Journal of Medicinal Chemistry, 1971, Vol. 14, No. 4 361
DIHYDROBENZOFURANSA N D AChE
TABLE IV PHARMACOLOGICAL ACTIVITIES KO.
,----Ulcer .4
B
indexaC
Dose, TC
mg/kg,
PO
LD50, mg/kg, i p
20 > 1000 41 1 . 7 3 . 0 1 . 0 2.0 20 61 1 . 6 1.7 2.5 2.0 20 >2000 42 2.1 1.4 2.5 1 . 9 20 > 1000 35 2.2 1.6 2.1 1.9 31 1.4 2.5 1.2 1.8 15 > 1000 20 > 1000 58 1.7 1 . 5 2.0 1.7 68 20 1.6 1.5 2.0 1.7 69 2 . 7 1 . 3 1 . 3 1.7 20 >1000 24 2 . 2 1 . 6 1.6 1 . 7 20 28 1.7 20 >lo00 2.2 1 . 2 2.2 63 1 . 6 1 . 5 2.0 1 . 7 20 48 1 . 8 1.6 1 . 4 1 . 6 20 > 1000 59 1 . 3 3 . 4 1.1 1 . 5 20 39 0.9 4.0 1 . 5 1.5 20 37 1.4 1.0 2.5 1.5 20 18 1 . 2 1 . 2 2 . 3 1.5 20 70 1 . 9 1 . 5 1.1 1 . 5 20 33 1 . 6 1.1 2 . 0 1 . 4 20 62 1 . 0 1.1 2.2 1.4 20 76 1.1 1 . 0 2.2 1.3 20 71 1 . 2 0.9 1 . 5 1 . 2 20 57 0.0 1 . 0 2.6 1.1 20 56 0 . 3 1.8 1 . 6 1 . 0 20 Oxymethalone 2.2 1 . 2 1 . 5 1 . 7 50 4 Values indicate the ratio to the value of the control animals without receiving drugs for ulcer remedy. the same manner as XI11 from XII, was dissolved in 22 ml of EtOH containing 7 ml of HzO and concd HCl (1.0 g, 0.01 mole)
and was hydrogenated in the presence of PtOz (1.0 g) at 40-50" and 6 atm pressure. After HZuptake was completed, the mixt was cooled and filtered from the catalyst. EtOH was removed under reduced pressure, and the solid which sepd from the soln was filtered off. To the filtrate was added 10 ml of lOy0aq NazCOS, and the soln was extd with CHCL and dried (NazSOc). After the removal of the solvent, the resulting solid was recrystd to give 27. 1-(3,4,5-Trimethoxybenzoyl)-3-aminopiperidine (XVIIb).-A soln of 2.2 g (0.0095 mole) of 3,4,5-trimethoxybenzoyl chloride in 5 ml of MeCN was added gradually to a soln of 2.2 g (0.0095 mole) of 27 and 0.6 g (0.0113 mole) of Na2C03 in 6 ml of HzO with vigorous stirring and cooling with an ice bath. After stirring 2 hr at room temp, the soln was extd with CHCla. The solvent was removed under reduced pressure, and the residue was hydrogenated with 10% Pd/C (0.2 g) in 100 ml of EtOH and concd HC1 (0.7 ml) at ordinary temp and pressure. -4fter HZuptake was completed, the mixt was filtered from the catalyst, the solvent was removed under reduced pressure, and the resulting solid was recrystd from EtOH-MeCN to give 1.6 g (65.27,) of an Anal. (C15HZ2N204.HCl. amorphous powder, mp 239-242'. HzO) C, H, N. 1-(3,4,5-Trimethoxybenzoyl)-3-( p-nitrobenzamido)piperidine [75,IVb, R = p-NOz; R, = 3,4,5-(Me0)3] was obtained from XVIIb by treating with p-nitrobenzoyl chloride as in method C. 1-(3,4,5-Trimethoxybenzoyl)-3-( p-aminobenzamido)piperidine [74,IVb, R = p-NH2; Rl = 3,4,5-(Me0)3] was obtained from 75 in the same manner as 42 from 44.
