U.S. patent number 10,662,213 [Application Number 15/308,491] was granted by the patent office on 2020-05-26 for gemcitabine prodrugs.
This patent grant is currently assigned to NuCana plc. The grantee listed for this patent is NuCana plc. Invention is credited to Valentina Ferrari, Hugh Griffith, Christopher McGuigan, Michaela Serpi, Magdalena Slusarczyk.
United States Patent |
10,662,213 |
Griffith , et al. |
May 26, 2020 |
Gemcitabine prodrugs
Abstract
This invention relates to a prodrug of the monophosphate
nucleotide of the well-known oncology drug gemcitabine.
Specifically, it relates to
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate when present as
a single phosphate diastereoisomer and, in particular, it relates
to the (S)-phosphate diastereoisomer which offers a remarkable and
unexpected increase in solubility relative to the
(R)-diastereoisomer. The (S)-phosphate epimer is also
preferentially taken up into cyclodextrin solutions over the
(R)-diastereoisomer.
Inventors: |
Griffith; Hugh (Edinburgh,
GB), McGuigan; Christopher (Cardiff South Glamorgan,
GB), Slusarczyk; Magdalena (Cardiff South Glamorgan,
GB), Serpi; Michaela (Cardiff South Glamorgan,
GB), Ferrari; Valentina (Cardiff South Glamorgan,
GB) |
Applicant: |
Name |
City |
State |
Country |
Type |
NuCana plc |
Edinburgh |
N/A |
GB |
|
|
Assignee: |
NuCana plc (Edinburgh,
GB)
|
Family
ID: |
53499027 |
Appl.
No.: |
15/308,491 |
Filed: |
June 25, 2015 |
PCT
Filed: |
June 25, 2015 |
PCT No.: |
PCT/GB2015/051857 |
371(c)(1),(2),(4) Date: |
November 02, 2016 |
PCT
Pub. No.: |
WO2015/198058 |
PCT
Pub. Date: |
December 30, 2015 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20170107246 A1 |
Apr 20, 2017 |
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Foreign Application Priority Data
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Jun 25, 2014 [GB] |
|
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1411253.6 |
Jun 25, 2014 [IN] |
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2050/MUM/2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P
43/00 (20180101); A61P 1/16 (20180101); A61P
15/00 (20180101); A61P 13/08 (20180101); C07H
19/10 (20130101); A61P 1/04 (20180101); A61P
1/18 (20180101); A61P 11/00 (20180101); A61P
13/10 (20180101); A61P 13/12 (20180101); A61P
35/02 (20180101); A61P 35/00 (20180101); A61K
31/7068 (20130101); A61P 7/00 (20180101) |
Current International
Class: |
A61K
31/7068 (20060101); C07H 19/10 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO-9937753 |
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Jul 1999 |
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WO |
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WO-2001/007454 |
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Feb 2001 |
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WO |
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WO-2005012327 |
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Feb 2005 |
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WO |
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WO-2006/081363 |
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Aug 2006 |
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WO |
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WO-2007/092620 |
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Aug 2007 |
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WO |
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WO-2010/063701 |
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Jun 2010 |
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WO |
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WO-2011/062503 |
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May 2011 |
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WO |
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WO-2013/107515 |
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Jul 2013 |
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WO |
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WO-2014076490 |
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May 2014 |
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WO |
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WO-2015/038596 |
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Mar 2015 |
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WO |
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WO-2015081133 |
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Jun 2015 |
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WO |
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WO-2015/198058 |
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Dec 2015 |
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WO |
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WO-2015/198059 |
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Dec 2015 |
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WO |
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WO-2016/012781 |
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Jan 2016 |
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WO |
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WO-2016/055769 |
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Apr 2016 |
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WO |
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WO-2016/181093 |
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Nov 2016 |
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WO |
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WO-2017/060661 |
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Apr 2017 |
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WO |
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WO-2017/098252 |
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Jun 2017 |
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WO |
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WO-2017/109444 |
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Jun 2017 |
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WO |
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WO-2017/109485 |
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Jun 2017 |
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WO |
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WO-2017/109486 |
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Jun 2017 |
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WO |
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Other References
Slusarczyk et al., Journal of Medicinal Chemistry, Feb. 2014, vol.
57, No. 4, pp. 1531-1542. cited by examiner .
Slusarczyk, M. et al., "Application of ProTide Technology to
Gemcitabine: A Successful Approach to Overcome the Key Cancer
Resistance Mechanisms Leads to a New Agent (NUC-1031) in Clinical
Development," Journal of Medicinal Chemistry, vol. 57, No. 4, p.
1531-1542, Feb. 27, 2014. cited by applicant .
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Application No. PCT/GB2015/051857. cited by applicant .
U.S. Appl. No. 16/065,369, Griffith et al. cited by applicant .
U.S. Appl. No. 16/065,476, Griffith et al. cited by applicant .
Harris et al., "Synthesis and antiviral evaluation of
phosphoramidate derivatives of
(E)-5-(2-bromovinyl)-2'-deoxyuridine," Antiviral Chem Chemother
12:293-300 (2001). cited by applicant .
Lackey et al., "Enxyme-catalyzed therapeutic agent (ECTA) design:
activation of the antitumor ECTA compound NB1011 by thymidylate
synthase," Biochem Pharmacol 61:179-189 (2001). cited by applicant
.
McGuigan et al., "Phosphoramidate derivatives of AZT as inhibitors
of HIV:studies on the carboxyl terminus," Antiviral Chem Chemother
4(2):97-101 (1993). cited by applicant .
McGuigan et al., "Synthesis and Evaluation of some masked phosphate
esters of the anti-herpesvirus drug 882C (Netivudine) as potential
antiviral agents," Antiviral Chem Chemother 9:233-243 (1998). cited
by applicant .
McGuigan, "A phosphoramidate ProTide (NUC-1031) and acquired and
intrinsic resistance to gemcitabine," J Clin Oncol, 29:E13540
(2011). cited by applicant .
McIntee et al., "Amino Acid Phosphoramidate Nucleosides: Potential
ADEPT/GDEPT Substrates," Bioorg Med Chem Letts 11:2803-2805 (2001).
cited by applicant .
Siddiqui, "Design and Synthesis of Lipophilic Phosphoramidate
d4T-MP Prodrugs Expressing High Potency Against HIV in Cell
Culture: Structural Determinants for in Vitro Activity and QSAR," J
Med Chem 42:4211-4128 (1999). cited by applicant .
Wu, et al., "Synthesis and Biological Activity of Gemcitabine
Phosphoramidate Prodrug," J Med Chem, 50(15): 3743-3746 (2007).
cited by applicant .
U.S. Appl. No. 15/279,611, McGuigan. cited by applicant .
International Search Report and Written Opinion for International
Application No. PCT/GB2004/003148 dated Jan. 20, 2005. cited by
applicant .
Bilir et al., "Acetaminophen and DMSO modulate growth and
gemcitabine cytotoxicity in FM3A breast cancer cells in vitro,"
Neoplasma 51(6):460-464 (2004). cited by applicant .
Uekama, "Novel Approach of Cyclodextrin-based Pharmaceutical
Formulation," Yakugaku Zasshi, 132(1):85-105 (2012). cited by
applicant.
|
Primary Examiner: McIntosh, III; Traviss C
Attorney, Agent or Firm: Gordon; Dana M. Foley Hoag LLP
Claims
The invention claimed is:
1. A pharmaceutical solution formulation, comprising water,
gemcitabine[phenyl-(benzoxy-L-alaninyl)]-(S)-phosphate 3:
##STR00005## 3 or a pharmaceutically acceptable salt or solvate
thereof, and a further component, wherein said further component is
selected from a polar organic solvent, a cyclodextrin, and both a
polar organic solvent and a cyclodextrin; wherein the
gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-(S)- phosphate 3 has a
diastereoisomeric purity of greater than about 90%, and the
pharmaceutical solution is for intravenous administration.
2. The pharmaceutical solution of claim 1, wherein the solution
comprises the polar organic solvent.
3. The pharmaceutical solution of claim 2, wherein the polar
organic solvent is selected from the group consisting of propylene
glycol, polyethylene glycol and ethanol.
4. The pharmaceutical solution of claim 3, wherein the polar
organic solvent is propylene glycol.
5. The pharmaceutical solution of claim 3, wherein the polar
organic solvent is polyethylene glycol.
6. The pharmaceutical solution of claim 5, wherein the polyethylene
glycol is PEG400.
7. The pharmaceutical solution of claim 3, wherein the polar
organic solvent is ethanol.
8. The pharmaceutical solution of claim 1, wherein the solution
comprises the cyclodextrin.
9. The pharmaceutical solution of claim 8, wherein the cyclodextrin
is selected from the group consisting of a-cyclodextrin,
(3-cyclodextrin, y-cyclodextrin, 2-hydroxypropyl-P-cyclodextrin,
sulfobutylether (3-cyclodextrin sodium salt, and partially
methylated 3-cyclodextrin.
10. The pharmaceutical solution of claim 1, wherein the
gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-(S)-phosphate 3 is in the
form of a free base.
