Note: Descriptions are shown in the official language in which they were submitted.
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DIAZONAMIDE A ANALOG
Technical Field
[0001] The invention relates to an analog of diazonamide A that has superior
antitumor
activity. A synthetic route to this compound is also disclosed.
Background Art
[0002] Diazonamide A is a mitotic spindle-disrupting agent first isolated from
the marine
organism Diazona angulata. Numerous atteinpts have been made to synthesize
this
compound and its analogs. PCT publication WO 03/106438 describes a putative
synthetic
route; however, the structure of diazonamide A provided in that publication is
incorrect.
U.S. patent 7,022,720 ('720) correctly discloses the structure of diazonamide
A and describes
the synthesis of some of its analogs through the combined use of catalytic
Heck
endocyclization, stereo-controlled ring-contracting pinnacol rearrangement,
and indole
arylation via internal photo-induced electron transfer. Generic structures of
some analogs are
provided. A daughter application claiming priority from this patent was filed
31 October 2005 and is published as 2006/0089397 and includes the structure of
the analog,
Compound J, claimed herein. The '720 patent does not specifically describe the
compound
of the present invention, which has surprisingly potent anti-mitotic activity.
Disclosure of the Invention
[0003) The present invention is directed to a compound of the formula
HN N 27
N O
HO = O O
O = H
NH
N
H
(Compound J) and the pharmaceutically acceptable salts thereof. The invention
is also
directed to pharmaceutical compositions containing this compound and/or its
salts, to
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modified forms of this compound coupled to stabilizing or targeting agents,
and to methods
of treating proliferative diseases, in particular Taxo1TM-resistant cancers,
using these
compounds and formulations.
[0004] In another aspect, the invention is directed to methods to synthesize
Compound J
and its salts.
Brief Description of Drawings
[0005] Figure 1 shows the ability of Compound J to inhibit the growth of
various cancer
cell lines in comparison to diazonamide A, a hydroxylated form of Compound J.,
paclitaxel,
and vinblastine.
[0006] Figure 2 shows a plot of survival fraction against concentration of
drug with
respect to PTX10 ovarian cancer cell lines for Compound J and paclitaxel.
[0007] Figure 3 is a graph showing the pharniacokinetics of Conipound J.
[0008] Figures 4A and 4B show the effect of Compound J as coinpared to
paclitaxel on
xenografts of HCTI 16 and PC-3 cells.
[0009] Figures 5A and 5B show that Compound J has no effect on neutrophil
proliferation or cell counts in vivo.
Modes of Carrying Out the Invention
[0010] Compound J has been shown, as described in the examples below, to have
potent
anti-mitotic activity with respect to certain cancers, in particular cancers
that are resistant to
TaxolTM. Compound J can be supplied in its free base fonn, or can be supplied
as a
pharmaceutically acceptable salt, or as a mixture of the depicted form and the
corresponding
salt. Suitable salts include those of inorganic acids such as hydrochlorides,
hydrobromides,
sulfates, hydrosulfates, and the like, or organic acid addition salts such as
the acetates,
formates, maleates, and the like.
[0011] In addition, Compound J may be coupled to moieties such as targeting
agents.
Among such targeting agents are antibodies or immunologically active fragments
thereof,
including single-chain antibody forms directed against tumor antigens or
against receptors or
integrins associated with tumors, peptidomimetics directed against these
moieties, and tlie
like. In addition, Compound J may be coupled to an excipient such as
polyethylene glycol
for altering pharmacokinetics.
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[0012] The formulations useful in the invention include standard formulations
such as
those set forth in Remington's Pharmaceutical Sciences, latest edition, Mack
Publishing Co.,
Easton, PA, incorporated herein by reference. Such formulations include those
designed for
oral delivery, slow release, topical administration, parenteral
administration, or any other
suitable route as determined by an attending physician or veterinarian. Thus
administration
may be systemic or local. Suitable vehicles or excipients include liposomes,
micelles,
nanoparticles, polymeric matrices, buffers, and the full range of
forinulations known to
practitioners.
[0013] Compound J is particular useful in treating proliferative diseases, in
particular,
tumors and malignancies associated with breast, ovary, lung, colon, prostate,
melanoma,
colon, pancreas, glioma, carcinoma, and the like.
[0014] The formulations that include Compound J and/or its salt and/or its
conjugates
may also be used in combination with other drugs, such as additional antitumor
agents or
other palliative compounds such as compounds that aid in nutrition or general
health.
[0015] Compound J is conveniently synthesized by treating the free amino
precursor
with S-2,5-dioxopyrrolidin-1-yl-2-hydroxy-3-methylbutanoate. This converts the
free amine
to the 3-methyl-2-hydroxybutylate.
[0016] Those skilled in the art will appreciate that this coupling reaction
can also be
accomplished with other activated esters of the 2-hydroxy3-methylbutanoate,
such as by way
of example only N-hydroxybenzotriazole ester, perfluorophenyl ester, N-
hydroxyphthalimide
esters, activated esters generated by the reaction of the carboxylic acid with
a carbodiimide,
and other activated esters conventionally used for acylation of an amine to
form amide
bonds; thus the invention provides a method to prepare Compound J by coupling
an activated
2-hydroxy-3-methylbutanoate derivative, which may optionally be protected at
the 2-
hydroxyl, with the above described amine. The amine may also optionally be in
protected
form, i.e. it may have protecting groups on either or both of the indole
nitrogen and the
indoline nitrogen. Suitable protecting groups for use on the hydroxyl include
acyl groups,
silyl groups, pyran acetals, and the like. Suitable protecting groups for use
on the ring
nitrogen atoms of the amine compound, which are not intended to react with the
hydroxybutanoate activated ester, may include acyl groups such as carbamates
or
trifluoroacetate, as well as silyl groups. Suitable protecting groups and
methods to attach and
remove them are well known in the art, and are described, for example, in T.H.
