CA1270248A - Imidazole compounds as immunomodulating agents - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

There are prepared compounds of the formulae:

(1) or

Description

~:70;~8 BACKGROUND OF THE INVENTION
-Simon U~S. patents 4,221,909 and 4,211,794 and Giner-Sorolla U.S. patent 4,221,glO and Faanes International Journal o Immunopharmacology, Vol. 2, No. 3, page 197 (1980), Florentine, International Journal of Immunpharmacology, Vol. 2, No. 3, page 240 (1980), Hadden, ~nternational Journal of Immuno?harmacology, Vol. 2, No. 3, page 198 (1980), Pahwa, International Journal of Immunophar~,2cology, Vo~. 2, No. 3, page 199 (1980), Wybran, Internatio..al Journal of Immunopharmacology, Vol 2, ~o. 3, pace 201 (1980) and Simon, 4th International Congress Or ~munology, Paris (1980) show that erythro-9-(2-hydroxy-3-nonyl)-hypoxanthine (~T 15392), as well as other members of the series described in the patents cited above are potent immunomodulating agents which have been demonstrated to enhance depressed immunity in both animals possessing tumors, Sato, International Journal of Immuno-pharmacology, Vol. 2, No. 3, page 200 (1980), as well as in humans with various tumors, Simon, hmerican Chemical Society ~ook of Abstracts, 182nd ~merican Chemical Society Meeting (1981). While the synthesis of these therapeutically useful agents can be carried out by the methods described in the patents cited above, their production, e.g.
the production of NPT 15392, on a large scale using those methods was cumbersome, costly, and tLme consuming.

SVMMARY OP THE INVENTION

There are prepared compounds of the formulae (1, ) C ~ ~N

CH3~fH-l - (CH2)n-cH3 OH H

(2.) lH

CH _~H-CH- (CH ) -CH3 OH

or 3 . 1 OH

/~ N
R
CH3-~H-CH^ ~CH2 ~ -C~
H

~270~48 .

where R is lower alkyl, e.g. of 1 to 4 carbon atoms such as methyl, ethyl, propyl, or butyl or halogen of atomic weight 35 to 80, i.e. chlorine or bromine.

The compound erythro-3-(2-hydroxy-3-nonyl)-4-amino-imidazole-5-carboxamide (NPT 15459) is useful in producing NPT 15392 and its homologues of formula (1) above are useful in making the corresponding homologues of NPT 15392. In addition to being useful in making NPT 15392 and its homo-loguesl ~PT 15459 and its homologues are useful in preparing novel analogues of NPT 15392 (and its homologues which contain various functional gro~ps in the 2-position of the purine ring, e.g. the compounds of formulae (2) and ~3). Such derivatives would be difficult to prepare without the aid of ~PT 15359 and its homologues.

The compounds of ~ormulae ~ 2), and ~3) also have immunomodulating ox immunopotentiating activity.

An immunopotentiator or immunomodulator is any agent which either restores depressed immune function, or enhances normal immune function, or both. Immune function is defines as the development and expression of humoral (anti~ody-mediated) immunity, cellular (thymocyte-mediated) immunity, or macrophage and granulocyte mediated resistance. It logically includes agents acting directly on the cells involved in the expression of immune response, or on cellular or molecular mechanisms which, in turn, act to modify the function of cells involved in immune response. Augmentation of immune function 70~48 may result from the action of an agent to abrogate suppressive mechanisms derived by negative-feedback influences endogenous or exogenous to the immune system. Thus, immune potentiators have diverse 5 mechanisms of action. Despite the diveristy of cell site of action and biochemical mechanism of action of immunopotentiators, their applications are essentially the same; that is, to enhance host resistance.

Applications of Immunopotentiators 1) The principal protective function of the immune system relates to resistance to invasion by pathogens, including viruses, rickettsia, myco-plasma, bacteria, fungi, and parasites of all types.
Thus, improvement of immune response, particularly when depressed, would calculatedly improve resis-tance in infection or infestation by any of the above pathogens. An immunopotentiator alone or in combination with anti-infective therapy can be applied to any and all infectious diseases.

2) ~ second protective function of the immune system is thought to be resistance to engraftment of foreign tissue, either natural as in the fetal-maternal relationship; or unnatural as performed by the transplant physician. Immunopo-tentiators can also be used to facilitate rejection of fetal or placental tissues or to modify or in~uce tolerance to grafts.

