Non-utilization of Radioactive lodinated Uracil, Uridine, and Orotic Acid by Animal Tissues in Vivo W. H. PRUSOFF,WL. HOLMES,tANDA. D. WELCH

Department of Pharmacology, School of Medicine, Western Reserve University, Cleveland, Ohio)

Adenine (1, 6), guanine (1, 3, 5), cytidine (13), lometric localization of brain tumors. Further desoxycytidine (18), thymidine (18), and orotic more, an I'3-labeled oxazine dye had a significant acid (2), can be utilized by certain mammalian or effect in prolonging the life of mice bearing trans ganisms for the synthesis of nucleic acids ; and the planted tumors (19). If an effective and easily syn rate of incorporation of many of these compounds thesized radioactive iodine-labeled compound into the nucleic acids of rapidly growing tissues, could be found, the possibility might be afforded of such as regenerating liver or neoplastic tissues, is the comparable use of compounds labeled with greater than into those of resting tissues (10, 21). eka-iodine (astatine2@), a potent emitter of alpha Although 8-azaguanine is not a naturally occur particles, although this element is prepared with ring compound, evidence that it can be incorpo difficulty and has the inconveniently short half rated to a small extent into mammalian nucleic life of 7.5 hours (12). acids has been presented (16). This analog of gua Three P3-labeled pyrimidines, iodouridine-5- nine markedly inhibited the growth of Tetrahy I'S', iodouracil-5-I'31, and iodoorotic acid-5-P31, mena geleii, a guanine-requiring protozoan, and of were synthesized, and their incorporation into nor certain experimental tumors. Kidder et al. (14) mal tissue, regenerating liver, and certain experi suggested that the carcinostatic properties of the mental tumors was investigated. The results re analog might be explained on the basis of a require vealed that none of these compounds is incorporat ment for guanine for the synthesis of nucleic acid ed to any significant degree into any of the tissues by cancer cells. However, Mitchell and his co studied. workers (16) and Bennett et al. (4) were unable to EXPERTh{ENTAL demonstrate preferential utilization for nucleic Synthesis of iodoorotii@acid-5-P31.—To a solution acid synthesis of 8-azaguanine-2-C'4 by a suscep of radioactive iodide (5 mc. ; 1 mjzg. ; 0.8 ml.), con tible tumor (E 0771). tamed in a 15 ml.-centrifuge tube, was added In view of the observations regarding the incor NaOH (10 per cent, 0.1 ml.) and carrier KI (4 poration of 8-azaguanine into nucleic acids and the mg.). The centrifuge tube was fitted with a stop fact that there is a much greater rate of nucleic per containing inlet and outlet tubes, and the solu acid synthesis in tumor tissue than in the corre tion was then concentrated to a volume of 0.2 ml. sponding normal tissue of the same animal, this by passing a jet of dry air over it while it was heat study was undertaken to investigate the possibili ed on the steam bath. The exhaust air was led ty of a preferential concentration by tumor tissue through a cold carbon tetrachlonide trap and final of P31-denivatives of naturally occurring pyrimi ly directly into the flue of the fume hood. dines. An incorporation of I'31-labeled pyrimidines The concentrated solution was cooled in an ice could be of diagnostic and possibly of chemothera bath, and the iodide was oxidized by the addition peutic value. Considerable success has been at of H2S04 (18 N, 0.2 ml.) and 0.005 M K103 (0.3 tained in the use of I'31-labeled diiodofiuorescein ml.). A solution of orotic acid (200 mg.) in NaOH derivatives (17), iodinated human serum albumin (3 N; 2 ml.) was added, and this was followed im (8), and sodium iodide (9) in the isotope-encepha mediately by iodine (320 mg.). The mixture was *This investigation was supported, in part, by a grant from heated on the steam bath for 15—20minutes. Dur the National Institutes of Health, U.S. Public Health Service. ing the heating, a further addition of NaOH (3 N) t Merck Postdoctoral Fellow of the National Research was made if necessary to effect complete solution Council of Canada. of the iodine. The reaction mixture was cooled in Received for publication September 22, 1952. ice; upon acidification with acetic acid a white 221

