[CANCER RESEARCH 51, 1866-1875. April 1, 1991]

Mechanistic Studies of a Novel Class of Trisubstituted Platinum(II) AntitumorAgents1

L. Steven Hollis, Wesley I. Sundquist, Judith N. Burstyn, Wendy J. Heiger-Bernays, Steven F. Hellem,Kazi J. Ahmed, Alan R. Amundsen, Eric W. Stern, and Stephen J. Lippard2

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 ¡W. I. S., J. N. B., W. J. H-B., S. F. B., K. J. A., S. J. L.],and Engelhard Corporation, Research and Development, Mento Park, Edison, New Jersey 08818 [L. S. H., A. R. A., E. W. S.J

ABSTRACT

Chemical and biological studies are presented for a new series ofplatinum)II) antitumor agents that violate the classical structure-activityrelationships established for platinum complexes. These new agents,which have demonstrated activity against murine and human tumorsystems, are ciî-|Pt(NH3)2(Am)Cl|'1'cations, in which Am is a derivative

of pyridine, pyrimidine, purine, or aniline. Members from this seriesblock simian virus 40 DNA replication vn vitro and inhibit the action ofDNA polymerases at individual guanine residues in replication mappingexperiments. Monoclonal antibodies that bind selectively to cisplatinlesions on calf thymus DNA were used in a competitive enzyme-linkedimmunosorbent assay study to show that the platinum-triamine complexes do not produce the type of intrastrand cross-links on DNA thatare characteristic of cisplatin and analogues with the general formula

TRISUBSTITUTED PLATINUM(ll) ANTITUMOR AGENTS

H,N

H.,N

pt

Am

Members of this series of compounds show activity against anumber of murine tumors, including S180a, P388, and L1210,and human tumor cell lines. Since these platinum-triaminecomplexes are cationic species that contain three nitrogen donors and only one leaving group, they cannot be considered asclassical cisplatin analogues. Without loss of NH? or Am fromthe platinum coordination sphere, such species would produceonly monofunctional lesions on DNA, as previously establishedfor closely related, but inactive, platinum-triamine complexessuch as [Pt(dien)Cl]+ (31, 32). Monofunctionally binding com

plexes have not proved to have antitumor activity (21). In orderto address the binding mode of the new platinum-triaminecomplexes, as well as other fundamental questions about theirmechanism of action, the studies described in the present articlewere carried out.

MATERIALS AND METHODS

Chemical Studies

Compound Preparation

Platinum-triamine complexes were prepared as described previously(30). Two additional compounds investigated here were obtained in thefollowing manner.

/rfl/M-|Pt(NH.,X4-Br-py)Cl2]. Method a: m-[Pt(NH3)2(4-Br-py)Cl]Cl(88 mg) was dissolved in 3.2 ml of PBS (pH 7.2; Sigma Chemical Co.)and the resulting solution was stored at 37°Cfor 14 days. Well-formed

crystals, suitable for X-ray diffraction studies, were collected by filtration, washed with water, and air dried (yield, 45 mg). Method b: cis-[Pt(NH.,)2(4-Br-py)Cl]Cl (1.0 g) was refluxed in 0.1 M HCI (50 ml) for1 h. The resulting yellow precipitate was collected by filtration, washedwith water, and dried under vacuum (yield, 0.53 g) (analysis: C, H, N).

m-|Pt(NH.,)(4-CH3-py)Cl2]. A solution of K[Pt(NH3)Cl3] (1.953 g)and 4-methylpyridine (0.509 g) in 25 ml of water was stirred at 50°Cfor 12 h. The solution was cooled to 25°Cand the resulting yellow

precipitate was collected by filtration, washed with water, and dried(yield, 1.2 g) (analysis: C, H, N).

Physical Studies

NMR Spectroscopy. 'H NMR spectra were obtained with a VarianXL-300 spectrometer. l95Pt and 15Nspectra were recorded in dimethylformamide with a Varian XL-200 spectrometer by using previouslydescribed methods (33). l95Pt chemical shifts are reported relative to0.1 M K2[PtCl4] at -1624 ppm, which is referred to H2PtCl6 at 0 ppm."N chemical shifts are reported relative to 15NH4NO, (0.5 g/3 ml H2O)at 0 ppm. il/.ï-[Pt('5NH3)2(Am)Cl](NO,)complexes were prepared fromc/s-[Pt(15NH;i)2Cl2]in dimethyl formamide according to published pro

cedures (30).X-Ray Crystallography. Single crystal X-ray diffraction studies of

fra/is-[Pt(NH3)(4-Br-py)Cl2] (1) were conducted with an Enraf-NoniusCAD-4F diffractometer with graphite-monochromated MoK,, radiation. Compound 1 crystallizes in the monoclinic space group P2,/c(C52h,No. 14), as determined from systemic absences (34) and axialphotographs, with the following unit cell parameters: a = 11.842 (1),b = 10.658 (1), c = 7.7453 (6) A; ß= 96.249 (7)°;V= 971.7 (3) A3; Z

