U.S. patent application number 17/212806 was filed with the patent office on 2021-07-15 for pyrimidine prodrugs for the treatment of viral infections and further diseases.
The applicant listed for this patent is Janssen Sciences Ireland Unlimited Company. Invention is credited to Jacques Armand Henri BOLLEKENS, Laurent Jacques Emile CALMUS, Werner Constant Johan EMBRECHTS, Jerome Emile Georges GUILLEMONT, Florence Marie HERSCHKE, Tim Hugo Maria JONCKERS, David Craig MC GOWAN, Pierre Jean-Marie Bernard RABOISSON, Bart Henri Theresia STOOPS.
Application Number | 20210214317 17/212806 |
Document ID | / |
Family ID | 1000005477979 |
Filed Date | 2021-07-15 |
United States Patent
Application |
20210214317 |
Kind Code |
A1 |
GUILLEMONT; Jerome Emile Georges ;
et al. |
July 15, 2021 |
PYRIMIDINE PRODRUGS FOR THE TREATMENT OF VIRAL INFECTIONS AND
FURTHER DISEASES
Abstract
This invention relates to pyrimidine prodrug derivatives,
processes for their preparation, pharmaceutical compositions, and
their use in therapy.
Inventors: |
GUILLEMONT; Jerome Emile
Georges; (Ande, FR) ; JONCKERS; Tim Hugo Maria;
(Heist-op-den-Berg, BE) ; RABOISSON; Pierre Jean-Marie
Bernard; (Wavre, BE) ; MC GOWAN; David Craig;
(Brussels, BE) ; EMBRECHTS; Werner Constant Johan;
(Beerse, BE) ; STOOPS; Bart Henri Theresia;
(Wechelderzande, BE) ; HERSCHKE; Florence Marie;
(Antwerp, BE) ; BOLLEKENS; Jacques Armand Henri;
(Ixelles, BE) ; CALMUS; Laurent Jacques Emile;
(Braine L'Alleud, BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Janssen Sciences Ireland Unlimited Company |
Co Cork |
|
IE |
|
|
Family ID: |
1000005477979 |
Appl. No.: |
17/212806 |
Filed: |
March 25, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
16338256 |
Mar 29, 2019 |
10968184 |
|
|
PCT/EP2017/074600 |
Sep 28, 2017 |
|
|
|
17212806 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 239/48 20130101;
C07D 405/12 20130101; C07F 9/6512 20130101 |
International
Class: |
C07D 239/48 20060101
C07D239/48; C07D 405/12 20060101 C07D405/12; C07F 9/6512 20060101
C07F009/6512 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 29, 2016 |
EP |
16191568.1 |
Claims
1. A compound of formula (I) ##STR00008## or a pharmaceutically
acceptable salt, solvate or polymorph thereof, wherein R.sub.1A is
hydrogen or a substituted or unsubstituted C.sub.1-3 alkyl;
R.sub.1B is selected from a substituted or unsubstituted C.sub.1-3
alkyl, heterocycle, or a substituted or unsubstituted
phosphoramidate; and R.sub.2 is a C.sub.1-8 alkyl optionally
substituted by a hydroxyl group, --OSi(C.sub.1-4 alkyl).sub.3, or
an organophosphate group.
2. A compound according to claim 1 wherein R.sub.1B is a
substituted or unsubstituted phosphoramidate and wherein R.sub.2 is
a C.sub.1-6 alkyl substituted by a hydroxyl group.
3. A compound according to claim 1 wherein R.sub.1A or R.sub.1B are
methyl and wherein R.sub.2 is a C.sub.6-alkyl substituted by a
hydroxyl group.
4. (canceled)
5. (canceled)
6. (canceled)
7. A compound according to claim 1, wherein R.sub.1A and R.sub.1B
are methyl and wherein R.sub.2 is a C.sub.6-alkyl substituted by a
hydroxyl group.
8. A compound according to claim 1, wherein R.sub.2 is a C.sub.1-8
alkyl optionally substituted by a hydroxyl group, --OTBDMS, or
--OP(.dbd.O)(OEt).sub.2.
