Note: Descriptions are shown in the official language in which they were submitted.
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PROCESSES FOR PREPARING (E)-STYRYLBENZYLSULFONE
COMPOUNDS AND USES THEREOF FOR TREATING PROLIFERATIVE
DISORDERS
FIELD OF THE INVENTION
The invention relates to methods for preparing (E)-styrylbenzylsulfone
compounds. The invention further relates to methods for use of such (E)-
styrylbenzylsulfone compounds in the treatment of proliferative disorders, and
protection from the cytotoxic effects of ionizing radiation and of cytotoxic
chemotherapeutic agents.
BACKGROUND OF THE INVENTION
Cancer is now believed to result from unlimited growth of a given cell,
which is often due to a block in the ability of cells to undergo
differentiation
and/or apoptosis. Most of our understanding of how cells grow and divide comes
from the study of cells grown in vitro. The cell cycle is typically divided
into four
phases, Gl, S, G2 and M. The periods associated with DNA synthesis (S phase)
and mitosis (M phase) are separated by gaps called G1 and G2 (Malumbres, M.;
Barbacid, M. Nat. Rev. Cancer 2001, 1, 222-231; Sherr, C.J., McCormick, F.
Cancer Cell 2002,2, 103-112; Grana, X,; Reddy, E. P. Oncogene 1995, 11,211-
219). The last two decades have seen a series of discoveries, which have
provided
us with a better understanding of the complexity of the control mechanisms,
which ensure ordered progression of cell cycle. It is becoming apparent that
the
order and timing of the cell cycle is critical for accurate transmission of
genetic
information, and consequently a number of biochemical pathways have evolved
to ensure that initiation of a particular cell cycle event is dependent on the
accurate completion of the others. These biochemical pathways have been termed
' Checkpoints. '
Most normal cells, unless they have received a stimulus to proliferate or
differentiate, remain in a resting state, termed Go. However, when the
organism
requires additional cells, extracellular stimuli induce the cells to enter the
G1
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phase of the cell cycle and become committed to cell division. It is at a late
point
in the G1 phase of the cell cycle that a potentially dividing cell reaches the
"restriction point," a time at which the cell must determine whether the
conditions
are suitable for continued proliferation (Blagosklonny, M. V.; Pardee, A. B.
Cell
Cycle 2002, 1, 103-105; Donjerkovic, D.; Scott, D. W. Cell Res. 2000, 10, 1-
16;
O'Connor, P. M. Cancer Surv. 1997, 29, 151-182). Provided that conditions are
conducive to proliferation, the cell proceeds past this checkpoint. An
absolute
prerequisite for cell growth is the duplication of its genetic material, which
occurs
during the S phase. Once the DNA has been replicated, the cell "ascertains"
whether this process has been correctly executed during the second checkpoint
during G2, and provided that it has, the cell divides during mitosis, or M
phase
(Millard, S. S.; Kof, , A. J. Cell Biochem. 1998, suppl. 30-31, 37-42). The
ordered
growth process seen in normal cells is a result of regulatory control
mechanisms
that restrain cell cycle machinery. The genetic changes seen in a malignant
cell
are primarily aimed at overriding this negative regulation and result in the
loss of
one or both of the intrinsic checkpoints that are normally used by their
normal
counterparts. While some of the oncogenes, such as ras, force progression
through G1, other genes such as Rb, which are termed tumor suppressor genes,
function as "gatekeepers" of these restriction points (Mc Donald, E. R.; El-
Diery,
W.S. Ann. Med. 2001, 33, 113-122; Ewen, M.E. Prog. Cell Cycle Res. 2000, 4, 1-
17). Cancer is characterized by a loss of one or more tumor suppressor genes,
which enables a malignant cell to ignore all of the safeguards that are aimed
at
preventing unwanted cell division.
An important rule associated with cell cycle progression (for both normal
and tumor cells) is the fact that once a cell crosses the "restriction point"
(which is
the G1 /S boundary), it has to either divide into two daughter cells or die4
due to
the fact that most eukaryotic cells can exist in S, G2 and M phases of the
cell
cycle for only a limited span of time. Most chemotherapeutic agents, such as
paclitaxel, that are currently used in cancer therapy function by blocking
cell
cycle progression at a point beyond Gi/S boundary (M phase in the case of
paclitaxel), resulting in the death of the tumor cell (Wang, T.; Wang, H.;
Soong,
Y. 88, 2619-2628). A major problem with many of the current drugs is their
inability to discriminate between normal and tumor cells. As a result, normal
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cells undergoing active cell division also become blocked at the mitotic phase
of
the cell cycle and enter programmed cell death pathways, the effects of which
are
often manifested as the toxic side-effects seen in patients treated by these
drugs.
A second problem appears to be the development of resistance to many of the
chemotherapeutic agents often due to over-expression of drug transporters. Our
quest was to design new chemical entities that exhibit reduced toxicity in
normal
cells and are not recognized by drug transporters that are over-expressed in
drug-
resistant tumor cells.
What are needed are methods of preparing effective antiproliferative,
radioprotective and chemoprotective activity agents. The methods and
compositions of the present invention satisfy these and other long felt needs
with
the following invention that provides the synthesis of a group of styryl
benzyl
sulfones which induce apoptotic death of a wide variety of human tumor cell
lines
at sub nanomolar concentrations while exhibiting relatively low toxicity to
normal
human cells. More importantly, compounds prepared by these methods were
found to be active against a wide variety of human tumor cell lines that are
resistant to the activity of many of the cytotoxic agents.
SUMMARY OF THE INVENTION
In one aspect of the invention, compounds, processes, pharmaceutical
compositions and therapeutic methods are provided. The biologically active
compounds are in the form of aromatic olefins, structurally linked via an
optionally substituted methylene sulfone, an optionally substituted methylene
sulfoxide, an optionally N-substituted sulfonamide, or an optionally N-
substituted
carboxamide linker, to a phenol or thiophenol functionality, or a derivative
of
such a phenol or thiophenol functionality.
According to one aspect of the invention, processes for preparing
compounds according to Formula I are provided,
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x (R3)b
(R )a
A
R1
wherein,
A is -S- or -0-;
R1 is selected from the group consisting of ¨H; halo(Ci-C6)alkyl,
preferably trifluoro(Ci-C6)alkyl, difluoro(Ci-C6)alkyl and chloro(Ci-C6)alkyl
more preferably trifluoro(Ci-C3)alkyl, difluoro(Ci-C3)alkyl and chloro(Ci-
C3)alkyl, most preferably ¨CF3, ¨CHF2 and ¨CH2C1; -C(=0)Rw; ¨S(=0)Rw;
-SO2Rw; -(C1-C6 hydrocarbylene)Rz, preferably ¨(Ci-C6)alkyleneRz, more
preferably ¨(C1-C6)alkylene-CORY, -P(=0)(01n2;
substituted and unsubstituted aryl, preferably substituted and unsubstituted
phenyl; substituted and unsubstituted heteroaryl, preferably substituted and
unsubstituted monocyclic heteroaryl; ¨SiRCi-C6)allcyli3, preferably, -Si(CH3)2-
C(CH3)3 (tert-butyldimethylsilyl); and ¨CH2CH2SiRCi-C6)alkylb, preferably
¨CH2CH2Si(CH3)2-C(CH3)3 and ¨CH2CH2Si(CH3)3;
each R" is independently selected from the group consisting of ¨H and
¨(C1-C7)hydrocarbyl, preferably -(Ci-C6)alkyl, more preferably 4C1-C3)alkyl,
most preferably ¨CH3 or ¨C2H5;
Rw is selected from the group consisting of ¨(CI-C7)hydrocarbyl,
preferably 4Ci-C6)alkyl, more preferably -(Ci-C3)alkyl, most preferably ¨CH3
or
¨C2H5; -N1r2; -OR"; halo(C1-C3 alkyl), preferably chloro(C1-C3 alkyl) and
trifluoro(Ci-C3 alkyl); -NleCR`Ra-C(=0)-Rn; -CleRa-N(R")-Rc; substituted and
unsubstituted aryl, preferably substituted and unsubstituted phenyl;
substituted
and unsubstituted aryl(CI-C3)alkyl, preferably substituted and unsubstituted
phenyl(C -C3)alkyl ; substituted and unsubstituted heteroaryl, preferably
substituted and unsubstituted monocyclic heteroaryl; substituted and
unsubstituted
heteroaryl(Ci-C3)alkyl, preferably substituted and unsubstituted monocyclic
heteroaryl(Ci-C3)alkyl; substituted and unsubstituted heterocyclyl;
substituted and
unsubstituted heterocyclyl(Ci-C3)alkyl, -(Ci-C3 alkylene)P(=0)(01n2; -(Ci-
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C3)perfluoroalkylene-N(CH3)2; -(C1-C3)alkylene-N+(Ci-C3)3; -(CI-C3)alkylene-
N+(CH2CH2OH)3; -(CI -C4alkylene)-C(=0)-halogen; -(C1-C4)perfluoroalkylene-
CO2R", -(Ci -C3alkylene)C(=0)ORY; and -(C1-C3alkyl ene)0C(=0)-(C -C3
alkyl ene)C(=0)RY;
RY is selected from the group consisting of ¨OR", -N11"2 and -(Ci-C6)alkyl;
Rz is selected from the group consisting of ¨C(=0)RY; -NR"CltyRa-C(=0)-
RI% -NW/2; -OR"; substituted and unsubstituted aryl, preferably substituted
and
unsubstituted phenyl; substituted and unsubstituted heteroaryl, preferably
substituted and unsubstituted monocyclic heteroaryl; and -C(=0)(C1-C3)alkyl;
each Ra is independently selected from the group consisting of ¨H; ¨(C1-
C6)alkyl; ¨(Ci-C6)heteroalkyl, particularly ¨CH2SH, -(CH2)2C(=0)-NH2, ¨CH2-
OH, -CH(OH)-CH3, ¨(CH2)4-NH2, and ¨(CH2)2-S-CH3; -(CH2)3-NH-
C(NH2)(=NH); ¨CH2C(=0)NH2; ¨CH2COOH; ¨(CH2)2COOH; substituted and
unsubstituted aryl, preferably substituted and unsubstituted phenyl;
substituted
and unsubstituted aryl(Ci-C3)alkyl, preferably substituted and unsubstituted
phenyl(Ci-C3)alkyl, more preferably substituted and unsubstituted benzyl,
particularly 4-hydroxybenzyl; substituted and unsubstituted heterocyclyl,
preferably substituted and unsubstituted heteroaryl, particularly ¨CH2-(3-
indoly1),
more preferably substituted and unsubstituted monocyclic heteroaryl; and
substituted and unsubstituted heterocyclyl(C1-C3)alkyl, preferably substituted
and
unsubstituted heteroaryl(CI-C3)alkyl, more preferably substituted and
unsubstituted monocyclic heteroaryl(C1-C3)alkyl, most preferably substituted
and
unsubstituted monocyclic heteroaryl-CH2-, particularly ¨CH2-inidazoly1;
each R11 is independently selected from the group consisting of ¨OR",
-NR"2, and an N-terminally linked peptidyl residue containing from 1 to 3
amino
acids in which the terminal carboxyl group of the peptidyl residue is present
as a
functional group selected from the group consisting of ¨CO2R" and
¨C(=0)NR"2;
each Rc is independently selected from the group consisting of ¨H and a
carboxy terminally linked peptidyl residue containing from 1 to 3 amino acids
in
which the terminal amino group of the peptidyl residue is present as a
functional
group selected from the group consisting of -NH2; ¨NHC(=0)(C1-C6)alkyl;
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-NH(Ci-C6)alkyl; -NH(C -C6 alky1)2 and ¨NHC(=0)0(C1-C7)hydrocarbyl,
preferably ¨NHC(=0)0(Ci-C6)alkyl and ¨NHC(=0)0-benzyl;
Q is aryl or heteroaryl;
each R2 and R3 are independently selected from the group consisting of
halogen; ¨(Ci-C7)hydrocarbyl, preferably -(Ci-C6)alkyl, more preferably -(Ci-
C3)alkyl, most preferably ¨CH3 and ¨C2H5; ¨C(=0)Ity; ¨NR"2; -NHC(=0)R";
-NHS021e1; -NH1e; -NHCIVRaC(=0)Rn; -NHS0212."; -C(=0)01V1;
-C(=0)NHR", -NO2; -CN; -OR"; -P(=0)(01:02; -C(=NH)NH2,
dimethylamino(C2-C6 alkoxy); -NHC(=NR")NHR"; -(Ci-C6)haloalkyl, preferably
trifluoro(Ci-C6)alkyl and difluoro(Ci-C6)alkyl, more preferably trifluoro(CI-
C3)alkyl and difluoro(CI-C3)alkyl, most preferably ¨CF3 and
¨CHF2; and -(C1-C6)haloalkoxy, preferably trifluoro(Ci-C6)alkoxy and
difluoro(Ci-C6)alkoxy, more preferably trifluoro(Ci-C3)alkoxy and difluoro(Ci-
C3)alkoxy, most preferably ¨0CF3 and ¨OCHF2;
wherein, the two R" groups on -P(=0)(01V)2 and ¨N1V2 may optionally
form a five- or six-membered heterocyclic ring, preferably a five-membered
ring,
which may further optionally be fused to an aryl or carbocyclic ring,
preferably an
aryl ring, more preferably a phenyl ring;
a is 0, 1, 2 or 3;
bis 0,1,2 or3;
wherein the sum of a and b is preferably at least 1;
the conformation of the substituents on the exocyclic carbon-carbon
double bond is either E- or Z-;
X is ¨C*H(Rx)Y- or
Y is ¨S(=0)- or ¨SO2-;
Z is ¨C(=0)- or ¨SO2-;
R' is selected from the group consisting of ¨H; ¨(Ci-C6)alkyl, preferably ¨
(C1-C3)alkyl, more preferably methyl and ethyl; and ¨C(=0)(C1-C6)alkyl,
preferably ¨C(=0) (Ci-C3)alkyl, more preferably acetyl and propionyl; and
* indicates that, when Rx is other than ¨H, the conformation of the
substituents on the designated carbon atom is (R)-, (S)- or any mixture of (R)-
and
(5)-; or
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a salt of such a compound, preferably a pharmaceutically acceptable salt
of such a compound;
provided that;
(a) when A is -0- and RI is ¨H;
b is greater than 0; and
R3 is other than (Ci-C6)alkyl, ¨OH and ¨NO2.
(b) when X is ¨1\1Ie-Z- and A is -0-;
Rz is other than ¨C(=0)R3', ¨NIVT2 and unsubstituted aryl; and
It' is other than ¨(Ci-C6)alkyl; and
(c) when X is ¨C*H(InY- and A is -0-;
RI is other than halo(C1-C6)alkyl and unsubstituted aryl;
Rz is other than ¨NR"2 and unsubstituted aryl; and
R" is other than ¨(Ci-C7)hydrocarbyl.
According to some embodiments of compounds of Formula I, Q is aryl,
preferably phenyl or naphthyl, more preferably phenyl.
According to other embodiments of compounds of Formula I, Q is
heteroaryl, preferably monocyclic heteroaryl.
According to some embodiments of compounds of Formula I, there are
provided compounds of Formula IE:
X
________________________________________________________ (R3)b
(R2)a ______________________
IE
A
R1
wherein the exocyclic carbon-carbon double bond is in the (E)-
configuration.
According to some embodiments of compounds of Formula I, RI is -H.
According to other embodiments of compounds of Formula I, RI is other
than -H.
Preferably, when one or more of Q, RI, Ra or le
is a monocyclic
heteroaryl group, the monocyclic heteroaryl group is independently selected
from
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the group consisting of pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, thienyl,
furyl,
pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, 1,2,3-
triazolyl,
1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-
oxadiazolyl,
1,3,4-thiadiazoly1 and 1,3,4-oxadiazolyl.
More preferably, when one or more of Q, RI, Ra, 11" or Rz is a monocyclic
heteroaryl group, the monocyclic heteroaryl group is independently selected
from
the group consisting of pyridyl, thienyl, furyl, pyrrolyl, imidazolyl,
thiazolyl,
oxazolyl, pyrazolyl, and isothiazolyl.
Most preferably, when one or more of Q, R1, Ra, ler or Rz is a monocyclic
heteroaryl group, the monocyclic heteroaryl group is independently selected
from
the group consisting of pyridyl, thienyl, and furyl.
Preferably, when one or more of Q, R1, Ra, Rw or Rz is a heteroaryl group
other than a monocycyclic heteroaryl group, the heteroaryl group is selected
from
the group consisting of indolyl, quinolyl, isoquinolyl, cinnolinyl,
quinoxalinyl,
quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin,
benzofuryl, 1,2-benzisoxazolyl, benzothienyl, benzoxazolyl, benzthiazolyl,
purinyl, benzimidazolyl, benztriazolyl, thioxanthinyl, carbazolyl, carbolinyl,
acridinyl, pyrrolizidinyl, and quinolizidinyl.
More preferably, when one or more of Q, RI, Ra, Rw or Rz is a heteroaryl
group other than a monocycyclic heteroaryl group, the heteroaryl group is
selected from the group consisting of indolyl, quinolyl, isoquinolyl,
benzofuryl,
benzothienyl, benzoxazolyl, benzthiazolyl, and benzimidazolyl.
Most preferably, when one or more of Q, RI, Ra, Rw or Rz is a heteroaryl
group other than a monocycyclic heteroaryl group, the heteroaryl group is
selected from the group consisting of indolyl, quinolyl, isoquinolyl,
benzofuryl
and benzothienyl.
Preferably, substituted aryl and heteroaryl rings in RI, Ra, Rw and Rz
groups are mono-, di- or tri-substituted, more preferably mono- or di-
substituted
by substituents selected from the group consisting of halogen; (C1-
C7)hydrocarbyl, preferably benzyl and (Ci-C6)alkyl, more preferably benzyl and
(C1-C3)alkyl, most preferably benzyl, methyl and ethyl; ¨NR.v2; ¨NO2; -CN;
heterocyclyl, preferably N-methylpiperazinyl, morpholinyl and thiomorpholinyl;
-
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Ole and ¨0(C1-
C7)hydrocarbyl, preferably -0(C1-C6)alkyl and ¨0-benzyl,
more preferably -0(Ci-C3)alkyl, most preferably benzyl, methoxy and ethoxy.
More preferably, substituted aryl and heteroaryl rings in RI, Ra, le' and le
groups are mono-, di- or tri-substituted, more preferably mono- or di-
substituted
by substituents selected from the group consisting of chloro; fluoro; bromo; -
(C1-
C6)alkyl, more preferably -(Ci-C3)alkyl, most preferably methyl and ethyl; -
NH2;
¨NO2; -CN; heterocyclyl, preferably N-methylpiperazinyl, morpholinyl and
thiomorpholinyl; -OH and -0(CI-C6)alkyl, more
preferably
-0(C i-C3)alkyl, most preferably methoxy and ethoxy.
Most preferably, substituted aryl and heteroaryl rings in R1, le, lei and le
groups are mono-, di- or tri-substituted, more preferably mono- or di-
substituted
by substituents selected from the group consisting of chloro, fluoro, bromo,
methyl, ¨NO2, -CN, -OH, and methoxy.
Preferably substituted heterocyclyl groups contained within Ra and le'
groups are mono-, di- or tri-substituted, more preferably mono- or di-
substituted,
by substituents selected from the group consisting of -(C1-C7)hydrocarbyl,
preferably benzyl and -(C1-C6)alkyl; more preferably methyl, ethyl and benzyl;
-
C(=0)(C1-C6)alkyl, preferably -C(=0)(Ci-C3)alkyl, more preferably acetyl; and
¨
(C1-C6)per-fluoroalkyl, preferably ¨(Ci-C3)perfluoroalkyl, more preferably
¨CF3.
More preferably substituted heterocyclyl groups contained within Ra and
R"' groups are mono-, or di-substituted, by substituents selected from the
group
consisting of -(C1-C6)alkyl; more preferably methyl and ethyl,
and -C(=0)(C1-C3)alkyl, more preferably acetyl.
According to some embodiments of the invention, the sum of a and b is at
least 2. According to other embodiments of the invention, the sum of a and b
is at
least 3. According to still other embodiments of the invention, the sum of a
and b
is at least 4. According to some embodiments of the invention, both a and b
are at
least 1. According to other embodiments of the invention, a is at least 1 and
b is
at least 2. According to other embodiments of the invention, b is at least 1
and a
is at least 2. According to still other embodiments of the invention, both a
and b
are at least 2.
According to preferred embodiments of compounds of Formula I:
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when b is 1, substitution of R3 groups on Q is at the ortho- or para-
position;
when b is 2, substitution of R3 groups on Q is at either ortho- and
para- positions, or at both ortho- positions; and
when b is 3, substitution of R3 groups on Q is at the para- position
and at both ortho- positions.
Preferably, for compounds according to Formula I, Q is aryl; b is 1, 2 or 3;
and each R2 is ¨OR" or halogen, which may be the same or different.
More preferably, for compounds according to Formula I, Q is phenyl; b is
2 or 3; and each R2 is ¨OR", which may be the same or different. Most
preferably, each R2 is ¨OCH3.
In one aspect of the invention, processes for preparing compounds
according to Formula IE are provided.
I Q _____ (R3)b
(R2).
IE
A
R1
wherein RI, R2, R3, A, Q, a and b, and X are as defined herein for
compounds of Formula I.
In one embodiment of the invention, processes for preparing (E)-
styrylbenzylsulfones compounds according to Formula IE wherein 12.1, R2, R3,
A,
Q, a and b are as defined herein for compounds of Formula I, and X is ¨CH*Rx-
Y-, are provided, comprising a synthesis reaction as shown in Scheme 1.
In another embodiment of the invention, processes for preparing (E)-
styrylbenzylsulfones compounds according to Formula IE wherein RI, R2, R3, A,
Q, a and b are as defined herein for compounds of Formula I, and X is ¨CH*Rx-
Y-, are provided, comprising Knoevenagel-type condensation synthesis reaction
as shown in Scheme 2.
In one preferred embodiment of the invention, processes for preparing (E)-
styrylbenzylsulfones compounds according to Formula IE wherein RI, R2, R3, A,
Q, a and b are as defined herein for compounds of Formula I, and X is ¨CH*Rx-
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are provided, comprising an aldehyde condensation synthesis reaction as
shown in Scheme 3.
In another preferred embodiment of the invention, processes for preparing
(E)-styrylbenzylsulfones compounds according to Formula IE wherein 11.1, R2,
R3,
A, Q, a and b are as defined herein for compounds of Formula I, and X is ¨
CH*Rx-Y-, are provided, comprising an Knoevenagel-type condensation
synthesis reaction as shown in Scheme 4.
In yet another preferred embodiment of the invention, processes for
preparing bioavailable water soluble prodrug (E)-styrylbenzylsulfones
compounds
according to Formula IE wherein R1, R2, R3, A, Q, a and b are as defined
herein
for compounds of Formula I, and X is ¨CH*Rx-Y-, are provided, comprising a
synthesis reaction as shown in Scheme 5.
In yet another preferred embodiment of the invention, processes for
preparing (E)-styrylbenzylsulfones compounds according to Formula IE wherein
le, R2, R3, A, Q, a and b are as defined herein for compounds of Formula I,
and X
is ¨CH*Rx-Y-, are provided, comprising a synthesis reaction as shown in Scheme
6.
In yet another preferred embodiment of the invention, a process for
preparing compound (E)-2,4,6-Trimethoxystyry1-3-Hydroxy-4-Methoxybenzyl
Sulfone (Compound ON 013100) is provided, comprising the synthesis reaction
shown in Scheme 3.
In yet another preferred embodiment of the invention, a process for
preparing compound (E)-2,4,6-Trimethoxystyry1-3-Hydroxy-4-Methoxybenzyl
Sulfone (Compound ON 013100) is provided, comprising the synthesis reaction
shown in Scheme 4.
In yet another preferred embodiment of the invention, a process for
preparing bioavailable water soluble prodrug compound (E)-2,4,6-
(Trimethoxystyry1)-3 -0-Phosphate Disodium-4-Methoxybenzyl Sulfone
(Compound ON 013105), is provided, comprising a synthesis reaction as shown
in Scheme 5.
In yet another preferred embodiment of the invention, a process for
preparing bioavailable water soluble prodrug compound (E)-2,4,6-
(Trimethoxystyry1)-3-0-Phosphate Disodium-4-Methoxybenzyl Sulfone
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(Compound ON 013105) is provided, comprising the synthesis reaction shown in
Scheme 6.
In yet another preferred embodiment of the invention, a process for
bioavailable water soluble prodrug compound (E)-2,4,6-(Trimethoxystyry1)-3-0-
Phosphate Disodium-4-Methoxybenzyl Sulfone (Compound 013105) is provided,
comprising the synthesis reaction shown in Scheme 7.
In yet another aspect of the invention, compounds, compositions and
methods for the treatment and/or prevention of cancer and other proliferative
disorders are provided.
In yet another aspect of the invention, compounds which are selective in
killing tumor cells at therapeutically useful concentrations are provided.
In yet another aspect of the invention, compounds, compositions and
methods for inducing neoplastic cells to selectively undergo apoptosis are
provided.
In yet another aspect of the invention, compounds, compositions and
methods which enable prophylactic treatment of proliferative disorders are
provided.
In yet another aspect of the invention, compounds, compositions and
methods for protecting normal cells and tissues from the cytotoxic and genetic
effects of exposure to ionizing radiation, in individuals who have incurred,
will in
the future incur, or are at risk for incurring exposure to ionizing radiation
are
provided. Exposure to ionizing radiation may occur in controlled doses during
the treatment of cancer and other proliferative disorders. Alternatively,
exposure
to ionizing radiation may occur in uncontrolled doses beyond the norm accepted
for the population at large during high risk activities or environmental
exposures.
In yet another aspect of the invention, compositions and methods for
protecting individuals from the cytotoxic side effects of mitotic phase cell
cycle
inhibitors and topoisomerase inhibitors, used in the treatment of cancer and
other
proliferative disorders are provided.
In yet another aspect of the invention, a method for treating cancer or
other proliferative disorders which reduces or eliminates cytotoxic effects on
normal cells is provided.
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In yet another aspect of the invention, compositions and methods for
enhancing the effects of mitotic phase cell cycle inhibitors and topoisomerase
inhibitors, used for the treatment of cancer or other proliferative disorders
are
provided.
In yet another aspect of the invention, a therapeutic program for treating
cancer or other proliferative disorder which includes administration of a
cytoprotective compound prior to administration of a chemotherapeutic agent,
which cytoprotective compound induces a reversible cycling quiescent state in
non-tumored tissues is provided.
In yet another aspect of the invention, a method for safely increasing the
dosage of mitotic phase cell cycle inhibitors and topoisomerase inhibitors,
used in
the treatment of cancer and other proliferative disorders is provided.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 Anti-
tumor effects of (E)-2,4,6-Trimethoxystyry1-3-
Hydroxy-4-Methoxybenzyl Sulfone (6aa). A. 6aa inhibits the growth of parental
(MES-SA) and Paclitaxel resistant (MES-SA/DX5) cell lines with equal
efficiency. The parental uterine sarcoma cells and the MDR positive (MES-
SA/DX5) cells were plated into 6 well dishes and treated with various
concentrations of 6aa and Paclitaxel for 96 h. The number of viable cells from
duplicate plates was determined by trypan blue exclusion. B. Soft Agar Assays.
MIA-PaCa-2 cells (1.0 x105) were plated in soft agar containing various
concentrations of each compound in triplicates. After three weeks of growth,
the
plates were stained for 48 h using 0.05% nitroblue tetrazolium solution.
Representative plates were photographed using an Olympus stereoscope mounted
with a Sony digital camera system (DKC5000, Sony Inc).
FIGURE 2
Preferential tumor cell killing activity of 6aa. A. Cell
Cycle analyses. Normal (HUVEC) and tumor cells (DU145) were treated with 20
nM concentration of 6aa and incubated in medium containing 10% fetal bovine
serum. At 24 hr intervals, the cells were fixed, stained with propidium iodide
and
analyzed for their DNA content by flow Cytometry. B. Induction of apoptosis in
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normal (HUVEC) and tumor cells (DU145) was assessed by western blot analysis
of cell lysates treated with 6aa for 24, 48 and 72 h. The Western blots were
probed with anti-PARP antibodies to assess the cleavage of the protein.
