U.S. patent application number 14/150655 was filed with the patent office on 2014-06-26 for methods and compositions of targeted drug development.
This patent application is currently assigned to Joseph P. Errico. The applicant listed for this patent is Joseph P. Errico. Invention is credited to John Allocco, Joseph P. Errico, William J. Feaver, Jeffrey D. Herron, Nicholas J. Lodato, Benjamin Mugrage, Jane Ong, Corey Pine, Matthew Sills, Ignatius Turchi, Pam Wines.
Application Number | 20140179681 14/150655 |
Document ID | / |
Family ID | 44306150 |
Filed Date | 2014-06-26 |
United States Patent
Application |
20140179681 |
Kind Code |
A1 |
Errico; Joseph P. ; et
al. |
June 26, 2014 |
METHODS AND COMPOSITIONS OF TARGETED DRUG DEVELOPMENT
Abstract
Provided herein are compounds having anti-proliferative effect.
Also provided are compounds that can modulate the activity of
multi-domain proteins comprising a dimerization arm and interdomain
tether, such as EGFR, where an untethered, extended conformation is
the active state and a tethered conformation is the inactive state,
resulting in an autoinhibited configuration. Also provided are
methods and pharmacophores for identifying such compounds. Other
aspects provide methods or therapeutic treatment for proliferative
diseases, disorders, or conditions, such as those associated with
EGFR.
Inventors: |
Errico; Joseph P.; (Warren,
NJ) ; Mugrage; Benjamin; (Cranbury, NJ) ;
Turchi; Ignatius; (Yardley, PA) ; Sills; Matthew;
(Berkeley Heights, NJ) ; Herron; Jeffrey D.;
(Bordentown, NJ) ; Lodato; Nicholas J.; (Union,
NJ) ; Ong; Jane; (Franklin Parik, NJ) ; Pine;
Corey; (Juniper, FL) ; Feaver; William J.;
(East Brunswick, NJ) ; Allocco; John; (Staten
Island, NY) ; Wines; Pam; (Manalapan, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Errico; Joseph P. |
|
|
US |
|
|
Assignee: |
Errico; Joseph P.
Warren
NJ
|
Family ID: |
44306150 |
Appl. No.: |
14/150655 |
Filed: |
January 8, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13521010 |
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PCT/US11/20418 |
Jan 6, 2011 |
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14150655 |
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61292776 |
Jan 6, 2010 |
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Current U.S.
Class: |
514/212.01 ;
514/252.12; 514/274; 514/275; 514/363; 514/415; 514/424; 514/471;
514/584; 540/604; 544/296; 544/297; 544/383; 548/138; 548/511;
548/542; 549/493; 564/23; 703/11 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 35/00 20180101; C07D 401/12 20130101; A61P 17/06 20180101;
A61K 31/47 20130101; A61P 9/00 20180101; C07D 239/52 20130101; C07D
403/12 20130101; C07C 335/26 20130101; G16B 15/00 20190201; A61P
17/08 20180101; C07D 409/06 20130101; C07D 215/26 20130101; C07D
405/06 20130101; C07D 307/54 20130101; C07D 239/47 20130101; C07D
209/08 20130101; A61K 31/496 20130101; C07D 285/135 20130101; C07D
239/42 20130101; A61K 31/4709 20130101; C07D 295/26 20130101; A61K
45/06 20130101; C07D 207/48 20130101; A61P 17/00 20180101; C07D
405/14 20130101; C07D 409/14 20130101; A61P 17/12 20180101; C07D
401/14 20130101; C07D 417/14 20130101; A61P 17/02 20180101 |
Class at
Publication: |
514/212.01 ;
564/23; 514/584; 544/297; 514/275; 514/274; 548/511; 514/415;
549/493; 514/471; 548/138; 514/363; 548/542; 514/424; 544/383;
514/252.12; 540/604; 544/296; 703/11 |
International
Class: |
C07D 403/12 20060101
C07D403/12; C07D 239/42 20060101 C07D239/42; C07D 239/47 20060101
C07D239/47; C07D 239/52 20060101 C07D239/52; G06F 19/16 20060101
G06F019/16; C07D 307/54 20060101 C07D307/54; C07D 285/135 20060101
C07D285/135; C07D 207/48 20060101 C07D207/48; C07D 295/26 20060101
C07D295/26; C07C 335/26 20060101 C07C335/26; C07D 209/08 20060101
C07D209/08 |
Claims
1. A compound having a formula of: ##STR00454## or a stereoisomer
or pharmaceutically acceptable salt thereof; wherein, R.sup.101 is
selected from the group consisting of: (i) straight chain or
branched C-1 to C-4 lower alkyl optionally containing unsaturation;
(ii) C-1 to C-6 cycloalkyl optionally containing unsaturation or
one oxygen or nitrogen atom; and (iii) aryl having an unsubstituted
phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or
6-position with one or more groups selected from the group
consisting of: (a) straight chain or branched C-1 to C-4 lower
alkyl optionally containing unsaturation; (b) C-1 to C-6 cycloalkyl
optionally containing unsaturation or one oxygen or nitrogen atom;
and (c) heteroaryl containing from 1 to 4 N, O, or S atoms;
R.sup.102 is hydrogen; R.sup.103 is selected from the group
consisting of: (i) hydrogen; (ii) straight chain or branched C-1 to
C-4 lower alkyl optionally containing unsaturation; (iii) C-1 to
C-6 cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; (iv) alkoxy --OR.sup.10 where R.sup.10 is selected
from the group consisting of: (a) a straight chain or branched C-1
to C-4 lower alkyl optionally containing unsaturation; and (b) a
C-1 to C-6 cycloalkyl optionally containing unsaturation or one
oxygen or nitrogen atom; and (v) halogen; R.sup.104 and R.sup.105
are independently selected from the group consisting of: (i)
hydrogen; (ii) straight chain or branched C-1 to C-4 lower alkyl
optionally containing unsaturation; (iii) C-1 to C-6 cycloalkyl
optionally containing unsaturation or one oxygen or nitrogen atom;
and (iv) alkoxy --OR.sup.10 where R.sup.10 is selected from the
group consisting of: (a) a straight chain or branched C-1 to C-4
lower alkyl optionally containing unsaturation; (b) a C-1 to C-6
cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; R.sup.106 is selected from the group consisting of:
(i) hydrogen; (ii) straight chain or branched C-1 to C-4 lower
alkyl optionally containing unsaturation; (iii) aryl having an
unsubstituted phenyl ring or a phenyl ring substituted at the 2-,
3-, 4-, 5- or 6-position with one or more groups selected from the
group consisting of: (a) straight chain or branched C-1 to C-4
lower alkyl optionally containing unsaturation; (b) C-1 to C-6
cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; and (c) heteroaryl containing from 1 to 4 N, O, or S
atoms; (d) aryl comprising a phenyl or heteroaryl containing from 1
to 4 N, O, or S atoms; (e) alkoxy --OR.sup.10 where R.sup.10 is
selected from the group consisting of: (1) a straight chain or
branched C-1 to C-4 lower alkyl optionally containing unsaturation;
and (2) a C-1 to C-6 cycloalkyl optionally containing unsaturation
or one oxygen or nitrogen atom; (f) trifluoromethyl; (g)
trifluoromethoxy; (h) difluoromethoxy; (i) 3,4-methylenedioxy; (j)
2,3-methylenedioxy; (k) nitro; and (l) halogen; and (iv) C-1 to C-3
substituted alkyl having at least one substitution group selected
from the group consisting of: (a) carboxyl --COOR.sup.10 where
R.sup.10 is selected from the group consisting of: (1) a straight
chain or branched C-1 to C-4 lower alkyl optionally containing
unsaturation; and (2) a C-1 to C-6 cycloalkyl optionally containing
unsaturation or one oxygen or nitrogen atom; (b) aryl having an
unsubstituted phenyl ring or a phenyl ring substituted at the 2-,
3-, 4-, 5- or 6-position with one or more groups selected from the
group consisting of: (1) straight chain or branched C-1 to C-4
lower alkyl optionally containing unsaturation; (2) C-1 to C-6
cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; (3) aryl having phenyl or heteroaryl containing from
1 to 4 N, O, or S atoms; (4) alkoxy --OR.sup.10 where R.sup.10 is
selected from the group consisting of: (A) a straight chain or
branched C-1 to C-4 lower alkyl optionally containing unsaturation;
and (B) a C-1 to C-6 cycloalkyl optionally containing unsaturation
or one oxygen or nitrogen atom; (5) trifluoromethyl; (6)
trifluoromethoxy; (7) difluoromethoxy; (8) 3,4-methylenedioxy; (9)
2,3-methylenedioxy; (10) nitro; and (11) halogen; and (c) aryloxy
(--OAr); R.sup.107 is selected from the group consisting of: (i)
hydrogen; (ii) straight chain or branched C-1 to C-4 lower alkyl
optionally containing unsaturation; (iii) C-1 to C-6 cycloalkyl
optionally containing unsaturation or one oxygen or nitrogen atom;
(iv) aryl having an unsubstituted phenyl ring or a phenyl ring
substituted at the 2-, 3-, 4-, 5- or 6-position with one or more
groups selected from the group consisting of: (a) straight chain or
branched C-1 to C-4 lower alkyl optionally containing unsaturation;
(b) C-1 to C-6 cycloalkyl optionally containing unsaturation or one
oxygen or nitrogen atom; (c) aryl comprising a phenyl or heteroaryl
containing from 1 to 4 N, O, or S atoms; (d) alkoxy --OR.sup.10
where R.sup.10 is selected from the group consisting of: (1) a
straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation; and (2) a C-1 to C-6 cycloalkyl optionally
containing unsaturation or one oxygen or nitrogen atom; (e)
trifluoromethyl; (f) trifluoromethoxy; (g) difluoromethoxy; (h)
3,4-methylenedioxy; (i) 2,3-methylenedioxy; (j) nitro; and (k)
halogen; and (v) C-1 to C-3 substituted alkyl having at least one
substitution group selected from the group consisting of: (a)
carboxyl --COOR.sup.10 where R.sup.10 is selected from the group
consisting of: (1) a straight chain or branched C-1 to C-4 lower
alkyl optionally containing unsaturation; and (2) a C-1 to C-6
cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; (b) aryl having an unsubstituted phenyl ring or a
phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with
one or more groups selected from the group consisting of: (1)
straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation; (2) C-1 to C-6 cycloalkyl optionally
containing unsaturation or one oxygen or nitrogen atom; (3) aryl
having phenyl or heteroaryl containing from 1 to 4 N, O, or S
atoms; (4) alkoxy --OR.sup.10 where R.sup.10 is selected from the
group consisting of: (A) a straight chain or branched C-1 to C-4
lower alkyl optionally containing unsaturation; and (B) a C-1 to
C-6 cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; (5) trifluoromethyl; (6) trifluoromethoxy; (7)
difluoromethoxy; (8) 3,4-methylenedioxy; (9) 2,3-methylenedioxy;
(10) nitro; and (11) halogen; and (c) aryloxy (--OAr); and Formula
(25) excludes a compound of Formula (24): ##STR00455## or a
stereoisomer or pharmaceutically acceptable salt thereof; wherein,
R.sup.108 is selected from the group consisting of: (i) straight
chain or branched C-1 to C-4 lower alkyl optionally containing
unsaturation; (ii) C-1 to C-6 cycloalkyl optionally containing
unsaturation or one oxygen or nitrogen atom; (iii) C-1 to C-3
substituted alkyl having at least one substitution group selected
from the group consisting of: (a) carboxyl --COOR.sup.10 where
R.sup.10 is selected from the group consisting of: (1) a straight
chain or branched C-1 to C-4 lower alkyl optionally containing
unsaturation; and (2) a C-1 to C-6 cycloalkyl optionally containing
unsaturation or one oxygen or nitrogen atom; (b) aryl having an
unsubstituted phenyl ring or a phenyl ring substituted at the 2-,
3-, 4-, 5- or 6-position with one or more groups selected from the
group consisting of: (1) straight chain or branched C-1 to C-4
lower alkyl optionally containing unsaturation; (2) C-1 to C-6
cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; (3) aryl having phenyl or heteroaryl containing from
1 to 4 N, O, or S atoms; (4) alkoxy --OR.sup.10 where R.sup.10 is
selected from the group consisting of: (A) a straight chain or
branched C-1 to C-4 lower alkyl optionally containing unsaturation;
and (B) a C-1 to C-6 cycloalkyl optionally containing unsaturation
or one oxygen or nitrogen atom; (5) trifluoromethyl; (6)
trifluoromethoxy; (7) difluoromethoxy; (8) 3,4-methylenedioxy; (9)
2,3-methylenedioxy; (10) nitro; and (11) halogen; and (c) aryloxy
(--OAr); (iv) aryl comprising a phenyl or heteroaryl containing
from 1 to 4 N, O, or S atoms; (v) amino; and (vi) hydroxyl;
R.sup.109 is hydrogen; R.sup.110 and R.sup.111 are independently
selected from the group consisting of: (i) hydrogen; (ii) straight
chain or branched C-1 to C-4 lower alkyl optionally containing
unsaturation; (iii) C-1 to C-6 cycloalkyl optionally containing
unsaturation or one oxygen or nitrogen atom; (iv) C-1 to C-3
substituted alkyl having at least one substitution group selected
from the group consisting of: (a) carboxyl --COOR.sup.10 where
R.sup.10 is selected from the group consisting of: (1) a straight
chain or branched C-1 to C-4 lower alkyl optionally containing
unsaturation; and (2) a C-1 to C-6 cycloalkyl optionally containing
unsaturation or one oxygen or nitrogen atom; (b) aryl having an
unsubstituted phenyl ring or a phenyl ring substituted at the 2-,
3-, 4-, 5- or 6-position with one or more groups selected from the
group consisting of: (1) straight chain or branched C-1 to C-4
lower alkyl optionally containing unsaturation; (2) C-1 to C-6
cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; (3) aryl having phenyl or heteroaryl containing from
1 to 4 N, O, or S atoms; (4) alkoxy --OR.sup.10 where R.sup.10 is
selected from the group consisting of: (A) a straight chain or
branched C-1 to C-4 lower alkyl optionally containing unsaturation;
and (B) a C-1 to C-6 cycloalkyl optionally containing unsaturation
or one oxygen or nitrogen atom; (5) trifluoromethyl; (6)
trifluoromethoxy; (7) difluoromethoxy; (8) 3,4-methylenedioxy; (9)
2,3-methylenedioxy; (10) nitro; and (11) halogen; and (c) aryloxy
(--OAr); (v) alkoxy --OR.sup.10 where R.sup.10 is selected from the
group consisting of: (a) a straight chain or branched C-1 to C-4
lower alkyl optionally containing unsaturation; and (b) a C-1 to
C-6 cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; and (vi) halogen; R.sup.112 is selected from the
group consisting of: hydrogen and straight chain or branched C-1 to
C-4 lower alkyl optionally containing unsaturation; R.sup.113 is
selected from the group consisting of: (i) straight chain or
branched C-1 to C-4 lower alkyl optionally containing unsaturation;
(ii) trifluoromethyl; (iii) aryl comprising a phenyl or heteroaryl
containing from 1 to 4 N, O, or S atoms; and (iv) amino; R.sup.114
is selected from the group consisting of: (i) hydrogen; (ii) acyl
--COR.sup.10 where R.sup.10 is selected from the group consisting
of: (a) a straight chain or branched C-1 to C-4 lower alkyl
optionally containing unsaturation; and (b) a C-1 to C-6 cycloalkyl
optionally containing unsaturation or one oxygen or nitrogen atom;
and (iii) carboxyl --COOR.sup.10 where R.sup.10 is selected from
the group consisting of: (a) a straight chain or branched C-1 to
C-4 lower alkyl optionally containing unsaturation; and (b) a C-1
to C-6 cycloalkyl optionally containing unsaturation or one oxygen
or nitrogen atom; R.sup.115 is selected from the group consisting
of: (i) straight chain or branched C-1 to C-4 lower alkyl
optionally containing unsaturation; (ii) trifluoromethyl; and (iii)
aryl comprising a phenyl or heteroaryl containing from 1 to 4 N, O,
or S atoms; and Formula (27) excludes a compound of Formula (24):
##STR00456## or a stereoisomer or pharmaceutically acceptable salt
thereof; wherein n is 1 or 2; R.sup.116 is selected from the group
consisting of: (i) straight chain or branched C-1 to C-4 lower
alkyl optionally containing unsaturation; and (ii) aryl comprising
a phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms;
R.sup.117 of is hydrogen; R.sup.118 is selected from the group
consisting of: (i) hydrogen, (ii) straight chain or branched C-1 to
C-4 lower alkyl optionally containing unsaturation; (ii) alkoxy
--OR.sup.10 where R.sup.10 is selected from the group consisting
of: (a) a straight chain or branched C-1 to C-4 lower alkyl
optionally containing unsaturation; and (b) a C-1 to C-6 cycloalkyl
optionally containing unsaturation or one oxygen or nitrogen atom;
and (iii) halogen; R.sup.119 is selected from the group consisting
of: (i) hydrogen; (ii) straight chain or branched C-1 to C-4 lower
alkyl optionally containing unsaturation; and (iii) aryl comprising
a phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms;
R.sup.120 is selected from the group consisting of: (i) hydrogen,
(ii) straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation; and (iii) alkoxy --OR.sup.10 where
R.sup.10 is selected from the group consisting of: (a) a straight
chain or branched C-1 to C-4 lower alkyl optionally containing
unsaturation; and (b) a C-1 to C-6 cycloalkyl optionally containing
unsaturation or one oxygen or nitrogen atom; R.sup.121 is selected
from the group consisting of: hydrogen and aryl comprising a phenyl
or heteroaryl containing from 1 to 4 N, O, or S atoms; and Formula
(28) excludes a compound of Formula (24): ##STR00457##
2. The compound of claim 1, wherein (A) the compound is according
to Formula (25) (10381-like Type D) and at least one of the
following features is satisfied R.sup.101 is selected from the
group consisting of methyl and phenyl; R.sup.103 is selected from
the group consisting of methyl, ethyl, chloro, methoxy, and ethoxy;
R.sup.104 is selected from the group consisting of hydrogen,
methyl, and methoxy; R.sup.105 is selected from the group
consisting of hydrogen, methyl, methoxy, and ethoxy; R.sup.106 is
selected from the group consisting of methyl and phenyl; or the
compound is selected from the group consisting of ##STR00458## (B)
the compound is according to Formula (27) (10381-like Type E) and
at least one of the following features is satisfied R.sup.108 is
selected from the group consisting of: methyl, phenyl, and
hydroxyl; R.sup.110 and R.sup.111 are independently selected from
the group consisting of: hydrogen, methyl, methoxy, and chloro;
R.sup.112 is methyl; R.sup.113 is selected from the group
consisting of: methyl, phenyl, and amino; R.sup.115 is selected
from the group consisting of: methyl, phenyl, and trifluoromethyl;
the compound is selected from the group consisting of ##STR00459##
or (C) the compound is according to Formula (28) (10381-like Type
F) and at least one of the following features is satisfied n is 2
and the compound has a formula of: ##STR00460## or the compound is
##STR00461##
3. The compound of claim 1 that inhibits EGFR activity comprising:
eight or more of functional groups F(IV)1, F(IV)2, F(IV)3, F(IV)4,
F(IV)5, F(IV)6, F(IV)7, F(IV)8, F(IV)9, and F(IV)10 of a Scheme IV
10381-like pharmacophore; wherein functional group F(IV)1 accepts a
hydrogen bond from receptor side chain OH of Thr239 of SEQ ID NO: 1
and has coordinates of r=49.686, .theta. (theta)=113.993, and .phi.
(phi)=-17.014 and a spherical radius of about 1.2 .ANG.; functional
group F(IV)2 accepts a hydrogen bond from receptor side chain OH of
Thr239 of SEQ ID NO: 1 and has coordinates of r=48.071, .theta.
(theta)=115.388, and .phi. (phi)=-16.211 and a spherical radius of
about 1.6 .ANG.; functional group F(IV)3 forms a hydrophobic
contact with a side chain of receptor Met576 and imidazolidine ring
of Pro242 of SEQ ID NO: 1 and has coordinates of r=50.781, .theta.
(theta)=113.121, and .phi. (phi)=-17.520 and a spherical radius of
about 1.2 .ANG.; functional group F(IV)4 forms a hydrophobic
contact with a side chain of receptor Met576 and imidazolidine ring
of Pro242 of SEQ ID NO: 1 and has coordinates of r=52.021, .theta.
(theta)=114.264, and .phi. (phi)=-15.878 and a spherical radius of
about 1.5 .ANG.; functional group F(IV)5 forms a hydrophobic
contact with a side chain of receptor Met576 and imidazolidine ring
of Pro242 of SEQ ID NO: 1 and has coordinates of r=50.322, .theta.
(theta)=111.253, and .phi. (phi)=-15.426 and a spherical radius of
about 1.2 .ANG.; functional group F(IV)6 forms a hydrophobic
contact with a side chain of receptor Met576 and imidazolidine ring
of Pro242 of SEQ ID NO: 1 and has coordinates of r=51.575, .theta.
(theta)=112.433, and .phi. (phi)=-13.827 and a spherical radius of
about 1.5 .ANG.; functional group F(IV)7 forms a hydrophobic
contact with a side chain of receptor Leu243 and a side chain of
Thr239 of SEQ ID NO: 1 and has coordinates of r=47.767, .theta.
(theta)=112.521, and .phi. (phi)=-10.196 and a spherical radius of
about 1.2 .ANG.; functional group F(IV)8 donates a hydrogen bond to
a backbone carbonyl of His280 of SEQ ID NO: 1 and has coordinates
of r=45.184, .theta. (theta)=112.328, and .phi. (phi)=-8.875 and a
spherical radius of about 1.2 .ANG.; functional group F(IV)9 forms
a hydrophobic contact with a side chain of receptor Met244 and
Leu243 of SEQ ID NO: 1 and has coordinates of r=49.512, .theta.
(theta)=108.007, and .phi. (phi)=-8.504 and a spherical radius of
about 1.2 .ANG.; functional group F(IV)10 forms a hydrophobic
contact with a side chain of receptor Met244 of SEQ ID NO: 1 and
has coordinates of r=50.431, .theta. (theta)=104.985, and .phi.
(phi)=-7.420 and a spherical radius of about 1.8 .ANG.; and the
compound substantially maintains a non-extended tether inactive
configuration of EGFR or substantially prevents stabilization of an
extended tether active configuration of EGFR.
4. A compound having a formula of: ##STR00462## or a stereoisomer
or pharmaceutically acceptable salt thereof; wherein, R.sup.23 and
R.sup.27 are hydrogen; R.sup.24 and R.sup.26 are independently
selected from the group consisting of: (i) hydrogen; (ii) C-1 to
C-6 lower alkyl; (iii) alkoxy --OR.sup.10 where R.sup.10 is a
straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation or a C-1 to C-6 cycloalkyl optionally
containing unsaturation or one oxygen or nitrogen atom; and (iv)
halogen; R.sup.25 is selected from the group consisting of: (i)
hydrogen; (ii) straight chain or branched C-1 to C-4 lower alkyl
optionally containing unsaturation; (iii) C-1 to C-6 cycloalkyl
optionally containing unsaturation or one oxygen or nitrogen atom;
(iv) C-1 to C-3 substituted alkyl having at least one substitution
group selected from the group consisting of (a) carboxyl
--COOR.sup.10 where R.sup.10 is a straight chain or branched C-1 to
C-4 lower alkyl optionally containing unsaturation or a C-1 to C-6
cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; (b) aryl having an unsubstituted phenyl ring or a
phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with
one or more groups selected from the group consisting of: straight
chain or branched C-1 to C-4 lower alkyl optionally containing
unsaturation; C-1 to C-6 cycloalkyl optionally containing
unsaturation or one oxygen or nitrogen atom; aryl having phenyl or
heteroaryl containing from 1 to 4 N, O, or S atoms; alkoxy
--OR.sup.10 where R.sup.10 is a straight chain or branched C-1 to
C-4 lower alkyl optionally containing unsaturation or a C-1 to C-6
cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; trifluoromethyl; trifluoromethoxy; difluoromethoxy;
3,4-methylenedioxy; 2,3-methylenedioxy; nitro; and halogen; and (c)
aryloxy (--OAr); (v) alkoxy --OR.sup.10 where R.sup.10 is a
straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation or a C-1 to C-6 cycloalkyl optionally
containing unsaturation or one oxygen or nitrogen atom; and (vi)
halogen; R.sup.28 is selected from the group consisting of: (i)
hydrogen; (ii) straight chain or branched C-1 to C-4 lower alkyl
optionally containing unsaturation; and (iii) aryl having an
unsubstituted phenyl ring or a phenyl ring substituted at the 2-,
3-, 4-, 5- or 6-position with one or more groups selected from the
group consisting of: (a) straight chain or branched C-1 to C-4
lower alkyl optionally containing unsaturation; (b) C-1 to C-6
cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; and (c) halogen. R.sup.29 is selected from the group
consisting of: (i) hydrogen; (ii) straight chain or branched C-1 to
C-4 lower alkyl optionally containing unsaturation; (iii) hydroxy;
(iv) alkoxy --OR.sup.10 where R.sup.10 is a straight chain or
branched C-1 to C-4 lower alkyl optionally containing unsaturation
or a C-1 to C-6 cycloalkyl optionally containing unsaturation or
one oxygen or nitrogen atom; and (v) halogen; R.sup.30 is selected
from the group consisting of: (i) hydrogen; (ii) straight chain or
branched C-1 to C-4 lower alkyl optionally containing unsaturation;
(iii) alkoxy --OR.sup.10 where R.sup.10 is a straight chain or
branched C-1 to C-4 lower alkyl optionally containing unsaturation
or a C-1 to C-6 cycloalkyl optionally containing unsaturation or
one oxygen or nitrogen atom; (iv) trifluoromethyl; and (v) halogen;
R.sup.31 is selected from the group consisting of: (i) hydrogen;
(ii) hydroxy; (iii) alkoxy --OR.sup.10 where R.sup.10 is a straight
chain or branched C-1 to C-4 lower alkyl optionally containing
unsaturation or a C-1 to C-6 cycloalkyl optionally containing
unsaturation or one oxygen or nitrogen atom; (iv) straight chain or
branched C-1 to C-4 lower alkyl optionally containing unsaturation;
(v) trifluoromethyl; and (vi) halogen; and R.sup.32 is selected
from the group consisting of: (i) hydrogen; (ii) straight chain or
branched C-1 to C-4 lower alkyl optionally containing unsaturation;
(iii) hydroxy; (iv) trifluoromethyl; and (v) halogen; and Formula
(13) excludes the compound of Formula (12): ##STR00463## or a
stereoisomer or pharmaceutically acceptable salt thereof; wherein,
R.sup.33 is selected from the group consisting of: (i) straight
chain or branched C-1 to C-4 lower alkyl optionally containing
unsaturation; (ii) C-1 to C-3 substituted alkyl having at least one
substitution group selected from the group consisting of: (a)
carboxyl --COOR.sup.10 where R.sup.10 is a straight chain or
branched C-1 to C-4 lower alkyl optionally containing unsaturation
or a C-1 to C-6 cycloalkyl optionally containing unsaturation or
one oxygen or nitrogen atom; (b) aryl having an unsubstituted
phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or
6-position with one or more groups selected from the group
consisting of: straight chain or branched C-1 to C-4 lower alkyl
optionally containing unsaturation; C-1 to C-6 cycloalkyl
optionally containing unsaturation or one oxygen or nitrogen atom;
aryl having phenyl or heteroaryl containing from 1 to 4 N, O, or S
atoms; alkoxy --OR.sup.10 where R.sup.10 is a straight chain or
branched C-1 to C-4 lower alkyl optionally containing unsaturation
or a C-1 to C-6 cycloalkyl optionally containing unsaturation or
one oxygen or nitrogen atom; trifluoromethyl; trifluoromethoxy;
difluoromethoxy; 3,4-methylenedioxy; 2,3-methylenedioxy; nitro; and
halogen; and (c) aryloxy (--OAr); and (iii) aryl having an
unsubstituted phenyl ring or a phenyl ring substituted at the 2-,
3-, 4-, 5- or 6-position with one or more groups selected from the
group consisting of: (a) straight chain or branched C-1 to C-4
lower alkyl optionally containing unsaturation; (b) C-1 to C-6
cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; (c) aryl having phenyl or heteroaryl containing from
1 to 4 N, O, or S atoms; (d) alkoxy --OR.sup.10 where R.sup.10 is a
straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation or a C-1 to C-6 cycloalkyl optionally
containing unsaturation or one oxygen or nitrogen atom; (e)
trifluoromethyl; (f) trifluoromethoxy; (g) difluoromethoxy; (h)
3,4-methylenedioxy; (i) 2,3-methylenedioxy; (j) nitro; and (k)
halogen; R.sup.34 is hydrogen; R.sup.35 is selected from the group
consisting of: (i) hydrogen; (ii) hydroxy; (iii) alkoxy --OR.sup.10
where R.sup.10 is a straight chain or branched C-1 to C-4 lower
alkyl optionally containing unsaturation or a C-1 to C-6 cycloalkyl
optionally containing unsaturation or one oxygen or nitrogen atom;
(iv) straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation; (v) trifluoromethyl; and (vi) halogen;
R.sup.36 is selected from the group consisting of: (i) hydrogen;
(ii) straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation; (iii) hydroxy; (iv) alkoxy --OR.sup.10
where R.sup.10 is a straight chain or branched C-1 to C-4 lower
alkyl optionally containing unsaturation or a C-1 to C-6 cycloalkyl
optionally containing unsaturation or one oxygen or nitrogen atom;
(v) trifluoromethyl; and (vi) halogen; and R.sup.37 is hydrogen;
and Formula (14) excludes a compound of Formula (12): ##STR00464##
or a stereoisomer or pharmaceutically acceptable salt thereof;
wherein, n is 1 or 2; X.sup.2 and X.sup.3 are independently
selected from the group consisting of: carbon, oxygen, nitrogen,
and sulfur; R.sup.38 is hydrogen; R.sup.39 is selected from the
group consisting of: (i) hydrogen; (ii) C-1 to C-6 lower alkyl;
(iii) alkoxy --OR.sup.10 where R.sup.10 is selected from the group
consisting of: (a) a straight chain or branched C-1 to C-4 lower
alkyl optionally containing unsaturation; and (b) a C-1 to C-6
cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; and (iv) halogen; R.sup.40 is hydrogen; R.sup.41 is
selected from the group consisting of: (i) hydrogen; (ii) straight
chain or branched C-1 to C-4 lower alkyl optionally containing
unsaturation; and (iii) aryl having an unsubstituted phenyl ring or
a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with
one or more groups selected from the group consisting of: (a)
straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation; (b) C-1 to C-6 cycloalkyl optionally
containing unsaturation or one oxygen or nitrogen atom; (c) aryl
having phenyl or heteroaryl containing from 1 to 4 N, O, or S
atoms; (d) alkoxy --OR.sup.10 where R.sup.10 is selected from the
group consisting of: (1) a straight chain or branched C-1 to C-4
lower alkyl optionally containing unsaturation; and (2) a C-1 to
C-6 cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; (e) trifluoromethyl; (f) trifluoromethoxy; (g)
difluoromethoxy; (h) 3,4-methylenedioxy; (i) 2,3-methylenedioxy;
(j) nitro; and (k) halogen; R.sup.42 is selected from the group
consisting of: (i) hydrogen; (ii) straight chain or branched C-1 to
C-4 lower alkyl optionally containing unsaturation; (iii) hydroxy;
(iv) alkoxy --OR.sup.10 where R.sup.10 is selected from the group
consisting of: (a) a straight chain or branched C-1 to C-4 lower
alkyl optionally containing unsaturation; and (b) a C-1 to C-6
cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; and (v) halogen; R.sup.43 is selected from the group
consisting of: (i) hydrogen; (ii) straight chain or branched C-1 to
C-4 lower alkyl optionally containing unsaturation; (iii) alkoxy
--OR.sup.10 where R.sup.10 is selected from the group consisting
of: (a) a straight chain or branched C-1 to C-4 lower alkyl
optionally containing unsaturation; and (b) a C-1 to C-6 cycloalkyl
optionally containing unsaturation or one oxygen or nitrogen atom;
(iv) trifluoromethyl; and (v) halogen; R.sup.44 is selected from
the group consisting of: (i) hydrogen; (ii) hydroxy; (iii) alkoxy
--OR.sup.10 where R.sup.10 is selected from the group consisting
of: (a) straight chain or branched C-1 to C-4 lower alkyl
optionally containing unsaturation; and (b) a C-1 to C-6 cycloalkyl
optionally containing unsaturation or one oxygen or nitrogen atom;
(iv) straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation; (v) trifluoromethyl; and (vi) halogen;
R.sup.45 is selected from the group consisting of: (i) hydrogen;
(ii) straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation; (iii) hydroxy; (iv) trifluoromethyl; and
(v) halogen; and Formula (15) excludes the compound of Formula
(12): ##STR00465## or a stereoisomer or pharmaceutically acceptable
salt thereof; wherein, n is 1 or 2; X.sup.4 and X.sup.5 are
independently selected from the group consisting of: carbon,
oxygen, nitrogen and sulfur; R.sup.46 and R.sup.50 are hydrogen;
R.sup.47 and R.sup.49 are independently selected from the group
consisting of: (i) hydrogen; (ii) straight chain or branched C-1 to
C-4 lower alkyl optionally containing unsaturation; (iii) alkoxy
--OR.sup.10 where R.sup.10 is selected from the group consisting
of: (a) a straight chain or branched C-1 to C-4 lower alkyl
optionally containing unsaturation and (b) a C-1 to C-6 cycloalkyl
optionally containing unsaturation or one oxygen or nitrogen atom;
(iv) halogen; and (v) R.sup.47 and R.sup.49 form a 5 or 6 membered
cycloalkyl ring; R.sup.48 is selected from the group consisting of:
(i) hydrogen; (ii) straight chain or branched C-1 to C-4 lower
alkyl optionally containing unsaturation; (iii) C-1 to C-6
cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; (iv) C-1 to C-3 substituted alkyl having at least
one substitution group selected from the group consisting of: (a)
carboxyl --COOR.sup.10 where R.sup.10 is selected from the group
consisting of: (1) a straight chain or branched C-1 to C-4 lower
alkyl optionally containing unsaturation; and (2) a C-1 to C-6
cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; (b) aryl having an unsubstituted phenyl ring or a
phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with
one or more groups selected from the group consisting of: (1)
straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation; (2) C-1 to C-6 cycloalkyl optionally
containing unsaturation or one oxygen or nitrogen atom; (3) aryl
having phenyl or heteroaryl containing from 1 to 4 N, O, or S
atoms; (4) alkoxy --OR.sup.10 where R.sup.10 is selected from the
group consisting of: (A) straight chain or branched C-1 to C-4
lower alkyl optionally containing unsaturation; and (B) a C-1 to
C-6 cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; (5) trifluoromethyl; (6) trifluoromethoxy; (7)
difluoromethoxy; (8) 3,4-methylenedioxy; (9) 2,3-methylenedioxy;
(10) nitro; and (11) halogen; and (c) aryloxy (--OAr); (v) alkoxy
--OR.sup.10 where R.sup.10 is selected from the group consisting
of: (a) a straight chain or branched C-1 to C-4 lower alkyl
optionally containing unsaturation; and (b) a C-1 to C-6 cycloalkyl
optionally containing unsaturation or one oxygen or nitrogen atom;
(vi) halogen; and (vii) R.sup.48 forms a 5 or 6 membered cycloalkyl
ring with R.sup.47; R.sup.51 is selected from the group consisting
of: (i) hydrogen; (ii) straight chain or branched C-1 to C-4 lower
alkyl optionally containing unsaturation; and (iii) aryl having an
unsubstituted phenyl ring or a phenyl ring substituted at the 2-,
3-, 4-, 5- or 6-position with one or more groups selected from the
group consisting of: (a) straight chain or branched C-1 to C-4
lower alkyl optionally containing unsaturation; (b) C-1 to C-6
cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; (c) aryl having phenyl or heteroaryl containing from
1 to 4 N, O, or S atoms; (d) alkoxy --OR.sup.10 where R.sup.10 is
selected from the group consisting of: (1) a straight chain or
branched C-1 to C-4 lower alkyl optionally containing unsaturation;
and (2) a C-1 to C-6 cycloalkyl optionally containing unsaturation
or one oxygen or nitrogen atom; (e) trifluoromethyl; (f)
trifluoromethoxy; (g) difluoromethoxy; (h) 3,4-methylenedioxy; (i)
2,3-methylenedioxy; (j) nitro; and (k) halogen; R.sup.52 is
selected from the group consisting of: (i) hydrogen; (ii) straight
chain or branched C-1 to C-4 lower alkyl optionally containing
unsaturation; (iii) hydroxy; (iv) alkoxy --OR.sup.10 where R.sup.10
is selected from the group consisting of: (a) straight chain or
branched C-1 to C-4 lower alkyl optionally containing unsaturation;
and (b) a C-1 to C-6 cycloalkyl optionally containing unsaturation
or one oxygen or nitrogen atom; and (v) halogen; R.sup.53 is
selected from the group consisting of: (i) hydrogen; (ii) straight
chain or branched C-1 to C-4 lower alkyl optionally containing
unsaturation; (iii) hydroxy; (iv) trifluoromethyl; and (v) halogen;
and Formula (16) excludes the compound of Formula (12):
##STR00466##
5. The compound of claim 4, wherein (A) the compound is according
to Formula (13) (1734-like Type P) and one or more of the following
features are satisfied: R.sup.24 and R.sup.26 are independently
selected from the group consisting of: hydrogen and alkoxy;
R.sup.25 is selected from the group consisting of: hydrogen or
alkoxy --OR.sup.10; R.sup.28 is selected from the group consisting
of: hydrogen or aryl; R.sup.28 is hydrogen; R.sup.30 is straight
chain or branched C-1 to C-4 lower alkyl optionally containing
unsaturation; R.sup.31 is selected from the group consisting of:
hydroxy or alkoxy --OR.sup.10; R.sup.32 is hydrogen; or the
compound is selected from the group consisting of ##STR00467## (B)
the compound is according to Formula (14) (1734-like Type Q) and
one or more of the following features are satisfied: R.sup.35 is
straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation; R.sup.36 is selected from the group
consisting of: (i) hydroxy and (ii) alkoxy --OR.sup.10 where
R.sup.10 is selected from the group consisting of: (a) a straight
chain or branched C-1 to C-4 lower alkyl optionally containing
unsaturation; and (b) a C-1 to C-6 cycloalkyl optionally containing
unsaturation or one oxygen or nitrogen atom; or the compound is
##STR00468## (C) the compound is according to Formula (15)
(1734-like Type R) and one or more of the following features are
satisfied: n is 1; X.sup.2 and X.sup.3 are independently selected
from the group consisting of: oxygen or carbon; R.sup.42 is
hydrogen; R.sup.43 is straight chain or branched C-1 to C-4 lower
alkyl optionally containing unsaturation; R.sup.44 is selected from
the group consisting of (i) hydroxy and (ii) alkoxy --OR.sup.10
where R.sup.10 is selected from the group consisting of (a) a
straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation and (b) a C-1 to C-6 cycloalkyl optionally
containing unsaturation or one oxygen or nitrogen atom; R.sup.45 is
hydrogen; or the compound is ##STR00469## or (D) the compound is
according to Formula (16) (1734-like Type S) and one or more of the
following features are satisfied: n is 1; X.sup.4 and X.sup.5 are
independently selected from the group consisting of oxygen and
carbon; R.sup.48 is selected from the group consisting of hydrogen
or straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation; R.sup.47 and R.sup.48 form a 5 membered
cycloalkyl ring; R.sup.51 is selected from the group consisting of
(i) hydrogen and (ii) aryl having an unsubstituted phenyl ring or a
phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with
one or more groups selected from the group consisting of: (a)
straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation, (b) C-1 to C-6 cycloalkyl optionally
containing unsaturation or one oxygen or nitrogen atom, (c) aryl
having phenyl or heteroaryl containing from 1 to 4 N, O, or S
atoms, (d) alkoxy --OR.sup.10 where R.sup.10 is selected from the
group consisting of (1) a straight chain or branched C-1 to C-4
lower alkyl optionally containing unsaturation, and (2) a C-1 to
C-6 cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom, (e) trifluoromethyl, (f) trifluoromethoxy, (g)
difluoromethoxy, (h) 3,4-methylenedioxy, (i) 2,3-methylenedioxy,
(j) nitro, and (k) halogen; R.sup.52 is hydrogen; or the compound
is selected from the group consisting of ##STR00470##
6. The compound of claim 4 that inhibits EGFR activity, the
compound comprising: five or more of functional groups F(II)1,
F(II)2, F(II)3, F(II)4, F(II)5, F(II)6, F(II)7, F(II)8, and F(II)9
of a Scheme II 1734-like pharmacophore; wherein functional group
F(II)1 donates an H-bond or forms a salt bridge to a carboxylate
side chain of receptor Asp553 of SEQ ID NO: 1 and has coordinates
of r=56.363, .theta. (theta)=94.368, and .phi. (phi)=-17.752 and a
spherical radius of about 1.2 .ANG.; functional group F(II)2 is a
donor and has coordinates of r=53.290, .theta. (theta)=101.494, and
.phi. (phi)=-23.244 and a spherical radius of about 1.0 .ANG.;
functional group F(II)3 forms a hydrophobic contact with a side
chain of receptor Val568, imidazolidine side chain of receptor
Pro552 and with a side chain of Met253 of SEQ ID NO: 1 and has
coordinates of r=53.726, .theta. (theta)=97.830, and .phi.
(phi)=-18.377 and a spherical radius of about 1.7 .ANG.; functional
group F(II)4 forms a hydrophobic contact with side chain of
receptor Val575, Met253, and with an imidazolidine ring of receptor
Pro552 of SEQ ID NO: 1 and has coordinates of r=53.647, .theta.
