U.S. patent application number 14/127675 was filed with the patent office on 2014-05-22 for compounds for the treatment of cancers associated with human papillomavirus.
This patent application is currently assigned to PIRAMAL ENTERPRISES LIMITED. The applicant listed for this patent is Veena R Agarwal, Gandhali Ashwin Deshpande, Muralidhara Padigaru, Swati Ajay Piramal. Invention is credited to Veena R Agarwal, Gandhali Ashwin Deshpande, Muralidhara Padigaru, Swati Ajay Piramal.
Application Number | 20140142159 14/127675 |
Document ID | / |
Family ID | 46579265 |
Filed Date | 2014-05-22 |
United States Patent
Application |
20140142159 |
Kind Code |
A1 |
Piramal; Swati Ajay ; et
al. |
May 22, 2014 |
COMPOUNDS FOR THE TREATMENT OF CANCERS ASSOCIATED WITH HUMAN
PAPILLOMAVIRUS
Abstract
The present invention relates to the pyrrolidine substituted
with flavone derivatives, represented by the compounds of Formula
(I) ##STR00001## or a pharmaceutically acceptable salt, a solvate,
a stereoisomer or a diastereoisomer thereof for use in the
treatment of cancers associated with human papillomavirus. The
present invention also relates to the pharmaceutical compositions
containing the compounds of Formula (I) for the treatment of
cancers associated with human papillomavirus.
Inventors: |
Piramal; Swati Ajay;
(Mumbai, IN) ; Padigaru; Muralidhara; (Mumbai,
IN) ; Agarwal; Veena R; (Mumbai, IN) ;
Deshpande; Gandhali Ashwin; (Mumbai, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Piramal; Swati Ajay
Padigaru; Muralidhara
Agarwal; Veena R
Deshpande; Gandhali Ashwin |
Mumbai
Mumbai
Mumbai
Mumbai |
|
IN
IN
IN
IN |
|
|
Assignee: |
PIRAMAL ENTERPRISES LIMITED
Mumbai
IN
|
Family ID: |
46579265 |
Appl. No.: |
14/127675 |
Filed: |
June 22, 2012 |
PCT Filed: |
June 22, 2012 |
PCT NO: |
PCT/IB2012/053166 |
371 Date: |
December 19, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61500799 |
Jun 24, 2011 |
|
|
|
Current U.S.
Class: |
514/422 |
Current CPC
Class: |
A61P 35/00 20180101;
C07D 405/04 20130101; A61K 31/4025 20130101; A61P 31/20 20180101;
A61K 45/06 20130101 |
Class at
Publication: |
514/422 |
International
Class: |
C07D 405/04 20060101
C07D405/04 |
Claims
1. A method for the treatment of human papillomavirus (HPV)
associated cancer comprising administering to the subject in need
thereof a therapeutically effective amount of a compound of Formula
(I), or a pharmaceutically acceptable salt, a solvate, stereoisomer
or a diastereoisomer thereof; ##STR00009## wherein Ar is a phenyl
group, which is unsubstituted or substituted by 1, 2, or 3
identical or different substituents selected from: halogen; nitro,
cyano, C.sub.1-C.sub.4-alkyl, trifluoromethyl, hydroxyl and
C.sub.1-C.sub.4-alkoxy.
2. The method according to claim 1 wherein, in the compound of
Formula (I) Ar is a phenyl group substituted by 1, 2 or 3 identical
or different substituents selected from chlorine, bromine,
fluorine, iodine, C.sub.1-C.sub.4-alkyl and trifluoromethyl.
3. The method according to claim 1 wherein, in the compound of
Formula (I) Ar is a phenyl group substituted by chlorine.
4. The method according to claim 1 wherein, in the compound of
Formula (I) Ar is a phenyl group substituted by chlorine and
trifluoromethyl.
5. The method according to claim 1, wherein the compound of Formula
(I) is a (+)-trans isomer represented by Formula (IA), or a
pharmaceutically acceptable salt or a solvate thereof; ##STR00010##
wherein Ar is a phenyl group, which is unsubstituted or substituted
by 1, 2, or 3 identical or different substituents selected from
halogen, nitro, cyano, C.sub.1-C.sub.4-alkyl, trifluoromethyl,
hydroxyl or C.sub.1-C.sub.4-alkoxy.
6. The method according to claim 5, wherein the compound of Formula
(IA) is
(+)-trans-2-(2-Chlorophenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-
pyrrolidin-3-yl)-chromen-4-one hydrochloride (compound A).
7. The method according to claim 5, wherein the compound of Formula
(IA) is
(+)-trans-2-(2-Chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydr-
oxymethyl-1-methylpyrrolidin-3-yl)-chromen-4-one hydrochloride
(compound B).
8. The method according to claim 1, wherein the HPV associated
cancer is selected from anal cancer, vulvar cancer, vaginal cancer,
penile cancer, cervical cancer, oropharyngeal cancer, cancer of the
oral cavity, lung cancer, non-melanoma skin cancer and cancer of
the conjunctiva.
9. The method according to claim 8, wherein the HPV associated
cancer is cervical cancer.
10. A method for the treatment of human papillomavirus (HPV)
associated cancer comprising administering to the subject in need
thereof a therapeutically effective amount of a pharmaceutical
composition comprising a therapeutically effective amount of the
compound of Formula (I) as defined in claim 1, or a
pharmaceutically acceptable salt, a solvate, a stereoisomer or a
diastereoisomer thereof and a pharmaceutically acceptable
carrier.
11. The method according to claim 10, wherein the pharmaceutical
composition comprises at least one further pharmaceutically active
compound, wherein the pharmaceutically active compound has
anticancer activity.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the pyrrolidine substituted
with flavone derivatives, represented by the compounds of Formula
(I) (as described herein) or pharmaceutically acceptable salts,
solvates, stereoisomers or diastereoisomers thereof for use in the
treatment of cancers associated with human papillomavirus (HPV).
The present invention also relates to pharmaceutical compositions
containing the compounds of Formula (I) for use in the treatment of
cancers associated with human papillomavirus.
BACKGROUND OF THE INVENTION
[0002] Human papillomavirus (HPV) is a circular, non-enveloped
dsDNA virus that infects squamous epithelial cells. HPV enters the
body, usually through a break in the skin, and then infects the
cells in the layers of the skin. HPV is transmitted by skin-to-skin
contact. HPV infections can be acquired through a cut or through
sexual activity with an infected person. This includes kissing or
touching the skin of the infected areas and having intercourse. A
mother with a genital HPV infection may also transmit the virus to
the infant during labour.
[0003] HPVs are a group of more than 120 related viruses, 33% of
which are known to infect genital tract (Microbiology and Molecular
Biology Reviews, 2004, 68 (2):362-372). Certain types of human
papillomavirus are able to transform normal cells into abnormal
ones which can go on to form cancer. Accordingly, these viruses are
classified as high-risk types and low-risk types. High-risk HPVs
are associated with cancers. Genital HPV infections are very common
and can lead to anogenital cancers. Persistent infections with
high-risk HPVs are the primary cause of cervical cancer. HPV
infections also cause anal cancer, vulvar cancer, vaginal cancer
and penile cancer (Int. J. Cancer, 2006, 118(12): 3030-44). The
high risk subtypes are HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56,
58, 59, 66 and 68, the most common being HPV 16, 18, 31, 33 and 45.
Further, several types of HPVs, particularly type 16, have been
found to be associated with HPV-positive oropharyngeal cancer, a
form of head and neck cancer (N. Engl. J. Med., 2007,
356(19):1944-56).
[0004] High risk HPVs produce two oncoproteins, E6 and E7, which
are necessary for viral replication. During the HPV infection in
humans, the HPV E6 protein binds and promotes the degradation of
tumor suppressor p53 by an ubiquitin-mediated pathway diminishing
the ability of the cell to undergo apoptosis. The HPV E7 protein
binds and degrades the retinoblastoma protein (pRb), preventing it
from inhibiting the transcription factor E2F, resulting in loss of
cell cycle control.
[0005] It has been estimated that HPV accounts for approximately 5%
of all cancers worldwide (Int. J. Cancer, 2006,
118(12):3030-3044).
