U.S. patent application number 09/986327 was filed with the patent office on 2002-05-02 for compounds having reversible inhibiting activity of carnitine palmitoyl-transferase.
This patent application is currently assigned to Sigma-Tau Industrie Farmaceutiche Riunite S.p.A.. Invention is credited to Arduini, Arduino, Chiodi, Piero, De Angelis, Francesco, Giannessi, Fabio, Marzi, Mauro, Minetti, Patrizia, Tinti, Maria Ornella.
Application Number | 20020052348 09/986327 |
Document ID | / |
Family ID | 11380036 |
Filed Date | 2002-05-02 |
United States Patent
Application |
20020052348 |
Kind Code |
A1 |
Giannessi, Fabio ; et
al. |
May 2, 2002 |
Compounds having reversible inhibiting activity of carnitine
palmitoyl-transferase
Abstract
Compounds of formula (I) 1 wherein the groups are as defined in
the description are disclosed. The compounds of formula (I) are
endowed with reversible inhibiting activity of carnitine
palmitoyl-transferase and are useful in the preparation of
medicaments useful in the pathologies related to a hyperactivity of
carnitine palmitoyl-transferase, such as hyperglycemia, diabetes
and pathologies related thereto, heart failure, ischemia.
Inventors: |
Giannessi, Fabio; (Pomezia
(RM), IT) ; Marzi, Mauro; (Rome, IT) ;
Minetti, Patrizia; (Rome, IT) ; De Angelis,
Francesco; (Rome, IT) ; Tinti, Maria Ornella;
(Roma, IT) ; Chiodi, Piero; (Ciampino, IT)
; Arduini, Arduino; (Rome, IT) |
Correspondence
Address: |
NIXON & VANDERHYE P.C.
8th Floor
1100 North Glebe Road
Arlington
VA
22201-4714
US
|
Assignee: |
Sigma-Tau Industrie Farmaceutiche
Riunite S.p.A.
|
Family ID: |
11380036 |
Appl. No.: |
09/986327 |
Filed: |
November 8, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09986327 |
Nov 8, 2001 |
|
|
|
09677328 |
Oct 2, 2000 |
|
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Current U.S.
Class: |
514/105 ;
514/114; 514/120; 514/331; 514/478; 514/561; 514/563 |
Current CPC
Class: |
C07D 257/04 20130101;
C07C 275/16 20130101; A61P 3/04 20180101; C07C 229/26 20130101;
C07D 295/15 20130101; C07C 311/06 20130101; C07D 453/02 20130101;
A61P 43/00 20180101; C07C 271/22 20130101; A61P 3/06 20180101; A61P
9/10 20180101; C07C 335/08 20130101; C07C 271/12 20130101; A61P
3/10 20180101; C07C 229/22 20130101; C07F 9/5407 20130101 |
Class at
Publication: |
514/105 ;
514/114; 514/120; 514/331; 514/478; 514/561; 514/563 |
International
Class: |
A61K 031/66; A61K
031/445; A61K 031/195 |
Foreign Application Data
Date |
Code |
Application Number |
May 15, 1998 |
IT |
MI98A001075 |
May 11, 1999 |
IT |
PCT/IT99/00126 |
Claims
1. Compounds of formula (I) 3wherein: X.sup.+ is selected from the
group consisting of N.sup.+(R.sub.1,R.sub.2,R.sub.3) and
P.sup.+(R.sub.1,R.sub.2,R.sub.3), wherein
(R.sub.1,R.sub.2,R.sub.3), being the same or different, are
selected in the group consisting of hydrogen and C.sub.1-C.sub.9
straight or branched alkyl groups, --CH.dbd.NH(NH.sub.2),
--NH.sub.2, --OH; or two or more R.sub.1, R.sub.2 and R.sub.3,
together with the nitrogen atom, which they are linked to, form a
saturated or unsaturated, monocyclic or bicyclic heterocyclic
system; with the proviso that at least one of the R.sub.1, R.sub.2
and R.sub.3 is different from hydrogen; Z is selected from
--OR.sub.4, --OCOOR.sub.4, --OCONHR.sub.4, --OCSNHR.sub.4,
--OCSOR.sub.4, --NHR.sub.4, --NHCOR.sub.4, --NHCSR.sub.4,
--NHCOOR.sub.4, --NHCSOR.sub.4, --NHCONHR.sub.4, --NHCSNHR.sub.4,
--NHSOR.sub.4, --NHSONHR.sub.4, --NHSO.sub.2R.sub.4,
--NHSO.sub.2NHR.sub.4, --SR.sub.4, wherein --R.sub.4 is a
C.sub.1-C.sub.20 saturated or unsaturated, straight or branched
alkyl group, optionally substituted with a A.sub.1 group, wherein
A.sub.1 is selected from the group consisting of halogen atom,
aryl, heteroaryl, aryloxy or heteroaryloxy group, said aryl,
heteroaryl, aryloxy or heteroaryloxy groups being optionally
substituted with one or more C.sub.1-C.sub.20 saturated or
unsaturated, straight or branched alkyl or alkoxy group and/or
halogen atom; Y.sup.- is selected from the group consisting of
--COO.sup.-, PO.sub.3H.sup.-, --OPO.sub.3H.sup.-, tetrazolate-5-yl;
with the proviso that when Z is --NHCOR.sub.4, X.sup.+ is
trimethylammonium, Y.sup.- is -COO.sup.-, then R.sub.4 is C.sub.20
alkyl; with the proviso that when Z is --NHSO.sub.2R.sub.4, X.sup.+
is trimethylammonium and Y.sup.- is --COO.sup.-, then R.sub.4 is
not tolyl; with the proviso that when Z is --NHR.sub.4, X.sup.+ is
trimethylammonium and Y.sup.- is --COO.sup.-, then R.sub.4 is not
C.sub.1-C.sub.6 alkyl. their (R,S) racemic mixtures, their single R
or S enantiomers, their pharmaceutically acceptable salts.
2. Compounds according to claim 1, wherein R.sub.1, R.sub.2 and
R.sub.3 are methyl.
3. Compounds according to claim 1, wherein the heterocyclic system
formed by R.sub.1, R.sub.2 and R.sub.3 together with nitrogen is
selected from the group consisting of morpholinium, quinuclidinium,
pyridinium, quinolinium and pyrrolidinium.
4. Compounds according to claim 1, wherein R.sub.1 and R.sub.2 are
H, R.sub.3 is selected from the group consisting of
--CH.dbd.NH(NH.sub.2), --NH.sub.2 and --OH.
5. Compounds according to any one of claims 1-4, wherein Z is
selected from the group consisting of ureido (--NHCONHR.sub.4) or
carbamate (--OCONHR.sub.4, --NHCOOR.sub.4), R.sub.4 is a
C.sub.7-C.sub.20 saturated or unsaturated, straight or branched
alkyl group.
6. Compounds according to claim 5, wherein R.sub.4 is a
C.sub.9-C.sub.18 saturated or unsaturated, straight or branched
alkyl group.
7. Compounds according to claim 1, selected from the group
consisting of
R,S-4-trimethylammonium-3-(nonylcarbamoyl)-aminobutyrate;
R,S-4-quinuclidinium-3-(tetradecyloxycarbonyl)-oxybutyrate;
R,S-4-trimethylammonium-3-(nonylcarbamoyl)-oxybutyrate;
R,S-4-trimethylammonium-3-(nonyloxycarbonyl)-oxybutyric acid
chloride;
R,S-4-trimethylphosphonium-3-(nonylcarbamoyl)-oxybutyrate;
R,S-4-trimethylammonium-3-(octyloxycarbonyl)-aminobutyrate;
R,S-4-trimethylammonium-3-(nonyloxycarbonyl)-aminobutyrate;
R,S-4-trimethylammonium-3-octyloxybutyrate;
R,S-4-trimethylammonium-3-tet- radecyloxybutyrate;
R,S-1-guanidinium-2-tetradecyloxy-3-(tetrazolate-5-yl)- -propane;
R,S-1-trimethylammonium-2-tetradecyloxy-3-(tetrazolate-5-yl)-pro-
pane;
R,S-3-quinuclidium-2-(tetradecyloxycarbonyl)-oxy-1-propanephosphonat-
e monobasic;
R,S-3-trimethylammonium-2-(nonylaminocarbonyl)-oxy-1-propanep-
hosphonate monobasic;
R,S-3-pyridinium-2-(nonylaminocarbonyl)-oxy-1-propan- ephosphonic
acid chloride; R-4-trimethylammonium-3-(tetradecylcarbamoyl)-a-
minobutyrate;
R-4-trimethylammonium-3-(undecylcarbamoyl)-aminobutyrate;
R-4-trimethylammonium-3-(heptylcarbamoyl)-aminobutyrate;
R,S-4-trimethylammonium-3-(nonylthiocarbamoyl)-aminobutyrate;
R-4-trimethylammonium-3-(nonylcarbamoyl)-aminobutyrate;
S-4-trimethylammonium-3-(nonylcarbamoyl)-aminobutyrate;
S-4-trimethylammonium-3-(tetradecylcarbamoyl)-aminobutyrate;
R,S-4-trimethylammonium-3-tetradecylaminobutyrate;
R,S-4-trimethylammonium-3-octylaminobutyrate;
R,S-4-trimethylammonium-3-(- decansulfonyl) aminobutyrate;
R,S-4-trimethylammonium-3-(nonylsulfamoyl) aminobutyrate;
S-4-trimethylammonium-3-(dodecansulfonyl) aminobutyrate;
R-4-trimethylammonium-3-(dodecansulfonyl) aminobutyrate;
S-4-trimethylammonium-3-(undecylsulfamoyl)aminobutyrate;
R-4-trimethylammonium-3-(undecylsulfamoyl)aminobutyrate;
R-4-trimethylammonium-3-(dodecylcarbamoyl)aminobutyrate;
R-4-trimethylammonium-3-( 10-phenoxydecylcarbamoyl)aminobutyrate;
R-4-trimethylammonium-3-(trans-.beta.-styrenesulfonyl)aminobutyrate.
8. A process for the preparation of compounds of claim 1, wherein Z
is carbonate (--OCOOR.sub.4), carbamate (--NHCOOR.sub.4,
--OCONHR.sub.4), thiocarbamate (--OCSNHR.sub.4, --NHCSOR.sub.4) or
thiocarbonate (--OCSOR.sub.4), comprising the reaction of
X+--CH.sub.2--CH(OH)--CH.sub.- 2--Y-, wherein X+ and Y- have the
same meanings as in claim 1, of the desired structure, optionally
protected on the acid Y- group, respectively with alkyl
chloroformates, alkyl isocyanates, alkyl isothiocyanates, alkyl
thiochloroformates, wherein the alkyl moiety is the desired R.sub.4
alkyl group.
9. A process for the preparation of the compounds of claim 1,
wherein Z is amide (--NHCOR.sub.4), thioamide (--NHCSR.sub.4),
carbamate (--NHCOOR.sub.4, --OCONHR.sub.4), thiocarbamate
(--NHCSOR.sub.4, --OCSNHR.sub.4) ureido (--NHCONHR.sub.4),
thioureido (--NHCSNHR.sub.4), sulfinamide (--NHSOR.sub.4),
sulfonamide (--NHSO.sub.2R.sub.4), sulfinamoylamino
(--NHSONHR.sub.4), and sulfamide (--NHSO.sub.2NHR.sub.4)- ,
comprising the reaction of
X.sup.+--CH.sub.2--CH(NH.sub.2)--CH.sub.2--Y.- sup.-, wherein
X.sup.+ and Y.sup.- have the same meanings as in claim 1, of the
desired structure, optionally protected on the acid Y.sup.- group,
respectively with acyl chlorides, thioacyl chlorides, alkyl
chloroformates, alkyl thiochloroformates, alkyl isocyanates, alkyl
thioisocyanates, alkyl sulfinyl chlorides, alkyl sulfonyl
chlorides, SOCl.sub.2 and alkyl amines, alkyl sulfamoyl chlorides
(or SO.sub.2Cl.sub.2 and alkyl amines), wherein the alkyl moiety is
the desired R.sub.4 alkyl group.
10. A process for the preparation of the compounds of claim 1,
wherein Z is --OR.sub.4 or --SR.sub.4 comprising a) the reaction of
carbonyl compounds of formula Hal--CH.sub.2--CO--CH.sub.2--COOR',
wherein Hal is a halogen atom and R' is the residue of a suitable
ester, with respectively alcohols and thiols R.sub.4OH or
R.sub.4SH, wherein R.sub.4 is as defined in claim 1, to give the
respective ketal or thioketal; b) transformation of the the
respective ketal or thioketal into the respective ether or
thioether; c) substitution of the Hal atom with a nucleophilic
group, and d) transformation of the nucleophilic group into the X+
group, wherein X.sup.+ is N.sup.+(R.sub.1,R.sub.2,R.sub.3) or,
alternatively e) step b) is followed by the substitution of the Hal
atom with a (R.sub.1,R.sub.2,R.sub.3)-substituted phosphine to
obtain the compounds of formula (I) wherein X.sup.+ is
P+(R.sub.1,R.sub.2,R.sub.3).
11. A process for the preparation of the compounds of claim 1,
wherein Z is --NHR.sub.4 comprising the reaction of
X.sup.+--CH.sub.2--CH(NH.sub.2)- --CH2--Y.sup.-, wherein X.sup.+
and Y.sup.- have the same meanings as in claim 1, of the desired
structure, optionally protected on the acid Y.sup.- group, with
alkane carbaldheydes, wherein the alkyl moiety is a one-term lower
homologue of the desired R.sub.4, and subsequent reduction.
12. Compounds according to claims 1-7, for use as medicaments.
13. Pharmaceutical composition comprising a therapeutically
effective amount of at least a compound of claims 1-7, in admixture
with pharmaceutically acceptable vehicles and excipients.
14. Pharmaceutical composition comprising a therapeutically
effective amount of at least a compound of claims 1-7, in admixture
with pharmaceutically acceptable vehicles and excipients and
optionally in combination with other active ingredients.
15. Use of a compound of claims 1-7, for the preparation of a
medicament useful for the treatment of pathologies related to a
hyperactivity of carnitine palmitoyl-transferase.
16. Use according to claim 15, wherein said pathology is selected
from the group consisting of hyperglycaemia, diabetes and
pathologies related thereto, heart failure, ischemia and ketonic
states.
17. Pharmaceutical composition according to claim 14, wherein said
other active ingredient is a suitable well-known active ingredient
for the treatment of diabetes.
18. Pharmaceutical composition according to claim 17, wherein said
other active ingredient suitable for the treatment of diabetes is
selected from the group consisting of sulfonylurea, L-carnitine,
fibrate and other agonists of peroxisomal proliferator activated
receptor (PPAR-.alpha.), agonists of 9-cis retinoic acid activated
receptor, HMG-CoA reductase inhibitor, .beta.-sitosterol inhibitor,
cholesterol acyltransferase inhibitor, biguanides, cholestyramine,
angiotensin II antagonist, melinamide, nicotinic acid, fibrinogen
receptor antagonists, aspirin, .alpha.-glucosidase inhibitors,
insulin secretogogue, insulin and glucagon-like peptides
(incretins) and agonists of PPAR-.gamma..
19. Use of the pharmaceutical composition any one of claims 17-18
for the treatment of diabetes.
20. Pharmaceutical composition according to claim 14, wherein said
other active ingredient is a suitable well-known active ingredient
for the treatment of obesity.
21. Pharmaceutical composition according to claim 20, wherein said
other active ingredient suitable for the treatment of obesity is
selected from the group consisting of fenfluramine,
dexfenfluramine, phentiramine, a .beta.-3-adrenergic receptor
agonist.
22. Use of the pharmaceutical composition any one of claims 20-21
for the treatment of obesity.
23. Pharmaceutical composition according to claim 14, wherein said
other active ingredient is a suitable well-known active ingredient
for the treatment of high triglyceridhemia.
24. Pharmaceutical composition according to claim 14, wherein said
other active ingredient suitable for the treatment of high
cholesterol levels and in modulating HDL plasma levels.
25. Pharmaceutical composition according to claim 24, wherein said
active ingredient suitable for the treatment of high cholesterol
levels and in modulating HDL plasma levels, is selected from the
group consisting of fibrates, and other PPAR-.alpha. agonists;
inhibitors of cholesterol biosynthesis, HMG-CoA reductase
inhibitors, statins, inhibitors of cholesterol absorption, acyl
CoA:cholesterol acyltransferase inhibitors, anion exchange resins,
nicotinyl alcohol, nicotinic acid or a salt thereof; vitamin E;
thyromimetics and L-carnitine.
26. Use of the pharmaceutical composition any one of claims 24-25
for the treatment of high cholesterol levels and related
diseases.
27. Use according to claim 26 for the treatment of hypertension,
obesity, atherosclerosis, diabetes and related conditions.
Description
[0001] The present invention relates to compounds having inhibiting
activity against carnitine palmitoyl transferase. The present
invention relates also to pharmaceutical compositions containing at
least one of these compounds active ingredients and to the use of
said compounds in the preparation of medicaments useful in the
treatment of pathologies related to a hyperactivity of carnitine
palmitoyl-transferase, in particular hyperglycaemic states, such as
diabetes and related pathologies and of congestive heart
failure.
BACKGROUND OF THE INVENTION
[0002] To date, hypoglycaemic therapy is based on the use of drugs
having different mechanism of action (Arch. Intern. Med., 1997,
157, 1802-1817).
[0003] Insulin and its analogues represent the most used therapy,
recurring to the direct hypoglycaemic action of this hormone.
[0004] Other compounds act indirectly by stimulating insulin
release (sulphonylureas). A different target of hypoglycaemic drugs
is represented by the reduction of glucose intestinal absorption
through the inhibition of intestinal glucosidases, or by reducing
insulin resistance.
[0005] Hyperglycaemia is also treated with gluconeogenesis
inhibitors, such as biguanides.
[0006] Some works have also stressed out the relationship between
gluconeogenesis and fatty acid oxidation.
[0007] The membrane bound long-chain acylcarnitine transferases,
also known as carnitine palmitoyltransferase (CPT), are widely
represented in organs and subcellular organelles (Bieber, L. L.
1988 Ann. Rev. Biochem. 57: 261-83). The well-established role of
this category of enzymes is the transport of activated long-chain
fatty acids through mitochondrial membranes. In this context, the
outer mitochondrial membrane CPT I catalyzes the formation of
long.-chain acylcarnitines that are transported across the
mitochondrial membrane by a specific carrier, and reconverted into
long-chain acyl-coenzyme A esters by CPT II, which resides in the
inner mitochondrial membrane. Long-chain acyl-CoAs are then
oxidised to acetyl-coenzyme A, which activates a key
gluconeogenetic enzyme: pyruvate carboxylase.
[0008] Other works report that diabetic patients have high blood
levels of fatty acids, whose liver oxidative fate gives rise to an
increase of acetyl-coenzyme A, ATP and NADH. High availability of
these compounds maximally stimulates gluconeogenesis, which is in
part responsible of the elevated glucose blood levels in diabetic
patients. CPT inhibition indirectly reduces the extent of liver
gluconeogenesis, and hence blood glucose levels.
[0009] CPT inhibitors have been disclosed in J. Med. Chem., 1995,
38(18), 3448-50 and in the corresponding European patent
application EP 0 574 355 as potential derivatives with
hypoglycaemic activity.
[0010] Aminocarnitines N-acylated with --COR residue, wherein R is
an aliphatic residue with 1 to 19 carbon atoms are disclosed in
WO85/04396 useful for investigating the role of transferases in the
body, in particular the specificity of carnitine
acyltransferase.
[0011] Emeriamine and its analogues are disclosed in EP 0 127 098
and J. Med. Chem. 1987, 30, 1458-1463.
