U.S. patent number 7,790,735 [Application Number 11/500,860] was granted by the patent office on 2010-09-07 for methanocarba cycloalkyl nucleoside analogues.
This patent grant is currently assigned to N/A, The United States of America as represented by the Department of Health and Human Services. Invention is credited to Kenneth A Jacobson, Victor E Marquez.
United States Patent |
7,790,735 |
Jacobson , et al. |
September 7, 2010 |
Methanocarba cycloalkyl nucleoside analogues
Abstract
The present invention provides novel nucleoside and nucleotide
derivatives that are useful agonist or antagonists of P1 and P2
receptors. For example, the present invention provides a compound
of formula A-M, wherein A is modified adenine or uracil and M is a
constrained cycloalkyl group. The adenine or uracil is bonded to
the constrained cycloalkyl group. The compounds of the present
invention are useful in the treatment or prevention of various
diseases including airway diseases (through A.sub.2B, A.sub.3,
P2Y.sub.2 receptors), cancer (through A.sub.3, P2 receptors),
cardiac arrhythmias (through A1 receptors), cardiac ischemia
(through A.sub.1, A.sub.3 receptors), epilepsy (through A.sub.1,
P2X receptors), and Huntington's Disease (through A.sub.2A
receptors).
Inventors: |
Jacobson; Kenneth A (Silver
Spring, MD), Marquez; Victor E (Montgomery Village, MD) |
Assignee: |
The United States of America as
represented by the Department of Health and Human Services
(Bethesda, MD)
N/A (N/A)
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Family
ID: |
22644097 |
Appl.
No.: |
11/500,860 |
Filed: |
August 8, 2006 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20060270629 A1 |
Nov 30, 2006 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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10169975 |
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7087589 |
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PCT/US01/00981 |
Jan 12, 2001 |
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60176373 |
Jan 14, 2000 |
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Current U.S.
Class: |
514/269;
544/309 |
Current CPC
Class: |
C07D
473/34 (20130101); A61P 29/00 (20180101); A61P
35/00 (20180101) |
Current International
Class: |
C07D
239/54 (20060101); A61K 31/513 (20060101) |
Field of
Search: |
;544/309 ;514/269 |
References Cited
[Referenced By]
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Apr 1997 |
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WO 94/03456 |
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Feb 1994 |
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WO |
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WO 94/25605 |
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Nov 1994 |
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WO |
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WO 94/25607 |
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Nov 1994 |
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WO |
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Feb 1995 |
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WO |
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WO |
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WO |
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WO |
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WO 97/27177 |
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Jul 1997 |
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WO |
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WO 98/05662 |
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Feb 1998 |
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WO |
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Primary Examiner: Rao; Deepak
Attorney, Agent or Firm: Leydig, Voit & Mayer, Ltd.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This is a divisional of co-pending U.S. patent application Ser. No.
10/169,975, which is the U.S. national stage of PCT/US01/00981,
filed Jan. 12, 2001, claiming the benefit of U.S. Provisional
Patent application No. 60/176,373, filed Jan. 14, 2000, the
disclosures of which are incorporated by reference.
Claims
What is claimed is:
1. A compound of the formula: ##STR00008## wherein R.sub.1 is
hydrogen, alkenyl, alkynyl, or aminoalkyl; R.sub.2 and R.sub.9 are
independently hydrogen, alkyl, alkenyl, alkynyl, or aminoalkyl;
R.sub.3, R.sub.4, and R.sub.5, are each independently hydrogen,
hydroxyl, alkoxy, alkyl, alkenyl, alkynyl, aryl, acyl, alkylamino,
arylamino, phosphoryl, or phosphonyl; R.sub.8 and R.sub.7 are each
independently sulfur or oxygen; and R.sub.10 is methylene,
dihalomethyl, carbonyl, or sulfoxide; or a salt of said compound;
with the proviso that when R.sub.1 is hydrogen, R.sub.2 is methyl,
R.sub.4 and R.sub.5 are hydroxyl, R.sub.7 and R.sub.8 are O,
R.sub.9 is hydrogen, and R.sub.10 is methylene, R.sub.3 is not
hydrogen.
2. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and a compound or salt of claim 1.
3. The compound or salt of claim 1, wherein the phosphoryl group is
a diphosphoryl group.
4. The compound or salt of claim 3, wherein R.sub.1, R.sub.2, and
R.sub.9 are hydrogen, R.sub.7 and R.sub.8 are oxygen, R.sub.10 is
methylene, R.sub.5 is diphosphoryl, and R.sub.3 and R.sub.4 are
hydrogen or hydroxyl.
5. The compound or salt of claim 4, wherein R.sub.3 and R.sub.4 are
hydroxyl.
6. The compound or salt of claim 4, wherein R.sub.3 is hydrogen and
R.sub.4 is hydroxyl.
7. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and a compound or salt of claim 3.
8. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and a compound or salt of claim 4.
9. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and a compound or salt of claim 5.
10. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and a compound or salt of claim 6.
11. A method of treating a mammal for epilepsy comprising
administering to the mammal an effective amount of a compound or
salt of claim 1.
12. A method of treating a mammal for epilepsy comprising
administering to the mammal an effective amount of a compound or
salt of claim 3.
13. A method of treating a mammal for epilepsy comprising
administering to the mammal an effective amount of a compound or
salt of claim 4.
14. A method of treating a mammal for epilepsy comprising
administering to the mammal an effective amount of a compound or
salt of claim 5.
15. A method of treating a mammal for epilepsy comprising
administering to the mammal an effective amount of a compound or
salt of claim 6.
16. A method of treating a mammal for Parkinson's disease
comprising administering to the mammal an effective amount of a
compound or salt of claim 1.
17. A method of treating a mammal for Huntington's disease
comprising administering to the mammal an effective amount of a
compound or salt of claim 1.
Description
TECHNICAL FIELD OF THE INVENTION
This invention pertains to a novel class of receptor ligands for P1
and P2 receptors and their therapeutic use. More specifically, the
invention pertains to nucleoside derivatives in which the sugar
moiety is replaced with a cycloalkyl group that is conformationally
constrained by fusion to a second cycloalkyl group.
BACKGROUND OF THE INVENTION
Purines such as adenosine have been shown to play a wide array of
roles in biological systems. For example, physiological roles
played by adenosine include, inter alia, modulator of vasodilation
and hypotension, muscle relaxant, central depressant, inhibitor of
platelet aggregation, regulator of energy supply/demand, responder
to oxygen availability, neurotransmitter, and neuromodulator.
(Bruns, Nucleosides & Nucleotides, 10(5), 931-934 (1991)).
Because of its potent actions on many organs and systems, adenosine
and its receptors have been the subject of considerable
drug-development research (Daly, J. Med. Chem., 25, 197 (1982)).
Potential therapeutic applications for agonists include, for
instance, the prevention of reperfusion injury after cardiac
ischemia or stroke, and treatment of hypertension and epilepsy
(Jacobson, et al., J. Med. Chem., 35, 407-422 (1992)). Adenosine
itself has recently been approved for the treatment of paroxysmal
supra ventricular tachycardia (Pantely, et al., Circulation, 82,
1854 (1990)). Adenosine receptor agonists also find use as
anti-arrhythmics, antinociceptives, anti-lipolytics,
cerebroprotectives, and antipsychotics.
P2 receptors, are present in heart, skeletal, various smooth
muscles, prostate, ovary, and brain and have been implicated in
certain aggregation processes associated with thrombosis and as
anti-hypertensive and anti-diabetic agents. Agonists that bind the
P2 receptor induce activation of phospholipase C, which leads to
the generation of inositol phosphates and diacyl glycerol with a
subsequent rise in intracellular calcium concentration and muscle
relaxation. P2 receptor antagonists block ADP-promoted aggregation
in platelets and thereby exert an anti-thrombotic effect.
All P1 and P2 receptor nucleoside ligands suffer from chemical
instability that is caused by the labile glycosidic linkage in the
sugar moiety of the nucleoside. However, it has been found that
relatively few ribose modifications are tolerated by the presently
known agonists and antagonists of P1 and P2 receptors.
New compositions are needed that have improved chemical stability
and that do not destroy the activity of such compounds.
The invention provides such compositions and methods of using them
in the treatment of disease. These and other advantages of the
present invention, as well as additional inventive features, will
be apparent from the description of the invention provided
herein.
BRIEF SUMMARY OF THE INVENTION
The present invention provides novel nucleoside and nucleotide
derivatives that are useful agonists or antagonists of P1 or P2
receptors. The invention is premised upon the novel combination of
adenine and uracil and their derivatives with a constrained
cycloalkyl group, typically a cyclopentyl group. The constraint on
the cycloalkyl group is introduced by fusion to a second cycloalkyl
group. In the case of cyclopentane, the fusion is typically with
cyclopropane. The present compounds retain a surprising binding
affinity despite the substitution for the ribose group. Moreover,
the absence of the glycosidic bond in the compounds assists in
improving the chemical stability of the compounds and aids in
overcoming the stabilit problem associated with the glycosidic bond
in previously known P1 and P2 receptor ligands.
The compounds of the present invention are useful in the treatment
or prevention of various airway diseases (through A.sub.2B,
A.sub.3, P2Y.sub.2 receptors), cancer (through A.sub.3, P2
receptors), cardiac arrhythmias (through A.sub.1 receptors),
cardiac ischemia (through A.sub.1, A.sub.3 receptors), epilepsy
(through A.sub.1, P2X receptors), Huntington's Disease (through
A.sub.2A receptors), Immunodeficient disorders (through A.sub.2,
A.sub.3 receptors), inflammatory disorders (through A.sub.3,
P.sub.2 receptors), neonatal hypoxia (through A.sub.1 receptors),
neurodegenerative (through A.sub.1, A.sub.3, P2 receptors), pain
(through A.sub.1, A.sub.3, P2X3 receptors), Parkinson's Disease
(through A.sub.2A receptors), renal failure (through A.sub.1
receptors), schizophrenia (through A.sub.2A receptors), sleep
disorders (through A.sub.1 receptors), stroke (through A.sub.1,
A.sub.3, P2 receptors), thrombosis (through P2Y.sub.1, P2Y.sub.AC
receptors), urinary incontinence (through P2X.sub.1receptors),
diabetes (through A.sub.1 receptors), psoriasis (through P2X
receptors), septic shock (through P2 receptors), brain trauma
(through A.sub.1 receptors), glaucoma (through A.sub.3 receptors)
and congestive heart failure (through P2 receptors).
The invention may best be understood with reference to the
accompanying drawings and in the following detailed description of
the preferred embodiments.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a new class of nucleoside and
nucleotide analogs that serve as selective agonists or antagonists
for P1 and P2 receptors.
Generally, the compounds of the present invention comprise two
basic chemical components designated "A" and "M" which are
covalently bonded to one another. Component A comprises adenine or
uracil, and component M includes a constrained cycloalkyl group.
Preferably the adenine and uracil are chemically modified or
substituted with moieties that allow the compound to bind to a P1
or P2 receptor. To that end any of a wide variety of chemical
groups can be used to modify adenine and uracil. Those groups are
well known to those of skill in the receptor art. Preferably, when
A is purine or a purine derivative, the linkage between A and M is
a chemical bond between the N9 purine nitrogen and the C1 carbon of
the cycloalkyl group. Where A is pyrimidine or a pyrimidine
derivative, the bond is between N1 pyrimidine nitrogen and the C1
carbon of the cycloalkyl group. The compounds of the present
invention have improved stability and surprising receptor binding
affinity.
While not wishing to be bound to any particular theory, it is
believed that the constrained cycloalkyl group assists in improving
chemical stability and receptor affinity. Preferably the cycloalkyl
groups are capable of adopting a conformation such that the
compound can bind to P1 or P2 receptors. As a result, preferred
cycloalkyl groups are those that tend to form energetically
favorable interactions with P1 and P2 receptors and avoid
energetically unfavorable ones, such as unfavorable ionic and/or
steric interactions. Further, the cycloalkyl group is derivatized
with a bridging group. The constraint restricts the cycloalkyl
group to certain conformations that are believed to be beneficial
to binding affinity. The preferred cycloalkyl group is a
cyclopentyl group. With cyclopentyl groups the preferred method for
introducing a conformational constraint is by derivatizing with a
fused cyclopropane bridge. With this modification the cyclopentane
ring is believed to be constrained to mimic the conformation of a
rigid furanose ring.
Compounds of the present invention include the compounds shown
below in Formulae I and II.
