Note: Descriptions are shown in the official language in which they were submitted.
~2~ 5
This invention relates to therapeutic compositions
which contain andenosine derivatives of anti-inflammatory,
analgecis and antipyretic activity, as their active principleO
This application is a divisional application of
copending application No. 375,784 filed April 21, 1981.
According to the present invention there is pro-
vided a therapeutic composition of anti-inflammatory, anal-
gesic and antipyretic activity, comprising as its active
principle at least one compound of general formula
. N~-R1
~ (Il)n (I)
1 2-S-R
L/\l
~ I
2 2
in which R is a linear or branched alkyl radical of 1 to 18
C atoms, or phenyl-alkylene in which the alkylene chain has
1 to 6 C atoms and n is 0 or 1 or a pharmaceutically accep-
table acid addition salt thereof and a pharmaceutically
acceptable carrier or dilent therefor.
Preferred meanings for R are~ methyl, ethyl,
propyl, isopropyl, butyl, isobutyl, sec.butyl, pentyl, hexyl,
heptyl, octyl, decyl, dodecyl, hexadecyl, octadecyl or
benzyl.
~g
-- 2
,_
~O9~L5
The preferred acid addition salts of the compounds
of formula (I) are: chloride, sulphate, phosphate, formate,
acetate, citrate, tartara-te, methanesulphonate or p-toluene-
sulphonate.
The compounds of formula (I) are prepared by
various methods according to the meaning of the various
radicals.
For preparing the group of compounds of formula:
NH2
~ ~ . (I3)
CH2--S-R
~\1
OH OH
. ~. .
. . .: .
~2~ S
in which R has the aforesaid meanings, the Legraverand method
(Legraverand M.Ibanez S., et al (1977) Eur. J. Med. Chem.
12, 105-108) has been followed, in which andenosine is con-
verted into 5'-chloro-5'-deoxyadenosine by reaction with
thionyl chloride in hexamethylphosphoramide.
The 5'-chloro-5'-deoxyadenosine is then converted
into the required thioether by reaction with the correspon-
ding mercaptan in a 2N sodium hydroxide solution at 80C.
The thioethers obtained are purified by recrys-
tal1isation from water or from lower aliphatic alcohols.
The compounds (Ia) can then be salified with the
stoichiometric quantity of the required acid.
..--.
~20~1~
The compounds of formula
~12
I
~ ~ O (Ib)
CH2--S-R
~\1
' ' 1~
OH H
in which R, Rl, R2 are as heretofore defined, are prepared
by oxidation of the corresponding thioethers, obtained by
the methods heretofore indicated, by means of bromine or
hydrogen peroxide in aqueous solution (Green Stein J.P.,
Winitz M. (l961)-Chemistry of the amino acids John
Wiley & Sons Inc. 2146). The products obtained are purified
by recrystallisation from water.
~O Of all the products prepared, the one which has
proved particularly interesting for the purpose of the pre-
sent invention is 5'-deoxy-5l-methylthioadenosine (MTA) of
formula
NH
~ ~ (II)
¦ ~ CH2 S-CH3
H
~2Q~15
which is a physiological compound already present in living
organisms.
The method has been found for preparing this pro-
duct which is particularly simple and economical from an
industrial viewpoint. The process which is the subject
matter of copending application No. 375,874 consists essen-
tially of carrying out hydrolysis of the S-adenosylmethionine
(SAME) under strictly controlled critical conditions, which
lead to practically total hydrolysis and complete crystal-
lisation of the MTA
.. ' NH2
I N
~ ~
NH C~ ~2
2 1 2 2
. ~ CH3
\ _ /
OH OH
N~
1 2
c}l2 ~3 cll3 N~12
HO-CH2-CH2-CH-COOH ~ H
OH OH
-- 6
~zo9~s
The controlled hydrolysis process can be 2pplied to SAi~
prepared in any mamler,
However, the method of preparation of the SA~ solution is also
an influencing factor in carrying out the new process in an
economically convenient manner.
The following operational stages provide the most economical
embodiment of the process:
a) Normal bread yeast is enriched in SAME by treatment with
methionine in accordance with the ~chlenk method (Schlenk F.
(1965) Enzymologie 29~ ~83)
b) The yeast cells suspended in water are lysised by treatment
with eth~l or methyl acetate at ambient tempe,ature (DT-OS P23
36401.4).
