Saturated and unsaturated pyridazino4,5-B! indolizines useful as antidementia agents

This invention relates to novel compounds which bind to central cholinergic muscarinic M1 receptors and may be useful for treatment of diseases attributed to cholinergic insufficiency such as presenile dementia, senile dementia of the Alzheimer's type, Parkinson's disease, Down's syndrome, and dementia pugilistica. The compounds useful in this invention have the formula ##STR1## or a 6, 7, 8, 9 tetrahydro analog thereof, where R.sup.1 is selected from H, C.sub.1 -C.sub.6 alkyl, C.sub.3 -C.sub.6 cycloalkyl, C.sub.1 -C.sub.6 alkoxy, cyano, halo, nitro, and --NR.sup.4 R.sup.5, where R.sup.4 and R.sup.5 are independently selected from H, C.sub.1 -C.sub.6 alkyl and phenyl, optionally substituted with halo, cyano, hydroxy, nitro, amino, mono or di C.sub.1 -C.sub.6 alkylamino, phenylamino, C.sub.1 -C.sub.6 alkyl or C.sub.1 -C.sub.6 alkoxy; PA1 R.sup.2 is CF.sub.3 --, CF.sub.3 CH.sub.2 --, or --CH.sub.2 --X--R.sup.6 where X is oxygen or NH and R.sup.6 is C.sub.1 -C.sub.6 alkyl, phenyl-(CH.sub.2).sub.n -- or --(CH.sub.2).sub.n --NR.sup.7 R.sup.8 where R.sup.7 and R.sup.8 are selected from H or C.sub.1 -C.sub.6 alkyl, or R.sup.2 is equal to R.sup.3, and when the compound is hydrogenated at positions 6, 7, 8, and 9, R.sup.2 can also be C.sub.1 -C.sub.6 alkyl, and PA1 R.sup.3 is --(CH.sub.2).sub.m --NR.sup.9 R.sup.10 where R.sup.9 and R.sup.10 are independently H, C.sub.1 -C.sub.6 alkyl, phenyl, phenyl-(CH.sub.2).sub.n --, or NR.sup.9 R.sup.10 forms a mono or bicyclic azacycloalkane group having from 5 to 10 members, one of which may be a heteroatom selected from O, S, or NR.sup.11 where R.sup.11 is C.sub.1 -C.sub.6 alkyl, phenyl, pyrimidinyl, pyridinyl, or pyrazinyl or R.sup.9 is H and R.sup.10 is a mono or bicyclic azacycloalkyl group having from 5 to 10 members where the nitrogen is either a bridgehead nitrogen or the nitrogen may be optionally substituted with C.sub.1 -C.sub.6 alkyl, phenyl-(CH.sub.2).sub.n -- or C.sub.3 -C.sub.6 cycloalkyl; PA1 n is 1-6 and m is 2-5; PA1 or a pharmaceutically acceptable salt thereof.

This patent application claims priority to the United States Provisonal 
Application Number 60/008,548 filed on Dec. 13, 1995. 
FIELD OF THE INVENTION 
This invention relates to novel compounds having CNS activity. More 
particularly it relates to novel pyridazino4,5-b!indolizine and 
6,7,8,9-tetrahydropyridazino4,5-b!indolizine compounds which bind to 
central cholinergic muscarinic receptors and, therefore, may be useful for 
treatment of diseases involving hypofunction of the cholinergic system. 
Neurological illnesses related to cholinergic deficiency include presenile 
dementia and senile dementia of the Alzheimer's type (SDAT), Parkinson's 
disease, Down's Syndrome, and dementia pugilistica. Cognitive disorders 
which occur with these conditions include forgetfulness, confusion, memory 
loss, attentional deficits, and deficits in visual perception. 
BACKGROUND OF THE INVENTION 
The "cholinergic hypothesis" R. T. Bartus, R. L. Dean III, B. Beer, A. S. 
Lippa, Science, (Jul. 30, 1982) 217:408-417, "The Cholinergic Hypothesis 
of Geriatric Memory Dysfunction" suggests that memory loss due to 
decreased levels of acetylcholine can be ameliorated by correcting the 
levels of acetylcholine in the brain using an acetylcholine releasing 
agent, an acetylcholine esterase inhibitor, or by using a drug which 
mimics acetylcholine (cholinomimetic). Marketing of the acetylcholine 
esterase inhibitor, tacrine, has demonstrated that improvement in memory 
can be shown in patients with mild to moderate Alzheimer's Disease M. 
Williams, Curr. Opin. Invest. Drugs (May 1993) 2(5):541-544, 
"Tacrine-recommendation for approval"!. The utility of this drug is 
limited, however, because of adverse side effects especially at the higher 
doses where it is most effective. Clinical studies using the natural 
alkaloid, arecoline, a cholinergic agonist, have also demonstrated memory 
improvement in patients with mild to moderate Alzheimer's Disease. Because 
of the short half-life of arecoline, the clinical study was done using 
continuous infusion of the drug over a 2 week period; In addition, a 
peripheral muscarinic antagonist, N-methylscopolamine, was also 
administered during the study to prevent potential autonomic side effects. 
T. T. Soncrant, K. C. Raffaele, S. Asthana, A. Berardi, P. P. Morris, and 
J. V. Haxby, Psychopharmacology (1993) 112:421-427, "Memory improvement 
without toxicity during chronic, low dose intravenous arecoline in 
Alzheimer's disease"! 
