KR19980701552A - THERAPEUTIC COMPOUNDS-FATTY ACID CONJUGATES - Google Patents

Abstract

The present invention relates to a series of therapeutic compounds conjugated to one to three fatty acid-derived acyl groups. Therapeutic compounds of the present invention include 1) a corticosterone drug; 2) opiates and opioid antagonists; 3) antiviral nucleosides such as AZT; 4) cyclosporine and related cyclopeptides; 5) folate antagonists including methotrexate, folic acid and folic acid analogues; 6) DOPA, catecholamine precursors such as dopamine, and catecholamines such as adrenaline, noradrenaline, its derivatives; And 7) a carboxylic acid containing alkylating agent such as chlorambucil, mepholan. The therapeutic compound of the present invention is conjugated to the fatty acid by a linking group containing a carboxylic acid such as tromethamine or an ethanolamine derivative. In particular, the present invention relates to altering the pharmacokinetic profile and delivery mode of these compounds by conjugating these therapeutic compounds with one, two, or three acyl derivatives of fatty acids.

Description

Therapeutic compound-fatty acid conjugate

The present invention relates to therapeutic compounds conjugated to one to three fatty acid-derived acyl groups. The therapeutic compounds of the present invention can be used in the following groups:

1. Corticosterone drugs;

2. opiates and opioid antagonists;

3. antiviral nucleosides such as AZT;

4. Cyclosporine and related cyclopeptides;

5. Folate antagonists, folic acid and folic acid analogues, including methotrexate;

6. DOPA, catecholamine precursors such as dopamine, and catecholamines such as adrenaline, noradrenaline and derivatives thereof; And

7. Chloramvsil and a carboxylic acid containing alkylating agent such as mepholan.

In particular, the present invention relates to altering the pharmacokinetic profile and delivery mode of these compounds by conjugating these therapeutic compounds with an acyl derivative of one to three fatty acids.

Background Art and Detailed Description of the Invention

1. Corticosterone drug

Corticosterone-based drugs based on naturally occurring hormones produced in the adrenal cortex are among the most commonly used treatments. There are two main groups of corticosteroid hormones with overlapping activities.

Glucocorticoids - Normal biological effects include carbohydrate

Metabolic regulation, and has a high level of anti-inflammatory activity.

Mineral corticoids - are associated with water and mineral metabolism.

Corticosterone is based on cholesterol molecules, both natural and synthetic, and generally has the following common structural features.

a) Hydroxyacetyl at position 17 (-CO-CH ₂ OH)

b) a ketone group (= O) at the 3-position,

c) a double bond between the 4 and 5 molecules

Except for the hydroxyl residue of hydroxyl acetyl at position 17 (hydroxyl at position 21), such as, for example, hydrocortisone acetate, this group is generally not modified in the active analog of the hormone.

An example of a glucocorticoid is hydrocortisone (cortisol or 17 hydroxycorticosterone)

An example of an inorganic corticode is aldosterone.

One embodiment of the invention is particularly concerned with the anti-inflammatory action (both natural hormones and synthetic drugs) of (but not limited to) the following glucocorticoids.

Cortisone

Hydrocortisone

Flooded cortisone

Prednisone

Prednisolone

Methylprednisolone

Triamcinolone

Dexamethasone

Betamethasone

Parameter hand

Fluorocinolone

The present inventors have found that the components of this series can be linked to one to three kinds of fatty acid acyl derivatives. These novel conjugated compounds are believed to be improved over unbonded therapeutic agents. In addition, these new compounds are believed to aid in the delivery of these drugs, such as oral, dermal penetration, intraarticular, intranasal, and / or guided routes.

Therefore, the first embodiment of the present invention is a compound represented by the following formula.

[Wherein, in the above formula

X is a corticosterone hormone or drug component, connected to Y through a hydroxy group,

Y is a spacer group,

AA is an amino acid, n is 0 to 5,

B is H or CH ₂ OR ₃ ,

R ₁ , R ₂ and R ₃ are the same or different and each is an acyl group derived from hydrogen, methyl, ethyl or fatty acid, and at least one of R ₁ , R ₂ and R ₃ is an acyl group derived from a fatty acid.

A second embodiment of the present invention is a compound represented by the following formula.

X - Y [AA] n - NH - CH 2 - CH 2 O - R 4

[Wherein, in the above formula

X is a corticosterone hormone or drug component, connected to Y through a hydroxy group,

Y is a spacer group,

AA is an amino acid, n is 0 to 5,

R ₄ is an acyl group derived from a fatty acid.]

A third embodiment of the invention is a method for sustaining or altering the activity of a corticosterone hormone or drug component comprising administering a compound of the general formula:

[Wherein, in the above formula

X is a corticosterone hormone or drug component, connected to Y through a hydroxy group,

Y is a linking group,

AA is an amino acid, n is 0 to 5,

B is H or CH ₂ OR ₃ ,

R ₁ , R ₂ and R ₃ are the same or different and each is an acyl group derived from hydrogen, methyl, ethyl or fatty acid, and at least one of R ₁ , R ₂ and R ₃ is an acyl group derived from a fatty acid.

A fourth embodiment of the present invention is a method for sustaining or modifying the activity of a corticosterone hormone or drug component by administering a compound of the general formula:

X - Y - [AA] n - NH - CH 2 - CH 2 O - R 4

[Wherein, in the above formula

X is a corticosterone hormone or drug moiety and is linked to Y through a hydroxy group,

Y is a spacer group,

AA is an amino acid, n is 0 to 5,

R ₄ is an acyl group derived from a fatty acid.]

Fatty acids may be saturated or unsaturated.

As noted, X is connected to connector Y through a hydroxy group.

Typically, such hydroxy groups are located at 17 or 21, but may be at other positions such as 16.

A linking group Y useful for linking a compound having a hydroxy group in the present invention to an amino group of Tris (when B is CH ₂ OR ₃ ) or between amino acids [AA, if present]

a) a linking group having a carboxyl group connected to the compound and having a carboxyl group connected to Tris (or an amino acid, if present) such as dicarboxylic acid through an anhydride such as succinic anhydride or maleic anhydride;

b) a linking group having a carboxyl group connected to the compound and having an aldehyde group connected to Tris (or amino acid, if present) such as glyoxylic acid (for example, in the presence of a reducing agent such as NaBH ₄ )

c) a linking group having a carboxy group linked to the compound and having a halide group linked to Tris (or amino acid, if present) such as chloroacetic acid

d) a linking group having a carboxy group linked to the compound and having an N = C = O group in which ethyl is linked to Tris (or amino acid, if present) such as socyanatoacetate.

X may be any component of a corticosterone compound, but currently X is preferably hydrocortisone or cortisone.

In another preferred embodiment of the present invention, Y is dicarboxylic acid, AA is absent or glycine or alanine, and the linkage is through the hydroxyl group at position 21.

As will be appreciated, R ₁ , R ₂ and R ₃ are either hydrogen or an acyl group of a fatty acid. It is also apparent to those skilled in the art that substituents other than methyl and ethyl are possible for R ₁ , R ₂ and R ₃ . It is the first essential condition that at least one of R ₁ , R ₂ and R ₃ must be an acyl group derived from a fatty acid.

When R ₁ , R ₂ and R ₃ are each an acyl group of a fatty acid, they are preferably the same group. It is also preferred that the acyl group of the fatty acid has a chain of 3 to 18 carbon atoms, more preferably 10 to 18.

Those skilled in the art will appreciate that the hydroxy groups located at various sites of the molecule can be modified into several components of other classes of steroid (or analog) hormones, such as male and female hormones.

The present invention also provides a therapeutic composition comprising the first or second embodiment of the present invention and a pharmaceutically acceptable carrier. The composition may also comprise a non-conjugated component of a corticosterone hormone or drug.

The therapeutic compositions may be administered by any suitable route known to those skilled in the art. Such routes include skin permeation, intraarticular, oral, intranasal, and guideway routes.

2. Opioids and opioid antagonists

Morphine is a classic example of a subclinical analgesic that acts on the CNS receptors of naturally occurring opiate peptides, enkephalin and endorphins similar to these. It is a powerful addictive drug used to relieve severe pain from severe pain associated with conditions such as heart attack, cancer, kidney stones or post-operative pain (colic) due to gallstones. This drug has a short biological half-life and is normally delivered orally or by injection. Related opiate analgesics or antagonists include hydro morphine, oxymorphin, levorphanol, levallopan, codeine, nalmefene, nalofine, naloxone, naltrexone, buprenorphine, butorphanol and nalbuphine.

Morphine has the following structure.

When the hydroxy group at the 3 or 6 position is transformed into a lipophilic group, the absorption and partitioning ratio of morphine changes to CNS in particular.

The present inventors have found that morphine and related opiate analgesics or antagonists (morphine based) can be ester linked to one to three acyl derivatives of fatty acids via a spacer at the 3-position hydroxy. These new conjugate mixtures appear to be better than unbonded therapeutic agents. It is also believed that similar linkages are possible through the hydroxyl group at the 6-position.

Therefore, a fifth embodiment of the present invention is a compound represented by the following formula.

[Wherein, in the above formula

X is a morphinic component and is connected to Y via a hydroxy group (e.g., a hydroxy group at the 3 or 6 position)

Y is a spacer group,

AA is an amino acid, n is 0 to 5,

B is H or CH ₂ OR ₃ ,

R ₁ , R ₂ and R ₃ are the same or different and each is an acyl group derived from hydrogen, methyl, ethyl or fatty acid, and at least one of R ₁ , R ₂ and R ₃ is an acyl group derived from a fatty acid.

A sixth embodiment of the present invention is a compound represented by the following formula:

X - Y - [AA] n - NH - CH 2 - CH 2 O - R 4

[Wherein, in the above formula

X is a morphine-based component and is connected to Y through a hydroxyl group at the 3- or 6-position,

Y is a spacer group,

AA is an amino acid, n is 0 to 5,

R ₄ is an acyl group derived from a fatty acid.]

A seventh embodiment of the present invention is a method of continuing or changing the activity of a morphine compound by administering a compound represented by the following general formula.

[Wherein, in the above formula

X is a morphine-based component and is connected to Y through a hydroxyl group at the 3- or 6-position,

Y is a linking group,

AA is an amino acid, n is 0 to 5,

B is H or CH ₂ OR ₃ ,

R ₁ , R ₂ and R ₃ are the same or different and each is an acyl group derived from hydrogen, methyl, ethyl or fatty acid, and at least one of R ₁ , R ₂ and R ₃ is an acyl group derived from a fatty acid.

An eighth embodiment of the present invention is a method of continuing or changing the activity of a morphine compound by administering a compound of the following formula:

X - Y - [AA] n - NH - CH 2 - CH 2 O - R 4

[Wherein, in the above formula

X is a morphine-based component and is connected to Y through a hydroxyl group at the 3- or 6-position,

Y is a spacer group,

AA is an amino acid, n is 0 to 5,

R ₄ is an acyl group derived from a fatty acid.]

Fatty acids may be saturated or unsaturated.

A linking group Y useful for linking a compound having a hydroxy group in the present invention to an amino group of Tris (when B is CH ₂ OR ₃ ) or between amino acids [AA, if present]

a) a linking group having a carboxyl group connected to the compound and having a carboxyl group connected to Tris (or an amino acid, if present) such as dicarboxylic acid through an anhydride such as succinic anhydride or maleic anhydride;

b) a linking group having a carboxyl group connected to the compound and having an aldehyde group connected to Tris (or amino acid, if present) such as glyoxylic acid (for example, in the presence of a reducing agent such as NaBH ₄ )

c) a linking group having a carboxy group linked to the compound and having a halide group linked to Tris (or amino acid, if present) such as chloroacetic acid

d) a linking group having a carboxy group linked to the compound and having an N = C = O group in which ethyl is linked to Tris (or amino acid, if present) such as socyanatoacetate.

In a preferred embodiment of the present invention, X is morphine in the 3 or 6 position, Y is dicarboxylic acid, AA is absent, or glycine or alanine.

As described above, R ₁ , R ₂ and R ₃ are preferably hydrogen or an acyl group of a fatty acid. It is also apparent to those skilled in the art that substituents other than methyl and ethyl are possible for R ₁ , R ₂ and R ₃ . It is the first essential condition that at least one of R ₁ , R ₂ and R ₃ must be an acyl group derived from a fatty acid.

When R ₁ , R ₂ and R ₃ are each an acyl group of a fatty acid, they are preferably the same group. It is also preferred that the acyl group of the fatty acid has a chain of 3 to 18 carbon atoms, more preferably 10 to 18.

The present invention also provides a therapeutic composition comprising the fifth or sixth embodiment of the present invention and a pharmaceutically acceptable carrier. The composition may also comprise unbonded morphine-based components.

The present therapeutic compositions may be administered by any suitable route acceptable to those skilled in the art. Such routes include skin permeation, intraarticular, oral, intranasal, and guideway routes.

3. Antiviral nucleosides

As described above in one embodiment of the present invention, the present invention provides therapeutic conjugates of AZT (azidinium or zidovudine) and other antiviral nucleosides (e.g., acyclovir, gancyclovir, And includes antiviral agents bound to one to three acyl groups via linking group (s), and includes the use of such compounds, including, but not limited to, . In particular, the present invention relates to the pharmacokinetics and / or alteration of modes of delivery and targeting of such drugs when binding to one to three fatty acid acyl derivatives.

AZT is an example of an antiretroviral drug. The drug is active against human immunodeficiency virus (HIV) and other mammalian retroviruses. This drug is a thymidine analog that is converted to a triphosphate derivative by normal cellular enzymes. In this form, the drug inhibits viral reverse transcription (synthesis of RNA-dependent DNA). The DNA chain is terminated by the binding of the modified thymidine. Because AZT is widely prescribed for AIDS and has a short biological half-life, it should be administered every four days. The use of this drug involves many side effects, ranging from zone to inhibition of the production of new blood cells, and is related to conditions.

AZT has the following structure.

The present inventors have found that AZT and similar drugs (hereinafter referred to as antiviral nucleoside analogues) can be bound to one to three fatty acid acyl derivatives. These new compounds are believed to improve delivery, ingestion, half-life and targeting of the drug within the cell after oral, intranasal, skin permeation, guidance and other modes of transport. In addition, the distribution of drug within the body can be altered to increase the percentage of drug delivered to lymphocytes in the CNS and lymphatic system.

Therefore, a ninth embodiment of the present invention is a compound represented by the following formula:

[Wherein, in the above formula

X is an antiviral nucleoside, connected to Y via a hydroxy group,

Y is a spacer group,

AA is an amino acid, n is 0 to 5,

B is H or CH ₂ OR ₃ ,

R ₁ , R ₂ and R ₃ are the same or different and each is an acyl group derived from hydrogen, methyl, ethyl or fatty acid, and at least one of R ₁ , R ₂ and R ₃ is an acyl group derived from a fatty acid.

A tenth embodiment of the present invention is a compound represented by the following formula.

X - Y - [AA] n - NH - CH 2 - CH 2 O - R 4

[Wherein, in the above formula

X is an antiviral nucleoside, connected to Y via a hydroxy group,

Y is a spacer group,

AA is an amino acid, n is 0 to 5,

R ₄ is an acyl group derived from a fatty acid.]