[0012] Notwithstanding the mechanism of activity above outlined, to
date, drugs inhibiting CPT capable to effectively counteract
hyperglycaemia do not exist. For some products, such as tetradecyl
glycidic acid, or etomoxir, myocardial hypertrophy have been
evidenced as side effects (Life Sci., 1989, 44, 1897-1906).
[0013] None of the therapies presently used in clinic is fully
satisfying, in particular due to the onset of unwanted side
effects, such as severe hypoglycaemia, allergic phenomena, oedema,
diarrhoea, intestinal disturbances, kidney toxicity, etc.
[0014] The necessity to obtain alternative effective therapies for
hyperglycaemia still remains.
ABSTRACT OF THE INVENTION
[0015] It has now surprisingly been found that compounds of general
formula (I): 2
[0016] wherein: X.sup.+ is selected from the group consisting of
N.sup.+(R.sub.1,R.sub.2,R.sub.3) and P.sup.+
(R.sub.1,R.sub.2,R.sub.3), wherein
[0017] (R.sub.1,R.sub.2,R.sub.3), being the same or different, are
selected in the group consisting of hydrogen and C.sub.1-C.sub.9
straight or branched alkyl groups, --CH.dbd.NH(NH.sub.2),
--NH.sub.2, --OH; or two or more R.sub.1, R.sub.2 and R.sub.3
together with the nitrogen atom, which they are linked to, form a
saturated or unsaturated, monocyclic or bicyclic heterocyclic
system; with the proviso that at least one of the R.sub.1, R.sub.2
and R.sub.3 is different from hydrogen;
[0018] Z is selected from
[0019] --OR.sub.4,
[0020] --OCOOR.sub.4,
[0021] --OCONHR.sub.4,
[0022] --OCSNHR.sub.4,
[0023] --OCSOR.sub.4,
[0024] --NHR.sub.4,
[0025] --NHCOR.sub.4,
[0026] --NHCSR.sub.4,
[0027] --NHCOOR.sub.4,
[0028] --NHCSOR.sub.4,
[0029] --NHCONHR.sub.4,
[0030] --NHCSNHR.sub.4,
[0031] --NHSOR.sub.4, p1 --NHSONHR.sub.4,
[0032] --NHSO.sub.2R.sub.4,
[0033] --NHSO.sub.2NHR.sub.4,
[0034] --SR.sub.4,
[0035] wherein --R.sub.4 is a C.sub.1-C.sub.20 saturated or
unsaturated, straight or branched alkyl group, optionally
substituted with a A.sub.1 group, wherein A.sub.1 is selected from
the group consisting of halogen atom, C.sub.6-C.sub.14 aryl,
heteroaryl, aryloxy or heteroaryloxy group, said aryl, heteroaryl,
aryloxy or heteroaryloxy groups being optionally substituted with
one or more C.sub.1-C.sub.20 saturated or unsaturated, straight or
branched alkyl or alkoxy group and/or halogen atom;
[0036] Y.sup.- is selected from the group consisting of
--COO.sup.-, PO.sub.3H.sup.-, --OPO.sub.3H.sup.-,
tetrazolate-5-yl;
[0037] with the proviso that when Z is --NHCOR.sub.4, X.sup.+ is
trimethylammonium, Y is --COO.sup.-, then R.sub.4 is C.sub.20
alkyl;
[0038] with the proviso that when Z is --NHSO.sub.2R.sub.4, X.sup.+
is trimethylammonium and Y.sup.- is --COO.sup.-, then R.sub.4 is
not tolyl;
[0039] with the proviso that when Z is --NHR.sub.4, X.sup.+ is
trimethylammonium and Y.sup.- is --COO.sup.-, then R.sub.4 is not
C.sub.1-C.sub.6 alky.
[0040] The present invention further comprises the use of the
compounds of the above-mentioned formula (I) as active ingredients
for medicaments, in particular for medicaments useful for the
treatment of pathologies related to a hyperactivity of carnitine
palmitoyl carnitine, such as and in particular hyperglycemic
states, diabetes and related pathologies, congestive heart failure
and dilatative cardiopathy.
[0041] The present invention comprises pharmaceutical compositions
containing compounds of formula (I) as active ingredients, in
admixture with pharmaceutically acceptable vehicles and
excipients.
[0042] The present invention comprises also processes for the
preparation of compounds of formula (I).
[0043]
DETAILED DESSCRIPTION OF THE INVENTION
[0044] Within the scope of the present invention, as examples of
C.sub.1-C.sub.20 linear or branched alkyl group, methyl, ethyl,
propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl,
dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl,
octadecyl, nonadecyl and eicosyl and their possible isomers are
meant, such as for example isopropyl, isobutyl, tert-butyl.
[0045] Examples of C.sub.1-C.sub.20 linear or branched alkenyl
group are methylene, ethylidene, vinyl, allyl, propargyl, butylene,
pentylene, wherein the carbon-carbon double bond, optionally in the
presence of other carbon-carbon unsaturations, can be situated in
the different possible positions of the alkyl chain, which can also
be branched within the allowed isomery.
[0046] Examples of (C.sub.6-C.sub.14) aryl group are phenyl, 1- or
2-naphthyl, anthryl, optionally substituted as shown in the general
definitions above-mentioned.
[0047] Examples of heterocyclic groups thienyl, quinolyl, pyridyl,
N-methylpiperidinyl, 5-tetrazolyl, optionally substituted as shown
in the general definitions above-mentioned.
[0048] As halogen atom it is intended fluorine, chlorine, bromine,
iodine.
[0049] The compounds of formula (I) can be also in the form of
inner salts.
[0050] A first group of preferred compounds comprises the compounds
of formula (I) wherein N.sup.+(R.sub.1,R.sub.2,R.sub.3) is
trimethyl ammonium.
[0051] A second group of preferred compounds comprises the
compounds of formula (I) wherein two or more R.sub.1, R.sub.2 and
R.sub.3, together with the nitrogen atom, which they are linked to,
form a saturated or unsaturated, monocyclic or bicyclic
heterocyclic system; for example morpholinium, pyridinium,
pyrrolidinium, quinolinium, quinuclidinium.
[0052] A third group of preferred compounds comprises the compounds
of formula (I) wherein R.sub.1 and R.sub.2 are hydrogen and R.sub.3
is selected from the group consisting of --CH.dbd.NH(NH.sub.2),
--NH.sub.2 and --OH.
[0053] Within the different embodiments of the present invention,
the R.sub.4 group is preferably a C.sub.7-C.sub.20 saturated or
unsaturated, straight or branched alkyl group. In fact, it has been
observed the length of the alkyl chain R.sub.4 significantly
increases the selectivity against CPT. Preferred R.sub.4 groups are
selected from the group consisting of heptyl, octyl, nonyl, decyl,
undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl,
heptadecyl, octadecyl, nonadecyl and eicosyl.
[0054] Preferred examples of Z group are ureido (--NHCONHR.sub.4),
and carbamate (--NHCOOR.sub.4, --OCONHR.sub.4) ones.
[0055] In particular, compounds of formula (I) wherein X.sup.+,
R.sub.1, R.sub.2, R.sub.3, have the above disclosed meanings, Z is
ureido (--NHCONHR.sub.4) or carbamate (--NHCOOR.sub.4,
--OCONHR.sub.4), R.sub.4 is a C.sub.7-C.sub.20, preferably a
C.sub.9-C.sub.18 saturated or unsaturated, straight or branched
alkyl group, are preferred.
[0056] The compounds of formula (I) have an asymmetry center on
carbon atom bound to a Z group. For the purposes of the present
invention, each compound of formula (I) can exist both as R,S
racemic mixture and as separated R/S isomeric form.
[0057] The compounds of formula (I) are quaternary ammonium or
phosphonium derivatives always containing a Y.sup.- anionic group.
Dependently on pH, each compounds of formula (I) can exist
indifferently as amphoion (inner salt) or as a compound wherein
Y.sup.- is present in the YH form. In such a case, X.sup.+ is
salified with a pharmacologically acceptable acid. Formula (I)
covers all these different possibilities. In case of nitrogen atoms
having basic character, the salts with pharmaceutically acceptable
acids, both inorganic and organic, such as for example,
hydrochloric acid, sulfuric acid, acetic acid, or, in the case of
acid group, such as carboxyl, the salts with pharmaceutically
acceptable bases, both inorganic and organic, such as for example,
alkaline and alkaline-earth hydroxides, ammonium hydroxide, amine,
also heterocyclic ones. Examples of pharmaceutically acceptable
salts are chloride; bromide; iodide; aspartate; acid aspartate;
citrate; acid citrate; tartrate; acid tartrate; phosphate, acid
phosphate; fumarate; acid fumarate; glycerophosphate;
glucosephosphate; lactate; maleate; acid maleate; mucate; orotate;
oxalate; acid oxalate; sulfate; acid sulfate; trichloroacetate;
trifluoroacetate; methanesulfonate; pamoate and acid pamoate.
[0058] A first group of particularly preferred compounds
comprises:
[0059]
R,S-4-trimethylammonium-3-(nonylcarbamoyl)-aminobutyrate;
[0060]
R,S-4-quinuclidinium-3-(tetradecyloxycarbonyl)-oxybutyrate;
[0061] R,S-4-trimethylammonium-3-(nonylcarbamoyl)-oxybutyrate;
[0062] R,S-4-trimethylammonium-3-(nonyloxycarbonyl)-oxybutyric acid
chloride;
[0063]
R,S-4-trimethylphosphonium-3-(nonylcarbamoyl)-oxybutyrate;
[0064]
R,S-4-trimethylammonium-3-(octyloxycarbonyl)-aminobutyrate;
[0065]
R,S-4-trimethylammonium-3-(nonyloxycarbonyl)-aminobutyrate;
[0066] R,S-4-trimethylammonium-3-octyloxybutyrate;
[0067] R,S-4-trimethylammonium-3-tetradecyloxybutyrate;
[0068]
R,S-1-guanidinium-2-tetradecyloxy-3-(tetrazolate-5-yl)-propane;
[0069]
R,S-1-trimethylammonium-2-tetradecyloxy-3-(tetrazolate-5-yl)-propan-
e;
[0070]
R,S-3-quinuclidinium-2-(tetradecyloxycarbonyl)-oxy-1-propanephospho-
nate monobasic;
[0071]
R,S-3-trimethylammonium-2-(nonylaminocarbonyl)-oxy-1-propanephospho-
nate monobasic;
[0072]
R,S-3-pyridinium-2-(nonylaminocarbonyl)-oxy-1-propanephosphonic
acid chloride;
[0073]
R-4-trimethylammonium-3-(tetradecylcarbamoyl)-aminobutyrate;
[0074]
R-4-trimethylammonium-3-(undecylcarbamoyl)-aminobutyrate;
[0075] R-4-trimethylammonium-3-(heptylcarbamoyl)-aminobutyrate;
[0076]
R,S-4-trimethylammonium-3-(nonylthiocarbamoyl)-aminobutyrate;
[0077] R-4-trimethylammonium-3-(nonylcarbamoyl)-aminobutyrate;
[0078] S-4-trimethylammonium-3-(nonylcarbamoyl)-aminobutyrate;
[0079]
S-4-trimethylammonium-3-(tetradecylcarbamoyl)-aminobutyrate;
[0080] R,S-4-trimethylammonium-3-tetradecylaminobutyrate;
[0081] R,S-4-trimethylammonium-3-octylaminobutyrate;
[0082] R,S-4-trimethylammonium-3-(decansulfonyl)aminobutyrate;
[0083] R,S-4-trimethylammonium-3-(nonylsulfamoyl)aminobutyrate;
[0084] S-4-trimethylammonium-3-(do decansulfonyl)aminobutyrate;
[0085] R-4-trimethylammonium-3-(dodecansulfonyl) aminobutyrate;
[0086] S-4-trimethylammonium-3-(undecylsulfamoyl)aminobutyrate;
[0087] R-4-trimethylammonium-3-(undecylsulfamoyl)aminobutyrate;
[0088] R-4-trimethylammonium-3-(dodecylcarbamoyl)
aminobutyrate;
[0089] R-4-trimethylammonium-3-(
10-phenoxydecylcarbamoyl)aminobutyrate;
R-4-trimethylammonium-3-(trans-.beta.-styrenesulfonyl)
aminobutyrate.
[0090] The compounds of formula (I) can be prepared with reactions
that are well known in the state of the art.
[0091] A process for the preparation of the compounds of claim 1,
wherein Z is --NHR.sub.4 comprising the reaction of
X.sup.+--CH.sub.2--CH(NH.sub.- 2)--CH.sub.2--Y.sup.-, wherein
X.sup.+ and Y.sup.- have the same meanings as in claim 1, of the
desired structure, optionally protected on the acid Y.sup.- group,
with alkane carbaldheydes, wherein the alkyl moiety is a one-term
lower homologue of the desired R.sub.4 and subsequent
reduction.
[0092] Generally, the compounds of formula (I), wherein Z is
carbonate (--OCOOR.sub.4), carbamate (--OCONHR.sub.4,
--NHCOOR.sub.4), thiocarbamate (--OCSNHR.sub.4, --NHCSOR.sub.4,) or
thiocarbonate (--OCSOR.sub.4), are obtained by reacting a compound
of formula X.sup.+--CH.sub.2--CH(OH)--CH.sub.2--Y.sup.-, wherein
X.sup.+ and Y.sup.- are as above defined, of the desired structure,
optionally protected on the acid Y.sup.- group, respectively with
alkyl chloroformates, alkyl isocyanates, alkyl isothiocyanates,
alkyl thiochloroformates, containing the desired R.sub.4 alkyl
group.
[0093] Compounds of formula (I), wherein Z is amide
(--NHCOR.sub.4), thioamide (--NHCSR.sub.4), carbamate
(--NHCOOR.sub.4, --OCONHR.sub.4), thiocarbamate
(--NHCSOR.sub.4--OCSNHR.sub.4,), ureido (--NHCONHR.sub.4),
thioureido (--NHCSNHR.sub.4), sulfinamide (--NHSOR.sub.4),
sulfonamide (--NHSO.sub.2R.sub.4), sulfinamoylamino
(--NHSONHR.sub.4), and sulfamide (--NHSO.sub.2NHR.sub.4), are
obtained by reacting X.sup.+--CH.sub.2--CH(N-
H.sub.2)--CH.sub.2--Y.sup.-, wherein X.sup.+ and Y.sup.- are as
above defined, of the desired structure, optionally protected on
the acid Y.sup.- group, respectively with acyl chlorides, thioacyl
chlorides, alkyl chloroformates, alkyl thiochloroformates, alkyl
isocyanates, alkyl thioisocyanates, alkyl sulfinyl chlorides, alkyl
sulfonyl chlorides, SOCl.sub.2 and alkyl amines, alkyl sulfamoyl
chlorides (or SO.sub.2Cl.sub.2 and alkyl amines), containing the
desired R.sub.4 alkyl group.
[0094] Compounds of formula (I), wherein Z is --OR.sub.4 or
--SR.sub.4 are obtained by the reaction of carbonyl compounds of
formula Hal--CH2--CO--CH2--COOR', wherein Hal is a halogen atom,
preferably chlorine, and R' is the residue of a suitable ester,
such as for example a lower alkyl ester (an ethyl or a tert-butyl
ester) with respectively alcohols and thiols R40H or R4SH, wherein
R4 is as above defined, to give the respective ketal or thioketal,
followed by the transformation of the respective ketal or thioketal
into the respective ether or thioether, subsequent substitution of
the Hal atom with a nucleophilic group, such as azido, phthalimido,
nitro, amino, alkyl amino group, and transformation of the
nucleophilic group into the X+ group, wherein X+ is
N.sup.+(R.sub.1,R.sub.2,R.sub.3) or, alternatively the Hal atom is
substituted with a (R.sub.1,R.sub.2, R.sub.3)-substituted phosphine
to obtain the compounds of formula (I) wherein X.sup.+ is
P.sup.+(R.sub.1,R.sub.2, R.sub.3).
[0095] Compounds of formula (I), wherein Z is --NHR.sub.4 are
obtained by reacting
X.sup.+--CH.sub.2--CH(NH.sub.2)--CH.sub.2--Y.sup.-, wherein X.sup.+
and Y.sup.- have the same meanings as in claim 1, of the desired
structure, optionally protected on the acid Y.sup.- group, with
alkane carbaldheydes, wherein the alkyl moiety is a one-term lower
homologue of R.sub.4, and subsequent reduction.
[0096] Regarding the various meanings of R.sub.4, present in the
different reactives, these reactives are available in the market,
or can be prepared according to well-known methods in literature,
which the experts in the field can resort to, completing with their
own knowledge of the argument.
[0097] Pharmaceutically acceptable salts are obtained with
conventional methods found in the literature, and do not
necessitate of further disclosure.
[0098] The compounds disclosed in the present invention have
reversible inhibiting activity of carnitine palmitoyl-transferase
(CPT). This activity allows their use as active ingredients in the
preparation of medicaments useful for the treatment and prevention
of hyperglycaemia, diabetes and disorders related thereto, such as,
for example diabetic retinopathy, diabetic neuropathy. The
compounds of the present invention are also useful as active
ingredient for the treatment and prevention of cardiovascular
disorders, such as congestive heart failure. The compounds of
formula (I) are also applicable for medicaments for the prevention
and treatment of ketonic states, wherein it is intended the
pathological conditions characterized by high levels of ketone
bodies in blood.
[0099] Inhibiting activity mainly occurs on the isoform I of
palmitoyl carnitine transferase (CPT-I).
[0100] A further object of the present invention relates to
pharmaceutical compositions comprising at least a compound of
formula (I), in an amount such as to produce a significant
therapeutical effect. The compositions according to the present
invention are conventional and are obtained with commonly used
methods in the pharmaceutical industry. According to the desired
administration route, the compositions shall be in solid or liquid
form, suitable to the oral, parenteral, intravenous or transdermal
route. The compositions according to the present invention comprise
together with the active ingredients at least a pharmaceutically
acceptable vehicle or excipient. Formulation co-adjuvants, for
example solubilizing, dispersing, suspending, emulsionating agents
can be particularly useful. Examples of suitable oral
pharmaceutical compositions are capsules, tablets, granulates,
powders, syrups, elixirs. Examples of suitable parenteral
pharmaceutical compositions are solutions, emulsions, suspensions.
Examples of suitable transdermal pharmaceutical compositions are
patches, subcutaneous implants.
[0101] The compounds of formula (I) can also be used in combination
with other well-known active ingredients.
[0102] The dose of the active ingredients will vary depending on
the kind of active ingredient used, the administration route, the
grade of pathology to be treated and the general conditions of the
subject. The dosage and posology shall be determined by the clinic
expert or the physician. Generally, a therapeutic effect can be
obtained at dosages comprised between 1 and. 100 mg/kg body
weight.
[0103] The compounds according to the present invention are useful
as medicaments with hypoglycaemic activity. A further object of the
present invention is the preparation of a pharmaceutical
composition comprising admixing at least a compound of formula (I)
with suitable pharmaceutically acceptable excipients and/or
vehicles.
[0104] The following examples further illustrate the invention.
EXAMPLE 1
R,S-4-trimethylammonium-3-(nonylcarbamoyl)-aminobutyrate (ST
1251)
Nonyl Isocyanate
[0105] A solution of decanoyl chloride (20 g, 104.8 mmoles) in
acetone (30 ml) was dropped into a solution of sodium azide (9.53
g, 146.6 mmoles) in water (30 ml), cooled in an ice bath. The
temperature of the azide solution was kept between 10 and
15.degree. C. after one hour, the solution was transferred in a
separatory funnel and the lower phase (the aqueous one) was
eliminated. The higher phase was transferred into a flask
containing 100 ml of toluene, previously warmed at 65.degree. C.