Acknowledgments.-The authors wish to thank Dr. S. Ogihara, President of Kyorin Pharmaceutical Co., Ltd., Tokyo, for his deep interest and continuous encouragement. We are grateful to Mr. A. Saito, Mr. N.Watanabe, and Mr. T. Umezawa for their expert technical assistance,
Synthesis and Pharmacological Activity of Dihydrobenzofuransl MATHIASP. MERTES,*LARRY J. POWERS, Department of Medicinal Chemistry, School of Pharmacy, University of Kansas, Lawrence, Kansas 66044 AND
MILOS11.HAVA
Department of Pharmacology, University of Kansas Medical Center, Kansas City, Kansas 66103 Received J u l y 3, 1970 The synthesis of the cis and trans isomers of -5- and 7-dimethylamino-3-hydroxy-2-methyl-2,3-dihydrobenzofuran methiodide (lla-d) started with nitration of 3-acetoxy-2-methyl-2,3-dihydrobenzofuran and subsequent separation of the isomers cis-5-, frans-.5-, cis-7-, and frans-7-nitro-3-acetoxy-2-methyl-2,3-dihydrobenzofurans (8a-d). Catalytic reduction of the respective nitro compounds in the presence of CHZOgave the corresponding dimethylamino compounds 9a-d. Deacetylation to the alcohols loa-d and treatment with Me1 yielded lla-d. Nitration of 2-methylcoumaran-3-one gave the 5- and 7-nitro ketones (5a and 5b). Reduction and concurrent methylation with CHZO followed by treatment of the separated isomers with Me1 afforded 5-dimethylamino-2methylcoumaran-3-one methiodide (6a)and the 7 isomer (6b). Using an excess of CHZOin the same sequence with 5a yielded the alcohol addition product, .i-dimethylamino-2-hydroxymethyl-2-methylcoumaran-3-one methiodide (7). Biological examination revealed muscarinic action (sa, 1/100 .4Ch) and nicotinic activity (sa, 1/20 nicotine, lla, 1/100 nicotine, llb, 1/200 nicotine). Both butyryl- and acetylcholinesterase were inhibited by 6a and 6b; the potency of 6a ( K , = 2.5 X 10-8) was reflectedin the LDjo (10 mg/kg). The remainder of the compounds displayed little or no activity and low toxicity (LD,o 50 to 200 mg/kg) with the exception of lla which was a weak muscarinic antagonist.
Acetylcholine (ACh) can assume an infinite number of conformations; based on this a great deal of research has been described that has restricted this freedom by the synthesis of rigid analogs of ACh.233 Agents wit,h a (1) Supported b y a n N D E h Title I V Predoctoral Fellowship, 1966-1969 t o L . J. P. a n d by G r a n t 1K3-CA-10739 from t h e National Cancer I n s t i t u t e of t h e National Institutes of Health. Abstracted in p a r t from t h e thesis of L. J. P. submitted t o t h e G r a d u a t e School, r n i r e r s i t y of Kansas, Lawrence. K a n . , in partial fiilfillment of t h e requirements for t h e Doctor of Philosophy. (2) Several recent examples of rigid acetylcholine analogs are noted. (a) J. R. Robinson, B. Belleau, a n d B. Cox, J . .?fed. Chem., 1 2 , 848 (1969). (b)
limited number of allowable conformations having both potent muscarinic and nicotinic effects are muscarine E. E . Smissman, W. L. Nelson, J. B. LaPidus, a n d J. Day, ibid., 9, 458 (1966). (c) E. E. Smissman a n d G. S.Chappell, ibid., 12, 429 (1969). (d) P. D. Armstrong, J. G . Cannon, a n d J. P. Long, .Vatura ( L o n d o n ) , 220, 65 (1968). (e) C . Y. Chiou, J. P. Long, J. G . Cannon, a n d P. D. .irmntrong, J . Pharmacol. E z p . Ther., 166, 243 (1969). (3) Several recent dioxolane analogs are cited. (a) M. h l a y a n d D. J. Triggle, J . Pharm. Sci., 67, 511 (1968). (b) D. R . Garrison, hl. M a y , H . F. Ridley, a n d D. J. Triggle, J . M P ~Chem., . 12, 130 (1969). ( c ) H. F. Ridley, s. S. Chatterjee, J. F. Morna, a n d D . J. Triggle, i b i d . , 12, 931 (1969).
