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Microscale syntheses of anti-tumour platinum compounds labelled with 191Pt
hr. J. AppI. Radiar. hoc. Vol. 36, No. 3. pp. IEI-184, 1985 Printed in Great Bntain. AU rights reserved
OOE-708X, 85 53.00 f 0.00 Copyright C 1985 Pergamon Press Ltd
Microscale Syntheses of Anti-Tumour Platinum Compounds Labelled With 19*Pt J. BAER,
R. HARRISON,‘? C. A. McAULIFFE,‘* H. L. SHARMA’ and A. G. SMITH’
A. ZAKI,’
‘Department of Chemistry, University of Manchester Institute of Science and Technology, Manchester M60 lQD, and ‘Department of Medical Biophysics, Stopford Building, University of Manchester, Oxford Road, Manchester Ml3 9PT. U.K. (Received 26 January 1984; in revisedform 1 October 1984) Several compounds of platinum have been found to have significant anti-tumour activity and are in various stages of clinical trials. Four such compounds: cis-PtCl,(NH,),, cis PtCl,(cyclopropylamine)2. cis,trans-PtC1,(OH)2(isopropylamine), and cti-Pt( 1, I-cyclobutanedicarboxylate)(NH,)1 were synthesised with radioactive platinum-191 as a label for the study of animal organ distribution, patient blood clearance, urinary excretion and renal uptake and clearance. This paper describes the microscale synthesis of these compounds. Purification and quality control procedures are also described.
Introduction
Although cis-dichlorodiammineplatinum(II), cisPtC12(NH3)2,(cis-DDP) has proved to be remarkably potent against several tumour types and has become established as a useful clinical agent,“’ it suffers from several disadvantages. Among these are adverse reactions including nephrotoxicity and occasional hearing loss, nausea and allergy. The renal toxicity is particularly unwelcome since it may be dose limiting and preclude further treatment using this drug.(k3’ Considerable effort has been expended worldwide in preparing and testing hundreds of analogues of cis-DDP in the hope that superior drugs might emerge. (4)As well as low nephrotoxicity a search for platinum drugs effective against tumours resistant to cis-DDP therapy is also a current aim. Several such (second generation) drugs have emerged which are proving to be as effective as, or superior to, cis-DDP in some respects; amongst these are cis-dichloro-rran.s-dihydroxo-ci.s-diisopropylamineplatinum(H) (CHIP)‘” and (1, l-cyclobutanedicarboxylate)diammineplatinum(II) (CBDCA).(@ Tne objective of this work was to develop procedures for microscale synthesis of such complexes in order to produce their radiolabelled counterparts. The radionuclide used for these syntheses is mainly 19’Pt (tliZ = 3 days) with small quantities of Ig3’“Pt (t,!? -4.3 days) and ls8Pt (t,,: = 10.2 days), being present. The production procedure for the radioisotope has been described briefly in an earlier *Author to whom all correspondence should be addressed. t Now at Amersham International, Amersham. *.a.,.
36,SA
18
publication”’ and the limitations imposed by the presence of ‘*8Pt has been reported by Robins and Leach.(8) These radiolabelled compounds have been used in organ distribution studies on normal and Yoshida tumor-bearing ratsc9) and for blood clearance and clearance via urine studies in selected human subjects.“‘.“’ For these tracer studies in humans and laboratory animals, radiopharmaceuticals had to be of high specific activity, because of the toxicity associated with these compounds, therefore the syntheses had to be carried out on a small scale (SO-100 mg). The present report is on the microscale synthesis procedures, some of which have been a refinement and optimization of the published work.‘“.‘3’ Experimental Microscale Flexes
syntheses
of the *Pt-radioiabelled
corn -
The procedure developed started with the addition of inactive Na,PtCl, to the carrier-free H,*PtCI, (* indicates radioactive platinum), the mass of the carrier-free material being negligible. Regular preparations have been carried out on 0.05 g Na,PtCl,. An outline of the microscale preparation of the *Pt-radiolabelled drugs is shown in Scheme 1. Considerable effort went towards optimising experimental conditions at each stage, and its yield. Transfer of the reaction mixtures and products between vessels, and filtrations, were avoided; the bulk of the preparation was carried out in one small centrifuge tube to minimise losses. Some features of interest to be noted are:
J. BAERet al.
182
Na,‘PtCI, i Na,‘PtCI, I Na,‘Ptl, I cis-[‘PtA,I,]a i
N2H,.ZHCI. pH=54
C-2’C.
5 mm: 65’C. 5mm
Scheme 2.
6Nal. O’C. 1 min
3A, 5 mm. OT; 50%
AgNO,,
15 min
10 min. ambent
temp.
cis-[‘Pti,(H,OM (NO,), I
NaCl or HCI
(0.1 N). 1-2 h. 35-4672
cis-[‘PtA,CI,]” i [‘PtA,(OH),Cl,]b
A=ammonia (cis-DDP, CBDCA) isopropylamine (CHIP) cyclopropylamine (CP)
mixed iodide/chloride complexes. Partial dissolution of the iodide complex was found to increase the rate of the reaction. (6) A considerable excess of NaCl or HCl (0.1 N) was used to precipitate excess silver and to maximise the rate of reaction. Quality control of the *Pt -radiolabelled complexes
H,O,.