(theta)=103.844, and .phi. (phi)=-20.990 and a spherical radius of
about 1.4 .ANG.; functional group F(II)5 donates an H-bond to a
side chain hydroxyl of Thr570 of SEQ ID NO: 1 and has coordinates
of r=51.093, .theta. (theta)=104.261, and .phi. (phi)=-25.552 and a
spherical radius of about 1.2 .ANG.; functional group F(II)6 is a
donor having directionality of F4 with respect to a backbone
carbonyl of receptor Thr570 of SEQ ID NO: 1 and has coordinates of
r=52.340, .theta. (theta)=103.980, and .phi. (phi)=-27.461 and a
spherical radius of about 1.5 .ANG.; functional group F(II)7
accepts an H-bond from a receptor backbone NH of Ala573 of SEQ ID
NO: 1 and has coordinates of r=51.383, .theta. (theta)=106.455, and
.phi. (phi)=-24.319 and a spherical radius of about 1.2 .ANG.;
functional group F(II)8 is an acceptor having directionality of F7
with respect to the backbone NH of receptor Ala573 of SEQ ID NO: 1
and has coordinates of r=52.861, .theta. (theta)=107.691, and .phi.
(phi)=-25.448 and a spherical radius of about 1.5 .ANG.; functional
group F(II)9 donates an H-bond to a backbone carbonyl of Asp563 and
forms a salt bridge to a side chain carboxylate of receptor Asp563
of SEQ ID NO: 1 and has coordinates of r=57.688, .theta.
(theta)=99.198, and .phi. (phi)=-21.588 and a spherical radius of
about 1.2 .ANG.; and the compound substantially maintains a
non-extended tether inactive configuration of EGFR or substantially
prevents stabilization of an extended tether active configuration
of EGFR.
7. A compound having a formula of: ##STR00471## or a stereoisomer
or pharmaceutically acceptable salt thereof; wherein, R.sup.54 is
selected from the group consisting of: (i) a 5 or 6 membered
heterocyclic containing from 1 to 4 N, O, or S atoms or any
combination of those atoms with carbon atoms to form a heterocyclic
aromatic ring optionally substituted with from 1 to 3 groups
selected from: (a) straight chain or branched C-1 to C-4 lower
alkyl optionally containing unsaturation; (b) C-1 to C-6 cycloalkyl
optionally containing unsaturation or one oxygen or nitrogen atom;
(c) alkoxy --OR.sup.10 where R.sup.10 is selected from the group
consisting of: (1) a straight chain or branched C-1 to C-4 lower
alkyl optionally containing unsaturation; and (2) a C-1 to C-6
cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; (d) trifluoromethyl, (e) trifluoromethoxy, (f)
substituted amino, (g) nitro; and (h) halogen; R.sup.55 is selected
from the group consisting of: hydrogen and straight chain or
branched C-1 to C-4 lower alkyl optionally containing unsaturation;
R.sup.56, R.sup.57, R.sup.58, and R.sup.59 are hydrogen; R.sup.60
is selected from the group consisting of: (i) straight chain or
branched C-1 to C-4 lower alkyl optionally containing unsaturation;
(ii) C-1 to C-6 cycloalkyl optionally containing unsaturation or
one oxygen or nitrogen atom; and (iii) C-1 to C-3 substituted alkyl
having at least one substitution group selected from the group
consisting of: (a) carboxyl --COOR.sup.10 where R.sup.10 is
selected from the group consisting of: (1) a straight chain or
branched C-1 to C-4 lower alkyl optionally containing unsaturation;
and (2) a C-1 to C-6 cycloalkyl optionally containing unsaturation
or one oxygen or nitrogen atom; (b) aryl having an unsubstituted
phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or
6-position with one or more groups selected from the group
consisting of: (1) straight chain or branched C-1 to C-4 lower
alkyl optionally containing unsaturation; (2) C-1 to C-6 cycloalkyl
optionally containing unsaturation or one oxygen or nitrogen atom;
(3) aryl having phenyl or heteroaryl containing from 1 to 4 N, O,
or S atoms; (4) alkoxy --OR.sup.10 where R.sup.10 is selected from
the group consisting of: (A) a straight chain or branched C-1 to
C-4 lower alkyl optionally containing unsaturation; and (B) a C-1
to C-6 cycloalkyl optionally containing unsaturation or one oxygen
or nitrogen atom; (5) trifluoromethyl; (6) trifluoromethoxy; (7)
difluoromethoxy; (8) 3,4-methylenedioxy; (9) 2,3-methylenedioxy;
(10) nitro; and (11) halogen; and (c) aryloxy (--OAr); and Formula
(18) excludes a compound of Formula (17): ##STR00472## or a
stereoisomer or pharmaceutically acceptable salt thereof; wherein,
R.sup.61, R.sup.62, and R.sup.64 are independently selected from
the group consisting of: hydrogen and straight chain or branched
C-1 to C-4 lower alkyl optionally containing unsaturation; R.sup.63
and R.sup.65 are independently selected from the group consisting
of: (i) hydrogen; (ii) straight chain or branched C-1 to C-4 lower
alkyl optionally containing unsaturation; and (iii) alkoxy
--OR.sup.10 where R.sup.10 is selected from the group consisting
of: (a) a straight chain or branched C-1 to C-4 lower alkyl
optionally containing unsaturation; and (b) a C-1 to C-6 cycloalkyl
optionally containing unsaturation or one oxygen or nitrogen atom;
R.sup.66 is selected from the group consisting of: hydrogen and
straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation; R.sup.67, R.sup.68, R.sup.69, and R.sup.70
are hydrogen; R.sup.71 is selected from the group consisting of:
(i) straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation; (ii) C-1 to C-6 cycloalkyl optionally
containing unsaturation or one oxygen or nitrogen atom; (iii) aryl
having an unsubstituted phenyl ring or a phenyl ring substituted at
the 2-, 3-, 4-, 5- or 6-position with one or more groups selected
from the group consisting of: (a) straight chain or branched C-1 to
C-4 lower alkyl optionally containing unsaturation; (b) C-1 to C-6
cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; (c) aryl having phenyl or heteroaryl containing from
1 to 4 N, O, or S atoms; (d) alkoxy --OR.sup.10 where R.sup.10 is
selected from the group consisting of: (1) a straight chain or
branched C-1 to C-4 lower alkyl optionally containing unsaturation;
and (2) a C-1 to C-6 cycloalkyl optionally containing unsaturation
or one oxygen or nitrogen atom; (e) trifluoromethyl; (f)
trifluoromethoxy; (g) difluoromethoxy; (h) 3,4-methylenedioxy; (i)
2,3-methylenedioxy; (j) nitro; and (k) halogen; (iv) C-1 to C-3
substituted alkyl having at least one substitution group selected
from the group consisting of: (a) carboxyl --COOR.sup.10 where
R.sup.10 is selected from the group consisting of: (1) a straight
chain or branched C-1 to C-4 lower alkyl optionally containing
unsaturation; and (2) a C-1 to C-6 cycloalkyl optionally containing
unsaturation or one oxygen or nitrogen atom; (b) aryl having an
unsubstituted phenyl ring or a phenyl ring substituted at the 2-,
3-, 4-, 5- or 6-position with one or more groups selected from the
group consisting of: (1) straight chain or branched C-1 to C-4
lower alkyl optionally containing unsaturation; (2) C-1 to C-6
cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; (3) aryl having phenyl or heteroaryl containing from
1 to 4 N, O, or S atoms; (4) alkoxy --OR.sup.10 where R.sup.10 is
selected from the group consisting of: (A) a straight chain or
branched C-1 to C-4 lower alkyl optionally containing unsaturation;
and (B) a C-1 to C-6 cycloalkyl optionally containing unsaturation
or one oxygen or nitrogen atom; (5) trifluoromethyl; (6)
trifluoromethoxy; (7) difluoromethoxy; (8) 3,4-methylenedioxy; (9)
2,3-methylenedioxy; (10) nitro; and (11) halogen; and (c) aryloxy
(--OAr); (v) heteroaryl containing a heterocyclic ring containing
from 1 to 4 N, O, or S atoms optionally substituted with one or
more groups selected from the group consisting of: (a) straight
chain or branched C-1 to C-4 lower alkyl optionally containing
unsaturation; (b) C-1 to C-6 cycloalkyl optionally containing
unsaturation or one oxygen or nitrogen atom; (c) aryl having phenyl
or heteroaryl containing from 1 to 4 N, O, or S atoms; (d) alkoxy
--OR.sup.10 where R.sup.10 is selected from the group consisting
of: (1) a straight chain or branched C-1 to C-4 lower alkyl
optionally containing unsaturation; and (2) a C-1 to C-6 cycloalkyl
optionally containing unsaturation or one oxygen or nitrogen atom;
(e) trifluoromethyl; (f) trifluoromethoxy; (g) nitro; and (h)
halogen; and Formula (19) excludes a compound of Formula (17):
##STR00473## or a stereoisomer or pharmaceutically acceptable salt
thereof; wherein, R.sup.72 is selected from the group consisting
of: hydrogen and straight chain or branched C-1 to C-4 lower alkyl
optionally containing unsaturation; R.sup.73 is selected from the
group consisting of: (i) straight chain or branched C-1 to C-4
lower alkyl optionally containing unsaturation; (ii) C-1 to C-6
cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; (iii) C-1 to C-3 substituted alkyl having at least
one substitution group selected from the group consisting of: (a)
carboxyl --COOR.sup.10 where R.sup.10 is selected from the group
consisting of: (1) a straight chain or branched C-1 to C-4 lower
alkyl optionally containing unsaturation; and (2) a C-1 to C-6
cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; (b) aryl having an unsubstituted phenyl ring or a
phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with
one or more groups selected from the group consisting of: (1)
straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation; (2) C-1 to C-6 cycloalkyl optionally
containing unsaturation or one oxygen or nitrogen atom; (3) aryl
having phenyl or heteroaryl containing from 1 to 4 N, O, or S
atoms; (4) alkoxy --OR.sup.10 where R.sup.10 is selected from the
group consisting of: (A) a straight chain or branched C-1 to C-4
lower alkyl optionally containing unsaturation; and (B) a C-1 to
C-6 cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; (5) trifluoromethyl; (6) trifluoromethoxy; (7)
difluoromethoxy; (8) 3,4-methylenedioxy; (9) 2,3-methylenedioxy;
(10) nitro; and (11) halogen; and (c) aryloxy (--OAr); and (iv)
acyl --COR.sup.10 where R.sup.10 is selected from the group
consisting of: (a) a straight chain or branched C-1 to C-4 lower
alkyl optionally containing unsaturation; (b) a C-1 to C-6
cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; R.sup.74, R.sup.75, R.sup.76, and R.sup.77 are
hydrogen; and R.sup.78 is selected from the group consisting of:
(i) straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation; (ii) C-1 to C-6 cycloalkyl optionally
containing unsaturation or one oxygen or nitrogen atom; (iii) C-1
to C-3 substituted alkyl having at least one substitution group
selected from the group consisting of: (a) carboxyl --COOR.sup.10
where R.sup.10 is selected from the group consisting of: (1) a
straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation; and (2) a C-1 to C-6 cycloalkyl optionally
containing unsaturation or one oxygen or nitrogen atom; (b) aryl
having an unsubstituted phenyl ring or a phenyl ring substituted at
the 2-, 3-, 4-, 5- or 6-position with one or more groups selected
from the group consisting of: (1) straight chain or branched C-1 to
C-4 lower alkyl optionally containing unsaturation; (2) C-1 to C-6
cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; (3) aryl having phenyl or heteroaryl containing from
1 to 4 N, O, or S atoms; (4) alkoxy --OR.sup.10 where R.sup.10 is
selected from the group consisting of: (A) a straight chain or
branched C-1 to C-4 lower alkyl optionally containing unsaturation;
(B) a C-1 to C-6 cycloalkyl optionally containing unsaturation or
one oxygen or nitrogen atom; (5) trifluoromethyl; (6)
trifluoromethoxy; (7) difluoromethoxy; (8) 3,4-methylenedioxy; (9)
2,3-methylenedioxy; (10) nitro; and (11) halogen; and (c) aryloxy
(--OAr); and Formula (20) excludes a compound of Formula (17):
##STR00474## or a stereoisomer or pharmaceutically acceptable salt
thereof; wherein, n is 1 or 2; R.sup.79, R.sup.81, R.sup.82,
R.sup.83, R.sup.84, R.sup.85, and R.sup.86 are hydrogen; R.sup.80
is selected from the group consisting of: (i) hydrogen, (ii)
straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation; (iii) alkoxy --OR.sup.10 where R.sup.10 is
a straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation or a C-1 to C-6 cycloalkyl optionally
containing unsaturation or one oxygen or nitrogen atom; (iv)
halogen; and (v) trifluoromethyl; R.sup.87 is selected from the
group consisting of: (i) straight chain or branched C-1 to C-4
lower alkyl optionally containing unsaturation; (ii) C-1 to C-6
cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; (iii) C-1 to C-3 substituted alkyl having at least
one substitution group selected from the group consisting of: (a)
carboxyl --COOR.sup.10 where R.sup.10 is selected from the group
consisting of: (1) a straight chain or branched C-1 to C-4 lower
alkyl optionally containing unsaturation; and (2) a C-1 to C-6
cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; (b) aryl having an unsubstituted phenyl ring or a
phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with
one or more groups selected from the group consisting of: (1)
straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation; \ (2) C-1 to C-6 cycloalkyl optionally
containing unsaturation or one oxygen or nitrogen atom; (3) aryl
having phenyl or heteroaryl containing from 1 to 4 N, O, or S
atoms; (4) alkoxy --OR.sup.10 where R.sup.10 is selected from the
group consisting of: (A) a straight chain or branched C-1 to C-4
lower alkyl optionally containing unsaturation; and (B) a C-1 to
C-6 cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; (5) trifluoromethyl; (6) trifluoromethoxy; (7)
difluoromethoxy; (8) 3,4-methylenedioxy; (9) 2,3-methylenedioxy;
(10) nitro; and (11) halogen; and (c) aryloxy (--OAr); and (iii)
aryl comprising a phenyl or heteroaryl five or six membered ring
containing from 1 to 4 N, O, or S atoms; and Formula (21) excludes
a compound of Formula (17): ##STR00475## or a stereoisomer or
pharmaceutically acceptable salt thereof; wherein, n is 3, 4, or 5;
R.sup.88 is selected from the group consisting of: hydrogen and
straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation; R.sup.89, R.sup.90, R.sup.91, R.sup.83,
and R.sup.92 are hydrogen; R.sup.93 is selected from the group
consisting of: (i) straight chain or branched C-1 to C-4 lower
alkyl optionally containing unsaturation; (ii) C-1 to C-6
cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; (iii) C-1 to C-3 substituted alkyl having at least
one substitution group selected from the group consisting of: (a)
carboxyl --COOR.sup.10 where R.sup.10 is a straight chain or
branched C-1 to C-4 lower alkyl optionally containing unsaturation
or a C-1 to C-6 cycloalkyl optionally containing unsaturation or
one oxygen or nitrogen atom; (b) aryl having an unsubstituted
phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or
6-position with one or more groups selected from the group
consisting of: (1) straight chain or branched C-1 to C-4 lower
alkyl optionally containing unsaturation; (2) C-1 to C-6 cycloalkyl
optionally containing unsaturation or one oxygen or nitrogen atom;
(3) aryl having phenyl or heteroaryl containing from 1 to 4 N, O,
or S atoms; (4) alkoxy --OR.sup.10 where R.sup.10 is selected from
the group consisting of: (A) a straight chain or branched C-1 to
C-4 lower alkyl optionally containing unsaturation; and (B) a C-1
to C-6 cycloalkyl optionally containing unsaturation or one oxygen
or nitrogen atom; (5) trifluoromethyl; (6) trifluoromethoxy; (7)
difluoromethoxy; (8) 3,4-methylenedioxy; (9) 2,3-methylenedioxy;
(10) nitro; and (11) halogen; and (c) aryloxy (--OAr); and (iv)
aryl comprising a phenyl or heteroaryl five or six membered ring
containing from 1 to 4 N, O, or S atoms; and Formula (22) excludes
a compound of Formula (17): ##STR00476## or a stereoisomer or
pharmaceutically acceptable salt thereof; wherein, R.sup.94 is a 5
or 6 membered heterocyclic aromatic ring containing from 1 to 4 N,
O, or S atoms or any combination of those atoms with carbon atoms
optionally substituted with from 1 to 3 of groups selected
from:
(i) straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation; (ii) C-1 to C-6 cycloalkyl optionally
containing unsaturation or one oxygen or nitrogen atom; (iii)
alkoxy --OR.sup.10 where R.sup.10 is selected from the group
consisting of: (a) a straight chain or branched C-1 to C-4 lower
alkyl optionally containing unsaturation; and (b) a C-1 to C-6
cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; (iv) trifluoromethyl; (v) trifluoromethoxy; (vi)
substituted amino; (vii) nitro; and (viii) halogen; R.sup.95 is
selected from the group consisting of: hydrogen and straight chain
or branched C-1 to C-4 lower alkyl optionally containing
unsaturation; R.sup.96, R.sup.97, R.sup.98, and R.sup.99 are
hydrogen; R.sup.100 is aryl comprising a phenyl or heteroaryl
containing from 1 to 4 N, O, or S atoms; and Formula (23) excludes
a compound of Formula (17): ##STR00477##
8. The compound of claim 7, wherein (A) the compound is according
to Formula (18) (1886-like Type J) and one or more of the following
features are satisfied: R.sup.54 is selected from the group
consisting of: 4,6-dimethyl-2-pyrimidine;
2,6-dimethoxy-4-pyrimidine; 6-methoxy-4-pyrimidine;
5-ethyl-2-(1,3,4-Thiadiazole); 5-methyl-3-Isoxazole;
3-methoxy-6-pyridazinamine, 2-thiazole; and 2-methoxy-3-pyrazine.
R.sup.55 is selected from the group consisting of hydrogen and
methyl; the compound is selected from the group consisting of:
##STR00478## (B) the compound according to Formula (19) (1886-like
Type K) is ##STR00479## (C) the compound according to Formula (20)
(1886-like Type L) is selected from the group consisting of:
##STR00480## (D) the compound according to Formula (21) (1886-like
Type M) is ##STR00481## (E) the compound of Formula (22) (1886-like
Type N) is selected from the group consisting of: ##STR00482## or
(F) the compound of Formula (23) (1886-like Type O) is selected
from the group consisting of: ##STR00483##
9. The compound of claim 7 that inhibits EGFR activity comprising:
at least seven or more of functional groups F(III)1, F(III)2,
F(III)3, F(III)4, F(III)5, F(III)6, F(III)7, F(III)8, and F(III)9
of a Scheme III 1886-like pharmacophore; wherein functional group
F(III)1 donates a hydrogen bond to a side chain carbonyl of
receptor Gln408 and accepts a hydrogen bond from the side chain
NH.sub.2 of receptor Gln384 of SEQ ID NO: 1 and has coordinates of
r=81.552, .theta. (theta)=41.243, and .phi. (phi)=45.369 and a
spherical radius of about 1.2 .ANG.; functional group F(III)2
accepts a hydrogen bond from a side chain OH of receptor Ser440 of
SEQ ID NO: 1 and has coordinates of r=87.287, .theta.
(theta)=40.739, and .phi. (phi)=51.781 and a spherical radius of
about 1.2 .ANG.; functional group F(III)3 accepts a hydrogen bond
from a side chain OH of receptor Ser440 of SEQ ID NO: 1 and has
coordinates of r=86.320, .theta. (theta)=41.915, and .phi.
(phi)=52.323 and a spherical radius of about 1.5 .ANG.; functional
group F(III)4 forms a favorable coulombic interaction with an
imidazole side chain of receptor His409 of SEQ ID NO: 1 and has
coordinates of r=85.870, .theta. (theta)=38.463, and .phi.
(phi)=41.650 and a spherical radius of about 1.2 .ANG.; functional
group F(III)5 forms a favorable coulombic interaction with an
imidazole side chain of receptor His409 of SEQ ID NO: 1 and has
coordinates of r=82.241, .theta. (theta)=37.431, and .phi.
(phi)=44.151 and a spherical radius of about 1.5 .ANG.; functional
group F(III)6 accepts a hydrogen bond from, or forms a salt bridge
to, NH.sup.3+ of receptor Lys465 of SEQ ID NO: 1 and has
coordinates of r=88.009, .theta. (theta)=37.822, and .phi.
(phi)=54.903 and a spherical radius of about 1.2 .ANG.; functional
group F(III)7 accepts a hydrogen bond from, or forms a salt bridge
to, NH.sup.3+ of receptor Lys465 of SEQ ID NO: 1 and has
coordinates of r=86.513, .theta. (theta)=36.889, and .phi.
(phi)=54.484 and a spherical radius of about 1.5 .ANG.; functional
group F(III)8 donates a hydrogen bond to a side chain carbonyl of
receptor Gln408 of SEQ ID NO: 1 and has coordinates of r=81.552,
.theta. (theta)=41.243, and .phi. (phi)=45.369 and a spherical
radius of about 1.2 .ANG.; functional group F(III)9 donates a
hydrogen bond to a side chain carbonyl of receptor Gln408 of SEQ ID
NO: 1 and has coordinates of r=79.652, .theta. (theta)=40.928, and
.phi. (phi)=44.528 and a spherical radius of about 1.6 .ANG.; and
the compound substantially maintains a non-extended tether inactive
configuration of EGFR or substantially prevents stabilization of an
extended tether active configuration of EGFR.
10. A compound having a formula of: ##STR00484## or a stereoisomer
or pharmaceutically acceptable salt thereof; wherein, R.sup.122 and
R.sup.126 are independently selected from the group consisting of:
(i) hydrogen, (ii) alkoxy --OR.sup.10 where R.sup.10 is selected
from the group consisting of: (a) a straight chain or branched C-1
to C-4 lower alkyl optionally containing unsaturation; and (b) a
C-1 to C-6 cycloalkyl optionally containing unsaturation or one
oxygen or nitrogen atom; (iii) trifluoromethyl; and (iv) halogen;
R.sup.123 and R.sup.125 are independently selected from the group
consisting of: (i) hydrogen; (ii) trifluoromethyl; (iii) halogen;
(iv) straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation; (v) acyl --COR.sup.10 where R.sup.10 is
selected from the group consisting of: (a) a straight chain or
branched C-1 to C-4 lower alkyl optionally containing unsaturation;
(b) a C-1 to C-6 cycloalkyl optionally containing unsaturation or
one oxygen or nitrogen atom; and (vi) alkoxy --OR.sup.10 where
R.sup.10 is selected from the group consisting of: (a) a straight
chain or branched C-1 to C-4 lower alkyl optionally containing
unsaturation; and (b) a C-1 to C-6 cycloalkyl optionally containing
unsaturation or one oxygen or nitrogen atom; R.sup.124 is selected
from the group consisting of: (i) hydrogen; (ii) straight chain or
branched C-1 to C-4 lower alkyl optionally containing unsaturation;
(iii) alkoxy --OR.sup.10 where R.sup.10 is selected from the group
consisting of: (a) a straight chain or branched C-1 to C-4 lower
alkyl optionally containing unsaturation; and (b) a C-1 to C-6
cycloalkyl optionally containing unsaturation or one oxygen or
nitrogen atom; (iv) acyl --COR.sup.10 where R.sup.10 is selected
from the group consisting of: (a) a straight chain or branched C-1
to C-4 lower alkyl optionally containing unsaturation; and (b) a
C-1 to C-6 cycloalkyl optionally containing unsaturation or one
oxygen or nitrogen atom; and (v) halogen; R.sup.127 and R.sup.131
are independently selected from the group consisting of: (i)
hydrogen, (ii) straight chain or branched C-1 to C-4 lower alkyl
optionally containing unsaturation; and (iii) alkoxy --OR.sup.10
where R.sup.10 is selected from the group consisting of: (a) a
straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation; and (b) a C-1 to C-6 cycloalkyl optionally
containing unsaturation or one oxygen or nitrogen atom; R.sup.128
and R.sup.130 are independently selected from the group consisting
of: hydrogen and halogen; R.sup.129 is selected from the group
consisting of: (i) hydrogen; (ii) halogen; (iii) alkoxy --OR.sup.10
where R.sup.10 is selected from the group consisting of: (a) a
straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation; and (b) a C-1 to C-6 cycloalkyl optionally
containing unsaturation or one oxygen or nitrogen atom; and (iv)
straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation; R.sup.132 is selected from the group
consisting of: hydrogen and straight chain or branched C-1 to C-4
lower alkyl optionally containing unsaturation; and Formula (32)
excludes a compound of Formula (31): ##STR00485## or a stereoisomer
or pharmaceutically acceptable salt thereof; wherein n is 1 or 2;
R.sup.133 and R.sup.137 are independently selected from the group
consisting of: hydrogen; halogen; and straight chain or branched
C-1 to C-4 lower alkyl optionally containing unsaturation;
R.sup.134 and R.sup.136 are independently selected from the group
consisting of: hydrogen; trifluoromethyl; and halogen; R.sup.135
selected from the group consisting of: hydrogen and halogen;
R.sup.138, R.sup.140, and R.sup.141 are hydrogen; R.sup.139 is
selected from the group consisting of: hydrogen and halogen;
R.sup.142 is selected from the group consisting of: hydrogen and
straight chain or branched C-1 to C-4 lower alkyl optionally
containing unsaturation; and Formula (33) excludes a compound of
Formula (31): ##STR00486##
11. The compound of claim 10, wherein (A) the compound is according
to Formula (32) (11091-like Type H) and one or more of the
following features are satisfied: R.sup.122 and R.sup.126 are
independently selected from the group consisting of hydrogen;
trifluoromethyl; methoxy; and chloro; R.sup.122 or R.sup.126 is
hydrogen; R.sup.123 and R.sup.125 are independently selected from
the group consisting of halogen and trifluoromethyl; R.sup.124
selected from the group consisting of halogen and straight chain or
branched C-1 to C-4 lower alkyl optionally containing unsaturation;
R.sup.127 and R.sup.131 are independently selected from the group
consisting of hydrogen, methyl, and methoxy; R.sup.128 and
R.sup.130 are independently selected from the group consisting of
hydrogen and chloro; R.sup.129 is selected from the group
consisting of hydrogen; chloro; methoxy; and methyl. R.sup.132 of
Formula (32) is selected from the group consisting of hydrogen;
methyl; and ethyl; or the compound is selected from the group
consisting of: ##STR00487## or (B) the compound is according to
Formula (33) (11091-like Type I) and one or more of the following
features are satisfied: R.sup.133 and R.sup.137 are independently
selected from the group consisting of: hydrogen; methyl; and
chloro. R.sup.134 and R.sup.136 are independently selected from the
group consisting of: hydrogen; trifluoromethyl; and chloro.
R.sup.135 is chloro. R.sup.139 is selected from the group
consisting of: fluoro and hydrogen. R.sup.142 is selected from the
group consisting of: hydrogen or methyl and the compound is
selected from the group consisting of ##STR00488##
12. The compound of claim 10 that inhibits EGFR activity
comprising: at least nine or more of functional groups F(V)1,
F(V)2, F(V)3, F(V)4, F(V)5, F(V)6, F(V)7, F(V)8, F(V)9, F(V)10, and
F(V)11 of a Scheme V 11091-like pharmacophore; wherein functional
group F(V)1 accepts a hydrogen bond from a side chain amino of
receptor Lys465 of SEQ ID NO: 1 and has coordinates of r=85.329,
.theta. (theta)=34.962, and .phi. (phi)=53.394 and a spherical
radius of about 1.2 .ANG.; functional group F(V)2 accepts a
hydrogen bond from a side chain amino of receptor Lys465 of SEQ ID
NO: 1 and has coordinates of r=87.180, .theta. (theta)=35.618, and
.phi. (phi)=53.267 and a spherical radius of about 1.5 .ANG.;
functional group F(V)3 accepts a hydrogen bond from a side chain
amino of receptor Lys465 of SEQ ID NO: 1 and has coordinates of
r=86.349, .theta. (theta)=34.560, and .phi. (phi)=55.424 and a
spherical radius of about 1.5 .ANG.; functional group F(V)4 donates
a hydrogen bond to a side chain carbonyl of receptor Gln411 of SEQ
ID NO: 1 and has coordinates of r=83.958, .theta. (theta)=35.296,
and .phi. (phi)=51.296 and a spherical radius of about 1.2 .ANG.;
functional group F(V)5 donates a hydrogen bond to a side chain
carbonyl of receptor Gln411 of SEQ ID NO: 1 and has coordinates of
r=82.060, .theta. (theta)=34.984, and .phi. (phi)=50.365 and a
spherical radius of about 1.5 .ANG.; functional group F(V)6 donates
a hydrogen bond to a side chain nitrogen of receptor Gln411 of SEQ
ID NO: 1 and has coordinates of r=83.884, .theta. (theta)=36.166,
and .phi. (phi)=49.227 and a spherical radius of about 1.2 .ANG.;
functional group F(V)7 donates a hydrogen bond to a side chain
nitrogen of receptor Gln411 of SEQ ID NO: 1 and has coordinates of
r=82.006, .theta. (theta)=35.523, and .phi. (phi)=49.076 and a
spherical radius of about 1.5 .ANG.; functional group F(V)8 accepts
a hydrogen bond to a side chain imidazole of receptor His409 of SEQ
ID NO: 1 and has coordinates of r=85.100, .theta. (theta)=38.590,
and .phi. (phi)=43.143 and a spherical radius of about 1.2 .ANG.;
functional group F(V)9 forms a favorable .pi.-.pi. interaction with
a phenyl ring of receptor Phe412 of SEQ ID NO: 1 and has
coordinates of r=84.418, .theta. (theta)=37.489, and .phi.
(phi)=46.968 and a spherical radius of about 1.2 .ANG.; functional
group F(V)10 forms a favorable .pi.-.pi. interaction with a phenyl
ring of receptor Phe412 of SEQ ID NO: 1 and has coordinates of
r=82.486, .theta. (theta)=38.027, and .phi. (phi)=47.246 and a
spherical radius of about 1.5 .ANG.; and functional group F(V)11
forms a favorable hydrophobic interaction with a side chain of
receptors Val417 and Ile448 of SEQ ID NO: 1 and has coordinates of
r=84.438, .theta. (theta)=40.753, and .phi. (phi)=48.557 and a
spherical radius of about 1.2 .ANG.; functional group F11 forms a
favorable hydrophobic interaction with a side chain of receptors
Val417 and Ile448 of SEQ ID NO: 1 and has coordinates of r=84.438,
.theta. (theta)=40.753, and .phi. (phi)=48.557 and a spherical
radius of about 1.2 .ANG.; and the compound substantially maintains
a non-extended tether inactive configuration of EGFR or
substantially prevents stabilization of an extended tether active
configuration of EGFR.
13. A method of treating a proliferative disease, disorder, or
condition, comprising: administering to a subject in need thereof a
composition comprising a therapeutically effective amount of (i) a
compound of any one of claims 1, 4, 7, or 10, or a stereoisomer or
pharmaceutically acceptable salt thereof; (ii) a compound selected
from the group consisting of: ##STR00489## or a stereoisomer or
pharmaceutically acceptable salt thereof; or (iii) a compound
selected from the group consisting of: ##STR00490## or a
stereoisomer or pharmaceutically acceptable salt thereof; and a
pharmaceutically acceptable carrier or excipient.
14. The method of claim 13, wherein the proliferative disease,
disorder, or condition is (i) selected from the group consisting
of: cancer; a blood vessel proliferative disorder; a fibrotic
disorder; a mesangial cell proliferative disorder; psoriasis;
actinic keratoses; seborrheic keratoses; warts; keloid scars;
eczema; and hyperproliferative diseases caused by a viral
infection; (ii) is a disease, disorder, or condition associated
with EGFR; or (iii) is a disease, disorder, or condition associated
with EGFR selected from the group consisting of: cancer; a blood
vessel proliferative disorder; a fibrotic disorder; a mesangial
cell proliferative disorder; psoriasis; actinic keratoses;
seborrheic keratoses; warts; keloid scars; eczema; and
hyperproliferative diseases caused by a viral infection.
15. A method for identifying an epidermal growth factor receptor
(EGFR) inhibitor, comprising: providing a pharmacophore comprising
a scheme selected from the group consisting of: Scheme II, Scheme
III, Scheme IV, or Scheme V as input to a 3-dimensional database;
comparing a three dimensional structure of a candidate compound to
the three dimensional structure of the pharmacophore; selecting a
candidate compound with a three dimensional structure that
substantially aligns with: five or more functional groups of Scheme
II (ADS-1734-like); seven or more functional groups of Scheme III
(AD4-1886-like); eight or more of functional groups of Scheme IV
(AD4-10381-like); or nine or more of functional groups of Scheme V
(AD4-11091-like); wherein similarity between the three-dimensional
structure of the candidate compound and the three-dimensional
structure of the pharmacophore is indicative of an ability of the
candidate compound to inhibit EGFR by substantially maintaining a
tethered inactive configuration of EGFR or substantially preventing
stabilization of the untethered active configuration of EGFR;
Scheme II (ADS-1734-like) comprises functional groups F(II)1,
F(II)2, F(II)3, F(II)4, F(II)5, F(II)6, F(II)7, F(II)8, and F(II)9;
wherein functional group F(II)1 donates an H-bond or forms a salt
bridge to a carboxylate side chain of receptor Asp553 of SEQ ID NO:
1 and has coordinates of r=56.363, .theta. (theta)=94.368, and
.phi. (phi)=-17.752 and a spherical radius of about 1.2 .ANG.;
functional group F(II)2 is a donor and has coordinates of r=53.290,
.theta. (theta)=101.494, and .phi. (phi)=-23.244 and a spherical
radius of about 1.0 .ANG.; functional group F(II)3 forms a
hydrophobic contact with a side chain of receptor Val568,
imidazolidine side chain of receptor Pro552 and with a side chain
of Met253 of SEQ ID NO: 1 and has coordinates of r=53.726, .theta.
(theta)=97.830, and .phi. (phi)=-18.377 and a spherical radius of
about 1.7 .ANG.; functional group F(II)4 forms a hydrophobic
contact with side chain of receptor Val575, Met253, and with an
imidazolidine ring of receptor Pro552 of SEQ ID NO: 1 and has
coordinates of r=53.647, .theta. (theta)=103.844, and .phi.
(phi)=-20.990 and a spherical radius of about 1.4 .ANG.; functional
group F(II)5 donates an H-bond to a side chain hydroxyl of Thr570
of SEQ ID NO: 1 and has coordinates of r=51.093, .theta.
(theta)=104.261, and .phi. (phi)=-25.552 and a spherical radius of
about 1.2 .ANG.; functional group F(II)6 is a donor having
directionality of F4 with respect to a backbone carbonyl of
receptor Thr570 of SEQ ID NO: 1 and has coordinates of r=52.340,
.theta. (theta)=103.980, and .phi. (phi)=-27.461 and a spherical
radius of about 1.5 .ANG.; functional group F(II)7 accepts an
H-bond from a receptor backbone NH of Ala573 of SEQ ID NO: 1 and
has coordinates of r=51.383, .theta. (theta)=106.455, and .phi.
(phi)=-24.319 and a spherical radius of about 1.2 .ANG.; functional
group F(II)8 is an acceptor having directionality of F7 with
respect to the backbone NH of receptor Ala573 of SEQ ID NO: 1 and
has coordinates of r=52.861, .theta. (theta)=107.691, and .phi.
(phi)=-25.448 and a spherical radius of about 1.5 .ANG.; and
functional group F(II)9 donates an H-bond to a backbone carbonyl of
Asp563 and forms a salt bridge to a side chain carboxylate of
receptor Asp563 of SEQ ID NO: 1 and has coordinates of r=57.688,
.theta. (theta)=99.198, and .phi. (phi)=-21.588 and a spherical
radius of about 1.2 .ANG.; Scheme III (AD4-1886-like) comprises
functional groups F(III)1, F(III)2, F(III)3, F(III)4, F(III)5,
F(III)6, F(III)7, F(III)8, and F(III)9; wherein functional group
F(III)1 donates a hydrogen bond to a side chain carbonyl of
receptor Gln408 and accepts a hydrogen bond from the side chain
NH.sub.2 of receptor Gln384 of SEQ ID NO: 1 and has coordinates of
r=81.552, .theta. (theta)=41.243, and .phi. (phi)=45.369 and a
spherical radius of about 1.2 .ANG.; functional group F(III)2
accepts a hydrogen bond from a side chain OH of receptor Ser440 of
SEQ ID NO: 1 and has coordinates of r=87.287, .theta.
(theta)=40.739, and .phi. (phi)=51.781 and a spherical radius of
about 1.2 .ANG.; functional group F(III)3 accepts a hydrogen bond
from a side chain OH of receptor Ser440 of SEQ ID NO: 1 and has
coordinates of r=86.320, .theta. (theta)=41.915, and .phi.
(phi)=52.323 and a spherical radius of about 1.5 .ANG.; functional
group F(III)4 forms a favorable coulombic interaction with an
imidazole side chain of receptor His409 of SEQ ID NO: 1 and has
coordinates of r=85.870, .theta. (theta)=38.463, and .phi.
(phi)=41.650 and a spherical radius of about 1.2 .ANG.; functional
group F(III)5 forms a favorable coulombic interaction with an
imidazole side chain of receptor His409 of SEQ ID NO: 1 and has
coordinates of r=82.241, .theta. (theta)=37.431, and .phi.
(phi)=44.151 and a spherical radius of about 1.5 .ANG.; functional
group F(III)6 accepts a hydrogen bond from, or forms a salt bridge
to, NH.sup.3+ of receptor Lys465 of SEQ ID NO: 1 and has
coordinates of r=88.009, .theta. (theta)=37.822, and .phi.
(phi)=54.903 and a spherical radius of about 1.2 .ANG.; functional
group F(III)7 accepts a hydrogen bond from, or forms a salt bridge
to, NH.sup.3+ of receptor Lys465 of SEQ ID NO: 1 and has
coordinates of r=86.513, .theta. (theta)=36.889, and .phi.
(phi)=54.484 and a spherical radius of about 1.5 .ANG.; functional
group F(III)8 donates a hydrogen bond to a side chain carbonyl of
receptor Gln408 of SEQ ID NO: 1 and has coordinates of r=81.552,
.theta. (theta)=41.243, and .phi. (phi)=45.369 and a spherical
radius of about 1.2 .ANG.; and functional group F(III)9 donates a
hydrogen bond to a side chain carbonyl of receptor Gln408 of SEQ ID
NO: 1 and has coordinates of r=79.652, .theta. (theta)=40.928, and
.phi. (phi)=44.528 and a spherical radius of about 1.6 .ANG.;
Scheme IV (AD4-10381-like) comprises functional group F(IV)1,
F(IV)2, F(IV)3, F(IV)4, F(IV)5, F(IV)6, F(IV)7, F(IV)8, and F(IV)9;
wherein functional group F(IV)1 accepts a hydrogen bond from
receptor side chain OH of Thr239 of SEQ ID NO: 1 and has
coordinates of r=49.686, .theta. (theta)=113.993, and .phi.
(phi)=-17.014 and a spherical radius of about 1.2 .ANG.; functional
group F(IV)2 accepts a hydrogen bond from receptor side chain OH of
Thr239 of SEQ ID NO: 1 and has coordinates of r=48.071, .theta.
(theta)=115.388, and .phi. (phi)=-16.211 and a spherical radius of
about 1.6 .ANG.; functional group F(IV)3 forms a hydrophobic
contact with a side chain of receptor Met576 and imidazolidine ring
of Pro242 of SEQ ID NO: 1 and has coordinates of r=50.781, .theta.
(theta)=113.121, and .phi. (phi)=-17.520 and a spherical radius of
about 1.2 .ANG.; functional group F(IV)4 forms a hydrophobic
contact with a side chain of receptor Met576 and imidazolidine ring
of Pro242 of SEQ ID NO: 1 and has coordinates of r=52.021, .theta.
(theta)=114.264, and .phi. (phi)=-15.878 and a spherical radius of
about 1.5 .ANG.; functional group F(IV)5 forms a hydrophobic
contact with a side chain of receptor Met576 and imidazolidine ring
of Pro242 of SEQ ID NO: 1 and has coordinates of r=50.322, .theta.
(theta)=111.253, and .phi. (phi)=-15.426 and a spherical radius of
about 1.2 .ANG.; functional group F(IV)6 forms a hydrophobic
contact with a side chain of receptor Met576 and imidazolidine ring
of Pro242 of SEQ ID NO: 1 and has coordinates of r=51.575, .theta.
(theta)=112.433, and .phi. (phi)=-13.827 and a spherical radius of
about 1.5 .ANG.; functional group F(IV)7 forms a hydrophobic
contact with a side chain of receptor Leu243 and a side chain of
Thr239 of SEQ ID NO: 1 and has coordinates of r=47.767, .theta.
(theta)=112.521, and .phi. (phi)=-10.196 and a spherical radius of
about 1.2 .ANG.; functional group F(IV)8 donates a hydrogen bond to
a backbone carbonyl of His280 of SEQ ID NO: 1 and has coordinates
of r=45.184, .theta. (theta)=112.328, and .phi. (phi)=-8.875 and a
spherical radius of about 1.2 .ANG.; functional group F(IV)9 forms
a hydrophobic contact with a side chain of receptor Met244 and
Leu243 of SEQ ID NO: 1 and has coordinates of r=49.512, .theta.
(theta)=108.007, and .phi. (phi)=-8.504 and a spherical radius of
about 1.2 .ANG.; and functional group F(IV)10 forms a hydrophobic
contact with a side chain of receptor Met244 of SEQ ID NO: 1 and
has coordinates of r=50.431, .theta. (theta)=104.985, and .phi.