11. The pharmaceutical solution of claim 1, wherein
gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-(5)-phosphate 3 has a
diastereoisomeric purity of greater than about 95%.
12. The pharmaceutical solution of claim 1, wherein
gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-(5)-phosphate 3 has a
diastereoisomeric purity of greater than about 98%.
13. The pharmaceutical solution of claim 1, wherein
gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-(5)-phosphate 3 has a
diastereoisomeric purity of greater than about 99.5%.
Description
RELATED APPLICATIONS
This application is a .sctn. 371 national stage application based
on Patent Cooperation Treaty Application serial number
PCT/GB2015/051857, filed Jun. 25, 2015; which claims the benefit of
priority to GB 1411253.6, filed Jun. 25, 2014; and IN
2050/MUM/2014, filed Jun. 25, 2014.
This invention relates to a prodrug of the monophosphate of the
well-known oncology drug gemcitabine. Specifically, it relates to
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate (chemical name:
2'-Deoxy-2',2'-difluoro-D-cytidine-5'-O-[phenyl
(benzoxy-L-alaninyl)] phosphate) when present as a single phosphate
diastereoisomer and, in particular, it relates to the (S)-phosphate
diastereoisomer which offers a remarkable and unexpected increase
in solubility relative to the (R)-diastereoisomer. The
(S)-phosphate diastereoisomer is also preferentially taken up into
cyclodextrin solutions over the (R)-diastereoisomer.
BACKGROUND
Gemcitabine (1; marketed as Gemzar.RTM.) is an effective nucleoside
analogue that is currently approved to treat breast, non-small cell
lung, ovarian and pancreatic cancers and widely used to treat a
variety of other cancers including bladder, biliary, colorectal and
lymphoma.
##STR00001##
Gemcitabine's clinical utility is limited by a number of inherent
and acquired resistance mechanisms. At the cellular level
resistance is dependent on three parameters: (i) the
down-regulation of deoxycytidine kinase, necessary for the
activation into the phosphorylated moiety; (ii) the reduced
expression of nucleoside transporters, in particular, hENT1
required for uptake by cancer cells; and (iii) the up-regulation of
catalytic enzymes especially cytidine deaminase that degrades
gemcitabine.
WO2005/012327 describes a series of nucleotide prodrugs for
gemcitabine and related nucleoside drug molecules. Among them
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate (NUC-1031; 2) is
identified as a particularly effective compound.
These prodrugs appear to avoid many of the inherent and acquired
resistance mechanisms which limit the utility of gemcitabine
(Application of ProTide Technology to Gemcitabine: A Successful
Approach to Overcome the Key Cancer Resistance Mechanisms Leads to
a New Agent (NUC-1031) in Clinical Development'; Slusarczyk et all;
J. Med. Chem.; 2014, 57, 1531-1542).
NUC-1031 2 is prepared as a mixture of two diastereoisomers,
epimeric at the phosphate centre.
##STR00002##
Unfortunately, NUC-1031 2 is extremely lipophillic and thus poorly
water soluble (by calculation: <0.1 mg/mL), and the ionisable
moieties, pyrimidine nitrogen and phenolic hydroxyl have calculated
pKas that lie out-side the pH range suitable for parenteral
administration. It is essentially insoluble in water, regardless of
salt content or pH, and this has implications for the development
of formulations for delivering the prodrug at sufficiently high
dosages for effective treatment. It also has implications for the
development of efficient manufacturing processes which will allow
NUC-1031 to be produced cost effectively.
It is an aim of certain embodiments of this invention to provide
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate (NUC-1031; 2) in
a form which can be formulated into a effective pharmaceutical
composition.
It is also an aim of certain embodiments of this invention to
provide a form of
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate (NUC-1031; 2)
which can be prepared and stored for an extended period of
time.
It is an aim of certain embodiments of this invention to provide
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate (NUC-1031; 2) in
a form which has a higher solubility than prior art forms.
It is an aim of certain embodiments of this invention to provide
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate (NUC-1031; 2) as
a single diastereoisomer at phosphorus.
Certain embodiments of this invention satisfy some or all of the
above aims.
The gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate of the
current invention is preferably of substantially the same activity
as gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate (NUC-1031;
2). It may, however, have slightly lower activity but have other
benefits as described in this specification if there is a
manufacturing or therapeutic benefit to the use of it in this
form.
BRIEF SUMMARY OF THE DISCLOSURE
In accordance with the present invention there is provided
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(S)-phosphate 3:
##STR00003## or a pharmaceutically acceptable salt or solvate
thereof. Preferably, the
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(S)-phosphate 3 is in a
substantially diastereoisomerically pure form.
The inventors have discovered a surprising and remarkable
difference in the solubilities of the two diastereoisomers. The
(S)-epimer 3 has sufficient solubility in mixtures of a number of
polar organic solvents with water to render it suitable for
formulation and administration as a therapeutic agent. The
(R)-epimer 4 is substantially insoluble in most of the solvent
mixtures measured. This remarkable difference in solubility had not
previously been identified and the potential benefits of this
property of the (S)-epimer had not been identified. In a number of
the solvent mixtures tested the difference in solubility between
the (S)-epimer and the (R)-epimer is over 100 fold.
Surprisingly, the (S)-epimer is also preferentially taken up into
cyclodextrin solutions over the (R)-epimer. This has not been
observed with other gemcitabine phosphate derivatives.
In a second aspect of the present invention is provided a
pharmaceutical formulation comprising
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(S)-phosphate 3, or a
pharmaceutically acceptable salt or solvate thereof, having a
diastereoisomeric purity of greater than about 90%, and at least
one pharmaceutically acceptable excipient.
The formulation may be for parenteral, e.g. for intravenous,
subcutaneous or intramuscular administration. Preferably, the
formulation is for intravenous administration.
The formulation may be an aqueous formulation which optionally also
comprises a polar organic solvent. In the case of parenteral (e.g.
intravenous) administration, the formulation preferably also
comprises a polar organic solvent.
The formulation may also comprise a cyclodextrin.
In a third aspect of the invention is provided
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(S)-phosphate 3, or a
pharmaceutically acceptable salt or solvate thereof, for medical
use.
In a fourth aspect of the invention is provided
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(S)-phosphate 3, or a
pharmaceutically acceptable salt or solvate thereof, for use in
treating cancer.
In a fifth aspect of the invention is provided
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(S)-phosphate 3, or a
pharmaceutically acceptable salt or solvate thereof, for use in the
manufacture of a medicament for treating cancer.
In a sixth aspect of the invention is provided a method of treating
cancer, the method comprising administering to a subject in need
thereof a therapeutically effective amount of
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(S)-phosphate 3, or a
pharmaceutically acceptable salt or solvate thereof.
A solvate will typically be a hydrate. Thus, the
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(S)-phosphate may be in
the form of a salt or hydrate. It may be that the
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(S)-phosphate is not in
the form of a salt and/or a solvate (e.g. hydrate). Preferably, it
is in the form of the free base.
The gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(S)-phosphate may have
a diastereoisomeric purity of greater than about 90%. It may have a
diastereoisomeric purity of greater than 95%, 98%, 99%, or even
99.5%. `Substantially diastereomerically pure` is defined for the
purposes of this invention as a diastereomeric purity of greater
than about 90%.
The cancer may be a cancer selected from: pancreatic cancer, breast
cancer, ovarian cancer, bladder cancer, colorectal cancer, lung
cancer, bladder cancer, prostate cancer, cholangiocarcinoma, renal
cancer, cervical cancer, thymic cancer, a cancer of an unknown
primary origin. The cancer may also be lymphoma or leukemia.
In a seventh aspect of the invention is provided a method of
providing at least one diastereoisomer of
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate in a
substantially diastereoisomerically pure form, the method
comprising the steps of: obtaining a mixture of
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(R)-phosphate 4 and
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(S)-phosphate 3;
subjecting the mixture to a separation technique; and once
separated, isolating
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(R)-phosphate 4 and/or
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(S)-phosphate 3 in a
substantially diastereoisomerically pure form.
In a eighth aspect of the invention is provided a method of
providing at least one diastereoisomer of
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate in a
substantially diastereoisomerically pure form, the method
comprising the steps of: obtaining a mixture of 3'-protected
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(R)-phosphate 4 and
3'-protected gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(S)-phosphate
3; subjecting the mixture to a separation technique; once
separated, isolating 3'-protected
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(R)-phosphate 4 and/or
3'-protected gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(S)-phosphate
3 in a substantially diastereoisomerically pure form; removing the
3'-protecting group from one or both of the separated
diastereoisomers to provide
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(R)-phosphate 4 and/or
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(S)-phosphate 3 in a
substantially diastereoisomerically pure form.
A 3'-protected gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate
is a derivative of
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate in which the
3'-hydroxy group features a hydroxyl protecting group. The
protecting group in question must be removable cleanly. Exemplary
protecting groups include silyl protecting groups (e.g.
tert-butyldimethylsilyl and triethylsilyl) in which case the
protecting group may be removed using a reagent selected from TFA,
HF, fluorosilicic acid and tetrabutylammonium fluoride. An
alternative protecting group would be a carbonate group (e.g.
tertbutylcarbonate) in which case the protecting group may be
removed using a Bronsted acid (e.g. TFA) or a Lewis acid (e.g.
