, 0.04 mol) was stirred in 50 ml concentrated
sulfuric acid for 1 hour. The reaction mixture was cooled in an ice bath
and diluted with 50 ml water. This mixture was extracted three times with
ethyl acetate. The ethyl acetate extracts were combined, dried over sodium
sulfate and the solvent removed in vacuo to yield 9.0 g (78% yield) of
3,3-Diphenylindanone (Compound 2) as a white solid having a melting point
of 119-123.degree. C.
6.10 Synthesis of 1-Hydroxy-3,3-Diphenylindane (Compound 3)
1-Hydroxy-3,3-Diphenylindane (Compound 3) was synthesized as follows: A
solution of 2 g (0.007 mol) 3,3-diphenylindanone (Compound 2) in 20 ml of
tetrahydrofuran was added dropwise to a solution of 0.34 g (0.009 mol)
LiAlH.sub.4 in 10 ml tetrahydrofuran at 0-5.degree. C. The mixture was
warmed to reflux and refluxed for 3 hr., cooled to 0-5.degree. C. and 30
ml of 1 M HCl added slowly. The mixture was then extracted three times
with 60 ml ethyl acetate. The ethyl acetate extracts were combined, washed
with a saturated aqueous solution of sodium bicarbonate and dried over
sodium sulfate. Evaporation of the solvent gave 0.9 g (45% yield) of
1-Hydroxy-3,3-Diphenylindane (Compound 3) as white crystals with a melting
point of 133-135.degree. C.
6.11 Synthesis of 1-N-Oxime-3,3-Diphenylindane (Compound 4)
1-N-Oxime-3,3-Diphenylindane (Compound 4) was synthesized as follows:
3,3-Diphenylindanone (Compound 2) (2.0 g, 0.007 mol) was combined with 2.4
g (0.035 mol) of hydroxylamine hydrochloride and 5.8 g (0.07 mol) of
sodium acetate and dissolved in 30 ml of methanol. The solution was
stirred at room temperture for 16 hr and then 100 ml of water was added.
The mixture was extracted with 100 ml ethyl acetate and the organic layer
dried over sodium sulfate. Evaporation of the solvent gave 1.9 g (90%
yield) of 1-N-Oxime-3,3-Diphenylindane (Compound 4) as a white solid
having a melting point of 138-141.degree. C.
6.12 Synthesis of
Spiro[3,3-diphenyl-2,3-dihydro(1H)indene-1,3'-2'-cyanooxirane] (Compound
5) and 2-Cyanomethyl-3,3-diphenylindanone (Compound 9)
Spiro[3,3-diphenyl-2,3-dihydro(1H)indene-1,3,-2'-cyanooxirane] (Compound 5)
and 2-cyanomethyl-3,3-diphenylindanone (Compound 9) were synthesized as
follows: 3,3-diphenylindanone (Compound 2), 5.0 g (0.0176 mole) and 2.62 g
(0.0229 mole) of potassium hydride were stirred at room temperature in 40
mL of tetrahydrofuran. After the gas evolution subsided (approx. 45 min),
1.5 mL (0.0215 mole) of bromoacetonitrile was added. The dark red mixture
was stirred for 1 hour and then 50 mL of water was added. The mixture was
extracted three times with 75 mL of ethyl acetate. The combined organic
extracts were concentrated in vacuo, loaded onto a silica gel column and
eluted with 10% ethyl acetate in hexane. Three fractions were collected.
After evaporation of the solvent, the first fraction yielded unreacted
starting material (3.5 g). The second fraction yielded 0.49 g (9% yield)
of spiro[3,3-diphenyl-2,3-dihydro(1H)indene-1,3'-2'-cyanooxirane]
(Compound 5) as a white solid. The third fraction yielded 1.05 g (18%
yield) of 2-cyanomethyl-3,3-diphenylindanone (Compound 9) as a yellow oil.
