Aminopyridine Carbamic Acid Esters: Synthesis and Potential as Acetylcholinesterase Inhibitors and Acetylcholine Releasers

Aminopyridine Carbamic Acid Esters: Synthesis and Potential as Acetylcholinesterase Inhibitors and Acetylcholine Releasers GREGORYM. SHUTSKE*',JOHN D. ToMER*, KEVINJ. KAPPLES~,NICHOLAS J. HRiB*, JOHN G. JURCAK*, GINAM. BORES', FRANCIS P. HUGER',WAYNE PETKO', AND CRAIG P. SMITH' Received January 18, 1991, from the Departments of *Chemical Research and 'Biological Research, Hoechst-Roussel Pharmaceuticals, Inc., Accepted for publication May 25, 1991. Somewille, NJ 08876.

tetramethylethylenediaminegives metalation exclusively at the 4-position,3 and he has further reported the ortho amination of lithiated tertiary benzamides by reaction with ptoluenesulfonyl azide, followed by reduction with sodium borohydride under phase-transfer conditions.4 These two methodologies were successfully combined to give 4-amino3-pyridyl NJVdiethylcarbamate (2a) in good yield.6 As predicted, no product derived from metalation a t the 2-position was detected in the reaction mixture. Unfortunately, as Snieckus and others have pointed out,6 this strategy is not applicable to the synthesis of the desired NJV-dimethylcarbamate (2b), because of the competing reaction in which the carbonyl group of 4b is attacked by the organolithium reagent (Scheme I). However, 2b could be obtained from 2a through the intermediacy of 4amino-3-pyridinol(5a). The highly water soluble 5a had been synthesized by the pereulfate oxidation of 4-aminopyridine,7but the availability As part of our continuing interest in centrally acting of 2a made possible a new and more convenient synthesis of cholinesterase inhibitors as potential therapeutic agents for 5a. The carbamate group of 2a was cleaved by hydrazine, and Alzheimer's disease,' we became interested in recent reports that the pyridylurea LF 14 { [l,l-dimethyl-3-(4-amino-3- then the reaction mixture was poured into cold acetone to produce analytically pure 58. Under these workup conditions, pyridy1)lurea; 1)is a cholinesterase inhibitor and an acetylthe hydrazine was destroyed and an aqueous phase (from choline releaser.* The desirability of combining these two which it would have been difficult to recover 5a) was avoided. properties in a single molecule prompted us to investigate the With a synthesis of 5a in hand, the desired 2b was anticholinesterase activity and acetylcholine-releasing propsynthesized by first reacting 5a with neat NJV-dimethylforerties of the carbamate analogues (2). The compounds repremamide dimethyl acetal, which protected the primary amine sented by 2 were also of interest because of their obvious as an amidine (68). Reaction of 6a with NJV-dimethylcarsimilarity to the potent cholineeterase inhibitor pyridostigbamyl chloride then yielded the carbamate 78. Exposure of ?a mine bromide, 3. to aqueous trifluoroacetic acid cleaved off the amidine to give 2b, which was isolated as its trifluoroacetate salt. Care had to Results and Discussion be taken so that the carbamates 2a and 2b were not exposed to basic conditions, because of their tendency to rearrange to Chemistry-The synthetic approaches to 2 are shown in the (3-hydroxy-4-pyridyl)ureass (Scheme I). Scheme I. On the basis of reports from Snieckus and coAcetylcholinesterase Inhibition-The aminocarbamates workers, 2 should be available through an ortho-directed (28 and 2b) were evaluated for cholinesterase inhibition metalation strategy. Snieckus has shown that the ortho(Table I). It was not surprising that the NJVdiethylcarbadirected metalation of 3-pyridyl NJVdiethylcarbamate (48) mate 2a did not exhibit potent cholinesterase inhibition: In with n-butyllithium at low temperature in the presence of cholinesterase inhibitors of the carbamate type, the carbonyl group is probably involved in acylating the active site, serine,* and as described above, the NJVdiethyl moiety was purposefully designed to be a sterically demanding group, tending to protect the carbonyl group from nucleophilic attack. As anticipated, the NJV-dimethylcarbamate 2b showed good anticholinesterase activity, consistent with other 3-pyridyl carbamatess although not in the range of the 1 2 highly potent pyridostigmine. The protected carbamate 7a was also tested for anticholinesterase activity and, somewhat surprisingly, demonstrated better activity than 2b (Table I). This result prompted us to synthesize a series of amidine carbamates analogous to 7a. The structures and physical properties of these compounds I Bi (7b-r) are included in Table I, and the properties of the 3 corresponding precursors (6a-m) are found in Table 11. Two

Abstract 0 4-Amino-3-pyridyl carbamates (2a-c)were synthesized as

potential acetylcholinesterase inhibitors and acetylcholine releasers on the basis of the reported activity of the analogous N(4-amino-3-pyridyl)WJ"dimethy1urea (1). Although 4-amino-3-pyridyl N,Ndimethylcarbamate (2b) showed good cholinesterase inhibition [concentration that elicited a 50% reduction in the maximal enzyme response (IC,) was 13.4 441, it had no effect on the stimulated release of [3H]acetylcholinefrom rat striatal slices. 4-[[(Dimethylamino)methylene]amino]-3-pyridylN,N dimethylcarbamate (7a), an intermediate in the synthesis of 2b, demonstrated surprisingly good cholinesterase inhibition (IC, was 9.4 44) but showed no activity as a releaser. A precursor to 7a, N(3-hydroxy4-pyridyl)-N',N-dimethylformamidine (a), showed some activity in release but was not an esterase inhibitor, whereas the precursor to 6a. 4-amino-3-pyridinol(5a),was a potent releaser. A new synthesis of 5a, based on an ortho-directed lithiation strategy, is also reported.