Cholinergic receptors which bind to and are activated by the alkaloid, 
muscarine, are called muscarinic receptors. Three pharmacologically 
defined subtypes of muscarinic receptors have been identified. They are 
referred to as M1, M2, and M3 based upon their affinity for the M1 
antagonist, pirenzepine, the M2 antagonist, AFDX-116, and the M3 
antagonist, 4-diphenylacetoxy N-methylpiperidine methiodide (4-DAMP). Five 
different human muscarinic receptors have been cloned. The Hm1 (human m1) 
receptor is found primarily in brain. T. I. Bonner, Trends in 
Pharmacological Sciences, supplement, Jul. 20-27 (1989) p 11-15, "New 
types of muscarinic acetylcholine receptors"!. Activation of the m1 
receptor results in an increase in phosphoinsoitide hydrolysis (PI 
turnover).K. Fukuda, et al. , Ibid., p. 4-10, "Selective effector 
coupling of muscarinic acetylcholine receptor subtypes"!. Carbachol, like 
muscarine, is able to fully activate m1 receptors. These compounds, 
however, contain a quaternary ammonium group and as a result are not able 
to cross the blood brain barrier and enter the CNS. On the contrary, 
compounds of this invention do not contain a quaternary ammonium group and 
thus can enter the CNS. The compounds of this invention bind to M1 
receptors in brain and are useful for treatment of symptoms of central 
cholinergic insufficiency. 
U.S. Pat. No. 4,985,560 discloses a class of pyridazino4,5-b!indolizines 
which bind to central cholinergic M1 receptors and are useful for 
treatment of diseases involving hypofunction of the cholinergic system. It 
has now been found that substitution in position 4 improves affinity to 
central M1 cholinergic receptors. Surprisingly, reduction of the pyridine 
ring of the pyridazino4,5-b!indolizine compounds of this invention 
significantly increases M1 affinity and selectivity for M1 versus M2 
receptors in brain. Examples of saturated pyridazino4,5-b!indolizines of 
this invention are not covered in the above patent. 
The synthesis of the tetrahydroindolizine ring by means of a 1,3-dipolar 
cycloaddition reaction has been reported as has its conversion to a 
tetrahydropyridazino4,5-b!indolizine ring. The latter resulted from the 
reaction of hydrazine with adjacent benzoyl substituents on the 
tetrahydroindolizine ring.Uchida, T., et al., J. Heterocyclic Chem. 15, 
1303 (1978)! 
SUMMARY OF THE INVENTION 
The compounds of the present invention are characterized by the general 
formula 
##STR2## 
or a 6, 7, 8, 9 tetrahydro analog thereof, where R.sup.1 is selected from 
H, C.sub.1 -C.sub.6 alkyl, C.sub.3 -C.sub.6 cycloalkyl, C.sub.1 -C.sub.6 
alkoxy, cyano, halo, nitro, and --NR.sup.4 R.sup.5, where R.sup.4 and 
R.sup.5 are independently selected from H, C.sub.1 -C.sub.6 alkyl and 
phenyl, optionally substituted with halo, cyano, hydroxy, nitro, amino, 
mono or di C.sub.1 -C.sub.6 alkylamino, phenylamino, C.sub.1 -C.sub.6 
alkyl or C.sub.1 -C.sub.6 alkoxy; 
R.sup.2 is CF.sub.3 --, CF.sub.3 CH.sub.2 --, or --CH.sub.2 --X--R.sup.6 
where X is oxygen or NH and R.sup.6 is C.sub.1 -C.sub.6 alkyl, 
phenyl-(CH.sub.2).sub.n -- or --(CH.sub.2).sub.n --NR.sup.7 R.sup.8 where 
R.sup.7 and R.sup.8 are selected from H or C.sub.1 -C.sub.6 alkyl, or 
R.sup.2 is equal to R.sup.3, and when the compound is hydrogenated at 
positions 6, 7, 8, and 9, R.sup.2 can also be C.sub.1 -C.sub.6 alkyl, and 
R.sup.3 is --(CH.sub.2).sub.m --NR.sup.9 R.sup.10 where R.sup.9 and 
R.sup.10 are independently H, C.sub.1 -C.sub.6 alkyl, phenyl, 
phenyl-(CH.sub.2).sub.n --, or NR.sup.9 R.sup.10 forms a mono or bicyclic 
azacycloalkane group having from 5 to 10 members, one of which may be a 
heteroatom selected from O, S, or NR.sup.11 where R.sup.11 is C.sub.1 
-C.sub.6 alkyl, phenyl, pyrimidinyl, pyridinyl, or pyrazinyl or R.sup.9 is 
H and R.sup.10 is a mono or bicyclic azacycloalkyl group having from 5 to 
10 members where the nitrogen is either a bridgehead nitrogen or the 
nitrogen may be optionally substituted with C.sub.1 -C.sub.6 alkyl, 
phenyl-(CH.sub.2).sub.n -- or C.sub.3 -C.sub.6 cycloalkyl; 
n is 1-6 and m is 2-5; 
or a pharmaceutically acceptable salt thereof. 
In the above definitions of terms, the term C.sub.1 -C.sub.6 alkyl includes 
straight and branched chain hydrocarbons such as methyl, ethyl, propyl, 
isopropyl, butyl, t-butyl and the like. A pharmaceutically acceptable salt 
is an acid addition salt formed by an invention compound and a 
pharmaceutically acceptable inorganic or organic acid such as hydrochloric 
acid, sulfuric acid, phosphoric acid, acetic acid, formic acid, fumaric 
acid, citric acid, maleic acid, or methanesulfonic acid. 
The preferred compounds of this invention are those of Examples 1-15. 
DETAILED DESCRIPTION OF THE INVENTION 
Compounds of this invention may be prepared by a variety of synthetic 
routes using conventional methods or commercially available starting 
materials (Scheme I). 
##STR3## 
Thus a substituted or unsubstituted 2-indolizinecarboxylic acid ester is 
treated with an acylating agent, such as an acid anhydride or acid 
chloride in the presence of a base such as triethylamine TEA!, and an 
acylation catalyst such as N,N-dimethylaminopyridine DMAP!, to give a 
substituted or unsubstituted 3-acyl-2-indolizinecarboxylic acid ester. The 
acyl ester is then allowed to react with hydrazine hydrate in a polar 
solvent, such as ethanol, to give a 
4-substituted-pyridazino4,5-b!indolizin-1-one. Treatment of the 
pyridazino4,5-b!indolizin-1-one with a chlorinating agent, such as 
phosphorus oxychloride, gives a 
1-chloro-4-substituted-pyridazino4,5-b!indolizine. The 
1-chloropyridazino4,5-b!indolizine is allowed to react with a di or 
triamine compound at elevated temperatures to give an unsubstituted or 
substituted 1-(di or triamino)-4-substituted-pyridazino4,5-b!indolizine 
of the present invention. Catalytic hydrogenation of these 
pyridazino4,5-b!indolizines using a catalyst, such as palladium on 
carbon, in a hydrogenation apparatus, such as a Parr apparatus, in an 
acidic polar mixture, such as acetic acid and methanol with or without 
addition of a stronger acid, such as sulfuric acid, gives the 
6,7,8,9-tetrahydropyridazino4,5-b!indolizines of this invention. 
