WO1994007904A1

WO1994007904A1 - Derivatives of 7,8-disubstituted guanosines

Info

Publication number: WO1994007904A1
Application number: PCT/US1993/009189
Authority: WO
Inventors: Allen B. Reitz
Original assignee: Mcneilab, Inc.
Priority date: 1992-10-01
Filing date: 1993-09-27
Publication date: 1994-04-14
Also published as: AU5165793A

Abstract

2',3'-cyclic derivatives of 7,8 disubstituted guanosines of general formula (I) are disclosed as potent immunostimulants.

Description

DERIVATIVES OF 7.8-DISUBSTITUTED GUANOSINES

BACKGROUND OF THE INVENTION

An animal's immune system is comprised of numerous elements that act in a fashion to counteract, to eliminate, or to neutralize agents that are recognized by that system as foreign to the animal host. Generally, but not necessarily, the agent or substance recognized as foreign by the immune system has its origin exogenous to the host. Exemplary of such exogenous substances are infectious bacteria and the by-products of their cellular activity, virus particles and their proteins, proteins injected by insect stings, and the like. In autoimmune diseases, such as rheumatoid arthritis, the host's immune system perceive host-made proteins or self-made proteins as if they were foreign.

The principal effectors of the immune system are the leukcvtes, which include the lymphocytes that mediate humoral immunity, cytotoxic T cells and T cell mediators of delayed-type hypersensitivity which are the principal effectors of cell mediated immunity.

T cells express important surface antigens designated CD 2, 3, 4, 5 and 8 that are related to T cell functions. Helper T cell precursors are of the CD 4+, 8", phenotype. Thi and T 2 subsets of CD 4+ 8- T cells normally participate in the activation and regulation of B cells. These helper T cells are known to assist in activation differentiation and regulation of immunoglobulin-secreting B cells after antigen presenting cells, such as B ceils, take up process, and present antigen in association with class II MHC molecules. The T i2 cells provide cell associated and soluble (LK) signals for B cell proliferation and differentiation.

Guanosine-3',5'-cyclic monophosphate (cGMP) was previously thought to be implicated as a naturally occurring agent for providing the required intracellular second message for B cell proliferation. 8-Bromoguanosine-3',5'- cyclic quanasons monophosphate (8 Br-cGMP, an analog of cGMP,) has been found to be a weak synthetic intracellular B lymphocyte mitogen. The immune response can be modified by artificial supression (herein called immunosupression) or enhancement (immunopotentiation or immunostimulation). Immunosuppression; i.e., artificially induced decreased responsiveness, can be achieved by six general methods: (1 ) administration o antigen, (2) administration of specific antisera or antibody, (3) use of other biologic reagents such as antilymphocyte antisera, (4) use of immuno- suppressive drugs or hormones, (5) radiation, and (6) surgical removal of lymphoid tissue. Immunopotentiation can be achieved by including the administration of an agent effecting an increase in the rate at which the immune response develops, an increase in the intensity or level of the response, a prolongation of the response, or the development of a response to an otherwise non-immunogenic substance.

The agents that are known to enhance immune responses are generally termed adjuvants and can be placed into two general categories: (1 ) those providing general potentiation; i.e., substances that enhance both cellular and humoral immune responses for a wide variety of antigens, and (2) those providing specific potentiation, i.e., substances which enhance specific responses to certain antigens only.

Substances that act as adjuvants can be grouped into the following categories: (1 ) water and oil emulsions, e.g., Freund's adjuvant, (2) synthetic polynucleotides, (3) hormones, drugs and cyclic nucleotides, (4) endotoxins, (5) proteinaceous lymphokines and monokines such as the interleukins and growth factors.

An example of a substance capable of specifically potentiating the immune response is a transfer factor, a dialyzable leukocyte extract (DLE) obtained from human peripheral leukocytes. It has been reported that the transfer factor exhibits some effectiveness in patients with immunodeficiencies and possible effectiveness in cancer patients and in patients with limited immunodeficiencies. However, much remains to be learned about this particular substance.

In some diseases and physiological conditions such as AIDS, X-linked agammaglobulinemias, senescence and drug-induced-immunosuppression, cell activation and differentiation is lacking and/or exists only at a reduced level, thereby lessening the immune response of the host. These diseases an conditions are representative of naturally and artificially induced immunosuppressed states. Here, enhanced activation and differentiation, if it can be effected, tends to beneficially lessen the disease manifestation and/or improve the patient's condition.

An immunopotentiated state can be illustrated by the bodily condition after vaccination. Here, the immune response, already enhanced due to antigenic stimulation could be beneficially enhanced still further to provide an improved degree and/or duration of immunity.

U.S. Patent No. 4,539,205 to Goodman and Weigle describes modulation of animal cellular responses with 8-substituted guanine derivatives bonded 9- 1 ' to an aldose having 5 or 6 carbon atoms in the aldose chain (ring). The cellular modulations described in that patent relate mostly to immunomodulation such as adjuvanticity in producing primary and secondary immune responses. Activity against certain neoplastic conditions is also disclosed as are T cell-replacing activity, an IL-1 like activity on thymocytes, and induction of the release of lysosomal enzymes from neutroohils. The 8- substitutents in those molecules have electron withdrav/ing inductive effects relative to hydrogen. Thus, halo, mercapto or its thioxo tautonsr, acyl mercapto, alkyl sulfido, nitro, cyano, keto, halomethyl and methyleneoxy alkyl and the like were disclosed as useful, while electron donating substituents such as an amino group were found to be inactive.