##STR00001##
Formulae I and II show compounds in which a derivatized or
underivatized adenine base is joined to a constrained cyclopentyl
group. For purposes of reference, the carbon atom of the
cyclopentyl group, M, that is joined to adenine, A, is the C1
carbon and the adenine is joined to M through its N9 nitrogen. In
the compounds of Formulae I and II the constrained cyclopentyl
group is derivatized with a fused cyclopropane bridge. In Formula I
the cyclopropyl group bridges carbon atoms C4 and C6. In Formula II
the cyclopropyl group bridges carbon atoms C6 and C1. These
distinct bridging patterns constrain the cyclopentyl group into
distinct conformations, specifically the N-(northern) conformation
as in Formula I and the S-(southern) conformation as in Formula II.
These two conformations are thought to mimic the two biologically
active conformations of furanose groups for P1 and P2 receptor
binding pockets.
The compounds described by Formulae I and II can be further defined
by a variety of suitable modifications to the adenine group. As
discussed above, any of a wide variety of chemical groups can be
used to form suitable adenine derivatives that comprise the novel
compounds of the present invention, provided that the resulting
compound is capable of binding to a P1 or P2 receptor. These
chemical groups are well known in the art and have been described,
for example in U.S. Pat. Nos. 5,284,834; 5,498,605; 5,620,676;
5,688,774; and Jacobson and Van Rhee, PURINERGIC APPROACHES IN
EXPERIMENTAL THERAPEUTICS, Chapter 6, p. 101 (Jacobson and Jarvis
eds., 1997); and Jacobson et al., THE P2 NUCLEOTIDE RECEPTORS, p.
81-107, in THE RECEPTORS (Turner et al. eds. 1998), which are
incorporated by reference herein. The combination of the chemically
modified adenine and the constrained cycloalkyl group provides a
surprising improvement in both chemical stability and binding
affinity.
By way of example and not in limitation of the present invention in
the compounds of Formulae I and II, R.sub.1 is hydrogen, alkyl,
cycloalkyl, alkoxy, cycloalkoxy, aryl, arylalkyl, acyl, sulfonyl,
arylsulfonyl, thiazolyl or bicyclic alkyl; R.sub.2 is hydrogen,
halo, alkyl, aryl, arylamino, aryloxide, alkynyl, alkenyl,
thioether, cyano, alkylthio or arylalkylthio; R.sub.3, R.sub.4, and
R.sub.5, are each hydrogen, hydroxyl, alkoxy, alkyl, alkenyl,
alkynyl, aryl, acyl, alkylamino, arylamino, phosphoryl, phosphonyl,
boronyl, or vanadyl, and they can be the same or different; R.sub.6
is hydrogen, alkyl, alkenyl, alkynyl, or aminoalkyl. R.sub.7 is a
methylene, dihalomethyl, carbonyl, or sulfoxide group. R.sub.8 is
carbon or nitrogen. At least one of R.sub.1, R.sub.2, and R.sub.6
is not hydrogen. It can be appreciated that various combinations of
the above groups are also within the invention provided that they
retain agonist or antagonist activity with a P1 or P2 type
receptor.
Where an alkyl, alkenyl, alkynyl group is referenced by itself or
as part of another group, the reference is to an uninterrupted
carbon chain consisting of no more than 20 carbon atoms. Aryl and
cycloalkyl groups contain no more than 8 carbons in the ring.
Reference to alkyl groups is further meant to include straight or
branched chain alkyls, arylalkyl, aminoalkyl, haloalkyl, alkylthio
or arylalkylthio groups. Alkyls specifically include methyl through
dodecyl. Where alkyl groups are present at position R.sub.6 in
adenine, it is preferred that the chain length be no longer than 6
carbons. Arylalkyl groups include, phenylisopropyl, phenylethyl.
Aminoalkyl groups can be any suitable alkyl group also containing
an amine. Similarly, haloalkyl groups can be any suitable alkyl
group that contains a halo substituent, such as bromo, chloro,
flouro, iodo. Alkylthio includes such moieties as thiomethyl,
thiopentyl, thiohexyl, thioheptyl, thiooctyl, thiodecyl,
thioundecyl, ethylthioethyl, or 6-cyanohexylthio groups. Alkylthio
also is meant to include arylalkylthio such as
2-(p-nitrophenyl)ethyl)thio, 2-aminophenylethylthio,
2-(p-nitrophenyl)ethylthio, or 2-aminophenylethylthio.
Cycloalkyls for example cyclopentyl, cyclohexyl,
hydroxycyclopentyl.
Alkoxys include for example methoxy groups.
Cycloalkoxys can include cyclopentoxy.
Aryl moieties can be arylalkyl, arylalkylthio, arylsulfonyl,
arylamino, aryloxide, heteroaryl, haloaryl, arylurea,
arylcarboxamido, heteroarylamino or sulfoaryl. Benzyl groups are
one species of aryl group. In addition, the arylalkyls include
R-phenylisopropyl or phenylethyl. Aryloxides can be phenyl,
R-phenylisopropyl, phenylethyl,
3,5-dimethoxyphenyl-2-(2-methylphenyl)ethyl and sulfophenyl.
Haloaryl can be iodobenzyl among other halogenated aryl groups.
Additionally, the heteroaryls include, for example, furans such as
tetrahydrofuran.
Acyl groups include carbonyls.
Alkenyl groups are analogous to alkyl groups but include at least
one carbon-carbon double bond. When present at the R.sub.6 group of
adenine it is preferred that the carbon chain length be from 2 to 6
carbons.
Similarly, alkynyls are analogous to alkenyl groups but contain at
least one triple carbon-carbon bond. As with other groups, when
present at the R.sub.6 position of adenine it is preferred that
they are not longer than 6 carbons.
Phosphoryl groups include diphosphoryl, triphosphoryl,
thiophosphoryl, thiodiphosphoryl, thiotriphosphoryl,
imidodiphosphate, imidotriphosphate, methylene diphosphate,
methylenetriphosphate, halomethylene diphosphate, halomethylene
triphosphate, boranophosphate, boranodiphosphate,
boranotriphosphate, or phosphorothioate-2-thioether for
example.
Thio groups include alkylthio, arylalkylthio, alkenylthio, or
arylthios. Alkylthio includes such groups as thiomethyl,
thiopentyl, thiohexyl, thioheptyl, thiooctyl, thiodecyl,
thioundecyl, ethylthioethyl, or 6-cyanohexylthio. Alkenylthio
includes 5-hexenylthio. Arylthios include
2-(p-nitrophenyl)ethyl)thio, 2-aminophenylethylthio,
2-(p-nitrophenyl)ethylthio, or 2-aminophenylethylthio.
One example of a suitable thiazolyl is
(benzothiazolyl)thio-2-propyl.
Examples of bicycloalkyls include s-endonorbornyl, or
carbamethylcyclopentane.
Halo groups include such elements as fluoro, bromo, chloro, or
iodo.
It will also be appreciated that any group that may be further
substituted can be, and still be within the scope of the invention.
For example, all of the R.sub.1 groups except hydrogen can be
further substituted. By way of illustration, when R.sub.1 is not
hydrogen, it can be further modified by substitutions with any of
the following chemical substituents including amino, cyano,
alkoxyl, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl,
acyl, halo, hydroxy, phosphoryl, sulfonyl, sulfonamido, carboxyl,
thiohydroxyl, sulfonamido, carboxyl, and carboxamido groups.
Similarly, for R.sub.2-R.sub.10 all of the groups other than
hydrogen can be substituted further. Multiple substitutions are
also contemplated.
In a preferred embodiment R.sub.1 can be either methyl,
cyclopentyl, cyclohexyl, phenyl, R-phenylisopropyl, benzyl, or
phenylethyl; R.sub.2 is chloride; and R.sub.6 can be a
C.sub.1-C.sub.6 alkylamino, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
alkenyl, C.sub.1-C.sub.6 alkynyl group.
Other compounds of the present invention include the compounds
shown below in Formulae III and IV. The Formulae show compounds in
which a derivatized or underivatized uracil base is joined to a
constrained cyclopentyl group.
##STR00002## The compounds defined by formulae III and IV can be
further defined by a variety of suitable modifications. For example
R.sub.1 can be hydrogen, or an alkyl group; R.sub.2 can be
hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkenyl,
C.sub.1-C.sub.6 alkynyl, or a C.sub.1-C.sub.6 aminoalkyl group;
R.sub.3, R.sub.4, R.sub.5, can each independently be the same as
discussed previously with respect to Formulae 1 and Formulae II.
R.sub.6 and R.sub.7 are each independently either sulfur or
oxygen.
Certain compounds of the present invention are ligands of P2
receptors. A variety of P2 receptors are known in the art and the
present compounds act at one or more of these, which include for
example, P2X and P2Y receptors. These Receptor ligands are
compounds that bind receptors, preferably in the binding pocket. In
certain embodiments the compound can be a P2 receptor agonist. In
other embodiments the compound can be a P2 receptor antagonist.
Certain compounds of the present invention are ligands for the P1
receptor. A variety of subclasses of P1 receptors are known and
various of present compounds act at one or more these species,
which include for example A.sub.1, A.sub.2, and A.sub.3 receptors.
Certain compounds act as P1 receptor agonists while others appear
to act as antagonists.
The compounds of the present invention are useful in the treatment
or prevention of various airway diseases (through A.sub.2B,
A.sub.3, P2Y.sub.2 receptors), cancer (through A.sub.3, P2
receptors), cardiac arrhythmias (through A.sub.1 receptors),
cardiac ischemia (through A.sub.1, A.sub.3 receptors), epilepsy
(through A.sub.1, P2X receptors), Huntington's Disease (through
A.sub.2A receptors), Immunodeficient disorders (through A.sub.2,
A.sub.3 receptors), inflammatory disorders (through A.sub.3,
P.sub.2 receptors), neonatal hypoxia (through A.sub.1 receptors),
neurodegenerative (through A.sub.1, A.sub.3, P2 receptors), pain
(through A.sub.1, A.sub.3, P2X3 receptors), Parkinson's Disease
(through A.sub.2A receptors), renal failure (through A.sub.1
receptors), schizophrenia (through A.sub.2A receptors), sleep
disorders (through A.sub.1 receptors), stroke (through A.sub.1,
A.sub.3, P2 receptors), thrombosis (through P2Y.sub.1, P2Y.sub.AC
receptors), urinary incontinence (through P2X.sub.1receptors),
diabetes (through A.sub.1 receptors), psoriasis (through P2X
receptors), septic shock (through P2 receptors), brain trauma
(through A.sub.1 receptors), glaucoma (through A.sub.3 receptors),
and congestive heart failure (through P2 receptors).
The present invention is further directed to a pharmaceutical
composition comprising a pharmaceutically acceptable carrier and at
least one compound selected from the group consisting of the
presently described compounds.
The pharmaceutically acceptable excipients described herein, for
example, vehicles, adjuvants, carriers or diluents, are well-known
to those who are skilled in the art and are readily available to
the public. It is preferred that the pharmaceutically acceptable
carrier be one that is chemically inert to the active compounds and
one that has no detrimental side effects or toxicity under the
conditions of use.
The choice of excipient will be determined in part by the
particular compound of the present invention chosen, as well as by
the particular method used to administer the composition.
Accordingly, there is a wide variety of suitable formulations of
the pharmaceutical composition of the present invention. The
following formulations for oral, aerosol, parenteral, subcutaneous,
intravenous, intramuscular, interperitoneal, rectal, and vaginal
administration are merely exemplary and are in no way limiting.
One skilled in the art will appreciate that suitable methods of
utilizing a compound and administering it to a mammal for the
treatment of disease states, which would be useful in the method of
the present invention, are available. Although more than one route
can be used to administer a particular compound, a particular route
can provide a more immediate and more effective reaction than
another route. Accordingly, the described methods are merely
exemplary and are in no way limiting.
The dose administered to an animal, particularly human and other
mammals, in accordance with the present invention should be
sufficient to effect the desired response. Such responses include
reversal or prevention of the bad effects of the disease for which
treatment is desired or to elicit the desired benefit One skilled
in the art will recognize that dosage will depend upon a variety of
factors, including the age, species, condition or disease state,
and body weight of the animal, as well as the source and extent of
the disease condition in the animal. The size of the dose will also
be determined by the route, timing and frequency of administration
as well as the existence, nature, and extent of any adverse
side-effects that might accompany the administration of a
particular compound and the desired physiological effect. It will
be appreciated by one of skill in the art that various conditions
or disease states may require prolonged treatment involving
multiple administrations.
Suitable doses and dosage regimens can be determined by
conventional range-finding techniques known to those of ordinary
skill in the art. Generally, treatment is initiated with smaller
dosages that are less than the optimum dose of the compound.