By adjusting the pH to between 4 and 6 and filtering, an aqueous
solution is obtained containing practically 811 the S~E present
in the ~nitial yeast
c) The so ution is concentrated under vacuum at 35_40C to about
1/10 cf its initial volume
d) The concentrate is boiled u~der reflux for about 30 minutes and
the pH adjusted to 7 with soda
e) The solution is left to stand at 0~5 C, and the precipitated MTA
is collected practicaily completely and at good purity. -
The stages cq d and et which as stated are critically necessary for
obtaini~g complete selective hydro ysis of SAi~E to ~A without
formstion of by-products~ are new.
The preparstion of some products used according to the present
invention are de.scribed hereinafter.
EX~i_LE 1
Preparation of 5'-deo~y-5'-methylthioadenosine (~
12~ r9~ 5
.
11 litres of ethyl acetate and 11 litres of water at ambient
temperature are added to 90 kg of bread yeast which has been
enriched in SAME by adding methionine until the SA~ content
is 6.88 ~ kg.
4fter energetic stirring for 30 minutes, the pH is adjusted to
4.5 with dilute H2S04, the mixture is filtered ~nd the residue
is ~lashed ~ith water io give 140 litres of solution with a SAME
content of 4.~0 ~/1, equal to 99.5,~ of the SA~E present in the
initial material.
The lysate thus obtained is concentrated under vacuum (30 mm Hg;
35_40C) to 3 volume cf about 14 litres. The concentrated solution
is boiled under reflux at normal pressure for 30 minutes. It is
cooled to 20~, the pH adjusted to 7 with 40,' ~oda, and left over-
night in a refrigeration cell (+3C).
A white precipitate is formed which is ~iltered5 dissolved in 10
litres of boiling distilled water and crystallised by cooling this
solutio~.
410 g of crystalline i~A of high purity are obtained, equal to a
yield of gOc,/ with respect to the SAi~ subjected to hydrolysis.
The characteristics of the product obtained coincide ~ith those
of pure li~A obtsined by other means.
E~AI~LE 2
Preparation of 5'-deox-J-5'-e-thylthioadenosine
1 kg of adenosine is dissol-~ed under a nitrogen atmosphere in 10 1
of hexamethylphosphoramide, and 705 1 of thionyl chloride are added
unàer cooling.
The mixture is left to react at ambient temperature for 20 hours.
10 1 of water are added, and the mixture neutralised with 2N NaOH.
S
g
The 5~-deoxy-5~-ch;oroadenosine which thus forms is allowed to
crystallise overnight at ~C. It is filtered off. 0~950 kg
of 5'-deoxy_5'-chloroadenosine are obtained (yield ~9%~.
0.950 kg of 5'-deoxy-5'-chloroadenosine are dissolved -.n 10 1
of 2N NaOH, and 200 ml of ethanethiol are added. The mixture
is heated to 80C and left to react for 1 hour. It is neutralised
with glacial acetic acid. The 5'-deoxy-5'-ethylthioadenosine which
thus forms is allowed to precipitate overnight at 3 C. It is
filtered off and recrystallised from water.
0.830 kg of product are obtained (yield 80% with respect to the
preceding stage).
E~MPLE ~
Preparation of other compounds of class Ia
The method as described in example 2 is carried out~ but using
propanethiol,b~tanethiol, isobutanethiol, pentanethiol~ hexanethiol
and benzylthiol respectively~ in place of the ethanethiol~
EXA~LE 4
Preparation of N ~ 2', 3'-triacetyl-5'-deoxy-5'-thioadenosine.
1 kg of l~rA is suspended in 10 1 of anhydrous pyridine, and 3 1
of acetic ~nhydride are added. The mixture is left to react for
4 hours, 20 1 of water are added, and the mixture concentrated
under vacuum to give an oily mass free from pyridine. This is
dissolved in a hot 1:1 mixture of petroleum ether/chloroform (10 1)
and left to crystsllise. The product is recrystallised from a
i:l petroleum ether/chloroform mixture. 1.140 kg of product are
obtained (yield 80',').