380 1 Journal of Pharmaceutical Sciences Vol. 81, No. 4, April 1992

OO22-3~9/92/04oO-0380$02.50/0 0 1992, American Pharmaceutical Association

Table C[e(Substituted)-3-pyrldlnyl] Carbamater

Compound (Method)s

Purification Method or Recrystallization Solvent

X

R2

mp, "C

Molecular Formulab

! , d 'l

confidence limits)c

CH, -(cH2)5CH3 CH3 CH3 CH3 CH, CH,

NH2 NH2 NH2 N=CHN(CH,), N=CHN(C2H5), N=CHN(CH(CH3)J2

Et0Ac:pentane 138-140 159-1 60 CH,OH:ether EtOAc 1 344 Distillation 64-66 Ether:hexane 73-74 Chromatography 7&79

65 54 58 87 37 34

CH3

CH3

N=CHN(CH2),CH2

EtOAc

99-100

CH,

CH,

N=CHN(CH,),CHZ

EtOAc

CH3

CH3

N=CHN(CH2)5CH2

CH3 CH3 CH,

CH3 CH3 CH,

N=CHN(CH2),0CH2CH2 N=CHN(CH3)(CH2)2CH3 N=CHN(CH3)CH2C8H5

CH3 CH3 CH,

CH3 CH3 CH,

N=CHN(CH3)CH(CH2)4CH2 CH2C12:EtOAc N= CHN[(CH2),CH3J2 Ether N=CHN[(CH,),CH& Ether:CH,CI,: hexane N=CHN(CH2)2SCH2CHz EtOAc N=CHN(CH,), EtOAc N=CHN(CH,), Ether N=CHN(CH,), EtOAc N=CHN(CH& EtOAc N=CHN(CH,), Chromatography

C2H5

CH,

C2H5

n

n n

m

Acetvlcholinesteras; Inhibition lc., (95%

C13H24N402

>100 13.4(10.8-16.5) >100 9.4(6.8-12.8) >100 >100

68

C13H16N402

9.4(6.9-12.7)

86-89

84

C14H20N402

47.1 (35.6-62.3)

Chromatography

68-70

86

C15HZ?N402

>100

EtOAc CH,CI,:EtOAc Et0Ac:pentane

127-1 29 130-1 33 146-1 47

74 25 38

C13H18N403

>100

157-1 59 84-85 123-1 25

56 70 46

156-158 90-91 67-69 137-1 39 138 (dec) 151 (dec)

68 63 63 66 57 82

cl OH,

5N302

C8HllN302* C2HF302' cl lH1SN3O2 cl1H16N402

C13H?ON402

Cl4H=N4O2 C4H404' 40.6(21.2-77.7) Cj7HmN402 * 1.5C4H404' 18.7(14.2-24.6) C16Hz4N402 * C4H404' ClSHd402 CZlHmN4O2 * C4H404'

46.4(35.6-60.1) 67.4(58.1-78.2) 20.2(10.4-39.4)

cl 3H1 8N402S

52.5 (44.6-61.7) >100 >100 12.6(5.9-27.0) 83.0(32.9-209.5) >100 0.032(0.024-0.042)

