JP2015164934A - Nucleotide analogs - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide, e.g., intermediates for phosphonomethoxy nucleotide analogs, in particular, intermediates suitable for use in efficient oral delivery of such analogs.SOLUTION: Novel compounds are provided that comprise esters of antiviral phosphonomethoxy nucleotide analogs with carbonates and/or carbamates having the structure -OC(R2)2OC(O)X(R)a, The compounds are useful as intermediates for the preparation of antiviral compounds or oligonucleotides, or are useful for administration directly to patients for antiviral therapy or prophylaxis (R2 is as described in the specifications ).

Description

The present invention relates to intermediates for phosphonomethoxynucleotide analogs, and in particular to intermediates suitable for use in the efficient oral delivery of such analogs.

  Various techniques for oral delivery of such analogs per se and these and other therapeutic compounds are known. WO 91/19721, WO 94/03467, WO 94/03466, WO 92/13869, U.S. Pat.Nos. 5,208,221, 5,124,051, DE 4138584A1, WO 94/10539, WO 94/10467, WO 96/18605, WO 95 / 07920, WO 9579/07919, WO 92/09611, WO 92/01698, WO 91/19721, WO 88/05438, EP0632048, EP0481214, EP0369409, EP0269947, U.S. Pat.Nos. 3,524,846 and 5,386,030, Engel Chem. Rev. .77: 349-367 1977, Farquhar et al., J. Pharm. Sci. 72: 324-325 1983, Starrett et al., Antiviral Res. 19: 267-273 1992, Safadi et al., Pharmaceutical Research 10 (9): 1350-1355 1993, Sakamoto et al., Chem. Pharm. Bull. 32 (6): 2241-2248 1984, and Davidsen et al., J. Med. Chem. 37 (26): 4423-4429 1994.

The present invention provides the following:
(1) Compounds having the following formula (1a) and salts, hydrates, tautomers and solvates thereof:

Where Z is —OC (R ² ) ₂ OC (O) X (R) _a , ester, amidate or —H;
A is a residue of an antiviral phosphonomethoxynucleotide analog;
X is N or O;
R ² is independently -H, C ₁ -C ₁₂ alkyl, C ₅ -C ₁₂ aryl, C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, C ₇ -C ₁₂ alkenyl aryl, C ₇ -C ₁₂ alkynylaryl, or C ₆ -C ₁₂ alkaryl, any one of which is unsubstituted or substituted with 1 or 2 halo, cyano, azido, nitro or —OR ³ , wherein R ³ Is C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl or C ₅ -C ₁₂ aryl;
R is independently -H, C ₁ -C ₁₂ alkyl, C ₅ -C ₁₂ aryl, C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, C ₇ -C ₁₂ alkenyl aryl, C ₇ -C ₁₂ alkynylaryl, or C ₆ -C ₁₂ alkaryl, any one of which is unsubstituted or 1 or 2 halo, cyano, azido, nitro, -N (R ⁴ ) ₂ or- Substituted with OR ³ , wherein R ⁴ is independently —H or C ₁ -C ₈ alkyl, provided that at least one R is not H; and a is 1 when X is O , 1 or 2 when X is N;
Provided that when a is 2 and X is N, (a) two N-bonded R groups can be combined to form a carbocycle or oxygen-containing heterocycle, and (b) one N-bond R may further be —OR ³ or (c) both N-linked R groups may be —H.
(2) The compound according to item 1, having the following formula (1), and salts, hydrates, tautomers and solvates thereof:

Where B is guanine-9-yl, adenine-9-yl, 2,6-diaminopurine-9-yl, 2-aminopurine-9-yl or their 1-deaza, 3-deaza, or 8- Whether it is an aza analog or
Or B is cytosin-1-yl;
R is independently -H, C ₁ -C ₁₂ alkyl, C ₅ -C ₁₂ aryl, C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, C ₇ -C ₁₂ alkenyl aryl, C ₇ -C ₁₂ Alkynylaryl, or C ₆ -C ₁₂ alkaryl, any one of which is unsubstituted or substituted with 1 or 2 halo, cyano, azido, nitro or —OR ³ , wherein R ³ is C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl or C ₅ -C ₁₂ aryl;
R ¹ is hydrogen, —CH ₃ , —CH ₂ OH, —CH ₂ F, —CH═CH ₂ , or —CH ₂ N ₃ , or R ¹ and R ⁸ are bonded to —CH ₂ — Forming;
R ² is independently hydrogen or C ₁ -C ₆ alkyl; and
R ⁸ is hydrogen or —CHR ² —OC (O) —OR, or R ⁸ combines with R ¹ to form —CH ₂ —. (3) The compound according to item 2, wherein R ² is —H.
(4) The compound according to item 3, wherein R ¹ is —CH ₃ .
(5) The compound according to item 1, wherein R ² is —H.
(6) The compound according to item 1, wherein one R ² is —CH ₃ and the other R ² is H.
(7) The compound according to item 1, wherein R ³ is C ₁ -C ₆ alkyl or phenyl.
(8) The compound according to item 1, wherein R ³ is —CH ₃ or —C ₂ H ₅ .
(9) The compound according to item 1, wherein X is O.
(10) The compound according to item 1, wherein X is N, and one R ³ is H.
(11) The compound according to item 4, wherein the compound is enriched or resolved at a carbon atom chiral center bonded to R ¹ .
(12) The compound according to item 4, wherein at least about 90% of the compound has the (R) configuration at the R ¹ moiety.
(13) The compound according to item 12, wherein B is adenine-9-yl.
(14) The compound according to item 13, wherein each R is ethyl.
(15) The compound according to item 13, wherein each R is isopropyl.
(16) The compound according to item 13, wherein each R is 3-pentyl or neopentyl.
(17) The compound according to item 13, wherein each R is t-butyl or isobutyl.
(18) The compound according to item 4, wherein B is 2,6-diaminopurin-9-yl.
(19) The compound according to item 3, wherein R ¹ is H.
(20) The compound according to item 19, wherein B is adenine-9-yl.
(21) The compound according to item 4, wherein R is C ₁ -C ₁₂ alkyl.
(22) The compound according to item 3, wherein R ¹ is —CH ₂ OH.
(23) The compound according to item 22, wherein B is cytosin-1-yl.
(24) The compound according to item 1, which is specified in Table B and compound groups 1 to 19.
(25) A compound according to item 22, wherein at least about 90% of the compound is in the (S) configuration at the R ¹ moiety.
(26) A method comprising orally administering a therapeutically effective amount of the compound according to item 1 to a patient who is infected with or at risk of viral infection. (27) A method for preparing a compound of formula (1a), wherein a phosphonomethoxynucleotide analog diacid is converted to L-CH (R ² ) OC (O) X (R) _n (where L is a leaving group) And the method of reacting with.
(28) A method for preparing a compound of formula (1), comprising reacting a compound of formula (6) below with L-CHR ² —OC (O) —OR and recovering a compound of formula (1) Comprising the steps of:

Where B is guanine-9-yl, adenine-9-yl, 2,6-diaminopurin-9-yl, 2-aminopurin-9-yl or their 1-deaza, 3-deaza, or 8 -Is an aza analog or B is cytosin-1-yl;
R ¹ is hydrogen, —CH ₃ , —CH ₂ OH, —CH ₂ F, —CH═CH ₂ , or —CH ₂ N ₃ , or R ¹ and R ⁸ are bonded to —CH ₂ — Forming;
R ⁸ is hydrogen or —CHR ² —OC (O) —OR, or R ⁸ combines with R ¹ to form —CH ₂ —;
R ² is -H, C ₁ -C ₁₂ alkyl, aryl, alkenyl, alkynyl, alkynyl aryl, alkynyl aryl, alkaryl, arylalkynyl, arylalkenyl or arylalkyl, which may be unsubstituted or Substituted with halo, azide, nitro or OR ³ , wherein R ³ is C ₁ -C ₁₂ alkyl;
R is independently H, C ₁ -C ₁₂ alkyl, aryl, alkenyl, alkynyl, alkynyl aryl, alkynylaryl, alkaryl, arylalkynyl, arylalkenyl or arylalkyl, which are unsubstituted or Alternatively substituted with halo, azide, nitro or OR ³ , provided that at least one R is not H; and L is a leaving group.
(29) A method according to item 30, comprising carrying out the reaction using at least about 1.0 equivalent of L-CHR ² —OC (O) —OR.
(30) The method according to item 31, comprising performing the reaction in an organic solvent at a reaction temperature of about 4 to 100 ° C. for about 4 to 72 hours in the presence of an organic base.
(31) The method according to item 28, wherein the compound of the formula (1) is recovered by forming a salt, precipitating the salt, and recovering the precipitated salt.
(32) A method according to item 31, wherein the salt is formed from sulfuric acid, phosphoric acid, lactic acid, or citric acid.

SUMMARY OF THE INVENTION According to the present invention, there are provided compounds having the following formula (1a) and salts, hydrates, tautomers and solvates thereof:

Wherein Z is independently —OC (R ² ) ₂ OC (O) X (R) _a , ester, amidate or —H, but at least one Z is —OC (R ² ) ₂ OC (O ) X (R) _a ;
A is a residue of an antiviral phosphonomethoxynucleotide analog;
X is N or O;
R ² is independently -H, C ₁ -C ₁₂ alkyl, C ₅ -C ₁₂ aryl, C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, C ₇ -C ₁₂ alkenyl aryl, C ₇ -C ₁₂ alkynylaryl, or C ₆ -C ₁₂ alkaryl, any one of which is unsubstituted or substituted with 1 or 2 halo, cyano, azido, nitro or —OR ³ , wherein R ³ Is C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl or C ₅ -C ₁₂ aryl;
R is independently -H, C ₁ -C ₁₂ alkyl, C ₅ -C ₁₂ aryl, C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, C ₇ -C ₁₂ alkenyl aryl, C ₇ -C ₁₂ alkynylaryl, or C ₆ -C ₁₂ alkaryl, any one of which is unsubstituted or 1 or 2 halo, cyano, azido, nitro, -N (R ⁴ ) ₂ or- Substituted with OR ³ , wherein R ⁴ is independently —H or C ₁ -C ₈ alkyl, provided that at least one R is not H; and a is 1 when X is O , 1 or 2 when X is N;
Provided that when a is 2 and X is N, (a) two N-bonded R groups can be combined to form a carbocycle or oxygen-containing heterocycle, and (b) one N-bond R may further be —OR ³ or (c) both N-linked R groups may be —H.

Further embodiments of the compounds of the invention are the following compounds of formula (1) and salts, hydrates, tautomers and solvates thereof:

Where B is guanine-9-yl, adenine-9-yl, 2,6-diaminopurine-9-yl, 2-aminopurine-9-yl or their 1-deaza, 3-deaza, or 8- Whether it is an aza analog or
Or B is cytosin-1-yl;
R is independently -H, C ₁ -C ₁₂ alkyl, C ₅ -C ₁₂ aryl, C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, C ₇ -C ₁₂ alkenyl aryl, C ₇ -C ₁₂ Alkynylaryl, or C ₆ -C ₁₂ alkaryl, any one of which is unsubstituted or substituted with 1 or 2 halo, cyano, azido, nitro or —OR ³ , wherein R ³ is C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl or C ₅ -C ₁₂ aryl;
R ¹ is hydrogen, —CH ₃ , —CH ₂ OH, —CH ₂ F, —CH═CH ₂ , or —CH ₂ N ₃ , or R ¹ and R ⁸ are bonded to —CH ₂ — Forming;
R ² is independently hydrogen or C ₁ -C ₆ alkyl; and
R ⁸ is hydrogen or —CHR ² —OC (O) —OR, or R ⁸ combines with R ¹ to form —CH ₂ —.

  Other embodiments include orally administering a therapeutically effective amount of a compound of formula (1a) or (1) to a patient who is infected with or at risk of viral infection.

Another embodiment of the present invention includes a process for the preparation of a compound of formula (1a), which comprises a phosphonomethoxynucleotide analog diacid and LC (R ² ) ₂ OC (O) X (R) _a ( Where L is a leaving group)
And the step of reacting.

In a particular embodiment of the invention, there is provided a process for the preparation of a compound of formula (1) comprising the following compound of formula (4):

And LC (R ² ) ₂ OC (O) X (R) _a is included.

DETAILED DESCRIPTION OF THE INVENTION The abbreviations NMP, DMF and DMPU mean N-methylpyrrolidinone, dimethylformamide and N, N′-dimethylpropylene urea, respectively.

