The present invention relates to tanaproget derivatives, metabolites, and uses thereof
BACKGROUND OF THE INVENTION
The invention particularly provides novel derivatives of tanaproget.
Tanaproget is a potent, non-steroidal progesterone receptor agonist being developed for
use in contraception as an alternative to currently available oral contraceptives. The
elimination of steroid progestins from contraceptive regimens may reduce the common
side effects of oral contraceptives.
SUMMARY OF INVENTION
The present invention provides metabolites of the active compound tanaproget. These
compounds are useful in methods and kits for monitoring therapy with tanaproget.
Among these metabolites, a rare S-linked glucuronide conjugate, S-glucuronide
tanaproget has been isolated and now synthesized. Having first been identified as a
metabolite, when a tanaproget glucuronide is delivered to a subject, it is a prodrug that is
enzymatically cleaved to tanaproget by glucuronidase in vivo. Thus, the invention
provides tanaproget glucuronide derivatives formulated for administration as a tanaproget
prodrug. In one embodiment, administration is by the oral route to maximize the
advantages of glucuronidase activity in the gut.
Other aspects and advantages of the invention will be apparent from the following
detailed description of the invention.
DETAILED DESCRIPTION OF THE INVENTION
The derivatives of the invention are unique synthetic metabolites that are believed to be
bioequivalent to the metabolites produced by a subject following administration of
tanaproget. Thus, the derivatives of the invention are useful as standards in kits for
monitoring tanaproget therapy, and for generating antibodies specific for tanaproget
metabolites. Such antibodies are useful for monitoring and studying the effects of
tanaproget therapy.
Further, the tanaproget glucuronide derivatives of the invention can be delivered
to a subject as a pro-drug, which is cleaved to the active form of tanaproget in vivo.
Thus, the invention further provides pharmaceutical compositions and kits containing the
tanaproget glucuronide derivatives of the invention, and methods of using same to
deliver tanaproget to a subject. A subject may include any mammal, preferably female,
including humans and non-humans.
As used herein, the PR agonist compound termed "NSP-989" or "tanaproget"
[Wyeth] is characterized by a core structure:
Methods for the synthesis of the illustrated core structure, 5-(4,4-dimethyl-2-thioxo-l,4-
dihydro-2//-3,l-benzoxazin-6-yl)-l-methyl-l/f-pyrrole-2-carbonitrile, are described in
US Patent No. 6,436,929, US Patent Application No. 11/113,794 (filed April 25, 2005),
and US Provisional Patent Application Nos. 60/675,550 (filed April 28, 2005);
60/675,551 (filed April 28, 2005); 60/675,599 (filed April 28, 2005); 60/675,737 (filed
April 28, 2005); and 60/675,738 (filed April 28, 2005), as are uses for this compound.
Other suitable synthetic methods for obtaining tanaproget will be readily apparent to one
of skill in the art. The present invention is not limited by the means for producing
tanaproget.
The present invention provides glucuronide derivatives of the above core
structure. The derivatives of the invention may contain one or more asymmetric centers
and may thus give rise to optical isomers and diastereoisomers. While shown without
respect to stereochemistry below, the present invention includes such optical isomers and
diastereoisomers; as well as the racemic and resolved, enantiomerically pure R and S
stereoisomers; as well as other mixtures of the R and S stereoisomers and
pharmaceutical ly acceptable salts thereof.
In one embodiment, the glucuronide moiety is attached through the N atom in the
oxindole ring. This derivative can be characterized by the structure:
In another embodiment, the glucuronide moiety \s attached through the S atom
bound to the oxindole ring. In one embodiment, this derivative is characterized by the
structure:
In other embodiments, these compounds have the following stereochemistry.
The tanaproget glucuronide derivatives of the invention may be produced
synthetically, using conventional techniques. For example, a solution of tanaproget in
anhydrous neutral solvent (e.g., DMF) is added dropwise under a nitrogen atmosphere to
a solution of a strong base (e.g., sodium hydride) diluted in the solvent and then cooled
using dry ice. After mixing, a solution of acetobromo-a-D-glucuronic acid ester is
added. The reaction solution is then warmed to room temperature, and stirred. After
about 8 to 24 hours, the reaction solution is partitioned between water and an organic
solvent, e.g., ethyl acetate. The aqueous layer is extracted. The combined organic layers
are washed with saturated sodium chloride (NaCl) solution (100 mL), dried, and the
solvent removed in vacua.
Alternatively, the glucuronide derivates of the invention can be produced in an
enzymatic system using suitable methods.
Conventional techniques can be used to recover the purified crude tanaproget
derivatives. In one embodiment, the tanaproget derivatives may be purified by the
means described in US Provisional Patent Application No. 60/675,738 (filed April 28,
2005), hereby incorporated by reference. In another embodiment, the crude extract can
be passed through an HPLC reverse phase column with a gradient of solvents to remove
unreacted tanaproget and the starting materials and reagents from the crude products.
Suitable solvents for use in the gradients of a column can be readily selected by one of
skill in the art. In the example herein, acetonitrile/methanol and acetonitrile/ammonium
acetate were used as the solvents. The fractions, which contain tanaproget derivatives,
are combined and the purified solvents are evaporated to provide the tanaproget
derivative mixture. Thin layer chromatography or other chromatographic methods
known in the art may be used for purification.
In order to isolate the individual derivatives, the purified mixture can be
subjected to further separation using chromatographic techniques. For example, high
performance liquid chromatography (HPLC) can be used. Suitable columns and
conditions for separation will be readily apparent to one of skill in the art given the
present disclosure.
In another aspect, this invention includes pharmaceutical compositions and
treatments which comprise administering to a subject (e.g., a female of child bearing age
for contraception or another mammal for therapeutic purposes) a pharmaceutical ly
effective amount of one or more glucuronide derivatives of tanaproget as described
above as agonists of the progesterone receptor.
The tanaproget glucuronide compounds of this invention, used alone or in
combination, can be utilized in methods of contraception, pre-menopausal, perimenopausal
and/or post-menopausal hormone replacement therapy, and the treatment
and/or prevention of skin disorders, dysfunctional bleeding, estrus synchronization,
uterine leiomyomata, endometriosis, polycystic ovary syndrome, and carcinomas and
adenocarcinomas of the endometrium, ovary, breast, colon, and prostate. Additional
uses of the invention include stimulation of food intake.