U.S. Patent No. 4,643,992 and its corresponding published European patent application No. 83306791.1 further disclose the use of derivatives of 8- hydroxyguanine (8-oxoguanine), 7-methyl-8-oxoguanine and 7-methyl-8- thioxoguanine in modulating animal cellular responses. Further results using guanine derivatives disclosed in U. S. Patent No. 4,539,205 are also disclosed in U.S. Patent No. 4,643,992, as are similar results us^qng guanine derivatives disclosed for the first time in that patent.

Still further, several papers and book chapters have been published by some of the present inventors and iheir co-workers relating to still further effects of compounds disclosed and claimed in U. S. Patent No. 4,643,992.

Exemplary of those published papers are Goodman, Rroc. Soc. Exp. Biol. Med., 179:479 (1985); Goodman, J. Immunol., 136:3335 (1986); Goodman and Weigle in Puriπe Metabolism In Man, Part 3, Nyhan and Thompson, eds., Plenum Press, New York, page 451 and 443 (1986); Goodman and Weigle, J. Immunol., 735:3284 (1985); Goodman and Wolfert, Immunol. Res., 5:71 (1986) Goodman, J. Immunol., 737:3753 (1986); and Goodman and Hennen, Cell. Immunol., 702:395 (1986).

U.S. Patent No. 5,011,828 describes certain 7,8 disubstituted guanine nucleosides that enhance an immune response in human and animal cells. The nucleosides described in this patent are improvements over the other guanosine derivatives mentioned previously in that they either provide a similar response at a lower dose or provide a greater enhancement of the response at a given dose.

It is an object of the present invention to develop other novel guanosine derivatives that possess either similar or improved activity as compared to the prior art guanosine derivatives.

SUMMARY OF THE INVENTION

It has been found that 2',3'-cyclic derivatives of 7,8-disubstituted guanosines of the following general formula I:

wherein R¹ , R², X, W and Y are as described hereinafter and comprise a class of guanosine derivatives that are potent immunostirnulants. Pharmaceutical compositions containing such compounds and methods of enhancing immune response in human and animal cells using such compounds were also discovered. DETAILED DESCRIPTION OF THE INVENTION

More particularly, the compounds of the present invention are represented by the general formula I:

wherein X is selected from any of O, S, NCN or Se. Most preferably, X is O.

W is selected from any of C, S, S(NH) or S(O). Most preferably, W is selected from any of C, S(O) or S.

Y is O. In addition, when W is C, Y may also be S.

R¹ is selected from any of H or C-i-Cβ acyl. More preferably, R¹ is H.

R² is selected from any of C1-C5 alkyl, C3-C5 alkylene such as ethylene, propenyl and 2-butenyl, aralkyl, wherein the alkyl portion is C1 to CQ and wherein the aryl ring may be optionally independently substituted with one or more substituents such as halogen, nitro, C1 to C2 alkyl, trifluoromethyl, thio, amino or substituted amino. Particularly preferred substituted amino groups are dimethyl amino, acetamido, phenylamino and sulfonamido. More preferably, R² is C3-C5 alkylene and most preferably allyl.

Particularly preferred compounds of formula I are those wherein X is O, R1 is H, R2 is allyl, W is C, S(O) or S and Y is O.

Examples of particularly preferred compounds of the present invention are 2-amino-7-(2-propenyl)-9-[β-D-ribofuranosyl-2',3'-carbonate]purine-6,8(1 H)- dione; 2-amino-7-(2-propenyl)-9-[β-D-ribofuranosyl-2',3'-sulfate]purine- 6,8(1 H)-dione and 2-amino-7-(2-propenyl)-9-[β-D-ribofuranosyl-2',3'- sulfite]purine-6,8(1 H)-dione.

When W=S and Y equals 0, there can be noninterconverting diastereomers with respect to the stereochemistry of the S atom.

A useful guanosine is substantially free from ionic charge at physiological pH values; i.e., about pH 7.0 to about pH 7.5, except for the ionic charges that might be provided by the relatively acidic 1 -position ring nitrogen atom. Thus, a useful molecule is free of acid and base-containing moieties that are not present in guanosine.

The guanines are acids, and as such can form base addition salts. Such salts are useful in providing storage stability and do not provide an added ionic charge to a guanine derivative used in a method of the invention because of the buffering effect provided by the host's blood and lymph systems or the buffer of a culture medium.

Pharmaceutically acceptable, non-toxic base addition salts of guanine derivatives are useful herein, and can be formed by treatment of the immune response-enhancing agent with an appropriate base, in a suitable solvent such as water or a lower alkyl alcohol such as methanol or ethano Exemplary inorganic bases include sodium hydroxide, potassium hydroxide, ammonium hydroxide and the like. Exemplary organic bases include tris-(hydroxymethyl)- aminomethane (TRIS), 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES) and the like. Conversely, the base addition salt form can be converted to the free guanosine form by treatment with acid.

As used herein unless otherwise noted alkyl whether used alone or part of a substituent group includes straight and branched chains such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, 2-methyl-3- butyl, 1-methylbutyl, 2-methylbutyl and neopentyl. Of course, if the alkyl substituent is branched there must be at least 3 carbons.