After 1.5 hours, the solution was evaporated to dryness, giving
13.37 g crude product, which after vacuum distillation gave 8.3 g
pure product in the form of colorless liquid.
[0106] Yield 47%.
[0107] .sup.1H-NMR (300 MHz; CDCl.sub.3):
[0108] .delta.: 3.3 (t, 2H), 1.6 (m, 2H), 1.45-1.2 (m, 12H),
0.9(brt, 3H).
R,S-4-trimethylammonium-3-(nonylcarbamoyl)-aminobutyrate
[0109] Nonyl isocyanate (15.42 g, 91.12 mmoles) was added to a
solution of aminocarnitine, inner salt (7.3 g, 45.56 mmoles) in
anhydrous DMSO (350 ml) and the solution was left to stand for 60
hours at 40.degree. C. The resulting mixture was transferred in a 3
l Erlenmeyer flask, containing ethyl ether (2.5 l) and the solvent
was separated by decanting the formed precipitate, which was then
transferred into a flask and precipitated again with ethyl ether.
The so obtained crude product was washed several times with ethyl
ether and purified on a silica gel chromatographic column, using a
CHCl.sub.3: MeOH 9:1 to CHCl.sub.3: MeOH 3:7 gradient until elution
of impurities with higher Rf, then eluting the product of interest
with MeOH only. 9.7 g of pure product were obtained.
[0110] Yield 68%.
[0111] M.p.: 145-147.degree. C.
[0112] .sup.1H-NMR (300 MHz; D.sub.2O):
[0113] .delta.: 4.4 (m, 1H), 3.45 (dd, 1H), 3.30 (d, 1H), 3.05 (s,
9H), 2.9 (t, 2H), 2.3 (d, 2H), 1.3 (m, 2H), 1.15 (brs, 12H), 0.8
(brt, 3H).
[0114] FAB Mass=330, [(M+H).sup.+].
[0115] Elemental analysis: responding to the expected formula
C.sub.17H.sub.35N.sub.3O.sub.3.
[0116] K.F.=2.5% water.
[0117] TLC silica gel CHCl.sub.3:iPrOH:MeOH:H.sub.2O:CH.sub.3COOH
42:7:28: 10.5: 10.5;
[0118] Rf=0.55.
[0119] HPLC: SGE-SCX column (5 .mu.m, 250.times.4 mm), T=30.degree.
C., mobile phase 0.2 M KH.sub.2PO.sub.4:CH.sub.3CN 85:15, pH as
such, flow 0.75 ml/min, detector: RI, UV 205 nm, RT=12.63 min.
EXAMPLE 2
R,S-4-quinuclidinium-3-(tetradecyloxycarbonyl)-oxybutyrate (ST
1265)
ter-Butyl R,S-4-guinuclidinium-3-hydroxybutyrate iodide
[0120] Quinuclidine (2.40 g, 21.60 mmoles) was added to ter-Butyl
R,S-4-iodo-3-hydroxybutyrate (6.18 g, 21.60mmoles) in acetonitrile
(60 ml) and the solution was warmed to 60.degree. C. for 20 hours
under stirring. After evaporation of the solvent, the residue was
dissolved in acetonitrile and precipitated with ethyl ether several
times to give 7.2 g of product, contaminated with about 13% by
weight of quinuclidine iodide (as from NMR). After repeated
crystallization from CH.sub.3CN/Et.sub.2O, 4.3 g of pure product
were obtained.
[0121] Yield 50%.
[0122] M.p.: 124-127.degree. C.
[0123] .sup.1H-NMR (300 MHz; D.sub.2O):
[0124] .delta.: 4.50 (m, 1H), 3.40 (m, 2H), 2.42 (m, 2H), 2.08 (m,
1H), 1.88 (m, 6H), 1.34 (m, 9H).
[0125] FAB Mass=270, [M.sup.+].
[0126] Elemental analysis: responding to the expected formula
[0127] C.sub.15H.sub.28 INO.sub.3.
[0128] K.F.=0.5% water.
[0129] The preparation of ter-butyl 4-iodo-3-hydroxybutyrate was
carried out as described in J. Pharm. Science 64/7, 1262-1264,
1975.
Tetradecyl Chloroformate
[0130] 29 ml of a 20% toluene solution of phosgene (55.98 mmoles)
was added to tetradecyl alcohol (4 g, 18.66 mmoles) and the
reaction mixture was left to stand for 20 hours under stirring at
room temperature. After solvent evaporation, the residue was taken
up with hexane and evaporated to dryness (several times) to give
5.1 g product as colorless liquid.
[0131] Yield 98%.
[0132] .sup.1H-NMR (300 MHz; CDCl.sub.3):
[0133] .delta.: 4.30 (t, 2H), 1.72 (m, 2H), 1.30 (m, 22H), 0.85
(brt, 3H).
ter-butyl R,S-4-guinuclidinium-3-(tetradecyloxycarbonyl)-oxy
butyrate chloride
[0134] Dimethylaminopyridine (922 mg, 755 mmoles) and tetradecyl
chloroformate (2.09 g, 7.55 mmoles) were added to ter-butyl
R,S-4-quinuclidinium-3-hydroxybutyrate (2 g, 5.03 mmoles) in
anhydrous CH.sub.2Cl.sub.2 (20 ml). The solution was left to stand
at room temperature for 20 hours under stirring. After this time,
the solution was diluted with CHCl.sub.3 saturated with NaCl, and
dried over anhydrous sodium sulfate. The dry residue obtained after
evaporation was taken up with ethyl ether and the undissolved
residue was filtered off. After solvent evaporation a crude product
was obtained. Flash-chromatography (CHCl.sub.3: MeOH 9:1) and
elution with MeOH on Amberlyst A-21 resin (activated in HCl from),
gave 1.6 g product as chloride.
[0135] Yield 58%.
[0136] M.p.: 59-60.degree. C.
[0137] .sup.1H-NMR (300 MHz; CDCl.sub.3):
[0138] .delta.: 5.50 (m, 1H), 4.55 (d, 2H), 3.80 (m, 7H), 2.90 (dd,
1H), 2.75 (dd, 1H), 2.22 (m, 1H), 2.05 (d, 6H), 1.65 (m, 2H), 1.41
(s, 9H), 1.25 (m, 22H), 0.85 (brt, 3H).
[0139] FAB Mass=510, [M.sup.+].
[0140] Elemental analysis: responding to the expected formula
[0141] C.sub.30H.sub.56 ClNO.sub.5.
[0142] K.F.=1.5% water.
R,S-4-guinuclidinium-3-(tetradecyloxycarbonyl)-oxybutyrate
[0143] Trifluoroacetic acid (6 ml) was added to ter-butyl
R,S-4-quinuclidinium-3-(tetradecyloxycarbonyl)-oxybutyrate chloride
(1.05 g, 1.92 mmoles) and the solution was left to stand for 1 hour
at room temperature under stirring. After vacuum-evaporation of
trifluoroacetic acid, the residue was taken up with cyclohexane and
evaporated to dryness several times, then transferred on an
Amberlyst IRA 402 resin (Cl.sup.-form) and eluted with water. The
crude product, obtained by freeze-drying was purified through
silica gel flash-chromatography (CHCl.sub.3: MeOH 8:2) giving 480
mg product as inner salt.
[0144] Yield 55%.
[0145] M.p.: 132-134.degree. C.
[0146] .sup.1H-NMR (300 MHz; D.sub.2O):
[0147] .delta.: 5.35 (m, 1H), 4.05 (m, 2H), 3.40 (m, 8H), 2.55 (dd,
1H), 2.35 (dd, 1H), 2.08 (m, 1H), 1.90 (m, 6H), 1.55 (m, 2H), 1.20
(m, 22H), 0.75 (brt, 3H).
[0148] FAB Mass=454, [(M+H).sup.+.
[0149] Elemental analysis: responding to the expected formula
C.sub.26H.sub.47NO.sub.5
[0150] K.F.=1.5% water.
[0151] TLC silica gel CHCl.sub.3:MeOH 7:3.
[0152] Rf=0.34.
[0153] HPLC: SGE-SCX column (5 .mu.m, 250.times.4 mm), T=30.degree.
C., mobile phase 0.05 M (NH.sub.4)H.sub.2PO.sub.4:CH.sub.3CN 60:40,
pH 4.0, flow 0.75 ml/min, detector: RI, UV 205 nm, RT=6.72 min.
EXAMPLE 3
R,S-4-trimethylammonium-3-(nonylcarbamoyl)-oxybutyrate (ST
1298)
Benzyl ester of
R,S-4-trimethylammonium-3-(nonylcarbamoyl)-oxybutyric acid
Perchlorate
[0154] Nonyl isocyanate (7.39 g, 43.36 mmoles) was added to a
solution of R,S-carnitine perchlorate, benzyl ester (7.69 g, 21.86
mmoles) in toluene (100 ml) and the solution was refluxed for 5
days under stirring. Nonyl isocyanate (1.84 g, 10.86 mmoles) was
further added and the reaction mixture was left under reflux for
other 5 days. The solvent was vacuum-evaporated and the residue was
washed with ethyl ether and subsequently taken up with chloroform,
washed with water and dried over anhydrous sodium sulfate. The oil
resulting from the evaporation of the organic phase was purified
through flash-chromatography column, using a gradient CHCl.sub.3 to
CHCl.sub.3: MeOH 95:5. 4.4 g product were obtained in the form of a
thick oil.
[0155] Yield 38.6%.
[0156] .sup.1H-NMR (200 MHz; CDCl.sub.3):
[0157] .delta.: 7.3 (s, 5H), 5.4 (m, 2H), 5.05 (m, 2H), 3.8 (dd,
1H), 3.55 (d, 1H), 3.15 (s, 9H), 3.05 (m, 2H), 2.75 (m, 2H), 1.4
(m, 2H), 1.2 (brs, 12H), 0.8 (brt, 3H).
[0158] TLC silica gel CHCl.sub.3: MeOH 9:1;
[0159] Rf=0.29.
R,S-4-trimethylammonium-3-(nonylcarbamoyl)-oxybutyrate
[0160] 10% Pd/C (0.44 g) was added to benzyl ester of
R,S-4-trimethylammonium-3-(nonylcarbamoyl)-oxybutyric acid
perchlorate (4.4 g, 8.44 mmoles) in MeOH (115 ml) and the mixture
was hydrogenated at 47 psi for 4 hours. After filtration on celite,
the solution was vacuum-concentrated and passed through an
Amberlyst A-21 resin, eluting with MeOH. After solvent evaporation,
2.47 g product were obtained.
[0161] Yield 88.7%.
[0162] M.p.: 151-153.degree. C.
[0163] .sup.1H-NMR (300 MHz; D.sub.2O):
[0164] .delta.: 5.4 (m, 1H), 3.75 (dd, 1H), 3.5 (d, 1H), 3.15 (s,
9H), 3.05 (t, 2H), 2.55 (dd, 1H), 2.40 (dd, 1H), 1.45 (m, 2H), 1.20
(brs, 12H), 0.8 (brt, 3H).
[0165] FAB Mass=331, [(M+H).sup.+].
[0166] Elemental analysis: responding to the expected formula
C.sub.17H.sub.34 N.sub.2O.sub.4.
[0167] K. F.=1.5% water.
[0168] TLC silica gel MeOH.
[0169] Rf=0.22.
[0170] HPLC: SPHERISORB-SCX column (5 .mu.m, 250.times.4 mm),
T=35.degree. C., mobile phase 50 mM KH.sub.2PO.sub.4:CH.sub.3CN
40:60, pH 4.0 with H.sub.3PO.sub.4, flow 0.75 ml/min, detector: RI,
UV 205 nm, RT=5.33 min.
EXAMPLE 4
R,S-4-trimethylammonium-3-(nonyloxycarbonyl)-oxybutyrate chloride
(ST 1297)
Benzyl ester of
R,S-4-trimethylammonium-3-(nonylcarbamoyl)-oxybutyric acid
chloride
[0171] Dimethylaminopyridine (3.8 g, 31.2 mmoles) and nonyl
chloroformate (6.45 g, 31.2 mmoles) were added to R,S-carnitine
perchlorate, benzyl ester (7.33 g, 20.8 mmoles) in anhydrous DMF
(50 ml) at 0.degree. C. The temperature was left to raise to room
temperature and the reaction mixture was left to stand for 3 days
under stirring. CHCl.sub.3 was added and the solution was washed
with 1N perchloric acid. The organic phase was dried over anhydrous
sodium sulfate and evaporated to dryness, to give 6.02 g crude
product, which was purified through flash-chromatography
(CHCl.sub.3: MeOH 85:15). 3.52 g a thick oil were obtained, which
were subsequently dissolved in MeOH and passed through an Amberlyst
A-21 resin (activated in HCl from), eluting with MeOH. After
vacuum-evaporation of the solvent, 3.1 g oily product were
obtained.
[0172] Yield 32.4%.
[0173] .sup.1H-NMR (200 MHz; CDCl.sub.3):
[0174] .delta.: 7.3 (s, 5H), 5.45 (m, 1H), 5.05 (s, 2H), 4.4 (d,
1H), 4.1 (t, 2H), 3.8 (dd, 1H), 3.4 (s, 9H), 2.9 (m, 2H), 1.55 (m,
2H), 1.2 (brs, 12H), 0.8 (brt, 3H).
[0175] Mutatis mutandis, the preparation of nonyl chloroformate was
carried out as disclosed in Example 2 for tetradecyl
chloroformate.
R,S-4-trimethylammonium-3-(nonyloxycarbonyl)-oxybutyric acid
chloride
[0176] 10% Pd/C (110 mg) was added to benzyl
R,S-4-trimethylammonium-3-(no- nyloxycarbonyl)-oxybutyric acid
chloride (1.1 g, 2.4 mmoles) in MeOH (10 ml) and the mixture was
hydrogenated at 47 psi for 2 hours. After filtration on celite, the
solution was vacuum-dried giving 883 mg product (yield 100%), which
was further purified by precipitation from CH.sub.3CN/Et.sub.2O.
600 g of product were obtained.
[0177] Yield: 68%.
[0178] M.p.: 150.degree. C. dec.
[0179] .sup.1H-NMR (300 MHz; D.sub.2O):
[0180] .delta.: 5.4 (m, 1H), 4.1 (m, 2H), 3.75 (dd, 1H), 3.55 (d,
1H), 3.1 (s, 9H), 2.7 (m, 2H), 1.5 (m, 2H), 1.20 (brs, 12H), 0.7
(brt, 3H).
[0181] FAB Mass=332, [M.sup.+].
[0182] Elemental analysis: responding to the expected formula
[0183] C.sub.17H.sub.34 ClNO.sub.5.
[0184] K.F.=1.7% water.
[0185] TLC silica gel CHCl.sub.3:MeOH 1:1;
[0186] Rf=0.10.
[0187] HPLC: SPHERISORB-C.sub.1 column (5 .mu.m, 250.times.4.6 mm),
T=30.degree. C., mobile phase 50 mM
(NH.sub.4)H.sub.2PO.sub.4:CH.sub.3CN 60:40, pH 3.0 with
H.sub.3PO.sub.4, flow 0.75 ml/min, detector: RI, UV 205 nm, RT=5.67
min.
EXAMPLE 5
R,S-4-trimethylphosphonium-3-(nonylcarbamoyl)-oxybutyrate (ST
1300)
Ethyl ester of R,S-4-trimethylphosphonium-3-hydroxybutyric acid
iodide
[0188] A 1M solution of trimethylphosphine in THF (93 ml) was added
to ethyl R,S-4-iodo-3-hydroxybutyrate (20 g, 77.5 mmoles) and the
reaction mixture was left to stand at room temperature for 5 days
under stirring. Ethyl ether was added, and the -precipitate formed
was separated by decantation. The precipitate was triturated with
Et.sub.2O and dried under vacuum, giving 18.5 g product.
[0189] Yield 71.3%.
[0190] M.p.: 105-107.degree. C.
[0191] .sup.1H-NMR (200 MHz; CDCl.sub.3):
[0192] .delta.: 4.6 (m, 1H), 4.15 (q, 2H), 3.1 (m, 1H), 2.75 (m,
3H), 2.2 (d, 9H), 1.3 (t, 3H).
[0193] The ethyl ester of
R,S-4-trimethylphosphonium-3-hydroxybutyric acid was prepared as
described in Tetrahedron 1990, 4277-4282, starting from
R,S-3-hydroxy-4-butyrolactone.
Ethyl ester of
R,S-4-trimethylphosphonium-3-(nonylcarbamoyl)-oxybutyric acid
iodide
[0194] Nonyl isocyanate (4.04 g, 23.86 mmoles) was added to the
ethyl ester of R,S-4-trimethylphosphonium-3-hydroxybutyric acid
iodide (4 g, 11.97 mmoles) in anhydrous DMF (80 ml) and the
solution was left to stand for 7 days at 110.degree. C. under
stirring. CHCl.sub.3 was added (300 ml) and the solution was washed
with water and dried over Na.sub.2SO.sub.4. The residue obtained
after evaporation of the solvent was taken up with acetonitrile,
the formed solid was filtered off and the filtrate was purified by
silica gel flash-chromatography, using CHCl.sub.3: MeOH 8:2. 2.07 g
of product in the form of a thick oil were obtained.
[0195] Yield 34.3%.
[0196] .sup.1H-NMR (200 MHz; CDCl.sub.3):
[0197] .delta.: 5.4 (m, 2H), 4.15 (q, 2H), 3.15 (m, 4H), 2.8 (d,
2H), 2.2 (d, 9H), 1.5 (m, 2H), 1.2 (brs, 12H), 0.8 (brt, 3H).
R,S-4-trimethylphosphonium-3-(nonylcarbamoyl)-oxybutyrate
[0198] Ethyl ester of
R,S-4-trimethylphosphonium-3-(nonylcarbamoyl)-oxybut- yric acid
iodide (2.07 g, 4.11 mmoles) was dissolved into 1 N HCl (200 ml)
and the solution was warmed to 70.degree. C. for 3 hours. The
residue obtained after solvent vacuum-evaporation was taken up with
MeOH and passed through Amberlyst A-21 resin, eluting with MeOH. A
crude product was obtained, which was purified by
flash-chromatography, eluting with MeOH and giving 700 mg
product.
[0199] Yield: 49%.
[0200] M.p.: 123-127.degree. C. dec.
[0201] .sup.1H-NMR (300 MHz; D.sub.2O):
[0202] .delta.: 5.3 (m, 1H), 3.1 (m, 2H), 2.80-2.45 (m, 4H), 1.85
(d, 9H), 1.4 (m, 2H), 1.2 (brs, 12H), 0.8 (brt, 3H).
[0203] FAB Mass=348, [(M+H).sup.+].
[0204] Elemental analysis: responding to the expected formula
C.sub.17H.sub.34 NO.sub.4P.
[0205] K.F.=3.4% water.
[0206] TLC silica gel MeOH;
[0207] Rf=0.18.
[0208] HPLC: SPHERISORB-SCX column (5 .mu.m, 250.times.4 mm),
T=25.degree. C., mobile phase 50 mM KH.sub.2PO.sub.4:CH.sub.3CN
40:60, pH 4.0 with H.sub.3PO.sub.4, flow 0.75 ml/min, detector: RI,
UV 205 nm, RT=5.18 min.
[0209] The following Examples 6 and 7 are further illustrated by
FIG. 1.