[0006] Cancer of the cervix uteri is the second most common cancer
among women worldwide. About 86% of the cases occur in developing
countries. Cervical cancer accounts to 13% of the cancers occurring
in females (World: Human Pappilomavirus and related cancers,
summary report, November 2010).
[0007] Persistent Human papillomavirus (HPV) infections are now
recognized as the cause of essentially all cervical cancers.
According to the American Cancer Society, in 2010, about 12,000
women in the United States would be diagnosed with this type of
cancer and more than 4,000 would die from it. Cervical cancer is
diagnosed in nearly half a million women each year worldwide,
claiming a quarter of a million lives annually.
[0008] Vulvar and Vaginal cancers account to about 3 to 5% and 1-2%
respectively of all gynecologic cancers and penile cancers accounts
to about 0.2% of all cancers in the United States. Despite their
infrequency, vulvar, vaginal and penile cancers remain important
diseases, because of their significant impact on sexuality. Though
there is no single etiologic factor, there is a strong association
with HPV infection. HPV is thought to be responsible for about 40%
of penile cancers. Many studies have shown the presence of HPV
types 16 and 18 in penile carcinoma (Hum. Pathol., 1991,
22.908-913). HPV is also responsible for about 65% of vaginal
(International Journal of Cancer, 2009, 124(7):1626-1636) and 50%
of vulvar cancers (Vaccine, 2006, 24(suppl 3): S11-S25) and HPV-16
accounted for most HPV positive cases for both the cancers (Obstet.
Gynecol., 2009, 113(4):917-24).
[0009] HPV infection is also associated with anal cancer. It is
estimated that about 1,600 new cases of HPV-associated anal cancers
are diagnosed in women and about 900 are diagnosed in men each year
in the United States. In general, HPV is thought to be responsible
for about 90% of anal cancers (International Journal of Cancer,
2009, 124(7):1626-1636). Notably HPV 16 seems to be responsible for
most of the anal cancer. According to a study HPV 16, was detected
in 84 percent of anal cancer specimens examined (New England
Journal of Medicine, 1997, 337(19):1350-8).
[0010] Cancer of head and neck typically refers to squamous cell
carcinomas of the head and neck. Head and neck cancers account for
approximately 3 percent of all cancers in the United States (A
Cancer Journal for Clinicians, 2010, 60(5): 277-300). Head and neck
cancers are identified by the area in which they begin. They are
typically classified as: cancers of oral cavity, salivary gland,
paranasal sinuses, nasal cavity, pharynx, nasopharynx, oropharynx,
hypopharynx, larynx and lymph nodes in the upper part of the neck.
Cancer of the oral cavity (the front two-thirds of the tongue, the
gingiva, the buccal mucosa, the floor of the mouth under the
tongue, the hard palate, and the small area behind the wisdom
teeth) and cancer of the oropharynx (the soft palate, the base of
the tongue, and the tonsils) are the most common types of cancer
caused by HPV. Studies have shown that about 60% of oropharyngeal
cancers are caused by HPV (Cancer Epidemiology, Biomarkers and
Prevention, 2005, 14(2):467-475), in particular HPV16, is a causal
factor for some head and neck squamous cell carcinoma (HNSCC).
[0011] HPV has also been associated with lung carcinomas. According
to the published articles the incidence of HPV in lung cancer was
24.5% (Lung Cancer, 2009, 65: 13-18). A study conducted in China
revealed that the risk of lung squamous cell carcinomas was 3.5
times higher among HPV-positive population compared with the
HPV-negative population and 16.9 times higher for patients with
positive HPV-16 than negative HPV-16 (Oncol. Rep., 2009,
21(6):1627-32).
[0012] A role for human papillomaviruses has also been proposed in
a diverse range of other malignancies, particularly, non-melanoma
skin cancer the commonest malignancy in fair-skinned populations
worldwide. Skin cancer is rarely fatal and is responsible for less
than 1% of all cancer deaths. However its impact on the public
health is nevertheless considerable. The involvement of HPV in
human skin cancer was first demonstrated in patients with the rare
hereditary disease epidermodysplasia verruciformis (Journal of
National Cancer Institute Monographs, 2003, No. 31).
[0013] HPV is also associated with intraepithelial neoplasia of the
conjunctiva (0-80%) and in 62-100% of invasive carcinomas of the
conjunctiva, eyelid and lacrimal sac (IARC Monographs, 64:130).
There is a strong association between HPV and conjunctival
papillomas. HPV type 6/11 is the most common HPV type in
conjunctival papilloma (Br. J. Ophthalmol., 2001, 85:785-787).
[0014] HPV 6, 11 and 13 are typically labeled as low-risk, because
the infection with these types has low oncogenic potential and
usually results in the formation of benign lesions such as genital
warts (technically known as condylomata acuminata) and mild
dysplasia of the cervix, HPV 6 and 11 are also associated with
conjunctival papilloma.
[0015] Despite the high incidence of genital HPV infection and its
association with malignant diseases, there is no effective
antiviral therapy for HPV infection. Gardasil.RTM. and
Cervarix.RTM. are the two vaccines currently on the market against
two of the most common high risk HPVs (HPV 16 and 18). However,
they are only of prophylactic type and do not treat the existing
HPV associated cancer. Moreover, the high cost, issues with social
acceptance, and limitations in health care systems through which
the vaccine can be provided, limits the availability of this
vaccine to women, particularly in developing countries where
HPV-associated anogenital cancers (relating to the anus and the
genitals) are most commonly found. Consequently there remains a
need to identify other, less expensive and more universally
available approaches for preventing and/or treating HPV associated
cancers.
[0016] The inventors have surprisingly found that pyrrolidine
substituted with flavone derivatives are effective against cancers
associated with HPV.
[0017] The invention described herein provides pyrrolidine
substituted with flavone derivatives represented by Formula (I) (as
described herein) for the treatment of human papillomavirus
associated cancers.
SUMMARY OF THE INVENTION
[0018] According to one aspect of the invention, there is provided
use of a compound of Formula (I), a pharmaceutically acceptable
salt, a solvate, a stereoisomer or a diastereoisomer thereof for
the treatment of a cancer associated with human papillomavirus.
[0019] According to another aspect of the invention, there is
provided use of a compound of Formula (I), a pharmaceutically
acceptable salt, a solvate, a stereoisomer or a diastereoisomer
thereof for the treatment of a cancer associated with HPV wherein
the cancer is anal cancer, vulvar cancer, vaginal cancer, penile
cancer, cervical cancer, head and neck cancer such as oropharyngeal
cancer and cancer of the oral cavity, lung cancer, non-melanoma
skin cancer or cancer of the conjunctiva.
[0020] According to yet another aspect of the invention, there is
provided use of a compound of Formula (I), a pharmaceutically
acceptable salt, a solvate, a stereoisomer or a diastereoisomer
thereof for the treatment of cervical cancer.
[0021] According to another aspect of the invention, there is
provided a method for the treatment of human papillomavirus
associated cancers, comprising administering to the subject in need
thereof a therapeutically effective amount of a compound of Formula
(I) a pharmaceutically acceptable salt, a solvate, a stereoisomer
or a diastereoisomer thereof.
[0022] According to another aspect of the invention, there is
provided a method for the treatment of a cancer associated with HPV
wherein the cancer is selected from anal cancer, vulvar cancer,
vaginal cancer, penile cancer, cervical cancer, head and neck
cancer such as oropharyngeal cancer and cancer of the oral cavity,
lung cancer, non-melanoma skin cancer or cancer of the conjunctiva
in a subject, comprising administering to the subject in need
thereof a therapeutically effective amount of a compound of Formula
(I), a pharmaceutically acceptable salt, a solvate, a stereoisomer
or a diastereoisomer thereof.
[0023] According to yet another aspect of the invention, there is
provided a method for the treatment of cervical cancer, comprising
administering to the subject in need thereof a therapeutically
effective amount of a compound of Formula (I), a pharmaceutically
acceptable salt, a solvate, a stereoisomer or a diastereoisomer
thereof.
[0024] According to another aspect of the invention there is
provided a pharmaceutical composition, comprising a therapeutically
effective amount of a compound of Formula (I) a pharmaceutically
acceptable salt, a solvate, a stereoisomer or a diastereoisomer
thereof in association with a pharmaceutically acceptable carrier
for the treatment of a cancer associated with HPV.
