Note: Descriptions are shown in the official language in which they were submitted.
33~
, . ~ ~
The invention relates to novel amides of L-aspartyl-
D-serine and L-aspartyl-D-O-methylserine which are especially
useful in view of their potent sweetening properties, novel
methods for their use in foods and edible composition containing
them.
In United States 3,492,131 certain alkyl esters of
L-aspartyl-L-phenylalanine were found to be up to 200 times as
sweet as sucrose and to be substantially free of bitter flavor
notes which detracted from earlier artificial sweeteners such
as saccharin. These compounds were subsequently found to have
only limited stability in a~ueous systems due to diketopiperazine
formation especially at the neutral-acidic p~ conditions
prevalent in most food systems.
Mazur et al., J. Med. Chem., 16, 1284 (1973) has disclosed
that lower alkyl esters of L-aspartyl-D-alamine and certain
homologs thereof, especially L-aspartyl-D-alanine isopropyl ester,
have sweetness potencies of up to 125 times sucrose.
Sukehiro et al., Seikatsu Ka~aku, 11, 9-16 (1977); Chem.
Abstr., 87, 168407h (1977) has disclosed certain amides of L-
aspartyl-D-alanine of the formula
,~3 - 1-
2 n
~1~ /NH~/ C\NHRl
COOH CH3
where Rl is methyl, ethyl, n-propyl, isopropyl, n-
butyl, isobutyl, secondary butyl, cyclohexyl or the
carbon residue of the methyl esters of glycine/ d
alanine or I-alanine. The most potent compounds were
those wherein R1 is one of the above butyl groups or
cyclohexyl, having respectively, 100-125 and 100 times
the sweetness of sucrose. Since the n-butylamide was
found to have 125 times the sweetness of sucrose and
the isobutyl and secondary butyl amides are 100 x
sucrose, it was concluded that the potency of these
amides is affected mainly by the number of carbon
atoms in the alkyl group, Rl, and that structural
isomerism in the alkyl group has little effect on the
sweetness potency.
Esters of L-aspartyl-D-serine and L-aspartyl-D-
threonine have been found by Ariyoshi et al., Bull.
C_ . Soc. Japan, 47, 326 (1974) to be sweeter than
the corresponding esters of L-aspartyl-D-alanine and
L-aspartyl-D-2-aminobutyric acid, respectively. The
most potent of these esters, L-aspartyl~D-serine n-
. propyl ester, was 320 times as sweet as a 5~ sucrosestandard.
United States 3,971,822 discloses esters o-f L~aspartyl-D-
alaninol with carboxylic acids, including 2-methyl-butyric,
cyclopropanecarboxylic, cyclobutanecarboxylic and 2-methylcyclo-
butanecarboxylic acids. The esters with cyclo-propane- and
cyclobutanecarboxylic acids were 200 x and 220 x sucrose,
respectively~ The ester with 2-methylcyclobutanecarboxylic acid
was only 160 x sucrose. Corresponding L-aspartyl-D-serinol esters
are also disclosed, the sweetest of which, the ester with propionic
acid, is 160 x sucrose.
United States 3,959,245 and United States 3,907,766 dis-
close, respectively, L-aspartylaminomalonic acid methyl 2-
methylcyclohexyl diester, and the corresponding alkyl fenchyl
diester. The former is reported to be 6600 x sucrose, the latter
4200-33,000 x sucrose. In a related publication by the inventors,
Chem. Pharm. Bull., 24, 2112 (1976), a series of L-aspartylamino-
malonic acid diesters is disclosed. one of the ester groups being
methyl or ethyl and the other being one of a variety of branched
alkyl and cycloalkyl groups.
In our related United States Patent No. 4,411,925 issued
October 25, 1983, it was found -that it is not merely the size of
the amide substituent that is critical for a high degree of
sweetness in L-aspartyl-D-alanine amides, but, to the contrary, it
is the precise spatial arrangement of the amide substituent, R,
that is critical. Certain L-aspartyl-D-alanine amides which are
branched at the alpha carbon atom (the carbon atom bearing the
amide nitrogen atom) and also branched again at one or bo-th of beta
and beta' carbon atoms were -found to have significant advantages.
- 3 -
a~l~
~ ~ v ~ ~
The present invention provides certain novel
branched amides of L-aspartyl-D-serine and L-aspartyl-
D-O-methylserine dipeptides which have unexpectedly
, high sweetness potency and are free from undesirable
flavor qualities at conventional use levels. They
have also been found to have surprisingly high stability
both in solid form and in aqueous systems over the pH
range found in most food systems even at the elevated
temperatures used in baking and conventional food
processing.
The novel compounds of the invention are the L-
aspartyl-D-amino acid dipeptide amides of the formula
NH 2 C
~ C~NH ~ ~ HR
COOH Ra
and the physiologically acceptable cationic and acid
addition salts thereof, wherein Ra is CH2OH or
CH2OCH3, and R is a branched member selected from the
group consisting of fenchyl, diisopropylcarbinyl, d-
methyl-t-butylcarbinyl, d-ethyl-t-butylcarbinyl, di-t-
butylcarbinyl, 2-methylthio-2,4-dimethylpentan-3-yl,
R3 ~ R3 R
, ~( CH2 ) m R6~ C~2 ) n~X
where at least one of R3, R4, R5, R6 is alkyl having
from one to four carbon atoms and the remainder are
hydrogen or alkyl having from one to four carbon
atoms; X is O, S, SO, SO2, C=O or CHOH; m is zero, l,
3~3 ~
2, 3 or 4; n and p are each zero; 1, 2 or 3 and the
sum of n ~ p is not greater than 3; the sum of the
carbon atoms in R3, R , R5 and R6 is not greater than
six and when both of R3 and R4 or R5 and R6 are alkyl
they are methyl or ethyl;
R7 R8
R9
(CH2)m
where m is as defined above, one of R7, R8, R9 is
alkyl having from one to four carbon atoms and the
remainder are hydrogen or alkyl having from one to
four carbon atoms and the sum of the carbon atoms in
R7, R~ and R9 is not greater than six;
n
~1_(CH2)
( CH2 ) q
where m and q are the same or different and each have
the values previously defined for m;
R R
~ (C72)t
O~Z
where each of R12 and R13 are methyl or ethyl, or
R12 is hydrogen and R13 is alkyl having from one to
four carbon atoms, Z is O or NH and t is 1 or 2;
3~
_<~CH2)2
R16~
where w i5 Or 1, 2, 3 or 4, R14 and R16 are each
alkyl having from one to four carbon atoms, R15 i~
hydrogen, OH or alkyl having from one to two carbon
atoms, where the sum of the carbon atcms in R14, R15
and R16 is not greater than six and when both of R14
and R15 are alkyl they are methyl or ethyl; and
Rl~
R2 0
whe:re R17 and Rl9 are alkyl having from one to four
carbon atoms, R18 and R20 are hydrogen or alkyl
ha~ing from one to two carbon atoms, taken separately,
A is OH and s is hydrogen, OH or methyl and when
taken together A and B are -CH20C-, -CH2NHC-, -OCCH2~,
O O O
-NHCCH2-, OC-, -NHC- or -OCO-, where the sum of the
~ 1l n n
O O O O
carbon atoms in R17, R18, Rl9 and R20 is not greater
than six and when both of R17 and R18 or Rl9 and R20
are alkyl they are methyl or ethyl.
33~
While the preferred sweeteners of the invention
are those dipeptide amides of formula ~I) wherein the
aspartylamino acid dipeptide moiety is derived from
L-aspartic acid and a D-amino acid, RaCH(NH2)COOH,
also included within the scope of the invention are
mixtures containing the most preferred L-aspartyl-D-
arnino acid amides of formula (I) wherein one or both
of the aspartyl or the other amino acid (i.e., serine
or O-methylserine) moieties is racemic such as e.g.,
DL-aspartyl-D-serine amides,
DL-aspartyl-DL-serine amides,
L-aspartyl-DL-serine amides,
L-aspartyl-DL-O-methylserine amides,
DL-aspartyl-DL-O-methylserine, and
DL-aspartyl-D-O-methylserine amides.
Those compounds of formula (I) wherein the
aspartyl moiety is entirely of the D configuration or
the other amino acid moiety is entirely of the L-
configuration have little or no sweetness.
An especially preferred group of L-aspartyl-D-
amino acid amides of formula (I) are those wherein R
is an acyclic member selected from the group consisting
of diisopropylcarbinyl, d-methyl-t-butylcarbinyl and
di-t-butylcarbinyl.
Another especially preferred group of L-aspartyl-
D-amino acid amides of formula (I) are those wherein
R is a member selected from the group consisting of
R3 ~ ~3 R4
(CH2)m 1 ~~(CH2)n~X
R6 ~ R6 ~ 2)p
33~3
--8--
R7 R8 ~ CH2~m
~ R ~ ,
`n (CH2)q
(CH2)m
R12 R13 R14 ~15
~ C72)t ' ~ (CE~2)W
o// Rl OH
and ~1 ~
B
R2~
wherein R3-R9, R12 R20, A, B, X, Z, m, n, p, q, t and
w are as defined above; and more particularly preferred
are those compounds of formula (I) wherein R has one
of the first four values of the group immediately
above.
Particularly preferred amides of formula (I) are
the L-aspartyl-D-serine amides, i.e., those wherein
Ra is CH20H.
Examples of the more valuable L-aspartyl-D-amino
acid dipeptide amides of the inventio.n include those
of for~ula (I) wherein R is-
- 9 -
(~)fenchyl,
diisopropylcarbinyl,
d-methyl-t-butylcarbinyl,
di-t-butylcarbinyl,
2,6-diethylcyclohexyl,
2-methylcyclopentyl,
2-ethyl-6-methylcyclohexyl,
2-ethylcyclohexyl,
2-methylcyclohexyl,
2,2-dimethylcyclohexyl,
2-ethylcyclopentyl,
2-methyl-6-isopropylcyclohexyl
2,2,6,6-tetramethylcyclohexyl,
2,2,4,4-tetramethyltetrahydrofuran-3-yl,
lS 2,2,6-trimethylcyclohexyl,
2-isopropylcyclohexyl,
2,5-dimethylcyclopentyl,
2,6-dimethylcyclohexyl f
2 isopropylcyclopentyl,
2,2,5,5-tetramethylcyclopentyl,
t-butylcyclopropylcarbinyl,
2,2,4,4-tetramethylthietan-3-yl,
2,2,4,4-tetramethyl~ dioxothietan-3-yl,
2,2,4,4-tetramethyltetrahydrothiophene-3-yl,
3,5-dimethyltetrahydrothiapyran-4-yl,
2-t-butylcyclohexyl or
dicyclopropylcarbinyl;
Especially valuable sweeteners include the above
compounds wherein R is:
di-t-butylcarbinyl,
2,2,6-trimethylcyclohexyl,
2-t-butylcyclohexyl,
2-isopropylcyclohexyl,
3~
--10--
2,6-dimethylcyclohexyl,
2,5-dimethylcyclopentyl,
2 isopropylcyclopentyl,
2,2,5,5-tetramethylcyclopentyl,
2,2,4,4-tetramethyltetrahydrothiophene-3-yl,
t-butylcyclopropylcarbinyl,
dicyclopropylcarbinyl,
2,2,4,4-tetramPthylthietane-3-yl or
2,2,4,4-tetramethyl-1,1-dioxothietan-3-yl.
More especially preferred are those compounds of
formula (I) wherein R is.
2,2,5,5-tetramethylcyclopentyl,
2,2,4,4-tetramethyltetrahydrothiophene-3-yl,
t-butylcyclopropylcarbinyl,
dicyclopropylcarbinyl,
2,2,4,4-tetramethylthietan-3-yl, and
2,2,4,4-tetramethyl-1,1-dioxothietan-3-yl.
Most particularly preferred compounds of formula (I)
are those wherein:
R is C~2OH and R is
dicyclopropylcarbinyl,
2,2,4,4-tetramethylthietan-3-yl,
2,2,4,4~tetramethyl-1,1-dioxothietan-3-yl, and
those wherein Ra is CH2OCH3 and R is
2,2,4,4-tetramethylthietan-3-yl and
2,2,4,4~tetramethyl-l,lrdioxothietan-3-yl, which
have sweetness potencies of from 200-1200 times that
of sucrose.
The invention further provides compositions for
sweetening edible materials which comprises a sweeten-
ing amount of a compound of formula (I) and a non-
toxic carrier. Most particularly preferred compositions
are those containing L-aspartyl~D-serine N-(dicyclo-
propylcar~inyl)amide, L-aspartyl-D-serine N-(2,~,4,4-
tetramethylthietane-3-yl)amide, the l,l-dioxo deriva~
tive of the latter.
Additionally, sweetened edible compositions
comprising an edible material and a sweetening amount
of a compound of the invention, are provided.
Also provided is a method for sweetening edible
compositions which comprises adding thereto a sweeten-
ing amount of a compound of the invention.
The invention further provides compositions for
sweetening edible materials which comprises a sweet-
ening amount of a mixture of a compound of formula (I)
and saccharin or a physiologically acceptable salt
thereof.
Especially preferred such mixtures are those
wherein in said compound of formula (I), Ra is CH2OH
and R is dicyclopropylcarbinyl, 2,~4,4-tetramethyl-
thietan-3-yl or 2,2,4,4-tetramethyl-1,1-dioxothietan-
3-yl. Most particularly preferred are mixtures of L-
aspartyl-D-serine N-(2,2,~,~-tetramethylthietan-3-
yl)amide and said saccharin, especially those wherein
said compound and said saccharin are present in a
weight ratio of from 1:1 to 1:8.
By physiologically acceptable salts of saccharin
is meant the salts of saccharin with physiologically
acceptable cations such as e.g., the sodium, potassium,
calcium or ammonium salts.
By physiologically acceptable cationic salts of
the compounds of the invention is meant the salts
formed by neutralization of the free carboxylic acid
group of the compounds of formula (I) by bases of
12-
physiologically acceptable metals, ammonia and
amines. Examples of such metals are sodium, potassium,
calcium and magnesium. Examples of such amines are
N-methylgluc~mine and ethanolamine.
By the term physiologically acceptable acid
addit:ion salts is meant those salts formed between
the fxee amino group of the compound of formula (I)
and a physiologically acceptable acid. Examples of
~uch acids are acetic, benzoic, hydrobromic, hydro-
chloric, citric, fumaric, gluconic, lactic, maleic,
malic, nitricr phosphoricr saccharic, succinic and
tartaric acids.
The invention still further provides valuable
novel intermediates, useful in preparation of the
invention compounds of formula (I). Said inter-
mediates are the D-amino acid amides of the formula
RaCHCONHR
NH~
where Ra is as previously defined and Rc is a member
selected from the group consisting of fenchyl, diiso-
propylcarbinyl, d-methyl-t-butylcarbinyl, d-ethyl-t
butylcarbinyl, di-t-butylcarbinyl, cyclopropyl-t-
butylcarbinyl, cyclopentyl-t butylcarbinyl, dicyclo-
propylcarbinyl,
R3 ~ 40
C 2)m
R R50
where ml is 1, 2 or 3 and
when ml is l: R30, R40, R50 and R60 are each methyl,
when ml is 2: R30 is methyl, ethyl or isopropyl and
R40, R50 and R60 are each hydrogen or R30 and
R50 are each methyl and R40 and R60 are each hydrogen,
and when m is 3:
(a) R is :isopropyl or t-butyl, R , R and
R60 are each hydrogen,
(b) R is ethyl, R is methyl, R40 and R
are each hydrogen, or
(c) R30 and R40 are each methyl and R50 and
R60 are each hydroyen or methyl, and
CH __~R
~ (CH2)n
~ C 2 ) P2
CH3 R
where when n2 and P2 are each zero: R41 and R61 are
each mPthyl and X2 is S, SO2, C=O or CHOH,
when n2 is zero and P2 is 1: R41 and R61 are each
methyl and X2 is O, S, or SO , and
when n2 is 1 and p~ is 1: R~l and R51 are each
hydrogen and X2 is S or SO2.
The suffix n car~inyl" as used herein denotes the
moiety -CH-. Thus, for example, diisopropylcarbinyl
is the group (i-C3H7)2-CH- and dicyclopropylcarbinyl-
amine is ( ~ )2CHNH2.
The instant dipeptide amides are convenientlymanufactured by methods suitable for coupling of
amino acids. A preferred method for preparing the
dipeptide amides of formula (I) is outlined below.
3~
-14-
NHQ Ra (l) condense
L- ~ + D-~IH2CHCOORll (2j H2O
COOR COOH
or carboxyl activated
derivative
NHQ Ra N~Q Ra
COO ~ ~ RNH2 ~ OHN ~ ONHR
(II) / (III)
/deprotect
(I)
In the above L-aspartic acid derivatives Q is one of
the well known amino-protecting groups which can be
selec~ively removed such as those described by
Boissonnas, Advances in _r~anic Chem., 3, 159-190
-
(1963). Particularly preferred amino-protecting
groups are benzyloxycarbonyl and tert-butyloxycarbonyl.
R10 is preferably an alkyl group having from one to
four carbon atoms or benzyl. The D-serine or D-O-
methylserine employed may be in the form of the free
amino acid wherein Rll is hydrogen, but is preferably
a carboxyl-protected derivative wherein Rll may be
the residue of an ester group such as methyl or
ethyl, but is preferably a silyl group such as
trialkylsilyl, having from three to twelve carbon
atoms. An especially preferred such group is trimethyl-
silyl for reasons of economy and efficiency.
In the first step of the above reaction sequence
the diprotected ~-aspartic acid is condensed with the
appropriate D-amino acid or a carboxy-protected
3~
derivative to provide the diprotected dipeptide of
formula ~II). While this step may be carried out with
the diprotected aspartic acid in the presence of
condensing agents such as, for example, dicyclohexyl-
carbodiimide, it is preferred to employ an alpha-
carboxyl activated derivative of the diprotected
aspartic acid. Preferred such carboxyl activated
derivatives are the chloride, bromide, anhydride or
mixed anhydride. Especially preferred for reasons of
efficiency are the mixed anhydrides of the above
diprotected-L-aspartic acids with esters of chloro-
carbonic acid, particularly the alkyl esters wherein
said alkyl has from one to four carbon atoms. Most
preEerred mixed anhydrides are those prepared from
the methyl and ethyl esters of chlorocarbonic acid
for reasons of economy.
In one preferred method for preparing the
compounds of formula (I), beta benzyl-N-benzyloxy-
carbonyl-L-aspartic acid is reacted with ethyl
chlorocarbonate to form the corresponding mixed
anhydride by methods known in the art. In a separate
vessel the D-amino acid, RaCH(NH2)COOH, which is
obtained from commercial sources or by resolution of
the racemic amino acid by known methods [see e.g.
Yamada ~,al., J. Or~. Chem., 38, 4408 (1973)], is
converted to the trimethylsilyl ester by contacting
the amino acid with an equimolar amount of trimethyl-
silyl chloride in the presence of a reaction inert
organic solvent; for the case when R is CH2OH, two
molar equivalents of silylating agent is ordinarily
employed. Suitable solvents for this purpose are,
for example, pyridine, dimethylformamide or dimethyl-
acetamide; especially preferred i5 dimethylformamide.
~3~Lr~
In a typical reaction according to this method,
the D-amino acid e.g., D-O-methylserine, dissolved in
dlmethylformamide and an equimolar amount of tri-
methylchlorosilane is added at room temperature. In
a separate flask beta-benzyl N-benzyloxycarbonyl-L-
aspartic acid and a molar excess of an acid binding
agent, preferably triethylamine are dissolved a
mixture of dimethylformamide and tetrahydrofuran and
an equimolar amount of ethylchlorocarbonate is added
at room temperature or below, preferably at about -25
to 25C. and especially at about -10 to 0C. to form
the mixed anhydride. To this is added the solution
of e.g./ D-O-methylserine trimethylsilyl ester,
preferably at a temperature within the sarne range.
Reaction is ordinarly complete within one to two
hours arter which the reaction mixture is poured into
water or agueous acid, for example hydrochloric acid,
and the product of formula (II) extracted with a
water immiscible solvent, typically chloroform,
methylene chloride or ethyl ether and isolated by
standard methods. The diblocked dipeptide (II) is
ordinarily of suf~icient purity for use in the next
step, but may be further purified if desired, for
example by columrl chromatography.
In the second step of this method the diblocked
dipeptide (II) is reacted with an equimolar arnount of
primary amine of formula RNH2 to provide the corres~
ponding di~locked dipeptide amide intermediate of
~ormula (III) wherein Ra, R, R10 and Q are as previously
defined. As in the first step, the carboxylic acid
forrn of the reactant (II) can be successfully employed
by use of condensing agents, for example dicyclohexyl- -
carbodiimide to provide the intermediates of formula (III).
However, it is preferred to convert the compound of
formula (II) to a carboxyl activated derivative, for
example the chloride, bromide or mixed anhydride, the
latter being preferred. Thus, employing the particular-
ly preferred compound of formula (II) wherein R10 isbenzyl and Q is benzyloxycarbonyl, the mixed anhydride
is prepared. As above, the preferred anhydrides are
those obtained from esters of chlorocarbonic acid and
the methyl or ethyl esters thereof are particularly
preferred. The mixed anhydrides of compound (II) are
prepared employing reactants and conditions described
above for the first step of this sequence. In a
typical reaction the compound of formula (II) and
triethylamine in approximately equimolar amounts are
combined in a reaction inert organic solvent, for
example tetrahydrofuran, the mixture cooled to about
-10C. and ethylchlorocarbonate added to obtain the
mixed anhydride. To this is then added an equimolar
amount of the amine of formula R~H2 or a solution
~0 thereof, for example in the same reaction inert
solvent and at a temperature in the range of from
about -50 to 25C. and preferably at from -35 to
-5~. After the addition of the amine is complete,
the reaction mixture is allowed to warm to about ~oom
temperature and maintained at this temperature until
reaction is substantially complete, ordinarily from
about 1 to ~0 hours. The desired intermediate of
formula ~II) is then isolated and purified, if desired,
by the same methods described above for compound (II).
In the final step of this method the carboxyl
protecting group, R10 and amino protecting group, Q,
are removed to provide the desired s~eeteners of
formula (I)G
-18-
The method selected for removal of protecting
groups from the dipeptide amide of formula (III) will
vary depending on a number of factors which will be
apparent to those of skill in the art. Two important
factors for such selection are the nature of the
protecting groups ~10 and Q, and the nature of the
amide substituent, R. For example, when R10 and Q
are, respectively, the especially preferred groups
benzyl and ~enzyloxycarbonyl and R does not contain
sulfur, a preferred method for removing said protect-
ing groups is, ordinarily, by hydrogenolysis.
However, when R10 is benzyl or alkyl as defined above
and Q is tert-butyloxycarbonyl and R has any of the
~alues above, it is ordinarly preferred to remove the
protecting groups by hydrolysis. A combination of
hydrolysis and hydrogenolysis is preferred in those
cases wherein R10 is alkyl, Q is benzylo~ycarbonyl
and R does not contain sulfur.
When hydrogenolysis is selected for removal of
protecting groups from the intermediate of formula (III)
it is preferred to carry out the reaction in the
presence of a catalytic amount of a noble metal
catalyst, palladium being especially preferred, and
in the presence of a reaction inert solvent. Examples
of such solvents include the lower alkanols, such as
methanol, ethanol, isopropanol and n-butanol; ethers,
such as tetrahydrofuran, ethyl ether, 1,2-dimethoxy-
ethane and diethyleneglycol dimethylether; esters
such as ethyl acetate, methyl propionate and dimethyl-
succinate; and dimethylformamide. Particularlypreferred such solvents are methanol and ethanol for
reasons of economy and efficiency. While the hydro-
genolysis may be carried out successfully at higher
pressures and temperatures, use of pressures of from
about 1-10 atmospheres and room temperature are
--19--
preferred for reasons of economy and convenience. ~t
the preferred temperature and pressure the reaction
is ordlnarily complete in from about 30 minutes to
about six hours, after which the catalyst is removed,
5 typically by filtration, the solvent evaporated and
the resulting product purified, if desired, by standard
methods, ~or example by recrystallization or column
chromatography.
When hydrolysis is selected for removal of one
or both of protecting groups R10 and Q any of the
well ~nown methods for alkaline hydrolysis or acid
hydrolysis of esters and the like may be employed
with some success. However, when blocking groups R10
are to be removed by hydrolysis, alkaline hydrolysis
is preferred, and especially preferred conditions are
use of at least an equivalent amount of a strong
base, for example, sodium hydroxide or potassium
hydroxide in the presence of water and a lower
alkanol, particularly methanol or ethanol, at or
about room tempera~ure. Under these preferred
conditions hydrolytic removal of the R10 group is
ordinarily complete in a few hours or less.
When the amino protecting group Q is tert-
butyloxycarbonyl it is preferred to use acid hydro-
lysis for its removal. Especially preferred is
dilute aqueous hydrochloric acid in the presence of
methanol or ethanol and heating the mixture at reflux.
