Inhibition of Phenylethanolamine N-Methylfransferase - 5 0 , [eiss0-95, PI^^^-^^^^, I ~ 1650. I ~ ~ ~ D-threo-(1R ,2R)-l-p-Bromophenyl-2dichloroacetamido-l,3propanediol (XIX): CD (c 1.042, Cupra A) [O]700 10, [8]6SO 90, [elsss0, [elslo-100, [e1470-4so0, [elz83!440, [el27B1200*. D-threo-(lR ,2R)-l-p-Iodophenyl-2d1chloroacetamido-l,3-150, [e1640 -205, propanediol(XX): CD (c 1.02, Cupra A) [ e ~ 0 , ~[ e 1 ~~ , ~~4[~14so-3so 4 ,- ~ 0~, [elzs7 ~ 2425, [elzes1625*;CD (c 0.102,Cupra A) [el35o160, [ 8 ] 2 6 9 7465, [el247560*. D-threo-(lR, 2R)- 1-p-Cyanophenyl-2dichloroacetamido1,3propanediol(XXI): CD (c 1.09,Cupra A) [O]700-165, [6]650 -200, 25, [ei430-390 0 , i e i 2 9 6 2070, [elz9o1470*; CD (c 0, 8065, [e]z725700*. 0.109,Cupra A) [e]33o240, D-threo-(1R ,2R)- 1-p-Methylsulfonylphenyl-2dichloroacetamido1,3-propanediol (XXII): CD (c 1.10, Cupra A) -140, [ e l 6 2 5 -2oO,ie1,,,-~~,0, [e],,,2335, [e],,,2000*;CD (c 0.110, Cupra A) [ 6 i n z s 500, I e l Z 6 6 9550, [ e l 2 4 8 1290. D-threo250 208-21 1 >250 2 35 -2 36 237-239 210-215 86-9 l g 217-220 170-1 72 189-191 >250 234-237 235 (sub) 158-160 173-175 148-149 190-192 202-205 195-196 207-209 190-192 176-178 141-143 109-1 11 2 38-240
i-PrOH + EtOAc i-PrOH + EtOAc i-PrOH + EtOAc i-PrOH iPrOH iPrOH + EtOAc i-PrOH + EtOAc EtOAc + MeOH EtOAc + MeOH i-PrOH + Me,CO EtOAc + MeOH EtOAc + MeOH i-PrOH + EtOAc EtOAc + MeOH i-PrOH + EtOAc i-PrOH + EtOAc EtOAc + MeOH EtOAc + MeOH EtOAc + MeOH i-PrOH + EtOAc i-PrOH + EtOAc i-PrOH + EtOAc iPrOH + EtOAc EtOAc + MeOH EtOAc i-PrOH + MeOH
% yield
FormulaC
68 55 60 22 38 63 41 54 57 23 58 73 50 68 9h 68 68 35 39 51 52 47 67 61 82 64 52
C,H7C1F,N*HCl C,H,F,N * HCI C,H,F,N .HCl C 7H,BIN * HCl C,H,F ,N * HCl C,H J .HCl C,H,ClF' ,N. HCl C ,H ,Cl,N .HCl C,H ,&IN C,H ,F' JN. HCl C,H,,ClN *HC1 CJi I J *HCl C,,H,,N~HCl 1
105
8 1JN
C,H,F ,N .HCI C1J ,N *HC1 C,H,Cl,N .HC1 C.$ ,,ClN .HC1 CJil,CIN.HC1 C$I 12ClN.HC1 C,H,Cl ,N .HC1 CJi I ,Cl,N .HCl C,H,Cl,NO *HC1 C,H,Cl,N *HCl C.$, ,Cl,N.HCl C,,,H13Cl,N.HCl C,H,Cl,NO .HC1
"Methods refer to the Experimental Section, the general procedure used, and preparation of starting material(s). bMelting points were detected in an open capillary in an oil bath and are uncorrected. CAllcompounds were analyzed for C, H, N, and halogen and are correct within 10.3% of their calculated values. dNitrile is commercially available. eAcetophenone is commercially available. fCyclopropy1 phenyl ketone is commercially available. gBoiling point (0.1 mm). hRecovered 88% or-trifluoromethylbenzyl alcohol.
