CONCEPTION DE STABILISATEURS DE POLYMERISATION DE LA TUBULINE

METHOD OF DESIGNING TUBULIN POLYMERIZATION STABILIZERS

Abrégé français

L'invention concerne un procédé permettant la conception de composés de remplacement du paclitaxel, qui stabilisent le processus de polymérisation de la tubuline. Ces composés en solution présentent les caractéristiques de conformation stérique du paclitaxel naturel et sont capables de se fixer à la protéine de tubuline au même site de liaison que le paclitaxel. Ces composés stabilisent la polymérisation de la tubuline par un mécanisme correspondant au mécanisme d'activité du paclitaxel. Comparés au paclitaxel naturel, ces composés présentent une solubilité accrue, sont plus faciles à synthétiser et offrent des possibilités de réactions combinatoires permettant une spécificité et d'une optimisation.

Abrégé anglais

A method for designing paclitaxel alternative compounds which stabilize the
tubulin polymerization process has been found. These compounds in solution
possess steric conformational properties of natural paclitaxel and are capable
of binding to the tubulin protein at the same site where paclitaxel is known
to bind. The compounds of the present invention stabilize tubulin
polymerization in a way that is mechanistically equivalent to activity
mechanism of paclitaxel. The compounds of the present invention have increased
solubility, simpler synthesis, and the possibility for specificity and
optimization due to the combinatorial reactions over natural paclitaxel.

Revendications

WE CLAIM:
1. A method for designing anti-tumor compositions, comprising:
(a) using molecular modeling software on a computer to create a plot of an
active conformation of a known anti-tumor composition, said active
conformation
representative of a three-dimensional conformation of said known anti-tumor
composition interacting with a target biological site, said plot providing a
first digital
representation of said active conformation, said first digital representation
depicting a
plurality of binding sites of said known anti-tumor composition;
(b) using said software to eliminate portions of said first digital
representation
while preserving the depiction of said binding sites;
(c) using said software to build a second digital representation of a created
composition, said created composition having a three-dimensional conformation
and
binding sites similar to said known anti-tumor composition.
2. The method of Claim 1, wherein said known anti-tumor composition has a
structure including a central skeleton which is depicted in said plot, and
wherein said
software is utilized to eliminate said central skeleton from said depiction
and to
substitute therefore a second central skeleton having desired characteristics.
3. The method of Claim 1, wherein said known anti-tumor composition has a
structure including a central skeleton and one or more original side chains
which are
depicted in said plot, and wherein said software is utilized to eliminate one
or more
original side chains from said depiction and optionally to substitute a
created side
chain for one or more of said original side chains.
4. The method of Claim 3, further comprising using said software to eliminate
said central skeleton from said depiction and to substitute therefore a second
central
skeleton having desired characteristics.
51

5. The method of Claims 1,2, 3 or 4 wherein a calculation is performed to
determine a binding energy for said created composition, and wherein said
created
composition is further modified to improve putative binding characteristics,
wherein
an improved binding characteristic is characterized by a higher binding
energy.
6. The method of Claims 1, 2, 3, 4, or 5 wherein said known anti-tumor
composition is paclitaxel.
7. A method for designing a paclitaxel alternative composition, which
alternative composition has a central skeleton structure composed of single or
multiple
ring groups which hold multiple functional groups in a fairly rigid alignment,
said
central skeleton structure having first, second, and third side chains;
wherein said first side chain is connected to said central skeleton with a
carbonyl group at a distance of about 1.5 to 5.5 Angstroms from said central
skeleton;
wherein said second side chain places an sp3 oxygen atom at a distance of
about 4.5 to 7.5 Angstroms from the skeleton and about 9 to 11 Angstroms from
the
carbonyl oxygen of said first side chain;
wherein said third side chain is placed in an energetically accessible
conformation that places an aromatic ring in a location that is simultaneously
about 4
to 6 Angstroms from a substitute for hexene and about 8 to 10 Angstroms from
the
oxygen in said second side chain, said third side chain selected to mimic the
steric and
binding properties of the C2 ester in paclitaxel;
said method comprising using molecular modeling software on a computer to
design said alternative composition.
8. The method of Claim 7, wherein said alternative composition further
comprises one or more bulking groups and wherein said bulking groups increase
the
size of said composition to mimic the overall size and shape of the paclitaxel
molecule.
9. The method of Claim 7, wherein said first side chain is selected and
positioned to mimic the isoserine group in taxane.
52

10. The method of Claim 7, wherein said sp3 oxygen is positioned in space to
simulate the position of the oxetane ring of paclitaxel.
11. The method of Claims 7-11 further comprising synthesizing said
alternative composition.
12. A paclitaxel compound having a chemical structure
<IMG>
wherein R1 and R2 are selected from the group consisting of hydrogen, methyl,
acetyl,
ethyl, C1-C4 aliphatic chain and substituted C1-C6 aliphatic chain; wherein
said substituted aliphatic chain is substituted once or twice with functional
groups selected from the group consisting of: amide, ketone, hydroxy; phenyl,
carboxylic acid, and an amino acid;
wherein X is selected tiom the group consisting of O; CH2; NH; S; S-CH2; O-
CH2; or
none;
wherein R3 is selected from the group consisting of:
<IMG>
53

<IMGS>
54

<IMGS>
55

<IMG>
wherein the aryl rings sure unsubstituted or singly, doubly, or triply
substituted with R selected from the goup consisting of H, OH, and an
electron withdrawing substituent;wherein R' is selected from the group
consisting of OH and H; wherein R" is selected from the group
consisting of: NHBOC and H; wherein R"' is selected from the group
consisting of: singly or doubly substituted aryl and fused aromatic ring;
wherein R4 is selected from the group consisting of:
<IMGS>
wherein the aromatic ring is singly, doubly, or triply
substituted with any electron-withdrawing substituent
compatible with the system which provides a lower
energy gap in an .pi.-.pi. interaction;
56

<IMG>
wherein R"" is a fixed aromatic ring or a fused aromatic
ring substituted any electron-withdrawing substituent
compatible with the system which provides a lower
energy gap in a .pi.-.pi. interaction;
<IMG>
wherein R""' is selected from the group consisting of: H,
cyclopropane, C1-C3 hydrocarbon chain, and C1-C3
substituted hydrocarbon chain wherein said substituted
hydrocarbon chain is substituted with any electron-
withdrawing substituent compatible with the system
which provides a lower energy gap in a .pi.-.pi. interaction;
wherein R5 is selected from the group consisting of:
<IMG>
H, CH3, C1-C5 hydrocarbon chain, C1-C5 substituted
hydrocarbon chain, small hydrocarbon ring or
heterocyclic ring, small substituted hydrocarbon ring or
heterocyclic ring, citric acid and derivatives thereof,
acetic acid and derivatives thereof, ascorbic acid and
derivatives thereof, glucouroic acid or derivatives
thereof, lactose, sialic acid, monosaccharides or
57

disaccharides of glyceraldehyde, erythrose, threose,
ribose, arabinose xylose lyxose, allose, altrose, glucose,
mannose, gulose, idose, galactose, talose, or their acidic
ketose, alditol or inositol forms, calcium chelating
molecule, oxygenated small molecule, any organic
molecule that exhibits calcium-binding properties
similar to tetracyclin
<IMGS>
58

<IMGS>
59

<IMG>
wherein R6 is selected from the group consisting of:
<IMGS>
H, CH3, OH, amine, C1-C5 carbo-aliphatic chain
substituted with two or three of the following groups
selected from the group consisting of keto, hydroxy,
sulfoxy, amide, an amino acid, and ethers of the form -
CH2-O-(CH2)n-CH3 where n=1-5, wherein the right hand
hydrocarbon chain is unsubstituted or substituted with 1
to 5 -OH or carbonyl groups.
60

13. A paclitaxel compound having a chemical structure
<IMG>
wherein R1 and R2 are selected from the group consisting of hydrogen, methyl,
acetyl,
ethyl, C1 - C4 aliphatic chain and substituted C1 - C6 aliphatic chain,
wherein
said substituted aliphatic chain is substituted once or twice with functional
groups selected from the group consisting of amide, ketone, hydroxy, phenyl,
carboxylic acid, and an amino acid;
wherein X is selected from the group consisting of O; CH2; NH; S; S-CH2; O-
CH2; or
none;
wherein R3 is selected from the group consisting of:
<IMGS>
61

<IMGS>
62

<IMGS>
wherein the aryl rings are unsubstituted or singly, doubly, or triply
substituted with R selected from the group consisting of: H, OH, and an
electron withdrawing substituent; wherein R' is selected from the group
consisting of: OH and H; wherein R" is selected from the group
63

consisting of: NHBOC and H; wherein R"' is selected from the group
consisting of: singly or doubly substituted aryl and fused aromatic ring;
wherein R4 is selected from the group consisting of:
<IMGS>
wherein the aromatic ring is singly, doubly, or triply
substituted with any electron-withdrawing substituent
compatible with the system which provides a lower
energy gap in a .pi. - .pi. interaction;
<IMG>
wherein R"" is a fixed aromatic ring or a fused aromatic
ring substituted with any electron-withdrawing
substituent compatible with the system which provides a
lower energy gap in a .pi. - .pi. interaction;
<IMG>
64

<IMGS>
66

<IMGS>
wherein R6 is selected from the group consisting of:
<IMG>
67

<IMGS>
H, CH3, OH, amine, C1-C3 carbo-aliphatic chain
substituted with two or three of the following groups
selected from the group consisting, of keto, hydroxy,
sulfoxy, amide, an amino acid, ethers of the form -CH2-
O-(CH2)n -CH3 where n=1-5, wherein the right hand
hydrocarbon chain is unsubstituted or substituted with 1
to 5 -OH or carbonyl groups.
14. A paclitaxel compound having a chemical structure
<IMG>
wherein R1 and R2 are selected from the group consisting of hydrogen, methyl,
acetyl,
ethyl, C1 - C4 aliphatic chain and substituted C1 - C6 aliphatic chain,
wherein
said substituted aliphatic chain is substituted once or twice with functional
groups selected from the group consisting of amide, ketone, hydroxy, phenyl,
carboxylic acid, and an amino acid;
wherein X is selected from the group consisting of: O; CH2; NH; S; S-CH2; O-
CH2; or
none;
wherein R3 is selected from the group consisting of:
68