ZnBr.sub.2).
The separation technique may be chromatography, e.g. column
chromatography, preparative thin layer chromatography or
preparative HPLC. Where the separation technique is preparative
HPLC, it may be carried out using a chiral column, e.g. one
comprising amylose tris (3,5-dimethylphenylcarbamate). An example
of a chiral column useful in the process of the invention is
Chiralpak AD.TM.; the stationary phase of which consists of a 20
.mu.m silica support onto which amylose tris
(3,5-dimethylphenylcarbamate) has been physically coated.
The separation technique may be selective uptake into a
cyclodextrin solution. This technique involves contacting the
mixture with a cyclodextrin solution such that one epimer is taken
up into the cyclodextrin solution in preference to the other
epimer, and then separating the cyclodextrin solution from the
undissolved solid. The cyclodextrin solution may be an aqueous
cyclodextrin solution. The separation of the solution from the
solid may be achieved by filtration.
The invention also provides
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(R)-phosphate 4:
##STR00004## or a pharmaceutically acceptable salt or solvate
thereof. The invention also provides a pharmaceutical formulation
comprising gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(R)-phosphate
4, or a pharmaceutically acceptable salt or solvate thereof, having
a diastereoisomeric purity of greater than about 90%, and a
pharmaceutically acceptable excipient, as well as medical uses of
compound 4 and methods of treatment using compound 4. These aspects
of the (R)-epimer correspond to those described in relation to
compound 3 in the third to sixth aspects of the invention described
above. The gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(R)-phosphate
may be in a substantially diastereoisomerically pure form. It may
be that the gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(R)-phosphate
is not a salt and/or a solvate (e.g. hydrate). Preferably, it is
present as the free base.
The R-epimer has been shown to have a half-life on incubation with
isolated human hepatic cells which is four times that of the
S-epimer (see Example 4). The longer half-life associated with
R-isomer indicates a lower intrinsic clearance and should result in
a different pharmacokinetic and pharmacodynamic profile to the
S-isomer. This profile may mean a higher and more prolonged
concentration of the R-isomer in the systemic circulation and hence
greater exposure to the R-epimer than would be achieved with the
S-epimer. The AUC for the R-isomer could therefore be higher,
resulting in greater and more prolonged exposure to the moiety, for
example, for oral route of administration where first pass effects
are more pronounced. This prolonged exposure to the R-epimer could
allow for substantively prolonged tumour exposure to the R-epimer
and may result in greater efficacy, where the reduced first pass
metabolism in the liver will result in higher drug concentrations.
This different property could also allow for targeting of specific
tumours where a longer PK profile may result in greater efficacy in
hard to access tumour sites where vasculature is poor. Prolonged
exposure to the R-epimer may ensure adequate drug concentration of
the active metabolite through more phases of the cell cycle,
including cell division.
BRIEF DESCRIPTION OF THE DRAWINGS
Embodiments of the invention are further described hereinafter with
reference to the accompanying drawings, in which:
FIG. 1 shows the chromatograph for separation of compounds 3 and 4
by HPLC using a Chiralpak AD column and a n-heptane/IPA gradient
solvent system
FIG. 2 shows the structure of compound 4 as determined by x-ray
diffraction
FIG. 3 shows the structure of compound 3 as determined by x-ray
diffraction
FIG. 4 shows the .sup.31P-NMR spectrum (202 MHz, D.sub.2O) of
NUC-1031 isomeric mixture (3.12 mM), after addition of HP-.beta.-CD
in a 1:2.3 molar ratio.
FIG. 5 shows the HPLC traces of NUC-1031 (3.12 mM) in MeOH (A) in
H.sub.2O after addition of HP-.beta.-CD in a 1:2.3 molar ratio
(B).
DETAILED DESCRIPTION
Throughout this specification, the term S-epimer or
S-diastereoisomer refers to
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(S)-phosphate. Likewise,
throughout this specification, the term R-epimer or
R-diastereoisomer refers to
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(R)-phosphate.
The compounds of the present invention can be used in the treatment
of the human body. They may be used in the treatment of the animal
body. In particular, the compounds of the present invention can be
used to treat commercial animals such as livestock. Alternatively,
the compounds of the present invention can be used to treat
companion animals such as cats, dogs, etc.
The compounds of the invention may be obtained, stored and/or
administered in the form of a pharmaceutically acceptable salt.
Suitable pharmaceutically acceptable salts include, but are not
limited to, salts of pharmaceutically acceptable inorganic acids
such as hydrochloric, sulphuric, phosphoric, nitric, carbonic,
boric, sulfamic, and hydrobromic acids, or salts of
pharmaceutically acceptable organic acids such as acetic,
propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric,
malic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic,
phenylacetic, methanesulphonic, toluenesulphonic, benzenesulphonic,
salicylic, sulphanilic, aspartic, glutamic, edetic, stearic,
palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric
acids. Suitable base salts are formed from bases which form
non-toxic salts. Examples include the aluminium, arginine,
benzathine, calcium, choline, diethylamine, diolamine, glycine,
lysine, magnesium, meglumine, olamine, potassium, sodium,
tromethamine and zinc salts. Hemisalts of acids and bases may also
be formed, for example, hemisulfate, hemioxalate and hemicalcium
salts. In certain embodiments, particularly those that apply to the
s-epimer, the compound is in the form of a HCl salt or a
hemioxalate salt.
Compounds of the invention may exist in a single crystal form or in
a mixture of crystal forms or they may be amorphous. Thus,
compounds of the invention intended for pharmaceutical use 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, or spray
drying, or evaporative drying. Microwave or radio frequency drying
may be used for this purpose.
For the above-mentioned compounds of the invention the dosage
administered will, of course, vary with the compound employed, the
mode of administration, the treatment desired and the disorder
indicated. For example, if the compound of the invention is
administered parenterally, then the dosage of the compound of the
invention may be in the range from 0.1 to 5 g/m.sup.2, e.g. from
0.5 to 2 g/m.sup.2. The size of the dose for therapeutic purposes
of compounds of the invention will naturally vary according to the
nature and severity of the conditions, the age and sex of the
animal or patient and the route of administration, according to
well known principles of medicine.
Dosage levels, dose frequency, and treatment durations of compounds
of the invention are expected to differ depending on the
formulation and clinical indication, age, and co-morbid medical
conditions of the patient.
A compound of the invention, or pharmaceutically acceptable salt
thereof, may be used on their own but will generally be
administered in the form of a pharmaceutical composition in which
the compounds of the invention, or pharmaceutically acceptable salt
thereof, is in association with a pharmaceutically acceptable
adjuvant, diluent or carrier. Conventional procedures for the
selection and preparation of suitable pharmaceutical formulations
are described in, for example, "Pharmaceuticals--The Science of
Dosage Form Designs", M. E. Aulton, Churchill Livingstone,
1988.
Depending on the mode of administration of the compounds of the
invention, the pharmaceutical composition which is used to
administer the compounds of the invention will preferably comprise
from 0.05 to 99% w (percent by weight) compounds of the invention,
more preferably from 0.05 to 80% w compounds of the invention,
still more preferably from 0.10 to 70% w compounds of the
invention, and even more preferably from 0.10 to 50% w compounds of
the invention, all percentages by weight being based on total
composition.
For oral administration the compounds of the invention may be
admixed with an adjuvant or a carrier, for example, lactose,
saccharose, sorbitol, mannitol; a starch, for example, potato
starch, corn starch or amylopectin; a cellulose derivative; a
binder, for example, gelatine or polyvinylpyrrolidone; and/or a
lubricant, for example, magnesium stearate, calcium stearate,
polyethylene glycol, a wax, paraffin, and the like, and then
compressed into tablets. If coated tablets are required, the cores,
prepared as described above, may be coated with a concentrated
sugar solution which may contain, for example, gum arabic,
gelatine, talcum and titanium dioxide. Alternatively, the tablet
may be coated with a suitable polymer dissolved in a readily
volatile organic solvent.
For the preparation of soft gelatine capsules, the compounds of the
invention may be admixed with, for example, a vegetable oil or
polyethylene glycol. Hard gelatine capsules may contain granules of
the compound using either the above-mentioned excipients for
tablets. Also liquid or semisolid formulations of the compound of
the invention may be filled into hard gelatine capsules.
Liquid preparations for oral application may be in the form of
syrups or suspensions, for example, solutions containing the
compound of the invention, the balance being sugar and a mixture of
ethanol, water, glycerol and propylene glycol. Optionally such
liquid preparations may contain colouring agents, flavouring
agents, sweetening agents (such as saccharine), preservative agents
and/or carboxymethylcellulose as a thickening agent or other
excipients known to those skilled in art.
For parenteral (e.g. intravenous) administration the compounds of
the invention may be administered as a sterile aqueous or oily
solution. The compounds of the invention are very lipophillic.
Aqueous formulations will typically, therefore, also contain a
pharmaceutically acceptable polar organic solvent.
Cyclodextrins have been shown to find wide application in drug
delivery (Rasheed et al, Sci. Pharm., 2008, 76, 567-598).