The compounds of the present invention may be synthesized according to the following general reaction scheme. REACTION SCHEME

IV

As shown, the reaction comprises reacting a 7,8-disubstituted guanosine

(II) with a silylchloride to protect the 5' hydroxyi of the guanosine nucleoside. This reaction is carried out in pyridine in the presence of t-butyldi methyl silylchloride and imidazole. This reaction requires about 9.5 hours at room temperature. The resulting compound of formula HI is then reacted with a suitable biselectrophile such as carbonyl dimidazole, sulfuryldimidazole or thionyl chloride often in the presence of a mild base such as pyridine to yield the compound of formula IV. The compound of formula IV is then re-; -sted with a suitable fluoride source such as tetra-n-butylammonium fluoride I" ΗF (about 1 to 20 hours, at about 20°C to 60°C) or cesuirή fluoride in THF or dioxane to yield the compound of formula V, which is a compound of the present invention with R¹ being H. Insertion of the other possible R¹ group Cf Cβ acyl can be effected by treatment with a conventional acylating agent such as acyl halides, acyl anhydrides or acyl imidazoles under standard conditions at which the acyl electrophile reacts with the 5- alcohol in the presence of a base such as triethylamine or pyridine.

The starting 7,8-disubstituted guanosine of formula II may be prepared by the techniques described in U.S. Patent No. 5,011 ,828 and by those processes described in Tetrahedron Lett. 1991, 32, 4823-4826.

A composition of this invention comprises a diluent amount of a physiologically tolerable carrier (also referred to herein as a vehicle or diluent) admixed with an immunopotentiating (immune response-enhancing or immunostimulating) effective amount of a substituted guanine nucleoside derivative or salt of this invention described before.

A composition for in vivo administration is typically provided for per oral or parenteral administration in customary unit dosage compositions. The term "unit dosage" and its grammatical equivalents as used herein refer to physically discrete units suitable as unitary dosages for human patients and other mammals, each unit containing a predetermined effective amount of the guanosine active ingredient calculated to produce the desired therapeutic effect in association with the required physiologically tolerable carrier, e.g. a diluent or a vehicle. The specifications for the unit dosage forms of this invention are dictated by and are directly dependent on (a) the unique characteristics of the active guanosine derivative ingredient and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active ingredient for therapeutic use in vitro, as well as in vivo in humans and other animals.

Examples of suitable unit dosage forms in accord with this invention are tablets, capsules, pills, powder packets, granules, wafers, and the like, segregated multiples of any of the foregoing, as well as liquid solutions, emulsions and suspensions. Liquid compositions can be administered in usual manners such as subcutaneousiy, intraperitoneally, intramuscularly, intravenously perorally or the like.

The amount of active ingredient that is administered in vivo as an effective immunostimulating amount depends on the age and weight of the patient, the

c particular condition to be treated, the frequency of administration, and the route of administration. The total daily dose range can be about 0.01 to about 400 milligrams per kilogram of body weight, more preferably about 0.1 to about 400 milligrams per kilogram of body weight. The human adult dose is in the range of about 70 to about 7000 milligrams daily, given either as a single dose or in 3 or 4 divided doses. Veterinary dosages correspond to human dosages with the amounts administered being in proportion to the weight and metabolic rate of the animal as compared to adult humans.

It will be appreciated by those skilled in the art that useful in vivo concentrations can vary from animal species to animal species. Those skilled workers also know that appropriate concentrations can be readily determined.

Concentrations for the in vitro contacting of animal cells are about 1x10^-6 molar to about 10^-3 molar for cell concentrations of about 10⁶-10⁷ cells per milliiiter. More preferably, the concentration is about 1x10"⁵ molar to about 3x10"⁴ molar. As will be seen from the Results Section hereinafter, the peak concentration; i.e., the cone ntration that provides the greatest adjuvanticity, for a given guanosine analog can vary as much as ten or more fold when studied in mouse as compared to human lymphocyte systems.

A composition can be solid or liquid. Physiologically tolerable carriers are well known in th& art. Exemplary of liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredient guanosine derivative and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate- buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose and other solutes. The latter carriers are exemplified by Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection and Lactated Ringer's Injection. Preferably, the solution should be isotonic unless it is an oral pediatric suspension.

Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional phases are glycerin, vegetable oils, such as sesame oil, cotton seed oil, and water-oil emulsions. Exemplary solid carriers include those materials usually used in the manufacture of pills, tablets or capsules, and include corn starch, lactose, dicalciυm phosphate, thickeners such as tragacanth gum and methylcellulose U.S. P., finely divided S.O2 polyvinylpyrrolidone, magnesium stearateiand the like. Additionally, the solid carrier can include biodegradable and non- biodegradable polymers, polypeptide carriers, affinity carriers such as AFFI- GEL 601 (phenyl boronate resin available from BIO-RAD Laboratories, Richmond, California), liposomes and synthetic polymers, as are known in the art. Antioxidants such as methylparaben and propylparaben can be present in both solid and liquid compositions, as can sweeteners such as cane or beet sugar, sodium saccharin, sodium cyclamate and the dipeptide aspartic- phenylalanine methyl ester sweetener sold under the tradename NUTRASWEETR (aspartame) by G. D. Searle Co.

