HETEROCYCLIC TETRAZOLES AS LIPOLYSIS ISHIBITORS
March 1967
149
Heterocyclic Tetrazoles, a New Class of Lipolysis Iiihibitorsl GERALD F. HOLLASD8 3 D JOSEPH s.PEREIR.1 3f edical Research Laboratories, Chas. Pfirer dl. Co., Inc., Groton, Connect icid
96.340
Received Sepfember 94)1966
A series of pyridyl-substituted 5-(3-pyridyl)tetrazoles and other 5-(heterocyclic)tetrazoles were prepared and evaluated for lipolysis inhibitory activity. The reaction between het,erocyclic nitriles and sodium azide in dimethylformamide provided a convenien nthetic procedure for most of these compounds. They were xreened for their ability t o inhibit the norepinep ie-induced release of free fatty acids ( F F h ) from isolated rat adipose tissue and for their ability to depress the fasting plasma F F A levels in the dog. The most active lipolysis inhibitor was 5-(3-pyridyl)tetrazole. Although 5-(3-pyridyl)tetrazole was a much weaker in t 4 r o lipulysis inhibitor than nicotinic acid, it depressed plasma F F A levels in the dog for a longer period of time. The relationship be M-een inhibiting lipid mobilization from adipose tissue and decreasing plasma lipid levels was developed.
During t,he past 5 years a vide variety of compounds of varying st'ructures have been examined in these laboratories for their ability to inhibit lipid mobilization (lipolysis inhibition) from adipose t'issue. Lipid mobilization involves the net release of free fat'ty acids (FPA) from the triglyceride-rich adipose tissue stores. It has been firmly established that the FFA are a primary source, uia hepatic synthesis, of plasma lipoproCHZOCOR
I
CHOCOR
1
CHZOCOR TG
CHZOH
+ RCOOH FFX
i + CHOH
-4 lipolysis inhibitor having the lipid same intrinsic activity in isolated adipose tissue as nicotinic acid, but of greater metabolic -tability. would be expected to depress plasma FFA for L: longer period of time. Such a prolonged depression of pla-ma FF-\ would be expected to reduce total pla-ma lipids more effectively than does nicotinic acid. The similarity between the acidic character of the carboxyl group and the tetrazole function ( i f 5-substituted tetrazoles (1) is well li11ow-n.~~For example,
Rl=r
I
CHZOH G
HNINGN I
teiri triglyceride^.^,^ Thus, inhibiting t'riglyceride the ionization constant of 5-phenyltetrnzole (pKa = hydrolysis in adipose t'issue would reduce t,he supply of 4.5) is slightly greater than that, of the corresponding plasma FFA to the liver, t,hereby reducing hepatic carboxylic acid, benzoic acid (pKa = 5.1).!4 *4lso of triglyceride synthesis. The reduced availability of import'ance, the tet'razole function appears to be mehepatic t,riglycerides would limit the completioii of the t,abolically stable. l5 A series of pyridyl- and other hetermajor cholest,erol transporting unit, the lipoprotein ocyclic tetrazoles were prepared in the hope 111' finding a complex. The decrease in the concent'rat'ion of this lipolysis inhibit'or having a longer duraticm of IT-\transport unit, in turn, restricts the removal of cholesdepressing activity than t'hat of nicotinic aciil. t,erol from the liver. -4 normally operative feedback Synthesis.-The general reaction between pyritlyl mechanism would t'hen depress hepatic cholesterol and ot'her heterocyclic nitriles (2) and .odium azide (3) syn t h e s i ~ . ~ ! ~ served as a convenient procedure for the syiit.hesis of The pyridine and related heterocyclic acids repretmhepyridyl and heterocyclic 5-t.etrnzoles (1). Origsent one structural type which we have extensively ininally a combinatmionof acetic acid anti 1-butanol \\-as vestigated. Kicotinic acid has been shown to depress used as t'he solvent for this Hon-ever! mi the level of plasma FFA after acut'e administration to improved procedure using dimethylforiiiamicle, in place 1iia11.