Greene,
PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 2"d ed,
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[0017] The following examples are offered to illustrate but not to limit the
invention.
Examples 1-16 describe the synthesis of Compound J. Examples 17-20 describe
its
biological activity.
Example I
7-Bromoindole
[0018] 2-Bromonitrobenzene (1.10 kg, 5.45 mol) was dissolved in
tetrahydrofuran (10 L)
at room temperature. This solution cooled with stirring in a bath maintained
at -78 C. When
the internal teinperature reached -40 C, vinylmagnesium bromide (16.3 L, 16.3
mol) was
added at such a rate as to maintain the internal temperature at -40 C during
the addition.
Upon complete addition, the reaction was removed from the bath and allowed to
warm
slowly to -30 C over the course of 45 rninutes. This required occasional
cooling. The -30 C
reaction solution was quenched by rapid addition of a slightly cool (-10 C)
solution of
saturated aqueous NH4C1(10 L). Slight foaming occurred. (Inverse quench into
the
ammonium chloride solution is also satisfactory.) This resulted in a biphasic
mixture with
some undissolved magnesium salts in the form of a gel. The mixture was stirred
for 30
minutes and separated. The aqueous layer was back extracted with
tetrahydrofuran (10 L).
The combined organic layers were evaporated at reduced pressure with a bath
temperature of
35 C and the resulting dark oil was taken up in metliylene cl-Aoride (5 L) and
dried with
Na2SO4. The mixture was filtered and concentrated. The resulting material was
chromatographed, eluting with 2% ethyl acetate-hexanes to give 7-bromoindole
(557 g, 52%
yield) of as an off-white solid. 'H NMR (CDC13): consistent with proposed
structure.
Example 2
2-Acetamido-3 -(7-bromo-1 H-indol-3 -yl)propanoic acid
[0019] To a 5 L, 3-neck round bottom flask, equipped with stir bar, nitrogen
atmosphere,
thermocouple and condenser, was added the title compound of Example 1 (252.1
g,
1.29 mol) followed by acetic acid (1.5 L) and acetic anhydride (760 mL, 8.04
mol). L-serine
(266.9 g, 2.53 mol) was charged after stirring for 20 min. This mixture was
stirred for 4h
before it was heated to 40 C. After most of the solids had dissolved, the
reaction was heated
to 90 C, followed by an excursion to 110 C. The reaction was then cooled to 80
C, stirred at
this temperature, and the reaction progress was monitored by HPLC. After 5
hours the
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reaction was complete as judge by the absence of 7-bromoindole in the
chromatogram. The
heat was removed, and the reaction was allowed to continue stirring overnight
at room
temperature.
[0020] Methanol (450 mL) was added and the reaction was concentrated in vacuo
at
-50 C to a thick, black tar. Methanol (3 L) was added to the residue and after
vigorous
agitation most of the residue went into solution, leaving behind a fine
precipitate. To this
mixture was added of H2SO4 (52.5 mL), and the reaction was stirred at reflux
overnight. The
reaction was cooled to room temperature and diluted with tetrahydrofuran (3
L). The
solution was charged to a 12 L separatory funnel containing saturated aqueous
NaHCO3
(4 L). This mixture was extracted with methyl t-butyl ether (3 x 4 L). The
organic layers
were combined and washed with brine and dried over Na2SO4, then concentrated
in vacuo to
afford a mixture of brown solid and brown oil. Methylene chloride (500 mL) was
added to
the crude product, and some white solids remained undissolved. These crystals
were filtered
to afford -10 g of product. Seed crystals were added to the filtrate, and
after 30 minutes, a
brown solid had precipitated. The new mixture was filtered, and more seed
crystals were
added to produce a third crop of precipitate, and this third mixture was also
filtered.
Addition of seed crystals to the filtrate produced no additional product. The
filtrate was
concentrated in vacuo to afford a brown foam, which was redissolved in
methylene chloride
(600 mL). Methyl t-butyl ether (MTBE) (1,250 mL) was slowly added to the
solution, which
precipitated a brown solid. The mixture was filtered, and the filtrate was
added to other
impure samples, then purified by column chromatography, eluting with methylene
chloride-hexanes. None of the fractions containing product were very pure
(range: 50% -
75%), so all were recrystallized with MTBE to afford pale yellow powders.
These samples
were combined with the recrystallization samples to afford the title compound
(139 g, 33%
yield, 75% purity). 1H NMR (CDC13): consistent with proposed structure.
Example 3
Methyl2-amino-3-(7-bromo-lH-indol-3-yl)propanoate hydrochloride
[0021] H2S04 (340 mL) was added slowly to a stirred mixture of the title
compound of
Example 2 (342 g, 1.05 mol) in methanol (3.4 L). The resulting dark brown
mixture was
heated to reflux for 16 hours at which time HPLC analysis indicated completion
of the
reaction. The reaction was cooled to room temperature and quenched slowly into
a stirred
mixture of water (4.8 L), sodium bicarbonate (342 g) and methylene chloride
(4.8 L).