= 4. Full details of the data collection and refinement are presented assupplementary material (Table SI).4 The Enraf-Nonius Structure De

termination Package (35) was used for data collection, structure solution, and refinement. The structure was solved by direct methods (36)and refined with 2067 reflections (28 < 54°).Full matrix least-squares

refinement with anisotropic thermal parameters for all non-H atomsconverged at RF = 0.026 and RwF= 0.033, where RF = £| |F0|-|FC| |/S|F0| and RwF= [£w (IFoHFcDVEwlF,,!2]"2. Further details of the

refinement, including a complete listing of atomic positional and thermal parameters, interatomic bond lengths and angles, and observed andcalculated structure factors, are available as supplementary material(Tables S1-S5).4

Synthesis and Platination of d(GpC) and dG. The dinucleoside monophosphate d(GpG) was synthesized by using the phosphotriestermethod according to the procedure of Gait (37). Purification wasaccomplished by flash chromatography on silica, DE-52 aniónexchangechromatography, and finally reverse phase HPLC on a C18 column inwhich a 0. l M NH4OAc/CH3CN mixture was used as the mobile phase.5The d(GpG) product (300 MM) was allowed to react with cis-[Pt(NH3)2(/V.i-cytosine)Cl]Cl at a formal platinum to dinucleotide ratioof 1.3:1 for about 200 h at 37°C.Reverse phase HPLC was used to

separate and purify the reaction products which were then analyzed forthe amount of bound platinum/d(GpG) by flameless atomic absorptionspectroscopy on a Varian 1475 spectrophotometer equipped with aGTA 95 graphite tube atomization system and programmable sampledispenser. Deoxyguanosine (1.7 IHM)was allowed to react with m-[Pt(NH3)2(yW-cytosine)Cl]Cl or c/s-[Pt(NH3)2(4-CH3-py)Cl]CI at a formal platinum to nucleoside ratio of 1.1:1 for about 100 h at 39°C.

Purification was achieved by HPLC as described above.Characterization of the Platinated Products. Two different enzymatic

digestion protocols were used in order to characterize the platinateddinucleotides. The first used DNase I (Sigma), PI nuclease, and alkalinephosphatase (both from Boehringer Mannheim Biochemicals) to digestthe material into constituent nucleosides (38) without disrupting cova-lent platinum-nitrogen bonds (39). The second protocol resembles thefirst but omits the final alkaline phosphatase step in order to leave the5'-phosphates intact. Approximately 20 /ig of d(GpG) was digested in120 /il at 37°Cfor 24 h, and the products were separated by reverse

phase HPLC (5-22.5% CH3CN gradient in 30 min at 1 mi/min flowrate). The proton NMR chemical shifts of the H8 resonances of themajor product, designated A, were studied as a function of pH in orderto determine the platinum coordination site. Samples were prepared inD2O and titrated with NaOD. pH readings are reported uncorrectedfor deuterium isotope effects.

Biological Studies

In Vitro Replication Assays

Preparation of Cytosolic Cell Extracts, SV40 T-Antigen, and PlasmiliDNAs. The human embryonic 293 cell cytoplasmic extract was prepared as described previously (28). SV40 T-antigen was immunopuri-fied from 293 cells infected with AdSSVRl 15 (40, 41). Plasmid DNAswere obtained as reported elsewhere (41); pSVOll contains the 0.2-kilobase pair Hindlll to Sphl fragment of SV40 in pUC18.

Platination of DNA. pSVOl 1 plasmid DNA was incubated at 37°C

for 16 h in the presence of platinum compound at several formal(D/N)r ratios in 3 mM NaCI, l mM Na2HPO4 (pH 7.4) or 10 mM Tris-Cl, 1 mM Na2EDTA (pH 7.6). Platinum complexes were freshly dissolved just prior to use. Unbound platinum was removed by ethanolprecipitation of the DNA. The amount of platinum bound/nucleotide,(D/N)b, was measured by atomic absorption spectroscopy.

Replication Assays and Analysis. Reactions were carried out in 50-M'solutions at 37°Cfor 60 min as described previously (28). Reactions

were terminated by addition of Na2EDTA (pH 8.0) to 10 mM, 5 fig ofdenatured, sheared calf thymus DNA and 1 ml of 8% trichloroaceticacid, 1% sodium pyrophosphate and allowed to stand for 15 min onice. The acid-insoluble material was collected by filtration onto Whatman GF-C glass fiber filters and then washed several times with 10 mlof ice-cold 8% trichloroacetic acid, 1% sodium phosphate and twice

* Data on file with the National Auxiliary Publication Service. 5W. I. Sundquist and M. V. Keck, unpublished results.

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TRISUBSTITUTED PLATlNUM(ll) ANTITUMOR AGENTS

with ice-cold ethanol. Radioactivity was determined by liquid scintillation counting.