The term "skin" is meant to describe the outer covering of a mammalian form
including, without limitation, the epidermis, dermis, and subcutaneous tissues.
Typically, the skin can include other components such as hair follicles and sweat glands.
Skin disorders include, e.g., acne and hirsutism.
The term "acne" is meant to include any skin disorder where a skin pore becomes
blocked and/or thereby becomes inflamed. The term acne includes without limitation
superficial acne, including comedones, inflamed papules, superficial cysts, and pustules;
and deep acne, including deep inflamed modules and pus-filled cysts. Specific acne
conditions can include, but are not limited to, acne vulgaris, acne comedo, papular acne,
premenstrual acne, preadolescent acne, acne venenata, acne cosmetica, pomade acne,
acne detergicans, acne excoriee, gram negative acne, acne rosacea, pseudofolliculitis
barbae, folliculitis, perioral dermatitis, and hiddradenitis suppurativa. The term
"hirsutism" is meant to describe a skin disorder where an overgrowth of hair growth is
observed in areas of the body which are not normally subject to excessive hair growth.
A number of skin disorders can be treated with the compounds of the present
invention, including skin disorders of the hair follicles and sebaceous glands. In one
embodiment, skin disorders such as acne and hirsutism, among others, can be treated
according to the present invention.
Other skin disorders including dry/chapped skin, seboria, psoriasis, or alopecia
can be treated using the compounds and compositions of the invention. The invention is
also useful for treating the skin against the effects of environmental conditions.
This invention also includes pharmaceutical compositions utilizing the
compounds herein, optionally in combination with a pharmaceutically acceptable carrier
or excipient. When the compounds are employed for the above utilities, they may be
combined with one or more pharmaceutically acceptable carriers or excipients, for
example, solvents, diluents and the like, and may be administered orally in such forms as
tablets, capsules, dispersible powders, granules, suspensions containing, for example,
from about 0.05 to 5% of suspending agent, syrups containing, for example, from about
10 to 50% of sugar, and elixirs containing, for example, from about 20 to 50% ethanol,
and the like, or parenterally in the form of sterile injectable solutions or suspensions
containing from about 0.05 to 5% suspending agent in an isotonic medium. Such
pharmaceutical preparations may contain, for example, from about 25 to about 90% of
the tanaproget derivative in combination with the carrier, more usually between about
5% and 60% by weight.
The effective dosage of tanaproget derivative employed may vary depending on
the particular tanaproget glucuronide derivative employed, the mode of administration
and the severity of the condition being treated. However, in general, satisfactory results
are obtained when the compounds of the invention are administered at a daily dosage of
from about 0.5 to about 500 mg/kg of animal body weight, optionally given in divided
doses one to four times a day, or in a sustained release form. For most large mammals,
the total daily dosage is from about 1 to 100 mg, or from about 2 to 80 mg. Dosage
forms suitable for internal use comprise from about 0.5 to 500 mg of the tanaproget
derivative in intimate admixture with a solid or liquid pharmaceutically acceptable
carrier. This dosage regimen may be adjusted to provide the optimal therapeutic
response. For example, several divided doses may be administered daily or the dose may
be proportionally reduced as indicated by the exigencies of the therapeutic situation.
These tanaproget glucuronide derivatives may be administered orally as well as
by intravenous, intramuscular, or subcutaneous routes. Solid carriers include starch,
lactose, dicalcium phosphate, microcrystalline cellulose, sucrose and kaolin, while liquid
carriers include sterile water, polyethylene glycols, non-ionic surfactants and edible oils
such as corn, peanut and sesame oils, as are appropriate to the nature of the tanaproget
derivative and the particular form of administration desired. Adjuvants customarily
employed in the preparation of pharmaceutical compositions may be advantageously
included, such as flavoring agents, coloring agents, preserving agents, and antioxidants,
for example, vitamin E, ascorbic acid, BHT and BHA.
The preferred pharmaceutical compositions from the standpoint of ease of
preparation and administration are solid compositions, particularly tablets and hard-filled
or liquid-filled capsules. Oral administration of the tanaproget glucuronide derivatives is
presently preferred.
These tanaproget derivatives may also be administered parenterally or
intraperitoneally. Solutions or suspensions of these tanaproget derivatives as a free base
or pharmacologically acceptable salt can be prepared in water suitably mixed with a
surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol,
liquid, polyethylene glycols and mixtures thereof in oils. Under ordinary conditions of
storage and use, these preparations contain a preservative to prevent the growth of
microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersions. In all cases, the form must be sterile and must be
fluid to the extent that easy syringe ability exits. It must be stable under conditions of
manufacture and storage and must be preserved against the contaminating action of
microorganisms such as bacterial and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol (e.g., glycerol, propylene glycol and
liquid polyethylene glycol), suitable mixtures thereof, and vegetable oil.
The present invention provides kits or packages of pharmaceutical formulations
designed for use in regimens described herein. In one embodiment, these kits are
designed for daily oral delivery over 21-day, 28-day, 30-day, or 31-day cycles, among
others, and for one oral delivery per day. When the compositions are to be delivered
continuously, a package or kit can include the composition in each tablet. When the
compositions are to be delivered with periodic discontinuation, a package or kit can
include placebos on those days when the composition is not delivered.
In one embodiment, the kits are organized to indicate a single oral formulation or
combination of oral formulations to be taken on each day of the cycle, including oral
tablets to be taken on each of the days specified, and in a further embodiment, one oral
tablet will contain each of the combined daily dosages indicated.
In one embodiment, a kit can include a single phase of a daily dosage of the
compound of the invention over a 21-day, 28-day, 30-day, or 31-day cycle.
Alternatively, a kit can include a single phase of a daily dosage of the compound of the
invention over the first 21 days of a 28-day, 30-day, or 31-day cycle. A kit can also
include a single phase of a daily dosage of the compound of the invention over the first
28 days of a 30-day or 31-day cycle.