Cyclodextrins are a family of cyclic oligosaccharides. They act as
a `molecular cage` which encapsulates drug molecules and alters
properties of those drug molecules such as solubility.
Cyclodextrins comprise (.alpha.-1,4)-linked .alpha.-D-glucopyranose
units. Cyclodextrins may contains 6, 7 or 8 glucopyranose units
(designated .alpha.-, .beta.- and .gamma.-cyclodextrins
respectively). Cyclodextrins used in pharmaceutical formulations
are often .beta.-cyclodextrins. The pendant hydroxyl groups can be
alkylated with a C.sub.1-C.sub.6 substituted or unsubstituted alkyl
group. Examples of cyclodextrins are .alpha.-cyclodextrin,
.beta.-cyclodextrin, .gamma.-cyclodextrin,
2-hydroxypropyl-.beta.-cyclodextrin (HP-.beta.-CD), sulfobutylether
.beta.-cyclodextrin sodium salt, partially methylated
.beta.-cyclodextrin.
The size of the dose for therapeutic purposes of compounds of the
invention will naturally vary according to the nature and severity
of the conditions, the age and sex of the animal or patient and the
route of administration, according to well known principles of
medicine.
Dosage levels, dose frequency, and treatment durations of compounds
of the invention are expected to differ depending on the
formulation and clinical indication, age, and co-morbid medical
conditions of the patient.
The present invention also includes all pharmaceutically acceptable
isotopically-labelled forms of compounds 3 or 4 wherein one or more
atoms are replaced by atoms having the same atomic number, but an
atomic mass or mass number different from the atomic mass or mass
number of the predominant isotope usually found in nature.
Examples of isotopes suitable for inclusion in the compounds of the
invention include isotopes of hydrogen, such as .sup.2H and
.sup.3H, carbon, such as .sup.11C, .sup.13C and .sup.14C, chlorine,
such as .sup.36Cl, fluorine, such as .sup.18F, iodine, such as
.sup.123I and .sup.125I, nitrogen, such as .sup.13N and .sup.15N,
oxygen, such as .sup.15O, .sup.17O and .sup.18O, phosphorus, such
as .sup.32P, and sulphur, such as .sup.35S.
Certain isotopically-labelled compounds, for example, those
incorporating a radioactive isotope, are useful in drug and/or
substrate tissue distribution studies. The radioactive isotopes
tritium, i.e. .sup.3H, and carbon-14, i.e. .sup.14C, are
particularly useful for this purpose in view of their ease of
incorporation and ready means of detection.
Substitution with heavier isotopes such as deuterium, i.e. .sup.2H,
may afford certain therapeutic advantages resulting from greater
metabolic stability, for example, increased in vivo half-life or
reduced dosage requirements, and hence may be preferred in some
circumstances.
Substitution with positron emitting isotopes, such as .sup.11C,
.sup.18F, .sup.15O and .sup.13N, can be useful in Positron Emission
Topography (PET) studies for examining substrate receptor
occupancy.
Isotopically-labelled compounds can generally be prepared by
conventional techniques known to those skilled in the art or by
processes analogous to those described using an appropriate
isotopically-labelled reagent in place of the non-labelled reagent
previously employed.
The method of treatment or the compound for use in the treatment of
cancer may involve, in addition to the compound of the invention,
conventional surgery or radiotherapy or chemotherapy. Such
chemotherapy may include the administration of one or more other
active agents.
Where a further active agent is administered as part of a method of
treatment of the invention, such combination treatment may be
achieved by way of the simultaneous, sequential or separate dosing
of the individual components of the treatment. Such combination
products employ the compounds of this invention within a
therapeutically effective dosage range described hereinbefore and
the one or more other pharmaceutically-active agent(s) within its
approved dosage range.
Thus, the pharmaceutical formulations of the invention may comprise
another active agent.
The one or more other active agents may be one or more of the
following categories of anti-tumour agents:
(i) antiproliferative/antineoplastic drugs and combinations
thereof, such as alkylating agents (for example cyclophosphamide,
nitrogen mustard, bendamustin, melphalan, chlorambucil, busulphan,
temozolamide and nitrosoureas); antimetabolites (for example
gemcitabine and antifolates such as fluoropyrimidines like
5-fluorouracil and tegafur, raltitrexed, methotrexate, pemetrexed,
cytosine arabinoside, and hydroxyurea); antibiotics (for example
anthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin,
epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin);
antimitotic agents (for example vinca alkaloids like vincristine,
vinblastine, vindesine and vinorelbine and taxoids like taxol and
taxotere and polokinase inhibitors); proteasome inhibitors, for
example carfilzomib and bortezomib; interferon therapy; and
topoisomerase inhibitors (for example epipodophyllotoxins like
etoposide and teniposide, amsacrine, topotecan, mitoxantrone and
camptothecin);
(ii) cytostatic agents such as antiestrogens (for example
tamoxifen, fulvestrant, toremifene, raloxifene, droloxifene and
iodoxyfene), antiandrogens (for example bicalutamide, flutamide,
nilutamide and cyproterone acetate), LHRH antagonists or LHRH
agonists (for example goserelin, leuprorelin and buserelin),
progestogens (for example megestrol acetate), aromatase inhibitors
(for example as anastrozole, letrozole, vorazole and exemestane)
and inhibitors of 5.alpha.-reductase such as finasteride;
(iii) anti-invasion agents, for example dasatinib and bosutinib
(SKI-606), and metalloproteinase inhibitors, inhibitors of
urokinase plasminogen activator receptor function or antibodies to
Heparanase;
(iv) inhibitors of growth factor function: for example such
inhibitors include growth factor antibodies and growth factor
receptor antibodies, for example the anti-erbB2 antibody
trastuzumab [Herceptin.TM.], the anti-EGFR antibody panitumumab,
the anti-erbB1 antibody cetuximab, tyrosine kinase inhibitors, for
example inhibitors of the epidermal growth factor family (for
example EGFR family tyrosine kinase inhibitors such as gefitinib,
erlotinib and
6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)-quinazol-
in-4-amine (Cl 1033), erbB2 tyrosine kinase inhibitors such as
lapatinib); inhibitors of the hepatocyte growth factor family;
inhibitors of the insulin growth factor family; modulators of
protein regulators of cell apoptosis (for example Bcl-2
inhibitors); inhibitors of the platelet-derived growth factor
family such as imatinib and/or nilotinib (AMN107); inhibitors of
serine/threonine kinases (for example Ras/Raf signalling inhibitors
such as farnesyl transferase inhibitors, for example sorafenib,
tipifarnib and lonafarnib), inhibitors of cell signalling through
MEK and/or AKT kinases, c-kit inhibitors, abI kinase inhibitors,
PI3 kinase inhibitors, PIt3 kinase inhibitors, CSF-1R kinase
inhibitors, IGF receptor, kinase inhibitors; aurora kinase
inhibitors and cyclin dependent kinase inhibitors such as CDK2
and/or CDK4 inhibitors;
(v) antiangiogenic agents such as those which inhibit the effects
of vascular endothelial growth factor, [for example the
anti-vascular endothelial cell growth factor antibody bevacizumab
(Avastin.TM.); thalidomide; lenalidomide; and for example, a VEGF
receptor tyrosine kinase inhibitor such as vandetanib, vatalanib,
sunitinib, axitinib and pazopanib;
(vi) gene therapy approaches, including for example approaches to
replace aberrant genes such as aberrant p53 or aberrant BRCA1 or
BRCA2;
(vii) immunotherapy approaches, including for example antibody
therapy such as alemtuzumab, rituximab, ibritumomab tiuxetan
(Zevalin.RTM.) and ofatumumab; interferons such as interferon
.alpha.; interleukins such as IL-2 (aldesleukin); interleukin
inhibitors for example IRAK4 inhibitors; cancer vaccines including
prophylactic and treatment vaccines such as HPV vaccines, for
example Gardasil, Cervarix, Oncophage and Sipuleucel-T (Provenge);
and toll-like receptor modulators for example TLR-7 or TLR-9
agonists; and
(viii) cytotoxic agents for example fludaribine (fludara),
cladribine, pentostatin (Nipent.TM.);
(ix) steroids such as corticosteroids, including glucocorticoids
and mineralocorticoids, for example aclometasone, aclometasone
dipropionate, aldosterone, amcinonide, beclomethasone,
beclomethasone dipropionate, betamethasone, betamethasone
dipropionate, betamethasone sodium phosphate, betamethasone
valerate, budesonide, clobetasone, clobetasone butyrate, clobetasol
propionate, cloprednol, cortisone, cortisone acetate, cortivazol,
deoxycortone, desonide, desoximetasone, dexamethasone,
dexamethasone sodium phosphate, dexamethasone isonicotinate,
difluorocortolone, fluclorolone, flumethasone, flunisolide,
fluocinolone, fluocinolone acetonide, fluocinonide, fluocortin
butyl, fluorocortisone, fluorocortolone, fluocortolone caproate,
fluocortolone pivalate, fluorometholone, fluprednidene,
fluprednidene acetate, flurandrenolone, fluticasone, fluticasone
propionate, halcinonide, hydrocortisone, hydrocortisone acetate,
hydrocortisone butyrate, hydrocortisone aceponate, hydrocortisone
buteprate, hydrocortisone valerate, icomethasone, icomethasone
enbutate, meprednisone, methylprednisolone, mometasone
paramethasone, mometasone furoate monohydrate, prednicarbate,
prednisolone, prednisone, tixocortol, tixocortol pivalate,
triamcinolone, triamcinolone acetonide, triamcinolone alcohol and
their respective pharmaceutically acceptable derivatives. A
combination of steroids may be used, for example a combination of
two or more steroids mentioned in this paragraph;
(x) targeted therapies, for example PI3Kd inhibitors, for example
idelalisib and perifosine; or compounds that inhibit PD-1, PD-L1
and CAR T.
