Note: Descriptions are shown in the official language in which they were submitted.
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
NH2
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
1133396
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.
- 4 -
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 1260
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,
-- 10 --
~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
-- 11 --
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.
- 12 -
~ -.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 :¢
-- 13 --
, .`,~
,
1133396
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
<~
NH2
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
- 14 -
1133396
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
- 15 -
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:
.
--16 -
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):
- 17 -
~1 .
113;~396
OH
~OOC-Ch~ O~H ~III)
NH2
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
- 18 -
1133396
o~
HOOC-CH - ~ - CH20H (I)
1H2
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
- 19 -
: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.
- 20 -
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
- 21 -
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:
3 3
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
- 22 -
~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
1133396
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,
700
(F) Preparation of the compound (forphenicinol)
of the formula:
/o
HOOC--CH~--CH 2 OH ( I )
NH2
- 24 -
~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:
- 25 -
~.~333~
ext. TM5
~ppm : 5.12 (2H, s), 5.18 (lH, s), 7.35 - 7.55
(2H), 7.82 (lH, d)
I
.~ :
;
. .
-- 26 --
