U.S. patent application number 11/031250 was filed with the patent office on 2005-06-09 for prodrugs of phosphonate nucleotide analogues.
Invention is credited to Becker, Mark W., Chapman, Harlan H., Clhlar, Tomas, Elsenberg, Eugene J., He, Gong-Xin, Kernan, Michael R., Lee, William A., Prisbe, Ernest J., Rohloff, John C., Sparacino, Mark L..
Application Number | 20050124583 11/031250 |
Document ID | / |
Family ID | 22821718 |
Filed Date | 2005-06-09 |
United States Patent
Application |
20050124583 |
Kind Code |
A1 |
Becker, Mark W. ; et
al. |
June 9, 2005 |
Prodrugs of phosphonate nucleotide analogues
Abstract
A novel method has led to the identification of novel mixed
ester-amidates of PMPA for retroviral or hepadnaviral therapy,
including compounds of structure (5a) 1 having substituent groups
as defined herein. Compositions of these novel compounds in
pharmaceutically acceptable excipients and their use in therapy and
prophylaxis are provided.
Inventors: |
Becker, Mark W.; (Belmont,
CA) ; Chapman, Harlan H.; (La Honda, CA) ;
Clhlar, Tomas; (Foster City, CA) ; Elsenberg, Eugene
J.; (San Carlos, CA) ; He, Gong-Xin; (Fremont,
CA) ; Kernan, Michael R.; (Pacifica, CA) ;
Lee, William A.; (Los Altos, CA) ; Prisbe, Ernest
J.; (Los Altos, CA) ; Rohloff, John C.;
(Mountain View, CA) ; Sparacino, Mark L.; (Morgan
Hill, CA) |
Correspondence
Address: |
DORSEY & WHITNEY LLP
INTELLECTUAL PROPERTY DEPARTMENT
4 EMBARCADERO CENTER
SUITE 3400
SAN FRANCISCO
CA
94111
US
|
Family ID: |
22821718 |
Appl. No.: |
11/031250 |
Filed: |
January 6, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11031250 |
Jan 6, 2005 |
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10798692 |
Mar 11, 2004 |
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10798692 |
Mar 11, 2004 |
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10354207 |
Jan 28, 2003 |
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10354207 |
Jan 28, 2003 |
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09909560 |
Jul 20, 2001 |
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60220021 |
Jul 21, 2000 |
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Current U.S.
Class: |
514/81 ;
544/244 |
Current CPC
Class: |
A61P 31/00 20180101;
A61P 35/00 20180101; A61P 31/18 20180101; A61P 43/00 20180101; A61P
31/20 20180101; C07F 9/65616 20130101; C07H 21/00 20130101; A61P
31/12 20180101; C07H 19/20 20130101; A61P 1/16 20180101; G01N
33/5011 20130101; C07H 19/10 20130101; C12Q 1/18 20130101 |
Class at
Publication: |
514/081 ;
544/244 |
International
Class: |
A61K 031/675 |
Claims
1. A fumarate salt of a nucleotide analog compound according to
structural formula (Ia): 34that comprises at least about 60% by
weight a diastereomer according to structure (Ib): 35
2. The fumarate salt of claim 1 in which the nucleotide analog
compound comprises at least about 80% by weight the diastereomer of
structure (Ib).
3. The fumarate salt of claim 1 in which the nucleotide analog
compound comprises at least about 95% by weight the diastereomer of
structure (Ib).
4. The fumarate salt of claim 1 in which the nucleotide analog
compound comprises at least about 99% by wieight the diastereomer
of structure (Ib).
Description
1. CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of Ser. No. 10/798,692,
filed Mar. 11, 2004, now pending, which is a continuation of Ser.
No. 10/354,207, filed Jan. 28, 2003, now pending, which is a
continuation of Ser. No. 09/909,560, filed Jul. 20, 2001, now
abandoned, which claims benefit under 35 U.S.C. .sctn. 119(e) to
Ser. No. 60/220,021, filed Jul. 21, 2000, all of which are
incorporated herein by reference.
2. FIELD
[0002] This application relates to prodrugs of methoxyphosphonate
nucleotide analogues. In particular it relates to improved methods
for making and identifying such prodrugs.
[0003] Many methoxyphosphonate nucleotide analogues are known. In
general, such compounds have the structure
A-OCH.sub.2P(O)(OR).sub.2 where A is the residue of a nucleoside
analogue and R independently is hydrogen or various protecting or
prodrug functionalities. See U.S. Pat. Nos. 5,663,159, 5,977,061
and 5,798,340, Oliyai et al, "Pharmaceutical Research"
16(11):1687-1693 (1999), Stella et al., "J. Med. Chem."
23(12):1275-1282 (1980), Aarons, L., Boddy, A. and Petrak, K.
(1989) Novel Drug Delivery and Its Therapeutic Application
(Prescott, L. F. and Nimmo, W. S., ed.), pp. 121-126; Bundgaard, H.
(1985) Design of Prodrugs (Bundgaard, H., ed.) pp. 70-74 and 79-92;
Banerjee, P. K. and Amidon, G. L. (1985) Design of Prodrugs
(Bundgaard, H., ed.) pp. 118-121; Notari, R. E. (1985) Design of
Prodrugs (Bundgaard, H., ed.) pp. 135-156; Stella, V. J. and
Himmelstein, K. J. (1985) Design of Prodrugs (Bundgaard, H., ed.)
pp. 177-198; Jones, G. (1985) Design of Prodrugs (Bundgaard, H.,
ed.) pp. 199-241; Connors, T. A. (1985) Design of Prodrugs
(Bundgaard, H., ed.) pp. 291-316. All literature and patent
citations herein are expressly incorporated by reference.
3. SUMMARY OF THE INVENTION
[0004] Prodrugs of methoxyphosphonate nucleotide analogues intended
for antiviral or antitumor therapy, while known, traditionally have
been selected for their systemic effect. For example, such prodrugs
have been selected for enhanced bioavailability, i.e., ability to
be absorbed from the gastrointestinal tract and converted rapidly
to parent drug to ensure that the parent drug is available to all
tissues. However, applicants now have found that it is possible to
select prodrugs that become enriched at therapeutic sites, as
illustrated by the studies described herein where the analogues are
enriched at localized focal sites of HIV infection. The objective
of this invention is, among other advantages, to produce less
toxicity to bystander tissues and greater potency of the parental
drug in tissues which are the targets of therapy with the parent
methoxyphosphonate nucleotide analogue.
[0005] Accordingly, pursuant to these observations, a screening
method is provided for identifying a methoxyphosphonate nucleotide
analogue prodrug conferring enhanced activity in a target tissue
comprising:
[0006] (a) providing at least one of said prodrugs;
[0007] (b) selecting at least one therapeutic target tissue and at
least one non-target tissue;
[0008] (c) administering the prodrug to the target tissue and to
said at least one non-target tissue; and
[0009] (d) determining the relative antiviral activity conferred by
the prodrug in the tissues in step (c).
[0010] In preferred embodiments, the target tissue are sites where
HIV is actively replicated and/or which serve as an HIV reservoir,
and the non-target tissue is an intact animal. Unexpectedly, we
found that selecting lymphoid tissue as the target tissue for the
practice of this method for HIV led to identification of prodrugs
that enhance the delivery of active drug to such tissues.
[0011] A preferred compound of this invention, which has been
identified by this method has the structure (1), 2
[0012] where Ra is H or methyl,
[0013] and chirally enriched compositions thereof, salts, their
free base and solvates thereof.
[0014] A preferred compound of this invention has the structure (2)
3
[0015] and its enriched diasteromers, salts, free base and
solvates.
[0016] In addition, we unexpectedly found that the chirality of
substituents on the phosphorous atom and/or the amidate substituent
are influential in the enrichment observed in the practice of this
invention. Thus, in another embodiment of this invention, we
provide diastereomerically enriched compounds of this invention
having the structure (3) 4
[0017] which are substantially free of the diastereomer (4) 5
[0018] wherein
[0019] R.sup.1 is an oxyester which is hydrolyzable in vivo, or
hydroxyl;
[0020] B is a heterocyclic base;
[0021] R.sup.2 is hydroxyl, or the residue of an amino acid bonded
to the P atom through an amino group of the amino acid and having
each carboxy substituent of the amino acid optionally esterified,
but not both of R.sup.1 and R.sup.2 are hydroxyl;
[0022] E is --(CH.sub.2).sub.2--, --CH(CH.sub.3)CH.sub.2--,
--CH(CH.sub.2F)CH.sub.2--, --CH(CH.sub.2OH)CH.sub.2,
--CH(CH.dbd.CH.sub.2)CH.sub.2--, --CH(C.ident.CH)CH.sub.2--,
--CH(CH.sub.2N.sub.3)CH.sub.2--, 6
[0023] --CH(R.sup.6)OCH(R.sup.6')--, --CH(R.sup.9)CH.sub.2O--or
--CH(R.sup.8)O--, wherein the right hand bond is linked to the
heterocyclic base;
[0024] the broken line represents an optional double bond;
[0025] R.sup.4 and R.sup.5 are independently hydrogen, hydroxy,
halo, amino or a substituent having 1-5 carbon atoms selected from
acyloxy, alkyoxy, alkylthio, alkylamino and dialkylamino;
[0026] R.sup.6 and R.sup.6' are independently H, C.sub.1-C.sub.6
alkyl, C.sub.1-C.sub.6 hydroxyalkyl, or C.sub.2-C.sub.7
alkanoyl;
[0027] R.sup.7 is independently H, C.sub.1-C.sub.6 alkyl, or are
taken together to form --O-- or --CH.sub.2--;
[0028] R.sup.8 is H, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
hydroxyalkyl or C.sub.1-C.sub.6 haloalkyl; and
[0029] R.sup.9 is H, hydroxymethyl or acyloxymethyl;
[0030] and their salts, free base, and solvates.
[0031] The diastereomers of structure (3) are designated the (S)
isomers at the phosphorus chiral center.
[0032] Preferred embodiments of this invention are the
diastereomerically enriched compounds having the structure (5a)
7
[0033] which is substantially free of diastereomer (5b) 8
[0034] wherein
[0035] R.sup.5 is methyl or hydrogen;
[0036] R.sup.6 independently is H, alkyl, alkenyl, alkynyl, aryl or
arylalkyl, or R.sup.6 independently is alkyl, alkenyl, alkynyl,
aryl or arylalkyl which is substituted with from 1 to 3
substituents selected from alkylamino, alkylaminoalkyl,
dialkylaminoalkyl, dialkylamino, hydroxyl, oxo, halo, amino,
alkylthio, alkoxy, alkoxyalkyl, aryloxy, aryloxyalkyl, arylalkoxy,
arylalkoxyalkyl, haloalkyl, nitro, nitroalkyl, azido, azidoalkyl,
alkylacyl, alkylacylalkyl, carboxyl, or alkylacylamino;
[0037] R.sup.7 is the side chain of any naturally-occurring or
pharmaceutically acceptable amino acid and which, if the side chain
comprises carboxyl, the carboxyl group is optionally esterified
with an alkyl or aryl group;
[0038] R.sup.11 is amino, alkylamino, oxo, or dialkylamino; and
[0039] R.sup.12 is amino or H;
[0040] and its salts, tautomers, free base and solvates.
[0041] A preferred embodiment of this invention is the compound of
structure (6),
9-[(R)-2-[[(S)-[[(S)-1-(isopropoxycarbonyl)ethyl]amino]phe-
noxyphosphinyl]methoxy]propyl]adenine, also designated herein
GS-7340. 9
[0042] Another preferred embodiment of this invention is the
fumarate salt of structure (5) (structure (7)),
9-[(R)-2-[[(S)-[[(S)-1-(isopropoxycarbo-
nyl)ethyl]amino]phenoxyphosphinyl]methoxy]propyl]adenine fumarate
(1:1), also designated herein GS-7340-2 10
[0043] The compounds of structures (1)-(7) optionally are
formulated into compositions containing pharmaceutically acceptable
excipients. Such compositions are used in effective doses in the
therapy or prophylaxis of viral (particularly HIV or hepadnaviral)
infections.
