of acetic a d a a z apparent, but quantitative i-olation. J>sleni
Dose le\ el, m y A #
Monomer act. (I)"
Polymer ;let. tII!"
IIa
SO Iiiactive Iiiac,livth 160 t nac.tive Inactive 320 Iiiact ivr .\ctive 640 Ciirativr .li*t.ive 1280 Ciirativr .lctive Ih, IIh 40 Iiiact ive Ciirative 160 Cur at ive 'rosic Toxic. 640 Curative IC, 111, 40 Inactive Clirative Ciirat ivr Clirative 160 640 Ciirativr ('iirat ive Aiitinialarial testing wit$ dolie by I)r. Leo I t m e at the Uiiiversity of Miami Medicit1 Srhool. Test, were carried out employing Plasmodium berghei in young ICIl/Ha Swiss mice. ', .%ctive = mice in a treated group survive at least 14 days: ciirative = mice in a treated group wrvive to :40 days: toxic = death- I JC riming through day 5 after infectioii arr attribiited t o d r ~ i p action: Control animal.*do n o t die M'ore d a y 6. I:t,
IKI
:ittempi ua. made to &chi?\v
Acknowledgments.-Thib work n a b supported I I I part by Public Health Service Grant 5 ROl-AI06662. We are indebted t o Dr. ,J, P. TI instead of 1.1 nil of 3 i r i aqueou.i HCI. The yield of produo1 softening a t 230-235' rvaa g (14.2(; ). The iiitrinaic. vincosit). a1 25" was 0.10; infrared data 0 i,:32:30 s, 3050 ni, 2860 iv, 2610 1395 *, 1500 m, 1445 i n , l;3i5 w. 165 8 . 11.53 q, 1095 ni, 1000 n'. $150 n'.
L
J,
.Incc(. Calcd for Cgljj,83&S: C:. -17.77: I f , 4.45: S , !?.: leported previously for a sulfap ine-formaldehyde cwpolymer.
LThen the reactioii mixture was made basic, the odor of amnioni:i The solution mi- evaporated t o dryiies:;. after rencidification. The residue was thoroughly washed with water t o remove the salt present. The brown solid left w:i- ideiitiral with the sulfonic acid hylrol I $ the sulfapyridirie-formald Hydrolysis of 1Ib.--IIb (6.S g ) a a , dissolved ~ iii 30 nil of 10' ( aqueous NaOH and refluxed for S hr. The solutioii wah cooled a ~ i dthe p H was adjusted t o 8 with dilute aqueour HC1. The gunimy precipitate (3.5 g ) was washed with HC1 after filtratioii and dried. The infrared apectrum of thi.4 material was comparable to that of IIa, and acid hydrolysi3 of this productl" yielded 111. The filtrate a t p H 8 was made stroiigly acidic with HCI, anti 2.2 g (96'G) of benzoic arid (identified h>- infrared romparisoiil was precipitated. Hydrolysis of He.-IIc (2.0 g) \va> dissolved ill 12 nil of 10( ( :iqueous NaOH arid refluxed 24 hr. The solutio11was cooled and carefully neutralized to pH S. A preciipitate appeared which was collected by filtration, waahed with w-ttter, aiid dried t u yield 1.45 g of material h ng aii iiifrared spectrum comparable t o Ira, and acid hydrolj of this product yielded 111. The filtratr a ( pH 8 was made strongly acid with aqueous HCI. The ( I ~ I I I , was apparent.
;?j = intrinsic
-
viscosity
M =average molecular weight K a n d a = empirical constants
Iri order to differentiate between the two possibilities, or to recognize the presence of both structures ill the product) the purified reaction product was treated with aqueous nitrous acid t o diazotize any free amino groups present. 1-pori wirmirig the solution and vel11) This \vurk na;. 1akt.u 111 i n 1 1 f r i ~ i n[tie thesis to be submitGed b y X i . .John R a i z a n o in partial fitlfillnient of t h e reqiiirrments for t h e I ' k l l . {legree . V i I . Chcm., 8 , 388 (19ti5) , (,2) (a) K ,J, L'ornril a i d I.. G. I ~ u r i a r r u n a .1. €L J. Cornel1 and L. G. Iliinarunla. .I. P o l y m e r S c i . , SA, 827 (1HfiRI; ( h i I.. G . Donanim? a n d ,101 n R a r r a n o , .I. lJw/. Chpni.. 9 , 2.58 (1966).
