U.S. patent application number 14/420487 was filed with the patent office on 2016-01-28 for 2,3'-anhydro-2'-deoxy-5-fluorouridine derivatives with cytotoxic activity, a manufacturing process and application.
This patent application is currently assigned to Adam Mickiewicz University. The applicant listed for this patent is ADAM MICKIEWICZ UNIVERSITY. Invention is credited to Lech CELEWICZ, Karol KACPRZAK, Marta LEWANDOWSKA, Piotr RUSZKOWSKI.
Application Number | 20160024133 14/420487 |
Document ID | / |
Family ID | 52778964 |
Filed Date | 2016-01-28 |
United States Patent
Application |
20160024133 |
Kind Code |
A1 |
CELEWICZ; Lech ; et
al. |
January 28, 2016 |
2,3'-ANHYDRO-2'-DEOXY-5-FLUOROURIDINE DERIVATIVES WITH CYTOTOXIC
ACTIVITY, A MANUFACTURING PROCESS AND APPLICATION
Abstract
The subject matter of the invention is novel
2,3'-anhydro-2'-deoxy-5-fluorouridine derivatives of general
formula 1 ##STR00001## where R is a trifluoromethyl,
2,2,2-trifluoroethyl, difluoromethyl, perfluoroethyl,
1-fluoroethyl, 2-fluoroethyl, 1,1 -difluoroethyl,
1,2-difluoroethyl, 2,2-difluoroethyl, 1,1,2-trifluoroethyl,
1,2,2-trifluoroethyl, 1,1,2,2-tetrafluoroethyl,
1,2,2,2-tetrafluoroethyl group. In a second aspect, the subject
matter of the invention is a process for the manufacture of
2,3-anhydro-2'-deoxy-5-fluorouridine derivatives of general formula
1. In a third aspect, the subject matter of the invention is an
application of 2,3'-anhydro-2'-deoxy-5-fluorouridine derivatives of
general formula 1 in the anticancer treatment of breast cancer,
cervical cancer, lung cancer and nasopharynx cancer.
Inventors: |
CELEWICZ; Lech; (Poznan,
PL) ; KACPRZAK; Karol; (Pecna, PL) ;
RUSZKOWSKI; Piotr; (Suchy Las, PL) ; LEWANDOWSKA;
Marta; (Srem, PL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ADAM MICKIEWICZ UNIVERSITY |
Poznan |
|
PL |
|
|
Assignee: |
Adam Mickiewicz University
Poznan
PL
|
Family ID: |
52778964 |
Appl. No.: |
14/420487 |
Filed: |
August 22, 2014 |
PCT Filed: |
August 22, 2014 |
PCT NO: |
PCT/PL2014/050050 |
371 Date: |
February 9, 2015 |
Current U.S.
Class: |
514/49 ;
536/26.8 |
Current CPC
Class: |
C07H 19/10 20130101;
A61P 35/00 20180101; C07F 9/6561 20130101; A61K 33/42 20130101;
C07F 9/24 20130101; C07H 19/073 20130101 |
International
Class: |
C07H 19/10 20060101
C07H019/10 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 24, 2014 |
PL |
P.408981 |
Claims
1. 2,3'-Anhydro-2'-deoxy-5-fluorouridine derivatives of general
formula 1 ##STR00011## where: --Ar is a phenyl, 4-chlorophenyl,
1-naphthyl, 2-naphthyl group or a phenyl group substituted at
position para, meta or ortho: with one F, Cl, Br or I atom, an
alkyl substituent containing from 1 to 12 carbon atoms, an alkoxy
group containing from 1 to 12 carbon atoms, a nitro or
trifluoromethyl group; or a phenyl group substituted at any
position with two identical or different substituents of the group
of F, Cl, Br or I, an alkyl substituent containing from 1 to 12
carbon atoms, an alkoxy group containing from 1 to 12 carbon atoms,
a nitro or trifluoromethyl group; and --R is a trifluoromethyl,
2,2,2-trifluoroethyl, difluoromethyl, perfluoroethyl,
1-fluoroethyl, 2-fluoroethyl, 1,1-difluoroethyl, 1,2-difluoroethyl,
2,2-difluoroethyl, 1,1,2-trifluoroethyl, 1,2,2-trifluoroethyl,
1,1,2,2-tetrafluoroethyl, 1,2,2,2-tetrafluoroethyl group.