C14H20N402

C13H20N402 C10H14N402

cll H16N402 C15H16N402 ~~~

~~~~~

Experimental Secrion. Combustion analyses of C, H, and N were within 20.4% of the calculated values. Data are concentrations that elicited a 50%reduction in the maximal (control) enzyme response. This procedure is described in full in the Experimental Section. a Trifluoroacetate. Maleate. 0 Sesquifumarate. a General synthetic methods are described in the

'

Tabk ICH(3-Hydroxy-epyridyl)-N',N'-dlalky~ormamldlner

Compound 60

6b 6c

6d 6e 61

ss 6h

Method'

A A A A A A

61

A B B

61 Sk 61 6m

B B B B

R,K,N 4N

R4

R3

CH3

CH3

C2H5

C2H5

CH(CH3)2

CH(CH3)2 -(cH2)44CH215-(cH2)6-(C~2)2-0-(C~2)2CH3 (CH2)3CH3 CH3 CH2C6H5

-

CH(CH2),CH2 (CH2)2CH3 (CH2)2CH3 (CH2)SCH3 (CH2)SCH3 -(CH2)2-S-(C~2)2(3.43

Recrystallization Solvent EtOAc:pentane Et0Ac:hexane Hexane CH,OH:ether EtOAc Ethwpentane CH30H Et0Ac:hexane -d

Et0Ac:hexane

-

-

d

CH30H

mp, "C

Yield, %

121-123 104-106 77-79 190 (dec) 1 71-1 73 117-1 18 192-1 93 83-82

-

127-1 29

55

C8Hl l N 3 0

62 53

cl OH, S N 3 0

36

CloHq3N30 * 2HCIC

55

c1 1H15N30

22

C12Hl

59 4 -1d 68

-

-

197-1 99

87

d

d

Molecular Formulab

C12H19N30

7"3O

C10H13N302

-

C11H,7N3$

C13H19N30

-d

d

-d

c1OH13N3OS

General synthetic methods are described in the Expermental Section. Combustion analyses for C, H, and N were within +0.4% of the calculated values, except where indicated. C: calcd., 45.47;found, 45.05. Used without further purification in the next reaction. a

methods, A and B, were used to synthesize the 3-pyridinol amidines (Scheme I and Table 11). Method A was analogous to the synthesis of 7s and consisted of heating 5a w i t h the appropriate amide acetal. Method B, o n the other hand,

involved an amidine exchange reactionlo with the dimethyl amidine 6a and a high-boiling amine in refluxing toluene. L i k e 68, each of the 3-pyridinol derivatives 6b-m was treated w i t h Nfl-dimethylcarbamyl chloride to give the correspondJournal of Pharmaceutical Sciences I 381 Vol. 81, No. 4, April 7992

uoyNR1R2 4a

4b

4a, A, = R, = CzH, 4b, R l = R 2 = CH,

a b c

-L--)

&0TNRlR2

a. b, c

/ i (2a)

2a. R, = R2 = C2H, 2b, R,

= Rz

-

Sa

CH,

3

I

2C, NRlRZ = N

1

h (7a and 7n) (method E)

$N(c2H5)* 0 OH

d

l e , R3

- -

R4 CH3 Pa)

R,R,N+N

R,R,N'+N I (melhod C)

(y0yNRIR2 4

8

-

or g (method 0 )

e, (method A)

cym -

6a g (Table I)

'la r (Table II)

6a

___)

-

6h m (Table I)

(method 6)

Scheme L ( a ) sec;.BuLi, tetramethylethylenediamine,THF, -65 "C; (b) 4-CH3C6H,S0,N3, ether; (c) NaBH,, mBu,N+HSO,-, R3R,NCH(OCH3),, A; (f) RlR2COCI, Et3N; (9) RINCO, NaH, THF; (h) CF&OOH, H,O; (I)rert-BuOK, THF.

ing NJV-dimethylcarbarnates 7b-m. Compound 6a was also reacted with 1-piperidinecarbonyl chloride to give 7n, which yielded the corresponding 4-aminod-pyridyl carbamate 2c after hydrolysis in aqueous trifluoroacetic acid. In addition, 2a was reacted with N,N-dimethylformamide dimethyl acetal to give the diethylcarbamate 70. Finally, 6a was reacted with methyl-, ethyl-, or phenylisocyanate to give the corresponding monoalkyl carbamates 7 p r . The amidine carbamates analogous to 7a (7b-r) were evaluated as cholinesterase inhibitors (Table I).As in the case of 2a and 2b, steric influences tended to predominate for 7b-r. In the amidine portion of the molecules, as the nitrogen substituents became larger than methyl groups (7b, 7c, and 7h-11, activity decreased. Even when these substituents were constrained in rings (7d-g and 7m), only the smallest (7d)had activity comparable with that of 7a. T w o analogues with relatively large substituents, 7i and 71, showed better activity than would have been predicted. A conformationally flexible, lipophilic binding site, which would bind large, suitably placed lipophilic groups, has been proposed for acetylcholinesterase;" such binding could account for the better than expected activity of 7i and 71. The same trends noted for the amidine portion of 7a-r held true for the carbamate functionality: The piperidine and diethyl carbamates (7n and 70) had weak activity (Table I), and the piperidine carbamate with an amino group in the 4-position (2c) was also a poor cholinesterase inhibitor (Table I). Similarly, among the monosubstituted carbamates ( 7 p r ) , only the methyl-substituted analogue (7p) had activity comparable with that of 7a (Table I). Acetylcholine Release-On the basis of published data for 1, some compounds were evaluated for their ability to enhance electrically stimulated release of acetylcholine12 from rat striatal slices. None of the aminocarbamates (2a-c) had releasing activity at 100 p M , nor did any of the amidine carbamates (7a-r). The known 5a, however, was a potent releaser, with activity similar to that of 4-aminopyridine (at 100 p.M) (Figure 1).Although this activity was not surprising, in view of the potent releasing effects of the homologous 3-methoxy-4-aminopyridine,13this paper is, to our knowledge, the first report of the acetylcholine-releasing effect of 5a. The data suggested that a free hydroxyl group a t the 3-position is necessary for releasing activity and prompted an investigation of the intermediate 4-[[(dialkylamino)methyl382 1 Journal of Pharmaceutical Sciences Vol. 81, No. 4, April 1992