6.13 Synthesis of 2-(2'-Propenyl)-1-(2'-propenoxy)-3,3-diphenylindane
(Compound 6)
2-(2'-Propenyl)-1(2'-propenoxy)-3,3-diphenylindane (Compound 6) was
synthesized as follows: 3,3-diphenylindanone (Compound 2) 2.0 g (0.007
mole) and 0.28 g (0.0084 mole) sodium hydride were stirred at room
temperature in 40 mL of dimethylformamide for 1 hour. The reaction mixture
was then added drop-wise to 0.64 mL (0.0078 mole) of allyl bromide at
-50.degree. C. The mixture was then warmed to reflux and refluxed for 1
hour. After cooling to room temperature, 50 mL of water was added. The
mixture was extracted with ethyl acetate, dried over sodium sulfate and
concentrated in vacuo. 2-(2'-propenyl)-1-(2'-propenoxy)-3,3-diphenylindane
(Compound 6) was isolated in 30% yield as the first fraction from a silica
gel column using 10% dichloromethane in hexane as eluate.
6.14 Synthesis of 1-Acetoxy-3,3-diphenylindane (Compound 7)
1-Acetoxy-3,3-diphenylindane (Compound 7) was synthesized as follows:
1-Hydroxy-3,3-diphenylindane (Compound 3) (0.06 g, 0.0021 mol) was
combined with 0.3 mL (0.0022 mol) triethylamine in 10 mL of
dichloromethane. The mixture was warmed to reflux with stirring to
dissolve all of the starting material. The heat was removed and 0.16 mL
(0.0022 mol) of acetyl chloride was added to the warm solution. The
mixture was returned to reflux and stirred at reflux for 1 h. After
cooling to room temperature, the reaction was quenched by adding 5 mL of
water. The reaction mixture was extracted with dichloromethane and the
organic layer dried over sodium sulfate. Evaporation of the solvent gave
0.008 g (11% yield) of 1-acetoxy-3,3-diphenylindanone (Compound 7) as an
off-white solid with a melting point of 90.degree. C.
6.15 Synthesis of 6-Chloro-3,3-di(4-chlorophenyl)indanone (Compound 8)
6-Chloro-3,3-di(4-chlorophenyl)indanone (Compound 8) was synthesized as
follows: 3,3,3-Tris(4-chlorophenyl) propionic acid (1.5 g, 0.004 mol) was
stirred in 10 mL of concentrated sulfuric acid at room temperature for 1.5
h. The reaction mixture was then poured into 10 mL of ice water and the
mixture extracted with dichloromethane. The solvent was evaporated and 0.8
g (54% yield) of 6-Chloro-3,3-di(4-chlorophenyl)indanone (Compound 8) was
collected as an off-white solid having a melting point of 134.degree. C.
6.16 Synthesis of 6-Chloro-2-cyanomethyl-3,3-di(4'-chlorophenyl)indanone
(Compound 10)
6-Chloro-2-cyanomethyl-3,3-di(4'-chlorophenyl)indanone (Compound 10) was
synthesized as follows: 6-Chloro-3,3-di(4'-chlorophenyl)indanone (Compound
8) (1.0 g, 0.0026 mol) was dissolved in 5 mL of tetrahydrofuran and 0.124
g (0.0031 mol) of sodium hydride was added. The reaction mixture was
stirred at room temperature for 1.5 h before 0.22 mL (0.0215 mol) of
bromoacetonitrile was added. After stirring overnight the reaction was
quenched with water and extracted with ethyl acetate. The extracts were
combined and the solvent removed in vacuo. The residue was purified on a
silica gel column using 5% ethyl acetate in hexane as the eluent. The
first fraction from the column was recovered starting material (1.05 g).
The second fraction contained undesired side reaction product. The third
fraction contained the desired product. After evaporation of the solvent,
0.179 g (16% yield) 6-Chloro-2-cyanomethyl-3,3-di(4'-chlorophenyl)indanone
(Compound 10) as a pale yellow solid was obtained.
