Note: Descriptions are shown in the official language in which they were submitted.
3~ 6~
2100 . 1~02
BACKGE~OU~ D Sl~ RY OF T~IE ~LNVENTION
.. .... _ .. . _ _ _
The free tetra acid, N-(phosphonoacetyl)-L-aspartic
acid (sometimes referred to herein as PAL~), is a Icno~
compound. The present invention relates to novel N-(p~osphono-
acetyl)-L-aspartic acid (PALA) compounds, especially disodium
PALA, and to methods for preparation o~ N-(phosphonoacetyl)~L~
aspartic acid compounds. A particular aspect of the present
in~ention is concerned ~ith the large-scale preparation o~ the
kno~m tetrasodium sal~ and the novel disodlum salt of N-(phdsphono~
acetyl)-L-aspartic acid.
Z66
The known tetra acid compound, N-IphosPhonoacetyl)-L-aspartlc
acid (PALA), was first prepared by Stark et al,, J. Biol. Chem., 246,
6599 (1971). The tetrasodium salt of PALA is a known antitumor agent,
as reported in the literature, particularly Cancer Research, 36, 2720
~1976). For example, the survival time of mice bearing intraperitoneal
- - P388 leukemia was prolonged by up to 64~ (when treated with P~LA tetra-
sodium salt in a dose range of 188 to 750 mg.~kg., i.p.). Lewis lung
sarcoma was highly sensitive to PALA tetrasodium salt in mice at i.p.
doses from 240 to 490 mg./kg. Mice bearing B16 melanoma survived 77 to
86~ longer than did controls when treated with PALA tetrasodium salt
(490 mg./kg., i.p.).
While the synthesis of the tetra acid PALA is straightforward,
the preparation of the tetrasodium salt, especially in kilogram quantities,
has proven to be a major problem. The methods of the present invention
are particularly well-suited for the production of such quantities.
,,~
~ _ 3 _
2~;6
DES~PTIO~ OI: THE ~'R~FRRrD EMBo-r)l~lENTs
The novel p~,T~ compounds o~ the invention are N-(phosphono-
acetyl)-L-aspartic acid, disodi~ salt, and ~he correspondin~
e~hyl ester and di~en~yl ester;
N-(phosphonoacetyl)-I.-aspartic acid~ dibenzyl ester~ and
the corresponding N,~'-dibenzylethylencdiamine salt and
cyclohexylarnine salt;
N-(phosphonoacetyl)-L-aspartic acid, tetraethyl ester
and -~he corresponding ~imethyl ~,P-diethyl ester;
N-(phos~honoacetyl)-L-aspartic acid, calcium salt; and
the piperazine salt and cyclohexylamine salt of N-
(phosphonoacetyl)-L-aspartic acid.
The novel compounds of the invention either possess anti-
tumor activity in v~vo or are intermedia`~es which can advantageously
be used ror the production of active anti,umor P~L~ compounds or,
unli~e the known P~LA tetrasodium salt, are relatively non-
hygroscopic substances or are mobile, free-flowing par~icula~e
sollds .
Some o these novel PALA compounds exlst in anhydrous
form, some in solvated (including hydra~ed) fonn. Bo~ ~orms
are su;tab]e for purposes of ~he invention. The clisodium P~L~
compound produced by ~he methods o~ the invention is a h~drate,
differing from one preparation to another in the content o water
of hydration, typically ~rom about 0.2 to 2 moles of water.
The compound also may contain as a solvate ethanol (e.g., from
about 0.1 to 0.5 mole), acetic acid (e.g., from about 0.03 to
0.~ mole) and sodium acetate (e.~., about 0.2 mole). The
acetic acid and ethanol can be removed by free~e-drying. Two
to three lyophilizations afford solvent ree material. The
disodium PALA compound produced by these methods may also contain
su~stantial amounts of trisodium PALA including as much as 30
to 40~ trisodium PALA~ All of such solvates of disodium PAL~
and mixtures o~ disodlu~ PALA and trisodium PA~ are intendecl
to be included within the term disodium PALA (or equivalent term)
as used ~erein since these product forms are interchangeable for
use as the active antitumor ingredient of formulations contemplated~
by the invention. For use as an antitumor agent~ the novel
~ 6 ~
disodium PA~A ;s substantially equivalent in activity and
to~icity ~:O tlle lCIIOWII ~:e~rasodium PAT~. Disodium PALA can be
used ~or its an~ ur,~or ac.ivity, according to the invention,
in the ~or~ of pharmace~tical con~positions and a compatible
p~rmace~ltioa 1 ly acceptable carrier The compos;tions may also
con~ain a~timicrobial agen~s and o~her antitumor agents. The
compositions may ~e made up in any pharmaceutical ~orm appropri2~e
for the rout-e of ad~irlistration in ques~ion. Examples of such
compositions include solid compositions for oral administration
such as ~abl~ts, capsules, pills, powders and granules, liquid
compositions ~or t:opical or oral admi~istra~ion such as solutions,
suspensions, syrups and elixirs, and preparations for parenteral
administration such as sterile solutions, suspensions or emulsions.
For use as an anti~umor agent, the compositions are administered
~n a dosage ~egimen such that the tumor growth is inhibited. A
suOgested dosage regi~en ~or use as an antitumor agent (especially
~or solid ~umors as described above) in mammalian species is 50
to 500 mg. of disodium PALA per kilogram ~or a single daily
parenteral (e.g., intravenous infusion, as a 2~ aqueous solution)
tre2tn7ent course. Thc novel calcium saLt of PALA
is e~uivalent pharmaceutically for the
,.
26~
purposes of ~he inventi.on to disoclium PA~ and thus can be
used in place of or in combination ~ith disodiùm PALA in the
above-described composi~ions. The calcium sal~ of l'AL~ has
favorable so]ubility properties; it di~solves ill water
permit~ing ready formula~ion yeL it dissolves rela~ively slowly
thus allowing for ~ater ~ash re~oval of soluble inorganic
i~urities.
The novel disodium PALA advantageously is a mobLle, free-
flowing par~iculate solid which can readily be han~led, analyzed
and weighed for formulation purposes. By co-,nparison with the
kno~Tn tetrasodium PALA, it is relatively nonhygroscopic. Tetra-
sodium PAL~ absorbs atmospheric moisture 1.5 times *as~er than
disodium PALA and is difficult to kandle and al~alyze. Whereas
tetrasodium PALA is water-soluble, disodium
PALA advantageously has a solubility in water of greater than
9~0 mg./ml. Disodium PALA as a 2~ (w./v.) solution in water
characteristically has a pH of about 4; comparable trisodium Pk.I~
and tetrasodium PALA solutions have a pH o about 6 and about 9,
respectively. Disodium P~LA is also charact:erized by a 60-~1c. nucl2ar
Z~I~
,lagnetic resonance (nmr) spect:rum ~Jhich typically shows a
doubJe~ corres~onding l:o t he methylene group (~CH2) ~hich is
alpha to t~e -Cll g, oup whereas tetr2sodiu~ P~LA characteristically
has an nmr spec~r~m ~71-~iCII shows a 3 ~ ine multipleL corl esponding
to the ment;.oIIed methylene group.
The P~ et~ae~hy.l e~;ter and PALA di~nethyl e~ter, P,P-
diethyl este~ are r~adi1.y obtained in an.hydrous fo~m and ~ive
~lemen~al analyses ui~ e~cell.ellt ag~eement l~et~een the ~ound
~nd calc~la~ed values~ For exal~lple, ~he maximu~ difference
bet~een calcula~ed and ound values of hydrclgen ~or ~hese tetra-
esters is 0 03~ comyared with a maximum 1~27~ di~fe~ence for
tet~asodium PALA
C~clohe~yla~ine and piperazine form solid PALA salts.
In addition, the cyclohe~ylamine salt is completely non-
hygroscopic.
One e~.bodimen~ o the inven~ion, in a ~etllod for the
preparation of PALA compour~ds, co~prises the steps o~ ~eacting
~-aspart:ic acid with ben~yl alcohol and p-t:oluenesulfonic acid
t:o obt~in T-aspartic ~cid, dibenzyl es~e~ p-toluenesul~onate,
2Çi6
reacting the l.-asyartic acîd, diben~yl ester p-toluenesulfonclte
wi~h trie~hylamine, ,~dding phosphonoacetyl chloride to produce
PAL~ dibenzyl es~er; and separating the PALA dibenzyl ester from
unreacted phosphonoacetyl chloride. The PALA dibenzyl ester is
se?arated in any sui~able way. Since it is insoluble in water,
the PALA dibenzyl ester and unreacted phosp~onoacetyl chloride
preferabl-y are separa~ed b~ ~7ashing the reaction mixture with water
to remove the phosphonoacetyl chloride.
~ ccording to another embodiment, the dibenzyl ester is
subjected to hydrolysis ~Ji~h aqueous sodium hydroxide ta obtain
a product Inixture containlng te~rasodium PAL~ hereinafter referred
to as the "product mixture".
Another embodiment comprises the steps of subjecting the
product mixture to an ion e~change procedure ~o obtain N-
(phosphonoacetyl)-L-aspartic acid in the free acid form, titrating
said free acid to a pH of 9.2 and recovering the purified tetra-
sodium salt.
Anothe~ embodiment comprises the steps of dissolving said
product mixture in glacial acetic acid, diluting the resulting
solution with etl~nol to precipitate the disodium PALA, and
recovering said disodium PALA.