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1953 American Association for Cancer Research. 222 Cancer Research precipitate of the sodium salt of 5-iodoorotic acid elementary analysis, ia position S of the pyrimidine was formed. The free acid was formed upon re ring. crystallization from 0.1 N HC1. The product was Synthesis of iodouridine-I'31.—Radioactive centrifuged off, and the supernate was discarded. iodide solution (10 mc.), as obtained from Oak In an attempt to remove any unreacted iodine, the Ridge, and a solution of KI (0.06 M, 0.3 ml.) were precipitate was washed with 1 : 1 ethanol-acetic pipetted into a 25-ml. round-bottomed flask (glass acid until the supernate was colorless, then once stoppered) and evaporated to 0.5 ml. by heating with ethanol and twice with ether. The iodoorotic on a steam bath while passing a stream of air into acid was then recrystallized twice from 50 per cent the flask. After the addition of HNO3 (8 N, 0.5 ml.) ethanol. to oxidize the iodide to free iodine, water (25 ml.), With rapid heating the free acid decomposed at uridine (200 mg.), iodine (200 mg.) and chloroform 270°—278°C. with the evolution of iodine vapors. (2ml.)wereadded,andtheresultantmixturewas In several syntheses the incorporation of radioac refluxed on a steam bath for 8 hours. All operations tive iodine ranged from 40 to 50 per cent of the were performed in the hood, and special precau theoretical value. The elementary analysis' was: tions were taken to minimize contamination of the found: C, 21.37; H, 0.97; N, 10.14; I, 44.89; calcu atmosphere with radioactive iodine. lated: C, 21.28; H, 1.06; N, 9.94; I, 45.03. When All joints were sealed with silicone, and air was the radioactive iodoorotic acid was chromato blown through the top of the condenser to carry graphed, using the iso-amyl alcohol-S per cent any iodine vapors into a CHCI3-trap which was Na2HPO4 system of Carter (7), the center of radi cooled in an ice bath ; emitted air was passed di oactivity, measured in a well-type scintillation rectly into the flue of a fume hood. During reflux counter, coincided with the ultraviolet absorbing ing, clusters of long white needles of iodouridine spot R,, 0.89, as located by a Mineralite ultraviolet were formed. After cooling overnight at 5@C., the lamp, model SL-2537. In NaOH (0.01 N), the ul chloroform and aqueous layers were discarded, and traviolet absorption spectrum showed a maximum the remaining crystals of iodouridine were washed at 303 m@z(e = 9,170) and a minimum at 255 mM; with ether until the solvent was colorless. The and in HC1 (0.01 N) a maximum at 287 m@ (e yield at this point was 200 mg., with 32 per cent of 6,460) and a minimum at 246 mjz were observed. the original I's' incorporated or 64 per cent of the The position of the iodine atom has been as ory. The compound was further purified by treat signed to carbon-S for the following reasons. Hy ment with Nuchar (C250) and by recrystallization droxy (oxy) groups are already attached to the from hot water. carbon atoms at the 2 and 4 positions, and a car With rapid heating the compound decomposed boxyl group is attached to carbon-6 (the new at 2050_2080 C. with the evolution of iodine va numbering system of Chemical Abstracts is used). pors. The point of decomposition varied with the If the iodine together with the carboxyl group was rate of heating. In NaOH (0.01 N) the ultraviolet attached to the carbon at position-6, the typical absorption spectrum showed a maximum at 278 ultraviolet absorption spectrum characteristic of a @/@Land a minimum at 253 m@, and in HC1 (0.01 pyrimidine structure would have been destroyed N) a maximum at 289 mj@ and a minimum at 249 because of inhibition of resonance caused by satu m@hwere observed. The elementary analysis' was: ration of the 5—6double bond. For a similar reason, found: C, 29.17, 29.14; H, 2.95, 3.03; N, 7.48, 7.53; the iodine together with a hydroxy group could not I, 34.09, 34.18; calculated: C, 29.10; H, 3.24; N, be attached to the carbon at either position-2 or 7.50; I, 34.33. -4. The possibility of substitution of either of the Further evidence that this compound is 5-iodo hydroxy groups at positions 2 and 4 is ruled out by uridine was given by its reaction to bromine water; the elementary analysis. The formation of a hydro whereas addition of the latter to an aqueous solu gen iodide complex by orotic acid is not only highly tion of uridine resulted in immediate decoloriza improbable, because of the absence of any basic tion of the bromine water, one drop of this reagent groups in the molecule, but, in addition, is also retained its color when added to an aqueous solu ruled out by the fact that the iodoorotic acid re tion of iodouridine. Hydrolysis of iodouridine with tamed the theoretical amount of iodine after four HC1O4 (12 N) in a boiling water bath for 1 hour re recrystallizations from HC1 (0.1 N). Therefore, the sulted in a compound having an R, similar to that only available position left for substitution by of uracil in two solvent systems (7, 20), as indicat iodine, which satisfies the spectral data and the ed in Table 1. Violet fumes, indicating a cleavage of the organic iodine, were formed during the hy ‘Theelementary analyses were performed by Dr. E. W. D. drolysis. Silver nitrate added to the acidified Huffman, Denver, Cob. (HNO3) aqueous solution of iodouridine gave no

