(12) STANDARD PATENT (11) Application No. AU 2011202075 B8 (19) AUSTRALIAN PATENT OFFICE
(54) Title Phosphoramidate alkylator prodrugs
(51) International Patent Classification(s) C07H 19/10 (2006.01) C07H 19/00 (2006.01)
(21) Application No: 2011202075 (22) Date of Filing: 2011.05.05
(43) Publication Date: 2011.05.26 (43) Publication Journal Date: 2011.05.26 (44) Accepted Journal Date: 2013.01.17 (48) Corrigenda Journal Date: 2013.05.30
(62) Divisional of: 2006263433
(71) Applicant(s) Threshold Pharmaceuticals, Inc.
(72) Inventor(s) Jiao, Hailong;Kaizerman, Jacob;Matteucci, Mark;Duan, Jian-Xin
(74) Agent / Attorney Spruson & Ferguson, L 35 St Martins Tower 31 Market St, Sydney, NSW, 2000
(56) Related Art US5472956 A MISIURA K. et al: Acta Biochim. Polonica, 2002, 49(1)169-176. 2011202075 05 May 2011 5
AH21 alone
(4064035
Phosphoramidate or
IkRTK in
combination PHOSPHORAMIDA
alkylator with
one
prodrugs or
more
TE Abstract
anti-neoplastic
ALKYLA can
be
used
TOR
to
agents.
treat
PRODRUGS
cancer
when
administered 2011202075 05 May 2011 The performing 5845c(4166805_1) Actual of Address Name Invention
following
Applicant
and Inventor(s):
it for
Title:
known Address statement
Service: :
to
me/us:
is
a
full
description COMPLETE FOR Threshold Jian-Xin Jacob (CCN Sydney 31 St Spruson Mark Hailong United Boulevard, Phosphoramidate
Martins Market PATENTS
A
Matteucci
3710000177)
Kaizerman States STANDARD
AUSTRALIA
Jiao NSW Duan &
of
Pharmaceuticals, Street Tower Ferguson 5th
SPECIFICATION this
2000 of
ACT
Floor,
America invention,
Level alkylator
1990 PATENT
Redwood
35
including prodrugs
Inc.,
City,
of
1300 the California,
best
S&FRef: Seaport
method
94063,
838876D1
of
2011202075 05 May 2011 30 25 20 15 10 5
.
the al., imidazo alkylation other released chemotherapeutic Pharmacological Cyclophosphamide Cyclophosphamide or DNA macromolecules alkylators. pharmacology, encompass Description present Field 60/695,755 [0004] other [0003] [0001] [0002]
an
formation supra}.
nucleophilic aziridinium hyperproliferative of alkylation
invention
groups Invention alkylates Phosphoramidate This The Alkylating
of
The a
filed of
healthy of diverse
PHOSPHORAMIDArE present application
Related
and
of
neighboring-heteroatom-stabilized chemotherapeutic
is such
29
cation. CROSS-REFERENCES Basis
generally nucleophilic agent
biomolecules moieties postulated
and and medicine.
Jun
agents cells
group
invention as
Art
Ifosfamide Ifosfamide, of
2005, disease DNA If
claims canand BACKGROUND
Therapeutics,
based
such of ( the relates “
alkylators to
chemicals
which under moieties
active
provides
conditions be of
as
benefit alkylators result
alkylators
to healthy an are the are
physiological is the
alkylators important
phosphate,
each
”
incorporated an in
such
2001, that of compositions or fields
unwanted important
TO non-cancerous
with
U.S. used “
activated
ALKYLATOR OF
mustards act have as
RELATED
1389-1399,
of
the phosphoramidate THE are
mechanism as provisional in
chemistry,
amino,
the onium
strong conditions
DNA cancer released
herein toxic subclass
in INVENTION ” ability
and )
the used
within
sulfhydryl, intermediates electrophiles, cells,
APPLICATIONS events therapy, McGraw-Hill, methods
by
liver
patent in
away
biology, to
in of
(see
reference.
the
PRODRUGS
alkylate can cancer chemotherapeutic the
and
in alkylator
from antitumor Hardman application such
patients get for tumor
hydroxyl, the molecular
treating alkylated. such for the biologically
as active
New
cells
prodrugs. example,
tumor,
(see
et activity as
No.
York, al.,
carboxyl alkylator an cancer
to biology,
Hardman
act Such aziridine DNA alkylators. The
vital
through of USA).
The as
and
a and and
et
2 2012
Dec
Cyclophosphamide ^nfj Ifosfamide 06
[0005] There remains a need for new phosphoramidate based alkylators that can be used to
treat cancer or other hyperproliferative disease conditions, preferably compounds less toxic to
normal cells. The present invention meets these needs and provides novel phosphoramidate alkylator prodrugs as well as methods of therapy employing them, as summarized in the following section.
2011202075 BRIEF SUMMARY OF THE INVENTION [0006] In one aspect the present invention provides compounds which are hypoxia activated
phosphoramidate alkylator prodrugs and methods of their synthesis. The phosphoramidate
alkylator prodrugs of the present invention can have the formula Alk-T wherein Aik is a
phosphoramidate alkylator, T is L-Z3 wherein L is a linker Z3 is a bioreductive group.
[0006a] In another aspect there is provided a compound having the formula:
wherein
L is selected from the group consisting of CH2, CHMe, CMe2,
1
and Z3 is selected from the group consisting of
6554197_ I.RTK 2a 2012
Dec
HO 06
2011202075
each R.g is independently selected from the group consisting of hydrogen, aryl, heteroaryl, C1-C4
alkyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, heterocyclyl, C1-C6 acyl, Ci-C6heteroacyl, aroyl,
heteroaroyl, C1-C6 alkoxycarbonyl, Cj-Cg alkylaminocarbonyl, di C1-C6 alkylaminocarbonyl and
C1-C6 alkoxy;
each X4 is selected from the group consisting of halo, alkylsulfonyloxy, heteroalkylsulfonyloxy,
cycloalkylsulfonyloxy, heterocyciylsulfonyloxy, arylsulfonyloxy and heteroarylsulfonyloxy;
or a pharmaceutically acceptable salt thereof.
[006b] In another aspect there is provided a method to synthesize a compound of the first
aspect comprising reacting a compound having the formula Z3-L-OH, a trisubstituted phosphine,
a compound of the formula:
6554I97_| RTK 2b 2012
Dec
06
and a dialkyl azodicarboxylate to yield the compound of claim 1.
[0007] In one aspect, the present invention provides phosphoramidate alkylator prodrugs of formula (I):
2011202075 Rs
(0
wherein
[0008] Y| is O, S, NR^. or NSO2R.6 wherein each R.6 is independently C1-C6 alkyl, C1-C6 heteroalkyl, aryl, or heteroaryl;
[0009] Y2 is O, S, NR^, NCOR6, or NSO2R6; [0010] each of R1-R5 independently is hydrogen, hydroxyl, amino, Ci-Ce alkyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, heterocyclyl, C1-C6 alkoxy, C1-C6 alkylamino, C1-C6
dialkylamino, aryl, heteroaryl, C1-C6 acyl, C1-C6 heteroacyl, aroyl, or heteroaroyl; or together
6554197 I RTK any two of R1-R5 form a C3-C10 heterocycle; or each of Ri — Rs independently is a Trigger T wherein T is L-Z3
[0011] L is selected from
-[C(Zl)2-Y3]v-[C(=O)-O]q-[C(Z1)2-Z2-Y4]u-[C(Zi)2]z-[-C(Z1)=C(ZI)]g-Z3and 5 -[C(Z1)2-Y3]v-(S(=O)2)q-[C(Zl)2-Z2-Y4]u-[C(Z1)2]2-[C(Z1>C(Z1)]g-Z3; wherein each z, v, q, u, and g independently is 0 or 1;
[0012] Y3 is S, O, or NR7 wherein each R7 is independently hydrogen, hydroxyl, C1-C6 alkyl, CpCe heteroalkyl, C3-Cg cycloalkyl, heterocyclyl, Ci-Cg alkoxy, Ci-C6alkylamino, Cr Ce dialkylamino, aryl, heteroaryl, Ci-Cg acyl, Ci-Ccheteroacyl, aroyl, or heteroaroyl;
10 [0013] Y4 is O, S, or -NR7-C(=O)-O-;
[0014] each Zj independently is hydrogen, halogen, Cj-C6 alkyl, C]-C6 heteroalkyl, aryl, heteroaryl, C3-C8 cycloalkyl, heterocyclyl, Ci-C6 acyl, CrC6 heteroacyl, aroyl, or hetero aroyl;
[0015] Z2 is C1-C6 alkylene, C]-C6 heteroalkylene,
15
wherein each Xi is independently N or CR8, each R8 is independently hydrogen, halogen, nitro, cyano, CO2H, C)-C6 alkyl, C]-C6heteroalkyl, Ci-C6 cycloalkyl, CrCe alkoxy, Cj-Ce alkylamino, CrC6 dialkylamino, aryl, CONCR-^, CrC6 acyl, Ci-C6heteroacyl, aroyl, or heteroaroyl;
20 [0016] X2 is NR7, S, or O; and
[0017] Z3 is selected from the group consisting of:
3 2011202075 05 May 2011 15 10 5
J
phosphoramidate ------pharmacophores, in 0.01 heteroalkylsulfonyloxyalkyl; heteroalkylsulfonyloxyalkyl; [0023] [0022] Ce alkylsulfonyloxyalkyl, [0021] [0020] [00191 [0018]
an
alkylsulfonyloxyalkyl,
nm.
oxidation-reduction
[0024] In an (iii) (ii) (i) with In
one individual at a at
NR2R3
related the least
least
embodiment,
alkylator a proviso
pharmaceutically
two
one
and embodiment, In
isomer 2-heteroalkylsulfonyloxyalkyl,
of
a of NR4R5
2-heteroalkylsulfonyloxyalkyl, reaction. that related
prodrugs R1-R5
R1-R5
and Z3
or in
both
is
a formula
are at embodiment, is
racemic a the
least
selected
having
bioreductive selected acceptable together
present
one (I):
or
IC50
from
of
from non-racemic
4
are"^; invention
the
NR2R3
salt, or
group the
the present GI50,
solvate,
group
2-arylsulfonyloxyalkyl,
group
and and
that
provides in 2-arylsulfonyloxyalkyl,
mixture invention
cells NR4R5
consisting
can hydrate, consisting
under accept
phosphoramidate
is of
'" provides
or isomers,
hypoxia, NC1;
of
one of
a
2-haloalkyl, prodrug
2-haloalkyl, or
or
and more bioisosteres,
no of
and
2
2- thereof. 50
alkylator electrons
2-
μΜ 2-Cj-
2-
to
) 2011202075 05 May 2011 30 25 20 15 10 5
phosphoramidate hyperproliferative or of acceptable comprising phosphoramidate [0028] [0029] treatement HCR nM alkylator HCR, detail hypoxia [0027] invention normoxic present by prodrugs up prodrugs [0026] alkylator tide another cytotoxicity of cellular [0025]
third such
a the to
compounds to compound
of
1000
further
measured
50 relative
therapy.
fine invention
related cytotoxicity 50 of
In
In In prodrug In In prodrugs which having
μΜ has
cells
excipient, of
to fold
50 the one one one a therapy, another is
later cancer related and a 10,000.
μΜ
measured IC50
phosphoramidate
as
embodiment, hypoxic in in aspect, less aspect, aspect are by In hypoxic
alkylator
alkylator of provides
measured a hypoxic diseases hypoxic
in to
having
one the values
HCR up
related
toxic
the
or is and the embodiment, carrier, 0.01
the measured to
ratio is the the
embodiment, present
cytotoxicity
application, by
other
of cytotoxicity 5000 a
in
nM
IC50
present
in prodrug present and present comprising phosphoramidate prodrug and relapsed embodiment,
by 25 clonogenic of corresponding
or hypoxic the
and
hyperproliferative
normoxic normoxic values, fold to normoxic the
diluent. invention
present alkylator by
100,000.
invention
which phosphoramidate invention invention relative
of
of
less antiproliferation cancer.
of of
the
and
the which the in administering
0.1 assays 10
toxic
invention
cancer is the cells. and cells
cells. invention
normoxic has present
prodrugs
to IC50 nM normoxic
up In In
provides
present are provides provides alkylator
hypoxic 100,000. in 5
a
under another to and
another
to hypoxic In
values treated corresponding up
diseases. 1000
invention 50
a provides using
to
or of
related
cells.
alkylator hypoxia, invention
assays μΜ a
cells. novel
a a a
prodrugs cytotoxicities,
one the related in fold therapeutically embodiment,
In is
pharmaceutical million method
cytotoxicity
the and
hypoxic
resistant a invention
In that
less embodiment, phosphoramidate related has phosphoramidate In and relative
another a
prodrug
embodiment, of
provides a
is hypoxia normoxic
having fold, toxic a of using related
50
known,
hypoxic and
treating to embodiment,
μΜ
and the Ci
up of and related first in
the normoxic effective of
0 an
0.1
embodiment, cytotoxicity formulation phosphoramidate
, to corresponding to cancer
a
defined the the C cells to
relative
line, IC50 cytotoxicity pharmaceutically
0.01 10,000 5 cancer
nM phosphoramidate o, a
cellular present embodiment,
alkylators patient alkylator or
as
in second
treated to nM. amount
cells.
C90
the measured in
cells IC$o and 5 fold,
greater
μΜ
ratio, values
In in the
other line,
of in value is
and
need of for and
a
0.1
the a
of
a
2011202075 05 May 2011 30 25 20 15 10 5
the herein chemical defined, invention, metastatic I. section invention organized and invention of the cancer Gemcitabine on the [0036] [0035] combination [0032] [0030] [0034] [0031] [0033] .
Definitions
treatment,
compounds tumor
H460 H460 biological
are
agent. is
all
The
The Figure Figure Figures Figure Figure and
growth xenograft xenograft applicable
is alone
intended into or cancer.
terms
organized
with
one therapies, on medical following detailed
assays sections of
or tumor 3 4 2 1
in
5, that
of the
CDDP demonstrates demonstrates demonstrates demonstrates in
In
to the 6 mouse mouse
DETAILED art,
to
combination;
invention
for another and is have
arts. description
as
growth BRIEF any
only administrations, H460
definitions known, notations
the
on follows: 7
model. model.
the aspect Tn demonstrate
for tumor compounds
xenograft embodiment,
some
in DESCRIPTION
meanings
and the the the the the is
the DESCRIPTION
of and of Section administered
and
growth are
methods effect convenience effect effect effect cases,
the
H460 the
other
provided
Section
mouse
and different
invention. the of commonly
of of of of
I
terms in
xenograft
the
provides the scientific
effect
formulations, Compound Compound Compound Compound for the
model. phosphoramidate
invention. IV OF
in
making
to of with
H460
aspects
OF
combination provides of
assist
THE the
understood
useful
mouse
Compound or THE commonly
reader, xenograft
25 25
them; 25 25 medical
DRAWINGS the
and
employing This
INVENTION (150 (50 dosed (100 definitions;
examples
model. reader.
embodiments
and Section
mg/kg with
detailed
by
mg/kg
mg/kg
alkylator 25 terms understood mouse
in
disclosure those
in combination at Unless
the of
)
combination
III least
or )
) Section
on
description model.
synthetic
dosed on
compounds of
describes terminology prodrug
tumor
of
skill otherwise another tumor
meanings
found
the
in II
in
with growth present describes methods
of growth
the
with methods is in anti
of the
any CDDP used are
the
in
in
2011202075 05 May 2011 30 25 20 15 10 5
electrophile macromolecules heteroaryl, hydroxyl, "Phosphoramidate carboxamido, ethenyl, [0040] further (C2-C6) monovalent heteroarylsulfonyloxy. and aryl, nitro, the herein alkylcarbox-amido, ( the prefix saturated definition [0039] can the Cg defined an [0038] [0037] “ D
integer
alkyl
prefix. radical ” like. be containing
), heteroaryl,
ethenyl,
is hydroxyl, should further optionally
alkenyl herein
ethynyl, includes not
"Alkylator" For "Alkenyl" As "Alkyl" monovalent
amino,
of from or alkyl
As
is
hydrocarbon included used
the
each
portion
-SO2NH2, not
present optionally ethynyl,
used for
at
includes, 2-20,
alkylsulfonyloxy,
via or term
Ci-C amino,
mono methyl, means least be herein, necessarily
of clarity alkylator"
heteroalkylsulfonyloxy,
means -SO2NH2, in
an
substituted
means the
thereof have to as hydrocarbon 6 or
this
one
CpCi electrophillic alkoxy,
or
indicate -OSO mono generally a
generated definitions
be radical ethenyl, and/or "a"
ethyl,
linear
di(C]-C
disclosure, a double the
substituted a
linear
means
will or
be alkoxy, reactive
2 -OSO same or -(Ci-C6)
C
n-propyl,
"an"
with for construed saturated having the
r
di(Cj-C6) have
propenyl, 6 understood bond, heteroalkylsulfonyloxy,
C6 monovalent ) by radical
ready
an herein 2 meaning.
alkyl number means
reaction -(Ci-C6)
substituents,
Ci-Ce
alkylthio, the intramolecular moiety
six alkylator
with
alkyl, the but
prefixes 2-propyl,
reference, amino, monovalent
or having to
(e.g.,
alkyl and
number "at alkylthio,
1,3-butadienyl no substituents, of
fewer represent
with 7 capable in alkyl, mono For
hydrocarbon
least
main
more
aryl
-COOH,
for the alkyl,
amino,
halo,
the
example, including (Ci-C a
main
n-butyl,
which of mono art. one" nucleophile or
and or
chain than cyclization. number
of
-COOH,
hydrocarbon
carbon alkenyl, heteroarylsulfonyloxy. C a di(Ci-Ce)
halo, the 2 substantial
forming
chain -CONH2, including
-C or ),
three
or an arylsulfonyloxy carbon and
2-butyl, Ci-qq (Ci-Cg) 6 for radical inclusion "one
di(C|-Cg)
alkenyl aziridine of C2-C6 atoms
carbon
-CONH2,
the alkoxy, double example,
carbon on
alkylsulfonamido, a or ,
atoms
or
like. mono-
covalent
for radical
the or
difference
alkyl, more." tert-butyl, alkenyl indicated
ether, Ci-C
of
atoms. a
or example, bonds.
alkylsulfonamido,
macromolecule. araalkyloxy),
atoms branched
Alkenyl such
in mono- deuterium
aziridinium or Cj-s qq
or or an cyano,
, alkyl
ether,
di(C[-C6)
wherein
a
definitions
(Ci-Ce)
indicated in alkyl over pentyl,
For alkyl, branched
or
deuterium the
can linkage
cyano, nitro, example, difCj-Ce)
the portion, aryl,
(“
prefix
when
or be
qq D
alkyl and
alkyl-
C ” in
), is
r to
a
2011202075 05 May 2011 30 25 20 15 10 5
More amino, alkyl, optionally ring naphthyl, specifically phenylalkyl), independently integer integer [0044] cycloalkyl, alkylamino, cycloalkyl, preferably heteroarylsulfonyloxy. alkyl, methyl-propylene, to of CH2CH2OCH2CH2-, cyano, deuterium to the one alkylsulfonamido, (Ci-C6)alkyl-carboxamido, [0041] [0042] [0043]
twelve twelve 6
alkylene
or carbons. to
specifically, optionally cycloalkyl,
more nitro, mono-alkylamino, 10 from from
"Cycloalkyl"
"Heteroalkylene" "Alkylene" "Aryl"
and carbon carbon
ring substituted ( one,
cycloalkylalkyl, cycloalkylalkyl, “
the
di-alkylamino,
heteroatoms biradical. D 0 0 ethenyl,
The the
hi ” selected to to atoms
), term two,
refers
substituted one
atoms atoms 5, 5, hydroxyl, substituted
cycloalkyl-alkyl,
the cycloalkyl aryl, pentylene,
R R
three, aryl
-CH2CH means
embodiment, which
’ ’ ethynyl,
independently
term
refers and to and
from For heteroaryl, or For optionally
includes,
a (i.e.
di-alkylamino,
a
four R R monovalent has cycloalkyl
example,
haloalkyl, -SO2NH2, amino, phenyl, phenyl example halo, branched a is
hydrogen, ”
” to forms
group 2 linear hexylene,
oxygen, N(CH3)CH
substituted C1-C6 are are
essentially a ot
monovalent
nitro,
independently independently five
substituted
but
R phenyl alkyl mono
thereof. or
can
or saturated
with x alkoxy, heteroalkylene alkylene
saturated haloalkoxy, and phenylalkyl) -OSO includes, sulfur, is substituents -C(O)R
alkyl,
monocyclic cyano, have
and
or not
hydroxyl,
2 or one,
independently
the R CH2-, or
heteroalkylsulfonyloxy,
2 y
the di(Ci-C6)alkyl limited -(Ci-C6)
C1-C6
phenylalkyl),
one together
nitrogen cycloalkyl, cyclic divalent
meaning with 8 includes
Z two, hydroxyl,
divalent (where for like.
hydrogen -CH hydrogen
or
heteroalkyl,
substituents selected alkylthio, example, or three alkoxy,
or hydrocarbon more includes, to,
2
alkyl,
CH -(CR is hydrocarbon
bicyclic and/or
methylene, given R phenyl,
hydrocarbon
cycloalkyl
with z or alkoxy, cycloalkylalkyl, 2
double is SCH2CH
or or
-(CR
’
amino, or from four mono R
hydrogen,
cyclopropyl,
above phosphorous) -COOH, ” alkyl, one alkyl,
-CH
optionally
)
COR aromatic including n biphenyl,
’ -CONR
substituents
R amino, deuterium
bonds radical
to
2 or ” ethylene,
halo,
OCH
2 or radical )
and and foran and
-,
eight n (where
di(Ci-Ce) radical -COOR
heterocyclyl. and -CONH2,
alkyl,
aryl R x R and hydrocarbon 2 C
acylamino,
of R
substituted O-,-
for
x alkylene phenyl 1 2 substituents,
cyclohexyl, is the
y having -C6 -naphthyl,
and
three
may
(
(where R propylene,
can
or having hydrogen, “ example,
selected
haloalkyl,
(where D
like. is
alkenyl
R ”
mono- also be
hydrogen, ), y to or
from are except alkyl,
present
n seven
mono from More
phenyl).
be
n is radical
and from
ether,
is
alkyl, 2- or an one
an one
2- that
di-
in
2011202075 05 May 2011 30 25 20 15 10 5
hydrogen haloalkoxy, hydroxyl, cycloalkyl, (CR alkyl, or (CR optionally ring attachment selected -S(O) indicating by 2-hydroxyethyl, hydrogen, radical atoms 2-methylsulfonyl-ethyl. alkylamino, (provided di-alkylcarbamoyl. cycloalkyl-alkyl, integer [0047] [0046] aryloxycarbonyl, substituents dimethylaminocarbonyl-cyclohexyl, cyclohexenyl, [0045]
four amino,
’ ’ atoms R R
P
and
” R ” in
is substituents, )n-CONR ) from
Z n
from
the
-COOR "Heteroaryl" In
portions. "Heteroalkyl"
through or
alkoxy, R that halo, substituted the alkyl,
having
point cycloalkyl-alkyl,
one heteroalkyl,
di-alkylamino, independently is alkyl, portion 0
N,
number
phenylcyclohexyl, n hydrogen, to
fluoro, embodiment,
is
2,3-dihydroxypropyl,
cycloalkyl,
O, of x aryl, 2 carboxamido, (where
a amino, R at
and 0),
),
carbon the y
or R selected of
least with (where x
independently alkyl,
means of
aralkyl,
S, R
is the
heteroaryl alkylamino, means
x -COR
For
n carbon the
hydrogen, alkyl, acylamino,
and one
the
heteroalkyl
is selected atom
or
cycloalkyl, cycloalkyl-alkyl,
phenyl n each from remaining
an a
R
understanding
R hydroxyalkyl. aromatic
is alkoxycarbonyl, an
monovalent (where x cycloalkyl, mono-
y
integer
atoms
of
an and 4-carboxycyclohexyl, are,
alkyl
of radical alkyl,
the
from integer di-alkylamino,
alkyl, or
with the and
mono-alkylamino, R independently
2-methoxyethyl, R group phenylalkyl). y or heteroalkyl
(e.g., radical
ring ring from cycloalkyl-alkyl,
together
above,
cycloalkyl, is the
cyano, will di-alkylcarbamoyl one cycloalkyl,
hydrogen, cycloalkyl-alkyl, from monocyclic,
containing atoms
like.
that Ci-Cio)
0 exclusive
Representative
aryl,
9 to be
as
to aryloxycarbonyl, R
-OR
eight
0
the on is w defined 5,
,
radical. to being
araalkyl, OH cycloalkyl
R
R an cycloalkyl-alkyl, of W point
refers
x In 5,
, alkyl, cycloalkyl-alkyl, ’ substituents, ,
-NR each aromatic of or and benzyloxymethyl, one, R
2-carboxamidocyclohexenyl,
R one di-alkylamino, bicyclic
y C, herein aryl,
’
the , alkoxy.
of
and to and x
R R
phenyl with R alkoxycarbonyl, other, two, embodiment, or phenyl examples
w attachment ” cyano, the
y
or araalkyl,
,
is are alkylsulfonyl.
R R
and with
ring.
carboxamido, ” or heterocyclyl. z the hydrogen,
or total
can
independently Additionally, hydrogen, are
preferably
or tricyclic three
-S(O)pR or
understanding -OR
one, halo,
phenylalkyl,
independently The number be
phenylalkyl), include,
haloalkyl, aryl amino,
of
W
further ring 2-cyanoethyl, R two ,
heteroaryl
nitro, the -NR x
or alkyl, z radical
and alkyl,
(where
one,
R heteroatoms or
araalkyl. of
heteroalkyl mono or for z x
the
R
substituted
three is
hydrogen cyano,
carbon R
mono- cycloalkyl,
y
two,
example, y , - hydrogen that
of
prefix or
together
or
p 2- ring
5
is
-
three the
and
to R
or an
y
is
12 or is
) 2011202075 05 May 2011 30 25 20 15 10 5
within portion piperazinyl, number hexahydro-lA piperidinyl, methylpyrrolidin-3-yl, includes, phenylalkyl), hydrogen phenylalkyl), [0049] cycloalkyl, independently haloalkoxy, halo, ring selected where P(=O)OR pyrazolotriazinyl, benzothienyl, benzoxazolyl, from cyclic triazolopyridinyl, pyrrolyl, limited is and isobenzofuranyl, constituent [0048]
cycloalkyl
the can
nitro, O,
the
radical one
of of to,
derivatives
NR from be
"Cj-Ce but pyrazolyl,
"Heterocyclyl" W or ring the
carbon
pyridyl, or ,
cycloalkylalkyl, cyano,
optionally ring morpholinyl,
and or -COR
alkyl, (where
is
or
or two -(CR cycloheteroalkyl 6 of
alkyl, indazolyl,
hydrogen quinolyl, -thiopyran-4-yl, can
S(O) not
heterocyclyl.
piperidin-2-only atoms. -(CR
Acyl" 3
benzothiazolyl,
pyrazolopyrimidinyl, atoms C
triazolotriazinyl, hydroxyl, to be
and ’ (where limited
furanyl, thereof. R
P pyridazinyl, R aryl, atoms
” (where 8 ’
any
R )
substituted is
2-pyrroIidon-l-yl, ring R n means
”
-COOR
tetrahydroquinolinyl,
(e.g., or hydrogen, pyrrolopyrymidinyl,
) or is
pyrrolidinyl, arylalkyl, n arrangement
R -CONR
to, can
alkyl, hydrogen, atoms "cycloheteroalkyl" thienyl, phenyl
Unless is p alkoxy,
pyridyl, -CO-(Ci-C« More C3-C10)
is hydrogen, optionally or tetrahydroimidazo
(n pyrimidinyl,
an benzoisothiazolyl, and R*
heterocyclyl independently in x
is R
heteroaryl, pyrazolotetrazinyl, or alkyl,
integer indicated thiazolyl,
specifically
and which y amino,
an the
phenylalkyl). refers (where alkyl,
tetrahydropyranyl, tetrahydrofuranyl, allowed
triazolopyrimidinyl,
integer
R
derivatives furyl, be alkyl, cycloalkyl,
y
alkyl), from one
are,
mono-alkylamino, cycloalkyl, replaced to
benzofuranyl, 10 otherwise, indolizinyl, isoquinolyl, n isothiazolyl,
group heteroarylalkyl, means
the
by quinolyl,
to is
from cycloalkyl,
independently the with 0
an
wherein [4,5-c] four
the to total
term benzotriazolyl,
More
thereof. integer
exclusive cycloalkylalkyl, 2), 0
by one, a
bonding
hexaaza-indenly, ring
to
saturated
number cycloalkylalkyl,
the
the
a
thienyl,
heteroaryl tetrahydrothiofuranyl, pyridinyl, pyrazolopyridinyl, N-methylpiperidin-3-yl,
5,
benzimidazolyl, two, carbonyl triazolyl, specifically
the
cycloalkylalkyl,
atoms
tetrahydrobenzofuranyl,
arrangement remaining R pyrrolotriazinyl, from The
’
term
characteristics of cycloalkyl, of di-alkylamino, and three
of
benzothienyl, or
each the 0 prefix are
carbon
indolyl, R
alkyl unsaturated imidazolinyl, to includes, group. imidazolyl,
or
” number
phenyl heteroatoms
5, the other, ring are
four
and
phenyl indicating R of is
atoms term independently ’ benzisoxazolyl cycloalkylalkyl,
The
as atoms isoindolyl, the
and phenyl
heptaaza-indenyl substituents
hydrogen, or
but
of of defined haloalkyl,
pyrrolidinyl,
hetero heterocyclyl phenylalkyl), non-aromatic heterocyclyl 1,1-dioxo- or R isoxazolyl,
heteroatoms. the
in is
”
being
N-
selected
the the or not are
atoms above.
alkyl,
C,
or
[0050] "C|-C6 Heteroacyl" means -CO-(Ci-C6 heteroalkyl), wherein the term heteroalkyl is as defined above.
[0051] "Aroyl" means -CO-aryl, wherein the term aryl is as defined above.
[0052] "Heteroaroyl" means -CO-heteroayl, wherein the term heteroaryl is as defined 5 above.
[0053] "Rsuisulfonyloxy" means Rsui-S(=O)2-O- including alkylsulfonyloxy, heteroakylsulfonyloxy, cycloalkylsulfonyloxy, heterocyclylsulfonyloxy, arylsulfonyloxy and heteroarylsulfonyloxy wherein Rsui is alkyl, heteroakyl, cycloalkyl, heterocyclyl, aryl and heteroaryl respectively, and wherein alkyl, heteroakyl, cycloalkyl, heterocyclyl, aryl and 10 heteroaryl are defined above. Examples of alkylsulfonyloxy include Me-S(=O)2-O-, Et- S(=O)2-O-, CF3-S(=O)2-O- and the like, and examples of arylsulfonyloxy include
'----- ' · '----- ' · '----- ' and the like. Alkylsulfonyloxy, heteroakylsulfonyloxy, cycloalkylsulfonyloxy, heterocyclylsulfonyloxy, arylsulfonyloxy, and heteroarylsulfonyloxy groups can be leaving groups in phosphoramidate 15 alkylators and can be replaced in a cell by nucleic acids such as DNA or RNA, and imidazoles, carboxylates, or thiols of proteins, causing alkylation and cell death. The rate of reaction of various Rsuisulfonyloxy groups with nucleic acids, proteins or water can be modulated depending on for example the electron withdrawing nature and the steric bulk of the Rjui moiety and can provide phosphoramidate alkylators and prodrugs thereof which are 20 more toxic to tumors in general and hypoxic zones of tumor in particular over healthy cells.
[0054] “Substituents” mean, along with substituents particularly described in the definition of each of the groups above, those selected from: deuterieum, -halogen, -OR’, -NR’R”, -SR’, -SiR’R”R’”, -OC(O)R’, -C(O)R’, -CO2R’, -CONR’R”, -OC(O)NR’R”, -NR”C(O)R’, -NR’-
C(O)NR”R’”, -NR”C(O)2R’, -NH-C(NH2)=NH, -NR’C(NH2)=NH, -NH-CtTJH^NR’, -
25 S(O)R’, -S(O)2R’, -S(O) 2NR’R”, -NR’SCO^R”, -CN and -NO2, -R’, -N3, perfluoro(C]-
C4) alkoxy, and perfluoro(C]-C4) alkyl, in a number ranging from zero to the total number
of open valences on the radical; and where R’, R” and R’” are independently selected from hydrogen, C]_g alkyl, C3_g cycloalkyl, C2_8 alkenyl, C2_g alkynyl, unsubstituted aryl and
heteroaryl, (unsubstituted aryl)-C]-4 alkyl, and unsubstituted aryloxy-Cj-4 alkyl, aryl
11 2011202075 05 May 2011 30 25 20 15 10 5
base, by present particular compounds or ( [0056] with atoms regioisomers invention encompassed (optical -NR from a substituents ring the the by membered nitrogen groups, substituted [0055] 2 C(O)-(CH (CH and Other 14 NR substituent
C). ηοζ
contacting
an
aryl aryl
q 2 ’
so radioactive
’ -
either 0 )r-B-,
-
is alkylene
that suitable All and
are or to invention
formed
or centers) or or
an The atom,
Certain a
substituents may 3,
2
isotopic intended
unsubstituted constitute -S(O) heteroaryl heteroaryl
ring.
integer single ) neat with
which wherein and on q
of
and term within -U the
substituents also
may tether they adjacent
the
3 or 2 isotopes, X or
1-3
-, For compounds neutral
contain individual NR
bond,
5
are
of "pharmaceutically variations in
wherein double contain formula
to is
optionally can A halogens, the
such example, ’
of ring a ring from - prepared -O-, found be
and suitable is
be from
and scope atoms
aryl-Cj-4
form
encompassed relatively
selected
such
may may compounds.
bonds;
-NR B 0 include combined
unnatural
T
-(CH
isomers
r on
to
2 of of
are
1-4 is
of
unsubstituted of
and be -NR of
as optionally
optionally ’ with inert 2.
the
-, the the an
independently
such 2
the
carbon the for replaced -S-, the )
from
Alternatively,
U alkyl
s integer each ’ compounds acidic present compounds
R -X
relatively (e.g.,
3 solvent.
present
example proportions aryl acceptable racemates, with
”
compounds -S(O)-, are
5 within
is For
-(CH hydrogen
of
groups. atoms.
meant
be be or independently
functionalities,
separate
of the
with the
invention example,
heteroaryl C]-g
2 replaced replaced invention.
from 12
)t-,
-S(O) Examples
the nitrogen tritium nontoxic above
described -CH2-, of
a
Two
to salts" diastereomers,
two
When
of where
double scope or alkyl,
with
the include 1 enantiomers) 2
atomic unsubstituted
to
-,
aryl
of of the possess
with ( with
present
3
is
or
ring atom 3. -Ο-,
a H),
R
acids the the The
of C] s
of
-NH-,
meant compounds bond.
sufficient
base herein. ’ substituents -S(O) and
One
the and a 1-pyrrolidinyl a
salts -g
iodine-125 isotopes substituents substituents may -NH-,
substituent substituent compounds to
or asymmetric
alkoxy invention,
t present
addition
R -O-,
of
form 2 are to Alternatively,
geometric
derived bases,
NR are optionally ”
When
the include Cj-g -S-, are
independently
amount
-CH2-
’ all at
a -.
may or
single
attached
attached
invention.
3-, ( -S(O)-, one depending
salts intended
The
l25 C[-g
of on on alkyl. of
from compounds whether
of
and
4-, carbon be
I) salts
isomers,
the the
or
or
adjacent adjacent of the be
bonds substituent or
can
radiolabeled 5-,
a thioalkoxy 4-morpholinyl.
more
the
two replaced formula -S(O)2-, formula
to to carbon-14 present single of
6-, be
to
radioactive atoms a
the desired integers
on the
of of
ring be
obtained of
or
atoms atoms of
the
same the the
bond, active
the
7-
-A- -T
the
with -S(O) R atom
new
2 ’
-
of
in of of
i
2011202075 05 May 2011 30 25 20 15 10 5
are unsolvated invention. solvated compound as form [0058] a benzenesulfonic, propionic, [0057] to Salts glucuronic the the dihydrogenphosphoric, nitric, trimethylamine, piperazine, glucosamine, pharmaceutically-acceptable addition either invention ethanolamine, dibenzylethylenediamine, occuring like. contacting secondary ferric,
base
be solubility
salts present like,
” of converted Salts ,
neat
or carbonic, ferrous, Journal
the
of
forms,
as salts
Certain The acid
amines
contain
isobutyric, Certain for amino and forms
the or or derived invention well compound piperadine,
in
neutral
galactunoric in include histidine, and
the
neutral ethylenediamine,
polar of lithium, tertiary
including
into
as
monohydrogencarbonic, a tripropylamine,
compounds
and p-tolylsulfonic,
and
Pharmaceutical acids purposes relatively isolating suitable
compounds the from
either
solvents,
forms
contain the
are malonic, those
form
salts differs
amines,
polyamine such magnesium, sulfuric, hydrabamine,
pharmaceutically-acceptable like, intended hydrated
diethylamine, base inert
acids
the of of
derived derived of basic
as
of
both inorganic
from
but such
the the
such
of parent
benzoic, including arginate
or the solvent.
N-ethylmorpholine, monohydrogensulfuric, and tromethamine
citric,
the present Science,
functionalities, compounds otherwise
acid basic to resins,
forms.
as the present
compounds from from
manganic,
the
be present
compound arginine, isopropylamine,
various addition bases
tartaric,
and encompassed and succinic, like
2-diethyl Examples
relatively phosphoric, inorganic substituted
procaine, invention.
In
1977, invention
the the acidic
(see, invention include 13 general, may
salt manganous, and betaine,
with like, methanesulfonic, salts. salts
66, in
acid
suberic, aminoethanol, e.g.,
be
functionalities forms of nontoxic the acids purines, the
amines,
N-ethylpiperidine, a and 1-19). are
aluminum, can
monohydrogenphosphoric,
pharmaceutically regenerated organic within the
addition
sufficient
lysine,
Berge, like.
may conventional caffeine,
hydriodic,
equivalent
like
salts
exist in solvated fumaric,
potassium, Certain theobromine, certain exist
When organic
cyclic the
hydrochloric,
methylglucamine, bases S.M., of
in salts
2-dimethylaminoethanol,
amount ammonium, scope
organic
choline,
unsolvated in
and that
by
forms
mandelic,
compounds or
physical
amines, to specific can include multiple
et
acids manner.
contacting
phosphorous the
allow the
al,
of sodium,
acceptable glucamine, be
of
acids are
N,N like. “ the
parent triethylamine,
like
Pharmaceutical obtained
the hydrobromic,
naturally- salts
compounds properties, the
equivalent forms
crystalline calcium, present phthalic,
’
The
- like acetic,
desired Also of
zinc
compounds
the
morpholine, form of
the
parent
acid as
acids
primary,
salt by and included
present
well
of copper,
acid,
such to
or
with of the
the and
as
amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.
[0059] As used herein, a "glucose analog" includes mono-, di- and tri-saccharides. The glucose analog includes sacchrides comprising glucosamine, N-acetyl-glucosamine; fructose; 5 mannose and mannose derivatives; glucose and glucose derivatives, including but not limited to 2-deoxyglucose (2-DG), N-acetyl-2-amino-2-deoxyglucose, 3-amino-3-deoxy-glucose, 2- amino-2-deoxy-glucose; and galactose and galactose derivatives including but not limited to D-2-deoxy-D-galactose, D-4-amino-4-deoxy-galactose and D-2-amino-2-deoxy-galactose. Thus, the glucose analog can differ from glucose or a derivative such as DG and glucosamine 10 in that it is an epimer thereof. In addition, the glucose analog can be a fluorinated derivative of any of the foregoing compounds. Moreover, the oxygen in the ring of any of the foregoing compounds can be substituted with an isostere selected from the group consisting of S, sulfone, and the like. For example, glucose analog can be 5-thio-D-glucose or a derivative thereof.
15 [0060] A wavy line " " means the point of attachment of one group or moiety to
another. For example, both indicate that the thio group is the point of attachment to another group or moiety.
[0061] The terms CO, C(O), C(=O), -CO- are used interchangeably herein. The terms CO2 and COO are used interchangeably herein.. The terms; SO2, S(O)2 are used interchangeably 20 herein. The terms SO and S(=O) are used interchangeably herein. The terms PO and P(=O) are used interchangeably herein.
[0062] As used herein, a "bioisostere" of a chemical moiety such as molecule, group, or atom means another chemical moiety having similar size and spatial disposition of electron pair or pairs. Bioisosteres and bioisosterism are well-known tools for predicting the 25 biological activity of compounds, based upon the premise that compounds with similar size, shape, and electron density can have similar biological activity. Known bioisosteric replacements include, for example, the interchangeability of-F, -OH, -NH2, -Cl, and -CH3; the interchangeability of -Br and -1-C3H7; the interchangeability of-I and -Z-C4H9; the interchangeability of -O-, -S-, -NH-, -CH2, and -Se-; the interchangeability of -N=, -CH=, and 30 -P= (in cyclic or noncyclic moieties); the interchangeability of phenyl and pyridyl groups; the
14 ) 2011202075 05 May 2011 30 25 20 15 10 5
one metabolized kept conditions, feasible results [0066] mono- need substituted [0065] group http://www.chemcomp.com/ known event circumstance the alkylators. identifies of identified, interchangeability [0064] similar functional identify limiting C(=R interchangeability "pharmacophore", [0063] 1996,
medicinal
program biological
at not
ar
or
optionally
Chem.