2. A process for the preparation of
2,3'-anhydro-2'-deoxy-5-fluorouridine derivatives of general
formula 1 ##STR00012## where Ar and R are as defined in claim 1,
wherein the process comprises reaction of a triazolide of general
formula 2 ##STR00013## where Ar is a phenyl, 4-chlorophenyl,
1-naphthyl, 2-naphthyl group or a phenyl group substituted at
position para, meta or ortho: with one F, Cl, Br or I atom, an
alkyl substituent containing from 1 to 12 carbon atoms, an alkoxy
group containing from 1 to 12 carbon atoms, a nitro or
trifluoromethyl group; or a phenyl group substituted at any
position with two identical or different substituents of the group
of F, Cl, Br or I, an alkyl substituent containing from 1 to 12
carbon atoms, an alkoxy group containing from 1 to 12 carbon atoms,
a nitro or trifluoromethyl group; with fluorinated amines of
general formula 3 R--NH.sub.2 (3) where R is a trifluoromethyl,
2,2,2-trifluoroethyl, difluoromethyl, perfluoroethyl,
1-fluoroethyl, 2-fluoroethyl, 1,1-difluoroethyl, 1,2-difluoroethyl,
2,2-difluoroethyl, 1,1,2-trifluoroethyl, 1,2,2-trifluoroethyl,
1,1,2,2-tetrafluoroethyl, 1,2,2,2-tetrafluoroethyl group, or amine
salt of general formula 4 R--NH.sub.3.sup.+X.sup.- (4) where R is
as defined above, and X.sup.- is an inorganic anion of the group
Cl.sup.-, Br.sup.-, HSO.sub.4 .sup.-, SO.sub.4.sup.2-,
NO.sub.3.sup.-, in the presence of aliphatic amines.
3. A method for treating at least one cancer selected from the
group consisting of breast cancer, cervical cancer, lung cancer and
nasopharynx cancer, comprising administering to a patient inflicted
with the cancer a therapeutic formulation comprising
2,3'-anhydro-2'-deoxy-5-fluorouridine derivatives of general
formula 1 ##STR00014## where Ar is para-chlorophenyl or phenyl and
R is --CF.sub.3 or --CF.sub.2--CF.sub.3.
4. The method according to claim 3, wherein the cancer is breast
cancer and the 2,3'-anhydro-2'-deoxy-5-fluorouridine derivative of
general formula 1 is 2,3'-anhydro-2'-deoxy-5-fluorouridine
5'-[N-(2,2,2-trifluoroethyl)-O-(4-chlorophenyl)]phosphate.
5. The method according to claim 3, wherein the cancer is cervical
cancer and the 2,3'-anhydro-2'-deoxy-5-fluorouridine derivative of
general formula 1 is 2,3'-anhydro-2'-deoxy-5-fluorouridine
5'-[N-(2,2,2-trifluoroethyl)-O-(4-chlorophenyl)]phosphate.
6. The method according to claim 3, wherein the cancer is lung
cancer and the 2,3'-anhydro-2'-deoxy-5-fluorouridine derivative of
general formula 1 is 2,3'-anhydro-2'-deoxy-5-fluorouridine
5'-[N-(2,2,2-trifluoroethyl)-O-(4-chlorophenyl)]phosphate.
7. The method according to claim 3, wherein the cancer is
nasopharynx cancer and the 2,3'-anhydro-2'-deoxy-5-fluorouridine
derivative of general formula 1 is
2,3'-anhydro-2'-deoxy-5-fluorouridine
5'-[N-(2,2,2-trifluoroethyl)-O-(4-chlorophenyl)]phosphate.
Description
[0001] The subject matter of the invention is novel
2,3',-anhydro-2'-deoxy-5-fluorouridine derivatives and a process
for the manufacture thereof and also their application as cytotoxic
agents.
[0002] Cancer diseases are one of the principal health disorders
reported in humans, having the highest mortality rates and
increasing numbers of new cases, primarily related to the increased
life expectancy and to lifestyle. The treatment of cancer diseases
is difficult, expensive and in many cases not efficacious.
Therefore, there is an urgent need for novel substances with
cytostatic activity. They may be sourced from natural products and
their derivatives as well as constitute synthetic compounds.
[0003] Derivatives or analogues of purine or pyrimidine bases and
modified nucleosides are a very important group of synthetic
cytostatic agents. These include compounds, such as 5-fluorouracil
and its derivatives, e.g. 5-fluoro-2'-deoxyuridine (floxuridine).