<IMGS>
69

<IMGS>
70

<IMG>
wherein the aryl rings are unsubstituted or singly, doubly, or triply
substituted with R selected from the group consisting of H, OH, and an
electron withdrawing substituent;wherein R' is selected from the group
consisting of OH and H; wherein R" is selected from the group
consisting of NHBOC and H; wherein R"' is selected from the group
consisting of singly or doubly substituted aryl and fused aromatic ring;
wherein R4 is selected from the group consisting of:
<IMGS>
wherein the aromatic ring is singly, doubly, or triply
substituted with any electron-withdrawing substituent
compatible with the system which provides a lower
energy gap in a .pi. - .pi. interaction;
71

<IMG>
wherein R"" is a fixed aromatic ring or a fused aromatic
ring substituted with R any electron-withdrawing
substituent compatible with the system which provides a
lower energy gap in a .pi.- .pi. interaction;
<IMG>
wherein R""' is selected from the group consisting of: H,
cyclopropane, C1-C3 hydrocarbon chain, and C1-C3
substituted hydrocarbon chain wherein said substituted
hydrocarbon chain is substituted with any electron-
withdrawing substituent compatible with the system
which provides a lower energy gap in a .pi. - .pi. interaction;
wherein R5 is selected from the group consisting of:
<IMG>
H, CH3, C1-C5 hydrocarbon chain, C1-C5 substituted
hydrocarbon chain small hydrocarbon ring or
heterocyclic ring, small substituted hydrocarbon ring or
heterocyclic ring, citric acid and derivatives thereof,
acetic acid and derivatives thereof, ascorbic acid and
derivatives thereof, glucouroic acid or derivatives
thereof, lactose, sialic acid, monosaccharides or
72

disaccharides of glyceraldehyde, erythrose, threose,
ribose, arabinose xylose lyxose, allose, altrose, glucose,
mannose, gulose, idose, galactose, talose, or their acidic
ketose, alditol or inositol forms, calcium chelating
molecule, oxygenated small molecule, any organic
molecule that exhibits calcium-binding properties
similar to tetracyclin
<IMGS>
73

wherein R''''' is selected from the group consisting of H,
cyclopropane, C1-C3 hydrocarbon chain, and C1-C3
substituted hydrocarbon chain wherein said substituted
hydrocarbon chain is substituted with any electron-
withdrawing substituent compatible with the system
which provides a lower energy gap in a .pi. - .pi. interaction;
wherein R5 is selected from the group consisting of:
<IMG>
H, CH3, C1-C5 hydrocarbon chain, C1-C5 substituted
hydrocarbon chain, small hydrocarbon ring or
heterocyclic ring, small substituted hydrocarbon ring or
heterocyclic ring, citric acid and derivatives thereof,
acetic acid and derivatives thereof, ascorbic acid and
derivatives thereof, glucouroic acid or derivatives
thereof, lactose, sialic acid, monosaccharides or
disaccharides of glyceraldehyde, erythrose, threose,
ribose, arabinose xylose lyxose, allose, altrose, glucose,
mannose, gulose, idose, galactose, talose, or their acidic
ketose, alditol or inositol forms, calcium chelating
molecule, oxygenated small molecule, any organic
molecule that exhibits calcium-binding properties
similar to tetracyclin
65

<IMGS>
74

<IMG>
wherein R6 is selected from the group consisting of:
<IMGS>
H, CH3, OH, amine, C1-C5 carbo-aliphatic chain substituted with two or three
of the following groups selected from the group consisting of: keto, hydroxy,
sulfoxy,
amide, an amino acid, ethers of the form -CH2-O-(CH2)n-CH:3 where n=1-5,
wherein
the right hand hydrocarbon chain is unsubstituted or substituted with 1 to 5 -
OH or
carbonyl groups.
15. A paclitaxel compound having a chemical structure
<IMG>
75

wherein R1 and R2 are selected from the group consisting of: hydrogen, methyl,
acetyl,
ethyl, C1 - C4 aliphatic chain and substituted C1 - C6 aliphatic chain,
wherein
said substituted aliphatic chain is substituted once or twice with functional
groups selected from the group consisting of amide, ketone, hydroxy, phenyl,
carboxylic acid, and an amino acid;
wherein X is selected from the group consisting of: O; CH2; NH; S; S-CH2; O-
CH2; or
none;
wherein R3 is selected from the group consisting of
<IMGS>
76

<IMGS>
77

<IMGS>
wherein the aryl rings are unsubstituted or singly, doubly, or triply
substituted with R selected from the group consisting of H, OH, and an
electron withdrawing substituent;wherein R' is selected from the group
consisting of: OH and H; wherein R'' is selected from the group
consisting of: NHBOC and H; wherein R''' is selected from the group
consisting of: singly or doubly substituted aryl and fused aromatic ring;
wherein R4 is selected from the group consisting of
78

<IMGS>
wherein the aromatic ring is singly, doubly, or triply
substituted with any electron-withdrawing substituent
compatible with the system which provides a lower
energy gap in a .pi. - .pi. interaction;
<IMG>
wherein R'''' is a fixed aromatic ring or a fused aromatic
ring substituted with any electron-withdrawing
substituent compatible with the system which provides a
lower energy gap in a .pi. - .pi. interaction;
<IMG>
wherein R''''' is selected from the group consisting of: H,
cyclopropane, C1-C3 hydrocarbon chain, and C1-C3
substituted hydrocarbon chain wherein said substituted
79

hydrocarbon chain is substituted with any electron-
withdrawing substituent compatible with the system
which provides a lower energy grip in a .pi. - .pi. interaction;
wherein R5 is selected from the group consisting of:
<IMG>
H. CH3, C1-C5 hydrocarbon chain, C1-C5 substituted
hydrocarbon chain, small hydrocarbon ring or
heterocyclic ring, small substituted hydrocarbon ring or
heterocyclic ring, citric acid and derivatives thereof,
acetic acid and derivatives thereof, ascorbic acid and
derivatives thereof, glucouroic acid or derivatives
thereof, lactose, sialic acid, monosaccharides or
disaccharides of glyceraldehyde:, erythrose, threose,
ribose, arabinose xylosc lyxose, allose, altrose, glucose,
mannose, gulose, idose, galactose, talose, or their acidic
ketose, alditol or inositol forms, calcium chelating
molecule, oxygenated small molecule, any organic
molecule that exhibits calcium-binding properties
similar to tetracyclin
80

<IMGS>
81

<IMGS>
wherein R6 is selected from the goup consisting of:
<IMG>
82

<IMGS>
H, CH3, OH, amine, C1-C3 carbo-aliphatic chain substituted with two or three
of the following groups selected from the group consisting of keto, hydroxy,
sulfoxy,
amide, an amino acid, ethers of the form -CH2-O-(CH2)n-CH3 where n=1-5,
wherein
the right hand hydrocarbon chain is unsubstituted or substituted with 1 to 5 -
OH or
carbonyl groups.
16. A paclitaxel compound having the following bicyclo-octane chemical
structure
<IMG>
wherein R1 and R2 are selected from the group consisting of hydrogen, methyl,
acetyl,
ethyl, C1 - C4 aliphatic chain and substituted C1 - C6 aliphatic chain,
wherein
said substituted aliphatic chain is substituted once or twice with functional
groups selected from the group consisting of: amide, ketone, hydroxy, phenyl,
carboxylic acid, and an amino acid;
wherein X is selected from the group consisting of: O; CH2; NH; S; S-CH2; and
O-
CH2;
wherein R3 is selected from the group consisting of:
83

<IMGS>
84

<IMGS>
85

<IMG>
wherein the aryl rings are unsubstituted or singly, doubly, or triply
substituted with R selected from the group consisting of H, OH, and an
electron withdrawing substituent; wherein R' is selected from the group
consisting of OH and H; wherein R" is selected: from the group
consisting of NHBOC and H; wherein R''' is selected from the group
consisting of singly or doubly substituted aryl sand fused aromatic ring;
wherein R4 is selected from the group consisting of
<IMGS>
wherein tine aromatic ring is singly, doubly, or triply
substituted with any electron-withdrawing substituent
compatible with the system which provides a lower
energy gap in a .pi.- .pi. interaction;
86

<IMG>
wherein R''''is a fixed aromatic ring or a fused aromatic
ring substituted with any electron-withdrawing
substituent compatible with the system which provides a
lower energy gap in a .pi.- .pi. interaction;
<IMG>
wherein R'''' is selected from the group consisting of: H,
cyclopropane, C1-C3 hydrocarbon chain, and C1-C3
substituted hydrocarbon chain wherein said substituted
hydrocarbon chain is substituted with any electron-
withdrawing substituent compatible with the system
which provides a lower energy gap in a .pi.- .pi. interaction;
wherein R5 is selected from the group consisting of
<IMG>
H, CH3, C1-C5 hydrocarbon chain, C1-C5 substituted
hydrocarbon chain, small hydrocarbon ring or
heterocyclic ring, small substituted hydrocarbon ring or
heterocyclic ring, citric acid and derivatives thereof;
acetic acid and derivatives thereof, ascorbic acid and
derivatives thereof, glucouroic acid or derivatives
thereof, lactose, sialic acid, monosaccharides or
87

disaccharides of glyceraldehyde, erythrose, threose,
ribose, arabinose xylose lyxose, allose, altrose, glucose,
mannose, gulose, idose, galactose, talose, or their acidic
ketose, alditol or inositol forms, calcium chelating
molecule, oxygenated small molecule, any organic
molecule that exhibits calcium-binding properties
similar to tetracyclin
<IMGS>
88