An alternate route to compounds of this invention involves catalytic 
reduction of the pyridine ring of 
4-substituted-pyridazino4,5-b!indolizin-1-ones using hydrogen and a 
catalyst, such as palladium on carbon, under pressure in an apparatus, 
such as a Parr apparatus, in an acidic polar solvent mixture, such as a 
mixture of water, acetic acid and ethanol containing sulfuric acid (Scheme 
II). The 6,7,8,9-tetrahydropyridazino4,5,-b!indolizin-1-ones are then 
treated with a chlorinating agent, such as phosphorus oxychloride to give 
1-chloro-4-substituted-6,7,8,9-tetrahydropyridazino4,5-b!indolizines. 
Reaction of these chloro compounds with di or triamines at elevated 
temperatures gives substituted or unsubstituted 1-(di or 
triamino)-4-substituted-6,7,8,9-tetrahydropyridazino4,5-b!indolizines of 
the present invention. 
##STR4## 
Treatment of intermediates of this invention, 
4-hydroxymethyl-pyridazino4,5-b!indolizin-1-ones, with a chlorinating 
agent, such as phosphorus oxychloride, gives 
1-chloro-4-chloromethylpyridazino4,5,-b!indolizines. Reaction of these 
dichloro compounds with di or triamines gives compounds of this invention 
(Scheme III). 
##STR5## 
2-Indolizinecarboxylic acid which may be variously substituted as desired 
Bragg et al., J. Chem. Soc., 3277 (1963)! is converted to an ester 
(methanol treated with an acid such as HCl) to give the intermediate 
2-carbomethoxyindolizine used in the following procedures. 
Alternatively, the indolizine-2-carboxylates used in Scheme I can be 
prepared from appropriately substituted 2-methylpyridines that are either 
commercially available, such as 5-butyl-2-methyl pyridine, 
6chloro-2-picoline, 2-amino-6-methyl pyridine, 2-cyano-6-methyl pyridine 
or 2,4-dimethylpyridine, or may be prepared according to standard 
literature synthetic procedures. 5-Methylindolizine-2-carboxylic acid is 
disclosed by Bode et al., J. Chem. Soc., Perk. Trans. 1, 3023-3027 (1994) 
and references therein. 5-Cyanoindolizine-2-carboxylic acid methyl ester 
is disclosed by Abramovitch and Mathur in Heterocycles 5(1), 91-94 (1976) 
and the 6-ethoxy and methoxy analogs of indolizine-2-carboxylic acid are 
disclosed by De and Saha in J. Pharm. Sci. 64(2), 249-252 (1975). The 
synthetic route to the indolizine-2-carboxylates from a 2-methylpyridine 
is shown in scheme IV. 
##STR6## 
The following specific examples are included for illustrative purposes and 
should not be construed as limiting this disclosure in any way. Invention 
compounds can be prepared by a person skilled in the art using chemicals 
and intermediates that are either commerically available or prepared 
according to literature procedures.

EXAMPLE 1 
N-4-(3-Azabicyclo3.2.2!non-3-yl)butyl!-4-(trifluoromethyl)pyridazino4,5b 
!indolizine-1-amine 
2-Carbomethoxyindolizine (3.00 g, 17.14 mmol) was dissolved in methylene 
chloride containing N,N-dimethylaminopyridine (DMAP, 0.230 g, 1.89 mmol) 
and triethylamine (TEA, 2.08 g, 20.57 mmol). Trifluoroacetic anhydride 
(3.96 g, 18.86 mmol) was added slowly (addition time: 45 minutes) and the 
reaction mixture was allowed to heat under reflux for 72 hours. The 
organic phase was washed with water and then passed through a pad of 
silica gel eluting the product with 20% hexane in methylene chloride. 
3-Trifluoroacetyl-2-carbomethoxyindolizine (I) (3.95 g) was obtained in 
85% yield as a yellow-green oil. 
A mixture of (I) (3.60 g, 13.3 mmol) and excess 85% aqueous hydrazine in 
ethanol (250 mL) was heated under reflux for 10 minutes (a precipitate 
formed). After cooling in a freezer overnight, the solid was collected by 
filtration and dried to give 
4-trifluoromethylpyridazino4,5-b!indolizin-1-one (II, 2.65 g, 79%) 
Compound II (2.62 g, 10.3 mmol) and phosphorus oxychloride (3.18 mL, 34 
mmoL) were combined and heated under reflux for 2 days. After cooling to 
room temperature, the reaction mixture was poured into ice water and made 
basic with aqueous sodium hydroxide (pH&gt;7). The product was collected by 
filtration and dried in a vacuum oven overnight to give 2.82 g (100%) of 
1-chloro-4-trifluoromethylpyridazino4,5-b!indolizine (III) 
A mixture of chloro compound III (620 mg, 2.28 mmol) and 
4-(3-azabicyclo3.2.2!nonyl)butylamine (896 mg, 4.57 mmol), 123 mg of 
ammonium chloride and 0.4 mL of triethylamine was heated under reflux for 
2 days. After cooling, the reaction mixture was dissolved in methylene 
chloride and the organic phase was washed three times with water. The 
organic phase was dried over magnesium sulfate, evaporated under reduced 
pressure and purified by flash column chromatography on silica gel eluting 
with 40:60 methanol:ethyl acetate. The title compound (561 mg, 57%) was 
converted to the hydrochloride salt using ethereal HCl and ether: m.p. 
