CA1133396A - Immunopotentiator comprising forphenicine and production of forphenicine - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE:
A new immunopotentiator is now provided, which comprises as the active ingredient forphenicine of the formula or a pharmaceutically acceptable salt thereof or a hydrate thereof. Forphenicine is now found to be readily produced by oxidizing a new compound of the formula (I)

Description

1~333~6 SUMMARY OF TH~. INVENTION
Field of the invention This invention relates to a new immunopotentiator comprising forphenicine as the active in~redient, a new process for the production of forphenicine and to a method of enhancing the immune response in living animals with the new immunopotentiator.
Description of the prior art Forphenicine is a known substance which is isolated and recovered from the culture broth of a strain of the genus Streptomyces by H. Umexawa et al., and it has been known that forphenicine is a potent inhibitor to alkaline phosphatase. Forphenicine has a very low toxicity as shown by the fact that it gives an LD50 value of more than 500 mg/kg.
- upon intraperitoneal injection in mice to estimate its acute toxicity. Moreover, it is confirmed that forphenicine is a compound represented by the formula OH

E~OOC-CH ~ CHO

2 ~

(see Japanese Patent Application Pre-publication "Kokai" No.
116685/75 and "Journal of Antibiotics "Vol. 31, No. 3 pp. 244-246 and Vol. 31, No. 5, pp. 483-484 (1978)).
We, the present inventors, have made extensive research on whether forphenicine is useful as a medicine for any purpose. In consequence, we have now found that forphenicine as well as its pharmaceutically acceptable salts and hydrate exhibit an immunopotentiatin~ activity in 1~33396 living animals.
DETAILED DESCRIPTION OF THE INVENTION
According to a first aspect of this invention, therefore, there is provided a pharmaceutical composition for use as immunopotentiator which comprises as the active ingredient an effective amount of forphenicine having the formula / OH

HOOC-fH ~ CHO

or a pharmaceutically acceptable salt hereof or a hydrate thereof in association with a pharmaceutically acceptable carrier for the active ingredient.
Forphenicine present in the pharmaceutical composition according to this invention may be any of a pharmaceutically acceptable salt thereof or a hydrate of its compound, or a salt of the hydrate.
The pharmaceutically acceptable salt of forphenicine includes an alkali metal salt, an alkaline earth metal salt and the like and trialkyl amine salt as the carboxylate ;~ of forphenicine as well as acid-addition salt of the amino group thereof, including such as hydrochloride, sulphate, methanesulfonate, trifluoroacetate and the like~
The pharmaceutical composition of this invention may be formulated into injectable solutions or suspensions by dissolving or suspending forphenicine at a suitable level of from 0.1% to 10% by weight into a physiological saline solution or other conventional pharmaceutically acceptable liquid vehicle such as Ringer 16 solution, with or without aid of a suitable dispersion agent, and together with one or more of pH-adjuster, buffer, stabilizer, excipient, isotonic adjuster, local anesthetic and the like. The injectable solution or suspension so prepared may be given, eg. by intravenous injection, intramuscular injection or intraperitoneal injection. The injectable solution or suspension so prepared may be lyophilized by a conventional procedure, if desired, to give a lyophilized, injection `~ 10 solution which may be dissolved in water just before use.
- The pharmaceutical composition of this invention may also be formulated as conventional orally administerable forms such as tablets, capsules, powders, solutions and suspensions, either by admixing an amount of forphenicine with a conventional pharmaceutically acceptable solid carrier such as starch, sucrose, talc and calcium carbonate or by dissolving or suspending an amount of forphenicine in a pharmaceutically acceptable liquid carrier such as ethanol and water together with ordinary excipient, stabilizer, binder, disintegrating agent, lubricant, polish, coloring agent, flavorants and the like. The proportion of forphenicine to the solid or liquid carrier may be chosen appropriately depending on the form of the orally administerable formulation prepared and usually may be in a ratio of from 1:1 to 1:100 by weight.
It will be appreciated that the actual preferred dosage of forphenicine used will vary according to the particular composition formulated for administration, the mode of administration and the particular disease to be treated. ~any factors that modify the action of the ~133396 pharmaceutical composition of this invention will be taken into account by the skilled in the art, for example, age, body weight, sex, diet, time of administration, route of administration, rate of excretion, drug combinations, reaction sensitivities and severity of the disease. Generally, about 0.02 mg to 200 mg of forphenicine may be given to an adult person as a units dosage once a day or twice or more a day according to the conditions of patients. Optimal dosages for a given set of conditions of a patient can be ascertained by the skilled in the art using conventional dosage determination tests in view of the above guidelines and in view of the past experiences as obtained when determining suitable dosages of the previously known immunopotentiating drugs.
According to a second aspect of this invention, therefore, there is provided a method of enhancing the immune response in living animals which comprises administer-; ing to a living animal an immunopotentiating composition comprising an effective amount of forphenicine having the formula / OH