3) A third protective function of the Lmmune system is thought to be resistance to malig-nant cell develop~ent as in cancer. The use of immunopotentiators can be used in cancer treatment to enhance tumor rejection and to inhibit t~mor recurrenceS followling other forms of therapy.

~27~

4) A fourth protective function involves the capacity to recognize foreignness and to maintain nonreactivity to self by positive suppressor mechanisms. In aut~-immune and related disorders, immune reactivity directed at self anti-gens or exaggerated, elevated responses are apparent which are self-destructive. Immunopotentiators can be used to restore normal suppressor ~echanis..s, induce tolerance, or otherwise promote a normal immune response.

Each of the protective functions of the i~une system can be modified by n~n-specific therapy with im~unopotentiators alone or in com~i-nation with other agents employed to im?rove resistance or to kill the invading pathogen. In addition, specific resistance can be augmente~ b~
use of immunopotentiators in conjunction with some form of antigen as in a vaccine employins, for example, virus, tumor cells, etc. This use can be to induce either specific immunity or tolerance.
The latter might be exempli'ied by use ~ith antisen in allersy or auto-Lmmune diseases. Vse of immuno-potentiators may be either therapeutic or prophy-lactic; the latter particularly in aging, where infection, auto-immunity, and cancer are more common. The timing of administration and routes are variable and may be critical in de erm,ining whether a positive or negative response results.
Any agent capable o augmenting i~une response may inhibit it depending on timing and dose; thus, under certain circumstances an immunopotentiator could be used as an immunosuppressive agent for use in allergy, auto-immunity and trans~lantation.

~2~

Various procedures for determining immunomodulating activity are shown in Simon E.P.O.
published application 0036077, Illustrative of compounds within the invention in addition to NPT 15459 are:
erythro-3-(2-hydroxy-3-amyl)-4-amino-imidazole-5-carboxamide, erythro-3-(2-hydroxy-3-hexyl)-4-amino-imidazole-S-carboxamide, erythro-3-(2-hydroxy 3-heptyl)-4-amino-imidazole-5-carbozamide, erythro-3-(2-hydroxy-3-octyl)-4-amino-imidazole-S-carboxamide, erythro-9-(2-hydroxy-3-nonyl)-2-amino-hypoxanthine, erythro-9-(2-hydroxy-3-amyl)-2-amino-hypoxanthine, erythro-9-(2-hydroxy-3-hexyl)-2-amino-hypoxanthine, erythro-9-(2-hydroxy-3-heptyl)-2-amino-hypoxanthine, erythro-9-(2-hydroxy-3-octyl)-2-amino-hypoxanthine, erythro-9-t2-hydroxy-3-nonyl)-2-chloro-hypoxanthine, erythro-9-(2-hydroxy-3-amyl)-2-chloro-hypoxanthine, erythro-9-(2~hydroxy-3-hexyl)-2-chloro-hypoxanthine, erythro-9-(2-hydroxy-3-heptyl)-2-chloro~
hypoxanthine, erythro-9-(2-hydroxy-3-octyl)-2-chloro-. 30 hypoxanthine, erythro-9-(2-hydroxy-3-nonyl)-2-bromo-hypoxanthine, erythro-9-(2-hydroxy-3-amyl)-2-bromo-hypoxanthine, erythro 9-(2-hydroxy-3-nonyl)~2-methyl-hypoxanthine, erythro-9-(2-hydroxy-3~myl)-2-methyl-hypoxanthine, erythro-9-(2-hydroxy-3-hexyl)-2-methyl-hypoxanthine, erythro-9-(2-hydroxy-3-heptyl)-2-methyl-hypoxanthine, erythro-9-(2-hydroxy-3-octyl)-2-methyl-hypoxanthine, erythxo-9-t2-hydroxy-3-nonyl)-2 ethyl-hypoxanthine, erythro-9-(2-hydorxy-3-hexyl)-2-ethyl-hypoxanthine, and erythro-9-(2-hydroxy-3-amyl)-2-ethyl-hypoxanthine.

BRIEF DESCRIPTION OF THE DRAh'lNGS

Figure 1 is the IR-spectrum of NPT 15459, and Figure 2 is the W -spectrum of NPT 15459.

