SUMMARY OF THE INVENTION The present invention is directed to novel couinarin derivatives, and more precisely bicoumarin derivatives derived from the etherification of two 7-hydroκycoumarin radicals with bivalent alcohols of an aliphatic or cycloaliphatic nature of a mixed character with regard to this series. The new derivatives are represented by the following formula I:

in which each of the substituents ^" B-,--^ and R ₇ -R*ι ₀ represent hydrogen or a εubstituent chosen from the group formed by:

- a halogen,

- a free or esterified carboxylic group,

- a free or esterified or etherified hydroxy group, - a lower alkyl or monocycloarylal yl or lower onocycloalkyl-alkyl radical, or a corresponding unsaturated radical, which can be substituted in the aliphatic portion by one or more free or esterified or etherified hydroxy groups, or by

oxo groups or by free or esterified carboxy groups, and in the aryl portion by one or more lower alkyl groups or halogens or lower hydroxy or alkoxy groups, and in the cycloaliphatic part by one or more lower alkyl groups,

- a onocycloalkyl radical or a corresponding unsaturated radical, unsubstituted or substituted by one or more lower alkyl groups, a monocycloaryl radical, unsubstituted or substituted by one or more lower alkyl groups or halogens or lower hydroxy or alkoxy groups,

- R ₁ and R ₆ represent an aza-alkyl or aza-raonocycloalkyl or aza-monocycloalkyl- -alkyl or aεa-alkyl-monocycloalkyl or aza-alkyl-monocycloalkyl-alkyl radical, or a corresponding unsaturated radical, with a maximum of 12 carbon atoms, and which may be interrupted in the carbon atom chain by the groups -NH-, -O—, or -S-, and/or may be substituted by free or esterified or etherified hydroxy groups, or by oxo or by lower alkyl groups, or by free or esterified carboxy groups. R _Λ and , may also represent these moieties, and

- -X- represents a bivalent hydrocarbyl radical chosen from the group formed by an alkylene, monocycloaryl-alkylene or monocyclσalkyl- -alkylene radical or a corresponding unsaturated radical, which may be interrupted in the carbon atom chain by heteroato s chosen from the group formed by -NH-, -0-, and -S-, or by a monocyclo- -arylene or monocycloalkylene radical, and may be substituted in the aliphatic or cyclo- aliphatic part by one or more halogens or free or esterified or etherified hydroxy groups, or by

T

lower amino, alkyl-, or d ^' ialkyl-amino groups or C ₅ . ₆ alkylene amino groups, optionally interrupted by -NH-, -O-, or -S- groups, by oxo groups or by free or esterified carboxy groups, and in the aromatic moiety by one or more lower alkyl or halogen groups or lower hydroxy or alkoxy groups, and a monocycloalkylene radical, unsubstituted or substituted by one or more lower alkyl groups or free or esterified or etherified hydroxy groups, or by amino, alkyl, or lower dialkyla ino groups, or by oxo groups or by free or esterified carboxy groups.

The novel bicoumarin derivatives of the invention have interesting pharmaceutical properties and can be used in therapy. The invention also encompasses the salts of said compounds, especially those with pharma¬ ceutically acceptable acids or bases. The present invention also encompasses pharma¬ ceutical preparations containing one or more of the aforesaid bicoumarin derivatives and the therapeutic use of such compounds- The invention is also directed to preparation methods for the novel compounds and their salts. Due to the close relationship between the free compounds and salts with regard to their pharmaceutical properties, which consititute the basis of the present invention, in the following description whatever is said of the free substances will be true also of their salts, where the meaning does not specifically exclude this possibility.

The new bicoumarin derivatives of formula I and their salts have an anti-thrombotic and an i- -hypertensive action and can be used in different vascular pathologies, for example peripheral

vascular pathologies, anginal afflictions and cerebral vascular pathologies.

In the new compounds of formula I, the halogen atoms, both as R..- ₅ and R ₇ -*R ₁₀ substituents and as substituents of the aromatic radicals optionally present in these substituents or in substituent X, are preferably fluorine, chlorine and bromine atoms, and of these preferably chlorine. The R ₂ -R ₅ and R* ₇ -R ₁₀ alkyl radicals and, with regard to the aliphatic moiety, the "lower" monocycloaryl-alkyl and monocycloalkyl-alkyl groups, or the corresponding unsaturated groups, have a maximum of 7 carbon atoms. Preference is given to alkyl groups having 1 to 4 carbon atoms and alkenyl groups having 2 to 7 carbon atoms. All these groups can have straight or branched chains.

The cycloaliphatic radicals R _j - _g and •R ₇ -R ₁₀ or those contained in such substituents are nionocyclic and have preferably from 3 to 7 carbon atoms in the ring and more particularly from 5 to 7 carbon atoms.

Of the unsaturated hydrocarbyl radicals, both aliphatic and alicyclic, special mention should be made of those with only one double bond, comprising alkenyl groups and cycloalkenyl groups. In the monocyclic alicyclic radicals, special mention should be made of the cyclohexane derivatives, comprising cyclohexyl and cyclohexenyl radicals. ^R ₂ ^_R ₅ ^{nd R} ₇ ~- ^R -ιo ^ar Y-l groups are monocyclic and therefore derive from benzene and can be substituted in the aforesaid manner, that is with lower alkyl, or halogen or lower hydroxy or alkoxy groups. The term ^• 'lower" employed herein and

indeed generally in the present description is meant to refer to groups with a maximum of 7 carbon atoms. This is also true of the corresponding alkoxy or alkenyl groups. Such groups have especially a maximum of 4 carbon atoms and are preferably methyl or ethoxy groups. The substituents of the aromatic groups are preferably no more than 3. The aryl groups have preferably a total of 9 carbon atoms. What is said herein about these aryl groups is true also of the substituting aliphatic groups, for example arylalkyl groups.

In the aza-alkyl, aza-monocycloalkyl, aza-mono- cycloalkyl-alkyl, aza-alkyl- onocycloalkyl or aza-alkyl- onocycloalkyl-alkyl radicals, R,, ₄ , R _ό or R, or in the corresponding unsaturated radicals, any methylene or methyl group can be substituted by the -NH-group and this group can therefore be present both in the aliphatic moiety and in the cyclic parts of the hydrocarbyl ^' radicals. These radicals can be interrupted also by other heteroatoms at other points of the hydrocarbyl chain or by other -NH-groups. Preferably, oxygen or sulfur atoms can interrupt the chain in the cyclic radicals. Regarding the number of carbon atoms and possible double bonds present, apart from the condition that such aza-hydrocarbyl groups can have up to 12 carbon atoms, preference is given to those moieties which correspond to the corresponding hydrocarbyl groups mentioned above for the substituents ^ ₂ ~^ ^{anc R} ₇ ~ ^R ιo' - ^Cπ particular, alkyl groups as substituents of cyclic groups have preferably a maximum of 7 carbon atoms, especially from 1 to 4 carbon atoms, and they are above all methyl groups. Cycloalkyl groups have preferably from 5 to 7

carbon atoms and especially 6 carbon atoms. Such groups can be interrupted by the -NH-group, and are therefore aza-cyclohexyl or aza-cyclopentyl groups, such as the piperidine and pyrrolidine groups and can be interrupted also by other heteroatoms, such as -NH-, -0-, and -S-, and can therefore be piperazine, morpholine or thiomorpholine residues.

The bivalent -X-radical has, as an alkylene group, preferably from 1 to 8 carbon atoms, and can however be interrupted or substituted by alicyclic or aromatic carbocyclic radicals. Preferably, in this case, only one monocyclic radical is present, corresponding preferably to the radicals pre¬ viously described as preferential forms of _j - s and R ₇ -R ₁₀ substituents. The total number of carbon atoms of the -X-substituent can therefore be more than 3, but preferably should not exceed 18 carbon atoms. This is true also in consideration of the hydrocarbyl functions or groups which ca ^* substitute both the aliphatic moiety, and the aromatic or alicyclic moiety. These substituents are preferably those which have in part already been specified for the R-*-R ₁₀ substituents and those specified hereafter, especially with regard to esters and ethers. The -X- group can also represent a cycloalkylene radical, and these radicals preferably correspond to those described above where these are not substituents of the alkylene group.

The modi ied functions optionally present in all the aforesaid radicals or present as the R*,-R ₁₀ substituents themselves are esterified carboxy groups, esterified or etherified hydroxy groups and alkyl-, or dialkyl-, or alkylene amino groups. These functions may, however, be in free form, for

exa ple as unsubstituted amino groups. The carboxy groups in free or esterified form are derived especially from the following acids: formic, acetic, propionic, butyric, tri ethylacetic, n-valerianic, capronic, succinic, phenylacetic, benzoic, trimethoxybenzoic, chlorobenzoic, alkylsulfonic acids containing from 1 to 4 carbon atoms, such as methanesulfonic acid or arylεulfonic acid, especially those containing only one benzene residue, for example p-toluene- sulfonic acid, of the inorganic acids, should be mentioned, for example, sulfuric acid or phosphoric acid.

Esterified carboxy groups are preferably those derived from monovalent or bivalent aliphatic alcohols, saturated or unsaturated, with a maximum of 7 carbon atoms or from monoaryl-aliphatic alcohols with a maximum of 9 carbon atoms. Etherified hydroxy groups are preferably those also derived from saturated or unsaturated aliphatic alcohols with a maximum of 7 carbon atoms or from monoaryl-aliphatic alcohols with a maximum of 9 carbon atoms, while esterified hydroxy groups are derived especially from carboxylic acids of the aliphatic, araliphatic, aromatic or alicyclic series having preferably from 1 to 9 carbon atoms. In the alkyl and dialkylamines the alkyl groups have a maximum of 7 carbon atoms and preferably between 1 and 4, in the alkyleneamino groups there are 5 or 6 carbon atoms and the azacyclo-alkyl ring can be interrupted by other heteroato s, in particular by the -NH-, -O-, and -S-groups.

Substituent amino groups are, for example, those derived from methyla ine, ethyla ine, propyl- a ine, dimethylamine, diethylamine, pyrrolidine, piperidine, piperazine or morpholine. Of the aforesaid alkyl groups, both as substit¬ uents of the bicoumarin residue, and of the aforesaid hydrocarbyl radicals, special mention should be made of the following groups: methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl; of the alkenyl groups: vinyl, allyl, propenyl, isobutenyl, 2-butenyl and 2 pentenyl. Likewise, special examples of cycloalkyl groups are: cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, and of cylcoalkenyl groups the cyclopentenyl and cyclohexenyl groups.

Examples of aralkyl groups are benzyl, phenethyl, phenylpropyl and cinnamyl groups. Cycloalkyl-alkyl radicals are, for example, the cyclopentyl, cyclo- hexylmethyl, cyclopentylethyl and cyclohexylethyl groups and examples of the cycloalkenyl-alkyl groups are

2-cyclohexenyl-methyl or 2-cyclohexenylethyl groups. Aryl groups, both as substituents of the bicoumar¬ in residue, and of the aforesaid aliphatic hydrocarbyl radicals are, for example, the phenyl, toluyl, di-and tri ethyl-phenyl, ethyl-phenyl, allyl-phenyl, chlorophenyl, bromophenyl, -fluoro¬ phenyl, trichlorophenyl, onohydroxy-phenyl, dihydrox -phenyl or tri ethoxy-phenyl groups. Particularly valuable are the compounds of formula I in which -X- represents an alkylene radical having from 1 to 6 carbon atoms, which can be unsubstituted or substituted by one or two functions chosen from the groups formed by free or esterified or etherified hydroxy groups, or free or

esterified carboxy groups o ^' r oxo groups or free amino groups or lower alkyl or dialkyla ino groups or C ₅ . _& -alkyleneamino groups, optionally inter¬ rupted by -NH-, -0-, or -s-groups, where the derivatives of such functions are preferably those mentioned above.

The R ₁ -R ₆ substituents may be identical or different from the corresponding _y -Rp- _Q substituents. Of particular importance are novel compounds of formula I wherein the molecule is symmetrical, the two coumarin residues being mirror images of each other, that is, compounds in which R**~R ₆ are equal to R ₇ -R ₁₀ , respectively.

Among these compounds, special mention should be made of those with the formula:

in which

A represents a saturated aza-alkyl, aza-mono- cycloalkyl-alkyl or aza-alkyl-monocycloalkyl radical with a maximum of 12 carbon atoms and in which the cycloalkyl group has 5 or 6 carbon atoms, optionally further interrupted in the carbon atom chain by one of the groups -NH-, -0-and -S-, and which can be substituted at the carbon atoms by alkyl groups with 1 or 2 carbon atoms or by lower

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hydroxy or alkoxy groups.

B, C and D may represent a hydrogen atom and B may also represent a lower alkyl or alkylene radical or a monocyclic aryl radical or a halogen atom, C may represent the same aza-hydrocarbyl radical as defined by the substituent A, or the same hydro¬ carbyl groups as defined for B, and D may repre¬ sent the same hydrocarbyl radicals as defined for B. The moiety -X- represents an alkylene radical having from 1 to 6 carbon atoms and which can be interrupted in the carbon atom chain by hetero- atoms chosen from the group formed by -NH-, -0-, and -S-, and/or which may be unsubstituted or substituted by one or two functions chosen from the group formed by halogens, hydroxy groups, free or esterified with carboxylic acids having from 1 to 9 carbon atoms or etherified with alcohols with 1 to 7 carbon atoms, and carboxy groups, free or esterified with alcohols having from 1 to 7 carbon atoms and oxo groups and lower alkylamino or dialkylamino groups.

In these selected derivatives the aryl groups may be substituted as in the case of the compounds of formula I and especially by 1 to 3 methyl or alkoxy groups. In substituent A the cycloalkyl groups not containing nitrogen may be unsubstituted or substituted by l to 3 alkyl groups with a maximum of 3 carbon atoms and likewise those containing the -NH-group. These hydrocarbyl substituents, like the hydroxy groups, are no more than 2. The azacyclic groups are, for example, those specified above, in particular the radicals derived from piperidine, piperazine, morpholine or thiomorpholine. In the hydrocarbyl- a ino groups of substituent -X-, these have

preferably a maximum of 4 carbon atoms.