HOOC-CH ~ / ~ CHO

or a pharmaceutically acceptahle salt thereof or a hydrate thereof.
The effect of forphenicine (FPC) on the immune responses was illustrated by the following experimental data.

113339~

1. Effect of FPC on antibody formation in vivo a) Effect of FPC on antibody formation to SRBC (sheep red blood cell) in mice Mice were immunized with 108SRBC by intravenous injection and simultaneously 1 mg, lOO~g, lO~g or l~g of FPC was injected by intraperitoneal injection, respectively.
Four days later, the number of antibody forming cells in spleen cells in term of the number of plaque forming cells (PFC) were enumerated by Jerne's hemolytic plaque assay (see N.K. Jerne, A.A. Nordin and C. Henrry: "The agar plaque technique for recognizing antibody-producing cells.
Cell-bound Antibodies." ed. B Amos and H. Koprowski pp.
109-122, Wister Institute Press. Philadelphia, 1963). As shown in Table 1 below, the intraperitoneal injection of 1 mg, lOO~g or lO~g/mouse of FPC augments the antibody formation to SRBC in mice.
Table 1 Effect of forphenicine (FPC) on antibody formation of SR~C in mice . PFC` ~er s~leen *
Immunlzed wlth - - -(+ S.E **) SRBC 111,000 + 3,500 108SRBC, FPC 1 mg, i.p. 187,000 + 11,200 : " , " lOO~g l 272,000 + 23,500 , ll lO~g l 222,000 + 42,900 1 , ll l~g " 113,000 + 12,900 * 4 days after immunization ** S.E. means standard error b) Effect of FPC on antibody formation in vitro The effect of FPC on antibody formation in vitro was ~ 5 ~

1~33396 determined according to the method described by Mishell &
Dutton. Each dose of FPC and 106SRBC as antigen were added at initiation of the cultivation to the cultures which contained 15 x 106 cells of dissociated spleen cells - from CDFl mice. Antibody formation was determined 4 days later by enumerating in terms of plaque forming cells (PFC).
As shown in Table 2 below, the addition of O.l~g to O.OOOl~g of FPC increased the number of antibody forming cells in the cultures.
Table 2 Effect of Forphenicine on Antibody Formation in vitro ,~ A,d,dition to cultures PFC/culture *

lO SRBC 2430 ln6S~BC + FPC, l~g 1700 " + " O.l~g 3240 " + " O.Ol~g 3120 " + " O.OOl~g 3200 " + " O.OOOl~g 2500 * Results in spleen cells cultured for 4 days.
The experiment was designed to determine on what time of the addition of FPC at different times in the period of cultures the strongest stimulatory effect is found. To the spleen cell cultures were added O.OOl~g/culture of PPC
at a time of initiation of cultures and 24 hours, 48 hours or 72 hours after start of cultures, respectively. The result is shown in Table 3 below. Among the additions of FPC at different times, the maximal stimulatory effect was ~1~3396 observed with the addition at the initiation of cultures, whereas the additions at 24 houxs or 48 hours or 72 hours after start of cultures were ineffective. As described by Pierce et al., the primary effect of FPC might be in activating macrophage or T-cells among cells concerning antibody formation to SRBC in vitro.
Table 3 Influence of Addition of Forphenicine at Varying Times in Period of Spleen Cell Culture for Antibody Formation to SRBC