An eleventh embodiment of the present invention is a method for sustaining or altering the activity of an antiviral nucleoside by administering an antiviral nucleoside of the general formula:

[Wherein, in the above formula

X is an antiviral nucleoside, connected to Y via a hydroxy group,

Y is a linking group,

AA is an amino acid, n is 0 to 5,

B is H or CH ₂ OR ₃ ,

R ₁ , R ₂ and R ₃ are the same or different and each is an acyl group derived from hydrogen, methyl, ethyl or fatty acid, and at least one of R ₁ , R ₂ and R ₃ is an acyl group derived from a fatty acid.

A twelfth embodiment of the present invention is a method for maintaining or changing the activity of an antiviral nucleoside by administering an antiviral nucleoside represented by the following general formula.

X - Y - [AA] n - NH - CH 2 - CH 2 O - R 4

[Wherein, in the above formula

X is an antiviral nucleoside, connected to Y via a hydroxy group,

Y is a spacer group,

AA is an amino acid, n is 0 to 5,

R ₄ is an acyl group derived from a fatty acid.]

Fatty acids may be saturated or unsaturated.

A linking group Y useful for linking a compound having a hydroxy group in the present invention to an amino group of Tris (when B is CH ₂ OR ₃ ) or between amino acids [AA, if present]

a) a linking group having a carboxyl group connected to the compound and having a carboxyl group connected to Tris (or an amino acid, if present) such as dicarboxylic acid through an anhydride such as succinic anhydride or maleic anhydride;

b) a linking group having a carboxyl group connected to the compound and having an aldehyde group connected to Tris (or amino acid, if present) such as glyoxylic acid (for example, in the presence of a reducing agent such as NaBH4)

c) a linking group having a carboxy group linked to the compound and having a halide group linked to Tris (or amino acid, if present) such as chloroacetic acid

d) a linking group having a carboxy group linked to the compound and having an N = C = O group in which ethyl is linked to Tris (or amino acid, if present) such as socyanatoacetate.

In a preferred embodiment of the present invention, X is preferably AZT, acyclovir, gancyclovir, vidarabine, idoxuridine, tripyridine, ddI, ddC, ddA or ribavirin. It is also preferred that Y is dicarboxylic acid, AA is absent, or glycine or alanine.

As described above, R ₁ , R ₂ and R ₃ are preferably hydrogen or an acyl group of a fatty acid. It is also apparent to those skilled in the art that substituents other than methyl and ethyl are possible for R ₁ , R ₂ and R ₃ . It is the first essential condition that at least one of R ₁ , R ₂ and R ₃ must be an acyl group derived from a fatty acid.

When R ₁ , R ₂ and R ₃ are each an acyl group of a fatty acid, they are preferably the same group. It is also preferred that the acyl group of the fatty acid has a chain of 3 to 18 carbon atoms, more preferably 10 to 18.

The present invention also provides a therapeutic composition comprising the ninth or tenth embodiment of the present invention and a pharmaceutically acceptable carrier. The composition may also comprise unconjugated antiviral nucleosides.

The present therapeutic compositions may be administered by any suitable route acceptable to those skilled in the art. Such routes include oral, intranasal, skin permeation, and routes to guide routes.

4. Cyclosporine and related cyclopeptides

Cyclosporine is a closely related cyclic peptide system that exhibits potent immunosuppressive activity. Cyclosporine is used extensively (often in combination with glucocorticoids such as prednisolone) to prevent rejection during organ transplantation. Cyclosporine reversibly inhibits the production of interleukin and interferon in helper T lymphocytes and / or acts to inhibit interleukins from binding to the receptor of killer T lymphocytes, and is useful for cell mediated It is believed to reduce the response. In animal studies, cyclosporin has been shown to inhibit a range of immune responses including delayed skin hypersensitivity, Freund ' s adjuvant induced arthritis and T cell dependent antibody production, and can be used in a wider range of applications than in recent applications For example, topical application of cyclophosphine is useful for the treatment of psoriasis and / or arthritis.

The structure of the cyclosporin system is shown below as the other cyclosporins A, B, C, D and G only in the side chain R. [

The present inventors propose that the components of this series can be bound to one to three kinds of fatty acid acyl derivatives. This can be done by linking to an unchanging hydroxy group or through a linking of the variable R, for example, cyclosporin C has a threonine side chain that can be used as a link to R. These novel compounds are believed to improve the delivery, ingestion, half-life and / or delivery mode of the cyclosporine drug. In addition, these novel compounds are believed to facilitate delivery of such drugs by oral, skin permeation, intranasal, parenteral, and / or guiding, by facilitating transport through lipophilic membranes.

Therefore, a thirteenth embodiment of the present invention is a compound represented by the following formula:

[Wherein, in the above formula

X is a component of a cyclosporine drug, which is linked to Y via a hydroxy group,

Y is a spacer group,

AA is an amino acid, n is 0 to 5,

B is H or CH ₂ OR ₃ ,

R ₁ , R ₂ and R ₃ are the same or different and each is an acyl group derived from hydrogen, methyl, ethyl or fatty acid, and at least one of R ₁ , R ₂ and R ₃ is an acyl group derived from a fatty acid.

A fourteenth embodiment of the present invention is a compound represented by the following formula:

X - Y - [AA] n - NH - CH 2 - CH 2 O - R 4

[Wherein, in the above formula

X is a component of a cyclosporine drug, which is linked to Y via a hydroxy group,

Y is a spacer group,

AA is an amino acid, n is 0 to 5,

R ₄ is an acyl group derived from a fatty acid.]

A fifteenth embodiment of the present invention is a method for administering a compound represented by the following general formula to maintain or modify the activity of a cyclosporin drug.

[Wherein, in the above formula

X is a component of a cyclosporine drug, which is linked to Y via a hydroxy group,

Y is a linking group,

AA is an amino acid, n is 0 to 5,

B is H or CH ₂ OR ₃ ,

R ₁ , R ₂ and R ₃ are the same or different and each is an acyl group derived from hydrogen, methyl, ethyl or fatty acid, and at least one of R ₁ , R ₂ and R ₃ is an acyl group derived from a fatty acid.

A sixteenth embodiment of the present invention is a method for administering a compound represented by the following general formula to maintain or modify the activity of a cyclosporine drug.

X - Y - [AA] n - NH - CH 2 - CH 2 O - R 4

[Wherein, in the above formula

X is a component of a cyclosporine drug, which is linked to Y via a hydroxy group,

Y is a spacer group,

AA is an amino acid, n is 0 to 5,

R ₄ is an acyl group derived from a fatty acid.]

When X is a component of a cyclosporine drug, it is linked to Y through the constant hydroxy moiety of this series or the hydroxy group of the threonine side chain of Cyclosporin C. Alternatively, new specific analogs can be generated with a series of reactive side chains in this position, whereas only via hydroxy.

Fatty acids may be saturated or unsaturated.

A linking group Y useful for linking a compound having a hydroxy group in the present invention to an amino group of Tris (when B is CH ₂ OR ₃ ) or between amino acids [AA, if present]

a) a linking group having a carboxyl group connected to the compound and having a carboxyl group connected to Tris (or an amino acid, if present) such as dicarboxylic acid through an anhydride such as succinic anhydride or maleic anhydride;

b) a linking group having a carboxyl group connected to the compound and having an aldehyde group connected to Tris (or amino acid, if present) such as glyoxylic acid (for example, in the presence of a reducing agent such as NaBH ₄ )

c) a linking group having a carboxy group linked to the compound and having a halide group linked to Tris (or amino acid, if present) such as chloroacetic acid

d) a linking group having a carboxy group linked to the compound and having an N = C = O group in which ethyl is linked to Tris (or amino acid, if present) such as socyanatoacetate.

X is preferably a cyclosporin-based component, and particularly preferably cyclosporin C. It is also preferred that Y is dicarboxylic acid, AA is absent or glycine or alanine.

As described above, R ₁ , R ₂ and R ₃ are preferably hydrogen or an acyl group of a fatty acid. It is also apparent to those skilled in the art that substituents other than methyl and ethyl are possible for R ₁ , R ₂ and R ₃ . It is the first essential condition that at least one of R ₁ , R ₂ and R ₃ must be an acyl group derived from a fatty acid.

When R ₁ , R ₂ and R ₃ are each an acyl group of a fatty acid, they are preferably the same group. It is also preferred that the acyl group of the fatty acid has a chain of 3 to 18 carbon atoms, more preferably 10 to 18.

The present invention also provides a therapeutic composition comprising a thirteenth or fourteenth embodiment of the present invention and a pharmaceutically acceptable carrier. The composition may also comprise components of the unconjugated cyclosporine drug or related cyclopeptides.

The present therapeutic compositions may be administered by any suitable route acceptable to those skilled in the art. Such routes include oral, skin permeation, intranasal, parenteral, and guideway routes.

5. Folate antagonists including methotrexate, folic acid and folic acid analogs

Methotrexate, an anti-metabolite, is an example of a folic acid antagonist drug. Because it acts as a competitive inhibitor of folate reductase, it inhibits the conversion of vitamin folate into its active form, polyphosphate, and reduces the proliferation of new cells. Methotrexate is prescribed for cancer therapy and is also used to reduce the proliferation of epithelial cells for the treatment of psoriasis that does not respond to other forms of treatment. Small doses of methotrexate are likely to be effective in inhibiting inflammatory cell responses, inhibiting the progression of rheumatoid arthritis, and relieving symptoms.

The inventors of the present invention have found that methotrexate-based components can bind to one to three kinds of fatty acid acyl derivatives. These new compounds are believed to improve delivery, ingestion, penetration into the joint, half-life and / or delivery mode and distribution to the CNS of these drugs. It is believed that these drugs are helpful in delivering orally, nasally, transdermally, intratumorally, parenterally, intraarticularly, and / or guided.

Methotrexate

Therefore, a seventeenth embodiment of the present invention is a compound represented by the following formula.

[Wherein, in the above formula

X is a folic acid antagonist component, which is linked to Y via a carboxy group,

Y is optionally a spacer group,

AA is an amino acid, n is 0 to 5,

B is H or CH ₂ OR ₃ ,

R ₁ , R ₂ and R ₃ are the same or different and each is an acyl group derived from hydrogen, methyl, ethyl or fatty acid, and at least one of R ₁ , R ₂ and R ₃ is an acyl group derived from a fatty acid.

An eighteenth embodiment of the present invention is a compound represented by the following formula:

X - Y - [AA] n - NH - CH 2 - CH 2 O - R 4

[Wherein, in the above formula

X is a folic acid antagonist component, which is linked to Y via a carboxy group,

Y is optionally a spacer group,

AA is an amino acid, n is 0 to 5,

R ₄ is an acyl group derived from a fatty acid.]

A nineteenth embodiment of the present invention is a method of continuing or changing the activity of a folate antagonist compound by administering a compound of the following formula:

[Wherein, in the above formula

X is a folic acid antagonist component, which is linked to Y via a carboxy group,

Y is optionally a spacer group,

AA is an amino acid, n is 0 to 5,

B is H or CH ₂ OR ₃ ,

R ₁ , R ₂ and R ₃ are the same or different and each is an acyl group derived from hydrogen, methyl, ethyl or fatty acid, and at least one of R ₁ , R ₂ and R ₃ is an acyl group derived from a fatty acid.

A twentieth embodiment of the present invention is a method of continuing or changing the activity of a folate antagonist compound by administering a compound of the following formula:

X - Y - [AA] n - NH - CH 2 - CH 2 O - R 4

[Wherein, in the above formula

X is a folic acid antagonist component, which is linked to Y via a carboxy group,

Y is optionally a spacer group,

AA is an amino acid, n is 0 to 5,

R ₄ is an acyl group derived from a fatty acid.]

Fatty acids may be saturated or unsaturated.

A linking group Y useful for linking a compound having a hydroxy group in the present invention to an amino group of Tris (when B is CH ₂ OR ₃ ) or between amino acids [AA, if present]

a) a linking group having an amino group linked to the compound and having a carboxyl group connected to Tris (or an amino acid, if present) such as an amino acid or an antibiotic

b) a linking group having an amino group linked to the compound and having a sulfonic acid group connected to Tris (or an amino acid, if present) such as 2-aminoethanesulfonic acid (taurine)

c) a linking group having a hydroxyl group connected to the compound and having a carboxyl group connected to Tris (or an amino acid, if present) such as glycolic acid, lactic acid and the like

d) a linking group having a hydroxyl group connected to the compound and having a sulfonic acid group connected to Tris (or an amino acid, if present) such as 2-hydroxyethanesulfonic acid (isethone acid)

e) a linking group having a hydroxyl group connected to the compound and having a reactive halide group connected to Tris (or amino acid, if present) such as 2-chloroethanol

f) a reaction between a compound having a reactive carboxy containing a compound system such as p-hydroxybenzaldehyde, 2-chloroacetic acid, 1,2-dibromoethane and ethylene oxide and an amino group of Tris (or an amino acid, if present) Other potentially suitable couplers are included.

In a preferred embodiment of the present invention X is methotrexate; Y is absent or is an amino acid, glycolic acid, 3-hydroxypropionic acid, or lactic acid, AA is absent or glycine or alanine, and the linkage is either an amide bond or an ester linkage, the? -Carboxy .

As described above, R ₁ , R ₂ and R ₃ are preferably hydrogen, methyl, ethyl or an acyl group of a fatty acid. It is also apparent to those skilled in the art that substituents other than methyl and ethyl are possible for R ₁ , R ₂ and R ₃ . It is the first essential condition that at least one of R ₁ , R ₂ and R ₃ must be an acyl group derived from a fatty acid.

When R ₁ , R ₂ and R ₃ are each an acyl group of a fatty acid, they are preferably the same group. It is also preferred that the acyl group of the fatty acid has a chain of 3 to 18 carbon atoms, more preferably 10 to 18.

The present invention also provides a therapeutic composition comprising the seventeenth or eighteenth embodiment of the present invention and a pharmaceutically acceptable carrier. The composition may also comprise components of the unconjugated folic acid antagonist system.

The present therapeutic compositions may be administered by any suitable route acceptable to those skilled in the art. Such routes include oral, nasal, skin permeation, intratumoral, parenteral, intraarticular, and guideway routes.

6. Catecholamine precursors and catecholamines

DOPA is a precursor of catecholamines, including adrenaline, noradrenaline and dopamine; Are important pharmacologically active groups of compounds including neurotransmitter amines which act as adrenergic stimulants and vasoconstrictors. DOPA and analogs (hereinafter referred to as DOPA-mediated) appear to increase the level of dopamine in the brain, possibly reducing incoordination in Parkinson's disease.

The present inventors have found that DOPA can be linked to one to three kinds of fatty acid acyl derivatives. These new compounds will improve the delivery of DOPA through the gastrointestinal tract and the blood-brain barrier, and will also improve its half-life. It is also believed that these novel compounds are helpful in delivering drugs to the oral, skin permeable, intranasal, parenteral and / or guided routes.

DOPA

Therefore, the twenty first embodiment of the present invention is a compound represented by the following formula.

[Wherein, in the above formula

X is a DOPA-based component, which is connected to Y via a carboxy group or an amino group,

Y is optionally a spacer group,

AA is an amino acid, n is 0 to 5,

B is H or CH ₂ OR ₃ ,

R ₁ , R ₂ and R ₃ are the same or different and each is an acyl group derived from hydrogen, methyl, ethyl or fatty acid, and at least one of R ₁ , R ₂ and R ₃ is an acyl group derived from a fatty acid.

