Note: Descriptions are shown in the official language in which they were submitted.
~ 3~ OB 7~/B 035
The present invention relates to synthetic phospholipids
and to a process for their manufacture and their use.
The process of the invention comprises the manufacture
of synthetic phospholipids which do not occur in nature and
have a varied phosphorus - nitrogen distance.
Naturally occurring phospholipids are fat-like triglycerides
containing two long-chained fatty acids and a phosphoric
acid radical to which a base is linked. They occur in
all animal and vegetable cells, especially in the brain,
heart, liver, yolk, as well as in soybeans. The most -
important ones among the naturally occurring phospholipids
are the cephalins and lecithins, in which colamine or ;
.
quoline are present as bases.
Lecithins and cephalins are widely used since they
have colloidal, surface-active, emulsifying, softening, anti-
oxidizing, purifying and physiological properties. Being
natural products they are physiologically acceptable in
foodstuffs and therefore superior to many synthetlc
substances of similar activity. They are added to
20 ~ margarine in order to assure a better linkage to water;
when lecithin is used in chocolate and coating masses it
brings about a better and quicker wetting of the mixing
constituents, a ~eduction o viscosity and thus a
considerable saving o expensive cacao butter. At the
~same time, the rancidity and the "fat-bloom" are prevented
during storage.
: ~ :
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, . .
'' ~ ~ ' .
., , : . :, - , . :. . :. . :, . : :
. .' . . :.
10~15 llor~ 7~/B 035
1:
Lecithin when used in sweets helps to emulsify sirup
with fat. At the same time, it prevents the fat from
getting rank and sugar from crystallizing. Bakery goods
can be more readily processed owing to the improved
wetting during the mixiny operation. Up to 20% of the
otherwise required fat can thus be saved, and the yi ld
can be increased up to 2 % owing to the better linkage
to water.
Large amounts of soybean lecithin are also added
to fodder material since this promotes the absorption
of the foodstuffs in the digestive tube and taken together
with fish- and meat-flour it counteracts the damaging
effect of cholesterine.
Used for cosmetic purposes and in the manufacture
of soap, small additives thereof improve the suppleness
and absorption of ointments, creams, tooth pastes, soaps
and the like.
In the leather and textile industries, lecithin emulsions
~are being used as adjuvants in the processing owing to
~20 ~ their antioxidizing effect. In paints the lecithin
prevents the pigments from settling and reduces the
viscosity, thus improving the processing conditions.
It is also possible to improve printing pastes and inks
for paper and textiles using lecithin. Lecithin emulsions
are also used in pesticides since these have a good
stability and adhesiveness.
.
'
- HO~ 7~/B 035
~)6~
Lecithins and cephalins have recently gained a particular
importance since it was found that they have important
functions on the cell oxidation and other cellular processes.
The function of the phospholipids in the cellular metabolism
has however little been elucidated and is therefore particularly ~
difficult since the isolated compounds are obtained in only -
low amounts and their syntheses involve great difficulties.
The syntheses of phospholipids often require many steps,
and the desired products are obtained in only small yields
(cf. A.J. Slotboom and P.P.M. Bonsen, Chem. Phys. Liquids -
(1970) S. 301.)
Lecithin and cephalin are obtained from natural
products, for example from yolk, cerebral matter, spinal
marrow and soybeans. The commercial products have very much
~15 differing properties, and it i5 therefore difficult in many
cases to use lecithin and cephalin for the various
applications owing to the differing content of phospholipids.
; It is an object of this invention to provide a proaess
for the synthesis of phospholipids which is simple and
easy to manage without requiring-the use of expensive
starting materials. Further object of this invention is
to prov1de new compounds which have a similar structure to
the naturally occurring phospholipids and possess similar or
~; ~ even better properties than the naturally occurrin~ phospholipids
~25 owing to the combination of lipophilic and hydrophilic as well
as acidic and basic groups in the same molecule.
~ 4 ~
~ .
,........................................................................ . .
:' .. -. . , . , . ~ ' ' ' ' , .. .
' -"." ' '''''; ' '', .. ,, ~'" ,.. " ,': ,.:. " ."' ".' ,"'' ' :
-. . . , :,
I~O~ 035
lO~ LS
The process for the manufacture of synthetic phospholipids
comprises
A) reacting a polyhydroxy compound having a free
hydroxy group and the other hydroxy groups of
which are protected with an ~r -halogenalkyl-phos-
phoric acid dichloriae of the general formula
,
Cl \ o
P-O-Alk-X
10~ : Cl /
in which X stands for fluorine, chlorine, bromine
or iodine, and Alk stands for an alkyl or
cycloalkyl group having at least 3 C-atoms,
and : -
. - .. ~
15~ B) reacting the resulting reaction product with
: an amine of the formula
R
~: 3
20~ in which Rl, R2 and R3 each stands for hydrogen
or methyl.