[0044] In a further embodiment, a method is provided for the facile
manufacture of 9-[2-(phosphonomethoxy)propyl]adenine (hereinafter
"PMPA" or 9-[2-(phosphonomethoxy)ethyl]adenine (hereinafter "PMEA")
using magnesium alkoxide, which comprises combining
9-(2-hydroxypropyl)adenine or 9-(2-hydroxyethyl)adenine, protected
p-toluenesulfonyloxymethylphospho- nate and magnesium alkoxide, and
recovering PMPA or PMEA, respectively.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0045] FIG. 1 shows HPLC/C-14 traces of PBMC extracts from human
blood incubated for 1 h at 37.degree. C. with TDF, GS-7340 and
PMPA.
[0046] FIG. 2 shows PMPA and Prodrug concentration in plasma and
PBMCs following oral administration of GS 7340-2 to Dogs at 10
mg-eq/kg.
[0047] FIG. 3 depicts Tenofovir exposure in PBMCs and plasma upon
administration of 10 mg-eq/kg in dogs.
5. DETAILED DESCRIPTION OF THE INVENTION
[0048] The methoxyphosphonate nucleotide analogue parent drugs for
use in this screening method are compounds having the structure
A-OCH.sub.2P(O)(OH).sub.2 wherein A is the residue of a nucleoside
analogue. These compounds are known 11
[0049] per se and are not part of this invention. More
particularly, the parent compounds comprise a heterocyclic base B
and an aglycon E, in general having the structure wherein the group
B is defined below and group E is defined above. Examples are
described in U.S. Pat. Nos. 4,659,825, 4,808,716, 4,724,233,
5,142,051, 5,130,427, 5,650,510, 5,663,159, 5,302,585, 5,476,938,
5,696,263, 5,744,600, 5,688,778, 5,386,030, 5,733,896, 5,352,786,
and 5,798,340, and EP 821,690 and 654,037.
[0050] The prodrugs for use in the screening method of this
invention are covalently modified analogues of the parent
methoxyphosphonate nucleotide analogues described in the preceding
paragraph. In general, the phosphorus atom of the parent drug is
the preferred site for prodrug modification, but other sites are
found on the heterocyclic base B or the aglycon E. Many such
prodrugs are already known. Primarily, they are esters or amidates
of the phosphorus atom, but also include substitutions on the base
and aglycon. None of these modifications per se is part of this
invention and none are to be considered limiting on the scope of
the invention herein.
[0051] The phosphorus atom of the methoxyphosphonate nucleotide
analogues contains two valences for covalent modification such as
amidation or esterification (unless one phosphoryl hydroxyl is
esterified to an aglycon E hydroxyl substituent, whereupon only one
phosphorus valence is free for substitution). The esters typically
are aryloxy. The amidates ordinarily are naturally occurring
monoamino acids having free carboxyl group(s) esterified with an
alkyl or aryl group, usually phenyl, cycloalkyl, or t-, n- or
s-alkyl groups. Suitable prodrugs for use in the screening method
of this invention are disclosed for example in U.S. Pat. No.
5,798,340. However, any prodrug which is potentially believed to be
capable of being converted in vivo within target tissue cells to
the free methoxyphosphonate nucleotide analogue parent drug, e.g.,
whether by hydrolysis, oxidation, or other covalent transformation
resulting from exposure to biological tissues, is suitable for use
in the method of this invention. Such prodrugs may not be known at
this time but are identified in the future and thus become suitable
candidates available for testing in the method of this invention.
Since the prodrugs are simply candidates for screening in the
methods their structures are not relevant to practicing or enabling
the screening method, although of course their structures
ultimately are dispositive of whether or not a prodrug will be
shown to be selective in the assay.
[0052] The pro-moieties bound to the parent drug may be the same or
different. However, each prodrug to be used in the screening assay
will differ structurally from the other prodrugs to be tested.
Distinct, i.e. structurally different, prodrugs generally are
selected on the basis of either their stereochemistry or their
covalent structure, or these features are varied in combination.
Each prodrug tested, however, desirably is structurally and
stereochemically substantially pure, else the output of the
screening assay will be less useful. It is of course within the
scope of this invention to test only a single prodrug in an
individual embodiment of the method of this invention, although
typically then one would compare the results with prior studies
with other prodrugs.
[0053] We have found that the stereochemistry of the prodrugs is
capable of influencing the enrichment in target tissues. Chiral
sites are at the phosphorus atom and are also found in its
substituents. For example, amino acid used in preparing amidates
may be D or L forms, and the phosphonate esters or the amino acid
esters can contain chiral centers as well. Chiral sites also are
found on the nucleoside analogue portion of the molecules, but
these typically are already dictated by the stereochemistry of the
parent drug and will not be varied as part of the screen. For
example the R isomer of PMPA is preferred as it is more active than
the corresponding S isomer. Typically these diasteromers or
enantiomers will be chirally enriched if not pure at each site so
that the results of the screen will be more meaningful. As noted,
distinctiveness of stereoisomers is conferred by enriching or
purifying the stereoisomer (typically this will be a diastereomer
rather than an enantiomer in the case of most methoxyphosphonate
nucleotide analogues) free of other stereoisomers at the chiral
center in question, so that each test compound is substantially
homogeneous. By substantially homogeneous or chirally enriched, we
mean that the desired stereoisomer constitutes greater than about
60% by weight of the compound, ordinarily greater than about 80%
and preferably greater than about 95%.
[0054] 5.1 Novel Screening Method
[0055] Once at least one candidate prodrug has been selected, the
remaining steps of the screening method of this invention are used
to identify a prodrug possessing the required selectivity for the
target tissue. Most conveniently the prodrugs are labeled with a
detectable group, e.g. radiolabeled, in order to facilitate
detection later in tissues or cells. However, a label is not
required since other suitable assays for the prodrug or its
metabolites (including the parent drug) can also be employed. These
assays could include mass spectrometry, HPLC, bioassays or
immunoassays for instance. The assay may detect the prodrug and any
one or more of its metabolites, but preferably the assay is
conducted to detect only the generation of the parent drug. This is
based on the assumption (which may not be warranted in all cases)
that the degree and rate of conversion of prodrug to antivirally
active parent diphosphate is the same across all tissues tested.
Otherwise, one can test for the diphosphate.
[0056] The target tissue preferably will be lymphoid tissue when
screening for prodrugs useful in the treatment of HIV infection.
Lymphoid tissue will be known to the artisan and includes CD4
cells, lymphocytes, lymph nodes, macrophages and macrophage-like
cells including monocytes such as peripheral blood monocytic cells
(PBMCs) and glial cells. Lymphoid tissue also includes non-lymphoid
tissues that are enriched in lymphoid tissues or cells, e.g. lung,
skin and spleen. Other targets for other antiviral drugs of course
will be the primary sites of replication or latency for the
particular virus concerned, e.g., liver for hepatitis and
peripheral nerves for HSV. Similarly, target tissues for tumors
will in fact be the tumors themselves. These tissues are all
well-known to the artisan and would not require undue
experimentation to select. When screening for antiviral compounds,
target tissue can be infected by the virus.
[0057] Non-target tissues or cells also are screened as part of the
method herein. Any number or identity of such tissues or cells can
be employed in this regard. In general, tissues for which the
parent drug is expected to be toxic will be used as non-target
tissues. The selection of a non-target tissue is entirely dependent
upon the nature of the prodrug and the activity of the parent. For
example, non-hepatic tissues would be selected for prodrugs against
hepatitis, and untransformed cells of the same tissue as the tumor
will suffice for the antitumor-selective prodrug screen.
[0058] It should be noted that the method of this invention is
distinct from studies typically undertaken to determine oral
bioavailability of prodrugs. In oral bioavailability studies, the
objective is to identify a prodrug which passes into the systemic
circulation substantially converted to parent drug. In the present
invention, the objective is to find prodrugs that are not
metabolized in the gastrointestinal tract or circulation. Thus,
target tissues to be evaluated in the method of this invention
generally do not include the small intestines or, if the intestines
are included, then the tissues also include additional tissues
other than the small intestines.
[0059] The target and non-target tissues used in the screening
method of this invention typically will be in an intact living
animal. Prodrugs containing esters are more desirably tested in
dogs, monkeys or other animals than rodents; mice and rat plasma
contains high circulating levels of esterases that may produce a
misleading result if the desired therapeutic subject is a human or
higher mammal.
[0060] It is not necessary to practice this method with intact
animals. It also is within the scope of this invention to employ
perfused organs, in vitro culture of organs (e.g. skin grafts) or
cell lines maintained in various forms of cell culture, e.g. roller
bottles or zero gravity suspension systems. For example, MT-2 cells
can be used as a target tissue for selecting HIV prodrugs. Thus,
the term "tissue" shall not be construed to require organized
cellular structures, or the structures of tissues as they may be
found in nature, although such would be preferred. Rather, the term
"tissue" shall be construed to be synonymous with cells of a
particular source, origin or differentiation stage.
[0061] The target and non-target tissue may in fact be the same
tissue, but the tissues will be in different biological status. For
example, the method herein could be used to select for prodrugs
that confer activity in virally-infected tissue (target tissue) but
which remain substantially inactive in virally-uninfected cells
(corresponding non-target tissue). The same strategy would be
employed to select prophylactic prodrugs, i.e., prodrugs
metabolized to antivirally active forms incidental to viral
infection but which remain substantially unmetabolized in
uninfected cells. Similarly, prodrugs could be screened in
transformed cells and the untransformed counterpart tissue. This
would be particularly useful in comparative testing to select
prodrugs for the treatment of hematological malignancies, e.g.
leukemias.
[0062] Without being limited by any particular theory of operation,
tissue selective prodrugs are thought to be selectively taken up by
target cells and/or selectively metabolized within the cell, as
compared to other tissues or cells. The unique advantage of the
methoxyphosphonate prodrugs herein is that their metabolism to the
dianion at physiological pH ensures that they will be unable to
diffuse back out of the cell. They therefore remain effective for
lengthy periods of time and are maintained at elevated
intracellular concentrations, thereby exhibiting increased potency.
The mechanisms for enhanced activity in the target tissue are
believed to include enhanced uptake by the target cells, enhanced
intracellular retention, or both mechanisms working together.
However, the manner in which selectivity or enhanced delivery
occurs in the target tissue is not important. It also is not
important that all of the metabolic conversion of the prodrug to
the parent compound occurs within the target tissue. Only the final
drug activity-conferring conversion need occur in the target
tissue; metabolism in other tissues may provide intermediates
finally converted to antiviral forms in the target tissue.
[0063] The degree of selectivity or enhanced delivery that is
desired will vary with the parent compound and the manner in which
it is measured (% dose distribution or parent drug concentration).
In general, if the parent drug already possess a generous
therapeutic window, a low degree of selectivity may be sufficient
for the desired prodrug. On the other hand, toxic compounds may
require more extensive screening to identify selective prodrugs.
The relative expense of the method of this invention can be reduced
by screening only in the target tissue and tissues against which
the parent compound is known to be relatively toxic, e.g. for PMEA,
which is nephrotoxic at higher doses, the primary focus will be on
kidney and lymphoid tissues.
[0064] The step of determining the relative antiviral activity of a
prodrug in the selected tissues ordinarily is accomplished by
assaying target and non-target tissues for the relative presence or
activity of a metabolite of the prodrug, which metabolite is known
to have, or is converted to, a metabolite having antiviral or
antitumor activity. Thus, typically one would determine the
relative amount of the parent drug in the tissues over
substantially the same time course in order to identify prodrugs
that are preferentially metabolized in the target tissue to an
antivirally or antitumor active metabolite or precursor thereof
which in the target tissue ultimately produces the active
metabolite. In the case of antiviral compounds, the active
metabolite is the diphosphate of the phosphonate parent compounds.
It is this metabolite that is incorporated into the viral nucleic
acid, thereby truncating the elongating nucleic acid strand and
halting viral replication. Metabolites of the prodrug can be
anabolic metabolites, catabolic metabolites, or the product of
anabolism and catabolism together. The manner in which the
metabolite is produced is not important in the practice of the
method of this invention.
[0065] The method of this invention is not limited to assaying a
metabolite which per se possesses antiviral or antitumor activity.
Instead, one can assay inactive precursors of the active
metabolites. Precursors of the antivirally active diphosphate
metabolite include the monophosphate of the parent drug,
monophosphates of other metabolites of the parent drug (e.g., an
intermediate modification of a substituent on the heterocyclic
base), the parent itself and metabolites generated by the cell in
converting the prodrug to the parent prior to phosphorylation. The
precursor structures may vary considerably as they are the result
of cellular metabolism. However, this information is already known
or could be readily determined by one skilled in the art.