Under these conditions hydrolysis is ordinarily
complete in a few hours or less.
Isolation of the products of formula (I) after
removal of protecting groups by an~ of the above
hydrolysis methods employs standard procedures known
in the art. For example, after acid hydrolysis the
reaction mixture is evaporated to remove solvent, the
3~
-20-
aqueous residue washed with a water immiscible non-
polar solvent, for example, ethyl ether or chloroform
after which the aqueous layer is made alkaline and
the product extracted with a water-immiscible solvent
such as, for example, ethyl acetate and the product
obtained by evaporation of solvent. If desired, the
product can be further purified, for example, by
recrystallization or column chromatography. ~en
alkaline hydrolysis to remove a protecting group
RlO is followed by hydrogenolysis to remove the amino
protecting group Q, the reaction mixture from the
alkaline hydrolysis is preferably neutralized by
addition of acid, for example, hydrochloric acid, and
the neutralized reaction mix~ure subjected to hydro-
genolysis as described above.
A second preferred method for manufacture of the
instant compounds of formula (I) is shown below.
Ra Ra
D-QNHCHCOOH + RNH2 QNHCHCONHR
or carboxyl activated ~ (IV)
derivative
NHQ
deprotect Ra COO ~ COOH
- - ~ NH2CHCONHR ~ (III)----~(I)
(V)
Ra, R, R and Q are as defined above.
The amino protected D-amino acid or its carboxyl
activated derivative is reacted with an equimolar
amount of amine RNH2 employing methods and conditions
dPscribed above for the preparation of intermediates
(II) and (III) to obtain an amino protected D-amino
acid amide of formula (IV). The protecting group Q
3~3~
-21-
is removed by hydrogenolysis or hydrolysis as described
above and the resulting free amino amide (V) is
condensed with a diblocked L-aspartic acid derlvative
or a carhoxyl activated derivative thereof, as
described above for the preparation of intermediates
of formula (II), to provide the diblocked dipeptide
amide of formula (III) from which the desired sweetener
of formula (I) is obtained as previously described.
In a modification of this method an intermediate
of fo~nula (IV) wherein R contains a cyclic or
acyclic sulfide moiety ~-S-) may be oxidized to the
corresponding sulfoxide or sulfone prior to its
conversion to intermediate (V) and subsequent reactions
as described above, to provide compounds of formula (I)
wherein R is a sulfoxide or sulfone.
In a third preferred method for preparing the
compounds of the invention the D-amino acid amide of
formula (V), described above, is reacted with L-
aspartic acid N-thiocarboxyanhydride to provide
directly the compounds of formula (I). In carrying
out this method the intermediate (V) in a suitable
solvent is contacted with an equimolar amount of L-
aspartic acid N-thiocarboxyanhydride at a mildly
alkaline pH at a temperature of from about -25 to
10C. to provide the compound of formula (I). The
alkaline p~ for this reaction is provided by means of
a strong base, for example, sodium hydroxide or
potassium carbonate. Suitable solvents for this
reaction are those that dissolve at least a portion
of the reactants under the reaction conditions
employed without reacting with either xeactant to an
appreciable extent and allow the products formed in
the reaction to be isolated with relative ease.
Examples of such solvents for this reaction are
-2~
water, tetrahydrofuran, 1,2-dimethoxyethane, diethyl-
eneglycol dimethylether, dimethylsulfoxide, dimethyl-
formamide and combinations thereof; preferred solvents
are water, and its mixtures with te-trahydrofuran. A
preferred alkaline pH range for this reaction is from
about 8 to 10 and a pH of about 9 is especially
preferred. An especially prefexred temperature is in
the range of about -10 to 0C.
Vnder the preferred conditions mentioned above
the reaction is ordinarily complete in one to two
hours. The product of formula (I) then isolated by
standard methods, for example, the pH of the reaction
mixture is a~justed to the isoelectric pH of the
product, ordinarily about pH 5.0-5.6, to precipitate
the product of formula (I), the bulk of the solvent
removed by evaporation or filtration and the crude
material slurried with an organic solvent, for
example, methanol, ethanol, ethyl ether, ethyl
acetate or mixtures thereof. ~he product of formula (I)
is then isolated, by filtration for example. It may
be further purified, if desired, by, e.g., recrystal-
lization or column chromatography.
The sweetness potency of the instant compounds
was determined by comparison of their gustatory
sweetnesses with sucrose. Aqueous solutions o~ the
compound of formula (I~ diluted to a suitable range
of concentrations were compared with a sucrose standard
by an expert taste p2nel. Comparisons were generally
made with aqueous sucrose solutions of 7-9%, i.e.,
7-9 g. per 100 ml. Higher sucrose concentrations
have a distinctive mouthfeel which may influence
results and lower sucrose concentration are no~ -
indicative of normal use situations. If, for example
a 0.014% solution of the compound of formula (I) is
judged to be equally as sweet as a 7% sucrose solution,
then the sweetness potency of that compound is
7/0.014 = 500 x sucrose. All of the sweetness
potency values stated herein for the compounds of the
invention were determined by this method. At threshold
concentrations (i.e., the lowest concentration at
whlch sweetness is first noticed, whlch for sucrose
is ordinarily at concentrations in the range of
2-3%), the potency of a sweetener, such as the
compounds of the invention, is generally twice that
observed by comparison of its gustatory sweetness
with 7-9% solutions of sucrose.
The requisite amines of formula RNH2 wherein R
is as previously defined are either commercially
available or can be obtained from readily available
precursors. For example, the 2-alkylcyclohexylamines
and 2,6-dialkylcyclohexylamines can be obtained by
catalytic hydrogenation of the corresponding alkyl
substituted anilines. Many of the amines are obtained
by reductive amination of the corresponding ketone
using a variety of conditions known in the art. For
example, reductive amination by the well known
Leuckhart reaction employing formic acid and formamide
as reducing agents, see for example, the review in
Or~anic Reactions, Wilev and Sons, New York, Vol. 5,
p. 301, 1949, may be employed. Alternatively, the
appropriate ketone can be reductively aminated
employing sodium cyanoborohydride and ammonium
acetate see for example, J. AmerO Chem. Soc., 93,
2897 (1971), or by means of ethanolic ammonia in the
presence of a hydrogenation catalyst such as Raney
nickel, platinum or palladium, see, for example,
Or ~nic Reactions, 4, 174 (1948). Many of the amines
of formula RNH2 are obtained from the corresponding
ketones by formation of an intermediate oxime formed
by reacting the ketone with hydroxylamine or its
-24-
salts under conditions well known in the art. The
oxime intermediate is then reduced by catalytic
hydrogenation or by means of sodium in the presence
of a lower alkanol at elevated temperatureO
particularly preferred method, especially useful for
reducing oximes of sulfur-containing ketones, employs
reduction of the oxime in ethanol and a molar excess
of sodium at the reflux temperature of the mixture.
The reguisite ketone precursors of the amines
RNH2 are ei~her commercially available, known in the
axt or prepared by known methods. For example, the
ketones of formula (VI) and (VII)
~ CH2)m ~ 2)p /
(VI) tVII)
where R , R , R5, R , X, m, n and p are as defined
above, except those of formula (VII) wherein X is
C=O, may be obtained by alkylation of the correspond-
ing compounds wherein R , R4, R5 and R6 are each
hydrogen to provide compounds of the above formula
wherein from one to all of R3, R4, R5, ~6 are alkyl
as defined above. The alkylation is carried out, for
~ example, employing alkylating agents such as the
appropriate alkyl hal:Lde or alkyl sulfate under
neutral or alkaline conditions provided by strong
bases, for example, sodium hydride or sodium amide.
~sing the same method compounds of the formula (VI)
and (VII) wherein only 1, 2 or 3 of the substituents
alpha to the keto group are alkyl can be converted to
compounds of the same formula wherein from two to
3~
rour of R3, R4, R5, R6 are alkyl. Gem-dialkyl
compounds of formula (VI) and (VII) wherein either R3
and R4 or R5 and R6 are said alkyl can be obtained
from the appropriate monoalkyl compound hy blocking
the unsubstituted alpha-position prior to alkylation
and subsequent removal of the blocking group. For
example, 2,2-dimethylcyclohexanone may be obtained by
condensation of 2-methylcyclohexanone with ethyl-
formate in the presence of sodium methoxide and the
resulting intermediate alkyiated as outlined below.
O O
- ~ ONa
I J .L HCO2C~H5 ~ C~ I
NaOCH3 \~
Na H2O, OH- ~
Ketones of formula (VI) or (VII) wherein one or
both of R3 and R5 are propyl or butyl may be obtained
by condensation of the corresponding alpha-unsubstitut-
ed compound with the appropriate aldehyde or ketone
under alkaline conditions to an intermediate alpha-
or alpha,alpha'-alkylidene ketone which can then be
hydrogenated to provide the-desired ketone.
The requisite cyclobutanones are obtained by
methods described by Conia et al., Bull. Soc. chim~
France, 726 (1963) and Conia, Ind. chim. Belge, 31,
981 (1966).
An alternative method for preparing the ketones
of formula (VI) and (VII) involves a cyclization of
an acyclic precursor. For example, by means of the
well known Dieckmann cyclization of dicarboxylate
YL i~ ~ ~
-26-
esters and subsequent hydrolysis and decarboxylation;
see e.~., Moaern Synthetic Reactions, ~. A. Benjamin,
~enlo Park, Cal., 1972, p. 740. The alpha-keto
esters produced, especially those with no other
alpha-substituent, can also be alkylated prior to
hydrolysis and decarboxylation, if desired. This
reaction can also be used to provide ketones (VI) and
tVII) which are unsubstituted at the carbons adjacent
to the carbonyl group which can be alkylated as
described above~
For preparation of diketones of formula (VII)
wherein X is C=O the keto group of acyclic keto-
dicarboxylate ester precursor is converted to a ketal
or thioketal, e.g., dimethyl ketal, diethylthio
ketal, ethylenedioxy ketal or ethylenedithio ketal,
prior to Dieckmann cyclization. Ester group hydrolysis
and decarhoxylation affords a keto-ketal which may be
converted to the corresponding amino ketal, by methods
described above, followed by hydrolysis of the ketal
group by methods well known in the art. The resulting
amino ketone can be hydrogenated, if desired, to the
corresponding hydroxyamine tX = C~OH) by known methods,
e.g. by reduction with sodium borohydride.
2,2,4,4-Tetraalkyl-3-hydroxycyclobutylamines are
prepared from the corresponding 1,3-diones by the
method of ~.S. 3,125,569.
The amines of formula
R ~
NH2--~--( C~2 ) n\
6,~t C~I2 ) /
R R
where X is CEIOH and R3-R6, n and p are as defined
above, or N~protected derivatives thereof e.g., N-
benzyloxycarbonyl derivatives, may be oxidized, e.g.
3~
-27-
by chromium trioxide, to the corresponding cornpounds
wherein X is C=O. Alternatively, the hydroxyamine
may be reacted first with e.g., a carbo~yl activated
derivative of an ~-protected D-O-methylserine and the
resulting intermediate of formula (IV) wherein R is
sald hydroxy-containing group, oxidized, e.g., with
chromium trioxide, to provide the corresponding
ketone. The resulting ketone of formula (IV) is then
converted to the desired product of formula (I) where
R is a ke-to-containing group as desired above.
Certain of the ketones of formula (VII) are also
obtained from acyclic precursors derived from ketones
of the formula (VIII) wherein R , R , R and R are
R6 ~ R3 ---(VIII)
R R
as previously define~. For example four-membered
ketones of formula (VII) where X is O or S are
obtained by bromination of (VIII) with two moles of
bromine and the resulting alpha,alpha'-dibromo
compound cyclized with, e.g., sodium hydroxide to
provide an oxetanone or hydrogen sulflde to provide a
thietanone. The corresponding five-membered ring
ketones (VII) are obtained when (VIII) is firs~
reacted with formaldehyde to provide an intermediate
alpha-hydroxymethyl compound which is then brominated
at the alpha'-position and cyclized with sodium
hydroxide or hydrogen sulfide to provide the corresponding
compounds of formula (VII) wherein X is O or S,
respectively.
~ertain of the tetrahydropyran-~ ones and tetra-
hydrothiapyran-4-ones of formula (VII) are obtained
by adding the elements of water or hydro~en sulfide
to the appropriately substituted divinylXetone.
-~8-
Ketone intermediates of Eormula (IX) which ~ay be
converted to amines via the oxime are obtained by
methods outlined below where R17, ~18, Rl9 and R are
as defined above~
Rl9 ~ 1 8 CH2O 1 ~ R18 PR1 ~ R17
COOC2H5 CO2C2 5 OH
(X) (XI) (IX)
The appropriately substituted acetoacetic ester (X)
is condensed with formaldehyde, e.g. under alkaline
conditions, and the resulting hydroxmethylated inter-
mediate (XI) is then cyclized, for example by heating
in the presence of a mild acid or base with removal
of ethanol as it forms.
Bromination of acetoacetic esters of formula (X)
and subsequent treatment of the product with, e.g.
sodium hydroxide, provides ketones of formula (XII)
which are converted to the corresponding amine as
described above.
X ~ R ~ R13 R20 ~ 17
C02C2H5
(XII)
Alternatively, the ketolactones (XII) can be
prepared by the method described in Zeit. Chemie, 13,
11 (1973~; Chem. Abstr., 78, 135596e (1973), by reaction
of the appropriate cyclobutan-1,3-dione with hydrogen
peroxide.
~29-
The dibromo derivative of (VIII), described
above, can also be treated with alkali hydroxides,
e.g. sodium hydroxide under mild conditions, to form
the corresponding 1,3-dihydroxyketone which is converted
to the corresponding 1,3-dioxane-2,5-dione of formula
~XIII) by reaction with phosgene.
VIII~ R20 ~ 17 - 20 ~ COCl2 R ~ R17
Rl B BrR OH O~ O~_,O
o
(XIII)
The 5-oximino intermediate of (XIII) upon treatment with
sodium in ethanol as described above, provides the
corresponding 5-amino compound.
Treatment of a monobromo derivative of ketones of
formula (VIII) with, e~g. ethyl malonate, and subsequent
hydrolysis, decarboxylation and esterification of
resulting product affords intermediates of fo~mula
(XIV) which serve as precursors of the ketones (XV)
as shown below, for example.
R2 ~ ~R ~ R18 ~ R
02C2H5 C02C2H5
(XIV) (XV)
The ketolactones (XV) are then converted to the
corresponding 4-amino compound, e.g. by reduction of
the oxime, as described above.
~r~
30-
The 1,3-dibromoketone derivatives of (VIII),
aescribed abo~e, also can be converted to the corres-
ponding 1,3-dimercaptoke-tone by reaction with at least
two moles of sodium hydrosulfide. Treating the
dimercaptoketone with reagents such as iodine, hydrogen
peroxide or hypochlorous acid under disulfide forming
conditions, well ~nown in the art provides the ketones
of forrnula XVI which are converted to amines by reduction
of the oxime employing, e.g., sodium in ethanol.
o
R6 L~R3
R5~\ ~R
S - S
(XVI)
~ nines of formula tXVII) are provided directly,
for example, by the method of Nagase et al., Chem.
Pharm. Bull. 17, 398 (1969) as shown below.
_
R ~ CHO CH20 ~ CHO NH4Cl R13 ~ CN
/ H20, K
~J~ 2 Rl 2 ~//
~ ~ 13
(XVII)
Use of ethylene oxide in place of formaldehyde
in the first step of the above reaction seguence affords
the corresponding 3-amino-2-pyrones,
-~Rl
O ~ R
3~
Lactarns corresponding to the above lactone inter-
mediates or those of formula (IX), (XII), (XV~ or
(XVII), are obtained by reaction of the appropriate
lactone with ammonia; for example, the above lactone
is contacted with an excess of anhydrous ammonia in
ethanol and the mixture allowed to stir overnight at
ambient temperature to provide compounds of the
forrnula
~ 2 R12
` ~ ~
HN
Alternatively, certain lactam intermediates are
provided by the following reaction sequence.
O
X ) CH 2 Rl ~RRl 7 H 2
(C6H5CH2)~NH C02C2H5 Pd/C
N(CH2C6H5)2
~ ~ R20~R173
H 2 2 5 N
The resulting ketones are then converted to the
requisite amines by methods described above.
The isomeric ketolactams are obtained by the
following reaction sequence:
o o o
R2 ~ l71.Br2 ~R ~ l ~Rl ~ ~ Rl7
Rl ~ R 8 (C6H5~H2)2NH R19 ~ IR18 R 9 ¦ ¦ R18
\ 3.H2, Pd/C NH ~ H
C02C2HS C02C2~5 o
The corresponding 5-membered lactams are also
obtalned by the method of U.S. 3,125,569:
R20 NOH PCl~ ~ O
Rl~/ ~ R18 poidyphosphoric Rl9~ ~ R18
Cyclic or open chain alpha-hydroxyketones or
alpha,alpha'--dihydroxyketones of the formula
Rl4 ~ 5 Rl ~ A
~ (CH2)m or O ~ B
R16X~ R20~
where R -R , m, A and B are as previously defined
are prepared by bromination with one or two moles of
bromine and treatment of the bromo or dibromo inter-
mediate with an hydroxylic base, e.g., sodium hydroxide
or potassium hydroxide as described above. The reaction
sequence is exemplified as follows:
-33-
O O
C~3~ ~ 3 _ 2 ~3 ~ OHH3
2. NaOH,
ethanol
P ~
2. NaOH, HO ¦ OH
ethanol ~
~ icycloalkylketones (XVIII) and alkylcycloalkyl-
ketones (XIX) are prepared by the reaction of the
appropriate acid halide and Grignard reagent employing
conditions and reagents well known in the art, e.g.,
as shown below.
COCl MgCl~ (CH2)m
+ ~ - ~ ~
~(CH2)q ~ (CH2)mL(CH2)q
t XVI I I )
R_ ~
(CH2)m
(XIX)
lQ A~ines of formula RNH2 where R is as pre~lously
defined are also obtained by the well known Hofmann
reaction by convexsion of t~e appropriate carboxamide
with alkali metal hypohalite. This procedure is
especially useful for the preparation of cyclopropyl-
amines. The corresponding cyclopropyl amides are
-34-
obtained and converted to amlnes, e.g. as shown below.
R6 R3 R6 R3
RS ~ R ~ N2CHCO2C2H5 ~ R ~ R4
R6 R3 CO2C2H5
1. NH R5 \ / R4
\ /
2. NaOBr y
N~2
The first step of the above sequence to form the
cyclopropylcarboxylic acid ester is known in the art,
see for example, Mescheryakov et al., Chem. Abstr.,
54, 2~436 (1960).
The compounds of formula (I) or intermediate
amides therefore, wherein R is
R3 R
2 ) n
R6 ~ (C 2)p
where X is SO or SO2 are obtained from the correspond-
ing compounds wherein X is S by oxidation employing
reagents and conditions known to form sulfoxides and
sulfones from sulfides. Alternatively, the appro-
priate ketone of formula (VII) where X is S or theamine derived from said ketone, as described above,
can be oxidized to the sulfoxide or sulfone prior to
coupling to form the dipeptide amide of formula (I).
Preferred reagents and conditions for such oxidation
of sulfides include use of hydrogen peroxide in a
solvent, for example, ace-tic acid or acetone. ~en
equimolar amounts of reactants are employed the product
is the sulfoxide, which is readily converted to the
3 i~
-35-
corresponding sulfone by an additional moie of peroxide.
O-ther ?referred oxidants are potassium permangante,
sodium metaperiodate or chromic acid, for pre?aration
of the sulfones, and m-chloroperbenzoic acid. The
latter reagent being especially use~ul for conversion
of the above thioketones (VII) to the corresponding
sulfoxide employing one mole of this reagent, or the
sulfone when two moles of the peracid are employed.
The compounds of formula (I) and the physio-
logically acceptable salts thereof provide advantages
as sweetening agents in view of their high potenc~,
their physical form and stability. They are, ordinarily,
crystalline, non-hygroscopic, water soluble solids.
They are uniquely characterized by possessing a sweet
taste, devoid of undesirable harsh or bitter flavor
qualities at ordinary use levels. They can be usefully
employed to impart sweetness to edible materials. The
term "edible materials" as used herein signifies all
non-toxic substances consumable by humans or other
animals, in solid or liquid form. Illustrative of
such substances are: foods, including foodstuffs,
prepared food items, chewing gum and beverages; food
additives, including flavoring and coloring agents as
well as flavor enhancers; and pharmaceutical preparations.
The compounds of the invention can be prepared in
a variety of forms suitable for utili2ation of sweeten-
ing agents. Typical forms which can be employed are
solid forms such as powders, tablets, granules and
dragees; and liquid forms such as solutions, suspensions,
syrups, emulsions as well as other commonly employed
forms par1icularly suited for combination with edible
materialsO These forms can consist of the compounds
of formula (I) or their physiologically acceptable
salts either apart or in association with non-toxic
sweetening agent carriers, i.e. non-toxic substances
~36-
commonlv ernployed in association with sweetening
agen-ts. Such suitable carriers include liquids such
as water, ethanol, sor~itol, glycerol, citric acid,
corn oil, peanut oil, soybean oil, sesame oil, propylene
glycol, co:rn syrup, maple syrup and liquid paraffin,
and solids such as lactose, cellulose, starch, dextrin,
modified s~arches, polysaccharides such as polydextrose
(see, e.g. ~.S. 3,76~,165 and U.S. 3,876,794), calcium
phosphate (mono-, di- or tri-basic) and calcium sulfate.
Likewise ~seful and compatible are compositions
containing a compound of the invention combined with
a known sweetening agent such as, for example, sucrose,
saccharin, cyclamate, L-aspartyl L-phenylalanine
me-thyl ester and the like, useful for sweetening
edible materials. Especially useful are the mi~tures
of compounds of formula (I) and saccharin or a physio-
logically acceptable salt thereof, e.g., the sodium,
potassium, calcium or ammonium salt of saccharin. In
said mixtures with saccharin the compounds of formula (I)
reduce or completely mask the well known, undesirable
bitter aftertaste of the saccharin.
Particularly useful such sweetener compositions
are those containing saccharin in admixture with
compounds o-E formula (I) which are at least 400 times
as sweet as sucrose, especially those wherein Ra is
CH20H and R is dicyclopropylcarbinyl, 2,2,4,4-tetra-
methylthietan-3-yl or 2,2,4,4-tetramethyl-1,1-dioxo-
thietan-3-yl. Most particularly preferred are such
mixtures of saccharin and L-aspartyl-D-serine ~-
(2,2,4,4-tetramethylthietan-3-yl)amide, especially
such mixtures which contain the latter compound of
formula (I) and saccharin in a weight ratio in the
range of from 1:1 to 1:8. These mixtures are not only
pleasantly sweet tasting and appreciably devoid of
-37-
bitter aftertaste, they a e, unexpectedly, significantly
sweeter than calculated by summation of sweetness of
the indlvidual components of the mixture. That is,
they exhibit a synergist effect, being up to 50~
sweeter than calculated. In mixtures of saccharin or
its salts and l,-aspartyl-D-serine N-(2,2,4,4-te-tramethyl-
thietan-3-yl)amide in ratios outside the above range
the synergist effect is consideraly reduced.
The invention also provides sweetened edible
compositions comprising an edible material and a
sweetening amount of a compound of formula (I), a
physiologically acceptable salt thereof alone or in
combination with a non-toxic carrier or known sweeten-
ing agent. Examples of specific edible materials
which provide such sweetened edible compositions
include: fruits, vegetables, juices, meat products
such as ham, bacon and sausage; egg products, fruit
concentrates, gelatins and gelatin-like products such
as jams, jellies, preserves, etc.; milk products such
as ice cream, sour cream and sherbet; icings, syrups
including molasses; corn, wheat, rye, soybean, oat,
rice and barley products such as bread, cereals,
pasta~ cake and cake mixes, fish, cheese and cheese
products, nut meats and nut products, beverages such
as coffee, tea, carbonated and non-carbonated soft
drinks, beers, wines and other liquors; confections
such as candy and fruit flavored drops, condiments
such as herbs, spices and seasonings, flavor enhancers
such as monosodium glutamate and chewing gum. The
instant sweeteners are also of use in prepared packaged
products such as dietetic sweeteners, liquid sweeteners,
granulated flavor mixes which upon reconstitution with
water provides non-carbonated drinks, instant pudding
mixes, instant coffee and tea, coffee whiteners,
malted milk mixes, pet foods, livestock feed, tobacco
and consumable toiletries such as mouth washes and toothpaste
as well as proprietary and non-proprietary pharmaceutical
preparations and o-ther products of -the food, pharmaceutical and
sundry industries.
Fspecially preferred sweetened edible composi-tions are
carbona-ted beverages con-taining one or more of the instant
sweeteners.
