GAMMA-EMITTING RECEPTOR-BINDING QUINUCLIDINYL BENZILATES;METHODS OF PREPARATION THEREOF AND IMAGING AND ASSAY METHODS UTILIZING SAME

Abstract: Compounds of the formula: (FORMULA) wherein R is phenyl, cyclopentyl or cyclohexyl, R<u1>u is H or lower alkyl preferably CH<u3>u, X is in the ortho-, meta-or para- position, and is selected from the group consisting of <s125>sI <s123>sI, I, <s18>sF, <s77>sBr, <s75>sBr, NH<u2>u, and -N=N-N (FORMULA) , R<u2>u is in the 2, 3 and 4 position and is selected from the group consisting of H and lower alkyl, and Z <s//c>s is an anion; or free amine form thereof; methods for their production and compositions for and methods of tissue radio-assay and radio-external imaging utilizing said radioisotope containing compounds.

Description: GAMMA-EMITTING RECEPTOR-BINDING QUINUCLIDINYL BENZILATES; METHODS OF PREPARATION THEREOF AND IMAGING AND ASSAY METHODS UTILIZING SAME BACKGROUND -OF THE INVENTION Field of the Invention The present invention relates to various muscarinic cholinergic receptor-binding compounds, gamma-emitting radio-isotope derivatives thereof, intermediates and methods for the production thereof and the use of the said radioisotope derivatives as radio-pharmaceuticals in the assay and external imaging of myocardial tissue and other organs containing muscarinic cholinergic receptors. It is known that muscarinic cholinergic receptors are concentrated in myocardial and other tissue. It is theorized that these receptors are involved in the action of drugs or biochemicals in the myocardial tissue and that changes in the concentration of the receptor in the myocardial tissue are a function of a disease state therein or in other tissue containing the receptors. It has been established that various compounds function both in vitro and in vivo as muscarinic cholinergic receptor binders or antagonists. These compounds have a high affinity for and competitively bind with the receptor. It has been established that various quinuclidinyl benzilates are effective muscarinic cholinergic receptor binders. It has further been suggested that tritium-labeled quinuclidinyl benzilates may be utilized as radiotracers for various assay procedures involving myocardial tissue. See Eckelman et al, J. Nucl. Med. 20, 350 (1979) and Gibson et al, J. Nucl. Med. 2 , 865 (1979) . . . OMPI There are numerous disadvantages inherent in the use of tritium-labeled radiotracers. For example, numerous problems are associated with "counting" beta- emissions of tritium-labeled, compounds. Liquid scintillator must be added to each sample which is a time-consuming and expensive procedure. Toluene is the typical scintillator liquid employed which is presently subject to strict, environmental sanctions thereby rendering its disposal problematical. In addition beta- counting procedures are plagued, with problems of chemluminescense and quenching which are absent in gamma-counting. Tl-201 is presently employed for the detection and quantification of myocardial infarcts. However, the radiohalogenides- such as 1-123 and Br-77 have better imaging characteristics in that their higher gamma energies can be detected with increased sensitivity and positional resolution as compared with the lower gamma energy of Tl-201 (80 Ke V X-rays). Since the size of the infarct is related to mortality and residual function, improved resolution will mean improved prognosis and evaluation of drug therapy. The radiohalogenides F-18 and Br-75 are positron emitters which offer the unique capabilities of reduction and quantification associated with such decay characteristics. It is an object of the present invention to provide various quinuclidinyl benzilates and gamma- emitting radioisotope containing, quinuclidinyl benzilates useful for muscarinic