)-Rar); in or
a molecule pharmacophores.
compound
other on circumstance temperature
As be, A
A "Optional" a disubstituted the
groups
this
stable with for combination An reasonable the
used
or present, 3D
chemistry, Rev. pharmacophore
can, an<
bioisosteric
property,
at information otherwise range example
i an Pharmacophore
mono-
can
least herein,
or
the
in but 96:3147-76;
of of and is alkyl
chemically
or the
and be of an -C=C- one of interchangeability
need
occurs a
with once quantitative
"optionally"
4 relative
week. bioisosteric or and made aromatic molecule. of
of
group. a
the converted
in °C Such
replacements “
di-
programs substituents
can not, prodrug
an which
the and as
description
or fdept/prodinfo.htm). and
of based substituted
alkyl and the
be methods
less,
feasible occur, pharmacophore
to phosphoramidate -S- search
nitrogen instances
used
itself. structural
the
Burger, As prediction
” to
means
(for equivalents on group in available means
an chemical
used and
the
to the from
or
known
of-CO-, compound. are example, includes
active
To with
identify
variables
(Rar-NiR^-Rar)
that that and absence spatial
in 1991,
herein,
well
a information produce the
15
which
of
compound to an
situations the in the
or or
and structure
Chemical the
known -SO-,
perform
A.
situations alkyl
the
alkylator benzene
arrangement other more pharmacophores description subsequently
such
of
is
Prog. binding one A it
art.
a
moisture permissible
does
stable and prodrug, group
molecules to active
an of
described
that, where is
pharmacophore
See,
Drug for Computing persons and
skill
-SO prodrugs arrangement where not not.
ability
” compound
an after
includes means for
form
thiophene); 2 of
or substantially
the in -. Res. a
described For unsaturated
a pharmaceutically
other the only example,
the These
containing
of
in administration, or heterocyclo in small
with
example, and
37:287-311.
that this ordinary heterocyclo
the a art Group
if
instances
molecule
chemically is
or
phosphoramidate
examples respect such will number -related function application the
event called the
chemically Patani
altered
carbon
(see alkyl the
be
a "heterocyclo skill
group
combination
or
able to
where a
same have
of
group
searches
et
of
is
can,
are reactive
at in
active when
(R
atoms
al.,
a
to least is the
been not ar
or the but
- is not
art
or
is
2011202075 05 May 2011 25 30 20 10 15 5
‘
bioreductive nitroimidazole these nitrothiophenes, nitrobenzenes, Prodrugs such electrochemical can herein in a oxidation-reduction “ under [0070] cytotoxicity are such cellular effect toxicity, drug, generated [0069] active certain form compound [0068] [0067] Chemistry normoxic
group
vivo
cells be
as
as, is classes
altered
on certain
phosphoramidate
reduced a
and/or biological
by less
that
growth
for phosphoramidate
residing or
For As As As cells. can
by
A enzymatic
(or cytotoxicity
improved ”
example,
active
can
used used used
Biochemical groups
of metabolic example, metabolic of biological
also
in
a
electron that
in
As moieties
and
means; a suitable nitrothiazoles, accept vitro·,
in
herein, herein herein, vivo conditions compound be
or
used may
multiplication. a reaction. include,
prepared inactive, hydrolysis, normoxic in flavor,
and/or (3)
and
”
deficient electrons,
stability
or or
be conditions a herein, alkylator.
“ precursor may of a a
hypoxic Approach, hypoxic
that
alkylator
other (5)
“ “ substituted as
a bioreductive
cytotoxic
but for such or
compound
be in described
The
that but using
can
nitrooxazoles,
agent a environment
biological
vitro or example optionally are nitrobenzoic
metabolism
“ hydrogen,
that the
can bioreductive cells tumor be cytostatic thereof) transport
generate
prodrug
not
compounds
Oxford
by reduced is
chemical
agent be with a ”
in
limited its pharmaceutically
or
DT-diaphorase, are
group zone, (see eliminated
more processes.
agent cytotoxicity is ” substituted,
a
is a and/or cells characteristics,
agent University
is in modified variety
16 etc. pharmaceutically
nitrofurans, acid the in
a
”
or
modification an group vivo
to,
prodrug detail
refers that vivo
is
residing reference
in
”
agent amides,
groups
or its
is
or vitro.
and/or
of are
and/or
an is A an
below, cytotoxicity
to chemically
in
such groups. on
Press,
a or prodrug
not that
thiols, hydride agent
a
vitro.
active group and in based
hypoxic nitroazoles, As
compound
group Nogrady, cleaved fewer
drugs in
that is a upon
one
used nitropyrroles, hypoxic New
vitro that effectively or active
As Other
(1) form on the
can ion;
that
bioreductive
side by but such
activation on
cells. herein inhibits used York, electron by that
under redox
have,
photochemical
(2)
1985,
of normoxic
accepts
that compound. which
examples effects a nitroimidazoles, environment
that
can
the biological herein that
As
pages
reversible “
produces hypoxia; potential
relative normoxic
or Medicinal
the compound be deficient where
used releases upon
can
electrons or suppresses
group
reduced,
“ modified
cells. 388-392). of hypoxic lower
be
herein
As activation
to each process,
(4)
a for in reduced under or
is the cells the toxic used
vivo
is that in
the a of i.e.,
” an
2011202075 05 May 2011 30 25 20 15 10 5
tissue capacity by tumors, polysaccharides, [0075] [0074] platinum derivatives, neoplasm, [0073] angiogenic or in agent but groups such nitrothiazole groups, [0072] achieve group, In group groups understand, bioreductive conditions [0071]
combination maintain the another
not
as
” growth,
uncontrolled , by
including
such to refers
limited lymphomas,
cyano, or the of
As As As
As coordination Generally,
modifying
provide
tumor
some example,
metastasis, of
agents,
desired
adrenocortical in used used in used used as
groups group
a to which
a
view
-CH2-haIogen sulfone, to tumor, with
state any
combination
chemoprotectants, but
or herein, herein, herein, herein, methyl,
a
alkylating
lies redox growth
cancer.
a
of one the may other that and agent not can
of bioreductive
complexes, by with
the
within sulfonamide,
remission nitropyrrole
of limited be non-solid “ group “ “
an
be
DT-diaphorase, potential. neoplasm methoxy, malignant
cancer compounds used disclosure suppressants,
skill and
benign loss
Anti-neoplastic “ substituted
anti-neoplastic
agents of
where a
to
spread
in
in of to range such ”
contain
refers
anthracenediones, of the
the group both cyano, cancers
or
” and
or
or For moiety cancer halogen
clinical carboxamide, herein, (neoplasia)
groups.
malignant.
treatment art and where of amine
alkylators,
certain with growth
this certain to
having
abnormal
can electron
and/or carboxamide,
can such ” one
can agents
agent
how purpose, refers
one symptoms
the 17 is groups, “
For alleviate,
tune
herb of
-F, and hormones
of be as
a by group or
or
to
” a
redox example, antimetabolite,
withdrawing
cancer. leukemia. , -Cl, ”
to include, substituted
or
cells
“ positional more group a
substitute or “
tumor the substituted
to anti-tumor thiol. cancer -CF strong
other
can or
or
achieve -CF reduce, redox potential that
electron
-Br, 3 of and diagnostic
”
, Such
undergo
but
nitrothiophene, 3
One
and refers plant electron cells , can more
and
control. these with antagonists, can potential
are groups,
the
ureas, ameliorate,
milder agent agents of take
certain that or
donating sulfonamide extracts.
to than be
not
skill an desired reduction and
cancers
abnormal markers withdrawing
used. the ”
lies
electron methylhydrazine limited
,
electron electron can
100 of or other in
natural form
within
a “
the anti-cancer
redox groups, nitrofuran, be prevent,
diseases anti-cancer
bioreductive that
bioreductive associated in
to, art used of withdrawing groups, new
withdrawing donating
the products, have
said
solid potential. anti- will
groups
including
alone cell hypoxic or caused
range.
the
and to place
or with
or
2011202075 05 May 2011 30 25 20 15 10 5
diminishment treatment therapy, cancer line administered does preventing [0081] administration likelihood Thus, drug provides administration of effect, drug palliation example, administration, beneficial beneficial [0080] grammatical equivalents [0078] of [0079] or [0077] [0076]
cancer one elimination ”
not that, chemotherapy. thaf
a
when e.g., or
therapeutically
don
necessarily
As
As As a As
As
As
more of in as
when when or
of or or patient
or
alleviation,
of
’ the used used a used
used used both used stabilization used t
equivalents in the desired desired
of
delaying cancer work,
this of of
of
symptoms
and one administered subject. administered extent onset
the a herein, herein, one herein, initial herein, herein, herein, herein, with
phrase) series
indirect
or occur
or
results, clinical symptom(s). “ which
dose, Third
more
(or the of effective stop a amelioration, treatment,
The prescription a of a a “ a
“ “ of of disease,
reoccurrence) of
administering by reduction treating onset physician “ means “
“
and
this has second
prophylactically working
therapeutically administration, doses.
the line administrations.
including results
cancer
full to to administration
failed can phrase) disease
a ” a (or amount
therapeutic subject, therapy decreasing subject delay ”
first-line
line Thus, occur a reoccurrence) ” or include,
who is
palliation
for of condition to
other clinical
” called.
state, includes
” or a
of respond can therapy
a
with instructs a or refers will only symptom
slowing drug
disease prophylactically
effective including
therapy,
hyperproliferative “
but be of
effective of effect 18 and administration
have results. cancer,
after or one
or
the administered
is
to are
both refers
to
other elimination patient
of administering
therapy
a
or
of
does severity
dose,
a or the
not
administration patient
and amount
disease
first the symptoms. direct disease
amount will symptoms
For
to beneficial intended limited
not
subsequent
act and
refers therapy
chemotherapy
have that purposes
or to
administration,
effective ” necessarily
of or of
in
” can progression,
of of
self-administer frequency
disease
is ’
of prescribing symptoms, to,
one to
the
a
the one prophylactic a
(and given
occur
results The
that
drug a taking drug
alleviation
of
drug intended
or
drug
of treatment, or
amount
a
is full grammatical more
conditions,
this
to
more
is series for only occur
given
described regimen is of
steps to
a an prophylactic amelioration,
or the an a invention,
subject including
the the
administrations.
after therapeutic amount drug.
manifestations
reducing or can effect, of a by
amount
to
for
treatment
symptom(s), second-line patient. drug
amelioration
doses.
obtain
be or
the
below. (and For
“ e.g., of and/or
first self
of
the
effect a
of a
a
1 i ■ i
2011202075 05 May 2011 30 25 20 15 10 5
the activated metastases, supply exhibit Cyclophosphamide then, [0086] 84(9):1280-5 cancer of exhibit or [0085] and and tumor the the receptors. based cancer is than Ila. example, therapies, [0084] [0083] [0082]
that solid a
difficult
patient energy blocked Compounds even lower
normal low
on
growth
not
cells and, slower cells median tumors
Both
The
in Most Some As the tumor the
their
rely
oxygen all
These required
and the consequently, to
resistant
can One
regions cells.
used
vascularized partitioning and hypoxic cancer
is
Cyclophosphamide rates
kill
on
replicating
drug-mediated oxygen liver falls cancer
poor
. oxygen be
often
reason
drugs Rofstad
poisons,
herein hypoxic partial
However,
as, and of
for
in
via of cells to survival.
disordered; areas
division.
or cells
the
concentrations the
therapy cell
Ifosfamide are levels intermediates is
“
pressure even of
divide LogP etal.,
growth regions DNA,
that normal called
tumor zone
effective, division
reside of the
such
cancer
are the more, The cancer
”
(see substance
Br Thus, of
more
microtutbule,
means
cytotoxic become
of
leaving subject associated range and tumor than
in drugs
can a J mechanisms
for
new and
tumor. Cancer. a insensitive because therapies,
cells
of to frequently Ifosfamide
the poorly exemplify
the
example,
a
of divide
almost represent yield less vasculature.
measure hypoxic to significant betwen median
corresponding 40 can agents,
intermittent
with
19 1999, than
vascularized cancer
to active
and
mutate slowly.
including
inevitably
to of
60
than
De
octanol are how
a oxygen
- 10 of a selective
such various activation
80(1 poor
mm they
significant
cells Jaeger phosphoramidate
regions lipophilicity prodrugs mm
normal
The
and these
As
cytotoxic 1):1697-707). Hg,
blockage.
response
have
phosphoramidate
and normal Hg.
concentration generally
do
new develop growth solid a
et
for
agents and
tumor
of
and not
cells water.
al., a
and
source dividing vasculature
the lower tumor,
fifty
kill
tissue, action
agents of Br to
factors
and can drug These tumor
can grows, divide
a therapy, J all
percent
substance oxygen
of alkylators, Not Cancer. be slowly-dividing not
are
of of resistance. and cells of as
metastases
under-vascularized under-vascularized and
oxidatively
- more
it only
unable the alkylator-based surprisingly, specifically normal that
the requires
increased targeting, of
growth
concentration cancer 2001,
supports ten
cells solid frequently determined
Alkylators to
cells. percent
Another
and
generate
in a factor tumors cells
blood
target for them
in
1 (cylophosphamide mustard) and 2 (ifosfamide mustard), respectively (see below). The charge neutral Hemiacetals 1 and 2, can have a half life of many minutes and can permeate in and out of the cell. In contrast, the anionic Alkylators 1 and 2 are much less cell membrane permeable and once formed extracellularly inefficiently kills the cell by alkylating cellular 5 DNA.
[0087] When the phosphoramidate alkylators reach the tumor, they generally kill cells in the fast growing, well vascularized, normoxic, outer zone of the tumor. However, these phosphoramidate alkylators are not as effective in permeating into the less vascularized, slower growing, progressively hypoxic inner tumor zones and in killing tumor cells therein. 10 Before any of these active alkylators reach the tumor, they can react with healthy cells and result in toxicity and/or cell death.
Cyclophosphamide Hemiacetal 1 Alkylator 1
Liver oxidation
Ifosfamide Hemiacetal 2 Alkylator 2
[0088] While the hypoxic tumor is difficult to treat, the hypoxic tumor zone can generate 15 reduced derivatives of a variety of chemical groups (see the reference Workman et al., 1993, Cancer and Metast. Rev. 12: 73-82), and prodrugs of cytotoxins can be developed to exploit such bioreductive environments (PCT Application Nos. US 04/009667 and US 05/08161; PCT/US2005/041959 and PCT/US2005/042095, all Matteucci et al.). Such a hypoxia reducable (or hypoxia activated) prodrug can be constructed by employing a bioreductive 20 group (Z3) together with an alkylator. The bioreductive group is employed as part of a Trigger moiety covalently bonded or attached to the phosphoramidate alkylator.
20 ) 2011202075 05 May 2011 30 25 20 15 10 5
hypoxia pharmacodynamics to understanding [0092] significant enantiomer increased (vii) presence attributes [0091] alkylator known present Med. embodiments, (iii) toxicity present Application 4,908,356; the activated linker wherein alkylator 155-85, Chem. [0090] [0089]
healthy
following
less providing
Chem.
L
2002,45: phosphoramidate
invention invention.
or activated isolated
toxic is Aik
For To
of released, Phosphoramidate The prodrugs.
trigger. (iv) aqueous normoxic
advance lower bonded
5,306,727;
specific
more
understand
Publication
2000, effective is
increased
compounds structure side
of the
effective a
compounds
phosphoramidate tumor value 3540-8; than
prodrugs
have
phosphoramidate
solubility (ii)
effect to
43·. In
of over
in cells In
a
some
prodrugs tumor
the vivo one
5,403,932; 2258-65; general, one bioreductive of
biology
hypoxia why
phosphoramide No.US
known
alkylator profile
compared
Hemick
IC50 nature of
bioreductive
of or alkylator metabolism. embodiments,
(vi) the therapy, of the
the more
or
which the provided particularly
2003/0008850;
anti-cancer
invention prodrug or 2001, selective increased
of
IC90, invention
5,190,929; et
derivatives
alkylator
some phosphoramidate
the
of to alkylator group
al., derivatives
a
are hypoxic the
44:
in
hypoxia linker
group J. alkylator
Alk-Trigger combination conjpounds herein
hypoxic
disclosed
cytoxicity
following the 69-73; Med. can Z3. stability
phosphoramidate are
under and
21
5,472,956; prodrugs
(L)
by: phosphoramidate moiety,
generally
In tumor less
and
activated
in are Chem. Trigger
and/or one prodrugs (i) tissue,
hypoxia 2001,44: particular
to active therein, Papot
reported
measured
characteristics: the
alkylator of
cells.
embodimenk liver of
or
of 2003,46:148-54;
the
be nature and the T these (ii)
some
prodrug
the et and
has
and
that microsomal present described
and
bioreductive In
74-7;
al., lower 6,656,926;
is
in present alkylator
prodrugs less
by attributes.
a some
helpful. pharmacokinetics, combination
of
the
are alkylator
are Curr. structure
larger
the should Hemick
toxic normoxic
references,
invention achiral the not (i)
embodiments,
invention
as phosphoramidate Med.
derivatives,
a Trigger
the
degradation US
of
HCR phosphoramidate
to higher
prodrugs be group In L-Z3, US
the
et and
normoxic
subject
Patent
of Chem., much some
cytoxicity, al. represent
Patent
values
invention
Borch
these avoid
differ T Z3,
hypoxic wherein J.
is
and
less Med.
of of
an (iii)
the 2002, a
and/or
Nos.
(v)
cells et hypoxia from the
the
toxic
a
the
al.,
and have the
2,
J.
2011202075 05 May 2011 30 25 20 15 10 5
(0093 oxygen, reduction bonded get the generate formed healthy reductase [0095] Z3 semiquinone a alkylator through reduced phosphoramidate hydroxylamino hydroxylamino invention hypoxic, hypoxia groups healthy phosphoramidate [0094] difficult the dissociation than environment
quinone
in
reduced radical hypoxic
hypoxic J the
in
can to tissue,
cells
and the to
to activated or In
The Trigger.
A
kills tumor
potential
enzyme the
employ the moiety
anion.
an
reach
release certain react number
in
Trigger
region yet
of
radical and
within oxygen Trigger
cells. NO2C-)
indole cells a in
the
group or cells. hypoxic another
with the hypoxic
toxic
in alkylator. alkylator
nitro compared prodrugs
For
an
embodiments,
of the the
of in
of T When
a solid anion. presence hydroquinone
sensitivity
amino
the T.
or
bioreductive the
oxygen and/or releases through example, phosphoramidate enzymes phosphoramidate
side containing
in the tumor
In bioreductive
solid tumor region
such a
is
effects the that
The
semiquinone zone group In to near
released
to of
the the
normoxic another tumor. zone hypoxic
a
can
cytoclirome are of
hypoxic tissue, revert
oxygen, of
prodrug the
having the
conjugated phosphoramidate
aromatic
and to the group
and
the much be
having
phosphoramidate the hypoxic
within group.
bioreductive
responsible These flow back reduction embodiment, solid
flow
tumor, alkylator alkylator.
patient. tumor 0.01
tissue.
the radical in less of
or
a of 1
to P450
of the tumor
N
22
% the phosphoramidate
π first Thus,
% tumor. toxic
indole an the ¢)2(-)
the
an
zone electron
oxygen,
invention
oxygen. Under anion tumor of
Thus
electron or
reductase
electron step bioreductive
for nitro The while
group to the
alkylator.
for
the
in can or quinone
healthy, the
alkylator
modulates respectively
the
bioreductive a zone
normoxia, aryl the example, active group system
another
have hypoxic minimizing
reduction varies
pair encounters
pair present
semiquinone
or enzymes
and
moieties
a
is cytotoxin. heteroaryl The
from normoxic
alkylators from group
of higher
prodrugs
in depending
reduced can
one tumor, the
the as
invention
a released
to
of
the
group zone the
more
in death
oxygen bioreductive can the and Trigger a
the concentration as
well NO2C-)
amino
hydroquinone
radical
an cells
hypoxic, to moiety can reduce bioreductive
of
The having
not bioreductive easily
causes
of partly indole an
phosphoramidate
the vascularized provides
sensitivity
kill
ultimately non-cancerous T
compared or
or present anion releases
the
covalently penetrate
0.1 cells
on
reducing a group quinone
nitro
% the of
in
group
the
of
to
the
can the or
is
2011202075 05 May 2011 30 25 20 15 10 5
alkylator embodiment, capable of prodrug phosphoramidate to phosphoramidate prodrug [0099] invention phosphoramidate phosphoramidate the of phosphoramidate electron the phosphoramidate reduction tissue. toxic reduced [0098] electron and reduced [0097] oxygen, semiquinone agents [0096]
the
an hypoxia
“ bioreductive
tissue. Methods
anticancer to present
”
)
of
without
the with rich and that prodrug withdrawing the According in The The A a potential
forming
by
hypoxia healthy
The bioreductive hypoxic
radical
release
the
linker
a respect radical invention, Trigger
ease of the Trigger
agent novel
altering Treatment cytochrome
group
can
alkylator alkylator alkylator alkyltor alkylator alkylator action
or
an of normoxic or anion
to activated the
cancer
anion to be
difficulty
T linker
the ammonium ensures an phosphoramidate
the
T easier
phosphoramidate
phosphoramidate
can modulated
in
its of group
containing electron
bioreductive
itself prodrug
present
one prodrug ” in
in
can in (Alk-H). glutathione cells cytotoxic
be or
section).
P450
to a a phosphoramidate tissue a that
an oxidized, βπώοίΐϊπιβηζ influence bioreductive hypoxia loses of hypoxia
can reduce
and
rich reducing electron
cation invention, that reductase the
by
can release
can
a some
tissue For properties.
quaternary prodrug
phosphoramidate The
employing
group is transferase compared
be
non-specific, reduce non-senselective
alkylator
under hydrolyzed, the in
example, alkylator alkylator,
used
withdrawing
or chemical the
the compared
the
group
oxidative, enzyme the a
23 and all
properly
phosphoramidate
is
bioreductive
physiological it clinic, alkylator to
volume
of
absorbed
in ammonium For is
prodrugs a to
“ (see, does release.
attachment
its Trigger tune influenced
the causing when nature
or or example, oxidation,
to hypoxic can
hydrolytic,
presence
” employed phosphoramidate other not e.g., thiolyzed
alkylator. the
of
prodrug, manner. the
release it
in
of distribution of
T
In react
healthy
group toxicity is
phophoramidate
pharmacokinetic
conditions. the group reducing
containing
the the
one
of covalently by specificity a
hydrolysis,
alkylator, high
of tissue
the or
present
linker
an
the and Trigger does Z3 embodiment Telcyta™, or
oxygen. can
reacts normoxic
to bioreductive
active
thiolytic
can linker volume can
healthy agents quickly.
not of
yield
and/or
an
invention be In alkylator bonded
when a in release slowly or
release
toxin
phosphoramidate or amino
(L), one measured
a If
can
a alkylator
(“ a of cells
property stability thiolysis.
normoxic
a prodrug the
of
reducing it
and
According NO
distribution
in
be
with to the group is
the
group the
can
and are
absence
so an 2 further the
(
present
-)
by
more of make easily
If of
to
or cells
the the in
the
an
a
compound of the invention having a high volume of distribution while avoiding possible endosomal trapping. In another emdodiment, a Trigger T comprising a carboxyl functionality will exist as the anionic carboxylate anion form. CChOin the extracellular space outside of normal healthy tissue and not pass easily through the normal cell membrane. The lower pH 5 in tumor extracellular space can convert the CO2C) to the uncharged “CO2H” form allowing the prodrug to pass through tumor cell membrane.
[0100] A phosphoramidate alkylator containing a hydroxyl, amino, mercapto, and/or a carboxyl group can be transformed into a prodrug by covalently attaching a Trigger T to one or more of these functional groups. During the transformation from a phosphoramidate 10 alkylator to a prodrug, a hydroxyl group in the phosphoramidate alkylator can be transformed, for example, to an ether or an acetal; an amino to an alkylamino, a carbamate, or an amide; a carboxyl group to an ester, and a mercapto group to a thioether or a thioacyl, as described in greater detail in the Method of Synthesis and the Experimental sections below. These transformations can yield a prodrug which is less polar or more lipophilic than the 15 corresponding phosphoramidate alkylator. Non polar phosphoramidate alkylator prodrugs may not be readily soluble in aqueous pharmaceutical carriers or diluents. Solubility enhancer groups like CO2H, amino, alkylamino, dialylamino, and hydroxyl can be employed in the Trigger T to modulate the solubility of the prodrug and overcome any problems encountered in preparing aqueous formulations of the phosphoramidate alkylator prodrugs.
20 [0101] Phosphoramidate alkylators of the present invention can have one or more N-(2- haloalkyl) or N-(2-haloethyl) and/or one or more aziridine (OTN\I) moiety covalently bonded
to a P=O moiety as shown below. Upon release of the anionic phosphoramidate alkylator moiety an aziridine or aziridium species forms which can alkylate DNA (See EXAMPLE section, Example 36). Depending upon the electron witlidrawing nature of R2 and R3 25 substituents, the aziridinium formation kinetics can vary. For example, as shown in the reaction sequence below, the rate of alkylation can increase when the NR2R3 moiety is
changed from NH2 to (see Engle et al., J. Med. Chem., 1987, 25:1347-57). Substituents on the nitrogen atoms can alter the geometry of the phosphoramidate alkylator, the delocalization of the lone electron pair on this nitrogen atom in the P=O moiety, the 30 availability of the nitrogen lone electron pairs for aziridinium or subsequent aziridine formation, and the aqueous solubility of the phosphoramidate alkylator prodrug and the phosphoramidate alkylator.
24 2011202075 05 May 2011 20 15 10 5
HCR Borch of unexpectedly unexpectedly Z3-CH(Z2)-O-P(=O)(NHCH(R group 2-nitrofuran-5-yl, nitroimidazol-5-yl), wherein choloroethyl) Z3-CH2-O-P(=O)(NHCH hypoxic [0103] containing in cells example, alkylator [0102]
formula cells
Phosphoramidate of values than
as
et
the under
Z2 cytotoxicity, al., Z3
In The prodrugs
Compounds in
present
(I): ifosfamide
is one of and
J. normoxic
high more methyl; present phosphoramidate hypoxia.
known Med.
aspect, ifosfamide
or invention
HCR
employing toxic
2-nitrothiophen-5-yl, 5-nitro Chem.,
invention
low Alkylator
phosphoramidate R
mustard 24
cells lone the 9 (See
values,
2 in is and
CH normoxic
present
mustard hydrogen, hypoxic pair
in imidazolyl,
andUS EXAMPLE
2 25
X4)2, 2-nitroimidazole-bioreductive
an 9
or
)CH Prodrug
mustard arises
on in were
anti-proliferation ifosfamide
anti-proliferation invention N
Z Patent 2
(see toxicity cells X4) 3
in methyl, -CH respectively,
alkylator
bioreductive 2
or part
e.g.,
;
section). R4 compared or 2 alkylator Phosphoramidate
No. cyclofosfamide -O-P(=O)(NHCH(R
and 2-nitrofuran-5-yl; (I) mustard
out provides 25
compounds or
6,656,926
high of isopropyl; derivatives
R3
Phosphoramidate cytotoxicity
400 the
to cell
groups
analogs HCR
phosphoramidate
normoxic discovery
to
cytotoxicity
both P4,
1000
mustard; and
Z3 (Z group having
Pl and
supra). and 3
9 is
), assay improved )CH fold
4-17, cells,
that
and l-N-methyl-2- having
%*?(+) Aziridinium each show
2-nitrothiophene-5-yl, 2
or more
alkylator 0(-)
assays, X4)
with phosphoramidate
N,N Pl
and/or an
X4 alkylators
2 9, unexpectedly
formulas: ,
solubility. indole
toxic ’
a and (tetrakis-2- and is
IC50 in
possessed Cl
prodrugs
dna P21-22, contrast
in
or quinonyl of
prodrugs hypoxic
Br
0.05 For
were high
to in
μΜ
the
2011202075 05 May 2011 25 20 15 10 5
Ceheteroacyl, Ce OH, heteroaryl, [0113] [0112] -[C(Z,) -[C(Z [0111] Cg wherein wherein dialkylamino, heteroalkyl, alkyl, any heteroalkyl, wherein [0110] [0109] [0108] [0107] [0106] [0105] [0104]
alkoxy, dialkylamino,
two OP(=O)(OH)
Cj-Cgheteroalkyl, I
) 2 2
Trigger of -Y3]v-(S(=O) -Y each X each Z each Y Y Yi Y3 L each
2 2 Ci-Ce
4 2 C3-C8 3 R1-R5 is
is ]v-[C(=O)-O] is C3-C8 aryl, is is is is
selected
z,
aroyl,
Xi
Zi Ci-Ce aryl, NR of O, O, S, O,
v,
is
Ri-Rj alkylamino,
aryl, independently O, or is cycloalkyl, 2 form S, S, S, 7
q,
, , cycloalkyl, L-Z3;
heteroaryl,
independently
nitro, or heteroaryl; S,
or NR NRg or alkylene, u, 2
heteroaryl,
) from
or heteroaroyl;
-NR NR CI a and independently 6 -[C(Z
C3-C8 , C3-C10
q
O; or cyano, NC0R -[C(Z 7
'
7 g NSO wherein
and -C(=O)-O-;
heterocyclyl, Ci-C
independently
1
Ci-Ce heterocyclyl, )
Ci-Ce 2 cycloalkyl,
heterocycle; 1 is -Z
)
s 2 CO 2 Ci-Ce
, R6
hydrogen, -Z
N 6 2 or
-Y4] dialkylamino,
2 heteroalkylene, 2
each or wherein
acyl, H, -Y4]u-[C(Z NSO
is
u CRs
acyl, hydrogen, C -[C(Z
R
r Ci-Ceheteroacyl, 2 CpCe heterocyclyl,
R 7
C6 Ci-Ce
is wherein or
halogen, is 6 26 Ci-Ceheteroacyl, each
;
1
alkyl, 0
) independently each 2
1 or
] ) acyl,
z 2
-[C(Z aryl, alkoxy,
]
Rg hydroxyl, 1 z
-[-C(Z
of ; Ci-C Re
is
C1-C6
Ci-Ceheteroacyl,
R1-R5 CON(R
1 independently is Ci-Cg )=C(Z
6
Ci-Ce independently
1 heteroalkyl, )=C(Z
aroyl, alkyl,
amino, independently hydrogen,
alkoxy, aroyl, 7 ! )] )
alkylamino, 2 g I ,
-;
)] or Ci-C« Ci-C
g
Cj-Ce
-;and or heteroaroyl;
C|-Ce Ci-Ce
Ci-C« heteroaroyl;
aroyl, 6 hydroxyl,
heteroalkyl, hydrogen, acyl,
alkyl,Ci-C
is
Ci-Ce alkylamino, alkyl,
cycloalkyl, or a Ci-
Trigger
heteroaroyl;
or
CpCe
Ci-C halogen,
together
aryl, 6
T 6
Cr Ci-
2011202075 05 May 2011 15 10 5
pharmacophores, heteroalkylsulfonyloxyalkyl; [0119] heteroalkylsulfonyloxyalkyl [0121] [0120] alkylsulfonyloxyalkyl, alkylsulfonyloxyalkyl, with [0118] [0117] [0116] [0115] [0114]
the
proviso In In In an (iii) (i) Z3 (ii)
one one one individual at is
at each
a least least
embodiment, embodiment, embodiment, bioreductive that
a
of pharmaceutically
two
one in NR2R3
isomer 2-heteroalkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl,
formula
of of
Ri-Rs
R1-R5 ;
and
group and R any z
or
2 is
(I) -R
NR4R5 a
are
at is one 1
racemic 5 .
:
selected least selected are
selected acceptable
of
not are
R one 2
or -R
the
^ from
from
27 of from s non-racemic
N is same.
\); NR2R3 salt,
the the
the and
solvate,
group
group
2-arylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, group
and
mixture
NR4R5
consisting consisting
hydrate, consisting
is of
or
isomers,
of: of
of
a
2-haloalkyl, prodrug 2-haloalkyl, or
and and bioisosteres, NO
2- 2- thereof. 2
2
2-
[0122] In one embodiment, the present invention provides hypoxia activated phosphoramidate alkylator prodrugs each employing two phosphoramidate alkylators. In one embodiment, the phosphoramidate alkylator prodrug of the present invention employs an 1 5 N-alkyl-2-nitroimidazol-5-yl moiety or a l-N-methyl-2-nitroimidazol-5-yl moiety as a bioreductive group or Z3. In one embodiment, the phosphoramidate alkylator prodrug of the present invention employs a 2-nitrofuran moiety as a bioreductive group or Z3.
[0123] In one embodiment, the present invention excludes the compounds:
O (CIH2CH2C)2N II N(CH2CH2CI)2
0 I (CIH2CH2C)2N^J 1^, NH2 °\ t____ ^NO: Me no2 h2c------
h A-J Me 2 3
10 Pl P2
O 0 (CIH2CH2C)2N II nh2
no2
>
P3 P4
28 2011202075 05 May 20 P9 PIO 29 h 3 ch 2 co Pll N(CH 2 CH 2 CI) 2 I 2011202075 05 May 2011 5 wherein
R a
is
H,
Br
(Pl P12 4),
NMe2
(Pl 5),
CN
(Pl 30 6),
or
CONH 2
(Pl 7), P13 N(CH NO NfCHzCHjCIfe 2 CH 2 2 CI) 2 2011202075 05 May 2011 o 2 n Br P20 31 Cl ο CM Cl 2011202075
P36 P37 wherein R is H, Me or allyl;
5 {0124] 3-(5-Methoxy-l-methyl-4,7-indolequinonyl)-methyl bis[N-methyl-N-(2- bromoethyl)] phosphorodiamidate (P27),
[0125] 3-(5-Methoxy-l -methyl-4,7-indolequinonyl)methyl N,N-bis(2-bromoethyl)- phosphorodiamidate (P28),
[0126] 2-(5-Methoxy-l -methyl-4,7-indolequinonyl)methyl bis[N-methyl-N-(2- 10 bromoethyl)]phosphorodiamidate (P29),
[0127] 2-(5-Methoxy-l -methyl-4,7-indolequinonyl)methyl N,N-bis(2-chloroethyl) phosphorodiamidate (P30),
[0128] 2-(5-Methoxy-l -methyl-4,7-indolequinonyl)methyl N,N-bis(2-bromoethyl)- phosphorodiamidate (P31),
15 [0129] 3-(5-Methoxy-l-methyl-4,7-indolequin-onyl)methyl N,N-bis(2-bromoethyl)- phosphorodiamidate (P32),
32 [0130] 2-(5-Methoxy-1 -methyl-4,7-indolequinonyl)methyl bis[N-methyl-N-(2- bromoethyl)]phosphorodiamidate (P33),
[0131] 2-(5-Methoxy-l -methyl-4,7-indolequinonyl)methyl N,N-bis(2-chloroethyl)- phosphorodiamidate (P34), and
5 [0132] 2-(5-Methoxy-1 -methyl-4,7-indolequinonyl)methyl N,N-bis(2-bromoethyl)- phosphorodiamidate (P35)
[0133] In a related embodiment, the present invention provides a compound of formula (I) with the proviso that
[0134] (i) at least one of R1-R5 are selected from the group consisting of 2- 10 alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyIoxyalkyl, and 2- heteroalkylsulfonyloxyalkyl and
[0135] at least one of R1-R5 are selected from the group consisting of 2-haloalkyl, 2- alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2- heteroalkylsulfonyloxyalkyl; or
15 [0136] (ii) at least one of R1-R5 is selected from the group consisting of 2-haloalkyl, 2-Ci- Cj alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2- heteroalkylsulfonyloxyalkyl; and at least one of NR2R3 and NR4R5 is VWN\}; or
[0137] (iii) each NR2R3 and NR4R5 are
[0138] In another related embodiment, the present invention provides a compound of 20 formula (I) with the proviso that the formula (I) excludes R2 and R3 together forming a morpholine ring or R4 and R5 together forming a morpholine ring.
[0139] In one embodiment, the present invention excludes a compound of the following structure:
O .Cl
Cl
33 wherein Z| is hydrogen or Ci-Ce alkyl.
[0140] In one embodiment, the present invention provides compounds wherein the Trigger T is:
5 [CCZjh-YaJ-ICiZjh-Zz-Y^ -[C(Z1)2]z-[C(Z1)=C(Zi)]-Z3; [C(Z1)2-Y3]-[C(Z1)2]z-[C(Z1)=C(ZI)]-Z3; [C(Z1)2-Y3 -[C(Z1)2]z-Z3; [C(Z1)2-Y3]-(C(=O)-O)-[C(Z1)2]z-[C(Z1)=C(Z1)]-Z3; [C(Z1)2-Y3]-(C(=O)-O)-[C(Z1)2]z-Z3; 10 [CiZjh-YaHC^OJ-O) -[CiZOJz-fCiZjhCiZOl-Zj; [C(Zi)2-Z2-Y4]-(C(Z1)2]z-[C(Z1)=C(ZI)]-Z3; -[C(Zi)2]z-[C(Z1)=C(Z1)]-Z3;
15 [0141] In an additional embodiment, Z3 is:
34 2011202075 05 May 20 10 5
CMe CH(Me)-O-, [0144] CH(CONH [0143] CH(CH 2 -NMe-.
2 NMe In In
2 one one )->
CH(Me)-S-; 2
)-, -CH(CF
embodiment embodiment,-C(Zj)
-C(CH 3 )-, 2 NMe
-CH(Me)-NMe-,
-CH(CHF
-Z 2 ) 2 2 -Y4-together -,
or 2 -Y 2 -C(CH )-, 3
- -C(Me)2-,
is:
-CH(Me)-NH-;
2 -CH CO 35
is: 2 2 H) -O-,
-C(Et) 2 -.
-CH 2 -, 2 -CMe -S-,
-CH(CH
-CH 2 -NMe-, 2 2 -NMe, NMe
-CMez-NMe-, 2
)-, -CH
-
2 -NH->
or-
j 2011202075 05 May 2011 10 5
(ΠΙ): wherein [0148] wherein [0147] heterocyclyl. In R12 dihalomethyl, from CH=C(CORi2)-, C(Ar)=CH-, [0146] [0145]
another
is
the
is
each each independently In In group In In
embodiment;
one another another one
-CH=CAr-, Ζ,γ Zi
trihalomethyl,
consisting embodiment, is independently embodiment,
wherein
hydrogen
embodiment, embodime^
hydrogen,
Trigger -C(COAr)=CH-,-CH=C(COAr)-,
Ar of
or
is OH, hydroxymethyl,
the -[C(Zj)=C(Zi)J-
is aryl Ci-Ce
is
present
Trigger H Trigger OMe, C^-Ce
optionally or
alkyl
Ci-Q
CF3,
allcyl, invention
is: is:
and
36 alkyl. O-CHF2, CO2H,
substituted Ci-C is:-CH=CH-,
R 8
is provides 6
heteroalkyl, CONH H,
OCF3,
OH,
with
-C(COR
2 compounds
-C(CN)=CH-, , or
NO CONMe2,
up
-OP(=O)(OH)
Ο 2
to ,
3 12 CN, -Ο
five )=CH- 8
cycloalkyl,
halo, of
and substituents
-CH=C(CN)-,
formulas
or
CONHMe;
halomethyl,
2 - .
or
(II)
selected
and and
-
Trigger and
(Π) (ΙΠ)
wherein each R2-R5 independently is selected from the group consisting of hydrogen, hydroxyl, Ci-Ce alkyl, Ci-C6heteroalkyl, C3-C8 cycloalkyl, heterocyclyl, Ci-Ce alkoxy, Ci-Ce 5 alkylamino, Ci-Ce dialkylamino, aryl and heteroaryl; or together any two of R2-R5 form a C3- C10 heterocycle; each Y1 independently is S or O; and each Trigger T is defined as in formula
(i);
[0149] with the proviso that in formulas (II) or (ΠΙ):
[0150] (i) at least two of R1-R5 are selected from the group consisting of 2-haloalkyl, 2- 10 alkylsulfonyloxyalkyl, 2.heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2 heteroalkylsulfonyloxyalkyl; or
[0151] (ii) at least one of R1-R5 is selected from the group consisting of 2-haloalkyl, 2-Ci- Ce alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2- '0; or heteroalkylsulfonyloxyalkyl; and at least one ofNR2R3 and NR4R5 is
ΧΛ/νΉ 15 [0152] (iii) each NR2R3 and NR4R5 are \l; and
[0153] an individual isomer or a racemic or non-racemic mixture of isomers, bioisosteres, pharmacophores, a pharmaceutically acceptable salt, solvate, hydrate, or a prodrug thereof.
[0154] In one embodiment, the present invention provides a compound of formula (II)
wherein Trigger T is -CH2-Z3, -CH(Zj)-Z3 , or-C(Zi)2-Z3 wherein Zi is Cj-alkyl and Z3 is:
20
with the proviso that in formula (U):
37 [0155] (i) one of R2 and R3 is H and one of R4 and R5 is H;
[0156] (ii) one of R2 and R3 is Ci-alkyl and one of R4 and R5 is C|-alkyl; or
[0157] (iii) at least one of R2-R5 is hydroxyl, amino, C3-C8 cycloalkyl, heterocyclyl, Ci-C6 alkoxy, C1-C6 alkylamino, C1-C6 dialkylamino, aryl, heteroaryl, C1-C6 acyl, C]-C6heteroacyl, 5 or aroyl or heteroaroyl.