[0066] If the prodrug being assayed does not exhibit antitumor or
antiviral activity per se then adjustments to the raw assay results
may be required. For example, if the intracellular processing of
the inactive metabolite to an active metabolite occurs at different
rates among the tissues being tested, the raw assay results with
the inactive metabolite would need to be adjusted to take account
of the differences among the cell types because the relevant
parameter is the generation of activity in the target tissue, not
accumulation of inactive metabolites. However, determining the
proper adjustments would be within the ordinary skill. Thus, when
step (d) of the method herein calls for determining the activity,
activity can be either measured directly or extrapolated. It does
not mean that the method herein is limited to only assaying
intermediates that are active per se. For instance, the absence or
decline of the prodrug in the test tissues also could be assayed.
Step (d) only requires assessment of the activity conferred by the
prodrug as it interacts with the tissue concerned, and this may be
based on extrapolation or other indirect measurement.
[0067] Step (d) of the method of this invention calls for
determining the "relative" activity of the prodrug. It will be
understood that this does not require that each and every assay or
series of assays necessarily must also contain runs with the
selected non-target tissue. On the contrary, it is within the scope
of this invention to employ historical controls of the non-target
tissue or tissues, or algorithms representing results to be
expected from such non-target tissues, in order to provide the
benchmark non-target activity.
[0068] The results obtained in step (d) are then used optimally to
select or identify a prodrug which produces greater antiviral
activity in the target tissue than in the non-target tissue. It is
this prodrug that is selected for further development.
[0069] It will be appreciated that some preassessment of prodrug
candidates can be undertaken before the practice of the method of
this invention. For example, the prodrug will need to be capable of
passing largely unmetabolized through the gastrointestinal tract,
it will need to be substantially stable in blood, and it should be
able to permeate cells at least to some degree. In most cases it
also will need to complete a first pass of the hepatic circulation
without substantial metabolism. Such prestudies are optional, and
are well-known to those skilled in the art.
[0070] The same reasoning as is described above for antiviral
activity is applicable to antitumor prodrugs of methoxyphosphonate
nucleotide analogues as well. These include, for example, prodrugs
of PMEG, the guanyl analogue of PMEA. In this case, cytotoxic
phosphonates such as PMEG are worthwhile candidates to pursue as
their cytotoxicity in fact confers their antitumor activity.
[0071] A compound identified by this novel screening method then
can be entered into a traditional preclinical or clinical program
to confirm that the desired objectives have been met. Typically, a
prodrug is considered to be selective if the activity or
concentration of parent drug in the target tissue (% dose
distribution) is greater than 2.times., and preferably 5.times.,
that of the parent compound in non-target tissue. Alternatively, a
prodrug candidate can be compared against a benchmark prodrug. In
this case, selectivity is relative rather than absolute. Selective
prodrugs will be those resulting in greater than about 10.times.
concentration or activity in the target tissue as compared with the
prototype, although the degree of selectivity is a matter of
discretion.
[0072] 5.2 Novel Method for Preparation of Starting Materials or
Intermediates
[0073] Also included herein is an improved method for manufacture
of preferred starting materials (parent drugs) of this invention,
PMEA and (R)-PMPA. Typically, this method comprises reacting
9-(2-hydroxypropyl)adenine (HPA) or 9-(2-hydroxyethyl)adenine (HEA)
with a magnesium alkoxide, thereafter adding the protected aglycon
synthon p-toluene-sulfonyloxymethylphosphonate (tosylate) to the
reaction mixture, and recovering PMPA or PMEA, respectively.
[0074] Preferably, HPA is the enriched or isolated R enantiomer. If
a chiral HPA mixture is used, R-PMPA can be isolated from the
chiral PMPA mixture after the synthesis is completed.
[0075] Typically the tosylate is protected by lower alkyl groups,
but other suitable groups will be apparent to the artisan. It may
be convenient to employ the tosylate presubstituted with the
prodrug phosphonate substituents which are capable of acting as
protecting groups in the tosylation reaction, thereby allowing one
to bypass the deprotection step and directly recover prodrug or an
intermediate therefore.
[0076] The alkyl group of the magnesium alkoxide is not critical
and can be any C.sub.1-C.sub.6 branched or normal alkyl, but is
preferably t-butyl (for PMPA) or isopropyl (for PMEA). The reaction
conditions also are not critical, but preferably comprise heating
the reaction mixture at about 70-75.degree. C. with stirring or
other moderate agitation.
[0077] If there is no interest in retaining the phosphonate
substituents, the product is deprotected (usually with
bromotrimethylsilane where the tosylate protecting group is alkyl),
and the product then recovered by crystallization or other
conventional method as will be apparent to the artisan.
[0078] 5.3 Heterocyclic Base
[0079] In the compounds of this invention depicted in structures
(3) and (4), the heterocyclic base B is selected from the
structures 12
[0080] wherein
[0081] R.sup.15 is H, OH, F, Cl, Br, I, OR.sup.16, SH, SR.sup.16,
NH.sub.2, or NHR.sup.17;
[0082] R.sup.16 is C.sub.1-C.sub.6 alkyl or C.sub.2-C.sub.6 alkenyl
including --CH.sub.3, --CH.sub.2CH.sub.3, --CH.sub.2C.ident.CH,
--CH.sub.2CH.dbd.CH.sub.2 and --C.sub.3H.sub.7;
[0083] R.sup.17 is C.sub.1-C.sub.6 alkyl or C.sub.2-C.sub.6 alkenyl
including --CH.sub.3, --CH.sub.2CH.sub.3, --CH.sub.2C.ident.CH,
--CH.sub.2CH.dbd.CH.sub.2, and --C.sub.3H.sub.7;
[0084] R.sup.18 is N, CF, CC1, CBr, CI, CR.sup.19, CSR.sup.19, or
COR.sup.19;
[0085] R.sup.19 is H, C.sub.1-C.sub.9 alkyl, C.sub.2-C.sub.9
alkenyl, C.sub.2-C.sub.9 alkynyl, C.sub.1-C.sub.9
alkyl-C.sub.1-C.sub.9 alkoxy, or C.sub.7-C.sub.9 aryl-alkyl
unsubstituted or substituted by OH, F, Cl, Br or I, R.sup.19
therefore including --CH.sub.3, --CH.sub.2CH.sub.3, --CHCH.sub.2,
--CHCHBr, --CH.sub.2CH.sub.2Cl,
[0086] --CH.sub.2CH.sub.2F, --CH.sub.2CCH, --CH.sub.2CHCH.sub.2,
--C.sub.3H.sub.7, --CH.sub.2OH, --CH.sub.2OCH.sub.3,
--CH.sub.2OC2H.sub.5, --CH.sub.2OCCH,
--CH.sub.2OCH.sub.2CHCH.sub.2, --CH.sub.2C.sub.3H.sub.7,
--CH.sub.2CH.sub.2OH, --CH.sub.2CH.sub.2OCH.sub- .3,
[0087] --CH.sub.2CH.sub.2OC.sub.2H.sub.5, --CH.sub.2CH.sub.2OCCH,
--CH.sub.2CH.sub.2OCH.sub.2CHCH.sub.2, and
[0088] --CH.sub.2CH.sub.2OC.sub.3H.sub.7;
[0089] R.sup.20 is N or CH;
[0090] R.sup.21 is N, CH, CCN, CCF.sub.3, CC.ident.CH or
CC(O)NH.sub.2;
[0091] R.sup.22 is H, OH, NH.sub.2, SH, SCH.sub.3,
SCH.sub.2CH.sub.3, SCH.sub.2C.ident.CH, SCH.sub.2CH.dbd.CH.sub.2,
SC.sub.3H.sub.7, NH(CH.sub.3), N(CH.sub.3).sub.2,
NH(CH.sub.2CH.sub.3), N(CH.sub.2CH.sub.3).sub.2,
NH(CH.sub.2C.ident.CH), NH(CH.sub.2CHCH.sub.2), NH(C.sub.3H.sub.7),
halogen (F, Cl, Br or I) or X wherein X is
--(CH.sub.2).sub.m(O).sub.n(CH.sub.2).sub.mN(R.sup.10).sub.2
wherein each m is independently 0-2, n is 0-1, and
[0092] R.sup.10 independently is H,
[0093] C.sub.1-C.sub.15 alkyl, C.sub.2-C.sub.15 alkenyl,
C.sub.6-C.sub.15 arylalkenyl, C.sub.6-C.sub.15 arylalkynyl,
C.sub.2-C.sub.15 alkynyl,
C.sub.1-C.sub.6-alkylamino-C.sub.1-C.sub.6 alkyl, C.sub.5-C.sub.15
aralkyl, C.sub.6-C.sub.15 heteroaralkyl, C.sub.5-C.sub.6 aryl,
C.sub.2-C.sub.6 heterocycloalkyl,
[0094] C.sub.2-C.sub.15 alkyl, C.sub.3-C.sub.15 alkenyl,
C.sub.6-C.sub.15 arylalkenyl, C.sub.3-C.sub.15 alkynyl,
C.sub.7-C.sub.15 arylalkynyl,
C.sub.1-C.sub.6-alkylamino-C.sub.1-C.sub.6 alkyl, C.sub.5-C.sub.15
aralkyl, C.sub.6-C.sub.15 heteroalkyl or C.sub.3-C.sub.6
heterocycloalkyl wherein methylene in the alkyl moiety not adjacent
to N.sup.6 has been replaced by --O--,
[0095] optionally both R.sup.10 are joined together with N to form
a saturated or unsaturated C.sub.2-C.sub.5 heterocycle containing
one or two N heteroatoms and optionally an additional O or S
heteroatom,
[0096] or one of the foregoing R.sup.10 groups which is substituted
with 1 to 3 halo, CN or N.sub.3; but optionally at least one
R.sup.10 group is not H;
[0097] R.sup.23 is H, OH, F, Cl, Br, I, SCH.sub.3,
SCH.sub.2CH.sub.3, SCH.sub.2C.ident.CH, SCH.sub.2CHCH.sub.2,
SC.sub.3H.sub.7, OR.sup.16, NH.sub.2, NHR.sup.17 or R.sup.22;
and
[0098] R.sup.24 is O, S or Se.
[0099] B also includes both protected and unprotected heterocyclic
bases, particularly purine and pyrimidine bases. Protecting groups
for exocyclic amines and other labile groups are known (Greene et
al. "Protective Groups in Organic Synthesis") and include
N-benzoyl, isobutyryl, 4,4'-dimethoxytrityl (DMT) and the like. The
selection of protecting group will be apparent to the ordinary
artisan and will depend upon the nature of the labile group and the
chemistry which the protecting group is expected to encounter, e.g.
acidic, basic, oxidative, reductive or other conditions. Exemplary
protected species are N.sup.4-benzoylcytosine- ,
N.sup.6-benzoyladenine, N.sup.2-isobutyrylguanine and the like.
[0100] Protected bases have the formulas Xa.1, XIa.1, XIb.1, XIIa.1
or XIIIa.1 13
[0101] wherein R.sup.18, R.sup.20, R.sup.21, R.sup.24 have the
meanings previously defined; R.sup.22A is R.sup.39 or R.sup.22
provided that R.sup.22 is not NH.sub.2; R.sup.23A is R.sup.39 or
R.sup.23 provided that R.sup.23 is not NH.sub.2; R.sup.39 is
NHR.sup.40, NHC(O)R.sup.36 or CR.sup.41N(R.sup.38).sub.2 wherein
R.sup.36 is C.sub.1-C.sub.19 alkyl, C.sub.1-C.sub.19 alkenyl,
C.sub.3-C.sub.10 aryl, adamantoyl, alkylanyl, or C.sub.3-C.sub.10
aryl substituted with 1 or 2 atoms or groups selected from halogen,
methyl, ethyl, methoxy, ethoxy, hydroxy and cyano; R.sup.38 is
C.sub.1-C.sub.10 alkyl, or both R.sup.38 together are 1-morpholino,
1-piperidine or 1-pyrrolidine; R.sup.40 is C.sub.1-C.sub.10 alkyl,
including methyl, ethyl, propyl, isopropyl, butyl, isobutyl,
t-butyl, pentyl, hexyl, octyl and decanyl; and R.sup.41 is hydrogen
or CH.sub.3.