FIGURE 3 In vivo Anti-tumor effects of (E)-2,4,6-Trimethoxystyry1-4-
Methoxybenzyl sulfone (6s) and (E)-2,4,6-Trimethoxystyry1-31-0-phosphate
disodium-4-methoxybenzyl Sulfone (6ab). Female athymic (NCr-nu/nu) mice
were injected subcutaneously with 0.5-1x107 ER-negative human breast tumor
cells (BT-20) in 0.2 mL of PBS and the tumors allowed to grow to a size of 100-
150 mm3 in size in about 14 days. The mice were then paired such that the
pairs
harbored equal sized tumors, which were then used to test the therapeutic
effects
of 6s and 6ab. A Of the pairs, the animals were treated with either 50 mg/kg
6s
following a Q4D schedule or 25 mg/kg using a Q2D schedule or vehicle (DMSO)
control. The tumor size was then measured on alternate days in two dimensions
and the volume determined using either of the following equations: 1: V=
(Lx(S2))7T/6); where L is the longer and S is the shorter of the two
measurements.
B. 6ab was dissolved in PBS and was administered intravenously (50 mg/kg)
through the tail vein on every alternate day. Tumor measurements were done as
in materials and methods.
FIGURE 4 Bone marrow toxicity profile of 6ab. To assess the toxicity
of 6ab, the phosphate salt of the drug was injected into mice (100 mg/Kg) and
bone marrow harvested from femur and tibia after 12, 24 and 36 h following the
injection of the drug. The bone marrow cells were cultured in methylcellulose
medium supplemented with a mixture of stem cell factor, GM-CSF, IL-3 and
erythropoietin for one week and colony forming units were determined.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
General
The term "individual" or "subject", includes human beings and non-
human animals. With respect to the disclosed radioprotective and
cytoprotective
methods, these terms refer, unless the context indicates otherwise, to an
organism
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that is scheduled to incur, or is at risk for incurring, or has incurred,
exposure to
ionizing radiation or exposure to one or more cytotoxic chemotherapeutic
agents.
The expression "effective amount" when used to describe therapy to a
patient suffering from a proliferative disorder, refers to the amount of a
compound
according to Formula I that inhibits the growth of tumor cells or
alternatively
induces apoptosis of cancer cells, preferably tumor cells, resulting in a
therapeutically useful and selective cytotoxic effect on proliferative cells
when
administered to a patient suffering from a cancer or other disorder which
manifests abnormal cellular proliferation. The term "effective amount" is
inclusive of amounts of a compound according to Formula I and its enantiomers,
metabolites, prodrugs, polymorphs, the crystalline form, and anhydrous and
hydtrated forms thereof that may be metabolized to an active metabolite in an
amount that inhibits the growth of tumor cells or induces apoptosis of cancer
cells.
The term "antibody" is intended to encompass not only intact antigen-
binding immunoglobulin molecules, but also to include antigen-binding
fragments
thereof such as Fab, Fab', F(ab1)2, and Fv fragments, capable of binding the
epitopic determinant or any other fragment retaining the antigen-binding
ability of
an intact antibody.
The expression "humanized antibody" refers to an antibody that has its
complementary determining regions (CDR's) derived from a non-human species
immunoglobulin, and the remainder of the antibody molecule derived from a
human immunoglobulin.
The expression "chimeric antibody" means an antibody comprising a
variable region and a constant region derived from different species.
The expression "humanized chimeric antibody" is meant a chimeric
antibody in which at least the constant region is human-derived.
The expression "monospecific polyclonal antibody" means an antibody
preparation comprising multiple antibody species having specificity for a
single
antigen.
The term "proliferative disorder" means a disorder wherein cells are made
by the body at an atypically accelerated rate.
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Radioprotection
As used herein, "ionizing radiation" is radiation of sufficient energy that,
when absorbed by cells and tissues, induces formation of reactive oxygen
species
and DNA damage. This type of radiation includes X-Rays, gamma rays, and
particle bombardment (e.g., neutron beam, electron beam, protons, mesons and
others), and is used for medical testing and treatment, scientific purposes,
industrial testing, manufacturing and sterilization, weapons and weapons
development, and many other uses. Radiation is typically measured in units of
absorbed dose, such as the rad or gray (Gy), wherein 1 rad = 0.01 Gy, or in
units
of dose equivalence, such as the rem or sievert (Sv), wherein 1 rem = 0.01 Sv.
The Sv is the Gy dosage multiplied by a factor that includes tissue damage
done. For example, penetrating ionizing radiation (e.g., gamma and beta
radiation) have a factor of about 1, so 1 Sv = ¨1 Gy. Alpha rays have a factor
of
20, so 1 Gy of alpha radiation = 20 Sv.
By "effective amount of ionizing radiation" is meant an amount of
ionizing radiation effective in killing, or in reducing the proliferation, of
abnormally proliferating cells in an individual. As used with respect to bone
marrow purging, "effective amount of ionizing radiation" means an amount of
ionizing radiation effective in killing, or in reducing the proliferation, of
malignant cells in a bone marrow sample removed from an individual.
By "acute exposure to ionizing radiation" or "acute dose of ionizing
radiation" is meant a dose of ionizing radiation absorbed by an individual in
less
than 24 hours. The acute dose may be localized, as in radiotherapy techniques,
or
may be absorbed by the individual's entire body. Acute doses are typically
above
10,000 millirem (0.1 Gy), but may be lower.
By "chronic exposure to ionizing radiation" or "chronic dose of ionizing
radiation" is meant a dose of ionizing radiation absorbed by an individual
over a
period greater than 24 hours. The dose may be intermittent or continuous, and
may be localized or absorbed by the individual's entire body. Chronic doses
are
typically less than 10,000 millirem (0.1 Gy), but may be higher.
By "at risk of incurring exposure to ionizing radiation" is meant that an
individual may intentionally, e.g., by scheduled radiotherapy sessions, or
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inadvertently be exposed to ionizing radiation in the future. Inadvertent
exposure
includes accidental or unplanned environmental or occupational exposure.
By "effective amount of a radioprotective compound" is meant an amount
of compound according to Formula I effective to reduce or eliminate the
toxicity
associated with radiation in normal cells of the individual, and also to
impart a
direct cytotoxic effect to abnormally proliferating cells in the individual.
As used
with respect to bone marrow purging, "effective amount" of the radioprotective
compound according to Formula I means an amount of compound effective to
reduce or eliminate the toxicity associated with radiation in bone marrow
removed from an individual, and also to impart a direct cytotoxic effect to
malignant cells in the bone marrow removed from the individual.
Cytoprotection
By "mitotic phase cell cycle inhibitor" is meant a chemical agent whose
mechanism of action includes inhibition of a cell's passage through any
portion of
the mitotic (M) phase of the cell cycle.
By "effective amount" of a mitotic phase cell cycle inhibitor or
topoisomerase inhibitor is meant an amount of said inhibitor effective in
killing or
reducing the proliferation of cancer cells in a host animal.
By "effective amount" of the cytoprotective compound according to
Formula I is meant an amount of compound effective to reduce the toxicity of
the
mitotic phase cell cycle inhibitor or topoisomerase inhibitor on normal cells
of the
animal.
The expression "cell cycle" refers to the usual description of cell
development in terms of a cycle consisting of a series of phases - interphase
and
M (mitotic) phase - and the subdivision of interphase into the times when DNA
synthesis is proceeding, known as the S-phase (for synthesis phase), and the
gaps
that separate the S-phase from mitosis. G1 is the gap after mitosis but before
DNA synthesis starts, and G2 is the gap after DNA synthesis is complete before
mitosis and cell division. Interphase is thus composed of successive G1 , s
and G2
phases, and normally comprises 90% or more of the total cell cycle time. The M
phase consists of nuclear division (mitosis) and cytoplasmic division
(cytokinesis). During the early part of the M phase, the replicated
chromosomes
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condense from their extended interphase condition. The nuclear envelope breaks
down, and each chromosome undergoes movements that result in the separation
of pairs of sister chromatids as the nuclear contents are divided. Two new
nuclear
envelopes then form, and the cytoplasm divides to generate two daughter cells,
each with a single nucleus. This process of cytokinesis terminates the M phase
and marks the beginning of the interphase of the next cell cycle. The daughter
cells resulting from completion of the M phase begin the interphase of a new
cycle.
By "topoisomerase" is meant an enzyme that catalyzes the conversion of
DNA from one topological form to another by introducing transient breaks in
one
or both strands of a DNA duplex.
By "topoisomerase inhibitor" is meant a chemical agent whose mechanism
of action includes interfering with the function of a topoisomerase.
"Topological isomers" are molecules that differ only in their state of
supercoiling. Type I topoisomerase cuts one strand of DNA and relaxes
negatively supercoiled DNA, but does not act on positively supercoiled DNA.
Type II topoisomerase cuts both strands of DNA and increases the degree of
negative supercoiling in DNA.
Chemical
The term "alkyl", by itself or as part of another substituent, e.g., alkoxy,
haloalkyl or aminoalkyl, means, unless otherwise stated, a saturated
hydrocarbon
radical having the number of carbon atoms designated (i.e. C1-C6 means one,
two,
three, four, five or six carbons) and includes straight, branched chain,
cyclic and
polycyclic groups. Examples include: methyl, ethyl, propyl, isopropyl, butyl,
isobutyl, tert-butyl, pentyl, neopentyl, hexyl, cyclohexyl, norbornyl and
cyclopropylmethyl. Preferred alkyl groups are -(Ci-C6)alkyl. Most preferred is
-
(Ci-C3)alkyl, particularly ethyl, methyl and isopropyl.
"Substituted alkyl" means alkyl, as defined above, substituted by one, two
or three substituents preferably independently selected from the group
consisting
of halogen, -OH, -0(C1-C4)alkyl, -NH2, -N(CH3)2, -CO2H, -0O2(Ci-C4)alkyl, -
CF3, -CONH2, -SO2NH2, -C(=NH)NH2, -CN and ¨NO2. More preferably, the
substituted alkyl contains one or two substituents independently selected from
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halogen, -OH, NH2, -N(CH3)2, trifluoromethyl and ¨CO2H; most preferably,
independently selected from halogen and -OH. Examples of substituted alkyls
include, but are not limited to, 2,2-difluoropropyl, 2-carboxycyclopentyl and
3-
chloropropyl.
The term "alkylene", by itself or as part of another substituent means,
unless otherwise stated, a divalent straight, branched or cyclic chain
hydrocarbon
radical having the designated number of carbons. A substitution of another
group
on alkylene may be at any substitutable carbon, i.e., the expression ¨C(=0)(C1-
C4
alkylene)Rw would include, for example:
0
0 0 0 Rw
Rw R ,Le_e_k/K Rw
H3C CH3
The term "alkoxy" employed alone or in combination with other terms
means, unless otherwise stated, an alkyl group having the designated number of
carbon atoms, as defined above, connected to the rest of the molecule via an
oxygen atom, such as, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy
(isopropoxy) and the higher homologs and isomers. Preferred are (CI -
C3)alkoxy,
particularly ethoxy and methoxy.
The term "amine" or "amino" refers to radicals of the general formula
-NRR', wherein R and R' are independently selected from hydrogen or a
hydrocarbyl radical, or wherein R and R' combined form a heterocycle. Examples
of amino groups include: ¨NH2, methyl amino, diethyl amino, anilino, benzyl
amino, piperidinyl, piperazinyl and indolinyl.
The term "aromatic" refers to a carbocycle or heterocycle having one or
more polyunsaturated rings having aromatic character (4n + 2) delocalized 7(
(pi)
electrons).
The term "aryl" employed alone or in combination with other terms,
means, unless otherwise stated, a carbocyclic aromatic system containing one
or
more rings (typically one, two or three rings) wherein such rings may be
attached
together in a pendent manner, such as a biphenyl, or may be fused, such as
naphthalene. Examples include phenyl; anthracyl; and naphthyl. Preferred are
phenyl and naphthyl, most preferred is phenyl.
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The term "aryl-(Ci-C3)alkyl" means a radical wherein a one to three
carbon alkylene chain is attached to an aryl group, e.g., -CH2CH2-phenyl.
Preferred is aryl(CH2)- and aryl(CH(CH3))-. The term "substituted aryl-(Ci-
C3)alkyl" means an aryl-(Ci-C3)alkyl radical in which the aryl group is
substituted. Preferred is substituted aryl(CH2)-. Similarly,
the term
"heteroaryl(Ci-C3)alkyl" means a radical wherein a one to three carbon
alkylene
chain is attached to a heteroaryl group, e.g., -CH2CH2-pyridyl. Preferred is
heteroaryl(CH2)-. The term "substituted heteroaryl-(Ci-C3)alkyl" means a
heteroaryl-(Ci-C3)alkyl radical in which the heteroaryl group is substituted.
Preferred is substituted heteroaryl(CH2)-.
The term "arylene" by itself or as part of another substituent means, unless
otherwise stated, a divalent aryl radical. Preferred are divalent phenyl
radicals,
particularly 1,4-divalent phenyl radicals.
The term "cycloalkyl" refers to ring-containing alkyl radicals. Examples
include cyclohexyl, cyclopentyl, cyclopropyl methyl and norbornyl.
The expression "exocyclic double bond," unless otherwise stated, refers
herein to a carbon-carbon double bond external to a chemical ring structure.
Specifically, the expression refers to the carbon-carbon double bond in
compounds of the invention, which is not contained in either the phenyl ring
or
the aromatic ring, Q, but rather is the double bond which is alpha to the
aromatic
ring, Q;
The terms "halo" or "halogen" by themselves or as part of another
substituent, e.g., haloalkyl, mean, unless otherwise stated, a fluorine,
chlorine,
bromine, or iodine atom, preferably, fluorine, chlorine, or bromine, more
preferably, fluorine or chlorine.
The term "haloalkyl" means, unless otherwise stated, an alkyl group as
defined herein containing at least one halogen substituent and no substituent
that
is other than halogen. Multiple halogen substituents, up to substitution of
all
substitutable hydrogens on the alkyl group may be the same or different.
Preferred haloalkyl groups include, for example, perfluoro(Ci-C4)alkyl, gem-
difluoro(Ci-C4)alkyl, and chloro(Ci-C4)alkyl. More preferred haloalkyl groups
include, for example, -CF3, -C2F5, -CH2CF3, -CHF2, CF2CH3, and -CH2C1.
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The term "heteroalkyl" by itself or in combination with another term
means, unless otherwise stated, a stable straight or branched chain radical
consisting of the stated number of carbon atoms and one or two heteroatoms
selected from the group consisting of 0, N, and S, and wherein, in the sulfur
heteroatoms may be optionally oxidized and the nitrogen heteroatoms may be
optionally quaternized or oxidized. The oxygens bonded to oxidized sulfur or
nitrogen may be present in addition to the one or two heteroatoms in the
heteroalkyl group. The heteroatom(s) may be placed at any position of the
heteroalkyl group, including between the rest of the heteroalkyl group and the
fragment to which it is attached, as well as attached to the most distal
carbon atom
in the heteroalkyl group. Examples include: -0-CH2-CH2-CH3, -CH2-CH2CH2-
OH, -CH2-CH2-NH-CH3, -CH2-S02-NH-CH3, --CH2-S-CH2-CH3, and
-CH2CH2-S(=0)-CH3. Up to two heteroatoms may be consecutive, such as, for
example, -CH2-NH-OCH3, or -CH2-CH2-S-S-CH3.
The term "heterocycle" or "heterocyclyr or "heterocyclic" by itself or as
part of another substituent means, unless otherwise stated, an unsubstituted
or
substituted, stable, mono- or multicyclic heterocyclic ring system which
consists
of carbon atoms and at least one heteroatom selected from the group consisting
of
N, 0, and S, and wherein the nitrogen and sulfur heteroatoms may be optionally
oxidized, and the nitrogen atom may be optionally quaternized. The
heterocyclic
system may be attached, unless otherwise stated, at any heteroatom or carbon
atom which affords a stable structure.
The term "heteroaryl" or "heteroaromatic" refers to a heterocycle having
aromatic character. A monocyclic heteroaryl group is a 5-, 6, or 7-membered
ring, examples of which are pyrrolyl, fury!, thienyl, pyridyl, pyrimidinyl and
pyrazinyl. A polycyclic heteroaryl may comprise multiple aromatic rings or may
include one or more rings which are partially saturated. Examples of
polycyclic
heteroaryl groups containing a partially saturated ring include
tetrahydroquinolyl
and 2,3-dihydrobenzofuryl. For compounds of Formula I, the attachment point on
ring Q is understood to be on an atom which is part of an aromatic monocyclic
ring or a ring component of a polycyclic aromatic which is itself an aromatic
ring.
The attachment point on ring Q may be a ring carbon or a ring nitrogen and
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includes attachment to form aromatic 'quaternary ammonium salts such as
pyridinium.
Examples of non-aromatic heterocycles include monocyclic groups such
as: aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine,
pyrroline,
imidazoline, pyrazolidine, dioxolane, sulfolane, 2,3-dihydrofuran, 2,5-
dihydrofuran, tetrahydrofuran, thiophane, piperidine, 1,2,3,6-
tetrahydropyridine,
1,4-dihydropyridine, piperazine, morpholine, thiomorpholine, pyran, 2,3-
dihydropyran, tetrahydropyran, 1,4-dioxane, 1,3-dioxane, homopiperazine,
homopiperidine, 1,3-dioxepane, 4,7-dihydro-1,3-dioxepin and
hexamethyleneoxide.
Examples of heteroaryl groups include: pyridyl, pyrazinyl, pyrimidinyl,
particularly 2- and 4-pyrimidyl, pyridazinyl, thienyl, furyl, pyrrolyl,
particularly
2-pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, particularly 3- and 5-
pyrazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl,
tetrazolyl,
1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazoly1 and 1,3,4-
oxadiazolyl.
Examples of polycyclic heterocycles include: indolyl, particularly 3-, 4-,
5-, 6- and 7-indolyl, indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl,
particularly 1- and 5-isoquinolyl, 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl,
quinoxalinyl, particularly 2- and 5-quinoxalinyl, quinazolinyl, phthalazinyl,
1,8-
naphthyridinyl, 1,4-benzodioxanyl, coumarin, dihydrocoumarin, benzofuryl,
particularly 3-, 4-, 1,5-naphthyridinyl, 5-, 6- and 7-benzofuryl, 2,3-
dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl, particularly 3-, 4-, 5-,
6-,
and 7-benzothienyl, benzoxazolyl, benzthiazolyl, particularly 2-benzothiazoly1
and 5-benzothiazolyl, purinyl, benzimidazolyl, particularly 2-benzimidazolyl,
benztriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl,
pyrrolizidinyl, and
quinolizidinyl.
The term "heteroarylene" by itself or as part of another substituent means,
unless otherwise stated, a divalent heteroaryl radical. Preferred are five- or
six-
membered monocyclic heteroarylene. More preferred are heteroarylene moieties
comprising divalent heteroaryl rings selected from pyridine, piperazine,
pyrimidine, pyrazine, furan, thiophene, pyrrole, thiazole, imidazole and
oxazole.
For compounds of the present invention, when an aromatic or
heteroaromatic ring is attached to a position and the ring comprises a
polycyclic
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ring which is partially saturated, the attachment point on the aromatic or
heteroaromatic ring is on a ring atom of an aromatic ring component of the
polycyclic ring. For example, on the partially saturated heteroaromatic ring,
1,2,3,4-tetrahydroisoquinoline, attachment points are ring atoms at the 5-, 6-
, 7-
and 8- positions.
The aforementioned listing of heterocyclyl and heteroaryl moieties is
intended to be representative, not limiting.
The term "hydrocarbyl" refers to any moiety comprising only hydrogen
and carbon atoms. Preferred hydrocarbyl groups are (Ci-Ci2)hydrocarbyl, more
preferred are (Ci-C7)hydrocarbyl, most preferred are benzyl and (Ci-C6)alkyl.
The term "hydrocarbylene" by itself or as part of another substituent
means, unless otherwise stated, a divalent moiety comprising only hydrogen and
carbon atoms. A substitution of another group on hydrocarbylene may be at any
substitutable carbon, i.e., the expression ¨(C1-C6 hydrocarbylene)Rw would
include, for example:
Rw Rw
Rw
H3C CH3
The expression "carboxy terminally-linked peptidyl residue" refers to a
peptide radical as a substituent on a molecule of Formula I. The radical is
bonded
through the carboxyl functionality of a peptidyl residue to form a
carboxamide,
carboxylic ester or acyl sulfide (-S-C(=0)-).
The amino acid residues comprising the carboxy terminally-linked
peptidyl residue may comprise natural or unnatural amino acids or a
combination
thereof. Unnatural amino acids are amino acids other than the twenty essential
amino acids. One example of an unnatural amino acid is a D-amino acid, i.e.,
an
amino acid having a stereochemistry opposite the stereochemistry of natural L-
amino acids. Another example of an unnatural amino acid is an amino acid
having a side chain that differs from the side chains occurring in the natural
amino
acids, for example a-ethyl glycine or a-phenyl glycine. A third example is an
amino acid having a backbone variation. Examples of amino acid backbone
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variations include 13-alanine and 13-turn mimetics such as Freidinger's
lactam. A
fourth example of an unnatural amino acid is an amino acid having two a-
substituents, e.g., a,a-dimethyl glycine.
The amino terminus of the carboxy terminally-linked peptidyl residue may
be an unsubstituted amino group, or may be substituted. Substitutions on the
amino terminus include mono- and di-(Ci-C6 alkyl), -C(=0)(C1-C6 alkyl), -
C(=0)0(Ci-C7)hydrocarbyl) and commonly employed nitrogen protecting groups
such as tert-butoxycarbonyl (BOC), carbobenxyloxy (CBZ), 2,4-dimethoxybenzyl
and fluorenylmethoxycarbonyl (FMOC).
The expression "amino terminally-linked peptidyl residue" refers to a
peptide radical as a substituent on a compound according to Formula I. The
radical is bonded through the terminal amino functionality of the peptidyl
residue
to form a carboxamide, sulfonamide, urea or thiourea.
The carboxy terminus of the amino terminally-linked peptidyl residue may
be a free carboxyl group or a salt thereof, or may be derivatized as an ester
or
amide. Suitable esters include alkyl, preferably (C1-C6) alkyl; and arylalkyl,
preferably benzyl esters. Suitable amides include the primary amide and
secondary and tertiary amides comprising one or two nitrogen substituents
independently selected from (C1-C3)alkyl, preferably methyl or ethyl; aryl,
preferably phenyl; and aryl(Ci-C3)alkyl groups, preferably benzyl or
substituted
benzyl.
As with the carboxy terminally-linked peptidyl residues, the amino acids
comprising the amino terminally-linked peptidyl residue may comprise natural
or
unnatural amino acids or a combination thereof.
The term "(Cx-Cy)perfluoroalkyl," wherein x < y, means an alkyl group
with a minimum of x carbon atoms and a maximum of y carbon atoms, wherein
all hydrogen atoms are replaced by fluorine atoms. Preferred is -(C1-
C6)perfluoroalkyl, more preferred is -(C1-C3)perfluoroalkyl, most preferred is
¨CF3.
The term "trifluoro(Cx-Cy)alkyl" means an alkyl group with a minimum of
x carbon atoms and a maximum of y carbon atoms, wherein the three hydrogen
atoms on a terminal carbon (-CH3) are replaced by fluorine atoms. Examples
include ¨CH2CF3, -(CH2)2-CF3 and ¨CH(CH3)-CF3.
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The term "difluoro (Cx-Cy) alkyl" means an alkyl group with a minimum
of x carbon atoms and a maximum of y carbon atoms, wherein one carbon atom is
geminally substituted with two fluorine atoms. The fluorine-substituted carbon
may be the any carbon in the chain having at least two substitutable
hydrogens,
including the a terminal -CH3 group and the proximal carbon through which the
difluoro(Cx-Cy)alkyl is bonded to the rest of the molecule. Examples include ¨
CH2CF2H, -(CH2)2-CF2H and ¨CF2-CH3 and 3,3-difluorocyclohexyl.
The term "substituted" means that an atom or group of atoms has replaced
hydrogen as the sub stituent attached to another group. For aryl and
heteroaryl
groups, the term "substituted" refers to any level of substitution, namely
mono-,
di-, tri-, tetra-, or penta-substitution, where such substitution is
permitted. The
substituents are independently selected, and substitution may be at any
chemically
accessible position.
The naming of compounds disclosed herein was done by employing the
structure naming programs included in ChemDraw software packages. The
compounds, except for the a,b-unsaturated sulfonamides, were named using the
"Structure to Name" program within ChemDraw Ultra Version 8.0 (CI 1985-2003,
CambridgeSoft Corporation, 100 Cambridgepark Drive, Cambridge, MA 02140
USA). The structures of the a,b-unsaturated sulfonamides disclosed herein were
named using the Nomenclator Plug-in for ChemDraw 7Ø
Compounds of Formula I may be prepared via synthetic organic chemistry
methods within the capability of a chemist of ordinary skill. Compounds of
Formula I wherein the exocyclic carbon-carbon double bond is (E)- is
preferably
prepared via procedures that are selective for the preparation of (E)-olefins
respectively. The synthetic routes for the preparation of a,13-Unsaturated
Sulfoxides and Sulfones of Formula IE are shown below in Schemes 1, 2, 3, 4,
5,
and 6.
A. Preparation of a,13-Unsaturated Sulfoxides and Sulfones of Formula I
(i) Preparation of Compounds of Formula IE
One preferred preparation of (E)-compounds of Formula I wherein X is ¨
C*H(Rx)S0- or --C*H(Rx)S02-, is by a Knoevenagel condensation according to
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the Scheme 1 below, wherein RI, R2, R3, Rx, A, Q, a, b, n and * are as defined
herein for Formula I.
In one preferred embodiment of the preparation of (E)-compounds of
Formula I, the synthesis route illustrated in Scheme 1 serves to produce the
compound of Formula 6, which is itself a compound of Formula I. In addition,
the compound of Formula 6 serves as advanced intermediate which may be
further derivatized to provide additional novel compounds of Formula I. A
preferred embodiment for synthesis route illustrated in Scheme 1 is depicted
in
Example 1, infra.
In Scheme 1, substituted benzyl bromides, 1, wherein R is defined as in
Table 1 infra, are reacted with thioglycollic acid 2, in the presence of mild
or
strong bases, to obtain compound 3. Suitable base agents include, e.g., NaOH,
Me0H, sodium-, potassium-, lithium hexamethyldisilazide, lithium diisopropyl
amide, and the like. The reaction is preferably performed at room temperature
or
higher, more preferably from 30 C to 50 C. Complete oxidation of 3, with
hydrogen peroxide in the presence of glacial acetic acid yields 4. The
oxidation
reaction is preferably performed at room temperature or higher, more
preferably
from 30 C to 50 C. Knoevenagel condensation of 4, with aromatic aldehydes 5,
wherein RI is defined as in Table 1 infra, in toluene in the presence of
catalytic
amounts of piperidine and benzoic acid yields styryl benzyl sulfones, 6,
wherein
R and RI as defined herein. The Knoevenagel condensation reaction is
preferably
performed at 120 C or higher. Alternately, the condensation between 4 and 5
may
be carried out in glacial acetic acid in the presence of a catalytic amount of
benzylamine to obtain 6. The condensation reaction is preferably performed at
118 C or higher. Prefereably, the reaction temperatures for condensation are
about 120 C to about 140 C.
Scheme 1. Synthesis of (E)-Styryl Benzyl Sulfones
CA 02646874 2008-12-17
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0
7Br
VSOH
7,70H a
R¨ + HS
R¨ ______________________________________________________________ =
0
1 2 3
0
02 02
7S
OH CHO N
cord
1
4 5 6
In another preferred embodiment of the preparation of (E)-compounds of
Formula I, the synthesis route illustrated in Scheme 2 also serves to produce
mono
substituted styryl benzyl sulfones such as the compound of Formula 6, which as
noted supra is itself a compound of Formula I and serves as an advanced
intermediate which may be further derivatized to provide additional novel
compounds of Formula I. A preferred embodiment for the synthesis route
illustrated in Scheme 1 is depicted in Example 1, infra.
In scheme 2, benzyl mercaptans with desired substituents, 7, wherein R is
defined as in Table 1 infra, are reacted with a-bromo substituted
acetophenones,
8, wherein RI is defined as in Table 1 infra, in absolute alcohol in the
presence of
a base to obtain 9. Suitable base agents include, e.g., NaOH, KOH, and Li0H.
