US 20020094.999A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2002/0094999 A1 Greig et al. (43) Pub. Date: Jul.18, 2002 (54) HIGHLY SELECTIVE Related U.S. Application Data BUTYRYLCHOLINESTERASE INHIBITORS FOR THE TREATMENT AND DAGNOSS OF (62) Division of application No. 09/254,494, filed on Jun. ALZHEMER'S DISEASE AND DEMENTAS 17, 1999, filed as application No. PCT/US98/14063, filed on Jul. 9, 1998. (76) Inventors: Nigel H. Greig, Phoenix, MD (US); Qian-Sheng Yu, Baltimore, MD (US); (60) Provisional application No. 60/052,087, filed on Jul. Arnold Brossi, Bethesda, MD (US); 9, 1997. Timothy T. Soncrant, Silver Spring, MD (US); Marvin Hausman, Publication Classification Stevenson, WA (US) (51) Int. Cl." ............................................ A61K 31/407 Correspondence Address: (52) U.S. Cl. .............................................................. 514/411 Intellectual Property Docket Administrator Gibbons, Del Deo, Dolan, Griffinger & (57) ABSTRACT Vecchione One Riverfront Plaza Newark, NJ 07102-5497 (US) The present disclosure relates to the discovery that highly Selective butyrylcholinesterase inhibitors can prevent or (21) Appl. No.: 10/071,488 treat cognitive impairments associated with aging or Alzhe imer's disease. A preferred butyrylcholinesterase inhibitor is (22) Filed: Feb. 7, 2002 cymserine. Patent Application Publication Jul.18, 2002 Sheet 1 of 7 US 2002/0094999 A1 Compound Structure No. Compounds ReferencesNo. in 1, R=CH Physostigmine 1. 2, R= (S1 Phenserine 21 122 O CH3 RHN? 3, R= SC Tolserine 3. 8? Ol 4, R=-C Cymserine 4, 192 CH, CH, 5, R= ?C 4-Methoxyphenserine CH 6, R-(C 4-Methylpherserine 13? 7, R= CC 2-Isopropylphenserine 182 O CH3 8, R=CH, N-Norphysostigmine 5 ech 9, R= C, N'-Norphenserine 6 1. CHN 'NH 10, R=CC N-Nortoiserine 7 11. Rs -(C N-Norcymserine 8 9 CH3 12, R=CH N-Benzylnorphysostigmine 9 “ch 13, R=C N-Benzyinorphenserine 10 N N 14, R=-- CC N'-Benzyinortolserine1- w 11 CH3 Bn i5, R-g N-Benzylnorcymserine 12 16, R=CH N-Phenethylnorphysostigmie 13' RHNC9 CH3 17, R= G N-Phenethylnorphenserine:- - 14141 C t N J 18,8, RR= CC N'-Phenethylnortolserinel 15 CHCH2CH2Ph 19, R-1C N-Phenethylnorcymserine 16 GURE A Patent Application Publication Jul.18, 2002 Sheet 2 of 7 US 2002/0094999 A1 Q CH3 20, Ri=CH3, R= -CH3CH=CH 73 N N 21, R=CH R=NHCH, 6 CH3 R2 E. ine O CH 22, R=CH3 Thiaphysoverinepny 3i *OthN S 24,are R=wo CC Thiatolserineas a11 4. CH3 25, R-1C Thiacymserine 134 26, R=CH Physovenine 2a CH3 5 ech 9a N O 28, R= CC Tolsveninew 2 CH 9 CH3 30, R=CH N-Benzyinorphysostigmie (-)9° RHNCON O 31, R= G N-Benzylnorphenserine (-)10 N 'N 32, R= CC N-Benzylnortolserine Bn CH 33, R-1C N-Benzylnorcymserine O CH 34, R=CH N-Norphysostigmie (-)11 RHNdo C 3. 35, R= G N-Norphenserine (-)12 N N 36, R= CC N-Nortoiserine H CH 3 37, R-1C N-Norcymserine FIGURE B Patent Application Publication Jul.18, 2002 Sheet 3 of 7 US 2002/0094999 A1 38, R=CH, NN-Bisnorphysostigmie (-)10 RHNd 39, R= Gr N,N-Bisnorphenserine (-)97 Ol 40, R= CC N,N-Bisnortoiserine H H 41, R-1C N,N-Bisnorcymserine 42, R=CH NN-Bisbenzylnorphysostignielar8 to: (-)197 *O CH3 43, R= C, N,N-Bisbenzynorphenserine(-)187 N N 44, R= CC N,N-Bisbenzylnortoiserine B B 45, Rs-C N,N-Bisbenzyinorcymserine FGRE C Patent Application Publication Jul.18, 2002. Sheet 4 of 7 US 2002/0094999 A1 Pheniserines Alter the Secretion of B-APP In Vitro Cells: Human Neuroblastoma cells (1 x 107, MR-32) Conditions: low serum media (0.5% FBS) incubated with drug 2 days Antibody: 6E10 (residues 1-28 of Af)-Shown 22C11 (arnino terminal, extracellular domain of 3-APP)-6E10 pattern Anti-HSP-70 (heat shock protein-70)-No different from unfreated CNT Drugs: Phenserine, Toiserine (AChE selective) Cymserine (BChE selective) 2 3 4. 5 6 7 8 (Mw) CNT PhenSeine folserine Cynserine 16 5 50 5 50 5 50 ugm Conditioned media 2 3. 4. 5 6 7 8 (MW) CNT Phenseline Tolserine Cymserine 16 5 50 5 50 5 50 g/ml 95 unaba u as a = 66 Cell lysate ReRE 2 Patent Application Publication Jul.18, 2002 Sheet 5 of 7 US 2002/0094999 A1 Naive (Non-lesion) Lesion - a gap H Bar, H : 150 2 is 2. 2 -|| 3333 332.2 22 3|333 3 222 2 Control Phenserine Cymserine Control Phenserine Cymserine p = 0.0025 versus total B-APP level in naive control. # p < 0.003 versus total B-APP level in lesion control, + p < 0.0003 versus 3-APR level in naive control. }{ p < 0.025 versus B-APR level in lesion control. Cymserine protects against a cholinergic forebrain lesion-induces increase in ?