  Heterocycle means aromatic and non-aromatic cyclic moieties. The heterocyclic moiety typically includes one ring or two fused rings, where the ring is a 5-membered or 6-membered ring, and typically 1 or 2 non-carbon atoms. (For example, oxygen, nitrogen or sulfur, usually oxygen or nitrogen).

As used herein, “alkyl” refers to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms unless otherwise indicated. Including C ₁ -C ₁₂ hydrocarbons in the form of linear, secondary, tertiary or cyclic structures. For example,

Cyclopropyl, cyclobutyl, cyclopropylmethyl, cyclopentyl, cyclobutylmethyl, 1-cyclopropyl-1-ethyl, 2-cyclopropyl-1-ethyl, cyclohexyl, cyclopentylmethyl, 1-cyclobutyl-1-ethyl, 2-cyclobutyl- 1-ethyl, 1-cyclopropyl-1-propyl, 2-cyclopropyl-1-propyl, 3-cyclopropyl-1-propyl, 2-
Examples include cyclopropyl-2-propyl and 1-cyclopropyl-2-propyl.

As used herein, “alkenyl” is a C ₁ -C ₁₂ hydrocarbon containing a linear, secondary, tertiary or cyclic structure unless otherwise indicated. An example is

1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, and 1-cyclohex-3- Enil.

As used herein, “alkynyl” is a C ₁ -C ₁₂ hydrocarbon containing a linear, secondary, tertiary or cyclic structure unless otherwise indicated. An example is

It is.

Salts include those derived from a combination of suitable anions (eg, inorganic or organic acids). Suitable acids include acids that have sufficient acidity to form stable salts, and are preferably low toxicity acids. For example, certain organic and inorganic acids (eg HF, HCl, HBr, HI, H ₂ SO ₄ , H ₃ PO ₄ ), or basic centers from organic sulfonic acids, organic carboxylic acids (typically amines The salts of the present invention can be formed from acid addition to Exemplary organic sulfonic acids include C ₆ -C ₁₆ aryl sulfonic acids, C ₆ -C ₁₆ heteroaryl sulfonic acids and C ₁ -C ₁₆ alkyl sulfonic acids (eg, phenyl, a-naphthyl, b-naphthyl, (S ) -Camphor, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, pentyl and hexyl sulfonic acid). Exemplary organic carboxylic acids include C ₁ -C ₁₆ alkyl, C ₆ -C ₁₆ aryl carboxylic acid and C ₄ -C ₁₆ heteroaryl carboxylic acid (eg, acetic acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, glutaric acid Acid, tartaric acid, citric acid, fumaric acid, succinic acid, malic acid, maleic acid, hydroxymaleic acid, benzoic acid, hydroxybenzoic acid, phenylacetic acid, cinnamic acid, salicylic acid, and 2-phenoxybenzoic acid). Salts also include salts of the compounds of the invention with one or more amino acids. Many amino acids, particularly naturally occurring amino acids found as protein components, are suitable, but amino acids are typically basic or acidic groups (eg, lysine, arginine or glutamic acid), or neutral groups (eg, , Glycine, serine, threonine, alanine, isoleucine, or leucine). The salt is usually biologically compatible or pharmaceutically acceptable, or non-toxic, particularly for mammalian cells. Biologically toxic salts are generally used with synthetic intermediates of the compounds of the invention. The salts of the compounds of the invention can be crystalline or non-crystalline.

A is a residue of a phosphonomethoxynucleotide analog. The parent compound has the structure AOCH ₂ P (O) (OH) ₂ . They are well known and have shown antiviral activity. As such, it is not part of the present invention. In general, A has the structure BQ, where B is a purine or pyrimidine base or an aza and / or deaza analog thereof, and Q is a cyclic or acyclic aglycone. B is attached to Q via the 9 position of purine or the 1 position of pyrimidine. Examples of these analogs are U.S. Pat. 935, WO93 / 07157, WO94 / 03467, and WO96 / 23801. Typically, A has the structure BCH ₂ CH (CH ₃ ) — or BCH ₂ CH ₂ —.

  The notation “a” is an integer of 1 or 2. When X is N, a is 2, and one R is usually H and the other is not H. When X is O, a is 1.

In general, B is guanine-9-yl, adenine-9-yl, 2,6-diaminopurin-9-yl, 2-
Aminopurin-9-yl or their 1-deaza, 3-deaza, or 8-aza analogs, or B is cytosyn-1-yl. Usually B is adenine-9-yl or 2,6-diaminopurin-9-yl. In the compound of formula (1a), one Z optionally includes an ester or amidate. Suitable esters or amidates are described, for example, in WO95 / 07920. Exemplary esters are phenyl, benzyl, o-ethoxyphenyl, p-ethoxyphenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, N-ethylmorpholino, C ₁ -C ₈ O-alkyl and C ₁ -C ₈ NH-alkyl. However, the compounds of all of the present invention comprises at least one _{^{-C (R 2) 2 OC (}} O) X (R) a moiety.

R ² is independently -H, C ₁ -C ₁₂ alkyl, C ₅ -C ₁₂ aryl, C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, C ₇ -C ₁₂ alkenyl aryl, C ₇ -C ₁₂ alkynylaryl, or C ₆ -C ₁₂ alkaryl, any one of which is unsubstituted or substituted with 1 or 2 halo, cyano, azido, nitro or —OR ³ , wherein R ³ Is C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl or C ₅ -C ₁₂ aryl. R ² is usually H or C ₁ -C ₆ alkyl, and typically only one R ² is other than H. In most embodiments, R ² is H in both cases. The carbon atom to which R ² is attached can be chirally substituted, in which case R ² is in the (R), (S) or racemic configuration. In most embodiments, when R ² is other than H, the compounds of the invention are chirally enriched or pure at this site. In general, however, production is somewhat cheaper if chirality at the R ² carbon can be avoided. Thus, R ² is H when it is desired to minimize the synthesis cost.

  X is O or N, typically O. Carbamates (where X = N) tend to be more stable in the biological environment than carbonates. When X is O, a is 1.

R is independently -H, C ₁ -C ₁₂ alkyl, C ₅ -C ₁₂ aryl, C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, C ₇ -C ₁₂ alkenyl aryl, C ₇ -C ₁₂ Alkynylaryl, or C ₆ -C ₁₂ alkaryl, any one of which is unsubstituted or in one or two halo, cyano, azide, nitro, —N (R ⁴ ) ₂ or —OR ³ Substituted, wherein R ⁴ is independently —H or C ₁ -C ₈ alkyl, provided that at least one R is not H. Generally, R is C ₁ -C ₆ secondary or straight chain alkyl, which is unsubstituted or substituted with OR ³ . When X is N, a is 2. In the latter case, one R is usually other than H. Alternatively, the two N-linked R groups are joined to form a carbocycle or O-containing heterocycle, which typically contains 3 to 5 carbon atoms in the ring. When R is unsaturated but not aryl, the site of unsaturation is not critical and is in the Z or E configuration. For example, an alkenyl chain of a naturally occurring unsaturated fatty acid is suitable as the R group. R also includes cycloalkenyl or cycloalkynyl containing one or two unsaturated bonds, typically one unsaturated bond. When R is unsaturated, usually R is alkenyl or alkynyl without aryl substitution.

When R is substituted with halo, cyano, azide, nitro or OR ³ , typically R contains one of these substituents. When substituted with two of these substituents, they are the same or different. Generally, the substituent found on R is OR ³ . An exemplary R group containing an OR ³ substituent is —CH ₂ C (CH ₂ OCH ₃ ) (CH ₃ ) ₂ .

When R comprises an aryl group, the aryl group is generally bonded directly to X or to X by methylene or ethylene. Aryl groups can include —N═ or —O— as a ring atom. Generally, aryl groups contain 5 or 6 carbons. If replaced
The aryl moiety is substituted with halo or OR ³ in the ortho, meta or para position, in this case R ³
Is typically C ₁ -C ₃ . An aryl group containing 5 carbons is typically 2-, 3- or 4-pyridyl. In general, if substituted, only one substituent is found on the aryl moiety. Exemplary aromatic and non-aromatic heterocyclic groups used herein include, for illustrative purposes, Paquette, Leo A .; "Principles of Modern Heterocyclic Chemistry" (WA Benjamin, New York, 1968), in particular Chapters 1, 3, 4, 6, 7, and 9; “The
Chemistry of Heterocyclic Compounds, A series of Monographs "(currently from John Wiley & Sons, New York, 1950), especially volumes 13, 14, 16, 19, and 28; and" J. Am. Chem. Soc. "82 : Heterocycle described in 5556 (1960), but is not limited thereto.

Examples of heterocycles include, for illustrative purposes, but are not limited to: pyridyl, thiazolyl, tetrahydrothiophenyl, sulfurized tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, Thianaphthalenyl, indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoxyl Norinyl, azosinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H, 6H-1,5,2-dithiazinyl, thienyl, thiantenyl,
Pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathiinyl, 2H-
Pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl (indazoly), purinyl, 4H-quinolidinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinrinylyl, apteridylyl, pteridinyl, pteridinyl, 4 -Carborinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl, isoclindolinyl, indolinyl Benzotriazolyl, benzisoxazolyl, oxindori , Benzoxazolinyl, and isatinoyl.

  The carbon-bonded heterocycle is, for illustrative purposes, 2, 3, 4, 5, or 6 position of pyridine, 3, 4, 5, or 6 position of pyridazine, 2, 4, 5, or 6 position of pyrimidine, 2, 3, 5, or 6 position, furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole 2, 3, 4, or 5 position, oxazole, imidazole or thiazole 2, 4, or 5 position, isoxazole, pyrazole , Or 3, 4, or 5 of isothiazole, 2 or 3 of aziridine, 2, 3, or 4 of azetidine, 2, 3, 4, 5, 6, 7, or 8 of quinoline or isoquinoline It is bonded at the 1, 3, 4, 5, 6, 7, or 8 position, but is not limited thereto. More typically, carbon-bonded heterocycles are 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2- Including pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

Nitrogen-linked heterocycles are for purposes of illustration aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazolin, 3-pyrazolin , Piperidine, piperazine, indole, indoline, 1-position of 1H-indazole, 2-position of isoindole or isoindoline, 4-position of morpholine, and 9-position of carbazole or b-carboline. It is not limited. Even more typically, nitrogen-bonded heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

R comprises the structure —C ₂ -C ₆ R ⁵ C ₂ -C ₆ , where each C ₂ -C ₆ is independently a 2, 3, 4, 5 or 6 carbon straight chain, branched A chain or cyclic alkyl moiety (eg, ethylene, ethyl, propylene, propyl, isopropylene, isopropyl, cyclohexyl, etc.) and R ⁵ is —O— or —NR ⁶ —, wherein R ⁶ is 1 It is a straight, branched or cyclic alkyl having 2, 3, 4, 5 or 6 carbon atoms.

An embodiment includes compounds wherein R ⁴ is —H or —CH ₃ .

R comprises the structure -C ₂ -C ₁₂ R ⁹ where each C ₂ -C ₁₂ is independently 2, 3, 4, 5, 6, 7, 8
, 9, 10, 11 or 12 carbon linear, branched or cyclic alkyl moieties, and R ⁹ is N-morpholino

N-piperidino, 2-pyridyl, 3-pyridyl or 4-pyridyl.

R is also —C (CH ₂ (X) _0-1 R ⁷ ) ₃ , —CH [C (CH ₂ (X) _0-1 R ⁷ ) ₃ ] ₂ and —CH ₂ (C (X) _{0- 1} R ⁷ ) ₃ wherein R ⁷ is a straight, branched or cyclic alkyl of 1, 2, 3, 4, 5 or 6 carbons, or R ⁷ is 5 or 6 carbons Is aryl. In these embodiments, typically one or two, usually one X is present, X is usually oxygen, and R ⁷ is typically methyl, ethyl, isopropyl, propyl or butyl. , Usually methyl.

  R is usually phenyl, methyl, ethyl, 1-propyl, 2-propyl, n-butyl, i-butyl, t-butyl, pentyl or 3-pentyl.

R ¹ is a substituent found in prior art phosphonomethoxynucleotide analogs. R ¹ is typically hydrogen, —CH ₃ , —CH ₂ OH, —CH ₂ F, —CH═CH ₂ , —CH ₂ N ₃ , or R ¹ and R ⁸ are a bond To form —CH ₂ —. R ¹ is usually H or methyl. When R ¹ and R ⁸ are combined to form methylene, B is typically cytosin-1-yl.