6.17 Synthesis of
6-Chloro-3,3-di(4'-chlorophenyl)-2-N-oxime-3,3-diphenylindane (Compound
11)
6-Chloro-3,3-di(4'-chlorophenyl)-2-N-oxime-3,3-diphenylindane (Compound 11)
was synthesized as follows: 6-Chloro-3,3-di(4'-chlorophenyl)indanone
(compound 8) (0.80 g, 0.0021 mol) was combined with 0.72 g (0.0103 mol) of
hydroxylamine hydrochloride and 1.69 g (0.0206 mol) of sodium acetate and
dissolved in 25 mL of methanol. The solution was stirred at room
temperature for 16 h and then water was added. The mixture was extracted
with ethyl acetate and the organic layer was dried over magnesium sulfate.
Evaporation of the solvent gave 0.85 g (100% yield) of
6-Chloro-3,3-di(4'-chlorophenyl)-2-N-oxime-3,3-diphenylindane (Compound
11) as a white solid having a melting point of 85.degree. C.
6.18 Synthesis of 2-Acetamide-3,3-diphenylindanone (Compound 12)
2-Acetamide-3,3-diphenylindanone (Compound 12) was synthesized as follows:
2-Cyanomethyl-3,3-diphenylindanone (0.685 g, 0.0021 mol) was combined with
10 mL of concentrated sulfuric acid and 10 mL of glacial acetic acid. The
solution was stirred at room temperature for 3 h and then water was added.
The mixture was cooled in an ice bath and neutralized to pH 7 with
concentrated ammonium hydroxide and then extracted with ethyl acetate. The
organic layer was dried over magnesium sulfate. Evaporation of the solvent
gave 0.77 g of a light orange solid. This solid was crystallized from a
mixture of ethyl acetate and hexane. 2-Acetamide-3,3-diphenylindanone
(Compound 12) was obtained as off-white crystals, 0.527g (73% yield),
having a melting point of 169-171.degree. C.
6.19 Synthesis of 2-Cyanomethyl-3,3-diphenylindanol (Compound 13)
2-Cyanomethyl-3,3-diphenylindanol (Compound 13) was synthesized as follows:
2-Cyanomethyl-3,3-diphenylindanone (Compound 2) (0.311 g, 0.001 mol) was
dissolved in 5 mL of ethanol at room temperature. Sodium borohydride
(0.437 g, 0.011 mol) was added and the mixture was stirred at room
temperature for 15 min. The mixture was diluted with ethyl acetate and the
pH was adjusted to 2 with 2N hydrochloric acid. The layers were separated
and the aqueous layer extracted twice with ethyl acetate. The combined
extracts were evaporated in vacuo and the crude product was purified on a
silica gel column using 20% ethyl acetate in hexane. The first fraction
was unreacted starting material. The second fraction, when the solvent was
evaporated, gave 0.16 g (51% yield) of 2-Cyanomethyl-3,3-diphenylindanol
(Compound 13) as a white solid having a melting point of 79-85.degree. C.
6.20 Synthesis of 2-Acetamide-3,3-diphenylindanol (Compound 14)
2-Acetamide-3,3-diphenylindanol (Compound 14) was synthesized as follows:
2-Acetamide-3,3-diphenylindanone (Compound 12) (0.100 g, 0.0003 mol) was
dissolved in 2 mL of ethanol and 0.5 mL of methanol at room temperature.
Sodium borohydride (0.136 g, 0.0004 mol) was added and the mixture was
stirred at room temperature for 3 hours. The mixture was quenched with 2N
hydrochloric acid to pH 1. The mixture was extracted with ethyl acetate
and the combined extracts dried over magnesium sulfate. Evaporation of the
solvent gave an off-white solid which was crystallized from a mixture of
ethyl acetate/hexane. 2-Acetamide-3,3-diphenylindanol (Compound 14) was
collected by filtration as a white solid (0.026 g, 25% yield) having a
melting point of 218-220.degree. C.