The pxesent invention moreover relates to the synthetic~ :
phospholipids prepared according to the new process and to
- ~
.
~ 5 _
.
. . . .
~ ~, , .,:
IIOE 74/B 035
5 - .
the use of the new compounds as stabilizers for enzyme
compositions as emulsifiers and as drugs.
The compounds prepared according to the inuention
may be used for the same purposes as the natural
phospholipids.
The compounds of the invention have valuable
pharmacological properties. The compounds which are
analogous to lecithin are potent surfactants and
as such have a great influence on natural cell
membranes and on the permeability conditions in
biomembranes. By selectively varying the phosphate-
trimethylammonium distance of the fatty acid used
and of the base used, the properties of
the cell membranes can be modified in a selective
manner.
When the surface activity of the cell membranes
are influenced by means of the compounds of the in-
vention, the activity of the pharmaceutical composition
is modified as well, i.e. their absorbabiliky and
their distribution in the organism.
Owing to the marked surface activity, the compounds
of the invention cause a change of the properties of
cell membranes when administered to warm-blooded animals
~ by the oral or intraperitoneal route. When administered
in even higher concentrations, cytolytical
phenomena are observed. The administration of sublytical
doses causes changes in the cellular membranes.
, . , . ~ : , .,, ..................... : , . . ...... . . :
.
IIOE 74/B 035
10~ 5
Compounds having saturated fatty acid esters of 16 and
more carbon atoms, for example palmitic acid, are -`
immunological adjuvants, whilst compounds having chain
lengths of less than 14 carbon atoms were found to cause
an inhibition (imuno-suppressant action) of the imuno- -
apparatus. These results were observed with phosphoric
acid quoline esters. The immunological adjuvant effect
reveals itself in a general increasa of the antibody
level.
The comprehensive variants of structure which were
brought about on the lysophospholipid molecule led to more
efficient adjuvants.
Enzyme in the cellular membranes which are dependent
on phosphoLipids contain natural phospholipid mixtures
`15 having a large number of unsaturated fatty acids. Due
to the instability of unsaturated fatty acids in the
presence of oxygen, stabilization of such enzyme compo-
sitions is difficult.
Enzyme compositions of this kind, however, may be
~20 ~delipidized, thus losing their enzymatic activity.
A reactivation of the enzyme can be obtained using
the phospholipids of the invention having no unsaturated
fatty acid radicals. Reactivation may be brought about
,
; by mixing the phospholipid in an adequate ratio with the
compound of the invention. It is thus possible to reactivate
and stabilize enzyme which depend on phospholipids.
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,,, ., ', . ', , . ,,. :, ::
i ' ,' ' , , ,, ' ' ' ~ ,
.. . . . . .
HOE 74/B 035
36~15
According to the concept of various authors, hybrid-
formation and the cell fusions are induced by lysolecithin.
Thus it is possible to produce cell hybrids in a manner
similar as with Sendai virus. A disadvantage is the great
cytolytic activity of the lysolecithins obtained from
egg lecithin which are used for these investigations.
The compounds of this invention having a finely varying
cytolytic activity allows the experiments on cell fusion
to be optimized, that is the cytolysis can be avoided.
As already mentioned above, the compounds of the
invention are good emulsifiers owing to the combination -
of lipophilic and hydrophilic as well as of acidic and
basic groups in the same molecule, and the~ form stable
emulsions at a pH ranging from 0 to 11. They may therefore
~advantageously be used ln detergents. In addition they
have the further advantage that owing to their
closed relation to natural phospholipids, they can
be degradated in a biological manner, thus avoiding
~ ~ ecological problems. Moreover, the compounds of
the invention having more than 6 carbon atoms between
the phosphorus and the nitrogen atoms, were found
to~be safe against the attack of phospholipases C and
D, so that their bactericidal and bacteriostatic activities
cannot be destroyed by these enzyme produced in the
~25 cells.
According to the process of the present invention
polyhydroxy compounds having a free hydroxy group are
~, ~ , .
,
.' ' ' . ., , ~' ',, ' ' ' ' , ' . , ,' ' ' ~ ' . ,
:"' ': . ' ''.. ' "'' ~ ' ' ': : , . '' ':'
' . ~ . ' ' ~ ~ ' .
,
-~!IOE ~ 35
reacted with a halogenalkylphosphoric acid dichloride.
The protection of the hydroxy groups in the polyhydroxy
compounds may be effected by etherification, esterification
or ketalization. For the process of the invention,
polyhydricaliphatic alcohols, such as erythritol,
pentitols and hexitGls, may generally be used as
the polyhydroxy compounds 1,2- or 1,3-diglycerides
and other glycerol derivatives. Examples of polyhydroxy ~ -
compounds which may be reacted according to the invention
are illustrated in detail later on in connection with
the compounds of the invention.