6o’C.
1Omin
Scheme I. “LOSSof activity in supematant and washing is IO-20x, depending upon iridium concentration. bYields: cis-DDP* 760/,; CP* 65%; CHIP* 70%; CBDCA* 70%. (1) Na,PtCl, was employed in preference to K2PtC1,, since it is more soluble, and thus very concentrated solutions can be used in the reduction and subsequent reactions, with a consequent shortening of reaction times. Hydrazine hydrochloride was chosen in preference to other reducing agents. (2) pH control was found to be important. At low pH, protonation of the amine competes with platinum-amine complexation and the rate and extent of reaction decreases. High pH conditions are also to be avoided since the hydroxyl ion has a high affinity for platinum, and this could interfere in the third step at which all four coordination sites need to be occupied by iodide ions. (3) The addition of six mole equivalents of sodium iodide enhances the stability of the [PtI$- ion and reduces the chance of PtI, precipitation. (4) Three mole equivalents of amine were used in order to increase the rate of cis-[PtA& formation. After reaction the diiodo product is washed thoroughly in order to remove unreacted platinum salts. Also, in the supernatant liquid was the iridium impurity produced by the initial activation of osmium plus the subsequent decay of any lE5Pt, ls6Pt, ‘s7Pt, “*Pt or ‘89Pt. Reduction of iridium(W) in step (1) gives the inert d6 iridium(III), which is not complexed, and so remains in solution. (5) Silver nitrate was added in 10% excess in order to ensure the complete precipitation of the silver iodide and thus avoid the eventual formation of Table I. Thin layer chromatography
Thin layer chromatography was found to be satisfactory as a method of purity control for these three *Pt-radiolabelled complexes. Only small quantities of the complex were required and the procedure was rapid and straightforward. Table 1 lists R, values obtained for samples of the cyclopropylamine complex (CP) and its iodide analogue, with various eluent systems using alumina as stationary phase. To allow for unambiguous identification of spots authentic compounds were always used as markers when analysing unknowns. The eluent mixture chosen for routine analysis of CP* was acetone: HCl(1 M) (19: 1) for both alumina/glass and alumina/plastic chromatography plates. Table 2 lists some data for CHIP and several potential platinum-containing impurities obtained with various eluents using alumina/glass plates. AcetonejHCl gives a good separation between CHIP and the other complexes. The purity of the recrystallised cis-DDP* was estimated by employing the TLC analysis on alumina/glass plates using acet0ne:O.l M HCl (7:3) as the eluent mixture (R/= 0.63); CBDCA was eluted using a 7:3 isopropanol/water mixture (R,= 0.45). The purity of the radiolabelled complexes was measured quantitavely by counting the activity of both pure and impurity spot(s) on the glass/plastic TLC plates using an autogamma counter. Impure samples (< 96%) were purified as follows: CHIP was purified via preparative TLC alumina/glass plates using the eluent mixture acetone:0.12 M HCl (9:1), and the recovery of the radiolabelled complex was achieved by extraction from the stationary phase with methanol; CP* was passed through a pressurised column silica gel as stationary phase and using acetone/CH;OH was eluent, this method being found to be both quick and efficient, and fractions were data for cyclopropylamine
complexes R,
Plates Alumina;glass Alumirwplastic
+Eluent
cis-~cll(cP,?J
ci.Y-[PtL(CP)I]
(19:l) Acetone:HCI(l M) (9: I) Acetone:H,O
0.84 0.82
0.96 0.95
(19:l) Acetone:HCl(I M) (9:l) Acetone:H,O
0.90 0.77
0.94 0.83
Acetone (9:l) Acetone:CHCI,
0.15 0.08
0.32 0.26
183
Microscale syntheses of anti-tumour platinum compounds Table 2. Thin layer chromatography Complex cir-,rranr-IPtCI,(OH)zcIP,zl
data for isopropylamine complexes Eluent
(CHIP)
rronr-.~rcurr-,~~~rr-[Pt(OH)P~(rPfZl rrans-[PtCI,(IP)J
cis-IPtCl*(IP)J
cir-N,(IP)?J
(9: I) Acetone:H,O
(9: I) Acetone:HC1(0.12 M) (9: 1) Ch,CI,:EtOH (9:l) Acetone:H,O (9: I) Ch,CII:EtOH (9: I) Acetone:HZO (9: 1) Acetone:HCI (9: I) CH&EtOH (9: 1) Acetone:HzO (9: I) Acetone:HCl (9: I) CH,CI,:EtOH (9: I) Acetone:H:O (9:l) Acetone:HCl
Rf 0.42 0.47 0.19 1.00 0.97
1.oo 0.96 0.92 0.94 0.84 0.65 0.97 0.98