(phi)=-7.420 and a spherical radius of about 1.8 .ANG.; and Scheme
V (AD4-11091-like) comprises functional group functional groups
F(V)1, F(V)2, F(V)3, F(V)4, F(V)5, F(V)6, F(V)7, F(V)8, F(V)9,
F(V)10, and F(V)11; wherein functional group F(V)1 accepts a
hydrogen bond from a side chain amino of receptor Lys465 of SEQ ID
NO: 1 and has coordinates of r=85.329, .theta. (theta)=34.962, and
.phi. (phi)=53.394 and a spherical radius of about 1.2 .ANG.;
functional group F(V)2 accepts a hydrogen bond from a side chain
amino of receptor Lys465 of SEQ ID NO: 1 and has coordinates of
r=87.180, .theta. (theta)=35.618, and .phi. (phi)=53.267 and a
spherical radius of about 1.5 .ANG.; functional group F(V)3 accepts
a hydrogen bond from a side chain amino of receptor Lys465 of SEQ
ID NO: 1 and has coordinates of r=86.349, .theta. (theta)=34.560,
and .phi. (phi)=55.424 and a spherical radius of about 1.5 .ANG.;
functional group F(V)4 donates a hydrogen bond to a side chain
carbonyl of receptor Gln411 of SEQ ID NO: 1 and has coordinates of
r=83.958, .theta. (theta)=35.296, and .phi. (phi)=51.296 and a
spherical radius of about 1.2 .ANG.; functional group F(V)5 donates
a hydrogen bond to a side chain carbonyl of receptor Gln411 of SEQ
ID NO: 1 and has coordinates of r=82.060, .theta. (theta)=34.984,
and .phi. (phi)=50.365 and a spherical radius of about 1.5 .ANG.;
functional group F(V)6 donates a hydrogen bond to a side chain
nitrogen of receptor Gln411 of SEQ ID NO: 1 and has coordinates of
r=83.884, .theta. (theta)=36.166, and .phi. (phi)=49.227 and a
spherical radius of about 1.2 .ANG.; functional group F(V)7 donates
a hydrogen bond to a side chain nitrogen of receptor Gln411 of SEQ
ID NO: 1 and has coordinates of r=82.006, .theta. (theta)=35.523,
and .phi. (phi)=49.076 and a spherical radius of about 1.5 .ANG.;
functional group F(V)8 accepts a hydrogen bond to a side chain
imidazole of receptor His409 of SEQ ID NO: 1 and has coordinates of
r=85.100, .theta. (theta)=38.590, and .phi. (phi)=43.143 and a
spherical radius of about 1.2 .ANG.; functional group F(V)9 forms a
favorable .pi.-.pi. interaction with a phenyl ring of receptor
Phe412 of SEQ ID NO: 1 and has coordinates of r=84.418, .theta.
(theta)=37.489, and .phi. (phi)=46.968 and a spherical radius of
about 1.2 .ANG.; functional group F(V)10 forms a favorable
.pi.-.pi. interaction with a phenyl ring of receptor Phe412 of SEQ
ID NO: 1 and has coordinates of r=82.486, .theta. (theta)=38.027,
and .phi. (phi)=47.246 and a spherical radius of about 1.5 .ANG.;
and functional group F(V)11 forms a favorable hydrophobic
interaction with a side chain of receptors Val417 and Ile448 of SEQ
ID NO: 1 and has coordinates of r=84.438, .theta. (theta)=40.753,
and .phi. (phi)=48.557 and a spherical radius of about 1.2
.ANG..
16. The method of claim 15, further comprising one or more of the
following features: determining identity and spatial orientation of
at least a portion of atoms of EGFR associated with stabilizing a
tethered configuration of domain II and domain IV of EGFR in an
inactive conformation; and constructing a pharmacophore, wherein
the pharmacophore comprises a pluralitiy of pharmacophoric features
that approximates the identity and the spatial orientation of the
at least a portion of atoms of EGFR associated with stabilizing a
tethered configuration of domain II and domain IV of EGFR in an
inactive conformation such that the pharmacophore structural
features are complementary to the inactive EGFR configuration.
determining identity and spatial orientation of at least a portion
of atoms of EGFR associated with stabilizing a tethered
configuration of domain II and domain IV of EGFR in an inactive
conformation by analysis of X-ray crystallographic data derived
from a crystalline form of EGFR in an inactive, tethered
conformation; wherein at least one pharmacophoric feature
approximates identity and spatial orientations of at least a
portion of atoms of domain II of EGFR in a tethered inactive
conformation; wherein at least one pharmacophoric feature
approximates identity and spatial orientations of at least a
portion of atoms of a cleft region between domain II and domain IV
of EGFR in a tethered inactive conformation; or determining a
docking affinity of the candidate molecule for the at least a
portion of atoms of EGFR associated with stabilizing a tethered
configuration of domain II and domain IV of EGFR in an inactive
conformation, wherein docking affinity is quantified by energy
gained upon interaction of the candidate molecule with the target
biomolecule, energy required to attain the docked conformation
relative to the lowest energy conformation, or a combination
thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. Nonprovisional
application Ser. No. 13/521,010 filed 6 Jul. 2012 (Int'l. filing
date 6 Jan. 2011); which claims the benefit of priority to Int'l.
Application No. PCT/US11/20418 filed 6 Jan. 2011 and U.S.
Provisional Application No. 61/292,776, filed 6 Jan. 2010; each of
which is incorporated herein by reference in its entirety.
INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING
[0002] The Sequence Listing, which is a part of the present
disclosure, includes a computer readable form comprising nucleotide
and/or amino acid sequences of the present invention. The subject
matter of the Sequence Listing is incorporated herein by reference
in its entirety.
FIELD OF THE INVENTION
[0003] The present invention generally relates to novel chemical
compounds for use in the treatment of disease, methods of
manufacture of such compounds and intermediates thereof, and
methods of identifying lead molecules for use in quasi-rational
drug design.
BACKGROUND OF THE INVENTION
[0004] Rational drug development is a process of developing lead
molecules, not by randomly screening thousands of molecules in the
blind hope of finding one that shows the desired activity, but
rather by deducing the active site of the target and devising a
chemical that interacts with that site in the appropriate
manner.
[0005] Epidermal Growth Factor Receptor (EGFR) is a member of the
ErbB (HER) family receptor tyrosine kinase (RTKs), which regulate
cell growth and differentiation and are implicated in many human
cancers. EGFR activation and dimerization is discussed in, for
example, Burgess et al. (2003) Moledular Cell 12, 541-552 and
Ferguson et al. (2003) Molecular Cell 11, 507-517.
[0006] EGF activates its receptor by inducing dimerization of the
extracellular region of EGFR. The activation of EGFR has been
described through results of disulfide bond mapping as well as
X-ray crystal structures. The crystal structures of ligand-bound
sEGFR showed that dimerization is receptor mediated, with two
individual ligand molecules present in the dimer. The dimerization
interface of activated EGFR is completely occluded by
intramolecular interactions, and is an autoinhibited configuration.
To activate the receptor, a large domain rearrangement that exposes
this occluded interface must accompany EGF binding where EGF does
not contribute to the EGFR dimer interface. The EGFR mechanism is
in sharp contrast to most other receptor tyrosine kinase activation
mechanisms in which the bound ligand contributes directly to the
receptor dimerization interface and does not dramatically alter the
conformation of the extracellular region of the receptor tyrosine
kinase.
[0007] EGFR contains four subdomain I, II, III, and IV. Almost all
receptor/receptor contacts observed in the crystal structures of
EGFR are mediated by domain II. At the center of the dimer
interface is a prominent loop (residues 242-259 of EGFR) that
extends from the second C1 module (module 5) of each domain II and
reaches across the interface to interact primarily with domain II
of its dimerization partner. This domain II loop, which is specific
to ErbB receptors, is the "dimerization arm". The dimerization arm
of domain II is completely occluded by intramolecular interactions
with domain IV (i.e., an autoinhibited configuration). There are
two smaller interaction sites in the dimer that involve side chains
from the second and the sixth disulfide-bonded modules of domain
II. And the dimer interface may extend into domain IV. While the
two receptor molecules approach one another very closely toward the
C terminus of domain IV, a well-defined, tight interface is not
observed.
[0008] Although EGF and TGF-.alpha. clearly do not span the dimer
interface, each ligand simultaneously contacts two separate binding
surfaces in the same EGFR molecule. The bound EGF or TGF-.alpha.
molecule resembles a wedge between domains I and III. The
relationship between domains I and II is essentially identical to
that seen in IGF-1R and in the activated sEGFR dimer, implying that
ligand binding does not greatly influence the relative orientation
of these two domains. But the relationship between domains II and
III differs dramatically in the activated and unactivated
structures. A direct intramolecular interaction between
cysteine-rich domains II and IV restrains the domain II/III
relationship that characterizes the unactivated configuration. This
interdomain "tether" is stabilized by essentially identical
interactions between the two cysteine rich domains (II and IV) in
inactive sErbB3 and sEGFR.
[0009] The intramolecular domain II/IV tether precisely buries the
dimerization arm of domain II against domain IV, so that the
tethered configurations of sErbB3 and sEGFR cannot dimerize and
thus appear to be autoinhibited. Moreover, the two ligand binding
surfaces on domain I and III are too far apart in the tethered
configuration for a single ligand to bind to both simultaneously.
Consequently, the tethered configuration can only form low-affinity
interactions with ligand, using just one of its ligand binding
surfaces at a time.
[0010] Switching between the unactivated and activated
configurations of sEGFR requires domains I and III to be drawn
toward one another through a 130.degree. rotation of the rigid
domain I/II pair in one plane and a 20.degree. rotation in another.
Only this extended configuration of sEGFR is capable of both
high-affinity ligand binding and efficient dimerization.
[0011] Based upon energetic calculations, it is currently thought
that at any given time, about 95% of sEGFR molecules will be
tethered and the remaining 5% will not. The presence of ligand and
subsequent binding to domains I and III of the non-tethered form
will drive the equilibrium toward the non-tethered form, trapping
receptor molecules in the extended state that can dimerize.
[0012] Exposure of the dimerization arm is not sufficient alone to
drive EGFR dimerization. Also required is additional contact sites
in modules 2 and 6 of domain III. These two additional contact
sites and the dimerization arm cooperate at the dimer
interface.
[0013] Known strategies of EGFR inhibition are directed to antibody
binding of domain III to provide steric hindrance of the required
configuration change (e.g., Erbitux). Other conventional strategies
are directed to antibody binding of domain II, specifically the
dimerization arm, so as to prevent dimerization (e.g., pertuzumab).
Still other conventional strategies are directed to antibody
binding of domain IV residues that participate in the
intramolecular tether (e.g., trastuzumab, Herceptin). But no
existing strategies are directed to the tethering mechanism of
activation.
SUMMARY OF THE INVENTION
[0014] Described herein are compounds and compositions having an
anti-proliferative effect, along with methods of therapeutic
treatment with such compounds. Also provided are methods of
discovery of such compounds. An approach described herein
identifies modulators of the activity of multi-domain proteins
comprising a dimerization arm and interdomain tether, such as EGFR,
where an untethered, extended conformation is the active state and
a tethered conformation is the inactive state, resulting in an
autoinhibited configuration.
[0015] One aspect of the invention provides small molecule
compounds.
[0016] In one embodiment, compounds have a formula of
##STR00001##
with constituents as defined herein, or a stereoisomer or
pharmaceutically acceptable salt thereof.
[0017] In one embodiment, compounds have a formula of
##STR00002##
with constituents as defined herein, or a stereoisomer or
pharmaceutically acceptable salt thereof.
[0018] In one embodiment, compounds have a formula of
##STR00003##
with constituents as defined herein, or a stereoisomer or
pharmaceutically acceptable salt thereof.
[0019] In one embodiment, compounds have a formula of
##STR00004##
with constituents as defined herein, or a stereoisomer or
pharmaceutically acceptable salt thereof.
[0020] In one embodiment, a 1734-like compound inhibits EGFR
activity and comprises at least five or more of functional groups
F(II)1, F(II)2, F(II)3, F(II)4, F(II)5, F(II)6, F(II)7, F(II)8, and
F(II)9 of a Scheme II 1734-like pharmacophore, with functional
groups as defined herein.
[0021] In one embodiment, compounds have a formula of
##STR00005##
with constituents as defined herein, or a stereoisomer or
pharmaceutically acceptable salt thereof.
[0022] In one embodiment, compounds have a formula of
##STR00006##
with constituents as defined herein, or a stereoisomer or
pharmaceutically acceptable salt thereof.
[0023] In one embodiment, compounds have a formula of
##STR00007##
with constituents as defined herein, or a stereoisomer or
pharmaceutically acceptable salt thereof.
[0024] In one embodiment, compounds have a formula of
##STR00008##
with constituents as defined herein, or a stereoisomer or
pharmaceutically acceptable salt thereof.
[0025] In one embodiment, compounds have a formula of
##STR00009##
with constituents as defined herein, or a stereoisomer or
pharmaceutically acceptable salt thereof.
[0026] In one embodiment, compounds have a formula of
##STR00010##
with constituents as defined herein, or a stereoisomer or
pharmaceutically acceptable salt thereof.
[0027] In one embodiment, a 1886-like compound inhibits EGFR
activity and comprises at least seven or more of functional groups
F(III)1, F(III)2, F(III)3, F(III)4, F(III)5, F(III)6, F(III)7,
F(III)8, and F(III)9 of a Scheme III 1886-like pharmacophore, with
functional groups as defined herein.
[0028] In one embodiment, compounds have a formula of
##STR00011##
with constituents as defined herein, or a stereoisomer or
pharmaceutically acceptable salt thereof.
[0029] In one embodiment, compounds have a formula of
##STR00012##
with constituents as defined herein, or a stereoisomer or
pharmaceutically acceptable salt thereof.
[0030] In one embodiment, compounds have a formula of
##STR00013##
with constituents as defined herein, or a stereoisomer or
pharmaceutically acceptable salt thereof.
[0031] In one embodiment, a 10381-like compound inhibits EGFR
activity and comprises at least eight or more of functional groups
F(IV)1, F(IV)2, F(IV)3, F(IV)4, F(IV)5, F(IV)6, F(IV)7, F(IV)8,
F(IV)9, and F(IV)10 of a Scheme IV 10381-like pharmacophore, with
functional groups as defined herein.
[0032] In one embodiment, compounds have a formula of
##STR00014##
with constituents as defined herein, or a stereoisomer or
pharmaceutically acceptable salt thereof.
[0033] In one embodiment, compounds have a formula of
##STR00015##
with constituents as defined herein, or a stereoisomer or
pharmaceutically acceptable salt thereof.
[0034] In one embodiment, a 11091-like compound inhibits EGFR
activity and comprises at least nine or more of functional groups
F(V)1, F(V)2, F(V)3, F(V)4, F(V)5, F(V)6, F(V)7, F(V)8, F(V)9,
F(V)10, and F(V)11 of a Scheme V 11091-like pharmacophore, with
functional groups as defined herein.
[0035] One aspect of the invention provides a method of treating a
proliferative disease, disorder, or condition using a compound
described herein.
[0036] In one embodiment, the method of treating a proliferative
disease, disorder, or condition comprises administering to a
subject in need thereof a composition comprising a therapeutically
effective amount of a compound with a formula selected from the
group consisting of: Formula (13); Formula (14); Formula (15);
Formula (16); Formula (18); Formula (19); Formula (20); Formula
(21); Formula (22); Formula (23); Formula (25); Formula (27);
Formula (28); Formula (32); and Formula (33), as defined herein, or
a stereoisomer or pharmaceutically acceptable salt thereof.
[0037] In one embodiment, the method of treating a proliferative
disease, disorder, or condition comprises administering to a
subject in need thereof a composition comprising a therapeutically
effective amount of a compound with a formula selected from the
group consisting of: AD4-1734 (Formula (12)); AD4-1886 (Formula
(17)); AD4-10381 (Formula (24)); and AD4-11091 (Formula (31)), or a
stereoisomer or pharmaceutically acceptable salt thereof.
[0038] In one embodiment, the method of treating a proliferative
disease, disorder, or condition comprises administering to a
subject in need thereof a composition comprising a therapeutically
effective amount of a compound with a formula selected from the
group consisting of:
##STR00016##
or a stereoisomer or pharmaceutically acceptable salt thereof.
[0039] In some embodiments, the composition further comprises a
pharmaceutically acceptable carrier or excipient.
[0040] In some embodiments, the treated proliferative disease,
disorder, or condition is selected from: cancer; a blood vessel
proliferative disorder; a fibrotic disorder; a mesangial cell
proliferative disorder; psoriasis; actinic keratoses; seborrheic
keratoses; warts; keloid scars; eczema; and hyperproliferative
diseases caused by a viral infection.
[0041] In some embodiments, the treated proliferative disease,
disorder, or condition is associated with EGFR. In some
configurations, the proliferative disease, disorder, or condition
associated with EGFR is selected from the group consisting of:
cancer; a blood vessel proliferative disorder; a fibrotic disorder;
a mesangial cell proliferative disorder; psoriasis; actinic
keratoses; seborrheic keratoses; warts; keloid scars; eczema; and
hyperproliferative diseases caused by a viral infection.
[0042] One aspect provides a method for identifying an EGFR
inhibitor.
[0043] In one embodiment, the method for identifying an EGFR
inhibitor comprises: providing a pharmacophore comprising a scheme
selected from the group consisting of Scheme II (as defined herein)
as input to a 3-dimensional database; comparing a three dimensional
structure of a candidate compound to the three dimensional
structure of the pharmacophore; and selecting a candidate compound
with a three dimensional structure that substantially aligns with
five or more functional groups of Scheme II (ADS-1734-like).
[0044] In one embodiment, the method for identifying an EGFR
inhibitor comprises: providing a pharmacophore comprising a scheme
selected from the group consisting of Scheme III (as defined
herein) as input to a 3-dimensional database; comparing a three
dimensional structure of a candidate compound to the three
dimensional structure of the pharmacophore; and selecting a
candidate compound with a three dimensional structure that
substantially aligns with seven or more functional groups of Scheme
III (AD4-1886-like).
[0045] In one embodiment, the method for identifying an EGFR
inhibitor comprises: providing a pharmacophore comprising a scheme
selected from the group consisting of Scheme IV (as defined herein)
as input to a 3-dimensional database; comparing a three dimensional
structure of a candidate compound to the three dimensional
structure of the pharmacophore; and selecting a candidate compound
with a three dimensional structure that substantially aligns with
eight or more of functional groups of Scheme IV
(AD4-10381-like).
[0046] In one embodiment, the method for identifying an EGFR
inhibitor comprises: providing a pharmacophore comprising a scheme
selected from the group consisting of Scheme V (as defined herein)
as input to a 3-dimensional database; comparing a three dimensional
structure of a candidate compound to the three dimensional
structure of the pharmacophore; and selecting a candidate compound
with a three dimensional structure that substantially aligns with
nine or more of functional groups of Scheme V (AD4-11091-like).
[0047] In some embodiments of the method for identifying an EGFR
inhibitor, similarity between the three-dimensional structure of
the candidate compound and the three-dimensional structure of the
pharmacophore is indicative of an ability of the candidate compound
to inhibit EGFR by substantially maintaining a tethered inactive
configuration of EGFR or substantially preventing stabilization of
the untethered active configuration of EGFR.
[0048] Some embodiments of the method for identifying an EGFR
inhibitor further comprise determining identity and spatial
orientation of at least a portion of atoms of EGFR associated with
stabilizing a tethered configuration of domain II and domain IV of
EGFR in an inactive conformation; and constructing a pharmacophore,
wherein the pharmacophore comprises a plurality of pharmacophoric
features that approximates the identity and the spatial orientation
of the at least a portion of atoms of EGFR associated with
stabilizing a tethered configuration of domain II and domain IV of
EGFR in an inactive conformation such that the pharmacophore
structural features are complementary to the inactive EGFR
configuration. In some configurations, determining identity and
spatial orientation of at least a portion of atoms of EGFR
associated with stabilizing a tethered configuration of domain II
and domain IV of EGFR in an inactive conformation comprises
analysis of X-ray crystallographic data derived from a crystalline
form of EGFR in an inactive, tethered conformation.
[0049] In some embodiments of the method for identifying an EGFR
inhibitor, at least one pharmacophoric feature approximates
identity and spatial orientations of at least a portion of atoms of
domain II of EGFR in a tethered inactive conformation. In some
embodiments of the method for identifying an EGFR inhibitor, at
least one pharmacophoric feature approximates identity and spatial
orientations of at least a portion of atoms of a cleft region
between domain II and domain IV of EGFR in a tethered inactive
conformation.
[0050] Some embodiments of the method for identifying an EGFR
inhibitor further comprise determining a docking affinity of the
candidate molecule for the at least a portion of atoms of EGFR
associated with stabilizing a tethered configuration of domain II
and domain IV of EGFR in an inactive conformation. In some
configurations, docking affinity is quantified by energy gained
upon interaction of the candidate molecule with the target
biomolecule, energy required to attain the docked conformation
relative to the lowest energy conformation, or a combination
thereof.
[0051] Other objects and features will be in part apparent and in
part pointed out hereinafter.
BRIEF DESCRIPTION OF THE DRAWINGS
[0052] Those of skill in the art will understand that the drawings,
described below, are for illustrative purposes only. The drawings
are not intended to limit the scope of the present teachings in any
way.
[0053] FIG. 1A shows pharmacophore 21_thr100_glu105 (see Example 3;
Table 1). FIG. F10A: Pharmacophore 21_thr100_glu105. FIG. F10B:
Pharmacophore 21_thr100_glu105 aligned to AD4-1886.
[0054] FIG. 1B shows AD4-1886 aligned to pharmacophore
21_thr100_glu105.
[0055] FIG. 2: AD4-11091 aligned to the pharmacophore model, Pharm
1886-6. (see Example 3; Table 3).
[0056] FIG. 3 shows the binding site at the interface of Domain II
and Domain IV of the Inactive Form of EGFr (1NQL.pdb) as determined
by the site finder in MOE. The Carbon Atoms of the Domain II
Residues are Colored Red and those of the Domain IV Residues are
Colored Blue.
[0057] FIG. 4 shows Pharm-1 nql-glue-6 aligned to the hit
AD4-1734.
[0058] FIG. 5 shows DockPharm 1505-2 aligned to the hit
AD4-10381.
[0059] FIG. 6A-FIG. 6F are a series of two-dimensional
representations of AD4-1886 and AD4-1886-like compounds docked with
the EGFR/cetuximab complex. Docking of compound AD4-1886 to EGFR is
depicted, for example, in FIG. 6A. Docking of compound AD4-11883
(an AD4-1886-like compound) to EGFR is depicted, for example, in
FIG. 6B. Docking of compound AD4-11975 (an AD4-1886-like compound)
to EGFR is depicted, for example, in FIG. 6C. Docking of compound
AD4-11409 (an AD4-1886-like compound) to EGFR is depicted, for
example, in FIG. 6D. Docking of compound AD4-11638 (an
AD4-1886-like compound) to EGFR is depicted, for example, in FIG.
6E. Docking of compound AD4-11645 (an AD4-1886-like compound) to
EGFR is depicted, for example, in FIG. 6F.
[0060] FIG. 7A-FIG. 7F are a series of two-dimensional
representations of AD4-11091 and AD4-11091-like compounds docked
with the EGFR/cetuximab complex. Docking of compound AD4-11091 to
EGFR is depicted, for example, in FIG. 7A. Docking of compound
AD4-12509 (an AD4-11091-like compound) to EGFR is depicted, for
example, in FIG. 7B. Docking of compound AD4-12423 (an
AD4-11091-like compound) to EGFR is depicted, for example, in FIG.
7C. Docking of compound AD4-12528 (an AD4-11091-like compound) to
EGFR is depicted, for example, in FIG. 7D. Docking of compound
AD4-12522 (an AD4-11091-like compound) to EGFR is depicted, for
example, in FIG. 7E. Docking of compound AD4-12504 (an
AD4-11091-like compound) to EGFR is depicted, for example, in FIG.
7F.
[0061] FIG. 8A-FIG. 8F are a series of two-dimensional
representations of AD4-1734 and AD4-1734-like compounds docked with
inactive EGFR. Docking of compound AD4-1734 to EGFR is depicted,
for example, in FIG. 8A. Docking of compound AD4-10631 (an
AD4-1734-like compound) to EGFR is depicted, for example, in FIG.
8B. Docking of compound AD4-10188 (an AD4-1734-like compound) to
EGFR is depicted, for example, in FIG. 8C. Docking of compound
AD4-10186 (an AD4-1734-like compound) to EGFR is depicted, for
example, in FIG. 8D. Docking of compound AD4-10633 (an
AD4-1734-like compound) to EGFR is depicted, for example, in FIG.
8E. Docking of compound AD4-10174 (an AD4-1734-like compound) to
EGFR is depicted, for example, in FIG. 8F.
[0062] FIG. 9A-FIG. 9F are a series of two-dimensional
representations of AD4-10381 and AD4-10381-like compounds docked
with inactive EGFR. Docking of compound AD4-10381 to EGFR is
depicted, for example, in FIG. 9A. Docking of compound AD4-11340
(an AD4-10381-like compound) to EGFR is depicted, for example, in
FIG. 9B. Docking of compound AD4-12632 (an AD4-10381-like compound)
to EGFR is depicted, for example, in FIG. 9C. Docking of compound
AD4-12681 (an AD4-10381-like compound) to EGFR is depicted, for
example, in FIG. 9D. Docking of compound AD4-12732 (an
AD4-10381-like compound) to EGFR is depicted, for example, in FIG.
9E. Docking of compound AD4-11511 (an AD4-10381-like compound) to
EGFR is depicted, for example, in FIG. 9F.
[0063] FIG. 10A-FIG. 10B are a series of line and scatter plots
showing % inhibition of EGFR as a function of concentration of
Tykerb (FIG. 10A) or Iressa (FIG. 10B) either alone or in
combination with AD4-10628. A shift in the dose-response curve to
the left indicates a more potent response.
[0064] FIG. 11 is a scatter plot showing Dose Reduction Index (DRI)
as a function of Fa for DRI Tykerb and DRI AD4-10628.
[0065] FIG. 12 is a histogram showing a summary of Combination
Index (CI) values at 90% inhibition (ED90) for a series of AD4
compounds in combination with Tykerb. Compound 4 is Iressa.
Compound 5 is Tarceva. The balance of compounds are AD4 compounds
described herein. Response below the dark middle line (i.e.,
CI<0.9) indicates synergism.
[0066] FIG. 13A-FIG. 13D are a cartoon depicting conformations of
EGFR. FIG. 13A shows EGFR as a tethered monomer. FIG. 13B shows
EGFR as an untethered monomer. FIG. 13C shows EGFR in a ligand
stabilized extended conformation. FIG. 13D shows EGFR as a ligand
induced activated dimer.
[0067] FIG. 14 is a cartoon depicting ligand-induced dimerization
and activation of the kinase domain of EGFR.
DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS
[0068] Described herein are compounds and compositions having an
anti-proliferative effect, along with methods of therapeutic
treatment with such compounds and methods of discovery of such
compounds. Various small molecule compounds described herein can
hold proteins of multiple domains together in a tethered, inactive
state. Also provided are methods to identify the structural
requirements of such inhibitors, screen for effective inhibitors,
optimize the structure of identified candidates, and utilize
identified small molecule compounds in therapeutic treatment
regimes.
[0069] One aspect of the invention is directed to small molecule
compounds efficacious in treating proliferative diseases or
conditions. Various embodiments of compounds described herein can
have an anti-proliferative effect. Various embodiments of compounds
described herein can hold multiple domain proteins in a tethered,
inactive state. Various embodiments of compounds described herein
can have an inhibitory effect on EGFR. Compounds described herein
have been demonstrated to be empirically effective in treating
proliferative diseases and conditions.
[0070] One aspect of the invention is directed to therapeutic
treatment of proliferative diseases and disorders using compounds
and compositions described herein.
[0071] One aspect of the invention is directed to compounds,
methods, and apparatuses for developing one or more drugs for one
or more targeted therapies. More specifically, the approach
described herein identifies modulators of the activity of
multi-domain proteins comprising a dimerization arm and interdomain
tether, where an untethered, extended conformation is the active
state and a tethered conformation is the inactive state, resulting
in an autoinhibited configuration. The pharmacophoric approach
described herein is based upon a mechanistic understanding of
conformation-dependent protein receptor activation mechanisms, thus
avoiding conventional combinatorial chemistry and high throughput
screening techniques.
[0072] Biomolecule Target Selection
[0073] Desirable target enzymes include those for which there
exists crystallography data sufficient to discern a ligand binding,
activation, and/or dimerization mechanism. The various methods of
the invention can be used to generate pharmacophore models for a
variety of multi-domain protein targets (crystallized with and/or
without ligand) having an interdomain tether associated with
activation state. Thus is provided compounds that can prevent
untethering and stabilization of the extended conformation, and
methods for identifying such compounds.
[0074] It shall be understood that the types of biomolecule target
for the lead molecules generated by the methods of the present
invention can include one or more of EGFR (i.e., ErbB1), HER2/c-neu
(ErbB-2), Her 3 (ErbB-3), and Her 4 (ErbB-4).
[0075] EGFR
[0076] Described herein is targeting of various portions of the
domains of EGFR so as to prevent stabilization of the untethered,
extended conformation. In other words, a small molecule inhibitor
can be used to hold proteins of domain II and IV together in the
tethered, inactive state. This strategy can provide for some
retention of the basal levels of EGFR signaling, retention of EGF
response, and/or reduce EGF-independent dimerization. Such a
therapeutic effect would slow rapid growth of cancer cells (which
are more sensitive given increased expression levels of EGFR) but
retain at least a portion of basal EGFR activity necessary for
healthy tissue function.
[0077] Known strategies of EGFR inhibition are directed to antibody
binding of domain III to provide steric hindrance of the required
configuration change (e.g., Erbitux). Other conventional strategies
are directed to antibody binding of domain II, specifically the
dimerization arm, so as to prevent dimerization (e.g., pertuzumab).
Still other conventional strategies are directed to antibody
binding of domain IV residues that participate in the
intramolecular tether (e.g., trastuzumab, Herceptin). But, in
contrast to the approach described herein, the above conventional
strategies do not prevent untethering or stabilization of the
extended conformation.
[0078] As described above, EGFR has an autoinhibited configuration
in which the dimerization arm of domain II is completely occluded
by intramolecular interactions with domain IV (see e.g., FIG. 13A).
EGF activates its receptor by inducing dimerization of the
extracellular region of EGFR (see e.g., FIG. 13D). Almost all
receptor/receptor contacts observed in the crystal structures of
EGFR are mediated by domain II, specifically, a prominent loop
(residues 242-259 of EGFR) that extends from the second C1 module
(module 5) of each domain II (i.e., the dimerization arm). The
unactivated configuration is characterized by a direct
intramolecular interaction between cysteine-rich domains II and IV,
which restrains the domain II/III relationship (see e.g., FIG.
13A). This interdomain "tether" is stabilized by essentially
identical interactions between the two cysteine rich domains (II
and IV) in inactive sEGFR. Switching between the unactivated and
activated configurations of sEGFR requires domains I and III to be
drawn toward one another through a 130.degree. rotation of the
rigid domain I/II pair in one plane and a 20.degree. rotation in
another (see e.g., FIG. 13B). Only this extended configuration of
sEGFR is capable of both high-affinity ligand binding (see e.g.,
FIG. 13C) and efficient dimerization (see e.g., FIG. 13D). In the
activated and dimerized configuration, the dimerization arm of
domain II reaches across the interface to interact primarily with
the corresponding domain II arm of its dimerization partner (see
e.g., FIG. 13D). EGFR dimerization also requires interaction of
contact sites in modules 2 and 6 of domain III. The presence of EGF
ligand and subsequent binding to domains I and III of the
non-tethered form will drive the equilibrium toward the
non-tethered form, trapping receptor molecules in the extended
state that can dimerize.
[0079] The approach described herein provides for some retention of
the basal levels of EGFR signaling. In healthy individuals there
exists a baseline signal from EGFR necessary for growth, with
enhanced EGF levels promoting accelerated growth in, for example,
wound recovery. But cancer cells have been demonstrated to exhibit
more EGFR, which increases the probability of the untethered
conformation and subsequent EGF binding to the unoccluded domain
I/III ligand binding site, thereby activating EGFR. An inhibitor
that holds multi-domain proteins of EGFR together in a tethered,
inactive state can allow for some basal levels of EGFR
signaling.
[0080] Furthermore, small molecule inhibitors that prevent
stabilization of the untethered state (see e.g., FIG. 14B depicting
untethered state, and FIG. 14C depicting stabilized untethered
state) of EGFR can be used in conjunction with other anti-EGFR
therapeutic agents. Use of small molecule inhibitors described
herein in conjunction with other anti-EGFR therapeutic modalities
can allow decreased dosage and/or increased maximal inhibition. For
example, use of such small molecule inhibitors can be used in
conjunction with Erbitux (which binds domain III blocking EGF),
which would allow a lower dosage of Erbitux and/or increased
maximal inhibition.
[0081] In various embodiments, domain II of EGFR, as existing in
the tethered state, is targeted so as to prevent opening (i.e., the
configuration change from tethered to open, see e.g., FIG. 13A-B
depicting tethered and untethered conformations). In various
embodiments, the cleft between domain II and domain IV is targeted
so as to prevent opening (i.e., the configuration change from
tethered to open). A single small molecule can be used to span the
two domains. Alternatively, a series of small molecules (e.g., at
least two small molecules) in several compartments can be used in
conjunction so as to span the two domains. In various embodiments,
domain II of EGFR, as existing in the untethered state, is targeted
so as to prevent stabilization of the untethered state (see e.g.,
FIG. 13B-C, depicting untethered and stabilized conformations). In
various embodiments, domain III of EGFR is targeted (e.g., modules
2 and 6 of domain III, which are interacting contact sites required
for EGFR dimerization) so as to prevent stabilization of the
untethered state.
[0082] Pharmacophoric Approach
[0083] One aspect is directed to a pharmacophoric approach for
developing a drug targeting a multi-domain protein having an
interdomain tether associated with activation state. Based upon the
activation and dimerization mechanism of a biomolecule of interest,
binding targets are identified and characterized. The mechanism
and/or binding target can be characterized, for example, via
crystallography data. The target binding domains can be expressed
as one or more pharmacophore features and/or compiled in a
pharmacophore model comprising one or more pharmacophore
features.
[0084] Pharmacophore generation can be according to software
designed for such a task. Candidate molecules (from, for example,
one or more chemical libraries) can be selected from those
molecules which align to the pharmacophore models. Preferably,
candidate molecules are docked and scored in silico for interaction
with the target binding domain. Again, docking and scoring can be
according to software designed for such a task. After selection of
molecules aligning to one or more pharmacophore models, with
optional docking and scoring in silico, the selected molecules can
be obtained, for example, by chemical synthesis or from a
commercial source. The selected molecules can be measured for
binding affinity and/or effect on function for the target
biomolecule. Such assessment can be according to a biological
assay. The tested molecules can be further selected according to
desirable measured parameters. The selected molecules and/or the
further selected molecules can optionally be further optimized.
[0085] Determining Structure Spatial Position
[0086] From the activation and dimerization model of the target
biomolecule, target regions can be identified and 3D binding
domains can be defined. Definition of the binding domain(s)
generally involves the determination of the specific spatial
position of the atoms of the portion of the target biomolecule
which plays a role in the activation and dimerization
mechanism.
[0087] Determination of the spatial position of the binding portion
can be achieved by means of various in silico techniques. For
example, software packages can be used that model the structure of
the binding surface and match it to a model of the active surface
of the target to assess levels of compatibility. Such software
includes CAMAL.
[0088] Determination of the spatial position of the binding portion
can be achieved by means of X-ray crystallography. X-ray
crystallography can be used to determine the structure of atoms
within a structure that is known to play a role in the activation
and dimerization mechanism, and to then use this structural
information to build a synthetic molecule that binds to one or more
of these components and interferes with configuration changes
and/or stabilization. Techniques for employing X-ray
crystallography for structural determination are known in the art
(see e.g., Messerschmidt (2007) X-Ray Crystallography of
Biomacromolecules: A Practical Guide, John Wiley & Sons,
ISBN-10: 3527313966; Woolfson (2003) An Introduction to X-ray
Crystallography, 2d Ed., Cambridge University Press, ISBN-10:
0521423597). Creation of X-ray crystal structures are also known in
the art (see e.g., U.S. Pat. No. 6,931,325 to Wall and U.S. Pat.
No. 6,916,455 to Segelke, each incorporated herein by reference).
Except as otherwise noted herein, therefore, the process of the
present invention can be carried out in accordance with such
processes.
[0089] Parameters derived from X-ray crystallography observed
diffraction data include, but are not limited to, hydrogen bonders,
apolar hydrophobic contacts, salt bridge interactions, polar
surface area of the domain, apolar surface area of the domain,
shape complementarily score for the antibody-target complex, and
explicitly placed water molecules. Also useful is characterization
of bonds between atoms. The distance between two atoms that are
singly bonded ranges from about 1.45 to about 1.55 .ANG.. Atoms
that are double bonded together are typically about 1.2 to about
1.25 .ANG. apart. Bonds that are resonant between single and double
bonds typically have an about 1.30 to about 1.35 .ANG.
separation.
[0090] Construction of Pharmacophores
[0091] A pharmacophore model can be constructed from structural
information of biomolecule components playing a role in activation
and dimerization, including definition of atom position. Small
molecules with complementary features to components of the target
biomolecule, such as a component playing a role in activation and
dimerization, have the potential to interfere with configuration
changes and/or stabilization necessary for activation and
dimerization and thus have therapeutic utility.
[0092] In various embodiments, in silico approaches can be used for
de novo structure design with a fragment based approach employing
contact statistics, 3D surface models, and docked ligands as
templates. From the spatial position information, and/or from other
parameters described above, one can derive 3D ligand-receptor
models (e.g., interaction pattern, pharmacophore schemes), surface
maps (e.g., topography/shape, electrostatic profile,
hydrophobicity, protein flexibility), and docking models (e.g.,
scoring system for ligand binding, minimum energy calculation).
[0093] Techniques for pharmacophore model construction are known in
the art and described extensively herein (see e.g., Examples 4-5).
Except as otherwise noted herein, therefore, the processes of the
present invention can be carried out in accordance with such
processes.
[0094] A pharmacophore model or scheme is generally a set of
structural features in a ligand that are related, preferably
directly related, to the ligand's recognition at a receptor site
and its biological activity. Pharmacophore features can be derived
from corresponding donor, acceptor, aromatic, hydrophobic, and/or
acidic or basic moieties of the corresponding target biomolecule,
especially those features on domains participating in dimerization
and activation mechanisms. It shall be understood that additional
information about the nature of the atoms in the target biomolecule
being used in a pharmacophore scheme, and not simply the spatial
location of the atoms, can assist in the modeling process of a new
chemical lead. These characteristics include, but are not limited
to, the pKa values of the atoms, the rotational rigidity of the
bonds holding the atoms in place, the nature of the bonds
themselves (single, double, resonant, or otherwise), the projected
directionality of hydrogen bond donors and acceptors, etc.
[0095] Typical feature components useful in generating a
pharmacophore scheme include, but are not limited to, atomic
position; atomic radii; hydrogen bond donor features; hydrogen bond
acceptor features; aromatic features; donor features; acceptor
features; anion features; cation features; acceptor and anion
features; donor and cation features; donor and acceptor features;
acid and anion features; hydrophobic features, hydrogen bond
directionality, and metal ligands (see e.g., Examples 4-5). Such
features can be located, for example, at a single atom, centroids
of atoms, or at a projected directional position in space.
[0096] It is contemplated that numerous pharmacophore queries can
be designed for any given target biomolecule. It is further
contemplated that these pharmacophore queries will be useful to
identify small molecule ligands which interact with the target
biomolecule at a site involved with dimerization and activation,
especially towards maintaining a tethered, inactive
conformation.
[0097] Exemplary resources for accomplishing such modeling and
queries include, but are not limited to MOE (CGG) (providing
pharmacophore query and visualization), Glide (Schrodinger)
(providing docking and scoring), Accord for Excel (Accelrys)
(providing organization of molecular information including chemical
structures and formulas), and the ZINC database (UCSF) (providing a
library of commercial compounds). One design tool for the
generation of pharmacophores from immune system protein-target
biomolecule structural binding characterization is MOE, or
Molecular Operating Environment (Chemical Computing Group). Model
generation uses geometrical and electronic constraints to determine
the 3D positions of features corresponding to the immune system
protein. The model of these embodiments consists of spherical
features in 3D space. The diameter of the spheres can be adjusted
(e.g., about 0.5 to about 3.0 .ANG.). Such models allow matches
and/or partial matches of the features.
[0098] Pharmacophoric structural features can be represented by
labeled points in space. Each ligand can be assigned an annotation,
which is a set of structural features that may contribute to the
ligand's pharmacophore (see e.g., Examples 4-5). In various
embodiments, a database of annotated ligands can be searched with a
query that represents a pharmacophore hypothesis (see e.g.,
Examples 6-7). The result of such a search is a set of matches that
align the pharmacophoric features of the query to the
pharmacophoric features present in the ligands of the searched
database (see e.g., Examples 6-7, Tables 11-15). The number of hits
within the database depends, at least in part, upon the size of the
database and the restrictiveness of the pharmacophore query (e.g.,
partial matches, number of features, etc.). Properties and
parameters of the molecules present within the search database are
used to focus the outcome of the query. For example, compounds with
a defined range of molecular weight (MW) or lipophilicity (log P)
can be present in the searched section of the library database of
compounds.
[0099] Candidate Molecules
[0100] The subject methods find use in the screening of a variety
of different candidate molecules (e.g., potentially therapeutic
candidate molecules). As described above, candidate molecules can
be searched using a pharmacophore query. Candidate molecules
encompass numerous chemical classes, though typically they are
organic molecules, preferably small organic compounds having a
molecular weight of more than 50 and less than about 2,500 Daltons.
Candidate molecules can comprise functional groups for structural
interaction with proteins, particularly hydrogen bonding, and can
include at least an amine, carbonyl, hydroxyl or carboxyl group,
preferably at least two of the functional chemical groups. The
candidate molecules can comprise cyclical carbon or heterocyclic
structures and/or aromatic or polyaromatic structures substituted
with one or more of the above functional groups.
[0101] In preferred embodiments, the candidate molecules are
compounds in a library database of compounds. One of skill in the
art will be generally familiar with, for example, numerous
databases for commercially available compounds for screening (see
e.g., ZINC database, UCSF, with 2.7 million compounds over 12
distinct subsets of molecules; Irwin and Shoichet (2005) J Chem Inf
Model 45, 177-182). One of skill in the art will also be familiar
with a variety of search engines to identify commercial sources or
desirable compounds and classes of compounds for further testing
(see e.g., ZINC database; eMolecules.com; and electronic libraries
of commercial compounds provided by vendors, for example:
ChemBridge, Princeton BioMolecular, Ambinter SARL, Enamine, ASDI,
Life Chemicals etc).
[0102] Candidate molecules for screening according to the methods
described herein include both lead-like compounds and drug-like
compounds. A lead-like compound is generally understood to have a
relatively smaller scaffold-like structure (e.g., molecular weight
of about 150 to about 350 D) with relatively fewer features (e.g.,
less than about 3 hydrogen donors and/or less than about 6 hydrogen
acceptors; hydrophobicity character x log P of about -2 to about 4)
(see e.g., Angewante (1999) Chemie Int. ed. Engl. 24, 3943-3948).