The polyhydroxy compounds are reacted with a halogen- ~-
alkylphosphoric acid dichloride of the general formula
Cl
P-O-Alk-X
Cl
in which X stands for fluorine, chlorine, bromine or
lodine, and Alk stands for an alkyl or cycloalkyl group
having at least 3 C-atoms. Preferably w -bromoalkyl-
phosphoric acid dichlorides are used. In the above
formula, Alk preferably contains 3 to 25 C-atoms,
. .
more preferably 3 to 16, and most preferably 3 to 12
C-atoms, in the case of alkyl groups, or 6 C atoms i~ the
case of cycloalkyl groups (cyclohexyl). The halogenalkyl-
phosphoric acid dichlorides of the desired chain len~th
_ 9 _ . .
..
~ . " .,, , , , - , . , , ~ , . " . , ,
IIO~ 7~/B 035
lO~ LS
are obtained by reacting halogenated alcohols of the
following formula
HO - Alk - X
of a corresponding chain length, that is Alk has the
same meaning as given above, with phosphorus oxy-
trichloride. The said halogenated alcohols may be
obtained from the corresponding diols. For cxample
the brominated alcohols may be prepared by introducing
a bromine atom per molecule of diol according to a
simple process. The reaction product having one
bromine atom is removed from the reaction medium
by extraction and thus further bromination is
excluded.
The reaction of halogenalkylphosphoric acid
dichloride with the polyhydroxy compound is preferably
carried out in an inert organic solvent, for example
halogenated hydrocarbons such as chloroform, carbon
tetrachloride, benzene, toluene, petroleum ether
and the like. The reactio~ should be carried out
under exclusion of moisture. The reaction temperature
generally ranges from -10 to 50C, preferably
rom 0 to 20C. The reaction is preferably carried
~ out in the presence of an inert base, for example
triethylamine or pyridine. The halogenalkylphosphoric
acid dichloride is generally dissolved in the inert
solvent, and the base is added. Whilst stiring , the
-- 1 0
:, ~ , . ~', :, :' , : : , :
. . .
. .
HOE 74/B 035
10~ 5
polyhydroxy compound, likewise dissolved in an inert solvent,
is added dropwise to the phosphorylation agent, where required -
while cooling.
Under these conditions, the reaction is smooth. It is
generally complete within a short time. However, it is
recommendable to continue stirring for some time to assure
complete reaction. Reaction periods ranging from half
an hour to five hours are usual. -
The reaction may be checked, for example, by thin-layer
chromatography. When the reaction is complete, the solvent
and excess base are eliminated at a low temperature, and the
reaction product may be separated by usual methods, for - ;
example by extraction. It is generally not necessary
to purify the reaction product but it may be reacted
immediately with the desired amine base without further
purification. ;
For this purpose, the reaction product is dissolved
in a suitable solvent, and an ethanolic or an aqueous
solution of the corresponding amine base is added thereto.
This reaction is carried out at room temperature or at
a slightly elevated temperature, for example at 55C,
for 5 to 20 hours, or or 1 to 6 hours. The reaction
procedure can be controlled by thin-layer chromatography.
Care should be taken that the necessary reaction
time be not exceeded since, after the reaction is
complete, the reaction product decomposes.
After it has been made sure that the reaction is
-
... - . . - - - .- - . - . , . , ,. . , .... . .. , .-,.. . . . .
,,', . ! ~ ' ' ' ' ' ' ' ' '
,, . , ' ' . ', . ' " ! " ; ~ i
- `~
~~" 110E _4/B 035
complete, the reaction product is isolated in a known
manner, for example it may be purified by column chro-
matography.
The yields of the products which are analytically
pure, generally, range from lO to 25 % of the theoretical
yield, calculated on the diglycerides used or on the other
corresponding starting materials.
Examples of compounds which may be prepared according
to the process of the invention are given in the following:
l. Lecithins and cephalins of the following fo~ulae
- .:
O ' ' : : .
Il .
H2C -- O - C Rl
p IV
HC - O - C - R2
~ 15 H2C - O - ~ - O - Alk - N - X2
:' ~ .o:~3 \X ... ::
iS~ : O
~ ` : H2C - O - C -- RlX
~, 20 1 ~O ~ / l
HC - O - P - O - Alk - N - X2 V
0~ \X
~O 3
' ~H2C - O - C R2
., .
~ ' ' ' '
.~J
~ - l2 -
" ,' .