water. Silver nitrate(192 pmol, 0.1 cm3) was then added and the mixture was stirred for 15 min at 40°C. To the solution containing the diaquo species (cis[pt(NH3)z(HzO)&N03)1), sodium chloride (0.2 cm3, 1040pmol) was added and the temperature held at 35-45”C for 15 min and the volume was reduced to 0.3 cm3. On cooling, cis-DDP crystallised; this was filtered and washed with distilled water (0.2 cm3), and finally recrystallised from 0.1 M HCl at 40%: Final Preparaiion procedures yield was 6&75x, based on platinum. Standard solutions were prepared and stored at 2. Cis-[‘9’PtCI, (cyclopropylamine)J, (CP*). The 5°C in the dark: N,H,.2HCl (4.541 g in 100cm’ procedure was identical to that followed for cis-DDP H20), NaI (19.62 g in 25 cm’ H,O), AgNOJ (3.261 g up to the formation of Na,*PtI,. At this point in 10 cm3 HzO), NaCl (15.30 g in 50 cm3 H,O), isocyclopropylamine (141 pmol, 0.1 cm)) was added and propylamine (2.2 cm3 in 7.8 cm’ H,O), cyclothe mixture was stirred for 5 min at ambient tempropylamine (4.5 cm’ in 20.5 cm3 H,O), ammonium perature and 15 min at 40°C. On cooling, cis-PtI,(cp)z hydroxide (_ 0.7 M). precipitated; this was filtered by centrifugation and washed with water (3 x 0.2 cm’), resuspended in Microscale syntheses of the radiolabelled complexes water (0.2 cm)) and acetone (0.2 cm3) and then silver 1. Cis-[‘9’PtC!,(1VH3,1, (cis-DDP*). The carriernitrate (192 pmol, 0.1 cm3) was added and the mixfree radionuclide as H,*PtCI, (in 0.1 M HCl) was ture was stirred for 10min at ambient temperature. transferred to a conical centrifuge tube (15 cm3). The Sodium chloride solution (1037 ,umol, 0.2 cm’) solusolution was evaporated to dryness using a boiling tion was added and the mixture stirred at 4O’C for 2 h water bath and by directing a gentle air stream to the during which time the acetone was evaporated and side cf the tube. To this was added Na,pt,*xH,O the volume was maintained by the addition of distilled water (0.2 cm3). The precipitates were washed (87pmol; 34.05% Pt; O.OSg) and the mixture diswith distilled water (2 x 2 cm3) and the product was solved in distilled water (0.2cm3). The solution was extracted with acetone (3 x 3 cm3) at 40°C. The acechilled in ice and a solution of hydra&e dihydrotone solution was filtered and evaporated to dryness chloride (43 pmol; 0.1 cm’) was added with conto recover CP*. Yields were typically 60-70x based tinuous stirring for 5 min at 0-2”C, followed by 5 min on platinum. at 85°C. The reaction was accompanied by evolution of nitrogen and the yellow-orange solution became Presswised column chromatography of CP*: A yellow-red in colour as the platinum(W) was reduced chromatography column (5.5 cm diameter, 14.0 cm depth of stationary phase) was prepared from silica to platinum(I1). The mixture was restored to room temperature and the pH adjusted by the very careful gel (Kieselgel 60, 230-4OOmesh, Merck Co.). The solvent used for packing was acetone:MeOH (19: 1). dropwise addition of saturated sodium carbonate CP (ls-20 mg) dissolved in eluent (15 cm9 with solution (-0.2 cm) until carbon dioxide evolution gentle warming (3&35”(Z), was carefully pipetted just ceased. Solutions containing sodium iodide onto the top of the column; the pipette was rinsed (525 pmol, 0.1 cm’) and ammonium hydroxide with eluent and this was carefully pipetted onto the (h 140 ,umol, 0.1 cm3) were added in quick succession and stirring was continued for 2 min at room temtop of the sample. After each addition a correspondperature and 15 min at 45°C. Distilled water (3 cm3) ing amount of eluent was drained off so that the upper level coincided with the top of the stationary was added to the contents of the centrifuge tube and then mixed on a vortex mixer, centrifuged, and the phase. The column was carefully filled with eluent supematant liquid was carefully removed. The and moderate pressure applied using compressed yellow-brown solid, cisPt11(NH3),, was washed with nitrogen. The passage of activity through the column distilled water (0.5 cm’), then resuspended in 0.2 ,cm3 was foliowed by the use of a hand-held Geiger-type
collected when elution of the major peak, as indicated by a hand-held monitor, was about to begin. CisDDP and CBDCA were recrystallised from 0.1 M HCI and water respectively. Chemical kuzlisation. Spots were visualised by spraying with a solution of SnClZ.2H20 (19 mmol, 2.26 g) in hot cont. HCI and making up to 100 cm’ with distilled water.
