The hydroxyl protons appear as a broad band at 6 2.7, HI is a miiltiplet at, 6.5, H 2 at 2.3, H3 at'4.5, H, at 4.0, and H:, a t 3 .7 .

I t was found that the 01 and p anomers of 12 could be separated by repeated chromatography on silica gel living CHC13 and CHCla-methaiiol as the solvents. The first fraction was 12a, [ L Y ] * ~ D f45" ( c 10.6, CH,OH), and the second fraction was 12b,

6-Trifluoromethyl-2-( 2'-deoxy-~-ribofuranosyl)-as-triazine- 3,5(2H,4H)-dione (5-Trifluoromethyl-6-aza-2'-deoxyuridine, 7). -The mixture of anomers 12a and 12b (0.10 g, 0.22 mmole), 10 ml of methanol, and approximately 50 mg of prereduced 5% palladium on carbon in methanol (20 ml) was hydrogenated a t room temperature and pressure. A4fter the mixture had taken up 10 ml of hydrogen (997, of theory), the reduction was stopped. The mixtiire was filtered aiid the filtrate was evaporated to dry- ness in caciio to yield a transparent, semisolid mass (81 mg) which was chromatographed on silica (Merck AG., silica gel, 0.05-0.2 p ) with CHC1,. The first fraction cont,ained dipheiiyl- methane (identified by iimrj. Changing t o 1 5 5 methanol in CHCls gave a frartion containing 54 mg (82';') of the anomers of 7 as a clear glass. After passing an aqlieoiis solution through a Ilowex 50 If' (H' form) column, the fractioiis absorbing in the lilt raviolet were evaporated, and the residiie was redissolved in methanol, filtered, and evaporated to give 7 as a glas.5, softening at 78". The presence of methanol was confirmed by nmr.

-Anal. Calcd for C S H ~ ~ F ~ S ~ O ~ ~ C H ~ O H : C, 36.48; H, 4.28: S, 12.76. Foiind ( b o samples): C, 36.83, 36.12; H, 3.86, 4.18; s, 13.2'2.

Similar rediictioii of the anomer 12a gave anomer 7a, purified 011 silica, [LY]"D +76" ( c 2.3, CH,OH), A,,, ( pH 1.0) 269 mp ( e 5000), ( p H 12.4) 264 mp (t 5000).

The anomer 12b gave 7b, [cu]*'D -36" ( e 2.4, CH,OH), Amax ( pH 1.0)269mp (e4900), (pH 12.4j264 mp(t4900),afterchroma- tography wit,h met,hanol. Compound 7b gives the following sig- nals in the sugar portion of the molecule: HI, 6 6.6; H?, 2 . 5 ; H,, 4.5: Ha, 4.0; arid Hj, 3.7. The integral is consistent with the structure.

Inhibition Studies. Dihydrofolate Reductase Assay.-The stock solution of XADPH was prepared by dissolving NADPH (Sigma Chemical Corp.) in 0.02 -11 Tris buffer at a pH of 8.5 to give a firial colicelitration of 0.5 pmole/ml. Dihydrofolic acid, prepared by dithionite redtiction of folic acid according to Futter- man,35 was dissolved in 0.005 M acetate buffer containing 0.01 31 mercaptoethanol at a p H of 4.5 t,o give a final concentration of 0.5 pmole/ml. The assay was that of Friedkin and co-norkers:j6 and cont,ained 0.2 ml of the SADPH stock solution, 0.1 ml of the

[ a ] * 4 ~ -10" ( C 4.26, CHBOH).

(35) S. Fotterman, J . Bid. Chem., 228, 1031 (1957). (36) 11. Friedkin, E. .J. Crairford, S. R. Humphreys, a n d A, Goldin,

Cancer Res. , 22, 600 (1962).

dihydrofolic acid stock solution, 0.75 ml of 0.0% 31 mercapto- ethanol in 0.1 M phosphate biiffer a t pH of 7 .5 and enzyme, inhibitor, or inhibitor solvent, and water to a total of 1.5 ml. Dihydrofolic acid --as replaced by 0.005 JP acetate biiffer coii- taining 0.01 M mercapt,oethaiiol at a pH of 4.5 in the referenre cell.

The change in absorbance at 340 mp (32') in a Beckinail 1)U spectrophotometer was recorded with 10 X expawion of the transmission scale and wa'i liiiear for 10 niiii. A4fter coiivei'- sion to absorbance units, 52% of the change in absorbance rep- resents the utilization of dihydrofolic acid, and this change way used in determining the specific activity of the enzyme.