~ .
i,:
~51 .''. '
HO~ 7~/B _35
1()6;~ 5
Starting materials are racemical or optically active
1,2- or 1,3-diglycerides having unsaturated, saturated
or branched fatty acids or fatty acids containing a
cycloalkane or an aromatic ring.
In the above formula, Xl, X2 and X3, independently of
one another, each stands for a hydrogen atom or a methyl
group, Alk is defined as above, Rl and R2 each stands
for a straight-chain or branched saturated or unsaturated
alkyl group which may be substituted by a cycloa~kyl group
or an aromatic group. The alkyl groups contain from 9 to
25 C-atoms, preferably 12 to 18 C-atoms, and most preferably
_ 14 to 18 C-atoms. The cycloalkyl groups may contain from
5 to 7, preferably 5 to 6, C-atoms. As aromatic groups,
for example phenyl groups or substituted phenyl groups may
be mentioned. Rl and R2 may preferably stand for fatty
- acid radicals, for example radicals or palmitic acid,
``~ and stearic acid.
, .... .. . ..
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., .
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, .
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.~ .
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,. . .. . ... .. , .,, , . .... ,. . , : :
,:, . . . ,, . . . . .,,, . .. . ., , " , . ,, . . j ., . . :. .
~ lO 74/B 035
~O~ lLS
2. Lyso compounds of compounds of the general formula
IV or V
Starting materials for the manufacture of the lyso
compounds of the general formula IV or V are, for
example, 1-acyl-2-bencylglycerol or 1-bencyl-2-acyl-
glycerol. The starting materials may also be pre-
pared biochemically from lecithins and cephalins by
enzymatic splitting with phospholipases A 1 and A 2.
3. Analogs with saccharic alcohols
,
. O
, 11
H C - O - C - R
O
,: .11
H-C - O - C - R
O IV
,~ 15 H-Ç - O - C - R
i.', I l
H-C - O - C - R
ol :
H-C - O - C - R / 1
H2C O ~ - O - Alk - N - X2
.
in which Alk, Xl, X2 and X3 are defined as above, and
R has the same meaning as given for Rl and R2. The
starting materials used are acylated saccharic alco-
hols containing z-l acyl radicals in the case of z
1~ hydroxy groups, z being an integer of from 2 to 7~
, . .
~ preferably 3 to 6, most preferably 4, 5 or 6. Cyclic
.. ,
~ saccharic alcohols may also be used.
:"~. . ', .
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- 14 -
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IIOE 74/B 035
6;~1S
4. Ether analogs and e-ther-ester analogs of the compounds
.
of Groups 1 to 3
For example
H 2 C - O
~-~ - o - R2 X
H2C - O - P - O - Alk - N / X2 VII
-.'
H C - O - R
21 ol ~ .
H-C - O - C - R2 X
p ~3/ 1 ':,
2c o 7 - O - Alk - N \ X2 VIII
- 15in which Rl, R2, Alk, Xl, X2 and X3 are defined as
further above.
: Starting materials are the 1,2- and 1,3-dialkylglyce-
. . ~ . .
.$ : rol ethers or ethyl glycerol alkyl ethers~
j~ 5. Dialkyl ketone glycerol phospholipids
"~ 20
~' H2C - \ ~ Rl
P2C - O - P - O - Alk - ~N9 / X IX
0~ X
-;l 25
.~ . . - 15 ~ . :~
. ~ , . ~ . .
~ 5 IIOE 7~/B 035
2 C O ~ ~ Rl
H-C - O 1l H / Xl
H2C - O - P - O - Alk - N \ X2 X
Q X3
In the above formula IX and X, Rl, R2, Alk, Xl, X2
and X3 are defined as above.
Starting substances are l,2- and l,3-dialkylketone-
glycerols or the correspondlng acetals which may be
o~tained from glycerol or 2-benzylglycerol by a re-
action with the corresponding ketone or aldehyde.
The ketone or aldehyde may also contain a cycloalkane
or aromatic ring in the lateral chain~
6. Cycloalkyl ketone glycerol phosphollpids
: ~ .
~ .
H2C - O~ _~ : . "
/ C( CH2 ) y ' '
H-C - O X
l XI
H C-O-P-O- Alk -N - X
2 O~ 2
: : X3
~': '
: ~ .
~ ~ H2f - ~
:~ I ~ 2 Y X .
~ HC - O ~ O / l
;: H2C - 7 - O - Alk -- N - X XII
: O 9
. . .
: , - 16 -
-, . . . ~: . . ., : . ~ . .
~- ' . :. , .:.,', :': ,.:'' . ., ' . ' ' '.. ' ':: .. . .: .'. ' ' : '' ' .. , ; . . '
.. , , ,, .,,, ,. s~ .. . .. .. .
:.;. .: . . ., :: . : . :~ ... .