Other suitable solvents include, e.g., methanol, ethanol, n-propanol,
isopropanol,
butanol, acetic acid and triethylamine. The reaction is preferably performed
at
room temperature or higher, more preferably from 30 C to 50 C. Oxidation of
9
with hydrogen peroxide in glacial acetic acid yields 10. The oxidation
reaction is
preferably performed at room temperature or higher, more preferably from 30 C
to 50 C. Reduction of the carbonyl group in 10 with sodium borohydride in
methanol yields corresponding alcohol, 11. Suitable reducing agents include
CA 02646874 2008-12-17
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hydride reducing agents, e.g., NaBH4 and NaBH3CN. Other suitable solvents
include, e.g., tetrahydrofuran (THF), methanol, ethanol, n-propanol,
isopropanol,
butanol or acetic acid. The reduction reaction is preferably performed at low
temperature, preferably from ¨40 C to 0 C. Elimination of water from 11 in
refluxing solution of p-toluenesulfonic acid in benzene afforded 6. The
reduction
reaction is preferably performed at 80 C or higher, more preferably from
about
120 C to about 140 C.
Scheme 2. Alternate Method for the Synthesis of (E)-Styryl Benzyl Sulfones
/SH
Br
+ ,¨Ri a
7 8 9
0 OH
02 02
R
_r-
11
02
R-
10 6
In another preferred embodiment of the preparation of (E)-compounds of
Formula I, the aldehyde condensation synthesis route illustrated in Scheme 3
serves to produce the compound of Formula 20, which is itself a compound of
Formula I. In addition, the compound of Formula 20 serves as advanced
intermediate which may be further derivatized to provide additional compounds
of Formula I. A preferred embodiment for the aldehyde condensation synthesis
route illustrated in Scheme 3 is depicted in Example 1, infra.
CA 02646874 2008-12-17
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In Scheme 3, isovanillin 12 was reacted with TBDMS-THF to produce
TBDMS protected isovanillin 13. Suitable
solvents include, e.g., DMF,
tetrahydrofuran (THF). The reaction is preferably performed at room
temperature
or higher, more preferably from 30 C to 50 C. TBDMS protected isovanillin 13
was reduced to alcohol 14 with sodium borohydride. Suitable reducing agents
include hydride reducing agents, e.g., NaBH4 and NaBH3CN. Suitable solvents
include, e.g., Me0H ethanol, n-propanol, isopropanol, butanol and acetic acid.
The reduction reaction is preferably performed at low temperature, preferably
from ¨40 C to 0 C. The benzyl alcohol 14 was converted to benzyl chloride 15
with SOC12. The reaction is preferably performed at low temperature,
preferably
from ¨40 C to 0 C. Benzyl chloride 15 was subsequently converted to 16 by
condensing with thioglycollic acid. Suitable base agents include, e.g. NaOH,
KOH, and Li0H. Other suitable solvents include, e.g., Me0H, ethanol, n-
propanol, isopropanol, butanol, triethylamine and tetrahydrofuran (THF). The
condensation reaction is preferably performed at room temperature or higher,
more preferably from 30 C to 50 C. Deprotection of TBDMS with tetrabutyl
ammonium fluoride and subsequent oxidation of 17 with hydrogen peroxide
provided 18. The deprotection reaction is carried out in tetrahydrofuran. The
deprotection reaction is carried out at room temperature or higher, more
preferably from 30 C to 50 C. Condensation of 18 with aldehyde 19 provided
styryl benzyl sulfone 20. Other suitable solvents for the condensation
reaction
include, e.g., toluene, triethylamine and glacial acetic acid in the presence
of
bases like piperidine,triethylamine and benzylamine. The deprotection reaction
is
carried out at 120 C or higher.
30
CA 02646874 2008-12-17
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Scheme 3 Synthesis of (E)-2,4,6-(Trimethoxystyry1)-3-Hydroxy-4-
Methoxybenzyl Sulfone
HO
CHO CHO
c
a 40 b
OH OTBDMS OTBDMS
0 0 0
12 13 14
0 0
CI S---.,
OH
____________________ 0 40
02 e
OTBDMS OTBDMS OH
0 0 0
16 17
S,
OH CHO
0 0 1 0
10 OH
+
0
g
---0-
0 5 S
02
0 1 OH
0
5 18 19
In another preferred embodiment of the preparation of (E)-compounds of
Formula I, the Knoevenagel-type condensation synthesis reaction as shown in
Scheme 4 serves to produce the compound of Formula 20, which is itself a
10 compound of Formula I. In addition, the compound of Formula 20 serves as
advanced intermediate which may be further derivatized to provide additional
compounds of Formula I. A preferred embodiment for the Knoevenagel-type
CA 02646874 2008-12-17
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condensation synthesis reaction illustrated in Scheme 4 is depicted in Example
1,
infra.
In Scheme 4, Isovanillin 12 was reacted with 4- toluenesulfonyl chloride
in the presence of pyridine to obtain compound 21. Other suitable solvents for
the
reaction include, e.g., substituted pyridines, trimethylamine, triethylamine
and
DIPEA. The reaction is preferably performed at 30 C to 50 C or higher.
Reduction of 21 with sodium borohydride gave 22. Suitable reducing agents
include hydride reducing agents, e.g., NaBH4 and NaBH3CN. Other suitable
solvents include, e.g., Me0H ethanol, n-propanol, isopropanol, butanol and
acetic
acid. The reduction reaction is preferably performed at 20 C. Treatment of 22
with thionyl chloride in benzene resulted in 23. Other suitable solvents for
the
reaction include, e.g., pyridine, substituted pyridines, trimethylamine,
triethylamine and DIPEA. The reaction is preferably performed at 15 C to 20
C
or higher. On condensation with thioglycollic acid, 23 yielded
benzylthioacetic
acid 24. Suitable base reagents include, e.g., NaOH, KOH, and Li0H. Other
suitable solvents include, e.g., Me0H ethanol, n-propanol, isopropanol,
butanol,
acetic acid, and triethylamine. The condensation reaction is performed at
about
65 C. Prefereably, the reaction temperatures for condensation are about 60 C
to
about 85 C. Oxidation of 24 with hydrogen peroxide yields corresponding
sulfonylacetic acid 25. Other suitable solvents for the oxidation include,
e.g.,
acetic acid, Me0H ethanol, n-propanol, isopropanol and butanol. The oxidation
reaction is preferably performed at room temperature or higher. Knoevenagel
type
condensation of 25 with 2,4,6-trimethoxy benzaldehyde 19 in the presence of a
base produced unsaturated sulfone 26. The Knoevenagel condensation reaction is
preferably performed at 80 C or higher. Prefereably, the reaction
temperatures for
condensation are about 80 C to about 140 C. Suitable base reagents include,
e.g.,
NaOH, KOH, Li0H, triethylamine and piperidine in benzoicacid. Suitable
solvents include, e.g., xylene, toluene,C6H6 and Me0H. Removal of tosyl group
by treating 26 with sodium hydroxide gave the styryl benzyl sulfone 20.
Suitable
base reagents include, e.g., NaOH, KOH, and Li0H. Other suitable solvents
include, e.g., Me0H ethanol, n-propanol, isopropanol, butanol, triethylamine
and
acetic acid. The reaction is preferably performed at 80 C or higher.
Prefereably,
the reaction temperatures are about 80 C to about 140 C.
CA 02646874 2008-12-17
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Scheme 4. Alternate Method for the Synthesis of (E)-2,4,6-(Trimethoxystyry1)-3-
Hydroxy-4-Methoxybenzyl Sulfone
CA 02646874 2008-12-17
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HO
CHO CHO
* OH a
r 0
OTs b
r
0 OTs c ____________________________________________________________ =
0 0 0
/ ,--
12 21 22
0 0
02
01 S S
OH OH
* d * e
). 0
OTs OTs OTs +
0 0 0
/
24 25
23
,,,..--o = o"%-õ,
CHO
0
$
S
0
70 O
g
,
f 02
_________________________ p
0
1 OTs
26
19
õ,---o 0 o'',....,
1
000 S
02
IOH
CA 02646874 2008-12-17
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In yet another preferred embodiment of the preparation of (E)-compounds
of Formula I, the synthesis reaction as shown in Scheme 5 is used for
preparing
the bioavailable water soluble sodium phosphate prodrug (E)-
styrylbenzylsulfone
compound of Formula 29, which is itself a compound of Formula I. In addition,
the compound of Formula 29 serves as advanced intermediate which may be
further derivatized to provide additional compounds of Formula I. A preferred
embodiment for the synthesis reaction illustrated in Scheme 5 is depicted in
Example 1, infra.
In Scheme 5, the prodrug was synthesized in three steps starting from
styryl benzyl sulfone 20. Phosphorylation of phenolic group in 20 employing
dibenzyl phosphite under basic conditions gave 0-dibenzyl phosphate 27.
Suitable base reagents include, e.g., KH2PO4, NaOH, KOH, and Li0H. Other
suitable solvents include, e.g., triethylamine, Me0H ethanol, n-propanol,
isopropanol, and butanol. The reaction is preferably performed at room
temperature or higher, more preferably from 30 C to 50 C. Cleavage of benzyl
groups with bromo trimethylsilane in acetonitrile produced 3-0- phosphate 28.
Suitable solvents include, e.g., dichloromethane, Me0H ethanol, n-propanol,
isopropanol, butanol, acetic acid and triethylamine. The reaction is
preferably
performed at room temperature or higher, more preferably from 30 C to 50 C.
Treatment of the phosphonate acid 28 with sodium hydroxide in anhydrous
ethylene glycol dimethyl ether yielded disodium- 0-phosphate 29. Suitable base
reagents include, e.g., KH2PO4, NaOH, KOH, and Li0H. Other suitable solvents
include, e.g., ethyleneglycoldimethyl ether, dichloromethane, Me0H ethanol, n-
propanol, isopropanol, butanol, acetic acid and triethylamine. The reaction is
preferably performed at room temperature or higher, more preferably from 30 C
to 50 C.
Scheme 5. Method for the Synthesis of (E)-2,4,6-(Trimethoxystyry1)-3-0-
Phosphate Disodium-4-Methoxybenzyl Sulfone
CA 02646874 2008-12-17
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---0 0 o-, . .,,c) . so
1 1
0 $ s
02
a ____________________________ p
0 0 S
02 0
b v
1 1 OBz
OH
2027
P-,....
OBz
0 . o. o I. o
1 1
0 le S
02 0
c
_____________________________ >
0 140 S
02 0
1
0 / 0 /
OH 1 0-Na+
. .,
P P
10 IC)
OH 0-Na+
28 29
In yet a more preferred embodiment of the preparation of (E)-compounds
of Formula I, the synthesis reaction as shown in Scheme 6 is used for
preparing
5 the bioavailable water soluble sodium phosphate prodrug (E)-
styrylbenzylsulfone
compound of Formula 29, which is itself a compound of Formula I. In addition,
the compound of Formula 29 serves as advanced intermediate which may be
further derivatized to provide additional compounds of Formula I. A preferred
embodiment for the synthesis reaction illustrated in Scheme 6 is depicted in
10 Example 2, infra.
In Scheme 6, Stage 1, isovanillin 12 is reacted with 4- toluenesulfonyl
chloride in the presence of pyridine to obtain compound 21. The reaction is
preferably performed at 70 C to 80 C. In Stage 2, reduction of 21 with
sodium
borohydride as reducing agent in methanol yields 22. The reduction reaction is
CA 02646874 2008-12-17
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preferably performed at 15-20 C. In Stage 3, treatment of 22 with thionyl
chloride
in benzene with subsequent washing in hexane results in 23. The reaction is
preferably performed at 15 C to 20 C. In Stage 4, on condensation with
thioglycollic acid, 23 yields benzylthioacetic acid 24. The condensation
reaction
is performed at 65 C. Prefereably, the reaction temperatures for condensation
are
about 60 C to about 80 C. In Stage 5, oxidation of 24 with hydrogen peroxide
yields corresponding sulfonylacetic acid 25. The oxidation reaction is
preferably
performed at room temperature or higher, more preferably from 30 C to 50 C.
In
Stage 6, Knoevenagel type condensation of 25 with 2,4,6-trimethoxy
benzaldehyde 19 in the presence of a base produces unsaturated sulfone 26.
Suitable base reagents include, e.g., NaOH, KOH, and LOH. Suitable solvents
include, e.g., Me0H, ethanol, n-propanol, isopropanol, butanol and acetic
acid.
The Knoevenagel condensation reaction is performed at about 80 C. Preferably,
the reaction temperatures for condensation are about 80 C to about 140 C. In
Stage 7, removal of tosyl group by treating 26 with sodium hydroxide gave the
styryl benzyl sulfone 20. The reaction is preferably performed at about 80 C
or
higher. Preferably, the reaction temperatures are about 80 C to about 140 C.
Other suitable base reagents include, e.g., KOH, and Li0H. Suitable solvents
include, e.g., Me0H ethanol, n-propanol, isopropanol and butanol . In Stage 8,
phosphorylation of the phenolic group in 20 employing dibenzyl phosphite in
KH2PO4 with triethylamine as solvent yields 0-dibenzyl phosphate 27. The
reaction is preferably performed at room temperature. Suitable base reagents
include, e.g., NaOH, KOH, and Li0H. Suitable solvents include, e.g., Me0H
ethanol, n-propanol, isopropanol, butanol and acetic acid. In Stage 9,
cleavage of
the benzyl groups with bromo trimethylsilane in acetonitrile yields 3-0-
phosphate 28. The reaction is preferably performed at room temperature. Other
suitable solvents include, e.g., Me0H ethanol, n-propanol, isopropanol and
butanol. In Stage 10, treatment of the phosphate 28 with sodium hydroxide in
anhydrous ethylene glycol dimethyl ether yielded disodium- 0-phosphate 29. The
reaction is preferably performed at room temperature or higher. Other suitable
base reagents include, e.g., KOH, and Li0H. Other suitable solvents include,
e.g.,
Me0H, ethanol, n-propanol, isopropanol and butanol.
CA 02646874 2008-12-17
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Scheme 6: Method for the Synthesis of (E)-2,4,6 -(Trimethoxystyry1)-3-0-
Phosphate Disodium-4-Methoxybenzyl Sulfone
STAGE ¨1
= CHO
CHO S,,
+ 110 Pyridine
0
0 70-80 C
0,
OH
02
12 21
STAGE ¨ 2:
CHO
10 NaBH4 OH
0 = 0
0,5
Me0H 0 I.
02 02
21 22
STAGE ¨ 3:
OH 110 Cl
SOC12
-J
0
0 Benzene
02 02
22 23
STAGE ¨ 4:
CA 02646874 2008-12-17
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O
. s T Cl
SHCH2COOH HO O
00 ____________________________________ o. ...o
CH3OH/NaOH
0,s 0,s .
02 02
23 24
STAGE - 5:
OH OH
= S Vr S7(
0 le 02
0 H202 0
0O2
, AcOH 0-s02
S
* 0
24 25
STAGE - 6:
e
OH
. S7( 0 H
02 0
. /
0 0 70 0 Benzene 1
0
0, + _________________________ )1, ,-,1
Benzoic acid k-i
SO2 Piperidine 02
S SO2
. A * (i)
25 26
STAGE - 7:
CA 02646874 2008-12-17
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0
1110
20% NaOH 0
O '
Methanol 0
02
0 SO2
HO SO2
26 20
STAGE -8
0*o o/
0 (C6H5CH20)P(0)H ), 0
HO
TEA, CC14,DMAP
SO2
0 SO2
Bz0 OBz
0
20 27
15
CA 02646874 2008-12-17
-40-
STAGE ¨9
O___
O___
*so 0
AO
I e (CH3)3SiC1
___________________________________________ > I O
a
0 .
CH3CN 0
SO2
0 0 SO2
I *I
-- P-... 1
Bz0 ii OBz -- P--..
0 HO ii OH
0
27 28
STAGE¨ 10
/
O o
O IP oo 410 o
I Vo NaOH I
0liw 0
. 1,2-Di methoxy
Ethane
SO2 * 0 SO2
0
I I
HO OH Na0 ti ONa
0 0
28 29
In yet a more preferred embodiment of the preparation of (E)-compounds
of Formula I, the synthesis reaction as shown in Scheme 7 is used for
preparing
the bioavailable water soluble sodium phosphate prodrug (E)-
styrylbenzylsulfone
CA 02646874 2008-12-17
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compound of Formula 29, which is itself a compound of Formula I. In addition,
the compound of Formula 29 serves as advanced intermediate which may be
further derivatized to provide additional compounds of Formula I. A preferred
embodiment for the synthesis reaction illustrated in Scheme 7 is depicted in
Example 3, infra.
In Scheme 7, phosphorylation of the phenolic group in styryl benzyl
sulfone 20 employing phosphorous oxychloride with triethylamine and THF as
solvent yields the 3-0-chloro phosphate compound 20. The reaction is
preferably
performed at about 0 C or higher. Preferably, the reaction temperatures are
about
0 C to room temperature. The 3-0-chloro phosphate compound 20 is then treated
with potassium hydroxide in THF. and then treated with dilute HC1 to yield the
phosphoric acid precipitate of compound 20. The reaction is preferably
performed
at about 0 C. Suitable base reagents include, e.g., NaOH, and Li0H. Suitable
solvents include, e.g., Me0H ethanol, n-propanol, isopropanol,and butanol.
Treatment of the phosphate compound 20 with sodium hydroxide in methanol
yielded disodium-O-phosphate compound 29. The reaction is preferably
performed at room temperature or higher. Other suitable base reagents include,
e.g., KOH, and Li0H. Other suitable solvents include, e.g., Me0H, ethanol, n-
propanol, isopropanol, butanol and triethylamine.
The phosphoric acid precipitate of compound 20 is then dissolved in
methanol and treated with sodium hydroxide to yield the disodium-O-phosphate
29. The reaction is preferably performed at room temperature or higher. Other
suitable base reagents include, e.g., KOH, and Li0H. Other suitable solvents
include, e.g., Me0H ethanol, n-propanol, isopropanol and butanol.
In yet another aspect of the present invention, compounds of Formula I
prepared by the methods disclosed herein are useful for the treatment of
proliferative disorders, non-cancer proliferative disorders, as well as
radioprotective treatment, and chemoprotective treatment.
I. Treatment of Proliferative Disorders
In yet another aspect of the present invention, compounds of Formula IE
and salts thereof prepared by the methods disclosed herein are believed to
selectively inhibit proliferation of cancer cells, and kill various tumor cell
types
CA 02646874 2008-12-17
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without killing (or with reduced killing of) normal cells. It is believed that
cells
are killed at concentrations where normal cells may be temporarily growth-
arrested but not killed.
A. Treatment of Cancer
The compounds of Formula 1E of the invention may be administered to
individuals (mammals, including animals and humans) afflicted with cancer.
The compounds of the invention are believed to inhibit the proliferation of
tumor cells and, for some compounds, to induce cell death. Cell death is
believed
to result from the induction of apoptosis. The compounds are believed
effective
against a broad range of tumor types, including but not limited to the
following:
ovarian cancer; cervical cancer; breast cancer; prostate cancer; testicular
cancer,
lung cancer, renal cancer; colorectal cancer; skin cancer; brain cancer;
leukemia,
including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoid
leukemia, and chronic lymphoid leukemia.
More particularly, cancers that may be treated by the compounds,
compositions and methods of the invention include, but are not limited to the
following:
cardiac cancers, including, for example sarcoma, e.g., angiosarcoma,
fibrosarcoma, rhabdomyosarcoma, and liposarcoma; myxoma; rhabdomyoma;
fibroma; lipoma and teratoma;
lung cancers, including, for example, bronchogenic carcinoma, e.g.,
squamous cell, undifferentiated small cell, undifferentiated large cell, and
adenocarcinoma; alveolar and bronchiolar carcinoma; bronchial adenoma;
sarcoma; lymphoma; chondromatous hamartoma; and mesothelioma;
gastrointestinal cancer, including, for example, cancers of the esophagus,
e.g., squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, and lymphoma;
cancers of the stomach, e.g., carcinoma, lymphoma, and leiomyosarcoma; cancers
of the pancreas, e.g., ductal adenocarcinoma, insulinoma, glucagonoma,
gastrinoma, carcinoid tumors, and vipoma; cancers of the small bowel, e.g.,
adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma,
hemangioma, lipoma, neurofibroma, and fibroma; cancers of the large bowel,
e.g.,
adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, and leiomyoma;
CA 02646874 2008-12-17
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genitourinary tract cancers, including, for example, cancers of the kidney,
e.g., adenocarcinoma, Wilm's tumor (nephroblastoma), lymphoma, and leukemia;
cancers of the bladder and urethra, e.g., squamous cell carcinoma,
transitional cell
carcinoma, and adenocarcinoma; cancers of the prostate, e.g., adenocarcinoma,
and sarcoma; cancer of the testis, e.g., seminoma, teratoma, embryonal
carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell
carcinoma, fibroma, fibroadenoma, adenomatoid tumors, and lipoma;
liver cancers including, for example, hepatoma, e.g., hepatocellular
carcinoma; cholangiocarcinoma; hepatoblastoma; angiosarcoma; hepatocellular
adenoma; and hemangioma;
bone cancer including, for example, osteogenic sarcoma (osteosarcoma),
fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma,
malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant
giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses),
benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and
giant cell tumors;
nervous system cancers including, for example, cancers of the skull, e.g.,
osteoma, hemangioma, granuloma, xanthoma, and osteitis deformans; cancers of
the meninges, e.g., meningioma, meningiosarcoma, and gliomatosis; cancers of
the brain, e.g., astrocytoma, medulloblastoma, glioma, ependymoma, germinoma
(pinealoma), glioblastoma multiform, oligodendroglioma, schwannoma,
retinoblastoma, and congenital tumors; and cancers of the spinal cord, e.g.,
neurofibroma, meningioma, glioma, and sarcoma;
gynecological cancers including, for example, cancers of the uterus, e.g.,
endometrial carcinoma; cancers of the cervix, e.g., cervical carcinoma, and
pre-
tumor cervical dysplasia; cancers of the ovaries, e.g., ovarian carcinoma,
including serous cystadeno carcinoma, mucinous cystadeno carcinoma,
unclassified carcinoma, granulosa-thecal cell tumors, Sertoli-Leydig cell
tumors,
dysgerminoma, and malignant teratoma; cancers of the vulva, e.g., squamous
cell
carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, and
melanoma; cancers of the vagina, e.g., clear cell carcinoma, squamous cell
carcinoma, botryoid sarcoma, and embryonal rhabdomyosarcoma; and cancers of
the fallopian tubes, e.g., carcinoma;
CA 02646874 2008-12-17
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hematologic cancers including, for example, cancers of the blood, e.g.,
acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic
leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple
myeloma, and myelodysplastic syndrome, Hodgkin's lymphoma, non-Hodgkin's
lymphoma (malignant lymphoma) and Waldenstrom's macroglobulinemia;
skin cancers including, for example, malignant melanoma, basal cell
carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi,
lipoma, angioma, dermatofibroma, keloids, psoriasis; and adrenal gland cancers
including, for example, neuroblastoma.
Cancers may be solid tumors that may or may not be metastatic. Cancers
may also occur, as in leukemia, as a diffuse tissue. Thus, the term "tumor
cell" as
provided herein, includes a cell afflicted by any one of the above identified
disorders.
B. Treatment of Non-Cancer Proliferative Disorders
The compounds of Formula IE prepared by the methods disclosed herein
are also believed useful in the treatment of non-cancer proliferative
disorders, that
is, proliferative disorders which are characterized by benign indications.
Such
disorders may also be known as "cytoproliferative" or "hyperproliferative" in
that
cells are made by the body at an atypically elevated rate. Non-cancer
proliferative
disorders believed treatable by compounds of the invention include, for
example:
hemangiomatosis in newborn, secondary progressive multiple sclerosis,
atherosclerosis, chronic progressive myelodegenerative disease,
neurofibromatosis, ganglioneuromatosis, keloid formation, Pagets Disease of
the
bone, fibrocystic disease of the breast, uterine fibroids, Peronies and
Duputren's
fibrosis, restenosis, benign proliferative breast disease, benign prostatic
hyperplasia, X-linked lymphoproliferative disorder (Duncan disease), post-
transplantation lymphoproliferative disorder (PTLD), macular degeneration, and
retinopathies, such as diabetic retinopathies and proliferative
vitreoretinopathy
(PVR)
Other non-cancer proliferative disorders believed treatable by compounds
of the invention include the presence of pre-cancerous lymphoproliferative
cells
associated with an elevated risk of progression to a cancerous disorder. Many
CA 02646874 2008-12-17
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non-cancerous lymphoproliferative disorders are associated with latent viral
infections such as Epstein-Barr virus (EBV) and Hepatitis C. These disorders
often begin as a benign pathology and progress into lymphoid neoplasia as a
function of time.
Treatment of tumor cells with the compounds of the invention is believed
to lead to inhibition of cell proliferation and induction of apoptotic cell
death.
II. Radioprotective Treatment
The compounds of the invention are also believed to protect normal cells
and tissues from the effects of acute and chronic exposure to ionizing
radiation.
Individuals may be exposed to ionizing radiation when undergoing
therapeutic irradiation for the treatment of proliferative disorders. The
compounds are believed effective in protecting normal cells during therapeutic
irradiation of abnormal tissues. The compounds are also believed useful in
protecting normal cells during radiation treatment for leukemia, especially in
the
purging of malignant cells from autologous bone marrow grafts with ionizing
radiation.
According to the invention, therapeutic ionizing radiation may be
administered to an individual on any schedule and in any dose consistent with
the
prescribed course of treatment, as long as the radioprotectant compound
according to the invention is administered prior to the radiation. The course
of
treatment differs from individual to individual, and those of ordinary skill
in the
art can readily determine the appropriate dose and schedule of therapeutic
radiation in a given clinical situation.
III. Chemoprotective Treatment
In addition, the compounds of Formula IE prepared by the methods
disclosed herein are believed to protect normal cells and tissues from the
effects
of exposure to mitotic phase cell cycle inhibitors and topoisomerase
inhibitors.
Mitotic phase cell cycle inhibitors include, by way of example and not
limitation, taxanes, such as paclitaxel and its analogs; vinca alkaloids such
as
vincristine and vinblastine; colchicine; estramustine; and naturally occurring
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macrolides such as rhizoxin, maytansine, ansamitocin P-3, phomopsin A,
dolastatin 10 and halichrondin B.
Paclitaxel is an anti-mitotic drug presently used as an initial treatment for
ovarian, breast and lung cancer, with moderate success. Vincristine is a well-
established anti-mitotic drug widely used for the treatment of breast cancer,
Hodgkin's lymphoma and childhood cancers.
Topoisomerase inhibitors include compounds that inhibit topoisomerase I,
compounds that inhibit topoisomerase II, and compounds that inhibit both
topoisomerase I and II.
Inhibitors of topoisomerase I include, for example, adriamycin, etoposide,
13-lapachone (Calbiochem No. 428022), AG-555 (Calbiochem No. 112270), 10-
hydroxycamptothecin (Calbiochem No. 390238), AG-1387 (Calbiochem No.
658520), rebeccamycin (Calbiochem No. 553700), nogalamycin (Calbiochem No.
488200), and topotecan (Calbiochem No. 614800).
Inhibitors of topoisomerase II include, for example, camptothecin,
irinotecan and topotecan, amsacrine (Calbiochem No. 171350),
aurintricarboxylic
acid (Calbiochem No. 189400), bruneomycin (Calbiochem No. 571120),
ellipticine (Calbiochem No. 324688), epirubicin (Calbiochem No. 324905),
etoposide (Calbiochem No. 341205), genistein (Calbiochem No. 345834), and
merbarone (Calbiochem No. 445800). .
Inhibitors of both topoisomerase I and II include, for example, aclarubicin
(Calbiochem No. 112270), congocidine (Calbiochem No. 480676), daunomycin
(Calbiochem No. 251800), ellagic acid (Calbiochem No. 324683), and suramin
(Calbiochem No. 574625).
The compounds of the present invention are believed to not only protect
normal cells, but are also to be operationally cytotoxic in tumor cells. In
normal
cells, the cytoprotective compounds of the invention are believed to induce a
reversible resting state rendering the normal cells relatively refractory to
the
cytotoxic effect of mitotic phase cell cycle inhibitors and topoisomerase
inhibitors.