The L-asparatyl-D-amino acid dipeptide amides of the
instan-t lnven-tion and -the corresponding dipeptide amides of our
rela-ted Uni-ted States Patent No. 4,411,925 issued October 25, 1983,
of the formula
HOOCCH2CHCONHCHCONHR
NH2 Rai
where R is as defined herein and Ra is methyl,
e-thyl, n-propyl or isopropyl, are also useful in the applications
disclosed in the art for L-aspartyl-L-phenylalanine methyl es-ter
and analogs thereof. For example, they are useful in -the same
functions disclosed in the following patents and paten-t appli-
cations for L-aspartyl-L-phenylalanine methyl. es-ter. In these
uses they have the advantages disclosed for the dipeptide ester
as well as their previously mentioned advantages in stability
and potency:
- 38 -
.æ~
-39-
U.S. Patent Nos.:
______
3,64 ,491 3,971,857 3,865,957
3,761,288 3,982,023
3,800,046 A,001,456
3,818,077 4,004,039
3,829,5~8 4,007,288
3,875,311 4,031,258
3,875,312 4,036,~92
3,886,295 4,051,268
3,922,369 4,059,706
3,934,048 4 r 122,195
3,947,600 4,139,636
3,955,000 4,143,170
3,956,507 4,153,737
5 Canadian Patent Nos.:
1,026,g87
1,027,113
1,028,197
1,043,~58
1,046,840
Netherlands Patent A~plicatlon Nos.
73-04,314
73-11,307
75-14,921
76-05,390
76-08,963
West German Offenle~ sschrift Nos.:
2,438,317
2,456,926
2,509,257
~,518,302
2,609,999
2,646,224
2,713,951
~40-
Bel lan Patent Nos -
830,020
838,938
863,138
882,672
Great Britian Patent No 1 454 571-
Ja~an Kokai No. 77-04,176;
French Patent No. 2,338,651 and
Swiss Patent No. 590 615
The invention is further illustrated by the
following examples.
-41~
EXAMPLE 1
L-Aspartyl-D-serine N-(dicyclopropylcarbinyl)amide
I, Ra = CH2OH, R =
A. D-HOCH2CHCOOH
NHC'.bz
5' A solution of 4.41 g. (0.042 mole) D-serine in
21 ml. of 2N sodium hydroxide was cooled to 5 to
10C., adjusted to pH 10.0-11.5 wi-th concentrated
hydrochloric acid and 6.9 ml. (0.048 mole) benzyl
chloroformate was added in increments over 1.5 hours
with simultaneous addition of 2N sodium hydroxide to
maintain the mixture within the above range of pH.
The mixture was stirred overnight at room temperature,
washed with ethyl ether and the aqueous phase acidified
(pH 2.5-3.0) with 6N hydroehloric acid. Extraetion
with ethyl acetate, washing the extracts with brine
and drying (MgSO4), afforded 3.14 g. of product as a
eolorless solid whieh was reerystallized from 20 ml.
ethyl acetate to yield 2.64 g. of product, Rf 0.93
[thin layer ehromatography (TLC), ethyl acetate/
hexane/acetie acid, 9:9:2, by volume~.
-~2-
B. D-~OCH2CHCONH
N~ICbz ~
To a slurry of 2.~ g. (0.01 mole) of N-Cbz-D-
serine, obtained in Part A, in 75 ml. chloroform was
added 1.1 ml. (0.01 mole) N-meth~lmorpholine. A
solution was obtained which was cooled to -12C. To
this was added 0.96 ml. (0.01 mole) ethyl chloro-
formate, the mixture stirred at -10C. ror five
minutes, a solution of 1.11 ~. (0.01 mole) dicyclo-
propylamine in 5 ml. chloroform was added and stirring
continued for five minutes at -15C. The reaction
mixture was allowed to warm to room temperature,
washed successively with 0.5N hydrochloric acid, 5%
sodium bicarbonate solution, water and the chloroform
evaporated ln vacuo. The aqueous washes were combined
and extracted with ethyl acetate. The ethyl acetate
extracts were combined with the residue obtained by
evaporation o~ chloroform and the ethyl acetate was
dried (MgSO4~ and evaporated ln vacuo to afford a
white solid which was dried in the vacuum oven overnight
to give 3.2 g. of the desired product, Rf 0.54 which
was used ln the next step.
3~:~
-~3-
C. D-HOCH~CHCONH
2 ~
The 3.2 g. (9.6 mmole) N-Cbz-amide, obtained in
Part B, was dissolved in 70 ml. methanol, 1.0 9. 5~
Pd/C catalyst added and the mixture hydrogenated at
60 psi (4.2 kg./cm.2) for 30 minutesO The catalyst
was removed by filtration and the filtrate evaporated
in vacuo to yield 1.93 g. of product as a soap-like
solid.
D. C6H5cH2ococ~2cHcoNHcH(c~2oH)coNHcH( ~ )2
NHCbz
A mixture of 3.4 g. (9.5 mmole) beta-benzyl N-
benzyloxycarbonyl-L-aspartate, loO ml. (9.5 mmole) N
methylmorpholine and 0.9 ml. (9.5 mmole) ethyl
chloroformate was stirred at -15 to -10C. for five
minutes and a solution of l.9 g. (9.5 mmole) D-serine
N-dicyclopropylcarbinylamide, obtained in Part C, in
10 ml. chloroform was added at -15C. The resulting
mixture was stirred at -10C. for five minutes,
allowed to warm to ambient temperature and stirred
for one hour. The reaction mixture was evaporated ln
vacuo to remove solvent, the residue taken up in
ethyl acetate (250 ml.), washed in turn with lN
hydrochloric acid, 5% sodium bicarbonate solution,
brine and dried over anhydrous magnesium sulfate.
The solvent was evaporated in vacuo to obtain a
gelatinous solid. This was taken up in 75 ml~ hot
ethyl acetate. Upon cooling a crystalline solid was
obtained which was dried in vacuo at 40C. to yield
2.75 g. of the desired diprotected dipeptide amide as
a fine white solid, Rf 0.30.
E. A mixl_ure of 2.75 g. of the diprotected di-
pep-tide amide, obtained in Part D, 200 ml. methanol
and 1.0 g. 5~ Pd/C catlayst was hydrogenated at
60 psi (4.2 kg./cm.2) for one hour during which
product precipitated. The catalyst/product mixture
was filtered, the filter cake slurried in 100 ml. hot
water and filtered again. The combined filtrates
were e~aporated to dryness, triturated with water,
filtered and dried ln vacuo to arford 260 mg. of
product as a fine white, fluffy solid, M.P. 252-254C.,
Rf 0.58 (TLC, n-butanol/water/acetic acid 4:1:1,
ninhydrin spray).
The filter cake from the hydrogenation was
slurried in 50 ml. of O.lN hydrochloric acid, the
mixture filtered through diatomaceous earth (Supercel),
the filtrate ~pH 1.6) was adjusted to pH 5.9 with
sodium hydroxide solution, and the precipitated
product collected by filtration and dried in vacuo to
yield an additional 800 mg. of product. Total step
yield, 68~.
Mass spectrum (m/e) 313 (M )~
Sweetness potency: 700 x sucrose.
~r~
-45-
EXAMPLE 2
L-Aspar-tyl-D-serine N-(2,4-dimethyl-3-pentyl)amide
I, Ra = C~2OH, R =
A. D-HOCH2CHCONH
NHCbz
A mixed anhydride was prepared as follows:
l.0 g. (4.3 mole) N-benzyloxycarbonyl-D~serine was
dissolved in 50 ml. tetrahydrofuran, cooled to -10C.
under a nitrogen atmosphere and 0.47 ml. (4.3 mmole)
N-methylmorpholine and 0.41 ml. (4~3 mmole) ethyl
chloroformate were added. The mi~ture was stirred at
-10C. for 30 minutes.
To the solution of mixed anhydride was added
495 mg. (4.3 mmole) 2,4-dimethyl-3-aminopentane
dissolved in a small amount of chloroform, the
mixture stirred at -10C. for 15 minutes and allowed
to warm to room temperature. Ethyl acetate (40 ml.)
was added and -the mixture was washed with lN hydro-
chloric acid, sodium bicarbonate solutionr brine and
the organic layer was dried over anhydrous magnesium
sulfate. Evaporation of solvent in vacuo gave
1.27 g. colorless solid which was triturated with
ethyl ether, filtered and air dried to afford l.0 g.
of colorless product, Rf 0.77 (TLC, ethyl acetate/
hexane, 7:3 by volume, vanillin spray).
46-
B. D~HOCH2CHCONH
__ 2
The above 1.0 g. of product was dissolved in
50 ml. methanol, 0.5 g. 5% Pd/C catalyst added and
the mixture hydrogenated at 50 psi (3.52 kg./cm.2)
until hydrogen uptake ceased. Filtration and evapora-
tion of filtrate gave 700 mg. of the desired product.
C. C6H5CH2OCOCH2CHCONHCH(CH2OH)CONH
NHCbz
A solution of 1~36 g. (3.8 mmole) beta-benzyl N-
benzyloxycarbonyl-L-aspartate in 10 ml. tetrahydrofuran
was cooled to -10C. and 0.42 ml. (3.8 mmole) N-
methylmorpholine was added. To this was added
dropwise 0.36 ml. (3.8 mmole) ethyl chloroformate and
the resulting mixture stirred at -10C. for S minutes.
Then 566 mg. (2.8 mmole) D-serine N-(2,4-dimethyl-3-
pentyl)amide (from Paxt B, above) in a few milliliters
of tetrahydroEuran was added dropwise and stirring
continued for 15 minutes. The reaction mixture was
allowed to warm to room temperature and evaporated in
vacuo to afford a solid residue. This was mixed with
ethyl acetate, washed with lN hydrochloric acid and
the organic phase washed with 5% aqueous sodium
bicarbonate, brine, dried (MgSO4) and the solvent
evaporated to afford 1.6 g. of product as an amorphous
solid which was used in the next step.
3~ 3~
-47-
D. To a solution of 2.7 g. of the diblocked di-
peptide amide (preparation as described in Part C,
above) in methanol was added 1.5 g. 5% Pd/C catalyst
and the mixture was hydrogenated at 50 psi (3.52 kg./cm. )
until hydrogen uptake ceased. The catalyst was
removed by filtration and the filtrate evaporated in
vacuo to a small volume and allowed to stand at room
temperature. The precipitated product was collected
by filtration and dried in vacuo to afford 342 mg. of
____
colorless solid.
Sweetness, 180 x sucrose.
-48-
EXAMPLE 3
L-Aspartyl-D-serine N-
(2,2,4,4-tetramethylthietan-3-yl)amide
C~3 CH3
I, Ra = CH2OH, R = ~ S
CH/ CH
A. D-HOCH2CHCOOH
NH_-Bo
The method is that described by Moroder et al.,
Z. Physiol. Chem. 357, 1651 (1976), for preparing t-
Boc-amino acids. To 10 ml. each of dioxane and water
was added 2.18 g. (10 mmole) di-t-butyl dicarbonate
1.6 ml. (11.5 mmole) triethylamine and 1.05 g.
(10 mmole) D-serine. The mixture was stirred for
30 minutes at room temperature and the dioxane
evaporated _ vacuo. The aqueous residue was cooled
in ice, ethyl acetate was added and the mixture
stirred while adding dilute potassium bisulfate
solution to pH 2-3. The aqueous layer was separated,
extracted twice with ethyl acetate and the combined
extracts washed with water, dried (Na2SO~) and the
solvent evaporated ln ~acuo to yield 1.7 g. of
product as a viscous paste.
_~9_
CH CH3
~<
B. D-HOCH2CHCONH ~ S
NH2 CH'`CH
A mixed anhydride was prepared from 2.85 g.
(14 mmole) of N-t-butoxycarbonyl-D-serine, 1.55 ml.
N-methylmorpholine, 1.34 ml. ethyl chloroformate ln
75 ml. methylene chloride at -12 to -10C. by the
method of Example l r Part B. To this mixture was
added 2.01 g. (14 mmole) of 3~amino-2,2,4,4-tetra-
methylthietane and stirring continued for five
minutes at -12C. The product was isolated as
described in Example l, Part B to afford 4 g. of a
viscous liquid residue. The residue was dissolved in
40 ml. methylene chloride, 12 ml. trifluoroacetic
acid (d = 1.480) was added and the mixture was
stirred at room temperature for three hours. The
reaction mixture was made alkaline with 40% sodium
hydroxide solution, the organic layer separated, the
aqueous layer was saturated with sodium chloride and
extracted with methylene chloride. The combined
extracts were dried (MgSO4) and concentrated to
dryness in vacuo to yield 2.21 g. amorphous off-white
solid. Crystallization from ethyl ether/hexane gave
1.92 g. of product as a fine r white solid.
-50-
(CH3)2
C~ t-ButylOC(:)CH2CIHCONH(~HCONH ~ S
NH-t-soc ( 3)2
A mixture of 2.3 g. (8.0 mmole) beta-t-butyl N~
t-butoxycarbonyl-L-asparate, 0.88 ml. (8.0 mmole) N-
methylmorpholine and 0.77 ml. (3.0 mmole) ethyl
chloroformate in 40 ml. methylene chloride was
stirred at -12C. for five minutes. ~ solution of
1.85 g. (8.0 mmole) D-serine N (2,2,4,4~tetramethyl-
thietan-3-yl)amlde in 5 ml. of the same solvent was
added and stirring continued at -12 to -10C. for ten
minutes. The mixture was allowed to warm to room
temperature, stirred for one hour at this temperature
and the solvent evaporated. The residue was taken up
in ethyl acetate, washed with dilute hydrochloric
acid, sodium bicarbonate solution, brine, dried
(MgSO4) and the ethyl acetate evaporated to afford
3.34 g. of amorphous solid. Crystalliæation from
ethyl ether/hexane gave 2.91 g. of colorless solid
product, Rf 0.70 (ethyl acetate/he~ane, 7:3).
D. A solution of 2.4 g. (4.78 mmole) of the product
obtained in Part C, above, in 60 ml. chloroform was
fitted with a gas inlet tube and anhydrous hydrogen
chloride bubbled through the solution. After f,ive
minutes, precipitation of solid was observed. The
hydrogen chloride addition was continued for ten
minutes, then the mixture was stirred at ambient
temperature for one hour and evaporated to dryness ln
vacuo. The residue was taken up in water, washed
with chloroform, the pH adjusted to 5.6, washed again
with chloroform, and the aqueous phase evaporated ln
vacuo. Ethanol was added to the residue and the
mixture evaporated to dryness in vacuo. The residue
was dissolved in 25 ml. hot water and allowed to
cool. The precipitated product was collected by
filtration and dried in vacuo to yield 1.12 g. (67~)
of product, M.P. 193-196C. Rf 0.32 (n-butanol/water/
acetic acid, 4:1:1).
Analysis Calculated for C14H25N3O5S:
C, 48.39; H, 7.25; N, 12.09; S, 9.23.
Found: C, 46.77; H, 7.48; N, 11.91; S, 8.82
Sweetness potency, 1200 x sucrose.
3'~
-52-
EXAMPLE 4
L-Aspartyl-D-serine N-(2,2,4,~-
tetramethyl~l,l-dioxothietan-3-yl)amide
I, R = CH2H' R = ~ SO2
C 3 C 3
__
A.3-~mino~2,2,4,4-tetramethylthietan-1,1-dioxide
A solution of 14.53 g. (0.1 mole) 3-amino-
2,2,4,4-tetramethylthietane and 64.17 g. (0.3 mole)
sodium m-periodate in 500 ml. water was stirred
overnight at room temperature. The reaction mixture
was adjusted to p~ 13 with sodium hydroxide solution
and the precipitated sodium iodate removed by filtra-
tion. The filtrate was washed with 100 ml. ethyl
ether, the aqueous phase extracted continuously with
methylene chloride over 18 hours, the extract dried
(MgSO4) and solvent evaporated ln vacuo. The residual
solid was recrystallized from ethyl acetate to
provide 8.5 9. of product, M.P. 104-106.5C. A
second crop of crystals was obtained, 2.7 g., MoP~
103-106C. Total yield, 63%.
-53-
B. D-Serine N- ( 2,2,4,4-tetramethyl-1,1-dioxo-
thietan-3-yl)amide
_ ~ ~ ~
By the method of Example 1, Part B, 1.33 g.
(6.5 mmole) N-benzyloxycarbonyl-D-serine, 0.715 ml.
N-methylmorpholine, 0.62 ml. ethyl chloroformate and
1.15 g. (6.5 mmole) 3-amino-2,2,4,4-tetramethyl-
thietane-l,l-dioxide gave 2.3 g. of N-benzyloxy-
carbonyl~D-serine N-(2,2,4,4-tetramethyl-1,1-dioxo-
thietan-3 yl)amide as a viscous li~uid, Rf 0.37
(ethyl acetate/hexane, 7:3). This liquid was dis-
solved in methanol, 750 mg. of 5% Pd/C catalyst added
and the mixture hydrogenated by the method of Example 1,
Part C. After filtering to remove the catalyst, the
methanol was evaporated in vacuo, the residue taken
up in lN hydrochloric acid and extracted with chloro-
form. The aqueous layer was made alkaline with
sodium hydroxide saturated with sodium chloride and
extracted continuously wlth chloroform overnight.
Evaporation of solvent gave 1.54 g. of product as a
viscous liquid which solidified upon standing~ Rf
0.32 (m-butanol/water/acetic acid, 4:1:1).
-5a_
6 5 2 2,E ' ~ H32
_ _____Cbz CH2OH CH3 CH
The diblocked dipeptide amide of the above
formula was prepared on a 4.7 millimolar scale
employing the method of Example 1, Part D, with
1.7 g. beta-benzyl N-benzyloxycarbonyl-L-aspartate,
0.51 ml. N-methylmorpholine, 0.45 ml. ethyl chloro-
formate and 1.24 g. D-serine N-(2,2,4,4~tetramethyl-
1,1-dloxothietan-3-yl)amide. The product, 2.45 g.,
was obtained as a colorl~ss amorphous solid. Two
grams of this material was purified by chromatography
on 60 g. of silica gel, eluting wlth ethyl acetate to
afford 1.2 g. of amorphous solid product, Rf 0.30
(ethyl acetate/hexane, 7:3).
D. A mi~ture of 1.2 g. of purified product from
Part C, above, 75 ml. methanol and 0.6 g. of 5% Pd/C
was hydrogenated at 80 psi (5.6 kg./cm.2). When
hydrogen uptake ceased, the catalyst was removed by
filtration, and the filtrate evaporated to afford a
colorless solid residue. The residue was taken up in
water, washed with chloroform and the aqueous layer
evaporated 1n vacuo. The residual solid was crystal-
lized from ethanol to afford 255 mg. of the desired
dipeptide amide as a fine white solid, M.P. 170-173,
Rf 0.20. An additional 180 mg. of product was
obtained by reworking the filter cake from the
hydrogenation.
Sweeetness potency: 850 x sucrose.
EXAMPLE 5
D-O-Methylser~ne
A. N-Chloroacetyl-dl-O-methylserine
.. ~. ... ~
To 125 ml. water was added 59.55 g. (0.5 mole)
dl-O-methylserine, the mixture was stirred and 20 g.
(0.5 mole) sodium hydroxide was added. The resulting
solution was cooled in ice and simultaneously, from
two dropping funnels was added over one hour, a
solution of 20 g. of sodium hydroxide in 125 ml.
water and 47.8 ml. 10.6 mole) chloroace-tyl chloride.
The addition rates were adjusted to maintain the
reaction mixture at pH 9.0-9.5. After the addition
was completed, the resulting mixture was stirred for
one hour at pH 9.0-9.5. The mixture was washed twice
with methylene chloride, the aqueous phase acidified
to pH 1.5 with concentrated hydrochloric acid while
cooling in ice, saturated with sodium chloride and
extracted several times with chloroform. The combined
extracts were dried (MgSO4) and the solvent evaporated
in vacuo. The residue was stirred with ethyl ether
.
to precipitate a yellow solid product which was
collected by filtration and dried, 69.38 g. (71%).
M.P. 104-]07C., Rf O.Z3 (ethyl acetate/hexane/acetic
acid, 9:9:2, phosphomolybdate spray). A second crop
was obtained from the aqueous phase by adding more
sodium chloride and extracting with chloroform.
Evaporation of chloroform gave 3.65 g. of product.
Total yield 75%.
3~
-56-
B. N-Chloroacetyl-~-O-methylserine
To 3000 ml. of water at 35-37C. was added
73.03 g. (0.37 mole) N-chloroacetyl dl-O-methylserine
and the mixture adjusted to pH 7.1g by addition of
concentrated ammonium hydroxide. Water was added to
make a total volume of 3700 ml. To this was added
17 mg. of commercial porcine kidney aminoacylase, N-
acylamino acid amidohydrolase; EC 3.5.1.14 (Acylase I)
having 1845 units/mg. (1 unit is defined as the
amount required ~o hydrolyze 1 micromole of N-acetyl-
L-methionine per hour at pH 7.0 and 25C.). The
amount of enzyme added was that calculated to hydro-
lyze the susceptible isomer in six hours. The
xesulting solution was maintained at 37-38C. for
28 hours with intermittent addition of ammonium
hydroxide to maintain the pH at 7.1 to 7.2. An
adclitional 5 mg. of enzyme was added after 24 hours.
The hydrolysis mixture was acidified to pH ~.5 with
glacial acetic acid, filtered through a 0.6 ~
millipore filter (type BD) and the filtrate evaporated
in vacuo below 35C. to reduce the total volume to
__
100-150 ml. The residual mixture was acidified to
pH 2.00 with hydrochloric acid and extracted several
times with ethyl acetate and further extracted with
chloroform.- The separate organic extracts were each
washed with water, dried ~MgSO4) and solvent evaporated
ln vacuo to afford a yellow liquid residue. Addition
of hexane and evaporation ln vacuo induced crystalli-
zation. The ethyl acetate extracts afforded 16.67 g.
(~6%) of N-chloroacetyl-D-O methylserine, M.P.
95-96C., [alpha]D - 15.5 (C = 1, lN NaOH). The
chloroform extracts gave 4.61 g. (13%) of the same
product, M.P. 90-34C. (odor of chloroacetic acid).
Both crops of product showed a single spot upon thin-
layer chromatography on silica gel plates, R~ 0.35;9:9:2 eth~l acetate/hexane/acetic acid, phospho-
molybdate spray.
33~
-57-
C. To 16.67 g. (0.085 mole) N-chloroacetyl-D-0-
methylserine was added 25 ml. of 2N hydrochloric acid
and the m:ixture heated a-t reflux for three hours.
The mixt~re was concentrated in vacuo, chasing any
residual chloroacetic acid with additional water.
The solid residue was washed with ethyl ether and
collected by filtration to afford 12.31 g. (93O) D-0-
methylserine hydrochloride, M.P. 188-190C.; [alpha]D -
16~7 (C = 0.7, CH30H).
3~
-58-
EXAMPLE 6
L-Aspartyl-3-O-methylserine
N-(dicyclopropylcarbinyl)amide
I, Ra CH2OCH3, R =
A. D-CH3OCH2C~ICOOH
NHCbz
-
A solution of 12.31 g. (0.079 mole) 3-O-methyl-
serine in 40 ml. water containing 6.32 g. (0.158
mole) sodium hyciroxide was cooled to 5-10C. and
11.74 ml. (0.0806 mole) benzyl chloroformate and 4M
sodium hydroxide were added simultaneously at pH
8-9. The resulting mixture was stirred until the pH
remained at 8 without further addition of base.
After washing with methylene chloride, the aqueous
phase was acidified with concentrated-hydrochloric
acid, extracted four times with methylene chloride~
the extracts dried (MgSO4) and solvent evaporated in
va_uo. The viscous liquid residue was stirred with
hexane and the precipitated solid collected by
filtration to yield 18.6 g. oE product (93%), [alpha]D ~
2.7 (C = 1, lN NaOH), Rf 0.43.
-59-
B. D-CH3OCH2CHCONH
NHCbz
To a solution of 3.80 g. (0.015 mole) N-benzyl-
oxycarbonyl-D-O-methylserine in 75 ml. m~thylene
chloride was added 1.68 ml. (0.015 mole) N--methyl~
morpholine and the mixture cooled to -15C. To this
was added 1.43 ml. (0.015 mole) ethyl chloroformate,
the mixture stirred at -20 to -15C. for ten minutes
and 1.68 g. (0.015 mole) dicyclopropylcarbinyl amine
was added. The mixture was allowed to warm to room
temperatu:re and stirred for -two hours. The resulting
mixture was washed twice with lN sodium hydroxide,
twlce with lN hydrochloric acid and dried over
magnesium sulfate. Evaporation of solvent in vacuo
gave 5.1 g. (98~) of the desired product, Rf 0.59
upon silica gel TLC in 1:1 ethyl acetate/hexane,
phosphomolybdate spray.