<IMGS>
89

<IMG>
wherein R6 is selected from the group consisting of:
<IMGS>
H, CH3;, OH, amine, C1-C5 carbo-aliphatic chain.
substituted with two or three of the following groups
selected from the group consisting of: keto, hydroxy,
sulfoxy, amide, an amino acid, ethers of the form -CH2-
O-(CH2)n-CH3 where n=1-5, wherein the right hand
hydrocarbon chain is unsubstituted or substituted with 1
to 5-OH or carbonyl groups.
17. The compound of claim 12, 13, 14, 15, or 16, wherein the amino acid
identity of R1 or R2 is selected from the group consisting of asparagine,
glutamine,
aspartic acid, glutamic acid, threonine, serine and tyrosine.
90

18. The compound of claim 12, 13, 14, 15, or 16, wherein R1 is chosen from
the group consisting of H and CH3.
19. The compound of claim 12, 13, 14, 15, or 16, wherein R2 is chosen from
the group consisting of CH3, CH2OCOCH3,
<IMG>
wherein R is H or singly, doubly, or triply substituted or
fused; and
<IMG>
wherein R' is selected from the group consisting of H
and CH3.
20. The compound of claim 12, 13, 14, 15, or 16, wherein R''' is selected from
the group consisting of imidazol ring, tryptophan unsubstituted or substituted
with
carboxylic acid derivatives.
91

21. The compound of claim 12, 13, 14, 15, or 16, wherein R3 is any group
derived from the 13 position in taxane's skeleton that exhibits activity
toward
inhibiting the depolymerization of microtubules or anticancer activity.
22. The compound of claim 12, 13,14, 15, or 16, wherein the oxygenated
small molecule of R5 is selected from the group consisting of dipeptides of
"ASP-
ASN", or "GLY-GLN" and the cyclic dipeptide of "PHE-GLN."
23. The compound of claim 12, 13, 14, 15, or 16, wherein the amino acid of
R6 is selected from the group consisting of serine, asparagine, and threonine.
92

Description

CA 02379676 2002-O1-14
WO 01/05779 PCT/US00/19524
METHOD OF DESIGNING TUBULIN POLYMERIZATION STABILIZERS
TECHNICAL FIELD OF INVENTION
This invention relates a method of designing paclitaxel alternative compounds.
BACKGROUND
Paclitaxel is a complex diterpenoid sold commercially as TAXOLo (Bristol-
Myers-Squibb). Paclitaxel and many of its derivative have been reported to
possess
potent antileukemia activity as well as significant anticancer activity
against
carcinomas of the ovaries, breast, lung, bladder, esophagus, head, and neck.
(Rowinsky, E.K. and Donehower, R.C. 1991. "The clinical pharmacology and use
of
antimicrotubule agents in cancer chemotherapeutics," Pharmacol Ther 52:35-84;
and
~ Rowinsky, E. K. 1994. "Update on the antitumor activity of paclitaxel in
clinical
trials," Ann Pharmacother 28(5 Supply: S 18-22).
Paclitaxel and its derivatives are represented by the following chemical
formula. For paclitaxel, R = acetyl; R, = OH; and RZ = NHCOC6H5. For the
derivative docetaxel, R = OH; R, = OH; RZ = NHCOOC(CH3)3.
R
O
R2 O ~.n3
~CH3
O'',. H~O
R~ -p O
OH ~CH3
O O//

CA 02379676 2002-O1-14
WO 01/05779 PCT/LJS00/19524
The structure for the taxane skeleton is represented as follows:
O
3
~CH3
O
R40 ,. O
C=O
CH3
The mechanism of action for paclitaxel has been previously determined.
(Derry, et al. 1997. "Taxol differentially modulates the dynamics of
microtubules
assembled from unfractionated and purified beta-tubulin isotypes,"
Biochemistry
36:3554-3562). Its anti-tumor activity is due to its ability to bind to beta-
tubulin in
assembled microtubules and stabilize them (Manfredi, J.J. and Horwitz, S.B.
1984.
"Taxol: an antimitotic agent with a new mechanism of action," Pharmacol Ther
25:83-
125; and Horwitz, S.B. 1992. "Mechanism of action of taxol, " Trends Pharmacol
Sci
13:134-136). In vivo, paclitaxel affects spindle function during mitosis,
resulting in
cell cycle arrest in G2/M phase. In vitro, paclitaxel promotes microtubule
assembly
and prevents their disassembly under conditions which would otherwise cause
depolymerization (Schiff, et al. 1979. "Promotion of microtubule assembly in
vitro by
taxol" Nature 277:665-667; and Pamess, J. and Horwitz, S.B. 1981 "Taxol binds
to
polymerized tubulin in vitro," J Cell Biol 91:479-487).
The x-ray structure of paclitaxel bound to its receptor has been previously
reported. (Nogales, et al. 1998. "Structure of the alpha beta tubulin dimer by
electron
crystallography," Nature 391:199-203; erratum published in Nature 393:191).
The
alpha-beta heterodimer is the structural subunit of microtubules, which are
cytoskeletal elements essential for intracellular transport and cell division
in
eukaryotes. The structures of alpha- and beta-tubulin are identical with each
monomer
being formed by a core of two beta-sheets surrounded by alpha-helices. The
monomer
structure is divided into three functional domains: the amino-terminal domain
containing the nucleotide-binding region, an intermediate domain containing
the
2

CA 02379676 2002-O1-14
WO 01/05779 PCT/US00/19524
paclitaxel-binding site, and the carboxy-terminal domain, which is reported to
constitute the binding surface for motor proteins.
Despite the anticancer and antileukemia activity of paclitaxel, there are
distinct
disadvantages of using paclitaxel as a therapeutic agent. For example, the
synthesis
and/or production of paclitaxel from natural sources is very complex and
costly.
Paclitaxel is highly toxic, often being toxic at therapeutic levels.
Paclitaxel's low
solubility causes complications in preparation and administration of
therapeutic
dosages.
Numerous patents have issued which disclose paclitaxel derivatives in which
substitutions have been made on the taxane skeleton. For example, U.S. Patent
No.
4,814,470 issued March 21, 1989, discloses paclitaxel derivatives which
reportedly
have anticancer activity greater than paclitaxel itself. Referring to the
chemical
formula given above, the chemical formulas for these derivatives consist of R
= H or
acetyl; R~ = OH or tent-butoxycarbonylamino; and R2 = OH when R~ = tert-
butoxycarbonylamino or R2 = tert-butoxycarbonylamino when R, = OH. U.S. Patent
No. 4,206,221 issued June 3, 1980, discloses paclitaxel derivatives for the
remission of
leukemia in animals. Referring to the chemical formula given above, the
chemical
formulas for these derivatives consist of R = acetyl; R, = OH; and R2 =
NHCOCCH3CHCH3. Among other examples are U.S. Patent No. 5,635,531
disclosing 3'aminocarbonyloxy paclitaxels, U.S. Patent No. 5,912,264
disclosing 6-
halo- or nitrate-substituted paclitaxels, U.S. Patent No. 6,017,935 disclosing
7-sulfur
substituted paclitaxels, and U.S. Patent No. 5,977,386 disclosing 6-thio-
substituted
paclitaxels. By making substitutions on the taxane skeleton, derivatives
demonstrating
anticancer activity have been found.
Compounds have also been disclosed which are not paclitaxel derivatives but
exhibit "paclitaxel activity", i.e., the inhibition of depolymerization of
microtubules.
A marine natural product (+)-discodermolide has been reported to possess
tubulin-
polymerizing and antitumor properties. (Hung, et al. 1996. "(+)-Discodermolide
binds
to microtubules in stoichiometric ratio to tubulin dimers, blocks taxol
binding and
3

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103GSI07402 CA 02379676 2002-O1-14
results in mitotic arrest," Chem Biol 3:287-293). The basic strunture of
discodermolide is as follows:
A synthetic anticancer agent known as GS-164 having the following chemical
structure has been reported to stimulate microtubule polymerization.
F-
O
N
OH
~O
F
Comparative conformational analysis reportedly indicated that the structure of
GS-164
mimics the nnininaum essential sites of TAXOL~ required to exhibit TAXOL~-like
properties. (Shintani, et u1. 1997. "GS-164, a small synthetic compound,
stimulates
tubulin polymerization by a similar mechanism to that of Taxol,." Cancer
Chemother
Yharmacol 40:513-520.)
Disadvantages have also been associated with some pacl.itaxel derivatives. For
example, many pactitaxel derivatives reported to date. have not had the steric
conformational properties of natural paclitaxel, nor has there been the
ability to change
the right side of the molecule by combinatorial chemistry with carbohydrates,
calcitun
chelators, or oxygenated small molecules.
Other compounds with similar structures to taxane are disclosed as possessing
numerous activities. In U.S. 4,349,552 S-fluorouracil derivatives useful for
treating
cancer are disclosed. In U.S. 5,302,589 heterocyclic inhibitors of 5-alpha-
testosterone
4
Emflfansszeit 5.Sev. 22:17
AMENDED SHEET
30 31 32