2; DETAILED DESCRIPTION

The synthesis of NP~ 15459 is carried out according to the scheme presented below:

(1) H2N \ / H N \ / C-H
HC + HC(OC2H5)3 2 HC
N ~C CH3CN N~ C2H5 I II III

(2) III -~ e:rythro CH3-fH-fH-(CH2)5-CH3 OH NH2~ /C N
H2N \~ ~>
IV ~ N

CH -fH-CH-(CH ) -CH
OH

V (NPT 15459) , ~2~X9~

The other compounds within formula (1) can be prepared in the same manner by replacing erythro-3-amino-2-nonan~l by the corresponding aminoalcohol, e.g. erythro-3-amino-2-pentanol and erythro-3-amino-2-octanol. ~n place of ethyl orthoformate there can be used other lower alXyl orthoformatesd eOg. methyl orthoformate, propyl orthoformate and butyl orthoformate.

Example Svnthesis of Erythro-3-(2-hydroxy-3-nonvl)-4-a~ino-imidazolë-5-carboxamice (~PT 9~59 (V)) _ _ For improved yield, reactions (1) and (2) above are carried out seque~tially without isolation of the intermediate product III. 2-amino-2-cyano-aceta~ide (1) (171 g, 1.71 moles) is suspended in 2200 ml of acetonitrile. Orthoformic acid triethyl-ester (II) (334 ml - 2.0 moles) and 2 ml of pyridine are added to the suspension with stirring. The suspension is heated to reflux temperature, using an oil bath preheated to 100C. The sus~ension is held at boiling temperature for 40 to 60 minutes.
III is produced in situ. 272.5 g of erythro~-3-amino-2-nonanol (IV) (1.71 moles) are then added over a 3 to 5 minute period and boiling is continued for an additional 10 to 15 minutes. The reaction is ouickly chilled to room temperature. The erythro-3-(2-hydroxy-3-nonyl)-4-amino-imidazole-5-carboxaride (~lPT 15459) (V) crystallizes. It is filtered b~
suction, washed with a small amount of acetonitrile and dried in vacuo at 70C.
~'ield: 330.4 g, 72~ of theory ~elting point (after recrystallization fro~ acetoni-trile`: 154-158C.

~L~,'7~324~3 ~10--SUMMARY OF CHEMICAL PROPERTIES OF (NPT 15459) Ch ical_or Physical Property Value M.P. 154-158C
C Theory: S8.18 Found: 57.80 N Theory: 20.87 Found: 21.00 Solubility - solubl e-i sopropanol - Yery soluble - HCl (Aq), acetic acid, methanol, ethanol - insoluble - benzene, water rLc sil;c2 gel (ethylacetate/methanGl:
8:2) Rf = 0.61 Mol . Wt . 268.35 Appearance Colorless and odorless powder _ ntity 1. IR-spectrum: (tig. 1) - bands at 3450 cm 1 (-~H2) 3400-3100 cm (several) (-CONH2 a.o.) 2950 cm 1 (-CH3 -, - CH2 -) 1660 cm 1 (-imidazole) 2. U~'-spectrum: (Fig. 2) - maximum at 267 nm minimum at 217 nm Foundi~o:~inal Valuf~:
Sulphate Ash nqt ponderable< 0,5 ~
-_~avy Metals < 50 ppm < 50 pp~
Found Loss of Dryinq not ponderable _ 2 ~
Pvrity (TLC) no side spots no side s,~cts Percentage of Side Products < O, 2 mole ~ < 10 mole Content 98, 7 X' '~5 - 105 - ~`2~

The reaction scheme described below illustrates the use of NPT 15459 in the synthesis of NPT 15392:

Reaction Scheme:

OH

N N~N~ 3 2~ ~N~
2 I reflux temp. N N
CH3-CIH-cH- (CH2) 5-CH3 CH3-fH-CH- (CH ) -CH
OH OH

V VI

268.35 27~3.35 The homologues of NPT 15392 can be prepared in the same manner Erom the corresponding homologues of NPT 15459. In place oE ethyl orthoforma-te -there can be employed -the other lower alkyl or-thoEormates, e.g. -the methyl, propyl, and butyl orthoformates. In forming NPT 15392 the acetic anhydride forms -the acetate of NPT 15392 and this is -then hydrolized with alkali, e.g.
sodium hydroxide.
In place of an orthoformic acid ester formic acid can also be used.

