WO1990000049A2

WO1990000049A2 - Immunosuppressive analogues and derivatives of succinylacetone

Info

Publication number: WO1990000049A2
Application number: PCT/US1989/002762
Authority: WO
Inventors: Danute E. Nitecki; Margaret Moreland; Lois Aldwin; Corey H. Levenson; Irwin Braude; David F. Mark
Original assignee: Cetus Corporation
Priority date: 1988-06-29
Filing date: 1989-06-23
Publication date: 1990-01-11
Also published as: AU4759990A; WO1990000049A3

Abstract

Succinylacetone derived or related medicaments and methods of synthesis of the same are shown wherein the medicaments consist of succinylacetonyl-proline-PEG, succinylacetonyl-NH-PEG, or compounds that have formula (I), wherein n = 1-6; R = CH3, CF3, -CO2RIV, (a), or (b); RI, RII = H, F, CH¿3?, or (c); R?III¿ = H, -CO¿2R?IV, (d), (e), or tetrazolyl; RIV = H, or alkyl; and that have immunosuppressive activity both in vivo and in vitro based on their activities in cellular immunologic assays and adjuvant induced arthritis in rats, respectively.

Description

IMMUNOSUPPRESSIVE ANALOGUES AND DERIVATIVES

OF SUCCINYLACETONE

Technical Field of the Invention

This invention is in the area of immunology, and specifically relates to immunopharmacology as applied to the development of immunosuppressive drugs that are useful in treating a wide variety of diseases arising from a dysfunctional hyperactive immune system. Compositions and methods of using the same that are particularly useful in treating autoimmune diseases are shown.

Background of the Invention

There is a continuous search for therapeutics that have immunosuppressive activity, and therefore facilitate organ tissue transplants, as well as being beneficially applied to the treatment of various autoimmune diseases. The basis of organ tissue rejection is known and resides in the histocompatibility two locus (H-2) in the mouse, and the human leucocyte antigen (HLA) complex in the human. Klein, J., et al. Ann. Rev. Immunol. 1:119 (1983). Both systems code for cell surface molecules that are recognized as foreign in a recipient host. Only in those instances where the donor and recipient are genetically identical, that is when the donor and recipient are identical twins, is there little or no chance of rejection of the transplanted organ. However, since the donor and recipient are rarely genetically identical, some degree of histocompatibility antigenic mismatch is present, and hence the application of immunosuppressive drugs is required. This is true, even where donors and recipients are HLA matched, that is, matched with the antigens of the major histocompatibility complex loci, since rejection of the transplant can still arise as a result of mismatching of minor genetic loci that are also involved in rejection.

Immunosuppressive drugs are also widely used in the area of graft versus host rejection, particularly bone mairow transplants. The graft from a donor contains a significant number of immunocompetent lymphoid cells that can mount an effective destructive reaction against host cells. Bone marrow transplants are often employed to treat various malignant diseases, including leukemia. Generally this involves immunologically crippling the leukemic patient, and then transplanting bone marrow from a donor. Unless the lymphoid cells in the donor marrow are suppressed they can react against recipient tissue antigens, often with dire consequences.

A variety of drugs, and antisera to lymphoid cells are used as

immunosuppressives. Particularly useful drugs are corticosieroids, othiopime, and cyclosporin. In addition, various monoclonal antibodies, alone or when coupled to a cytotoxic agent are available for ridding the donor marrow of lymphoid cells. P. S. Russell et al., Annual Review Medicine 25:63 (1984). In addition, U.S. Patent 4,670,467 claims succinylacetone (SA, 4, 6-dioxoheptanoic acid) as an

immunosuppressive medicament. Succinylacetone is a seven carbon organic ketoacid.

In addition to being used in the area of organ tissue transplantation, and graft versus host disease, immunosuppressive drugs are widely sought after to treat autoimmune diseases. Autoimmunity can generally be defined (Smith, H. and

Steinberg. A. Autoimmunity - A Perspective Ann. Rev. Immunol. 1:175 (1983)) as the generation of an immune response against a person's own self components.

Autoimmune diseases generally develop spontaneously in humans. Both a person's genetic predisposition, as well as environmental factors may play a role. Those agents which are thought to initiate autoimmunity are poorly defined. However,

autoimmunity can be induced in experimental animals by suitable immunization procedures with known antigens. Classical examples of experimentally induced autoimmune diseases in animals are experimental or allergic encephalomyelitis, and adjuvant induced arthritis. The former is induced by immunization with a myelin basic protein, and induces an autoimmune disease having neurological symptoms involving partial or complete paralysis of the hind legs of animals. The latter entails use of any microbacterium for induction of arthritis in rats. Examples of autoimmune diseases in humans include various forms of diabetes systemic lupus erythematosus, myasthenia gravis, chronic thyroiditis, hemolytic anemia, and multiple sclerosis. Additionally, rheumatoid arthritis is often considered an autoimmune disease.

Many autoimmune diseases arc thought to occur by different immune mechanisms. This includes a cytotoxic mechanism whereby antibody reacts with antigen, and the resulting complex becomes membrane associated. Often this initiates complement mediated lysis of the involved cells. A second mechanism involves the interaction of lymphoid cells, rather than antibodies and complement, with antigen. Often this results in an inflammatory response, such as that seen in rheumatoid arthritus.

Immunosuppressive drugs are used to treat autoimmune diseases, much as they are used to treat organ tissue transplants and graft versus host disease. In the clinical arena, cyclosporin A has been shown to be effective in treating various experimentally induced autoimmune diseases. Shevach, E. Ann. Rev. Immunol. 2-397 (1985). These include experimental allergic encephalomyelitis, and an autoimmune form of diabetes which develops in the BB rat strain. Similarly, cyclosporin A has been applied in the clinical setting, and used to treat patients with posterior uveitis. Similarly, it has been υsεά to treat type-1 diabetes mellitus in humans. Despite these results, however, cyclosporin A has side effects which has limited its use in the clinical setting. In addition to cyclosporin A, other immunosuppresshe drugs have been shown to have a degree of efficacy when used to treat various autoimmune disease. For example. corticosteroids are often used to treat rheumatoid arthritis. Hereto, however, corticosteroids are by no means a cure, but rather provide temporary relief, all-be-it with severe toxic side effects.

Summary of the Invention

An object of the invention is to provide immunosuppressive compounds that are useful in treating patients suffering from diseases associated with hyperactive immune systems.

Another object of the invention is to provide analogues or derivatives of succinylacetone that have hithertofore unknown immunosuppressive activity when applied to the treatment of graft versus host disease, or autoimmune diseases.

A third object of the invention is to provide derivatives of succinylacetone that are maintained in a patient's circulation for longer times than succinylacetone.

A fourth object of the invention is the description of immunosuppressive compounds that have the formula:

wherein

A further object of the instant invention is to describe methods whereby derivatives of succinylacetone can be administered to patients in effective amounts to control or eliminate graft host disease or various autoimmune diseases.

Other objects and advantages of the present invention will become apparent upon a reading of the detailed description below.

Brief Description of the Tables

Table 1 presents data showing the effects of succinylacetonyl-proline derivatized polyethylene glycol 4000 on IL-2 and IFN-ɣ production, as well as the incorporation of tritiated thymidine into human lymphocytes.

Table 2 shows the effect of succinylacetonyl-proline derivatized with polyethylene glycol 4000 in a phytohemagglutinin assay.

Table 3 shows the effect of succinylacetone methyl-ester on the production of IL-2 and IFN-ɣ, and the incorporation of tritiated thymidine in a mixed lymphocyte reaction. Table 4 shows the effect of methyl 4-acetyl-5-oxohexanoate on the production of IL-2 and IFN-ɣ, and the 13 human lymphoid cells in a mixed lymphocyte reaction.

Table 5 shows the effect of succinylacetone methyl ester and methyl 4 acetyl-5-oxohexanoate in a phytohemagglutinin assay.