R ³ is C ₁ -C ₁₂ alkyl, but typically C ₁ -C ₆ alkyl.

Compounds of structure (1) typically have B as adenine-9-yl, R ¹ as methyl or H, R ⁸ as —CHR ² —OC (O) —OR and R, R ² and R ³ as above It is a compound which is as follows.

The compounds of the present invention are enriched or resolved at the carbon atom chiral center attached to R ¹ according to previous findings regarding optimal antiviral activity in configuration at this site, as appropriate. Thus, when R ¹ is methyl, the compound is in the (R) configuration at this center;
And substantially free of the (S) enantiomer.

Other embodiments include compounds of structures (10) and (11), wherein R and each R ² are independently selected and R ² is C ₁ -C ₆ alkyl:

  Exemplary embodiments include the compounds specified in Table B. Each compound in Table B is shown as a compound having the following formula (8):

The compounds specified in Table B use the structure numbered as shown in Table A, and the following conversion BRR ¹ .R ²
According to the numbers assigned to B, R, R ¹ and R ² . Thus, the compound designated 1.2.3.4 is adenin-9-yl for B, —CH ₂ CH ₃ for both R groups, —CH ₂ OH for R ¹ , and — (CH ₂ CH ₂ for both R ² groups. ) Identified as ₂ CH ₃

  Exemplary embodiments include the following numbered groups of compounds:

1 Each compound specified in Table B having a hydroxyl group bonded to a phosphorus atom in place of only one carbonate moiety and a second carbonate moiety (ie, B—CH ₂ —CHR ¹ —O—CH ₂ —P ( O) (OH) -O-CHR 2 -OC (O) -OR). Thus, the compound of group 1 designated 1.4.1.1 in Table B has the structure: adenine-9-yl-CH ₂ —CH (CH ₃ ) —O—CH ₂ —P (O) (OH) —O— CH ₂ —OC (O) —OCH (CH ₃ ) ₂

2 Compounds specified in Table B having only one carbonate moiety and having only the R ¹ moiety, # 3 (—CH ₂ OH), where R ⁸ of the compound of formula (1) is bound to R 1 It is modified to form a - -CH ₂ and. Thus, Group 2 compounds designated 1.4.3.1 in Table B have the structure: adenine-9-yl-CH ₂ -CH (CH _2- ◇) -O-CH ₂ -P (O) (O- ◇ ) —O—CH ₂ —OC (O) —OCH (CH ₃ ) _2, where the symbol ◇ represents a covalent bond that joins the oxygen and carbon atoms together.

3 Compounds designated in Table B and compounds designated by Compound Groups 1 and 2, wherein each purine base listed in Table A is a 3-deaza analog (eg, 3-deazaadenine-9-yl). Accordingly, the compounds of Group 3 with the specified 1.4.1.1 in defined and Table B Table A has the structure: 3-deazaadenine-9-yl _{-CH 2 -CH (CH 3) -O} -CH 2 -P ( O) (— O—CH ₂ —OC (O) —OCH (CH ₃ ) ₂ ) ₂ . 1.4.1.1 in compound group 1 as defined in Table A
The compounds of Group 3 designated with the structure: 3-deazaadenine-9-yl _{-CH 2 -CH (CH 3) -O} -CH 2 -P (O) (OH) -O-CH 2 -OC (O ) -OCH (CH ₃ ) ₂ .

4 Compounds specified in Table B and compounds specified by Compound Groups 1 and 2, wherein each purine base listed in Table A is a 1-deaza analog (eg, 1-deazaadenine-9-yl). Thus, the group 4 compounds defined in Table A and designated 1.4.1.1 in Table B have the structure: 1-deazaadenine-9-yl-CH ₂ —CH (CH ₃ ) —O—CH ₂ —P ( O) (— O—CH ₂ —OC (O) —OCH (CH ₃ ) ₂ ) ₂ . 1.4.1.1 in compound group 1 as defined in Table A
The compounds of group 3 designated as follows have the structure: 1-deazaadenine-9-yl-CH ₂ —CH (CH ₃ ) —O—CH ₂ —P (O) (OH) —O—CH ₂ —OC (O ) -OCH (CH ₃ ) ₂ .

  5 Compounds designated in Table B and compounds designated by Compound Groups 1 and 2, wherein each purine base listed in Table A is an 8-aza analog (eg, 8-azaadenine-9-yl).

6 Compounds designated in Table B as well as compounds designated by Compound Groups 1-5, wherein R moieties 1-25 listed in Table A are replaced with the following groups:
1-cyclopropyl (cyclopropyl replaces —CH _3, which is the R moiety 1 in Table A)
2-CH ₂ -cyclopropyl 3-(CH ₂ ) ₂ -cyclopropyl 4-(CH ₂ ) ₃ -cyclopropyl 5-(CH ₂ ) ₄ -cyclopropyl 6 -cyclobutyl 7 -CH ₂ -cyclobutyl 8-(CH ₂ ) ₂ -cyclobutyl 9- (CH ₂ ) ₃ -cyclobutyl 10-(CH ₂ ) ₄ -cyclobutyl 11 -cyclopentyl 12 -CH ₂ -cyclopentyl 13-(CH ₂ ) ₂ -cyclopentyl 14-(CH ₂ ) ₃ -cyclopentyl 15- (CH ₂ ) ₄ -Cyclopentyl 16 -cyclohexyl 17 -CH ₂ -cyclohexyl 18-(CH ₂ ) ₂ -cyclohexyl 19-(CH ₂ ) ₃ -cyclohexyl 20-(CH ₂ ) ₄ -cyclohexyl 21 -CH (CH ₃ ) CH ₂ -cyclopropyl 22 -CH (CH ₃ ) CH ₂ -cyclobutyl 23 -CH (CH ₃ ) CH ₂ -cyclopentyl 24 -CH (CH ₃ ) CH ₂ -cyclohexyl 25-(CH ₂ ) _0-4- Cyclooctyl, therefore, group 6 compounds defined in Table A and designated 1.16.1.1 in Table B , Structure: adenine-9-yl _{-CH 2 -CH (CH 3) -O} -CH 2 -P (O) (- O-CH 2 -OC (O) -O- cyclohexyl) has _two. Compounds of Group 6 defined in Table A and designated 1.16.1.1 in Compound Group 1 have the structure: adenine-9-yl-CH ₂ —CH (CH ₃ ) —O—CH ₂ —P (O) ( OH) —O—CH ₂ —OC (O) —O-cyclohexyl. Group 6 compounds defined in Table A and designated 1.16.1.1 in Compound Group 3 have the structure: 3-deazaadenine-9-yl-CH ₂ —CH (CH ₃ ) —O—CH ₂ —P (O ) (— O—CH ₂ —OC (O) —O-cyclohexyl) ₂ .

7 Compounds designated in Table B as well as compounds designated by Compound Groups 1-5, wherein R moieties 1-25 listed in Table A are replaced with the following groups:
1 7 carbon alkyl ^* 4 10 carbon alkyl 7-(CH ₂ ) ₂ C ₆ H ₅
2 8 carbon alkyl 5 11 carbon alkyl 7-(CH ₂ ) ₃ C ₆ H ₅
3 9 carbon alkyl 6 12 carbon alkyl 9-(CH ₂ ) ₄ C ₆ H ₅
10 -C (CH ₃ ) ₂ CH (CH ₃ ) ₂
11 -CH (CH ₃ ) C (CH ₃ ) ₃

^* Alkyl groups are linear, branched, cyclic or monounsaturated (-C = C-).

  8 Compounds designated in Table B as well as compounds designated by Compound Groups 1-5, wherein R moieties 1-25 listed in Table A are replaced with the following groups:

  9 Compounds designated in Table B as well as compounds designated by Compound Groups 1-5, wherein R moieties 1-25 listed in Table A are replaced with the following groups:

  10 Compounds designated in Table B as well as compounds designated by Compound Groups 1-5, wherein R moieties 1-25 listed in Table A are replaced with the following groups:

  11 Compounds designated in Table B as well as compounds designated by Compound Groups 1-5, wherein R moieties 1-25 listed in Table A are replaced with the following groups:

  12 Compounds designated in Table B as well as compounds designated by Compound Groups 1-5, wherein R moieties 1-25 listed in Table A are replaced with the following groups:

25 -CH ₂ CH (C ₂ H ₅ ) OC ₄ H ₉
13 Compounds designated in Table B as well as compounds designated by Compound Groups 1-5, wherein R moieties 1-25 listed in Table A are replaced with the following groups:
1- (CH ₂ ) ₂ O-cyclopropyl 2-(CH ₂ ) ₂ O-cyclobutyl 3-(CH ₂ ) ₂ O-cyclopentyl 4-(CH ₂ ) ₂ O-cyclohexyl 5-(CH ₂ ) ₂ OCH ₂ -Cyclopropyl 6- (CH ₂ ) ₂ OCH ₂ -cyclobutyl 7-(CH ₂ ) ₂ OCH ₂ -cyclopentyl 8-(CH ₂ ) ₂ OCH ₂ -cyclohexyl 9-(CH ₂ ) ₂ O- (CH ₂ ) ₂ cyclopropyl _{10 - (CH 2) 2 O-} (CH 2) 2 cyclobutyl _{11 - (CH 2) 2 O-} (CH 2) 2 cyclopentyl _{12 - (CH 2) 2 O-} (CH 2) 2 cyclohexyl 13 - ( CH ₂ ) ₃ O-cyclopropyl 14-(CH ₂ ) ₃ O-cyclobutyl 15-(CH ₂ ) ₃ O-cyclopentyl 16-(CH ₂ ) ₃ O-cyclohexyl 17-(CH ₂ ) ₃ OCH ₂ -cyclopropyl 18-(CH ₂ ) ₃ OCH ₂ -cyclobutyl 19-(CH ₂ ) ₃ OCH ₂ -cyclopentyl 20-(CH ₂ ) ₃ OCH ₂ -cyclohexyl 21 -CH (CH ₃ ) CH ₂ O-cyclopropyl 22 -CH ( CH ₃ ) CH ₂ O-cyclobutyl 23 —CH (CH ₃ ) CH ₂ O-cyclopentyl 24 —CH (CH ₃₎ CH ₂ O-cyclohexyl _{25 -CH (CH 3) CH 2} OCH 2 - compound named in cyclohexyl 14 Table B and compounds named by compound groups 1-5, wherein, R moieties listed in Table A 1-25 are replaced with the following groups:

  15 A group of compounds AJ below.

  A Compounds designated in groups 8-14, wherein the oxygen atom (—O—) in the R moiety is replaced with —NH—.

B Compounds designated in groups 8-14, wherein the oxygen atom in the R moiety is replaced with —N (CH ₃ ) —.

Compounds named in C groups 8-14 where the oxygen atom in the R moiety -N (C ₂ H ₅₎ - is replaced by.

D Compounds designated in groups 8-14, wherein the oxygen atom in the R moiety is replaced with —N (CH ₂ CH ₂ CH ₃ ) —.

E Compounds designated in groups 8-14, wherein the oxygen atom in the R moiety is replaced with —N (CH (CH ₃ ) ₂ —.

F Compounds designated in groups 8-14, wherein the oxygen atom in the R moiety is replaced with n-butyl substituted nitrogen (—N (CH ₂ ) ₃ CH ₃ ) —).

  G Compounds designated in groups 8-14, wherein the oxygen atom in the R moiety is replaced with i-butyl substituted nitrogen.

  H Compounds designated in groups 8-14, wherein the oxygen atom in the R moiety is replaced with t-butyl substituted nitrogen.

  I Compounds designated in groups 8-14, wherein the oxygen atom of the R moiety is replaced with a linear, branched or cyclic 5-carbon alkyl substituted nitrogen.

J Compounds specified in groups 8-14, wherein the oxygen atom in the R moiety is replaced with a straight, branched or cyclic 6 carbon alkyl substituted nitrogen.
Thus, compounds of group 15B defined in Table A and designated 1.1.1.1 in compound group 8 have the structure: adenine-9-yl-CH ₂ —CH (CH ₃ ) —O—CH ₂ —P (O ) (— O—CH ₂ —OC (O) —O— (CH ₂ ) ₂ N (CH ₃ ) ₂ ) ₂ . Compounds of group 15B defined in Table A and designated 1.1.1.1 in compound group 1 have the structure: adenine-9-yl-CH ₂ —CH (CH ₃ ) —O, as specified in group 8. having _{-CH 2 -P (O) (OH} ) -O-CH 2 -OC (O) -O- (CH 2) 2 N (CH 3) 2. The compounds of group 15B defined in Table A and designated 1.16.1.1 in compound group 3 have the structure 3-deazaadenine-9-yl-CH ₂ —CH (CH ₃ ) as specified in group 8. _{-O-CH 2 -P (O)} (- O-CH 2 -OC (O) -O- (CH 2) 2 N (CH 3) 2) having a _2.

