J. Med. Chem. 1992,35, 3141-3147 were placed on a horizontal wire ring 5.5 cm in diameter, which was attached to a 16-cm vertical rod. The hind paws and fore paws were placed a t opposite sides of the ring. I t is important that the ambient temperature is maintained a t 30 OC and that the environment be free of auditory stimuli and bright lights. The criteria for immobility are detailed in ref 22. The response is calculated as the fraction of time the mouse is immobile over a 5-min test period. Measurements were always done between 2 and 4 p.m. and the animals were used only once.
3141
Acknowledgment. We thank NIDA for partial support
(Grants DA02052, DA06481 and a Research Scientist
Award to SIB) and Prof. M. A. Tius, University of Hawaii, for helpful correspondence. Registry No. Ib,113418-02-3; 3a,137945483; 3b,137945-49-4; 3c,13794550-7; 3d,137945-51-8;3e,13794552-9; 4a, 137945-53-0; 4b,137945-54-1;4c,137945-552; 5a,13794556-3; 5b,13794557-4; 5c, 137945-58-5; 6a, 137945-59-6; 6b,137945-60-9.
Synthesis and Cholinergic Properties of N-Aryl-2-[[[5-[ (dimethylamino)methyl]-2-furanyl]methyl]t hiolethylamino Analogs of Ranitidine Matthew J. Valli,t Yunzhao Tang,* J. W. Kosh,l James M. Chapman, Jr.,*J and J. Walter Sowell, Sr.l Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of South Carolina, Columbia, South Carolina 29208, and Department of Pharmacology, Shanxi Medical College, Taiyuan, Shanxi 030001, People's Republic of China. Received January 30, 1992
A series of N-aryl-2-[[ [5-[(dimethylamino)methy1]-2-furanyl]methyl]thio]ethylamino analogs of the Hz-antagonist, ranitidine, was synthesized and the abilities of the compounds to alleviate the cholinergic deficit characteristic of Alzheimer's disease evaluated. The compounds were initially tested for their ability to inhibit human erythrocyte acetylcholinesterase activity in vitro. Selected compounds were further evaluated for butyrylcholinesterase inhibition, M1 and M2cholinergic receptor binding, potentiation of ileal contractions, and the ability to elevate brain acetylcholine levels in mice. The analogs were compared to tetrahydroaminoacridine and to a recently reported series of bis[ [(dimethylamino)methyl]furans]. The N-aryl-2-[[ [5-[(dimethylamino)methy1]-2-furanyl]methyl]thio]ethylamine derivatives were generally comparable to tetrahydroaminoacridine and the bis[ [(dimethy1amino)methyllfurans] in acetylcholinesteraseinhibition, M1/Mz receptor binding, and the potentiation of ileal contractions, while being more potent inhibitors of acetylcholinesterase than butyrylcholinesterase. The 4-nitro-3-pyridazinyl analog, 26,was notable in demonstrating a potent and selective binding to the M2 receptor, with an M2 ICSO/M1ICm of 0.060. Compounds in which the substituents on the dinitro-N-aryl moiety were relatively small were the best at inhibiting acetylcholinesterase in vitro. The N-aryl-24 [[5-[(dimethylamino)methy1]-2-furanyl]methyl]thio]ethylamines in general, and those with small N-aryl substituents in particular, were superior to the bis[ [ (dimethylamino)methyl]furans] in elevating brain ACh levels in mice, probably due to enhanced distribution into the CNS. The 1,5-difluoro2.4-dinitrophenyl analog, 8, resulted in the largest elevation in brain acetylcholine levels, affording a 53% increase at 88 mg/kg.
In a previous communication,we described the synthesis and cholinergic properties of a series of bis[ [(dimethylamino)methyl]furan] analogs of ranitidine.' These compounds possessed the general structure shown in Figure 1,and demonstrated potent acetylcholinesterase (AChE) inhibitory activity in vitro for a wide variety of substituents, "Z". Compound 1 (ICm = 0.03 pM) was the most potent AChE inhibitor in the series and was found to be approximately 6 times more potent than tetrahydro-9aminoacridine (THAI, which is currently undergoing extensive clinical investigation in the treatment of Alzheimer's disease (AD). A number of these analcgs also exhibited an enhanced selectivity for AChE inhibition vs butyrylcholinesterase (BChE) inhibition, possessed M,/M2 muscarinic receptor affinities similar to THA, and potentiated acetylcholine-induced contractions of isolated rat ileum. The bia[[(dimethylamino)methyl]furans], however, showed little ability to elevate mouse brain acetylcholine levels in vivo, with the most potent compound, 2, demonstrating a 22% increase at 80% of its approximate lethal dose. T h e relatively high molecular weights of these compounds and the presence of two very basic tertiary, aliphatic amino groups probably limited distribution into the central nervous system (CNS).' Current address: Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, IN 46285. t
Shanxi Medical College. University of South Carolina.
