ITMI971863A1 - AMIDOALKYLPIPERAZINE ACTIVE ON LOW URINARY TRACT - Google Patents
AMIDOALKYLPIPERAZINE ACTIVE ON LOW URINARY TRACT Download PDFInfo
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- ITMI971863A1 ITMI971863A1 ITMI971863A ITMI971863A1 IT MI971863 A1 ITMI971863 A1 IT MI971863A1 IT MI971863 A ITMI971863 A IT MI971863A IT MI971863 A1 ITMI971863 A1 IT MI971863A1
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- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Description
DESCRIZIONE dell’invenzione avente per titolo: DESCRIPTION of the invention entitled:
"AMMIDOALCHILPIPERAZINE ATTIVE SULLE BASSE VIE URINARIE" "AMIDOALKYLPIPERAZINES ACTIVE ON THE LOWER URINARY TRACT"
AMBITO DELL’INVENZIONE SCOPE OF THE INVENTION
L’Invenzione riguarda ammidoalchilplperazine, le composizioni farmaceutiche che le contengono e gli usi di tali derivati e composizioni. The invention relates to amidoalkylplperazines, the pharmaceutical compositions that contain them and the uses of such derivatives and compositions.
Il flavossato, l’ossibutinina e l’imipramlna sono principi attivi rappresentativi di tre diverse classi di composti attualmente usati nella terapia dell’Incontinenza urinaria.Tali sostanze sono state sperimentate 1n modelli animali che hanno confermato la loro attività’. Flavoxate, oxybutynin and imipramlna are active ingredients representative of three different classes of compounds currently used in the treatment of urinary incontinence. These substances have been tested in animal models that have confirmed their activity '.
I composti dell’invenzione, riportati di seguito, non condividono quasi nessuna caratteristica strutturale con le suddette sostanze, esclusa la presenza nella molecola d1 un atomo di azoto terziario basico. The compounds of the invention, shown below, share almost no structural characteristics with the aforementioned substances, except for the presence in the molecule of a basic tertiary nitrogen atom.
I composti dell’Invenzione evidenziano una maggiore efficacia rispetto ai suddetti principi nei test farmacologici indicativi dell’attività’ sulle basse vie urinarie, in particolare dell’attività’, contro le disfunzioni neuromuscolari delle basse vie urinarie,e sono dotati di un’affinità’ potente e selettiva per il recettore serotoninergico 5-HT!A· The compounds of the invention show greater efficacy than the aforementioned principles in pharmacological tests indicative of the activity 'on the lower urinary tract, in particular the activity', against neuromuscular dysfunctions of the lower urinary tract, and are endowed with an affinity ' potent and selective for the 5-HT serotonin receptor! A
Sotto un certo aspetto, l’invenzione riguarda i composti di formula I: From a certain point of view, the invention concerns the compounds of formula I:
<D <D
dove where is it
R rappresenta un gruppo cicloalchile o eteroarile, R represents a cycloalkyl or heteroaryl group,
R1 rappresenta un atomo di idrogeno o un gruppo alchile inferiore, A rappresenta un gruppo eteroarile, R1 represents a hydrogen atom or a lower alkyl group, A represents a heteroaryl group,
Z rappresenta un gruppo CH2, CHJCHJ, CH2C(O),CH2CH(OH),0,OCH2, C(0), ciascuno dei quali e’ raffigurato con l’estremità’ sinistra che e’quella che si collega all’anello piperazinico e l’estremità’ destra che e’ quella che si collega al gruppo B, B rappresenta un gruppo alchile, cicloalchile, arile ed eteroarile sostituiti e non, Z represents a group CH2, CHJCHJ, CH2C (O), CH2CH (OH), 0, OCH2, C (0), each of which is shown with the left end which is the one that connects to the piperazine ring and the right end which is the one that connects to group B, B represents a substituted and unsubstituted alkyl, cycloalkyl, aryl and heteroaryl group,
n e’ 1 o 2. n is 1 or 2.
L’invenzione riguarda anche i diastereoisomeri, gli enantiomeri, gli N-ossidi, i polimorfi, i solvatati e i sali farmaceuticamente accettabili di tali composti. The invention also relates to the diastereomers, enantiomers, N-oxides, polymorphs, solvates and pharmaceutically acceptable salts of these compounds.
L’invenzione riguarda inoltre le composizioni farmaceutiche comprendenti i composti di formula I o i diastereoisomeri, gli enantiomeri, gli N-ossidi, i polimorfi, i solvatati e i sali farmaceuticamente accettabili di tali composti in miscela con diluenti e veicoli farmaceuticamente accettabili. The invention also relates to pharmaceutical compositions comprising compounds of formula I or diastereoisomers, enantiomers, N-oxides, polymorphs, solvates and pharmaceutically acceptable salts of such compounds in admixture with pharmaceutically acceptable diluents and vehicles.
In un altro aspetto l’Invenzione riguarda l’uso delle composizioni per il trattamento dei pazienti affetti da disfunzioni neuromuscolari delle basse vie urinarie, in particolare mediante riduzione della frequenza delle contrazioni vescicali dovute alla distensione della vescica ed aumento della capienza della vescica, tale uso prevede la somministrazione al paziente di una quantità’ terapeuticamente efficace di uno o piu’ composti selezionati di formula I. In another aspect, the invention relates to the use of the compositions for the treatment of patients suffering from neuromuscular dysfunctions of the lower urinary tract, in particular by reducing the frequency of bladder contractions due to distension of the bladder and increasing the capacity of the bladder. provides for the administration to the patient of a therapeutically effective quantity of one or more selected compounds of formula I.
Sotto ancora un altro aspetto, l’Invenzione riguarda metodi di Interazione con i recettori serotonlnergici 5-HT1A e quindi, in virtù’ dell 'attività’ svolta a livello di questo recettore, il possibile impiego nel trattamento di disturbi del sistema nervoso centrale quali l ’ansia e la depressione, l’ipertensione, i disturbi del ciclo sonno/veglia, il comportamento alimentare e/o la funzionalità’ sessuale e 1 disturbi cognitivi nel mammiferi , in particolare nell’uomo. Under still another aspect, the invention relates to methods of interaction with the 5-HT1A serotoninergic receptors and therefore, by virtue of the activity carried out at the level of this receptor, the possible use in the treatment of central nervous system disorders such as anxiety and depression, hypertension, sleep / wake cycle disorders, eating behavior and / or sexual function, and cognitive disorders in mammals, particularly in humans.
DESCRIZIONE DETTAGLIATA DELL’INVENZIONE DETAILED DESCRIPTION OF THE INVENTION
Composti preferiti dell ' invenzione Preferred Compounds of the Invention
I gruppi cicloalchile R e B preferiti sono quelli con 5-7 atomi di carbonio. I gruppi eteroarile R, A e B sono preferibilmente gruppi aromatici mono o biciclici con 5-12 atomi di cui uno o piu’ sono eteroatoml (per esemplo azoto, ossigeno e zolfo) e i restanti sono atomi di carbonio. Preferred cycloalkyl groups R and B are those with 5-7 carbon atoms. The heteroaryl groups R, A and B are preferably mono or bicyclic aromatic groups with 5-12 atoms of which one or more are heteroatom (for example nitrogen, oxygen and sulfur) and the remaining are carbon atoms.
I gruppi alchile B sono preferibilmente gruppi alchile C2-C6 lineari ο ramificati. I gruppi arile B sono preferibilmente gruppi aromatici mono o biciclici con 6-12 atomi di carbonio (per esemplo gruppi fenile o naftUe). I sostituenti del gruppi arile ed eteroarlle possono contenere uno o piu’ atomi di alogenoo gruppi alchlle, alcossl, alogenoalcossi, ciano, carbamolle, adle, nitro, ammino, acilammlno, alchllsolfonllammino o alchllammlno. The alkyl B groups are preferably linear or branched C2-C6 alkyl groups. The aryl groups B are preferably mono or bicyclic aromatic groups with 6-12 carbon atoms (for example phenyl or naphthu groups). The substituents of the aryl and heteroarl groups may contain one or more halogen atoms or alkyl, alkoxy, haloalkoxy, cyano, carbamolle, adle, nitro, amino, acylamol, alkylsulfonllamino or alkylamino groups.
Sintesi del composti dell'Invenzione Synthesis of the Compounds of the Invention
In generale i composti dell'Invenzione possono essere preparati con 1 metodi Illustrati nel seguenti schemi d1 reazione, o versioni modificate degli stessi, ricorrendo a materie prime e reagenti facilmente reperibili e procedimenti di sintesi tradizionali. In general, the compounds of the invention can be prepared with the methods illustrated in the following reaction schemes, or modified versions thereof, by resorting to readily available raw materials and reagents and traditional synthesis processes.
Se non diversamente specificato, 1 diversi sostituenti del composti e del materiali presenti negli schemi di reazione sono definiti nello stesso modo in cui sono definiti sopra nella formula I. Unless otherwise specified, the different substituents of the compounds and materials present in the reaction schemes are defined in the same way as they are defined above in formula I.