Inhibitors were dissolved in water, alcohol, or 0.02 31 S a O H to give stock sollitions containing 1 prnole/O.l ml. [Then ba-e or alcohol was iised as the colvent the effect, of the solvent oii the uninhibited reaction was examined, and theGe ratei were com- pared to the inhibited reaction.

Thymidylate Synthetase.-The assay solution contained 0.05 pmole of deoxyuridine 5'-nioiiophosphate (Sigma Chemical Corp.), 0.22 pmole of tetrahydrofolic acid (General Biochemicals Corp.), 15 pmoles of formaldehyde, 25 pmoles of AIgCI?, 130 pmoles of mercaptoethanol, 0.9 pmole of disodium ethylenedi- aminetetraacetic acid, 45 pmoles of Tris biiffered at a pH of 7.4, and enzyme to a total volume of 1.2 ml. Tetrahydrofolic acid was added to 1 M mercaptoethanol and adjusted to pH 7.4 aiid this solution containing approximat,ely 20 pmolei/rnl wa,s di- vided into small fractions and frozen. Deoxyuridine 5'-mono- phosphate was dissolved in s-ater to give a stock solution con- taining approximately 1 pmole/ml and \vas stored, frozen. The aisay stock solution (minus mix) contained 12 mmoles of mer- captoethanol, 27 pmoles of tetrahydrofolic acid, 1.8 mmoles of formaldehyde, and 3 mnioles of XgCI2 in a total volume of 30 ml. The assay sample cell contained 0.25 ml of the minus mix and 0.05 ml of the deoxyiiridine j'-monophosphate stock sollition and enzyme, inhibit'or solution, or inhibitor solvent and buffer d were added to a total of 1.2 ml. Deoxyuridine 5'-monophosphate was replaced by water in the reference cell.

The change in absorbance at 340 mp waq read at 30" in a Beckman DB spectrophot,ometer arid recorded v i th 10 X ex- pansion of the transmission scale and converted to absorbance. Under these conditions the change in absorbance iiwally was linear for the first 10 min.

Acknowledgment.-The authors wish t'o acknowledge t'he assistance of Xrs . William Riggs for enzyme studies, JIessrs. Larry Hare, Donald Thompson, and Quentin Gilman for synthesis of starting materials, and Professor Morris Friedkin arid associates for hell)- ful discussions.

1,4,5,6-Tetrahydro-as-triazines. I. Sulfuric Acid Catalyzed Condensation of Nitriles and Hydrazino Alcohols'

DOSALU L. TREPASIEH, EUGEXE R. WAGNER, GVY HARIZIS, AXD ALLAN D. RUDZIK

C'heriiiitr y lZestarch and Pharniacology Deparlrizents, Human Health Research and Development Cento , The DOE Chemzcal Company, Inclmnapolis, Indiana

\~:~riowlj. S I I ~ ) S ( i t riled 1,4,.i,ti-~e~r~rh~clro-us-iriazines \\-ere prepared by treating iiitrile~ ivith hydrazilio :tlcc~licil~ i n the presence of concentrated sulfuric acid. Th(, spectral properties :tnd pharmacological activity of the compounds are discussed. trans-( - )-3-(o-ChlorophenyI)- 1,6-dimethyl-~-phenyl-l,4,5,6-tetrahydro-us-triazine was found to possess analgetic activity as measiired by the inhibition of hydrochloric acid induced writhing and by a modified hot plate procedure. Five other compounds were found to inhibit maximal electroshock seizures in mice indicating anticonviilsant activity.

The scope and mechanism of this reaction was invest'igated.

We observed that S-amino-( -)-ephedrine (I) reacted ambient temperature to give t~ans-(+)-1,6-dimethyl- with benzonitrile in concentrated sulfuric acid at 3,5-diphenyl-l,4,5,6-tetrahydro-as-triazine (11). .I

literature search revealed the absence of any r e p r t r on the acid-catalyzed condensation of hydrazino dcoliols und nitriles. In fact, thc only cxainplc of :i

(1) Presented in par t before the Diyision of hledirinal Chemistry a t {lie 152nd National Meeting of the .\inerican Cheinical Society. New York, N. Y,, iel,L 1988.

h-<L

I 2

4

(i

1

)

- I

\

!) I O I I 12 1:: I4 I .i lti 17 IS I !I

"I 21 .? L'

' I 1'mr hpectra were obtained at 6Cl JLc with a. Varian ~ 1 - G O speclroriieter. Spectra for 4, 7, a i d 15 were obirtined for 10' I)?() m l i i t i o i t b

uiiir(aining a trace of 3-(trimethyls -l)-l-propaiie;;~ilforiic acid sodirini salt (TLISPS) :i> an internal standard and spectra fo r all rhe other compounds were obtained for 10 ~ ~ I t r n v i ~ i l e t CDCl, solutions containing n, trace of tetramethylsilane as an internal standard.