A twenty-second embodiment of the present invention is a compound represented by the following formula:

X - Y - [AA] n - NH - CH 2 - CH 2 O - R 4

[Wherein, in the above formula

X is a DOPA-based component, which is connected to Y via a carboxy group or an amino group,

Y is optionally a spacer group,

AA is an amino acid, n is 0 to 5,

R ₄ is an acyl group derived from a fatty acid.]

A twenty-third embodiment of the present invention is a method for continuously or changing the activity of a DOPA compound by administering a compound represented by the following general formula:

[Wherein, in the above formula

X is a DOPA-based component, which is connected to Y via a carboxy group or an amino group,

Y is optionally a spacer group,

AA is an amino acid, n is 0 to 5,

B is H or CH ₂ OR ₃ ,

R ₁ , R ₂ and R ₃ are the same or different and each is an acyl group derived from hydrogen, methyl, ethyl or fatty acid, and at least one of R ₁ , R ₂ and R ₃ is an acyl group derived from a fatty acid.

A twenty-fourth embodiment of the present invention is a method of continuing or changing the activity of a DOPA compound by administering a compound represented by the following general formula.

X - Y - [AA] n - NH - CH 2 - CH 2 O - R 4

[Wherein, in the above formula

X is a DOPA-based component, which is connected to Y via a carboxy group or an amino group,

Y is optionally a spacer group,

AA is an amino acid, n is 0 to 5,

R ₄ is an acyl group derived from a fatty acid.]

Fatty acids may be saturated or unsaturated.

A linking group Y useful for linking a compound having a carboxy group (such as DOPA) with an amino group of Tris (when B is CH ₂ OR ₃ ) or an amino acid between [AA, if present]

a) a linking group having an amino group linked to the compound and having a carboxyl group connected to Tris (or an amino acid, if present) such as an amino acid or an antibiotic

b) a linking group having an amino group linked to the compound and having a sulfonic acid group connected to Tris (or an amino acid, if present) such as 2-aminoethanesulfonic acid (taurine)

c) a linking group having a hydroxyl group connected to the compound and having a carboxyl group connected to Tris (or an amino acid, if present) such as glycolic acid, lactic acid and the like

d) a linking group having a hydroxyl group connected to the compound and having a sulfonic acid group connected to Tris (or an amino acid, if present) such as 2-hydroxyethanesulfonic acid (isethone acid)

e) a linking group having a hydroxyl group connected to the compound and having a reactive halide group connected to Tris (or amino acid, if present) such as 2-chloroethanol

f) a reaction between a compound having a reactive carboxy containing a compound system such as p-hydroxybenzaldehyde, 2-chloroacetic acid, 1,2-dibromoethane and ethylene oxide and an amino group of Tris (or amino acid, if present) Other potentially suitable couplers are included.

An unrestricted example of a linking group Y useful in linking a compound having an amino group (such as DOPA) with an amino group of Tris (when B is CH ₂ OR ₃ ) or an amino acid between [if present]

a) a linking group having a carboxyl group connected to the compound and having a carboxyl group connected to Tris (or an amino acid, if present) such as dicarboxylic acid through an anhydride such as succinic anhydride or maleic anhydride; Two sulfonic acid groups or similar compounds with two reactive halide groups can be used.

b) a sulfonic acid group having a carboxyl group connected to the compound and connected to Tris (or amino acid, if present) such as hydroxyethanesulfonic acid (isethone acid), or having a sulfonic acid group connected to the compound, (If present) having a carboxyl group

c) has a reactive halide group connected to a Tris (or amino acid, if present) such as 2-chloroethanol, or has a reactive halide group linked to the compound, and has an amino acid A linking group having a carboxyl group connected to the linking group

d) have a reactive halide group linked to the compound and have a sulfonic acid group connected to Tris or an amino acid (if present), or have a sulfonic acid group connected to the compound and connected to Tris or an amino acid between them These functional compounds, such as linking groups with reactive halides, are included.

In a preferred embodiment of the present invention, X is DOPA. Y is absent or is an amino acid, glycolic acid, 3-hydroxypropionic acid or lactic acid, AA is absent or glycine or alanine, and the linkage is either an amide bond or an ester bond to the carboxy group.

As described above, R ₁ , R ₂ and R ₃ are preferably hydrogen or an acyl group of a fatty acid. It is also apparent to those skilled in the art that substituents other than methyl and ethyl are possible for R ₁ , R ₂ and R ₃ . It is the first essential condition that at least one of R ₁ , R ₂ and R ₃ must be an acyl group derived from a fatty acid.

When R ₁ , R ₂ and R ₃ are each an acyl group of a fatty acid, they are preferably the same group. It is also preferred that the acyl group of the fatty acid has a chain of 3 to 18 carbon atoms, more preferably 10 to 18.

The present invention also provides a therapeutic composition comprising the 21st or 22nd embodiment of the present invention and a pharmaceutically acceptable carrier. The composition may also comprise components of the unbonded DOPA system.

The present therapeutic compositions may be administered by any suitable route acceptable to those skilled in the art. Such routes include oral, skin permeation, intranasal, parenteral, and guideway routes.

7. Carboxylic acid group containing alkylating agent

An example of this class of compounds is chlorambucil. Chlorambucil is a functional alkylating agent that cross-links DNA strands to inhibit cell replication, thus acting as a cytotoxic drug. Recently, the drug has been applied to the treatment of Hodgkin's disease, certain types of non-Hodgkin's lymphoma, certain leukemia, ovarian cancer and some breast cancers.

The present inventors have found that chlorambucil and similar drugs can be linked to one to three fatty acid acyl derivatives. These novel compounds are believed to improve the delivery, ingestion, half-life and / or delivery mode of the drug. In addition, these novel compounds are believed to aid in the delivery of oral, intranasal, dermal, parenteral, intratumoral, and / or guide routes.

Chlorambucil

Therefore, the twenty-fifth embodiment of the present invention is a compound represented by the following formula:

[Wherein, in the above formula

X is a chlorambucyl-based component, connected to Y through a carboxy group,

Y is optionally a spacer group,

AA is an amino acid, n is 0 to 5,

B is H or CH ₂ OR ₃ ,

R ₁ , R ₂ and R ₃ are the same or different and each is an acyl group derived from hydrogen, methyl, ethyl or fatty acid, and at least one of R ₁ , R ₂ and R ₃ is an acyl group derived from a fatty acid.

A twenty-sixth embodiment of the present invention is a compound represented by the following formula:

X - Y - [AA] n - NH - CH 2 - CH 2 O - R 4

[Wherein, in the above formula

X is a chlorambucyl-based component, connected to Y through a carboxy group,

Y is optionally a spacer group,

AA is an amino acid, n is 0 to 5,

R ₄ is an acyl group derived from a fatty acid.]

A twenty-seventh embodiment of the present invention is a method for maintaining or changing the activity of a chlorambucil system compound by administering a compound represented by the following general formula:

[Wherein, in the above formula

X is a chlorambucyl-based component, connected to Y through a carboxy group,

Y is optionally a spacer group,

AA is an amino acid, n is 0 to 5,

B is H or CH ₂ OR ₃ ,

R ₁ , R ₂ and R ₃ are the same or different and each is an acyl group derived from hydrogen, methyl, ethyl or fatty acid, and at least one of R ₁ , R ₂ and R ₃ is an acyl group derived from a fatty acid.

A twenty-eighth embodiment of the present invention is a method of administering a compound represented by the following general formula to thereby maintain or modify the activity of a chlorambucil system compound.

X - Y - [AA] n - NH - CH 2 - CH 2 O - R 4

[Wherein, in the above formula

X is a chlorambucyl-based component, connected to Y through a carboxy group,

Y is optionally a spacer group,

AA is an amino acid,

n is from 0 to 5,

R ₄ is an acyl group derived from a fatty acid.]

Fatty acids may be saturated or unsaturated.

A linking group Y useful for linking a compound having a carboxy group (such as chlorambucil) with an amino group of Tris (when B is CH ₂ OR ₃ ) or an amino acid between [AA, if present]

a) a linking group having an amino group linked to the compound and having a carboxyl group connected to Tris (or an amino acid, if present) such as an amino acid or an antibiotic

b) a linking group having an amino group linked to the compound and having a sulfonic acid group connected to Tris (or an amino acid, if present) such as 2-aminoethanesulfonic acid (taurine)

c) a linking group having a hydroxyl group connected to the compound and having a carboxyl group connected to Tris (or an amino acid, if present) such as glycolic acid, lactic acid and the like

d) a linking group having a hydroxyl group connected to the compound and having a sulfonic acid group connected to Tris (or an amino acid, if present) such as 2-hydroxyethanesulfonic acid (isethone acid)

e) a linking group having a hydroxyl group connected to the compound and having a reactive halide group connected to Tris (or amino acid, if present) such as 2-chloroethanol

f) a reaction between a compound having a reactive carboxy containing a compound system such as p-hydroxybenzaldehyde, 2-chloroacetic acid, 1,2-dibromoethane and ethylene oxide and an amino group of Tris (or an amino acid, if present) Other potentially suitable couplers are included.

In a preferred embodiment of the present invention, X is chlorambucil. Y is absent or is an amino acid, glycolic acid, 3-hydroxypropionic acid or lactic acid, AA is absent or glycine or alanine, and the linkage is either an amide bond or an ester bond to the carboxy group.

As described above, R ₁ , R ₂ and R ₃ are preferably hydrogen or an acyl group of a fatty acid. It is also apparent to those skilled in the art that substituents other than methyl and ethyl are possible for R ₁ , R ₂ and R ₃ . It is the first essential condition that at least one of R ₁ , R ₂ and R ₃ must be an acyl group derived from a fatty acid.

When R ₁ , R ₂ and R ₃ are each an acyl group of a fatty acid, they are preferably the same group. It is also preferred that the acyl group of the fatty acid has a chain of 3 to 18 carbon atoms, more preferably 10 to 18.

The present invention also provides a therapeutic composition comprising the 25th or 26th embodiment of the present invention and a pharmaceutically acceptable carrier. The composition may also comprise a non-bonded chlorambucyl system component.

The present therapeutic compositions may be administered by any suitable route acceptable to those skilled in the art. Such routes include oral, intranasal, skin permeation, parenteral, intratumoral, and guideway routes.

In order to more clearly understand the features of the present invention, preferred embodiments thereof will be described with reference to the following examples.

The abbreviation was used as follows.

AZT 3'-azido-3'-deoxy thymidine

DCC 1,3-dicyclohexylcarbodiimide

DCM dichloromethane

DCU N, N'-dichlorohexylurea

DIEA N, N'-diisopropylethylamine

DMAP 4-Dimethylaminopyridine

DMF dimethylformamide

DOPA 3- (3,4-dihydroxyphenyl) -alanine

DSC N, N'-disuccinimidyl carbonate

EtOAc ethyl acetate

Gly glycine

GTP ₁ glycine-Tris-monopalmitate

GTP ₂ Glycine-Tris-dipalmitate

GTP ₃ Glycine-Tris-tripalmitate

HOSu N-hydroxysuccinimide

HPLC High Performance Liquid Chromatography

17-b hydrocortisone 17-butyrate-hydrocortisone

MeOH Methanol

Mor morphine

MTX methotrexate

NMR nuclear magnetic resonance

Suc Succinic acid

TBDMS tert-butyldimethylsilyl

TFA Trifluoroacetic acid

THF tetrahydrofuran

TLC thin layer chromatography

TPTU o- (1,2-dihydro-2-oxo-1-pyridyl) -N, N, N ', N'-tetramethyl-uronium tetrafluoroborate

Tris 2-amino-2-hydroxy-methyl-1,3-propanediol

TSTU o- (N-succinimidyl) -N, N, N ', N'-tetramethyl-uronium tetrafluoroborate

Z Benzyloxycarbonyl

Analytical HPLC

C18 reverse phase column (Radial Pack).

System I - for compounds without fatty acid residues

Buffer A - 0.1% TFA in water

Buffer solution B - 80% acetonitrile: 0.1% TFA containing 20% water

A concentration gradient program from 30% B to 100% B maintained to 8 'over 5'; Flow at 2 ml / min

Latency - RtI

System II - for hydrophobic compounds typically containing fatty acid residues

Buffer A - 50% acetonitrile: 0.1% TFA in 50% water

Buffer B - 50% acetonitrile: 0.1% TFA with 50% THF

A concentration gradient program from 20% B to 100% B maintained at 8 'over 5'; Flow at 2 ml / min

Latency - RtII

Preparation of Gly-Tris

40 pa. The Z-Gly-Tris solution in ethanol was hydrogenated in a Parr hydrogenation reactor in the presence of pressure, palladium on carbon (10%) to give the title compound. The removal of the Z group was monitored by HPLC. Filtered and washed with ethanol to remove the catalyst. The solvent was evaporated to give the title compound in 95% yield. The preparation of Z-Gly-Tris is described in Whittaker, R. G., Hayes, P. J., and Bender, V. J. (1993). Peptide Research 6; 125 and Australian Patent No. 649242.

Example 1

Synthesis of hydrocortisone-Suc-Gly-Tris- (palmitate) _n : wherein n = 1, 2 or 3.

I. hydrocortisone-succinate

Succinic anhydride (1.65 g, 15 mmol) and DIEA (1.7 ml, 10 mmol) were added to a solution of hydrocortisone (3.65 g, 10 mmol) in acetonitrile (450 ml) and the reaction mixture was stirred at room temperature for 36 h. HPLC analysis of the reaction mixture showed 93% of the title compound. The solvent was evaporated and the residue was redissolved in ethyl acetate and washed with water. The ethyl acetate phase was evaporated under reduced pressure, and the residue was triturated in diethyl ether to obtain 4.4 g of a white powder having a yield of 95%. R _t I 5.94 '.

II. Hydrocortisone-Suc-OSu

To a solution of hydrocortisone-succinate (4.0 g, 8.65 mmol) in acetonitrile (120 ml) was added DSC (4.43 g, 17.3 mmol) in 30 ml of DMF and DIEA (1 ml). After 30 minutes, a white precipitate formed and analyzed by HPLC. As a result, a new peak appeared at 6.7 'at 90%. The precipitate was filtered to give 4 g of the title compound (100% purity by HPLC). The filtrate was evaporated and the residue was triturated in acetonitrile and diethyl ether to give additional 0.6 g of the title compound. Total yield: 95%, R _t I 6.7 '.

III. Hydrocortisone-Suc-Gly-Tris

To a solution of hydrocortisone-Suc-OSu (3.9 g, 7 mmol) in 20 ml of DMF was added Gly-Tris (1.78 g, 10 mmol) in 20 ml of DMF and the reaction mixture was stirred at room temperature. HPLC analysis after 4 hours showed that the yield of the title compound (R _t I 4.98 ') was 69%. The solvent was removed under reduced pressure and the residue was redissolved in 50 ml of ethyl acetate and washed with 100 ml of water. The aqueous phase was evaporated to 20 ml and the title compound was extracted with 200 ml of ethyl acetate (3 times) and analyzed by HPLC to give 2.4 g of the title compound in 95% purity: yield 56%, R _t I 4.98 '.