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Stirring was continued for 1.5 hours. The layers were separated and the
aqueous layer was
back extracted twice with methylene chloride (3.0 L). The combined extracts
were dried
with sodium sulfate and concentrated to a black tar. The material was
dissolved in
methylene chloride (3.0 L). 1N HCI in diethyl ether (1.1 L) was added slowly
with external
cooling. The suspension was cooled to ice bath temperature and filtered. The
solids were
washed twice with metliylene chloride (500 mL) and three times with hexanes
(500 mL).
The solids were dried to constant mass in a vacuum oven at 32 C to give the
title compound
(267.4 g, 76% yield). 1H NMR (CDC13): consistent with proposed structure.
Example 4
Methyl2-((S)-2-(benzyloxycarbon lamino)-
3 -methylbutanamido)_3-(7-bromo-1 H-indol-3 -yl)propanoate
[0022] The title compound of Example 3 (256.6 g, 770 mmol), TBTU (296.4 g, 1.2
eq)
and Cbz-L-valine (212.7 g, 1.1 eq) were dissolved in dimethylforinamide (DMF,
anhydrous,
2,700 mL) and cooled to 0 C for 30 min. Diisopropylethylamine (DIEA, 268 mL)
was
added slowly and the solution was allowed to warm to room temperature.
Stirring was
continued for 4 hours at which time HPLC indicated completion of reaction. The
reaction
was diluted with ethyl acetate (11 L) and water (7.5 L). The mixture was
stirred for 1 hour
and allowed to separate. The organic layer was washed once with water (7.5 L),
twice with
brine (7.5 L) and twice with saturated NaHCO3 (7.5 L). The material was dried
with sodium
sulfate and concentrated to brown black solid. The material was taken up in
methylene
chloride (7.5 L) and combined with 22.2 g of another lot of similar quality
material and silica
gel (400 g). The solvent was removed to support the crude compound on silica.
This
material was divided in half and each half was chromatographed on a 6 in x 4
ft silica gravity
column. Each was eluted with methylene chloride (20 L) followed by 5% acetone
in
methylene chloride (20 L) followed by 8% acetone in methylene chloride (30 L)
to give the
title compound (383 g, 89% yield). 1H NMR (CDC13): consistent with proposed
structure.
Example 5
Methyl2-((S)-1-(benzyloxycarbon lamino)-2-methylpropyl)-
-(7-bromo-1 H-indol-3 -yl)oxazole-4-carboxylate.
[0023] Dichlorodicyanoquinone (340.8 g, 1,500 mmol) was added to a stirred
solution of
the title compound of Example 4 (361.4 g, 681 mmol) dissolved in
tetrahydrofuran (15 L)
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and heated to reflux for 6 hours at which time HPLC indicated complete
reaction. The
reaction was concentrated to '/4 its volume and diluted with ethyl acetate (12
L). The
resulting black solution was washed three times with saturated aqueous NaHCO3
(5.5 L).
The organic layer was dried over sodium sulfate and concentrated to give the
title compound
as a black solid (392 g, 100% yield). 'H NMR (CDC13): consistent with proposed
structure.
Example 6
Methyl2-((S)-1-amino-2-methy_lpropyI)-
5-(7-bromo-lH-indol-3-yl)oxazole-4-carboxylate hydrobromide
[0024] To 33% HBr in acetic acid (1.33 L) was added the title compound of
Example 5
(403.4 g, 766 mmol) and the stirred vigorously for 1 hour and 20 min. The
mixture was
slowly and carefully added to MTBE (12 L) with external cooling and strong
agitation. The
mixture was stirred for 1 hour at 0 C and filtered under N2. The hygroscopic
solids were
washed with MTBE (1 L) and dried to constant mass in a vacuum oven to yield
the title
compound (277.5 g, 76.5%) as a fine brown solid. 'H NMR (CDC13): consistent
with
proposed structure.
Example 7
Meth yl 2-((S)-1-((S)-2-(benzylo carbon l~nino)-3-(4-h d~roxyphenyl
propanamido)-
2-methylpropyl)-5-(7-bromo-1 H-indol-3-yl)oxazole-4-carboxylate
[0025] To diisopropylethylainine (225 mL, 1,290 mmol) stirred at 0 C was added
a
solution of the title compound of Example 6 (277.5 g, 586.5 mmol), Cbz-L-
tyrosine (194.2 g
615.9 mmol) and TBTU (207.2 g, 1.1 eq) in dimethylformamide (anhydrous, 2.77
L). The
reaction was allowed to warm to room temperature and stirred for 16 hours at
which time
HPLC indicated completion of reaction. The reaction solution was slowly poured
into
saturated aqueous NaHCO3 (12.0 L) and stirred for 30 min. The precipitate was
filtered and
the filter cake was tlioroughly washed with water. The resulting brown
material was dried to
constant mass in a vacuum oven at 40 C to yield the title compound (435 g).
The title
compound was further purified by recrystallization. The title compound was
dissolved in
isopropanol (9.0 L) at 70 C. The insoluble material was removed by filtration
and the filtrate
was heated while slowly adding hexanes (9.0 L). The suspension was allowed to
cool to
room temperature at which time an ice bath was applied. Once cooled the
mixture was
stirred at ice bath temperature for 30 min and filtered. The solid was washed
with hexanes
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and dried to constant mass at 40 C in a vacuum oven giving pure title compound
(264 g,
61 % yield). 1H NMR (CDC13): consistent with proposed structure.
