J. Whiting, Y.K. Au-Young and B. Belleau- A Convenient Synthesis of L(+)-Muscarine

8/3/2019 J. Whiting, Y.K. Au-Young and B. Belleau- A Convenient Synthesis of L(+)-Muscarine

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A Convenient Synthesis of L(+)-M uscarineDepartment oj' Chemistry, Uniuersity of Ottawa, O t tarva, Canada

Received May 1 , 1972A new total synthesis of L( +)-muscarine is described. Performic acid oxidation of N-acetylcrotylglycinegave the corresponding 4,5-diol which was resolved at the a-carbon by stereospecific hydrolysis of theN-acetyl gr ou p of the L-isomer with hog kidney acylase I. The resulting cc-L(+)-aminoacid diol (3: Cha rt 1 )was deaminated with nitrous acid to give 4-hydroxy-5-methyl-2-te trahydrofuran carboxylic acid with reten-tion of configuration at position 2. Reaction of the corresponding methyl ester with dimethylamine gave twomajor amide constituents in 65% yield. Reduction of these two N,N-dimethylamides (5a and h) with li thiumaluminum hydride fol lowed by quaternizat ion wi th methyl iodide afforded a pu re i somer of L(+)-m uscar ine(6a) and L (+)-m usca rine (66) in high yields. Th e biological act ivity of the lat ter was the sam e as that of thenatural alkaloid.Une nouvel le synthtse totale de la L(+ )-mu scar ine est decri te. L 'oxydat ion par I 'acide performique duN-acCtyl crotylglycine do nne le diol-4,5 corre spon dant qui a CtC resolu au niveau d u carb one cc par hydrolysestCrCospCcifique du gr ou pe N- acityle de I'isomere L par I 'acylase I , provenant d e rein de porc. L 'a-L-(+ )amino

acide diol (3, part ie I) r t sul tan t , aprt s desam inat ion par I 'acide ni t reux, condu i t a I'acide hydroxy-4 mCthyl-5tetrahydrofurane-2 carboxylique avec rCtention de configuration sur le carbone 2. La reaction de I'estermi thyl ique correspond ant avec la dimt thylamine donn e essent iel lement d eux composes amides avec unrendement de 65%. La rCduction d e ces deux N.N-dimCthylamides (5a et h) pa r I'hydrure de li thium d'alu-minu m suivie d'un e tran sform ation en sel quate rnair e par I'iodure de methyle, a permis I'obtention d'unisomtre pur de la L (+ ) muscarine (60) et de la L ( + ) muscarine (6h ), avec de bons rendements. L 'activi tCbiologique de cette derniere est la m&mequ e celle de I'alcaloide naturel. [Traduit par le journal]Canad ian Journal o f Chemis t ry, 50, 3322 (1972)

IntroductionAs part of our research studies on the natureand function of the acetylcholine receptor pro-tein of excitable membranes (1, 2, 3), access tothe potent cholinomimetic alkaloid L( +)-muscarine (6b: Chart 1) (4) an d its radioactiveform became essent ial h'oider to discriminatebetween pharmacologically specific and non -specific binding sites. Extraction of the alkaloidfrom natural sources (5 ) is impractical at bestand most of the recent biological studies withthis qu atern ary base and its stereoisomers havebeen carried o ut w ith totally synthetic materials( 6 ) . The synthesis, chemistry, and stereo-chemistry of muscarine have been reviewed byWilkinson (7) and a lso by Eugster (8) who haspioneered much of the relevant work in thisfundamental area of medicinal chemical re-search. Th e X-ray structure and absolute con-figuration of the alkaloid h ave been elucidatedby Jellinek (9). Subsequently, Pauling (10) hasdrawn analogies between its conformation andthat of acetylcholine in the crystalline states.'Author to whom correspondence should be addressed,Department of Chemist ry, McGil l Universi ty, Montreal ,Quebec.

