An Investigation of the Value of Lead Compounds in the Treatment of Malignant Tumours

AN INVESTIGATION OF THE VALUE OF LEAD COMPOUNDS IN THE TREATMENT OF MALIGNANT TUMOURS

MEMBERS OF THE LIVERPOOL MEDICAL RESEARCH ORGANIZATION DIRECTOR, M. DATNOW, M.D., F.R.C.S.E. Organic Chemistry by B. Beileneolin, P1i.D. ; Q. F. Hownrd, P1i.D.; Dircctor, Professor I. M. Hcilbron. Pharmncology by T. N. A. Jeffconte, M.D., F.R.C.S.E.; Director, Professor W. J. Dilling

(From the University of Liverpool) Some encouraging results that had been obtained in the treatment of cancer by injecting the lead salts of various organic acids were described by Blair-Bell (1). His researches have been followed up, and this paper deals with the results of the main therapeutic investigations that have so far been undertaken. It has been recorded (2) that favourable reaults were obtained with lead salts of several aliphatic amino-acids, either in aqueous solution or in a suspended or super- saturated state, this last condition being attained readily by the addition of gum acacia (2 per cent) or gelatin (2 per cent), to the aqueous solution of the lead salt (3). While endeavouring to lower the toxicity of these preparations, our attention was drawn to the value of sodium thiosulphate for reducing the poisonous effects of heavy metals; this is enhanced by the property of this salt to form with lead salts complex ions which increase consider- ably the solubility of the lead salt. It was found, also, that substitution of the amino-group was frequently beneficial, the most efficacious sub- stitute being the aryl-sulphonyl radicle. A number of such compounds have, therefore, been prepared and examined with the object of deter- mining to what extent curative power is dependent upon structure. Unfortunately, the results have so far afforded little helpful evidence, since all such compounds, with the exception of p-toluene sulphonylglycine, are about equal in their therapeutic value. After successful pharmacological tests, the lead compounds were tested out for their carcinotropic action on rabbits on which the Brown- Pearce rabbit tumour had been grafted. In our hands this tumour has behaved with extraordinary uniformity. In no case out of some thou- sand inoculations was the tumour found to retrogress after it had been satisfactorily established. The percentage of takes has been over 88 per cent. The usual span of life for a rabbit after grafting is between three and six weeks, although a few have survived up to three months. At the commencement of our investigations, Rections were always taken prior to treatment so as to substantiate the presence of active cancer, and at the same time a laparotomy was performed upon the rabbits in order to observe the extent of the growth. Later our experience taught us that this was unnecessary, and a selection was made of the rabbits with the most advanced growths for treatment while the re- 531 532 LIVERPOOL MEDICAL RESEARCH ORQANIZATION mainder of each group was kept for control purposes. In no case was retrogression observed in an untreated control animal in which the tumour had been established. The rate of spread and period of survival of the treated animals were compared with those in animals not injected. Details will be given under the appropriate compounds. The lead preparations were all injected intravenously. Inhibition of the growth and survival of the rabbits for periods longer than the controls were taken as evidence of beneficial effect and good carcinotropic action. This was further confirmed by histological examinations. Our attention was drawn to a preparation (R.232) by Dr. Collier (4) of the Koch Institute, Berlin, which is the lead potassium complex of 2 :6 dithiol pyridine-4-carboxylic acid (see H.152). We have investi- gated several analogous compounds in which lead is attached to a mercapto-group, but the results obtained are as yet insufficient to war- rant any deductions as to their value. In the preparation of these lead complexes, the usual aim is a lead content of 0.5 per cent, which is similar to that of the original (5) colloidal lead (S.7), and which facilitates comparisons of the results obtained from different compounds. The method of administration to cancer patients has been intra- venous, commencing with initial doses of 3 to 5 C.C. of the 0.9 per cent Rolutions, subsequent dosage being entirely dependent on the response of the patient to treatment, which is estimated chiefly by the numbers of stippled red cells in the blood and by the renal excretion. The in- tervals between injections vary from one to eight weeks. They are fre- quent at first and are then gradually lengthened; patients are asked to report every six or eight weeks for six months, then every nine to twelve weeks for a year, aiid every twelve weeks after this. No untoward reactions have been noticed. EXPERIMENTAL Lead acetylglycinate (H.71), prepared by double decomposition of sodium acetylglycinate aiid lead acetate in an aqueous solution (1 per cent gelatin), forms a colloidal suspension, which is stable for several days. Lead thiosulphute (H.80), with the addition of one, two, or three molecular proportions of sodium thiosulphate, yields a satisfactory solution containing complex lead sodium thiosulphate. Leud benzene suZphoiL?lZgZ~cin.at~(H.126) : Benzene sulphonylglycine, C,H,SO,. NH. CHrCOOH, was prepared in 85 per cent yield according to the method which has been described by Ihrfelt (6). Freshly distilled benzene sulphochloridc (8.8 ,m.) was mixed with aqueous potassium amino-acetate (from 3.8 gm. glyciiie in 100 C.C. water) and the whole warmed to between 50-60", dilute potassium hydroxide being added, with stirring, at such a rate that the solution remained faintly alkaliiie throughout. On completion of the reaction (one and a half hours), the filtered solution was acidified with hydrochloric acid, when almost pure benzene sulplioiiylglyciiic separated out. The solid, after LEAD COMPOUNDS IN TREATMENT OF MALIGNANT TUMOURS 533 recrystallisation from dilute alcohol, formed hard, colourless rhombs, m.p. 169". An aqueous solution of the lead salt, prepared by double decomposition of sodium benzene sulphonylglycinate and the equivalent amount of lead acetate, at a concentration equal to 0.5 per cent lead, was unsatisfactory from the pharmacological point of view. In view of the results obtained by the addition of sodium thiosulphate to lead thiosulphate (see H.80), it was decided to treat the compound under discussion in a similar manner. Experience proved the following procedure to be the most efficacious manner of dispensing this arylsulphonylglycinate and the analogous ones described in this paper. For convenience all solutions 'of sodium arylsulphonylglycinates referred to in the following pages contain that weight of sodium salt in 100 C.C. solution which is equivalent to 5 gm.