The one or more other active agents may also be antibiotics.
Throughout the description and claims of this specification, the
words "comprise" and "contain" and variations of them mean
"including but not limited to", and they are not intended to (and
do not) exclude other moieties, additives, components, integers or
steps. Throughout the description and claims of this specification,
the singular encompasses the plural unless the context otherwise
requires. In particular, where the indefinite article is used, the
specification is to be understood as contemplating plurality as
well as singularity, unless the context requires otherwise.
Features, integers, characteristics, compounds, chemical moieties
or groups described in conjunction with a particular aspect,
embodiment or example of the invention are to be understood to be
applicable to any other aspect, embodiment or example described
herein unless incompatible therewith. All of the features disclosed
in this specification (including any accompanying claims, abstract
and drawings), and/or all of the steps of any method or process so
disclosed, may be combined in any combination, except combinations
where at least some of such features and/or steps are mutually
exclusive. The invention is not restricted to the details of any
foregoing embodiments. The invention extends to any novel one, or
any novel combination, of the features disclosed in this
specification (including any accompanying claims, abstract and
drawings), or to any novel one, or any novel combination, of the
steps of any method or process so disclosed.
The reader's attention is directed to all papers and documents
which are filed concurrently with or previous to this specification
in connection with this application and which are open to public
inspection with this specification, and the contents of all such
papers and documents are incorporated herein by reference.
The following abbreviations are used in this specification:
DMF--N,N-dimethylformamide DMSO--dimethylsulfoxide IPA--isopropyl
alcohol NMP--N-methylpyrroldinone PEG--polyethylene glycol
TBDMS--tert-butyldimethylsilyl TFA--trifluoroacetic acid
Example 1
The (R) and (S) isomers were separated by HPLC under the following
conditions: Equipment: Agilent 1200.TM. series with DAD detector
Flow rate: 1.0 mL/min Column: Chiralpak AD.TM.; 250.times.4.6 mm ID
(normal phase) Temperature: ambient Particle size: 20 .mu.m Feed:
dissolved in MeOH; 10 g/L Solvent: n-heptane/IPA 10->50% IPA The
chromatogram is shown in FIG. 1. The (S)-epimer eluted at 8.6 min
and the (R)-epimer eluted at 10.3 minutes.
Characterisation Methods and Materials: Proton (.sup.1H), carbon
(.sup.13C), phosphorus (.sup.31P) and fluorine (.sup.19F) NMR
spectra were recorded on a Bruker Avance 500 spectrometer at
25.degree. C. Spectra were auto-calibrated to the deuterated
solvent peak and all .sup.13C NMR and .sup.31P NMR were
proton-decoupled. The purity of final compounds was verified to be
>95% by HPLC analysis using Varian Polaris C18-A (10 .mu.M) as
an analytic column with a gradient elution of H.sub.2O/MeOH from
100/0 to 0/100 in 35 min. The HPLC analysis was conducted by Varian
Prostar (LC Workstation-Varian prostar 335 LC detector).
2'-Deoxy-2',2'-difluoro-D-cytidine-5'-O-[phenyl(benzyloxy-L-alaninyl)]-(S)-
-phosphate 3
(ES+) m/z, found: (M+Na.sup.+) 603.14.
C.sub.25H.sub.27F.sub.2N.sub.4O.sub.8NaP required: (M.sup.+)
580.47.
.sup.31P NMR (202 MHz, MeOD): Op 3.66
.sup.1H NMR (500 MHz, MeOD): .delta..sub.H 7.58 (d, J=7.5 Hz, 1H,
H-6), 7.38-7.32 (m, 7H, ArH), 7.26-7.20 (m, 3H, ArH), 6.24 (t,
J=7.5 Hz, 1H, H-1'), 5.84 (d, J=7.5 Hz, 1H, H-5), 5.20 (AB system,
J.sub.AB=12.0 Hz, 2H, OCH.sub.2Ph), 4.46-4.43 (m, 1H, H-5'),
4.36-4.31 (m, 1H, H-5'), 4.25-4.19 (m, 1H, H-3'), 4.07-4.00 (m, 2H,
H-4', CHCH.sub.3), 1.38 (d, J=7.2 Hz, 3H, CHCH.sub.3).
.sup.19F NMR (470 MHz, MeOD): .delta..sub.F -118.0 (d, J=241 Hz,
F), -120.24 (broad d, J=241 Hz, F).
.sup.13C NMR (125 MHz, MeOD): .delta..sub.C 174.61 (d,
.sup.3J.sub.C-P=5.0 Hz, C.dbd.O, ester), 167.63 (C--NH.sub.2),
157.74 (C.dbd.O base), 152.10 (d, .sup.2J.sub.C-P=7.0 Hz, C--Ar),
142.40 (CH-base), 137.22 (C--Ar), 130.90, 129.63, 129.39, 129.32,
126.32 (CH--Ar), 124.51 (d, .sup.1J.sub.C-F=257 Hz, CF.sub.2),
121.47, 121.43 (CH--Ar), 96.67 (CH-base), 85.92 (broad signal,
C-1'), 80.31 (C-4'), 71.27 (apparent t, .sup.2J.sub.C-F=23.7 Hz,
C-3'), 68.03 (OCH.sub.2Ph), 65.73 (d, .sup.2J.sub.C-P=5.30 Hz,
C-5'), 51.66 (CHCH.sub.3), 20.42 (d, .sup.3J.sub.C-P=6.25 Hz,
CHCH.sub.3).
Reverse HPLC, eluting with H.sub.2O/MeOH from 100/0 to 0/100 in 35
min, showed one peak of diastereoisomer with t.sub.R=22.53 min.
2'-deoxy-2',2'-difluoro-D-cytidine-5'-O-[phenyl(benzyloxy-L-alaninyl)]-(R)-
-phosphate 4
(ES+) m/z, found: (M+Na.sup.+) 603.14.
C.sub.25H.sub.27F.sub.2N.sub.4O.sub.8NaP required: (M.sup.+)
580.47.
.sup.31P NMR (202 MHz, MeOD): .delta..sub.P 3.83
.sup.1H NMR (500 MHz, MeOD): .delta..sub.H 7.56 (d, J=7.5 Hz, 1H,
H-6), 7.38-7.31 (m, 7H, ArH), 7.23-7.19 (m, 3H, ArH), 6.26 (t,
J=7.5 Hz, 1H, H-1'), 5.88 (d, J=7.5 Hz, 1H, H-5), 5.20 (s, 2H,
OCH.sub.2Ph), 4.49-4.46 (m, 1H, H-5'), 4.38-4.34 (m, 1H, H-5'),
4.23-4.17 (m, 1H, H-3'), 4.07-4.01 (m, 2H, H-4', CHCH.sub.3), 1.38
(d, J=7.2 Hz, 3H, CHCH.sub.3).
.sup.19F NMR (470 MHz, MeOD): .delta..sub.F -118.3 (d, J=241 Hz,
F), -120.38 (broad d, J=241 Hz, F).
.sup.13C NMR (125 MHz, MeOD): .delta..sub.C 174.65 (d,
.sup.3J.sub.C-P=5.0 Hz, C.dbd.O, ester), 167.65 (C--NH.sub.2),
157.75 (C.dbd.O base), 152.10 (d, .sup.2J.sub.C-P=7.0 Hz, C--Ar),
142.28 (CH-base), 137.50 (C--Ar), 130.86, 129.63, 129.40, 129.32,
126.31 (CH--Ar), 124.50 (d, .sup.1J.sub.C-F=257 Hz, CF.sub.2),
121.44, 121.40 (CH--Ar), 96.67 (CH-base), 85.90 (broad signal,
C-1'), 80.27 (C-4'), 71.30 (apparent t, .sup.2J.sub.C-F=23.7 Hz,
C-3'), 68.02 (OCH.sub.2Ph), 65.50 (C-5'), 51.83 (CHCH.sub.3), 20.22
(d, .sup.3J.sub.C-P=7.5 Hz, CHCH.sub.3).
Reverse HPLC, eluting with H.sub.2O/MeOH from 100/0 to 0/100 in 35
min, showed one peak of diastereoisomer with t.sub.R=21.87 min
X-ray diffraction data was also obtained for the two isomers and
the resulting images are shown in FIGS. 2 and 3. The corresponding
diffraction data and methodology is provided in Tables 1 to 4
below.