[0158] In one embodiment, the present invention provides a compound of formula (II) wherein Z3 is a bioreductive group selected from:
with the proviso that in formula (I):
10 [0159] (i) at least one of Rj-Rs are selected from the group consisting of 2- alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2- heteroalkylsulfonyloxyalkyl and
[0160] at least one of R[-R5 are selected from the group consisting of 2-haloalkyl, 2- alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2- 15 heteroalkylsulfonyloxyalkyl; or
[0161] (ii) at least one of R1-R5 is selected from the group consisting of 2-haloalkyl, 2-Ci- Ce alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2-
heteroalkylsulfonyloxyalkyl; and at least one of NR2R3 and NR4R5 is
[0162] (iii) each NR2R3 and NR4R5 are
20 [0163] In one aspect, the present invention provides phosphoramidate alkylator prodrugs of formula (I):
38 wherein
[0164] Rj is a -[C(Zl)2-Y3]v-[C(=0)-O]q-[C(Z1)2-Z2-Y4]u-Z3 or -[C(Zi)2-Y3]v-(S(=O)2)q-[C(Zi)2-Z2-Y4]u-Z3) wherein each v, q, and u independently is 0 or 1; 5 and Z3 is a glucose or an analog thereof with the proviso that it excludes glucose conjugates of phosphoramidate alkylators described in the reference Wiessler et al., US Patent No. 5,622,936;
[0165] each of R2-R5 independently is hydrogen, hydroxyl, amino, Cj-Q alkyl, Ci-Ce heteroalkyl, C3-C8 cycloalkyl, heterocyclyl, C|-C6alkoxy, Ci-Ce alkylamino, Cj-C^ 10 dialkylamino, aryl and heteroaiyl, CrC6 acyl, Cj-Cgheteroacyl, aroyl, or heteroaroyl; or together any two of R1-R5 form a C3-C10 heterocycle;
[0166] with the proviso that in formula (I):
[0167] (i) at least two of R2-R5 are selected from the group consisting of 2-haloalkyl, 2- alkylsulfonyloxyalkyl, 2.-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2- 15 heteroalkylsulfonyloxyalkyl;
[0168] (ii) at least one of R2-R5 is selected from the group consisting of 2-haloalkyl, 2-Ci- Ci alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2-
\ΛΛΤΝ I heteroalkylsulfonyloxyalkyl; and at least one of NR2R3 and NR4R5 is or
[0169] (iii) each NR2R3 and NR4R5 are ; and
20 [0170] an individual isomer or a racemic or non-racemic mixture of isomers, bioisosteres, pharmacophores, a pharmaceutically acceptable salt, solvate, hydrate, or a prodrug thereof.
[0171] In another embodiment, the present invention provides the compounds:
39 Phosphoramidate alylator prodrugs
O o
%/*■
/ —N
Κ» Phosphoramidate alkylator cytotoxins
[0172] In another embodiment, the present invention provides the compounds:
X4
wherein X4 and Z3 are defined as above.
5 [0173] In another embodiment, the present invention provides the compounds:
40 2011202075 05 May 2011 Phosphoramidate alkylator wherein [0175] In [0174]
one
embodiment,
R
The prodrug In 2
-
another R5
following
alkylator
are
to
defined
yield
embodiment,
prodrug
scheme is
the
-(N-CHzCHzXjh.
as
corresponding
in
exemplifies
formula
the
present
(Π).
hypoxic phosphoramidate 41
invention
reduction
provides
alkylator.
of
the
the
compounds:
phosphoramidate active
alkylator
J 2011202075 05 May 2011 10 5 (IV) Phosphoramidate [0178] alkylator wherein [0177] [0176]
-(VII)
R2
In prodrug The In
one - another
R5
alkylator following
embodiment, are
to
defined
yield embodiment, prodrug - NR4R5 nr
scheme 2
r the 3 N
as \II/
(IV)
the corresponding
in
hypoxic exemplifies
present formula
the N(R
^ present
reduction
invention
(II).
hypoxic phosphoramidate 42
invention
provides
reduction
provides (V)
the
alkylator.
of
compounds
the
the NR4R
compounds: nr
phosphoramidate 2 s r 3
of
the active
formulas
0 alkylator p I» \
NR4R5
(VI) (VII)
wherein each R9 independently is hydrogen, deuterium, aryl, heteroaryl, Cj-Q alkyl, Ci-Ce heteroalkyl, C3-C8 cycloalkyl, heterocyclyl, Ci-Ci acyl, Cj-Ciheteroacyl, aroyl, heteroaroyl, 5 Ci-Ce alkoxycarbonyl, Ci-Ce alkylaminocarbonyl, di CrCe alkylaminocarbonyl, or C1-C6 alkoxy, or together two R9 groups form a heterocycle; each Rio is hydrogen, Ci-Ce alkyl, Cr Cg heteroalkyl, C3-C8 cycloalkyl, heterocyclyl aroyl or heteroaroyl, or together two Rio groups form a heterocycle;
[0179] Rn is independently is hydrogen, deuterium, aryl, heteroaryl, Ci-Cg alkyl, Cj-Ce 10 heteroalkyl, C3-C8 cycloalkyl, heterocyclyl, Cj-Ci acyl, Ci-Ceheteroacyl, aroyl, heteroaroyl, Ci-Ce alkoxycarbonyl, Cj-Ce alkylaminocarbonyl, di Ci-Ce alkylaminocarbonyl, or C1-C6 alkoxy, or together two R9 groups form a heterocycle with the proviso that when Rn is Ci-Cg
alkyl or Ci-C6 heteroalkyl then Rn excludes χ4 ; or together two Ri i groups form a heterocycle;
15 [0180] X4 is Cl, Br, alkylsulfonyloxy, heteroalkylsulfonyloxy, arylsulfonyloxy, or heteroalkylsulfonyloxy; and
[0181] Trigger T is [QZ^-Y3]v-(C(=O)-O)q-[C(Z1)2-Z2-Y4]u-[C(Z1)2]2-[C(Z1)=C(ZI)]g-Z3.
[0182] In a related embodiment, in formulas (TV) - (VII), each R9 is independently hydrogen, deuterium, C1-C3 alkyl, CpCi heteroalkyl, C3-C« cycloalkyl, heterocyclyl, aryl or 20 heteroaryl. In another embodiment, each R9 is independently hydrogen, deuterium, or Ci-Ce alkyl. In another related embodiment, each R9 is independently methyl, ethyl, propyl, isopropyl, isobutyl, tertiary butyl, or cyclopropyl.
43 ) 2011202075 05 May 2011 10 5 [0186] wherein [0185] heteroaromatic wherein [0184] [0183]
X4 In one In In In
one
another and another one
of
embodiment, moiety R
Rio embodiment, jo
is is embodiment, embodiment,
-(CH2) defined
capable
e the
-Intercalator
the as
of
present
in
the present
the intercalating
formula
present present
invention
invention
wherein (VIII)
44 (IV).
invention invention
between
provides
an provides
Intercalator
provides provides
a
nucleic
the
a
compound
compound
the the
acid is
compound: compound: an
base
aromatic
of
of
pair.
formula
formula
or
(IV)
(VIII):
2011202075 05 May 2011 15 10 5
methyl, (X-A), and wherein [0189] (X): wherein [0188] (IX): and wherein [0187]
each
N(R
(X-B) cyclopropyl,
each
In
Ri each 10 In each In )
one 1
2
one one
is is and R9
R9
R9
independently selected embodiment,
βηύχ^ίηιβηζ independently
embodiment,
independently (X-C): is
methoxy, independently
from
the
NH hydrogen,
the and the
is
is
present 2 hydrogen,
present
hydrogen, , present hydroxyl; hydrogen, < NHMe, / \ xh
methyl, invention
invention invention
NMe
methyl,
or methyl, (X) 45 (ix) methyl, I °\
j
together
2 ethyl, Trigger ,
Trigger
NEt rA provides
provides
provides
ethyl,
ethyl, . ethyl,
2 propyl, ,
Λ two
propyl,
propyl, ,
propyl,
the
Ri
the the
isopropyl,
j
compounds
compound form compound
isopropyl,
isopropyl, isopropyl,
a
heterocycle.
benzyl,
of of of
or
or or
the
the the
cyclopropyl;
cyclopropyl.
cyclopropyl; substituted
formula formula formulas
2011202075 05 May 20 10 5
(XI)-(XV): [0190] (TV), wherein
(VI)
X In
2 and
one and (X
4 (VII). H
X4, embodiment, 2 X R CH 4 n'
are 2 C) 2
defined N
χ xA
0 Ϊ 11
the
as ” N(CH
present 2 in
formula 2 CH \
2 invention X4)2 2 - (X-A) 46
(I), X 2
and
provides
Rio,
and
the
Rn
compounds
are Me Me
defined NO NO
of NO 2 2
, as
and the 2
in
formula formulas
I
2011202075 05 May 20 10
is isopropyl. in heterocycloalkylsulfonyloxy, is propyl, wherein
Cl, compounds
Br,
cyclopropyl,
each
alkylsulfonyloxy,
(ΧΠΙ) (XI) In
one Rn
of (XV)
N(R
formulas
independently embodiment, io
methoxy, )2
(XII),
heteroalkylsulfonyloxy,
arylsulfonyloxy,
and
a
is
(XIV), compound
hydrogen, hydroxyl; and
and
of (XV),
47 (XIV) and methyl
or
formula
heteroarylsulfonyloxy. X|,
when
cycloalkylsulfonyloxy,
(ΧΠ) or X
2 substituted (X) ,
X4 and
excludes is
Z3
Cl
are
or
methyl,
defined Br
a
compound then
In
benzyl,
one Rn as
above;
embodiment, excludes
wherein
isopropyl,
and
X4 Z3
2011202075 05 May 2011 20 15 10
5
NR CH wherein (XVI) prodrug of [0193] oxidatively prodrug cyclopropyl. [0192] present XII, embodiment, or (XIV), [0191]
2 12
the XV CH2(-X6-CH2CH2)f,
wherein XIV,
invention
include
K
or
invention compared wherein In In
In
is (XV) or
metabolizing one
one
Ci-Qalkylene one
XV each
In the
compounds
embodiment,
provide embodiment wherein general,
embodiment,
Rn
wherein present
excludes R12 to
is known
is
C3-C8
or
a independently
a each proteins invention
pharmacokinetically
R CH cyclopropyl
or the 5,
h phosphoramidate
the
the
2 cycloalkyl. 7, Cj-C6 Ri excludes the
(-X6-CH2)f compound:
8,9,
i present present
in present is
provides
heteroalkylene. hydrogen. the 10,
group propyl defined
cell,
invention invention 13,
invention
In wherein (XVI)
48 another phosphoramidate particularly
14,15, can
alkylator
or
improved as Examples
isopropyl. be
above.
provides e provides
provides
In
19, more is embodiment,
one 1-10,
prodrugs. 23,24,25,26,
in phopsphoramidate
of stable
embodiment
the
a
the
In compounds f a
phopsphoramidate
is
alkylator
compound
another liver
compounds
0-3, than
the
the
and an cycloalkyl
32,
embodiment,
prodrugs
K prodrug alkyl of
of
Xe
is 34,
formula
of
alkylator formula
(C(R is
and group
the
O,
compounds alkylator is of
I2 36. formula S,
)2)
ΧΠ, formulas
to the (XII),
or
c
In ,
XIV,
one
2011202075 05 May 2011 10 5
compounds [0196] compound or wherein or wherein [0195] (XVII-(XVIII) [0194]
heteroarylsulfonyloxy. heteroarylsulfonyloxy;
e e In In
In
is
is of
one
of
one
one 0-4, 0-4,
formula
formulas
embodiment,
embodiment, embodiment,
and X4
is X4
(XIX)
Cl
described is
or Χό In Cl,
Br, wherein
a is the
the
Br, the related
O, alkylsulfonyloxy,
present
present present herein.
alkylsulfonyloxy, S,
or e embodiment,
is
NR12
invention
invention 1 Ha invention .
(XIX) See 49
wherein
EXAMPLE
heteroalkylsulfonyloxy,
provides
provides
provides the heteroalkylsulfonyloxy,
R12
present
is
the
defined the the section (XVIII)
invention
compound
compound compounds
as for
above.
examples
provides
arylsulfonyloxy,
of of
arylsulfonyloxy, of
formula
formula
the
of a formula
(XX): (XIX):
2011202075 05 May 2011 5
[0198] provides heteroalkylsulfonyloxy, [0197] analog wherein
includes
R In a In
g
compound
one
is one
glucose
embodiment, mono, embodiment,
of or
di
arylsulfonyloxy,
formula a and
glucose
the
the tri
saccharides. present
XX present
analog;
wherein
invention invention or
e
heteroarylsulfonyloxy.
(XX) is
50
In e
0-4,
is
a
1 related provides
provides . and
X4
embodiment,
is
the
the Cl,
compounds:
compounds: Br,
As alkylsulfonyloxy,
used the
present
herein,
invention
a
glucose
2011202075 05 May 2011 10 5
hydrolyzed [0201] alkylator wherein [0200] (XXI) wherein [0199]
Yi In
R In conjugate: In
9
enzymatically one
is another and one
S
embodiment,
or X4 embodiment,
O;
are
βπύ^ΐπιεηζ
and
defined
to Trigger
produce
such
the
as
the present in
T an
is formula
present oxime-phosphoramidate
defined
invention (XXI) 51
VI. invention
as Br
Trigger in
provides formula Br.
provides
(I). the
alkylator
the compound
oxime-phosphoramidate
conjugate
of
the
formula can -
be
.) 2011202075 05 May 2011 20 15 10 5
pharmacophores, [0209] an [0208] 4C(Z at 2-heteroalkylsulfonyloxyalkyl, [0207] heteroalkylsulfonyloxyalkyl, 4C(Z [0206] with independently heteroaryl; [0205] hydrogen, wherein (XXII): [0203] [0204] [0202]
least individual
the
I 1
) ) one 2 2
-Y3]v-[C(=0)-O] proviso -Y3]Y-(S(=O)2) each each (iii) (i) (ii) Ri each In
hydroxyl,
or
of
at another -
at isomer
each
t
Z NR2R3 together
R5, least R1-R5
is
least independently
R independently
that a 2 a
/ki Yi,
NR
Trigger pharmaceutically
two
one aspect,
or CpCe in and
and 2 and
R r R2
q a formula -[C(Z of 3 3
of
racemic
q Ri
NR and and -[C(Z Y2
R1-R5 T
R1-R5 alkyl,
the *-R
f
selected 2-arylsulfonyloxyalkyl, 2 K
are
U'K 1 1 is NR R2* 1 *R3*
)2-Z2-Y4]u-[C(Z
is 2-arylsulfonyloxyalkyl, present 5
1 (XXII): *
1 )2-Z2-Y4]u-[C(Z
defined
C,
and 2 CpCealkoxy, or
and independently or *R form R
' i
. is
4 S, 1
non-racemic
acceptable 2;
3 from R vwn
Ri *
or ^(zo invention i both a *-R
*-R
as
P; heterocycle;
'\l;or the
: in 5 t
s ) 52 wv*N * r
*
-K 1 formula group are
)2]
is
(ΧΧΠ) Ci-C salt,
1
is
)2] provides mixture z — 2-haloalkyl,
-[C(Z 2-haloalkyl,
selected z
-[-C(Z
solvate, 6 (Z0 or consisting
alkylamino,
; or
(I); or
and 2 1 t )=C(Z
)
each r
z heteroalkylsulfonyloxyalkyl;
of 2-heteroalkylsulfonyloxyalkyl; / the 1
>C(Z from Ν R
I isomers,
hydrate, 4 . \ΙΙ/
‘ compounds 2-Cj-Ce
R1-R5
2-alkylsulfonyloxyalkyl, I
)] of Y2 P x- the g
1
-Z Ci-Cedialkylamino, )] \
g
3 ζ
group and
-Z · bioisosteres, -; or R Ri* Ν
I alkylsulfonyloxyalkyl,
3 3 \
*
a
and R
prodrug of
i R consisting *-R or
2 the * 3
* formula
thereof.
of
aryl, or
2.
and
I I I I I I
2011202075 05 May 2011 20 15 10 5
heteroalkylsulfonyloxyalkyl, 4C(Z,) 4C(Z with [0218] Trigger heteroaryl; hydrogen, wherein [0217] [0216] (ΧΧΙΠ): bonded ofY [0215] [0213] [0214] arylene, [0212] [0211] [0210]
the 4,Υί, 1
)
2 2
T -Y3]v-(S(=O)2) proviso -Y3]v-[C(=O)-O] to
(i) or each Ri
In each each
each K each selected and
hydroxyl,
the
or
heteroarylene, at is - another
together
Ye least R
R1-R5
same selected Y4/Y5, R9 m r
s that independently ,
has independently is
Yi from
two
independently carbon aspect, Cj-Ce , in and
to and and
R2
q
from formula
-[C(Z of be the
q Ri*-R
and -[C(Zi)2-Z2-Y4]
Y
Y
R2-R5
(C(R O, alkyl,
group atom 2 the 6
the 2-arylsulfonyloxyalkyl,
are
independently l S, R )2-Z2-Y4]u-[C(Z
is 5
present 9 2
(ΧΧΠΙ) * is )2) group or
*
defined and
Ci-Ce or 0
independently consisting form
1-4; n Ci-Ce NR7. or
; adjacent
and R2*-Rs*
1 consisting
invention ; alkoxy, a
as (Y alkyl u heterocycle; -[C(Z
(XXIII) in 5 is
-(C(R9)2) carbon of 53
1 Trigger
formula O, are )2]
or
C|-Ce 1 is )2]
z provides of S, heteroalkyl,
-[C(Z 2-haloalkyl,
selected z
NR -[-C(Z Ci-C
atoms or
m alkylamino,
or (I); -Y4-(C(R9)2)in-Y6)n
2 7 1 )=C(Z ,
each 6 heteroalkyl-sulfonyloxyalkyl; or
alkylene,
the I are
)=C(Z from
a
bond;
or R1-R5* compounds cycloalkyl 2-alkylsulfonyloxyalkyl, 1 )]
together the g
1 -Z3-; )] Cj-Cedialkylamino,
CrCe
g
with group -Z3and
independently
the or when
heteroalkylene,
of
wherein consisting
heterocyclyl;
the proviso
covalently
formula
n
is is that
of
aryl, a
1-8; 2-
one and
[0219] (ii) at least one of R2-R5 and R2*-Rs* is 2-haloalkyl, 2-Ci-Cealkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, or 2-heteroalkylsulfonyloxyalkyl; and
one of NR2R3 and NR2*R3* is or
[0220] (iii) NR2R3 and NR2*R3* together are both or NR4R5 and NR^Rs*
I 5 together are both ^1; and
[0221] an individual isomer or a racemic or non-racemic mixture of isomers, bioisosteres, pharmacophores, a pharmaceutically acceptable salt, solvate, hydrate, or a prodrug thereof.
[0222] L2 is
10 wherein X is defined as above.
[0223] In another embodiment, the present invention provides the compound of the formula (XXIV):
f3 V4 r4* r3* I ii I 0 < r2^ (ZO2)rK-(ZO2)r-^N\H//N\ I °\ 0 Trigger ^Trigger
(XXTV)
15 wherein R2, R3, R4, R2*, R3*, R4*, Z, K and Trigger are as defined in Formula (ΧΧΠ).
[0224] In another embodiment, the present invention provides the compounds of formula (XXTV) having the structure of formula (XXV) or (XXVI):
54 (XXV) (XXVI)
[0225] In another embodiment, the present invention provides the compound of the formula (XXVI):
(XXVI)
wherein Xb X2, X4, and e are defined as in formula (XXV).
[0226] In another aspect, the present invention provides the compound of the formula (XXVII):
Trigger^^W^^Tngg^ . 10
(XXVII)
wherein R2-Rs, r, k, Yi, and Trigger T are defined as in formula (XXIV).
[0227] In one embodimenL the the present invention provides a compound of Formula:
55 2011202075 05 May 2011 5 consisting [0228] wherein Li
is
CH
T 2 In ,
of: CHMe, is
another
L-Z3;
C(Me)
embodiment, 2 ,
CH 2 OCH2,
the
present
(CH 2 )
56 3 invention ,
CH 2 S(CH
provides 2 )2,
CH
2 a S(CH
Z3
selected 2 ) 3 ,
from
the
group [0229] In another embodiment, the present invention provides a moiety having the formula:
R3
Rs °x selected from the group consisting of:
57 2011202075 05 May 20 α 58 2011202075 05 May 20 OP(=O)(OH)2. methyl, wherein consisting [0230]
or each In
ethyl; of:
another
Zj,
Within
R R 7 7,
is
embodiment, and
methyl,
this
Re
embodiment,
is
trifluoroethyl, defined
the
present
as
above.
59 ethyl, invention
Within
propyl,
provides
this
and
embodiment,
cyclohexyl;
a
T
selected
Zj
and
from is
Re hydrogen,
the is
OH
group
or
2011202075 05 May 2011 5 and wherein or embodiment, wherein [0231]
methanesulfonyloxy.
Z3
is
T each In
is
another
defined
R9 R9
is is
hydrogen, hydrogen
embodiment,
as
above
or
methyl, or
Ci-Cg
more
the
present
ethyl, particularly alkyl
60 and isopropyl,
invention
each
T
is X4
or L-Z3 provides
is
isobutyl; X
halo 4
wherein
or a
compound R and su
L HO i S(=O)2O-
X4 CH
is 2 ,
chloro,
CHMe, of
formula: .
In
bromo, another CMe2,
2011202075 05 May 2011 15 5
determining described synthesis phosphoramidate of pharmacokinetic resonance plasma, phosphoramidate the tumor wherein and [0232]
phosphoramidate
likes,.
deuterated
tissue
can X4
of
in In methods However,
dosage,
is a the be
one as
octadeuterated-compound halo
determined
phosphoramidate their EXAMPLE
aspect or
alkylator alkylators
or
and pharmacodynamic
frequency alkylators
non-deuterated the
R
SU the such
presence |S(=O)2O.
prodrugs. more present
section. and deuterated
and/or of
their efficiently
dosing, alkylator
of
invention In or
such Pharmacokinetic
phosphoramidate prodrugs
another properties hydrogenated
analogs 25
and
deuterated
prodrugs
and compared
61 provides similar
embodiment,
are
octadeuterated can
of
equally
the be of
administration analogs analogs,
to deuterated and
useful formula alkylator phosphoramidate
their
or cytotoxic
X4
pharmacodynamic in
in corresponding such isofosfamide
is vivo,
determining
prodrugs
phosphoramidate Cl
as
related
with or for
compounds
Br.
example
alkylators respect
is
parameters. Such
alkylator used
information
deuterated to
in in 25,36
alkylators
and/or hypoxic blood
is
The
and
2011202075 05 May 2011 invention selective [0233]
groupings In include:
another
of group
the
compounds of
embodiments,
of
the
the
62 EXAMPLES.
present
invention
Examples
provides
of
compounds
the
individual
of
the
and
2011202075 05 May 2011 63 2011202075 05 May 2011 64 r 2011
Cl May
05
2011202075
65 Γ i 2011
May
05
2011202075
66 2011202075 05 May 2011 15 10
146 145 141 140
67 147 2011202075 05 May 20 normoxic as
Z3
and
shows tisue.
hypoxic
tumor
specific
toxicity 68
while
being
much
less
toxic
to
healthy,
2011202075 05 May 20 10 5
phosphoramidate alkylator benzenesulfonyloxy, (VII), wherein [0236] phosphoramidate alkylator [0235]
and
X4
prodrug In la prodrug In
ionized
one is one
OH defined
embodiment, embodiment,
alkylators
which
alkylator which forms
or
as
para-toluenesulfonyloxy.
upon thereof. upon in
formula
the bioreduction the bioreduction Cl
present In present
(I), a
related
and
invention invention
releases
releases R9, OH 69 lb embodiment,
Rio,
provides
provides the the and
corresponding corresponding
Rn,
X4
>
and
a a are
novel is novel
Cl, defined
phosphoramidate Br, phosphoramidate
novel novel
methanesulfonyloxy,
as
in
or or OH OH ii
If formulas
OH known known
lc
(IV)-
and ionized forms thereof; wherein N(R)0)2 is selected from the group consisting of NH2, NHMe, NMe2, NEt2,
each Ri i is independently hydrogen, Me, ethyl, cyclopropyl, isopropyl, propyl, benzyl, 5 substituted methyl, cyclopropyl, methoxy, and hydroxyl; or together two Rn form a heterocycle.
[0237] The anti-cancer agent Cyclophosphamide metabolizes to Id (Rio is hydrogen) and Ifosfamide metabolizes to le (each Rn is hydrogen), when used in cancer treatment. Glufosfamide, which is being evaluated in the clinic for cancer treatment, releases an 10 alkylator of formula le (each Rn is hydrogen, see Wiessler et al., US Pat. No. 5,622,936; PCT application No. US05/03370 entitled “Anti Cancer Therapies”, US Pat. Appl. No. 60/638995 entitled “Glufosfamide Combination Therapy” and Attorney docket No. 021305- OO59OOUS filed on 11 May 2005 entitled “Glufosfamide Combination Therapy”). Telcyta™ which is being evaluated in the clinic for cancer treatment, releases 1 f (Rosen et al., Clin 15 Cancer Res. 2004, 10(11):3689-98).
[0238] Known phosphoramidate alkylator prodrugs such as ifosfamide and cyclophosphamide metabolize to produce cytotoxic by products such as acrolein and chloroacetaldehyde which cause undesirable patient side-effects such as hemorrhagic cystitis, coma or death. In one embodiment, the present invention provides a phosphoramidate 20 alkylator prodrug which upon metabolism produces less toxic by products per treatment as compared to those produced by the metabolism of ifosfamide and/or cyclophosphamide. In one embodiment, the phosphoramidate alkylator prodrugs of the present invention do not produce acroleiiy by in vivo metabolism. Examples of toxic by products resulting from metabolism of the prodrugs of the invention include chloro, bromo, alkylsulfonyloxy, 25 heteroalkylsulfonyloxy, arylsulfonyloxy, orheteroarylsulfonyloxy-acetaldehyde, (for metabolic production of chloroacetaldehyde from ifosfamide see the reference Hardman et al., supra, page 1396). In another embodiment, the present invention provides a phosphoramidate alkylator prodrug which upon oxidative metabolism produces 5-95% as much chloroacetaldehyde or an equivalent as defined above, per treatment, as produced by 30 ifosfamide metabolism.
70 2011202075 05 May 2011 30 25 20 15 10 5
formula: [C(Z [0243] [CiZ^HCi^j-OHqZjh-^-Y^H; modified [0242] -[CXZ^Jz-tcizo^zoj-Zj [CfZ^-Zz^] [C(Z,) [CCZ.h-YaHCiOj-OXCiZ^h-ZjH.od; a [C(z [C(Z [C(Z [CCZjji-YJ-fCCZ^-ZrYd Z [C(Z alkylator- hypoxic hypoxic [0241] alkylator above. phosphoramidate bonded bioreductive Alk-Triggermod a different [0240] alkylator [0239]
bystander 3
phosphoramidate .mod> I I 1 I ) 1 ) h-Y3]-[C(Zj) ) 2 )
2 2 2 2
-Y
-Y -Y to -Y3]-[C(Zi) -Y
In
to
In In tumor enough
Z
3 Upon 3 3 yield of In from The prodrug 3 ]-H;
T ]-(C(=O)-O)-[C(Z ]-(C(=O)-o)-[C(z 3 one ]-(C(=O)-O)-[C(Z
the one
another .
M effect
the one
group
or
phosphoramidate phosphoramidate
-[C(Z embodiment, the a zone reduction
embodiment, present upon
Aik- embodiment, to modified
upon alkylator
and
2 2 phosphoramidate
activate embodiment, ]z-[C(Z ] forms alkylator
z which I
-Z ) reduction T
2 is
]
M 3 activation invention z
.
-[C(Z n
termed conjugate. of
,o<
a phosphoramidate
J
I can
or )=C(Z the -mod -[CiZjhjaCiZ^ZOj-Zj^od; ; modified the
the
1 Trigger prodrug 1 1
)
) ) the some 2 1 2 2
)=C(Z prodrug
-Z
activate of ] ] bioreductive wherein alkylator bystander alkylator z a z
the
-[C(Z -[C(z
2 to present I
in modified the -Y )]-Z
other
diffuse hypoxic alkylator T
In 4
T
phosphoramidate 1 bioreductive can ] )]-Z3- m 3
1 I
-[C(Z includes . one )=C(Z compounds )=c(z these
mod Z is
derivative
invention modified can yield 3
[C(Zi>2-Y3]-[C(Zi) effect selected .
;
mO m phosphoramidate
embodiment or alkylator
71 od; group tissue
1
d prodrugs. undergo ) being
1 I
penetrate 2 )]-Z3.
)]-Z3H„od; is
a ]
and
the 2
allows modified bioreduced -[C(Z
group
phosphoramidate (Z
by
provides from:
of protected formed following m
o 3 prodrug
d degradation incorporating ) the
1 alkylator ;
)=C(Z
within a into
the
(Z modified
invention phosphoramidate
3 upon
T ). tumor
or a 2
alkylator
and
-Z 1 m such linkers compound )]-Z the
When
otherwise
is 2 prodrug,
-Y4]-H; the
the
selected Trigger
3 to
phosphoramidate zones . as
mod
but
a
reduction alkylator
yield
reduction phosphoramidate
(L) phosphoramidate linker
prodrug. ;
reside
and having which which the reduced
T either from:
alkylator (L) is
linker
nearby prodrug. of modified
of
are demonstrates
as For
the the
Z
or the
described 3
(L) not
example, can
prodrug the
to be
-[C(Z1)2-Y3]-(C(=O)-O)-[C(Z1)2-Z2-Y4]-[C(Z1)2]z-[C(Zi)=C(ZJ)]-; -[C(Z,)2-Y3]-[C(ZI)2-Z2-Y4]-[C(Z1)2]z-[C(ZI)=C(Z1)]-; -[C(Z1)2-Y3]-[C(Z1)2]z-[C(Z1)=C(Zl)]-;-[C(Z1)2-Y3]-[C(Z1)2]z-;
-[C(Z1)2-Y3]-(C(=O)-O)-[C(Z1)2]z-[C(Z1)=C(Zi)]-;-[C(Z1)2-Y3]-(C(=O)-O)-C(Z1)2-; 5 -[C(Zj)2-Y3]-(C(=O)-O) -[QZOJrtQZO^iZj)]-; and -[C(Zi)2-Z2-Y4] -[CiZihktCiZO^Zj)]-; -[C(Z1)2]z-[C(Z1)=C(ZI)J- and -[C(Zt)2]z-.
[0244] In one embodiment, the present invention provides a Trigger T which upon
bioreduction is modified to TriggerMod or Tm and the phosphoramidate alkylator is separated
from Tm in less than 0.1 second. In another embodiment, the phosphoramidate alkylator is
10 separated from Tm in between 0.01 to 0.10 second. In another embodiment, the
phosphoramidate alkylator is separated from Tm in between 0.1 to 1.0 second. In another
embodiment, the active phosphoramidate is separated from Tm in between 1.0 to 10.0
seconds. In another embodiment, the phosphoramidate alkylator is separated from Tm in between 10.0 to 100.0 seconds.
15 [0245] In a related embodiment, upon activation or reduction, a phosphoramidate alkylator
prodrug yields a prodrug with a modified Trigger T (Tm) which subsequently releases the phosphoramidate alkylator 20 to 500 pm from the site of activation or reduction; or 20 to 100 pm from the site of activation or reduction. Bystander effect of a phosphoramidate alkylator prodrug of the present invention can be measured using cellular spheroids and multilayer 20 cellular assay (for example of such assays see Kyle et al., Cancer Res. 2004,64(17):6304-9 and West et al., Cancer Chemother. Pharmacol., 1987,20(2):109-14); and as described in greater detail in Examples 35 and 37. Tumor cells can be grown in culture as multicellular spheroids to create an in vitro model of the tumor microenvironment in solid tumors containing a hypoxic region and a quiescent cell population responding to the environmental 25 stresses of limited nutrients and increased waste production. These spheroids have the unique property of developing gradients of oxygen and nutrients as the aggregate of cells continue to divide and grow outward. After the viable rim reaches approximately 150 pm in size, a hypoxic region develops, that drives the cells in this region into a quiescent state and eventually to cell death. A necrotic core develops as a result of the dying cells. The spheroid 30 can be divided into 4 distinct compartments for modeling the effectiveness of a hypoxic activated prodrug: 1) the outer aerobic and actively dividing region; 2) a region of intermediate hypoxia; 3) a region of hypoxia where cells are not cycling; 4) and a necrotic core containing dead cells and cellular debris. The response of a drug will depend on a
72 2011202075 05 May 20 30 25 20 15 10 5
prodrug, phosphoramidate die least prodrugs from the and [0248] phosphoramidate prodrugs efficiently concentrations molecular determining vasculature. prodrug second, with by HAP hypoxic monolayer [0247] aerobic equilibrating number reactivity [0246] effect). activated number spheroid.
phosphoramidate hypoxic knowing the
ten a a ’
s
modification
half
cell
can
between cells and
the
which of The of In that fraction Anti that
In
drug
of kill weights The
different
factors; life
one
membrane culture be
selectivity
cytoxicity
one the up In
the
the
assessment or are gas can
cancers tumor
combined
hypoxic one to upon ofless
activation to
embodiment,
bystander are hypoxic
embodiment,
activated
safer
alkylator
of 1.0
alkylator and reach leave one
that versus
embodiment, the able
at the
levels.
alkylator to activation cells.
therefore
the million-fold
than than drugs
of permeability and
ability
impede the 10.0 spheroid hypoxic tumor
to
fraction of
with the
of site
intact
effect prodrug drug
hypoxic used target site 0.1 the
For the
The
seconds, hypoxic can
phosphoramidate
the
of the prodrugs of other zones
second; corresponding
effectiveness diffusion such change from in
example,
in
spheroids.
cancer
effect
bind are compound attachment can present the
the and
present the
a releases safer..
cytotoxicity
chemotherapeutic
of
hypoxia described
as present that where be
entire activated the and to cell
the of
the
between bioreduction
cells are modulated
tissue
can and invention
expected
for the invention between
in
prodrug
In ratio
spheroid.
converted it The the
to
of
which invention
away of a
73 be not one compound activated
was
phosphoramidate
penetrate hypoxia in prodrug surrounding alkylator
the
alkylator/cytotoxic
a
of up HAP of 0.01
reach greater compound
embodiment, activated
the Trigger from cell the provides and 10.0 by
to it provides
releases
to was
The can into 150
varying
agents aerobic
phosphoramidate provides
prodrug
kill in
the (HAP) activated
to 0.10
the
into and alone detail
therapeutically
used activated; -
expected
the 100.0
T of phosphoramidate
the 200 and
vasculature.
phosphoramidate
can
its
the second, a the (activation that
each
and corresponding can by the
phosphoramidate
in as vasculature the
plays
rate kill phosphoramidate alkylators
μΜ
deepest phosphoramidate therefore seconds.
phosphoramidate
Examples
nitroreductases; either a
concentration
hypoxic
be of increased
agent).
monotherapy.
cell nearby of and
away
between
an the compared
cellular
kill target
important the of alkylator regions
effective monotherapies. Some
be from formed
and/or In compartments. the cells
can ability 35
alkylator safety alkylator evaluated
either 0.1 only alkylator
alkylation,
and
methods
the
be to of alkylator (bystander of
to
alkylator
prodrug. the have
in
alkylator The cells role
alkylator predicted of O
the the 37. results
event,
1.0 2 vivo
the
of impact
in
outer
to
on high in The
for
the
(at
a
2011202075 05 May 2011 30 25 20 15 10 5
prodrug or ability prodrugs cytotoxicity IC50, LC10, cytotoxic alkylator proliferation recognize without released anoxic [0251] corresponding fold, yields prodrug, or example, phosphoramidate [0250] fold and least alkylator. alkylator, phosphoramidate resulting hypoxic [0249]
LC99
LC
can IC90, less
and about IC90
jo of
a
the or is
oxygen
phosphoramidate of
from show
of
prodrug the
To tissues any than
typically activity In larger hypoxic
In in that for
LC50, or in higher
the This
the
ratio
10
one its prodnig determine one
assay
LC90, a phosphoramidate
a
the the cytotoxicity
up corresponding phosphoramidate
manner
fold phosphoramidate present removal
(anoxia) than;
masking LC?o, version, by
embodiment,
(HCR)
of as to is
compounds
cytotoxic
alkylator.
alkyl conditions,
is about
or
less
virtue its
measured the about
relative expressed
that
IC99
or
hypoxic that invention. if
ator.
phosphoramidate cytotoxic
and
and
or
compounds LC99 a 100 effect
determined
of alkylator
a or
of phosphoramidate
masks with
activity
million
the
the can
Typically, to phosphoramidate to LC99. a
under cells
the in determined
phosphoramidate alkylator
by
can cell healthy
about
concomitant
activation very alkylator be alkylator a
activity
Trigger or The the
clonogenic
are
prodrug vary
of a selective fold The hypoxic
reduces provided
measure little based IC50; 10,000-fold,
the larger exposed
normoxic the
ratio or and that
alkylator than
or T corresponding with
in
or oxygen
less phosphoramidate
and that
on
is
alkylator modified 74 conditions. can the or normoxia can
toxicity the reduction
of
survival herein of
covalently the
alkylator IC90 an cytotoxic to subsequent
the
cytotoxicity is
cytotoxic HCR depend be alkylator the
cells.
the
IC corresponding
at (hypoxia). prodrug less
cytotoxicity or linked
hypoxia 5
least
and include
o,
drug prodrug
phosphoramidate of LC90.
experiment
and active of IC90,
The
prodrug on phosphoramidate
the activity. bonded greater
the
about prodrug
activity
to either release hypoxia is the
HCR
phosphoramidate
as alkylator or
as selective a bioreductive about as
One
is
Trigger
cytotoxic measured
LC50 phosphoramidate
of
a
10-fold
selectively can phosphoramidate the with
to
cytotoxic
of determined In is or a as of
the
is 100
phosphoramidate
hypoxic
expressed be the of one generation
measured
skill
called prodrug air
cytotoxicity
T phosphoramidate
alkylator 1 1 fold
to
phosphoramidate
activity in version, (normoxic) nM, for μΜ
alkylator. in group about
a agent active
hypoxia to
the example tumor
manner
or
based
the alkylator
will
about as in
of greater. art
(Z 1,000,000- of
that
alkylator
the than IC50, IC10
an under
alkylator,
the of show 3
the killing
will ), on
As anti or
that
10,000 is
the
by
or
the
IC90,
IC99
one
at
2011202075 05 May 2011 30 25 20 15 10 5
phosphoramidate prodrug for [0256] formula present measure Such about provides prodrug, [0255] alkylators, invention 1,000,00; further adjacent provides folds than phosphoramidate normoxic prodrug [0254] such present hypoxic [0253] section). related invention [0252]
i.v.
phosphoramidate the
as, more
0.5-0.6%
or
invention away
having embodiment, XV invention
the
In having corresponding for under to The phosphoramidate cytotoxicity
Tumors a i.p. In In
provides 10-10,00; In
toxicity. has
phosphoramidate 5-1,000,00; one than the
lipophilicity wherein one example, a
another
injection. from logP
related a
vasculature, a a
oxygen embodiment,
hypoxic hypoxic the
embodiment,
log? variety
alkylator provides
have alkylator
has
of the phosphoramidate
In and corresponding
each related
a the
of embodiment,
the a greater vasculature
another alkylator
phopsphramidate a
10-10,00; normoxic concentrations. hypoxic Another
25-5,000 0.1
of
gradient toxicity cytotoxicity or phosphoramidate
compounds
Ri
prodrug a alkylator
oxygen prodrug the to
nM
phosphoramidate embodiment,
i alkylator
the
than is
the 0.5% embodiment,
hydrophilicity
to H prodrugs
cytotoxicity example
present which
and of
toxicity. folds present
and
0. 50 and normoxic having concentrations. the
having
in prodrugs oxygen
alkylator
of 25-5,000 In μΜ as
prodrug
near tissues
phosphoramidate can
more
is
0.1
one described invention alkylator
invention of
and can the 5
a In
alkylator be
hypoxic the nM
to
logP concentration the such
embodiment,
75 of
toxicity. toxic another
phosphoramidate
generate
be about easily of alkylator a
prodrugs
1,000,000 having folds necrotic
0.1 to HCR present
the hydrophilic,
between prodrugs
prodrug 50
in provides than
nM
provides In
toxicity
prodrug prodrug. 150 formulated more
Examples embodiment, μΜ of
hypoxic one phosphoramidate
to prodrug the core. invention
that
25
μΜ alkylator
folds
and
5 0 the that toxic embodiment,
of are
corresponding to
μΜ a which
and
can
away, of
a
In
the phosphoramidate such a present
100,000 In
phosphoramidate more compounds can toxicity
alkylator
HCR the
29,30
one as having than and generate 4
one present
provides
prodrug
such
an vary is as the present and
embodiment, than a
the
embodiment, of
10
a aqueous
invention
HCR
and present (see
which
prodrug
lower
as
a from
10 the to
alkylators, corresponding
prodrug
invention the IogP phosphoramidate
prodrug, those
of
31. 24,25 invention to 10,000 a
EXAMPLE
of present
corresponding
the 10%, in 100,000. is
50 invention less formulation
alkylator having
provides 25 exemplified tissues alkylator
present
of
to
and the
folds
than the
in to can under
invention
5 the 10,000
has
present tissues 5,000
36.