A method of enhancing the immune response of leukocytes is also contemplated. Preferably, the immune response is an antigen-specific response. In accordance with this method, leukocytes such as lymphocyte preparations, B cells, T cells, NK cells, LAK cells, neurophils and macrophages are contacted separately or in combination in an aqueous medium with a before-described composition containing an immunostimulating effective amount of a before-described guanine nucleoside derivative.

The method can be practiced in vivo in humans, laboratory mammals such as mice, rats and guinea pigs or in veterinary animals and pets such as pigs, horses, cattle, dogs and cats. The method can also be practiced in vitro i cell cultures such as in hybridoma culture for the production of monoclonal antibodies.

The leukocytes are contacted in an aqueous medium regardless of whether the composition of guanosine derivative is itself a solid or liquid, or whether or not the liquid of the composition is aqueous. For the in vivo method the aqueous medium is supplied at least in part by the water of the blood of lymph. For in vitro methods, the aqueous medium is supplied at least in part b the culture medium used.

Contact between the composition and leukocytes is maintained for a time period sufficient for the contacted cells to manifest the enhancement of their immune response. Said immunostimulation can itself be manifest in cellular

A \ _Λ proliferation, enhanced antibody secretion, enhanced T helper activity, enhanced cytotoxic activity, enhanced cytokine production from T cells and macrophages, enzyme secretion from neutrophils, and the like.

The specific results discussed hereinafter illustrate a non-specific mitogenic response of murine spleen B cells, NK cell mediated cytotoxicity as well as the preferred antigen-specific antibody responses of murine B cells and of human peripheral blood lymphocytes which can, but need not be, depleted of T suppressor cells. Additional illustrative antigen-specific immunoenhancements that can be achieved using a method of the invention include proliferation of T ceils, the in vitro reconstitution of the primary immune response in murine or human immunodeficient B cells, T cell-replacing activity in normal murine or human B cells, and an in vivo enhancement of-murine antibody production. Any or all of the above are also contemplated as participating in enhancement of anti-tumor immune defense mechanisms.

For use in vivo, contact between leukocytes and a composition is typically maintained for a time period sufficient for the animal to clear the guanosine derivative from its body as by metabolism, excretion or both processes. That time period can be longer than that required for immunostimulation to be manifest. Contact with an individual unit dose is typically maintained for a time period of hours to about a week or more, depending, for a given compound, upon the carrier or vehicle used.

Contact in vitro can be maintained for a period of time sufficient for one of the before-described immunostimulations to become manifest as determined by standard assay techniques. Such maintenance times typically take about one to about seven days of time, and more usually about 2 to about 6 days.

Certain compounds of the present invention were tested to determine thei ability to enhance the immune response. The tests which were carried out are described l ; einafter. In each of the tests 7-allyl-8-oxoguanosine (loxoribine) was used as a standard. The results of each of the tests are reported in the Table that follows the description of the tests. In the Table, the compound numbers refer to the compounds made in Examples which are reported hereinafter. NK ASSAY

A. Animals

Male CBA/J and C3H/HeJ mice were obtained from Jackson Laboratories,

Bar Harbor, ME. All mice were used at 8 to 12 weeks of age. They were fed Purina rodent laboratory chow and tap water ad libitum.

B. Tissue Culture Medium

Culture medium was RPMI 1640 medium (flow Laboratories, McLean, VA) supplemented with 5% fetal bovine serum (FBS, GIBCO, Grand Island, NY or HyClone Laboratories, Logan, Utah) and 100 lU/ml penicillin, 100 μg/ml streptomycin, 2mM L-glutamine (all from Flow Laboratories, McLean, VA).

C. Cell Lines

The NK-sensitive cell line YAC-1 , was originally obtained from Dr. John Roder, Mount Sinai Hospital, Toronto, Ontario. It was maintained in basic culture medium in a 37°C, 5% CO2 incubator. Cell lines were tested periodically for mycoplasma and found to be negative.

D. Loxoribine

For these in vitro experiments 10.2 mg loxoribine were dissolved in 0.1 ml

1 N NaOH. This was further diluted with 9.9 ml culture medium to give a 3 mM stock solution. The stock solution was filtered through 0.22 μM millipore filters and diluted further with sterile medium. Vehicle controls contained equivalent concentrations of NaOH.

Lymphoid Cell Preparation

Mice were killed by cervical dislocation and spleens were removed aseptically and pressed through a stainless steel mesh. Cells were centrifuged for 10 min at 220g and resuspended in culture medium at 10⁷ cells/ml.

*^s F. In Vitro Culture of Lymphoid Cells with Loxoribine

Spleen cells at 4 concentrations, each in triplicate (10⁶, 5x10⁵, 2.5x10⁵, 1.25x10⁵ spleen cells) were cultured in 96 well, round bottom culture trays (Linbro, Flow Laboratories, McLean, VA) in a volume of 100 μl. Compounds 1 - 3 were added to the wells in a volume of 100 μl to give fin.^*-* concentrations of 3, 10, 30, 100, 300 μM of drug. Cultures were incubated fcr 18 hr & 7°C in 5% C0₂.