Table 6 shows the effect of succinylacetone methyl ester on the incorporation of tritiated thymidine by human lymphoid cells in a secondary mixed lymphocyte reaction.

Table 7 shows the effect of methyl 4-acetyl-5-oxohexanoate on the

incorporation of tritiated thymidine by human lymphoid cells in a secondary mixed lymphocyte reaction.

Table 8 shows the effect of succinylacetone methyl ester on the viability of peripheral blood lymphocytes, with, or without stimulation by phytohemagglutinin.

Table 9 shows the effect of methyl 4-acetyl-5-oxohexanoate on the viability of peripheral blood lymphocytes, with, or without stimulation by phytohemagglutinin.

Table 10 shows the effects of succinylacetone and derivatives in a adjuvant induced arthritis animal model system.

Figure 1 shows analogues and derivatives of succinylacetone.

Figure 2 shows the Sephadex G-50 chromatographic separation of

succinylacetonyl-proline-PEG.

Figure 3 shows the absorbance spectra of fractions 15-34 from the Sephadex G- 50 column.

Figure 4 shows the absorbance spectrum of PEG-4000 NH

Description of the Invention

The present invention describes several novel immunosuppressive molecules that are analogues or derivatives of succinylacetone (SA).

Most of the compounds can be described by the following formula:

wherein

Specific examples of preferred embodiments of the subject immunosuppressive compounds are shown in Figure 1, along with the structure of succinylacetone.

Figure 1

Succinyl Acetone (SA)

Methyl ester-SA

Methyl 4 acetyl-5-oxohexanoate

Figure 1 Continued

SA-pro-NH-PEG(4K)

Butyl SA

n-octvlamide-SA

n-butylamide-SA

t-butylamide-SA

glutarylacteone methyl ester

glutarylacetone (acid)

adipoylacctone methyl esser

Figure 1 Continued

SA-hexylamide

6-hydroxy-4-oxo heptanoic acid derivative

4-hydroxy-4-oxo heptanoic acid derivatives

4-6-dioxo 7,7,7-trifluoro heptanoic acids

4,6-dioxo 5,5-difluoro heptanoic acid

4,6-dioxo 5,5-dimethyl heptanoic acid

4,6-dioxohexanoic acid derivatives

4,6-dioxopimelic acid derivatives

Figure 1 Continued

4,6,8-trioxononanoic acid derivatives

3,5-dioxohexanoic phosphonic acid derivatives

5-(3,5-dioxohexyl) tetrazole

4,6-dioxoheptyl alcohol and its phosphate ester

4,4-diacetyl pimelic acid derivatives

N,N-diacetyl β-alanine

N,N-diacetyl-ɣ-amino-butyric acid

Methyl 4-acetyl-5-oxohexanoate

The immunosuppressive properties of the instant molecules can be described both as to in vitro and in vivo efficacy in experimental systems. It is important to point out that the results obtained from the in vitro systems arc directly predictive of the in vitro immunosuppressive properties of the derivatives of succinylacetone described herein. The in vitro mixed lymphocyte assay described below is presently employed in the clinical setting as an indicator of histocompatibility, and is premised on the transformation of resting genetically dissimilar lymphocytes into cells which synthesize DNA and undergo proliferation. It has been demonstrated that

incompatibility at the major histocompatibility complex is mainly responsible for this phenomenon.

A second assay widely used to study immune responsiveness is mitogenic stimulation of thymocytes with antigenic substances of plant origin. The most wideh used plant molecule is phytohemagglutinin (PHA). Although PHA stimulates DNA synthesis nonspecifically in a large number of lymphocytes, unlike true antigenic stimulation which causes mitogenesis of sub-populations of lymphocytes, the susceptibility of a patient's lymphocytes to PHA stimulation has been shown to correlate with the overall immune responsiveness of the patient.

Thus, it will be appreciated as to both the mixed lymphocyte and PHA assa\ that they are valuable for identifying immune suppressive molecules in vitro, and that the results obtained therefrom are predictive of their in vivo effectiveness.

The following definitions will assist in understanding the invention.

Derivatives of succinylacetone, in addition to those shown in Figure 1 , may include succinylacetone conjugated to. polyethylene glycol (PEG), polypropylene glycol (PPG) or other polymer molecules known to extend the in vivo circulation time of medicaments.

The polymer need not have any particular molecular weight, but it is preferred that the molecular weight be between about 300 and 100,000, more preferably between 350 and 40,000, depending, for example, on the particular succinylacetone derivative.

Preferably the PEG homopolymer is unsubstituted, but it may also be substituted at one end with an alkyl group. Preferably the alkyl group is a C₁-C₄ alkyl group, and most preferably a methyl group. Most preferably, the polymer is an unsubstituied homopolymer of PEG, a monomethyl-substituted homopolymer of PEG or polyoxyethylated glycerol, and has a molecular weight of about 350 to 40,000.

Pegylated proline succinylacetone (succinylacetonyl-proline-PEG) is preferred in the invention and has the structure

Succinylacetonyl-proline-PEG, the methyl ester derivative, and glutarylacetone, and adipoylacetone, were synthesized as described in detail below. Methyl 4 acetyl-5- oxohexanoate was purchased from Aldrich Chemical Corp. The synthesis of

is described in Journal of Labelled Compounds and Radiopharmacetuicals. 1985. 22(9):869-881, and in U.S. Patent No. 4,011,321.

The synthesis of

and 4, 4-diacεtyl pimelic acid are described in Japanese Patent No. 57/203030, and in

Australian Journal of Chemistrv. 1967, 20:123-130, respectively.

Also, N, N-diacetyl β-alanine and 6-hydroxy 4-keto heptanoic acid methyl ester are described in the Journal of Medicinal Chemistry. 1985, 28:9-12 and in Journal of

Organic Chemistry. 1988, 52:4893, respectively. All of the above publications are hereby incorporated by reference in their entireties.

Furthermore, the following compounds may be synthesized as described below:

Succinyl Trifluoroacetone Methyl Ester

Succinyl Trifluoroacetone

Acetoacetonyl Methyl Benzoate

2-Acetoacetonyl Benzoic Acid

Synthesis of 3,5-Dioxohexylacyanide

Synthesis of 5-(3,5-Dioxobexyl) Tetrazo/e Synthesis of Beta-Keto t-Butyldimethylsilyl Enol Esters of SA Methyl Ester

Synthesis of Beta-Keto Isopropyl Enol Esters of SA Methyl Ester

Synthesis of Beta-Hydroxy Keto Derivatives of SA Methyl Ester

Synthesis of 4,6,8-Trioxo Methyl Nonanoate

Derivatives of succinylacetone were tested for immunosuppressive activity in several different types of assay systems. The first consisted of measuring the amounts of interleukin-2 (IL-2), and interferon-ɣ (IFN-ɣ) produced by human lymphoid cells in a mixed lymphocyte reaction. Low levels of these molecules in the reaction mixture indicate that the immune system is being suppressed by the subject succinylacetone derivatives.

IL-2 and IFN-ɣ levels were determined as follows:

IL-2 was measured by enumerating the number of viable HT-2 cells found afier an 18-24 hours period. HT-2 cells are a mouse IL-2 dependent cell line which die in the absence of IL-2. The assay is described by Gillis et al., J. of Immunol. 120:2027 (1978).

IFN-ɣ was determined by measuring the amount of protection conveyed on human WISH cells against the cytopathic effects of encephalomyocarditis virus (EMC). This assay is described by I. A. Braude. J. of Immunol. Methods 63:237 (1983). Both publications are hereby incorporated by reference.