C. 2,3-Dichloroacetophenone. A solution of 2,3dichlorobenzonitrile (0.25 mol, 43 g) in 500 ml of Et,O was added dropwise to a stirred solution of methyl Grignard (0.5 mol) in 500 ml of Et,O and refluxed 2 hr. The mixture was carefully added to 250 nil of concentrated HC1 in ice and allowed to stand overnight. The aqueous layer was extracted (Et,O) and the extract washed (H,O, 10% Na,CO,, saturated NaCl soh) and dried (Na,SO,). Evaporation yielded 45 g (96%) of a dark liquid which was used without purification (nmr showed no starting nitrile): ir C=O (1700 cm-I), no C=N absorption. D. N-Cyclopropylbenzamide. The amide was prepared according to method of Aeberli, et al.,'" in 62% yield, mp 96-98' (lit.'" mp 95-97"). Anal. (C,,HllNO). E. N-Methyl-2,3-dichlorobenzamide.Oxalyl chloride (0.25 mol, 22 ml) was added slowly to a stirred solution of 2,3dichlorobenzoic acid (0.05 mol, 9.55 g) in 100 ml of C,H,. The solution was dissolved in 150 ml of Et,O at 0", dry CH,NH, introduced, and the resultant mixture stirred at room temperature 4 hr. The mixture was washed (2 N HCI, 10% Na,CO,, saturated NaCl solution), dried (Na,SO,), and evaporated. The resulting amide was recrystallized (C,H,-cyclohexane) to give 5.5 g (54%), mp 118-121". Anal. (C,H 7C1,NO). 2,3-Dichloro-a-bromotoluene. A solution of 2,3dichlorotoluene (0.31 mol, 50 g),N-bromosuccinimide (0.34 mol, 60.5 g), and benzoyl peroxide (150 mg) in 1 1. of CC1, was refluxed 16 hr. The mixture was cooled to room temperature and the resulting succinimide filtered. The filtrate was washed (H,O), dried (Na,SO,), and evaporated to yield 74 g (99%) of yellow liquid which was used without further purification (nmr showed no starting material). F. 2,3-Dichlorophenylacetonitrile. A stirred solution of 2,3dichloro-a-bromotoluene (0.15 mol, 36 g) in 150 ml of DMF-100 ml of H,O at 0" was treated with KCN (0.225 mol, 14.6 g), which was added in portions. The mixture was allowed to warm to room temperature, stirred for 16 hr,and then diluted with 250 ml of H,O and extracted (Et,O). The Et,O layer was washed (H,O, saturated NaCl solution), dried (Na,SO,), and evaporated. The residue was distilled at 92-95" (0.5 mm) to yield 12.6 g (45%) of colorless liquid. A recrystallized sample (hexane) had mp 72-74". Anal. (C,H,Cl,N). Ethyl 2,3-Dichlorobenzylcyanoacetate. A solution of ethyl cyanoacetate (0.32 mol, 36 g) in 50 ml of C,H, was added dropwise to a suspension of NaH (0.24 mol, 5.8 g) in 150 ml of DMF-50 ml of C,H,. The mixture was allowed to stir at room temperature until
gas evolution ceased. A solution of 2,3dichloro-ol-bromotoluene (39 g, 0.162 mol) in 50 ml of C,H, was then added dropwise and the mixture stirred 48 hr. The excess NaH was destroyed with the addition of a few milliliters of EtOH. The mixture was then diluted with 500 ml of H,O and extracted (C,H,) and the extracts were washed (H,O, saturated NaCl solution), dried (Na,SO,), and evaporated. The resulting residue was distilled at 144-150" (0.1 mm) to yield 16.9 g (37%) of product. Anal. (C,,H,,Cl,NO,). 2,3-DichlorobenzylcyanoaceticAcid. A solution of ethyl 2,3dichlorobenzylcyanoacetate (0.06 mol, 16.9 g) in 250 ml of MeOH260 ml of 10% Na,CO, (0.24 mol) was heated at 65" for 16 hr. The MeOH was evaporated and the aqueous mixture diluted with 200 ml of H,O. The aqueous layer was washed (Et,O), acidified (concentrated HCl), and extracted (Et,O). The extracts were washed (H,O, saturated NaCl solution), dried (Na,SO,), and evaporated. The resulting solid was recrystallized (C,H,) to yield 9.2 g (62%), mp 159-162". Anal. (C,,H,Cl,NO,). G. 3-(2,3-DichlorophenyI)propionitrile. 