-APPY and, additionally, reduces levels in normal animals FIGURE 3 Patent Application Publication Jul.18, 2002 Sheet 6 of 7 US 2002/0094999 A1 CYMSERINE, 1 MG/KG LV. N. RAT O 1. - FIGURE 4 Patent Application Publication Jul.18, 2002 Sheet 7 of 7 US 2002/0094999 A1 -L- 09. e-I Lued so II US 2002/0094999 A1 Jul.18, 2002 HIGHLY SELECTIVE organo phosphates. AS above, no specific indication is given BUTYRYLCHOLINESTERASE INHIBITORS FOR as to which type of inhibitors would be used in which THE TREATMENT AND DIAGNOSIS OF Specified disorder. ALZHEMER'S DISEASE AND DEMENTAS 0004 Geula and Mesulam in a paper entitled “Cholinest BACKGROUND OF THE INVENTION erases and the Pathology of Alzheimer's Disease”, Alzhe imer's Disease and Associated Disorders, Vol. 9, Suppl. 2, 0001 Defects in the cholinergic system have been Sug pp. 23-28 (1995) make the following observations in sum gested to underlie cognitive impairments associated with mary: "Alzheimer's Disease (AD) is accompanied by a normal aging and Alzheimer's disease (Bartus et al., Science marked loss of acetylcholinesterase (AChE) activity asso 217:408-417 (1982); Fisher et al., Neurobiol. Aging 13:9-23 ciated with cortical cholinergic axons and cholinoceptive (1992)). Much research has focused on the development of neurons. Simultaneous with this loss, cholinesterase (ChE) cholinomemetic replacement therapy as a potential treat activity emerges in AD cortex in the form of AChE and ment of these impairments. Among them, cholinesterase BChE activity associated with plaques, tangles, and amyloid inhibitors, Such as physostigmine (Phy) and tetrahydroami angiopathy. Our observations have shown that the ChE’s noacridine (Tha) have been investigated for memory-en associated with the pathological lesions of AD (ADChEs) hancing effects in both animals (Rupniak et al., Neurobiol. possess different enzymatic properties and quite possibly are Aging 11:09-613; 1990); Murray et al., Psychopharmacol of a different Source as compared with the ChES associated ogy 105:134-136(1991) and human patients (Mohs et al., J. with normal neurons and axons. The ADChEs most likely Am. Geriatr. Soc. 3:749-757 (1985); Summers et al., N. have noncholinergic functions involved in the pathogenesis Engl. J. Med. 315:1241-1245(1986)). of AD. In a further section the authors at p.26 state: “These observations indicate that glia are a likely Source of the ChE, 0002 Other agents have been proposed as selective and particularly the BChE, associated with the pathological inhibitors of acetylcholinesterase (AChE). Thus heptyl-phy lesions of AD. They also suggest that a high ratio of BChE SoStigmine (Heptyl-Phy) was described as having greater to AChE positive glia may play a permissible or causative lipophilicity, longer inhibitory action on cholinesterase and role in the neuropathology of this disease. It is possible that more persistent increases in acetylcholine in brain with leSS other pools of ChE exist with enzymatic properties similar toxicity than the parent compound (Brufani et al., Pharma or identical to those of AD ChEs. This possibility remains col. Biochem. Behav. 26:625-629 (1987)). There is concern, unexplored.” however, as to whether the therapeutic window of heptyl Phy is wide enough for clinical use. Phenserine ((-)-N- 0005 Workers in the art have indicated that BChE is phenylcarbamoyl eseroline) has been identified as a Supe found in Significantly higher quantities in AD plaques than rior, Selective AChE inhibitor and thus Suited as an agent for in plaques from age-matched non-demented brains. More the therapy for cognitive impairments associated with aging over, BChE was found to alter the aggregation of beta and Alzheimer's disease. (U.S. Pat. No. 5,409,948, issued amyloid peptide (AB). It has been hypothesized that Since Apr. 25, 1995). In U.S. Pat. No. 5,171,750 issued Dec. 15, AChE is inhibited by high concentrations of acetylcholine 1992, a series of substituted phenserines are disclosed which (ACh), while BChE remains unaffected, it may well be that are indicated to be either selective inhibitors of AChE or BChE may play an important role in the in Vivo regulation butyrylcholinesterase (BChE). The cumylcarbamate (4'-iso of synaptic concentrations of ACh in the brain of AD propylphenylcarbamate) derivative of (-)-phySovenol was patients. BChE inhibitor, instilled into the brain has pro noted to have a reverse enzyme Specificity, i.e., it inhibited duced a significant increase in the level of extracellular Ach. BChE selectively over AChE. The patent indicates that the (Giacobini, et al., Proc. Soc. Neurosci., 22; 203, 1996.) compounds of the invention are useful “for treating cholin ergic disorderS Such as glaucoma, Myasthenia Gravis, 0006. It has also been found that in 3 AD specimens Alzheimer's disease and as an antidote against poisoning plaques, which were of the compact or neuritic type, were with organo phosphates.” There is no indication as to which almost always associated with intense BChE activity.