Replication Mapping of Platinum-Triamine Complexes

Preparation and Platination of Plasmid DNA. Single-stranded viralDNA was purified from bacteriophage M13mpl8 by precipitation,followed by phenol/chloroform extraction, and separated from anydouble-stranded contaminants by hydroxylapatite chromatography.Replicative form M13mpl8 DNA was isolated by the cleared lysatemethod. DNA, either single or double stranded, was allowed to reactwith the platinum complexes at a (D/N)f ratio of 1.25 x IO"' for 16 hat 37°Cin 3 mM NaCl, l mM Na2HPO„,pH 7.4. Following ethanol

precipitation and careful washing to remove the excess platinum complex, the DNA was quantitated by UV absorbance and the level ofplatinum was determined by atomic absorption spectroscopy.

Replication Mapping on Adducted Templates. The platinated DNAwas primed with an oligonucleotide and used as a template for DNAreplication. For replication experiments on single-stranded DNA, anequimolar amount of a 17-mer primer (Universal primer; New EnglandBiolabs) was added and the template was primed by heating to 85°C

and cooling to room temperature. Double-stranded M13mpl8 DNAwas first denatured with 2 N NaOH and a 2-fold excess of a 24-merprimer (New England Biolabs; -40 primer or reverse primer) was added

just prior to neutralization with ammonium acetate and ethanol precipitation. The 24-mer primer was necessary to ensure adequate annealingto the platinated templates. Replication was initiated by addition ofSequenase enzyme (United States Biochemical) or the Klenow fragmentof DNA polymerase I (Boehringer-Mannheim Biochemicals) to theprimed template in the presence of «-35S-thio-dATP(5 ¿tCi,600 ^Ci/Mmol)and unlabeled dGTP, dCTP, and dTTP (0.9 MM)in 25 mM Tris-Cl (pH 7.6), 6.5 mM dithiothreitol, 12 mM MgCl2, 30 mM NaCl. Thismixture was incubated for 10 min at room temperature. An excess ofall four dNTPs was then added to a total dNTP concentration of 40MM(single-stranded templates) or 70 MM(double-stranded templates)and the incubation was continued for 15 min at 37°C.The reaction was

stopped by the addition of 90% formamide, 5 mM EDTA-dye mixture.The samples were denatured by heating to 85°Cfor 3 min and the

products analyzed by electrophoresis on 8% polyacrylamide/7 M ureagels. Sequencing reactions were carried out according to the Sequenaseprotocol (United States Biochemical) and electrophoresed in parallel.The gels were dried and autoradiographed at room temperature for 2-

5 days.

Immunochemical Studies

Preparation of Platinated DNA

Native calf thymus DNA (Sigma) was extracted with 24:1 chloro-form/isoamyl alcohol (3 times), precipitated with ethanol (2 times),and dialyzed against low salt buffer (3 mM sodium chloride, 1 mMNa2HPO4, pH 7.4) prior to platination. DNA samples were subsequently incubated in low salt buffer with either m-DDP, cis-[Pt(NH3)2(4-CH3-py)Cl]Cl, c/i-[Pt(NH3)2(W-cytosine)Cl]Cl, or cw-[Pt(NH3)(4-CH3-py)Cl2] at a formal drug to nucleotide ratio of 0.05(37°C,24 or 168 h). Under these conditions, the binding of all four

platinum complexes was essentially quantitative at both time points.Platinum-modified DNA samples were precipitated from ethanol to

stop the platination reactions and to remove any unbound platinum.The precipitated DNA was washed with 70% ethanol and redissolvedin PBS buffer (0.15 M sodium chloride, 10 mM sodium phosphate, pH7.2). DNA nucleotide concentrations were determined by UV spectroscopy (assuming t = 6600 M~' cm~') and bound platinum was measured

by atomic absorption spectroscopy.

ELISA

ELISA experiments were performed as described previously (42).Briefly, rá-DDP-DNA, (D/N)b = 0.040, was bound to microtiter plates(Dynatech) coated with poly-/-lysine (Sigma) and the plates were subsequently blocked with poly-/-glutamate (Sigma) and 1% bovine serum

albumin (Sigma). Unbound reagents were removed with successivewashes of buffer (150 mM sodium chloride, 10 mM Tris-HCl, pH 7.2).The monoclonal antibody CPT2 was diluted 1:100 from culture supernatant and preincubated for 60 min with DNA competitors prior toaddition to the microtiter wells. Unbound antibody was removed fromthe wells with 0.2% Tween 20/PBS washes (3 times) and the remainingbound antibody was detected using an alkaline phosphatase-conjugatedrabbit anti-mouse immunoglobulin antibody (Zymed). The latter antibody was quantitated by measurement of absorbance at 405 nm, 60min after addition of the alkaline phosphatase substrate p-nitrophen-ylphosphate (1 mg/ml in 50 mM sodium carbonate, 2 mM magnesiumchloride, pH 9.5; 37°Cincubation). The specificity and avidity of CPT2

have been described previously (42) and the antibody was used at aconcentration that gave absorbances of 1-2 AU (405 nm) after 60 minin the absence of competitor.