05-09-2001 9/5/01 3:17: PAGE 005/47 l~ightFAX US001952~
CA 02379676 2002-O1-14
10365107402
reductase are disclosed. Novel succinimides and maleimides that inhibit
cytokines are
described in U.S. 5,658,940. Lastly, bicyclooctane and bicycloheptane
derivatives
reportedly act as ligands to CCK and gastrin receptors in U.S. 5,674,905.
In addition to there being compounds with a similar stn;iciure to taxane,
there
are also existing computational methods to study paclitaxel's binding site
with tubulin
and its therapeutic elect. Because epothilones compete against paclitaxel for
microtubule binding, the paclitaxel binding site of tubulin was modeled with
suitable
epothilone conformers to better understand how epothilonc interacts with
tubulin
(Wang, M., et al. 1999. "A unified and quantitative receptor model for the
microtubule
binding of paclitaxel and eopthilone," Orgarac Letters 1:43-46;i. The in vitro
synergistic effects on teratocarcinoma of paclitaxel with other drugs used to
treat
cancer were modeled computationally to propose new doseage:; when these drugs
are
co-administered (Chou, T-C., et al. 1994. "Computerized quani.itation of
synergism
and antagonism of Taxol, Topotecan, and Cisplatin against hun:~an
teratocarcinoma
cell growth: a rational approach to clinical protocol design," JJVatI Can Inst
86: I5I7-
1524).
A method has been found by which compounds exhibiting paclitaxel-like
activity and having distinct advantages over paclitaxel and known derivatives
can be
synthesized.
4/1
Emofangsieit 5.Sep. 22:11
AMENDED SHEET

CA 02379676 2002-O1-14
WO 01/05779 PCT/US00/19524
SUMMARY OF THE INVENTION
In one aspect, the present invention is a method for designing anti-tumor
compositions, comprising: (a) using molecular modeling software on a computer
to
create a plot of an active conformation of a known anti-tumor composition, the
active
conformation representative of a three-dimensional conformation of the known
anti-
tumor composition interacting with a target biological site, the plot
providing a first
digital representation of the active conformation, the first digital
representation
depicting a plurality of binding sites of the known anti-tumor composition;
(b) using
the software to eliminate portions of the first digital representation while
preserving
the depiction of the binding sites; and (c) using the software to build a
second digital
representation of a created composition, the created composition having a
three-
dimensional conformation and binding sites similar to the known anti-tumor
composition. Preferably, the known anti-tumor composition has a structure
including
a central skeleton which is depicted in the plot, and the software is utilized
to
eliminate the central skeleton from the depiction and to substitute therefore
a second
central skeleton having desired characteristics. More preferably, the known
anti-
tumor composition has a structure including a central skeleton and one or more
original side chains which are depicted in the plot, and the software is
utilized to
eliminate one or more original side chains from the depiction and optionally
to
substitute a created side chain for one or more of the original side chains.
The method
can further comprise using the software to eliminate the central skeleton from
the
depiction and to substitute therefore a second central skeleton having desired
characteristics. Preferably, a calculation is performed to determine a binding
energy
for the created composition, and the created composition is further modified
to
improve putative binding characteristics, wherein an improved binding
characteristic
is characterized by a higher binding energy. An exemplary known anti-tumor
composition is paclitaxel.
In another aspect, the invention is a method for designing a paclitaxel
alternative composition, which alternative composition has a central skeleton
structure
composed of single or multiple ring groups which hold multiple functional
groups in a
fairly rigid alignment, the central skeleton structure having first, second,
and third side

CA 02379676 2002-O1-14
WO 01!05779 PCT/US00/19524
chains; wherein the first side chain is connected to the central skeleton with
a carbonyl
group at a distance of about 1.5 to 5.5 Angstroms from the central skeleton;
herein the
second side chain places an spa oxygen atom at a distance of about 4.5 to 7.5
Angstroms from the skeleton and about 9 to 11 Angstroms from the carbonyl
oxygen
of the first side chain; wherein the third side chain is placed in an
energetically
accessible conformation that places an aromatic ring in a location that is
simultaneously about 4 to 6 Angstroms from a substitute for hexene and about 8
to 10
Angstroms from the oxygen in the second side chain, the third side chain
selected to
mimic the steric and binding properties of the C2 ester in paclitaxel; wherein
the
method comprises using molecular modeling software on a computer to design the
alternative composition. The alternative composition can further comprise one
or
more bulking groups and wherein the bulking groups increase the size of the
composition to mimic the overall size and shape of the paclitaxel molecule.
Preferably, the first side chain is selected and positioned to mimic the
isoserine group
in taxane. Preferably, the spa oxygen is positioned in space to simulate the
position of
the oxetane ring of paclitaxel. Preferably, the method further comprises
synthesizing
said alternative composition.
In another aspect, the invention is a paclitaxel compound having a chemical
structure selected from one of the following norbonyls
Rt R2 Ri R2
x- R3 x- R3
Rs RSV ~ Rs
R4 R4
Rt R2 Ri R2
x- R3 x- R3
/ Rs _ / Rs
R6 Ra CH2 R6 Ra CH2
6

CA 02379676 2002-O1-14
WO 01/05779 PCT/US00/19524
wherein Rl or RZ or both Rl and R2 are hydrogen, methyl, acetyl, ethyl, short
aliphatic
chain (C1 - C4), or substituted aliphatic chain (C, - C6) where substitution
includes in one or two of the Rl organic functional groups such as an amide;
ketone; hydroxy; phenyl; carboxylic acid; an amino acid, for example,
asparagine, glutamine, aspartic acid, glutamic acid, threonine, serine or
tyrosine. Preferable chemical structures are obtained with the following:
Ri = H or CH3;
RZ = CH3; CHZOCOCH3; or
CH2
R
CH3CH2
R
wherein R is H or singly, doubly, or triply substituted or
fused; or
-O CH2-CH2\~ O
~ CH2
O , . ~CH3
O\C O
R'
\CH3
wherein R' is H or CH3;
wherein X = O; CH2; NH; S; S-CH2; O-CHZ or none;
wherein R3 is one of the following:
7

CA 02379676 2002-O1-14
WO 01/05779 PCT/US00/19524
O O CH3
-C-CHOH(r/s)CH(r/s)CH2NHCOCH2C-CH3
CH3
R
O O
II II
-C-CHOH(r/s)CH(r/s)CH2C
R
R
O O
II II
-C-CHOH(r/s)CHNHC
R
R
O
I I
-C-CHR'(r)CH(s)R"
R
wherein R' = OH when R" = NHBOC; R' = H when R" _
NHBOC; R'=OH whenR"=H; R'=HwhenR"=H
(These substituents are still active in paclitaxel per
Guenard, et al. 1993. "Structure-activity relationships of
taxol and taxotere analogues," J Natl Cancer Inst
Monogr 15:79-82.);
8

CA 02379676 2002-O1-14
WO 01/05779 PCT/US00/19524
O
I I
-C-CHR'(r)CHR"'
wherein R' is as given above and R"' can also be
substituted aryl (single or double) or fused aromatic ring
as in tryptophan or imidazol ring, or substituted
tryptophan; preferably, the aromatic ring can be
substituted with carboxylic acid derivatives;
O
I I
-C-C=C
R (trans)
or
O O
-C-CHOH(r)CH(s)NHCC=CHCH3
CH3
\R (cephalomannine)
or
O O
NH-C-C C-NH-C-O
I
OH
N
or
9

CA 02379676 2002-O1-14
WO 01/05779 PCT/US00/19524
O O
-NH-C-C C-NH-C-O
I I
OH CH2
COOH
O O CH3
-NH-C-C C-NH-C-O-C-CH3
OH CH2 \CH3
COOH
or
O O CH3
NH-C-C C-NH-C-O-C-CH3
~CH3
OH CH2
COOH
or
O O CH3
-NH-C-C C-NH-C-O-C~ CH3
OH CH3
N
For the aryl groups in R3, R can be H or singly, doubly, or triply
substituted OH or preferably with electron withdrawing substituents
such as fluoro (F') or trifluoromethyl (CF3 -). R3 can also be any group
derived from the 13 position in taxane's skeleton that exhibits activity
toward inhibiting the depolymerization of microtubules and/or
anticancer activity;
wherein R4 is one of the following:

CA 02379676 2002-O1-14
WO 01/05779 PCT/US00/19524
COOH
S
I I
CH2 X C
O
II
-CH2-O-C
R
O
I I
CH2-NH-C
R
when the aromatic ring is singly, doubly, or triply
$ substituted;
or
O
I I
CH2-O-C- R""
where R"" is a fixed aromatic ring or substituted fused
aromatic ring; or
O
I I
-C-O- R""'
wherein R""' can be H or a short nonsubstituted or
substituted hydrocarbon chain C~ wherein n = 1-3 or
cyclopropane;
One or more subsititution can be made on the aromatic ring of R4.
1 S Preferably, the substituent(s) on the substituted aromatic ring is an
electron withdrawing substituent. Examples include fluoro- and
chloro-substitution, but any electron-withdrawing substituent
11

CA 02379676 2002-O1-14
WO 01/05779 PCT/US00/19524
compatible with the system may be used which provides a lower energy
gap in a rr - rr interaction between the composition and aromatic amino
acids of proteins;
wherein RS is one of the following:
OH
-CH2~C~ ,O
C'
O
CH3-C-O, CH3
O
OH
or H; or CH3; or small nonsubstituted or substituted
hydrocarbon C" where n = 1-5; or small nonsubstituted
or substituted hydrocarbon ring or heterocyclic ring; or
citric acid and derivatives thereof; or acetic acid and
derivatives thereof; or ascorbic acid and derivatives
thereof; or glucouroic acid or derivatives thereof; or
lactose, sialic acid, or monosaccharides or disaccharides
of glyceraldehyde, erythrose, threose, ribose, arabinose
xylose lyxose, allose, altrose, glucose, mannose, gulose,
idose, galactose, talose, or their acidic ketose, alditol or
inositol forms; or calcium chelating molecule or
oxygenated small molecule, i.e., small carboxylic acids;
or a dipeptide such as "ASP-ASN" or "GLY-GLN", a
cyclic dipeptide such as "PHE-GLN", or small organic
molecules that mimic the functional properties of these
peptides; or any organic molecule that exhibits calcium-
binding properties similar to tetracyclin as given below
12

CA 02379676 2002-O1-14
WO 01/05779 PCT/US00/19524
HO~CH3 N(CH3)2
_0H
C ~ B~ A~ , NH2
H O .OH
Ca++~
or
O
-CH2-O-C
or
S
-CH2-O-C
or
NH-COCH3
-CH2-S-C~ COOH
H
or
OH
i
H2C~
CH2
~O
CH2 -CH2
or
O
I I
CH2- NH-C-NH -OH
13