Un procedimento per sintetizzare 1 composti di formula I e' rappresentato nello Schema 1. A process for synthesizing the compounds of formula I is represented in Scheme 1.
Le eteroarllammlne II (Y = NH2) possono essere alchllate con Idonei alcanl α,e -disostituiti III per dare gli Intermedi IV. Le reazioni possono essere eseguite 1n solvente aprotlco polare come N,N-d1metilformamm1de (DHF), tetraldrofurano (THF), diossano, acetone, acetonltrile o solventi clorurati come dlclorometano o cloroformio, a temperatura compresa tra 0°C e 120°C, 1n presenza di un accattone protonlco come trietilamm1na (EtjN), diisopropiletilemmina, ecc., eventualmente 1n presenza di potassio Ioduro. Negli Intermedi III, X e X, possono essere cloro, bromo, lodo, alchl1- o ariIsolfonllessi. Heteroarllamines II (Y = NH2) can be alkylated with suitable alkyl α, and -disubstituted III alkyls to give IV intermediates. The reactions can be carried out in a polar aprotlc solvent such as N, N-d1methylformamide (DHF), tetraldrofuran (THF), dioxane, acetone, acetonltril or chlorinated solvents such as dlchloromethane or chloroform, at a temperature between 0 ° C and 120 ° C, 1n presence of a protonic beggar such as triethylamine (EtjN), diisopropylethylamine, etc., possibly the presence of potassium iodide. In Intermediates III, X and X, they can be chlorine, bromine, lodo, alkyl1- or ariIsulfonllessi.
Schema1. Scheme 1.
Un altro metodo per sintetizzare gli intermedi IV utilizza II (Y = alogeno) come materie prime da far reagire con composti III in cui X e X.sono rispettivamente NH2 e OH. Queste reazioni di alchilazione sono eseguite in solvente polare aprotico come DMF,toluene ecc., o protico come n-BuOH, a temperatura compresa tra 40‘C e 140°C, normalmente usando un equivalente di eccesso di reagente III con X = NH2 quale accettore protonico, come da G. Doleschall et al., Tetrahedron, 32, 57-64 (1976). I suddetti amminoalcoli IV (X,= OH) sono fatti reagire con un agente clorurante come POC13, SOCl2 o PC15 per dare gli intermedi IV (Χ1 = Cl). Le reazioni sono eseguite in solvente aprotico come cloroformio, DMF, piridina, a temperatura compresa tra 50‘C e temperatura d1 ricadere. Another method for synthesizing intermediates IV uses II (Y = halogen) as raw materials to be reacted with compounds III in which X and X are NH2 and OH, respectively. These alkylation reactions are performed in aprotic polar solvent such as DMF, toluene etc., or protic such as n-BuOH, at a temperature between 40'C and 140 ° C, normally using an equivalent of excess reagent III with X = NH2 as proton acceptor, as by G. Doleschall et al., Tetrahedron, 32, 57-64 (1976). The above IV aminoalcohols (X, = OH) are reacted with a chlorinating agent such as POC13, SOCl2 or PC15 to give the IV intermediates (Χ1 = Cl). The reactions are carried out in an aprotic solvent such as chloroform, DMF, pyridine, at a temperature between 50'C and a reflux temperature.
Questi intermedi sono usati nell’alchilezione di idonei derivati piperazlnici V per dare gli intermedi VI. Le alchilazioni possono essere eseguite in solvente clorurato come diclorometano, cloroformio o 1,2-dicloroetano,o in solvente aprotico polare come DMF,THF, acetone, acetonitrile, ecc., o protico come n-butanolo,o in solvente apolare come toluene,benzene, n-eptano, ecc., a temperatura compresa tra 0°C e 120°C, eventualmente in presenza di un accettore protonico come Et3N, 4-dimet1lamminopiridina, potassio carbonato, cesio carbonato, eventualmente in presenza di potassio ioduro. These intermediates are used in the alkylation of suitable piperazlnic derivatives V to give the intermediates VI. The alkylations can be performed in chlorinated solvent such as dichloromethane, chloroform or 1,2-dichloroethane, or in polar aprotic solvent such as DMF, THF, acetone, acetonitrile, etc., or protic such as n-butanol, or in non-polar solvent such as toluene, benzene, n-heptane, etc., at a temperature between 0 ° C and 120 ° C, possibly in the presence of a proton acceptor such as Et3N, 4-dimeth1laminopyridine, potassium carbonate, cesium carbonate, possibly in the presence of potassium iodide.
Gli intermedi VI possono essere ottenuti per reazione di alchilazione di II (Y = NH2) con derivati VII, in cui B, R1, Z,X e n hanno lo stesso significato di cui sopra. Tali reazioni possono essere eseguite a temperatura di fusione senza solvente o in solvente aprotico come diclorometano, cloroformio, DMF,THF, acetone, acetonitrile,ecc., o protico come n-butanolo, a temperatura compresa tra 0°C e 160°C, eventualmente in presenza di un accettore protonico come Et3N, potassio o cesio carbonato, 4-dimetilamminopiridina ed eventualmente in presenza di potassio ioduro. Intermediates VI can be obtained by alkylation reaction of II (Y = NH2) with derivatives VII, wherein B, R1, Z, X and n have the same meaning as above. These reactions can be performed at melting temperature without solvent or in aprotic solvent such as dichloromethane, chloroform, DMF, THF, acetone, acetonitrile, etc., or protic such as n-butanol, at a temperature between 0 ° C and 160 ° C, possibly in the presence of a proton acceptor such as Et3N, potassium or cesium carbonate, 4-dimethylaminopyridine and possibly in the presence of potassium iodide.
Gli intermedi di formula VI sono acilati con Idonei acil cloruri per dare I in solventi aprotici come diclorometano, cloroformio, 1,2-dicloroetano, DMF, acetone, acetonitrile, toluene, ecc., a temperatura compresa tra 0°C e 100<*>C, eventualmente in presenza di una base organica quale accettore protonico come trietilamina, The intermediates of formula VI are acylated with suitable acyl chlorides to give I in aprotic solvents such as dichloromethane, chloroform, 1,2-dichloroethane, DMF, acetone, acetonitrile, toluene, etc., at a temperature between 0 ° C and 100 <* > C, possibly in the presence of an organic base as a proton acceptor such as triethylamine,
4-dimetilammino-piridina. 4-dimethylamino-pyridine.
Gi intermedi IV possono essere alternativamente adlati con metodi tradizionali con RC(0)C1 o altri reagenti di acilazione disponibili per dare gli intermedi IV’ che si fanno reagire con composti V, come detto sopra, per dare composti I. Gli intermedi V e/o VI o VII in cui Z - CH2CHCOH) sono ottenuti dagli stessi intermedi in cui Z = CH2C(O) per riduzione. Queste reazioni possono essere eseguite ricorrendo a idruri metallici come LiAlH^ (Rickborn J., J.Org. Chem. The IV intermediates can be alternatively adlated with traditional methods with RC (0) C1 or other available acylation reagents to give the IV intermediates which are reacted with compounds V, as mentioned above, to give compounds I. The intermediates V and / or VI or VII in which Z - CH2CHCOH) are obtained from the same intermediates in which Z = CH2C (O) by reduction. These reactions can be performed using metal hydrides such as LiAlH ^ (Rickborn J., J.Org. Chem.
35, 1041 (1970)), NaBH4, Na(0Ac)jBH (Gribble N.,Tetrahedron Lett. 24, 4287 (1983)), Zn(BH4)2 (Chakrabarty R.,Tetrahedron Lett. 31, 7663 (1990)),ecc., come agenti riducenti. Come solventi di reazione si possono impiegare dletiletere, THF,metanolo,etanolo,diossano e miscele degli stessi o altri idonei a temperatura tra 10°C e temperatura di ricadere. 35, 1041 (1970)), NaBH4, Na (0Ac) jBH (Gribble N., Tetrahedron Lett. 24, 4287 (1983)), Zn (BH4) 2 (Chakrabarty R., Tetrahedron Lett. 31, 7663 (1990) ), etc., as reducing agents. Dlethylether, THF, methanol, ethanol, dioxane and mixtures of the same or other suitable at temperatures between 10 ° C and reflux temperature can be used as reaction solvents.
In alternativa,queste riduzioni possono essere eseguite con i composti di formula I in cui Z = CH2C(0), a condizione che si usino litio tri-t-butossialluminio idruro (Endy L.,J.Org. Chem. 35, 549 (1970)) o altri agenti riducenti selettivi. Alternatively, these reductions can be carried out with the compounds of formula I in which Z = CH2C (0), provided that lithium tri-t-butoxyaluminium hydride is used (Endy L., J.Org. Chem. 35, 549 ( 1970)) or other selective reducing agents.