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1953 American Association for Cancer Research. PRUSOFF et al.—Non-utilization of Radioactive lodinated Pyrimidines 223 precipitate, an indication that the iodine atom was iodinated pyrimidine derivatives into regenerating organically bound. liver or experimental tumors was observed ; the Synthesü of iodouracil-I'31.—The method used tumors employed were Sarcoma 180, Brown was a modification of that of Johnson and Johns Pearce carcinoma, adenocarcinoma E 0755, 4-di (11). Radioactive iodide solution (5 mc.) was methylaminoazobenzene-induced hepatoma, and pipetted into a 50-ml. centrifuge tube and placed an x-ray-induced bone tumor.3 Both thyroid and in an ice bath. A solution of KI (0.06 M, 0.1 ml.) gastric tissues usually contained a higher concen was added, and then H2S04 (18 N, 0.05 ml.) and a tration of radioactive iodine than did the other tis solution of KIO3 (0.005 M, 0.8 ml.) in order to oxi sues which were investigated. Since these tissues dize the iodide to free iodine. After the addition of are known to concentrate the iodide ion, the prob a solution of uracil (200 mg.) in NaOH (2.5 N, 1.4 ability of the partial hydrolysis of these organic ml.), an 12K1 solution was added until a definite iodo-compounds in vivo is indicated. Tumors occa excess of free iodine was present. Upon warming sionally had some activity, but never more than and adding additional IrKI solution to maintain that of other tissues. The values shown for the dis an excess of iodine, iodouracil began to crystallize. tribution of radioactivity among the various tis The suspension was cooled in an ice bath for I sues were obtained as part of a screening program; hour, and then the crystallized iodouracil was re hence, because of the limited number of animals moved by centrifugation. The residue was sus studied, these results should not be interpreted as pended in water, acidified with acetic acid (9 N), being a statistically acceptable tissue distribution. and again centrifuged. The residue was washed with acidified alcohol and then S times with boil TABLE 1 ing ether. The compound was recrystallized from THE R@ VALuES OF HYDROLYTIC PRODUCTS OF water and then from alcohol. The yield was 276 SOME IODINATED PYRIMIDINES mg. of iodouracil with 82 per cent incorporation of SATURATED5I8O.AMTL ALCOHOL n-BUTANOL the original 1131added or 64 per cent of the theo N)RpPER CENT Na2HPO4WITE HCI (0.1 afterCoa@ownhydrolysisbefore Ry afterRp before Rp retical value. hydrolysis5lodouracil0.52 hydrolysis5hydrolysis Determination of radioactivity.2—The concen 0.60Uracil0.69 0.52055 tration of I's' in the tissues studied was determined 0.82lodouridine0.70 0.720.82 0.32Uridine0.80 0.690.34 with the aid of a scintillation counter constructed 0.710.18 0.34