In a further embodiment, a kit can include a single combined phase of a daily
dosage of the compound of the invention and an estrogen over a 21-day, 28-day, 30-day,
or 31-day cycle. Alternatively, a kit can include a single combined phase of a daily
dosage of the compound of the invention and an estrogen over the first 21 days of a 28-
day, 30-day, or 31-day cycle. A kit can also include a single combined phase of a daily
dosage of the compound of the invention and an estrogen over the first 28 days of a 30-
day or 31-day cycle.
In another embodiment, a 28-day kit can include a first phase of from 14 to 28
daily dosage units of the compound of the invention; a second phase of from 1 to 11
daily dosage units of an estrogen; and, optionally, a third phase of an orally and
pharmaceutically acceptable placebo for the remaining days of the cycle.
In yet a further embodiment, a 28-day kit can include a first phase of from 14 to
21 daily dosage units of the compound of the invention; a second phase of from 1 to 11
daily dosage units of an estrogen; and, optionally, a third phase of an orally and
pharmaceutical ly acceptable placebo for the remaining days of the cycle.
In another embodiment, a 28-day kit can include a first phase of from 18 to 21 daily
dosage units of a compound of the invention; a second phase of from 1 to 7 daily dosage
units of an estrogen; and, optionally, an orally and pharmaceutically acceptable placebo
for each of the remaining 0 to 9 days in the 28-day cycle.
In another embodiment, a 28-day kit can include a first phase of 21 daily dosage
units of a compound of the invention; a second phase of 3 daily dosage units for days 22
to 24 of an estrogen; and, optionally, a third phase of 4 daily dosage units of an orally
and pharmaceutically acceptable placebo for each of days 25 to 28.
In still another embodiment, the daily dosage of each pharmaceutically active
component of the regimen remains fixed in each particular phase in which it is delivered.
It is further preferable that the daily dose units described are to be delivered in the order
described, with the first phase followed in order by the second and third phases. To help
facilitate compliance with each regimen, the kits may also contain the placebo described
for the final days of the cycle.
A number of packages or kits are known in the art for the use in dispensing
pharmaceutical agents for oral use. In one embodiment, the package has indicators for
each day of the 28-day cycle, and may be a labeled blister package, dial dispenser
package, or bottle.
Metabolites and uses thereof
The tanaproget glucuronide derivatives of the invention are useful for monitoring therapy
with tanaproget or with a tanaproget prodrug (e.g., a S-glucuronide tanaproget
compound) in a subject. Additionally, the invention provides other tanaproget
metabolites useful for monitoring therapy with tanaproget and its prodrugs. When used
as a reagent and/or a standard, the tanaproget metabolite compounds may be labeled,
e.g., with a radioactive, fluorescent, or colorimetric tag.
In one embodiment, the invention further provides an isolated tanaproget
metabolite, which can be enzymatically or synthetically produced. Such a metabolite can
be selected from among a tanaproget glucuronide derivative. Other suitable metabolites
contain the tanaproget core structure and optional substitutions, including, e.g.,
tanaproget having a sulfate moiety located on the thiocarbonyl group; tanaproget having
a hydroxy group located on the pyrrole ring; tanaproget having a hydroxy group located
on the phenylpyrrole ring; and tanaproget having a carbamate in place of the
thiocarbonyl group.
One or more of these tanaproget metabolites can serve as a standard, i.e., for
comparison purposes, in a method for detecting the presence of a tanaproget metabolite
in a sample. A "sample" as used herein refers to a biological sample, such as, for
example, tissue or fluid isolated from an individual (including without limitation plasma,
serum, cerebrospinal fluid, urine, lymph, tears, saliva and tissue sections) or from in vitro
cell culture constituents, as well as samples from the environment.
In another embodiment, one or more of the tanaproget metabolites can be used to
generate an antibody or antibodies that are used to detect the presence of the tanaproget
metabolites in a sample. Suitably, the antibody is a monoclonal or polyclonal antibody
specific for a tanaproget derivative. In one desirable embodiment, such an antibody
selectively binds to the tanaproget derivative of the invention, and distinguishes that
metabolite from tanaproget and other metabolites thereof.
The term "antibody" as used herein is intended to include fragments thereof
which are specifically reactive with tanaproget and/or its metabolites, e.g., an Fv
fragment and a F(ab)2 fragment.
An antibody specific to a tanaproget derivative of the invention can be prepared
using standard techniques wherein the antigen is a derivative of the invention. See, e.g.,
Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold
Spring Harbor, NY.
The polyclonal and monoclonal antibodies to specific sites of a tanaproget
metabolite may be used for development of immunoassays or therapeutic drug
monitoring (TDM) kits. Such assays could include, but are not limited to, direct,
inhibition, competitive or sandwich immunoassays (ELISA or other assay systems), RIA,
solid or liquid phase assays or automated assay systems.
Where a competitive assay is used, the competitor for the antibody may be a
tanaproget derivative of the invention bound to the assay plate, or a labeled derivative,
e.g., a fluorolabeled derivative, a radiolabeled derivative, or a tritiated derivative.
Where desired, a kit can be used to facilitate the methods of the invention.
A kit of the invention may contain an appropriately labeled tracer, an antibody, standard,
instructions for use, and packaging. The label for the tracer may be any suitable label,
e.g., a radioactive, fluorescent or calorimetric label. Where convenient, the components
of the kit may be in lyophilized form.
The assay procedure of the invention has the advantages that it may be carried
out rapidly and simply using standard bioanalytical equipment to give accurate and
reproducible results. Also, whole blood may be used without the need for extraction.
The invention also provides an assay kit suitable for detecting the amount of
tanaproget metabolite in a sample (e.g., blood or urine). In one embodiment, the kit
comprises a binding competitor that displaces the pharmaceutical from tanaproget
metabolite in the sample; and an antibody that binds to the pharmaceutical but not
significantly to the binding competitor.
The following examples are provided to illustrate the invention and do not limit
the scope thereof. One skilled in the art will appreciate that although specific reagents
and conditions are outlined in the following examples, modifications can be made which
are meant to be encompassed by the spirit and scope of the invention.
The following examples are illustrative of the methods for generating compounds
of the invention.
Example 1 - Preparation of Tanaproget S-Glucuronide from Rat Liver
Microsomes
The glucuronide of tanaproget was prepared in male rat liver microsomes, and the
structure was identified as a S-glucuronide conjugate by liquid chromatography
(LC)/mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy.
