Note: Descriptions are shown in the official language in which they were submitted.
-- 2 --
HOE 83/F 192
A large number of processes is known for the pre-
paration of carboxamide and peptide bonds tsee, for
example, Houben-Weyl, Methoden der organ;schen Chemie
(Methods of Organic Chemistry~, Vol. XV, Part II, pages
1-364. Also Angew~ Chemie _ , 129 ~1980)). All these
processes aim, w;th vary;ng success, at ensuring the cri-
ter;a, wh;ch are necessary for the synthesis of pepticles,
of freedom from racemization, and of straightforward pro-
cedure under mild conditions w;th high yields and ~lith
10 readily accessible starting ma.erials.
A process for the preparation of carboxamides is
known from European Patent A1~56,618, in which compounds
containing carboxyl groups are reacted with compounds
~Ihich contain a free amino group in the presence of
15 dialkylphosph;nic anhydrides. The dialkylphosphinic acid
which is liberated during the reaction is bouncl by an
excess of an organic base or a basic buffer which is added
to the reaction mixture at the start.
The present process represents a new way OT opti-
20 mi~ing tlle said conditions for an economic syntheo;s of
pept;des and amides~
It has been found that compounds containin~ car-
boxamide groups, in particular oligopep~ides, can be pre-
pared under mild conditions and in ~ood yield by reacting
,~ 25 compounds which contain a ~ree amino group~ in particular
? aminocarboxylic acid derivatives or pept;des whose carboxyl
., .
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;!;
/,
` ~
~ 5~
-- 3 --
group is protected, in the presence of the anhydride of a
dialkylphosphinic acid, with compounds which contain a
free carboxyl group, in particular aminocarboxyl;c acids
or peptides whose am;no ~roup is acylated. The ne~ pro-
cess comprises mainta;ning the hydrogen ion concentrationof the react;on m;xture within a defined range during the
reaction by metering in a base.
The radicals introduced to protect the functiona
groups can, in the case of synthesis of peptides~ subse-
1û quently be el;minated ;n a customary manner.
Anhydr;des of dialkylphosph;n;c acids are to be
understood to be compounds of the formula I
R R
- O - P ~ O -- P. =. O ~ I
t t
R R
.
in wh;ch R denotes alkyl. The substituents R shown in the
formula can be identical or different. Anhydrides in ~Jh1ch
both P atoms have identical substituents are preferred~
~ Jith;n the scope of the ;nvention, anhydrides o-f
the formula I in which R ;s in each case a lower alkyl,
prefcrably one havin~ 1 to l~ carbon atolns, are particu
2n larly suitable~
The dialkylphosph;nic anhydridès use~ accordin0 to
the invent;on are colorless liquids. They are stable at
room temperature ancl can be d;stilled under reduced pressure
witllout decompos-ition. They are readily soluble in most
~ ~5~
-- 4 --
non-aqueous solvents, especially in lipid solvents, such
as chloroform or rnethylene chloride, but also in polar
solvents, such as DMF and DMA.
Examples of anhydrides of dialkylphosphinic acid
~hich may be mentioned are: methylethylphosphinic anhy-
dride, methylpropylphosphinic anhydride, methylbutylphos-
phinic anhydride, diethylphosphinic anhydride, di-n-propyl-
phosphinic anhydride and di-n-butylphosphinic anhydride.
The preparation of the dialkylphosphinic anhydrides
can be carried out in a manner known per se by, for exam
ple, rcact;on of the d;alkyLphosph;noyl chlorides with
alkyl dialkylphosphinates at 150-160C ~Houben-Weylr
Methoden der Organischen ~hemie, G. Thieme Verl., Stuttgart
~963r Vol. XII, pages 266 et seq.). Processes in which
1S dialkylphosphinic acids, their salts or esters are reacted
with phosgene are particularly preferred (German Patent
2,129,583, German Offenlegungsschrift 2~225,545). ~'
The process according to the invention is pre-
ferably carr;ed out in a mixed aqueous, single- or 2-phase
Z0 system~ within a narrow pH range, preferably at approxi-
mately constan~ pH. It is possible for the pH of the reac~
tion mixtures to be 5-10 and, advantageously, it should be
;n the neutral or weakly acid range; pH values between '~
5.0 and 7.0 are particularly preferred. However, it ;s
Z5 also possible to carry out the syntheses ;n the ~leakly ~
alkaline range. The p~l ;s preferably controlled by metered ~',
addi.ion o-f concentrated aqueous solutions of alkali metal ~;~
hydrox;des~ but it i5 also po~sible to use organic bases, 5j~
such as N-ethylmorpholirle~ triethylamine or trialkylamine ';~
i~:
- ~ . - : .