245.degree.-246.degree. C. 
Anal. Calc'd for C.sub.23 H.sub.28 F.sub.3 N.sub.5 .multidot.2 HCl 
.multidot.1.75 H.sub.2 O: C, 50.41; H, 6.36; N, 12.48 
Found: C, 50.27; H, 6.42; N, 12.75 
EXAMPLE 2 
N-3-(3-Azabicyclo3.2.2!non-3-yl)propyl-4-(trifluoromethyl)pyridazino4.5- 
b!indolizine-1-amine 
According to the procedure of Example 1, chloro compound III was allowed to 
react with 3-(3-azabicyclo3.2.2!non-3-yl)propylamine to give the title 
compound which was converted to the hydrochloride salt, 
m.p.252.degree.-253.degree. C. 
Anal. Calcd for C.sub.22 H.sub.26 F.sub.3 N.sub.5 .multidot.2 
HCl.multidot.1.75 H.sub.2 O: C, 50.63 H, 6.08; N, 13.42 
Found: C, 50.85; H, 6.48; N, 13.26. 
EXAMPLE 3 
N,N-Diethyl-N'-4-(methoxymethyl)pyridazino4,5-b!indolizin-1-yl!-1.3-propa 
nediamine 
2-Carbomethoxyindolizine (15.0 g, 85.6 mmol) was dissolved in 
dichloroethane containing N,N-dimethylaminopyridine (DMAP, 1.15 g, 
9.45mmol) and triethylamine (TEA, 10.40 g, 103 mmol). Methoxyacetyl 
chloride (9.44 g, 87 mmol) was added and the reaction mixture was allowed 
to heat under reflux in a nitrogen atmosphere for 72 hours. The organic 
phase was washed with water, aqueous sodium hydroxide, and dilute HCl. The 
organic phase was dried (M.sub.g SO.sub.4) and evaporated to give a 
residue which was purified by high pressure liquid chromatography (silica 
gel, methanol in methylene chloride ) to give 
2-carbomethoxy-3-methoxymethylacetylindolizine IV (7.06 g, 33%). 
A mixture of (IV) 7.06 g, 30.5 mmol! and excess 85% hydrazine hydrate in 
ethanol (300 mL) was heated under reflux (a precipitate formed). After 
cooling, the solid was collected by filtration and dried to give 
4-methoxymethylpyridazino4,5-b!indolizin-1-one (V, 6.81 g, 97%) 
Compound V (6.3 g, 27.5 mmol) and phosphorus oxychloride (100 mL) were 
combined and heated under reflux for 4 hours. Most of the POCl.sub.3 was 
removed using a rotary evaporator. The residue was poured into ice water 
and the aqueous phase was extracted with methylene chloride using a 
continuous extractor apparatus. Evaporation of the solvent gave a residue 
which was purified by flash chromatography on silica gel eluting with 3:7 
isopropanol:methylene chloride to give the chloro compound, VI (6.8 g, 
81%). 
A mixture of chloro compound VI (0.98 g, 3.95 mmol), 4 equivalents of 
N'-diethylaminopropylamine (2.04 g, 15.8 mmol) , and 1 equivalent of 
triethylamine (0.61 mL) was heated in N-methylpyrrolidinone at 120.degree. 
C. overnight. After cooling, the reaction mixture was dissolved in 
methylene chloride and the organic phase was washed three times with 
water. The organic phase was dried over magnesium sulfate, evaporated 
under reduced pressure and purified by flash column chromatography on 
silica gel eluting with 93:5:2 methylene chloride:methanol:triethylamine. 
The title compound (1.14 g mg, 85%) was converted to the hydrated 
dihydrochloride salt using ethereal HCl and ether: m.p. 
111.degree.-112.degree. C. 
Anal. Calcd for C.sub.19 H.sub.27 N.sub.5 O.multidot.2 HCl.multidot.2 
H.sub.2 O: C, 50.67; H, 7.38; N, 15.47 
Found: C, 50.82; H, 7.24; N, 15.08 
EXAMPLE 4 
N-3-(3-Azabicyclo3.2.2!non-3-yl)propyl!-4-(methoxymethyl)pyridazino4.5-b 
!indolizin-1-amine 
Following the procedure of Example 3, chloro compound VI (0.50 g, 2.02 
mmoL) was allowed to react with 3-(3-azabicyclo3.2.2!non-3-yl)propylamine 
(0.911 g, 5 mmoL) in N-methylpyrrolidinone containing TEA. The crude 
product was purified by flash column chromatography on silica gel eluting 
with 10-15% methanol in methylene chloride containing 1% ammonium 
hydroxide. The title compound was converted to the hydrated 
dihydrochloride salt, m.p. 229.degree.-232.degree. C. 
Anal. Calcd for C.sub.23 H.sub.31 N.sub.5 O.multidot.2 HCl.multidot.1/2 
H.sub.2 O.multidot.1/4 CHCl.sub.3 : C, 55.26; H, 6.83; N, 13.86 
Found: C, 55.04; H, 6.76; N, 13.51 
EXAMPLE 5 
N-4-(3-Azabicyclo3.2.2!non-3-yl)butyl!-4-(methoxymethyl)pyridazino4.5-b! 
indolizin-1-amine 
Following the procedure of Example 3, chloro compound VI (1.00 g, 4.04 
mmoL) was allowed to react with 4-(3-azabicyclo3.2.2!non-3-yl)butylamine 
(2.21 g 12.12 mmoL) in N-methylpyrrolidinone containing TEA and ammonium 
chloride (1 equiv). The crude product was purified by flash column 
chromatography on silica gel eluting with 10-15% methanol in methylene 
chloride containing 1% ammonium hydroxide. The title compound was 
converted to the hydrated dihydrochloride salt, m.p. 220.degree. C. 
(dec.). 
Anal. Calc'd for C.sub.24 H.sub.33 N.sub.5 O.multidot.2 HCl.multidot.0.75 
H.sub.2 O: C, 57.83; H,7.48; N, 14.05 
Found: C, 58.35; H,7.45; N, 14.18. 