6.21 Synthesis of 3,3-Diphenylindanone-2-methyl acetate (Compound 15)
3,3-Diphenylindanone-2-methyl acetate (Compound 15) was synthesized as
follows: 3,3-Diphenylindanone (Compound 2) (3.84 g, 0.0135 mol) was
dissolved in 30 mL of tetrahydrofuran at room temperature. Potassium
hydride (1.85 g, 0.0162 mol) was added and the mixture was stirred at room
temperature for 1 hour. Methyl chloroformate (1.25 mL, 0.0162 mol) was
added and the mixture was stirred at room temperature for 1 hour. The
mixture was quenched with water and extracted with ethyl acetate. The
combined extracts were dried over magnesium sulfate. Evaporation of the
solvent gave an dark brown solid which was purified on a silica gel column
using 5% ethyl acetate in hexane as eluent. The product was collected in
the second fraction off the column. Evaporation of the solvent gave a
slightly wet, pink solid which was stirred in hexane.
3,3-Diphenylindanone-2-methyl acetate (Compound 15) was collected by
filtration as an off-white solid (2.06 g, 45% yield) having a melting
point of 140-142.degree. C.
6.22 Synthesis of 3,3-Diphenyl-1-indanyl 2-naphthylmethyl ether (Compound
16)
3,3-Diphenyl-l-indanyl 2-naphthylmethyl ether (Compound 16) was synthesized
as follows: 1-Hydroxy-3,3-diphenylindane (Compound 3) (0.25 g, 0.87 mmol)
was dissolved in 10 mL of dimethylformamide and cooled to 0.degree. C.
with stirring. Sodium amide (0.042 g, 1.04 mmol) was added and the
reaction stirred for 0.5 h at 0.degree. C. before 0.23 g (1.04 mmol) of
2-bromomethylnaphthalene was added. The reaction mixture was allowed to
warm to room temperature and stirred for 15h. An equal volume of water was
added to the mixture and this was extracted twice with 50 mL of ethyl
acetate. After drying over magnesium sulfate the solvent was evaporated
and the resultant solid was purified on a silica gel column using 2% ethyl
acetate in hexane as the eluent. The second fraction collected was the
desired product. Evaporation of the solvent gave 0.300 g (81% yield) of
3,3-Diphenyl-1-indanyl 2-naphthylmethyl ether (Compound 16) as an
off-white, sticky solid.
6.23 Synthesis of 3,3-Diphenyl-l-indanyl .alpha.-(4-methyltoluate) ether
(Compound 17)
3,3-Diphenyl-1-indanyl .alpha.-(4-methyltoluate) ether (Compound 17) was
synthesized as follows: 1-Hydroxy-3,3-diphenylindane (Compound 3) (0.505
g, 1.8 mmol) was combined with 0.069 g (2.9 mmol) of sodium amide in 10 mL
of dimethylformamide and stirred at room temperature for 1.5 h before
0.667 g (2.9 mmol) of methyl 4-(bromomethyl)benzoate was added. The
reaction mixture was stirred for 18 h. The reaction mixture was poured
into 50 mL of water and extracted four times with 25 mL of ethyl acetate.
The combined extracts were washed with brine, dried over sodium sulfate
and the solvent evaporated to yield a yellow oil. The oil was purified by
vacuum distillation to give 0.370 g (47% yield) of 3,3-Diphenyl-1-indanyl
.alpha.-(4-methyltouate) ether (Compound 17) as a yellow solid having a
melting point of 50-52.degree. C.
6.24 Synthesis of 3,3-Diphenyl-1-indanyl .alpha.-(2-chlorotoluyl) ether
(compound 18)
3,3-Diphenyl-1-indanyl .alpha.-(2-chlorotoluyl) ether (Compound 18) was
synthesized as follows: 1-Hydroxy-3,3-diphenylindane (Compound 3) (0.503
g, 1.8 mmol) was combined with 0.075 g (3.1 mmol) of sodium amide in 10 mL
of dimethylformamide and stirred at room temperature for 1.5 h before 0.40
mL (3.2 mmol) of 2-chlorobenzyl chloride was added. The reaction mixture
was stirred for 21 h. The reaction mixture was poured into 50 mL of water
and extracted four times with 25 mL of ethyl acetate. The combined
extracts were washed with brine, dried over sodium sulfate and the solvent
evaporated to yield a yellow oil. The oil was purified by vacuum
distillation to give 0.520 g (70% yield) of 3,3-Diphenyl-1-indanyl
.alpha.-(2-chlorotoluyl) ether (Compound 18) as a solid having a melting
point of 27-29.degree. C.