` IIOE 74/B 035
1~)6~ti1S ~
In the above formula XI and XII, Alk, Xl, X2 and X3
are defined as above, y stands for an integer of from
5 to 32, preferably 5 to 18, more preferably from
5 to 16, most preferably from 5 to 12, i.e. 5, 6, 7,
8, 9, 10, 11, 12 and so on. In the above formula, Alk
may also contain 2 carbon atoms.
Starting substances are 1~2- and 1,3-cycloalkyl ketone
glycerols which may be obtained from glycerol or
2-benzyl glycerol by a reaction with the corresponding
cycloalkanone.
7. Desoxy lysolecithins and cephalins
:~ 1 . . ',:
H C - O - C ~ R
2~
I H X
~15 'f 2)m ll ~ / 1 VIII
H2C - O - P - O - Alk N 2
:: :
~ H3l ~
~20 ~ ~ ~ (fH2)m 0 xl
H-~ - O - - R ~ / XIV
2C - o - p _ o _ Alk - N - X
o ~3 \
~ 25 ;
:'; :
~ 17 -
-
-
:
:,:: ::: :,.:,. - ,:-,, :.:, , . ; .-, . :. : ,: -: . . .
,, i;~, :,'', ..' ;'','', , '',' ' '' ,' ., .' '', ~,;, ' " ' ' ' ''
. ~ . , , ., -, , . , j , ~ ,
,. , : . :
~~ IIOE 7~/B 035
lS
H3l
(fH2) p
H-C - O - -- R p + q = m XV
(I 2)q I Xl
H2C ~ O - P - O - Alk
o~ X3
,
In the above formulae, Alk, Xl, X2, X3, R are defined
i as above, m stands for zero or for an integer of
from 1 to 14, preferably 1 to 8, most preferably for
2, 3, 4, 5 or 6. The sum of p and q makes up m.
',~ ' .
i Starting substances for the compounds of the general
,
formula XIII, XIV and XV are the corresponding mono-
~15~ acyl alkane diols, preferably ~r,~3 '-monoacyl alkane
iols. The alkane diols may be saturated, unsaturated
or branched and may also contain a cycloalkane or an
aromatic ring.
~ ~ .
8. Ether analogs of the compounds of Group 7
20~ ~ The above classes of compounds are examples of com-
pounds obtained according to the process of the in-
vention. The process of the invention is generally
-
; ~ applicable and may be used for the synthesis of many
compounds.
. . . . . . .
- 18 -
- ,
~: ', : ' .
.
~r~
HOE 74/B 035
ltJ~
The compounds obtained according to the process of the
invention may be isolated generally by column chromato- '
graphy on silica gel. The analytically pure products
are white, amorphous powders having an uncharacteristical
melting point. Therefore, characteri~ation is ,' -
generally made by thin-layer chromatography and ele-
mentary analysis.
The following examples illustrate the invention.
lQ '' ~ '
E X A M P L E 1:
.
Preparation of brominated alcohols of different
chain lengths according to a simplified method: ~'~
The compounds of the following type were synthe-
15 sized:
Br - (CH2jn OH
Alk having 4 to 10 carbon atoms, i.e. n is an integer of 4 to 10. ~-
;~ ; ; Starting products were diols of the corresponding
~2~0 chain length having terminal alcohol functions. Since
in each case, only one bromine atom per diol molecule
was to be,introduced, a process had to be found where-
n the reaction product was eliminated immediately
rom the proper reaction medium and thus further re-
~25 ,action was excluded. For this purpose, an extraction
method was suitable.
~Diol and hydrobromic acid were placed in a round
flask. The starting products were overlaid with petro- '-
1 9
.
, ",, ,. . , , . , . ,, .. , ,,, . , ~ , .- ,, . , , , . ~ . .
,: : ,, :, , , ,- , , : ,: , ,. , ,, :.. :. : :
-
HOE 7~/B 035
1~6~1S
leum benzine or with benzene/petroleum benzine. The selec-
tion of the extraction agent depended on the insolubility
of the diol and the good solubility of the reaction pro-
duct therein. The round flask was equipped with a reflux
condenser. While very vigorously stirring by means of a
magnetically operated stirrer, the mixture was refluxed
by means of an adequate heating device until the re-
action of the starting product was complete. The pro-
ceeding reaction was checked by means of thin-layer
0 chromatography.
The extraction medium phase was then separated and
dried with calcium sulfate. After the siccative had been
filtered off, the extraction medium was eliminated in
the rotary evaporator. The residue was subjected to
fractionated distillation in an oil pump vacuum.
The yield ranged from about B0 to 95 percent of the
theoretical yield, calculated on the diol used.