  16 Compounds designated in Table B as well as compounds designated by Compound Groups 1-5, wherein R moieties 1-25 listed in Table A are replaced with the following groups:

In part 1 to 10, R ⁹ is N- morpholino, in moieties 11 to 20, R ⁹ is 2-pyridyl and in moieties 21 to 25, R ⁹ is 3-pyridyl.

  17 Compounds designated in Table B as well as compounds designated by Compound Groups 1-5, wherein R moieties 1-25 listed in Table A are replaced with the following groups:

In part 1 to 5, R ⁹ is 4-pyridyl, in moieties 6 to 9, R ⁹ is N- morpholino and in moieties 9 to 13, R ⁹ is N- piperidyl.

  18 A group of compounds AJ below.

  A Compounds designated in Table B and compounds designated by groups 1-17, wherein compound (8) is replaced by compound (9)

Where one R ² is as specified in Table A and the other R ² is —CH ₃ .

B Compounds designated in Table B and compounds designated by Compound Groups 1-17, wherein Compound (8) is replaced with Compound (9), wherein one R ² is as specified in Table A And the other R ² is —CH ₂ CH ₃ .

C Compounds designated in Table B and compounds designated by Compound Groups 1-17, wherein Compound (8) is replaced with Compound (9), wherein one R ² is as specified in Table A And the other R ² is — (CH ₂ ) ₂ CH ₃ .

D Compounds designated in Table B and compounds designated by Compound Groups 1-17, wherein Compound (8) is replaced with Compound (9), wherein one R ² is as specified in Table A And the other R ² is —CH (CH ₃ ) ₂ .

E Compounds designated in Table B and compounds designated by Compound Groups 1-17, wherein Compound (8) is replaced with Compound (9), wherein one R ² is as specified in Table A And the other R ² is — (CH ₂ ) ₃ CH ₃ .

F Compounds designated in Table B and compounds designated by Compound Groups 1-17, wherein Compound (8) is replaced with Compound (9), wherein one R ² is as specified in Table A And the other R ² is — (CH ₂ ) ₄ CH ₃ .

G Compounds designated in Table B and compounds designated by Compound Groups 1-17, wherein Compound (8) is replaced with Compound (9), wherein one R ² is as specified in Table A And the other R ² is —CH ₂ CH (CH ₃ ) ₂ .

H Compounds designated in Table B and compounds designated by compound groups 1-17, wherein compound (8) is replaced with compound (9), wherein one R ² is as specified in Table A And the other R ² is —C (CH ₃ ) ₃ .

I Compounds designated in Table B and compounds designated by Compound Groups 1-17, wherein Compound (8) is replaced with Compound (9), wherein one R ² is as specified in Table A And the other R ² is —C ₅ H ₁₁ .

J Compounds designated in Table B and compounds designated by compound groups 1-17, wherein compound (8) is replaced with compound (9), wherein one R ² is as specified in Table A And the other R ² is —C ₆ H ₁₃ . Thus, the group 18A compounds defined in Table A and designated 1.4.2.3 in Table B have the structure: adenine-9-yl-CH ₂ —CH ₂ —O—CH (CH ₃ ) —P (O) _{(-OC (C 2 H 5)} (CH 3) -OC (O) -O-CH (CH 3) 2) having a _2. Compounds of Group 18A defined in Table A and designated 1.4.1.1 in Compound Group 1 have the structure: adenine-9-yl-CH ₂ —CH (CH ₃ ) —O—CH ₂ —P (O) ( _{OH) -O-CH (CH 3} ) -OC (O) -O-CH (CH 3) having _2. Compounds of group 18A defined in Table A and designated 1.1.1.1 in compound group 3 have the structure: 3-deazaadenine-9-yl-CH ₂ —CH (CH ₃ as specified in compound group 8 _{) -O-CH 2 -P (O} ) (- OCH (CH 3) -OC (O) -O- (CH 2) 2 OCH 3) having a _2.

  19 Compounds designated in Table B and compounds designated by compound groups 1-18, wherein compound (8) and compound (9) are replaced by compound (10) and compound (11), respectively.

Here both R moieties are the same. Thus, the group 19 compounds defined in Table A and designated 1.4.1.1 in Table B have the structure: adenine-9-yl-CH ₂ —CH (CH ₃ ) —O—CH (CH ₃ ) —P (O) (— O—CH ₂ —OC (O) —N—CH (CH ₃ ) ₂ ) ₂ Group 19 compounds defined in Table A and designated 1.4.1.1 in Compound Group 1 have the structure: adenine-9-yl-CH ₂ —CH (CH ₃ ) —O—CH ₂ —P (O) ( OH) —O—CH ₂ —OC (O) —N—CH (CH ₃ ) ₂ . The group 19 compounds defined in Table A and designated 1.1.1.1 in compound group 3 have the structure 3-deazaadenine-9-yl-CH ₂ -CH (CH ₃ as specified in compound group 8 _{) -O-CH 2 -P (O} ) (- OCH 2 -OC (O) -N- (CH 2) 2 OCH 3) having a _2.

The compounds of the present invention are chemically stable to varying degrees. In order to ensure a sufficient expiration date and appropriate biodistribution upon oral administration, the compound is preferably chemically stable. Generally, it has a t1 / 2 longer than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours at pH 7.4, preferably further 1, 10, or 100 hours at pH 2.0 An embodiment with a longer t1 / 2 is selected. For example, t-butyl carbonate found in Table 1 has a t1 / 2 that is more unstable than these parameters and is therefore not preferred. Furthermore, the optimal compound of the present invention should have a bioavailability of greater than about 20%, preferably about 30% in beagle dogs (detailed below).

Synthetic Methods The carbamates and carbonates of the present invention are prepared from the phosphonomethoxynucleotide analog diacid and the synthon LCH (R ² ) OC (O) X (R) _a . Although L is a leaving group such as Cl, it is understood that any conventional leaving group used in organic chemistry in nucleophilic substitution reactions can be successfully used in place of chloro. In particular, leaving groups include halides (eg, Cl, Br and I), and sulfonate esters (eg, methane, benzene or toluene sulfonate esters). Synthons are prepared by reacting LCH (R ² ) OC (O) L with HOR for the preparation of carbonate synthons or HNR ₂ for the preparation of carbamate synthons. The synthon is then reacted with a selected nucleotide analog (typically PMPA) to form the desired carbamate or carbonate adduct. Carbamates are prepared by reacting synthons with nucleotide analogs under typical nucleophilic attack conditions (eg, in Et ₃ N / DMF at room temperature). Carbonates are formed by reacting an appropriate synthon with a nucleotide analog in the presence of an organic base (typically an amine base). In addition, masked leaving groups such as thioethers, which can be activated, for example, by oxidation and coupled directly to the phosphonic acid moiety, can be used. Intermediates can thus be made using other leaving groups such as diphenylphosphinic acid and others known in form acetal and glycosylation chemistry.

Compounds with X═N and R═OR ³ can be prepared by alkylation with the appropriate haloalkyl, O-alkyl carbamate. N, O-dialkylhydroxylamines are known in the literature and can be prepared by alkylation of hydroxylamine or by reductive amination of aldehydes or ketones using alkylhydroxylamines. Dialkylhydroxylamines can be acylated with appropriately substituted haloalkyl chloroformates under conditions similar to those used to prepare unsubstituted chloromethyl carbamates. The phosphonate can then be alkylated with haloalkyl, O-alkyl carbamates under the conditions used for carbonates and carbamates to give prodrugs. Of course, leaving groups other than chloride can be used everywhere.

In an exemplary method, the carbonate compound of the present invention is prepared by reacting L-CHR ² —OC (O) —OR with (4) to produce a compound of formula (1).

The reaction typically proceeds in two simultaneous steps, where a monoester is first formed and then a diester is formed when the reaction is allowed to proceed for an extended period of time. In this situation, the monoester is typically not isolated as an intermediate.

To make diesters containing different carbonate or carbamate functionality, the monoester intermediate is recovered from the initial reaction and then reacted using, for example, a second L-CHR ² —OC (O) —OR reagent. Is completed, thereby being replaced with a second ester different from the first ester.

If necessary, the carbonate synthesis reaction is performed using at least about 1.0 and typically 2 equivalents of L-CHR ² —OC (O) —OR. The reaction is carried out in the presence of an organic base in an organic solvent at a reaction temperature of about 4 to 100 ° C. for about 4 to 72 hours. Exemplary suitable organic bases include triethylamine or Hunig base. Exemplary suitable organic solvents include DMF, DMPU, or NMP.

Monoester or diester products are purified by standard methods, including flash column chromatography or salting out. Suitable salts for purification and / or formulation of the final product are sulfuric acid, phosphoric acid, lactic acid, diesters or monoesters of structure (1) or (1a),
Includes fumaric acid or citric acid salts or complexes.

Utility The compounds of the present invention are useful for the treatment or prevention of one or more viral infections in humans or animals. Examples of viral infections include DNA viruses, RNA viruses, herpes viruses (CMV, HSV1, HSV2, VZV, etc.), retroviruses, hepadnaviruses (eg, HBV), papillomaviruses, hantaviruses, adenoviruses, and Infection caused by HIV. Other infections to be treated here with compounds include MSV, RSV, SIV, FIV, MuLV and other retroviral infections of rodents and other animals. The prior art describes that the antiviral specificity and parental drug specificity of nucleotide analogues is provided by the compounds of the present invention. Dosages, viral targets and appropriate routes of administration to attack the site of infection are well known in the art for parent drugs. The determination of the appropriate dose is determined by the physician, given the molecular weight of the compounds of the invention and, if they are administered orally, the bioavailability in those animals (or inferred in clinical trials in humans). It's simple for me. The oral dosage in humans of the compounds of the invention is in the range of approximately 0.5-60 mg / Kg / day, usually in the range of 1-30 mg / Kg / day for antiviral treatment, and typically The range is approximately 1.5 to 10 mg / Kg / day.

The compounds of the present invention are also useful as intermediates in the preparation of detectable labels for oligonucleotide probes. By conventional enzymatic or chemical means, the compound is hydrolyzed to give the diacid, diphosphorylated, and incorporated into the oligonucleotide. Bases incorporated from the compounds of the present invention can participate in base pairing and therefore do not substantially interfere with binding of the oligonucleotide to its complementary sequence (E. De Clercq Rev. Med. Virol. 3). : 85-96 1993). However, when interfering with the binding of an oligonucleotide, including an analog, to its complementary sequence, the compounds of the invention may optionally contain a 3 ′ terminal base, a non-nociceptive position, and a conventional site for oligonucleotide labeling. Is incorporated into the oligonucleotide. The aglycone provided by the nucleotide analog incorporated into the oligonucleotide is detected by any means (eg, by binding to an antibody specific for the NMR or nucleotide analog).

Pharmaceutical Formulations The compounds of the invention and their pharmacological, ie physiologically acceptable salts (hereinafter collectively referred to as active ingredients) can be administered by any route to the condition to be treated. Administered. Suitable routes are oral, rectal, nasal, topical (including eye, buccal, and sublingual), vaginal, and parenteral (subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and hard) Including the outside of the membrane). In general, the compounds of the invention are administered orally. However, if the embodiment is not sufficiently orally bioavailable, it can be administered by any other route described above.

  While it is possible for the active ingredients to be administered as the pure compound, it is preferable to present them as a pharmaceutical formulation. The formulations of the present invention comprise at least one active ingredient, as defined above, with one or more acceptable carriers and optionally other therapeutic ingredients. The carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.

Formulations include those suitable for topical or systemic administration, oral, rectal, nasal, buccal, sublingual, vaginal, and parenteral (subcutaneous, intramuscular, intravenous, intradermal, intrathecal, And administration (including epidural). The formulation is in unit dosage form and is prepared by any method well known in the pharmaceutical art. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. Formulations are generally prepared by combining the active ingredient uniformly and intimately with a liquid carrier or a finely divided solid carrier or both and then molding the product if necessary. Is done.