18-l
J. BAERet al.
mini monitor and fractions (10 cm’) collected as elution of the major peak was about to begintypically after the flow of cu. 24Ocm” eluent. The activity in each fraction was determined by using a scintillation counter. The most active fractions were evaporated to dryness to obtain pure crystalline CP*. TLC was used to check final purity. Immediately before use the compounds were dissolved in saline and millipore filtered into sterile vials. Three samples of each compound were tested for the absence of pyrogens before use in human subjects. 3. Cis-trans-[‘9’PtCI,(OH),(isopropylumine),], (CHIP *). The initial procedure was identical to those followed for both cis DDP* and CP* up to Na,PtI,. At this stage isopropylamine (142 pmol, 0.1 cm3) was added and stirring continued for 2 min at ambient temperature and for 15 min at 50°C. Distilled water (2cm’) was added to the centrifuge tube and the contents rotamixed, centrifuged, and the liquor was carefully removed. The yellow-brown diiodide precipitate was washed twice more. and then the material was resuspended in water (0.2 cm3) and silver nitrate solution (192 pmol, 0.1 cm-‘) was added and the mixture was stirred for 15 min, during which time the temperature was raised from 50 to 70°C. Without cooling, sodium chloride solution (1040 pmol, 0.2 cm3) was added and the temperature raised and held at 8O’C for 1.5min. On cooling to room temperature the supematant liquid was removed and the precipitate washed with distilled water (3 x 1 cm’). The precipitate was resuspended in water (0.2cm3) and hydrogen peroxide (100 vol, 0. I cm3) was added, and the mixture heated to 80°C for 10min. This oxidation step was twice repeated with periodic mixing on a vortex mixer and centrifugation to counteract foaming. The solution was evaporated to dryness in a flow of air and washed with the minimum amount of acetone (cu. 0.2cm3) to remove a deeply coloured highly soluble impurity. The product was recovered by evaporating to dryness the filtered methanol extract of the remaining precipitate. Yields 60-70x based on platinum. Preparatire TLC of CHIP* (i) Plates: A slurry of silica gel (Fluka, Kieselgel GF 254) containing CaSO, binder in water was used to deposit a 2 mm layer upon glass plates (20 x 20cm). Activation was carried out by heating to 120°C for 2 h. (ii) Sample Application: Crude CHIP* (20-25 mg) was dissolved in methanol (1 cm’) and applied by multiple spotting the base line. The eluent was nalong propanol:MeOH (4:l). For recovery, the area containing CHIP* was then recovered by extraction with methanol and evaporation of the resultant solution. 3. cis-[‘9’Pt (1,l -cyclobutanedicarbqxylate)diam mine], (CBDCA*). The procedure was identical to that followed for cis DDP* up to the formation of cis-PtI,(NHj)z. Two equivalents of silver nitrate solution were added to a suspension of the diiodide in
0.2 cm3 of water and the mixture stirred for 25 min at 40%. Water (1 mL) was added to the mixture and 2. j equivalents of 1,l -cyclobutanedicarboxylic acid (adjusted to pH 6) were added. Stirring continued for 2 min at ambient temperature after which the silver salts were removed by centrifugation. The solution was gently evaporated to dryness at 4O’C over a period of 1 h and the solid washed with the minimum volume of (i) ice-cold methanol-water (ii) acetone. The compound was recrystallised from water at 40°C. Yields were 60-70x based on platinum. Analytical rhin layer chromatography
The alumina/glass plates used were Merck 60 Fz3, (Type E) 20 x 20 cm(O.25 mm; the alumina/plastic plates used were Eastman Kodak 13252, 20 x 20 cm 0.1 cm. Before use the chromatography tanks were saturated with eluent vapour by vigorous shaking and then allowed to stand for several hours. A standard quantity (50cm3) of eluent was always used. Compounds were applied by disposable 5 PL capillary pipettes upon a starting line 1.5 cm above the base of the plate. Development was completed when the solvent front had travelled 1Ocm from this line. Acknow/edgemenfs-The technical assistance of Miss A. IM. McNeilly is gratefully acknowledged.
References 1. Durant I. R. In Cisplatin: Current Sratus and New Developments(Eds Prestayko, A. W., Crooke S. T. and
Carter S. K.) p. 3 17 (Academic Press, New York, 1980). DeConti R. C., Toftness B. R., Lange R. C. and Creasey W. A. Cancer Res. 33, 1310 (1973). Rozenecweig M., Von Hoff D. D., Pento J. S. and Muggia F. M. J. Clin. Hematol. Oncol. 7, 672 (1977). Wilkinson R.. Cox P. J.. Jones M. and Harran K. R. Biochimie 60,’ 85 1 ( I978 j. Bradner W. T., Rose W. C. and HuftaIen J. B. Cisplatin: Current Status and New Decelopments (Eds Prestayko A. W., Crooke S. T. and Carter S. K.) p. 171 (Academic Press, New York, 1980). 6. Harrap K. R., Jones M., Wilkinson C. R., Clinks H. IMcD., Sparrow S., Mitchley B. C. V., Clark S. and Veasey A. Cisplatin: Current Status and New DaTelopments (Eds Prestayko A. W.. Crooke S. T. and Carter S. K.) D. 193 (Academic Press. New York. 1980). 7. Sharrrra H. L. and Smith A. G. J. RadioanaL Chek 64, 249 (1981). 8. Robins A. B. and Leach M. 0. Cancer Treat. Rep. 67(3), 245 (1983). 9. Harrison R., McAuhffe C. A., Zaki A., Baer J., Sharma H., Smith A., Jackson H. and Fox B. W. Cancer Chemother. Pharmacol. 10, 90 (1983). 10. Thatcher N.. Sharma H.. Harrison R.. Smith A.. Zaki A.. McAuliffe C. A., Crowther D. and Fox B. W. Cancer Chemother. Pharmacol. 9, 13 (1982). II. Sharma H., Thatcher N., Baer, J., Zaki A., Smith A.. McAuliffe C. A., Crowther D., Owens S. and Fox B. W. Cancer Chemorher. Pharmaccl. 11, 5 (1983). 12. Fernelius W. C. (Ed.) InorganicSynthesis,Vol. 2, p. 250 McGraw-Hill, New York, 1946). 13. Dhara S. C. Ind. J. Chem. 8, 193 (1970).