I composti finali I in cui Z = 0, OCH2 e B = arile possono essere preparati dai corrispondenti derivati N-ossidi dei composti I in cui l - legame o CH2 per riarrangiamento termica (isomerizzazione di Meisenheimer): The final compounds I in which Z = 0, OCH2 and B = aryl can be prepared from the corresponding N-oxide derivatives of compounds I in which l - bond or CH2 by thermal rearrangement (Meisenheimer isomerization):
Questa reazione può essere eseguita in solvente aprotico polare come diossano (Kbuthier A.H. et al.,J.Org. Chem.52, 1710-1713 (1987) e riferimenti bibliografici ivi citati), a temperatura compresa tra 60<*>C e temperatura di ricadere. This reaction can be carried out in a polar aprotic solvent such as dioxane (Kbuthier A.H. et al., J.Org. Chem. 52, 1710-1713 (1987) and references cited therein), at a temperature between 60 <*> C and temperature to relapse.
Un altro metodo per sintetizzare i composti di formula I e’ raffigurato nello schema 2. Another method for synthesizing the compounds of formula I is shown in diagram 2.
Schema 2. Scheme 2.
Gli intermedi IV,ottenuti come descritto nello Schema 1, possono essere usati per alchilare le piperazine monoprotette Vili,dove P rappresenta t-butossicarbonile, benzilossicarbonile, per dare gli intermedi IX. Parecchi esempi di protezione e deprotezione di diversi gruppi reattivi possono essere trovati in T. W. Greene: "Protective Groups in Organic Synthesis", Wiley Interscience (1991). The intermediates IV, obtained as described in Scheme 1, can be used to alkylate the monoprotected piperazines VIII, where P represents t-butoxycarbonyl, benzyloxycarbonyl, to give the intermediates IX. Several examples of protection and deprotection of different reactive groups can be found in T. W. Greene: "Protective Groups in Organic Synthesis", Wiley Interscience (1991).
Le condizioni di reazione per preparare gli intermedi IX sono le stesse riportate per la preparazione degli Intermedi VI nello schema 1. Analogamente, le reazioni di acilazione degli intermedi IX al fine di preparare gli Intermedi X possono essere eseguite nelle stesse condizioni adottate per slntetlzare i composti finali I nello Schema 1. The reaction conditions for preparing the intermediates IX are the same reported for the preparation of the Intermediates VI in scheme 1. Similarly, the acylation reactions of the intermediates IX in order to prepare the Intermediates X can be carried out under the same conditions adopted for the preparation of the compounds endings I in Scheme 1.
I derivati piperazinici XI ottenuti dalla deprotezione degli intermedi X con metodi noti possono essere alchilatl con B-Z-X, tranne per Z = 0, 0CH2 per dare i composti finali I. Queste reazioni possono essere eseguite in solvente aprotlco polare come DMF, acetone, acetonitri le, ecc. , o protico come n-BuOH o in solvente clorurato come dici orometano, cloroformio o 1 , 2— dici oroetano, a una temperatura compresa tra 10<*>C e 120’C e in presenza di un accettore protonico come potassio o cesio carbonato, Et3N, dimeti lammlnopi ridina, diisopropiletilammina. The piperazine derivatives XI obtained from the deprotection of the intermediates X by known methods can be alkylated with B-Z-X, except for Z = 0, 0CH2 to give the final compounds I. These reactions can be carried out in an aprotlcopolar solvent such as DMF, acetone, acetonitrile, etc. , or protic such as n-BuOH or in chlorinated solvent such as di oromethane, chloroform or 1,2 - diy goldethane, at a temperature between 10 <*> C and 120'C and in the presence of a proton acceptor such as potassium or cesium carbonate , Et3N, dimethylammylnopi ridine, diisopropylethylamine.
Un altro metodo per sintetizzare 1 composti di formula I in cui R1 = H e’ raffigurato nello Schema 3. Another method to synthesize 1 compounds of formula I in which R1 = H is shown in Scheme 3.
I composti eteroarile II (Y = alogeno) sono usati per al chi lare le amminoalchi laldeidi protette XII (X = NH2) per dare i corrispondenti acetall di eteroari lamminoalchl 1- aldeidi XIII. La reazione può’ essere eseguita in solvente aprotico polare come piridina, DMF, ecc. , o apolare come toluene a temperatura compresa tra 40 °C e 120°C, eventualmente in presenza di una base come Et3N. The heteroaryl compounds II (Y = halogen) are used for alkylating the protected aminoalkylaldehydes XII (X = NH2) to give the corresponding acetals of heteroarylaminoalkl 1- aldehydes XIII. The reaction can be performed in a polar aprotic solvent such as pyridine, DMF, etc. , or apolar as toluene at a temperature between 40 ° C and 120 ° C, possibly in the presence of a base such as Et3N.
Una tecnica di reazione alternativa per preparare gli intermedi XIII consiste nell’alchilare i composti eteroarile II (Y = ΝΗ;) con i composti protetti XII (X = Br) con metodi tradizionali o mediante l’aza-anione di II,ottenuto usando una base (per esempio n-butil litio, litio diisopropilammide, litio esametildisililammide, sodio idruro, sodio ammide) in solvente aprotico apolare o polare (per esempio toluene,THF). An alternative reaction technique for preparing intermediates XIII is to alkylate the heteroaryl compounds II (Y = ΝΗ;) with the protected compounds XII (X = Br) by traditional methods or by the aza-anion of II, obtained using a base (e.g. n-butyl lithium, lithium diisopropylamide, lithium hexamethyldisilylamide, sodium hydride, sodium amide) in apolar or polar aprotic solvent (e.g. toluene, THF).
Gli intermedi XIII possono essere acilati con RCOCI per dare gli intermedi d1 formula XIV usando le stesse condizioni di acilezione riportate piu’ sopra (Schemi 1 e 2). The intermediates XIII can be acylated with RCOCI to give the intermediates d1 formula XIV using the same conditions of acylection reported above (Schemes 1 and 2).
Questi intermedi sono stabili e sono deprotetti con metodi comuni (per esempio idrolisi acida) appena prima di essere usati nei passaggi successivi. These intermediates are stable and are deprotected by common methods (e.g. acid hydrolysis) just before being used in subsequent steps.
L’aldeide XV ottenuta dalla deprotezione di XIV può’ essere fatta reagire con idonea piperazina N-sostituita V in condizioni di amminazione riduttiva per dare i composti finali I (R1 = H). The aldehyde XV obtained from the deprotection of XIV can be reacted with suitable N-substituted piperazine V under conditions of reductive amination to give the final compounds I (R1 = H).
Queste reazioni possono essere eseguite in solvente polare come metanolo,etanolo o solvente clorurato come diciorometano, cloroformio, usando alcali boroidrurl come NaBH4, NaBH2CN, NaBH(OAc)3, eventualmente in presenza di un promotore acido come acido acetico a temperatura compresa tra 10 "C e 100’C.Quando si usa V (Z = CH2CO) si può’ ottenere la contemporanea riduzione della funzione chetonica ad alcolica usando NaBH4. These reactions can be performed in polar solvent such as methanol, ethanol or chlorinated solvent such as dichoromethane, chloroform, using borohydrurl alkali such as NaBH4, NaBH2CN, NaBH (OAc) 3, optionally in the presence of an acid promoter such as acetic acid at a temperature between 10 " C and 100'C. When using V (Z = CH2CO) it is possible to obtain the simultaneous reduction of the ketone function to alcohol using NaBH4.
In alternativa s1 possono far reagire le aldeidi XV con idonea piperazina N-protetta Vili usando le stesse condizioni riducenti riportate sopra per dare i composti finali I ed ottenendo gli intermedi X (R,= H). Alternatively, s1 can react the aldehydes XV with suitable N-protected piperazine VIII using the same reducing conditions described above to give the final compounds I and obtaining the intermediates X (R, = H).
I derivati piperazlnici XI ottenuti dalla deprotezione di X (R3 = H) con metodi comuni possono essere alchilati con B-2-X,tranne quando Z = 0,OCH2 per dare i composti finali I (R’,= H). Queste reazioni possono essere eseguite nelle condizioni riportate sopra per le reazioni dello Schema 2. The piperazlnic derivatives XI obtained from the deprotection of X (R3 = H) with common methods can be alkylated with B-2-X, except when Z = 0, OCH2 to give the final compounds I (R ', = H). These reactions can be performed under the conditions listed above for the reactions of Scheme 2.
Vengono qui di seguito forniti alcuni esempi che hanno il solo fine di meglio illustrare l’Invenzione in oggetto dimostrandone i vantaggi e l’appUcabillta’, senza tuttavia costituire una limitazione della stessa. Some examples are provided below which have the sole purpose of better illustrating the invention in question by demonstrating its advantages and the appUcabillta ', without however constituting a limitation thereof.