H 2 0 : (d) H2NNH,; (e)

enelaminol-3-pyridinols as releasers. Again, a strong steric influence was in evidence: The dimethyl and pyrrolidine analogues 6a and 6d had moderate potency, with activity significantly greater than that of control at 100 but other, more sterically demanding analogues had much weaker activity. On the basis of the activity of 5a, the alkyl analogues 5b-d were synthesized to evaluate their activity as acetylcholine releasers (Scheme 11).Compound 2a was acylated smoothly with acid chlorides to give the amides 9a and 9b; reduction with borane-dimethyl sulfide complex gave the 4-alkylamino-3-pyridyl carbamates 10a and 1Oc. Alternatively, reaction of 2a with propionic acid and sodium borohydride gave 10b directly. Cleavage of 10a-c with hydrazine, as described earlier, gave the corresponding 4-alkylamino-3-pyridinols (5b-d). Again, analogues with increasingly larger substituents on the 4-amino group showed significantly poorer activity as releasers (5b, Figure 1).

a,

0

9a, R = CH3 9b, R = (CH,)$Hs

R"NH

Scheme Il-(a) RCOCI, Et3N, CH,CI,; BH3 * (CH,),S, THF; (d) HPNNH2.

(b) RCOOH, NaBH,; (c)

250

1

n

So

5b

6a

6d

60

69

6m

4-AP

Flgure 1-Enhancement of electrically stimulated release of rH]acetylcholine from rat striatal slices, compared with 4-aminopyridine (4-AP). Data are from single determinations and are expressed as percent increase over control values. Details of the experimental conditions are given in the Experimental Section. Key: (B) 10 @; ( 0 )100 @.

Conclusions Carbamate analogues of 1 were synthesized and studied as acetylcholine releasers and cholinesterase inhibitors. Some NJV-dimethylcarbarnates (including some amidines that were designed as protected primary amines) were good cholinesterase inhibitors, although no compounds had both anticholinesterase and acetylcholine-releasing activities. A new synthesis of 5a was reported, based on an ortho-lithiation strategy, and this compound was a potent releaser.

Experimental Section Melting points (mp) were determined on a Thomas-Hoover capillary mp apparatus and are uncorrected. IR spectra were recorded on a Perkin-Elmer 547, and NMR spectra were taken on a Varian XL-200. Chemical shifta are reported in parta per million relative to tetramethylsilane as an internal standard. Mass spectral data were determined by direct insertion at 70 eV with a Finnigan 4000 GC-MS, equipped with an INCOS data system. Merck 230-400-mesh silica gel was used for flash chromatography. Elemental analyses were performed by Oneida Research Services, Whitesboro, NY; data from combustion analysis indicated by elemental symbols were within 50.4% of calculated values. Tosyl azide was synthesized according to a literature pr~cedure.'~ Because of the potential shock sensitivity of this reagent,6 it was synthesized only in the amount required by the experimental scale and it was used immediately after synthesis (it was never stored). N,N-Dimethylformamide dimethyl acetal was purchased from Aldrich Chemical Company and was used without further purification; the other amide acetals used in method A were synthesized according to a patented procedure.16 4-Amino-3-pyridyl NJ-Diethylcarbamate (2akCompound 4a (9.70 g, 50.0 mmol) and tetramethylethylenediamine (6.39 g, 55.0 mmol) were dissolved in dry tetrahydrofuran (THF; 100 mL) and chilled in a dry ice:acetone bath. sec-Butyllithium (42 mL of a 1.3 M solution in cyclohexane, 54.6 mmol) was then added in a dropwise manner, and the reaction mixture was stirred in the cold for 1h. At the end of this time, tosyl azide (10.80 g, 55.0 mmol) in ether (100mL) was added, and the reaction was allowed to come to mom temperature. Tetra-n-butylammonium hydrogen sulfate (2.55 g, 7.5 mmol) was then added, followed by the dropwise addition of a solution of sodium borohydride (5.85 g, 155 mmol) in HzO (15 mL, rate of addition adjusted to control foaming). The resulting mixture was stirred for 30 min at mom temperature, and then 10% HCl was added to obtain a pH of 1-2 (rate of addition again adjusted to control foaming). After being stirred overnight, the reaction mixture was filtered from the precipitated boric acid, and then the filtrate was washed three times with CH2Clz. The aqueous phase was then made basic with 10% NaOH and extracted with CH2C12.Drying (MgSO,) and evaporation gave a residue that was purified by flash chromatography (5% triethylamine in EtOAc) and recrystallization to give pure 2a (see Table I for physical data); IR (KBr): 3420, 3320 (NH,),