4-Bromo-butanol-(l) and 5-bromo-pentanol-(l) were pre-
pared as follows:
0 22.5 g (0.25 mol) of 1,4-butane diol or
26 g of 1,5-pentane diol (0.025 mol)
were refluxed together with
80 g of hydrobromic acid (47 percent strength, 0.48 mol),
~ 500 ml o~ benzene and
S~ 50 ml of petroleum benzine (boiling point 100 to 140C)
for 6.5 and 6 hours, respectively.
2~ -
, . . .
"' . '~ ".' ' .." ," ' ' '" ' . ' ' ,, ' ', ' , '' , , ' , ' '''' ' .", ' , ': , ' ,' , ':
,, " " . .. . .
~ lS ~ OE 74/B 035
The rest of the brominated alcohols were prepared as follows:
29~6 g (0.25 mol) of the corresponding diol were re-
fluxed together with
g (0.48 mol) of hydrobromic acid (47 percent
strength),
1500 ml of petroleum benzine (boiling point 100 to 140C).
The following reaction products were prepared:
.. . .
T a b 1 e
10 Reaction product Reaction period Physical constants
4-bromobutanol-(1) 6.5 hours boiling poink (0.7mm~g)
58 - 60C
5-bromopentanol-~1) 6 hours boiling point (0.5 mmHg)
72 - 74C
6-bromohexanol-(1) 1.5 hours boiling point (0.6 mmHg) -
85 - 87C
7-bromoheptanol-(1) l.S hours boiling point (0.5 mmHg)
87 - 89C
8-bromooctanol-(1) 1 hour boiling point (0.5 mmHg)
110 - 112C
. .
9-bromononanol-(1) 1 hour boiling point (0.4 mmHg)
112 - 114C
10-bromodecanol-(1~ 30 minutes boiling point (0.3 mmHg)
124 - 1~6C -
: .
25 Up to the 8-bromooctanol-(1), the reaction products were
colorless liquids. 9-bromononano1-(1) and l0-bromodecanol-(1)
were white solid products at room temperature. Brominated
alcohols of greater chain length may p~incipally be
- 21 -
.: .. . . : . ~ ; .. , .. ., ,. ; . , . :,.. ...
:: ;- . ~ , , , - , ;;, . . .
'',~. ',,,' ' , '' ""' ''',' '.'~, ', ' ,;, ' :'
;, , : , . . .. .
~~ HO~ 74/B 035
prepared also according to this method. Since all these
reaction products were solid substances, they were puri-
fied by recrystallization.
: '
:
.:
.
. . .
; i , .
:~ : : . .
.
, ~ '
,
- 22 -
. , ,,. . : ., , , - , . , : ,.. . ...
- . .. . . . .
:::, : ,
... .... . . . .. . ... . .
~\
1063~5 HOE 74/B 035
Example 2
Preparation of lecithins having a modified phosphorus-nitrogen
distance in the polar head:
A. ur-bromoalkylphosphoric acid dichloride:
.
32 mmols = 30 ml of phosphorus oxytrichloride (freshly dis-
tilled, boiling point 105 to 107C) in
70 ml of absolute chloroform (distilled for 90 minutes
with circulation over P2O5)
were placed in a round flask. At room temperature, nitrogen was
fed into this solution for a short time to expel air. The flask
was equipped with a dropping funnel and sealed airtight. While
stirring with a magnetically operated stirrer, 20 mmols of the
brominated alcohol of desired chain length in 50 ml of absolute
chloroform were slowly added dropwise at room temperature with
the exclusion of moisture. Stirring was continued for about 12
hours. The hydrogen chloride resulting from the reactian as well - -
~
: ' '
~as excess phosphorus oxytrichloride and chloroform were elimi-
20~ nated at 30C in a rotary evaporator. To eliminate any trace of
~`~ phosphorus oxytrichloride, toluene was added and likewise
drained off.
The conversion rate was 95 to 100 ~, and the reaction was
checked by means of thin-layer chromatograms.
B. Phosphorylation:
~ ' -
~ 23
: . :
'' ' . ,
:, .. . ., . :.. , .. -.. .. . . , , . ; : .
, ~ : . : . .
~: . . .. . ..
. . .. . . .
- ,
llO~, 7~/B 035
The ~ -bromoalkylphosphoric acid dichloride obtained sub A. was
taken up in 60 ml of absolute chloroform, and the solution was
cooled to 0C. ~,~hile stirring by means of a magnetically ope-
rated stirrer, 10 ml of triethylamine (dried over lithium-alu-
minum-hydride and freshly distilled) were added. 14 mmols of
the corresponding diglyceride, for example
SN-1,2-dipalmitoylglycerol,
SN-1,2-dimyristoylglycerol,
1,2-dipentadecylketoneglycerol
or another of the above-mentioned starting substances in 60 ml
of absolute chloroform were slowly added dropwise at 30 to 35C
while stirring by means of a magnetically operated stirrer and
with the exclusion of atmospheric moisture.