  Formulations of the present invention suitable for oral administration are in discrete units, for example as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as powders or granules; solutions in aqueous or non-aqueous liquids Or as a dispersion; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient can also be provided as a bolus, electuary or paste.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets are prepared in a suitable machine with the active ingredient in free-flowing form (eg powders or granules), optionally with binders, lubricants, inert diluents, preservatives, surfactants and dispersants. It can be prepared by mixing and compressing. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. Tablets can be coated or scored as necessary and can be formulated to provide slow or controlled release of the active ingredient therein.

For infections of the eye or other external tissues (eg mouth and skin), the formulation is preferably 0.1% w / w, for example, in the range of 0.01-10% w / w (between 0.1% and 5%) in increments of, for example, 0.6% w / w, 0.7% w / w, etc.), preferably in an amount of 0.2-3% w / w, and most preferably 0.5-2 Applied as a topical ointment or cream containing the active ingredient in an amount of% w / w. When formulated in an ointment, the active ingredient can be used with a paraffinic or water-miscible ointment base. Alternatively, the active ingredient can be formulated in a cream with an oil-in-water cream base.

If desired, the cream-based aqueous phase may be a polyhydric alcohol having two or more hydroxyl groups (eg, propylene glycol, butane 1,3-diol, mannitol, sorbitol, glycerol, for example, at least 30% w / w And polyethylene glycol (including PEG 400) and mixtures thereof). Topical formulations may desirably contain compounds that enhance the absorption or penetration of the active ingredient into the skin or other affected areas. Examples of such transdermal penetration enhancers include dimethyl sulfoxide and related analogs.

  The oily phase of the emulsions of the present invention can be composed of known ingredients in a known manner. The phase may simply comprise an emulsifier (otherwise also known as an emulgent), which desirably comprises at least one emulsifier and a fat or oil or a mixture of both fat and oil. . Preferably, the hydrophilic emulsifier is included with a lipophilic emulsifier that acts as a stabilizer. It is also preferred to include both oil and fat. The emulsifier with or without stabilizers constitutes an emulsifying wax, and the wax, together with oil and fat, constitutes an emulsifying ointment base, which forms the oily dispersed phase of the cream formulation.

Emulsions and emulsion stabilizers suitable for use in the formulations of the present invention include Tween ^(R) 60, Span ^(R) 80, cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glycerol monostearate, and lauryl. Contains sodium sulfate.

The selection of suitable oils and fats for the formulation is based on achieving the desired cosmetic properties. Thus, the cream is preferably non-greasy, non-staining and washable with an appropriate consistency to avoid leakage from tubes or other containers Should be a good product. Linear or branched, mono- or dibasic alkyl esters (eg, di-isoadipate, isocetyl stearate, propylene glycol diester of palm fatty acid, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate A blend of branched esters known as 2-ethylhexyl palmitate or Crodamol CAP may be used, the last three being the preferred esters, used alone or in combination, depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and / or liquid paraffin or other mineral oils can be used.

Formulations suitable for topical administration to the eye also include eye drops. Here, the active ingredient is dissolved or suspended in a suitable carrier for the active ingredient, in particular an aqueous solvent. In such formulations, the active ingredient is suitably present at a concentration of 0.01-20%, in some embodiments 0.1-10%, and in other embodiments about 1.0% w / w.

  Formulations suitable for topical administration in the mouth include lozenges containing the active ingredient in a scented base (usually sucrose and acacia or tragacanth); active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia Including pastilles; and mouthwashes containing the active ingredients in a suitable liquid carrier.

  Formulations suitable for rectal administration may be presented as a suppository containing, for example, cocoa butter or salicylate with an appropriate base.

Formulations suitable for nasal or inhalation administration when the carrier is a solid are for example particle sizes in the range 1 to 500 microns (particle sizes in the range between 20 and 500 microns increasing by 5 microns, for example 30 microns). , Including 35 microns etc.). Formulations suitable for administration as a nasal spray or nasal nose when the carrier is a liquid include aqueous or oily solutions of the active ingredients. Suitable formulations for aerosol administration can be prepared according to conventional methods and delivered with other therapeutic agents. Inhalation therapy is easily administered via a metered dose inhaler.

  Formulations suitable for intravaginal administration may be provided as pessaries, tampons, creams, gels, pastes, foams or spray formulations that contain, in addition to the active ingredient, a carrier such as those known to be suitable in the art. .

  Formulations suitable for parenteral administration are sterile and include aqueous and non-aqueous infusion solutions, which include antioxidants, buffers, bacteriostats, and solutes (these are intended for formulations) Can be made isotonic with the blood of the recipient); and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickeners. Formulations can be provided in unit dose or multi-dose containers, eg, with elastomeric stoppers, sealed ampoules and vials, and in a sterile liquid carrier (eg, water) immediately prior to use for injection. It can be stored in freeze-dry conditions that require only addition. Improvised infusion solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Preferred unit dosage formulations are those containing a daily dose or unit daily sub-dose of the active ingredient, as described above, or an appropriate fraction thereof.

  In addition to the ingredients specifically described above, the formulations of the present invention may include other conventional agents in the art related to the type of formulation in question. For example, those suitable for oral administration can include flavoring agents.

  The present invention further provides veterinary compositions comprising at least one active ingredient as described above together with a veterinary carrier therefor.

  Veterinary carriers are materials useful for purposes of administering the composition to cats, dogs, horses, rabbits, and other animals, and can be solid, liquid or gaseous materials, which are In addition, it is inert or acceptable in the veterinary field and is compatible with the active ingredient. These veterinary compositions can be administered orally, parenterally, or by any other desired route.

  The compounds of the invention can be used to provide controlled release pharmaceutical formulations comprising a matrix or absorbent material and one or more compounds of the invention as active ingredients. Here, the release of the active ingredient can be controlled and regulated, allowing less frequent administration or improving the pharmacokinetic or toxicity profile of the compound. Controlled release formulations adapted for isolated unit oral administration containing one or more compounds of the invention can be prepared according to conventional methods.

  All citations found herein are incorporated by reference.

  The following examples further illustrate the invention. However, this should not be construed as limiting the invention.

Example 1
PMPA synthesis

Process Summary PMPA is prepared as follows: (S) -glycidol is reduced to (R) -1,2-propanediol by catalytic hydrogenation. This is then reacted with diethyl carbonate to give (R) -1,2-propylene carbonate. Carbonate reacts with adenine and a catalytic amount of a base (eg, sodium hydroxide) to give (R) -9- [2- (diethylphosphonomethoxy) propyl] adenine, which is isolated without lithium alkoxide. (Alkyls containing 1, 2, 3, 4, 5 or 6 carbon atoms such as n-hexoxide, n-pentoxide, n-butoxide, i-butoxide, t-butoxide, n-propoxide, i-propoxy , Ethoxide, methoxide) and diethyl p-toluenesulfonyl-oxymethylphosphonate (prepared by reacting diethyl phosphite with paraformaldehyde and tosylating the product in situ). The resulting (R) -9- [2-diethylphosphonomethoxypropyl] adenine is deesterified with bromotrimethylsilane to obtain crude PMPA. This is then purified by precipitation from water while adjusting the pH. The product is further purified by recrystallization with water to give PMPA monohydrate.

This process uses a small amount of base, such as NaOH, in step 1. This increases the reaction rate by a factor of about 10 compared to the same reaction without base. Step 1 also uses hydrogen gas instead of generating hydrogen in situ using a reagent such as HCO ₂ NH ₄ . This process uses lithium alkoxide in step 4b. This slowly exotherms upon addition to the reaction mixture. The use of highly reactive bases such as NaH results in an exothermic reaction that generates hydrogen gas during the reaction and is difficult to control. The use of NaH thus requires more effort and care than lithium alkoxide. Lithium alkoxide bases also have an improved by-product profile compared to that obtained with NaH (eg, overalkylated, typically obtained from the use of less starting material, or lithium alkoxide). Product with product).

  The scale of the following method is proportionally reduced or increased if desired. The scheme and process steps show the synthesis of (R) -PMPA. The process may be carried out with chirally impure starting materials (eg (R, S) -glycidol) to obtain a chiral mixture of intermediates or final products.

If desired, the scale of the process steps described below can be increased or decreased. The scheme and process steps show the synthesis of (R) -PMPA and (R) -bis (POC) PMPA. The process is carried out with a chirally impure starting material (eg (R, S) -glycidol) to give a chiral mixture of intermediates, eg 1,2-propylene carbonate, PMPA or bis (POC) PMPA A chiral mixture of
Step 1. (R) -1,2-propanediol:
(S) -glycidol (1.0 kg, 13.5 moles) was added to (i) an inert atmosphere (eg, nitrogen) and (ii) denatured ethyl alcohol (7.85 kg EtOH and 54 g, 16.7% containing 2 mole% sodium hydroxide). To a reactor containing a stirred suspension of 5% palladium catalyst (100 g) on activated carbon (50% wet) in NaOH solution). Prior to the addition of (S) -glycidol, the contents of the inert reactor containing the catalyst and ethanol solution are typically cooled to about 0 ° C. (usually −5 ° C. to 5 ° C.). Hydrogen gas, only 20 psi, is then introduced into the inerting reaction vessel containing the reactants at a temperature of only 25 ° C. The mixture is stirred for about 4-5 hours until hydrogen consumption ceases. The completion of the reaction is monitored by TLC (no (S) -glycidol left or trace remains). The mixture is then filtered (eg, diatomaceous earth (about 150 g)), the solids are removed, and the filtrate is concentrated in vacuo at a temperature of no more than 50 ° C. until volatiles are collected or very slowly. To obtain an oil containing the crude product. The crude product is used directly in the next step. The yield of the title compound is about 100%.

Step 2. (R) -1,2-propylene carbonate:
Sodium ethoxide in diethyl carbonate (1.78 kg, 15.1 moles) and modified ethyl alcohol (210 g of sodium ethoxide in 21% w / w ethanol) was used as (R) -1,2-propanediol (used in step 1 above). Theoretically based on the amount of (S) -glycidol made 1.0 kg) and the solution is heated to 80-150 ° C. to distill off the ethanol. If necessary to achieve completion of the reaction, additional diethyl carbonate (0.16 kg) is added to the reaction mixture and then distilled to remove the ethanol. Reaction completion is monitored by TLC indicating that there is no or trace of detectable (R) -1,2-propanediol. The residue is fractionally distilled at 120 ° C. and 10-17 mm Hg to give the title compound as a colorless liquid. The purity of the product is typically 96% or more as determined by GC analysis.

Step 3. Diethyl p-toluenesulfonyloxymethylphosphonate:
In a reactor containing an inert atmosphere, such as nitrogen, a mixture of diethyl phosphite (0.80 kg), paraformaldehyde (0.22 kg), and triethylamine (0.06 kg) in toluene (0.11 kg) at about 87 ° C. Heat for 2 hours. The mixture is then refluxed for about 1 hour until the reaction is complete, as monitored by TLC indicating no detectable or trace amounts of diethyl phosphite. An inert atmosphere is maintained during the reaction. Toluene is necessary to moderate the reaction and can explode if not used. Completion of the reaction is confirmed by ¹ H NMR if necessary (a peak of δ8.4-8.6 ppm diethyl phosphite is no longer detected). Cool the solution to about 1 ° C. (typically about −2 ° C. to 4 ° C.) and add p-toluenesulfonyl chloride (1.0 kg), then slowly add triethylamine (0.82 kg) at about 5 ° C. While (exothermic addition), the temperature is maintained at only about 10 ° C. (typically 0 ° C. to 10 ° C.).
The resulting mixture is warmed to 22 ° C. and stirred for at least about 5 hours (typically about 5 hours) until the reaction is complete as indicated by TLC (no or little detectable p-toluenesulfonyl chloride). 4.5 hours to 6.0 hours), confirmed by ¹ H NMR as necessary (δ7.9 ppm p-toluenesulfonyl chloride doublet is no longer detected). The solid is removed by filtration and washed with toluene (0.34 kg). Combine the wash and filtrate twice with water (1.15 kg for each wash) or water (1.15 kg), 5% aqueous sodium carbonate (3.38 kg) in sequence, if necessary, then water (1.15 kg) Wash twice with either. After each wash, the reactor contents are briefly stirred and allowed to settle, then the lower aqueous phase is discarded. If the reaction results in an emulsion, brine (0.23 kg water containing 0.08 kg NaCl) can be added to the initial organic / water mixture, then the reactor contents are stirred to allow the solids to settle and lower layers , Add 1.15 kg of water, stir, allow the solids to settle, and again discard the lower aqueous layer. Distill the organic phase in vacuo at a temperature of only 50 ° C (only 10% at 110 ° C and water content is only 0.3% by KF titration relative to LOD) Except yielding about 60-70% yield of the title compound as an oil of about 85-95% purity.