1710 (carbamate C=O) cm-'; 'H NMR (CDCl,): 6 1.21 (dt, 6H, N(CH,CH,),), 3.42 (dq, 4H, N(CH,CH,),), 4.36 (broad 8, 2H, exchanges with D,O, NH2), 6.62 (d, J = 7 Hz, l H , H-61, 8.08 (d, J = 7 Hz,lH, H-6), 8.13( 8 , l H , H-2); MS: m/e 209 (M9. Anal. (CloHl6N,O2) C, H, N. 4-Amino-3-pyridinol (la)-Compound 2a (36.29 g, 0.173 mol) was suspended in anhydrous hydrazine (150 mL), and this mixture was warmed on a steam bath for 1h. At the end of this time, the resulting solution was added slowly to acetone (1.2 L) which was chilled to -10 "C (ice:CH,OH bath). After the addition was complete, anhydrous ether (400 mL) was added, and the mixture was refrigerated overnight. The colorless, crystalline product was filtered off and washed with ether to give 16.8 g (88%) of analytically pure 5a: mp 220 "C (dec) (reported7 mp 240-242 "C); 'H NMR [dimethyl sulfoxide (DMSO)d6]: 6 5.40 (broad s,2H, exchanges with D20, NH,), 6.48 (d, J = 7 Hz,l H , H-5), 7.60 (d, J = 7 Hz,lH, H-6), 7.69 (8, lH, H-2), 8.50 (broad s, lH, exchanges with D20, OH); M S m/e 110 (M+). Anal. Compound 6a w& s y n t h e s k d by method A,-Scheme I. Compound 5a (1.10 g, 10.0 mmol) was refluxed for 30 min in dimethylformamide dimethyl acetal(10 mL). At the end of this time, the reaction mixture was concentrated under reduced pressure, and the residue was passed over a short column of florisil and eluted with 5% CH30H in EtOAc. Evaporation and recrystallization gave analytically pure 6a (see Table I1 for physical data); IR (CHCl,): 3400 (OH), 1645 (amidine C=N), 1600 (pyridine) cm-'; 'H NMR (CDCI,): 6 3.08 (8, 3H, N(CH3)2), 3.12 (8, 3H, N(CH&), 6.75 (broad 8, l H , exchanges with DzO, OH), 6.80 (d, J = 7 Hz,lH, H-5), 7.80 (8, lH, N-CH), 8.01 (d, J = 7 Hz, l H , H-61, 8.20 ( 8 , l H , H-2); M S mle 165 (M+). Data for the compounds prepared in an analogous fashion (6b-g) are presented in Table II. 4-[[(3-Hydroxy-4-pyridyl)iminolmethyl]thiomorpholine (6m)Compound 6m was synthesized by method B,Scheme I. Compound 6a (5.14 g, 31.2 mmol) was reflwed overnight in dry toluene (120 mL) containing thiomorpholine (distilled from CaH,: 10.5 mL, 10.8 g, 104 mmol). At the end of this time, the reaction mixture was concentrated, and the residue was triturated with ether. The crude solid obtained in this manner was dissolved in CH,OH, adhered to silica gel, and then flash chromatographed (10% CH30H in EtOAc). Evaporation of the productsontaining fractions and recrystallization of the resulting residue gave 6m (see Table I1 for physical data); IR (KBr): 3400 (OH), 1635 (amidine C=N), 1590 (pyridine) cm-'; 'H NMR (DMSO4): 6 2.63 (m, 4H, (CH,)S(CH,)), 3.68 (m, 2H, N(CH2)), 3.96 (m, 2H, N(CH,)), 6.90 (d, J = 7 Hz,l H , H-5), 7.83 (d, J = 7 Hz, lH, H-6), 7.97 (8, lH, N = O , 8.00 (s, IH, H-2), 8.45 (broad 8 , lH, exchanges with D,O, OH); MS: m/e 223 (M? Data for compounds prepared in a n analogous fashion (6h-I) are presented in Table 11. 4-[[(Dimethylamino)methylenelamino]-3-pyridyl Nfl-Dimethylcarbamate (7akCompound 7a was synthesized by method C, Scheme I. Compound 6a (1.65 g, 10.0 mmol) was refluxed for 1 h in benzene (20 mL) containing triethylamine (1.10 g, 11.0 mmol) and NJ-dimethylcarbamyl chloride (1.10 g, 10.0 mmol). At the end of this time, the reaction mixture was applied directly to a silica column and eluted with 5% triethylamine in EtOAc. Evaporation of the product-containing fractions gave chromatographically pure product, which was distilled in a bulb-to-bulb apparatus (oven temperature at 175 "C) to give analytically pure 7a (seeTable I for physical data); IR (CHCl,): 1720 (carbamate C=O), 1645C=N), 1590 (pyridine) cm-'; 'H NMR (CDCI,): 63.00 (s,6H, amidine NCH, and carbamate NCH,), 3.03 (8, 3H, amidine NCH,), 3.12 ( 8 , 3H, carbamate NCH,), 6.81 (d, J = 7 Hz,lH, H-5), 7.61 (8, l H , N=CH), 8.26 (d, J = 7 Hz,l H , H-6), 8.30 (8, IH, H-2); MS: mle 236 (M?. Data for compounds prepared in an analogous fashion ( 7 b n ) are presented in Table I. 4-[[(Dimethylamino)methylene]amino]-3-pyridyl N-Methylcarbamate (7p)-Compound 7p was synthesized by method D, Scheme I. To a hot suspension of 6a (7.0 g, 42.0 mmol) in dry THF (200 mL) was added NaH (0.168 g of a 60% oil dispersion, 42.0 mmol) and methyl isocyanate (2.6 mL, 44.0mmol). The reaction was stirred at ambient temperature overnight, and then it was chilled in an ice: MeOH bath, giving a precipitate that was filtered off and washed with ether. The solid was distributed between CH,C12 (250 mL) and saturated aqueous NH,C1(30 mL), and then the aqueous phase was extracted twice more with CHzC12(200 mL). The combined organic phase was Journal of Pharmaceutical Sciences I 383 Vol. 81, No. 4, April 1992