The structure of the product was confirmed by
~-NMR spectroscopy.
3~
--60--
C . D-CH3OCH2CH ( NH2 ) CON~
. . . _
The 5.1 9. of N-benzyloxycarbonyl-D-O~methylserine
N-(dicyclopropylcarbinyl)amide, obtained in Part B,
above, was hydrogenated by the procedure of Example 1,
Part C, to afford 2O96 g. (95%) D-O-methylserine N-
(dicyclopropylcarbinyl)amide as a liquid, Rf 0.39;
[alpha]D ~ 23.2 (C = 0.7, lN HCl). The structure was
confirmed by lH-NMR spectroscopy.
D. C6H5CH2OCOCH2CH(NHCbz)CONHCH(CH2OCH3)CONHCH( ~ )2
. . . _ _ .
A mixture of 4.97 g. (13.9 mmole) beta-benzyl N-
benzyloxycarbonyl-L-aspartate, 1.55 ml. (13.9 mmole)
N-methylmorpholine and 1.33 ml. (13.9 mmole) ethyl
chloroformate were reacted as described in Example 1,
Part Dl to provide 7.43 g. (97~) of the diblocked
dipeptide amlde which was recrystallized twice from
e-thyl acetate to g:ive 4.25 g. of colorless product,
Rf 0.45 (7:3 ethyl acetate/hexane). The structure
was verified by 1~-NMR.
E. Hydrogenation of the 4.25 g. of purified di~
blocked dipeptide amide obtained in Part D, above, by
the procedure of Example 1, Part E, gave 2.4 g. (95%)
of the desired dipeptide amide, M.P. 215~217C.
(dec.); [alpha]D + 37.6 (C = 0.8, 1.2N HCl); Rf
0.41.
Sweetness potency, 85 x sucrose.
3~
-61-
EXAMPLE 7
_
L~Aspar-tyl-D-O-methylserine N-
2,2,4,4-tetramethylthietan-3-yl)amide
3~/ 3
I, Ra = CH2OCH3, R = ~ s
3 3
A. D-CH3OCH2CHCOOH
NHt-Boc
. =
To a solution of 2.89 g. (18.6 mmole) D-O~
methylserine hydrochloride in 11 ml. water was added
6.48 mlO (46.5 mmole) triethylamine, 5.10 g. (20.7
mmole) 2-(t~butoxycarbonyloxyimino)-2-phenylaceto-
nitrile ("BOC-ON"~ and 11 mi. tetrahydrofuran. The
mixture was stirred at room temperature overnight,
diluted with 25 ml. water and washed with ethyl
acetate. The aqueous layer was acidified to pH 1.8
with lM hydrochloric acid, extracted with ethyl
acetate (3 x 75 ml.), dried (MgSO4) and evaporated in
vacuo to afford 4.2 g. of product as a viscous
liquid, Rf 0.65 ~9:9:2 ethyl acetate/hexane/acetic
acid, phosphomolybdate spray).
B. N-t-Boc-D-0-Methylserine N-(2,2,4,4-tetramethyl-
thietan-3-yl)amide
~o a solution of 4.2 g. (18.6 mmole) N-t-Boc-D-
0-methylserine in 90 ml. me-thylene chloride was added
2.08 ml. N-methylmorpholine, the rnixture cooled
to -15C. and 1.78 ml. ethyl chloroformate added.
After stirring for 8 minutes at -20 to -15C.,
2.70 g. (18.6 mole) 3-amino-2,2,4,4-tetramethyl-
thietane dissolved in 10 ml. methylene chloride was
added at the same temperature and the mixture allowed
to warm to room temperature. After stirring for two
hours the mixture was washed with dilute sodium
hydroxide, dilute hydrochloric acid, dried over
anhydrous magnesium sulfate and the solvent evaporated
_ vacuo to yield 6.04 g. (94%) of colorless solid,
Rf 0.35 (3:7 ethyl acetate/hexane, phosphomolybdate
spray~. The structure was verified by H-NMR.
C. D-O-~ethylserine N-(2,2,4,4-tetramethylthietan-
3-y~_amide
To a solution of 6.04 g. (17.4 mmole) of N-t~
Boc-D~O-methylserine N-(2,2,4,~-tetramethylthietan-3-
yl)amide in 13.4 ml. methylene chloride was added
6.7 ml. (87 mmole) trifluoroacetic acid (sp. gr.
1.480) and the mixture was stirred at room tempera-
ture for three hours. An additional 1.0 ml. in ? ml.
methylene chloride was added and stirring continued
for one hour. The reaction mixture was made alkaline
with 40% (w/w) sodium hydroxide solution, the organic
layer separated and the aqueous layer extracted
several times with fresh methylene chloride. The
combined extracts were dried (MgSO~) and solvent
evaporated _ vacuo to give 4.29 gO of crude liquid
product. This was taken up in 20 ml. lN hydrochloric
acid, washed with ethyl ether, the aqueous layer made
alkaline with sodium hydroxide (40% w/w), saturated
with sodium chloride and extracted with methylene
chloride. Evaporation of the extracts afforded
3.21 9. (75~) of the desired product, Rf 0.41;
[alpha~D - lg.8 (C = 0.8, lN HCl). The structure of
this product was verified by its H-NMR spectrum.
-64-
3 3
D. t-Butyl OCOCH2CH CONH CH CONH ~ S
NH-t-BccH20cH3 CH3CH3
By employing 3.76 g. of product obtained by the
procedure of Part C, above, the procedure of Example 3,
Part C, was repeated on a 13 millimolar scale using
50 ml. methylene chloride as solvent to afford 5~0 g~
(74%) of the desired diblocked dipeptide amide as a
brittle foam, Rf 0.40 (ethyl acetate/hexane, 1:1;
phosphomolybdate spray). The structure was verified
by the 1H-NMR spectrum of the product.
E. Anhydrous hydrogen bromide was bubbled through a
solution of 5.0 g. (9.7 mmole) of the diblocked
dipeptide amide provided in Part D, above, while
stirring at room tempexature for one hour. The
resulting mixture was evaporated to dryness in vacuo
15 and the resulting yellow solid residue dissolved in
water. The solution was washed twice with ethyl
ether, once with methylene chloride, the aqueous
phase adjusted to pH 5.8 with sodium hydroxide
solution and evaporated to dryness 1n vacuo. The
20 residual solid was dissolved in 10 ml. 95% ethanol
and ethyl ether added to precipitate the title
compound in two crops:
1.66 g., [alpha]D + 13.4 (C = 0.9, 1~2N HCl),
M.P. 85-90C.;
1~20 9~ r [alpha]D + 13.9 (C = 0~8r 1 o2N HCl) ~
TLC of each crop showed product spot at Rf 0.51 with
small amount of material of Rf 0.44.
-6~-
F. Purification via p~toluenesulfonate salt
To 10 ml. water was added 1.39 g. (3.85 mmole)
of the combined crops of product obtained above and
0.66 g. ~3.83 mmole) p-toluenesulfonic acid. The
resul~ing solution was stirred at room temperature
for two hours. The precipitated solid was collected
by filtration and washed with a small amount of water
to afford 0.94 g. of p-.oluenesulfonate salt. The
salt was combined with 3 ml. of liquicl anion exchange
resin (Amberlite LA-l~), 6 ml. hexane, 2 ml. water
and the mixture stirred for two hours. The aqueous
phase was separated, washed with hexane and evaporated
to dryness in vacuo to give 0.72 g. of off-white
solid, [alpha]D ~ 22.43 (C = 0.8, 1.2N HCl); Rf
0.48.
Sweetness potency: 320 x sucrose. The sweet
taste was judged to be unusually clean, free of off
flavor notes and to have a quick sweetness impact.
~ A registered trademark of Roh~ and Haas Co.
E~AMPLE 8
L Aspartyl-D-O-methylserine N-(2,2,4,4-
tetramethyl-l,l-dioxothietan-3-yl)amide
__
A. D CH3OC~2CHCONH ~ S
NHCbz CH3 H3
I~ 75 ml. methylene chloride was dissolved
3.8 g. (15 mmole) ~-benzyloxycarbonyl-D-O-methyl
serine. N-methylmorpholine (1.68 ml.) was added, the
solution cooled to -15C. and 1.43 ml. ethyl chloro-
forrnate added. The resulting mixture was stirred ior
8 minutes at -15C., then 2.18 g. (15 mmole) 3-amino-
2,2,4,4~tetramethylthietane was added and the mixture
allowed to warm to room temperature. Stirring was
continued for two hours at room temperature, the
reactlon mixture was~ed with dilute sodium hydroxide,
dilute hydrochloric acid, dried (MgSO4) and the
solvent evaporated ln vacuo to give 6.11 g. of liquid
product, Rf 0.58 (ethyl acetate/hexane 1:1; phosphomolybdate
spray).
3~
-67-
B. Oxidatlon to l,l-dioxide
The product from Part A, above, 6.11 g., was
dissolved in 75 ml. chloroform and cooled in ice
while adding 7.12 g. m-chloroperbenzoic acid in
portions. The reaction mixture was allowed to warm
to room temperature and stirred for three hours.
Additional chloroform (75 ml.) was added and the
solution washed twice each with 5~ (w/v) sodium
carbonate solution, 0.5N sodium thiosulfate and lN
hydrochloric acid. After drying the organic phase
(MgSO4) and evaporation of solvent, 6.24 g. of
product was obtained as a viscous liguid, Rf 0.22
(ethyl acetate/hexane, 1:1; phosphomolybdate spray)
with traces of starting material and sulfoxide. The
H-NMR spectrum was in agreeement with the structure
for the desired sulfone with a small amount of
chloroform.
-68-
C . D-CH30CH2CHCONH~ S2
N~2 ~ 3
A mixture of 6.11 g. of N-benzyloxycarbonyl-D-O-
methylserine N-(2,2,4,4-tetramethyl-1,1-dioxothietan-
3-yl)amide, 250 ml. methanol and 3.0 g. 5% palladium-
on-carbon catalyst was hydrogenated at 50 psi (3.52
km./cm.~) for two hours. The catalyst was removed by
filtration, the filtrate was evaporated 1n vacuo and
the residue taken up in 35 mlO lN hydrochloric acid.
The acidic solution was washed three times with
chloroform, made alkaline with solid sodium hydroxide,
saturated with sodium chloride and extracted with 3 x
50 ml. chloroform. The eombined extracts were dried
(MgSO~) and solvent evaporated in vacuo to give
3.37 g. (80%) of the alpha-amino amide product as a
colorless liquid, Rf 0.29; [alpha]D - 15i7 (C = 0.8,
lN HCl). The structure was verified by ~-NMR
spectroscopy.
--69--
CH~H3
D . C 6H 5CH 20COCH2Cfl ( NHCbz ) CONHCH ( CH 20CH3 ) CONH S<50 2
- CH3 CH3
Employing 3.37 g. (12 mmole) of the product
obtained in Part C, above, as starting material, in
the procedure of ~xample 1, Part D, the desired
diblocked dipeptide amide was obtained as a clear
glass, 6073 g. (91%), Rf 0.28 (ethyl acetate/hexane,
70:30). The lH-NMR spectrum was in agreement with
the structure for this compound.
E. A mixture of 6.73 g. beta-benzyl N-Cbz-L-aspartyl-
D-O-methylserine N-(2,2,4,4-tetramethyl-1,1-dioxothietan-
3-yl)amide, 250 ml. methanol and 2.0 g. 5% Pd/C
catalyst was hydrogenated by the procedure of Part C,
above. The residue remaining after evaporation of
solvent was stirred overnight in ethyl ether and the
solid product collected by filtration and dried in
the vacuum oven to yield 3.3 g. (77~) of the desired
dipeptide amide, Rf 0.23; M.P. 140-150C. (dec.);
[alpha]D + 20.3 (C = 1, 1.2N HCl).
Sweetness potency: 200 x sucrose.
3~
-70-
EXAMPLE 9
L-Aspartyl-D-serine N-(dl-cls,trans-
2,6-dimethylcyclohexyl)amide
CH3
I, Ra = C~I2OH, R -
- 3
A. dl-cis,trans-2,6-Dimethylcyclohexylamine
A solution of 2.1 g. trans-2,6-dimethylcyclo-
hexanone oxi~e in 30 ml. dry ethanol was heated at
reflux. TG this was added in portions 3.1 g. of
metallic sodium. When the addition was complete, the
mixture was maintained at reflux fox 30 minutes and
allowed to cool to room temperature. The resulting
gel was dissolved in water, adjusted to pH 2.0 with
hydrochloric acid and washed with ethyl ether. The
aqueous phase was made alkaline with sodium hydroxide,
extracted wlth ether, the extracts dried (MgSO4) and
evaporated to provide the desired amine as a colorless
liquid.
B. D-Serine N-(dl-cls,trans-2,6-dimethylcyclohexyl)amide
Employing 1.47 g. (6.15 mmole3 N-Cbz-D serlne,
775 mg. (6.15 mmole) _ -cis,trans-2,6-dimethylcyclo-
hexylamine and equimolar amounts of N-methylmorpholine
and ethyl chloroformate and subsequent removal of
amino-protecting group by catalytic hydrogenation by
the procedures of Examples 1, Parts B and C, afforded
0.70 g. of the desired D-serine amide as a white
solid, Rf 0.64 (ethyl acetate/hexane, 7:3).
C. ~ t~ tide Amide
By employirlg 600 mg. (2.8 mmole) of D-serine-N-
(dl-cis,trans-2,6~dimethylcvclohexyl)amide in the
procedure of Example 1, Part D, the corresponding
beta-benzyl N-benzyloxycarbonyl-L-aspartyl-D-serine
amlde, 1.2 g., was obtained as a colorless solid.
Recrystallization from isopropyl ether gave 1.0 g.,
Rf 0.35 (ethyl acetate/hexane, 7:3).
D. Catalytic hydrogenation of the diprotected
dipeptide amide provided in Part C, above, (1.0 g.)
in methanol in the presence of 0.6 g. 5% palladium/
carbon catalyst by the procedure of Example 1,
Part E, yielded 435 mg. of the title compound as an
off-white crystalline solid.
Sweetness potency: 200 x sucrose.
-72-
EXAMPLE 13
Beta-Benzyl N-benzyloxycarbonyl-
L-AspartyI-D-O-methylserine
C~H5cH2occH2cHcoNHcHcooH
O NH CH2OCH3
Cbz
D~O-Methylserine (~.65 g., 56.1 mmole) ls dis-
solved in 100 ml. of ~,N-dimethylformamide (DMF) and
to the solution is added dropwise at room temperature
6.7~ g. (62.4 mmole) OL trimethylchlorosilane. In a
separate flask is placed beta-benzyl N-benzyloxycar-
bonyl-L-aspartate (18.0 g., 50.~ mmole), triethylamine
(12.35 g., 122 mmole) and 110 ml. each of ~MF and
tetrahydrofuran and the resulting solution cooled
to -15C. ~o the solution is added ethyl chloro-
formate (5.95 g., 55.1 mmole) and the resultin~
mixture stirred for ten minutes at -10C. To this is
then added dropwise the DMF solution of silylated D-O-
methylserine prepared above while maintaining the
mixture at -5 to -10C. The mixture is stirred
at -5C. for one hour, 0.2 N hydrochloric acid added
until the mixture is acidic and the resulting mixture
extracted with chloroform. The chloroform extracts
are combined and washed several times with dilute
hydrochloric acid to remove remaining DMF. The
solvent is evaporated in vacuo to provide the title
compound.
When the procedure is repeated, but employing D-
serine in place of D-O-methylserine and twice the
above amount of trimethylchlorosilane, beta-benzyl N-
Cbz-L-aspartyl-D-serine is obtained in like manner.
'3~
EXAMPLE 11
Beta-Methyl-N-benzylo~ycarbonyl-
L-aspartyl-D-ser ne
A suspension of 80.7 g. ~0.78 mole) D~serine in
200 ml. of DMF is cooled to lO~C., 184 g. (1.70 mole)
of trimethylchlorosilane is added in portions and the
resulting mixture s-tirred at 25C. for one hour.
In a separate flask is placed a solution of
158 g. 10.86 mole) of beta-methyl L-aspartic acid
hydrochloride in one liter of water. To this is added
34.5 g. ~0086 mole) of sodium hydroxide followed by
80 g. of sodium bicarbonate and the resulting mixture
stirred vigorously. After cooling to 5-10C. 161 g.
(0.94 mole) of benzyloxycarbonyl chloride is added in
portions and stirring continued for two hours at this
temperature. The reaction mixture is washed with
100 ml. of ethyl acetate, acidified by addition of
80 ml. of concentrated hydrochloric acid and extracted
with ethyl acetate (2 x 450 ml.). The extract (900 ml.)
is found to contain 218 g. (0.78 mole, 90~ yield~ of
beta-methyl-N-benzyloxycarbonyl-L-aspartate. It is
used in the next step without further purification.
The ethyl acetate extract is cooled to -20C.,
165 g. (1.63 mole) of triethylamine and 84 g. (0.78
mole) ethyl chloroformate are added. The solution is
stirred at -15 to -20C. for 30 minutes then treated
quickly with the DMF solution of silylated D-serine
prepared above and the resulting mixture is allowed to
warm to ambient temperature over one hour with stirring.
The reaction mixture is washed with water (3 x 500 ml.),
the organic layer dried over sodium sulfate and
evaporated _ vacuo to afford the title compound.
-74
When the procedure is repeated, but employing D-
O-methylserine in place of D-serine and only half the
above amount of trimethylchlorosilane, beta-methyl N-
Cbz-L-aspartyl-D~O-methylserine is obtained.
By use of DL-serine or DL-O-methylserine in the
procedures of Examples 10 and 11 the corresponding
diblocked L-aspartyl-DL--amino acid dipeptides of the
formula below are obtained.
RlOOOCCH2CHCONHCHCOOH
NHCbz CH20Rb
where Cbz is CcH2c6H5~ R is H or CH3 and R is CH3
2 6 5-
~_ 1 3 ~L ~
-75-
EXAMPLE I2
L-Aspzrtyl-D-serine
N-(trans-2-methylcyclohexyl)amide
_
A. To a solution of 228 g. (0.62 mole) of beta-
methyl-N-benzyloxycarbonyl-L-aspartyl-D-serine in one
liter of ethyl acetate is added 69 g. (0~68 mole)
triethylamine, the mixture is cooled to -20C. and
67 g. (0.62 mole) of ethyl chloroformate is added.
The resulting solution is stirred for 30 minutes
at -15 to -20C~, then treated with 76.5 g. (0.68 mole~
of trans-2-methylcyclohexylamine and stirring continued
for 30 minutes. After allowing to warm to room
temperature the mixture is washed twice with 500 ml.
portions of water containing 15 ml. of concentrated
hydrochloric acid, twice with 500 ml. of 5~ aqueous
sodium bicarbonate, then water. The organic layer is
dried (Na2SO~), concentrated in vacuo to about 200 ml.
and 400 ml. of hexane was added whereupon beta-methyl-
N-benzyloxycarbonyl-L-aspartyl-D-serine N-(trans-2-
- 20 methylcyclohexyl)amide precipitated.
. . _ ~3~J
-76-
B. ~o 230 g. (0.50 mole) of the product obtained in
Part A, above, dissolved in S00 ml. of methanol is
added a solution of 24 g. (0.60 mole) of sodium
hydro~ide in 500 ml. of water. The mixture is stirred
at 30C. for o~e hour, neutralized to about pH 7 with
dilute hydrochloric acid and charged into an autoclave.
Two grams of 5% palladium/carbon catalyst is added and
the mixture hydrogenated at 25C., 3.5 kg./cm.2
~50 psi) for one hour. The catalyst is removed by
filtration, the filtrate evaporated ln vacuo to
200 ml., the concentrate acidified to pH 5.2 with
concentrated hydrochloric acid, then granulated at
5C. for one hour. The resulting precipitate is
collected by filtration, the wet cake ~issolved in a
mixture of water and concentrated hydrochloric acid,
carbon treated, filtered and the filtrate adjusted to
pH 5.2 with 50% (w/w) sodium hydroxide solution.
After granulation at 5C., filtering, washing with
cold water and drying, the desired product is obtained.
By employing the appropriate beta-methyl~N-Cbz-L-
aspartyl-D (or DLj-amino acid dipeptide in the above
procedures the corresponding dipeptide amides of the
formula below, where R is CH2OH or CH2OCH3, are
obtained in like manner.
/ CH2--CH CONHCHCONH~
COOH NH R
2 CH3
EXAMPIE I3
L-Aspartyl-D-O-methylserine N-(dl-
t-butylcyclopropylcarbinyl)amide:
, ( 3)3~ 2 3
.. ~ . . . .
A. t-Butylcyclopropylcarbinylamine
To 0.5 mole each of cyclopropanecarbonyl chloride
and cuprous chloride in 500 ml. of dry ethyl ether was
added dropwise under a nitrogen atmosphere 238 ml.
(0.5 mole) of 2.1 M t-butylmagnesium ehloride in the
same solvent at -10C. The reactlon mixture was
poured into a mixture of 250 ml. of 3 M hydroehloric
aeid and 700 g. of iee, the organic layer separated,
washed with water, sodium biearbonate solution, brine
and dried over anhydrous magnesium sulfate. The ether
was eYaporated at reduced pressure and the residue
distilled at atmospherie pressure to provide 45 g.
(72%) of t-butyleyelopropylketone, B.P. 145-153C.
The 45 9. (0.36 mole) of ketone was reacted with
hydroxylamine hydroehloride and sodium aeetate in 1:1
ethanol/water by the method of Preparation Q. After
heating at reflux overnight the reaetion mixture was
eooled and the preeipitated oxime colleeted and
washed with cold ethanol to obtain 23.5 g. of t-
butylcyclopropylketoxime. An additional 7.7 g. was
obtained from the mother liquors. The eombined crops
were reerystallized from 1~1 ethanol/water to provide
25.2 g. (50%) of oxime, M.P. 113.5-114C.
-78-
To a solution of 5.0 g. (0.035 mole) of oxime in
80 ml. of ethanol was added 8.0~ g. (0.35 mole) of
sodium and the reaction carried out and product
isolated as described in Preparation Q, to afford
3.31 g. of crude dl-t-butylcyc]opropylcarbinylamine.
This was distilled at atmospheric pressure to yield
2.01 g. (45~) of product boiling at 153-155C.
/C(CH3)3
B. C6H5CH2OCOCH2CHCONHCHCONHCH ~
NE~Cbz C~I20CH3 V
. . .
To a 250 ml. three-necked flask fitted with a
stopper, thermometer, drying tube and magnetic stirring
bar was added 2.82 g. (6.6 mmole) of beta-benzyl-N-
benzyloxycarbonyl-L-aspartyl-D-O-me-thylserine, 50 ml.
tetrahydrofuran and 1.0 ml. (7.0 mmole) triethylamine.
The mixture was cooled to -10C., 0.69 ml. (7.0 mmole)
ethyl chloroformate was added, stirred for 20 minutes,
cooled to -35C. and 0.76 g. (0.66 mmole) of dl-t-
butylcyclopropylcarbinylamine added. The reaction
mixture was allowed to warm slowly to room temperature
and stirred overnight. The mixture was poured into
100 ml. water, extracted with 170 ml. of ethyl acetate
and the organic phase washed with 5% aqueous sodium
bicarbonate (2 x 50 ml.), 3M hydrochloric acid (2 x
50 ml.), brine (1 x 70 ml.) and dried over anhydrous
magnesium sulfate. The dried extract was evaporated
to dryness ln vacuo to provide the crude diblocked
dipeptide amide which was purified by column chromato-
graphy on silica gel.
3~
-79-
C. The purified product from Part ~, 2.33 g., was
hydrogenated over a palladium-on-carbon catalyst as
described in Example 1, Part E, to afford the desired
dipeptide amide.
T'ne compound of formula ~I) wherein R ls
/(CH3)3
CH ~ ~ and R is CH20H is similarly obtained from
the appropriate diblocked dipeptide.
-80
EXAMPLE 14
By employing the appropriate amine or formula
RNH2 i.n the procedure of the preceding Examples the
following L-aspartyl-D-amino acid amides are provided
in lik.e manner.