9. A compound according to claim 8, wherein R.sub.2 is a C.sub.1-8
alkyl optionally substituted by a hydroxyl group or R.sub.2 is
selected from the group consisting of: ##STR00009##
10. A pharmaceutical composition comprising a compound according to
claim 1, or a pharmaceutically acceptable salt, solvate or
polymorph thereof, together with one or more pharmaceutically
acceptable excipients, diluents or carriers.
11. A method for treating a viral infection, comprising
administering to a subject in need thereof the compound according
to claim 1 in an effective amount to treat the viral infection.
12. The method of claim 11, wherein the viral infection is a
hepatitis B infection.
13. A method for treating an immune disorder, comprising
administering to a subject in need thereof the compound according
to claim 1 in an effective amount to treat the immune disorder.
14. A method for treating cancer, comprising administering to a
subject in need thereof the compound according to claim 1 in an
effective amount to treat the cancer.
15. A compound selected from the group consisting of: ##STR00010##
or a pharmaceutically acceptable salt thereof.
16. A pharmaceutical composition comprising the compound according
to claim 15, or a pharmaceutically acceptable salt thereof,
together with one or more pharmaceutically acceptable excipients,
diluents or carriers.
17. A method for treating a viral infection, comprising
administering to a subject in need thereof the compound according
to claim 15 in an effective amount to treat the viral
infection.
18. The method of claim 17, wherein the viral infection is a
hepatitis B infection.
19. A method for treating an immune disorder, comprising
administering to a subject in need thereof the compound according
to claim 15 in an effective amount to treat the immune
disorder.
20. A method for treating cancer, comprising administering to a
subject in need thereof the compound according to claim 15 in an
effective amount to treat the cancer.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/338,256, filed on Mar. 29, 2019, which is a
35 U.S.C. .sctn. 371 filing of International Application No.
PCT/EP2017/074600, filed Sep. 28, 2017, which application claims
priority to European Application No. 16191568.1, filed Sep. 29,
2016. The entire contents of these applications are incorporated
herein by reference in their entirety.
[0002] The present invention relates to pyrimidine prodrug
derivatives and the use of said pyrimidine prodrug derivatives in
the treatment of viral infections, immune disorders, and cancer, or
as a vaccine adjuvant, whereby the induction of a T helper 1 (Th1)
immune response is desired.
[0003] For instance, in the treatment of chronic hepatitis B (HBV),
a Th1 response to reinvigorate exhausted virus-specific CD8.sup.+ T
cells in the infected organs would be highly beneficial (Science
1999, 284, 825-829; J. Virol. 2003, 77, 68-76). Such a response is
induced by the innate immune system, in particular by stimulating
Toll-like receptors (TLR) such as TLR3, 4, 7, 8 and 9 (Clin.
Transl. Immunol. 2016, 5(5):e85). TLR8 agonists induce one of the
strongest Th1 response in human cells, via the secretion of
IL-12p70 and the upregulation of CD40 or OX40L activation markers
and indirectly IFN.quadrature. (J Immunol. 2006, 176(12):7438-46;
Hum Immunol. 2011 January; 72(1):24-31; J Leukoc Biol. 2012,
91(1):105-17), which have shown their potential ex vivo to treat
chronic HBV (PLoS Pathog. 2013, 9, e1003208; J. Exp. Med. 2014,
211, 2047-2059; PLoS Path. 2013, 9, e1003490; PLoS Pathog. 2014,
10, e1004210).
[0004] Only one TLR8 agonist, administered by subcutaneous
injection, is currently in development for cancer indications
(Clin. Cancer Res. 2014, 20, 3683; Cancer Immunol. Immunother.
2013, 62, 1347; WO2012/045089. See also US20080234251,
US20100029585). Therefore, there exists a strong need for
orally-available TLR8 agonists to treat infections such as chronic
HBV.