XOTES
September 1967 lecting the evolved nitrogen, it waa found that the volume of N2 collected, per unit weight of I1 used, indicated that only I1 was present. If the product had been a mixture of I and 11, less nitrogen would have evolved, and if I were the structure, no IS2would have been produced. Thus, it appears that the copolymers possess the structure exemplified by 11. The intrinsic viscosity of I1 in DMSO at 25' was 0.34 and by light-scattering measurements in DMSO, its average molecular weight was found to be 4700 f 10%. The polymer system (11) appeared to be monodisperse since fractions of differing inherent viscosities could not be obtained by fractional precipitation from either DJIF or DAIS0 with water or aqueous saline solutions. Because we wished to establish a viscosity-molecular weight relationship for this DDS-formaldehyde copolymer system, the identical synthetic reaction for the preparation of I1 was reemployed utilizing a 0.5 and 1.0 M excess of formaldehyde, respectively. The reactions went smoothly to yield products similar in all respects to I1 except that the intrinsic viscosities at 25' in DMSO and the average molecular weights by light-scattering measurements in DMSO proved to be 0.38 and 7600 f 10% and 0.45 and 10,000 f lo%, respectively. Having obtained intrinsic viscosity and molecular weight data for three similar samples, we were in a position to obtain the Staudinger constants (see eq 2 ) which relate viscosity to molecular eight.^ By plotting the logarithm of [v] us. log -17the . slope of the curve obtained and the intercept of the curve were log a and log K , respectively, and, for this system, a = 0.37 and K = 0.0145. Thus, we can now determine absolute weight-average molecular weights directly from viscosity measurements, and we are notv capable of determining the relationship of molecular weight t o biological activity in a much more quantitative fashion than previously. zh Both DDS and I1 were screened for antimalarial activity under identical conditions a. reported previouq1y,2band the result!: are shown in Table I. TABLE I
965
DDS, and dried or, the crude product was dissolved in DMF and reprecipitated by the addition of water, filtered, and air dried. The yield of product softening a t 235" was 2.52 g. The intrinsic viscosity of the product in DMSO a t 25' was 0.34, and the molecular weight of the product by light-scattering measurements in DMSO was 4700 f 10%; infrared data (cm-'): 3540 w, 3480 w, 3380 s, 3245 w, 3070 w, 2835 w, 1600 s, 1535 s, 1470 w. 1435 w, 1375 w, 1330 s, 1300 s, 1285 s, 1210 w, 1160 s, 1120 s, 1090 m, 1060m, 1040 w, 970 w, 845 m, 825 w, 740 m, 710 s. Anal. Calcd for C13H12N2SO?: C, ,59.99; H, 4.62; N, 10.iR: S, 12.30. Found: C, 59.81; H, 4.92; N, 10.20; S, 12.13. By carrying out the experiment above employing 1.2 ml of 37% aqueous formaldehyde solution (0.5 M excess) instead of 0.8 ml, 2.50 g of product softening a t 245' was obtained. This material had an intrinsic viscosity of 0.38 in DMSO a t 25' and a molecular weight (determined by light scattering measurements in DMSO) of 7600 + 10%; infrared data (cm-I): 3545 w, 3480 w, 3370m, 3250w, 3065w, 2860w,1600s, l520s, 1475m, 1420 w, 1330 m, 1300 s, 1275 s, 1210 w, 1165 8, 1120 s, 1090 m, 1055 w, 1020~,970~,8m 4 5, 8 2 5 m , 7 4 0 m , 710m. dnal. Calcd for C13H12N2S02: C, 59.99; H, 4.62; N, 10.75; S, 12.30. Found: C, 59.76; H, 4.62; N, 10.53; S, 12.05. Again, by repeating the experiment for the preparation of I1 employing 1.6 ml of 377, aqueous CHtO solution (1.0 M excess) in place of 0.8 ml, 2.53 g of product