Example 6
R,S-4-trimethylammonium-3-(octyloxycarbonyl)-aminobutyrate chloride
(ST 1253) (2a, FIG. 1)
Step A
[0210] 3 g (0.012 mmoles) aminocarnitine isobutyl ester were
dissolved into 20 ml anhydrous CH.sub.2Cl.sub.2. 2.48 ml (0.1078
moles) triethylamine and 3.6 g (0.0178 moles) octyl chloroformate
(previously prepared by reacting the alcohol with a toluene
solution of phosgene) were added to the solution. The reaction
mixture was left to stand for 4.5 hours at room temperature. Then
the solvent was evaporated off and the resulting solid was
dissolved into ethyl acetate and filtered. The solvent was
vacuum-evaporated to dryness and the resulting solid was purified
on silica gel, eluting with 100% CHCl.sub.3, then with
CHCl.sub.3:MeOH 95:5 and 90:10. The product was obtained with a 50%
yield.
[0211] TLC silica gel (CHCl.sub.3 42/MeOH 28/isopropyl alcohol
7/water 10.5/acetic acid 10.5)/acetone 7:3;
[0212] Rf=0.8.
[0213] HPLC: SPHERISORB-SCX column (5 .mu.m, 250.times.4 mm),
mobile phase 50 mM (NH.sub.4)H.sub.2PO.sub.4:CH.sub.3CN 60:40, pH
4.0, detector: RI, UV 205 nm, RT=8.6 min.
[0214] .sup.1H-NMR (300 MHz; CD.sub.3OD):
[0215] .delta.: 4.56-4.46 (m, 1H), 4.12-4.02 (m, 2H), 3.94-3.88 (m,
2H), 3.66-3.5 (s, 9H), 3.4 (s, 9H), 2.74-2.66 (m, 2H), 2-1.86 (m,
1H), 1.68-1.56 (t, 2H), 1.4-1.2 (m, 12H), 0.97-0.7 (d, 6H), 0.6-0.3
(t, 3H).
[0216] Elemental analysis: responding to the expected formula
[0217] C.sub.20H.sub.41 N.sub.2O.sub.4Cl.
Step B
[0218] The ester obtained in step A was hydrolysed on Amberlyst IRA
402 resin (OH- activated form) eluting with water. Water was
evaporated to dryness; the resulting solid was triturated with
acetone and subsequently filtered. A white solid was obtained.
[0219] Yield 94%.
[0220] M.p.=170.degree. C. dec.
[0221] .sup.1H-NMR (300 MHz; CD.sub.3OD):
[0222] .delta.: 4.4 (m, 1H), 4.05 (t, 2H), 3.5 (d, 2H), 3.2 (s,
9H), 2.4 (d, 2H), 1.6 (m, 2H), 1.4-1.2 (m, 12H), 0.95-0.85 (t,
3H).
[0223] FAB Mass=454, [(M+H).sup.+.
[0224] Elemental analysis: responding to the expected formula
C.sub.16H.sub.32N.sub.2O.sub.4
[0225] K.F.=1.74% water.
[0226] TLC silica gel (CHCl.sub.3 42/MeOH 28/isopropyl alcohol
7/water 10.5/acetic acid 10.5)
[0227] Rf=0.65.
[0228] HPLC: SGE-SCX column (5 .mu.m, 250.times.4 mm), mobile phase
0.05M (NH.sub.4)H.sub.2PO.sub.4:CH.sub.3CN 60:40, detector: RI, UV
205 nm, RT=9.0 min.
EXAMPLE 7
R,S-4-trimethylammonium-3-(nonyloxycarbonyl)-aminobutyrate (ST
1285) (2b, FIG. 1)
Step A
[0229] The product was prepared as disclosed in Example 6, step A,
using nonyl chloroformate
[0230] Yield: 50%.
[0231] TLC silica gel (CHCl.sub.3 42/MeOH 28/isopropyl alcohol
7/water 10.5/acetic acid 10.5)/acetone 7:3
[0232] Rf=0.71.
[0233] HPLC: SGE-SCX column (5 .mu.m, 250.times.4 mm), mobile phase
50 mM (NH.sub.4)H.sub.2PO.sub.4:CH.sub.3CN 60:40, pH 4.0, detector:
RI, UV 205 nm, RT=10.417 min.
[0234] .sup.1H-NMR (300 MHz; CD.sub.3OD):
[0235] .delta.: 4.54-4.44 (m, 1H), 4.1-4.02 (m, 2H), 3.96-3.86 (m,
2H), 3.6-3.5 (m, 2H), 3.2 (s, 9H), 2.72-2.66 (m, 2H), 2-1.86 (m,
1H), 1.66-1.56 (m, 2H), 1.38-1.26 (m, 14H), 0.96-0.94 (d, 6H),
0.92-0.86 (t, 3H).
Step B
[0236] The product was prepared as disclosed in Example 6, step
B.
[0237] Yield 80%.
[0238] M.p.=160.degree. C. dec.
[0239] .sup.1H-NMR (300 MHz; CD.sub.3OD):
[0240] .delta.: 4.5-4.35 (m, 1H), 4.1-4.0 (t, 2H), 3.55-3.45 (d,
2H), 3.2 (s, 9H), 2.45-2.35 (d, 2H), 1.7-1.5 (m, 2H), 1.4-1.2 (m,
14H), 0.9-0.8 (t, 3H).
[0241] Elemental analysis: responding to the expected formula
C.sub.17H.sub.34N.sub.2O.sub.4
[0242] K.F.=1.3% water.
[0243] TLC silica gel (CHCl.sub.3 42/MeOH 28/isopropyl alcohol
7/water 10.5/acetic acid 10. 5);
[0244] Rf=0.62.
[0245] HPLC: SGE-SCX column (5 .mu.m, 250.times.4 mm), mobile phase
0.05M (NH.sub.4)H.sub.2PO.sub.4:CH.sub.3CN 60:40, detector: RI, UV
205 nm, RT=7.56 min.
[0246] The following Examples 8-9 are further illustrated by FIG.
2.
EXAMPLE 8
R,S-4-trimethylammonium-3-octyloxybutyrate (ST 1207) (6a, FIG.
2)
Step A
[0247] 39 g (0.3 moles) octyl alcohol were dissolved in 25 ml
toluene and 14.5 ml (0.107 moles) ethyl chloroacetate and 8 ml
Thionyl chloride were added thereto at -15.degree. C. At the end of
the addition, the reaction mixture was left to stand for 4 hours at
room temperature Ethyl acetate was then added and the solution was
washed three times with 1N NaOH and subsequently with water. The
organic phase was treated with anhydrous sodium sulfate, filtered
and vacuum-evaporated to dryness. The product was purified on
silica gel chromatographic column, eluting with gradient from
hexane alone to hexane/ethyl ether 95:5. The product was obtained
with 80% yield.
[0248] TLC silica gel hexane/ ethyl ether 85:15;
[0249] Rf=0.75.
[0250] .sup.1H-NMR (300 MHz; CDCl.sub.3):
[0251] .delta.: 4.2-4.09 (q, 2H), 3.80 (s, 2H), 3.4-3.5 (dd, 2H),
2.85 (s, 2H), 1.60-1.58 (m, 2H), 1.4-1.2 (m, 10H), 0.90-0.80 (t,
3H).
[0252] Elemental analysis: responding to the expected formula
[0253] C.sub.22H.sub.33 ClO.sub.4.
Step B
[0254] 9 ml BF.sub.3.Et2O were dropped to a mixture of 26.8 g
(0.066 moles) of the product obtained in the preceding step A and
13.5 ml triethylsilane at 0.degree. C. At the end of the addition,
the reaction mixture was refluxed for 4 hours. After cooling, ether
was added and the solution was washed twice with NaOH 1N, then
water; the organic phase was dried over anhydrous sodium sulfate,
filtered and vacuum-evaporated to dryness. An oil was obtained,
which was purified on silica gel chromatographic column, eluting
with gradient from hexane alone to hexane/ethyl ether 95:5. The
product was obtained with a 70% yield.
[0255] TLC silica gel hexane/ethyl ether 90:10;
[0256] Rf=0.47. .sup.1H-NMR (300 MHz; CDCl.sub.3):
[0257] .delta.: 4.2-4.09 (dd, 2H), 4.0-3.85 (m, 1H), 3.62-3.40 (m,
4H), 2.70-2.50 (dd, 2H), 1.55-1.50 (m, 2H), 1.4-1.2 (m, 1OH),
0.90-0.80 (t, 3H).
[0258] Elemental analysis: responding to the expected formula
C.sub.14H.sub.27ClO.sub.3
Step C
[0259] 5.2 g (0.08 moles) NaN.sub.3 and a catalytic amount of
tetrabutyl ammonium bromide were added to a solution of 11.4 g
(0.041 moles) product obtained in the preceding step B. The
reaction mixture was left for three nights at 60.degree. C. The
solution was vacuum-evaporated to dryness. A thick dark solution
was obtained, which was purified on silica gel chromatographic
column, eluting with gradient from hexane alone to hexane/ethyl
ether 95:5. The product was obtained with a 83% yield.
[0260] TLC silica gel hexane/ethyl ether 95:5;
[0261] Rf=0.23.
[0262] .sup.1H-NMR (300 MHz; CDCl.sub.3):
[0263] .delta.: 4.2-4.09 (dd, 2H), 4.0-3.80 (m, 1H), 3.60-3.40 (dd,
2H), 3.40-3.20 (dd, 2H), 2.70-2.40 (dd, 2H), 1.60-1.40 (m, 2H),
1.4-1.1 (m, 10H), 0.90-0.80 (t, 3H).
[0264] Elemental analysis: responding to the expected formula
C.sub.14H.sub.27N.sub.3O.sub.3
Step D
[0265] The product obtained in the preceding step C (15.39 g, 0.054
moles) was dissolved in 31 ml of acetic acid and the resulting
solution was subjected to catalytic hydrogenation with 10% Pd/C at
60 psi for 7 hours. The reaction progress was checked by TLC, until
disappearance of the starting product (hexane/ethyl ether 95:5).
Thereafter, formaldehyde was added (4.6 ml, 0.167 moles) followed
by 10% Pd/C and the mixture was hydrogenated at 30 psi for 2 days.
The catalyst was filtered off and the mixture was vacuum-dried. A
pale yellow liquid was obtained, which was taken up with methylene
chloride, washed with 1N NaOH, then water, then NaCl saturated
solution; the organic phase was dried over anhydrous sodium
sulfate, filtered and vacuum-evaporated to dryness. A thick oil was
obtained. The product was obtained with a 98% yield.
[0266] TLC silica gel AcOEt/MeOH/NH.sub.3 90:10:3;
[0267] Rf=0.42.
[0268] .sup.1H-NMR (300 MHz; CDCl.sub.3):
[0269] .delta.: 4.2-4.09 (dd, 2H), 3.85-3.80 (m, 1H), 3.60-3.40
(dd, 2H), 2.65-2.40 (dd, 2H), 2.40-2.20 (dd, 2H), 2.20 (s, 6H),
1.60-1.40 (m, 2H), 1.4-1.1 (m, 10H), 0.90-0.80 (t, 3H).
[0270] Elemental analysis: responding to the expected formula
C.sub.16H.sub.36NO.sub.3
Step B
[0271] The product obtained in the preceding step D (15.21 g, 0.053
moles) was dissolved in 98 ml THF and 8 ml methyl iodide were added
thereto. The reaction progress was left overnight at room
temperature. The mixture was vacuum-evaporated to dryness. A thick
oil was obtained. The product was obtained with a 98% yield.
[0272] TLC silica gel AcOEt/MeOH/NH.sub.3 90:10:3;
[0273] Rf=0.1 0.
[0274] .sup.1H-NMR (300 MHz; CDCl.sub.3):
[0275] .delta.: 4.45-4.3 (m, 1H), 4.2-4.09 (dd, 2H), 3.75-3.30 (m,
2H), 3.5 (s, 9H), 2.75-2.60 (dd, 2H), 1.60-1.45 (m, 2H), 1.30-1.15
(m, 10H), 0.90-0.80 (t, 3H).
[0276] Elemental analysis: responding to the expected formula
C.sub.16H.sub.39INO.sub.3
Step F
[0277] The product obtained in the preceding step E, was hydrolysed
on Amberlyst IRA 402 resin (OH.sup.- activated form) eluting with
water. Water was evaporated to dryness; the resulting solid was
treated with isopropyl alcohol three times. A white solid was
obtained.
[0278] Yield=93%
[0279] M.p.=106.degree. C. dec.
[0280] .sup.1H-NMR (300 MHz; MeOD):
[0281] .delta.: 4.30-4.15 (m, 1H), 3.70-3.60 (dd, 1H), 3.50-3.40
(m, 2H), 3.20 (s, 9H), 2.75-2.65 (dd, 1H), 2.20-2.10 (dd, 1H),
1.60-1.50(m, 2H), 1.40-1.20 (m, 10H), 0.9-0.8 (t, 3H).
[0282] Elemental analysis: responding to the expected formula
C.sub.15H.sub.31NO.sub.3.
[0283] K.F.=5.7% water.
[0284] TLC silica gel (CHCl.sub.3 42/MeOH 28/isopropyl alcohol
7/water 10.5/acetic acid 10.5);
[0285] Rf=0.7.
[0286] HPLC: SGE-SAX column (5 .mu.m, 250.times.4 mm), mobile phase
0.025M (NH.sub.4)H.sub.2PO.sub.4:CH.sub.3CN 30:70, detector: RI, UV
205 nm, flow=0.75 ml/min, RT=5.85 min.
[0287] MS-FAB+glycerol matrix=274.
EXAMPLE 9
R,S-4-trimethylammonium-3-tetradecyloxybutyrate (ST 1228) (6b, FIG.
2)
Step A
[0288] The product was prepared as in example 8, step A using
tetradecyl alcohol. The product was obtained with 73% yield.
[0289] TLC silica gel hexane/ethyl ether 95:5;
[0290] Rf=0.63.
[0291] .sup.1H-NMR (300 MHz; CDCl.sub.3):
[0292] .delta.: 4.2-4.09 (q, 2H), 3.80 (s, 2H), 3.4-3.5 (dd, 2H),
2.85 (s, 2H), 1.60-1.58 (m, 2H), 1.4-1.2 (m, 22H), 0.90-0.80 (t,
3H).
[0293] Elemental analysis: responding to the expected formula
C.sub.34H.sub.67ClO.sub.4.
Step B
[0294] The product was prepared as in example 8, step B. The
product 2b, shown in FIG. 2, was obtained with a 72% yield.
[0295] TLC silica gel hexane/ethyl ether 95:5;
[0296] Rf=0.4.
[0297] .sup.1H-NMR (300 MHz; CDCl.sub.3):
[0298] .delta.: 4.2-4.09 (dd, 2H), 4.0-3.85 (m, 1H), 3.62-3.40 (m,
4H), 2.70-2.50 (dd, 2H), 1.55-1.50 (m, 2H), 1.4-1.2 (m, 22H),
0.90-0.80 (t, 3H).
[0299] Elemental analysis: responding to the expected formula
C.sub.20H.sub.39O.sub.3
Step C
[0300] The product was prepared as in example 8, step C. The
product was obtained with 79% yield.
[0301] TLC silica gel hexane/ethyl ether 90:10;
[0302] Rf=0.36.
[0303] .sup.1H-NMR (300 MHz; CDCl.sub.3):
[0304] .delta.: 4.2-4.09 (dd, 2H), 4.0-3.80 (m, 1H), 3.60-3.40 (dd,
2H), 3.40-3.20 (dd, 2H), 2.70-2.40 (dd, 2H), 1.60-1.40 (m, 2H),
1.4-1.1 (m, 22H), 0.90-0.80 (t, 3H).
[0305] Elemental analysis: responding to the expected formula
C.sub.20H.sub.39N.sub.3O.sub.3
Step D
[0306] The product was prepared as in example 8, step D. The
product was obtained with a 98% yield.
[0307] TLC silica gel AcOEt/MeOH/NH.sub.3 90:10:3;
[0308] Rf=0.72.
[0309] .sup.1H-NMR (300 MHz; CDCl.sub.3):
[0310] .delta.: 4.2-4.09 (dd, 2H), 3.85-3.80 (m, 1H), 3.60-3.40
(dd, 2H), 2.65-2.42 (dd, 2H), 2.38-2.20 (dd, 2H), 2.18 (s, 6H),
1.60-1.40 (m, 2H), 1.4-1.1 (m, 22H), 0.90-0.80 (t, 3H).
[0311] Elemental analysis: responding to the expected formula
C.sub.22H.sub.45NO.sub.3.
Step E
[0312] The product was prepared as in example 8, step E. The
product was obtained with a 99% yield.
[0313] TLC silica gel AcOEt/MeOH/NH.sub.3 90:10:3;
[0314] Rf=0.15.
[0315] .sup.1H-NMR (300 MHz; CDCl.sub.3):
[0316] .delta.: 4.45-4.3 (m, 1H), 4.2-4.09 (dd, 2H), 3.75-3.30 (m,
2H), 3.5 (s, 9H), 2.75-2.60 (dd, 2H), 1.60-1.45 (m, 2H), 1.30-1.15
(m, 22H), 0.90-0.80 (t, 3H).
[0317] Elemental analysis: responding to the expected formula
C.sub.23H.sub.48INO.sub.3.
Step F
[0318] The product was prepared as in example 8, step F. The
product was obtained with a 99% yield.
[0319] M.P.=106.degree. C. dec.
[0320] .sup.1NMR (300 MHz; DMSO-D6):
[0321] .delta.: 4.10-4.0 (m, 1H), 3.60-3.20 (m, 4H), 3.05 (s, 9H),
2.40-2.30 (dd, 1H), 1.80-1.70 (dd, 1H), 1.50-1.40 (m, 2H),
1.30-1.15 (m, 22H), 0.9-0.8 (t, 3H).
[0322] Elemental analysis: responding to the expected formula
C.sub.21H.sub.43NO.sub.3.
[0323] K.F.=6.4% water.
[0324] TLC silica gel (CHCl.sub.3 42/MeOH 28/isopropyl alcohol
7/water 10.5/acetic acid 10.5);
[0325] Rf=0.6.
[0326] HPLC: SGE-SCX column (5 .mu.m, 250.times.4 mm), mobile phase
0.05M (NH.sub.4)H.sub.2PO.sub.4:CH.sub.3CN 40:60, detector: RI, UV
205 nm, flow=0.75 ml/min, RT=4.38 min.
[0327] MS-FAB+glycerol matrix=358.3
[0328] The following Examples 10-11 are further illustrated by FIG.
3a-b.
EXAMPLE 10
R,S-1-guanidinium-2-tetradecyloxy-3-(tetrazolate-5-yl)propane (ST
1263) (10, FIG. 3b)
Step A
[0329] 6.65 g (0.0179 moles) of the intermediate prepared in
Example 9, step C were dissolved in 10 ml of methanol and 10 ml of
4N NaOH were added to the solution. The reaction was left to stand
for 16 hours at room temperature. 20 ml 6N HCl were added to the
solution, which was extracted with ethyl acetate. The organic phase
was dried over anhydrous sodium sulfate, filtered and vacuum
concentrated. The product was obtained as a white solid with a
95.6% yield.
[0330] TLC silica gel hexane /ethyl ether 1:1;
[0331] Rf=0.5.
[0332] M.p.=42-45.degree. C.
[0333] .sup.1H-NMR (300 MHz; CD.sub.3OD):
[0334] .delta.: 3.9-3.8 (m, 1H), 3.56-3.48 (m, 2H), 3.42-3.26 (dd,
2H), 2.68-2.5 (m, 2H), 1.6-1.5 (m, 2H), 1.4-1.2 (s, 22H), 0.90-0.80
(t, 3H).
[0335] Elemental analysis: responding to the expected formula
C.sub.18H.sub.35N.sub.3O.sub.3.