[0102] For bases of structures XIa.1 and XIb.1, if R.sup.39 is
present at R.sup.22A or R.sup.23A, both R.sup.39 groups on the same
base will generally be the same. Exemplary R.sup.36 are phenyl,
phenyl substituted with one of the foregoing R.sup.36 aryl
substituents, --C.sub.10H.sub.15 (where C.sub.10H.sub.15 is
2-adamantoyl), --CH.sub.2--C.sub.6H.sub.5, --C.sub.6H.sub.5,
--CH(CH.sub.3).sub.2, --CH.sub.2CH.sub.3, methyl, butyl, t-butyl,
heptanyl, nonanyl, undecanyl, or undecenyl.
[0103] Specific bases include hypoxanthine, guanine, adenine,
cytosine, inosine, thymine, uracil, xanthine, 8-aza derivatives of
2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine,
hypoxanthine, inosine and xanthine; 7-deaza-8-aza derivatives of
adenine, guanine, 2-aminopurine, 2,6-diaminopurine,
2-amino-6-chloropurine, hypoxanthine, inosine and xanthine; 1-deaza
derivatives of 2-aminopurine, 2,6-diaminopurine,
2-amino-6-chloropurine, hypoxanthine, inosine and xanthine; 7-deaza
derivatives of 2-aminopurine, 2,6-diaminopurine,
2-amino-6-chloropurine, hypoxanthine, inosine and xanthine; 3-deaza
derivatives of 2-aminopurine, 2,6-diaminopurine,
2-amino-6-chloropurine, hypoxanthine, inosine and xanthine;
6-azacytosine; 5-fluorocytosine; 5-chlorocytosine; 5-iodocytosine;
5-bromocytosine; 5-methylcytosine; 5-bromovinyluracil;
5-fluorouracil; 5-chlorouracil; 5-iodouracil; 5-bromouracil;
5-trifluoromethyluracil; 5-methoxymethyluracil; 5-ethynyluracil and
5-propynyluracil.
[0104] Preferably, B is a 9-purinyl residue selected from guanyl,
3-deazaguanyl, 1-deazaguanyl, 8-azaguanyl, 7-deazaguanyl, adenyl,
3-deazaadenyl, 1-dezazadenyl, 8-azaadenyl, 7-deazaadenyl,
2,6-diaminopurinyl, 2-aminopurinyl, 6-chloro-2-aminopurinyl and
6-thio-2-aminopurinyl, or a B' is a 1-pyrimidinyl residue selected
from cytosinyl, 5-halocytosinyl, and
5-(C.sub.1-C.sub.3-alkyl)cytosinyl.
[0105] Preferred B groups have the formula 14
[0106] wherein
[0107] R.sup.22 independently is halo, oxygen, NH.sub.2, X or H,
but optionally at least one R.sup.22 is X;
[0108] X is
--(CH.sub.2).sub.m(O).sub.n(CH.sub.2).sub.mN(R.sup.10).sub.2
wherein m is 0-2, n is 0-1, and
[0109] R.sup.10 independently is H,
[0110] C.sub.1-C.sub.15 alkyl, C.sub.2-C.sub.15 alkenyl,
C.sub.6-C.sub.15 arylalkenyl, C.sub.6-C.sub.15 arylalkynyl,
C.sub.2-C.sub.15 alkynyl,
C.sub.1-C.sub.6-alkylamino-C.sub.1-C.sub.6 alkyl, C.sub.5-C.sub.15
aralkyl, C.sub.6-C.sub.15 heteroaralkyl, C.sub.5-C.sub.6 aryl,
C.sub.2-C.sub.6 heterocycloalkyl,
[0111] C.sub.2-C.sub.15 alkyl, C.sub.3-C.sub.15 alkenyl,
C.sub.6-C.sub.15 arylalkenyl, C.sub.3-C.sub.15 alkynyl,
C.sub.7-C.sub.15 arylalkynyl,
C.sub.1-C.sub.6-alkylamino-C.sub.1-C.sub.6 alkyl, C.sub.5-C.sub.15
aralkyl, C.sub.6-C.sub.15 heteroalkyl or C.sub.3-C.sub.6
heterocycloalkyl wherein methylene in the alkyl moiety not adjacent
to N.sup.6 has been replaced by --O--,
[0112] optionally both R.sup.10 are joined together with N to form
a saturated or unsaturated C.sub.2-C.sub.5 heterocycle containing
one or two N heteroatoms and optionally an additional O or S
heteroatom,
[0113] or one of the foregoing R.sup.10 groups is substituted with
1 to 3 halo, CN or N.sub.3; but optionally at least one R.sup.10
group is not H; and
[0114] Z is N or CH, provided that the heterocyclic nucleus varies
from purine by no more than one Z.
[0115] E groups represent the aglycons employed in the
methoxyphosphonate nucleotide analogues. Preferably, the E group is
--CH(CH.sub.3)CH.sub.2-- or --CH.sub.2CH.sub.2--. Also, it is
preferred that the side groups at chiral centers in the aglycon be
substantially solely in the (R) configuration (except for
hydroxymethyl, which is the enriched (S) enantiomer).
[0116] R.sup.1 is an in vivo hydrolyzable oxyester having the
structure --OR.sup.35 or --OR.sup.6 wherein R.sup.35 is defined in
column 64, line 49 of U.S. Pat. No. 5,798,340, herein incorporated
by reference, and R.sup.6 is defined above. Preferably R.sup.1 is
aryloxy, ordinarily unsubstituted or para-substituted (as defined
in R.sup.6) phenoxy.
[0117] R.sup.2 is an amino acid residue, optionally provided that
any carboxy group linked by less than about 5 atoms to the amidate
N is esterified. R.sup.2 typically has the structure 15
[0118] wherein
[0119] n is 1 or 2;
[0120] R.sup.11 is R.sup.6 or H; preferably
R.sup.6.dbd.C.sub.3-C.sub.9 alkyl; C.sub.3-C.sub.9 alkyl
substituted independently with OH, halogen, O or N; C.sub.3-C.sub.6
aryl; C.sub.3-C.sub.6 aryl which is independently substituted with
OH, halogen, O or N; or C.sub.3-C.sub.6 arylalkyl which is
independently substituted with OH, halogen, O or N;
[0121] R.sup.12 independently is H or C.sub.1-C.sub.9 alkyl which
is unsubstituted or substituted by substituents independently
selected from the group consisting of OH, O, N, COOR.sup.11 and
halogen; C.sub.3-C.sub.6 aryl which is unsubstituted or substituted
by substituents independently selected from the group consisting of
OH, O, N, COOR.sup.11 and halogen; or C.sub.3-C.sub.9 aryl-alkyl
which is unsubstituted or substituted by substituents independently
selected from the group consisting of OH, O, N, COOR.sup.11 and
halogen;
[0122] R.sup.13 independently is C(O)--OR.sup.11; amino; amide;
guanidinyl; imidazolyl; indolyl; sulfoxide; phosphoryl;
C.sub.1-C.sub.3 alkylamino; C.sub.1-C.sub.3 alkyldiamino;
C.sub.1-C.sub.6 alkenylamino; hydroxy; thiol; C.sub.1-C.sub.3
alkoxy; C.sub.1-C.sub.3 alkthiol; (CH.sub.2), COOR.sup.11;
C.sub.1-C.sub.6 alkyl which is unsubstituted or substituted with
OH, halogen, SH, NH.sub.2, phenyl, hydroxyphenyl or
C.sub.7-C.sub.10 alkoxyphenyl; C.sub.2-C.sub.6 alkenyl which is
unsubstituted or substituted with OH, halogen, SH, NH.sub.2,
phenyl, hydroxyphenyl or C.sub.7-C.sub.10 alkoxyphenyl; and
C.sub.6-C.sub.12 aryl which is unsubstituted or substituted with
OH, halogen, SH, NH.sub.2, phenyl, hydroxyphenyl or
C.sub.7-C.sub.10 alkoxyphenyl; and
[0123] R.sup.14 is H or C.sub.1-C.sub.9 alkyl or C.sub.1-C.sub.9
alkyl independently substituted with OH, halogen, COOR.sup.11, O or
N; C.sub.3-C.sub.6 aryl; C.sub.3-C.sub.6 aryl which is
independently substituted with OH, halogen, COOR.sup.11, O or N; or
C.sub.3-C.sub.6 arylalkyl which is independently substituted with
OH, halogen, COOR.sup.11, O or N.
[0124] Preferably, R.sup.11 is C.sub.1-C.sub.6 alkyl, most
preferably isopropyl, R.sup.13 is the side chain of a naturally
occurring amino acid, n=1, R.sup.12 is H and R.sup.14 is H. In the
compound of structure (2), the invention includes metabolites in
which the phenoxy and isopropyl esters have been hydrolyzed to
--OH. Similarly, the de-esterified enriched phosphonoamidate
metabolites of compounds (5a), 5(b) and (6) are included within the
scope of this invention.
[0125] Aryl and "0" or "N" substitution are defined in column 16,
lines 42-58, of U.S. Pat. No. 5,798,340.
[0126] Typically, the amino acids are in the natural or l amino
acids. Suitable specific examples are set forth in U.S. Pat. No.
5,798,340, for instance Table 4 and col. 8-10 therein.
[0127] Alkyl as used herein, unless stated to the contrary, is a
normal, secondary, tertiary or cyclic hydrocarbon. Unless stated to
the contrary alkyl is C.sub.1-C.sub.12. Examples are --CH.sub.3,
--CH.sub.2CH.sub.3, --CH.sub.2CH.sub.2CH.sub.3,
--CH(CH.sub.3).sub.2, --CH.sub.2CH.sub.2CH.su- b.2CH.sub.3),
--CH.sub.2CH(CH.sub.3).sub.2, --CH(CH.sub.3)CH.sub.2CH.sub.3- ,
--C(CH.sub.3).sub.3, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3,
--CH(CH.sub.3)CH.sub.2CH.sub.2CH.sub.3,
--CH(CH.sub.2CH.sub.3).sub.2, --C(CH.sub.3).sub.2CH.sub.2CH.sub.3),
--CH(CH.sub.3)CH(CH.sub.3).sub.2,
--CH.sub.2CH.sub.2CH(CH.sub.3).sub.2),
--CH.sub.2CH(CH.sub.3)CH.sub.2CH.s- ub.3,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3,
--CH(CH.sub.3)CH.sub.2CH.sub.2CH.sub.2CH.sub.3,
--CH(CH.sub.2CH.sub.3)(CH- .sub.2CH.sub.2CH.sub.3),
--C(CH.sub.3).sub.2CH.sub.2CH.sub.2CH.sub.3,
--CH(CH.sub.3)CH(CH.sub.3)CH.sub.2CH.sub.3,
--CH(CH.sub.3)CH.sub.2CH(CH.s- ub.3).sub.2,
--C(CH.sub.3)(CH.sub.2CH.sub.3).sub.2,
--CH(CH.sub.2CH.sub.3)CH(CH.sub.3).sub.2,
--C(CH.sub.3).sub.2CH(CH.sub.3)- .sub.2, and
--CH(CH.sub.3)C(CH.sub.3).sub.3. Alkenyl and alkynyl are defined in
the same fashion, but contain at least one double or triple bond,
respectively.
[0128] Where enol or keto groups are disclosed, the corresponding
tautomers are to be construed as taught as well.
[0129] The prodrug compounds of this invention are provided in the
form of free base or the various salts enumerated in U.S. Pat. No.
5,798,340, and are formulated with pharmaceutically acceptable
excipients or solvating diluents for use as pharmaceutical products
also as set forth in U.S. Pat. No. 5,798,340. These prodrugs have
the antiviral and utilities already established for the parent
drugs (see U.S. Pat. No. 5,798,340 and other citations relating to
the methoxyphosphonate nucleotide analogues). It will be understood
that the diastereomer of structure (4) at least is useful as an
intermediate in the chemical production of the parent drug by
hydrolysis in vitro, regardless of its relatively unselective
character as revealed in the studies herein.