[0005] In accordance with the present invention a compound of
formula (I) is provided
##STR00001##
or a pharmaceutically acceptable salt, solvate or polymorph
thereof, wherein R.sub.1A is selected from hydrogen, a substituted
or unsubstituted C.sub.1-3 alkyl, heterocycle, or a substituted or
unsubstituted phosphoramidate, R.sub.1B is selected from hydrogen,
a substituted or unsubstituted C.sub.1-3 alkyl, heterocycle, or a
substituted or unsubstituted phosphoramidate, with the exception
that R.sub.1A and R.sub.1B are not both hydrogen, R.sub.2 is a
C.sub.1-8 alkyl, which is optionally substituted by a hydroxyl
group.
[0006] The present invention concerns a prodrug composition of a
pharmaceutical compound. The pharmaceutical compound is
characterized by bioavailability of 50% or less and a molecular
weight in the range of 100-1000 Daltons. Also described is a method
of delivering a pharmaceutical compound to an individual including
the step of orally administering said prodrug to an individual. The
prodrug moiety is attached to the pharmaceutical compound wherein
the modification allows the TLR8 agonist potential to be
attenuated. The prodrug is enzymatically cleaved prior to or at the
site of the liver to yield the pharmaceutical compound, such that
the pharmaceutical compound is delivered to the individual limiting
TLR8 agonism prior to the liver.
[0007] In a first embodiment the present invention provides
compounds of formula (I) wherein R.sub.1A and/or R.sub.1B are a
substituted or unsubstituted phosphoramidate and wherein R.sub.2 is
a C.sub.1-6 alkyl preferably substituted by a hydroxyl group.
[0008] In a second embodiment the present invention provides
compounds of formula (I) wherein R.sub.1A and/or R.sub.1B are
methyl and wherein R.sub.2 is a C.sub.6-alkyl substituted by a
hydroxyl group.
[0009] The compounds of formula (I) in any stereochemical form and
their pharmaceutically acceptable salt, solvate or polymorph
thereof have activity as pharmaceuticals, in particular as inducers
of interferon.
[0010] So, in a further aspect the present invention provides a
pharmaceutical composition comprising a compound of formula (I) or
a pharmaceutically acceptable salt, solvate or polymorph thereof
together with one or more pharmaceutically acceptable excipients,
diluents or carriers.
[0011] Furthermore a compound of formula (I) or a pharmaceutically
acceptable salt, solvate or polymorph thereof according to the
current invention, or a pharmaceutical composition comprising said
compound of formula (I) or a pharmaceutically acceptable salt,
solvate or polymorph thereof can be used as a medicament.
[0012] Another aspect of the invention is that a compound of
formula (I) or a pharmaceutically acceptable salt, solvate or
polymorph thereof, or said pharmaceutical composition comprising
said compound of formula (I) or a pharmaceutically acceptable salt,
solvate or polymorph thereof can be used accordingly in the
treatment of a disorder in which the induction of interferon is
involved.
[0013] The term "alkyl" refers to a straight-chain or
branched-chain saturated aliphatic hydrocarbon containing the
specified number of carbon atoms.
[0014] "Heterocycle" refers to molecules that are saturated or
partially saturated and include tetrahydrofuran, tetrahydropyran,
dioxane or other cyclic ethers. Heterocycles containing nitrogen
include, for example azetidine, morpholine, piperidine, piperazine,
pyrrolidine, and the like. Other heterocycles include, for example,
thiomorpholine, dioxolinyl, and cyclic sulfones.
[0015] Pharmaceutically acceptable salts of the compounds of
formula (I) include the acid addition and base salts thereof.
Suitable acid addition salts are formed from acids which form
non-toxic salts. Suitable base salts are formed from bases which
form non-toxic salts.
[0016] The compounds of the invention may also exist in unsolvated
and solvated forms. The term "solvate" is used herein to describe a
molecular complex comprising the compound of the invention and one
or more pharmaceutically acceptable solvent molecules, for example,
ethanol.
[0017] The term "polymorph" refers to the ability of the compound
of the invention to exist in more than one form or crystal
structure.