We wish to report on a new synthetic approacwhich provides access to L( +)-muscarine moconveniently and efficaciously than beforAdap tat ion of the method to the preparat ion the radioactive analo g can be readily visualizeThe synthetic scheme illustrated in Chartwas adopted because of the following broaadvantages that it offers: (a) the intermediaof an N-acylated a-aminoacid provides fready enzymatic resolution to a n optically puL (+ )-aminoacid (1 1); (b) ni t rous acid deamintion of a-aminoacids proceeds with complerentention of configuration (1 1) and , in a favoable case such as 3 (Ch art l ) , r ing closure totetrahydrofuran ring should compete favorabwith a-substitution by the solvent (12). In thmann er, the desired natu ral absolute configurt ion abou t C-2 of 4 (Chart 1) would be securwithout ambiguity or difficulty. From there,should be a simple matter of adaptin g Eugstetechniques (8).Performic acid oxidat ion of predominanttrans-N-acetyl-DL-crotylglycine (1 ) (13) folowed by mild alkaline hydrolysis gave 2high yield as a mixture of stereoisomerIncubation of the lat ter mixture at pH 8 wi


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W HI T I NG E T AL . : SYNTHESIS OF MU S C A R IN ECH,CH=CHCH,CHCO,H CH,CH-CHCH,CHCO,HI +HNA, I I IOH OH HNA,

1 2

1 cylase IHO HNO,; CH,CH-CHCH,CHCO~

MeOH t t IH3C OH OH !H3

hog kidney acylase 1 led to the isolation in 25%yield of a pure crystalline L(+)-a-aminoacid(3) unresolved about its hydroxyl bearingcarbons. This optically active mixture of pre-dominantly threo-diols was treated with nitrousacid and the resulting ring-closed acid esterifiedwith methanolic hydrogen chloride to giveoptically active 4 as a clear oil in 42% yield(based on 3). Reaction of the latter withethanolic dimethylamine according to the litera-ture (8 and refs. cited therein) gave a mixtureof amides in good yield. Given that configura-tional integrity is retained at position 2, thereare four possible stereoisomers about C-4 and-5 but two should predominate since trans-1should yield mostly threo-diols. All four wereactually detected and isolated by chroma-tography on silicic acid but only two majorcrystalline fractions (5a and b) in 1 :1 ratioaccounted for 65% of the total amide mixture.

These two predominant isomers were reducedwith lithium aluminum hydride and the result-ing amines converted to their correspondingcrystalline quaternary iodides. The first majorisomer (from 5a) was shown to possess structure6a by high resolution n.m.r. spectroscopy (220MHz) (see Fig. 1). However, the relative con-figuration about positions 4 and 5 was notdetermined although we are inclined to favorthe allo-arrangement (ma) on the basis that thepredominantly trans-configuration of the start-ing crotylglycine (1) should lead to a pre-dominance of the DL-threo-diol (2) which inturn should ultimately give equal amounts ofthe two possible trans-enantiomers aboutpositions 4 and 5 as the major products. Inagreement with this expectation, Mr. BruceLippert of our laboratories established that,whereas the first major isomer 6a possessedvery weak activity on the guinea-pig ileum (in


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3324 C A N A D I A N J O U R N A L OF CH E M IS T RY . VOL. 50. 1972

I I I I4.6 41 4.0 3.6L 4 0 LO O

F I G . 1. The n.m.r. spectru m of 60 at 220 MHz in I : I DMSO-benzene (sweep t ime 100 s ; sweep width 500 Hz; sweoffset variable as indicated). In the absence of benzene, H 4 and H S are poorly resolved. Irradiat ion of H4 col lapsed the Oto a singlet but i rradiat ion of H S did not . Irradiat ion of O H caused part ial col lapse of H 4 but not of H S .Only structureis consistent wi th these data (suppl ied by Dr. A. H. Grey of the Canadian 220 MHz n.m.r. center at Sheridan ParOntar i o ) .

agreement with published data for DL-allo-muscarine (6 ) ) , the other major isomer 66surpassed acetylcholine in potency by a factorof 2. Since of all the possible stereoisomers ofmuscarine only the natural alkaloid displayssuch uniquely high activity ( 6 ) , it follows thatthe second major isomer is L(+)-muscarine(66). Therefore, the other major isomer maybe tentatively assigned the allo-configuration6a which is enantiomeric with 6b at positions4 and 5. Access to the rare L(+)-muscarinehas become significantly easier than before.Experimental