Pra. 1. EFFECTOF H. 186 (LEADBENZENE SULPHONYLULYCINATE) ON BWD PRESSURE, SIXOWINGSLIGHT FALL AND RAPID RECOVERYA~EB INJECTION INTRAVENOUSLYOF 5 C.C. lead. The sodium thiosulphate solution employed contains 248 gm. per litre of the pentahydrate. The preparation of H.126 which is typical of all such compounds is, therefore, carried out as follows :

Solution A. 16 C.C. 5 per cent gum acacia solution. 4 e.c. lead acetate solution (containing 10 per cent lend). 20 C.C.distilled water. Solution B. 16 c.c. gum acacia solution. 8 c.e. stock sodium salt solution. 8 c.c. stock sodium thiosulphate solution. 8 C.C. distilled water. Equal volumes of solutions A and B are mixed thoroughly (usually in 3 C.C. portions) just before injection. The mixed solutions, on stand- ing for some days, gradually deposit lead sulphide, which renders them unsuitable for clinical use. Lead ions are undoubtedly present in these 534 LIVERPOOL MEDICAL RESEARCH ORGANIZATION preparations ; this is proved by the immediate formation of precipitates on addition of sodium phosphate or chromate to the mixture, but these precipitates show a decided difference in colour from the corresponding precipitates obtained with a simple lead salt. Potassium iodide does not precipitate lead iodide from these preparations. The compound was satisfactory from the pharmacological point of vicw (Fig. l),as it caused no material variation in the pulse or respiration, and the blood pressure changes were comparatively small, the fall tiveraging 15 mm. Hg and the recovery taking place rapidly in about 20 lo 30 seconds. These facts warranted the application of thiR preparation for therapeutic purposes, but it was first tested on rabbits in which the Brown-Pearcc tumour had been satisfactorily established and in many cases had reached a very advanced stage. A batch of 23 rabbits was used. Of these, 4 were completely cured, as no active growth was found post morfcm, although sect ions taken before treatment werc undoubtedly positive. The carciiiotropic .properties of this preparation are good as far as the Brown-Pearce turnour is concerned. The dos- ages and the histological findings will be recorded elsewhere, but it is of interest to note here that one of the rabbits lived for one year and eight months after inoculation with the growth, the usual survival period being three to six weeks. When treatment was commenced 011 this mimal, it had nodules in its peritoneal cavity, found at exploratory laparotomy, and there were secondary deposits in the left eye. An initial dose (10 c.c.) of H.126 was given intravenously and was followed eight days later by a further 5 C.C. The animal remained well for one year and seven months, when the eye turnour appeared to become active again. A fnrther dose of 10 C.C. was then given intravenously but the rabbit eventually died. Post mortem the rabbit was found to be free from growth except for its eye. Several other interesting phenomena occurred in the treated rabbits as a result of the healing of the malignant processes, such as intestinal obstruction and hydronephrosis. When treatment was initiated in one rabbit of this group, there was a large mass of growth in the pelvis and auotlicr in the trmcutum; there were also some nodules of growth in the left kidney. Under treatment the masses were observed to diminish in size, and the rabbit appeared to be cloiiig well. Subsequently, however, it was seen to become progrcssivcly thinner and eventually it had to bc destroyed. Post mortem no active growth was found; white plaques of fibrous tissue marked the situations of the tumour masses. The hcal- iiig of the mass in the pelvis had produced partial constriction of the lower end of the ureter, and this led to hydronephrosis with completc destruction of the kidney substance. The nodules in the kidney were replaced by fibrotic calcified white patches. A great disadvantage of this preparation is its tendency to producc marked stippling of the erythrocytes. In all the animals treated, definite histological changes attributable to the lead preparation were found ; these were characterised by cloudy swelling, fatty degeneration, arid necrosis of the caiicer cells. This necrosis was quite different in LEAD COMPOUNDS IN TREATMENT OF MALIGNANT TUMOURS 535 character from that found normally in this rabbit tumour, as it was not associated with the usual marked round-cell infiltration. The success of the experimental investigation with H.126 prompted us to apply it to the treatment of patients sufferiiig from cancer, and this is now in progress, with encouraging results. Lead p-toluene sulphonylglycinate (H.136) : p-Toluene sulphonylglycine, CH, . C,H,SO,-NH-CII,COOH, was prepared from equimo- lecular proportions of p-toluene sulphonyl chloride and glycine in a manner analogous to that employed in the preparation of H.126. The reaction mixture, after filtration, was rendered faintly acid by the addition of hydrochloric acid, and the voluminous white precipitate recrystallised from dilute alcohol, when colourless needles, m.p. 196- 198", were obtained.