[0018] The compounds of the present invention may be administered
as crystalline or amorphous products. They may be obtained for
example as solid plugs, powders, or films by methods such as
precipitation, crystallization, freeze drying, spray drying, or
evaporative drying. They may be administered alone or in
combination with one or more other compounds of the invention or in
combination with one or more other drugs. Generally, they will be
administered as a formulation in association with one or more
pharmaceutically acceptable excipients. The term "excipient" is
used herein to describe any ingredient other than the compound(s)
of the invention. The choice of excipient depends largely on
factors such as the particular mode of administration, the effect
of the excipient on solubility and stability, and the nature of the
dosage form.
[0019] The compounds of the present invention or any subgroup
thereof may be formulated into various pharmaceutical forms for
administration purposes. As appropriate compositions there may be
cited all compositions usually employed for systemically
administering drugs. To prepare the pharmaceutical compositions of
this invention, an effective amount of the particular compound,
optionally in addition salt form, as the active ingredient is
combined in intimate admixture with a pharmaceutically acceptable
carrier, which carrier may take a wide variety of forms depending
on the form of preparation desired for administration. These
pharmaceutical compositions are desirably in unitary dosage form
suitable, for example, for oral, rectal, or percutaneous
administration. For example, in preparing the compositions in oral
dosage form, any of the usual pharmaceutical media may be employed
such as, for example, water, glycols, oils, alcohols and the like
in the case of oral liquid preparations such as suspensions,
syrups, elixirs, emulsions, and solutions; or solid carriers such
as starches, sugars, kaolin, diluents, lubricants, binders,
disintegrating agents and the like in the case of powders, pills,
capsules, and tablets. Because of their ease in administration,
tablets and capsules represent the most advantageous oral dosage
unit forms, in which case solid pharmaceutical carriers are
obviously employed. Also included are solid form preparations that
can be converted, shortly before use, to liquid forms. In the
compositions suitable for percutaneous administration, the carrier
optionally comprises a penetration enhancing agent and/or a
suitable wetting agent, optionally combined with suitable additives
of any nature in minor proportions, which additives do not
introduce a significant deleterious effect on the skin. Said
additives may facilitate the administration to the skin and/or may
be helpful for preparing the desired compositions. These
compositions may be administered in various ways, e.g., as a
transdermal patch, as a spot-on, as an ointment. The compounds of
the present invention may also be administered via inhalation or
insufflation by means of methods and formulations employed in the
art for administration via this way. Thus, in general the compounds
of the present invention may be administered to the lungs in the
form of a solution, a suspension or a dry powder.
[0020] It is especially advantageous to formulate the
aforementioned pharmaceutical compositions in unit dosage form for
ease of administration and uniformity of dosage. Unit dosage form
as used herein refers to physically discrete units suitable as
unitary dosages, each unit containing a predetermined quantity of
active ingredient calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier.
Examples of such unit dosage forms are tablets (including scored or
coated tablets), capsules, pills, powder packets, wafers,
suppositories, injectable solutions or suspensions and the like,
and segregated multiples thereof.
[0021] Those of skill in the treatment of infectious diseases will
be able to determine the effective amount from the test results
presented hereinafter. In general it is contemplated that an
effective daily amount would be from 0.01 mg/kg to 50 mg/kg body
weight, more preferably from 0.1 mg/kg to 10 mg/kg body weight. It
may be appropriate to administer the required dose as two, three,
four or more sub-doses at appropriate intervals throughout the day.
Said sub-doses may be formulated as unit dosage forms, for example,
containing 1 to 1000 mg, and in particular 5 to 200 mg of active
ingredient per unit dosage form.
[0022] The exact dosage and frequency of administration depends on
the particular compound of formula (I) used, the particular
condition being treated, the severity of the condition being
treated, the age, weight and general physical condition of the
particular patient as well as other medication the individual may
be taking, as is well known to those skilled in the art.
Furthermore, it is evident that the effective amount may be lowered
or increased depending on the response of the treated subject
and/or depending on the evaluation of the physician prescribing the
compounds of the instant invention. The effective amount ranges
mentioned above are therefore only guidelines and are not intended
to limit the scope or use of the invention to any extent.