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VI. Conjugates of Formula I Compounds
Compounds according to Formula I may be reacted to form conjugates
with a carrier molecule. The carrier may comprise any molecule sufficiently
large
to be capable of generating an immune response in an appropriate host animal.
One such preferred carrier is keyhole limpet haemocyanin (KLH). Additionally,
structural components of substituents on the phenyl or Q rings of compounds of
the invention (e.g., as peptidyl substituents) can potentially provide
antigenic
activity sufficient to raise antibodies to the compounds of Formula I.
Alternately,
a Formula I compound may be conjugated to an antibody (Ab). Antibodies for
conjugation to Formula I compounds preferably comprise monoclonal antibodies
and monospecific polyclonal antibodies or fragments thereof, most preferably
tumor-specific antibodies, or fragments thereof. The antibody (Ab) may be
covalently linked to compounds of Formula I, via a covalent linker (L) to form
a
conjugate of the Formula I-L-Ab.
The compounds of Formula I can readily be covalently bonded to
antibodies, preferably tumor-specific monoclonal antibodies (Mab) via a
suitable
bifunctional linker (¨L¨) to yield a conjugate of general Formula, I¨L¨Ab. A
general synthetic route for preparing compounds of the present invention of
general Formula I¨L¨Ab is shown in Scheme 14 of U.S. Patent Application No.
20080058290.
The covalent linker (L) between a compound according to Formula I and
an antibody (Ab) to form a conjugate of the Formula I-L-Ab may, in its
simplest
faint, comprise a single covalent bond connecting the compound according to
Formula I to the antibody. More commonly, the compound according to Formula
I is attached to the antibody using a suitable bifunctional linking reagent.
The
term "bifunctional linking reagent" refers generally to a molecule that
comprises
two reactive moieties which are connected by a spacer element. The term
"reactive moieties" in this context, refers to chemical functional groups
capable of
coupling with an antibody or a compound according to Formula I by reacting
with
functional groups on the antibody and the compound according to Formula I.
An example of a covalent bond formed as a linker between a compound
according to Formula I and an antibody is a disulfide bond formed by the
oxidation of an antibody and a compound according to Formula I, wherein a
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substituent on the phenyl ring or Q-ring of Formula I comprises a peptidyl
moiety
containing one or more cysteine amino acids. The cysteine residues can be
oxidized to form disulfide links by dissolving 1 mg of the a suitable compound
according to Formula I and 0.5 equivalents of the desired antibody in 1.5 mL
of
0.1% (v/v) 17.5 mM acetic acid, pH 8.4, followed by flushing with nitrogen and
then 0.01 M K2Fe(CN)6. After incubation for one hour at room temperature, the
adduct peptide is purified, e.g., by HPLC.
Another example of a suitable covalent bond formed as a linker between a
compound according to Formula I and an antibody is an amide bond formed by
reacting an amino group on a compound of the invention with a carboxylic acid
group which forms part of the primary structure of the antibody (Ab) (e.g.,
for
example a glutamic or aspartic amino acid residue). Alternately, an amide bond
could be formed if the reacting moieties were reversed, i.e., the compound
according to Formula I contains a carboxylic acid functionality and reacts
with an
amino functionality within the Ab structure.
Alternatively, a compound according to Formula I and an antibody Ab
may be covalently linked using a bifunctional linking reagent. In one such
embodiment of the present invention, a compound according to Formula I,
wherein a substituent on the phenyl ring or Q-ring of Formula I comprises a
peptidyl moiety, is coupled to an antibody using a bifunctional linking
reagent.
For example, adducts can be prepared by first preparing S-(-N-
hexylsuccinimido)-modified derivatives of an antibody and of a compound
according to Formula I, according to the method of Cheronis et al., J Med.
Chem.
37: 348 (1994),
N-hexylmaleimide, a precursor for the modified antibody and
compound according to Formula I, is prepared from N-
(methoxycarbonyl)maleimide and N-hexylamine by mixing the two compounds in
saturated NaHCO3 at 0 C according to the procedure of Bodanszky and
Bodanszky, The Practice of ,Peptide Synthesis; Springer-Verlag, New York, pp.
29-31 (1984).
The product of the resulting reaction mixture is isolated by extraction into a
suitable solvent, e.g., ethyl acetate, followed by washing with water, dried
over
Na2SO4. The extract is then concentrated in vacua to produce N-hexylmaleimide.
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S-(N-hexylsuccinimido)-modified antibody and Formula I compound are then
prepared from a cysteine-containing peptide and N-hexylmaleimide by mixing
one part peptide with 1.5 parts N-hexylmaleimide in DMF (3.3 mL/mM peptide)
followed by addition to 30 volumes of 0.1 M ammonium bicarbonate, pH 7.5.
The S-alkylation reaction carried out in this manner is complete in 30 mm. The
resulting S-(N-hexylsuccinimido)-modified peptide monomer is purified by
preparative reverse-phase HPLC, followed by lyophilization as a fluffy, white
powder.
Bis-succinimidohexane peptide heterodimers (wherein one peptide is the
antibody and the other peptide is a Formula I compound wherein a substituent
on
the phenyl or Q ring of Formula I comprises a peptidyl moiety), may be
prepared
according to the method of Cheronis et al., supra from cysteine-substituted
peptides. A mixture of one part bis-maleimidohexane is made with two parts
peptide monomer in DMF (3.3mL/mM peptide) followed by addition to 0.1
ammonium bicarbonate, pH 7.5. The reaction mixture is stirred at room
temperature and the reaction is usually complete within 30 mm. The resulting
bis-succinimidohexane peptide dimer is purified by preparative reverse-phase
HPLC. After lyophilization the material is a fluffy, white powder.
Covalently linked adducts of the Formula I¨L¨Ab may be prepared by
utilizing homo-bifunctional linking reagents (wherein the two reactive
moieties
are the same), such as, for example, disuccinimidyl tartrate, disuccinimidyl
suberate, ethylene glycolbis-(succinimidyl succinate), 1,5-difluoro-2,4-
dinitrobenzene ("DFNB"), 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene
("DIDS"), and bis-maleimidohexane ("BMH"). The linking reaction occurs
randomly between the Ab and a compound according to Formula I having a
peptidyl moiety as part of at least on substituent on the phenyl ring or the Q
ring
of Formula I.
Alternatively, hetero-bifunctional linking reagents may be employed.
Such agents include, for example, N-succinimidy1-3-(2-pyridyldithio)propionate
("SPDP"), sulfosuccinimidy1-2-(p-azidosalicylamido)ethy1-1-3'-dithiopropionate
("SASD", Pierce Chemical Company, Rockford, IL), N-maleimidobenzoyl-N-
hydroxy-succinimidyl ester ("MBS"), m-maleimidobenzoylsulfosuccinimide ester
("sulfo-MBS"), N-succinimidy1(4-iodoacetypaminobenzoate
("STAB"),
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succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate ("SMCC"),
succinimidy1-4-(p-maleimidophenyl)butyrate ("SMPB"), sulfosuccinimidy1(4-
iodoacetypamino-benzoate ("sulfo-SIAB"), sulfosuccinimidyl 4-(N-maleimido-
methyl)cyclohexane-1-carboxylate ("sulfo-SMCC"), sulfosuccinimidyl 441)-
maleimidopheny1)-butyrate ("sulfo-SMPB"), bromoacetyl-p-aminobenzoyl-N-
hydroxy-succinimidyl ester, iodoacetyl-N-hydroxysuccinimidyl ester, and the
like.
For hetero-bifunctional linking, a compound according to Formula I is
derivatized with, for example, the N-hydroxysuccinimidyl portion of the
bifunctional reagent, and the resulting derivatized compound is purified by
chromatography. Next, a suitable antibody is reacted with the second
functional
group of the bifunctional linking reagent, assuring a directed sequence of
binding
between components of the desired adduct
Typical hetero-bifunctional linking agents for forming protein-protein
conjugates have an amino-reactive N-hydroxysuccinimide ester (NHS-ester) as
one functional group and a sulfhydryl reactive group as the other functional
group. First, epsilon-amino groups of surface lysine residues of either the
antibody or the Formula I compound are acylated with the NHS-ester group of
the
cross-linking agent. The remaining component, possessing free sulfhydryl
groups, is reacted with the sulfhydryl reactive group of the cross-linking
agent to
form a covalently cross-linked dimer. Common thiol reactive groups include for
example, maleimides, pyridyl disulfides, and active halogens. For example, MBS
contains a NHS-ester as the amino reactive group, and a maleimide moiety as
the
sulfhydryl reactive group.
- 25 Photoactive
hetero-bifunctional linking reagents, e.g., photoreactive
phenyl azides, may also be employed. One such reagent, SASD, may be linked to
either an antibody or to a Formula I compound wherein at least one substituent
on
Q or the phenyl ring of Formula I comprises a peptidyl moiety, via its NHS-
ester
group. The conjugation reaction is carried out at pH 7 at room temperature for
about 10 minutes. Molar ratios between about 1 and about 20 of the cross-
linking
agent to the compounds to be linked may be used.
Numerous bifunctional linkers, useful as linkers (-L-), exist which have
been used specifically for coupling small molecules to monoclonal antibodies,
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and many of these are commercially available. Examples include
N-
succinimidy1-3-(2-pyridyldithio)-propionate (SPDP), 2-iminothiolanc (2-IT), 3-
(4-carboxamidophenyldithio)propionthionnidate (CDPT), N-succinimidyl-
acetyltbioacetate (SATA), ethyl-S-acetyl-propionthioimidate (AMPT) and N-
succinirnidy1-3-(4-carboxamidophenyldithio)propionate (SCDP). Procedures for
preparation of immunoconjugates using these linkers is detailed in Toxin-
Targeted Design for Anticancer Therapy. II: Preparation and Biological
Comparison of Different Chemically Linked Gelonin-Antibody Conjugates
(Cattel, et al, J. Pharm. Sci., 82:7, p699-704, 1993).
According to one embodiment of the invention the antibody comprises a
tumor-specific antibody, more preferably a tumor-specific monoclonal antibody
or a tumor-specific monospecific polyclonal antibody.
Monoclonal antibodies (Mabs) may be advantageously cleaved by
proteolytic enzymes to generate fragments retaining the antigen-binding site.
For
example, proteolytic treatment of IgG antibodies with papain at neutral pH
generates two identical so-called "Fab" fragments, each containing one intact
light chain disulfide-bonded to a fragment of the heavy chain (Fd). Each Fab
fragment contains one antigen-combining site. The remaining portion of the IgG
molecule is a dirner known as "Fe". Similarly, pepsin cleavage at pH 4 results
in
the so-called F(ab')2 fragment.
Methods for preparation of such fragments are known to those skilled in
the art. See, Goding, Monoclonal Antibodies Principles and Practice, Academic
Press (1983), p. 119-121 Fragments of the anti-DBF-MAF monoclonal
antibodies containing the antigen binding site, such as Fab and F(ab')2
fragments,
may be preferred in therapeutic applications, owing to their reduced
immunogenicity. Such fragments are less immunogenic than the intact antibody,
which contains the immunogenic Fe portion.
The effects of sensitization in the therapeutic use of animal origin
monoclonal antibodies in the treatment of human disease may be diminished by
employing a hybrid molecule generated from the same Fab fragment, but a
different Fe fragment, than contained in Mab's previously administered to the
same subject. It is contemplated that such hybrid molecules formed from the
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monoclonal antibodies of the invention may be used in therapy. The effects of
sensitization are further diminished by preparing animaUhuman chimeric
antibodies, e.g., mouse/human chimeric ant-bodies, or humanized (i.e. CDR-
grafted) antibodies. Such monoclonal antibodies comprise a variable region,
i.e.,
antigen binding region, and a constant region derived from different species.
Chimeric animal-human monoclonal antibodies may be prepared by
conventional recombinant DNA and gene transfection techniques well known in
the art. The variable region genes of a mouse antibody-producing myeloma cell
line of known antigen-binding specificity are joined with human
imrnunog,lobulin
constant region genes. When such gene constructs are transfected into mouse
myeloma cells, antibodies are produced which are largely human but contain
antigen-binding specificities generated in mice. As demonstrated by Morrison
et
al., Proc. Natl. Acad. Sci. USA 81, 6851-6855, 1984, both chimeric heavy chain
V region exon (VH)-human heavy chain C region genes and chimeric mouse light
chain V region exon (V*)-human * light chain gene constructs may be expressed
when transfected into mouse myeloma cell lines. When both chimeric heavy and
light chain genes are transfected into the same myeloma cell, an intact H21,2
chimeric antibody is produced. The methodology for producing such chimeric
antibodies by combining genomic clones of V and C region genes is described in
the above-mentioned paper of Morrison et al., and by Boulianne et al., Nature
312, 642-646, 1984. Also see Tan et al., J. Immunol. 135, 3564-3567, 1985 for
a
description of high level expression from a human heavy chain promotor of a
human-mouse chimeric chain after transfection of mouse myeloma cells. As an
alternative to combining genomic DNA, cDNA clones of the relevant V and C
regions may be combined for production of chimeric antibodies, as described by
Whitte et al., Protein Eng. 1, 499-505, 1987 and Liu etal., Proc. NatL Acad.
Sci.
USA 84, 3439-3443, 1987.
For examples of the preparation of chimeric antibodies, see the following
U. S. Patents: 5,292,867; 5,091,313; 5,204,244; 5,202,238; and 5,169,939.
Any of these
recombinant techniques are available for production of rodent/human chimeric
anti-DBP-MAF monoclonal antibodies.
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To further reduce the immunogenicity of murine antibodies, "humanized"
antibodies have been constructed in which only the minimum necessary parts of
the mouse antibody, the complementarity-determining regions (CDRs), are
combined with human V region frameworks and human C regions (Jones et al.,
Nature 321, 522-525, 1986; Verhoeyen et al., Science 239, 1534-1536, 1988;
Reichmann et al., 322, 323-327, 1988; Hale et al., Lancet 2, 1394-1399, 1988;
Queen et al., Proc. Natl. Acad. Sci. USA 86, 10029-10033, 1989).
This technique results in the reduction of the xenogeneic elements in the
humanized antibody to a minimum. Rodent antigen binding sites are built
directly into human antibodies by transplanting only the antigen binding site,
rather than the entire variable domain, from a rodent antibody. This technique
is
available for production of chimeric rodent/human antibodies of reduced human
inununogenicity.
Several such monoclonal antibodies, chimeric animal-human monoclonal
antibodies, humanized antibodies and antigen-binding fragments thereof have
been made available. Some examples include:
Satumomab Pendetide (by Cytogen, a murine Mab directed against TAG-
72);
Igovomab (by CIS Bio, a murine Mab fragment Fab2 directed against
tumor-associated antigen CA 125);
Arcitumomab (by Immunomedics, a murine Mab fragment Fab directed
against human carcinoembryonic antigen CEA);
Capromab Pentetate (by Cytogen, a murine Mab directed against tumor
surface antigen PSMA);
Tecnemab KI (by Sorin, murine Mab fragments (Fab/Fab2 mix) directed
against HMW-MAA);
Nofetumomab (by Boehringer Ingelheim/NeoRx, murine Mab fragments
(Fab) directed against carcinoma-associated antigen);
Rituximab (by Genentecla/IDEC Pharmaceuticals, a chimeric Mab directed
against CD20 antigen on the surface of B lymphocytes);
Trastuzumab (by Genintech, a humanized antibody directed against
human epidermal growth factor receptor 2 (HER 2));
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Votumumab (by Organon Teknika, a human Mab directed against
cytokeratin tumor-associated antigen);
Ontak (by Seragen/Ligand Pharmaceuticals, an IL-2-diphtheria toxin
fusion protein that targets cells displaying a surface IL-2 receptor);
IMC-C225 (by Imclone, a chimerized monoclonal antibody that binds to
EGFR);
LCG-Mab (by Cytoclonal Pharmaceutics Monoclonal antibody directed
against lung cancer gene LCG)
ABX-EGF (by Abgenix, a fully human monoclonal antibody against the
epidermal growth factor receptor (EGFr)); and
Epratuzumab (by Immunomedics, a humanized, anti-CD22 monoclonal
antibody).
VII. Salts of compounds of the invention
The compounds of the present invention may take the form of salts. The
term "salts", embraces salts commonly used to form alkali metal salts and to
form
addition salts of free acids or free bases. The term "pharmaceutically-
acceptable
salt" refers to salts which possess toxicity profiles within a range so as to
have
utility in pharmaceutical applications. Pharmaceutically unacceptable salts
may
nonetheless possess properties such as high crystallinity, which have utility
in the
practice of the present invention, such as for example utility in a synthetic
process. Suitable
pharmaceutically-acceptable acid addition salts may be
prepared from an inorganic acid or from an organic acid. Examples of such
inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic,
sulfuric and phosphoric acid. Appropriate organic acids may be selected from
aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and
sulfonic classes of organic acids, example of which are formic, acetic,
propionic,
succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic,
glucuronic,
maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic,
salicyclic, salicyclic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic
(pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, 2-
hydroxyethanesulfonic, toluenesulfonic, sulfanilic, cyclohexylaminosulfonic,
stearic, algenic, B-hydroxybutyric, salicyclic, galactaric and galacturonic
acid.
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Examples of pharmaceutically unacceptable acid addition salts include, for
example, perchlorates and tetrafluoroborates.
Suitable pharmaceutically acceptable base addition salts of compounds of
the invention include for example, metallic salts made from calcium,
magnesium,
potassium, sodium and zinc or organic salts made from /V,Ni-
dibenzyl ethyl enediamine, chloroprocaine, choline,
diethanol amine,
ethylenediamine, meglumine (N-methylglucamine) and procaine. Examples of
pharmaceutically unacceptable salts include lithium salts and cyanate salts.
All of
these salts may be prepared by conventional means from the corresponding
compound according to Formula I by reacting, for example, the appropriate acid
or base with the compound according to Formula I.
VIII. Administration of compounds of the invention
The compounds may be administered by any route, including oral and
parenteral administration. Parenteral administration includes, for example,
intravenous, intramuscular, intraarterial, intraperitoneal, intranasal,
rectal,
intravaginal, intravesical (e.g., to the bladder), intradennal, topical or
subcutaneous administration. Also contemplated within the scope of the
invention is the instillation of drug in the body of the patient in a
controlled
formulation, with systemic or local release of the drug to occur at a later
time.
For example, the drug may localized in a depot for controlled release to the
circulation, or for release to a local site of tumor growth.
One or more compounds useful in the practice of the present inventions
may be administered simultaneously, by the same or different routes, or at
different times during treatment.
The active agent is preferably administered with a pharmaceutically
acceptable carrier selected on the basis of the selected route of
administration and
standard pharmaceutical practice. The active agent may be formulated into
dosage forms according to standard practices in the field of pharmaceutical
preparations. See Alphonso Gennaro, ed., Remington '.s' Pharmaceutical
Sciences,
18th Ed., (1990) Mack Publishing Co., Easton, PA. Suitable dosage forms may
comprise, for example, tablets, capsules, solutions, parenteral solutions,
troches,
suppositories, or suspensions.
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For parenteral administration, the active agent may be mixed with a
suitable carrier or diluent such as water, an oil (particularly a vegetable
oil),
ethanol, saline solution, aqueous dextrose (glucose) and related sugar
solutions,
glycerol, or a glycol such as propylene glycol or polyethylene glycol.
Solutions
for parenteral administration preferably contain a water soluble salt of the
active
agent. Stabilizing agents, antioxidizing agents and preservatives may also be
added. Suitable antioxidizing agents include sulfite, ascorbic acid, citric
acid and
its salts, and sodium EDTA. Suitable preservatives include benzalkonium
chloride, methyl- or propyl-paraben, and chlorbutanol. The composition for
parenteral administration may take the form of an aqueous or nonaqueous
solution, dispersion, suspension or emulsion.
For oral administration, the active agent may be combined with one or
more solid inactive ingredients for the preparation of tablets, capsules,
pills,
powders, granules or other suitable oral dosage forms. For example, the active
agent may be combined with at least one excipient such as fillers, binders,
humectants, disintegrating agents, solution retarders, absorption
accelerators,
wetting agents absorbents or lubricating agents. According to one tablet
embodiment, the active agent may be combined with carboxymethylcellulose
calcium, magnesium stearate, mannitol and starch, and then formed into tablets
by
conventional tableting methods.
The specific dose of a compound according to the invention to obtain
therapeutic benefit for treatment of a proliferative disorder will, of course,
be
determined by the particular circumstances of the individual patient
including, the
size, weight, age and sex of the patient, the nature and stage of the
proliferative
disorder, the aggressiveness of the proliferative disorder, and the route of
administration of the compound.
For example, a daily dosage of from about 0.05 to about 50 mg/kg/day
may be utilized. Higher or lower doses are also contemplated.
A. Radioprotection
The compounds of the invention are further believed useful in the
protection of normal cells from the cytotoxic and genetic effects of exposure
to
radiation, in individuals who have incurred, who will in the future incur and
who
are at risk for incurring exposure to ionizing radiation.
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The specific dose of compound according to the invention to obtain
therapeutic benefit for radioprotection will, be determined by the particular
circumstances of the individual patient including, the size, weight, age and
sex of
the patient, the type, dose and timing of the ionizing radiation, and the
route of
administration of the compound of the invention.
For example, a daily dosage of from about 0.05 to about 50 mg/kg/day
may be utilized. Higher or lower doses are also contemplated.
Exposure to radiation by an individual may comprise therapeutic radiation
administered to the individual or in some indications, to bone marrow removed
from the individual.
An individual may also be exposed to ionizing radiation from occupation
or environmental sources, as discussed in the Background of the Invention,
above.
For purposes of the invention, the source of the radiation is not as important
as the
type (i.e., acute or chronic) and dose level absorbed by the individual. It is
understood that the following discussion encompasses ionizing radiation
exposures from both occupational and environmental sources.
Individuals suffering from effects of acute or chronic exposure to ionizing
radiation that are not immediately fatal are said to have remediable radiation
damage. Such remediable radiation damage can be reduced or eliminated by the
compounds and methods of the present invention.
An acute dose of ionizing radiation which may cause remediable radiation
damage includes a localized or whole body dose, for example, between about
10,000 millirem (0.1 Gy) and about 1,000,000 millirem (10 Gy) in 24 hours or
less, preferably between about 25,000 millirem (0.25 Gy) and about 200,000 (2
Gy) in 24 hours or less, and more preferably between about 100,000 millirem (1
Gy) and about 150,000 millirem (1.5 Gy) in 24 hours or less.
A chronic dose of ionizing radiation which may cause remediable
radiation damage includes a whole body dose of about 100 millirem (.001 Gy) to
about 10,000 millirem (0.1 Gy), preferably a dose between about 1000 millirem
(.01 Gy) and about 5000 millirem (.05 Gy) over a period greater than 24 hours,
or
a localized dose of 15,000 millirem (0.15 Gy) to 50,000 millirem (0.5 Gy) over
a
period greater than 24 hours.
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(i) Radioprotection: Therapeutic Ionizing Radiation
For radioprotective administration to individuals receiving therapeutic
ionizing radiation, the compounds of the invention should be administered far
enough in advance of the therapeutic radiation such that the compound is able
to
reach the normal cells of the individual in sufficient concentration to exert
a
radioprotective effect on the normal cells. The pharmacokinetics of specific
compounds may be determined by means known in the art and tissue levels of a
compound in a particular individual may be determined by conventional
analyses.
The compound may be administered as much as about 24 hours,
preferably no more than about 18 hours, prior to administration of the
radiation.
In one embodiment, the therapy is administered at least about 1, 2, 3, 4, 5,
6, 7, 8,
9, 10, 11 or 12 hours before administration of the therapeutic radiation. Most
preferably, the compound is administered once at about 18 hours and again at
about 6 hours before the radiation exposure.
One or more compounds of Formula I may be administered
simultaneously, or different compounds of Formula I may be administered at
different times during the treatment.
Where the therapeutic radiation is administered in serial fashion, it is
preferable to intercalate the administration of one or more radioprotective
compounds within the schedule of radiation treatments. As above, different
radioprotective compounds of the invention may be administered either
simultaneously or at different times during the treatment. Preferably, an
about 24-
hour period separates administration of the radioprotective compound and the
therapeutic radiation. More preferably, the administration of the
radioprotective
compound and the therapeutic radiation is separated by about 6 to 18 hours.
This
strategy will yield significant reduction of radiation-induced side effects
without
affecting the anticancer activity of the therapeutic radiation.
For example, therapeutic radiation at a dose of 0.1 Gy may be given daily
for five consecutive days, with a two-day rest, for a total period of 6 - 8
weeks.
One or more compounds of Formula I may be administered to the individual 18
hours previous to each round of radiation. It should be pointed out, however,
that
more aggressive treatment schedules, i.e., delivery of a higher dosage, is
contemplated according to the present invention due to the protection of the
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normal cells afforded by the radioprotective compounds. Thus,
the
radioprotective effect of the compound increases the therapeutic index of the
therapeutic radiation, and may permit the physician to safely increase the
dosage
of therapeutic radiation above presently recommended levels without risking
increased damage to the surrounding normal cells and tissues.
(ii) Radioprotection: Radiation-treated Bone Marrow
The radioprotective compounds of the invention are further useful in
protecting normal bone marrow cells from radiologic treatments designed to
destroy hematologic neoplastic cells or tumor cells which have metastasized
into
the bone marrow. Such cells include, for example, myeloid leukemia cells. The
appearance of these cells in the bone marrow and elsewhere in the body is
associated with various disease conditions, such as the French-American-
British
(FAB) subtypes of acute myelogenous leukemias (AML), chronic myeloid
leukemia (CML), and acute lymphocytic leukemia (ALL).
CML, in particular, is characterized by abnormal proliferation of immature
granulocytes (e.g., neutrophils, eosinophils, and basophils) in the blood,
bone
marrow, spleen, liver, and other tissues and accumulation of granulocytic
precursors in these tissues. The individual who presents with such symptoms
will
typically have more than 20,000 white blood cells per microliter of blood, and
the
count may exceed 400,000. Virtually all CML patients will develop "blast
crisis",
the terminal stage of the disease during which immature blast cells rapidly
proliferate, leading to death.
Other individuals suffer from metastatic tumors, and require treatment
with total body irradiation (TBI). Because TBI will also kill the individual's
hematopoietic cells, a portion of the individual's bone marrow is removed
prior to
irradiation for subsequent reimplantation. However, metastatic tumor cells are
likely present in the bone marrow, and reimplantation often results in a
relapse of
the cancer within a short time.
Individuals presenting with neoplastic diseases of the bone marrow or
metastatic tumors may be treated by removing a portion of the bone marrow
(also
called "harvesting"), purging the harvested bone marrow of malignant stem
cells,
and reimplanting the purged bone marrow. Preferably, the individual is treated
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with radiation or some other anti-cancer therapy before the autologous purged
bone marrow is reimplanted.
Thus, the invention provides a method of reducing the number of
malignant cells in bone marrow, comprising the steps of removing a portion of
the
individual's bone marrow, administering an effective amount of at least one
radioprotective compound according to the present invention and irradiating
the
treated bone marrow with a sufficient dose of ionizing radiation such that
malignant cells in the bone marrow are killed. As used herein, "malignant
cell"
means any uncontrollably proliferating cell, such a tumor cell or neoplastic
cell.
The radioprotective compounds protect the normal hematopoietic cells present
in
the bone marrow from the deleterious effects of the ionizing radiation. The
compounds also exhibit a direct killing effect on the malignant cells. The
number
of malignant cells in the bone marrow is significantly reduced prior to
reimplantation, thus minimizing the occurrence of a relapse.
Preferably, each compound according to Formula I is administered to the
bone marrow in a concentration from about 0.25 to about 100 micromolar; more
preferably, from about 1.0 to about 50 micromolar; in particular from about
2.0 to
about 25 micromolar. Particularly preferred concentrations are 0.5, 1.0 and
2.5
micromolar and 5, 10 and 20 micromolar.
The radioprotective compounds may be added directly to the harvested
bone marrow, but are preferably dissolved in an organic solvent such as DMSO.
Pharmaceutical formulations of compounds of Formula I, such as are described
in
more detail below may also be used.