In more 0.
a
a
a
by formulas XIV; XX and XV wherein each Rn is methyl or, cyclopropyl, and administered in a patient can pass the cell membrane to penetrate inside cancer cells. Another example a prodrug having a logP between 0 and is 5, 6, 7, or 16. (for measured logP of phopsphramidate alkylator prodrugs of the present invention see EXAMPLES section).
5
lib. Method of synthesis
[0257] The present invention arises in part out of the discovery that compound 36, which could not be isolated by reacting
10 l-N-methyl-2-nitroimidazole-5-methanol, and n-butyl lithium in a suitable solvent, was readily synthesized by employing a Mitsunobu-type reaction wherein l-N-methyl-2- nitroimidazole-5-methanol was activated by the addition of triphenylphosphine and
diisopropyl azodicarboxylate, and reacted with to yield compound 36.
[0258] Thus, in one aspect the present invention provides a method of synthesizing a 15 phosphoramidate compound comprising reacting a phosphoramidic or a phosphordiamidic acid and an alcohol to yield a phosphoramidate. In another aspect, the present invention provides methods of synthesizing the novel phosphoramidate alkylator prodrug compounds of the invention or those that are known. In one embodiment, the present invention provides a method of synthesizing a phosphoramidate alkylator prodrug comprising reacting, a novel 20 or known phosphoramidate alkylator, a Trigger-OH, a trisubstituted phosphine, and a dialkyl azodicarboxylate to yield a novel or known phosphoramidate alkylator prodrug. In one embodiment of the method, in a first step the Trigger-OH is reacted with the trisubstituted phosphine and the dialkyl azodicarboxylate to yield an intermediate, and in a second step, the phosphoramidate alkylator is added to the intermediate obtained from the first step to yield
76 2011202075 05 May 20 10
phosphoramidate phosphoramidate wherein [0259] known the L-Z
product. 3 ,
wherein
phosphoramidate R In 9
is one
as Such
Z3
embodiment, defined
alkylators: alkylator
is: a
Mitsunobu
above.
alkylator
prodrug
the
type
present
prodrugs comprising
reaction
invention o
or 77
is
reacting derivatives,
particularly
provides
each
Alk-Trigger,
suitable a
of
method
novel
for
or of
synthesis
known
wherein synthesizing
Trigger
of
novel
a
is
or
)
2011202075 05 May 2011 10 5
comprising heteroalkylsulfonyloxyalkyl; alkylsulfonyloxyalkyl, compound wherein [0260] respectively, with Trigger
a
(i) Trigger-OH,
X4, wherein
In at
reacting of
one least R5,
formula:
R7, embodiment,
R2-R5 two
(a) and a
of trisubstituted
2.-heteroalkylsulfonyloxyalkyl, a
are Rg
R novel r
are defined R
5 the
as or are
defined known present Trigger
selected
R5R4N. phosphine, as R5R4N.
in
formula phosphoramidate invention as
Ο
II from
in 78 OH O P^ OTrigger || O P^
||
I formula
μ
and
the /
(I) , ,NR
nr a provides
/ group with
dialkyl
2 2 2-arylsulfonyioxyalkyl,
^Rg r (I). R 3 3
the
X ^R consisting alkylator
4
azodicarboxylate a
proviso
method 9
HO Trigger-. of
that of
to
formula: —
2-haloalkyl,
synthesize ο Ρ II
—
Ο
to N(R and —
yield O
P 1 11 2- o
— ) a
2 2-
N(R 10 ) 2 2011202075 05 May 2011 10 5
is [0262] is (b) (c) heteroalkylsulfonyloxyalkyl; [0261] alkylsulfonyloxyalkyl,
selected selected
a a
dialkyl Trigger-OH
In In (iii) (ii)
another one from
from at
NR2R3
azodicarboxylate
least embodiment,
the the
wherein embodiment,
one
group and group
of 2-heteroalkylsulfonyloxyaIkyl, NR4R5
R1-R5 consisting
consisting Trigger
the
and
to
both
the compound
is yield
at
is selected
group
RsR^^JI^NRzRg R5R4N
together least
ofi defined of:
the
one of
compound
of from
formula: are~' 79 as
of formula: OTrigger O OH O || I
in
NR2R3 the
N Formula ^; NR
group
of
2 2-arylsulfonyloxyalkyl,
R and
formula: 3
consisting
(I), NR4R5
a
trisubstituted
is
of
2-haloalkyl, or
phosphine, and
2-
2-CrCe
and 2011202075 05 May 20 Cl 80 Γ 2011
May
05
Br 2011202075
Cl
[0263] In another embodiment, the reaction includes a solvent such as THF, dioxane, a Cj- C6 alkyl acetate, chloroform, dichloromethane, acetonitrile and the like. In another βπύκχΐΗηεηζ each substituent in the trisubstituted phosphine is independently selected from a 5 Ci-Q alkyl, CrCe heteroalkyl, C3-Cg cycloalkyl, heterocyclyl, aryl, heteroaryl, and Ci-Cg alkoxy substituent. In another embodiment, Trigger T- is
wherein Xb X2, Z], and Z2 are defined as in formula (I).
10 [0264] In another embodiment, the present invention provides a method to synthesize a phosphoramidate alkylator prodrug comprising
81 [0265] (i) reacting in a solvent selected from THF, dioxane, dichloromethane, chloroform, ethyl acetate, propyl acetate, butyl acetate, or acetonitrile a compoumd of formula:
wherein each Ri i is independently hydrogen, cycloprpyl, methyl, ethyl, benzyl, or methoxy; 5 each R9 is independently hydrogen, methyl, ethyl, propyl, or cyclopropyl; and X4 is halo, methylsulfonyloxy, phenylsulfonyloxy, 4-methylphenylsulfonyloxy, and 4- halophenylsulfonyloxy;
(ii) a trisubstituted phosphine selected from triphenylphosphine, tributylphosphine, tributylphosphite; and
10 (iii) diethyl or diisopropyl azodicarboxylate;
to yield a product of formula:
[0266] In another embodiment, the present invention provides a method of synthesizing a compound of formula:
comprising the steps of:
82 (i) reacting in an aprotic solvent, a Trigger-OH, wherein Trigger is defined as in Formula (I); a trisubstituted phosphine; and a dialkyl azodicarboxylate to yield an Intermediate (i);
(ii) reacting the Intermediate (i) obtained from step (i) with a compound of formula
Π R\1
5
wherein each R9, Rn, and X4 is defined as in Formula (I), to yield the compound of formula:
[0267] In another embodiment, the trisubstituted phosphine is P(Ri2)3 wherein each Rj2 is H, Ci-Ce alkyl, Ci-Ce heteroalkyl, C3-Cs cycloalkyl, heterocyclyl, aryl, or heteroaryl. In 10 another embodiment, the trisubstituted phosphine is a polymer supported trisubstituted phosphine. In another embodiment, the trisubstituted phosphine is triphenylphosphine, tributylphosphine, tripropylphosphine, triethylphosphine, or trimethylphosphine. In another embodiment, the trisubstituted phosphine is a polymer supported triphenyl phosphine. Polymer supported trisubstituted phosphines are commercially available, for example, from 15 Varian Inc. of Palo Alto, California. In another embodiment, the present invention provides a method of synthesizing the compounds wherein each Rn is hydrogen. In another embodiment, the present invention provides a method of synthesizing the compounds
83 2011202075 05 May 2011 10
haloethyl-N-(Ri3)ammonium {0271] yield hydrogen, phosphoramidate [0270] [0269] compound [0268]
a
dichlorophosphoramidate
(b) (a) In
In Ci-Ci
wherein
one
reacting refluxing another
embodiment,
alkyl,
alkylator
the
embodiment, the
POClj
Ci-Cgheteroalkyl,
Trigger
dichlorophosphoramidate
prodrug
salt, with the
selected
intermediate;
present
wherein a the
comprising N-2-haloethyl-N-(Ri3)ammonium
present
from
invention C3-C8
R13
84
invention
the is
the
cycloalkyl,
hydrogen,
group
intermediate steps provides
provides
consisting of:
Ci-Ce heterocyclyl,
a
method
in
the
alkyl,
step of:
method
to salt,
(a)
CpCeheteroalkyl,
synthesize aryl,
with
wherein
of
heteroaryl,
making a
N-2-
R a
n
a is
to
2011202075 05 May 2011 25 20 15 10 5
phosphoramidate cycloalkyl, (Ri3)ammonium to [0278] phosphoramidate [0277] diisopropylamide. [0275] diglyme, is before [0276] separated tetrahydrofuran diisopropylethylamine. employed embodiment, from In and separated Trigger-OH [0274] monochlorophosphoramidate [0273] Cj-Cg [0272]
yield lithium,
another
then
the
cycloalkyl, subjecting
a
0>)
(a) In In diethylether, rest distilling dichlorophosphate In In (c)
from from
in sodium, heterocyclyl,
embodiment,
one one
reacting reacting one one
and reacting step
of the
the
the
embodiment, embodiment, (THF) the salt,
embodiment,
embodiment,
alkylator a
base alkylator
it (b)
heterocyclyl,
base the rest In or rest to reaction
in the
wherein
include
the
one potassium
or the dichlorophosphoramidate employed
or
of a of
in
In
aryl, dichlorophosphate
the
THF. solvent monochlorophosphoramidate
dioxane. reaction the embodiment, the
a prodrug. prodrug one
solvent intermediate; mixture separation
trialkyl
heteroaryl, the reaction the R13 reaction the intermediate; the
embodiment,
aryl,
hexaallcyldisilazide;
in
about present monochlorophosphoramidate
is phosphoramidate dichlorophosphoramidate
in
comprising step hydrogen, to
by heteroaryl, amines, step
mixture yield mixture
is 1
the flash
(b)
and invention equivalent
performed
(c). and
solvent
intermediate is and the
85
the
a such column a
In by Ci-Ce before base
the tertiary phosphoramidate
under and
solvent one
flash
as,
steps provides employed
alkylator
in each
by
a
sodium
alkyl,
embodiment, chromatography
triethyl subjecting
base
a reduced
intermediate
column amine. first
solvent employed of: in
of
in Ci-Q step
intermediate POCI3, removing
a prodrug
or
amine in a
method
intermediate pressure.
chromatography Suitable solvent
potassium (a) to
step it
alkylator heteroalkyl,
to in yield
the with
a
obtained
or
(c)
the step Trigger-OH, of
to excess
on base
to
tertiary step is
the a synthesize of reaction
silica
yield N-2-haloethyl-N- (b)-is
hydride; dimethoxyethane, prodrug.
step
useful
of
(c) in
POCI3 C3-Cg
step
gel.
a
step amines is (a) on
in
separated in and
or silica (b)
is
a step In
(b) in
step
lithium
a
one is vacuo
with
base
(b).
gel (c)
2011202075 05 May 2011 20 15 10
5
schematically [0286] acetate. embodiment, prodrug. [0282] [0285] intermediate; [0284] to phosphoramidate [0283] heterocyclic wherein [0281] the another (0280] (0279]
yield
temperature
a
In Various embodiment, X4 In (a) (c) (b)
In monochlorophosphamide In In
another
one
=
one reacting oxidizing
reacting another one phopsphoramidate
the below:
and Br
embodiment,
embodiment,
of embodiment, solvent l-N-alkyl-2-aminoimidazole-5-carboxylate or
alkylator
embodiment,
step Cl;
embodiment,
PCI3 the
step the
e (a).
used monochlorophosphamide =
intermediate with
(b) prodrug 1-3
the
in the is steps
a
ankylator
step performed Trigger-OH N,N-di(2-haloethyl)ammonium
base
present the
derivative; comprising (a)
(c)
present used
in and
is
invention step prodrugs
dimethoxyethane,
in (b) at
86
is
invention
step a (b)
are
step the temperature
to
(b) performed derivative
steps of provides
(c) yield
the is
is provides
triethylamine. of:
present the
between
a can
diglyme, with phosphoramidate at
method
temperatures
be a salt invention
Trigger-OH method
synthesized
and 20-100°C
to or In
synthesize a a
another
for
base C1-C6 as
below
shown synthesizing
to
higher alkylator in as
alkyl yield
a described
a solvent 0°C.
below:
than
an
In
/ 2011202075 05 May 2011 25 20 15 10 5
prodrug resistant provided EXAMPLE phosphoramidate present “ [0292] patient alkylator are [0291] present Illa. Nos. al., methods [0290] Appl. bioreductive N-alkyl-2-amino-5-hydroxymethylimidazole below. 1122; [0289] [0288] [0287] Hypoxia NHRCH
provided PCT
Methods 2004/254103
Pub. Denny
in
of invention invention
to
Cancer by 2 prodrug Appl.
In of
Examples The Synthesis The need Activated COOMe
the
these
No.
one section. the the
by
group
et present synthetic of 1
of
Pub.
-N-alkyl-2-aminoimidazole-5-carboxylates present present WO the embodiment,
al., therapy
alkylating Treatment
alkylator has employed therapy are
and
references of
of Z US
anti-Cancer Nos. 04/009667,
3 provided the invention
. bioreductive methods
2005/043244, invention invention methods Pat.
with
formula
WO thereof
prodrug agents.
in Nos.
alkylatin the
Borch 04/85421 treating
in
to or
Agents and
are
present by 5,750,782; can further selected is
synthesize one
Alkylating employed groups
selected
provided
administering Hypoxia
et be and
g that cancer
” al.,
agents
adapted and ;
detail invention
Borch deGroot
from
is and supra.
from
WO 5,780,585;
phosphoramidate 87
known.
derivatives in in activated according
agents phosphoramidate
in can (I)
treating further et from
04/85361; H+ the the
In
et — to al., provides lead
(XXVII).
al., one
“ the can compounds Known
the EXAMPLES
(supra).
produgs
detail
to 5,872,129;
to
cancer
2001, employed patient embodimenL
kill references
NH development can the
and a
phosphoramidate cancer 2 in
method
methods CN be alkylator Current
In
according
US
Lin the a alkylator
reduced
exemplified one phosphoramidate
and in
” Pat.
EXAMPLES et
Matteucci cells section
the of embodiment,
al.,
the
Med. 6,251,933;
provided
Appl. of prodrugs
treating
present
prodrugs,
to in to phosphoramidate US cancers
the yield the below. Chem.
in Pat. entitled.
et alkylators
more methods
the
cancer by invention al.,
of section
various
Davis Appl. that
the
alkylator 8: and the the
PCT
rapidly 1093-
are
in
et Pub.
1- a
as
2011202075 05 May 2011 30 25 20 15 10 5
treating one phosphoramidate other as provides compounds combination administering busulfan, melphalan, with present prodrugs [0295] alkylators drug [0294] alone supra). treatment one resistant other for pathway, Alkyltransferase, [0293] transported and dividing slower
a
embodiment, second cancer
embodiment,
can
the anti-cancer having
therapies. or
cancer invention growing
produce alkylators
a in
In cancer or In which thiotepa, The
and/or by method prodrug
resistance
line combination chlorambucil,
of one one higher
drugs substantially with
one
as
prodrugs
the by
therapy embodiment,
are embodiment, cells
hypoxic
a
agents. Slowly
the
alkylator
the cells
or provides other administering present
glutathione, the
first such
of cisplatin, is cyclophosphamide, oxygen
not
more
mismatch
present
treating administered to and present
resistant
line
anti-cancer cross-resistant of as
the
alkylators with dividing
In
cancer
strains dacarbazine, no invention alkylators
mechlorethamine the
prodrug phosphoramidate
containing therapy compounds one
carboplatin, invention nephrotoxicity.
a another invention the present the
glutathione
repair metastatic
to
embodiment,
as region.
and
present
cancer present
such
(for
with a
of agents.
the
alone
by
thir
are
proteins,
anticancer invention the cancer
with
example, provides ifosfamide, temozolomide,
compounds alkylators. administering provides of carboplatin. d
The killed cells
and invention
invention invention
fine or cancer
the transferases,
In known
and
in
alkylators 88 latter region oxaliplatin. In
the
in present
another therapy
and by combination
are
one a see, the melphalan, agent.
a by present
method the
method
Increased cells decreased glufosfamide,
alkylators. provides of
provides effective
is
hypoxic
administering as embodiment Hardman
prodrugs
the
invention administered the embodiment,
the carmustine,
compared prodrugs the
In survive
invention
compounds
present
of
compounds
one of nucleotide
are
with
a phosphoramidate cancer treating
in activity permeation
treating
method
In et embodime^
of
treating
postulated the alone
which
al.,
mechlorethamine, to another other the invention
the
as streptozocin,
provides
in treatment zone the the
pages
cancer phosphoramidate of
a present
of a
combination excision or of of
first
anti-cancer cancer are
cancers cancer present guanine-O
the
in
treating the act of embodiment,
to
not
1393 combination
alone fine present actively by
a as present a
be
invention. by
method resistant
alkylators cells
repair
cross-resistant
administering a
invention
resistant
responsible
therapy bendamustin,
alkylators
and source
cancer or 6
agents. with -
invention in
in 1433,
the of
to
of a the
by with to In
In
invention alone or in combination with other anti-cancer agents. In one embodiment, the present invention provides a method of treating cancer by administering after a prior treatment with surgery and/or radiation therapy the compounds of the present invention alone or in combination with other anti-cancer agents. In one embodiment, the present invention 5 provides a method of treating cancer, the cancer having relapsed after prior chemotherapy, sugery, radiation or any combination of them, by administering the compounds of the present invention alone or in combination with other anti-cancer agents.
[0296] In methods for treating cancer provided by the present invention, an effective amount of phosphoramidate alkylator prodrugs is administered to the subject. Generally, the 10 subject can be any human or non-human mammal. The preferred subject is a human subject. Other particular subjects include but are not limited to non-human primates, dogs, cats, farm ’ animals and horses. In one version, the phosphoramidate alkylator prodrug is administered alone. In one version the phosphoramidate alkylator prodrug is administered in combination with one or more additional anti-cancer agents. In one version the phosphoramidate alkylator 15 prodrug is administered in conjunction with a therapeutic cancer treatment, including but not limited to surgery and radiation. The phosphoramidate alkylator prodrug will typically be administered in a pharmaceutical composition. Various pharmaceutical compositions that can be used are described in the Formulations section infra.
[0297] The phosphoramidate alkylator prodrug and their pharmaceutical compositions can 20 be used to treat any type of cancer in a subject, particularly in a human subject Cancers that can be treated include but are not limited to leukemia, breast cancer, skin cancer, bone cancer, liver cancer, brain cancer, cancer of the larynx, gallbladder, pancreas, rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma of both ulcerating and papillary type, metastatic 25 skin carcinoma, osteosarcoma, Ewing's sarcoma, veticulum cell sarcoma, myeloma, giant cell tumor, small-cell lung tumor, gallstones, islet cell tumor, primary brain tumor, acute and chronic lymphocytic and granulocytic tumors, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuronms, intestinal ganglioneuromas, hyperplastic comeal nerve tumor, marfanoid habitus tumor, Wilm's tumor, seminoma, 30 leiomyomater tumor, cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion, mycosis fungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and other sarcoma, malignant
89 2011202075 05 May 2011 30 25 20 15 10 5
including been thiotepa, regression agents, cisplatin) [0300] (CAVP-1 (CAV); variety phosphoramidate can chemotherapy death. States, of [0299] combination 22 often illustrative Attorney and invention types entirety head multiforma, hypercalcemia, limited cancers [0298]
illustrative December
be
PCT alkylating
and involves of
used and most Current
of
etoposide to
herin containing
cisplatin, cancers Likewise, the
6). Lung treatment Pat. Docket The
neck combination are of lung
as purposes
as
Modest
of combinations
leukemias, ovarian therapies
discussed provided
cancers Pub. by 2005, phosphoramidate
with
a the
cancer cancer, protocols who agents,
cancer,
renal
single
that
reference No.
alkylator and and
simultaneous
No. several have significant or
die
and survival
below.
cancer. can
cell
021305-005900PC;
containing
cisplatin without affects
carboplatin.
as including ovarian chemotherapy agent
WO in especially
within
further lymphomas,
PCT
been
for be provided tumor, the
consisting
different prodrug, Tidmarsh
2005/076888. treated
Those the
or
benefits
The more
following Pat.
reported
radiotherapy areas
cancer, 1
(VP-16); below, in
alkylator or treatment
year polycythermia cyclophosphamide, hypoxic
non-small most Appl.
combination
by
successive than of
with
Current cytotoxic
of examples
malignant
et of
of regimens from
the
skill
pancreatic
a
hypoxic for
active al., references,
cyclophosphamide, diagnosis, entitled 100,000 phosphoramidate the
and
prodrug
methods
regions and
of
non-small
in Several combination
PCT or
combination phosphoramidate
cell
90
cyclophosphamide, lung
the
drugs drugs US administration surgery. of
with have melanomas,
tissue.
vera,
“ Pat. lung
males
combination
art cancer, Glufosfamide
Pat. amenable can
cancer
described making of each
have in
existing will
been
ifosfamide, Appl. cell
adenocarcinoma, cancer, particularly these
App.
the Such and A
chemotherapy
of
therapies appreciate and involve produced lung
phosphoramidate reported alkylator treatment
it
which No.
50,000 cancers No. to doxorubicin and
cancers herein. combination the of breast prostate
therapies alkylator treatment cancer.
PCT/US2005/0473
combination a 60/760,599
epidermoid leading melphalan,
doxorubicin
the variety is be
for
females for
at
prodrug are that cancer,
incorporated
of include used Thus, integration
least cancer.
ovarian
small
glioblastoma (etoposide
ovarian discussed prodrugs
are
cancer with
cause and of
in therapies.
temporary in
in
also
colon anti-cancer
and alkylator
carcinomas. can
the chlorambucil, but cell therapy vincristine
a and the the Examples
cancer
of chemotherapy cancer
described. 60/719,787 treatment
be
of are
of lung
in
description cancer for
United cancer, VP-16 plus
14
used
the its not ”
A
prodrug
, include filed
cancer,
have
in of
of
on
2011202075 05 May 2011 30 25 20 15 10 5
prodrug limited body-directed (VDEPT), be meeting [0303] colon combination benefit, likelihood alone Agents chemotherapy United agent(s) invention [0302] treating and prednimustine, cancer, have has and replace described methods [0301] six cisplatin
used
to been 5-fluorouracil, is
been or cancer
eight
the
to
States in for
therapy including
5-fluorouracil the an prostate in currently In
Cancer the or reported for
Cancer
is various “
of second
herein reported the combination agent
gene-directed one
unmet cycles. carboplatin used
with using
treating reducing foregoing
and enzyme
in treatment
” version and
cancer
(BDEPT). of patients or
of is
in the
most treatment known is
used.
to need
a
melphalan,
the used
for cisplatin. in the such The
likewise prodrug,
consist ovarian compounds
prodrug measurable is addition
large use
treatment common prostate of in for in with
of the compounds
as combinations, with approaches enzyme
the which combination
in prostate
better a
with
most The primarily
bowel
other
single cancer a
can late
treatment
Combination
advanced therapy cancer and to
is
cause
estramustine general
a
methods.
and the prodrug offer treatment effective
stage tumor the
phosphoramidate drugs, hydroxyurea.
is agent cancer
in
and
to agent(s)
methods the most characterized ”
of
which of
significant
with
disease
cancer (ADEPT), either methods,
colorectal methods uses masses hypoxic
cancer but second or
therapy
include
treatment
common chemotherapy
methods in cyclophosphamide 91
is a
of currently phosphate to
described
existing therapy
phosphoramidate following associated
a death by replace most
The tissue.
a
described ” phosphoramidate
therapeutic “ the
cancer
phosphoramidate (GDEPT), 50 virus-directed by
alkylator malignancy for for
present
alkylators common in percent
hypoxic including used. such
this
an plus this herein, men
Several
relapse
has is with
agent herein
combination disease. also
disease.
prednimustine
invention
proven prodrug benefit above
and or
only regions. at
cause
and
estramustine
in
alkylator but used chemotherapy after more. or
3-
enzyme “ provide
men
bacteria-directed
alkylator
in
age the a to
not
alkylator to and
5-Fluorouracil of
to hormonal of
15 addition
4-week
provides
be
methods
in Using 55, cancer
limited
treat While
the to therapy,
potential
prodrug
prodrug the prodrug of
20
and and present
prodrug
only prostate phosphate,
United
prodrug
percent 5-FU intervals
to death protocols
cisplatin, to this treatment.
methods
for
the either
marginal
as “ for therapy forms
anti
cancer treating is in
enzyme
in
States are
useful can
the for to
not and for
.
/ 2011202075 05 May 2011 30 25 20 15 10 5
ankyiosis a on multisystem are multiple another psoriasis, elevated, by In administered vasculitis disorders, diabetes, [0305] duodenum, periodontal adhesions, syndrome, neoplastic disease, hemolytic bronchitis hypersensitivity demyelinating tube hyperproliferative atrophic consisting abnormally cancer [0304] compound
one a the administered
prosthesis
diseases,
cellular embodiment,
hyperproliferative embodiment,
sclerosis, multiple
gastritis, of scaly In
ulcerative multiple n of
vasculitis
anemia, vasculitis, and
primary of
stenosis
another joints
some small disease, increased of
chronic, the to
allergic
hyperproliferation giant
implanted lesions. the neuropathies, chronic
treat
central to vasculitis
sclerosis,
bowel,
embodiments
benign affected.
a
present sclerosis, disease
prevent
inflammatory
or sclerosing aspect, colitis, disease cell
the associated polycystic
relapsing, a
the pancreatitis,
angiitis
restenosis, rate
hyperproliferative
obstructive
nervous In hyperpriliferative arteritis,
hyperproliferative
and in
prostatic
diseases
invention.
another treated or
(Henoch-Schonlein ulcerative the myocarditis, a
In
characterized a rheumatoid
hyperproliferative dermatomyositis,
colon), amount
subject and
another
cholangitis,
present
with kidney inflammatory
of system,
graft of neutropenia, scleritis,
granulomatosis
according pemphigus
embodiment, the hyperplasia, airway characterized
keratinocytes
congenital
silicosis
by proctitis, of
In
invention, embodiment, rejection, invention disease,
cellular myositis, and another arthritis, coating
disease
by
disease disease, scleroderma, rheumatoid
diseases
to Wegener's progressive
92
disease and
vulgaris,
inflammatory
purpura),
eczema vasculitis the polyarteritis
deficiencies bullous
disease
proliferation).
the the hypersensitivity provides
embodiment, by
a selected
restenosis, (Churg-Strauss other treated
which nasal
chronic compound present
cellular
hyperproliferative the prosthesis treated
that
arthritis,
primary including
pemphigoid, resulting compounds granulomatosis. forms polyps, strictures
irritant builds
demyelination associated
is from a can
method sinusitis,
method psoriasis, of nodosa, is hyperproliferation
bowel and
of lead
of rheumatoid
the the
with
the
up In glomerulonephritis, nasolacrimal serum dermatitis,
the
pneumoconiosis, benign
pneumonitis, from atopic
disease), of
hyperproliferative
one
complement
on is to group
disease,
of of with
a polyangitis present Crohn's bile
coeliac
selected
destruction composition the a
sickness, embodiment, treatment cellular the disease
disease
dermatitis, in prostatic
ducts,
connective skin consisting
present arthritis,
the asbestosis,
inflammatory
Kawasaki's
invention disease,
disease,
duct from
to brain. proliferation treated
(e.g.,
strictures
characterized system,
surgical overlap
of
form
and
hyperplasia.
diseases, invention type
containing non eustachean the
a
psoriasis,
the
of an tissue chronic disease
In
is asthma,
is
group
I
(of
2011202075 05 May 2011 30 25 20 15 10 5
physician. (e.g., agent the product amount among administration, [0308] type prodrug and will gut. intramuscular, administration oral must formulations, only hypoxic modes a can using pharmaceutical Illb. [0307] [0306] treated abnormally
phosphoramidate
cancer,
corresponding be
recognize routes, of
molecular If
ready-for-injection released Fonnulations, be
a
other
such cancer effected of
of comprising is
phosphoramidate prior
rehydrated region The
Administration A
administration, benign the localization
intraduodenal
phosphoramidate
activation and factors.
amount to phosphoramidate a by
being
that or intravascular
the
weighfl phosphoramidate by of
formulations its thereby
a prostatic a
a
formulation phosphoramidate severity
any
a
entry formulation that
alkylator or modes
tumor.
treated, phosphoramidate
of
diluted,
is
of method
solubility dose,
a
formulations block
into not
of
the formulations, phosphoramidate alkylator routes,
hyperplasia,
of of
Many a or
the
desired,
prodrug
cancer, for the phosphoramidate will administration, alkylator
the
respectively,
urine infusion),
of that alkylator that phosphoramidate
administration
large
parenteral alkylator and
cancer, the
be cancer
prodrug
enables results
alkylator flow. the dependent
then
phosphoramidate can hypoxic but
alkylator
intestine
prodrug a and
rate
prodrug disease
topical, be drugs and
also the
prodrug
in
delivery described alkylator prior dosages injection
formulated
93 of dosages
absorption the practitioner
prodrug, lyophilized and
alkylator
will are or administration, on prodrug to
in
and to
age, contained
alkylator
a colon. normoxic
the
administered which can
injection. typically subject that of
prodrug
(including rectal herein. weight,
subject
the and alkylator be
for
may
prodrug can of
The
can prostate or
formulated prodrugs
prodrug the a routes. such
in
be
concentrated cytotoxicity),
Described
phosphoramidate be be
administered,
employ and being actual In the
discretion the used
intravenous,
activated formulated
by prodrug
administration, addition for
dose condition epithelial
disposition
Those
intravenous selected
to
treated, the when route
for
a the
administered route in
treatment
will
administration
of
to
by
formulations of
this site of treating the
and
as
the of cells these
the for
bacteria skill subcutaneous,
administration of depend
of section cytotoxic of
alkylator the
prescribing thus injection, severity including
action,
the grow in
of
patient, cancers
the
the
and prodrug
cancer in on
are
that
the art
the by the of the
and not
2011202075 05 May 2011 30 25 20 15 10 5
of over phosphoramidate range [0312] embodiment, administered dose [0311] administered treatement (combination) daily, 0.5 about invention or about invention single to 35 treatement throughout side central kg [0310] adequate, human, some a [0309]
a
patient
divided about
mg/kg/day, mg/kg/day body phosphoramidate
two
in effect;
of
0.25
either 2 instances,
or
In the
catheters
to about this In
weekly
10 weight, divided
wherein In
one In for for
while
about
of doses. range to another
of
the mg/kg/day; larger
one
as
one would
on a
once
treatement cancer about treatement
embodiment, cancer
5 with
dose in a about
day
once
to cycles. embodiment,
in dosage monotherapy
embodiment, 8 of alkylator (PICC or single doses.
In an doses a mg/kg/day;
about
other embodiment,
standard
amount 2.5 by about in about
week.
and
one and effective alkylator 0.5 every
the
infusion
mg/kg/day;
or about
line)
levels
of can of other In cases 30 embodiment, to other
3 range 0.1
prodrug divided
cancer three cancer one a to about to
mg/kg; of also
larger and
to
the 0.5 about
about the hyperproliferative (compound below dosage
about prodrug still
hyperproliferative
care
for embodiment, of
about
for days
be
portable effective to 20
present
and
and
doses.
about is larger an
about
dose
about therapies.
divided
about 0.05 20
treatment 2
mg/kg/day; the
administered
is hour
for 35 other to other the
is mg/kg;
typically
is
lower
0.5 about
to not 0.25 doses mg/kg/day two
invention In 10 intravenous of effective
8
dose
administered or
about
hyperproliferative mg/kg/day, into hyperproliferative
94
one to
to the
greater the
continuously weeks. of to
In
limit 4 about about about
can
of
present several about
embodimenζ
mg/kg/day; effective about human diseases
one diseases in 7
once a
be dose provides g/day,
the
of compound in
than
6 8 embodiment,
15
bag In
employed
0.5 the single to mg/kg/day 1
range
a and
small patients, invention intermittently mg/kg; of another mg/kg/day
week 500 is about
is dose about and to
aforesaid
using a
in
about in
a and
compound
about
or of
mg/kg
doses
the method pump. the diseases diseases
the
of for of
0.2 10
or
divided about
without embodiment, about a the
a
dose range the
alone)
is 1 range
peripherally mg/kg;
four the 12.5
15
compound
to to
range for
administered and patient maximum
present
mg/kg/day;
0.001 of
(less about
about effective 2
is is is
administration weeks. mg/kg
of
as doses.
of
about mg/kg/day causing
or cancer administered in in
can
or the described
about
frequently); in the the
weight 5 to
2.5
8 invention
be
conjunction present a
mg/kg/day of of
mg/kg 0.25
about
inserted
For daily range
In range dose dose treatment g/day.
any
more
the 0.1 the
about for one
a and, to in
present earlier to for harmful O.lg dose in
70
5 of is of
about single
i.v.
than
for days
about In the a
0.5
kg
in
per in
2011202075 05 May 20 20 15 10 5
’ HED standard animal
the humans animal prodrug prodrug prodrug [03131 another In prodrug In mg/kg phosphoramidate embodiment, of of accordingly,
another another a
about Baboon Marmoset Squirrel Monkeys Rabbit Dog Mini-pig Micro-pig Ferret Mouse Guinea Rat Hamster Animal table
phosphoramidate =
b dose a animal to can
To
embodiment, ranges, and is is is is For below:
1 Guidance about
about pig about embodiment, embodiment, mg about about
be convert monkey
by 13
other example,
a
the
converted dose of phosphoramidate HED
500 human
25 25 1.25
10
a therapeutically
alkylator mammalian
phosphoramidate
animal concerning in mg/kg
mg/kg mg/kg mg/kg convertion
the mg/kg
mg/kg alkylator cynomolgus,
equivalent
the the
to therapeutically
dose
to to to of the prodrug
therapeutically therapeutically
to
x about about about the
animals.
corresponding administration (animal
about
prodrug/kg factor. in
alkylator effective | body
dose mg/kgto
50 250 is rhesus, 150
Human alkylator
12.5 administered
mg/kg
weight
weight
A
For (HED) mg/kg mg/kg
effective
therapeutically prodrug dose mg/kg
of effective HED effective species
or Equivalent 95
human of
patient can
prodrug/kg
of
in
stumptail. can of of is
body
the kg/human (assumes of
a the the
dose also is
be daily not in
body
patient dose dose administered
equivalent body body weight.
weight
calculated a
be
of
listed Dose daily
dose
effective
provided weight a of of of
a weight weight
phosphoramidate 12.3 weight 5.3 6.2 3.1 3.1 4.6 1.8 5.3 6.2 7.4 to 1.8 1.4 1.1
patient a a 60
dose or of (HED)
of In phosphoramidate phosphoramidate be
kg
for the dose
a one from
of
treated.
in of of phosphoramidate
in
dose
human) in by
weights the patient weight
a embodiment, conversion the the the
(HED) kg)
the and daily
patient determined
patient patient range
0J3
formula: In from
in to to
outside dose
.
as
another
alkylator mg/kg, be about
to
of
described
studies alkylator to alkylator to factor treated.
in be about
be be a
500
for the
the alkylator treated.
divide treated. treated.
8
an range in
5
mg
In
in
2011202075 05 May 2011 30 25 20 1 10 5 5
continued prodrug [0316] alternatively, at weeks within prodrug six weeks [0315] within six prodrug time. in dose), administration used. administration will administrations. administered smaller dose administration). administration phosphoramidate related days, time. one [0314]
least
any
consecutive consecutive
be embodiment,
can
or For within Administration within every a Typically, a event,
one
apparent
embodiments,
is is doses
six-month is six-month In at Administration
be To
for
administered example, administered administered
month least one
administered other
multiple achieve at a a a twice through
once phosphoramidate
twelve-month is selected twelve-month least weeks, embodiment, weeks,
10
to For not daily
alkylator or a day,
period, period,
one
consecutive daily, whether phosphoramidate
every
one,
at limited therapeutic example,
once days,
administration
the
or, administration
or, or
least
of at by
at at schedules
two,
less or once
least alternatively, least skill third course alternatively, least or, or,
the
every prodrug typically
two, divided administration to
the
period. period.
alternatively,
alternatively, frequently,
three, practitioner
the in daily,
one 3 10 2 day, days.
alkylator
administration effectiveness,
days of three, days other the
days dose other
administration
is
a
or into
four,
for is art or
In alkylator day In for Depending usually per per
once
day
per for four, can
for the for
than
one of one at
three
at
in
(including
five prodrug week week
and
at least
cancer
month, for at is (qod) at
daily for be least
one
a embodiment
96 embodiment,
five
consecutive
least
daily least least administered
week
or
smaller divided the prodrug the at at
of
for three for
embodiment
3
dose at is least or least on
treatment
per
convenience the 5 therapeutically is
consecutive optionally two,
two,
(including,
particularly least at
at can
at consecutive by the repeatedly
consecutive
least therapeutically
day least can doses two, least two, into
is
infusion three, be three,
dose, six
daily a administered
but be a
appropiate two, phosphoramidate two multiple two,
three,
three, six
phosphoramidate that, months and
administered a
at
four, can formulation, four,
as
days phosphoramidate administration consecutive
of
smaller
convenienL administered
least
three,
three,
with days,
administered
as
noted,
four,
four, effective the include days,
five five
used will times in
20
effective a patient,
in
four,
at four,
a
daily five pump five
doses
or or and a
days
be 6-month some least herein,
divided multiple to
in at at
and
daily
months, employed. or
or five and five
often
for the
alkylator
least least
over multiple per
or and the alkylator
Ί can thrice
at at instances,
dose
route
consecutive
or intravenous a
patient. or “ least
dose least alkylator
month,
entire
daily period.
period
daily for also six a six
at at
period is or, daily.
of
least least at
weeks weeks of six six
” be
In
daily least
a but
of for In
of
It
2011202075 05 May 20 30 25 20 15 10 5
concentration administration the one consecutive phosphoramidate [0319] a the wherein patient of prodrug phosphoramidate quided multiple-round period [0318] twice, more two period some administrations, and can phosphoramidate administration. be for five [0317]
phosphoramidate
patient
present
cancer administered month,
several be
such to to
times,
embodiments,
or
ranging ten weight) ranging by a ten
the in dminis In
In
three three Consistent weight,
in this
administrations, invention,
the consecutive
at
optimizing
days, days,
one administration
sometimes
the
curve least
can
ranges
disclosure.
or
tered to from from for schedules
aspect, patient For
and a
a alkylator
alkylator more ten
10 typically alkylator be phosphoramidate
a
10 alkylator week, (AUC)
specified example, one a once with consecutive one
such
selected or
the stated
days phosphoramidate
a
times days, “
with
five phosphoramidate
administering to to dose
Illustrative
a administration for two, daily
prodrug
of prodrug
over per 25 more month,
prodrug several several
(e.g.,
prodrug
to
a or
with administration
a
to
minimum in to
and
no-treatment month, three
ten phosphoramidate
for
for the
achieve
some often
150 days, 1
a
frequency
day two ” weeks
at include weeks mg a
course
can no-treatment alkylator
refers therapeutically or for
week, mg least
and
embodiments, three
at a treatments months, to five
be
alkylator
number 3 regimens a therapeutically of
least
between
between 1
alkylator maximal
consecutive
three at to
of administered
those
or a to
(with g to will
97
of least phosphoramidate
prodrug (i) treatment
of
for ten 3 ten
three
a
to days a administering
of be
described alkylator
prodrug phosphoramidate (with a
several
can administrations phosphoramidate
of 20 administrations,
each ten, each
phosphoramidate days
prodrug sustained apparent effective
other a months,
per
days be in
days, phosphoramidate or a
multiple-day multiple-day effective
in
within accordance
The repeated
phosphoramidate
week, weeks
at is
prodrug per multiple anticancer herein,
least treatment administered
5
to
theoretically dose area six
month. consecutive
a
alkylator
the a
at or phosphoramidate
specified months,
dose five
qod
once,
least
under regimens
such more. or has skilled
alkylator
alkylator “ with
rounds alkylator
treatment.
treatment. agents,
five
to
can regimen
or
a
3
twice, as
ten prodrug
alkylator the therapeutic
regimen
days the every
Thus,
or
to be
practicioner optimal administration alkylator days, time
”
consecutive
plasma for
a
ten prodrug
present prodrug repeated a of
year per
prodrug) or according
other a period,
a 7 Other
Similarly, per
three
patient
prodrug of week alkylator
dosing or
prodrug)
effect
methods a kg
day per
longer.
once,
or
of for days,
can
to kg of for
on in
2011202075 05 May 2011 30 25 20 15 10 5
prodrugs will prodrug remain [0322] present 30 remain another 20-80% prodrugs embodiment, (update prodrug method [0321] alkylator require between In compounds invention alkylator invention, of observed, with prodrug, determine regimen [0320] for
minutes.
one the
be any
a
present
lower. greater embodiment, phosphoramidate greater
invention
an embodiment, with of of single unchanged
can
In of
will 10 In that prodrug as prodrug. In
can
the
or infusion
administration, and efficacy.
the a
another to measured of one In increase
human related
for the remain
result
invention, have than invention than
30 administration. the present another then
embodiment, the
present
which
minutes. having
present
In 80% time
80% when a
in
embodiment, resumed. example embodiment, convenience
the
As
up the in the
one by
a
invention
embodiment,
vivo
when maximal
is
to compounds
employed in unchanged unchanged
its invention maximum alkylator present a AUC,
incubated
embodiment, and invention understood
20% longer treatment
A therapeutically
half
and
short
incubated the the A
while unchanged
upon invention
life
higher of exposure data the pharmacokinetics
half the kind
prodrug
provides for
half tolerated
with include when the
of can
examples of that present
bioreductive
minimizing
in the if life. reduction/activation
the of
between
patient,
the life
C with have
available) the
mouse is
cancer ma treatment incubated
present
can
provides
of effective when
phosphoramidate A longer
x of phosphoramidate 1,25,
art
98
dose invention
will mouse
the a short
the be of
in for without liver
1
that
incubated tumor
phosphoramidate
suspended contribute
vivo the
and (MTD) phosphoramidate
to invention
than protein group other
half
of of
phosphoramidate with
liver dose
300 microsomal
is maximal
36.
the half cancer
provides
that treated
cells departing life
cancer
of minutes.
mouse
microsomal
and for
phosphoramidate
The for
in
life
with
required the
of
to can temporarily
to
alkylator that
a can alkylator undesirable 30
the
with plasma toxicity
of higher hypoxic
phosphoramidate a
therapeutic
phosphoramidate have liver mouse
minutes. phosphoramidate
protein
prodrug. between determine
from phosphoramidate
In alkylator
the alkylator
for
one
microsomal alkylator a protein
the prodrug concentration
prodrugs while
the
phosphoramidate in
liver tumor a
if for
MLM phosphoramidate embodiment,
patient vivo
3
In toxicity scope
drugs,
alkylator
the
to prodrugs
30
microsomal
prodrug the another for
zone
half of prodrugs 10
minutes. dose,
stability
that
AUC
of 30
alkylator
side
the alkylator protein treatment minutes.
alkylator
is
life
form the
alkylator minutes
(C
remain
prodrug present
the can
effects of
will
ma of the
that
the of x)
a In for
a
2011202075 05 May 2011 30 25 20 15 10 5
a formulations magnesium binding various tablets ingredients organic symptoms administration conventional dosage [0325] as or formulation, form phosphoramidate [0324] defined indirectly phosphoramidate be prior longer plasma were phosphoramidate
[0323] clearance Various diffuse similar
a emulsion;
temporarily
suppository.
suitable measured treatment.
containing
contains forms disintegrants,
agents solvents.
and stability, not
type pharmacokinetic Suitable A In
by and (CL),
such stearate,
formulation
only
reach
re-treatment
of
solution, for pharmaceutical
can cessation suitable for
such normal (drug
a
stopped
and significant
as topical
area
phosphoramidate by In
oral be
and The A various pharmaceutical
alkylators alkylator other alkylator-
flavorings,
as any
specific formulation listed sodium holiday) employed
under such
Cmax
administration pharmaceutical for and organ sucrose,
of
administration as event, parts of
regimens, single excipients,
symptoms).
parameters in suspension;
severe as
a of
concentrations released curve
released lauryl
phosphoramidate days T clearance
the carrier
starch, phosphoramidate of can
m
binders,
as in gelatin
administration the conjugate. of EXAMPLES
toxicity soft be symptoms carriers but (AUC),
sulfate, alkylator
a
the
tumor
or therefrom
alginic ended phosphoramidate as
therefrom.
such
individualized such
and compositions
for of and Therefore, excipient. as excipients, dose a
tablet, a is
an
include
mouse
hard parenteral
as of or
and acacia. as phosphoramidate at
The observed
acid,
prodrug
are can
ointment 99
citric
a other
the volume
section
alkylator
(which phosphoramidate talc filled of alkylator
observed. capsule, phosphoramidate be
liver
time and
inert
precise an
and acid tumors
Additionally, can
adjusted
can,
by
injection intermittent described
gelatin
during certain
of (see or microsomal
when can
the alkylator
diluents be
can drug prodrug
distribution prodrugs if pill cream;
dosages
used be like. in
also desired, The
be
the
to
capsules. repeat powder, holidays
alkylator complex the measured
as
employed
reflect herein. Hardman to period or
Thus first
prodrug
and can, alkylator case lubricating a dosing
prepare
alkylator- fillers, and of sterile
stability
administration, contain
for under organ
the
for
of sustained
for patient that
silicates, prodrug of will
Preferred Solid or
rectal
a
period present
example,
can
together temporary et oral water
solution,
metastatic prodrug the determined are
steady of typically
(MLM additional al.,
T
agents
be
compositions
tolerance
toxicity administration, m tablet based administration
or supra). release can and and
in conjugate
invention materials,
a state
with
be unit or stability), suspension
dosing
such
be
various with halting forms
on include disease.
a in no
(Vss), of
a
as
the
can can of
of of
a
2011202075 05 May 2011 3 25 20 15
10 0 5
[0329] prodrug Hie. alkylator other generated and in the drug-factory to prodrug leading containing the the Company, disclosure. polyethylene [0328] of phosphoramidate suitably When [0327] desired, ethanol, can therefore, [0326]
the normal
active
tumor
hypoxic present
start Combination be
anticancer
normoxic
aqueous
combined to can
In
can buffered, to propylene emulsifying prodrug,
The drug Methods
Exemplary
from can tissues.
a
include
Philadelphia, accordance increasing
divide. invention
be
higher For region
diffuse to
methods glycols, reduce
are
co-administered
a produce
suspensions region agents examples,
phosphoramidate
therapies The with
as alkylator
known, if
lactose
of
concentration of At glycol, from
desired.
described
parenteral toxic
agents preparing
are
populations a propylene
use
this
with of various or of
tumor.
within
Pa.,
effective
the cancer the cytoxins or
or of point, side-effects see
glycerin,
prodrug
the or or
milk
will the hypoxic 17 tumor
Remington
sweetening in suspending elixirs
a methods in Once th various administration
prodrug treatment tumor glycol
such of be the
Edition
combination sugar of
in alkylator administered
the in are or apparent,
rapidly killing
following released
cells are
cells sterile
arising combinations phosphoramidate an
pharmaceutical or destroyed, and
of
to desired (1984).