CA 02379676 2002-O1-14
WO 01/05779 PCT/US00/19524
or
NH2
N'
N
N
O
OH OH
or
JCH2 -N=N-CH3
O
OH
or
O
CH3
HN
O' -N
-O O
OH
or in some cases can also be any of the R4 groups;
wherein R6 and/or R6' , which can be the same or different, is one of the
following:
O
I I
-C-CH20H
14

CA 02379676 2002-O1-14
WO 01/05779 PCT/US00/19524
O O
II II
-C-CH20-C-CH3
-C-O-CH3
or H; or CH3; or OH; or amine or short carbo-aliphatic
chain, substituted with two or three of the following:
keto, hydroxy, sulfoxy, amide, or an amino acid residue
such as serine, asparagine, or threonine; or ethers of the
form -CH2-O-(CH2)~-CH3 where n=1-5 and the right
hand hydrocarbon chain may be substituted with up to
five -OH or carbonyl groups;
In anther aspect the invention is a paclitaxel compound having the following
bicyclo-octane chemical structure
R2 , Rs
Rl X
RS
wherein R, or R2 or both R1 and RZ are hydrogen, methyl, acetyl, ethyl, short
aliphatic
chain (C, - C4), or substituted aliphatic chain (C ~ - C6) where substitution
includes in one or two of the R, organic functional groups such as an amide;
ketone; hydroxy; phenyl; carboxylic acid; an amino acid, for example,
asparagine, glutamine, aspartic acid, glutamic acid, threonine, serine or
tyrosine. Preferable chemical structures are obtained with the following:
R~ = H or CH3;
R2 = CH3; CHZOCOCH3; or

CA 02379676 2002-O1-14
WO 01/05779 PCT/CTS00/19524
CHZ
R
CH3CH~
R
wherein R is H or singly, doubly, or triply substituted or
fused; or
CH2-CH2 O
~ //
-O O-C~CH2
O .. ~CH3
O\C O
R'
CH3
wherein R' is H or CH3;
wherein X = O; CH2; NH; S; S-CH2; O-CH2 or none;
wherein R3 is one of the following:
O O CH3
-C-CHOH(r/s)CH(r/s)CH2NHCOCH2C-CH3
CH3
R
O O
II II
-C-CHOH(r/s)CH(r/s)CH2C
R
v
R
16

CA 02379676 2002-O1-14
WO 01/05779 PCT/US00/19524
O O
II II
C-CHOH(r/s)CHNHC
R
R
O
I I
-C-CHR'(r)CH(s)R"
R
wherein R' = OH when R" = NHBOC; R' = H when R" _
NHBOC; R'=OH whenR"=H; R'=HwhenR"=H
(These substituents are still active in paclitaxel per
Guenard, et al. 1993. "Structure-activity relationships of
taxol and taxotere analogues," JNatl Cancer Inst
Monogr 15:79-82.);
O
I I
-C-CHR'(r)CHR"'
wherein R' is as given above and R"' can also be
substituted aryl (single or double) or fused aromatic ring
as in tryptophan or imidazol ring, or substitut;.d
tryptophan; preferably, the aromatic ring can be
substituted with carboxylic acid derivatives;
O
I I
-C-C=C
R (trans)
17

CA 02379676 2002-O1-14
WO 01/05779 PCT/US00/19524
or
O O
II II
C-CHOH(r)CH(s)NHCC=CHCH3
CH3
'R (cephalomannine)
or
O O
NH-C-C C-NH-C-O
I
OH
or
O O
-NH-C-C C-NH-C-O
I I
OH CHZ
COOH
O O CH3
-NH-C-C C-NH-C-O-C-CH3
~CH3
OH CHZ
COOH
or
O O CH3
-NH-C-C C-NH-C-O-C-CH3
OH CH2 \CH3
I
COOH
or
18

CA 02379676 2002-O1-14
WO 01/05779 PCT/US00/19524
O O CH3
-NH-C-C C-NH-C-O-C~ CH3
OH CH3
N
For the aryl groups in R3, R can be H or singly, doubly, or triply
substituted OH or preferably with electron withdrawing substituents
such as fluoro (F') or trifluoromethyl (CF3 -). R3 can also be any group
derived from the 13 position in taxane's skeleton that exhibits activity
toward inhibiting the depolymerization of microtubules and/or
anticancer activity;
wherein R4 is one of the following:
COOH
S
I I
CH2 X C
O
II
-CH2-O-C
R
O
I I
-CH2-NH-C
R
when the aromatic ring is singly, doubly, or triply
substituted;
or
O
I I
-CH2-O- C- R""
19

CA 02379676 2002-O1-14
WO 01/05779 PCT/US00/19524
where R"" is a fixed aromatic ring or substituted fused
aromatic ring; or
O
I I
-C-O- R""'
wherein R""' can be H or a short nonsubstituted or
substituted hydrocarbon chain C" wherein n = 1-3 or
cyclopropane;
One or more subsititution can be made on the aromatic ring of R4.
Preferably, the substituent(s) on the substituted aromatic ring is an
electron withdrawing substituent. Examples include fluoro- and
chloro-substitution, but any electron-withdrawing substituent
compatible with the system may be used which provides a lower energy
gap in a n - rr interaction between the composition and aromatic amino
acids of proteins;
wherein RS is one of the following:
OH
-CHI
C~~ O
O
II CH3
CH3-C-O
O
OH
or H; or CH3; or small nonsubstituted or substituted
hydrocarbon C~ where n = 1-5; or small nonsubstituted
or substituted hydrocarbon ring or heterocyclic ring; or
citric acid and derivatives thereof; or acetic acid and
derivatives thereof; or ascorbic acid and derivatives
thereof; or glucouroic acid or derivatives thereof; or
lactose, sialic acid, or monosaccharides or disaccharides

CA 02379676 2002-O1-14
WO 01/05779 PCT/US00/19524
of glyceraldehyde, erythrose, threose, ribose, arabinose
xylose lyxose, allow, altrose, glucose, mannose, gulose,
idose, galactose, talose, or their acidic ketose, alditol or
inositol forms; or calcium chelating molecule or
oxygenated small molecule, i.e., small carboxylic acids;
or a dipeptide such as "ASP-ASN" or "GLY-GLN", a
cyclic dipeptide such as "PHE-GLN", or small organic
molecules that mimic the functional properties of these
peptides; or any organic molecule that exhibits calcium-
binding properties similar to tetracyclin as given below
HO~CH3 N(CH3)2
_0H
C ~B~A~ ~NH2
H O .OH O O
Ca++~
or
O
II
-CH,-O-C
or
S
-CH2-O-C
or
NH-COCH3
-CH2-S-C~ COOH
H
21

CA 02379676 2002-O1-14
WO 01/05779 PCT/US00/19524
or
OH
i
H2C~
CH2
~O
-CH2 -CH2
or
O
II
-CH2-NH-C-NH -OH
or
NH2
N'
N
N
O
OH OH
or
OH
HZ O OCH2 -N=N-CH3
O
OH ~ ~OH
OH
or
22

CA 02379676 2002-O1-14
WO 01/05779 PCT/US00/19524
O
CH3
HN
O' -N
-O O
OH
or in some cases can also be any of the R4 groups;
wherein R6 and/or R6' , which can be the same or different, is one of the
following:
O
I I
C-CH20H
O O
II II
-C-CH20-C-CH3
C-O-CH3
or H; or CH3; or OH; or amine or short carbo-aliphatic
chain, substituted with two or three of the following:
keto, hydroxy, sulfoxy, amide, or an amino acid residue
such as serine, asparagine, or threonine; or ethers of the
form -CH2-O-(CHz)"CH3 where n=1-5 and the right
hand hydrocarbon chain may be substituted with up to
five -OH or carbonyl groups;
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. lA-lE depict the five steps of the computational drug design method used.
Fig. 1A depicts the structure of paclitaxel in the tubulin binding site with
the paclitaxel
skeleton and non-active side chains removed and the position of the modified
side
chain, C3 ester group and oxetane oxygen atom maintained. Fig. 1 B depicts the
23

CA 02379676 2002-O1-14
WO 01/05779 PCT/US00/19524
addition of a selected central skeleton, bicyclo[3.2.1)octane. Fig. 1C depicts
the
connection of the ester group using a CHZ group. Fig. 1D depicts the addition
of a
chain that allows the remaining oxygen to be in the correct location. Fig. 1E
depicts
the addition of an acetyl group to the new skeleton to mimic the paclitaxel C
10 acetyl.
Fig. 2 depicts the synthesis scheme resulting in the dime D which is the
reactant for the Diels-Alder reaction.
Fig. 3 depicts the Diels-Alder reaction between Substance (D) and Substance
(Hl) followed by the combinatorial addition of chlorinated carbohydrate and
subsequent removal of the t-butyl-dimethyl-silyl protective group (TBDMS) to
yield
the substituted norbornyl Product (Vi).
Fig. 4 depicts the Diels-Alder reaction between Substance (D) and Substance
(H2) followed by the combinatorial addition of chlorinated carbohydrates and
other
small functionalized Ryodow carbon chains and rings, and subsequent removal of
the
t-butyl-dimethyl-silyl protective group (TBDMS) to yield the substituted
norbornyl
Product (V2).
Fig. 5 depicts the Diels-Alder reaction between Substance (D) and Substance
(H3) followed by the combinatorial addition of chlorinated carbohydrate and
subsequent removal of the t-butyl-dimethyl-silyl protective group (TBDMS) to
yield
the substituted norbornyl Product (V3), wherein n=2 or 3.
Fig. 6 depicts the preparation of norbornene-1,4-diester (I). Two methods of
preparation are presented: Route A beginning from 1,4-dicarboxymethyl-ester-
cyclohexadiene and Route B beginning from munoic acid.
Fig. 7 depicts the preparation of the diol diester Substance (K6) starting
from
norbornene-1,4-diester, Substance (I).
Fig. 8 depicts the preparation of the substituted norbornyl Product (V1) from
the diol diester Substance (K6).
24