Step B
[0336] At 0.degree. C., 4.96 ml TEA were dropped into a solution
containing 2.79 g (8.19 mmoles) of the compound obtained in step A,
aminopropionitrile (0.58 g, 8.2 mmoles) and DEPC
(diethylphosphocyanydate ) (1.71ml) in 4.2 ml of anhydrous DMF. The
reaction was left to stand for 1 hour at room temperature. The
solvent was evaporated and the residue was dissolved in ethyl
acetate, washed twice with water, then with a NaCl saturated
solution. The organic phase was dried over anhydrous sodium
sulfate, filtered and vacuum concentrated. The product was obtained
and purified through a silica gel column with hexane: ethyl ether
(7:3/1:1/3:7).
[0337] Yield: 71%.
[0338] TLC silica gel ethyl ether 100%;
[0339] Rf=0.42.
[0340] .sup.1H-NMR (300 MHz; CDCl.sub.3):
[0341] .delta.: 6.6-6.4 (m, 1H), 3.9-3.8 (m, 1H), 3.60-3.4 (m, 5H),
3.3-3.2 (dt, 1H), 2.7-2.6 (t, 2H), 2.6-2.4 (dd, 2H), 1.6-1.5 (m,
2H), 1.4-1.2 (m, 22H), 0.90-0.80 (t, 3H).
[0342] Elemental analysis: responding to the expected formula
C.sub.21H.sub.39N.sub.5O.sub.2
Step C
[0343] 2.99 g (0.0114 moles) triphenylphosphine and 0.2 ml water
were added to a solution containing 2.99 g (7.62 mmoles) of the
compound obtained in step B. The reaction was left to stand
overnight at room temperature. The solvent was evaporated off and
the product was obtained and purified through a silica gel column
with ethyl acetate 100%, then ethyl acetate:methanol:ammonia
7:3:0.3.
[0344] Yield: 65%.
[0345] TLC silica gel ethyl acetate:methanol:ammonia 7:3:0.3;
[0346] Rf=0.26.
[0347] .sup.1H-NMR (300 MHz; CD.sub.3OD):
[0348] .delta.: 3.78-3.7 (m, 1H), 3.58-3.48 (m, 4H), 2.8-2.7 (dd,
2H), 2.7-2.6 (m, 2H), 2.5-2.3 (dd, 2H), 1.6-1.5 (m, 2H), 1.4-1.3
(m, 22H), 0.90-0.80 (t, 3H).
[0349] Elemental analysis: responding to the expected formula
C.sub.21H.sub.41N.sub.3O.sub.2
Step D
[0350] 1.69 g (4.6 mmoles) of the compound obtained in step C were
treated with 1.2 g (5.2 mmoles) (BOC).sub.2O and 9.2 ml 1N NaOH for
30 minutes at room temperature. The reaction mixture was poured
into ethyl acetate and washed four times with IN HCl, then water
and a saturated NaCl solution. The organic phase was dried over
anhydrous sodium sulfate, filtered and vacuum concentrated to
dryness. The product was obtained as a white solid.
[0351] Yield: 100%.
[0352] TLC silica gel ethyl ether 100%;
[0353] Rf=0.26.
[0354] M.p.=83-84.degree. C.
[0355] .sup.1H-NMR (300 MHz; CDCl.sub.3):
[0356] .delta.: 7.2-7.0 (m, 1H), 4.9-4.8 (m, 1H), 3.8-3.6 (m, 1H),
3.5-3.4 (dt, 4H), 3.2-3.0 (m, 2H), 2.6 (t, 2H), 2.4 (d, 2H), 1.5
(m, 2H), 1.4 (s, 9H 1.4-1.2 (m, 22H), 0.90-0.80 (t, 3H).
[0357] Elemental analysis: responding to the expected formula
C.sub.26H.sub.49N.sub.3O.sub.4.
Step E
[0358] The product obtained in step D (1.19 g, 2.56 mmoles) was
dissolved into 12 ml of anhydrous THF, under argon atmosphere, then
3.062 g of triphenylphosphine, 1.54 ml of triethylsilylazido and
4.9 ml of DEAD (diethylazodicarboxylate) were dropped at 0.degree.
C. within three days, until disappearance of the starting product.
The mixture was then treated with an aqueous solution of cerium
ammonium nitrate and diluted with CH.sub.2Cl.sub.2. The reaction
was left to stand for 2 hours, the organic phase was washed with a
saturated NaCl solution, dried over anhydrous sodium sulfate and
vacuum-dried. The residue was purified through a silica gel column
with hexane/ethyl acetate (9:1/8:2/7:3). The product was obtained
with a 66% yield.
[0359] TLC silica gel hexane/AcOEt 1:1;
[0360] Rf=0.34.
[0361] .sup.1H-NMR (300 MHz; CDCl.sub.3):
[0362] .delta.: 4.95-4.8 (m, 1H), 4.7-4.5 (m, 2H), 3.9-3.8 (m, 1H),
3.50-3.40 (m, 1H), 3.40-3.31 (m, 1H), 3.3-3.2 (m, 1H), 3.22-3.0
(dd, 2H), 3.10-3.0 (m, 3H), 1.45-1.35 (m, 1H), 1.2 (m, 22H),
0.90-0.80 (t, 3H).
[0363] Elemental analysis: responding to the expected formula
C.sub.25H.sub.48N.sub.6O.sub.3
Step F
[0364] The product obtained in step E (0.969 g, 1.97 mmoles) was
dissolved into 13.09 ml anhydrous THF, then 13.1 ml of 3N HCl were
added. The reaction mixture was left to stand for 2 hours, at
50.degree. C. under stirring. The reaction mixture was
vacuum-dried, the residue was taken up with CH.sub.2Cl.sub.2 and
treated with a 1 N NaOH solution. The organic phase was separated,
dried over anhydrous sodium sulfate and vacuum-dried. The product
was obtained with a 92% yield.
[0365] TLC silica gel AcOEt/MeOH/NH.sub.3 9:1:0.3
[0366] Rf=0.31.
[0367] .sup.1H-NMR (300 MHz; CDCl.sub.3):
[0368] .delta.: 4.78-4.58 (m, 2H), 3.8-3.7 (m, 1H), 3.5-3.4 (m,
1H), 3.30-3.24 (m, 1H), 3.24-3.18 (m, 4H), 3.05-3.0 (dd, 2H),
3.0-2.6 (dd, 2H), 1.4 (m, 2H), 1.2 (m, 22H), 0.90-0.80 (t, 3H).
[0369] Elemental analysis: responding to the expected formula
C.sub.21H.sub.40N6O
Step G
[0370] The product obtained in step F (2.78 g, 7.1 mmoles) was
dissolved into 20 ml anhydrous MeOH, then 2.34 g
iminomethanesulfonic acid (prepared with well-known methods) were
added within 3 days. The obtained suspension was
vacuum-concentrated, then treated with IN NaOH and left under
stirring for 30 minutes. The solid was filtered, washed with water,
then acetone. The title product was obtained with a 45% yield.
[0371] TLC silica gel AcOEt/MeOH/NH.sub.3 7:3:0.3;
[0372] Rf=0.22.
[0373] M.p.=240.degree. C. dec.
[0374] .sup.1H-NMR (300 MHz; CD.sub.3OD):
[0375] .delta.: 3.90-3.75 (m, 1H), 3.6-3.4 (m, 2H), 3.40-3.20 (m,
2H), 3.20-3.10 (dd, 1H), 2.95-2.85 (dd, 1H), 1.4 (m, 2H), 1.2 (s,
22H), 0.90-0.80 (t, 3H).
[0376] Elemental analysis: responding to the expected formula
C.sub.19H.sub.39N.sub.7O.
[0377] HPLC: Spherisorb-C1 (5 .mu.m, 250.times.4.6 mm), mobile
phase 0.05 M KH.sub.2PO.sub.4:CH.sub.3CN 35:65, pH=3, flow 0.75
ml/min, detector: UV 205 nm, RT=5.51 min.
[0378] MS-FAB+glycerol matrix=382.
EXAMPLE 11
R,S-1-trimethylammonium-2-tetradecyloxy-3-(tetrazolato-5-yl)propane
(ST 1287) (9, FIG. 3b)
Steps A-F
[0379] The compounds were prepared as in steps A-F of Example
10.
Step H
[0380] 2.79 g (7.14 mmoles) of the compound prepared in Example 10,
step F were suspended in 18 ml water and 1.47 ml HCOOH and 1.57 ml
H.sub.2CO were added thereto. The reaction mixture was refluxed
overnight, then was allowed to cool down and methylene chloride was
added; pH was adjusted to 9 with 0.5 N NaOH. The mixture was
extracted three times with methylene chloride. The organic phase
was washed with 0.5 N NaOH, water and dried over anhydrous sodium
sulfate, filtered and vacuum concentrated. The product was obtained
as a solid with a 100% yield.
[0381] TLC silica gel AcOEt/MeOH/NH.sub.3 9:1:0.3;
[0382] Rf=0.58.
[0383] .sup.1H-NMR (300 MHz; CDCl.sub.3):
[0384] .delta.: 4.7-4.5 (m, 1H), 3.8-3.7 (m, 1H), 3.5-3.4 (m, 1H),
3.30-3.20 (m, 2H), 3.10 (m, 3H), 2.45-2.35 (m, 2H), 2.30 (s, 6H),
1.4-1.3 (m, 2H), 1.2-1.0 (m, 22H), 0.90-0.80 (t, 3H).
[0385] Elemental analysis: responding to the expected formula
[0386] C.sub.23H.sub.44N.sub.6O.
Step I
[0387] 2.99 g (7.14 mmoles) of the compound obtained in step H were
dissolved in THF and 2.5 ml of CH.sub.3I were added thereto. The
reaction was left to stand for 3 hours at room temperature. The
solvent was evaporated off and the solid residue was washed with
hot ether, left overnight under stirring, then filtered. The
product was obtained.
[0388] Yield: 100%.
[0389] TLC silica gel CHCl.sub.3:iPrOH:MeOH:H.sub.2O:CH.sub.3COOH
42:7:28:10.5:10.5;
[0390] Rf=0.73.
[0391] .sup.1H-NMR (300 MHz; CDCl.sub.3):
[0392] .delta.: 4.90-4.80 (m, 2H), 4.70-4.55 (m, 1H), 4.40-4.25 (m,
1H), 3.80-3.60 (m, 2H), 3.60-3.40 (m, 3H), 3.30 (s, 9H), 3.30-3.10
(m, 2H), 1.60-1.40 (m, 2H), 1.3-1.1 (m, 22H), 0.9-0.8 (t, 3H).
[0393] Elemental analysis: responding to the expected formula
C.sub.24H.sub.47IN.sub.6O.
[0394] MS-FAB+glycerol matrix=436.
Step L
[0395] The product obtained in step I (2.99 g, 5.33 mmoles) was
dissolved in MeOH, then passed through IRA 402 resin in OH.sup.-
form, conditioned in MEOH. The title product was obtained as a
solid, which was subsequently triturated with AcOEt.
[0396] Yield=88%.
[0397] TLC silica gel CHCl.sub.3:iPrOH:MeOH:H.sub.2O:CH.sub.3COOH
(42:7:28: 10.5: 10.5)/acetone 8:2;
[0398] Rf=0.73.
[0399] TLC silica gel CHCl.sub.3:iPrOH:MeOH:H.sub.2O:CH.sub.3COOH
42:7:28:10.5: 10.5;
[0400] Rf=0.73.
[0401] M.p.=180.degree. C. dec.
[0402] .sup.1H-NMR (300 MHz; CDCl.sub.3):
[0403] .delta.: 4.30-4.20 (m, 1H), 3.90-3.70 (m, 2H), 3.60-3.55 (m,
1H), 3.50-3.30 (m, 4H), 3.25 (m, 1H), 3.0-2.9 (m, 1H), 1.60-1.40
(m, 2H), 1.3-1.1 (m, 22H), 0.9-0.8 (t, 3H).
[0404] Elemental analysis: responding to the expected formula
C.sub.21H.sub.43N.sub.5O.
[0405] MS-FAB+glycerol matrix=382.
[0406] K.F.=1% water
[0407] HPLC: Spherisorb-C1 (5 .mu.m, 250.times.4.6 mm), mobile
phase 0.05 M KH.sub.2PO.sub.4:CH.sub.3CN 35:65, pH=3, flow 0.75
ml/min, detector: UV 205 nm, RT-5.18 min.
[0408] The following Examples 12-14 are further illustrated by FIG.
4.
EXAMPLE 12
R,S-3-quinuclidinium-2-(tetradecyloxycarbonyl)-oxy-1-propanephosphonate
monobasic (ST 1260)
Step A
[0409] In anhydrous environment, -70.degree. C., a hexane solution
of 1.6 M BuLi (14 ml, 0.022 moles) was dropped into a solution of
dibenzyl phosphite (5.8 g, 0.022 mmoles) in THF. After 15 minutes,
1.8 ml (0.022 moles) of epibromhydrine, dissolved in 5 ml THF, were
added. After the addition, etherated BF.sub.3 (3.6 ml, 0.022 moles)
was dropped very slowly. The reaction was left for further 3 hours
at -70.degree. C. A saturated ammonium chloride solution was added;
then the temperature was left to raise to room temperature. This
solution was extracted several times with AcOEt and the gathered
organic phases were treated with saturated NaHCO.sub.3, and dried
over anhydrous sodium sulfate, filtered and vacuum concentrated. An
oil was obtained, which after purification on silica gel
chromatography (AcOEt/ Hexane 1:1), gave 1.1 g of unreacted
dibenzylphosphite and 5.3 g of product of interest.
[0410] Yield=60%.
[0411] TLC silica gel AcOEt/Hexane 7:3;
[0412] Rf=0.54.
[0413] .sup.1H-NMR (300 MHz; CD.sub.3OD):
[0414] .delta.: 7.4-7.2 (m, 10H), 5.1-4.9 (m, 4H), 4.2-4.0 (m, 1H),
3.5-3.3 (dd, 2H), 2.2-2.0 (m, 2H).
[0415] Elemental analysis: responding to the expected formula
C.sub.17H.sub.20BrO.sub.4P.
[0416] MS-FAB+glycerol matrix=399, 400, 401, 402.
Step B
[0417] 2 g (5. mmoles) of the compound obtained in step A were
dissolved at 10% concentration and the solution cooled down to
0.degree. C. 1.4 ml TEA (10 mmoles) and 0.62 g (5 mmoles) DMAP
(dimethylaminopyridine) were dropped thereto. Immediately after,
5.2 mmoles tetradecyl chloroformate were added and the temperature
was left to raise to room temperature. The reaction progress was
checked on TLC and worked up at the disappearance of the starting
compound. Further chloroform was added and the reaction mixture was
washed with 1N HCl and water. After drying over anhydrous sodium
sulfate, the solvent was evaporated off and an oil was obtained,
which was purified through flash-chromatography using hexane/AcOEt
7:3 as eluant. The product was obtained.
[0418] Yield: 75%.
[0419] TLC silica gel hexane/AcOEt 7:3;
[0420] Rf=0.31.
[0421] .sup.1H-NMR (300 MHz; CDCl.sub.3):
[0422] .delta.: 7.4-7.2 (m, 10H), 5.1-4.9 (m, 5H), 4.1-3.9 (m, 2H),
3.6-3.4 (dd, 2H), 2.4-2.2 (m, 2H), 1.6-1.4 (m, 2H), 1.3-1.1 (m,
22H), 0.9-0.7 (t, 3H).
[0423] Elemental analysis: responding to the expected formula
C.sub.32H.sub.48BrO.sub.6P.
Step D
[0424] The product obtained in step B (6.39 g, 10 mmoles) was
dissolved in 12 ml DMF, then quinuclidine was added (2.2 g, 20
mmoles) together with TBAI (tetrabutyl ammonium iodide) in
catalytic amounts (1% by weight with respect to the substrate). The
reaction was carried out at a temperature of 50.degree. C., until
the starting product disappeared. At the end of reaction, the
mixture was concentrated under high vacuum, obtaining a semisolid
containing the product. The latter was purified through silica gel
flash-chromatographychromatography, using CHCl.sub.3/MeOH 8:3. The
product was obtained.
[0425] Yield=15%.
[0426] TLC silica gel CHCl.sub.3:iPrOH:MeOH:H.sub.2O:CH.sub.3COOH
(42:7:28:10.5:10.5)/-acetone 8:2;
[0427] Rf=0.8.
[0428] .sup.1H-NMR (300 MHz; MeOD):
[0429] .delta.: 7.4-7.1 (m, 50H), 5.3-5.1 (m, 1H), 4.9-4.8 (d, 2H),
4.1-4.0 (m, 2H), 3.8-3.4 (m, 2H), 3.4-3.2 (m, 6H), 2.2-1.7 (m, 9H),
1.6-1.4 (m, 2H), 1.3-1.1 (m, 22H), 0.9-0.7 (t, 3H).
[0430] Elemental analysis: responding to the expected formula
C.sub.32H.sub.54NO.sub.6P.
[0431] MS-FAB+glycerol matrix=580.
Step E
[0432] The product obtained in step D was dissolved in MeOH, then
10% Pd/C (5% by weight with respect to the substrate) was added;
the dispersion was hydrogenated (60 psi) at room temperature for 18
hours. At the end, the dispersion was filtered through celite and
concentrated to dryness. The title product was obtained without
further purifications.
[0433] Yield=99%.
[0434] TLC silica gel CHCl.sub.3:iPrOH:MeOH:H.sub.2O:CH.sub.3COOH
(42:7:28: 10.5: 10.5)/acetone 8:2;
[0435] Rf=0.57.
[0436] .sup.1H-NMR (300 MHz; D.sub.2O):
[0437] .delta.: 5.5-5.3 (m, 1H), 4.2-4.1 (m, 2H), 4.0-3.4 (m, 8H),
2.2-1.7 (m, 9H), 1.60-1.40 (m, 2H), 1.3-1.1 (m, 22H), 0.9-0.7 (t,
3H).
[0438] Elemental analysis: responding to the expected formula
C.sub.25H.sub.48NO.sub.6P.
[0439] MS-FAB+glycerol matrix=490.
[0440] K.F.=7% water
[0441] HPLC: Spherisorb-C1 (5 .mu.m, 250.times.4.6 mm), mobile
phase 0.075 M KH.sub.2PO.sub.4:CH.sub.3CN 60:40, flow 0.75 ml/min,
detector: RI, UV 205 nm, RT=16.53 min.
EXAMPLE 13
R,S-3-trimethylammonium-2-(nonylaminocarbonyl)-oxy-1-propanephosphonate
monobasic (ST 1286)
Step A
[0442] The product was prepared as disclosed in step A of Example
12.
Step C
[0443] The product obtained in the previous step (4 g, 10 mmoles)
was dissolved in CH.sub.2Cl.sub.2 (10% solution) and etherated
BF.sub.3 (1.6 ml) and nonyl isocyanate (3.38 g, 20 mmoles) were
added at room temperature. The reaction was worked up after 30
minutes, firstly adding further CH.sub.2Cl.sub.2, then washing the
organic phase with 1N NaOH several times. The product was purified
on silica gel flash-chromatography (Hexane/AcOEt 7:3).
[0444] Yield=85%.
[0445] TLC silica gel AcOEt/Hexane 6:4;
[0446] Rf=0.28.
[0447] .sup.1H-NMR (300 MHz; CDCl.sub.3):
[0448] .delta.: 7.4-7.2 (m, 10H), 5.1-4.9. (m, 5H), 4.6-4.2 (m,
1H), 3.7-3.5 (dd, 2H), 3.2-3.0 (m, 2H), 2.4-2.2 (m, 2H), 1.5-1.3
(m, 2H), 1.3-1.1 (m ,12H), 0.9-0.7 (t, 3H).
[0449] Elemental analysis: responding to the expected formula
C.sub.27H.sub.40BrNO.sub.5P.