Utilizing compound 1 as a prototype, we have therefore synthesized a series of N-aryl derivatives of 2-[ [ [5-[(dimethylamino) methy11- 2-furanyl]methyl]thio]ethylamine with the general structure shown i n Figure 2. We have also synthesized N-substituted analogs of 2-[ [ [5-[(dimethylamino)methyl]-2-furanyl]methyl] thiolethylamine, containing 2-cyano-3-fluoropheny1,2-nitro-4-fluoropheny1 and 4-nitro-3-pyridazinyl moieties. In comparison to compound 1, the series of compounds in the present work had lower molecular weights and generally possessed a single, tertiary, aliphatic amino group. It was postulated that these changes would augment distribution across the blood-brain barrier, thus allowing these compounds t o display greater cholinergic effects within t h e CNS. Chemistry N-Arylation of the primary amine2 3 with either 2,4dinitrofluorobenzene, 1,5-difluoro-2,4-dinitrobenzene, 2,6-difluorobenzonitrile,or 2,5-difluoronitrobenzene in acetonitrile in the presence of anhydrous sodium carbonate yielded compounds 4-7, respectively. These products were (1) Sowell, J. W., Sr.; Tang, Yunzhao; Valli, M. J.; Chapman, J.
M., Jr.; Usher, L. A.; Vaughn, C. M.; Kosh, J. W. Synthesis and cholinergic properties of bis[ [(dimethylamino)methyl]furanyl] analogs of ranitidine. J . Med. Chem. 1992, 35, 1102-1108. (2) Price, B. J.; Clitherow, J. W.; Bradshaw, J. Patentschrift (Switz.) 647,517. Furanalkanamide derivatives. Chem. Abstr. 1986, 104, P 168352f.
0022-2623/92/1835-3141$03.00/00 1992 American Chemical Society
Valli et al.
3142 Journal of Medicinal Chemistry, 1992, Vol. 35, No. 17 ( C H & N C H ; ! ~0C H , S - - Z
Scheme 11"
-SCH2--CH;!N(CH& 0
3
2, Z = -(CH;!)d-
Figure
-
CHNO2 ( H ~ C ) ; ! N C H ~ ~ C H z S C H 2 C H 2 NIIH - ~ - S C Hb~
1.
24 CHNO, ( H ~ C ) , N C H z ~ C H 2 S C H , C H z N H - C -IIN H N H ,
Figure 2.
C.d
0
Scheme I"
25
26
" Reagents: (a) 1,l-bis(methy1thio)-2-nitroethylene,CH,CN; (b) HpNNHp,absolute C,H,OH; (c) 40% aqueous glyoxal; (d) triton B, toluene, water. 4 -7
5-23
"P
XR 8 NH:, 9 NHCH3 10 NHC3H7n 11 NHCSHlln 12 NHCHzCH2N(CH3)2
comp
XR
16 17 15 19 20 21
NHCH~C~HS NHNH:, NHN=C(CH3);! NH6H5 NHC6H4-p-F NHCeH4-O-CN
comp R' R2 R3 R4
4 5 6 7
NO2 H NO;! H CN F NO2 H
NO;! NO;! H F
H F H H
15 N H C H ,-a 0 Reagents: (a) Na2C03, CH,CN; (b) 2,4-dinitrofluorobenzene, 1,5-difluoro-2,4-dinitrobenzene, 2,6-difluorobenzonitrile,or 2,5-difluoronitrobenzene; (c) Na2C03 or NaH; (d) primary amine or phenol.