H h 1

kN C6HG I1

1 ,A, 5,Ci-t e traliydro-as-triazine is 3-1net hoxy-.i,6-diphen- yl-1,~,3,Ci-tetrahydro-as-triazine." Related heterocy- cles, such as 1,2,5,6-tetrahydro-, 2,3,4,j-tetrahydro-,4 3-thion0-2,3,4,5-tetrahydro-,~ 3,5-dioxo-2,3,4,5-tetrahy- dro-,6 3-0~0-2,3,4,5tetrahydro-,~ 3-oxo-l,4,5,6-tetrahy- dro-,* arid G-oxo-l,.2,5,G-tetrahydro-as-triazir~esg have been reported.

Recause of tlic uniqueness of both the heterocyrlic riiig aiid its fornintiori via a modified Ititter reaction :iritl

t)ccausc cc>rtnin isosteric 3,G-dihydro-4H-l,3,-l-oxacli- :tziiic:s li~ivc been reported'O to possess biological activ-

iiiitiated to deterrniiie the scope of the synthesize a series of variously sub-

('2) 31. I'olonolski, 31. Pesson, and 11. Kajzinan, C o m p t . Rend., 283, ll:34

) C . H. Grundmann and R. F. W. Ratz, C'lis?n. Ber., 91, l i 6 6 (1968). ) 31. Busch. G. Friedenberger. and rV. Tischbein, i h i d . , 67, 1783 (1924). ) M. Gianturro and A. Romeo, Gazz. Chim. Ital., 83, 429 (1953).

t i ) 1,;. Cattelain, Cowp1. Rend. , 316, 257 (1942). 7 ) .\. Slirl iad, ./. / ' f .akt. Chern.. [2] 60, 456 (18!49). j ( ', lI:irriPs, Ciirm. Bur . . 28, 1223 (1895). ) I<. T. \ \ ' idnuinn, ibzd., 26, 2612 [18!#:3),

I 10) I). 1.. 'l'repnnier, 1'. I:. lirieeer, ani1 .J. N. ICble, . I . . l f v d . Chctu., 8, bO'2 I ! J t i.-~ I ,

883

Chemical shifts, -ppm--- NCII3

"81 2.88 2 ,80 2.90 2 .82 2.81 2.89 2 . 79 2.83 2.83 2.77 2 . 79 2,%3 2 . SO 2.95 2.87 2.84 2.90 2.70 2.65 2.70 2.75

iY €1

4.61 4.72 4.61

4 . 6 2 4 .63

4.74 5.00 4.23 4.66 4.74 4.79 4 .75

4.75 4.68 4.78 4.10 4 . 5 5.54 4.60

. . .

. . .

. . .

R3

7.26 7.27 7.27 7.50 7.26 7.28 7.51 7.29 7.27 1.29 7 . 3 7,3.5 7.35 7.35 1.48 7.34 7.35 7.35 1.39 7.29 7.30 7.29

Rz 4 21 4 22 4 24 4 69 4 24 4 25 4 68 4 75 4 27 1 29 4 79 4 80 4 81 4 78 1 48 4 32 4 29 4 44 1 39 4 62 4 69 4 69

R1 1 . 03 1.00 1.03 1.18 1.05 1.05 1.14 3.10 1.05 2.48 i3.10 3.14 3.14 3.12 3.13 1.07 1.05 1.09 2.36 2.98 3.06 3.07

6- H 2.52 2.50 2.49 3.37 2.50 2.50 3.35 2.57 2.56 2.48 2.62 2 . .56 2.64 2.60 3.13 2 60 2.54 2 66 2.36 2.43 2.41 2 45

Couuling, cws Ha-Ha

6 . 8 6.68 7 . 0 7.75 6.85 6 . 8 7 . 9 * 0 . 4 7 . 4 6.65

7 . 1 7 .0 i. 0 . 5 7.15 6 . 9

6 . 6 6 . 8 6 . 2 5

. . .

. . .

. . . 7.35 7 . 5 -7

Ha-114

1 . 6 1 58 1 . 4

1.55 . . .

1 . 4

1 . 5 1.65

1 . 7 >0 1 . 6 1 . 8

. . .

. . .

. . 1.75 l . 2 * 0 2 1 .9

< 2 1.2 -1

. . .