.Among the aforesaid bicoumarin derivatives of formula II according to the present invention, of special interest are those in which B is one of the aforesaid nitrogen-free hydrocarbyl groups, C and D are hydrogen atoms or one of said nitrogen-free hydrocarbyl groups or a halogen. In these compounds A is, for example, the diethylamino-ethyl or diiso- propylamino-ethyl radical or optionally their corresponding quaternary groups, obtainable for example by reaction of the corresponding compounds having a tertiary nitrogen with lower alkyl halogen- ides, especially with methyl bromide. Moreover, A is especially the morpholino-methyl, piperidinyl- -methyl, thiomorpholinyl-methyl, or piperazinyl- - ethyl radical. B is especially the methyl or phenyl group, C and D represent hydrogen, allyl or chlorine and X is for example the trimethylene, 3-hydroxy-trimethylene, hexa ethylene group or the 2-di-carbethoxy-trimethylene group.

Hereafter is a list of representative compounds of the invention:

1, 3-bis[3-(β-diethylaminoethyl)-4-methylcoumarin-7- -yloxy] ropane ^• l , 3-biε [ 3- (β-diethylaminoethyl) -4-methyl-8-chloro- coumarin-7-yloxy 3 propane

1 , 3 -bis [ 3 - ( β-diethylaminoethyl ) -4-phenylcoumarin-7-

-yloxy] ropane

1 , 3-bis [ 3- (morpholinomethyl) -4-phenyl-6-chloro-8- -methy lcoumarin-7-yloxy] propane

1 , 3 -bis [ 3 - ( orpholinomethyl ) - -methy 1-6 , 8-diallyl- coumarin-7-yloxy] propane

1 , 3-bis [ 3- (morpholinomethyl ) - , 8-dimethyl-6-allyl- coumarin-7-yloxy]propane

1,3-bis[3-(morpholinomethyl)-4-methyl-6-chloro-8-

-allylcoumarin-7-yloxy]propane

1,3-bis[3-(morpholinomethyl)-4,8-dimethyl-6-*chloro- coumarin-7-yloxy]propane 1,6-bis[3-(β-morpholinoethyl)-4-methylcoumarin-7-

-yloxy]hexane bis[3-(β-diethylaminoethyl)-4-methylcoumarin-7-

-yloxy] carbethoxy methane bis[3-(β-diethylaminoethyl)-4-methyl-8-chloro- coumarin-7-yloxy]carbethoxy methane bis[3-(β-diethylaminoethyl)-4-ρhenylcoumarin-7-

-yloxy] carbethoxy methane bis[3-(morpholinomethyl)- -methy1-6,8-diallyl- coumarin-7-yloxy]carbethoxy methane 1,3-bis[3-(β-diethylaminoethyl)-4-methyl-8-chloro- coumarin-7-yloxy]-2-hydroxypropane

1,3-bis[3-(morpholinomethyl)-4-raethyl-6,8-diallyl- coumarin-7-yloxy]-2-hydroxypropane l,3-biε[3-(morpholinomethyl)-4-phenyl-*6-chloro-8- -allylcoumarin-7-yloxy]-2-hydroxypropane

1,3-bis[3-(β-morpholinoethyl)-4-methylcoumarin-7-

-yloxy]-2-hydroxypropane

1,3-bis[3-(β-morpholinoethyl)-4-methyl-8-chloro- coumarin-7- loxy]-2-hydroxypropane and the others listed in the following illustra¬ tive Examples.

Other specific compounds include the following:

1,4-bis[3-(β-diethylaminoethyl)-4-methylcoumarin-7-

-yloxy]cyclohexane l, -bis[3-(β-diethylaminoethyl)-4-methyl-6,8-

-diallylcouταarin-7-yloxy]-2,3-dimethylcyclohexane

1, 3-bis[3-(β-diethylaminoethyl)-4-methylcoumarin-7-

-yloxy]-2-phenylpropane

The novel bicoumarin derivatives described above may optionally be salified, if they possess basic or acid functions. It is thus possible to prepare, on the one hand, salts with organic or metal bases and, on the other hand, salts obtained by the addition of acids or alkyl or aryl halogenides or the corresponding sulfonic acids. Particularly important salts are those which are therapeutically acceptable, and the new compounds of the invention can be used in this form. The salts can also be derived from bases or from acids which cannot be used for therapeutic purposes and in this case they serve, for example, as intermediate compounds for the purification of the novel products of the invention. The following are examples of therapeutically acceptable acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and methanesulfonic acid, malic acid, tartaric acid and succinic acid. Picric and picrolinic acid are particularly suitable for the formation of salts which may serve for the purification of the compounds. The amino groups can therefore be transformed into ammonium salts, in particular the tertiary amino groups can be transformed into quaternary groups, especially quaternary ammonium groups, such as tetramethy1ammonium chloride or bromide. Of the metal salts special mention should be made of alkali metal salts, alkaline earth or magnesium salts, for example potassium, sodium, ammonium and calcium salts, but also those with organic bases, such as primary, secondary or tertiary amines which are aliphatic or aromatic or heterocyclic, such as methylamine, ethylamine, propyl- amine, piperidine, morpholine, ephedrine, fur-

furylamine, choline, ethylenediamine or amino- ethanol.

The antithrombotic and antihyper ensive activity of a compound in view of a possible application in vascular pathology, also in hypertensive subjects, can be deduced from in vitro and in vivo experi¬ ments according to the following criteria:

1. effects on platelet aggregation in vitro

2. effects on certain leukocyte functions in vitro: studies on the adhesion and activation of poly orphonucleate leukocytes

3. effects in vivo in a model of arterial throm¬ bosis in dog . effects in vivo in models of arterial hyper- tension in rat.

The platelet antiaggregating activity of the new products according to the invention can be demonstrated by the following study on human platelets in the presence of various aggregating agents: adenosine diphosphate (ADP) , platelet activating factor (PAF) and arachidonic acid (AA) .

The study was performed with the following bicoumarin derivatives.

1) 1, 3-bis[3-(β-diethylaminoethyl)-4-methylcoumarin-7 -yloxy]propane hydrochloride

2) bis[3-(β-diethylaminoethyl)-4-methyl-coumarin-7- -yloxy]dicarbethoxy methane hydrochloride

3) bis[3-(β-diethylaminoethyl)-4-methyl-coumarin-7- -ylox ]carbethoxy methane hydrochloride 4) 1,3-bis[3-(β-diethylaminoethyl)-4-phenylcoumarin-7 -yloxy3propane hydrochloride 5) bis[3-(β-diethylaminoethyl)-4-methy1-8-chloro- coumarin-7-yloxy carbethoxy methane hydro¬ chloride

6) bis[3-(β-diethylaminoethyl) -4-methyl-8-chloro- coumarin-7-yloxy3dicarbethoxy methane hydro¬ chloride

7) 1,3-bis[3-(morpholinomethyl)-4-methyl-6, 8-di- llylcoumarin-7-yloxy3-2-hydroxypropane hydro¬ chloride

8) 1,3-bis[3-(morpholinomethyl)-4,8-di-methyl-6- -allylcoumarin-7-yloxy3propane hydrochloride

9) 1, 3-bis[3-(morpholinomethyl)-4-methyl-6-allyl-8- -chlorocoumarin-7-yloxy3-2-hydroxypropane hydrochloride

10) l,3-bis[3-(β-diethylaminoethyl)-4-methylcoumarin- 7-yloxy3-2-hydroxypropane hydrochloride

11) 1, 3-bis[3-(β-diethylaminoethyl)-4-methyl-8- -chlorocoumarin-7-yloxy3-2-hydroxypropane hydrochloride

The products were εolubilized in saline solution (lOμl) and added 1 minute before the aggregating agents. Concentrations ranging between 1-100 μM were tested.

Two specific antagonists for the PAF receptor were also tested, namely: L - 652,731 (Merck Sharp & Dohme) and SRI 63-675 (Sandoz Res. Inst.) .

Platelet aggregation

Blood was drawn from healthy volunteers who had taken no drugs for at least two weeks. It was gathered on 3.8% citrate in a ratio of 9:1. Plate¬ let rich plasma (PRP) was obtained by centrifuga- tion at 190 rpm for 15 minutes at room tempera¬ ture. Aggregation was induced by Bom's method (Born G. V. R. : Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature 194: 926-927, 1962), using an Ξlvi Logos 840

aggregometer.

The platelets were stimulated in the aggregometer with threshold concentrations of aggregating agent (TAC) so as to induce irreversible aggregation. (The threshold concentration is defined as the lowest concentration at which the biphasic wave has at least 60% optic density variation) . TAC was therefore calculated after incubation (37"C for 1 minute) of 250 μl of PRP while being constantly stirred at 1000 rpm and subsequent stimulation with the aggregating agents (ADP, PAF, A at concentrations of l-io μM, 100-500 nM and 0.7-3 M respectively).

Aggregation was then monitored for 3 minutes- The compounds were added to the PRP so as to obtain micromolar concentrations. 250 μl of PRP were incubated with the test products (37*C for 1 minute while being stirred) and then stimulated with the aggregating agents. Threshold inhibition concentrations (TIC) were assessed for each compound, that is, the lowest concentration able to inhibit aggregation induced by TAC of the various aggregating agents.

Results

The data obtained (Table 1) show that the test products were efficient platelet antiaggregating agents from concentrations of 0.1-2.5 μM. They were active against all the aggregating agents tested (unlike other antiplatelet drugs) .

Table 1 :

Effect of the bicoumarin derivatives on aggregation induced by various aggregating agents on human platelets, Values expressed as minimum concentrations (μM) able to inhibit aggregation induced by TAC of the aggregating agents.

(data are means of 3 or 4 replications for each experiment. The results were comparable to at least two other experi- mentε, performed on platelets from two different donors) .

The effects of the bicoumarin derivatives on some leukocyte functions can be studied in vitro by measuring the adhesion and activation of poly- morphonucleate leukocytes.

Aεsessments relative to two specific functions of PMN are reported hereafter:

1. PMN adhesion to endothelial cells (EC)

2. production of εuperoxide anion by PMN Materials and Methods

Test substance (solubilization and concentrations) Bicoumarin derivatives 1-4 of those previously listed were tested.

The products were solublized in saline immediately before the experiment. The cells (PMN) were then pretreated with increasing concentrations (0.1-500 μM) for 15 minutes at 37'C.

Indomethacin, nordihydroguaiaretic acid, BN 52021, diltiazem, dexamethazone and Ibuprofen were used for comparison.

Endothelial cells: preparation of the culture and treatment with interleukin-l ( L-1

Endothelial cells (EC) were isolated from umbilical cord and cultivated in culture medium 199 with a supplement of 20% fetal calf serum, in the presence of 50 μg/ml of specific growth supplement (Sigma Chemical) and 100 μg of pig intestine heparin. The cells were used at confluence (eighth passage) . In the experiments to assess adhesion, the growth medium was removed and the cells (1-1.5 x 10 ^s in a 2-cm ² well) were washed once with 1 ml of Hank's balanced saline solution (HBSS) and incubated at 37'C with 600 μl of 199 medium containing 0.25% of bovine serum albumin (BSA, Sigma) in the presence or absence of IL-1 (purified human natural IL-1, Ultrapure IL-1,

Genz me) , 10 U/ml. After 4 hours at 37 ^* C the medium was removed, the cells washed twice with 1 ml HBSS -f- 0.25% BSA and used to test adhesion.

PMN: preparation and labelling

Immediately before the experiment, PMN were removed from whole, anticoagulated blood taken from healthy volunteers, using dextran sedimentation and Ficoll-Hypaque gradients (Zimmermann G. A. et al.: Granulocyte adherence in pulmonary and systemic arterial blood samples from patients with adult respiratory distress syndrome. A . Respir. Pis. 129: 798-804, 1984). The isolated PMN (95-100%) (10 ⁷ cells/ml) were suspended in HBSS, in the absence of Ca/Mg, containing HEPES 20 mM (pH 7.4) and labelled for 15 minutes at room temperature with 5 μCi/ml 111 Indium-oxine (Amersham) . The cells were radiolabelled and then washed twice with HBSS, in the absence of Ca/Mg, containing 0.25% of BSA and resuspended in the same buffer.

PhfN-EC adhesion test

Adhesion was assessed by incubating endothelial cells (in the presence or absence of IL-1) with known quantities of labelled PMN (500 μl of suspension of PMN at a final concentration of 1.5 x 10 ⁶ PMN/dish) .

10 μl aliquots of CaCl ₂ and MgCl ₂ (final concentrations of 1-1.5 mM, respectively) were added immediately and the cells were incubated for 15 minutes at 37 ^* C. At the end of incubation the upper phase was carefully removed, the dishes washed twice with 1 ml of HBSS + 0.25% BSA to remove the non-adhered PMN, incubated for at least 10 minutes with 250 μl of NaOH M + 1% SDS (sodium-dodecylsul ate) .

The radioactivity associated with the cells was assessed by means of a gamma counter.

Superoxide anion production (O.,", bv stimulated PMN

Experiments were performed in two different conditions of PMN stimulation: - PMN stimulated by TPA (12-o-tetradecanoyl-

-phorbol-13-acetate)

P N stimulated by FMLP (N-formylmethionyl-

-leucyl-phenylalanine) .

The εuperoxide anion (0 ₂ ^" ) was measured by cytochrome C reduction, according to the method described by Johnston (Johnston R. : Secretion of superoxide anion. Methods Stud. Mononucl. Phago¬ cytes. : 489-497, 1981) modified by Del Maschio (Del Maschio et al. : Measurement of ionized cytoplasmic calcium mobilization with the photoprotein aequorin in human polymorphonuclear leukocytes activated by platelet activating factor (PAF) . Journal of lipid mediators 1, 25-36, 1989) and then assessed by a colorimetric test. In brief, PMN reεuspended in HBSS (in the presence of Ca ^2* and Mg ^2* ) were preincubated at 22 ^* C for 5 min in the presence of cytochrome C (10 mg/ml) and then exposed to the agonist for 40 minutes. The cell suspension was centrifuged (1 minute) in Eppendorf vials and aliquots of the upper phases (200 μl) were transfered to a 96-well dish. Absorbance was measured at 550 and 540 n simultaneously with a Multiskan spectrophotometer (Titertek, Flow Laboratories, Scotland) and the reduction in cytochrome C was calculated in relationship to the complete reduction in cyto¬ chrome C induced by dithionite.

