Pyridine and Dihydropyridine Compounds Soc., 125, 2175 (1924). (14) R. F. Shonk, D. D. Miller, and D. R. Feller, Biochem. Pharmacol.. 20.3403 (1971). (15) D. R. Feller, R. F. Shonk, and D. D. Miller, J . Pharm. Pharmacol., 22,869 (1970). (16) G. Fassina, C. E. Toth, and R. Santi, Prog. Biochem. Pharma-
Journal ofMedicina1 Chemistry, 1975, Vol. 18, No, 5
457
col., 3,377 (1967). (17) R. T. Brittain, D. Jack, and A. C. Ritchie, Adu. Drug Res., 5 , 197 (1970). (18) D. Arunlakashana and H. Schild, Brit. J . Pharmacol., 14, 48 (1959). (19) T. Nash, Biochem. J., 55,416 (1953).
Porphyria-Inducing Activity of a Series of Pyridine and Dihydropyridine Compounds. Investigation in a Cell Culture System M. W. Roomi Department of Pharmacology, Queen’s University, Kingston, Ontario, Canada. Received July 22,1974 A series of analogs of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) was prepared and tested for porphyria-inducing activity in chick embryo liver cells. One of the analogs tested, viz. 3,5-di-tert-butoxycarbonyl1,4-dihydro-2,6-dimethylpyridine, was found to be highly active despite the %bsence of a 4-alkyl substituent. It was concluded that tert- butoxycarbanyl groups are resistant to enzymic hydrolysis and that compounds containing such groups are resistant to inactivation by chick embryo liver cells. Several analogs of DDC were found with considerably higher activity. These should be useful in inducing high levels of 6-aminolevulinic acid synthetase prior to undertaking the isolation of the enzyme.
benzyloxycarbonyl-l,4-dihydro-2,6-dimethyl (If) a n d t h e T h e overproduction of porphyrins in liver cells induced corresponding pyridine 2f and their activity compared with by a variety of drugs results from a n enhanced synthesis of t h a t of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylt h e first enzyme in t h e porphyrin biosynthetic pathway, pyridine (DDC) a n d 3,5-diethoxycarbonyl-2,4,6-trimethylviz. 6-aminolevulinic acid (ALA) ~ y n t h e t a s e . l - As ~ a result pyridine (OX-DDC, 2a). For comparative purposes t h e anof recent study it was concluded t h a t for a chemical t o inalogs containing 4-methyl substituents were synthesized duce porphyria it must remain in the liver for a period of a t ( l g , li, 2g, 29. least several hours in order to induce a n d maintain high For further progress in studies of the mechanism of ALA levels of ALA synthetase.j Consequently it is clear t h a t a synthetase induction by drugs, it would be desirable to isoporphyria-inducing drug must possess chemical features late this enzyme in pure form. This would enable a n antit h a t prevent it from being rapidly metabolized a n d inactibody to be prepared to this enzyme a n d would facilitate divated by the liver. Previous studies on t h e relationship berect measurement of ALA synthetase. Before attempting tween chemical structure and porphyria-inducing activity t h e isolation of this enzyme it would be desirable to induce of a series of compounds related to 3,5-diethoxycarbonylhigh levels of this enzyme. For this reason a study of DDC 1,4-dihydro-2,4,6-trimethylpyridine (DDC, l a ) a n d the reanalogs was undertaken to find a drug of high activity. T h e lated pyridine 2a led t o the suggestion t h a t the underlying following drugs were synthesized a n d their porphyrin-incritical feature for activity was a n ester group which is sterducing activity measured: 3,5-dimethoxycarbonyl-1,4-dihyically hindered from h y d r o l y ~ i s . ~Recently -~ Parker and dro-2,4,6-trimethylpyridine( l e ) a n d t h e corresponding Weinstocklo have studied the metabolism of 1,4-dihydropyridine 2e. 2,6-dimethyl-4-(2-trifluoromethylphenyl)-3,5-pyridinediIn previous studies t h e porphyria-inducing activity of carboxylic acid diethyl ester in t h e dog. Principle urinary DDC analogs was assessed by a qualitative procedure metabolites were isolated a n d identified and showed t h a t which involved the observation of the fluorescence intensit h e above compound is aromatized a n d hydrolyzed during t y of chick embryo liver cells 20 hr after incubation with a biotransformation. T h e two 0 - methyl substituents