~ 9 _
Another embodiment c~prises the steps of reacting PAL~
dibenzy-l estex with ~IjN~dibenzyl~tilylenediamine to produce the
N~NI-dibenzylethylenediamine sal.t of the PAL~ diben~yl ester,
subjec~:ing said sal~ to hydrolysis ~o produce a produc'c mi~ture
contain:ing the P~.LA teLrasodium salt~ dissolving said product
~i~ture in glacial 2Ce~iC acid, diluting t-he resulting solu~ion
with ethanol ~o precipitate the PAIJA disodium salt, and recovering
said PA]~ disodium salt.
~ nother embodi~en~ comprises using perchloroethyleIle ~s the
es~e-rificatlon medium in the reaetion o L~aspartic acid with
benzyl alcohol and p-toluenesulfonic acid mono~ydra~e. In
prepari.ng di.ben7:y~ aspartate, perchlox~e~hylene was the solven~ of
choice. It fo~ms an excellent azeotrope with water; it boils
high enough that the large~scale esterification is soon c~npleted
(within 3 hours) and, a~ the same time, the product stabi.lity is
not afected by the ~e~nperature- at least during this s'nor~ ti~le
o thermal con~act.
-- 10 ~
Z66
Another embodi~en~--in a method for the preparation of PAL~
compounds, including ~:he steps of reacting PA~ dibenzyl ester,
cyclohexylami.ne salt with sodium hydroxide to produce L-aspartic
acid, N-(phosphonoacetyl)-, tetrasodium salt, and reacting said
tetrasodium salt with acetic acid to produce disodium PA~ c~prises
preci.pitating ~he obtained disodium salt twice from water, the
second precipitation including adding an aqueous solutior of said
~ 6~
disodium salt drop~ise ~o a vortex oE vigo~ously stirred e~hanol,
~hereby removing ;mpuri.ties in ~he ~orm of acetic acid and sodium
acetate.
~ le production o disodium ~LA according to the in~ention
ofers the ollo~ing advarltages: (1) a shor~er ~ime is required
to synthesize the ~llaterial due to (a) complete elimination of ion
e~change col.umns, ~nd ('o) substantial reduction in the volume o~
wate~ required to be evaporated; (2) the-projected cost for 5 kg.
amoun~s of n~terial is at least 30~ less than t~at or 5 kg. o~
tetr~sodium PAL~; (3) the consistently lo~ hydrogen analyses
associated
- 12 -
Z6i~
v7itll ~:et:raso~iu~] PALA ~re no longer a problem; (4) the synthesis
is -readil~ adaptable to scale-up; (5) ~he produc~ obl:ained is less
hyy,roscopic t:han ~etrasodi.u~ P~A and, unlike tetrasodiu~ PALA,
is a ~nobile, free floT7in~ particulate solid whi.ch can be readily
handled and weighed -for orm~1at-ion purposes; and (6) the ~a~erial
is extremely water soluble.
Another embodj~nt, in ~ method or the preparation o a P~L~
compourld, coml-,rises the steps o reac~;n~ ~-aspartic acid,
dibenzyl es~er p-toluenesulfonate with triethylamine, adding
phosphonoace~yl chlorîde to produce PAL~. dibenzyl ester, and
reac~ing saic~ clibenzyl ester with cyclohexyla~ine to produce
the cyclohexylamine sal~ of said dibenzyl ester.
The following examples are illustrative of the present
inventi.on.
` lll~iZ6~
Example I
L-Aspartic acid, dibenzyl ester p-toluenesulfonate (I)
A stirred mixture of L-aspartic acid (399 g.;
3.00 moles), benzyl alcoho] (1.95 kg.; 18.0 moles),
p-toluenesulfonic acid monohydrate (582 g.; 3.06 moles),
and dry benzene (1.2 1.) was heated at reflux for 16 hours.
The water formed in the reaction (145 ml.) was removed by
means of a Dean-Stark trap. The resulting solution was
cooled to room temperature, then diluted with benzene (1.2 1.)
and ether (3.6 1.). The resulting solid was collected on
a filter, washed with ether (7.0 1.), and dried; yield, 1195
g. (82%). The crude product was recrystallized from methanol
(1720 ml.) to give 959 g. (80% recovery) of purified (I);
m.p., 158-159.5; literature m.p., 158-160.
Additional reactions were caried out to give a
total of 4.27 kg. ofproduct suitable for further transfor-
mation.
- 14 -
jvb/
.
111~26~
Phosphonoacetic acid (II)
A stirred solution of triethyl phosphonoacetate
(900 g.; 4.01 moles) in 6 M hydrochloric acid (6.1 1.)
was heated at reflux for 6.5 hours. The solution was con-
centrated _ vacuo, then last traces of water were removed
by co-evaporation with benzene (2 x 300 ml.). The solid
residue was recrystalli~ed twice from 1.0 ]. of glacial acetic
acid to give 342 g. (60.8%) of acid (II); m.p., 140-141 ;
literature m.p., 143 . Additional reactions were carried
out to give a total of 1020 g. of product suitable for
further transformation.
,
' .
- 15 -
jv~/
111~
h~ noacetyl c~!loride (III~
A stirrcd mixture of phosphonoacetic acid (II)
(35'; g., 2.54 mo]es) ana thionyl chloride (1770 ml.)
was heated'~t S0-SS or 4 5 hours~ 'I'he resulting
solution was concentrated in vacuo ~C35; aspirator
press~re then 1 rQm. Hg) to yive 395.7 g. (98.3%) of
product. The yellow, oily matcrial was used in the
~ollowiny reaction s~ithout further characteriza~ion.
~-Aspartic acid, N-(phosphonoacetvl~-, albenzYl ester (IV)
To a cool (15~3, s~irred suspension of L-aspartic
acid, dibenzyl es~er p-toluenesulfonate (I3 (812 g.;
1.67 moles) in dry dioxane (5 2 1.) ~as added, drop-
wise, ~riethylamine (486 g.; 4 ~0 moles) during 30
minutes.` The resulting solution was stirred at 15
or 30 minutes, then phosphonoacetyl chloride (III)
(395.7 ~.; Z.~00 moles) dissolved in dry dioxane (600
ml.) s~as added, dropwise, durin~ 1 hour. The tempera-
ture was maintained below 15 during the addition.
l'he cooling ~ath s~as removed, and the reaction mixture
was stirred for 1 hour. The insolubles were filtered
off and washed ~ith dioxane (2.0 1.). The fil~rate
s~as concentrated in ~acuo, then the oily residue ~as
dissolved in benzene (10.1 1.). The org~nic solution
was washed s7ith ~ater (6 Y~ 4.0 1 ), driea over
- 16 -
.. . ...... .... . .. ....... ..... . . ... ....
2~i
.
magne~i~n sul~at~, then evaporated in vac~o to ~ive
568 g. (78.1~) of product as a yellow, crusty solid
sui~able for fur~h~r trarlsformation
:.
L~Aspartic acid, N-tphosphonoacetyl)-~ tetrasodium salt
4-$ ~2 ~PALA) (VI~)
. .,. ~
To a cool (10), stirred solution of sodium
h~droxide ~312 g.; 7.80 moles) in 10.0 1. of water was
added, in one portion, L-aspartic acid~ N-(phosphono-
acet~ , dibenzyl ester (IV) tS68 q.; 1.30 moles).
The mix~ure was stir~ed at 10-lS for 6 hours~ then
the insolubles were filtcred of~. The iltrate was
concentrated in vacuo to a volume o 3.0 1. then -
extxactea with methylene chloride ~1 x ~.3 1.) and
ethex (1 x 1.3 1.). The aqueous solution was added to
12.0 1. o~ ethanol resulting in the precipitation
o~ a semi-solid. Ater aecanta~ion, the material was
dissolved in ~ater (680 ml.), and equal portions of the
solution were applied to two AG50~-X~ th~drogen form~
cation exchange resin columns (9.~ cm~ x 25 cm.).
Each column was eluted with 2.0 1. o~ wa~er (20 ~rac-
tions ~f 100 ml. each~. Fractions 7-14 o~ each
column, which contained the ~esired product as deter-
mined by TLC, were combined and evapo~ated in vacuo
(bath temperature ~ 30). ~he oily r~sidue was
.
.. . .. . . . , , ., . . ~ . . .. . . . .. . . . .... . . .. . ...... . ..
2~
dissolved in acetoDe (2.0 1.), charcoal (50 g.) was added,
and the mixture was stirred at room temperature for 18 hours.
The insolubles were filtered off, then the filtrate was evapor-
ated in vacuo. The semi-solid residue (tetraacid; 227.1 g.)
was dissolved in 2.0 1. of water. The stirred solution was
cooled to lO then titrated to pH 9.2 with 1 N aqueous sodium
hydroxide (3123 ml.). The basic solution was concentrated at
reduced pressure (1-2 mm. Hg; ~ 30 ), and the oily residue was
triturated with acetone (5.8 l.). The solid material was par-
tially dried in vacuo then triturated with acetone (2.0 1.)
and ether (2.0 1.). The resulting powder was dried at reduced
pressure over phosphorus pentoxide for 9 days at room tempera-
ture to give 272.2 g. of the desired product.
Calc'd. for C6H6N08P C H N P Na
4Na 4.5 H20 16.99 3.56 3.30 7.30 21.68
Found 16.76 2.29 3.46 7.31 21.61
Spectral Data:
Nuclear Magnetic Resonance (D20)
2.29 (m, 4, -CH2 c~to P+ CH2 ~ to -~H);
204.13 (m, 1, -CH)
Optical Rotation
Observed Literature
C~ D5 + 9~44 (c, 3.7~3 [d~ D + 10.30 (c, 3.798
in water) in water)
- 18 -
jvb/
216~ 1
Chromatograph~:
Thin Layer Chromato~phy
(Cellulose, Quanta/Gram Q2F Glass Plates)
Solvent System Rf Value
1. Ethanol-ammonium hydroxide- 0.13
water (6:1:3)
2. n-Butanol-acetic acid-water 0.30
(5:2:3) (tailing)
3. Lithium chloride ~0.6M)- 0.61
ethanol-ammonium hydroxide
(5:5:1)
4. Ethanol-water (203) 0.81
Quantity Spotted: 112 g.