OOE-708X, 85 53.00 f 0.00 Copyright C 1985 Pergamon Press Ltd
Microscale Syntheses of Anti-Tumour Platinum Compounds Labelled With 19*Pt J. BAER,
R. HARRISON,‘? C. A. McAULIFFE,‘* H. L. SHARMA’ and A. G. SMITH’
A. ZAKI,’
‘Department of Chemistry, University of Manchester Institute of Science and Technology, Manchester M60 lQD, and ‘Department of Medical Biophysics, Stopford Building, University of Manchester, Oxford Road, Manchester Ml3 9PT. U.K. (Received 26 January 1984; in revisedform 1 October 1984) Several compounds of platinum have been found to have significant anti-tumour activity and are in various stages of clinical trials. Four such compounds: cis-PtCl,(NH,),, cis PtCl,(cyclopropylamine)2. cis,trans-PtC1,(OH)2(isopropylamine), and cti-Pt( 1, I-cyclobutanedicarboxylate)(NH,)1 were synthesised with radioactive platinum-191 as a label for the study of animal organ distribution, patient blood clearance, urinary excretion and renal uptake and clearance. This paper describes the microscale synthesis of these compounds. Purification and quality control procedures are also described.
Introduction
Although cis-dichlorodiammineplatinum(II), cisPtC12(NH3)2,(cis-DDP) has proved to be remarkably potent against several tumour types and has become established as a useful clinical agent,“’ it suffers from several disadvantages. Among these are adverse reactions including nephrotoxicity and occasional hearing loss, nausea and allergy. The renal toxicity is particularly unwelcome since it may be dose limiting and preclude further treatment using this drug.(k3’ Considerable effort has been expended worldwide in preparing and testing hundreds of analogues of cis-DDP in the hope that superior drugs might emerge. (4)As well as low nephrotoxicity a search for platinum drugs effective against tumours resistant to cis-DDP therapy is also a current aim. Several such (second generation) drugs have emerged which are proving to be as effective as, or superior to, cis-DDP in some respects; amongst these are cis-dichloro-rran.s-dihydroxo-ci.s-diisopropylamineplatinum(H) (CHIP)‘” and (1, l-cyclobutanedicarboxylate)diammineplatinum(II) (CBDCA).(@ Tne objective of this work was to develop procedures for microscale synthesis of such complexes in order to produce their radiolabelled counterparts. The radionuclide used for these syntheses is mainly 19’Pt (tliZ = 3 days) with small quantities of Ig3’“Pt (t,!? -4.3 days) and ls8Pt (t,,: = 10.2 days), being present. The production procedure for the radioisotope has been described briefly in an earlier *Author to whom all correspondence should be addressed. t Now at Amersham International, Amersham. *.a.,.
36,SA
18
publication”’ and the limitations imposed by the presence of ‘*8Pt has been reported by Robins and Leach.(8) These radiolabelled compounds have been used in organ distribution studies on normal and Yoshida tumor-bearing ratsc9) and for blood clearance and clearance via urine studies in selected human subjects.“‘.“’ For these tracer studies in humans and laboratory animals, radiopharmaceuticals had to be of high specific activity, because of the toxicity associated with these compounds, therefore the syntheses had to be carried out on a small scale (SO-100 mg). The present report is on the microscale synthesis procedures, some of which have been a refinement and optimization of the published work.‘“.‘3’ Experimental Microscale Flexes
syntheses
of the *Pt-radioiabelled
corn -
The procedure developed started with the addition of inactive Na,PtCl, to the carrier-free H,*PtCI, (* indicates radioactive platinum), the mass of the carrier-free material being negligible. Regular preparations have been carried out on 0.05 g Na,PtCl,. An outline of the microscale preparation of the *Pt-radiolabelled drugs is shown in Scheme 1. Considerable effort went towards optimising experimental conditions at each stage, and its yield. Transfer of the reaction mixtures and products between vessels, and filtrations, were avoided; the bulk of the preparation was carried out in one small centrifuge tube to minimise losses. Some features of interest to be noted are:
J. BAERet al.
182
Na,‘PtCI, i Na,‘PtCI, I Na,‘Ptl, I cis-[‘PtA,I,]a i
N2H,.ZHCI. pH=54
C-2’C.
5 mm: 65’C. 5mm
Scheme 2.
6Nal. O’C. 1 min
3A, 5 mm. OT; 50%
AgNO,,
15 min
10 min. ambent
temp.
cis-[‘Pti,(H,OM (NO,), I
NaCl or HCI
(0.1 N). 1-2 h. 35-4672
cis-[‘PtA,CI,]” i [‘PtA,(OH),Cl,]b
A=ammonia (cis-DDP, CBDCA) isopropylamine (CHIP) cyclopropylamine (CP)
mixed iodide/chloride complexes. Partial dissolution of the iodide complex was found to increase the rate of the reaction. (6) A considerable excess of NaCl or HCl (0.1 N) was used to precipitate excess silver and to maximise the rate of reaction. Quality control of the *Pt -radiolabelled complexes
H,O,.
6o’C.