’
dextrose
alkylator s or agents, employing
and
the
can
generate
Pharmaceutical high the prodrug 100 dividing
aqueous flavoring
in
to due
forms invention,
with kill
section. be most
in
the those molecular for
together
killed a to
combination the solutions.
for
other hypoxic hypoxic
of oral the
thereof. normal include difficult
cells. solutions,
a
skilled
cancer agents,
killing this compositions phosphoramidate
phosphoramidate
by
administration, alkylator a anti-cancer
with
phosphoramidate invention weight The the Sciences,
cell
region region solutions
in
cells to Such adjacent coloring
phosphoramidate
with diluents hypoxic for
kill this toxicity.
within polyethylene in
dosage example,
can a
cancer
art the
with phosphoramidate agents adjacent or Mack
or
normoxic
matters in become
those such phosphoramidate the
region
suspensions alkylator
the
alkylator a view After
forms
cells
specific Publishing prodrug
(
alkylator
“ tumor, aqueous
as
known, anticancer regions
or
of acts
normoxic cancer water, glycols. growing
alkylators cancer
can dyes
this prodrug
within
amount as relative therein
be
of
or
a and, cell
a
cells by
in
of
if
2011202075 05 May 2011 30 25 20 15 10 5
phosphoramidate and continued to example administered other phosphoramidate [0332] administration with administration alkylator another embodiment, region, [0331] the the of agent palliation sensitivity survival diminishment prodrug [0330] agent Co-administration cancer administration agent
administration
cells
treatment regions known
a cancer
”
to alone,
phosphoramidate
).
.
therapies, the
anticancer otherwise
combination In
be or and
prodrug As The Without after or
of
phosphoramidate anti-cancer one of other adminstered
therapy.
used
contemporaneously stabilization e.g., the with
a cancer
co-administration
tumors
of of
of the
phosphoramidate can embodiment,
of anticancer alkylator
beneficial extent alkylator the the
herein, such
and greater
resistent a intending
cessation agent
provides
in the
phosphoramidate
cells Agent Agent, In
therapies that Agent some
agents as,
Agent,
other of
alkylator to
(also
a alleviation
of
to are for prodrug
disease, prodrug phosphoramidate the
to
agent (i.e.,
therapeutic
cases
a of
(i.e., to the the
are alkylator
a
embodiments, the not in example better
referred
patient and
be phosphoramidate administration as
disease of
anticancer general administered
the
alkylator
with
anticancer the has prodrug efficiently provide described bound
treatment a is delay is
therapeutic course phosphoramidate or
initiation first
a administered
alkylator or the
prodrugs
to co-administration amelioration synergistic results.
state,
targets
allowing by or herein
initiation administered
one is prodrug
agent,
of
slowing
any
in
agent 101 a
killed continued with alkylator
partial phosphoramidate as of
or the EXAMPLE of
prodrug
the
result of particular as,
part the more alkylator
the
allowing a other an
or
effect the by
is
after rapidly of phosphoramidate “
of of Agent.
Agent
or otherwise anticancer of other first
the
than the prodrug invention alkylator of
disease throughout one
prior complete
therapy). the
is
the
on of
several
anticancer other administered mechanism prodrug continued
” dividing cancer section. lower administration or
same a ) initiation induction
In
to when phosphoramidate
more
progression, is one refractory
administration cancer prodrug alkylator
agent
target
advantages “ remission, course In doses
therapy), co-administered
the
is
embodiment, a
symptoms cells another
agent
during
phosphoramidate
first
alkylator
or
to of
or course the therapy.
prior of
increases of completion be
cancer
effect, in
prodrug
to
administered
of the hypoxic alone.
therapy. used
the
and amelioration, part prolonged embodiment, treatment.
over the
to
of anticancer
of of
prodrug
alkylator normoxic
treatment such cell
See of anticancer for
a
the the
known is the ”
cells the
with death. first treatment of
for
In Agent, cancer, co
the
one prior is While
in
a
2011202075 05 May 2011 25 20 15 10 5
an therapies. radiotherapy, alkylator is [0335] days a administered viewed alkylator administration [0334] receptor receptor superoxide. with present agent tissue superoxide with period completed inhibitors, (0333]
phosphoramidate
co-administered effective
during
a oxygen
is
from
chemoprotective of
as
invention ” ”
a
prodrug, In Anticancer prodrug,
In
from generated administration thiol administration
a prior
amount one
in each the the
phosphoramidate In in an to
noimoxic
of
reacting or
toxic another version any yield presence appropriate
to cycle.
the
provides a a each with
alkylator
the of phosphoramidate
or disulfide.
from drug
anti-cancer effects superoxide
one
all
administration
cycle the of
embodiment,
with In agent
tissues
of
of of of therapy a
the or
one Agent a phosphoramidate NHOH(+)
the
a oxygen,
surgery the of
prodrug
phosphoramidate proteins
more of
second method
or In
alkylator
aspect anticancer Agent.
can multiple administration
agent(s). a one and
today according
chemoprotectant chemotherapeutic
lead
procedure,
the
is drug. Z embodiment,
the
alkylator and of
of of 3
For administered
.
prodrug typically radical
to the
the Superoxide treating the cycles
nucleic drugs.
In
unwanted certain A
second invention, 102 to
chemoprotectant
alkylator the
phosphoramidate
alkylator
prodrug
a (as
or anion of
schedule administration
cancer context In involves
acid
any
treatment well drugs, the
drug. one
is
on Chemoprotective
agents, side formed
combination (see prodrug chemoprotectant
a a
is as
a prodrug
using
embodiment,
phosphoramidate cytotoxin
of
such
daily administered repeated effects. a multiple
below).
administering complete
with
an
upon
can
the
as
and
basis can alkylator effective
administered
certain the react a
In
and
the at rounds, prevent of be phosphoramidate
for one
other each set the
such
initiated reduction agents
the
with in can
at
topoisomerase
of prodrug amount embodiment,
chemoprotective
combination a
production
round. alkylator
least Agent;
“ or additional
reduce phosphoramidate
cycles) the Michael-
protect
in “
cycles, and
“
two combination of
Michael-
of
can
in
Z can
prodrug or
3 general, healthy
be of ” reacts
the with more
be of
2011202075 05 May 20 30 25 20 15 10 5
mitoxantrone, 2-ethylhydrazide, interferon-alpha, elfomithine, amsacrine, asparaginase, cytarabine, 6-mercaptopurine, tubercidin, olivomycin, melphalan, daunorubicin, mitobronitol, azaserine, carmustine, estramustine, triethylenethiophosphoramide, phosphoramidate altretamine, analogs improsulfan, section. administration. with phosphoramidate administered administering be of [0338] [0337] agent, [0336]
the administered
some
additional
the
thereof The Chemotherapeutic
When
two
bestrabucil,
ubenimex, dideoxyuridine, time
novembichin,
chlorozotocin, peplomycin, elliptinium imatinib,
piposulfan, pulmozyme,
mitolactol, via ifosfamide, mopidamol, Agents
daunomycin,
the agents
(refrence
between a See interferon-beta,
at
therapies, procarbazine, the
alkylator phosphoramidate alkylator agents thiamiprine,
the
for same
can triethylenemelamine, can zinostatin, cactinomycin,
bisantrene, same
acetate,
example
benzodepa,
plicamycin, simultaneously administrations.
apps), gefitinib, be phenesterine,
be
nitracrine, aceglatone, or
prodrug prodrug a doxifluridine, 6-diazo-5-oxo-l-norleucine,
agents
time administered
administered phosphoramidate different
trimethylolomelamine, thioguanine, razoxane,
glufosfamide,
interferon-gamma,
combination or
carboplatin,
mechlorethamine,
that
of alkylator
is
can
carubicin,
porfiromycin,
aclacinomycins,
administered aldophosphamide routes. the
prednimustine, can
be and
sizofiran,
enocitabine,
invention as One simultaneously flutamide,
administered denopterin, be triethylenephosphoramide,
ancitabine,
103
the sequentially prodrug
therapies
gemcitibine, used defofamide, alkylator of carzinophilin,
same
phenamet,
skill 2-deoxy-D-glucose,
with spirogermanium, in interleukin-2, puromycin, include,
gallium is
mechlorethamine
or
combination chlorambucil, floxuridine, in actinomycin trofosfamide,
pteropterin, azacitidine,
as used
prodrug mycophenolic
glycoside,
separately. an different
the
and
or described
demecolcine, erlotinib,
additional
but can
art in
nitrate, chromomycin, possible
combination
dacaibazine, streptonigrin, will
are be and
lentinan, formulations
5-fluorouracil,
aminolevulinic
with
trimetrexate, F(l), 6-azauridine, administered
not
understand in
For chlomaphazine, uracil hydroxyurea, additional meturedepa, paclitaxel,
lonidamine chemotherapeutic
time
the acid,
oxide
limited
the podophyllinic
example,
diaziquone, anthramycin,
EXAMPLE
mitoguazone, mustard, periods
a
nogalamycin, with dactinomycin, mannomustine,
hydrochloride, streptozocin,
and
therapy to,
methods tamoxifen,
sequentially
caimofur, one
tegafur, uredepa, and
if
busulfan,
acid, between can
a
or
acid,
be can
more
of
L-
) CM Ο 2011202075 05 May 30 25 20 15 10 5
tetraazabicyclo-[8, methoxyestradiol pleurotin PD98059 (SNP) KF58333; AAG), shock (lS,4S,10S,21R)-7-[(Z)-ethylidene]-4,21-diisopropyl-2-oxa-I2,I3-dithia-5,8,20,23- kinase suitable whose phosphoramidate alpha anti-cancer factor prodrug [0341] an carboplatin. antiangeogenisis with treated similar [0340] alkylator combination oxoplatin. used vinblastine, erlotonib, (0339]
anti-cancer
an
include
(HIF1 protein and
or inhibitors; expression and
with
therapeutics. for antiangeogenisis
with
PX-478; EGFR
In In (2'-amino-3-inethoxyflavone); prodrug. In
genistein; other teniposide,
other
use agent
a) another another one cyclophosphamide, an
In with platinum 90 another
or agent
in antiangeogenisis
one receptor. NO
version,
(Hsp90)
geldanamycin
to
or inhibitor, 7,
that LY294002; alkylator or this quinoxaline an
6]-tricos-16-ene-3, In
inhibit
version, 2-ME2), version embodiment,
donors; indanone; that activity
antiangeogenisis
In
chemotherapeutic acts, one tenuazonic
version derivatives,
one
a acts, inhibitor inhibitors
EGFR
version
phosphoramidate
a either prodrug the
of
microtubule protein version a is
vincristines,
rapamycin;
either phosphoramidate of
these method 1 staurosporin; increased analogs,
,4-dioxides;
and inhibitor
the inhibitors
directly acid,
a
of
and including
phosphoramidate of
such
or directly combination of methods vincristine.
the
6,9,19,
the inhibitor triaziquone, subsequently is enzyme, the
agent,
and combination
PX-12
as used upon inhibitors
histone or taxanes, invention and
combination
suitable
geldanamycin,
indirectly, sodium 104 alkylator protein radicicol
or 22-pentanone but
and
subsequently to including increased
indirectly,
including (1-methyipropyl such alkylator
not treat Other
deacetylase methods
compositions
2,2 such
for
butyrate include
kinase- treated
limited
as
alkylator
treatment breast prodrug and ’
,2"-trichlorotriethylamine, to chemotherapeutic coadministration
treatment a
as but HEFla
prodrug glucose inhibit but
to radicicol
of
17-allylamino-geldanamycin (FR901228,
novobiocin,
1 with treated
gefitinib
to cancer. not
inhibit
(NaB);
treatment not (MEK-1) inhibitors
described
prodrug cis
discodeimolide, methods, levels.
described
limited hypoxia-inducible
2-imidazolyl a limited
transporter methods,
is platinum, phosphoramidate
with
derivatives
the administered sodium and
HIFla
using inhibitors is
epidermal panzem depsipeptide); such
to
a
herein with
to a erlotonib.
agents administered phosphoramidate
herein
Cisplatin,
subject a A
carboplatin, nitropurruside
as
or
subject an vastin a inhibitors disulfide);
such
can
[(E)-
VEGF,
and (2-
that include
urethan, such with growth
is
be factor
and
alkylator
as and is can treated
used
heat
as
an with
(17- and
be P
1 13
in
2011202075 05 May 20 30 25 20 15 10 5
"Evaluation Chemotherapy, Selection example, “ cancer variety Agents of agent anti-angiogenic treat mimics [0344] combination Naprosyn), [0343] (Clinoril), (Orudis, (Voltaren), lavendustin VEGF, compositions inhibitors tecogalan, group anti-angiogenic derivatives 67(8):947-954, camptothecins; [0342] synergistically
microtubule
cancer. F
selected
drugs. consisting
of
described
Lr and batimastat,
In Oruvail), the of In
In synergistic
for tolmetin 8 thalidomide,
of addition
etodolac aspirin, New
addition, of glycolytic
A,
another / In
reference with Those
purposes described
In
from
any
U assembly, ” Williams another medroxyprogesterone
incorporated agent and
agent,
with
Agents Vivo above,
of iji
an
ketoralac of
captopril,
(Tolectin), and the
U of angiostatin, YC-1, to version,
(Lodine), anti-angiogenic
combinations because
the
a and
Tumor
b Vendetti, inhibitors
skill
the including
version, of group phosphoramidate
acetaminophen herein thrombospondin,
the
X and for foregoing;
to
the another
combination a
ci
in
treat present (Toradol), Clinical
compound
a herein Wilkins,
cartilage Models pyruvic combination
b consisting
the
rofecoxib fenoprofen phosphoramidate include
a
an tii
"Relevance
like but
cancer.
phosphoramidate
art anti-cancer
tf of agent
by methods coumarins;
not
Trial," compound halopyruvates, can JI.
acetate,
for a acid Baltimore,
derived Cox-2 reference, oxaprozin
phosphoramidate (Tylenol). of described
limited of
(Vioxx), readily that Predicting alkylator
TNP-470,
(Nalfon),
a therapies plays
alkylators,
Pharmacological phosphoramidate 105 of
and
inhibits recombinant inhibitors agent,
inhibitor,
Transplantable
barbituric
and to
alkylator
an determine
compositions
and (Daypro), 1975, in ibuprofen
prodrug anti-angiogenic
alkylator
important
indomethacin and Clinical provided including Biochem. including a
or its
phosphoramidate Cisplatin,
and
like
Avastin. otherwise genistein, derivatives.
alkylator
prodrug and
human
as the
nabumetone Simpson celecoxib (Advil), Activity prodrug
Basis
by
role alkylator described thiobarbituric Pharmacol., bromopyruvate, but
Animal-Tumor
anti-cancer described
Carboplatin, the Other
platelet
(Indocin), is not
in antagonizes endostatin, prodrug agents
of
present
administered naproxen
angiogenesis, is Herren for
limited Cancer (Celebrex),
prodrug useful
administered alkylator herein.
(Relafen),
Anticancer
herein selected factor
drugs
15 and
methods
acid ketoprofen et
and to
interleukin, angiogenesis
Apr Systems (Aleve, the can
al., other
with 4, provides For
a that analogs; prodrug
inhibitors with
cytotoxic diclofenac
from
action
be Taxol, 2001, pyruvate 1985, sulindac
Drugs,"
the with act and anti used
an
to
the
of
a
to the an
in
2011202075 05 May 2011 30 25 20 15 10 5
therapies later methods minus alkylator the [0348] a d administered paclitaxel, [0347] and can cancer absence delivered embodiments, phosphoramidate continued indicative to Differences synergy" the [0346] combination methods Proc. another describe [0345]
imini phosphoramidate
the standard
involve, sum for approved)
net shing
initiation Am.
treatment,
a
by
described of anticancer The of
prodrug)
These
longer methods When of While log can at
throughout of docetaxel, Assoc.
a more the
unwanted dosages described a
in
for phosphoramidate
administration in therapeutic dose
of be lower
the drugs cell single
methods
accordance
of
a period example, tumor
synergy
defined
effectively
wherein phosphoramidate
alkylator
or Cancer drug herein to regimen
therapy
alkylator kill agent.
dose,
employed
are aid at
doxorubicin, Agents
the herein,
side
cell
of of co-administered lower
less can in
is synergy. as
can
course
greater time administering Res. and there
Two
with
effects kill
prodrug not the of with net
of
prodrug, killing be
than
therefore at
doses
in
the
optionally
alkylator a by together determination 26: required for
log
used the optimal drugs
is
phosphoramidate one the of
than
two those
the synergy
of
those 330, cisplatin,
of
other
treatment alkylator cancer
will,
(than methods
embodiment,
to the
the
optimal tumor can drugs ten-fold
allow
improve
each
with standard or for prodrug with an
Agents
other
at for drug
additional be currently
between maximum
cells
anti-cancer least therapeutic
produces
106 cell or longer
of a prodrug said
the a of
described with
dose
or therapy. phosphoramidate
(one phosphoramidate
carboplatin,
which
whether or
( patient
kill in administration. physician dosages.
drugs to
i.e.,
the
alkylator
the
stopping
some of
used),
periods, a log) possess anti-cancer by
phosphoramidate tolerated is
present a the when is other
drug,
benefit
When and
significantly herein. the used
outcomes
are incorporated two
embodiments,
most thus
to optimum
administration
at prodrug growth
used drug than considered therapeutic
including with drugs
invention treat employed
a
doses. in ameliorating active
agent(s)
lower
alkylator
alkylator Such would or without accordance
over another
cancer
act
of drugs.
in better
combination
single herein
The
than
“ synergistically. but
cancer
accordance alkylator
currently
conclusively
provides low be
synergy be in is
a
prodrug with
anti-cancer
prodrug
will not dosed "degree In administered combination tumor the approved phosphoramidate
dose Agent some
by
with some
cell
limited
case typically existing
reference,
if prodrug
” practiced a
using
cell
regimen the
or
therapies as
a method
will of with
in
all
well dose
kill agent, the to
either be
(or
of with the prior be
and
than
as
of
a
the toxic side effects of such drugs. The exact dosage for a given patient varies from patient to patient, depending on a number of factors including the drug combination employed, the particular disease being treated, and the condition and prior history of the patient, but can be determined using only the skill of the ordinarily skilled artisan in view of the teachings 5 herein.
[0349] Specific dose regimens for known and approved chemotherapeutic agents or antineoplastic agents (i.e., the recommended effective dose) are known to physicians and are given, for example, in the product descriptions found in the Physician's Desk Reference 2003, (Physicians' Desk Reference, 57th Ed) Medical Economics Company, Inc., Oradell, N.J 10 and/or are available from the Federal Drug Administration. Illustrative dosage regimens for certain anti-cancer drugs are also provided below.
[0350] Cancer drugs can be classified generally as alkylators, anthracyclines, antibiotics, aromatase inhibitors, bisphosphonates, cyclo-oxygenase inhibitors, estrogen receptor modulators, folate antagonists, inorganic aresenates, microtubule inhibitors, modifiers, 15 nitrosoureas, nucleoside analogs, osteoclast inhibitors, platinum containing compounds, retinoids, topoisomerase 1 inhibitors, topoisomerase 2 inhibitors, and tyrosine kinase inhibitors. In accordance with the methods described herein, a phosphoramidate alkylator prodrug can be co-administered with any anti-cancer drug from any of these classes or can be administered prior to or after treatment with any such drug or combination of such drugs. In 20 addition, a phosphoramidate alkylator prodrug can be administered in combination with a biologic therapy (e.g., treatment with interferons, interleukins, colony stimulating factors and monoclonal antibodies). Biologies used for treatment of cancer are known in the art and include, for example, trastuzumab (Herceptin), tositumomab and ,3II Tositumomab (Bexxar),
rituximab (Rituxan).
25 [0351] Alkylators useful in the practice of the methods described herein include but are not limited to busulfan (Myleran, Busulfex), chlorambucil (Leukeran), ifosfamide (with or without MESNA), cyclophosphamide (Cytoxan, Neosar), glufosfemide, melphalan, L-PAM (Alkeran), dacarbazine (DTIC-Dome), and temozolamide (Temodar). In accordance with the methods described herein a phosphoramidate alkylator prodrug is co-administered with an 30 alkylator to treat cancer. In one version, the cancer is chronic myelogenous leukemia, multiple myeloma, or anaplastic astrocytoma.
107 [0352] In one embodiment, the present invention provides a method of treating cancer treatable by administering an alkylator by administering the phosphoramidate alkylator prodrugs of the present invention or those alone or in combination with at least another alkylator or a prodrug thereof. Alkylators, such as, for example, cyclophosphamide,
5 ifosfamide, glufosfamide, mechlorethamine, melphalan, chlorambucil, dacarbazine, temozolomide, carmustin'e, streptozocin, bendamustin, busulfan, thiotepa, cisplatin, carboplatin, and oxaliplatin, and types of cancers treated using any one of such alkylators alone or in combination with other anti cancer or chemoprotective agents are described for example in the reference Hardman et al., (supra).
10 [0353] In one embodiment, the present invention provides a method of treating cancer by coadministering a phosphoramidate alkylator prodrug with at least the alkylator Cyclophosphamide, in the treatment of Stages ID and IV malignant lymphomas, multiple myeloma, leukemia, mycosis fungoides, neuroblastoma, ovarian adenocarcinoma, retinoblastoma, and carcinoma of the breast Cyclophosphamide is administered for 15 induction therapy in doses of 1500-1800 mg/m2 that are administered intravenously in divided doses over a period of three to five days; for maintenance therapy, 350-550 mg/m2 are administered every 7-10 days, or 110-185 mg/m2 are administered intravenously twice weekly. In accordance with the methods described herein, a phosphoramidate alkylator prodrug is co-administered with cyclosphosphamide administered at such doses or at lower 20 doses and/or for a longer duration than normal for administration of Cyclosphosphamide alone.
[0354] In one embodiment, the present invention provides a method of treating cancer by administering a phosphoramidate alkylator produg of the invention together with a cancer treatment regimen using at least the alkylator Mechlorethamine. For example, 25 Mechlorethamine is used in the combination chemotherapy regimen MOPP (mechlorethamine, Oncovin (vincristine), procarbazine, and prednisone) in patients with Hodgkin’s disease and administered by intravenous bolus administration is doses 6mg/m2 on
days 1 and 8 of the 28 day cycles of each course of treatment.
[0355] In one embodiment, the present invention provides a method of treating cancer by 30 administering a phosphoramidate alkylator produg of the invention with a cancer treatment regimen using at least the alkylator Ifosfamide. Ifosfamide is used to treat pediatric and adult sarcomas, carcinomas of cervix and lung, and in combination with other drugs for germ cell
108 π 2011 testicular cancer. Ifosfamide is used as part of the ICE (Ifosfamide, Carboplatin, and Etoposide) ans RICE (Rituxan and ICE) regimens for treating lymphomas (see Hardman et May
al., supra). 05 [0356] In one embodiment, the present invention provides a method of treating cancer by
5 administering a phosphoramidate alkylator produg of the invention with a cancer treatment regimen using at least the alkylator Glufosfamide. Glufosfamide is in the clinic for the treatment of pancreatic cancer or Gemzar resistant pancreatic cancer. Glufosfamide can be used for treating breast cancer, Morbus Hodgkin, gastrointestinal tract cancer, or as part of the GCE (Glufosfamide, Carboplatin, and Etoposide) or RGCE (Rituxan and GCE) regimen,
2011202075 10 for treating lymphomas. (Tidmarsh et al., PCT Pat. Appl. No. PCT/US2005/047314 filed on 22 December 2005, and PCT Pat. Appl. entitled “Glufosfamide combination therapy”, Attorney Docket No. 021305-005900PC; and US Pat. App. No. 60/760,599 and 60/719,787 and PCT Pat. Pub. No. WO 2005/076888, incorporated in their entirety herin by reference).
[0357] In one embodiment, the present invention provides a method of treating cancer by 15 administering a phosphoramidate alkylator produg of the invention with a cancer treatment regimen using at least an alkylator selected from the group consisting of ethylenimines and methylmelamines. In another embodiment, the ethylenimine is Triethylenemelamine or Thiotepa.
[0358] Thiotepa can be used to treat adenocarcinomas of the breast, ovary, and bladder, 20 malignant lymphomas, branchiogenic carcinomas, and Wilms’ tumor. Thiotepa was used at high doses in combination chemotherapy with cyclophosphamide in patients with refractory ) malignancies treated with autologous bone transplantation and to treat a variety of cancers including bladder, ovarian, breast, lung, brain, and lymphomas (see, International Agency for Research on Cancer Monographs on the Evaluation of Carcinogenic Risk of Chemicals to 25 Humans, 1975, 9 : 286, Lyon, France; International Agency for Research on Cancer Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Humans, 1990,50 : 415, Lyon, France; and MEDLINEplus, 2003, Drug Information: Thiotepa, National Library of Medicine). The methylmelamine Altretamine is used to treat advanced ovarian cancer after failure of first round therapies.
30 [0359] In one embodiment, the present invention provides a method of treating cancer by administering a phosphoramidate alkylator produg of the invention with a cancer treatment regimen using at least the alkylator Melphalan, Chlorambucil, or Bendamustine. Melphalan
109 ) 2011202075 05 May 20 30 25 20 15 10 5
regimen Doxorubicin is bladder, cancer. of platinum regimen administering sarcoma. can Dacarbazine. [0363] administering to myelomas, [0362] gastrointestinal alkylator regimen administering before regimen combination leukemia [0361] administering example, is [0360] developed treat
used
childhood. treat used
be
chronic
used to
bone
pancreatic
Combination to using head using
using using
coordination In In
treat
is In Γη
In non-Hodgkin and treat
by
malignant
one Carmustine. to one
one one to marrow another
with
lyphocytic
and
a at Cisplatin
Dacarbazine a chronic Salmedix a at
advanced treat a
at at treat
multiple phosphoramidate
phosphoramidate
tumors. embodiment, phosphoramidate phosphoramidate embodiment,
embodiment, embodiment, least least
least least neck,
Cyclophosphamide islet
malignant
ovarian
embodiment,
transplantation.
chemotherapy
a
a astrocytomas, complex myelogenous a
the
cell
’ platinum alone
endometrium,
s nitrosourea myolema Inc. testicular leukemia In
Carmustine lymphoma, alkylator
is another carcinoma.
carcinoma. can
used
the or the the gliomas. the
alkylator alkylator.
with
be
present coordination
alkylator present
alkylator present
present alkylator
alkylator and cancer; and the to
embodiment,
Busulfan. used alkylator. of leukemia. metastatic
treat can cyclophosphamide
chronis
triazene small to primary can
Cisplatin
treat
is to
invention be invention
invention invention
and malignant be
In
produg
produg Cisplatin. produg produg treat
used cell 110
one
lymphocytic administered patients
with
complex Busulfan alkylator
In macroblobulinemia. High tumors
hematological with carcinoma
the another embodiment, to
of
of
provides of of provides
one provides provides treat
nitrosourea doses melanoma,
the
Bleomycin, the
the the Cisplatin with
of
of alkylator. is is
is
invention
Hodgkin invention invention the embodiment, invention
Paclitaxel, Temozolomide.
used
leukemia,
used
of orally. acute of
a
a a a brain,
busulfan
method the method method method
malignancies, the
to
can
to Hodgkin
is myelogenous
Etoposide, lung, ’
treat
In Chlorambucil s
treat triazene
Streptozocin with
with
with melanoma,
with be Bendamustine, disease,
and one Cyclophosphamide,
of of
of of
can used
the
chronic
and advanced
a
a a
a
treating multiple
treating treating embodiment, ’ treating
cancer
cancer s cancer
cancer be nitrosourea
Temozolomide
alkylator
disease, some
to
lymphomas,
and
such used
treat
leukemia and granulocytic
which
is
treatment
treatment cancer treatment Vinblastine treatment
cancer
cancer cancer neoplasms myeloma. ovarian
as,
in used
cancer and
is
for
is the
by to adult by by
by
used
or
of
2011202075 05 May 2011 30 25 20 15 10 5
but lymphocytic [0367] herein cancer. and limited IJS05/008161. [0366] described doses). prior methods three intervals; bronchogenic dose carcinoma, been soft acute naphthacenedione, antibiotic lyxo-hexopyranosyl)oxy]-8-glycoloyl-7,8,9, breast methods Kaposi idarubicin anthracycline are [0365] [0364]
are
daunomycin
not tissue
in to successive used
lymphoblastic
a
not
cancer. In to
’ the limited phosphoramidate and Cyclic s
Aromatase Antibiotics
described
described As one dactinomycin, herein weekly Anthracyclines
sarcoma,
isolated limited
successfully and
(Idamycin), range
thyroid
continuing leukemia,
one
version, carcinoma,
to
bone
Anthracycline to,
(Cerubidine, days are
treat
example of
intravenous
to
from doxorubicin
herein,
prostate more
carcinoma, herein
inhibitors
30-75 provided sarcomas, useful anastrozole leukemia,
repeated
cancer.
the other
after to valrubicin
cultures actinomycin
commonly
useful
alkylator
and
produce cancer the a a
mg/m in
cancer,
phosphoramidate phosphoramidate leukemias, the
DanuoXome). by cytotoxin In the injection compound gastric useful
every breast lymphomas acute (Adriamycin,
in
administration
2 one of (Arimidex)
the
is practice
as
(Valstar), regression
the
prodrug Streptomycespeucetius bladder a
known reference version, a in myeloblastic
four carcinoma. cancer carcinoma, D
practice single
at
the prodrugs and (Cosmegen),
(8S,1OS)-10-[(3-Amino-2,3,6-trideoxy-alpha.-L-
doses weeks. of
practice
of
cancer, is
as
Kaposi and 111
selected 1 and
the intravenous
the In Doxil,
in 0-tetrahydro-6,8,
alkylator both co-administered alkylator
Matteuci doxorubicin,
of of
accordance of
disseminated
methods
letroazole cancer ovarian useful In
Doxorubicin
the
doxorubicin
leukemia, 20
Hodgkin ’ metastatic
of
s accordance Rubex),
from methods bleomycin sarcoma. mg/m
the
prodrug
prodrug
et is in
injection carcinoma, described
methods
acute al.,
the the var.
(Ellence). (Femara). 2 with is
; and Wilm's mitoxantrone
or neoplastic
a carcinoma
PCT practice described group 11 at with is
caesius. with cytotoxic
is is
nonlymphocytic
30
(Blenoxane),
non-Hodgkin the -trihydroxy- such typically
co-administered co-administered
administered
described
mg/m Patent herein
an
methods tumor,
transitional consisting the
In
In doses
antibiotic of
accordance herein accordance conditions methods
Doxorubicin of 2
the anthracycline
Aplication administered include doses (Novantrone),
neuroblastoma,
the (or
methods herein 1
described
daunorubicin,
-methoxy-5, types,
include
ovary, of at
at
cell on leukemia, to
of
lower acute but 2
with
starting
such treat
1-day with each include with the
bladder
No.
has and are
but
an in
as
the of
the
not
a
12-
.) 2011202075 05 May 2011 30 25 20 15 10 5
intramuscular courses such of treatment of drug methylamino]benzoyl]-L-glutamic treat herein breast [0372] are [0371] is administered accordance include cancerous oxygenase include described [0370] cancer. the biphosphonate described include methods [0369] aromatase [0368]
mycosis patients breast
not
group
courses that cancer.
cancer. a
of limited
but but phosphoramidate but
cancer
As has Folate of Estrogen described Cyclo-oxygenase Bisphosphonate
therapy.
herein consisting herein with
condition
fungoides. inhibitor
inhibitor
with are
are are advanced one
repeated injections
been In with
to
not inhibitor chorioadenoma not one not antagonists or
example,
the a methotrexate a
used
an
the
phosphoramidate receptor phosphoramidate
limited For limited limited herein
version,
to methods to
known
of
estrogen as
stages
treatment Methotrexate
treat treat of leukemias, in
multiple
needed to
doses the alkylator the inhibitors
a
to
to treat to
inhibitors
modulators useful
cancer. cancer. as phosphoramidate
the
of
tamoxifen described treatment celecoxib compound zoledronate
receptor
familial
and
malignant destruens
with of
cancer is cancer. myeloma,
in administered
twice acid,
15
tremetrexate. prodrug
In useful is
In alkylator alkylator rest the
useful to
administered
one one adenomatous modulator
is useful
herein of
(Celebrex). commonly
(Nolvadex)
30 practice
In weekly N-[4-[[(2,4-diamino-6-pteridinyl)methyl
period
and osteosarcoma. (Zometa). lymphoma
gestational
in bone version, version, one
mg
is in 112
the hydatiform
prodrug alkylator in prodrug
co-administered a
the
are
version,
to
of phosphoramidate
intramuscular metastases
the of practice In
to practice prevent
one
administered
known
the
the the
as and
In accordance practice treat polyposis.
In and choriocarcinoma
is is
follows.
accordance or
prodrug cancer methods cancer
accordance the
fulvestrant
co-administered
co-administered
mole. in of more cancer.
the as of
cancer from
the
the
of
methotrexate, the
occurrence
is is injections with
weeks the
is treatment
methods with
For
It daily described
breast
colon methods solid
alkylator In co-administered
is
with is
methods
with (Faslodex).
a
choriocarcinoma,
also one the a
folate
for
interposed
tumors, cancer cancer
and cancer. the
the
methods
version,
described
are
useful with with
a or of
described herein
methods five-day in prodrug
is
antagonist methods described
reoccurrence advanced administered
selected the
an or
a a
or
in In cyclo
a
between include the prostate treatment described
pre-
the with herein
is course, herein
cancer
co herein
from cases to
an
but
of
in
2011202075 05 May 2011 30 25 20 15 10 5
in prodrug with with sarcoma, oncolytic treatment obtained the phosphoramidate cancer, cancer. Application methods Anti binding 008400US epothilone methods (Velban), interferes treat but herein [0374] alkylator trimetrexate) methotrexate trimethoxyphenyl)-amino]methyl]-2,4-quinazolinediamine methods [0373] mg doses
weekly
compound
are
vincristine or, the
Cancer cancer.
of
a
Kaposi
alternatively, is not methods In
anticancer
phosphoramidate from rhabdomyosarcoma,
of intravenous described
agents 30 of
described Microtubule
Inorganic prodrug. not
paclitaxel with
one
and B
limited the
No. acute
mg/m Agents
or
In administered
is administered the 22-oxo-vincaleukoblastine,
’
version, s the 021305-004520US). present administered
D in another one
PCT/US2005/042095; sarcoma,
described
alkylator
common
leukemia. 2
or
the assembly drugs to . herein
herein,
arsenates
and (Taxol, version,
For
a of doses arsenic
inhibitors
treatment
derivative invention the
25 antifolate Prodrugs
mycosis and
include and
alkylator
prodrug
mg prior periwinkle herein, a cancer of at
Paxene),
It or phosphoramidate at
the
prodrugs trioxide useful neuroblastoma, such
2
has metastatic
are such disassembly
mg/m
of (as to cancer
are
of
fungoides,
Thereof but
drug is also administered
a
doses Hodgkin's treatment is
prodrug used either, in phosphoramidate doses. ovarian provided In
vinorelbine co-administered (Trisenox).