; Direction: Phospray (A commercial reagent used to visuali~e
phosphorus containing compounds).
Results: The compound moves as one spot in each of the
solvent systems. TLC of the free acid, liberated
from the tetrasodium salt with hydrochloric acid,
gave a negative test for aspartic acid when
sprayed with ninhydrin.
-- 19 --
jvb/
2~
Example 2
L-Aspartic acid, N-(phosphonoacetyl)-, disodium salt 1.1 H20
L-Aspartic acid, N-(phosphonoacetyl)-, tetrasodium
salt 4.5 H20 (VI~)~0.0 g.; 0-236 mole) was dissolved in hot
(90), glacial acetic acid (125 ml.). Celite (5 g.) was added
to the hot, cloudy solution, then the insolubles were filtered
off. The clear, cooled, dark yellow filtrate was diluted with
ethanol (300 ml.), and the resulting mixture was stirred at
room temperature for 30 minutes. The precipitated solid was
collected on a filter, washed by resuspension in ethanol
(3 x 300 ml.) and ether (1 x 300 ml.), then dried to give
5.7 g. (80.8%) of the disodium salt as a white powder. An
additional 5.0 g. of product was prepared in a similar manner.
The combined material (10.7 g.), contaminated with acetic acid
and ethanol (determined by N.M.R.), was dissolved in water
(250 ml.). The aqueous solution was clarified by filtration
then freeze-dried. The lyophili~ing process was repeated
two more times, then the product was dried to constant weight
in vacuo at 40 over phosphorus pentoxide; yield of analytic-
ally pure product, 9.0 g. (84.1% recovery).
Anal.
Calc'd. for C6H8N08P 2 Na 1.1 H20
C H N P Na
22.60 3.22 ~1.39 9.71 14.42
Found 22.68 3.21 4.36 9.62 14.37
- 20 -
- jvb/ -
111~2~ib~
I
; Spectral Data:
Nuclear Magnetic Resonance (D20)
~ 2.75 (d, 2, J=20 J7Z, -CH2 ~ to P); 2.80
(d, 2, -~H2 ~ to -CH); 4.53 (t, 1, -CH)
.. : ,
Optical Rotation:
Observed
r~ D + 15.95 ~c, 2.000 in water)
Chromatography:
Thin Layer Chromatography
(Cellulose, Quanta/Gram Q2F Glass Plates)
Solvent System Rf Value
1. Lithium chloride (0.6 M)-ethanol- 0.47
ammonium hydroxide (5:5:1)
2. Ethanol-water (2:3) 0.75
3. n-Butanol-acetic acid-water 0.19
(5:2:3)
Detection: Phospray ( A commercial spray reagent used to
visualize phosphorus-containing compounds).
Results: The compound moves as one spot in each of the
solvent systems.
Example 3
L-Aspartic acid, N-(phosphonoacetyl)-, disodium salt, mono-
hydrate 0.3 acetic acid 0.1 ethanol
To 2.0 1. of hot (85 ), glacial acetic acid was
added, in one portion, L-aspartic acid, N-(phosphonoacetyl)-,
tetrasodium salt, tetrahydrate (VII) (214 g.; 0.516 mole).
jvb/
111(~26~
After stirring the mixture at 85-90 for 30 minutes, Celite
(50 g.) was added, then the insolubles were filtered off. The
clear, dark yellow filtrate was cooled to room temperature and
liluted with ethanol (4.5 1.). The resulting mixture was
stirred for 30 minutes, then the precipitated solid was collected
on a filter. The material was washed by resuspension in
ethanol (2 x 3.5 1.) and ether ( 1 x 1.2 1.) then dried
in vacuo at 55 over phosphorus pentoxide to give 111.8 g.
(63.8%) of the analytically pure disodium salt.
Anal.
Calc'd. for C6H8NO8P 2 Na H2o 0 3 C2H42 0.1 C2H6O
C H N P Na
24.04 3.50 4.12 9.12 13.53
Found 24.19 3.56 4.14 8.96 13.48
Spectral Data:
Nuclear Magnetic Resonance (D2O)
~ 0.92 (t, 0.3, -CH3 of ethanol); 1.83 (s, 0.9, -CH3
of acetic acid); 2.55 (d, 2, J=20 Hz, -~H2 ~ to P);
2.58 (d, 2, -CH2 oC to -CH); 3.33 (q, 0.2, -~H2 of
ethanol); 4.32 (t, 1, -CH)
Optical Rotation:
Observed
__ _
t~D + 15.31 (c, 2.103 in water)
- 22 -
jvb/
266
. .
Chromatography:
Thin Layer Chromatography
(Cellulose, Quanta/Gram Q2F Glass Plates)
Solvent System Rf Value
1. Lithium chloride (0.6 M)-ethanol- 0.54
ammonium hydroxide (5:5:1)
2. Ethanol-water (2:3) 0.69
3. Ethanol-ammonium hydroxide-water 0.18
(6:1:3) (elongated)
10 4. n-~utanol-acetic acid-water0.20
(5:2:3) (tailing)
Detection: Phospray (A commercial spray reagent used to
visualize phosphorus-containing compounds).
Results: The compound moves as one spot in each of the
solvent systems.
Example 4
L-Aspartic acid, N-(phosphonoacetyl)-, disodium salt, mono-
hydrate 0.3 acetic acid 0.1 ethanol
L-Aspartic acid, N-(phosphonoacetyl)-, tetrasodium
20 salt 4.5 H20 (VII) (716.8 g.; 1.690 moles) was added, in one
portion, to hot (95~, glacial acetic acid ~7.0 1.). The
mixture was stirred at 90-95 for 45 minutes, then Celite
(250 g.) was added. After stirring the hot (90-95) mixture
for 20 minutes, the insolubles were collected on a filter and
washed with acetic acid (0.5 1.)~ The clear, dark orange
filtrate was cooled to room temperature and diluted with
ethanol (16.1 1.). The resulting mixture was stirred for 1
hour, then the precipitated solid was collected on a filter.
, l - 23 -
,
Z6~i
The material was suspended in ethanol (7.5 1.), and the sus-
pension was vigorously stirred for 3 hours. The solid was
collected on a filter then washed as above with ethano]
(2 x 7.5 1.) and ether ( 1 x 7.5 1.). The material was dried
in vacuo at 50-55 over phosphorus pentoxide to give 426.5 g.
(74.3%) of the analytically pure disodium salt.
~nal.
Calc'd. for C6H8N08P 2 Na H2o 0 3 C2H42 0.1 C2H6
C H N P Na
24.04 3.50 4.12 9.12 13.53
Found24.35 3.46 4.19 8.78 13.44
Spectral Data:
Nuclear Magnetic Resonance (D20)
~ 0.98 ~t, 0.3, -~H3 of ethanol); 1.88 (s, 0.9,
- CH3 of acetic acid); 2.61 (d, 2, J=20 Hz, -~H2
~ to P); 2.63 (d, 2, -CH2 d to -CH); 3.44
(q, O.2, -CH2 of ethanol); 4.36 (t, 1, -~H)
Optical Rotation:
Observed
~ D ~ 14.86 (c, 1.998 in water)
Chromatography:
Thin Layer Chromatography
(Cellulose, Quanta/Gram Q2F Glass Plates)
- 24 -
jvb/
X
Solvent SystemRf Value
1. Lithium chloride (0.6 M) -ethanol- 0.60
ammonium hydroxide (5:5:1)
2. Ethanol-water (2:3) 0.78
3. Ethanol-ammonium hydroxide-water 0.19
(6:1:3)
- 4. n-Butanol-acetic acid-water0.22
(5:2:3) (elongated)
Quantity Spotted: 80 ~ g.
Detection: Phospray (A commercial spray reagent used to
visualize phosphorus-containing compounds).
Results: The compound moves as one spot in each of the
solvent systems.
Example 5
L-Aspartic acid, N-(phosphonoacetyl)-, dibenzyl ester,
' N,N' - dibenzylethylenediamine Salt (XII)
To a cool (5), stirred solution of L-aspartic
acid, N-(phosphonoacetyl)-, dibenzyl ester (IV) (2449 g.;
5625 moles) in methylene chloride (9.5 1.) was added, drop-
wise, N,N'-dibenzylethylenediamine (1488 g.; 6.191 moles)
dissolved in methylene chloride (1.65 1.) during 3.0 hours.
The temperature was maintained below 15 during the addition.
- After removing the cooling bath, the reaction solution was
stirred at room temperature for 16 hours then concentrated
in vacuo to an oil. The residue was dissolved in acetone
(5.0 1.), and the solution was stored overnight (18 hours) at
room temperature. A finely divided, white solid, which had
precipitated from solution, was filtered off, then the filtrate
was evaporated at reduced pressure. The crude material was
- 25 -
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lll(~Z~
dissolved in ethyl acetate (12.0 1.). The organic solution was
washed with water (3 x 3.5 1.), dried over magnesium sulfate,
stirred with Norit A (125 g.) fGr 45 minutes, then spin-
evaporated in vacuo. The residue ("glass") was triturated to
a powder by vigorous stirring with ether-petroleum ether
(b.p., 30-60) (5.0 1.: 7.0 1.). The solid product was collected
on a filter then dried to give 1609 g of the tan colored salt;
m.p., ~ 300 . An additional reaction was carried out in a
similar manner to give a total of 2308.5 g. of (XII) suitable
for the further transformation.