Preferably, the radioprotective compound is added to the harvested bone
marrow about 20 hours prior to radiation exposure, preferably no more than
about
24 hours prior to radiation exposure. In one embodiment, the radioprotective
compound is administered to the harvested bone marrow at least about 6 hours
before radiation exposure. One or more compounds may be administered
simultaneously, or different compounds may be administered at different times.
Other dosage regimens are also contemplated.
If the individual is to be treated with ionizing radiation prior to
reimplantation of the purged bone marrow, the individual may be treated with
one
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or more radioprotective compounds prior to receiving the ionizing radiation
dose,
as described above.
(iii) Radioprotection: Environmental or Occupational Radiation Exposure
The invention also provides a method for treating individuals who have
incurred remediable radiation damage from acute or chronic exposure to
ionizing
radiation, comprising reducing or eliminating the cytotoxic effects of
radiation
exposure on normal cells and tissues by administering an effective amount of
at
least one radioprotective compound. The compound is preferably administered in
as short a time as possible following radiation exposure, for example between
0 -
6 hours following exposure.
Remediable radiation damage may take the form of cytotoxic and
genotoxic (i.e., adverse genetic) effects in the individual. In another
embodiment,
there is therefore provided a method of reducing or eliminating the cytotoxic
and
genotoxic effects of radiation exposure on normal cells and tissues,
comprising
administering an effective amount of at least one radioprotective compound
prior
to acute or chronic radiation exposure. The compound may be administered, for
example about 24 hours prior to radiation exposure, preferably no more than
about 18 hours prior to radiation exposure. In one embodiment, the compound is
administered at least about 6 hours before radiation exposure. Most
preferably,
the compound is administered at about 18 and again at about 6 hours before the
radiation exposure. One or more radioprotective compounds may be administered
simultaneously, or different radioprotective compounds may be administered at
different times.
When multiple acute exposures are anticipated, the radioprotective
compounds of the invention may be administered multiple times. For example, if
fire or rescue personnel must enter contaminated areas multiple times,
radioprotective compounds of the invention may be administered prior to each
exposure. Preferably, an about 24-hour period separates administration of the
compound and the radiation exposure. More preferably, the administration of
radioprotective compounds and the radiation exposure is separated by about 6
to
18 hours. It is also contemplated that a worker in a nuclear power plant may
be
administered an effective amount of a radioprotective compound of the
invention
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prior to beginning each shift, to reduce or eliminate the effects of exposure
to
ionizing radiation.
If an individual is anticipating chronic exposure to ionizing radiation, the
radioprotective compound may be administered periodically throughout the
duration of anticipated exposure. For example, a nuclear power plant worker or
a
soldier operating in a forward area contaminated with radioactive fallout may
be
given the radioprotective compound every 24 hours, preferably every 6 - 18
hours, in order to mitigate the effects of radiation damage. Likewise, the
radioprotective compound may be periodically administered to civilians living
in
areas contaminated by radioactive fallout until the area is decontaminated or
the
civilians are removed to a safer environment.
B. Chemoprotection
The compounds of the invention are believed useful in protecting
individuals from the cytotoxic side effects of mitotic phase cell cycle
inhibitors
and topoisomerase inhibitors, used in the treatment of cancer and other
proliferative disorders.
The specific dose of a compound according to the invention to obtain
therapeutic benefit for chemoprotection will be determined by the particular
circumstances of the individual patient including, the size, weight, age and
sex of
the patient, the type and dose of the administered chemotherapy, the nature
and
stage and cell damage, and the route of administration of the compound of the
invention.
For example, a daily dosage of from about 0.05 to about 50 mg/kg/day
may be utilized. Higher or lower doses are also contemplated.
For providing cytoprotection from cytotoxic effects of chemotherapeutic
agents, the schedule of administration of the cytotoxic drug, i.e., mitotic
phase
cell cycle inhibitor or topoisomerase inhibitor, can be any schedule with the
stipulation that the compound according to Formula I is administered prior to
the
cytotoxic drug. The cytoprotective compound should be administered far enough
in advance of the cytotoxic drug such that the former is able to reach the
normal
cells of the patient in sufficient concentration to exert a cytoprotective
effect on
the normal cells. Again, individual drug pharmacokinetics and blood levels of
a
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specific drug in a specific patient are factors that may be determined by
methods
known in the art.
The cytoprotective compound is administered at least about 1 hour,
preferably, at least about 2 hours, and more preferably, at least about 4
hours,
before administration of the cytotoxic drug. The compound may be administered
as much as about 48 hours, preferably no more than about 36 hours, prior to
administration of the cytotoxic drug. Most preferably, the compound is
administered about 24 hours before the cytotoxic drug. The compound may be
administered more or less than 24 hours before the cytotoxic effect, but the
protective effect of the compounds is greatest when administered about 24
hours
before the cytotoxic drug. One or more cytotoxic drugs may be administered.
Similarly, one or more of the compounds of Formula I may be combined.
Where the cytotoxic drug or drugs is administered in serial fashion, it may
prove practical to intercalate cytoprotective compounds of the invention
within
the schedule with the caveat that a 4-48 hour period, preferably a 12-36 hour
period, most preferably a 24 hour period, separates administration of the two
drug
types. This strategy will yield partial to complete eradication of cytotoxic
drug
side effects without affecting anticancer activity.
For example, the mitotic inhibitor may be given daily, or every fourth day,
or every twenty-first day. The compound according to Formula I may be given
24 hours previous to each round of inhibitor administration, both as a
cytoprotective agent and as an antitumor agent.
The compounds of the invention may be administered for therapeutic
effect by any route, for example enteral (e.g., oral, rectal, intranasal,
etc.) and
parenteral administration. Parenteral administration includes, for example,
intravenous, intramuscular, intraarterial, intraperitoneal, intravaginal,
intravesical
(e.g., into the bladder), intradermal, topical, subcutaneous or sublingual
administration. Also contemplated within the scope of the invention is the
instillation of drug in the body of the patient in a controlled formulation,
with
systemic or local release of the drug to occur at a later time. For anticancer
use,
the drug may be localized in a depot for controlled release to the
circulation, or
local site of tumor growth. When more than one compound according to Formula
I is administered, or when one or more compounds of Formula I are administered
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in addition to one or more cytotoxic drugs, the different compounds may be
administered by the same or different routes.
IX. Pharmaceutical Compositions
The compounds of the invention may be administered in the form of a
pharmaceutical composition, in combination with a pharmaceutically acceptable
carrier. The active ingredient in such formulations may comprise from 0.1 to
99.99 weight percent. By "pharmaceutically acceptable carrier" is meant any
carrier, diluent or excipient which is compatible with the other ingredients
of the
formulation and to deleterious to the recipient.
The active agent is preferably administered with a pharmaceutically
acceptable carrier selected on the basis of the selected route of
administration and
standard pharmaceutical practice. The active agent may be formulated into
dosage forms according to standard practices in the field of pharmaceutical
preparations. See Alphonso Gennaro, ed., Remington 's Pharmaceutical Sciences,
18th Ed., (1990) Mack Publishing Co., Easton, PA. Suitable dosage forms may
comprise, for example, tablets, capsules, solutions, parenteral solutions,
troches,
suppositories, or suspensions.
For parenteral administration, the active agent may be mixed with a
suitable carrier or diluent such as water, an oil (particularly a vegetable
oil),
ethanol, saline solution, aqueous dextrose (glucose) and related sugar
solutions,
glycerol, or a glycol such as propylene glycol or polyethylene glycol.
Solutions
for parenteral administration preferably contain a water-soluble salt of the
active
agent. Stabilizing agents, antioxidizing agents and preservatives may also be
added. Suitable antioxidizing agents include sulfite, ascorbic acid, citric
acid and
its salts, and sodium EDTA. Suitable preservatives include benzalkonium
chloride, methyl- or propyl-paraben, and chlorbutanol. The composition for
parenteral administration may take the form of an aqueous or nonaqueous
solution, dispersion, suspension or emulsion.
For oral administration, the active agent may be combined with one or
more solid inactive ingredients for the preparation of tablets, capsules,
pills,
powders, granules or other suitable oral dosage forms. For example, the active
agent may be combined with at least one excipient such as fillers, binders,
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humectants, disintegrating agents, solution retarders, absorption
accelerators,
wetting agents absorbents or lubricating agents. According to one tablet
embodiment, the active agent may be combined with carboxymethylcellulose
calcium, magnesium stearate, marmitol and starch, and then formed into tablets
by
conventional tableting methods.
The practice of the invention is illustrated by the following non-limiting
examples.
EXAMPLE 1
Design, Synthesis and Biological Evaluation of (E)-Styryl Benzyl sulfones as
Novel Anticancer Agents.
Introduction:
In this Example, the synthesis and Structure Activity Relationship of a
group of cytotoxic molecules that selectively induce growth arrest of normal
cells
in the G1 phase, while inducing a mitotic arrest of tumor cells resulting in
selective killing of tumor cell populations with little or no effect on normal
cell
viability is described. The broad spectrum of anti-tumor activity in vitro and
xenograft models, lack of in vivo toxicity and drug resistance suggests
potential
for use of these agents in cancer therapy.
Materials and Methods:
Chemistry. General methods. All reagents and solvents were obtained
from commercial suppliers and used without further purification unless
otherwise
stated. Solvents were dried using standard procedures and reactions requiring
anhydrous conditions were performed under N2 atmosphere. Reactions were
monitored by Thin Layer Chromatography (TLC) on precoated silica gel F254
plates (Sigma-Aldrich) with a UV indicator. Column chromatography was
performed with Merck 70- 230 mesh silica gel 60A . Yields were of purified
product and were not optimized. Melting points were determined using an
Electrothermal Mel-Temp 3.0 micro melting point apparatus and are
uncorrected.1H NMR and 13C NMR spectra were obtained with a Bruker AM 300
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and 400 MHz spectrometer. The chemical shifts are reported in parts per
million
(6) dovvnfield using tetramethyl silane (Me4Si) as internal standard and CDC13
as
the solvent except where indicated. Spin multiplicities are given as s
(singlet), d
(doublet), hr s (broad singlet), m (multiple , and q (quartet). Coupling
constants
(J values) were measured in hertz (Hz). Combustion analyses were performed
with a Perkin-Elmer 2402 series II CHNS/O analyzer by Quantitative
Technologies Inc. White House, New Jersey. All the compounds gave satisfactory
combustion analysis results (C, H, N within 0.4% of calculated values).
Benzylsulfonylacetic acids were prepared according to the procedure reported
in
the literature.13
General Procedure for the Preparation of (E)-Styryl Benzyl Sulfones (6):
Method A (Schemel):
A mixture of benzylsulfonyl acetic acid 4 (10 mmol), araldehyde 5 (10
mmol), acetic acid (10 mL), and a catalytic amount of benzylamine (150 L) was
refluxed for about 2-8 h. After completion of the reaction (TLC monitoring,
CHC13 on silica gel plate), coold the contents to room temperature, the
precipitated product was filtered and washed with 2-propanol. If solid was not
formed, the reaction mixture was diluted with ether, washed with saturated
NaHCO3, dilute hydrochloric acid and water. The organic layer was dried over
sodium sulfate, filtered and concentrated under vacuum to obtain the desired
crude product 6. The crude product was recrystallized in 2-propanol, to yield
an
analytically pure sample of 6. The following (E)-styryl benzyl sulfones 6 were
prepared using the above procedure.
(E)-4-Methoxystyry1-4-Methoxybenzyl sulfone (6a). The title compound
was obtained from 4-methoxybenzylsulfonylacetic acid and 4-
methoxybenzaldehyde following the procedure as described in method A. Yield:
51%; white solid, mp 150-152 C. 1H NMR: 6 3.65 (s, 3H, OCH3), 3.68 (s, 3H,
OCH3), 4.07 (s, 2H, CH2), 6.36 (d, 1H, J= 14.4 Hz, = CH), 6.71- 6.76 (m, 4H,
Ar-
H), 7.09- 7.22 (m, 5H, Ar-H+ vinylic). Anal.
(Ci7F11804S): C, H.
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(E)-4-Fluorostyry1-4-Methoxybenzyl sulfone (6b). The title compound
was obtained from 4-methoxybenzylsulfonylacetic acid and 4-fluorobenzaldehyde
following the procedure as described in method A. Yield: 60%; white solid, mp
148-149 C. 1H NMR: 6 3.81 (s, 3H, OCH3), 4.25 (s, 2H, CH2), 6.61 (d, 1H, J=
14.0 Hz, = CH), 6.91- 7.45 (m, 9H, Ar-H + vinylic). Anal. (C161115F03S): C, H.
(E)-4-Chlorostyry1-4-Methoxybenzyl sulfone (6c). The title compound
was obtained from 4-methoxybenzylsulfonylacetic acid and 4-
chlorobenzaldehyde following the procedure as described in method A. Yield:
66%; white solid, mp 176-177 C. 1H NMR: 6 3. 56 (s, 3H, OCH3), 4.00 (s, 2H,
CH2), 6.40 (d, 1H, J= 16.0 Hz, = CH), 6.63- 7.14 ( m, 9H, Ar-H + vinylic).
Anal.
(Ci6Hi5C103S): C, H.
(E)-4-Nitrostyry1-4-Methoxybenzyl sulfone (6d). The title compound was
obtained from 4-methoxybenzylsulfonylacetic acid and 4-nitrobenzaldehyde
following the procedure as described in method A. Yield: 56%; light yellow
solid,
mp 179-181 C. 1H NMR: 6 3.76 (s, 3H, OCH3), 4.24 (s, 2H, CH2), 6.78 (d, 1H,
J= 15.6 Hz, = CH), 6.84 (m, 2H, Ar-H), 7.20-7.24 (m, 2H, Ar-H), 7.41 (d, 1H,
J=
15.6 Hz, CH=), 7.52 (d, 2H, J= 13.3Hz, Ar-H), 8.19 (d, 2H, J= 11.1Hz, Ar-H).
Anal. (Ci6Hi5NO5S): C, H, N.
(E)-4-Aminostyry1-4-Methoxybenzyl sulfone (6e). In a 100 mL round-
bottomed flask fitted with condenser, 5% Pd/C (0.073 g) was taken and 20 mL
ethanol was added slowly. Compound 6d (0.5 g, 1.4 mmol) and hydrazine hydrate
(1.24 g, 38.7 mmol) were added to the content of the flask and refluxed for 6
h.
Completion of the reaction was monitored by TLC (CHC13 on silica gel plate),
filtered the reaction mixture through celite and concentrated the reaction
mixture
to 50% volume. The concentrated mixture was poured onto crushed ice, the solid
formed was filtered, washed with cooled water, dried to get the desired
product
6e. Yield: 52%; light yellow solid mp 164-168 C. 1H NMR: 6 3. 80 (s, 3H,
OCH3), 4.22 (s, 2H, CH2), 4.04 (br s, 2H, NH2), 6.42 (d, 1H, J=15.6 Hz, = CH),
6.60-7.31 (m, 9H, Ar-H + vinylic). Anal. (Ci6Hi7NO3S): C, H, N.
(E)-2-Methoxystyry1-4-Methoxybenzyl sulfone (60. The title compound
was obtained from 4-methoxybenzylsulfonylacetic acid and 2-
methoxybenzaldehyde following the procedure as described in method A. Yield:
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53%; white solid mp 115-117 C. 1H NMR: 6 3.75 (s, 3H, OCH3), 3.81 (s, 3H,
OCH3), 4.18 (s, 2H, CH2), 6.82- 7.27 (m, 9H, Ar-H + vinylic) 7.56 (d, 1H, J=
15.6
Hz, CH=). Anal. (C17H1804S): C, H.
(E)-2-Chloro-4-fluorostyry1-4-Methoxybenzyl sulfone (6g). The title
compound was obtained from 4-methoxybenzylsulfonylacetic acid and 2-chloro-
4-fluorobenzaldehyde following the procedure as described in method A. Yield:
56%; white solid mp 154-155 C. 1H NMR: 8 3. 75 (s, 3H, OCH3), 4.22 (s, 2H,
CH2), 6.62 (d, 1H, J= 15.6 Hz, = CH), 6.83- 7.41 (m, 7H, Ar-H), 7.68 (d, 1H,
J=
15.6 Hz). Anal. (Ci6Hi4C1F03S): C, H.
(E)-2,4-Dimethylstyry1-4-Methoxybenzyl sulfone (6h). The title
compound was obtained from 4-methoxybenzylsulfonylacetic acid and 2,4-
dimethylbenzaldehyde following the procedure as described in method A. Yield:
55%; white solid mp 126-128 C. 1H NMR: 8 2.21 (s, 3H, CH3), 2.27 (s, 3H,
CH3), 3. 74 (s, 3H, OCH3), 4.19 (s, 2H, CH2), 6.49 (d, 1H, J= 15.4 Hz, = CH),
6.83- 7.24 (m, 7H, Ar-H), 7.55 (d, 1H, J= 15.4 Hz). Anal. (C181-12003S): C, H.
(E)-4-Fluoro-3-methoxystyry1-4-Methoxybenzylsulfone (6i). The title
compound was obtained from 4-methoxybenzylsulfonylacetic acid and 4-fluoro-
3-methoxybenzaldehyde following the procedure as described in method A.
Yield: 55%; white solid mp 105-107 C. 1H NMR: 8 3.69 (s, 3H, OCH3), 3. 75 (s,
3H, OCH3), 4.20(s, 2H, CH2), 6.55 (d, 1H, J= 15.5 Hz, = CH), 6.82- 7.31 (m,
8H,
Ar-H + vinylic). Anal. (Ci7Hi7F04S): C, H.
(E)-3,4-Dimethoxystyry1-4-Methoxybenzyl sulfone (6j). The title
compound was obtained from 4-methoxybenzylsulfonylacetic acid and 3,4-
dimethoxybenzaldehyde following the procedure as described in method A.
Yield: 54%; white solid mp 160-161 C. 1H NMR: 8 3.23 (s, 3H, OCH3), 3.55 (s,
3H, OCH3), 3.81 (s, 3H, OCH3), 4.37 (s, 2H, CH2), 6.71 (d, 1H, J= 15.4 Hz, =
CH), 6.64- 7.05 (m, 7H, Ar-H), 7.55 (d, 1H, J= 15.4 Hz). Anal. (Ci8H2005S): C,
H.
(E)-3,5-Dimethylstyry1-4-Methoxybenzyl sulfone (6k). The title
compound was obtained from 4-methoxybenzylsulfonylacetic acid and 3,5-
dimethylbenzaldehyde following the procedure as described in method A. Yield:
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58%; white solid mp 127-130 C. 1H NMR: 8 2.08 (s, 6H, 2XCH3), 3.57 (s, 3H,
OCH3), 3.99(s, 2H, CH2), 6.40 (d, 1H, J= 15.5 Hz, = CH), 6.83- 7.24 (m, 7H, Ar-
H), 7.11 (d, 1H, J= 15.5 Hz). Anal.
(Ci8H2003S): C, H.
(E)-2,6-Dimethylstyry1-4-Methoxybenzyl sulfone (61). The title compound
was obtained from 4-methoxybenzylsulfonylacetic acid and 2,6-
dimethylbenzaldehyde following the procedure as described in method A. Yield:
53%; white solid mp 99-101 C. 1H NMR: 6 2.15 (s, 3H, CH3), 2.27 (s, 3H, CH3),
3.75 (s, 3H, OCH3), 4.13 (s, 2H, CH2), 6.41 (d, 1H, J= 15.4 Hz, = CH), 6.83-
7.35
(m, 7H, Ar-H), 7.55 (d, 1H, J= 15.4 Hz). Anal. (Ci8H2003S): C, H.
(E)-2,6-Dimethoxystyry1-4-Methoxybenzyl sulfone (6m). The title
compound was obtained from 4-methoxybenzylsulfonylacetic acid and 2,6-
dimethoxybenzaldehyde following the procedure as described in method A.
Yield: 51%; white solid mp 136-138 C. 1H NMR: 6 3.81(s, 3H, OCH3), 3.85(s,
3H, OCH3), 3.91 (s, 3H, OCH3), 4.23 (s, 2H, CH2), 7.19 (d, 1H, J= 15.4 Hz, =
CH), 6.64- 7.05 (m, 7H, Ar-H), 7.90 (d, 1H, J= 15.4 Hz). 13C NMR: 8 160.6,
160.3, 135.9, 132.9.9, 132.6, 126.4, 120.9, 114.5, 110.7, 104.0, 61.7, 56.2,
55.7.
Anal. (Ci8H2005S): C, H.
(E)-2,4-Dimethoxystyry1-4-Methoxybenzyl sulfone (6n). The title
compound was obtained from 4-methoxybenzylsulfonylacetic acid and 2,4-
dimethoxybenzaldehyde following the procedure as described in method A.
Yield: 59%; white solid mp 161-162 C. 1H NMR: 6 3.73 (s, 3H, OCH3), 3.77 (s,
6H, 2 x OCH3), 4.14 (s, 2H, CH2), 6.71 (d, 1H, J= 15.5 Hz, = CH), 6.37- 7.23
(m,
7H,Ar-H), 7.42 (d, 1H, J= 15.5 Hz). Anal. (Ci8H2005S): C, H.
(E)-2,5-Dimethoxystyry1-4-Methoxybenzyl sulfone (6o). The title
compound was obtained from 4-methoxybenzylsulfonylacetic acid and 2,5-
dimethoxybenzaldehyde following the procedure as described in method A.
Yield: 54%; white solid mp 105-107 C. 1H NMR: 8 3.71(s, 3H, OCH3), 3.75 (s,
3H, OCH3), 3.76 ( s, 3H, OCH3), 4.18 (s, 2H, CH2), 6.78- 7.26 (m, 8H, Ar-H +
vinylic), 7.52 (d, 1H, J= 15.6 Hz). Anal. (Ci8H2005S): C, H.
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(E)-3,5-Dimethoxystyry1-4-Methoxybenzyl sulfone (6p). The title
compound was obtained from 4-methoxybenzylsulfonylacetic acid and 3,5-
dimethoxybenzaldehyde following the procedure as described in method A.
Yield: 62%; white solid mp 119-121 C. 1H NMR: 8 3.70 (s, 6H, 2 x OCH3), 3.71
(s, 3H, OCH3), 4.15 (s, 2H, CH2), 6.55 (d, 1H, J= 15.5 Hz, = CH), 6.42- 7.20
(m,
7H, Ar-H), 7.23 (d, 1H, J= 15.5 Hz). Anal. (Ci8H2005S): C, H.
(E)-2,4,5-Trimethoxystyry1-4-Methoxybenzyl sulfone (6q). The title
compound was obtained from 4-methoxybenzylsulfonylacetic acid and 2,4,5-
trimethoxy-benzaldehyde following the procedure as described in method A.
Yield: 66%; white solid mp 146-148 C. 1H NMR: 8 3.73 (s, 3H, OCH3), 3.78 (s,
6H, 2 x OCH3), 3.81 (s, 3H, OCH3), 4.20 (s, 2H, CH2), 6.86 (m, 4H, Ar-H), 7.00
(d, 1H, J= 15.6 Hz, = CH), 7.31 (d, 2H, J= 8.9 Hz), 7.61 (d, 1H, J= 15.6 Hz,
CH¨). Anal. (C19H2206S): C, H.
(E)-2,3,4-Trimethoxystyry1-4-Methoxybenzyl sulfone (6r). The title
compound was obtained from 4-methoxybenzylsulfonylacetic acid and 2,3,4-
trimethoxy-benzaldehyde following the procedure as described in method A.
Yield: 54%; white solid mp 154-156 C. 1H NMR: 8 3.75 (s, 3H, OCH3), 3.79 (s,
6H, 2 x OCH3), 3.83 (s, 3H, OCH3), 4.18 (s, 2H, CH2), 6.76 (d, 2H, J= 8.9 Hz,
Ar-
H), 6.86 (d, 2H, J= 9.0Hz, Ar-H), 6.99 (d, 1H, J= 15.6 Hz, = CH), 7.31 (d, 2H,
J-
8.9 Hz), 7.61 (d, 1H, J= 15.6 Hz, CH=). Anal. (C19H2206S): C, H.
(E)-2,4,6-Trimethoxystyry1-4-Methoxybenzyl sulfone (6s). The title
compound was obtained from 4-methoxybenzylsulfonylacetic acid and 2,4,6-
trimethoxy-benzaldehyde following the procedure as described in method A.
Yield: 34%; white solid mp 143-145 C. 1H NMR: 8 3.80 (s, 3H, OCH3), 3.82 (s,
6H, 2 x OCH3), 3.85 (s, 3H, OCH3), 4.20 (s, 2H, CH2), 6.08 (s, 2H, Ar-H), 6.88
(d, 2H, J 9.2 Hz, Ar-H), 7.00 (d, 1H, J= 15.6 Hz, = CH), 7.31 (d, 2H, J= 8.8
Hz),
7.81 (d, 1H, J= 15.6 Hz, CH=). 13C NMR: 8 164.2, 161.8, 160.2, 135.9, 132.6,
123.0, 121.1, 114.4, 104.2, 90.9, 61.8, 56.1, 55.8, 55.6. Anal. (Ci9H2206S):
C, H.
(E)-3,4,5-Trimethoxystyry1-4-Methoxybenzyl sulfone (6t). The title
compound was obtained from 4-methoxybenzylsulfonylacetic acid and 3,4,5-
trimethoxy-benzaldehyde following the procedure as described in method A.
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Yield: 54%; white solid mp 138-141 C. 1H NMR: 8 3.76 (s, 3H, OCH3), 3.80 (s,
6H, 2 x OCH3), 3.83 (s, 3H, OCH3), 4.16 (s, 2H, CH2), 6.78 (d, 2H, J= 9.1 Hz,
Ar-H), 6.91 (d, 2H, J= 8.9 Hz, Ar-H), 7.04 (d, 1H, J= 15.6 Hz, = CH), 7.37 (d,
2H, J= 8.8 Hz), 7.79 (d, 1H, J= 15.6 Hz, CH=). Anal. (C19H2206S): C, H.
(E)-2,6-Dimethoxy-4-hydroxystyry1-4-Methoxybenzyl sulfone (6u). The
title compound was obtained from 4-methoxybenzylsulfonylacetic acid and 2,6-
dimethoxy-4-hydroxybenzaldehyde following the procedure as described in
method A. Yield: 58%; white solid mp 134-136 C. 1H NMR: 8 3.47 (s, 6H, 2 x
OCH3), 3.55 ( s, 3H, OCH3), 3.98 (s, 2H, CH2), 5.77 (s, 2H, Ar-H), 6.63 (d,
2H,
J= 8.5 Hz, Ar-H), 6.73 (d, 1H, J= 15.6 Hz, = CH), 7.05 (d, 2H, J= 8.5 Hz),
7.55
(d, 1H, J= 15.6 Hz, CH=). Anal. (Ci8H2006S): C, H.
Preparation of 4-Fluoro-2,6-dimethoxybenzaldehyde: Phosphorous
oxychloride (1.8 mL, 19.3 mmol) was added slowly to a well-stirred mixture of
1-fluoro-3,5-dimethoxy benzene (2.6 mL, 19.25 mmol) and N, N-
dimethylformamide (2.5 mL, 20 mmol) while temperature was kept below -5 C.
Stiring was continued at room temperature for 1.5 h and at 60 C for another
2h.
Reaction completion was monitored by TLC. The reaction mixture was cooled
and hydrolyzed with ice-water (60 mL). The resulting suspension was
neutralized
by addition of 5N NaOH, extracted with ethyl acetate (2 x 30 mL), the aqueous
phase was adjusted to pH 10 by 5N NaOH and re-extracted with ethyl acetate (2
x
mL). The combined organic phases were washed with saturated aqueous
NaHCO3 solution (30 mL) and brine (30 mL) and dried over anhydrous sodium
sulfate. The dried solution was concentrated to get the crude product which on
purification by column chromatography afforded a colorless pure product.
Yield:
25 75%; white
solid mp 79-81 C. 11-1 NMR: 8 3.85 (s, 3H, OCH3), 3.89 (s, 3H,
OCH3), 6.25 (S, 2H, Ar-H), 10.24 (s, 1H, CHO). Anal. Calcd for Ci8H0F05S: C,
58.70, H, 4.92. Found: C, 58.64, H, 4.91.