Both 5-fluorouracil and 5-fluoro-2'-deoxyuridine have similar
cytostatic activity, and are since many years used in the treatment
of cancer, such as breast cancer, gastric cancer, colorectal
cancer, ovarian cancer and the like, either in monotherapy or in a
combination with other agents. 5-Fluoro-2'-deoxyuridine is also
used in the treatment of hepatic cancer owing to better hepatic
metabolism compared to 5-fluorouracil. Difficulties with using
5-fluorouracil and 5-fluoro-2'-deoxyuridine are related to the
development of cancerous cell resistance to those agents due to
their long-term intake. A significant limitation in the use of
5-fluorouracil is its relatively high toxicity resulted in
undesired neurotoxic and cardiotoxic effects. Because
5-fluorouracil and 5-fluoro-2'-deoxyuridine are not selective with
respect to cancerous and normal cells, their application in therapy
is considerably limited.
[0004] As shown by Srivastav, 2,3'-anhydro-2'-deoxy-5-fluorouridine
has weak antiviral activity against HBV and negligible cytotoxic
activity against the Huh-7 line (IC.sub.50>200 .mu.g/mL)
(Srivastav, M. Mak, B. Agrawal, D. L. J. Tyrrell, R. Kumar Bioorg.
Med. Chem. Lett., 2010, 20, 6790-6793).
[0005] The objective of the invention has been to develop novel
cytotoxic compounds being 2,3'-anhydro-2'-deoxy-5-fluorouridine
derivatives showing high selectivity with respect to cancer cells
and application thereof in cancer treatment.
[0006] The subject matter of the invention is
2,3'-anhydro-2'-deoxy-5-fluorouridine derivatives of general
formula 1
##STR00002##
where: [0007] Ar is a phenyl, 4-chlorophenyl, 1-naphthyl,
2-naphthyl group or a phenyl group substituted at position para,
meta or ortho with one F, Cl, Br or I atom, an alkyl substituent
containing from 1 to 12 carbon atoms, an alkoxy group containing
from 1 to 12 carbon atoms, a nitro or trifluoromethyl group; or a
phenyl group substituted at any position with two identical or
different substituents of the group of F, Cl, Br or I, an alkyl
substituent containing from 1 to 12 carbon atoms, an alkoxy group
containing from 1 to 12 carbon atoms, a nitro or trifluoromethyl
group; [0008] R is a trifluoromethyl, 2,2,2-trifluoroethyl,
difluoromethyl, perfluoroethyl, 1-fluoroethyl, 2-fluoroethyl,
1,1-difluoroethyl, 1,2-difluoroethyl, 2,2-difluoroethyl,
1,1,2-trifluoroethyl, 1,2,2-trifluoroethyl,
1,1,2,2-tetrafluoroethyl, 1,2,2,2-tetrafluoroethyl group.
[0009] In a second aspect, the subject matter of the invention is a
process for the manufacture of
2,3'-anhydro-2'-deoxy-5-fluorouridine derivatives of general
formula 1
##STR00003##
where Ar and R are as defined above, which involves a reaction of a
triazolide of general formula 2, where
##STR00004## [0010] Ar is a phenyl, 4-chlorophenyl, 1-naphthyl,
2-naphthyl group or a phenyl group substituted at position para,
meta or ortho with one F, Cl, Br or I atom, an alkyl substituent
containing from 1 to 12 carbon atoms, an alkoxy group containing
from 1 to 12 carbon atoms, a nitro or trifluoromethyl group; or a
phenyl group substituted at any position with two identical or
different substituents of the group of F, Cl, Br or I, an alkyl
substituent containing from 1 to 12 carbon atoms, an alkoxy group
containing from 1 to 12 carbon atoms, a nitro or trifluoromethyl
group; [0011] with fluorinated amines of general formula 3,
where
[0011] R--NH.sub.2 (3) [0012] R is a trifluoromethyl,
2,2,2-trifluoroethyl, difluoromethyl, perfluoroethyl,
1-fluoroethyl, 2-fluoroethyl, 1,1-difluoroethyl, 1,2-difluoroethyl,
2,2-difluoroethyl, 1,1,2-trifluoroethyl, 1,2,2-trifluoroethyl,
1,1,2,2-tetrafluoroethyl, 1,2,2,2-tetrafluoroethyl group [0013] or
amine salts of general formula 4,
[0013] R--NH.sub.3.sup.+X.sup.- (4)
where R is as defined above, and X.sup.- is an inorganic anion of
the group Cl.sup.-, Br.sup.-, HSO.sub.4.sup.-, SO.sub.4.sup.2-,
NO.sub.3.sup.- in the presence of aliphatic amines, preferably
triethylamine.
[0014] The reaction starts when a solution containing the
triazolide of general formula 2 is treated with fluorinated amine
of general formula 3 in a quantity of from 0.1 to 5 equivalents
with respect to the triazolide, most preferably 3 equivalents.