EXPERIMENTAL SECTION
Preparation of 2 and 3
##STR00002##
[0024] In a closed vessel, diethyl chlorophosphate (0.3 mL, 2.08
mmol) was added dropwise to a suspension of 1 (For synthesis of 1
see WO2012/136834)(0.5 g, 2.08 mmol) in CHCl.sub.3 (20 mL) at room
temperature for 5 min, and then Et.sub.3N (0.38 mL, 2.71 mmol) was
added dropwise. The reaction mixture was heated to 55.degree. C.
The solvent was removed under reduced pressure. The crude was
purified by reverse phase chromatography. First purification (start
95% [0.1% HCOOH]-5% [CH.sub.3CN:CH.sub.3OH 1:1] and finished 0%
[0.1% HCOOH]-100% [CH.sub.3CN:CH.sub.3OH 1:1). Second purification
(start 81% [25 mM NH.sub.4HCO.sub.3]-19%[100% CH.sub.3CN] and
finished 45% [25 mM NH.sub.4HCO.sub.3]-55% [100% CH.sub.3CN]. The
best fractions were pooled and the solvents removed to afford
compounds:
2, LC-MS ES.sup.+ m/z=377.1; Rt. 2.01 min, method A.
[.alpha.].sub.D.sup.23+7.6 (c 0.64, MeOH). mp 161.4.degree. C.
.sup.1H NMR (300 MHz, CD.sub.3OD) .delta. ppm 0.91 (m, 3H),
1.26-1.43 (m, 10H), 1.57 (m, 1H), 1.67 (m, 1H), 3.60 (d, J=5.1 Hz,
2H), 3.82 (s, 3H), 4.13 (m, 4H), 4.24 (m, 1H), 7.41 (s, 1H). 3,
LC-MS ES.sup.+ m/z=513.0; Rt. 2.49 min, method A.
[.alpha.].sub.D.sup.23+32.1 (c 0.29, MeOH). .sup.1H NMR (300 MHz,
CD.sub.3OD) .delta. ppm 0.91 (m, 3H), 1.22-1.44 (m, 16H), 1.68 (m,
2H), 3.83 (s, 3H), 3.98-4.24 (m, 10H), 4.41 (m, 1H), 7.46 (s,
1H).
Preparation of 5
##STR00003##
[0026] An aqueous solution of dimethylamine (6.67 mL, 132.8 mmol)
and copper (I) iodide (19.28 mg, 0.13 mmol) were added to a
solution of
(R)-2-((2-chloro-5-methoxy-pyrimidin-4-yl)amino)hexan-1-ol (For
synthesis see WO2012/136834) (500 mg, 1.92 mmol) in 1,4-dioxane (5
mL) in a steel reactor. The reaction mixture was heated to
160.degree. C. overnight. The reaction was cooled and filtered
through packed Celite and the solvent was evaporated to dryness to
give a crude that was purified by column chromatography on silica
gel to yield
(R)-2-((2-(dimethylamino)-5-methoxypyrimidin-4-yl)amino)hexan-1-ol
(250 mg, 0.93 mol). LC-MS ES.sup.+ m/z=269.1; Rt: 2.00 min, method
A. .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 0.90 (m, 3H),
1.25-1.47 (m, 4H), 1.58 (m, 1H), 1.67 (m, 1H), 3.05 (s, 6H), 3.61
(m, 2H), 3.77 (s, 3H), 4.21 (m, 1H), 7.35 (br s, 1H).
[.alpha.].sub.D.sup.23-8.25 (c 0.64, CH.sub.3OH). mp 86.1.degree.
C.
Preparation of 6
##STR00004##
[0028] Compound 6 was prepared analogous to the procedure to
prepare 5 with the exception that methylamine was used. LC-MS
ES.sup.+ m/z=255.1; Rt: 1.85 min, method A. .sup.1H NMR (300 MHz,
CD.sub.3OD) .delta. ppm 0.91 (m, 3H), 1.26-1.44 (m, 4H), 1.56 (m,
1H), 1.66 (m, 1H), 2.81 (s, 3H), 3.60 (m, 2H), 3.76 (s, 3H), 4.18
(m, 1H), 7.32 (br s, 1H). [.alpha.].sub.D.sup.23+1.8 (c 0.59,
CH.sub.3OH). mp 86.1.degree. C.