Thereafter, the dosage is increased by small increments until the
optimum effect under the circumstances is reached. The present
inventive method typically will involve the administration of about
0.1 to about 300 mg of one or more of the compounds described above
per kg body weight of the individual.
The following examples further illustrate the present invention
but, of course, should not be construed as in any way limiting its
scope. In the examples, unless otherwise noted, compounds were
characterized and resonances assigned by 300 MHz proton nuclear
magnetic resonance mass spectroscopy using a Varian GEMINI-300
FT-NMR spectrometer. Also, unless noted otherwise, chemical shifts
are expressed as ppm downfield from tetramethylsilane. Synthetic
intermediates were characterized by chemical ionization mass
spectrometry (NH.sub.3) and adenosine derivatives by fast atom
bombardment mass spectrometry (positive ions in a noba or m-bullet
matrix) on a JEOL SX102 mass spectrometer. Low resolution
CI--NH.sub.3 (chemical ionization) mass spectra were carried out
with Finnigan 4600 mass spectrometer and high-resolution EI
(electron impact) mass spectrometry with a VG7070F mass
spectrometry at 6 kV. Elemental analysis was performed by Atlantic
Microlab Inc. (Norcross, Ga.). NMR and mass spectra were consistent
with the assigned structure.
EXAMPLE 1
In all of the potent adenosine agonists previously developed, the
ribose moiety is present, and consequently, these agonists are
subject to deglycosylation and other pathways of metabolic
degradation in vivo. In order to design non-glycosyl adenosine
agonists and thereby increase biological stability and potential
receptor selectivity, carbocyclic modifications of the ribose
moiety have been introduced. In previous studies of adenosine
analogues it was found that if adenosine derivatives having
carbocyclic modifications of the ribose ring (compounds 1-4, below)
bind to adenosine receptors it is only with greatly reduced
affinity.
In the present study we have incorporated a complex carbocyclic
modification of ribose for use with adenosine agonists. This
modification, wherein only one isomeric form retains high affinity
and receptor selectivity, is the "methanocarba" ring. In this
modification a fused cyclopropane ring constrains the accompanying
cyclopentane moiety to mimic the conformation of a rigid furanose
ring. The furanose ring of nucleosides and nucleotides in solution
is known to exist in a rapid, dynamic equilibrium between a range
of Northern and opposing Southern conformations as defined in the
pseudorotational cycle. For methanocarba analogues, the
bicyclo[3.1.0]hexane ring can constrain the cyclopentane ring into
a N-, 2'-exo envelope pucker, and a S-, 3' exo form.
##STR00003##
These two extreme forms of ring pucker usually define biologically
active conformations. This example shows that nucleoside binding to
P1-(adenosine) receptors, is favored when the fixed ring-twist
conformation is in the N-- conformation.
Chemical Synthesis.
Nucleosides and synthetic reagents were purchased from Sigma
Chemical Co. (St. Louis, Mo.) and Aldrich (St. Louis, Mo.).
2,6-Dichloropurine was obtained from Sigma. m-iodobenzyl bromide
was purchased from Aldrich (St. Louis, Mo.).
4-(6-Aminopurin-9-yl)-1-hydroxymethyl-bicyclo[3.1.0]hexane-2,3-diol
(1) and compounds 5c and 5d were obtained from Dr. Victor Marquez.
Compounds 7a and 9a were synthesized in our laboratory.
The synthetic strategy used in this example is shown below. The
synthesis of N6-substituted N-methanocarba adenosine derivatives
optimized for interaction with A1 (CP=cyclopentyl) or A3
(IB=3-iodobenzyl) receptors. Reagents: a) DEAD, Ph.sub.3P; b) MEOH,
rt; c) BCl.sub.3; d) H2/Pd; e) 3-iodobenzyl bromide, 50.degree. C.,
DMF, 2 days; f) NH.sub.40H, MEOH, 80.degree. C., 3 days.
##STR00004##
(1'R, 2R, 3'R, 4'R,
1'aR)-2,3-(dihydroxy)-4-(hydroxymethyl)-1-(6-cyclopentylaminopurine-9-yl)-
bicyclo(3.1.0)hexane) (6c)
A solution of 8c (4 mg, 0.01 mmol) in methanol (0.5 ml) was
hydrogenated at atmospheric pressure over 10% Pd/C (1 mg) to
furnish the product 6c (83% yield). H'NMR (CD.sub.30D): .delta.
0.7-0.8 (m, IH, 6'-CHH), 1.46-1.88 (m, 1OH, 6'CHH, 1'aH,
4CH.sub.2),2.01-2.20 (m, 1H, NCH), 3.34 (d, 1H, J=9.77 Hz, 5'CHH),
3.88 (d, 1H, J=6.84 Hz, 3'CH), 4.26 (d, 1H, J=9.77 Hz, 5'CHH),
4.66-498 (m, 2H, 2'CH, 1'CH), 8.28 (s, 1H, 2CH), 8.5 (s, 1H, 8CH).
HRMS(FAB): Cal: 346.1879 Found: 346.1879
(1'R, 2'R, 3'R,
4'R)-2,3-(dihydroxy)-4-(hydroxymethyl)-1-(6-(3-idobenzylamino)purine-9-yl-
)cyclopentane (7b)
A mixture of aristeromycin (3.5 mg, 0.013 mmol) and
3-iodobenzybromide (12 mg, 0.039 mmol) in anhydrous DMF was heated
for 3 days, and solvent was removed under vacuum. The excess
3-iodobenzylamine was removed from the reaction mixture by adding
ether to the reaction mixture, and stirring was continued for 5
min. followed by decantation of the supernatant ether phase. The
residue was dried, suspended in methanol (1 ml) and ammonium
hydroxide (0.5 ml), and heated at 80.degree. C. in a closed tube
for 1 h. Solvent was removed under vacuum, and the residue obtained
was purified by flash column chromatography using 7/3
chloroform/methanol to furnish 3.0 mg (47%) of the product.
H.sup.1NMR(CD.sub.30D) .delta. 1.86-1.96 (m, 1H, 1'CHH), 2.14-2.30
(m, 1H, 1'CHH), 2.38-2.48 (m, 1H, 4'CH), 3.3-3.38 (m, 1H, 5'CHH),
3.67 (d, 1H, J=6.84 Hz, 5'CHH), 3.96-4.06 (m, 1H, 3'CH), 4.43-4.48
(m, 1H, 2'CH), 4.73-4.82 (m, 1H, 1'CH), 5.26 (s, 2H, ArCH.sub.2),
7.12 (t, 1H, J=7.82 Hz, ArH), 7.32 (d, 1H, J=7.82 Hz, ArH), 7.66
(d, 1H, J=7.82 Hz, ArH), 7.73 (s, 1H, ArH), 8.06 (s, 1H, 2CH). 8.08
(s, 1H, 8CH).
Preparation of
4-[6-(3-iodobenzylamino)-purin-9-yl]-1-hydroxymethyl-bicyclo[3.1.0]hexane-
-2,3-diol (7c, (N)-Methanocarba-N.sup.6-(3-iodobenzyl)adenosine) by
Dimroth rarrangement:.sup.1
To a solution of
4-(6-amino-purin-9-yl)-1-hydroxymethyl-bicyclo[3.1.0]hexane-2,3-diol
(5c, 20 mg, 0.0721 mmol) in DMF (0.5 mL) was added m-iodobenzyl
bromide (64 mg, 0.216 mmol), and the mixture was stirred at
50.degree. C. for 2 days. DMF was then removed under a stream of
N.sub.2. To the resulting syrup 0.5 mL of acetone and 1 mL of ether
were added and the syrup solidified. The solvents were removed by
decantation, and again ether was added and removed. The solid was
dried and dissolved in 1 mL MEOH. NH.sub.4OH (1.5 mL) was added and
the mixture was stirred at 80.degree. C. for 3 days. After cooling
down to room temperature, the solvents were removed under reduced
pressure and the residue was purified by preparative TLC (silica
60; 1 000 .mu.m; Analtech, Newark, Del.; ethyl
acetate-i-PrOH--H.sub.2O (8:2:1)) to give 26 mg of the product
(7c), yield: 73%. .sup.1H NMR (CDCl.sub.3): .delta. 0.82 (t, J=6.0
Hz, 1 H), 1.41 (t, J=4.8 Hz, 1 H), 1.72 (dd, J=8.5, 6.0 Hz, 1 H),
3.36 (d, J=10.8 Hz, 1 H), 4.05 (d, J=6.9 Hz, 1 H), 4.33 (m, 1 H),
4.80-4.88 (m, 3 H), 5.21 (d, J=6.9 Hz, 1 H), 6.25 (m, br, 1), 7.07
(t, J=7.8 Hz, 1 H), 7.35 (d, J=7.8 Hz, 1 H), 7.61 (d, J=7.8 Hz, 1
H), 7.74 (s, 1), 7.93 (s, 1 H), 8.33 (s, 1 H). MS(FAB): m/z 494
(M.sup.++I).
(1'R, 2'R, 3'R, 4'R,
1'aR,)-2,3-(dihydroxy)-4-(hydroxymethyl)-1-(2-chloro-6-cyclopentylaminopu-
rine-9-yl)bicyclo(3.1.0)hexane) (8c):
To a solution of 15 (36 mg, 0.076 mmol) in anhydrous
dichloromethane was added BCl.sub.3 (1M solution in
dichloromethane, 0.23 ml, 0.23 mmol) at 0.degree. C. The reaction
mixture was warmed to room temperature and stirred for 10 min. To
this mixture was added methanol (1 ml) followed by ammonium
hydroxide (0.5 ml). The mixture was concentrated under vacuum, and
the residue obtained was purified by flash column chromatography
using 9/1 chloroform-1/methanol as eluent to furnish 14 mg of the
product 8c (48% yield) as a solid.
H.sup.1NMR(CDCl.sub.3): .delta..o.65-0.9 (m, IH, 6'CHH), 1.1-1.4
(m, 2H, 6'CHH, 1'aH), 1.4-1.9 (m, 8H, 4CH.sub.2), 2.0-2.2 (m, 1H,
N.sup.6CH), 3.34 (d, 1H, J=7.2 Hz, 5'CHH), 3.97 (d, 1H, J=4.6 Hz,
3'CH), 4.25 (d, 1H, J=7.2 Hz, 5'CHH),4.687 (s, 1H, 1'CH), 5.11 (d,
1H, J 4.6, 2'CH), 7.85 (s, 1H, 8CH). HRMS(FAB): Cal: 380.1489
found: 380.1498
(1'R, 2'R, 3'R, 4'R,
1'aR)-2,3-(dihydroxy)-4-(hydroxymethyl)-1-(2-chloro-6-(3-idobenzylamino)p-
urine-9-yl)bicyclo(3.1.0)hexane) (9c) was synthesized by the same
method as 8c in 53% yield.
H.sup.1NMR(CD.sub.3OD): .delta. 0.70-0.78 (m, 1H, 6'CHH), 1.50-1.63
(m, 2H, 6, CHH, 1'aH), 3.33 (d, 1H, J=11.72 Hz, 5'CHH), 3.88 (d,
1H, J=6.84 Hz, 3'CH), 4.26 (d, 1H, J=11.72 Hz, 5'CHH), 4.71-4.83
(m, 2H, 1'CH, 2'CH), 7.1 (t, 1H, J=7.82 Hz, ArH), 7.40 (d, 1H,
J=7.82 Hz, ArH), 7.61 (d, 1H, 7.82 Hz, ArH), 7.78 (s, 1H, ArH),
8.54 (s, 1H, 8CH). HRMS(FAB): Cal: 528.0299 Found: 528.0295
(2R, 3R, 4R, 1'aR,
1S)-2,3-(O-isopropylidine)-4-(methylenebenzyloxy)-1-(2,6dichloropurine-9--
yl)bicyclo(3.1.0)hexane) (12)
To a solution of triphenyl phosphine (260 mg, 1 mmol) in anhydrous
THF (2 ml) was added DEAD (0.16 ml, 1 mmol) dropwise at 0.degree.
C., and stirring was continued for 20 min. To this solution was
added a solution of 2,6-dichloropurine in THF (4 ml) followed by
the addition of 11 (145 mg, 0.5 mmol) in THF (4 ml). The reaction
mixture was warmed to room temperature, and stirring was continued
for 6 h. Solvent was evaporated under vacuum, and the residue
obtained was purified by flash chromatography using 7/3
petroleumether/ethylacetate as eluent to furnish 141 mg of the
product (12) (70% yield) as a gum.