Alternatively, the reaction of the triazolide of general formula 2
may be carried out with fluorinated amine salts of general formula
4, most preferably with amine hydrochlorides. If so, a solution
containing the triazolide is treated with fluorinated aliphatic
amine salt of general formula 4 in a quantity of from 0.1 to 5
equivalents with respect to the triazolide, most preferably 3
equivalents, and a tertiary aliphatic amine or pyridine, most
preferably triethylamine, in a quantity of from 0.1 to 5
equivalents with respect to the triazolide, most preferably 3
equivalents. The further reaction procedure is identical
irrespective of whether an amine of general formula 3 or its salt
of general formula 4 is used. The reaction is conducted for 10
minutes to 10 hours, most preferably for 2 hours. The reaction
medium is a solvent of the group of lower aliphatic nitriles,
anhydrous DMF or DMSO. Most preferably it is acetonitrile. The
reaction proceeds at temperatures between 0.degree. C. and
70.degree. C.; due to practical reasons, however, the reaction is
most preferably carried out at room temperature. The resulting
product is isolated from the reaction mixture by removal of the
solvent and purification using column chromatography on silica gel,
preferably using a chloroform-methanol mixture as the mobile phase,
containing between 0.5% and 50% by volume of methanol, most
preferably 1%.
[0015] Considering time savings and the commercial availability of
reagents, a preferable option for the synthesis of
2,3'-anhydro-2'-deoxy-5-fluorouridine derivatives of general
formula 1 is to perform all reactions in single reaction vessel (so
called one-pot synthesis). If so, the synthesis consists of three
steps carried out one by one so that intermediates do not have to
be isolated, according to Scheme 1.
##STR00005##
[0016] In step 1, an aryldichlorophosphate of general formula 5 is
reacted,
##STR00006##
where Ar is a phenyl, 4-chlorophenyl, 1-naphthyl, 2-naphthyl group
or a phenyl group substituted at position para, meta or ortho with
one F, Cl, Br or I atom, an alkyl substituent containing from 1 to
12 carbon atoms, an alkoxy group containing from 1 to 12 carbon
atoms, a nitro or trifluoromethyl group; or a phenyl group
substituted at any position with two identical or different
substituents of the group of F, Cl, Br or I, an alkyl substituent
containing from 1 to 12 carbon atoms, an alkoxy group containing
from 1 to 12 carbon atoms, a nitro or trifluoromethyl group, with
1,2,4-triazole in a quantity of from 2 to 4 equivalents, most
preferably three equivalents, in the presence of triethylamine in a
quantity of from 2 to 4 equivalents, most preferably 2.5
equivalents, in a solvent of the group of lower aliphatic nitrites,
anhydrous DMF or DESO, most preferably in acetonitrile, e.g.
following a procedure reported in Lewandowska, M., Ruszkowski, P.,
Baraniak, D., Czarnecka, A., Kleczewska, N., Celewicz, L. (2013)
Eur. J. Med. Chem., 2013, 67, 188-195. This step results in a
formation of a bis-triazolide of general formula 6
##STR00007##
where Ar is as defined above.
[0017] In step 2, the resulting bis-triazolide of general formula 6
is reacted with 2,3'-anhydro-2'-deoxy-5-fluorouridine (AdFU) which
forms a triazolide of general formula 2, where Ar is as defined
above.
[0018] This step is conducted by adding
2,3'-anhydro-2'-deoxy-5-fluorouridine (AdFU) to the reaction
mixture in a quantity of from 0.1 to 1 equivalent, most preferably
0.66 equivalent, with respect to the aryldichlorophosphate of
general formula 5 used in step 1. An amine, most preferably
pyridine, is a necessary additive used in this step which improves
AdFU solubility, used in a quantity of from 1 to 3 equivalents with
respect to AdFU, most preferably 2 equivalents. The process stage
is conducted for 10 minutes to 10 hours, preferably for 3 hours and
most preferably for one hour.