ESEMPIO 1 EXAMPLE 1
1-[N-cicloesilcarbonil-N-(2-piridil)-2-aminoetil]-4-(2,5-d1clorobenz1l)piperazina 1- [N-cyclohexylcarbonyl-N- (2-pyridyl) -2-aminoethyl] -4- (2,5-d1chlorobenz1l) piperazine
a) /tf-(2.2-d1metoss1etn)-N-(2-p1r1dn)c1cloesancarbossamm1de (1A) Ad una soluzione d1 5,97 g d1 2—(2,2-d1metoss1etilamm1no)-plrldina, preparata come descritto da Kaye I.A. et al., J. Am. Chem. Soc. 1951, 73, 5467, 1n 40 mL of tetraldrofurano, agitata a 0°C 1n atmosfera di azoto, furono aggiunti per gocciolamento 13,1 mL di butil litio (2,5 M 1n esano). Dopo rimozione del bagno refrigerante, la miscela fu agitata per 1 h a temperatura ambiente. Successivamente furono aggiunti per gocciolamento 4,46 mL di dcloesancarbonil cloruro. Dopo 5,5 h in agitazione a temperatura ambiente, furono aggiunti 2 mL d1 metanolo. La soluzione fu evaporata a secchezza sotto vuoto a dare un olio marroncino, che fu purificatomediante cromatografia flash (cloroformio-et1le acetato 7:3). Le frazioni contenenti il prodotto del titolo furono evaporate a secchezza sotto vuoto a dare 8,3 g (resa: 78* d.t.) del composto del titolo, utilizzabile senza ulteriore purlfIcazione 1n reazioni successive. a) /tf-(2.2-d1methox1etn)-N-(2-p1r1dn)c1cloesancarboxamm1de (1A) To a solution d1 5.97 g d1 2— (2,2-d1methox1ethylamm1no) -plrldine, prepared as described by Kaye I.A. et al., J. Am. Chem. Soc. 1951, 73, 5467, 1n 40 mL of tetraldrofuran, stirred at 0 ° C in nitrogen atmosphere, 13.1 mL of butyl lithium (2.5 M 1n hexane) were added dropwise. After removal of the cooling bath, the mixture was stirred for 1 h at room temperature. Subsequently 4.46 mL of dcloesancarbonyl chloride were added dropwise. After 5.5 h under stirring at room temperature, 2 mL of methanol was added. The solution was evaporated to dryness in vacuo to give a brownish oil, which was purified by flash chromatography (chloroform-ethyl acetate 7: 3). The fractions containing the title product were evaporated to dryness in vacuo to give 8.3 g (yield: 78 * a.d.) of the title compound, usable without further purification in subsequent reactions.
Attività del composti dell'Invenzione Activity of the Compound of the Invention
L'attività del composti dell’invenzione come Inibitori della frequenza dellaminzione e per aumentare la capacità vesciale il rende efficaci per la terapia delle disfunzioni neuromuscolari delle basse vie urinarle nel mammiferi, tra le quali, senza limitazioni, disuria, Incontinenza ed enuresi. The activity of the compounds of the invention as inhibitors of the frequency of amination and to increase the vascular capacity makes it effective for the therapy of neuromuscular dysfunctions of the lower urinary tract in mammals, including, without limitation, dysuria, incontinence and enuresis.
Le caratteristiche del composti dell’Invenzione conferiscono loro una potenza nettamente maggiore rispetto al farmaci di riferimento flavossato e imipremine, e un profilo d’azione diverso da quello dell’osslbutlnina. Tali dati sono stati raccolti sperimentando i suddetti composti in un modello di ratto in cui e’ stata Indotta la contrazione ritmica della vescica riempiendola con soluzione The characteristics of the compounds of the invention give them a much greater potency than the flavoxate and imipremin reference drugs, and an action profile different from that of oxlbutylin. These data were collected by experimenting with the aforementioned compounds in a rat model in which the rhythmic contraction of the bladder was induced by filling it with solution
fisiologica ed e’ stato valutato l’effetto dei composti in esame e degli standard di riferimento sulla frequenza e l’ampiezza delle contrazioni stesse, con particolare riguardo alla potenza nell'indurre la scomparsa delle contrazioni ritmiche. physiological and the effect of the compounds in question and of the reference standards on the frequency and amplitude of the contractions themselves was evaluated, with particular regard to the power in inducing the disappearance of rhythmic contractions.
Prima della presente invenzione, il trattamento delle disfunzioni neuromuscolari delle basse vie urinarie comportava la somministrazione di composti che agiscono direttamente sulla muscolatura della vescica, come il flavossato, farmaco spasmolitico attivo anche sul centro pontino della minzione, composti anticolinerglci come l’ossibutlnlna, e farmaci ad azione mista come l’lmipramina. Before the present invention, the treatment of neuromuscular dysfunctions of the lower urinary tract involved the administration of compounds that act directly on the bladder muscles, such as flavoxate, a spasmolytic drug also active on the pontoon center of urination, anticholinergic compounds such as oxybutylnlna, and drugs mixed action such as lmipramine.
Tuttavia, le terapie che comportano l’inibizione diretta della muscolatura pelvica (compreso Π detrusore) possono avere effetti collaterali indesiderabili quali svuotamento incompleto o paralisi da accomodazione, tachicardia e secchezza della bocca, e i farmaci come l’lmipramina possono avere effetti tossici rilevanti, in particolare sull’apparato cardiovascolare (ipotensione ortostatica, aritmia ventricolare), alle dosi terapeutiche. However, therapies involving direct inhibition of the pelvic musculature (including Π detrusor) may have undesirable side effects such as incomplete voiding or accommodation paralysis, tachycardia and dry mouth, and drugs such as imipramine may have significant toxic effects, particularly particularly on the cardiovascular system (orthostatic hypotension, ventricular arrhythmia), at therapeutic doses.
Sarebbe quindi auspicabile aumentare il numero di farmaci a disposizione del medico per il trattamento delle disfunzioni It would therefore be desirable to increase the number of drugs available to doctors for the treatment of dysfunctions
neuromuscolari delle basse vie urinarie. Anche gli effetti dei farmaci disponibili attualmente (flavossato, ossibutinina e imlpramina) sul modello di ratto e’ riportato nella Tabella 1. neuromuscular of the lower urinary tract. The effects of currently available drugs (flavoxate, oxybutynin and imlpramine) on the rat model are also shown in Table 1.
I composti dell’invenzione evidenziano una maggiore durata d’azione (per esempio durata della quiescenza della vescica senza contrazioni) rispetto al flavossato, all'ossibutinina e all’imipramina. The compounds of the invention show a longer duration of action (for example, duration of bladder quiescence without contractions) compared to flavoxate, oxybutynin and imipramine.
Inoltre, diversamente dall’ossibutinlna, 1 composti dell’invenzione non incidono sull'ampiezza delle contrazioni, a indicazione che non c’e’ compromissione della contrattilità’ della vescica.. Furthermore, unlike oxybutynlna, the compounds of the invention do not affect the amplitude of contractions, indicating that there is no compromise in the contractility of the bladder ..
Inoltre, l’effetto positivo sulle basse vie urinarie dei composti dell’Invenzione e’ stato dimostrato anche in un modello cistometrico nel ratto cosciente, in cui i composti dell’invenzione si comportano sempre favorevolmente rispetto al farmaci di riferimento, in Furthermore, the positive effect on the lower urinary tract of the compounds of the invention was also demonstrated in a cystometric model in the conscious rat, in which the compounds of the invention always behave favorably with respect to the reference drugs, in
particolare 1’ossibutinina (Tabella 2). Contrariamente a questo farmaco, infatti, 1 composti della presente invenzione non incidono sostanzialmente sulla pressione di minzione, escludendo il rischio di urina residua in una vescica a scarsa contrattilità’. particular 1 oxybutynin (Table 2). Contrary to this drug, in fact, the compounds of the present invention do not substantially affect the urination pressure, excluding the risk of residual urine in a bladder with poor contractility '.
Infine, la presenza di un’affinità’ elevata per il recettore 5-HT1A (Tabella 3) indica un ruolo importante di questo recettore nell’azione dei composti dell’Invenzione. Finally, the presence of a high affinity for the 5-HT1A receptor (Table 3) indicates an important role of this receptor in the action of the compounds of the invention.
I test farmacologici (e le tabelle) suddetti sono riportati nella sezione Dati Farmacologici. The above pharmacological tests (and tables) are reported in the Pharmacological Data section.
Applicazioni terapeutiche Therapeutic applications
I pazienti che necessitano di trattamento con questi composti e composizioni sono affetti da disfunzioni neuromuscolari delle basse vie urinarie, trattate da E.J.McGuire in "Campbell’s UROLOGY" 5° Ed., 616-638, 1986, W.B. Saunders Company, e sono inoltre quelli affetti dalle disfunzioni associate alla compromissione della funzionalità’ del recettore 5-HT1A. Patients requiring treatment with these compounds and compositions are affected by neuromuscular dysfunctions of the lower urinary tract, treated by E.J. McGuire in "Campbell's UROLOGY" 5th Ed., 616-638, 1986, W.B. Saunders Company, and are also those affected by the dysfunctions associated with impaired functionality of the 5-HT1A receptor.