Animal Studies

Antitumor Screening

The platinum complexes /rani-[Pt(NH3)(4-Br-py)Cl2] and cis-[Pt(NH3)(4-CHrpy)Cl2] were screened versus Sarcoma 180 ascites (i.p.-i.p.) in female CFW mice by using previously described methods (30).Screening data for these compounds are presented as supplementarymaterial (Table S6).4

RESULTS AND DISCUSSION

Biochemical Studies of the m-|Pt(NH.,)2(Am)CI|+ Complexes

Inhibition of DNA Replication. Since cisplatin is known tobind DNA and inhibit replication in vitro and in vivo (28, 43-45), studies were conducted to determine whether the platinum-triamine complexes behave in a similar fashion. An in vitrosystem described previously (28) was used to measure theinhibitory effects of two platinum-triamine complexes on SV40

DNA replication. This system utilizes cytosolic extracts fromthe human embryonic kidney cell line 293 in combination withSV40 origin-containing plasmiti templates, SV40 large T-anti-gen, and nucleotide triphosphates to replicate the SV40 genome. The extracts contain all the necessary enzymes to supportDNA synthesis with the exception of large T-antigen, a virallyencoded protein supplied exogenously. The conditions closelymimic those required in eukaryotic cells for replication ofnuclear DNA (41,46).

In separate experiments, SV40 origin-containing plasmidDNAs were platinated at low drug/nucleotide levels [(D/N)b =1.4-1.7 x IO"3]with the platinum-triamine complexes and two

control compounds, cisplatin and [Pt(dien)Cl]]Cl (28). Themodified templates were subjected to the in vitro replicationassay and DNA synthesis was monitored by following theincorporation of [a-12P]dAMP into the newly synthesized DNA.

Table 1 Effect of platinum complexes on in vitro DNA replication from pSVOiltemplates in 293 cytosolic extracts at (D/N)b levels indicated'

ModificationUnmodifiedas-DDPcis-DDPc/s-DDP[Pt(dien)Cir

[Pt(dien)Circis-[Pt(NH,)2(4-Me-py)Cir

c/i-[Pt(NHj)2(4-Br-py)Cir(D/N),0.01.7

x10-0.9x10-0.4x10"3.4

x 10-0.9 x10-1.7

x 10-1.4 x 10-pmol

dAMP/h56.82.62.810.435.0

45.68.6

8.5%

control1004.64.918.361.6

80.315.1

15.0°Except for the last two entries, the data in this table are reproduced from

Ref. 11.

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The results of these studies (Table 1) show that the two biologically active platinum-triamine complexes, m-[Pt(NH.1)i(4-CH,-py)CI]Cl and cw-[Pt(NH,),(4-Br-py)Cl]Cl, are nearly aseffective as cisplatin (85 versus 95% inhibition) in blockingDNA replication in vitro. In contrast, the related triaminecomplex, [Pt(dien)Cl]Cl, which displays no antitumor activity,does not effectively inhibit DNA synthesis under similarconditions.

Since m-[Pt(NH3)2(Am)Cl]+ complexes and [Pt(dien)Cl]+ are

both expected to bind to DNA in a monofunctional fashion, itis difficult to explain why these compounds differ in their abilityto inhibit DNA replication. Previously, platinum complexesthat produce monofunctional lesions on DNA were found to beinactive as antitumor agents (21). Since the m-[Pt(NH.,)2(Am)Cl]+ complexes display activity, it is important to understand the nature of their Pt-DNA adducts. In particular, is itthe monofunctional DNA adducts produced by the platinum-triamine complexes that are ultimately responsible for thedisruption of DNA synthesis, or do ligand exchange reactionsoccur leading to bifunctional cross-links having properties similar to those produced by cisplatin? This question is addressedby the experiments described below.

Sequence Specificity of Platinum-Triamine Complex Bindingto DNA. Replication mapping studies were carried out to determine the DNA regiospecificity of the platinum-triaminecomplexes (45). DNA from bacteriophage M13, both single-stranded and double-stranded forms, was modified with cis-DDP, m-[Pt(NH3)2(4-Br-py)Cir, m-[Pt(NH,)2(4-CH,-py)Cl]+,or [Pt(dien)Cl]+ to a level of approximately one adduci/1000nucleotides, (D/N)b ~ 0.001. The adducted DNA was primed

with an oligonucleotide and used as a template for secondstrand synthesis by either T7 DNA polymerase (Sequenase) orthe Klenow fragment of DNA polymerase I. The stop sitesresulting from the presence of the platinum-triamines werecompared directly with those resulting from the presence of theactive antitumor drug c/s-DDP and also with the inactive platinum-triamine [Pt(dien)Cl]+.

The triamine complexes m-[Pt(NH3)2(4-Br-py)Cl]+ and cis-[Pt(NH3)2(4-CH3-py)Cl]+ both block replication, as evidenced

by the strong bands observed when second strand synthesisproducts are run on sequencing gels (Fig. 1). The same resultswere obtained when double-stranded DNA was modified withthe triamine complexes and replicated in a similar manner orwhen the Klenow fragment of DNA polymerase I was substituted for T7 DNA polymerase (data not shown). The inhibitionof replication by the platinum-triamines in this experimentcorroborates the results obtained for SV40-platinated templatesreplicated in the cell extracts, in which incorporation of dATPwas inhibited by these complexes (vide supra).