Step 4. (R) -9- [2- (Diethylphosphonomethoxy) propyl] adenine:
In a reactor containing an inert atmosphere, such as nitrogen, adenine (1.0 kg), sodium hydroxide (11.8 g), (R) -1,2-propylene carbonate (0.83 kg), and N, N-dimethyl A mixture of formamide (6.5 kg) was monitored at 130 ° C. (typically about 125-120 ° C) until the reaction was complete as needed by monitoring that area normalized HPLC showed only about 0.5% adenine remaining. 138 ° C) for about 18-30 hours. The resulting mixture is cooled to about 25 ° C. (typically about 20-30 ° C.) and contains a stage I intermediate, (R) -9- (2-hydroxypropyl) adenine, which At this point it may precipitate. After cooling, lithium t-butoxide (3.62 kg), 2.0 M in tetrahydrofuran, was added to the stage I intermediate to produce the lithium salt of (R) -9- (2-hydroxypropyl) adenine in a mild exothermic reaction To do. The slurry is treated with diethyl p-toluenesulfonyloxymethylphosphonate (1.19 kg) and the mixture is heated to a temperature of about 32 ° C. (typically about 30-45 ° C.) and at least about 2 hours (typically For about 2-3 hours). Meanwhile, the mixture becomes uniform. Additional diethyl p-toluenesulfonyloxymethiolphosphonate (1.43 kg) is added and the mixture is at a temperature of about 32 ° C (typically about 30-45 ° C) for at least about 2 hours (typically about 2 to 2 ° C). (3 hours) Stir. Additional lithium t-butoxide (0.66 kg), 2.0 M in tetrahydrofuran and diethyl p-toluenesulfonyloxymethylphosphonate (0.48 kg) were added two more times, and after each time the mixture was at least about 2 hours at a temperature of about 32 ° C. Stir. The completion of the reaction is monitored as necessary by area normalized HPLC showing that only 10% of stage I intermediate remains. If the reaction is incomplete, additional lithium t-butoxide (0.33 kg), 2.0 M in tetrahydrofuran, and diethyl p-toluenesulfonyloxymethylphosphonate (0.24 kg) are added and the reaction mixture is at a temperature of about 32 ° C. Maintain for at least about 2 hours to achieve complete reaction. The mixture is then cooled to about 25 ° C. (typically about 20-40 ° C.) and then glacial acetic acid (0.5 kg) is added. The resulting mixture is concentrated in vacuo under a final maximum mixture temperature of about 80 ° C., about 29 inches of Hg (in Hg) vacuum. The residue is cooled to about 50 ° C. (typically about 40-60 ° C.) and water (1.8 kg) is added and the reaction is rinsed into additional water (1.8 kg). The solution is continuously extracted with dichloromethane (about 35 kg) for 12-48 hours and glacial acetic acid (0.2 kg) is periodically added to the aqueous phase after about 5 hours and about 10 hours after the continuous extraction time. Completion of the extraction, if necessary, indicates that area-normalized HPLC shows only about 7% (R) -9- [2-diethylphosphonomethoxy) propyl] adenine remains in the aqueous phase. It is confirmed. The combined dichloromethane extracts are first concentrated at atmospheric pressure and then in vacuo at an extraction temperature of only about 80 ° C. to give the title compound as a viscous orange oil. The title compound yield is about 40-45 wt% (normalized HPLC) and its purity is typically 60-65% by area normalized HPLC. The actual weight of the title compound after concentration is approximately 1.6 times the theoretical amount (ie 3.8 times the expected yield). The additionally observed weight is due to impurities and / or solvents remaining after continuous extraction and concentration.

Step 5. (R) -9- [2- (phosphonomethoxy) propyl] adenine, crude product:
Bromotrimethylsilane (1.56 kg) was added to crude (R) -9- [2- (diethylphosphonomethoxy) propyl] adenine (1.0 kg based on the adenine input from step 4 above) and acetonitrile (0.9 kg). To the reactor containing the mixture is added with cooling to maintain a temperature of only about 50 ° C. The tubing was rinsed with acetonitrile (0.3 kg) and the mixture was refluxed at about 60-75 ° C. for about 2-4 hours, preventing splashing of the reactor contents with moderate stirring. Reaction completion is monitored by area normalization HPLC showing residual monoethyl PMPA and diethyl PMPA remaining of only about 3%. When the reaction is complete, additional bromotrimethylsilane (0.04 kg) is placed in the reactor and the reaction is refluxed with moderate stirring for at least about 1 hour. Volatiles are removed by distillation at a temperature of only about 70 ° C, first at atmospheric pressure and then in vacuum (about 24-27 inches Hg (in Hg)) at a temperature of only about 70 ° C. The reactor is then cooled to about 20 ° C. (typically about 15-25 ° C.) and water (1.9 kg) is added to the residue while maintaining the temperature at only about 50 ° C. (exothermic addition) )did. The mixture is cooled to 20 ° C. and washed with dichloromethane (1.7 kg) with stirring for about 30 minutes. The isolated aqueous phase is then filtered through a 1 μm cartridge filter, diluted with water (3.2 kg), heated to about 40 ° C. (typically about 35-50 ° C.), and about 45 ° C. While maintaining the temperature, the pH is adjusted to about 1.1 (typically about 0.9 to 1.3) with an aqueous sodium hydroxide solution (about 0.15 kg NaOH as a 50% solution). PMPA seed crystals are added to the mixture and the pH is reduced to about 45 ° C. (requires about 0.15 kg NaOH) while maintaining the temperature at about 45 ° C. (typically about 35 ° C. to 50 ° C.). Adjust to 2.8 (typically about 2.6-3.0). Cool the solution to about 22 ° C. (typically about 15-25 ° C.) over about 3-20 hours while preventing slow splashing of the contents with slow to moderate stirring. Meanwhile, the product begins to precipitate at about 35 ° C. Adjust the pH of the slurry to about 3.2 (typically about 3.1-3.3) (usually using 50% aqueous sodium hydroxide or concentrated hydrochloric acid as needed). Cool the slurry to about 5 ° C. (typically about 0-10 ° C.) and stir slowly at that temperature range for at least about 3 hours. The solid is collected by filtration and washed successively with cold water (0.35 kg) and acetone (0.3 kg) to give crude PMPA as a wet solid, typically about 97% pure. The product is heated to about 50 ° C. and dried under vacuum to a water content of less than 10%. The amount of diethyl PMPA is calculated from the amount of adenine used in the previous step of synthesis (estimated yield 100%) and calculated from the net weight of crude diethyl PMPA (which may contain other compounds) Not.

Step 6. (R) -9- [2- (phosphonomethoxy) propyl] adenine, pure substance:
While stirring at moderate to high speed, a suspension of crude PMPA in water (1.00 kg corrected for water content) (product of step 5) (approximately 5 ° C) at about 100 ° C (typically) until all solids are dissolved , About 95-110 ° C.). The resulting solution is then clarified by hot filtration and rinsed with additional hot water (1 kg, about 95-110 ° C.). The filtrate is heated to 100 ° C. and then cooled. Cooling is continued to about 30 ° C. (typically about 20-25 ° C.) over about 3-5 hours with slow stirring and then to about 10 ° C. (typically about 5-15 ° C.). After holding at about 10 ° C. for at least about 3 hours, the solid is collected by filtration and washed successively with cold water (1.5 kg, about 0-10 ° C.) followed by acetone (1 kg). The wet cake is dried in vacuo at about 50 ° C. (typically about 40-60 ° C.) to a moisture content of about 5.9% (typically about 3.9-7.9%) and pure PMPA monohydrate Get a Japanese product. The purity of the product by both area normalization and weight normalization HPLC is typically greater than 98%. If the chemical purity is insufficient, the product can be re-purified by repeating this process.

Recrystallization as needed:
0.75 g PMPA (Preparation A) was recrystallized from H ₂ O (11.3 mL, 15: 1 weight ratio) and the suspension was heated to 95-100 ° C. After cooling to room temperature, the crystallized PMPA was cooled in a freezer. After 3 hours, the crystals are filtered on a coarse frit equipped with Tyvek ^™ and the filter cake is ice-cold H
Rinse with ₂ O and acetone and air dry to give a wrinkled white solid (Preparation B) as a constant weight. Recovery was 0.64 g (85.3%). HPLC showed 98.5-98.9% pure PMPA. No impurity of 14.7 minutes was observed. The recrystallized liquid (1039-91-23) showed 71.4% purity PMPA with 4.8 minutes (26.9%) (probably solvent) of the main impurities.
14.7 min Impurity = 0.05% Preparation of B PMPA was recrystallized again from 9.6 mL H ₂ O heated to 95-100 ° C. (15: 1 weight ratio). After cooling to room temperature, the crystallized PMPA was cooled in the freezer overnight. Filter the PMPA on a coarse frit equipped with Tyvek ^™ , rinse the filter cake with ice-cold H ₂ O and acetone, then suction dry to give a wrinkled white solid (Preparation C) as a constant weight. Obtained. Recovery was 0.52 g (81.3%). HPLC (JH52807, JH52810) showed PMPA with a purity of 99.3-99.5%. The largest impurity was 0.22% at 19 minutes. The recrystallized liquid showed 64.9% pure PMPA with 0.01% impurity at 14.7 minutes and 0.09% impurity at 19 minutes.

Preparation of C PMPA (0.50 g) was recrystallized again from about 7.5 mL of boiling H ₂ O (15: 1 weight ratio). Cool to room temperature and filter the PMPA on a coarse frit equipped with Tyvek ^™ . The filter cake was rinsed with ice-cold H ₂ O and acetone, then sucked dry to give a wrinkled white solid (Preparation D) as a constant weight. The filtrate was also concentrated to give a white solid (Preparation E).

Recovery: filter cake: 0.41 g (82%), filtrate: 0.08 g, combined 0.49 g (98%). HPLC analysis indicated that the filtrate (Preparation E) was 99.9% pure. PMPA prepared in this manner is used to produce the compounds of the present invention.

Step 7. Bis (POC) PMPA fumarate:
In a reactor having an inert atmosphere, such as nitrogen, a mixture of 1-methyl-2-pyrrolidinone (4.12 kg), PMPA monohydrate (1.00 kg), triethylamine (0.996 kg) is about 15-45. Stir for minutes (typically about 30 minutes). Next, chloromethyl-2-propyl carbonate (2.50 kg)
And the mixture is heated to about 55-65 ° C. (typically about 60 ° C.) and about 3-6 hours, typically about 4 hours, as required by HPLC (present To do (POC) PMPA 15%
Until the reaction is complete, stir so that the contents do not splash. The mixture is diluted with isopropyl acetate (10.72 kg), cooled to about 15-30 ° C. (typically about 25 ° C.) as rapidly as possible, and the reactor contents are about 15-30 ° C. (typically The mixture is stirred for about 20-60 minutes (typically about 30 minutes) while maintaining at about 25 ° C. The solid is removed by filtration and washed with isopropyl acetate (4.44 kg). The combined organic phases are about 15-30 ° C. (typically about 25 ° C.) with water (3.28 kg) with moderate stirring for about 1-10 minutes to avoid the formation of an emulsion. Extract twice. The phases are then separated. The combined aqueous phases are back extracted twice with isopropyl acetate (3.56 kg) (about 15-30 ° C., typically about 25 ° C.). Water (2.20 kg) (about 15-30 ° C., typically about 25 ° C.) with moderate stirring for about 1-10 minutes to combine all organic phases and avoid emulsion formation Wash with. The combined organic phases (which are about 25-43 ° C but only 45 ° C) are then concentrated in vacuo (about 26.5-28 inches Hg) to about 30% of the initial volume (about 10-121 / kg PMPA monohydrate). After polishing filtration using an in-line 1 μm filter, the organic phase is concentrated to a pale yellow oil at a temperature of about 20-38 ° C., but only 40 ° C. under vacuum (about 28 inches Hg). Continue until is left. About 0.5-2.0 hours until the solid is dissolved in a solution of fumaric acid (0.38 kg) in 2-propanol (6.24 kg) warmed (about 45-55 ° C., typically about 50 ° C.) Dissolve with vigorous stirring. The warmed solution is then filtered using an in-line 1μ filter, if necessary, with minimal cooling of the solution. The filtrate at about 34-50 ° C., typically about 40 ° C., is stirred with the minimum degree of stirring necessary to obtain a homogeneous solution. The resulting solution is seeded with a small amount of bis (POC) PMPA fumarate (about 100 mg), if necessary, with minimal agitation over about 30 minutes and about 30-33 ° C. , Typically to about 32 ° C. and to about 12-18 ° C., typically about 15 ° C., over about 1-2 hours, typically over about 1 hour. If crystal formation begins before the seed crystal is added, the seed crystal may not be necessary. Crystals can form over a range of about 12-33 ° C. as the solution is cooled. If the solution is further cooled, crystallization occurs at a lower temperature (eg, from about −10 to about 11 ° C.). When crystal formation begins, stirring is interrupted. The mixture is allowed to stand at about 15 ° C. for at least about 12 hours (typically about 12-30 hours). The resulting slurry is filtered (Tyvek) and the filter cake is washed with a premixed solution (1: 4 v / v) in isopropyl acetate (0.70 kg) in butyl ether (2.44 kg). Filter cake, which is only 40 ° C, is dried in a vacuum for about 1-3 days, and the dried product is optionally ground (Fitzmill M5A with a 0.050 inch screen) and bis (POC) PMPA fumarate white Of fine, powdery crystals (purity about 97.0-99.5%).