~ I E R T EPOWERS, S, AND 15 ~
362 Journal of Jledicinal Chemisfry, 1971, Val. 14,NO.4
(1) arid the keto derivative, muscarone (2). The subtle differences in structure are difficult t o explain, for example, F V a ~ e has r ~ ~referred to the freedom of rotation of the trimethylanimoniummethyl group of muscarine (1) and muscarone (2) in diff ereiitiating muscarinic and nicotinic action. OH 0 , / 'Me
2a, L-(+)-muscarone, R, H; R1= N+Me,Ib, 5R-allomuscarone, Ri =NMe:I; R-= H
1
E
V A
derivative 6b (benzomuscarone). In addition, t Iir) :~nalogous3 isomers l l a , l l b , :tnd 6a 11 we ' ~ 1 3 0prepirrd. Titration of 2-inethylcoumarari-:I-onc to givcb 5- a i i d 7-riitro-"-methylcouniaran-~~-oIi[~ (5a : i r d 5b) 1i:i\ l ) k > o i i described ~arlier.' Reductive allcyhiion of 5a u-ing excesb CH2O yielded the :ddol additioii product 7. SCHEML I
m0- R 1 y p l-H , \
Me
RSa, R, =NO,; R-= H b, R1=H, RL=NO-
3
4
RL
+ 6a, R, = NMe,.l-; RL= H The unusual reversal of biological act'ivity found in + the muscarone analogs (2a) has prompted speculations b. R, H; R,= iiMe '1on the nature of the ketone in linking to the r e ~ e p t o r . ~ , " Analogy to h C h can be noted if the CO of muscarone is considered to bind a t the same site as the AChCO, then the %atom chain, CH2CjCA of muscarone (2a) is comparable to the CH,CH,O chain spanning the quaternary N and the CO of ACh. Furthermore, C, of L - ( + ) muscarone (2) and D ( - )- (R)-acetyl-P-methylcholine have similar relative corifigurntioiis and the same apHoitever, if only 2 equiv of CH20\\ere used, rather than plies to the (5R)-alloniuscarotie (2b) and L-( +)-(S)the large excess, the condensation at C-2 could b r prracet~l-~-methj-lchoIinc. Support for this analogy vented. -Inother problem uhich arose in tlic \>vicomes from the biological activity of 2a, 2b, and (+)thesis of 6 was the inqtability of the intermediate tlior ( - )-acetyl-p- methylcholine which have pronounced methylamino ketones; for thib rea5oii the reduction I$ a s muscarinic and nicotinic e f f e ~ t s . ~However, ,~ the choperformed in I'hH. After 5 equiv of HS had beeii ahliiiesteriise activity of these isomers varies, thc L - ( + ) sorbed, the reaction mixture M as filtered, the filtrate (S)-acet yl-p-methylcholine is :i fair substrate for the a i dried, and an excets of AIf>ITT as added to give I he enzyme while the I)-( - ) isomer inhibits the enzyme. methiodide salt5 of 3-dimetliylamino-"-methylc~~~iL-( fj- m d I)-( - )-niuscaroIie and ( f)-allomuscarone maran-3-one (6a) arid the 7 isomer (6b, benzomu3are potent muscarinic agents (-4-10 times ACh). caroiie) The ppt \\ hich formed i n the preparsiioii of The inhibitory activity agniriit cholinesteras~:is \ve:il;; difficult to dry completel> -Ittempts t o rcboth ( +)-allomuscaroiie arid (i )-muscarone have simcrj stallize by the uwal nianner resulted intlecompu~iiion ilar potency (Ki M) which indicates the 2-11? An anal. sample n a s obtained by di-solving the ppt 111 group neither enhances nor inhibits binding. These JIeOH at 2.5' and slonl> adding EtnOt o give crys 6b and other correlations suggest the OC&',C;CH?S +A Tea The synthebi5 of the isomeric amino alcoholz (11 fragment of muscarones is interacting \\-ith the musScheme 11) required a sequence that nould givc a re'tcarinic receptor and esterase site. >onable meaiis of beparating tlie cis and t ran\ i-oin('1\ The synthesis of rigid analogs of muscarine has r i o t As described earlier' the iiomers of +erie, 8 \\ere p 1 ~ been as successful as the synthesis of rigid aiialogs of pared by reductiori of the Letone, acet? latiori of tlic .2Ch. Hardegger and Halder6 reported the attempted resulting alcohol, :md wparat ion of t h e iwrncr5 t o of the bicj-clic muscarine aiialoga 3 and 4 ~ i a give 8a-d. Conversion into the respective dimcth) 1a1 llainiich reactioii on normuscaroiie. \Vhile nmirioacetates 9a-d I\ :is :tccomplishecl ti\. reduct IVP neither bicyclic analog \\-:is obtained b>-this method, 4 alkylation,* hon ever, large amounts of catah i t \\('re ithesized by an alternate route. In this \vork used to avoid long reaction time ixice the sloii reduch t o report the synthesis of ~ians-'i-dirnc- tion of 8b using a limited amount of c:ttalyst gave :L t liylaniiiio-3-hydrox~-2-methyl-2,3 - dihydrobenzofuran significant amount of I lie eliniinatioii product, k h methiodide ( l l d , benzomuscarine), the cis isomer l l c methylamiiiobeiizofuraii. Alkaline hydrolyses of the (beiizoepimuscarine) descrihed earlier.'a and tlie kcto series 9a-d to the tilcohols loa-d 11 as folloned ti>- treatment nit11 lIeI to give lla-d. The phj iical constmts ( 4 ) (a) 1:or references bee -1.H . 13eckett, 1..J . Harper, a n d J. \V. Ciitliero\r-, J . I'hnrm. i'harmocol., 1 6 , 362 (1 7-?;I\Ie2 10d Tians 7-ShIe2 lla Cis 3-SlIeaI Ilb Trans 5-xhIe31 llc CIS 7-SSIe31 Ild Trans 7-SJleJ Analyw for C, €1, and N theoretical.