This metabolite was also identified as a major metabolite in both male and female rat,
dog, and human liver microsomes, and it was also the major drug related component
found in rat, dog, and human plasma. This S-glucuronide can also be synthetically
prepared simultaneously with a N-glucuronide. The two synthetic glucuronides could be
separated by HPLC and their structures were all characterized by LC/MS and NMR
spectroscopy. In the following scheme, the NSP-989 terminology is used in the place of
tanaproget.
A. Incubation of Tanaproget And Extraction OfTanaproget Glucuronide
Liver microsomes from Sprague-Dawley rats were prepared in-house
using a differential ultracentrifugation method described by Lake [Lake, B. In
Biochemical Toxicology: A Practical Approach, Snell, K, Mullock, B (eds), IRL Press:
England, 1987, 183-215] with slight modifications. Microsomal protein and cytochrome
P450 content were determined by the method of Bradford [Bradford, MM Anal.
Biochem. 1976; 72:248-254] and Omura and Sato [Omura, T, Sato, R J. Biol. Chem.
1964; 238:2370-2378], respectively. The protein concentration and P450 content were
50.9 mg/mL and 0.42 nmol/mg protein, respectively. Ammonium acetate, magnesium
chloride, and uridine diphosphoglucuronic acid (UDPGA) were purchased from Sigma
Chemical Company (St. Louis, MO). The solvents used for extraction and for
chromatographic analysis were HPLC grade or ACS Reagent Grade (Mallinckrodt
Baker, Phillipsburg, NJ).
Incubations (100 mL) were performed with tanaproget (40 yM), UDPGA
(5 mM), magnesium chloride (10 mM), and male rat liver microsomes (1.5 mg/mL), in
0.1 M potassium phosphate buffer, pH 7.4 at 37°C. The samples were pre-incubated for
1 minute at 37°C, and the reactions were initiated by the addition of UDPGA. The
sample was cooled using an ice bath to stop the reaction after 3 hours. Unreacted
tanaproget was removed from the samples by two extractions with diethyl ether (200 mL
Tanaproget glucuronide and remaining unreacted tanaproget were
extracted by solid phase extraction using C-18 cartridges and elution with methanol
(10 mL). The methanol eluent was dried by rotary evaporation under vacuum at room
temperature. The residues were extracted with 50% acetonitrile in water (5 mL) and
centrifuged at 3500 rpm for 15 min. Aliquots (800 uL) of supernatants were analyzed by
a Waters 2690® HPLC system with a semi-preparative column. Separation of the
tanaproget metabolites was accomplished on a Phenomenex Luna™ column (C18, 250 x
10 mm ID, 5 um particle size) (Phenomenex, Torrance, CA) and metabolites were
detected by monitoring UV absorbance at 310 nm. The autosampler temperature was set
to 6 °C, while the column was at room temperature. Ammonium acetate (10 mM, pH
4.5) and acetonitrile were used as mobile phase A and B, respectively. The following
gradient at a flow rate of 2 mL/min with a 5 min post run re-equilibration was employed:
0 min 20% B, 1 min 20% B, 10 min 40% B, 20 min 70% B, 25 min 95% B, 28 min 95%
B, 30 min 20% B. Under these conditions, the tanaproget glucuronide peak (Ml) at
15.5 min was collected for NMR spectroscopic analysis. Fractions containing the
glucuronide conjugate peak were collected into clean tubes and frozen on dry ice
immediately after collection. All glucuronide fractions were combined and acetonitrile
was removed by rotary evaporation. The glucuronide was extracted from the aqueous
eluates by solid phase extraction using C-18 cartridges. Water (2 mL) was used to wash
residual buffer and 50% methanol in water was used to elute the glucuronide conjugate.
The methanol/water eluates were dried by rotary evaporation under vacuum at room
temperature. The remaining aqueous solutions were transferred to a 5 mL conical vial to
remove water by lyophilization. Drying of the solid residue was continued overnight to
remove additional moisture prior to NMR spectroscopic analysis.
B. HPLC/MS Analysis Conditions:
A Micromass Quattro Ultima™ triple quadrupole mass spectrometer
(Waters Corp., Milford, MA) was used in this work. It was equipped with electrospray
ionization (ESI) interface and operated in both the positive and negative ionization
modes. Settings for the mass spectrometer were: ESI spray 2.5 KV, cone 50 V, mass
resolution 0.7 Da ± 0.2 Da width at half height, desolvation gas flow 900-1000 L/h, cone
gas flow 50-80 L/h, source block temperature 80 °C, desolvation gas temperature 250
°C. LC/MS data were analyzed with Micromass MassLynx software (Waters Corp.,
versions 3.5 and 4.0).
Solvents used for chromatographic analysis of tanaproget glucuronide
isolated from rat liver microsomes were HPLC grade or ACS Reagent Grade
(Mallinkrodt Baker, Phillipsburg, NJ and EMD Chemicals, Gibbstown, NJ). The HPLC
system in conjunction with the mass spectrometer was a Waters Alliance model 2695™
HPLC system. It was equipped with a built-in autosampler and a model 996 diode array
UV detector set to monitor 210-350 nm. Separations were accomplished on a
Phenomenex Luna C18(2) column (150 x 2 mm, 5 um) (Phenomenex, Torrance, CA)
with a Deltabond™ C18 guard column (10 x 2 mm) (ThermoElectron Corp., Bellefonte,
PA). The flow rate was 0.3 mL/min. During LC/MS sample analysis, up to 10 min of
the initial flow was diverted away from the mass spectrometer prior to evaluation of
metabolites. Mobile phase A was 10 mM ammonium acetate in water, pH 4.5, (diluted
from a 0.5 M stock solution of equal molar amounts of ammonium acetate and acetic
acid) and mobile phase B was acetonitrile. The linear mobile phase gradient used was: 0
min 10% B, 1 min 10% B, 10 min 15% B, 35 min 17.5% B, 36 min 25% B, 50 min 30%
B, 55 min 50% B, 60 min 90% B, 62 min 90% B, 65 min 10% B, 75 min 10% B.