FIQ.2. EFFEPTOF H. 136 (LFADP-TOLUENE-SIJLPHONYMLYCINATE) ON RWPIRATIONAND BLOODPRFS€iU&E, (JHOWINQFALL OF 10 MM. HQ IN BLOODPBEIEUBE, WITHOUT PULSE WEAKENINQ AFTE& THE FIFTHINTRAVENOUS INJECl'lON OF 5 C.C.

In a solution of gum acacia alone, a suspension of the lead salt, equivalent to 0.5 per cent Pb, and prepared by double decomposition of sodium p-toluene sulphonylglycinate and lead acetate, was unstable to some extent. The addition of sodium thiosulphate to the solution in the proportions outlined in the case of H.126 resulted in a satisfactory preparation (H.136). The pharmacological tests were satisfactory : initial injections caused no effects on either the pulse or respiration, but after the fifth injection slight falls in blood pressure, of not more than 15 mm. Hg, took place (Fig. 2). In a group of ten rabbits bearing Brown-Pearce tumours, and treated with H.136, there appeared to be no inhibition of the growth. Twenty per cent of the treated animals succnmbed before the controls, and we therefore conclude that this preparation has no carcinotropic action. 536 LIVEWOOL MEDICAL RESEARCH ORGANIZATION

Lead p-iodobetzzene sul2'7~oi~?jl,~l?jcinate(H.138) : p-Iodobcnzene sulphonylglycinc, I.C,H, . SO,.NH . CH, . COOH, was prepared as follows : p-Iodobenzeiie sulphochloride (1 mol.) was condensed with glycine (1 mol.) in thc prcscncc of alkali (7) in the manner already dc- scribed under the preparation of H.136. The crude solid, obtained by acidification of the reaction mixture, separated from aqueous alcohol as colourless needles, m.p. 199-200". (Found : S, 9.5 ; I, 37.2. C,H,O,NST requires S, 9.4; I, 37.2%.) A stable suspension of the lcad salt in gum acacia (0.5 per cent Pb) could not be obtained without tlie addition of thiosulphate solution, the mixture so obtained slowly depositing solid. Pharmacological tests showed that there were no respiratory effects and only negligible falls

FIG. 3. EFFECTOF H. 138 (LEADP.IODOBENZZNZ-SULPHONYLGLYCINATE) UWN REBPIEATION, PI~LBE,AND BLOODPREBSUBE, SHOWING NEULIGIBLE RESULT OF INJECTING5 C.C. INTRAVENOUSLY(FAST DRUM)

in blood prcssiirc, amounting to betwceii 4 and 15 mm. Hg; these are due to a very slight weakciiiiig in tho pulsc wave (Fig. 3). A batch of 14 tumour-betiring rabbits was treated with H.138. Thi.cc of the animals died bcforc thc controls, and three at about the same time. Of these latter, one showed no growth post mortem, death beiiig. due to some causc other than canccr. Most of the other animals died 2 to 12 weeks after the CoiitrolH, Hiid one animal is alive and appears normal seven months aftcr inoculation. This substance seems, therefore, a favourable one for cliiiical trial. Lead tolzie9te-2: 4-disul~~iioii?~l,~l?~ciriate(H.140) : Toluene-2 :4-disul- phonylglycine, CH,C,H,( S0,NH. C'HXOOH ),, was prepared by a modification of the method of Rosciigrcii (8). Toluene-2 :4-disulphonyl chloride (5.8 p.)dissolved in cther wtis mixed with aqueous sodium amino-acctate (from 3 gm. glycinc) and the reaction carried out in the manner described above. The solution, after careful acidification, was thoroughly cxtractcd with ether, the residual oil after ovaporation of the solvent slowly solidifying. Xecrystallisation from ethyl acetate yielded tlie pure compouiid (m.p. 185"). A satisfactory solution of the LEAD COMPOUNDS IN TREATMENT OF MALIGNANT TTTMOURS 537 lead salt (H.140), prepared as described under H.126, could be obtained only by the addition of sodium thiosnlphate solution. Pharmacological tests revealed no pulse or respiratory effects ; the blood pressure falls were quite moderate (5-15 mm. Hg.) aiid within thc limits of safety for injections of foreign substaiices (Fig. 4). Twelve rabbits treated with H.140 survived the control animals, two actually being alive and well twelve months after the tumour was origi- nally established. This preparation has very favourable carcinotropic properties and is being utilised to treat patients suffering from malignant disease. Lead m-carboxybenzene sulphon~tlglycinate (H.142) : m-Carboxy- benzene sulphonylglycine, COOH .C,H,SO,NH . CH,COOH, (9) was