The mixed lymphocyte reaction assay can be carried out by techniques well known to those skilled in the art. L. Hudson and F. C. Huy, Practical Immunology, p. 260, Blackwell Scientific Publications (1976). Briefly this consists of isolating lymphocytes from human peripheral blood by standard density gradient centrifugation techniques from two separate individuals. Approximately 2 X 10⁵ lymphocytes of each individual are combined to yield a total of 4 X 10⁵ lymphocytes per culture. All procedures are carried out under sterile conditions, and the lymphocytes are isolated and kept in physiologically compatible solutions. To the mixture of lymphocytes is added a desired amount of an appropriate succinylacetone derivative, and after different times an aliquot of the mixture is removed and assayed for IL-2 or IFN-ɣ, or ³H-T is measured. Succinylacetonyl-Proline-NH-PEG

Table 1 shows that there is a considerable diminution in the amount of IL-2 and IFN-ɣ produced as a function of the concentration of succinylacetonyl-proline-NH-PEG. It is worth noting that when compared to cell culture media, Roswell Park Memorial Institute media (RPMI), not containing succinylacetonyl-proline-NH-PEG. that there is more than about a three fold decrease in the amount of either IL-2, or IFN-ɣ. It will be appreciated that Table 1 presents the results from two separate experiments.

Table 1 also presents a control experiment wherein the effects of PEG-4,000-NH₂ on IL-2 and IFN-ɣ production is shown. Although PEG-4000-NH₂ does inhibit the production of these molecules, inhibition, however, is well below that observed for succinylacetonyl-proline-NH-PEG.

As mentioned above, one measure of an immune response as reflected in a mixed lymphocyte assay is the stimulation of DNA synthesis, and a concomitant increase in cell number. Thus, to further ascertain the immunosuppressive activity of succinylacetonyl-proline-NH-PEG, its effect on inhibition of thymocyte growth, as detected by uptake of tritiated thymidine, was assayed. Table 1 shows the results. A 50% reduction in thymidine uptake is observed at a concentration of about 625 μM succinylacetonyl-proline-PEG. This inhibition should be compared to about 2 % observed for PEG-4000-NH₂.

TABLE I

Succinvlacetonyl-oroline- NH-PEG vs PEG-4000-NH₂

SAMPLE IL-2 IFN -ɣ CPM(% INHIBITION) SA-pro-NH-PE G Exp.I Exp.II Exp a Exp.II Exp.I EXP.II

cpm cpm %lnhib. cpm % Inhib.

10mM <7 <6 <945 <1125 26 99.95 127 99.7

5.0mM <7 <6 <945 <1125 1162 97.9 182 99.6

2.5 mM <7 <6 <945 <1125 598 98.9 1467 96.9

L25mM <7 <6 4980 <1125 9729 82.7 8787 81.3

625mM <7 <6 5603 1041 31490 38.5 25471 45.8

313mM <7 <6 4150 2802 41599 18.8 34570 26.5

156mM 8 8 4980 2179 44129 21.7 29907 36.4

78mM <7 5 5603 2179 47326 16.0 33977 27.7

RPMI media 24 19 2802 1868 56358 - - - 47016 - - -

PEG-4000-NH₂

10mM 8 10 1972 1556 18545 70.7 23733 43.1

5.0mM 10 10 3113 1868 32003 49.5 28961 30.5

2.5mM 15 17 3113 1868 50295 20.6 33330 20.1

1.25mM 14 17 3424 1972 56850 10.2 45587 9.3

625μM 19 17 3113 1868 59571 5.9 42578 2.1

313μM 20 26 5188 1868 56774 10.3 42970 3.1

156μM 21 41 4047 1547 67188 6.1 36185 13.2

78μM 18 23 3424 1688 61969 2.1 47187 -13.2

RPMI media 18 25 1972 1778 63327 - - - 41694 - - - - Table 2 shows the effect of succinylacetonyl-proline-NH-PEG in a

phytohaemagglutinin assay. The procedure for carrying out the phytohaemagglutinin assay is also well known to those skilled in the an. Briefly, it consists of isolating human lymphocytes as described above, and incubating about 1.0 X 10⁶ cells per ml in a suitable physiological solution with phytohaemagglutinin, and the appropriate concentration of succinylacetone-proline-NH-PEG. Tritiated thymidine is added 48 hours later and allowed to incubate for twenty-four hours before the cells are counted. Table 2 shows that there is more than an 80% inhibition of tritiated thymidine uptake at a concentration of 8.3 mM, and that this inhibition decreases rapidly to about 10% at a concentration of 925 μM. In a second phytohemagglutinin assay, also shown in Table 2. wherein lymphocytes isolated from a second individual were tested, it is apparent that the level of inhibition is even greater. Approximately 95% inhibition is observed at a concentration of 8.3 mM succinylacetonyl-proline-NH-PEG, and this diminishes to 38.7% at a concentration of 925 μM.

TABLE II

Succinylacetonyl-proliine-NH-PEG-4000 in pHA Assay

SAMPLE

% %

CPMI±SD Inhib. CPMI±SD Inhib.

8.3mM 8386 1242 (83.6) 2748 786 (95.2)

2.8mM 35839 2352 (29.9) 23869 1410 (59.0)

925μM 45744 1129 (10.6) 35678 12393 (38.7)

309μM 49754 4294 47690 9003 (7.2)

102μM 49805 12018 40562 9677

34μM 51149 6031 30116 720

11.3μM 50680 2000 34898 3607

RPMI MEDIA 51149 5542 58159 4858

PHA + 47165 1524 51378 8890

PHA - 126 56 135 64 Succinylacetone Methyl Ester

A second derivative of succinylacetone which was shown to have considerable immunosuppressive activity is succinylacetone methyl ester. Table 3 shows the effect of varying the concentration of succinylacetone methyl ester on the production of IL-2 or IFN-ɣ in a mixed lymphocyte reaction. The results from duplicate experiments are shown. The concentration of succinylacetone methyl ester that inhibits the production of both of these substances by about 50% is 733 μM.

Table 3 also shows the inhibition of tritiated thymidine uptake in a primary mixed lymphocyte reaction as a function of succinylacetone methyl ester concentration. Duplicate experiments are also shown. The concentration that inhibits tritiated thymidine uptake by one-half is approximately 733 μM. Succinylacetone methyl ester was also tested in a phytohaemagglutination assay to further determine the potency of it immunosuppressive activity, and to ascertain whether this assay correlates with the results obtained in the primary mixed lymphocyte reaction assay. Table 5 shows two mixed lymphocyte assays conducted in duplicate using the lymphoid cells from two individuals. The amount of succinylacetone methyl ester that displays a 50% inhibition of phytohaemagglutination stimulation is between 103-309 μM in one assay, and about 103 μM in the second assay.

TABLE III

Methyl Ester SA-1º MLR

SAMPLE IL-2 IFN-ɣ CPM ± SD

Expl ExpII Expl ExpII Expl ExpII

20rnM <6 <8 <630 <375 164 28 90 38

6.7mM <6 <8 <630 <375 89 16 126 2

2.2mM <6 <8 788 <375 3185 486 33497

733μM 14 23 1890 688 30535 3068 40700 10026

244μM 19 28 1575 938 66159 2795 78306 7352

81μM 24 28 1628 1094 80182 9420 87595 1161

27μM 25 34 2713 1032 75211 2332 97879 1 123

9μM 28 38 1860 1000 87101 7114 110425 4941

3μM 41 34 2558 1313 81740 114428 1060

RPMI 30 54 2325 1313 84707 299 109628 9886

Methyl 4 Acetvl-5-Oxohexanoate

An analogue of succinylacetone was shown to have immunosuppressive activity, and this is methyl 4-acetyl-5-oxohexanoate. Table 4 summarizes its immunosuppressive effects in a primary mixed lymphocyte reaction assay where the parameters measured were IL-2 or IFN-ɣ production. Approximately 2.2 mM methyl 4 acetyl-5-oxohexanoate causes a 50% reduction in the production of either of these molecules. Table 4 also shows the effects of these compounds on the incorporation of tritiated thymidine in a mixed lymphocyte assay. Again, it was observed that the concentration of methyl 4-acetyl-5-oxohexanoate that causes a 50% inhibition in thymidine uptake is about 2.2 mM.