! Addition to cultures PFC/culture *

106SRBC, FPC 0 hr 2500 " " 24 hr 1380 " " 48 hr 1720 " " 72 hr 1500 . . _ * Results in spleen cells cultured for 4 days.
2. EEfect of FPC on cellular immunity The effect of FPC on cellular immunity was tested by employing delayed-type hypersensitivity (D'.T.H.) to SRBC
according to the method described by Lagrange et al. (see P.H. Lagrange, G.B. Mackaness and T.E. Mille: "J. Exp. Med.", 139, 1529-1539 (1974)). Mice were immunized with 108SRBC
by subcutaneous injection to right hind footpad. 4 Days later, they were received an eliciting dose of 103SRBC in left hind footpad. D.T.H. response was measured as the increase of left hind footpad in thickness at the end of 24 hous thereafter. FPC was injected intraperitoneally at 1~3339~

a time of immunization or at a time of eliciting injection.
As shown in Table 4 below, each injection of lOO~g, lO~g/
mouse of FPC at a time of immunization augmented establish-ment of D.T.H. and the injection at a time o~ the eliciting also augmented the response. Since the injection of FPC to mouse footpad which were not immunized with SRBC did not show any inflammatory response, it is clear that FPC is a strong stimulator for both of antibody formation and cellular immunity.
Table 4 Effect of Forphenicine on D.T.H. to SRBC
in Mice i increase immunized with elicited with thickness (x 0.1 ~nn) 108SRBC 108SRBC 8.3 + 0.8 10 SRBC, FPC lOO~g, i.p. 10 SRBC 11.5 + 0.9 " " lO~g " " 10.3 + 0.9 " " l~g " " 13.6 + 1.8 " " O.l~g " " g.o + 0.4 108SRBC 108SRBC, FPC lOO~g, i.p. 14.5 + 0.8 " " " lO~g10.6 ~ 1.3 " " " l~g12.0 + 0.9 " " " O.l~g7.6 + 0.8

3. Effect of FPC on transplantable animal tumors a) Effect of FPC on transplantability of L-1210 in CDF
mice - It is known thatmice given 10 leu~emia L-1210 cells/
mouse intravenously were died with resulting infusion of ~ ~33396 L-1210 cells in whole body within 11 to 12 days. At a time of transplantation, L-1210 cells were mixed with l~g of FPC and then injected to mice intravenously. ~s shown in Table 5, mice given FPC-mixed cells were survived 4 out of 10 mice more than 60 days. It is observed that l~g of FPC
does no~ affect growth of L-1210 cells.
Table 5 Effect of FPC on Transplantability of L-1210 by Intravenous Injection in CDFl Mice M.S .D. * ILS %** surviYvors r 103L-1210 cells, i.v. 12.3 - 0/5 10 L-1210 cells + FPC l`~g, i.v. >28.0 >128 2/5 * M.S.D. denotes Mean Survival Days.
** ILS ~ (percentage of Interval of ~ife~ Span) was calculated according to the following equation:

Number of mean survival days for the - ILS~ ) = treated grou~
Number of mean survival ~ays for the -control group x 100)-100 b) ~ffect of FPC on Gardner lymphoma in C3H/He mice Gardner lymphoma cells which were maintained in C3H/
He mice by a successive transplantation every 6 days were inoculated into inguinal region of mice subcutaneously, and then 100 g/mouse of FPC was iniected daily for 10 consecutive days intraperitoneally. Thirty days thereafter, tumor was taken out, weighed and the growth rate was estimated. Comparing to non-treated control, as shown in Table 6, tumor growth of FPC-treated group was inhibited ~ ~333~6 about 67% in a mean value and the tumors in 4 out of lO
mice w~re inhibited more than 90%.
Table 6 Effect of FPC on Gardner Lymphoma in C3H/He Mice mean weight Inhibition ~ of of tumor tumor growth Control (l x 105 cells, s.c.) 741 _ FPC, lOO~g/m, i.p. * 246 66.8 * injected daily for 5 days after implantation of tumor cells.
From all of the experiment mentioned above, it is indicated that FPC, which is a potent inhibitor for alkaline phosphatase of chicken intestines, primarily activates macrophage and stimulates imrnune responses. Thus, this agent are very useful for therapy of many immunodeficient disease, i.e. rheumatoid diseases, thyroiditis, multi-plesclerosis, systemic 1upus erythematosus etc. and immunological deficiency of tumor bearing host and immuno-therapy for tumors.
Now, we have succeeded to provide a new process for the production of forphenicine, starting with a new compound represented by the formula (I) H

HOOC-IH~ CH2 ~I) According to a third aspect of this invention, ~133396 therefore, there is provided a process for the preparation of forphenicine which co~prises oxidizing a compound of the formula (I) OH

HOOC-CH - ~ - CH2H (I) l H2 In carrying out the process of this invention, the starting compound (I) is oxidized with a known oxidizing agent to convert its hydroxymethyl group into aldehyde group and thereby to give forphenicine. In this case, the starting compound (I) should preferably be subjected to oxidation reaction usually in the form of an amino-protected derivative thereof. Any amino-protecting group may be used as the amino-protecting group available in the starting compound (I) employed in the oxidation reaction of the present process,provided that, after the oxidation reaction, it may easily be removed under such a condition which will not decompose forphenicine. For example, a preferred amino-protecting group includes an alkoxycarbonyl group such as t-butyloxycarbonyl and the like.
Any known oxidizing agent which is conventionally used to oxidize an aromatic hydroxymethyl group to an aldehyde group may be used as the oxidizing agent in the present process. Namely, use may be made of activated manganese dioxide, chromic acid, lead tetraacetate, ruthenium tetraoxide, selenium dioxide, halogen and the like. Any organic solvent which is inert to the oxidation reaction, such as methylene chloride, acetic acid may be 113~396 used as thc reaction medium. Water m~y be also used when the oxidizing ayent used is water-soluble. The oxidation reaction can usually be conducted at room temperature or at an elevated temperature. Subsequent to the oxidation reaction, the amino-protecting group may be removed from the amino-protected forphenicine in the conventional procedure to give forphenicine. Alternatively, the oxidation product, i.e. the amino-protected forphenicine may be isolated and recovered by the conventional means prior to the removal of the amino-protecting group, but the oxidation product may be subjected to the reaction of removing the amino-protecting group without any particular isolation.
Subsequent to the removal of the amino-protecting group, forphenicine may be purified and isolated using a method for the purification of forphenicine described in Japanese Patent Application prepublication "Kokai" No. 116685/75, `~ for example, using SP-Sephadex~C-25 (a product of Pharmacia - Fine Chemicals Co., Sweden) and the like.
A method for the preparation of the starting compound (I) starting from a hydroxyterephthalic acid di-methyl ester is summarized in the following reaction scheme. In the reaction scheme below, Rl represents methyl group, and the group ~C ~R forms a divalent hydroxyl-protecting group where R2 and R3 are each methyl group.