EXAMPLE 6 
4-(Phenylmethoxy)methyl!-N-2-4-(2-pyrimidinyl)-1-piperazinyl!ethyl!pyrid 
azino4,5-b!indolizin-1-amine 
Following the procedure of Example 3, 2-carbomethoxyindolizine was reacted 
with benzyloxyacetyl chloride to give 
3-benzyloxyacetyl-2-carbomethoxyindolizine (VII) Cyclization of VII with 
hydrazine and reaction of the resulting pyridazino4,5-b!indolizin-1-one 
with POCl.sub.3 gave the corresponding chloro compound (VIII). The 
reaction of VII with 4-(2-pyrimidinyl)-1-piperizinyl)ethyl amine gave the 
title compound. Treatment with ethereal HCl in ether gave the product as 
the trihydrochloride salt: m.p. 140.degree. C. 
Anal. Calc'd for C.sub.28 H.sub.30 N.sub.8 O.multidot.3HCl.multidot.0.75 
H.sub.2 O: C, 54.46; H,5.63; N, 18.15 
Found: C, 54.24; H,5.81; N, 18.36. 
EXAMPLE 7 
N,N-Diethyl-N'-4-3-(diethylamino)propyl!amino!methyl!-pyridazino4.5-b! 
indolizin-1-yl!-1,3-propanediamine 
Following the procedure of Example 3, reaction of 2-carbomethoxyindolizine 
with acetoxyacetyl chloride gave 
3-acetyloxyacetyl-2-carbomethoxyindolizine (IX). Reaction of IX with 1.5 
equiv of anhydrous hydrazine in dry ethanol gave 
4-hydroxymethylpyridazino4,5-b!indolizin-1-one which upon refluxing with 
excess POCl.sub.3 gave 1-chloro-4-chloromethylpyridazino4,5-b!indolizine 
(X). Compound X (1.0 g, 4.0 mmol) was combined with excess 
N,N-diethylaminopropylamine (2.68 mL, 17 mmoL) in N-methylpyrrolidinone (2 
mL) containing TEA (0.74 mL, 1.2 equiv.) and 1.2 equiv. of ammonium 
chloride (vigorous initial reaction with gas evolution). When the reaction 
subsided the stirred reaction mixture was heated at 100.degree. C. 
overnight. After cooling to room temperature, the volatiles were removed 
by vacuum distillation. The residue was dissolved in methylene chloride 
and extracted with water (3 times), dried (MgSO.sub.4), and the solvent 
evaporated. The residue was purified by flash column chromatography on 
silica gel eluting with 20% methanol in methylene chloride containg 2% 
ammonium hydroxide. The isolated free base was converted to the tetra 
hydrochloride salt using ethereal HCl in ether m.p. 
120.degree.-123.degree. C. 
Anal. Calcd for C.sub.25 H.sub.41 N.sub.7 .multidot.4 HCl.multidot.1.5 
H.sub.2 O: C, 49.02; H,7.90; N, 16.00 
Found: C, 49.15; H,7.95; N, 15.89. 
EXAMPLE 8 
N,N-Diethyl-N'-6,7,8,9-tetrahydro-4-(methyl)-pyridazino4,5-b!indolizin-1- 
yl!-1.3-propanediamine 
4-(Methyl)-N,N-diethyl-N'-pyridazino4,5-b!indolizin-1-yl!-1,3-propanediam 
ine (2.75 g, 8.83 mmoL) was hydrogenated in a Parr appartus with 10% Pd on 
charcoal (0.54 g) in a 1:1 mixture of acetic acid and methanol at 55 psi 
and room temperature. Filtration through Solka Floc to remove the catalyst 
and evaporation of the filtrate gave a crude product which was passed 
through a pad of alumina eluting with 5% methanol in methylene chloride to 
give the free base of the title product in quantitative yield. The free 
base was converted to the monofumarate salt: m.p.171.degree.-172.degree. 
C. 
Anal. Calc'd for C.sub.18 H.sub.29 N.sub.5 .multidot.C.sub.4 H.sub.4 
O.sub.4 .multidot.0.5 H.sub.2 O: C, 59.98 H,7.78; N,15.90 
Found: C, 59.99; H,7.93; N,15.76. 
EXAMPLE 9 
N-2-(3-Azabicyclo3.2.2non-3-yl)ethyl!-6,7,8,9-tetrahydro-4-(methyl)pyrida 
zino4,5-b!indolizin-1-amine 
4-Methylpyridazino4,5-b!indolizin-1-one (5.08 g, 25.5 mmol) was 
hydrogenated at room temperature and 55 psi using a Parr apparatus in a 
mixture containing 100 mL each of water, ethanol and acetic acid, 3 mL of 
concentrated sulfuric acid, and 10% Pd on charcoal (500 mg). A white 
precipitate was observed. The precipitate and catalyst were filtered 
through Solka Floc and the solids were washed with methanol and methylene 
chloride. The washes were evaporated to give 
6,7,8,9-tetrahydro-4-(methyl)pyridazino4,5-b!indolizin-1-one (XI). The 
reduction filtrate was evaporated in vacuo to remove most of the 
volatiles, then filtered to isolate additional XI. The aqueous phase was 
then washed with methylene chloride to extract the remaining XI. The 
product from all three fractions was combined and dried in vacuo to give 
3.2 g (67%) of XI. 