[0025] According to another aspect of the invention, there is
provided a method for the manufacture of medicaments, comprising a
compound of Formula (I), a pharmaceutically acceptable salt, a
solvate, a stereoisomer or a diastereoisomer thereof which are
useful for the treatment of a cancer associated with HPV.
[0026] Other aspects and further scope of applicability of the
present invention will become apparent from the detailed
description to follow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1A depicts the reproductions of Western blots showing
significant upregulation of p53 in the cells treated with compound
A and compound B.
[0028] FIG. 1B depicts the reproductions of Western blots showing
significant downregulation of expression of E6 and E7 in SiHa cells
treated with compound A and compound B.
[0029] FIG. 2 depicts the reproductions of RT-PCR showing
significant downregulation of expression of E6 and E7 in SiHa and
HeLa cells treated with compound A and compound B at the
transcription level.
[0030] FIG. 3A is a graphical representation of p53-EGFP nuclear
translocation in SiHa cells.
[0031] FIG. 3B is a graphical representation of p53-EGFP nuclear
translocation in HeLa cells.
[0032] FIG. 4 is a graphical representation of tumor growth profile
in SiHa xenograft animals after administration of Compound A.
[0033] FIG. 5 is a graphical representation of tumor growth profile
in SiHa xenograft animals after administration of Compound B.
DETAILED DESCRIPTION OF THE INVENTION
[0034] The general terms used hereinbefore and hereinafter
preferably have within the context of this disclosure the following
meanings, unless otherwise indicated. Thus, the definitions of the
general terms as used in the context of the present invention are
provided herein below:
[0035] The singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise.
[0036] It will be understood that "substitution" or "substituted
with" includes the implicit proviso that such substitution is in
accordance with permitted valence of the substituted atom and the
substituent, as well as represents a stable compound, which does
not readily undergo transformation such as rearrangement,
cyclization, elimination, etc.
[0037] The term "C.sub.1-C.sub.4alkyl" refers to the radical of
saturated aliphatic groups, including straight or branched-chain
containing from 1 to 4 carbon atoms. Examples of alkyl groups
include but are not limited to methyl, ethyl, propyl, butyl,
isopropyl, isobutyl, sec-butyl, tert-butyl and the like.
[0038] The term "C.sub.1-C.sub.4alkoxy" refers to an alkyl group as
defined above attached via oxygen linkage to the rest of the
molecule. Examples of alkoxy include, but are not limited to
methoxy, ethoxy, propoxy, butoxy, tert-butoxy and the like.
[0039] The term "halogen" refers to fluorine, chlorine, bromine and
iodine.
[0040] The term "hydroxy" or "hydroxyl" as used herein, refers to
--OH group.
[0041] The term "therapeutically effective amount", as used herein
refers to the amount of a compound represented by Formula (I), a
pharmaceutically acceptable salt, a solvate, a stereoisomer or a
diastereoisomer thereof, that, when administered to a subject in
need of such treatment, is sufficient to inhibit the activity of
human papillomavirus (HPV) such that the disease mediated by HPV is
reduced, treated or alleviated.
[0042] The term "HPV" or "Human papillomavirus" as used herein
refers to a member of the papillomavirus family of viruses that is
capable of infecting mammals. The term includes both high-risk type
and low risk type HPVs unless otherwise indicated.
[0043] The term "subject" as used herein, refers to an animal,
preferably a mammal, most preferably a human, who is in the need of
treatment of diseases mediated by HPV. The term subject may be
interchangeably used with the term patient in the context of the
present invention.
[0044] The term "mammal" as used herein is intended to encompass
humans, as well as non-human mammals which are susceptible to
infection by human papillomavirus. Non-human mammals include but
are not limited to domestic animals, such as cows, pigs, horses,
dogs, cats, rabbits, rats and mice, and non-domestic animals.
[0045] The term "treat" or "treatment" or "treated" with reference
to HPV associated cancer in a subject, preferably a mammal, more
preferably a human include: (i) inhibition of cancer i.e.,
arresting the development of the cancer; (ii) reduction in the
regression of the cancer or slowing down of the cancer; (iii)
amelioration of the cancer i.e., reducing the severity of the
symptoms associated with the cancer (iv) relief, to some extent, of
one or more symptoms associated with cancer.
[0046] As used herein the term "pharmaceutically acceptable" is
meant that the carrier, diluent, excipients, and/or salt must be
compatible with the other ingredients of the formulation, and not
deleterious to the recipient thereof. "Pharmaceutically acceptable"
also means that the compositions or dosage forms are within the
scope of sound medical judgment, suitable for use for an animal or
human without excessive toxicity, irritation, allergic response, or
other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0047] The present invention furthermore includes all solvates of
the compounds of the formula (I), for example hydrates, and the
solvates formed with other solvents of crystallization, such as
alcohols, ethers, ethyl acetate, dioxane, dimethylformamide or a
lower alkyl ketone, such as acetone, or mixtures thereof. Certain
compounds of the present invention can exist in unsolvated forms as
well as solvated forms, including hydrated forms. Certain compounds
of the present invention may exist in multiple crystalline or
amorphous forms. In general, all physical forms are equivalent for
the uses contemplated by the present invention and are intended to
be within the scope of the present invention.
[0048] The term "p53" refers to a nuclear phosphoprotein which acts
as a tumor suppressor.
[0049] The term "pRb" or "retinoblastoma protein" refers to a tumor
suppressor protein that is dysfunctional in many types of
cancer.
[0050] The term "downregulation" refers to reducing, partially or
totally, the indicated activity or expression. In the context of
the present invention the term "downregulation of the oncoproteins
E6 and E7" refers to the decrease in the expression level of E6 and
E7 respectively. The level may be determined by any suitable method
in the art, including Wester blot assay.
[0051] The term "upregulation of p53" refers to increase in the
expression level of the tumor suppressor protein 53.
[0052] The term "senescence" refers to the stage in which the cells
lose their ability to divide.
[0053] The term "apoptosis" refers to the natural process of
programmed cell death. It is a process of self-destruction, in
which the cell uses specialized cellular machinery to kill itself.
The cells disintegrate into membrane-bound particles that are then
eliminated by phagocytosis. Apoptosis is a mechanism that enables
metazoans to control cell number and eliminate cells that threaten
the animal's survival.
[0054] The term "transcription" refers to a process of creating a
complementary RNA copy of a sequence of DNA.
[0055] The term "cell cycle arrest" refers to a regulatory process
that halts the progression through the cell cycle during one of the
normal phases (G1, S, G2, M).
[0056] The compound with anti-cancer activity refers to a substance
which is capable of inhibiting cell proliferation or is capable of
inducing cell death. Non-limiting examples of anti-cancer agent
suitable for use in the compositions of the invention include (i)
microtubule disrupting agents such as taxanes and paclitaxel (ii)
kinase inhibitors such as imatinib, erlotinib and BAY-43-9006,
(iii) mTOR inhibitors such as rapamycin (iv) antineoplastic agents
such as carboplatin, cisplatin, oxaliplatin, etoposide and
Dacarbazine and (xvi) topoisomerase inhibitors such as topotecan
and irinotecan (vi) antimetabolites such as cytarabine,
fluorouracil, gemcitabine, topotecan, Hidroxyurea, Thioguanine,
Methotrexate (vii) antibiotics such cytotoxic agents doxorubicin,
bleomycin and dactinomycin.
[0057] According to one aspect of the present invention there is
provided a compound of Formula (I),
##STR00002##
wherein Ar is a phenyl group, which is unsubstituted or substituted
by 1, 2, or 3 identical or different substituents selected from:
halogen, nitro, cyano, C.sub.1-C.sub.4-alkyl, trifluoromethyl,
hydroxyl or C.sub.1-C.sub.4-alkoxy; or a pharmaceutically
acceptable salt, a solvate, a stereoisomer or a diastereoisomer
thereof, for use in the treatment of a cancer associated with
HPV.