The PMNs were stimulated by 0.5 μg/ml of the tumor promotor 12-0-tetradecanoyl-phorbol-13-acetate (TPA Sigma) or by FMLP (10 ^*7 -10 ^" *M) .

Results Preincubation of the PMNs with the bicoumarin derivatives tested was clearly effective in reducing adhesion of these cells to the cultured endothelium (both in basal conditions and after exposure to a strong stimulant such as IL-1) . Moreover, the bicoumarin derivatives tested reduce superoxide anion production induced in human PMN'ε both by the chemotactic peptide FMLP and by the phorbol ester. In particular:

1. PMN adhesion to the endothelial cells (± IL-1 The data (Table 2) on the effect of preincubation of PMN with compounds 1 and 4 indicate that: compound 1 proved to be active both on control endothelium and that treated with IL-1. compound 4 induced total inhibition at -500 μM. 2. Superoxide anion production <O _z ^~ ) by stimulated PMN The data obtained indicate that

0 ₂ " production from TPA-εti ulated PMN (Table 3) was markedly inhibited after preincubation with compound 1 and compound 4

Comparison substances such as: indomethacin, nordihydroguaiaretic acid, BN 52021, diltiazem, dexa ethasone proved inactive up to 500 μM. θ ₂ ^' production from FMLP-stimulated PMN (Table 4) was inhibited after preincubation with compound 4.

Table 2 :

Effect of bicoumarin derivatives on PMN adhesion to endothelial cells treated or not treated with IL-1

Inhibitors + IL-1 - IL-1

% of inhibition

-7 -10

21

21 ^• 55

68

94

The endothelial cells were incubated (4 hrs at 37*C) with medium containing 10 u/ml of IL-1 or the equivalent volume of saline, then washed and used for the adhesion test. The labelled PMN treated with saline or increasing concentrations of product (15 min at 37*C) were incubated with the ndothelial cells (+ IL-1) . PMN adhesion was assessed after 15 minutes at 37 ^* C (see Materials and Methods) .

Table 3 :

Effect of the bicoumarin derivatives on TPA-induced superoxide production in polymorphonucleate leukocytes .

% of inhibition

50 50

0 0

0 0 0 0 100

The PMNs were activated with TPA (0.5 μg/ml) and the superoxides measured 40 minutes later (see Materials and Methods) Data are means of 3 or 4 replications per experiment (as indicated in Table 1)

Table 4 :

Effect of the bicoumarin derivatives on superoxide production induced by FMLP in polymorphonucleate leukocytes (expressed as % of inhibition) .

Compound μM FMLP 10 -- FMLP 10 - ⁷ M

10%

100%

100% ^• 100%

The PMNs were incubated for 15 minutes at 37 ^β C with saline or increasing concentrations of the compound. FMLP ( lO^-lO ^' -^) was then added and the 0 ₂ " production was measured after 40 minutes (see Materials and Methods)

Data are means of 3 or 4 replications per experiment (as indicated in Table 1)

Effects in vivo of the bicoumarin derivativeε in an arterial thrombosis model in dog

Objective The objective of this experiment was to assess the antithrombotic action of some bicoumarin derivatives in an acute model of peripheral thrombosis in dog. In particular, the experiment focused on the efficacy in inhibiting arterial platelet thrombus formation (induced by critical stenosis of the femoral artery) which is the cause of this thrombotic process and consequent ischemia.

The pharmacological characterization of this mo-del has already been described (Prosdocimi M. et al.: Stenosis and vascular damage as a cause of thrombosis in dog femoral artery. Naunvn-Schmiedeberg's Arch. Pharmacol.: 338: 430-437, 1988). The close causal relationship between arterial thrombosis and the onset of acute occlusion of various kinds of arteries including the coronarieε makes it a very interesting model (Davies M. J. et al. : Thrombosis and acute coronary-artery lesions in sudden cardiac ischemic death. N. Engl.' J. Med. : 310: 1137-1140, 1984). It is therefore highly predictive of the therapeutic application of new drugs in arterial thrombosis. Materials and Methods

Test substances (solubilization and concentrations) Bicoumarin derivatives 1, 4, 5 were tested. The products were solubilised in saline and administered intravenously (i.v.) in εingle doses (0.5 - 3 mg/kg) .

Compariεon drugs were ASA, chlorpromazine, Ketanserin, dazmegrel, heparin, dipyridamole, and prazosin (versuε saline) . In vivo experiments Male beagle dogs (weighing 9-12 kg) were used in a total of 79 experiments. The animals were anesthetized with pentobarbital at an initial dose of 35 mg/kg i.v. followed by slow i.v. infusion to ensure constant anesthesia. All the animals were artificially fanned with air to maintain a constant level of 3.5% C0 ₂ in the exhalant, which was constantly monitored with a gas analyzer (LB2,- Beckman Instruments) . Catheters were inserted into the femoral veins of both hindlimbs for administration of anesthetic and drug and withdrawal of blood samples (Gould USA) . ECG was monitored by subcutaneous electrodes and the heartbeat with a cardiotachometer. These variables were recorded on a polygraph and visualized on a screen (Battaglia Rangoni, Italy) .

About 2 cm. of the femoral artery were isolated from the surrounding tissueε. An electromagnetic transducer was placed around the artery (2.0-2.5 mm) and the vessel was stenosed with a plastic tube (Lexan). The inner diameter of the tube measured 1.6-1.8 mm. Whenever this procedure failed to cause the desired cyclic blood flow variations (CBFV) , the vessel was compressed for 10 minutes with a clamp, as damage to the vesεel endothelium favors thrombi formation. The cylinder was placed at the site of compression and in each case CBFV was subsequently observed. The test products were administered 30 minutes after stabilization of the CBFV _*

The effects of the products were quantified as scores on a fixed scale of 0 to 4 (see Table 5) as described in the literature (Aiken J. W. et al.: Endogenous prostacyclin contribvites to the efficacy of a thro boxane synthetase inhibitor for preventing coronary artery thrombosis. J. Pharmacol. Exp. Ther. 219: 299-308, 1981).

Results The results obtained (Table 5) indicate that the compounds tested reduce thrombi formation. A marked antithrombotic efficacy was observed in the case of compound 4 (100% responders after 0.5 mg/kg i.v.) and compound 1 (albeit to a slightly lesser degree: 90% responders after 3 mg/kg i.v.).

Table 5:

Antithrombotic effect of the bicoumarin derivatives

No. indicateε the number of experiments * corresponds to 50 I.U./kg

Score 0 •== no effect

Score 1 = slight reduction in occlusion frequency Score 2 - reduction in occlusion frequency

Score 3 = reduction in occlusion frequency and increase in minimum flow observed Score 4 = no occlusion

Effects in vivo of bicoumarin compound 1 in models of arterial hypertension in rat

Objective: The antihypertensive activity of compound 1 in two specific models of arterial hypertension was assessed to confirm the results of the preliminary screening of the test product.

In particular, the effects of 1 were studied in the following experimental conditions:

1. model of hypertension induced by renal stenosis (Goldblatt's model)

2. model of hypertension induced by deoxycorticosterone acetate (DOCA) .

Materials and Methods Test: substance (solubilizations and concentrations)

Compound l was tested in comparison to papaverine. The products were suspended in 0.5% aqueous tragacanth and administered orally (os) at a doεe of 100 mg/kg/die (in a volume of 10ml/kg) . Model of ,hypertension induced by renal stenosis (Goldblatt's model)

30 male Wistar rats (120-140 gr) were used, having been rendered hypertensive by means of stenosis according to the method described by Goldblatt et al . (Goldblatt H. et al. : Studies on experimental hypertension. I. The production of persistent elevation of systolic blood pressure by means of renal ischaemia. J. Exo. Med.. 59, 347-379, 1934). After anesthesia, an incision was made in the lumbocostal region and the left kidney was displaced towards the abdomen. The renal pedunculus was exposed, the artery isolated and compressed

with a clamp in the vicinity of the abdominal aorta. The right kidney was removed through a second incision. The wall was sutured, the skin incisions closed with clips and the animal was left to recover.

All animals were then treated i.ui. with 30,000 TI of benzyl penicillin procaine and per os for 4 consecutive days with 100 mg/kg/day of compound 1 in parallel with papaverine. Systolic blood pressure was measured on the 1st and 4th days, before and afte.r treatment (1st and 4th hrs) . (Only those animals with systolic blood ^' pressure of over 150 m Hg were included in the study) Model of hypertension induced by deoxycortisone acetate (DOCA)

" 30 male Wistar rats weighing 90-120 g, were rendered hypertensive by a method similar to that described by Green et al. (Green D. M. , Saunders F. J. , Wahlgren N., Craig R. L. : Self-sustaining, post-DCA hypertensive cardiovascular diseaεe. Am. J. Phvsiol.: 170, 94-106, 1952). An incision was effected under an anesthetic on the left side of the abdominal wall, ^" and the left kidney was removed. A 50-mg DOCA pellet was implanted ε.c.. The abdominal wall was sutured, the skin incision closed with clips and the animal left to recover. Drinking water was substituted with a solution containing 0.8% of sodium chloride and 0.1% of potassium chloride.

The animals then underwent treatment and systolic blood pressure readings, as previously described (Goldblatt's model).

Results The data obtained show an interesting anti-hypertensive activity of compound 1, in particular: 1. Renal εtenosiε model (Goldblatt)

As reported in Table 6, compound 1 effectively reduces pressure values. This effect was observed on all readings made on the fourth day of the study. Papaverine, on the other hand, had only a modest, not significant, effect on all readings made after treatmen .

2. Model of hypertension induced by DOCA In this model too, treatment with compound 1 proved equally active, with a reduction in pressure values on the 4th day (Table 7).

TE SHEET

Table 6:

Effect of repeated doses of compound 1 and papaverine on systolic blood pressure of rats rendered hypertensive according to Goldblatt's method

Group Treatment Mean systolic blood pressure (mmHg) (10)* (Eig/kg) per group on

in brackets the number of animals per group

Table 7:

Effect of repeated doses of compound 1 and papaverine on systolic blood pressure of rats rendered hypertensive by administration of DOCA/saline

Group Treatment Mean blood pressure ( mHg) per (10)* (mg/kg) croup on

Day 1 ay 4

Pre-dose 1 h 4 h Pre-dose 1 h 4 h

1 Vehicle 206 218 205 204 209 206

2 comp. 1 (100) 203 197 198 158 179 174

3 Papaverine(100) 201 184 191 186 193 203

* in brackets the number of animals per group

The novel bicoumarin derivatives of formula I of the invention can be prepared in the known way, and more precisely by a - rocedure which comprises treating a 7-hydroxy-coumarin of the. formula:

in which R^ ^ are the same substituents as defined for formula I or groups convertible thereto or a metal salt, with a compound of the formula:

Z.-X-Z ₂ (IV)

where Z, and Z ₂ are the same or are different from each other, and each represents a reactive group with respect to phenol etherification (al ylation) and -X- has the same εignificance as in formula I or it signifies a substituent convertible in the same, or treating the aforesaid cou arin compound III with a compound of the formula:

Z^ -Z- _j (V)

where Z ₁ and -X- have the same significance aε in the previous case and Z3 stands for a non-reactive group with regard to phenol etherification (alkylation) , but is convertible to a reactive group with regard to this reaction, or a hydrogen atom of the unsaturated H-C= structure of the terminal hydrocarbyl group of -X- , and then converting Z ₃ in the obtained compound of the formula:

to a reactive group with regard to phenol etherification, and treating the coumarin compound obtained with a 7-hydroxy-coumarin of the of formula:

or one of its metal εalts, in which R ₆ -R ₁₀ have the same εignificance as the _! -R ₅ substituents of formula III, but are not necessarily identical to them, and converting the obtained bicoumarin compound R*,-R ₁₀ and -X- substituents, differing from those corresponding to their significance in the compounds of formula I, into substituents of the same formula, and if desired, converting the products obtained into their metal or acid addition salts, or into their quaternary ammonium salts.

The procedure which involves treating the compound of formula III with the compound of formula IV in one single step is preferred when bicoumarin com¬ pounds of formula I having a symmetrical structure are to be prepared, in which substituents ^Rs are identical to substituents R ₆ -R ₁₀ . In this case stoichio etric quantities in a ratio of 2 to 1 of compounds III and IV are used.

To obtain compounds with an asymmetric structure, in which at least one of the substituents R ₆ -R ₁₀ differs from the corresponding substituent among R- _j -Rs the compound of formula III is etherified with the compound of formula V, in which Z ₃ is a non-reactive group with respect to phenol etherification, but convertible into a reactive group. In a second step, when the Z ₃ group has been converted into the reactive group, the coumarin compound, obtained with the 7-hydroxy-coumarin of formula VII, is etherified. To obtain asymmetrical compounds it is also possible to exploit the different reactivity of groups Z^ and Z ₂ by treating compound III with reactive compound IV, etherifying in a first step in a stoichiometric ratio of about 1 to 1, or in higher ratios, that is, with an excess of compound III, to obtain subεtantially the compound of the formula:

which can be iεolated from the reaction mixture, and then itself etherified with a 7- hydroxy-cou arin of type VII, different from the compound of formula III. A reactive group with regard to phenol etherification, such as Z, and Z ₂ , may be a reactive functional group and as such it is essentially a functionally modified hydroxy group, especially a hydroxy group esterified with appropriate acids,

known in the literature, for example with hydracids or with inorganic or organic oxygenated acids, for example εulfonic acids. The esters with the hydracids are alkylene halogenides, especially bromides, iodides and chlorides of the Br-X-Br type, the two reactive groups optionally differing one from the other. Of the oxygenated esters should be mentioned the esters of sulfuric or εulfurous acid, and especially the esters with alkyl- or aryl-sulfonic acids, such as methanesulfonic or p-toluenesulfonic acid.