Compound XI (3.00 g, 15 mmol) was heated under reflux with excess 
POCl.sub.3 (25 mL) until no starting material remained (TLC, silic gel, 5% 
methanol in methylene chloride), then poured over ice, made basic with 
aqueous sodium hydroxide and the aqueous phase extracted with methylene 
chloride to isolate the product. The organic phase was dried (MgSO.sub.4), 
filtered, and evaporated to give a residue which was purified by flash 
column chromatography (silica gel, 5% methanol in methylene chloride) to 
give 1-chloro-4-methyl-6,7,8,9-tetrahydropyridazino4,5-b!indolizine XII 
(3.00 g, 92%) 
A mixture of compound XII (0.51 g, 2.31 mmoL), 
2-(3-azabicyclo3.2.2!non-3-yl)ethylamine 0.970 g, 2.5 equiv.) , ammonium 
chloride (1 equiv.) and N-methylpyrrolidinone were heated under reflux in 
a nitrogen atmosphere overnight. No starting material remained by TLC 
(silica gel, 20% methanol in methylene chloride). The reaction mixture was 
diluted with methylene chloride and washed successively with sat. sodium 
bicarbonate and water. The organic phase was dried (MgSO.sub.4), filtered, 
evaporated and the residue purified by flash column chromatography on 
silica gel eluting with methanol in methylene chloride containing a 
gradient of ammonium hydroxide. The title compound (0.52 g, 67%) was 
recovered and converted to the dihydrochloride salt: m.p. 
272.degree.-274.degree. C. 
Anal. Calc'd for C.sub.21 H.sub.31 N.sub.5 .multidot.2 HCl.multidot.0.5 
H.sub.2 O: C, 57.93; H, 7.87; N, 16.08 
Found: C, 58.30; H, 7.86; N, 16.02 
EXAMPLE 10 
N-3-(3-Azabicyclo3.2.2!non-3-yl)propyl!-6,7,8,9-tetrahydro-4-(methyl)pyri 
dazino4,5-b!indolizin-1-amine 
Following the procedure in Example 9, compound XII was allowed to react 
with 3-(3-azabicyclo3.2.2!non-3-yl)propylamine to give the title compound 
which was converted to the dihydrochloride salt: 
m.p.278.degree.-279.degree. C. 
Anal. Calcd for C.sub.22 H.sub.33 N.sub.5 .multidot.2 HCl.multidot.2H.sub.2 
O: C, 55.46; H, 8.25; N, 14.70 
Found: C, 55.27; H, 8.31; N, 14.46. 
EXAMPLE 11 
N-4-(3-Azabicyclo3.2.2!non-3-yl)butyl!-6,7,8,9-tetrahydro-4-methylpyridaz 
ino4,5-b!indolizin-1-amine 
Following the procedure in Example 9, compound XII was allowed to react 
with 4-(3-azabicyclo3.2.2!non-3-yl)butylamine to give the title compound 
which was converted to the dihydrochloride salt: m.p.210.degree. C. 
(dec.). 
Anal. Calc'd for C.sub.23 H.sub.35 N.sub.5 .multidot.2 
HCl.multidot.2H.sub.2 O: C, 56.32; H, 8.43; N, 14.28 
Found: C, 56.28; H, 8.35; N, 14.28. 
EXAMPLE 12 
N-4-(3-Azabicyclo3.2.2!non-3-yl)butyl!-6,7,8,9-tetrahydro-4-(trifluoromet 
hyl)-pyridazino4,5-b!indolizin-1-amine 
The title compound of Example 1 (360 mg, 0.72 mmol), 10% Pd on charcoal (72 
mg), ethanol and acetic acid (about 50 mL each), and 1.5 mL of 
concentrated sulfuric acid were hydrogenated at 55 psi in a Parr apparatus 
overnight. The reaction mixture was filtered through a pad of Solkafloc 
and the filtrate was evaporated under reduced pressure. The residue was 
dissolved in water and the aqueous solution was adjusted to pH 7 with 
aqueous sodium hydroxide and then extracted several times with methylene 
chloride. The organic phase was dried and the solvent removed under 
reduced pressure to give the title compound. Treatment of the title 
compound with ethereal HCl in ether gave the hydrated dihydrochloride salt 
as a tan solid: m.p.140.degree. C. 
Anal. Calc'd for C.sub.23 H.sub.32 F.sub.3 N.sub.5 .multidot.2 
HCl.multidot.1 H.sub.2 O: C, 52.47; H, 6.89; N, 13.30 
Found: C, 52.49; H, 7.16; N, 13.59. 
EXAMPLE 13 
N,N-Diethyl-N'-6,7,8,9-tetrahydro-4-(methoxymethyl)-pyridazino4,5-b!indol 
izin-1-yl!-1.3-propanediamine 
Following the procedure in Example 12, the compound in Example 3 was 
converted to the title compound. The product was treated with ethereal HCL 
in ether to obtain the hydrated dihydrochloride salt as a yellow solid: 
m.p.145.degree.-147.degree. C. 
Anal. Calc'd for C.sub.19 H.sub.31 N.sub.5 O.multidot.2 
HCl.multidot.H.sub.2 O: C, 52.29; H, 8.08; N, 16.04 
Found: C, 52.45; H, 7.99; N, 15.59. 
EXAMPLE 14 
N-4-(3-Azabicyclo3.2.2!non-3-yl)butyl!-6,7,8,9-tetrahydro-4-(methoxymethy 
l)-pyridazino4,5-b!indolizin-1-amine 
Following the procedure in Example 12, the compound in Example 5 was 
converted to the title compound. The product was treated with ethereal HCl 
in ether to obtain the hydrated dihydrochloride salt as a yellow solid: 
m.p. 185.degree. C. (dec.). 
Anal. Calc'd for C.sub.24 H.sub.37 N.sub.5 O.multidot.2 
HCl.multidot.H.sub.2 O.multidot.0.1C.sub.4 H.sub.10 O: C, 57.47; H, 8.30; 
N, 13.74 
Found: C, 57.71; H, 8.23; N, 13.50. 
EXAMPLE 15 
6,7,8,9,-Tetrahydro-N-4-(4-morpholinyl)butyl!-4-(phenylmethoxy)methyl!-py 
radazino4,5-b!indolizin-1-amine 
Following the procedure in Example 12, 
N-4-(4-morpholinyl)butyl!-4-(phenylmethoxy)methyl!-pyridazino4,5-b!indo 
lizin-1-amine was converted to the title compound as a light yellow solid: 
m.p. 144.degree.-145.degree. C. 