EXAl`IPL~ 5
Preparation of other compounds of class Ib
The method described in example 4 is carried out~ but using other
~2~9~L5
-- 10 --
thioethers or propionic anhydride, butyric anhgdride, benzoy~
chloride or tosyl chloride instea~ of I~
EXAMPLE 6
Preparation of 51-deoxy-2'~3'-isopropylidene-5'-methylthioadenosine.
l kg of I~A are suspended in 25 l of anhydrous acetone~ and 2.5 kg
of molten ZnCl2 are added. Reaction is carried out under reflux
for 5 hours. The mixture is then concentrated under vacuum to
l/3 of its initial volume~ ar.d 7.5 kg of barium hydroxide octahydrate
in aqueous suspension are added. Carbon dioxide is then bubbled
through until neutral. The mixture i5 filtered and the residue
washed with acetone. The filtrate is concentrated under vacuum
to give a syrupy residue~ It is taken up in 3 hot l:l chloroform/
petroleum ether mixture (lO l)~ filtered and left to crystallise.
The product is recrystallised from l:l chloroform~petroleum ether~
to give 0.795 kg of product (yield 70%).
EXAIvlPLE 7
Prepara~ion of other compounds of class Ic.
The method as described i~ example 6 is carried out~ but s~arting
from the corresponding adenosine derivatives instead of l~A.
EXAIV1PLE 8
Preparation of l~iTA sulphoxide.
l kg of lviTA are suspended in lO l of water~ and bromine is added
under cooling~
The aqueous solution containing bromine is immediately decoloured
by the oxidation of the ~A to sulphoxide.
Addition of bromine is continued until the solution does not
decolourise furthe .
The solution is decolourised by further addition of small quantities
of ~iTA.
2~9~15
11 --
The aqueous solution is treated with ~mberlite IRA 9~ resin
(registered trademark cf Rohm and Haas for a weakly basic ion
exchange resin with a polystyrene matrix) until the reaction
of the bromide ions disappears.
The mixture is filtered and the residue washed with water.
The aqueous solution is concentrated to 10 1, trested with
activsted carbon (100 g) and lyophilised.
0.950 kg of product are obtained (yield 90~).
EXA~PLE 9
Preparation of other compounds of class Id
The method described in example 8 is followed, but starting from
the corresponding adenosine derivatives instead of ~ITA.
As initially stated~ it has been found that the co~pounds of
formula I possess strong antiinflammatory activity, accompanied
by analgesic and antipyretic action.
The antiinflammatory activity was demonstrated initially for some
terms of the class by the test of experimental adema in the rat
bycarragen9 by determining the percentage protection by the Winter
method (J. Pharm. exper. Therap. 1411 369 1963). The values
obtained are shown in Table 1.
12~9~5
- 12 -
T A B L E
Compound of formula (I) Dosa ad.min- Percent protec-
~stered tion calculated on
orally edema development
m~/kg
n = 0 R = -CH2, ~1 = R2 = H 37 5
3 1 Z 23 (a) 50
n = 0 R = -CH2-c~H5~ R1 R2 lO
/ CH3
n = 0 R = -CH2-CH ' 1 2 5 62
~ CH3
n = 0 R = -(CH2)6~cH3~ R1 R2 20
n = 0 R - -(CH2)ll~cH3' R1 R2 112 10
n = 0 R = -(CH2)4-CH3- Rl R2 25
n = O R = -CH2-cH3~ Rl R2 80 44
n = Q R = -(CH2)2~cH3~ R1 R2 80 53
/ CH3
n = 0 R = -CH , R1 = R2 = H 80 45
~3
n = 0 R = -(CH2)3~cH3 ' R1 2 85 39
n = 0 R = C1H~CH2~CH3 ' R1 R2 85 35
n = 0 R = -(CH2)7~cH3~ Rl R2 100 l~7
n = 0 R = -(CH2)9-cH3~ Rl-R2 106 33
3 1 2 156 50
3' 1 2 8.6 (a) 50
3' 1 2 3 714 47
12(~9~S
- 13 -
n = 0 R = -CH3, R1 = R2 = tosyl 204 15
n = 0 R = -CH3, R1 = R2 = -C-c6 5 164 10
n - 0 R = -CH3, R1 = H, R2-R1 = isopropyl 91 20
Indo~ethacine 9 50
(a) signifies that the product was ad~inistered intramuscularly.
.4s can be seen from this table, the ~D50 of the ~A is 37 mg/kg,
and is thus the lowest of those of tne compound~ tested when
administered orally.