G. ⁵¹ Chromium Release Assay

YAC-1 target cells (5x10⁵) were incubated with 100 μCi sodium ⁵¹Cr (Amersham Canada, Oakville, Onta o) for 1 hr at 37°C, in 5% CO2, washed three times with. Phosphate Buffered Saline (PBS), and suspended in culture medium at 5 x 10⁴/ml. The plates containing cells to be tested for cytotoxic activity were centrifuged at 220g for 10 min, 100 μl supernatant were removed from each well, and 100 μl target cells were added to the wells. The plates were centrifuged for 5 min at 150g, incubate 1 at 37°C in 5% CO2 for 4 hr, and centrifuged for 5 min at 300g. Supematants were removed from the wells using the Skatron Supernatant Collection System (Skatron, Sterling, VA) and the samples were counted in a Beckman gamma counter. Maximum lysis was obtained by counting the total CPM in 5 x 10³ target cells. Minimum release, obtained from target cells cultured with medium alone, was always lower than 10% of the maximum CPM. Percent Lysis was calculated as:

Sample CPM - Minimum CPM X 100

Maximum CPM - Minimum CPM

Each time a compound was tested, loxoribine was tested as well for direct comparison purposes. The ED50 value, taken as a measure of potency, varied from 15-34 μM for loxoribine (see Table). The maximal response or loxoribine was assigned the value of 100%. The activity for compounds 1-3 is given in Table as a direct comparison to that of loxorbine. For example, the 145% response of compound 1 , indicates that it exhibited 145% of the same response as loxoribine on that give day. MURINE AND HUMAN ADJUVANTICITY

In Vitro Adiuvanticitv

Contacting animal antibody-producing cells with a composition useful herein provides an adjuvant effect on the primary antibody response to SRBC (sheep red blood cells) and other immunogens when evaluated in vitro. The immune response-modulating composition and effective amount of immunogen SRBC, are typically admixed to contact the cells substantially simultaneously. The words "antigen" and "immunogen" are used interchangeably.

At optimal concentration, a composition containing an effective amount of a useful guanosine analog derivative enhances the response to SRBC by at least about 2-6-fold. The effect is dose dependent.- Enhancement of the antibody response cannot be accounted for by the additive effectscof the specific response to SRBC and the polyclonal response tθ vth©1ga§nosine analog derivative. . GA ■aie-. . BC-ar

The adjuvant effect of compositions containing a useful guanosine analog derivative is exerted on immunogen-experienced (primed) as well as on naive cells. Both responses are enhanced by contacting the cells with compositions containing an effective amount of guanosine analog derivative. This adjuvant effect is dependent upon the concentration of immunogen added to culture. Thus, the primary IgM as well as the secondary IgM and IgG responses to immunogen (antigen) are augmented by contacting B cells with a composition containing an effective amount of an guanosine analog derivative as active ingredient, and maintaining that contact as discussed herein.

While immune responses; i.e., responses of B lymphocytes (or B cells) ar observed to be enhanced at all immunologically effective doses of immunogen administered in an appropriately supportive cellular milieu, the degree of enhancement is usually greatest at optimal or near optimal immunogen concentrations. Additionally, adjuvanticity of guanosine analog derivatives is synergistic with immunogen and not just due to the sum of independent immunogen-specific and polyclonal (nonspecific) responses. In summary, an enhanced immune response can thus be obtained by contacting B cells substantially simultai -ously with an effective, priming amount of immunogen and an immune response-enhancing composition useful herein, followed, after a primary immune response is obtained, by an additional contacting of the primed cells with a further effective amount of immunogen (antigen) alone or substantially simultaneously with a further amount of immune response-enhancing composition.

Guanosine analog derivative-containing compositions useful herein are thought to enhance the primary humoral immune response by acting directly upon the B cell and/or the immunogen-presenting cell. Thus, one of these derivatives enhances the antibody response mounted against T-independent antigens; i.e., responses that involve B cells and immunogen-presenting cells. In addition, compositions containing a guanosine analog derivative can replace the need of B cells for T helper cells, as discussed hereina :.r, and therefore exert their adjuvant effect in cultures initiated in the absence of intact, functional T cells. A replacement of T cells with T cell helper activity contained in mixed lymphocyte culture (MLC) supernates or other T cell df "βd lymphokines does not diminish the ability of a guanosine analog derivative to augment the antibody response.

Still further, the synergy observed between the soluble T cell signal contained in MLC supemate and the guanosine analog derivative-co_ taining composition indicates that the signal supplied by each is qualitative .y distinct. This synergy is observed over a range of supernate concentrations, indicating that the guanosine analog derivative is not simply providing more of the same "signal" that T cells provide. A comparable degree of synergy can be observed when such B cell cultures are supplemented with T cells rather than with T cell- like supernates (which are in fact T cell derived), and are contacted r the presence of immunogen with a guanosine analog derivative-containing composition useful in this invention.

In summary, T cell-mediated effects of the adjuvanticity of guanosine analog derivatives are not ruled out by the observation of T-independence for that adjuvanticity, i.e., the existance of a T cell-independent phase. Thus, more substantial enhancement can be observed from a composition containing the guanosine analog derivative under conditions of stimulation with low doses of T-dependent and T-independent type 2 antigens (T cell dependent situations) than with T-independent type 1 antigens (more completely T cell-independent), which suggests the presence of a T cell-dependent component. Moreover, guanosine analog derivatives are thought to act (either directly or indirectly) on precursors of T helper cells to increase the ability of a population of such cells to support (i.e., help) an antibody response to immunogen.