L-Aspartic acid, N-(phosphonoacetyl)-, tetrasodium salt (V)
To a cool (14), stirred solution of sodium hydro-
xide (240 g.; 6.00 moles) in water (7.8 1.) was added, in
portions, thoroughly pulverized L-aspartic acid, N-(phosphono-
acetyl~, dibenzyl ester, N,N'-dibenzylethylenediamine salt
(XII) (675.7 g.) during 5 minutes. The reaction mixture was
stirred at 10-15 for 6 hours, Celite (250 g.) was added,
then the insolubles were filtered off. The filtrate was ex-
tracted with methylene chloride (2 x 1.5 1.) and ether
(1 x 1.5 1.) then concentrated in vacuo (C40; 3-5 mm. Hg ).
The aqueous solution (3.8 1. volume) was clarified by filtra-
tion and diluted with ethanol (13.5 l.)resulting in the preci~
pitation of an oil. After standing for 18 hours at room tempera-
ture, the aqueous ethanol solution was removed leaving 600 ml.
of crude (V) as an orange oil. Additional hydrolyses were
carried out in a similar manner to give a total of 1350 ml.
of oil suitable for further transformation.
- 26 -
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. ,!
L-Aspartic acid, N-(phosphonoacetyl)-, disodium salt
0.2 H20 0.2 sodium acetate 0.4 acetic acid 0.15 ethanol
__
I.-Aspartic acid, N-(phosphonoacetyl)-, tetrasodium
salt (V) (1350 ml. of oil) was dissolved in glacial acetic
acid (6.5 1.) at room temperature. The orange solution was
stirred for 30 minutes, clarified by filtration, then diluted
with ethanol (18.0 1.). The resulting mixture was stirred for
; 1 hour, then the solvent was removed using filter candles. The
solid was suspended in ethanol (10.5 1.) and the mixture was
vigorously stirred for 1 hour. The ethanol was drawn off as
above, then the material was washed twice more with ethanol
tlO.5 1. then 6.0 1.). The solid was collected on three filters,
under a nitrogen atmosphere, washed with ether (2 x 1.0 1./
funnel), then partially dried by spin~evaporation at reduced
pressure (30-45 ; aspirator then 3-5 mm Hg). The lumpy solid
was thoroughly pulverized, under nitrogen, then dried in vacuo
over phosphorus pentoxide (33.5 hours at room temperature and
12.5 hours at 50) to give 945.1 g. of the analytically pure
desired product.
Anal.
Calc'd. for C6H8N08P 2 Na 0.2 H20 0.2 C2H302Na
0 4 C2H42 0.15 C2 6
C H N P Na
25.74 3.31 4.00 8.85 14.45
Found 25.55 3.35 4.06 9.19 14.30
- 27 -
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X
%Çi~
Spectral Data:
Nuclear Magnetic Resonance (D20)
S 0.89 (t, 0.45, -CH3 of ethanol); 1.78 (s, 1.8,
-CH3 of acetate + acetic acid); 2.54 (d, 2,
-CH2 ~ to -CH); 2.54 (d, 2, J=20.3 Hz,
-~H2 ~ to P); 3.36 (q, 0.30, -CH2 of ethanol);
4.28 (t, 1, -CH)
Optical Rotation:
Observed
10 C~D + 16.39 (c, 1.885 in water)
Chromatography:
Thin Layer Chromatography
(Cellulose, Quanta/Gram Q2F Glass Plates)
; Solvent System Rf Value
1. Lithium chloride (0.6 M)-ethanol-- 0.64
; ammonium hydroxide (5:5:1~
2. Ethanol-water (2:3) 0.78
3. Ethanol-ammonium hydroxide-water - 0.24
~6:1:3) (elongated)
4. n-Butanol-acetic acid-water 0.28
(5:2:3) (tailing)
Detection: Phospray (A commercial spray reagent used to
visualize phosphorus-containing compounds).
Results: The compound moves as one spot in each of the
solvent systems.
- 28 -
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Z~
.
Example 6
L-Aspartic acid, N-(phosPhonoacetyl)-, dibenzyl ester (IV)
To a cool (10 ), stirred suspension of L-aspartic
acid, dibenyl ester p-toluenesulfonate (I) (5785 g.; 11.91
moles) in dry dioxane (30.0 1.) was added, in one portion,
triethylamine (3238 g.; 32.00 moles). The resulting solution
was stirred for 1 hour then phosphonoacetyl chloride (III)
(2236 g.; 14.11 moles2 dissolved in dry dioxane (5.5 1.) was
added, dropwise, during 3 hours. The temperature was maintained
below 30 during the addition. The cooling bath was removed,
and the reaction mixture was stirred for 1 hour. The insolubles
were filtered off and washed with dioxane (12.0 1.). The
filtrate was concentrated in vacuo, then the oily residue was
dissolved in methylene chloride (64.0 1.). The organic solu-
tion was washed with water (6 x 19.0 1.), dried over
sodium sulfate, then evaporated at reduced pressure to a
volume of 5.0 1.
L-Aspartic acid, N-(phosphonoacetyl)-, tetrasodium salt (V)
To a cool (15), stirred solution of sodium hydro-
xide (1323 g.; 33.08 moles) in water ~43.0 1.) was added, in
one portion, 2.4 1. of the above prepared methylene chloride
solution of L-aspartic acid, N-(phosphonoacetyl)-, dibenzyl
ester (IV) (2.4 1. = 2400 g.; 5.512 moles). The reaction
mixture was stirred at 10-15 for 8 hours, Celite (825 g.)
was added, then the insolubles were filtered off (600 g.
Celite pad). The filtrate was extracted with me~hylene chloride
(2 x 9.0 1.) and ether (1 x 9.0 1.). The aqeuous solution was
combined with that from an identical run and concentrated
in vacuo ( ~ 35 ; 3-5 mm. Hg). The solution (25.0 1. volume)
~j, ,.
- 29 -
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Z~i~
was clarified by filtration (400 g. Celite pad) and diluted
with ethanol (88.0 1.) resulting in the precipitation of an
oil. After standing for 7.5 hours at room temperature, the
aqueous ethanol solution was removed leaving 4.4 1. of crude
(V) as an orange oil. An additional 6695 g. of (IV) was
hydrolyzed in a similar manner to give a total of ~.98 1. of
oil suitable for further transformation.
L-Aspartic acid, N-(phosphonoacetyl~-, disodium salt
L-Aspartic acid, N-(phosphonoacetyl)-, tetrasodium
salt ~V) (4.4 1. of oil) was dissolved in glacial acetic acid
(14.0 1.) at room temperature. The orange solution was stirred
for 1 hour, clarified by filtration, then diluted with ethanol
(44.0 1.). The resulting mixture was stirred for 1.5 hours,
! then the solvent was removed USillg filter candles. The solid
was suspended in ethanol (30.0 1.), and the mixture was
vigorously stirred for 2 hours. The ethanol was drawn off as
above, then the material was washed by resuspension in ethanol
(2 x 30.0 1.) and ether (1 x 14.0 1.). The solid was collected
on two filters, under a nitrogen atmosphere, then partially
dried by spin-evaporation in vacuo (30-45 ; aspirator pressure
then 3-5 mm Hg). An additional 2.93 1. of oil (V) was reacted
in a similar manner. The combined lumpy material was thoroughly
pulverized, under nitrogen, then dried in vacuo over phosphorus
pentoxide (40 hours at room temperature and 17 hours at 45-50)
to give 4562.5 g. of a light yellow powder. The nuclear
magnetic resonance spectrum and elemental analysis of this
material revealed the presence of sodium acetate (0.1 mole),
acetic acid (0.5 mole), and ethanol (0.15 mole). A 2000 g.
30 -
jvb/
Zi6
. .
portion of the product was added, in portions, to 11.5 1. of
vigorously stirred glacial acetic acid during 20 minutes. The
mixture was stirred at room temperature for 1 hour, then the
solution was clarified by filtration. The filtrate was diluted
with etnanol (26.0 1.), and the resulting mixture was stirred
for 2 hours. The solvent was removed (filter candles), then
the solid was wasned twice by resuspension in ethanol (7.0 1.
then 15.0 1.), collected on a filter, and dried by spin-
evaporation at reduced pressure. The white solid (1989 g.)
was determined by nuclear magnetic resonance to contain acetic
acid (1.25 moles) and ethanol (0.24 mole). A 1974 g. quantity
of the material was dissolved in water (4.0 1.), and the
aqueous solution was diluted with ethanol (16.0 1.). The
resulting mixture was stirred for 30 minutes, then the precipi-
tated oil was allowed to settle. The aqueous ethanol solution
was removed, and the oil was washed once with ethanol (3.5 1.).
This material, which contained acetic acid (0.03 mole) and
ethanol (0.8 mole) as determined by nuclear magnetic resonance,
was dissolved in water (32.0 1.~. The aqueous solution was
clarified by filtration then freeze-dried to give 1512.4 g.
of a flocculant, yellow solid.
Anal.