In contrast, a drug-like compound is generally understood to have a
relatively larger scaffold (e.g., molecular weight of about 150 to
about 500 D) with relatively more numerous features (e.g., less
than about 10 hydrogen acceptors and/or less than about 8 rotatable
bonds; hydrophobicity character x log P of less than about 5) (see
e.g., Lipinski (2000) J. Pharm. Tox. Methods 44, 235-249).
Preferably, initial screening is performed with lead-like
compounds.
[0103] When designing a lead from spatial orientation data, it can
be useful to understand that certain molecular structures are
characterized as being "drug-like". Such characterization can be
based on a set of empirically recognized qualities derived by
comparing similarities across the breadth of known drugs within the
pharmacopoeia. While it is not required for drugs to meet all, or
even any, of these characterizations, it is far more likely for a
drug candidate to meet with clinical success if it is
drug-like.
[0104] Several of these "drug-like" characteristics have been
summarized into the four rules of Lipinski (generally known as the
"rules of fives" because of the prevalence of the number 5 among
them). While these rules generally relate to oral absorption and
are used to predict bioavailability of compound during lead
optimization, they can serve as effective guidelines for
constructing a lead molecule during rational drug design efforts
such as may be accomplished by using the methods of the present
invention.
[0105] The four "rules of five" state that a candidate drug-like
compound should have at least three of the following
characteristics: (i) a weight less than 500 Daltons; (ii) a log of
P less than 5; (iii) no more than 5 hydrogen bond donors (expressed
as the sum of OH and NH groups); and (iv) no more than 10 hydrogen
bond acceptors (the sum of N and O atoms). Also, drug-like
molecules typically have a span (breadth) of between about 8 .ANG.
to about 15 .ANG.. It will be understood that a candidate molecule,
or even a selected molecule, may not meet all, or even any, of
these characterizations. Nonetheless, the above guidelines are
helpful in drug screening and design.
[0106] As explained above, the number of molecules identified as
hits to the pharmacophore depend, at least in part, on the size of
the database and the restrictiveness of the pharmacophore query.
The number of molecules identified as hits from a pharmacophore
query can be reduced by further modeling of fit to the binding site
of the target biomolecule. Such modeling can be according to
docking and scoring methods, as described below.
[0107] Docking and Scoring
[0108] Candidate molecules identified as being complementary to
certain features of a target biomolecule as compared to a
pharmacophore model (e.g., through a pharmacophore query as
described above) can be further selected according to docking
affinity for the target biomolecule (see e.g., Examples 6-7). In
addition to pharmacophore model generation for database queries, a
second sequential and complementary method for compound
identification and design can be employed. Pharmacophore queries
can filter out compounds quickly and docking and scoring can
evaluate ligand-target biomolecule binding more accurately. In the
case of protein or enzyme target biomolecules, amino acid residues
of different domains in an inactive conformation can be used to
define the docking site.
[0109] In various embodiments, selected compounds from the
pharmacophore queries are docked to the target binding site using
software designed for such analysis (e.g., Glide (Schrodinger, NY).
Docking affinity can be calculated as numerical values (e.g.,
"Glide score") based upon, for example, energy gained upon
interaction of the molecule with the protein (e.g., "g_score")
and/or energy required to attain the docked conformation relative
to the lowest energy conformation (e.g., "e_model") (see e.g.,
Examples 6-7). For these particular examples, the more negative the
score, the better the docking. Preferably, the g_score is less than
about -5. Preferably, the e_model score is less than about -30. It
is contemplated that the desirable numerical quantification of
docking can vary between different target biomolecules.
[0110] In various embodiments, a threshold docking score (e.g.,
g_score and/or e_model score) can be chosen so as to manage the
number of molecules for acquisition and further testing. For
example, in some docking studies, a g-score of negative 5.0 (or
greater magnitude in a negative direction) is considered a
desirable docking score and the cut off is adjusted accordingly. As
another example, in some docking studies, a g-score of negative 7.5
(or greater magnitude in a negative direction) is considered a
desirable docking score and the cut off is adjusted accordingly.
Thus, the magnitude of the g_score can be used to adjust a number
of hits to a workable number that can be acquired and tested. As an
example, if the total number of compounds identified from a
pharmacophore query was about 1,000 to about 3,000, the docking
scores can be used to rank such compounds so as to select about 100
to about 200 for further testing. It is contemplated the number of
compounds to be selected for further testing could be lower or
higher than these estimates. Preferably, magnitude of the g_score
is used as a selection criteria, but it is contemplated that
e_model score could be similarly used, especially where e_model
score is of low magnitude. It is further contemplated that the
selection criteria can be based upon both g_score and e_model
score, preferably weighted toward g_score.
[0111] Docking and scoring can result in a group of compounds with
multiple conformers. Using suitable modeling software (e.g., MOE),
3D structures can be converted to 2D and duplicates thereby
removed. The resulting list of preferred chemical structures can
used to search for commercial vendors using, for example, search
engines designed for such a task (e.g., eMolecules.com).
[0112] Effect on Target Biomolecule
[0113] Candidate molecules selected according to pharmacophore
query and/or further selected according to docking analysis can be
tested for effect on the target biomolecule. Assessment of effect
of a molecule on biomolecule function (e.g., inhibition of
enzymatic activity) can be assessed by various methods known in the
art (see e.g., Examples 1-3). For example, inhibitory effect of a
candidate molecule on the catalytic activity of a target enzyme can
be assessed by known activity assays specific for the target enzyme
(see e.g., Reymond, ed. (2006) Enzyme Assays: High-throughput
Screening, Genetic Selection and Fingerprinting, John Wiley &
Sons, 386 p., ISBN-10: 3527310959; Eisenthall and Danson, Ed.
(2002) Enzyme Assays, 2d edition, Oxford University Press, 384 p.,
ISBN-10: 0199638209). As described herein, an in-cell Western (ICW)
screening protocol can be used to evaluate candidate compounds (see
e.g., Example 1; Chen et al. (2005) Analytical Biochemistry 338,
136-142). Also as described herein, a MTT Cell Proliferation Assay
can be used to evaluate candidate compounds (see e.g., Example 2).
Also as described herein, an EGF inhibitor assay can be used to
evaluate candidate compounds (see e.g., Example 3; Mukku (1984) J.
Biol. Chem. 259, 6543-6546; Duh et al. (1990) World J. Surgery 14,
410-418; Lokeshwar et al. (1989) J. Biol. Chem. 264(32),
19318-19326).
[0114] Further Refinement
[0115] Further refinement of candidate molecules can be conducted.
For example, data from biological assays can be correlated with the
docking model so as to further refine lead-like molecules and/or
drug-like molecules. Various software packages (e.g., MOE) can be
employed to visualize active compound interaction with a target
biomolecule to identify sites on the template suitable for
modification by de novo design. Analogs of active compounds can be
identified using similarity and sub-structure searches (see e.g.,
SciFinder; eModel). Available analogs can be analyzed according to
docking and scoring procedures described above. Analogs with
desirable docking scores can be acquired and further tested for
biological effect on the target biomolecule according to methods
described herein. One skilled in the art will understand these, and
other, methods of refining and further developing candidate
molecules identified by the methods presented herein.
[0116] Pharmacophores
[0117] Provided herein are a series of pharmacophores that can be
used to identify small molecules that can substantially maintain a
non-extended tether inactive configuration of EGFR or substantially
prevent stabilization of an extended tether active configuration of
EGFR. Pharmacophores include, but are not limited to, a Scheme II
pharmacophore (AD4-1734-like), a Scheme III pharmacophore
(AD4-1886-like), a Scheme IV pharmacophore (AD4-10381-like), and
Scheme V pharmacophore (AD4-11091-like).
[0118] Scheme II Pharmacophore (AD4-1734-Like)
[0119] A Scheme II pharmacophore (AD4-1734-like) can include
functional groups F(II)1, F(II)2, F(II)3, F(II)4, F(II)5, F(II)6,
F(II)7, F(II)8, and F(II)9.
[0120] Functional group F(II)1 donates an H-bond or forms a salt
bridge to a carboxylate side chain of receptor Asp553 of SEQ ID NO:
1 and has coordinates of r=56.363, .theta. (theta)=94.368, and
.PHI. (phi)=-17.752 and a spherical radius of about 1.2 .ANG.,
[0121] Functional group F(II)2 is a donor and has coordinates of
r=53.290, .theta. (theta)=101.494, and .PHI. (phi)=-23.244 and a
spherical radius of about 1.0 .ANG..
[0122] Functional group F(II)3 forms a hydrophobic contact with a
side chain of receptor Val568, imidazolidine side chain of receptor
Pro552 and with a side chain of Met253 of SEQ ID NO: 1 and has
coordinates of r=53.726, .theta. (theta)=97.830, and .PHI.
(phi)=-18.377 and a spherical radius of about 1.7 .ANG..
[0123] Functional group F(II)4 forms a hydrophobic contact with
side chain of receptor Val575, Met253, and with an imidazolidine
ring of receptor Pro552 of SEQ ID NO: 1 and has coordinates of
r=53.647, .theta. (theta)=103.844, and .PHI. (phi)=-20.990 and a
spherical radius of about 1.4 .ANG..
[0124] Functional group F(II)5 donates an H-bond to a side chain
hydroxyl of Thr570 of SEQ ID NO: 1 and has coordinates of r=51.093,
.theta. (theta)=104.261, and .PHI. (phi)=-25.552 and a spherical
radius of about 1.2 .ANG..
[0125] Functional group F(II)6 is a donor having directionality of
F4 with respect to a backbone carbonyl of receptor Thr570 of SEQ ID
NO: 1 and has coordinates of r=52.340, .theta. (theta)=103.980, and
.PHI. (phi)=-27.461 and a spherical radius of about 1.5 .ANG..
[0126] Functional group F(II)7 accepts an H-bond from a receptor
backbone NH of Ala573 of SEQ ID NO: 1 and has coordinates of
r=51.383, .theta. (theta)=106.455, and .PHI. (phi)=-24.319 and a
spherical radius of about 1.2 .ANG..
[0127] Functional group F(II)8 is an acceptor having directionality
of F7 with respect to the backbone NH of receptor Ala573 of SEQ ID
NO: 1 and has coordinates of r=52.861, .theta. (theta)=107.691, and
.PHI. (phi)=-25.448 and a spherical radius of about 1.5 .ANG..
[0128] Functional group F(II)9 donates an H-bond to a backbone
carbonyl of Asp563 and forms a salt bridge to a side chain
carboxylate of receptor Asp563 of SEQ ID NO: 1 and has coordinates
of r=57.688, .theta. (theta)=99.198, and .PHI. (phi)=-21.588 and a
spherical radius of about 1.2 .ANG..
[0129] A selected candidate compound can substantially align with
at least one of functional groups F(II)1, F(II)2, F(II)3, F(II)4,
F(II)5, F(II)6, F(II)7, F(II)8, and F(II)9. For example, a selected
candidate compound can substantially align with at least two, at
least three, at least four, at least five, at least six, at least
seven, at least eight, or at least nine of functional groups
F(II)1, F(II)2, F(II)3, F(II)4, F(II)5, F(II)6, F(II)7, F(II)8, and
F(II)9. Preferably, a selected candidate compound can substantially
align with at least five of functional groups F(II)1, F(II)2,
F(II)3, F(II)4, F(II)5, F(II)6, F(II)7, F(II)8, and F(II)9.
[0130] Scheme III Pharmacophore (AD4-1886-Like) A Scheme III
pharmacophore (AD4-1886-like) can include functional groups
F(III)1, F(III)2, F(III)3, F(III)4, F(III)5, F(III)6, F(III)7,
F(III)8, and F(III)9.
[0131] Functional group F(III)1 donates a hydrogen bond to a side
chain carbonyl of receptor Gln408 and accepts a hydrogen bond from
the side chain NH.sub.2 of receptor Gln384 of SEQ ID NO: 1 and has
coordinates of r=81.552, .theta. (theta)=41.243, and .PHI.
(phi)=45.369 and a spherical radius of about 1.2 .ANG..
[0132] Functional group F(III)2 accepts a hydrogen bond from a side
chain OH of receptor Ser440 of SEQ ID NO: 1 and has coordinates of
r=87.287, .theta. (theta)=40.739, and .PHI. (phi)=51.781 and a
spherical radius of about 1.2 .ANG..
[0133] Functional group F(III)3 accepts a hydrogen bond from a side
chain OH of receptor Ser440 of SEQ ID NO: 1 and has coordinates of
r=86.320, .theta. (theta)=41.915, and .PHI. (phi)=52.323 and a
spherical radius of about 1.5 .ANG..
[0134] Functional group F(III)4 forms a favorable coulombic
interaction with an imidazole side chain of receptor His409 of SEQ
ID NO: 1 and has coordinates of r=85.870, .theta. (theta)=38.463,
and .PHI. (phi)=41.650 and a spherical radius of about 1.2
.ANG..
[0135] Functional group F(III)5 forms a favorable coulombic
interaction with an imidazole side chain of receptor His409 of SEQ
ID NO: 1 and has coordinates of r=82.241, .theta. (theta)=37.431,
and .PHI. (phi)=44.151 and a spherical radius of about 1.5
.ANG..
[0136] Functional group F(III)6 accepts a hydrogen bond from, or
forms a salt bridge to, NH.sup.3+ of receptor Lys465 of SEQ ID NO:
1 and has coordinates of r=88.009, .theta. (theta)=37.822, and
.PHI. (phi)=54.903 and a spherical radius of about 1.2 .ANG..
[0137] Functional group F(III)7 accepts a hydrogen bond from, or
forms a salt bridge to, NH.sup.3+ of receptor Lys465 of SEQ ID NO:
1 and has coordinates of r=86.513, .theta. (theta)=36.889, and
.PHI. (phi)=54.484 and a spherical radius of about 1.5 .ANG..
[0138] Functional group F(III)8 donates a hydrogen bond to a side
chain carbonyl of receptor Gln408 of SEQ ID NO: 1 and has
coordinates of r=81.552, .theta. (theta)=41.243, and .PHI.
(phi)=45.369 and a spherical radius of about 1.2 .ANG..
[0139] Functional group F(III)9 donates a hydrogen bond to a side
chain carbonyl of receptor Gln408 of SEQ ID NO: 1 and has
coordinates of r=79.652, .theta. (theta)=40.928, and .PHI.
(phi)=44.528 and a spherical radius of about 1.6 .ANG..
[0140] A selected candidate compound can substantially align with
at least one of functional groups F(III)1, F(III)2, F(III)3,
F(III)4, F(III)5, F(III)6, F(III)7, F(III)8, and F(III)9. For
example, a selected candidate compound can substantially align with
at least two, at least three, at least four, at least five, at
least six, at least seven, at least eight, or at least nine of
functional groups F(III)1, F(III)2, F(III)3, F(III)4, F(III)5,
F(III)6, F(III)7, F(III)8, and F(III)9. Preferably, a selected
candidate compound can substantially align with at least seven of
functional groups F(III)1, F(III)2, F(III)3, F(III)4, F(III)5,
F(III)6, F(III)7, F(III)8, and F(III)9.
[0141] Scheme IV Pharmacophore (AD4-10381-Like)
[0142] A Scheme IV pharmacophore (AD4-10381-like) can include
functional groups F(IV)1, F(IV)2, F(IV)3, F(IV)4, F(IV)5, F(IV)6,
F(IV)7, F(IV)8, F(IV)9, and F(IV)10.
[0143] Functional group F(IV)1 accepts a hydrogen bond from
receptor side chain OH of Thr239 of SEQ ID NO: 1 and has
coordinates of r=49.686, .theta. (theta)=113.993, and .PHI.
(phi)=-17.014 and a spherical radius of about 1.2 .ANG..
[0144] Functional group F(IV)2 accepts a hydrogen bond from
receptor side chain OH of Thr239 of SEQ ID NO: 1 and has
coordinates of r=48.071, .theta. (theta)=115.388, and .PHI.
(phi)=-16.211 and a spherical radius of about 1.6 .ANG..
[0145] Functional group F(IV)3 forms a hydrophobic contact with a
side chain of receptor Met576 and imidazolidine ring of Pro242 of
SEQ ID NO: 1 and has coordinates of r=50.781, .theta.
(theta)=113.121, and .PHI. (phi)=-17.520 and a spherical radius of
about 1.2 .ANG..
[0146] Functional group F(IV)4 forms a hydrophobic contact with a
side chain of receptor Met576 and imidazolidine ring of Pro242 of
SEQ ID NO: 1 and has coordinates of r=52.021, .theta.
(theta)=114.264, and .PHI. (phi)=-15.878 and a spherical radius of
about 1.5 .ANG..
[0147] Functional group F(IV)5 forms a hydrophobic contact with a
side chain of receptor Met576 and imidazolidine ring of Pro242 of
SEQ ID NO: 1 and has coordinates of r=50.322, .theta.
(theta)=111.253, and .PHI. (phi)=-15.426 and a spherical radius of
about 1.2 .ANG..
[0148] Functional group F(IV)6 forms a hydrophobic contact with a
side chain of receptor Met576 and imidazolidine ring of Pro242 of
SEQ ID NO: 1 and has coordinates of r=51.575, .theta.
(theta)=112.433, and .PHI. (phi)=-13.827 and a spherical radius of
about 1.5 .ANG..
[0149] Functional group F(IV)7 forms a hydrophobic contact with a
side chain of receptor Leu243 and a side chain of Thr239 of SEQ ID
NO: 1 and has coordinates of r=47.767, .theta. (theta)=112.521, and
.PHI. (phi)=-10.196 and a spherical radius of about 1.2 .ANG.
[0150] Functional group F(IV)8 donates a hydrogen bond to a
backbone carbonyl of His280 of SEQ ID NO: 1 and has coordinates of
r=45.184, .theta. (theta)=112.328, and .PHI. (phi)=-8.875 and a
spherical radius of about 1.2 .ANG.
[0151] Functional group F(IV)9 forms a hydrophobic contact with a
side chain of receptor Met244 and Leu243 of SEQ ID NO: 1 and has
coordinates of r=49.512, .theta. (theta)=108.007, and .PHI.
(phi)=-8.504 and a spherical radius of about 1.2 .ANG..
[0152] Functional group F(IV)10 forms a hydrophobic contact with a
side chain of receptor Met244 of SEQ ID NO: 1 and has coordinates
of r=50.431, .theta. (theta)=104.985, and .PHI. (phi)=-7.420 and a
spherical radius of about 1.8 .ANG..
[0153] A selected candidate compound can substantially align with
at least one of functional groups F(IV)1, F(IV)2, F(IV)3, F(IV)4,
F(IV)5, F(IV)6, F(IV)7, F(IV)8, F(IV)9, and F(IV)10. For example, a
selected candidate compound can substantially align with at least
two, at least three, at least four, at least five, at least six, at
least seven, at least eight, at least nine, or at least ten of
functional groups F(IV)1, F(IV)2, F(IV)3, F(IV)4, F(IV)5, F(IV)6,
F(IV)7, F(IV)8, F(IV)9, and F(IV)10. Preferably, a selected
candidate compound can substantially align with at least eight of
functional groups F(IV)1, F(IV)2, F(IV)3, F(IV)4, F(IV)5, F(IV)6,
F(IV)7, F(IV)8, F(IV)9, and F(IV)10.
[0154] Scheme V Pharmacophore (AD4-11091-Like)
[0155] A Scheme V pharmacophore (AD4-11091-like) can include
functional groups F(V)1, F(V)2, F(V)3, F(V)4, F(V)5, F(V)6, F(V)7,
F(V)8, F(V)9, F(V)10, and F(V)11.
[0156] Functional group F(V)1 accepts a hydrogen bond from a side
chain amino of receptor Lys465 of SEQ ID NO: 1 and has coordinates
of r=85.329, .theta. (theta)=34.962, and .chi. (phi)=53.394 and a
spherical radius of about 1.2 .ANG..
[0157] Functional group F(V)2 accepts a hydrogen bond from a side
chain amino of receptor Lys465 of SEQ ID NO: 1 and has coordinates
of r=87.180, .theta. (theta)=35.618, and .PHI. (phi)=53.267 and a
spherical radius of about 1.5 .ANG..
[0158] Functional group F(V)3 accepts a hydrogen bond from a side
chain amino of receptor Lys465 of SEQ ID NO: 1 and has coordinates
of r=86.349, .theta. (theta)=34.560, and .PHI.(phi)=55.424 and a
spherical radius of about 1.5 .ANG..
[0159] Functional group F(V)4 donates a hydrogen bond to a side
chain carbonyl of receptor Gln411 of SEQ ID NO: 1 and has
coordinates of r=83.958, .theta. (theta)=35.296, and .PHI.
(phi)=51.296 and a spherical radius of about 1.2 .ANG.
[0160] Functional group F(V)5 donates a hydrogen bond to a side
chain carbonyl of receptor Gln411 of SEQ ID NO: 1 and has
coordinates of r=82.060, .theta. (theta)=34.984, and .PHI.
(phi)=50.365 and a spherical radius of about 1.5 .ANG..
[0161] Functional group F(V)6 donates a hydrogen bond to a side
chain nitrogen of receptor Gln411 of SEQ ID NO: 1 and has
coordinates of r=83.884, .theta. (theta)=36.166, and .PHI.
(phi)=49.227 and a spherical radius of about 1.2 .ANG..
[0162] Functional group F(V)7 donates a hydrogen bond to a side
chain nitrogen of receptor Gln411 of SEQ ID NO: 1 and has
coordinates of r=82.006, .theta. (theta)=35.523, and .PHI.
(phi)=49.076 and a spherical radius of about 1.5 .ANG..
[0163] Functional group F(V)8 accepts a hydrogen bond to a side
chain imidazole of receptor His409 of SEQ ID NO: 1 and has
coordinates of r=85.100, .theta. (theta)=38.590, and .PHI.
(phi)=43.143 and a spherical radius of about 1.2 .ANG..
[0164] Functional group F(V)9 forms a favorable .pi.-.pi.
interaction with a phenyl ring of receptor Phe412 of SEQ ID NO: 1
and has coordinates of r=84.418, .theta. (theta)=37.489, and .PHI.
(phi)=46.968 and a spherical radius of about 1.2 .ANG..
[0165] Functional group F(V)10 forms a favorable .pi.-.pi.
interaction with a phenyl ring of receptor Phe412 of SEQ ID NO: 1
and has coordinates of r=82.486, .theta. (theta)=38.027, and .PHI.
(phi)=47.246 and a spherical radius of about 1.5 .ANG.. and
[0166] Functional group F(V)11 forms a favorable hydrophobic
interaction with a side chain of receptors Val417 and Ile448 of SEQ
ID NO: 1 and has coordinates of r=84.438, .theta. (theta)=40.753,
and .PHI. (phi)=48.557 and a spherical radius of about 1.2
.ANG..
[0167] Functional group F11 forms a favorable hydrophobic
interaction with a side chain of receptors Val417 and Ile448 of SEQ
ID NO: 1 and has coordinates of r=84.438, .theta. (theta)=40.753,
and .PHI. (phi)=48.557 and a spherical radius of about 1.2
.ANG..
[0168] A selected candidate compound can substantially align with
at least one of functional groups F(V)1, F(V)2, F(V)3, F(V)4,
F(V)5, F(V)6, F(V)7, F(V)8, F(V)9, F(V)10, and F(V)11. For example,
a selected candidate compound can substantially align with at least
two, at least three, at least four, at least five, at least six, at
least seven, at least eight, at least nine, at least ten, or at
least eleven of functional groups F(V)1, F(V)2, F(V)3, F(V)4,
F(V)5, F(V)6, F(V)7, F(V)8, F(V)9, F(V)10, and F(V)11. Preferably,
a selected candidate compound can substantially align with at least
nine of functional groups F(V)1, F(V)2, F(V)3, F(V)4, F(V)5, F(V)6,
F(V)7, F(V)8, F(V)9, F(V)10, and F(V)11.
[0169] One aspect provides a method for identifying an epidermal
growth factor receptor (EGFR) inhibitor comprising: providing a
pharmacophore comprising any one of Schemes II, III, IV, or V as
input to a 3-dimensional database; comparing a three dimensional
structure of a candidate compound to the three dimensional
structure of the pharmacophore; selecting a candidate compound with
a three dimensional structure that substantially aligns with six or
more functional groups of any one of Schemes II, III, IV, or V;
wherein, similarity between the three-dimensional structure of the
candidate compound and the three-dimensional structure of the
pharmacophore is indicative of an ability of the candidate compound
to inhibit EGFR by substantially maintaining a tethered inactive
configuration of EGFR or substantially preventing stabilization of
the untethered active configuration of EGFR.
[0170] In some embodiments, the method further comprises
determining identity and spatial orientation of at least a portion
of atoms of EGFR associated with stabilizing a tethered
configuration of domain II and domain IV of EGFR in an inactive
conformation; and constructing a pharmacophore, wherein the
pharmacophore comprises a pluralitiy of pharmacophoric features
that approximates the identity and the spatial orientation of the
at least a portion of atoms of EGFR associated with stabilizing a
tethered configuration of domain II and domain IV of EGFR in an
inactive conformation such that the pharmacophore structural
features are complementary to the inactive EGFR configuration.
[0171] In some embodiments, determining identity and spatial
orientation of at least a portion of atoms of EGFR associated with
stabilizing a tethered configuration of domain II and domain IV of
EGFR in an inactive conformation comprises analysis of X-ray
crystallographic data derived from a crystalline form of EGFR in an
inactive, tethered conformation.
[0172] In some embodiments, at least one pharmacophoric feature
approximates identity and spatial orientations of at least a
portion of atoms of domain II of EGFR in a tethered inactive
conformation. In some embodiments, at least one pharmacophoric
feature approximates identity and spatial orientations of at least
a portion of atoms of a cleft region between domain II and domain
IV of EGFR in a tethered inactive conformation.
[0173] In some embodiments, the method further comprises
determining a docking affinity of the candidate molecule for the at
least a portion of atoms of EGFR associated with stabilizing a
tethered configuration of domain II and domain IV of EGFR in an
inactive conformation; wherein docking affinity is quantified by
energy gained upon interaction of the candidate molecule with the
target biomolecule, energy required to attain the docked
conformation relative to the lowest energy conformation, or a
combination thereof.
[0174] Compounds
[0175] Another aspect of the present invention includes small
molecule compounds, identified by the methods described herein.
Compounds described herein can have an anti-proliferative effect
useful in, for example, treating a proliferative disease, disorder,
or condition. Compounds described herein can be useful for the
treatment of diseases, disorders, or conditions related to a target
biomolecule according to which they were identified from. Various
embodiments of compounds described herein can hold multiple domain
proteins in a tethered, inactive state. For example, it is well
known that inhibition of growth factor proteins has a benefit in
treatment of certain conditions in oncology. As another example,
inhibition of EGFR has a benefit in treatment of certain conditions
associated with EGFR, as discussed further below. Compounds
described herein can have an EGFR inhibitory effect useful in, for
example, treating a proliferative disease or disorder associated
with EGFR. Compounds described herein have been demonstrated to be
empirically effective in treating proliferative diseases and
conditions.
[0176] Various compounds, including AD4-1734, AD4-1886, AD4-10381,
and AD4-10381, were identified as EGFR inhibitors through the
pharmacophoric approach described herein (see e.g., Examples 4-5).
Such compounds, and derivatives thereof, have utility as
therapeutic agents for treatment of proliferative diseases or
conditions. For example, compounds described herein can be used as
a therapeutic agent for the treatment of an EGFR-associated
disease, disorder, or condition. Analogs and derivatives of such
compounds are expected to have the same or similar
anti-proliferative effects and utility (see e.g., Examples 6-7).
Identified compounds and analogs and derivatives thereof are
further discussed below.
[0177] While under no obligation to provide an underlying mechanism
and in no way limiting the present invention by doing so, it is
presently thought that at least a portion of activity of compounds
described herein arise from inhibition of EGFR. It is further
contemplated that the presently described compounds may have
additional modes of action in their effectiveness in treating a
proliferative disease, disorder, or condition. Regardless of the
underlying mechanism, compounds described herein have been
demonstrated to be empirically effective in treating proliferative
diseases and conditions.
[0178] The following definitions are provided to better define the
present disclosure. Unless otherwise noted, terms are to be
understood according to conventional usage by those of ordinary
skill in the relevant art.
[0179] The expression "alkyl", unless specifically limited, denotes
a C.sub.1-12 alkyl group, suitably a C.sub.1-6 alkyl group, e.g.
C.sub.1-4 alkyl group. Alkyl groups may be straight chain or
branched. Suitable alkyl groups include, for example, methyl,
ethyl, propyl (e.g. n-propyl and isopropyl), butyl (e.g. n-butyl,
iso-butyl, sec-butyl and tert-butyl), pentyl (e.g. n-pentyl), hexyl
(e.g. n-hexyl), heptyl (e.g. n-heptyl) and octyl (e.g. n-octyl).
The expression "alk", for example in the expressions "alkoxy",
"haloalkyl" and "thioalkyl" should be interpreted in accordance
with the definition of "alkyl". Exemplary alkoxy groups include
methoxy, ethoxy, propoxy (e.g. n-propoxy), butoxy (e.g. n-butoxy),
pentoxy (e.g. n-pentoxy), hexoxy (e.g. n-hexoxy), heptoxy (e.g.
n-heptoxy) and octoxy (e.g. n-octoxy).
[0180] The expression "cycloalkyl", unless specifically limited,
denotes a C.sub.3-10 cycloalkyl group (i.e., 3 to 10 ring carbon
atoms), more suitably a C.sub.3-8 cycloalkyl group, for example, a
C.sub.3-6 cycloalkyl group. Exemplary cycloalkyl groups include
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and
cyclooctyl. A preferred number of ring carbon atoms is three to
six.
[0181] The expression "aryl", unless specifically limited, denotes
a C.sub.6-12 aryl group, suitably a C.sub.6-10 aryl group, more
suitably a C.sub.6-8 aryl group. Aryl groups will contain at least
one aromatic ring (e.g. one, two or three rings). An example of a
typical aryl group with one aromatic ring is phenyl. An example of
a typical aryl group with two aromatic rings is naphthyl.
[0182] The expression "heteroaryl", unless specifically limited,
denotes an aryl residue, wherein one or more (e.g., 1, 2, 3, or 4,
suitably 1, 2 or 3) ring atoms are replaced by heteroatoms selected
from N, S and O, or else a 5-membered aromatic ring containing one
or more (e.g., 1, 2, 3, or 4, suitably 1, 2 or 3) ring atoms
selected from N, S and O. Exemplary monocyclic heteroaryl groups
having one heteroatom include: five membered rings (e.g., pyrrole,
furan, thiophene); and six membered rings (e.g., pyridine, such as
pyridin-2-yl, pyridin-3-yl and pyridin-4-yl). Exemplary monocyclic
heteroaryl groups having two heteroatoms include: five membered
rings (e.g., pyrazole, oxazole, isoxazole, thiazole, isothiazole,
imidazole, such as imidazol-1-yl, imidazol-2-yl imidazol-4-yl); six
membered rings (e.g., pyridazine, pyrimidine, pyrazine). Exemplary
monocyclic heteroaryl groups having three heteroatoms include:
1,2,3-triazole and 1,2,4-triazole. Exemplary monocyclic heteroaryl
groups having four heteroatoms include tetrazole. Exemplary
bicyclic heteroaryl groups include: indole (e.g., indol-6-yl),
benzofuran, benzothiophene, quinoline, isoquinoline, indazole,
benzimidazole, benzthiazole, quinazoline and purine.
[0183] A saturated group is generally understood as having no
double or triple bonds. For example, in a saturated linear
hydrocarbon, each carbon atom is attached to two hydrogen atoms,
except those at the ends of the chain, which bear three hydrogen
atoms. For example, an unsaturated hydrocarbon is generally
understood as a carbon structure containing one or more double or
triple bonds.
[0184] The term "halogen" or "halo" includes fluorine (F), chlorine
(Cl) bromine (Br) or iodine (I).
[0185] The term "amino" refers to the group --NH.sub.2.
[0186] All possible stereoisomers of the claimed compounds are
included in the present disclosure. Where a compound described
herein has at least one chiral center, it may accordingly exist as
enantiomers. Where a compound possess two or more chiral centers it
may additionally exist as diastereomers. It is to be understood
that all such isomers and mixtures thereof are encompassed within
the scope of the present disclosure.
[0187] In view of the close relationship between the free compounds
and the compounds in the form of their salts, whenever a compound
is referred to in this context, a corresponding salt is also
intended, provided such is possible or appropriate under the
circumstances. The pharmaceutically acceptable salt can take a form
in which a basic side chain is protonated with an inorganic or
organic acid. Representative organic or inorganic acids include
hydrochloric, hydrobromic, perchloric, sulfuric, nitric,
phosphoric, acetic, propionic, glycolic, lactic, succinic, maleic,
fumaric, malic, tartaric, citric, benzoic, mandelic,
methanesulfonic, hydroxyethanesulfonic, benzenesulfonic, oxalic,
pamoic, 2-naphthalenesulfonic, p-toluenesulfonic,
cyclohexanesulfamic, salicylic, saccharinic or trifluoroacetic
acid. Alternatively it may take the form in which an acidic side
chain forms a salt with a metal ion (e.g., sodium, potassium ions
and the like) or other positive ion such as ammonium. All
pharmaceutically acceptable acid addition salt forms of the
compounds described herein are intended to be embraced by the scope
of this disclosure.
[0188] Some of the crystalline forms of the compounds may exist in
more than one polymorphic form and as such all forms are intended
to be included in the present disclosure. In addition, some of the
compounds may form solvates with water (i.e., hydrates) or common
organic solvents, and such solvates are also intended to be
encompassed within the scope of this disclosure. The compounds,
including their salts, can also be obtained in the form of their
hydrates, or include other solvents used for their
crystallization.
[0189] The present disclosure further includes within its scope
prodrugs of the compounds described herein. In general, such
prodrugs will be functional derivatives of the compounds which are
readily convertible in vivo into the desired therapeutically active
compound. Thus, in these cases, the methods of treatment of the
present invention, the term "administering" shall encompass the
treatment of the various disorders described with prodrug versions
of one or more of the claimed compounds, but which converts to the
above specified compound in vivo after administration to the
subject.
[0190] As used herein, the term "composition" is intended to
encompass a product comprising a claimed compound(s) in a
therapeutically effective amount, as well as any product which
results, directly or indirectly, from combinations of the claimed
compounds.
[0191] AD4-1734
[0192] AD4-1734 is identified as an inhibitor of epidermal growth
factor binding to its receptor (see e.g., Example 5).
##STR00017##
[0193] As described herein, a pharmacophore model was utilized to
identify small molecules that are AD4-1734-like.
[0194] Type P AD4-1734-Like
[0195] One structure derived from the AD4-1734-like pharmacophore
is "Type P" as follows:
##STR00018##
[0196] In the above structure, R.sup.23 and R.sup.27 of Formula
(13) can be hydrogen.
[0197] R.sup.24 and R.sup.26 of Formula (13) can be independently
selected from represent hydrogen, lower alkyl (one to six carbon,
straight chain, branched, or optionally containing unsaturation),
alkoxy (--OR.sup.10 where R.sup.10 is defined as a lower alkyl
group or cycloalkyl group in the above definition), or halogen (F,
Cl, Br, or I). Preferably, R.sup.24 and R.sup.26 of Formula (13)
are independently selected from hydrogen or alkoxy.
[0198] R.sup.25 of Formula (13) can represent hydrogen, lower
alkyl, cycloalkyl, substituted alkyl, alkoxy or halogen.
Preferably, R.sup.25 of Formula (13) is selected from hydrogen or
alkoxy. Substituted alkyl is defined as a hydrocarbon chain with
1-3 carbons which is substituted with additional functional groups
from the following list: carboxyl defined as --COOR.sup.10,
(R.sup.10 is a lower alkyl group or cycloalkyl group as in the
above definition), aryl, aryloxy (--OAr). Aryl is defined as an
unsubstituted phenyl ring or a phenyl ring substituted at the 2-,
3-, 4-, 5- or 6-position with one or more of the following groups:
lower alkyl defined as C-1 to C-4, straight chain, branched, or
optionally containing unsaturation; cycloalkyl, which is defined as
C-1 to C-6 optionally containing unsaturation; Aryl including
phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms;
Alkoxy (--OR.sup.10 where R.sup.10 is defined as a lower alkyl
group or cycloalkyl group in the above definition);
Trifluoromethyl, Trifluoromethoxy, Difluoromethoxy,
3,4-methylenedioxy, 2,3-methylenedioxy, Nitro or Halogen (F, Cl,
Br, I).
[0199] R.sup.28 of Formula (13) can be selected from hydrogen,
lower alkyl or aryl. Preferably, R.sup.28 of Formula (13) is
selected from hydrogen or aryl. More preferably, R.sup.28 of
Formula (13) is hydrogen. Aryl represents an unsubstituted phenyl
ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or
6-position with one or more of the following groups: lower alkyl
defined as above, cycloalkyl defined as above, or halogen.
[0200] R.sup.29 of Formula (13) can be selected from hydrogen,
lower alkyl, hydroxy, alkoxy or halogen. Lower alkyl, alkoxy, and
halogen are as defined above.
[0201] R.sup.30 of Formula (13) can be selected from hydrogen,
lower alkyl, alkoxy, trifluoromethyl, or halogen. Preferably,
R.sup.30 of Formula (13) is lower alkyl. Lower alkyl, alkoxy, and
halogen are as defined above.
[0202] R.sup.31 of Formula (13) can be selected from hydrogen,
hydroxy, alkoxy, lower alkyl, trifluoromethyl or halogen.
Preferably, R.sup.31 of Formula (13) is selected from hydroxy or
alkoxy. Lower alkyl, alkoxy, and halogen are as defined above.
[0203] R.sup.32 of Formula (13) can be selected from hydrogen,
lower alkyl, hydroxy, trifluoromethyl or halogen. Preferably,
R.sup.32 of Formula (13) is hydrogen. Lower alkyl and halogen are
as defined above.
[0204] In some embodiments, the compound(s) are the enantiomeric
isomers of Formula (13).
[0205] In some embodiments, the compound of Formula (13) is
AD4-10631.
##STR00019##
[0206] In some embodiments, the compound of Formula (13) is
AD4-10174.
##STR00020##
[0207] In some embodiments, the compound(s) of Formula (13)
excludes compound AD4-1734, Formula (12).
[0208] Type Q AD4-1734-Like
[0209] One structure derived from the AD4-1734-like pharmacophore
is "Type Q" as follows:
##STR00021##
[0210] In the above structure, R.sup.33 of Formula (14) can be
selected from lower alkyl, substituted lower alkyl, or aryl.
Preferably R.sup.33 of Formula (14) is aryl. Lower alkyl,
substituted lower alkyl, and aryl are as defined above.
[0211] R.sup.34 of Formula (14) can be selected from hydrogen.
[0212] R.sup.35 of Formula (14) can be selected from hydrogen,
hydroxy, alkoxy, lower alkyl, trifluoromethyl or halogen.
Preferably R.sup.35 of Formula (14) is lower alkyl. Lower alkyl,
alkoxy, and halogen are as defined above.
[0213] R.sup.36 of Formula (14) can be selected from hydrogen,
lower alkyl, hydroxy, alkoxy, trifluoromethyl or halogen.
Preferably, R.sup.36 of Formula (14) is selected from hydroxy or
alkoxy. Lower alkyl and halogen are as defined above.
[0214] R.sup.37 of Formula (14) can be selected from hydrogen.
[0215] In some embodiments, the compound(s) are the enantiomeric
isomers of Formula (14).
[0216] In some embodiments, the compound of Formula (14) is
AD4-10188.
##STR00022##
[0217] In some embodiments, the compound(s) of Formula (14)
excludes compound AD4-1734, Formula (12).
[0218] Type R AD4-1734-Like
[0219] One structure derived from the AD4-1734-like pharmacophore
is "Type R" as follows:
##STR00023##
[0220] In the above structure, X.sup.2 and X.sup.3 of Formula (15)
can be independently selected from carbon, oxygen, nitrogen or
sulfur atom. Preferably, X.sup.2 and X.sup.3 of Formula (15) are
independently selected from oxygen or carbon. In Formula (15), n
can be one or two resulting in a 5 or 6 membered ring. Preferably,
n of Formula (15) is n=1.
[0221] R.sup.38 of Formula (15) can be selected from hydrogen.
[0222] R.sup.39 of Formula (15) can be as defined for R.sup.24.
[0223] R.sup.40 of Formula (15) can be as defined for R.sup.23.
[0224] R.sup.41 of Formula (15) can be selected from hydrogen,
lower alkyl or aryl.
[0225] R.sup.42 of Formula (15) can be selected from hydrogen,
lower alkyl, hydroxy, alkoxy, or halogen. Preferably, R.sup.42 of
Formula (15) is hydrogen. Lower alkyl, alkoxy and halogen are as
defined above.
[0226] R.sup.43 of Formula (15) can be selected from hydrogen,
lower alkyl, alkoxy, trifluoromethyl, or halogen. Preferably
R.sup.43 of Formula (15) is lower alkyl. Lower alkyl, alkoxy and
halogen are as defined above.
[0227] R.sup.44 of Formula (15) can be selected from hydrogen,
hydroxy, alkoxy, lower alkyl, trifluoromethyl or halogen.
Preferably, R.sup.44 of Formula (15) is selected from hydroxy or
alkoxy. Lower alkyl, alkoxy and halogen are as defined above.
[0228] R.sup.45 of Formula (15) can be selected from hydrogen,
lower alkyl, hydroxy, trifluoromethyl, or halogen. Preferably,
R.sup.45 of Formula (15) is hydrogen. Lower alkyl and halogen are
as defined above.
[0229] In some embodiments, the compound(s) are the enantiomeric
isomers of Formula (15).
[0230] In some embodiments, the compound of Formula (15) is
AD4-10633.
##STR00024##
[0231] In some embodiments, the compound(s) of Formula (15)
excludes compound AD4-1734, Formula (12).
[0232] Type S AD4-1734-Like
[0233] One structure derived from the AD4-1734-like pharmacophore
is "Type S" as follows:
##STR00025##
[0234] In the above structure, X.sup.4 and X.sup.5 of Formula (16)
can be independently selected from carbon, oxygen, nitrogen or
sulfur atom. Preferably, X.sup.4 and X.sup.5 of Formula (16) are
independently selected from oxygen or carbon. In Formula (16), n
can be one or two resulting in a 5 or 6 membered ring. Preferably,
n of Formula (16) is n=1.