Lvmphocvte cultures

The serum-containing culture medium was prepared to contain the following per 100 mililiters: 91.9 milliliters RPM1 1640 (Flow Laboratories, Inc., Rockville, MD.), 0.1 milliliters of 100 x glutamine, 1.0 milliliter of 100 x sodium pyruvate, 1.0 milliliter of 50 x nonessential amino acids, 1.0 milliliter of water containing 10⁴ units of penicillin G and 10⁴ micrograms of streptomycin, and 5.0 milliliters of a supportive lot of fetal calf serum (FCS). These ingredients were admixed to apparent homogeneity. Spleen cell suspensions and populations enriched for splenic B cells were prepared as described in Goodman et al., J. Immunol., 121 :1905 (1978).

For evaluation of the primary humoral immune response to sheep erythrocytes (SRBC), 5x10⁶ to 10⁷ murine spleen cells were cultured in 1.0 milliliter of 5% FCS-containing medium for 4 or 5 days in the presence and absence of immunogen. Cells were incubated in culture trays (Costar, Cambridge, MA.) at 37°C, in a humidified atmosphere of 10% CO2 in air using tissue culture boxes (CBS Scientific, Del Mar, CA) that were rocked at a frequency of 7 cycles per minute. Pooled SRBC are available from the Colorado Serum Co., Denver CO.

Human peripheral blood lymphocytes (PBL) were prepared from normal heparinized venous blood by Ficoll-diatrizoate density gradient centrifugation. PBL were depleted of suppressor T cells bearing the histamine type 2 receptor by adhering them to the surfaces of histamine-rabbit serum albumin-coated plastic petri dishes (Cell-ect No. 2 kit; Seagen, Boston, MA) and by recovering the nonadherent cells by panning as described by Wysocki and Sato, Proc. Natl. Acad. Sci. USA, 75:2844 (1978) and modified by Cavagnaro and Osband, Biotech niques, January/February:30 (1983). The tissue culture medium employed in these studies for human lymphocytes was prepared as follows: One hundred milliliters (mL) contained 87.9 mL RPMI 1640 (Flow Laboratories, Rockville, MD), 0.1 mL 10Oxglutamine, 1.0 mL of 1.0 M HEPES buffer (Microbiological Associates, Betheseda, MD), 1.0 mL of water containing 10⁴ U of penicillin G and 10⁴ micrograms of streptomycin, and 10 ml of fresh autologous heat-inactivated plasma. For evaluation of the primary humoral immune response to SRBC, lymphoid cells prepared as above were cultured at a density of 2 x 10⁶/mL in a volume of 1.0 ml containing 5x10⁶ SRBC as antigen (Colorado Serum Co., Denver, CO) together with IL-2 (a partially purified preparation of human IL-2 that was free of interferon-gamma activity was obtained from Electro-Nucleonics, Inc., Silver Spring, MD) and the guanosine analog derivative.

Assay of human olaoue forming cells (PFC1

PFC secreting antibodies against SRBC were evaluated after 4 or 5 days of culture using a modification of the hemolytic plaque assay of Jerne and Nordin, Science, 140:405 (1963). The cells were brought up in complete medium before plaquing; they were plaqued in standard low M_r agarose (Bio- Rad Laboratories, Richmond, CA and were incubated in SRBC-absorbed guinea pig complement for one hour after a 1.5 hour incubation without complement.

Mice

CBA/CaJ mice, 8-16 weeks of age, are purchased from the Jackson Laboratory, Bar Harbor, ME. A breeding nucleus of CBA/N mice was provided by the Animal Production Section, National Institutes of Health, Bethesda, MD. SJL, BDFi and C57BL/6J mice 8-16 weeks old were obtained from the mouse breeding facility at Scripps Clinic and Research Foundation, La Jolla, CA. All mice were maintained on Wayne Blox F6 pellets (Allied Mills, Inc., Chicago, IL) and chlorinated water acidified with HCI to a pH value of 3.0

Cell preparations

Spleen and thymus cell suspensions were prepared as described in Goodman et al., J. Immunol., 121 :1905 (1978). B cell-enriched populations were prepared by treating 10⁸ spleen cells with a 1 :1000 dilution of monoclonal anti-Thy .1.2 antibody (New England Nuclear, Boston, MD) for 30 minutes at 4°C. Treated cells were centrifuged at 280 x gravity for 10 minutes, antibodies were removed, and the cells were resuspended in a 1 :6 dilution of CBA RBC-absorbed guinea pig complement at 37°C for 45 minutes. Cells were then washed andjβultured as-described before.

The human and mouse adjuvanticity assays were run on compounds 1 -3. In addition, loxoribine was tested on the same day as compounds 1-3.

MITOGENESIS ASSAY

Lvmphocvte cultures

Murine spleen cells were cultured in microculture plates (No. 3546, Costar, Cambridge, MA) at a cell density of 4 x 10⁶ viable cells milliliter in a volume of 0.1 milliliter, together with incremental concentrations of guanosine compounds 1 -3. Microcultures were incubated at 37°C in a humidified atmosphere of 10% CO2 in air. Cultures were fed daily with 8 microliter of nutritional cocktail, Mishell and Dutton, J. Exp. Med. 126:423 (1967).