H.sup.1NMR (CDCl.sub.3): .differential. 1.0 (m,1H, 6'CHH), 1.24 (s,
3H, CH.sub.3), 1.27-1.38 (m, 1H, 6'CHH), 1.55 (s, 3H,
CH.sub.3),1.62 (dd, 1H, J=4.88, 9.77 Hz, 1'aH), 3.34 (d, 1H, J=9.77
Hz, 5'CHH), 3.97 (d, 1H, J=9.77 Hz, 5'CHH), 4.50 (d, 1H, J=6.84 Hz,
3'CH), 4.57-4.68 (qAB, 2H, J=12.7 Hz, ArCH.sub.2), 5.17 (s, 1H,
1'CH), 5.32 (d, 1H, J=6.84 Hz, 2'H), 7.27.4 (m, 5H, Ar), 8.63 (s,
1H, 8CH).
(2R, 3R, 4R, 1'aR,
1S)-2,3-(O-isopropylidine)-4-(methylenebenzyloxy)-1-(2-chloro-6-cyclopent-
ylaminopurine-9-yl)bicyclo(3.1.0)hexane) (15):
To a solution of 12 (42 mg, 0.105 mmol) in methanol (2 ml) was
added cyclopentylamine at room temperature, and stirring was
continued for 6 hr for complete reaction. Solvent was removed under
vacuum, and the residue obtained was purified by flash column
chromatography using 7/3 petroleum ether/ethylacetate as eluent to
furnish 45 mg of the product 15 (90% yield) as a gum.
H.sup.1NMR(CDCl.sub.3): .delta. 0.92-0.96 (m, 1H. 6'CHH), 1.14-1.01
(m, 1H, 6'CHH), 1.23 (s, 3H, CH.sub.3),1.42-1.81 (m, 9H, 1'aH,
4CH.sub.2),1.54 (s, 3H, CH.sub.3), 2.08-2.21 (m, 1H, N.sup.6CH),
3.44 (d, 1H, J=9.76 Hz, 5'CHH), 3.90 (d, 1H, J=9.76 Hz, 5'CHH),
4.51 (d, 1H, J=6.84 Hz, 3'CH), 4.57-4.67 (qAB, 2H, J=12.7 Hz,
ArCH.sub.2), 5.04 (s, 1H, 1'CH), 5.32 (d, 1H, J=6.84 Hz, 2'CH),
7.2-7.4 (m, 5H, Ar), 8.18 (s, 1H, 8CH).
(1'R, 2'R, 3'R, 4'R,
1'aR)-2,3-(O-isopropylidine)-4-(methylenebenzyloxy)-1-(2-chloro-6-(3-idob-
enzylamino)purine-9-yl)bicyclo(3.1.0)hexane) (16) was synthesized
in 70% yield by the same method as 15, except using
3-iodobenzylamine hydrochloride and two equivalents of
triethylamine.
H.sup.1NMR(CDCl.sub.3): .delta. 0.87-0.91 (m, 1H, 6'CHH), 1.10-1.29
(m, 1H, 6'CHH), 1.17 (s, 3H, CH.sub.3),1.42-1.56 (m, 1H, 1'aH),
1.47 (s, 3H, CH.sub.3), 3.37 (d, 1H, J=9.77 Hz, 5'CHH), 3.84 (d,
1H, J=9.77 Hz, 5'CHH), 4.44 (d, 1H, J=6.84 Hz, 3'CH), 4.50-4.60
(qAB, 2H, J=11.72 Hz, ArCH.sub.2), 4.70 (bs, 1H, NH), 4.98 (s, 1H,
1'CH), 5.24 (d, 1H, J=6.84 Hz, 2'CH), 7.0 (t, 1H, J=7.82 Hz, ArH),
7.2-7.34 (m, 6H, ArH), 7.55 (d, 1H, J=7.82, ArH), 7.65 (s, 1H,
ArH), 8.08 (s, 1H, 8CH).
Pharmacological Analyses.
Materials
F-12 (Ham's) medium, fetal bovine serum (FBS) and
penicillin/streptomycin were from Gibco BRL (Gaithersburg, Md.).
[.sup.125 I]AB-MECA (1000 Ci/mmol) and .sub.[.sup.35S]guanosine
5'-(.gamma.-thio)triphosphate (1000-1500 Ci/mmol) were from DuPont
NEN (Boston, Mass.). Adenosine deaminase (ADA) was from Boehringer
Mannheim (Indianapolis, Ind.). All other materials were from
standard local sources and of the highest grade commercially
available.
Cell Culture and Membrane Preparation
CHO cells stably transfected with either human A.sub.1 or A.sub.3
receptors (gift of Dr. Gary Stiles and Dr. Mark Olah, Duke
University Medical Center) were cultured as monolayers in medium
supplemented with 10% fetal bovine serum. Cells were washed twice
with 10 ml of ice-cold phosphate buffered saline, lysed in lysis
buffer (10 mM Tris.HCl buffer, pH 7.4, containing 2 mM MgCl.sub.2
and 0.5 mM EDTA), and homogenized in a Polytron homogenizer in the
presence of 0.2 U/ml adenosine deaminase. The crude membranes were
prepared by centrifuging the homogenate at 1000.times.g for 10 min
followed by centrifugation of the supernatant at 40,000.times.g for
15 min. The pellet was washed once with the lysis buffer and
recentrifuged at 40,000.times.g for 15 min. The final pellets were
resuspended in 50 mM Tris.HCl buffer, pH 7.4, containing 10 mM
MgCl.sub.2 and 0.1 mM EDTA and stored at -70.degree. C.
Radioreceptor Binding
Determination of binding to adenosine A.sub.1, A.sub.2A and
A.sub.2B receptors was carried out as reported. Determination of
A.sub.3 adenosine receptor binding was carried out using
[.sup.125I]AB-MECA. Briefly, aliquots of crude transfected CHO cell
membranes (approximately 40 .mu.g protein/tube) were incubated with
0.5 nM [.sup.125I]AB-MECA, 10 mM MgCl.sub.2, 2 units/ml adenosine
deaminase, 50 mM Tris.HCl (pH 7.4) at 37.degree. C. for 60 min. The
total volume of the reaction mixture was 125 .mu.l. Bound and free
ligands were separated by rapid filtration of the reaction mixture
through Whatman GF/B glass filters. The filters were immediately
washed with two 5 ml-portions of ice-cold 50 mM Tris.HCl buffer (pH
7.4). The radioactivity bound to the filters was determined in a
Beckman gamma counter. Specific binding was defined as the amount
of the radioligand bound in the absence of competing ligand minus
the amount of that bound in the presence of 100 .mu.M NECA.
Ki-values were calculated using the K.sub.d for [.sup.125I]AB-MECA
binding of 0.56 nM.
Determination of [3'S]GTP.gamma.S Binding
[.sup.35 S]GTP.gamma.S binding was determined by the method of
Lorenzen et al. The incubation mixture contained in a total volume
of 125 .mu.l, 50 mM Tris.HCl (pH 7.4), 1 mM EDTA, 10 mM MgCl.sub.2,
10 .mu.M guanosine 5'-diphosphate, 1 mM dithiothreitol, 100 mM
NaCl, 0.2 units/ml adenosine deaminase, 0.16 nM [.sup.35 S]
GTP.gamma.S (about 50,000 cpm) and 0.5% BSA. The CHO cell membranes
expressing A.sub.1 or A.sub.3 receptors were preincubated with the
above-mentioned assay mixture at 37.degree. C. for 1 h and further
incubated for 1 hr after the addition of [.sup.35 S]GTP.gamma.S.
Incubations were terminated by rapid filtration of the samples
through glass fiber filters (Whatman GF/B), followed by two 5 ml
washes of the same buffer. After transferring the filters into a
vial containing 3 ml of scintillation cocktail, the radioactivity
was determined in a scintillation counter.
Data Analysis. Analyses of saturation binding assays and
concentration-response curves were carried out using the GraphPad
Prism (GraphPad Software Inc., San Diego, Calif.). Comparisons
between groups were carried out using the unpaired Student's
test.
Results
Chemical Synthesis
The methanocarbocyclic 2'-deoxyadenosine analogues, shown below in
Table 1, in which a fused cyclopropane ring constrains the
cyclopentane ring into a rigid envelope configuration of either a
N-- or S-- conformation, were synthesized in a manner similar as
shown above. The N-methanocarba analogues of various
N.sup.6-substituted adenosine derivatives, including cyclopentyl
and iodobenzyl, in which the parent compounds are potent and
selective agonists at either A.sub.1 or A.sub.3 receptors,
respectively, were prepared. 2,6-Dichloropurine, 10, was condensed
with the cyclopentyl derivative, 11, using the Mitsunobu reaction,
followed by substitution at the 6-position and deprotection to give
8c or 9e. The 2-chloro substitution of compound 8c was removed by
catalytic reduction to give 6c. This allowed the incorporation in
the N-configuration series of the 2-chloro modification of adenine,
which was of interest for its effect on adenosine receptor
affinity. An N.sup.6-(3-iodobenzyl) group could also be introduced
in either aristeromycin, 5b, or N-methanocarba-adenosine, 5c, by
the Dimroth rearrangement, to give 7b and 7c.
Biological Activity
A pair of methanocarba analogues of adenosine, 5c and 5d,
corresponding to N-- and S-- conformations of ribose, were tested
in binding assays, the results of which are shown in Table 1 below,
at four subtypes of adenosine receptors. The more synthetically
challenging S-isomer (5d) was available only as the racemate and
therefore was tested as such. At rat A1, rat A2A, and human A3
subtypes, the N-analogue proved to be of much higher affinity than
the S-analogue. At the human A2B receptor, binding was carried out
using [3H]ZM 241,385, however the affinity was too weak to
establish selectivity for a specific isomer. Affinity of
N-methanocarba-adenosine, 5c, vs. adenosine, 5a, was particularly
enhanced at the A3 receptor subtype, for which the ratio of
affinities of N-- to S-analogues was 150-fold. Although a poor
substrate for adenosine deaminase (ADA), the binding curve for 5c
was shifted in the presence of ADA, therefore the affinity values
for 5c and 5d obtained in the absence of ADA are entered in Table
1, below. The South confomer, 5d, is even a worse substrate of ADA
(100-fold less) which explains why the curves in the presence and
absence of ADA for 5d are virtually the same. Aristeromycin, 5b,
bound weakly to adenosine receptors, with slight selectivity for
the A.sub.2A subtype. Compound 5c was more potent than
aristeromycin, 5b, in binding to A1 (4-fold) and A3 (4500-fold)
adenosine receptors.
Compounds 6c and 8c are patterned after A1 receptor-selective
agonists, while compounds 7c and 9c are patterned after A3
receptor-selective agonists. Compounds 6 and 7 are unsubstituted at
the 2-position, while compounds 8 and 9 contain the potency
enhancing 2-chloro substituent. The N6-cyclopentyl N-methanocarba
derivative, 6c, based on CPA, 6a, maintained high selectivity for
A1 receptors, although the affinity of 6c at rat A1 receptors was
3-fold less than for 6a. In one series it was possible to compare
ribose, cyclopentyl, and N-methanocarba derivatives having the same
N6-substitution. The N6-(3-iodobenzyl) derivative, 7c, based on a
5'-hydroxy analogue, 7a, of IB-MECA, with a Ki value of 4.1 nM was
2.3-fold more potent at A3 receptors than the ribose-containing
parent. Thus, the selectivity of 7c for human A3 versus rat AI
receptors was 17-fold. The aristeromycin analogue, 7b, was
relatively weak in binding to adenosine receptors.
Among 2-chloro-substituted derivatives, the N-methanocarba
analogue, 8c, was less potent at A1 and A2A receptors than its
parent 2-chloro-N6-cyclopentyladenosine, 8a, and roughly equipotent
at A3 receptors. Thus, 8c was 53-fold selective in binding to rat
A1 vs. human A3 receptors. The N-methanocarba analogue, 9c, of
2-chloro-N6-(3iodobenzyl)adenosine, 9a, had Ki values (nM) of 141,
732, and 2.2 at A1, A2A, and A3 receptors, respectively. Thus, the
2-chloro group slightly enhanced affinity at A3 receptors, while
reducing affinity at A1 receptors.
The receptor binding affinity upon replacement of ribose with the
N-methanocarba moiety was best preserved for the A3 subtype, at
which differences were small. At A1 receptors the loss of affinity
for structures 6-9 was between 3- and 8-fold. At A2A receptors the
loss of affinity was between 6- and 34-fold.