~ In addition, it inhibits t,he norepiiiephrine-inof both acetic acid and 1-butanol, gave. oti the whole, duced release of FFA from isolated adipose t ' i s ~ u e . ~ , ~ It has been suggested that the hypocholesteremic effect DAIF IZCS + Sa'?; --+ 1 of nicot'inic acid follows from its lipolysis inhibitory ac2 3 tivity.' The known rapid metabolism of nicotiiiic acid10 could account for the observed short duration of higher yields in shorter reaction IILone case, plasma FFA depression which, in turn, could account in the preparation of 5-(3-pyridylmethyl) tetrnzole, the for t8helarge doses of nicotinic acid required for plasma react,ion between 3-pyridylacetonitrile :?lid sodium azide was only successfully carried out in the ncetic acid and (1) G. F. Holland, F. A . Hochsrein, E. R . Pinson, Jr., and J. N . Pereira, presented in part a t the session on Recent Developments in Medicinal 1-butanol solvent combination. Chemistry. 10th National Medicinal Chemistry Symposium, Bloomington, Ind., June 28,1966. (2) A comprehensive r e r i e x of adipose tissue can be found in "Handhook of Physiology, Section 5 : Adipose Tissue," A . E. Renold and G. F. Cahill, Jr., Ed., American Physiological Society, \Vashington, D . C., 1965. (3) R. J. H a d , Metab. Clin. Ezptl., 10, 1031 (1961). (4) R . J. H a w 1 and A. Goldfien, J . Lipid Res., 2, 389 (1961). (5) C. B. Tal-lor and R. G . Gould, Circulation, 2, 467 (1950). (6) E. P. N a d h a r a Bhattathiry and A f . D. Siperstein, J . Clin. I n c e s t . , 42, 1613 (1963). Carlson and L. Ore, Acta M e d . Scand., 172, 641 (1962). ( 8 ) L. A. Carlson, ihid.. 173, 719 (1963). (9) R. P. Eaton, Proc. Soc. Ezptl. B i d . .Wed., 114, 599 (1963). (10) E . Ginoulhiac, L. T . Tenconi, and F. RI. Chiancone, S a t u r e , 193, 948 (1962).
(11) R . Altschul, -1,Hoffer, and J. 1). Stephen, A r c ' . B 64, ,658 (1955).
(12) W-.B. Parsons, Jr., R . \\-. P. .ichor, K , G. Rerge. B. F. lIcIieneie, and N. W Barker, Proc. S t a f Meetings M a y o Clinic, 31, 377 (1956). (13) R. M. Herbst in "Essays in Biochemistry," 9. Graff, Ed.. .John \Yiley and Sons, Inc., New York, N. Y., 1956, p p 141-155. (14) R . M. Herbst and IGOY0)of plasma FFA. The structural requirements for in vivo lipolysis inhibitory activity among the pyridyl- and other heterocyclic tetrazoles were found to be quite restrictive. Within the pyridyl-substituted 5-(3-pyridyl)tetrazole series (Table I), 5-(3-pyridyl)tetrazole (R = H) was the most active. It gave a maximal reduction of plasma F1:h in the dog, like nicotinic acid, with a dose of 10 mg/kg. A number of otber members of this series (R = 5-CHR, 5-F, 5-NH2, G-XHCOCH,, 5-COOH, and 2-SCH8) were moderately active and produced less than a maximal reduction (30-607,) of plasma FFd. The 2-pyrazinyl, 3-pyridylmethyl, and 3-pyridyl Noxide members of the 5-(heterocyclic) tetrazole series (Table 11) had modest FFh depressing activity. They gave less than a maximal reduction of plasma FF-1 (30-607,). S o member of this series produced a
1.-i2
2000(
PLASMA FFA , uEq/L
niaxiniul reduction o f plxhma E'Y.1. Similar to the finding.: obtained in the in ritro testing procedure, loration of the widic. tetrazole function a t either the 2 or 4 pu~ition on the pyridine nurleus, namely, .5-(2-pyr-
;\larch 1967
HETEROCYCLIC TETRAZOLES AS L I P O L Y ~IKHIBITORS IS
The improved duration of action of 5-(3-pyridyl)tetrazole over nicotinic acid, in spite of a decreased activity in the isoIated adipose tissue system, is probably attributable to its greater metabolic stability.se The extended duration of FFA-depressing activity of 5-(3-pyridyl)tetrazole in the dog prompted a clinical evaluation of this compound. Preliminary results show that it lowers plasma FFh and cholesterol on repeated administration to man.*' A more detailed analysi- of the pharmacological and clinical profiles of t5-(3-pyridyl)tetrazole will be published elsewhere.