IV. Hydrocortisone-Suc-Gly-Tris- (palmitate) _n ; Wherein n = 1, 2 or 3

Palmitic acid (2.46 g, 9.63 mmol) and a catalytic amount of DMAP were added to a solution of hydrocortisone-Suc-Gly-Tris (2,4 g, 3,85 mmol) in 100 ml of DCM and 10 ml of DMF and the reaction mixture was cooled to 0 & . DCC (2.06 g, 10.02 mmol) in 20 ml of DCM was added to the reaction mixture dropping funnel. The reaction mixture was stirred at 0 < 0 > C for 30 minutes and then at room temperature. After 4 hours of reaction, a mixture of the title compounds was formed with 1,2 or 3 palmitic acid (36%, 29%, 3.2%, respectively). The solvent was evaporated and the residue redissolved in DCM. The DCU was filtered and the filtrate was evaporated to dryness. The residue was redissolved in a 1: 1 mixture of acetonitrile and THF and separated by preparative HPLC using a C18 column (40 x 100 mm) to give 800 mg of hydrocortisone-Suc-Gly-Tris-monopalmitate, 1700 mg of hydrocortisone-Suc-Gly-Tris-dipalmitate and 230 mg of hydrocortisone-Suc-Gly-Tris-tripalmitate. Each conjugate was further purified on a silica column with a gradient of concentration from ethyl acetate: petroleum ether (60:40) to ethyl acetate: petroleum ether (90:10).

Example 2

Synthesis of 17-butyrate (17-b) hydrocortisone-21-Suc-Gly-Tris- (palmitate) _n : wherein n = 1, 2 or 3.

I. 17-Butyrate (17-b) Hydrocortisone-21-Succinate

Succinic anhydride (1.25 g, 12.5 mmol) and DIEA (0.34 ml, 5 mmol) were added to a solution of 17-b hydrocortisone (2.16 g, 5 mmol) in acetonitrile (80 ml) and the reaction mixture was stirred at room temperature for 40 hours . The reaction mixture was subjected to HPLC analysis to find that the title compound was 98%. The solvent was evaporated and the residue was redissolved in ethyl acetate and washed with water. The ethyl acetate phase was evaporated under reduced pressure and the residue was triturated in diethyl ether to give 2.6 g of a white powder having a yield of 95%.

II. 17-b hydrocortisone-21-Suc-OSu

To a solution of 17-b hydrocortisone-21-succinate (1.5 g, 2.8 mmol) in acetonitrile (40 ml) was added DSC (2.16 g, 7 mmol) in 30 ml of DMF and DIEA (0.47 ml). After reaction at room temperature for 1 hour, analysis by HPLC showed that about 85% of the title compound was formed. (100% purity by HPLC). The solvent was evaporated and the next step was carried out without further purification.

III. 17-b-hydrocortisone-21-Suc-Gly-Tris

To 20 ml of a DMF solution of 17-b hydrocortisone-21-Suc-OSu (2.8 mmol) was added Gly-Tris (1.5 g, 8.4 mmol) in 20 ml of DMF and the reaction mixture was stirred at room temperature. After 5 hours HPLC analysis yielded 80% yield of the title compound (R _t I 6.09 '). The solvent was removed under reduced pressure and the residue was redissolved in 20 ml of 50:50 water / acetonitrile and purified by preparative HPLC (Waters Prep4000 using a C18 column). 0.75 g of the title compound was obtained.

IV. 17-b hydrocortisone-21-Suc-Gly-Tris- (palmitate) _n ; Wherein n = 1, 2 or 3

To the solution of 17-b hydrocortisone-21-Suc-Gly-Tris (0.51 g, 0.74 mmol) in 20 ml of DCM was added palmitic acid (0.57 g, 2.22 mmol) and a catalytic amount of DMAP and the reaction mixture was cooled to 0 & . DCC (0.45 g, 2.22 mmol) in 10 ml of DCM was added dropwise to the reaction mixture. The reaction was stirred at 0 < 0 > C for 30 min and then at room temperature. After 2 hours, a mixture of the title compounds was formed with 1,2 or 3 palmitate groups (7%, 47%, 46%, respectively, by HPLC). The DCU was filtered, and the filtrate was evaporated to dryness. The residue was redissolved in a 1: 1 mixture of acetonitrile and THF. Through HPLC, the solution was treated with a mixture of the title compounds: mono; di; tripalmitate; 7:16:69. This mixture was separated by preparative HPLC using a C18 column (40 x 100 mm) to obtain 850 mg of 17-b hydrocortisone-21-Suc-Gly-Tris-tripalmitate, 17-b hydrocortisone-21-Suc 150 mg of -Gly-Tris-dipalmitate and 120 mg of monopalmitate. The mono and dipalmitate conjugates need to be further purified by silica chromatography with a gradient of concentration from ethyl acetate: petroleum ether (60:40) to ethyl acetate: methanol (90:10), but the tripalmitate conjugate is purified by preparative HPLC The post-purity was 100%.

HPLC analysis indicated that the title product was highly pure and had no parent drug and no other derivatives.

Example 3

Synthesis of morphine-Suc-Gly-Tris-di and tripalmitate

The present inventors have found that a phenolic hydroxyl group at the C-3 position of morphine (Mor) is protected by Gly-Tris-dipalmitate or Gly-Tris-dipalmitate via a succinic acid (Suc) linkage without protecting the secondary hydroxyl group at the C- Tris-tripalmitate. ≪ / RTI > Synthesis of Mor-Suc-Gly-Tris-dipalmitate was two-step, with a total yield of 54%.

I. Preparation of morphine-succinate

Under nitrogen at 0 ° C, triethylamine (9.584 ml, 69.45 mmol) was added dropwise to a suspension of morphine sulphate (9.286 g, 27.78 mmol) in dry DMF (140 ml). After stirring for 10 min, succinic anhydride (2.781 g, 27.78 mmol) was added portionwise to the reaction. The reaction _{TLC (50% EtOH / H 2} 0) and analysis HPLC (RtI of Mor-Suc was 5.06 ") was monitored by. The reaction was complete within 24-48 hours and the product precipitated. The precipitate was filtered off and washed with a small amount of cold DMF and THF. ¹ H NMR and HPLC all showed that the product had sufficient purity (Mor-Suc 6.11 g; yield 57%) for the next reaction.

It was found through preliminary ¹ H NMR that succination occurred at phenolic hydroxyl at the C-3 position.

II. Production of morphine-Suc-Gly-Tris-dipalmitate

Mor-Suc-Gly-Tris- (palmitate) _n .

DCC (0.506 g, 2.45 mmol) and N-hydroxysuccinimide (0.308 g, 2.68 mmol) were added to a suspension of Mor-Suc (0.860 g, 2.23 mmol) in dry THF (44 ml) at 0 ° C under nitrogen. The resulting mixture was slowly warmed to room temperature and refluxed overnight. The reaction was monitored by HPLC (System I); The retention time of the active ester, Mor-Suc-OSu, was 5.45 min. After the reaction was complete, the mixture was cooled to 0 C and the precipitate was filtered and washed with dry DCM. The filtrate was vigorously stirred at room temperature and directly added to Gly-Tris-dipalmitate (1.459 g, 2.23 mmol). Aminolysis of Mor-Suc-OSu was monitored by HPLC (System II) and TLC (10% MeOH / DCM). HPLC and TLC all indicated that the reaction was complete after stirring overnight. The solvent was removed under vacuum and the residue was redissolved in DCM and washed several times with a pH of 7. Dry the organic phase (MgSO ₄₎ and evaporated to give a light yellow solid. The crude product was purified by flash chromatography (silica, 10% MeOH / DCM) to give the title compound (2.16 g) in good yield (94.7%).

III. Preparation of morphine-Suc-Gly-Tris tripalmitate

Following the general method set out in the above, Mor-Suc (0.101g, 0.26mmol ) to give the GTP and ₃ successfully coupled to _{Mor-Suc-GTP 3 (0.215g} ) in 65.6% isolated yield. The product was purified by flash chromatography using 10% MeOH / EtOAc eluant on alumina.

HPLC (System II), Mor-Suc-GTP ₂ and Mor-Suc-GTP ₃ were high purity and no parent drug.

Example 4

Synthesis of AZT-Gly-Tris- (palmitate) _n : wherein n = 1, 2 or 3.

AZT was reacted with succinic anhydride to form AZT-succinic acid. This was reacted with a mixture of Gly-Tris-mono, di, and tripalmitate (GTP _n ) prepared by catalytic hydrogenation of Z-GTP _{n by} the DCC / HOSu method. Column chromatography on silica gel and preparative HPLC were used to separate the final compound.

1. AZT-succinic acid

AZT (1.068 g, 4 mmol), succinic anhydride (0.440 g, 4.4 mmol) and DMAP (0.015 g) were placed in a 25 ml flask equipped with a condenser. DMF (10 ml) was added and the mixture was stirred for 5 minutes and soaked in the oil bath preheated to 90 < 0 > C for 1.5 h until HPLC verification indicated completion of the reaction. DMF was evaporated in vacuo (40 < 0 > C) and the residue was used directly in the subsequent reaction. The residue can be purified by chromatography on silica gel and the product can be separated, if necessary, into 87% yield and 97% purity.

II. AZT-Suc-Gly-Tris- (palmitate) _n : wherein n = 1, 2 or 3.

AZT-succinic acid (1.468 g, 4 mmol) was dissolved in DCM (30 ml) and HOSu (0.552 g, 4.8 mmol) was added. The mixture was stirred for 5 minutes then DCC (0.988 g, 4.8 mmol) was added in one portion. Stirring was continued for 1 hour until HPLC analysis indicated completion of the reaction. The reaction mixture was filtered and insoluble DCU was washed with DCM (35ml). The combined filtrate was collected in a reaction vessel, then GTP _n (2.5 g) was added and stirred overnight. The reaction was monitored by HPLC. Additional GTP _n was added until all active OSu esters were consumed. The reaction mixture was evaporated in vacuo. The product was purified on silica by column chromatography using hexane / ethyl acetate and ethyl acetate / methanol as eluent to separate AZT-Suc-Gly-Tris mono, di and tripalmitate. The product was further purified by preparative HPLC (C18 column).

Example 5

Preparation method 1 of cyclosporin-Suc-Gly-Tris- (palmitate) _n ; Wherein n = 1, 2 or 3.

1. Cyclosporin-Suc

The title compound can be prepared by reacting succinic anhydride with cyclosporin A, B, D and G in the presence of triethylamine in DMF. The threonine side chain in Cyclosporin C needs to be protected before carrying out this reaction.

II. Cyclosporin-Suc-Gly-TRIS

Cyclosporin-Suc was reacted with Gly-Tris in the presence of DCC and HOSu to give the title compound.

III. Cyclosporin-Suc-Gly-TRIS mono, di, and tripalmitate

The cyclosporin-Suc-Gly-Tris was reacted with palmitic acid in a molar ratio of 1 to 2 in the presence of DCC to give the title compound. The three title compounds could then be separated via preparative HPLC or silica gel chromatography eluting with an organic solvent. Side chain protection of cyclosporin C was removed prior to purification.

Production method 2 of cyclosporin-Suc-Gly-Tris- (palmitate) n; N = 1, 2 or 3;

I. Preparation of benzyl esters of Suc-Gly-Tris-TriOTBDMS

Suc-monobenzyl ester can be prepared by reacting succinic anhydride with benzyl alcohol in the presence of a strong base. The remaining unmodified carboxy group can then be reacted with Gly-Tris in the presence of DCC and HOSu to give Bzl-Suc-Gly-Tris. Subsequently, the three hydroxyl groups of tris can be protected by reaction with TBDMS chloride in the presence of imidazole to give the title compound.

II. Cyclosporin-Suc-Gly-Tris-mono, di, and tripalmitate

The benzyl ester of Bzl-Suc-Gly-Tris-TriOTBS can be removed using hydrogen in the presence of palladium on carbon to yield the unprotected carboxyl group. This unprotected carboxyl group can react with the hydroxy group of the cyclosporin (A, B, D and G; cyclosporin C requires side chain protection as above) in the presence of DCC to form an ester bond. In this compound, the TBDMS-protected three hydroxyls of Tris can be deprotected by an acetic acid reaction and reacted with palmitic acid in the presence of DCC to produce a mixture of the title compounds. The three title compounds can then be separated by preparative HPLC or silica gel chromatography.

Example 6

Synthesis of methotrexate-Gly-Tris-di and tripalmitate

Synthetic Reaction Overview

A method devised is to remove glutamyl residues of methotrexate (MTX) by enzymatic cleavage with Carboxypeptidase G and replace it with glutamic acid (? -Tetrabutyl; gamma -methyl) in which two carboxyl groups are selectively esterified . The methyl ester was then selectively removed to provide the attachment site of Gly-Tris to be palmitated. The dipalmitate derivative (and minor amount of tripalmitate) was isolated by silica chromatography and / or extraction.

I. [[(2,4-diamino-6-pteridinyl) methyl] methylamino] benzoic acid

Glutamic acid was separated from carboxypeptidase G from methotrexate as described in J. Med. Chem. 24, 1450-1455 (1981) to give the title compound. The yield of I obtained was substantially quantitative.

II. alpha-tert-butyl-gamma-methyl L-glutamate

Justus Liebigs Ann. Chem. 646, 134 (1961)], the title compound was prepared from? -Methyl L-glutamate. Yields vary widely, but are typically in the 30-50% range. Compound II obtained as an oil was used directly in the subsequent coupling reaction.

III. α-tert-butyl-γ-methyl methotrexate

J. Med. Chem. 24, 1450-1455, (1981)], the title compound was obtained. The yield was generally 50 to 60%.

IV. alpha-tert-butyl methotrexate

J. Med. Chem. 24, 1450-1455, (1981)], the title compound was obtained by hydrolysis of III with Ba (OH) ₂ . As can be seen by < ¹ > H NMR, compound IV was generally sufficiently pure that it did not need to be purified by ion exchange chromatography. The yield was generally in the range of 75-80%.

V. Methotrexate-a-tert-butyl? -Gly-Tris

HOSu (21 mg, 0.22 mmol) was added to IV (100 mg, 0.20 mmol) in DMF / acetonitrile (1: 1, 6 ml) and DCC (45 mg, 0.22 mmol) was added. The reaction mixture was stirred at room temperature and monitored by HPLC (System I). After 5 hours, the production of OSu ester was completed to 85%, and HPLC analysis showed that the residue was IV and a small amount of decomposition product. Gly-Tris solution [prepared by hydrogenating Z-Gly Tris (1.5 equivalents in 3 ml of DMF) in DMF on 10% Pd / C] was added and the reaction was then subjected to HPLC. The reaction was completed within 30 to 60 minutes. Next, after the reaction mixture was diluted with H ₂ 0 (3-5ml), allowed to stand for 10 minutes and filtered for DCU. The solvent was removed from the filtrate and the residue was lyophilized to remove the solvent and then purified by preparative HPLC to give the title compound as a light yellow solid. The yield was 30 mg, 23%. On a larger scale, a yield of 69% V was obtained using 800 mg IV. The purity of the product was found to be 97% by HPLC.