Example 8
CbzHNHN N
0
0 COzMe
0 I=IH Br
[0026] The title compound of Example 7 (45.0 g, 65 mmol) was dissolved in
tetrahydrofuran (325 mL) and was added rapidly to a-20 C solution of Phl(OAc)2
(20 g,
62 mmol) and LiOAc (12.7 g, 196 mmol) in 2,2,2- trifluoroethanol (13.0 L). The
solution
was stirred at -20 C for 25 minutes at which time solid NaHCO3 (117.5 g) was
added. The
cold bath was removed and stirring was continued for an additional 30 min. The
mixture
was filtered at 10 C and the filtrate was concentrated. The residue, 94.6 g,
was taken up in
CHCl3-tetrahydrofuran (3:1, 300 mL) and sonicated for 10 minutes. The
precipitated
undesired diastereomer was removed by filtration and the filtrate was
concentrated to give
crude title compound. The above operations were performed a total of 3 times
to give a
combined total of 223.6 g of crude title compound. The crude title compound
was initially
purified by silica plug filtration eluting witli 100% ethyl acetate. This
resulted in 179.9 g of
material which was further purified by multiple coluinn chromatography. A
first column was
eluted using 6:1 methylene chloride-tetrahydrofuran. This resulted in 71.1 g
of material,
which was taken up in 6:1 methylene chloride-tetrahydrofuran (200 mL) and
refrigerated
overnight. The precipitated undesired diastereomer was removed by filtration
and the filtrate
was concentrated to give 66.6 g of compound. This material was chromatographed
for an
additional 2 times eluting with 15:1 methylene chloride-tetrahydrofuran to
give the title
compound in two fractions: (16.8 g and 9.3 g). 1H NMR (DMSOd-6): consistent
with
proposed structure. Mass spectrum (ESI) m/z: 687 (M+1).
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Example 9
Me Me
HN
COzH
CbzHN,,,
Br
N
H
[0027] To a flask containing the compound synthesized in Example 8 (102 mg,
0.148 mmol) was added methanol (3.6 mL). The solution was cooled in ice-water
bath for
15 minutes. A stock solution of LiOH in water (3 5.4 nig/0.6 mL, 1.48 mmol)
was added
dropwise at 0 C. The mixture was warmed to room temperature (all precipitate
dissolved)
and stirred for 4 hours. Less than 1% of the starting material remained after
checking by
LCMS. About 10 g of ice was added to the reaction mixture and the temperature
was
decreased to 0 C. Aqueous HCl (1N, 1.6 mL) was added dropwise at 0 C to adjust
the pH of
the reaction mixture to between 2 and 3. Ethyl acetate (2 x 20 mL) was used to
extract the
desired acid. The combined organic layers were washed with water (10 mL),
brine (10 mL)
and dried over Na2SO4. The solution was concentrated to afford 100 mg of the
title
compound which was used in next step without further purification. 1H NMR
(DMSOd-6):
consistent with proposed structure.
Example 10
Me Me
O O
HN NH
CbzHN,,,,,, O / HN
OH
Br
N
H
[0028] To a dry flask containing the compound synthesized in Example 9 (100
mg) was
added 2-amino-l-(7-hydroxy-lH-indol-3-yl)ethanone hydrochloride (50.3 mg,
0.222 mmol)
and anhydrous DMF (0.5 mL). Triethylamine (31 l, 0.222 mmol) was added at
room
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temperature under N2. A pre-made yellow solution of DHOBt (8.45 mg, 0.0518
mmol),
EDC'HCI (42.6 mg, 0.222 mmol) and triethylamine (31 l, 0.222 mmol) in
anhydrous DMF
(2.0 mL) was added to the solution at room temperature. The mixture was
stirred at 41 to
42 C under N2 for 6liours. The reaction mixture was diluted with ethyl acetate
(30 mL)
followed by washing with water (10 mL), 10% aqueous NaHSO~ (10 mL), water
(2 x 10 mL), saturated aqueous NaHCO3 (10 mL), water (2 x 1-0 mL), and brine
(10 mL).
The solution was dried over Na2SO4, filtered and evaporated to give the title
compound (130
mg). 'H NMR (DMSOd-6): consistent with proposed structure.
Exam lp e 11
Me Me
o 0
HN
NH
CbzHN,, O
''', O IiN OAc
Br
N
H
[0029] To a dry flask containing the compound synthesized in Example 10 was
added
anhydrous tetrahydrofuran (0.9 mL) and CHZCl2 (2.7 mL). The resulting solution
was cooled
in ice-water bath for 15 minutes. Acetic anhydride (42 ul, 0.444 mmol) and
pyridine (18 ul,
0.222 mmol) were added at 0 C. Then the mixture was warmed to room temperature
and
stirred for 3.5 hours under N2. The reaction was monitored via LCMS. The
reaction solution
was diluted with ethyl acetate (30 mL) followed by washing with water (10 mL)
and brine
(10 mL) and drying over Na2SO4. After concentration, 136 mg of crude product
was
obtained. Flash chromatography, eluting with ethyl acetate/CH2C12, 30/70 -
35/65) afforded
the title compound (80 mg, 61 % overall yield over the last three steps). 'H
NMR
(DMSOd-6): consistent with proposed structure.