Step F
[0450] The compound obtained in the preceding step (5.68 g, 10
mmoles) was dissolved in DMF (11 ml), together with TBAI
(tetrabutyl ammonium iodide) in catalytic amounts (1% w/w with
respect to the substrate). This solution was saturated with gaseous
trimethylamine. The reaction was carried out at 50.degree. C.,
until the starting compound disappeared. At the end of the
reaction, the solution was high vacuum-concentrated, obtaining a
semisolid, containing the product. The latter was isolated and
purified through silica gel flash-chromatography using a gradient
from CH.sub.2Cl.sub.2 only to CH.sub.2Cl.sub.2:MeOH 1.1. The
product was obtained.
[0451] Yield: 25%.
[0452] TLC silica gel CHCl.sub.3:iPrOH:MeOH:H.sub.2O:CH.sub.3COOH
(42:7:28:10.5:10.5)/acetone 8:2;
[0453] Rf=0.73.
[0454] .sup.1H-NMR (300 MHz; CDCl.sub.3):
[0455] .delta.: 7.5-7.2 (m, 5H), 5.5-5.4 (m, 1H), 4.9-4.8 (m, 4H),
4.0-3.6 (m, 2H), 3.2-3.1 (s, 9H), 2.2-2.1 (s, 9H), 2.0-1.8 (m,.2H),
-1.5-1.4 (m, 2H), 1.4-1.2 (m, 12H), 0.9-0.7 (t, 3H).
[0456] Elemental analysis: responding to the expected formula
C.sub.27H.sub.42N.sub.2O.sub.5P.
[0457] MS-FAB+glycerol matrix=457.
Step G
[0458] The product obtained in step F was dissolved in MeOH, then
10% Pd/C (5% by weight with respect to the substrate) was added;
the dispersion was hydrogenated (60 psi) at room temperature for 18
hours. At the end, the dispersion was filtered through celite and
concentrated to dryness. The title product was obtained without
further purifications.
[0459] Yield=99%.
[0460] TLC silica gel CHCl.sub.3:iPrOH:MeOH:H.sub.2O:CH.sub.3COOH
(42:7:28:10.5:10.5)/acetone 8:2;
[0461] Rf=0.31.
[0462] .sup.1H-NMR (300 MHz; D.sub.2O):
[0463] .delta.: 5.6-5.5 (m, 1H), 4.1-3.5 (m, 2H), 3.2-3.1 (s, 9H),
3.1-3.0 (m, 2H), 2.2-1.7 (m, 2H), 1.5-1.4 (m, 2H), 1.4-1.2 (m,
12H), 0.9-0.7 (t, 3H).
[0464] Elemental analysis: responding to the expected formula
C.sub.15H.sub.35N.sub.2O.sub.5P.
[0465] MS-FAB+glycerol matrix=367.
[0466] K.F.=3% water.
[0467] HPLC: Spherisorb-C1 (5 .mu.m,. 250.times.4.6 mm), mobile
phase 0.05 M (NH.sub.4)H.sub.2PO4:CH.sub.3CN 35:65, flow 0.75
ml/min, detector: RI, UV 205 nm, RT=7.31 min.
EXAMPLE 14
R,S-3-pyridinium-2-(nonylaminocarbonyl)-oxy-1-propanephosphonic
acid chloride (ST 1268)
Step A
[0468] The product was prepared as disclosed in step A of Example
12.
Step C
[0469] The product was prepared as disclosed in step C of Example
13.
Step H
[0470] The compound obtained in the preceding step (5.68 g, 10
mmoles) was dissolved in anhydrous pyridine (50% solution),
together with TBAI (tetrabutyl ammonium iodide) in catalytic
amounts (1% w/w with respect to the substrate). The reaction was
carried out at 50.degree. C., until the starting compound
disappeared. At the end of the reaction, the solution was high
vacuum-concentrated, obtaining a semisolid, containing the product,
which was isolated and purified through silica gel
flash-chromatography using a gradient from CH.sub.2Cl.sub.2 only to
CH.sub.2Cl.sub.2:MeOH from 9:1 to 1:1.
[0471] Yield: 20%.
[0472] TLC silica gel CHCl.sub.3:iPrOH:MeOH:H.sub.2O:CH.sub.3COOH
(42:7:28:10.5:10.5)/acetone 8:2;
[0473] Rf=0.73.
[0474] .sup.1H-NMR (300 MHz; CDCl.sub.3):
[0475] .delta.: 9.4-9.3 (d, 2H), 8.2-8.1 (t, 1H), 7.9-7.8 (t, 2H),
7.3-7.1 (m, 5H), 5.3-5.1 (m, 3H), 4.9-4.8 (m, 2H), 3.0-2.9 (m, 2H),
2.2-1.6 (m, 2H), 1.4-1.2 (m, 2H), 1.3-1.1 (m, 12H), 0.9-0.7 (t,
3H).
[0476] Elemental analysis: responding to the expected formula
C.sub.24H.sub.38N.sub.2O.sub.5P.
[0477] MS-FAB+glycerol matrix=477.
Step I
[0478] The product obtained in step H (4.76 g, 10 mmoles) was
dissolved in 100 ml CH.sub.2Cl.sub.2 and 20 mmoles TMSI
(trimethylsilyl iodide) were added to the resulting solution. After
30 minutes, the reaction was finished; 0.5 ml water were added to
the mixture, which was concentrated to dryness. The final product
was purified and isolated by RP-18 silica gel chromatography, using
a gradient water/methanol 9:1 to methanol 100%. The solid was
dissolved in water and passed through IRA 402 resin (Cl.sup.-
activated). ST 1268 was obtained.
[0479] Yield=80%.
[0480] M.p.=202-204.degree. C.
[0481] TLC silica gel CHCl.sub.3:iPrOH:MeOH:H.sub.2O:CH.sub.3COOH
(42:7:28: 10.5:10.5)/acetone 8:2;
[0482] Rf=0.48.
[0483] .sup.1H-NMR (300 MHz; D.sub.2O):
[0484] .delta.: 9.4-9.3 (d, 2H), 8.2-8.1 (t, 1H), 7.9-7.8 (t, 2 H),
5.5-5.4 (m, 1H), 5.2-4.8 (m, 2H), 3.0-2.9 (m, 2H), 2.2-2.0 (m, 2H),
1.4-1.1 (m, 14H), 0.9-0.7 (t, 3H).
[0485] Elemental analysis: responding to the expected formula
C.sub.18H.sub.32N.sub.2 ClO.sub.5P.
[0486] MS-FAB+glycerol matrix=387.
[0487] K.F.=6% water.
[0488] HPLC: Spherisorb-C1 (5 .mu.m, 250.times.4.6 mm), mobile
phase 0.050 M KH.sub.2PO.sub.4:CH.sub.3CN 35:65, flow 0.75 ml/min,
detector: RI, UV 205 nm, RT=5.61 min.
EXAMPLE 15
R-4-trimethylammonium-3-(tetradecylcarbamoyl)-amino butyrate (ST
1326)
[0489] The product was prepared as disclosed in Example 1, starting
from tetradecyl isocyanate and R-aminocarnitine, inner salt, except
the crude product was obtained by precipitation with ethyl ether,
from the reaction mixture, directly washed with ethyl ether and
purified on a silica gel chromatographic column.
[0490] Yield 57%.
[0491] M.p.: 160-162.degree. C.
[0492] [.alpha.].sub.20.sup.D=-21.1.degree. (c=0.5, MeOH).
[0493] .sup.1H-NMR (300 MHz; CD.sub.3OD):
[0494] .delta.: 4.52 (m, 1H), 3.60 (dd, 1H), 3.48 (d, 1H), 3.20 (s,
9H), 3.10 (t, 2H), 2.40 (m, 2H), 1.45 (m, 2H), 1.28 (brs, 22H), 0.8
(brt, 3H).
[0495] ESI Mass=400, [(M+H).sup.+.
[0496] Elemental analysis: responding to the expected formula
C.sub.22H.sub.45N.sub.3O.sub.3.
[0497] K.F.=2.5% water.
[0498] TLC silica gel CHCl.sub.3:iPrOH:MeOH:H.sub.2O:CH.sub.3COOH
42:7:28:10.5:10.5;
[0499] Rf=0.50.
[0500] HPLC: SGE-SCX column (5 .mu.m, 250.times.4 mm), T=30.degree.
C., mobile phase 0.05 M (NH.sub.4)H.sub.2PO.sub.4:CH.sub.3CN 75:25,
pH=4.9 (as such), flow 0.75 ml/min, detector: RI, UV 205 nm,
RT=13.63 min.
EXAMPLE 16
R-4-trimethylammonium-3-(undecylcarbamoyl)-aminobutyrate (ST
1327)
[0501] The product was prepared as disclosed in Example 1, starting
from undecyl isocyanate and R-aminocarnitine, inner salt, purified
on a silica gel chromatographic column and further purified by
precipitation from acetonitrile.
[0502] Yield 50%.
[0503] M.p.: 149-150.2.degree. C.
[0504] [.alpha.].sub.20.sup.D=-21.16.degree. (c=1, MeOH).
[0505] .sup.1H-NMR (300 MHz; CD.sub.3OD):
[0506] .delta.: 4.52 (m, 1H), 3.60 (dd, 1H), 3.48 (d, 1H), 3.20 (s,
9H), 3.10 (t, 2H), 2.40 (m, 2H), 1.45 (m, 2H), 1.28 (brs, 16H), 0;8
(brt, 3H).
[0507] ESI Mass=358, [(M+H).sup.+;
[0508] Elemental analysis: responding to the expected formula
C.sub.19H.sub.39N.sub.3O.sub.3.
[0509] K.F.=2.3% water.
[0510] TLC silica gel CHCl.sub.3:iPrOH:MeOH:H.sub.2O:CH.sub.3COOH
42:7:28:10.5:10.5.
[0511] Rf=0.50.
[0512] HPLC: SGE-SCX column (5 .mu.m, 250.times.4 mm), T=30.degree.
C., mobile phase 0.05 M (NH.sub.4)H.sub.2PO.sub.4:CH.sub.3CN 80:20,
pH=4.9 (as such), flow 0.75 ml/min, detector: RI, UV 205 nm,
RT=17.37 min.
EXAMPLE 17
R-4-trimethylammonium-3-(heptylcarbamoyl)-aminobutyrate (ST
1328)
[0513] The product was prepared as disclosed in Example 1, starting
from heptyl isocyanate and R-aminocarnitine, inner salt,. purified
on a silica gel chromatographic column and further purified by
precipitation from acetonitrile.
[0514] Yield 47%.
[0515] M.p.: 149-150.degree. C.
[0516] [.alpha.].sub.20.sup.D=-34.0.degree. (c=0.97, MeOH).
[0517] .sup.1H-NMR (300 MHz; CD.sub.3OD):
[0518] .delta.: 4.52 (m, 1H), 3.60 (dd, 1H), 3.48 (d, 1H), 3.20 (s,
9H), 3.10 (t, 2H), 2.40 (m, 2H), 1.45 (m, 2H), 1.30 (brs, 8H), 0.8
(brt, 3H).
[0519] ESI Mass=302, [(M+H).sup.+;
[0520] Elemental analysis: responding to the expected formula
C.sub.15H.sub.31N.sub.3O.sub.3
[0521] K.F.=6.17% water
[0522] TLC silica gel CHCl.sub.3:iPrOH:MeOH:H.sub.2O:CH.sub.3COOH
42:7:28:10.5:10.5.
[0523] Rf=0.50.
[0524] HPLC: SGE-SCX column (5 .mu., 250.times.4 mm), T=30.degree.
C., mobile phase 0.05 M (NH.sub.4)H.sub.2PO.sub.4:CH.sub.3CN 85:15,
pH=6 (H.sub.3PO.sub.4), flow 0.75 ml/min, detector: RI, UV 205 nm,
RT=7.16 min.
EXAMPLE 18
R,S-4-trimethylammonium-3-(nonylthiocarbamoyl)-aminobutyrate (ST
1329)
[0525] The product was prepared as disclosed in Example 1, starting
from nonyl isothiocyanate and R,S-aminocarnitine, inner salt.
Chromatography was carried out with a CHCl.sub.3/MeOH gradient from
8:2 to 2:8.
[0526] Yield 53%
[0527] M.p.: 104-107.degree. C.
[0528] .sup.1H-NMR (200 MHz; CD.sub.3OD):
[0529] .delta.: 5.45 (brm, 1H), 3.75 (dd, 1H), 3.55-(d, 1H), 3.45
(brm, (2H), 3.22 (s, 9H), 2.48 (m, 2H), 1.55 (m, 2H), 1.30 (brs,
-12H), 0.90 (brt, 3H).
[0530] ESI Mass=346, [(M+H).sup.+;
[0531] Elemental analysis: responding to the expected formula
C.sub.17H.sub.35N.sub.3O.sub.2S
[0532] K.F.=2.6% water;
[0533] TLC silica gel CHCl.sub.3:iPrOH:MeOH:H.sub.2O:CH.sub.3COOH
42:7:28:10.5:10.5.
[0534] Rf=0.74;
[0535] HPLC: SGE-SCX column (5 .mu.m, 250.times.4 mm), T=30.degree.
C., mobile phase 0.05 M (NH.sub.4)H.sub.2PO.sub.4:CH.sub.3CN 85:15,
pH=6.0 (H.sub.3PO.sub.4), flow 0.75 ml/min, detector: RI, UV 205
nm, RT=8.87 min.
EXAMPLE 19
R-4-trimethylammonium-3-(nonylcarbamoyl)-aminobutyrate (ST
1283)
[0536] The product was prepared as disclosed in Example 1, starting
from nonyl isocyanate and R-aminocarnitine, inner salt.
[0537] M.p.: 146-147.degree. C.
[0538] [.alpha.].sub.20.sup.D=-13.4.degree. (c=0.5, H.sub.2O).
[0539] Elemental analysis: responding to the expected formula
C.sub.17H.sub.35N.sub.3O.sub.3
[0540] K.F.=2.8% water.
[0541] Remaining physico-chemical data were coincident with those
of racemic ST1251 (Example 1).
EXAMPLE 20
S-4-trimethylammonium-3-(nonylcarbamoyl)-aminobutyrate (ST
1338)
[0542] The product was prepared as disclosed in Example 1, starting
from nonyl isocyanate and S-aminocarnitine, inner salt.
[0543] M.p.: 146-147.degree. C.
[0544] [.alpha.].sub.20.sup.D=+16.7.degree. (c=0.43, H.sub.2O).
[0545] .sup.1H-NMR (300 MHz; CD.sub.3OD):
[0546] .delta.: 4.52 (m, 1H), 3.60 (dd, 1H), 3.45 (d, 1H), 3.18 (s,
9H), 3.10 (t, 2H), 2.40 (m, 2H), 1.45 (m, 2H), 1.28 (brs, 12H),
0.90 (brt, 3H).
[0547] ESI Mass=330, [(M+H).sup.+;
[0548] Elemental analysis: responding to the expected formula
C.sub.17H.sub.35N.sub.3O.sub.3
[0549] K.F.=1.8% water.
[0550] Remaining physico-chemical data were coincident with those
of racemic ST1251 (Example 1).
EXAMPLE 21
S-4-trimethylammonium-3-(tetradecylcarbamoyl)-aminobutyrate (ST
1340)
[0551] The product was prepared as disclosed in Example 1, starting
from tetradecyl isocyanate and S-aminocarnitine, inner salt, except
the crude product was obtained by precipitation with ethyl ether,
from the reaction mixture, directly washed with ethyl ether and
purified on a silica gel chromatographic column.
[0552] Yield=57%;
[0553] M.p.: 166-167.degree. C.
[0554] [.alpha.].sub.20.sup.D=+20.7.degree. (c=0.5, MeOH).
[0555] Elemental analysis: responding to the expected formula
C.sub.22H.sub.45N.sub.3O.sub.3
[0556] K.F.=1.7% water.
[0557] Remaining physico-chemical data were coincident with those
of racemic ST1326 (Example 15).
EXAMPLE 22
Isobutyl R,S-4-trimethylammonium-3-tetradecylamino-aminobutyrate
(ST 1252)
R,S-4-trimethylammonium-3-tetradecylamino-aminobutyrate isobutyl
Ester Acetate
[0558] Isobutyl ester of racemic aminocarnitine (5 g, 0.0198 moles)
and tetradecanal (4.6 g, 0.0217 moles) were dissolved into 250 ml
methanol. Glacial acetic acid (1.13 ml, 0.198 moles) and 1 g 10%
Pd/C were added. The mixture was hydrogenated at 30 psi overnight.
After filtration on celite, the solution was vacuum-concentrated. A
pale yellow oil was obtained, which was purified through a silica
gel column, eluting firstly with AcOEt, then AcOEt/MeOH 9:1. 4 g of
product were obtained.
[0559] Yield=47%;
[0560] TLC silica gel (CHCl.sub.3 42/MeOH 28/isopropyl alcohol
7/water 10.5/acetic acid 10.5)/methyl acetate 7:3
[0561] Rf=0.74.
[0562] .sup.1H-NMR (300 MHz; CD.sub.3OD):
[0563] .delta.: 3.92-3.90 (d, 2H), 3.64-3.58 (m, 1H), 3.50-3.30 (m,
2H), 2.80-2.50 (m, 4H), 2.0-1.9 (m, 1H), 2.6-2.4 (m, 2H), 1.3 (s,
22H), 0.98-0.82 (m, 9H).
[0564] R,S-4-trimethylammonium-3-tetradecylamino-aminobutyrate
[0565] The isobutyl ester of
R,S-4-trimethylammonium-3-tetradecylamino-ami- nobutyric acid,
acetate salt, (3.3 g) was hydrolysed on Amberlyst IRA 402 resin
(OH.sup.- activated form) and eluted with water. Water was
evaporated to dryness under reduced pressure; the resulting white
solid was washed with methanol, filtered and vacuum-dried. 1.95 g
of product were obtained.
[0566] Yield 70%
[0567] M.p.=160.degree. C. dec.
[0568] .sup.1H-NMR (300 MHz; CD.sub.3OD):
[0569] .delta.: 4.4 (m, 1H), 3.40-3.35 (m, 3H), 3.2 (s, 9H),
2.80-2.72 (m, 1H), 2.56-2.42 (m, 2H), 2.27-2.16 (m, 1H), 1.55-1.40
(m, 2H), 1.3 (s, 22H), 0.92-0.85 (t, 3H).
[0570] Elemental analysis: responding to the expected formula
C.sub.21H.sub.44N.sub.2O.sub.2
[0571] K.F.=1.93% water.
[0572] TLC silica gel (CHCl.sub.3 42/MeOH 28/isopropyl alcohol
7/water 10.5/acetic acid 10.5)
[0573] Rf=0.5.
[0574] HPLC: SGE-SCX column (5 .mu.m, 250.times.4 mm), mobile phase
0.05M (NH.sub.4)H.sub.2PO.sub.4:CH.sub.3CN 60:40, pH=4, flow=0.75
ml/min; detector: RI, UV 205 nm, RT=30.017 min.
EXAMPLE 23
R,S-4-trimethylammonium-3-octylaminobutyrate (ST 1254)
R,S-4-trimethylammonium-3-octylamino-aminobutyrate Isobutyl Ester
Acetate
[0575] Isobutyl ester of racemic aminocarnitine chloride, (5 g,
0.0198 moles) and octanaldehyde (2.79 g, 0.0217 moles) were
dissolved into 250 ml methanol. Glacial acetic acid (1.13 ml, 0.198
moles) and 1 g 10% Pd/C were added. The mixture was hydrogenated at
30 psi overnight. After filtration on celite, the solution was
vacuum-concentrated. 8.5 g product were obtained, subsequently
purified through a silica gel column, eluting firstly with AcOEt,
then AcOEt/MeOH (9:1; 8.5:1.5). 3 g of product were obtained.