cholinergic receptor assays, and imaging of the myocardium and other muscarinic cholin¬ ergic assay and tissue imaging techniques. Summary of the Invention These and other objects of the present invention are achieved by providing compounds of the formula: Wherein R is phenyl, cyclopentyl or cyclohexyl, R is H or lower alkyl, preferably CH,, X is in the ortho-, meta- or para- position, and is selected from the 123 τ 1, 1 τ , -N in the 2, 3, or 4 position and is selected from alkvl , and, (?) #-' is an anion; or the corresponding free amines, i.e., the non-protonated or non-quaternary salt form. The radio-labeled compounds are gamma-emitting and are useful in formulating compositions suitable for the in vitro assay of muscarinic cholinergic receptors in e.g. , myocardial tissue and for formulating compositions suitable for use and the method for external imaging or radio-assay of myocardial tissue and other tissue containing muscarinic cholinergic receptors. The iodo, amino- and triazeno- derivatives are valuable as intermediates in the preparation of the radio- labeled analogs. OMPI /,, WIPO Detailed Description of the Invention The aminobenzil may be prepared according to the method of Augl et al, Arinu. Conf. , SPE, Reinf. Plastic/ Compos. Div. Proc, 26th, 19D, 1 (1971) to produce 4-Aminobenzil. The 4-Aminobenzil is rearranged to yield 4-aminobenzilic acid which is in turn esterfied to produc the ethyl ester. The ethyl ester of the 4-aminobenzilic acid is then reacted with quinuclidin-3-ol to give the expected 3-quinuclidinyl 4-aminobenzilate (4-amino-QNB) . The purified 4-amino QNB ' is then converted to the 4-triazeno-QNB according to the procedure reported by Tewson et al, J. Nucl. Med. 2_ , 671 (1979). The purified 4-triazeno-QNB is then reacted with iodide or a desired gamma-emitting radionuclide to produce the desired produc In the above structural formula the anion Z ^ may be any pharmaceutically acceptable anion such as Cl , Br " , or I " , S0 4 = , HCOO " , CH- j COO " , etc. The following examples illustrate the preparation of the claimed compounds. Example 1 4-Aminobenzilic Acid A solution of 25 g of NaOH in 50 ml of H^O was placed in a water bath kept at 95°C. To this magneticall stirred solution was added 11.26 g (50 mol)' of 4-amino- benzil (I) in small portions. After the addition was completed the mixture was stirred for 5 hours at 95°C. Water was added during the reaction to maintain the volum at about 100 ml. After 5 hours the heating was discontinued and the reaction mixture was transferred to separatory funnel and extracted twice with 50 ml of ethyl ether. The aqueous layer was cooled to 0° acidified with cone. HpSO. until turbid, and extracted with ethyl acetate (AcOEt) (100ml). The aqueous layer was acidified and extracted with AcOEt again. The combined AcOEt layers were washed with water (2x) , dried over MgSO, , filtered and spin evaporated in AcOEt and filtered. The combined precipitates were recrystallized from water/acetone 25/75. Yield 8.9 g(73%). Yellow crystals, mp dec. 150°C; Silica gel TLC in acetone, Rf 0.25; HPLC Bondapak μC lg in " MsOH/H^O (75/25) pH 4 (formic acid). IR, UV, mass spec, were consistent with the structure. Elemental Analysis calc for C I Λ H I T NO " 2/3 H 2 0, Calc. C 67.28, H 5.78, N 5.60; Found C 67.45, 67,64, H 5.49, 5.41, N 5.52, 5.51. Example 2 4-Aminobenzilic Acid Ethyl Ester 4.8 g (19.7 mmol) of 4-aminobenzilic acid was dissolved in 200 ml of absolute ethanol (EtOH) saturated with dry HC1 gas. The reaction mixture was refluxed for 24 hours then spin evaporated to dryness in vacuum. The residue was dissolved in water and neutralized with NaHC0 3 , extracted with 2 x 50 ml of AcOEt, washed with water (2x) and the AcOEt extract filtered through a siliconized filter and