2,3-Dichlorobenzylcyanoacetic acid (0.04 mol, 9.2 g) was heated at 180" for 2 hr until evolution of gas ceased. The resulting residue was distilled at 103106" (0.1 mm) to yield 4.1 g (53%) of colorless liquid. Anal. (C,H ,Cl,N). H. 2,3- and 3,4-Dichlorophenoxyacetonitriles. The respective phenol (0.1 mol, 16.3 g), CICH,CN (7.5 g, 0.1 mol), and K,CO,(O.l mol, 13.8 g) wererefluxed in 100 ml of acetone for 16 hr. The cooled mixture was diluted with H,O, basified (NaOH), and extracted (EtOAc). The extract was washed (5 N NaOH, H,O, saturated NaCl solution), dried (MgSO,), and evaporated. The resulting solid was recrystallized (C,H,-cyclohexane) to yield 2,3dichloro- [ 18 g (89%), mp 86-89'. Anal. (C,H,Cl,NO)] and 3,4dichlorophenoxyacetonitrile [ 17.8 g (88%), mp 59-61", Anal (C,H$12NO)l. p-(3,4-Dichlorophenyl)propionicAcid. Hydrogenation of 3,4dichlorocinnamic acid (0.46 mol, 100 g) in dioxane over Pd/C catalyst yielded 80 g (80%) of the recrystallized (hexane) propionic acid, mp 86-89". Anal. (C,H,Cl,O,). I. p-(3,4-Dichlorophenyl)propionamide. A solution of p-(3,4dichloropheny1)propionic acid (0.1 mol, 21.9 g) in 200 ml of C,H, was treated at room temperature with oxalyl chloride (0.5 mol, 64 g). The mixture was refluxed 3 hr, cooled, and evaporated in uacuo The residue was dissolved in 250 ml of Et,O and the ethereal solution saturated with dry NH, at 0". The mixture was stirred at room temperature 4 hr, washed (H20, 2 N HCl, 10% Na,CO,, saturated
106
Journal of Medicinal Chemistry, 1973, Vol. 16, No. 2
NaCI), and dried (Na,SO,). Evaporation (Et,O) and recrystallization (C,H,-cyclohexane) yielded 17.5 g (81%) of the amide, mp 76-78", Anal. (C,H,CI,NO). 3-(3,4-DichlorophenyI)propanol-l. A solution of p-(3,4dichloropheny1)propionic acid (0.2 mol, 43.8 g) in 250 ml of THF was added dropwise to a solution of BH, (0.6 mol) in 600 ml of THF at 0" under N,. The mixture was refluxed 3 hr and cooled to On, and the excess BH, was destroyed with 2 NHCI. The THF was removed in vacuo, the aqueous layer was neutralized (2 N NaOH) and extracted (Et,O). The extracts were washed (saturated NaHCO,, saturated NaCl solution), dried (Na,SO,), and evaporated. The resulting residue was distilled at 121-122" (0.5 mm) to yield 37.6 g (91%). Anal. (C,H,,CI,O). J. 4-(3,4-DichlorophenyI)butyronitrile. The propanol (0.1 mol, 20.5 g) was dissolved in 75 ml of dry C,H,N and CH,SO,CI (0.11 mol, 8.4 ml) was added dropwise, while maintaining the temperature below 20". The mixture was stirred at room temperature for 3 hr and then added to 75 ml of concentrated HCI in ice. The aqueous layer was extracted (Et,O) and the extract washed (10% Na,CO,. saturated NaCl solution), dried (Na,SOJ, and evaporated. The resulting 28 g (99%) of light-yellow liquid sulfonate ester was immediately dissolved in 100 ml of DMF-25 ml of H,O and cooled to 0". Solid KCN (0.15 mol, 9.75 g) was added and the mixture stirred at room temperature 72 hr. The mixture was diluted with 100 ml of H,O and extracted (Et,O). The Et,O extracts were washed (H,O, saturated NaCI) and dried (Na,SO,). Evaporation of solvent yielded a red liquid which was distilled at 115-1 18" (0.1 mm) to yield 18.1 g (84%) of colorless liquid.Ana1. (C,,H,Cl,N). Enzyme Assay. The enzyme preparation and the method of enzyme assay have been previously described in detail." 4 n (NH,),SO, fraction of the high-speed centrifugal supernatant fraction from a homogenate of whole adrenal glands from rabbits was used as the enzyme." L-Norepinephrine (40 pM) was the substrate.
Fuller, Roush, Snoddy, M o ~ ~ o J ~ Enzyme activity was calculated as picomoles of epinephrine formed per 30 min of incubation. Several concentrations of each inhibitor were tested for enzyme inhibition; the concentrations were spaced on a log basis, e.g., 1, 3, 10, 32, 100. 317 KM, and were selected t o achieve a range of inhibition on both sides of 50%. By interpolation, the molar concentration required for 50% inhibition was determined. The negative log of this concentration is defined as the pl,, value.