by Maclntyre (15). Since I's' emits highly pene * With 1% N HC1O4 for 1 hr. in boiling water bath. trating gamma radiations, the tissue concentration of I's' could be determined directly without frac Prior to the administration of iodoorotic acid, tionation or isolation. In the earlier studies each an investigation was conducted of its toxicity in tissue was dissolved in NaOH (6 N) to give a final white mice. Eighteen mice in groups of three each volume of S ml. in a calibrated test tube. With a were given intraperitoneal injections of NaHCO3 specially constructed test tube holder, to maintain (1 per cent, 0.2 ml.) containing 0, 1, 5, 10, 15, or 20 constant geometrical relationships, the radioac mg. of iodoorotic acid. No toxic effect was ob tivity was determined by comparing it to the same served in any of the animals. volume of a radioactive iodide solution of known Radioactive iodine derivatives of orotic acid, specific activity. In order to eliminate the necessi uridine, and uracil do not appear to be of diagnos ty of dissolving the tissue and also to improve the tic value for the localization of centers of rapid nu geometric advantage, tissues were examined sub cleic acid synthesis, and, under the conditions of sequently by placing individual samples in a these experiments, no chemotherapeutic activity planchet and locating this directly beneath the was evidenced. crystal or the scintillation counter. SUMMARY lodouridine, iodoorotic acid, and iodouracil, la RESULTS AND DISCUSSION beled with J131,were synthesized. The syntheses of The results obtained from injections of iodo orotic acid, iodouridine, and iodouracil labeled a We are grateful to Dr. Harold P. Rusch of the McArdle Memorial Laboratory of the University of Wisconsin for rats with iodine-131 into various animal species, nor bearing 4-dimethylaminoazobenzene-induced hepatoma and mal and otherwise, as indicated, are shown in mice carrying Sarcoma 180; to Dr. Alfred Gellhorn of Co Table 2. No preferential incorporation of these lumbia University for donor rabbits bearing the Brown-Pearce carcinoma in the anterior chamber of the eye and donor mice 2We are grateful to Dr. H. L. Friedell, Dr. W. J. Mac bearing adenocarcinoma E 0755; and to Dr. Simon Koletsky Intyre, and Mr. J. S. Krohrner for advice and assistance in the oftheAtomic Energy ResearchProjectandtheDepartment of measurements of radioactivity and for supplying the I―, allo PathologyofthisSchoolofMedicineformaking availablearat cated to them by the Atomic Energy Commission. bearing an x-ray-induced bone tumor.

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1953 American Association for Cancer Research. TABLE 2

ADMINISTRATION AND LOCALIZATION OF RADIOACTIVE IODINE DERIVATIVES OP NATURALLY OCCURRING PYRAMIDINES

MITBOD CoMPonIm OP AD CONCENTRATIONOF ACTIVITY AND Mfl(ISTW TIME OF Microcuriea per SOLVENT ANIMAL CoanIrIoN TION TOTAL DOSE SACRIFICE Tissue gram wet weight 5-lodouri Rat Partially IPt 100 pc. (6.25 mg.) in 16 hr. after Thyroid 0.46±0.023 dine-I― (1)@ hepatecto 5 divided doses at last injec Stomach 0.01±0.001 (in 2 per mized 2-hr. intervals tion Remainder <0.005 cent starting18hr. NaHCO3) after hepatectomy Mouse Sarcoma IP 65 sic. (4.10 mg.) in 24 hr.after Thyroid and 0. 054±0.03; 0. 90±0.09 (2) 180 5 divided doses last in trachea during a 48-hr. jection Stomach 1.10±0.05;0.10±0.01 period Tumor 0. 25±0.01;0. 18±0.01 Liver 0.04 ±0.002; 0.01±0.005 Muscle 0.06± 0.003; 0.01±0.005 Rabbit Anterior IV@ 750 @uc.(47.0mg.) in a Thyroid 6 .4±0.32 (2) chamber 5 divided doses Tumor <0.005 transplant over a 48-hr. pe Stomach 0.89±0.045 of Brown riod Pearce car cinoma 5-Iodoura Rat Normal IF 10 pc. (1.73 mg.) 48 hr.after Alltissues <0.005 cil_11h1(in (1) injection propylene glycol)