TABLE IV

Methyl 4-Acetyl-5-Oxohexanoate- 1 MLR

Sample Expl ExpII Expl ExpII Expl ExpII

20mM <6 <12 <285 <469163 2 109 3 6.7mM <6 <12 <285 <469 135 2 21280 2448

2.2mM 9 35 285 703 29389 2138 84363 4217

733μM 17 70 570 1172 67787 3233 113732 5345

244μM 17 52 9981953 48284 122672 2128

81μM 42 70 13131560 81881 2876 113054 3782 27μM 30 52 12601248 77634 8627 112463 7009

9μM 46 85 12601248 77627 6627 117109 3074

3μM 29 70 12601144 74259 4938 114713 2136

RPMI 23 77 12601248 77397 81 114230 1186

MEDIA Methyl 4-acetyl-5-oxohexanoate was also tested in a phytohemagglutination assay, and these results are shown in Table 5. Two assays were conducted usinglymphoid cells from two individuals. In both assays the concentration of methyl 4- acetyl-5-oxohexanoate that caused about a 50% inhibition in phytohaemagglutination stimulation was about 309 μM.

TABLE V

PHA - Stimulation of Succinylacetone Methyl Ester And

Methyl 4-Acetyl-5-Oxohexanoate

Succinyl- Methyl 4

Acetone Acetyl-5- Methyl Oxohexa- Ester noate

EXP. I EXP. II EXP. I EXP. II cpm ±SD cpm ±SD cpm ±SD cpm ±SD

25mM 113 20 112 33 25 mM 200 108 200 55

83mM 79 29 107 53 83 mM 192 33 156 28

2.8mM 126 38 65 12 2.8mM 116 41 717 91

926μM 565 36 6949,234. 926μM 2276 154 5600 1195

12 431

309μM 5927 124 5292 891 309μM 14098 92 19713 3109

103μM 17595 2625 17733 6322 103μM 18677 1409 30569 3168

34μM 16549 1394 28614 5959 34μM 19399 547 34006 1 101 l lμM 18100 862 28645 4692 11μM 19401 1995 33417 7177

3.8μM 20292 1643 37936 2487 3.8μM 19888 3900 33799 1633

RPMI 21587 89 36957 3908 18405 1777 38185 6925 MEDIA

Secondary Mixed Lvmphocyte Assays

In addition to testing the immunosuppressive activities of succinylacetone derivatives in a primary mixed lymphocyte reaction, or a phytohemagglutinin stimulation assay, two of the derivatives, succinylacetone methyl ester and methyl 4-acetyl-5-oxohexanoate, were tested in a secondary mixed lymphocyte reaction assay. The secondary mixed lymphocyte assays differs from the primary mixed lymphocyte reaction assays in that they employ many more primed responder cells that are responsive to the primary stimulating cells. The presence of such responsive cells is a reflection of immunological memory in an ongoing immunological response. The protocol for carrying out a secondary mixed lymphocyte assay involves performing a primary lymphocyte assay as described above, and recovering viable cells about 9- 10

days after the primary mixed lymphocyte reaction exhibits little or no cell proliferation. Generally between 10% to 50% of the original input cells are recovered in viable condition. These cells are then used in die secondary mixed lymphocyte reaction. It is worth noting that because a secondary mixed lymphocyte reaction is greatly enriched for responsive cells, fewer cells can be used in the secondary assay.

The procedure for carrying out a secondary mixed lymphocyte reaction is described by T. Meoen, Immunological Methods. Eds I. Lefkoivits and B. Pemis, Economic Press, New York (1979). It will be appreciated that described therein is a method for carrying out secondary lymphocyte reactions using mouse cells, however such methods are generally applicable to performing secondary mixed lymphocyte reactions using human cells with modifications that are well known to those skilled in the art. Tables 7 and 8 show immunosuppressive effects of succinylacetone methyl ester, and methyl 4-acetyl-5-oxohexanoate on the uptake of tritiated thymidine in secondary mixed lymphocyte assay. The assays were conducted in duplicate involving lymphoid cells isolated from two individuals. It is impoπant to note that the

immunosuppressive effects of both chemicals are similar to those observed in the primary mixed lymphocyte reaction assay, and therefore are correlative of their suppressive effects described in that system. For succinylacetone methyl ester or methyl 4-acetyl-5-oxohexanoate the concentration that causes about a 50% reduction in tritiated thymidine uptake is between about 2.2 mM and 740 μM.

TABLE VI

Sυccinvlacetone Methvl Ester 2º-MLR

Exp I Exp II

Sample CPM ±SD % Inhib. CPM ±SD % inhib

20mM 64 29 (99.9) 87 13 (99.9)

6.7mM 51 18 (9.99) 62 4 (9.99)

2.2mM 11778 2205 (85.4) 29851 6242 (68.0)

740μM 67456 - - (16.3) 85447 12061 (8.5)

247μM 87337 479 125345 35225

MEDIA 80578 8523 93241 9086

PBL 11658 122 50760 1581

EBV 196 35 153 62

TABLE VII

Methyl 4-Acetyl-5-Oxohexanoate

2º-MLR

Exp I EXD II

Sample CPM ±SD % Inhib. CPM ±SD % inhib

20mM 76 9 (9.99) 134 51 (9.99)

6.7mM 112 35 (9.99) 4402 2242 (95.3)

2.2mM 1995 - - (97.5) 43264 10237 (53.7)

740μM 61568 8943 (23.6) 88958 2804 (4.8)

247μM 73805 13480 86893 4332

MEDIA 80578 8523 93421 9086

PBL 11658 122 50760 1581

EBV 196 35 153 62 Viability Testing

In order to insure that the immunosuppressive effect of the succinylacetone derivatives was not being caused by generalized cytotoxicity, the effect of succinylacetone methyl ester, and methyl 4-acetyl-5-oxohexanoate on the viability of peripheral blood lymphocytes in a phytohaemagglutination stimulation assay was determined. Viability was determined using the trypan blue exclusion method well known in the art Tables 8 and 9 show the results for succinylacetone methyl ester and methyl 4-acetyl-5-oxohexanoate, respectively. The assays were conducted in duplicate wherein peripheral blood lymphocytes were isolated from two separate individuals, and then treated with or without phytohemagglutinin in the presence of various concentrations of the appropriate succinylacetone derivative. Table 8 shows there is little viability at a concentration of about 6.7 mM succinylacetone methyl ester. However, viability is greatly increased, and is nearly 100%, at a concentration ranging between 2.2 mM and 733 μM. These concentrations are those which were shown to be effective in the immunosuppressive assays described above.

Surprisingly, methyl 4-acetyl-5-oxohexanoate exhibits no toxicity even at

concentrations as high as 20 mM (Table IX).