Some terminal epoxide groups present in the -X-radical between the terminal carbon atom and the one next to it are also reactive* groups which may react with the phenol group of 7-hydroxycoumarin to form the ether and a hydroxyl group at the carbon atom next to the terminal one. The phenol etherification reaction is effected under known conditions which depend above all on the nature of the reactive groups Z, and Z ₂ and on the fact that the 7-hydroxycoumarin≤ themselves or their metal salts are used as starting compounds, such as in particular the alkaline metal salts, especially sodium, potaεεiu or cesium salts. Starting with the εalts it is possible to etherify with the compound Z^X-Z.,, Z ₁ and Z ₂ being reactive functional groups, in a neutral organic solvent, such as an alcohol, an ether, for example ethyl or methyl alchol, or dioxane or tetrahydrofuran or a ketone, such as acetone or methyl ethyl ketone, or in amides, such as dimethylformamide, or sulfoxides, such as dimethylsulfoxide, or N-methylpyrrolidone, at room temperature or more and especially at a higher temperature, such as between

E-E

50* and 150"C. In this case ^" alkylene halogenides of the Br-X-Br type or the corresponding chlorides or the corresponding compounds having only one reactive group substituting the X are preferable. Starting with the 7-h droxycou arins, the etherification reaction is performed with said reactive functional groups in the presence of a basic compound, such as especially an inorganic base, for example an alkaline hydroxide or an alkaline carbonate, or a suitable azotated base, such as pyridine or collidine in one of the aforesaid solvents. This is especially true when said alkylene halogenides are used or also the esters of oxygenated acids, such as alkyl- or arylsulfonateε.

A non-reactive Z ₃ group substituting the alkylene -X- radical, but convertible into a reactive group, is for example a hydroxyl group, free or esterified with particular acids, such as ^' mono-esters of carbonic acid, such as the benzyloxycarbonyl group. Once they have been introduced into the 7-hydroxycoumarin molecule by the -X- radical, such groups can be converted into said reactive functional groups for example in the known way, e.g., in the case of the benzyloxycarbonyl esters by reduction in the known way and subsequent function conversion at the hydroxy group, for example by conversion into tos late.

Z ₃ may also stand for the hydrogen atom of an unsaturated H-c= structure of the terminal

I hydrocarbyl group of the -X- radical. This unsaturated structure may be converted into the

I i

H-C--C- epoxide group in the known way, for example b

by reaction with peroxides, ^" such as hydrogen peroxide or organic peracids, such as perbenzoic acid or perphthalic acid, thus obtaining a group capable of etherifying (alkylating or arylating) the phenol group in the 7-position of the coumarin compound. Conversion of said unsaturated compound in the epoxide compound can be effected preferably in a neutral organic solvent such as an ether or in an aromatic hydrocarbon, for example benzene or toluene. After conversion of Z ₃ into a reactive group, etherification of the second stage is; effected with a compound of formula VII under the conditions described above.

In the bicoumarins obtained according to the described procedure, R-R-*,*. groups and -X- radicals are converted, which are modifications of the groups intended for the desired compounds, for example functional derivatives of substituents present in such groups, in the intended groupε. Thus, for example, free hydroxy or carboxy groups present in said substituents may be functionally modified at the end of the procedure, for example esterified or etherified. This is also true for the R.,-R*, ₀ substituents, wherever theεe represent functional groupε, for example carboxy groups.

Vice-versa, modified functional groups can be converted into free functional groups. .Amino groups can be alkylated and also converted into quaternary ammonium salts. All of these reactions can be performed in the knov/n way.

The bicoumarin compounds obtained by the aforesaid procedure can be converted into their salts. This can be done in the known way, for example by preparing metal salts of compounds containing

carboxy functions by treatment with set quantities of a hydroxide or alkaline carbonate or of an organic base, for example one of those mentioned above. Compounds which present amino functions in the alkylene -X- radical can be transformed into salts obtained by acid addition, for example those mentioned above, or into quaternary ammonium salts. The invention also includes modifications of the aforesaid procedure, in which it is interrupted at any one stage or in which one starts with an intermediate compound and the remaining steps are carried out, or in which the starting products ^' are formed in situ.

Another object of the present invention is directed to pharmaceutical preparations containing as active substance one or more of the new bicoumarin derivatives or their salts, and in particular those mentioned above. Such pharmaceutical preparations can be for oral, rectal, parenteral, local or tranεdermal use. They can therefore be in solid or semisolid form, for example pills, tablets, gelatin capsules, capsules, suppositories, or soft gelatin capsules. For parenteral use it is possible to use those forms intended for intramuscular, subcutaneous or transdermal administration, or suitable for infusions or intravenous injections and can therefore be presented as solutions of the active compounds or as freeze-dried powders of the active compounds to be mixed with one or more pharmaceutically acceptable excipients or diluents, convenient for the above uses and with an osmolarity compatible with physiological fluids. For local use, preparations in the form of sprays, creams or

ointments for topical use may be employed, or suitably treated sticking plasters for transdermal administration. The preparations of the invention can be administered to humans or animals. They contain preferably about 0.01 to 10% by weight of active component for the solutions, sprays, ointments and creams, and between 100% and preferably between 5 and 50% by weight of the active compound for the preparations in solid form. Doses to be administered depend on individual needs, on the desired effect and on the chosen route of administration. An average daily dose of 10-30 mg by the intravenous route or 100-400 mg by the oral route is usually advised for humans for the treatment of vascular pathologies (of a thrombotic or hypertensive nature) . The following disorders are particularly indicated in connected with the present invention: peripheral vasculopathies, arteriopathies of the lower limbs, anginal complaints and cerebral vasculopathies. The following Examples illustrate the invention:

EXAMPLE 1:

1,3-bis[3-(β-diethylaminoethyl)-4-methylcoumarin-7-

-yloxy3propane- hydrochloride 70.0 g of AD 112 3-(β-diethylaminoethyl)-4-methyl-

-7-hydroxycoumarin are suspended in 500 ml of ethanol and to this are added 28.5 g of 50% KOH in water. It is left to stand for 1 hour at 50*C, then concentrated and vacuum-dried. It is gathered with 500 ml of 2-butanone and 70.3 g of K ₂ CO _j are added. It is heated while being shaken, and then 25.7 g of 1,3-dibromopropane are added and it is left to react for 12 hours.

The solvent is evaporated and the residue is dissolved with 500 ml of ethyl acetate; it is then washed with a IN solution of NaOH. The solvent is anhydrated and concentrated. The product is purified by chromatography with Prep LC System Waters, using as eluent a mixture of CHCl ₃ -CH ₃ OH-NH<OH (30%) in a gradient range of 95:5:0.2 to 70:30:0.2. The pure fractions are concentrated and crystallized from toluene. The crystallized product is dissolved in ethyl acetate, anhydrated and treated with HCl in ethanol until a Congo red indicator change. It is filtered and crystallized from isopropyl alcohol. Thiε is filtered and vacuum-dried, obtaining 42.2 g of a compound to which elementary analysiε (C,H,N) and nuclear magnetic reεonance εpectroεcopy (protons) both attribute a structure of 1,3-bis[3-(β-diethylaminoethyl)-4-methylcoumarin-7- -yloxy3propane hydrochloride.

EXAMPLE 2:

1,3-bis[3(.β-diethylaminoethyl)--4-methyl-8-chloro- coumarin- -yloxy3propane hydrochloride

70.0 g of 3-(β-diethylaminoethyl)-4-methy1-7-hy- droxy-8-chlorocoumarin are suspended in 500 ml of ethanol and to this are added 25.4 g of 50-% KOH in water. It is left to stand for 1 hour at 50 ^* C and then concentrated and vacuum-dried. It is gathered with 500 ml of 2-butanone and to this are added 31.2 g of I-^CO _j . It is heated while being shaken, and then to this are added 22.8 g of 1,3-dibromopropane and it is left to react for 12 hours.

The solvent is evaporated and the residue is dissolved with 500 ml of ethyl acetate. It is washed with a IN solution of NaOH. The solvent is anhydrated and concentrated. It is purified by chromatography with Prep LC System Waters, using as eluent a mixture of CHCl ₃ -CH ₃ OH-NH ₄ OH (30%) in a gradient range of 95:5:0.2 to 70:30:0.2. The pure fractions are concentrated and crystallized from toluene.

The crystallized product is dissolved in ethyl acetate, anhydrated and treated with HC1 in ethanol until a Congo red indicator change. It is filtered and cryεtallized from isopropyl alcohol. This is filtered and vacuum-dried, obtaining 36.4 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance εpectroscopy (protons) both attribute a εtructure of

1,3-bis[3 (β-diethylaminoethyl)-4-methyl-8-chloro- cou arin-7-yloxy3propane hydrochloride.

EXAMPLE 3:

1, 3-bis[3-(β-diethylaminoethyl)-4-phenylcoumarin-7-

-yloxyjpfopane hydrochloride

63.5 g of 3-(β-diethylaminoethyl)-4-ρhenyl-7-hy- droxycou arin hydrochloride are suspended in 500 ml of ethanol and to this are added 38.0 g of 50% KOH in water. It is left to stand for 1 hour at 50 ^* C and then concentrated and vacuum-dried.

It is gathered with 500 ml of 2-butanone and to this are added 23.5 g of K ₂ C0 ₃ . The resultant is heated while being shaken, and then to this are added 17.1 g of

1,3-dibromopropane and it is left to react for 12 hours.

The solvent is evaporated and the residue is dissolved with 500 ml of ethyl acetate. It is

washed with a IN solution of NaOH. The solvent is anhydrated and concentrated. It is purified by chromatography with Prep LC System Waters, using as eluent a mixture of CHC^-CH-OH-NH^OE (30%) in a gradient range of 98:2:0.2 to 85:15:0.5. The pure fractions are concentrated and crystallized from toluene.

The crystallized product is dissolved in ethyl acetate, anhydrated and treated with HCl in ethanol until a Congo red indicator change. It is lyophilized, obtaining 9.2 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a structure of 1,3-bis[3-(β-diethyl-aminoethyl)-4-phenyl- coumarin-7-yloxy]propane hydrochloride.

EXAMPLE 4:

1,3-bis[3-(morpholinomethyl)-4-phenyl-6-chloro-8-

-methylcoumarin-7-yloxy3propane hydrochloride 30.0 g of 3-morpholinometh l-4-phenyl-6-chloro- -7-hydroxy-S-methylcoumarin) are suspended in 500 ml of ethanol and to this are added 8.7 g of 50% KOH in water. It is left to stand for 1 hour at 50 ^e C and then concentrated and vacuum-dried. It is gathered with 500 ml of 2-butanone and to this are added 13.9 g of K ₂ C0 ₃ . It is heated while being shaken, and then to this are added 7.8 g of 1,3-dibromoproρane (0.0389 mole) and it is left to react for 12 hours. The solvent is evaporated and the residue is dissolved with 500 ml of ethyl acetate; it is washed with a IN solution of NaOH. The solvent is anhydrated and concentrated, and the product is crystallized from toluene-

-45-

The crystallized product is dissolved in ethyl acetate, anhydrated and treated with HCl in ethanol until a Congo red indicator change. It is filtered and crystallized from CH.OH-H ₂ 0 90:10. This is filtered and vacuum-dried, obtaining 4.8 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroεcopy (protons) both attribute a structure of 1,3-bis[3- -(morpholinomethyl)-4-phenyl-6-chloro-8-methyl- coumarin-7-yloxy)propane hydrochloride.

EXAMPLE 5:

1, 3-bis[3-(morpholinomethyl)-4-methyl-6,8-diallyl- coumarin-7-yloxy propane hydrochloride 20.0 g of 3-morpholinomethyl-4-methyl-6,8-diallyl- -7-hydroxycoumarin) are εuspended in 200 ml of ethanol and to this are added 6.3 g of 50% OH in water. It is left to stand for 1 hour at 50 ^β C and then concentrated and vacuum-dried. The resultant is gathered with 200 ml of 2-butanone and to this are added 7.7 g of K ₂ C0 ₃ . It is heated while being shaken, and then to this are added 5.65 g of 1,3-dibromopropane and it is left to react for 12 hours.

The solvent is evaporated and the residue is dissolved with 200 ml of ethyl acetate; it is washed with a IN solution of NaOH. The solvent is anhydrated and concentrated. It is purified by chromatography with Prep LC system Waters, using as eluent a mixture of CH ₂ Cl ₂ -EtAc-CH ₃ OH in a gradient range of 85:15:0 to 80:20:0.5. The pure fractions are concentrated. The residue is gathered in ethyl acetate, anhydrated and treated with HCl in ethanol until a Congo red indicator change. It is filtered, washed and vacuum-dried, giving 12.4 g

of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a structure of 1,3-bis[3-(morpholinomethyl)-4-methyl-6,8-di- allylcoumarin-7-yloxy] ropane hydrochloride. EXAMPLE 6:

1,3-bis[3-(morpholinomethyl)-4,8-dimethyl-6-allyl- coumarin-7-yloxy3propane hydrochloride

16.0 g of 3-morpholinomethyl-4,o-dimethyl-6-allyl- -7-hydroxycoumarin are suspended in 200 ml of ethanol and to this are added 5.4 g of 50% KOH in water. It is left to stand for 1 hour at 50 ^* C and then concentrated and vacuum-dried. It is gathered with 200 ml of 2-butanone and to this are added 6.6 g of K _g CO--,. It is heated while being shaken, and then 4.85 g of

1,3-dibromopropane are added and the solution is left to react for 12 hours.

The solvent is evaporated and the residue is dissolved with 200 ml of ethyl acetate; it is washed with a IN solution of NaOH. The solvent is anhydrated and concentrated. It is purified by chromatography with Prep LC System Waters, using as eluent a mixture of CH ₂ Cl ₂ -EtAc-CH ₃ OH 70:30:5. The pure fractions are concentrated and crystallized from ethyl acetate.

The crystallized product is dissolved in warm ethyl acetate, anhydrated and treated with HCl in ethanol until a Congo red indicator change. It is filtered and washed in ethyl alcohol. This is filtered and vacuum-dried, obtaining 5.0 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance εpectroscopy (protons) both attribute a structure of l,3-biε[3-(morpho-linomethyl)-4,8- dimethyl-6-allylcoumarin-7-ylox 3-propane hydrochloride.

EXAMPLE 7 :

1,3-bis[3-(morpholinomethyl)-4-methyl-6-chloro-8-

-allylcoumarin-7-yloxy3propane hydrochloride

20-0 g of 3-morpholinomethyl-4-methyl-6-chloro-7- -hydroxy-8-allylcoumarin are suspended in 200 ml of ethanol and to this are added 6.4 g of 50% KOH in water. It is left to stand for 1 hour at 50*C and then concentrated and vacuum-dried. It is gathered with 200 ml of 2-butanone and to this are added 7.9 g of K ₂ C0 ₃ . It is heated while being shaken, and then to this are added 5.75 g of 1,3-dibromopropane and it is left to react for 12 hours.