R
Ac Ac Ac Ac H H H H H H H H was
Formulaa
Rlp,
O C
C13H18C1N03 157-1.58 C13H1gCIN03 144-145 C~~HI~CIN 144 O 5-146 ~ 5 C13H1gC1N03 132 3-133 CI1H~,NOP 90-91 C I ~ H ~ G C ~ N144-143 OI C I I H & l ~ O L 142-143 C1,H1&1NO, 129-130 Cl2H18IS02 190 5-192 C12HJSOZ 171 5-172 5 ClnHlsIN02 191-192 C~~HI~IXO 182-183 , fouiid to be within 1 0 4% of
acetates 8 with regard to the protons at C-2 and C-X7 The coupling constants were always in the order JCls-2,3 > Jt7ana-2,3, and the C-3 proton of the cis isomers was alnays deshielded more than the C-3 proton of the trans isomers. The coupling constants were approximately 2 Hz in the trans isomers and 6 Hz in the cis isomers throughout the series. Biological Results.-The muscarinic activity was tested on the guinea pig ileum by the cumulative doseresponse method using ACh.CI- as the reference.
12
the axis of the C5-CH2 bond. However, other reasons such as interference by the P h ring or a change in the nature of the quaternary X in going from trimethylalkyl to trimethylaryl substitution could also prevent receptor interaction. Potent anticholinesterase activity was observed in both the 5- and 7-substituted ketones 6a and 6b. The 5 isomer 6a was a strong competitive inhibitor of both Ac and butyrylcholinesterase, with K , E 3 X lo-* X for both purified enzyme preparations. The 7-substituted analog 6b was competitive but less inhibitory against butylrylcholinesterase (Ki = 2.5 X 10-j AI) and AChE ( K , = 3,s X 10-hLJf). The high anticholinesterase activity of 6a was reflected in the toxicity studies where the animals died with symptoms of cholinesterase blockade. The relatively lower toxicity of l l a can be attributed to the associated atropine-like action observed in the muscarinic assay. Recently Chothia and Paulingg proposed an active conformation for ilCh that describes the interaction with AChE. Analysis of the conformation of the (9) C. Cliothia a n d P. Pauling, .Vature (London), 22S, 919 (1969).
364
Journal of Medicinal Chemistry, 1971, Vol. 14,No. 4
~IERTE POWERS, S, A N D Havs
TABLE I1 BIOLOGICAL RESULTS
Compd
Isomer
Muscarinic activity," ACh = 1.0
Nicotinic activity,b nicotine
---
= 1.0
Butyrylcholinesterase inhibition--Horse serum,c R a t serum Ki
K,
Ach lla
cis4
Weak antagonist
0.01
Weakd
=
1 lJ
-----.IChE erythrocytes
inliibltion,--------.
LD P O . K,
rug /lie
K m = 1 x 10-'.11
4 X 10-'111 Weakd
200
l l b trans-5 Weakd .j0 0.005 Weakd 0 7 r c a t 2 x 10-a 'lf 6a 3 0 x 10-8 J.1 3 0.01 0 05 100% at 2 x 10-6 111 . 2 5 x 10-8 Jf 10 Weakd l l c cis-7 Weakd 100 Weakd Wenkd l l d trans-7 100 SO'; inhib at lo-' -11 3 8 X Jl 6b 7 507, at 1 x 10-4 51 2 5 x 10-5 M 100 3Ieasured on the guinea pig ileum. b hleasured on the frog rectus abdominis muscle arid the chicken biventer cervicia mwclc. Weak irihibitioii (10-20';) K , ax determined by the titrimetric technique plotting l / s t's. l / v . (Correlation coefficient > 0.96). was noted at 10-3 JI.