dried over MgSO.. The dried solution was filtered and spin evaporated in vacuum. Yellow oil, 2.8 g (52%). SGTLC in acetone Rf 0.8. HPLC Bondapak μC lg MeOH 1/H 2 0 (75.25) pH 4 (formic acid). Elemental Analysis Calc. for C. fi H, 7 NO,. Calc. N 5.16; Found N 5.04, 5.26. Example 3 4-Aminobenzilate of 3- (R,S)-quinuclidinol 5.16 g (40 mmol) of 3- (R,S) -quinuclidinol was dissolved in 50 ml of dry benzene and 20 ml of benzene distilled off. A clean 100 mg piece of sodium was added and the magnetically stirred suspension, protected from moisture and C0 2 (NaOH trap), refluxed for 24 hrs.2.8 g (10.3 mmol) of ethyl 4-aminobenzilate was dissolved in 50 ml of dry benzene and 20 ml of benzene removed by distillation. Both solutions were combined and refluxed, protected from moisture and CO for 24 hours. The solution was spin evaporated, the residue suspended in water and extracted twice with 50 ml of AcOEt. The AcOEt extract was washed repeatedly with water, filtered through a siliconized filter paper and dried over MgSO^. The dried solution was filtered and spin evaporated in vacuum. The residue was dissolved in CH,CN and charged on a silica gel column (2.8 x 100 cm) eluted with CH,CN. Pure fractions crystallized on standing. The product recrystallized from CH,CN, 1.5 g (41%) white crystals; SGTLC n-butanol, acetic acid, water 4:1:1, Rf 0.4; HPLC Bondapak μC 18 MeOH/H 2 0 40/60, pH 4 formic acid. IR, UV, mass spec, were consistent with the structure of the product. Calc. for C21 H 24 N 2°3 ' Calc. C 71.57, H 6.86, N 7.95; Found C 71.27, H 6.87, N 8.19. Example 4 4- [2- (3-methylpiperid-l-yl) -1,2- diaza-ethylen-1-yl] -benzilate of 3- (R,S)-quinuclidinol Sodium nitrite (75 mg, 1.08 mmol) was added to a cooled (0°C) solution of 190 mg (54 mmol) of 4-amino QNB (IV) in 6 ml of 10% H 2 S0 4 and acetone (5:1). The mixture was stirred for 15 min. at 0° then treated with 65 mg (1.08 mmol) of urea. The diazonium salt slurry was added to a cooled (0°C) solution of 536 mg (5.4 mmol) of 3-methylpiperidine in 5 ml of water. The mixture was stirred at 0°C for 20 min. then made basic with 4N NaOH to pH 12 and extracted with CHC1, (3x5 ml) . The combined extracts were washed repeatedly with water and dried over MgSO.. The solution was filtered and spin evaporated in vacuum. The residue was extracted with petroleum ether (3 x 10 ml) and the combined extracts evaporated and dried in high vacuum to remove the traces of 3-methylpiperdine. Obtained was 240 mg (96%) of viscous yellow oil. SGTLC in 2% NH 4 0H in MeOH, Rf 0.5. HPLC Bondapak μ lg 5 mM 1- hexanesulfonic acid pH 4 (H 2 S0 4 ) in MeOH/H 2 0, 60/40. UV, IR, mass spec, were consistent with the product structure. Elemental analysis for C- 7 H, 4 4 0, • 2H-0. Calc. 65.03, H 7.68, N 11.23; Found C 65.35, H 7.44, N 10.62. Example 5 (R,S) -Quinuclidin-3-ol-4-iodo benzilate A solution of 5 mg (11 μ ol) of QNB-triazene (V) and 1.6 g (11 μmol) of sodium iodide in 1 ml of trifluoro- ethanol was treated with 6.4 mg of methane-sulfonic acid. The reaction mixture was heated on a water -bath for 45 min. , cooled, 5 ml of water added and extracted with 2 x 5 ml of AcOEt. The aqueous layer was then neutralized with 4.N NaOH and extracted with 3 x 5 ml of AcOEt. The organic layer was washed three times with 1 ml of water, filtered through a siliconized paper and dried over Na 7 S0 4 . After spin evaporation in vacuum, a yield of 2.7 mg (54%) was obtained as a yellow oil. UV, IR, mass spec, were consistent SGTLC in n-BuOH:AcOH:H 2 0, 60/40. Bondapak μ, g 5mM 1-octanesulfonic acid, pH 4, MeOH/H 2 0, 60/40. Example 6 (R,S) -Quinuclidin-3-ol-4-bromo or 4-iodo or 4-fluorobenzilate (radio nuclides) The compounds are prepared using the above pro¬ cedure to produce the iodo-benzilate substituting sodium radio iodide or