Anal. Calcd for C.sub.26 H.sub.35 N.sub.5 O.sub.2 .multidot.2 
HCl.multidot.2.8 H.sub.2 O: C, 54.50; H, 7.49; N,12.22 
Found: C, 54.43; H, 7.49; N,12.21. 
Pharmacology 
1. Tritiated Pirenzapine Binding Inhibition 
The selective muscarinic binding by the compounds of this invention was 
established by determining the percentage of inhibition of .sup.3 
H!pirenzepine (PZ) binding to rat brain tissue at a 10 micromolar 
concentration. The cortex of rat brains obtained from male Sprague-Dawley 
rats (300 g) are homogenized using a hand-held teflon-coated pestle in 20 
volumes of 0.32M sucrose. After centrifugation (747.times.g for 10 min at 
4.degree. C.), the resultant supernatant is decanted and recentrifuged 
(18677.times.g for 20 min at 4.degree. C.). The resultant pellet is 
resuspended in the original volume of 0.32M sucrose and frozen. After 
thawing, the suspension is diluted (1:1 v/v) with 10 mM Na.sub.2 HPO.sub.4 
/KH.sub.2 PO.sub.4 buffer (pH=7.4). .sup.3 H!PZ (0.5 nM, 0.04 .mu.Ci) is 
then incubated in multiple tubes with a 100 .mu.l sample of the tissue 
suspension and 10 .mu.l of a test compound solution (10 .mu.M in water or 
ethanol) or vehicle (for control) in a sufficient quantity of the above 
phosphate buffer to give a total volume of 1 ml. One half of the tubes 
also contain 2 .mu.M atropine sulfate which binds non-specifically with 
muscarinic receptors and provides for correcting the data for binding with 
other muscarinic receptors which may be present. After 60 minutes of 
incubation as 25.degree. C. in the dark, the binding is terminated by 
vacuum filtration onto Whatman GF/B filters (pre-soaded for 60 minutes in 
0.1% (w/v) polyethylenimine to reduce non-specific binding). After three 
washes with the phosphate buffer (4.degree. C., 3 ml/wash) the vacuum is 
allowed to continue for two minutes before the filter-trapped 
radioactivity is assayed by liquid scintillation spectroscopy. Specific 
.sup.3 H!PZ binding is defined as total binding minus binding in the 
presence of 2 .mu.M atropine sulfate. Data are pesented in Table I below 
as the percent inhibition (control-test!/control.times.100%) of .sup.3 
H!PZ binding by the test or reference compound. 
2. Phosphoinositide Turnover 
The ability of the compounds of this invention to stimulate hydrolysis of 
phosphoinositide (PI) in Chinese Hampster Ovary (CHO) cells which had been 
transfected with CMV vector containing cDNA expressing M1 acetylcholine 
receptors was determined in accordance with the procedure of El-Fakahany 
et al., J.Pharmacol. Exp. Ther. 257, 938 (1991), whereby PI hydroxysis is 
performed in reaction tubes, each containing 880 .mu.L Kreb's Buffer, 10 
.mu.L of 1.0M LiCl solution, 10 .mu.l of the compound representative of 
this invention or control vehicle, and 100 .mu.L of CHO cell suspension in 
Kerb's Buffer (1,000,000 cells per mL). The tubs are incubated for one 
hour at 37.degree. C. The reaction is quenched with chloroform. Phase 
separation is assured with the addition of methanol and water followed by 
centrifugation. The tritiated inositol phosphates are recovered on BioRad 
AG 1-X8 anion exchange resin in the formate cycle. After washing the resin 
with water and myo-inositol, the inositol phosphates are eluted with 
ammonium formate/formic acid, collected and subjected to liquid 
scintillation spectroscopy. The results are expressed as a percentage of 
the mean value obtained for carbachol (EC.sub.50 =8.0 .mu.M). 
Table I summarizes the results of the above in -vitro procedures. 
TABLE I 
______________________________________ 
IN VITRO PHARMACOLOGY 
M1 Binding PI Turnover 
% Inhib. of % above 
COMPOUND .sup.3 H! Pz @ 10 .mu.M 
baseline 
______________________________________ 
Example 3 98.4 
Example 4 100.5 
Example 5 100.5 
Example 6 98.3 
Example 7 99.3 
Example 8 98 
Example 11 101 @ 1 .mu.M 
102.5 
Example 13 109 
Example 14 101 73 
Example 15 100 
carbachol 100 
______________________________________ 
Current evidence indicates the presence of M.sub.1 and M.sub.2 muscrinic 
receptors in the central nervous system. M2 receptors are presynaptic and 
function on cholinergic terminals as "autoreceptors" to modulate the 
release of acetylcholine. Activation of the M2 receptors inhibits 
acetylcholine release and is not a therapeutically desirable result for 
the treatment of a disease characterized by cholinergic hypofunction. M1 
receptors are localized on the postsynaptic nerve cell. While the central 
nervous system contains a high proportin of M1 receptors, muscarinic 
receptors in the periphery are mainly of the M2 type. For these reasons, 
pharmacotherapy designed to directly enhance central cholinergic function 
should be targeted toward the M1 receptor, rather than the M2 receptor. In 
order to determine the selectivity of a test compound for M1 versus M2 
receptors, it is necessary to test compounds not only for their ability to 
affect M1 muscarinic binding, but for their ability to affect M2 
muscarinic bindings as well. Triatiated quinuclidinyl benzylate (.sup.3 
H!QNB) binds selectively with M2 receptors. 
3. M2 Binding With .sup.3 H!QNB 
Male Sprague-Dawley rat brain cerebellum which contain a high proportion of 
M2 receptors are dissected and homogenized using a hand-held teflon-coated 
pestle in 20 volumes of 0.32M sucrose. After centrifugation (747.times.g 
for 10 min at 4.degree. C.), the resultant supernatant is decanted and 
recentrifuged (18677.times.g for 20 min at 4.degree. C.). The resultant 
pellet is resuspended in the original volume of 0.32M sucrose and frozen. 