It has previously been shown that rà -DDP inhibits replication by coordinating to multiple guanine sequences (45). Thetendency of the modified T7 polymerase to stop at pairs ofadjacent guanines on DNA modified by m-DDP is clearly seenin Fig. 1, lane 2. The pattern of stop sites is quite different forDNA modified with m-[Pt(NH.,)2(4-Br-py)Cl]+ and cis-[Pt(NH,)2(4-CH.,-py)Cl]+ (Fig. 1, lanes 4 and 5). The principal

stop sites on DNA modified with these triamines are at singleguanine residues with no preference for the multiple guaninesites. This observation suggests that cw-[Pt(NH3)2(4-Br-py)Cl]+and rà -[Pt(NH3)2(4-CH3-py)Cir bind monofunctionally to isolated guanine residues. It is clear from the differences betweenthe mapping pattern produced by c/s-DDP and those affordedby the c/s-[Pt(NH3)2(Am)Cl]+ complexes that the platinum-

12345

I•

r

r I:

>u/

Fig. 1. Mapping of the replication blockage sites on single-stranded M13mp 18DNA modified with platinum compounds with the use of T7 DNA polymerase(Sequenase). The sequence of the adducted strand is read from 5' (top) to 3'(bottom). The direction of DNA synthesis is from 5' (bottom) to .V (top) on thecomplementary strand. Lane I, unmodified DNA control: lane 2. cisplatin-modified DNA ((D/N)b = 0.00094); lane 3, |Pt(dien)CI)CI-modified DNA ((D/N)b = 0.00118); lane 4, f/s-[Pt(NH3)2(4-CH.,-py)CI]CI «D/N)b= 0.00121); lane5, os-|Pt(NH,)2(4-Br-py)CI)CI ((D/N)b = 0.00102).

triamine complexes do not bind to DNA in the same manneras c/s-DDP, nor do they form bifunctional complexes containing the c/s-|Pt(NH3)2j2+ fragment through the loss of the pyri-

dine ligand prior to binding to DNA.The aromatic amine ligand appears to be essential for the

antitumor activity of the platinum-triamine complexes. Therelated triamine complexes, [Pt(dien)Cl]+ and [Pt(NH3).,Cl]+,

also form monofunctional lesions on DNA yet are completelyinactive as antineoplastic agents. The monofunctional adductsformed when [Pt(dien)Cl]+ is used to modify DNA do not block

replication either in the mammalian cell extract system (Table1) or in the replication mapping assay (Fig. 1, lane 3). Theseobservations indicate the requirement for the aromatic amineligand for replication inhibition by these complexes and suggest

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that disruption of DNA synthesis might be an important component of the antitumor activity of this class of platinumcomplexes.

Immunochemical Studies. The foregoing results indicate thatplatinum-triamine complexes do not form bifunctional adductson DNA through loss of an amine ligand. This conclusion wasfurther supported by using antibodies raised against the double-stranded DNA adducts of ds-DDP. These antibodies have beenfound to be quite useful for probing the structures of DNAadducts formed by a number of different platinum complexes(47-50). One such monoclonal antibody, CPT2, binds selectively to ds-bidentate platinum cross-links of adjacent purinenucleotides,^.,m-[Pt(NH,):(N7-,N7-GpG)]andm-[Pt(NH,)2-(N7-, N7-ApG)]), but not to DNA adducts formed by trans-DDP or the monofunctional complex [Pt(dien)Cl]Cl. This antibody is relatively insensitive to the chemical nature of theinert platinum amine ligands, however, and therefore binds toDNA adducts formed by complexes such as [Pt(en)Cl2] and[Pt(dach)Cl2] in addition to ds-DDP itself (47). We have usedthese properties of CPT2 specificity to investigate whetherplatinum-triamine complexes form ds-bidentate cross-linkswith adjacent purine nucleotides in DNA.

The initial DNA binding of platinum-triamine complexessuch as ds-[Pt(NH.i)2(4-CH.i-py)Cl]Cl is expected to occur viasubstitution of the relatively labile chloride ligand to yieldmonofunctional adducts (51). Closure of these monofunctionalplatinum adducts could then, in principal, occur to form twodifferent types of cis-bidentate adducts depending upon whichof the ligands is replaced (NHi or 4-CH.i-py). In either case,however, the resulting platinated DNA should be recognized bythe CPT2 antibody since the two types of adducts would differonly in the nature of their inert amine ligands. It is thereforepossible to test directly whether platinum-triamine complexesdo, in fact, close to form ds-bidentate adducts since any suchspecies can be detected immunochemically.