6.25 Synthesis of 3-(3',3'-diphenyl-2'-indanyl-1'-one)propanol (Compound
19)
3-(3',3'-diphenyl-2'-indanyl-1'-one)propanol (Compound 19) was synthesized
as follows: 3,3-Diphenylindanone (Compound 2) (2 g, 0.007 mol) was
dissolved in 10 mL of tetrahydrofuran, cooled in an ice bath, and 0.97 g
(0.0085 mol) of potassium hydride was added. The reaction mixture was
stirred at room temperature for 0.5 h before 0.72 mL (0.0077 mol) of
3-bromo-1-propanol was added. After stirring overnight the reaction was
quenched with water and extracted with ethyl acetate. The combined
extracts were dried over magnesium sulfate and the solvent removed in
vacuo. The residue was purified on a silica gel column using 15% ethyl
acetate in hexane as the eluent. The first fraction from the column was
recovered starting material (1.05 g). The second fraction contained the
product. After evaporation of the solvent, 0.84 g (35% yield) of
3-(3',3'-diphenyl-2'-indanyl-1'-one)propanol (Compound 19) as a beige
solid having a melting point of 98.degree. C. was obtained.
6.26 Synthesis of 2-(Ethyl-2'-(1,3-dioxolane))-1-hydroxy-3,3-diphenylindene
(Compound 20)
2-(Ethyl-2'-(1,3-dioxolane))-l-hydroxy-3,3-diphenylindene (Compound 20) was
synthesized as follows: 3,3-Diphenylindanone (Compound 2) (4.0 g, 0.0141
mol) was dissolved in 30 mL of tetrahydrofuran at room temperature.
Potassium hydride (2.4 g, 0.0175 mol) was added and the mixture was
stirred at room temperature for 0.5 h. 2-(2-Bromoethyl)-1,3-dioxolane (2.0
mL, 0.0170 mol) was added and the mixture was continued stirring overnight
at room temperature. The mixture was quenched with water and extracted
with ethyl acetate. The combined extracts were purified on a silica gel
column using 8% ethyl acetate in hexane followed by 10% ethyl acetate in
hexane as eluent. The product was collected in the second fraction off the
column. Evaporation of the solvent gave
2-(Ethyl-2'-(1,3-dioxolane))-1-hydroxy-3,3-diphenylindene (Compound 20) as
an off-white solid (0.47 g, 9% yield) having a melting point of
124-126.degree. C.
6.27 Other Compounds
Other compounds of the invention can be synthesized by routine modification
of the above-described syntheses, or by other methods that are well known
in the art. Compound 1 is available from Maybridge Chemical Company
(distributor: Ryan Scientific, S.C.).
7. EXAMPLE
In Vitro Activity
This Example demonstrates the ability of several exemplary compounds of
structural formula (I) to inhibit the Gardos channel of erythrocytes
(Gardos Channel Assay) and/or mitogen-induced cell proliferation
(Mitogenic Assay) in vitro. The assays are generally applicable for
demonstrating the in vitro activity of other compounds of structural
formula (I).
7.1 Experimental Protocol
The percent inhibition of the Gardos channel (10 .mu.M compound) and the
IC.sub.50 were determined as described in Brugnara et al., 1993, J. Biol.
Chem. 268(12):8760-8768. The percent inhibition of mitogen-induced cell
proliferation (10 .mu.M compound) and the IC.sub.50 were determined or
described in Benzaquen et al. (1995, Nature Medicine 1:534-540) with NIH
3T3 mouse fibroblast cells (ATCC No. CRL 1658). Other cell lines, e.g.,
cancer cells, endothelial cells and fibroblasts, as well as many others,
may be used in the cell proliferation assay. Selection of a particular
cell line will depend in part on the desired application, and is well
within the capabilities of an ordinarily skilled artisan.
7.2 Results
The results of the experiment are provided in TABLE 1, below. Clotrimazole
is reported for purposes of comparison. Most of the compounds tested
exhibited significant activity in both assays. All of the compounds tested
exhibited significant activity in at least one of the assays.