The solvent is evaporated and the residue is dissolved with 200 ml of ethyl acetate; it is washed with a IN solution of NaOH. The solvent is anhydrated and concentrated. It is crystallized from acetone until a clean product is obtained. The crystallized product is disεolved in ethyl acetate, anhydrated and treated with HCl in ethanol until a Congo red indicator change. It is filtered and crystallized from ethyl alcohol. This is filtered and vacuum-dried, obtaining 6.7 g of a compound to which elementary analysis (c,H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a structure of l,3-bis[3-(morpho-linomethyl)-4-methyl-6- chloro-8-allylcoumarin-7-yloxy3propane hydrochloride.

EXAMPLE 8:

1,3-bis[3-(morpholinomethyl)-4,8-dimethyl-6-chloro- coumarin-7-ylox ]propane hydrochloride

15.0 g of 3-morpholinomethyl-4,8-dimethyl-6- -chloro-7-hydroxycoumarin are suspended in 200 ml of ethanol and to this are added 5.2 g of 50% KOH in water. It is left to stand for l hour at 50 ^* c and

then concentrated and vacuum-dried. It is gathered with 200 ml of 2-butanone and to this are added 6.4 g of K _j CO- _j . It is heated while being shaken, then to this are added 4.8 g of 1,3-dibromopropane and it is left to react for 12 hours.

The solvent is evaporated and the reεidue is dissolved with 200 ml of ethyl acetate? it is washed with a IN solution of NaOH. The solvent is anhydrated and concentrated. It is purified by chromatography with Prep LC System Waters, using as eluent a mixture of CHCl ₃ --EtAc-CH ₃ OH 70:30:2. The pure fractions are concentrated and crystallized from ethyl acetate.

The crystallized product is dissolved in ethyl acetate-chloroform, anhydrated and treated with HCl in ethanol until a Congo red indicator change. It is filtered and crystallized from ethyl alcohol- Thiε is filtered and vacuum-dried, obtaining 4.0 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance εpectroscopy (protons) both attribute a structure of l,3-bis[3- -(morpholinomethyl)-4,8-dimethyl-6-chlαrocoumarin- -7-yloxy3propane hydrochloride.

EXAMPLE 9: l,3-bis[3-(morpholinomethyl)-4-methyl-6-allyl-8- -chlorocoumarin-7-yloxy3propane hydrochloride

22.0 g of 3-morpholinomethyl-4-methyl-6-allyl-7- -hydroxy-8-chlorocoumarin are suspended in 200 ml of ethanol and to this are added 7.1 g of 50% KOH in water. It is left to stand for 1 hour at 50*C and then concentrated and vacuum-dried. It is gathered with 200 ml of 2-butanone and to this are added 8.7 g of K ₂ C0 ₃ . It is heated while being shaken, and then to this

are added 6.5 g of 1,3-dibromopropane and it is left to react for 12 hours.

The solvent is evaporated and the residue is dissolved with 200 ml of ethyl acetate; it is washed with a IN solution of NaOH. The solvent is anhydrated and concentrated. It is purified by crystallization from CH ₃ OH-EtAc.

The crystallized product is dissolved in ethanol and treated with HCl in ethanol until a Congo red indicator change. It is filtered and crystallized from ethyl alcohol. This is filtered and vacuum-

-dried, obtaining 5.0 g of a compound to which ^' elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a structure of 1,3-bis[3-(morpholinomethyl)-4-

-methyl-6-allyl-8-chlorocoumarin-7-yloxy3propane hydrochloride.

EXAMPLE 10: l,3-bis[3-(morpholinomethyl)-4-phenyl-6-chloro-8- -allylcoumarin-7-ylox 3propane hydrochloride

40.0 g of 3-morpholinomethyl-4-phenyl-6-chloro-7-

I

-hydroxy-8-allylcoumarin are suspended in 500 ml of ethanol and to this are added 10.9 g of 50% KOH in water. It is left to stand for 1 hour at 50 ^* C and then concentrated and vacuum-dried. It is gathered with 500 ml of 2-butanone and to this are added 13.4 g of K ₂ C0 ₃ . It is heated while being shaken, and then to this are added 10.0 g of l,3-dibromopropane and it is left to react for 12 hours.

The solvent is evaporated and the residue is dissolved with 500 ml of ethyl acetate; it is washed with a IN solution of NaOH. The solvent is anhydrated and concentrated. It is purified by

chromatography with Prep LC System Waters, using as eluent a mixture of CH ₂ Cl ₂ -EtAc 80:20. The pure fractions are concentrated.

The residue is dissolved in ethyl acetate, anhydrated and treated with HCl in ethanol until a Congo red indicator change. It is filtered and washed. It is vacuum-dried, obtaining 4.0 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance εpectroscopy (protons) both attribute a structure of 1,3-bis[3- -(morpholinomethyl)-4-phenyl-6-chloro-8-allyl- coumarin-7-yloxy3propane hydrochloride.

EXAMPLE 11: 1,3-bis[3-(morpholinomethyl)-4-phenyl-6-all l-8- -methylcoumarin-7-yloxy3propane hydrochloride

50.0 g of 3-morpholinomethyl-4-phenyl-6-allyl-7- -hydroxy-8-methylcoumarin are suspended in 500 ml of ethanol and to this are added 14.3 g of 50% KOH in water. It is left to stand for 1 hour at 50 ^* C and then concentrated and vacuum-dried. It is gathered with 500 ml of 2-butanone and to this are added 17.6 g of l^CO- _j . It is heated while being shaken, then to thiε areadded 12.9 g. of 1,3-dibromopropane and it is left to react for 12 hours.

The solvent is evaporated and the residue is disεolved with 500 ml of ethyl acetate; it is washed with a IN solution of NaOH. The solvent is anhydrated and concentrated. It is purified by chromatography with Prep LC System Waters, using as eluent a mixture of CH ₂ Cl ₂ -EtAc-CH ₃ OH 70:30:10. The pure fractions are concentrated and crystallized from ethyl acetate/n-hexane 1:2.5.

The crystallized product is dissolved in ethyl acetate, anhydrated and treated with HCl in ethanol until a Congo red indicator change. It is filtered and crystallized from ethyl alcohol al 98%. This is filtered and vacuum-dried, obtaining 14.6 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a structure of 1,3-bis[3-(morpho¬ linomethyl)-4-phenyl-6-allyl-8—methylcoumarin-7- ylox 3propane hydrochloride.

EXAMPLE 12:

1,3-bis[3-(morpholinomethyl)-4-phenyl-6-allyl-8-

-chlorocoumarin-7- loxy3propane hydrochloride 30.0 g of 3-morpholinomethyl-4-phenyl-6-allyl-7- -hydroxy-8-chlorocoumarin are εuspended in 300 ml of ethanol and to thiε are added 8.2 g of 50% KOH in water. It is left to stand for 1 hour at 50°C and then concentrated and vacuum-dried. It is gathered with 300 ml of 2-butanone and to this are added 10.1 g of

.^CO _j . It is heated while being shaken, then to this are added 7.4 g of 1,3-dibromopropane and it is left to react for 12 hours.

The solvent is evaporated and the residue is dissolved with 300 ml of ethyl acetate; it is washed with a IN solution of NaOH. The solvent is anhydrated and concentrated. It is purified by chromatography with Prep LC System Waters, using as eluent a mixture of CH _∑ Cl ₂ -EtAc 90:10. The pure fractions are concentrated and crystallized from 95% ethanol.

The crystallized product is disεolved in ethyl acetate, anhydrated and treated with HCl in ethanol until a Congo red indicator change. It is filtered and

crystallized from ethanol/ chloroform 4 : 3 . This is filtered and vacuum-dried, obtaining 11.1 g of a compound to which elementary analysis (C, H, N) and nuclear magnetic resonance spectroscopy (protons) both attribute a structure of

1 , 3 -bis [ 3 - (morpholinomethyl) -4-phenyl-6-allyl- -8 -chlorocoumarin-7 -yloxy 3 propane hydrochloride.

EXAMPLE 13 : 1,3-bis[3-(morpholinomethyl)-4-phenyl-6,8-diallyl- coumarin-7-ylox 3propane hydrochloride

30.0 g of 3-morpholinomethyl-4-phenyl-6,8-diallyl- -7-hydroxycoumarin are suspended in 300 ml of ethanol and to this are added 7-8 g of 50% KOH in water. It is left to stand for 1 hour at 50"C and then concentrated and vacuum-dried. It is gathered with 300 ml of 2-butanone and to this are added 9.9 g of I^CO- _j . It is heated while being shaken, and then to this are added 7.2 g of 1, 3-dibromopropane and it is left to react for 12 hours.

The solvent is evaporated and the residue is dissolved with 300 ml of ethyl acetate; it is washed with a IN solution of NaOH. The solvent is anhydrated and concentrated. It is purified by chromatography with Prep LC System Waters, using as eluent a mixture of CH ₂ Cl ₂ -EtAc 80:20. The pure fractions are concentrated.

The residue is dissolved in ethyl acetate, anhydrated and treated with HCl in ethanol until a Congo red indicator change. ^' It is filtered and crystallized from isopropyl alcohol. This is filtered and vacuum-dried, obtaining 10.0 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a

εtructure of l,3-bis[3-(morpholinomethyl)-4-phenyl- 6,8-diallylcoumarin-7-yloxy3-propane hydrochloride.

EXAMPLE 14: 1, 3-bis[3-(β-morpholinoet.hyl)-4-methylcoumarin- -7-ylox 3propane hydrochloride

19.0 g of 3-(β-morpholinoethyl)-4-methyl-7- -hydroxycoumarin are suspended in 300 ml of ethanol and to this are added 5.5 g of 50% KOH in water. It is left to stand for 1 hour at 50*C and then concentrated and vacuum-dried. It is gathered with 300 ml of 2-butanone and to this are cidded 9.0 g of K _j CO- _j . It is heated while being shaken, and then to this are added 6.6 g of 1,3-dibromopropane (0-0328 mole) and it is left to react for 12 hours.

The solvent is evaporated and the residue is dissolved with 300 ml of ethyl acetate; it is washed with a IN solution of NaOH. The solvent is anhydrated and concentrated. It is crystallized from methanol.

The crystallized product is dissolved in ethyl acetate, anhydrated and treated with HCl in ethanol until a Congo red indicator change. It is filtered and washed. It is further filtered and vacuum-dried, obtaining 5.8 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a structure of l,3-bis[3-(β-roorpholino- ethyl)-4-methylcoumarin-7-yloxy]propane hydro- chloride.

EXAMPLE 15:

1,3-bis[3-(β-morpholinoethyl)-4-methyl-8-chloro- coumarin-7-yloxy3propane hydrochloride

30.0 g of 3-(β-morpholinoethyl)-4-methyl-7- -hydroxy-8-chlorocoumarin hydrochloride are suspended in 300 ml of ethanol and to this are added 18.7 g of 50% KOH in water. It is left to stand for 1 hour at 50*C and then concentrated and vacuum-dried. It is gathered with 300 ml of 2-butanone and to this are added 11.5 g of K ₂ C0 ₃ . It is heated while being shaken, and then to this are added 8.4 g of l,3-dibromopropane and it is left to react for 12 hours.

The solvent is evaporated and the residue is dissolved with 300 ml of ethyl acetate; it is washed with a IN solution of NaOH. The solvent is anhydrated and concentrated. It is washed with warm CH ₃ OH.

The product is dissolved in chloroform/ thyl ace¬ tate, anhydrated and treated with HCl in ethanol until a Congo red indicator change. It is filtered and washed. This iε filtered and vacuum-dried, obtaining 22.0 g of a compound to which elementary analyεis (C, H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a structure of l , 3-bis [ 3- (β-morpholinoethyl) -4-methyl-8- -chlorocoumarin-7-yloxy3 propane hydrochloride.

EXAMPLE 16 :

1,6-bis[3-(β-morpholinoethyl)-4-methylcoumarin-7-

-yloxyjhexane hydrochloride

25.0 g of 3-(β-morpholinoethyl)-4-methy1-7- -hydroxycoumarin sulfate are suspended in 500 ml of ethanol and to this are added 22 g of 50% KOH in H ₂ 0. It is left to stand at 50"C for 1 hour and then concentrated and vacuum-dried. It is gathered in 2-butanone and to this are added 9.0 g of K^O- _j . It is

heated while being shaken. To this are then added 8.0 g of 1.6 dibromohexane and it is left to react for 20 hours. The solvent is evaporated and the residue is dissolved with 500 ml of CHC1 ₃ and washed with H ₂ 0. The solvent is concentrated and the product precipitated by adding methanol. It is gathered with a few ml of CHC1- and it is reprecipitated with ethyl acetate (EtAc) . It is dissolved in a mixture . of chloroforra/methanol and HCl in ethanol is added until a Congo red indicator change.

The product is concentrated slightly and precipitated by adding ethyl acetate. This is filtered and vacuum- -dried, obtaining 10.7 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a structure of 1,6-bis [3-(β-morpholinoethyl)-4- -methylcoumarin-7-yloxy3hexane hydrochloride.

EXAMPLE 17: Bis(3-(β-diethylaminoethyl)-4-methylcoumarin-7-yloxy3 dicarbethoxy methane hydrochloride

50.0 g of 3-(β-diethylaminoethyl)-4-methyl-7- -hydroxy-coumarin are placed in a 500-ml reactor equipped with a shaking and separating apparatus. 300 ml of toluene and 49.7 g of I-^CO _j are added. It is heated, while separating and eliminating the reaction water. The toluene is then concentrated to about 50 ml, and it is cooled to room temperature and to this are added 100 ml of DMSO. One hour later 31.7 g of the diethyl ester of dibromomalonic acid are added, and the mixture is left to react for 12 hours. It is gathered with toluene and washed with H ₂ 0 and a IN solution of NaOH. It is anhydrated and the solvent is concentrated. It is purified by chromatography with Prep

LC System Waters using as eluent a mixture of CH ₂ Cl ₂ -CH ₃ OH-NH _ι OH (30%) 95:7:0.2.