Preparation of 8
##STR00005##
[0029] Step 1. Preparation of 7. TBDMSCI (7.53 g, 49.9 mmol) was
added to a solution of 1 (10.0 g, 41.6 mmol) and Et.sub.3N (11.6
mL, 83.2 mmol) in DMF (120 mL). The mixture was stirred at rt for
70 h. EtOAc and a 10% aq. NaHCO.sub.3 was poured into the solution.
The layers were separated and the organic layer was washed with
brine (twice). The organic layer was dried over MgSO.sub.4, the
solids were removed by filtration, and the solvent of the filtrate
was removed under reduced pressure to give an orange oil, purified
by silica column chromatography using the following gradient: from
CH.sub.2Cl.sub.2 100% to 90%, CH.sub.3OH 0 to 10% (with 5% aq.
NH.sub.3) to afford 7, as a colorless oil. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 0.01 (s, 6H), 0.80-0.88 (m, 12H),
1.17-1.34 (m, 4H), 1.45 (m, 1H), 1.61 (m, 1H), 2.48-2.52 (m, 12H),
3.49 (dd, J=10.00, 6.50 Hz, 1H), 3.58 (dd, J=10.00, 4.70 Hz), 3.66
(s, 3H), 4.09 (m, 1H), 5.44 (s, 2H), 5.84 (d, J=9.09 Hz, 1H), 7.37
(s, 1H). LC-MS ES.sup.+ m/z=355.1, Rt: 3.76, Method: B).
[.alpha.].sub.D.sup.20+53.33 (c 0.3, DMF). Step 2. Under N.sub.2,
bis(2-oxo-3-oxazolidinyl)phosphinic chloride (1.05 g, 4.11 mmol)
was added portionwise to a solution of
((hydroxyphosphoryl)bis(oxy))bis(methylene)
bis(2,2-dimethylpropanoate) (1.61 g, 4.93 mmol) and DIPEA (2.83 mL,
16.4 mmol) in anhydrous 2-methyltetrahydrofuran/DMF (20 mL/14.5
mL). The mixture was stirred at rt for 2 h. Step 3. Then 7 (729 mg,
2.06 mmol) in anhydrous 2-methyltetrahydrofuran (17 mL) was added
and the mixture was stirred at rt for 2h. The reaction mixture was
diluted with EtOAc and water, the organic layer was washed with
brine (twice), dried over MgSO.sub.4, the solids were removed by
filtration, and the solvent of the filtrate was removed under
reduced pressure, then purified by silica column chromatography
using a mobile phase gradient: from heptane/EtOAc (80/20 to 0/100)
to give 290 mg of 8 as a colorless oil. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 0.84 (t, J=6.82 Hz, 3H), 1.12 (s, 18H),
1.18-1.34 (m, 4H), 1.41-1.65 (m, 2H), 3.42 (m, 2H), 3.74 (s, 3H),
4.03 (m, 1H), 4.61 (t, J=5.56 Hz, 1H), 5.60 (m, 4H), 6.44 (s, 1H),
7.45 (s, 1H), 8.77 (m, 1H). LC-MS ES.sup.+ m/z=547.6, Rt: 3.02,
Method: B
Preparation of
(2R)-2-((5-methoxy-2-((tetrahydrofuran-2-yl)amino)pyrimidin-4-yl)-amino)h-
exan-1-ol (9)
##STR00006##
[0031] In a Schlenk flask, DOWEX(R) 50WX2 (100-200 mesh) resin (one
spatula) was added at room temperature to a mixture of 1 (4 g, 11.3
mmol), 2,3-dihydrofuran (1031 .mu.L, 13.5 mmol, 1.2 eq.) in
anhydrous dichloroethane (99 mL). The mixture was stirred at
80.degree. C. for 20 h. Additional 2,3-dihydrofuran (688 .mu.L,
9.03 mmol, 0.8 eq.) was added and the mixture was stirred at
80.degree. C. for 20 h. An extraction was performed with
CH.sub.2Cl.sub.2 and water, and the layers were separated. The
organic layer was dried over MgSO.sub.4, filtered, and the solvent
was removed under reduced pressure to afford the titled
compound.