Calc'd for C6H6N08P 2 Na . ~12 0 03 C2H42 0.35 C2H60
C H N P Na
24.23 3.68 4.18 9.24 13.72
Found 24.39 3.56 4.15 9.03 13.44
- 31 -
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266
Spectral Data:
Nuclear Magnetic Resonance (D20)
~ 1.19 (t, -CH3 of ethanol); 2.11 (s, -CH3 of
acetic acid~; 2.83 (d, 2, J=20 Hz, -~H2 ~ to P);
2-87 (d, 2, -~H2 ~ to -CH~; 3.67 (q, -~H2 of
ethanol); 4.61 (t, 1, -CH)
Otpical Rotation:
Observed
~D + 14.68 (c, 1~873 in water)
Chromatography:
Thin Layer Chromatography
(Cellulose, Quanta/Gram Q2F Glass Plates)
Solvent SystemRf Value
l. Lithium chloride (0.6 M)-ethanol- 0.65
ammonium hydroxide (5:5:1)
2. Ethanol-water (2:3) 0.83
3. Ethanol-ammonium hydroxide-water 0.31
- (6:1:3) (elongated)
4. n-Butanol-acetic acid-water0.28
(5:2:3)
Detection: Phospray (A commercial spray reagent used to
visualize phosphorus-containing compounds).
Results: The compound moves as one spot in each of the
solvent systems.
.~ jvb/
%6~i
Scale-up development for the preparation of the
pure salt has overcome some potentially serious manipulative
- problems. The cyclohexylamine salt of dibenzyl PALA is pre-
- pared by adding from about 0.9 to about 1.0 equivalent of
cyclohexylamine to an acetone solution of dibenzyl PALA.
The product is insoluble in acetone whereas a large percentage
of impurities remain in solution. The purity of the product
is upgraded to an acceptable level by recrystallization from
absolute methanol.
Difficulties are encountered in the use of dioxane
for the preparation of dibenzyl PALA. Triethylamine hydrochlor-
ide is an insoluble by-product of the reaction, and large
volumes of solvent are required in order to maintain sufficient
stirring. In addition, the reaction is exothermic, and the use
of dioxane limits the extent of cooling to -~12, which is the
temperature at which dioxane freezes.
The solvent substituted for dioxane in this reaction
was methylene chloride. This solvent offers the following
advantages: (a) It is nonflammable; (b) It allows for a
lower cooling temperature; (c) The volume of solvent is reduced
in half; (d) The removal of triethylamine hydrochloride by
- filtration is eliminated since it is soluble in the reaction
mixture; and (e) The evaporation of the solvent prior to
work-up is no longer necessary.
Additional process improvements include the fact
that the cyclohexylammonium salt is hydrolyzed directly to
the tetrasodium PALA. This eliminates the extra manipulation
of releasing the dibenzyl PALA from the amine salt prior to
! jvb/
?Z~6
~, .
hydrolysis.
A final point is that the volume of water required
for the hydrolysis has been reduced by 63% over that used in
the initial synthetic work. This, of course, allows for larger
scale runs to be made using the same size equipment. At the
bench scale, using as a maximum 50 1. flasks, this procedure
has been used to prepare disodium PALA in ~2 kg. lots. Incor-
porating all of the described modifications, a run using 50
and 100 gallon Pfaudlers has been successfully carried out.
At full scale, ~15 kg. of the target material can be produced
per run using this size equipment. The process, as currently
developed, is limited only by the siæe of the equipment.
By the present methods, the purity of the desired
disodium PALA material has been upgraded to a level satisfactory
for parenteral administration in a suitable vehicle for treat-
ment of human cancer, particularly for investigatlonal purposes
on a large scale. This was accomplished by (1) completely
eliminating acetic acid and sodium acetate through a turbulent
flow precipitation; and (2) isolating dibenzyl PALA as the
cyclohexylammonium salt. In addition, the procedure has been
optimized for ease of scale-up, and the problems of process
manipulations have been solved.
Example 7
Phosphonoacetyl chloride (III)
To a stirred mixture of phosphonoacetic acid (II)
(2000 g.; 14.28 moles), N, N-dimethylformamide (208.8 g.;
2.856 moles), and dioxane (7.15 1.) was added, dropwise,
- 34 -
X jvb/
thionyl chloride (3568 g.; 29.99 moles) during 1.5 hours.
The temperature was maintained below 30 during the addition.
The resulting solution was heated at 45 for 2.5 hours then
cooled to 5 . Water (283 ml.; 15.7 moles)dissolved in dioxane
~2.5 1.) was then added, dropwise, over a period of 2 hours.
The temperature was kept below 10 during the addition. This
solution of acid chloride (III) was stirred at 5-10 for 40
minutes then used in the following reaction without further
characterization. A second chlorination was carried out con-
currently, under the same conditions, using identical quantities
of reactants.
L-Aspartic acid, N-~pho_phonoacetyl)-, dibenzyl ester (IV)
A stirred suspension of L-aspartic acid, dibenzyl
ester p-toluenesulfonate (I) (4625 g.; 9.525 moles) in dioxane
(20.0 1.) was cooled to 15, then triethylamine (4820 g.;
47.63 moles~ was added, in a thin stream, during 1 hour. The
resulting solution was stirred for 20 minutes, then the above
solution of phosphonoacetyl chloride (III), prepared from 14.28
moles of the corresponding acid, was added, dropwise, over a
period of 5 hours. The temperature was maintained below 20
during the addition. Additional triethylamine (1162 g.;
11.48 moles) was added and the reaction mixture was stirr-ed for
1 hour. After standing for g hours at room temperature, the
mixture was diluted with acetone (5.5 1.), stirred for 15
minutes, then the insolubles were collected on a filter and
washed with dioxane (10.0 1.~. A second reaction was carried
out concurrently, under the same conditions, using identical
amounts of materials. The filtrates from tlle two runs were
jvb/
2~6
combined and spin-evaporated in vacuo. The residue (orange,
viscous oil) was dissolved in methylene chloride (110.0 1.),
then the organic solution was gently washed with water (6 x
30.0 1.). After drying the solution over sodium sulfate
(11.3 kg.) and magnesium sulfate (2.3 kg.), the insolubles
were filtered off (Celite pad), and the filtrate was evaporated
in vacuo to constant weight; yield of dibenzyl PALA (IV)
7970 g. (96.1%l. This yellow, viscous oil was suitable for
further transformation.
L-Aspartic acid, N-(phosphonoacetyl)-, dibenzyl ester,
cyclohexylamine salt
Cyclohexylamine (1815 g.; 18.30 moles) was added,
dropwise, to a cold (7), stirred solution of L-aspartic acid,
N-(phosphonoacetyl)-, dibenzyl ester (I~) (7970 g.; 18.30
moles) in acetone ~24.0 1.) during 1.25 hours. The temperature
was maintained below 15 during the addition. The co~ling
bath was removed, and the resulting mixture was stirred for
1 hour. The mixture was stored at room temperature for 6
hours, t~hen the precipitated solid was collected on a filter,
washed with acetone (15.0 1.), and dried: yield, 4932 g.;
m.p., 176.5-177.5 . This material was recrystallized from
boiling methanol (35.0 1.) then dried to give 1663 g. of the
purified salt; m.p., 178-181; The mother liquor was concen-
trated in vacuo to a volume of 20.0 1. The solution was
diluted with acetone (16.0 1.) and cooled (-10) to give an
additional 967 g. of product; m.p., 177-180. A third crop of
material (429 g.) was obtained by evaporating the above methanol-
- 36 -
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1~11;t;26~
acetone filtrate to near dryness and suspending the residue
in acetone (5.0 1.); total amount of the purified amine salt
suitable for further transformation, 3059 g. (62.0% recovery).
L-Aspartic acid, N-(phosphonoacetyl)-, tetrasodium salt (V)
To a cold (5), stirred solution of sodium hydroxide
(1291 g.; 32.28 moles) in water (20.5 1.) was added, in por-
tions, during 30 minutes, L-aspartic acid, N-(phosphonoacetyl)-,
dibenzyl ester, cyclohexylamine salt (3059 g.; 5.378 moles).
The reaction mixture was stirred at 5-15 for 3.5
hours, then extracted with methylene chloride t2 x 8.5 1.)
and ether (1 x 8.5 1.~. The aqueous solution was clarified
by filtration, concentrated in _acuo ( ~35; 3-5 mm. ~Ig) to
a volume of 14.6 1., then diluted with ethanol (51.4 1.).
The resulting mixture was stirred for 1 hour and stored at room
temperature for 12 hours. The aqueous ethanol solution was
removed giving crude (V) as a light yellow oil suitable for
further transformation.
L-Aspartic acid, N-(phosphonoacetyl)-, disodium salt
Glacial acetic acid (8.0 1.) was added to the above
precipitated oil ~crude L-aspartic acid, N-(phosphonoacetyl)-,
tetrasodium salt (V) prepared from 3059 g. of the amine salt~ .
The mixture was stirred at room temperature for 30 minutes,
then a gelatinous insoluble was filtered off. The clear, light
yellow filtrate was diluted with ethanol (24.0 1.). The
resulting mixture was stirred for 1.75 hours, then the pre-
cipitated material was collected on a filter. The solid was
suspended in ethanol (14.5 1.), and the mixture was vigorously
X 37
jvb/
l~l.(~Z6~i ~
stirred for 1 hour. The prod~sct was collected on four filters
then partially dried by spin-evaporation in vacuo (30-45 ;
aspirator pressure then 3-5 mm. Hg). The lumpy material
(2870 g.) was dissolved in water (5.25 1.), the solution was
clarified by filtration, then the filtrate (~6.9 1. volume)
was diluted with ethanol (21.0 1.). The resulting mixture was
stirred for 30 minutes, then the precipitated oil was allowed
to settle (1 hour). The aqueous ethanol solution was removed,
and the oil was washed once with ethanol (4.3 1.). This material
was dissolved in water (8.15 1.), and the solution (9.8 1.)
was divided into three portions (two of 4.0 1.; one of 1.8 1.).