CA 02379676 2002-O1-14
WO 01/05779 PCT/US00/19524
Fig. 9A-9G depicts schemes for synthesizing substituted bicyclo-octanes.
DETAILED DESCRIPTION
A method of synthesizing tubulin polymerization stabilizers has now been
found. These stabilizing compounds in solution possess steric conformational
properties of natural paclitaxel and are capable of binding to the tubulin
protein at the
same site where paclitaxel is known to bind and with the same or stronger
binding
energies as paclitaxel. These compounds stabilize tubulin polymerization in a
way
that is mechanistically equivalent to activity mechanism of paclitaxel. The
compounds made by the present invention are paclitaxel-alternative molecules
and are
referred to hereinafter as the "EB compounds." These compounds have increased
solubility, simpler synthesis, and the possibility for specificity and
optimization due to
the combinatorial reactions over natural paclitaxel.
Computational molecular modeling studies were used to define the properties
of the EB compounds necessary to mimic the shape and binding of paclitaxel in
tubulin. These studies were performed using the molecular mechanics force
fields
CHARMM and MMFF; however, it would be understood by one of skill in the art
that
other force fields could also be used. Conformational search and docking
procedures
were used to compute both the conformational energies and binding in the
tubulin
active site. These technques are commercially available in software packages
that
include but are not limited to QUANTATM (Molecular Simulations, Inc., San
Diego
CA, U.S.A.), Insight II~ and associated modules (Molecular Simulations, Inc.,
San
Diego CA, U.S.A.), Cerius2~ and associated modules (Molecular Simulations,
Inc.,
San Diego CA, U.S.A.), Molecular Operating Environment (MOE; Chemical
Computing Groups, Inc., Montreal, Quebec, Canada), and CHARMm~ (Molecular
Simulations, Inc., San Diego CA, U.S.A.).
The EB compounds of the present invention are designed using the following
computational modeling technique.

CA 02379676 2002-O1-14
WO 01/05779 PCT/US00/19524
Step I: The structure of a compound known to have anti-tumor activity is
obtained in its active conformation, e.g. in the tubulin binding site;
Step 2: The central skeleton is eliminated from the structure, along with any
side chains known not to be crucial to its activity;
S Step 3: A new central skeleton is built that will hold the side chains not
removed in Step 2 in the same orientation. Additional functional groups can be
optionally added to mimic the sterics of the active compound (i.e., its
overall shape);
Step 4: Conformation search techniques are used to find an energetically
accessible conformer of the compound with a conformation similar to that of
the
active conformer;
Step ~: A docking calculation is used to determine the binding energy for the
new compound, in the conformation from Step 4, in the tubulin binding site;
Step 6: Further changes are optionally made to the structure in order to
improve the binding characteristics.
The EB compounds of the present invention are all characterized by the
following structural and physicochemical features:
( 1 ) Central skeleton: a central skeleton structure composed of single or
multiple ring groups which include norbornyls, borane, noradamantane,
adamantane,
biotin, cyclopropane, cyclobutane, cyclopentane, cyclohexane, cyclohexene,
cycloheptane, and bicyclo-octane. The skeleton holds multiple functional
groups in a
fairly rigid alignment equivalent to the taxane skeleton. Structurally, the
skeleton
replaces the six membered hexene ring in paclitaxel. For the biotin skeleton,
the two
fused five membered rings act to structurally replace the paclitaxel hexene
ring. The
central skeleton structure has various side chains labeled as R groups.
(2) Side Chain 1: a side chain connected to the central skeleton with a
carbonyl group at a distance of about 1.5 to 5.5 Angstroms from the skeleton.
This is
the taxane iso-serine group or R3;
(3) Side Chain 2: a side chain that places an spa oxygen atom at a distance of
about 4.5 to 7.5 Angstroms from the skeleton. One conformation of this side
chain
should allow this oxygen atom to be oriented in space similar to that of the
oxygen in
26

CA 02379676 2002-O1-14
WO 01/05779 PCT/US00/19524
the oxetane ring of paclitaxel. Specifically, the oxygen should be about 9 to
11
Angstroms from the carbonyl oxygen mentioned in Side Chain 1 above in one of
the
energetically accessible conformers of the molecule. This is the RS group and
in some
casesthe R2 group;
S (4) Side Chain 3: a group that mimics the steric and binding properties of
the
C2 ester in paclitaxel. This is R7 or R4 or some of the larger RZ groups. The
groups
should be able to adopt an energetically accessible conformation that places
an
aromatic ring in a location that is simultaneously about 4 to 6 Angstroms from
the
hexene substitute, about 8 to 10 Angstroms from the carbonyl oxygen specified
in part
(2) above, and about 4 to 6 Angstroms from the oxygen specified in Side Chain
2
above; and
(5) additional groups that allow the overall size and shape of the molecule to
be similar to paclitaxel. These are the groups Ri, R2, R6 and in some cases
R4.
Representative chemical structures for the EB compounds are given below:
norbornyls
Rt R2 Rt Rz
x- R3 x- R3
Rs ~~ ~ Rs
Ra Ra
Rt R2 Rt R2
x- R3 x- R3
Rs , Rs
~S ~CH2 ~ R CH2
R4 4
27

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borane
noradamantane
S adamantane
biotin
cyclopropane
R3
Rs
R3
Rs
R3
Rs
R6 Ra
R3
R
Rs
X- R3
R~ ~, '1~~~-Rs
28

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cyclobutane
X- R3
Ra ' ,~X- Rs
cyclopentane
X- R3
,. Rs
cyclohexane
X- R3
.,~,Rs
cyclohexene
X- R3
,.
,,Rs
CH3 CH3
Rs
~CH~
H3C Ov
R4
R3
29

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cycloheptane
X~ R3
.,Rs
bicyclo[3,2,1]octane
R2 , R3
R1 X
Rs
R4
R6
bicyclo[2,1,1]hexane
R2
Rt X-R3
R6 ~ Rs
R4
bicyclo[1,1,1]pentane
R2
R X- R3
i
Rs
R4

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heterocyclic compound I
O
R
CH3
heterocyclic compound II
O
OH II
HO~CH3~C,C-O~CH
Rt X~R3 O /
Rs CH3
and heterocyclic compound III
O
OH II
HO~CH3~ IC,C-CH2-,CH2
\C=O
R1 O
Rs
wherein R~ or R2 or both R, and R2 are hydrogen, methyl, acetyl, ethyl, short
aliphatic
chain (C1 - C4), or substituted aliphatic chain (Ci - C6) where substitution
includes in one or two of the R1 organic functional groups such as an amide;
ketone; hydroxy; phenyl; carboxylic acid; an amino acid, for example,
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asparagine, glutamine, aspartic acid, glutamic acid, threonine, serine or
tyrosine. Preferable chemical structures are obtained with the following:
Rl = H or CH3;
R2 = CH3; CHZOCOCH3; or
CH2
R
CH3CHz
R
wherein R is H or singly, doubly, or triply substituted or
fused; or
-O CH2-CH2\~ O
~CH2
O ..~CH3
O\C O
R'
CH3
wherein R' is H or CH3;
wherein X = O; CH2; NH; S; S-CH2; O-CHZ or none;
wherein R3 is one of the following:
O O CH3
C-CHOH(r/s)CH(r/s)CH2NHCOCHZC-CH3
CH3
R
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O O
II II
-C-CHOH(r/s)CH(r/s)CH2C
R
R
O O
II II
-C-CHOH(r/s)CHNHC
R
R
O
I I
-C-CHR'(r)CH(s)R"
R
wherein R' = OH when R" = NHBOC; R' = H when R" _
NHBOC; R'=OH whenR"=H; R'=HwhenR"=H
(These substituents are still active in paclitaxel per
Guenard, et al. 1993. "Structure-activity relationships of
taxol and taxotere analogues," J Natl Cancer Inst
Monogr 15:79-82.);
O
I I
-C-CHR'(r)CHR"'
wherein R' is as given above and R"' can also be
substituted aryl (single or double) or fused aromatic ring
as in tryptophan or imidazol ring, or substituted
33

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tryptophan; preferably, the aromatic ring can be
substituted with carboxylic acid derivatives;
O
I I
-C-C=C
(trans)
or
O O
II II
-C-CHOH(r)CH(s)NHCC=CHCH3
CH3
'R (cephalomannine)
or
O O
NH-C-C C-NH-C-O
I
OH
N
or
O O
-NH-C-C C-NH-C-O
I I
OH CH2
COOH
O O CH3
-NH-C-C C-NH-C-O-C-CH3
OH CH2 \CH3
I
or COON
34

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O O CH3
NH-C-C C-NH-C-O-C~ CH3
OH CH2 CH3
COOH
or
O O CH3
NH-C-C C-NH-C-O-C~ CH3
OH CH3
N
For the aryl groups in R3, R can be H or singly, doubly, or triply
substituted OH or preferably with electron withdrawing substituents
such as fluoro (F') or trifluoromethyl (CF3 ~). R3 can also be any group
derived from the 13 position in taxane's skeleton that exhibits activity
toward inhibiting the depolymerization of microtubules and/or
anticancer activity;
wherein R4 is one of the following:
COOH
S
I I
-CH2- X-C
O
I I
-CH2-O-C
R