[0235] R.sup.46 and R.sup.50 of Formula (16) can be independently
selected from hydrogen.
[0236] R.sup.47 and R.sup.49 of Formula (16) can be independently
selected from hydrogen, lower alkyl, alkoxy or halogen. Lower
alkyl, alkoxy and halogen are as defined above.
[0237] R.sup.48 of Formula (16) can be selected from hydrogen,
lower alkyl, cycloalkyl, substituted alkyl, alkoxy, or halogen.
Preferably, R.sup.48 of Formula (16) is selected from hydrogen or
lower alkyl. Lower alkyl, cycloalkyl, substituted alkyl, and alkoxy
are as defined above. Halogen is F, Cl, Br, or I.
[0238] Alternatively, R.sup.47 and R.sup.48 of Formula (16) can be
joined to form a ring as described for Formula (15). Preferably,
R.sup.47 and R.sup.48 of Formula (16) are joined to form a ring a
5-membered ring.
[0239] R.sup.51 of Formula (16) can be selected from hydrogen,
lower alkyl, or aryl. Preferably, R.sup.51 of Formula (16) is
selected from hydrogen or aryl. Lower alkyl or aryl are as defined
above.
[0240] R.sup.52 of Formula (16) can be selected from hydrogen,
lower alkyl, hydroxy, alkoxy or halogen. Preferably, R.sup.52 of
Formula (16) is hydrogen. Lower alkyl, alkoxy and halogen are as
defined above.
[0241] R.sup.53 of Formula (16) can be selected from hydrogen,
lower alkyl, hydroxy, trifluoromethyl or halogen. Preferably,
R.sup.53 of Formula (16) is hydrogen. Lower alkyl and halogen are
as defined above.
[0242] In some embodiments, the compound(s) are the enantiomeric
isomers of Formula (16).
[0243] In some embodiments, the compound of Formula (16) is
AD4-10639.
##STR00026##
[0244] In some embodiments, the compound of Formula (16) is
AD4-10179.
##STR00027##
[0245] In some embodiments, the compound(s) of Formula (16)
excludes compound AD4-1734, Formula (12).
[0246] AD4-1886
[0247] AD4-1886 is identified as an inhibitor of epidermal growth
factor binding to its receptor (see e.g., Example 4).
##STR00028##
[0248] As described herein, a pharmacophore model was utilized to
identify small molecules that are AD4-1886-like.
[0249] Type J AD4-1886-Like
[0250] One structure derived from the AD4-1886-like pharmacophore
is "Type J" as follows:
##STR00029##
[0251] In the above structure, R.sup.54 of Formula (18) is a 5 or 6
membered heterocyclic containing from 1 to 4 N, O, or S atoms or
any combination of those atoms with carbon atoms to form a
heterocyclic aromatic ring which is optionally substituted with
from 1 to 3 of the following groups: lower alkyl, cycloalkyl,
alkoxy (--OR.sup.10 where R.sup.10 is defined as a lower alkyl
group or cycloalkyl group in the above definition),
trifluoromethyl, trifluoromethoxy, substituted amino, nitro or
halogen (F, Cl, Br, or I). Lower Alkyl is defined as one to six
carbon, straight chain, branched, or optionally containing
unsaturation. Cycloalkyl is defined as C-1 to C-6 optionally
containing unsaturation. As preferred examples, R.sup.54 of Formula
(18) can represent: 4,6-Dimethyl-2-pyrimidine,
2,6-Dimethoxy-4-pyrimidine, 6-Methoxy-4-pyrimidine,
5-Ethyl-2-(1,3,4-Thiadiazole), 5-Methyl-3-Isoxazole,
3-Methoxy-6-pyridazinamine, 2-Thiazole or 2-Methoxy-3-pyrazine.
[0252] R.sup.55 of Formula (18) can be selected from hydrogen or
lower alkyl. Preferably, R.sup.55 of Formula (18) is selected from
hydrogen or methyl. Lower alkyl is as defined above.
[0253] R.sup.56, R.sup.57, R.sup.58, and R.sup.59 of Formula (18)
can be independently selected from hydrogen.
[0254] R.sup.60 of Formula (18) can be selected from lower alkyl,
cycloalkyl, or substituted alkyl. Lower alkyl and cycloalkyl are as
defined above. Substituted alkyl is defined as a hydrocarbon chain
with 1-3 carbons which is substituted with additional functional
groups from the following list: carboxyl defined as --COOR.sup.10
(R.sup.10 is a lower alkyl group or cycloalkyl group as in the
above definition), aryl, aryloxy (--OAr) where aryl is defined as
an unsubstituted phenyl ring or a phenyl ring substituted at the
2-, 3-, 4-, 5- or 6-position with one or more of the following
groups: lower alkyl defined as C-1 to C-4, straight chain,
branched, or optionally containing unsaturation, cycloalkyl defined
as C-1 to C-6 optionally containing unsaturation, aryl including
phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms,
alkoxy (--OR.sup.10 where R.sup.10 is defined as a lower alkyl
group or cycloalkyl group in the above definition),
Trifluoromethyl, Trifluoromethoxy, Difluoromethoxy,
3,4-methylenedioxy, 2,3-methylenedioxy, Nitro or Halogen (F, Cl,
Br, I).
[0255] In some embodiments, the compound(s) are the enantiomeric
isomers of Formula (18).
[0256] In some embodiments, the compound of Formula (18) is
AD4-12158.
##STR00030##
[0257] In some embodiments, the compound of Formula (18) is
AD4-12267.
##STR00031##
[0258] In some embodiments, the compound of Formula (18) is
AD4-11384.
##STR00032##
[0259] In some embodiments, the compound(s) of Formula (18)
excludes compound AD4-1886, Formula (17).
[0260] Type K AD4-1886-Like
[0261] One structure derived from the AD4-1886-like pharmacophore
is "Type K" as follows:
##STR00033##
[0262] In the above structure, R.sup.61, R.sup.62, and R.sup.64 of
Formula (19) can be independently selected from hydrogen or lower
alkyl. Lower alkyl is as defined above.
[0263] R.sup.63 and R.sup.65 of Formula (19) can be selected from
hydrogen, lower alkyl or alkoxy. Lower alkyl and alkoxy are as
defined above.
[0264] R.sup.66 of Formula (19) can be selected from hydrogen or
lower alkyl. Lower alkyl is as defined above.
[0265] R.sup.67, R.sup.68, R.sup.69, and R.sup.70 of Formula (19)
can be independently selected from hydrogen.
[0266] R.sup.71 of Formula (19) can be selected from lower alkyl,
cycloalkyl, aryl, substituted alkyl, or heteraryl. Lower alkyl,
cycloalkyl, aryl, and substituted alkyl are as defined above.
Hereoaryl is defined as a heterocyclic ring containing from 1 to 4
N, O, or S atoms which has optional substitution with one or more
of the following functional groups: lower alkyl defined as C-1 to
C-4, straight chain, branched, or optionally containing
unsaturation, cycloalkyl defined as C-1 to C-6 optionally
containing unsaturation, aryl including phenyl or heteroaryl
containing from 1 to 4 N, O, or S atoms, alkoxy (--OR.sup.10 where
R.sup.10 is defined as a lower alkyl group or cycloalkyl group in
the above definition), trifluoromethyl, trifluoromethoxy, nitro or
halogen (F, Cl, Br, I).
[0267] In some embodiments, the compound(s) are the enantiomeric
isomers of Formula (19).
[0268] In some embodiments, the compound of Formula (19) is
AD4-11409.
##STR00034##
[0269] In some embodiments, the compound(s) of Formula (19)
excludes compound AD4-1886, Formula (17).
[0270] Type L AD4-1886-Like
[0271] One structure derived from the AD4-1886-like pharmacophore
is "Type L" as follows:
##STR00035##
[0272] In the above structure, R.sup.72 of Formula (20) can be
selected from Hydrogen or Lower Alkyl. Lower alkyl is as defined
above.
[0273] R.sup.73 of Formula (20) can be selected from lower alkyl,
cycloalkyl, substituted alkyl, or acyl. Lower alkyl, cycloalkyl,
and substituted alkyl are as defined above. Acyl represents a
substituted ketone --COR.sup.10 where R.sup.10 is defined as a
lower alkyl group or cycloalkyl group in the above definition.
[0274] R.sup.74, R.sup.75, R.sup.76, and R.sup.77 of Formula (20)
can be independently selected from hydrogen.
[0275] R.sup.78 of Formula (20) can be selected from lower alkyl,
cycloalkyl, and substituted alkyl. Lower alkyl, cycloalkyl, and
substituted alkyl are as defined above.
[0276] In some embodiments, the compound(s) are the enantiomeric
isomers of Formula (20).
[0277] In some embodiments, the compound of Formula (20) is
AD4-10608.
##STR00036##
[0278] In some embodiments, the compound of Formula (20) is
AD4-10609.
##STR00037##
[0279] In some embodiments, the compound(s) of Formula (20)
excludes compound AD4-1886, Formula (17).
[0280] Type M AD4-1886-Like
[0281] One structure derived from the AD4-1886-like pharmacophore
is "Type M" as follows:
##STR00038##
[0282] In the above structure, n of Formula (21) can be 1 or 2,
which represents one or two methylene groups resulting in either a
five or six membered fused ring system.
[0283] R.sup.79, R.sup.81, R.sup.82, R.sup.83, R.sup.84, R.sup.85,
and R.sup.86 of Formula (21) can be independently selected from
hydrogen.
[0284] R.sup.80 of Formula (21) can be selected from hydrogen,
lower alkyl, alkoxy, halogen, or trifluoromethyl. Lower alkyl and
alkoxy are as defined above. Halogen is defined as F, Cl, Br, or
I.
[0285] R.sup.87 of Formula (21) can be selected from lower alkyl,
cycloalkyl, substitute alkyl, or aryl. Lower alkyl, cycloalkyl,
substitute alkyl, and aryl are as defined above.
[0286] In some embodiments, the compound(s) are the enantiomeric
isomers of Formula (21).
[0287] In some embodiments, the compound of Formula (21) is
AD4-10626.
##STR00039##
[0288] In some embodiments, the compound(s) of Formula (21)
excludes compound AD4-1886, Formula (17).
[0289] Type N AD4-1886-Like
[0290] One structure derived from the AD4-1886-like pharmacophore
is "Type N" as follows:
##STR00040##
[0291] In the above structure, n of Formula (22) can be 3, 4 or 5,
which represents 3, 4, or 5 methylene groups resulting in either a
five, six, or seven membered ring (pyrrolidine, piperidine, or
hexahydro-1H-azepine).
[0292] R.sup.88 of Formula (22) can be selected from hydrogen or
lower alkyl. Lower alkyl is as defined above.
[0293] R.sup.89, R.sup.90, R.sup.91, R.sup.83, and R.sup.92 of
Formula (22) can be independently selected from hydrogen.
[0294] R.sup.93 of Formula (22) can be selected from lower alkyl,
cycloalkyl, substituted alkyl, or aryl. Lower alkyl, cycloalkyl,
substituted alkyl, and aryl are as defined above.
[0295] In some embodiments, the compound(s) are the enantiomeric
isomers of Formula (22).
[0296] In some embodiments, the compound of Formula (22) is
AD4-11970.
##STR00041##
[0297] In some embodiments, the compound of Formula (22) is
AD4-12107.
##STR00042##
[0298] In some embodiments, the compound(s) of Formula (22)
excludes compound AD4-1886, Formula (17).
[0299] Type O AD4-1886-Like
[0300] One structure derived from the AD4-1886-like pharmacophore
is "Type O" as follows:
##STR00043##
[0301] In the above structure, R.sup.94 of Formula (23) is a 5 or 6
membered heterocyclic containing from 1 to 4 N, O, or S atoms or
any combination of those atoms with carbon atoms to form a
heterocyclic aromatic ring which is optionally substituted with
from 1 to 3 of the following groups: lower alkyl, cycloalkyl,
alkoxy (--OR.sup.10 where R.sup.10 is defined as a lower alkyl
group or cycloalkyl group in the above definition),
trifluoromethyl, trifluoromethoxy, substituted amino, nitro or
halogen (F, Cl, Br, or I). Lower alkyl is defined as one to six
carbon, straight chain, branched, or optionally containing
unsaturation. Cycloalkyl is defined as C-1 to C-6 optionally
containing unsaturation.
[0302] R.sup.95 of Formula (23) can be selected from hydrogen or
lower alkyl. Lower alkyl is as defined above.
[0303] R.sup.96, R.sup.97, R.sup.98, and R.sup.99 of Formula (23)
can be independently selected from hydrogen.
[0304] R.sup.100 of Formula (23) can be selected from aryl or
heteroaryl. Aryl and heteroaryl are as defined above.
[0305] In some embodiments, the compound of Formula (23) is
AD4-11883.
##STR00044##
[0306] In some embodiments, the compound of Formula (23) is
AD4-11638.
##STR00045##
[0307] In some embodiments, the compound of Formula (23) is
AD4-11645.
##STR00046##
[0308] In some embodiments, the compound(s) of Formula (23)
excludes compound AD4-1886, Formula (17).
[0309] AD4-10381
[0310] AD4-10381 is identified as an inhibitor of epidermal growth
factor binding to its receptor (see e.g., Example 5).
##STR00047##
[0311] As described herein, a pharmacophore model was utilized to
identify small molecules that are AD4-10381-like.
[0312] Type D AD4-10381-Like
[0313] One structure derived from the AD4-10381-like pharmacophore
is "Type D" as follows:
##STR00048##
[0314] In the above structure, R.sup.101 of Formula (25) can be
selected from lower alkyl, cycloalkyl or aryl. Preferably,
R.sup.101 of Formula (25) is selected from methyl or phenyl. Lower
alkyl is defined as one to six carbon, straight chain, branched, or
optionally containing unsaturation. Cycloalkyl is defined as three
to six membered carbocyclic ring optionally containing
unsaturation. Aryl represents an unsubstituted phenyl ring or a
phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with
one or more of the following groups: lower alkyl defined as above,
cycloalkyl defined as above, or heteroaryl containing from 1 to 4
N, O, or S atoms.
[0315] R.sup.102 of Formula (25) can be selected from hydrogen.
[0316] R.sup.103 of Formula (25) can be selected from hydrogen,
lower alkyl, cycloalkyl, alkoxy, or halogen. Preferably, R.sup.103
of Formula (25) is selected from methyl, ethyl, chloro, methoxy, or
ethoxy. Lower alkyl is defined as above. Cycloalkyl is defined as
above. Alkoxy is defined as --OR.sup.10 where R.sup.10 is defined
as a lower alkyl group or cycloalkyl group in the above
definition.
[0317] R.sup.104 of Formula (25) can be selected from hydrogen,
lower alkyl, cycloalkyl, or alkoxy. Preferably, R.sup.104 of
Formula (25) is selected from hydrogen, methyl, or methoxy. Lower
alkyl is defined as above. Cycloalkyl is defined as above. Alkoxy
is defined as above.
[0318] R.sup.105 of Formula (25) can be selected from hydrogen,
lower alkyl, cycloalkyl, or alkoxy. Preferably, R.sup.105 of
Formula (25) is selected from hydrogen, methyl, methoxy, or ethoxy.
Lower alkyl is defined as above. Cycloalkyl is defined as above.
Alkoxy is defined as above.
[0319] R.sup.106 of Formula (25) can be selected from hydrogen,
lower alkyl, aryl, or substituted alkyl. Preferably, R.sup.106 of
Formula (25) is selected from methyl or phenyl. Lower alkyl is
defined as above. Substituted alkyl is defined as lower alkyl group
with substitution of additional groups such as an aryl thioether,
--S--Ar', where Ar' is defined as phenyl, substituted phenyl or an
unsubstituted heteroaryl five or six membered ring containing from
1 to 4 N, O, or S atoms, or a heteroaryl five or six membered ring
containing from 1 to 4 N, O, or S atoms which has one or more
optional substitution with the substituent defined as one or more
of the following groups: lower alkyl defined as above, cycloalkyl
defined as above, unsaturation, bicyclic heterocycles such as
benzimidazoles, benzoxazoles, benzthiazoles, or benzopyrazoles.
Aryl is defined as an unsubstituted phenyl ring or a phenyl ring
substituted at the 2-, 3-, 4-, 5- or 6-position with one or more of
the following groups: lower alkyl defined as C-1 to C-4, straight
chain, branched, or optionally containing unsaturation, Cycloalkyl
defined as C-1 to C-6 optionally containing unsaturation, aryl
including phenyl or heteroaryl containing from 1 to 4 N, O, or S
atoms, Alkoxy (--OR.sup.10 where R.sup.10 is defined as a lower
alkyl group or cycloalkyl group in the above definition),
trifluoromethyl, trifluoromethoxy, difluoromethoxy,
3,4-methylenedioxy, 2,3-methylenedioxy, nitro or halogen (F, Cl,
Br, I).
[0320] R.sup.107 of Formula (25) can be selected from hydrogen,
lower alkyl, cycloalkyl, aryl, or substituted alkyl. Preferably,
R.sup.107 of Formula (25) is methyl. Lower alkyl is defined as
above. Cycloalkyl is defined as above. Aryl is defined as above.
Substituted alkyl is defined as above.
[0321] In some embodiments, the compound(s) are the enantiomeric
isomers of Formula (25).
[0322] In some embodiments, the compound(s) of Formula (25)
excludes compound AD4-10381, Formula (24).
[0323] In some embodiments, the compound of Formula (25) is
AD4-11511.
##STR00049##
[0324] In some embodiments, the compound of Formula (25) is
AD4-12632.
##STR00050##
[0325] In some embodiments, the compound of Formula (25) is
AD4-10381.
##STR00051##
[0326] In some embodiments, the compound of Formula (25) excludes
AD4-10381.
[0327] Type E AD4-10381-Like
[0328] One structure derived from the AD4-10381-like pharmacophore
is "Type E" as follows:
##STR00052##
[0329] In the above structure, R.sup.108 of Formula (27) can be
selected from lower alkyl, substituted alkyl, cycloalkyl, aryl,
amino, or hydroxyl. Preferably, R.sup.108 of Formula (27) is
selected from methyl, phenyl, or hydroxyl. Lower alkyl is defined
as above. Substituted alkyl is defined as above. Cycloalkyl is
defined as above. Aryl is defined as above.
[0330] R.sup.109 of Formula (27) can be selected from hydrogen.
[0331] R.sup.110 of Formula (27) can be selected from hydrogen,
lower alkyl, substituted alkyl, cycloalkyl, alkoxy, or halogen.
Preferably, R.sup.110 of Formula (27) is selected from hydrogen,
methyl, methoxy, or chloro. Lower alkyl is defined as above.
Substituted alkyl is defined as above. Alkoxy is defined as above.
Halogen is defined as F, Cl, Br, or I.
[0332] R.sup.111 of Formula (27) can be selected from hydrogen,
lower alkyl, substituted alkyl, cycloalkyl, alkoxy, or halogen.
Preferably, R.sup.111 of Formula (27) is selected from hydrogen,
methyl, methoxy, or chloro. Lower alkyl is defined as above.
Substituted alkyl is defined as above. Cycloalkyl is defined as
above. Alkoxy is defined as above. Halogen is defined as above.
[0333] R.sup.112 of Formula (27) can be selected from hydrogen or
lower alkyl. Preferably, R.sup.112 of Formula (27) is methyl. Lower
alkyl is defined as above.
[0334] R.sup.113 of Formula (27) can be selected from lower alkyl,
trifluoromethyl, aryl, or amino. Preferably, R.sup.113 of Formula
(27) is selected from methyl, phenyl, or amino. Lower alkyl is
defined as above. Aryl is defined as above.
[0335] R.sup.114 of Formula (27) can be selected from hydrogen,
acyl or carboxyalkyl. Acyl represents a substituted ketone
--COR.sup.10 where R.sup.10 is defined as a lower alkyl group or
cycloalkyl group in the above definition. Carboxyalkyl is defined
as a substituted ester group COOR.sup.10 where R.sup.10 is defined
as a lower alkyl group or cycloalkyl group in the above
definition.
[0336] R.sup.115 of Formula (27) can be selected from lower alkyl,
trifluoromethyl, or aryl. Preferably, R.sup.115 of Formula (27) is
methyl, phenyl or trifluoromethyl. Lower alkyl is defined as above.
Aryl is defined as above.
[0337] In some embodiments, the compound(s) are the enantiomeric
isomers of Formula (27).
[0338] In some embodiments, the compound of Formula (27) is
AD4-12681.
##STR00053##
[0339] In some embodiments, the compound of Formula (27) is
AD4-12679.
##STR00054##
[0340] In some embodiments, the compound(s) of Formula (27)
excludes compound AD4-10381, Formula (24).
[0341] Type F AD4-10381-Like
[0342] One structure derived from the AD4-10381-like pharmacophore
is "Type F" as follows:
##STR00055##
[0343] In the above structure, n of Formula (28) can be 1 or 2,
which represents one or two methylene groups resulting in either a
five or six membered carbocyclic ring. The six membered ring
contains optional unsaturation resulting in a fused benzo-ring
system as shown below (see Formula (29)).
##STR00056##
[0344] R.sup.116 of Formula (28) or Formula (29) can be selected
from lower alkyl or aryl. Preferably, R.sup.116 of Formula (28) or
Formula (29) are independently methyl or phenyl. Lower alkyl is
defined as above. Aryl is defined as above.
[0345] R.sup.117 of Formula (28) or Formula (29) can be selected
from hydrogen.
[0346] R.sup.118 of Formula (28) or Formula (29) can be selected
from hydrogen, lower alkyl, alkoxy, or halogen. Lower alkyl is
defined as above. Alkoxy is defined as above. Halogen is defined as
above.
[0347] R.sup.119 of Formula (28) or Formula (29) can be selected
from hydrogen, lower alkyl, or aryl. Lower alkyl is defined as
above. Aryl is defined as above.
[0348] R.sup.120 of Formula (28) or Formula (29) can be selected
from hydrogen, lower alkyl, or alkoxy. Lower alkyl is defined as
above. Alkoxy is defined as above.
[0349] R.sup.121 of Formula (28) or Formula (29) can be selected
from hydrogen or aryl. Aryl is defined as above.
[0350] In some embodiments, the compound(s) are the enantiomeric
isomers of Formula (28) or Formula (29).
[0351] In some embodiments, the compound of Formula (28) is
AD4-11436.
##STR00057##
[0352] In some embodiments, the compound(s) of Formula (28) and
Formula (29) excludes compound AD4-10381, Formula (24).
[0353] AD4-11091
[0354] AD4-11091 is identified as an inhibitor of epidermal growth
factor binding to its receptor (see e.g., Example 4).
##STR00058##
[0355] As described herein, a pharmacophore model was utilized to
identify small molecules that are AD4-11091-like.
[0356] Type H AD4-11091-Like
[0357] One structure derived from the AD4-10381-like pharmacophore
is "Type H" as follows:
##STR00059##
[0358] In the above structure, R.sup.122 and R.sup.126 of Formula
(32) can be independently selected from hydrogen, alkoxy,
trifluoromethyl or halogen. Preferably, R.sup.122 and R.sup.126 of
Formula (32) are independently selected from hydrogen,
trifluoromethyl, methoxy, or chloro. More preferably, one or both
of R.sup.122 and R.sup.126 of Formula (32) is hydrogen. Alkoxy and
halogen are as defined above.
[0359] R.sup.123 and R.sup.125 of Formula (32) can be independently
selected from hydrogen, trifluoromethyl, halogen, lower alkyl, acyl
or alkoxy. Preferably, R.sup.123 and R.sup.125 of Formula (32)
independently selected from halogen or trifluoromethyl. Halogen,
lower alkyl, acyl and alkoxy are as defined above.
[0360] R.sup.124 of Formula (32) can be selected from hydrogen,
lower alkyl, alkoxy, acyl or halogen. Preferably, R.sup.124 of
Formula (32) is lower alkyl or halogen. Lower alkyl, alkoxy, acyl
and halogen are as defined above.
[0361] R.sup.127 and R.sup.131 of Formula (32) can be independently
selected from hydrogen, lower alkyl, or alkoxy. Preferably,
R.sup.127 and R.sup.131 of Formula (32) are independently selected
from hydrogen, methyl, or methoxy. Lower alkyl and alkoxy are as
defined above.
[0362] R.sup.128 and R.sup.130 of Formula (32) can be independently
selected from hydrogen or halogen. Preferably, R.sup.128 and
R.sup.130 of Formula (32) are independently selected from hydrogen
or chloro. Halogen is as defined above.
[0363] R.sup.129 of Formula (32) can be selected from hydrogen,
halogen, alkoxy, or lower alkyl. Preferably, R.sup.129 of Formula
(32) is selected from hydrogen, chloro, methoxy, or methyl.
Halogen, alkoxy, and lower alkyl are as defined above.
[0364] R.sup.132 of Formula (32) can be selected from hydrogen or
lower alkyl. Preferably, R.sup.132 of Formula (32) is selected from
hydrogen, methyl, or ethyl. Lower alkyl is as defined above.
[0365] In some embodiments, the compound(s) are the enantiomeric
isomers of Formula (32).
[0366] In some embodiments, the compound of Formula (32) is
AD4-12509.
##STR00060##
[0367] In some embodiments, the compound of Formula (32) is
AD4-12522.
##STR00061##
[0368] In some embodiments, the compound of Formula (32) is
AD4-12528.
##STR00062##
[0369] In some embodiments, the compound(s) of Formula (32)
excludes compound AD4-11091, Formula (31).
[0370] Type I AD4-11091-Like
[0371] One structure derived from the AD4-10381-like pharmacophore
is "Type I" as follows:
##STR00063##
[0372] In the above structure, n of Formula (33) can be 1 or 2,
which represents one or two methylene groups resulting in either a
five or six membered carbocyclic ring.
[0373] R.sup.133 and R.sup.137 of Formula (33) can be independently
selected from hydrogen, lower alkyl or halogen. Preferably,
R.sup.133 and R.sup.137 of Formula (33) are independently selected
from hydrogen, methyl or chloro. Lower alkyl and halogen are as
defined above.
[0374] R.sup.134 and R.sup.136 of Formula (33) can be independently
selected from hydrogen, trifluoromethyl, or halogen. Preferably,
R.sup.134 and R.sup.136 of Formula (33) are independently selected
hydrogen, trifluoromethyl, or chloro. Halogen is as defined
above.
[0375] R.sup.135 of Formula (33) can be selected from hydrogen or
halogen. Preferably, R.sup.135 of Formula (33) is chloro. Halogen
is as defined above.
[0376] R.sup.138, R.sup.140, and R.sup.141 of Formula (33) can be
independently selected from hydrogen.
[0377] R.sup.139 of Formula (33) can be selected from hydrogen or
halogen. Preferably, R.sup.139 of Formula (33) is selected from
fluoro or hydrogen. Halogen is as defined above.
[0378] R.sup.142 of Formula (33) can be selected from hydrogen or
lower alkyl. Preferably, R.sup.142 of Formula (33) is selected from
hydrogen or methyl. Lower alkyl is as defined above.
[0379] In some embodiments, the compound(s) are the enantiomeric
isomers of Formula (33).
[0380] In some embodiments, the compound of Formula (33) is
AD4-12846.
##STR00064##
[0381] In some embodiments, the compound of Formula (33) is
AD4-12239.
##STR00065##
[0382] In some embodiments, the compound(s) of Formula (33)
excludes compound AD4-11091, Formula (31).
[0383] Synthesis
[0384] One aspect provides methods for the synthesis of compounds
described herein (see Example 9).
[0385] An AD4-1734-like compound can be synthesized as follows:
##STR00066##
[0386] An AD4-1886-like compound can be synthesized as follows:
##STR00067##
[0387] An AD4-11091-like compound can be synthesized as
follows:
##STR00068##
[0388] An AD4-11511-like compound can be synthesized as
follows:
##STR00069##
[0389] Further methodology for synthesis of AD4-12632, which is an
AD4-11511-like compound, is provided in Example 9.
[0390] The above reactions can include any condition or combination
of conditions disclosed in Example 9.
[0391] Pharmaceutical Formulations
[0392] Embodiments of the compositions of the invention include
pharmaceutical formulations of the various compounds described
herein. A compound described herein can be employed in pure form
or, where such forms exist, in pharmaceutically acceptable salt
form and with or without a pharmaceutically acceptable excipient.
The compounds described herein can be formulated by any
conventional manner using one or more pharmaceutically acceptable
carriers and/or excipients as described in, for example,
Remington's Pharmaceutical Sciences (A. R. Gennaro, Ed.), 21st
edition, ISBN: 0781746736 (2005). Such formulations will contain a
therapeutically effective amount of the agent, preferably in
purified form, together with a suitable amount of carrier so as to
provide the form for proper administration to the subject. The
formulation should suit the mode of administration. The agents of
use with the current invention can be formulated by known methods
for administration to a subject using several routes which include,
but are not limited to, parenteral, pulmonary, oral, topical,
intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, intranasal, epidural, ophthalmic, buccal, and rectal.
The individual agents may also be administered in combination with
one or more additional agents of the present invention and/or
together with other biologically active or biologically inert
agents. Such biologically active or inert agents may be in fluid or
mechanical communication with the agent(s) or attached to the
agent(s) by ionic, covalent, Van der Waals, hydrophobic,
hydrophillic or other physical forces.
[0393] Controlled-release (or sustained-release) preparations may
be formulated to extend the activity of the agent and reduce dosage
frequency. Controlled-release preparations can also be used to
effect the time of onset of action or other characteristics, such
as blood levels of the agent, and consequently affect the
occurrence of side effects.
[0394] Combination with Kinase Inhibitors
[0395] Compounds described herein can be used with, or formulated
with, known therapeutic compounds. Combination therapy is
understood as a therapeutic regimen comprising, e.g., an
anti-proliferative compound described herein and a second agent. An
anti-proliferative compound and a second agent can be formulated
for separate administration or may be formulated for administration
together.
[0396] Compounds described herein can be combined with another
anti-prolifative compound, such as the EGFR kinase inhibitors,
Tykerb, Iressa, and Tarceva, or Erbitux, a humanized monoclonal
antibody to the EGF receptor, to produce a greater therapeutic
effect than either agent alone. As shown herein, when AD4 compounds
were evaluated in a cell proliferation assay with Tykerb, Iressa,
Tarceva or Erbitux, the effect of the combination of agents to
inhibit cell proliferation was greater than the effect of any of
the agents alone (see e.g., Example 8). Specifically, compounds
described herein were evaluated with Tykerb, Iressa, Tarceva or
Erbitux at a fixed concentration ratio, which was ascertained from
the results of dose-response curves of each agent alone.
[0397] An compound described herein, such as an EGFR inhibitor, can
be used with, or formulated and used with a second agent that
inhibits vascularization of a tumor. Vascularization of a solid
tumor generally refers to formation of blood vessels in a solid
tumor. An agent that inhibits the vascularization of a tumor can
inhibit vessel initiation, development, or maintenance leading to,
for example, the reduction in the number or density of vessels in a
tumor.
[0398] A compound described herein can be used with, or formulated
and used with a second agent that modifies, for example increasing,
permeability of a solid tumor. Permeability of a solid tumor
generally refers to the permeability of a solid tumor to a
therapeutic. A solid tumor may be said to be permeable to a
therapeutic if the therapeutic is able to reach cells at the center
of the tumor.
[0399] A compound described herein can be used with, or formulated
and used with, a chemotherapeutic second agent. A chemotherapeutic
agent refers to a molecule or composition used to treat a
malignancy. Such agents can be used in combination with a compound
described herein or with a combination therapy described herein.
Chemotherapeutic agents include agents that can be conjugated to a
compound described herein or can be used in combination with the
combination therapy in unconjugated form.
[0400] A compound described herein can be used with, or formulated
and used with a second agent that is a biological agent. A
biological agent, also called a biologic, are generally understood
as a product of a biological system, e.g., an organism, cell, or
recombinant system. Examples of such biologic agents include, but
are not limited to, nucleic acid molecules (e.g., antisense nucleic
acid molecules), interferons, interleukins, colony-stimulating
factors, antibodies (e.g., monoclonal antibodies), and
cytokines.
[0401] A compound described herein can be used or formulated with
an EGFR inhibitor approved for treatment of an EGFR-related
condition or disorder. For example, compounds described herein can
be used with or formulated with one or more of Tykerb, Iressa,
Tarceva, or Erbitux. Tykerb, Iressa, and Tarceva are kinase
inhibitors that block EGFR tyrosine kinase activity. Erbitux is a
humanized monoclonal antibody that binds to an extracellular
epitope on EGFR. Erbitux blocks activation of the receptor by
preventing both ligand binding and receptor dimerization. In
various embodiments, a compound described herein can lock EGFR into
a dimerization incompetent conformation. Thus, compounds described
herein and known EGFR inhibitors, such as those described above,
can act in a complementary or synergistic fashion.
[0402] A compound described herein, such as AD4-1734-like
compounds, AD4-1886-like compounds, AD4-10381-like compounds, or
AD4-11091-like compounds, can be used or formulated with Tykerb.
Compounds described herein, such as AD4-1734-like compounds,
AD4-1886-like compounds, AD4-10381-like compounds, or
AD4-11091-like compounds, can be used or formulated with Iressa. A
compound described herein, such as AD4AD4-1734-like compounds,
AD4-1886-like compounds, AD4-10381-like compounds, or
AD4-11091-like compounds, can be used or formulated with or
Tarceva.
[0403] Therapeutic Use
[0404] Another aspect is a process of treating a proliferative
disease, disorder, or condition with a compound described herein.
In various embodiments, a proliferative disease, disorder, or
condition is associated with a target biomolecule having an
interdomain tether associated with activation state, such as EGFR.
The therapeutic method can include administration of a
therapeutically effective amount of a compound of the invention to
a subject in need thereof. In some embodiments, the compound is a
compound described herein having anti-proliferative effects. In
some embodiments, the compound is a compound described herein EGFR
inhibitory activity. In some embodiments, the compound is an EGFR
inhibitor that acts to bind one or more domains of EGFR so as to
prevent tether extension and maintain an inactive conformation.
[0405] In various embodiments, the therapeutic method includes
administration of one or more compounds described herein.
[0406] For example, the therapeutic method can include
administration of one or more compounds according to a formula
selected from the following: Formula (13); Formula (14); Formula
(15); Formula (16); Formula (18); Formula (19); Formula (20);
Formula (21); Formula (22); Formula (23); Formula (25); Formula
(27); Formula (28); Formula (32); and Formula (33), as defined
above, or a stereoisomer or pharmaceutically acceptable salt
thereof.
[0407] As another example, the therapeutic method can include
administration of one or more compounds selected from the
following: AD4-1734 (Formula (12)); AD4-1886 (Formula (17));
AD4-10381 (Formula (24)); and AD4-11091 (Formula (31)), or a
stereoisomer or pharmaceutically acceptable salt thereof.
[0408] As another example, the therapeutic method can include
administration of one or more compounds selected from the
following:
##STR00070##
or a stereoisomer or pharmaceutically acceptable salt thereof.
[0409] Methods described herein are generally performed on a
subject in need thereof. For example, a subject in need of the
therapeutic methods described herein can be diagnosed with a
proliferative disease, disorder, or condition, or at risk thereof.
As another example, a subject in need of the therapeutic methods
described herein can be diagnosed with a disease, disorder, or
condition associated with EGFR, or at risk thereof. A determination
of the need for treatment can be assessed by a history and physical
exam consistent with the disease, disorder, or condition at issue.
Diagnosis of the various conditions treatable by the methods
described herein is within the skill of the art. The subject can be
an animal subject, preferably a mammal, more preferably horses,
cows, dogs, cats, sheep, pigs, mice, rats, monkeys, guinea pigs,
and chickens, and most preferably a human.
[0410] Examples of proliferative diseases or conditions treatable
with compositions described herein include, but are not limited to,
cancer; blood vessel proliferative disorders; fibrotic disorders;
mesangial cell proliferative disorders; psoriasis; actinic
keratoses; seborrheic keratoses; warts; keloid scars; eczema; and
hyperproliferative diseases caused by virus infections, such as
papilloma virus infection.
[0411] While under no obligation to provide an underlying mechanism
and in no way limiting the present invention by doing so, it is
presently thought that at least a portion of activity of compounds
described herein arise from inhibition of EGFR. It is further
contemplated that the presently described compounds may have
additional modes of action in their effectiveness in treating a
proliferative disease, disorder, or condition. Regardless of the
underlying mechanism, compounds described herein have been
demonstrated to be empirically effective in treating proliferative
diseases and conditions.
[0412] Various compounds described herein can be effective for
inhibiting EGFR, and thus, effective against diseases or conditions
associated with EGFR, such as include, but are not limited to,
proliferative diseases. In some embodiments, the proliferative
disease treated by a compound described herein is a condition
caused by excessive growth of cancer or non-cancer cells that
express a member of the EGFR family of receptors. The excess cells
generated by a proliferative disease can express EGFR at normal
levels or can overexpress EGFR. Particularly suitable diseases or
conditions associated with EGFR can be those stimulated by a ligand
of EGFR or mutations of such ligands. Examples of such ligands that
stimulate EGFR include, but are not limited to, EGF, TGF-alpha,
heparin-binding growth factor (HBGF), .beta.-cellulin, and
Cripto-1. Examples of proliferative disease associated with EGFR
include, but are not limited to, cancer; blood vessel proliferative
disorders; fibrotic disorders; mesangial cell proliferative
disorders; psoriasis; actinic keratoses; seborrheic keratoses;
warts; keloid scars; eczema; and hyperproliferative diseases caused
by virus infections, such as papilloma virus infection.
[0413] Cancer, or neoplasia, refers generally to any malignant
neoplasm or spontaneous growth or proliferation of cells. A subject
having "cancer", for example, may have a leukemia, lymphoma, or
other malignancy of blood cells. In certain embodiments, the
subject methods are used to treat a solid tumor. Exemplary solid
tumors include but are not limited to non small cell lung cancer
(NSCLC), testicular cancer, lung cancer, ovarian cancer, uterine
cancer, cervical cancer, pancreatic cancer, colorectal cancer
(CRC), breast cancer, as well as prostate, gastric, skin, stomach,
esophageal, and bladder cancer.
[0414] Treatment of cancer or treating a subject having cancer can
include inhibition of replication of cancer cells, inhibition of
spread of cancer, reduction in tumor size, lessening or reducing
the number of cancerous cells in the body of a subject, or
amelioration or alleviation of symptoms of cancer. A treatment can
be considered therapeutic if there is a decrease in mortality or
morbidity, and can be performed prophylactically, or
therapeutically.
[0415] Methods described herein can be used to treat (e.g., reduce
tumor size, decrease the vascularization, and/or increase the
permeability of) an established tumor. An established tumor is
generally understood as a solid tumor of sufficient size such that
nutrients, e.g., oxygen, can no longer permeate to the center of
the tumor from the subject's vasculature by osmosis and therefore
the tumor requires its own vascular supply to receive nutrients.
Methods described herein can be used to treat a solid tumor that is
not quiescent and is actively undergoing exponential growth.
[0416] A therapeutic protocol can be modified according to
permeability of a solid tumor. Permeability of a solid tumor
generally refers to the permeability of a solid tumor to a
therapeutic. A solid tumor may be said to be permeable to a
therapeutic if the therapeutic is able to reach cells at the center
of the tumor. An agent that increases the permeability of a tumor
may for example, normalize, e.g., maintain, the vasculature of a
solid tumor. Tumor vascularization or tumor permeability can be
determined by a variety of methods known in the art, such as, e.g.
by immunohistochemical analysis of biopsy specimens, or by imaging
techniques, such as sonography of the tumor, computed tomography
(CT) or magnetic resonance imaging (MRI) scans.
[0417] Different types of psoriasis display characteristics such as
pus-like blisters (pustular psoriasis), severe sloughing of the
skin (erythrodermic psoriasis), drop-like dots (guttae psoriasis)
and smooth inflamed lesions (inverse psoriasis). The treatment of
all types of psoriasis (e.g., psoriasis vulgaris, psoriasis
pustulosa, psoriasis erythrodermica, psoriasis arthropathica,
parapsoriasis, palmoplantar pustulosis) is contemplated by the
invention.
[0418] 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 proliferation disorders
include, but are not limited to, arthritis, where new capillary
blood vessels invade the joint and destroy cartilage, and ocular
diseases, like diabetic retinopathy, where new capillaries in the
retina invade the vitreous, bleed and cause blindness. Disorders
related to the shrinkage, contraction or closing of blood vessels,
such as restenosis, are also implicated.
[0419] Fibrotic disorders refer to the abnormal formation of
extracellular matrix. Examples of fibrotic disorders include, but
are not limited to, hepatic cirrhosis and mesangial cell
proliferative disorders. Hepatic cirrhosis is characterized by the
increase in extracellular matrix constituents resulting in the
formation of a hepatic scar. Hepatic cirrhosis can cause diseases
such as cirrhosis of the liver. An increased extracellular matrix
resulting in a hepatic scar can also be caused by viral infection
such as hepatitis. Lipocytes appear to play a major role in hepatic
cirrhosis. Other fibrotic disorders implicated include
atherosclerosis.
[0420] Mesangial cell proliferative disorders refer to disorders
brought about by abnormal proliferation of mesangial cells.
Mesangial proliferative disorders include, but are not limited to,
various human renal diseases, such as glomerulonephritis, diabetic
nephropathy, malignant nephrosclerosis, thrombotic microangiopathy
syndromes, transplant rejection, and glomerulopathies.
[0421] EGFR (Tuzi et al., 1991, Br. J. Cancer 63:227-233; Torp et
al., 1992, APMIS 100:713-719) HER2/neu (Slamon et al., 1989,
Science 244:707-712) and the PDGF-R (Kumabe et al., 1992, Oncogene
7:627-633) are known to be overexpressed in many tumors and/or
persistently activated by autocrine loops. Overexpression of the
receptor and autocrine loops have been demonstrated in most common
and severe cancers (see e.g., Akbasak and Suner-Akbasak et al.,
1992, J. Neurol. Sci. 111:119-133; Dickson et al., 1992, Cancer
Treatment Res. 61:249-273; Korc et al., 1992, J. Clin. Invest.
90:1352-1360; Lee and Donoghue, 1992, J. Cell. Biol.