softening a t 253" was obtained. This material had a n intrinsic viscosity of 0.45 in DMSO at 25" and a molecular weight (determined by light-scattering measurements in DMSO) of 10,000 f 10%; infrared data (em-1): 3.590 w, 3490 w, 3375 m, 3205 w, 3025 w, 2850 w, 1600 s, 1515 m, 1470 w, 1420 w, 1330 m, 1300 s, 1275 s, 1203 w, 1160 s, 1120 s, 1085 m, 1050 w, 1020 w, 960 w, 840 m, 820 w, 740 w, 710 m. Anal. Calcd for C13H12NnS02: C, 59.99; H, 4.62; N, 10.75; S,12.30. Found: C, 59.73; H, 4.83; N,10.56; S, 12.38. Reaction of I1 with Nitrous Acid.-A known weight of I1 was dissolved in enough concentrated HC1 to dissolve the copolymer a t 25" and enough ice was added to the solution to initiate precipitat,ion. Sufficient concentrated HC1 was added to redissolve the precipitate which appeared, and a cold saturated aqueous solut,ion of NaNOt was added unt,il the copolymer solution gave a positive test for HNOn with starch-iodide paper. The solution was carefully warmed, and N?was rapidly evolved and collected over n't-saturated water in a closed system. When S2 evolution ceasd, the reaction flask was cooled to Oo, and the volume of collected Xt was measured. After corrections for temperature and pressure were made, it was found thitt, 0.9.5 mole of Kt was evolved per mole of I1 employed.
Acknowledgment.--We are indebted to the Public Health Service Service for partial support of this work under Grant no. 5ROlXIOGG62 and t'o Drs. lIilt80n Kerker and Josip Kratohvil for the use of their lightscatt'ering equipment.
ANTIMAL.~RIIL ACTIVITY" OF UUS ASD ITSFORMALDEHYDE COPOLYMER (11) 1)ose level. me/kg of hods l i t
D D i 8Ct.b
I I act.b
40 Curative ; nontoxic Inactive ; nontoxic 160 Curative: nontoxic Active; nontoxic 640 Curative; toxic Curative; nontoxic Antimalarial testing done by Dr. Leo Rane at' the University of Miami AIedical School. Tests were carried out employing Plasmodium berghei in young I C R / H a Swiss mice. * Active, when mice in a treated group survive a t least 14 days; curative, when mice in a treated gronp survive to 30 days; toxic, deaths occurring through day 3 after infection are attributed to drug action. Control animals do not die before day 6. 11
Experimental Section Copolymerization of DDS with Formaldehyde.-A mixture of 2.44 g (0.0094 mole) of DDS, 250 ml of water, and 5 ml of 4% aqueous HCl was heated to reflux, and 0.8 ml (0.0098 mole) of 377, aqueous formaldehyde solution was added. The mixture was refluxed for 9 hr, and the precipitated product was removed by filtration, extracted with boiling water to remove unreacted (3) F. W. Rillmeyer, "Textbook of Polymer Chemistry," Interscience Publishers, Inc., New York, N. Y.. 1957.
N-Thymidylglycine and Ethyl p-N-Thymidylaminobenzoate' ?VIATHI.ISP. LIERTES
AKD
QUENTIN GILJIAS~'~
Department of Medicinal Chemistry, School of Pharmacy, University of Kansas, Lawrence, Kansas 66044 Received April 27, 1967
The biological activity and tumor inhibition noted in folate and pyrimidine analogs have been widely explored. All available evidence suggests that the action of the fluoropyrimidines is exerted by inhibition of thyinidylate synthetase, the enzyme catalyzing the conversion of deoxyuridine 5'-monophosphat'e (dURIP) to thymidine 5'-monophosphate (TAIP).2 Recently, (1) (a) This work was generously supported by G r a n t CA-7522 of the National Cancer Institute, National Institutes of Health, U. S. Public Health Ssrvice, Bethesda, Md. (b) National Szience Foundation Undergraduate Research Participant. (2) C. Heidelberger, Progr. Nucleic Acid Res. M o l . B i d . , 4, 1 (1965).