In the same test, the ED50 of indomethacine is 9 mg/kg. At
tkese doses, there is the appearance of serious gastric lesions,
whereas at the ED50 doses the ~A gives rise to no seeondary
effect on the gsstro-intest-nal system. It should also be
noted that the LD of indomethacine in the rat is 12 m ~kg
(Martelli A. in Aspetti di farmacologia dell'infiammazione, page
73, published by Tamburini - Milan 1973), whereas the LD50 of
MTA in the rat is > 200 m~k ~ oa.
The following therapeutical indices are therefore obtained:
Indomethacine TI = 1.3
MTA TI = ~ 54.05
The compounds according to the invention were also subjected to
a series of pharmacological tests for the purpose of confirming
their antiinflammator~ activity and for demonstrating their
analgesic and antipyretic activity.
The results obtained in some of these tests with MTA are given
hereinafter~ this being a product which in all cases proved to
be the most active when administered orally~ and which is
~09'.9~5
- 14 _
certainly the safest as it is a compound physiologically present
in the organism, as alregdy stated.
Again from the industrial production viewpoint, the method for
producing i~A from SA~ as discovered is by far the most simple
and economical, and enables it to be marketed at a particularly
low price.
As can be seen from the dats of table 1~ methylthioadenosine
sulphoxide is particularly active when administered intrgmuscularly.
The g~eater activity of said compound on intramuscular administration
~as confirmed in all the tests carried out. Some significsnt dsta
regarding ~A sulphoxide are also given, however it should be noted
that all the compounds tested were in all cOsses shown to be active,
although at different levels,
A - Antiinflammatory activity
~he products were tested by pleuritis induced in the rat by
carragan in accordance with the Velo method (Velo G.P., DUNN
C~J. et al. (1973) J. Path. 111, 149).
~TA at a dose of 75 mg/kg by oral administration gave a
protection of 42.4,S calculated on the volume of the exu~ate~
and 48.8% calculated on the total number of cells present in
the exudate,
A comparable protection wa,s obtained with 10 mg/kg of indomethacine,
i~e. with a dose much closer to the LD50. In the same test~
the MTA sulphoxide givss a protection of 75.3C,S calculated on
the volume of the exudate, and 76~4~o calculated on the total
number of cells present in the exudate when administered
intramusculsrly at a dose of 80 m~kg.
B _ Antiinflammatory activity
In the granuloma test in the rat by cotton pellets (~inter C.A.,
,, . ~
126~9~J 5
- 15 -
Riseley E.A. et al ~1963) J. Pharm. Exper. Ther. 141, 369), which
is significant for chronic inflammation~ the ~TA gave a protection
of 30,~ with an oral dose OI 9 mg/kg, with a TI of 222.
C - The analgesic activity of the products was tested by two tests
considered very significant.
- In the hot plate test on the mouse according to Roberts (Roberts
E. Simonsen D~Go (1966) Biochem. Pharmac. 15, 1875) the MT~ gives
a protection of 50C,' with an oral dose of 37 m~kg. An approx-
imately equivaler.t protection of 58,' is o~tzined with 100 mg/kg
of amidopyrine admin-stered orally.
In the same test MTA sulphoxide gives a protection of 50,' at a
dose of 20 m~kg when administered intramuscularly, and at a
dose of 100 mg/kg when administered orally.
- In the stretching test by phenylquinone (Siegmund ~., Cadmus R.,
GO_LU (1957) Proc. Soc. Exp. Biol. Med. 951 729), the ~A gives
a protection of 51% at an oral dose of 37 m~kg.
In the same test~ M~A sulphoxide has an ED50 of 10 mg/kg when
administered intramuscularly.
D _ Antipyretic activity.
This was measured ror the new products by means of fever induced
in the rat by beer yeast (Winder C.U. et al (1961) J. Pharmacol.
Exp. Ther. 133~ lI7).
The antipyretic effect evaluated one hour after oral administration
of r~TA at a dose of 300 mg/kg gave a temperature reduction of
4 .59C~ with respect to the controls~ which were treated only with
yeast. This percentage corresponded to a temperaturs lowering
from 38.8C to 37~4 C.