FIO. 4. EFFECTOF H. 140 (LEADTOLURNE-E : 4-Dl8ULPHONYLOLYCINATE) ON aESPIRATION, . PULSE, AND n1mD PRESSURE, SIIOWINO FALLOF PRESSURE AFTER 5 C.0. INJECTED INTRAVENOUSLY prepared in 100 per cent yield by coiideiisiiig m-carboxybenzcne sul- phony1 chloride with glycine according to the general method described by Abderhalden (10). The pure compound melted at 194" after recrystallisation from dilute alcohol. (Found : 8, 12.2 ; N, 5.6. C,H,O,- NS requires S, 12.4; N, 5.476.) The normal lead salt (H.142) formed a satisfactory suspension in gum without the addition of sodium thiosulphate solution, and in this form gave satisfactory pharmacological results: the effects were slight; there was 110 weakening of either pulse or respiration arid the falls of blood pressure were usually about 8 mm. Hg (Fig. 5). 'Twelve rabbits treated with H.142 survived the control animals, and there was evidence of inhibition of growth in a11 the rabbits, the growth 538 LIVERPOOL MEDICAL RESEARCH ORGANIZATION extending very slowly as compared with the controls. Three of the treated rabbits died of iiitercurreiit disease and no growth was found post mortem. One is still alive, but the growth is progressing. This compound appears, therefore, to have fair carcinotropic properties. Lead 4-acetylaminophennetole-8-sz~lphonylglycinatc(H.151) : The preparation of 4-acetylaminophenetole-2-sulplionylglycine,CH,CO .- NH . (&H3(OC,H,) . S0,NH. CH, . COOH, (11), by condensing phenace- tin-2-sulphonyl chloride with glycine, was carried out in the usual manner. After acidification of the reaction mixture with acetic acid and recrystallisation of the crude product from hot water, the pure 4-acetylamiiiopheiietole-2-sulphonylglycinemelted at 166" (Found : 5,

FIG. 5. EFFECTOF H. 142 (LEAD M-CNLBOXYBENZENE-EULPK~NYMLPCINATE)ON RESPIRATION, PULSE, AND BLOODPaEsSUItE AFTER 5 C.C. INJECTED INT&AVENO[JSLY 10.3. CltHlaO,N,Srequires S, 10.2%). The lead salt (H.151) forms u. satisfactory suspension in gum acacia solution. The pharmacological tests were satisfactory; the injections causing very slight changes in blood pressure (maximal fall 8 mm. Hg), pulse, or respiration (Fig. 6). Thirteen cancer-bearing rabbits were treated with H.151 and most of them died withiii a month of their being grafted with the tumour. It is concluded, therefore, that the preparation has no carcinotropic properties. Lead calcium phosphate [Pb,Ca,( PO,),] (H.146) : The preparation of this substance in colloidal form for clinical purposes requires the following stock solutions : (1) lead acetate solution containing 10 per cent Pb; (2) trisodium phosphate solution containing 122.2 gm. of crystals (Na,PO, .12H,O) per litre ; (3) calcium acetate solution containing 42.51 gm. per litre calcium acetate (CR(AC)~.H~~). LEAD COMPOUNDS IN TREATMENT OF MALIGNANT TUMOURS 539 Two further solutions were made up from these, solution A consist- ing of: Lead acetate solution ...... 2.5 C.C. Calcium acetate solution ...... 5 C.C. 5 per cent gum acacia ...... 10 C.C. Distilled water ...... 7.5 C.C. while the second comprised : 8odium phosphate solution ...... 5 C.C. Gum acacia solution ...... 10 C.C. Distilled water ...... 10 C.C. These two solutions, when mixed, with stirring, yielded an opalescent

FIO. 6. ACTIONOF H. 151 (LEaD-4-ACErPLnMINOPHENETOLE-2-SULPHONYMLYClNATE)ON RESPIRATION,PULSE AND BLOODPILESSUBE, SHOXTN(~ NEGLI~IBLE EFFECTS AFTER 5 C.C. INJECTEDINTRAVENOUSLY fluid, a small amount of white precipitate being separated by centrifuging. The resulting colloidal solution was stable for several days. Pharmacological experiments showed that this preparation was very suitable for injection because it caused only slight falls in the blood pressure (average 12 mm. Hg) and no weakening of the pulse or respiration. Only three out of a series of ten rabbits bearing Brown-Pearce tumour treated with H.146 survived for longer than six weeks, and post mortem these three were found to have numerous metastatic deposits in various parts of the body. It is therefore concluded that this substance has poor carcinotropic properties. Lead fumarate (H.149) (12) : As a solution of the normal lead salt, containing 0.5 per cent Pb, tended to deposit crystals, recourse was had to the addition of sodium thiosulphate as already described for H.126. The resulting mixture was quite satisfactory. Lead fumarate, when injected, causes very slight reductions in blood pressure (average 10 mm. Bg) without any weakening of the 540 LIVERPOOL MEDICAL RESEARCH ORGANIZATION

TABLEI: H.15.9 Protocole -___ Date No. o tumour Injections Date of Result anima inoculated given death _-- I 30.432 i C.C. 8.6.32 6.7.32 Right kidney hydronephrotic. Small masses 5 C.C. 20.6.32 of growth on cord and several large glands in lumbar region. Slight delay of fatal issue.

2 20.5.32 5 C.C. 8.6.32 22.6.32 Rabbit became paralysed after injection and had faecal and urinary incontinence. It had to be destroyed. The growth was very necrotic.

3 3 1.5.32 5 C.C. 15.6.32 14.7.32 Generalized dhmination of growth. There 5 C.C. 29.6.32 seemed to be no beneficial effect of the injections.

4 31.5.32 5 C.C. 15.6.32 12.7.32 Death due to extensive pleurisy with effusion. 5 C.C. 29.6.32 No metastases found; primary growth necrotic.