118:1057-1070). Overexpression of EGFR is known to be associated
with cancers of the bladder, brain, head and neck, pancreas, lung,
breast, ovary, colon, prostate, and kidney. (see e.g., Atalay et
al., 2003, Ann. Oncology 14:1346-1363; Herbst and Shin, 2002,
Cancer 94:1593-1611; Modjtahedi et al., 1996, Br. J. Cancer
73:228-235). Overexpression of EGFR can be correlated or associated
with poor prognosis of the patients (see e.g., Herbst and Shin,
2002, Cancer 94:1593-1611; Modjtahedi et al., 1996, Br. J. Cancer
73:228-235). HER2 has been associated with breast, ovarian,
gastric, lung, pancreas and bladder cancer.
[0422] An inhibitor compound described herein can be used
therapeutically either as exogenous materials or as endogenous
materials. Exogenous agents are those produced or manufactured
outside of the body and administered to the body. Endogenous agents
are those produced or manufactured inside the body by some type of
device (biologic or other) for delivery to within or to other
organs in the body.
[0423] According to the methods described herein, administration
can be parenteral, pulmonary, oral, topical, intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, epidural, ophthalmic, buccal, or rectal
administration.
[0424] An effective amount of a compound described herein is
generally that which can exhibit an anti-proliferative effect to an
extent such as to ameliorate the treated condition. For example, an
effective amount of a compound described herein may inhibit EGFR to
an extent such as to ameliorate the treated condition. In some
embodiments, an effective amount is that amount of therapy (or
combination therapy) that is sufficient to affect a desired result
on a cancerous cell or tumor, including, but not limited to, for
example, reducing tumor size, reducing tumor volume, decreasing
vascularization of a solid tumor, or increasing the permeability of
a solid tumor to an agent, either in vitro or in vivo. In certain
embodiments, an effective amount of therapy (or combination
therapy) is the amount that results in a percent tumor inhibition
of more than about 55%, about 60%, about 65%, about 70%, about 75%,
about 80%, about 85%, about 90%, about 95%, about 99%, or about
100%. In certain embodiments, an effective amount of therapy (or
combination therapy) is sufficient to achieve a desired clinical
result, including but not limited to, for example, ameliorating
disease, stabilizing a subject, preventing or delaying the
development of, or progression of cancer in a subject. An effective
amount of therapy (or combination therapy) can be determined based
on one administration or repeated administration. Methods of
detection and measurement of the indicators above are known to
those of skill in the art. Such methods include, but are not
limited to measuring reduction in tumor burden, reduction of tumor
size, reduction of tumor volume, reduction in proliferation of
secondary tumors, decreased solid tumor vascularization, expression
of genes in tumor tissue, presence of biomarkers, lymph node
involvement, histologic grade, and nuclear grade.
[0425] In some embodiments, tumor burden can be determined. Tumor
burden, also referred to as tumor load, generally refers to a total
amount of tumor material distributed throughout the body of a
subject. Tumor burden can refer to a total number of cancer cells
or a total size of tumor(s), throughout the body, including lymph
nodes and bone barrow. Tumor burden can be determined by a variety
of methods known in the art, such as, for example, by measuring the
dimensions of tumor(s) upon removal from the subject, e.g., using
calipers, or while in the body using imaging techniques, e.g.,
ultrasound, computed tomography (CT) or magnetic resonance imaging
(MRI) scans. Tumor size can be determined, for example, by
determining tumor weight or tumor volume.
[0426] When used in the methods of the invention, a therapeutically
effective amount of a compound described herein can be employed in
pure form or, where such forms exist, in pharmaceutically
acceptable salt form and with or without a pharmaceutically
acceptable excipient. For example, the agents of the invention can
be administered, at a reasonable benefit/risk ratio applicable in a
sufficient amount sufficient to inhibit the target biomolecule for
which the compound is specific for the treatment or prophylaxis of
a disease, disorder, or condition associated with the target
biomolecule.
[0427] Toxicity and therapeutic efficacy of such compounds, and
pharmaceutical formulations thereof, can be determined by standard
pharmaceutical procedures in cell cultures and/or experimental
animals for determining the LD.sub.50 (the dose lethal to 50% of
the population) and the ED.sub.50, (the dose therapeutically
effective in 50% of the population). The dose ratio between toxic
and therapeutic effects is the therapeutic index that can be
expressed as the ratio LD.sub.50/ED.sub.50, where large therapeutic
indices are preferred.
[0428] The amount of a compound described herein may be combined
with a pharmaceutically acceptable carrier to produce a single
dosage form will vary depending upon the host treated and the
particular mode of administration. It will be appreciated by those
skilled in the art that the unit content of agent contained in an
individual dose of each dosage form need not in itself constitute a
therapeutically effective amount, as the necessary therapeutically
effective amount could be reached by administration of a number of
individual doses.
[0429] Administration of a compound described herein can occur as a
single event, a periodic event, or over a time course of treatment.
For example, modulators can be administered daily, weekly,
bi-weekly, or monthly. As another example, a compound can be
adminstered in multiple treatment sessions, such as 2 weeks on, 2
weeks off, and then repeated twice; or every 3rd day for 3 weeks.
One of ordinary skill will understand these regimes to be exemplary
and could design other suitable periodic regimes. For treatment of
acute conditions, the time course of treatment will usually be at
least several days. Certain conditions could extend treatment from
several days to several weeks. For example, treatment could extend
over one week, two weeks, or three weeks. For more chronic
conditions, treatment could extend from several weeks to several
months or even a year or more.
[0430] The specific therapeutically effective dose level for any
particular subject will depend upon a variety of factors including
the disorder being treated and the severity of the disorder;
activity of the specific compound employed; the specific
composition employed; the age, body weight, general health, sex and
diet of the patient; the time of administration; the route of
administration; the rate of excretion of the composition employed;
the duration of the treatment; drugs used in combination or
coincidental with the specific compound employed; and like factors
well known in the medical arts (see e.g., Koda-Kimble et al. (2004)
Applied Therapeutics: The Clinical Use of Drugs, Lippincott
Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic
Clinical Pharmacokinetics, 4.sup.th ed., Lippincott Williams &
Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics
& Pharmacokinetics, McGraw-Hill/Appleton & Lange, ISBN
0071375503). For example, it is well within the skill of the art to
start doses of the composition at levels lower than those required
to achieve the desired therapeutic effect and to gradually increase
the dosage until the desired effect is achieved. If desired, the
effective daily dose may be divided into multiple doses for
purposes of administration. Consequently, single dose compositions
may contain such amounts or submultiples thereof to make up the
daily dose. It will be understood, however, that the total daily
usage of the compounds and compositions of the present invention
will be decided by an attending physician within the scope of sound
medical judgment.
[0431] Compounds of the invention that inhibit a target biomolecule
can also be used in combination with other therapeutic modalities.
Thus, in addition to the therapies described herein, one may also
provide to the subject other therapies known to be efficacious for
particular conditions linked to the target biomolecule. Treatment
in accord with the methods described herein can be performed prior
to, concurrent with, or after conventional treatment modalities for
a disease, disorder, or condition associated with a target
biomolecule for which the compound is specific.
[0432] Kits
[0433] Also provided are kits. Such kits can include the
compositions of the present invention and, in certain embodiments,
instructions for administration. Such kits can facilitate
performance of the methods described herein, for example, treatment
methodologies or screening methodologies. When supplied as a kit,
the different components of the composition can be packaged in
separate containers and admixed immediately before use. Components
include, but are not limited to one or more compounds described
herein, vectors, diagnostic reagents, assay reagents, and/or
combinations thereof. Such packaging of the components separately
can, if desired, be presented in a pack or dispenser device which
may contain one or more unit dosage forms containing the
composition. The pack may, for example, comprise metal or plastic
foil such as a blister pack. Such packaging of the components
separately can also, in certain instances, permit long-term storage
without losing activity of the components.
[0434] Kits may also include reagents in separate containers such
as, for example, sterile water or saline to be added to a
lyophilized active component packaged separately. For example,
sealed glass ampules may contain a lyophilized component and in a
separate ampule, sterile water, or sterile saline, each of which
has been packaged under a neutral non-reacting gas, such as
nitrogen. Ampules may consist of any suitable material, such as
glass, organic polymers, such as polycarbonate, polystyrene,
ceramic, metal or any other material typically employed to hold
reagents. Other examples of suitable containers include bottles
that may be fabricated from similar substances as ampules, and
envelopes that may consist of foil-lined interiors, such as
aluminum or an alloy. Other containers include test tubes, vials,
flasks, bottles, syringes, and the like. Containers may have a
sterile access port, such as a bottle having a stopper that can be
pierced by a hypodermic injection needle. Other containers may have
two compartments that are separated by a readily removable membrane
that upon removal permits the components to mix. Removable
membranes may be glass, plastic, rubber, and the like.
[0435] In certain embodiments, kits can be supplied with
instructional materials. Instructions may be printed on paper or
other substrate, and/or may be supplied as an electronic-readable
medium, such as a floppy disc, mini-CD-ROM, CD-ROM, DVD-ROM, Zip
disc, videotape, audio tape, and the like. Detailed instructions
may not be physically associated with the kit; instead, a user may
be directed to an Internet web site specified by the manufacturer
or distributor of the kit.
[0436] Compositions and methods described herein utilizing
molecular biology protocols can be according to a variety of
standard techniques known to the art (see, e.g., Sambrook and
Russel (2006) Condensed Protocols from Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10:
0879697717; Ausubel et al. (2002) Short Protocols in Molecular
Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook
and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed.,
Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J.
and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier
(2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005)
Production of Recombinant Proteins: Novel Microbial and Eukaryotic
Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004)
Protein Expression Technologies, Taylor & Francis, ISBN-10:
0954523253).
[0437] Definitions and methods described herein are provided to
better define the present disclosure and to guide those of ordinary
skill in the art in the practice of the present disclosure. Unless
otherwise noted, terms are to be understood according to
conventional usage by those of ordinary skill in the relevant
art.
[0438] In some embodiments, numbers expressing quantities of
ingredients, properties such as molecular weight, reaction
conditions, and so forth, used to describe and claim certain
embodiments of the present disclosure are to be understood as being
modified in some instances by the term "about." In some
embodiments, the term "about" is used to indicate that a value
includes the standard deviation of the mean for the device or
method being employed to determine the value. In some embodiments,
the numerical parameters set forth in the written description and
attached claims are approximations that can vary depending upon the
desired properties sought to be obtained by a particular
embodiment. In some embodiments, the numerical parameters should be
construed in light of the number of reported significant digits and
by applying ordinary rounding techniques. Notwithstanding that the
numerical ranges and parameters setting forth the broad scope of
some embodiments of the present disclosure are approximations, the
numerical values set forth in the specific examples are reported as
precisely as practicable. The numerical values presented in some
embodiments of the present disclosure may contain certain errors
necessarily resulting from the standard deviation found in their
respective testing measurements. The recitation of ranges of values
herein is merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range. Unless otherwise indicated herein, each individual value is
incorporated into the specification as if it were individually
recited herein.
[0439] In some embodiments, the terms "a" and "an" and "the" and
similar references used in the context of describing a particular
embodiment (especially in the context of certain of the following
claims) can be construed to cover both the singular and the plural,
unless specifically noted otherwise. In some embodiments, the term
"or" as used herein, including the claims, is used to mean "and/or"
unless explicitly indicated to refer to alternatives only or the
alternatives are mutually exclusive.
[0440] The terms "comprise," "have" and "include" are open-ended
linking verbs. Any forms or tenses of one or more of these verbs,
such as "comprises," "comprising," "has," "having," "includes" and
"including," are also open-ended. For example, any method that
"comprises," "has" or "includes" one or more steps is not limited
to possessing only those one or more steps and can also cover other
unlisted steps. Similarly, any composition or device that
"comprises," "has" or "includes" one or more features is not
limited to possessing only those one or more features and can cover
other unlisted features.
[0441] All methods described herein can be performed in any
suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g. "such as") provided with respect to
certain embodiments herein is intended merely to better illuminate
the present disclosure and does not pose a limitation on the scope
of the present disclosure otherwise claimed. No language in the
specification should be construed as indicating any non-claimed
element essential to the practice of the present disclosure.
[0442] Groupings of alternative elements or embodiments of the
present disclosure disclosed herein are not to be construed as
limitations. Each group member can be referred to and claimed
individually or in any combination with other members of the group
or other elements found herein. One or more members of a group can
be included in, or deleted from, a group for reasons of convenience
or patentability. When any such inclusion or deletion occurs, the
specification is herein deemed to contain the group as modified
thus fulfilling the written description of all Markush groups used
in the appended claims.
[0443] Citation of a reference herein shall not be construed as an
admission that such is prior art to the present disclosure.
[0444] Having described the present disclosure in detail, it will
be apparent that modifications, variations, and equivalent
embodiments are possible without departing the scope of the present
disclosure defined in the appended claims. Furthermore, it should
be appreciated that all examples in the present disclosure are
provided as non-limiting examples.
EXAMPLES
[0445] The following non-limiting examples are provided to further
illustrate the present invention. It should be appreciated by those
of skill in the art that the techniques disclosed in the examples
that follow represent approaches the inventors have found function
well in the practice of the invention, and thus can be considered
to constitute examples of modes for its practice. However, those of
skill in the art should, in light of the present disclosure,
appreciate that many changes can be made in the specific
embodiments that are disclosed and still obtain a like or similar
result without departing from the spirit and scope of the
invention.
Example 1
EGFR Inhibition Assay
[0446] The following example describes general EGFR in-cell Western
(ICW) screening protocol. Methods are according to Chen et al.
(2005) Analytical Biochemistry 338, 136-142 (incorporated herein by
reference), except as otherwise noted.
[0447] Cell Plates: A431 cells (ATCC # CRL-1555) were grown in
Dulbecco's Modified Eagle Medium (DMEM; ATCC #30-2002) supplemented
with 10% fetal bovine serum, 105 units/ml pen/strep (Invitrogen
#15140155) and 2.1 mM L-glutamine (ATCC #30-2214). Cells were
seeded into 96 well tissue culture plates (BD Falcon #353948) at a
density of 30,000 cells per well and incubated at 37.degree. C.
with 5% CO.sub.2 overnight. Two cell plates were prepared for each
compound plate.
[0448] Serum Starvation: The cells were serum starved prior to
compound addition. The media was removed by aspiration and the
cells washed with PBS (200 .mu.l/well; Invitrogen #20012-027). The
PBS was removed by aspiration and replaced with 200 .mu.l of DMEM
(ATCC #30-2002) supplemented with 105 units/ml pen/strep
(Invitrogen #15140155) and 2.1 mM 1-glutamine (ATCC #30-2214). The
cell plates were incubated at 37.degree. C./5% CO.sub.2 for 2
hours.
[0449] Compound Plate Preparation: The test compounds were solvated
in 100% DMSO (Sigma #472301-2L) at a concentration of 25 mM.
Compounds found not to be completely soluble in 100% DMSO at 25 mM
were diluted to 10 mM with 100% DMSO and TFA (Fluka #91699) added
to a final concentration of 0.2%. 40 .mu.l of test compound was
added to the appropriate well of a 96 well plate (Falcon #351190).
As controls, 6 .mu.M EGFR kinase inhibitor PD168393 (EMD/Calbiochem
#513033) in 100% DMSO and DMSO alone were added to various wells.
Prepared compound plates were stored at RT prior to use and at
4.degree. C. long term.
[0450] Compound Dilution Plate Preparation: 250 .mu.l of DMEM
supplemented with 1 mg/ml BSA (Sigma A3059-10G) was added to the
appropriate wells of a 96 well plate to prepare the compound
dilution plates. Using a multichannel pipettor, 1.25 .mu.l of
compound from the compound plate was transferred into the compound
dilution plate. This dilution rate will give a compound
concentration in the assay of 125 .mu.M.
[0451] Compound Addition: The starve media was removed from the
cell plates by aspiration. Using a multichannel pipettor, the
compound dilution plates were mixed by pipetting up and down three
times. 50 .mu.l of mixed, diluted compound was added to each of two
rows/columns on each of two cell plates. The cell plates with
compound were incubated at 37.degree. C./5% CO.sub.2 for 4
hours.
[0452] EGF Addition: 20 ng/ml EGF (Upstate #01-107) was prepared in
DMEM supplemented with 1 mg/ml BSA. 50 .mu.l of 20 or 0 ng/ml EGF
was added to the appropriate wells without the removal of compound.
The compound and EGF were mixed by pipetting up and down three
times. The plates were incubated at 37.degree. C./5% CO.sub.2 for
10 min. In some screening assays the final concentration of EGF
used for simulation was 6.6, or 12.5 ng/ml rather than 10
ng/ml.
[0453] Fixation and Triton Washing: The EGF+compound was removed by
aspiration and 150 .mu.l of freshly prepared Fixation Solution
(1.times.PBS, Sigma P3813-10PK, and 4% Formaldehyde, Pierce #28908)
was immediately added. The plates were incubated at RT for 20 min
without shaking. The Fixation Solution was removed by aspiration
and the plates washed four times with 200 .mu.l each of Triton Wash
Solution (1.times.PBS, Sigma P3813-10PK, and 0.1% Triton X-100,
T8787-50ML) for 5 min with gentle shaking.
[0454] Blocking and Probing: Following the last Triton Wash, the
plates were blocked for 1.5 h at RT with shaking using 150 .mu.l of
Odyssey Blocking Buffer (LI-COR #927-40000). The block was removed
by aspiration and 50 .mu.l of diluted primary Ab mix was added. The
plates were incubated at 4.degree. C. overnight with gentle
shaking. The plates were washed five times with 200 .mu.l of PBST
(1.times.PBS, Sigma P3813-10PK, and 0.1% Tween-20, Sigma
P9416-50ML) for 5 min each with shaking 50 .mu.l of diluted
secondary Ab mix was added and the plates incubated at RT for 1 h
with shaking. The plates were washed 5.times. with 200 .mu.l of
PBST for 5 min each with shaking. One final wash with 200 .mu.l of
PBS (Sigma P3813-10PK) for 5 min with shaking was performed prior
to scanning.
[0455] Primary Ab Mix contained: 0.1% Tween-20 (Sigma P9416-50ML);
1/500 dilution anti-total EGFR (Invitrogen #AHR5062); 1/800
dilution anti-phospho EGFR (Tyr 1173; Cell Signalling #4407); and
Odyssey Blocking Buffer (LI-COR #927-40000). Some screening assays
used a 1/100 dilution of anti-phospho EGFR (Tyr1045; Cell
Signalling #2237) rather than the anti-phospho EGFR Ab indicated
above.
[0456] Secondary Ab Mix contained: 0.2% 10% Tween-20 (Sigma
P9416-50ML); 1/1200 dilution anti-mouse IR680 conjugate (LI-COR
926-32220); 1/1200 dilution anti-rabbit IR800CW conjugate (LI-COR
926-32211); and Odyssey Blocking Buffer (LI-COR #927-40000). In
some screening assays, the dilution of the secondary Ab conjugates
was 1/800 rather than 1/1200.
[0457] Plate Scanning. The plates were scanned on an Odyssey
Infrared Imaging System from LI-COR Biosciences. The focus offset
was set at 3.5 mm and the scanning intensity set at 3 for the 700
channel and 7 for the 800 channel.
[0458] Data Analysis: The % of Maximum and % Inhibition values were
calculated as follows. 700 Channel=signal for total EGFR (used to
control for variation in cell number). 800 Channel=signal for
phosphorylated EGFR. 800.sub.-EGF=basal level EGFR
phosphorylation+non-specific signal (no compound).
800.sub.+EGF=800.sub.-EGF+EGF dependent EGFR receptor
phosphorylation (no compound). 700/800.sub.com=700 or 800 channel
signals with compound. %
Maximum={[(800.sub.com/700.sub.com)-(800.sub.-EGF/700.sub.-EGF)]/[(800.su-
b.+EGF/700.sub.+EGF)-(800.sub.-EGF/700.sub.-EGF)]}.times.100%. %
Inhibition=100%-(% Maximum).
Example 2
MTT Assay
[0459] The following example describes the MTT Cell Proliferation
Assay. The MTT Cell Proliferation Assay served as a secondary
screen to evaluate active compounds from the primary cell based ICW
screening protocol, described above. The MTT assay was used to
evaluate toxicity through viability and proliferation effects, and
compares the growth of an epithelial carcinoma A431 cell line
(American Type Culture Collections (ATCC) cat #CRL-155) and an MDBK
line (ATCC cat #CCL-22) derived from a healthy bovine kidney after
a three day compound treatment and incubation.
[0460] The MTT Cell Proliferation Assay is a colorimetric assay
system that measures the reduction of the tetrazolium salt MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) into
insoluble formazan crystals, produced by the mitochondria of viable
cells. After incubation of the cells with the MTT, the formazan
crystals formed are solubilized by the addition of a detergent or
DMSO (ACS Reagent grade, Sigma cat#472301). The color can then be
quantified by spectrophotometric means. Among the applications for
the method are drug sensitivity, cytotoxicity, response to growth
factors, and cell activation. The reduction of tetrazolium salts is
now recognized as a safe, accurate alternative to radiometric
testing.
[0461] Methods are according to manufacturer instructions for MTT
Cell Proliferation Assay, ATCC, Cat. No. 30-1010K, except as
otherwise noted.
[0462] Each MTT assay tested 11 compounds and 1 standard in a dose
response curve with 5 concentrations, and 2.5 fold dilutions. Two
replicates on each cell plate and 2 duplicate plates gave 4
individual replicates of each concentration, in each cell line.
Concentrations were diluted in 100% DMSO from 25 mM stocks. Initial
concentrations used and final concentrations tested were as
follows: 20 mM to 100 .mu.M; 8 mM to 40 .mu.M; 3.2 mM to 16 .mu.M;
1.3 mM to 6.4 .mu.M; and 0.51 mM to 2.6 .mu.M.
[0463] Twelve 96 well plates of each cell line were plated. Plating
was done by dislodging cells as in passage protocol, counting on a
hemocytometer, re-suspending in standard growth media, and plating
using a multichannel pippetor.
[0464] Standards: AD4-10289 was used as the dose response standard,
run at one concentration lower than the test compounds; 40, 16,
6.4, 2.6, and 1.0 .mu.M final concentrations. 10289 was run in
place of 1 test compound, with the same number of replicates. 16
.mu.l of 25 mM stock+34 .mu.l DMSO=8 mM starting concentrations,
and serial diluted as with test compounds in same plate. AD4-1734
was used at 2 concentrations, 31.125 .mu.M and 23.5 .mu.M final
concentrations. These concentrations were used on each plate to
give 100%, and less than 100% activity in A431, while also giving
less than 50% and 0 activity in DMBK cells. Two replicates of each
concentrations was run on each cell plate. All compound addition
and cell treatments were done using aseptic technique under a
laminar flow aseptic hood.
[0465] Control: 0.5% DMSO in treatment media was used as the
control. The assay was demonstrated to tolerate up to 1.0% DMSO
with no significant growth differences.
[0466] MTT dye (Thiazolyl Blue Tetrazolium Bromide, Sigma cat
#M5655) stock was prepared in bulk and stored at 4.degree. C. for
use: 5 mgs/ml sterile PDS, shielded from light. 4 ul of this stock
per 100 uls treatment media per well was used. 130 ml of
MTT/treatment was prepared fresh to dye 12 plates; 124.8 ml
treatment media+522 mls of MTT stock.
[0467] Cells (harvested from 95% confluent flasks) were plated at
densities of 7500 cells per well. Plates were incubated overnight
prior to treatment, with treatment day=Day 0. Compound treatments
were prepared prior to washing cells to minimize time cells were
dry at room temperature after final aspiration. Compounds were
diluted in 100% DMSO in 96 well polypropylene plates: 40 .mu.l 25
mM stock+10 .mu.l DMSO=20 mM; 20 .mu.l of 20 mM+30 .mu.l DMSO=8 mM;
20 .mu.l of 8 mM+30 .mu.l DMSO=3.2 mM; 20 .mu.l of 3.2 mM+30 .mu.l
DMSO=1.3 mM; 20 .mu.l of 1.3 mM+30 .mu.l DMSO=0.51 mM.
[0468] 5.0 .mu.l/ml were added to treatment media, in sterile, 2.2
ml deep well plates=0.5% dmso final. Ten .mu.l of each dmso
concentration was added to 2.0 ml of treatment media and then mixed
using a 1200 .mu.l multichannel pipettor. Cell plates were treated
from the deep well plates. Treatment media was standard growth
media containing 0.5% FBS and standard additions. This reduced FBS
concentration allowed for a slower growth rate in cells. In plate 1
of each cell line, standard AD4-10289 was run in lane 3 (see above
for rates).
[0469] Media was aspirated from cell plates, and washed with 200
.mu.l/well sterile PBS. Aspirated PBS wash, and cell plates were
treated with 200 .mu.l of compound/treatment media per well, and
incubated at 37.degree. C., 5% CO.sub.2 for 3 days.
[0470] Prior to harvesting, visual observations were made under an
inverted microscope. Plates were harvested by: aspirating the
growth media; adding back MTT dye/100 .mu.l treatment media; and
incubating plates at 37.degree. C. for 1 hour. MTT dye was
aspirated. 100 .mu.l/well dmso was added, and then shaken on a
Bellco plate shaker for 5 minutes, at 4.5 setting.
[0471] Plates were read in a Tecan Sunrize UV plate reader, at 560
nm. Settings: Read mode, Outside Normal 2 sec; Shake settle time 3
sec. Data is reported as % inhibition, calculated for individual
plates using control values from that plate.
Example 3
Testing of Identified Compounds from Pharmacophores for EGFR
Inhibition
[0472] Identified compounds, representing various pharmacophore
models, were tested for ability to inhibit EGFR at 25 .mu.M.
[0473] AD4-compounds were identified using pharmacophore models
(see Example 3, Example 4) and then were docked with the binding
site of EGFR (SEQ ID NO: 1) that is recognized by defined CDRs of
cetuximab. The inhibition of epidermal growth factor binding by
AD4-compounds was then determined (NovaScreen BioSciences, Hanover,
Md.). Inhibition of EGF binding was determined at 25 .mu.M
concentration.
[0474] For the inhibitor assays, K.sub.D (binding affinity) was
1.04 nM, while B.sub.max (receptor number) was 43.0 fmol/mg tissue
(wet weight). Receptor source was rat liver membranes. The
radioligand was [.sup.125I]EGF (150-200 Ci/ng) at a final ligand
concentration of 0.36 nM. A non-specific determinant was used as
EGF--[100 nM]. The reference compound and positive control was EGF.
Reactions were carried out in 10 mM HEPES (pH 7.4) containing 0.1%
BSA at 25.degree. C. for 60 minutes. The reaction was terminated by
rapid vacuum filtration onto glass fiber filters. Radioactivity
trapped onto the filters was determined and compared to control
values to ascertain any interactions of test compounds with the EGF
binding site. The EGF inhibitor assays were modified from, for
example, Mukku (1984) J. Biol. Chem. 259, 6543-6546; Duh et al.
(1990) World J. Surgery 14, 410-418; Lokeshwar et al. (1989) J.
Biol. Chem. 264(32), 19318-19326.
Example 4
Generation of Pharmacophores for Target Inhibition from
EGFR/Cetuximab Crystal Structure
[0475] The following example describes analysis of the target
protein-antibody crystal structure and generation of pharmacophores
for EGFR inhibition.
[0476] The protein crystal structure of cetuximab complexed to EGFR
has been reported by Ferguson et al. (Cancer Cell, 2005, 7, 301-311
and the crystallographic data deposited in the Protein Data Bank as
PDB code 1YY9. Structural information which define the position of
the atoms of cetuximab was utilized to construct a pharmacophore
model used to identify small molecules having similar atoms in
similar positions. Small molecules having similar features to the
antibody can demonstrate similar biological activity and thus
similar therapeutic utility.
[0477] The pharmacophore feature generation and pharmacophore
virtual screening module of the Molecular Operating Environment
(MOE) software from Chemical Computing Group (CCG) (Montreal,
Quebec, Canada) was used in the pharmacophore definitions described
below. MOE's pharmacophore applications use a general notion of a
pharmacophore being a set of structural features in a ligand that
are directly related to the ligand's recognition at a receptor site
and its biological activity.
[0478] In MOE, pharmacophoric structural features are represented
by labeled points in space. Each ligand is assigned an annotation,
which is a set of structural features that may contribute to the
ligand's pharmacophore. A database of annotated ligands can be
searched with a query that represents a pharmacophore hypothesis.
The result of such a search is a set of matches that align the
pharmacophoric features of the query to the pharmacophoric features
present in the ligands of the searched database. The MOE software
suite provides for interactive modifications (positions, radii, as
well as other characteristics of the pharmacophoric query can be
interactively adjusted); systematic matching (all possible matches
of the ligand and the query are systematically examined); partial
matching (the search algorithm is capable of finding ligands that
match only a portion of the query); and volume filtering (the query
can be focused by adding restrictions on the shape of the matched
ligands in the form of a set of volumes).
[0479] The pharmacophore features of this example were generated
using the Pharmacophore Query Editor in MOE. All hydrogen bond
donor features are spheres of 1.2 Angstroms in radius and are
colored purple. All hydrogen bond acceptor features are spheres of
1.2 Angstroms in radius and are colored cyan. All aromatic features
are spheres of 1.2 Angstroms in radius and are colored green. All
combined acceptor-anion pharmacophore features are spheres of 1.2
Angstroms in radius and are colored grey. All combined
donor-acceptor features are spheres of 1.2 Angstroms in radius and
are colored pink. All combined donor-cation features are spheres of
1.2 Angstroms and are colored red. All donor, acceptor, aromatic,
combined acid-anion, and combined donor-acceptor directionality
features are spheres of 1.5 Angstroms in radius and colored dark
grey for donors, dark cyan for acceptors, dark green for aromatics,
dark cyan for combined acid-anions, and dark grey for combined
donor-acceptors. A feature that is marked essential in the
pharmacophore query must be contained in the ligand in order for
that ligand to be a hit.
[0480] All of the pharmacophore features were derived from the
corresponding donor, acceptor, aromatic and acid moieties of the
corresponding antibody in complex with its receptor (e.g.,
cetuximab complexed with EGFr, pdb accession number 1YY9) taken
from crystal structures deposited in the protein databank with two
exceptions. In some cases two methods provided the MOE software are
used to place pharmacophore features. These are explained
below.
[0481] The Contact statistics calculated, using the 3D atomic
coordinates of a receptor, preferred locations for hydrophobic and
hydrophilic ligand atoms using statistical methods. Using this
method hydrophobic-aromatic and H-bonding features were placed, as
noted in the individual pharmacophore definitions.
[0482] The MultiFragment Search essentially places a relatively
large number of copies of a fragment (e.g., 200 copies of ethane)
into a receptor's active site. The fragments are placed randomly
around the active site atoms and are assumed not to interact with
each other; no regard is paid to fragment overlap. Next, a special
energy minimization protocol is used to refine the initial
placement: the receptor atoms feel the average forces of the
fragments, while each fragment feels the full force of the receptor
but not of the other fragments. Using this technique it was
possible to place hydrophobic, H-bond donors, acceptors and anions
and cations in favorable positions within the receptors for use as
MOE pharmacophore features.
[0483] Excluded volumes were generated for the pharmacophores
defined below except when indicated. These were derived from the
position of the receptor atoms near the antibody binding site.
Excluded volumes are positions in space where ligand atoms must be
excluded in order to avoid bumping into the receptor. They were
generated in MOE by selecting the receptor residues within 5
Angstroms from the antibody and selecting "union" from the
pharmacophore query editor in MOE.
[0484] In the Individual Pharmacophore Definitions described below,
abbreviations were as follows: F=pharmacophore feature; Donor=Don,
Acceptor=Acc, Anion=Ani, Cation=Cat, Acceptor and
Anion=Acc&Ani, Donor and Cation=Don&Cat, Donor and
Acceptor=Don&Acc, Aromatic=Aro, Hydrophobe=Hyd.
[0485] EGFR Complexed with Antibody Cetuximab (1YY9.Pdb)
[0486] The crystal (1YY9.pdb) of protein EGFR (SEQ ID NO: 1)
complexed with antibody cetuximab (SEQ ID NO: 5 and SEQ ID NO:6)
was analyzed according to the procedures described above. Results
showed that two sets of residues of the antibody cetuximab make
contact with the receptor. These are Gly54-Asp58 and
Thr100-Glu105.
[0487] Hit AD4-1886 from Pharmacophore Model 21_Thr100_Glu105
[0488] Pharmacophore model 21_thr100_glu105 (described below in
Table 1 and 2 and depicted in FIG. 1A-FIG. 1B) produced the hit,
AD4-1886.
[0489] In the Individual Pharmacophore Definitions described below,
abbreviations were as follows: F=pharmacophore feature; Donor=Don,
Acceptor=Acc, Anion=Ani, Cation=Cat, Acceptor and
Anion=Acc&Ani, Donor and Cation=Don&Cat, Donor and
Acceptor=Don&Acc, Aromatic=Aro, Hydrophobe=Hyd.
TABLE-US-00001 TABLE 1 21_thr100_glu105 F(III)1 Derived from the
side chain OH of Partial match, antibody Tyr102. This OH donates
ligand must an H-bond to the side chain match at least 7 carbonyl
of receptor pharmacophore Don&Acc Gln408 and accepts an H-bond
from features. the side chain NH2 of receptor FIG. 1A-FIG. 1B
Gln384. F(III)2 Derived from the backbone carbonyl Acc of antibody
Tyr102 accepting an H-bond from the side chain OH of receptor
Ser440. F(III)3 Directionality of F2 with respect Acc2 to the OH of
Ser440. F(III)4 Derived from side chain phenol ring Aro of the
antibody Tyr101 forming a favorable coulombic interaction with the
imidazole side chain of the receptor His409. F(III)5 Directionality
of F4 with respect Aro2 to the imidazole of His409. F(III)6 Derived
from the side chain Acc&Ani carboxylate of antibody Asp103.
Acceptor accepts an H-bond from or Anion forms a salt bridge to
NH3+ of receptor Lys465. F(III)7 Directionality of F6 with respect
Acc2 to the side chain of Lys465. F(III)8 Derived from the side
chain OH of Don antibody Tyr102. This OH donates an H-bond to the
side chain carbonyl of receptor Gln408. This feature is marked
essential. F(III)9 Directionality with respect to the Don2 side
chain carbonyl of receptor Gln408. V1 Excluded volume
TABLE-US-00002 TABLE 2 Cartesian and spherical coordinates of
features for pharmacophore 21_thr100_glu105. sphere sphere .THETA.
.PHI. radius volume Preferred Feature X Y Z r (theta) (phi) (A)
(A.sup.3) features F(III)1 37.771 38.261 61.320 81.552 41.243
45.369 1.2 7.2 Don&Acc F(III)2 35.242 44.754 66.137 87.287
40.739 51.781 1.2 7.2 Acc F(III)3 35.245 45.639 64.234 86.320
41.915 52.323 1.5 14.1 Acc2 F(III)4 39.910 35.496 67.237 85.870
38.463 41.650 1.2 7.2 Aro F(III)5 35.866 34.818 65.306 82.241
37.431 44.151 1.5 14.1 Aro2 F(III)6 31.030 44.156 69.520 88.009
37.822 54.903 1.2 7.2 Acc&Ani F(III)7 30.168 42.269 69.193
86.513 36.889 54.484 1.5 14.1 Acc2 F(III)8 37.771 38.261 61.320
81.552 41.243 45.369 1.2 7.2 preferred Don F(III)9 37.200 36.592
60.180 79.652 40.928 44.528 1.6 17.2 Don2
[0490] Hit AD4-11091 from Pharmacophore Model Pharm-1886-6
[0491] Pharmacophore model Pharm-1886-6, which was derived from the
Glide docked pose of AD4-1886, afforded the hit AD4-11091. This is
a partial match model. The ligand must match at least 9
pharmacophore features recited in Table 3 and 4.
TABLE-US-00003 TABLE 3 Pharm-1886-6 F(V)1 Ligand accepts an H-bond
from side chain Partial match, Acc&Ani amino of receptor Lys465
ligand must F(V)2 Directionality of F1 with respect to the match at
least 9 Acc2 side chain amino of receptor Lys465 pharmacophore
F(V)3 Directionality of F1 with respect to the features. Acc2 side
chain amino of receptor Lys465 FIG. 2 F(V)4 Ligand donates an
H-bond to the side Don chain carbonyl of receptor Gln411. This
feature is marked essential F(V)5 Directionality of F4 with respect
to the Don2 side chain carbonyl of Gln411 F(V)6 Ligand donates an
H-bond to the side Don chain nitrogen of receptor Gln411. This
feature is marked essential. F(V)7 Directionality of F6 with
respect to Don2 the side chain nitrogen of Gln411 F(V)8 Ligand
accepts an H-bond to the side Acc chain imidazole of receptor
His409 F(V)9 Ligand forms a favorable .pi.-.pi. interaction Aro
with the phenyl ring of receptor Phe412 F(V)10 Directionality of F9
with respect to the Aro2 phenyl ring of receptor Phe412 F(V)11
Ligand forms favorable hydrophobic Aro interactions with the side
chains of receptor Val417 and Ile448. This feature is marked
essential V1 Excluded volume
TABLE-US-00004 TABLE 4 Cartesian and spherical coordinates of
features for pharmacophore Pharm-1886-6. sphere sphere .THETA.
.PHI. radius volume Preferred Feature X Y Z r (theta) (phi) (A)
(A.sup.3) features F(V)1 29.158 39.252 69.930 85.329 34.962 53.394
1.2 7.2 Acc&Ani F(V)2 30.366 40.690 70.870 87.180 35.618 53.267
1.5 14.1 Acc2 F(V)3 27.798 40.331 71.112 86.349 34.560 55.424 1.5
14.1 Acc2 F(V)4 30.334 37.857 68.525 83.958 35.296 51.296 1.2 7.2
preferred Don F(V)5 30.012 36.233 67.233 82.060 34.984 50.365 1.5
14.1 Don2 F(V)6 32.328 37.488 67.721 83.884 36.166 49.227 1.2 7.2
preferred Don F(V)7 31.212 36.002 66.743 82.006 35.523 49.076 1.5
14.1 Don2 F(V)8 38.730 36.298 66.517 85.100 38.590 43.143 1.2 7.2
Acc F(V)9 35.061 37.556 66.983 84.418 37.489 46.968 1.2 7.2 Aro
F(V)10 34.495 37.311 64.976 82.486 38.027 47.246 1.5 14.1 Aro2
F(V)11 36.483 41.320 63.964 84.438 40.753 48.557 1.2 7.2 preferred
Aro
[0492] Using the above described methodology, one can generate
pharmacophore models for a variety of protein targets (crystallized
with ligand).
Example 5
Generation of Pharmacophores for Target Inhibition from Inactive
EGFR Crystal Structure
[0493] The following example describes analysis of the target
protein crystal structure and generation of pharmacophores for EGFR
inhibition. The ligands found by this methodology will interact
with residues from Dom II and Dom IV of EGFR and thereby yielding
an inactive form of the receptor.
[0494] Methods are according to those described in Example 4,
except as noted otherwise. Abbreviations for Individual
Pharmacophore Definitions are as described above.
[0495] The protein crystal structure of the inactive conformation
of EGFR has been reported by Ferguson et al. (Ferguson, K. M.,
Berger, M. B., Mendrola, J. M., Cho, H., Leahy, D. J., Lemmon, M.
A. (2003) EGF activates its receptor by removing interactions that
auto-inhibit ectodomain dimerization Mol. Cell. 11: 507-517). The
binding site was determined using the site finder module in the MOE
software. It consists of the interface of residues from Domain II
(23 residues, Lys227, Phe228, Lys 235, Asp236, Thr237, Cys238,
Pro239, Pro240, Leu241, Met242, Tyr244, Tyr249, Gln250, Met251,
Gly257, Lys258, Tyr259, Ser260, Cys265, Val275, His278, Gly279 and
Ser280) and Domain IV (16 residues, Arg548, Gly549, Pro550, Asp551,
Asn552, Asp561, His564, Val566, Thr568, Cys569, Pro570, Ala571,
Gly572, Val573, Met574 and Leu580) (see e.g., FIG. 3).
[0496] Structural information which was derived from contact
statistics and MFSS in the program MOE was used to construct
pharmacophore models used to identify small molecules having
similar atoms in similar positions.
[0497] Two methods provided in the MOE software are used to place
pharmacophore features which correspond to hit AD4-1734. These are
contact statistics and MFSS, as described above. Both the contact
statistics and MFSS algorithms were applied to the Domain II-Domain
IV interface binding site of the inactive form of EGFr (1NQL.pdb,
see figure above).
[0498] Excluded volumes were generated in MOE by selecting the
receptor residues of the binding site at the Domain II and Domain
IV interface described above and selecting "union" from the
pharmacophore query editor in MOE.
[0499] In the Individual Pharmacophore Definitions described below,
abbreviations were as follows: F=pharmacophore feature; Donor=Don,
Acceptor=Acc, Anion=Ani, Cation=Cat, Acceptor and
Anion=Acc&Ani, Donor and Cation=Don&Cat, Donor and
Acceptor=Don&Acc, Aromatic=Aro, Hydrophobe=Hyd.
[0500] Hit AD4-1734 from Pharmacophore Model Pharm-1nql-Glue-6
[0501] Pharmacophore model Pharm-1 nql-glue-6 (see TABLE 5 and 8;
FIG. 4) produced the hit AD4-1734. This is a partial match model.
The ligand must match at least 5 pharmacophore features.
TABLE-US-00005 TABLE 5 Pharm-1nql-glue-6 F(II)1 Derived from MFSS
(see above). Partial match, Don&Cat Ligand donates an H-bond or
forms a ligand must salt bridge to the carboxylate side match at
least 5 chain of receptor Asp553 pharmacophore F(II)2 Derived from
MFSS. This feature is features. Don ignored in this pharmacophore
model FIG. 4 F(II)3 Derived from hydrophobic contact Hyd
statistics. Ligand forms hydrophobic contacts with side chain of
receptor Val568, imidazolidine side chain of receptor Pro552 and
with the side chain of Met253 F(II)4 Derived from hydrophobic
contact Hyd statistics. Ligand forms hydrophobic contacts with side
chain of receptor Val575, Met253, and with the imidazolidine ring
of receptor Pro552. This feature is marked essential F(II)5 Derived
from MFSS. Ligand donates an Don H-bond to the side chain hydroxyl
of Thr570 F(II)6 Directionality of F4 with respect to Don2 the
backbone carbonyl of receptor Thr570 F(II)7 Derived from MFSS.