Example 2
Preparation of alkyl chloromethyl carbonate

Alcohol (73 mmol) and chloromethyl chloroformate (Fluka, 6.23 ml, 70 mmol
) In diethyl ether was cooled to 0 ° C. under argon. Pyridine (5.7 ml, 70 mmol) was added dropwise with stirring over 10 minutes. The solution was stirred at 0 ° C. for 1 hour, then warmed to room temperature and stirred for an additional 3 hours. The ether solution was filtered, washed with 1M HCl, dried over MgSO ₄ , filtered and concentrated on a rotary evaporator. 0.1torr
Was applied for a short time to give alkyl chloromethyl carbonate in 85-95% yield. Ethyl chloromethyl carbonate is somewhat volatile and does not remain long on the rotovap, i.e. adversely affects yield (87-35%).

Example 3
Preparation of bis-ethyloxymethyl carbonate of PMPA

R = Et. Anhydrous PMPA (5 g, 16 mmol) and DIEA (Hunig's base) (11.5 ml, 66 mmol)
Was placed in anhydrous DMF (50 ml). Chloromethyl carbonate (49 mmol) was then added and the suspension was heated to 50 ° C. under argon with rapid mechanical stirring. After 1 hour, the reaction was clear and the temperature dropped to 35 ° C. The reaction was then stirred for 48 hours. DMF was removed on a rotary evaporator and the reaction was partitioned between CH ₂ Cl ₂ and water. The CH ₂ Cl ₂ layer was dried over magnesium sulfate, filtered and concentrated on a rotary evaporator. The residue was taken up in methylene chloride and applied to a silica gel column (150 g, SiO ₂ ). This was eluted with 500 ml of isopropanol in methylene chloride at 0, 3, 6, 9, 12, 15, 18% (v / v) respectively, then 21% and 2000 mL. Appropriate fractions were pooled and evaporated to give the desired product.

Example 4
Preparation of PMPA bis-n-butyloxymethyl carbonate

Butyl chloromethyl carbonate. A solution of butyl alcohol (50 mmol) and chloromethyl chloroformate (4.5 mL, 50 mmol) in diethyl ether (200 mL) was cooled to 0 ° C. under argon. Pyridine (5.7 mL, 50 mmol) was added dropwise with stirring over 5 minutes. The solution was stirred at 0 ° C. for 15 minutes, then warmed to room temperature and stirred for an additional 3 hours. The ether solution was filtered, washed with 1M HCl, then twice with water, dried over MgSO ₄ , filtered and concentrated on a rotary evaporator to give butyl chloromethyl carbonate (7 g, 85% ).

Dibutyl PMPA carbonate. Anhydrous PMPA (4 g, 13 mmol) and DIEA (10.5 mL, 60 mmol) were placed in anhydrous DMF (40 mL). Then butyl chloromethyl carbonate (40 mmol) was added and the suspension was stirred at room temperature for 48 hours. The reaction was then heated to 50 ° C. for 18 hours. DMF was removed on a rotary evaporator. The reaction was then partitioned between CH ₂ Cl ₂ (250 mL) and water (250 mL). The CH ₂ Cl ₂ layer was washed once with saturated aqueous NaHCO ₃ solution, dried over magnesium sulfate, filtered and concentrated on a rotary evaporator. The residue was taken up in methylene chloride and applied to a silica gel column (150 g, SiO ₂ ). This was eluted with 500 mL isopropanol in methylene chloride at 0, 3, 6, 9, 12, 15, 18% (v / v) respectively, followed by 21% and 2000 mL isopropanol in methylene chloride. Appropriate fractions were pooled and evaporated to give the desired product.

Example 5
Synthesis of PMPA bis-n-propyloxyethyl carbonate

Preparation of propyl-1-chloroethyl carbonate. A solution of propyl alcohol (70 mmol) and 1-chloroethyl chloroformate (7.6 mL, 70 mmol) in diethyl ether (200 mL) was cooled to 0 ° C. under argon. Pyridine (70 mmol) was added dropwise with stirring over 5 minutes. The solution was stirred at 0 ° C. for 15 minutes, then warmed to room temperature and stirred for an additional 4.5 hours. The ether solution was filtered, washed with 1M HCl, then twice with water, dried over MgSO ₄ , filtered and concentrated on a rotary evaporator to give propyl-1-chloroethyl carbonate (9.8%). g, 84%). Anhydrous PMPA (0.3 g, 1 mmol) and DIEA (0.7 mL, 4 mmol) were placed in anhydrous DMF (2 mL) under argon. Propyl-1-chloroethyl carbonate (3 mmol) was then added and the suspension was stirred at 50 ° C. for 20 hours. DMF was removed on a rotary evaporator. The reaction was then partitioned between CH ₂ Cl ₂ and water. The CH ₂ Cl ₂ layer was dried over magnesium sulfate, filtered and concentrated on a rotary evaporator. The residue was taken up in methylene chloride and applied to a silica gel column (25 g, SiO ₂ ). This was eluted with 100 mL of CH ₂ Cl ₂ , 3, 6, 9, 12, 15, 18% (v / v) isopropanol in methylene chloride, 50 mL, then 21%, 200 mL isopropanol in methylene chloride, respectively. Appropriate fractions were pooled and evaporated to give the desired product.

Example 6
Synthesis of chloromethyl isopropyl carbonate To a cold solution (about 10 ° C.) of chloromethyl chloroformate (65 mL) in diethyl ether (1.4 L) was added isopropanol (56 mL), followed by dropwise addition of pyridine (60 mL). After the addition, the cold bath was removed and the reaction mixture was stirred for 18 hours. The reaction mixture was poured into a separatory funnel containing cold water (100 mL). The ether layer was separated and washed with water (100 mL × 2) and then dried over Na ₂ SO ₄ . Chloromethyl isopropyl carbonate (107 g, 95%) was worked up by evaporation of the solvent. Chloromethyl isobutyl carbonate, chloromethyl neopentyl carbonate, chloromethyl tert butyl carbonate and chloromethyl 3-pentyl carbonate are synthesized in a similar manner.

Example 7
Synthesis of bisisopropyloxymethyl carbonate of PMPA
To a stirred suspension of PMPA (7.26 g, 0.026 mmol) in DMF (100 mL) at 50 ° C. was added Et ₃ N (10.8 mL, 0.0778 mmol). The reaction mixture became homogeneous and chloromethyl isopropyl carbonate (12.1 g, 0.0778 mmol) was added to the reaction mixture and stirring was continued at 50 ° C. (oil bath temperature) for 20 hours. The solvent was removed under reduced pressure and the crude product was chromatographed on a silica gel column. Elution with 10% isopropanol in CH ₂ Cl ₂ removed all non-polar impurities. Further same elution with the same solvent mixture gave 1.3 g (about 10%) of the prodrug.

Example 8
Synthesis of bisisopropyloxymethyl carbonate of PMPA
To a stirred suspension of PMPA (1 g, 3.57 mmol) in DMF (5 mL), Et ₃ N (1.5 mL, 10.71 mmol) is added and chloromethyl isopropyl carbonate (1.67 g, 10.71 mmol) is added to the reaction mixture. Added. The reaction mixture was then diluted with ethyl acetate (100 mL) and filtered. The filtrate was washed with water (2 × 50 mL) and finally with brine (10 mL). The obtained crude product from which the solvent had been removed was dried under vacuum. The resulting oil was dissolved in isopropanol (7 mL) and citric acid (260 mg) was added. The mixture was stirred for 16 hours at room temperature and then cooled to 0 ° C. The product was crystallized and the crystals were filtered and dried. Mp 76-81 ° C.

Example 9
Preparation of chloromethyl carbamate

  A solution of amine (24 mmol), DIEA (30 mmol), and DMAP (5 mmol) in methylene chloride (5 mL) was added to a cold (0 ° C.) solution of chloromethyl chloroformate (25 mmol) in methylene chloride (45 mL). Added dropwise over a period of minutes. The solution was warmed to room temperature over 1.5 hours. The solution was diluted in ethyl acetate (100 mL) and washed with saturated sodium bicarbonate, 1M HCl, and saturated sodium chloride. This was then dried over magnesium sulfate, filtered and evaporated to give the desired chloromethyl carbamate.

Example 10
Synthesis of bismorpholinooxymethyl carbamate of PMPA

Anhydrous PMPA (0.3 g, 1 mmol) and DIEA (1 mL, 6 mmol) were placed in anhydrous DMF (2 mL). Chloromethylmorpholinocarbamate (3 mmol) was then added and the suspension was then stirred at room temperature for 3 days. The reaction was partitioned between CH ₂ Cl ₂ / isopropanol and 0.1 M citrate buffer (pH 6). The CH ₂ Cl ₂ layer was washed with water, dried over magnesium sulfate, filtered and concentrated on a rotary evaporator. The residue was taken up in methylene chloride and applied to a silica gel column (5 g, SiO ₂ ). This is 0, 3, 6, 9, 12,
Elution was performed with 25 mL of isopropanol in 15,18% (v / v) methylene chloride, followed by 21%, 100 mL of isopropanol in methylene chloride. Appropriate fractions were pooled and evaporated to give the desired product.

Example 11
Synthesis of bispiperidinooxymethyl carbamate of PMPA

Anhydrous PMPA (0.3 g, 1 mmol) and DIEA (0.7 mL, 4 mmol) were placed in anhydrous DMF (2 mL). Chloromethylpiperidinocarbamate (3 mmol) was then added and the suspension was then stirred at room temperature for 3 days. Additional DIEA (4 mmol) and chloromethylpiperidinocarbamate (100 μL) were added and the reaction was stirred for 27 hours. The reaction was partitioned between CH ₂ Cl ₂ / isopropanol and 0.1 M citrate buffer (pH 6). The CH ₂ Cl ₂ layer was dried over magnesium sulfate, filtered and concentrated on a rotary evaporator. The residue was taken up in methylene chloride and applied to a silica gel column (5 g, SiO ₂ ). This is 0 for each
Elute with 25 mL of isopropanol in 3, 6, 9, 12, 15, 18% (v / v) methylene chloride, followed by 21%, 100 mL of isopropanol in methylene chloride. Appropriate fractions were pooled and evaporated to give the desired product.

Example 12
Other carbamate intermediates
To a solution of chloromethyl chloroformate (4.16 mL) in CH ₂ Cl ₂ (30 mL) was added tertbutylamine (4.9 mL) and proton sponge (10 g). The reaction mixture was stirred for 18 hours, then it was poured into a separatory funnel containing cold 0.5N HCl (100 mL). The CH ₂ Cl ₂ layer was separated and washed with water (100 mL × 2) and then dried over Na ₂ SO ₄ . Solvent evaporation gave chloromethyl tertbutylcarbamate (8 g). Chloromethyl n-butylcarbamate (R = n-butyl) and chloromethyldimethylcarbamate (R = Me) were prepared in the same manner.