radio bromide or tetrabutyl ammonium radio fluoride for sodium iodide. To prepare .the protonated or quaternary derivative, the product of Example 5 or 6 is dissolved in a solution of the appropriate salt and recrystallized therefrom. OMPI IPO Ex am le 7 The radiohalogenide containing compound is utilized for imaging as follows.: The radio labeled tracer (I-123-QNB) is injected intravenously in the amount of 1 to 10 mCi at a specific activity to exceed 1000 Ci/mmol. Images of the distribution of radioactivity in the target organ are obtained as a function of time and the data stored in a computer. The data is then analyzed using an appropriate pharmacokinetic model to determine the concentration of receptors as a function of time. Alternately the information can be used to determine the blood flow to th target organ. For positron-emitting radiotracers such as Br-75 and F-18, coincidence counting is used. The images are collected over 2 hours and the target organ can be any locus of muscarinic cholinergic receptors such as the brain, the heart or the pancreas. Example 8. The radiohalogenide containing compound can be use for the radio assay of tissue as follows: Heart microsomal preparations are obtained as described by Harden et al [Mol Pharmacol 12: 1-15, 1976J. The heart from a rabbit is removed after the animal has been killed with ether. The heart is dissected free from atria and large vessels, minced with scissors and homogenized in buffer using a Brinkman Polytron. The ho ogenate is certrifuged at 10,000 g for 20 min. and the supernatent discarded. The pellet is resuspended in buffer and recentrifuged. The pellet is then suspended i buffer. This homogenate is centrifuged over a layer of 0.2 M. sucrose above a layer of 1.72 M sucrose. Membranes are collected at the interface of the two sucrose layers and used immediately for the radio- receptor assay. O Aliquots of the muscarinic cholinergic receptor isolated above, 1-125 IQNB and the test drugs are incubated at 37°C with agitation. Incubations are carried out routinely for 60 min. Each incubation is terminated within 10 sec. by filtering the suspension through a GF/B glass fiber filter positioned over a vacuum flask. The filter is rinsed four times with buffer. The filter is then counted in a NaΙ(Tl) scintillation counter. Specific binding is experimentally determined from the difference between counts bound in the absence and presence of 1 μM atropine. This same procedure can be used for any of the radio-labeled derivatives of the invention.

Claims: CLAIMS Wherein R is phenyl, cyclopentyl or cyclohexyl, R., is H or lower alkyl, X is in the ortho-, meta- or para- position, and is selected from the group consi •s.t-i■ng of - 125I τ , 123I τ , I τ , 18 F, 77 Br, 75 Br, NH 2 , and ■ N = N P wherein R 2 is the 2, 3 or 4 position and is selected from th group consisting of H and lower alkyl and Z ^ is an anion; or the free amine form thereof. „>... WIP 2. A compound of the formula; Wherein R is phenyl, cyclopentyl or cyclohexyl R χ is H or CH 3 , I is in the ortho-, eta- or para- position, and -2-^ is an anion; or the free amine form thereof. 3. The compound of Claim 2, wherein said I is the meta-position and R, is H. 4. The compound of Claim 2 wherein said I is in the 5. The compound of Claim 2 wherein said I is in the para-position and R. is H. 6. The compound of Claim 2 wherein said I is in the para-position and R, is CH-.. 7. The compound of Claim 2 wherein I is in the ortho- position and R, is H. 8. The compound of Claim 2 wherein I is the in the ortho-position and R- is CH,. OMPI tΑ & Wherein R is phenyl, cyclopentyl or cyclohexyl, R 1 is H or CH 3 , 125 I is in the ortho-, meta- or para^ position, and 2-^ is an anion; or the free amine form thereof. 