TABLE-US-00001 TABLE 1 Crystal data and structure refinement for
(R)-epimer 4. Identification code shelx Empirical formula
C.sub.25H.sub.27F.sub.2N.sub.4O.sub.8P Formula weight 580.47
Temperature 296(2) K Wavelength 1.54184 .ANG. Crystal system
Monoclinic Space group C 2 Unit cell dimensions a = 19.2280(3)
.ANG. .alpha. = 90.degree.. b = 10.22330(10) .ANG. .beta. =
97.966(2).degree.. c = 13.6911(2) .ANG. .gamma. = 90.degree..
Volume 2665.34(6) .ANG..sup.3 Z 4 Density (calculated) 1.447
Mg/m.sup.3 Absorption coefficient 1.541 mm.sup.-1 F(000) 1208
Crystal size 0.584 .times. 0.095 .times. 0.051 mm.sup.3 Theta range
for data collection 3.259 to 73.477.degree.. Index ranges -23 <=
h <= 23, -12 <= k <= 12, -17 <= l <= 13 Reflections
collected 9684 Independent reflections 5150 [R(int) = 0.0239]
Completeness to theta = 67.684.degree. 99.9% Absorption correction
Semi-empirical from equivalents Max. and min. transmission 1.00000
and 0.61719 Refinement method Full-matrix least-squares on F.sup.2
Data/restraints/parameters 5150/549/437 Goodness-of-fit on F.sup.2
1.065 Final R indices [I > 2sigma(I)] R1 = 0.0646, wR2 = 0.1759
R indices (all data) R1 = 0.0681, wR2 = 0.1793 Absolute structure
parameter 0.039(10) Extinction coefficient n/a Largest diff. peak
and hole 0.477 and -0.917 e .ANG..sup.-3
TABLE-US-00002 TABLE 2 Atomic coordinates (.times.10.sup.4) and
equivalent isotropic displacement parameters (.ANG..sup.2 .times.
10.sup.3) for (R)-epimer 4. U(eq) is defined as one third of the
trace of the orthogonalized U.sup.ij tensor. x y z U(eq) N(1)
746(2) 9565(4) 4120(3) 35(1) C(1) 1373(2) 10267(5) 4426(4) 40(1)
C(2) 779(3) 12200(5) 4030(4) 40(1) C(3) 131(3) 11526(5) 3717(4)
45(1) C(4) 141(3) 10216(5) 3785(4) 39(1) C(5) 723(2) 8163(4)
4257(4) 33(1) C(6) 1301(2) 6686(5) 5412(3) 34(1) C(7) 1730(2)
6675(4) 4553(3) 33(1) C(8) 1228(3) 7319(5) 3735(4) 37(1) C(9)
1755(3) 6761(6) 6387(4) 43(1) C(10) 1727(3) 3180(6) 7861(5) 50(1)
C(11) 1763(4) 2447(7) 7027(5) 61(2) C(12) 1751(4) 1097(8) 7088(7)
75(2) C(13) 1724(5) 533(8) 7970(9) 94(3) C(14) 1685(6) 1247(9)
8799(8) 97(3) C(15) 1693(5) 2625(8) 8758(6) 78(2) P(1) 1047(1)
5375(1) 7561(1) 43(1) F(1) 1535(2) 7981(3) 3064(2) 54(1) F(2)
828(2) 6372(3) 3227(2) 54(1) N(2) 792(3) 13496(4) 3968(4) 53(1)
N(3) 1370(2) 11570(4) 4362(4) 44(1) O(1) 1897(2) 9608(4) 4734(4)
54(1) O(2) 868(2) 7849(4) 5276(3) 42(1) O(3) 1921(2) 5414(4)
4294(3) 42(1) O(4) 1354(2) 6700(4) 7211(3) 48(1) O(5) 1755(2)
4556(4) 7796(3) 51(1) O(6) 514(2) 4743(5) 6869(4) 63(1) N(4) 789(3)
5715(6) 8600(5) 69(1) O(7) 595(5) 5712(9) 10681(7) 76(1) O(8)
1614(5) 4778(9) 10851(7) 73(1) C(16) 1339(8) 6191(13) 9486(10)
72(1) C(17) 1345(8) 7650(14) 9611(10) 75(1) C(18) 1162(8) 5543(19)
10418(11) 73(1) C(19) 1420(3) 4227(5) 11796(4) 74(1) C(20) 1213(3)
2833(5) 11556(4) 74(1) C(21) 1737(3) 1889(5) 11701(4) 74(1) C(22)
1562(3) 570(5) 11626(4) 74(1) C(23) 863(3) 195(5) 11406(4) 73(1)
C(24) 339(3) 1139(5) 11261(4) 74(1) C(25) 514(3) 2458(5) 11336(4)
74(1) N(4A) 789(3) 5715(6) 8600(5) 69(1) O(7A) 643(8) 6651(15)
10878(10) 71(1) O(8A) 1156(10) 4892(18) 10491(12) 73(1) C(16A)
1034(13) 6570(20) 9296(16) 72(1) C(17A) 797(12) 7900(20) 9187(16)
74(2) C(18A) 945(13) 6100(20) 10317(17) 72(1) C(19A) 974(5) 4324(9)
11444(7) 74(1) C(20A) 986(5) 2873(9) 11442(7) 74(1) C(21A) 1654(5)
2345(9) 11684(7) 74(1) C(22A) 1747(5) 996(9) 11687(7) 74(1) C(23A)
1172(5) 176(9) 11448(7) 74(1) C(24A) 504(5) 704(9) 11206(7) 74(1)
C(25A) 411(5) 2052(9) 11203(7) 74(1)
TABLE-US-00003 TABLE 3 Crystal data and structure refinement for
(S)-epimer 3. Identification code shelx Empirical formula
C.sub.25.29H.sub.26.44F.sub.2N.sub.4.14O.sub.8P Formula weight
585.44 Temperature 293(2) K Wavelength 1.54184 .ANG. Crystal system
Monoclinic Space group P 2.sub.1 Unit cell dimensions a =
11.3844(3) .ANG. .alpha. = 90.degree.. b = 34.8283(7) .ANG. .beta.
= 111.282(4).degree.. c = 15.1260(6) .ANG. .gamma. = 90.degree..
Volume 5588.5(3) .ANG.3 Z 8 Density (calculated) 1.392 Mg/m3
Absorption coefficient 1.477 mm-1 F(000) 2434 Crystal size 0.249
.times. 0.072 .times. 0.042 mm3 Theta range for data collection
3.135 to 73.481.degree.. Index ranges -13 <= h <= 12, -42
<= k <= 42, -17 <= l <= 18 Reflections collected 43816
Independent reflections 21792 [R(int) = 0.0582] Completeness to
theta = 67.684.degree. 100.0% Absorption correction Semi-empirical
from equivalents Max. and min. transmission 1.00000 and 0.62509
Refinement method Full-matrix least-squares on F2
Data/restraints/parameters 21792/2/1478 Goodness-of-fit on F2 1.022
Final R indices [I > 2sigma(I)] R1 = 0.0628, wR2 = 0.1597 R
indices (all data) R1 = 0.0921, wR2 = 0.1794 Absolute structure
parameter 0.031(13) Extinction coefficient n/a Largest diff. peak
and hole 0.513 and -0.413 e .ANG.-3
TABLE-US-00004 TABLE 4 Atomic coordinates (.times.104) and
equivalent isotropic displacement parameters (.ANG.2 .times. 103)
for the (S)-epimer 3. U(eq) is defined as one third of the trace of
the orthogonalized Uij tensor. x y z U(eq) C(1) -2481(7) 1599(2)
6585(6) 35(2) C(2) -4079(6) 1825(2) 7037(5) 36(2) C(3) -3292(7)
2138(2) 7558(6) 40(2) C(4) -2138(7) 2159(2) 7573(6) 40(2) C(5)
-417(6) 1916(2) 7113(6) 37(2) C(6) 1520(6) 2176(2) 7885(6) 37(2)
C(7) 1182(7) 2311(2) 6853(6) 41(2) C(8) -198(7) 2183(2) 6399(6)
42(2) C(9) 2187(7) 2464(2) 8638(6) 40(2) C(10) -428(7) 3331(2)
8699(6) 42(2) C(11) -661(8) 3061(2) 9279(7) 52(2) C(12) -1894(8)
2980(2) 9149(8) 53(2) C(13) -2880(8) 3159(3) 8495(7) 59(2) C(14)
-2631(9) 3426(4) 7913(9) 71(3) C(15) -1403(9) 3517(3) 8012(7) 61(2)
C(16) 3026(7) 3528(2) 8008(6) 44(2) C(17) 4074(8) 3825(2) 8156(7)
52(2) C(18) 3226(7) 3213(2) 7405(6) 46(2) C(19) 2875(10) 3054(3)
5787(8) 65(3) C(20) 4128(8) 3142(2) 5690(6) 46(2) C(21) 4412(9)