obtained in yields ranging from 38 to 90% (Scheme I). Compound 5 served as the immediate precursor to compounds 8-23. The remaining fluorine atom of compound 5 was displaced by either ammonia, primary aliphatic amines, hydrazine, aniline, substituted anilines, or p-nitrophenol to yield the desired products. Typically, these compounds were easily purified by flash chromatography and yields were generally greater than 75%. Compound 243 was prepared by condensation of the primary amine 3 with 1,l-bis(methylthio)-Znitroethylene in refluxing acetonitrile under an inert atmosphere (Scheme 11). Displacement of methyl mercaptan in compound 24 by hydrazine was facile, yielding compound 25. Utilizing the procedure of Hamberger, et al.,4 the pyridazine 26 was obtained in an excellent yield by the conden(3) Martin-Smith, M.; Price, B. J.; Bradshaw, J.; Clitherow, J. W. Eur. Patent Appl. 2,930. Amine derivatives and pharmaceutical compositions containing them. Chem. Abstr. 1980, 92, P76145w. (4) Hamberger, H.; Reinshagen, H.; Schulz, G.; Sigmund, G. Polar Ethylene I: The synthesis of 4-nitropyridazine. Tetrahedron Lett. 1977, 4 1 , 3619-3622.
sation of the hydrazine 25 with glyoxal. Results and Discussion With several significant exceptions, compounds 4-26 denote a novel series of potent acetylcholinesterase inhibitors which are pharmacologically similar to both THA and the previously reported' bis[ [ (dimethylamino)methyllfurans] 1 and 2. All of the agents were initially tested for in vitro AChE (human erythrocyte) inhibitory activity to determine their potential to reverse the cholinergic deficit characteristic of AD. Compounds that demonstrated AChE inhibitory concentrations (IC5,,'s) of lo4 M or less were further examined for additional cholinergic properties. AChE Inhibition. With the exception of compound 6, all the analogs presented in Table I were more potent in vitro inhibitors of AChE than ranitidine (IC50= 2.3 pM).l In addition, the most active compounds, 5,8,9, and 17 were slightly more potent inhibitors of the enzyme than THA (ICso= 0.18 pM). Furthermore, compounds 8 and 17 possessed inhibitory activities similar to those of the more potent bis[ [ (dimethylamino)methyl]furanyl]derivatives previously reported, 1 and 2.' It is interesting to note that the most active agents, 5 (R= F), 8 (R = NHJ, 9 (R= NHCHJ, and 17 (R = NHNHJ all contained small R Substituents (Table I). Activity, in fact, decreased as the size of the alkyl substituent on nitrogen, R, increased in compounds 8-11. Compounds possessing aromatic R substituents were not generally as active as those containing small alkyl substituents, although the 3-(pyridylmethyl) analog 15 (ICm= 0.31 pM) and the p-nitrophenyl ether 23 (ICm = 0.27 pM)demonstrated moderate AChE inhibitory activity. Deviation from the 1,3-diamino-4,6dinitrophenyl moiety resulted in the least active compound, 6, although the 4-fluoro-2-nitrophenyl analog 7 (IC5o= 0.65 pM)and the 4-nitro-3-pyridazinyl analog 26 (IC50= 0.47 pM) showed moderate activity. Finally, it should be noted that compound 5 retains an aromatic fluorine capable of undergoing nucleophilic displacement in this in vitro assay as well as the other pharmacological assays detailed below. Therefore, compound 5 may deviate significantly in mechanism from the other compounds presented herein. Butyrylcholinesterase Inhibition. The ability of the compounds to selectively inhibit AChE rather than BChE wm assessed in vitro. Compounds 4-26 were all much less
Journal of Medicinal Chemistry, 1992, Vol. 35, No. 17 3143
Ranitidine Analogs
Table I. Physical and Biological Data of N-Aryl Derivatives of 2-[[[5-[ (Dimethylamino)methy1]-2-furanyl]methyl]thio]ethylamine
n-
-
n
compounds 6 , 7 , 2 6
compounds 4,5,8-23
compd R physostigmine tetrahydro-9-aminoacridine(THA) a H 5 F 6
-Q CN
AChE-Ia ICw (PM) 0.043 0.18 0.20 0.12 2.1
yield (%)
-
recryat solvent
mp ("C)
ethef
49-51
C,H,N,S
anal.*
-
84 90 38
F
0.65
68
0.083 0.16 0.26 1.2 0.52 0.37
95 69 90 87 73 78
14
1.3
70
ethef
124-126
C,H,N,S
15
0.31
41
ethef
107-108
C,H,N,S
16
0.69 0.076 0.44 0.64 0.68 0.63 0.65
53 29 41 89 93 24 13
ethef MeOH EtOH ethef ethere ethef ethef
91-93 146-148 101-103 104-106 118-120 119-121 110-112
C,H,N,S C,H,N,S C,H,N,S C,H,N,S C,H,N,S CJWS C,H,NS
0.27 0.47
40 80
7 Nb,
8
9 10 11 12 13
NH2 NHCHS NHC3H7n NHC6Hlln NHCH&H2N(CH&
17 18 19 20 21 22
23d
NHN&(CH3I2 NHCeH6 NHCeHd-p-F NHCsH4-o-CN
"Human acetylcholinesterase (Sigma Type XIII) was used and the procedure of Ellman' was followed. Experimenta were conducted at room temperature and substrate (acetylthiocholine) concentration was 5.0 X lob M. 'Carbon, hydrogen, nitrogen, and sulfur analyses were within &0.4% of theoretical. 'Product obtained after flash chromatography was triturated with ether. dIsolated, characterized, and evaluated as the p-toluenesulfonic acid salt.