C. LC/MS Results
Tanaproget glucuronide produced protonated and deprotonated molecular
ions ([M+H]+ and [M-H]~, at m/z 474 and 472, respectively), which indicated a
molecular weight of 473. This was 176 Da larger than tanaproget. Loss of 176 Da from
m/z 474 in the positive ionization mass spectrum and from m/z 472 in the negative
ionization mass spectrum generated the fragment ions at m/z 298 in the positive
ionization mode and m/z 296 in the negative ionization mode, which are assigned as
tanaproget. The glucuronic acid ion fragment was observed at m/z 175 in negative
ionization mode. The m/z 339 fragment ion appears to be from fragmentation of the
glucuronic acid ring. These data were consistent with glucuronic acid conjugation of
tanaproget.
D. NMR Spectroscopy
Deuterated dimethylsulfoxide (DMSO-de) was used for all NMR samples.
For the metabolite samples isolated from rat liver microsomes, dissolution was done in a
glove bag under argon to reduce absorption of atmospheric water. Typically 50 jaL
aliquots of a total of 200 nL of DMSO-de were used to rinse the vial containing the
vacuum dried sample and then transferred into a 3 mm NMR tube. NMR spectra were
obtained at 500 (Varian Inova™ instrument), and 600 (Bruker Avance™ instrument)
MHz. The bulk of the experimental NMR work was performed on the Varian Inova
500™ MHz instrument equipped with a Varian™ 3 mm !H observe indirect detection
probe. Chemical shifts 8 (ppm) are reported relative to internal TMS (80.0) for !H and
13C. In DMSO-de, *H chemical shifts are referenced to residual protonated DMSO at
82.49, and I3C chemical shifts are referenced to internal DMSO-de at 839.5. Chemical
shifts for 15N are reported relative to liquid ammonia (80.0) and referenced to external
formamide at 8112.0. Proton multiplicities are reported as s = singlet, d = doublet, dd =
doublet of doublets, and m = multiplet. J values are given as 'H - *H coupling constants
in Hz.
General parameters for 'H-NMR experiments include a 5000 Hz spectral width,
32K data points, a 45° pulse width, a 1 second relaxation delay and the averaging of
from 32 to 1000 scans, depending on sample concentration. Line broadening (~ 0.5
Hz) or gaussian processing routines were used to increase the signal-to-noise (S/N).
General parameters for a 13C-NMR experiment include a 25,000 Hz spectral width, 64K
data points, a 45° pulse width, a 1 second relaxation delay and averaging of at least
10,000 scans to achieve optimal S/N. In addition, 2 Hz line broadening was applied to
increase S/N. All spectra were acquired at 25 °C.
Several types of 2D NMR experiments were utilized to determine !H-'H and *HI3C
connectivities. These included a gCOSY experiment for the determination of threebond
'H-'H connectivities, gHSQC and gHMBC experiments for the determination of
one-, two-, three-, and four-bond 'H-13C- connectivities, and a NOESY or ROESY
experiment for the determination of through-space connectivities.
Despite several attempts at using freshly isolated tanaproget glucuronide samples,
the purity and concentration of the samples were typically low (< 70% pure and an
estimated total amount of < 20 ug in solution) which hampered acquisition of complete
2D NMR datasets. In addition, during the process of the microsomal incubation and
subsequent isolation steps, the solution tended to rapidly produce the carbamate analog
of tanaproget (the sulfur of tanaproget replaced by oxygen). Nevertheless, sufficient
chemical shift, coupling, and 2D NMR correlations were obtained to make nearly
complete *H and 13C assignments of tanaproget glucuronide isolated from rat liver
microsomes. However, the location of the glucuronide attachment from the
microsomally derived sample alone could not be determined. Only one key protoncarbon
heteronuclear correlation was observed in the gHMBC NMR spectrum of the rat
liver microsome incubation sample, that from H-l' (5.10 ppm) to C-6 (161.63 ppm).
However, this same crosspeak was expected whether the metabolite was an N-or an
HPLC/MS data for the synthetic compounds were acquired using a
Waters Alliance 2695™ HPLC coupled to a Waters ZQ™ mass spectrometer. In general,
the samples were analyzed using an open-access LC/MS method as previously described
[Mallis, LM, Sarkahian, AB, Kulishoff, JM, Jr., Watts, WL, Jr. J. Mass Spectrom. 2002;
37:889-896].
Deuterated dimethylsulfoxide (DMSO-dg) was used for all NMR samples
except for 2, which was dissolved in CDCb (deuterated solvents from Aldrich,
Milwaukee, WI). NMR spectra were obtained at 300 (Bruker DPX™ instrument), 400
(Varian Inova™ instrument), 500 (Varian Inova™ instrument), and 600 (Bruker
Avance™ instrument) MHz. The bulk of the experimental NMR work was performed on
the Varian Inova 500™ MHz instrument equipped with a Varian™ 3 mmH observe
indirect detection probe. Chemical shifts 8 (ppm) in DMSO-de are reported as described
previously for tanaproget glucuronide. In CDCb, *H chemical shifts are referenced to
residual protonated CHCls at 67.27, and 13C chemical shifts are referenced to internal
CDCb at 877.7. Chemical shifts for 15N are reported as described previously for
tanaproget glucuronide.
General parameters for ^-NMR and I3C-NMR experiments are as
described previously for tanaproget glucuronide. All spectra were acquired at 25 °C.
2D NMR experiments used to determine 'H-'H and 'H-13C connectivities were gCOSY,
gHSQC, gHMBC, NOESY and ROESY.
B. Preparation of the Protected S-Glucuronide Acid (2) of Tanaproget
A solution of tanaproget (0.292 g, 1.0 mmol) in anhydrous dimethyl
formamide (DMF) (10 mL) was added dropwise under a nitrogen atmosphere to a
solution of sodium hydride (NaH) (0.052 g, 2.2 mmol) in DMF (50 mL) that was cooled
to approximately -70°C (dry ice). After stirring for 10 min, a solution of acetobromo-ot-
D-Glucuronic acid methyl ester (0.396 g, 1 mmol) in DMF (10 mL) was added dropwise.