The pure fractions are concentrated, gathered with ethyl acetate, anhydrated and the hydrochloride is precipitated with HCl in ethanol. 9.0 g of a compound are obtained to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a structure of bis(3-(β-di¬ ethylaminoethyl)-4-methylcoumarin-7-yloxy dicarb- ethoxy methane hydrochloride.

EXAMPLE 18:

Bis[3-(β-diethylaminoethyl)-4-methyl-8-chlorocou- marin-7-ylox 3dicarbethoxy methane hydrochloride 57.0 g of 3-(β-diethylaminoethyl)-4-methyl-7- -hydroxy-3-chlorocoumarin are placed in a 500-ml reactor equipped with a shaking and separating apparatus. 300 ml of toluene and 49.7 g of K ₂ C0 ₃ are added. It is heated, separating and eliminating the reaction water. The toluene is concentrated to about 50 ml, cooled to room temperature and to this are adde'd 100 ml of DMSO. One hour later to this are added 31.7 g of the diethyl ester of dibromomalonic acid and it is left to react for 12 hours. The resultant is gathered with toluene and washed with H ₂ 0 and a IN solution of NaOH. The solvent is anhydrated and concentrated. It is purified by chromatography with Prep LC System Waters using as eluent a mixture of CH ₂ Cl ₂ -CH ₃ OH-NH ₄ OH (30%) 98:2:0.2. The pure fractions are concentrated, gathered with ethyl acetate, anhydrated, and the hydrochloride is precipitated with HCl in ethanol. 13.0 g of a compound are obtained to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons)

both attribute a structure of bis[3-•( -diethyl¬ aminoethyl) -4-methyl-8-chlorocoumarin-7-yloxy3- dicarbethoxy methane hydrochloride.

EXAMPLE 19:

Bis[3-(β-diethylaminoethyl)-4-phenylcoumarin-7- -yloxy}dicarbethoxy methane hydrochloride

25.0 g of 3-(β-diethylaminoethyl)-4-phenyl-7- -hydroxy-coumarin are placed in a 500-ml reactor equipped with a shaking and separating apparatus. 300 ml of toluene and 27.7 g of K ₂ C0 ₃ are added. It is heated, separating and eliminating the reaction water. The toluene is then concentrated to about 50 ml , cooled to room temperature and to this are added 100 ml of DMS0. One hour later 12.7 g of the diethyl ester of dibromomalonic acid are added, and it is left to react for 12 hours. It is gathered with toluene and waεhed with H ₂ 0 and a IN solution of NaOH. The solvent is anhydrated and concentrated. It is purified by chromatography with Prep LC Syεtem Waters using as eluent a mixture of CH ₂ Cl ₂ -crf ₃ OH-NH _i OH (30%) 95:5:0.2.

The pure fractions are concentrated, gathered with ethyl acetate, ^' anhydrated, and the hydrochloride is precipitated with HCl in ethanol. 12.5 g of a compound are obtained to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a structure of bis[3-(β-diethylaminoethyl)-4-phenylcoumarin-7- -yloxy]dicarbethoxy methane hydrochloride.

EXAMPLE 20:

Bis[3-(β-diethylaminoethyl)-4- ethyl-coumarin-7-

-yloxy]carbethoxy methane hydrochloride

81.0 g of 3-(β-diethylaminoethyl)-4-methy1-7-hydroxy— -coumarin are placed in a 500- l reactor equipped with a shaking and separating apparatuε. 300 ml of toluene and 48.4 g of K _j CO _j are added. It iε heated, separating and eliminating the reaction water. The toluene is concentrated to 50 ml, cooled to room temperature and to this are added 100 ml of DMSO. One hour later 23.55 g of ethyl dichloro- acetate are added and it is left to react for 48 hourε.

The reaction mixture iε gathered with ethyl acetate and washed with H ₂ 0 and a IN solution of NaOH. ^' The solvent iε anhydrated and concentrated. It is purified by chromatography with Prep LC System Waters using a mixture of CHCl- _j -CIL^OH 80:20 as eluent. The pure fractions are evaporated. This iε gathered in ethyl acetate, anhydrated, and the hydrochloride is precipitated with HCl in ethanol. It is filtered, washed with ethyl acetate and vacuum-dried, obtaining 28.0 g of a compound to which elementary analysis (C,H,N) and nuclear magnet.lc resonance spectroscopy (protons) both attribute a structure of bis[3-(β-diethylaminoethyl)-4-methyl- -coumarin-7-ylόxy]carbethoxy methane hydrochloride.

EXAMPLE 21:

Bis [3- (β-diethylaminoethyl) -4-methyl-8-chlorocou- marin-7-yloxy 3 carbethoxy methane hydrochloride

50 .0 g of 3 -(β-diethylaminoethyl) -4 -methyl-7- -hydroxy-8-chlorocoumarin are placed in a 500-ml reactor equipped with a shaking and separating apparatus .

300 ml of toluene and 26.7 g of K ₂ CC> ₃ are added. It is heated, separating and eliminating

the reaction wa er. The toluene is concentrated to 50 ml, cooled to room temperature and to this are added 100 ml of DMSO. One hour later 12.6 g of ethyl dichloroacetate are added, and it is left to react for 48 hours.

The reaction mixture is gathered with ethyl acetate and washed with H ₂ 0 and a IN solution of NaOH. The solvent is anhydrated and concentrated. It is purified by chromatography with Prep LC System Waters using a mixture of CHC^-CH _j OH-NH^OH (30%) 95:5:0.2 as eluent. The pure fractions are evaporated. It is gathered in ethyl acetate, anhydrated, and the ^' hydrochloride is precipitated with HCl in ethanol. It is filtered, washed with ethyl acetate and vacuum-dried, obtaining 17.0 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a structure of bis[3-(β-diethylamino¬ ethyl)-4-methyl-8-chlorocoumarin-7-yloxy)carbethoxy methane hydrochloride.

EXAMPLE 22:

Bis[3-(β-diethylaminoethyl)-4-phenylcoumarin-

-7-yloxy3carbethoxy methane hydrochloride 25.0 g of 3-(β-diethylaminoethyl)-4-ρhenyl-7- -hydroxycoumarin hydrochloride are placed in a 500-ml reactor equipped with a shaking and separating apparatus. 300 ml of toluene and 27.7 g of K ₂ C0 ₃ are added. It is heated, separating and eliminating the reaction water. The toluene is concentrated to 50 ml, cooled to room temperature and to this are added 100 ml of DMSO. One hour later 6.26 g of ethyl dichloroacetate are added, and it is left to react for 48 hours.

The reaction mixture iε gathered with ethyl acetate and washed with H ₂ 0 and a IN solution of NaOH. The solvent is anhydrated and concentrated. It is purified by chromatography with Prep LC System Waters using a mixture of CHC1 ₃ -CH ₃ 0H-NH ₄ 0H (30%) 95:5:0.2 as eluent. The pure fractions are evaporated. It is gathered in ethyl acetate, anhydrated, and the hydrochloride is precipitated with HCl in ethanol, obtaining 5.5 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a structure of bis[3-(β-diethylaminoethyl)-4-phenylcoumariή-7- -yloxy3carbethoxy methane hydrcchloride.

EXAMPLE 23:

Bis[3-(morpholinomethyl)- -methyl-6,8-diallyl- coumarin-7-yloxy3carbethoxy methane hydrochloride

20.0 g of 3-(morpholinomethyl)-4-methyl-6,8- -diallyl-7-hydroxycoumarin are placed in a 500-ml reactor equipped with a shaking and separating apparatus. 300 ml of toluene and 11.6 g of K ₂ C0 ₃ are added. It is heated, εeparating and eliminating the reaction water. The toluene is concentrated to 50 ml, cooled to room' temperature and to thiε are added 100 ml of DMSO. One hour later to this are added 4.39 g of ethyl dichloroacetate and it is left to react for 43 hours.

The reaction mixture is gathered with ethyl acetate and washed with H ₂ 0 and a IN solution of NaOH. The solvent is anhydrated and concentrated. It is purified by chromatography with Prep LC System Waters using a mixture of CHCl ₃ -CH ₃ OH-NH _t OH (30%) 95:5:0.2 as eluent. The pure fractions are evaporated. This is gathered in ethyl acetate, anhydrated, and the

hydrochloride is precipitated with HCl in ethanol. 5.6 g of a compound are obtained to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a structure of .bis[3-(morpholinomethyl)-4-methyl- -6,8-diallylcoumarin-7-yloxy]carbethoxy methane hydrochloride.

EXAMPLE 24: Bis[3-(morpholinomethyl)-4-methyl-6-chloro-8-allyl- coumarin-7-yloxy3carbethoxy methane hydrochloride

30.0 g of 3-(morpholinomethyl)-4-methyl-6-chloro- -7-hydroxy-8-allylcoumarin are placed in a 500-ml reactor equipped with a shaking and separating apparatuε. 300 ml of toluene and 16.5 g of K ₂ C0 ₃ are added. It is heated, separating and eliminating the reaction water. The toluene is concentrated to 50 ml, cooled to room temperature and to this are added 100 ml of DMSO. One hour later to this are added 6.75 g of ethyl dichloroacetate and it is left to react for 48 hours.

The reaction mixture is gathered with ethyl acetate and washed with H ₂ 0 and a IN solution of NaOH. The solvent is anhydrated and concentrated. It is purified by chromatography with Prep LC System Waters using a mixture of CHCl ₃ -CH ₃ OH-NH ₄ OH (30%) 95:5:0.2 as eluent. The pure fractions are evaporated. This is gathered in ethyl acetate, anhydrated, and the hydrochloride iε precipitated with HCl in ethanol. It iε filtered, washed with ethyl acetate and vacuum-dried, obtaining 10.0 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a structure of bis[3-(morpholinomethyl)-

-4-methyl-6-chloro-S-allylcoum£irin-7-yloxy3 carbethoxy methane hydrochloride.

EXAMPLE 25: Bis[3-(morpholinomethyl)-4,8-dimethyl-6-allyl- coumarin-7-ylox 3carbossimetano hydrochloride

30.0 g of 3-(morpholinomethyl)-4,8-dimethyl-6- -allyl-7-hydroxycoumarin are placed in a 500-ml reactor equipped with a shaking and separating apparatus. 300 ml of toluene and 25.2 g of K ₂ C0 ₃ are added thereto. It is heated, separating and eliminating the reaction water. The toluene is concentrated to 50 ml, cooled to room temperature and to this are added 100 ml of DMSO. One hour later to thiε are added 7.2 g of ethyl dichloroacetate and it is left to react for 48 hours.

The reaction mixture is gathered with ethyl acetate and washed with H ₂ 0 and a IN solution of NaOH. The solvent is anhydrated and concentrated. It is purified by chromatography with Prep LC System Waters using a mixture of CH ₂ Cl ₂ -EtAc-CH ₃ OH 70:30:2 as eluent. The pure fractions are evaporated and gathered in ethyl acetate, anhydrated, and the hydrochloride is precipitated with HCl in ethanol. It is filtered, washed with ethyl acetate and vacuum-dried, obtaining 9.1 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a structure of bis[3-(morpholinomethyl)- - ,8-dimethyl-6-allylcoumarin-7-yloxy3carboxy- methane hydrochloride.

EXAI-TPLE 26:

Bis[3-(morpholinomethyl)-4-methyl-6-allyl-8-chloro- coumarin-7-yloxy3carbethoxy methane hydrochloride

30.0 g of 3-(morpholinomethyl)-4-methyl-6-allyl- -7-hydroxy-8-chlorocoumarin are placed in a 500-ml reactor equipped with a shaking and separating apparatuε. 300 ml of toluene and 17.8 g of KJCO-J are added. It is heated, separating and eliminating the reaction water. The toluene is concentrated to 50 ml, cooled to room temperature and to this are added 100 ml of DMSO. One hour later 6.75 g of ethyl dichloroacetate are added thereto, and it iε left to react for ^' 48 hours.

The reaction mixture is gathered with ethyl acetate and washed with H ₂ 0 and a IN solution of NaOH. The solvent iε anhydrated and concentrated. It is purified by chromatography with Prep LC System Waters using a mixture of CH ₂ Cl ₂ -EtAc-CH ₃ OH 70:30:1 as eluent. The pure fractions are evaporated, gathered in ethyl acetate, anhydrated and the hydrochloride is precipitated with HCl in ethanol. It is filtered, washed with ethyl acetate and vacuum-dried, obtaining 9.9 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a structure of bis[3-(morpholinomethyl)-4-methyl-6-allyl- 8-chlorocoumarin-7-yloxy3carbethoxy methane hydrochloride.

EXAMPLE 27:

1, 3-biε[3-(β-diethylaminoethyl)-4-methyl-8-chloro- coumarin-7-yloxy]-2-hydroxypropane hydrochloride

50.0 g of 3-(β-diethylaminoethyl)-4-methyl-7- -hydroxy-8-chlorocoumarin are suspended in 500 ml

of ethanol and to this are added 18.1 g of 50% KOH in H ₂ o. It is left to stand at 50*C while shaking for one hour and then concentrated and vacuum-dried. It is gathered with 500 ml of 2-butanone and to this are added 22.0 g of I-^CO- _j . It is heated while being shaken, then to this are added 9.9 g of epibromohydrin and it iε left to react for 3 days. The solvent is evaporated and the residue iε dissolved with 500 ml of ethyl acetate. It is washed with a IN solution of NaOH. The solvent is anhydrated and concentrated. It is purified by chromatography with Prep LC System Waters using as eluent a mixture of CH ₂ cl ₂ ~CH ₃ OH-NHOH (30%) 95:5:0.2. The pure fractions are evaporated, crystallized from ethanol, gathered in ethyl acetate, anhydrated, and the hydrochloride is precipitated with HCl in ethanol. The hydrochloride is crystallized from ethanol. This is filtered and vacuum-dried, obtaining 6.8 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a εtructure of 1,3-bis[3-(β-diethyl¬ aminoethyl)-4-methyl-8-chlorocouraarin-7-yloxy]-2- -hydroxypropane hydrochloride.