By comparisor.~ amidopyrine administered orally at a dose of 200 m~ kg
produced a temperatu~e reduction of 4069i~, and intramuscular
9~5
administration of MTA at a dose of 80 mg/Kg gave a tem-
perature reduction of 2.35%.
E- Platelet antiaggregation activity.
The compounds of the invention have also been
evaluated with respect to their possible platelet
antiaggregat on capacity.
Platelet aggregation is known to be a complex
phenomenon which can be divided into a primary stage due to
the direct action of a stimulus (for example adenosine di-
phosphate, i.e. ADP, or epinephrin) and a secondary stage
due to the aggregation induced by a ADP released by the
platelets. In this respect, when the platelets come into
contact with the subendothelial collagen, the collagen
initiates an entire series of reactions which lead to the
release of ADP by the platelets. It is this ADP which
causes the second wa~e of platelet aggregation. The fol-
lowing tests wexe carried out in order to evaluate the
antiaggregation effect of the new compounds:l) "in vitro"
tests on platelet aggregation induced by ADP and collagen,
in the presence of the new products; 2) "in vitro" tests on
platelet aggregation induced by arachidonic acid (AA); 3 )
"in vivo"`tests on platelet aggregation induced by ADP and
collagen in persons treated with the new products.
In this case the most significant results were
again obtained with MTA, because of which the results ob-
tained using this product are given as indicative of the
behaviour of the entire class.
1) "in vitro" tests.
Blood was withdrawn without stasis, and an anti-
coagulant (3.8% sodium citrate) was added to give a blood:
citrate ratio of 9:1. Plasma rich in plateletes and plasma
poor in platelets were obtained by centrifuging at ambient
temperature. The platelet aggregation was estimated using
, ~ 16 -
~,
~209~5
the Born & Cross method (G.V.R. Born and M.~. Cross, J.
Physiol., Lond. 168, 178, 1963) on the plasma fraction rich
in platelets. The aggregating agents were used in the
following concentrations: ADP (Sigma) 1 ~M; collagen (Horn)
5 ~g/ml; arachidonic acid 4xlO 4M.
Adenosine at a concentration of lxlO 5M was used
as the antiaggragation activity reference substance.
The results obtained with ADP are shown on the graphs of
Fig. 1, in which the abscissa indicates the time in minu-
tes and the ordinate the precentage aggregation.
Curve 1 relates to the controls, curve 2 to the
samples treated with lxlO 5 adenosine, and curve 3 to the
samples treated with 5xlO M MTA.
From the curve pattern it is apparent that MTA
strongly reduces primary platelet aggregation due to ADP,
and as a consequence inhibits the 2nd aggregation wave.
The same tests carried out with collagen gave negative
results, i.e. MTA showed no inhibiting power towards
platelet aggregation induced by collagen which was worthy
of note.
2) Fig. 2 shows the effects of various MTA concentrations on
platelet aggregation induced by AA at a concentration of
4.xlO M. Curve 1 relates to the controls, curve 2 to MTA
at a concentration of 2.5xlO 4M, curve 3 to MTA a~ a concen-
tration of 5xlO 4M, and curve 4 to MTA at a concentration of
10 3M. As is apparent, the platelet aggregation inhibiting
effect of the MTA is proportional to its concentration. The
capacity of MTA to increase the inhibiting effect of pros-
tacyclin (PGI2) in aggregation induced by AA was also inves-
tigated. In Fig. 3, curve 1 relates to the controls, curve
2 to MTA at a concentration of 5xlO 4M, curve 3 to PGI2 at a
concentration of 5xlO 9M, and curve 4 to a mixture consist-
;~ ~ - 17 -
~ .~
1209~5
ing of SxlO 4M MTA and 5xlO 9M PGI2. It ls apparent from
Fig. 3 that when used in mixture there is a strong increase
in the antiaggregation action at concentrations which are in
themselves ineffective.
3) "in vivo" tests.
Three apparently healthy volunteer subjects aged
35, 42 and 48 years respectively, and who had not taken any
drug for at least 15 days, were submitted to aggregation
tests before and after consuming the new products at a dose
of 100 mg every 8 hours for 3 days, these tests then being
evaluated. The blood sample for the determination of the
platelet aggregation was taken 2 hours before consuming
the last dose of product under test.
Fig. 4 shows the results obtained with MTA.