Measurement of DNA synthesis. During the final 24 hpurs of culture, cells were radiolabeied with 1.0 micro Ci of [ ³H]TdR/culture (5 Ci/mM, Amersham Radiochemicals, Amersham, England). The microcultures were harvested with a Brandel cell harvester, Model M24V (Biological Research and Development Laboratories, Rockville, MD) onto glass fiber filter strips. Filter disks were transferred to plastic scintillation vials, covered with liquid scintillation cocktail, and counted in a Beckman LS-230 liquid scintillation counter.

Since there is some variability between runs of the same compound, such as loxoribine, some of the data in the Table are presented qualitatively relative to loxoribine . Thus, the full activity of loxoribine is represented by four pluses

(++++), whereas activity less than loxoribine is represented by fewer than four.

In the case of compound 1 , a numerical value of 145% is given with 100% being assigned to loxoribine as the base value. TABLE

NK Activation Murine Human

(Potency) Mitogenicity

Cmpd. # W & Y Values Adjuvanticity Adjuvanticity

1 W- S. Y-O 145% (33 μ ) 145% 46%

W- S(0), Y«0 33% (140 μM) 36%

W-= C, Y= 0 50%

100% (21 μM)

Loxoribine 100% (15-34 μM) 100% (standard)

As is apparent from the results reported in the Table Compounds 1 and 3 are extremely potent compounds with compound 2 showing potency, but not at the levels of the standard and compounds 1 or 3.

The preparation of compounds 1-3 reported in the Table, will now be described in the following Examples:

EXAMPLE 1 : 2-Amino-7-(2-propenvh-9-fR-D-ribofuranosvl-2'.3'- cart?Qnate]-purine-6,g(1 H)-dione (1 )

A solution 2-amino-7-(2-propenyl)-9-[5'-(t-butyldimethylsilyl)-β-D- ribofuranosyl]-purine-6,8(1H)-dione (1.28 g, 2.82 mmol) and 1 ,1- carbonyldiimidazole (0.91 g, 5.64 mmol) in dry THF (10 mL) was allowed to sti overnight under nitrogen atmosphere. Most of the THF was evaporated, and water was added. The product crystallized upon standing overnight. The crystals were filtered, washed with water and ether, and then air-dried to give 1.21 g (90%) of the desired material. This was purified further by flash silica gel chromatography (methylene chloride/methanol/ammonium hydroxide; 90:9:1 ) to give 0.63 g (47%) of white crystalline powder. The majority of this material (0.52 g, 1.08 mmol) and tetrabutylammonium fluoride (2.1 mL of a 1 M solution in THF) were added to THF (10 mL), and the solution was allowed to stir at room temperature overnight. The THF was then largely removed, and the residue treated with water. The crystals which formed were then collected and washed with water, ether, isopropanol, and then air-dried to give 0.29 g (74%) of fluffy white crystals of 1 (m.p. 323-325°C, dec). High-field H-1 NMR and mass spectral analysis supported the assigned structure.

Anal. Calcd. for Ci H₁₅N₅O.0.2H2θ: C, 45.58; H, 4.20; N, 18.98; H₂0, 0.98 Found: C, 45.89; H, 4.42; N, 18.80; H₂0, 0.99

The 2-amino-7-(2-propenyl)-9-[5¹-(t-butyldimethylsilyl)-β-D-ribofuranosyl]- purine-6,8(1 H)-dione was prepared as follows:

A solution of t-butyldimethylsilyl chloride (0.5O g, 3.3 mmol), imidazole (0.22 g, 3.3 mmol), and dry pyridine (5 mL) was stirred for 5 min. 7-Allyl-8- oxoguanosine (1.00 g, 2.96 mmol) was then added. The guanosine derivative slowly dissolved in the pyridine. After 5 h, the solution was poured into 35 mL of water. A small amount of ether was added. After stirring overnight, the crystals which had deposited were filtered, washed well with water, then thrice with ether, and air dried to give 0.95 g of a white powder. This material was purified by flash silica gel chromatography (methylene chloride/methanol/ ammonium hydroxide; 90:9:1). After recrystallization from MeOH/EtOAc there was obtained 0.60 g (45%) of white fluffy needles of the title compound (mp 257-259°C). High-field H-1 NMR and mass spectral analysis supported the assigned structure.

Anal. Calcd for Ci9H₃₁ N5θ₆Si: C, 50.31 ; H, 6.89; N, 15.44 Found: C, 50.37; H, 6.77; N, 15.57 EXAMPLE 2: 2-Amino-7-(2-propenvn-9-(β-D-ribofuranosγl-2'.3'-sulfate)- pμrine-6,8(1 H)-dione (2).

A solution of 2-amino-7-(2-propenyl)-9-[5'-(t-butyldimethylsilyl)-β-D- ribofuranosyl]-purine-6,8(1 H)-dione (3.55 g, 7.8 mmol), sulfuryidiimidazole (1.55 g, 7.8 mmol), LiH (0.16 g, 20.1 mmol) and DMF (1.6 mL) in pyridine (16 mL) was allowed to stir under an argon atmosphere for 4.5 h. Since some of the starting guanosine material was still present by TLC, an additional 50 mg of LiH was added. After a total of 7 h, the solution was cooled and treated with HOAc (15 mL). After stirring for 15 min, the reaction mixture was added dropwise to ca. 200 mL of water, with vigors ,-s stirrinq. The pale orange solid which formed was collected and washed with wate" -id allowed to air dry overnight. The product was purified by flash silica gel chromatography (methylene chloride/methanol/ammonium hydroxide; 95:4.5:0.5) to give 1.68 g (42%) of a white foamy glass. High-field H-1 NMR and mass spectral analysis supported the structure as being the 5'-(t-butyldimethylsilylether) of 2. A sample of this substance (1.10 g, 2.13 mmol) was dissolved in MeOH/methylene chloride (1 :1 , 10 mL). This solution was filtered to remove some black particulates, and then the solution was treated with p- toluenesulfonic acid hydrate (0.40 g, 2.13 mmol) and stirred. After 1 hour, the solution was chilled in an ice/water bath. The crystals that formed were collected and washed with cold MeOH and ether. The resultant solid (0.72 g, 85%) was air-dried and then dried under vacuum at room temperature to give 2 (m.p. 300°C with decomposition). High-field H-1 NMR supported the assigned structure.