Ligand accepts an Acc H-bond from receptor backbone NH of Ala573
F(II)8 Directionality of F7 with respect to Acc2 the backbone NH of
receptor Ala573 F(II)9 Derived from MFSS. Ligand donates an
Don&Cat H-bond to the backbone carbonyl of Asp563 and forms a
salt bridge to the side chain carboxylate of receptor Asp563. This
feature is marked essential V1 Excluded volume
TABLE-US-00006 TABLE 6 Cartesian and spherical coordinates of
features for pharmacophore Pharm-1nql-Glue-6. sphere sphere .THETA.
.PHI. radius volume Preferred Feature X Y Z r (theta) (phi) (A)
(A.sup.3) features F(II)1 53.523 -17.135 -4.293 56.363 94.368
-17.752 1.2 7.2 Don&Cat F(II)2 47.983 -20.609 -10.619 53.290
101.494 -23.244 1 4.2 optional Don F(II)3 50.511 -16.780 -7.319
53.726 97.830 -18.377 1.7 20.6 Hyd F(II)4 48.632 -18.658 -12.837
53.647 103.844 -20.990 1.4 11.5 preferred Hyd F(II)5 44.675 -21.359
-12.586 51.093 104.261 -25.552 1.2 7.2 Don F(II)6 45.067 -23.421
-12.644 52.340 103.980 -27.461 1.5 14.1 Don2 F(II)7 44.906 -20.294
-14.555 51.383 106.455 -24.319 1.2 7.2 Acc F(II)8 45.475 -21.640
-16.064 52.861 107.691 -25.448 1.5 14.1 Acc2 F(II)9 52.952 -20.952
-9.221 57.688 99.198 -21.588 1.2 7.2 preferred Don&Cat
[0502] Hit AD4-10381 from Pharmacophore Model DockPharm 1505-2
[0503] Pharmacophore model DockPharm 1505-2 (see TABLE 7 and TABLE
8; FIG. 5), afforded the hit AD4-10381. This is a partial match
model. The ligand must match at least 8 pharmacophore features.
TABLE-US-00007 TABLE 7 DockPharm1505-2 F(IV)1 Derived from MFSS.
Ligand accepts an Partial match, Acc H-bond from receptor side
chain OH of ligand must Thr239. This feature is marked essential
match at least 8 F(IV)2 Directionality of F1 with respect to
pharmacophore Acc2 side chain OH of Thr239. This feature features.
is marked essential FIG. 5 F(IV)3 Derived from MFSS. F Ligand forms
Aro hydrophobic contacts with side chain of receptor Met576 and
imidazolidine ring of Pro242 F(IV)4 Directionality of F3 with
respect to Aro2 side chain of receptor Met576 and imidazilidine
ring of Pro242 F(IV)5 Derived from MFSS. Ligand forms Aro
hydrophobic contacts with side chain of receptor Met576 and
imidazolidine ring of Pro242 F(IV)6 Directionality of F5 with
respect to Aro2 side chain of receptor Met576 and imidazolidine
ring of Pro242 F(IV)7 Derived from MFSS. Ligand forms Aro
hydrophobic contacts with side chain of receptor Leu243 and side
chain of Thr239 F(IV)8 Derived from MFSS. Ligand donates an Don
H-bond to the backbone carbonyl of His280. This feature is marked
essential F(IV)9 Derived from hydrophobic contact Aro statistics.
Ligand forms hydrophobic contacts with side chains of receptor
Met244 and Leu243. This feature is marked essential F(IV)10 Derived
from MFSS. Ligand forms Hyd hydrophobic contacts with side chain of
receptor Met244 V1 Excluded volume
TABLE-US-00008 TABLE 8 Cartesian and spherical coordinates of
features for pharmacophore DockPharm 1505-2. sphere sphere .THETA.
.PHI. radius volume Preferred Feature X Y Z r (theta) (phi) (A)
(A.sup.3) features F(IV)1 43.406 -13.282 -20.203 49.686 113.993
-17.014 1.2 7.2 preferred Acc F(IV)2 41.702 -12.124 -20.610 48.071
115.388 -16.211 1.6 17.2 preferred Acc2 F(IV)3 44.536 -14.059
-19.940 50.781 113.121 -17.520 1.2 7.2 Aro F(IV)4 45.616 -12.975
-21.378 52.021 114.264 -15.878 1.5 14.1 Aro2 F(IV)5 45.210 -12.475
-18.241 50.322 111.253 -15.426 1.2 7.2 Aro F(IV)6 46.291 -11.393
-19.681 51.575 112.433 -13.827 1.5 14.1 Aro2 F(IV)7 43.427 -7.811
-18.296 47.767 112.521 -10.196 1.2 7.2 Aro F(IV)8 41.296 -6.448
-17.166 45.184 112.328 -8.875 1.2 7.2 preferred Don F(IV)9 46.569
-6.963 -15.306 49.512 108.007 -8.504 1.2 7.2 preferred Aro F(IV)10
48.308 -6.291 -13.040 50.431 104.985 -7.420 1.8 24.4 Hyd
Example 6
Ligand Docking and Scoring to EGFR Protein Complexed with Antibody
Cetuximab
[0504] The compounds selected for docking to the target protein,
crystal (1YY9.pdb) of protein EGFR (SEQ ID NO: 1) complexed with
antibody cetuximab (SEQ ID NO: 5 and SEQ ID NO:6), were those which
were found to align to the pharmacophore models generated in the
MOE modeling software (see Example 3). These compounds were
obtained in MOE database format. The 3-dimensional atomic
coordinates of these compounds were written to a structure data
format (*.sdf) file using the export command in the MOE database
window without adding hydrogens.
[0505] The LigPrep software module of Maestro modeling software
(Schrodinger LLC, NY, N.Y.) was next employed to prepare the
compounds for docking. The *.sdf file was converted into Maestro
format using LigPrep. Hydrogens were then added and any charged
groups neutralized. Ionization states were generated for the
ligands at 7.0+/-1.0 pH units. After this, tautomers were generated
when necessary, alternate chiralities were generated and low energy
ring conformers were produced. This was followed by removing any
problematic structures and energy minimizing the resulting ligands
using MacroModel software module. Finally a Maestro file (*.mae)
was written of the ligands which were now ready for docking. All of
these steps were automated via a python script supplied by
Schrodinger, LLC.
[0506] The following describes protein preparation. First the
protein structure 1 YY9 of EGFR complexed with antibody cetuximab
was imported into Maestro in PDB format. Hydrogens were added and
any errors such as incomplete residues were repaired. The protein
structure was checked for metal ions and cofactors. Charges and
atom types were set for metal ions and cofactors as needed. Ligand
bond orders and formal charges were adjusted if necessary. The
binding site was determined by picking the ligand (for 1YY9 it is
either the Thr100-Tyr101-Tyr102-Asp103-Tyr104-Glu105 or
Gly54-Gly55-Asn56-Thr57-Asp58 pieces of the antibody) in Maestro
(Glide). The program determines the centroid of the picked ligand
and draws a 20 Angstrom box which is the default setting with the
centroid of the ligand at the center of the box. The box was the
binding site for the ligands to be docked. The protein preparation
facility, which is automated in Glide, consists of two components,
preparation and refinement. The preparation component added
hydrogens and neutralized side chains that are not close to the
binding site and do not participate in salt bridges. The refinement
component performed a restrained minimization of the
co-crystallized complex which reoriented side-chain hydroxyl groups
and alleviated potential steric clashes.
[0507] The following describes receptor grid generation. Glide
searches for favorable interactions between one or more ligand
molecules and a receptor molecule, usually a protein. The shape and
properties of the receptor are represented on a grid by several
different sets of fields including hydrogen bonding, coulombic
(i.e., charge-charge) interactions hydrophobic interactions, and
steric clashes of the ligand with the protein. In the first step
the receptor must be defined. This was done by picking the ligand.
The unpicked part of the structure was the receptor. The ligand was
not included in the grid calculation but was used to define the
binding site as described above. Scaling of the nonpolar atoms of
the receptor was not included in the present docking runs. The
grids themselves were calculated within the space of the enclosing
box. This is the box described above and all of the ligand atoms
must be contained in this box. No pharmacophore constraints were
used because the Glide extra precision scoring function performs
better without these constraints.
[0508] To use Glide, each ligand must be a single molecule, while
the receptor may include more than one molecule, e.g., a protein
and a cofactor. Glide can be run in rigid or flexible docking
modes; the latter automatically generates conformations for each
input ligand. The combination of position and orientation of a
ligand relative to the receptor, along with its conformation in
flexible docking, is referred to as a ligand pose. All docking runs
are done using the flexible docking mode. The ligand poses that
Glide generates pass through a series of hierarchical filters that
evaluate the ligand's interaction with the receptor. The initial
filters test the spatial fit of the ligand to the defined active
site, and examine the complementarity of ligand-receptor
interactions using a grid-based method. Poses that pass these
initial screens enter the final stage of the algorithm, which
involves evaluation and minimization of a grid approximation to the
OPLS-AA nonbonded ligand-receptor interaction energy. Final scoring
is then carried out on the energy-minimized poses. By default,
Schrodinger's proprietary GlideScore multi-ligand scoring function
is used to score the poses. If GlideScore was selected as the
scoring function, a composite Emodel score is then used to rank the
poses of each ligand and to select the poses to be reported to the
user. Emodel combines GlideScore, the nonbonded interaction energy,
and, for flexible docking, the excess internal energy of the
generated ligand conformation. Conformational flexibility is
handled in Glide by an extensive conformational search, augmented
by a heuristic screen that rapidly eliminates unsuitable
conformations, such as conformations that have long-range internal
hydrogen bonds.
[0509] The settings used in the docking runs of this example were
as follows. Grid file was read in. Extra precision (XP) scoring
function was used. Docked using conformational flexibility. 5000
poses per ligand for the initial Glide screen were kept (default).
Scoring window for keeping initial poses was 100.0 (default). Best
800 poses per ligand for the energy minimization was kept
(default). For the energy minimization, a distance dependent
dielectric constant of 2.0 was used and maximum number of conjugate
gradient steps was 100 (defaults). The ligand file was then loaded.
Molecules with >120 atoms and/or >20 rotatable bonds were not
docked (default). Van der Waals radii of ligand atoms with partial
charges <0.15 were scaled by 0.80. This was done to mimic
receptor flexibility. Constraints and similarity were not used.
Poses with Coulomb plus Van der Waals energies >0.0 were
rejected. To ensure that poses for each molecule were
conformationally distinct, poses with RMS deviation <0.5 and/or
maximum atomic displacement of 1.3 Angstroms were discarded.
[0510] The following describes Glide Scoring. The choice of
best-docked structure for each ligand was made using a model energy
score (Emodel) that combines the energy grid score, the binding
affinity predicted by GlideScore, and (for flexible docking) the
internal strain energy for the model potential used to direct the
conformational-search algorithm. Glide also computed a specially
constructed Coulomb-van der Waals interaction-energy score (CvdW)
that was formulated to avoid overly rewarding charge-charge
interactions at the expense of charge-dipole and dipole-dipole
interactions. This score was intended to be more suitable for
comparing the binding affinities of different ligands than is the
"raw" Coulomb-van der Waals interaction energy. In the final data
work-up, one can combine the computed GlideScore and "modified"
Coulomb-van der Waals score values to give a composite score that
can help improve enrichment factors in database screening
applications. The mathematical form of the Glide score is:
GScore=0.065*EvdW+0.130*Coul+Lipo+Hbond+Metal+BuryP+RotB+Site
[0511] where EvdW is van der Waals energy (calculated with reduced
net ionic charges on groups with formal charges, such as metals,
carboxylates, and guanidiniums); Coul is the Coulomb energy
(calculated with reduced net ionic charges on groups with formal
charges, such as metals, carboxylates, and guanidiniums); Lipo is
the lipophilic contact term (rewards favorable hydrophobic
interactions); HBond is the hydrogen-bonding term (separated into
differently weighted components that depend on whether the donor
and acceptor are neutral, one is neutral and the other is charged,
or both are charged); metal is the metal-binding term (only the
interactions with anionic acceptor atoms are included; if the net
metal charge in the apo protein is positive, the preference for
anionic ligands is included; if the net charge is zero, the
preference is suppressed); BuryP is the penalty for buried polar
groups; RotB is the penalty for freezing rotatable bonds; and Site
is polar interactions in the active site (polar but
non-hydrogen-bonding atoms in a hydrophobic region are
rewarded).
[0512] The following describes generation of the virtual compound
library that was screened. The lead-like compounds from a free,
virtual database of commercially available compounds was downloaded
in structure data format (sdf, Molecular Design Limited) from the
ZINC database (Irwin and Shoichet (2005) J. Chem. Inf. Model.
45(1), 177-182). The lead-like database is comprised of
approximately 890,000 compounds divided into 33 segments. This was
used to generate the database of conformers for screening by MOE.
Hydrogens were then added. For a pharmacophore search, a database
of low energy conformers must be generated. The Conformation Import
command was applied to the sdf file above. After the conformers
were generated, preprocessing of the conformer database was
applied. This step, called feature annotation, determined the types
of pharmacophore features in each molecule/conformation and their
geometrical relationships. This was then compared with the query
and those molecules/conformations that matched the query within the
given tolerance were saved as hits.
[0513] AD4-1886-Like
[0514] Analysis of compounds from the ZINC database against
pharmacophore 21_thr100_glu105 identified from the 1YY9.pdb crystal
of protein EGFR (SEQ ID NO: 1) complexed with antibody cetuximab
(SEQ ID NO: 5 and SEQ ID NO:6) (see e.g., Example 3) according to
the methods described above identified the compound AD4-1886.
##STR00071##
[0515] The compounds in TABLE 9 were identified via AD4-1886
structure similarity searches and were docked to the 1YY9.pdb
binding site (erbitux residues thr100_glu105) to obtain their Glide
and Emodel scores. Also depicted in TABLE 9 is ICW assay and MTT
assay results (see Example 1, Example 2, Example 7).
TABLE-US-00009 TABLE 9 Glide Score, E-Model score, ICW assay, and
MTT Assay Results for AD4-1886-like Compounds. Glue Glue- Erbitux-
Erbitux- Sub- G- E- ICW MTT G- E- AD4-1886 by ICW Inhibition Class
Score Model Screen Screen Score Model ##STR00072## AD4- 11883 O
-6.72 -56.32 83.13 19.51/ non- conver- gent -3.22 -50.29
##STR00073## AD4- 11975 K -5.19 -59.024 75.826 17.52/ 17.16 (0.98)
-5.78 -36.79 ##STR00074## AD4- 11409 K -5.51 -61.783 74.94 17.18/
11.66 (0.68) -2.96 -34.87 ##STR00075## AD4- 11638 O -5.68 -63.037
68.754 11.19/ 14.90 (1.33) -3.24 -50.39 ##STR00076## AD4- 11645 O
-7.29 -61.027 66.452 45.60/ 47.02 (1.03) -3.56 -39.45 ##STR00077##
AD4- 10608 L -6.31 -65.526 62.846 6.33/ 5.35 (1.18) -3.57 -38.88
##STR00078## AD4- 10676 K -4.99 -35.282 59.637 18.58/ 8.84 (0.48)
-3.48 -39.56 ##STR00079## AD4- 12119 K -4.87 -59.115 55.94 18.64/
14.62 (0.78) -1.86 -39.07 ##STR00080## AD4- 10706 K -5.17 -38.332
55.497 15.12/ 23.11 (1.53) -0.32 -35.65 ##STR00081## AD4- 11416 K
-5.11 -57.963 53.517 15.37/ 13.54 (0.88) -5.3 -41.96 ##STR00082##
AD4- 12148 O -5.18 -66.098 51.845 42.82/ 39.24 (0.92) -3.95 -43.35
##STR00083## AD4- 12152 K -6.63 -69.497 50.528 21.02/ 16.20 (0.77)
-3.38 -44.49 ##STR00084## AD4- 10626 M -7.03 -54.119 50.05 5.31/
19.57 (3.68) -4.2 -40.24 ##STR00085## AD4- 10624 L -5.71 -66.733
49.448 25.64/ 16.76 (0.65) -2.01 -33.65 ##STR00086## AD4- 12097 K
-7.24 -55.243 48.543 15.08/ 9.85 (0.65) -3.72 -36.73 ##STR00087##
AD4- 12040 K -5.67 -59.95 47.822 17.13/ 14.82 (0.87) -4.17 -33.64
##STR00088## AD4- 10705 K -4.28 -36.375 47.764 23.09/ 25.20 (1.09)
-0.07 -35.1 ##STR00089## AD4- 11367 O -6.03 -60.038 46.872 43.33/
43.63 (1.01) -3.56 -32.87 ##STR00090## AD4- 11413 K -5.42 -52.398
46.23 31.40/ 29.77 (0.95) 7 -4.38 -41.06 ##STR00091## AD4- 10623 M
-6.86 -51.962 46.185 25.78/ 38.34 (1.49) -4.83 -44.1 ##STR00092##
AD4- 11871 O -5.5 -63.42 45.776 49.72/ 41.84 (0.84) -2.83 -36.58
##STR00093## AD4- 12164 K -5.78 -50.998 45.64 50.58/ non- conver-
gent -4.3 -45.59 51.37/ 549.1 (1.07) ##STR00094## AD4- 11968 K
-7.13 -59.876 45.095 6.49/ 6.29 (0.97) -3.78 -36.75 ##STR00095##
AD4- 11422 K -5.81 -57.435 44.982 29.46/ 9.23 (0.31) -3.75 -38.46
##STR00096## AD4- 11971 K -5.84 -56.864 44.908 41.54/ 41.10 (0.99)
-2.14 -36.33 ##STR00097## AD4- 10609 L -5.54 -63.839 43.828 6.84/
6.34 (0.93) -3.09 -40.73 ##STR00098## AD4- 11369 O -5.24 -57.914
43.546 48.64/ 47.17 (0.97) -3.5 -43.41 ##STR00099## AD4- 12041 L
-5.79 -47.552 43.515 6.61/ 14.29 (2.16) -1.92 -34.81 ##STR00100##
AD4- 11970 N -5.62 -44.739 43.246 non- conver- gent -0.26 -35.75
##STR00101## AD4- 12175 K -3.1 -60.769 42.36 39.99/ 9.56 (0.24)
-3.36 -29.19 ##STR00102## AD4- 12107 N -6.12 -53.813 41.933 14.08/
non- conver- gent -3.53 -38.84 ##STR00103## AD4- 12280 K -4.17
-55.921 41.439 10.23/ 17.68 (1.73) -1.38 -41.29 5.76/ 21.24 (3.69)
##STR00104## AD4- 12139 K -4.11 -63.237 41.23 18.56/ 5.30 (0.29)
-5.93 -41.9 ##STR00105## AD4- 12104 N -5.48 -48.445 41.204 38.98/
37.55 (1.30) -4.1 -36.8 ##STR00106## AD4- 11642 O -6.28 -65.521
40.922 9.89/ 9.46 (0.96) -3.16 -44.18 ##STR00107## AD4- 12146 N
-2.65 -54.642 40.916 26.57/ 25.76 (0.97) -3.96 -31.29 ##STR00108##
AD4- 11417 K -5.19 -59.747 40.711 25.47/ 49.29 (1.94) -4.49 -46.82
##STR00109## AD4- 11985 L -5.94 -48.648 40.346 62.51/ 39.85 (0.64)
-3.44 -30.5 ##STR00110## AD4- 11419 K -5.01 -63.607 40.289 31.32/
38.81 (1.24) -2.95 -44.3 ##STR00111## AD4- 12262 O -5.64 -55.994
40.146 42.85/ 43.44 (1.01) -3.46 -49.03 ##STR00112## AD4- 10120 L
-6.73 -62.371 39.873 22.84/ 39.27 (1.72) -4.84 -37.89 ##STR00113##
AD4- 11979 L -4.83 -50.876 39.775 9.31/ 16.16 (1.74) -2.24 -30.07
##STR00114## AD4- 11648 O -5.95 -54.648 38.362 46.74/ 45.07 (0.96)
-3.43 -42.24 ##STR00115## AD4- 10611 L -6.72 -63.131 38.285 30.94/
20.04 (0.65) -2.77 -42.62 ##STR00116## AD4- 11646 O -5.63 -58.436
37.949 19.43/ 21.11 (1.09) -3.11 -43.16 ##STR00117## AD4- 12126 L
-5.72 -62.255 37.795 14.90/ 41.85 (2.81) -2.57 -35.52 ##STR00118##
AD4- 12183 K -3.58 -64.491 37.695 25.56/ 17.25 (0.46) -2.96 -34.21
##STR00119## AD4- 11880 O -4.5 -62.253 37.604 47.28/ 47.33 (1.00)
-2.65 -45.91 ##STR00120## AD4- 12171 L -5.28 -49.354 37.375 38.92/
37.13 (0.95) -4.18 -43.51 ##STR00121## AD4- 10708 K -5.35 -35.677
37.233 16.64/ 35.92 (2.16) -4 -36.76 ##STR00122## AD4- 11421 N
-5.75 -53.081 37.181 41.46/ 43.51 (1.05) -3.85 -34.1 ##STR00123##
AD4- 12049 L -5.44 -48.47 36.966 32.27/ 11.96 (0.37) -2.43 -24.87
##STR00124## AD4- 12158 J -6.58 -71.788 36.727 5.01/ 4.58 (0.91)
-4.16 -49.79 ##STR00125## AD4- 12165 K -6.81 -66.767 36.632 63.62/
12.03 (0.19) -3.94 -47.61 ##STR00126## AD4- 11415 L -6.25 -70.703
35.841 15.74/ 10.52 (0.67) -4.75 -47.34 ##STR00127## AD4- 12288 K
-5.92 -54.007 35.584 non- conver- gent -4.78 -40.58 ##STR00128##
AD4- 10613 L -5.8 -59.786 35.514 13.92/ 11.84 (0.85) -3.36 -37.53
##STR00129## AD4- 12173 O -4.83 -49.002 35.393 37.26/ 105.8 (2.84)
-2.76 -34.16 55.76/ 54.00 (0.97) ##STR00130## AD4- 12122 O -5.82
-52.564 35.068 non- conver- gent/ 40.29 -2.89 -44.52 ##STR00131##
AD4- 11538 L -6.95 -65.791 34.978 5.48/ 5.19 (0.95) -3.36 -40.64
##STR00132## AD4- 12064 N -4.99 -54.071 34.778 47.78/ 42.73 (0.89)
-2.6 -34.85 ##STR00133## AD4- 12042 K -5.83 -56.582 34.592 non-
conver- gent -2.07 -29.6 ##STR00134## AD4- 12016 L -4.01 -54.12
34.399 15.78/ 23.53 (1.49) -2.74 -30.33 ##STR00135## AD4- 12196 K
-6.07 -70.658 34.379 18.01/ 36.80 (2.04) -1.74 -49.22 ##STR00136##
AD4- 12044 K -5.61 -54.634 34.334 63.18/ 33.11 (0.52) -2.09 -37.5
##STR00137## AD4- 12060 N -5.79 -59.463 34.272 48.53/ 40.47 (0.83)
-3.38 -36.21 ##STR00138## AD4- 12062 K -6.61 -55.772 34.268 64.51/
18.83 (0.29) -3.49 -36.09 ##STR00139## AD4- 11881 O -6.34 -65.317
34.08 63.31/ 46.45 (0.73) -3.58 -44.41 ##STR00140## AD4- 11418 K
-4.89 -66.222 34.067 11.91/ 10.25 (0.86) -3.64 -51.66 ##STR00141##
AD4- 12010 K -7.06 -56.321 33.772 6.68/ 6.16 (0.92) -3.62 -37.4
##STR00142## AD4- 11402 N -4.4 -60.537 33.635 11.82/ 17.36 (1.47)
-3.56 -43.59 ##STR00143## AD4- 11977 N -4.93 -42.763 33.588 32.51/
non- conver- gent -3.11 -28.88 ##STR00144## AD4- 11637 O -5.26
-60.687 33.491 non- conver- gent/ 17.76 -3.04 -46.33 ##STR00145##
AD4- 12149 K -5.38 -67.463 33.331 21.88/ 36.21 (0.60) -2.96 -49.52
##STR00146## AD4- 12273 O -4.61 -53.119 33.104 31.74/ 42.94 (1.35)
-2.59 -34.17 ##STR00147## AD4- 11874 O -6.4 -52.192 32.474 34.98/
38.97 (1.11) -3.3 -48.79 ##STR00148## AD4- 11867 O -4.91 -55.985
32.432 51.01/ 48.09 (0.94) -3.11 -43.51 ##STR00149## AD4- 11885 O
-5.44 -52.547 32.38 37.83/ 48.34 (1.28) -2.88 -40.04 ##STR00150##
AD4- 12003 K -5.54 -53.098 32.007 11.53/ 12.79 (1.11) -4.01
-31.62
##STR00151## AD4- 11412 K -6.18 -58.012 31.958 11.42/ 20.02 (1.75)
-4.65 -40.44 ##STR00152## AD4- 12153 N -6.74 -58.542 31.36 15.58/
21.77 (0.72) -3.2 -49.88 ##STR00153## AD4- 12024 K -5.06 -59.351
31.244 6.77/ 6.34 (0.97) -3.51 -37.04 ##STR00154## AD4- 12265 N
-5.64 -51.979 31.19 38.12/ 35.06 (0.92) -4.14 -42.09 ##STR00155##
AD4- 11870 O -6.19 -57.173 30.849 55.07/ 47.30 (0.86) -3.16 -44.1
##STR00156## AD4- 10612 L -6.39 -64.392 30.841 11.68/ 13.21 (1.13)
-1.5 -37.91 ##STR00157## AD4- 10313 L -2.31 -36.062 30.774 20.85/
17.62 (0.85) -3.5 -38.52 ##STR00158## AD4- 11396 K -5.9 -61.408
30.745 25.09/ 21.23 (0.85) -4.36 -46.45 ##STR00159## AD4- 12145 N
-6.3 -51.616 30.469 40.50/ 41.75 (1.03) -1.35 -41.29 ##STR00160##
AD4- 12029 K -5.31 -45.509 30.376 25.32/ 51.15 (2.02) -3.9 -42.48
##STR00161## AD4- 12267 J -5.05 -43.093 30.36 non- conver- gent
-3.71 -30.79 ##STR00162## AD4- 11861 O -6.6 -58.006 30.242 -2.03
-43.96 ##STR00163## AD4- 11420 N -4.8 -54.408 30.052 -3.02 -32.21
##STR00164## AD4- 12039 L -6.45 -54.185 29.977 -3.16 -35.07
##STR00165## AD4- 12074 K -5.08 -55.471 29.545 16.24/ 32.28 (1.99)
-2.98 -39.72 ##STR00166## AD4- 12093 K -6.31 -46.102 29.436 non-
conver- gent -2.96 -39.97 ##STR00167## AD4- 12073 L -5.45 -59.824
29.313 19.54/ 32.28 (1.65) -5.06 -41.61 11.59/ 16.97 (1.46)
##STR00168## AD4- 10103 M -5.14 -48.394 29.295 -4.54 -44.4
##STR00169## AD4- 12266 J -5.78 -68.303 29.289 49.64/ 45.88 (0.92)
-4.54 -53.86 ##STR00170## AD4- 11385 J -5.17 -53.196 28.892 -2.89
-29.47 ##STR00171## AD4- 12051 L -5.4 -55.888 28.852 -3.34 -31.94
##STR00172## AD4- 12168 K -6 -59.724 28.819 non- conver- gent -3.48
-33.83 60.47/ 19.68 (0.33) ##STR00173## AD4- 11424 N -5.82 -59.614
28.422 -3.64 -28.82 ##STR00174## AD4- 12059 N -4.54 -58.82 28.33
32.25/ 37.69 (1.17) -3.42 -40.97 ##STR00175## AD4- 11384 J -5.62
-60.184 28.127 -3.68 -47.04 ##STR00176## AD4- 11410 L -5.68 -61.822
28.101 -3.84 -40.3 ##STR00177## AD4- 12071 L -4.56 -58.755 27.713
-4.19 -41.48 ##STR00178## AD4- 11407 K -5.89 -52.032 27.585 -3.55
-37.01 ##STR00179## AD4- 11536 J -6.57 -53.881 27.431 -3.46 -46.82
##STR00180## AD4- 12017 K -6.1 -52.279 27.373 6.53/ 5.93 (0.91)
-5.41 -35.72 ##STR00181## AD4- 11863 O -5.54 -59.966 27.36 -1.92
-39.12 ##STR00182## AD4- 11994 K -6.23 -55.137 27.18 -1.85 -35.39
##STR00183## AD4- 11973 N -4.87 -45.457 27.176 -3.5 -42.87
##STR00184## AD4- 12147 N -3 -57.789 26.967 -0.46 -40.6
##STR00185## AD4- 11976 L -2.46 -54.259 26.918 -2.33 -34.3
##STR00186## AD4- 11406 N -7.04 -54.126 26.851 -4.09 -39.79
##STR00187## AD4- 12286 N -4.81 -52.846 26.829 15.46/ 29.94 (1.94)
-4.26 -32.26 ##STR00188## AD4- 12157 L -4.38 -52.787 26.73 -1.28
-39.47 ##STR00189## AD4- 12163 J -6.08 -59.91 26.671 -3.35 -43.82
##STR00190## AD4- 11405 J -6.94 -58.643 26.497 -4.41 -53.71
##STR00191## AD4- 11888 O -6.74 -63.413 26.355 -3.37 -41.13
##STR00192## AD4- 11395 J -6.11 -55.753 26.301 -4.63 -49.78
##STR00193## AD4- 12103 K -7.21 -63.637 26.212 -3.1 -44.09
##STR00194## AD4- 11523 K -6 -59.162 26.098 -3.83 -33.07
##STR00195## AD4- 12075 L -6.25 -65.453 25.956 19.24/ 40.11 (2.08)
-7.04 -41.76 ##STR00196## AD4- 12098 K -5.89 -53.46 25.946 23.36/
16.41 (0.70) 0.121 -33.78 ##STR00197## AD4- 11879 O -4.9 -68.161
25.689 -2.48 -47.6 ##STR00198## AD4- 12014 K -5.59 -65.343 25.611
-3.5 -34.96 ##STR00199## AD4- 11401 N -5.59 -56.827 25.557 -3.9
-51.43 ##STR00200## AD4- 11978 L -3.01 -51.532 25.53 -2.15 -31.77
##STR00201## AD4- 11986 L -5.62 -53.997 25.368 -4.32 -35.72
##STR00202## AD4- 10622 L -5.32 -69.992 25.347 -5 -49.48
##STR00203## AD4- 11980 J -4.48 -57.693 25.235 49.82/ 24.40 (0.49)
-2.39 -48.62 ##STR00204## AD4- 12291 M -7.05 -58.86 25.191 -3.47
-34.09 ##STR00205## AD4- 12133 L -5.78 -42.682 25.058 -0.57 -35.65
##STR00206## AD4- 12092 L -4.77 -52.761 25.019 -1.58 -29.14
[0516] AD4-11091-Like
[0517] The AD4-11091-like compounds in TABLE 10 were identified via
Glide docked pose of AD4-1886. Depicted in TABLE 10 is Glide score,
E-model score, and ICW assay and MTT assay results (See Examples
1-3).
TABLE-US-00010 TABLE 10 Glide Score, E-Model score, ICW assay, and
MTT Assay Results for AD4-11091-like Compounds. Glue- Glue- Erbitux
Erbitux AD4- G- E- ICW -G- -E- Pharmacophore AD4-11091 LIKE by ICW
Inhibition No. Score Model assay MTT assay Score Model Query
##STR00207## AD4- 12509 -6.15 -54.56 140.975 5.49/5.24(0.95) -2.169
-37.161 1YY9-AD4- 11091 LIKE ##STR00208## AD4- 12423 -5.16 -62.7
129.155 6.35/5.77(0.91) 4.23/2.92(0.69) -4.008 -31.651 1YY9-AD4-
11091 LIKE ##STR00209## AD4- 12528 -7.06 -54.89 123.946
5.43/5.30(0.98) -4.226 -40.6 1YY9-AD4- 11091 LIKE ##STR00210## AD4-
12522 -5.62 -59.43 123.893 6.20/5.33(0.86) -4.336 -39.519 1YY9-AD4-
11091 LIKE ##STR00211## AD4- 12851 -6.66 -56.44 118.637
6.51/5.39(0.83) -3.534 -35.395 1YY9-AD4- 11091 LIKE ##STR00212##
AD4- 12504 -5.98 -54.77 118.46 11.09/15.61(1.41) -3.478 -32.583
1YY9-AD4- 11091 LIKE ##STR00213## AD4- 12835 -4.47 -54.41 116.579
6.15/5.89(0.96) -2.989 -24.087 1YY9-AD4- 11091 LIKE ##STR00214##
AD4- 12833 -4.64 -56.3 115.634 5.94/5.96(1.00) -3.931 -36.628
1YY9-AD4- 11091 LIKE ##STR00215## AD4- 12494 -5.78 -61.9 112.965
5.64/5.71(1.01) -3.005 -37.074 1YY9-AD4- 11091 LIKE ##STR00216##
AD4- 11091 -4.2 -43.59 112.858 non-convergent -3.987 -42.271
1NQL-AD4- 1734 top 3 ##STR00217## AD4- 12844 -5.23 -51.72 112.768
6.80/6.13(0.90) -1.121 -36.357 1YY9-AD4- 11091 LIKE ##STR00218##
AD4- 12436 -6.93 -61.5 108.826 5.47/5.53(1.01) -3.738 -34.38
1YY9-AD4- 11091 LIKE ##STR00219## AD4- 12505 -5.92 -59.81 93.3284
5.69/6.02(1.06) -3.807 -37.664 1YY9-AD4- 11091 LIKE ##STR00220##
AD4- 12487 -6.35 -56.12 84.5602 6.45/5.50(0.85) -3.209 -35.185
1YY9-AD4- 11091 LIKE ##STR00221## AD4- 12424 -6.87 -61.52 82.3761
4.08/4.73(1.16) -3.929 -44.784 1YY9-AD4- 11091 LIKE ##STR00222##
AD4- 12430 -5.64 -61.33 82.0877 4.07/5.54(1.36) -4.089 -37.858
1YY9-AD4- 11091 LIKE ##STR00223## AD4- 12842 -5.06 -58.74 80.8965
6.72/5.55(0.83) -4.203 -41.175 1YY9-AD4- 11091 LIKE ##STR00224##
AD4- 12503 -6.81 -57.7 79.4624 4.05/5.43(1.34) -4.398 -46.889
1YY9-AD4- 11091 LIKE ##STR00225## AD4- 12442 -5.97 -64.21 79.1185
5.59/5.76(1.03) -3.929 -36.195 1YY9-AD4- 11091 LIKE ##STR00226##
AD4- 12468 -6.39 -57.69 77.7928 4.04/5.41(1.34) -3.531 -39.573
1YY9-AD4- 11091 LIKE ##STR00227## AD4- 12389 -5.83 -54.1 72.5659
12.15/6.07(0.50) -1.372 -36.929 1YY9-AD4- 11091 LIKE ##STR00228##
AD4- 12846 -4.89 -53.74 71.4924 71% at 2.6 microMolar
5.55/5.38(0.97) -4.09 -40.727 1YY9-AD4- 11091 LIKE ##STR00229##
AD4- 12501 -6.53 -53.93 71.3823 4.06/4.06(1.00) -4.39 -49.596
1YY9-AD4- 11091 LIKE ##STR00230## AD4- 12400 -6.67 -56.3 69.4793
4.05/5.77(1.42) -4.487 -36.418 1YY9-AD4- 11091 LIKE ##STR00231##
AD4- 12239 -4.79 -57.44 67.8291 58% at 2.6 microMolar
16.13/13.77(0.85) -3.787 -33.019 1YY9-AD4- 11091 LIKE ##STR00232##
AD4- 12472 -4.86 -50.57 64.2647 6.23/5.42(0.87) -4.241 -30.519
1YY9-AD4- 11091 LIKE ##STR00233## AD4- 12529 -7.76 -56.15 63.9249
6.35/6.47(1.02) -4.728 -39.186 1YY9-AD4- 11091 LIKE ##STR00234##
AD4- 12395 -4.85 -55.34 57.5206 7.92/6.96(0.88) -4.241 -44.652
1YY9-AD4- 11091 LIKE ##STR00235## AD4- 12783 -6.55 -55 57.4272
7.00/7.62(1.09) -4.334 -34.188 1YY9-AD4- 11091 LIKE ##STR00236##
AD4- 12433 -5.66 -62.36 54.4095 29.06/38.50(1.32) -3.35 -37.559
1YY9-AD4- 11091 LIKE ##STR00237## AD4- 12438 -6.13 -61.5 53.245
5.69/5.90(1.04) -4.367 -31.989 1YY9-AD4- 11091 LIKE ##STR00238##
AD4- 12461 -5.04 -52.58 53.1391 4.06/5.91(1.46) -3.308 -39.144
1YY9-AD4- 11091 LIKE ##STR00239## AD4- 10700 -6.44 -62.8 52.5996
14.69/14.35(0.98) -1.113 -36.75 1YY9-AD4- 1886 Dockpharm
##STR00240## AD4- 12822 -4.19 -52.61 51.4082 6.92/7.04(1.02) -3.072
-33.644 1YY9-AD4- 11091 LIKE ##STR00241## AD4- 12403 -5.67 -58.34
50.7196 4.03/5.30(1.32) -2.864 -39.727 1YY9-AD4- 11091 LIKE
##STR00242## AD4- 12798 -6.21 -53.39 49.531 4.74/5.69(1.20) -3.7
-41.52 1YY9-AD4- 11091 LIKE ##STR00243## AD4- 12820 -6.64 -59.05
47.471 5.98/5.93(0.99) -3.737 -43.758 1YY9-AD4- 11091 LIKE
##STR00244## AD4- 11432 -6.34 -60.05 47.0006 7.80/7.82(1.00) -2.138
-42.697 1YY9-AD4- 11091 LIKE ##STR00245## AD4- 12829 -4.49 -36.92
46.4407 36.97/37.27(1.01) -4.396 -24.523 1YY9-AD4- 11091 LIKE
##STR00246## AD4- 12836 -5.44 -57.16 46.1658 9.32/8.43(0.90) -4.633
-35.888 1YY9-AD4- 11091 LIKE ##STR00247## AD4- 12827 -6.59 -56.14
45.9206 6.49/6.40(0.99) -4.353 -37.272 1YY9-AD4- 11091 LIKE
##STR00248## AD4- 12521 -5.59 -54.87 45.7824 6.99/6.54(1.22) -3.596
-28.511 1YY9-AD4- 11091 LIKE ##STR00249## AD4- 11427 -6.36 -57.89
44.904 11.97/14.48(1.21) -3.173 -34.903 1YY9-AD4- 11091 LIKE
##STR00250## AD4- 12443 -5.91 -55.75 44.7896 6.71/6.91(1.03) -3.756
-40.035 1YY9-AD4- 11091 LIKE ##STR00251## AD4- 12477 -5.1 -56.94
44.2167 6.65/5.44(0.82) -4.704 -45.689 1YY9-AD4- 11091 LIKE
##STR00252## AD4- 12843 -4.42 -54.07 43.3347 74% at 2.6 microMolar
6.26/12.23(1.95) -3.616 -45.969 1YY9-AD4- 11091 LIKE ##STR00253##
AD4- 12460 -6.63 -60.84 42.5115 6.33/5.61(0.87) -3.956 -34.204
1YY9-AD4- 11091 LIKE ##STR00254## AD4- 12560 -6.6 -67.91 42.3275
8.88/5.23(0.59) -2.493 -38.312 1YY9-AD4- 11091 LIKE ##STR00255##
AD4- 12850 -4.74 -59.3 42.2628 31.78/35.77(1.13) -5.454 -36.419
1YY9-AD4- 11091 LIKE ##STR00256## AD4- 12481 -6.81 -56.97 42.006
10.38/11.04(1.06) -4.203 -42.388 1YY9-AD4- 11091 LIKE ##STR00257##
AD4- 12435 -5.71 -52.17 41.4281 6.67/6.15(0.92) -3.596 -34.513
1YY9-AD4- 11091 LIKE ##STR00258## AD4- 12854 -6.57 -55.99 38.7055
6.64/8.19(1.23) -3.134 -34.386 1YY9-AD4- 11091 LIKE ##STR00259##
AD4- 12246 -6.97 -57.98 38.3844 8.36/8.01(0.96) -3.234 -36.483
1YY9-AD4- 11091 LIKE ##STR00260## AD4- 12391 -6.13 -58.89 37.4191
11.09/9.09(0.82) -1.131 -37.349 1YY9-AD4- 11091 LIKE ##STR00261##
AD4- 12853 -5.17 -52.29 36.92 8.76/7.51(0.86) -4.265 -38.827
1YY9-AD4- 11091 LIKE ##STR00262## AD4- 12413 -5.58 -54.77 35.1946
6.83/6.35(0.93) -4.789 -34.803 1YY9-AD4- 11091 LIKE ##STR00263##
AD4- 12837 -6.48 -49.2 35.1542 6.28/5.68(0.90) -3.525 -37.042
1YY9-AD4- 11091 LIKE ##STR00264## AD4- 11344 -5.87 -60.29 34.1727
15.06/15.26(1.01) -2.857 -32.85 1YY9-AD4- 11091 LIKE ##STR00265##
AD4- 12525 -6.67 -61.47 34.1311 14.75/12.15(0.82) -3.692 -39.056
1YY9-AD4- 11091 LIKE ##STR00266## AD4- 12411 -6.38 -56.96 34.0556
6.70/5.18(0.77) -3.267 -31.845 1YY9-AD4- 11091 LIKE ##STR00267##
AD4- 12485 -5.32 -49.68 33.3993 6.98/7.19(1.03) -3.785 -35.542
1YY9-AD4- 11091 LIKE ##STR00268## AD4- 12828 -4.75 -55.94 32.6456
26.61/63.39(2.38) -4.427 -40.661 1YY9-AD4- 11091 LIKE ##STR00269##
AD4- 12527 -6.11 -56.16 31.9587 26.91/20.01(0.74) -4.98 -40.481
1YY9-AD4- 11091 LIKE ##STR00270## AD4- 12517 -6.51 -58.67 31.5308
30.39/20.01(0.66) -3.452 -41.81 1YY9-AD4- 11091 LIKE ##STR00271##
AD4- 12465 -5.59 -56.91 31.3476 9.92/6.54(0.66) -3.435 -35.487
1YY9-AD4- 11091 LIKE ##STR00272## AD4- 12823 -3.94 -58.13 30.8167
4.07/4.06(1.00) -1.408 -47.23 1YY9-AD4- 11091 LIKE ##STR00273##
AD4- 12537 -6.29 -58.32 30.7051 35.37/38.65(1.09) -3.88 -38.727
1YY9-AD4- 11091 LIKE ##STR00274## AD4- 11428 -6.22 -59.58 29.8673
20.77/15.46(0.74) -3.692 -42.78 1YY9-AD4- 11091 LIKE ##STR00275##
AD4- 12577 -6.85 -61.65 29.4151 6.62/5.92(0.89) -4.289 -30.521
1YY9-AD4- 11091 LIKE ##STR00276## AD4- 12250 -6.65 -62.41 29.3683
12.68/11.57(0.91) -3.033 -43.246 1YY9-AD4- 11091 LIKE ##STR00277##
AD4- 12832 -3.94 -58.13 29.02 7.71/16.30(2.11) -1.408 -47.23
1YY9-AD4- 11091 LIKE ##STR00278## AD4- 12512 -5.28 -56.81 28.9126
6.40/5.83(0.91) -3.555 -34.118 1YY9-AD4- 11091 LIKE ##STR00279##
AD4- 12482 -7.87 -59.36 28.3382 6.54/6.05(0.93) -5.834 -34.551
1YY9-AD4- 11091 LIKE ##STR00280## AD4- 11430 -6.25 -48.49 27.1482
19.83/16.84(0.85) -3.823 -33.209 1YY9-AD4- 11091 LIKE ##STR00281##
AD4- 10693 -6.87 -60.55 26.8206 48.24/35.85(0.74) -2.528 -44.456
1YY9-AD4- 1886 Dockpharm ##STR00282## AD4- 12825 -5.72 -54.54
26.7866 6.78/5.46(0.81) -4.103 -37.363 1YY9-AD4- 11091 LIKE
##STR00283## AD4- 12431 -5.95 -50.11 26.7815 6.40/5.57(0.87) -4.12
-36.705 1YY9-AD4- 11091 LIKE ##STR00284## AD4- 12813 -6.36 -51.7
26.1431 11.76/13.63(1.16) -5.202 -33.055 1YY9-AD4- 11091 LIKE
##STR00285## AD4- 12499 -5.38 -58.36 26.0767 6.99/6.58(0.94) -2.372
-38.545 1YY9-AD4- 11091 LIKE ##STR00286## AD4- 12824 -4.66 -56.6
25.9501 Not Active -2.832 -37.478 1YY9-AD4- 11091 LIKE
##STR00287## AD4- 12502 -5.63 -60.71 25.1182 6.97/5.46(0.78) -2.992
-38.026 1YY9-AD4- 11091 LIKE ##STR00288## AD4- 12855 -4.57 -57.09
25.0542 6.83/6.25(0.92) -4.8 -33.063 1YY9-AD4- 11091 LIKE
[0518] Two-dimensional representations of the docked pose of
AD4-1886, along with AD4-1886-like compounds were produced. Docking
of compound AD4-1886 to EGFR is depicted, for example, in FIG. 6A.