(E)-2,6-Dimethoxy-4-fluorostyry1-4-Methoxybenzyl sulfone (6v). The title
compound was obtained from 4-methoxybenzylsulfonylacetic acid and 2,6-
30 dimethoxy-4-
fluorobenzaldehyde following the procedure as described in method
A. Yield: 55%; white solid mp 146-148 C. 11-1 NMR: 8 3.47 (s, 6H, 2 x OCH3),
3.55 (s, 311, OCH3), 3.98 (s, 2H, CH2), 5.77 (s, 2H, Ar-H), 6.63 (d, 2H, J=
8.5 Hz,
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Ar-H), 6.73 (d, 1H, J= 15.6 Hz, = CH), 7.05 (d, 2H, J= 8.5 Hz), 7.55 (d, 1H,
J=
15.6 Hz, CH=). Anal.(C181-119F05S): C, H.
(E)-2,4,6-Trimethylstyry1-4-Methoxybenzyl sulfone (6w). The title
compound was obtained from 4-methoxybenzylsulfonylacetic acid and 2,4,6-
trimethylbenzaldehyde following the procedure as described in method A. Yield:
51%; white solid mp 97-99 C. 1H NMR: 8. 2.16 (s, 3H,CH3), 2.28 (s, 6H,
2XCH3), 3.76 (s, 3H, OCH3), 4.13 (s, 2H, CH2), 6.08 ( s, 2H, Ar-H), 6.42 (d,
1H,
J= 15.4 Hz, = CH), 6.82 (m, 4H, Ar-H), 7.56 (d, 1H, J= 15.4 Hz, CH=). Anal.
(Ci9H2203S): C, H.
(E)-2,4,6-Trimethoxystyry1-4-Trifluoromethoxybenzyl sulfone (6x). The
title compound was obtained from 4-trifluoromethoxybenzylsulfonylacetic acid
and 2,4,6-trimethoxybenzaldehyde following the procedure as described in
method A. Yield: 52%; white solid mp 133-135 C. 1H NMR: 8 3.82 (s, 6H, 2 x
OCH3), 3.87 (s, 3H, OCH3), 4.26 (s, 2H, CH2), 6.10 (s, 2H, Ar-H), 6.99 (d, 1H,
J= 15.6 Hz, = CH), 7.20-7.48 (m, 4H, Ar-H), 7.78 (d, 1H, J= 15.6 Hz, CH=).
Anal. (C19H0F306S): C, H.
(E)-3,4,5-Trimethoxystyry1-3-Hydroxy-4-Methoxybenzyl Sulfone (6y).
The title compound was obtained from 3-hydroxy-4-methoxybenzylsulfonylacetic
acid and 3,4,5-trimethoxybenzaldehyde following the procedure as described in
method A. Yield: 60%; white solid mp 118-120 C. 1H NMR: 8 3.83 (s, 6H, 2 x
OCH3), 3.85 (s, 3H. OCH3), 3.89 (s, 3H. OCH3), 4.16 (s, 2H, CH2), 5.60 (s, 1H,
OH), 6.09 (s, 2H, Ar-H), 6.82-6.96 (m, 3H, Ar-H), 7.05 (d, 1H, J=15.6 Hz,
=CH),
7.85 (d, 1H, J= 15.6 Hz, CH¨). Anal. (C19H2207S): C, H.
(E)-2,6-Dimethoxy-4-hydroxystyry1-3-Hydroxy-4-Methoxybenzyl
Sulfone (6z). The title compound was obtained from 3-hydroxy-4-
methoxybenzylsulfonylacetic acid and 2,6-dimethoxy-4-hydroxybenzaldehyde
following the procedure as described in method A. Yield: 54%; white solid mp
123-125 C. 1H NMR: 8 3.77 (s, 6H, 2 x OCH3), 3.81 ( s, 3H, OCH3), 4.28 (s,
2H,
CH2), 6.10 (s, 2H, Ar-H), 6.71-6.92 (m, 3H, Ar-H), 7.00 (d, 1H, J=15.5 Hz,
=CH), 7.59 (d, 1H, J= 15.5 Hz, CH¨). Anal. (Ci8H2007S): C, H.
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(E)-2,4,6-Trimethoxystyry1-3-Hydroxy-4-Methoxybenzyl Sulfone (6aa).
The synthesis of the title compound was described in the preparation of 20
(Scheme 3). Yield: 63%; white solid mp 125-127 C. 1H NMR: 8 3.83 (s, 6H, 2 x
OCH3), 3.85 (s, 3H. OCH3), 3.89 (s, 3H. OCH3), 4.16 (s, 2H, CH2), 5.60 (s, 1H,
OH), 6.09 (s, 2H, Ar-H), 6.82-6.96 (m, 3H, Ar-H), 7.05 (d, 1H, J=15.6 Hz,
=CH),
7.85 (d, 1H, J= 15.6 Hz, CH=). 13C NMR: 8 164.1, 161.9, 147.3, 145.9, 135.7,
123.4, 123.1, 122.2, 117.5, 111.1, 105.5, 104.4, 90.9, 62.0, 56.3, 56.1, 55.8.
Anal.
(Ci9H2207S): C, H.
(E)-2,4,6-Trimethoxystyry1-31-0-phosphate disodium-
4-methoxybenzyl
Sulfone (6ab). The synthesis of the title compounds was described in the
preparation of 29 (Scheme 5). Yield: 98%; white solid mp 152-154 C. 11-1 NMR
(D20): 8 3.68 (s, 6H, 2 x OCH3), 3.71 (s, 3H. OCH3), 3.78 (s, 3H. OCH3), 4.35
(s,
2H, CH2), 5.92 (s, 2H, Ar-H), 6.91 (s, 2H, Ar-H), 6.97 (d, 1H, J=15.6 Hz,
=CH),
7.39 (s, 1H, Ar-H), 7.43 (d, 1H, J= 15.6 Hz, CH=). 13C NMR (D20): 8 164.4,
161.7, 151.1, 143.8, 136.8, 125.9, 123.4, 120.6, 120.2, 113.1, 103.4, 91.1,
61.0,
56.4, 56.2, 55.9. Anal. (Ci9H21010Na2PS): C, H.
(E)-2,4,6-trimethoxystyry1-3,4,5-Trimethoxybenzyl sulfone (6ac). The
title compound was obtained from 3,4,5-trimethoxybenzylsulfonylacetic acid and
2,4,6-trimethoxybenzaldehyde following the procedure as described in method A.
Yield: 53%; white solid mp 151-153 C. 1H NMR: 8 3.81(s, 6H, 2 x OCH3), 3.83
(s, 6H, 2 x OCH3), 3.84 (s, 3H, OCH3), 3.85 ( s, 3H, OCH3), 4.19 (s, 2H, CH2),
6.10 (s, 2H, ArH), 6.60 (s, 2H, ArH), 7.03 (d, 1H, J= 15.6 Hz, = CH), 7.83 (d,
1H, J= 15.6 Hz, CH=). Anal. (C211-12608S): C, H.
(E)-2,4,6-Trimethoxystyry1-2,3,4-Trimethoxybenzyl sulfone (6ad). The
title compound was obtained from 2,3,44.timethoxybenzylsulfonylacetic acid and
2,4,6-trimethoxybenzaldehyde following the procedure as described in method A.
Yield: 52%; white solid mp 94-96 C.1H NMR: 8 3.77, 3.83, 3.84, 3.86, 3.90 (
s,
6x 3H, OCH3), 4.32 (s, 2H, CH2), 6.08 (s, 2H, aromatic), 6.67 (d, J = 8.4Hz,
1H,
aromatic), 7.11 (d, J= 15.6 Hz, 1H, = CH), 7.16 ( d, J = 8.4Hz, 1H, aromatic),
7.79 (d, J= 15.6 Hz, 1H, CH=). Anal. (C21112608S): C, H.
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(E)-2,4,6-trimethoxystyry1-4-chlorobenzyl sulfone (6ae). The title
compound was obtained from 4-chlorobenzylsulfonylacetic acid and 2,4,6-
trimethoxybenzaldehyde following the procedure as described in method A.
Yield: 60%; white solid mp 181-184 C. 1H NMR: 6 3.83 (s, 2 x 3H, OCH3), 3.85
(s, 3H, OCH3), 4.22 (s, 2H, CH2), 6.09 (s, 2H, Ar-H), 6.99 (d,1H, J= 15.5 Hz,
=
CH), 7.29 (s, 4H, Ar-H), 7.76 (d, 1H, J= 15.5 Hz, CH=). Anal. (C181-119C105S):
C,
H.
(E)-2,4,6-trimethoxystyry1-4-Nitrobenzyl sulfone (6af). The title
compound was obtained from 4-nitrobenzylsulfonylacetic acid and 2,4,6-
trimethoxybenzaldehyde following the procedure as described in method A.
Yield: 60%; light yellow solid mp 179-184 C. 1H NMR: 8 3.83 (s, 2 x 3H,
OCH3), 3.86 ( s, 3H, 0C143), 4.35 (s, 2H, CH2), 6.09 (s, 2H, Ar-H), 7.01 (d,
1H,
J= 15.5 Hz, = CH), 7.57 (d, 2H, J =9.0 Hz, Ar-H), 7.76 (d, J= 15.5 Hz, 1H,
CH¨),
8.21 (d, 2H, J = 9.0 Hz, Ar-H). Anal. (Ci8Hi9N07S): C, H, N.
(E)-2,4,6-trimethoxystyry1-4-Cyanobenzyl sulfone (6ag). The title
compound was obtained from 4-cyanobenzylsulfonylacetic acid and 2,4,6-
trimethoxybenzaldehyde following the procedure as described in method A.
Yield: 58%; white solid mp 140-142 C. 1H NMR: 8 3.81 (s, 3H, 2 x OCH3),
3.87 (s, 3H, OCH3), 4.21 (s, 2H, CH2), 6.00 (s, 2H, Ar-H), 6.78 (d, 2H, J =
8.5
Hz, Ar-H), 7.07 (d, 1H, J= 15.6 Hz, = CH), 7.29 (d, 2H, J = 8.5 Hz, Ar-H),
7.86
(d, 1H, J= 15.6 Hz, CH=). Anal. (C19H19NO5S): C, H, N.
(E)-2,4,6-trimethoxystyry1-4-Carboxybenzyl sulfone (6ah). The title
compound was obtained from 4-carboxybenzylsulfonylacetic acid and 2,4,6-
trimethoxy-benzaldehyde following the procedure as described in method A.
Yield: 60%; white solid mp 143-145 C. 1H NMR: 6 3.83 (s, 6H, 2 x OCH3), 3.88
(s, 3H, OCH3), 4.11 (s, 2H, CH2), 6.01 (s, 2H, Ar-H), 6.68 (d, 2H, J = 8.5 Hz,
Ar-
H), 6.97 (d, 1H, J 15.6 Hz, = CH), 7.13 (d, 2H, J = 8.5 Hz, Ar-H), 7.76 (d,
1H,
J= 15.6 Hz, CH=). Anal. (C19H2007S): C, H.
(E)-2,4,6-trimethoxystyry1-4-Hydroxybenzyl sulfone (6ai). The title
compound was obtained from 4-tert-Butoxybenzylsulfonylacetic acid and 2,4,6-
trimethoxy-benzaldehyde following the method A procedure. During the
condensation the protective tert-butoxy group cleaved to hydroxy group. Yield:
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52%; white solid mp 141-143 C. 1H NMR: 8 3.73 (s, 6H, 2 x OCH3), 3.77 (s, 3H,
OCH3), 4.11 (s, 2H, CH2), 4.36 (s, 1H, OH), 6.01 (s, 2H, Ar-H), 6.68 (d, 2H, J
=
8.5 Hz, Ar-H), 6.97 (d, 1H, J= 15.6 Hz, = CH), 7.13 (d, 2H, J = 8.5 Hz, Ar-H),
7.76 (d, 1H, J= 15.6 Hz, CH=). Anal. (Ci5H2006S): C, H.
Method B (Scheme 2):
Preparation of Phenacyl Benzyl Sulfones (10). General Procedure
To a cooled solution of sodium hydroxide (100 mmol) in absolute
methanol (50 mL), taken in a 250 mL round-bottomed flask, benzyl thiol 7 ( 100
mmol) was added slowly through a dropping funnel and the reaction mixture was
stirred for 5 mm. An appropriate phenacyl bromide 8 (100 mmol) was added in
portions to the contents of the flask and stirred for 3-4 h. After completion
of the
reaction, the contents of the flask were poured into crushed ice and the
compound
formed was washed with ice-cold water and dried to get phenacyl benzyl sulfide
9.
The above crude phenacyl benzyl sulfide 9 (50 mmol) in glacial acetic
acid (100 ml) was taken in a 250 mL round-botomed flask and 30% hydrogen
peroxide (60 mL) was added in portions at frequent intervals. Then the
reaction
mixture was stirred at room temperature for 24 h. The solid, if any formed was
separated by filtration and the filtrate was poured onto crushed ice. The
compound separated was filtered, washed with water, dried and added to the
first
crop, if any. The total product on recrystallization from methanol afforded
pure
phenacyl benzyl sulfone (10).
4-Methoxyphenacy1-4-methoxybenzyl sulfone (10a). The title compound
was obtained from 4-methoxybenzyl thiol and 4-methoxyphenacyl bromide
followed by oxidation of the resultant compound. Yield: 80%; white solid mp
128-130 C. 1H NMR : 8 3.78( s, 3H, OCH3), 3.88 ( s, 3H, OCH3), 4.60 (s, 2H,
CH2), 4.89 (s, 2H, CH2), 6.98 (d, 2H, J=8.7Hz, Ar-H), 7.09(d, 2H, J= 8.9 Hz,
Ar-
H), 7.36 (d, 2H, J=8.7 Hz, Ar-H), 8.04 (d, 2H, J= 8.9 Hz, Ar-H). Anal. Calcd
for
Ci7H1805S: C, 61.06, H, 5.42. Found: C, 61.09, H, 5.40.
4-Chlorophenacy1-4-methoxybenzyl sulfone (10b). The title compound
was obtained from 4-methoxybenzyl thiol and 4-chlorophenacyl bromide
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followed by oxidation of the resultant compound. Yield: 82%; white solid mp
141-143 C. 1H NMR : 8 3.89( s, 3H, OCH3), 4.50 (s, 2H, CH2), 4.79 (s, 2H,
CH2), 7.06 (d, 2H, J= 8.3 Hz, Ar-H), 7.47 (d, 2H, J=8.4 Hz, Ar-H), 7.60 (dd,
4H,
J= 8.2, 9.4 Hz, Ar-H). Anal. Calcd for CI6H15C104S: C, 56.72, H, 4.46. Found:
C,
56.69, H, 4.42.
Preparation of 13- Hydroxy Benzyl Sulfones (11). General Procedure
To an ethanolic solution (20 mL) of phenacyl benzyl sulfone 10 (10
mmol) maintained at 0 C, was added NaBH4 (10 mmol) slowly under N2
atmosphere. The reaction mixture was maintained at 0 C for 0.5 h. After
completion of the reaction, monitored by TLC, the contents were poured on to
crushed ice. The solid separated out was filtered, washed with water and dried
under vacuum to yield 11.
2-(4-Methoxybenzylsulfony1)-1-(4-methoxyphenyl)ethanol (11a). The title
compound was obtained by the reduction of 10a with sodium borohydride.
Yield: 70% ; white solid mp 112-114 C. NMR: 8. 3.43 (dd, 2H, J=9.9 and 4.6
Hz), 3.76( s, 3H, OCH3), 3.78 ( s, 3H, OCH3), 4.47 (dd, 2H, J=15.7 and 13.6
Hz),
5.09 (m, 1H, CHOH), 6.00 (d, 1H, J= 4.2 Hz, OH), 6.93 (d, 2H, J=8.6 Hz, Ar-
H), 6.99(d, 2H, J= 8.6 Hz, Ar-H), 7.35 (t, 4H, J=8.8 Hz, Ar-H). Anal. Calcd
for
C17H2005S: C, 60.69, H, 5.99. Found: C, 60.73, H, 5.97.
1-(4-Chloropheny1)-2-(4-methoxybenzylsulfonypethanol (1 lb). The title
compound was obtained by the reduction of 10b with sodium borohydride.
Yield: 78% ; white solid mp 130-132 C. 1H NMR : 8 3.63 (dd, 2H, J=10.1 and
4.4 Hz),), 3.93 ( s, 3H, OCH3), 4.75 (dd, 2H, J=13.5 and 10.6 Hz), 5.26 (d,
1H, J=
8.3 Hz, CHOH), 6.17 (br s, 1H, OH), 7.10 (d, 2H, J=8.3 Hz, Ar-H), 7.51(d, 2H,
J= 8.3 Hz, Ar-H), 7.64 (dd, 4H, J=9.4 and 8.2 Hz, Ar-H). Anal. Calcd for
C16H17C104S: C, 56.39, H, 5.03. Found: C, 56.34, H, 4.99.
Preparation of (E)- Styryl Benzyl Sulfones (6).
p-Toluenesulfonic acid (1 mmol) was added in one portion to a mixture of
p-hydroxy benzyl sulfone 11 (5 mmol) in anhydrous benzene (25 mL) at room
temperature under N2 atmosphere. The temperature was raised to 80 C, and the
mixture was refluxed for 3 h using Dean-Stark water separator. After
completion
of the reaction monitored by TLC, the reaction mixture was concentrated under
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reduced pressure and then quenched by the addition of water (25 mL). The
aqueous layer was neutralized with a saturated aqueous solution of sodium
hydrogen carbonate and extracted with dichloromethane (3 x 25mL). The
combined organic extracts were washed with brine (2 x 25 mL) dried over
Na2SO4, filtered and the solvent was evaporated under reduced pressure to
afford
crude product, which on recrystallization in 2- propanol afforded the desired
product 6 in excellent yield.
(E)-4-Methoxystyry1-4-Methoxybenzyl sulfone (6a). The title compound
was obtained by dehydration of 11a as described in the above procedure. Yield:
65%; white solid, mp 151-153 C. Analytical data is same as 6a obtained by
method A.
(E)-4-Chlorostyry1-4-Methoxybenzyl sulfone (6c). The title compound
was obtained by dehydration of 1 lb as described in the above procedure.
Yield:
69%; white solid, mp 175-177 C. Analytical data is same as 6c obtained by
method A.
Synthesis of (E)-2,4,6-Trimethoxystyry1-3-Hydroxy-4-Methoxybenzyl Sulfone,
6aa: (Scheme 3).
Preparation of 3 - [(tert-Butyldimethylsilypoxy]-4-methoxyb enzaldehyde (13).
To a cooled solution of 3-hydroxy-4-methoxybenzaldehyde 12 (10.0 g,
65.7 mmol) in dry N,N-dimethyl formamide (75 mL) was added
diisopropylethylamine (16.99 g, 131.4 mmol). Before the addition of 1.0 M
solution of tert-butyldimethylsilyl chloride in tetrahydrofuran (11.89 g or
78.9
mL, 78.85 mmol) the mixture was stirred under nitrogen for 10 min. After
complete addition over 30 min, the reaction mixture was left overnight (12-16
h).
Reaction completion was checked by TLC (chloroform on silica gel plate). Water
was added to the reaction mixture, extracted with dichloromethane and the
organic layer was washed with a saturated sodium bicarbonate solution, water
and
dried. Removal of solvent in vacuo yielded as an oil which was subjected to
column chromatography (eluant: chloroform) to afford as an yellow viscous oil
13
(100%). 1H NMR: 8 0.21 (s, 6H, 2 x CH3), 1.01(s, 9H, 3 x CH3), 3.91 (s, 3H,
OCH3), 6.94 (d, 1H, J=8.5 Hz, 5H), 7.36 (d, 1H, J=2Hz, 2H), 7.47 (dd, 1H, J=
8.5,
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2 Hz, 6H), 9.89 (s, 1H, CHO). Anal. Calcd for CHH2203Si: C, 63.12, H, 8.32.
Found: C, 63.09, H, 8.30.
Preparation of 3-[(tert-Butyldimethylsilypoxy]-4-methoxybenzyl Alcohol (14).
To a cooled solution of 3-Rtert-Butyldimethylsilypoxy]-4-
methoxybenzaldehyde 13 (17.5 g, 65.7 mmol) in methanol (100 mL) under
nitrogen, sodium borohydride (2.98 g, 78.8 mmol) was added and stirred at room
temperature for 30 min. After the reaction was complete as indicated by TLC
(chloroform on silica gel plate), ice was added to the reaction mixture and
extracted with ethyl acetate. The organic layer was separated, washed with
water
and dried. Removal of solvent in vacuo yielded on yellow oil 14 (73.5%). 1H
NMR: 8 0.18 (s, 6H, 2 x CH3), 1.00 (s, 9H, 3 x CH3), 1.90 (hr s, 1H, OH), 3.82
(s,
3H, OCH3), 4.56 (s, 2H, CH2OH), 6.94 (hr s, 3H, Ar-H). Anal. Calcd for
C14H2403Si: C, 62.64, H, 9.01. Found: C, 62.59, H, 9.03.
Preparation of 3-[(tert-Butyldimethylsilypoxy]-4-methoxybenzyl Chloride (15).
To a cooled solution of 3-[(tert-Butyldimethylsilypoxy]-4-methoxybenzyl
alcohol 14 (9.5 g, 35.4 mmol) in benzene (50 mL) under nitrogen, thionyl
chloride (6.32 g or 3.87 mL, 53.1 mmol) dissolved in benzene (5 mL) was added
over 10 min and maintained the temperature at 0 C for 2h. The completion of
reaction was checked by TLC (chloroform on silica gel plate). Ice was added to
the reaction mixture and the solution was extracted with ethylacetate. The
organic
layer was washed with saturated bicarbonate solution and water and dried over
anhydrous sodium sulfate. Removal of the solvent in vacuo afforded 15. Yield:
98.5%; yellow oil. 1H NMR: 8 0.16 (s, 6H, 2 x CH3), 1.00 (s, 9H, 3 x CH3),
3.80
(s, 3H, OCH3), 4.44 (s, 2H, CH2), 6.70-7.01 (m, 3H, Ar-H). Anal. Calcd for
Ci4H23C102Si: C, 58.62, H, 8.08. Found: C, 58.69, H, 8.03.
Preparation of 3-Rtert-Butyldimethylsilypoxy]-4-methoxybenzylthio acetic acid
(16).
To a solution of sodium hydroxide (2.79 g, 69.7mmol) in methanol (30
mL) was added mercaptoacetic acid (3.21 g or 2.42 mL, 34.9 mmol) slowly and
stirred under nitrogen for 10
minutes. 3-[(tert-Butyldimethylsilypoxy]-4-
methoxybenzyl chloride 15 (10.0 g, 34.9 mmol) was added slowly to the reaction
mixture and stirred at room temperature for 3 h. Reaction completion was
checked by TLC (chloroform on silica gel plate). The reaction mixture was then
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poured into ice and neutralized with concentrated HC1. The resulting material
was
extracted with ethyl acetate. The ethyl acetate solution was washed with water
and dried over anhydrous sodium sulfate. Removal of the solvent in vacuo
afforded 16. Yield: 75%; white solid mp 57-59 C. 11-1 NMR: 8 0.18 (s, 6H, 2 x
CH3), 1.02 (s, 9H, 3 x CH3), 3.34 (s, 2H, CH2), 3.84 (s, 3H, OCH3), 4.04 (s,
2H,
CH2), 6.80-7.01 (m, 3H, Ar-H). Anal. Calcd for C16H2604SiS: C, 56.10, H, 7.65.
Found: C, 56.08, H, 7.61.
Preparation of 3-Hydroxy-4-methoxybenzylthio acetic acid (17).
To a cooled solution of 34(tert-Butyldimethylsilypoxy]-4-
methoxybenzylthio acetic acid 16 (8.75 g , 25.5 mmol) in tetrahydrofuran (40
mL) was added 1.0 M solution of tetra-n-butyl ammonium fluoride in
tetrahydrofuran (6.68 g or 25.54 mL, 25.5 mmol) slowly and stirred under
nitrogen for 2 h at room temperature. The progress of the reaction was
monitored
by TLC (9:1- chloroform:methanol on silica gel plate). Water was added to the
reaction mixture and extracted with ethyl acetate. The organic layer was
washed
with water and dried. Removal of the solvent in vacuo yielded a semi solid
that
was subjected to column chromatography (initial with chloroform and finally
with
ethyl acetate) afforded the pure product 17. Yield: 50%; white solid mp 128-
130
C. 1H NMR: 6 3.34 (s, 2H, CH2), 3.84 (s, 3H, OCH3), 4.04 (s, 2H, CH2), 6.80-
7.01 (m, 3H, Ar-H). Anal. Calcd for Ci0H1204S: C, 52.62, H, 5.30. Found: C,
52.58, H, 5.35.
Preparation of 3-Hydroxy-4-methoxybenzylsulfonyl acetic acid (18).
To a solution of 3-Hydroxy-4-methoxybenzylthio acetic acid 17 (2.9 g,
12.7 mmol) in glacial acetic acid (15 mL) was added 6 mL 30% hydrogen
peroxide and stirred over night (18 h). The completion of the reaction was
determined by TLC. The mixture was then poured into ice water and extracted
with ethyl acetate. The organic layer was washed with water and dried. Removal
of the solvent in vacuo afforded pure product 18. Yield: 60%; white solid mp
164-
165 C. 114 NMR: 8 3.84 (s, 3H, OCH3), 4.04 (s, 2H, CH2), 4.29 (s, 2H, CH2),
6.85-7.11 (m, 3H, Ar-H). Anal. Calcd for CI0H1206S: C, 46.15, H, 4.65. Found:
C, 46.21, H, 4.69.
Preparation of (E)-2, 4, 6-Trimethoxystyryl ¨3-Hydroxy-4-Methoxybenzyl
Sulfones (20, 6aa).
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A mixture of 3-hydroxy-4-methoxy benzyl sulfonyl acetic acid 18 (1.9 g,
7.3 mmol), 2,4,6-trimethoxybenzaldehyde 19 (1.58 g, 8.0 mmol), benzoic acid
(0.134 g, 1.1 mmol) and piperidine (0.081 g, 0.95mmol) in toluene (50 mL) was
refluxed for 2-3 h with continuous removal of water using a Dean-Stark water
separator. Reaction completion was determined by TLC (9:1
chloroform:methanol on silica gel plate). The reaction mixture was then cooled
to
room temperature, water was added and extracted with ethyl acetate. The
organic
layer was washed with saturated sodium bicarbonate solution, dilute
hydrochloric
acid, water and dried. Removal of the solvent in vacuo yielded a crude product
which on recrystalization from 2-propanol resulted pure product 20/6aa.
Alternate Method for the Synthesis of (E)-2,4,6-Trimethoxystyry1-3-Hydroxy-4-
Methoxybenzyl Sulfone 6aa (Scheme 4):
Preparation of 3-[(p-Toluenesulfonyl)oxy]-4-methoxybenzaldehyde (21).
A mixture of 3-hydroxy-4-methoxybenzaldehyde 12 (5.0 g, 32 mmol)and
p- toluenesulfonyl chloride (10.0 g, 52.5 mmol) was dissolved in pyridine
(12.5
mL, 154 mmol). The reaction mixture stirred for 5 mm and maintained at 70-80
C. The clear reaction mixture becomes turbid and becomes slurry. The stirring
was continued for a period of 2 h at 70-80 C. The reaction completion was
determined by TLC and the contents of the flask were cooled to room
temperature
and poured in to cold water. The white crystalline solid formed was filtered,
washed successively with 5 mL 1:1 HC1: H20, 5 mL of 5% NaOH solution and
water till the filtrate is free from pyridine, dried to constant weight to get
the
desired product 21. Yield: 98%; white solid mp 148-151 C. 11-1 NMR: 8 2.40
(s,
3H, CH3), 3.71 (S, 3H, OCH3), 6.91-7.83 (m, 7H, Ar-H), 9.83 (s, 1H, CHO).
Anal. Calcd for CI5H1405S: C, 58.81, H, 4.61. Found: C, 58.79, H, 4.59.
Preparation of 3-[(p-Toluenesulfonyl)oxy]-4-methoxybenzyl Alcohol (22).