adjacent t o each ethoxycarbonyl group of 3,5-diethoxycarbonyl-1,4- drug. In t h e present study t h e amount of porphyrin in chick embryo liver cells a n d growth medium was measured dihydro-2,4,6-trimethylpyridinea n d related compounds quantitatively. were thought t o protect t h e ethoxycarbonyl group from hydrolysis to yield the inactive free diacid. This explained the inactivity of 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimeth-Experimental Section ylpyridine ( l b ) . If this interpretation was correct, i t apAll melting points are uncorrected. The structures of all compeared possible t h a t active analogs of 3,5-diethoxycarbonpounds are supported by their ir, uv, and NMR spectra. Spectra were recorded on Perkin-Elmer Model 173E, Unicam SP800 and a yl-1,4-dihydro-2,4,6-trimethylpyridine might be obtained Varian A-60 spectrometer (Me&). All compounds were analyzed in which t h e ester group could be protected from enzymic for C, H, and N and the results were within f0.4% of the theoretihydrolysis by other means t h a n two o-alkyl substituents. I t cal value. Benzyl acetoacetate was obtained as a gift from Dr. S. F. was thought t h a t replacement of the ethyl ester substituent MacDonald, N.R.C., Ottawa. tert-Butyl acetoacetate and methyl by more bulky groups such as tert-butyl ester or benzyl acetoacetate were purchased from Aldrich Chemical Co. ester groups might yield active analogs in compounds in Culture of Chick Embryo Cells on Petri Dishes. The procedure of Granick4 was used with the following modifications. The which the 4-methyl substituent of 3,5-diethoxycarbonylenzyme solution for preparing the cell suspension, from four chick 1,4-dihydro-2,4,6-trimethylpyridine was replaced by hyembryo livers, consisted of 5 ml of 2.5% trypsin in saline (Microbidrogen. T o test this idea t h e following four analogs of 3,sological Associates) and 5 ml of magnesium and calcium free diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine were Earle’s solution. The suspension was centrifuged at low speed and synthesized: 3,s-di-tert- butoxycarbonyl- 1,4-dihydro-2,6the supernatant discarded. The sediment was resuspended in culdimethyl ( l h ) a n d the corresponding pyridine 2h, 3,5-diture medium (Eagle basal medium, Microbiological Associates,
458 Journal of Medicinal Chemistry, 1975, Vol. 18, No. 5
Hoomi
T a b l e I. Structure and Physical Properties of Porphyria-Inducing Dihydropyridines and Pyridines
la-i
No.
la lb IC
Id
le If 1g lh li 2a 2b 2c 2d 2e 2f 2g 2h 2i
R
Ri
Mp o r bp (mni), 'C 130-131" 17 8-1 81 1O8-llOc 22 5-22Id 155-1 5 7" 119-121 125-127 142-1 44 166-1 68 138 (0.4)' 71-72' 130 (0.45)h 102-1 04 7 8-8 0" 8 8-9 0 50-52 108-1 10 53-55
C r p s t n solvent MeOH E tOH E tOH MeOH MeOH E tOH E tOH E tOH E tOH
2a-i
Yield,
-
78 80 70
68 72 60 63 66 65 75
E tOH
62 65
E ther-ripentane E ther-n-pentane E tOH E ther-n-pentane MeOH E ther-n-pentane
73 78 50
53 35 40
aLit. mp 131": A . Hantzsch, Justus Liebigs Ann. Chem., 215, 1 (1882). bLit.13 mp 175-180". cLit. mp 110": F. Engelmann, Justus Liebigs Ann. Chem., 231, 37 (1885). dLit. mp 219-220": T. B. H. McMurry and M . T. Richardson, J . Chem. SOC.C, 1804 (1967). e T . J . vanBergen and R. K. Kellogg, J . A m . Chem. .Soc., 94,8451 (1972).fLit.6 138" (0.4 mm). gLit.14 130" (0.45 mm). ILit. mp 100-102": T. J. van Bergen and R . M. Kellogg. J . Org. Chem., 36, 978 (1971). Inc.). Bovine serum (Pentax Inc., Winley-Morris Co. Ltd.) was dines were oxidized to the corresponding pyridines with chloranil according to the procedure of Braude.I4 The properties of the pyriused throughout our experiments. dines are reported in Table I. Measurement of t h e Porphyria-Inducing Effect of Drugs. The culture medium was removed from each petri dish after 24 hr Results and Discussion of incubation and replaced with 4 ml of fresh medium. Porphyriainducing drugs were dissolved in 95% redistilled ethanol (5 11) and T h e first analogs investigated for porphyria-inducing acadded to the liver cells by means of disposable microliter pipets. tivity were 3,5-di-tert-butoxycarbonyl-1,4-dihydro-2,4,6After the addition of porphyria-inducing drugs the petri dishes