Example 13
Other oxymethylalkyl carbamate prodrugs of PMPA
To a stirred suspension of PMPA (4.51 g, 0.016 mmol) in DMF (50 mL) was added Et ₃ N (6.7 mL, 0.048 mmol). The reaction mixture became homogeneous and chloromethyl tertbutyl carbamate (8 g, 0.048 mmol) was added to the reaction mixture and stirring was continued at room temperature for 3 days. The solvent was removed under reduced pressure and the crude product was chromatographed on a silica gel column. Elution with 10% isopropanol in CH ₂ Cl ₂ removed all less polar impurities. Further identical elution with the same solvent mixture gave 1.25 g of prodrug. n-Butyl and methyl carbamate were prepared in the same manner from the intermediates of the previous examples.

Example 14
Chemical stability of PMPA carbonate
PMPA carbonate solution stability at pH 7.4 at 37 ° C with 10 mM buffer (NaH ₂ PO ₄ and Na ₂ HPO
_{In 4} ), the total ionic strength was adjusted to 0.15M with KCl. The assay was performed at 37 ° C. by adding 200 μL of PMPA carbonate stock solution (about 1 mg / mL in DMSO) to 10 mL of preequilibrated buffer. Samples were removed at specific time points and analyzed by HPLC. Chemical t1 / 2 is expressed as the number of hours required to hydrolyze 50% of the carbonate at a specific pH.

Example 15
Oral bioavailability of PMPA and PMPA carbonate in beagle dogs PMPA (9-[(R) -2- (phosphonomethoxy) propyl] adenine) and PMPA carbonate were tested to determine the pharmacokinetics of PMPA in beagle dogs. The effect of dose was determined, in particular the bioavailability of PMPA after oral administration to beagle dogs.

  PMPA monohydrate was synthesized by Gilead Sciences. Tetrabutylammonium hydrogen phosphate (TBAHP) was obtained from Fluka (Ronkonkoma, NY). Acetonitrile was obtained from Baxter (Muskegon, MI). Dibasic potassium phosphate, monobasic potassium phosphate, and sodium acetate trihydrate were obtained from Mallinckrodt (Paris, KY). Chloroacetaldehyde and trifluoroacetic acid (TFA) were obtained from Aldrich (Milwaukee, WI).

The intravenous formulation used as a standard was an isotonic aqueous solution containing 50 mg / mL PMPA. The compound was adjusted to pH 7.4 by adding 10 mL of WFI (water for injection from Abbott Laboratory) and 1N NaOH. The solution was diluted to 15 mL with WFI and sterile filtered using a 0.2 μm filter. The PMPA dose was 10 mg / kg (0.2 mL / kg).

  An intravenous formulation for the 1 mg / kg dose was prepared as described above except that only 75 mg of PMPA was added to WFI and the final concentration was 5 mg / mL. The dose was 1 mg / kg (0.2 mL / kg). An oral formulation of carbonate was prepared in 20-40% PEG 400/50 mM citric acid and adjusted to pH 2.2. The dose is 6.2-10 mg equivalent of PMPA / kg and is shown in Table 1.

Two groups of 5 adult male beagle dogs were used. The average body weight at the first dose was 9.6 ± O.4 kg (range 9.2 to 10.2). Dogs were fasted 12-18 hours before dose and 6 hours after dose. For pre-treatment with pentagastrin, the dog was treated with pentagastrin (Peptavlon 0.25
6 mg / mL, Ayerst Laboratories, Inc., Philadelphia, PA)
μg / kg given 20 minutes before dosing. Water was given freely.

  Each formulation was administered at a single dose to 5 male Beagle dogs. Individual vials of each formulation were given to each animal. Intravenous formulations were administered from the blood vessels in the head. The oral suspension was administered using gavage and then washed twice with 10 mL water. At least a one week washout period was allowed between doses.

Blood samples (4.0 mL) were collected from each animal into heparinized tubes by the direct carotid route. The animals remained conscious during the sample collection period. The blood was immediately processed for plasma by centrifugation at 2000 rpm for 10 minutes. Plasma samples were frozen and kept at ≦ −20 ° C. until analysis.

  Urine samples were collected for periods of 0-24 and 24-48 hours. Urine samples from 0-24 and 24-48 hours are divided into aliquots, mixed and analyzed based on collected volume, and the amount of PMPA recovered from urine between 0-48 hours It was determined.

PMPA in plasma and urine was measured as follows. PMEA (9- (2-phosphono-methoxyethyl) adenine; adefovir) was used as an internal standard for both analyses. The total concentration of PMPA in canine plasma or urine samples was determined by derivatizing PMPA and PMEA with chloroacetaldehyde to obtain highly fluorescent N ¹ _, N ⁶ -ethenoadenine derivatives as described ( Russell, J. et al. (1991) Determination of 9-[(2-phophonylmethoxy) -ethyl] Adenine in Rat Urine by High-Performance Liquid Chromatography with Fluorescence Detection.J Chromatogr. (Netherlands), 572,
321-326.).

  Sample extraction for PMPA in plasma and urine was performed as follows. Plasma (200 μL) and internal standard (20 μL, 10 μg / mL PMEA, giving a final PMEA concentration of 1 μg / mL) were mixed with 400 μL acetonitrile containing 0.1% TFA to precipitate the protein. The sample was then evaporated at room temperature and dried under reduced pressure (Savant SpeedVac). Urine samples (20 μL) and internal standards (30 μL, 10 μg / mL PMEA, giving a final PMEA concentration of 1.5 μg / mL) were used directly for derivatization without drying.

Samples were derivatized as follows for analysis. Dry plasma or urine samples are reconstituted or mixed in 200 μL derivatization cocktail (0.34% chloroacetaldehyde, pH 4.5 in 100 mM sodium acetate), stirred and Eppendorf Centrifuge for 10 minutes at 14,000 rpm Centrifuge in 5402. The supernatant was then transferred to a clean screw-capped test tube and incubated at 95 ° C. for 40 minutes. The derivatized sample was quenched on ice and evaporated to dryness at room temperature under reduced pressure. The dried sample was reconstituted in 200 μL mobile phase A (see below), stirred, and centrifuged in an Eppendorf Centrifuge 5402 at 14,000 rpm for 10 minutes. The supernatant was then transferred to an automatic injection vial for HPLC analysis.

  Plasma and urine samples were analyzed by HPLC with Fluorescence Detection. The HPLC system was a Model P4000 solvent delivery system (Thermo Separation. San Jose, Calif.) Equipped with a Model AS3000 auto-injector and a Model F2000 fluorescence detector. The column is a Zorbax RX-C18 (5 μm, 150 × 4.6 mm) with a Brownlee RP-18 Newguard guard column (7 μm, 15 × 3.2 mm) (Alltech, Deerfield. IL) (MAC-MOD. Chadds Ford, NY) Met. The mobile phases used were: A, 2% acetonitrile and 5 mM TBAHP in 25 mM potassium phosphate buffer, pH 6.0; B, 65% acetonitrile and 5 mM TBAHP in 25 mM potassium phosphate buffer, pH 6.0. The flow rate was 1.5 mL / min and the column temperature was maintained at 35 ° C. by the column oven. The gradient profile returns to 100% A 2.0 minutes, then linear gradient to 100% B, 13.0 minutes immediately back to 100% A. Detection was by fluorescence excited at 236 nm and emitting at 420 nm, and the injection volume was 50 μL. The total cycle time between infusions was 25 minutes. Data were acquired and stored with the Peak Pro data acquisition system (Beckman, Palo Alto, CA).

  Pharmacokinetic parameters for intravenous and oral formulations of PMPA and PMPA carbonate were evaluated using a non-compartmental method. Intravenous data was analyzed using PCNONLIN Model 201 (5). Oral data was analyzed using Model 200. Additional pharmacokinetic parameters are calculated as follows: CL = dose / AUC (O-∞); where CL is the total plasma clearance. Vss = CL × MRT; where Vss is the apparent dispensing volume at steady state. MRT is the average residence time.

Initial plasma concentration (Co) was determined by extrapolation of log transformed data to zero time. Bioavailability is expressed as follows:

Urine collection is represented by:

  Comparison of oral bioavailability of t-Bu, 3-pentyl, isopropyl, Et carbonate parameters by one-sided t-test (StatView (R) Version 4.0, Software for the Statistical Analysis. Abacus Concepts, Inc., Berkeley, CA) did. A P value of ≦ 0.05 was considered significant.

Biological t1 / 2: Dog livers were freshly obtained from Pharmakon USA (Waverly, PA). Liver homogenate was prepared according to the following standard protocol. Dog liver was rinsed 3 times with ice cold 50 mM sodium phosphate / potassium buffer and homogenized with Tekmar Tissumizer homogenizer (VWR 33995-060). The homogenate was centrifuged at 9000 g for 20 minutes at 4 ° C. (11,000 rpm, Eppendorf Centrifuge 5402; Brinkmann Instruments, Westbury, NY). The supernatant is called the S9 fraction. The protein concentration in the S9 fraction was determined using the Bio-Rad Protein Assay Kit II using bovine serum albumin as a standard. Esterase activity was determined using o-nitrophenyl butyrate as a substrate, and activity was calculated based on the increase in absorbance at 420 nm after 1 minute incubation. The homogenate was stored as -1.0 mL aliquots at -70 ° C.

Intestinal homogenate: Canine intestinal segment (jejunum / ileum), Pharmakon USA (Waverly
, PA) and intestinal homogenates were prepared as described for the liver. Intestinal homogenate was stored as -1.0 mL aliquots at -70 ° C.

  Human intestinal homogenate (S9) was obtained from Keystone Skin Bank (Exton, PA) at a concentration of 20 mg protein / ml.

  Study design: Enzymatic stability studies on plasma and intestinal homogenates were performed using 90% biological fluids.

Stability measurement: One blank (no drug) incubation was performed for each biological fluid. All biological fluid test tubes (open) were pre-incubated without PMPA prodrug in a shaking bath at 37 ° C. and 100 vibrations / minute for 5 minutes. PMPA prodrug was added to the test incubation (final concentration: 20 μg / mL), mixed and maintained at 37 ° C. with 100 vibrations / minute. Samples (50 μL) were withdrawn at 0, 30, and 60 minutes and the reaction was quenched with 100 μL of 0.1% trifluoroacetic acid (TFA) in acetonitrile. The quenched sample was centrifuged for 5 minutes at 14,000 rpm in an Eppendorf Centrifuge 5402, and the supernatant was used for HPLC analysis.

Calculation: For each incubation, the rate constant observed for degradation was calculated by plotting the log of the peak area of the PMPA prodrug versus the time of incubation (minutes). The slope was observed as a rate constant (k _obs ). The half-life was calculated by the following equation:

When the observed rate constant for decomposition was less than 0.01 min- ¹ , t1 / 2 was expressed as stable.

  The results of the beagle study are shown in Table 1 below.

Example 16
Antiviral activity of PMPA and PMPA carbonate in tissue culture
PMPA (9-[(R) -2- (phosphonomethoxy) propyl] adenine) and PMPA carbonate were tested to determine their activity against HIV-1. The antiviral activity of carbonates 5a, 5c-g against HIV-1 (IIIB) was determined in MT-2 cells, and IC ₅₀ (50% inhibitory concentration) and CC ₅₀ (concentration killing 50% cells) values. It was measured. Carbonate prodrugs showed increased potency (about 2.5-500 times) compared to PMPA (Table 2). The cytotoxicity of the prodrug was also increased, but the selection index was also improved compared to PMPA. Increased activity can result in increased cellular uptake of the prodrug, followed by intracellular conversion to effective PMPA. PMPA undergoes subsequent phosphorylation and becomes an antiviral active diphosphate metabolite. t-Butyl carbonate 5d showed only a 2.5-fold increase in activity over PMPA, with reduced selectivity, presumably due to chemical instability. Antiviral activity data showed good permeation of alkyl methyl carbonate prodrugs into cells. This is probably due to their increased lipophilicity. The partition coefficient value supports this hypothesis, and all prodrugs are more lipophilic (logP = 0.6-3.2) compared to PMPA (logP = −2.5).

Table 2. Antiretroviral activity of PMPA and PMPA prodrugs against HIV-1

^a IC ₅₀ -50% inhibitory concentration; ^b CC ₅₀ -50% cell killing concentration; ^c SI-selected reading (CC ₅₀ / IC ₅₀ ); nd-not determined; DMSO was used as a control.

Claims (1)

Invention described in the specification.