1Omin
Scheme I. “LOSSof activity in supematant and washing is IO-20x, depending upon iridium concentration. bYields: cis-DDP* 760/,; CP* 65%; CHIP* 70%; CBDCA* 70%. (1) Na,PtCl, was employed in preference to K2PtC1,, since it is more soluble, and thus very concentrated solutions can be used in the reduction and subsequent reactions, with a consequent shortening of reaction times. Hydrazine hydrochloride was chosen in preference to other reducing agents. (2) pH control was found to be important. At low pH, protonation of the amine competes with platinum-amine complexation and the rate and extent of reaction decreases. High pH conditions are also to be avoided since the hydroxyl ion has a high affinity for platinum, and this could interfere in the third step at which all four coordination sites need to be occupied by iodide ions. (3) The addition of six mole equivalents of sodium iodide enhances the stability of the [PtI$- ion and reduces the chance of PtI, precipitation. (4) Three mole equivalents of amine were used in order to increase the rate of cis-[PtA& formation. After reaction the diiodo product is washed thoroughly in order to remove unreacted platinum salts. Also, in the supernatant liquid was the iridium impurity produced by the initial activation of osmium plus the subsequent decay of any lE5Pt, ls6Pt, ‘s7Pt, “*Pt or ‘89Pt. Reduction of iridium(W) in step (1) gives the inert d6 iridium(III), which is not complexed, and so remains in solution. (5) Silver nitrate was added in 10% excess in order to ensure the complete precipitation of the silver iodide and thus avoid the eventual formation of Table I. Thin layer chromatography
Thin layer chromatography was found to be satisfactory as a method of purity control for these three *Pt-radiolabelled complexes. Only small quantities of the complex were required and the procedure was rapid and straightforward. Table 1 lists R, values obtained for samples of the cyclopropylamine complex (CP) and its iodide analogue, with various eluent systems using alumina as stationary phase. To allow for unambiguous identification of spots authentic compounds were always used as markers when analysing unknowns. The eluent mixture chosen for routine analysis of CP* was acetone: HCl(1 M) (19: 1) for both alumina/glass and alumina/plastic chromatography plates. Table 2 lists some data for CHIP and several potential platinum-containing impurities obtained with various eluents using alumina/glass plates. AcetonejHCl gives a good separation between CHIP and the other complexes. The purity of the recrystallised cis-DDP* was estimated by employing the TLC analysis on alumina/glass plates using acet0ne:O.l M HCl (7:3) as the eluent mixture (R/= 0.63); CBDCA was eluted using a 7:3 isopropanol/water mixture (R,= 0.45). The purity of the radiolabelled complexes was measured quantitavely by counting the activity of both pure and impurity spot(s) on the glass/plastic TLC plates using an autogamma counter. Impure samples (< 96%) were purified as follows: CHIP was purified via preparative TLC alumina/glass plates using the eluent mixture acetone:0.12 M HCl (9:1), and the recovery of the radiolabelled complex was achieved by extraction from the stationary phase with methanol; CP* was passed through a pressurised column silica gel as stationary phase and using acetone/CH;OH was eluent, this method being found to be both quick and efficient, and fractions were data for cyclopropylamine
complexes R,
Plates Alumina;glass Alumirwplastic
+Eluent
cis-~cll(cP,?J
ci.Y-[PtL(CP)I]
(19:l) Acetone:HCI(l M) (9: I) Acetone:H,O
0.84 0.82
0.96 0.95
(19:l) Acetone:HCl(I M) (9:l) Acetone:H,O
0.90 0.77
0.94 0.83
Acetone (9:l) Acetone:CHCI,
0.15 0.08
0.32 0.26
183
Microscale syntheses of anti-tumour platinum compounds Table 2. Thin layer chromatography Complex cir-,rranr-IPtCI,(OH)zcIP,zl
data for isopropylamine complexes Eluent
(CHIP)
rronr-.~rcurr-,~~~rr-[Pt(OH)P~(rPfZl rrans-[PtCI,(IP)J
cis-IPtCl*(IP)J
cir-N,(IP)?J
(9: I) Acetone:H,O
(9: I) Acetone:HC1(0.12 M) (9: 1) Ch,CI,:EtOH (9:l) Acetone:H,O (9: I) Ch,CII:EtOH (9: I) Acetone:HZO (9: 1) Acetone:HCI (9: I) CH&EtOH (9: 1) Acetone:HzO (9: I) Acetone:HCl (9: I) CH,CI,:EtOH (9: I) Acetone:H:O (9:l) Acetone:HCl
Rf 0.42 0.47 0.19 1.00 0.97
1.oo 0.96 0.92 0.94 0.84 0.65 0.97 0.98