,~
~ o ~

Example 2 - UTILIZATION OF NPT 15459 TO P~ODUCE NPT 153g2.
(Erythro-9-(2-hydroxy-i-nonyl)hypoxanthine) -415 g of erythro-3-t2-hydroxy-3-nonyl)-4-amino-imidazole-5-carboxamide (V~ (1.55 moles) are suspended in 385 ml of orthoformic acid triethylester.
220 ml of acetic acid anhydride are added wi~h stirring. The suspension that is obtained is heated with stirring up to 100-105C. An exothermic -reaction ensues with liberation of ethanol. The liberated ethanol is separated by distillationO The temperature of the reaction mixture is increased slowly up to 130-140C. Stirring is continued for about 3 hours, while the liberated ethanol is continuousl.y separated by distillation. The reaction mixture is concentrated to a viscous syrup, which is s~irred into a mixture of 415 ml of 30% aqueous NaOH and 1.68 liter of water. The brown solution is slightly acidified by addition of a solution of 123 ml of acetic acid in 1.5 liter of water.
Erythro-9-~2-hydroxy-3-nonyl)-hypoxanthine (VI) crystallizes, is f.iltered by suction, washed with water and dried in vacuo at 60C.
Yield: 350 g corresponding to 81.4~ of theory Melting point (after repeated recrystallization from aqueous ethanol): 200-201C
Content by titration: 99.4%
Content of threo isomer: 0.9~

NPT 15459 and its homologues possess the a~ility to react with a ~ariety of reagents which 12'70Z~8 could lead to ring closure and the production of novel purine derivatives. By ~he judicious choice of reagents, it is possible to produce a number of 2-substituted derivatives of NPT 15392 and its homologues tha~ could not be produced by other means.
The scheme provided in the following diagram illus-trates a number of such examples.

C~ > ~--COC~
CH,~Ç~ H~')5 C~3 .
OAC
~7~-~r ~ O

H~

"c - N ~--N
R, C~
.J c~ >

~,C~ ~ ~1 C-N~i N O
~ (B~) C~ Ni o I ~
~, H.~N9 C~ ÇH- CH-(CH~)S ~H3 OH
e~2~ (2-hydr~ 3-rx~uyl)-hypoxauth~ne ~:70~48 In the reaction scheme just set forth Rl is the group 3 7H CH-(CH2)5-CH3 OH

The 2, 6-dichloro-purine compound in the abo~e scheme can be converted to the 2-chloro hypoxanthine using conventional procedures, e.g. by refluxing with sodium hydroxide in the manner sho~
in Simon U.S. patent 4,221,909 ~ethod D. The latter csmpound can then be converted to the corresponding 2-amino-hypoxanthine by reacting with methanolic ammonia in a manner analogous to that shown in Simon U.S. patent 4,221,909 Method B.

Compounds of formula ~3) where R is methyl can be made by reacting NPT 15459 or its l; homologues with ethyl orthoacetate rather than ethyl orthoformate. Compounds of formula (3) where R is ethyl can be made in similar manner by reacting ~PT 15459 with ethyl orthopropionate.

The Lmmunomodulating activity of ~PT
15459 was determined. The results are set forth below in Tables 1 and 2.

In Table 1 the effect of NPT 15459 on con A Induced Proliferation is described. The procedure is described in Simon U.S. patent 4,221,909, column 24, line 12 to column 25, line 10.

In Table 1 the effect of NPT 15459 on LPS
Induced Proliferation is described~

In Table 2 the SRBC Induced Antibody Formation procedure employed was that shown in Simon EPO application 0036077 page 46.

TAB!E 1 IMMUNOMODULATING ACTIVITY NPT ~Se59 Mitogen Induced Lymphocyte Proliferation Concentration Test X Percent Change (DOS2ge) from Contrsl CDm Con.rol Con A Induced 3i,0i9 Q
Proliferation .020 ~/ml " " 35,7~7 12 .5~ " " " 30,920 -12

5.0 " " " 20,09& 43a 10.0 " " " 12,736 66a Control LPS Induced 7044 0 Proliferation .02 ~g/ml ' " 61&4 -12 .5 " " " 6676 -6 .0 " " " 4128 ~2a 10.0 " " " 2449 -6 .