Each portion was added, during 13 hours, to the vorte~ of
vigorously stirred ethanol (10 x aqueous volume: 2 x 40.0 1.;
1 x 18.0 1.). After stîrring the mixtures for 2 hours, the
water-ethanol solutions were siphoned off, and the solid from
the three precipitations was combined. The material was stirred
for 30 minutes in ethanol (10.0 1.), collected on a filter,
then dried to constant weight in vacuo at room temperature
over phosphorus pentoxide. The dried product (1748.0 g.)
was passed through a 150,f~ , stainless steel sieve and thoroughly
blended to give the disodium PALA as a white powder.
Anal.
Calc'd for C6H7 6N08P2.4 Na 2 H20 0.5 C2H60
C H N P Na
22.91 4.01 3.82 8.44 15.04
Found 23.16 3.76 3.79 8.57 15.18
.
- 38 -
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,,~
~1 .
26~
Sodium analysis indicates a composition of
60% di-Na P~LA
40% tri-Na PALA
Based on the empirical formula,
% H2O = 9.8%
% EtOH = 6.3%
Spectral Data:
Nuclear Magnetic Resonance (D2O)
~ 1.17 (t, 1.5, -CH3 of ethanol); 2.74 (d, 2, -CH2
C~to -CH); 2.77 (d, 2, J=20 Hz, -CH2 CX to P);
3.63 (q, 1, -CH2 of ethanol); 4.48 (t, 1, -CH)
Optical Rotation:
! Observed
~D + 14.73 (c, 2.098 in water)
Chromatography:
Thin Layer Chromatography
(Cellulose, Quanta/Gram Q2F Glass Plates)
Solvent SystemRf Value
1. Lithium chloride (0.6 M)-ethanol- 0.52
ammonium hydroxide (5:5:1)
2. Ethanol-water (2:3) 0.72
3. Ethanol-ammonium hydroxide-water 0.16
(6:1:3) (elongated)
4. n-Butanol-acetic acid-water0.22
(5:2:3) (tailing)
~ 39 -
jvb/
:
. - lll~Z6~
.
Detection: (a) Ninhydrin
(b) Phospray
Results: The compound moves as one phospray positive spot
in each of the solvent systems. No aspartic acid
was observed on spraying with ninhydrin.
Example 8
Phosphonoacetic acid, P,P-diethyl ester
Triethyl phosphonoacetate (89.7 g.; 0.400 mole) was
added, in one portion, to potassium hydroxide (0.408 mole;
26.1 g. of 87.6% pure material) dissolved in ethanol (450 ml.)
and water (150 ml.). The solution was stirred at room tempera-
ture for 22 hours then spin-evaporated in vacuo (bath tempera-
ture ~ 25; 3-5 mm. Hg). The residue was suspended in ether
(400 ml.). The crystalline material, potassium salt, was
collected on a filter then dissolved in water (300 ml.). The
stirred solution was cooled to 5, then concentrated hydro-
chloric acid (33 ml.) was added, dropwise, during 15 minutes.
The temperature was maintained below 10 during the addi~ion.
The solution was stirred at 5-10 for 30 minutes then concen-
trated in vacuo (bath temperature ~ 25; 3-5 mm. Hg). The
residue was suspended in acetone (350 ml.). The insoluble
potassium chloride was filtered off, and the filtrate was
evaporated at reduced pressure. Ether (500 ml.) was added to
the residual oil. The resulting mixture was cooled to 10 ,
and the insolubles were filtered off. The filtrate was deco]orized
with charcoal, dried over magnesium sulfate, then concentrated
in acuo to give 69.3 g. (88.2~) of product as a pale yellow
- 40 -
X jvb/
lil~
oil. This material was suitable Lor further transformation.
Phosphonoacetyl chloride, P,P-diethyl ester
Oxalyl chloride (6305 g.; 0.500 mole) dissolved in
dry benzene (100 ml.) was added, dropwise, to a cool (10 ),
stirred solution of phosphonoacetic acid, P,P-diethyl ester
(19.6 g.; 0.100 mole~ in dry benzene (350 ml.) during 45
minutes The temperature was maintained at 5-10 during the
addition. The cooling bath was removed, and the solution was
stirred for 2 hours. The volatiles were removed in vacuo,
then the residue was co-evaporated with benzene (2 x 50 ml.)
to give the acid chloride as a yellow liquid. This material
was used in the following reaction without further characteri-
zation.
L-Aspartic acid, N-(phosphonoacetyl)-, dibenzyl ester,
P,P-diethyl ester
To a cool (15), stirred suspension of L-aspartic
acid, dibenzyl ester p-toluenesulfonate (53.4 g.; 0.110 mole)
in dioxane (325 ml.) was added, dropwise, triethylamine (21.3 g.;
0.210 mole) during 10 minutes. The resulting solution was
stirred at 10-15 for 15 minutes, then a solution of acid
chloride, prepared from 0.100 mole of acid in dioxane (100 ml.)
was added, dropwise, during 50 minutes. The temperature was
maintained below 15 during the addition. The cooling bath
was removed, and the reaction mixture was stirred for 2 hours.
The insoluble material was collected on a filter then washed
with dioxane (50 ml.) and ether (100 ml.). The filtrate was
spin-evaporated in vacuo, and the residue was dissolved in
- 41 -
~? jvb/
i266
benzene (500 ml.). The organic solution was washed with water
~4 x 150 ml.), decolorized with charcoal (10 g.), dried over
magnesium sulfate, then concentrated in vacuo to an oil;
yield, 51.6 g. ( ~ 100%2. A 22.8 g. portion of the crude
product was dissolved in ethanol (80 ml.). The solution was
added, in one portion, to AG50W-X8 (hydrogen form) cation
exchange resin (550 ml.) suspended in ethanol (200 ml.),
and the mixture was stirred at room temperature for 9 hours.
The resin was collected on a filter and washed with ethanol
(250 ml.), then the filtrate was evaporated in vacuo. The
oily residue was dissolved in ben~ene (400 ml.), then the
solution was dried over magnesium sulfate and spin-evaporated
at reduced pressure. The material was dried to constant weight
in vacuo to give 19.7 g. (86.5% recovery) of tlc homogeneous
product (silica gel; acetone-petroleum ether (b.p., 30-60 )
(2:3) or ethyl acetate). This viscous, yellow oil was suitable
for further transformation.
L-Aspartic acid, N-(phosphonoacetyl)-, P-ethyl ester, disodium
salt, monohydrate
To a cool (10 ), stirred solution of L-aspartic
acid, N-(phosphonoacetyl)-, dibenzyl ester, P,P-diethyl ester
(19:7 g.; 0.0400 mole2 in absolute ethanol (lO0 ml.) was added,
dropwise, sodium hydroxide (5.00 g.; 0.125 mole) dissolved in
absolute ethanol (60 ml.) during 20 minutes. The temperature
was maintained below 15 during the addition. The solution
was stirred at 10-15 for 45 minutes then heated at reflux for
30 minutes. An additional 3.2 g. (0.080 mole) of sodium
hydroxide dissolved in ethanol (200 ml.j was added, and the
jvb/
zl~6
mixture was refluxed for 2.5 hours. The resulting solid was
collected on a filter, washed by suspension in ethanol (70 ml.)
and ether (150 ml.), then dried. This tan powder (16.4 g.)
was dissolved in hot (75), glacial acetic acid (30 ml.).-
The solution was cooled to room temperature, diluted with
ethanol (50 ml.), then clarified by filtration. Ethanol
(300 ml.) was added to the filtrate, and the resulting mixture
was cooled. The precipitated solid was collected on a filter,
successively washed by suspension in ethanol (350 ml.), acetone
(250 ml.), and ether (250 ml.), then dried; yield of product,
9.2 g. (66.6%). An 8.3 g. portion of this material, contaminated
with acetic acid (determined by N.M.R.), was suspended in
ethanol (200 ml.). The stirred suspension was heated at reflux
for 15 minutes then cooled to room temperature. This heating-
cooling process was repeated two more times, then the solid was
collected on a filter. The above washing procedure was per-
formed a total of three times. The white solid was dried to
constant weight in vacuo at 40 over phosphorus pentoxide to
give 7.2 g. (86.7% recovery) of analytically pure product,
diso-dium PALA, P-ethyl ester H20. The compound moves as one
spot on cellulose (Quanta/Gram Q2F glass plates) developed
with ethanol-water (2:3~ (Rf = 0.80), ethanol-ammonium hydroxide-
water (6:1:3) (Rf = 0.42), or n-butanol-acetic acid-water
(5:2:3) (Rf = 0.40); detection by Phospray.
Anal.
8 12 8 2
C H N P Na
27.84 4.09 4.06 8.97 13.32
Found 27.71 4.13 4.07 8.86 13.45
- 43 -
jvb/
.