As shown in Fig. 2, no antibody detectable adducts wereformed when DNA was platinated for 24 h at 37°Cwith the

platinum-triamine complexes ds-[Pt(NH.,)2(4-CH.,-py)Cl]Cl or

ito

BO

70

60

so

w-

Legend

a c/«-[Pl(NH3)(4-Mt-Py)CI2]

•ci»-[Pt(NHJJN3-Cytoiin»)CI]CI3 Z

o c/«-[Pt(NH.L(4-M«-Py)CI]CI3 2

0.1 1 10 100

[Ft] Bound (nM)1000

Fig. 2. Recognition of Pt-DNA adducts by the monoclonal antibody CPT2 asdetermined in a competitive ELISA. Competitive inhibition of antibody bindingto immobilized rii-DDP-DNA |(D/N)b = 0.040)) is plotted as a percentage ofantibody binding in the absence of competitor (ci control). Concentrations ofthe competitors m-|Pt(NH3)2(4-CH,-py)CI]CI-DNA [(D/N)b = 0.045], cis-[Pt(NH,h(,V.}-cytosine)Cl]Cl-DNA [

TRISUBSTITl'TED PLATINUM(Il) ANTITUMOR AGENTS

site of platination. As demonstrated previously (52, 53), coordination of a platinum atom to the N7 position of the guaninering lowers the pKu of the HI proton. Titration of this protoncauses a shift in the H8 proton resonance, an effect which isreadily monitored. Platinum complexes of the d(GpG) ligandare expected to display two H8 resonances, one for the 5'- andone for the 3'-guanosine. Interestingly, the 'H NMR spectrum

of product A contains four H8 resonances of equal intensity,which titrate in two groups of two, as shown in Fig. 3. Thetitration behavior of two of these resonances, denoted 3 and 4,is consistent with unplatinated guanosines. Resonances 1 and2 display a completely different titration behavior, titrating atpH ~8. This lowered pKa is characteristic of platinum bindingto the N7 position of guanine (52, 53). From this behavior itcan be concluded that m-[Pt(NH.,)2(/W-cytosine)Cl]Cl bindsonly to one of the two guanine bases in d(GpG).

Further insight into the nature of the platinum adduct inproduct A was provided by enzymatic digestion analyses (38).The platinated d(GpG) was first treated with DNase I and PInuclease to cleave the DNA phosphodiester backbone leavingnucleotides with 5'-phosphates. Alkaline phosphatase was thenadded to remove the 5'-phosphate to yield the corresponding

nucleosides. An HPLC analysis of the products of this enzymatic digestion procedure is shown in Fig. 4a. Peak 3 is freedeoxyguanosine nucleoside, assigned by coinjection with a sample of pure dG. The c/s-[Pt(NH1):(ArJ-cytosine)(dG)]:+ cation,prepared by reaction of c/s-[Pt(NH,):(A'3-cytosine)Cl]Cl with

dG. was used to assign peak 2 by coinjection. Since these arethe only products observed in the enzymatic digestion analysis,it can be concluded that product A is a monofunctional adduct.

The assignment of product A as a single monofunctionaladduct does not explain the presence of four H8 resonances inits 'H NMR spectrum, however. The occurrence of two sets of

resonances suggests either that product A is a mixture of twochromatographically inseparable compounds or that it is asingle compound which can exist in either of two slowly inter-converting conformations. Thus, product A might be a 50:50mixture of compounds in which platinum bound either at the3'- or the 5'-guanosine of d(GpG). Alternatively, it could be a

chemically pure compound with platinum bound to only oneguanosine, present in two slowly interconverting forms. Theformer of these two possibilities was ruled out by repeating theenzymatic digestion but omitting the alkaline phosphatase step.This "modified digest" leaves the 5'-phosphate attached to the3'-guanosine, in effect labeling it and allowing it to be differentiated from the 5'-guanosine. which will not have such aphosphate group. If platinum were bound only to the 5'-

guanosine, one would expect to see two products appear in themodified digestion, namely. m-[Pt(NH.,):(^-cytosine)(dG)]:+and d(pG). If platinum were coordinated only to the 3'-nucle-

oside, the modified digestion would produce two completelydifferent products, free dG and m-|Pt(NH,)2(A'.?-cyto-sine)(dpG))+. The modified digest of a mixture of platinumbound to both the 3' and 5' positions would yield all four of

these products.Fig. 4b displays an HPLC trace of the modified enzymatic

digestion of product A. Peak 2 is the same as found in thecomplete enzymatic digestion procedure, c/s-[Pt(NH.

TRISUBSTITUTED PLATINUM(M)ANTITUMOR AGENTS

Table 2 mPt and "Ar chemical shifts (ppm) and coupling constants (Hz) for cis-fP, and related complexes"

'J('"Pt-"N)

Complex[Pt("NH3)3Cirm-[Pt("NH3)2(py)Cirrà -[Pt("NH3)2(4-CH3-py)Cirm-[Pt(15NH3)2(4-Br-py)CI]+i-/s-[Pt('5NH3)2(l-CH,-cytosine)Cirm-[Pt("NH3)2(dC)Cirrá-[Pt("NH3)2(dG)Cirâ„¢-[Pt('5NH3)(n-butyl-NH2)Clprij-[Pt('5NH3)2CI2]cis-Ã-

Pt( '5NH3)2(4-Br-py)(dG)l2**cÃ-Ã--(Pt(15NH3)2(4-Br-py)(H20)]J*'16("5Pt)-2357-2277-2277-2275-2345-2341-2304-2410-2088-2433-2055cis

toAm-68.7-61.3-61.2-61.2-65.4-65.5-64.1-67.1-65.9-63.9-82.1transtoAm-65.5-68.9-68.6-69.2-67.6-67.7-68.4-65.4-67.2-66.8cistoAm312323323322323322317318303312384transtoAm278276278284290291293266288298