After thawing, the suspension is diluted(1:2 v/v) with 10 mM Na.sub.2 
HPO.sub.4 KH.sub.2 PO.sub.4 buffer (pH=7.4). .sup.3 H!quinuclidinyl 
benzylate (0.23 nM, 0.01 .mu.Ci) is then incubated in multiple tubes with 
a 100 .mu.l sample of the tissue suspension and 10 .mu.l of a test 
compound solution (10 .mu.M in water or ethanol) or vehicle (for control) 
in a sufficient quantity of the above phosphate buffer to give a total 
volume of 1 ml. The assays are done in duplicate. One half of the tubes 
also contain 100 .mu.M atropine sulfate which binds non-specifically with 
muscarinic receptors and provides for correcting the data for binding with 
other muscarinic receptors which may be present. After 60 minutes of 
incubation as 25.degree. C. in the dark, the binding is terminated by 
vacuum filtration onto Whatman GF/B filters. After three washes with the 
phosphate buffer (4.degree. C., 3 ml/wash) the vacuum is allowed to 
continue for two minutes before the filter-trapped radioactivity is 
assayed by liquid scintillation spectroscopy. Specific .sup.3 H!QNB 
binding is defined as total binding minus binding in the presence of 100 
.mu.M atropine sulfate. Comparison of the effects of test compounds on M2 
binding with effects of M1 binding (above) will indicate the relative in 
vitro selectivity of test compounds toward M1 versus M2 receptors. Table 
II shows the selectivity of the compound of Example 11 for binding to 
muscarinic M1 receptors as compared to binding to muscarinic M2 receptors. 
TABLE II 
______________________________________ 
M2/M1 
COMPOUND M1 Binding M2 Binding Ki ratio 
______________________________________ 
Example 11 Ki = 1.34 nM Ki = 13.35 nM 
10 
______________________________________ 
4. M1 Binding With Chinese Hampster Ovary Cells 
The binding affinity of the compounds of this invention at muscarinic 
receptor subtypes was determined by incubating triplicate samples of 
homogenized Chinese Hamster Ovary (CHO) cells which had been transfected 
with CMV vector containing cDNA expressing individual muscarinic receptor 
subtypes, for one hour at 37.degree. C. with 0.23 nM radiolabeled 
quinuclidinyl benzilate .sup.3 H!QNB, a representative compound of this 
invention, and a volume of 10 mM phosphate buffer to obtain a final 
incubation volume of 1000 .mu.L. Vehicle and 2 .mu.M atropine sulfate are 
substituted for the test solution to determine total and non-specific 
bindings, respectively. After incubation, the solutions are filtered and 
the filter paper is subjected to scintillation spectroscopy for 
radioactivity counting. Specific binding in the presence of the compound 
of this invention is expressed as a percentage of the atropine-sensifive 
binding. A concentration-response evaluation is obtained through 
non-linear regression analysis to obtain an IC.sub.50 and/or K.sub.i 
value. This procedure is based on hat of Tonnaer et al., Life Sci., 40, 
1981 (1987) The results obtained with several invention compounds is shown 
in Table III. 
TABLE III 
______________________________________ 
Human m1 receptors in CHO cells: 
COMPOUND Ki (nM) 
______________________________________ 
Example 1 240 
Example 2 140 
Ekample 9 80 
Example 10 9 
Example 12 206 
______________________________________ 
Pharmaceutical Composition 
Compounds of this invention may be administered net or with a 
pharmaceutical carrier to a patient in need thereof. The pharmaceutical 
carrier may be solid or liquid. 
Applicable solid carriers can include one or more substances which may also 
act as flavoring agents, lubricants, solubilizers, suspending agents, 
fillers, glidants, compression aids, binders or tablet-disintegrating 
agents or an encapsulating material. In powders, the carrier is a finely 
divided solid which is in admixture with the finely divided active 
ingredient. In tablets, the active ingredient is mixed with a carrier 
having the necessary compression properties n suitable proportions and 
compacted in the shape and size desired. The powders and tablets 
preferably contain up to 99% of the active ingredient. Suitable solid 
carriers include, for example, calcium phosphate, magnesium stearate, 
talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl 
cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine, low 
melting waxes and ion exchange resins. 
Liquid carriers may be used in preparing solutions, suspensions, emulsions, 
syrups and elixirs. The active intredient of this invention can be 
dissolved or suspended in a pharmaceutically acceptable liquid carrier 
such as water, an organic solvent, a mixture of both or pharmaceutically 
acceptable oils or fat. The liquid carrier can contain other suitable 
pharmaceutical additives such a solubilizers, emulsifiers, buffers, 
preservatives, sweeteners, flavoring agents, suspending agents, 
thickeningk agents, colors, viscosity regulators, stabilizers or 
osmo-regulators. Suitable examples of liquid carriers for oral and 
parenteral administration include water (particularly containing additives 
as above, e.g., cellulose derivatives, preferable sodium carboxymethyl 
cellulose solution), alcohols (including monohydric alcohols and 
polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., 
fractionated coconut oil and raachis oil). For parenteral adminstration 
the carrier can also be an oily ester such as ethyl oleate and isopropyl 
myristate. Sterile liquid carriers are used in sterile liquid form 
compositions for parenteral admininstration. 
Liquid pharmaceutical compositions which are sterile solutions or 
suspensions can be utilized by, for example, intramuscular, 
intraperitoneal or subcutaneous injection. Sterile solutions can also be 
administered intravenously. Oral administration may be either liquid or 
solid composition form. 
Preferably the pharmaceutical composition is in unit dosage form, e.g., as 
tablets or cpsules. In such form, the comosition is sub-divided in unit 
dose containing appropriate quantities of the active ingredient; the unit 
dosage form can be packaged compositions, for example packed powders, 
vials, ampoules, prefilled syringes or sachets containing liquids. The 
unit dosage form can be, for example, a capsule or tablet itself, or it 
can be the appropriate number of any such compositions in package form. 
The dosage to be used in the treatment of a specific patient suffering from 
cerebral acetylcholine insufficiency must be subjectively determined by 
the attending physician. The variables involved include the severity of 
the dysfunction, and the size, age, and response pattern of the patient.