~CH2-CHCONHCHCONHR
COOH NH2 R
where R~ is CH2OH: R -
_ _
2,5-dimethylcyclopentyl, 2,6-diisopropylcyclohexyl,
2,5-diethylcyclopentyl, 2,2-dimethylcyclohexyl,
2,5-dilsopropylcyclopentyl, 2,2,6 trimethylcyclohexyl,
2-methyl-5-1sopropylcyclo- 2,2,6,6 tetramethylcyclo-
pentyl, hexyl,
2,2,5-trimethylcyclopentyl, l-fenchyl,
15 trans-2-ethylcyclohexyl, dl-~enchyl,
trans,t.rans-2-methyl-5- 2-methylcyclopentyl,
ethylcyclohexyl, 2-ethylcyclopentyl,
2,2,5,5-tetramethylcyclo- 2-isopropylcyclop~ntyl,
pentyl 2-t-butylcyclopentyl t
2,2,4,4-tetramethyltetra- t-butylcyclopentylcarbinyl,
hydrofuran-3-yl, diiscpropylcarbinyl,
2-methy:1-6 ethylcyclohexyl, d-methyl-t-butylcarbinyl,
2,6-diethylcyclohexyl, dl-methyl-t-butylcarbinyl,
2 isopropylcyclohexyl, di-t-butylcarbinyl,
2-t-~utylcyclohexyl, isopropyl-t-butylcarbinyl,
2-met~yl~6-t-butylcyclohexyl, methyl-isobutylcarbinyl,
dicyclobutylcarbinyl, 2,2,3,3-tetramethylcyclo-
dicyclopentylcarbinyl, propyl,
dicyclohexylcarblnyl, 2,2,4,4-tetramethylcycl butyl, .
-81-
where R. is CH2OH:
_ R _ __ R _ __
dieyclc,heptylcarbinyl, 2-methyloxetan-3~yl,
cyclobutylcyclopropyl- 2,2-dimethyloxetan-3-yl
5earbi.nyl 2-t-butyl~4-methyloxetan-3-
eyclobutylcycloheptyl- yl,
earbi.nyl 2,4-diethyl-2,4-dimethyl-
cyelopentyleyclopropyl- oxetan-3-yl,
earbi.nyl 2,4-dimethyloxetan-3-yl,
2,2,4,4-tetramethyl-3-oxo- 2,2-diethyl-4,4-dimethyl-
eyelobu-tyl oxetan-3-yl,
2,2,4,4-tetramethyl-3- 2-see-butyleyelopentyl,
hydroxyeyelobutyl, 2,2-di-n-propyleyelopentyl,
2-methyleyelobutyl, 2,4-dimethyltetrahydrofuran-
15 2,4-dimethyleyelobutyl, 3-yl,
2,2-dimethyl-4-ethylcyelo- 2-methyltetrahydrofuran-3-yl,
butyl, 2-t-butyltetrahydrofuran-3-
2,2,4,~l-tetramethyleyelo- yl,
buty:L 2-ethyl-4-n-butyltetrahydro-
2,2-diethyl-4~4-dimethyl- furan-3-yl,
eyelobutyl, 2-n-butyl-4-ethyltetrahydro-
2,4 di:isopropyleyelobutyl, Euran-3-yl,
2-t-bul:yleyelobutyl, 3,5-dimethyltetrahydropyran-
2-methyleyeloheptyl, 4-yl,
25 2-isopropyleycloheptyl, 3,5-diisopropyltetrahydro-
2-t-bu~:yleyeloheptyl, pyra-n-4-yl,
2,7-dirnethyleyeloheptyl, 3,3,5,5-tetramethyltetra-
2,7-diisopropyleyeloheptyl, hydropyran-4-yl
3-t-butyl-5-rnethyltetrahydro- 2,2,4,4-tetrahydropyran-3-yl,
pyran-4-yl 4,4-dimethyltetrahydropyran-
2-methyltetrahydropyran-3-yl, 3-yl,
_ _
-82-
where Ra is CH20H: ¦ ~
4-methyltetrahydropyran-3-yl, 2,2,5,5-tetramethyl-3-cyclo-
4-sec-bu1:ylte~rahydropyran- pentenyl,
3-yl, 2,5-dimethyl-3-cyclopentenyl,
2-isopropyltetrahydropyran- 2-methyl-3-cyclopentenyl,
3-yl, 2,5-diisopropyl-3-cyclo-
2,4-diisopropyltetrahydro- pentenyl,
pyran-3-yl, 4-methyloxepane-3-yl,
2-methyloxepane 3-yl, 2,2,4,4-tetramethyloxepane-
2,4-dimethyloxepane-3-yl, 3-yl,
2,4-diisopropyloxepane-3-yl, 2,2-dimethyloxepane-3-yl,
3-methyloxepane-4-yl, 5,5-dimethyloxepane-4-yl,
3,3-dimeth.yloxepane-4-yl, 3-isopropyloxepane-4-yl,
3,5-diisopropyloxepane-4-yl, 2,4-dimethyltetrahydropyran-
5-isopropyloxepane-4-yl, 3-yl,
2-isopropylcyclopropyl, 2-t-butylcyclopropyl,
2,2-dimethylcyclopropyl, ethylcyclopropylcarbinyl,
lsopropylcyclopropylcarbinyl
-83-
where Ra is CH OCH :
_ 2 - 3-
R
2,2-dimethyl-5-t-butylcyclopentyl,
2-isobutylcyclohexyl,
2-n-butyl-6-ethylcyclohexyl,
2,2-diethylcyclohexyl,
2-t-buty~-6-methylcyclohexyl,
2,4-diethyl-2,4-dimethyltetxahydrofuran-3-yl,
2,4-dimethyltetrahydrofuran-3-yl,
2,2,4,4-tetramethyltetrahydrofuran~3-yl,
3,5-dimethyltetrahydropyran-4-yl,
3,3,5,5-tetramethyltetrahydropyran-4-yl,
2,2,4,4-tetramethyltetrahydropyran 3-yl,
4,4-dimethyltetrahydropyran-3-yl,
2,2-dimethyltetrahydropyran-3-yl,
3,3,5,5~tetramethyloxepane~4-yl,
2,3-diisopropylcyclopropyl,
- 2-t-butylcyclopropyl,
isopropylcyclopropylcarbinyl,
d-methyl-t-butylcarbinyl,
diisopropylcarbinyl,
di-t-butylcarbinyl,
l-fenchyl,
2,2,5-trimethyl-3-cyclopentenyl.
-84-
R R R
~ CH2)2 ~ CH3 ~ C2H5)3
CH3 CH3
CH3 ,CH3
(COH ) ~ ~C (CH2)3cH3
3 C~3
CH2CH3
~CH~CH(CH3)2]2~CH~C(C~3)3/CH(CH3)2
'b b ~
(C~3)2
f -C(CH3)3 ~ ( 2 3)3~ C 2 3
b \a ~CH2,3
( 2C 3)2 /C 2CH3 C(CH3)3
b ~CH2,3
~ (CH3)3~ CH(CH3)2 ~ C (C 2CH3)2
b ~ 2)3
CH2CH2CH3 Fl
<~ 12 ( CH2 ) 2CH3 _<~H ( CH3 ) 2 <b
3~
--85--
R R
CH ( CH3 ) 2 CH2CH3 ,a
b _<~CH2)3CH3b
CH3 o
~CH(CH2)3CH3 ~C(CH3)3
b `O b
_~CH ( CH2CH2CH3 ) 2 </( CE~2 ) 4C 3 ~ CH2 ) 3
`O b
5 ~ ~0
CH3
c~o3,2 -~b ~3)2
CH(CH3)2
~0 C~ C~33
CH3 CH3
_ ~3 ) 3 C~3 C:2 35
-86~
EXAMPLE 15
L Aspartyl-D-O-methylserine N-(3~5-
dimethyltetrahydrothiopyran-4 yl)amide:
C~
I, R = ~ , Ra = CH2OCH3
C~3
mixture of cis/trans and trans/trans isomers
_
A. 3,5-Dimethyltetrahydrothiopyran-4-one
A mixture of 2 g. of sodium acetate and 25 ml. of
ethanol was saturated with hydrogen sulfide gas. To
this was added 7.0 g. (0.063 mole) diisopropenylketone
while cooling in an ice bath until the reaction was no
longer exothermic. The mixture was stirred at room
temperature while passing hydrogen sulfide through the
mixture for four hours then allowed to stand overnight.
The ethanol and excess H2S were evaporated in vacuo
and the residue taken up in ethyl ether, washed in
turn with water, potassium carbonate solution, dilute
hydrochloric acid, and water again. The ether extracts
were dried (Na2SO4) and evaporated to provide 6.8 g.
of oil. This was distilled in vacuo through a 10 cm.
Vigreaux column to provide 1.67 g. of product, B.P.
83-86C./9 mm. which was used in the next step without
further purification.
-87-
3. 4-Oximino 3!~ dlmet yltetrahydrothiop~ran
A mixture o~ 1.67 g. (0.011 mole) of the cyclic
kel:one obtained in Part ~, 1.6 g. (0.023 mole) hydroxyl-
amine hydrochloride and 1.9 g. ~0.023 mole) sodium
acetate in 30 ml. of water and 10 ml. of ethanol were
heated at reflux for three hours, cooled and the
precipitate recovered by filtration. After recrystal-
lization from 1:1 methanol-water 1.5 g. of oxime was
obtained as a white solid, M.P. 60-85C. which is a
mixture of isomers of suitable purity for use in the
next step.
C. trans/trans and cis/trans-4-Amino-3,5-dimethyl-
tetrahydrothiopyran
To a solution of 1.45 g. (0.009 mole) of the
oxime obtained in Part B in 15 ml. of ethanol wasadded in portions 5 g. of sodium shot followed by an
add:itional 25 ml. of ethanol and the resulting mixture
heated at reflux for about 30 minutes. The reaction
mixture was diluted with water, extracted with ethyl
ether, and the extracts washed with water. The ether
layer was extracted with dilute hydrochloric acid and
the aqueous layer washed with fresh ether. The
a~ueous layer was made alkaline by addition of sodium
hydroxide solution and extracted with ether again.
The organic layer was dried (MgSO4) and the ether
evaporated to obtain 1.1 g. of residual colorless oil.
Gas--liquid chromatography (OV-l column with temperature
programming from 30 to lOO~C.) showed the product to
contain two major components in a 60/40 ratio. lH-NMR
(CDC13) indicated the product to be a mixture of 4-
amino-3-trans-5-trans-dimethyltetrahyarothiopyran and
. .
the corresponding 3-cis-5-trans-isomer.
--g8--
D. N(3~5-Dimetllyltetrahydrothiopyran 4 yl) t-
butoxycarbonyl-D-O-methylserine amide
C~
t-Boc-NH-CHCONH ~ ~
CH20CH3F
H3
_ _ _ _ _ _ .
Vnder anhydrous conditions, to a mixture of
1.96 g. (8.9 mmole) of N-t-Boc-D-O-methylserine
obtained in Example 7, Part A, 1.98 g. (19 mmole)
triethylamine and 40 ml. of tetrahydrofuran, cooled
to -10C., is added dropwise 0.96 g. (8.9 mmole) ethyl
chloroformate and the resulting mixture stirred at
this temperature for 20 minutes. To this is added
1.1 g. (7.5 mmole) of the mix-ture of lsomers of 4-
amlno-3,5-dimethyltetrahydrothiopyran obtained in
Paet C and the resulting mix-ture stirred at ~lO~C.
for 10 minutes then allowed to warm to room temperature.
The reaction mixture is diluted with water and extracted
with ethyl acetate. The organic layer is washed with
sodium bicarbonate solution, dilute hydrochloric acid,
water, brine then dried (Na2SO4) and the solvent
evaporated at reduced pressure to obtain the product.
~
-89-
E. N-(3,5-Dimethylketrahydrothiopyran-4-yl)-D-
O-methylserine amidec~
NH 2 CHCONH~S
CH20CH3 /
CH3
The t-Boc-amide obtained in Part D is dissolved
in 15 ml. of ethanol and a mixture of 5 ml. of concen-
trated hydrochloric acid and 10 mlO o water are added.
The resulting mixture is heated at reflux for 30
minutes, cooled and the ethanol removed by evaporation
in vacuo. The aqueous residue is washed with ethyl
ether, made alkaline with sodium hydroxide solution,
extracted with ether and the extracts dried (Na2SO4).
Evaporation of solvent provides the desired amino amide.
F. Coupling of D-O-methylserine amide with L-aspartic
acld N-thiocarboxyanhydride
The D-O-methylserine amide provided in Part E,
1.25 g. (5.1 mmole) is dissolved in 5 ml. of tetra-
hydrofuran and 5 ml. of water was added. The clear
solution is cooled in ice and 0.89 g. (5.1 mmole) of
L-aspartic acid N-thiocarboxyanhydride is added in one
portion. To this is added as required, 0.5 M sodium
hydroxide to maintain the mixture at pH 9 . After
stirring 30 minutes the reaction mixture is washed
with ethyl ether then ethyl acetate and the washes
discarded. The aqueous phase is acidified with dilute
hydrochloric acid to pH 5.6 and evaporated to dryness
at reduced pressure. The residue is taken up in hot
methanol (100 ml.), filtered and the methanol evaporated.
The residue was taken up a~ain in hot methanol, filtered
and the filtrate decolori~ed with activated carbon,
3~3
90-
filtered through diatomaceous earth and the filtrate
evaporated to obtain the crude product. The crude
product is dissolved in hot water (11 ml.) and filtered,
evaporated under a stream of nitrogen to 5 ml. and
cooled in ice to precioitate the product which is
collected by filtration and dried.
~ se of t-Boc-D-serine, t-Boc-DL-serine or t-Boc-
DL-O-methylserine in place of t-Boc-D-O-methyls2rine
in the procedure of Part D, above, and reacting the
resulting N-t-Boc-D ~or DL)-amino acid in the procedures
of Parts D, E and F, provides the corresponding compounds
of formula (I) wherein R is 3,5-dimethyltetrahydrothio-
pyran-4-yl and Ra is CH20~ or C~20C~3.
EXAMPLE 16
L-Aspartyl-D-serine N-(2,2,4,4-
tetramethyltetrahydrothiophene-3-yl)a~ide:
3 ~ 3
I, R - ~ ~ , Ra = C~2OH
CH3 CH3
... . ~
3 ~ 3
A. t Boc-NHCH-CONH ~ ~
3 C~3
To a solution of 2.26 g~ ~11 mmole) of N-t-
butoxycarbonyl-D-serine in 75 ml. of tetrahydrofuran
is added 1.47 ml. (10 mmole) of triethylamine and the
mixture cooled to -10C. At this temperature is added
0.96 ml. (10 mmole) of ethyl chloroformate and stirring
continued for 15 minutes. After cooling to ~20C.,
1.6 g. (10 mmole) of dl-3-amino-2,2,4,4-tetramethyl-
tetrahydrothiophene is added and the resulting mixture
is allowed to warm to room temperature. ~thyl acetate
lS is added and the mixture is washed twice with 50 ml.
portions of 5% ~by weight) aqueous citric acid,
aqueous sodium bicarbonate (1 x 50 ml.) and saturated
brine (1 x 50 ml.). The organic layer is dried
(Na2SO4) and evaporated to dryness at reduced pressure
to afford N(2,2,4,4-tetramethyltetrahydrothiophene-3-
yl)-t butoxycarbonyl-D-serine amide. This product is
used without further purification in the next step.
-92-
~ 3
B. HocH2~H(~H2)cONH ~ 1
>~S
CH3 CH3
. _
To 3 g. of the product from Part A i5 added 5 ml.
of methanol and 30 ml. of lM hydrochloric acid and the
rnixture is heated on the steam-bath for 30 minutes.
S The methanol is removed by evaporation and the xesidue
extracted with ether. The ether is discarded and the
aqueous phase is adjusted to pH 11~0 with sodium
hydroxide solution, extracted with ethyl acetate, the
extracts dried (Na2SO4) and evaporated to dryness to
11~ obtain D-serine N(2,2,4,4-tetramethyltetrahydrothio-
phene-3-yl)-amide.
C. Coupllng to form dipeptide amide
The D-~serine amide obtained in Part B, 1.03 g.
(4.25 mmole) is mixed with 10 ml. of water, cooled in
l!; ice and the pH of the mixture adjusted to 9.2 with
0.5 N sodium hydroxide solution. To this is added
portionwise with stirring 0.8 g. (4.25 mmole) of L-
aspartic acid N-thiocarboxyanhydride while maintaining
the mixture at pH 9 with sodium hydroxide solution
(0.5 N). When the addition is completed the resulting
mixture is stirred at O~C. for 45 minutes, adjusted to
pH 5.2 wit:h hydrochloric acid and evaporated to
dryness ln vacuo. The residue is slurried with
methanol, filtered to remove precipitated solids and
2S methanol :removed from the filtrate by evaporation at
reduced pressure. The resulting crude product is
purified by column chromatography on silica gel.
-93-
EXAMPLE 17
Employing the procedures of Examples 3, 7, 8,
15 and 16, corresponding L-aspartyl-D-amino acid
amides (I) wherein Ra is CH20H or CH20CH3 and R is as
defined below are prepared from the appropriate
starting rnaterials via D-RaCH(NH2)CONHR intermediates.
The corresponding L-aspartyl-DL-amino acid amides are
similarly provided when a t-Boc-DL-amino acid is
employed in place of the D-enantiomer. Likewise, use
of DL-aspartic N-thiocarboxyanhydride in the coupling
step affords the DL-D or DL-DL compounds of formula (I).
HOOCCH CHCONHCHCONHR -~
2NH2 Ra where R = CH20H or
CH20CH3
R R R
- ~3 ~ CH3)3 ~ ~O
C~13 CH2CH3
CH3 C(CH3)3 C2H ~ CH3
~ S ~ S ~ 2
C(CH3)3 C2 5 C 3
CH3 ~CH2;3C3; ~ S
(CH3)2 3~ C 3 C ~ 3
CH ( CH3 ) 2 ~5CH3 ) 2 ~S
CH(CH3)2 CH(CH3)2
-
~94--
R R
CH~I C ~ CH3 ) 3 -~52
CH3 CEI3 CH3
C~ ~ 3 ,~
_~ ~S ~ ,SO
`r ( CH2 ) 2 ,>~( CH2 ) 2
CH~ l H3
S (CH2)2 S
CH3 C 3 3
_~CH3 --~ 2 CH~H3
H2)2 ~
CE~2C~H3 CH3 CH2CH3
CH(t H3)2 CH3 CH3 C 3
~ CH2 ) 3 >~
CH ( CH3 ) 2 3 3
t~H~103 ~SCH2 ) 3CH~
3 3 3
C;~CH3 ~5 ~
CH3 3 CH2CH3 C 3 3
-95~
R R R
C ~ CH2 ~ CH3)2 ~ 2 3
3 3 CH2)3
CH(CH3)2
3)2 C ~ CK3
3 3
CH ~ H3 ~ ~ oCH3
3 3 CH2CH3
CH(CH3)2 CH ~ CH3
~ CH(CH3)2 C ~ CH3
CH ~ I3 C ~ CH3 ~ 3)3
3 3 C(~H3)3
CH(CH3)2/C~CH3)3 CH(CH3)2
~0 ~ ~0
~7 1
V CH(CH3~2
3)2 CH3C ~ H2CH3 ~ 2
CH3
~S02'
CH3 CH3
35~
-96-
EXAMPLE 18
L~Aspartyl-D-serine N-(2-methylthio-
_ 2,4-dimethylpentan-3-yI)amide
A. 2-Methylt_io-2,4-dlmethylpentan-3-one
A solution of 200 ml. of methanol containing 9.2
g. (0.40 mole) sodium metal was cooled in an ice-bath
and saturated with gaseous methyl mercaptan. To this
was added 77.2 g. (0.40 mole) of 2-bromo-2,4-dimethyl-
pentan-:3-one at room temperature and the resulting
mixture stirred for two hours. The reaction mixture
was diluted with water, extracted with ethyl ether,
the extracts washed with water, brine and dried over
anhydrous sodium sulfate. The ether was evaporated
and the residue distilled i vacuo to afford 50.4 g.
of prodllct, B.P. 76 (20 mm.).
B. 2-Methy~thio~2,4-dimethyl-3-aminopentane
A e;olution of 6.0 g. (0.038 mole) 2-methylthio
2,4-dime!thylpentan-3-one, 9.9 g. formamide and 2.1 g.
of 100% formic acid was heated at reflux while removing
water formed in the reaction by means of a fraction-
ating heacl. After 12 hours an additional 2.5 g. of
formic acid was added and reflux continued for another
24 hours in the same manner, by which time the reaction
mixture reached a temperature of 190C. The mixture
was cooled, diluted with water and extracted with
ethyl acetate. The extracts were washed with water and
evaporated to dryness at reduced pressure to provide
- 5.3 g. of residual oil. The oil was refluxed with 40
ml. o 6N hydrochloric acid for six hours, diluted
with water, washed with ether and the aqueous phase
3~
-97-
made strongly alkaline with sodium hydroxide. After
extracting with ethyl ether and evaporation of the
extract, 3.3 g. (56%) of colorless amine was obtained
which gave a single peak by gas-liquid chromatography
on a six foot OV-l column at 110C.; retention time
412 seconds.
C. 3-Serine N-(2 methylthio-2,4-dimethylpentan-
3-yl)amide
To a solution of 3.47 g. (0.017 mole) N-t-
butoxycarbon~yl~D-serine and 2.5 g. (0.017 mole)
triethylamine in 100 ml. of tetrahydrofuran at -15C.
is added 1.63 ml. of ethyl chloroformate. After
stirring fox 15 minutes, 2.49 g. (0.017 mole) 2-
methylthio-2,,4-dimethyl-3-aminopentane is added and
the mixture stirred for one hour. The reaction mixture
is diluted with ethyl acetate, washed with water, 5%
aqueous citric acid (w/v), sodium bicarbonate solution
and brine. The organic phase is evaporated to dxyness.
The residue is taken up in 100 ml. methanol~ 60 ml. of
concentrated hydrochloric acid added and the mixture
refluxed for one hour. After evaporation of methanol,
the residue is taken up in water, washed with ether,
the aqueous phase adjusted to pH 12 with sodium
hydroxide and extracted with ethyl ether. Evaporation
of the extracts affords the desired D-serine amide.
839
~98-
D. A solution of 3.3 g. (0.013 mole) of the D-serine
amlde, obt.ained in Part C, in 30 ml. acetone and 17
ml. water is adjusted to pH 9.9 with sodium hydroxide
solution a.nd cooled to -2C. To this is added 2.78 g.
(0.013 mole) L-aspartic N-thiocarboxyanhydride in
small portions over 20 minutes while maintaining the
p~I at 9.9 with lN sodium hydroxide. When the addition
is compl~ted, the resulting mixture is stirred for 30
minutes at -2C., washed with ethyl acetate acidified
to pH 2 with hydrochloric acid and washed again with
ethyl acet.ate. The aqueous phase is then adjusted to
pH 5.2 and. evaporated to dryness. The crude dipeptide
amide is obtainea by slurrying the residue in methanol,
filtering, treatment of the filtrate with ether and
filtering to obtain a second crop.
The c:rude product is purified by preparative
layer chromatography on silica gel plates (20 x 20 x 2
mm.) eluting with butanol/water/acetic acid, 4:1:1 by
volume. The product zone was cut out and eluted with
methanol l:o give the purified L-aspartyl-D-serine amide.
When N-t-butoxycarbonyl-D-O-methylserine is
employed :in the procedure of Part C, above, in place
of the N~t-Boc-D~serine used therein, and the resulting
product traated by the procedure of Part D, above, the
corresponding L-aspartyl-D-O-methylserine amide is
obtained.
-
- 99 -
EXAMPLE_19
L-Aspartyl-D-serine N-(2-
hydroxy-2,4-dlmethyl-3-pentyl)amide,
/CH(CH3)2
I, Ra = CH2OH, R = ~
\C(CH3)2
OH
A. ~ oxy~2,4-dim ~ r~ o~
To a s1:irred solution of 28.3 ml. (0.2 mole) 2,4-
dimethyl-3-pentanone in 100 ml. chloroform was added
dropwise 10 3 ml. (0.2 mole) bromine in 30 ml. of the
same solvent. The resulting mixture was stirred for
a few minutes, the sol~ent evaporated in acuo, the
residue taken up in 100 ml. ethanol. Water, 50 ml.,
and 10 M sodium hydroxide, 50 ml., added. The resulting
mixture was stirred at reflux for one hour, diluted
with 200 ml. water and extracted with 3 x 50 ml. ethyl
ether. The extracts were dried (MgSO4) evaporated to
dryness and the residue distilled to obtain 15.95 g.
(61%) of the hydroxy-ketone, b.p. 60-62C.jl8 mm.
B. 3-Amino-2~hydroxy-2,4-dimethylpentane
The hydroxy ketone from Part A, 15 g. (0.115 mole~
was reduced in refluxing mixture of formamide and
formic acid by the method of Example 13, Part D, to
obtain 4.5 g. (30~) of the hydroxy amine, b.p. 80-81C./
17 mm.
--100--
C. _blocked dipeptide amide
To a solution of 2.22 g. (5.0 mmole) beta-
benzyl-N-benzyloxycarbonyl-L-aspartyl-D-serine in
35 ml. tetrahydrofuran cooled to -15C. is added
5 0.55 ml. (5.0 mmole) N-methylmorpholine and 0.48 ml.
(5.0 mmole) ethyl chloroformate. The mixture is
stirred at -15 to -10C. for two minutes and 0.66 g.