~ -.i`~J ' 1~33396 o, ~
, ~ o o \ o~ ~
r~ ~
~ a) h .~ O
O' ' X I ~N
O
- ~ "

S i \
O :rl . O
~ ) rl o 1' ~ ,~ o ~
O ~ R

\ / o o '~ ,~f u~

O ~ oU
Z :¢

, .`,~

, This invention is now illustrated with reference to the following Examples.
Example 1 (a) Protection o-f an amino group in the compound of the formula (I):

o <~

379 mg of the compound tI) was admixed with 5 ml.of water and 0.42 ml. of triethylamine to prepare a solution of the compound (I). To this solution was added a solution of 528 mg of t-butyl S-4,6-dimethylpyrimidine-2-ylthiolcarbonate in 5 ml. of dioxane. The admixture so obtained was allowed to stand at ambient temperature for 23 hours to effect the reaction for protection of the amino group of the compound (I) by t-butoxycarbonyl group. The reaction solution so formed was diluted with 30 ml. of water and then extracted twice with lO ml. of ethyl acetate to remove the unreacted carbonate. The aqueous layer was then cooled to 0cand adjusted to pH 2.0 by addition of lN hydrochloric acid. The reaction solution was extracted five times with 10 ml. of ethyl acetate for recovery oftheproduct, and the extracts were combined together, washed once with water and then distilled under reduced pressure to remove the solvent and thereby to give 672 mg of pale yellow viscous oily product. The residue so obtainedw,as 'admixe'd-~with'me~thylene chloride, and the resultant colorless precipitate was filtered off, washed with methylene chloride and dried in vacuo to give 436 mg of a colorless powder.
This colorless powder consisted of an amino-protected derivative of the compound (I) in which an amino group is protected with t-butoxycarbonyl group and represented by the formula (II):

OH

HOOC-fH -- < ~ ~ - CH2OH
IH
CO tII) H3C-C~CH3 Melting point: 143 to 145C
Infrared absorption spectrum:
. vmax 3525, 3225, 3080, 2990, 2740, 2625, 1735, 1650, 1595, 1505, 1~87, 1435, 1405, 1375, 1300, 1250, 1230, 1210, 1190, 1165, 1120, 1060, 1038, 980, 920, 860, 830, 805, 780, 720, 695 Nuclear magnetic resonance spectrum:
(60 MHz, in deutero-methanol solution) ~ ppMSm : 1.43 (9H, s), 5.10 (lH, s), 6.67 (2H, s), 6.88 (lH, d), 6.89 (lH, dd), 7.29 (lH, d).
(b) Oxidization of the aminc-protected derivative of the compound (I) 60 mg of the compound (II) obtained in the preceding step (a) of this Example was dissolved in 0.5 ml. of acetic acid, and to the resulting solution was added 250 mg of chromium (IV) oxide~pyridine complex (Collins reagent) at 0C. The mixture so obtained was subjected to the reaction at 0C for five minutes and then at ambient temperature for 3~;

one hour and su~sequently concen~rated to dryness under reduc~d pressure. The resultant residue was dissolved in 30 ml. of water and 5 ml. of butanol, and the resulting solution was extracted four times with 5 ml. of butanol.
The butanol phases were combined together, washed once with 5 ml. of water and distilled under reduced pressure to remove butanol.
The resultant residue was admixed with 1 ml. of trifluoroacetic acid and the resulting admixture was allowed to stand at ambient temperature for 30 minutes to effect the removal of the amino protecting group and then concentrated to dryness under reduced pressure. The solid residue obtained was dissolved in a small volume of water, the insoluble matter was filtered off, and the filtrate was ; then passed through a column of 10 ml. of SP-Sephadex~C-25 (H -form, a product of Pharmacia Fine Chemicals Co., Sweden).
The resin column was then eluted with water. The fractions containing the eluted forphenicine were combined together, concentrated to dryness under reduced pressure and dried in vacuo to give 12 mg of slightly yellow microcrystals of forphenicine.
The inhibitory activity to alkaline phosphatase of this crystal exhibited IC50 = 0 075 mcg/ml.
Melting point: ~300 Infrared absorption spectrum:
~ mBax : 2960, 2900, 2830, 2640, 1665, 1650, 1620, 1580, 1520, 1500, 1430, 1390, 1370, 1355, 1310, 1290, 1275, 1235, 1205, 1160, 1150, 1128, 1045, 950, 910, 905, 865, 805, 795, 735, 680 Nuclear magnetic resonance spectrum:
.