°All NMR spectra were obtained in dimethyl formamide, except where indicated.* Measured in H2O.

order to evaluate further the stereochemical origin of thisbarrier to rotation, cw-[Pt(NH,)2(Ar3-cytosine)(dG)]2+ and cis-[Pt(NH2)2(4-CH,-py)(dG)]2+ were synthesized by allowingdeoxyguanosine to react with c/s-[Pt(NH,)2(A'3-cytosine)Cl]Cland m-[Pt(NH,)2(4-CH,-py)Cl]Cl, respectively. If the rotational barrier in product A were due to steric interactionsbetween substituents on the 5'-guanosine base and either the

carbonyl or exocyclic amino groups of the coordinated cytosine,then a ligand lacking these groups, such as pyridine, should notexhibit such a barrier to rotation. The H8 resonance of the 'HNMR spectrum of m-[Pt(NH,)2(A'^-cytosine)(dG)]2+ is split,

owing to the presence of two conformational isomers. Whenthis sample was heated to 70°C,the resonances broadened, but

the sample temperature could not be raised high enough tocause them to coalesce. The temperature dependent behaviorof the 'H NMR resonances is consistent with the presence of arotational barrier. The H8 resonance in the 'H NMR spectrumof m-[Pt(NH,)2(4-CH.,-py)(dG)]2+ is a sharp singlet. We inter

pret the greater simplicity of the latter spectrum to be due tothe absence of a rotational barrier in this complex. Similarbarriers have been reported for a variety of square planarplatinum nucleobase complexes (54-59). Thus, product A,formed between the antitumor active compound m-[Pt(NH,)2(A'^-cytosine)Cl]Cl and d(GpG), contains platinumcoordinated monofunctionally to the 5'-guanosine N7 position,

an adduci with a barrier to rotation that is measurable on theNMR time scale.

While this work was in progress, the 'H NMR spectrum ofthe reaction product of c/s-[Pt(NH,)2(4-CH,-py)Cl]Cl wjthd(GpG) was reported (60). Only sharp singlets appeared for theH8 resonances, consistent with the absence of a barrier torotation that could be measured on the NMR time scale. In themajor reaction product, the site of platination was assigned toN7 of the 5'-guanosine, and minor products containing platinum bound at N7 of the 3'-guanosine and at both guanosines

were also observed. These results are consistent with independent observations in our laboratory."

Chemical Reactivity of m-|Pt(15NH3)2(Am)Clp Complexes.

Several experiments were carried out to investigate whether theplatinum-triamine complexes might spontaneously eliminatean amine ligand, affording species that could form bifunctionaladducts with DNA. Since previous NMR studies of l5N-labeledplatinum(II) complexes have shown that 195Ptand I5N chemicalshifts and 195Pt-'5N coupling constants reflect the donor properties of ligands bound to platinum (33, 61-66), a series of15NH,-substituted triamine complexes, c¿s-[Pt('5NH.,)2(Am)Cl]+,was prepared to evaluate the relative donor properties of se-

* S. F. Bellon and S. J. Lippard. unpublished results.

lected Am ligands. The results of an NMR study of a series ofeight complexes of the form m-fPtO^NHj^AmJCl]* are sum

marized in Table 2. From the coupling constant data, it isapparent that chloride is the weakest donor ligand in each case,the '95Pt-'5N coupling constant for an ammine trans to chlorinebeing 24-50 Hz greater than for the corresponding couplingconstant trans to Am. The relative donor strength of each Amligand can also be estimated from the data. Based on the l95Pt

chemical shifts, the Am donor strength decreases in the following order: /i-butylamine > NH3 > 1-CHj-cytosine, dC > dG >py, 4-CH,-py, 4-Br-py. In contrast, the 195Pt-'5N-coupling con

stant data suggest a similar but somewhat different ordering:n-butylamine > NH,, py, 4-CH.,-py > 4-Br-py > 1-CH,-cytosine, dC, dG. Although the donor properties of the heterocyclicAm ligands appear somewhat different, as judged by theseparameters, both series indicate that these ligands are slightlyweaker donors than NH.,.

Although the NMR studies of these complexes reveal thatNH, is a stronger donor than Cl~, it has similar donor proper

ties to the heterocyclic Am ligands. Consequently, labilizationof ammonia at the position trans to the Am ligand should notbe highly favored in m-[Pt(NH.,)2(Am)Cl]+ complexes in theabsence of Cl~ substitution. The results suggest that the first

step in the reaction of the triamine complexes with DNA isformation of monofunctional c/'s-[Pt(NH,)2(Am)(DNA-base)]2+adducts. Based on the ll}5Pt-l5N-coupling constant data obtainedon the dG complex cw-[Pt('5NH.,)2(4-Br-py)(dG)]2+, the 4-Br-

py ligand is a slightly better donor than deoxyguanosine. Sinceeach of the amine groups in this species has similar donorstrengths, it is difficult to predict from these data alone whichligand would be the leaving group if substitution by a secondDNA base were to occur. Kinetic and steric effects would playan important role in such ligand exchange reactions.