' .~ : .
:
:~ 2~
-- 5 --
having up to 6 carbon atorns.
For the preparation of oligopeptides by the pro-
cess 2ccording to the ;nvent;on, the starting mater;als
used are, on the one hand~ an am;noacid or a pept;de hav;ng
a protected carboxy( group and, on the other-hand, an
am;noacid or a peptide having a protected amino group.
It ;s poss;ble to use for the protection of the
carboxyl groups all the protective ~roups customary in
peptide synthesis. Esters of straight-chain or branched
aliphatic alcohols, such as methanol, ethanol and tert~-
butanol, are particularly suitable. Esters of aral;phatic
alcohols, s~ch as benzyl alcohol or diphenylmethylcarb;nol,
can also be used~
~ikew;se, it ;s possible to use for the protection
of the amino groups all the protective groups custom3ry in
peptide synthesis~ Examples of particularly suitable
groups ~Ihich may be mentioned are the carboben~oxy radical
and the carbo~tert.-butyloxy radical.
It is poss;ble to use as the solvent all the
anhydrous ;nert solvents customary in pept;de synthesis~
for example methylene chloride, chloroform, d~methyl,or~
mamide, dimethylacetamide~ d;oxane or tetrahydrofurarl.
Solvents wh;ch can be used in the sin~le phase
mixed a~lueous procedure for the reaction are mixtures of
water and an organic solvent which is misc;ble with water~
such as, for example, d;oxane, tetrahydrofuran, dimethyl-
formamide or dime~hylacetamide. The use of systems of
this type ;s especially advantageous when linking pep~i~es
~hich ~re ma;nly solubLe in wrter.
.
For the two-phase mixed aqueous procedure for the
react,on, it is possible to use systems such as~ for
example, ethyl acetate, propyl acetate, n-butyl acetate,
methylene chloride, glycol dimethyl ether, 3-methyltetra-
S hydrofuran and chlorof~rm, each ;n a heterogeneous mix-
ture with water.
As a rule, the reaction takes place suff;c;ently
rap;dly at room temperature. Gentle warm;ng is not
injur;ous. Higher tcmperatures~ say above 30Cr are not
10 adv;sable, espec;ally ;n pept;de synthes;s, because of
the dar1ger of racemization. A reaction temperature between
0 and 3~C is preferred, and one between 5 and ?5C is
particularly preferred.
The process accord;ng to the ;nvent;on makes it
possible for the first time to follo~ the course of the
reaction by the consumption of the base which is used when
the pH is approx;mately constant. For this purposer the
reaction is preferably carried out in an automatic record~
ing pi~-stat, advarlta~eously with v;gorous mixin~ of the
m;xed aqueous system conta;n;ng the reactants, the carboxyl
component, thc am;rle component and the d;alkylphosphin1c
anhydride. The graph provided by the recorder of the con~
sumptiol1 of base as a function of t;me sho~s a curvc which
becoll1es asymptotic toward the end of the reaction ~cf. the
curves repl~oduced ;n the Fig.). Thus, tl)e process accord-
ing to the invention makes it possible to detect the end-
point of the synthet;c react;on ;n a s~raightforward manner~
It ought to be noted that the "apparent" pH va~ues in
these m;xed aqueous systems measured us;ng the rnethod
.
~ "
,
' : `
~2~
descr;bed can differ from the true pH values in these
systems.
When buf-fer solutions are used in a known nanner
to trap the d;all;ylphosphinic acid, then it is unavoidable
5 that excess salts, composed of inorganic salts, occur ;n
the mother l;quors of the batches. When carrying out the
process industrially, they make it difficult to recover
the dialkylphosphinic acid produced from the condensing
agent, and they can give rise to effluent problems.
Durins isolatiorl of the reaction procluGt by extrac
tion from alkali solution with a lipoid phase which is mis-
cible l~ith water to only a limited extent, the alkali
~etal salts wll;ch are forrned dur;ng the course of the re-
action when alkali metal hydroxides are used as the bases
1S in this variant of the process according to the ;nvention
are, because of their relat;ve~y h;gh hyclroph;licity and
in contr~st to the salts resulting when some tertiary
organic bases are used, relatively easy to remove.
Furthcrmore~ contam;nation of the lipo;d phase by er~tracted
20 tert;ary base ;s avoided in this procedure.