The agonist-induced stimulation of binding of guanine nucleotides
to activated G-proteins has been used as a functional assay for a
variety of receptors, including adenosine receptors. Binding of
[.sup.35S]GTP-.gamma.-S was studied in membranes prepared from CHO
cells stably expressing human A1 or A3 receptors (Table 2). The
non-selective adenosine agonist NECA (5'-N-ethyluronamidoadenosine)
caused a concentration-dependent increase in the level of the
guanine nucleotide bound. Compound 6c was highly selective and a
full agonist at human A1 but not rat A1 receptors. Both 7c and 9c
stimulated the binding of [.sup.35S]GTP-.gamma.-S, however the
maximal stimulation was significantly less than that produced by
either NECA or N6(3-iodobenzyl)adenosine, 7a, both being full A3
agonists. Compounds 7c and 9c resulted in relative stimulation of
[.sup.35S]GTP-.gamma.-S binding of only 45% and 22%, respectively,
indicating that the efficacy of the N-methanocarba analogue at A3
receptors was further reduced upon 2-chloro modification. The
potency of compounds 7c and 9c, indicated by the EC50 values in
this functional assay, was greater than the potencies of either
NECA or compound 7a (Table 2). Thus, the N-methanocarba
N6-(3-iodobenzyl) analogues appear to be highly potent and
selective partial agonists at human A3 receptors
TABLE-US-00001 TABLE I Affinities of Adenosine Derivatives.sup.1
##STR00005## K.sub.1 (nM) or % displacement Compound R' R
RA.sub.1.sup.a RA.sub.2A.sup.b hA.sub.3.sup.c 1a H cyclopentyl 0.59
462 274 .+-. 20 CPA 240 (r) 1B H cyclopentyl 5.06 .+-. 0.51 6800
.+-. 1800 170 .+-. 51 1781 1c H cyclopentyl 5110 .+-. 790 15% at 10
.mu.M 1783 2a H 3-iodobenzyl 20.0 .+-. 8.5 17.5 .+-. 0.5 9.5 .+-.
1.4 (r) IB0-ADO, 541 2b H 3-iodobenzyl 69.2 .+-. 9.8 601 .+-. 236
4.13 .+-. 1.76 1743 3a Cl yclopentyl 0.6 950 237 (r) CCPA 3b Cl
cyclopentyl 8.76 .+-. 0.81 3390 .+-. 520 466 .+-. 58 1761 3c Cl
cyclopentyl 3600 .+-. 780 45 .+-. 5% at 100 .mu.M 1782 4a Cl
3-iodobenzyl 18.5 .+-. 4.7 38.5 .+-. 2.0 1.41 .+-. 0.17 (r) 542 4b
Cl 3-iodobenzyl 141 .+-. 22 732 .+-. 207 2.24 .+-. 1.45 1760 4c Cl
3-iodobenzyl 8730 .+-. 370 25,400 .+-. 3800 1784 Compound R.sub.2
rA.sub.1.sup.a rA.sub.2A.sup.b hA.sub.2B.sup.b hA.sub.3.s- up.b
A.sub.1/A.sub.3 5a H Estd. 10.sup.d estd. 30.sup.d <10% at 100
.mu.M estd. 1000 (r).sup.d,e 100 5b H 6260 .+-. 730 2150 .+-. 950
47,300 .+-. 10,600 20,000 .+-. 7900 (r).sup.e 0.31 5c H 1680 .+-.
80 .sup. 22,500 .+-. 100 (h).sup.e,f 35 .+-. 2% at 50 .mu.M.sup.f
404 .+-. 70.sup.f 4.2 5d H 15% at 100 .mu.M >100,000 (h).sup.e,f
20 .+-. 4% at 50 .mu.M.sup.f 62,500 .+-. 2900.sup.f >1 (racemic)
6a CP 1.50 .+-. 0.51 857 .+-. 163 21,200 .+-. 4300 .sup. 274 .+-.
20,240 (r).sup.e 0.0055 6c CP 5.06 .+-. 0.51 6800 .+-. 1800 139k
.+-. 19k 170 .+-. 51 0.030 7a IB 20.0 .+-. 8.5 17.5 .+-. 0.5 3570
.+-. 100 9.5 .+-. 1.4 (r).sup.e 2.1 7b IB 25,900 .+-. 1600 <10%
100 .mu.M n.d. 1960 .+-. 370 13 7c IB 69.2 .+-. 9.8 601 .+-. 236
12,100 .+-. 1300 4.13 .+-. 1.76 17 8a CP 1.33 .+-. 0.19 605 .+-.
154 20,400 .+-. 1200 237 (r).sup.e 0.0056 8c CP 8.76 .+-. 0.81 3390
.+-. 520 27 .+-. 7% at 100 .mu.M 466 .+-. 58 0.019 9a IB 18.5 .+-.
4.7 38.5 .+-. 2.0 5010 .+-. 1400 1.41 .+-. 0.17 (r).sup.e 13 9c IB
141 .+-. 22 732 .+-. 207 41,000 .+-. 700 2.24 .+-. 1.45 63
.sup.1(a) simple carbocyclic, (b) and methanocarba-adenosine,
(N)-conformation, (c) and S-conformation, (d) derivatives in
radioligand binding assays at rat A.sub.1,.sup.a rat
A.sub.2A,.sup.b human A.sub.2B,.sup.b and human A.sub.3 receptors,
.sup.c unless noted..sup.e
TABLE-US-00002 TABLE II Effect of ligands to stimulate
[.sup.35S]GTP.gamma.S binding to membranes of cells expressing the
cloned hA.sub.1AR or hA.sub.3AR or in rat cerebral cortical
membranes containing the A.sub.1AR cloned hA.sub.1AR % Maximal
rA.sub.1AR % Maximal cloned hA.sub.3AR % Maximal Ligand EC.sub.50
(nM).sup.a Stimulation.sup.c EC.sub.50 (nM).sup.a Stimulation.sup.c
EC.sub.50 (nM).sup.a Stimulation.sup.c NECA n.d. n.d. 155 .+-. 15
100 6a 4.15 .+-. 0.90 100 20.3 .+-. 13.1 100 7980 .+-. 60 100 6c
21.5 .+-. 2.3 102 .+-. 1 100 .+-. 17 75 .+-. 6 >10,000 14 .+-.
2% at 10 .mu.M 7a 43.1 .+-. 10.4 91 .+-. 1 340 .+-. 98 95 .+-. 4
5.16 .+-. 0.71 100 7b >10,000 5 .+-. 2% at 10 .mu.M n.d.
>10,000 15 .+-. 5% at 10 .mu.M 7c 218 .+-. 18 86 .+-. 2 940 .+-.
114 55 .+-. 5 0.70 .+-. 0.16 45.3 .+-. 6.8 8c 31.2 .+-. 3.3 97 .+-.
1 145 .+-. 35 96 .+-. 2 n.d. 9c 142 .+-. 24 91 .+-. 1 684 .+-. 75
48 .+-. 3 0.67 .+-. 0.19 22.0 .+-. 2.8 .sup.aEC.sub.50 for
stimulation of basal [.sup.35S]GTP-.gamma.-S binding by agonists in
membranes from transfected CHO cells (.+-.S.E.M.), n = 3. n.d. not
determined.
Discussion
Nearly all of the thousands of known adenosine agonists are
derivatives of adenosine. Although molecular modeling of adenosine
agonists has been carried out, there has been no direct evidence
from this for a conformational preference of the ribose ring in the
receptor binding site. In the present study, methanocarba-adenosine
analogues have defined the role of sugar puckering in stabilizing
the active receptor-bound conformation. The S-methanocarba analogue
of adenosine, 5d, was only weakly active, presumably because of a
disfavored conformation that decreases receptor binding. In
contrast, the methanocarba analogues constrained in the
N-conformation, e.g. 5c-9c, displayed high receptor affinity,
particularly at the A3 receptor. In binding assays at A1, A2A, and
A3 receptors, N-methanocarba-adenosine proved to be of higher
affinity than the S-analogue, with an N:S-affinity ratio of 150 at
the human A3 receptor. Thus, the biological potency and efficacy of
this series of nucleosides appears to be highly dependent on ring
puckering, which in turn would influence the orientation of the
hydroxyl groups within the receptor binding site.
The structure activity relationship (SAR) of adenosine agonists
indicates that the ribose ring oxygen may be substituted with
carbon, as in 5b and 7b, however much affinity is lost. As
demonstrated with the aristeromycin derivative, 7b, simple
carbocyclic substitution of the ribose moiety of otherwise potent,
N6-subsituted adenosine agonists greatly diminishes affinity, even
in comparison to aristeromycin, 5b.
In comparison to the ribose analogues, the N-methanocarba
N6-subsituted adenosine agonists were of comparable affinity at A3
receptors, but less potent at A1, A2A, and A2B receptors. The
N-methanocarba N6-cyclopentyl derivatives were A1
receptor-selective and maintained high efficacy at human
recombinant but not rat brain A1 receptors, as indicated by
stimulation of binding of [.sup.35S]GTP.gamma.S. This may be
related to either species differences or heterogeneity of G
proteins, since the degree of agonist efficacy of a given compound
may be highly dependent on the receptor-associated G protein.
N-Methanocarba N6-(3-iodobenzyl)adenosine and the 2-chloro
derivative had Ki values of 4.1 and 2.2 nM at A3 receptors,
respectively, and were selective partial agonists. As for the
ribose parents, additional 2-chloro substitution was favorable for
receptor selectivity. However, unlike the ribose forms, efficacy
was reduced in N6-(3-iodobenzyl) analogues, such that partial A3
receptor agonists 7c and 9c were produced.
Partial agonists are possibly more desirable than full agonists as
therapeutic agents due to potentially reduced side effects in the
former. Partial agonists may display in vivo specificity for sites
at which spare receptors are present, and the drug would therefore
behave with apparent "full" efficacy. Thus, for compounds 7c and
9c, partial agonism combined with unprecedented functional potency
at A3 receptors (<1 nM) may give rise to tissue selectivity.
Thus, at least three of the four adenosine receptors favor the
N-conformation. For another member of the GPCR superfamily, the
P2Y1 receptor, we recently reported that the ribose N-conformation
of adenine nucleotides also appears to be preferred at the receptor
binding site. Thus, the P1 and at least one of the P2 purinoceptors
share the preference for the N-conformation. This may suggest a
common motif of binding of nucleoside moieties among these GPCRS.
The insights of this conformational preference may be utilized in
simulated docking of adenosine agonists in a putative receptor
binding site and to design even more potent and selective
agents.
At the binding site of ADA, the N-isomer is also preferred,
although the carbocyclic adenosine analogues are relatively poor
substrates (relative rates of deamination are: 5a, 100; 5b, 0.99;
5c, 0.58; 5d, 0.010. N6-substituted analogues, such as 6c-9c, would
not be expected to be substrates for ADA. Other enzymes, such as
HIV reverse transcriptase and Herpes thymidine kinase (HSV-1 TK)
are also able to discriminate between the two antipodal
conformations of restricted methanocarba thymidine analogues.
In conclusion, we have found that the introduction of a
methano-carbocyclic modification of the ribose ring of purine
agonists represents a general approach for the enhancement of
pharmacodynamic and because of the absence of the glycosyl bond,
potentially of pharmacokinetic properties. This approach could
therefore be applied to the development of cardioprotective,
cerebroprotective, and anti-inflammatory agents.
EXAMPLE 2
Introduction
P2 receptors, which are activated by purine and/or pyrimidine
nucleotides, consist of two families: G protein-coupled receptors
termed P2Y, of which 5 mammalian subtypes have been cloned, and
ligand-gated cation channels termed P2X, of which 7 mammalian
subtypes have been cloned. The P2Y.sub.1 receptor, which is present
in the heart, skeletal and various smooth muscles, prostate, ovary,
and brain, was the first P2 subtype to be cloned. The nomenclature
of P2 receptors and their various ligand specificities is well
established.
Nucleotide agonists binding at P2Y.sub.1 receptors induce
activation of phospholipase C (PLC), which generates inositol
phosphates and diacylglycerol from phosphatidyl
inositol-(4,5)-bisphosphate, leading to a rise in intracellular
calcium. A P2Y.sub.1 receptor antagonist may have potential as an
anti-thrombotic agent, while a selective P2Y.sub.1 receptor agonist
may have potential as an anti-hypertensive or anti-diabetic
agent.