More presicely, the full-line curves relate to the
values obtained with blood samples from untreated patients,
whereas the dashed-line curves related to the values ob-
tained for the same patients treated with MTA.
It is apparent that MTA strongly inhibits platelet
aggregation induced by ADP (l~M) "in vivo". The same tests
repeated on adding 5~g/ml of collagen to the blood demon-
strated that MTA is not effective in inhibiting platelet
aggregation induced by collagen, but only lengthens the
latency time of the phenomenon.
The fact that MTA (and in a more or less compar-
able manner the other products of the same class) strongly
inhibits platelet aggregation induced by ADP, whereas it has
practically no effect on aggregation induced by collagen,
indicates that MTA inhibits the 1st aggregation wave,
whereas it has a negligible direct effect on the 2nd
aggregation wave.
~2Q9!~L5
Its use in association with other known antiaggre-
gation drugs which are yenerally active towards the 2nd wave
whereas are only poorly effective towards the 1st wave is
therefore particularly interesting.
s
The demonstrated activity suggests the use of MTA
(and of the other compounds of the series, even if less
effective) not only as a platelet antiaggregation drug, but
also as an antithrombotic and antiatherosclerotic drug,in
that, besides being the basis of thrombogenesis mechanisms,
the altered relationship between pla-telets and vasal walls
also plays a primary role in atherosclerotic illness.
-- 19 --
~209!~.5
-20
Sleep inducing activity.
The ~lorris test was used (Morris R.W.(1966) Arch. int. Pharmacodyn
161, ~o. 2, 380~
In this test, the MTA increased by 8~' the duration of the sleep
induced by pentobarbital in the mouse~ at an intramuscular dose
of 20 mg/kg.
Acute toxicity.
The compounds of the present invention are practically free from
acute toxicity when administered orally. They are practically
free from toxicity st therapeutic doses for any method of
sdministration,
The following values are obtained for MTA and MTA sulphoxide~
M~A - DL50 in the mouse orally ~ 2000 mg/kg
intravenously 360 mg/kg
MTA sulphoxide - DL50 in the mouse
orally > 2000 mg/kg
intravenously 400 m~kg
The adenosine derivatives of formula (I) can be administered,
diluted with suitable pharmacologically acceptaole excipients,
in any therapeutically useful form, orally, parenterally or by
venous or rectal means~ They can also be used in products for
external use by topical application.
Some examples of typical pharmaceutical compositions with ~ITA
are given hereinafter by way of example:
..
~209~LS
_ 21 -
100 mg capsules
~A 100.2 mg
Mannitol 195.0 mg
Magnesium stearate5.0 mg
300.2 mg
50 mg capsules
MTA 50.1 mg
Mannitol 100~0 mg
Magnesium stearate3.0 mg
153.0 mg
100 mg tablets
I`~A 100.2 mg
Starch 100.0 mg
~iagnesium stearate15.0 mg
Lactose 85.o mg
3.Z mg
50 mg tablets
MTA 50.1 mg
Starch 120.0 mg
Magnesium stearate15.0 mg
Lactose 115.0 mg
300~1 mg
100 mg sup~ositories
MTA 100~2 mg
Suppository mass1,700.0 mg
1,800.2 ~g
~0~5
2~ -
50 m~ su~ositories
~TA 50.1 mg
Suppository mass1,450.0 mg
1,500.1 mg
_0 mg injactable_phial
MTA . HCl (56.15 mg basically
equiv31ent) 50 mg
Lidocain HCl 25 mg
l~ater to make up to 3 ml
25 mg in,iectable phial
MTA ~ HCl (28.07 mg basically
equivalent) 25 mg
1idocai.n HCl20 mg
:~ater to ma~e up to 2 ml
100 mg oral dose
MTA . HCl (112.3 mg bas.ically
equivalent) 100 mg
Citrus flavouring0.025 mg
Sugar 1 g
Antifermenting agent 50 mg
Water to make up to 5 ml
50 mg oral dose
i~lTA , ~Cl (56,15 mg basically
equivalent) 50 mg
Citrus flavouring0.015 mg
Sugar -5 g
Antifermenting agent 30 mg
Water to ma~e up to 5 ml
O~ S
. ~ 23 -
- 100 g ointment
~A 5 g
Base for water-soluble
ointment, to make up to 100 g
Antioxid~nt 0.1 g
,
.