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O
I I
CH2-NH-C
R
when the aromatic ring is singly, doubly, or triply
substituted;
or
O
I I
-CH2-O-C- R""
where R"" is a fixed aromatic ring or substituted fused
aromatic ring; or
O
I I
-C-O- R""'
wherein R""' can be H or a short nonsubstituted or
substituted hydrocarbon chain C~ wherein n = 1-3 or
cyclopropane;
One or more subsititution can be made on the aromatic ring of R4.
Preferably, the substituent(s) on the substituted aromatic ring is an
electron withdrawing substituent. Examples include fluoro- and
chloro-substitution, but any electron-withdrawing substituent
compatible with the system may be used which provides a lower energy
gap in a rr - rt interaction between the composition and aromatic amino
acids of proteins;
wherein RS is one of the following:
36

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OH
-CH2~C~ ~O
C'
O
I I CH3
CH3-C-O,
O
OH
or H; or CH3; or small nonsubstituted or substituted
hydrocarbon C" where n = I-5; or small nonsubstituted
or substituted hydrocarbon ring or heterocyclic ring; or
S citric acid and derivatives thereof; or acetic acid and
derivatives thereof; or ascorbic acid and derivatives
thereof; or glucouroic acid or derivatives thereof; or
lactose, sialic acid, or monosaccharides or disaccharides
of glyceraldehyde, erythrose, threose, ribose, arabinose
xylose lyxose, allose, altrose, glucose, mannose, gulose,
idose, galactose, talose, or their acidic ketose, alditol or
inositol forms; or calcium chelating molecule or
oxygenated small molecule, i.e., small carboxylic acids;
or a dipeptide such as "ASP-ASN" or "GLY-GLN", a
cyclic dipeptide such as "PHE-GLN", or small organic
molecules that mimic the functional properties of these
peptides; or any organic molecule that exhibits calcium-
binding properties similar to tetracyclin as given below
HO CH3 N(CH3)2
~ ~ ,OH
C ~ B~ A ~ , NH2
H O .OH O O
Ca++~
37

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or
O
-CH2-O-C
or
S
CH2-O-C
or
NH-COCH3
-CH2-S-C~ COOH
H
or
OH
i
H2C~
CH2
~O
-CH2 -CH?
or
O
II
-CH2-NH-C-NH-OH
or
38

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NH2
N'
N
N
O
OH OH
or
OH
HZ O OCH2 -N=N-CH3
O
OH ~ ~ OH
OH
or
O
CH3
HN
O/ 'N
-O O
OH
or in some cases can also be any of the R4 groups;
wherein R6 and/or R6' , which can be the same or different, is one of the
following:
O
I I
-C-CH20H
O O
II II
-C-CH20-C-CH3
39

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-C-O-CH3
or H; or CH3; or OH; or amine or short carbo-aliphatic
chain, substituted with two or three of the following:
keto, hydroxy, sulfoxy, amide, or an amino acid residue
such as serine, asparagine, or threonine; or ethers of the
form -CH2-O-(CHZ)"-CH3 where n=1-5 and the right
hand hydrocarbon chain may be substituted with up to
five -OH or carbonyl groups;
wherein R7 is O-R4, or NN, or NN-(C=O~phenyl, or NN-(C=S)-phenyl; and
wherein Rg is the following:
O
-O-C
R
The EB compounds of the present invention can be synthesized using
conventional techniques using available starting materials. Representative
synthesis
schemes are presented in Fig. 2-l0E and some of the examples given below. The
synthesis methods provided result in racemic mixtures; however, it is within
the skill
of one in the art to prepare and separate the diasteromers to isolate the
preferred chiral
compound. All such forms of these compounds are expressly included in the
present
invention.
Example 1: Designing A Representative EB Compound
Using Computational Technique
The bicyclo[3.2.1] octane compound having the chemical structure given
below was designed using the following procedure:
Step l: The structure of docetaxel in the tubulin binding site was obtained.
The docetaxel skeleton and non-active side chains were removed, while holding
the

CA 02379676 2002-O1-14
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modified side chain, C3 ester group and oxetane oxygen atom in place. This
results in
the structure illustrated in Fig. 1A.
CH3
CH3
O CH3
OH
OH
Step 2: A skeleton was selected which will hold these side chains. Selecting
the bicyclo[3.2.1 ] octane skeleton results in the structure illustrated in
Fig. 1 B;
Step 3: The ester group was connected to the skeleton. Using a CH2 group to
connect them results in the structure illustrated in Fig. 1 C;
Step 4: A chain was constructed that will allow the remaining oxygen atom to
be in the correct location. This results in the structure illustrated in Fig.
1D;
Step 5: Functional groups were added so that the new skeleton will take up
about the same amount of space as the bulkier docetaxel skeleton. In this
case, an
acetyl group was added to match the location of the docetaxel C 10 acetyl,
resulting the
structure illustrated in Fig. 1 E;
Step 6: Existing conformational search techniques were used to compute a list
of conformers of the molecule and their associated conformational energies. If
an
energetically accessible conformer that is similar in shape to docetaxel in
its binding
conformation was found, that conformer was used in Step 7. Othterwise the
process
was repeated with different skeleton and R groups;
Step 7: The energetically accessible conformers were used in a docking
calculation to determine the binding energy of the compound in the tubulin
active site
as shown in Example 2; and
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Step 8: Compound showing an acceptable binding energy in Step 7 were then
synthesized and assayed.
Example 2: Computation of Binding Energies
The binding energies were obtained computationally for the EB compound
given in Example 1 by the following procedure:
Step I: The molecular structure was obtained according to Example 1.
Step 2: Computational comformation searches on the molecule were
performed using MOE software. The MMFF94 force field was used to compute
energies. Both the RIPS and Hybrid Monte Carlo conformation search algorithms
were used. This yielded a number of conformers of the molecule along with
their
relative conformational energies. Some of the lowest energies obtained were:
E, = 235.0108 kcal/mol
EZ = 237.0030 kcal/mol
E3 = 237.8409 kcal/mol
E4 = 239.0317 kcal/mol
ES242.6232 kcal/mol
=
E6= 243.2933
kcal/mol
E~243.6304 kcal/mol
=
Eg243.7124 kcal/mol
=
Step 3: The conformation of these conformers were examined by overlaying
the computed conformer with the structure of a compound known to have a drug
activity in its active conformation. For this study, the paclitaxel derivative
docetaxel
(TAXOTERE~, Rhone-Poulenc Rorer) in its active conformation was obtained from
the protein data bank. In this study, the lowest energy compound had a
comformation
very similar to the active conformation of docetaxel. This conformer was used
in the
subsequent steps.
Step 4: A model of the binding site was obtained. The structure of tubulin
with docetaxel bound in the active site was obtained from the protein data
bank. The
structure of the tubulin/docetaxel complex was minimized using the MMFF force
42