.
It was made sure by thin layer chromatography that the reaction
was almost complete during the dropwise addition. During the re-
action, the light yellow solution turned into a dark-brown col-
~ oured solution. Stirring was continued for another 3 to 5 hours.Chloroform and triethylamine were then eliminated at 35C in a
. .
rotary evaporator. The reaction product was taken up in 100 ml
of tetrahydrofuran. While stirring lM sodium acetate solution
, , .
of pH 8.4 was added until the solution remained weakly alkaline.
100 ml of diisopropylether were then added to the reaction pro-
duct thus hydrolyzed, and the mixture was stirred for 1 hour.
After the phases had been separated, extraction was repeated
using 50 ml of ether. The combined ether phases were stirred
- 2~ -
, , . , - " :,1 . : : .. , , , , , , , . . . .::
,,.,, , . , . ,.. ,.. , ., , . . . . . ,, .. . :, " .
, , . . , ., , . : . , ,. . , , : , : .. : . , , .. ::, .
,~ ,,., , ,. : , . . ,.. , , , :, :: : ,: .. .
IIOE 74/B 03_
over sodium carbonate for 1 hour, filtered, and ether was then
eliminated in the ro~ary evaporator.
The subsequent reactions were carried out without further puri-
fication of this reaction product.
C. Reaction with an amino base:
The reaction product obtained sub B. was taken up in 150 ml of
10 ` butanone for further reaction yielding lecithin~s. When a
cephalin was to be prepared, it was dissolved in 50 ml of chloro-
form and in 100 ml of methanol. 100 ml Of acetonitrile and 100
ml of an ethanolic or aqueous solution of the correspondlng
amino base were added thereto~
: .
':. -
The reaction vessel was sealed airtight and maintalned for 1 to
6 hours at 55C, or for 5 to 20 hours at room temperature. The
reaction procedure was checked by thin-layer chromato~raphy~
:
When the necessary reaction time was exceeded, the reaction pro- ~ ~
duct decomposed, which was manifested by a stxong reduction in ~ -
. .
yield. The volatile constituents of the reaction mixture were
then eliminated at 50C in a rotary evaporator. The residue was
taken up in 150 ml of chloroform, 100 ml of 2 % formic acid, and
200 ml of methanol were added, and the mixture was shaken. The
reaction product was in the chloroform phase and was treated
with 100 ml of O.lM sodium acetate solution of pH 5.6 and 200 ml
; ~ of methanol for neutralisation purposes. Upon repeated separa-
tion of the phases, the chloroform phase was dried over 10 g
- 25 -
- - - ",; , , :: - , . ,. , , , ~ . , , ;. . .
. :;. , .. . : -, ,. ,, :, .. , . , :, ; . , . :, . . , :
" ~ , . : - . : , . .
~6~5 HOE 74/B 035
of sodiumsulfate and the chloroform was eliminated in the rotary
evaporator.
The so-obtained crude product was purified by means of column
chromatography. For this purpose, a column was charged with a
suspension obtained from 100 g of silica gel (Mallinckrodt AR
p.a.) in a solvent system of chloroform/methanol/ammonia = ~-
200/15/l. The product dissolved in 10 to 15 ml of solvent was
then applied to the top o~ this column, and contaminants were
then eluted by means of the above-said system. The reaction pro-
duct was then eluted with chloroform/methanol/ar~monia = 65/15/l
and 65/30/3, respectively. The fractions were checked by thin-
layer chromatography as for their purity.
':
The yields of the analytically pure products ranged from 10 to
25~ of the theoretical yield, calculated on the diglycerides
used or on other corresponding starting substances.
..'
The compounds cited in the following with their individual ~na-
lytical data were prepared:
Group 1: SN-1,2-dipalmitoylglycerol-3-phosphoric acid-5-
trimethylaminopentylester C43H88NOgP
mol wei~ht: 794.15
calculated: C 65.03 % H 11.17% N 1.76% P 3.90 %
found: C 64.36 % H 11.04% N L.84% P 3.91 %
. . -.
- 26 - ~ ~
. ~ , , ., ... ,. , . : , :,
.: . ...... ...
., . : : , ~, . . : , .,, ' , , ,: , . : ,
lt~ 5 HOE 74/B 035
Group 2: SN-l-palmitoylglycerol-3-phosphoric acid-5-trimethyl-
aminopentylester C27H58NgP
mol. weight: 537.72 - --
calculated: C 58.35 % H 10.52 % N 2.52 % P 5O57 %
The analytical data obtained complied with the cal-
culated values. -
Group 3: 1,2,3,4,5-pentamyristoyl-D-mannitol-6-phosphoric acid-
7-trimethylaminoheptylester C92Hl66NOl4P
mol. weight: 1,541.31
calculated: C 71.69 % H 10.86 % N 0.91 % P 2.01 %
The analytical data obtained complied with the cal-
culated values.