[0019] In step 3, the triazolide of general formula 2 obtained in
step 2 is reacted with fluorinated amines of general formula 3 or
salts thereof of general formula 4, most preferably with
hydrochlorides, in a presence of triethylamine to yield the final
product of general formula 1. This step is conducted by adding a
fluorinated amine of general formula 3 to the reaction mixture
containing the resulting compound of general formula 2, in a
quantity of from 0.1 to 5 equivalents with respect to
2,3'-anhydro-2'-deoxy-5-fluorouridine (AdFU) used in step 2, most
preferably 3 equivalents. Alternatively, the reaction of the
triazolide of general formula 2 prepared in step 3 may be carried
out with fluorinated amine salts of general formula 4, most
preferably with hydrochlorides. If so, a fluorinated aliphatic
amine salt of general formula 4 is added to a solution containing
the triazolide of general formula 2 in a quantity of from 0.1 to 5
equivalents with respect to 2,3'-anhydro-2'-deoxy-5-fluorouridine
(AdFU) used in step 2, most preferably 3 equivalents, and a
tertiary aliphatic amine or pyridine, most preferably
triethylamine, in a quantity of from 0.1 to 5 equivalents with
respect to 2,3'-anhydro-2'-deoxy-5-fluorouridine (AdFU), most
preferably 3 equivalents. The further reaction procedure is
identical irrespective of whether an amine of general formula 3 or
its salt of general formula 4 is used. The reaction is conducted
for 10 minutes to 10 hours, most preferably for 2 hours at
temperatures between 0.degree. C. and 70.degree. C.; due to
practical reasons, however, the reaction is most preferably carried
out at room temperature. The resulting product is isolated from the
reaction mixture by removal of the solvent and purified using
column chromatography on silica gel, preferably using a
chloroform-methanol mixture as the mobile phase, containing between
0.5% and 50% by volume of methanol, most preferably 1%.
[0020] In a third aspect, the subject matter of the invention is an
application of 2,3'-anhydro-2'-deoxy-5-fluorouridine derivatives of
general formula 1, where Ar is para-chlorophenyl or phenyl and R is
--CF.sub.3 or --CF.sub.2--CF.sub.3, in particular
2,3'-anhydro-2'-deoxy-5-fluorouridine
5'-[N-(2,2,2-trifluoroethyl)-O-(4-chlorophenyl)]phosphate--FEAdFU
of the invention in the treatment of breast cancer, cervical
cancer, lung cancer or nasopharynx cancer. In vitro studies on
cancer cell lines of breast cancer, cervical cancer, lung cancer
and nasopharynx cancer confirmed strong cytotoxic effects with
activity higher than that of 2'-deoxy-5-fluorouridine (5FdU) and
5-fluorouracil already employed in therapy, when tested in
identical conditions. Furthermore, FEAdFU has a favourable and high
selectivity index (SI) which shows that it has high and selective
cytotoxicity against cancer cells with low toxicity against normal
cells.
[0021] Cytotoxic activity tests were performed using the following
cancer cell lines: MCF-7 (breast cancer), HeLa (cervical cancer)
A549 (lung cancer) and KB (nasopharynx cancer) and normal cells
(human dermal fibroblasts, HDF) obtained from ECACC (European
Collection of Cell Cultures).
[0022] Cytotoxicity tests were carried out using a standard
procedure with sulphorhodamine B. They involved incubation of the
cancer cell lines in the logarithmic growth phase for 72 hours with
the compound tested and, subsequently, spectrophotometric
determination of the degree of cell growth inhibition using
adsorption of a dye (sulphorhodamine B) which binds cellular
proteins. The determination was carried out according to a
procedure reported in: Vichai, V., Kirtikara, K. Nature Protocols,
2006, 1, 1112.
[0023] Determination of Cytotoxicity
[0024] Preparation of Cells for the Experiment:
[0025] Cells of the cell line tested in the logarithmic growth
phase were seeded onto 24-well plates in a quantity of 20.000
cells/2 mL of the growth medium per well and, subsequently,
incubated in an incubator at 37.degree. C., in the 5% CO.sub.2
atmosphere for 24 hours.
[0026] Solutions of the test compounds were prepared in DMSO in the
following concentrations: 0.05; 0.1; 0.5; 1; 5; 10; 50; 100; 500
.mu.M.
[0027] The cells of the lines tested were treated with the
solutions of the test compounds in a laminar-flow chamber which
ensured sterile working conditions according to the following
procedure: the first three wells were used as a control: they
contained 20 .mu.L of DMSO only; successive solutions of the test
compound were added to subsequent wells (20 .mu.L), starting with
the lowest concentration (three wells for each concentration
level). Subsequently, the plates were placed in an incubator at
37.degree. C. for 72 hours. After the end of incubation, the
adhered cells were fixed by adding 500 .mu.L of cold (4.degree. C.)