trimethylpyridine ( l i ) , 3,s-di-tert- butoxycarbonyl- 1,4-diwere returned to the incubator for approximately 24 hr. The porhydro-2,6-dimethylpyridine ( l h ) , 3,5-diethoxycarbonylphyrin content of cells and media was determined. After determin1,4-dihydro-2,4,6-trimethylpyridine (DDC, l a ) , and 3,sing the protein contents of cells" the results were expressed as Wg of porphyrin/mg of protein. Each drug was tested in triplicate a t a diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyriparticular concentration. dine (IC).T h e results which are shown i n Figure 1 reveal Preparation of Dihydropyridines. Dihydropyridines were that the tert- butoxycarbonyl compounds are considerably synthesized according t o the procedure of Loev and Snader" by more potent than DDC ( l a ) a n d its closely related 4-ethyl condensation of a P-oxo ester (0.1 mol) with an aldehyde (0.05 mol) analog IC. T h e high activity of the dihydropyridine ( l h ) and concentrated ammonium hydroxide (14.3 N , 10 ml) in boiling which lacks a I-methyl s u b s t i t u e n t was of particular interethanol (20 ml) for 2 hr. In some cases when only small quantities est i n view of t h e fact that all previous aromatic comof the d-oxo ester were available the reactions were carried out on a smaller scale. Upon cooling, the solution was poured into cold pounds which we have shown to be active contained ethyl water (1.50 ml) whereupon the dihydropyridine separated from soether groups protected by two o-alkyl substituents. It lution. The dihydropyridine was extracted with ether. The ether would t h u s appear that t h e t e r t - butoxycarbonyl groups a r e solution washed successively with 10% sodium hydroxide, water, resistant to enzymic hydrolysis a n d that compounds con5% hydrochloric acid, and water. The ether solution was dried (sotaining these groups resist inactivation by liver cells. In our dium sulfate) and evaporated and the residue crystallized twice. In the case of 3,~-dimethoxycarbonyl-1,4-dihydro-2,4-dimethylpyri- next series of experiments (Figure 2) the activity of t h e cordine, the crude product separated from the ethanol solution after responding pyridines (2i, 2h, 2a, a n d 2 c ) was investigated. cooling and was removed by filtration and crystallized. The propAll compounds showed marked activity and i t was of conerties of the dihydropyridines are reported in Table I. siderable interest that t h e tert- butoxycarbonyl derivative Conversion of Dihydropyridine t o Pyridines. 3,5-Dimethoxy2 h which lacked a 4-methyl substituent showed marked accarbonyl-1,4-dihydro-2,6-dimethylpyridineand 3,5-dimethoxytivity. 1,4-dihydro-2,4,6-trimethylpyridine were oxidized to the corresponding pyridines with a mixture of glacial acetic acid and sodiReplacement of t h e ethoxycarbonyl substituents of DDC um nitrite by the method of Loev and Snader.12 3,5-Dibenzyloxy(la) and t h e corresponding pyridine derivative 2 e with carbonyl-1,4-dihydro-2,6-dimethylpyridine and 3,5-dibenzyloxybenzyloxycarbonyl substituents (1 and 2g) led t o decreased carbonyl-1,4-dihydro-2,4,6-trimethylpyridine were oxidized to the activity (Figure 3). W h e n t h e 4-methyl substituents of bencorresponding pyridine with a mixture of nitric acid, sulfuric acid, zylcarbonyl compounds (1 a n d 2g) were replaced by hydroand water by the procedure of Singer and M~E1vain.l~ gen, t h e r e was virtually n o retention of activity i n these a n 3,5-Di-tert-butoxycarbonyl-1,4-dihydro-2,6-dimethylpyridine and 3,j-di-tert-butoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyri- alogs (1 a n d 2f). I t t h u s appears likely that benzylcarbonyl
Journal of Medicinal Chemistry,1975, Vol. 18, No. 5 459
Pyridine and DihydropyridineCompounds
1
1.4 h
c .0)
//
t 0
L
/
n
;1.0
/ /
I
/
\
/
0
i
/
Y
/
C
/
.-0
/
c
0
/
3
/
E 0
/
0.5
/
0
/
/
c
0.1
Log
Dose
(pg/ml)
butoxycarbonylFigure 1. Dose-response curve for 3,5-di-tert1,4-dihydro-2,6-dimethylpyridine(lh, A-A); 3,5-di-tert-butoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine(li, 0-0); 3,5-diethoxycarbonyl-l,4-dihydro-2,4,6-trimethylpyridine(la, 0-0); and 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine (IC, 0-0).