2 are
plant
the
thereof
been
for US that accordance is
herein, (or cancer not
refractory also practice
’
children
and
Patent In
(Attorney can
(Vinca
of 113 shown is weekly cancer, at
with limited
and
one disease,
in which alkylator
microtubules) co-administered commonly lower
discodermolide be twice
of a the
(Navelbine),
Wilm's Applications
In version,
a “ co-administered
of
breast
rosea, with microtubule to and
microtubule
breast reference intramuscular acute
accordance to can
doses).
the
Ref weekly. be alkylator with lymphosarcoma,
vincristine
prodrug 1.4 the
useful methods be
tumor. or
Linn.) known Nos. promyelocytic
a
cancer,
a (commonly mg/m
methods used
phosphoramidate ovary
5-Methyl-6-[[(3,4,5- microtubule useful
Matteucci In docetaxel
or 021 entitled with prodrug
in
inhibitor
is with inhibitor, and Vincristine
accordance
2 as in
its
described
combination origin. non-small (Oncovin), co-administered injections for 305-008500US, with
in the vincristine,
an
derivatives. is
described the
adults. the
useful “ inorganic known reticulum-cell practice
is
a Tubulin ”
(Taxotere),
et
methods
leukemia
inhibitor
phosphoramidate practice is co-administered As such
al.,
herein
is
with any
of
cell
one in vinblastine
alkylator In
administered
as PCT
herein with
doses as is of
the
agent accordance Binding
arsenate lung
Tubulin
the
example, an described a
of
the
to (APL). include with 021305-
taxane, Patent other
alkaloid the
treat
of
a
that
50
to
i 2011202075 05 May 20 30 ( 25 20
15 10 5
lymphocytic administered accordance cladribine floxuridine thioguanine, but recurrent to [0377] described not BiCNU, uptake organic [0376] alkylator the deoxyglucose increase incorporated cell the compound cancer. but alkylator treat limited microtubule simultaneously [0375]
treat
are limited therapeutic
compound rather, to
colorectal
not take of
cancer.
to
nitrite
Gliadel In Nucleoside 2-deoxyglucose
Nitrosoureas glioblastoma
prodrug prodrug
Modifiers herein (Leustatin, glucose.
Leucovorin
limited with
(FUDR), one administration to that up
leukemia 6-TG
with inhibitor. herein
procarbazine
and or 2-deoxyglucose N-hydroxyurea.
methods version,
In increases cancer.
with the Wafer), a
a
a
is
is one phosphoramidate to (Thioguanine), a
spermineNONOate,
nucleoside
methods
by useful co-administered phosphoramidate
co-administered fludarabine analogs mercaptopurine, or 2-CdA),
(CLL),
useful version, (Wellcovorin), multifoime.
reference),
within the In
provided
and uptake the
of
in accordance
(Matulane), cancer
useful
described
a estramustine ability in the hairy
gemcitabine
phosphoramidate
analog a the (see N-hydroxyurea the
or few (Fludara), practice
herein.
hydroxyurea
and cancer is
to practice cell in
the of alkylator
with days colon with
treat 6-MP which to alkylator the
with administration a
herein reference to leukemia, lomustine,
cell
treat
of practice
is nitric In treat to a (Emcyt).
leukemia (Gemzar),
azacytidine cancer.
the
modifier of
the prostate (Purinethol), 114 is a to another
prodrug cancer.
a week
the
used
cancer, has (Hydrea), take
methods prodrug phosphoramidate methods oxide alkylator
Smith
adenocarcinoma methods
of
CCNU
been
In up
with
after
In together
cancer such
the
and
and of In or is
one
as accordance glucose, (Vidaza),
et
as
reported described N-hydroxyurea co-administered
methods one a
described
other
another initiation
cytarabine fluorouracil, the prodrug
version, capecitabine
(CeeBU), al., nitric version,
described
described or
version, latter with
1999, glioblastoma, drugs
including oxide
pentostatin
to anti-cancer alkylator
described
is
administration
herein a with herein of of compounds the
enhance phosphoramidate
Cancer (Cytosar-U, carmustine administered herein
such
herein the treatment the
precursor,
5-FU modifier (Xeloda).
the at
with
pancreas, a cancer
include but
as
levels prodrug
phosphoramidate
methods is
herein Letters
include (Adrucil),
the including (Nipent), 5-fluorouracil agent
not one a
stimulate
with
is nitrosourea (BCNU, ability
is
DepoCyt), such reported
of
limited but
In
a
version
B-cell include
to
is 141:
2- but a
treat are
co
as
of
are
85,
the an not to
to a of
to
metastatic breast cancer, non-small cell lung cancer, or metastatic colorectal carcinoma. As one example, the compound 5-fluoro-2,4(lH,3H)-pyrimidinedione, also commonly known as 5-fluorouracil, is an antimetabolite nucleoside analog effective in the palliative management of carcinoma of the colon, rectum, breast, stomach, and pancreas in patients who are 5 considered incurable by surgical or other means. 5-Fluorouracil is administered in initial therapy in doses of 12 mg/m2 given intravenously once daily for 4 successive days with the
daily dose not exceeding 800 mg. If no toxicity is observed at any time during the course of the therapy, 6 mg/kg are given intravenously on the 6th, 8th, 10th, and 12th days. No therapy is given on the Sth, 7th, 9th, or 11th days. In poor risk patients or those who are not in an 10 adequate nutritional state, a daily dose of 6 mg/kg is administered for three days, with the daily dose not exceeding 400 mg. If no toxicity is observed at any time during the treatment, 3 mg/kg.can be given on the 5th, 7th, and 9th days. No therapy is given on the 4th, 6th, or 8th days. A sequence of injections on either schedule constitutes a course of therapy. In accordance with the methods described herein, a phosphoramidate alkylator prodrug is co 15 administered with 5-FU administered at such doses or with the prodrug form Xeloda with correspondingly adjusted doses. As another example, the compound 2-amino-l,7-dihydro- 6H-purine-6-thione, also commonly known as 6-thioguanine, is a nucleoside analog effective in the therapy of acute non-pymphocytic leukemias. 6-Thioguanine is orally administered in doses of about 2 mg/kg of body weight per day. The total daily dose can be given at one 20 time. If after four weeks of dosage at this level there is no improvement, the dosage can be cautiously increased to 3 mg/kg/day. In accordance with the methods described herein, a phosphoramidate alkylator prodrug is co-administered with 6-TG administered at such doses (or at lower doses).
[0378] Osteoclast inhibitors useful in the practice of the methods described herein include 25 but are not limited to pamidronate (Aredia). In accordance with the methods described herein a phosphoramidate alkylator prodrug is co-administered with an osteoclast inhibitor to treat cancer, ha one version, the cancer is osteolytic bone metastases of breast cancer, and one or more additional anti-cancer agents are also co-administered with a phosphoramidate alkylator prodrug.
30 [0350] Platinum compounds useful in the practice of the methods described herein include but are not limited to cisplatin (Platinol) and carboplatin (Paraplatin). In accordance with the methods described herein a phosphoramidate alkylator prodrug is co-administered with a platinum compound to treat cancer. In one version, the cancer is metastatic testicular cancer,
115 2011202075 05 May 2011 30 25 20 15 10 5
treat phosphoramidate include etoposide administration administration version, prodrugs the phosphoramidate prodrug. useful [0353] cancer. accordance include from treatment metastatic administered limited administered can administered described of Cisplatin, administered accordance example, [0352] [0351]
doses
reference be
cancer. the
in
co-administered
but to
but As
the
of
Topoisomerase group the Topoisomerase Retinoids thereof
the As phosphate herein, of
tretinoin, when ovarian
are noted 50-70 with with are cancer
transitional
palliative one In with Matteucci with with compound at
of of not
not
consisting
one these
used the
the
useful
example, a alkylator a a above, alkylator mg/m
a a a
cancer,
limited phosphoramidate is limited phosphoramidate
topoisomerase useful
ATRA
version,
topoisomerase phosphoramidate retinoid methods methods (Etopophos).
metastatic for doses
treatment
with in 2
et
cis-Diaminedichloroplatinum cell however, 2 advanced once 1
of
in the al.,
ovarian
to
inhibitors to prodrug inhibitors Platinol, prodrug. (Vesanoid), (or
to
the bladder the the APL,
etoposide,
described topotecan described practice
PCT
every
treat at
carcinoma platinum cancer practice of
lower
1 carcinoma,
in Kaposi In bladder
Patent 1 metastatic is
Blenoxane,
inhibitor cancer.
three
As cancer useful alkylator inhibitor useful alkylator one
accordance co-administered
alkylator of
is
alitretinoin herein
herein (Hycamtin) doses). VP-16
another of
the
version
a compound ’ Application to
s
cancer,
of
cancer the
in which in In
sarcoma, four methods
116 but
the
testicular and to the prodrug prodrug the a one a
methods (Vepesid),
prodrug.
and phosphoramidate
phosphoramidate One example,
treat
is
with
weeks. of ovary,
practice
practice
transitional
is selected is
version, (Panretin),
not
and
Velbam the
not or and administered
cancer. of and with No. (II),
the
either is
administered
more
methods irinotecan and
described
colon,
the
amenable a co-administered
In
T-cell methods Platinol
teniposide,
of of
PCT/US2005/041959. phosphoramidate
commonly from the
a
ovarian present accordance
can
the topoisomerase precedes the additional
Topoisomerase cell and
cancer or
be the lymphoma. methods methods described
rectum, alkylator alkylator
bladder bexarotene herein and in
(Camptostar). to
described
co-administered
invention
tumors,
group concurrently
intravenous surgery
VM-26
known is Adriamycin or
anti-cancer with a
include
follows,
described with described or cancer cancer.
consisting
herein, prodrug prodrug
2
and
alkylator
small
the herein
or
as (Vumon), inhibitors (Targretin). inhibitor are
cisplatin
radiotherapy.
cisplatin, for
methods
but
injections selected
In provided
therewith. or
In
As cell
can
agents
is is the herein herein
with a
both, one
are
of
one co co
lung be
to
and and
not
a
is co
In
in
Γ 2011 refractory testicular tumors, refractory acute lymphoblastic leukemia (ALL), and small cell lung cancer. As noted above, however, in one version of the methods described herein, May
administration of a phosphoramidate alkylator prodrug either precedes or follows, or both,
05 administration of a topoisomerase 2 inhibitor but is not administered concurrently therewith.
5 [0354] Tyrosine kinase inhibitors useful in the practice of the methods described herein include but are not limited to imatinib (Gleevec). In accordance with the methods described herein a phosphoramidate alkylator prodrug is co-administered with a tyrosine kinase inhibitor to treat cancer. In one version, the cancer is CML or a metastatic or unresectable malignant gastrointestinal stromal tumor.
2011202075 10 [0355] Lonidamine analogs useful in the practice of the present invention are provided in the Matteucd et al. U.S. Pat. Appl. Nos. 11/346632; 60/764,427; 60/764,438; and applications entitled “Heterocyclic Lonidamine Analogs” (Attorney Docket No. 021305-007220US; 021305-007900US) and PCT Publication Nos. WO 2006/015191, WO 2006/015263 and WO 2006/01007 A2.. 15 [0356] Thus, described herein are methods of treating cancer in which a phosphoramidate alkylator prodrug or a pharmaceutically acceptable salt thereof and one or more additional anti-cancer agents are administered to a patient. Specific versions of such other anti-cancer agents include without limitation 5-methyl-6-[[(3,4,5-trimethoxyphenyl)amino]-methyl]-2,4- quinazolinediamine or a pharmaceutically acceptable salt thereof, (8S,10S)-10-(3-amino- 20 2,3,6-trideoxy-alpha-L-lyxo-hexopyranosyl)oxy]-8-glycoloyl-7,8,9,10-tetrahydro-6,8,ll- trihydroxy-l-methoxy-5,12-naphthacenedione or a pharmaceutically acceptable salt thereof; 5-fluoro-2,4(lH,3H)-pyrimidinedione or a pharmaceutically acceptable salt thereof; 2-amino- 1,7-dihydro-6H-purine-6-thione or a pharmaceutically acceptable salt thereof; 22-oxo- vincaleukoblastine or a pharmaceutically acceptable salt thereof; 2-bis[(2- 25 chloroethyl)amino]tetrahydro-2H-l ,3,2-oxazaphosphorine, 2-oxide, or a pharmaceutically acceptable salt thereof; N-[4-[[(2,4-diamino-6-pteridinyl)methyl]-methylamino]benzoyi]-L- glutamic acid, or a pharmaceutically acceptable salt thereof; or cisdiamminedichloro platinum (II).
30 IV. EXAMPLES
117 [0357] In the following examples, any reference to a compound designated by a letter is a reference to the structure shown next to or above that letter in the corresponding reaction schemes.
Synthesis
5 [0358] Methods to synthesize the phosphoramidate alkylator prodrugs of the present invention are provided in section lib. Starting materials used in the synthesis of the phosphoramidate alkylator prodrugs of the present invention were bought, when available, from commercial manufacturers, such as, for example, the Sigma-Aldrich Co. 1-N-Methyl- 2-nitroimidazole-5-methanol was purchased from Syngene, India. Non-commercially 10 available starting materials can be synthesized in via standard literature procedures. Such procedures can be identified via literature search tools such as SciFinder available from the American Chemical Society or Beilstein, available from MDL Software.
[0359] Reactions with moisture sensitive compounds, such as, for example, POC13 and PCI3, and their mono and dichloro derivatives were performed employing anhydrous solvents 15 and under nitrogen or argon. Separation of a product from the reaction mixture was performed employing a work-up where necessary, followed by vacuum distillation, crystallization, column chromatography, or preparative thick layer chromatography. A suitable eluent for the column chromatography of a compound can be determined by reading this disclosure and/or by determining the Rf of the compound by thin layer chromatography 20 and choosing a solvent which allows separation of the desired compound from undesired compounds. The choice of a particular eluent can depend, among other factors, on the polar nature of the compound, existence of other closely eluting compounds, type of stationary phase such as silica gel or alumina used, and the amount of pressure used to elute the solvent through the stationary phase. In practice, different compositions of solvents can be used to 25 separate the same compound.
[0360] Separated compounds were analyzed for their purity by standard analytical techniques, such as, TLC, NMR spectroscopy, and LC-MS, and stored in a freezer or a fridge, avoiding moisture, light, or air. Stock solutions of phosphoramidate alkylator prodrtfg compounds were prepared in DMSO and stored in a freezer.
30 Example 1 Synthesis of Compound 23
118 2011202075 05 May 2011 15 10 5
by was spectroscopy ml) hour, [0362] temperature chloroethylamine (TEA, 78°C concentrated. aqueous [0361]
LC/MS
was distilled
POCI3 the
0.22
refluxed layer
A To reaction
and
suspension ml,
HCI
was was a out
to
was solution Compound NMR
1.54 be
under
raised added (135°C)
+ hydrochloride mixture
pure. extracted
POCI3 mmol). + spectroscopy
vacuum of of to in POCI
N-Methyl-2-chloroethylammonium over 5-nitrofurfuryl
room 23 one was
with
Temperature 3 was
portion
night.
quenched
as temperature
was
DCM separated Synthesis
light Cl-P-N to TEA THF
be added
After followed O Cl
11
I
yellow
and pure.
5i
alcohol with (-jci(+)H Example
was /
removing followed by
of the (rt), 119
water
oil Compound LiN(TMS)2 flash increased
TEA, by combined DME
Cl the (200 3
N^ and
a 2
and THF column dropwise reaction
by HN^z^CI
excess mg, analyzed — / DIEA,
TEA
the to
CI
organic 5 1.4
-30°C
organic
chromatography
POCI3
was THF
(1 chloride mmol) addition
by
ml, HCI
continued
in solution
*H
under layer 7 one
Π in
mmol). . and
(10 of
THF
hour
triethylamine was
vacuum
31 gm) was
P for O
II
(10 5
and
23
and O-P-L^CI NMR separated.
After
in dried one
/ Cl s O
ml) NH
POC1 analyzed then
product
more
H the
and at Cl
2-
3
-
(40
The
5i
2011202075 05 May 2011 15 10 5
preparation by mmol) methanol was one was dimethoxyethane organic chloride was [0363] [0365] [0364]
flash
hour, wanned dried added
was
layer
chromatography (0.62
To To
Compounds
in the over
at
slowly
of
DCM solution a
up -78°C. reaction was
solution compound_5. gm,
MgSO
to
(DME)
dried
yielded -20°C, 4.75 added
After of
4 of mixture
of
and
over mmol) 5i
8
yielding lithium and TV-methyl diluted
5 (1 and 5.
concentrated.
min, MgSO
gm, the
was 16 in
with reaction bis(trimethylsilyl)amide THF 4.75
were 5ii compound
diluted 4 2-nitroimidazole-5-methanol
and (2.9
ethyl mmol)
at synthesized
concentrated
-78°C mmol, mixture
Purification with
acetate
120
5ii and LiN(TMS) LiN(TMS)
DME ethyl diisopropylethylamine
DME
as 770
JV-Methyl-2-chloroethylammonium was
oil. employing and
mg) acetate
by to warmed 2
2 washed
yield
flash was (3.2
and
added
mmol, a the
chromatography
to with
residue
washed (0.5
rt. procedure
brine. and
After 3.2 g,
(DIEA,
which 3.2
the with ml,
stirring The mmol)
reaction used 1
brine.
was M 1.65
with organic
in for
in at separated
ml,
THF)
6-12% The
mixture rt the
for 9.5 layer
i I
2011202075 05 May 2011 15 10 5
phosphorylation white refluxed was Additional remove filtered to g, in employed dried dibromopropane wise [0367] [0366] H
200 rt, DMF 2
N^ coevaporated
poured at
with solid
mmol) HBr, 120
rt through
the
(38 (4
A To
and
in
Na °C
aqueous product h). 48% solution aqueous
0H
mL) into a the
2
the
was solution SO
The
a
next provided
(10
water a reaction 4 celite
+ with
added
and solution
35b
reaction portion of portion
g, reaction.
compound_35a of
ethanol concentrated (250 50
pad. was
to
ethanolamine
below. mixture mmol).
the (5 of
(about mixture mL), filtered The
ml) p-toluenesulfonyl
thrice, reaction Synthesis
filtrate
and was was
After 20
and to was
and ml), extracted
(5 removed heated yield
(6.03 mixture,
washed
heating was TsCI, g) Example cooled
following
and
of in
compound_35a 121 DMF concentrated mL,
Compound up aqueous K
the
2
with
by
to chloride CO with for followed to
100 3 reaction
rt,
distillation
3
120°C two
addition ethyl
acetone diluted
mmol) HBr
more
(19 35
by to (bath acetate.
mixture
(48%, as 130
POCI
dryness g, of with dropwise
and twice
hours, and yellow
100 a temperature). °C
large 3
K2CO3
water 50 the
The
was and mmol)
the to
ml)
oil reaction
addition
volume
organic yield
refluxed employed (20 reaction
35a
which (13.8 was
35 was
mL),
a distilled
K2CO3 mixture residue g,
of
added layer of was
for was
100
and acetone
in 1,3-
40
the was
cooled mmol) (27.6 drop
to
which
was h.
a
[0368] A suspension of compound_35b (1 g) in POC13 (14 mL) was heated at 130°C for about 14 h and excess POCI3 removed under vacuum at 130°C (bath temperature). The residue was purified by column chromatography on silica gel employing 10-80% ETOAc/hexane to yield product 35c which was convereted to compound 35 of the present 5 invention employing the same procedure as provided in Example 2 employing for column chromatographic separation silica gel andl0-80% acetone/toluene as the eluent.
Example 4 Synthesis of Compound 7 10 [0369] Compound 7 was prepared by employing N-cyclopropyl-2-chloroethylammonium chloride as provided below
i) /THF/NaOH Cl
>-nh2
ii) HCI/dioxanc iii) 7j SOC12
Cl POCl3/reflux
>-NH2(+)CI(-)
7i Cl !__ / Ϊ>-ΝΗ2(+)α(-) Cl
D^n,o p-ci DIEA/THF Cl' 7ii Cl
A P—N C|- 1 LiN(TMS)2 . ( 7111 Cl
[0370] To a solution of cyclopropylamine (25g) in dry THF (30 ml) a solution of 2- bromoethanol (17.6g, 0.141 mol) in 30 ml THF was added dropwise over 35 minutes. The 15 reaction mixture was stirred for 1 hour at rt, and heated at 50°C for 75 minutes. After cooling, the reaction mixture was concentrated to yield an orange oil to which was added a
122 2011202075 05 May 2011 30 25 20 15 10 5
flash (110 was layers into After added. solution dissolved 78°C g, with was minutes, (47% for yield) 7.5 cooled solution vacuo dried through 95% colorless solution [0374] [0373] [0372] [0371]
2.11
1.5 hours.
separated 25 refluxed
chromatography mg, dry
yield)
(MgSO 15 and of were
which
at
to hours ml mmol) After
a of
theoretical) of of minutes
ether 53-56°C and
N-methyl-2-nitroimidazole-5-methanol
To 7i 7ii 51% liquid
0°C DIEA in
short water
The lithium
HC1 sodium which dried
(3.00g,
dry (0.50
a
was 4 extracted and (6
15
)
by and solution yield) (100 were
reaction and path
which h), in THF
minutes, (0.545
and
the flash
over concentrated analyzed
(1 was g,
SOC1 dioxane
bis(trimethylsilyl)amide
hydroxide cooled,
ml), 19.2 evaporated
combined
of distillation mm 2.11 reaction
as extracted
over (2
was MgSO chromatography
analyzed
4
of pale g,
a
2 mixture
ml) mmol) filtered,
times
Hg) yellow
(6.50g, mmol) the a 4.22
silica
by (4.0M, analyzed and
solution
yellow under
4
intermediate mixture
to
(7g)
*H in and 3 with to mmol)
to
concentrated
was
apparatus
by yield using
was times oil
and dry and 54.9 NMR give give
in 18.3 argon.
concentrated
oil *H
which ethyl of added by
THF residual concentrated water N-cyclopropyl-2-chloroethylamine
an was
mmol) added an 0-50% NMR
an with which 7iii
ml, in LC/MS to
intermediate
orange
orange acetate
to
0-10% be The under slowly alcohol to was (172
73.2 (50 in ethyl
yield
to slowly to 123
pure. POC1 was was volatiles
of THF
reaction
analyzed ml). be yield
and mg,
mmol) to
hexane
argon. oily
oily (75 (76.8
acetate methanol
warmed
added analyzed
7ii pure. (3.7 and give 3
(1.6M, by 1H
0.538 (15 The
ml). a residue.
residue. as alcohol
removed the residue.
mg,
g,
syringe, mixture NMR
was a
in The a ml)
by
by
(30
reaction
yellow 36.6
clear,
resulting The
to mmol)
ethyl 0.306
0.489 in to
syringe.
LC-MS
added.
and reaction ml). rt,
be to (5.94 DCM
The mmol) The combined
The
stirred warmed was pale
in
be
acetate refluxed
oily 7iii
ml, mmol) mixture in
The
vacuo
residue
residue
pure.
oil g,
The
and residue
to cooled 2 The
yellow by
0.489 residue. mixture
in 42%
ml combined for yield distilled
to MS.
dry reaction
to 'H
hydrochloride was
to reaction organic under THF
was
2 give was
was
rt mmol) yield)
NMR yield to
was hours, THF oil compound_7
partially slowly,
was
-78°C
The
stirred
was
separated distilled 315 (3.6g, in nitrogen
triturated
mixture
organic 7i layer
(30
as mixture to
vacuo cooled
was
poured residue
added. mg (5.42g,
be a
and stirred
ml)
for 79% clear,
was
pure. (0.33
in
for
a by was 10 to a
-
Example 5 Synthesis of Compounds 6 and 15 .Cl Tv* TEA OOH θ2Ν·^Ν Cl PC13 + I __ . I DCM
6
[0375] To a suspension of bis(2-chloroethyl)ammonium chloride (1.43 g, 8.01 mmol) in 5 dichloromethane (DCM) phosphorus trichloride (0.32 ml, 3.64 mmol) was added at rt followed by addition of TEA (3.05 ml, 21.84 mmol). The reaction mixture was stirred at rt for 30 minutes and then //-methyl 2-nitroimidazolyl methanol (0.474 g, 3.31 mmol) in DME was added. After stirring for 0.5 hour, the reaction mixture was cooled to -20°C and tert- butyl hydroperoxide (0.7 ml, 3.82 mmol, 5.5 M in Decane) was added. The reaction mixture 10 was warmed to rt over a period of one hour, and poured into 10% aqueous HC1. The Organic layer was separated and the aqueous layer was extracted with DCM. The combined organic solution was dried with MgSO4 and concentrated to yield a residue which was purified by flash chromatography with 6-12% methanol in DCM yielding 6.
[0376] Compound 15 was synthesized using the method described for the synthesis of 15 Compound 6 above.
TEA OOH HCI DCM
15
Example 6 Synthesis of Compounds 23, 26 and 36
Cl
I 20 J
124 [0377] To a solution of 7V-methyl-2-nitroimidazole-5-methanol (180 mg, 1.14 mmol), triphenylphosphine (300 mg, 1.14 mmol), and isophosphoramide mustard (lc, 127 mg, 0.57 mmol) in THF (10 ml) diisopropyl azodicarboxylate (DIAD, 0.22ml, 1.14 mmol) was added dropwise at rt. After two hours reaction mixture was concentrated and the residue separated 5 by flash chromatography with 30-100% acetone in toluene yielding compound_36.
[0378] Compounds 23 and 26 were synthesized employing the procedure of Example 6.
Example 7 Synthesis of Compound 1
10
[0379] N-methyl-2-nitroimidazole-5-methanol (50 mg, 0.318 mmol) was dissolved in dry THF (2 ml) under nitrogen. The solution was cooled to -78°C and a solution of lithium bis(trimethylsilyl)amide (IM in toluene, 0.35 ml, 0.35 mmol) was added by syringe. After 5 minutes a solution of bis(chloroethyl) phosphoramidic dichloride (91 mg, 0.35 mmol) in THF 15 (2 ml) was added. After stirring at 78°C for 30 minutes, the temperature was reduced to - 20°C employing a NaCl/ice bath and anhydrous ammonia was bubbled through the reaction mixture for 5 minutes. The reaction mixture was purged with nitrogen, wanned to rt, poured into 25 ml water and extracted with ethyl acetate (4 x 25 ml). The combined organic layers were dried (MgSO4) and concentrated to give pale yellow oil which was separated by flash 20 chromatography over silica gel using 0-10% methanol in dichloromethane yielding compound 1 (32 mg, 28 % yield) of an oil which soldified on standing and was analyzed by LC/MS and 1H NMR to be pure.
Example 8 Synthesis of Compounds 25, 26 25 [0380] To a solution of 2-bromoethylammmonium bromide (19.4 g) in DCM (90 mL) at - 10°C was added a solution of POClj (2.3 mL) in DCM (4 mL) followed by addition of a solution of TEA (14.1 mL) in DCM (25 mL). The reaction mixture was filtered, the filtrate concentrated to ca. 30% of the original volume and filtered. The residue was washed with DCM (3x25 mL) and the combined DCM portions concentrated to yield a solid to which a
125 mixture of THF (6 mL) and water (8 mL) was added. THF was removed in a rotary evaporator, the resulting solution chilled overnight in a fridge. The precipitate obtained was filtered, washed with water (10 mL) and ether (30 mL), and dryed in vacuo to yield 2.1 g of:
O II H HO-P-N^ Br
5 [0381] Isophosphoramide mustard
O II H HO-P-N I ^Cl
can be synthesized employing the method provided in Example 8, substituting 2- bromoethylammmonium bromide with 2-chloroethylammmonium chloride. Synthesis of Isophosphoramide mustard has been described (see for example Wiessler et al., supra).
10 [0382] The phosphoramidate alkylator toxin:
O II H HO-P-N^
was transformed into compounds 24 and 25, employing the method provided in Example 6 and the appropriate Trigger-OH.
Example 9
15 Synthesis of Compounds 37-105 [0383] The following compounds 37-105 were synthesized employing the Mitsunobu type coupling described for the synthesis of 25 or 36 above, and upon appropriate substitution of the Trigger-OH and the ifosfamide mustard analog employed. For example, for the synthesis of compounds 40, 81, 83, 87, 89,95,96,100, and 104, the ifosfamide mustard analog 20 employed was HOP(=O)(NHCH2CH2C1)2; in compounds 50, 53, 55,56, 58 - 65, 68 - 71,73 - 75,77 - 80, 82,, 84 - 86, 88, 90 - 92, 94, 97 - 99, 101 - 103, and 105, the ifosfamide mustard analog employed is HOP(=O)(NHCH2CH2Br)2; in compounds 37, 39, 52, 54, and
126 2011202075 05 May 2011 10 5
nitrothiophene-2-methanol; methanol, HOP(=O)(NHCH(CHMe HOP(=O)(NHCHMeCH2Cl) and analog analog 93 [0384] 105,
,
the 49
included
employed employed The the ifosfamide
l-N-methyl-5-nitroimidazole-2-methanol, ifosfamide
various
the
was is following
mustard
Trigger-OH the
the mustard
S
2 enantiomer S )CH2Cl)
analog
enantiomer Trigger-OH 2 ;
in analog
compounds
compounds
2 employed ;
and
of
employed
of
HOP(=O)(NHCHMeCH
compounds: in
HOP(^)(NHCH(CHMe2)CH
compounds
employed 127
is 38,41,51,
the
was
R
5-nitrofuran-2-methanol, the enantiomer l-N-methyl-2-nitroimidazole-5-
in 46
and R
the
-
enantiomer
48,
57 synthesis
the the
2 of Cl)2;
ifosfamide ifosfamide
in of of
2
compounds Compounds Cl)2.
5-
mustard mustard
43
37
-
-
45
2011202075 05 May 20 10 [0385]
The
following
compounds
were
according· 128
to
the
method
described
in
Example
6. 2011202075 05 May 2011 129 2011202075 05 May 20 130 68 2011202075 05 May 20 15
synthesis Examples
of
10
phosphoramidate
—
26 88
describes
the
alkylator
synthesis Synthesis 89
prodrugs Example
of
of
various 131
Compound
of
10
the
Trigger-OH
invention.
52i
compounds 90
employed
in
the
Γ 2011
PdCl2(dppf)
I KOAc, DMF May OH 52ii 52iii 05 [0386] A solution of compound 52ii (100 mg, 0.48 mmol), 52iii (73 mg, 0.48 mmol), and
KOAc (190 mg, 1.92 mmol) in DMF (5 ml) was degassed thrice and PdCl2(dppf) (36 mg, 0.048 mmol) added to it at rt under an argon atmosphere. The reaction mixture was heated at 5 60°C for two hours, diluted with ethyl acetate (EA) and washed with brine. The organic layer was dried, concentrated, and the residue separated by column chromatography on silica gel employing as eluent EA/Hex (0 - 80%) to yield 52i.
2011202075 [0387] Compounds 55i, 63i, 59i, 65i, and 68i were prepared in a similar manner as described
PdCl2(dppf)
KOAc, DMF 55i
PdCl2(dppf)
KOAc, DMF 63ii 52iii 63i
PdCl2(dppf)
KOAc, DMF ϊ 1°
PdCl2(dppf)
KOAc, DMF 52iii
Example 11
DIEA
132 [0388] To a solution of compound 68ii (100 mg, 0.31 mmol) and 3-amino-l-propanol (0.047 ml, 0.62 mmol) in THF (2.5 ml), DIEA (0.162 ml, 0.93 mmol) was added at rt. The reaction mixture was stirred overnight and concentrated to yield a residue which was separated by column chromatography on silica gel employing as eluent EA/Hex (0-80%) to
5 yield compound 68i.
[0389] Compound 69i was made similarly as depicted in the scheme below.
69i
Example 12
K2co3
Acetone
10 [0390] To a solution of compound 70ii (100 mg, 0.87 mmol) and compound 70iii (112 mg, 0.87 mmol) in acetone (8 ml), was added K2CO3 (78.6 mg, 0.87 mmol) at rt. The reaction mixture was heated at 60°C with stirring for 1 h, filtered, and concentrated to yield a residue which was separated by column chromatography on silica gel employin (EA\Hex) 0-60% to 15 yield compound 70i. [0391] Compound 51 i was made similarly as depicted in the scheme below.
K7CO,
HO
51iii
Example 13 20 (HAP triggers - check #s)
PdCl2(dppf)
KOAc,DMF
[0392] A solution of compound 59ii (200 mg, 0.96 mmol) and 59iii (127 mg, 0.96 mmol) in DMF (3 ml) was degassed thrice and PdCl2(dppf) (50 mg, 0.07 mmol) was added to ϊζ
133 2011202075 05 May 2011 20 Ι 10
5 5
phenyldichlorophosphate employing compound syringe under added DCM. rt was TEA 100%) was [0393] eluent followed to atmosphere
for yield PhO-P-CI
separated diluted
(0.75
1 hydrogen
platinum(IV)oxide
EA\Hex to
The h,
filter, o Cl a I
To
by yield poured residue
67iii. a ml,
combined
with and Mitsunobu
a Cui
the . o-s by
suspension
+
5.38 compound
(0-70%)
for
the
Compound
(8.5 O O EA,
filtrate into column II II which -s-cr'X/NKjHCl
— 0.5
mmol) reaction O 2 II
+
mg,
brine, washed organic
h.
O
type
to was concentr
(0.2 2 67i
chromatography (20 of N 0.043 67ii. The
yield
and
Synthesis
the 67iii
67i mixture reaction
separated
ml, N-y mg),
with
layers
reaction
stirring.
organic To
mmol) (472 compound
ated was 1.34 TT
the brine, a „,
were nH was solution
mg,
as
reacted under of mmol)
reaction
by
-20°C
TEA, mixture
and
layer described Example The
Compoundsl06 Example
THF, the
heated
2.69 PPh column on dried
TEA
58i. vacuum
134 organic DCM
reaction
silica separated, at tort with 3 of
0°Ctort , mmol) mixture
DIAD was -20°C,
with compound
at (0.27
15 chromatography
for 14
l-N-methyl2-nitroimidazole-5-methanol 60 gel
diluted
and layer
MgSO»
mixture the °C in
ml, employingas followed degassed,
and
DCM coevaperated and for synthesis
separated,
1.92
67ii
with
the
two and
107 was
[
x\^O (20
mmol),
aqueous (42
by
MeOH, hours.
concentrated. and
67
wanned
ml) on of the
mg)
eluent dried,
vigorously
silica Compound —
with
was
dropwise at o O
S II II
The in layer filtered —
rt,
up EA/hexane EtOH
and added toluene gel
under reaction
to pto extracted
EtOH employing concentrated The
rt, through addition
stirred (5 36. 2,
argon
stirred
to h
ml) residue
2 mixture
o O S·
II II yield (ΙΟ
with was
a of
at as
2011202075 05 May 20 15 10 5
was [0395] ml, to separated twice, with addition (phenylsulfonyl)ethylamine added [0394]
100%)
1.54 synthesized
water
POCI3 the
of mmol).
Compounds
To
to by combined
and TEA
yield
a
column (0.13
solution 108
POC the
using (1
The product
organic *3 ml, ml,
organic
chromatography
+ reaction 108-112, a
of 1.4 7
similar Ο
mmol).
2 5-nitro 106.
Ν-ζΚ/ mmol) layer
hydrochloride layers
temperature
shown method. Compound
furfuryl separated.
The at
were
-78°C, ΟΗ
on below reaction
dried, alcohol silica TEA,
(832 was
107: The -78 followed 135 :
was THE gel concentrated
warmed °c'
mg,
aqueous (200
employing
warmed
109 3.5
by mg,
up mmol)
-10°C
the layer
1 to
.4 to dropwise to
-10°C as
mmol)
tort rt,
was yield added
eluent
stirred
extracted
in a
in
to addition residue
1
THF acetone\toluene for it, h,
2- followed
1
with (10
h, which
of
quenched
ml)
TEA DCM
by
was was
(0.216
the (30
110 111
112 5 were synthesized employing the procedure described for the synthesis of compound 35 in Ts Ts Example 3 and substituting HO OH with
Example 16 Synthesis of Compounds 113-117
1, LiN(TMS)2 9 ^Cl THF, -78 °C o2n OH + Cl—Ρ-Νχ Cl ^CI 2, NH3, -20 °C 10 113ii li [0396] Compound 113 was synthesized following a procedure described in Example 7 as described here. To a solution of 113ii (181 mg, 1.16 mmol) in THF (8 mL) was added dropwise LiN(TMS)2 (1.2 mL, 1 M THF solution, 1.2 mmol) at -78 °C, followed by the addition of li. The reaction mixture was warmed up to -20°C and NH3 bubbled through the 15 reaction mixture for 5 minutes. Water (20 mL) was added to the reaction mixture and the reaction mixture extracted thrice with EA (30mL). The combined organic layers were dried and concentrated to yield a residue which was separated by column chromatography on silica gel employing acetone\toluene (30-100%) to yield compound 113.
[0397] Compounds 114-117 were synthesized according to the method described for
O2N OH 20 Compound 13 and substituting with the appropriate Trigger-OH as starting material.
136 Γ 2011
O °—ρ-ν
May νη2
117 05
Example 17 5 Synthesis of octadeutereated ifosfamide and Compound 64 (octadeuterated-Compound 25)
165oC 2011202075
DIAD, PPh3 „ O/C°2 d2cs Br
64iii
[0398] 48% HBr (60 mL) was added dropwise to diethanolamine at 0°C. The reaction mixture was stirred for lhr at rt and then gently refluxed and slowly distilled, 16 mL liquid being collected in 2 hrs until 155°C (oil bath). This was replaced twice with 60 mL of 48% 10 HBr and the distillation continued for an additional 5 hr. 90 mL liquid was collected. The resultant solution was heated at 165°C for 2hr and evaporated under vacuum. The residue was recrystalled from an absolute ethol (10 mL)-ethyl acetate (30 mL) to 11.3 g of <74-2- bromoethamine hydrobromide (compound 64i). Compound 64i (19.5 mmol, 1.0 eq.) was added dropwise to a suspension of J4-2-bromoethamine hydrobromide (40.0 mmol, 2.05 eq.) 15 in dry DCM (100 mL) under argon,at -20°C, followed by the dropwise addition of TEA (81.9 mmol, 4.2 eq.) at -20°C. The reaction mixture was stirred at ~20°C for 0.5 h, and at rt for 2 h, poured into water, and extracted twice with DCM (30 mL). The combined organic layers were washed with brine, dried over Na2SO4> and concentrated under reduced pressure to yield a residue which was separated by column chromatography on silica gel employing as eluent 20 Hexane/EA (100:70(v/v)) to yield 7.0 g of compound 64ii. PtCh (0.7 g) was added to a solution of compound 64ii (7.0 g) in MeOH (160 mL), the reaction mixture degassed and exchanged with H2 thrice, stirred under H2 for 3 h at it, and diluted with MeOH until the
137 138
white solid in the reaction mixture dissolved. The diluted reaction mixture was filtered, the 2012
filtrate concentrated under reduced pressure to yield a residue which was washed with anhydrous
ether twice to yield 2.9 g of compound 64iii. To a suspension of compounds 64iii (1.92 g 1.0 Dec
eq), l-N-methyl-2-nitroimidazolemethanol (1.01 g, 1.1 eq.), and PPh3 (2.39 g, 1.5 eq.) in THF
06 (20 mL) was added DIAD (1.76 ml, 1.5 eq.), under argon, at 0°C. The reaction mixture was
stirred for 2 hours while being warmed up from 0°C to rt, following which volatiles were
removed under vacuum to yield a residue. The residue was separated by flash chromatography on silica gel employing as eluent Acetone/Toluene (100:70(v/v)) to yield 1.35 g of compound 64.
Example 18 2011202075 Synthesis of Compound 2i
2ii 2iii [0399] The vinyl derivative, 2iii, was synthesized according to the reference Cavalleri et al., J. Het. Chem., 1972, 9:979, and oxymercurated as follows. Hg(OAc)2 (208 mg, 0.653 mmol) was
dissolved in water (0.7 mL) and THF (0.7 mL), followed by the addition of compound 2iii (100 mg, 0.653 mmol). The reaction mixture was stirred at rt for 1.5 h, NaBH4 (25 mg) added to it in portions, and after stirring for 15 min the reaction poured into water, extracted with EA, the EA
layer dried and concentrated to yield a residue which was separated by silica gel column
chromatography employing as eluent EA/Hexane (0-100%) to yield compound 2i (16 mg).
Example 19 Synthesis of Compound 94i
Bn Bn OAc hno3 rX/0Ac o2n^ NaBH4 < Ac2O/AcOH OAc OAc MeOH 94iii 94ii [0400] A solution of 94iii (7.1 g) in Ac2O (9.7 mL) was added dropwise into a solution of Fuming nitric acid (1.5 mL) was added into AcOH (12mL) at 0°C. The reaction mixture was
warmed up to rt, stirred 1 hr, fuming nitric acid (1 mL) was added dropwise into it and stirred for
1.5 h. The reaction mixture was poured into water, extracted with EA, the EA layer dried and concentrated to yield a residue which was separated by silica gel column chromatography
6554197 1 RTK 139
employing as eluent EA/Hexane (0-100%) to yield compound 94ii. Compound 94ii (600 mg, 2012
1.77 mmol) was suspended in methanol (10 mL) at 0°C followed by the addition of NaBH4 (141
mg) in portions into the reaction mixture over 5 min. NaBH4 (lOOmg) was added once every Dec
hour thrice, the reaction mixture was stirred for 3.5 h, poured into water, extracted with EA, the
06 EA layer dried and concentrated to yield a residue which was separated by silica gel column chromatography employing as eluent EA/Hexane (0-100%) to yield compound 94i (289 mg) as a
yellow solid.
Example 20 Synthesis of Compound 96i
2011202075 NC\
ac NaBH4 FT,° ------O2N^sJryZ OAc DMF OAc MeOH OH 96ii 96iii 96i [0401] A mixture of A (1.4 g), CuCN (0.56g) and DMF (25 mL) was stirred at 140°C for 35
min and on 300 mL of crushed ice and stirred for 10 min. The reaction mixture was then filtered
and the residue was separated by column chromatography employing as eluent Hexane:EA (1:0 to 2:3) to yield compound 96iii as yellow oil (617 mg). Compound 96iii was converted to alcohol 96i and separated by column chromatography, following a similar method as employed for compound 94iii in Example 19 and using THF instead of MeOH as solvent in the reaction.