50% trichloroacetic acid (TCA) and incubated at 4.degree. C. for 1
hour. Subsequently, each well was rinsed with sterile water and
dried. The rinsing was repeated five times. The fixed cells were
stained for 30 minutes by adding 500 .mu.L of 0.4% dye solution
(sulphorhodamine B) dissolved in 1% acetic acid. Any unbound dye
was removed by decanting it from the plate, and the cells were
washed 4 times with 1% acetic acid. Subsequently, the plates were
dried in air for approx. 5 minutes. Bounded dye was dissolved by
adding 1500 .mu.L of 10 mM Tris-base buffer
(trishydroxymethylaminomethane) to each well and shaken using an
orbital shaker for 5 minutes. Subsequently, 200 .mu.L of solution
from each well was transferred to each of two wells on a new
96-well plate and absorption of the solutions was determined
spectrophotometrically at a wavelength of 490-530 nm using a plate
reader. Percentage inhibition of cell growth by the test compound
was calculated assuming the absorption of the control solution as
100%.
[0028] Cytotoxicity tests for the other compounds and cell lines
were performed following an identical procedure.
[0029] Depending on the type of the cell line, the following growth
media were used:
[0030] The MCF-7 line was grown in Dulbecco's Modified Eagle's
Medium (DME) from Sigma (cat. no. D5796), while the HeLa, A549 and
KB lines were grown in RPMI-1640 Medium from Sigma (cat. no.
R8758).
[0031] IC.sub.50 values, being concentration of a compound needed
to obtain 50% inhibition of cell growth, were determined for all
the derivatives tested. Derivatives for which IC.sub.50<4
.mu.g/mL are generally assumed as active (abbreviated as A),
derivatives with values in an IC.sub.50 range of 4-30 .mu.g/mL are
considered medium active (abbreviated as MA), while those for which
IC.sub.50>30 .mu.g/mL are considered non-active (abbreviated as
NA).
[0032] To enable comparison, identical tests were performed using
known cytotoxic agents: 5-fluoro-2'-deoxyuridine and
5-fluorouracil.
[0033] In addition, selectivity indexes (SI) were calculated for
FEAdFU, defined as: SI=IC.sub.50 for a normal cell line (HDF
fibroblasts)/IC.sub.50 for a cancer cell line. A favourable high
selectivity index (SI) shows that the activity of a compound
against cancer cells is higher than its toxicity against normal
cells.
[0034] The results of cytotoxic activity tests for the compounds of
general formula 1 are shown in Table 1. The values are average
results of three independent determinations.
TABLE-US-00001 TABLE 1 Cytotoxic activity, IC.sub.50 Line MCF-7
HeLa A549 KB Fibroblasts (breast cancer) (cervical cancer) (lung
cancer) (nasopharynx cancer) (HDF) Compound [.mu.g/mL] [.mu.M] SI
[.mu.g/mL] [.mu.M] SI [.mu.g/mL] [.mu.M] SI [.mu.g/mL] [.mu.M] SI
[.mu.g/mL] [.mu.M] FEAdFU 0.12 (A) 0.24 50 0.18 (A) 0.36 33.3 0.2
(A) 0.4 30 0.2 (A) 0.4 30 6.0 12.0 MFEAdFU 11.8 (MA) 25.4 3.7 16.0
(MA) 34.5 2.7 -- -- -- 12.2 (MA) 26.3 3.5 43.1 92.9
2,3'-anhydro-2'- 3.7 (A) 16.22 -- 11.0 (MA) 48.21 -- -- -- -- 11.0
(MA) 48.21 -- -- -- deoxy-5- fluorouridine (AdFU) 5-fluoro-2'- 2.81
(A) 11.4 -- 3.20 (A) 13.0 -- 3.30 (A) 13.4 -- 3.37 (A) 13.7 -- --
-- deoxyuridine 5-fluorouracil 2.37 (A) 18.2 -- 2.73 (A) 21.0 --
2.78 (A) 21.4 -- 2.86 (A) 22.0 -- -- --
[0035] The cytotoxicity of 2,3'-anhydro-2'-deoxy-5-fluorouridine
5'-[N-(2,2,2-trifluoroethyl)-O-(4-chlorophenyl)]phosphate (FEAdFU)
being the subject matter of the application was tested as highly
active. Furthermore, the compound has a high selectivity index
(SI), defined above, which shows highly selective activity against
cancer cells with low activity against normal cells
(fibroblasts).
[0036] The cytotoxicity and selectivity index of the MFEAdFU
derivative, where Ar is para-chlorophenyl and R is
--CH.sub.2--CH.sub.2F, are considerably lower with respect to
FEAdFU.
[0037] The subject matter of the invention is in particular the
application of FEAdFU for the manufacture of drugs used in breast
cancer chemotherapy. It was confirmed in the tests that FEAdFU had
very high activity against breast cancer cells (MCF-7 line), with
an IC.sub.50 of 0.24 .mu.M and more than 47-fold higher activity
than 5FdU and more than 75-fold higher activity than 5FU. It also
had 67-fold higher activity than the original
2,3'-anhydro-2'-deoxy-5-fluorouridine (AdFU). Furthermore, the
compound had a very high selectivity index (SI=50) which showed
effective activity against cancer cells of that line and low
activity against fibroblasts.