dried (MgSO,) and concentrated under reduced pressure to give crude 7p, which was recrystallized to yield analytically pure 7p (see Table I for physical data); IR (CHCl,): 3480 (NH), 1750 (carbamate C=O), 1645 (amidine C=N), 1590 (pyridine) cm-'; 'H NMR (CDCI,): 6 2.80 (d, J = 6 Hz,3H, NHCH,), 3.02 ( 8 , 6H, N(CH3)2),5.78 (q, J = 6 Hz, lH, exchanges with D,O, NHCH,), 6.82 (d, J = 7 Hz, lH, H-5),7.61 (8, lH, N=CH), 8.23 (a, lH, H-2), 8.25 (d, J = 7 Hz,lH, H-6); M S rnle 222 (M?. Data for compounds prepared in an analogous fashion (7q and 7r) are presented in Table I. 4-Amino-3-pyridyl NJV-Dimethylcarbamate Trifluoroacetate (%b)--Compound2b was synthesized by method E, Scheme I. Compound 7a (7.4 g, 31.0 mmol) was refluxed for 30 rnin in trifluoroacetic acid (30 mL) and HzO (15 mL). The reaction mixture was then concentrated under reduced pressure, filtered over a pad of basic alumina, eluted sequentially with CH2C12, 30% EtOAc in CHzCl,, and EtOAc. Because of the small amount of basic alumina that was used, the excess trifluoroacetic acid was neutralized, but the trifluoroacetate salt passed through intact. Evaporation of the productcontaining fractions gave chromatographically pure 2b, which was recrystallized to yield pure 2b (see Table I for physical data); IR (KBr): 3370,3210 (NH,), 1725 (amidine C=O) cm-'; 'H NMR (DMSO-4): 6 2.91 (8,3H, NCH,), 3.06 (8,3H, NCHJ, 7.00 (d, J = 7 Hz,lH, H-51, 8.08 (broad s,2H, exchanges with D,O, NH,), 8.12 (d, J = 7 Hz,lH, H-6), 8.37 ( 8, lH, H-2), 14.0 (broad 8 , lH, exchanges with D,O, COOH); M S d e 181 (M9. 4-(Acetylamino)-3-pyridyl NJV-Diethylcarbamate (9abCompound 2a (4.18 g, 20.0 mmol) was dissolved in CH,C12 (75 mL) containing triethylamine (2.22 g, 22.0 mmol). The reaction mixture was chilled in an ice:H,O bath, and then acetyl chloride (1.57 g, 20.0 mmol) was added in a dropwise manner. f i r stirring in the cold for 15 min, the mixture was concentrated under reduced pressure to a volume of -20 mL. This suspension was applied directly to a flash chromatography column and eluted with 5% triethylamine in EtOAc. The product-containing fractions were evaporated to give 3.76 g (75%) of chromatographically pure 9a. Analytically pure 9a was obtained by recrystallization from Et0Ac:pentane: mp 102-104 "C; IR (CHCl,): 3450 (NH), 1720 (amidine and amide C=O), 1600 (pyridine) cm-'; 'H NMR (CDCI,): 6 1.25 (dt, 6H, N(CH2CH3)2),2.20 ( 8 , 3H, COCH,, 3.48 (dq, 4H, N(CH,CH,),), 7.80 (broad 8 , lH, exchanges with D20, NHCO), 8.21 (d, J = 7 Hz, lH, H-5), 8.19 (8, lH, H-2) 8.20 (d, J = 7 Hz,lH, H-6); M S d e 251 (M+).Anal. (C12H17N303)C, H, N. 4-(Ethylamino)-3-pyridyl NJV-Diethylcarbamate (IOakCompound 9a (3.43g, 13.6 mmol) was dissolved in THF (75 mL), and then the reaction mixture was chilled in an ice:H,O bath. Boranemethyl sulfide was added (3.25 mL, 2.60 g, 34.2 mmol), and the reaction mixture was stirred at 0°C (30 m i d . At the end of this time, additional borane:methyl sulfide was added (3.0 mL), and stirring was continued for 3 h. The reaction mixture was then poured into ice:concentrated HCl, stirred for 30 min, basified with concentrated aqueous NH,, and then extracted with EtOAc. The organic phase was then dried (MgS04),evaporated, and purified by flash chromatography (EtOAc:CH,Cl,:CH,OH at 5.0:4.5:0.5) to give 0.86 g (27%) of chromatographically pure 10a. Recrystallization from hexane gave analytically pure 10a: mp 107-108 "C; IR (CHCl,): 3440 (NH), 1720 (carbamate C-O), 1605 (pyridine) cm-'; 'H NMR (CDCl,): 6 1.22 (m, 9H, N(CH,CH,), and NHCH,CH,), 3.20 (quintet, 3H, NHCH2CH3), 3.42 (dq, 4H, N(CH,CH,),), 4.20 (t, lH, exchanges with DzO, NHCHZCH,), 6.58 (d, J = 7 Hz, lH, H-S), 8.08 (8, lH, H-2), 8.13 (d, J = 7 Hz,lH, H-6); MS: d e 237 (M+). Anal. (ClzHlJ'I302) C, H, N. 4-(Propylamino)-3-pyridylNfl-Diethylcarbamate (lOb)-A solution of propionic acid (58.5 mL, 704 mmol) in benzene (100mL) was treated portionwise with NaBH, (9.6 g, 253 mmol). f i r the frothing Subsided, 2a (5.30 g, 25.0 mmol) was added, and the mixture was heated at 80 "C for 2 h. The reaction was then poured into dilute 5% NaOH. and the aqueous phase was extracted three times with EtOAc. The combined organic phase was washed with H20, dried (MgSO,), and purified by flash chromatography (8% triethylamine in toluene). Evaporation of the product-containing fractions and recrystallization from hexane gave 3.2 g (51%) of analytically pure 1 0 b mp 75-78 "C; IR (CHCl,): 3450 (NH), 1720 (carbamate C=O), 1610 (pyridine) cm-'; 'H NMR (CDC1,): 6 1.00 (t, J = 6Hz, 3H, NHCH2CH2CH3),1.22 (m,6H, N(CH,CH,),), 1.67 (sextet, J = 6 Hz,2H, NHCHzCH,CH3), 3.12 (q, J = 6 Hz,2H, NHCH,CH,CH,), 4.16 (t, lH, exchanges with DZO, NHCH&H&H3), 6.58 (d, J = 7 Hz,lH, H-5),8.06 (8, lH, H-2), 8.12 (d, J = 7 Hz,lH, H-6); MS: rnle 251 (M+h Anal. (Cl3HllN3O2)C,