XIolar absorptionh x ~ ~ ~ ~ ~ ~ , mp ( 6 x 1 0 - 3 )

230 (17.5), 304 (4.T) d

239 (14, 7), 279 ( 6 . .5) r I

289 (4 .2) 227 (16. l), 289 (4.8)

292 (4 .2) 314 ( 4 . 0 )

f

293 (4 .0 ) f

238(14.8), 280(6.7) 317 (3 .9) 230 ( i5 .9) , 300 (4.0) 260 (4.3) 224 (6.6) , 322 (3 .4) 287 (4 .3) 26.5 (13.5) , 396 ( 4 . 2 )

d d d d

[aln, deg (c in g i100 ml,

C"3oH)'

$35.9 (4.004)

+42.1(3.776)

+27 .6 (4.010) + 3 3 . 4 (4.004)

- 132.7 (4.018)

-138.2 (4.004)

+30.4(4 .006)

+26.G(4.014) + 3 0 . 2 (4,010) - I09 .:5 (4,010)

absorption maxima and molar absorption were measured on a Beckman DU spectrophotometer. Ritdolah laboratorv polarimeter Model No. 62 at room temperature.

0 Optical rotation waq measured on a Not measured. e Hvdrochloride. Data were not obtained

I -

hecaiise the wavelength of maximum absorbance n-as 220 mfi or less.

interaction does not prevent coplanarity of the rings, it may be responsible for lack of ultraviolet absorption maximum at 280-320 mp because (a) when the chloro or methyl group is in the p a m position (6 and 14) there is a maximum at 280-320 mp, and (b) both the isosteric oxa- and thiadiazines having o-chlorophenyl or o-tolyl substituents exhibit a maximum in the 280-320-mp- region and Stewart-Briegleb models indicate free rota- tion about the oxa- and thiadiazine-phenyl bond, since there is a lack of steric interaction of the o-chloro or o-methyl group with either the oxygen or sulfur atoms of the oxa- or thiadiazine

The nmr spectra exhibited the expected chemical shifts and coupling constants (Table I). The 3- substituted 1,6-diniethyl-5-phenyl-1,4,5,6-tetrahydro- us-triazines in Table I exhibited J s , ~ - 7 cps. This coupling indicates these protons most likely are trans- axial-axial. Comparing with the isosteric oxidiazines where both the cis and trans isomers were available it is seen that the couplings compare favorably with the trans-oxadiazine which has a J5,6 - 7.2 cps and not with the cis-oxidiazine which has a J6,6 - 2.5 cps.13 Thus, all of the 3-substituted 1,6-dimethyl-5-pheny1-1,4,5,6- tetrahydro-us-triazines in Table I are trans isomers. The double bond is probably between N-2 and C-3 rather than between C-3 and S-4 because of the com- plex splitting of the C-5 proton which indicates a proton at N-4 and because the Hs-H, splitting is -1.6 cps. Placement of the double bond between C-3 and K-4 is very unlikely because the consequent

( I ? ) D. 1,. Treltanier and V Spranrmanis, J . O r y . Chem., 19, 2161 (1964).

Hydrobromide.

coupling between H-5 and H-2 would be expected to be unobservably small.

In order to determine the scope of the reaction various hydrazino alcohols, such as, N-amino-( -)-ephedrine, N-amino-( +)-pseudoephedrine, 1-( 1-methy1hydrazino)- 2-propqno1, 1-methylhydrazino-t-butyl alcohol, and 0- (1-methylhydrazinomethyl) benzyl alcohol, were al- lowed to react with a variety of aliphatic and aromatic nitriles in concentrated sulfuric acid according to method B. Rydrazino alcohols possessing a tertiary aliphatic or secondary benzyl hydroxyl, ie., a type that yields a relatively stable carbonium ion in concentrated H2SO4, condensed with a variety of nitriles to give triazines. Hydrazino alcohols containing a secondary or primary aliphatic hydroxyl mould not condense with a nitrile in concentrated to give a triazine. Instead the nitrile was converted to an amide. Treat- ment of the diastereoisomeric hydrazino alcohols, X- amino-( -)-ephedrine (I) and N-amino-(+)-pseudo- ephedrine (111)) with benzonitrile in concentrated H2S04 yielded trans- (+ ) -1 , 6-dimet hyl-3,5-diphenyl- 1 , - 4,5,6-tetrahydro-as-triazine (11), exclusively. The necessity of the hydrazino alcohol possessing a hydroxyl group that forms a relatively stable carbonium ion and the fact that both I and 111, which are diastereo- isomeric hydrazino alcohols that differ only in configura- tion about the hydroxyl-bearing carbon atom, reacted with benzonitrile to give 11, exclusively, indicate that the reaction most likely proceeds via attack of the nitrilium sulfate upon the carbonium ion resulting from the dehydration of the hydrazino alcohol (Scheme J).

s o .