5 31.5.32 5 c c. 15.6.32 4.7.32 Bilateral hydronephrow. Several meta- 5 c.c 29.6.32 static nodulm present but not actively growing.

6 28.7.32 7 C.C. 7.8.32 23.8.32 Marked involvement of both kidneys. No obvious effect on the growth.

7 m.7.32 7.5. C.C. 7.8.32 5.9.32 Tumour developed more slowly than in controls, but rabbit died eventually of metas- tssee. 8 17.E.32 5 C.C.2.9.32 7.9.32 This rabbit had marked diarrhoea and the Erowth appeared to be extensively necrotic. Toxic absorption may account for the death of this animal.

9 17.8.32 5 C.C. 2.9.33 7.9.32 This rabbit had marked diarrhoea and the growth appeared to be extensively necrotic. Toxic absorption may account for the death of this animal. pulse ; there occurs, however, from repeated injections, a progressive slowing of the respiratory rate. The results obtained with n series of 17 rabbits bearing Brown- l'earcc carcinoma were very unsatisfactory, as most of the animals died without showing any retardation of growth due to the lead compound. Lead 2: G difhiolisonicotinnfe (H.152), COOK( C,H,N)S,Pb: For this preparation (13), 2 : 6-dicliloro-citraziiiic acid (5 gm.) with a solution of potassium hydrosulphide (from 5 gm. potassium hydroxide) in methyl alcohol (50 c.c.) was heated under pressure for five hours at 135O, after which the reaction mixture was filtered and the orange-red filtrate concentrated by evaporation under reduced pressure at 50-60'. LEAD COMPOUNDS IN TREATMENT OF MALIGNANT TUMOURS 641

TABLEI (Cdinupd) - Date No. a tumour Injectione Date of Result anid inoculated given death

10 25.9.32 5 C.C. 12.11.32 29.1.33 No growth found post mortem. No definite 5 C.C. 22.11.32 cause of death ascertained. 5 C.C. 8.12.32 5 C.C. 5. 1.33 5 C.C. 27. 1.33

11 25.9.32 5 C.C. 12.11.32 13.11.32 Sections show more necrosis than before treatment. No marked dissemination of growth.

12 18.10.32 5 C.C. 12.22.32 14.11.32 Same as 11, with somewhat more diseemina- tion.

13 25.9.32 5 C.C. 12.11.32 13.1.33 Cause of death not obvious; may be due to toxic sbeorption. Bection showed marked necrosis of all the growth.

14 8.12.32 10 C.C. 10.1.33 12.1.33 Death from peritonitis. Sections show marked nccrosis of the growth.

16 22.12.32 10 C.C. 10.1.33 5.2.33 Sc3ctions showcd a good deal of necrosis. 7 C.C. 20.1.33 10 C.C. 3.2.33

16 12.1.33 13 C.O. 27.1.33 7.3.33 Death due to diarrhoea. Sections showed to 15.2.33, in mmive necrosis of the growth. ten intraven- ow do=, at first daily; t h e n every other day.

17 12.1.33 12.5 C.C. 27.1.3: 1.5.33 There was a large mass in the pelvis to which -15.2.33. the intestines were adherent. Sections 1 C.C. 25.3.33 show only necrotic material. 5 C.C. 21.4.33

18 12.1.33 15.5 C.C. 27.1% 18.2.33 Jections show a great deal of necrosis. -10.2.33