Example 21 Synthesis of Compound 99i
NaBH4 OAc PdCl2(PPh3)2 ------1 Cul/TEA MeOH 99ii [0402] A mixture of 99ii (500 mg), PdC12(PPh3)2 (208 mg), and Cui (56.4 mg) was suspended in TEA (15 mL), the reaction mixture was degassed and flushed with Ar 6 times each. Propyne was bubbled through the reaction mixture for 15 min, and the reaction continued under a propyne atmosphere at 50°C bath for 2 h. The reaction mixture was poured into EA, filtered,
the filtrate concentrated to yield a residue which was separated by
65S4I97 I RTK 2011202075 05 May 2011 25 20 15 10 5
yield combined K2CO3 more solution solution (88 oil residue h. which residue with and flask. 585.7 silica [0403] compound
After to
g, stirred
the 48
than
which
gel mmol,
1.07 was The
to washed which
g concentrated which flask,
the
Ethyl
column
a Synthesis ethyl of Synthesis 45°C.
dissolved reaction mol). at 99i pH
was reaction
1 grounded rt
-N-methyl-2-amino
was
NaH (286
was of formate for acetate with
added
The
The chromatography 8-9
triturated
about
cooled, mixture
of (60%
mg).
of
2
in
mixture
concentrated to and
reaction
x
l-N-methyl-2-amino
layers into l-N-methyl-2-amino
10% ethanol (500
1/5 25
14 extracted oil
its mL powder
its was
h. aqueous
twice mL)
suspension, cooled were
pH mixture
original
(400 Volatiles of
cooled
was
adjusted imidazole-5-carboxylic ethanol. employing with
reaction
dried
with prior
down, mL) HO
added
was volume hexane in
Example
EA Example Ac, over
were to 54 and
an
to
the The mixture reaction) stirred
imidazole-5-carboxylic
imidazole-5-carboxylic g, 140 to (5
H2NCN
ice-water
as 1 cone MgSC>4,
removed
white 1.35 sarcosin (500 x using
using
filtrate eluent
200
23
22
at HC1
was mol) mL)
90-1 precipitate contained a concentrated mL (45
filtered, bath, EA/Hexane rotary
was
using methyl (50 carefully
to added
00°C followed g, acid
mL) evaporated
yield 1.07 stirred,
evaporator a
ethyl
and for in ester rotary
slowly
was
and
mol),
a neutralized a
HC1
1.5 by
sticky
volatiles (0-100%)
acid 1-L acid
a ester. hydrochloride filtered stirred
gas evaporator 3 h
during and to
and
round-bottomed x to
ethyl ethyl at
yield light outlet 50
yield
a sodium the
at
removed
off
mL). temperature to
by
ester a
ester 1
brown resulting
a
10°C period
yield
and connected
a addition
thick to
clear
The
(82 acetate
yield the
for
to paste
brown of
g,
1.5
2 of not a
h,
CHO Ο
ONa
1.HC1, EtOH
2. NCNH2> AcONa
[0404] Ethyl formate (850 mL) was added to sarcosine methyl ester HC1 salt (205 g, 1.46 mol, grounded into powder prior to use), potassium carbonate (205 g, 1.48 mol), and EtOH (800 mL,), stirred overnight at rt, and filtered. The filtrate was concentrated in a rotary 5 evaporator during which the residue separated into two layers. The upper layer was separated and the lower layer was extracted with EA. Combined EA layers and the upper layer was dried over MgSO47 filtered, and concentrated to yield 185 g (81%) of N-formyl sarcosine methyl ester which was used for the following reaction. NaH (60% oil suspension, 16.0 g, 0.4 mol) was carefully added in several portions in 1 h to a mixture of N-formyl sarcosine methyl 10 ester (50 g, 0.34 mol) and ethyl formate (160 mL) cooled in an ice-water bath. The reaction mixture was stirred, the temperature raised to rt, and the stirring continued overnight. The reaction mixture was triturated twice with hexane (100 mL each time), the residue dissolved in EtOH (100 mL) and concentrated HC1 (60 mL), and the reaction mixture stirred at 110°C. After 1 h, the reaction mixture was cooled down, filtered, the residue washed with EtOH and 15 the filtrate concentrated to yield a thick brown oil. The oil was added to 10% HO Ac in water (200 mL), NH2CN (35 g) and sodium acetate (90 g), stirred at 95°C. After lh the reaction mixture was concentrated to 1/3 its original volume in a rotary evaporator and its pH adjusted to about 9 by addition of sodium carbonate. The reaction mixture was then extracted with EA (8 x 100 mL), the combined EA layers dried, filtered, and concentrated to yield a residue 20 which was purified by recrystallization to yield l-N-methyl-2-amino imidazole-5-carboxylic acid ethyl ester (“amino ester”).
Example 24
Synthesis of l-N-methyl-2-nitroimidazole-5-carboxylis acid ethyl ester
141 [0405] A solution of the amino ester (36.94 g, 0.218 mol) in 200 ml of acetic acid was added drop wise to a solution of sodium nitrite (100 g, 1.449 mol) and water (300 ml) cooled in an ice-water bath, and stirred. The temperature of the reaction mixture, which was 5 measured to be around -5 - 10°C was raised to rt and and the reaction mixture stirred overnight. The reaction mixture was extracted with DCM (3 x 150 mL). The combined DCM layers were dried and evaporated to yield a reddish residue which was separated by column chromatography on silica gel employing as eluent EA/hexane (30%) to yield 1-N- methyl-2-nitroimidazole-5-carboxylic acid ethyl ester (“nitro ester”) as a light brown solid 10 (27 g, yield 62%).
[0406] This method described in Example 24 and employing aqueous acetic acid is an improvement of the method using about 7% sulfuric acid (v/v) for the diazonium ion formation from the amino ester. Using aqueous sulfuric acid, the reaction volume becomes large causing difficulty in stirring the reaction mixture effectively. For example, a reaction 15 involving 150 g of the amino ester required a reaction mixture volume of about 12 L. The sticky nitro ester formed as product in aqueous sulfuric acid and disrupted the stirring of the reaction mixture.
Example 25
Synthesis of l-N-methyl-2-nitroimidazole-5-carboxylis acid
/N 20 CO2Et CO2H
[0407] A suspension of the nitro ester (39.2 g, 196.9 mmol) in IN NaOH (600 mL) and water (200 mL) was stirred at rt for about 20 h to give a clear light brown solution. The pH of the reaction mixture was adjusted to about 1 by addition of cone. HC1 and the reaction mixture extracted with EA (5 x 150 mL). The combined ethyl acetate layers were dried over 25 MgSO4 and concentrated to yield l-N-methyl-2-nitroimidazole-5-carboxylis acid (“nitro acid”) as a light brown solid (32.2 g, 95%).
142 2011202075 05 May 2011 20 15 10 5
mmol) mmol) DME, gel reaction nitroimidazole-5-methanol washed stirred dry (67 acetate. less dropwise [0408] [0409]
(0
mg, ice-acetonitrile than
-100%
LiN(TMS)2 for was in To
with 0.32 mixture
A
0°C. addition anhydrous
1 a
mixture added
h
suspension THF. mmol)
acetone\toluene) followed The
Synthesis was
drop
of
was reaction bath
The
of
was THF
water concentrated
the
wise added by
of 119i combined (temperature
added
(360
nitro
the
of l-N-methyl-2-nitroimidazole-5-methanol as during mixture
to
1
an at
addition
-N-methyl-2-nitroimidazole-5-carboxylis
to mL) this and acid
α
Synthesis -78°C orange
yield
a THF
and
cooled LiNflMSh
the period was was (30.82 Examples
< DME
with
the
of
Compound reaction -20
portions
solid stirred Example warmed Example
sodium
of
reaction residue
of
g, °C). vigorous
143
Compound (25 1 180.23
28A-28V
while
h
mixture were Isobutyl
up
O while
borohydride g)
26 - 27 was °2N
2 mixture 119.
N
which to
stirring. mmol)
the evaporated ζ
x separated 0°C. maintaining ,υ
was
reaction ,N chloroformate 119 119
was
during ch The
and warmed
After (36 2
recrystallized oh
solid by triethylamine
to
mixture
g, a
chromatography yield a period 10 no (50
947
to temperature was
2 min,
rt. (37.8 acid mg,
a
mmol)
l-N-methyl-2-
was filtered
of After
compound
0.32
from
mL,
10 (140 cooled
and
min
1
mmol)
off
around 288 ethyl h,
mL,
on
and the and in
11
silica
285 a in
or 9i
I 2011 [0410] Compounds 134 to 155 were synthesized by employing the corresponding substituted phosphormamidate and hydroxy substituted Trigger (Trigger-OH), according to May
the procedures described in Examples 1-27 above. 05 Example 29A
5 [0411] The solubility of the following compounds is as listed below :
Compound Solubility (in saline at room temperature)
10 10 mg/mL
25 15 mg/mL 2011202075 73 10 mg/mL
155 <1 mg/mL
Example 29B
Antiproliferation Assay
[0412] To determine the effect of phosphoramidate alkylator prodrugs on cell proliferation, 10 the antiproliferative activity of these compounds was tested in a multi-well Alamar Blue based assay. Cell growth in the presence and absence of the test compound was compared, as measured by a fluorescence plate reader at excitation 550nm and emission 590nm (see Biosource International Inc., Tech Application Notes, Use of Alamar Blue in the measurement of Cell Viability and Toxicity, Determining IC50). The following cell lines were 15 tested with 20,000 cells/well/500pL medium: NCI-H460 cells (ATCC HTB-177, RPMI medium (Gibco Products, Invitrogen Corporation, Carlsbad, CA)), HT29 cells (ATCC HTB- 38, RPMImedium (Gibco)), MES-SA cells (ATCC CRL-1976, McCoy’s 5amedium (ATCC)), MES-SA/Dx5 cells ((ATCC CRD-1977), McCoy’s 5a medium (ATCC)), ACHN cells (ATCC CRL-1611, Minimum essential medium, Eagle (ATCC)), PC3 cells (ATCC 20 CRL-1435, Ham’s F12K. medium (ATCC)). The cells were seeded in glass inserts placed in each well of a 24-well plate in the density and medium as specified above one day prior to compound testing. After 24 hours, these plates were divided into two groups - anoxia group and air group. A test compound was added to each well (200pL volume) in the treatment groups at concentrations varying from 100, 30,10, 3, 1, 0.3, 0.1, 0.03, to 0.01 μΜ. All test
144 ο CM £ compounds were serially diluted in complete medium with final DMSO concentrations less s than or equal to 1% in each well. The cells in the anoxia treatment group were incubated for 2 hours in a Bactron II anaerobic chamber. The cells in the air treatment group were o incubated for 2 hours in standard tissue-culture incubators. Following the 2 hour treatment 5 with a test compound, the test compound was removed from each well, cells were washed with 500pL medium, and incubated for 3 days in 500pL fresh medium. After 3 days, cells were stained with 10% Alamar Blue for 2 hours after which the capacity of cells to proliferate was measured (as mentioned above), and the 50% growth inhibitory concentration (GIso (also referred to IC50 herein)) of test compounds was calculated and tabulated in Table X below.
2011202075 10 Table X: IC50 values (uM)
Compound H460 HT29 MES-SA MES- ACHN PC3 Anoxi a/Air Anoxia/Air Anoxia/ SA/Dx5 Anoxia/ Anoxia/Air Air Anoxia/Air Air P2 44/>100 1 0.4/72 50/>100 1 / >100 23 0.04/5 7.5/- 23 0.1/14 154 0.9/2 139 16/100 140 5/-65 2 8/>100 5 0.05 / 6 10/>100 22 0.7/16 3 >20/>100 142 >40/>100 4 40/>100 143 4.5/3.5 6 0.7/>100 22/>100 5/>100 144 7/>100 145 >100/ >100. 147 >100/ >100 7 0.14/25 7/>100 0.59/83 11 5.2/>100 12 1.7/>100 9 >10/>100 - 8 0.013/0.6 36 0.88/ 55/>100 5/>100 7.5/>100 >100 149 50/>100 15 0.08 /1 16 1.6/>100
145 2011202075 05 May 20 110 109 108 107 106 119 118 150 38 90 89 88 87 37 86 27 24 25 82 85 84 81 79 78 77 76 75 74 35 33 32 28 31 26 83 80 35 34 10 14 18 17 0.03 0.03 100/>100 0.3/>100 0.3/>100 0.003 8.5/>100 0.075/50 22/>100 0.06/2.8 0.09/3.5 0.01/3.4 0.33 83/>100 0.05 0.01/1.8 l.l/>100 0.7/100 0.05 0.15/86 0.15/20 9/>100 25/100 3.5/3.5 50/100 0.3 0.3 1.6/5.5 0.01/4 0.1 >100/ >100/ >100/ >100/ 3.4/9 1.4/3 0.33/ 0.36/ 9/26 >100 >100 8/46 >100 >100 >100 >100 -/21 1/9
/
1 / /
/ / /
/
0.53 0.02
21 / 13 35 3.7 0.3
55 40
1.8/>100 16/>100 0.8
/
57 0.13/10 1/100 0.1/2 >100 0.9/ 146
0.3/>100 0.1/0.8 0.2
/
62 0.6/>100 Ο co 39 0.2/5 91 -/>100 92 >100/ May
>100
05 41 -/7 42 0.5/9 93 0.1/3.8 94 0.3/2 95 -/2.7 96 0.1/0.1 120 0.3 / 50 121 0.04/1 122 0.04/1.3 43 2/60 2011202075 44 3/100 45 6/>100 46 5/>100 47 4/>100 48 -/>100 97 0.01 / 0.1 49 -/>100 50 0.1/3 98 0.1/2 51 3/7 52 15/20 53 3/10 99 0.1/1 100 0.5 / 35 54 1/60 55 5/12 56 0.5/10 123 100/>100 57 14/100 124 -/0 125 -/100 126 -/0 111 50/100 58 5/10 59 2/6 60 15/15 61 0.3/4 62 2/45 63 1/8 127 0.02 / 5 128 0.02/10 112 70/>100 103 0.02/2 113 1/100
147 2011202075 05 May 2011 10 5
antiproliferative were medium (HT29 and desired cells/well/500pL assay [0413] Compound
105 air. 129 114 133 132 131 117 116 115 104 130
70 69 68 67 66 65 72 71 incubated
23 as 1
cells)
oxygen
The previously (Gibco)) To
determine below. calculated 100/>100
48/>100 22/>100 71 concentrations for 0.3/100 0.1/0.1 activity <0.1/1 0.4/12 0.5/12 0.7/20 0.5/15 25/75 >100/ 1/100
0.5/5
2/65 medium 8/70 >100 1/80
or 3/3 />100 0.05
0.3 2
n described
HT29 hours 2
the
Antiproliferation
ICso of
Table in
oxygen these (ATCC in 0.1%
values glass
a (see
varying
Bactron 5
Y1
compounds
Oz
inserts
Example dependence
HTB-38, : (μΜ)
IC™
from 0.3%
II
Example
Assay values are in anaerobic 10
29). 24-well
anoxia, RPMI was 148 Oz tabulated
of
- (uM)
NCI-H460 Oxygen tested
phosphoramidate 30 0.6%
medium
plates 0.1%, chamber
6 in 7 in
in
Oz H460
Table
Dependence
a one 0.3%,
multi-well (Gibco)) cells
flushed
cells day l%Oz Y1
50
0.6%, 5 (ATCC
prior (H460 alkylator
were with
Alamar 1%,
to
HTB-177,
10%
cells)
gasses seeded testing.
10%
prodrugs,
Oz Blue-based
or
oxygen,
of at
Table The
the RPMI 20,000
Air the 100 cells 5
Y2
2011202075 05 May 2011 100 110 109 108 106 119 99 54 53 52 98 49 51 50 97 47 46 44 24 38 37 35 26 25 88 84 34 36 16 10 5 0.006 0.007 >100 0.03 0.05 0.01 0.03 0.5 0.3 0.3 0.1 0.4 0.3 0.5 0.3 0.3 0.1 1 0 1 1 10>100 >100 30 10 3 1 1 -0.5 6 3 1 149 >100 >100 0.01 0.85 0.5 100 0.5 30 0.2 0.7 10 45 10 60 50 45 25 60 10 10 5 7 3 3 3 5 6 5 1 >100 >100 40 60 10 10 5 >100 100 25 10 5 >100 >100 >100 >100 >100 >100 >100 0.02 100 100 100 60 0.5 35 20 10 25 >1 40 40 50 40 55 15 7 2 3 1 4 5
' 2011202075 05 May 2011 15 10 5
with in before of 200ppm (ATCC)). HTB-38, were cells clonogenic (0414] Compound
aluminum oxygenation
pre-calibrated
per 25 tested: 114 127 113 128 123 70 66 63 61 56 62 55
the
O
dish To RPMI
2 test A ) survival
NCI-H460
was determine
vessels solution in
and
between
medium 5mL
achieved n
2 >100
added 0.02 0.02 gasses 2
0.3 0.3 0.5
assay 2 2 (see 1 1 5 1
of of
the
cells
medium)
200ppm the
(Gibco)), Example directly
0.1% Table
prior was Clonogenic
by oxygen
test
(ATCC
exposing
perfonned.
to O
Y2: compound
and
2
to 2
experimentation.
PC3 dependence 33)
days
cells
IC 0.3% HTB-1 air
Assay
for 5 the cells o
were
prior Example
(2mL values
2
O Cells glass was
hours. 77,
2
(ATCC 150 -
achieved to Oxygen
of
volume). RPMI
made
0.6%
(μΜ) dishes were compound
phosphoramidate
31
25 For For
>100 >100 CRL-1435,
O 20 30 in plated 30 15 in medium 4 5 8 8
1 1
Dependence
by in the 2
the
complete HT29
Anoxia
a flushing
anaerobic
Bactron aluminum 1%O testing. in
(Gibco)), 60 cells
Ham
2 or
medium mm
the
alkylator
hypoxia Π The
chamber, ’
s
anaerobic vessels, 10% glass anaerobic
F12K
HT29 following
immediately
O
dishes
(less prodrugs, 2
medium
anoxia desired cells
chamber chamber
than
>100 (5x1 cell Air
(ATCC
or
>100 >100
levels a
100 lines 65 0 80 45 10 10 12
4 5 8 5
or
2011202075 05 May 2011 20 10 15 5
aluminum below. hypoxia 90% the checked number to 50 were Levels a and evacuation a exposure after
37°C series
Compound (Cell a 70(H460) 37 25 35 24 23 25 cells 25
drug
plated shaker
killing,
which stained (HT29) (H460) (H460) (H460) (H460) (H460) (PC3)
of water
of
Line) were of was using was
to oxygenation jig
five
for evacuations. in and
one drug,
with achieved 10% washed
bath that counted
a
clonogenic
an
rapid
flushing, 37 more
oxygen allowed the
survival) crystal on degree
evacuations
off a
glass evacuation (see
by
between shaking 0.07
The 0.2 0.5 0.3 0.3 N2 0.2 0.1 the the
violet
2 exposing electrode survival C
for
Example
dishes of
incubator
platform cells oxygen
monitoring
test
platform (0.25% 200
Table
and and
by
were compounds in
the (Anima,
ppm 33).
rinsing concentrations plastic
flushing flushings
(with Electrochemistry
for
glass
Y3:IC removed in
0.1%
Example (controls
and
The of
the 95%
0.4
water
both Petri Phoenixville, with dishes
151 remainder
air
90% 90 02 were was
with ethanol),
values were
from
gas medium.
dishes. bath 32 are
calculated
growth in
performed,
in 95%
and
flushed pre-warmed, achieved
the the
and (μΜ)
of
and
nitrogen liquid Ten
chamber PA)
medium
jigs) 0.6% the inhibitory The
0.25 colonies
0.6 and as
to 5 8 3 3
5 in 1
and
by phases.
was cells
to well). 02
14
a
tabulated
plus
varying
2 specially or and air
the days
shaken hour
were
concentration
aluminum containing
tight
jigs
5% gas After Following
later,
drug
then
the
carbon phases were
aluminum in
for modified
the
Table degree
the
exposure. trypsinized 5 Air
20 40 90 30 50 30
14 vessels
transferred more fifth 5 minutes,
dishes dioxide were
the (ICgo,
Y3
and jigs
than
and
to in
r~ 2011 [0415] To determine the electrochemical properties and reduction potentials of phosphoramidate alkylator prodrugs, cyclic voltammograms of these compounds were May
generated by Bioanalytical Systems, Inc. All experiments were conducted with glassy carbon
05 (3.0mm diameter) working electrodes, Ag/AgCl reference electrodes, and platinum wire auxiliary electrodes. Compounds were dissolved in ImL methanol to make final drug 5 concentrations between 0.5 and 1.5mM after the addition of 9mL Phosphate Buffered Saline (PBS). The solution was added to an electrochemical cell vial and sparged with Argon for 5 minutes to remove most of the oxygen. Cyclic voltammetry was performed at 100 mV/sec and at 10,000 mV/sec scan rates at a glassy carbon working electrode. One test run was performed at a CGME mercury electrode (CGME in SMDE mode, 150pm bore capillary, 2011202075 size 8 drop), but little difference was observed between mercury and glassy carbon voltammograms, so the mercury electrode was not used further. The single electron or multiple electron reduction potentials of compounds were generated at each scan rate and are tabulated in the table below.
Table: Reduction Potentials (mV)
Compound 100 mV/sec 10,000 mV/sec 1 -596 -638 5 -606 -634
36 -609 -634 25 -594 -626
24 -568 -636
34 -584 -663
78 -704 -746
82 -428,-610 -414,-769
88 -559 -629
108 -614 -593
103 -638, -769 , -875 -756
2-NO2-Imidazole -634 -693
5-NC>2-Furan -487 -638
4-NO2-Benzene -712, -1106 -735, -1268
Example 33
152 Γ 2011
Clonogenic Survival Assay
May [0416] The phosphoramidate alkylator prodrugs of the invention were tested in the assay as
follows. Exponentially growing human H460 cells (obtained from the ATCC) were seeded 05 into 60mm notched glass plates at a density of between 2.5 and 5 xlO5 cells per plate and
5 grown in RPMI medium supplemented with 10 % fetal bovine serum for 2 days prior to initiating drug treatment. On the day of the test, drug stocks of known concentrations were prepared in complete medium, and 2 ml of the desired stock added to each plate. To achieve complete equilibration between the surrounding gas phase and the liquid phase, the lid of the glass plate was removed and the plate shaken for 5 minutes on an orbital shaker. The plates
2011202075 10 were recovered and stored inside a glove-box. The glove-box was evacuated and gassed with either a certified anoxic gas mixture (95% nitrogen and 5% carbon dioxide) or with an aerobic (normoxic) gas mixture (95% air and 5% carbon dioxide). Cells were then incubated with the drug for 2 hours at 37°C.
[0417] At the end of prodrug treatment, plates were removed from each vessel, and the 15 prodrug was promptly removed from the cells. Plates were washed with phosphate buffered saline and a solution of trypsin-EDTA and then trypsinized for 5 minutes at 37°C. Detached cells were neutralized with medium plus serum and collected by centrifugation for 5 min at lOOxg. Cells were resuspended at approximately lxlO6 cells/ml and diluted 10 fold to yield stock concentrations for plating. The concentration of each stock was determined by 20 counting with a Coulter Z2 particle counter. Known numbers of cells were plated, and the plates were placed in an incubator for between 7 and 10 days. Colonies were fixed and stained with a solution of 95% ethanol and 0.25% crystal violet. Colonies of greater than 50 cells were counted, and the surviving fraction was determined.
[0418] HT 29 and cell based clonogenic assays were performed in the same way as 25 described above and in Example 31.
[0419] Cytotoxicity of compounds (Tables 1A and IB) were determined in hypoxia and in normoxia by clonogenic assay employing H460 and HT29 cell lines as provided in Examples 31 and 33 and expressed as IC90 in μΜ, and by anti-proliferation assay performed by modifying a multi-well assay described by Hay et al., J. Med. Chem., 2003,46:169-82 30 employing H460, HT29, HCT116, and DX-5 cell lines and expressed as IC50 in μΜ (see Example 29). The ratio of IC50 or IC90 determined in normoxia and hypoxia is called hypoxia
153 2011202075 05 May 20 22 21 20 18 17 16 9 P22 15 14 8 7 6 4 2 prodrugs 12 5 P3 11 10 3 P2 cytotoxicity S2 SI 1 Cpd#
0.5 0.9 1.4 1 of logP
the ration
present 0.7 0.25 3.4 1 0.08 8.5 5.2 >10 1 1.6 4.5 0.01 0.14 0.7 1.7 4.5 40 >20 0.4 >40 100 7 8 44.0
(HCR) P Hypoxia
invention.
and 0.2 .572.0 0.35 0.25
C can
be 7.8 9 8.5 9 >100 >100 >100 >100 16 >100 1 100 0.6 25 3.5 >100 3.5 >100 >100 >100 >100 >100 >100.0 10
a
measure P Nonnoxia Table 154 35
75.0
of 1A C
the
hypoxia 23 26 3 8 9 >12 >50 60 >20 >1 >10 60 12 ca. >140 >5 >12 >2 >14 >3 >5 180 1 180 HCR
1 P
selective 175 200 40 C
cytotoxicity
of
the
ο CN & 23 0.04 0.2 5 10 125 50 s 24 0.01 4 400 in o 25 0.05 50 1000 26 0.1 35 350 m 27 2.5 100 40 r- o 31 83 >100 >1 CM o 32 50 100 2 CM 34 <0.01 1.8 >180 o 625 CM 35 0.075 50 36 -0.1 0.88 0.2 >100 >100 >110 >500
a
5 Table IB j Comp HT29 DX-5 HCT116 No. P C HCR P HCR P HCR H N H N P C H N H N 1 50 100 0.4 100 >2 >100 1 >100 >100 23 7.5 2 5 10 >100 >10 6 55 >100 >2 5 >100 >20 7 7(100) >100 >5(1) 0.6 83 140 36 55(35) >100 3 >2 7.5 >100 >13 5 >100 >20 25 16 >100 >6 1 >100 >100 0.9 >100 >100 34 0.8 57 70 0.13 10 77
P = Proliferation; C = Clonogenic; H = Hypoxia; N = Normoxic
Example 34
155 2011202075 05 May 2011 15 5
oxygen- determined twice, per (Guava, centrifuged, concentrations [0420] ΓμΜ
60mm 5 0.5 0.05 0.005 0.0005 0 μΜ 50 5 0.5 0.05 0.005 0 μΜ
and
Hayward, and Cells
dish. incubated
using
and concentration-dependent |
Nitrogen Air Nitrogen
Air Nitrogen Nitrogen Air Nitrogen Air Air Nitrogen Nitrogen Nitrogen Air Air Air Nitrogen Nitrogen Nitrogen Air Air Air Nitrogen Air for (H60,
After fixed
Guava
2 CA).
h
for PC3 Effect
under in 24
additional Cell The 75% h
and
attachment, | of
either
G 35 21 data 55 55 43 55 56 59 Cycle 52 55 60 54 Go/G! 47 33 42 23 07 58 30 58 50 38 56 14 59 Go/G, ethanol
HT29) Compound q ’ /G
demonstrate
i normoxia 22
reagent
were manner at h
cells
IS in least HT29 H460 38 33 PC3 12 13 12 S
12 11 12 6 13 10 13 31 12 S 14 15
19 11 12 11 11 12 18 25 fresh
seeded
(Guava,
156
were (air)
on for
in
cells
that cells cells
medium. multiple
Cell 24
or exposed | at
Compound G^A
65 40 28 59 59 28 42 26 30 G h 38 11 17
Hayward, 26 46 anoxia 32 44
32 33 32 34 31 28 33 32 g a Cycle
2
at 2 density /M /
m -20°C.
human Cells
to
(nitrogen). Distribution
Compound
of CA)
25
were Cell
cancer 1.0
induces
by
x cycle trypsinized,
flow 1
Cells cell 0
6 25
cell cells/3ml distribution
cytometry lines. at
were
cycle the
indicated washed
medium arrest
was
in
Ο CM
0 Air 50 14 36 Nitrogen 47 13 39
May 12 35 0.005 Air 52 Nitrogen 46 14 40 05 0.05 Air 50 15 35 Nitrogen 37 11 52 0.5 Air 48 14 37 Nitrogen 8 8 84 5 Air 47 13 39 Nitrogen 14 50 36 5 Air Nitrogen
2011202075 Example 35 Spheroid Model [0421] Two human cancer cell lines were used in these spheroid studies to determine the 5 efficacy of the hypoxic activated phophoramidate alkylator prodrugs. HT29 colorectal adenocarcinoma (colon carcinoma) cells were seeded directly into a 125 ml spinner flask at 10,000 cells/ mL and grown in RPMI medium supplemented with 10% FBS and antibiotics. As these cells divided, they adhered to each other and formed spheroids. H460 lung carcinoma cells were seeded into a flask coated with a non-adherent surface to form small 10 balls of cells that can be seeded into a spinner flask. To initiate H460 cell seeds, 150 cm2
tissue culture flasks were coated with 1% agarose and then 10,000 cells per flask were added and allowed to grow in RPMI medium supplemented with 10% FBS and antibiotics for 3 to 5 days before seeding into spinner cultures. For both cell lines, growth medium was changed every day after the spheroids became visible to the eye. 15 [0422] In order to, determine the morphology and the location of hypoxic regions within an intact spheroid, whole spheroids were prepared for histology. For frozen sections, intact spheroids were washed in phosphate buffered saline (PBS) and embedded in OCT and rapidly frozen in a dry ice/2-methylbutane solution before being stored at -80°C. For paraffin embedded sections, intact spheroids were fixed in a freshly prepared solution of 4% 20 paraformaldehyde in PBS and subsequently embedded and sectioned. [0423] To assess the ability of a phosphoramidate alkylator prodrug to penetrate to the inner hypoxic cancer cells, become activated, release the phosphoramidate alkylator, and kill those inner cancer cells, the clonogenic survival of spheroids exposed to drug for 2 h was measured.
157 Γ 2011
[0424] Spheroids were placed in a new growth medium and incubated for at least 1 h before beginning experiments. Spheroids between 500 and 600 pm were isolated by filtering May the spheroid culture through a series of sterile mesh filters of defined size. Between 10 and 05 20 spheroids were placed on a siliconized notched 60 mm Pyrex dish in 3 mL of medium
5 with the desired concentration of the test compound. The dishes were placed in sealed aluminum vessels and exposed to a series of evacuations and gassings with certified gases containing 5% CO2 and a defined amount of O2 (0% O2, 3% O2,10% O2 or air). Spheroids were incubated in a shaking water bath to ensure both the equilibrium of the dissolved O2 in solution and the integrity of the spheroids in solution for 2 h. The test compound was 10 removed and the spheroids were washed before being completely digested with trypsin. 2011202075 Since the necrotic core contains cellular debris a treatment with DNase I was required to yield a uniform single cell-suspension. Cells were resuspended at 106/mL and plated for
clonogenic survival.
[0425] Initial dose response experiments were performed in monolayer cells under nitrogen, 15 0.6% O2, or air to establish the appropriate dose range and the oxygen dependence of phosphoramidate alkylator release from a phosphoramidate alkylator prodrug. Clonogenic survival was the end point and the data are summarized by the IC90 or C90 values (the inhibitory concentration required to kill 90% of the cells and yield 10% survival). Daunorubicin and cisplatin, each of which penetrates into speroids to a different extent, were 20 employed to kill the outer aerobic cancer cells of the spheroid. Daunorubicin was used to penetrate the outer layers of a multicellular spheroid due to its high affinity toward cells and cisplatin was used at doses appropriate kill only the outer aerobic cancer cells. As a control for a bioreductive drug that killed cells under hypoxia in monolayer cultures, but not in multicellular cell culture due to its high reactivity and poor penetration, Tirapazamine was 25 used both in monolayer based experments and in spheroids as tabulated below for H460 cells exposed for 2 h.
IC90 values for H460 cells exposed as monolayers or spheroids Drug Monolayer Spheroid N2 0.6% 02 Air 10% 02 Cisplatin 4.2μΜ 7.7μΜ 7.3 μΜ 8.0μΜ Daunorubicin 0.16μΜ 19μΜ Tirapazamine 14μΜ 27μΜ >100μΜ >200μΜ
158 2011202075 05 May 2011 15 10
lying another measurement cisplatin spheroids determine [0429] activated, [0428] [0427] [0426] IC90
cells for
Cisplatin Daunorubicin Cisplatin Compound Daunorubicin 97 Compound Compound Compound 35 70 24 25 25 Compound 36 Compound 1 Phosphoramidate
Similar agent
or H460 The A
as and their in
daunorubicin series
tabulated
the phosphoramidate
of that kill
cells results
ability +
clonogenic spheroid
of targets Compound 25 25 the
Ν2 0.2μΜ 0.35 n 0.1 0.07 1 1.25μΜ
Antiproliferation exposed phosphoramidate +
2
μΜ hypoxic
below: for to
Compound μΜ
μΜ μΜ alkylator penetrate
and aerobic the and
survival.
as
the efficacy
25
kill cells.
3μΜ 0.6% alkylator monolayers Monolayer 0.25 0.6 0.6% Monolayer
spreroids cancer
hypoxic prodrugs 25 into 2.3 IC50 12 14
μΜ
02 The
μΜ
The Assay of alkylator Ο
the
2 2.3 9 IC50
17 cells. Example
prodrug Compound (μΜ) results
results
cancer inner exposed
or
demonstrate - 159 Air 40μΜ
4μΜ 20 Air 75 (μΜ) 100
DNA spheroids
prodrugs
are μΜ μΜ lying
was
are μΜ 36 cells IC99 5.4
for
tabulated Mutant
25 tabulated
combined hypoxic
alone
2 were (μΜ)
to the h 29μΜ Spheroid were 10%
22 »100 »100μΜ 25.5 3 Spheroid 13 15 10% to phosporamidate
Repair μΜ ability
demonstrated and
the
μΜ μΜ μΜ below.
02
tested below:
cancer
μΜ
Ο simultaneously
combination, in 2 μΜ
to Cells combination
in
penetrate cells,
spheroids
in
prodrugs become
the
followed
into with
with HT29 to
the
for
inner
by 2
h. 2011202075 05 May 2011 20 15 10 5
25 well retested Dulbecco from results. present initially (ATCC antibiotics: exposed removed, [0430] [0433] and and [0432] [0431] Compound
under
the 36
as 25 36 ATCC.
under Only The with The Chinese
invention
specific 0^1860),
with Cells screened to
hypoxia.
’ and s
TJV135 Comopund EM9 AA8 IrslSF UV41 Cell AA8
following Modified following
the
cell
the Cell
anoxic The were the IrslSF UV41
AA8
hamster homologous line gene
clonogenic (Anoxia lines cells
2/>100
8/>100 with
cells for Line following
Since AA8 exposed Air)
UV135
or
2 defect:
Eagle table
table
(ATCC defective Nucleotide Nucleotide None Homologous Homologous Mutant Base an 25 assayed. h aerobic
ovary
under
UV41
anti-proliferation / under
lists lists
Medium excision to
(Wild assay cell cells recombination
pathway CRL-1859),
cells selected
hypoxic conditions
participates
in the anoxic the (Anoxia The 0.02 0.02 0.85 4/>100 lines 7/>100
n
(ATCC excision excision
(as EM9 homologous Air) type) 2
IC90 effect mutant recombination recombination
repair (Gibco) following
previously were
IC90 doses
or or
values /
CRL-1
of aerobic
and
aerobic
repair EM9 repair
assay
to tested repair in (μΜ)
160 exposure
supplemented of
specific both assayed
table recombination for (Anoxia 0.03/20
phosphoramidate 0.2/95
cells
867), described) and UV41
and
with conditions. Air) pathway, >300 conditions,
clonogenic Air the 20 17
lists
those
(ATCC
DNA of
IRS1SF by
2,500 nucleotide
/
XPG XRCC1 XRCC3 XPF (None) Gene
various the
proliferation
demonstrating
with to Compound repair
cell
or
CRL-1861),
the
were survival confirm defect (Anoxia cells.
2/>100 6/>100 UV135 3,000
10% cell
lines,
Air) test alkylator excision
pathways
sensitive
lines
All compound fetal cells/well/500pL
the as the for / 25
cell
measured
sensitivity
UV41 to proliferation pathway possibly selected
bovine repair prodrugs
were
Compounds to lines (Anoxia 2/>100 IRS1SF 0.3
Compound
Air) was
cells pathway
obtained serum / were
cells
mutated, by also 59
of were
/
IC50.
the
and
25
as
produced a significant amount of monoadducts. However, UV135 which is also involved in nucleotide excision repair was not sensitive to Compound 25. The predominant lesions produced by Compound 25 were DNA interstrand crosslinks. These results were confirmed in UV41 and irslSF cells with the clonogenic assay. The exposure under aerobic conditions 5 produced the same spectrum of sensitivities as seen under hypoxia, indicating that the aerobic toxicity was also caused by DNA interstrand crosslink formation. Compound 36 exhibited a similar pattern of sensitivity in the mutant cell lines, indicating that Compound 36 also produced DNA interstrand crosslinks.
10 Example 37 Multilayered Cell Culture Assay [0434] This example demonstrates the effect of Compound 25 on tissue penetration using multilayered cell culture (MCC) and to assess any bystander effect. MCCs were incubated with oxygenated media (20% O2 & 5% O2) or hypoxic media (approximately 0% O2) and the 15 test compound was exposed from one side (exposed surface, normoxic side) while the other side was temporarily closed off (far side, hypoxic side). When MCC’s are incubated in media at 20% O2 or 5% O2 a gradient in oxygen develops from the surface exposed to the media towards the far surface of the culture. The furthest 50 pm of tissue becomes depleted of oxygen. The extent of O2 depletion is greater with 5% than the 20% O2 gassed media; 20 incubation with 5% O2 reflects the in vivo situation most closely. Incubating MCCs with media at 0% O2 models perfusion limited hypoxia, where tumor blood vessels become completely depleted of oxygen and test compound must penetrate extensive distances to reach all cells. This situation therefore poses a greater barrier to drug penetration, if binding of activated drug acts to limit its penetration. 25 [0435] MCC based experiments were carried out with media gassed with 0, 5 or 20% O2 for 45 minutes prior to and during incubation with the test compound. HCTI16 cells were grown to a thickness of 150pm on a solid support and one side of the culture was clamped off to develop diffusion limited hypoxia. Cultures were exposed to test compound for 1 hr under 0% O2,5% O2 or 20% O2 and efficacy assessed by measuring the inhibition of BrdU 30 incorporation. The cultures were incubated for a second hour in fresh media at 20% 02 and removed from the apparatus and returned to a normal growth chamber, where media flows over both sides of the MCC. Cultures were incubated for 24 hours prior to BrdUrd labeling and subsequent cryosectioning. BrdUrd labeling on the exposed and far sides of the MCC
161 were analyzed using immunohistochemical staining, microscope imaging and computer image analysis to assess the effect of Compound 25 on cell proliferation.
[0436] When cultures were exposed to graded doses of Compound 25 under 20% O2, 5 fold less compound was required on the far (hypoxic) side compared to the exposed (normoxic) 5 side to produce comparable results, demonstrating penetration and hypoxic activation of Compound 25. When MCC’s were exposed to test compound under a more physiologically relevant condition of 5% O2, Compound 25 was 10 fold more effective at inhibiting BrdU incorporation on the hypoxic side as compared to the normoxic side. Normoxic sides of cultures at 5% & 20% O2 were equally affected by exposure to Compound 25. 10 [0437] Compound 25 is more effective on the hypoxic side of cultures under 5% O2 than with 0% O2. Comparison of normoxic versus hypoxic sides of cultures under 5% (¾ demonstrated that Compound 25 penetrates effectively through relatively well oxygenated tissue. Compound 25 is capable of killing hypoxic cells located about 150pm from functional blood vessels. Approximately 3-fold reduction in exposure to Compound 25 to the hypoxic 15 side was observed under 0% O2 relative to the exposure under 5% O2 conditions. Bystander effect was observed only at the highest concentration.