The reaction solution was then warmed to room temperature, and stirred for a total of 24
hours. After 24 hours, the reaction solution was partitioned between water (100 mL) and
ethyl acetate (100 mL). The aqueous layer was extracted again with ethyl acetate
(100 mL). The combined organic layers were washed with saturated sodium chloride
(NaCl) solution (lOOmL), dried (magnesium sulfate, MgSC>4), and the solvent removed
in vacua. Aliquots of the crude material were chromatographed under low-pressure
reversed-phase CIS (RediSep/ISCO, 125 x 25 mm ID, 40 |im particle size) conditions
with a gradient of 50 - 95% acetonitrile /water. Fractions eluting with 75 - 80%
acetonitrile /water contained the S-protected (D)-Glucuronic acid derivative (2) of
tanaproget. A total of approximately 200 mg of (2) was obtained at 95% purity based on
NMR spectroscopic analysis (32% isolated yield). All chemicals were purchased from
Aldrich (Milwaukee, WI) and used without further purification or drying.
C. Preparation of N- (3) and S-Glucuronic Acid (4) Derivatives of
Tanaproget
In a separate reaction, a solution of tanaproget (0.146 g, 0.5 mmol) in
anhydrous DMF (5 mL) was added dropwise under a nitrogen atmosphere to a solution
of NaH (0.027 g, 1.1 mmol) in DMF (25 mL) that was cooled to approximately -70°C
(dry ice). After stirring for 10 min, a solution of acetobromo-cc-D-glucuronic acid
methyl ester (0.198 g, 0.5 mmol) in DMF (5 mL) was then added dropwise. The reaction
solution was then warmed to room temperature, and stirred for a total of 8 hours. The
reaction solution was partitioned between water (100 mL) and ethyl acetate (100 mL).
The aqueous layer was extracted again with ethyl acetate (lOOmL). The combined
organic layers were washed with saturated NaCl solution (100 mL), dried (MgSC^), and
solvent removed in vacua to yield 0.350 g of crude reaction material. To a portion of
this material (0.210 g) was added a solution of MeOH / Hunig's base ((iso-Pr)2NEt) /
F^O (5 mL / 2 mL / 2 mL) and the solution was stirred for 8.5 hours at room
temperature. The pH of the reaction solution was then adjusted to 2.5 using HC1
(concentrated, approximately 1.5 mL) and chromatographed by semi-preparative
reversed-phase HPLC using YMC-Pack CN 150 x 20 mm, S-5 (am column with a
gradient of 15 - 35% acetonitrile in 10 mM (aqueous) ammonium acetate. From repeated
injections, approximately 5 mg of the N-(D)-glucuronic acid derivative (3) (3% isolated
yield from tanaproget) and 15mg of the S-(D)-glucuronic acid derivative (4) (10%
isolated yield from tanaproget) was purified to > 98% purity for NMR analysis and
LC/MS analysis and comparison.
Example 3 - Comparison of Synthetic Compounds to M1
From extensive comparison of the spectral and chromatographic data of the
microsomally-derived metabolite and the synthetic compounds, the metabolite has been
determined to be the S-(P)-D-glucuronide of tanaproget.
The positive ionization mode LC/MS spectrum (not shown) of synthetic
compound 2, the protected S-glucuronide of tanaproget, gave a protonated molecular ion
[M+H]+ at 614, indicating a molecular weight of 613. Loss of 297 Da gave m/z 317,
which is assigned to [M+H - tanaproget]*. An ion signal at m/z 257 is assigned to m/z
317 - acetic acid (C2H4O2), and an observed ion at m/z 197 is assigned to m/z 257 -
acetic acid ^FLtCh). Also, an ion at m/z 155 is assigned to m/z 197 - acetyl (CiHiO) +
H. The protonated and deprotonated LC/MS spectra (not shown) of the S-glucuronide 4
gave an [M+H]+ ion at 474 and an [M-H]" ion at m/z 472, respectively, (as was also the
case for 3, spectra not shown), indicating glucuronidation of tanaproget. Also observed
for 4 in the positive and negative ionization mode mass spectra are the ions m/z 298 and
m/z 296, assigned as tanaproget, that is, the loss of glucuronic acid. In addition, a
fragment at m/z 175, assigned as glucuronic acid, was also detected in the negative
ionization spectrum.
HPLC comparisons were also made to confirm whether the major synthetic
glucuronide of tanaproget, S-glucuronide 4, was identical to tanaproget glucuronide, now
proposed to be the S-glucuronide from the NMR and mass spectral results described
above. Five different types of reverse phase HPLC columns were used under two
different mobile phase conditions. These ten HPLC conditions covered a wide range of
selectivity as evidenced by the change of elution order of the major and minor
components in the samples. To compare and match the retention time of the major peaks
in the two samples, spiking experiments were performed. The synthetic glucuronide that
was determined to be S-glucuronide 4 was found to have identical retention times as the
tanaproget glucuronide metabolite under all ten HPLC conditions.
Key NMR correlations in the synthetic compounds 3 and 4 which located the site
of glucuronidation in tanaproget are described below. The proton chemical shift and
coupling constant for the anomeric proton (H-l5) with p-stereochemistry on the Nglucuronide
is 6.31 ppm, and is a doublet with a coupling constant of 9.5 Hz. The carbon
chemical shift of the anomeric carbon (C-T) is 90.2 ppm. The carbon chemical shift of
the benzoxazine-2-thione carbon (C-6) of the N-glucuronidated metabolite of tanaproget
is 188.0 ppm; this compared with the thiocarbonyl carbon of the benzoxazine-2-thione
group observed in the parent molecule, tanaproget, at 182.8 ppm.
There are four important 3-bond correlations observed in the HMBC spectrum
(not shown) of this molecule. They are from the anomeric proton (H-T) to the
benzoxazine-2-thione carbon (C-6) and to the sp2 aromatic carbon at 131.5 ppm (C-4),
and from the protons H-7 and H-9, observed at 7.50 ppm and 7.57 ppm, respectively, to
C-4. The proton chemical shift of the proton (H-10) that is in a position peri- to the Nglucuronide
has moved downfield to 7.80 ppm, compared to 7.13 ppm in the parent
molecule, indicating the proximity of the glucuronic acid group (and possibly the
carboxylic acid moiety) to this proton. *H-15N gHMBC experiments were run on all
compounds studied, but signals from the key nitrogen N-5 were not observed for any
compound except tanaproget (5144.81), which had an HMBC crosspeak to H-10. The
other nitrogen expected, N-15, was only observed in tanaproget (5154.99) and in 3
(6155.40), and in each compound had gHMBC correlations to H-17, H-18, and H-19.