10~
Spectral Data:
Infrared (Nujol)
Major bands: 3300, 2950, 2920, 2860, 1700, 1640,
1600, 1455, 1405, 1375, 1215, 1045,
935, 760 cm
Nuclear Magnetic Resonance (D20)
1.28 (t, 3, -CH3 of ethyl group); 2.82 (d, 2, -~H2
to -CH); 2.85 (d, 2, J = 20.0 Hz, -~H2 ~ to P);
4.00 (5 line m, 2, -~H2 of ethyl group); 4.57
(t, 1, -~H)
Example 9
L-Aspartic acid, N-(phosphonoacetyl)-, dibenzyl ester disodium
salt, tetrahydrate
A solution of L-aspartic acid, N-(phosphonoacetyl)-,
i dibenzyl ester (26.7 g.; 0.610 mole) and triethylamine (25.2 ml.;
0.180 mole) in acetone (180 ml.) was stirred at room temperature
for 1 hour. The volatiles were removed in vacuo, then the
semi-solid residue was dissolved in acetone (300 ml.). Sodium
oxide (27.0 g.; 0.180 mole) dissolved in acetone (150 ml.) was
then added, and the reaction solution was heated at reflux
for 3 hours. The solvent was removed by spin-evaporation at
reduced pressure, then the residue was suspended in water
(300 ml.). The solid material was collected on a filter, washed
with methanol (200 ml.) and acetone ~00 ml.)~ then dried to
constant weight to give 7.7 g. (26%) of ana]ytically pure
product; m.p., 7 300 . The compound either streaks or remains
at the base line on tlc. A sample was therefore converted to
the free acid with hydrochloric acid. This material moves as
X - 44 -
jvb/
.- ~
Z66
.
one spot on cellulose (Quanta/Gram Q2F glass plates) developed
with methanol-water (9:1), ethanol-acetone-water (5:4:1), or
ethanol-ether (1:1).
Anal.
20 20 8 2
C H N P Na
43.575.122.545.62 8.34
Found43.954.692.505.57 8.31
Spectral Data:
Infrared (Nujol)
__ .
Major bands: 3600, 3360, 2960, 2920, 2860~ 1740,
1720, 1635, 1540, 1450, 1375, 1340, 1280,
1200, 1130, 1045, 965, 725 cm 1
Example 10
L-Aspartic acid, diethyl ester, hydrochloride
Hydrogen chloride was bubbled into a stirred sus-
pension of L-aspartic acid (133 g.; 1.00 mole) in absolute
ethanol (1.95 1.) at room temperature for 2 hours. The resulting
solution was heated at reflux for 5 hours, cooled to room
temperature, then evaporated in vacuo. The residue was dissolved
in benzene (500 ml.). The solution was heated to reflux, and
the water present was removed by means of a Dean-Stark trap.
The solution was then concentrated at reduced pressure to an
oil which slowly crystalli~ed on standing. The solid was sus-
pended in ether (1.3 1.), collected on a filter, washed with
- 45 -
jvb/
Z6~
ether (800 ml.), then dried; yield of L-aspartic acid, diethyl
ester, hydrochloride, 216 g. (95.7%); m.p., 99-103. This
material was recrystallized from 1.25 1. of acetone-ether
~4:1) to give 164.6 g. ~76.2% recovery) of product suitable
for further transformation; m.p., 106.5-107.5 .
L-Aspartic acid, N-(phosphonoacetyl)-, tetraethyl ester
_ _ .
To a cool (10 ), stirred suspension of L-aspartic
acid, diethyl ester, hydrochloride (19.2 g.; 0.0850 mole) in
dioxane (350 ml.~ was added, dropwise, triethylamine (18.2 g.;
0.180 mole) during 20 minutes. The mixture was stirred at 10
for 15 minutes, then a solution of phosphonoacetyl chloride,
P,P-diethyl ester, prepared from 0.090 mole of acid, in dioxane
(90 ml.) was added, dropwise, during ] hour. The temperature
was maintained at 8-10 during the addition. The cooling bath
! was removed, and the reaction mixture was stirred for 3.5
hours. The insoluble material was collected on a filter then
washed with dioxane (100 ml.~ and ether (200 ml.). The filtrate
was spin-evaporated in vacuo, and the residue was dissolved in
ethyl acetate (300 ml.). The organic solution was washed with
water (3 x 100 ml.), dried over magnesium sulfate, then con-
centrated in vacuo to an oil; yield of L-aspartic acid,
N-(phosphonoacetyl)-, tetraethyl ester, 31.1 g. (99.6%). A
25.9 g. portion of the product was dissolved in ether (500 ml.),
and the solution was extracted with water (5 x 150 ml.). The
aqueous extracts were combined and concentrated at reduced
pressure to an oil. This pale yellow material was dried
in vacuo over phosphorus pentoxide to give 20.1 g. (77.6%
recovery) of analytically pure product. The compound moves
, - 46 -
jvb/
as a single spot on silica gel (Eastman Chromogram Sheet 13181)
developed with acetone, chloroform, or ethyl acetate; detection
by iodine vapors.
Anal.
Calc'd. for C14H26NO8P
C H N P
45,777.133.818.43
Found 45.887.113.818.50
Spectral Data:
Infrared (Neat)
Major bands: 3260, 2980, 2920, 2900, 1730, 1670,
1545, 15255 1440, 1390, 1365, 1335,
1230, 1200, 1090, 1040, 1015, 955,
850 cm 1
Nuclear Magnetic Resonance (CDC13)
S 1.23 (m, 12, -CH3 of ethyl groups); 2.83
(d of d, 2, -CH2 ~ to -CH); 2.85 (d, 2, J=20.0
Hz, -CH2 ~ to P); 4.08 (m, 8, -CH2 of ethyl groups);
4.77 (broad m, 1, -CH); 7.37 (broad d, 1, -NH)
Optical Rotation
Observed
~CL~D4 - 6.05 (c, 3.849 in water)
- 47 -
jvb/
. ~ ,. ' :- - '
'2~
_xample 11
L-Aspartic acid, dimethyl ester, hydrochloride
To absolute methanol (1.90 1.; 1.50 kg.; 46.8
moles) being stirred at -5 was added, dropwise5 thionyl chloride
(357 g.; 3.00 moles) during 2 hours. The temperature was
maintained between 0 and -5 during the addition. The solu-
tion was stirred at 0 to -5 for 1.5 hours, then L-aspartic
acid (133 g.; 1.00 mole) was added, in portions, over a period
of 35 minutes. The solution was stirred at -5 for 2.5 hours
and at room temperature for 16 hours. The volatiles were
removed in vacuo, then the oily residue was evaporated at reduced
pressure with benzene (4 x 175 mlO~. The resulting solid was
collected on a filter, washed with ether (400 ml.), and dried.
The crude material (190.4 g.) was recrystallized from 3.5 1.
of boiling acteone to give 135 g. (68.3%) of product suitable
for further transformation; m.p., 117-119.
L-Aspartic acid, N-(phosphonoacetylt-, dimethyl ester,
P,P-diethyl ester
To a cool (10 ), stirred suspension of L-aspartic
acid, dimethyl ester, hydrochloride (18.8 g.; 0.9050 mole) in
dioxane (350 ml.) was added, dropwise, triethylamine (20.2 g.;
0.200 mole) during 15 minutes. The mixture was stirred at 10
for 15 minutes, then phosphonoacetyl chloride, P,P-diethyl
ester, prepared from 0.100 mole of the acid, dissolved in
dioxane (100 ml.) was added, dropwise, during 1 hour. The
temperature was maintained at 5-10 during the addition. The
cooling bath was removed, and the reaction mixture was stirred
for 1.25 hours. The insolubles were collected on a filter,
- 48 -
jvb/
111C~2~
,
washed with dioxane (100 ml.) and ether (200 ml.), then the
filtrate was concentrated in vacl~o. The oily residue was
dissolved in benzene (100 ml.) and ether (400 ml.), then the
organic solution was washed with water (4 x 150 ml.). The
aqueous solutions were combined, saturated with sodium chloride,
and extracted with ethyl acetate (3 x 500 ml.). The combined
extracts were dried over magnesi-lm sulfate then evaporated
in vacuo to give 33.2 g. (103%) of crude product as a yellow
oil. An 18.0 g. portion of the material was dissolved in water
(50 ml.), and the solution was applied to an AG50W-X8 (hydrogen
form) cation exchange resin column (3.8 cm. x 15 cm.). The
column was eluted with 500 ml. of water. Fractions containing
the desired product, as determined by tlc, were combined and
spin-evaporated in vacuo. The residue was dissolved in chloro-
form (120 ml.), and the solution was dried over magnesium
sulfate. The solvent was removed at reduced pressure, then the
pale yellow oil was dried in vacuo over phosphorus pentoxide
to give 14.4 g. (80% recovery) of analytically pure L-aspartic
acid, N-(phosphonoacetyl)-, dimethyl ester, P,P-diethyl ester.
The compound moves as a single spot on ~silica gel (Eastman
Chromagram Sheet 13181) developed with acetone, chloroform,
or ethyl acetate; detection by iodine vapors.
Anal.
Calc'd for C12H22N08P
C H N P
42.48 6.544.12 9.13
Found 42.50 6.514.09 9.06
~ 49 ~
jvb/
lll(aZl~f~
Spectral Data:
Infrared (Neat) -
Major bands: 3260, 2980, 2950, 1735, 1665, 1530,
1435, 1365, ~220, 1160, 1040, 1015,
955 cm
Nuclear Magnetic Resonance (CDCl3)
~ 1.35 (t, 6, -CH3 of ethyl groups); 2.88
(d, 2, -CH2 ~ to -CH); 2.90 (d, 2, J=21.5 Hz.
-CH2 ~ to P); 3.65 (s, 3, -CH3); 3.70 (s, 3,
-CH3); 4.08 (5 line m. 4, -CH2 of ethyl groups);
4.80 (broad m. 1, -CH); 7.~7 (broad d, 1, - NH)
Optical Rotation - I
Observed
Corl 24 _ 6.89 (c, 3.183 in water)
Example 12
L-Aspartic acid, N-(phosphonoacetyl)-, piperazine salt
(1:2.5), tetrahydrate 0.5 C H OH
2 5 _
To a cool (10 ), stirred solution of L-aspartic
acid, N-~phosphonoacetyl)-, (10.2 g.; 0.0400 mole) in water
(60 ml.) was added, dropwise, piperazine (15.2 g.; 0.176 mole)
dissolved in water (100 ml.) during 35 minutes. The temperature
was maintained below 15 during the addition. The solution
was stirred at 10-15 for 1 hour then spin-evaporated in vacuo
( C 35; 2-5 mm. Hg). The semi-solid residue was triturated
with ethanol (2 x 200 ml.) then dissolved in wate~ (60 ml.).