[0058] According to one aspect of the invention there is provided a
(+)-trans isomer of the compound of Formula (I), as indicated in
Formula (IA) below,
##STR00003##
wherein Ar is a phenyl group, which is unsubstituted or substituted
by 1, 2, or 3 identical or different substituents selected from
halogen, nitro, cyano, C.sub.1-C.sub.4-alkyl, trifluoromethyl,
hydroxyl or C.sub.1-C.sub.4-alkoxy; or a pharmaceutically
acceptable salt thereof or a solvate thereof for use in the
treatment of a cancer associated with HPV.
[0059] According to another aspect of the invention there is
provided a compound of Formula (I), a pharmaceutically acceptable
salt, a solvate, a stereoisomer or a diastereoisomer thereof,
wherein Ar is phenyl group substituted by 1, 2, or 3 identical or
different substituents selected from chlorine, bromine, fluorine,
iodine, C.sub.1-C.sub.4-alkyl or trifluoromethyl, for use in the
treatment of a cancer associated with HPV.
[0060] According to another aspect of the invention there is
provided a compound of Formula (I), a pharmaceutically acceptable
salt, a solvate, a stereoisomer or a diastereoisomer thereof,
wherein Ar is phenyl group substituted by 1, 2, or 3 identical or
different halogens selected from chlorine, bromine, fluorine or
iodine, for use in the treatment of a cancer associated with
HPV.
[0061] According to another aspect of the invention there is
provided a compound of Formula (I), a pharmaceutically acceptable
salt, a solvate, a stereoisomer or a diastereoisomer thereof,
wherein Ar is phenyl group substituted by chlorine, for use in the
treatment of a cancer associated with HPV.
[0062] According to another aspect of the invention there is
provided a compound of Formula (I), a pharmaceutically acceptable
salt, a solvate, a stereoisomer or a diastereoisomer thereof,
wherein Ar is phenyl group substituted by chlorine and
trifluromethyl, for use in the treatment of a cancer associated
with HPV.
[0063] It will be appreciated by those skilled in the art that the
compounds of Formula (I) contain at least two chiral centres and
hence, exists in the form of two different optical isomers (i.e.,
(+) or (-) enantiomers), two different geometric isomers (cis and
trans) and 4 different diasteroisomers. All such enantiomers,
geometric isomers, diasteroisomers and mixtures thereof including
racemic mixtures are included within the scope of the invention.
The enantiomers of the compound of Formula (I) can be obtained by
methods disclosed in PCT Application Publication Nos. WO2004004632,
WO2007148158 and WO2008007169 incorporated herein by reference or
the enantiomers of the compound of Formula (I) can also be obtained
by methods well known in the art, such as chiral HPLC and enzymatic
resolution. Alternatively, the enantiomers of the compounds of
Formula (I) can be synthesized by using optically active starting
materials.
[0064] The manufacture of the compounds of Formula (I), which may
be in the form of pharmaceutically acceptable salts, and the
manufacture of pharmaceutical composition suitable for oral, rectal
and/or parenteral administration containing the above compounds are
generally disclosed in PCT Application Publication No.
WO2004004632, which is incorporated herein by reference.
[0065] As indicated herein above the compound of Formula (I) may be
used in the form of their salts. Preferred salt of compounds of
Formula (I) include acetates, alginates, ascorbates, aspartates,
benzoates, benzenesulfonates, bisulfates, borates, cinnamates,
citrates, ethanesulfonates, fumarates, glucuronates, glutamates,
glycolates, hydrochlorides, hydrobromides, hydrofluorides,
ketoglutarates, lactates, maleates, malonates, mesylate, nitrates,
oxalates, palmoates, perchlorates, phosphates, picrates,
salicylates, succinates, sulfamate, sulfates, tartrates, tosylate,
trifluoroacetic acid salt and other acid addition salts known to
the person skilled in the art.
[0066] Accordingly, an aspect of the invention, the compound of
Formula (IA) for use in the treatment of HPV associated cancer is
selected from
(+)-trans-2-(2-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxy-methyl-1-methyl--
pyrrolidin-3-yl)-chromen-4-one hydrochloride (referred to herein as
compound A) or
(+)-trans-3-[2[(2-Chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hyd-
roxymethyl-1-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride
(referred to herein as compound B). Compounds A and B are disclosed
in WO2007148158 and specifically as Example 10 and Example 44,
respectively.
[0067] The compound of Formula (IA) for use in the treatment of HPV
associated cancer can be
(+)-trans-2-(2-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxy-methyl-1-methyl--
pyrrolidin-3-yl)-chromen-4-one hydrochloride (compound A).
[0068] The compound of Formula (IA) for use in the treatment of HPV
associated cancer can be
(+)-trans-3-[2[(2-Chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hyd-
roxymethyl-1-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride
(compound B).
[0069] The cancer associated with HPV is selected from the group
consisting of anal cancer, vulvar cancer, vaginal cancer, penile
cancer, cervical cancer, head and neck cancer such as oropharyngeal
cancer and cancer of the oral cavity, lung cancer, non-melanoma
skin cancer and cancer of the conjunctiva.
[0070] A compound of Formula (I), a pharmaceutically acceptable
salt, a solvate, a stereoisomer or a diastereoisomer thereof can be
used for the treatment of cervical cancer.
[0071] A method for the treatment of human papillomavirus
associated cancer, comprising administering to the subject in need
thereof a therapeutically effective amount of a compound of Formula
(I) a pharmaceutically acceptable salt, a solvate, a stereoisomer
or a diastereoisomer thereof is described.
[0072] HPV associated cancers selected from the group consisting of
anal cancer, vulvar cancer, vaginal cancer, penile cancer, cervical
cancer, head and neck cancer such as oropharyngeal cancer and
cancer of the oral cavity, lung cancer, non-melanoma skin cancer
and cancer of the conjunctiva in a subject, can be treated by
administering to the subject in need thereof a therapeutically
effective amount of a compound of Formula (I), a pharmaceutically
acceptable salt, a solvate, a stereoisomer or a diastereoisomer
thereof.
[0073] Cervical cancer can be treated by administering to the
subject in need thereof a therapeutically effective amount of a
compound of Formula (I), a pharmaceutically acceptable salt, a
solvate, a stereoisomer or a diastereoisomer thereof.
[0074] The invention provides a method of inhibiting HPV associated
cancer; comprising contacting the HPV infected cells with a
compound of Formula (I).
[0075] According to an aspect of the invention there is provided a
method of inhibiting high risk HPV selected from HPV 16, 18, 31,
33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68, comprising
contacting the HPV infected cells with a compound of Formula
(I).
[0076] In an embodiment there is provided a method of inhibiting
HPV 16, 18 and 31, comprising contacting the HPV infected cells
with a compound of Formula (I).
[0077] According to this invention, the oncoproteins on HPV can be
downregulated. The oncoproteins may be the oncoproteins E6 and E7
on HPV. The downregulation can be carried out by, administering to
the subject a therapeutically effective amount of a compound of
Formula (I). Inhibiting oncoproteins E6 and E7 on HPV is a means of
inducing cell cycle arrest, senescence or apoptosis.
[0078] The oncoproteins E6 and E7 on HPV can be downregulated at
transcriptional level.
[0079] The phosphoprotein p53 can be activated by administering a
therapeutically effective amount of a compound of Formula (I) to
the subject in need thereof.
[0080] According to yet another embodiment of the present invention
there is provided a method of treatment of a cancer associated with
HPV 16 comprising administering to the subject in need thereof a
therapeutically effective amount of a compound of Formula (I), a
pharmaceutically acceptable salt, a solvate, a stereoisomer or a
diastereoisomer thereof.
[0081] According to another aspect of the invention, there is
provided a method for the manufacture of medicaments, comprising a
compound of Formula (I), a pharmaceutically acceptable salt, a
solvate, a stereoisomer or a diastereoisomer thereof which are
useful for the treatment of a cancer associated with HPV.
[0082] There is provided a pharmaceutical composition which
comprises a therapeutically effective amount of compound of Formula
(I), a pharmaceutically acceptable salt, a solvate, a stereoisomer,
or a diastereoisomer thereof in association with a pharmaceutically
acceptable carrier. The composition can include at least one
further pharmaceutically active compound, wherein the further
pharmaceutically active compound has anticancer activity. The
pharmaceutically active compound can be selected from, but not
limited to, bleomycin, cispaltin, topotecan hydrochloride,
imiquimod, podofilox, trichloroacetic acid and the like.