water. Silver nitrate(192 pmol, 0.1 cm3) was then added and the mixture was stirred for 15 min at 40°C. To the solution containing the diaquo species (cis[pt(NH3)z(HzO)&N03)1), sodium chloride (0.2 cm3, 1040pmol) was added and the temperature held at 35-45”C for 15 min and the volume was reduced to 0.3 cm3. On cooling, cis-DDP crystallised; this was filtered and washed with distilled water (0.2 cm3), and finally recrystallised from 0.1 M HCl at 40%: Final Preparaiion procedures yield was 6&75x, based on platinum. Standard solutions were prepared and stored at 2. Cis-[‘9’PtCI, (cyclopropylamine)J, (CP*). The 5°C in the dark: N,H,.2HCl (4.541 g in 100cm’ procedure was identical to that followed for cis-DDP H20), NaI (19.62 g in 25 cm’ H,O), AgNOJ (3.261 g up to the formation of Na,*PtI,. At this point in 10 cm3 HzO), NaCl (15.30 g in 50 cm3 H,O), isocyclopropylamine (141 pmol, 0.1 cm)) was added and propylamine (2.2 cm3 in 7.8 cm’ H,O), cyclothe mixture was stirred for 5 min at ambient tempropylamine (4.5 cm’ in 20.5 cm3 H,O), ammonium perature and 15 min at 40°C. On cooling, cis-PtI,(cp)z hydroxide (_ 0.7 M). precipitated; this was filtered by centrifugation and washed with water (3 x 0.2 cm’), resuspended in Microscale syntheses of the radiolabelled complexes water (0.2 cm)) and acetone (0.2 cm3) and then silver 1. Cis-[‘9’PtC!,(1VH3,1, (cis-DDP*). The carriernitrate (192 pmol, 0.1 cm3) was added and the mixfree radionuclide as H,*PtCI, (in 0.1 M HCl) was ture was stirred for 10min at ambient temperature. transferred to a conical centrifuge tube (15 cm3). The Sodium chloride solution (1037 ,umol, 0.2 cm’) solusolution was evaporated to dryness using a boiling tion was added and the mixture stirred at 4O’C for 2 h water bath and by directing a gentle air stream to the during which time the acetone was evaporated and side cf the tube. To this was added Na,pt,*xH,O the volume was maintained by the addition of distilled water (0.2 cm3). The precipitates were washed (87pmol; 34.05% Pt; O.OSg) and the mixture diswith distilled water (2 x 2 cm3) and the product was solved in distilled water (0.2cm3). The solution was extracted with acetone (3 x 3 cm3) at 40°C. The acechilled in ice and a solution of hydra&e dihydrotone solution was filtered and evaporated to dryness chloride (43 pmol; 0.1 cm’) was added with conto recover CP*. Yields were typically 60-70x based tinuous stirring for 5 min at 0-2”C, followed by 5 min on platinum. at 85°C. The reaction was accompanied by evolution of nitrogen and the yellow-orange solution became Presswised column chromatography of CP*: A yellow-red in colour as the platinum(W) was reduced chromatography column (5.5 cm diameter, 14.0 cm depth of stationary phase) was prepared from silica to platinum(I1). The mixture was restored to room temperature and the pH adjusted by the very careful gel (Kieselgel 60, 230-4OOmesh, Merck Co.). The solvent used for packing was acetone:MeOH (19: 1). dropwise addition of saturated sodium carbonate CP (ls-20 mg) dissolved in eluent (15 cm9 with solution (-0.2 cm) until carbon dioxide evolution gentle warming (3&35”(Z), was carefully pipetted just ceased. Solutions containing sodium iodide onto the top of the column; the pipette was rinsed (525 pmol, 0.1 cm’) and ammonium hydroxide with eluent and this was carefully pipetted onto the (h 140 ,umol, 0.1 cm3) were added in quick succession and stirring was continued for 2 min at room temtop of the sample. After each addition a correspondperature and 15 min at 45°C. Distilled water (3 cm3) ing amount of eluent was drained off so that the upper level coincided with the top of the stationary was added to the contents of the centrifuge tube and then mixed on a vortex mixer, centrifuged, and the phase. The column was carefully filled with eluent supematant liquid was carefully removed. The and moderate pressure applied using compressed yellow-brown solid, cisPt11(NH3),, was washed with nitrogen. The passage of activity through the column distilled water (0.5 cm’), then resuspended in 0.2 ,cm3 was foliowed by the use of a hand-held Geiger-type
collected when elution of the major peak, as indicated by a hand-held monitor, was about to begin. CisDDP and CBDCA were recrystallised from 0.1 M HCI and water respectively. Chemical kuzlisation. Spots were visualised by spraying with a solution of SnClZ.2H20 (19 mmol, 2.26 g) in hot cont. HCI and making up to 100 cm’ with distilled water.