3504(2) 5462(6) 48(2) C(22) 5504(9) 3580(3) 5340(7) 59(2) C(23)
6360(9) 3281(3) 5425(8) 67(3) C(24) 6068(10) 2917(3) 5643(9) 73(3)
C(25) 4979(10) 2851(3) 5777(7) 62(2) N(1) -1680(6) 1893(2) 7097(5)
36(1) N(2) -3652(6) 1572(2) 6561(5) 38(1) N(3) -5247(6) 1787(2)
7003(6) 50(2) N(4) 3092(6) 3390(2) 8954(5) 44(2) O(1) -2035(5)
1369(2) 6152(5) 49(1) O(2) 360(5) 2074(1) 7991(4) 39(1) O(3)
1997(5) 2165(2) 6434(5) 50(2) O(4) 1475(5) 2819(1) 8445(4) 39(1)
O(5) 805(5) 3441(1) 8776(4) 41(1) O(6) 2374(5) 3056(2) 10168(4)
43(1) O(7) 3851(6) 2925(2) 7682(5) 51(1) O(8) 2644(6) 3304(2)
6471(5) 57(2) P(1) 1979(2) 3167(1) 9166(1) 38(1) F(1) -960(4)
2496(2) 6274(4) 56(1) F(2) -474(5) 2023(2) 5530(4) 62(1) C(26)
-2061(6) 6176(2) 4177(5) 33(1) C(27) -3538(6) 5940(2) 4770(6) 35(2)
C(28) -2710(7) 5634(2) 5256(6) 38(2) C(29) -1565(7) 5637(2) 5238(6)
38(2) C(30) 84(6) 5918(2) 4754(5) 32(1) C(31) 1757(6) 5545(2)
4799(6) 34(2) C(32) 2320(6) 5811(2) 5667(5) 34(2) C(33) 1125(7)
6014(2) 5697(6) 38(2) C(34) 2224(7) 5144(2) 4906(6) 35(2) C(35)
882(8) 4453(2) 6664(7) 50(2) C(36) 404(11) 4719(4) 7049(10) 78(3)
C(37) -867(11) 4722(4) 6911(10) 88(4) C(38) -1658(11) 4449(4)
6353(11) 91(4) C(39) -1148(11) 4166(4) 5937(10) 84(4) C(40) 101(10)
4166(3) 6074(9) 69(3) C(41) 4806(7) 4794(2) 7341(6) 43(2) C(42)
6103(8) 4628(3) 7868(8) 59(2) C(43) 4987(7) 5214(3) 7111(7) 48(2)
C(44) 5647(11) 5817(3) 7891(9) 71(3) C(45) 7060(10) 5779(2) 8217(8)
60(2) C(46) 7713(12) 5808(4) 7623(9) 79(3) C(47) 8991(14) 5754(5)
7926(10) 98(4) C(48) 9642(12) 5675(4) 8866(10) 82(3) C(49) 9007(11)
5644(3) 9451(9) 74(3) C(50) 7752(11) 5689(3) 9171(8) 67(3) N(5)
-1179(5) 5908(2) 4744(5) 34(1) N(6) -3210(5) 6187(2) 4213(5) 35(1)
N(7) -4662(6) 5986(2) 4836(5) 43(2) N(8) 4159(6) 4572(2) 6488(5)
41(1) O(9) -1723(5) 6398(1) 3670(4) 40(1) O(10) 423(4) 5538(1)
4567(4) 36(1) O(11) 3246(5) 6065(2) 5588(4) 41(1) O(12) 2070(5)
4969(1) 5727(4) 37(1) O(13) 2175(5) 4448(2) 6814(4) 43(1) O(14)
2253(5) 4294(1) 5142(4) 41(1) O(15) 5126(5) 5327(2) 6402(4) 47(1)
O(16) 5079(6) 5439(2) 7854(5) 59(2) P(2) 2638(2) 4552(1) 5976(1)
36(1) F(3) 848(4) 5870(2) 6437(3) 53(1) F(4) 1260(4) 6399(1)
5827(4) 52(1) C(51) 1164(6) 6520(2) 10539(5) 33(2) C(52) -905(6)
6744(2) 9936(6) 35(2) C(53) -566(7) 7031(2) 9380(6) 40(2) C(54)
599(7) 7038(2) 9410(6) 36(2) C(55) 2751(6) 6779(2) 9946(5) 32(1)
C(56) 4386(6) 7140(2) 9914(5) 32(1) C(57) 4124(6) 6867(2) 9056(5)
34(2) C(58) 2905(7) 6665(2) 9004(5) 34(2) C(59) 4765(6) 7544(2)
9783(6) 35(2) C(60) 1742(7) 8055(2) 7650(7) 47(2) C(61) 1252(10)
7835(3) 6826(8) 68(3) C(62) 84(11) 7676(4) 6616(10) 82(3) C(63)
-591(8) 7726(3) 7205(9) 64(3) C(64) -89(8) 7946(2) 7989(8) 54(2)
C(65) 1092(8) 8113(2) 8209(7) 52(2) C(66) 5145(7) 7905(2) 7409(6)
41(2) C(67) 6043(9) 8071(3) 6954(7) 53(2) C(68) 5468(8) 7483(2)
7619(6) 42(2) C(69) 5155(10) 6864(3) 6889(8) 60(2) C(70) 6264(9)
6846(2) 6585(7) 53(2) C(71) 6155(10) 6982(3) 5686(8) 59(2) C(72)
7172(12) 6977(3) 5408(9) 71(3) C(73) 8315(12) 6830(3) 5991(10)
75(3) C(74) 8438(12) 6699(4) 6868(10) 81(3) C(75) 7418(11) 6703(3)
7169(8) 67(3) N(9) 1491(5) 6777(2) 9943(5) 34(1) N(10) -10(5)
6508(2) 10508(5) 35(1) N(11) -2054(6) 6705(2) 9902(5) 41(2) N(12)
5259(6) 8126(2) 8254(5) 41(1) O(17) 2023(4) 6309(1) 11077(4) 39(1)
O(18) 3243(4) 7154(1) 10110(4) 35(1) O(19) 5136(5) 6622(1) 9151(4)
39(1) O(20) 3850(4) 7708(1) 8941(4) 37(1) O(21) 2929(5) 8222(2)
7826(4) 46(1) O(22) 4481(5) 8389(1) 9527(4) 41(1) O(23) 6271(6)
7362(2) 8322(4) 49(1) O(24) 4745(6) 7264(2) 6883(5) 53(2) P(3)
4158(2) 8129(1) 8706(1) 36(1) F(5) 1915(4) 6800(2) 8253(3) 50(1)
F(6) 2937(5) 6285(1) 8908(4) 53(1) C(76) 3540(7) 11117(2) 8649(6)
35(2) C(77) 1484(7) 10884(2) 8173(6) 39(2) C(78) 1778(7) 10570(2)
7671(6) 40(2) C(79) 2942(7) 10551(2) 7677(5) 35(2) C(80) 5100(7)
10796(2) 8131(5) 35(2) C(81) 6303(6) 10530(2) 7384(6) 35(2) C(82)
6961(7) 10396(2) 8421(6) 40(2) C(83) 6006(7) 10527(2) 8865(6) 41(2)
C(84) 6246(7) 10233(2) 6633(6) 39(2) C(85) 3528(7) 9348(2) 6559(6)
38(2) C(86) 2822(8) 9666(2) 6114(7) 48(2) C(87) 1628(8) 9720(2)
6144(7) 48(2) C(88) 1173(9) 9470(3) 6643(9) 67(3) C(89) 1910(11)
9160(4) 7098(11) 94(5) C(90) 3074(10) 9099(3) 7046(9) 71(3) C(91)
7615(8) 9178(2) 7289(7) 49(2) C(92) 8505(9) 8866(2) 7193(9) 64(3)
C(93) 8450(7) 9499(3) 7889(6) 47(2) C(94) 9666(9) 9667(3) 9478(7)
62(3) C(95) 10946(8) 9549(3) 9561(6) 50(2) C(96) 11759(10) 9775(3)
9326(8) 61(2) C(97) 12923(11) 9654(4) 9378(10) 84(4) C(98)
13298(11) 9271(4) 9690(10) 84(4) C(99) 12498(11) 9039(3) 9906(8)
70(3) C(100) 11361(10) 9177(3) 9876(8) 63(3) N(13) 3848(5) 10818(2)
8143(5) 34(1) N(14) 2369(6) 11140(2) 8647(5) 38(1) N(15) 343(6)
10931(2) 8180(5) 48(2) N(16) 6823(6) 9309(2) 6359(5) 44(2) O(25)
4392(5) 11344(2) 9089(4) 46(1) O(26) 5042(5) 10633(1) 7266(4) 39(1)
O(27) 8180(5) 10544(2) 8843(5) 51(2) O(28) 5706(5) 9886(1) 6841(4)
39(1) O(29) 4702(5) 9262(1) 6535(4) 39(1) O(30) 4937(5) 9639(1)
5117(4) 41(1) O(31) 8836(5) 9768(2) 7578(5) 50(1) O(32) 8700(6)
9428(2) 8821(5) 62(2) P(4) 5510(2) 9535(1) 6125(1) 36(1) F(7)
5347(5) 10213(1) 8974(4) 55(1) F(8) 6542(5) 10684(2) 9723(4) 61(1)
C(101) 6810(20) 9411(8) 10080(20) 93(8) C(102) 6300(20) 9155(6)
9290(15) 83(7) N(17) 7140(30) 9630(9) 10710(20) 148(12)
Example 2
The solubilities of NUC-1031 and its diastereoisomers were
determined in a range of pharmaceutically acceptable solvent
systems. The protocol adopted was as follows:
A small volume, 1-2 mL, of each solvent system was prepared and a
weight of the compound in question was added. The solutions were
stirred for approximately 4 hours and then 0.45 .mu.L membrane
filtered. The concentration of the compound in question in the
filtrate was then determined by HPLC assay.