TABLE VIII

Viability Methyl Ester SA

(-) PHA (+)PHA (-) PHA (+) PHA

Sample* Cells/ml %Via Cells/ml %Via Cells/ml %Via Cells/ml %Via

20mM NO 0 ND 0 ND 0 ND 0

6.7mM 0.15 7 0.13 8 0.23 4 0.24 6

22mM 0.36 94 0.3 89 0.42 94 0.42 93

733μM 0.34 96 0.4 90 0.48 95 0.39 94

244μM 0.35 100 0.38 97 0.46 98 0.52 96

81μM 0.37 100 0.4 96 0.53 100 0.55 98

27μM 0.60 100 0.37 97 0.43 100 0.40 94

9μM 0.36 100 0.32 100 0.51 100 0.50 100

3μM 0.60 100 0.48 100 0.5 100 0.50 100

MEDIA 0.5 100 0.52 98 0.4 100 0.48 100 *X10⁶

TABLE IX

PBL Viability Methyl 4-Acetyl-5-Oxohexanoate

(-) PHA (+)PHA (-) PHA (+) PHA Sample* Cells/ml %Via Cells/ml %Via Cells/ml %Via Cells/ml

%Via

20mM 0.5 100 0.49 100 0.5 100 0.55 100

6.7mM 0.57 100 0.46 100 0.6 100 0.50 100

22mM 0.57 100 0.49 100 0.54 100 0.50 100 733μM 0.37 100 0.38 100 0.41 100 0.40 100

244μM 0.40 100 0.44 100 0.5 100 0.54 100

81μM 0.42 100 0.38 100 0.46 100 0.40 100

27μM 0.44 100 0.46 100 0.50 100 0.50 100

9μM 0.55 100 0.47 100 0.52 100 0.50 100 3μM 0.39 100 0.45 100 0.44 100 0.40 100

MEDIA 0.44 100 0.38 100 0.43 100 0.48 100

*X10⁶

In Vivo Results

The immunosuppressive effects of two of the derivatives of succinylacetone were determined in a rheumatoid arthritis experimental animal model system, and compared to the effects of succinylacetone. The derivatives were succinylacetone methyl ester and methyl 4-acetyl-5-oxohexanoate. The experimental model system was adjuvant induced arthritis in rats, and the protocol is described by J. Holoshitz, et al., Science 219:56 (1983), or by B. Waksman and C. Wennersten, Int. Arch. Allergy Appl. Immnnol. 23:129 (1963). Induction of the disease can be caused by a single injection, generally intradermally, of a suspension of killed Mycobacterium

tuberculosis in complete Freund's adjuvant (CFA). The route of injection can vary, but in the instant invention rats were injected at the base of the tail with an adjuvant mixture. The procedure for testing the effects of succinylacetone methyl ester and methyl 4-acetyl-5-oxohexanoate consisted of intradermally injecting killed

Mycobacterium tuberculosis in CFA followed by eitiier immediate treatment every other day with the derivatives or subsequent administration starting at 4 or 14 days later. The derivatives were administered intraarticularly into the right paw at concentrations of 325 μg, 150 μg, and 560 μg per kilogram of body weight for succinylacetone, succinylacetone methyl ester, and 4-acetyl-5-oxohexanoate, respectively.

Table 10 shows the effects of succinylacetone methyl ester and methyl 4- acetyl-5-oxohexanoate along with succinylacetone. Experiment I consisted of administering Mycobacterium in CFA and die appropriate compound on the same day, and every other day until day 24. Experiments II and III were similarly performed with the difference that succinylacetone or the derivatives were administered every other day starting at 4 or 14 days respectively, after

Mycobacterium/CFA administration until day 24. At die appropriate times, 14, 16, 18, 20 and 22 days after injection of Mycobacterium CFA, an overall arthritis score was obtained as described by J. Holoskitz above. It is apparent that the methyl ester derivative is most effective when it is administered 14 days after induction of the disease, (Exp. III, Table X). Moreover, succinylacetone is also effective when administered 14 days subsequent to injection of Mycobacterium tuberculosis plus CFA. In contrast, 4 acetyl-5-oxohexanoate is apparently most active when administered every other day from day 4 to day 24.

TABLE X

COMPOUND TIME (DAYS)

14 16 18 20 22 24 26

Succinylaceione Exp. I 0.6±1.3 0.8±1.8 1.4+2.6 3±5 3.8 ±5 4±3.9 5 ±4.3

Exp. II 3.2±3.8 3.2±3.8 4.8±3.6 7±2.3 6.2 ±2.4 6 ±2.7 7.8±3 Exp.lII 2.0±3.9 2.0±3.9 2.0±2.9 2.4±2.9 4.0 ±3.7 2.8 ±2.8 3.4±2.7

Succinylaceione

Methyl Ester Exp. I 2.4±2.5 2.8+2.4 3.4±1.9 6.4 ± 3.6 6.8±4.0 7.2±4.1 6.2±3.7

Exp. MI 1.6±2.6 2+2.8 3.2+4.0 4.0±4.6 4.8±4.1 4.8+4.1 5.6±4.7

Exp.lll 1.2±1.8 1.4+1.3 1.4±2.2 4.8±3.8 4.4±3.6 4.4±3.6 3.0±3.3

Methyl

4 Acctyl-5 oxo Exp. I 3.2±2.2 3.4±2.2 5.0±3.2 7.0±4.0 8.2 ±4.9 7.2±4.2 7.2±4.2 Hexanoate Exp. II 1.0±1.4 2.2±2.5 1.8+1.8 2.2±2.0 5.0 ±3.5 3.4±3.6 4.0±3.7

Exp.II! 2.0±2.0 2.4±1.5 3.4±2.6 5.6±2.7 7.4±4.8 7.4±5.3 7.4±5.3

Synthesis

A number of succinylacetone derivatives described herein were produced as follows. For preparation of succinylacetonyl-proIine-NH-FEG, an attempt was made first to generate an active ester of succinylacetone. Succinylacetone was dissolved in an aptotic organic solvent, (preferably dimethyl formamide, chloroform, or dichlσromethane, among others), and converted to an ester by addition of an equimolar amount of an appropriate condensing agent, such as a carbodiimide, (preferably dicyclohexyl carbodiimide, or 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide (EDAC)), and a slight molar excess of an appropriate ester forming alcohol, (preferably p-nitrophenol, 4-hydroxyl-3-nitro-benzene sulfonate, or N-hydroxysuccinimide, among others). It was expected that this reaction would produce an active ester from the alcohol and succinylacetone. However, subsequent experiments revealed that an enol lactone is, indeed, an intermediate in the reaction, and this was confirmed by subsequent NMR analyses of succinylacetone reacted with carbodiimide in die absence of an alcohol. Enol-lactone, in turn, reacts with both primary and secondary amines to produce the desired amides. Concomitant with the formation of the enol-lactone is the formation of urea from the carbodiimide.

When succinylacetone is treated as described-above with a secondary amine such as proline, the product formed is succinylacetonyl-proline. Succinylacetonyl-proline can be converted to succinylacetonyl-proline-NH-PEG by combining it with a condensing agent, in the presence of a suitable ester forming alcohol to form an active ester of the carboxylic acid group of proline. The preferred alcohol is p-mtrophenol, although other alcohols such as, for example, N-hydroxysuccinimide, 4-hydroxy-3-nitro-benzenesulfonate, among others may be used. The final step in this reaction sequence is to react the succinylacetonyl-prolyl-nitrophenyl active ester with PEG-NH₂ which results in the desired product, succinylacetonyl-proline-NH-PEG.

The succinylacetone active esters, or other activated species, formed in the step described above are also reactive with primary amines, which thus affords a synthetic route towards realizing succinylacetone PEG, which was employed as a control in a number of the experiments. Succinylaoetonyl-NH-PEG is realized by combining succinylacetone active species, probably the enol-lactone, or other activated species with PEG-NH₂ which produces succinyiacetonyl-NH-PEG. Succinylacetonyl-Proline-NH-PEG

Succmvlacetonvl-Proline

In more detail, succinylacetonyl-proline-NH-PEG was synthesized by first generating succinylacetonyl-proline. This consisted of dissolving 1 gm of

succinylacetone (6.3 mmole) and 1.6 gm (6.6 mmole) of l-hydroxyl-2-nitro-benzene-4-sulfonic acid in 10 ml dimethyl formamide. Next a suitable condensing agent was added, particularly useful is dicyclohexylcarbodiimide. About 1.6 g of

dicyclohexylcarbodiimide (6.3 mmole) was added to die mixture. After the reaction was stirred at room temperature for a short time (10 minutes), dicyclohexylurea began to precipitate. Dicyclohexylurea was removed from the reaction mixture by filtering, followed by adding the filtrate to a dimethyl formamide solution containing 0.74 g proline (6.3 mmole). The combined solutions were stirred overnight

The reaction was diluted with an equal volume of water, and extracted ten times with 10 ml of ethyl acetate. The combined organic extracts were dried over MgSO₄, filtered to remove MgSO₄ hydrate, and concentrated on a rotary evaporator to produce a thick slurry. Succinylacetone was separated from succinylacetonyl-proline using a 4 mm Chromatotron spinning plate silica gel chromatogram equilibrated with a solvent consisting of chloroform : acetic acid in the ratio of 90 : 10. Succinylacetone eluted at the solvent front. Succinylacetonyl-proline eluted with a more polar solvent consisting of chloroform : acetic acid:methanol in the ratio of 90 : 10 : 3. Those fractions containing succinylacetonyl-proline were pooled, concentrated with a rotary

evaporator and the remaining acetic acid solution diluted with water, followed by quick freezing and lyophilization. The final product was a yellow oil.