6. EXAMPLES
[0130] The invention will be more fully understood by reference to
the following examples:
Example 1a
[0131] 16
[0132] Adenine to PMEA using Magnesium Isopropoxide. To a
suspension of adenine (16.8 g, 0.124 mol) in DMF (41.9 ml) was
added ethylene carbonate (12.1 g, 0.137 mol) and sodium hydroxide
(0.100 g, 0.0025 mol). The mixture was heated at 130.degree. C.
overnight. The reaction was cooled to below 50.degree. C. and
toluene (62.1 ml) was added. The slurry was further cooled to
5.degree. C. for 2 hours, filtered, and rinsed with toluene
(2.times.). The wet solid was dried in vacuo at 65.degree. C. to
yield 20.0 g (90%) of 9-(2-hydroxyethyl)adenine as an off-white
solid. Mp: 238-240.degree. C. 17
[0133] 9-(2-Hydroxyethyl)adenine (HEA) (20.0 g, 0.112 mol) was
suspended in DMF (125 ml) and heated to 80.degree. C. Magnesium
isopropoxide (11.2 g, 0.0784 mol), or alternatively magnesium
t-butoxide, was added to the mixture followed by diethyl
p-toluenesulfonyloxymethylphosphonate (66.0 g, 0.162 mol) over one
hour. The mixture was stirred at 80.degree. C. for 7 hours. 30 ml
of volatiles were removed via vacuum distillation and the reaction
was recharged with 30 ml of fresh DMF. After cooling to room
temperature, bromotrimethylsilane (69.6 g, 0.450 mol) was added and
the mixture heated to 80.degree. C. for 6 hours. The reaction was
concentrated to yield a thick gum. The gum was dissolved into 360
ml water, extracted with 120 ml dichloromethane, adjusted to pH 3.2
with sodium hydroxide, and the resulting slurry stirred at room
temperature overnight. The slurry was cooled to 4.degree. C. for
one hour. The solids were isolated by filtration, washed with water
(2.times.), and dried in vacuo at 56.degree. C. to yield 20 g
(65.4%) of 9-[2-(phosphonomethoxy)et- hyl]adenine (PMEA) as a white
solid. Mp: >200.degree. C. dec. .sup.1H NMR (D.sub.2O) .delta.
3.49 (t, 2H); 3.94 (t, 2H); 4.39 (t, 2H); 8.13 (s, 1H); 8.22 (s,
1H).
Example 1b
[0134] 18
[0135] Adenine to PMPA using Magnesium t-Butoxide. To a suspension
of adenine (40 g, 0.296 mol) in DMF (41.9 ml) was added
(R)-propylene carbonate (34.5 g, 0.338 mol) and sodium hydroxide
(0.480 g, 0.012 mol). The mixture was heated at 130.degree. C.
overnight. The reaction was cooled to 100.degree. C. and toluene
(138 ml) was added followed by methanesulfonic acid (4.7 g, 0.049
mol) while maintaining the reaction temperature between
100-110.degree. C. Additional toluene (114 ml) was added to create
a homogeneous solution. The solution was cooled to 3.degree. C.
over 7 hours and then held at 3.degree. C. for one hour. The
resulting solid was isolated by filtration and rinsed with acetone
(2.times.). The wet solid was dried in vacuo at 80.degree. C. to
yield 42.6 g (75%) of (R)-9-[2-(hydroxy)propyl]adenine (HPA) as an
off-white solid. Mp: 188-190.degree. C. 19
[0136] (R)-9-[2-(hydroxy)propyl]adenine (HPA) (20.0 g, 0.104 mol)
was suspended in DMF (44.5 ml) and heated to 65.degree. C.
Magnesium t-butoxide (14.2 g, 0.083 mol), or alternatively
magnesium isopropoxide, was added to the mixture over one hour
followed by diethyl p-toluenesulfonyloxymethylphosphonate (66.0 g,
0.205 mol) over two hours while the temperature was kept at
78.degree. C. The mixture was stirred at 75.degree. C. for 4 hours.
After cooling to below 50.degree. C., bromotrimethylsilane (73.9 g,
0.478 mol) was added and the mixture heated to 77.degree. C. for 3
hours. When complete, the reaction was heated to 80.degree. C. and
volatiles were removed via atmospheric distillation. The residue
was dissolved into water (120 ml) at 50.degree. C. and then
extracted with ethyl acetate (101 ml). The pH of the aqueous phase
was adjusted to pH 1.1 with sodium hydroxide, seeded with authentic
(R)-PMPA, and the pH of the aqueous layer was readjusted to pH 2.1
with sodium hydroxide. The resulting slurry was stirred at room
temperature overnight. The slurry was cooled to 4.degree. C. for
three hours. The solid was isolated by filtration, washed with
water (60 ml), and dried in vacuo at 50.degree. C. to yield 18.9 g
(63.5%) of crude(R)-9-[2-(phosphon- omethoxy)propyl]adenine (PMPA)
as an off-white solid.
[0137] The crude(R)-9-[2-(phosphonomethoxy)propyl]adenine was
heated at reflux in water (255 ml) until all solids dissolved. The
solution was cooled to room temperature over 4 hours. The resulting
slurry was cooled at 4.degree. C. for three hours. The solid was
isolated by filtration, washed with water (56 ml) and acetone (56
ml), and dried in vacuo at 50.degree. C. to yield 15.0 g (50.4%) of
(R)-9-[2-(phosphonomethoxy)propy- l]adenine (PMPA) as a white
solid. Mp: 278-280.degree. C.
Example 2
Preparation of GS-7171 (III)
[0138] 2021
[0139] A glass-lined reactor was charged with anhydrous PMPA, (I)
(14.6 kg, 50.8 mol), phenyl (9.6 kg, 102 mol), and
1-methyl-2-pyrrolidinone (39 kg). The mixture was heated to
85.degree. C. and triethylamine (6.3 kg, 62.3 mol) added. A
solution of 1,3-dicyclohexylcarbodiimide (17.1 kg, 82.9 mol) in
1-methyl-2-pyrrolidinone (1.6 kg) was then added over 6 hours at
100.degree. C. Heating was continued for 16 hours. The reaction was
cooled to 45.degree. C., water (29 kg) added, and cooled to
25.degree. C. Solids were removed from the reaction by filtration
and rinsed with water (15.3 kg). The combined filtrate and rinse
was concentrated to a tan slurry under reduced pressure, water
(24.6 kg) added, and adjusted to pH=11 with NaOH (25% in water).
Fines were removed by filtration through diatomaceous earth (2 kg)
followed by a water (4.4 kg) rinse. The combined filtrate and rinse
was extracted with ethyl acetate (28 kg). The aqueous solution was
adjusted to pH=3.1 with HCl (37% in water) (4 kg). Crude II was
isolated by filtration and washed with methanol (12.7 kg). The
crude II wet cake was slurried in methanol (58 kg). Solids were
isolated by filtration, washed with methanol (8.5 kg), and dried
under reduced pressure to yield 9.33 kg II as a white powder:
.sup.1H NMR (D.sub.2O) .delta. 1.2 (d, 3H), 3.45 (q, 2H), 3.7 (q,
2H), 4 (m, 2H), 4.2 (q, 2H), 4.35 (dd, 2H), 6.6 (d, 2H), 7 (t, 1H),
7.15 (t, 2H), 8.15 (s, 1H), 8.2 (s, 1H); .sup.31P NMR (D.sub.2O)
.delta. 15.0 (decoupled).
[0140] GS-7171 (III). (Scheme 1) A glass-lined reactor was charged
with monophenyl PMPA, (II), (9.12 kg, 25.1 mol) and acetonitrile
(30.7 kg). Thionyl chloride (6.57 kg, 56.7 mol) was added below
50.degree. C. The mixture was heated at 75.degree. C. until solids
dissolved. Reaction temperature was increased to 80.degree. C. and
volatiles (11.4 kg) collected by atmospheric distillation under
nitrogen. The pot residue was cooled to 25.degree. C.,
dichloromethane (41 kg) added, and cooled to -29.degree. C. A
solution of (L)-alanine isopropyl ester (7.1 kg, 54.4 mol) in
dichloromethane (36 kg) was added over 60 minutes at -18.degree. C.
followed by triethylamine (7.66 kg, 75.7 mol) over 30 minutes at
-18 to -11.degree. C. The reaction mixture was warmed to room
temperature and washed five times with sodium dihydrogenphosphate
solution (10% in water, 15.7 kg each wash). The organic solution
was dried with anhydrous sodium sulfate (18.2 kg), filtered, rinsed
with dichloromethane (28 kg), and concentrated to an oil under
reduced pressure. Acetone (20 kg) was charged to the oil and the
mixture concentrated under reduced pressure. Acetone (18.8 kg) was
charged to the resulting oil. Half the product solution was
purified by chromatography over a 38.times.38 cm bed of 22 kg
silica gel 60, 230 to 400 mesh. The column was eluted with 480 kg
acetone. The purification was repeated on the second half of the
oil using fresh silica gel and acetone. Clean product bearing
fractions were concentrated under reduced pressure to an oil.
Acetonitrile (19.6 kg) was charged to the oil and the mixture
concentrated under reduced pressure. Acetonitrile (66.4 kg) was
charged and the solution chilled to 0 to -5.degree. C. for 16
hours. Solids were removed by filtration and the filtrate
concentrated under reduced pressure to 5.6 kg III as a dark oil:
.sup.1H NMR (CDCl.sub.3) .delta. 1.1 (m 12H), 3.7 (m, 1H), 4.0 (m,
5H), 4.2 (m, 1H), 5.0 (m, 1H), 6.2 (s, 2H), 7.05 (m, 5H), 8.0 (s,
1H), 8.25 (d, 1H); .sup.31P NMR (CDCl.sub.3) .delta. 21.0, 22.5
(decoupled).
Alternate Method for GS-7171(III)
[0141] 22
[0142] Monophenyl PMPA (II). A round-bottom flask with reflux
condenser and nitrogen inlet was placed in a 70.degree. C. oil
bath. The flask was charged with anhydrous PMPA (I) (19.2 g, 67
mmol), N,N-dimethylformamide (0.29 g, 3.3 mmol), and tetramethylene
sulfone (40 mL). Thionyl chloride (14.2 g, 119 mmol) was added over
4 hours. Heating was increased to 100.degree. C. over the same
time. A homogeneous solution resulted. Phenoxytrimethylsilane (11.7
g, 70 mmol) was added to the solution over 5 minutes. Heating in
the 100.degree. C. oil bath continued for two hours more. The
reaction was poured into rapidly stirring acetone (400 mL) with
cooling at 0.degree. C. Solids were isolated by filtration, dried
under reduced pressure, and dissolved in methanol (75 mL). The
solution pH was adjusted to 3.0 with potassium hydroxide solution
(45% aq.) with cooling in ice/water. The resulting solids were
isolated by filtration, rinsed with methanol, and dried under
reduced pressure to 20.4 g II (Scheme 2) as a white powder.
[0143] GS-7171 (III). Monophenyl PMPA (II) (3 g, 8.3 mmol),
tetramethylene sulfone (5 mL), and N,N-dimethylformamide (1 drop)
were combined in a round bottom flask in a 40.degree. C. oil bath.
Thionyl chloride (1.96 g, 16.5 mmol) was added. After 20 minutes
the clear solution was removed from heat, diluted with
dichloromethane (10 ml), and added to a solution of (L)-alanine
isopropyl ester (5 g, 33 mmol) and diisopropylethylamine (5.33 g,
41 mmol) in dichloromethane (20 mL) at -10.degree. C. The reaction
mixture was warmed to room temperature and washed three times with
sodium dihydrogenphosphate solution (10% aq., 10 mL each wash). The
organic solution was dried over anhydrous sodium sulfate and
concentrated under reduced pressure to a oil. The oil was combined
with fumaric acid (0.77 g, 6.6 mmol) and acetonitrile (40 mL) and
heated to reflux to give a homogeneous solution. The solution was
cooled in an ice bath and solids isolated by filtration. The solid
GS-7171 fumarate salt was dried under reduced pressure to 3.7 g.
The salt (3.16 g, 5.3 mmol) was suspended in dichloromethane (30
mL) and stirred with potassium carbonate solution (5 mL, 2.5 M in
water) until the solid dissolved. The organic layer was isolated,
then washed with water (5 mL), dried over anhydrous sodium sulfate,
and concentrated under reduced pressure to afford 2.4 g III as a
tan foam.
Example 3
Diastereomer Separation by Batch Elution Chromatography
[0144] The diastereomers of GS-7171 (III) were resolved by batch
elution chromatography using a commercially available Chiralpak AS,
20 .mu.m, 21.times.250 mm semi-preparative HPLC column with a
Chiralpak AS, 20 .mu.m, 21.times.50 mm guard column. Chiralpak.RTM.