[0032] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 0.84 (t,
J=6.57 Hz, 3H), 1.18-1.33 (m, 4H), 1.46 (m, 1H), 1.58 (m, 1H), 1.74
(m, 2H), 1.96 (m, 2H), 3.41 (m, 2H), 3.61 (m, 1H), 3.64-3.73 (m,
4H), 4.04 (m, 1H), 4.65 (t, J=5.31 Hz, 1H), 5.73 (m, 1H), 6.00 (d,
J=9.09 Hz, 1H), 6.54 (m, 1H), 7.42 (s, 1H). LC-MS ES.sup.+
m/z=311.3, Rt: 2.30, Method: B)
Preparation of
(2R)-2-((5-methoxy-2-((tetrahydro-2H-pyran-2-yl)amino)pyrimidin-4-yl)-ami-
no)hexan-1-ol (10)
##STR00007##
[0034] Compound 10 was prepared according to the procedure to
prepare 9, with the exception that 3,4-dihydro-2H-pyran was
used.
[0035] .sup.1H NMR (400 MHz, DMSO-de) .delta. ppm 0.85 (m, 3H),
1.17-1.66 (m, 12H), 1.82 (br d, J=4.55 Hz, 1H), 3.35-3.50 (m, 3H),
3.70 (s, 3H), 3.77 (m, 1H), 4.02 (m, 1H), 4.66 (m, 1H), 5.01 (m,
1H), 6.02 (d, J=8.59 Hz, 1H), 6.46 (t, J=10.11 Hz, 1H), 7.43 (m,
1H). LC-MS ES.sup.+ m/z=325.1, Rt: 2.45, Method: B)
Analytical Methods
TABLE-US-00001 [0036] Method code Instrument Column Mobile phase
Gradient Flow ( mL .times. / .times. min ) .times. Col T .function.
( .degree. .times. .times. C . ) .times. ##EQU00001## Run time
(min) A Agilent 1100- DAD-MSD YMC-pack ODS-AQ A: 0.1% HCOOH in From
95% A to 5% A in 4.8 min, 2.6 3 .times. 5 ##EQU00002## 6.0 G1956A
C18 (50 .times. H.sub.2O held for 1.0 min, 4.6 mm, B: CH.sub.3CN to
95% A in 0.2 3 .mu.m) min B Waters Acquity BEH C18 (1.7 .mu.m, A:
95% 7 mM NH.sub.4OAc/5% From 84.2 % A and 15.8% B 0 . 3 .times. 4
.times. 3 4 .times. 0 ##EQU00003## 6.07 2.1 .times. 100 CH.sub.3CN
(hold for 0.49 mm) B: CH.sub.3CN min) to 10.5% A and 89.5% B in
2.18 min, hold for 1.94 min, and back to the initial conditions in
0.73 min, hold for 0.73 min
[0037] Supporting ADME Data.
[0038] Intrinsic clearance (CL.sub.int). CL.sub.int was determined
in rat and human liver microsomes. Incubations were performed at
37.degree. C., at a concentration of 1 .mu.M of compound, and a
microsomal protein concentration of 1 mg/mL. Serial samples were
removed at intervals of up to 60 min and analyzed for the
concentration of compound to determine its intrinsic clearance
rate. Compound was incubated in rat and human hepatocytes (10.sup.6
cells/mL) at 1 .mu.M for 0, 10, 20, 40, 60 and 120 min. Serial
samples were removed at intervals of up to 120 min and analyzed for
the concentration of compound to determine its intrinsic clearance
rate.
[0039] Permeability and Efflux in vitro. The in vitro permeability
and potential to be transported by P-glycoprotein (P-gp) was
determined using an MDCK cell line transfected with human MDR1
(P-glycoprotein). Compound was added to the apical (A) or
basolateral (B) side of a confluent monolayer of MDCK-MDR1 cells.