"a"~ significant by 't test; p < 0.00 7(~

~ABLE 2 IM~UNOM~DULATING AC~IV~TY NP~ l54~9 Antib~dy Formation Ccncentrati~n ~est X Percer~t C~,~n3e (D~s2ge) ~r~m Cor.trol PFC/l o6 Cel 15 Saline SRBC Induced ~3 G
Antibody For~,aticn .Q5 m~kg l57 ll96 .5 " 12G ~l26 5.0 " ` 66 ~2 Fcrmu~atio~s ~ he compounds of the presert ir.~-en.io., can be fed to a mammal at a dosa-,e of 1-1000 ~?/i;s of body weight and could be anticipatec to be active at levels as low as 0.0005 ms/'~.s.

It is anticipated they may be ad~ir.iste~c~
in tablet or capsule form to humans and whe.e solubility permits in the form of an aqueous syru?, or as solutions in oil, or where insoluble ac a supension. Typical pharmaceutical formul2ticns are described below:
Capsule:
NPT 15459 0.1-500 ms Avicel~pH 101 (microcrystalline cellulose) to make 800 ms.

- 127~3;~48 Suspension Aqueous s~spensions can be made with a number of suspending agents incorporated with the active drug substances. Included as suspending agents are such substances as sodium carboxymethyl-cellulose, Na alginate gum, tragacanth, Avicel RC-591 (microcellulose), methylcellulose, ~eegum, xanthan gum. In addition to a suspending agen~
such substances as swee~ners, flavors, colorants, preservatives, pr~tective colloids, and dispersant~
may be added.

S~rup For~ulation h'~T 15~5 O.Q5 - 2;G ~ (or at ~axi~Lm le~el Df s~l~bil~ty) Corn Sug~r 3.25 g.
Di sti 11 ed ~er . 05 g .
FD 2nd C Red ~0 ,00175 9.
Sodi um Sa cch2 . i n . 00250 9 .
Al c~hol U. S. P. . 0~ g.

~ethyl parzben U.S.P. .005 9.

~lycerin .001 g.

Cherry flavor .31225 9.

Fruit fl2~0r . û0825 9.

Distilled ~a~Pr g.s.ad ~ ml.

Ta bl e t Formu l a t i on _ .

NPT 154~9 0.1 - 500 mg Av;cel~pH 101 130 mg Starch, modified 20 mg Ma~nesium stear2te U.S.P. ~ 9 Polyvinylpyrrolidone22 mg Ste~r;c acid U.S.P. 30 mg

Claims (9)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. Process for preparing a hypoxanthine derivative of the formula:

wherein n is an integer from 1 to 5 where R is H or a C1-4 alkyl, which comprises:
reacting a compound of the formula (1):
(1) wherein n is an integer from 1 to 5, with either an equimolar amount of C1-4 alkyl orthoester of a C1-4 fatty acid or with formic acid in the presence of acetic anhydride hydrolyzing the corresponding acetate with an alkali.

2. A process according to Claim 1 for preparing a compound wherein R is hydrogen, which comprises reacting a compound of the formula (1):
(1) wherein n is an integer from 1 to 5 with an equimolar amount of a C1-4 alkyl orthoformate or formic acid in the presence of acetic anhydride hydrolyzing the corresponding acetate with an alkali and recovering the compound wherein R is hydrogen.

3. A process according to Claim 2, wherein there is employed a C1-4 alkyl orthoformate.

4. A process according to Claim 2, wherein there is employed formic acid.

5. A process according to Claim 1, wherein there is employed a C1-4 alkyl orthoacetate to provide a compound of the formula wherein R is methyl.

6. A process according to Claim 5, wherein the C1-4 alkyl orthoacetate is ethyl orthoacetate.

7. A process according to Claim 1, wherein there is employed a C1-4 alkyl orthopropionate to provide a compound of the formula wherein R is ethyl.

8. A process according to Claim 7, wherein the C1-4 alkyl orthopropionate is ethyl propionate.

9. A process according to Claim 1 for preparing a compound of the formula comprising removing the alcohol formed in the reaction and adding alkali after removing the alcohol to neutralize the acetic anhydride.