In a thin layer chromatography plate developed in 90 : 10 chloroform:acetic acid succinylacetone showed an R_f of 0.78 while the product succinylacetonyl-proline has an R_f of 0.23, and proline remains at the origin.

Succinylacetonyl-proline-NH-PEG

Succinylacetonyl-proline was converted to succinylacetonyl-proline-NH-PEG by adding succinylacelonyl-proline to a condensing agent in the presence of p-nitrophenol in chloroform. The reaction was carried out in 10 ml of chloroform by adding 0.55 g (4.0 mmole) of p-nitrophenol to 0.64 g (3.9 mmole) of

succinylacetone-proline. Next, 0.80 g (3.3 mmole) of the condensing agent, dicyclohexylcarbodiimide was added. The latter was dissolved in 2 ml chloroform, and added slowly to die mixture with stirring. Within 10 minutes, a white precipitate of dicyclohexylurea formed, and a spectrophotometric determination [L. Aldwin and D.E. Nitecki, 1987, Anal. Biochem.. 164 : 494-501] of the amount of ester present at that time indicated that there was approximately 61.5% ester present A similar determination two hours later revealed no change in die amount of ester formed. Dicyclohexylurea was removed by filtering the solution, followed by die addition of 0.45 g (1.1 mmole) FEG-4000-NH₂ to the filtrate. The reaction mixture was allowed to stir at room temperature overnight. The product, succinylacetonyl-proline-NH-PEG was purified as follows.

The reaction mixture had a granular appearance, and tiierefore was filtered, followed by removal of the organic solvent with a rotary evaporator. This left a thick oil in the rotary evaporator flask which was not completely soluble in water. It was extracted three times with ethyl ether, to remove impurities which were not water soluble. PEG is completely insoluble in ethyl ether, and tiierefore

succinylacetonyl-proline-PEG was expected to remain in the aqueous phase. The aqueous phase was applied to a G-50 Sephadex sizing column (3 cm diameter × 41 cm high) that had previously been equilibrated with distilled water. The column was washed with distilled water and 4 ml fractions collected and monitored

spectrophotometrically to produce die elution profile shown in Figure 2. This chromatographic step primarily separates small molecular weight molecules such as succinylacetonyl-proline from larger ones such that PEG-4000-NH₂ and

succinylacetonyl-proline-NH-PEG co-elute. Spectrophotometric analysis of fractions 16-34 from the Sephadex G-50 column revealed that this material absorbs between 250-340 nm. This is in contrast to PEG-4000-NH₂ which absorbs in die range of about 210-225 nm. These results are shown in Figures 3 and 4. Figure 3 shows the spectrum of PEG-4000-NH₂, while Figure 4 shows the spectra of some of the

Sephadex G-50 fractions.

Fractions 15-34 were pooled and lyophilized to a white powder. Thin layer chromatography plates developed in 90 : 10 chloroform:acetic acid indicated that most of the ninhydrin reacting compound remained at the origin. No succinylacetone was detectable on die plate, but trace amounts of succinylacetonyl-proline could be seen. This method was used to produce the compounds for the bioassays shown in Table 1. The mixture was later repurified over a G-50 Sephadex column so that no trace of succinylacetone-proline could be detected on TLC plates. The purer succinylacetone- pτoline-NH-PEG-4000 was used in the bioassays shown in Table 2.

Succinylacetonyl-NH-PEG

Approximately 0.158 grams (1.0 mMole) of succinylacetone was combined witii 1.0 gram of PEG-NH₂ (molecular weight 10K) in 10 ml chloroform. Next 0.206 grams of dicyclohexylcarbodiimide was added in a small amount of chloroform. The reaction was allowed to proceed at room temperature overnight and was shielded from exposure to light. The major by-product of die reaction, dicyclohexylurea, was removed by filtration, and die slightly yellow filtrate concentrated by rotary evaporation. The remaining oil was dissolved in H₂O and dialyzed against dilute acetic acid, and subsequently water. After three changes of dilute acetic acid and diree changes of water, the contents of the dialysis bag were lyophilized. The white powder was redissolved in distilled water and was chromatographed over a gel filtration column, Sephadex G-50 (available from Pharmacia Corp.), to remove small molecular weight impurities. Fractions contaiiing succinylacetonyl-NH-PEG were identified spectrophotometrically, and shown to contain succinylacetonyl-NH-PEG by thin layer chromatography.

Succinylacetone Methyl Ester

Succinylacetone metiiyl ester, methyl 4-6-dioxoheptanoate, was synthesized as described by A. R. Battersby, et al., J.C.S. Perkin I. 1981 : 2786-2792.

Glutarylacetone Methyl Ester

Glutarylacetone methyl ester, (methyl 5, 7-dioxohexanoate), was syndiesized by reacting a magnesium complex of t-butyl acetoacetate with methyl 4-(chloroformyl) buryrate. The magnesium complex of t-butyl acetoacetate, in turn, was synthesized as described by Battersby, 1981, J. Chem. Soc.. Peririn Transactions I, p.2786-2799. Briefly, methanol (250 mL) was added to a mixture of magnesium shavings (15.91 gm; 654.5 mmole) and 0.5 mL of carbon tetrachloride. The mixture began to reflux as the metal dissolved and a cold water bath was used to control the reaction. After die metal had completely dissolved in the alcohol, and the mixture had cooled, t-butyl acetoacetate (106.7 mL; 101.8 gm; 664.2 mmole) was added dropwise over ten minutes. The mixture was refluxed for one hour, cooled, and filtered. The magnesium complex of t-butyl acetoacetate was collected, washed with cold methanol and dried under vacuum.

To a suspension of the magnesium complex of t-butyl acetoacetate (75 gm;

353 mmole) in 125 mL of ether was added methyl 4-(chlσroformyl) butyrate (54 mL; 64.31 gm; 391 mmole). The addition was made dropwise, with stirring under a reflux condenser. After the addition was complete, the mixture was refluxed for thirty minutes and allowed to cool. To the mixture was added 125 mL of 1.8M sulfiiric acid and die two-phase mixture transferred to a one liter separatory funnel. The aqueous layer was extracted with ether (3X100 mL) and die combined etiier extracts were washed witii brine (4X50 mL). The ether was evaporated and to die residue was added 300 mg of p-toluenesulfonic acid monohydrate. The mixture was heated to 170ºC and evolution of gas monitored with a bubbler. When gas evolution had subsided, die dark mixture was allowed to cool and 250 mL of ether was added. The solution was transferred to a one liter separatory funnel and extracted with ice-cold 2M sodium hydroxide (100, 50, 25 and 25 mL). The extracts were run direcdy into ice-cold 1.8M sulfuric acid (250 mL). The acidic suspension was extracted with methylene chloride (3X150 mL). The organic extracts were pooled and washed with 5% sodium bicarbonate (2X50 mL) and brine (2X50 mL), dried over sodium sulfate, filtered and die solvent removed under reduced pressure to yield 40 gm of dark syrup. This was taken up in 200 mL methylene chloride and adsorbed onto 60 gm silica gel by evaporation of the solvent and fractionated on a 600 mL column of silica gel by flash chromatography using ethyl acetate:hexane (1:4) as eluant Fractions containing the product were pooled and concentrated to a pale yellow oil weighing 33.08 gm. The R_f value (silica gel; ethyl acetate±exane 1:4) was 0.31, which is consistent with die product being glutarylacetone methyl ester as assessed by elemental analysis and die NMR spectrum of me product Adipoylacetone Methyl Ester