The process accord;ng to the invent;on ;s stra;~ht-
forward ~o carry out and provides pep~ides of high op~ical
pur;ty and in hi~h yield~ In addition, ;t is economical ar1d
env;ror1lnentally acceptable.
Z5 The dialkylphosphirl;c anhydr;des have low Molecular
weights, are readily obtained and pur;fied and have a high
proportion o~ reac~ive groups per unit wei~ht and have
h;gh lipophil;c;tyu The dialkylphosphin;c anhydr;des and
the corresponding dialkylphosphinic acids are lipicl~
,
-- 8 --
soluble. Th`i 5 nlakes it possible to work up water-soluble
peptide derivat;ves via a first precipitation scep using
suitable lipid solvents7
The dialkylphosphinic acid obtained from the
d;alkylphosphin;c anhydr;de dur;ng the course of the pep
t;de synthesis can be recovered fro~n the solut;ons remain-
in3 after the synthet;c react;on~ Recovery of the ~ialkyl-
phosph;nic acids from a relatively large amount of aqueous
mother liquors from the synthesis can be carried out by
extract;on w;th solvents such as chloroform and isobutanol
followed by work-up by distillation. In this context, it
;s of particular industrial interest that the dialkyl~
phosphinic ac;ds can be d;stilled in vacuo without decom-
pos;t;on~ The d;a~kylphosph;n;c acids thus recovered from
the ~lork-up can then readily be converted ;nto the corres-
pondin~ dialkylphos~hinic anhydrides by the process of
German Offenlegungsschrift 2,225,545.
In the mixed aqueous procedure described, it is
possible to replace the organic base by alkali metal
hydroxide solutions, and this considerably simplifies the
recovery of the dialkylphosphinic acids during the work~up
after the synthes;s as clescri~ed above. In th;s case, it
is pos3;ble to d1spense w;th the extract10n step. After
liberationr the dialkylpho3phinic ac;d can then be dis-
t;lled directly out of the evaporated mother liquor fromthe synthesis or, ;n the two-phase procedure, it js
recovered From the aqueous phase, after removal of the
l;poid phase, by acidification and extraction. The neutral
inorgarlîc salts then relnain in the aqueous phase.
~ . ~
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g
Example 1:
Ethy~ ester of carbobenzoxyglycine:
__ ____~
7.0 g (~.OS mole) of H-Gly-OCH3.HCl are added, with
stirring at -5C, to a solution of 10.5 9 (0.05 Inole~ oF
5 carbobenzoxyglyc;ne in 120 ml of ethyl acetate. 20 9 of
methylethyLphosphinic anhydride are added dropwise to the
resulting suspension~ The pH of the reaction mixture is
then adjusted to 7.0 at a temperature of 0 to ~10C by
dropuise adcJition of 4 N NaOH using an autotitrator, and
10 the batch~ ~Ihlch is thoroughly mixed by h;gh-speed stirr
ing, is maintailled a~ this pH un~il the recorder connected
to the autotitrator shows that the curve of consumption
of sodium hydroxide SOtUtiOIl has become asymp.otic with
the time axis. This is the case after about 60 r,linutes.
15 The ethyl acetate phase is now removed, extracted twice
w;th 50 rnl of saturated sodium bicarbonate sotution, dried
with sodium sulfate, and, a~ter removal o-f the solvent in
vacuo at room temperature, 1~.25 g (84% of theory) of Z~
dipeptide ester having a meltin~ point of 81C are
20 obta;ned.
Example 2:
Carboben,oxyphenyla~anine Gyclohexylam;de:
3.0 ~t ~0~01 mole~ of Z-Phe-0!t and 1nO ~ ~0~01 nnole,
1~2 nnl) of cyclohexylalnine are dissolve(l in a nlixture of
25 2n ml of tetrahydrofuran and 5 Ml oF water~ After cool;ng
the reac~ion rnixture to ~-5C~ 4 y of methylethylphos
phinic anhydride are added, with stirting, and the p~l of
~he reaction solution is acljusted to 6.0 with ~ N NaOH~
and is maintained constant throughout the react;on time by
: ,
.. ' . ' ,
- 10 -
metered addition of sod;um hydroxide solution as described
in Example 1~ lhe reaction is virtually complete after
60 minutes, as can be seen from the consumption of alkali
metal hydroxide. The reaction sqlution is evaporated in
S vacuo at room temperature, the residue is taken up with
ethyl acetate, and the ethyl acetate solution is washed
with 5% strength potassium bisulfate soLut;on, saturated
sodium b;carbona-te solution and water, dried over sod;um
sulfate and, after rernoval of the solven~ in vacuo at room
telnperature and dr~ of the product in vacuo over P20S~
3.0 g of final product of melting point 167C are obtained,
Ca~g = -3~0 ~c - 1, DM~).