AS is a proprietary packing material manufactured by Diacel and
sold in North America by Chiral Technologies, Inc. (U.S. Pat. Nos.
5,202,433, RE 35,919, 5,434,298, 5,434,299 and 5,498,752).
Chiralpak AS is a chiral stationary phase (CSP) comprised of
amylosetris[(S)-.alpha.-methylbenzyl carbamate] coated onto a
silica gel support.
[0145] The GS-7171 diastereomeric mixture was dissolved in mobile
phase, and approximately 1 g aliquots of GS-7171 were pumped onto
the chromatographic system. The undesired diastereomer, designated
GS-7339, was the first major broad (approx. 15 min. duration) peak
to elute from the column. When the GS-7339 peak had finished
eluting, the mobile phase was immediately switched to 100% methyl
alcohol, which caused the desired diastereomer, designated GS-7340
(IV), to elute as a sharp peak from the column with the methyl
alcohol solvent front. The methyl alcohol was used to reduce the
over-all cycle time. After the first couple of injections, both
diastereomers were collected as a single large fractions containing
one of the purified diastereomers (>99.0% single diastereomer).
The mobile phase solvents were removed in vacuo to yield the
purified diastereomer as a friable foam.
[0146] About 95% of the starting GS-7171 mass was recovered in the
two diastereomer fractions. The GS-7340 fraction comprised about
50% of the total recovered mass.
[0147] The chromatographic conditions were as follows:
1 Mobile Phase (Initial) GS-7171 - Acetonitrile:Isopropyl Alcohol
(90:10) (Final) 100% Methyl Alcohol Flow 10 mL/minute Run Time
About 45 minute Detection UV at 275 nm Temperature Ambient Elution
Profile GS-7339 (diastereomer B) GS-7340 (diastereomer A; (IV))
[0148] Diastereomer Separation of GS-7171 by SMB Chromatography
[0149] For a general description of simulated moving bed (SMB)
chromatography, see Strube et al., "Organic Process Research and
Development" 2:305-319 (1998).
[0150] GS-7340 (IV). GS-7171 (III), 2.8 kg, was purified by
simulated moving bed chromatography over 10 cm by 5 cm beds of
packing (Chiral Technologies Inc., 20 micron Chiralpak AS coated on
silica gel) (1.2 kg). The columns were eluted with 30% methanol in
acetonitrile. Product bearing fractions were concentrated to a
solution of IV in acetonitrile (2.48 kg). The solution solidified
to a crystalline mass wet with acetonitrile on standing. The
crystalline mass was dried under reduced pressure to a tan
crystalline powder, 1.301 kg IV, 98.7% diastereomeric purity: mp
117-120.degree. C.; .sup.1H NMR (CDCl.sub.3) .delta. 1.15 (m 12H),
3.7 (t, 1H), 4.0 (m, 5H), 4.2 (dd, 1H), 5.0 (m, 1H), 6.05 (s, 2H),
7.1 (m, 5H), 8.0 (s, 1H), 8.2 (s, 1H); .sup.31P NMR (CDCl.sub.3)
.delta. 21.0 (decoupled).
[0151] Diastereomer Separation by C18 RP-HPLC
[0152] GS-7171 (III) was chromatographed by reverse phase HPLC to
separate the diastereomers using the following summary
protocol.
2 Chromatographic Phenomenex Luna .TM. C18(2), 5 .mu.m, 100 .ANG.
column: pore size, (Phenomenex, Torrance, CA), or equivalent Guard
column: Pellicular C18 (Alltech, Deerfield, IL), or equivalent
Mobile Phase: A - 0.02% (85%) H.sub.3PO.sub.4 in water:acetonitrile
(95:5) B - 0.02% (85%) H.sub.3PO.sub.4 in water:acetonitrile
(50:50) Mobile Phase Gradient: % Mobile Phase % Mobile Phase Time A
B 0 100 0 5 100 0 7 70 30 32 70 30 40 0 100 50 0 100 Run Time: 50
minutes Equilibration Delay: 10 min at 100% mobile phase A Flow
Rate: 1.2 mL/min Temperature: Ambient Detection: UV at 260 nm
Sample Solution: 20 mM sodium phosphate buffer, pH 6 Retention
Times: GS-7339, about 25 minutes GS-7340, about 27 minutes
[0153] Diastereomer Separation by Crystallization
[0154] GS-7340 (IV). A solution of GS-7171 (III) in acetonitrile
was concentrated to an amber foam (14.9 g) under reduced pressure.
The foam was dissolved in acetonitrile (20 mL) and seeded with a
crystal of IV. The mixture was stirred overnight, cooled to
5.degree. C., and solids isolated by filtration. The solids were
dried to 2.3 g IV as white crystals, 98% diastereomeric purity
(.sup.31P NMR): .sup.1H NMR (CDCl.sub.3) .delta. 1.15 (m 12H), 3.7
(t, 1H), 3.95 (m, 2H), 4.05 (m, 2H), 4.2 (m, 2H), 5.0 (m, 1H), 6.4
(s, 2H), 7.1 (m, 5H), 8.0 (s, 1H), 8.2 (s, 1H); .sup.31P NMR
(CDCl.sub.3) .delta. 19.5 (decoupled). X-ray crystal analysis of a
single crystal selected from this product yielded the following
data:
3 Crystal Color, Habit colorless, column Crystal Dimensions 0.25
.times. 0.12 .times. 0.08 mm Crystal System orthorhombic Lattice
Type Primitive Lattice Parameters a = 8.352(1) .ANG. b = 15.574(2)
.ANG. c = 18.253(2) .ANG. V = 2374.2(5) .ANG..sup.3 Space Group
P2.sub.12.sub.12.sub.1 (#19) Z value 4 D.sub.calc 1.333 g/cm.sup.3
F.sub.000 1008.00 .mu.(MoK.alpha.) 1.60 cm.sup.-1
Example 4
Preparation of Fumarate Salt of GS-7340
[0155] GS-7340-02 (V). (Scheme 1) A glass-lined reactor was charged
with GS-7340 (IV), (1.294 kg, 2.71 mol), fumaric acid (284 g, 2.44
mol), and acetonitrile (24.6 kg). The mixture was heated to reflux
to dissolve the solids, filtered while hot and cooled to 5.degree.
C. for 16 hours. The product was isolated by filtration, rinsed
with acetonitrile (9.2 kg), and dried to 1329 g (V) as a white
powder: mp 119.7-121.1.degree. C.; [.alpha.].sub.D.sup.20
-41.7.degree. (c 1.0, acetic acid).
Example 5
Preparation of GS-7120 (VI)
[0156] 23
[0157] A 5 L round bottom flask was charged with monophenyl PMPA,
(II), (200 g, 0.55 mol) and acetonitrile (0.629 kg). Thionyl
chloride (0.144 kg, 1.21 mol) was added below 27.degree. C. The
mixture was heated at 70.degree. C. until solids dissolved.
Volatiles (0.45 L) were removed by atmospheric distillation under
nitrogen. The pot residue was cooled to 25.degree. C.,
dichloromethane (1.6 kg) was added and the mixture was cooled to
-20.degree. C. A solution of (L)-.alpha. aminobutyric acid ethyl
ester (0.144 kg, 1.1 mol) in dichloromethane (1.33 kg) was added
over 18 minutes at -20 to -10.degree. C. followed by triethylamine
(0.17 kg, 1.65 mol) over 15 minutes at -8 to -15.degree. C. The
reaction mixture was warmed to room temperature and washed four
times with sodium dihydrogenphosphate solution (10% aq., 0.3 L each
wash). The organic solution was dried with anhydrous sodium sulfate
(0.5 kg) and filtered. The solids were rinsed with dichloromethane
(0.6 kg) and the combined filtrate and rinse was concentrated to an
oil under reduced pressure. The oil was purified by chromatography
over a 15.times.13 cm bed of 1.2 kg silica gel 60, 230 to 400 mesh.
The column was eluted with a gradient of dichloromethane and
methanol. Product bearing fractions were concentrated under reduced
pressure to afford 211 g VI (Scheme 3) as a tan foam.
Example 5a
Diastereomer Separation of GS-7120 by Batch Elution
Chromatography
[0158] The diastereomeric mixture was purified using the conditions
described for GS-7171 in Example 3A except for the following:
4 Mobile Phase (Initial) GS-7120 - Acetonitrile:Isopropyl Alcohol
(98:2) (Final) 100% Methyl Alcohol Elution Profile: GS-7341
(diastereomer B) GS-7342 (diastereomer A)
Example 6
Diastereomer Separation of GS-7120 by Crystallization
[0159] A 1 L round bottom flask was charged with monophenyl PMPA,
(II), (50 g, 0.137 mol) and acetonitrile (0.2 L). Thionyl chloride
(0.036 kg, 0.303 mol) was added with a 10.degree. C. exotherm. The
mixture was heated to reflux until solids dissolved. Volatiles (0.1
L) were removed by atmospheric distillation under nitrogen. The pot
residue was cooled to 25.degree. C., dichloromethane (0.2 kg) was
added, and the mixture was cooled to -20.degree. C. A solution of
(L)-.alpha. aminobutyric acid ethyl ester (0.036 kg, 0.275 mol) in
dichloromethane (0.67 kg) was added over 30 minutes at -20 to
-8.degree. C. followed by triethylamine (0.042 kg, 0.41 mol) over
10 minutes at up to -6.degree. C. The reaction mixture was warmed
to room temperature and washed four times with sodium
dihydrogenphosphate solution (10% aq., 0.075 L each wash). The
organic solution was dried with anhydrous sodium sulfate (0.1 kg)
and filtered. The solids were rinsed with ethyl acetate (0.25 L,
and the combined filtrate and rinse was concentrated to an oil
under reduced pressure. The oil was diluted with ethyl acetate
(0.25 L), seeded, stirred overnight, and chilled to -15.degree. C.
The solids were isolated by filtration and dried under reduced
pressure to afford 17.7 g of GS-7342 (Table 5) as a tan powder:
.sup.1H NMR (CDCl.sub.3) .delta. 0.95 (t, 3H), 1.3 (m, 6H), 1.7,
(m, 2H), 3.7 (m, 2H), 4.1 (m, 6H), 4.4 (dd, 1H), 5.8 (s, 2H), 7.1
(m, 5H), 8.0 (s, 1H), 8.4 (s, 1H); .sup.31P NMR (CDCl.sub.3)
.delta. 21 (decoupled).
Example 7
Diastereomer Separation of GS-7097
[0160] The diastereomeric mixture was purified using the conditions
described for GS-7171 (Example 3A) except for the following:
5 Mobile Phase (Initial) GS-7120 - Acetonitrile:Isopropyl Alcohol
(95:5) (Final) 100% Methyl Alcohol Elution Profile GS-7115
(diastereomer B) GS-7114 (diastereomer A)
Example 8
Alternative Procedure for Preparation of GS-7097
[0161] GS-7097: Phenyl PMPA, Ethyl L-Alanyl Amidate. Phenyl PMPA
(15.0 g, 41.3 mmol), L-alanine ethyl ester hydrochloride (12.6 g,
83 mmol) and triethylamine (11.5 mL, 83 mmol) were slurried
together in 500 mL pyridine under dry N.sub.2. This suspension was
combined with a solution of triphenylphosphine (37.9 g, 145 mmol),
Aldrithiol 2 (2,2'-dipyridyl disulfide) (31.8 g, 145 mmol), and 120
mL pyridine. The mixture was heated at an internal temperature of
57.degree. C. for 15 hours. The complete reaction was concentrated
under vacuum to a yellow paste, 100 g. The paste was purified by
column chromatography over a 25.times.11 cm bed of 1.1 kg silica
gel 60, 230 to 400 mesh. The column was eluted with 8 liters of 2%
methanol in dichloromethane followed by a linear gradient over a
course of 26 liters eluent up to a final composition of 13%
methanol. Clean product bearing fractions were concentrated to
yield 12.4 g crude (5), 65% theory. This material was contaminated
with about 15% (weight) triethylamine hydrochloride by .sup.1H NMR.