Permeability in the A.fwdarw.B direction in absence and presence of
GF120918 and in the B.fwdarw.A direction in absence of GF120918 was
measured by monitoring the appearance of the test compound on the
opposite side of the membrane using a specific LC-MS/MS method. The
efflux ratio (B.fwdarw.A-GF120918/A.fwdarw.B-GF120918) was
calculated to determine whether the test compound was a P-gp
substrate.
[0040] Plasma stability. Plasma stability is measured in rat and
human plasma for 7 or 24 h at 37.degree. C. Compounds are incubated
at 1 .mu.M. Incubations are performed manually in a 24-well plate
and serial samples (100 .mu.L) are removed at various time points
prior to quenching with acetonitrile.
[0041] Samples were analyzed by LC-MS/MS (without internal
standard). Percentage remaining at each time point is calculated
relative to the average peak area of the t=0 sample. Half-life
(t1/2 in hours) is calculated from those data.
[0042] SGF stability and FASSIF stability. The stability of the
prodrugs in simulated gastric fluid (SGF) in presence of pepsin and
in fasted simulated intestinal fluid (FASSIF) supplemented with
pancreatine and esterase was measured. Test compounds were
incubated in vitro for up to 2 hours and samples were taken at
different time points. Samples were analyzed for parent compound
disappearance by LC-MS/MS. Percentage remaining at each time point
is calculated relative to the average peak area of the t=0 sample.
Half-life (t.sub.1/2 in hours) is calculated from those data.
[0043] Pharmacokinetics in rat. Prodrugs were administered orally
to SD rats using aqueous solution at 5 mg/kg eq dose. Plasma
samples were collected at different time points and the
concentration of parent and prodrug was analyzed using LC-MS/MS. PK
parameters were calculated using Phoenix WinNonlin 6.3. C.sub.max
(ng/mL) and AUC.sub.(0-last) (ngh/mL) were determined for both
prodrug and parent.
TABLE-US-00002 TABLE 1 Permeability in MDCK-MDR1 cell line. Papp A
.fwdarw. B + Papp A .fwdarw. B - Efflux ratio Entry GF120918
GF120918 B .fwdarw. A/A .fwdarw. B 2 1.6 0.4 54 3 1.6 <0.2
>61 5 47 20 3.2 6 27.5 6.8 10 8 10 <1 >43 10 17 1.8 33
TABLE-US-00003 TABLE 2 Intrinsic clearance (CLint) in liver
microsomes and hepatocytes. CL.sub.int CL.sub.int CL.sub.int
CL.sub.int rat liver human liver hepatocytes hepatocytes Entry
microsomes microsomes rat human 2 18 <7.7 20 <1.9 3 178* 297*
110 17 5 141 14 280 80 6 25 18 120 33 8 >347 n.d. >280
>280 9 27 115 n.d. n.d. 10 >347* 88-90 n.d. n.d. n.d. = not
done, *an average of two data points
TABLE-US-00004 TABLE 3 Stability in simulated gastric fluid (SGF)
and fasted simulated intestinal fluid (FASSIF) and stability in rat
and human plasma. t.sub.1/2 SGF t.sub.1/2 FASSIF t.sub.1/2 rat
t.sub.1/2 human Entry (h) (h) plasma (h) plasma (h) 2 >6 >6
>30 >30 3 >6 >6 >30 >30 5 >6 >6 >30
>30 6 >6 >6 >30 >30 8 3.9 0.1 <0.4 1 9 1 1.1
<0.4 <0.4 10 0.3 0.8 1.4 1.4
TABLE-US-00005 TABLE 4 Oral administration of 1 and prodrugs in
rat. C.sub.max AUC.sub.(0-last) C.sub.max AUC.sub.(0-last) Entry
prodrug prodrug Compound 1 Compound 1 1 n.a. n.a. 177 486 2 1360
2567 BQL BQL 5 88 144 37 60 6 129 206 47 114 n.a. = not
applicable
* * * * *