La presente invenzione comprende le formulazioni farmaceutiche che contengono i composti sopra elencati, nonché’ i metodi che impiegano tali formulazioni per il trattamento delle disfunzioni neuromuscolari delle basse vie urinarie quali disuria, incontinenza ed enuresi. La disuria comprende la frequenza urinaria, la nicturia e l’impellenza. Le sindromi d’incontinenza comprendono incontinenza da sforzo, incontinenza da impellenza e incontinenza da travaso. L’enuresi riguarda il passaggio involontario d1 urina la notte o durante 11 sonno. The present invention includes the pharmaceutical formulations that contain the compounds listed above, as well as the methods that use these formulations for the treatment of neuromuscular dysfunctions of the lower urinary tract such as dysuria, incontinence and enuresis. Dysuria includes urinary frequency, nocturia and urgency. Incontinence syndromes include stress incontinence, urge incontinence and transfer incontinence. Bedwetting concerns the involuntary passing of urine at night or during sleep.
Senza voler essere vincolati dalla teoria, si ritiene che la somministrazione di antagonisti del recettore 5-HT1A prevenga Without wishing to be bound by theory, the administration of 5-HT1A receptor antagonists is believed to prevent
l'attività’ indesiderabile dell’arco riflesso sacrale e/o dei the undesirable activity of the sacral reflex arch and / or
meccanismi corticali che regolano la minzione. Si prevede quindi che un’ampia gamma di disfunzioni neuromuscolari delle basse vie urinarie possano essere trattate con i composti della presente invenzione. cortical mechanisms that regulate urination. It is therefore expected that a wide range of neuromuscular dysfunctions of the lower urinary tract can be treated with the compounds of the present invention.
La"quantità’ efficace” di composto per trattare i disturbi urinari e’ una quantità’ che produce un miglioramento apprezzabile di almeno un sintomo o parametro del disturbo. The "effective amount" of compound to treat urinary disorders is a quantity that produces an appreciable improvement in at least one symptom or parameter of the disorder.
I disturbi delle vie urinarie e i relativi sintomi sono urgenza, frequenza, incontinenza, perdita di urina, enuresi, disuria, Urinary tract disorders and related symptoms are urgency, frequency, incontinence, urine loss, enuresis, dysuria,
difficolta’ di minzione e difficolta’ di svuotamento della vescica. difficulty urinating and difficulty emptying the bladder.
Un ulteriore parametro e’ il volume di urina. La quantità’ efficace per trattare il disturbo può’ essere trovata con esperimenti noti nell’arte, come stabilire una matrice di dosi e frequenze e confrontare un gruppo di unita’ sperimentali o soggetti in Another parameter is the volume of urine. The effective amount to treat the disorder can be found with experiments known in the art, such as establishing a matrix of doses and frequencies and comparing a group of experimental units or subjects in
corrispondenza di ciascun punto della matrice. La quantità’ esatta da somministrare al paziente può’ variare a seconda dello stato e della gravita’ del disturbo e della condizione fisica del paziente, il miglioramento apprezzabile dei sintomi e dei parametri può’ essere stabilito da un medico esperto nell’arte o segnalato dal paziente al medico. E’ sottinteso che l’attenuazione clinicamente o statisticamente rilevante di sintomi e parametri rientra negli scopi dell’invenzione. Attenuazione clinicamente rilevante significa percettibile al paziente e/o al medico. correspondence of each point of the matrix. The exact quantity to be administered to the patient can vary according to the state and severity of the disorder and the physical condition of the patient, the appreciable improvement of symptoms and parameters can be established by a doctor skilled in the art or reported by the patient to the doctor. It is understood that the clinically or statistically significant attenuation of symptoms and parameters falls within the scope of the invention. Clinically relevant attenuation means perceptible to the patient and / or physician.
I composti della presente invenzione possono essere formulati in forme farmaceutiche liquide con un veicolo fisiologicamente accettabile come, per esempio, soluzione fisiologica tamponata con fosfato o acqua delonlzzata. La formulazione farmaceutica può’ contenere anche eccipienti, compresi conservanti e stabilizzanti, che sono ben noti nell’arte. I composti possono essere formulati in unita’ farmaceutiche solide orali o non orali quali, per esempio, compresse, capsule, polveri e supposte, e possono inoltre contenere eccipienti, compresi senza limitazioni lubrificanti, plasticizzanti, coloranti, promotori dell’assorbimento, battericidi e slmili. The compounds of the present invention can be formulated in liquid pharmaceutical forms with a physiologically acceptable carrier such as, for example, phosphate buffered saline or delonated water. The pharmaceutical formulation may also contain excipients, including preservatives and stabilizers, which are well known in the art. The compounds may be formulated in oral or non-oral solid pharmaceutical units such as, for example, tablets, capsules, powders and suppositories, and may also contain excipients, including without limitation lubricants, plasticizers, dyes, absorption promoters, bactericides and similar. .
I modi di somministrazione sono la via orale ed enterale, endovenosa, intramuscolare, sottocutanea, transdermica, transmucosica (compreso boccale e rettale) e per inalazione. Preferibilmente, si usa la via orale o transdermica (ossia, rispettivamente con formulazioni orali solide o liquide o con cerotti cutanei). The methods of administration are oral and enteral, intravenous, intramuscular, subcutaneous, transdermal, transmucosal (including mouth and rectal) and inhalation routes. Preferably, the oral or transdermal route is used (i.e., with solid or liquid oral formulations or with skin patches, respectively).
La quantità’ d1 agente da somministrare può’ variare da circa 0,01 a circa 25 mg/kg/die, preferibilmente da circa 0,1 a circa 10 mg/kg/die e molto preferibilmente circa 0,2-5 mg/kg/die. E’ inteso che le formulazioni farmaceutiche della presente invenzione non necessitano di per se’ di iontenere la quantità’ intera di agente efficace nel trattamento del disturbo, poiché’ tale quantità’ efficace può’ essere raggiunta con la somministrazione di una pluralità’ di dosi delle formulazioni farmaceutiche stesse. The amount of agent to be administered can range from about 0.01 to about 25 mg / kg / day, preferably from about 0.1 to about 10 mg / kg / day and most preferably about 0.2-5 mg / kg. / day. It is understood that the pharmaceutical formulations of the present invention do not need per se to retain the entire amount of agent effective in treating the disorder, since this effective amount can be achieved by administering a plurality of doses of the pharmaceutical formulations themselves.
In una delle versioni preferite della presente invenzione, i composti sono formulati in capsule o compresse, ciascuna preferibilmente contenente 50-200 mg di composti, e sonomolto preferibilmente somministrati al paziente alla dose giornaliera totale di 50-400 mg, preferibilmente 150-250 mg, e molto preferibilmente 200 mg, per la cura dell'incontinenza urinaria e delle disfunzioni assodate alla compromissione dei recettori 5—HT1A. In one of the preferred versions of the present invention, the compounds are formulated in capsules or tablets, each preferably containing 50-200 mg of compounds, and are most preferably administered to the patient at a total daily dose of 50-400 mg, preferably 150-250 mg, and very preferably 200 mg, for the treatment of urinary incontinence and established dysfunctions due to the compromise of 5-HT1A receptors.
I metodi, le tabelle e gli esempi forniti d1 seguito hanno lo scopo di illustrare piu' esaurientemente le versioni preferite dell’invenzione dimostrandone i vantaggi e 1’applicabillta’, ma senza limitarne la portata. The methods, tables and examples provided below are intended to more fully illustrate the preferred versions of the invention, demonstrating the advantages and applicability, but without limiting its scope.