(5.Q mmole) 3-amino-2-hydroxy-2,4-dimethylpentane is
added. The mixture is allowed to warm to room tempera-
lO ture, stirred overnight and worked-up as described in
Example 13, Part B, to obtain the diprotected dipeptide
amide which is used directly in the next step.
D. The product from Part C, above, in 250 ml.
methanol is hydrogenated over l.0 y. 5% Pd/C at
15 60 psi (17 kg./cm.2) for two hours. The catalyst is
removed by filtration and the solvent evaporated ln
vacuo. The residue is dissolved in methanol and ethyl
ether is slowly added with stirring to precipitate the
title compound which is collected by filtration, and
dried in vacuo.
._
~ ~ \
3~
--101--
EXAMPLE 20
L-Aspar1:yl-D-O-methylserine N-(DL-2-amino-3,3-
dlmethy:L-4-hydroxybutanoic acid lactone)amide,
I, Ra = CH2OCH3, R =
CH3 CH3
A. DL-2-Amino-3,3-dimethyl-4-hydrogybutyric acid
lactone hydrochloride
Prepared by the method of Wieland, Chem. Ber.,
8l, 323 (1948)-
2-Keto-3 r 3-dimethyl-4-hydroxybutyric acid lactone,
3.5 g. was neutralized with dilute sodium hydroxide
and the aqueous solution evaporated ~o dryness in
vacuo. The residue was taken up in lO0 ml. warm
ethanol, filtered hot and a solution of 700 mg. sodium
metal in lO ml~ ethanol containing 2 g. hydroxylamine
hydrochloride was added. The sodium salt of 3,3-
dimethyl-4-hydroxy-2-oximinobutyric acid lactone, 5 g.
precipitated and ~as recrystallized from methanol.
The oxime wa; formed by decomposition of the sodium
salt in 2 N 'hydrochloric acid, from which it slowly
crystallized. After recrystallization from benzene-
hexane, M.P. l60C.
A solut:ion of 25 g. of the oxime in lO0 ml.
ethanol was added in portions to 5 g. platinum oxide
suspended in 150 ml. 2 N hydrochloric acid and the
mix~ure hydrogenated at atmospheric pressure for
2 days. Th.e catalyst was filtered off, the filtrate
evaporated and the residue taken up in 150 ml. ethanol.
3~
-102-
Treatment with 500 ml. ethyl ether precipitated DL-2-
amino-3,3-~imethyl-4-hydroxybutyric acid lactone
hydrochloride, 22 g., which was recrystallized from
ethanol/ether, M..P. 208-212C.
B. Dibloccked dipeptide amide
The aminolactone hydrochloride from Part A,
1.65 g. (0.010 mole) in 10 ml. methylene chloride and
an equimola.r amount of triethylamine is employed in
the procedure of Example 13, Part B, to provide
C6H5cH2ococE[2cH-coNHcH(cH2ocH3)coNH
NHC02CH2C6H5 C E~3
C. The product from Part B, above, (2.5 g.) is
dissolved in 200 ml, methanol, 0.2 g. of 5~ Pd/C
catalyst added, the mixture hydrogenated and the
product isolated as described in Example 13, Part C,
to afford the! desired dipeptide amide.
--103--
EXAMPLE 2 I
L-Aspartyl-D-serine N- ( 2, 2, 4, 4-
tetr~methyl -3 -hydroxycyclobutyl)amide,
~CE~3)2
I, Ra = CH2OH, R = ~OH
... . ... .,,, ,,, ,, ~CH3)2
..... .... .. . .. . .. . .
(~CH3 ) 2
A. D-HOCH2CHCONH~OH
-NHCbz (CH8~2 -
N-Benzyloxycarbonyl-D-serine (0.1 mole), is
reacted with cis/trans 2,2,4,4~tetramethyl-3-hydroxy-
cyclobutylamine by the method of Example 8, Part A, to
provide the N-Cbz-serine amide.
B. Hydrogenation of N-Cbz-serine amide by the
method of æxample 8, Part C provides the corresponding
2-amino compo~md, D-serine N (2,2,4,4-tetramethyl-3-
hydroxycyclobutyl)amide. The latter compound is
converted to the title compound by the procedures of
Example 8, Parts D and E.
The corresponding L-aspartyl-D-O-methylserine
amide is obtained in like manner.
-104-
EXAMPLE 22
L-Aspartyl-D-serine N-(2,2,4,4-
tetramethyl-3-oxocyclobutyl~amide
~CH3)2
`I, Ra = CH2OH, R = ~
~ CH3 ) 2
~H3)2
Ao D-HOCH2CHCONH ~
N~IC~z (CH3)2
N-Benzyloxycarbonyl-D-serine N-(2,2,4,4-tetra-
methyl-3-hydroxycyclobu~yl)amide prepared in Example 21,
Part A, 36.4 g., ~0.10 mole) dissolved in 1500 ml.
acetone is cooled to -10C. under a dry nitrogen
atmosphere and 42 ml. (0.11 mole) 2.67 M chromic
anhydride in di.lu~ed sulfuric acid is added. After
stirring for 15 mi.nutes at 10C.I the solvent is
evaporated in v.acuo, the residue poured into an ice-
_,
water mixture, neutralized with sodium hydroxide
solution and ext:racted with ethyl ether. The ether
extracts are dri.ed IMgSO~) and evaporated to dryness
to obtain the crude product which may be purified, if
desired, by colu;mn chromatography on silica gel.
B. Hydrogenation of the product of Part A, above, by
the method of Example 8, Par~ C provides D-serine N-
(2,2,4,4~tetramet:hyl~3-oxocyclobutyl)amide. This is,
in turn, converted to the title compound by the
methods described in Example 8, Parts D and E~
~6~ ~ 3
-105-
EXAMPLE 23
Employing the appropriate amine of formula RNH2
in the above procedures the compounds of formula ~I)
below, where R ic. CH2OH or CH2OCH3, are similarly prepared
NH2
2 ~
HOOC CONHCHCONHR ( I )
R R
(CH3)2
~OH ~OH
3)2 (CH3)2
~3)2 ~ 3
~0 ~ (CX2)4
~ CEI3 ) 2
CEI~HH3 _f~CH5 ) 2
>~H H H
CH3 3 2 5
CH3~ OH ~ )
~ ~ ~ I
>~/ HO~C2H5
CH 3 OH
H>~:~C2H5 n C3~7
~JH ~
H 2 5 HO n-C3E~7
~.
3~
--106--
R R
E.10 n-C~Hg (~C2H5 ) 2
C~>CE $~C52 ) 4
HO~E13 ~H3
~ ~0
n-C3H7
~E.13 ~3
_C_OH rl
~0
~OH C~H5
~0~ ~)
C2~5 2 5
_$rOH ~2
2H5 CH3)2
(CH3)2 ~
~OH n~C3H7
( H3)2
3~t
-107
R
~-C3H7 3
~-C3037 ~
_~OH n C4 9
\CrH3cH2 ) 2 ~CH2 ) 2
n-C4Hg c4 9
~COH2 ) 2 -~CH2 ) 2
CH3 CH3
n-C4Hg HO CH3
~(CH2~2 ~
~30H n-C4Hg
~3 ) 2 HO~CH3
~t) ~
2H5 ) 2
(J~3 ) 2 HO C2H5
~:NH ` C2H5
~5 ) 2 H~)H3
3~
--10~--
R R
HO~ E~ 5
O n-C4H9
n~C3H7 ~ 3
~H ~CH2)4
O CH3
Hg n C3H7
OH
C3H7
_~) 2 f~H~I3 ) 2
H3 ) 2
(~5 ) 2 ~H3
~OH
0/ C~13
~OH2
~ ~OH
o~ (CH3)2
~Hg foHH2)3cH3
~;IH ~OH
C2H5
3~
--109--
R R
HO C:H3 CH3
~CH3
HO CH3
HO l-C,~lHg C2H5
2~> _~LOH
/\ ~n-C H
HO CH3
HH g
H2CH3
HO `n-C3H7
HO~,CH3 _~n-C4Hg
S ~ ~OH
HO `CH C2H5
--3~X2 ) ~ CH~3H33
HO~C 3 _~3 7
~10 CH3 3x7
ro3 ) 2
HOki-C3H7 C 3
3~
--110--
R R
$~ ~OH
HO~C4H9 ~3CH3
i-C4Hg n C3H7
r OH _~OH
\rOH ~ 3 ) 3
( CH3 ) 2
C2H5 C2H5
~~ ~ OH
5)2 (C 3)3
C~H 5 _~ ) 2
2H5 ( CH3 )
C2~5
_~H ~O
CH3)3 CH3)2
n-C4~9 ~ ) 2
~)H ~O
CH3)2 (C2H5)2
~L9~ 3~
R R
C2H5 ~Ho3 ) 2
_~LOH ~C=O
~CH3 (CH3)2
c2~s
CH3 CH3
_¢~co ~C3P
CH3
C~C 2 H 5 _~
H
CH3 C21~I5 CH3
~3 2 5 C~
~2 5 ( CEI3 ) 2
_~3 7 ~Lo3 ) 2
~3H7 C~3
c~3 C3 7
-~FO
i-C3EI7 3 7
--1 12~
R R
~0 4 9
~ ~
CH~C H C 2 H 5
~LH~H) 2 ~o4 9
o
3)2 ~CH3)2
3)2 (C 3)2
<LN~O ~
~H ( C2H5 ) 2
C2~5 ( C2H5 ) 2
~0 ~0
~C2H5)2 CH3)2
n-C4Hg CH3 C2~I5
0
H3 CH3 C2H5
~H3 n C3 7
_~H ~0
~3 n-C3H7
3ffl
--1 13--
R R
3)2 (CH3)2
~-0 {~
( CE3 ) 2 c~3
(CH3) 2 (CE~3) 2
~2 ~(C(~=5)2
C~5 _~) 2
CH3 C2H5 ~ CH3 ) 2
CH3
~ ~9
I CH3 ) 2 n~4H9
$~ ~H
2 S (C 3)2
3~
--114--
R R
( CH3 ) 2 CH3 C2H5
~ ~0
~ ,k
( CH3 ) 2 CH C H
i-C4Hg . .
~H
F2~
3~
-115-
EXAMPLE 24
Carbonated CoIa Beverage
A carbonated cola beverage was prepared according
to the composition given below. The resulting beverage
5 was judged to have sweetness intensity comparable to
a control beverage containing 11% sucrose.
In~redient ~, weiqht
Caffeine (1% aqueous solution) 0.700
L-Aspartyl-D-serine N-(cis,t _ -2,6-
dimethylcyclohexyl)amide (10~ aqueous) 0.540
Cola flavor concentrate 0.080
Phosphoric acid (50% aqueous) 0~040
Citric acid (50% aqueous) 0.066
Sodium citrate (25% aqueous) 0.210
Caramel color (25% aqueous) 0.370
Lemon oil extract 0.012
Lime oil extract 0.021
Carbonated water (3.5 volumes carbon dioxide) 9 ~ _
100.000
Replacement of the L-aspartyl-D-serine N-(cis,
trans-2,6-dimethylcyclohexyl)amide in the above
formulation with 0.090% of 10% aqueous L-a~partyl-D-
sexine acid N-(dicyclopropylcarbinyl)amide or 1.35% of
10% aqueous L-aspartyl-D-O-methylserine N-(dicyclo-
propylcarbinyl)amide affords carbonated cola beverages
of like qualityO
-116-
EXAMPLE 25
Dietetic Hard Cand~
A hard candy is prepared according to the rollowing
i.ormulation and procedure:
]n~redi nts Grams
I,-Aspartyl-D-serine N-(dicyclopropylcarbinyl)-
amide 0-59
Water ~.oo
FD and C Red ~40 (10~ aqueous) 0.30
Cherry flavor 0.6Q
Citric acid 6.00
Polydextrose* 420.00
Water 180.00
In a small beaker dissolve the sweetener in
15 water, add color, flavor and citric acid and mix well
to dissolve. In a separate beaker combine polydextrose
cmd water. Stir while heating to 140C. then allow
t:o cool to 120-125C. Add other ingredients from
small beaker and mix or knead thoroughly. Transfer
mass to an oil coated marble slab and allow to cool
1:o 75-80C. Extract the mas~ through an oil coated
:Lmpression roller.
Use of 0.49 gO of L-aspartyl-D-serine N-(2,2,4,4-
tetramethyl-l,1-dioxothietan-3-yl)amide or 2.33 g. of
L-aspartyl-D-serine N~(2,2,4,4-tetramethyl~3-pentyl)amide
as sweetening agent in place of L-aspartyl-D-serine
N-(dicyclopropylcarbinyl)amide affords similar results.
*U.S. 3,766,165
3 ~"
-117~
EXAMPLE 26
A gelatin dessert is prepared according to the
following composition and procedure.
Ingredients Grams
__
Gelatin 225 Bloom 7.522
Citric acid 1.848
Sodium citrate 1.296
Strawberry flavor 0.298
L~Aspartyl-D-serine N-(2,2,4,4-tetramethyl-
thietan-3-yl)amide 0.036
Boiling water 240.000
Cold water 240.000
491.000
Premix the first five ingredients, add to boiling
water and stir to dissolve completely. Add cold
water and stir briskly. Transfer to serving dishes
and refrigerate until set.
.83~
-118
EXAMPLE 27
_
Low calorie table sweeteners are prepared according
to the following formulations:
A. A powder form of sweetener is prepared by blending
the following ingredients.
In~redients ~L_wei~ht
L-Aspartyl-D-serine N-(2,2,4,4-tetramethyl-
1,1-dioxothietan-3-yl)amide 0042
Crystalline sorbitol 49.52
10 Dextrin (dextrose equivalent 10) 50.00
Monosodium glutamate 0.02
Glucono-delta-lactone 0.02
Sodium citrate 0.02
100.00
15 One gra~ of the resulting blend is equivalent in
sweetness to about three grams of sucrose.
B. A table sweetener in liquid form is prepared as
follows.
Ingredients ~, weight
20 L-Aspartyl-D-serine N-(dicyclopropylcarbinyl)-
amide 0.17
Water gg.73
Sodium benzoate 0.10
100.00
25 One gram of the resulting solution is equivalent
in sweetness to about 1.2 grams of crystalline sucrose.
When the sweetener of formula (I) employed in
Part A, above, is 0.83 g~ of a 1:4 mixture of L-
aspartyl-D-serine N~2,2,4,4-tetramethylthietan-3-yl)-
amide and sodium saccharin comparable results are
obtained. Similarly when the L-aspartyl-D-serine N-
(dicyclopropylcarbinyl)amide employed in Part B,
above, is replaced by 0.34 g. of a 1:6 by weight
mixture of the same compound and sodium saccharin a
comparable liquid table sweetener is obtained.
33~
-119-
EXAMPI.E 28
. ~ _
Frozen Dessert
.~ __ . .
A vanilla sugarless fro~en dessert is prepared
according to the following formulation by conventional
practice.
In~redients %, weight
Heavy ,-ream (35% butterfat) 23.00
Nonfat milk solids 10.50
Mono and diglyceride emulsifier0.25
Polydextrose* 11.20
Water 54.49
L-Aspartyl-D-O-methylserine N-~2,2,4,4-
tetramethyl-l t l-dioxothietan-3-yl)amide 0.06
Gelatin (225 Bloom) 0.50
100.00
*U.S. 3,766,165
3~
-120-
EXAMPLE 29
Canned Pears
-- .
Fresh pears are washed, peeled, cored, sliced
into pieces and immersed in an aqueous solution
containi.ng 0.05~ by weight of ascorbic acid. The
sliced fruit is packed into screw-cap jars and the
jars filled with a syrup containi~g the following
ingredients:
Sorbitol 25.000
L-Aspartyl-D~serine N-(2,2,4,4-tetramethyl-
thietan-3-yl)amide 0O025
Citric acid 0.125
Water T q- 5 -
~00.000
The jars are capped loosely and placed in an
autoclave containing hot water and processed at
100C. for 45 minutes. The jars are removed, immediate
ly sealed by tightening the caps and allowed to cool.
33~
-121-
EXAMPLE 30
Powder Beveraqe Concentrate
Ingredients ~, Welght
Citric acid 31.78
5 Sodium citrate 5.08
Strawberry flavor 57.72
Strawberry FD and C color 0.54
L-Aspartyl-D-O-methylserine N-(2,2,4,4-tetra-
methylthietan-3-yl~amide 2.44
10 Carboxymethyl cellulose 2.44
100.00
Combine all ingredients in a blender and blend
until homogeneous. For use, 1.73 g. of powder beverage
concentrate is dissolved in 4 fluid ounces (118 ml.)
of water.
3~
-122-
EXAMPLE 31
Baked Cake
A highly acceptable vanilla cake was prepared
employing the following recipe:
Ingr~dients Grams
Emulsified shortening 16.09
Water 20.83
Eggs 23.00
Sodium bicarbonate l~lO
Vanilla extract, single fold 0.28
Glucono-delta-lactone 1.75
Polydextrose*, 70% aqueous solution 80.00
Nonfat dry milk 2.50
Cake flour 56u20
~hole milk powder 0.80
Wheat starch 1.40
L-Aspartyl~D-serine N-(2,2,4,4-tetxamethyl-
thietan-3-yl)amide 0.05
204.00
Combine nonfat dry milk, whole milk powder,
polydextrose solution and emulsified shortening. ~ix
at low speed until creamy and smooth (about 3 minutes),
add eggs and beat until a homogeneous creamy mix is
obtained. Dissolve sweetener in water, add to creamy
homogenate and mix 2-3 minutes. Add remaining ingre-
dients and mix until creamy and smooth ~3-5 minutes).
Place 120 g. of batter in small pregreased pan and
bake at 350F. (176C.) for 30 minutes.
*~.S. 3~766,165
-123
EXAlqPL~S 3 2
Synergistic Mixtures of L-Aspartyl-D-serine
N-(2,2,4,4-tetramethylthietan-3~yl)amide,
3/ 3
~I), R = CH2OH, R = ~ S ] and Saccharin
/
CH CH,
3 J
Blends of L-aspartyl-D-serine N-(2,~,4,4-tetra-
methylthietan-3-yl)amide and sodium saccharin were
prepared and evaluated for taste acceptability and
sweetness intensity by comparison with aqueous sucrose
standardsv Sweetness potency factors of sodium
saccharin and the invention compound (I, Ra = CH2OHt
(C 3)2
R = ~ S ) of 300 and 1200 x sucrose, respectively,
~CH3~,~2
were used t:o calculate the theoretical sweetness of
the blends. A series of taste panel evaluations were
carried out comparing aqueous solutions of the experi-
mental blends with sucrose solutions ranging 6 to 12%
(w/v) and 0,.C33% sodium saccharin solutionO Results
are tabulated below.
.
3~
-124-
Blend,Parts by Weight
[I, R =CH2OH,
~3)2
R= ~ ~ ] Sweetness
y~ Sodium PotencY x Sucrose ~ Taste
~ I3)2 Saccharin (T)Theory ~A)Actual Syner~y Qual itY
1 : 1 750 1125 50 clean,
sweet,
not
bitter
1 : 2 600 900 50 same
1 : 4 480 720 50 same
1 : 6 430 580 35 same
1 : 8 400 520 30 sweet,per-
ceptible
metallic
taste
1 : 9 390 sweet,
slight
` metallic
21) taste
1 : 10 381 sweet,
slight to
- moderate
metallic
taste
The ~ synergy was calculated according to the
following formula-
% Synergy = ATT x 100
where A i5 the actual sweetness determined by averaging the
taste panel results and T is the theoretical sweetness
determined from the composition of the mixtures by
weight e.g., for the 1:4 blend the theoretical sweetness
is (1/5 x 12003 + (~/5 ~ 300) = 480.
- From the results it is seen that with mixtures of
from l:i to 1:8 there is an unexpected increase in
sweetness potency of 30-50~. While there appeared to
be synergy at the higher ratios, the metallic taste
due to saccharin interfered with an accurate determina-
tion. Furthermore there is complete masking of the
-125-
well known bitter aftertalste of saccharin with blends
of from 1:1 to 1:6 and effective masking of bitterness
in blends containing up to one part L-aspartyl-D-
seri~e N-(2,2,4,4 tetramethylthietan~3-yl)amide and
5 8 parts sodium saccharin.
The 1:8 blend of L-aspartyl-D-serine N~(2,2,4,4-
tetramethylthietan-3-yl)amide/sodium saccharin at a
concentration of 0.0192% (w/v3 was found to be equi-
valent in sweetness to a 10~ (w/v) sucrose solution
and to 1:10 saccharin/cyclamate at 0.1177% (w/v).
In further sensory evaluation, a triangle test in
which trained taste panel members were presented three
samples. consisting of aqueous solutions of the above
1:8 blend of invention compound/sodium saccharin at
0.0192% and 1:10 saccharin/cyclamate at 0.1177%.
Panelists were asked to match the two like samples and
to indicate any preference. Seven of ten taste panel
members were not able to correctly differentiate the
two sweetener blends. Of those that corre.ctly paired
the like samples, the degree of difference between the
two blends was rated as being "very slight" or "just
perceptible".
These resu.lts indicate that there is no signi-
ficant difference between the 1:8 blend of invention
compound/saccharin and the 1:10 blend of saccharin/
cyclamate.
-126-
When the above procedure is repeated ~ut the
invention compound employed is of the formula (I)
wherein Ra is CH20CH3 and R is
( 3)2 (C 3)2
S or ~ ~S02
( 3)2 ~CH3)2
or wherein, Ra is CH20H and R is
(CH3)2
2 or -CH-( ~ )2
(C~3)2
similar results are obtained.
3~
~ 7--
EXAMPLE 33
Ssdium 5alt of L-Aspartyl-D-serine
N-(dicyclopropylcarbin ~)amide _
To a solution of 3.12 g. (0.01 mole) L-aspartyl-
D-serine N~(dicyclopropylcarbinyl)amide in 100 ml. of
ethanol is added 2 ml. of 5 N sodium hydroxide. The
resulting mixture is stirred for ten minutes at room
temperature then evaporated to dryness in vacuo. The
residue is triturated with anhydrous ethanol, filtered
and air dried.
When the sodium hydroxide employed above is
replaced with an equivalent amount of potassium
hydroxide, calcium hydroxide, magnesium hydroxide or
ammonium hydroxide the corresponding potassium,
calcium, magnesium and ammonium salts are formed in
like manner.
The remaining L-aspartyl-amino acid dipeptide
amides of formula (I) are also converted to carboxylate
salts as described above.
-128-
EXAMPLE 34
Acid Addition Salts
The L-aspartyl-D-amino acid dipeptide amide of
formula (I) is slurried in a small amount of water and
an equivalent amount of an acid such as hydrochloric,
phosphoric, sulfuric, acetic, maleic, fumaric, lactic,
tartaric, citric, gluconic or saccharic acid is added.
The resulting mixture is stirred for 15-30 minutes
then evaporated to dryness or precipitated by addltion
of a cosolvent such as methanol or ethanol.
--129--
PRE PARAT I ON A
Alkylcycloalkylcarbinylamines and
dicycloa_ ~ carbinylamlnes _
i. To a mixture of 118.5 g. (1.0 mole) of
cyclobutylcarbonyl chloride and 99 g. (1.0 mole)
cuprous chloride in 1000 ml. oE dry ether under a
nitrogen atmosphere is added dropwise 478 ml. (1.0
mole) of 2 M t-butylmagnesium chloride in the same
solvent. The addition is carried out at -5 to -15C.
The resulting mixture is poured into 500 ml. of 3 M
hydrochloric acid and 700 g. ice, the organic layer
is separated and washed successively with water,
sodium bicarbonate solution, brine and dried (MgSO4).
The dried ether extract is evaporated at reduced
pressure and the residue distilled to provide t-
butylcyclobutylketone.
ii. The ketone, 105 g. (0.75 mole), is mixedwith hydroxylamine hydrochloride 38.3 g. ~1.16 mole)
and sodium acetate, 123 g. (1.50 mole), in sufficient
water to effect solution, heated on the steam-bath
for A one hour, cooled and the mixture adjusted to p~
7.5 with sodium hydroxide solution. After extracting
the mixture with ether, the extracts are dried
(MgSO~) and evaporated to dryness to afford the
oxime. The oxime is dissolved in anhydrous ethanol
(about two liters per mole of oxime) and the solution
heated at reflux. Sodium metal (about 10 moles per
mole of oxime) is added in portions at a rate sufficient
to maintain reflux temperature. Wh~n all the sodium
is added the resulting mixture is cooled and 200 ml.
of ethanol Eollowed by 300 ml. of water is added.