II1.0.
a
E“
2 2.0
\
n
0
%
E
Y
\ 0,
3 1.5/
Y
c
K
/
.-0
/
c 0
/
3 1.0-
3
E a
0 0
0
0
0
/ /
/ /
“ I
0
1
/
L
0.3
0.7 Log
1.0 Dose
I.4 (rg/.mIl
2.0
Figure 2. Dose-response curves for 3,5-di-tertbutoxycarbonyl2,6-dimethylpyridine (2h, A-A); 3,5-di-tert-butoxycarbonyl2,4,6-trimethylpyridine (2i,0-0);3,5-diethoxycarbonyl-2,4,6-trimethylpyridine (2a, 0-0); and 3,5-diethoxycarbonyl-2,6-dimethyl-4-ethylpyridine (2c,0-0).
1.0 Log
Dose
2.0
(Ag ml)
Figure 4. Dose-response curves for 3,5-dimethoxycarbonyl-l,4dihydro-2,6-dimethylpyridine(Id, 0 - 0 ) ; 3,5-dimethoxycarbonyl1,4-dihydro-2,4,6-trimethylpyridine (le, A-A); 3,5-dimethoxycarbonyl-2,6-dimethylpyridine(2d, 0-- -0); and 3,5-dimethoxycarbonyl-2,4,6-trimethylpyridine(2e,0-- -0).
160
Journal ojMedicinal Chemistry, 1975, Vol. 18, N o . 5
groups of compounds (1 a n d 2f) are hydrolyzed by liver enzymes to inactive diacids. T h e difference in activity of pyridines and dihydropyridines containing benzyl a n d tertbutyl ester groups is probably d u e t o their different hydrolysis mechanism and rate.15 Replacement of t h e ethoxycarbonyl substituents of D D C ( l a ) and t h e corresponding pyridine derivative 2a with t h e methoxycarbonyl substituent ( 1 and 2e) resulted in decreased activity (Figure 4). When t h e 4-methyl substituent of methoxycarbonyl compounds was replaced by hydrogen ( 1 a n d 2d) t h e compounds showed no activity (Figure 4). This inactivity was similar t o t h a t previously found when the 4-methyl substituents of DDC ( l a ) and OX-DDC (2a) were replaced by hydrogen.3 It was concluded t h a t t h e tert- butoxycarbonyl compounds would induce higher levels of ALA synthetase in chick embryo t h a n D D C ( l a ) . For this reason these compounds should be valuable in inducing higher levels of ALA synthetase in chick embryo liver prior t o attempting t o isolate this enzyme. A c k n o w l e d g m e n t . T h i s investigation was supported by grant from t h e Medical Research Council, Canada, to Dr. (;, S. hlarks. T h e author wishes to acknowledge t h e assisB
Nouinson et al.
tance of Drs. D. L. J . Tyrrell a n d G. S. Marks with t h e cell culture technique used in this work. References (1)
S.Granick and G. Urata, J . Bioi.Chem., 238,821 11963).