Dati farmacologici Effetti sulle contrazioni ritmiche di svuotamentovesdcale indottedal volume nel ratto anestetizzato A. Metodi: Pharmacological data Effects on rhythmic volume-induced voiding contractions in the anesthetized rat A. Methods:
Sono stati usati ratti Sprague Dawley femmine del peso di 225-275 g (Cri: CDo BR, Charles River, Italia). Gli animali sono stati alloggiati con libero accesso a cibo e acqua e mantenuti 1n un ciclo forzato di 12 ore di alternanza luce/bu1o a 22-24‘C per almeno una settimana, tranne durante l’esperimento. L’attività’ sulle contrazioni ritmiche di svuotamento vescicale e’ stata valutata secondo il metodo di Dray (J. Pharmacol. Methods, 13:157, 1985), con alcune modifiche come in Guarneri (Pharmacol. Res., 27:173, 1993). In breve, i ratti sono stati anestetizzati con iniezione sottocutanea di 1,25 g/kg (5 ml/kg) di uretano, dopo di che e’ stata cateterizzata la vescica attraverso l’uretra usando un tubo di polietilene PE 50 riempito di soluzione fisiologica. Il catetere e' stato fissato in posizione con una legatura attorno all’orifizio uretrale esterno e collegato a un comune trasduttore di pressione (Statham P23 ID/P23 XL). La pressione endovescicale e’ stata visualizzata 1n continuo su un registratore a nastro (Battaglia Rangoni KV 135 con amplificatore DC1/TI). E’ stata quindi riempita la vescica attraverso il catetere di registrazione con volumi incrementali di soluzione fisiologica tiepida (37°C) fino a comparsa delle contrazioni riflesse di svuotamento (in genere 0,8-1,5 mi). Per l’Iniezione endovenosa (i.v.) dei composti allo studio, e’ stato inserito nella vena giugulare un tubo di polietilene PE 50 riempito di soluzione fisiologica. Female Sprague Dawley rats weighing 225-275 g were used (Cri: CDo BR, Charles River, Italy). The animals were housed with free access to food and water and maintained in a forced cycle of 12 hours of light / dark alternation at 22-24 'C for at least one week, except during the experiment. The activity on rhythmic contractions of bladder emptying was evaluated according to the Dray method (J. Pharmacol. Methods, 13: 157, 1985), with some modifications as in Guarneri (Pharmacol. Res., 27: 173, 1993) ). Briefly, rats were anesthetized with subcutaneous injection of 1.25 g / kg (5 ml / kg) of urethane, after which the bladder was catheterized through the urethra using a solution-filled PE 50 polyethylene tubing. physiological. The catheter was fixed in place with a ligature around the external urethral orifice and connected to a common pressure transducer (Statham P23 ID / P23 XL). Intravesical pressure was displayed continuously 1n on a tape recorder (Battaglia Rangoni KV 135 with DC1 / TI amplifier). The bladder was then filled through the registration catheter with incremental volumes of warm saline (37 ° C) until the reflex contractions of emptying appeared (generally 0.8-1.5 ml). For the intravenous injection (i.v.) of the compounds under study, a PE 50 polyethylene tube filled with physiological solution was inserted into the jugular vein.
Dal cistometrogramma sono stati ricavati il numero di contrazioni registrate 15 minuti prima (valore basale) e dopo il trattamento e l’ampiezza media delle contrazioni stesse (altezza media del picco in mmHg). The number of contractions recorded 15 minutes before (baseline value) and after treatment and the average amplitude of the contractions themselves (average height of the peak in mmHg) were obtained from the cystometrogram.
Poiché’ la maggior parte dei composti produce un effetto che insorge con relativa rapidità’ e porta alla completa cessazione delle contrazioni vescicali, la bioattivita’ e’ stata valutata facilmente misurando la durata della quiescenza della vescica (ossia, il tempo in cui non vi sono contrazioni). E’ stato inoltre registrato il numero di animali esaminati che evidenziano una riduzione del numero di Since 'most compounds produce an effect that occurs relatively rapidly' and lead to the complete cessation of bladder contractions, bioactivity has been easily assessed by measuring the duration of bladder quiescence (i.e., the time in which there are no contractions). The number of animals examined was also recorded, showing a reduction in the number of
contrazioni < 30% rispetto a quelle osservate nel periodo basale. contractions <30% of those observed in the baseline period.
Per confrontare la potenza dei composti sperimentati nell'inibire le contrazioni di svuotamento vescicale, sono state calcolate le dosi equiefficaci che producono 10 minuti di tempo di scomparsa (ED1min) mediante analisi della regressione lineare col metodo dei minimi quadrati, nonché’ le dosi estrapolate che inducono una riduzione del numero d1 contrazioni < 30% nel 50% dei ratti trattati (ED50, frequenza) con il metodo di BUss (Bliss C.I., Quart. J. Pharm. Pharmacol. H, 192-216, 1938). Dopo la cessazione dell'effetto dell’iniezione di farmaco, e’ stata confrontata l’altezza dei picchi con quella precedentemente registrata dopo la somministrazione endovenosa del solo veicolo. E’ stata valutata la potenza dei composti esaminati (valore ED50: dosi estrapolate che inducono il 30% di riduzione dell’ampiezza delle contrazioni nel 50% dei ratti trattati) su base quantitativa col metodo di Bliss (BUss C.I., Quart. J. Pharm. Pharmacol. 11., 192-216, 1938). To compare the potency of the tested compounds in inhibiting bladder emptying contractions, the equieeffective doses that produce 10 minutes of time to disappearance (ED1min) were calculated by linear regression analysis with the least squares method, as well as' the extrapolated doses that induce a reduction of the number of contractions <30% in 50% of the rats treated (ED50, frequency) with the method of BUss (Bliss C.I., Quart. J. Pharm. Pharmacol. H, 192-216, 1938). After the cessation of the effect of the drug injection, the height of the peaks was compared with that previously recorded after the intravenous administration of the vehicle alone. The potency of the compounds examined was evaluated (ED50 value: extrapolated doses that induce a 30% reduction in the amplitude of contractions in 50% of the treated rats) on a quantitative basis with the Bliss method (BUss C.I., Quart. J. Pharm . Pharmacol. 11, 192-216, 1938).
B. Risultati B. Results
La rapida distensione della vescica nel ratto anestetizzato con uretano produce una serie di contrazioni ritmiche di svuotamento le cui caratteristiche sono pubblicate (Maggi et al., Brain Res., 380:83, 1986; Maggi et al., J. Pharmacol. Exp. Ther., 230:500, 1984). La frequenza delle contrazioni e’ associata al braccio sensitivo afferente del riflesso della minzione e all’integrità’ del centro della minzione, mentre la loro ampiezza e’ una proprietà’ del braccio efferente del riflesso. In questo modello, i composti che agiscono principalmente sul sistema nervoso centrale (come la morfina) provocano un blocco delle contrazioni di svuotamento, mentre i farmaci che agiscono a livello del detrusore, come l ’ossibutinlna, riducono l ’ampiezza delle contrazioni. Rapid distension of the bladder in the urethane anesthetized rat produces a series of rhythmic voiding contractions whose characteristics are published (Maggi et al., Brain Res., 380: 83, 1986; Maggi et al., J. Pharmacol. Exp. Ther., 230: 500, 1984). The frequency of contractions is associated with the afferent sensitive arm of the urination reflex and the integrity of the urination center, while their amplitude is a property of the efferent reflex arm. In this model, the compounds that act mainly on the central nervous system (such as morphine) cause a block of the emptying contractions, while the drugs that act at the detrusor level, such as oxybutynlna, reduce the amplitude of the contractions.
I risultati ottenuti con i composti in esame sono riportati nella Tabella 1. The results obtained with the compounds under examination are reported in Table 1.
TABELLA 1 TABLE 1
Effetti sulle contrazioni ritmiche di svuotamento yescicale dopo soirani ni strazione endovenosa Effects on rhythmic contractions of yesical emptying after intravenous soirani nostration
I dati rappresentano i valori di ED1 min (dose estrapolata che induce 10 minuti di scomparsa delle contrazioni); i valori di ED50 (frequenza) (dose estrapolata che induce una riduzione del numero di contrazioni < 30* nel 50% dei ratti trattati) e i valori di ED50 (ampiezza) (dose estrapolata che induce una riduzione del 30% dell’ampiezza delle contrazioni nel 50% dei ratti trattati) The data represent the values of ED1 min (extrapolated dose that induces 10 minutes of disappearance of contractions); the values of ED50 (frequency) (extrapolated dose that induces a reduction in the number of contractions <30 * in 50% of the rats treated) and the values of ED50 (amplitude) (extrapolated dose that induces a 30% reduction in the amplitude of contractions in 50% of treated rats)
n.a. = non attivo; nessuna riduzione Importante dell’altezza del picchi I composti della presente invenzione si dimostrano nettamente piu’ potenti degli standard di riferimento (imipramina, flavossato, ossibutinina) nell'inibire le contrazioni vescicali. Inoltre non incidono sulla contrattilità’ della vescica (nessuna riduzione dell’ampiezza delle contrazioni), diversamente dall'ossibutinina e dall’imipramlna, escludendo il rischio della presenza di notevoli volumi residui di urina. n.a. = not active; no Important reduction in peak height The compounds of the present invention prove to be clearly more powerful than the reference standards (imipramine, flavoxate, oxybutynin) in inhibiting bladder contractions. They also do not affect the contractility of the bladder (no reduction in the amplitude of contractions), unlike oxybutynin and imipramlna, excluding the risk of the presence of large residual volumes of urine.