The solution so obtained was acidified by the careful addition of dilute hydrochloric acid, the yellow precipitate washed thoroughly with water, and finally dried over sulphuric acid in an atmosphere of nitrogen. The crude acid was converted into the mono-potassium salt, and its warm aqueous solution treated with a slight excess of lead carbonate or oxide. The clear solution obtained after filtration was faintly alkaline in reaction, and was restored to neutrality by the addition of 5 C.C. of N/100 hydrochloric acid. Evaporation of the now neutral solution, preferably in a vacuum desiccator, yielded the potassium-lead complex as a yellow, extremely soluble powder. An aqueous solution of this subatance gave no precipitate on addition of sodium phosphate solution. On pharmacological test, this preparation (H.152) caused moderate 542 LIVERPOOL MEDICAL IlESEAHCH OHGANlZATION falls of blood pressure (average 14 to 18 mm. Hg). There was, however, no marked weakening or any irregularity in the cardiac contrac- tions and no respiratory weakening. Further tests on rabbits with tumours showed that, although the preparation had some definite in- hibitory power on the rate of tumour growth, it was very irritant locally when injected and caused thrombosis in the veins. It also produced cloudy swelling in the renal and hepatic cells. It appears, therefore, unsuitable for human application. Sodium lead complex of o-thiolbemoic acid (H.153) : This preparation is obtained by dissolviiig sodium o-thiolbenzoate (from 15.4 gm. acid) in 200 C.C. water, warming the solution to about 80" in an atmosphere of nitrogen, and adding freshly precipitated lead hydroxide or carbonate until no more is dissolved. After half an hour, the solution is filtered and concentrated to small bulk under reduced pressure. The concentrated solution is then taken to dryness in a vacuum desiccator, whereby a readily soluble yellow did is obtained. Analytical data show that this preparation is not the normal mercaptide (Pb content 37 per cent) but a complex containing 9 per cent lead. No ionic lead reaction is given 011 addition of sodium phosphate solution, but lead sulphide is precipitated by hydrogen sulphide. This preparation is dispensed as a solution containing 0.1 per cent Pb. It was tested pharmacologically at first as a solution containing 0.5 per cent Pb. In this strength it caused severe reductions in blood pressure (average 40 mm. Hg) and, although initial injections caused no apparent weakening of the pulse, this effect was very marked after three injections, and there occurred also a temporary inhibition of respiration followed by compensatory increase in rate and depth. On dilution of the preparation, it was found that it produced negligible effects when injected in similar doses (5 c.c.) of 0.1 per cent Pb, and in this strength was recorded as safe for injection. H.153 proved to have fairly good carcinotropic properties. It produced necrosis of the growth in most of a series of 18 tumour-bearing rabbits, 3 of which were completely cured (see Table I). This preparation has been employed for the treatment of human beings and has been found to be non-toxic. It is interesting to note that the urine is stained yellow by the excretion of the substance through the kidneys. It has been found in the urine a few minute8 after its injection into the vein. We have frequently noted that the rabbits develop diarrhoea and die when extensive necrosis is present. This is probably due to toxic absorption. Lead pheqgyl thioglycollnte (H.160) : Phenyl thioglycollic acid, C,H,S. CH, . COOH, was prepared in excelleiit yield by the method of Osoki (14). The lead salt (H.160) forms a satisfactory suspension when dispensed in the usual way (see H.126). Pharmacological tests show that, although initial injections cause only slight falls (8 to 15 mm. Hg) in blood pressure, these increased (30 to 40 mm. Hg) after the foiirth injection, and the respiratory rate became gradually slower. The effect on blood pressure is cardiac in LEAD COMPOUNDS IN TREATMENT OF MALIGNANT TUMOURS 543 origin; there are evident weakening of the pulse and occasional cardiac irregularity. The preparation is regarded as safe for single injections. H.160 has some carcinotropic action on the Brown-Pearce tumour. It definitely retarded growth in 6 out of a series of 9 rabbits treated, but none was cured, and dl the rabbits died of malignant disease eventually. Partidly Tested Lead Compoulzds The following preparations have not yet been tested with regard to their influence on the Brown-Pearce tumour : Lead o-carbozyphenyZthiogZyco2late (H.161) : o-Carboxyphenyl- thioglycollic acid, COOH . C,H,S . CH,COOH, was prepared according to the directions of Friedlaender (15). The lead salt (H.161) dispensed in the usual way formed a suitable preparation without the addition of sodium thiosulphate solution. Pharmacological experiments showed this preparation to be safe for injection; it caused a slight rise succeeded by a slight fall in blood pressure and no respiratory effect. Lead complex of m-thidbenxoic acid (H.l64), waa prepared according to the method of Smiles and Stewart (M), or by reduction of m- carboxybenzene sulphochloride with tin and hydrochloric acid. The sodium salt was prepared in exactly the same manner as H.153, which it resembles very closely. Analysis shows the presence of approxi- mately 17 per cent lead, which, as in the case of H.153, is present in a very complex form. This preparation, when injected, produced falls in blood pressure (average 18 mm. Hg) which are regarded as within limits of safety; the recovery is somewhat slow, but this should not be of serious import. The respiration is gradually slowed by repeated injections, but this effect is compensated by an increase in depth. The potassium salt iR probably a better preparation pharmacologically, as the recovery from the fall in blood pressure is more rapid. Lead sodium thioglycollate (H.166) : This salt, prepared as described by Rosenheim and Davidsohn (17), has been shown to have the normal structure Pb(-S-CH,. COONa),, a statement which we have confirmed. A saturated aqueous solution of this salt, equivalent to 0.3 per cent lead, gave none of the usual reactions for ionic lead, but deposited lead sulphide on addition of sodium sulphide solution. The pharmacological tracings show only slight falls in blood pressure succeeded in early injections by a small rise in the level. There is no weakening of the pulse or respiration, and the preparation appears very suitable for injection. Lead quinoEinc?-s-sulpJo~~?~lgl~~~n~te(H.158): For the preparation of quinoline-8-sulphonylglycine,C,H,N . SO,NH .CH,COOH, a mixture of finely powdered quinoline-8-sulphochloride (18) (7.6 gm.) and gIycine (2.5 gm.) in water (50 c.c.) was warmed to about 60°, and potassium hydroxide was added with vigorous stirring over one and a half hours. The solution was cooled, filtered, and made acid to Congo red by the careful addition of dilute sulphuric acid. The precipitated 544 LIVEHPOOL MEDICAL RESEARCH ORGANIZATION oil, on cooling and scratching, solidified to a pale yellow crystalline solid, which separated from ethyl acetate in hard, transparent prisms, m.p. 136", easily soluble in alcohol, but almost insoluble in benzene or ligroin (Found: N, 10.5. C,,H,,SO,N, requires N, 10.5%). The lead derivative of this acid was dispenRed in exactly the same way as H.126. This compound causes reductions (average 12 to 15 111111. Hg) in blood pressure which are within limits of safety; there occurs slight weakening of the pulse after the seventh injection, but no respiratory effects. Lead o-ftuorobenzoate (H.130) : o-Fluorobenzoic acid, F.CmH,. - COOH, (19) was readily prepared by the oxidation of o-fluorotoluene by means of potassium permaiiganate according to the method of M. Holleman (20 ; 21 ) . The lead salt (H.130) formed a stable solution on dissolving in water. Pharmacologically, this was satisfactory ; it caused slight falls (10 mm. Hg) in blood pressure, but no weakening of the pulse or respiration. Unsatisfactorp Lead Compounds In addition to the foregoing, several other preparations have been examined and reject ed owing to their unsatisfactory pharmacological act ions. Lead diph~v~ylet7aer-rJ.iszilp~~~a~l.~lycinate (H.144) : Diphenylether- disulphonylglyciiie, HOOC .H,C . HN. 0,s .C,,H,O . C,H,SO,NH . CH,- COOH, was prepared as follows : Diphenylether-disulphonic acid (Fit- ting, 22 ; Hoffmeister, 23) was converted into the disulphon-chloride by the action of phosphorus pentachloride (50 gm.) for five hours at 140" on the finely powdered barium salt (55.8 gm.). After addition of water to the cold reaction mixture, the insoluble disulphochloride was separated off, dried and crystallised from benzene-ligroin, separating as colourless rhombs, m.p. 124-125" (Found : Cl, 19.4. C,,H,O,S,Cl, requires C1, 19.4%). The foregoing disulpho-chloride (7.4 gm.) was condensed with glycine (3 gm.) in the usual manner. Acidification of the reaction mixture yielded a colourless solid, insoluble in benzene or alcohol, but soluble in hot water, from which it was crystallised. The solid thus obtained melted at 210" (decornp.) (Found: S, 14.1; N, 6.4. C,,H,,O,- N,S, requires S, 14.4 ; N, 6.3%) . The lead salt (H.144), formed in the usual way, yielded a satisfactory preparation only on the addition of sodium thiosulphate solution. Pharmacological tests iiidicatcd that the preparation was not safe. There were severe falls of blood pressure (from 50 to 90 mm. Hg), with weakening of the pulse and very slow recovery. The respiration was also affected; in one tracing it is increased by about three times in depth, about one minute after the injection-a type of dyspnea-and in another it became irregular after several injections and finally failed (Fig. 7). LEAD COMPOUNDS IN TREATMENT OF MALIGNANT TUMOURB 545