-130-
The mixture is acidified with hydrochloric acid,
evaporated to remove ethanol and the residue made
alkaline IpH 12-13) with 10 M sodium hydroxide. The
alkaline mixture is extracted several times with
ether and the combined extracts dried (MgS04). Dry
hydrogen chlorine is passed through the dried extracts
until precipitation is complete. The precipitated
hydrochloride salt is collected by filtration, washed
with ether and air dried. The salt is converted to
the free base by means of aqueous sodium hydroxide,
extraction with ethyl ether and evaporation of the
extracts. The product, t-butylcyclobutylcarbinylamine
is of suitable purity for use in preparing the amides
of the invention but may be further purified, if5 desired, e.g. by distillation or column chromatography.
iii. By employing the appropriate acid halide
and Grignard reagent in the above procedure in place
of cyclobutylcarbonyl chloride and t-butylmagnesium
chloride the following amines are obtained in like
manner.
3 ~ 3~
-131-
39
~H2 ~
(CH2)m
m R7 R R9
0 H H H
0 CH3 H H
0 C~3 CH3 H
3 2 3 2 3C 2
0 CH3 n~C4H7 H
( 3)2 (CH3)2CH H
0 CH3 CH3 C(CH3)3
3 2 3CH2 H
1 n-C3H7 H H
1 CH3 c~3 H
1 CH3 n~C4H7 H
1 n-C3H7 n~C3H7 H
3 2 ( 3)3 H
2 CH3 CH3 CH3*
2 3 2 H
3C 2 C 3C 2 CH3CH~
2 CH3 H H
2 CH3 CH3 H
2 n-C3H7 ( 3~2 H
3 2 n~C4H7 H
3 CH3 CH3 C~I3
3 n~C3H7 ~ H
3 CH3 C~I3 H
4 CH3 H H
4 CH3 CH3 CH3
4 CH3CH2 CH3CH2 H
*B.P. 80-90~C. (21 mm.)
33~
-- L3 2
iv., The amines of the following formula are also
providecl in like manner.
~ 2)m
NH2~
(CH2)q
m q
0
0 2
0 3
0 4
1 2
1 3
1 4
2 2
2 3
2 4
3 3
3 4
4 4
The following amines are also prepared by this
method:
2,2-dimethyl-3-aminopentane r B.P. 123-126C.,
atmospheric pressure;
2,2,4-trimethyl-3-aminopentane, B.P. 149-150C.,
atmospheric pressure.
3~
-133-
PREPARATION B
2,2-Dimethyl~clohexylamine
i. 2,2- _ clohexanone
To a suspension of 13.5 g. (0.25 mole) sodium
methoxide in 500 ml. of ethyl ether was added 30.8 g.
(0.28 mole) 2-methylcyclohexanone and 20.3 g. (0.28
mole) ethyl formate. The mixture was stirred at room
temperature for 12 hours, fil-tered under a nitrogen
atmosphere, the solids washed with ethyl ether and
dried in the vacuum oven at 75C. The dried cake was
ground in a mortar and pestle to a fine powder to
obtain 17.5 g. (43~) of sodium 2-formyl-6-methylcyclo-
hexanone which was used in the next step.
The above product, 17.5 g. (0.11 mole) was added
to a mixture of 2.88 g. (0.13 mole) sodium shot, 500
ml. anhydrous ammonia and about 0.1 g. ferric chloride.
The resulting gray suspension was cooled to -45C.
and stirred for one hour at the reflux temperature of
the system. To this was added 20.86 g. (0.15 mole)
methyl iodide, the mixture stirred three hours at
reflux and allowed to evaporate while warming to room
temperature overnight. The residue was suspended in
300 ml. ethyl ether, refluxed to expell traces of
ammonia and water added to dissolve the solids. The
ether was extracted with water (3 x 100 ml.), the
combined aqueous layers treated with 6 g. of solid
sodium hydroxide and heated to steam distill the
ketone. The steam distillate was extracted with
ethyl ether, the extracts washed with brine, dried
and ether e~aporated to provide 2,2-dimethylcyclo-
hexanone as a colorless liquid, 2.0 g.
-13~-
- ii. The ketone provided above is converted to
the oxime and the latter reduced with sodium in
ethanol as described in Preparation A, Part ii, to
provide 3.1 g. of 2,2-dimethylcyclohexylamine.
The following 2,2-d.isubstituted ketones are pre-
pared and converted to amines by the above method in
like manner.
2,2-di.methylcyclopentanone
2,2-di.ethylcyclopentanone
2,2-di-n-propylcyclopentanone
2,2-diethylcyclohexylamine
3,3-dimethylthiepane-4-one
3,3-dimethyloxepane-4-one
4,4-dil~ethyloxepane-5-one
-135-
PREPARATION C
2,2,6,6-Tetramethylcyclohexylamine
i. 2,2,6,6-Tetramethylcyclohexanone
A 50~ suspension of sodium hydride in mineral
oil, 14.3 g. (0.30 mole), was suspended in tetra-
hydrofuran, the liquid decanted and the solid resuspended
and decanted again to remove the oilO Then 15 g.
(0.12 mole) of 2,6-dimethylcyclohexanone was added
followed by dropwise addition of a mixture o 11 g.
t-butanol and 20 ml. of tetrahydrofuran (vigorous
hydrogen evolution) and the resulting mixture refluxed
until hydrogen evolution was complete. To this was
added dropwise 37.8 g. 10.30 mole) methylsulfate and
the mixture heated at reflux for 24 hours. After
dilution with water, extraction with ethyl ether,
washing the extracts with water, drying and evaporation
of solvent below 40C., 17 g. of tetramethylketone
was obtained. This was distilled to obtain 14.6 g.
of produc~, B.P. 62-64C. ~15 mm.).
ii. The 2,2,6,6-tetramethylcyclohe~anone (8 g.)
obtained above was converted to the oxime and the
latter compound reduced by the procedure of Preparation A,
Part ii, to provide 1.4 g. of the desired amine as a
colorless liquid which was of suitable purity for use
as intermediate.
,.
- 1 3 ~--
PREPARATI ON D
i. 2,2,5,5-Tetrameth ~cyclo~enta one
To a slurry of 2.0 moles of sodium hydride
(washed to remove oil) in tetrahydrofuran was added
l90 ml. (2.0 mole) methyl sulfate at a fast rate.
Simultaneously, 35.7 g. (0.425 mole) cyclopentanone
in 50 ml. of the same solvent was added at a slow
rate. The reaction mixture warmed spontaneously to a
gentle reflux and hydrogen evolution was vigorous.
When the addition was completed, the mixture was
allowed to stir overnight at ambient temperature.
After heating to reflux for two more hours a mixture
of t-butanol in tetrahydrof~lran was added and reflux
continued for three hours. The reaction mixture was
diluted with water, extracted with ethyl ether, the
extracts washed with water, brine, dried over anhydrous
MgSO4 and the solvent evaporated to yield 48.2 g. of
crude product. This was distilled to afford 2~.2 g.
of tetramethylketone, B.P. 63-68C., 40 mm.
By employing a lower mole ratio of methyl
sulfate to cyclopentanone, the same method affords 2-
methylcyclopentanone, 2,5-dimethylcyclopentanone and
2,2,5-trimethylcyclopentanone.
ii. The following ketones are prepared in like
manner when the appropriate starting materials are
employed in the procedures of Part i, above, and
Preparation C. The alpha-propyl and alpha-butyl-
ketones are prepared using ~ ~ , the appropriate
alkylbromide as alkylating agent.
4 ~ 3g~
-137-
2,2,6-trimethylcyclohexanone
2~ethylcyclopentanone
2,2,4,4-tetramethylcyclobutanone
2-methylcyclobutanone
2,2-dimethylcyclobutanone
2,4-diisopropylcyclobutanone
2-t-butylcyclopentanone
2,2-dimethyl-5-t-butylcyclopentanone
2,5-diisopropylcyclopentanone
2-sec-butylcyclopentanone
2-isobutylcyclohexanone
2-methylcycloheptanone
2-t-butylcycloheptanone
2,7 dimethylcycloheptanone
2,7-diisopropylcycloheptanone
3,5-dimethyltetrahydro-4H-pyran-4-one
3,5-diisopropyltetrahydro-4H-pyran-4-one
3,3,5,5-tetram~thyltetrahydro-4H-pyran-4-one
3-mPthyl~5-t-butyltetrahydro-4H-pyran-4-one
3,3,5,5-tetramethyltetrahydro-4H-thiapyran-4-one
3-isopropyltetrahydro-4H~thiapyran-4-one
3,5-diisopropyltetrahydro-4H-thiapyran~4-one
3,-t-butyltetrahydro-4H-thiapyran-4-one
2-methyltetrahydro-4H-thiapyran-3-one
2,4-di~ethyltetrahydro-4H-thiapyran-3-one
2-methylthiepane-3-one
4-methylthiepane-3-one
2,4-diethylthiepane-3-one
2,4-di~sopropylthiepane-3-one
3,5-dimethylthiepane-4-one
3,3,5,5-tetramethylthiepane-4 one
4-methyltetrahydro-4H-pyran-3-one
4- _ -butyltetrahydro-4H-pyran-3-one
33~
-138-
2-isopropyltetrahydro-4H-pyran-3-one
2,4-diisopropyltetrahydro-4H-pyran-3-one
2,4-dimethyltetrahydro-4H-pyran-3-one
2-methyloxepane-3~one
4-methyloxepane-3-one
2,4-dimethyloxepane-3-one
2,2,4,4-tetramethyloxepane-3-one
3-methyloxepane-4-one
5-methyloxepane-4-one
3jS-dimethyloxepane-4-one
3,3,S,5-tetramethyloxepane-4-one
3,5-diisopropyloxepane-4-one
3-t-butyloxepane-4-one
5-t-butyloxepane-4-one
The ketones provided above are converted to the
corresponding amines by conversion to the oxime and
reduction with sodium in ethanol as described in
Preparation ~, Part ii, or Leuckart reduction o~ the
ketone as described in Preparation G, Part ii.
--13 9--
PREPARATION E
2,2,5,5-Tetramethylcyclopentylamine
A flask was charged with 35 g n (0.61 mole) of
40% sodium dispersion in mineral oil. The oil was
removed by washing with ethyl ether and decantation.
The sodium was then mixed with 400 ml. of ether and a
mixture of 32.8 g~ (0.20 mole) 2,2,5,5~tetramethyl~
adiponitrile, prepared by the method of Coffman et
alD, J. Am. Chem~ Soc., 80, 2868 ~1957), and 400 ml.
of tetrahydrofuran was added slowly. The resulting
mixture was stirred at room temperature for 4 hours,
the excess sodium decomposed by dr~pwise addition of
saturated aqueous ammonium chloride, the organic
layer washed with water, dried (Na2SO4) and evaporated
to afford 25 D 1 g ~ of crude 2,2,5,5-tetramethylcyclo~
pentylimine. The imine was dissolved in 75 ml. of
ethanol and added dropwise to a flask containing 23.3
9. (l mole) sodium shot. An additional 75 ml.
ethanol was added and the mixture heated at reflux
until the remaining sodium metal was consumed. The
reaction mixture was diluted with water, acidified to
pH 1 with concentrated hydrochloric acid, the aqueous
phase washed with ether then made strongly basic by
addition of sodium hydroxide. The organic layer was
extracted with ether, washed with brine, dried
(Na2SO4) and evaporated to dryness. The residue was
di~tilled in vacuo to afford 6.6 g. (23%) of the
_
desired amine, B.P. 60-61C. (20 mm.).
8~3~
-140-
PREPARATI0N P
2 kyl- and 2,6-Dialkylcyclohexylamines
To a solut:ion of 25 g. of 2,6-diisopropylaniline
iII 250 ml. each of ethanol and water was added 10 y.
of dry 5% ruthenium-on-carbon catalyst. The mixture
was hydrogenated in an autoclave at 100C., 1000 psi
(70.4 kg./cm.2) until hydrogen uptake ceased. The
catalyst was removed by filtration and the filtrate
evaporated to remove solvent. The residue was
distilled ln vacuo to obtain 11.2 g. of 2,6-diiso-
propylcyclohexylamine as a mixture of cis,trans and
trans,trans-isor~ers, B.P. 122-124C. a~ 22 mm~
____ _
By employing the appropriate 2-alkylaniline or
2,6-dialkylaniline as starting material and hydro-
genating by the above method the following cyclo
hexylamines are also obtained.
2-methyl-6~ethylcyclohexylamine, B.P. 82-87C.
at 19 mmO (50% ~ield);
2-methyl-6-isopropylcyclohexylamine, B.P. 86 at
14 mm. (45% yield);
2-n-butylcyclohexylamine;
2-ethyl-6-n-butylcyclohexylamine;
2-methyl-6-t-butylcyclohexylamine;
2 t-butylcyclohexylamine;
2,6-dimethylcyclohexylamine;
trans-2-ethylcyclohexylamine, B.P. 77 78 (23 mm.);
2,6-diethylcyclohexylamine, B.P. 96C., (17 mm.);
trans-2,-isopropylcyclohexylamine;
2-isobutylcyclohexylamine;
2 methyl-6-n-butylcyclohexylamine.
33~
PREPARATION G
2-t~Butylcy~lohexylamine
i. 2 t-Butylcyclohexanone
A solution of 31.25 g. (0.20 mole) t-butylcyclo-
hexanol in ~0 ml. of ethyl ether was cooled to 10C.
To this was added dropwise, with stirring, a solution
of 21.0 g. (0.07 mole) sodium dichromate dihydxate
and 15.75 m]~ (0.30 mole) concentrated sulfuric acid
in 100 mlO water whil~ maintaining the reaction
mixture below 25~C. The mixture was then warmed to
room temperature, stirred for two hours, poured onto
ice-water, ether layer sepaxated, the agueous phase
eætrac~ed again with ether and the combined extracts
washed with water, sodium bicarbonate and dried
(MgSO4). Evaporation of the ether afforded 30.6 g.
(99~) of the desired ketone.
iio Leuckart Reductio~`of Xetone
, . . . . .. ..
A mixture of 2-t-butylcyclohexanone 30.6 g.
(0.20 mole), formamide 50 ml. (1.2 mole) and formic
acid (10 m].) was heated at reflux while removing
water as it: formed in the reaction while returning
the ketone to the reaction vessel. Formic acid (10
ml.) was added as needed to control deposition of
ammonium c~rbonate in the condenser. After four
hours the reaction temperature reached 197C. and
distillation ceased. The mixture was cooled, diluted
with water (50 ml.) and extracted with ethyl acetate
(75 ml.). The organic layer was evaporated, concen-
trated hydrochloric acid added (50 ml. per 100 ml. of
residue), the mixture boiled overnight, cooled and
washed with 50 ml. of ethyl ether. The aqueous phase
.
3~
-142-
was adjusted to pH 11 with sodium hydroxide, cooled,
extracted with et:her (2 x 40 ml.) and the extracts
dried over sodium hydroxide pellets. The solvent was
evaporated and the residue distilled through a 10 crn.
column to obtain 21.93 g. of the title amine (71%),
B.P. 86-8BC. (21 mm.) as a mixture of cls and trans-
isomers.
iii. dl-Fenchone and l-fenchone are reduced to
the corresponding fenchylamines by the Leuckart
xeduction method of Part ii, above. (-)Fencylamine
is obtained as a water white liquid, B.P. 55-60C.
(6 mm.), [alpha]D -21.9 in 30% yield.
3~
143-
PREPARAT I ON H
2,4-Dimethyl-3-aminopentane
In a shaker bottle was placed 0.2 g. platinum
dioxide and 10 ml. water. The slurry was hydrogenated
at 50 psi (3.5 kg./cm2.) for 15 minutes. To the
resulting slurry of platinum black was added 34.26 g.
(0.30 mole) 2,4-dimethyl-3-pentanone, 20.0 g. (0.37
mole) ammonium chloride, 225 ml. ammonia saturated
methanol and 25 ml. concentrated ammonium hydroxide.
The reslllting slurry was hydrogenated at 60 psi
(~.2 kg./cm.2) and room temperature ~or 20 hours,
filtered, refluxed for for 1 hour and cooled. The
mixture was adjusted to p~ 2.0 with concentrated
hydrochlorlc acid and the volume was reduced by
evaporatioll at reduced pressure. After washing with
75 ml. ethyl ether, the aqueous solution was brought
to pH 13 with 10 M sodium hydroxide solution and
extracted with three 100 ml. portions of ether. The
extracts were combined, dried over anhydrous MgSO4
and saturated with gaseous hydrogen chloride. The
precipitated amine hydrochloride was collected by
filtration, air dried and decomposed with 75 ml. 10 M
sodium hydroxide solutionD The oily amine layer was
separated and distilled at atmospheric pressure, B.P.
129-132~C, yielding 17.6 g.
3~
--1 4 4 -
PRE PARATI ON
trans-2-Ethylcy_~lopentylamine
i. 2-Ethy~_yclopentanone
In a three-necked flask 5.0 g. of sodium metal
was dissolved in 250 ml. of dry ethanol and 31.24 g.
(0.20 mole) 2-carboethoxycyclopentanone added. To
the resulting yellow solution 18.4 ml. (0.23 mole)
ethyl iodide was added dropwise and the mixture
heated at reflux for two hours. After cooling, 250
ml. of brine and 50 ml. of water were added and the
mix-ture extracted with ethyl ether (2 x 100 ml.).
After drying (MgSO4) and evaporation of solvent 36.5
g. (99%) of 2-ethyl-2-car~oethoxycyclopentanone was
obtained.
This was decarboxylated by heating at reflux
with a mixture of 200 ml. of concentrated hydrochloric
acid and 100 rnl. of water. After four hours at
reflux carbon dioxide evolution was complete. The
mixture was cooled, saturated with sodium chloride,
extracted with ethyl ether, the extracts dried (MgSO4)
and ether evaporated. The residue was distilled to
obtain 12.62 g. (56%) of 2-ethylcyclopentanone, B.P.
97-98C. 1100 mm.).
ii. The product obtained above was converted to
trans-2-ethylcyclopentylamine by the procedure of
Preparation ~, Part ii, B.P. 150-151C. in 35% yield.
The identity of the product was verified by its
H~NMR spectrum.
By emp3Oying the appxopriate 2-carbethoxycyclo-
3~ alkanone or a corresponding hetercyclic ketone (pre-
pared by the well known Dieckmann cyclization of the
appropriate dicarboxylate ester, see e.g., H. O.
EIouse, "Modern Synthetic Reactions", W. A. Benjamin,
Menlo Park, Cal., 1972, p. 740.) and the appropriate
alkyl halide in place of ethyl iodide in the above
procedure the following amines of formula R N~2 are
prepared in like manner.
3~
-145-
R3
Where R is ~ ( 21m
R3
m
1 CH3
2 5
2 t-C4Hg
2 CH3
2 , sec-C4a9
4 CH3
4 t-C4Hg
Where R is ~ ~CH2)n- X
X ~ n p R3
O 1 0 . 2-CH3
O 1 0 4-CH3
O 1 0 2-t-C4~9
0 o 2 2-CH
O 0 2 4~CH3
O 0 2 4-sec-C4~Ig
O 0 2 2 ' C
O 0 3 2-C~3
0 0 3 4-CH3
O 1 2 3-CH3
O 1 2 5-CH3
O 1 - 2 5-t-C4Hg
O 1 2 3-t-C4Hg
S O 1 2-CH3
S O 1 4-CH3
S 1 1 3H7
S2 1 1 ~ C4~9
S 0 2 2-CH3
S 3 2-CH3
S O 3 4-CH3
3~3
--146--
PRE PARAT I ON J
trans 2 Isoprop~cycIopentylamine
i. 2-Isopropy]c~clopentanone
To a solution of 10 g. of sodium metal in 670
ml. of ethanol was added dropwise a mixture of 100 g.
(1.19 mole) cyclopentanone and 60 g. (1.03 mole)
acetone and the resulting mixture refluxed for 1.5
hours. The solvent was evaporated ln va o, the
residue taken up in ether, the solution washed wit~
3 M hydrochloric acid (5 x 200 ml.), 5% sodium
bicarbonate (3 x 200 ml.), brine (1 x 200 ml.) and
dried (MgSO4~. The ether was evaporated with mild
heating to afford 97 g. of dark liquid which was
distilled in vacuo to obtain 55 g. of 2-isopropylidene-
cyclopentanone, B.P. 96-100 (2.7 mm.).
To 12.75 g. of the above product in 250 ml. of
methanol was added 2.0 g. 5% palladium-on-carbon
catalyst and the mixture hydrogenated at 50 psi
(3.S kg./cm2.). After one hour the hydrogen uptake
was complete. The catalyst was removed and solvent
evaporated in vacuo to afford 12.75 g. of colorless
liquid. This was distilled to obtain 9.64 g. of 2-
isopxopylcyclopentanone, B.P. 74-76C. (20 mm.).
Reducti~n of 2-isopropylcyclopentanone by the
method of Preparation A, Part ii afforded the corres-
ponding amine, B.P. 167 (atm.) in 31~ yield.
`147-
PRE:PARAT I ON K
____
2,2-DlmethyI _-aminobutane
In a 500 ml. flask was placed 10.0 g. (0.10
mole) 2,2-dimethyl-3-butanone, 250 ml. methanol,
76.94 g. (1.,0 mole) ammonium acetate and 4O37 g.
(0.07 mole) sodium cyanoborohydride, and the mixture
was allowed to stir at room temperature for 24 houxs.
The pH was adjusted to 2.0 with concentrated hydro~
chloric acid and the methanol removed at reduced
pressure. The residual solid was dissolved in 500
ml. water and washed with three 100 ml. portions of
ether. The pH of the aqueous solution was adjusted
to 13 with 10 M sodium hydroxide and the mixture
extracted with three 100 ml. portions of ether. The
extracts were combined, dried over anhydrous MgSO4,
filtered and distilled. The amine (2.4 g.) distilled
at 102-103C at atmospheric pressure.
The racemic amine was resolved by the Polari-
metric Control method described by Bruck et al. J J.
Chem. Soc., 921 ~1956) employing the amlne hydrogen
tartarates and crystallizing from 70:30 methanol/water
~by volume) to obtain dextrorotatory amine of 93 ~ 4
purity and levorotatory amine of 80 + 4% purity.
When an equivalent amount of 2,2-dimethyl-3-
pentanone is employed in place of 2,2-dimethyl 3-
butanone in the above procedure 2,2-dimPthyl-3-amino-
pentane is obtained and resolved into its enantiomers.
3~
-148-
PREPARATION L
L-Aspartic acid N-thiocarboxyanh~
A. L-Aspartic acid (582 g., 4.29 mole) was added
gradually with stirring to 350.9 g. (8058 mole) of 50%
S sodium hydroxide solution at 0C. Methyl methyl
xanthate t550 g., 4.51 mole) in 405 ml. of methanol
was then added as rapidly as possible. The mixture
was heated at 45C. for 1.5 hours, cooled to room
tempera-ture, and washed with two portions of methylene
chloride. The methylene chloride washPs were discarded
and the aqueous phase acidified with concentrated
hydrochloric acid at 0C. The solution was extracted
with three portions of ethyl acetate~ and the combined
extracts washed with brine and dried over anhydrous
lS magnesium sulfate. The solvent was evaporated ln
vacuo to gi~e a yellow oil which crystallized upon
addition of ethylene dichloride and n-hexane. The N-
methoxy-thiocarbonyl-L-aspartic acid was collected by
filtration, washed with fresh n-hexane, and dried (420
g., 47%)-
M.P. 128-130C; lH-NMR (DMSO~d6), (delta) 2.73
(d, 2H, J = 6 ~z~, 3.63 (s, 3H), 4.43 (dt, lH, J =
6 Hz, 8 Hz), 6.63 (d, lH, J = 8 Hz); infrared spectrum
(KBr) 1715, 1515 cm 1.
3~ `
-
-149-
B. N-methoxythiocarbonyl-L-aspartic acid (207.0 g.,
1.00 mole) was dissolved in 1200 ml. ethyl acetate at
0C. and phosphorous tribromide (47 ml., 0.50 mole)
was added in one portion. The cooling bath was removed
and the temperature allowed to rlse spontaneously to
35C. The solution was stirred for 10 minutes after
which time a granular white precipitate had formed.
The reaction mixture was cooled to 0-5~C.~ the product
collected by filtration, washed with a small volume of
ether, and dried. The yield of analytically pure L-
aspartic acid N-thiocarboxyanhydride was 157.4 g.
(90%) .
M.P. 200-205C. (dec.); [alpha~25 = -109.5 (C =
1, THF); infrared spectrum (KBr) 3225, 1739, 1724,
1653, 1399 cm 1; lH-NMR (DMSO-d6) ppm (delta) 2.83 (d,
2H, J = 5.0 Hz), 4.70 (t, lH, J = 5.0 Hz), 9.23 (bs,
2H, ex); mass spectrum (m/e) 175 (M ), 87, 60.
3~ .
-150-
PR~PARATION M
~ =
i. Ethyl 2,2,3,3-Tetramethylcyclo~ropanecarboxy~te
The method of Mesheheryakov, Chem. Abstr., 54,
24436d (1960) was employed. To a mixture of 19 g.