[0438] The following table lists the effect of e exposed to graded doses of Compound 25 as measured by IC50 (concentration to inhibit BrdU incorporation by 50%). Side 0%Ο2(μΜ) 5%Οζ(μΜ) 20%Ο2(μΜ) Hypoxic -1.1 0.7 2.6 Normoxic ~1.7 8.0 >10
20 Example 38 Metabolism of Compound 25 By Human and Mouse Microsomal Protein [0439] An in vitro assessment of metabolic stability of a phosphoramidate alkylator 25 prodrug (Compound 25) was performed using human (HLM), rat (RLM) and mouse (MLM) liver microsomal proteins containing cytochrome P450 enzymes. A solution of Compound 25 (500pL, 5μΜ) was prepared by diluting a DMSO stock solution 100 fold in a water:methanol bridge solution, adding microsomal protein (1 mg/mL) in PBS/MgCl2, and enzymatic reactions initiated by adding an NADPH solution. 50 μΐ of the reaction mixture 30 was withdrawn at 0,10, 20, and 30 minutes after addition of the NADPH solution, the
162 2011202075 05 May 2011 5
26 34 25 23 6 7 No. 16 5 Comp. 1 proteins used study amount
as
compared
positive
of were 28 56 92 71 40 0 0 min) 5(20) 90 (%at30 (MLM) stability Metabolic
Compound Testosterone Nifedipine Testosterone Nifedipine 25 25
precipitated
HLM controls. Compound Compound (at 85 min) 85 (at 84 85 60 (85) 102 30 (% stability Plasma 100 100
min)
20 at 20
25 to
RLM by
250 250 The (mg/ml) MTD with
reversed
first
(Tables acetonitrile min) (6.7 0.11 0.15 0.08 0.26 1 (hr) Intravenous
1/2 study
phase
2A
27.5 7.8 compared Cmax 35 (pg/ml) 16.9
Metabolic Metabolic administration
and and HLM 127% RLM LC-MS/MS. 22% 65% 84% 163 0% 6%
Table Table
Table
2B) the 2.3 4.5 x (gg/ml AUC 12.5
hr)
clear
RLM 2A 2B
in
1 stability stability
mice
3.3
(Vkg) Vss supernatant 1.48 1.25
Nifedipine to
MLM
(% (% 368 /kg) 4.0 CL (ml/min 185
at at
(Table
30 30
MLM 137% RLM was
33% and 89% 2% 4% 6%
min) min) min) (11 0.18 0.16 0.25 (hr) 1 in Intraperitoneal 1/2
mice testosterone analyzed
1)
and 22.9 8.5 3 29.9 (pg/ml) Cmax
the
administration
for
second
were
3.5 the x (pg/mJ AUC 1.0
hr)
2011202075 05 May 2011 10 5
(mg/kg) determined [0441] were (mg/kg) 35 36 [0440] 150 Dose Drug 150 100 128 50 50 24 37 85 37 23 23 36 36 l l 5 5 a a “
a ·* determined ’
a Balb/c Various ... Various 90 56 Route (mg/kg)
i.v. i.p. i-P- i.P· in Dose 25 50 25 20 20 50 50 50 50 50 50 50 50
CD-I mice
in plasma Iv plasma 30%PEG/70% 30%PEG/70% 60 min) 85
CD-I Vivo Formulation mice Route i-P- i.p. i.p. i.v. i.p. i.p. i.v. i.v. i.p. i.v. i.v. i.p. ί·Ρ· Saline Saline Saline Saline
Iv
or pharmacokinetic Pharmacokinetics except mice 400
Vivo tumor Cremophore: CremophorerEthanol
except
25%PEG/75% 25%PEG/7S% 25%PEG/75% 25%PEG/75% 25%PEG/75% 25%PEG/75% 25%PEG/75% 25%PEG/75% where Pharmacokinetics
pharmacokinetic 0.24 Formulation (min) 5.00 5.00 Tmax 15.0
15.0 10% 10% 2.0 where
noted (1:2:7) (1:2:7) Saline 27.7
Ethanol parameters
PEG PEG
of Example Example 164
noted as (pg'mL)
Table Phosphoramidate Table
Saline Saline Saline Saline Saline Saline Saline Saline 27.5 22.9 Cmax 3.38 90.1 15.8 listed 10.8 : :
Saline Saline
as of parameters
4 3
40 39 listed Compound below of
phosphoramidate
1.27 (pg-h/mL)
(min) below 5.00 2.00 5.00 5.00 2.00 5.00 5.00 5.00 Tmax 5.00 5.00 5.00 15.0 15.0 AUC in 1239 325 438 784 307
of Table 77.4 Alkylator
25 Compound
in (pg/mL)
Table
4. 7.60 3.85 3.93 7.80 44.0 Cmax 3.00 35.0 29.9 27.7 8.50 15.0 12.6 16.9 0.18 Half-life (min)
95.2 58.7
11.0 Prodrugs ND alkylator 6.7
3.
25 44 h/mL) AUC
64.0 1439 89.1 57.4 (pg- 12.5 270 210 646 102 172 196 136 were
-
prodrugs
(%)
F* - - - - - 26.1
(min) Half 4.10 9.00 2.56 9.60 23.2 4.67 8.31 8.87 10.0 15.3 11.0 14.1 life -
2011202075 05 May 20 25 20 15 10 5
performed midazolam, for analyzed formic suitable enzyme/substrate prepared test 25) CYPC2 inhibitor phosphate phosphate, 100 100 [0442]
25 50 50 50 50 CYP2C9,
were compound /zL mL
acid
Eight ‘ ‘
of Tumor 19, Bioavailabilitv incubation of
by for (such prepared ude
a in
dehydrogenase, cofactor
pH
quinidine P·
in i.p. i.p. i.p. i.v. testosterone pre-warmed
adding
the
duplicate. 0 reaction (S)-mephenytoin
mice water
Cytochrome
· 7.4, PK ranges as
metabolite
solution. furafylline
along with
30% 30%
at 2.6 solution 50
(400
for
37 10% wells
from
mL
mM H460 Saline Saline Saline Saline Saline
A
PEG/70% PEG/70%
and °C, with enzyme/substrate CYP2D6 mL
summary
of andl PEG
P450
Isoform forms A
0.0229
NADP+, with
to the nifedipine 2C19 tumor for
2C9 1A2 of 10% eight control
for inactivate
:3
reactions acetonitrile
CYP1A2, Inhibition 100
of
serial CYPC219,
and formic /zM wells
of the
6.6 /zL reaction
45.0 5.00 15.0 15.0
2.0 ketoconazole the for to
dilutions probe
Example of mM
the of
were 200 acid
Table solution. IC50
sulfaphenazole CYP3A4)
of
a 1 for
165
enzymes,
:3
solution
glucose-6-phosphate,
dextromethorphan
Control the /zM. with substrate
(400
terminated serial 0.20 2C19).
6.0 8.6 values 6.33 0.93 27.5 38.5
5 9.2
of
Metabolism 41
no
IC50( The mL
the A for
using
dilutions
containing
inhibitor
then zero
are
The test of (phenacetin CYP3A4). reactions 'mM)
for
by
acetonitrile listed
Compound
HPLC/MS/MS. adding compound time-point reactions
40.4 the
247 635 CYP2C9, 177 of
~10
of NI - NI 25 was
for below. Compound 50
addition
a were
100
suitable 0.8
The mM also for CYP2D6
were
for
control (such
U/mL mL initiated CYP1A2,
N-benzylnirvanol
concentrations prepared. potassium
2C1 of 4.43 25.7 7.91
10.1
prepared of positive
Each
25 50 as
of
and 9) reaction
Compound mL by
glucose-6- in
assay diclofenac
.After adding
water
of
control 13.6 and - - -
10%
was
of was
a
to
for
2011202075 05 May 2011 25 20 15 10 5
parameters metabolite(s) be mg/kg Dawley monitored parent high-performance administration whether hepatocytes [0444] collected from spectrometry 60 [0443] Determination
determined and
100
compound Compound Pharmacokinetic Compound 120
rats each
and to
by
are minutes
at 520 are
in (LC/MS/MS). potential compared comparing
in a of computed.
plasma determined concentration
Determination amu. beagle of
20
(Compound liquid 25. 25
the by Formed
mg/kg
is NI
The The metabolite 3A4 quenching In 3A4
are
3A4 to dogs incubated parameters
chromatography the =
Vivo
the
No
pharmacokinetics product
determined Compound
Testosterone following
in Midazolam peak Potential Nifedipine
and 2D6
product
25) significant of Mouse,
of Pharmacokinetics
10
cynomologus
heights with is and the with
ion
Rat,
of Mass μΜ. related
Potential single metabolites
the ion
Compound Rat, acetonitrile by spectra 25.
mouse,
Dog
Example inhibition Example
(HPLC) The
at Balance appearance
a spectrum
Dog Concentrations
to LC/MS/MS
each Example
intravenous
166 and of
reactions
Compound
of
rat, Metabolites 0.049 monkeys
0.03 0.21 0.10 Compound and
Monkey
time the
of 42 detected 43
in are
25
Study prior dog,
of
Compound Human
conjunction potential
and 44
identified of point the
are
monkey
to method potential
administration
in
following its
25.
parent
centrifugation
of stopped
of Rats 25
acquired. Hepatocytes metabolite(s)
Compound
The metabolites Compound >50 >50 >50 and
NI
by 25
and and with
compound metabolites
disappearance performing
its and single at
mean human
metabolite(s) tandem 0
its of (pre-incubation),
and 25
25
intravenous
in are Metabolite pharmacokinetic 5,20,
to cryopreserved
and Sprague
analysis mass over
subsequently
determine full
50
its of
scans time
will and the
(s) by
are
100 also
in 30,
2011202075 05 May 2011 30 25 20 15 10 5
using Compound 30 samples is three bladder images prostate [0447] contents), intestine marrow, lens), phosphorimaging (brown of plasma, (LSC). times 25 Storm CM carcasses 25. [0445] [0446]
determined
minutes the
as
At 3600
concentrations
human
a Harderian and
image
820
The
specified are single
and (and
are and
gland, cecum and Normal
Sprague-Dawley
are cryomicrotome. small concentrations
produced or
at 25) protein
then white),
the
ultrafiltrate contents) analysis
contents,
embedded 2500 using 860. intravenous
salivary and
and blood intestine
gland, equilibrated times,
and plates
x binding The analysed
adrenal ultrafiltration. contents, with
for
g. software. bile-cannulated
and
are liver,
heart, gland, one concentration
A
in along each
standards
(and of Compound
measured dose. rats plasma 75
2%
Collected
gland, in
rat
total lung, using
Quantitative
to
kidney Plasma animal. epididymis, pL mouse,
contents), seminal with
CMC, are per
37°C. Exposed
Blood radioactivity aliquot
lymph blood, are
administered LC/MS/MS. for Ultrafiltration time 14
by
(cortex,
25
sections C
Protein
frozen
Sprague-Dawley rat, of analyzed the vesicles, The
plasma,
image autoradiographic point
into
spleen, radioactivity of
node brain screens
dog, Whole calibration esophagus,
Centrifree® the
Example
Example into 167 a
medulla, Binding
is
are
are analysis. Centrifree® submaxillary), monkey
(cerebrum,
ultrafilitrate
for
urine, skeletal euthanized. a spinal The
Body a
are
is freeze-dried, single determined
block
concentration performed
ultrafiltrates scanned
curve.
46 eyeball
in of 45 feces
papilla
cord, Autoradiography rats and
apparatus muscle, Autoradioluminographs intravenous
select Compound standards and
cerebellum,
device
are human is
and
trachea, Blood
sectioned
using (Uveal pancreas, by and
spiked
by tissues mounted administered
are
skin,
liquid
of
are aliquoting entire in is
plasma for is a collected radioactivity.
25 dose centrifuged
triplicate.
tract, thyroid
with Molecular
analyzed centrifuged stomach including
subsequent medulla)
at scintillation
pituitary
and section),
40 of
the aqueous of
exposed plasma 14 1
pm and
at Compound 4C-Compound I.S.
C-Compound
(and and
All
specified
adipose Dynamics bone,
at in and gland,
urinary
large to
(deuterated Frozen calibration
37°C
quantified
a plasma humor,
counting obtain
spiked
digital Leica on
bone
for 25
rat
at
2011202075 05 May 2011 25 20 10 15 5
5% American pathogen-free reduced measured mg/kg mm weeks compared Compound growth (L xenograft PBS) supplemented cancer caliper only in [0448] Compound and [0449] [0451] [0450]
the
x CO2.
3
PEG
W 5FU (day
at
peritoneal
of
Group of employing 2 twice Administration compared
)
tumor a
Example
Female The /2, 4 6 5 2 The 3 age, (lOmL/kg
5FU daily twice was at 8), Type to
36 36
where 10
vehicle.
per each body HT-29
were
with conditions. monitored and (in at growth
mg/kg dose weekly. Culture
subcutaneous week. CB17/SCID a
to
saline)
a
group 47
daily 5FU
L weight
10% allowed
HT-29 each),
administration Treatment of
cells is demonstrates In
to (Group
of
20,
the resulted
Tumor Collection.
dose addition fetal
of a
60 20 (Group by Compound
were
Human
Group of greater 60, human length
10
to 200 + + externally 60 20
each bovine of mice
8) 5FU 5FU acclimatize mice space. or
(mg/kg) volume harvested
20, in
as 5,6 combinations 200
1),
and degree colon mouse colon
greater
(purchased
the of
tabulated 60, was
The
Compound serum.
36 and
mg/kg
When vehicle W
usefulness measuring
(V)
and
at carcinoma
administered carcinoma cell
is than 7, was
from
and for 20,
the was
200 respectively) Example
the
(Groups 168 Cells lines below.
at alone. recorded
Group 5FU dose 60,
from width culture of
determined tumors mg/kg 46.9 least 35.7 20.2 34.6 36 16.1
tumors of 60 were and were
% alone.
cell alone xenograft related
Charles
a
and Administration
of three 1 2, 47
Inhibition for
compound 200
and
given twice
line cultured grew
maintained 3 a
200
in and three (dissolved xenograft. and
mg/kg/day inoculated days, inhibition
by HT-29 two River,
to per 2-3
mg/kg mouse compared Group 4,
the weeks,
an 13.3
3.3 vs dimensions in
respectively),
and week. hours of - - -
following average RPMI Cambridge, was in
this
in of model. 8 of handled Tumor
of
at each a
vehicle
30%
Compound
after a
obtained 3
37°C to tumor Growth invention
combination
1640
x
a volume reduced
cyclodextrin using 10 group volumes a
equation: incubator under
dose alone 6 and
growth media MA),
cells/
of from
a
in
receiving
36
each of
of tumor
digital
(saline
treating
7-8
were
animal
100 10 the of V
with
in =
2011202075 05 May 20 20 15 10 5
peritoneal [0454] inhibition. in occasional American pathogen-free weeks Example Compound (day the Compound 5% cancer [0452] [0453]
other
procedure EtOH,
(n= 8), Group
lb* la* of employing Associated 4 9 Ί 6 2 8 5 3
groups each Female
10)
Example 47 7 age, 8
subcutaneous
Type
5% mortality,
25). 25
above.
group
were described
conditions. Not (2.5 Cremophor
as
Culture CB17/SCID The
Compound well. Compound Compound Compound Compound a Compound
48 with
Applicable mg/ml
allowed of NCI Treatment
body particularly Vehicle demonstrates
Saline Taxol Taxol Taxol mice
these space. Collection. in Overall, 200 H460,
in Human and weight Example
was 5FU to
10% + mice
anti-tumor 25 25 25 25 25 25
acclimatize (NA) 90%
5FU When human
administered
Compound in
PEG; and colon (purchased
the
the saline; 47 The Treatment
the tumor
colon usefulness
and group
administration
carcinoma Example effects, (mg/kg) cell
tumors
Dose
for NA NA NA NA 100 100 administration 25 50 25 inoculated 50 10 10
36 169 volumes lines carcinoma
for treated at from
protocol showed least there 38.7 58.2
grew three
of 48 were
cell Charles
a
with three
were was
compound at to weeks, route
line cultured varying xenograft
1 an
- (q2d (q2d (q2d (qld (qld (qld the
(q7d (q2d (q7d (q2d (qld some x days,
measured
River, NCI i.v.
average - 1
high 0
as i.p.)
6 x x x x x x
2 x x x x x
rates
degree and
23 cells/
H460 and
tabulated 3) 3) 3) 5) 5) 5) h - of 3)/week
3)/week 5)/week l)/week l)/week
mouse
Cambridge,
Regimen dose and
after /week /week /week
/week /week /week
this
harvested volume
handled of NA as
animal
was
Taxol
of
tumor of described invention administration
model.
x
x x x x x weight x x x x
x in Compound 2 2 2 2 obtained
2
2 2 2 2 2 2 of
the under
in (1 weeks weeks weeks weeks weeks
weeks weeks weeks weeks weeks weeks growth MA), according
100
the
mg/ml table
loss
in in
mm
from 7-8 treating
and below: 36
of in 3
to
but
the
2011202075 05 May 20 25 20 15 10 5
weeks model. particularly benefit when cancer doses. administered comparisons saline therapeutically indicate some [0458] every similar [0455] [0457] [0456]
degree vehicle
day
or
of
as
qld/qd Groups to degrees
Female that no
Example Using Results The
age, demonstrated
that dosing
lung treatment
Compound of
in
with Group results treated were provided
=
effective
la this the weight of CB17/SCID 9 7 4 6 3 8 5
cancer, every are provided
a and tumor 49
mouse
allowed analysis. decreasing
presented demonstrate mice
describes
were
Taxol lb,
loss day; employing by
25 dose alone growth
n
had to the
additional
is but added
to q2d =
mice HED
of
efficacious
5;
acclimatize standard or reached
in frequency not the
Group
= about inhibition
in
Table that
65.9 46.7 52.6 63.1 50.1 (purchased in
a conversion, 46 52 every usefulness large
combination
H460, order
benefit. %
all
2 2
a
chemotherapeutic X2
to Inhibition
volume enough second three in of
for to
and about non-small based 10 this Example
dosing 170
indicate
from at
of
Each
regimens Compound that
model
least
day; to a of with
8 on Group
compound vs
mg/kg/day, Charles
cause
combination 946 38.8 for 31.7 combination 12.5
tumor
q7d three - - - any - 49 lung Taxol™,
of
higher
mm
for
6
any lung vehicle = agent,
(q2d
25
carcinoma days,
River,
volume 3 dosing every .
mortality. of can
doses Groups cancer
for x wherein
this therapy, taxol.
therapy and
3) effects be
Cambridge, the
seventh
Compound measurement
/week
compared
invention administered handled
and xenograft treatment of
Overall
the
but was particularly 5
provides
x day. mice
daily
2 are
associated
under
MA), weeks in 25 to
the mouse of not receiving
treating on dose lower
provided at
cancer,
additional
results
used
day 7-8
a with
can
with
29
for
be
2011
pathogen-free conditions. Human non-small lung carcinoma cell line NCI H460 was obtained from the American Type Culture Collection. The cell lines were cultured and
May harvested as described in Example 47 above, and inoculated at 3 x 106 cells/ animal in the
05 peritoneal subcutaneous space. When the tumors grew to an average volume of 100 mm3
5 (day 8), each group of mice (ten per group) was administered for three weeks, as tabulated in I
the table below: Compound 25 (2.5mg/ml in 10% PEG; administration route - i.p.); Compound 24 (0.3,0.1mg/mI in 10%PEG, administration route - i.p.) and Taxol (1 mg/ml in 5% EtOH, 5% Cremophor and 90% saline; administration - i.v. 2 h after administration of the test compound).
10 Treatment protocol 2011202075 No. of Dose Group Treatment Regimen Mice (mg/kg) 1 10 Vehicle* NA (ql d x 5d)/week x 3week 2 8 Taxol 10 (q2d x3)/week x 2week 3 8 Compound 24 3 (qld x 5d)/week x 3week Compound 24 1 (qld x 5d)/week x 3week 4 9 Taxol 10 (q2d x3)/week x 2week Compound 24 3 (qld x 5d)/week x 3week 5 8 Taxol 10 (q2d x3)/week x 2week Compound 25 25 (qld x 5d)/week x 3week 6 8 Taxol 10 (q2d x3)/week x 2week * - 50% PEG [0459] The body weight and tumor volume was determined as described in Example 47 above. Results for tumor growth inhibition measured on day 27 are as tabulated below. Comparisons were made on day 27 because that was the last day of measurements for the ) 15 vehicle group and those animals were sacrificed.
Group % Inhibition vs Group 1 Group 2 3 39.9 - 4 16 -32.7 5 51.5 23.3 6 56.8 31.7 2 36.7 -
[0460] These results demonstrate that daily doses of 3 mg/kg of Compound 24 and 25 mg/kg of Compound 25 inhibited tumor growth and that Compound 25 had a slightly greater benefit both as monotherapy and in combination with taxol. These effects were accompanied 20 by mild weight reductions, particularly in the Compound 25 + taxol group.
171 2011202075 05 May 2011 20 10 15 5
cancer, therapeutically Female lung therapeutically alone. was tumors age, cancer saline) above, conditions. [0461] Culture [0462] 10%PEG), *-
administered were cancer,
tumor
as and on grew alone CB17/SCID Collection. 7** 6** 4** 3* 2* 8** 5** Group 12** 11** 10** 1*
Using Example
demonstrated
allowed the
administration inoculated
Human
alone location
to
or days
effective effective an the in
4 9 9 # 9 9 9 9 9 9 8 8 8
for
to average or 50 combination
the mouse mice
colon The
mice
acclimatize
in flank; three
describes
at combination
employing combination
dose dose 3 cell
(purchased route carcinoma
to
x No volume No CDDP Compound CDDP Compound Test weeks, Compound 5-FU Compound CDDP Vehicle Compound 5-FU 5-FU Saline
**
10 lines HED
of of
treatment treatment - 6
the
- Article with
tumor cells/ 0.25 2
for
i.p.,
mg/kg/day, were
as conversion,
of a usefulness
(saline) therapy
at HT-29,
Taxol™,
Treatment with tabulated cell mg/kg/day,
from
100
anima] administrated
24 24 24 24 24 location least
cultured
line
Taxol™. mm
Charles
was three Example human
172
in N/A HT29 N/A
5 6 5 3 50 6 6 3 50 Dose 5 (mg/kg) 50 3 10 10 for
of and
in Compound
(day
peritoneum;
protocol the
scheduled; and
ml/kg
ml/kg a
for the the days,
compound Compound
River,
peritoneal
colon
was
8), 2
harvested the table
treatment h 50
each and
before treatment obtained
Cambridge, carcinoma below: 25 N/A iv, ip, iv, N/A ip, iv, ip, ip, ip, iv, iv, ip, iv, Routes, iv,
handled 5FU
Q3d group
can of once once (qld q3d (qld once q3d (qld (qld (qld q3d (qld q3d
24 subcutaneous
as 5-FU of
this
alone
=
can described
be
from Compound cancer, x
x x x of
Regimens x x x of x x x every
4 4 under 4 4
invention administered
or xenograft cancer, 5)/week 5)/week 5)/week 5)/week 5)/week 5)/week be
mice
MA),
(in
the cisplatin
administered
third
particularly
saline),
pathogen-free
American
(ten in
space. 7-8 particularly
x x 24
x x x x
Example
day in
mouse 2week 3 3 3 3week 3
per
weeks (CDDP;
week week week (in week treating
or at
group) When
a
CDDP Type
lung
at model.
of 47
a
in
the
r 2011
[0463] In control groups, tumors were implanted in two locations as part of separate study of effect of location on control group tumor growth. These results had no impact on the
May interpretation of the study and all treatments were compared to the vehicle group with tumors
on the same are of the body. The body weight and tumor volume were measured as described 05 5 in Example 47. Tumor growth inhibition measured on day 25 when vehicle tumors had
reached the maximal size and animals in that group were sacrificed is tabulated below
Group % Inhibition vs Group 4 Group 6 7 44.1 - 8 42.1 - 2011202075 9 71.1 28.9 10 53.2 24.8 11 50.7 20.9 5 59.3 - 6 37.4 -
[0464] The results demonstrate that Compound 24 as monotherapy resulted in tumor 10 growth inhibition of slightly more than 40% whereas combining Compound 24 administered in combination with CDDP or 5FU provided about 50-70% growth inhibition. According to this Example, the most therapeutically effective combination was that of Compound 24 and 5FU. The effects on tumor growth were associated with minor decreases in weights of the mice during treatment; however the mice recovered the lost weight after the end of treatment.
15 [0465] Using the mouse to HED conversion, Compound 24 can be administered at a therapeutically effective dose of about 0.25 to about 0.50 mg/kg/day, for the treatment of cancer, particularly colon cancer, alone or in combination with 5FU or CDDP.
Example 51 20 [0466] Example 51 describes the usefulness of a compound of this invention in treating cancer as demonstrated employing a H460, non-small lung carcinoma xenograft mouse model. Female CB17/SCID mice (purchased from Charles River, Cambridge, MA), 7-8 weeks of age, were allowed to acclimatize for at least three days, and handled under pathogen-free conditions. Human non-small cell lung carcinoma cell line NCI H460 was 25 obtained from the American Type Culture Collection. The cell lines were cultured and harvested as described in Example 47 above, and inoculated at 3 x 106 cells/ animal in the
173 2011202075 05 May 2011 30 25 20 15 10 5
therapy administration was tumors and Female Culture conditions. age, cancer lower particularly [0469] can therapeutically regimens demonstrate that delay 25. [0468] 25 times times treatment peritoneal [0467] CDDP 6
mg/kg
are
inoculated be administered were employed
with per doses. (Group
illustrated as
grew administered
(Groups is CB17/SCID Collection.
(Groups Example
Using These
demonstrated
allowed scheduled add week
was
subcutaneous a
Human lung
to daily significant
to 5)
route
initiated
at for
an
effective
the results for
6
the
or
cancer,
3 in for
2
and average to the dose
52
x the two
mouse and
colon the The - effects
mice with and
acclimatize three
10
describes 50 i.p., 7,
performed combination
in 6 employing of weeks Figure 3, dose
cell doses respectively). CDDP space. alone cells/ mg/kg
a
(purchased which carcinoma
respectively, 50 volume administrated weeks,
to
decreasing of
lines
related HED mg/kg,
CDDP
(Group
animal or of
1.
the (in daily When
groups for
in about Figure were
as with
of
a conversion, usefulness saline,
of combination
at decrease HT-29, Treatment
tabulated cell and
from in
100 dose.
in 4), frequency
the least each
a Results
cultured IV 4 this
of
Example 2 the saline
2
line 100 to mm Compound
tumors
IV). one h
shows 10 Charles
dose three These about model. peritoneal before human 174
mg/kg HT29
3 in mice
of
in
time), (day
Compound formulated for protocol
tumor and
a
of of the
with
days,
similar grew
52
8
data compound groups
River, received
CDDP
dosing Compound 8), colon was
mg/kg/day,
harvested with
table 25 Compound
5FU
volume
subcutaneous each and also to
at obtained
results
less
Cambridge,
an carcinoma below:
receiving
on version 25
for 100 or
handled
demonstrate vehicle group average
the of can frequent CDDP,
higher
as
and mg/kg
25
for
for this
25
described days from Compound
be
with of of
increase
the under at
100 50 (Group
invention xenograft space. administered
Compound volume doses mice
wherein
every MA),
50
the dosing
the mg/kg treatment
either mg/kg
that
mg/kg
pathogen-free combination
American
(ten
in
compared in 1), three When 7-8
both 25
of
3 Example compared
of the tumor
in
of mouse per CDDP
or
weeks 100
in in
Compound 25
of treating
Compound days daily dosing
6 at
the saline saline, group)
cancer,
mg/kg mm
a growth Type
to model.
at
for
of 47 dose
3 to
5 3 ,
5
or of
2011202075 05 May 2011 10 5
in ended. inhibition in cancer Compound [0471] growth sufficient Data 9 8 4 2 7 6 5 3 [0470] 10 1 (n Group
the saline,
=
are 9) combination
in
are
The based this at
The
of size Compound CDDP Compound CDDP Compound CDDP CDDP Compound Compound Compound Compound Compound CDDP Saline
25 tabulated 50mg/kg/day
tumor
Test model. results
body
and Group to
on 10
2 9 7 8 6 5 4 3 require
Article
tumor CDDP
groups;
growth weight
demonstrate below. These
25 25 25 25 25 25 25 25
that
volumes
and enhanced
the in and
effects Group
the 5 5 5 5 50 5 100 50 this 100 100 100 100 100 lOml/kg 100 24.2 36.2 44.2 51.8 50.7 48.1 31.3 30.1 28.2
mice (mg/kg) tumor Dose
mice
that %
model mgZkg/day at
were
1
the Inhibition recovered day
volume monotherapy be
effectiveness
of 25
accompanied sacrificed. Once q7dx2 Once q3dx5 Once (q2dx3)/week Once (qdx5)/week (q2d Q7dx2 Q3dx5 (qdx5)/week Once (qd
colon when 175
Group Administration with
was
x the vs 21.5 33.2 27.5 30.8
x
5)/week - - - - -
cancer, 3)/week
tumors lost determined a
2
administering variety
The of
body by
Compound
x x and results in x2week x2week
modest
x 2week 2week
of the weights
2week
Regimens that
as dose
vehicle
of described
body
treatment Compound
inhibition regimens 25
after
for weight group
the
treatment in
combination
treatment results of Example 25, had
loss,
tumor
formulated reached
more
of in
47. colon
of
so
r 2011 [0472] Using the mouse to HED conversion, Compound 25 can be administered at a therapeutically effective doses of about 4 to about 8 mg/kg/day, for the treatment of cancer, May
particularly non-small cell lung cancer, alone or in combination with CDDP, wherein the
05 daily dose can be administered with a decreasing frequency of dosing for higher doses 5 compared to lower doses.
Example 53 [0473] Example 53 describes the usefulness of a compound of this invention in treating cancer as demonstrated employing a H460, non-small lung carcinoma xenograft mouse
2011202075 10 model. Female CB17/SCID mice (purchased from Charles River, Cambridge, MA), 7-8 weeks of age, were allowed to acclimatize for at least three days, and handled under pathogen-free conditions. Human colon carcinoma cell line NCI H460 was obtained from the American Type Culture Collection. The cell lines were cultured and harvested as described in Example 47 and inoculated at 3 x 106 cells/ animal in the peritoneal subcutaneous space. 15 When the tumors grew to an average volume of 100 mm3, treatment was initiated in which
groups of 10 mice received vehicle (Group 1), CDDP at 6 mg/kg (IV one time, Group 2), Compound 25 at 150 mg/kg in saline, once a week for two weeks (i.p., Group 3), or the combination of the two agents (Group 4).
[0474] The results shown in Figure 3 demonstrate that 150 mg/kg per week of Compound 20 25 provided greater reduction in tumor growth than CDDP alone and that the combination of the two agents resulted in added benefit. These results also indicate that during the two week period of dosing mean tumor volume did not change indicating complete inhibition of tumor growth. These data indicate that Compound 25 administered at 150 mg/kg/day as monotherapy, once a week, is the most effective all the dosing regimens described in the 25 preceding examples (Examples 47-52). Little change in body weight was observed suggesting reduced toxicity with this dosing regimen.
[0475] Using the mouse to HED conversion, Compound 25 can be administered at a therapeutically effective dose of about 12 mg/kg/day, for the treatment of cancer, optionally administered at a frequency of once every week, particularly non-small cell lung cancer, 30 alone or in combination with CDDP.
Example 54
176 Γ 2011
(0476] Example 54 describes efficacy of Compound 25 via an ip bolus injection or ip infusion alone or in combination with Cisplatin in H460 xenografts mouse model. Female
May Nu-Foxnlnu homozygous nu/nu mice (purchased from Charles River, Cambridge, MA), 6
weeks of age, were allowed to acclimatize for at least three days, and handled under 05 5 pathogen-free conditions. Human colon carcinoma cell line HT29 was obtained from the American Type Culture Collection. The cell lines were cultured and harvested as described in Example 47 and inoculated at 3 x 106 cells/ animal in the peritoneal subcutaneous space. When the tumors grew to an average volume of 100 mm3 (day 8), each group of mice (ten per
group) was administered for three weeks, as tabulated in the table below: compound 10 Compound 25 (formulated as a 15 mg/ml saline solution, administration route - ip.,
2011202075 administrated 2 h before CDDP on the days the combination therapy is scheduled and CDDP in saline, IV.
Treatment protocol
Group Test Article Dose Regimens Dose Concentration: (mg/kg) Dose Volume 1 Saline 10 Q7dx2 0 mg/mL : 10 mL/kg 2 CDDP 6 Q7dx2 0.6 mg/mL: 10 mL/kg 3 Compound 25 150 Q7dx2 15 mg/mL : 10 mL/kg 4 Compound 25 150 q7dx2 15 mg/mL: 10 mL/kg CDDP 6 q7d x 2 0.6 mg/mL: 10 mL/kg 5 Compound 25 150 Q7d x 2 10 mg/mL: 15 mL/kg 6 Saline 0.2 ml 200 pL -1 week x2* 0 mg/mL : 1 pL/hr 7 Compound 25 15 mg/ml 200 pL -1 week x2* 15 mg/mL: 1 pL/hr * - Alzet pump, 200 pL for 1 week x 2 (re-implant new pump at the end of one week). 15 [0477] The body weight and tumor volume was determined as described in Example 47. The results are indicated in Figure 4. The data indicate that, while continuous application of Compound 25 alone or in combination with CDDP is efficacious, intermittent, such as once a week dosing can provide greater therapeutic benefit in the treatment of certain cancers such as non-small cell lung cancer.
20
Example 55 Compound 25 And Gemcitabine Combination Therapy (0478] A combination of Compound 25 and gemcitabine was administered to nude mice that were carrying tumors derived from type MiPaca2 human pancreatic cancer cells. 25 MiaPaca-2 tumor is a highly invasive, rapidly growing tumor that results in death within 20-
177 ) 2011202075 05 May 20 25 20 15 10 5
significantly administered stand gemcitabine cured. and treatment provided survival Group [0481] body survival. [0480] 5) [0479] powder. manufacturer 25 Compound Compound fluorescent 30
and
were days the
of images
4 2 6 3 resulted 5 Group 5 These 1 tumors *
care,
in compared
demonstrated formulated Four
Tumors modest Groups qd Gemcitabine and Group
untreated
protein.
24 25,
is = greater
with were
’ gemcitabine. results
within
s of
every of
were were
in Compound instructions. Gemcitabine Compound Gemcitabine Compound Gemcitabine Compound Compound Vehicle Compound
2
these reduction
3
greater
were
a 100%
significantly obtained to and
combination
than
in
administered considered day; demonstrate Mice a animals. Group
significantly tumors short was saline imaged 4
lethality
benefit that resulted qw
were
These in
obtained 24,
24 25 25 to 24 period 2.
=
and in
tumor The
remained
confirm
once or every
Five reduced
to animals administered
in
of
by
provided results that in gemcitabine/
i.p.,
tumor be
200 200 6 30 30 200 Dose 6 this lOml/kg
failed
Compound reduced
minor day size weekly commercially out
week. Treatment cured.
combination
as
effects. model
tumor at
treated of
30
(mg/kg) demonstrate but
cells tabulated to
zero effects
by
8
(Figure
until emit tumor no 178
No tumors
doses
Threshold of had
volume
Compound 25 In fluorescence
with additional
protocol
tumors
cancer fluorescence the
on
Group been
at below and growth treatment
qw (qd qw (qd 6). (qd (qd qw (qd of Schedule
in gemcitabine 30
end tumor that
vehicle
’ prepared
and
’ Group x x
x x x x transfected
mg/mg/day compared
x in x
Pharmaceuticals,
of
1, 5)/week 5)/week 5)/week 5)/week (8
tumor 3 3
5)/week effects
and 3 Group
25
prolonged
weeks weeks the
volume the mice/group).
weeks until with
combinations
control, 5 (Figure significantly
tumors study
regressed freshly reduction
2
on as
for for the for for Compound
to
for
with
were
and and
a survival. monotherapy 2
2 2 2 end at
survival.
single
2 7). gemcitabine,
grew
weeks weeks weeks weeks
according had which gemcitabine the weeks
considered
of rapidly Compounds Inc. in
prolonged
gene or
little
rapidly
the agent. animals
25
In time as gemcitabine/
Group
experiment
and
for contrast, a effect
after
to
with
dry
open
(Figure
to red
is
6 24 be
on
the
and
[0482] Using the mouse to HED conversion, Compound 25 can be administered at a therapeutically effective doses of about 2.5 mg/kg/day, for the treatment of cancer, particularly pancreatic cancer, in combination with gemcitabine.
5 Example 56 [0483] It is recognized that efficacious molecules for treatment of human diseases including cancer maybe toxic at doses near or sometime much greater than doses necessary to achieve beneficial effects. To determine appropriate dose and route of administering such a compound, it is necessary to understand its toxicity. Routinely, initial approaches to 10 determining the toxic dose involve the use of rodents such as mice to provide preliminary data that might support the design of similar studies in larger animals and humans. Test compounds (Compounds 24,25 and 36) were tested in mice as preliminary experiments for determining doses to be used in larger animals. Compound 25 was tested at doses as high as 300 mg/kg as a single dose and found to cause renal toxicides such as tubular necrosis and 15 protein spillage into the urine. Transient reductions in white blood cells were also observed. However, little toxicity was noticed at lower doses (100 and 200 mg/kg). These doses selected represent an approximation of doses that might be used in larger animals such as rats and dogs for the purpose of confirming that such toxicities exist and for predicting if renal function should be measured in humans.
20 [0484] Although the present invention has been described in detail with reference to specific embodiments, those of skill in the art will recognize that modifications and improvements are within the scope and spirit of the invention, as set forth in the claims which follow. All publications and patent documents (patents, published patent applications, and unpublished patent applications) cited herein are incorporated herein by reference as if each 25 such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an admission that any such document is pertinent prior art, nor does it constitute any admission as to the contents or date of the same. The invention having now been described by way of written description and example, those of skill in the art will recognize that the invention can 30 be practiced in a variety of embodiments and that the foregoing description and examples are for purposes of illustration and not limitation of the following claims.
179 180
CLAIMS 2012
1. A compound having the formula: Dec
06
wherein
2011202075 L is selected from the group consisting of CH2, CHMe, CMe2,
(6890905_2):RTK 181 2012
Dec
06
2011202075 each R.9 is independently selected from the group consisting of hydrogen, aryl,
heteroaryl, C1-C4 alkyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, heterocyclyl, C1-C6 acyl,
C|-C6heteroacyl, aroyl, heteroaroyl, C1-C6 alkoxycarbonyl, C1-C6
alkylaminocarbonyl, di C1-C6 alkylaminocarbonyl and C1-C6 alkoxy;
each X4 is selected from the group consisting of halo, alkylsulfonyloxy,
heteroalkylsulfonyloxy, cycloalkylsulfonyloxy, heterocyclylsulfonyloxy,
arylsulfonyloxy and heteroarylsulfonyloxy;
or a pharmaceutically acceptable salt thereof.
2. The compound of claim 1 wherein Z3 is selected from the group consisting of:
(6890905_2):RTK 182 2012
Dec
06
2011202075 each Rg is independently selected from the group consisting of hydrogen, aryl,
heteroaryl, C1-C4 alkyl, Ci-Ce heteroalkyl, C3-C8 cycloalkyl and heterocyclyl.
3. The compound of claim 2 wherein Z3 is selected from the group consisting of:
and X4 is selected from the group consisting of Br and Cl.
4. A compound of claim 1 having a formula selected from the group consisting of:
(6890905_2):RTK 183 2012
Dec
06
2011202075
(6890905_2):RTK 184 2012
Dec
06
2011202075
0 /\^Br
Br 5
(6890905 2) RTK 185 2012
Dec
06
2011202075
(6890905_2):RTK 186
5. A pharmaceutical formulation comprising the compound of any one of claims 1
2012 to 4, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable
excipient, carrier, or diluent. Dec
06 6. The pharmaceutical formulation of claim 5, wherein the diluent is water,
ethanol, propylene glycol, glycerin, or combinations thereof.
7. A method of treating cancer comprising administering to a patient in need of
therapy thereof, the pharmaceutical formulation of claim 5 or 6.
8. Use of a compound according to claim 1 for the manufacture of a 2011202075 pharmaceutical formulation for treating cancer.
9. The method of claim 7 or the use of claim 8, wherein the cancer is selected from
the group consisting of a solid tumor cancer, a soft tissue sarcoma, a leukemia,
multiple myeloma, prostate cancer, pancreatic cancer, breast cancer, lung cancer,
colorectal cancer, head and neck cancer, ovarian cancer, brain cancer, an astrocytoma
and a renal cell tumor.
10. The method of claim 7 or the use of claim 8, wherein the compound is
11. The method of claim 7 or the use of claim 8, wherein the compound is
(6890905_2):RTK 187
Br 2012
Dec
06
and the cancer is colorectal cancer.
12. The method of claim 7 or the use of claim 8, wherein the compound is 2011202075
and the cancer is ovarian cancer.
13. The method of claim 7 or the use of claim 8, wherein the compound is
and the cancer is lung cancer.
14. The method of claim 7 or the use of claim 8, wherein the compound is
(6890905_2):RTK 188 2012
Dec
06
15. The method of claim 7 or the use of claim 8, wherein the compound is 2011202075
16. The method of claim 7 or the use of claim 8, wherein the compound is
17. The method of claim 7 or the use of claim 8, wherein said method or use further
comprises treating the cancer in combination with another anti-cancer agent.
18. The method or use of claim 11, wherein said method or use further comprises
treating the cancer in combination with daunorubicin or cisplatin.
(6890905_2)RTK 189
19. The method or use of claim 13, wherein said method or use further comprises
2012 treating the cancer in combination with daunorubicin, paclitaxel or cisplatin.
Dec 20. The method or use of claim 15, wherein said method or use further comprises
06 treating the cancer in combination with gemcitabine.
21. The method of claim 7 or the use of claim 8, wherein the compound is used in a
dosage amount of 0.001 to 0.1 g/kg/day in single or divided doses.
22. The method of claim 7 or the use of claim 8, wherein the compound is used in a
dosage amount of 0.1 to 35 mg/kg/day in single or divided doses. 2011202075
23. The method of claim 7 or the use of claim 8, wherein the compound is used in a
dosage amount of 0.5 to 15 mg/kg/day in single or divided doses.
24. A method to synthesize a compound of claim 1 comprising reacting a compound
having the formula Z3-L-OH, a trisubstituted phosphine, a compound of the formula:
O
and a dialkyl azodicarboxylate to yield the compound of claim 1.
Dated 5 December 2012
Threshold Pharmaceuticals, Inc.
Patent Attorneys for the Applicant/Nominated Person
SPRUSON & FERGUSON
(6890905_2): RTK Ο (Μ Figure 1. 1/7
Compound 25 (50mg/kg) Efficacy in H460 Xenograft May
05
2011202075
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