To a cooled solution of 3-[(p-Toluenesulfonyl)oxy]-4-
methoxybenzaldehyde 21 (9.0 g, 29 mmol) in methanol (25 mL) was added
sodium borohydride (0.55 g, 14.5 mmol) in methanol (2.5 mL) over a period of 5-
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min maintaining the temperature around 15-20 C. The reaction mixture was
maintained at that temperature for further 30 min and checked TLC for
completion of the reaction. Water was added to the reaction mixture and the
solid
formed was filtered, washed with water and dried to afford 22. Yield: 97%;
white
5 solid mp 88-90 C. 1H NMR: 6 2.41 (s, 3H, CH3), 3.59 (s, 3H, OCH3), 4.57
(s,
2H, CH2), 6.80-7.79 (m, 7H, Ar-H). Anal. Calcd for C15H1605S: C, 58.43, H,
5.23. Found: C, 58.49, H, 5.19.
Preparation of 3-[(p-Toluenesulfonypoxy]-4-methoxybenzyl Chloride (23).
To a cooled solution of 3-[(p-Toluenesulfonyl)oxy]-4-methoxybenzyl
10 alcohol 22 (8.0 g, 26 mmol) in benzene (25 mL) was added thionyl
chloride (1.9
mL, 26 mmol) slowly over a period of 5-10 min maintaining the temperature
around 15-20 C. The reaction was maintained at those conditions for 2 h and
checked TLC for the completion. The flask is connected to a high vacuum
through a trap containing formic acid under mild heating to remove excess
thionyl
chloride. The slurry formed after complete removal of thionyl chloride and
benzene, filtered, washed with hexane and dried to afford 23. Yield: 90%;
white
solid mp102-105 C. 1H NMR: 6 2.43 (s, 3H, CH3), 3.61 (s, 3H, OCH3), 4.50 (s,
2H, CH2), 6.80-7.79 (m, 7H, Ar-H). Anal. Calcd for Ci5H15C104S: C, 55.13, H,
4.63. Found: C, 53.20, H, 4.59.
Preparation of 3-[(p-Toluenesulfonypoxy]-4-methoxybenzylthio acetic acid (24).
To a solution of sodium hydroxide (1.72 g, 42.9 mmol) in methanol (30
mL) was added mercaptoacetic acid (1.5 mL, 21.5 mmol) in portions and stirred
under nitrogen atmosphere for 10 min. 3-[(p-Toluenesulfonyl)oxy]-4-
methoxybenzyl chloride 23 (7.0 g, 21.5 mmol) was then added slowly to the
reaction mixture and stirred at reflux temperature for 5 h. The reaction
mixture
was then poured into ice containing concentrated HC1. The white crystalline
solid
separated was filtered, washed with water and dried to get the desired product
24.
Yield: 93%; white solid mp 116- 120 C. 1H NMR: 6 2.41 (s, 3H, CH3), 3.03 (s,
2H, SCH2), 3.61 (s, 3H, OCH3), 3.79 (s, 2H, CH2S), 6.80-7.81 (m, 7H, Ar-H).
Anal. Calcd for C17H1806S2: C, 53.39, H, 4.74. Found: C, 53.33, H, 4.71.
Preparation of 3-[(p-Toluenesulfonyl)oxy]-4-methoxybenzylsulfonyl acetic acid
(25).
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To a solution of 3-[(p-Toluenesulfonypoxy]-4-methoxybenzylthio acetic
acid 24 (7.0 g, 18.4 mmol) in glacial acetic acid (35 mL) was added 21 mL 30%
hydrogen peroxide and stirred over night (18 h). The reaction mixture was
poured
into ice water and the solid separated was filtered, washed with cold water
and
dried to get pure 25. Yield: 80%; white solid mp 142-146 C. 1H NMR: 8 2.50
(s,
3H, CH3), 3.61 (s, 3H, OCH3), 4.03 (s, 2H, SCH2), 4.60 (s, 2H, CH2S), 7.09-
7.74
(m, 7H, Ar-H), 13.4 (br s, 1H, OH). Anal. Calcd for Ci7E11808S2: C, 49.27, H,
4.38. Found: C, 49.22, H, 4.34.
Preparation of (E)-
2,4,6-Trimethoxystyry1-3-[(p-Toluenesulfonyl)oxy]-4-
Methoxy- benzyl Sulfone (26).
A mixture of 3-[(p-Toluenesulfonypoxy]-4-methoxybenzylsulfonyl acetic
acid 25 (6.0 g, 14.5 mmol ), 2,4,6- trimethoxybenzaldehyde 19 (2.85 g, 14.5
mmol), benzoic acid (0.27 g, 2.2 mmol) and piperidine (0.19 mL, 1.9 mmol) in
benzene (50 mL) was refluxed for 4-5 h with continuous removal of water using
a
Dean-Stark water separator. The reaction completion was checked by TLC (9:1
chloroform: methanol on silica gel plate). The reaction mixture was then
cooled to
room temperature and the crystalline solid formed was filtered, washed with
cold
benzene and dried to get the desired product 26. Yield: 65%; white solid mp
159-
166 C. 1H NMR: 8 2.42 (s, 3H, CH3), 3.61(s, 3H. OCH3), 3.81 (s, 3H, 3 x
0CH3), 4.21 (s, 2H, CH2), 6.06 (s, 2H, Ar-H), 6.80-7.74 (m, 7H, Ar-H), 7.01
(d,
1H, J=15.5 Hz, =CH), 7.83 (d, 1H, J= 15.5 Hz, CH=). Anal. Calcd for
C26H2809S2: C, 56.90, H, 5.14. Found: C, 56.83, H, 5.11.
Preparation of (E)-2,4,6-Trimethoxystyry1-3-Hydroxy-4-Methoxybenzyl Sulfone
(6aa).
A mixture of (E)-2,4,6- Tiimethoxystyry1-3-[(p-Toluenesulfonypoxy]-4-
methoxy-benzyl sulfones 26, (5.0 g, 9.1 mmol), 50 mL (20%) sodium hydroxide
solution and methanol (50 mL) were taken in a round bottomed flask and
refluxed
until the reaction mixture is clear without any turbidity (3-4 h). The
progress of
the reaction was monitored by TLC. The reaction mixture was cooled to room
temperature and neutralized with cold dilute MC! solution. The precipitate
separated after neutralization was filtered, washed with water and dried to
get the
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crude product which on recrystallization from 2-propanol resulted analytically
pure sample 6aa.Yield: 95%; white solid mp 124-127 C.
Synthesis of (E)-2,4,6-Trimethoxystyry1-3-0-phosphate disodium-
4-
methoxybenzyl Sulfone (6ab): (Scheme 5).
Preparation of (E)-2,4,6-Trimethoxystyryl- 3-0-Bis(benzyl)phosphoryl- 4 ¨
methoxybenzyl Sulfone (27).
To a stirred solution of (E)-2,4,6-Trimethoxystyry1-3-Hydroxy-4-
methoxybenzyl Sulfone 6aa (3.8 g, 9.6 mmol) in acetonitrile (48 mL) under
nitrogen atmosphere was added carbon tetrabromide (3.88 g, 11.72 mmol) and
triethylamine (1.46 g, 14.4 mmol) and stirring was continued for 10 mm.
Dibenzyl phosphite (3.20 g, 11.6 mmol) dissolved in acetonitrile (32 mL) was
added to the reaction mixture slowly. After the addition, the reaction mixture
was
stirred for 2 h, checked the TLC for completion of the reaction. The
phosphorylation was terminated by drop wise addition of potassium dihydrogen
phosphate (20 mL, 0.5 M) to the reaction mixture over a period of 10 min. The
solution was then extracted with ethyl acetate (3 x 60 mL). The organic
extracts
were combined and washed with water, dried and concentrated in vacuo. The
thick liquid obtained after concentration was purified on silica column using
chloroform: methanol with increasing polarity. The purified product was
concentrated in vacuo to afford pure dibenzyl ester 27. Yield: 73%; semi
solid. 111
NMR: 8 3.68 (s, 6H, 2 x OCH3), 3.71(s, 3H, OCH3), 3.74 (s, 3H, OCH3), 4.07 (s,
2H, CH2), 4.96-5.04 (m, 4H, OCH2), 5.98 (s, 2H, Ar-H), 6.60-7.42 (m, 14H, Ar-
H+ vinylic), 7.71 (d, 1H, J= 15.6 Hz, CH=). Anal. Calcd for C33H35010PS: C,
60.54, H, 5.39. Found: C, 60.48, H, 5.44.
Preparation of (E)-2,4,6-Trimethoxystyry1-3-0-phosphory1-4-methoxybenzyl
Sulfone (28).
To a stirred solution of the above dibenzyl ester 27 (4.36 g, 6.7 mmol) in
anhydrous dichloromethane (40 mL) under nitrogen at 0 C, was added
bromotrimethylsilane (2.14 g, 14.1 mmol). The stirring was continued for 45
min
at the same temperature and checked the TLC for completion of the reaction.
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Sodium thiosulfate (1 %, 50 mL) was added to the reaction mixture and stirring
was continued for an additional 5 min. The separated aqueous phase was
extracted with ethyl acetate (3 x25 mL). The organic extracts were
concentrated
in vacuo to afford the crude phosphoric acid 28, which was purified on a
silica
column using chloroform: methanol with increasing polarity. The purified
product was concentrated in vacuo to afford pure acid 28. Yield: 44.3%; White
solid mp 202-205 C.11-1NMR (DMS0- d6): 8 3.78(s, 6H, 2 x OCH3), 3.85(s, 3H,
OCH3), 3.86 (s, 3H, OCH3), 4.34 (s, 2H, CH2), 6.30 (s, 2H, Ar-H), 7.02 (m, 2H,
Ar-H), 7.12 (d, 1H, J= 15.6 Hz, = CH), 7.32 (s, 2H, OH), 7.52 (s, 1H,Ar-H),
7.61
(d, 1H, J= 15.6 Hz, CH=). Anal. Calcd for Ci9H23010PS: C, 48.10, H, 4.89.
Found: C, 48.14, H, 4.92.
Preparation of (E)-2,4,6-Trimethoxystyry1-31-o-phosphate disodium-4-methoxy-
benzyl Sulfone ( 6ab):
To a stirred solution of the above phosphoric acid 28 (1.35 g, 2.85 mmol)
in ethylene glycol dimethyl ether (125 mL) was added 2N sodium hydroxide
(0.27 g dissolved in 13.66 mL H2O, 6.8 mmol) and stirred for 3 h. The solid
formed was filtered, washed with acetone (2x 25 mL) and dried under vacuum to
get the product 6ab. Yield: 98%; white solid mp 152-154 C. 1H NMR (D20): 8
3.68 (s, 6H, 2 x OCH3), 3.71 (s, 3H. OCH3), 3.78 (s, 3H. OCH3), 4.35 (s, 2H,
CH2), 5.92 (s, 2H, Ar-H), 6.91 (s, 2H, Ar-H), 6.97 (d, 1H, J=15.6 Hz, =CH),
7.39
(s, 1H, Ar-H), 7.43 (d, 1H, J= 15.6 Hz, CH=). 13C NMR (D20): 8 164.4, 161.7,
151.1, 143.8, 136.8, 125.9, 123.4, 120.6, 120.2, 113.1, 103.4, 91.1, 61.0,
56.4,
56.2, 55.9. Anal. (C19H21010Na2PS): C, H.
Biology
Tissue Culture and Reagents: Paclitaxel was purchased from Sigma. Cell
lines were purchased from ATCC. Cell lines were routinely gown in DMEM or
RPM1 (CellGro) supplemented with 10% fetal bovine serum (Atlas) and 1
unit/mL penicillin-streptomycin (Gibco).
Cytotoxicity Assay
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A number of tumor cell lines were tested using a dose response end point
assay system. The cells were grown in either DMEM or RPMI supplemented
with 10% fetal bovine serum and lunit/mL Penicillin-Streptomycin solution. The
tumor cells were plated into 6 well dishes at a cell density of 1.0 x 105
cells/mL/well and compounds were added 24 h later at various concentrations.
Cell counts were determined from duplicate wells after 96 h of treatment. The
total number of viable cells was determined by trypan blue exclusion.
Soft Agar Assay
The soft agar plates were prepared as described by Cosenza, et al
(Cosenza, S.C. et al. In Cell Growth, Differentiation and Senescence,
Studzinski,
G.P., Ed.; Oxford University Press: 1999, 161-176.). Briefly, Noble bottom
agar
(0.8%) was plated onto 60 mm tissue culture plates. Exponentially growing MIA-
PaCa-2 cells (1.0 x105) were mixed with growth medium with various
concentrations of each compound and mixed with Noble agar to a final
concentration of 0.4%. Each concentration was plated in triplicate. The top
agar
was allowed to solidify and the plates were then incubated at 5% CO2 at 37 C
for
3 weeks. The plates were then stained with 0.05% nitroblue tetrazolium (NBT)
solution and representative plates were photographed using an Olympus
stereoscope mounted with a Sony digital camera system (DKC5000, Sony Inc).
Flow Cytometry
Human prostate tumor cells, DU145 cells, and normal diploid human lung
fibroblasts, HFL-1 cells, were grown in DMEM (Cellgro) supplemented with 10%
fetal bovine serum and 1 unit/mL penicillin-streptomycin. The cells were
plated
onto 100 tmn2 dishes at a cell density of 1.0 x 106 cells/dish, and 24 h
later, they
were treated with 2.5 1.1M of the compound. The cells were harvested 24, 48
and
72 h after treatment. The cells were removed from the plate by trypsin
digestion
and combined with the non-attached cells found in the medium. The cell pellets
were washed in phosphate buffered saline (PBS), and fixed in ice cold 70%
ethanol for at least 24 h. The fixed cells were then washed with room
temperature
PBS and stained with propidium iodide (50 ptg/mL) and RNase A (0.5 mg) for 30
min at 37 C. The stained cells were then analyzed on a Becton-Dickinson (BD)
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(FACScan) flow cytometer and the data analyzed by cell cycle analysis software
(Modfit, BD).
PARP Western
BT20 cells were plated at a density of 3.0 x 106 cells per 150 mm2 plate
and treated 24 h later with either DMSO or 6aa. The cells were collected at
the
indicated time points and cell pellets were frozen. The frozen cell pellets
were
lysed in 1% NP40/PBS lysis buffer containing protease inhibitors. Equal
amounts
of total cellular protein was then resolved on a 10%-SDS-polyacrylamide gel.
The gels were transferred onto nitrocellulose paper (S/S), hybridized with
anti-
PARP antibodies (BD) and developed using ECL (Perkin-Elmer, MA) solution.
Nude mouse assay
Female athymic (NCR-nu/nu, Taconic) nude mice were injected with 0.5-
1.0 x107 BT20 cells subcutaneously in the hind leg using a 1 mL tuberculin
syringe equipped with a 271/2 gauge needle. Approximately 14 days later, mice
were paired (N=8) and injected with 6ab or Phosphate buffered saline as the
vehicle control. The intravenous injections were performed in the mouse tail
vein
using a 1 mL tuberculin syringe equipped with a 30 gauge needle. The animals
were injected following a Q2D x3 schedule. Tumor measurements (two
dimensions) were done three times per week using traceable digital vernier
calipers (Fisher). Tumor volume was calculated using the following equation:
V= (Lx(S2)7c/6, where L is the longer and S is the shorter of the two
dimensions.
Body weight was determined during each measurement. The animals were
observed for signs of toxicity. The time of tumor volume doubling was
calculated
and the T-C value (difference in the average times post treatment for tumors
of
the treated groups to attain a doubling in volume compared to the average of
the
control group) was determined. Body weight loss of more than 10% was not
observed in any group nor were there any animal deaths. All studies were
performed under the guidelines of Temple University IACUC.
Bone Marrow Harvest and Colony Formation Assay
Bone marrow was harvested from femur and tibia of CD-1 mice injected
with 200 III, of PBS or 6ab [10 mg/mL:100 mg/kg dose] at 12, 24, or 48 h
before
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the sacrifice. The bone marrow cells were cultured in Methylcellulose medium
supplemented with 50 ng/mL rmStem Cell Factor, 10 ng/mL rmIL-3, 10 ng/mL rh
IL-6, 200 ug/mL human Transfeffin, and 3 units/mL rhErythropoietin (Stem Cell
Technologies, Vancouver, BC, Canada). Cultures were seeded in duplicates using
35 mm plastic petri dishes and colony forming units were determined after one
week.
Results
Structure-Activity Relationships (SAR)
Following the synthesis of this group of compounds, their in vitro
cytotoxicity was assessed using four different human tumor cell lines derived
from human breast (BT20), prostate (DU145), lung (H157) and colorectal
(DLD1) cancers. The results of this study are presented in Table 1. These
studies
show that the cytotoxic activity of the styryl benzyl sulfones is completely
dependent on the nature and position of the substituents on the two aromatic
rings.
In a majority of the compounds described here, a methoxy group was kept
constant at the 4th position on the aromatic ring of the benzyl moiety. A
moderate
cytotoxic activity was seen when a fluorine atom was present at 4-position
(6b) on
the styryl aromatic ring. Changing the
25
Titble 1. In Vitro Cytotoxicity of Styryl Benzyl Sulfones
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02
,.'s-...
1
R
compd R R1 BT20 DU145 H157 DLD1
ICso ( M) 1050 (11M) 1050 GAD
1050 (pM)
6a 4-0Me 4-0Me >20 >20 >20 >20
6b 4-0Me 4-F 1.5 1.25 2.5 2.0
6c 4-0Me 4-CI 2.0 2.5 3.5 3.0
6d 4.10DMe 4-NO2 5.0 7.5 7.5 5.0
6e 4-0Me 4-NH2 >20 >20 >20 >20
6f 4-0Me 2-0Me 5.0 3.5 5.0 7.5
6g 4-0Me 2-C1,4-F 2.5 2.0 2.5 4
6h 4-0Me 2,4-(CH3)2 15 12 15
15
6i 445Me 2-0Me,4-F 15 10 20
15
6j 4-0Me 3,4-(0Me)2 >20 >20 >20 >20
6k 4-0Me 3,5-(CH3)2 >20 >20 >20 >20
61 4-0Me 2,6-(CH3)2 >20 >20 >20 >20
6m 4-0Me 2,6-(0Me)2 0.40 0.25 0.70 0.70
6n 420Me 2,4-(0Me)2 >20 >20 >20 >20
6o 4-0Me 2,5-(0Me)2 >20 >20 >20 >20
6p 4-0Me 3,5-(0Me)2 >20 >20 >20 >20
6q 4-0Me 2,4,5-(0Me)3 >20 >20 >20 >20
6r 4-0Me 2,3,4-(0Me)3 >20 >20 >20 >20
6s 423Me 2,4,6-(0Me)3 0.020 0.025 0.030
0.040
6t 4-0Me 3,4,5-(0Me)3 >20 >20 >20 >20
6u 4-0Me 2,6-(0Me)2,4-0H >20 >20 >20 >20
6v 4-0Me 2,6-(0Me)2,4-F 0.75 0.5 0.5 0.75
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6w 4-0Me 2,4,6-(CH3)3 >20 >20 >20 >20
6x 4-0CF3 2,4,6-(0Me)3 3 4 6 4
6y 4-0Me,3-0H 3,4,5-(0Me)3 7.5 5 10 10
6z 4-0Me,3-0H 2,6-(0Me)2,4-0H 0.25 0.5 0.5 0.5
6aa 4-6Me,3-0H 2,4,6-(0Me)3 0.010 0.003 0.004
0.003
6ab 4-0Me,3-0P03Na22,4,6-(0Me)3 0.005 0.0025 0.007
0.007
6ac 3,4,5-(0Me)3 2,4,6-(0Me)3 >20 >20 >20 >20
6ad 2,3,4,-(0Me)3 2,4,6-(0Me)3 15 12.5 15 15
6ae 4-C1 2,4,6-(0Me)3 10 7.5 15 15
6af 44NO2 2,4,6-(0Me)3 15 15 20 15
6ag 4-CN 2,4,6-(0Me)3 12.5 7.5 15 20
6ah 4-COOH 2,4,6-(0Me)3 15 10 17.5 15
6ai 4-0H 2,4,6-(0Me)3 15 7.5 20 20
15 fluorine atom at the 4-positon with a chlorine (6c), a nitro
(6d), a methoxy (6a) or
an amino (6e) group gradually decreased the activity of the molecules. By
changing the position of the methoxy group from the 4 to 2-position (6f) on
the
styryl aromatic ring, the molecule partially recovered the lost activity. The
introduction of a chlorine atom in the 2-position (6g) in 6b retained
cytotoxic
20 activity, whereas a methoxy group in the same position (6i)
resulted in the loss of
activity. Dimethyl substitutions on the styryl aromatic ring with a methoxy
group
at 4-position on benzyl aromatic ring (6h, 6k and 61) resulted in the
molecules
possessing a low level of cytotoxicity. Whereas the results are quite
surprising for
the molecules that are disubstituted with methoxy groups (6j, 6m, 6n, 6o and
6p)
25 on the styryl aromatic ring, the results obtained in
cytotoxicity assays using these
compounds (6j, 6m, 6n, 6o and 6p) clearly show that the methoxy group, when
present at the 2,6-positions (6m), enhances the activity of the molecule by
greater
than 40 fold when compared to other disubstituted methoxy sulfones (6j, 6n, 6o
and 6p). Because the introduction of two methoxy groups on the styryl aromatic
30 ring enhanced the biological activity, some trimethoxy styryl analogs were
synthesized to determine if this further enhances their cytotoxic properties.
Analysis of these compounds (6q, 6r, 6s and 6t) in the cell-killing assays
showed
that 4-methoxybenzy1-2,4,6-trimethoxystyryl sulfone (6s) is 20-fold more
active
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than 6m, where as the other trisubstituted styryl sulfones (6q, 6r and 6t)
were
totally inactive at the highest concentration (20 M) tested. These results
show that
when the 2,4 and 6-positions on the styryl aromatic ring are occupied by
methoxy
groups, the molecules attain optimum biological activity. To validate whether
methyl groups at those positions can replace the methoxy groups and retain the
activity, 2,4,6-trimethyl styryl sulfone (6w) was prepared, which was found to
be
inactive in cell killing assays. To further assess the significance of the
methoxy
group on the 4-position of styryl ring in 6m, the methoxy group at that site
was
replaced with a hydroxy (6u) or a fluoro (6v) substituent. Both of these
replacements resulted in either a reduced level or total loss of activity.
Once the
methoxy subtituents are fixed at the 2, 4 and 6 positions of the styryl ring,
to
further enhance the activity of the molecule, the effect of other substituents
on the
benzyl aromatic ring was then determined. Replacing the methoxy group at the 4-
position on the benzylic aromatic ring of 6s by chloro (6ae), nitro (6af),
cyano
(6ag), carboxy (6ah) and hydroxy (6ai) resulted in molecules that
substantially lost
activity. These results show that the methoxy group is indispensable at the 4-
position of the benzyl aromatic ring of 6s with respect to its biological
activity. To
analyze the effect of the additional substituents on the benzyl aromatic ring,
a
number of analogs were synthesized containing 4-methoxy-3-halo, nitro, cyano,
carboxy, methoxy (data not shown), hydroxy (6aa), 3,4,5-trimethoxy (6ac),
2,3,4-
trimethoxy (6ad) benzyl sulfones. Cytotoxicity analyses of these analogs on
four
cancer cell lines showed that the compound with the hydroxy substituent at
position-3 (6aa) exhibited the best activity in the entire series. This
compound, 6aa,
is almost 8 to 10 fold more active than 6s in all four cell lines. Further,
the
introduction of a hydroxy group at the third position not only enhanced the
potency
of the molecule, but also created a method to generate a water-soluble analog
(6ab), which is critical for intravenous administration of the compound. The
conversion of the hydroxyl group in 6aa to a disodium phosphate 6ab derivative
did not alter the potency of the molecule.
Table 2. Tumor cell killing (IC5o) concentrations ( M) of 6s and 6aa
Cell Line Tumor Type 6s 6aa
T47D BREAST (ER+) 0.025 0.006
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MCF-7 BREAST (ER+) 0.003 0.004
DU145 PROSTATE (AR-) 0.025 0.007
PC-3 PROSTATE (AR+) 0.03 0.008
OV-CAR-3 OVARIAN 0.008 ' 0.006
Sk-OV-3 OVARIAN ND 0.006
MIA-PaCa2 PANCREATIC 0.008 0.004
U87 GLIOBLASTOMA 0.04 0.007
H157 NSCLC 0.03 0.007
A549 NSCLC 0.02 0.01
H187 SCLC 0.015 0.007
- N417 SCLC 0.008 0.005
AGS GASTRIC 0.02 0.007
RF1 GASTRIC 0.008 0.006
RF48 GASTRIC 0.01 0.005
COLO-205 COLO-RECTAL 0.015 0.009
DLD-1 COLO-RECTAL 0.04 0.008
HCT-116 COLO-RECTAL ND 0.009
HCT-15 COLO-RECTAL 0.02 0.008
SW480 COLO-RECTAL ND 0.007
SK-MEL-28 MELANOMA 0.04 0.007
CEM LEUKEMIC 0.03 0.009
K562 CML ND 0.004
T-
MOLT-4 0.009 0.005
lymphoblastic:ALL
Burkitt's Lymphoma
Namalwa (B-cell) 0.015 0.006
Burkitt's Lymphoma
Daudi (B-cell) 0.008 0.007
Burkitt's Lymphoma
Raji (B-cell) 0.009 0.004
MES-SA SARCOMA 0.01 0.006
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RESISTANT
*MES SAJDX5 0.01 0.005
SARCOMA
CEM LEUKEMIC 0.03 0.01
RESISTANT
*CEM/C2 0.01 0.01
LEUKEMIC
2008 Ovarian ND 0.005
*2008/17/4 Resistant Ovarian ND 0.004
* These cell lines constitute multi-drug resistant cell lines and show up-
regulation
of MDR and in the case of CEM/C2, additional mutations in the Topo-2 gene
(Harker, W. G.; et al. Cancer Res. 1985, 45, 4091-4096; Fujimori, A. et al
Cancer
Res. 1995, 55, 1339-1346).
Biological Results and Discussion
In vitro anti-tumor effects of 6s and 6aa compounds
The activity of two of the most active compounds listed in Table 1 was
then tested against 94 different human tumor cell lines and surprisingly, they
were
found to induce apoptosis of all of these cell lines with very similar GI50
values
(selected data shown in Table 2). Some of these compounds (such as 6s, 6aa)
were also tested by the National Cancer Institute, USA, through its
Developmental Therapeutics Program (DTP) against their panel of 60 human
cancer cell-lines (Greyer, M. R et al. Seminars in Oncology 1992, 19, 622-
663).
The results showed that these compounds exhibited broad-spectrum activity and
inhibited the growth of all of the tested cell lines, including drug-resistant
cell-
lines, at nanomolar concentrations. Notably, the GI50 and LC50 values for many
of these cell lines were similar, indicating that they induced apoptosis in
these
cells. Statistical comparison (using the NCI algorithm COMPARE) revealed that
these drugs are mitotic blockers of tumor cells.
6s and 6aa compounds are highly active against drug resistant tumor cell lines
Development of resistance to classical chemotherapeutic agents is widely
observed in patients who have not responded or have relapsed after first round
therapy and is the primary cause of treatment failure. In the initial
screening
experiments, it was observed that both the Onconova cell panel and the NCI
panel
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included several cell lines which are multi-drug resistant but were highly
sensitive
to the pro-apoptotic effects of this series of compounds (Harker, W. G. et al.
Cancer Res. 1985, 45, 4091-4096; Fujimori, A. et al. Cancer Res. 1995, 55,
1339-
1346). To further investigate the activity of these compounds against MDR
positive tumor types, the IC50 values of 6s and 6aa were determined using two
classical MDR positive cell lines. The results shown in Figure 1 a show a 96 h
dose response of the uterine sarcoma cell line MES-SA and the multidrug
resistant subline MES-SA/DX5 treated with 6aa (Harker, W. G. et al. Cancer
Res.
1985, 45, 4091-4096). This cell line has been shown to express high levels of
P-
glycoprotein and is resistant to a number of drugs including doxorubicin,
paclitaxel, vincristine, vinblastine, etoposide, mitoxantrone, dactinomycin,
and
daunorubucin. The activity of our compounds was then compared to the activity
of paclitaxel (MDR sensitive drug). The results show that the parental cell
line
was very sensitive to Paclitaxel (IC50 4 nM) but the MDR positive subline was
greater than 100 fold resistant (IC50 750 nM). When the two cell lines were
treated with 6aa, both the parental and the MDR positive cell lines were
equally
sensitive to the cell killing activity of the compound. It was also
investigated as
to whether atypical multidrug resistant cell are sensitive to 6aa. For these
studies,
the parental leukemic cell line CEM and its MDR subline CEM/C2 were
employed (Fujimori, A. et al. Cancer Res. 1995, 55, 1339-1346). CEM/C2 was
selected and subcloned for resistance to camptothecin and has cross resistance
to
etoposide, dactinimycin, bleomycin, mitoxantrone, doxorubicin, and
daunorubicin. The results show that the campothecin-resistant subline, CEM/C2,
was highly sensitive to the styryl benzyl sulfone series of compounds
suggesting
that these compounds do not share any cross resistance to classical MDR and
atypical MDR cell lines (Table 2).