The contamination was removed by dissolving the product in 350 mL
ethyl acetate, extracting with 20 mL water, drying the organic
solution over anhydrous sodium sulfate, and concentrating to yield
11.1 g pure GS-7097 as a white solid, 58% yield. The process also
is employed to synthesize the diastereomeric mixture of GS-7003a
and GS-7003b (the phenylalanyl amidate) and the mixture GS-7119 and
GS-7335 (the glycyl amidate). These diastereomers are separated
using a batch elution procedure such as shown in Example 3A, 6 and
7.
Example 9
In Vitro Studies of Prodrug Diastereomers
[0162] The in vitro anti-HIV-1 activity and cytotoxicity in MT-2
cells and stability in human plasma and MT-2 cell extracts of
GS-7340 (freebase) and tenofovir disoproxil fumarate (TDF), are
shown in Table 1. GS-7340 shows a 10-fold increase in antiviral
activity relative to TDF and a 200-fold increase in plasma
stability. This greater plasma stability is expected to result in
higher circulating levels of GS-7340 than TDF after oral
administration.
6TABLE 1 In Vitro Activity and Stability Stability T 1/2 (min)
HIV-1 MT-2 Activity Cytotoxicity Human Cell IC.sub.50 .mu.M
CC.sub.50 .mu.M Plasma Extract (P/MT-2) GS 7340 0.005 >40 90.0
28.3 3.2 TDF 0.05 70 0.41 70.7 0.006 Tenofovir 5 6000 -- -- --
[0163] In order to estimate the relative intracellular PMPA
resulting from the intracellular metabolism of TDF as compared to
that from GS-7340, both prodrugs and PMPA were radiolabeled and
spiked into intact human whole blood at equimolar concentrations.
After 1 hour, plasma, red blood cells (RBCs) and peripheral blood
mononuclear cells (PBMCs) were isolated and analyzed by HPLC with
radiometric detection. The results are shown in Table 2.
[0164] After 1 hour, GS-7340 results in 10.times. and 30.times. the
total intracellular concentration of PMPA species in PBMCs as
compared to TDF and PMPA, respectively. In plasma after 1 hour, 84%
of the radioactivity is due to intact GS-7340, whereas no TDF is
detected at 1 hour. Since no intact TDF is detected in plasma, the
10.times. difference at 1 hour between TDF and GS-7340 is the
minimum difference expected in vivo. The HPLC chromatogram for all
three compounds in PBMCs is shown in FIG. 1.
7TABLE 2 PMPA Metabolites in Plasma, PBMCs and RBCs After 1 h
Incubation of PMPA Prodrugs or PMPA in Human Blood. Total
Metabolites (% of Total Peak Area) C-14 Recovered, PMPA PMPAp,
PMPApp, Met. X, Met. Y, GS 7340, Compound Matrix .mu.g-eq % % % % %
% GS-7340 Plasma/ 43.0 1 -- -- 2 13 84 FP (60 .mu.g-eq) PBMC 1.25
45 16 21 18 -- -- RBC/FP 12.6 8 -- -- 24 11 57 Mono- PMPA PMPAp
PMPApp POC GS-4331 GS-4331 Plasma/ 48.1 11 -- -- 89 -- (TDF) FP (60
.mu.g-eq) PBMC 0.133 50 25 18 7 -- RBC/FP 10.5 93 7.0 -- -- -- PMPA
PMPAp PMPApp PMPA Plasma/ 55.7 100 -- -- FP (60 .mu.g-eq) PBMC
0.033 86 14 -- RBC/FP 3.72 74 10 16
[0165] Met. X and Met Y (metabolites X and Y) are shown in Table 5.
Lower case "p" designates phosphorylation. These results were
obtained after 1 hour in human blood. With increasing time, the in
vitro differences are expected to increase, since 84% of GS-7340 is
still 10 intact in plasma after one hour. Because intact GS-7340 is
present in plasma after oral administration, the relative clinical
efficacy should be related to the IC.sub.50 values seen in
vitro.
[0166] In Table 3 below, IC.sub.50 values of tenofovir, TDF,
GS-7340, several nucleosides and the protease inhibitor nelfinivir
are listed. As shown, nelfinavir and GS-7340 are 2-3 orders of
magnitude more potent than all other nucleotides or
nucleosides.
8TABLE 3 In Vitro Anti-HIV-1 Activities of Antiretroviral Compounds
Compound IC.sub.50 (.mu.M) Adefovir (PMEA) 13.4 .+-. 4.2.sup.1
Tenofovir (PMPA) 6.3 .+-. 3.3.sup.1 AZT 0.17 .+-. 0.08.sup.1 3TC
1.8 .+-. 0.25.sup.1 d4T 8 .+-. 2.5.sup.1 Nelfinavir 0.006 .+-.
0.002.sup.1 TDF 0.05 GS 7340 0.005 .sup.1A. S. Mulato and J. M.
Cherrington, Antiviral Research 36, 91 (1997)
[0167] Additional studies of the in vitro cell culture anti-HIV-1
activity and CC.sub.50 of separated diastereomers of this invention
were conducted and the results tabulated below.
9TABLE 4 Effect of Diastereomer Diastereomer Fold A/B CC.sub.50
Compound residue IC.sub.50 (.mu.M) change activity (.mu.M) PMPA --
5 1.times. -- 6000 Ala-methylester Mixture 1:1 0.025 200.times.
20.times. 80 GS-6957a A 0.0075 670.times. GS-6957b 0.15 33.times.
Phe-methylester Mixture 1:1 0.03 170.times. 10.times. 60 GS-7003a A
0.01 500.times. GS-7003b B 0.1 50.times..times. Gly-ethylester
Mixture 1:1 0.5 10.times. 20.times. GS-7119 A 0.05 100.times.
>100 GS-7335 B 1.0 5.times. Ala-isopropyl Mixture 1:1 0.01
500.times. 12.times. GS-7340 A 0.005 1,000.times. 40 GS-7339 B 0.06
83.times. >100 ABA-ethyl Mixture 1:1 0.008 625.times. 7.5.times.
>100 GS-7342 A 0.004 1,250.times. GS-7341 B 0.03 170.times.
Ala-ethyl Mixture 1:1 0.02 250.times. 10.times. 60 GS-7114 A 0.005
1,000.times. GS-7115 B 0.05 100.times..times.
[0168] Assay reference: Arimilli, M N, et al., (1997) Synthesis, in
vitro biological evaluation and oral bioavailability of
9-[2-(phosphonomethoxy)- propyl]adenine (PMPA) prodrugs. Antiviral
Chemistry and Chemotherapy 8(6):557-564.
[0169] "Phe-methylester" is the methylphenylalaninyl monoamidate,
phenyl monoester of tenofovir; "gly-methylester" is the
methylglycyl monoamidate, phenyl monoester of tenofovir.
[0170] In each instance above, isomer A is believed to have the
same absolute stereochemistry as GS-7340 (S), and isomer B is
believed to have the same absolute stereochemistry that of
GS-7339.
[0171] The in vitro metabolism and stability of separated
diastereomers were determined in PLCE, MT-2 extract and human
plasma. A biological sample listed below, 80 .mu.L, was transferred
into a screw-capped centrifuge tube and incubated at 37.degree. C.
for 5 min. A solution containing 0.2 mg/mL of the test compound in
a suitable buffer, 20 .mu.L, was added to the biological sample and
mixed. The reaction mixture, 20 mL, was immediately sampled and
mixed with 60 .mu.L of methanol containing 0.015 mg/mL of
2-hydroxymethylnaphthalene as an internal standard for HPLC
analysis. The sample was taken as the time-zero sample. Then, at
specific time points, the reaction mixture, 20 .mu.L, was sampled
and mixed with 60 .mu.L of methanol containing the internal
standard. The mixture thus obtained was centrifuged at 15,000 G for
5 min and the supernatant was analyzed with HPLC under the
conditions described below.
[0172] The biological samples evaluated are as follows.
[0173] (1) PLCE (porcine liver carboxyesterase from Sigma, 160 u/mg
protein, 21 mg protein/mL) diluted 20 fold with PBS
(phosphated-buffered saline).
[0174] (2) MT-2 cell extract was prepared from MT-2 cells according
to the published procedure [A. Pompon, I. Lefebvre, J.-L. Imbach,
S. Kahn, and D. Farquhar, "Antiviral Chemistry & Chemotherapy",
5:91-98 (1994)] except for using HEPES buffer described below as
the medium.
[0175] (3) Human plasma (pooled normal human plasma from George
King Biomedical Systems, Inc.)
[0176] The buffer systems used in the studies are as follows.
[0177] In the study for PLCE, the test compound was dissolved in
PBS. PBS (phosphate-buffered saline, Sigma) contains 0.01 M
phosphate, 0.0027 M potassium chloride, and 0.137 M sodium
chloride. pH 7.4 at 37.degree. C.
[0178] In the study for MT-2 cell extracts, the test compound was
dissolved in HEPES buffer. HEPES buffer contains 0.010 M HEPES,
0.05 M potassium chloride, 0.005 M magnesium chloride, and 0.005 M
dl-dithiothreitol. pH 7.4 at 37.degree. C.
[0179] In the study for human plasma, the test compound was
dissolved in TBS. TBS (tris-buffered saline, Sigma) contains 0.05 M
Tris, 0.0027 M potassium chloride, and 0.138 M sodium chloride. pH
7.5 at 37.degree. C.
[0180] The HPLC analysis was carried out under the following
conditions.
10 Column: Zorbax R.sub.x-C.sub.8, 4.6 .times. 250 mm, 5.mu.
(MAC-MOD Analytical, Inc. Chadds Ford, PA) Detection: UV at 260 nm
Flow Rate: 1.0 mL/min Run Time: 30 min Injection Volume: 20 .mu.L
Column Temperature: Ambient temperature Mobile Phase A: 50 mM
potassium phosphate (pH 6.0)/CH.sub.3CN = 95/5 (v/v) Mobile Phase
B: 50 mM Potassium phosphate (pH 6.0)/CH.sub.3CN = 50/50 (v/v)
Gradient Run: 0 min 100% Mobile Phase A 25 min 100% Mobile Phase B
30 min 100% Mobile Phase B
[0181] The results are shown below in Table 5 (also including
selected IC.sub.50 data from Table 4).
11TABLE 5 In Vitro Metabolism of Isomers A and B of PMPA
monoamidate at 37.degree. C. Human HIV MT-2 extract Plasma PMPA
monoamidate IC.sub.50 PLCE hydrolysis hydrolysis rate Stability No.
structure (.quadrature.M) rate and product and product (HP) 1 24
0.005 t1/2 = 2.9 min Met. X & PMPA t1/2 = 2.9 min Met. X &
PMPA t1/2 = 148 min Met. Y 2 25 0.05 t1/2 = 8.0 min Met. X &
PMPA t1/2 = 150.6 min Met. X & PMPA t1/2 = 495 min Met. Y 3 26
0.005 t1/2 = 3.3 min Met. X & PMPA t1/2 = 28.3 min Met. X &
PMPA t1/2 = 90.0 min Met. Y 4 27 0.06 t1/2 = 10.1 min Met. X &
PMPA t1/2 > 1000 min t1/2 = 231 min Met. Y 5 28 0.004 t1/2 = 3.9
min Met. X t1/2 = 49.2 min Met. X & PMPA t1/2 = 103 min Met. Y
6 29 0.03 t1/2 = 11.3 min Met. X t1/2 > 1000 min t1/2 = 257 min
Met. Y 7 30 0.05 t1/2 < 0.14 min MonoPOC PMPA t1/2 = 70.7 min
monoPOC PMPA t1/2 = 0.41 min monoPOC PMPA Met. X: 31 Met. Y: 32 A =
33
Example 10
Plasma and PBMC Exposures Following Oral Administration of Prodrug
Diastereomers to Beagle Dogs
[0182] The pharmacokinetics of GS 7340 were studied in dogs after
oral administration of a 10 mg-eq/kg dose.