VI Methotrexate α-tert-butyl ester γ-Gly-Tris (palmitate) n; In this case, n = 2 or 3

DMM (30 mg), palmitic acid (500 mg, 2 eq.) And DCC (400 mg, 2 eq.) Were added to a suspension of V (650 mg, 0.97 mmol) in DCM (50 ml) . The mixture was stirred at RT for 36 h, then diluted with DCM (80 ml) and cooled in ice for 10 min. The mixture was filtered and the filtrate was extracted with 0.01 M HCl (100 ml) and brine (2x100 ml). The solvent was dried (MgSO ₄ ) and the residue was applied to a silica column and eluted sequentially with DCM, DCM / 5% MeOH, DCM / 10% MeOH. Through this, the tree and dipalmitate of IV were ejected from the column as a yellow band. The pure fractions by TLC (DCM / isopropanol (30%)) were combined and the solvent removed to give the title compound as a light yellow solid.

VII. Methotrexate-γ-Gly-Tris-dipalmitate

VI (200 mg, 0.17 mmol) was added TFA (5 ml). The mixture was stirred at room temperature for 20 to 30 minutes and the solvent was removed by evaporation. The residue was partitioned between DCM (50ml) and H ₂ O (50ml). TEA was added to the aqueous phase to pH 7. Acetic acid was added to pH 3-4. The organic phase was collected, washed with H ₂ O (50 ml), dried (Na ₂ SO ₄ ) and the solvent was removed. The pure product by TLC (butanol / acetic acid / water, 4: 2: 1) was washed with ethanol and dried to give the title compound as a gold solid.

Example 7

Synthesis of L-DOPA-Gly-Tris- (palmitate) _n ; Wherein n = 1, 2 or 3.

Synthetic Reaction Overview

The active ester of Z ₃ -DOPA was prepared by protecting the amino group as well as the two phenolic hydroxyl groups of DOPA. The active ester of the fully protected compound was best formed using TPTU, presumably due to the structural barrier properties of this compound. This was reacted with Gly-Tris-dipalmitate to obtain Z ₃ -DOPA-Gly-Tris-dipalmitate.

In a different synthesis method, Z ₃ -DOPA-Gly-Tris was palmitylated (prepared by the active ester method from Z ₃ -DOPA and Gly-Tris). The product was hydrogenated to obtain the desired DOPA-Gly-Tris-dipalmitate and DOPA-Gly-Tris-tripalmitate in good yield.

I. N, O'O'-tricarbobenzoxy-L-DOPA [Z ₃ -Dopa]

Examples of the preparation method of Z ₃ -DOPA include Felix et al. 1974 was a method (J.Med.Chem. 17 .422-426). L-DOPA (7 g, 35.5 mmol) was added to a pre-cooled solution of 1 M NaOH (35.5 ml) and water (74 ml) under nitrogen blanket in a three necked flask at -10 ° C to give the title compound. Carbobenzoxychloride (20.2 g, 116.5 mmol) in 1M NaOH (100 ml) and diethyl ether (100 ml) was added dropwise and the solution stirred vigorously at -10 ° C over 1 hour. Stirring was continued at -10 ° C for 1 hour, then at 0 ° C for 1 hour and finally at 20 ° C for 2 hours. The precipitated sodium salt was collected by filtration, washed with diethyl ether, and partitioned between diethyl ether and 1M citric acid (100 ml each). The ether layer was washed with water, dried (Na ₂ SO ₄₎ and removal of the solvent in a vacuum oil tank (crude) - to give the title product (18g, yield 84.6%).

The title compound was further purified by preparative HPLC (reverse phase C18 column) to give the chromatographically pure title product. R _t II 4.77 '.

II. Z ₃ -DOPA-Gly-Tris-dipalmitate

Acetonitrile (30ml) of TPTU (1.68g, 5.62mmol) solution in acetonitrile (20ml) and DIEA (550ul a pH 8.50) of Z ₃ -DOPA Z ₃ was added to a stirred solution (1.68g, 2.8mmol) -DOPA ≪ / RTI > was prepared. The reaction was followed by HPLC and monitored at 300 nm. After 10 minutes the reaction was complete and the solvent and base were evaporated in vacuo. The residue was redissolved in DCM and re-evaporated, and this was repeated twice to remove the base completely. The residue solution in DCM (30 ml) was added to a stirred solution of GTP ₂ (2.0 g, 3 mmol) in DCM (15 ml) under a dark nitrogen atmosphere.

HPLC was monitored at 260 nm and the reaction was found to be complete after 30 minutes and a single product was produced. The reaction mixture was diluted with dry DCM (120ml) and washed with water _{(3x120ml) (Na 2 SO 4} ). The DCM layer contained the desired title compound which displayed a single peak with HPLC (R _t II 8.70 '). The solvent was removed in vacuo and the residue was washed repeatedly with cold acetonitrile to afford 3.40 g of the title compound as a white fluffy precipitate, 98% yield. HPLC, TLC and NMR spectroscopy showed the title compound to be of high purity.

An aliquot of the product was hydrogenated in ethanol for 5 hours in the presence of a catalytic amount of 10% palladium on carbon to give L-DOPA-GTP ₂ , RtII 8.05 ', the maximum absorption at 285 nm. The yield was 785 mg, 83.5%.

HPLC analysis indicated that the title compound was 99% pure and free of parent drug.

III. Preparation of Z ₃ -DOPA-Gly-Tris

A solution of Z ₃ -DOPA (800 mg, 1.33 mmol) in dry acetonitrile (10 ml) was reacted with a solution of TPTU (1 g, 3.4 mmol) in acetonitrile (5 ml) and DIEA (350 ul at pH 8.3) The formation of active ester by HPLC was monitored. The reaction was complete after 10 minutes and yielded the active ester with 89% yield as HPLC, RtII 5.19 '. The solvent and DIEA were repeatedly evaporated from DCM to give an oil residue. This was redissolved in acetonitrile (15 ml) and added dropwise with stirring to a freshly distilled solution of Gly-Tris (1 g, 5.6 mmol) in dry DMF (5 ml). The reaction was monitored by HPLC at 300 nm and 260 nm, indicating that the title compound was formed in both HPLC systems, I and II (Rt 7.11 'and 3.05', respectively).

The solvent was removed in vacuo and the title product was purified by preparative HPLC to give 655 mg of Z ₃ -DOPA-Gly-Tris (yield 60.4%). The structure of the product was confirmed by NMR spectroscopy.

IV. Z ₃ -DOPA-Gly-Tris-di and tripalmitate

A solution of Z ₃ -DOPA-Gly-Tris (600 mg, 1 mmol) in DCM (10 ml) was reacted with palmitic acid (312 mg, 1.22 mmol) and DCC (250 mg, 1.2 mmol) as described above. After the reaction, additional palmitic acid was added in an aliquot of 10 mg when necessary and subjected to HPLC. After addition of 40 mg of palmitic acid, it was found by HPLC that Z ₃ -DOPA-Gly-Tris-mono and dipalmitate were formed in a 1: 1 analogy. After filtration to remove DCU, the solvent was removed in vacuo and the mixture was separated by preparative HPLC using a C18 column.

During the reaction, most of the product was converted to the di- and tripalmitate forms which were separated to give 260 mg of dipalmitate and 520 mg of tripalmitate and the mixture was traced HPLC to calculate the percentage (21% P ₂ ; 35% P ₃ ).

Hydrogenation in ethanol in the presence of Pd / C catalyst gave L-DOPA-GTP ₂ and GTP ₃ to obtain the desired title product.

Example 8

Synthesis of chlorambucil-Gly-Tris- (palmitate) n; Where n = 1, 2 or 3.

The active ester of chlorambucil was prepared and reacted with a mixture of Gly-Tris mono, di, and tripalmitate (GTPn) produced by hydrogenating Z-GTPn to form the title compound. The obtained product was purified by column chromatography and preparative HPLC.

I. chlorambucil-Gly-Tris- (palmitate) _n ; Where n = 1, 2 or 3.

Chlorambucil (0.913 g, 3 mmol) and HOSu (414 mg, 3.6 mmol) were placed in a 50 ml flask with a magnetic stirring bar. DCM (15 ml) was added and the solution was stirred for 5 minutes. DCC (742 mg, 3.6 mmol) in DCM (15 ml) was added within 5 min. It was found that the reaction was completed after 1 hour through analytical HPLC.

The solution was filtered and the residue was washed with DCM (10 ml). The combined filtrates were collected in a flask and stirred to give solid GTP _n (4 g). Stirring was continued overnight and fresh GTP _n was added (300 mg). After 2 h, it was found by HPLC that there was no chlorambucil-OSu ester indicating the completion of the reaction.

The mixture was evaporated in vacuo (40 < 0 > C). The product was chromatographed on silica gel using hexane: ethyl acetate and then subjected to preparative HPLC to give a high purity product.

II. Chlorambucil-Gly-Tris-monopalmitate

DCC (0.726 g, 3.5 mmol) and HOSu (0.387 g, 3.3 mmol) were added portionwise to the chlorambucil (0.972, 3.2 mmol) solution in DCM (32 ml) at 0 ° C. The resulting mixture was stirred overnight at room temperature. After completion of the reaction according to HPLC (System I), the precipitate (DCU) was filtered off and washed with dry DCM (10 ml). To the filtrate, Gly-Tris-monopalmitate (1.208 g, 2.9 mmol) was added portionwise with vigorous stirring at room temperature overnight. When the reaction was complete, the product obtained was diluted with DCM and washed several times with H ₂ O. The organic phase was dried (MgSO ₄ ) and evaporated in vacuo to give crude product. The crude product was purified by flash chromatography (80% ethyl acetate / hexane, 2.5% MeOH / ethyl acetate) to yield the title compound (0.950 g) as a light yellow semi-solid in 46.6% yield.

III. Chlorambucil-Gly-Tris-tripalmitate

To a solution of chlorambucil (0.240 g, 0.789 mmol) in DCM (16 ml) was added DCC (0.171 g, 0.828 mmol) and DMAP (0.005 g, 0.039 mmol) at 0 ° C followed by Gly-Tris- tripalmitate g, 0.828 mmol). The resulting mixture was stirred overnight at room temperature. The precipitate was filtered and the filtrate was washed with a 5% aqueous acetic acid solution. It was then washed three times with H ₂ O to pH 7. The organic phase was dried (MgSO ₄ ) and concentrated in vacuo . The crude product was purified by flash chromatography (30%, 40% ethyl acetate / hexane) and recrystallized from ethyl acetate / hexane to give the title compound (0.45 g) as a white solid in 48.2% yield.

Example 9

Anti-inflammatory effects of hydrocortisone and hydrocortisone fatty acid conjugates in UVB model

Drugs Used

Sigma Chemicals, Lot no. 13H0525. Hydrocotisson was purchased from H-4001. Hydrocortisone-Suc-GTP ₂ , hydrocortisone-Suc-GTP ₃ and 17b hydrocortisone-Suc-GTP ₃ were synthesized as described above.

UVB erythema black

One type of i / b Skh-1 hairless albinosome female pre-exposed to UVB was used for all experiments. They were divided into three groups (mean weight 30g). Erythema (inflammation) was induced using an FS40 light source with one UVB tube. Mice were exposed to UVB for 15 min, corresponding to exposure to 2 MED (minimum erythema dose). The irradiated mice were obtained by uniformly dispersing 100 μl of a solution of hydrocortisone, hydrocortisone-Suc-GTP ₂ or hydrocortisone-Suc-GTP ₃ in ethanol (vehicle), using a Gilson pipettor, Irradiated, or pretreated several days before UVB exposure. The mice were left for 24 hours and skin folds were measured using a hand held micrometer.

Experiment

The effects of hydrocortisone on UVB-induced inflammation were investigated at the beginning of the study. After irradiation of the Skh-1 mouse group with UVB 2MED, hydrocortisone in ethanol was locally applied. After 24 hours, the net skin fold thickness increase (NSFT) was determined. In the concentration curves obtained, the optimum amount of hydrocortisone required to produce the best anti-inflammatory protective action against 2M ED exposure of UVB was determined. Administration of 0.5 mg per mouse showed almost 100% protection against erythema and species. This was used in subsequent experiments to determine the effect of the hydrocortisone conjugate.

Its protective action against hydrocortisone-succinate fatty acid conjugates and UVB-induced species (oedema).

The lifespan of locally applied hydrocortisone-succinate fatty acid conjugates was determined. Hydrocortisone with concentrations of 0.5% and 1% W / V (corresponding to 0.5 mg and 1.0 mg, respectively, per mouse) was used and the results were as follows. Test conjugates were applied 5 and 3 days before UV exposure. The conjugate was applied after UV exposure on day 0 to prevent UV absorption (sunscreen effect).

0.5 mg / mouse The experimental results are shown in Table 1.

The mean pure skin fold thickness (10 ^-2 mm) for 0.5 mg / mouse hydrocortisone and conjugates (HC-Suc-GTP ₂ and GTP ₃ and 17-b HC-Suc-GTP ₃ ). drug 5 days 3 days 0 days 0.5 mg / mouse hydrocortisone 102.7 ± 12.1 100.9 ± 7.3 10.4 ± 8.19 0.5 mg / mouse HC-Suc-GTP ₂ ^* 104.2 ± 14.7 94.3 ± 10.3 60.6 ± 8.6 0.5 mg / mouse HC-Suc-GTP ₃ ^* 93.8 ± 9.8 86.7 ± 9.4 55.8 ± 11.7 0.5 mg / mouse 17-b HC-Suc-GTP ₃ 90.7 ± 14.8 42.7 ± 13.7 Vehicle 112.6 + - 10.4 ^{* If the} HC content is 0.5 mg

result

Three types of hydrocortisone-succinate conjugates, hydrocortisone -Suc-Gly-Tris- di-palmitate _{(HC-Suc-GTP 2)} , hydrocortisone -Suc-Gly-Tris- tree palmitate (HC-Suc-GTP ₃ ) And 17-b HC-Suc-GTP ₃ , when applied on day 0 after UVB exposure, accounted for about 50% of hydrocortisone activity for UVB-induced species. It was also observed that when the conjugate was applied to mice 3 days before UVB exposure, the two conjugates still had a protective effect and slightly better effect than hydrocortisone alone. There was smoothing in the response to the conjugate indicating a modified delivery profile.

At higher concentrations of 1 mg per mouse, the hydrocortisone conjugate exhibited an enhanced protective effect, and on day 3 all of the experimental compounds showed better protection (Table 2). The delayed action of the hydrocortisone conjugate, which presents a more sustained and consistent delivery profile, has once again appeared.

Mean Pure Skin fold thickness for 1% W / V hydrocortisone and conjugate. drug 5 days 3 days 0 days 1 mg / mouse hydrocortisone 108.3 ± 5.1 62.9 ± 4.0 7.9 ± 4.4 1 mg / mouse HC-Suc-GTP ₂ ^* 60.8 ± 8.8 57.5 ± 10.9 1 mg / mouse HC-Suc-GTP ₃ ^* 103.8 ± 3.0 62.9 ± 4.0 27.9 ± 7.5 Vehicle 114.0 8.0 ^* About the HC content

conclusion

The efficacy of the hydrocortisone-succinate fatty acid conjugate was determined in a mouse UVB inflammation model. The overall biological result is that hydrocortisone in the form of a synthetic fatty acid conjugate is similar to hydrocortisone in biological action when measuring the protective action against UVB-induced inflammation and species. In particular, the fatty acid conjugate appears to have a modified delivery profile on the epidermis, because the total activity is delayed at day 0.

This result shows that using a fatty acid conjugated molecule to hydrocortisone may have several advantages over compartments with improved skin permeation transport. In addition, this altered transport reduces side effects caused by down regulation of the H-P-A axis when using hydrocortisone in extended use.