Recently, progress in the synthesis of selective P2 receptor
antagonists has occurred. Adenosine 3',5'- and 2',5'-bisphosphates
were recently shown to be selective antagonists or partial agonists
at P2Y.sub.1 receptors, and other classes of P2 antagonists include
pyridoxal phosphate derivatives, isoquinolines, large aromatic
sulfonates related to the trypanocidal drug suramin and various
dyestuffs, and 2',3'-nitrophenylnucleotide derivatives. Synthesis
of analogues of adenosine bisphosphates has resulted in
N6-methyl-2'-deoxyadenosine-3',5'-bisphosphate (1a, MRS 2179), a
competitive antagonist at human and turkey P2Y.sub.1 receptors,
with a KB value of approximately 100 Nm. The presence of an
N.sup.6-methyl group and the absence of a 2'-hydroxyl group both
enhanced affinity and decreased agonist efficacy, thus resulting in
a pure antagonist at both turkey and human P2Y.sub.1 receptor. The
corresponding 2-Cl analogue (1b, MRS 2216) was slightly more potent
than 1a as an antagonist at turkey P2Y.sub.1 receptors, with an
IC.sub.50 value of 0.22 .mu.M in blocking the effects of 10 nm
2-methylthioadenosine-5'diphosphate (2-MeSADP). MRS2179 (compound
1a) was inactive at P2Y.sub.2, P2Y.sub.4, and P2Y.sub.6 subtypes,
at the adenylyl cyclase-linked P2Y receptor in C6 glioma cells and
at a novel avian P2Y receptor that inhibits adenylyl cyclase.
However, the selectivity of this series of nucleotides for the
P2Y.sub.1 receptor is not absolute, since 1a also displayed
considerable activity at P2X.sub.1 receptors (EC.sub.50 1.2 .mu.M),
but not at P2Y.sub.2-4 receptors.
In order to move away from the nucleotide structure of 1a and
thereby increase biological stability and selectivity for the
receptors in the present study, further structural modifications of
the ribose moiety have been carried out. We have explored the SAR
of these two series and introduced major modifications of the
ribose moiety. These modifications include fixing the ring pucker
conformation in the carbocyclic series using a bridging
cyclopropane ring, ring enlargement with introduction of a nitrogen
atom, and ring contraction.
Results
Chemical synthesis
The methanocarbocyclic 2'-deoxyadenosine analogues in which the
fused cyclopropane ring fixes the conformation of the carbocyclic
nucleoside into a rigid northern or southern envelope conformation,
as defined in the pseudoroational cycle, were synthesized as
precursors of nucleotides 4 and 5 by the general approach of
Marquez and coworkers. Again, the N.sup.6-methyl group was
introduced by the Dimroth rearrangement, as shown below.
##STR00006## Position adenine modifications were further introduced
in the N-configuration series as shown below.
##STR00007## Biological Activity
Adenine nucleotides markedly stimulate inositol lipid hydrolysis by
phospholipase C in turkey erythrocyte membranes, through activation
of a P2Y.sub.1 receptor. The agonist used in screening these
analogues, 2-MeSADP, has a higher potency than the corresponding
triphosphate for stimulation of inositol phosphate accumulation in
membranes isolated from [.sup.3H]inositol-labeled turkey
erythrocytes.
The deoxyadenosine bisphosphate nucleotide analogues prepared in
the present study were tested separately for agonist and antagonist
activity in the PLC assay at the P2Y.sub.1 receptor in turkey
erythrocyte membranes, and the results are reported in Table 3.
Concentration-response curves were determined for each compound
alone and in combination with 10 nM 2-MeSADP.
Marquez and coworkers have introduced the concept of
ring-constrained carbocyclic nucleoside analogues, based on
cyclopentane rings constrained in the N-(Northern) and S-(Southern)
conformations by fusion with a cyclopropane (methanocarba) ring. In
the present studies the series of ring-constrained N-methanocarba
derivatives, the 6-NH.sub.2 analogues, 4a was a pure agonist of
EC.sub.50152 nM and 88-fold more potent than the corresponding
S-isomer, 5, also an agonist. Thus, the ribose ring N-conformation
appeared to be favored in recognition at P2Y.sub.1 receptors. The
N.sup.6-methy- and 2-chloro-N.sup.6-methyl-N-methanocarba
analogues, 4b and 4c, were antagonists having IC.sub.50 values of
276 and 53 nM, respectively.
Molecular Modeling
To better understand the role of the sugar puckering on the human
P2Y.sub.1 agonist and antagonists activities, we carried out a
molecular modeling study of this new generation of ribose-modified
ligands. Such modifications include cyclopentyl rings constrained
in the N-- and S-- conformations with cyclopropyl (methanocarba)
groups, six-membered rings (morpholino and anhydrohexitol
analogues), and cyclobutyl nucleotides. We have recently developed
a model of the human P2Y.sub.1 receptor, using rhodopsin as a
template, by adapting a facile method to simulate the
reorganization of the native receptor structure induced by the
ligand coordination (cross-docking procedure). Details of the model
building are given in the Experimental Section. We have also
reported the hypothetical molecular basis for recognition by human
P2Y.sub.1 receptors of the natural ligand ATP and the new potent,
competitive antagonist
2'-deoxy-N.sup.6-methyladenosine-3',5'-bisphosphate. Both ATP and
1a are present in the hypothetical binding site with a N-sugar ring
conformation. In the present work, the sterically constrained N--
and S-methanocarba agonist analogues, 4a and 5, respectively, were
docked into the putative binding site of our previously reported
P2Y.sub.1 receptor model. According to their structural similarity,
the cross-docking procedure demonstrated that the receptor
architecture found for binding the ATP and 1a was energetically
appropriate also for the binding of both 4a and 5. However,
N-methanocarba/P2Y.sub.1 complex appeared more stable by
approximately 20 kcal/mol than S-methanocarba/P2Y.sub.1 complex. In
the lowest energy docked complex of N-methanocarba agonist in the
proposed ligand binding cavity the side chain of Gln307 is within
hydrogen bonding distance of the N.sup.6 atom at 1.8 .ANG., and the
side chain of Ser314 is positioned at 2.0 .ANG. from the N.sup.1
atom and at 3.4 .ANG. from the N.sup.6 of the purine ring. As
already reported, another three amino acids are important for the
coordination of the phosphate groups in the antagonist: Arg128,
Lys280 and Arg310. Lys280 may interact directly with both
3'-5'-phosphates (1.7 .ANG., O3' and 1.7 .ANG., O5'), whereas
Arg128 and Arg310 are within ionic coupling range to both the O2
and 03 atoms of the 5'-phosphate. In molecular modeling studies
poor superimposition (rms=1.447) between the -- and S-methanocarba
agonist analogues has been found inside the receptor binding
domain. In particular, the adenine moiety and 5' phosphate of the
S-methanocarba derivative are shifted out position relative to with
the N-methanocarba isomer, decreasing the stability of the
S-methanocarba/PSY.sub.1 complex. This fact might be correlated
with the difference of their biological activity as seen in Table 4
below.
Using the information that a common binding site could be
hypothesized among these deoxyadenosine bisphosphate analogues, a
superimposition analysis of the energy-minimized of the more potent
antagonists has been performed. In this analysis we have used 1a as
a reference compound, and we have defined three matching pairs of
atoms, corresponding to N.sup.1 atom of the purine ring and the P
atoms of both 3' and 5' phosphate groups, to carry out the
superimposition analysis. As reported in Table 4, acceptable RMS
values have been obtained for all the antagonists compared with the
1a structure. As shown in FIG. 4A, this superimposition study
suggested that the two phosphate groups may occupy a common
receptor regions, and a general pharmacophore model for
bisphosphate antagonists binding to the human PSY.sub.1 receptor
can be extrapolated.
Discussion
In conclusion the present study has identified new pharmacological
probes of PSY.sub.1 receptors, including full agonists, partial
agonists, and antagonists. The SAR of 1a indicates that the ribose
ring oxygen may be readily substituted with carbon. Furthermore,
analogues of constrained conformation, e.g. the methanocarba
analogues, display enhanced receptor affinity. Additional 2-chloro
and N.sup.6-methyl substitution is favorable for affinity at
PSY.sub.1 receptors, and nearly pure antagonism is maintained
provided that the N.sup.6-methyl group is present.
Thus, the biological potency and efficacy of this series of
bisphosphates appears to be highly dependent on subtle
conformational factors, which would influence the orientation of
the phosphate groups within the receptor binding site.
The sugar moiety of nucleosides and nucleotides in solution is
known to exist in a rapid, dynamic equilibrium between extreme
2--exo/3'-endo (N--) and 2'-endo/3'-exo (S--) conformations as
defined in the pseudorotational cycle. While the energy gap between
N-- and S-conformation is in the neighborhood of 4 kcal/mol, such a
disparity can explain the difference between micromolar and
nanomolar binding affinities. Using a molecular modeling approach,
we have analyzed the sugar conformational requirements for a new
class of bisphosphate ligands binding to the human PSY.sub.1
receptor. As experimentally shown, the ribose ring Northern
conformation appeared to be favored in recognition at human
PSY.sub.1 receptor (see Table 4). We have found new support to our
recently presented hypothesis in which three important recognition
regions are present in the bisphosphate molecular structures; The
N.sup.1 atom of the purine ring and the P atoms of both 3' and 5'
phosphate groups. The N-conformation seems to be essential to
maximize the electrostatic interactions between the negatively
charged phosphates and the positively charged amino acids present
in the receptor binding cleft, as well Arg128, Lys280, and
Arg310.
Interestingly, the electrostatic contacts also appear to be crucial
for the recognition of bisphosphate antagonists. Using
superimposition analysis, a general pharmacophore model for the
bisphosphate antagonists binding to the PSY.sub.1 receptor has been
proposed. According to the pharmacophore map, recognition of the
bisphosphates antagonists at a common region inside the receptor
binding site and, consequently, a common electrostatic potential
profile is possible. As well for the agonists, the Northern
conformation seems to be essential to maximize the electrostatic
interactions between the negatively charged phosphates and the
positively charged amino acids presents in the receptor binding
cleft. As we predicted using the previously reported PSY.sub.1
receptor model, sugar moiety does not seen to be crucial for the
ligand recognition process.
As already described, the simple addition of the N.sup.6-methyl
group in several cases converted pure agonists to antagonists. From
a pharmacological point of view, this is really a unique situation.
With the addition of the N.sup.6-methyl group it is not possible to
have a double hydrogen-bonding interaction and, consequently, the
activation pathway is blocked. However, for all the N.sup.6-methyl
antagonists the possibility to participate in at least one of the
two possible hydrogen bonds appears to be very important for the
increase in affinity at the PSY.sub.1 receptor.
Chemical Synthesis
Nucleosides and synthetic reagents were purchased from Sigma
Chemical Co. (St. Louis, Mo.) and Aldrich (St. Louis, Mo.).
6-Chloro-2'-deoxypurine riboside was obtained from Sigma. Several
2'-deoxynucleosides, including an anhydrohexitol-adenine nucleoside
and 2'-deoxyaristeromycin were also synthesized.
Purity of compounds was checked using a Hewlett-Packard 1090 HPLC
apparatus equipped with an SMT OD-5-60 RP-C18 analytical column
(250.times.4.6 mm; Separation Methods Technologies, Inc., Newark,
Del.) in two solvent systems. System A: Linear gradient solvent
system: 0.1 M TEAA/CH.sub.3CN from 95/5 to 40/60 in 20 min and the
flow rate was of 1 mL/min. System B: linear gradient solvent
system: 5 mM TBAP/CH.sub.3CN from 80/20 to 40/60 in 20 min and the
flow rate was of 1 mL/min. Peaks were detected by UV absorption
using a diode array detector. All derivatives showed more than 95%
purity in the HPLC systems.
Purification of most of the nucleotide analogues for biological
testing was carried out on DEAE-A25 Sephadex columns as described
above. However, compounds 7b and 8a-c required HPLC purification
(system a, semi-preparative C18 column) of the reaction
mixtures.
General Procedure of Phosphorylation.
Method A: The nucleoside (0.1 mmol) and Proton Sponge.RTM. (107 mg,
0.5 mmol) were dried for several h in high vacuum at room
temperature and then suspended in 2 mL of trimethyl phosphate.
Phosphorous oxychloride (Aldrich, 37 .mu.L, 0.4 mmol) was added,
and the mixture was stirred for 1 h at 0.degree. C. The reaction
was monitored by analytical HPLC (eluting with a gradient
consisting of buffer: CH.sub.3CN in the ratio 95:5 to 40:60, in
which the buffer was 0.1 M triethylammonium acetate (TEAA); elution
time was 20 min; flow rate was 1 mL/min; column was SMT OD-5-60
RP-C18; detector was by UV in the E.sub.max range of 260-300 nm).
The reaction was quenched by adding 2 mL of triethylammonium
bicarbonate buffer and 3 mL of water. The mixture was subsequently
frozen and lyophilized. Purification was performed on an
ion-exchange column packed with Sephadex-DEAE A-25 resin, a linear
gradient (0.01 to 0.5 M) of 0.5 M ammonium bicarbonate was applied
as the mobile phase, and UV and HPLC were used to monitor the
elution. All nucleotide bisphosphates were collected, frozen and
lyophilized as the ammonium salts. All synthesized compounds gave
correct molecular mass (high resolution FAB) and showed more than
95% purity (HPLC, retention times are reported in Table 4).