Effetto sui parametri cistometrici nel ratto conscio Effect on cystometric parameters in the conscious rat
A. Metodi A. Methods
Sono stati usati ratti Sprague Dawley maschi (Cri: CDo BR) del peso di 250-350 g. Gli animali sono stati alloggiati con libero accesso a cibo e acqua e mantenuti in un ciclo forzato di alternanza luce/buio di 12 ore a 22-24"C per almeno una settimana, tranne durante lo svolgimento dell’esperimento. Per quantificare i parametri urodinamici nel ratto conscio, sono stati eseguiti studi cistometrografici con tecniche riportate in precedenza (Guarneri et al., Pharmacol. Res., 24:175, 1991). I ratti maschi sono stati anestetizzati con nembutal (30 mg/kg) e cloralio idrato (125 mg/kg) i.p. e posti in posizione supina. Male Sprague Dawley rats (Cri: CDo BR) weighing 250-350 g were used. The animals were housed with free access to food and water and kept in a forced 12-hour light / dark cycle at 22-24 "C for at least one week, except during the experiment. To quantify urodynamic parameters. in the conscious rat, cystometrographic studies were performed with previously reported techniques (Guarneri et al., Pharmacol. Res., 24: 175, 1991). Male rats were anesthetized with nembutal (30 mg / kg) and chloral hydrate ( 125 mg / kg) i.p. and placed in the supine position.
E’ stata fatta un’incisione di circa 10 mm lungo la linea mediana nella parete addominale rasata e detersa. La vescica e’ stata An incision of about 10 mm was made along the midline in the shaved and cleansed abdominal wall. The bladder was
delicatamente liberata dai tessuti aderenti, svuotata e quindi incannulata attraverso un’incisione nella cupola con una cannula di polietilene (Portex PP30) suturata permanentemente con filo di seta. La cannula e’ stata esteriorizzata attraverso un tunnel sottocutaneo nella regione retroscapolare, dove e’ stata collegata a un adattatore in plastica per evitare il rischio di asportazione da parte dell’animale. gently freed from adherent tissues, emptied and then cannulated through an incision in the dome with a polyethylene cannula (Portex PP30) permanently sutured with silk thread. The cannula was externalized through a subcutaneous tunnel in the retroscapular region, where it was connected to a plastic adapter to avoid the risk of removal by the animal.
Per l’iniezione endovenosa (i.v.) dei composti in esame e’ stato inserito nella vena giugulare un tubo di polietilene PE 50 riempito di soluzione fisiologica esteriorizzandolo nella regione retroscapolare. I ratti sono stati utilizzati esclusivamente un giorno dopo l’impianto. Il giorno dell’esperimento 1 ratti sono stati messi in gabbie di Bollman; dopo un periodo di stabilizzazione di 20 minuti, l’estremità’ libera del catetere vescicale e’ stata collegata tramite tubo a T a un trasduttore di pressione (Bentley T 800/Marb P 82) e a una pompa peristaltica (Gllson minipuls 2) per l’Infusione continua nella vescica di soluzione fisiologica alla velocita’ costante di 0,1 ml/min.. Il segnale della pressione endoluminale durante l’infusione e’ stato registrato in continuo con un poligrafo (Battaglia Rangoni K0380 con amplificatore ADC1/T). Sono stati valutati due parametri urodinamici: la capienza della vescica (BVC) e la pressione di minzione (MP). Per BVC (in mi) s1 intende il volume minimo infuso dopo il quale interviene la contrazione del detrusore (seguita dalla minzione). Per MP (1n mmHg) si intende la pressione endovescicale massima indotta dalla contrazione del detrusore durante la minzione. I valori basali di BVC e MP sono stati calcolati come media dei primi due cistometrogramnii registrati. A questo punto, l’infusione e’ stata interrotta e sono stati For the intravenous injection (i.v.) of the compounds under examination, a PE 50 polyethylene tube filled with physiological solution was inserted into the jugular vein, exteriorizing it in the retroscapular region. The rats were used only one day after implantation. On the day of the experiment 1 rats were placed in Bollman's cages; after a stabilization period of 20 minutes, the free end of the bladder catheter was connected via T-tube to a pressure transducer (Bentley T 800 / Marb P 82) and to a peristaltic pump (Gllson minipuls 2) for Continuous infusion of physiological solution into the bladder at a constant rate of 0.1 ml / min. The signal of the intraluminal pressure during the infusion was recorded continuously with a polygraph (Battaglia Rangoni K0380 with ADC1 / T amplifier). Two urodynamic parameters were evaluated: bladder capacity (BVC) and urination pressure (MP). By BVC (in mi) s1 means the minimum volume infused after which contraction of the detrusor occurs (followed by urination). MP (1n mmHg) refers to the maximum intravesical pressure induced by the contraction of the detrusor during urination. Baseline values of BVC and MP were calculated as the mean of the first two recorded cystometrograms. At this point, the infusion was stopped and they were
somministrati 1 composti in esame. Quindici minuti dopo la administered 1 test compound. Fifteen minutes after the
somministrazione endovenosa sono stati registrati altri due two more were recorded intravenously
cistometrogrammi per ciascun animale e calcolati i valori medi dei due parametri cistometrografid. La rilevanza statistica delle differenze tra i valori dei parametri urodinamici e’ stata analizzata mediante test t di Student per dati appaiati. cystometrograms for each animal and the mean values of the two cystometrografid parameters calculated. The statistical significance of the differences between the values of the urodynamic parameters was analyzed using Student's t test for paired data.
B. Risultati B. Results
L’effetto delle diverse dosi del composti esaminati e’ riportato nella tabella 2. Il composto dell’Es. 1 presenta un profilo slmile al flavossato nell'aumentare la BVC. La contrattilita’ della vescica non risulta compromessa non osservandosi variazioni rilevanti della MP. Al contrarlo, l’ossibutlnlna riduce sensibilmente e 1n modo dosedlpendente la MP senza effetti sulla BVC. The effect of the different doses of the compounds examined is shown in table 2. The compound of Ex. 1 exhibits a similar profile to flavoxate in increasing BVC. The contractility of the bladder is not compromised since no significant changes in PD are observed. By contracting it, oxybutylin significantly and dose-reducing significantly reduces MP without effects on BVC.
TABELLA 2 TABLE 2
Effetti sul cistometrogramma nel ratto conscio Effects on the cystometrogram in the conscious rat
*=p<0,05, **=P<0,01 rispetto al valori basali; * = p <0.05, ** = P <0.01 compared to baseline values;
rispetto al valori basali; compared to baseline values;
test t diStudent per dati appaiati Student's t test for paired data
I dati rappresentano i valori medi ± S.E.della capienza vesdcale (BVC,mi) e della pressione di minzione (MP,mmHg) prima,e 15 minuti dopo iniezione endovenosa dei composti. The data represent the mean ± S.E. values of vesdcal capacity (BVC, mi) and urination pressure (MP, mmHg) before, and 15 minutes after intravenous injection of the compounds.
Legame radiorecettoriale al recettore 5-ΗΤμ e altri siti di legame a diversi neurotrasmettltori Radioreceptor binding to the 5-ΗΤμ receptor and other binding sites to various neurotransmitters
A. Metodi A. Methods
Recettori 5HT1A ricombinanti umani Human recombinant 5HT1A receptors
La codifica genomica del clone G-21 del recettore serotoninergico 5-HT1A umano e’stabilmente trasferita 1n una linea cellulare umana (HeLa).Sono state coltivate delle cellule HeLa in monostrati in terreno Eagle modificato di Dulbecco (DMEM) integrato con siero di feto di vitello al 10% e gentamlcina (100 mg/ml),CO2 al 5% a 37'C. Le cellule sono state staccate dalla fiasca d1 coltivazione al 95% di confluenza con un raccoglitore cellulare e sottoposte a Usi in tampone ghiacciato Tris-HCl 5mM-EDTA 5mM (pH 7,4).Gli omogenati sono stati centrifugati a 40000 x g per 20 minuti e le membrane sono state risospese in un piccolo volume di tampone ghiacciato Tris-HCl 5mM-EDTA 5mM (pH 7,4) e immediatamente congelate e conservate a -70°C fino al momento dell’uso. Il giorno dell’esperimento le membrane cellulari sono state risospese in tampone legante: Tris-HCl 50mM (pH 7,4),MgCl2 2,5mM, pargilina 10μΜ (Fargln et al., Nature 335, 358-360, 1988). Le membrane sono state incubate in un volume finale di 1mi per 30 minuti a 30 "C con [<3>H]8-0H-DPAT 0,2-1 nM in assenza o presenza di sostanze allo studio; il legame non specifico e’ stato stabilito in presenza di 5-HT 10 μΜ. L’incubazione e’ stata arrestata con l’aggiunta di tampone Tris-HCl ghiacciato e filtrazione rapida attraverso filtri Whatman GF/B o Schleicher & Schuell GF52 protrattati con poiietilenimmina 0,2%. Genomic encoding of human 5-HT1A serotonin receptor G-21 clone is stably transferred to human cell line (HeLa) HeLa cells were grown in monolayers in Dulbecco's modified Eagle medium (DMEM) supplemented with fetal serum veal at 10% and gentamicin (100 mg / ml), CO2 at 5% at 37'C. Cells were detached from the 95% confluence cultivation flask with a cell collector and subjected to 5mM Tris-HCl ice-cold buffer 5mM-EDTA 5mM (pH 7.4). The homogenates were centrifuged at 40,000 x g for 20 minutes and the membranes were resuspended in a small volume of ice-cold 5mM-EDTA 5mM Tris-HCl buffer (pH 7.4) and immediately frozen and stored at -70 ° C until use. On the day of the experiment, the cell membranes were resuspended in binding buffer: Tris-HCl 50mM (pH 7.4), MgCl2 2.5mM, pargiline 10μΜ (Fargln et al., Nature 335, 358-360, 1988). Membranes were incubated in a final volume of 1ml for 30 minutes at 30 "C with [<3> H] 8-0H-DPAT 0.2-1 nM in the absence or presence of substances under study; non-specific binding and was established in the presence of 5-HT 10 μΜ. Incubation was stopped with the addition of ice-cold Tris-HCl buffer and rapid filtration through Whatman GF / B or Schleicher & Schuell GF52 filters extended with 0.2% polyethyleneimine. .