[0576] Yield=40%;
[0577] TLC silica gel (CHCl.sub.3 42/MeOH 28/isopropyl alcohol
7/water 10.5/acetic acid 10.5)
[0578] Rf=0.54.
[0579] .sup.1H-NMR (300 MHz; CD.sub.3OD):
[0580] .delta.: 3.92-3.90 (d, 2H), 3.64-3.58 (m, 1H), 3.50-3.30 (m,
2H), 2.80-2.50 (m, 4H), 2.0-1.9 (m, 1H), 2.6-2.4 (m, 2H), 1.3 (s,
10H), 0.98-0.82 (m, 9H).
R,S-4-trimethylammonium-3- octylaminobutyrate
[0581] The isobutyl ester of
R,S-4-trimethylammonium-3-tetradecylamino-ami- nobutyric acid,
acetate salt, (2.8 g, 0.00719) was hydrolysed on Amberlyst IRA 402
resin (OH.sup.- activated form) and eluted with water. Water was
evaporated to dryness under reduced pressure; the resulting white
solid was washed with methanol, filtered and vacuum-dried. 1.8 g of
product were obtained.
[0582] Yield 70%
[0583] M.p.=140.degree. C. dec.
[0584] .sup.1H-NMR (300 MHz; CD.sub.3OD):
[0585] .delta.: 3.42-3.30 (m, 3H), 3.2 (s, 9H), 2.85-2.70 (m, 1H),
2.60-2.40 (m, 2H), 2.30-2.20 (m, 1H), 1.55-1.40 (m, 2H), 1.3 (s,
10H), 0.92-0.85 (t, 3H).
[0586] Elemental analysis: responding to the expected formula
C.sub.15H.sub.32N.sub.2O.sub.2
[0587] K.F.=2.8% water.
[0588] TLC silica gel (CHCl.sub.3 42/MeOH 28/isopropyl alcohol
7/water 10.5/acetic acid 10.5)
[0589] Rf=0.32.
[0590] HPLC: SGE-SCX column (5 .mu.m, 250.times.4 mm), mobile phase
0.05M (NH.sub.4)H.sub.2PO.sub.4:CH.sub.3CN 40:60, pH=4, flow=0.75
ml/min; detector: RI, UV 205 nm, RT=43.20 min.
EXAMPLE 24
R,S-4-trimethylammonium-3-(decansulfonyl)aminobutyrate (ST
1364)
Aminocarnitine Isobutyl Ester Chloride Hydrochloride
[0591] Isobutyl ester of aminocarnitine, inner salt (3 g, 18.72
mmoles), was dissolved in isobutanol (120 ml) and ice-bath cooled.
Gaseous HCl was bubbled into the solution until complete saturation
and clearing of the mixture. The solution was refluxed (bath
temperature 130.degree. C.) overnight. The solvent was
vacuum-evaporated and the residue was triturated with Et.sub.2O.
5.1 g of white solid were obtained.
[0592] Yield=95%;
[0593] .sup.1H-NMR (200 MHz; D.sub.2O):
[0594] .delta.: 4.3 (m, 1H), 4.0 (d, 2H), 3.8 (d, 2H), 3.2 (s, 9H),
3.1 (m, 2H), 2.0 (m, 1H), 0.9 (d, 6H).
[0595] Elemental analysis: responding to the expected formula
Cl.sub.11H.sub.26Cl.sub.2N.sub.2O.sub.2.
[0596] K.F.=1% water.
R,S-4-trimethylammonium-3-(decansulfonyl)-aminobutyrate
[0597] The isobutyl ester of R,S-aminocarnitine chloride,
hydrochloride (1 g, 3.46 mmoles) in anhydrous dichloromethane (5
ml) was added with triethylamine (2.65 ml, 19 mmoles) and
decansulfonyl chloride (2.1 g, 8.65 mmoles) suspended in 3 ml
anhydrous dichloromethane, at 0.degree. C. The mixture was left
under stirring for 3 days at room temperature. The solvent was
evaporated to dryness, the residue was taken up with ethyl acetate
and the white precipitate of triethylamine hydrochloride was
separated by from the solution by vacuum-filtration. The ethyl
acetate solution was vacuum-dried to give 2.8 g of a yellow oil. 71
ml 1N NaOH were added to hydrolize the isobutyl ester, leaving the
suspension under stirring overnight at room temperature. The
suspension was evaporated and vacuum-dried, and the solid residue
was completely dried under oil-vacuum, taken up with methanol and
purified through silica gel chromatographic column, using methanol
as eluant. 555 mg of product were obtained.
[0598] Yield 44%
[0599] M.p.=158.degree. C. dec.
[0600] .sup.1H-NMR (300 MHz; CD.sub.3OD):
[0601] .delta.: 4.3 (m, 1H), 3.45 (m, 2H), 3.25 (s, 9H), 3.15 (m,
2H), 2.45 (d, 2H), 1.8 (m, 2H), 1.45 (m, 2H), 1.4 (brs, 12H), 0.9
(brt, 3H).
[0602] Elemental analysis: responding to the expected formula
C.sub.17H.sub.36N.sub.2O.sub.4S
[0603] Mass ESI =365 [(M+H).sup.+], 387[(M+Na).sup.+]
[0604] K.F.=3% water.
[0605] TLC silica gel (CHCl.sub.3 42/MeOH 28/isopropyl alcohol
7/water 10.5/acetic acid 10.5)
[0606] Rf=0.62.
[0607] HPLC: Spherisorb-C.sub.1 column (5 .mu.m, 250.times.4.6 mm),
mobile phase 0.05M K.sub.2H.sub.2PO.sub.4:CH.sub.3CN 35:65, pH as
such, flow=0.73 ml/min; temperature=30.degree. C., detector: RI, UV
205 nm, RT=7.0 min.
EXAMPLE 25
R,S-4-trimethylammonium-3-(nonylsulfamoyl)aminobutyrate (ST
1362)
[0608] The isobutyl ester of R,S-aminocarnitine chloride,
hydrochloride (2 g, 6.9 mmoles) in anhydrous dichloromethane (40
ml) was added with triethylamine (3.8 ml, 27.6 mmoles) and dropped
with SO.sub.2Cl.sub.2 in dichloromethane (1.7 ml in 10 ml final
solution) at 0.degree. C. The mixture was left under stirring for 3
days at room temperature, triethylamine (1.9 ml, 13.8 mmoles) and
nonylamine (2.5 ml, 13.8 mmoles) were added and the reaction
mixture was left under stirring overnight at room temperature. The
solvent was vacuum-evaporated, the residue was taken up with ethyl
acetate (100 ml) and the precipitate of triethylamine hydrochloride
was separated from the solution by vacuum-filtration. The ethyl
acetate solution was vacuum-dried to give 4.8 g of a yellow oil, to
which were added 105 ml 1N NaOH to hydrolize the isobutyl ester.
The mixture was left under stirring overnight at room temperature
and vacuum-dried. The residue was completely dried under
oil-vacuum. The yellow semisolid was crystallized from chloroform.
1.26 g of product were obtained.
[0609] Yield 50%
[0610] M.p.=152.degree. C. dec.
[0611] .sup.1H-NMR (300 MHz; CD.sub.3OD):
[0612] .delta.: 4.1 (m, 1H), 3.48 (d, 2H), 3.25 (s, 9H), 2.95 (m,
2H), 2.5 (t, 2H), 1.55 (t, 2H), 1.45 (brs, 12H), 0.9 (brt, 3H).
[0613] Elemental analysis: responding to the expected formula
C.sub.16H.sub.35N.sub.3O.sub.4S
[0614] Mass ESI=366 [(M+H).sup.+], 388[(M+Na).sup.+]
[0615] K.F.=5.8% water.
[0616] TLC silica gel (CHCl.sub.3 42/MeOH 28/isopropyl alcohol
7/water 10.5/acetic acid 10.5)
[0617] Rf=0.34.
[0618] HPLC: Spherisorb-C.sub.1 column (5 .mu.m, 250.times.4.6 mm),
mobile phase 0.05M KH.sub.2PO.sub.4:CH.sub.3CN 35:65, pH as such,
flow=0.75 ml/min; temperature=30.degree. C., detector: RI, UV 205
nm, RT=6.68 min.
EXAMPLE 26
S-4-trimethylammonium-3-( dodecansulfonyl)aminobutyrate (ST
1391)
[0619] The product was prepared as disclosed in Example 24,
starting from isobutyl ester of S-aminocarnitine chloride,
hydrochloride and dodecansulfonyl chloride, to give 600 mg of
product.
[0620] Yield 44%
[0621] M.p.=156.degree. C. dec.
[0622] [.alpha.].sub.D.sup.20=+6.degree. (c=0.245%, H.sub.2O)
[0623] .sup.1H-NMR (300 MHz; CD.sub.3OD):
[0624] .delta.: 4.3 (m, 1H), 3.45 (m, 2H), 3.25 (s, 9H), 3.15 (m,
2H), 2.45 (d, 2H), 1.8 (m, 2H), 1.45 (m, 2H), 1.4 (brs, 16H), 0.9
(brt, 3H).
[0625] Elemental analysis: responding to the expected formula
C.sub.19H.sub.40N.sub.2O.sub.4S
[0626] K.F.=8.6% water.
[0627] TLC silica gel (CHCl.sub.3 42/MeOH 28/isopropyl alcohol
7/water 10.5/acetic acid 10.5)
[0628] Rf=0.65.
[0629] HPLC: Spherisorb-C1 column (5 .mu.m, 250.times.4.6 mm),
mobile phase 0.05M KH.sub.2PO.sub.4:CH.sub.3CN 40:60, pH as such,
flow=0.75 ml/min; temperature=30.degree. C., detector: RI, UV 205
nm, RT=8.5 min.
EXAMPLE 27
R-4-trimethylammonium-3-(dodecansulfonyl)aminobutyrate (ST
1420)
[0630] The product was prepared as disclosed in Example 24,
starting from isobutyl ester of R-aminocarnitine chloride,
hydrochloride and dodecansulfonyl chloride, to give 450 mg of
product.
[0631] Yield 34%
[0632] M.p.=158.degree. C. dec.
[0633] [.alpha.]D.sup.20=-7.degree. (c=0.265%, H.sub.2O)
[0634] .sup.1H-NMR (300 MHz; CD.sub.3OD):
[0635] .delta.: 4.3 (m, 1H), 3.45 (m, 2H), 3.28 (s, 9H), 3.15 (m,
2H), 2.45 (d, 2H), 1.8. (m, 2H), 1.45 (m, 2H), 1.3 (brs, 16H), 0.9
(brt, 3H).
[0636] Elemental analysis: responding to the expected formula
C.sub.19H.sub.40N.sub.2O.sub.4S
[0637] K.F.=6.9% water.
[0638] TLC silica gel (CHCl.sub.3 42/MeOH 28/isopropyl alcohol
7/water 10.5/acetic acid 10.5)
[0639] Rf=0.66.
[0640] HPLC: Spherisorb-Cl column (5 .mu.m, 250.times.4.6 mm),
mobile phase 0.05M KH.sub.2PO.sub.4:CH.sub.3CN 40:60, pH as such,
flow=0.75 ml/min; temperature=30.degree. C., detector: RI, UV 205
nm, RT=8.11 min.
EXAMPLE 28
S-4-trimethylammonium-3-(undecylsulfamoyl)aminobutyrate (ST
1427)
[0641] The product was prepared as disclosed in Example 25,
starting from isobutyl ester of S-aminocarnitine chloride,
hydrochloride and undecyl amine, except the crude product was
purified on a silica gel chromatographic column, using a gradient
CHCl.sub.3: MeOH 9:1 to 1:9. The product was further purified on a
silica gel chromatographic column, using MeOH. 0.7 g of pure
product were obtained.
[0642] Yield 38%
[0643] M.p.=153.degree. C. dec.
[0644] [.alpha.].sub.D.sup.20=+4.degree. (c=0.25%, H.sub.2O,
pH=2)
[0645] .sup.1H-NMR (300 MHz; CD.sub.3OD):
[0646] .delta.: 4.1 (m, 1H), 3.48 (d, 2H), 3.25 (s, 9H), 2.95 (m,
2H), 2.5 (m, 2H), 1.55 (brt, 2H), 1.45 (brs, 16H), 0.9 (brt,
3H).
[0647] Elemental analysis: responding to the expected formula
C.sub.18H.sub.39N.sub.3O.sub.4S
[0648] K.F.=2.9% water.
[0649] TLC silica gel (CHCl.sub.3 42/MeOH 28/isopropyl alcohol
7/water 10.5/acetic acid 10.5)
[0650] Rf=0.68.
[0651] HPLC: Spherisorb-C1 column (5 .mu.m, 250.times.4.6 mm),
mobile phase 0.05M KH.sub.2PO.sub.4:CH.sub.3CN 60:40, pH as such,
flow=0.7 ml/min; temperature=30.degree. C., detector: RI, UV 205
nm, RT=8.384 min.
EXAMPLE 29
R-4-trimethylammonium-3-(undecylsulfamoyl)aminobutyrate (ST
1428)
[0652] The product was prepared as disclosed in Example 25,
starting from isobutyl ester of S-aminocarnitine chloride,
hydrochloride and undecyl amine, except the crude product was
purified on a silica gel chromatographic column, using a gradient
CHCl.sub.3: MeOH 9:1 to 1:9. The product was further purified on a
silica gel chromatographic column, using MeOH. 0.5 g of product
were obtained.
[0653] Yield 32%
[0654] M.p.=158.degree. C. dec.
[0655] [.alpha.].sub.D.sup.20=-4.degree. (c=0.25%, H.sub.2O,
pH=2)
[0656] .sup.1H-NMR (300 MHz; CD.sub.3OD):
[0657] .delta.: 4.1 (m, 1H), 3.48 (d, 2H), 3.25 (s, 9H), 2.95 (m,
2H), 2.5 (m, 2H), 1.55 (brm, 2H), 1.45 (brs, 16H), 0.9 (brt,
3H).
[0658] Elemental analysis: responding to the expected formula
C.sub.18H.sub.39N.sub.3O.sub.4S
[0659] K.F.=4.77% water.
[0660] TLC silica gel (CHCl.sub.3 42/MeOH 28/isopropyl alcohol
7/water 10.5/acetic acid 10.5)
[0661] Rf=0.68.
[0662] HPLC: Spherisorb-C.sub.1 column (5 .mu.m, 250.times.4.6 mm),
mobile phase 0.05M KH.sub.2PO.sub.4:CH.sub.3CN 60:40, pH as such,
flow=0.7 ml/min; temperature=30.degree. C., detector: RI, UV 205
nm, RT=8.379 min.
EXAMPLE 30
R-4-trimethylammonium-3-(dodecylcarbamoyl)aminobutyrate (ST
1375)
[0663] The product was prepared as disclosed in Example 1, starting
from R-aminocarnitine inner salt and dodecylisocyanate. The crude
product obtained after washing with diethyl ether was purified on a
silica gel chromatographic column to give 4.8 g of product.
[0664] Yield 55%
[0665] M.p.=147.degree. C. dec.
[0666] [.alpha.].sub.D.sup.20=-24.6.degree. (c=0.48%, MeOH)
[0667] .sup.1H-NMR (300 MHz; CD.sub.3OD):
[0668] .delta.: 4.51 (m, 1H), 3.60 (dd, 1H), 3.45 (dd, 1H), 3.2 (s,
9H), 3.1 (t, 2H), 2.4 (m, 2H), 1.45 (m, 2H), 1.3 (brs, 18H), 0.9
(t, 3H).
[0669] Elemental analysis: responding to the expected formula
C.sub.20H.sub.41N.sub.3O.sub.3
[0670] K.F.=5.4% water.
[0671] TLC silica gel (CHCl.sub.3 42/MeOH 28/isopropyl alcohol
7/water 10.5/acetic acid 10.5)
[0672] Rf=0.6.
[0673] HPLC: Spherisorb-C1 column (5 .mu.m, 250.times.4.6 mm),
mobile phase 0.05M KH.sub.2PO.sub.4:CH.sub.3CN 65:35, pH=5.6,
flow=0.75 ml/min; temperature=30.degree. C., detector: RI, UV 205
nm, RT=8.5 min.
EXAMPLE 31
R-4-trimethylammonium-3-( 10-phenoxydecylcarbamoyl)aminobutyrate
(ST 1449)
10-Phenoxydecyl isocyanate
[0674] A solution of 11-phenoxyundecanoyl chloride (31.1 g, 104.8
mmoles) in acetone (30 ml) was dropped into a solution of sodium
azide (9.53 g, 146.6 mmoles) in water (30 ml), cooled in an ice
bath, keeping the solution temperature between 10 and 15.degree. C.
After one hour, the solution was transferred in a separatory funnel
and the lower phase (the aqueous one) was eliminated. The higher
phase was transferred into a flask containing 100 ml of toluene,
previously warmed at 65.degree. C. After 1.5 hours, the solution
was evaporated to dryness, giving 13.37 g of crude product, which
could be used as such in the subsequent reaction.
[0675] .sup.1H-NMR (300 MHz; CDCl.sub.3):
[0676] .delta.: 7.2 (m, 2H), 6.9 (m, 3H), 3.9 (t, 2H), 3.6 (t, 2H),
1.4 (m, 2H), 1.3 (m, 10H).
R-4-trimethylammonium-3-(10-phenoxydecylcarbamoyl)-amino
butyrate
[0677] 10-phenoxydecylisocyanate (25.0 g, 91.12 mmoles) was added
to a solution of aminocarnitine, inner salt (7.3 g, 45.56 mmoles)
in anhydrous DMSO (350 ml) and the solution was left to stand for
60 hours at 40.degree. C. The resulting mixture was transferred in
a 3 l Erlenmeyer flask containing ethyl ether (2.5 l) and the
solvent was separated by decantation of the formed precipitate,
which was then taken with few chloroform, transferred into a flask
and precipitated again with ethyl ether. The so obtained crude
product was washed several times with ethyl ether and purified on a
silica gel chromatographic column, using a gradient CHCl.sub.3:
MeOH 9:1 to CHCl.sub.3: MeOH 3:7 gradient until elution of
impurities with higher Rf, then eluting the product of interest
with MeOH only. 13.5 g of pure product were obtained.
[0678] Yield 68%
[0679] .sup.1H-NMR (300 MHz; CD.sub.3OD):
[0680] .delta.: 7.2 (m, 2H), 6.9 (m, 3H), 4.5 (m, 1H), 3.9 (t, 2H),
3.6 (dd, 1H), 3.4 (dd, 1H), 3.2 (s, 9H), 3.1 (t, 2H), 2.4 (m, 2H),
1.8 (m, 2H), 1.6 (m, 2H), 1.4 (m, 2H), 1.3 (m, 10H).
[0681] FAB Mass=436, [(M+H).sup.+;
[0682] Elemental analysis: responding to the expected formula
C.sub.24H.sub.41N.sub.3O.sub.4
[0683] K.F.=2.3% water.
EXAMPLE 32
R-4-trimethylammonium-3-(trans-.beta.-styrenesulfonyl)aminobutyrate
(ST 1448)
R-aminocarnitine Isobutyl Ester Chloride Hydrochloride
[0684] R-aminocarnitine inner salt (3 g, 18.72 mmoles) was
dissolved in isobutanol (120 ml) and ice-bath cooled. Gaseous HCl
was bubbled into the solution until complete saturation and
clearing of the mixture. The solution was refluxed (bath
temperature 130.degree. C.) overnight. The solvent was
vacuum-evaporated and the residue was triturated with Et.sub.2O.
5.1 g of white solid were obtained.