Experimental Seetion 2s 5-(3-Pyridyl)tetrazole.-A stirred mixture of 1500 ml of dry (molecular sieve) DNF, 234 g (2.24 moles) of 3-cyanopyridine, 195 g (3 moles) of NaN3, 162 g (3 moles) of SH4C1, and 3 g o LiCl was heated to 12.5' for 12 hr. The insolubles, aft,er cooling to rooni temperature, were removed by suction filtration, and the DAIF was distilled in z'ucuo. The crude product remaining after the aolvent %-a$removed was dissolved in 4 1. of water and the pH was adjusted to 4 with HC1. There was obtained 166 g of product, mp 234" dec. Adjiisting the aqueous filtrate to pH 2 gave an additional 20 g, mp 234" dec. Recrystallization of both crops from water led to 160 g (49% yield) of purified product, nip 238" dec. A11 the pyridyl- and heterocyclic tetrazoles prepared from the corresponding cyano compounds were made by essentially the same procedure. 3-Cyanopyridines.-The folloming 3-cyanopyridines were available or prepared by literatiire procedures: 3-cyanopyridine,*9 3-cyano-i-methylpyridine, 3-cyano-5-methylpyridine,*9 2-methosy-3-cyaiiopyridiiie,5'3-cyaii0-6-methosypyridine,~23-cyano-63-cyniet,hylthiopyridine, 3-~yano-6-methylsulfonylpyridine,~~ aii0-6-an~iiiopyridiiie,~~ 3-cyan0-6-acetamidopyridine,~~ 3-cyano34,6-dimethylpyridine,"j 3-cyano-6-methy1-2(lH)-pyrid0ne,~~ :3-cyano-5-methylisoxazole,38 and 3-methyl-5~yanoyuiiioline,3~ cyaiioisoxazole.3* 3-Cyano-6-methylpyridine.-A mixture of 14.6 g (0.096 mole) of 2-chloro-3-cyano-6-methylpyridine36 and 14.2 ml(O.106 mole) of triethylamine in 400 ml of methanol containing 2 g of 5% Pd-C wa.; shaken under 2.8 kg of hydrogen/cmz at, 25'. After 1 hr the theoretical amoiuit of hydrogen was absorbed, the suspension was filtered, and the residue was washed well with methanol. The solvent was removed in L'UCZLO, 100 ml of water was added, aiid after filtering there was obtained 5.3 g of product, mp 80Sl"."9 The aqueoii.: filtrate was satiirated with NaCl and extracted with ether. An additional 2.3 g, mp 80-81", was obtained by removiirg the ether in I ' U C U O . The total yield was 7.6 g ( 6 5 ? i ) . 3-Cyano-4-trifluoromethylpyridine.-To a mixture of 90 g (1.07 mole.) of cyaiioacetamide and 91 g (1.07 moles) of piperidine (26) Dr. M. Scliach yon iyittenau of these laboratories has shown t h a t d-(:i-pyridyi)tetrazole is excreted by the dog essentially unchanged over a 24-lir period. ( 2 7 ) Cnpublished elxerrations of Drs. d. Gilgore and 8. DeFelice of o u r Clinical Pliarmacology Department. 128) Melting points were determined on a Thomas-Hoover capillary melting point apparatus and are uncorrected. Boiling points are uncorrected. Tile analyses were carried out hy the Physical Measurements Laboratory of C'has. Pfizer 6: Co., Inc. ! 