“ X-ray-in Jugular 25 ic. (4.8 mg.) 24 hr. after All tissues <0.005 duced vein injection bone tumor Rat 4-Dimethyl IV 60 tic. (10.5 mg.) in 48 hr. after Thyroid 0 .90±0.09; 0 .25±0.13 (2) aminoazo 3 divided doses at last in Tumor 0.01±0.005; 0.06±0.01 benzene 24-hr. intervals jection Stomach 0.03±0.01; 0.03±0.01 induced Liver <0.005; 0. 08 ±0.006 hepatoma Spleen <0 .005; 0 .03 ±0.015 Remainder <0.005; <0.005 Rat a 150 tic. (25.0mg.) in Liver 0.25±0.13; <0.005 (2) 3 divideddosesat Tumor 1.36 ±0.06;0.14±0.07 24-hr.intervals Thyroid 1.64±0.16;3.52±0.&7 Stomach 2. 16±0.02; 0.02±0 .01 Remainder 0.25±0.13; <0.005 5-lodoura- Mouse Adenocarci IP 20 pc.(2.46mg.)in 3 hr. after Thyroid and 0.27±0.14 cil-I'31(in (1) noma 2 divided doses at last in trachea ipercent E0755 24-hr.intervals jection Tumor 0. 15±0. 015 Na,CO,) Testicle 0.31±0.03 Liver 0.04±0.004 Muscle 0.27±0.003 @ a a a a @c.(0.86 mg.) 2 hr. after Thyroid 0.14±0.07 injection Tumor 0. 10±0.01 Testicle 0.07±0.007 Spleen 0.08±0.04 Stomach 0 .02±0.01 Rabbit Brown IV 50 ,.tc.(8.6 mg.) 24 hr. after Thyroid 0.84±0.03 (1) Pearce injection Remainder <0.005 carcinoma 5-Iodoorotic Rat Partially IF 96 sic.(14.4mg.)in 19 hr. after Thyroid 0.90±0.30; 0.52±0.025 acid-I― (2) hepatec 3 divided doses at last in- Stomach <0.005; 0.03±0.015 (in1 per tomized 4-hr. intervals per jection Remainder <0.005; <0.005 cent day, for 2 days, NaHCO,) starting 24 hr. after partial hepa tectomy Mouse Sarcoma IP 10,uc.(1.Smg.) 3, 6 and 24 8-hr. tissues (3) 180 hr.after Thyroidandtra- 0.10±0.05 lastin chea jection Liver 0.01±0.005 Tumor 0.03±0.015 Muscle 0.08±0.008 Bone marrow 0.88±0.088 Remainder <0.005 6and24-hr.tissue <0.005

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1953 American Association for Cancer Research. PRUSOFF et al.—Non-utiization of Radioactive lodinated Pyrimidines 225

TABLE 2—Continued

METHOD CoSipouND OF AD CONCENTRATIONOF ACTIVITY AND MINISTRA Thez OF Microcuries per SOLVENT ANIMAL CONDITION TION TOTAL DOSE SACRIFICE Tissue gram wet weight a a Adenocarci a a a 3-hr. tissues noma Thyroidandtra- 0.16±0.08 E 0755 chea Tumor 0.08±0.04 Muscle 0.05±0.005 Bone marrow 0.07±0.02 Remainder <0.005 6 and 24-hr. tissue <0.005 a Rabbit Anterior IV 8O0@ic.(120mg.)in 24 hr. after Thyroid 0.24±0.003 (1) chamber 4 divided doses last in Eye tumor <0.005 transplant at 24-hr. intervals jection Remainder <0.005 of Brown Pearce car cinoma S Number of animals. t IP = intraperitoneal injection. IV —intravenous injection.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1953 American Association for Cancer Research. Non-utilization of Radioactive Iodinated Uracil, Uridine, and Orotic Acid by Animal Tissues in Vivo

W. H. Prusoff, W. L. Holmes and A. D. Welch

Cancer Res 1953;13:221-225.

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