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Example 12
Me Me
HN N
CbzHN,,,,,
, O
N
H OAc
Br
N
H
[0030] Triphenylphosphine (474 mg, 1.81 mmol) and hexachloroethane (428 mg,
1.81 mmol) were added to a dry flask equipped with stir bar. Anhydrous CH2C12
(18.5 mL)
was added and the resulting solution was cooled well in ice-water bath under
N2.
Triethylamine (351 ul, 2.52 mmol) was added slowly to the solution, followed
by stirring for
minutes at 0 C. The solution of the compomid synthesized in Example 11 (160
mg,
0.180 nunol) in anhydrous CH2Cl2 (9.5 mL) was added dropwise and the
temperature was
kept at 0 C to 2 C. After addition, reaction mixture was stirred at 0 C for 10
ininutes (total
time should be less than 15 minutes). Water (34 L) was added to quench the
reaction. All
solvent was evaporated at 15 C under reduced pressure. Ethyl acetate (5 mL)
was added to
precipitate triphenylphosphine oxide. After filtering, the filtrate was
concentrated again and
the procedure above was repeated twice to remove additional triphenylphosphine
oxide. The
filtrate was concentrated followed by purification via flash chromatography
eluting with
ethyl acetate-toluene (60:40) to yield the title compound (110 mg, 70% yield.)
'H NMR
(DMSOd-6): consistent with proposed stiucture.
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Example 13
Me Me
HN
N
CbzHN,,, C
0
NH
\ / \ \
OH
N
H
[0031] The solution of the compound synthesized in Example 12 in acetoiiitrile
(10.0 mgl4 mL) was added to a quartz test tube followed by sparging with N2
for 30 minutes.
A stock degassed solution of LiOH in H20 (0.596 mg/0.60 mL) was added
dropwise. The
reaction solution turned dark yellow and was degassed with N2 for 30 minutes.
The quartz
test tube was placed in photoreactor illuminated with 300 nm light bulbs. The
reaction
solution was radiated for 45 minutes and sparged with N2. This reaction was
repeated 12
times. The combined reaction mixtures were diluted with ethyl acetate (200 mL)
followed
by washing with sat. NH4Cl (50 mL), H20 (50 mL), brine (50 mL) and drying over
Na2SO4
to give the title compound.
Exam lp e 14
Me Me
HN
N
CbzHNO
0
NH
N OTf
H
[0032] To a dry flask containing the compound synthesized in Example 13 (0.150
mmol)
was added dry K2C03 (61 mg) and anhydrous DMF (4 mL). A solution of 4-
nitrophenyl
trifluoromethanesulfonate in anhydrous DMF (61 mg/1.3 mL) was added to the
reaction
mixture at room temperature. The resulting yellow-brown solution was stirred
at room
temperature under N2 for 1 hour. Then the mixture was diluted with ethyl
acetate (100 mL)
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followed by washing with saturated aqueous NH4C1(2 x 20 mL), H20 (5 x 20 mL),
brine
(2 x 20 mL) and dried over NaZSO4. After concentration, the product was
purified via flash
chromatography, eluting with ethyl acetate-CH2Cl2 (30:70) to afford the title
compound
(50 mg, 38% overall yield for the last two steps). 'H NMR (DMSOd-6):
consistent with
proposed structure.
Example 15
Me Me
HN
N
HZN''''", O
O
- ~ I NH
\ ~ \ \
N 1 ~
H
[0033] To a flask containing the compound synthesized in Example 14 (50 mg,
0.057 inmol) was added MeOH (5 mL) and triethylamine (29 L, 0.205 mmol)
followed by
purging with N2. Then Pd(OH)2/C (95 mg) was added under N2. A balloon filled
with
hydrogen gas was added immediately and the flask was purged with hydrogen 4
times. The
reaction was allowed to proceed for 3 hours. The mixture was filtered through
a pad of
Celite and the residue was washed by MeOH (2 x 10 mL). The filtrate was
concentrated and
diluted with etllyl acetate (50 mL). The solution was washed with H20 (3 x 10
mL) and
brine (10 mL), and dried over Na2SO4. After concentration, the title compound
was obtained
(33 mg); it was used in the next step without further purification.
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Example 16
Me Me
OH HN
= N
H
O
- ~~ NH
\ / \ \
N
H
[0034] To synthesize compound J of formula (1) shown above, to a dry flask
containing
the compound synthesized in Example 15 (2 mg, 0.0469 mmol) was added anhydrous
tetrahydrofuran (1.1 mL). A solution (S)-2,5-dioxopyrrolidin-l-yl
2-hydroxy-3-methylbutanoate (11.1 mg, 0.0516 mmol) in anhydrous
tetrahydrofuran (0.3
mL) was added to the reaction mixture at room temperature. The reaction
mixture was
stirred at room temperature under N2 for 2 hours. The reaction mixture was
diluted with
CH2C12 (50 mL) followed by washing with sat. NaHCO3 (2x 5 mL), H20 (5 mL) and
brine
(5 mL), and drying over Na2SO4. After concentration, the final product was
purified via
flash chromatography, eluting with MeOH-CH2C12 (3:9) to afford the title
compound (19 mg,
58% yield). Mass spectrum (ESI) m/z: 697.2 (M+1). 1H NMR (DMSOd-6): consistent
with
proposed structure.