10. The compound of Claim 9 wherein said 125I is in the meta-position and R-, is H. 11. The compound of Claim 9 wherein 1?SI is in the meta-position and R^ is CH,, 12. The compound of Claim 9 wherein said 12"5I. is in the para-position and R-, is H. 13. The compound of Claim 9 wherein said 125I is in the para-position and R, is CH,. 14. The compound of- Claim 9 wherein 125I is in the ortho-position and P, is H. 15. The compound of Claim 9 wherein 125I is in the ortho-position and P.. is CH-,. OM WIP 16. A compound of the formula: 1 Wherein R is phenyl, cyclopentyl or cyclohexyl, R χ is H or CH 3 , 125 '.I is in the ortho-, meta- or para- josition, and -zr is an anion; or the free amine form thereof. 17. The compound of Claim 16 wherein said 123I is in the meta-position and R-, is H. 18. The compound of Claim 16 wherein said 123I is in the meta-position and R, is CH,. 19 The compound of Claim 16 wherein said 123I. is in the para-position and R. is H, 20. The compound of Claim 16 wherein .said 123I. is in the para-position and R, is CH,. 21. The compound of Claim 16 wherein said 1 3I is in the ortho-position and P, is H. 22. The compound of Claim 16 wherein said I is In the ortho-position and P^ is CH,. OMPI /■_, VIPO „« 23. A compound of the formula: Wherein R is phenyl, cyclopentyl or cyclohexyl, R., is H or CH 3 , 18 'F. is in the ortho-, eta- or para- position, and i-^ is an anion; or the free amine form thereof. 24. The compound of Claim 23 wherein said 18F is in the meta-position and R, is H. 25. The compound of Claim 23 wherein said 18F, is in the meta-position and R, is CH,. 26. The compound of Claim 23 wherein said 18F is in the para-pcsition and R, is H. 27. The compound of Claim 23 wherein said 18 F is in the para-position and R, is CH,. 28 The compound of Claim 23 wherein said 18F is in the- ortho - pos i tion and R , is H. 29. The compound of Claim 23 wherein said 18F is in the ortho-position andR, is- CH,. 30, A compound of the formula: Wherein R is phenyl, cyclopentyl or cyclohexyl, R 1 is H or CH 3 , 75 *B,r is in the ortho-, meta- or para- position, and ■ τ ^ is an anion; or the free amine form thereof. 31. The compound of Claim 30 wherein said 75Br is in the meta-position andRi is H. 32. The compound of Claim 30 wherein said 75,Br is in the meta-position andR is CH,. 33. The compound of Claim 30 wherein said 75Br is in the para-position andR. is H. 34. The compound of Claim 30 wherein said Br is in the para-position andR. is CH,. 35. The compound of Claim 30 wherein said 75Br is in the ortho-position andR, is H. 36. The compound of Claim 30 wherein said Br is in the ortho-position andR. is CH,. 37. A compound of the formula: Wherein R is phenyl, cyclopentyl or cyclohexyl, R-, is in the ortho-, meta- or para- position, and %• ^ is an anion; or the free amine form thereof. 38. The compound of Claim 37 wherein said 77Br is in the meta-position and R-, is H. 39. The compound of Claim 37 wherein said 77Br is in the meta-position and R-, is CH, . 40. The compound of Claim 37 wherein said 77Br is in the para-position and R-, is H. 41. The compound of Claim 37 wherein said 77Br is in the para-position and R, is CH. 42, The compound of Claim 37 wherein said 77,Br is in the ortho-position and R, is H. 43. The compound of Claim 37 wherein said 77Br is in the ortho-position and R is CH,. 44. A compound of the formula: Wherein R is phenyl, cyclopentyl or cyclohexyl, R χ is H or CH 3 , the triazene group is in the ortho-, meta- or para-position, R 2 is H or lower alkyl in the 2, 3, or 4 position, and ■ δ-^ is an anion; or the free amine form thereof. 45. The compound of Claim 44 wherein said triazene group is in the meta-position andR, is H. 46. The compound of Claim 44 wherein said triazene group is in the meta-position and R, is CH 3 . 