[0083] The pharmaceutical preparations may contain about 1 to 99%,
for example, about 5 to 70%, or from about 5 to about 30% by weight
of the compound of the Formula (I) or pharmaceutically acceptable
salt thereof. The amount of the active ingredient of the Formula
(I) or pharmaceutically acceptable salt thereof in the
pharmaceutical preparations normally is from about 1 to 1000
mg.
[0084] The compound of Formula (I) may be administered orally,
intravaginally, vulvovaginally, rectally, topically or parenterally
(including intravenous, subcutaneous, intramuscular, intravascular
or infusion). The compound of Formula (I) may have to be
administered by any route appropriate to the condition to be
treated. It will be appreciated that the preferred route may vary
with the condition of the patient.
[0085] Compositions intended for pharmaceutical use may be prepared
according to any method known in the art for the manufacture of
pharmaceutical compositions, e.g. Remington--The Science and
Practice of Pharmacy (21.sup.st Edition) (2005), Goodman &
Gilman's The Pharmacological Basis of Therapeutics (11.sup.th
Edition) (2006) and Ansel's Pharmaceutical Dosage Forms and Drug
Delivery Systems (9.sup.th Edition), edited by Allen et al.,
Lippincott Williams & Wilkins, (2011), Solid-State Chemistry of
Drugs (2.sup.nd Edition) (1999), each of which is hereby
incorporated by reference."
[0086] The compositions described herein may be in a form suitable
for oral administration, for example as a tablet or capsule; for
parenteral injection (including intravenous, subcutaneous,
intramuscular, intravascular or infusion) for example as a sterile
solution, suspension or emulsion; for topical administration for
example as an ointment, cream, gel, lotions or collodion; for
rectal, vaginal or vulvovaginal administration for example as a
suppository, tampons, pessaries, creams, gels, paste, foam or
vaginal ring. The composition can be administered topically to
external surfaces of skin surface, such as vulva and/or to
surrounding areas of skin. In addition or alternatively, the
composition can be administered intravaginally.
[0087] For oral use, the compound of Formula (I) may be
administered, for example, in the form of tablets or capsules,
powders, dispersible granules, or cachets, or as aqueous solutions
or suspensions. In the case of tablets for oral use, carriers which
are commonly used include lactose, corn starch, magnesium
carbonate, talc, and sugar, and lubricating agents such as
magnesium stearate are commonly added. For oral administration in
capsule form, useful carriers include lactose, corn starch,
magnesium carbonate, talc and sugar.
[0088] For intramuscular, intraperitoneal, subcutaneous and
intravenous use, sterile solutions of compound of Formula (I) are
usually employed, and the pH of the solutions should be suitably
adjusted and buffered.
[0089] For ointments, creams, the compound of Formula (I) is
formulated in oil-in-water or water-in-oil base. A vaginal cream
can be administered to contact a mucosal surface in the vaginal
cavity.
[0090] For rectal or vaginal use, the compound of Formula (I) can
be administered in the form of suppositories. A suppository
comprises of compound of Formula (I), a suitable suppository base
and additives such as preservatives, antioxidants, emulsifiers and
the like. Suitable suppository bases include natural or synthetic
triglycerides or paraffin hydrocarbons. The vaginal use the
compound of Formula (I) can also be administered in the form of
vaginal cream.
[0091] Compositions for oral delivery may be in the form of
tablets, lozenges, aqueous or oily suspensions, granules, powders,
emulsions, capsules, syrups, or elixirs. Orally administered
compositions may contain one or more optional agents, for example,
sweetening agents such as fructose, aspartame or saccharin;
flavoring agents such as peppermint, oil of wintergreen, or cherry;
coloring agents; and preserving agents, to provide a
pharmaceutically palatable preparation. Selectively permeable
membranes surrounding an osmotically active driving compound are
also suitable for oral administration of compounds of present
invention. Oral compositions can include standard vehicles such as
mannitol, lactose, starch, magnesium stearate, sodium saccharine,
cellulose, magnesium carbonate, etc. Such vehicles are preferably
of pharmaceutical grade.
[0092] Further, the effect of the compounds of the present
invention contained in the pharmaceutical composition may be
delayed or prolonged by proper formulation. For example, a slowly
soluble pellet of the compound may be prepared and incorporated in
a tablet or capsule. The technique may be improved by making
pellets of several different dissolution rates and filling capsules
with a mixture of the pellets. Tablets or capsules may be coated
with a film which resists dissolution for a predictable period of
time. Even the parenteral preparations may be made long-acting, by
dissolving or suspending the compound in oily or emulsified
vehicles which allow it to disperse only slowly in the serum.
[0093] Compositions for rectal administration or vaginal
administration are suppositories. Suppositories are solid bodies
for insertion into the rectum or vagina which melt or soften at
body temperature releasing one or more pharmacologically or
therapeutically active ingredients. Pharmaceutically acceptable
substances utilized in rectal suppositories are bases or vehicles
and agents to raise the melting point. Examples of bases include
cocoa butter (theobroma oil), glycerin-gelatin, carbowax
(polyoxyethylene glycol) and appropriate mixtures of mono-, di- and
triglycerides of fatty acids. Combinations of the various bases may
be used. Agents to raise the melting point of suppositories include
spermaceti and wax. Suppositories may be prepared either by the
compressed method or by molding.
[0094] Effective dose of the compound of Formula (I) depends at
least on the nature of the condition being treated, the mode of
delivery, and the pharmaceutical formulation, and will be
determined by the clinician using conventional dose escalation
studies. It can be expected to be from about 0.1 to about 100 mg/kg
body weight per day; particularly, from about 0.1 to about 10 mg/kg
body weight per day; more particularly, from about 0.1 to about 5
mg/kg body weight per day.
[0095] Compounds of Formula (I) may be prepared according to the
methods disclosed in PCT Patent Publication No. WO2004004632 and
PCT Patent Publication No. WO2007148158 which are incorporated
herein by reference.
[0096] The general process for the preparation of compounds of
Formula (I), or a pharmaceutically acceptable salt thereof,
comprises the following steps:
(a) treating the resolved enantiomerically pure (-)-trans
enantiomer of the intermediate compound of Formula VIA,
##STR00004##
with acetic anhydride in the presence of a Lewis acid catalyst to
obtain a resolved acetylated compound of Formula VIIA,
##STR00005##
(b) reacting the resolved acetylated compound of Formula VIIA with
an acid of Formula ArCOOH or an acid chloride of Formula ArCOCl or
an acid anhydride of Formula (ArCO).sub.2O or an ester of Formula
ArCOOCH.sub.3, wherein Ar is as defined hereinabove in reference to
the compound of Formula (I), in the presence of a base and a
solvent to obtain a resolved compound of Formula VIIIA;
##STR00006##
(c) treating the resolved compound of Formula VIIIA with a base in
a suitable solvent to obtain the corresponding resolved
.beta.-diketone compound of Formula IXA;
##STR00007##
wherein Ar is as defined above; (d) treating the resolved
.beta.-diketone compound of Formula IXA with an acid such as
hydrochloric acid to obtain the corresponding cyclized compound of
Formula XA,
##STR00008##
(e) subjecting the compound of Formula XA to dealkylation by
heating it with a dealkylating agent at a temperature ranging from
120-180.degree. C. to obtain the (+)-trans enantiomer of the
compound of Formula (I) and, optionally, converting the subject
compound into its pharmaceutically acceptable salt.
[0097] The Lewis acid catalyst utilized in the step (a) above may
be selected from: BF.sub.3, Et.sub.2O, zinc chloride, aluminium
chloride and titanium chloride.
[0098] The base utilized in the process step (b) may be selected
from triethylamine, pyridine and a DCC-DMAP combination
(combination of N,N'-dicyclohexyl carbodiimide and
4-dimethylaminopyridine).
[0099] It will be apparent to those skilled in the art that the
rearrangement of the compound of Formula VIIIA to the corresponding
.beta.-diketone compound of Formula IXA is known as a
Baker-Venkataraman rearrangement (J. Chem. Soc., 1933, 1381 and
Curr. Sci., 1933, 4, 214).
[0100] The base used in the process step (c) may be selected from:
lithium hexamethyl disilazide, sodium hexamethyldisilazide,
potassium hexamethyldisilazide, sodium hydride and potassium
hydride. A preferred base is lithium hexamethyl disilazide.