Exam lp e 17
Inhibition of Various Cell Lines by Compound J
[0035] A number of tumor cell lines representing breast, lung, colon, ovarian
and
prostate cancer as well as melanoma were assayed under standard growth
conditions in
presence of varying amounts of diazonamide A, a hydroxylated form of Compound
J,
Compound J, paclitaxel and vinblastine. The results are graphed as the
concentration that
diminishes growth by a factor of two (GI50) in Figure 1. In the box shown in
figure 1, AB-4
is the hydroxylated form of Compound J and AB-5 is Compound J. As shown, all
drugs
tested had very low GI50 concentrations in the breast tumor cell line MDA-MB-
435 but
diazonamide A, Compound J and vinblastine were more effective than the
remaining drugs
with respect to MDA-MB-23 1. In lung and colon, the hydroxylated form of
Compound J
was less effective than the remaining drugs. All of the drugs tested had very
low (single-
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digit nM) GI50's in the ovarian cell line OVCAR3 but paclitaxel was
significantly less
effective than the remaining four drugs against the ovarian cell lines IGR-OV1
and
SKMEL-2. In melanoma, Compound J and its hydroxylated form were less effective
than
the remaining drugs, but in the prostate cell lines PC-3 and LnCAP, all drugs
performed well.
(The hydroxylated form of Compound J was less active against PC-3.)
[0036] Additional experiments testing the effect of Compound J and paclitaxel
on the
survival of PTX10 ovarian tumor cell line are shown in Figure 2. Plotted
against
concentration added, these results show that Compound J is more effective than
paclitaxel in
lowering the percentage of cells surviving. In Figure 2, the circles represent
Compound J
and the diamonds represent paclitaxel.
Example 18
Pharmacokinetics
[0037] Compound J was injected intravenously into mice containing xenograft
tumors
grown from MDA-MB-435 breast cancer cells. The intratumoral concentration of
Compound J was measured as a function of time. The results are shown in Figure
3. As
summarized, the terminal half-life of this compound is 297.6 minutes, the area
under the
curve is 3010224 min* ng/ml and the volume of distribution is 2.9 1/kg.
Example 19
Activity in vivo
[0038] As shown in Figures 4A and 4B, Coinpound J is comparable to paclitaxel
in
inhibiting the growth of HCT116 xenografts and PC-3 xenografts.
[0039] Details ofthe preparation of murine models containing xenografts of
HCT116
cells or PC-3 cells are described in the section following these examples.
After the tumors
were grown, therapy utilizing Compound J or paclitaxel was initiated. As shown
in
Figure 4A, 20 mg/lcg of Compound J administered IV slows tumor growth in a
manner
similar to paclitaxel at the same concentration. In Figure 4A, the *'s
represent control mice,
the squares represent 5 mg/kg Compound J; the diamonds represent 20 mg/kg
Compound J;
and the slanted line represents 20 mg/kg paclitaxel. As seen, paclitaxel and
Compound J at
similar concentrations give similar curves of growtlz inhibition. In addition,
four of 10
animals used in the study were free of tumors for over five months and
experienced no
weight loss.
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[0040] In a similar model using PC-3 cells as the xenograft, again 20 mg/kg of
either
Compound J or paclitaxel significantly inhibited tumor growth. In Figure 4B,
the squares are
the control, the dark circles are the data-points for Compound J and the light
diamonds are
data-points for paclitaxel.
Example 20
Effect on Neutrophil Proliferation and Cell Counts
[0041] As shown in Figure 5, although paclitaxel results in a reduction of
neutrophil
counts in bone marrow and in peripheral blood at dosages of 5 mg/lcg and 20
mg/kg,
Compound J at these concentrations has no effect on these cell counts.
[0042] In addition to measuring the affect on tumors, the effect of paclitaxel
and
Compound J on neutrophils in blood marrow and blood were obtained. As shown in
Figure 5A, although paclitaxel at 20 mg/kg significantly lowered neutrophil
counts in bone
marrow, a coinparable dose of Compound J had no effect. The data are provided
as the
number of GR1+ cells in one femu.
[0043] In Figure 513, similarly, the neutrophil count in peripheral blood is
greatly
lowered by both 5 mg/kg and 20 mg/kg of paclitaxel, but is not significantly
affected by
similar amounts of Coinpound J. These data are tabulated as ANC (cell/ml).
[0044] The data obtained for growth inhibition in xenografts for Compound J as
compared to paclitaxel is shown below.