[0038] In another aspect, the subject matter of the invention is in
particular the application of FEAdFU for the manufacture of drugs
used in cervical cancer chemotherapy. It was confirmed in the tests
that FEAdFU had very high activity against cervical cancer cells
(HeLa line), with an IC.sub.50 of 0.36 .mu.M and more than 36-fold
higher cytotoxicity than 5FdU, more than 58-fold higher than 5FU
and more than 133-fold higher than AdFU. Furthermore, the compound
had a very high selectivity index (SI=33) which showed effective
activity against cancer cells of that line and low activity against
fibroblasts.
[0039] In another aspect, the subject matter of the invention is in
particular the application of FEAdFU for the manufacture of drugs
used in lung cancer chemotherapy. It was confirmed in the tests
performed that FEAdFU had very high activity against lung cancer
cells (A549 line), with an IC.sub.50 of 0.4 .mu.M, and more than
33-fold higher cytotoxicity than 5FdU and more than 53-fold higher
than 5FU. Furthermore, the compound had a very high selectivity
index (SI=30) which showed effective activity against cancer cells
of that line and low activity against fibroblasts.
[0040] In the final aspect, the subject matter of the invention is
in particular the application of FEAdFU for the manufacture of
drugs used in nasopharynx cancer chemotherapy. It was confirmed in
the tests that FEAdFU had very high activity against nasopharynx
cancer cells (KB line), with an IC.sub.50 of 0.4 .mu.M, and more
than 34-fold higher cytotoxicity than 5FdU, more than 55-fold
higher than 5FU and 120-fold higher than AdFU. Furthermore, the
compound had a very high selectivity index (SI=30) which showed
effective activity against cancer cells of that line and low
activity against fibroblasts.
[0041] The subject matter of the invention will be explained using
certain embodiments which illustrate the invention, without
limiting its scope.
[0042] Solvents and other chemical reagents were obtained from
Aldrich, Merck and POCh and used as received. Column chromatography
was performed with silica gel 60H used as the stationary phase
(0.045-0.075 mm/200-300 mesh) from Merck.
[0043] .sup.1H NMR, .sup.13C NMR and .sup.19F NMR spectra of the
compounds were recorded using Varian-Gemini (300 MHz) and Bruker
Avance (600 MHz) spectrometers with the following internal
standards: tetramethylsilane (TMS) when recording .sup.1H NMR and
.sup.13C NMR spectra and trichlorofluoromethane (CFCl.sub.3) for
.sup.19F NMR spectra.
EXAMPLE 1
[0044] Synthesis of FEAdFU
##STR00008##
2,3'-Anhydro-2'-dideoxy-5-fluorouridine (1.07 mmol, 0.244 g) in
pyridine (4 mL) was added to a solution of
4-chlorophenyldi(1,2,4-triazole)phosphate (1.07 mmol, 0.332 g) in
acetonitrile (4 mL). The reaction was stirred at room temperature
for 1 hour; subsequently, 2,2,2-trifluoroethylamine hydrochloride
(5.35 mmol, 0.725 g) and triethylamine (2.0 mL) were added to the
reaction mixture and the reaction was stirred at room temperature
for 1 hour. Reaction progress was monitored using thin-layer
chromatography (TLC) with CHCl.sub.3/MeOH (10:1) as the eluent.
When the substrates were completely consumed, saturated NaHCO.sub.3
solution (10 mL) was added to the reaction mixture, followed by
extraction with chloroform. The organic phase was dried over
anhydrous MgSO.sub.4 and evaporated twice with toluene to remove
any traces of pyridine. The resulting product was purified on a
chromatographic column with silica gel using a chloroform/methanol
mixture (100:1, v/v) as the eluent. Yield: 40%.
[0045] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 2.33-2.40 (m,
2H, N--CH.sub.2); 2.49-2.53 (m, 2H, H-2', H-2''); 3.03 (m, 1H,
H-4'); 3.46 (m, 2H, H-5',H-5''); 4.22-4.35 (m, 1H, H-3'); 5.93
(pseudo t, 1H, J=5.8 Hz, H-1'); 6.45 (m, 1H, NH--C--C); 7.23, 7.27
(d, 2H, J=8.7 Hz, 4-ClPh); 7.43, 7.48 (d, 2H, J=8.7 Hz, 4-ClPh);
8.14, 8.17 (d, 1H, J=5.2 Hz, H-6).