Example 10

Contact Hypersensitivity Assay.

Were assayed by the method described in Current Protocols in Immunology (Vol 1, Section 4.2 Eds Coligan et al. (1991) NIH). The sensitizer was oxalaurone. The simplest way to do this is as follows:

UVB radiation. Mice were irradiated with a single FL40SE UVB fluorescent tube (Oliphant) in a reflective batten. The UVB radiation measured at the target distance was 2.6x10 < 4 > W / cm < ² > using an in-line JITER IL1700 (International light IL1700) radio meter with a sensitive UVB detector at 250-315 nm. The mice were exposed for 3 consecutive days to remove the iron tower and exposed to 0.1179 J / cm ² UVB constituting the above-mentioned 1 minimum erythemal dose (MED). Accumulated quantities showed proper erythema without blisters. By measuring the middorsal skinfold thickness using a spring micrometer (Mercer, St. Albans, UK) at 24 hours after the primary UVB exposure, ie, the time at which the reaction is maximized, ie just before the third UVB exposure The amount of erythema as a species component of the reaction was measured.

Induction of Systemic Contact Hypersensitivity.

The backs of the experimental mice were exposed to 1 MED UVB radiation (or no UVB radiation) for 1, 2 and 3 days at the same time. On days 8 and 9, 0.1 ml of freshly prepared 3% (W / V) oxazolone (Sigma Chemical Co., St Louis, MO) was applied to the abdominal skin of mice by ethanol application. On day 15, the ear thickness was measured before stimulation using a spring micrometer, and then the mouse was stimulated by applying 5 ul of a freshly prepared 3% oxazolone / ethanol solution to the surface of both auricles. The ear thickness was repeatedly measured at 16 to 24 hours. The peak was recorded on the glans. For each treatment group, pure swelling of the ear was calculated as the difference between the average before stimulation and the average ear thickness after stimulation. With Student's test, the statistical significance of differences in ear swelling between the treatment groups was assessed.

Mice were coated with vehicle, 0.5 mg / mouse HC, 0.5 mg / mouse HC-Suc-GTP ₂ , 1 mg / mouse 17-b HC-Suc-GTP ₃ or 17-b HC within 10 minutes of UVB exposure. A total of 3 MEDs of unshielded UVB were used (Table 3).

Contact sensitization result Experimental material Pure swelling of the ear (10 ^-2 mm) Standard Deviation Vehicle control 17.7 6.94 0.5% HC-Suc-GTP ₂ 29.8 5.05 0.5% hydrocortisone 32.0 9.30 1% 17b-hydrocortisone 38.6 4.41 0.5% HC-Suc-GTP ₃ 32.8 10.28 Control without UVB 42.1 6.08

conclusion

All the samples tested showed protection against UVB-induced immunosuppression and systemic responses to sensitizers showed increased ear thickness. There is no difference in parent drug and conjugate in terms of ability to change immunosuppression due to UVB. However, it is important that the conjugate appears to be just like the parent drug in a systematic reaction.

Example 11

AZT

Cytotoxicity of AZT and AZT-fatty acid conjugates was tested for cytotoxicity and anti-HIV activity.

I. Cytotoxicity

The cytotoxicity of AZT and AZT-fatty acid conjugates was tested in vitro with JURKAT (acute T-cell leukemia) and peripheral blood mononuclear cells (PBMC). Cells were cultured with the drug and the survival rate was determined by a colorimetric proliferation test.

Way

a) Cell culture conditions

JURKAT cells inoculated with 1.6 × 10 ⁴ cells / 200㎕ complete RPMI in a 96-well plate at a density of 1640 ± drug and incubated 64 hours.

Human peripheral blood mononuclear cells (PBMC) were obtained from three healthy donors and separated by centrifugation on a Ficoll-Paque (Pharmacia). The cells 2 × 10 ⁵ cells / 200㎕ complete RPMI at a density of ± drug inoculated in a 96-well plate and cultured for 40 hours.

b) Delivery of medication

The drug was dissolved in ethanol and added to the culture medium. The final ethanol concentration in the medium was 1% (v / v).

c) Survival rate test

Survival rates were determined using the MTS assay (CellTiter 96TM AQueous Non-Radioactive Cell Proliferation Assay) from Promega. Reagent (40)) was added to each well, and absorbance was measured at 490 nm after 1 to 4 hours elapsed. The effect of drug on cell viability was compared with medium (100% survival rate) which was set to be free of drug. Wells containing no cells were used as a measure of basal absorbance (0% survival). Experiments were conducted for each concentration condition using more than 3-wells. The concentration (IC50) required to inhibit the growth of control cells by 50% was calculated by linear regression.

result

AZT-Suc-GTP ₁ was 40 times more cytotoxic to JURKAT cells than the parent drug (Table 4). This conjugate was also more cytotoxic to PBMC than AZT.

Cytotoxicity of AZT and AZT-Suc-GTP ₁ on PBMC and JURKAT cells Cell type COMPOSITION IC50 ([mu] M) JURKAT AZT 1720.8 AZT-Suc-GTP ₁ 43.0 PBMC AZT 105 AZT-Suc-GTP ₁ 26.7

II. Anti-HIV activity

The anti-HIV activity of AZT-Suc-GTP ₁ and AZT-Suc-GTP ₂ was also tested. Infected cells were incubated with the compound for 5 days. The compounds were dissolved in DMSO and used 5 times diluted. The following parameters were measured: happosite formation, viral antigen gp120 production, cell survival. Cytotoxicity was also measured in uninfected cells.

Two cell lines were used:

HIV-1 MN-infected C8166 (human T-lymphoblastoid cell)

HIV-IU4550-infected JM (semi-mature human T-cell to lymphoblastoid leukemia)

AZT showed little activity in JM cells, presumably because of insufficient phosphorylation, which was used to confirm the mode of action of the compound.

result

Effect of AZT-Suc-GTP ₁ , AZT-Suc-GTP ₂ , AZT and ddI on cell growth and HIV infection (Ag gp120 production) in JM and C8166 cells COMPOSITION JM C8166 EC50 IC50 EC50 IC50 AZT-Suc-GTP ₁ 8 μM 100 μM 0.012 μM 100 μM AZT-Suc-GTP ₂ 200 [mu] M 200 [mu] M 0.032 μM 200 [mu] M AZT 100 μM 5000 μM 0.016 μM 5000 μM ddI 1 μM 100 μM EC50 = concentration that reduces Ag gp120 by 50% in infected cells IC50 = concentration that reduces cell growth by 50%

The AZT-Suc-GTP ₁ conjugate showed cytotoxicity 50 times greater than AZT for both cell lines (Table 5); This result is consistent with the experimental results for PBMC and JURKAT cells (Table 4). The cytotoxicity of P ₂ conjugates was observed using JM and C8166 cells (25 times more cytotoxic than AZT).

AZT: ratio of conjugate EC50 (using data in Table 5) Cell line AZT: AZT-Suc-GTP ₁ AZT: AZT-Suc-GTP ₂ JM 12.5 About 0.5 C8166 1.3 0.5

The virus in the JM cell line was 12.5 times more sensitive to AZT-Suc-GTP ₁ than AZT (Table 6). This increase in toxicity to the virus has not been reflected in the pattern of HIV infection (happosite formation or growth profile of infected cells); This effect is only observed by detection of the antigen. The JM cell line did not metabolize AZT to the same extent as in C8166, but the level of triphosphate in the cell is higher because of the increased delivery of AZT through the P ₁ junction.

Review

AZT-Suc-GTP ₁ showed higher cytotoxicity to PBMC, JURKAT, C8166 and JM cells than AZT, suggesting increased cellular uptake. The kinase of the cell converts AZT to AZT-triphosphate, the latter inhibits HIV reverse transcriptase (RT), and inhibits α-DNA polymerase of cells at 100-fold concentration. The intracellular uptake is increased by the formation of the junction, and the cytotoxicity is also increased (Table 5). In infected JM cells, AZT-Suc-GTP ₁ is effective in reducing the amount of Ag gp120 produced by HIV, but due to the cytotoxicity of this compound itself, the cells did not regain normal growth.

This result is positive because it is consistent with the fact that the conjugate is absorbed and metabolized by the cells and AZT is released in a normal manner. Their cytotoxicity (which, if properly targeted, may also have a beneficial effect) is higher than unmodified AZT, but lower than the levels that interfere with use.

Based on these findings, we can re-examine the major limitations of current AZT therapies, such as fast clearance, low bioavailability, and a low delivery rate to the lymphatic system, the main organ of the added virus. According to Charman and Porter (1996 Advanced Drug Delivery Reviews in press), an important advantage of a hydrophobic prodrug is that it can: i) avoid the liver first-pass effect after oral administration; and ii) There is a possibility of targeting. Furthermore, the behavior of hydrophobic prodrugs such as AZT-Suc-GTP ₁ indicates a sustained release profile. Although the cytotoxicity of the conjugate is higher than that of AZT, this feature may reduce the overall required dose of AZT, thereby reducing side effects.

Example 12

Chlorambucil

The cytotoxicity of chlorambucil and chlorambucilic fatty acid conjugates (CGTP ₁ , P ₂ and P ₃ ) was examined in vitro using JURKAT cells (human acute T cell leukemia). After the cells were incubated with the drug, the survival rate was determined by a colorimetric proliferation test. Cytotoxicity to other cell lines was also studied (PC3 human prostate cancer cells, B16 mouse melanoma cells and human peripheral blood mononuclear cells).

Way

a) Cell culture conditions

JURKAT cells to 1.6 × 10 ⁴ cells / 200㎕ complete RPMI at a density of ± drug inoculated in a 96-well plate and incubated 64 hours. A 1: 3 split of adherent PC3 cells in a 75 cm ² flask was inoculated into 96-well plates and cultured for 48 h in complete RPMI. The medium was removed and complete RPMI + drug was added to the wells and further incubated for 72 hours.

A 1: 8 split of coalesced B16 cells in a 75 cm ² flask was inoculated into 96-well plates and cultured in complete EMEM for 48 hours. The medium was removed and complete EMEM + drug was added to the wells and further cultured for 48 hours.

Human peripheral blood mononuclear cells (PBMC) were obtained from one healthy donor and separated by centrifugation on a Ficoll-Paque (Pharmacil). The cells 2 × 10 ⁵ cells / 200㎕ complete RPMI at a density of ± drug inoculated in a 96-well plate and cultured for 40 hours.

b) Delivery of medication

Two types of delivery were tested:

i) Ethanol. The drug was dissolved in ethanol and added to the culture medium. The final ethanol concentration in the medium was 1% (v / v). This delivery method was used for experiments using JURKAT, PBMC, PC3 and B16 cell types.

ii) Plate coating. The drug was dissolved in ethanol and a fraction (40 ㎕ or less) was added to 90% (v / v) isopropanol. Then, the solution (50 μl) was added to the wells of the 96-well plate, and the solvent was evaporated by heating at 37 ° C., followed by addition of culture medium ± cells. This delivery method was used in several experiments using JURKAT cells.

c) Survival rate test

Survival rates were determined using the MTS assay (CellTiter 96TM AQueous Non-Radioactive Cell Proliferation Assay) from Promega. Reagent (40)) was added to each well, and absorbance was measured at 490 nm after 1 to 4 hours elapsed. The effect of drug on cell viability was compared with medium (100% survival rate) which was set to be free of drug. Wells that did not contain cells were used as a measure to demonstrate background absorbance (0% survival). Experiments were conducted for each concentration condition using more than 3-wells. The concentration (IC50) required to inhibit the growth of control cells by 50% was calculated by linear regression.

result

a) Cytotoxicity of drug

For all tested cell types, the cytotoxicity of CGTP ₁ was greater than that of chlorambucil (FIG. 7).

The concentration of chloramphenicol and CGTP ₁ (IC ₅₀ ) required to inhibit cell growth of control cells by 50% in different cell types Cell type IC50 The ratio of chlorambucil to CGTP ₁ Chlorambucil (uM) CGTP ₁ (uM) JURKAT 100.6 6.5 15.5 PC3 160.2 10.7 15.0 B16 36.5 23.1 1.6 PBMC (1) 45.0 7.1 6.3 PBMC (3) 22 5.5 About 4 () = Number of donors who separated PMBC

15-fold increase in activity was observed in JURKAT cells. The toxicity of CGTP ₁ to normal cells (PBMC) did not increase that much (about 5-fold). JURKAT cells appear to be more resistant to chloramphenicol than PBMCs. CGTP ₁ was toxic to both cells at similar concentrations. GTP ₁ conjugates also showed greater toxicity for PC3 and B16 cell types.

CGTP ₁ showed greater toxicity to JURKAT cells than mixtures of equimolar amounts of chlorambucil and GTP ₁ . GTP ₁ had no activity. This suggests that a process of conjugating chlorambucil to GTP ₁ is required for increased toxicity as observed (Tables 7, 8 and 9).

b) Effect of vehicle

Both of the vehicles (ethanol and isopropanol) showed no cytotoxicity in the dose range tested. Coating the plates to deliver the compounds increased the IC50 of chlorambucil and CGTP ₁ (Table 8), but the activity of CGTP ₂ and CGTP ₃ , which were not observed when delivering these compounds to the ethanol medium, (Table 9). This effect appears to be due to the reduced stability of the P ₂ and P ₃ conjugates in aqueous in vitro test conditions.

Plate coating and IC50 comparison of chloramphenicol and CGTP ₁ when delivered to JURKAT by addition of ethanol to the medium COMPOSITION IC50 (uM) ethanol Coated Chlorambucil 100.6 218.6 CGTP ₁ 6.5 8.6

Cytotoxicity of chlorambucil and fatty acid conjugates on JURKAT cells. The drug is coated on the plate. COMPOSITION IC50 (uM) CGTP ₁ 8.4 CGTP ₂ 17.7 CGTP ₃ 30.0 Chlorambucil 218.7

Review

Increased biological activity of the P ₁ conjugate of chlorambucil has been demonstrated in a variety of cell types. CGTP ₂ and CGTP ₃ also showed improved cytotoxicity against JURKAT cells.

Example 13

morphine

The biological activity of morphine (Mo) and myoxidase conjugate was tested by an antinociceptive assay using a mouse. The various doses of this drug and the persistence of the pain relief effect were examined.

Way

In the experiment, Quackenbush strain mice (male, 5 weeks old, about 30 g in vivo) were used. Before and during the experiment, the animals were free to access feed and water. The experimental animals were divided into 12 groups and weighed. The average weight for each group was calculated. The animals were then placed on a metal block heated to 50 ° C, and the time taken for the animal to show signs of discomfort was measured (baseline response; control group). Various doses of morphine or morphine-fatty acid conjugates were then injected into the abdominal cavity of mice (ip). Morphine is the H ₂ O solution, Morphine-fatty acid is 10% glyceryl monooleate and 21% Miglyol (Miglyol), 6% ethanol, containing 24% Tween 80 (33%) and 39% H ₂ O An injection was prepared with the emulsion. 0.2 ml of the appropriate solution was injected into each mouse with ip. The reaction on the hot plate was measured at regular intervals after the injection. Three doses of morphine and morphine-fatty acids and two time points were tested in each experiment. One mouse was used for each treatment condition, and the whole experiment was repeated three times. All animal responses were photographed and, in some cases, the behavior of the mice was assessed by an independent panel that did not know about the treatment of the mice. Members of the panel rated the behavior of the mice according to the following criteria:

0 = Mouse in pain with excitement

1 = mild discomfort

2 = Not excited and uncomfortable but bordered

3 = calm and calm; I do not seem to be nervous at all

The panel results were averaged.