Effect of 6aa and 6ab on soft agar colony formation
The anti-tumorigenic activity of 6aa and 6ab was then tested in soft agar.
For soft agar assays, three different cell lines, BT20, DU145 and MIA-PaCa-2,
representing breast, prostate and pancreatic cancers, respectively, were used.
In
all cases complete inhibition of the growth of tumor cells in a dose dependent
manner was observed for the individual compounds (Figure lb shows data for
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MIA-PaCa-2). In these assays, paclitaxel was used as a positive control, which
showed a slightly lower potency than that of 6aa and 6ab (Figure lb).
Effects of 6s and 6aa on cell cycle progression of normal and tumor cells
The effect of these compounds on normal and tumor cell cycle progression
was then examined using FACS analysis. Figure 2a show the effect of 6aa on the
cell cycle progression of human vascular endothelial cells (HUVEC) and DU145
(prostate cancer) cells. The results of this study show that the addition of
the 6aa
to HUVEC cells resulted in a block of their cell cycle progression in the G1
phase,
causing growth arrest without a loss of viability. On the other hand, tumor
cells
treated with this compound gradually accumulated in the G2/M phase of the cell
cycle and appeared to be unable to exit from this phase, leading to the
activation
of apoptotic pathways as judged by PARP [Poly(ADP-ribose) polymerase-1]
cleavage which is a marker for caspase activation (Figure 2b)( Soldani, C. et
al.
Apoptosis 2002, 7, 321-328). No PARP cleavage was observed in HUVEC cells
following similar treatment with 6aa compounds (Figure 2b).
In vivo anti-tumor effects of 6s and 6aa of compounds
In order to determine in vivo efficacy, the nude mouse model system was
utilized. A highly aggressive human estrogen negative breast carcinoma cell
line
(BT20) was xenografted into athymic nude mice. The animals were treated with
either 50 mg/kg of 6s using a Q4D schedule or 25 mg/kg using a Q2D schedule.
The animals were treated when the tumors were approximately 70 mm3 in size.
Figure 3a shows that intraperitonial (IP) injections of 6s using either of the
two
schedules were able to inhibit the growth of the tumors. The vehicle control
treated tumors, on average, increased in volume over the 22 day period by 5
fold
(62 mm3-335.5 mm3), while the Q2D 6s treated tumors increased in volume by
only 2.5 fold (67.5 mm3-165 mm3). The Q4D 6s treated tumors also had
significant but slightly less tumor growth inhibition, whereby these tumors
increased in average volume by only 2.9 fold (74 mm3-217 mm3). 6s was well
tolerated at these doses as determined by body weights and physical
observations.
These studies show that 6s is efficacious against human tumor xenografts while
showing no signs of toxicity at the schedules tested under this study.
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Because 6s is poorly water-soluble, 6ab was synthesized which was highly
water-soluble and allowed intravenous administration (Pettit, G. R. et al Anti-
Cancer drug. Des. 2000, 15, 203-216; Pettit, G. R. et al Anti-Cancer Drug.
Des.
2001, 16, 185-193; Pettit, G. R. et al. J. Med. Chem. 2002, 45, 2534-2542). To
test the effects of 6ab in vivo, two groups of mice were used. One group
received
the vehicle alone, while the second group received the compound by intravenous
(IV) injection into the tail vein (3b). The tumor size was then measured on
alternate days and the total length of the experiment was 21 days. The results
presented in Figure 3b show that 6ab readily inhibited tumor growth in this
xenograft model system. Of the 8 mice included in each group, 100% of the
control mice (placebo administered) showed a doubling or tripling of the total
tumor volume. On the other hand, the majority of the mice administered with 6s
or the phosphate salt of 6aa showed growth arrest or a gradual reduction in
their
tumor volume, suggesting that these compounds, with proper formulation can be
valuable anti-cancer therapeutics.
In vivo Toxicity studies in mice
To asses the in vivo toxicity profile, 100 mg/kg of 6aa was intravenously
administered into mice and its effect on the in vitro hematopoietic colony
formation of bone marrow cells was determined at 12, 24 and 48 h intervals
following the injection of the drug. These studies (Figure 4) show that there
was
no reduction in total, myeloid or lymphoid colony formation. Single dose and
repeat-dose (28 daily injections) toxicology studies and detailed biochemical
and
cellular analysis of one of the water-soluble analogs (6ab) revealed that
unlike
most other cytotoxic agents, this drug did not cause hematotoxicity (no
myleosuppression), liver damage or detectable neurotoxicity in these animals.
High, dose dependent, drug levels were sustained in circulation suggesting
that
therapeutic levels could be achieved without overt toxicity.
Acute and repeat toxicity studies in rats
Single dose and repeat-dose (28 daily injections) toxicology studies were
carried out with one water-soluble form of this series (6ab) to assess the
safety of
intravenously administered drug in rats. Detailed biochemical and cellular
analysis revealed that this drug did not cause hematotoxicity (no
myleosuppression), liver damage or any detectable neurotoxicity in these
animal
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studies. High dose dependent drug levels were sustained in circulation
suggesting
that therapeutic levels could be achieved without overt toxicity.
Conclusion
In this communication, the synthesis of a group of styryl benzyl sulfones
was described which induce apoptotic death of a wide variety of human tumor
cell lines at sub nanomolar concentrations while exhibiting relatively low
toxicity
to normal human cells. The studies show that the cytoxic activity of the
styryl
benzyl sulfones is completely dependent on the nature and position of the
substituents on the two aromatic rings. In a majority of the compounds
described
here, a methoxy group constant at the 4th position was retained on the
aromatic
ring of the benzyl moiety. These structure function studies show that when the
2,
4 and 6-positions on the styryl aromatic ring are occupied by the methoxy
groups,
the molecules attain optimum biological activity (6s). This activity could be
further enhanced by the introduction of a hydroxyl group at the third position
of
the benzylic ring (6aa and 6ab). Biological evaluation of the activity of
these
compounds show that these compounds are highly active against a wide variety
of
human tumor cell lines including those that are resistant to the activity of
many of
the currently used chemotherapeutic agents.
The low toxicity profile, both in vitro and in vivo and their potent tumor
inhibitory
activity as seen in soft agar and nude mouse xenograft assays point to the
potential value of these compounds as safe therapies for cancer, lacking many
of
the side effects normally associated with current chemotherapeutic agents.
Recent
studies with 6s, 6aa and 6ab show that these compounds altered the growth and
cell cycle status of mantle cell lymphoma cell lines and potently inhibited
the
expression of several important proteins, including cyclin-dependent kinase 4,
p53, mouse double minute 2 (MDM2), and cyclin D (Park, I.W. et al Oncogene
2007, 26, 5635-5642). Since 6s, 6aa and 6ab are highly effective in various
combinations with conventional chemotherapy; the lack of overt hematotoxicity
of these compounds may be beneficial for testing novel combinations for
advanced cancers, including tumors resistant to conventional chemotherapy. In
addition, their safety profile seen with normal hematopoietic cells suggest
that
these compounds have a potential use in in vitro purging of tumor cells from
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patient bone marrow for use in autologous bone marrow transplantation.
Clinical
and preclinical studies currently underway will reveal the best way to utilize
these
compounds in cancer therapy.
References
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(4) Blagosklonny, M. V.; Pardee, A. B. The Restriction Point of the Cell
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(9) Millard, S. S.; Kof , A. Cyclin-dependent kinase inhibitors in
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(10) Mc Donald, E. R.; El-Diery, W.S. Check point genes in cancer. Ann.
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(11) Ewen, M.E. Relationship between Ras pathways and cell cycle
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(13) (a) Reddy, M.V. R.; Reddy, S. Synthesis of a, 13-Unsaturated Suflones.
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Reddy, S.; Reddy, M. V. R.; Balasubramanyam, S. Preparation of
styryl benzyl sulfones and 1, 2-bis- (styrylsulfonyl-methyl)- 4,5-
dimethylbenzenes. Org. Prep. Proc. Int. 1988, 20. 205-212.
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P.; Vikramadithan, R. K; Rajagopalan, R. Novel Antidiabetic and
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Chroman Derivatives. J.Med.Chem. 1999, 42, 3265-3278.
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synthesis of 2-(arylsulfony1)-1-pheny1-3-ary1-2-propen-1-ones. Sulfur
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Renee, F.; Richard Francis, L. Novel Knoevenagel condensation of a
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EXAMPLE 2
Preparation of ON 013105
STAGE ¨ 1: Protection of Phenolic-OH group
(a) 500g of Isovanilin and 1000g of p-Toluenesulfonyl chloride are
dissolved
in 1250 ml of Pyridine in a 20 L glass reactor kept in a water bath. The
reaction
mixture is stirred well for about 15 minutes. The clear solution gradually
becomes turbid and becomes slurry. Stirring is continued for a period of 2
hrs.
The water bath is maintained at 70-80 degrees throughout the reaction.
(b) After two hours, the reaction mixture is allowed to attain room temp
and
cold water is added to the reactor to get a white crystalline solid. The solid
is
filtered and washed successively with 500 ml 1:1 HC1, 500 ml of 5% NaOH
solution and water till the filtrate is free from Pyridine. The precipitate is
dried
and weighed.
(c) The weight of the compound is 985 grams, i.e. 98%. The melting point
range 148¨ 151 C.
(d) TLC shows the absence of isovanillin and the presence of a spot moving
faster than isovanillin spot.
Remarks: 1. Yield depends upon the purity of p-Toluene sulfonyl chloride.
2. Color of the compound varies from off-white to white.
3. Washing and treatment are the key procedures to decide the
quality of the compound.
4. Reaction completes within 15 min. But to get the pure
compound stirring and heating should be continued up to 1.5 to 2
hrs.
All the chemicals used in this process are commercial samples.
Ratio of Reactants: IV : PTSC : Pyr.
1: 2 :2.5
Abbreviations: N: Isovanilin, PTSC: P-Toluenesulfonyl Chloride, Pyr: Pyridine
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02 CHO
CH+0 Cl Pyridine 1401
s- 0
0 70-80 C
OH -HC1 0, 10
02
STAGE ¨2: Stage 1 compound --) Mol wt: 306
MP range: 148-151 C
The Stage-1 compound 985g (3.2189 moles) is taken in 2.5 L of methanol in a
20L glass reactor maintained in a cold water bath (20 C). The reducing agent
sodium borohydride 60g (0.58 moles) in methanol is added slowly over a period
of 20 to 30 minutes. Reaction is over by the time the addition is completed.
During the addition of sodium borohydride, the solution in the reaction
becomes
clear without any solid. The stirring is continued. During the stirring
process
white crystalline solid separates in methanol. After two hours, water is added
to
the reaction mixture to ensure complete precipitation in the reactor. After 2
hours
the precipitate formed is filtered off and washed thoroughly with water. A
white
crystalline solid that weighs 965g (after drying) 97% is obtained. The melting
range of the compound is 88-90 C. Mol wt: 308
Remarks: 1. Sodium borohydride should be quickly made as fine powder
(if
crystalline) and to dissolve in cool methanol so that the vigorous
and exothermic nature of the reaction can be controlled.
2. During addition, small portions of NaBH4 methanol solution is
preferable, but quick enough.
3. Addition must be continuous and the heat developed in the
addition flask can be avoided by taking chilled methanol.
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4. Proper outlet (condenser) is required for the reactor to avoid
the development of the pressure inside.
5. Temperature should be controlled throughout the reaction
between 15 and 20 degrees Centigrade
The solvent and the NaBH4 are commercial samples.
Key words: Sodiumborohydride (SBH) MR: Melting range
RM: Reaction mixture
0 CHO
OH
NaBH4 *
0,PPJ0
s
02 02
STAGE ¨3: Stage 2 compound --> Mol wt: 308
MP range: 148-151 degrees C
Stage-2 compound 965g (3.13 moles) is taken in 2L of benzene in a 10L reactor
which is maintained at 15 and 20 C in a water bath. Thionyl Chloride 227 ml
(3.13 moles) in 500 ml of benzene is added dropwise to the reaction mixture
under vigorous stirring. The product starts separating from the clear reaction
mixture within 30 min. After completion of the addition the reaction mixture
is
allowed under stirring for about 2 hrs. The reactor is connected to vacuum
through a trap containing formic acid. Under mild heating and high vacuum
thionyl chloride and some portion of benzene is collected in the trap. The
process
is continued till all the unreacted thionyl chloride is distilled off The
slurry left
over is filtered and washed with hexane (hexane is added to the reaction
mixture
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in the reactor and allowed under stirring 15 minutes). The filtrate is
concentrated
under vacuum and the resulting precipitate is washed with hexane. Both the
precipitates are combined and dried under vacuum. The dried compound
weighed 925g (approx. 90%). The melting range is 102 ¨ 105 C. Completion of
the reaction is monitered by T.L.C.
Remarks: 1. Thionyl chloride should be handled in an effective Fume
hood.
It should be handled with gloves, goggles, and nose mask.
2. Benzene volume can be minimized and the addition can be
made fast.
3. Stirring with hexane will give pure product.
OH
Cl
SOC12 *
00
Benzene
0, 10
's
02
02
STAGE ¨4: Stage 3 compound 4 Mol wt: 326
MP range: 102-105 C
Sodium hydroxide 226g (2.83 X 2 moles) is added to 4 lit of Methanol taken in
a
20 lit glass reactor. To the clear solution of sodium methoxide, 197 ml (2.83
moles) of 100% Thioglycollic acid is added portion wise so that the
methanollic
solution reflux gently. Then the Stage-3 compound 925g (2.38 moles) is added
to
the methanolic solution in portions and the reaction mixture is subjected to
refluxion for about 5 hrs. The reaction is monitored by TLC. The reaction
mixture is cooled to room temperature under stirring. The cold water is added
to
the reactor to destroy methanol. This solution is neutralized with cold dilute
hydrochloric acid. A white crystalline solid separated is filtered and washed
with
water till no smell of thioglycollic acid is observed. The dry thioacetic acid
weighed 993g (93%). The melting range is 116-120 C.
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Remarks: 1. Na0H/CH3OH and NaSCH2COOH. This reaction is exothermic.
Hence, care should be taken to arrest the evaporation of CH3OH.
2. Plenty of cold water should be used to eliminate impurities
which interfere at this stage.
3. During neutralization the reactor should not develop
temperature.
4. Washing with water under vacuum is a must to remove the
unreacted thioglycollic acetic acid.
5. Work up should end without any smell of thioacetic acid.
HOT()
1
* Cl 110
SHCH2COOH 1
0 0
0, CH3OH/NaOH
0, 1.1
02 02
STAGE ¨5: Stage 4 compound 4 Mol wt: 381
MR: 116-120 degrees C
Stage 4 compound 993g (2.6062 moles) is taken in 4L of acetic acid contained
in
a 20L glass reactor. Hydrogen peroxide (30%) 3L is added to the contents of
the
reactor. The reactor is warmed just enough to dissolve Stage-4 compound in
acetic acid. The reactor is allowed at RT under stirring for 24 hrs. The
precipitate
formed in the reactor is separated, filtered, washed with water, and dried.
The
filtrate is diluted with cold water and the precipitate formed is filtered,
washed
with cold water, and dried. The two dried solids are added and weighed. The
weight of the solid is 937g (87%). This crude is further washed with benzene
to
give the pure product, which weighed 861g (80%). MP range 142-146 C.
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Remarks: 1. Acetic acid and Hydrogen peroxide are in the ratio of 5 : 3
to the
compound.
2. Reaction on heating is vigorous. Hence, dissolution of stage 4
compound should be done carefully under mild warming.
3. On completion of reaction the white crystalline solid separates
under stirring. For better quality, the precipitate and the filtrate
should be treated with cold water separately.
4. All the impurities carried over through all stages can be totally
removed by washing the precipitate with benzene. Hence the yield
of the pure compound varies from 80% to 90%.
OH OH
7(
1101 S7(
0 02 0
0 H202 0
0, AcOH O2 002S
STAGE ¨6: Stage 5 compound 4 Mol wt: 414
MP ranage: 142-146 degrees C
In a twenty litre glass reactor which is maintained in an oil bath is taken 8
lit of
benzene. To the contents of the flask 409g (2.0797 moles) of 2,4,6-trimethoxy
benzaldehyde is added and and the reaction mixture is stirred to dissolve the
aldehyde. To the aldehyde solution, 75g (0.6 moles) of benzoic acid and 60 ml
of
piperidine are added. Later Stage-5 compound 861g is added to the reaction
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mixture and the solution is refluxed. The Dean-Stark apparatus is fixed to the
reactor to remove the water, which forms during the reaction from time to
time.
Reaction is completed in 5 hrs. The reaction mixture is brought to the room
temperature under stirring. Crystalline substance starts forming and the
reaction
mixture is allowed to stand overnight to have good quality of the product. The
precipitate is filtered off and washed with benzene. The washings are
concentrated and the resulting precipitate is further washed with benzene. The
first and second crops were collected.
The melting range is 159-166 degrees C.
Remarks: 1. Stage 5 compound is soluble completely only after the
addition
of piperidine and at refluxing temparature.
2. Temperature more than 105 C may cause decarboxylation prior
to dehydration resulting in the formation of methyl benzyl sulfone
more in percentage. This will decrease the percentage of benzyl
stylyl sulfone.
3. Second crop should be washed with either ethylacetate or
Methanol to remove methylated compound.
0
OH
S 0 H
02 0
0\
0 Benzene 0 0
0
Benzoic acid
2
SO2 Piperidine 0
* S,0 SO2
STAGE ¨7: Stage 6 compound -4 Mol wt: 548
MP range: 159-166 C
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Stage-6 compound 740g (1.3503 moles) is taken in 7.4 lit of 20% sodium
hydroxide solution. To this 7.4 lit of methanol is added. The reactor is
heated to
reflux the solution under stirring. The compound is not completely soluble. As
time goes on the clear solution is obtained. TLC shows the completion of
reaction when the solution becomes very clear. The time taken for the
completion
of the reaction is 2 hrs. But to ensure complete conversion, stirring is
continued
further for a period of one to one and one half hours. The reaction mixture is
cooled to room temperature and the solution is neutralized with cool dilute
hydrochloric acid solution. The white (or light green) precipitate starts
separating. The precipitate is filtered, washed with water, and dried. The
crude
dry precipitate weighed 505 g (95%) which, has melting point range 124-127 C.
Remarks: 1. During work up ice and cold water should be used plenty to
get
neat crystalline compound. Otherwise a paste like compound
which sticks to the walls of the container will be obtained.
2. After completion of the reaction the reaction mixture should be
allowed to settle unreacted sulfone at the bottom. The solution
should be either decanted or allowed to pass through a filter paper.
The precipitate can be used along with the other batch of sulfone.
/
0 0.
--.. * .-
I 0 0'
20% NaOH __________________________________________ I 0
', * /
0
0 ).-
02
*0 Methanol 0
0 ,S SO2
0 HO SO2
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STAGE 8
Stage 7 compound 4 Mol wt: 394
MP range: 124-127 C
Stage-7 compound 500g (1.2690 moles) is taken for a 20L glass reactor.
Acetonitrile 5L is taken into the reactor. Stage-7 compound is not soluble
completely in acetomitrile. Carbon tetrachloride (AR) 790 ml (6 equivalents),
Triethylamine (AR) 921 ml (6 equivalents) and dimethylamino pyridine (0.1
equivalent) are added successively to the reactor under stirring. Stirring is
continued for about 10 minutes. Dibenzylphosphite, 421 ml (1.5 equivalent) is
added to the contents of the reactor at room temperature for a period of 30
minutes. During the addition, the the reaction mixture becomes clear solution,
and proceeds exothermically. By TLC analysis, the reaction is completed in an
hour after the addition. The reaction is allowed two more hours under stirring
and
allowed overnight.
To the contents of the reactor, 2.5L of 0.5M KH2PO4 solution is added and
stirred
5 minutes. The reaction mixture is allowed to settle. The organic layer is
separated and the aqueous layer is extracted with methylene chloride twice.
The
organic layers are combined and concentrated under vacuum. The viscous dense
organic layer is taken in the separating funnel. The heavy thick layer from
the
bottom is separated from the upper layer. The upper layer is again treated
with
DCM and the DCM layer is collected. The other layer mainly contains
impurities. Hence discarded. The thick organic layer and the DCM layer are
combined and concentrated once again. The yield is 829gy assumed as 100%.
Remarks: 1. Addition of DBP within 30 minutes generates heat in the
reactor. This heat energy is sufficient to move the reaction towards
right. Hence quick addition is needed.
2. All the impurities which are noticed at the base in TLC are
separated in the treatment of upper layer with DCM.
3. The viscous liquid can be used as it is for the next step. The
yield can be assumed as 100%.
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O____ O_'_=_
(1101 *
0 0
(C6H5CH20)P(0)H 0 0
0
SO2 TEA, CC14,DMAP 0. 0
I. SO2
HO 0
Bz0 OBz
0
STAGE 9 Stage 8 compound --> Mol wt: 626
MP range: dense liquid
Assuming 100% reaction in Stage-8, the syrupy compound is dissolved in 4L of
acetonitrile. To this solution 399 grams (2.1 equivalent) of sodium iodide is
added under stirring. The reactor is maintained in Nitrogen atmosphere and 344
ml of bromo/chloro trimethylsilane (2.1equivalent) is added drop wise in 45
minutes. The reaction mixture is stirred at room temperature. The reaction
mixture develops color (pink/red) during the addition of
bromo/chlorotrimethylsilane. Reaction completes in an hour after the addition
of
bromo/chlorotrimethylsilane. The reaction is further continued for about 2
hours.
To the reaction mixture, 4L of 1% hypo solution is added and the stirring is
maintained for 10 minutes. The organic layer is separated from the aqueous
layer.
The aqueous layer is washed twice with DCM. The combined organic layers are
concentrated under vacuum. The thick syrup that is left after concentration is
washed thoroughly with hexane and ethyl acetate successively. All the
impurities
are removed in washings. The compound is now left as pure and is subjected to
vacuum drying. Highly gummy semisolid is obtained. The isolated and pure
compound decomposes above 200 C. Assuming 100% reaction in Stage 9, the
compound is subjected to Stage 10.
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Remarks: 1. Bromotrimethylsilane/chlorotrimethylsilane is highly
hygroscopic and irritant. It should be drawn and injected into the
reactor with much caution.
2. Important ¨ During washing with hexane and ethyl acetate, the
fume hood should be closed for effective withdrawal of
lachrymatory vapors.
3. If stirring is not continuous, the reaction becomes stalemate.
0
0
1110
I 0
(CH3)3SiC1 [el
0
0
SO2 CH3CN =
0
0 SO2
=
Bz0== OBz
o HO OH
0
STAGE 10 Stage compound - Mol wt: 474
MP range: >200 C
Assuming 100% reaction in Stage-9, 601 gr. of the compound is dissolved in
7.5L
of 1,2- dimethoxyethane (Ethyleneglycol dimethylether). Sodium hydroxide
solution 1262 ml (8 % solution) is added in portions with vigorous stirring.
The
clear
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solution turns turbid and finally the precipitate comes out of the reaction
mixture.
At this stage, the pH is maintained between 7.0-7.5. The contents are stirred
for
four more hours. The reaction mixture is allowed to settle the precipitate.
The
precipitate is filtered and washed with acetone and dried under vacuum. The
dried compound weighs 510 grams (77%).
REMARKS: 1. The compound of Stage 9 is soluble in ether only with vigorous
shaking.
2. Addition of NaOH should be slow. For every addition, the RM
should be shaken thoroughly and each time PH should be checked
3. Before addition, the reaction mixture is acidic and is slowly
brought to 7.5 with NaOH solution.
4. Drying should be carried out under vacuum with temperature
not more than 30 C.
0 0
1101 *
0 0
8% NaOH I0 0
0
0
SO2 0 0
SO2
HO it OH Na0 ONa
0 0
25
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EXAMPLE 3
PHOSPHORYLATION OF COMPOUND 013100 USING POC13
The following Example is directed to the preparation of Compound
013105 by phosphorylation of Compound 013100 using POC13.
Scheme 7 below depicts the method for the synthesis of (E)-2,4,6-
(trimethoxystyry1)-3-0-phosphate di sodium-4-methoxybenzyl sulfone
(Compound 013105).
50 grams of compound 20 is dissolved in 500 ml of THF. To the solution
125 ml of Triethylamine is added. The solution is filtered to get the clear
solution.This is taken into a pressure equalizing dropping funnel.
Into a 2 lit three necked flask 50 ml of Phosphorous oxychloride in 150 ml
of THF is taken. The reactor is fitted with a mechanical stirrer and a reflux
condenser and kept in an ice bath to attain 0 C degree. To this cooled
solution, the
solution in the pressure equalizing funnel (Compound 013100 +Et3N+THF) is
added drop-wise for a period of 90 minutes (TLC shows the completion of
reaction).
The ice bath is allowed to attain room temperature and the stirring is
continued for 2 1/2 hrs. At this stage the reaction mixture develops light
yellow
color with the formation of water soluble precipitate.
The reaction mixture is again taken into the pressure equalizing dropping
funnel. Into the 2 lit flask 500 ml of ice cubes are taken. The flask is
cooled in an
ice bath. The reaction mixture is added to the ice cubes over a period of 45
minutes. After completion of the addition, stirring is continued for 5 to 7
hrs. To
the contents of the flask 200 ml of 50% potassium hydroxide solution (85%) is
added slowly. During the addition the flask is maintained in an ice bath.
After the
addition of KOH solution stirring is continued for a period of 40 minutes.
The reaction mixture is allowed to settle for the separation of organic
layer. The organic layer is separated and tested for the presence of Compound
013100 and the other impurity that appeared above the Compound 013100 in TLC
The aqueous layer is washed twice with THF (2 x 400 ml portions) and
each time the organic layer is tested for the impurities and discarded. At
this stage
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the aqueous layer is totally free from the impurities. Finally the aqueous
layer is
washed with 500 ml of ethyl acetate.
The aqueous layer is taken in a two litre RB which is maintained in an ice
bath. To this dilute HC1 solution (1:1, 135 ml) is added drop-wise under
moderate
stirring. During the addition the phosphoric acid of Compound 20 starts
separation as light yellow gummy solid. Stirring is continued for 3 1/2 hrs
to get
light yellow granulated solid. The solid compound is filtered dried and
weighed
(50 grams)
The solid compound is dissolved in 400 ml of methanol and the solution is
filtered to remove the insoluble and suspended impurities. The clear solution
is
taken in a conical flask equipped with a magnetic stirrer. The assembly is
kept in
a water bath. To the solution, 32 ml of 25% sodium hydroxide solution is added
drop-wise under stirring. During the addition a white phosphate salt formation
is
observed. After completion of the addition of NaOH solution, the PH of the
solution is found to be 8. Stirring is continued for 6 hrs for the completion
of
phosphate salt formation.
The phosphate salt is filtered and the conical flask is washed with 100 ml
of acetone to transfer on to the filter funnel. The precipitate is washed with
2 x
100 ml portions of acetone. Finally the salt is dried under vacuum. The dried
salt
weighed 50 gams. The Compound 013105 is found to be matching with the
standard sample.
30
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Scheme 7 : Method for the Synthesis of (E)-2,4,6-(Trimethoxystyry1)-3-0-
Phosphate Disodium-4-Methoxybenzyl Sulfone
0013
OCH3
ii3C0 OCH3
Ii3C0 OCH3
H3C0
H3C0
POCI3
OS 2
SO2 0 lir
if0 1
0****Cr ij KOH
HC1
0013
.ctis
HAW 0013
ii3C0 0083
Iva
H3co _ NaOH
SO2
SO2
0 lir ? Off
..,0Na
--0Na OH
The scope of the claims should not be limited by the preferred
embodiments set forth in the examples, but should be given the broadest
interpretation consistent with the description as a whole.