TABLE 1
______________________________________
Pharmacological Activities of Various Compounds
(% Inhibition measured at 10 .mu.M)
Mitogenic Assay Gardos Channel Assay
Compound IC.sub.50
Inhibition IC.sub.50
Inhibition
Number (.mu.M) (%) (.mu.M) (%)
______________________________________
Clotrimazole
0.626 93.0 0.046
99.3
(1) 0.700 97.0 0.419 98.0
(2) 1.300 99.0 1.006 100.0
(3) 1.100 90.0 0.819 100.0
(4) 2.600 99.0 1.350 100.0
(5) -- 29.0 -- 67.3
(6) 3.400 90.0 -- 35.0
(7) 3.400 98.0 1.152 88.0
(8) 2.000 97.0 0.176 30.0
(9) -- 45.0 0.505 100.0
(10) 3.300 98.0 -- 49.5
(11) 3.400 99.0 -- 50.0
(12) -- 31.0 0.189 99.5
(13) -- 12.0 1.590 99.5
(14) -- 3.0 2.961 90.5
(15) 7.500 80.0 2.901 54.8
(16) -- 75.0 -- 0
(17) -- 76.0 -- 0
(18) -- 73.0 -- 0
(19) 1.500 99.0 5.952 43.7
(20) -- 81.0 -- 0
______________________________________
8. EXAMPLE
Activity In Cancer Cell Lines
This Example demonstrates the antiproliferative effect of several exemplary
compounds of formula (I) against a variety of cancer cell lines. The
assays are generally applicable for demonstrating the antiproliferative
activity of other compounds of formula (I).
8.1 Growth of Cells
The antiproliferative assays described herein were performed using standard
aseptic procedures and universal precautions for the use of tissues. Cells
were propagated using RPMI 1640 media (Gibco) containing 2% N 5% fetal
calf serum (Biowhittaker) at 37.degree. C., 5% CO.sub.2 and 95% humidity.
The cells were passaged using Trypsin (Gibco). Prior to addition of test
compound, the cells were harvested, the cell number counted and seeded at
10,000 cells/well in 100 .mu.l 5% fetal calf serum (FCS) containing RPMI
medium in 96-well plates and incubated overnight at 37.degree. C., 5%
CO.sub.2 and 95% humidity.
On the day of the treatment, stock solutions of the test compounds (10 mM
compound/DMSO) were added in 100 .mu.l FCS containing medium to a final
concentration of 10-0.125 .mu.M and the cells were incubated for 2, 3 or 5
days at 37.degree. C., 5% CO.sub.2 and 95% humidity.
Following incubation, the cellular protein was determined with the
ulforhodamine B (SRB) assay (Skehan P et al., 1990, J. Natl. Cancer Inst.
82:1107-1112). Growth inhibition, reported as the concentration of test
compound which inhibited 50% of cell proliferation (IC.sub.50) was
determined by curve fitting.
Values for VP-16, a standard anti-cancer agent, are provided for
comparison.
Except for MMRU cells, all cancer cell lines tested were obtained from the
American Type Culture Collection (ATCC, Rockville, MD). The ATCC assession
numbers were as follows: HeLa (CCL-2); CaSki (CRL-1550); MDA-MB-231
(HTB-26); MCF-7 (HTB-22); A549 (CCL-185); HTB-174 (HTB-174); HEPG2
(HB-8065); DU-145 (HTB-81); SK-MEL-28 (HTB-72); HT-29 (HTB-38); HCT-15
(CCL-225); ACHN (CRL-1611); U-118MG (HTB-15); SK-OV-3 (HTB-77).
MMRU cells (Stender et al., 1993, J. Dermatology 20:611-617) were a gift of
one of the authors.
8.2 Results
The results of the cell culture assays are presented in TABLES 2 and 3,
below.