[0101] The dealkylating agent used in process step (e) for the
dealkylation of the compound of Formula IXA may be selected from:
pyridine hydrochloride, boron tribromide, boron trifluoride
etherate and aluminium trichloride. A preferred dealkylating agent
is pyridine hydrochloride.
[0102] Preparation of the starting compound of Formula VIA involves
reacting 1-methyl-4-piperidone with a solution of
1,3,5-trimethoxybenzene in glacial acetic acid, to yield
1-methyl-4-(2,4,6-trimethoxyphenyl)-1,2,3,6-tetrahydropyridine,
which is reacted with boron trifluoride diethyl etherate, sodium
borohydride and tetrahydrofuran to yield
1-methyl-4-(2,4,6-trimethoxyphenyl)piperidin-3-ol. Conversion of
1-methyl-4-(2,4,6-trimethoxyphenyl)piperidin-3-ol to the compound
of Formula VIA involves converting the hydroxyl group present on
the piperidine ring of the compound,
1-methyl-4-(2,4,6-trimethoxyphenyl) piperidin-3-ol to a leaving
group such as tosyl, mesyl, triflate or halide by treatment with an
appropriate reagent such as p-toluenesulfonylchloride,
methanesulfonylchloride, triflic anhydride or phosphorous
pentachloride in the presence of oxygen nucleophiles such as
triethylamine, pyridine, potassium carbonate or sodium carbonate,
followed by ring contraction in the presence of oxygen nucleophiles
such as sodium acetate or potassium acetate in an alcoholic solvent
such as isopropanol, ethanol or propanol.
[0103] It is to be understood that the invention may assume various
alternative variations and step sequences, except where expressly
specified to the contrary.
[0104] Moreover, other than in any operating examples, or where
otherwise indicated, all numbers expressing, for example,
quantities of ingredients used in the specification and claims are
to be understood as being modified in all instances by the term
"about".
[0105] Accordingly, unless indicated to the contrary, the numerical
parameters set forth in the following specification and attached
claims are approximations that may vary depending upon the desired
properties to be obtained by the present invention. At the very
least, and not as an attempt to limit the application of the
doctrine of equivalents to the scope of the claims, each numerical
parameter should at least be construed in light of the number of
reported significant digits and by applying ordinary rounding
techniques.
[0106] Those skilled in the art will recognize that several
variations are possible within the scope and spirit of this
invention. The invention will now be described in greater detail by
reference to the following non-limiting examples. The following
examples further illustrate the invention but, of course, should
not be construed as in any way limiting its scope.
EXEMPLIFICATION
[0107] In the following examples and elsewhere, abbreviations have
the following meanings:
TABLE-US-00001 List of abbreviations ATCC American Type Cell
Culture bp Base pairs BF.sub.3 boron trifluoride CO.sub.2 carbon
dioxide cDNA complementary DNA DMEM Dulbecco's Modified Eagle
Medium DMSO dimethyl sulfoxide DNA deoxyribonucleic acid dsDNA
double stranded Deoxyribonucleic Acid dNTPs Deoxynucleotide
Triphosphates e.e enantiomeric excess EGFP enhanced green
fluorescent protein Et.sub.2O diethyl ether FBS fetal bovine serum
g Gram H hour(s) HCl hydrochloric acid HPLC high performance liquid
chromatography HRP horseradish peroxidase ip intraperitoneally IPA
isopropyl alcohol MeOH Methanol .mu.g Microgram .mu.L microlitre mg
milligram mL milliliter mm millimetre mmol or mM millimolar mpk
milligram per kilogram MgCl.sub.2 magnesium Chloride MMLV-RT
Moloney Murine Leukemia virus Reverse Transcriptase
Na.sub.2CO.sub.3 sodium carbonate ng nanogram NP-40 nonyl
phenoxypolyethoxylethanol NaF sodium fluoride Na.sub.3VO.sub.4
sodium orthovanadate NaCl sodium chloride nm Nanometers pmol
Picomolar PCR polymerase chain reaction p.o per oral RNA
ribonucleic Acid RT-PCR Reverse transcription polymerase chain
reaction SDS-PAGE sodium dodecyl sulfate polyacrylamide gel
electrophoresis TFA trifluoroacetic acid UV Ultraviolet
Example 1
A) Preparation of
(+)-trans-2-(2-Chlorophenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-py-
rrolidin-3-yl)-chromen-4-one hydrochloride (Compound A)
[0108] Molten pyridine hydrochloride (4.1 g, 35.6 mmol) was added
to
(+)-trans-2-(2-chloro-phenyl)-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl-
)-5,7-dimethoxy-chromen-4-one (0.4 g, 0.9 mmol) and heated at
180.degree. C. for 1.5 h. The reaction mixture was cooled to
25.degree. C., diluted with MeOH (10 mL) and basified using
Na.sub.2CO.sub.3 to pH 10. The mixture was filtered and the organic
layer was concentrated. The residue was suspended in water (5 mL),
stirred for 30 minutes filtered and dried to obtain the compound,
(+)-trans-2-(2-chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-p-
yrrolidin-3-yl)-chromen-4-one.
[0109] Yield: 0.25 g (70%);
[0110] IR (KBr): 3422, 3135, 1664, 1623, 1559 cm.sup.-;
[0111] 1H NMR (CDCl.sub.3, 300 MHz): .delta. 7.56 (d, 1H), 7.36 (m,
3H), 6.36 (s, 1H), 6.20 (s, 1H), 4.02 (m, 1H), 3.70 (m, 2H), 3.15
(m, 2H), 2.88 (m, 1H), 2.58 (s, 3H), 2.35 (m, 1H), 1.88 (m, 1H); MS
(ES+): m/z 402 (M+1);
[0112] Analysis: C.sub.21H.sub.20ClNO.sub.5; C, 62.24 (62.71); H,
5.07 (4.97); N, 3.60 (3.48); Cl, 9.01 (8.83).
[0113] The compound (0.2 g, 0.48 mmol) as obtained above was
suspended in IPA (5 mL) and 3.5% HCl (25 mL) was added. The
suspension was heated to get a clear solution. The solution was
cooled and filtered to obtain the compound,
(+)-trans-2-(2-Chlorophenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-
-methyl-pyrrolidin-3-yl)chromen-4-one hydrochloride.
[0114] Yield: 0.21 g (97%); mp: 188-192.degree. C.; [.alpha.]
D25=+21.3.degree. (c=0.2, methanol);
[0115] 1H NMR (CD.sub.3OD, 300 MHz): .delta. 7.80 (d, 1H), 7.60 (m,
3H), 6.53 (s, 1H), 6.37 (s, 1H), 4.23 (m, 1H), 3.89 (m, 2H), 3.63
(m, 1H), 3.59 (dd, 1H), 3.38 (m, 1H), 2.90 (s, 3H), 2.45 (m, 1H),
2.35 (m, 1H); MS (ES+): m/z 402 (M+1)(free base).
[0116] This compound was subjected to chiral HPLC. Chiral HPLC was
done using column Chiralcel OD-H (250.times.4.6 mm) and solvent
system haxane:ethanol (92:08) with TFA (0.4%). The results are
recorded at 264 nm with solvent flow rate of 1 mL/minute The chiral
HPLC showed 100% e.e of the compound,
(+)-trans-2-(2-chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxy-methyl-1-methyl--
pyrrolidin-3-yl)-chromen-4-one hydrochloride.
B) Preparation of
(+)-trans-2-(2-chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hydrox-
ymethyl-1-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride
(Compound B)
[0117] A mixture of the compound,
(+)-trans-2-(2-Chloro-4-trifluoromethylphenyl)-8-(2-hydroxymethyl-1-methy-
l pyrrolidin-3-yl)-5,7-dimethoxy-chromen-4-one (0.25 g, 0.5 mmol),
pyridine hydrochloride (0.25 g, 2.16 mmol) and a catalytic amount
of quinoline was heated at 180.degree. C. for a period of 2.5 h.