Lead bemefiesuZphonyZ-,5-am.in.ova2erianate (H.141) : Benzenesul- phonyl-5-aminovalerianicacid, C,H,SO,NH( CH,),COOH, was prepared by the method of Schotten and Schliimann (24). The lead derivative (H.141) could only be obtained in a stable form by the addition of sodium thiosulphate solution. Experiments proved this preparation to be unsatisfactory because it caused large and rapid falls in blood pressure (from 30 to 60 111111. Hg), which remained below normal for several minutes, and also showed a progressive decline on repeated injections. The initial falls are due to cardiac weakening, but there is also a dilator effect on the vessels.

FI~.7. EFFECTOF H. 144 (LmDIPAENYIXTHER-DIBULPHONYLGLY(XNATE) UPON REBPIRA- "ION, PULSE AND BLOODPREBBURE, SHOWING SEWFALL (76 MM. HG) OF BLOODPsEsRURE AND IBREWLAIUTYQF BE~PIEATION AFTER 5 C.C. INJECTID INTRAVENOUSLY

Lead Naphthalene-I-sulph,onyl!jglyciwate ( H.148) : Naphthalene-l- sulphonylglycine, C,,H,SO,NH .CHXOOH, was prepared as follows. Naphthalene-1-sulphochloride (1 mol.) and glycine (1 mol.) were condensed in the usual manner. The resulting naphthalene-l-sulphonylglycine, after recrystallisation from benzene or aqueous alcohol, formed colourless needles, m.p. 141". The sodium salt is not very soluble in water (Found: S, 11.1. Cl,H,,,O,NSNa requires S, 11.15%). This substance forms a lend derivative (H.148) which can be dispensed in a satisfactory form only by the addition of sodium Ihio- sulphate solution. The pharmacological tests were unsatisfactory. There was marked weakening of the pulm, with at first little change in the respiration, but 546 LIVERPOOL MEDICAL RESEARCH ORGANIZATION the effects were cumulative and later injections caused increasingly large falls in blood prewure (30 to 50 mm. Ha) and progressive slowing and weakening of respiration (Fig. 8). 7-11ydrO$:y-2:3-diphenyl clwomone (H.65): This compound was examined in the hope that its lead salt might be capable of gel formation, an noted in the cases of the sodium and potaclsium salts (Robinson and Baker, 25). On mixing equivalent amounts of the chromone potassium salt and lead acetate solution at concentrations such that the resulting mixture contained 0.5 per cent Pb, a certain amount of precipitation took place. After filtration, the clear solution contained 0.45 per cent lead.