384 I Journal of Pharmaceutical Sciences Vol. 81, No. 4, April 1992

H, N. 4-(Ethylamino)-3-pyridinol Hydrochloride (BbbCompound 9a (7.50 g, 30.0 mmol) was dissolved in dry THF (100 mL), and borane-methyl sulfide (40 mL of 2.0 M, 80.0 rnmol) was added. The reaction mixture was refluxed for 30 min and then CH30H (10 mL) was added. After the reaction with the residual diborane had subsided, the solvents were removed under reduced pressure, and the residue was redissolved in CH,OH (50 mL). This solution was made strongly acidic with ethereal HCl and then refluxed for 1h. At the end of this time, the solvents were again removed under reduced pressure, and anhydrous hydrazine (25 mL) was added. This mixture was warmed at 80 "C for 30 min and then concentrated under reduced pressure to give a residue that was purified by flash chromatography (5% triethylamine in EtOAc, then 20% CH,OH in CH2Cl,). After the product-containing fractions were evaporated, the free base was taken up in a minimum volume ofethanol, and the hydrochloride was formed with ethereal HCl. Recrystallization from ethano1:ether gave 2.31 g (44%) of analytically pure 5b: mp 168-169°C; 'H NMR (DMSO-4):6 1.18 (t, J = 6 Hz,3H, NHCH,CH3), 3.39 (quintet, J = 6 Hz,2H, NHCHZCH,), 6.83 (d, J = 7 Hz,lH, H-5), 7.72 (t, J = 6 Hz, lH, exchanges with DzO, NHCH2CH3),7.90 (8, lH, H-2), 8.00 (d, J = 7 Hz,lH, H-6), 12.5 (broad s,2H, exchanges with D,O, OH and HCl); MS: rnle 138 ( M 9 . Anal. (C7HlazO * HCl) C, H, N. 4-(Propylamino)-3-pyridinolHydrochloride (5c)-Compound 10b (7.3 g, 29.0 mmol) was heated in anhydrous hydrazine (20 mL) at 80 "C for 3 h. The reaction was then chilled in ice and poured into excess acetone. Concentration under reduced pressure and purification of the resulting oil via flash chromatography (10% CH30H in CH2Cl2) gave crude 5c free base as an oil. The hydrochloride was formed in CH30H with ethereal HCl to give 2.00 g of 5c (37%): mp 167-170 "C; 'H NMR (DMSO4): 6 0.91 (t, J = 6 Hz, 3H, CHJ, 1.58 (sextet, J = 6 Hz,2H, CH2CH3),3.30 (q, J = 6 Hz,2H, NHCH,), 6.88 (d, J = 7 Hz, l H , H-5),7.77 (t,J = 6 Hz,lH, exchanges with D,O, NH), 7.83 (a, IH, H-2), 7.98 (d, J = 7 Hz, lH, H-6), 12.5 (broad 8, 2H, exchanges with DzO, OH and HCU; MS: mle 152 (M+). Anal. (CBH12NzO * HCl) C, H, N. 4-(Butylamino)-3-pyridinolHydrochloride ( 5 d b I n a procedure similar to that for 9a above, chromatographically pure butyramide (9b) was synthesized in 86% yield from 2a (6.40 g, 31.0 mmol) and butyryl chloride (3.52 g, 33.0 mmol) in CHzClz (100 mL) containing triethylamine (3.33 g, 33.0 mmol). The compound 9b obtained in this way (0.027 mol) was treated as above for 5b with boran+methyl sulfide, and then the crude 1Oc was warmed in anhydrous hydrazine at 80 "C to give 2.42 g of analytically pure 5d (54% from 9b) &r chromatography and recrystallization of the hydrochloride from ethano1:ether: mp 176177°C; 'H NMR (DMSO-4): 6 0.91 (t, J = 6 Hz,3H, CH3), 1.37 (sextet, 2H, CHZCH,), 1.56 (quintet, 2H, NHCH,CH,), 3.31 (9, J = 6 Hz,2H, NHCH,), 6.83 (d, J = 7 Hz,1H. H-5), 7.72 (t, J = 6 Hz,lH, exchanges with DzO,NH), 7.88 ( 8 , lH, H-2), 7.98 (d, J = 7 Hz, l H , H-61, 12.5 (broad 8, 2H, exchanges with DzO, OH and HCl); MS: rnle 166 (M+). Anal. (C$I14NZO * HCl) C, H, N. NJV-Diethyl-N'-(3-hydroxy-4-pyridinyl)urea( 8 b A solution of 2a (5.16 g, 24.7 mmol) in THF (100 mL) was treated with potassium t-butoxide (3.05 g, 27.1 mmol) and stirred at mom temperature for 1.5 h. At the end of this time, the resulting suspension was solubilized with MeOH and made acidic with an ethereal HCI solution. This solution was then neutralized with methanolic NH,, and the solution was paseed over a column of alumina and eluted with Et0Ac:MeOH. The solid obtained by evaporation of the product-containing fractions was purified via flash chromatography sequential elution first with CHzClz and then with 15% MeOH in CHzClzto give a slightly yellow solid. T w o recrystallizations from CH,CN:CH,OH gave 2.22 g (43%) of 8: mp 158-159 "C (dec); IR (KBr):1680 (urea C=O) cm-'; 'H NMR (DMSO4): 6 1.16 (t, 6H, CHzCH3), 3.38 (9, 4H, CH2CH,), 7.71 (d, J = 6 Hz, l H , H-5), 7.85 (broad 8 , lH, exchanges with D,O, OH), 7.90 (d, J = 6 Hz, H-6), 8.00 (a, lH, H-2), 10.6 (broad 8, lH, NH); M S mle 209 (M+). Anal. (C1Jil6N,O2) C, H, N. Acetylcholinesteraee Inhibition-Acetylcholinesterase activity was based on the method of Ellman et a1.16 Rat striatal homogenates (1:20, wlv, in 0.05 M NaH,PO, and 0.05 M Na2HP0,, adjusted to pH 7.2) were incubated a t 37 "C for 10 min with drug in 5,5'dithiobis(2nitrobenzoic acid), and a separate solution of acetylthiocholine was prepared in an identical buffer. Aliquots of drug solution and acetylthiocholine solution were added to cuvettes to yield final concentrations of 0.25 mM 5,5'-dithiobis(2-nitrobenzoicacid) and 5