47. The compound of Claim 44 wherein said triazene group is In the para-position and R-, is H. 48. The compound of Claim 44 wherein said triazene group is in the para-position and R. is CH, . 49. The compound of Claim 44 wherein said triazene group is in the ortho-position and P is H. 50. The compound of Claim 44 wherein said triazene group is in the ortho-position andR, is CH,. 51. A compound of the formula: entyl or cyclohexyl, R 1 is H or CH 3 , NH 2 is in the ortho, meta- or para- position, and ■ 3-^-' is an anion; or the free amine form thereof. 52. The compound of Claim 51 wherein said NH-, is in the meta-position and R, is H. 53. The compound of Claim 51 wherein said NH ~ is in the meta-position andR. is CH,. 54. The compound of Claim 51 wherein said NH 7 is in the para-position andR, is H. 55. The compound of Claim 51 wherein said NH-, is in the p r ara-p r osition andR-1 is CH,o. 56. The compound of Claim 51 wherein said NΗ ? is in the ortho-position and R. is H. 57. The compound of Claim 51 wherein said NH 2 is in the ortho-position and R., is CH... 58. A method for preparing a compound of Claim 51 comprising reacting a derivative of an aminobenzilic acid with a quinuclidin-3-ol to pτoduce a quinuclidinyl aminobenzilate. 59. A method for preparing a compound of Claim 44 comprising reacting a derivative of an aminobenzilic acid with a quinuclidin-3-ol to produce a quinuclidinyl aminobenzilate and converting the free amine group to a triazene group. 60. A method for preparing a compound of Claim 1 comprising reacting a derivative of an aminobenzilic acid with a quinuclidin-3-ol to produce a quinuclidinyl aminobenzilate, converting the free amino group to a triazene group, reacting the triazene derivative with a halogenide or radiohalogenide to form 3-quinuclidinyl halo benzilate. 61. A composition suitable for the assay of muscarinic cholinergic receptors in tissue comprising a gamma- emitting radioisotope containing compound of Claim 1 and a phara ceutically acceptable carrier therefor. 62. A composition of Claim 61 wherein said gamma- emitter is ~ l . 63. The composition of Claim 61 wherein said gamma- emitter i -s 123I τ . 64. The ition of Claim 61 wherein said gamma- OMPI '" .VIPO . •:;;ATIO^> 65. The composition of Claim 61 wherein said gamma- emitter i •s 5B_r. 66. The composition o£ Claim 61 wherein said gamma- emitter is Br. 67. A composition in unit dosage form suitable for use in a method for the external imaging or radio-assay of tissue containing muscarinic cholinergic receptors comprising a gamma-emitting radioisotope containing compound of Claim 1 and a pharmaceutically acceptable carrier therefor. 68. In a method for the. external imaging of or the radio-assay of muscarinic cholinergic receptors in tissue wherein a composition comprising a gamma-emitting radio¬ isotope containing muscarinic cholinergic receptor binding compound is administered to an animal or human patient and the gamma-emissions from the receptor-bound radioisotope containing compound are converted to visible images of the tissue or are otherwise quantitatively analyzed to determine muscarinic cholinergic receptor concentration in said tissue, the improvement wherein said administered compound is a gamma-emitting radioisotope containing compound of Claim 1. 69. The method of Claim 68 wherein said gamma-emitter is 123 I. 70. The method of Claim 68 wherein said gamma-emitter is 12S I. 71. The method of Claim 68 wherein said gamma-emitter 72. The method of Claim 68 wherein said gamma-emitter is Br. 73. The method of Claim 68 wherein said gamma-emitter is 77'Br.

Inventors: ECKELMAN W, GIBSON R, REBA R, RZESZOTARSKI W
Applicants: RESEARCH CORP, RES CORP
Application year: 1981
Main classification: A61K49
IPC codes: A61K51, C07D453, G01N33