1 2

>

4 )

0

- I

S

9 10 11 11'

13

14 a Hydrochloride. Optical antipode of 3.

theze ratio..

Toxicity LDsQ, me >400 >200 >200

>400 >200

>.50

>200 >400

>lo0 >loo >200 >2no

>200

>200 Compound 3 is trans-( - ) and 14 is trans-( +)

After the addition was completed, the mixture was poured onto crushed ice and the product was isolated as described in method A.

Method C.-To 200 ml of cooled (5 ' ) , stirred concentrated H2S04 was added, dropwise, 19.6 g (0.2 mole) of 3-dimethyl- aminopropionitrile. To t,his mixture was added, dropwise, a solution of 33.2 g (0.2 mole) of 01-( 1-methylhydrazinomethy1)- benzyl alcohol in 15 ml of CH2C12. The mixture was poured onto crushed ice, basified ( Na2C03, NaOH), and extracted with a 1 : 1 methylene chloride-ether mixture. The dried (NarSOa) or- ganic estrart wa, evaporated in vacuo leaving 34 g of an orange oil. Distillation of this oil yielded 6.3 g (16Yc) of l-methyl-5- phenyl-:l-vinyl-l,4,5,6- tetra hydro-as-t riazine : bp 145 O (1.5 mm) ; nmr (CDC1,) 2.2-2.7 (CH?, multiplet, two protons), 2.7 (SCHJ, singlet, three protons), 4.3-4.9 (CH and S H miiltiplet, two protons), 5.12 and ,527 (C=CH, two doublets, two protons), 5.9-6.5 (C=CH- miiltiplet, one proton), aiid 7.28 ppm (five aro- matic protons).

dnai . Calcd for Cl1.H1,S3: C, 71.61; H, 7.51; K, 20.89. Found: C, 71.21: H, 7.82: S, 20.68.

Attempted Preparation of 1,5-Dimethyl-3-phenyl-l,4,5,6- tetrahydro-as-triazine.-A solution of 20 g (0.18 mole) of I-( 1- methy1hydrazino)S-propanol in 10 ml of CHzCl? was added, dropwise, over a period of 2 hr, to a cooled (lo'), stirred solution of 20.6 g (0.2 mole) of benzonitrile in 250 ml of concentrated

H\drochloric acid M a x Hexobarbltol

testC shock testC time testC n rithing electric sleep

0/4 0/4 206/53 0/4 0/4 52/48 4/4 2/4 210/32

1 /4 0/4 140/32 3/4 3/4 1 68/32

7/10 4/4 3/4 2 1 3 / 3 3 6/10 10/10 1 /4 1 /4 137/45 2/4 4/4 300/33 3/10 9/10 1 /4 1 /4 87/32 1 /4 0/4 33/30 1 /4 0/4 27/30 3/4 0/4 6h/:30 6/10 4/4 0/4 44/30 8/10 1 /4 4/4 350/53

10/10 8/10

See Phaimacology for an explanation of

H804. After the addition was completed, the mixture was poured onto crushed ice and extracted thoroughly with chloro- form. The washed (H20) and dried (Na2S04) chloroform solution was evaporated zn zacuo leaving 22 g ( 9 0 7 ) of benzamide, nip

Condensation of Benzonitrile and N-Amino-( + )-pseudoephe- drine in Concentrated H2S04.-A solution of 36 g (0.2 mole) of S-amino-( +)-pseudoephedrine in 50 ml of CH2C1, was added, dropwise, to cooled (5 ' ) , stirred concentrated HzSOI (250 ml). After the addition was completed, 20.6 g (0.2 mole) of benzo- nitrile was added and the mixture was stirred at ambient tempera- ture overnight. The mixture was poured onto crushed ice, washed with chloroform, basified with Na2CO3 solution and the precipitated'solid was removed by burtion filtration. The solid was recrystal1ized:from i\opropyl alcohol to yield 18.5 g of white cryqtalline solid, mp 146-147". An admixtiire with ailthentic trans - 1,6 - dimethyl - 3,s -diphenyl - 1,4,5,6 - tetrahydro-as - triazine melted a t 146-147'.

127-128".

Also, the nmr qpectra were identical.

Acknowledgment.-The authors are grateful to Drs. TV. J. Potts and J. Heewhen for recording the interpret- ing infrared and nnir data, respectively, and to Dr. J . 11. Dunham and ;\liss J r . F. Crider for obtaining ultra- violet absorption and optical rotation data.