Adipoylacetone methyl ester (methyl 6, 8-dioxononanoate) was generated by reacting the magnesium complex of t-butyl acetoacetate with methyl adipoyl chloride. t-Butyl acetoacetate magnesium complex was synthesized as described above, and to a suspension of the complex (10.6 gm; 50 mmole) in 25 mL of ether was added methyl adipoyl chloride (9.83 gm; 55 mmole). The addition was made dropwise with stirring under a reflux condenser. After the addition was complete, the mixture was refluxed for thirty minutes and allowed to cool. To the mixture was added 25 mL of 1.8M sulfuric acid and die two-phase mixture transferred to a 125 mL separatory funnel. The aqueous layer was extracted with ether (3X25 mL) and die combined ether extracts were washed witii brine (4X10 mL). The ether was evaporated and to die residue was added 100 mg of p-toluene sulfonic acid monohydrate. The mixture was heated to 170 degrees and evolution of gas monitored with a bubbler. When gas evolution had subsided, the dark mixture was allowed to cool and 50 mL of ether was added. The solution was transferred to a one liter separatory funnel and extracted with ice-cold 2M sodium hydroxide (20, 10, 5 and 5 mL). The extracts were run direcdy into ice-cold 1.8M sulfuric acid (25 mL). The acidic suspension was extracted witii methylene chloride (3X30 mL). The organic extracts were pooled and washed with 5% sodium bicarbonate (2X10 mL) and brine (2X10 mL), dried over sodium sulfate, filtered and die solvent removed under reduced pressure to yield 7.05 gm of dark syrup. This was taken up in 50 mL methylene chloride and adsorbed onto 15 gm silica gel and fractionated on a column of silica gel by flash chromatography using ethyl acetate:hexane (1:4) as eluant Fractions containing the product as determined by TLC were pooled and concentrated to a pale yellow oil weighing 5.39 gm. The R_f value (silica gel; ethyl acetate:hexane 1:4) was 0.30.

Elemental analysis and NMR spectrum of the product was consistent with the expected material. Glutarylacetone

Glutaiylacetone, (5,7-dioxo octanoic acid), was generated from glutarylacetone methyl ester by hydrolysis. Briefly, glutarylacetone metiiyl ester (1.0 g) was hydrolyzed in 10 ml of 4.0 M HCl at 90º for 30 minutes. The solution was cooled and extracted with four 25 ml portions of methylene chloride. After drying over magnesium sulfate and evaporation of the solvent, the residue was re-crystallized from ether-hexane to give 0.3 g of crystalline 5, 7-dioxo octanoic acid.

Additional Synthesis

In addition to die above compounds, it will be appreciated that other succinylacetone analogs or derivatives may be made according to die following proposed synthetic schemes.

Succinyl Trifluoroacetone Methyl Ester (Methyl 7,7,7-trifIuoro 4.6-dioxoheptanoic acid) Methanol (250 mL) is added to magnesium shavings (15.91 gm; 654.5 mmole) containing 0.5 mL of carbon tetrachloride. The mixture refluxes as the metal dissolved, and a cold water bath is used to control the reaction. After the mixture has completely dissolved and cooled, t-butyl 3-oxo-4,4,4-trifluorobutanoate (140.9 gm; 664.2 mmole) is added dropwise over ten minutes. This mixture was refluxed for one hour, cooled, filtered and die resulting solid collected and washed with cold methanol and dried under vacuum.

The magnesium complex of t-butyl 3-oxo-4,4,4-trifluorobutanoate (266.5 gm;

635 mmole) is suspended in 250 ml of anhydrous ether in a three-neck one liter round bottom flask and carbomethoxypropionyl chloride (100 gm: 664 mmole) added dropwise over fifteen minutes while die mixture is stirred from overhead. The dropping funnel is removed and die mixture refluxed for thirty minutes. The mixture is cooled and 250 ml of 2N aqueous sulfuric acid slowly added. The mixture is transferred to a one liter separatory funnel and die layers separated. The aqueous layer is extracted with ether (2 X 150 ml) and die combined ether extracts washed with water (4 X 100 ml). The ether is evaporated and to die residual red oil is added 800 mg of toluene sulfonic acid monohydrate. The mixture is heated in an oil bath (with reflux condenser) to 175 degrees until evolution of gas ceases. The mixture is cooled and taken up in 500 ml of ether. The ether extracted with ice-cold 2M aqueous sodium hydroxide (175, 90, 50, and 35 ml), and the extracts run from the separatory funnel direcdy into 250 ml of ice-cold 1.8M aqueous sulfuric acid. The acidic suspension is extracted with methylene chloride (3 X 150 ml) and die combined organic extracts were washed with 5% aqueous sodium bicarbonate (2 X 100 ml) and brine (100 ml). The residue after evaporation of die methylene chloride may be purified by silica gel flash column chromatography using as eluant chloroform : methanol 97:3.

Snccinyl Trifluoroacetone From The Methyl Ester

The methyl ester (2.63 gms; 11.62 mmole) is suspended in 18 mL water and 2 mL of cone. HCI added. The mixture is heated with stirring at 50 degrees for two hours or until all the material dissolved. The solvent is removed under reduced pressure (aspirator at 50 degrees) and the residue taken up in 18 mL water and 2 mL of cone. HCI added. Again, the solution is heated at 50 degrees for two hours at which time TLC (methylene chloride:metiιanol 97:3) should show no ester remained. (UV and ferric chloride spray reagent; 2.7% w/v in 2N HCI). The solvent is once again removed under reduced pressure (water pump, 50 degrees) and 20 mL water added. The water is removed under reduced pressure (water pump, 50 degrees) and residual water removed by azeotroping with 50 mL of toluene. To the residue is added 50 mL edier and die solvent removed on die roto-vap. Finally, the residue is taken up in 10 mL of hot ether and filtered. Addition of four mL of hexane to the filtrate initiates crystallization and the flask is chilled. Crystals may be removed by filtration and dried.

Methyl 2(2,4-Dioxobutyl) Benzoate

The magnesium complex of t-butyl acetoacetate (135 gm; 635 mmole) is suspended in 250 ml of anhydrous ether in a three-neck one liter round bottom flask and mono-methyl phthaloyl chloride (192 gm: 664 mmole) is added over fifteen minutes while the mixture is stirred from overhead. The dropping funnel is removed and die mixture refluxed for thirty minutes. The mixture is cooled and 250 ml of 2N aqueous sulfuric acid slowly added. The mixture is transferred to a one liter separatory funnel and die layers separated The aqueous layer is extracted with ether (2 X 150 ml) and die combined ether extracts washed with water (4 X 100 ml). The ether is evaporated and to die residual red oil is added 800 mg of toluene sulfonic acid monohydrate. The mixture is heated in an oil bath (with reflux condenser) to 175 degrees until evolution of gas has ceased. The mixture is cooled and taken up in 500 ml of ether. The ether is extracted with ice-cold 2M aqueous sodium hydroxide (175, 90, 50, and 35 ml), and die extracts run from the separatory funnel direcdy into 250 ml of ice-cold 1.8M aqueous sulfuric acid. The acidic suspension is extracted with methylene chloride (3 X 150 ml) and die combined organic extracts washed with 5% aqueous sodium bicarbonate (2 X 100 ml) and brine (100 ml). The residue after evaporation of the methylene chloride may be purified by silica gel flash column chromatography to yield die product

2(2.4-Dioxobutyl) Benzoate From The Methyl Ester

The methyl ester (2.56 gms; 11.62 mmole) is suspended in 18 mL water and 2 mL of cone. HCI added. The mixture is heated with stirring at 50 degrees for two hours (all material had dissolved). The solvent is removed under reduced pressure (aspirator at 50 degrees) and the residue taken up in 18 mL water and 2 mL of cone. HCI added. Again, the solution is heated at 50 degrees for two hours at which time TLC (methylene chloride:metiιanol 97:3) will show no ester remaining. (UV and ferric chloride spray reagent; 2.7% w/v in 2N HCI). The solvent is once again removed under reduced pressure (water pump, 50 degrees) and 20 mL water added. The water is removed under reduced pressure (water pump, 50 degrees) and residual water removed by azeotroping with 50 mL of toluene. To the residue is added 50 mL ether and die solvent removed on the roto-vap. Finally, the residue is taken up in 10 mL of hot ether and filtered. Addition of four mL of hexane to the filtrate initiates crystallization and die flask is chilled. Crystals may be removed by filtration and dried.