.,
- 50 - ,
jvb/
lll~Z~i
Charcoal (1 g.2 was added, and the mixture was stirred at room
temperature for 25 minutes. The inso]ubles were filtered off,
then the filtrate was added, dropwiseg to vigorously stirred
ethanol (2.0 1.) during ~0 minutes. The resulting solid was
collected on a filter, washed with acetone (200 ml.) and dried
to give 5.8 g. (25.6%) of L-aspartic acid, N-(phosphonoacetyl)-,
piperazine salt (1:2.5), tetrahydrate 0-5 C2H5H; m-p-
92-95 ; 111-115 (sealed capillary). The compound moves as one
major spot on cellulose (Quanta/Gram Q2F glass plates)
developed with ethanol-ammonium hydroxide-water (6:1:3)
(Rf = 0.34), n-butanol-acetic acid-water (5:2:3) (Rf = 0.25),
or ethanol-water (2:3) (Rf = 0.79), detection by Phospray.
Anal.
Calc~d. for C6HloN08P 2-5 C4H10 2 2 2 5
C H N P
36.108.2014.86 5.48
Found35.747.2014.62 5.48
Spectral Data:
Infrared (Nujol)
Major bands: 3340, 3270, 2950, 2920, 2850, 2720
1645, 1625, 1580, 1460, 1380, 1295,
1050, 950 cm 1
Nuclear Magnetic Resonance (D O)
_ 2
~ 0.88 (t, 1.5, -CH3 of EtOH); 2.40 (3 line m.
4, -CH2 ~ to P ~ -CH2 of aspartate); 3.12 (s. 20,
-CH2 of piperazine ring); 3.33 (q. 1. -CH2 of
EtOH); 4.10 (broad t, 1, -CH of aspartate)
- 51 -
jvbt
lll(~Z;~i I
Example 13
L-Aspartic acid, N-(phosphonoacetyl)-
- To a cool (10 ), stirred solution of sodium hydro-
xide (289 g.; 7.23 moles) in-9~35 ]. of water was added, in
one portion, L-aspartic acid, N-(phosphonoacetyl)-, dibenzyl
ester (523 g.; 1.20 moles). The mixture was stirred at
10-15 for 7 hours, then the insolubles were filtered off~
The filtrate was concentrated in vacuo to a volume of 3.0 1.
then extracted with methylene chloride (1 x 1.36 1.) and ether
(1 x 1.36 1.). The aqueous solution was added to 12.0 1. of
ethanol resulting in the precipitation of a semi-solid. After
decantation, the material was dissolved in water (680 ml.),
and the solution was applied to an AG50W-X8 (hydrogen form)
cation exchange resin column (8 cm. x 46 cm.). The column
was eluted with 2.7 1. of water (18 fractions of 150 ml. each).
Fractions 8-16, which contained the desired product as deter-
mined by tlc, were combined and evaporated in vacuo (bath
temperature C 30 ). The oily residue was dissolved in acetone
(2.0 1.), charcoal (50 g.~ was added, and the mixture was
stirred at room temperature for 18 hours. The insolubles were
filtered off, then the filtrate was spin-evaporated at reduced
pressure. Tne residue was dried in vacuo at room temperature
over phosphorus pentoxide for 18 hours to give 220 g. (71.8%)
of the tetraacid. This semi-solid material was suitable
for further transformation.
- 52 -
~, jvb/
26~i
L-Aspartic acid, N-(phosphonoacetyl)-, calcium salt (1:1.5)
2.5 H20
To a cool (10 ), stirred solution of L-aspartic
acid, N-(phosphonoacetyl~- (20.4 g.; 0.0800 mole) in water
(300 ml.) was added, in portions, calcium carbonate (17.6 g.;
0.176 mole) during 30 minutes. The reaction mixture was stirred
at room temperature for 21 hours, then the insoluble material
(7.2 g.) was collected on a filter and washed with water
(50 ml.). Charcoal (5 g.) was added to the aqueous filtrate,
and the mixture was stirred for 35 minutes at room temperature.
The insolubles were filtered ofE, and the filtrate was diluted
with acetone (600 ml.). The resulting solid was collected on
a filter, washed with acetone (200 ml.) and ether (200 ml.),
then dried. This material (24.7 g.) was suspended in water
(125 ml.), and the mixture was vigorously stirred for 10
minutes. The insolubles were filtered off, then the filtrate
was added to acetone (350 ml.). The precipitated solid was
collected on a filter, washed with acetone (200 ml.), then
dried to give 10.8 g. (37.8%~ of analytically pure L-aspartic
acid, N-(phosphonoacetyl)-, calcium salt 2.5 H20; m.p-,
~ 280.
Anal.
Calc'd. for C6H7N08P 1.5 Ca 2.5 H20
C H N P Ca
20.17 3.39 3.92 8.67 16.83
Found 20.28 3.09 3.88 8.63 16.85
- 53 -
jvb/
'
6~
Spectral Data:
Infrared (Nujol)
Major bands: 3530, 3380, 2950,2920, 2850, 1590,
1455, 1375, 1]40,1110, 1070, 975 cm
Nuclear Magnetic Resonance (D O)
S 2.62 (3 line m, 4; -CH2 0~ to P + -CH2
~to -~H); 4.35 (t, 1, -CH)
Optical Rotation:
Observed
~ D2 + 8.38 (c, 3.1I3 in water)
Chromatography:
Thin Layer Chromatography
. .
(Cellulose, Quanta/Gram Q2F Glass Plates) - `
Solvent System Rf Value Rf Value
(Calcium Salt) (Free Acid)
1. n-Butanol-acetic acid- 0.18 0.29
water (5:2:3)
2. Ethanol-ammonium hydroxide- 0.00 0.13
water (6:1:3)
3. Ethanol-water (2:3) 0.84 0.75
Detection: Phospray (A commercial spray reagent used to
visualize phosphorus containing compounds).
Results: The free acid was liberated from the calcium salt
by acidification with hydrochloric acid. A base
',
- 54 -
jvb/
.,
266
line spot was observed for the acid in each of
the solvent systems and for the calcium salt in
system 3.
Example 14
L-Aspartic acid, N-(phosphonoacetyl)-, cyclohexylamine
salt dihydrate _
To a cool (15 ), stirred solution of L-aspartic
acid, N-(phosphonoacetyl)-, (5.1 g.; 0.020 mole) in water
(50 ml.) was added, dropwise, cyclohexylamine (8.7 g.; 0.088
mole) dissolved in water (15 ml.) during 15 minutes. The
temperature was maintained below 20 during the addition. The
solution was stirred at room temperature for 30 minutes then
i diluted with acetone (850 ml.). The resulting solid was
collected on a filter, washed with acetone (200 ml.), and air
dried. The crude product was dissolved in water (100 ml.),
charcoal (5 g.) was added, and the mixture was stirred at room
temperature for 1.5 hours. The insolubles were filtered off,
then the filtrate was added to vigorously stirred acetone
(1.3 1.). The precipitated solid was collected on a filter,
washed with acetone (400 ml.), then dried to give 8.0 g.
(65.7%) of L-aspartic acid, N-(phosphonoacetyl)-, cyclohexylamine
salt dihydrate; m.p., 167.5-170.5 . The compound moves as
one spot on cellulose (Quanta/Gram Q2F glass plates) developed
with ethanol-ammonium hydroxide-water (6:1:3) (Rf = 0.38),
n-butanol-acetic acid-water (5:2:3) (Rf = 0.33), or ethanol-
water (2:3) (Rf = 0.82); detection by Phospray.
- 55 -
jvb/
2E;6
Anal.
Calc'd. for C6HloNOgP 3-2 C6HllN 2 2
C H N P
49.74 9.219.67 5.09
Found 50.12 8.379.44 5.07
Spectral Data:
In f rared (Nujol)
Major bands: 3210, 3050, 2950, 2920, 2860,
2670, 2620, 1640, 1575, 1545,
1460, 1450, 1400, 1380, 1300,
1145, 1115, 1040 cm
Nuclear Magnet Resonance (D20~
1-50 (m, 32, -CH2 of cyclohexane ring); 2.57
(3 line m, 4, -CH2 C~ to P + -CH2 of aspartate);
2.97 (broad s, 3.2, -CH of cyclohexane ring);
4.22 (d of d, l, -CH of aspartate)
Example 15
L-Aspartic acid, N-(phosphonoacetyl)-, dibenzyl ester, cyclo-
hexylamine salt
138 g. (0.317 mole) of L-aspartic acid, N-(phosphono-
acetyl)-, dibenzyl ester was dissolved in acetone (500 ml.).
The stirred solution was cooled to lO , then cyclohexylamine
(69.2 g.; 0.700 mole) was added, dropwise, during 30 minutes.
The resulting suspension was stirred at room temperature for
18 hours. The precipitated solid was collected on a filter,
- 56 ~
jvb/
2~;6
washed with acetone (500 ml.) and ether (400 ml.), then dried
to give 47.2 g. of N-(phosphonacetyl-L-aspartic acid, dibenzyl
ester, cyclohexylamine salt. A 24.8 g. portion of the material
was recrystallized from boiling methanol (500 ml.) to give
18.3 g. (73.8% recovery) of the purified cyclohexylamine salt;
m.p., 186-188.
- 57 -
jvb/