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Table 1
SUMMARY OF COMPOUND J EFFICACY DATA
,_.__ .._ ._ . . __... . :.:_ --._ ......_ .. ....~.___...,.;.. -, _ . _.. -
.~_ ,.....~;
Tumor Compound Delivery IoMaxT/Ca Log10Tumor #Tumor
Route Cell Killb Free (day
HCT1 16 Campound J {20trt9Ik9} IV 0.2 1.8 215(243+)
(colon) Compound J {5m9[le9) IV 26.6 0.54 o15
Campaund J (5rngfkg) IP 41.9 0.37 0/5
Paclitaxo1 {20mglkg} IV 0.2 3.2 115 {243+}
PC3 C*miroundJ {20mgfkg} IV 0.3 3.4 215 (157+)
(prostate) Paclitax01 (20rrsglko) IV 0 3.4 4I5(157+)
tviC7A-MB-435 Compound J(20mglkg) IP 6.8 0.6 016
(breast) Vinblastine (0.7mg/kg) IF' 14,1 0.6 0/6
tu1DA-MB-435 Compound J(20mg/kg) IP 3.1 1.0 0/0
(breast) Vinblastine (0.7mg/kg) IP 41.1 0.2 0/6
HCT116 Compound J (20mg/kg) IP 9,3 1.0 015
(colon) V'inblastine (0.45 mglkg) lP 56.0 0.2 0/5
%Max T/C - median treated turnor+rveightt Log,o Tumor Cell Ki ll - [(T - C) X
0.301]ft'd
median control tumor weight T= time in days for treated group to reach 300 mg
C=time in days for controi group to reach 300 mg
Td = tumor doubling time
[0045] The details of the protocol for Examples 19 and 20 are as follows:
Materials Required:
= PC3 prostrate tumor cells and HCT116 colon tumor cells (acquired from the
tumor
repository Division of Cancer Treatment and Diagnosis (DCTD) of the NCI
= Complete RPMI Media
- RPMI (Cat# 11875-085, Invitrogen)
- 10% Heat-inactivated (30' 56 C) Fetal Bovine Serum (Cat# 100-106,
Gemini Bio-Products
- 2mM L-glutamine (Cat# 25030-081, Invitrogen)
- 0.1mM MEM Non-Essential Amino Acids Solution (Cat#1 1140-050,
Invitrogen)
- 10 U/ml Penicillin/10 g/ml Streptomycin (Cat#15140-122, Invitrogen)
- 1 mM Sodium Pyruvate (Cat411360-070)
= Trypsin-EDTA (Cat#25300-054, Invitrogen)
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= CaZ+/Mga+-free Phosphate Buffered Saline, 5% dextrose in water
= Cell Culture Dish 150 mm X 25 mm (Cat# 430599, Corning, Inc.)
= 10 and 25 ml pipets, standard pipet-aid, p200 and p1000 pipette tips and
standard
pipetmen
= Athymic NCr-nu/nu nude mice at 6-12 weeks of age, either sex (Strain code:
01B74,
NCI Frederick Animal Production Program - suppliers include Charles River Labs
and Taconic Farms). Female mice of approximately 7 weeks of age were utilized
for=
existing experiments.
= Standard 1 cc tuberculin syringe, 30G1/2 and 25G5/8 needles (Becton
Dickinson)
= Falcon 5 ml tubes (Cat#352054)
= 50 and 15 ml disposable plastic conical tubes (Falcon or similar vendor)
= Trypan Blue Solution (0.4%) (Cat#T8154, Sigma)
= Hausser phase contrast hemacytometer (Cat# 02-671-54, Fisher)
= Ice bucket
= Balance for weighing mice (accurate to 0.2g)
= Vernier calipers
= Vivarium facility free from known pathogens (nude mice should be housed in
isolation from conventional mice if possible - separate rooms or micro-
isolator cages
equipped with individual air flow; all animal housing, bedding, water should
be
autoclaved prior to use; standard irradiated mouse chow should be used;
animals
should be changed out of cages within laminar flow hood; investigators
handling
mice should be suitably garbed- disposable gown, bonnet, booties, gloves,
mask)
= AB-5 (Compound J) compound
= Ethyl alcohol, 200 proof (Aaper, MFD 041205)
= Cremaphor (Cat# C5135, Sigma)
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= Paclitaxel (Cat#ANP0010, Polynled Therapeutics)
Procedure:
1) Rapidly tliaw PC3 or HCT116 cells (if necessary) in 37 C water bath.
Transfer
contents to 9m1 complete RPMI media. Spin 5' at 1200 rpm (240Xg). Discard
media
and resuspend pellet in 8 ml complete RPMI media. Plate in 100 mm X 25 mm cell
culture dish in 5% C02, 37 C humidified incubator.
2) Allow to grow for 5-10 days, splitting (aspirate media, wash with 3-5 ml
PBS, add 1
ml trypsin-EDTA, incubate 3-5' at 37 C, add media to desired volume and
transfer to
multiple plates) 1:8 to 1:10 every 3-4 days as necessary to allow cells to
enter phase
of logarithmic growth.
3) When cells are growing well, begin to expand to 150 mm X 25 inm dishes. We
use
20 ml of media in a 150 mm dish and use 2 ml of trypsin for splitting. When
nearly
confluent, there should be 2X107 cells/150 mm dish.
4) Plan ahead to time arrival of nu/nu mice such that they can acclimate to
animal
facility for 1 week prior to injection of tuinor cells. During this time, ear
tag or mark
in accord with standard practice for vivarium).
5) Harvest PC3 or HCT116 cells when you anticipate having sufficient cells for
total
number of mice in study (Total number of mice = 7 mice/group X total #groups -
suggested groups: vehicle control, Paclitaxel positive control, AB-5 + up to 5
additional mice to allow for variation in take rate of xenograft; i.e. 26
mice). Plan to
inject 10 million cells/inouse and allow for loss of cells/volume during
injection
process. That is, if 30 mice are to be injected, plan to harvest cells
sufficient for at
least 35 injections - 35 X 10 million cells = 3.5 X 108 cells. In the existing
HCT116
experiment, 7.5 X 106 cells were injected per= mouse. Five mice per group were
utilizedfor AB-5 treatment and four= mice per group weye utilized foj= the
control and
paclitaxel tf eattnents. In the existing PC3 experiment, 107 cells were
injected.. Six
mice per group were utilized for the control tr=eatment and five mice per
group were
utilized for the AB-5 and paclitaxel trAeatments. Smallet= group sizes were
necessary
due to limitations in AB-5 availability.