The coordination of the mice after treatment was evaluated by placing the treated animals on a rotor-rod at 1.6 rpm. The ability of the experimental animals to stay on the rod was evaluated.

result

Experiments 1. Effect of morphine and morphine-Suc-GTP ₂ (5, 10 20 mg morphine / kg) at 1 hour and 4 hours after injection into mice

The same experiment was repeated four times and analyzed by analysis of variance. There was no significant difference between the different experimental doses. However, when different doses of each drug were collected, differences were found (Table 10).

Morphine and morphine-Suc-GTP ₂ mice were ip injected with 1 hour and 4 hours of response and behavior scores. Results are expressed as mean ± SEM. Twelve mice per group. COMPOSITION Time response ± SEM (sec) Behavior score ± SEM 1 hours 4 hours 1 hours 4 hours Morten 32.7 ± 2.74 28.2 ± 2.74 2.46 ± 0.166 1.93 + 0.166 Morphine-Suc-GTP ₂ 26.7 ± 2.74 31.2 ± 2.74 2.34 ± 0.166 2.35 ± 0.166

The results were analyzed by analysis of variance.

P = 0.13 for time response; For behavior scores, p = 0.11.

In both time-response and behavioral data, the morphine-Suc-GTP ₂ case shows a longer pain-inhibiting effect than the unconjugated morphine. Integrity experiments were also performed on mice treated with morphine-Suc-GTP ₂ . As with the morphine treated mice, these mice were able to stay on the rotor-rod after 1 hour and 4 hours after injection. Suggesting that morphine-Suc-GTP ₂ has a true pain-inhibiting effect without affecting the ability of the mouse to integrate.

Experiment 2. Time course of morphine and morphine-Suc-GTP ₂ activity of 5 mg Mo / kg

There was almost no activity at 1 hour after injection but increased after 4 hours and 6 hours (Table 11), indicating that morphine was slowly released from morphine-Suc-GTP ₂ . Morphine showed maximal activity after 1 hour and then decreased with time.

Morphine or morphine-Suc-GTP ₂ (5 mg Mo / kg) ip injection, and after 1, 4 and 6 hours elapsed, Time (hr) Average Time Response (sec) Morten Mo-Suc-GTP ₂ One 27.8 (4) 24.0 (4) 4 23.9 (8) 30.5 (8) 6 20.0 (4) 27.8 (4)

(n) = number of repetitions

Experiments 3. Effect of morphine and Mo-Suc-GTP ₃ (1 and 10 mg morphine / kg) in mice after 4 and 6 hours

Mo-Suc-GTP ₃ showed a longer lasting effect than morphine (Table 12).

Morphine or morphine-Suc-GTP ₃ ip after 4 and 6 hours after ip injection. Results are expressed as average time response (sec). Repeat the same experiment 4 times. Capacity (mg Mo / kg) Morten Mo-Suc-GTP ₃ 4 hours 6 hours 4 hours 6 hours One 23.2 20.8 23.5 27.8 10 23.0 24.2 29.8 29.0

Standard error of each mean = ± 2.86

The conjugation of morphine to GTP ₂ resulted in pain-inhibiting activity in mice and did not affect global locomotor activity. Morphine-GTP ₂ showed a sustained delivery-inhibiting effect and sustained delivery profile over morphine, the parent drug. Morphine-Suc-GTP ₃ showed similar effects.

Example 14

L-DOPA

The biological activity of L-DOPA and L-DOPA-GTP ₂ was tested in animal models of Parkinson's disease, a condition caused by low levels in the brain of neurotransmitter dopamine. Mice were pretreated with lesser amounts of dopamine depleted and less sensitive to animals. The ability of L-DOPA and L-DOPA-GTP ₂ to switch these conditions was measured.

Way

(Male, 5 weeks old, about 30 g in a living body). Before and during the experiment, the animals were free to access feed and water. Mice were intraperitoneally injected with (ip) lesser fins (5 mg / kg) and tested for tension after 4 hours. The mice were judged to be strained if they could stay on a 4.5 cm diameter stopper for 5 minutes. Immediately prior to use and observation, strained mice were ip injected with L-DOPA or L-DOPA-GTP ₂ suspended in miglyol (200-400 uL / mouse).

result

Experiment 1. Antagonist activity of L-DOPA

When dosing at doses of 364 mg / kg or more, all animals became active after administration of L-DOPA to the tensed mice (Table 13). The activity level was higher than that of normal mice. Animals were spastic and showed toxic effects of L-DOPA on the hind legs such as rearing, salivating and jumping.

Anti-Lesser Pin Activity of L-DOPA L-DOPA residue (mg / kg) Reaction at 15 min Mouse 1 Mouse 2 9.1 - - 91 - + 364 + + 637 + + 910 + + - = persistent tension + = very active

Experiment 2. Antirepressor activity of L-DOPA and L-DOPA-GTP ₂

As previously observed in Experiment 1, L-DOPA makes the tense mice very active (Table 14). But not with miglyol alone or with small doses of L-DOPA-GTP ₂ (9.1 and 91 mg DOPA / kg). Mice receiving high doses of L-DOPA-GTP ₂ (364 and 637 mg DOPA / kg) showed signs of recovery at 7 and 5 hours after each dose, respectively. These animals moved slowly, sporadically, and did not exhibit toxic effects due to treatment with high activity levels and L-DOPA. These results indicate that L-DOPA-GTP ₂ is capable of producing dopamine in the stressed mouse brain. Slow release due to the time it takes to react and the nature of the reaction (overactive mice at 20 min compared to slowing at 5 and 8 h)

Anti-Lesser Activity of L-DOPA and L-DOPA-GTP ₂ DOPA Residue (mg / kg) 20 minutes 2 hours 5 hours 6 hours 7 hours 8 hours 0 (3) --- --- --- --- --- --- L-DOPA 364 (1) + 637 (1) + DOPA-GTP ₂ 9.1 (2) - - - - - - 91 (2) - - - - - - 364 (2) - - - - M- MM 637 (2) - - M M M M - = no reaction; + = Very active; M = slow movement (n) = number of animals treated

discussion

L-DOPA-GTP ₂ has the ability to switch to Lesser Pine-Induced Tension in mice. After the administration of L-DOPA-GTP ₂ , a slow release effect was observed, and no toxic effect was observed in L-DOPA.

Example 15

Tumor cytotoxicity model: Testing of methotrexate and chlorambucil and its fatty acid conjugates

protocol

C57 black mice were given an initial dose of 2 X 10 < ⁵ > B16 melanoma cells by intradermal injection. Cells were suspended in MEM without serum. The abdomen was epilated and 100 μl of cell + MEM was injected. After 8 to 10 days of injection, growth of B16 tumor appeared at the injection site. Immediately prior to injecting the drug, the tumor was measured in micrometers and photographed (8-10 days).

The cytotoxic drug and its fatty acid conjugate were dissolved or suspended in 4: 1 soybean oil: ethyl oleate. 10 mg / ml or 20 mg / ml of solution of each mother drug or fatty acid conjugate was given at the same molar concentration, and the mother liquor solution was then injected at the same site as the tumor with a dose of 0.5 mg or 1 mg. The tumors were then photographed, measured, and re-injected every 2 or 3 days.

Tumor growth rates are shown in Table 15, and a statistical analysis of the effects on drug and drug conjugates is presented in Table 16.

Effect of Drug and Drug Conjugate in B5 Tumor Volume (mm ³ ) in C57 Mice process 1 day 3 days 5 days 6 days 7 days Drug dose Vehicle / 1 6.3 26 54 99.3 174 0 Vehicle / 2 14.1 142 138.7 261.8 419 0 Vehicle / 3 19.8 14.1 254.1 410.3 780 0 Vehicle / 4 22.4 55.9 158.1 290.4 473 0 MTX / 1 0.5 13.6 73.5 125.1 190 0.5 mg MTX / 2 3.8 4.2 14.1 14.1 21 0.5 mg MTX / 3 14.1 30.4 86.0 215.0 234 0.5 mg MTX / 4 26 86 219.4 266.5 515 0.5 mg MTXGTP _2/1 0.5 0 0 0 0 0.5 mg MTXGTP _2/2 0.5 0 11.8 19.8 26 0.5 mg MTXGTP _{2 of} 3 2.7 0 0 0 0 0.5 mg MTXGTP _2/4 16.6 16.6 64.5 77.6 113 0.5 mg MTXGTP _2/5 0.1 0.1 0.5 0.5 14 1.0 mg MTXGTP _2/6 8.2 10.8 21.8 46.8 50 1.0 mg MTXGTP _2/7 8.2 0 0 0 0 1.0 mg MTXGTP _2/8 10.8 30.4 142.5 261.8 379 1.0 mg Chlor / 1 0.5 0.5 0 0 0 0.5 mg Chlor / 2 1.8 0 0 0 0 0.5 mg Chlor / 3 8.2 21.8 7.7 7.7 5.9 0.5 mg Chlor / 4 16.6 38.3 108.4 125.1 174.1 0.5 mg CGTP _2/1 0.5 0.3 11.8 19.8 21.8 0.5 mg CGTP _2/2 1.0 One 19.1 13.6 35.1 0.5 mg CGTP _2/3 2.7 0 0.0 0 0 0.5 mg CGTP _2/4 14.1 27.4 82.8 167.4 152.8 0.5 mg CGTP _2/5 5 0 0 0 0 1.0 mg CGTP _2/6 5 7.7 38.3 61.6 92.6 1.0 mg CGTP _2/7 8.2 32.7 111.9 108.4 254.1 1.0 mg CGTP _2/8 38.3 73.5 174.1 240.3 215.0 1.0 mg ** MTX = Methotrexate Chlor or (C) = chlorambucil ** GTP2 = Gly-Tris- di-palmitate ** / 1 to / 8 indicates the number of each mouse, for example, CGTP _2/4 = CGTP ₂ mouse number 4

Mean and standard error for B16 tumor volume in C57 mice. The values for each measurement date are adjusted for various initial volumes. process 13/11/95 15/11/95 16/11/95 17/11/95 Vehicle 3.58 (0.533) 5.36 (0.8340) 6.69 (1.00) 8.33 (1.21) Chlorambucil (0.5 mg) 3.01 (0.610) 3.29 (0.955) 3.46 (1.15) 3.65 (1.38) CGTP ₂ (0.5 mg) 2.39 (0.523) 4.07 (0.881) 4.84 (1.06) 4.90 (1.28) CGTP ₂ (1.0 mg) 2.58 (0.523) 3.89 (0.819) 4.23 (0.98) 4.84 (1.19) MTXGTP ₂ (0.5 mg) 2.04 (0.565) 3.37 (0.884) 3.76 (1.06) 4.03 (1.28) MTXGTP ₂ (1.0 mg) 2.30 (0.536) 3.40 (0.839) 4.13 (1.01) 4.89 (1.22) Methotrexate (0.5 mg) 3.58 (0.610) 5.47 (0.955) 6.07 (1.15) 7.24 (1.38)

Unit is a statistically modified tumor volume; The figure in parentheses is SD. The results are all at a 95% significance level, except when MTX is not significantly different from the control. All of the rest are significantly different from the control but not different from each other.

The results show the following.

There was no significant difference between vehicle and methotrexate (MTX) in the treated mice.

2. There was a significant difference between vehicle / MTX and MTXGTP ₂ junctions at two different concentrations, but there was no difference between the two conjugate groups.

3. There was a significant difference between vehicle and chlorambucil, CGTP ₂ (0.5 mg) and CGTP ₂ (1.0 mg).

4. There was no significant difference between chlorambucil and two conjugate groups or between them.

5. High toxicity was observed in the chlorambucil test group (3 out of 7 mice died); No toxicity was seen in the other test groups.

6. The cytotoxicity of chlorambucil conjugates does not have obvious advantages compared to free chlorambucil, but the low toxicity of the conjugates provides therapeutic advantages.

conclusion

The present inventors have found that when applied to skin permeation, the nonsteroidal anti-inflammatory drug exhibits sustained activity as compared with unmodified parent drug when it is modified by one to three kinds of fatty acid acyl derivatives International Patent Application PCT / AU94 / 00440, incorporated by reference in its entirety). The present inventors have now found that when other therapeutic agents are modified in a similar manner, they are biologically active and exhibit altered activity as compared to unmodified drugs.

It will be apparent to those skilled in the art that various modifications and / or variations can be made in the present invention without departing from the spirit and scope of the invention as broadly described, as shown in the specific embodiments. Therefore, this embodiment should be considered both in the described aspects and in the non-limiting aspects.

Claims (68)