The reaction mixture was diluted with methanol (25 mL) and basified
with solid Na.sub.2CO.sub.3 to pH 10. The reaction mixture was
filtered, and washed with methanol. The organic layer was
concentrated and the residue purified by column chromatography
using 0.1% ammonia and 4.5% MeOH in chloroform as eluent to yield
the compound,
(+)-trans-2-(2-chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxy-
-methyl-1-methylpyrrolidin-3-yl)-chromen-4-one, as a yellow
solid.
[0118] Yield: 0.15 g (63.7%);
[0119] .sup.1H NMR (CDCl.sub.3, 300 MHz): .delta. 7.99 (m, 2H),
7.83 (d, 1H), 6.65 (s, 1H), 6.41 (s, 1H), 4.24 (m, 1H), 3.90 (m,
2H), 3.70 (m, 1H), 3.60 (m, 1H), 3.41 (m, 1H), 2.99 (s, 3H), 2.54
(m, 1H), 2.28 (m, 1H); MS (ES+): m/z 470 (M+1).
[0120] The compound (0.1 g, 0.2 mmol) as obtained above was
suspended in methanol (2 mL) and treated with ethereal HCl and the
organic solvent evaporated to yield the compound,
(+)-trans-2-(2-chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hydrox-
ymethyl-1-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride.
[0121] Yield: 0.1 g (92.8%);
[0122] 1H NMR (CDCl.sub.3, 300 MHz): .delta. 8.02 (d, 2H), 7.83 (d,
1H), 6.64 (s, 1H), 6.41 (s, 1H), 4.23 (m, 1H), 3.73 (m, 2H), 3.68
(m, 1H), 3.51 (m, 1H), 3.39 (m, 1H), 2.99 (s, 3H), 2.54 (m, 1H),
2.31 (m, 1H).
In Vitro Studies
[0123] Materials and Methods: Two cervical cancer cell lines SiHa
and HeLa (ATCC), positive for HPV genome were used for evaluation
of Compound A and Compound B. The cell lines were transfected with
a plasmid vector expressing p53 protein fused with a fluorescent
protein EGFP using lipofectamine (Invitrogen, CA, USA). After 48 h
of transfection the cells were maintained for 30-45 days in DMEM
(Sigma Aldrich, catalogue no. D5546) supplemented with 10% FBS and
800 .mu.g/mL Geneticin (Gibco BRL Life Technologies, Inc) for 30-45
days. The stably expressing cells were expanded and evaluated for
the presence of fusion construct under fluorescent microscopy.
After 24 h of transfection, the cells were treated separately with
Compound A and Compound B (henceforth referred to as treated cells)
for 24 h. Cells were harvested and were used for analysis of status
of E6, E7, p53 by western blot and by RT-PCR. Cells not treated
with the Compound A and Compound B (henceforth referred to as
untreated cells) were used as controls. HPV negative C33A cell was
used as negative control.
[0124] The terms fresh medium and culture medium refer to DMEM
(catalogue no. D5546).
Example 2
Analysis of Status of E6, E7, p53 by Western Blot
[0125] To prepare whole cell extracts for Western blot, the treated
cells were lysed in lysis buffer (50 mM Tris-HCl (pH 7.4), 1%
NP-40, 40 mM NaF, 10 mM NaCl, 10 mM Na.sub.3VO.sub.4, 1 mM
phenylmethylsulfonyl fluoride, and 10 mM dithiothreitol and 1
.mu.g/ml each of leupeptin and aprotinin). The cell lysates (50
.mu.g) were resolved by SDS-PAGE and the separated proteins (E6, E7
and p53) were transferred to polyvinylidene difluoride membrane
(PVDF) by wet transfer method using Bio-Rad electro-transfer
apparatus. After blocking with 10% non-fat milk in Tris buffered
saline containing 0.2% Tween-20, the membrane was incubated with
the primary antibody, followed by HRP-conjugated secondary
antibody. Proteins were visualized by 3,3'-Di amino benzidine (DAB)
method. Similar procedure was followed for untreated cells.
[0126] The following antibodies were used for western blot with the
indicated dilutions.
Primary Antibodies
TABLE-US-00002 [0127] Sr. Source No. Antibody Organism Company
Dilution 1 p53 Antibody Rabbit Cell Signaling (9282) 1:500 2 HPV16
E7(NM2) Mouse SantaCruz (sc-65711) 1:100 3 HPV E76 (N-17) Goat
SantaCruz (sc-1584) 1:100 4 Beta actin Mouse Sigma (A5316)
1:1000
Secondary Antibodies
TABLE-US-00003 [0128] Sr. No. Antibody Company Dilution 1 Rabbit
anti goat HRP Molecular Probes (R21459) 1:2000 2 Anti mouse HRP
Sigma (A9044) 1:2000 and 1:4000 3 Anti rabbit HRP Sigma (A0545)
1:2000
[0129] Results: It was observed that cells treated with Compound A
and Compound B showed increased expression of p53 protein in a dose
dependant manner in both SiHa and HeLa cells. However no expression
was observed in untreated cells. The expression of E6 was down
regulated in SiHa cells by both the compounds at the concentration
of 50 .mu.g/mL. Similarly the complete down regulation of E7 was
seen in cells treated with 25 .mu.g/mL of Compound A and also
complete down regulation of E7 was seen in cells treated with 25
.mu.g/mL of Compound B compared to control (FIG. 1A and FIG.
1B).
Example 3
Analysis of Status of E6, E7, p53 by RT-PCR
[0130] The expression status for gene E6 and E7 at the
transcription level was detected by Reverse Transcriptase PCR
(RT-PCR). The RNA was isolated from the treated and untreated cells
using Qiagen AllPrep DNA/RNA Mini Kit (Cat. No. 80204). The RNA
integrity was checked by visualization under UV after agarose gel
electrophoresis. The quantity and purity of RNA was analysed by
Nano Drop (Thermo Scientific). The 500 ng of RNA was converted into
cDNA using 0.1 .mu.g/.mu.l of Oligo dT (Promega), MMLV-RT, dNTPs,
RNA guard and RT buffer (Promega) and incubated at 37.degree. C.
for 1 h. The reaction was terminated by heating the reaction
mixture at 90.degree. C. for 3 minutes and used for PCR
amplification of E6 and E7 genes. The PCR was conducted as
follows:
TABLE-US-00004 5x Buffer 3 .mu.l DNA taq polymerase 0.3 .mu.l
Antisense primer (10 pmol) 1 .mu.l Sterile water 5.45 .mu.l 10 mM
dNTPs 0.8 .mu.l Sense primer (10 pmol) 1 .mu.l MgCl.sub.2 (50 mM)
0.45 .mu.l cDNA 3 .mu.l
The sequence of the primers are shown below
TABLE-US-00005 Gene Primer Sequence Size .beta.-Actin sense
5'-AGACTTCGAGCAGGAGATG-3' (SEQ ID NO: 1) 256 bp Antisense
5'-CTTGATCTTCATGGTGCTAGG-3' (SEQ ID NO: 2) 16 E6 sense
5'-TGAGGTATATGACTTTGCTTTTC-3' (SEQ ID NO: 3) 297 bp Antisense
5'-CAAGACATACATCGACCGGTCC-3' (SEQ ID NO: 4) 16 E7 sense
5'-AAATGACAGCTCAGAGGAGGAG-3' (SEQ ID NO: 5) 209 bp Antisense
5'GTTTCTGAGAACAGATGGGGCAC-3'(SEQ ID NO: 6)
[0131] Results: RT-PCR quantification revealed significant
inhibition of transcripts for both E6 and E7 in both SiHa and HeLa
cells treated with 50 .mu.g/mL of either Compound A or Compound B.
The .beta.-actin was used as a house keeping gene to normalize the
data in RT-PCR assay. The specificity of gene specific PCR was
determined using C33A cells which are negative for E6 and E7 genes
(FIG. 2).
Example 4
p53 Nuclear Translocation Assay
[0132] Materials and methods: Two cervical cancer cell lines SiHa
and HeLa (ATCC), positive for HPV genome were used. The cell lines
were transfected with the expression vector containing p53-EGFP
using lipofectamine (Invitrogen, CA, USA). After 48 h of
transfection the cells were maintained for 30-45 days in DMEM
(Sigma Aldrich, catalogue no. D5546) supplemented with 10% FBS and
800 .mu.g/mL Gen eticin (Gibco BRL Life Technologies, Inc) for
30-45 days. The stably expressing cells were used for the p53
Nuclear Translocation Assay.