mM acetylthiocholine. Absorbance changes were monitored with a Beckman DU-50 spectrophotometer, and slope values were determined with the Kindata program. Values for percent inhibition were calculated relative to a control sample, and the concentrations that elicited a 50% reduction in the maximal (control) enzyme response (ICm) were calculated by log probit analysis. Acetylcholine Releas-The method was a modification of the method of James and Cubeddu.17 Male Wistar rats were decapitated, striatal tissue was removed over ice, and coronal slices (0.4 mm thick) were prepared. After a 30-min preincubation period, slices were incubated with 0.1 pM [3H]choline (80 Ci/mmol) for 60 min at 35 "C with fresh buffer containing NaCl(118 mM), KC1 (4.7 mM), MgSO, (1.2 mM), KH,PO, (2.2 mM), NaHC03 (24.91, CaC1, (1.3 mM), and dextrose (11.1mM) and saturated with 95:5 02:C02 at pH 7.4. The slices were placed in glass superfusion chambers containing platinum electrodes and perfused a t 0.7 d m i n in buffer (see above) containing 20 pM hemicholinium-3 and 2 pM sulpiride, to maintain stoichiometry of releaee and block dopaminergic inhibition. Stimulation periods (2 Hz,2 min, 2-mi3 duration) occurred at 1-h intervals. Drugs were introduced between stimulations. The amount of tritium released per 7-min sample was expressed as a fraction of the total tritium content of tissue at the onset of the respective collection period (percent fractional release). The overflow of tritium induced by electrical stimulation was calculated as the total increase in radioactivity above the resting outflow obtained in the sample immediately preceding the onset of stimulation (Sl, in the absence of drug and 52, in the presence of drug). Drug effects were determined by first calculating the S2:Sl ratio for control and drug-treated s l i m and then normalized by expressing the drug-treated S2:Sl value as a percentage increase over the control S2:Sl value.

References and Notes 1. Shutske, G. M.; Pierrat, F. A.; Cornfeldt, M. L.; Szewczak, M. R.; Huger, F. P.; Bores, G. M.; Haroutunian, V.; Davis, K. L. J . Med. Chem. 1989,31, 1278. 2. (a)Potter, P. E.; Nitta, S.; Chaudhry, I.; Lalezari, I.; Goldiner, P.; Foldes, F. F. Neumhem. Znt. 1989 14, 433. (b) Foldes, F. F.; Ludvig, N.; Nagashima, H.;Vizi, E. 6.Neumhem.Res.1988,13, 761. 3. Miah, M. A. J.; Snieckus, V. J . Org. Chem. 1985,50,5436.

4. Reed, J. N.; Snieckus, V. Tetmhedron Lett. 1983,24, 3795. 5. Caution! Although tosyl azide has been reporfed to be stable (ref la), a report of its shock sensitivity (ref 19)indicates that extreme caution should be exercised in its use (seeExperimental Section). 6. Beak, P.; Snieckus, V. Acc. Chem. Res. 1982,15,306. 7. Boyland, E.; Sims, P. J . Chem. Soc. 1958,4198. 8. Ca-mon,.J. G. In Bur er's Medicinal Chemistry, part III,4th ed.; Wolff, M. F., Ed.; J. &ley & Sons: New York, 1981; pp 354-358. 9. Amal, F.; Cote', L. J.; Ginsburg, S.; Lawrence, G. D:{Naini, A.; Sano, M. Neurochem. Res. 1990,15, 587 (norpyridostigmine is reported to inhibit acetylcholinesterase by 50% at 1.5 x M in vitro). 10. Gautier, J.-A.; Miocque, M.; Farnoux, C. C. In The Chemis Amidines andzmidates; Patai, S., Ed.; J. Wiley & Sons:! : % a L 1975; pp 310-311. 11. Steinber G. M.; Mednick, M. L.; Maddox, J.; Rice, R.; Cramer, J. J . Me$: Chem. 1975,18, 1056. 12. Stimulated release consists solely of [3H]acetylcholine, whereas basal release is 80-90% [3H]choline (ref 20 and unpublished results from these laboratories). 13. Berger, S. G.; Waser, P. G.; Sin-Ren, A. C. Neuropharmacology 1989,28, 191. 14. Doering, W. v. E.; De Puy, C. H. J . Am. Chem. Soc. 1953, 75, 5955. 15. Hoffman, H.; Hammann, I.; Unterstenhtifer, G. U. S. Patent 3 949 022,1976. 16. Ellman, G.L.; Courtney, K. D.; Andres, V., Jr.; Featherstone, R. M. Biochem. Pharmacol. 1961, 7,88. 17. James, M. K.; Cubeddu, L. X. J . Pharmacol.Exp. Ther. 1987,240, 204. 18. Regitz, M. Synthesis 1972,351. 19. Guntrum, M. In Reagents for Organic Synthesis, Vol. 2; Fieser, M.; Fieser, L. F., Eds.; Wiley-Interscience: New York, 1969; p 468. 20. (a) Meyer, E. M.; Otero, D. B. J . Neurosci. 1985, 5, 1202; (b) Richardson, I. W.; Szerb, J. C. Br. J . Phurmacol. 1974,52, 499.

Acknowledgments We are indebted to Dana Hallberg for assistance in obtaining NMR spectra and to Margaret Hill for assistance in searching the literature.

Journal of Pharmaceutical Sciences I 385 Vol. 81, No. 4, April 1992