2 Y j Reilly T a r and Chemical Corp., Indianapolis, Ind. (:io) .I. .\I. Bobbitt and D. A . Scola, J . O r g . Chem., 25, 560 (1960). l:il) E. c'. Taylor, J r . , and .\. J. Crovetti, J . Am. Chem. Soc.. 78, 214 t 19.56). (32) H. 5 . Forrest and J. IYaIker, J . Chem. SOC.,1939 (1948). (:U) \V. T . Caldn.ell, F.T . Tyson. and L. Lauer, J . .4m. Chem. Soc., 66, 1 4 i 9 (lY.44). (:H) 21. Lipp, F. Dallacker. and J. Thoma, Monatsh. Chem., 91, 595
(1960). ( 3 5 ) N . 'perber. 21. Sherlock, D. Papa, and D. Kender, J . A m . Chem. S o c . . 81, TO4 (1959). (36) L . -1.Perez-Medina, R . P. >lariella, and S. 11. lIcElvain, i b i d . , 69, 2.571 (1947). (37) H. Gilman and S. h l . Spats, i h i d . , 63, 1553 (1941). (38) A. Quilico, L. Panizzi, and U. Cavazzuti, G a n . Chim. Ital., 68, 625
(1938). (39) PI. -4. Plattner, JY.Keller, and A . Boller, Heir. Chim. Acta, 57, 1379 '19541, have reported mp 8344.5' for 3-cyano-6-rnethylpyridine.
153
in 700 ml of absolute ethanol at 70" was added dropwise 17s g (1.04 moles) of ethyl 4,4,4-t~rifluoroa~etoacetate.~~ After heatiiig to reflux for 12 hr, t,he misture was cooled and filtered. The residue wa3 dissolved in 2 1. of water, and the solution was acidified with dilrite HC1. After filtering, the crude 3-cyano-4-trifluorometh\-l-6-hydroxy-2( lH)-pyridone was recrystallized from 1 1. of water to give 165 g (83% yield), mp 192-195.5".'O Thi. material was iiot purified further biit treated with POCl,, under the Same coiiditions used for the preparation of 3-cy&lio-2,6dichlor0-4-methylpyridine,~~ to give 3-cyano-2,6-dichloro-4-trifl~ioromethylp\-ridine,25 g (63% yield), bp 68-71' (0.5 mmj, nip 39-40". Anal. Calcd for C7HC1,F3S;Y: C, 34.88: H, 0.42: S , 11.62. Found: C, 34.99: H, 0.46: X, 11.20. Dehalogeiiation by the same procediire used for the preparatiuii of 3-cyaiio-6-methylpyridiiie gave 3-cyano-btrifluoromethylpyridiiie in 5Uc; yield, bp 70" (0.07 mm). Anal. Calcd for C,H,F&;Y: C, 48.84: H, 1.75: S , 16.27. Found: C, 4S.72: H, 2.04: S, 15.91. 3-Cyano-5-fluoropyridine.-A mixture of 2.5 g of 5-fluoronicotiiiamide'l and 5 g of P,O: were intimately mixed and heated at 230" under 0.01 mm of pressiire. During a period of 1 hr 1.39 g (64% yield) of product, distilled, mp 54-55.5". d n a l . Calcd for CsH3F?;?: C, 59.O'L; H, 2.48: S , 22.95. 1: H, 2.26: N, 23.05. P-Methylthio-3-cyanopyridine.--The same sequence of reactions used iii the preparation of 3-c~-ano-6-methylthiopyridiiie,~? but starting with 2-chloro-3-cyaiiopyridine,'? was carried out, mp 87.5-89.5O. 4nal. Calcd for C7H~lr'nS: C , 56.00; H, 4.03: S , 1X.66. Found: C, 55.79: H, 3.82: S, 18.53. 3-Cyano-5-aminopyridine.-A solution of 45 g of SiiCl;Y.2H20 in 90 ml of concentrated HCl was added to 50 ml of ether containing 10 g (0.054 mole) of 2-chloro-3-cyan0-5-nitroppridine.