2-Ace~arnido-4,5-di/1ydroO~yhee~a~~oiccid ( 2 )To a w arm so lution of 5.5 g of N-acetyl crotylglycine (13)in 22 ml of 98 % formic acid was added slowly 5.5 ml of 30%hydrogen peroxide. After the addition was completed, themixture was stirred for 5 11 at 40-30' and rl~en vapora(ed (odryrtess in cacrto. The residue was dissolved in 25 ml of 10%aqueous sodium carbonate, the solut ion al lowed to stand15-17 h. followed by acidification and evaporation to dry-ness in vocuo. The residue was extracted with 2-propanoland the combined ext racts t reated wi th 25 g of Dowex 50Wx 8 resin. After vigorous shakin g for 30 min, the mixtu rewas fil tered and the fil trate evaporated to dryness in vac~toto yield a viscous yellowish oil (5 g) whose n.m.r. spectrumwas consistent with structure 2 or the corresponding lac-

tones. A substantial proportion was in the lactone form judged from the 1.r. spectrum.Enzymatic Resolution o f 2A solution of 4 g of the preceding materia l in 290 ml pure water was t reated wi th 2 N LiOH unti l the p H rmained constan t at 8.0 (lactone opening is gradual so thLiOH is consu med o ver a period of several minutes). Amg portion of hog kidney acylase I (sigma) was added anthe volume of the solution broug ht to 1600 ml and gentst i rred at 38" for 20 h while adding appro priate quant i t ies 2 N LiOH so a s to keep the p H as close to 8 as possible. Tp H of the solution was then adjusted to 5 with acetic acithe mixture fil tered through Norit , and the fil trate concetrated in vacuo to a volume of abo ut 800 ml followed btreatment with 100 g of Dowex SOW x 8 (50-100 mesresin in the acid fo rm. After sha king for 30 min, the suspesion was fil tered, the resin washed with water, and thsuspe nded in 200 ml of a 2 M solution of triethylamine 20% aqueous methanol . The suspension was fi l tered, tresin washed with another 200 ml portion of the same soltion and the combined fil trates evaporated to drynessvacuo. The residue was first crystallized from 2-propanand then from ethan ol-water to give 1.0 g of white crystam.p. 185-187" (dec.) , [a]As 4.3" (H,O).Anal . Cal cd . fo r C 6H , , N 04 : C , 44 .17 ; H , 8 .00 . FounC, 44.3 1 ; H, 7.88.The fil trate from the Dowex resin was evaporated to drness in vaclro and the residue crystallized rrom methanoether to give 1.0 g of white needles, m.p. 182-185"; [a ]43.2" (H,O). Th is compoun d proved to be the unhydrolyzN-acetyl derivative enriched in the D-isomer.


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WHITING E T AL.: SYNTHESIS OF MUSCARINE 3325Anal. Ca l cd . fo r C8H , ,N 0, : C , 46 .83 : H . 7 .31 . Foun d:C, 46.61 ; H , 7.42.

Rittg Closure to 4T o a solut ion of 7.1 g of the preceding L(+) -amin oacidin 18 ml of water c ontain ing 8.5 g of silver nitrite wasadded d ropwise 56 ml of I N HCI while stirring vigorouslyat 0". After 1 h, the mixture was allowed to warm to roomtemperature and continuously stirred for 15-17 h. A fewdrops of dilute HCI were added and the silver chloridefiltered off. Th e fi l trate was evaporated to dryness it1 uucuoand the residue dissolved in dry methanolic hydrogenchloride. After 15 h at room temperature. the solution wasevap orate d an d the pro duct distil led to give 2.90 g (42%yield) of a colorless oil , b.p. 94-95"/0.4 mm; i .r.: 3440v(OH), 1720 v(C= O), 1190 and 1050 cm -' v(C-0);[ a l p - 0 .4 ' (H20) .Anal. Ca l cd . fo r C7H 120 , : C , 52 .50 ; H , 7 .50 . Fou nd: C .52.36: H. 7.62.Arnit~olysis f 4 to 5A solution of 8.5 g of the preced ing ester in 125 ml of a33% ethanolic solution of dry dimethylamine was heated at100" in a steel bomb for 24 h. The solution was evaporatedin vacuo and the residual oil extracted repeatedly with ether .Th e ether was evaporated and the l ight brown oi l appl ied ina li t t le chlor oform to a colum n of 1800 g of Silicar CC 7(chromatographic grade). Elution with chloroform (severalli ters) led to se para tion (as continuously monitored by t .1.c.)of the crude am ide into four fract ions: f irst to be eluted was-0.9 g of an oil followed in sequence by two major crystal-line fractions of 2.2 g each a nd finally by an impu re oilyfourth fraction. Th e first major fraction (5) was recrystal-lized from ether to give colorless needles, m.p. 63-65":[a]:, 46.4' (E tO H ) (ho mo gen eou s by t.1.c. and n.m.r.).Anal. Ca l cd . fo r C8 H, ,N 03 : C , 55 .47 ; H , 8 .73 ; N , 8 .09 .Found: C. 55.36; H, 8.57: N. 7.81.The second major fraction (56) was recrystallized fromether to give colorless needles, m.p. 57" (sublimation);[a];, 4.5' (E tO H) (ho mo gen eou s by t.1.c. an d n.m .r.).Anal. Ca l cd . fo r C ,H , ,N 03 : C , 55 .47; H , 8 .73 ; N , 8 .09.Foun d: C , 55 .63 ; H , 8 .62 ; N , 7.91.L (+ )-Muscuritte attd L(+)-Allotnusc orbreThe preceding amides 50 and b (300 mg) were added in15 ml of ether, after heating under reflux for 2 h, to suspen-sions of 1.2 g of LiAIH, in 60 ml of eth er: the m ixtureswere hydrolyzed at 0" wi th 12 ml of 30% aqueo us KO H.After work-up in the usual manner, the colorless oil amineswere treated with excess methyl iodide in ether whereuponwhite crystals of 6a iodide and 66 iodide (95 and 97%yields respectively) separated. They were recrystallized