Based on the gemcitabine dosage schedule used in the treatment of
pancreatic cancer, the molecular weight adjusted dose of NUC-1031
would be about 3200 mg, given as an infusion once weekly. As an
indication of the level of solubility required, taking a notional
target of a 500 mL infusion volume, the required solubility of the
NUC-1031 would be >6 mg/ml in the infusion fluid. However, this
solubility level is just an indication and solubilities below can
still provide effective therapies.
Table 5 shows the solubility of a
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate 2 epimeric
mixture in a range of solvents suitable for intravenous
administration.
TABLE-US-00005 Solvent Appearance Assay (mg/ml) Ethanol Solubilised
quickly, after 30 minutes precipitated out to white paste Glycerol
API evident Propylene Precipitation evident after 30 minutes 371
glycol PEG 400 Precipitation evident after 120 minutes 385 NMP
Clear solution >207 DMSO Clear solution >217
Table 6 shows the solubility of the two
gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate epimers 3 and 4
in a range of solvent/water mixtures.
TABLE-US-00006 Solvent (percentage by Solubility (R)- Solubility
(S)-epimer volume) epimer (mg/mL) 4 (mg/mL) 3 PROPYLENE GLYCOL 0
0.23 (10%) PROPYLENE GLYCOL 0.11 11.29 (50%) PEG400 (10%) 0 0.46
PEG400 (50%) 0.27 32.13 ETHANOL (10%) 0 0.27 ETHANOL (50%) 0.69
34.79
As can be seen from Table 6, the (R)-epimer 4 is substantially
insoluble in 10% mixtures of polar organic solvents in water. The
(S)-epimer 3, on the other hand, shows a significantly improved
solubility. In 50% mixtures of polar organic solvents in water, the
(S)-epimer 3 can be over 100 fold more soluble than the (R)-epimer
4. The (S)-epimer can thus provide a potentially very convenient
and effective therapy.
Example 3
To evaluate the differential uptake of the (R)- and (S)-epimers
into cyclodextrin, .sup.31P NMR spectra of NUC-1031 isomers mixture
after treatment with HP-.beta.-CD in D.sub.2O was recorded.
NMR studies. .sup.1H NMR (500 MHz) and .sup.31P NMR (202 MHz) were
recorded on a Bruker Avance 500 MHz spectrometer at 25.degree. C.
Chemical shifts (.delta.) are quoted in parts per million (ppm)
relative to internal D.sub.2O (.delta. 4.9 .sup.1H NMR) or external
85% H.sub.3PO.sub.4 (.delta. 0.00 .sup.31P NMR). Both HPLC and NMR
studies were carried out at room temperature
HPLC studies. Analytical High Performance Liquid Chromatography
(HPLC) analysis was performed using a ThermoScientific system.
Reverse-phase HPLC analyses were carried out on a SCIENTIFIC
Hypersil Gold C18, 5.mu., 150.times.4.6 mm eluting with
H.sub.2O/CH.sub.3CN from 90/10 to 0/100 in 30 min at a flow rate of
1 mL/min and at the detection wavelength of 280 nm. The retention
times of NUC-1031 epimers (dissolved in MeOH) are observed
respectively at 13.58 min for the (S)-epimer 3 and at 13.44 min for
the (R)-epimer 4 under these conditions (FIG. 5A).
2.36 mg of NUC-1031 isomer mixture (1:1.1 (S):(R)) was weighed and
transferred into a NMR tube. 13.3 mg of HP-.beta.-CD was then
dissolved in 1.3 mL of deuterium oxide and this was solution added
to the NMR tube (1:2.3 molar ratio NUC1031: HP-.beta.-CD) (NOTE:
not all the solid dissolved in the solution).
The .sup.31P NMR spectrum shows that HP-.beta.-CD is able to
enhance the solubility of NUC-1031 (S)-epimer 3 (4.14 Hz) relative
to the (R)-epimer (4.00 Hz), with the observed ratio of (S)- and
(R)-epimers in solution being 6.6:1 in favour of the (S)-epimer
(FIG. 4).
0.5 mL of the D.sub.2O solution from the NMR study was diluted to 1
mL by addition of 0.5 mL of water (1.15 mg/mL). 20 .mu.L of this
solution were injected into the HPLC.
HPLC analysis of the diluted NMR sample confirmed that the
(S)-epimer 3 is taken up into solution better than the (R)-epimer
4, with the observed ratio of (S)- and (R)-epimers in solution
being 5:1 in favour of the (S)-epimer, broadly in agreement with
the NMR data (FIG. 5B).
Similar studies conducted with another gemcitabine phosphate
derivative showed no difference between the uptake of the (S)- and
(R)-epimers of that derivative into a cyclodextrin solution.
Example 4
The clearance and bioavailability of most medicines are strongly
influenced by their first-pass metabolism in the liver. It is
possible to estimate the relative hepatic "metabolic stability" in
vitro by incubating compounds with cryopreserved hepatocytes and
determining the initial vs. the final amount of the test compound
in the incubation mixtures.
The following procedure is an HPLC-MS/MS assay using pooled human
cryopreserved hepatocyte suspension.
Assay Matrix
Human hepatocytes: mixed gender and pool of 10
Final cell density: 1 million (10.sup.6) viable cells/mL
Experimental Protocol
Pooled cryopreserved hepatocytes are thawed, washed, and
resuspended in Krebs-Heinslet buffer (pH 7.3). The reaction is
initiated by adding the test compound (1 .mu.M final concentration)
into cell suspension and incubated in a final volume of 100 .mu.L
on a flat-bottom 96-well plate for 0 minute and 120 minutes,
respectively, at 37.degree. C./5% CO.sub.2. The reaction is stopped
by adding 100 .mu.L of acetonitrile into the incubation mixture.
Samples are then mixed gently and briefly on a plate shaker,
transferred completely to a 0.8 mL V-bottom 96-well plate, and
centrifuged at 2550.times. g for 15 minutes at room temperature.
Each supernatant (150 .mu.L) is transferred to a clean cluster
tube, followed by HPLC-MS/MS analysis on a Thermo Electron
triple-quadrupole system.
This assay was modified for half-life determination. In this case,
the sampling time points are 0, 30, 60, 90, and 120 minutes.
Reference Compounds
Four reference compounds (1 .mu.M) were tested simultaneously with
the test compounds. Propranolol is relatively stable with human
hepatocytes, whereas flurazepam, naloxone, and terfenadine are
relatively unstable with human hepatocytes.
Analytical Methods
Samples are analyzed via (RP)HPLC-MS/MS using selected reaction
monitoring (SRM). The HPLC conditions consist of an HP1100 binary
pump with autosampler, a C-12 mixed-mode, 2.times.20 mm column, and
a gradient.
Data Analysis
Peak areas corresponding to the test compound are recorded by
HPLC-MS/MS. The metabolic stability, expressed as percent of the
test compound remaining, is calculated by comparing the peak areas
of the test compound at 2 hours to time zero. In case of half-life
determination, the half-life is estimated from the slope of the
initial linear range of the logarithmic curve of the test compound
remaining (%) vs. time, assuming first order kinetics.
The results are shown in Table 7.
Table 7 shows intrinsic clearances of the S epimer, the R epimer
and a mixture of the two epimers
TABLE-US-00007 Test Incubation Concentration Time % Compound
Remaining Half-Life (minute) (M) (minutes) 1st 2nd 3rd Mean 1st 2nd
3rd Mean Clint R/S Mix 1.0E-06 0 100.0 100.0 100.0 100.0 72.9 72.5
82.2 76 13.10 1.0E-06 30 64.3 68.7 74.3 69.1 13.10 1.0E-06 60 56.5
56.3 60.3 57.7 13.10 1.0E-06 90 59.6 55.9 45.2 53.6 13.10 1.0E-06
120 36.9 56.8 53.9 49.2 13.10 S 1.0E-06 0 100.0 100.0 100.0 100.0
40.5 36.5 35.0 37 26.60 1.0E-06 30 47.2 57.6 52.9 52.6 26.60
1.0E-06 60 35.8 32.0 30.4 32.7 26.60 1.0E-06 90 25.0 26.0 30.2 27.0
26.60 1.0E-06 120 31.0 30.6 35.6 32.4 26.60 R 1.0E-06 0 100.0 100.0
100.0 100.0 471.6 >120 >120 >120 <8.2 1.0E-06 30 80.2
86.9 94.3 87.1 <8.2 1.0E-06 60 97.4 {2.1} {1.3} 97.4 <8.2
1.0E-06 90 73.6 91.6 88.0 84.4 <8.2 1.0E-06 120 83.8 108.5 111.4
101.2 <8.2
* * * * *