The proton chemical shift and coupling constant for the anomeric proton (H-T)
with p-stereochemistry on the synthetic S-glucuronide 4 is 5.11 ppm, and is a doublet
with a coupling constant of 10.2 Hz. The carbon chemical shift of the anomeric carbon
(C-T) is 85.0 ppm. The carbon chemical shift of the derivatized benzoxazine-2-thione
carbon (C-6) of this S-glucuronidated metabolite of tanaproget is observed at 161.3 ppm,
quite upfield shifted from the observed thiocarbonyl carbon chemical shift (182.8 ppm)
of the benzoxazine-2-thione group observed in the parent molecule, tanaproget. There is
a 3-bond correlation observed in the HMBC spectrum of this molecule between the
anomeric proton (H-F) of the p-glucuronic acid and the derivatized benzoxazine-2-
thione carbon (C-6). Finally, there is a 2-bond correlation observed in the HMBC
l§
spectrum between the gem-dimethyl protons (H-l 1 and H-12) observed at 1.59 ppm and
1.70 ppm and the carbon (C-2) to which this group is attached, observed at 81.5 ppm.
These data confirm that the benzoxazine-2-thione (N(C=S)O) group of tanaproget did
not rearrange to a thiolcarbamate (N(C=O)S) group before S-glucuronidation.
Comparison of the !H NMR spectra and the NMR data derived from tanaproget
glucuronide and the two synthetic compounds 3 and 4, revealed that tanaproget
glucuronide must be the S-glucuronide derivative of tanaproget. The HI' chemical shift
for tanaproget glucuronide is 5.10 ppm, and 5.11 ppm for 4, compared to H-l' at 6.31
ppm observed for 3. Also, C-6, in the benzoxazine-2-thione part of tanaproget, resonates
at -161 ppm for both tanaproget glucuronide and 4, but at 188.0 ppm for 3 and at 182.8
ppm for tanaproget.
Example 4 - Enzymatic Hydrolysis of Tanaproget Glucuronide conjugate
metabolite
In order to confirm that that the tanaproget glucuronide derivative is a prodrug
when delivered to patients, the ability of the glucuronide conjugate to be enzymatically
cleaved by a glucuronidase which is native to the gastrointestinal tract in humans was
used in the following assay.
Pooled urine samples (4-8 hr) from healthy women were hydrolyzed by
Glusulase®. Aliquots (1 mL) of pooled urine were adjusted to pH 5 with 0.5 mL of 0.6
M sodium acetate buffer. The diluted urine was mixed with Glusulase® (9,000
units/mL, 100 u.L) and incubated at 37°C for 1 hr with gentle shaking. The reaction was
stopped by the addition of 2 mL of acetone and the precipitate was removed by
centrifugation. The supernatant was dried under nitrogen in a TurboVap™ (Caliper Life
Sciences, Hopkinton, MA). The residues were reconstituted with 1 mL of 60% methanol
in water and subsequently analyzed by HPLC and LC/MS, which confirmed the
conversion of glucuronide into parent drug tanaproget. Control incubations were
conducted under the same conditions, but without adding Glusulase®, or with
Glusulase® and lOmM of saccharolactone (|3-glucuronidase inhibitor) and no conversion
of glucuronide conjugate into tanaproget was observed.
Example 5 - Pharmacology
Tanaproget glucuronides are a prodrug of tanaproget, a first-in-class nonsteroidal
progesterone receptor agonist for use primarily in contraception. The effect of
tanaproget glucuronides on alkaline phosphase activity in T47D cells is analyzed as
follows.
A. Reagents:
Culture medium: DMEM:F12 (1:1) (GIBCO, BRL) supplemented with
5% (v/v) charcoal stripped fetal bovine serum (not heat-inactivated), 100 U/mL
penicillin, 100 ng/mL streptomycin, and 2 mM GlutaMax (GIBCO, BRL).
Alkaline phosphatase assay buffer: I. 0.1M Tris-HCl, pH 9.8, containing
0.2% Triton X-100, 0.1M Tris-HCl, pH 9.8, containing 4 mM p-nitrophenyl phosphate
(Sigma).
B. Cell Culture And Treatment:
Frozen T47D cells are thawed in a 37 °C water bath and diluted to
280,000 cells/mL in culture medium. To each well in a 96-well plate (Falcon, Becton
Dickinson Labware), 180 u,l of diluted cell suspension is added.
Twenty u,l of reference or test compounds diluted in the culture medium is
then added to each well. The cells are incubated at 37 °C in a 5% CC>2 humidified
atmosphere for 24 hours. For high throughput screening, one concentration of each
compound will be tested at 0.3 u,g/mL. Based on an average molecular weight of 300
g/mol for the compounds in the library, the concentration is approximately 1 uM.
Subsequently, active compounds will be tested in dose response assays to determine
ECSO.
C. Alkaline Phosphatase Enzyme Assay:
At the end of treatment, the medium is removed from the plate. Fifty ul of
assay buffer I is added to each well. The plates are shaken in a liter plate shaker for 15
min. Then 150 ul of assay buffer II is added to each well. Optical density measurements
are taken at 5 min intervals for 30 min at a test wavelength of 405 nM.
D. Analysis of dose-response data.
For reference and test compounds, a dose response curve is generated for
dose vs. the rate of enzyme reaction (slope). Square root-transformed data are used for
analysis of variance and nonlinear dose response curve fitting for both agonist and
antagonist modes. Huber weighting is used to down-weight the effects of outliers. EC50
values are calculated from the retransformed values. JMP software (SAS Institute, Inc.)
is used for both one-way analysis of variance and non-4 linear dose response analysis in
both single dose and dose response studies.
E. Results
Tanaproget S-glucuronide had 0.1 nM with 60% efficacy as compared to
progesterone.
Tanaproget can be regenerated by enzymatic hydrolysis experiments, such
as described in Example 4 above.