Method B: Nucleoside (0.1 mmol) dried for several h in high vacuum
at room temperature was dissolved in 2 mL of dry THF. Lithium
diisopropylamide solution (Aldrich, 2.0 M in THF, 0.4 mmol) was
added slowly at -78.degree. C. After 15 min tetrabenzyl
pyrophosphate (Aldrich, 0.4 mmol) was added and the mixture was
stirred for 30-60 min at -78.degree. C. The reaction mixture was
warmed to 0.degree. C.-rt and stirred for an addition period
ranging from 2 h to 24 h. Chromatographic purification (pTLC,
CHCl.sub.3:CH.sub.3OH(10:1) gave the tetrabenzyl phosphorylated
compound. This compound (20 mg) was dissolved in a mixture of
methanol (2 mL) and water (1 mL) and hydrogenated over a 10%
Pd-on-C catalyst (10 mg) at rt for 62 h. The catalyst was removed
by filtration and the methanol was evaporated. The residue was
treated with ammonium bicarbonate solution and subsequently frozen
and lyophilized. Purification, if necessary, was by the same
procedure as in method A.
(N-Methanocarba-2'-deoxyadenosine-3',5'-bis(diammonium phosphate)
(4a)
[(IR,2S,4S,5S)-1-[(phosphato)methyl]-4-(6-aminopurin-9-yl)bicyclo[3.1.0.]-
-hexane-2-phosphate tetraammonium salt]
Starting from 16 mg (0.06 mmol) of
(N)-methanocarba-2'deoxyadenosine and following the general
phosphorylation procedure A we obtained 1.8 mg (0.0037 mmol, 5.5%
yield) of the desired compound.
.sup.1H-NMR (D.sub.2O) .differential. 0.90 (1H, m, CH.sub.2-6),
1.10 (1H, m CH.sub.26'), 1.82 (1H, m, CH-5), 1.91 (1H, m,
CH.sub.2-3') 2.23 (1H, m, CH.sub.2-3'), 3.49 (1H, d, J=11.7 Hz,
CH.sub.2--OH), 4.16 (1H, d, J=6.9 Hz, CH.sub.2-2'), 8.39 (1H, s,
H-2), 8.54 (1H, s, H-8).
.sup.31P-NMR (D.sub.2O) .differential. 0.43 (s, 5'P); -0.19 (s,
3'P).
(N)-Methanocarba-N.sup.6-methyl-2'deoxyadenosine-3',5'-bis(diammonium
phosphate) (4b)
(1R,2S,4S,5S)-1-[(phosphato)methyl]-4-(6-methylaminopurin-9-yl)bicyclo[[3.-
1.0.]-hexane-2-phosphate tetraammoniun salt]
13.5 mg (0.0170 mmol) of compound 18 was converted to the
corresponding phosphoric acid analog using hydrogenation following
the general procedure B. Purification was performed on an
ion-exchange column packed with Sephadex-DEAF A-25 resin, linear
gradient (0.01 to 0.5 M) of 0.5 M ammonium bicarbonate was applied
as the elan to give 3.0 mg (0.0060 mmol, 35.3% yield) of the
desired compound.
.sup.1H-NMR (D.sub.2P) .differential. 0.93-0.98 (1H, m,
CH.sub.2-6'), 1.17 (1H, m, CH.sub.2-6'), 1.86-1.88 (1H, m, CH5'),
1.94-1.98 (1H, m, CH.sub.2-3'), 2.23-2.31 (1H, m, CH.sub.2-3'),
3.09 (3H, bs, N.sup.6--CH.sub.3), 3.61-3.64 (1H, m, CH.sub.2OH),
4.51-4.55 (1H, m, CH.sub.2OH), 5.01-5.03 (1H, m, CH-4'), 5.19-5.21
(1H, m, CH-2'), 8.22 (1H, s, H-2), 8.51 (1H, s, H-8). 31P-NMR
(D.sub.2O) .differential. 1.26, 1.92 (2s, 3'-P, 5'-P).
(N)-Methanocarba-N.sup.6-methyl-2-chloro-2'-deoxyadenosine-3',5'-bis(diamm-
onium phosphate) (4c)
[(1R,2S,4S,5S)-1[(phosphato)methyl]-4-(2-chloro-6-aminopurin-9-yl)bicyclo
[3.1.0]-hexane-2-phosphate tetraammonium salt]
The nucleoside, compound 23, reacted with tetrabenzyl
pyrophosphate, as in Method B, followed by an alternative
deprotection procedure. Starting from 10 mg (0.0323 mmol) of
(N)-methanorcarba-N.sup.6-methyl-2-chloro-2'-deoxyadenosine and
following the general phosphorylation procedure (Method B) we
obtained 9.5 mg (0.0114 mmol, 35.3% yield) of the desired compound,
(N)-methanocarba-N.sup.6-methyl-2-chloro-2'-deoxyadenosine-3',5'-bis(dibe-
nzyl phosphate).
.sup.1H-NMR (CDCl.sub.3).differential. 0.75-0.81 (H,
m,CH.sub.2-6'), 103-1.08 (1H, m, CH.sub.2-6'), 1.49-1.51 (1H, m,
CH-5'), 1.84-1.94 (1H, m, CH.sub.2-3'), 1.99-2.10 (1H, m,
CH.sub.2-3'), 3.12 (3H, bs, N.sup.6--CH.sub.3), 4.11-4.20 (1H, m,
CH.sub.2OH), 4.50-4.55 (H, m, CH.sub.2OH), 4.90-4.98, (8H, m,
--OCH.sub.2), 4.99-5.01 (1H, m, CH-4'), 5.23-5.30 (1H, m, CH-2'),
5.90 (1H, BS, NH), 7.20-7.29 (20H, m, C.sub.6H.sub.5), 7.82 (1H, s,
H-8)
.sup.31P-NMR (D.sub.2O) .differential. -0.58 (s,5'P); -1.06
(s,3'P).
MS(Cl--NH.sub.3) (M+1) 830 HRMS (FAB-) (M+Cs) Calcd. 962.1252;
Found 962, 1252
9.5 mg (0.0114 mmol) of the tetrabenzyl-protected intermediate
added to dry CH.sub.2Cl.sub.2 (1.0 mL) was cooled to -78.degree. C.
under argon and treated with 100 .mu.L of boron trichloride
solution (1M in CH.sub.2Cl.sub.2) and 100 .mu.L of anisole. The
reaction mixture was stirred for 12 hr at 0.degree. C. to rt and
extracted with triethylamine solution. Purification was performed
on an ion-exchange column packed with Sephadex-DEAE A-25 resin,
linear gradient (0.01 to 0.5 M) of 0.5 M ammonium bicarbonate was
applied as the eluent to give 0.4 mg (0.0007 mmol, 6.52 yield) of
the desired compound 4c.
.sup.1H-NMR (D.sub.2O) .differential.0.91-0.96 (1H, m,
CH.sub.2-6'), 1.12-1.16 (1H, m, CH.sub.2-6'), 1.80-1.84 (1H, m,
CH-5'), 1.85-1.98 (1H, m, CH.sub.2-3'), 2.20-2.50 (1H, m,
CH.sub.2-3'), 3.08 (3H, bs, N.sup.6--CH.sub.3), 3-57-3.60 (1H, m,
CH.sub.2OH), 4.52-4.67 (1H, m, CH.sub.2OH), 4.94-4.96 (1H, m,
CH-4'), 5.18-5.21 (1H, m, CH-2'), 8.52 (1H, s,H-8)
.sup.31P-NMR (D.sub.2O) .differential. 1.82, 2.52 (2s, 3'-P,
5'P)
(S)-Methanocarba-2', deoxyadeonosine-3',5'-bis(diammonium phosphate
(5)
[(1S,3S,4R,5S)-4-[(phosphato)methyl]-1-(6-aminopurin-9-yl)bicyclo[3.1.0]--
hexane-3-phosphate tetraammonium salt]
Starting from 16 mg (0.06 mmol) of
(S)-methanocarba-2'deoxyadenosine and following the general
phosphorylation procedure A, we obtained 2.1 mg (0.0043 mmol, 7.55
yield) of the desired compound 5.
.sup.1H-NMR (D.sub.2O) .differential. 1.36 (1H, m, CH.sub.2-6'),
1.53 (1H, t, J=4.8 Hz, CH.sub.2-6'), 2.05 (1H, m, CH.sub.2-5'),
2.30 (1H, m, CH-4'), 2.46 (2H, m, CH.sub.2-2'), 3.97 (2H, m,
CH.sub.2OH), 4.45 (1H, d, J=6.6 Hz, CH-3'), 8.16 (1H, s, H-2), 8.30
(1H, s, H-8). .sup.31P-NMR (D.sub.2O) .differential. 0.85 (bs,
5'P); 0.31 (bs, 3'P).
[(1S,3S,4R,5S)-1-[(Hydroxy)methyl]-2-hydroxy-4-(6-methylaminopurin-9-1yl)b-
icyclo[3.1.0]-hexane (17b)
The Dimroth rearrangement (Scheme 2) was carried out on
(N)-methanocarba-2'-deoxyadenosine. Specifically, the
(N)-methanorcarba-2'-deoxyadenosine (17a, 50.0 mg, 0.191 mmol) was
heated at 40.degree. C. with methyl iodide (71.5 .mu.L, 1.15 mmol)
in dry DMF (2.0 mL) for 48 h. The solvent was evaporated under
reduced pressure, and the residue was heated at 90.degree. C. with
ammonium hydroxide (4.0 mL) for 4 h. The water was evaporated, and
the residue was purified by pTLC using MeOH; CHCl.sub.3 (1:9) to
afford compound 17b as a colorless solid (40 mg, 0.15 mmol,
76%).
.sup.1H-NMR (CD.sub.3OD) .differential. 0.77.-0.81 (1H, m,
CH.sub.2-6'), 1.03-1.07 (1H, m, CH.sub.2-6'), 1.68-1.72 (1H, m,
CH-5'), 1.79-1.89 (1H, m, CH.sub.2-3'), 2.00-2.07 (1H, m,
CH.sub.2-3'), 3.12 (3H, bs, N.sup.6--CH.sub.3), 3-33 (1H, d,
J=CH.sub.2OH), 4.29 (1H, d, J=11.7 Hz, CH.sub.2OH), 4.89-4.92 (1H,
m, CH-4'), 5.02 (1H, d, J=6.9 Hz, CH-2'), 8.24 (1H, s, H-2), 8.49
(1H, s, H-8).
MS(CI--NH.sub.3): 276 (M+1) 830 HRMS(FAB-) (M+Cs) Calcd. 275.1382;
Found 275.1389.
(N)-Methanocarba-N.sup.6-methyl-2'-deoxyadenosine-3',5'-bis(dibenzylphosph-
ate) (18)
[(1S,2S,4S,5S)-1-[(dibenzylphosphato)methyl]-4-(6-methylaminopurin-9-yl)bi-
cyclo[3.1.0]-hexane-2-dibenzylphosphate]
Starting from 20.0 mg (0.0726 mmol) of
N-methanorcarba-N.sup.6-methyl-2'-deoxyadenosine 17b and following
the general phosphorylation procedure (Method B we obtained 13.5 mg
(0.0170 mmol, 23.4% yield) of the desired protected intermediate,
18 as shown in Scheme 2.
.sup.1H-NMR (CDCl.sub.3) .differential. 0.73-0.78 (1H, m,
CH.sub.2-6'), 0.94-0.98 (1H, m, CH.sub.2-6'), 1.53-1.54 (1H, m,
CH-5'), 1.81-1.91 (1H, m, CH.sub.2-3'), 2.05-2.13 (1H, m,
CH.sub.2-3'), 3.15 (3H, bs, N.sup.6--CH.sub.3), 3-70-3.83 (1H, m,
CH.sub.2OP), 4.49-4.55 (1H, m, CH.sub.2OP), 4.89-5.00 (8H, m,
OCH-.sub.2), 5.02-5.06 (1H, m, CH-4'), 5.27-5.32 (1H, m, CH-2'),
5.86 (1H, bs, NH), 7.21-7.23 (20H, m, C.sub.6H.sub.5), 7.86 (1H,
s,H-2), 8.31 (1H, s,H-8). .sup.31P-NMR (D.sub.2O) .differential.
-0.56, -1.05 (2s, 3'-P, 5'P) HRMS (FAB-) (M-Cs) Calcd. 928.1641;
Found 928.1700.