18-l
J. BAERet al.
mini monitor and fractions (10 cm’) collected as elution of the major peak was about to begintypically after the flow of cu. 24Ocm” eluent. The activity in each fraction was determined by using a scintillation counter. The most active fractions were evaporated to dryness to obtain pure crystalline CP*. TLC was used to check final purity. Immediately before use the compounds were dissolved in saline and millipore filtered into sterile vials. Three samples of each compound were tested for the absence of pyrogens before use in human subjects. 3. Cis-trans-[‘9’PtCI,(OH),(isopropylumine),], (CHIP *). The initial procedure was identical to those followed for both cis DDP* and CP* up to Na,PtI,. At this stage isopropylamine (142 pmol, 0.1 cm3) was added and stirring continued for 2 min at ambient temperature and for 15 min at 50°C. Distilled water (2cm’) was added to the centrifuge tube and the contents rotamixed, centrifuged, and the liquor was carefully removed. The yellow-brown diiodide precipitate was washed twice more. and then the material was resuspended in water (0.2 cm3) and silver nitrate solution (192 pmol, 0.1 cm-‘) was added and the mixture was stirred for 15 min, during which time the temperature was raised from 50 to 70°C. Without cooling, sodium chloride solution (1040 pmol, 0.2 cm3) was added and the temperature raised and held at 8O’C for 1.5min. On cooling to room temperature the supematant liquid was removed and the precipitate washed with distilled water (3 x 1 cm’). The precipitate was resuspended in water (0.2cm3) and hydrogen peroxide (100 vol, 0. I cm3) was added, and the mixture heated to 80°C for 10min. This oxidation step was twice repeated with periodic mixing on a vortex mixer and centrifugation to counteract foaming. The solution was evaporated to dryness in a flow of air and washed with the minimum amount of acetone (cu. 0.2cm3) to remove a deeply coloured highly soluble impurity. The product was recovered by evaporating to dryness the filtered methanol extract of the remaining precipitate. Yields 60-70x based on platinum. Preparatire TLC of CHIP* (i) Plates: A slurry of silica gel (Fluka, Kieselgel GF 254) containing CaSO, binder in water was used to deposit a 2 mm layer upon glass plates (20 x 20cm). Activation was carried out by heating to 120°C for 2 h. (ii) Sample Application: Crude CHIP* (20-25 mg) was dissolved in methanol (1 cm’) and applied by multiple spotting the base line. The eluent was nalong propanol:MeOH (4:l). For recovery, the area containing CHIP* was then recovered by extraction with methanol and evaporation of the resultant solution. 3. cis-[‘9’Pt (1,l -cyclobutanedicarbqxylate)diam mine], (CBDCA*). The procedure was identical to that followed for cis DDP* up to the formation of cis-PtI,(NHj)z. Two equivalents of silver nitrate solution were added to a suspension of the diiodide in
0.2 cm3 of water and the mixture stirred for 25 min at 40%. Water (1 mL) was added to the mixture and 2. j equivalents of 1,l -cyclobutanedicarboxylic acid (adjusted to pH 6) were added. Stirring continued for 2 min at ambient temperature after which the silver salts were removed by centrifugation. The solution was gently evaporated to dryness at 4O’C over a period of 1 h and the solid washed with the minimum volume of (i) ice-cold methanol-water (ii) acetone. The compound was recrystallised from water at 40°C. Yields were 60-70x based on platinum. Analytical rhin layer chromatography
The alumina/glass plates used were Merck 60 Fz3, (Type E) 20 x 20 cm(O.25 mm; the alumina/plastic plates used were Eastman Kodak 13252, 20 x 20 cm 0.1 cm. Before use the chromatography tanks were saturated with eluent vapour by vigorous shaking and then allowed to stand for several hours. A standard quantity (50cm3) of eluent was always used. Compounds were applied by disposable 5 PL capillary pipettes upon a starting line 1.5 cm above the base of the plate. Development was completed when the solvent front had travelled 1Ocm from this line. Acknow/edgemenfs-The technical assistance of Miss A. IM. McNeilly is gratefully acknowledged.
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Carter S. K.) p. 3 17 (Academic Press, New York, 1980). DeConti R. C., Toftness B. R., Lange R. C. and Creasey W. A. Cancer Res. 33, 1310 (1973). Rozenecweig M., Von Hoff D. D., Pento J. S. and Muggia F. M. J. Clin. Hematol. Oncol. 7, 672 (1977). Wilkinson R.. Cox P. J.. Jones M. and Harran K. R. Biochimie 60,’ 85 1 ( I978 j. Bradner W. T., Rose W. C. and HuftaIen J. B. Cisplatin: Current Status and New Decelopments (Eds Prestayko A. W., Crooke S. T. and Carter S. K.) p. 171 (Academic Press, New York, 1980). 6. Harrap K. R., Jones M., Wilkinson C. R., Clinks H. IMcD., Sparrow S., Mitchley B. C. V., Clark S. and Veasey A. Cisplatin: Current Status and New DaTelopments (Eds Prestayko A. W.. Crooke S. T. and Carter S. K.) D. 193 (Academic Press. New York. 1980). 7. Sharrrra H. L. and Smith A. G. J. RadioanaL Chek 64, 249 (1981). 8. Robins A. B. and Leach M. 0. Cancer Treat. Rep. 67(3), 245 (1983). 9. Harrison R., McAuhffe C. A., Zaki A., Baer J., Sharma H., Smith A., Jackson H. and Fox B. W. Cancer Chemother. Pharmacol. 10, 90 (1983). 10. Thatcher N.. Sharma H.. Harrison R.. Smith A.. Zaki A.. McAuliffe C. A., Crowther D. and Fox B. W. Cancer Chemother. Pharmacol. 9, 13 (1982). II. Sharma H., Thatcher N., Baer, J., Zaki A., Smith A.. McAuliffe C. A., Crowther D., Owens S. and Fox B. W. Cancer Chemorher. Pharmaccl. 11, 5 (1983). 12. Fernelius W. C. (Ed.) InorganicSynthesis,Vol. 2, p. 250 McGraw-Hill, New York, 1946). 13. Dhara S. C. Ind. J. Chem. 8, 193 (1970).