TABLE 2
______________________________________
SRB ASSAY RESULTS
(5% FCS, 5 Day Incubation)
Test Compound IC.sub.50 (.mu.M)
Cancer Type
Cell Line VP-16 8 11
______________________________________
Cervical HeLa <1.25 >10 5.1
CaSki 1.8 6.8 7
Breast MDA-MB-23 <1.25 >10 >10
MCF7 <1.25 5.5 4.4
Lung A549 <1.25 8.9 8.8
HTB174 <1.25 >10 5.9
Hepatocel HEPG2 <1.25 6.4 5.8
Prostate DU-145 <1.25 >10 >10
Melanoma SK-MEL-28 <1.25 >10 5.5
MMRU <1.25 >10 6.2
Colon HT29 <1.25 8.3 6.8
HCT-15 1.3 >10 6.6
Renal ACHN <1.25 >10 >10
CNS U118MG 2.2 >10 >10
Ovary SK-OV-3 >10
Normal HUVEC <1.25 >10 6.4
human GM 1.4 >10 >10
3T3 >10 >10
mouse L929 <1.25 >10 8.6
______________________________________
TABLE 3
__________________________________________________________________________
SRB RESULTS
Conditions
Test Compound IC.sub.50 (.mu.M) in Various Cell Lines
Compound
% FCS/days
A5A9
HT29
MMRU
MCF7
HEPG2
U118MG
__________________________________________________________________________
VP-16 2%/3 days
2.3 20 <2.5
<2.5
3 5%/2 days >10 >10 5.8
4 2%/3 days 8.5 <2.5 8.2 <2.5
8 5%/3 days >10 >10 3.3 >10 7.8 >10
__________________________________________________________________________
9. EXAMPLE
Formulations
The following examples provide exemplary, not limiting, formulations for
administering the compounds of the invention to mammalian, especially
human, patients. Any of the compounds described herein, or pharmaceutical
salts or hydrates thereof, may be formulated as provided in the following
examples.
9.1 Tablet Formulation
Tablets each containing 60 mg of active ingredient are made up as follows:
______________________________________
Active Compound 60 mg
Starch 45 mg
Microcrystalline 45 mg
Cellulose
Sodium carboxymethyl 4.5 mg
starch
Talc 1 mg
Polyvinylpyrrolidone 4 mg
(10% in water)
Magnesium Stearate 0.5 mg
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 powders which are then passed through a No. 14
mesh U.S. sieve. The granules are dried at 50.degree.-60.degree. 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
by a tablet machine to yield tablets each weighing 150 mg.
Tablets can be prepared from the ingredients listed by wet granulation
followed by compression.
9.2 Gelatin Capsules
Hard gelatin capsules are prepared using the following ingredients:
______________________________________
Active Compound 250 mg/capsule
Starch dried 200 mg/capsule
Magnesium Stearate 10 mg/capsule
______________________________________
The above ingredients are mixed and filled into hard gelatin capsules in
460 mg quantities.
9.3 Aerosol Solution
An aerosol solution is prepared containing the following components:
______________________________________
Active Compound 0.25% (w/w)
Ethanol 29.75% (w/w)
Propellant 22 77.00% (w/w)
(Chlorodifluoromethane)
______________________________________
The active compound is mixed with ethanol and the mixture added to a
portion of the propellant 22, cooled to -30.degree. 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.
9.4 Suppositories
Suppositories each containing 225 mg of active ingredient are made as
follows:
______________________________________
Active Compound 225 mg
Saturated fatty acid 2,000 mg
glycerides
______________________________________
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 mixture is then poured into a suppository mold of nominal 2 g capacity
and allowed to cool.
9.5 Suspensions
Suspensions each containing 50 mg of medicament per mL dose are made as
follows:
______________________________________
Active Compound 50 mg
Sodium 50 mg
carboxymethylcellulose
Syrup 1.25 mL
Benzoic acid solution 0.10 mL
Flavor q.v.
Color q.v.
Purified water to 5 mL
______________________________________
The active ingredient 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 some color are diluted with some of
the water and added, with stirring. Sufficient water is then added to
produce the required volume.
The foregoing written specification is considered to be sufficient to
enable one skilled in the art to practice the invention. Various
modifications of the above-described modes for carrying out the invention
which are obvious to those skilled in the pharmaceutical arts or related
fields are intended to be within the scope of the following claims.
All cited references are hereby incorporated in their entireties by
reference herein.
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