Recettori a1-adrenergici nativi (tessuti animali) Native a1-adrenergic receptors (animal tissues)
Gli studi di legame sui recettori a, adrenergici nativi sono stati eseguiti con membrana di corteccia cerebrale di ratto. Sono stati uccisi per lussazione cervicale dei ratti maschi Sprague Dawley (200-300 g, Charles River, Italia) e sono state dissecate, immediatamente congelate e conservate a -70‘C fino al momento dell’uso le cortecce cerebrali. I tessuti sono stati omogenati (2 x 20 sec.) in 50 volumi di tampone Tris-HCl 50mM freddo pH 7,4 con un omogeneizzatore Politron (velocita’ 7). Gli omogenati sono stati centrifugati a 48000 x g per 10 minuti, risospesi in 50 volumi dello stesso tampone, incubati a 37°C per 15 minuti e centrifugati e risospesi altre due volte. Le membrane finali sono state sospese 1n 100 volumi di tampone Tris-HCl 50mM pH 7,4 contenente pargilina 10μΜ e acido ascorbico 0,1%. Le membrane sono state incubate in un volume finale di 1 mi per 30 minuti a 25°C con [H]prazosina 0,1-0,5nM, in assenza o presenza di sostanze concorrenti. Il legame non specifico e' stato stabilito in presenza di fentolamina 10μΜ. L’incubazione e’ stata fermata con l’aggiunta di tampone Tris-HCl 50mM ghiacciato e filtrazione rapida attraverso filtri Whatman GF/B o Schleicher & Schuell GF52 pretrattati con Binding studies on native α-adrenergic receptors were performed with rat cerebral cortex membrane. Male Sprague Dawley rats (200-300 g, Charles River, Italy) were killed by cervical dislocation and the cerebral cortices were dissected, immediately frozen and stored at -70'C until use. The tissues were homogenized (2 x 20 sec.) In 50 volumes of cold 50mM Tris-HCl buffer pH 7.4 with a Politron homogenizer (speed 7). The homogenates were centrifuged at 48000 x g for 10 minutes, resuspended in 50 volumes of the same buffer, incubated at 37 ° C for 15 minutes and centrifuged and resuspended two more times. The final membranes were suspended in 100 volumes of 50mM pH 7.4 Tris-HCl buffer containing 10μΜ pargiline and 0.1% ascorbic acid. The membranes were incubated in a final volume of 1ml for 30 minutes at 25 ° C with 0.1-0.5nM [H] prazosin, in the absence or presence of competing substances. Non-specific binding was established in the presence of 10μΜ phentolamine. Incubation was stopped with the addition of ice-cold 50mM Tris-HCl buffer and rapid filtration through Whatman GF / B or Schleicher & Schuell GF52 filters pretreated with
polietllenimmina 0,2*. I filtri sono stati poi lavati con tampone ghiacciato e la radioattivita’ trattenuta dai filtri e’ stata valutata mediante spettrometria per scintillazione liquida. polyethyleneimine 0.2 *. The filters were then washed with ice-cold buffer and the radioactivity retained by the filters was evaluated by liquid scintillation spectrometry.
Recettori a2-adrenergid nativi (tessuti animali) Native a2-adrenergid receptors (animal tissues)
Gli studi di legame sui recettori adrenergici nativi (Diop L. et al., J. Neurochem. 41· 710-715, 1983) sono stati eseguiti con membrana di corteccia cerebrale di ratto. Dei ratti maschi Sprague Dawley (200-300 g, SO Harlan/Nossan, Italia) sono stati uccisi per lussazione cervicale e ne sono state asportate, Immediatamente congelate e conservate a ~70'C fino al momento dell’uso le cortecce cerebrali. I tessuti sono stati omogenati (2 x 20 sec.) in 50 volumi di tampone Tris-HCl 50mM freddo pH 7,4 con un omogeneizzatore Politron (velocita’ 7). Gl1 omogenati sono stati centrifugati a 49000 x g per 10 minuti, risospesi in 50 volumi dello stesso tampone, incubati a 37°C per 15 minuti e centrifugati e risospesi altre due volte. Le membrane finali sono state sospese in 100 volumi di tampone Tris-HCl 50mM pH 7,4 contenente pargilina 10μΜ e acido ascorbico 0,1%, Le membrane sono state incubate in un volume finale d11mi per 30 minuti a 25”C con [ H]Rauwolscina 0,5-1,5nM, in assenza o presenza di sostanze in esame. Il legame non specifico e’stato stabilito in presenza di fentolamina 10μΜ. L’incubazione e’ stata fermata con l’aggiunta di tampone Tris-HCl 50mM ghiacciato e filtrazione rapida attraverso filtri Whatman GF/B o Schleicher & Schuell GF52 pretrattati con poiietilenimmina 0,2%. I filtri sono stati poi lavati con tampone ghiacciato e la radioattivita’ trattenuta dai filtri e’ stata valutata mediante spettrometria per scintillazione liquida. Binding studies on native adrenergic receptors (Diop L. et al., J. Neurochem. 41 · 710-715, 1983) were performed with rat cerebral cortex membrane. Male Sprague Dawley rats (200-300 g, SO Harlan / Nossan, Italy) were killed by cervical dislocation and the cerebral cortices were removed, immediately frozen and stored at ~ 70'C until use. The tissues were homogenized (2 x 20 sec.) In 50 volumes of cold 50mM Tris-HCl buffer pH 7.4 with a Politron homogenizer (speed 7). The homogenates were centrifuged at 49000 x g for 10 minutes, resuspended in 50 volumes of the same buffer, incubated at 37 ° C for 15 minutes and centrifuged and resuspended two more times. The final membranes were suspended in 100 volumes of Tris-HCl 50mM pH 7.4 buffer containing 10μΜ pargiline and 0.1% ascorbic acid. The membranes were incubated in a final volume d11mi for 30 minutes at 25 "C with [H ] Rauwolscina 0.5-1.5nM, in the absence or presence of test substances. The non-specific bond was established in the presence of 10μΜ phentolamine. Incubation was stopped with the addition of ice-cold 50mM Tris-HCl buffer and rapid filtration through Whatman GF / B or Schleicher & Schuell GF52 filters pretreated with 0.2% polyethyleneimine. The filters were then washed with ice-cold buffer and the radioactivity retained by the filters was evaluated by liquid scintillation spectrometry.
Analisi dei dati Data analysis
del legame specifico dei radloleganti da parte delle sostanze allo studio per mezzo del programma d1 Interpolazione non lineare Alìflt (De Lean et al., Am. J. Physlol. 235, E97-E102, 1978). Il valore IC50 e’ stato convertito 1n costante di affinità' (K1) permezzo dell’equazione di Cheng & Prusoff (Cheng Y.C., Prusoff W.H., Blochem. Pharmacol. 22, 3099-3108, 1973). of the specific binding of radloligands by the substances under study by means of the program d1 Nonlinear interpolation Alìflt (De Lean et al., Am. J. Physlol. 235, E97-E102, 1978). The IC50 value was converted to 1n affinity constant (K1) by means of the Cheng & Prusoff equation (Cheng Y.C., Prusoff W.H., Blochem. Pharmacol. 22, 3099-3108, 1973).
B. Risultati B. Results
I risultati riportati nella tabella 3 Indicano che 1 composti dell’Invenzione possiedono un’affinità’ per 11 recettore 5-ΗΤ1A, affinità’ che e’ molto Inferiore per 1 ricettori alfa adrenerglcl rispetto al recettore serotonlnergico. The results shown in table 3 indicate that the compounds of the invention possess an affinity for 11 5-ΗΤ1A receptor, an affinity which is much lower for 1 alpha adrenergcl receptor than for the serotonergic receptor.
TABELLA 3 TABLE 3
Affinità’ di legame per il recettore 5-HT1A e altri siti recettorlall i dati sono espressi come K1 (nM) Binding affinity for the 5-HT1A receptor and other receptor sites all the data are expressed as K1 (nM)
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JP2000505141A JP2001512110A (en) | 1997-08-01 | 1998-07-31 | 1,4-disubstituted piperazine |
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PCT/EP1998/004796 WO1999006382A1 (en) | 1997-08-01 | 1998-07-31 | 1,4-disubstituted piperazines |
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