3.5-Dioxohexylcyanide

The magnesium complex of t-butyl acetoacetate (135 gm; 635 mmole) is suspended in 250 ml of anhydrous ether in a three-neck one liter round bottom flask and beta-cyano propionyl chloride (78.05 gm: 664 mmole) added dropwise over fifteen minutes wlύle the mixture is stirred from overhead. The dropping funnel is removed and the mixture refluxed for thirty minutes. The mixture is cooled and 250 ml of 2N aqueous sulfuric acid is slowly added. The mixture is transferred to a one liter separatory funnel and die layers separated. The aqueous layer is extracted with ether (2 X 150 ml) and die combined etiier extracts washed with water (4 X 100 ml). The ether is evaporated and to die residual red oil is added 800 mg of toluene sulfonic acid monohydrate. The mixture is heated in an oil bath (with reflux condenser) to 175 degrees until evolution of gas has ceased. The mixture is cooled and taken up in 500 ml of ether. The ether is extracted with ice-cold 2M aqueous sodium hydroxide (175, 90, 50, and 35 ml). The extracts were run from the separatory funnel direcdy into 250 ml of ice-cold 1.8M aqueous sulfuric acid. The acidic suspension is extracted with methylene chloride (3 X 150 ml) and die combined organic extracts washed with 5% aqueous sodium bicarbonate (2 X 100 ml) and brine (100 ml). The residue after evaporation of die methylene chloride may be purified by silica gel flash column chromatography using ethyl acetate: hexane 1:4 as eluant

5-(3.5-Dioxohexy ) Tetrazole

A mixture of the 3,5-dioxohexylcyanide (2.78 gm; 20 mmole), lithium azide (1.08 gm; 22 mmole) and sulfuric acid (one drop) in 10 ml dry dimethylformamide is heated to 100-120 degrees. Progress of die reaction may be checked by TLC (silica gel; chlσrofoπmmethanol 97:3). When no starting material remaines, the reaction is cooled and 20 ml of water added. The reaction is taken to dryness under reduced pressure and die residue fractionated by silica gel flash column chromatography using methylene chloride: glac. acetic acid: methanol 100:3:3 as eluant Fractions containing the product may be pooled and concentrated. Beta-Keto t-Bntyldim ethylsilyl Enol Ethers Of SA Methyl Ester

To a solution of sucάnyl acetone methyl ester (3.44 gm; 20 mmole) in 50 ml THF is added a 50% dispersion of sodium hydride in mineral oil (960 mg; 20 mmole) and die mixture stirred until gas evolution ceases. To the mixture of enolates is added a solution of t-butyl dimethylsilyl chloride (332 gm; 22 mmole) in 50 ml THF dropwise with stirring. Sodium chloride is removed by filtration and die residue fractionated by silica gel flash chromatography using ethyl acetate: hexane as eluant

Beta-Keto Isopropyl Enol Esters Of SA Methyl Ester

To each of the purified t-butyldimethylsilyl enol ethers of SA methyl ester (2.87 gm; 10 mmole) in THF cooled to -78 degrees is added tetrabutylammomum fluoride in THF (11 mmole). After thirty minutes at -78, 2-iodoρropane (1.87 gm; 11 mmole) is added to die mixture and die reaction allowed to slowly reach room temperature. The solvent is removed under reduced pressure and die residue purified by silica gel flash chromatography to yield die isopropyl enol ethers. Beta-Hvdroxy Keto Derivatives Of SA Metiiyl Ester

To a solution of each of die t-butyldimεthylsilyl enol ethers of SA methyl ester (2.87 gm; 10 mmole) in 50 ml of isopropanol at -20 degrees is added sodium borohydride (420 mg; 11 mmole). When no starting material remains

tetrabutylammonium fluoride (2.88 gm; 11 mmole) is added and reaction progress followed by TLC Work-up by silica gel column chromatography should yield the hydroxy keto derivatives.

It will be appreciated by those skilled in die art that an alternative approach to these compounds would be via catalytic hydrogenation of the enol double bond in the t-butyldimethylsilyl enol ether of SA methyl ester followed by fluoride mediated silyl ether cleavage to yield die desired hydroxy keto derivatives.

Methyl 4.6.8-Trioxo Nonanoate

To the diMiium salt of 2,4-pentanedione (224 gm; 20 mmole) in 50 ml dry THF is added carbomethoxypropionyl chloride (3.31 gm: 22 mmole) dropwise with stirring at zero degrees. When TLC shows no starting material remaining, the solvent is removed under reduced pressure and die residue fractionated by flash chromatography on silica gel using ethyl acetate : hexane as eluant

It will be apparent to those skilled in the art that the above-described methyl esters could be converted to the corresponding free acids by appropriate methods.

Having described what applicants believe is their invention, it is important to note that it will be apparent to those skilled in the art that there are many possible substitutions for the specific materials and methods shown above. It will be understood that these substitutions are intended to come within die scope of the following claim.

Claims

WHAT IS CLAIMED IS:

1. A method of treating disease comprising suppressing the effectiveness of a patients immune system comprising administering to a patient a therapeutically effective amount of an analogue or derivative of succinylacetone.

2. The method as described in claim 1, wherein said analogue or derivative of succinylacetone comprises the formula:

wherein

3. The method as described in claim 2, wherein n = 2-6.

4. The method as described in claim 1, wherein said analogue or derivatives of succinylacetone are selected from the group consisting of

succinylacetonyl-proline-PEG, succinylacetone methyl ester, methyl-4-acetyl-5-oxohexanoate, succinylacetonyl-NH-PEG, glutarylacetone, and adipoylacetone.

5. The method of claim 4, wherein said effective amount of said analogue or derivatives of succinylacetone is about 18 micrograms-800 mg/kg body weight.

6. The method of claim 5, wherein said succinylacetone analogue or derivatives are administered with pharmaceutically acceptable excipients.

7. The method of claim 6, wherein said disease is an autoimmune disease.

8. The method of claim 7, wherein said disease is die rejection of organ or tissue transplants.

9. The method of claim 6, wherein said disease is graft versus host disease.

10. Immunosuppressive compositions useful for treating diseases

comprising an analogue or derivative of succinylacetone.

11. Immunosuppressive compounds as described in claim 10, wherein said analogue or derivative of succinylacetone comprise the formula:

wherein

12. Immunosuppressive compounds as described in claim 11, wherein n = 2-6.

13. Immunosuppressive compositions as described in claim 10, wherein said derivatives of succinylacetone have the formula:

wherein X is primary or secondary amino group.

14. Immunosuppressive compositions as described in claim 11, wherein said analogue or derivatives of succinylacetone are selected from the group consisting of succinylacetonyl-proline-NH-PEG, succinyl-acetonyl-NH-PEG, succinylacetone methyl ester, methyl-4-acetyl-5-oxohexanoate, glutarylacetone and adipoylacetone.

15. Immunosuppressive compositions as described in claim 14 further including a physiologically acceptable excipient.