CA 02379676 2002-O1-14
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field. The docetaxel was then removed from the structure and the resuling
geometry
for tubulin was used for the docking calculations.
Step S: Docking calculations were run using the docking algorithm in the
MOE software and the MMFF94 force field. The binding energies that are
reported
are the sum of the intermolecular electrostatic term and the intermolecular
nonbond
(or van der Waals) term in the MMFF force field. The binding energy for our
compound was -138.7597 kcal/mol, as compared to the binding energy for
docetaxel
of -134.0277 kcal/mol when compared by the same method.
Step 6: Finding a new compound with an energetically accessible
conformation that has a binding energy similar to that of a known active
compound is
considered a positive result for the computational prediction of new
compounds.
Compounds showing preferred binding energies were then synthesized.
Example 3: Designing A Representative EB Compound Using
Computational Technique and Computation of Binding Energies
The following compound was designed as given in Example 1 and the binding
energies were determined as given in Example 2.
OH ~O O
C-C O
C
O O
O~ ~NH O O
CI ~ CH3
O C=O
H C~Cv C=O
3H3C CH3
CH3
The binding energy for this compound was determined to be -165.7084
kcal/mol.
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Example 4: Synthesis Of A Substituted Norbornyl
Synthesis of Diene Reactant (Substance D) for Diels - Alder Reaction
Now refernng to Fig. 2, which sets forth the synthesis scheme resulting in
Diene Substance (D), the starting material was a 2-cyclopenten-1-one molecule
which
can be protected in its 5 position by reacting with carboxyl-trimethyl-silane
in the
presence of lithium di-isopropylamide (LDA) and tetrahydrofuran(THF) to yield
Substance (A). Substance (A) then was condensed via a Michael condensation
with I-
t-butyl-dimethyl-silyloxy-acetic acid-methyl ester to yield condensation
Substance
(B). An additional Michael condensation (O-alkylation) with a series of acid
chlorides
substituted with R3 would yield the dime Substance (C). Selective desilylation
(removal of the TMS-trimethyl-silyl groups) followed by decarboxylation to
yield
Substance (D) which is the dime reactant for the Diels Alder reaction.
Synthesis of Dienophile (Substance H) for Diels Alder Reaction
The dienophile Substance (H) (Hi in Fig. 3; H2 in Fig. 4; H3 in Fig. 5) was
obtained from a Wittig reaction of dihydroxy acetone and phosphonyl-yilide
followed
by mono esterification with benzoic acid (or any R4) to yield Substance (H).
Diets-Alder Reaction
Substance (D) and Substance (H) were mixed under conditions known to the
art for Diets-Alder reactions and published in Corey, et al. 1969. JAm Chem
Soc
91:5675. The product of the Diets-Alder Reaction was an addition product,
Substance
(G) (G, in Fig. 3; GZ in Fig. 4; G3 in Fig. 5).
Combinatorial Addition of Carbohydrates or Chelatin~ Agents
Chlorinated carbohydrates or calcium chelators may be reacted with Substance
(G) (G, in Fig. 3; GZ in Fig. 4; G3 in Fig. 5). Shown in Fig. 3 is the
reaction of a
chlorinated carbohydrate ("sugar-chloride") to yield the Adduct (L) (L~ in
Fig. 3; L2
in Fig. 4; L3 in Fig. 5), which was then reacted with potassium fluoride salt
to remove
the t-butyl-di-methyl-silyl(t-BuDMSi) protective group to yield the
substituted
norbornyl Product (V) (V 1 in Fig. 3; V2 in Fig. 4; V3 in Fig. 5).
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CA 02379676 2002-O1-14
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Example 5: Synthesis Of Substituted Norbornyl
Preparation ofNorbornene-1,4-diester
Now referring to Fig. 6, two alternatives are shown for the synthesis of
norbornene-1,4-diester Substance (I): Route A and Route B. In Route A, 1,4,-
dicarboxymethyl-ester-cyclohexadiene, Substance (E), is reacted with
diazomethane,
according to the procedure of Guha et al, Chem. Abstr. 34:2822 (1940) to yield
Substance (I). In Route B, munoic acid, Substance (.n, was reacted with
diazomethane and heated to undergo a rearrangement as described in Guha et
al.,
Berichte 70:2109 (1937) to yield cyclopentene dicarboxymethylester, Substance
(F).
Substance (F) was then reacted with lithium dimethyl amide (LDA) according to
Della
et al, Austral. J. Chem 38:1705 ( 1985) and 1-bromo-2-chloro-ethane to yield
Substance (I).
Preparation of Diol ester
As shown in Fig. 7, Substance (I) was reacted with catecholborane, as
described in Brands et al., Tetrahedron Lett. 33:5887 (1992), followed by
Jones
oxidation to yield Substance (K~). Substance (K1) was reacted with lithium and
pyridine according to Meinwald et al, J. Org. Chem. 35:1891 (1970); see also
McMurry, Org. Reactions 24:188. Substance (K1) underwent selective
nucleophilic
demethylation to yield the monoester Substance (K2). Substance (K2) was
reacted
with phosphonyl-ylide (Wittig reaction) according to Organophosphorus Reagents
in
Organic Synthesis. 1979. J.LG. Coutogan (ed), Academic Press, London, pp. 17-
153
and Maryanoff, et al. 1989. Chem Rev 89:863 to yield Substance (K3). Sustance
(K3)
was reacted with the reagent (COCI)2 followed by tri-trimethylsilyloxy
ethylene,
according to Wissner, J.Org.Chem. 44:4617(1979) and Kende et al., Tetrahedron
Lett.
23:1751 (1982), to yield the Substance (K4). The hydroxy group of Substance
(K4)
was then protected with a t-butyl-dimethylsilylchloride (TBDMSCI) to yield
Substance (KS). The carboxyethylester group in position 1 of the norbornyl
skeleton
in Substance (KS) then underwent two reactions: formation of the acid chloride
carboxyl inversion and O-acylation with the appropriate R3 group, followed by
oxidation of the methylene at the norbornyl 5th position (exo attack) with
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CA 02379676 2002-O1-14
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tetraoxide (0s04) and esterification with benzoyl chloride (or any other R4
acid
chloride group) to yield Substance (K6).
Combinatorial addition of carbohydrate or chelatine went
Referring to Fig. 8, Substance (K6) was then reacted with any carbohydrate
chloride (also known as "sugar chloride" or "chlorinated carbohydrate") or
parallel
reagent to yield Substance (L1) which was then reacted with HF in pyridine to
yield
Product (V1).
Example 6: Tubulin Polymerization Assays
Various assays can be used to demonstrate binding of the EB compounds of
the present invention to tubulin.
Tubulin Polymerization Assay.
Tubulin polymerization activity is assayed using the Microtubule/Tubulin
Biochem Kit purchased from Cytoskeleton. The assay mixture contains the
following:
101 GTP (100mM)
21 ~l DMSO
200q1 Tubulin (5 mg/ml)
130 ~1 Test compound (1.5 mM)
670 ~.1 PEM (80mM Piperazine-N,N'-bis[2-
ethanesulfonic acid] sequisodiumsalt; 0.5
mM Magnesium chloride; 1 mM Ethylene
glycol-bis(b-amio-ethyl ether) N,N, N', N'
tetra-acetic acid, pH6.9) _ _ _
10001 Total Volume
The assay mixture is immediately placed in a Beckman DU640 UV/VIS
spectrophotometer at 24°C. Tubulin polymerization is monitored by
measuring the
absorbance at 340 mn every 60 seconds for one hour. The absorbance plots are
then
46

CA 02379676 2002-O1-14
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compared to a paclitaxel standard in order to compare relative tubulin
polymerization
activities.
Tubulin Polymerization at 37 °C/Incubation with Test Compound.
The absorbance at 340 nm of the mixture listed in the table above is initially
read at 24 °C. The mixture is then transferred to a 37 °C water
bath and incubated for
20 minutes. The absorbance at 340 nm is read then read using a Beckman DU640
spectrophotometer. Further readings are collected every 20 minutes up to a
total of 80
minutes. The mixture remain at 37 °C between readings. The absorbance
trend is
compared to a standard containing 130 ~1 of 30% DMSO in place of the test
compound.
Tubulin Polymerization in the Presence of Paclitaxel
In order to determine whether or not the test compounds compete for the
paclitaxel binding site on the tubulin protein without enhancing
polymerization, the
above mixture containing the test compound isincubated at 37 °C for 20
minutes. At
1 S this time, the mixture istransferred to a Beckman DU640 spectrophotometer
and 10
~M paclitaxel added. The absorbance at 340 nm is measured every 60 seconds for
one
hour and compared to a standard containing 130 ~l of 30% DMSO in place of the
test
compound along with 10 ~M paclitaxel.
Colorimetric assaYfor cell viability and proliferationof BT-20 breast cancer
cells
The following colorimetric assay is used to detect and measure cell viability
,
activation and proliferation of BT-20 breast cancer cells after incubation
with and
without the paclitaxel compounds of the present invention. Sodium 3'-
[1 [(phenylamino)-carbonyl]-bis(4-methoxy-6-nitro) benzene-sulfonic acid
hydrate
was used in a colorimetric assay for cell viability and proliferation by BT-20
breast
cancer cells. Cleavage of XTT by dehydrogenase enzymes of metabolically active
cells yields a highly colored formazan product which is water soluble.
Bioreduction
of XTT can be potentiated by the addition of electron coupling agents such as
phenazine methosulfate (PMS) or menadione (MEN). Optimal concentrations of PMS
47

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or MEN were determined. Assays were performed essentially as in Roehm, N.W.;
Rodgers, G.H.; Hartfield, S.M.; Glasebrook, A.L.; Journal of immunological
Methods,
142 (1991) 257-265. Briefly, solutions of 1 mg/mL XTT in 60°C DMEM
media and
25 mM PMS in deionized water are prepared (the XTT solution must be prepared
S fresh each day, although PMS is stable at 2-8°C in the dark for at
least three months).
Then thiol - free DMEM is warmed to 60°C and subsequently 1 S ~L of
the 25 mM
stock solution is mixed with 3 mL of XTT/media giving a PMS concentration of
125
~M. Immediately 25 ~L of XTT/PMS solution is added to 100 ~L of culture in
each
well giving final concentrations of 0.2 mg/mL XTT and 25 ~M PMS. Cells were
incubated at 37°C for 4-8 hours before reading the absorbance at 470
nm.
Tubulin depolymerization assay
Screening compounds for microtubule destabilization activity are
accomplished with the CytoDYNAMIX ScreenT"" 6 (Cytoskeleton, Inc.). Briefly,
tubulin are polymerized to steady state by the addition of one volume G-PEM
(80 mM
piperazine-N,N'-bis[2-ethanesulfonic acid] sequisodium salt, 0.5 mM magnesium
chloride, 1 mM ethylene glycol-bis(b-amino-ethyl ether) N,N,N'N'-tetraacetic
acid pH
= 6.9, and 10 mM GTP) plus 10% glycerol and incubating at 37°C for 30
minutes.
Test compounds are then prepared in 120 ~L G-PEM in a 96-well plate at
37°C. The
test compound and polymerized tubulin are mixed in a 3:1 ratio. The absorbance
at
340 nm is measured over time and compared to the paclitexal standard.
The EB compounds of the present invention can be applied to therapeutic
treatments of diseases such as cancer of various type, polycystic kidney
disease, and
inflammation and related uses. Generally, any disease which involves cell
division
can be adressed by these novel molecules.
It should be understood that the compounds of this invention may be modified
by appropriate functionalities to enhance selective biological properties.
Such
modifications are known in the art and include those which increase biological
penetration into a given biological system (e.g., blood, lymphatic system,
central
nervous system), increase oral availability, increase solubility to allow
administration
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CA 02379676 2002-O1-14
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by injection, alter metabolism and alter rate of excretion. In addition, the
compounds
may be altered to pro-drug form such that the desired compound is created in
the body
of the patient as the result of the action of metabolic or other biochemical
processes on
the pro-drug.
The compounds of this invention may be employed in a conventional manner
for the treatment of diseases. Such methods of treatment, their dosage levels
and
requirements would be understood by one of ordinary skill in the art from
available
methods and techniques. For example, a compound of this invention may be
combined with a pharmaceutically acceptable adjuvant for administration to a
patient
suffering from cancer in a pharmaceutically acceptable manner and in an amount
effective to lessen the severity of that disease.
The EB compounds may be employed in pharmaceutical compositions either
alone or together with other compounds of this invention. The compounds of
this
invention may also be co-administered either concommitantly or sequentially
with
1 S other therapeutic drugs to increase the effect of therapy. The
pharmaceutical
compositions can be administered orally, parenterally, by inhalation spray,
topically,
rectally, nasally, buccally, vaginally or via an implanted reservoir. The
pharmaceutical compositions are preferably administered by intravenous
infusion in
water, sodium chloride or any other suitable intravenous infusion solution.
REFERENCES
1. Guha and Hazra, Chem. Abstr. 1940, 34, 2822.
2. Guha and Sankaran, Berichte 1937, 70, 2109.
3. Cf. Della and Tsanaktsidls, Austral, J. Chem, 1985, 38, 1705.
4. Brands and Kende, Tetrahedron Lett. 1992, 33, 5887.
5. Meinwald and Putzig, J. Org. Chem. 1970
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