active inhibitom of BCM than THA or physostigmine and also generally poesegsed significantlymore favorable B C M IC,/ACM ICso ratios (Table 11). In fact, 11 of the 19 compounds tested had ratios greater than 10. The pfluorophenyl analog, 20, with a ratio of 1.6, was the least favorable, whereas the hydrazino derivative, 17, with a ratio of 67, was the most favorable. Ileal Activity. The effect of the compounds on acetylcholine-induced contractions in isolated rat ileum was determined in order to initially assess their distribution and overall cholinergic activity in an intact tissue. Nine of the compounds tested potentiated acetylcholine-induced rat ileal contraction by at least 20% at a concentration of 0.1 p M or less (Table 11). Compounds 5,12, and 20 were the most active in this assay and were comparable to THA, possessing activity at a concentration of 0.01 pM or less. Compounds 7,9, 15, 16, 19, and 21 were inactive, possibly reflecting poor distribution of these compounds into the intact ileal tissue. Furthermore, no contractions were observed immediately following the addition of any of the
test compounds, thereby denoting the absence of significant direct agonist activity. Muscarinic MI and M2Receptor Binding Studies. Whereas, agonist activity at MI-cholinergic receptor sites is believed to be of benefit in alleviating the cognitive deficit characteristic of AD, s i m i i activity at M2-receptor sites may inhibit ACh release.s-6.7 Therefore, the binding affinities of the teat compounds were determined for these two muscarinic receptors (Table 11). (5) Mash, D. C.; Flynn, D. D.; Potter, L. T. Losa of Mz muscarine receptors in the cerebral cortex in Alzheimer's disease and experimental cholinergic denervation. Science 1985, 228, 1115-1117. (6) Potter, L. T. Muscarine receptors in the cortex and hippocampus in relation to the treatment of Alzheimer's disease. In International Symposium on Muscarinic Cholinergic Mechanisms; Cohen, S., Sokolousky, M., Eds.; Freund Publishing Ltd.: London, 1987;pp 294-301. (7) McCormick, D. A.; Prince, D. A. Two types of muscarinic response to acetylcholine in mammalian cortical neurons. Proc. Not. Acad. Sci. U.S.A. 1985,82, 6344-6348.
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Journal of Medicinal Chemistry, 1992, Vol. 35, No. 17
Valli et al.
Table 11. Cholinergic-Related Activities of Selected N-Aryl-2-[[ [5-[ (dimethylamino)methyl]-2-furanyl]methyl]thio]ethylamineAnalogs of
Ranitidine compd Dhvsostiemine THA 4 5
7 8 9 10 12 13 14 15 16 17
18 19 20 21 22 23 26
BChE-I" ICw (pM) 0.035 0.023 1.8 3.8 3.3 2.4 3.3 3.8 1.4 4.7 5.2 6.1 1.3 5.1 8.4 1.6 1.1 1.8 18 1.1 10
BChE-I/ AChE-Ib 0.80 0.10 9.2 33 5.1 28 21 15 2.8 13 4.1 19 18 67 19 2.5 1.6 2.8 28 4.1 22
MIC ICW(pM) 37 2.0 1.4 1.8 4.0 3.8 0.43 3.2 0.67 0.53 1.5 0.38 2.0 3.8 3.1 6.4 2.1 3.3 -
M$ ICW(pM) 160 2.1 5.7 7.0 2.4 6.8 3.1 3.5 0.38 0.30 2.4 0.40 3.3 4.7 3.5 6.4 4.4 3.7 -
M2-ICW/ ileal' M1-ICw activity (pM) 4.3