[0183] Formulations. The prodrugs were formulated as solutions in
50 mM citric acid within 0.5 hour prior to dose. All compounds used
in the studies were synthesized by Gilead Sciences. The following
lots were used:
12 Amidate Amino Diastereo- GSI acid AA Ester isomer Lot Number
GS-7340-2 Alanine i-Propyl Isomer A 1504-187-19 GS-7339 Alanine
i-Propyl Isomer B 1509-185-31 GS7114 Alanine Ethyl Isomer A
1509-181-26 GS7115 Alanine Ethyl Isomer B 1509-181-22 GS7119
Glycine Ethyl Isomer A 1428-163-28 GS7342 .quadrature.-Aminobutyric
Ethyl Isomer A 1509-191-12 Acid GS7341 .quadrature.-Aminobutyric
Ethyl Isomer B 1509-191-7 Acid
[0184] Dose Administration and Sample Collection. The in-life phase
of this study was conducted in accordance with the recommendations
of the "Guide for the Care and Use of Laboratory Animals" (National
Institutes of Health publication 86-23) and was approved by an
Institutional Animal Care and Use Committee. Fasted male beagle
dogs (10.+-.2 kg) were used for the studies. Each drug was
administered as a single dose by oral gavage (1.5-2 ml/kg). The
dose was 10 mg-equivalent of PMPA/kg. For PBMCs, blood samples were
collected at 0 (pre-dose), 2, 8, and 24 h post-dose. For plasma,
blood samples were collected at 0 (pre-dose), 5, 15, and 30 min,
and 1, 2, 3, 4, 6, 8, 12 and 24 h post-dose. Blood (1.0 ml) was
processed immediately for plasma by centrifugation at 2,000 rpm for
10 min. Plasma samples were frozen and maintained at 70.degree. C.
until analyzed.
[0185] Peripheral Blood Mononuclear Cell (PBMC) preparation. Whole
blood (8 ml) drawn at specified time points was mixed in equal
proportion with phosphate buffered saline (PBS), layered onto 15 ml
of Ficoll-Paque solution (Pharmacia Biotech) and centrifuged at
400.times.g for 40 min. PBMC layer was removed and washed once with
PBS. Formed PMBC pellet was reconstituted in 0.5 ml of PBS, cells
were resuspended, counted using hemocytometer and maintained at
70.degree. C. until analyzed. The number of cells multiplied by the
mean single-cell volume was used in calculation of intracellular
concentrations. A reported value of 200 femtoliters/cell was used
as the resting PBMC volume (B. L. Robins, R. V. Srinivas, C. Kim,
N. Bischofberger, and A. Fridland, Antimicrob. Agents Chemother.
42, 612 (1998).
[0186] Determination of PMPA and Prodrugs in plasma and PBMCs. The
concentration of PMPA in dog plasma samples was determined by
derivatizing PMPA with chloroacetaldehyde to yield a highly
fluorescent N.sup.1, N.sup.6-ethenoadenine derivative (L. Naesens,
J. Balzarini, and E. De Clercq, Clin. Chem. 38, 480 (1992).
Briefly, plasma (100 .mu.l) was mixed with 200 .mu.l acetonitrile
to precipitate protein. Samples were then evaporated to dryness
under reduced pressure at room temperature. Dried samples were
reconstituted in 200 .mu.l derivatization cocktail (0.34%
chloroacetaldehyde in 100 mM sodium acetate, pH 4.5), vortexed, and
centrifuged. Supernatant was then transferred to a clean screw-cap
tube and incubated at 95.degree. C. for 40 min. Derivatized samples
were then evaporated to dryness and reconstituted in 100 .mu.l of
water for HPLC analysis.
[0187] Before intracellular PMPA could be determined by HPLC, the
large amounts of adenine related ribonucleotides present in the
PBMC extracts had to be removed by selective oxidation. We used a
modified procedure of Tanaka et al (K. Tanaka, A. Yoshioka, S.
Tanaka, and Y. Wataya, Anal. Biochem., 139, 35 (1984). Briefly,
PBMC samples were mixed 1:2 with methanol and evaporated to dryness
under reduced pressure. The dried samples were derivatized as
described in the plasma assay. The derivatized samples were mixed
with 20 .mu.L of 1M rhamnose and 30 .mu.L of 0.1M sodium periodate
and incubated at 37.degree. C. for 5 min. Following incubation, 40
.mu.L of 4M methylamine and 20 .mu.L of 0.5M inosine were added.
After incubation at 37.degree. C. for 30 min, samples were
evaporated to dryness under reduced pressure and reconstituted in
water for HPLC analysis.
[0188] No intact prodrug was detected in any PBMC samples. For
plasma samples potentially containing intact prodrugs, experiments
were performed to verify that no further conversion to PMPA
occurred during derivatization. Prodrug standards were added to
drug-free plasma and derivatized as described. There were no
detectable levels of PMPA present in any of the plasma samples, and
the projected % of conversion was less than 1%.
[0189] The HPLC system was comprised of a P4000 solvent delivery
system with AS3000 autoinjector and F2000 fluorescence detector
(Thermo Separation, San Jose, Calif.). The column was an Inertsil
ODS-2 column (4.6.times.150 mm). The mobile phases used were: A, 5%
acetonitrile in 25 mM potassium phosphate buffer with 5 mM
tetrabutyl ammonium bromide (TBABr), pH 6.0; B, 60% acetonitrile in
25 mM potassium phosphate buffer with 5 mM TBABr, pH 6.0. The flow
rate was 2 ml/min and the column temperature was maintained at
35.degree. C. by a column oven. The gradient profile was 90% A/10%
B for 10 min for PMPA and 65% A/35% B for 10 min for the prodrug.
Detection was by fluorescence with excitation at 236 nm and
emission at 420 nm, and the injection volume was 10 .mu.l. Data was
acquired and stored by a laboratory data acquisition system
(PeakPro, Beckman, Allendale, N.J.).
[0190] Pharmacokinetic Calculations. PMPA and prodrug exposures
were expressed as areas under concentration curves in plasma or
PBMC from zero to 24 hours (AUC). The AUC values were calculated
using the trapezoidal rule.
[0191] Plasma and PBMC Concentrations. The results of this study is
shown in FIGS. 2 and 3. FIG. 2 shows the time course of GS 7340-2
metabolism summary of plasma and PBMC exposures following oral
administration of pure diastereoisomers of the PMPA prodrugs.
[0192] The bar graph in FIG. 2 shows the AUC (0-24 h) for tenofovir
in dog PBMCs and plasma after administration of PMPA s.c., TDF and
amidate ester prodrugs. All of the amidate prodrugs exhibited
increases in PBMC exposure. For example, GS 7340 results in a
.about.21-fold increase in PBMC exposure as compared to PMPA s.c.
and TDF; and a 6.25-fold and 1.29-fold decrease in plasma exposure,
respectively.
[0193] These data establish in vivo that GS 7340 can be delivered
orally, minimizes systemic exposure to PMPA and greatly enhances
the intracellular concentration of PMPA in the cells primarily
responsible for HIV replication.
13TABLE 6 PMPA Exposure in PBMC and Plasma from Oral Prodrugs of
PMPA in Dogs PBMC/Plasma PMPA AUC in Plasma PMPA AUC in PBMC
Prodrug Exposure GS# Moiety Mean StDev N Mean StDev N in Plasma
Ratio GS-7114 Mono-Ala-Et-A 5.8 0.9 2 706 331 5 YES 122 GS-7115
Mono-Ala-Et-B 6.6 1.5 2 284 94 5 YES 43 GS-7340-2 Mono-Ala-iPr-A
5.0 1.1 5 805 222 5 YES 161 GS-7339 Mono-Ala-iPr-A 6.4 1.3 2 200 57
5 YES 31 GS-7119 Mono-Gly-Et-A 6.11 1.86 2 530 304 5 YES 87 GS-7342
Mono-ABA-Et-A 4.6 1.2 2 1060 511 5 YES 230 GS7341 Mono-ABA-Et-B 5.8
1.4 2 199 86 5 YES 34
Example 11
Biodistribution of GS-7340
[0194] As part of the preclinical characterization of GS-7340, its
biodistribution in dogs was determined. The tissue distribution of
GS-7340 (isopropyl alaninyl monoamidate, phenyl monoester of
tenofovir) was examined following oral administration to beagle
dogs. Two male animals were dosed orally with .sup.14C=GS-7340
(8.85 mg-equiv. of PMPA/kg, 33.2 .mu.Ci/kg; the 8-carbon of adenine
is labeled) in an aqueous solution (50 mM citric acid, pH 2.2).
Plasma and peripheral blood mononuclear cells (PBMCs) were obtained
over the 24-hr period. Urine and feces were cage collected over 24
hr. At 24 h after the dose, the animals were sacrificed and tissues
removed for analysis. Total radioactivity in tissues was determined
by oxidation and liquid scintillation counting.
[0195] The biodistribution of PMPA after 24 hours after a single
oral dose of radiolabelled GS 7340 is shown in Table 4 along with
the data from a previous study with TDF (GS-4331). In the case of
TDF, the prodrug concentration in the plasma is below the level of
assay detection, and the main species observed in plasma is the
parent drug. Levels of PMPA in the lymphatic tissues, bone marrow,
and skeletal muscle are increased 10-fold after administration of
GS-7340.
[0196] Accumulation in lymphatic tissues is consistent with the
data observed from the PBMC analyses, since these tissues are
composed primarily of lymphocytes. Likewise, accumulation in bone
marrow is probably due to the high percentage of lymphocytes (70%)
in this tissue.
14TABLE 7 Excretion and Tissue Distribution of Radiolabelled
GS-7340 in Dogs (Mean, N = 2) Following an Oral Dose at 10 mg-eq.
PMPA/kg. Tissue Conc. GS-4331 GS-7340 Ratio of GS Conc. Conc. 7340
Tissue/Fluid % Dose (ug-eq/g) % Dose (ug-eq/g) to GS-4331 Liver
12.40 38.30 16.45 52.94 1.4 Kidney 4.58 87.90 3.78 80.21 0.9 Lungs
0.03 0.53 0.34 4.33 8.2 Iliac Lymph Nodes 0.00 0.51 0.01 5.42 10.6
Axillary Lymph Nodes 0.00 0.37 0.01 5.54 14.8 Inguinal Lymph Nodes
0.00 0.28 0.00 4.12 15.0 Mesenteric Lymph 0.00 1.20 0.04 6.88 5.7
Thyroid Gland 0.00 0.30 0.00 4.78 15.8 Pituitary Gland 0.00 0.23
0.00 1.80 7.8 Salivary Gland (L + R) 0.00 0.45 0.03 5.54 12.3
Adrenal Gland 0.00 1.90 0.00 3.47 1.8 Spleen 0.00 0.63 0.17 8.13
12.8 Pancreas 0.00 0.57 0.01 3.51 6.2 Prostate 0.00 0.23 0.00 2.14
9.1 Testes (L + R) 0.02 1.95 0.02 2.01 1.0 Skeletal Muscle 0.00
0.11 0.01 1.12 10.1 Heart 0.03 0.46 0.15 1.97 4.3 Femoral Bone 0.00
0.08 0.00 0.28 3.5 Bone Marrow 0.00 0.20 0.00 2.05 10.2 Skin 0.00
0.13 0.00 0.95 7.2 Abdominal fat 0.00 0.16 0.00 0.90 5.8 Eye (L +
R) 0.00 0.06 0.00 0.23 3.7 Brain 0.00 <LOD 0.00 <LOD n.d.
Cerebrospinal Fluid 0.00 <LOD 0.00 0.00 n.d. Spinal Cord 0.00
<LOD 0.00 0.04 n.d. Stomach 0.11 1.92 0.26 2.68 1.4 Jejunum 1.34
3.01 0.79 4.16 1.4 Duodenum 0.49 4.96 0.44 8.77 1.8 Ileum 0.01 0.50
0.16 4.61 9.2 Large Intestine 1.63 5.97 2.65 47.20 7.9 Gall bladder
0.00 3.58 0.04 25.02 7.0 Bile 0.00 9.63 0.22 40.48 4.2 Feces 40.96
n.d. 0.19 n.d. n.a. Total GI Tract Contents 5.61 n.d. 21.64 n.d.
n.a. Urine 23.72 n.d. 14.73 n.d. n.a. Plasma at 24 h 0.00 0.20 0.00
0.20 1.0 Plasma at 0.25 h n.a. 3.68 n.a. 3.48 0.9 PBMC* 0.00 n.d.
0.00 63.20 n.d. Whole Blood 0.00 0.85 0.16 0.20 0.2 Total Recovery
81.10 68.96 Calculated using typical recovery of 15 .times.
10.sup.6 cells total, and mean PBMC volume of 0.2 picoliters/cell
n.s. = no sample, n.a. = not applicable, n.d. = not determined.
* * * * *