A compound having the general formula: [Wherein, in the above formula X is a corticosterone hormone or drug component, connected to Y through a hydroxy group, Y is a spacer group, AA is an amino acid, n is 0 to 5, B is H or CH ₂ OR ₃ , R ₁ , R ₂ and R ₃ are the same or different and each is an acyl group derived from hydrogen, methyl, ethyl or fatty acid, and at least one of R ₁ , R ₂ and R ₃ is an acyl group derived from a fatty acid. 2. The compound according to claim 1, wherein X is hydrocortisone or cortisone. 3. The compound according to claim 1 or 2, wherein Y is dicarboxylic acid, AA is absent or glycine or alanine, and the linkage is via a hydroxyl group at the 21-position. A compound having the general formula: X - Y - [AA] n - NH - CH 2 - CH 2 O - R 4 [Wherein, in the above formula X is a corticosterone hormone or drug component, connected to Y through a hydroxy group, Y is a spacer group, AA is an amino acid, n is 0 to 5, R ₄ is an acyl group derived from a fatty acid.] 5. The compound according to claim 4, wherein X is hydrocortisone or cortisone. 6. The compound according to claim 4 or 5, wherein Y is dicarboxylic acid, AA is absent or glycine or alanine, and the linkage is through a hydroxyl group at the 21-position. A method of sustaining or altering the activity of a corticosterone hormone or drug component by administering a compound of the general formula: [Wherein, in the above formula X is a corticosterone hormone or drug component, connected to Y through a hydroxy group, Y is a linking group, AA is an amino acid, n is 0 to 5, B is H or CH ₂ OR ₃ , R ₁ , R ₂ and R ₃ are the same or different and each is an acyl group derived from hydrogen, methyl, ethyl or fatty acid, and at least one of R ₁ , R ₂ and R ₃ is an acyl group derived from a fatty acid. 8. The method of claim 7, wherein X is hydrocortisone or cortisone. 9. The method according to claim 7 or 8, wherein Y is dicarboxylic acid, AA is absent or glycine or alanine, and the linkage is via a hydroxyl group at position 21. A method of sustaining or altering the activity of a corticosterone hormone or drug component by administering a compound of the general formula: X - Y - [AA] n - NH - CH 2 - CH 2 O - R 4 [Wherein, in the above formula X is a corticosterone hormone or drug moiety and is linked to Y through a hydroxy group, Y is a spacer group, AA is an amino acid, n is 0 to 5, B is H or CH ₂ OR ₃ , R ₄ is an acyl group derived from a fatty acid.] 11. The method of claim 10, wherein X is hydrocortisone or cortisone. The method according to claim 10 or 11, wherein Y is dicarboxylic acid, AA is absent or glycine or alanine, and the linkage is through a hydroxyl group at position 21. A compound having the general formula: [Wherein, in the above formula X is a morphinic component and is connected to Y via a hydroxy group (e.g., a hydroxy group at the 3 or 6 position) Y is a spacer group, AA is an amino acid, n is 0 to 5, B is H or CH ₂ OR ₃ , R ₁ , R ₂ and R ₃ are the same or different and each is an acyl group derived from hydrogen, methyl, ethyl or fatty acid, and at least one of R ₁ , R ₂ and R ₃ is an acyl group derived from a fatty acid. 14. The compound of claim 13, wherein X is morphine modified at the 3 or 6 position, wherein Y is dicarboxylic acid, AA is absent or glycine or alanine. A compound having the general formula: X - Y - [AA] n - NH - CH 2 - CH 2 O - R 4 [Wherein, in the above formula X is a morphine-based component and is connected to Y through a hydroxyl group at the 3- or 6-position, Y is a spacer group, AA is an amino acid, n is 0 to 5, R ₄ is an acyl group derived from a fatty acid.] 16. The compound of claim 15, wherein X is morphine modified at the 3 or 6 position, and Y is dicarboxylic acid, AA is absent or glycine or alanine. A method of sustaining or altering the activity of a morphine based compound by administering a compound of the general formula: [Wherein, in the above formula X is a morphine-based component and is connected to Y through a hydroxyl group at the 3- or 6-position, Y is a linking group, AA is an amino acid, n is 0 to 5, B is H or CH ₂ OR ₃ , R ₁ , R ₂ and R ₃ are the same or different and each is an acyl group derived from hydrogen, methyl, ethyl or fatty acid, and at least one of R ₁ , R ₂ and R ₃ is an acyl group derived from a fatty acid. 18. The method of claim 17, wherein X is morphine modified at the 3 or 6 position, wherein Y is dicarboxylic acid, AA is absent or glycine or alanine. A method of sustaining or altering the activity of a morphine based compound by administering a compound of the general formula: X - Y - [AA] n - NH - CH 2 - CH 2 O - R 4 [Wherein, in the above formula X is a morphine-based component and is connected to Y through a hydroxyl group at the 3- or 6-position, Y is a spacer group, AA is an amino acid, n is 0 to 5, R ₄ is an acyl group derived from a fatty acid.] 20. The method of claim 19, wherein X is morphine modified at the 3 or 6 position, wherein Y is dicarboxylic acid, AA is absent or glycine or alanine. A compound having the general formula: [Wherein, in the above formula X is an antiviral nucleoside, connected to Y via a hydroxy group, Y is a spacer group, AA is an amino acid, n is 0 to 5, B is H or CH ₂ OR ₃ , R ₁ , R ₂ and R ₃ are the same or different and each is an acyl group derived from hydrogen, methyl, ethyl or fatty acid, and at least one of R ₁ , R ₂ and R ₃ is an acyl group derived from a fatty acid. 22. The compound of claim 21, wherein X is AZT. 23. The compound according to claim 21 or 22, wherein Y is dicarboxylic acid, AA is absent or glycine or alanine. A compound having the general formula: X - Y - [AA] n - NH - CH 2 - CH 2 O - R 4 [Wherein, in the above formula X is an antiviral nucleoside, connected to Y via a hydroxy group, Y is a spacer group, AA is an amino acid, n is 0 to 5, R ₄ is an acyl group derived from a fatty acid.] 25. The compound of claim 24, wherein X is AZT. 26. The compound according to claim 24 or 25, wherein Y is dicarboxylic acid, AA is absent or glycine or alanine. A method of sustaining or altering the activity of an antiviral nucleoside by administering an antiviral nucleoside of the general formula: [Wherein, in the above formula X is an antiviral nucleoside, connected to Y via a hydroxy group, Y is a linking group, AA is an amino acid, n is 0 to 5, B is H or CH ₂ OR ₃ , R ₁ , R ₂ and R ₃ are the same or different and each is an acyl group derived from hydrogen, methyl, ethyl or fatty acid, and at least one of R ₁ , R ₂ and R ₃ is an acyl group derived from a fatty acid. 28. The method of claim 27, wherein X is AZT. 29. The method according to claim 27 or 28, wherein Y is dicarboxylic acid, AA is absent or glycine or alanine. A method of sustaining or altering the activity of an antiviral nucleoside by administering an antiviral nucleoside of the general formula: X - Y - [AA] n - NH - CH 2 - CH 2 O - R 4 [Wherein, in the above formula X is an antiviral nucleoside, connected to Y via a hydroxy group, Y is a spacer group, AA is an amino acid, n is 0 to 5, R ₄ is an acyl group derived from a fatty acid.] 31. The method of claim 30, wherein X is AZT. 32. The method of claim 30 or 31, wherein Y is dicarboxylic acid, AA is absent or glycine or alanine. A compound having the general formula: [Wherein, in the above formula X is a component of a cyclosporine drug, which is linked to Y via a hydroxy group, Y is a spacer group, AA is an amino acid, n is 0 to 5, B is H or CH ₂ OR ₃ , R ₁ , R ₂ and R ₃ are the same or different and each is an acyl group derived from hydrogen, methyl, ethyl or fatty acid, and at least one of R ₁ , R ₂ and R ₃ is an acyl group derived from a fatty acid. 34. The compound of claim 33, wherein X is cyclosporin C. 34. The compound according to claim 33 or 34, wherein Y is dicarboxylic acid, AA is absent or glycine or alanine. A compound having the general formula: X - Y - [AA] n - NH - CH 2 - CH 2 O - R 4 [Wherein, in the above formula X is a component of a cyclosporine drug, which is linked to Y via a hydroxy group, Y is a spacer group, AA is an amino acid, n is 0 to 5, R ₄ is an acyl group derived from a fatty acid.] 38. The compound of claim 36, wherein X is cyclosporin C. 38. The compound according to claim 36 or 37, wherein Y is dicarboxylic acid, AA is absent or glycine or alanine. A method of continuing or altering the activity of a cyclosporine drug by administering a compound of the general formula: [Wherein, in the above formula X is a component of a cyclosporine drug, which is linked to Y via a hydroxy group, Y is a linking group, AA is an amino acid, n is 0 to 5, B is H or CH ₂ OR ₃ , R ₁ , R ₂ and R ₃ are the same or different and each is an acyl group derived from hydrogen, methyl, ethyl or fatty acid, and at least one of R ₁ , R ₂ and R ₃ is an acyl group derived from a fatty acid. 40. The method of claim 39, wherein X is cyclosporin C. 41. The method according to claim 39 or 40, wherein Y is dicarboxylic acid, AA is absent or glycine or alanine. A method of continuing or altering the activity of a cyclosporine drug by administering a compound of the general formula: X - Y - [AA] n - NH - CH 2 - CH 2 O - R 4 [Wherein, in the above formula X is a component of a cyclosporine drug, which is linked to Y via a hydroxy group, Y is a spacer group, AA is an amino acid, n is 0 to 5, R ₄ is an acyl group derived from a fatty acid.] 43. The method of claim 42, wherein X is cyclosporin C. 44. The method according to claim 42 or 43, wherein Y is dicarboxylic acid, AA is absent or glycine or alanine. A compound having the general formula: [Wherein, in the above formula X is a folic acid antagonist component, which is linked to Y via a carboxy group, Y is optionally a spacer group, AA is an amino acid, n is 0 to 5, B is H or CH ₂ OR ₃ , R ₁ , R ₂ and R ₃ are the same or different and each is an acyl group derived from hydrogen, methyl, ethyl or fatty acid, and at least one of R ₁ , R ₂ and R ₃ is an acyl group derived from a fatty acid. 46. The method of claim 45 wherein X is methotrexate and Y is absent or is an amino acid, glycolic acid, 3-hydroxypropionic acid, or lactic acid, AA is absent, glycine or alanine, Is an amide bond or an ester bond linked to? -Carboxy of the glutamate residue of methotrexate. A compound having the general formula: X - Y - [AA] n - NH - CH 2 - CH 2 O - R 4 [Wherein, in the above formula X is a folic acid antagonist component, which is linked to Y via a carboxy group, Y is optionally a spacer group, AA is an amino acid, n is 0 to 5, R ₄ is an acyl group derived from a fatty acid.] 48. The method of claim 47 wherein X is methotrexate and Y is absent or is an amino acid, glycolic acid, 3-hydroxypropionic acid, or lactic acid, AA is absent, glycine or alanine, Is an amide bond or an ester bond linked to? -Carboxy of the glutamate residue of methotrexate. A method of continuing or altering the activity of a folate antagonist compound by administering a compound of the general formula: [Wherein, in the above formula X is a folic acid antagonist component, which is linked to Y via a carboxy group, Y is optionally a spacer group, AA is an amino acid, n is 0 to 5, B is H or CH ₂ OR ₃ , R ₁ , R ₂ and R ₃ are the same or different and each is an acyl group derived from hydrogen, methyl, ethyl or fatty acid, and at least one of R ₁ , R ₂ and R ₃ is an acyl group derived from a fatty acid. 50. The conjugate of claim 49, wherein X is methotrexate, Y is absent or is an amino acid, glycolic acid, 3-hydroxypropionic acid or lactic acid, AA is absent or glycine or alanine, Is an amide bond or an ester bond linked to? -Carboxy of the glutamate residue of methotrexate. A method of continuing or altering the activity of a folate antagonist compound by administering a compound of the general formula: X - Y - [AA] n - NH - CH 2 - CH 2 O - R 4 [Wherein, in the above formula X is a folic acid antagonist component, which is linked to Y via a carboxy group, Y is optionally a spacer group, AA is an amino acid, n is 0 to 5, R ₄ is an acyl group derived from a fatty acid.] 51. The method of claim 51, wherein X is methotrexate and Y is absent or is an amino acid, glycolic acid, 3-hydroxypropionic acid, or lactic acid, AA is absent, glycine or alanine, Is an amide bond or an ester bond linked to? -Carboxy of the glutamate residue of methotrexate. A compound having the general formula: [Wherein, in the above formula X is a DOPA-based component, which is connected to Y via a carboxy group or an amino group, Y is optionally a spacer group, AA is an amino acid, n is 0 to 5, B is H or CH ₂ OR ₃ , R ₁ , R ₂ and R ₃ are the same or different and each is an acyl group derived from hydrogen, methyl, ethyl or fatty acid, and at least one of R ₁ , R ₂ and R ₃ is an acyl group derived from a fatty acid. 54. The conjugate of claim 53, wherein X is DOPA and Y is absent or is an amino acid, glycolic acid, 3-hydroxypropionic acid, or lactic acid, AA is absent, glycine or alanine, Lt; / RTI > is a bond or an ester bond. A compound having the general formula: X - Y - [AA] n - NH - CH 2 - CH 2 O - R 4 [Wherein, in the above formula X is a DOPA-based component, which is connected to Y via a carboxy group or an amino group, Y is optionally a spacer group, AA is an amino acid, n is 0 to 5, R ₄ is an acyl group derived from a fatty acid.] 56. The method of claim 55, wherein X is DOPA and Y is absent or is an amino acid, glycolic acid, 3-hydroxypropionic acid, or lactic acid, AA is absent, glycine or alanine, Lt; / RTI > is a bond or an ester bond. A method of continuing or altering the activity of a DOPA compound by administering a compound of the formula: [Wherein, in the above formula X is a DOPA-based component, which is connected to Y via a carboxy group or an amino group, Y is optionally a spacer group, AA is an amino acid, n is 0 to 5, B is H or CH ₂ OR ₃ , R ₁ , R ₂ and R ₃ are the same or different and each is an acyl group derived from hydrogen, methyl, ethyl or fatty acid, and at least one of R ₁ , R ₂ and R ₃ is an acyl group derived from a fatty acid. 57. The conjugate of claim 57 wherein X is DOPA and Y is absent or is an amino acid, glycolic acid, 3-hydroxypropionic acid, or lactic acid, AA is absent, glycine or alanine, Bond or an ester bond. A method of continuing or altering the activity of a DOPA compound by administering a compound of the formula: X - Y - [AA] n - NH - CH 2 - CH 2 O - R 4 [Wherein, in the above formula X is a DOPA-based component, which is connected to Y via a carboxy group or an amino group, Y is optionally a spacer group, AA is an amino acid, n is 0 to 5, R ₄ is an acyl group derived from a fatty acid.] 59. The conjugate of claim 59 wherein X is DOPA and Y is absent or is an amino acid, glycolic acid, 3-hydroxypropionic acid, or lactic acid, AA is absent, glycine or alanine, Bond or an ester bond. A compound having the general formula: [Wherein, in the above formula X is a chlorambucyl-based component, connected to Y through a carboxy group, Y is optionally a spacer group, AA is an amino acid, n is 0 to 5, B is H or CH ₂ OR ₃ , R ₁ , R ₂ and R ₃ are the same or different and each is an acyl group derived from hydrogen, methyl, ethyl or fatty acid, and at least one of R ₁ , R ₂ and R ₃ is an acyl group derived from a fatty acid. 62. The method of claim 61, wherein X is chlorambucil and Y is absent or is an amino acid, glycolic acid, 3-hydroxypropionic acid, or lactic acid, AA is absent, glycine or alanine, Linked amide bond or an ester bond. A compound having the general formula: X - Y - [AA] n - NH - CH 2 - CH 2 O - R 4 [Wherein, in the above formula X is a chlorambucyl-based component, connected to Y through a carboxy group, Y is optionally a spacer group, AA is an amino acid, n is 0 to 5, R ₄ is an acyl group derived from a fatty acid.] 66. The method of claim 63, wherein X is chlorambucil and Y is absent or is an amino acid, glycolic acid, 3-hydroxypropionic acid, or lactic acid, AA is absent, glycine or alanine, Linked amide bond or an ester bond. A method of continuing or altering the activity of a chlorambucil system compound by administering a compound of the general formula: [Wherein, in the above formula X is a chlorambucyl-based component, connected to Y through a carboxy group, Y is optionally a spacer group, AA is an amino acid, n is 0 to 5, B is H or CH ₂ OR ₃ , R ₁ , R ₂ and R ₃ are the same or different and each is an acyl group derived from hydrogen, methyl, ethyl or fatty acid, and at least one of R ₁ , R ₂ and R ₃ is an acyl group derived from a fatty acid. 66. The method of claim 65, wherein X is chlorambucil and Y is absent or is an amino acid, glycolic acid, 3-hydroxypropionic acid, or lactic acid, AA is absent, glycine or alanine, Linked amide bond or an ester bond. A method of continuing or altering the activity of a chlorambucil system compound by administering a compound of the general formula: X - Y - [AA] n - NH - CH 2 - CH 2 O - R 4 [Wherein, in the above formula X is a chlorambucyl-based component, connected to Y through a carboxy group, Y is optionally a spacer group, AA is an amino acid, n is from 0 to 5, R ₄ is an acyl group derived from a fatty acid.] 66. The method of claim 67, wherein X is chlorambucil and Y is absent or is an amino acid, glycolic acid, 3-hydroxypropionic acid, or lactic acid, AA is absent, glycine or alanine, Linked amide bond or an ester bond.