esterase substrates, L-( -)-(8) and D-(+)-(R)-acetyla-methylcholine and L-( +)-(8)-acetyl-fi- methylcholirie suggested the substrate model exists in an antiplanar SC,C;Ol (13, 7 150') and antiplanar CJ2;OlCc structure (14, T 1 8 0 O ) . The authors noted that the con-
+
N
13, rNC,C,O,
+150-
14, +C,C,O,C,
+BO"
figuration of I)-( +)-(R)-acetyl-P-methylcholine w ould prevent adoption of the T of =15O0 for S C 4 C s 0due to the iteric hinderance in the partial eclipse of the @--\le group and K . If it can be aqwmed that the binding of inhibitors t o the esterase require5 the qimilar complimentation of the receptor site then I)-( -)-(R)acetyl-$ rnethylcholine should have low afinit y for the eaterace iite and should not be n substrate or an inhibitor of tlie enzyme. Hov ever, the I)-( - ) - ( R ) - @ - l k isomer IS known t o inhibit the enzyme; the \veal; inhibition noted could be due to nonbpecific binding, perhaps through the quaternary S since the alcohol choline also i, L: \wnk inhibitor ( K , = 4 X as are a variety uf quaternary ammonium compound4 such a s phen) I trimethylammonium iodide ( K , .lI).11n'2 The 5trong inhibition of =1CliE IIY the 3-subbtit ut et1 lietone 6a is prciunied to bdue t o binding at CO and S The inactivity of both thr 5-iubstituted cit and tran5 alcohols ( l l a arid l l b ) tuggeati that the ether 0 contributes little t o binding. l'urthermore, sinccl the OH arialogs are inactive I\ liereai the ketone iz itrongly hound i t is poctulated that the birding to the enzyme ic through ari at traction of a nucleophilic cite (imidnmle or w i n e OH) of the enzyme for the electrophilic CO.I3 Thetc motleh of binding exclude H bonding with the CO of 6a a 4 the donor mainly bpcauSe the slight differencr in energy gained from bonding to a C 0 compared to :t
disubstituted (OH) 0 in l l a or l l b (about 1kcallmole) could not account for the vast difference in biological activity. l 4 The planar structure of the active inhibitor 6a can be accommodated in t h e model proposed by Cliothia and Paulingg for the substrate of the esterase \vith a minor change. The optimal SC4C,02torsional angle i i proposed t o be 130" whereas the analogous atoms KC,C4C3,in 6a are antiplanar ( I S O O ) . Examination of models shon-i the 7-substituted ketone 6b to have the same distance betneen the CO ant1 the S as found in the most active isomer 6a. Thih isomer (6b) n a3 1000-fold le-s active in inhihit ion of the esterase. Examination of t h i t model revedb ti\ (J obvious factors that may account for the differencc If the atoms analogout t o the buhttrxte AIChare CYamined, the S C i C j a C d a tor.iona1 angle 1': lh0" :inti C 7 C ; a C 3 a C 3 i i alro 180" a fully eut ended model bet\\ (>en S and CO a t .een in 6b and the proposed h C h nioilcl. HOT\ever, further ttudj of tho mock11 reveal5 cliffcienceb that c a n tte related 10 tlie acet) I - ~ - n i r t h ; \ ~ l c l i o l i ~ i t ~ ~ If .iCh interact. u i t h the e.tei 111 the extt~llc~etl forn1 t h r n thc tnotleli c a n be oriented a\ .jhon 11 it1 Schciuc 111 Both iwiiif>r\ ui ucet\l-a-methJ Icliol~iit~ :tie
-
(10) ( a ) 11. D. I3aldridge, I V . .J. AIcCarville, a n d S . I., Friess, J . B m a r . C h e m . S a c . , 77, 7:39 (19551, (1,) S.I,. Fricss a n d 11. D . l\aldridge, ibid., 78, 2482 (1926). (11) (a) 1'. 13ergtnann a n d It, Sepal, B i o c h ~ m .. I . , 58, 092 ( 1 9 % ) . tlij
gerimentellea I'iiarinacologie," 1). L:iclller a n d .I. Farali, Ed., 0 . 13. Iioelle, s u b e d i t o r , Val. 15, S p r i n g e r Vprlag, Herlin, 1963, p 37-1. (13) I ? , 11. Kriiiika a n d I