E~!

~33396 ~100 ~z, deutero-trifluoroacetic acid solution) ~ pTpMSm : 5.51 (1~l, s), 7.2 - 7.5 (2~), 7.91 (lH, d), 10.02 (lH, s).
Exam~le 2 119 mg of the compound (II) obtained in the preceding step (a) of Example l was dissolved in 18 ml. of ethyl acetate, and to the resulting solution was added 1.5 g of activated manganese dioxide. The mixture so obtained was allowed to stand at ambient temperature for 4 hours to effect the oxidation. When the reaction was completed, manganese dioxide is filtered off and washed with 30 ml. of a mixed solvent of water-methanol (l:l by volume) and then with 30 ml. of methanol. The filtrate and washing liquids were combined together and concentrated to dryness under reduced pressure. The residue obtained was taken up into l ml. of trifluoroacetic acid and the mixture was subjected to the reaction at ambient temperature for 30 minutes to effect the removal of the amino-protecting t-buto~ycarbonyl group and then concentrated to dryness under reduced pressure. The residue obtained was taken up into 10 ml. of water, and the resulting solution was passed through a column of 50 ml. of SP-Sephadex~C-25 (identical to that used in the preceding step (b) of Example l) for the adsorption of the desired product. The column was eluted with water. The eluate was collected in 200 drop-fractions.
The compound of the formula (III):

~1 .

113;~396 OH

~OOC-Ch~ O~H ~III) was eluted in the fractions Nos. 6 - 10, the desired forphenicine in the fractions Nos. 18 - 29 and the unreacted compound (I~ in the fractions Nos. 30 - 48. The fractions of the eluate were respectively combined together, concentrated to dryness under reduced pressure and dried in ~acuo, recovering 5 mg of the compound (III), 34 mg of forphenicine and 11 mg of the compound (I), respectively.
The inhibitory activity to alkaline phosphatase of forphenicine so obtained exhibited IC50 = 0 057 mcg/ml.
Th~a physico-chemical properties of forphenicine thus o~tained was perfectly identical to those of forphenicine - obtained in the preceding step (b) of Example 1.
Example 3 - ~ .
1 g of forphenicine was dissolved in distilled water to a 1000 ml. volume. ThP resulting aqueous solution was sterilized in the conventional procedure, poured into vials in 2 ml.-portions and lyophilized. In use, this lyophilized preparation may be diluted with distilled water to give an injectable solution.
Example 4 below illustrates the preparation of the compound of the formula o~

HOOC-CH - ~ - CH20H (I) which is used as the starting compound in the process according to the third aspect of this invention and which is a new compound now denominated forphenicinol.
Example 4 (A) Preparation of the compound having the formula OH

H3COOC~ CHZH (V) Into a three-necked flask of 300 ml. capacity fitted with a cooling tube were placed 1577 mg of dimethyl hydroxyterephthalate of the formula H3COOC~ COOCH3 (IV) together with 50 ml. of methanol.
5675 mg of sodium borohydride was slowly added to the contents of the flask under stirring, during whi~ch effervescence, heat evolution and reflux began to take place. After completion of the addition over about 2 hours, the reaction mixture was refluxed for further one hour to complete the reduction. 100 ml of water was then :1133396 added to the reaction solution, which was adjusted to pH 2 with 6N hydrochloric acid, followed by addition of 50 ml.
of butanol. The methanol solvent was then distilled off under reduced pressure and the residue was extracted three times with 50 ml. of butanol. The hutanol extracts were combined together and washed with 20 ml. of water.
Removal of the butanol by evaporation under reduced pressure gave 1548 mg of a colorless powder. The powder was taken up in benzene and the solution was passed through a column of 100 ml. of silica gel (Kieselgel 60, 70 - 230 meshes, made by Merck Co.) which was charged as a suspension ~- in benzene, for adsorption of the desired compound. The i column was washed with 500 ml. of a mixture of benzene-ethyl acetate (9 : 1 by volume) and then eluted with a mixture of benzene-ethyl acetate (4 : 1 by volume). The eluate was collected in fractions of each 15 g and fraction Nos. 35 - 65 containing the desired compound were combined together and evaporated under reduced pressure to remove the solvent, yielding 1224 mg of colorless crystals.
Recrystallization from a mixture of methanol-benzene-ethylether gave 914 mg of the titled compound of formula (V) as colorless crystals with a melting point of 104.5 - 105C.
(B) Preparation of the compound having the formula:

\3 / CH3 , 0/\o COOC~ ~2 (VI) 1.

1~33396 1224 mg of the compound (V) obtained in the stage (A) was suspen~ed in 30 ml. of dry benzene, to which were then added 5 ml. of 2,2-dimethoxypropane and 50 ml. of p-toluenesulfonic acid monohydrate to introduce isopropylidene group as the hydroxyl-protecting group. The resultant mixture was allowed to stand at room temperature for 3 hours to effect the reaction. The clear reaction solution was passed through a column of silica gel (the same one as described in Stage (A)). The column was then washed with benzene and the washings were combined with the effluent from the column. The combined solution (about 250 ml. in total) was evaporated under reduced pressure to remove the solvent, leaving 1462 mg of a colorless oil. On standing at room temperature the oil was solidified into colorless crystals of the desired compound (VI) having a melting point of 47.5 - 48C.
(C) Preparation of the compound of the formula:

C ~ CH3 HOH2C~ 2 (VII) 1462 mg of the compound (VI) obtained in Stage (B) was dissolved in 20 ml. of dry benzene. To the solution was added dropwise with ice cooling 2.8 ml. of 70% sodium ,~ dihydro-bis(2-methoxyethoxy)aluminate (a reducincJ agent made by Wako ~unyaku X.K.) solution in benzene and the mixture was allowed to stand at ambient temperature for 2 hours to effect the reduction. The reaction mixture was then ice-cooled and adjusted to pH 7 with lN hydrochloric aci~. The white precipitate formed was filtered off, the filtrate was extracted with benzene and the extract was evaporated under reduced pressure to remove the benzene, affording 1213 mg of colorless crystals. Recrystallization from benzene-cyclohexane gave 92~ mg of the desired compound (VII) as colorless needles with a melting point of 73.5 - ~5.5~C.
(D) Preparation of the compound of the formula:

r OX

OHC-- ~--/ 2 (vIII) 250 mg of chromium trioxide was suspended in 10 ml.
of dry methylene chloride, to which was then adaed 240 mg of 3,5-dimethylpyrazol. The mixture was agitated at room temperature for 2 hours, followed by addition of a solution of the compound (VII) from Stage (C) in 2 ml. of dry methylene chloride. The resultant mixture was allowed to stand at ambient temperature for 2 hours to effect the oxidation. Thereafter, 100 ml. of ethyl ether was added to - the reaction solution and the precipitate formed was filtered off and washed with ethyl ether. The filtrate and the washings were combined together and evaporated under reduced pressure to remove the solvent. The residue was taken up in benzene and the solution was passed through a column of 50 ml. of silica gel (the same one as described in ~33396 Stage (A) which was charged while being suspended in benzene. The column was washed with 150 ml. of benzene and then eluted with a mixed solvent of benzene-ethyl acetate (50 : 1 by volume). The eluate was collected in fractions of each 15 g and fraction Nos. 13 - 22 containing the desired compound was combined together and evaporated under reduced pressure to remove the solvent. There was thus obtained 164 mg of the desired compound (VIII) as colorless viscous liquid.
I.R. spectrum:

max (cm ) : 2975, 2950, 2850, 2760, 1695, 1615, 1580, 1505, 1438, 1400, 1383, 1365, 1320, 1290, 1258, 1210, 1148, 1112, 1060, 1000, 985, 952, 880, 848, 830, 812, 775, 735, 705, 670 (E) Preparation of the aminonitrile compound of the formula:

C~ CH 3 NC-CH ~ ~ < ---C 2 (IX) 216 mg of sodium cyanide was dissolved in 5 mln of concen~rated aqueous ammonia (25 - 28%), to which were then added with ice-cooling under stirring 236 mg of ammonium chloride and a methanolic solution of 206 mg of the compound (VIII) obtained in Stage (D). The mixture was allowed to stand at room temperature for 4.5 hours to complete the reaction. Subsequently, the reaction solution was evaporated under reduced pressure to remove the ammonia and methanol, followed by dilution with 30 ml. of water and extraction with butanol (3 x 10 ml.). The extracts were combined together and evaporated to dryness under reduced pressure. The residue was taken up in methanol and silica gel was added to the solution and dried in vacuo (the same one as described in Stage (A)) and then suspended in benzene. The suspension was superposed on a column of 50 ml. of silica gel (the same on as above~ which was charged while being suspended in benzene. The column was washed with benzene and then eluted with a mixed solvent of benzene-ethyl acetate (4 : 1 by volume). The eluate was collected in fractions of each 15 g and fraction Nos.
14 - 35 containing the desired compound were combined to~ether and evaporated under reduced pressure to remove the solvent. There was thus obtained 198 mg of the titled compound (IX).
I.R. spectrum:
vKBar : 3400, 3320, 2980, 2950, 2870, 2220, 1623, 1585, 1505, 1435, 1388, 1380, 1363, 1315, 1288, 1258, 1200, 1142, 1120, 1055, 980, 950, 900, 870, 850, 820, 760, 735, (F) Preparation of the compound (forphenicinol) of the formula:

/o HOOC--CH~--CH 2 OH ( I ) ~133396 198 mg o~ the aminonitrile compound (IX) obtained in Stage (E) was dissolved in 5 ml. of ethanol, and 3 ml. of water and 2 g of barium hydroxide were added to the solution. The resultant mixture was refluxed for 5 hours to effect the hydrolysis. The reaction solution was then cooled to room temperature, diluted with water and adjusted to pH 2.0 with lN sulfuric acid, followed by reflux for further 30 minutes (for removal of the hydroxyl-protecting, isopropylidene group). A~ter completion of the reaction, the reaction mixture was subjected to centrifugation (3000 rpm.) for 10 rninutes to remove the barium sulfate produced and the supernatant was concentrated by evaporation under reduced pressure. The concentrate was passed through a column of 10 ml. of SP-Sephadex~C-25 (H form, made by Pharmacia Fine Chemicals Co., Sweden) to adsorb thereon the desired compound. The column was then eluted with water and the eluate was collected in fractions. Those fractions which contained the object compound were combined together and evaporated to dryness under reduced pressure to yield 89 mg of a pale yellow powder. The powder was crystallized from water to give 62 mg of forphenicinol, namely the compound (I) as colorless crystals. This compound has a melting point of 200C but gradually decom~oses above 200C
to discolor into brown.
I.R. spectrum:
vKmBax : 3450~ 3030, 2950, 2920, 2875, 2710, 2610, 2320, 2080, 1640, 1620, 1592, 1530, 1495, 1440, 1410, 1380, 1350, 1320, 1303, 1258, 1200, 1165, 1135, 1120, 1038, 980, 970, 940, 910, 872, 830, 780, 760, 738, 675 . . .
N.M.R. spectrum:

~.~333~

ext. TM5 ~ppm : 5.12 (2H, s), 5.18 (lH, s), 7.35 - 7.55 (2H), 7.82 (lH, d) I

.~ :

;

. .

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A pharmaceutical composition for use as immunopotentiator which comprises as the active ingredient an effective amount of forphenicine having the formula or a pharmaceutically acceptable salt thereof or a hydrate thereof in association with a pharmaceutically acceptable carrier for the active ingredient.