An example of the importance of the kinetic trans effect inthe substitution reactions of platinum-triamine complexes canbe seen when m-[Pt('5NH,)2(4-Br-py)Cl]Cl is placed in chloride

containing media. Dissolution of this complex in PBS followedby incubation at 37°Cover a period of 14 days afforded ammonium ion ("NH4+) which was detected in solution by "NNMR spectroscopy. The product of this reaction is trans-[Pt(NH,)(4-Br-py)Cl2], as revealed by single crystal X-ray dif

fraction studies (Fig. 5). Ammonia release thus occurs at thesite trans to the chloride ligand. This reaction could also beconducted on a preparative scale by heating c¿s-[Pt(NH.,)2(4-Br-py)Cl]Cl in 0.1 M HC1. A similar reaction occurs when cis-[Pt(NH,)2(l-CH,-cytosine)Cl]Cl is heated in aqueous solution(67). While in principle such a reaction might occur in vivobefore the platinum complex reaches DNA, it is unlikely that

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TRISUBSTITUTED PLATINUM(Il) ANTITUMOR AGENTS

CM

Fig. 5. Illustration of rrans-[Pt(NH3)(4-Br-py)Cl2] displaying non-hydrogen;m>m -id', thermal ellipsoids. H atoms are depicted as spheres with B assignedas 1 AJ for clarity.

the resulting trans complex would be responsible for the observed antitumor response. In fact, independent preparationand screening studies show that frans-[Pt(NHj)(4-Br-py)Cl2]does not display antitumor activity against the Sarcoma 180aascites in CFW mice (Table S6).4 While trans complexes of this

type are known to produce bifunctional lesions on DNA (45),these complexes have yet to demonstrate antitumor activity inmurine tumor systems.

Ammonia Loss Studies. In view of the forgoing results, studiesof the interaction between c/s-[Pt(15NH3)2(4-Br-py)Cl]Cl and

excess dG were carried out to determine whether ammoniarelease might occur during this model nucleobase reaction. The195Ptand 15NNMR spectra of the product of this reaction, cis-[Pt(l5NH3)2(4-Br-pyridine)(dG)]2"1", reveal that ammonia is not

liberated in the presence of excess dG, even after prolongedreaction times (7 days at 37°C).

Mechanistic Implications

The results of these studies, a preliminary account of whichwas given previously (68), demonstrate for the first time thatc/i-[Pt(NH3)2(Am)Cl]Cl platinum-triamine complexes inhibitDNA synthesis and specifically block DNA polymerases atplatinated guanosine residues. NMR spectroscopic, enzymaticdigestion, monoclonal antibody analysis, and replication mapping studies further revealed that these compounds bind DNAin a monofunctional manner, a conclusion confirmed by independent work that appeared while this manuscript was in preparation (60). Several questions remain, however, concerning themechanism by which DNA synthesis is inhibited. For example,what structural features of the monofunctional lesions are required for blocking DNA polymerase activity? Do the aromaticamine ligands themselves interact with DNA, perhaps by intercalation, forming a secondary, noncovalent interaction thatmimics the structural distortions produced by cis- or trans-DDPadducts?

While further studies are required to examine the details ofthe mechanism of inhibition at a molecular level, it is clear thatthe platinum-triamines represent a new class of platinum anti-tumor agents. Since these complexes bind DNA in a monodentate fashion, a binding mode previously expected not to resultin antitumor activity, it is likely that the details of their mech

anism of action will differ in some respects from that of cis-platin, which forms mainly intrastrand d(GpG) and d(ApG)cross-links. Recently, a cellular DRP that senses the structuraldistortions in DNA caused by cisplatin binding has been identified (69). This DRP does not bind to DNA damaged eitherby m-[Pt(NHj)2(Am)Cl]Cl platinum-triamine complexes or bytrans-DDP. One interesting hypothesis to account for the an-

ticancer activity of cisplatin is that the DRP recognizes andbinds m-DDP lesions on DNA, protecting them from repair(69). Consistent with this hypothesis is the fact that trans-DDPadducts are more efficiently repaired than those of cisplatin(28). The inability of cisplatin-damaged DNA to be replicatedor transcribed is presumably a major cause of tumor cell lethality. Perhaps the platinum-triamine complexes, although notrecognized by the cisplatin DRP, are not efficiently repairedand, consequently, their DNA adducts are toxic to tumor cells.Whatever the detailed mechanism, however, these complexesare unique from a mechanistic point of view and, therefore,worthy of additional biological testing in the hope that theymight also display a unique spectrum of activity. Further studiesshould reveal the extent to which these compounds hold promise as clinically useful antitumor drugs.

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1991;51:1866-1875. Cancer Res L. Steven Hollis, Wesley I. Sundquist, Judith N. Burstyn, et al. Platinum(II) Antitumor AgentsMechanistic Studies of a Novel Class of Trisubstituted

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