Anal. Calcd. for C₁₃H₁₅N5θ8S: C, 38.90; H, 3.77; N, 17.45;

Found: C, 39.23; H, 3.86; N, 17.27

EXAMPLE 3: 2-Amino-7-(2-propenyn-9-fβ-D-ribofuranosyl-2'.3'- sulfitel-Purine-6.8M H dione. 0.25 Hydrate (3^

A solution of 2-amino-7-(2-propenyl)-9-[5'-(t-butyldimethylsilyl)-β-D- riibofuranosyl]-purine-6,8(1 H)-dione (0.50 g, 1.1 mmol) in pyridine (5 mL) was chilled to 0°C and then treated with SOCI2 (0.16 mL, 2.2 mmol). After stirring for 1 hour, MeOH (ca. 1.5 mL) was added, and the solution was then treated with tetrabutylammonium fluoride (4 mL of a 1 M solution in THF). After stirring overnight, an additional 4 mL of the fluor - reagent was added. The solvents were mostly evaporated in vacuo, and the pale, yellow residue was treated with water (ca. 12 mL). After 1 hour with cooling, the solids were filtered and then washed with isopropanol and ether and then air dried to give 0.31 g (74%) of white powder. The product was recrystallized from DMSO/water to give 0.22 g of white powder, dried in vacuo at room temperature for two days (m.p. 275- 278°C). High-field H-1 NMR and mass spectral analysis support the assigned structure as a 3.4:1 mixture of exoendo diastereomers with respect to the sulfite functionality. This compound was stable to storage at room temperature for at least a year and a half.

Anal. Calcd. for Ci3H₁₅N₅O7S-0.25H₂0: C, 40.05; H, 4.00; N, 17.96; H₂0, 1.1 Found: C, 40.02; H, 3.92; N, 17.68; H₂0, 0.9

Claims

WHAT IS CLAIMED:

1. A compound of the formula I:

wherein X is selected from any of O, S or Se;

W is selected from any of C, S, S(NH) or S(O);

Y is O and when W is C, Y may also be S;

R¹ is selected from any of H or C-i-Cs acyl;

R² is selected from any of C₁-C5 alkyl, C3-C5 alkylene, aralkyl, wherein the alkyl portion is C₁ to CQ and wherein the aryl ring is either unsubstituted or substituted; and

a pharmaceutically acceptable acid addition salt thereof.

2. The compound of claim 1 , wherein the aryl ring is substituted with one or more substituents selected from any of halogen, nitro, C-i to C2 alkyl, trifluoromethyl, amino, substituted amino.

3. The compound of claim 1 , wherein X is O.

4. The compound of claim 1 , wherein W is selected from any of C, S(O) or S.

5. The compound of claim 1 , wherein R¹ is H.

6. The compound of claim 1 , wherein R² is C₃-C5 alkylene.

7. The compound of claim 6, wherein R² is allyl.

8. The compound of Claim 1 , wherein X is O; R¹ is H; R² is C3-C5 alkylene;

W is C, S (O) or S and Y is O.

9. The compound of Claim 1, having the name 2-amino-7-(2-propenyl)-9-[β D-ribofuranosyl-2\3'-carbonate]-purine-6,8(1 H)-dione.

10. The compound of Claim 1, having the name 2-amino-7-(2-propenyl)-9-[β- D-ribofuranosyl-2',3'-sulfite]-purine-6,8(1 H)-dione.

11. The compound of Claim 1, having the name 2-amino-7-(2-propenyl)-9-[β- D-ribofuranosyl-2',3'-sulfate]-purine-6,8(1 H)-dione.

12. A composition comprising a diluent amount of a physiologically acceptable carrier admixed with an immunopotentiating effective amount of th immune response-enhancing compound of the formula I as recited in Claim 1.

13. A method of enhancing an immune response which comprises contacting leukocytes in an aqueous medium with a compound of the formula I as recited in Claim 1 , in an amount sufficient to enhance the immune response.

14. The method of Claim 13, wherein the cells are contacted in vitro in a culture medium.

15. The method of Claim 13, wherein the cells contacted include B cells.

16. The method of Claim 15, wherein the B cells are human B cells.

17. The method of Claim 13, wherein the cells contacted include NK cells.

18. The method of Claim 17, wherein the NK cells are human NK cells.

19. The method of Claim 13, wherein the cells are T cells.

20. The method of Claim 13, wherein the cells are neutrophils.

2"» . The method of Claim 8, wherein the contacting is carried out in vivo by administering one or more unit doses of the composition to a mammal.