from acetonitrile-acetone and dried over P,O, at 100" forup to 24 h. Th e m. p. varied co nsiderably (from 131 -150")depending on crystal form an d drying t ime. Fo r 60, the m .p.was 131-132" on several occasions: [ a l p 29" (EtOH ); i tsn.m.r. spect rum (220 MH z) confi rmed the st ructural assign-ment (Fig. I) .Anal . Ca l cd. fo r C9H ,oI N0 , : C , 35 .89; H , 6 .69 ; N , 4 .64 .Foun d: C , 35 .68 : H , 6 .51 ; N , 4 .31.Fo r 6b iodide, the m.p. was 148-150" (li t . (6) 149 "); [a];,3.1 (EtO H) (l it . (6) for the chloride, 6.7 (H, O) ): the n.m.r.spect rum showed the product to be homogeneous and wasconsistent in every detail with the structure.Anal. Ca l cd . fo r C9 Hz0 INO 2:C, 35.89; H , 6.69: N, 4.64.Foun d: C . 35 .92 : H , 6 .76 ; N , 4 .47 .The cholinomimetic activity (guinea-pig ileum) of thisisomer was ident ical to that reported for L(+)-muscarineiodide (6).

We are grateful to the Nat ional Research Counci l ofCanada and Bristol Laboratories of Canada for the finan-cial support of this work. The skilful assistance of A.Gauthier in the preliminary phases of this work was highlyappreciated. We are also indebted to Dr. A. H . Grey of theCanadian 220 M Hz n.m.r. center (Sheridan Park) an d Dr.Arthu r Perl in for the n.m.r. spect ra an d thei r interpretat ion.

1. B. BELLEAU.Advances in chemistry series 108. R. F.Co uld . editor. American C hemical Society, 1971, p. 141.2. B. BELLEAUnd V. D I T U L L I O .Can. J . Biochem. 49,1131 (1971).3. B. BELLE AU. n Fundam ental concepts in drug-receptor interact ions. J . F. Daniel l i . J . F. Moran, andD. J. Triggle, edi tors. Academic Press, New York.1970. p. 121.4. C . H. EUGSTER nd P. W ASER. Experient ia, 10, 298(1954).5. C. H . EUG STER. Helv. Chim . Acta. 39. 1002 (19561.6. P. G. W ASER. Pharm acol . Rev. 13, 465 (1961). '7. S. WILKINSON. uart . Rev. 15, 153 (1961).8 . C . H . EUGSTER. Adv . Org . Chem. 2, 427 (1960).9 . F . JE L L I N E K .Acta Cryst. 10. 277 (1959).10 . P . PAULING. Itt Structural chemistry and molecularbiology. A. Rich and N. Davidson, edi tors. Freeman,San Francisco. 1968. p. 555.11. J . Greenstein and M. Wini tz. Chemist ry of aminoacids. John Wiley, New York. 1961.12. E. HARDEGGERnd F . LOHSE. Helv. Chim . Acta, 40,2383 (1957).13 . C . G . SKINNER,. EDELSON, nd W. SHIVE. J. Am.Chem. Soc. 83 , 2284 (1961).

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J. Whiting, Y.K. Au-Young and B. Belleau- A Convenient Synthesis of L(+)-Muscarine