Group 4:
:: ~
a3 1-palmitoyl-2-hexadecyletherglycerol-3-phosphoric acid-
9-trimethylaminononylester C47H98NO8P
mol. weight: 836.27
calculated: C 67.50 % H 11.~1 ~ N 1.67~i P 3.70 %
The analytical data obtained corresponded to the cal-
culated valuesO
:: :
b) 1,3-dioctyletherglycerol-2-phosphoric acid-6-trimethyl-
aminohexylester C2~H62NO7P
mol. weight: 555.78
calculated: C 60.51 % H 11.24 % N 2.52 % P 5.57 %
The analytical data obtained corresponded to the cal-
~ ~25 culated values.
- - 27 -
~: ': . .
: . , ,, . . . , :: :
,, , :, ' , ' ':, "
.
. .
~ S HOE 74/B 035
Group 5: 1,2-dipentadecylketoneglycerol-3-phosphoric acid-6-
trimethylaminohexylester C43HgoNO7P
mol. weight: 750.12
calculated: C 67.25 % H 11.83 % N 1.87 ~ P 4.13 % ~ -
found: C 67.28 % H 11.87 % N 1.82 % P 4.14 %
Group 7: 1-myristoylpropanediol-3-phosphoric acid-4-trimethyl- -
aminobutylester C24H52NO7P
mol. weight: 497~65
calculated: C 57.92 % H 10.53 % N 2.81 % P 6.22 % - -
The analytical data obtained corresponded to the ~al-
culated values.
. :
Group 8: l-t tradecyletherpropanediol-3-phosphoric acid-4-tri-
~ : .. ..
15 ~ meth~laminobutylester C24H54NO6P
mol. weight: 483.67
calculated: ~ 59.60 % H 11.25 ~ N 2.90 % P 6.40 %
The analyticaI data obtained corresponded to the cal-
culated ualues.
20 ~ ~
::::
Group 1: SN-1,2-dipalmitoylglycerol-3-phosphoric acid-6-tri-
methylaminohexylester, mol. weight~ 808.1
C44HgoNOgP
calcuIated: C 65.39 % H 11.23 % N 1.73 % P 3.83 %
25~ ~ found: C 66.66 % H 11.45 % N 1.80 % P 4.08 %
:: :
.,: .
- 28 -
., , . ~., , . .,, . , . i,,: , , ,., ,. , .. ,., : . , :., . . j . . . .. .
: :: .: : , : .. . " . ~ . . , .~ ., :,.,, :. ; . ., . : : , : , ,: .
, .: . ' . , : , , ' . , , . , " ; . ': . : , . ' . , ,, .:. ' : : .
SN-1,2-dipalmitoylglycerol-3-phosphoric acid-7-tri-
methylaminoheptylester, mol. weight: 822.20
C45H92N9
calculated: C 65.74 % H 11.28 % N 1.70 % P 3.77 %
found: C 64.90 % H 11.16 % N 2.02 % P 4.59 %
SN-1~2-dipa ~ toylglycerol-3-phosphoric acid-8-tri-
methylaminooctylester, mol. weight: 836.23
C46H94N9P
calculated: C 66.07 % H 11.33 % N 1.68 % ;P 3.70 %
found: C 64.15 % H 10.91 % N 2.30 % P 4.60 %
SN-1,2-dipalmitoylglycerol-3-phosphoric acid-9-tri-
methylaminononylester, mol. weight: 850.26
C47 96 9
calculated: C 66.39 % H 11.38 % N 1.65 % P 3.64 %
found: C 66.28 % H 11.43 % N 1.85 % P 3.85 %
.
Group 5: 1,2-dipentadecylketoneglycerol-3-phosphoric acid-5-tri-
methylaminopentylester, mol. weight: 736.11
42H88 7
calculated: C 66.90 % H 11.78 % N 1.90 % P 4.21 %
found: C 67.06 % H 11.78 % N 2.06 ~ P 4.22 %
; ~ ' '~. ';
1,2-dipentadecylketoneglycerol-3-phosphoric acid-8-tri-
methylaminooctylester, mol. weight: 778.19
45H94NO7P
caleulated: C 67.91 ~ H 11.92 % N 1.80 % P 3.98 %
found: C 68.21 ~ H 11.93 ~ N 1.89 % P 3.95 %
~, -- 2g --
, .- -- - . , :