(2) (3) (4) (5)
S. Granick, J . Biol. Chem., 238, PC 2247 (1963). S. Granick, Ann. h'.Y. Acad. Sei., 123, 188 (1965). S. Granick, J . Bioi.Chem., 241, 1359 (1966). W. J. Racz and G. S. Marks, Biochern. Pharmacol., 21, 143
(1972). (61 G. S. Marks, E. G. Hunter, U. K. Terner, and D. Schneck. Biochem. Pharmacol., 14,1077 (1965). (71 G. H. Hirsch, J. D. Gillis, and G. S. Marks, Riochpm. Pharmacol., 15, 1006 (1966). ( 8 ) G. H. Hirsch, G. L. Bubbar, and G. S. Marks. Hirichem. Pharmacoi., 16, 1455 (1967). (9) D. W. Schneck, W. J. Racz, G. H. Hirsch, G. I,. Bubbar, and G . S. Marks, Biochem. Pharmacol., 17,1385 (1968). (10) S. E. Parker and J . Weinstock,J. Med. Chem.. 16,34 (1973). i l l ) 0. H . Lowry, N.,J. Rosebrough, A. L. Farr. and R. J. Randall, .I Bioi. Chem., 193, 265 (1951). (12) B. Loev and K. M.Snader, J . Org. ('hem., 30,1914 (1965). (13) A. Singer and S. M. McElvain, Org. S y n t h . , 14, 30 (1934). ( 1 4 ) E. Braude, J. Hannah, and R. Linstead. J. Chem. S o c . , 3249 (1960). (1,5) J . Hine, "Physical Organic Chemistry". 2nd ed, McGraw-Hill, New York, N.Y., 1961. p 279.
Adenosine Cyclic 3',5'-Monophosphate Phosphodiesterase Inhibitors. 2.3-Substituted 5,7-Dialkylpyrazolo[ 1,5-a]pyrirnidines Thomas Novinson,* J o n P. Miller, Mieka Scholten, Roland K. Robins, Lionel N. Simon, Darrell E. O'Brien, a n d Rich B Meyer, J r . I ( \ Phatniai ( u t i t a l \ Inc
Yucleic Acid Research Institute Iruine, California 92664 RecezLed August 5 , 1974
A number of %bromo-, %nitro-, and 3-ethoxycarbonyl-5,7-dialkylpyrazolo[l,~-a]pyrimidines were synthesized and screened as in vitro CAMP phosphodiesterase inhibitors. The condensation of 3-aminopyrazole with symmetrical 3diketones (acetylacetone, heptane-3,5-dione, etc.) afforded symmetrical dialkylpyrazolo[ 1,5-a]pyrimidines ( 5 ) . The reaction ot' 3-aminopyrazole with unsymmetrical P-diketones (hexane-2,4-dione, heptane-3,5-dione, etc.) gave a mixture of 5-methyl-i-alkylpyrazolo[l,5-a]pyrimidine ( 3 ) and 5-alkyl-7-methylpyrazolo[1,5-a]pyrimidines 14). The technique for the separation of 3 from 4 is described. The inhibition constants, a (the ratio of the molar Is0 of theophylline to the molar 150 of the test compounds), were subjected to a Hansch correlation analysis. The results indicated that PDE isolated from beef heart tissue was most sensitive to changes in the length of the alkyl group in the 5 posit i o n of' the pyrazolo[l,5-a]pyrimidinering, whereas the PDE isolated from rabbit lung tissue was more sensitive to changes in the length of the 7-alkyl group. Experimentally and theoretically, the n-propyl group was found to approximate the ideal size for the alkyl group in both the 5 and 7 positions; 5,7-di-n-propyl-3-ethoxycarbonylpyrazolo [135alpyrimidine (5e) was the most potent inhibitor of both lung and heart PDE.
I t has been reported t h a t t h e pharmacological effects of theophylline (1) might be due t o the inhibition of 3',5'AMP phosphodiesterase ( P D E ) by this compound.l Rose and coworkers2 demonstrated t h a t certain dialkyl derivatives of t h e triazolo[2&c]pyrirnidines (6) [which bear some structural resemblance t o theophylline (111 inhibited P D E from lung tissue t o a greater extent t h a n 1. More importantly. derivatives of 6 effectively protected guinea pigs from histamine-induced bronchospasms. Bronchial constriction was known t o be accompanied by changes in t h e intracellular concentrations of 3',5'-AMP. Others have ex-
I CH
1
CH
R
2
3
plored t h e inhibition of P D E by certain pyrazolo[3,4-b]pyridines which were also found t o be effective as in vivo a n tidiabetic agents.?-j
s
s
CH 4 4
R' 5
k 6
We reported on t h e in vitro P D E inhibition of t h e dimethyl derivatives of the structurally similar pyrazolo[l.5alpyrimidine (2) in a recent publication.6 I t was found t h a t one of t h e better P D E inhibitors, 3-bromo-5,7-dimethylpyrazolo[l,5-~]pyrimidine(2d, ICN 3009), produced a n i m mediate a n d moderately prolonged increase in t h e cardiac o u t p u t of anaesthetized dogs.: T h e observed lack of