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6) Harvest cells as for splitting in steps 2 and 3 above. It is helpful to
harvest only 10
plates at a time and place harvested cells on ice as one will need to process
a large
number of plates for a single experiment. Wash plates with additional media 1-
2
times in addition to media used to dilute trypsin and harvest cells initially.
Cells from
plates with associated washes will comfortably fit in one 50 ml conical. All
steps
should be conducted using asceptic technique and cells and centrifuge should
be kept
cold tliroughout procedure.
7) Once all cells have been harvested, spin down 5' 1200 rpm (240Xg).
Resuspend
pellets in 5-10 ml of plain (serum- and additive-free) RPMI. Pool pellets and
then
count pooled cell population diluted to concentration suitable for counting.
Count 50
l + 50 l trypan blue on a standard hemacytometer. Spin down cells as before
and
repeat serum-free wash an additional two times to ensure complete removal of
serum.
It may be helpful to divide cells back into multiple 50 ml conical tubes for
counting
and washes, but be sure to pool back into a single tube for final spin.
8) Resuspend cell pellet, taking into account volume of pellet itself, at 4 X
107/ml in
serum free media. Pipeting or gentle vortexing is acceptable.
9) Take cells on ice to vivarium where mice should be already in cages ready
for
injection.
10) Draw up 0.8-0.9 ml of cells into lcc tuberculin syringe. Attach 25G needle
and
carefully knock out bubbles so that 0.7 ml of cells remain in syringe ready to
inject.
Leave remaining cells on ice.
11) Manually restrain mice with left hand, holding tail and skin behind the
head firmly
so mouse cannot move, but is still capable of breathing. Wipe skin to be
injected
with alcohol wipe and pull up an area of loose skin. Witll bevel up on needle,
gently
insert syringe and needle containing cells just under the skin of the mouse
and slowly
inject 0.2 ml of cells (1 X 107 cells). Area of injection should form a raised
area
under the skin. Replace mice in cages.
12) Begin measurements of tumor size with vernier calipers (measure length and
width
and calculate volume as (L X W2 )/2 three days following injection. Also
measure
mouse weight. In general, mouse weiglits should be measured at the same time
every
day (within 1-2 hours) to avoid expected daily fluctuation. Measure tumor
volume at
least one additional time at 2-3 day intervals to malce sure tumor volume is
increasing. Therapy can be initiated when tu.mor volume is between 150-250
mm3.
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This point should be reached within 5-10 days of initial injection. Do not use
mice
whose tumors fall significantly out of this range. Therapy was initiated 5
days after
injection of tumorfor the existing PC3 experiment and 8 days aftef= injection
of tumoy
foN the existing HCTI 16 experiment.
13) Prior to first day of therapy, aliquot paclitaxel and AB-5/Compound J
compounds
into individual dose aliquots. It should be possible to estimate weight of
mice on first
day of therapy based on weights measured during initial tumor growth period.
Aliquot sufficient compound per tube for the number of mice in treatment group
+
1.5-2 extra doses. Both paclitaxel and Compound J are given at 20 mg/kg. To
minimize compound loss and maximize accuracy, we weigh 20-30 mg of compound
and dissolve in ethanol. Aliquots of desired compound amount are then pipeted
into
individual tubes and dried down under vacuum. Aliquots are stored dessicated
at -
20 C in Teflon or glass tubes.
14) On the first day of therapy, randomly group mice such that the average
tumor volume
per group is consistent. If using males, multiple cages may need to be
utilized as it is
inadvisable to re-group male nu/nu mice with new nuhlu mice because of
fighting.
15) Prepare a stock solution of 1:1 cremaphor:ethanol. Prepare Compound J and
paclitaxel for injection by first dissolving in 20 l/dose cremaphor:ethanol
wit11
vigorous vortexing or sonnication as necessary. Dilute in 5% dextrose such
that final
concentration of cremaphor:ethanol is 10% (i.e. add 180 l of 5% dextrose per
dose).
Vortex to mix. Solution will be slightly viscous. Paclitaxel may require
slightly
higher concentration of excipient to remain in solution (up to 20% final). In
the
existing HCT116 and PC3 experiments, 20% excipient was utilizedfor the
paclitaxel
formulation and 10%for the AB-5 formulation.
16) Warm tail using a 150W heat lamp. We choose to warm only the tail but
standard
techniques used by vivarium are fine. Place mouse in Tailveiner. Wipe vein
with
alcohol wipe and slowly inject 0.2ml over 5 seconds.
17) Six doses should be given at an interval of every other day (q2dX6). In
the existing
HCT1 16 and PC3 experiments, doses were given Mon/Wed/Fri for two weeks (q2-
3dX6).
18) Mice should be weighed and dose recalculated for each dose.
19) Tumor volume and weight should be measured every 3-4 days (Monday/Friday)
for
several months following tumor injection. Control mice should reach 20 mm in
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longest diameter (point necessitating sacrifice at our facility) within 30-40
days.
Both paclitaxel and Compound J should cause significant regression with cures
in 40-
80% of mice.
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