References (1) J. Axelrod,J. Biol. Chem., 237, 1657 (1962). (2) R. W. Fuller and J. M. Hunt,Biochem. Pharmacol., 14, 1896 (1965). (3) L. R. Krakoff and J. Axelrod, ibid., 16, 1384 (1967). (4) R. W. Fuller, J . Mills, and M. M. Marsh, J. Med. Chem., 14. 322 (1971). (5) A. E. Kitabchi and R. H. Williams, Biochim. Biophps. Acta, 178,181 (1969). (6) R. J. Connett and N. Kirshner, J . Bioi. Chem., 245, 329 (1970). (7) L. R. Mandel, C. C. Porter, F . A . Kuehl, Jr., N. P. Jensen, S. M. Schmitt, T. B. Windholz, T. B. Beattie, J. A . Carty, B. G. Christensen, and T. Y . Shen, J. Med. Chert?., 13. 1043 (1970). (8) N. P. Jensen,S. M. Schmitt, T. B. Windholz, T. Y . Shen, L. R. Mandel, B. Lopez-Ramos. and C. C. Porter, ihid.. 15, 341 (1972). (9) B. R. Baker. R. E. Schaub, J. P. Joseph, F. J. McEvoy, and J. H. Williams, J. Org. Chem., 17, 141 (1952). (10) P. Aeberli, J. Gogerty, and W.J . Houlthan, J . Med. CherTi,, IO, 636 (1967). (11) R. W. Fuller and J. M . Hunt, Anal. Blochem, 16, 349 (1966). (12) G. N. Wiikinson,Biochem. J . , 80, 324 (1961).
Inhibition of Phenylethanolamine N-Methyltransferase by Benzylamines. 2. In Vitro and In Vivo Studies with 2,3-Dichloro-cu-methylbenzylamine Ray W. Fuller,* Betty Warren Roush, Harold D. Snoddy, and Bryan B. Molloy The Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46206. Received July 10, 15 72
2,3-Dichloro-cx-methylbenzylamine (DCMB) was a potent inhibitor of phenylethanolamine N-methyltransferase in vitro. The (+) isomer was more active than the (-) isomer, and the inhibition was reversible and competitive with respect to norepinephrine. The calculated Ki for DCMB was 0.09 compared to the K , of 1 2 pA4 for 1-norepinephrine as the substrate. The &-methyl was required for biological stability of benzylamines. DCMB localized in tissues in the following order of concentration when injected intraperitoneally into rats: lung > kidney > liver > brain > adrenal > spleen > heart > blood. The half-life of the compound in the adrenal gland after injection of a dose of 0.2 mmol/kg was 3.4 hr. The concentration of the drug in the adrenal glands bore a nearly linear relation to dose over the dose range of 0.1-0.4 mmol/kg. Treatment of rats with DCMB prior to forced exercise caused a significant reduction of epinephrine, indicative of phenylethanolamine N-methyltransferase inhibition in vivo.
w,
A specific inhibitor of phenylethanolamine N-methyitransferase (PNMT) in the adrenal medulla would suppress the formation of the adrenal medullary hormone, epinephrine, without directly interfering with norepinephrine biosynthesis. Such an inhibitor may find medicinal application, especially if some pathological states are associated with overproduction of epinephrine. Recently we have found that a group of benzylamines are unexpectedly potent inhibitors of PNMT in vitro. This paper describes further in vitro studies with one of the most active members of that group, 2,3-dichloro-a-methylbenzylamine, and also reports the results of the initial in vivo studies with this compound. In Vitro Studies. Figure 1 shows a comparison of the inhibition caused by the stereoisomers of 2,3-dichloro-amethylbenzylamine. The (+) isomer was more active than the racemic mixture and hence much more active than the (-) isomer. The difference between the isomers was more
'
than tenfold, the pIs0 value for the (+) isomer being 6.81 and that for the (-) isomer being 5.72. To determine whether the inhibition by 2,3-dichloro-amethylbenzylamine was reversible, we measured the ability of dialysis in Visking tubing to remove the inhibitor and thereby restore enzyme activity after the inhibitor and enzyme were mixed in vitro. The results in Table I show that dialysis for 24 hr nearly completely restored enzyme activity, indicating that the amount of inhibitor which was sufficient to cause 62-65% inhibition had been removed by dialysis. Thus, the inhibitor appears to form a reversible complex with the enzyme. Figure 2 shows a Lineweaver-Burk plot. The inhibition by 2,3-dichloro-a-methylbenzylamine was competitive with respect to the methyl-accepting substrate, norepinephrine. From the data in Figure 2 , kinetic constants were calculated by the method of Wilkinson.2 The V,, was not altered by the inhibitor, being 57 k 6 for the control and 58 f 2 and