[(1S,2S,42,5S)-1-[(Benzyloxy)methyl]-2-benzyloxy-4-(2-6-dichloropurin-9-yl-
)bicyclo[3.1.0]-hexane (21)
To an ice cold solution of triphenylphosphine (278 mg, 1.06 mmol)
in dry THF (2 mL) was added diethylazadicarboxylate (170, 1.06
mmol) dropwise under a nitrogen atmosphere, and the mixture was
stirred for 20 min until the solution turned red orange (Scheme 3).
This mixture was added dropwise to a cold stirred mixture of the
starting alcohol (135 mg, 0.417 mmol) and 2.6-dichloropurine (157
mg, 0.883 mmol) under a nitrogen atmosphere. The reaction mixture
was stirred in an ice bath for 30 min and then allowed to warm to
room temperature, and stirring continued for 12 h. Solvent was
removed by nitrogen purge, and the residue was purified by pTLC
using EtOAc petroleum ether (1:1) to afford a thick liquid (132 mg,
0.263 mmol, 64%).
.sup.1H NMR: (CD.sub.3OD) .delta. 0.85 (m, 1H), 1.13 (m, 1H), 1.59
(m, 1H), 1.68 (m, 1H), 2.06 (m, 1H), 3.17 (d, J=10.8 Hz, 1H),
4.11-4.57 (m, 5H), 5.20 (d, J=6.9 Hz, 1H), 6.6 (bs, 1H), 7.23-7.37
(m, 10H), 8.98 (s, 1H).
MS: (EI) 494 (M+).
[(1R,2S,4S,5S)-1-[(Benzyloxy)methyl]-2-benzyloxy-4-(2-chloro-6-methylamino-
purin-9-yl)bicyclo[3.1.0]-hexane (22)
Compound 21 (100 mg, 0.202 mmol) was dissolved in methylamine in
methanol (30% solution, 3 mL) and was stirred at rt for 12 h under
a nitrogen atmosphere. The solvent was evaporated, and the crude
product was purified by pTLC using EtOAc:petroleum ether (6:4) to
afford 22 as a light yellow solid (86 mg, 0.176 mmol, 88%).
.sup.1H NMR: (CD.sub.3OD) .delta. 0.70 (m, 1H), 1.06 (m, 1H), 1.50
(m, 1H), 1.76 (m, 1H), 1.96 (m, 1H), 3.01 (s, 3H), 3.08 (m, 2H),
4.03 (m, 4H), 4.45 (bs, 1H), 5.02 (bs, 1H), 8.38 (s, 1H).
MS: (Cl): 490 (M+1).
[(1R,2S,42,5S)-1-[(Hydroxy)methyl]-2-hydroxy-4-(2-chloro-6-methylaminopuri-
n-9-yl)bicyclo[3.1.0]-hexane (23)
Compound 22 (40 mg 0.0816 mmol) was dissolved in dry
CH.sub.2Cl.sub.2 (1.0 mL), and hydrogenated using BCl.sub.3 (1M in
CH.sub.2Cl.sub.2, 175 .mu.L) for 50 min at -78.degree. C. under
argon. The solvent was evaporated, and the crude product was
purified by pTLC using CHCl.sub.3: MeOH (10:1) to afford 23 as a
light yellow solid (10.0 mg, 0.0323 mmol, 39.6%).
.sup.1H NMR: (CD.sub.3OD) .differential. 0.77-0.81 (1H, m,
CH.sub.2-6'), 1.02-1.05 (1H, m, CH.sub.2-6'), 1.65-1.68 (1H, m,
CH-5'), 1.78-1.91 (1H, m, CH.sub.2-3'), 1.99-2.07 (1H, m,
CH.sub.2-3'), 3.08 (3H, bs, N.sup.6--CH.sub.3), 3.37 (1H, d, J=11.7
Hz, CH.sub.2OH), 4.27 (1H, d, J=11.7 Hz, CH.sub.2OH), 4.89-4.91
(1H, m, CH-4), 4.97 (1H, d, J=6.8 Hz, CH-2'), 8.46 (1H, s,
H-8).
MS: (CI--NH.sub.3): 310 (M+1), HRMS (FAB-): Calcd 309.0992, Found
309.0991.
Pharmacological Analyses.
P2Y.sub.1 receptor promoted stimulation of inositol phosphate
formation by adenine nucleotide analogues was measured in turkey
erythrocyte membranes as previously described. The K.sub.0.5 values
were averaged from 3-8 independently determined
concentration-effect curves for each compound. Briefly, 1 mL of
washed turkey erythrocytes was incubated in inositol-free medium
(DMEM; Gibco, Gaithersburg Md.) with 0.5 mCi of
2-[.sup.3H]myo-inositol (20Ci/mmol: American Radiolabelled
Chemicals, Inc., St. Louis Mo.) for 18-24 h in a humidified
atmosphere of 95% air/5% CO.sub.2 at 37.degree. C. Erythrocyte
ghosts were prepared by rapid lysis in hypotonic buffer (5 mM
sodium phosphate, pH 7.4, 5 mM MgCl.sub.2, 1 mM EGTA) as described.
Phospholipase C activity was measured in 25 .mu.L of
[.sup.3H]inositol-labeled ghosts (approximately 175 .mu.g of
protein, 200-500,000 cpm/assay) in a medium containing 424 .mu.M
CaCl.sub.2, 0.91 mM MgSO.sub.4, 2 mM EGTA, 115 mM KCl, 5 mM
KH.sub.2PO.sub.4, and 10 mM Hepes pH 7.0. Assays (200 .mu.L final
volume) contained 1 .mu.M GTP.gamma.S and the indicated
concentrations of nucleotide analogues. Ghosts were incubated at
30.degree. C. for 5 min, and total [.sup.3H]inositol phosphates
were quantitated by anion exchange chromatography as previously
described..sup.7,36
Data Analysis.
Agonist potencies were calculated using a four-parameter logistic
equation and the GraphPad software package (GraphPad, San Diego,
Calif.). EC.sub.50 values (mean.+-.standard error) represent the
concentration at which 50% of the maximal effect is achieved.
Relative efficacy (%) was determined by comparison with the effect
produced by a maximal effective concentration of 2-MeSADP in the
same experiment.
Antagonist IC.sub.50 values (mean.+-.standard error) represent the
concentration needed to inhibit by 50% the effect elicited by 10 nM
2-MeSADP. The percent of maximal inhibition is equal to 100 minus
the residual fraction of stimulation at the highest antagonist
concentration.
All concentration-effect curves were repeated in at least three
separate experiments carried out with different membrane
preparations using duplicate or triplicate assays.
TABLE-US-00003 TABLE 3 Stimulation of PLC at turkey erythrocyte
P2Y.sub.1 receptors (agonist effect) and the inhibition of PLC
stimulation elicited by 10 nM 2-MeSADP (antagonist effect), for at
least two separate determinations. Agonist Antagonist Effect,
Effect, % of % of maximal maximal IC.sub.50, .mu.M.sup.b Compound
increase.sup.a EC.sub.50, .mu.M.sup.a inhibition.sup.b (n)
1a.sup.c,e NE 99 .+-. 1 0.331 .+-. 0.059 (MRS 2179) (5) 1b.sup.e NE
95 .+-. 1 0.206 .+-. 0.053 1c.sup.e 4 d 96 .+-. 2 1.85 .+-. 0.74
1d.sup.e 6 .+-. 2 d 94 .+-. 2 0.362 .+-. 0.119 4a 92 .+-. 5 0.155
.+-. 0.021 NE 4b NE 100 0.157 .+-. 0.060 4c NE 100 0.0516 .+-.
0.0008 5 41 .+-. 13 13.3 34% at small decrease 100 .mu.M
.sup.aAgonist potencies were calculated using a four-parameter
logistic equation and the GraphPad softaware package (GraphPad, San
Diego, CA). EC.sub.50 values (mean .+-. standard error) represent
the concentration at which 50% of the maximal effect is achieved.
Relative efficacies (%) were determined by comparison with the
effect produced by a maximal effective concentration of 2-MeSADP in
the same experiment. Small increase refers to <10% at 100 .mu.M.
.sup.bAntagonist IC.sub.50 values (mean .+-. standard error)
represent the concentration needed to inhibit by 50% the effect
elicited by 10 nM 2-MeSADP. The percent of maximal inhibition is
equal to 100 minus the residual fraction of stimulation at the
highest antagonist concentration. .sup.c1a, MRS 2179; 4c, MRS 2279.
d EC.sub.50 was not calculated for increases of .ltoreq.10% at 100
.mu.M. .sup.evalues from refs. 17, 19. NE no effect at 100
.mu.M.
TABLE-US-00004 TABLE 4 Synthetic data for nucleotide derivatives,
including structural verification using high resolution mass
spectroscopy and purity verification using HPLC. FAB (M - H.sup.+)
HPLC (rt; min).sup.a No Formula Calcd Found System A System B
Method, Yield (%).sup.b 2 C.sub.10H.sub.15O.sub.9N.sub.5P.sub.2
410.0267 410.0269 3.53 10.72 B, 21- .7 3b
C.sub.12H.sub.19O.sub.8N.sub.5P.sub.2 422.0631 422.0664 3.41 8.21
B, 8.- 0 4a C.sub.12H.sub.17O.sub.8N.sub.5P.sub.2 420.0474 420.0482
3.92 7.30 A, 5.- 5 4b C.sub.13H.sub.19O.sub.8N.sub.5P.sub.2
434.0631 434.0622 5.91 7.83 B, 8.- 3 4c
C.sub.13H.sub.18O.sub.8N.sub.5P.sub.2Cl 468.0241 468.0239 8.05 8.54
B, - 2.3 5 C.sub.12H.sub.17O.sub.8N.sub.5P.sub.2 420.0474 420.0481
4.02 6.84 A, 7.5- 6 C.sub.11H.sub.16O.sub.8N.sub.5P.sub.2Cl
442.0084 442.0070 6.67 6.82 A, 2- 4.3 7b
C.sub.12H.sub.20O.sub.12N.sub.5P.sub.3 518.0237 518.0243 4.98 12.74
A, - 1.8 7c C.sub.12H.sub.19O.sub.9N.sub.5P.sub.2 438.0580 438.0580
4.63 9.36 B, 50- .1 7d C.sub.12H.sub.18O.sub.9N.sub.5P.sub.2Cl
472.0201 472.0190 5.67 9.97 B, - 31.3 8a
C.sub.12H.sub.20O.sub.8N.sub.6P.sub.2 437.0740 437.0721 2.37 8.78
8.0 8b C.sub.12H.sub.21O.sub.11N.sub.6P.sub.3 517.0403 517.0404
2.,42 9.23 7.2- 8c C.sub.12H.sub.22O.sub.14N.sub.6P.sub.4 597.0066
597.0053 2.96 10.02 4.0- .sup.aPurity of each derivative was
.gtoreq.95%, as determined using HPLC with two different mobile
phases. System A: gradient of 0.1M TEAA/CH.sub.3CN from 95/5 to
40/60 and System B: gradient of 5 mM TBAP/CH.sub.3CN from 80/20 to
40/60. .sup.bPhosphorylation methods: Method A refers to use of
phosphorous oxychloride, and Method B refers to use of tetrabenzyl
pyrophosphate/lithium diisopropylamide followed by hydrogenation.
The percent yields refer to overall yield for each phosphorylation
sequence. For the method of synthesis of 8 refer to Experimental
Section.
Abbreviations AIBN, 2,2'-azobisisobutyronitrile; ATP, adenosine
5'-triphosphate; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DCTIDS,
1,3-dichlorotetraisopropyl-1,1,3,3,-disiloxane; DEAD,
diethylazadicarboxylate; DEAE, diethylaminoethyl; DMAP,
4-dimethylaminopyridine; DMF, dimethylformamide; DMSO,
dimethylsulfoxide; FAB, fast atom bombardment (mass spectroscopy);
HPLC, high pressure liquid chromatography; MS, mass spectroscopy;
HRMS, high resolution mass spectroscopy; LDA, lithium
diisoproylamide; 2-MeSADP, 2-methylthioadenosine-5'-diphosphate;
TBAP, tetrabutylammonium phosphate; TEAA, triethylammoniun acetate;
THF, tetrahydrofuran;
All of the references cited herein, including patents, patent
applications, and publications, are hereby incorporated in their
entireties by reference.
While this invention has been described with an emphasis upon
preferred embodiments, it will be obvious to those of ordinary
skill in the art that variations of the preferred embodiments may
be used and that it is intended that the invention may be practiced
otherwise than as specifically described herein. Accordingly, this
invention includes all modifications encompassed within the spirit
and scope of the invention as defined by the following claims.
* * * * *
References