EXAMPLE 28: l,3-bis[3-(morpholinomethyl)-4-methyl-6,8-diallyl- coumarin-7-yloxy3-2-h droxypropane hydrochloride 50.0 g of 3-(morpholinomethyl)-4-methyl-6,8-diallyl-7-

-hydroxycoumarin are suspended in 500 ml of ethanol and to this are added 15.0 g of 50% KOH in H ₂ 0. It is left to stand at 50 ^* C while shaking for one hour and

then concentrated and vacuum-dried. It is gathered with 500 ml of 2-butanone and to this are added 24.8 g of K _∑ C0 ₃ . It is heated while being shaken, then to this are added 9.5 g of epibromohydrin and it is left to react for 3 days. The solvent is evaporated and the residue is dissolved with 500 ml of ethyl acetate. It is washed with a IN solution of NaOH. The solvent is anhydrated and concentrated. It iε purified by chromatography with Prep LC System Waters using as eluent a mixture of CH ₂ Cl ₂ -EtAc-CH ₃ OH 70:30:1.

The pure fractions are evaporated, gathered in ethyl acetate, anhydrated, and the hydrochloride is precipitated with HCl in ethanol. The hydro¬ chloride is crystallized from a mixture of ethan- ol-ethyl acetate or acetone. This is filtered and vacuum-dried, obtaining 16.3 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a structure of l,3-bis[3-(morpholino- methyl)-4-methyl-6,8-diallylcoumarin-7-yloxy3-2- -hydroxypropane hydrochloride.

EXAMPLE 29:

1,3-bis[3-(morpholinomethyl)-4-methyl-6-chloro-8- -allylcoumarin-7-yloxy3-2-hydroxypropane hydro¬ chloride

20.0 g of 3-(morpholinomethyl)-4-methyl-6-chloro- -7-hydroxy-8-allylcoumarin are suspended in 200 ml of ethanol and to this are added 6.4 g of 50% KOH in H ₂ θ. It is left to stand at 50 ^* C while being stirred for one hour, and then concentrated and vacuum-dried. It iε gathered with 200 ml of 2-butanone and 7.9 g of K ₂ C0 ₃ are added. It. is heated while being shaken, then to this are added 3.8 g of

epibromohydrin and it is left to react for 3 days. The solvent is evaporated and the residue is dissolved with 500 ml of ethyl acetate- It is washed with a IN solution of NaOH. The solvent is anhydrated and concentrated.

The reaction product is crystallized from ethyl ether/ethyl acetate, 10:1. It is gathered in ethyl acetate, anhydrated, and the hydrochloride is precipitated with HCl in ethanol. The hydrochloride is crystallized from ethanol. This is filtered and vacuum-dried, obtaining 6.0 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a structure of l,3-biε[3-(morpholinomethyl)- 4-methy1-6-chloro-8-allylcoumarin-7-yloxy]-2- hydroxypropane hydrochloride.

EXAMPLE 30:

1,3-bis[3-(morpholinomethyl)-4-phenyl-6-allyl-8- -methylcoumarin-7-yloxy3-2-hydroxypropane hydro¬ chloride

50.0 g of 3-(morpholinomethyl)-4-phenyl-6-allyl-7- -hydroxy-8-methylcoumarin are suspended in 500 ml of ethanol and to this are added 14.3 g of 50% KOH in H ₂ 0. It is left to stand at 50'C while being shaken for one hour, and then concentrated and vacuum-dried. It is gathered with 500 ml of 2-butanone and to this are added 26.5 g of K ₂ C0 ₃ . It is heated while being shaken, then to this are added 8.7 g of epibromohydrin and it is left to react for 3 days. The solvent is evaporated and the residue is dissolved with 500 ml of ethyl acetate. It is washed with a IN εolution of NaOH. The solvent is anhydrated and concentrated. It is purified by

chromatography with Prep LC System Waters using as eluent a mixture of CH ₂ Cl ₂ -EtAc~CH ₃ OH 70:30:1.

The pure fractions are evaporated, gathered in ethyl acetate, anhydrated, and the hydrochloride is precipitated with HCl in ethanol. It is washed, filtered and vacuum-dried, obtaining 5.0 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic reεonance spectroεcopy (protons) both attribute a structure of l,3-bis[3-(morpho- linomethyl)-4-phenyl-6-allyl-8-methylcoumarin-7- -ylox 3-2-hydroxypropane hydrochloride.

EXAMPLE 31:

1,3-bis[3-(morpholinomethyl)-4,8-dimethyl-6-allyl- coumarin-7-yloxy3-2.-hydroxypropane hydrochloride

30.0 g of 3-(morpholinomethyl)-4,8-dimethyl-6- -allyl-7-hydroxycoumarin are suspended in 500 ml of ethanol and to this are added 10.2 g of 50% KOH in H ₂ 0. It is left to stand at 50'C while being shaken for one hour, and then concentrated and vacuum-dried- It is gathered with 500 ml of 2-butanone and to this are added 12.6 g of K ₂ C0 ₃ . It is heated while being shaken, then to this are added 6.2 g of epibromohydrin. and it is left to react for 3 days. The solvent is evaporated and the residue is dissolved with 500 ml of ethyl acetate. It is washed with a IN solution of NaOH. The εolvent iε anhydrated and concentrated. It is purified by chromatography with Prep LC System Waters using as eluent a mixture of CH ₂ Cl ₂ -EtAc-CH ₃ OH 70:30:3.

The pure fractions are evaporated, gathered in ethyl acetate , anhydrated, and the hydrochloride is precipitated with HCl in ethanol . The hydrochloride is crystallized from a mixture of

ethanol -ethyl acetate . This is filtered and vacuum-dried, obtaining 9. 2 g of a compound to which elementary analysis (C, H,N) and nuclear magnetic resonance spectroεcopy (protonε) both attribute a εtructure of 1, 3-bis [3- (morpholino¬ methyl) - , 8-dimethyl-6-allylcoumarin-7-yloxy3 -2- -hydroxypropane hydrochloride.

EXAMPLE 32 : 1,3-bis[3-(morpholinomethyl)-4-methyl-6-allyl-8- -chlorocoumarin-7-yloxy3-2-hydroxyproρane hydro¬ chloride

20.0 g of 3-(morpholinomethyl)-4-methyl-6-allyl- -7-hydroxy-8-chlorocoumarin are suspended in 200 ml of ethanol and to this are added 5.8 g of 50% KOH in H ₂ 0. It is left to stand at 50*C while being shaken for one hour, then concentrated and vacuum-dried. It is gathered with 300 ml of 2-butanone and to this are added 7.9 g of K ₂ C0 ₃ . It is heated while being shaken, and then to this are added 3.8 g of epibromohydrin and it is left to react for 3 days. The solvent is evaporated and the residue is dissolved with 500 ml of ethyl acetate. It is washed with a IN solution of NaOH. The solvent is anhydrated and concentrated. It is purified by chromatography with Prep LC System Waters using as eluent a mixture of CH ₂ Cl ₂ -CH ₃ OH-NH ₄ OH (30%) 98:2:0.2.

The pure fractionε are evaporated, gathered in ethyl acetate, anhydrated and the hydrochloride is precipitated with HCl in ethanol. The hydrochloride iε crystallized from ethanol. This is filtered and vacuum-dried, obtaining 3.3 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both

attribute a εtructure of 1,3-bis[3-(morpho¬ linomethyl) -4-meth l-6-allyl-8-chlorocoumarin-7- -yloxy3-2-hydroxypropane hydrochloride.

EXAMPLE 33:

1,3-bis[3-morpholinomethyl)-4-phenyl-6-chloro-8- -allylcoumarin-7-yloxy3-2-hydroxypropane hydro¬ chloride

50.0 g of 3-(morpholinomethyl)-4-phenyl-6-chloro- -7-hydroxy-8-allylcoumarin are εuspended in 500 ml of ethanol and to this are added 13.5 g of 50% KOH in H ₂ 0. It iε left to εtand at 50"C while being shaken for one hour and is then concentrated and vacuum-dried. It is gathered with 500 ml of 2-butanone and to this are added 16.6 g of K ₂ co ₃ . It is heated while being shaken, and then to this are added 8.2 g of epibromohydrin and it is left to react for 3 days. The solvent is evaporated and the residue is dissolved with 500 ml of ethyl acetate. It is washed with a IN solution of NaOH. The εolvent is anhydrated and concentrated. It iε purified by chromatography with Prep LC System Waters using as eluent a mixture of CH ₂ Cl ₂ -EtAc 95:5.

The pure fractions are evaporated, gathered in ethyl acetate, anhydrated, and the hydrochloride is precipitated with HCl in ethanol-ethyl acetate. This is filtered and vacuum-dried, obtaining 7.7 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a εtructure of 1,3-bis[3-

-morpholinomethyl) -4-phenyl-6-chloro-8-allylcoumarin- 7-yloxy 3 ~2-hydroxypropane hydrochloride.

EXAMPLE 34 :

1,3-bis[3-(morpholinomethyl)-4-phenyl-6,8-diallyl- coumarin-7-yloxy]-2-hydroxypropane hydrochloride 30.0 g of 3-(morpholinomethyl)-4-phenyl-6,8- -diallyl-7-hydroxycoumarin are suspended in 300 ml of ethanol and to thiε are added 7.8 g of 50% KOH in H ₂ o. It is left to εtand at 50 ^* C while being εhaken for one hour and is then concentrated and vacuum-dried. It is gathered with 300 ml of 2-butanone and to thiε are added 9.9 g of ϊ^CO _j . It is heated while being shaken, and then to this are added 4.9 g of epibromohydrin and it iε left to react for 3 days. The solvent is evaporated and the residue is dissolved with 500 ml of ethyl acetate. It is washed with a IN solution of NaOH. The solvent is anhydrated and concentrated. It is purified by chromatography with Prep LC System Waters using as eluent a mixture of CH ₂ Cl ₂ -EtAc 80:20.

The pure fractions are evaporated, gathered in ethyl acetate, anhydrated, and the hydrochloride iε precipitated with HCl in ethanol. The hydro¬ chloride 'is freeze-dried, obtaining 5.0 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a structure of l,3-bis[3-(morpho¬ linomethyl)-4-phenyl-6,8-diallylcoumarin-7- loxy3- -2-hydroxypropane hydrochloride.

EXAMPLE 35: 1,3-bis[3-(morpholinomethyl)-4-phenyl-6-allyl-8- -chlorocoumarin-7-yloxy3-2-hydroxypropane hydro¬ chloride

33.0 g of 3-(morpholinomethyl)-4-phenyl-6-allyl-

-7-hydroxy-S-chlorocoumarin are suspended in 300 ml of ethanol and to this are added 8.8 g of 50% KOH in H ₂ 0. It is left to stand at 50*C while being shaken for one hour and then concentrated and vacuum-dried. It is gathered with 300 ml of 2-butanone and to this are added 11.1 g of K ₂ C0 ₃ . It is heated while being shaken, then to this are added 5.5 g of epibromohydrin and it iε left to react for 3 days. The solvent is evaporated and the residue is dissolved with 300 ml of ethyl acetate. It is washed with a IN solution of NaOH. The solvent is anhydrated and concentrated. It is purified by ^' chromatography with Prep LC System Waters using as eluent a mixture of CH ₂ Cl ₂ -EtAc 80:20. The pure fractions are evaporated, gathered in ethyl acetate, anhydrated, and the hydrochloride is precipitated with HCl in ethanol. The hydro¬ chloride iε crystallized from ethanol. Thiε iε filtered and vacuum-dried, obtaining 5.4 g of a compound to which elementary analyεis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a εtructure of 1,3-bis[3-(morpho¬ linomethyl) -4-phenyl-6-allyl-8-chlorocoumarin-7- -yloxy3~2-hydroχypropane hydrochloride.

EXAMPLE 36:

1, 3-bis[3-(β-morpholinoethyl)-4-methylcoumarin-7-

-yloxy3-2-hydroxypropane hydrochloride

25.0 g of 3-(β-morpholinoethyl)-4-methyl-7- -hydroxycoumarin sulfate are suspended in 300 ml of ethanol and to this are added 22.0 g of 50% KOH in H ₂ 0. It is left to stand at 50 ^* C while being shaken for one hour and then concentrated and vacuum-dried. It is gathered with 300 ml of 2-butanone and to

this are added 9.0 g of . It is heated while being shaken, and then to this are added 4.5 g of epibromohydrin and it is left to react for 3 days. The solvent is evaporated and the residue is dissolved with 300 ml of ethyl acetate. It is washed with a IN solution of NaOH. The solvent is anhydrated and concentrated. It is crystallized from CH ₃ OH, filtered, gathered in ethyl acetate, anhydrated, and the hydrochloride is precipitated with HCl in ethanol. It is washed, filtered and vacuum-dried, obtaining 9.0 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a structure of 1,3-bis[3-(β-morpholino- ethyl)-4-methylcoumarin-7-yloxy3-2-hydroxyprop ne hydrochloride.

EXAMPLE 37:

1,3-bis[3-(β-morpholinoethyl)-4-methyl-8-chloro- coumarin-7-yloxy]-2-hydroxypropane hydrochloride 46.0 g of 3-(β-morpholinoethyl)-4-methyl-7- -hydroxy-8-chlσrocoumarin hydrochloride are suspended in 500 ml of ethanol and to this are added 28.6 g of-50% KOH in H ₂ 0. It is left to stand at 50°C while being shaken for one hour and then concentrated and vacuum-dried. It is gathered with 500 ml of 2-butanone and to this are added 17.6 g of K ₂ C0 ₃ . It is heated while being shaken, then to this are added 8.9 g of epibromohydrin and it is left to react for 3 days. The solvent is evaporated and the residue is dissolved with 300 ml of ethyl acetate. It is washed with a IN solution of NaOH. The solvent is anhydrated and concentrated. It is purified by chromatography with Prep LC System Waters using as

eluent a mixture of CH ₂ Cl ₂ -EtAc-CH ₃ OH 70:30:10. The pure fractions are evaporated, gathered in CHCl ₃ -CH ₃ OH 2:1 and the hydrochloride is precipitated with HCl in ethanol. This iε filtered and vacuum-dried, obtaining 12.0 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a structure of l,3-bis[3-(β-morpholino- ethyl)-4-methyl-8-chlorocoumarin-7-yloxy3-2-hy- droxypropane hydrochloride.