[0133] Biological Studies: The stably expressing cells were seeded
on 96 well imaging plates (Becton Dickinson, USA) and allowed to
grow for 48 h in a humidified CO.sub.2 incubator at 37.degree. C.
The cell nucleus were stained with vital fluorescent nuclear dye
Hoechst 22334 (0.5 .mu.g/mL) for 5 minutes followed by fresh medium
replacement. A stock solution of 50 mg/mL was prepared in DMSO and
further dilutions were made to obtain the concentration of 2-50
.mu.g/mL were made in the culture medium containing 5% FBS. Cells
were treated separately with different concentrations (2-50
.mu.g/mL) of Compound A and Compound B. The imaging was carried out
at every 12 h using pathway Bio imager (BD, USA) using 20.times.
objective. The cells were segmented based on the nuclear channel
and number of cells positive for nuclear--EGFP were calculated to
score activity. Alternatively the cells were also imaged with Nikon
Ti inverted fluorescent microscope equipped with CCD camera Retiga
Exi and NIS element software. The bright field images were also
collected.
[0134] Results: Microscopic imaging for nuclear translocation of
EGFP linked p53 protein demonstrated that addition of Compound A
and Compound B resulted in increased nuclear translocation of
EGFP-p53 protein in both HeLa and SiHa cells. Further it was also
noticed that nuclear translocation was initiated at 12 h post
treatment and increased up to 24 h at 25 and 50 .mu.g/mL
concentration of Compound A as well as Compound B. The
representative graph showing the percentage p53 EGFP nuclear
positive cell for each drug for the different concentration of drug
is shown in (FIG. 3A and FIG. 3B).
Example 5
Senescence Assay
[0135] This assay was performed to detect senescence associated
.beta.-galactosidase staining to check the senescence status in the
treated cells.
[0136] Materials and methods: Two cervical cancer cell lines SiHa
and HeLa (ATCC), positive for HPV genome were used. The cell lines
were transfected with the expression vector containing p53-EGFP
using lipofectamine (Invitrogen, CA, USA). After 48 h of
transfection the cells were maintained for 30-45 days in DMEM
(Sigma Aldrich, catalogue no. D5546) supplemented with 10% FBS and
800 .mu.g/mL Geneticin (Gibco BRL Life Technologies, Inc) for 30-45
days. The stably expressing cells were used for the Senescence
assay.
[0137] Biological Studies: The stably expressing cells were treated
with Compound A and Compound B separately and fixed for 5 minutes
in 3% formaldehyde, washed, and incubated at 37.degree. C. with
5-bromo-4-chloro-3-indolyl .beta.-D-galactopyranoside solution (1
mg/ml), (prepared by dissolving in a solution containing 40 mM
citric acid (pH 6.5), 5 mM potassium ferrocyanide, 5 mM potassium
ferricyanide, 150 mM NaCl, and 2 mM MgCl.sub.2). After overnight
incubation at 37.degree. C., tissues or cells were visualized by
microscopy. The intense blue staining in the cytosol indicated the
induction of senescent like features in the cells.
Example 6
In Vivo Assay
[0138] All experiments were carried out in accordance with the
guidelines of Committee for the Purpose of Control and Supervision
of Experiments on Animals (CPCSEA) and with the approval of
Institutional Animal Ethics Committee (IAEC) in Piramal Healthcare
Limited, Goregoan, Mumbai, India.
[0139] In vivo efficacy of the compounds of the present invention
for the treatment of HPV associated cancer was studied by using
SiHa (HPV 16 positive cervical cancer) cell line.
SiHa Human Cervical Cancer Xenograft Mouse Model
[0140] Animals used: Nude Nu/J, Homozygous for FOXn1(Nu) male mice,
4 to 6 weeks old, weighing 22 to 25 g (Harlan Laboratories, US).
Animals were housed in animal isolator under specified
pathogen-free conditions maintained at 22 to 25.degree. C. and 55
to 75% humidity, with a 12-hour light/12-hour dark cycle. The mice
were acclimatized for a period of seven days before
experimentation. Animals were handled in a laminar flow hood. All
food and water was autoclaved.
Generation of Subcutaneous Xenograft Tumors in Nude Mice
[0141] Step 1: Preparation of a Single-Cell Suspension for
Injection into Nude Mice
[0142] The adherent SiHa cells (SiHa cell line-ATCC--HBT 35) were
harvested using Trypsin-EDTA solution and suspension was
centrifuged at 800 rpm for 5 minutes. The supernatant was removed
and the cell pellet was re-suspended in 1 mL serum-free MEM medium.
The cell count was obtained using 1:100 dilution. The cells were
diluted to obtain 5.times.10.sup.6 cells per 0.2 mL of
suspension.
[0143] Step 2: Generation of Subcutaneous Xenograft Tumors in Nude
Mice
[0144] On the day of tumor cell injection, cell suspension of step
1 was stored on ice in the laminar airflow hood. Each nude mouse
was injected with 0.2 mL of the cell suspension subcutaneously on
the right flank. The animals were observed after 5 days for tumor
growth by palpation around the injection site.
Conditions for Storage of the Compounds and Dose Preparation
[0145] Compound A: 3.5 mg/mL; vehicle: dextrose (5%) prepared in
water.
[0146] Topotecan Hydrochloride (used as standard): 0.2 mg/mL;
vehicle: methyl cellulose (0.25%).
[0147] Compound B: 20 mg/mL; vehicle: methyl cellulose (0.25%).
[0148] Cisplatin (used as standard): 0.6 mg/mL; vehicle: methyl
cellulose (0.25%).
[0149] All the compounds including the standard were stored at
4.degree. C. to 8.degree. C.
Dosing
[0150] Nude mice were housed in a group of 8 per cage (filter-top
cages) with autoclaved husk bedding and free access to food and
water was provided. Animals were handled as per the standard
guidelines. Treatment was initiated when tumor size volume was
about 100 mm.sup.3. The tumor-bearing mice were randomized (n=8) in
the following groups of treatment: [0151] i) Group 1: Control
group: Tumor-bearing mice administered with vehicle. [0152] ii)
Group 2: Tumor-bearing mice administered once daily with 35 mg/kg
of compound A intraperitoneally. [0153] iii) Group 3: Tumor-bearing
mice were administered with a single dose of 2 mg/kg of Topotecan
(standard) intraperitoneally. [0154] iv) Group 4: Tumor-bearing
mice were administered once daily with 200 mg/kg of Compound B p.o.
[0155] v) Group 5: Tumor-bearing mice were administered with a
single dose of 6 mg/kg of Cisplatin (standard)
intraperitoneally.
Treatment
[0156] Nude mice of Group 1, Group 2 and Group 4 were treated on
days 1-6 and 12-17. Group 3 and Group 5 mice were treated on day 1.
On the 23.sup.rd day animals from all groups were scarified and
samples were harvested for further analysis.
[0157] The volume administered to all the above Groups was 10
mL/kg.
Observations and Measurement
[0158] Following parameters were observed during treatment:
1. Gross animal health was observed everyday 2. Body weight was
observed everyday 3. Tumor was measured 2-3 days apart, using
vernier caliper.
[0159] Tumor volume in mm.sup.3 was calculated using the formula
for a prolate ellipsoid:
Tumor volume (mm.sup.3)=Length (mm).times.[Breadth
(mm).sup.2].times.0.5 [0160] assuming specific gravity of tumor as
1 and .pi. as 3
[0161] Treated to control ratio (.DELTA.T/.DELTA.C %) on a given
day was calculated using the formula:
.DELTA. T / .DELTA. C % on Day X = Tumor size Compound Day X -
Turmor size Compound Day 0 Tumor size control Day X - Tumor size
control Day 0 .times. 100 ##EQU00001##
[0162] Growth inhibition (GI) was calculated using the formula
GI on Day X=100-.DELTA.T/.DELTA.C% on Day X
[0163] Tumor growth inhibition results are given in FIG. 4 and FIG.
5.
[0164] Conclusion: Compound A and Compound B showed significant
tumor growth inhibition in HPV mediated cervical cancer xenograft
model.
* * * * *