(0.226 mole) of 2,3-dime~hyl-2-butene and 2 g. of
cupric sulfate is added at reflux a mixture of 51 g.
(0.447 mole) ethyl diazoacetate and 19 g. o~ 2,3-
dimethyl-2-butene. The resulting mixture is heated at
reflux for 3 hours, cooled, filtered and distilled to
afford 19.8 g. (26%) of the desired cyclic ester, B.P.
76-77 (15 mm.~.
ii. To 300 ml. of ethanol containing 40 g. of
ammonia is added 17 g. (0.10 mole) of the ester
obtained above and the resulting mixture allowed to
stand overnight. After heating at reflux for one hour
the ethanol was evaporated in vacuo to obtain 2,2,3,3-
tetramethylcyclopropanecarhoxamide.
A solution of 2.82 g. (O.02 mole) of the amide in
8 ml. tetrahydrofuran and 4 ml. of water is cooled to
5C. and 10 ml. of 2 M sodium hypochlorite added drop-
wise followed by 8 ml. of 20% (w/v) sodium hydroxide.
The two phase mixture is stirred at 5C. for 30 minutes
then at ~0C for one hour. The organic layer is
extracted with ether, the ether layer e~tracted with
2 M hydrochloric acid (3 x 20 ml.), ~he aqueous acidic
layer is made strongly alkaline with sodium hydroxide
and extracted with ether. The extracts are dried
(Na2SO4) and the ether evaporated at 25 (50 mm.) to
give 0.67 g. (25%) 2,2,3,3-tetramethylcyclopropyl-
- amine~ NMR (CDC13) ppm tdelta~:
0.95 t6H, singlet); 1.00 (6H, singlet); 1.83 (lH,
multiplet); 1.7 (2H, multiplet).
3~
-151
iii. The following substituted cyclopropylamines
are prepared in an analogous fashion from the appro-
priate olefin.
NH2
R ~ R
R6/ \ R
R3 R R5 R6
__
CH3 H CH3 H
CH3 H H H
i_CH ~ H H H
i_C3H H i_C H H
CH3 CH3 H H
t-C4H~ H H H
CH3 CH3 t-C4Hg H
Y33~
-152~
PREPARATION N
3-Amino-2,2,4~4-tetramethyloxetane
To 13.6 g. (0.12 mole) of diisopropylketonP is
added 0.2 ml. of phosphorus tribromide. To this is
added dropwise at 10C., 38.4 g. (0.24 mole) bromine
and the mixture warmed to 55-60C. and held at this
temperature for 1.5 hours. After cooling and partion-
ing between chloroform and water, the organic layer is
washed with sodium carbonate solution until neutral,
dried and the solvent evaporated to obtain 2,4-dibromo-
2,4-dimethylpentan-3-one.
To 0;,1 mole of the dibromoketone in 160 ml. of
ethanol is added a solution of 8 g. of sodium hydroxide
in 80 ml. water and the resulting mixture is stirred
at room temperature for 30 minutes. After diluting
with water the reaction mixture is extracted with
ethyl et7ner, the extracts washed with water, brine and
dried (MgS04). The ether is evaporated to provide
2,4-dihydroxy-2,4-dimethyl-3-pentanone. This is
dissolved in 50 ml. chloroform and 1.5 ml. concentrated
sulfuric acid added dropwise. The resulting mixture
is heated at reflux for five hours while removing
water clS its azeotropic mixture with chloroform. When
no more water is evolved the reaction mixture is
washed with water, the organic layer dried (MgS04~ and
solvenl_ evaporated to provide 2,2,4,4-tetramethyloxetane~
3-one whiGh is purified by distillation.
The ketone is converted to the oxime and reduced
~ith sodium/ethanol by the procedure of Preparation A,
Part ii.
3~
-153-
PP~EPARATION O
3-Amino-2,2-dimet Yloxetane
3-~ydroxy-3-methyl-2-butanone, 0.20 mole, is
treated dropwise with a equimolar amount of bromine at
room temperature and the resulting mixture stirred for
three hours. The mixture is taken up in chloroform,
washed wit:h sodium carbonate solution until neutral,
dried and solvent evaporated to obtain l-bromo~3-
hydroxy 3-methyl-2-butanone.
i0 To 0.1 mole of the bromoketone in 160 ml. of
ethanol is added a solution of 4 g. of sodium hydroxide
in ~ ml. water and the mixture stirred at room
temperature for 30 minutes. The mixture is diluted
with water, extracted with ether, the extracts washed
with water, brine and dried (MgSO4). The solvent is
evaporated and the residue taken up in 50 ml. of
chloroform. To this is added dropwise 1~5 ml. of
concentrated sul furic acid and the resulting mixture
heated at reflux while removing water as its azeotrope
with chloro~orm. When water evolution is complete the
resulting ketone is isolated and converted to the
desired amine as described in Preparation N.
3.~ 33~
-154-
PREP~RATION P
Employing the procedures of Preparation N and O
but starting with the appropriate ketone or alpha-
hydroxykelone in each case the ~ollowing amines are
prepared :Ln like manner.
R~ R4
R3 R4 R5 R6
CH3 H H H
CH3 H t-C H H
CH3 C2H5 CH3 C2H5
C~3 H CH3 H
CH3 CH3 C2H5 C2H5
-C H H H H
-C H H i_C H H
C2H5 C2H5 H H
-155
PREPARATION Q
~ .
Dicyclopro~~ c~ i ~n:--
In a 500 ml. round bottom flask was placed 41.7 g.(0.60 mole) hydroxylamine hydrochloride and 80 ml.
water. With stirring, 44 ml. of lOM sodium hydroxide
solution and 44.4 g. tO.40 mole) dicyclopropyl ketone
were added. The mixture was stirred at reflux for
three hours. After cooling, 60 ml. methylene chloride
was added and the mixture stirred until all oxime had
dissolved. The methylene chloride layer was separated
and dried over anhydrous magnesium sulfate. The
solvent was removed by evaporation at reduced pressure
and the residue recrystallized from 55 mlO hexane,
yielding 40.3 g. dicyclopropylketoxime, M.P. 69-72C.
In a 500 ml., three-necked round bottom flask was
placed 18.8 g. (0.15 mole) dicyclopropylketoxime and
150 ml~ anhydrous ethanol. With efficient stirring
19.2 g. (0.83 mole) sodium spheres was added in
portions as rapidly as possib]e, maintaining reflux
throughouk the addition. Following dissolution of the
sodium, the reaction was cooled to 60C. and 60 ml.
water was added. After cooling, 78 ml. concentrated
hydrochloric acid was added dropwise with stirring.
Ethanol was distilled at reduced pressure and 50 ml.
~S water addad to dissolve salts~ The mixture was
adjusted to p~ 13 with 10 M sodium hydroxide solution
and extracted with three 40 ml. portions of methylene
chloride. The extracts were combined, dried over
anhydrous magnesium sulfate, filtered and evaporated
at reduced pre~;sure. The residual amine was distilled
- at 88-90C./95 mm Hg, yielding 11.0 g. of the desired
product.
156~
PREPARATION R
2-Amino-3,3-dimethyl~
qamma--butyxoIactone Hydrochloride
The melhod is that of Nagase et al., Chem.
Pharm. Bull , 17, 398 (1969).
To a st:irred solution of 2,2-dimethylhydroacryl~
aldehyde [prepared from sec--butyraldehyde and form-
aldehyde by the method of Stiller, et al., J. Am.
Chem. Soc., 62, 1785 (1940)] 5.11 g. in methanol
(25 ml.), a solution of ammonium chloride, 2.94 g.,
and sodium cyanide, 2. 9 g., in water ~40 ml.) is added
dropwise. After stirring for three hours the mixture
is saturatecl with ammonia gas and allowed to stand at
room tempera~ture overnight. The resulting mixture is
concentrated~ ln vacuo to a small volume and 40 ml. of
concentrated~ hydrochloric acid is added. After
refluxing for three hours the mixture is evaporated in
vacuo and thle residue crystallized from ethanol-ethyl
ether and th~en from ethanol to give 2.2 g. of the
title compound, M.P~ 214-215C. (dec.).
Use of homologs of 2,2-dimethylhydracrylaldehyde
in the above procedure affords the corresponding
compounds of the formula
~ 12
where one of R12 and R13 is alkyl having from one to
four carbon atoms and the other is hydxogen or alkyl
having from one to four carbon atoms.
33~
--157-
PREPARAT I ON S
4-Amino-3,3, 5, 5-tetramethyl~tetrahydro-4H-pyran-2-one
i. Methyl 5-HYdroxy-2,~,4,4-tetramethyI-3-keto-
valerate
A mixture of 172 g. (loO mole) methyl 2,2,4-
trimethyl-3~ketovalerate, 5.4 g. (0.10 mole) sodium
methoxide and 33 g. (0O36 mole) paraformalde in
250 ml. methanol is heated at reflux for eight hours.
The mixture is cluenched by addition of water, neutralized
with hydrochloric acid, extracted wi~h ethyl ether,
washed with water, brine and solvent evaporated. The
residue is purified by vacuum distillation or chromato-
graphy on silica gel to provide the purified product.
ii . 2 r 2 r 4,4-tetramet~y~-2,4 dioxotetrahydro-4H-pyran
A solution of 101 g. (0.50 mole) of the above
product in 200 ml. methanol and 20 ml. concentrated
hydrochloric acid is heated at reflux for two hours,
cooled, poured into ice~water, extracted with ethyl
ether, the extracts washed with sodium bicarbonate
solution, water, clried and evaporated to dryness. The
residue was heatecl ln vacuo at 80-100C. for two hours
to obtain product of suitable purity for use in the
next step.
iii. The ketolactone obtained above is converted
to the corresponding 4-oximino derivative and this
reduced to the tit:le compound by the procedure of
Preparation Q.
.,
3~
--158--
PREPARATI ON T
4-Amino-3,3~5,~-tetra_ethyl 2-~iperidone
i. Methyl 5-Dibenzylamino-2,2,4,4-tetramethyl-3-
-
ketovalerate hydrochloride
_ _ ,
To a mixture of 86 g. (0. 5 0 mole) methyl ~,2,4-
trimethyl-3-ketovalerate, 117 g. (0.64 mole) dibenzyl-
amine hydrochloride and 19O8 g. (0.22 mole) paraform-
aldehyde is added a solution of 1 ml. of concentrated
hydrochloric acid in 150 ml. 95~ ethanol and the
mixture is heated at reflux for four hours. The
mixture is filtered, 500 ml. of hot acetone added to
the filtrate and the resulting mixture cooled then
refrigerated overnight. The precipitated product is
collec~ed by filtration, washed with acetone and
lS dried.
ii. 3,3,5,5-tetramethylpiperidin-2,4 dione
The above hydrochloride salt is parti ioned
between 0.1 N sodium hydroxide solution and ethyl
ether. The ether extracts are dried (MgSO4) evapor-
ated to dryness and the residue taken up in methanol.
To the methanol solution is added 1 g. of 10~ Pd/C and
the mixture hydrogenated at 3-4 atmospheres pressure
until hydxogen uptake is complete. The catalyst is
removed by filtration, the filtrate heated at reflux
for two hours, solvent evaporated and the residue
heated at 70-80C. in vacuo for two hours. The
_
residual product is purified by chromatography on
silica gel.
iii. The piperidinedione obtained above is
conve~ted to the 4-oximino derivative and this reduced
to the title 4-amino analog by the procedure of
Preparation Q.
-159-
PREPARATION U
3,3,5,5-Tetramethylpyrrolidin-2,4-dione
A mixture of 80 g. of 2,2,4,4-tetramethyl-1,3-
cyclobutanedione monoxime, prepared by the method of
U.S. 3,125,569, and 250 ml. 98~ (w/w) sulfuric acid
was warmed at S0-60C. for one hour and allowed to
stand overnight at room temperature. The reaction
mixture was poured onto 800 g. ice, extracted with
methylene chloride, the extracts washed with sodium
bicarbonate so]ution, water, dried (MgSO4) and evapor-
ated to remove solvent. The resulting mixture of
products was purified by column chromatography on
silica gel and the fractions containing the title
compound combined and evaporated to dryness.
The ketolactam thus obtained is converted to 3-
amino-3,3,5,5-t:etramethyl-2-pyrrolidone by methods
described above.
3~
--160--
PREPARATION V
3-Amino~2,2,4,4-tetramethylthietan and its 1,l-Dioxide
A~ 2 _ Dibro~o-2,4-dimet~xIpentan-3-one
To 136 g. (1.2 mole) of diisopropylketone was
added 2 ml. of phosphorus tribromide and the mixture
cooled to 10C. To this was added dropwise 384 g.
t2.4 mole) of bromine, the mixture allowed to warm to
room temperature. After two hours at this temperature
the mixture was warmed at 55-60Co for one hour then
cooled and partioned between chloroform and water.
The water was discarded and the organic layer washed
with sodium carbonate solution until neutral. The
organic layer was dried (MgSP~) and solvent evaporated
to obtain 316 g. (97~) of the desired product.
B. 2,2,4,4-Tetramet~y1-3-oxo h etane
Sodi.um metal, 23 g. (1.0 mole), was dissolved in
S00 ml. of dry methanol and the resulting mixture
cooled to 10C. Hydrogen sulfide gas was passed
through t:he mixture until it was saturated. Then
136 g. (t).5 mole) of the dibromoketone obtained in
Part A WclS added dropwise while continuing to allow
hydrogen sulfide to pass through the reaction mixture.
After the addition was completed the mixture was
stirred for two hours at 10C., allowed to warm to
room temperature and stirred overnight. After pouring
the reaction mixture into water, it was extracted with
ethyl ether and the extracts washed with dilute
hydrochloric acid and brine. After drying over
magnesium sulfate the ether was evaporated, the
residue slurried with methanol, cooled and filtered ~o
obtain 46 g. 164%) of solid product which was used
without purification in the next step.
3~
-161-
C. Reducti~le amination of ketone
.o 75 m;L. of dry methanol was added 4.5 g.
(0.031 mole) of 2,2,4,4-tetramethyl-3-oxothietane,
2,3.9 g. (0.31 mole) ammonium acetate and 1.36 g.
(0.0217 mole) sodium cyanoborohydride and the resulting
mixture heated at reflux for four hours. Additional
sodium cyano~orohydride (1.36 g.) was added and reflux-
ing continued for three days with a third increment of
the same reagent added at the start of the third day.
The resulting mixture was acidified to pH 2 with
hydrochloric acid and evaporated to dryness on the
rotary evaporator at reduced pressure. The residue was
dissolved in water, washed with ethyl ether, the
aqueous phase adjusted to pH 11 with sodium hydroxide
solution and extracted with ethyl ether. The extracts
were washed with brine, dried IMgSO4) and evaporated
to dryness to obtain 1.9 g. 142~) of the desired amine
as a crystal]ine solid. The structure of the product
was verified by its lH-NMR spectrum.
A~
-162-
D. 3-Amino-2,2,4,4-tetrameth~hietane-1,1-dioxide
The amine obtained in Part C, above, 29 g.
(0.2 mole) was dissolved in 50 ml. acetonitrile and
250 ml. water added. While maintaining the mixture at
5 pH 10 with sodium hydroxide, 35.8 g. (0.21 mole)
carbobe!nzoxy chloride was added over 30 minutes, the
mixture! stirred for one hour, filtered, the precipitate
washed with water and dried in acuo at 50C. to
provide the NCbz-amine, Rf 0.7 (hexane/ethyl acetate
4:1 v/v, phosphomolybdic acid spray), 52.1 g. (93.4%).
This was dissolved in 700 ml. methylene chloride,
77 g. (0.372 mole) m-chloroperbenzoic acid was added
slowly while maintaining the temperature belo~ 45C.
(20-42C.). The precipltated solid was collected by
filtration, the filtrate was washed with lN hydro-
chloric acid, aqueous sodium bicarbonate solution,
dried (,MgSO4) and the solvent evaporated. The residue
was crystallized from acetone-water to obtain 42 g.
(73%) of the Cbz-protected amine l,l~dioxide, Rf 0.7
(hexane/ethyl acetate 1:1 v/v, phosphomolybdic acid
spray).
The protecting group was removed by hydrogeno-
lysis of 5 g. of Cbz-amine in 250 ml. methanol, 5 ml.
concentrated hydrochloric acid and 2 g. of 5% Pd/C
(50% wet). The product was isolated in the usual
manner. Yield: 2.4 g. (85%), Rf 0.6. The retention
time upon gas-liquid chromatography on a 1 meter,
OV 1 column at 180C. was 1.3 minutes. The overall
yield for the three steps starting from 3-amino-
2,2,4,4-tetramethylthietane was 65%.
By employing equivalent amounts of amine and m-
chloroperbenzoic acid in the above procedure the
corresponding sulfoxide is obtained in like manner.
3~
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E. Employing the appropriate ketone of formula
R3R4cHCoc~R5R6 in place of diisopropylketone in the
procedures of Parts A-C affords the corresponding
amines of the formula shown below.
R5~ ~ R43
R3 R4 R5 R6
CH3 H CH3 H
CH3 H H X
C~H5 H H H
1-C3H7 ~ H H
l-C3H7 H i-C H H
t-C4Hg H H H
t-C~Hg H t-C4Hg H
n-C4Eg H ~-C4H9
C2H5 2 5 H
The corresponding sulfoxides and sulfones are
prepared ~y the procedure of Part D above.
3~
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PREPARATIOU W
3-Amino-2,2,4,4-tetramethyltetrahydrothiophene
A. 1-~ ~
To sodium methoxide pxepared from 7.5 g. of
sodium metal and 250 ml. of methanol was added 72.5 g.
(2.4 moles) paraformaldehyde followed by 250 g. (2.2
moles) diisopropylketone and the resulting mixture
heated at re~lux for three hours. The reaction was
quenched with water, neutralized with hydrochloric
acid, extracted with ethyl ether, washed with water,
brine ancl the solvent evaporated. The residual oil
(90 g.) was distilled ln vacuo to obtain 28 g. of the
desired product boiling at 92-98C. at 16-20 mm. GLC
on OV-l column at 107C., retention time 314 sec., 96%
pure.
When the above procedure was repeated on the same
scale but the reaction mixture refluxed for 16 hours,
31 g of product was obtained of 96% purity by GLC.
B. 4-Bromo-l-hydro~y-2,2,4-trimethylpenta_-3-one
To a stirred, refluxing solution of 69 g. (0.48
mole) of l--hydroxy-2,2,4-trimethylpentan-3-one in
500 ml. of chloroform wàs added dropwise a solution of
77 g. (0.48 mole) bromine in 100 ml. of chloroform.
When the addition was completed the mixture was stirred
at reflux for one hour, allowed to cool and stand
overnight at: room temperature. Evaporation of solvent
at reduced pressure afforded 127 g. of product which
was used in the next step without purification.
" ,
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C. 2,2,4,4-'retramethyltetrahydrothiophen-3-one
The product obtained in Part B, 79 g. (0.3 mole)
was dissolved in 300 ml. of dry pyridine, cooled to
0C. and 114 g. (0.6 mole) of p-toluenesulfonyl chloride
was added in portions at 0C. The resulting mixture
was stirred at this temperature for 3 hours, 15 minutes,
poured into ice/water and extracted with ethyl ether.
The extracts were washed with dilute hydrochloric
acid, water and brine then dried over anhydrous
magnesium sulfate. The solvent was evaporated to
provide 111 g. (98%) of crystalline tosylate.
The tosylate, 94 g. (0.25 mole) was dissolved in
one liter of pyridine, 180 g. (0.75 mole) of sodium
sulfide monohydrate added and the mixture heated to
75C. and held at this temperature for one hour and
allowed to stand at room temperature overnight. Water
was added and the mixture was extracted with ether.
The extracts washed with hydrochloric acid, brine,
dried (MgSO4) and the solvent evaporated to obtain
35 g. of the title compound, 89~ yield. The product
showed only one spot upon silica gel TLC, eluting with
ethyl acetate/hexane (1:4 by volume, R~ 0.5. The
H-NMR spectrum was in agreement with the structure
for the title compound.
3'~
-166-
D. Leuckart reduction of ketone
To a 100 ml. round-bottomed three-necked flask
fitted with stirrer, thermometer and condenser with
fractionating head was added 10.0 g. (0.063 mole) of
2,2,4,4-tetramethyltetrahydrothiophen-3-one r 15.2 ml.
(0.38 mole) formamide and 3.5 ml. (0.092 mole) formic
acid and the mixture heated at reflux (163C.) while
removing water. The reaction mixture was maintained
at 160--180C. for 20 hours with addition of formic
acid (10 ml.) at intervals. The pot temperature
increased to 200C. over this period. The reaction
mixture was cooled, water added and the mixture
extracted with ethyl acetate. The extracts were
evaporated _ vacuo. The residue was refluxed with
20 ml. of 6N hydrochloric acid for two hours, cooled,
the resulting mixture washed with ethyl ether, the
aqueous phase adjusted to pH 11 with sodium hydroxide
solution and extracted with ethyl ether. The extracts
were dried and evaporated to obtain 2 g. of 3-amino-
2,2,4,4-tetramethyltetrahydrothiophene which was
identifiecl by lH-NMR and appeared homogeneous upon
silica gel ~LC.
E. By employing the appropriate ketone as starting
material iII place of diisopropylketone in the above
procedures and that of Preparation X, the following
amines are similarly obtained.
.. i ~` "
-167
N ~
X J 4
X R3 R4 R5 R6
S CH3 H CH3 H
S H H CH3 H
S CH3CE[2~ CH3CH2 H
( 3)2 ~ 3)2CH
S CH3 CH3 H H
3 2 3 2 H
CH3 H CH3
O H H CH3 H
O CH3 H H H
O M H (CH3)3C H.
O C~3CH2 H n-C4H H
- 15 CH3 C~3CH2 CH3 CH3CH~
When the t:etrahydrothiophenes of the above
formula are contacted with an equimolar amount of
hydrogen perox:ide or m-chloroperbenzoic acid the
corresponding ,sulfoxide (X = SO) is formed in each
case. Treatment of the same starting material or the
sulfoxide with a-molar excess of the same reagents or
potassium permanganate affords the corresponding
sulfone (X = SO2).
3~
--16~--
PREPARATI ON_X
3-Amino-2,2,4,4-tetramethyltetrahydrofuran
A. 2,2,4,4-Tetramethyltetrahydrofuran-3-one
4-Bromo-1-hydroxy-2,2,4-trimethylpentan-3-one
(prepared as described in Preparation W, Parts A and
B) 25 g. (0.1 mole) was dissolved in 160 ml. of
ethanol and a solution of 3 g. (0.2 mole) sodium
hydroxide in 80 ml. of water was added. The resulting
mixture was stirred at room temperature for 30 minutes,
diluted with water, extracted with ethyl ether, the
extracts washed with water, brine and dried over
anhydrous magnesium sulfate. ~he solvent was evapor-
ated to afford 17.7 g. of 2,2,4-trimethylpentan-1,4-
diol as a colorless liguid which was identified by
lH-NMR. The diol was dissolved in 50 ml. of chloroform,
1.5 ml. of concentrated sulfuric acid added dropwise.
The mixture was heated at reflux for 3 hours, while
distilling water/chloroform azeotrope from the mixture.
After standing overnight at room temperature the
reaction mixture was washed with water, the organic
layer dried (MgSO4) and solvent evaporated ln vacuo to
provide 13.9 g. of colorless liquid. Distillation
afforded 8.3 g. of the desired product, B.P. 70-72
(50 mm.), overall yield 58~.
B The k~tone obtained in Part A, 8.0 g. (0O05~
mole), hydroxylamine hydrochloride, 8.0 g. (0.113
mole) and sodium acetate, 2.3 g. (0.113 mole), were
combined with 85 ml of ethanol and the mixture heated
at reflux for 48 hours. ~he resulting mixture was
diluted with water, extracted with ethyl ether, the
extracts washed with water, dried and evaporated to
yield 9.O g. of a mixture of ~y_- and anti-oximes,
identified by its l~-NMR spectrum.
-159-
The oxlme obtained above, 1.3 g. (8.28 mmole) was
dissolved i31 70 ml~ of dry ethanol, 1.9 g. of sodium
metal added and the mixture warmed to reflux and held
at this temperature for 15 minutes. Heating was
continued for two hours with addition of two more
increments (1.9 g. each) of sodium. The reaction
mixture was then diluted cautiously with water,
extracted with ethyl ether. The ether layer extracted
with dilute hydrochloric acid, the aqueous phase made
alkaline with sodium hydroxide and re-extracted with
ether. The extracts were dried (MgSO4) and evaporated
to dryness and the residue distilled to obtain the
desired am:ine, B.P. 68-69C. (15 mm). After further
purification by precipitation of the hydrochloride
salt from ethyl-ether-methanol, basifying the salt and
extracting again with ether 0.87 g. of amine of 93%
purity by gas chromatography (OV-1 column) was obtained.