EXAMPLE 38:

1,3-bis[3-(β-diisopropylaminoethyl)-4-methylcou- marin-7-yloxy)-2-hydroxypropane hydrochloride 25.0 g of 3-(β-diisopropylaminoethyl)-4-methyl-7- -hydroxycoumarin are suspended in 300 ml of ethanol and to this are added 8.2 g of 50% KOH in H ₂ 0. It is left to stand at 50*C while being shaken for one hour and then concentrated and vacuum-dried. It is gathered with 300 ml of 2-butanone and to this are added 10.0 g of K ₂ C0 ₃ . It is heated while being shaken, 5.0 g of epibromohydrin are added and it is left to react for 3 days. The solvent is evaporated and the residue iε dissolved with 300 ml of ethyl acetate. It is washed with a IN solution of NaOH. The solvent is anhydrated and concentrated. It is purified by chromatography with Prep LC System Waters using as eluent a mixture of CH ₂ Cl ₂ -CH ₃ OH-NH _Λ OH (30%) 95:5:0.2. The pure fractions are evaporated, gathered in ethyl acetate, anhydrated, and the hydrochloride is precipitated with HCl in ethanol. The hydrochloride is crystallized from isopropyl alcohol. This is filtered and vacuum-dried, obtaining 5.5 g of a compound to which

elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a structure of 1,3-bis[3-(β-diisopropylamino¬ ethyl)-4-methylcoumarin-7-yloxy)-2-hydroxypropane hydrochloride.

EXAMPLE 39:

1,3-bis[3-(β-diethylaminoethyl)-4-methylcoumarin-

-7-yloxy3-2-hydroxypropane hydrochloride 35.0 g of 3-(β-diethylaminoethyλ)-4-methyl-7-

-hydroxycoumarin are suspended in 500 ml of ethanol and to this are added 12.6 g of 50% KOH in H ₂ 0.' It is left to stand at 50"C while being shaken for one hour, and then it iε concentrated and vacuum-dried. It is gathered with 500 ml of 2-butanone and to this are added 15.5 g of K ₂ C0 ₃ . It is heated while being shaken, then to this are added 6.9 g of epibromohydrin and it is left to react for 3 days. The solvent is evaporated and the residue dissolv- ed with 300 ml of ethyl acetate. It is washed with a IN solution of NaOH. The solvent iε anhydrated and concentrated. It is purified by chromatography with Prep LC System Waters using as eluent a mix¬ ture of CH ₂ Cl ₂ -CH ₃ OH-NH ₄ OH (30%) 90:10:0.4. The pure fractions are evaporated, gathered in ethyl acetate, anhydrated, and the hydrochloride is precipitated with HCl in ethanol. The hydro¬ chloride is crystallized from ethanol. This is filtered and vacuum-dried, obtaining 18.0 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a εtructure of 1,3-bis[3-(β-di¬ ethylaminoethyl)-4-methylcσumarin-7-yloxy] -2- -hydroxypropane hydrochloride.

EXAMPLE 40 : l-[ -(β-diethylaminoethyl) -4-methylcoumarin-7- -yloxy3-3-[3-(β-diethylaminoethyl)-4-methyl-8- -chlorocoumarin-7-yloxy3propane hydrochloride 35.0 g of 3-(β-diethylaminoethyl)-4-methyl-7-hy- droxy-cou arin are suspended in 500 ml of ethanol and to this are added 14.2 g of 50% KOH in water. It is left to stand for 1 hour at 50 ^* C and then concentrated and vacuum-dried. It is gathered with 500 ml of 2-butanone and to this are added 35.1 g of K ₂ C0 ₃ . It iε heated while being shaken, then to this are added 25.7 g of 1.3 dibromopropane and it is left to react for 12 hours. The solvent is evaporated and the residue is dissolved with 500 ml of ethyl acetate. It is washed with a 1 N solution of NaOH. The solvent is anhydrated, concentrated and crystallized from isopropyl alcohol. It is filtered and the l-[3-(β-diethylaminoethyl)-4-methylcoumarin-7-yloxy]- 3-bromopropane crystals are dried. At the same time, the potassium salt of 3-(β-diethylamino¬ ethyl)-4-methyl-7-hydroxy-8-chlorocoumarin is prepared 'by treating 39.0 g of this compound with 14.2 g of 50% KOH in water in 500 ml of ethanol. This mixture is left to stand for l hour at 50 ^* C and then it is concentrated and vacuum-dried.

The potassium salt is gathered with 500 ml of 2-butanone and to this are added 35.1 g of K ₂ C0 ₃ , It is heated while being shaken, and then to this are added the 1-[3-(β-diethylaminoethyl)- -4-methylcoumarin-7-yloxy3-3-bromopropane crystals and it is left to react for 12 hours. The solvent is evaporated and the residue is dissolved with 500 ml of ethyl acetate. It is washed with a IN solution of NaOH. The solvent is anhydrated

and concentrated. The reaction product is purified by chromatography with Prep LC System Waters, using as eluent a mixture of CHCl ₃ -CH ₃ OH~NH ₄ OH (30%) at a gradient of 95:5:0.2 to 70:30:0.2. The pure fractions are concentrated and crystallized from toluene. The crystallized product is dissolved in ethyl acetate, anhydrated and treated with HCl in ethanol until a Congo red indicator change is noted. It is filtered and crystallized from isopropyl alcohol. This is filtered and vacuum-dried, obtaining 36.4 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroεcopy (protons) both attribute a structure of l-[3-(β-diethylaminoethyl)-4-methylcoumarin-7-yl- oxy3-3-[3-(β-diethylaminoethyl)-4-methyl-8-chloro- coumarin-7-yloxy3propane hydrochloride.

EXAMPLE 41:

1-[3-(β-diethylaminoethyl)-4-methylcoumarin-7-yl- oxy3-6-[3-(β-diethylaminoethyl)-4-methyl-8-chloro- coumarin-7-yloxy3hexane hydrochloride

35.0 g of 3-(β-diethylaminoethyl)-4-methyl-7- -hydroxycoumarin are suspended in 500 ml of ethanol and to this are added 14.2 g of 50% KOH in water. It is left to stand for 1 hour at 50'C and then concentrated and vacuum-dried. It is gathered with 500 ml of 2-butanone and to this are added 35.1 g of K ₂ C0 ₃ - It is heated while being shaken, then to this are added 31.0 g of 1.6 dibromohexane and it is left to react for 12 hours. The solvent is evaporated and the residue is dissolved with 500 ml of ethyl acetate. It is washed with a 1 N solution of NaOH. The solvent is anhydrated, concentrated and crystallized by ethyl alcohol. It is filtered and the l-[3-(β-di-

ethylaminoethyl)-4-methylcoumarin-7-yloxy -6-bromo- hexane crystals are dried.

At the same time, the potassium salt of 3-(β-di¬ ethylaminoethyl)-4-methyl-7-hydroxy-8-chloro coumarin iε prepared by treating 39.0 g of this compound with 14.2 g of 50% KOH in water in 500 ml of ethanol. It is left to stand for 1 hour at 50 ^* C and then concentrated and vacuum-dried. The potasεium Scilt is gathered with 500 ml of 2-butanone and to this are added 35.1 g of K ₂ C0 ₃ . It is heated while being shaken, and then to this are added the 1-[3-(β-diethylaminoethyl)- -4-methylcoumarin-7-ylox 3-6-bromohexane crystals and it is left to react for 12 hours. The solvent is evaporated and the residue is dissolved with 500 ml of ethyl acetate; it iε washed with a IN solution of NaOH. The solvent iε anhydrated and concentrated. It is purified by chromatography with Prep LC System Waters, using as eluent a mixture of CHCl ₃ -CH ₃ OH-NHOH (30%) at a gradient of 95:5:0.2 to 70:30:0.2.

The pure fractions are concentrated and precipitated by methahol. The precipitate is dissolved in a mixture of chloroforra/methanol and treated with HCl in ethanol until a Congo- red indicator change is observed. It is slightly concentrated and then precipitated by the addition of ethyl acetate. This is filtered and vacuum-dried, obtaining 35.7 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a εtructure of l-[3-(β-diethyl-amino- ethyl)-4-methylcoumarin-7-yloxy3-6-[3-(β-diethyl- -aminoethyl)-4-methyl-δ-chlorocoum£irin-7-yloxy3- hexane hydrochloride.

EXAMPLE 42: l-£3-(β-diethylaminoethyl)- -methylcoumarin-7-yl¬ oxy3~l-[3-(β-diethylaminoethyl)-4-methy1-8-chloro- coumarin-7-yloxy3dicarbethoxy methane hydro- chloride

25.0 g of 3-(β-diethylaminoethyl)-4-methyl-7- -hydroxycoumarin are placed in a 500-ml reactor having shaking and separating equipment. 300 ml of toluene and 24.8 g of K ₂ C0 ₃ are added. It iε heated, separating and eliminating the reaction water; the toluene is concentrated to about 50 ml, cooled to room temperature and to this are added 100 ml of DMSO. One hour later 31.7 g of dibromomalonic ester are added thereto, and it is left to react for 12 hours. The potassium salt of 3-(β-diethylamino¬ ethyl)-4-methyl-7-hydroxy-8-chlorocoumarin iε added, having been previously prepared by treating 28:5 g of the 8-chlorocoumarin compound with 10-1 g of 50% KOH in water for 1 hour in 250 ml of ethanol at 50 ^* C and evaporating to dryness under high vacuum.

The reaction mixture is left to react for 24 hours, and then gathered in toluene and washed with water and a IN solution of NaOH. The solvent is anhydrated and concentrated. 'It is purified by chromatography with Prep LC System Waters using as eluent a mixture of CH ₂ Cl ₂ -CH-OH-NH ₄ OH (30%) 96:4:0.2.

The pure fractions are concentrated, gathered with ethyl acetate, anhydrated, and the hydrochloride is precipitated with HCl in ethanol. A pulp is obtained which is freeze-dried, obtaining 11.2 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both attribute a structure of l-[3-(β- -diethylaminoethyl)-4-methylcoumarin-7-yloxy3-1-

-[3-(β-diethylaminoethyl)-4-methy1-8-chlorocoumarin-7 -yloxy)dicarbethoxy methane hydrochloride.

EXAMPLE 43: l-[3-(β-diethylaminoethyl)-4-methylcoumarin-7-yl¬ oxy3-l-[3-(β-diethylaminoethyl)-4-methyl-8-chloro- coumarin-7-yloxy)carbethoxy methane hydrochloride 25.0 g of 3-(β-diethylaminoethyl)-4-methyl-7- -hydroxy-cou arin are placed in a 500-ml reactor equipped with a shaking and separating apparatus. 300 ml of toluene and 24.8 g of K _j C^ are added. It is heated, separating and eliminating the reaction water; the toluene is concentrated to about 50 ml, cooled to room temperature and to this are added 100 ml of DMSO. One hour later 15.7 g of ethyl dichloroacetate are added, and it is left to react for 12 hours. The potassium salt of 3-(β-diethylamino¬ ethyl)-4-methyl-7-hydroxy-8-chlorocoumarin is added, having been previously prepared by treating 23.5 g of the 8-chlorocoumarin compound with 10.1 g of

50% KOH in water for 1 hour in 250 ml of ethanol at 50 ^* c and evaporating to dryness under high vacuum.

The reaction mixture iε left to react for 24 hours, and then gathered in toluene and washed with water and a IN solution of NaOH. The solvent is anhydrated and concentrated. It is purified by chromatography with Prep LC System Waters using as eluent a mixture of CH ₂ Cl ₂ -CH ₃ OH-NH ₄ OH (30%) 95:5:0.2.

The pure fractions are concentrated, gathered with ethyl acetate, anhydrated, and the hydrochloride is precipitated with HCl in ethanol. It is filtered, washed with ethyl acetate and vacuum-dried, obtaining 15.4 g of a compound to which elementary analysis (C,H,N) and nuclear magnetic resonance spectroscopy (protons) both

attribute a structure of l-[3-(β-diethylaminoethyl)-4-methylcoumarin-7- -yloxy)-l-[3-(β-diethylaminoethyl)-4-methyl-8-chloroc oumarin-7-yloxy3carbethoxy methane hydrochloride.

EXAMPLE 44:

Examples of pharmaceutical compositions for inj ection :

Preparation No. 1: one 2-ml ampoule contains:

- active principle 30 mg

- mannitol 100 mg

- water for injection q.ε. ad 2 ml

Preparation No. 2: one 2-ml ampoule containε:

- active principle 40 mg

- mannitol 100 mg

- water for injection q.s. ad 2 ml

Preparation No. 3: one 30-mg freeze-dried vial plus a 5-ml ampoule of solvent in which: a) one freeze-dried vial contains:

- active principle 30 mg

- mannitol 30 mg b) one 5-ml ampoule of solvent contains:

- sodium chloride 45 g

- water for injection q.s. ad 5 ml

Preparation No. 4: one 40-mg freeze-dried vial plus one 5-ml ampoule of solvent in which: a) one freeze-dried vial contains:

- active principle 40 mg

- mannitol 25 mg

b) one 5-ml ampoule of solvent contains:

- sodium chloride 45 mg

- water for injection q.s. ad 5 ml

EXAMPLE 45:

Examples of pharmaceutical compositions for oral use:

Preparation No. l: one 100-mg capsule contains:

- active principle 100.00 mg - saccharose 92.77 mg

- corn starch 30.93 mg

- magnesium εtearate 34.60 mg

- povidone 25.48 mg

- monobasic potassium phosphate 20.80 mg - trimellitate cellulose acetate 95.42 mg

- gelatin capsule 77.00 mg

Preparation No. 2: one 200-mg capsule contains:

- active principle 200-00 mg - saccharose 92.77 g

- corn starch 30.93 mg

- magnesium stearate 34.60 mg

- povidone 25.48 mg

- monobasic potassium phosphate 20.80 mg - trimellitate cellulose acetate 95.42 mg

- gelatin capsule 77.00 mg

Preparation No. 3: one 100-mg capsule contains:

- active principle 100.00 mg - vegetable oil 187.00 mg

- hydrogenated εoybean oil 2-40 mg

- hydrogenated vegetable oils 0.60 mg

- soybean lecitin 1.00 mg

- yellow wax 0.60 mg

- ethyl vanillin 0.30 mg

- gelatin 79-00 mg

- glycerol 30.00 g

- titanium bioxide E 171 1.10 mg

- cupric chlorophyll E 141 0.10 mg

- yellow iron oxide E 172 0.60 mg

- brown iron oxide E 172 0.03 mg

- sodium ethyl p-hydroxybenzoate 0.30 mg

- sodium propyl p-hydroxybenzoate 0.20 mg