Example 6 - Additional Tanaproget Metabolites
Using the methods described in Example 1 for obtaining the tanaproget
glucuronide metabolites, additional tanaproget metabolites were observed in the male rat
liver microsomal preparations and in male and female monkey liver microsome
preparations using known techniques.
Metabolite M2 was observed in male rat liver microsomal preparations. This
metabolite produced a [M-H]-at m/z 312. The product ion at m/z 58 from NCS-,
indicating an unchanged thioamide group, was also observed for NSP-989. Product ions
at m/z 159 and 195 indicated the pyrrole ring as the site of metabolism. Therefore,
metabolite M2 was proposed to be a hydroxy-NSP-989 with the hydroxy group at the
pyrrole moiety.
Metabolite M3 was observed in male rat and male and female monkey liver
microsome preparations. This metabolite produced a [M-H]-at m/z 312. The product ion
at m/z 58 from NCS-, indicating an unchanged thioamide group, was also observed for
NSP-989. The product ion at m/z 237, observed for NSP-989 at m/z 220, indicated
oxidation of either the phenyl or pyrrole ring. Product ions at m/z 195 and 252 were
consistent with oxidation of the phenyl or pyrrole ring. Therefore, metabolite M3 was
proposed to be a hydroxy-NSP-989 with the hydroxy group at the phenyl or pyrrole
moiety.
Metabolite M4 was observed in all in vitro metabolism samples. This metabolite
produced a [M-H]-at m/z 280, which was 16 atomic mass units (amu) less than NSP-989.
The lack of a product ion at m/z 58 and the 16 amu shift in molecular weight indicated a
modified thioamide group. Product ions, observed at m/z 129, 220 and 234 were also
present for NSP-989. Metabolite M4 also had the same HPLC retention time and product
ion spectrum (data not shown) as synthetic NSP-989-carbamate. Therefore, metabolite
M4 was identified as NSP-989-carbamate.
Metabolite M6 was observed in dog and in rat liver microsome preparations. This
metabolite produced a [M-H]-at m/z 344. The product ions at m/z 80 and 81 indicated
the presence of a sulfate group. Product ions at m/z 220 and 234 indicated that the nonthiocarbonyl
portions of the NSP-989 molecule were unchanged. Therefore, metabolite
M6 was identified as NSP-989 sulfate (6-(5-Cyano-l-methyl-lH-pyrrol-2-yl)-4,4-
dimethyl-4H-benzo[d][l ,3]oxazine-2-sulfonic acid).
These metabolites can be purified from microsome preparations using methods
described above for the glucuronide derivatives. Alternatively, these metabolites can be
generated using convention synthetic techniques.
All patent, patent publications, and other publications listed in this specification
are incorporated herein by reference. While the invention has been described with
reference to particular embodiments, it will be appreciated that modifications can be
made without departing from the spirit of the invention. Such modifications are
intended to fall within the scope of the appended claims.

WE CLAIM:
1. A synthetic glucuronide derivative of tanaproget.
2. The synthetic glucuronide derivative of tanaproget as claimed in claim 1,
said tanaproget having the core structure:
3. The synthetic glucuronide derivative of tanaproget as claimed in claim 1,
wherein said compound is an S-glucuronide derivative of tanaproget.
4. The synthetic glucuronide derivative of tanaproget as claimed in claim 3,
wherein said compound is an S-p-(D)-glucuronide derivative of tanaproget.
5. The derivative as claimed in claim 1 characterized by the structure:
6. The derivative as claimed in claim 1 characterized by the structure
7. A kit for monitoring therapy with tanaproget, said kit comprising a
standard comprising a derivative as claimed in any of claims 1 to 6.
8. A method for detecting metabolites of tanaproget, said method
comprising comparison of a sample to a derivative as claimed in any of claims 1 to 6.
9. An antibody generated using a derivative as claimed in claim 1, said
antibody being specific for tanaproget or a derivative thereof.
10. A kit for monitoring therapy with tanaproget, said kit comprising an
antibody as claimed in claim 9.
11. A method for detecting metabolites of tanaproget, said method
comprising detecting binding to an antibody as claimed in claim 9.
12. A composition useful in an assay for monitoring tanaproget therapy in a
subject comprising a tanaproget metabolite selected from the group consisting of:
(a) an enzymatically derived tanaproget glucuronide derivative;
(b) tanaproget having a sulfate moiety located on the thiocarbonyl
group;
(c) tanaproget having a hydroxy group located on the pyrrole ring;
(d) tanaproget having a hydroxy group located on the
phenylpyrrole ring; and
(e) tanaproget having a carbamate located on the thiocarbonyl
group.
13. A kit for detecting metabolites of tanaproget, said kit comprising
packaging and a standard eomprising a composition as claimed in claim 12.
14. A method for detecting metabolites of tanaproget, said method
comprising comparison of a sample to a tanaproget derivative selected from the group
consisting of:
(a) an enzymatically derived tanaproget glucuronide derivative;
(b) tanaproget having a sulfate moiety located on the thiocarbonyl
group;
(c) tanaproget having a hydroxy group located on the pyrrole ring;
(d) tanaproget having a hydroxy group located on the
phenylpyrrole ring; and
(e) tanaproget having a carbamate located on the thiocarbonyl
group.
15. An antibody generated using a tanaproget derivative selected from the
group consisting of:
(a) an enzymatically derived tanaproget glucuronide derivative;
(b) tanaproget having a sulfate moiety located on the thiocarbonyl
group;
(c) tanaproget having a hydroxy group located on the pyrrole ring;
(d) tanaproget having a hydroxy group located on the
phenylpyrrole ring; and
(e) tanaproget having a carbamate located on the thiocarbonyl
group.
16. A kit for monitoring therapy with tanaproget, said kit comprising an
antibody as claimed in claim 15.
17. A method for detecting metabolites of tanaproget, said method
comprising detecting binding to an antibody as claimed in claim 15.
18. A composition comprising a pharmaceutically acceptable carrier and a
glucuronide derivative of tanaproget as defined in any one of claims 1 to 6.
19. Use of a glucuronide derivative of tanaproget as defined in any one of
claims 1 to 6, or a pharmaceutically acceptable salt thereof, in preparing a medicament
useful for contraception in a mammal.
20. The invention substantially such as herein described.