Docking of compound AD4-11883 (an AD4-1886-like compound) to EGFR
is depicted, for example, in FIG. 6B. Docking of compound AD4-11975
(an AD4-1886-like compound) to EGFR is depicted, for example, in
FIG. 6C. Docking of compound AD4-11409 (an AD4-1886-like compound)
to EGFR is depicted, for example, in FIG. 6D. Docking of compound
AD4-11638 (an AD4-1886-like compound) to EGFR is depicted, for
example, in FIG. 6E. Docking of compound AD4-11645 (an
AD4-1886-like compound) to EGFR is depicted, for example, in FIG.
6F.
Example 7
Ligand Docking and Scoring to Inactive EGFR Protein
[0519] The compounds selected for docking to the target protein,
the inactive folded conformation of EGFr (PDB accession number
1NQL) were those which were found to align to the pharmacophore
models generated in the MOE modeling software (see Example 4).
[0520] Methods are according to those described in Example 6,
except as otherwise indicated.
[0521] The protein crystal structure of EGFr in its inactive state
(1NQL.PBD) was imported into Maestro in PDB format. The binding
site was determined by picking a ligand, ZINC3304802 which was one
of the pharmacophore hits found by MOE, in Maestro (Glide).
[0522] Analysis of compounds from the ZINC database against the
pharmacophores identified from the 1NQL.PDB crystal structure of
protein EGFR according to the methods described above identified
compounds AD4-1734, and AD4-10381.
##STR00289##
[0523] The compounds in the following tables were identified via
structure similarity searches and were docked to the binding site
to obtain their Glide and Emodel scores.
[0524] AD4-1734-Like
[0525] The compounds in TABLE 11 were identified via AD4-1734
structure similarity searches and were docked to the 1NQL.PDB
binding site to obtain their Glide and Emodel scores. Also depicted
in TABLE 11 is ICW assay and MTT assay results (See Example 1,
Example 2, Example 7).
TABLE-US-00011 TABLE 11 Glide Score, E-Model score, ICW assay, and
MTT Assay Results for AD4-1734-like Compounds. Sub- E- G- ICW
Pharmacophore AD4 No. Class Model Score Screen MTT Screen MTT
Follow-up Query ##STR00290## AD4- 10631 P -4.999 -46.538 104.48
35.64/37.47 (1.05) 1NQL-Glue-AD4- 1734 LIKE ##STR00291## AD4-1734 R
-6.985 -47.431 65.9 22.75/42.50 (1.87) 22.1/40.2(1.82) 1NQL-Glue
##STR00292## AD4- 10188 Q -6.118 -47.713 53.35 9.05/16.32 (1.80)
1NQL-Glue-AD4- 1734 LIKE ##STR00293## AD4- 10186 P -5.204 -49.183
47.518 40.10/45.74 (1.14) 1NQL-Glue-AD4- 1734 LIKE ##STR00294##
AD4- 10633 P -5.019 -48.58 46.398 5.20/non- convergent Not
Convergent 1NQL-Glue-AD4- 1734 LIKE ##STR00295## AD4- 10174 R
-7.534 -48.44 45.85 2.26/8.58 (3.80) 4.42/101.2 (22.9)
1NQL-Glue-AD4- 1734 LIKE ##STR00296## AD4- 10639 S -5.635 -48.32
43.695 42.59/non-convergent 27% Max & -12% Max 1NQL-Glue-AD4-
1734 LIKE ##STR00297## AD4- 10176 Q -7.195 -47.579 42.066 50% Max
& 11% Max 1NQL-Glue-AD4- 1734 LIKE ##STR00298## AD4- 10182 P
-5.62 -40.619 39.51 6.78/43.31 (6.39) 86% Max & 15% Max
3.75/non- convergent 1NQL-Glue-AD4- 1734 LIKE ##STR00299## AD4-
10171 Q -6.276 -48.614 39.389 16.52/21.02(1.27) 1NQL-Glue-AD4- 1734
LIKE ##STR00300## AD4- 10178 Q -6.311 -41.019 34.907 22% Max &
-19% Max 1NQL-Glue-AD4- 1734 LIKE ##STR00301## AD4- 10179 S -5.484
-53.221 34.742 13.16/13.87(1.05) 38% Max & 20% Max
1NQL-Glue-AD4- 1734 LIKE ##STR00302## AD4- 10181 R -5.427 -45.217
34.604 2.61/15.96 (6.11) 38% Max & 5% Max 1NQL-Glue-AD4- 1734
LIKE ##STR00303## AD4- 10180 P -6.42 -43.904 32.1 16.72/36.31(2.17)
70% Max & 13% Max 1NQL-Glue-AD4- 1734 LIKE ##STR00304## AD4-
10637 R -6.977 -48.57 30.057 20.81/41.33(1.99) 1NQL-Glue-AD4- 1734
LIKE ##STR00305## AD4- 10491 R -5.677 -45.143 29.223
22.22/40.61(1.83) 1NQL-Glue-AD4- 1734 LIKE ##STR00306## AD4- 10192
-5.691 -47.96 16.772 16.51/43.30 (2.62) 24% Max & 19% Max
1NQL-Glue-AD4- 1734 LIKE ##STR00307## AD4- 10217 -5.043 -45.23
-1.007 14.02/non-convergent 44% Max & 6% Max 1NQL-Glue-AD4-
1734 LIKE ##STR00308## AD4- 10618 -5.33 -55.695 -3.062
18.43/non-convergent 86% Max & 13% Max 1NQL-Glue-AD4- 1734 LIKE
##STR00309## AD4- 10209 -5.614 -42.711 -10.6 87% Max & 39% Max
2.50/50.15(20.0) 1NQL-Glue-AD4- 1734 LIKE ##STR00310## AD4- 10619
-6.255 -55.21 -44.55 87% @ 2.6 .mu.M 2.97/3.06(1.03) 1NQL-Glue-AD4-
1734 LIKE
[0526] AD4-10381-Like
[0527] The compounds in TABLE 12 were identified via AD4-10381
structure similarity searches and were docked to the 1NQL.PDB
binding site to obtain their Glide and Emodel scores. Also depicted
in TABLE 12 is ICW assay and MTT assay results (See Example 1,
Example 2, Example 7).
TABLE-US-00012 TABLE 12 Glide Score, E-Model score, ICW assay, and
MTT Assay Results for AD4-10381-like Compounds. AD4 Sub- G- E- ICW
ICW ICW MTT Assay MTT Follow- Structure No. Class Score Model 1 2
Avg IC.sub.50(A431/MDBK) up ##STR00311## 12632 D -5.83 -34.77 89.66
92.56 91.11 5.67/5.59(0.99) ##STR00312## 12681 E -4.03 -45.89 89.93
89.02 89.48 15.63/25.40(1.63) ##STR00313## 12732 D -5.1 -47.5 79.12
86.41 82.77 11.07/14.95(1.35) ##STR00314## 10381 + TFA D -6.02
-50.11 87.04 78.48 82.76 2.10/3.01(1.43) ##STR00315## 11511 D -4.58
-48.54 77.05 87.95 82.50 95% at 2.6 mM 1.26/0.57(0.45) ##STR00316##
11462 D -5.28 -51.09 77.14 77.49 77.32 14.60/46.58(3.19)
##STR00317## 11772 + TFA D -5.5 -43.52 80.68 73.50 77.09
9.75/9.04(0.93) ##STR00318## AD4- 12652 + TFA D -5.33 -42.7 82.35
64.55 73.45 NOT ACTIVE ##STR00319## AD4- 12641 + TFA D -5.24 -47.81
70.26 74.15 72.20 NOT ACTIVE ##STR00320## 11484 + TFA F -5.62
-44.77 67.84 73.46 70.65 11.34/9.72(0.86) ##STR00321## 12679 E
-4.15 -50.62 73.98 67.01 70.50 15.14/42.02(2.78) ##STR00322## 11741
D -3.89 -52.17 66.55 69.55 68.05 2.44/2.44 51.51/51.89 (1.01)
##STR00323## 12723 + TFA D -5.93 -44 67.34 65.91 66.62
9.33/9.99(1.07) ##STR00324## 11829 E -4.74 -36.8 62.56 68.79 65.68
19.57/16.18 ##STR00325## 12645 + TFA D -6.1 -43.6 60.00 69.95 64.97
6.49/6.29(0.97) ##STR00326## 12735 + TFA D -5.8 -44.56 69.46 59.98
64.72 17.92/15.32(0.85) ##STR00327## 11636 E -5.64 -48.26 61.28
63.84 62.56 26.75/14.70 ##STR00328## 11492 D -5.66 -48.59 53.35
71.23 62.29 non-convergent ##STR00329## 12660 + TFA D -5.55 -47.79
49.87 73.40 61.63 41.54/41.10(0.99) ##STR00330## 11445 + TFA D
-4.56 -56.29 59.54 61.02 60.28 9.61/45.14(4.70) ##STR00331## 11783
+ TFA D -5.05 -44.32 66.95 53.44 60.19 18.22/14.99(0.82)
##STR00332## 11436 + TFA F -4.46 -43.34 59.16 60.14 59.65
5.86/2.80(0.48) ##STR00333## 11472 D -5.57 -43.39 51.74 66.90 59.32
24.68/19.35 ##STR00334## 12612 + TFA D -5.87 -46.47 69.21 45.51
57.36 48.51/79.16 ##STR00335## 12693 + TFA E -4.92 -42.18 40.36
74.00 57.18 9.01/6.88(0.76) ##STR00336## 11812 E -4.52 -47.03 51.90
61.77 56.84 14.65/31.61 ##STR00337## 11601 D -6.11 -52.7 58.11
54.06 56.08 1.29/2.62 0.738/2.61(normal- ized) 3.67/48.56 (13.23)
##STR00338## 11825 E -4.03 -50.59 52.17 55.87 54.02 4.06/17.94
##STR00339## 12673 + TFA D -5.89 -44.35 44.70 60.90 52.80
33.08/22.52(0.68) ##STR00340## 12613 D -5.37 -45.92 47.72 56.65
52.18 17.58/11.50(0.65) ##STR00341## 11583 + TFA D -5.58 -53.92
47.93 54.26 51.10 47.28/47.33(1.00) ##STR00342## 12604 + TFA D
-3.38 -55.15 55.87 46.08 50.98 19.30/29.98(1.55) ##STR00343## 11316
E -5.01 -42.35 47.95 53.57 50.76 44.42/19.89(0.45) ##STR00344##
11450 D -2.82 -58.11 53.35 48.02 50.68 6.98/6.83 ##STR00345## 11582
D -6.23 -55.72 59.77 40.01 49.89 2.43/2.44 49.26/55.85 (1.13)
##STR00346## 11315 E -4.77 -43 48.33 50.82 49.58 very selective
MDBK 20% at 100 .mu.M ##STR00347## 11498 D -5.12 -47.66 42.15 56.48
49.32 5.39/8.77 ##STR00348## 12742 D -5.3 -45.02 33.49 64.92 49.20
19.60/18.95(0.97) ##STR00349## 12636 E -4.58 -41.6 49.55 48.49
49.02 15.88/34.63(2.18) ##STR00350## 11758 D -5.73 -43.23 53.63
41.93 47.78 53.81/576.2 ##STR00351## 11771 + TFA D -4.55 -44.55
51.58 42.32 46.95 18.17/42.13(2.32) ##STR00352## 12611 + TFA D
-5.46 -53.71 52.69 38.76 45.72 5.32/5.31(1.00) ##STR00353## 12738 +
TFA D -5.5 -46.21 53.67 37.02 45.35 45.36/24.62(0.54) ##STR00354##
11753 + TFA D -5.13 -57.6 42.44 47.28 44.86 NOT ACTIVE ##STR00355##
12665 + TFA D -5.51 -43.87 51.06 37.29 44.17 17.52/17.16(0.98)
##STR00356## 11448 D -4.63 -45.4 50.06 37.38 43.72 3.45/11.8(3.27)
##STR00357## 12736 D -5.39 -46.96 32.35 54.51 43.43 7.14/9.82(1.38)
##STR00358## 11607 + TFA D -6.52 -43.34 43.69 41.73 42.71
10.08/8.03(0.80) ##STR00359## 12608 + TFA D -5.19 -51.11 46.64
38.07 42.36 43.13/17.03(0.80)_ ##STR00360## 12737 D -5.3 -47.17
38.90 45.65 42.27 14.72/6.33(0.43) ##STR00361## 12609 D -5.81
-43.86 42.02 40.07 41.04 11.19/23.49(2.10) ##STR00362## 12683 + TFA
D -5.91 -44.79 54.37 27.38 40.88 63.42/45.31(0.71) ##STR00363##
12674 D -5.8 -34.96 29.69 51.69 40.69 10.67/8.41(0.79) ##STR00364##
11707 + TFA D -4.94 -55.26 43.70 37.31 40.51 15.70/non-convergent
##STR00365## 11474 D -4.35 -53.39 32.19 48.71 40.45
3.57/28.48(7.98) ##STR00366## 11471 + TFA D -4.39 -57.49 37.43
42.40 39.92 36.91/44.14(1.20) ##STR00367## 11712 + TFA D -4.74
-48.22 32.96 46.11 39.53 18.74/17.32(0.92) ##STR00368## 11730 + TFA
D -5.24 -46.64 47.15 30.67 38.91 14.48/10.05(0.69) ##STR00369##
12226 + TFA D -5.67 -51.38 42.24 35.23 38.74 42.50/49.01(1.15)
##STR00370## 12603 + TFA D not docked not docked 48.06 29.15 38.61
16.20/10.11(0.62) ##STR00371## 11442 D -5.57 -51.14 50.14 25.76
37.95 15.63/118.5 ##STR00372## 12778 D -3.8 -52.22 31.05 44.08
37.56 4.66/5.34(1.15) ##STR00373## 11455 E -5.64 -40.8 48.80 25.90
37.35 10.84/23.41(2.16) ##STR00374## 11826 + TFA D -5.44 -59.4
40.52 33.80 37.16 49.43/43.30(0.88) ##STR00375## 11449 E -3.52
-42.05 48.00 26.22 37.11 6.41/5.40(0.84) ##STR00376## 11755 D -5.33
-64.06 40.96 33.21 37.09 non-convergent 14.14/3.24 (0.23)
##STR00377## 11309 E -5.51 -42.35 33.31 40.70 37.00 ##STR00378##
12666 + TFA D -5.29 -49.45 48.16 25.37 36.77 14.48/17.13(1.18)
##STR00379## 12688 + TFA E -5.44 -42.43 45.86 26.95 36.40
5.04/3.58(0.71) ##STR00380## 12654 + TFA D -5.9 -44.23 37.92 34.22
36.07 15.95/12.62(0.79) ##STR00381## 11742 + TFA D -5.95 -57.7
33.84 37.63 35.74 16.09/non-convergent ##STR00382## 11456 D -5.61
-49.17 33.74 37.33 35.54 5.84/5.48(0.94) ##STR00383## 11785 + TFA D
-3.71 -54.57 41.06 30.01 35.54 10.35/9.15(0.88) 24.82/25.91 (1.04)
##STR00384## 12661 D -4.69 -40.42 29.34 41.14 35.24
43.15/38.19(0.89) ##STR00385## 12214 + TFA D -5.35 -48.28 42.29
27.52 34.91 32.43/45.47(1.40) ##STR00386## 11706 + TFA F -1.8
-49.31 37.65 30.99 34.32 17.60/25.45(1.45) ##STR00387## 12625 + TFA
D -5.41 -45.41 30.45 38.10 34.27 21.20/17.03(0.80) ##STR00388##
12739 D -5.22 -42.52 28.51 39.54 34.03 15.90/9.17(0.58)
##STR00389## 11476 D -5.09 -57.08 36.71 31.11 33.91
17.86/13.29(0.74) ##STR00390## 12741 + TFA D -5.68 -44.51 42.10
25.04 33.57 63.20/28.04(0.44) ##STR00391## 11795 + TFA D -4.61
-48.34 35.23 31.81 33.52 26.80/12.87(0.48) ##STR00392## 11773 + TFA
D -5.37 -48.14 43.74 23.30 33.52 39.36/20.99(0.53) ##STR00393##
11294 D -6.17 -45.01 29.81 37.13 33.47 6.43/19.78(3.08)
##STR00394## 11513 + TFA D -5.96 -56.74 32.64 34.26 33.45
6.73/6.32(0.94) ##STR00395## 11800 + TFA D -4.31 -58.11 34.26 32.02
33.14 6.71/5.86(0.87) ##STR00396## 12620 D -4.98 -47.62 32.26 33.77
33.01 5.17/5.30(1.03) ##STR00397## 11809 + TFA D -6.46 -44.94 31.42
34.02 32.72 39.28/10.88(0.28) ##STR00398## 12746 D -5.22 -48.45
18.02 47.28 32.65 17.86/11.89(0.67) ##STR00399## 12607 + TFA D
-5.11 -44.98 38.40 26.85 32.62 16.57/11.83(0.71) ##STR00400## 11759
+ TFA D -5.06 -46.66 38.43 26.39 32.41 14.20/7.36(0.52)
##STR00401## 11503 D -5.4 -50.16 25.88 38.53 32.21 6.71/19.02(2.83)
##STR00402## 12667 D -5.45 -48.78 29.45 34.27 31.86
36.05/19.55(0.54) ##STR00403## 12655 D -5.54 -42.42 30.00 33.36
31.68 11.59/7.89(0.68) ##STR00404## 11610 F -5.54 -43.39 22.50
40.84 31.67 4.98/17.38 ##STR00405## 12610 + TFA D -5.54 -43.39
41.25 21.42 31.33 50.40/19.62(0.39)
##STR00406## 11727 + TFA D -5.45 -54.36 37.00 25.09 31.05
35.11/16.14(0.46) ##STR00407## 11726 + TFA D -5.33 -54.88 35.72
26.02 30.87 NOT ACTIVE ##STR00408## 12777 D -5.54 -42.95 24.37
37.17 30.77 12.81/8.96(0.70) ##STR00409## 12689 + TFA E -4.12
-45.52 44.07 17.08 30.58 44.11/43.55(0.99) ##STR00410## 11792 D
-4.91 -45.07 28.62 32.50 30.56 5.36/13.57(2.53) ##STR00411## 11754
+ TFA D -5.38 -53.48 34.09 25.85 29.97 5.58/7.55(1.35) ##STR00412##
11681 + TFA F -4.75 -41.75 30.46 29.26 29.86 5.47/4.08(0.75)
##STR00413## 11483 + TFA F -5.7 -45.23 29.55 28.99 29.27
##STR00414## 12615 D -5.86 -44.34 31.59 26.88 29.24 5.12/5.93(1.16)
##STR00415## 11617 D -6.05 -62.56 27.71 30.66 29.18
6.01/19.73(3.28) ##STR00416## 11592 D -5.13 -65.33 24.41 33.91
29.16 21.82/18.28(0.84) ##STR00417## 11725 + TFA D -5.93 -41.53
26.65 31.20 28.93 13.00/1.11(0.85) ##STR00418## 11457 E -4.91
-34.84 38.67 18.48 28.58 27.40/48.97(1.79) ##STR00419## 12623 + TFA
D -5.2 -54.89 21.11 35.88 28.49 37.25/17.02(0.46) ##STR00420##
12740 + TFA D -5.62 -56.42 28.77 27.91 28.34 49.36/23.63(0.48)
##STR00421## 12684 + TFA D -5.56 -49.03 40.81 15.75 28.28
25.72/19.52(0.76) ##STR00422## 12213 G -5.11 -42.88 28.35 27.66
28.01 39.11/38.60(0.99) ##STR00423## 12227 G -4.6 -45.64 31.47
23.08 27.27 16.42/17.07(1.04) ##STR00424## 11680 + TFA F -4.78
-38.29 32.59 21.89 27.24 21.86/17.38(0.80) ##STR00425## 11609 D
-6.98 -54.01 28.83 25.63 27.23 35.98/24.31(0.68) ##STR00426## 12614
+ TFA D -5.79 -46.31 20.34 34.09 27.21 19.42/7.56(0.39)
##STR00427## 11663 + TFA G -6.6 -52.02 31.73 21.73 26.73
non-convergent ##STR00428## 11816 + TFA E -4.77 -41.3 39.76 13.58
26.67 14.15/28.12(1.99) ##STR00429## 12622 D -5.8 -48.37 21.30
31.34 26.32 20.80/11.52(0.55) ##STR00430## 11624 D -5.58 -47.58
22.54 29.61 26.08 14.04/22.37(1.59) ##STR00431## 12630 + TFA D
-5.66 -41.51 17.06 34.97 26.02 11.70/8.75(0.75) ##STR00432## 12229
+ TFA E -3.71 -32.18 16.57 35.37 25.97 19.23/20.86(1.08)
##STR00433## 12629 + TFA D -5.13 -42.48 19.83 31.95 25.89
12.66/10.71(0.85) ##STR00434## 11777 G -3.31 -42.7 37.92 13.81
25.87 6.60/10.66(1.62) ##STR00435## 11679 + TFA G -5.35 -46.98
31.02 20.64 25.83 12.55/8.73(0.70) ##STR00436## 12676 E -3.44
-42.61 28.01 23.61 25.81 12.93/18.79(1.45) ##STR00437## 11746 + TFA
D -5.48 -45.63 29.51 21.86 25.68 33.53/14.36(0.43) ##STR00438##
12743 + TFA D -4.99 -46.31 9.17 42.12 25.65 6.88/9.86(1.43)
##STR00439## 11300 + TFA D -7.97 -65.69 25.11 26.12 25.61
26.34/7.95(0.30) ##STR00440## 11751 D -6.76 -54.92 25.87 25.14
25.51 9.88/6.31(0.64) ##STR00441## 12669 + TFA D -5.44 -42.18 11.73
39.10 25.42 49.32/36.71(0.74) ##STR00442## 11807 + TFA G -5.01
-42.97 30.73 20.02 25.37 5.31/17.78(3.35) ##STR00443## 11673 + TFA
G -5.6 -42.41 33.30 17.39 25.35 non-convergent ##STR00444## 12776 +
TFA D -5.61 -43.06 21.34 29.30 25.32 29.74/19.03(0.64) ##STR00445##
11670 + TFA G -5.37 -39.67 34.91 15.63 25.27 13.52/24.32(1.80)
##STR00446## 11469 D -5.08 -60.97 26.79 23.71 25.25
10.12/11.47(1.13) ##STR00447## 11625 + TFA D -5.97 -44.48 31.51
18.94 25.22 49.03/34.70(0.71) ##STR00448## 12662 D -6.33 -40.08
21.64 28.79 25.21 ##STR00449## 12644 + TFA D -5.99 -43.95 16.45
33.67 25.06 46.89/30.86(0.66) ##STR00450## 11674 + TFA G -4.7
-38.24 31.91 18.16 25.04 20.20/35.53(1.76)
[0528] Two-dimensional representations of the docked pose of
AD4-1734, and AD4-10381 compounds, along with AD4-1734-like,
AD4-10381-like compounds were produced.
[0529] Docking of compound AD4-1734 to EGFR is depicted, for
example, in FIG. 8A. Docking of compound AD4-10631 (an
AD4-1734-like compound) to EGFR is depicted, for example, in FIG.
8B. Docking of compound AD4-10188 (an AD4-1734-like compound) to
EGFR is depicted, for example, in FIG. 8C. Docking of compound
AD4-10186 (an AD4-1734-like compound) to EGFR is depicted, for
example, in FIG. 8D. Docking of compound AD4-10633 (an
AD4-1734-like compound) to EGFR is depicted, for example, in FIG.
8E. Docking of compound AD4-10174 (an AD4-1734-like compound) to
EGFR is depicted, for example, in FIG. 8F.
[0530] Docking of compound AD4-10381 to EGFR is depicted, for
example, in FIG. 9A. Docking of compound AD4-11340 (an
AD4-10381-like compound) to EGFR is depicted, for example, in FIG.
9B. Docking of compound AD4-12632 (an AD4-10381-like compound) to
EGFR is depicted, for example, in FIG. 9C. Docking of compound
AD4-12681 (an AD4-10381-like compound) to EGFR is depicted, for
example, in FIG. 9D. Docking of compound AD4-12732 (an
AD4-10381-like compound) to EGFR is depicted, for example, in FIG.
9E. Docking of compound AD4-11511 (an AD4-10381-like compound) to
EGFR is depicted, for example, in FIG. 9F.
Example 8
Combinatorial Studies
[0531] Studies were initiated to evaluate the ability of various
compounds disclosed herein to synergize with several compounds
known to inhibit the function of the EGF receptor in a cell
proliferation assay (MTT assay). These compounds include Tarceva,
Tykerb (non-selective inhibitor of EGFR and HER2 tyrosine kinases),
Iressa (selective inhibitor of EGFR kinase), and a mouse antibody
homolog of Erbitux (clone 225; inhibits binding of EGF to the EGF
receptor). The rationale for this hypothesis is based on the idea
that the AD4 compounds interact with a different site than the EGFR
antibody, Erbitux, and have a different mechanism of action than
the EGFR kinase inhibitors, Tykerb, Iressa or Tarceva.
[0532] Methods are according to Examples 1-7 except as indicated
otherwise.
[0533] Known EGFR inhibitors Tykerb (AD4-0003), Iressa (AD4-0004),
Tarceva (AD4-0005) and Clone 225 (from which Erbitux was derived)
were titrated in the absence or presence of a fixed concentration
of AD4 compound. The EGFR kinase inhibitors and AD4 compound were
pre-diluted in 100% DMSO (DMSO+0.2% TFA for AD4 10381) as necessary
such that a 1/200 dilution into DMEM+BSA resulted in 2.times. the
final concentration desired. Clone 225 was diluted similarly with
the exception that the pre-dilution was made in DMEM+BSA rather
than DMSO. The EGFR inhibitor and AD4 compound dilutions were then
mixed 1:1 in a 96 well plate. 50 .mu.l of the mix was then added to
the cell plate.
[0534] For the Clone 225 Combination Experiment, Clone 225 (Lab
Vision/Thermo Scientific; #MS-269) was tested at concentration of
1, 0.5, 0.25, 0.125, 0.0625 and 0 .mu.g/ml. The EGF concentration
used for stimulation was 10 ng/ml. For the Tarceva Combination
Experiment, Tarceva was tested at concentrations of 156, 63, 25,
10, 4 and 0 nM. The EGF concentration used for stimulation was 5
ng/ml. For the Tykerb Combination Experiment, Tykerb was tested at
concentrations of 78, 31.25, 12.5, 5, 2 and 0 nM. The EGF
concentration used for stimulation was 5 ng/ml. For the Iressa
Combination Experiment, Iressa was tested at concentrations of 156,
63, 25, 10, 4 and 0 nM. The EGF concentration used for stimulation
was 5 ng/ml.
[0535] The concentration of AD4 compound used in each experiment
are provided in the graphs and data tables. A shifting of the
inhibitor curve to the left indicates an increase in the
effectiveness of the AD4-compound.
[0536] In these studies, the ability of an AD4 compound and the
known compound (e.g. Tykerb), either alone or combined in a fixed
constant ratio, were evaluated for their ability to inhibit cell
proliferation in the MTT assay. From these studies, the following
values were calculated: IC.sub.50 values for the AD4 compound
alone, for Tykerb (or other test compound) alone, and for each
compound when combined; the Combination Index (CI), which reflects
the degree of antagonism or synergism (see TABLE 13 below); and the
Dose Reduction Index (DRI), which is a measure of how many fold the
dose of each drug in a synergistic combination may be reduced at a
given effect level when compared with the doses of each drug
alone.
TABLE-US-00013 TABLE 13 Combination Index KEY: Range of CI
Description Graded Symbols <0.1 Very strong synergism + + + + +
0.1-0.3 Strong synergism + + + + 0.3-0.7 Synergism + + + 0.7-0.85
Moderate synergism + + 0.85-0.9 Slight synergism + 0.9-1.10 Nearly
additive +/- 1.10-1.20 Slight antagonism - 1.20-1.45 Moderate
antagonism - - 1.45-3.3 Antagonism - - - 3.3-10 Strong antagonism -
- - - >10 Very strong antagonism - - - - -
[0537] Results showed the following. A series of AD4-Pharma
compounds produce a synergistic effect with Tykerb, Iressa and
Erbitux to enhance their effect in a cell proliferation assay.
These synergistic effects were demonstrated by significant changes
in the Dose Reduction Index, the Combination Index and shifts in
the dose-response curves.
[0538] The effects of a number of compounds appear to involve
positive cooperativity because the effect increases as the
concentration of the compound increases. Compounds that
demonstrated the greatest degree of positive co-operativity usually
demonstrated high DRI values. Some of the compounds that
demonstrate the greatest degree of positive co-operativity also
show synergistic behavior as evidenced by a low CI value. Iressa
and Tarceva, another selective inhibitor of EGFR kinase, did not
produce synergistic effects with Tykerb. Compounds acting at the
same target (i.e., EGFR kinase) should not be synergistic.
[0539] An example of a shift in the dose-response curve is shown in
FIG. 10A-FIG. 10B, where AD4-10628 produced a leftward shift
(higher potency) in the dose-response curves for both Tykerb and
Iressa. The effect is more evident at higher concentrations of the
compound, indicating a positive co-operativity effect may be
involved in the compound's action.
[0540] Results for several of the more potent compounds are
summarized in TABLE 14. AD4-10628 AD4-11511 produce a leftward
shift (i.e., greater potency) in Tykerb's dose-response curve, and
demonstrate very high DRI values. Although significant shifts in
the dose-response curves for the EGFR kinase inhibitors are
produced, these effects are not translated into significant shifts
in the IC.sub.50 values (50% inhibition), since the effects of the
AD4 compounds are observed to a greater degree at higher
concentrations.
TABLE-US-00014 TABLE 14 Summary of Median Effect Analysis Studies
IC.sub.50 Tykerb IC.sub.50 Tykerb + DRI @ CI @ Compound (nM)
Compound (nM) ED97 ED90 AD4-10628 178 59 126 0.55 AD4-11511 189 118
84 0.87 Iressa 307 160 2 1.15 IC.sub.50 Iressa IC.sub.50 Iressa +
DRI @ CI @ (nM) Compound (nM) ED97 ED90 AD4-10628 207 93 105 0.91
AD4-11511 326 245 96 0.93
[0541] DRI values were calculated for the effect of the test
compound on Tykerb, as well as for Tykerb's effect on the test
compound. In general, most compounds enhanced the effect of Tykerb,
as demonstrated by a high DRI, whereas Tykerb usually had a minimal
effect on the test compound. As a result, the reported DRI is for
the effect of the test compound on Tykerb. This can best be
observed by plotting DRI as a function of Fa, or percent effect,
ranging from 5% to 97% (see e.g., FIG. 11). AD4-10628 had a
significant effect on the activity of Tykerb, which becomes greater
at higher activity levels (or doses), whereas Tykerb has little
effect on AD4-10628 (see e.g., FIG. 11). As shown in TABLE 14,
AD4-10628 and AD4-11511 all produced a fairly high DRI for both
Tykerb and Iressa.
[0542] CI values were calculated where the combined effect of the
compounds produces 50%, 90%, 95% and 97% (i.e., ED.sub.50,
ED.sub.90, ED.sub.95 and ED.sub.97) inhibition of cell
proliferation. A graph showing the CI values at 90% inhibition is
displayed in FIG. 12. Response below the red line (i.e CI<0.9)
indicates synergism. As indicated in FIG. 12, a number of compounds
demonstrated synergism. For example, the CI values for AD4-10628
(see TABLE 14) demonstrated significant synergy with Tykerb. In
contrast, only AD4-1505 shows synergy with Iressa based on the CI
value (data not shown). Since Iressa and Tykerb have similar
mechanisms of action, minimal interaction would be expected between
these two compounds. As shown in TABLE 14, little interaction is
observed based on the DRI and CI values for Iressa.
[0543] These results show that the AD4 compounds produce a
significant effect on EGF receptor-mediated cell proliferation
through a site that is distinct from either EGF receptor kinase or
the EGF receptor. Furthermore, based on their synergistic effect,
the compounds may provide a unique method to achieve the same or
enhanced therapeutic effect while using a lower therapeutic dose of
the marketed compounds, Tykerb, Iressa or Erbitux.
Example 9
Synthesis of Compounds
[0544] The following example describes synthesis of AD4-12632,
which is an AD4-11511-like compound.
[0545] Step 1 was as follows (see e.g., Hamann 1988 J. Med. Chem.
41(4), 623-639):
##STR00451##
[0546] m-Toluidine (across, 32.1 g, 0.3 mol) was dissolved in 250
ml of acetone and treated with 100 g of MgSO4, iodine (2.6 g, 0.01
mol) and a catalytic amount (200 mg) of t-butyl catechol. The
mixture was stirred and heated to reflux temperature overnight.
After cooling to room temperature the dark mixture was filtered
through Celite which was rinsed with EtOAc. The solution was
treated with decolorizing charcoal, filtered, and concentrated
using a rotovap. The dark residual oil was purified by fractional
distillation. The fraction boiling between 90-95.degree. C. @ 0.05
mm/Hg contained the desired product. The light yellow oil
solidified after cooling in the refrigerator to give the free base
as an off-white waxy solid. The free base was converted to the
hydrochloride salt as follows. The freebase was dissolved in
Et.sub.2O and cooled in an ice bath. A solution of hydrogen
chloride in Et.sub.2O was prepared and a slight molar excess (1.1
equivalents) was added from a dropping funnel. The mixture was
stirred at 0.degree. C. for 1 h and then filtered to collect
1,2-dihydro-2,2,4,7-tetramethylquinoline hydrochloride as a white
solid (MP 213-215.degree. C.).
[0547] Step 2 was as follows (see e.g., Web 2003 Bioorg. Med. Chem.
11, 77-85):
##STR00452##
[0548] 1,2-dihydro-2,2,4,7-tetramethylquinoline hydrochloride (9.35
g, 0.004 mol) was suspended with stirring in 30 ml water and 20 ml
of EtOH was added followed by 2-cyanoguanidine (Acros, 3.4 g, 0.004
mol). The heterogeneous mixture was heated to 100.degree. C. for 6
h and then cooled to room temperature. The solid was collected by
filtration and washed with 500 ml of cold water followed by 100 ml
of 2-propanol. The off-white solid was dried and then dissolved by
heating to 150.degree. C. in DMSO. Upon cooling to room temperature
a white solid formed and was collected by filtration. The solid was
rinsed with Et2O and dried to give
4,7-dimethylquinazoyl-2-guanidine hydrochloride as a white solid
(MP 329-331.degree. C.). The hydrochloride salt was suspended in 5N
aqueous sodium hydroxide and stirred at room temperature for 5
days. The white solid was collected by filtration and rinsed with
2.times.250 ml water and 100 ml of ice-cold 2-propanol. The solid
was dried to give 4,7-dimethylquinazoyl-2-guanidine free base as an
off white solid (MP 260-261.degree. C.).
[0549] Step 3 was as follows (see e.g., Shikhaliev 2002 Chem. Het.
Compounds 38(11), 1368-1370):
##STR00453##
[0550] 4,7-dimethylquinazoyl-2-guanidine (2.15 g, 0.01 mol) and
ethyl-2-methyl acetoacetate (1.58 g, 0.011 mol) were stirred in 15
ml DMSO to produce a suspension. The solution was heated to
130.degree. C. overnight to give a clear brown solution and then
cooled to room temperature. The solid that formed was collected by
filtration. The solid was dissolved in EtOH at room temperature
with stirring, treated with decolorizing charcoal and filtered
through a bed of Celite. The EtOH solution was cooled to 0.degree.
C. to give an off white solid that is collected by filtration (MP
226-228.degree. C.). The resulting product was the AD4-12632
compound.
Sequence CWU 1
1
11613PRTHomo sapiensEpidermal Growth Factor Receptor 1Glu Glu Lys
Lys Val Cys Gln Gly Thr Ser Asn Lys Leu Thr Gln Leu 1 5 10 15 Gly
Thr Phe Glu Asp His Phe Leu Ser Leu Gln Arg Met Phe Asn Asn 20 25
30 Cys Glu Val Val Leu Gly Asn Leu Glu Ile Thr Tyr Val Gln Arg Asn
35 40 45 Tyr Asp Leu Ser Phe Leu Lys Thr Ile Gln Glu Val Ala Gly
Tyr Val 50 55 60 Leu Ile Ala Leu Asn Thr Val Glu Arg Ile Pro Leu
Glu Asn Leu Gln 65 70 75 80 Ile Ile Arg Gly Asn Met Tyr Tyr Glu Asn
Ser Tyr Ala Leu Ala Val 85 90 95 Leu Ser Asn Tyr Asp Ala Asn Lys
Thr Gly Leu Lys Glu Leu Pro Met 100 105 110 Arg Asn Leu Gln Glu Ile
Leu His Gly Ala Val Arg Phe Ser Asn Asn 115 120 125 Pro Ala Leu Cys
Asn Val Glu Ser Ile Gln Trp Arg Asp Ile Val Ser 130 135 140 Ser Asp
Phe Leu Ser Asn Met Ser Met Asp Phe Gln Asn His Leu Gly 145 150 155
160 Ser Cys Gln Lys Cys Asp Pro Ser Cys Pro Asn Gly Ser Cys Trp Gly
165 170 175 Ala Gly Glu Glu Asn Cys Gln Lys Leu Thr Lys Ile Ile Cys
Ala Gln 180 185 190 Gln Cys Ser Gly Arg Cys Arg Gly Lys Ser Pro Ser
Asp Cys Cys His 195 200 205 Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro
Arg Glu Ser Asp Cys Leu 210 215 220 Val Cys Arg Lys Phe Arg Asp Glu
Ala Thr Cys Lys Asp Thr Cys Pro 225 230 235 240 Pro Leu Met Leu Tyr
Asn Pro Thr Thr Tyr Gln Met Asp Val Asn Pro 245 250 255 Glu Gly Lys
Tyr Ser Phe Gly Ala Thr Cys Val Lys Lys Cys Pro Arg 260 265 270 Asn
Tyr Val Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly Ala 275 280
285 Asp Ser Tyr Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys Cys
290 295 300 Glu Gly Pro Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly
Glu Phe 305 310 315 320 Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile
Lys His Phe Lys Asn 325 330 335 Cys Thr Ser Ile Ser Gly Asp Leu His
Ile Leu Pro Val Ala Phe Arg 340 345 350 Gly Asp Ser Phe Thr His Thr
Pro Pro Leu Asp Pro Gln Glu Leu Asp 355 360 365 Ile Leu Lys Thr Val
Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala 370 375 380 Trp Pro Glu
Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile 385 390 395 400
Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val 405
410 415 Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile
Ser 420 425 430 Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys
Tyr Ala Asn 435 440 445 Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser
Gly Gln Lys Thr Lys 450 455 460 Ile Ile Ser Asn Arg Gly Glu Asn Lys
Cys Lys Ala Thr Gly Gln Val 465 470 475 480 Cys His Ala Leu Cys Ser
Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg 485 490 495 Asp Cys Val Ser
Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp 500 505 510 Lys Cys
Lys Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser 515 520 525
Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile 530
535 540 Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His
Tyr 545 550 555 560 Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala
Gly Val Met Gly 565 570 575 Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala
Asp Ala Gly His Val Cys 580 585 590 His Leu Cys His Pro Asn Cys Thr
Tyr Gly Cys Thr Gly Pro Gly Leu 595 600 605 Arg Gly Cys Pro Thr
610
* * * * *