[0685] Yield=95%;
[0686] .sup.1H-NMR (200 MHz; D.sub.2O):
[0687] .delta.: 4.3 (m, 1H), 4.0 (d, 2H), 3.8 (d, 2H), 3.2 (s, 9H),
3.1 (m, 2H), 2.0 (m, 1H), 0.9 (d, 6H).
[0688] Elemental analysis: responding to the expected formula
C.sub.11H.sub.26Cl.sub.2N.sub.2O.sub.2.
[0689] K.F.=1% water.
R-4-trimethylammonium-3-(trans-.beta.-styrenesulfonyl)-aminobutyrate
[0690] The isobutyl ester of R-aminocarnitine chloride,
hydrochloride (1 g, 3.46 mmoles) in anhydrous dichloromethane (5
ml) was added with triethylamine (2.65 ml, 19 mmoles) and
trans-.beta.-styrenesulfonyl chloride (1.753 g, 8.65 mmoles)
suspended in 3 ml anhydrous dichloromethane, at 0.degree. C. The
mixture was left under stirring for 3 days at room temperature. The
solvent was evaporated to dryness, the residue was taken up with
ethyl acetate (100 ml) and the white precipitate of triethylamine
hydrochloride was separated by from the solution by
vacuum-filtration. The ethyl acetate solution was vacuum-dried,
then 71 ml 1N NaOH were added to hydrolize the isobutyl ester,
leaving the suspension under stirring overnight at room
temperature. The suspension was evaporated and vacuum-dried, and
the solid residue was completely dried under oil-vacuum, taken up
with methanol and purified through silica gel chromatographic
column, using methanol as eluant. 565 mg of product were
obtained.
[0691] Yield 50%
[0692] .sup.1H-NMR (300 MHz; CD.sub.3OD):
[0693] .delta.: 7.8 (d, 1H), 7.5 (m, 5H), 7.3 (d, 1H), 4.3 (m, 1H),
3.4 (m, 2H), 3.2 (s, 9H), 2.4 (d, 2H).
[0694] Elemental analysis: responding to the expected formula
C.sub.15H.sub.22N.sub.2O.sub.4S
[0695] ESI Mass=327 [(M+H).sup.+]
Pharmacological Activity
Determination of CPT Inhibiting Activity
[0696] CPT inhibition was evaluated essentially as described in
Kerner, J. & Bieber, L. L. (1990) Biochemistry 29: 4326-34 on
fresh mitochondrial preparations obtained from normally fed Fischer
rat liver or heart. Mitochondria were isolated from liver or heart
and suspended in 75 mM saccharose buffer, 1 mM EGTA, pH 7.5. 100
.mu.l mitochondrial suspension, containing 50 .mu.M [.sup.14C]
palmitoyl-CoA (specific activity 10,000 DPM/mole) and 10 mM
L-carnitine, were incubated at 37.degree. C., in the presence of
scalar concentrations of the test product (0-3 mM). Reaction time:
1 minute.
[0697] Table 1 shows the IC.sub.50 determined.
[0698] The compounds of the present invention have higher
inhibiting activity than the one of the reference compound
SDZ-CPI-975, Example 1, disclosed in EP 0 574 355.
1TABLE 1 IC.sub.50 of inhibition CPT1 curve in rat liver
mitochondria IC.sub.50 Compound (.mu.M/I) SDZ-CPI-975 17.4 ST1326
0.75 ST1327 3.2
Determination of Oleate-stimulated .beta.-hydroxybutyrate
Production
[0699] .beta.-hydroxybutyrate production is an index of CPT
activity. In fact, the production of ketone bodies, final products
of mitochondrial .beta.-oxidation, is related to CPT activity.
[0700] Mithocondrial preparations, obtained according to the method
by Venerando et al. (Am. J. Physiol. 266:C455-C461, 1994), were
used. Hepatocytes are incubated at 37.degree. C. in KRB bicarbonate
buffer at pH 7.4, 6 mM glucose, 1% BSA in O.sub.2/CO.sub.2 95/5
atmosphere at 2.5.times.10.sup.6 cells/ml. After 40 min incubation
with the test compound at different concentrations, the first set
of samples was taken (T.sub.0 min) and oleate was added (1 mM final
in KRB+BSA 1.4%). After 20 minutes, the second withdrawal was made
(T.sub.20 min)
[0701] Table 2 shows the results. The data are the mean of three
different experiments, twice carried out.
[0702] The compounds of the present invention have higher
.beta.-hydroxybutyrate inhibiting activity than the one of the
reference compound SDZ-CPI-975, Example 1, disclosed in EP 0 574
355.
2TABLE 2 IC.sub.50 of inhibition CPT1 curve of
.beta.-hydroxybutyrate production in rat hepatocytes IC.sub.50
Compound (.mu.M/I) SDZ-CPI-975 3.7 ST1251 0.5 ST1253 0.9 ST1285
1.9
Glucose and .beta.-hydroxybutyrate in Serum Fasted Rats Treated
With CPT Inhibitors
[0703] Normally fed Fischer rats were starved for 24 hours and
subsequently treated with the test compounds. One hour after the
treatment, the animals were sacrificed and serum concentrations of
glucose and .beta.-hydroxybutyrate were determined.
[0704] Table 3 shows the results. For the compound ST1326 were used
doses of 14.5 mg/2 ml/kg, for other test compounds, the doses are
equivalent to ST1326 one.
3TABLE 3 .beta.-hydroxybutyrate and glucose serum concentration in
24 hours-starved rats, after one hour from intraperitoneal
treatment. SDZ control CPI-975 ST1251 ST1253 ST1326 ST1327 ST1328
.beta.- OHB Mean 1867 119.9 99.8 118.8 133.1 93.0 169.2 s.e. 240
12.8 8.3 20.4 12.4 8.7 26.7 p< -- 0.001 0.001 0.001 0.001 0.001
0.001 Glu Mean 108.8 87.6 76.9 88.2 84.2 84.9 79.5 s.e. 6.7 1.0 2.3
3.9 2.4 1.6 1.6 p< -- 0.05 0.01 0.05 0.05 0.05 0.05
Glucose and Insulin Levels in Diabetic Animals Treated With CPT
Inhibitors
[0705] C57BL/6J male rats, 5-weeks old, were provided by Ch. River.
After 10 days of acclimatisation in standard conditions
(22.+-.2.degree. C.; 55.+-.15% humidity; 15-20/h air changes; 12
hours light-dark cycle, with 700-1900 lux) and with standard diet
with 4RF21 feedstock (Mucedola), glycaemia was controlled in
post-absorption state (starving from 8.30 a.m. to 4.30 p.m.). Blood
withdrawal was carried out cutting the tail end. Glucose was
analysed in blood acid supernatant (HCLO4 0,375 N) with
autoanalyzer Cobas Mira S with Glucose GDH Kit (Roche).
[0706] The animals were divided in two groups, 26 mice each and fed
with a high-fat and a low-fat diet, respectively.
[0707] After 2 months from the start of the diet, glycaemia was
tested, according to the starting method. After about 3 months from
the start of the diet, glycaemia was tested, according to the
starting method and plasma insulin levels were also determined
(with blood withdrawal from end tail cutting) using [125I] Rat
Insulin Kit (Amersham).
[0708] One 10 mice group fed with low-fat diet and two 10-mice
groups fed with high-fat diet were selected One of the two high fat
diet was administered with ST 1327 at the dose of 45 mg/Kg in
deionised H2O (p.o., twice a day, 8.30 a.m. and 5.30
p.m.).administration volume was 10 ml/Kg. the two remaining groups
was treated with vehicle only. High-fat or low-fat diets were
continued during the treatment.
[0709] After 20 days of treatment, glycaemia and plasma insulin
were measured. After 43 days of treatment, the animals were
sacrificed by decapitation in post-absorption state (fasting 8.30
a.m.-4.30 p.m.), 8 hours after the last treatment. Blood was
withdrawn and serum was separated by centrifugation and stored at
-80 .degree. C. Liver, heart and skeletal muscle (upper limbs) were
also extracted, frozen in dry ice-acetone and kept at -80 .degree.
C.
[0710] High-fat diet determined an increase of body weight,
glycaemia and insulin, with respect to low-fat diet.
[0711] After 20 days of treatment with ST 1327, glucose and insulin
levels significantly decreased.
[0712] Table 4 shows the results.
4TABLE 4 Glucose and insulin levels in rats fed with fat-rich diet.
High Fat diet High Fat diet Low fat diet Compound Control Treated
Control Glucose 248.5 .+-. 11.03 181.4 .+-. 9.63* 207.3 .+-. 6.84**
mg/dl (10) (9) (9) Insulin 1.632 .+-. 0.246 0.621 .+-. 0.117**
0.549 .+-. 0.050* ng/ml (10) (9) (9)
[0713] Student's t test, * and ** indicate p<0.001 and
p<0.01, respectively, against high fat diet; ( ) indicates the
number of cases.
[0714] These results shows that the compounds according to the
present invention are effective in controlling glycaemia in fasting
conditions. This is an important aspect in the treatment of
diabetes, wherein hepatic gluconeogenesis occurs during fasting
periods (i.e. nocturnal rest).
The Effect of CPT Inhibitors on Myocardial Ischemia
[0715] The compounds of the present invention are also effective in
the treatment of ischemia, in particular myocardial ischemia.
[0716] To this end, male Wistar rats, weighing 200-225 g, provided
by Charles-River, were kept at constant temperature of
23.degree.+/-1.degree. C., 50+/-10% relative humidity, 12 hours
light-dark cycle, fed with pellet 4RF21 (Mucedola) tap water ad
libitum.
[0717] The animals were anaesthetised with sodium Pentobarbital at
the dose of 70 mg/Kg i.p.. Hearts were rapidly removed and put in a
cold Krebs-Henseleit solution, before incannulation of aorta e
subsequent perfusion according to Langendorff technique at
37.degree. C. with a pressure of 100 cm water.
[0718] Perfusion medium (Krebs-Henseleit) at pH 7.4 consists in:
128 mM NaCl, 4.7 mM KCl, 1 mM MgCl2, 0.4 mM Na2HP04, 20.2 mM
NaHCO3, 1.3 mM CaCl2, 5 mM glucose. The medium was constantly
oxygenated with carbogen (95% O2, 5% CO2).
[0719] After a 10 min "conditioning" period, hearts were perfused
in a recirculant apparatus for 20 min. with the same medium
containing 0.6 mM palmitate complexed with albumine (fraction V,
fatty acid free), with or without the CPT inhibitor according to
the present invention. By way of example ST 1364 was used at
concentrations of 1 and 5 .mu.M. After such a period ischemia was
induced by reducing perfusion hydrostatic pressure from 100 cm to
20 cm for a period of 30 min. Reperfusion was started
re-establishing the starting pressure conditions (100 cm).Hearts
were controlled for 20 min. the inhibitor is present also during
reperfusion phase.
[0720] Lactate dehydrogenases (LDH) release was monitored in the
effluent in normal oxygenation conditions, during ischemia, with a
withdrawal of medium at 30', and during reperfusion, with
withdrawals at 1.5, 10, 15 and 20 minutes.
[0721] LDH release in the effluent is remarkably reduced, during
reperfusion results significantly reduced in the presence of ST1364
at the dose of 5 .mu.M (FIG. 1). This result indicates a lower
entity of cellular damage from reperfusion of the treated with
respect to the controls.
[0722] Statistical analysis was carried out with Student's "t" test
for non-paired data.
[0723] The number of the cases for each group is six (n=6).
[0724] The following Table 5 reports the results.
5TABLE 5 LDH release in perfusate (mU/ml/min) ST1364 ST1364 Control
1 .mu.M* 5 .mu.M** Basal 280 275 220 Ischemia 30' 200 220 200
Reperfusion 1' 640 480 410 Reperfusion 5' 660 500 380 Reperfusion
10' 670 495 380 Reperfusion 15' 700 510 320 Reperfusion 20' 720 580
325
[0725] Statistical analysis was carried out with Student's "t" test
for non-paired data. *p<0.05 vs controls; **p<0.01 vs
controls.
[0726] The number of the cases for each group is six (n=6).
[0727] LDH release in the effluent is remarkably reduced, during
reperfusion results significantly reduced in the presence of ST1364
at the dose of 5 .mu.M (FIG. 1). This result indicates a lower
entity of cellular damage from reperfusion of the treated with
respect to the controls.
[0728] In another aspect, the present invention provides a
combination of at least a compound of formula (I) with at least
another active ingredient suitable for the treatment of the disease
of interest.
[0729] In the treatment or prevention of diabetes, the present
invention provides a compound of formula (I), optionally in
combination with a suitable well-known active ingredient, such as
for example a sulfonylurea, L-carnitine, fibrate and other agonists
of peroxisomal proliferator activated receptor (PPAR-.alpha.),
agonists of 9-cis retinoic acid activated receptor, such as RXR, in
particular .alpha., .beta.- and .gamma.-isoforms, HMG-CoA reductase
inhibitor, .beta.-sitosterol inhibitor, cholesterol acyltransferase
inhibitor, biguanides, cholestyramine, angiotensin II antagonist,
melinamide, nicotinic acid, fibrinogen receptor antagonists,
aspirin, .alpha.-glucosidase inhibitors, insulin secretogogue,
insulin and glucagon-like peptides (incretins) and agonists of
PPAR-.gamma. (such as thiazolidinediones or others).
[0730] In the treatment or prevention of obesity, the present
invention provides a compound of formula (I), optionally in
combination with an suitable well-known active ingredient, such as
for example fenfluramine, dexfenfluramine, phentiramine, a
.beta.-3-adrenergic receptor agonist.
[0731] In the treatment or prevention of high triglyceridhemia, the
present invention provides a compound of formula (I), optionally in
combination with an suitable well-known active ingredient.
[0732] The compounds according to the present invention are also
useful in the treatment or prevention of high cholesterol levels
and in modulating HDL plasma levels, thus resulting beneficial in
the treatment or prevention of the diseases related with these
altered plasma levels. Examples of related diseases are
hypertension, obesity, atherosclerosis, diabetes and related
conditions. The medicaments containing at least a compound of the
present invention may contain in combination at least another
active ingredient effective in the treatment or prevention of the
above mentioned diseases. Examples of other active ingredient are
fibrates, such as clofibrate, bezafibrate and gemfibrozil and other
PPAR-.alpha. agonists; inhibitors of cholesterol biosynthesis, such
as HMG-CoA reductase inhibitors, such as statins, namely
lovastatin, simvastatin and pravastatin; inhibitors of cholesterol
absorption for example beta-sitosterol and (acyl CoA:cholesterol
acyltransferase) inhibitors for example melinamide; anion exchange
resins for example cholestyramine, colestipol or a
dialkylaminoalkyl derivatives of a cross-linked dextran; nicotinyl
alcohol, nicotinic acid or a salt thereof; vitamin E; thyromimetics
and L-carnitine.
[0733] The compounds of the present invention may be orally
administered in the form of a pharmaceutical composition,
comprising a therapeutically effective amount of at least a
compound of formula (I) in admixture with a pharmaceutically
acceptable vehicle and/or excipient. Examples of oral
pharmaceutical compositions are hard or soft capsules, tablets,
including sublingual administration, ampoules, sachets, elixirs,
suspensions, syrups, and the like. Alternatively, the active
ingredients according to the present invention may be incorporated
directly with the food of the diet. The amount of active compound
in such therapeutically useful compositions is such that an
effective dosage will be obtained. The active compounds can also be
administered intranasally as, for example, liquid drops or
spray.
[0734] The tablets, pills, capsules, and the like may also contain
a binder such as gum tragacanth, acacia, corn starch or gelatin;
excipients such as dicalcium phosphate; a disintegrating agent such
as corn starch, potato starch, alginic acid; a lubricant such as
magnesium stearate; and a sweetening agent such as sucrose, lactose
or saccharin. When a dosage unit form is a capsule, it may contain,
in addition to materials of the above type, a liquid carrier such
as a fatty oil.
[0735] Various other materials may be present as coatings or to
modify the physical form of the dosage unit. For instance, tablets
may be coated with shellac, sugar or both. A syrup or elixir may
contain, in addition to the active ingredient, sucrose as a
sweetening agent, methyl and propylparabens as preservatives, a dye
and a flavoring such as cherry or orange flavor.
[0736] These active compounds may also be administered
parenterally. Solutions or suspensions of these active compounds
can be prepared in pyrogen-free water.
[0737] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions.
[0738] If desired, or deemed necessary, the pharmaceutical
compositions may be in the controlled- release form. Various
techniques for preparing these forms are known.
[0739] General reference for pharmaceutical compositions can be
made to "Remington's Pharmaceutical Sciences Handbook", Mack Pub.
N.Y. USA.
[0740] The effective dosage of active ingredient employed may vary
depending on the particular compound employed, the mode of
administration, the condition being treated and the severity of the
condition being treated.
[0741] The compositions are formulated and administered in the same
general manner as detailed below. The compounds of the present
invention can be used effectively alone or in combination with one
or more additional active agents depending on the desired target
therapy. Combination therapy includes administration of a single
pharmaceutical dosage formulation which contains a compound of
formula I and one or more additional active agents, as well as
administration of a compound of formula I and each active agent in
its own separate pharmaceutical dosage formulation. For example, a
compound of formula I and an HMG-CoA reductase inhibitor can be
administered to the patient together in a single oral dosage
composition such as a tablet or capsule, or each agent administered
in separate oral dosage formulations. Where separate dosage
formulations are used, a compound of formula I and one or more
additional active agents can be administered at essentially the
same time, i.e., concurrently, or sequentially; combination therapy
is understood to include all these regimens.
[0742] An example of combination treatment or prevention of
atherosclerosis is wherein a compound of formula I is administered
in combination with one or more of the following active agents: an
antihyperlipidemic agent; a plasma HDL-raising agent; an
antihypercholesterolemic agent such as a cholesterol biosynthesis
inhibitor, for example an HMG-CoA reductase inhibitor, an HMG-CoA
synthase inhibitor, a squalene epoxidase inhibitor, or a squalene
synthetase inhibitor (also known as squalene synthase inhibitor);
an acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitor
such as melinamide; probucol; nicotinic acid and the salts thereof
and niacinamide; a cholesterol absorption inhibitor such as
beta-sitosterol; a bile acid sequestrant anion exchange resin such
as cholestyramine, colestipol or dialkylaminoalkyl derivatives of a
cross-linked dextran; an LDL (low density lipoprotein) receptor
inducer; fibrates such as clofibrate, bezafibrate, fenofibrate, and
gemfibrozil and other PPAR-.alpha. agonists, L-carnitine; vitamin
B.sub.6 and the pharmaceutically acceptable salts thereof; vitamin
B.sub.12; anti-oxidant vitamins such as vitamin C and E and beta
carotene; a beta-blocker; an angiotensin II antagonist; an
angiotensin converting enzyme inhibitor; and a platelet aggregation
inhibitor such as fibrinogen receptor antagonists (i.e.,
glycoprotein IIb/IIIa fibrinogen receptor antagonists) and aspirin.
The compounds of formula I can be administered in combination with
more than one additional active agent.
[0743] Another example of combination therapy can be seen in
treating obesity or obesity-related disorders, wherein the
compounds of formula I may be effectively used in combination with
for example, fenfluramine, dexfenfluramine, phentiramine and
.beta.-3 adrenergic receptor agonist agents and L-carnitine.
[0744] Another example of combination therapy can be seen in
treating diabetes and related disorders wherein the compounds of
formula I can be effectively used in combination with for example
sulfonylureas, biguanides, .alpha.-glucosidase inhibitors, other
insulin secretogogues, insulin and glucagon-like peptides
(incretins) and agonists of PPAR-.gamma. (such as
thiazolidinediones or others) as well as the active agents
discussed above for treating atherosclerosis.
* * * * *