[0046] .sup.13C NMR (DMSO-d.sub.6): .delta.31.26, 43.42, 59.38,
77.52, 85.42, 87.34, 122.12, 122.58, 125.59, 129.72, 139.18,
144.27, 149.12, 151.70, 162.93.
[0047] .sup.19F NMR (DMSO-d.sub.6): .delta. -158.26 (d, 1F, J=5.0
Hz); -71.40, -71.36 (t, 3F, J=10.0 Hz, N--C--CF.sub.3).
[0048] .sup.31P NMR (DMSO-d.sub.6) .delta.5.05; 5.20.
[0049] MS-ESI m/z: 500, 502 [M+H].sup.+; 522, 524 [M+Na].sup.+;
538, 540 [M+CL].sup.-. 498, 500 [M-H].sup.-; 534, 536, 538
[M+Cl].sup.-.
EXAMPLE 2
[0050] Synthesis of FEAdFU--one-pot procedure
##STR00009##
[0051] 1,2,4-Triazole (5.53 mmol, 0.382 g) and triethylamine (0.62
mL) were added to a solution of 4-chlorophenyldichlorophosphate
(1.84 mmol, 0.452 g) in acetonitrile (4.5 mL). The reaction was
stirred at room temperature for 30 minutes. After the end of the
first step, 2,3'-anhydro-2'-dideoxy-5-fluorouridine (0.88 mmol,
0.200 g) and pyridine (4.5 mL) were added to the reaction mixture.
The reaction was stirred at room temperature for 1 hour.
Subsequently, 2,2,2-trifluoroethylamine hydrochloride (2.64 mmol,
0.120 g) and triethylamine (1.5 mL) were added to the reaction
mixture and the reaction was stirred at room temperature for 2
hours. Reaction progress was monitored using thin-layer
chromatography (TLC) with CHCl.sub.3/MeOH (10:1) as the eluent.
When the substrates were completely consumed, saturated NaHCO.sub.3
solution (10 mL) was added to the reaction mixture, followed by
extraction with chloroform. The organic phase was dried over
anhydrous MgSO.sub.4 and evaporated twice with toluene to remove
any traces of pyridine. The resulting product was purified on a
chromatographic column with silica gel using a chloroform/methanol
mixture (100:1, v/v) as the eluent. Yield: 42%.
[0052] The product had identical spectral characteristics as that
discussed in Example 1.
EXAMPLE 3
[0053] Synthesis of MFEAdFU
##STR00010##
[0054] Using a procedure identical as in Example 2, a reaction
between phosphorylated 2,3'-anhydro-2'-dideoxy-5-fluorouridine
(0.88 mmol, 0.200 g) and 2-fluoroethylamine hydrochloride (263 mg;
2.64 mmol) was performed. Following chromatographic purification, a
product (MFEAdFU) was obtained with 34% yield.
[0055] .sup.1H NMR (DMSO-d.sub.6) .delta.: 2.56-2.75 (m, 2H, H-2',
H-2''), 3.00-3.23 (m, 2H, N--CH.sub.2--C), 3.87-4.20 (m, 1H, H-4'),
4.54 (m, 2H, H-5', H-5''), 4.68-4.79 (m, 2H, N--C--CH.sub.2), 5.54
(m, 1H, H-3'), 6.04 (pseudo t, J=6.2 Hz, 1H, H-1'), 6.72 (m, 1H,
NH--C--C), 7.24, 7.28 (d, 2H, J=8.6 Hz, 4-ClPh), 7.44, 7.48 (d, 2H,
J=8.6 Hz, 4-ClPh), 8.15, 8.18 (d, 1H, J=5.6 Hz, H-6), 11.99 (br s,
1H, 3-NH).
[0056] .sup.13C NMR (DMSO-d.sub.6) .delta.: 33.12, 45.51, 63.89,
77.12, 82.48, 83.24, 87.58, 121.74, 125.97 (d, J.sub.C--F=37.3 Hz),
128.12, 131.07, 143.74 (d, J.sub.C--F=232.3 Hz), 149.05, 151.48,
162.99 (d, J.sub.C--F=26.0 Hz).
[0057] .sup.19F NMR (DMSO-d.sub.6) .delta.: -71.17 (m, 1F), -158.19
(m, 1F).
[0058] .sup.31P NMR (DMSO-d.sub.6) .delta. 5.96; 6.01.
[0059] MS-ESI m/z: 464, 466 [M+H].sup.+; 486, 488 [M+Na].sup.+;
502, 504 [M+K].sup.+; 462, 464 [M-H].sup.-; 498, 500, 502
[M+Cl].sup.-.
* * * * *