%'lo. 8. ACTIONOF €1. 148 (LEALINAPHTHALENE-~-BULPHONYLOLYCINATE) ON RESPIRATION PULBE AND BLOQD PRESBURE, [JHOWINO FALLOD' 56 MM. HO AND RESPIRATORY 8LoWINO dFmcB 5 C.C. INJECTED INTJ&AVENOUBLY

The falls in the blood pressure (average 30 mm. Hg) caused by injections are beyond limits of safety, although the recoveries are re- markably rapid. The dicrotic wave in the pulse tracing suggests that the falls in pressure are due to arterial dilatation and not to cardiac impairment. The respiration is not influenced. The preparation is probably unsafe for clinical treatment. Lead sozoiodolate (H.111) : Lead sozoiodolate, prepared by dc- compositioii of lead acetate and sodium sozoiodolate (0.7619 gm.),was triturated with sodium thiosulphate pentahydrate (2.28 gm.),and the resulting sticky mixture dried in air. The dry solid, after being dissolved in 50 c.~.distilled water, was suitable for testing. This is a fairly satisfactory preparation, pharmacologically. It causes falls (10 to 20 mm. Ha) of blood pressure with slow recoveries ; there is slight wcakeiiirig of the pulse wave and also slight acceleration of respiration. LEAD COMPOUNDS IN TREATMENT OF MALIGNANT TUMOURS 547 The substance was very toxic to rabbits and produced definite necrosis of the liver with haemorrhages in the kidney cortex. It did not have any choriotropic action on the placentae of pregnant does. p-Carboxyyheuyll orthoylmykoric acid: The normal lead salt of this acid formed a satisfactory suspension in gum acacia (H.76). The isomeric m-carboxy derivative yielded a red, translucent colloid in the presence of gum acacia (H.79). This is an unsatisfactory preparation. It produces reductions (20 to 40 mm. Hg) in blood pressure, associated with weakening of pulse and slow recoveries. It has no respiratory effect. Amidophosphoric acid (H.96) (Stokes, 26) yielded, on treatment of its disodium salt with one equivaleiit of lead acetate in gum acacia solution, a stable suspension, from which only a trace of insoluble matter could be separated by centrifuging. Pharmacologically this is a highly satisfactory preparation, having practically no effect on blood pressure, pulse, or respiration. It produced haemorrhages and necrosis throughout the medulla and kidney cortex. In the livers of the treated rabbits cloudy swelling and necrosis were found but were not localised to any particular area.

SUMMARY 1. The general method employed in finding therapeutically active and safe lead compounds for the treatment of malignant disease is outlined. 2. The details of the preparation of 27 organo-lead compounds are described. 3. The pharmacological tests of those preparations which were chemically satisfactory are discussed and illustrated. 4. A summary of the results obtained by treating Brown-Pearce tumour-bearing rabbits with the pharmacologically satisfactory compounds is given. REWRENCE~ 1. BLAIR-BELL,W. : Some Aspects of the Cancer Problem, Baillihre, Tinclall & Cox, Lon- don, 1930, p. 417. 2. BLAIB-BELL, W. : Some Aspects of the Cancer Problem, Baillihre, Tindall & Cox, Lon- don, 1930, p. 388. 3. DIOKINBON,R.: J. Chem. Soc., London, 1 : 358,1929. 4. COLLIER, W. A.: Ztschr. f. Hyg. u. Infektionskr. 110: 236, 1929. 5. BLAIR-BELL,W. : Some Aspects5 of the Cancer Problem, Baillihre, Tinclall & Cox, Lon- don, 1930, p. 247. 6. IHRFELT:Berichte d. deutsch. chem. Gesellsch. 22: Ref. 692, 1889. 7. TROWER, J., AND HURDELBRINK,F.: J. prakt. Chemie 65: 83, 1902. 8. RQSENQREN,L. F.: Berichte d. deutsch. chem. Oesellsoh. 27: 888, 1894, Band 4. 9. SMILES,S., AND STEWART,J.: J. Chem. SOC., London 2: 1792, 1921. 10. ABDERHALDEN,E. : Fermentforsch. 12 : 180, 1930. 11. JOHNSON,R. N., AND SMILES,S.: J. Chem. Soc., London 2: 2384, 1923. 12. FOTJRNIFJ~:Con@s Francais de MBdecine, 1931. 13. BEHRMANN,A,, AND HOFMANN,A. W. : Berichte d. deutsch. chem. Gesellseh. 17 : 2681, 1884. 14. OBOKI: J. Chem. SOC.,Japan 52 : 460,1931. 548 LIVERPOOL MEDICAL RESEARCH ORGANIZATION

15. FHIEDLAENDER,P., AND CHWALA, A.: Monatsh. f. Chemie 28: 270,1907. 16. SMILEB,S., AND STEWART,J.: J. Chem. Soc., London 2: 1792, 1921. 17. ROBENHEIM, A., AND DAVIDBOHN, J. : Ztschr. f. anorg. Chem. 41 : 231, 1904. 18. EDINGER,A. : Berichte d. deutsch. chcm. Qesellsch. 41 : 937, 1908. 1% HAM, Q., AND SCHIEMANN, G. : Berichte d. deutsch. chem. Qesellsch. 60 : 1186, 1927. 20. HOLLEMAN,M. : Rec. d. Trav. chim. d. Pays-Bas et Belg. 24: 26, 1905. 21. HOLLEMAN,A. F.: Rec. d. Trav. chim. d. Pays-Bas et Belg. 25: 332, 1906. 22. FITTIC?,R. : Annalen der Chemie u. Pharm. 125 : 329,1863. 23. HOFFMEIBTER, w.: Annalen der Chemie u. Pharm. 159 : 204,1871. 24. SOHOTTEN,C., AND SCHL~~MANN,W.: Berichte d. deutsch. chem. Clesellnch. 24: 3600, 1891. 25. ROBINBON, P., AND BAKER,w.: J. Chem. soc., London 2: 1981,1925. 26. STOKEB,H. N.: Am. Chem. J. 15: 198, 1803.