~~ The initial reaction was exothermic: the misttire was s h e d vigorously until the temperature had fallen to 30°, diluted with 200 ml of water, made strongly basic with 40C0 SaOH, cooled, and filtered. There was obtained 7.9 g (93% yield) of 2-chloro-3cyano-5-amiiiopyridine, mp 192.5-194. O An analytical sample was prepared by a recrystallization from methanol, mp 193.5194". Anal. Calcd for C6H,C1?;,: C, 46.92; H, 2.63: S , 27.36. Found: C, 46.65: H, 2.64; N, 27.00. Dehalogenation by the same procediire used for the preparatioii of 3-cyano-6-methylpyridine gave 3-cyan0-~5-aminopyridine,3.3 g ( 5 5 7 , yield), mp 118-123". An analytical sample was prepared by a recrystallization from chloroform-hexane, mp 125-127.5'. Anal. Calcd for C&N3: C, 60.49: H, 4.23; N, 35%. Found: C, 60.53; H, 3.93: S , 35.52. 3 4 5-Tetrazolyl)pyridine-5-carboxylicAcid.-Over a period of 6 hr, 175 ml of a warm aqueous solut,ion of 1 AI KMn04 was added dropwise to a sollition, maintained at, 90-100", of 4 g (0.025 mole) of 5-[3-(5-methylpyridyl)]tetrazole in 150 nil of water. This misture was refluxed for an additional 16 hr. The filtrate, after cooling and filtering, was concentrated in vacuo to a volume of about 50 ml. The prodrict separated after acidification to pH 3-1 with HC1. After washing with water there waa obtained 1.1 g (247, yield) of product, mp 279-283" dec. TWJ recrystallizations from met.hano1-ether raised the melting point to 284-285" dec. 3-Cyano-4-trifluoromethyl-6-methylpyridine.-To a misture of 90 g (1.07 moles) of cyarioacetamide and 14 ml (0.14 mole) of piperidine in 750 ml of absolute ethanol at 75" was added dropwise 150 g (0.97 mole) of l,l,l-trifl~ioro-2,4-peiitanedione.1~ After heatiiig to reflux for 3 hr, 750 ml of water was added and the product, after cooling, was collected by suction filtratioii, 147 g, mp 231-233°.20~21 .4n additional 19 g was obtained from the mother liquor after acidification with acetic acid. The total yield of 3-cyano-4-t~rifluoromethyl-6-met,hyl-2 (1H)-pyridone was 8 5 9 , and this material was used in the next step witho(it further purification. A mixture of 27.9 g (0.136 mole) of the pyridone, 31.2 g (0.15 mole) of PCla, and 125 ml of PoC13 wa5 heated under gentle reflux for 18 hr. ilfter cooling, 60 ml of toluene was added, and the mixture was concentrated in cacuo to constant weight. The residue was cooled and 30 ml of ethanol (40) Portnoy*l has reported t h a t anhydrous 3-cyano-6-hydroiy-4-trifluoromethyl-Z(IH)-pyridone melts a t 246-219' dec. (41) G. F. Haa.kins and A. Roe, J . Org. Chem., 14, 328 (1949). (42) E. C.Taylor, Jr.. and -4.J. Crovetti, ibid., 19, 1633 (1954). (43) P. E. Fanra and R. A . Stein, J . Am. Chem. Soc., 77,1045 (195.5).