Example 3:
Z-Trp-Gly-OCH
3r35 B (0~01 mole) of Z Trp-~ll are dissolved in
20 ml of isopropyl acetate, 1.25 9 ~0.01 mole) of H~Gly-OCH~
is added, ~he vigorously stirred suspension is cooled to
-5C, and 4.n ml of methylethylphosphinic anhydride are
added at this temperature, at the same time adjustin~ the
pH to 5.7 by metered addition of 4 N NaOH using an auto-
titrator as described in Exa0ple 1. ~Ihen the phases are
thoroughly mixed~ the react;on is finished within ~0 min-
utes. The ~thyl ace~ate phase is rernoved and ~lorked up as
described in ~xampLe 2. ~ield: 3.53 g ~86% of theory)
~a~D-13.5 ~c ~ 0~1, glacial acet1c acid).
Exam~_e 4:
Z-Phe-Arq-Trp-Gly-OCH7
1.55 g (0.005 mole) of H Trp-Gly-OCH~ cl is added
to a solution o~ 2~Z6 ~ ~0~005 rnole) o~ Z Phe-Ar~-O~ in
~-
:'-.' ' , ~ - " ' '
- ' -
,: .
. . .
25 ml of isopropyl acetate, and the v;gorously st;rred
suspens;on ls cooled to -5C. Then ~ ml of nlethylethyl-
phosphinic ar,hydride are added dropwise, maintaining the
pH constant at 5.2 using 4 N NaOH as describeà in
Example 1. After 15 minutes, the temperature of the
reaction m;xture is allowed to reach room temperaturen
The reaction ;s virtually complete after ~0 m;nutes as is
shown by the graph of the consumption of NaOH with reaction
time. The ethyl acetate phase is no~l removed, washed with
water and saturated sodium bicarbonate solution, and the
reaction product is isolated from the dried solution by
evaporation in vacuo at room temperature and di0estion of
the residue with absolute diethyl ether~
Yield after recrystallizatiorl -From ethanol/ether:
; 15 3.0 9 (84.5% of theol~y~, Cu~2DO~ -25.7 (c ~- n.1~ DMF)~ ;
Example 5
_
Z-Lys(~oc~-va~-T~ 3
-1~91 ~ tO.OOS mole~ of Z-Lys(Boc)-OH are dissolved
;n 2S ml of butyl acetate ~Ihich is saturated with ~later~
and 1.65 ~ ~0~005 mole) of H~Val-Tyr~OCH~.HCl are added,
the ~igorously stirred reaction mixture is cooled to -5C~
and the pH of the rapidly stirred mixture is brought to 7.0
ith ~) N NaOH as descr;L)ed in Example 1, and it is main~
ta;ned at this value throu~llout the reaction time ~70 mirl-
utes). The temperature during the first 10 minutes of ther~act;on t;me ;s maintained at 5C ~o 0C, and is th~n
~llowed to ~arm to room temperature. The worl;ing up of
the but~l acetate phase containing the final product is
carried out as indicated in Example 2~
-.
'
~L2'~
~- 12 -
Yield after recrystallization from ethanol/ether:
3.0 9 (91% of theory), Ca~ = ~25 (c = 1~ ethanol)r
Example 6
Z-Gly-Leu-Arg-OCH
-- ~ 3
1.3 ml of methylethylphosph;nic anhydride is added
to a so!.ution of 642 mg of Z-Gly-Leu-OH and 449 mg of H-
Arg-OMe.HCl in 3 ml of dimethylacetamide and 0.5 ml of
water, ancl th~ pll dur;ng the reaction which no~ takes
place is maintained at 7.2 with a mixture of N ethylmor-
phol;ne and wa~er t1~1, vollvol) us;ng a pH-statO The
reaction is co~plete after 40 minutes accord-in~ to the
graph on the recorder~ The work;ng up is carried out as
described in Example 2~ but without the extraction of the
ethyl acetate phase with 5% potassium bisulfate solution
mentioned there~
Yield: 700 mg (71X of theory~
C~X]2DO = -2404 (C = 1, CH30H)
