History, Mechanism, and Synthesis of Cisplatin and Related Analogs
Michelle Livings
Chemical Information Retrieval, CHEM-767
Final Project, due 4 December 2010

Contents
(I) Introduction
(II) History of Cisplatin
(III) How Does Cisplatin Work?
(IV) Syntheses of Cisplatin
(V) Analogs of Cisplatin
(VI) Conclusions


Since 1978 cisplatin has been proven to be one of the most effective platinum-based anti-cancer agents for treating a variety of solid tumor cancers. In addition to its high effectiveness and its capacity to treat multiple types of cancers, the drug is very popular because it is relatively quick and simple to synthesize. Unfortunately cisplatin causes very serious violent side effects, including permanent hearing loss and toxicity to the kidneys and the nervous system. Thus a plethora of novel analogs based on cisplatin are being researched, several of which have reached Phase III clinical trials, and several others that have already been clinically approved for widespread public use. Research is still being undertaken to find a “miracle treatment” that will have little to no side effects and will be as effective (if not more effective) than cisplatin when administered orally.


Introduction
cis-diamminedichloroplatinum(II) (CDDP), the compound cis-PtCl2(NH3)2, more commonly known as cisplatin, is a popular anti-cancer agent used to treat a variety of different cancers including lymphomas (cancer of the lymphatic in the immune system); sarcomas (connective tissue cancers); certain carcinomas (cancers caused by transformed epithelial cells, such as ovarian cancer); and germ cell tumors. Cisplatin is a platinum(II)-based drug that is intravenously injected as an infusion in saline solution as a chemotherapy drug for direct treatment of solid tumors. It was the first platinum(II)-based anti-cancer agent (1); this class now also includes drugs such as carboplatin (cis-diammine(cyclobutane-1,1-dicarboxylate-O,O')platinum(II)) (2) and oxaliplatin ([(1R,2R)-cyclohexane-1,2-diamine](ethanedioato-O,O')platinum(II)) (3); each of these platinum complexes binds to DNA in vivo and causes a crosslinking of the DNA bases, ultimately resulting in programmed cell death, or apoptosis (4). Cisplatin is still among the most highly effective anti-cancer drugs; however it displays toxicity to the kidneys and the nervous system (5), thus researchers are constantly looking for an adequate replacement that is equally effective while being significantly less toxic.

History of Cisplatin
Cisplatin was first synthesized by Michel Peyrone in 1845. The structure of the compound was correctly proposed by Alfred Werner in 1893, upon the proposal of Werner’s theory of coordination chemistry, when he identified the square planar configuration and distinguished between cisplatin and transplatin; respectively, the cis and trans isomers of the square planar compound. (Werner won the Nobel Prize for Chemistry for this theory of coordination chemistry in 1913.) In 1965 Barnett Rosenberg at the University of Michigan discovered that cisplatin effectively inhibited cellular division of E. coli bacteria. Rosenberg, a biophysicist, was researching whether electrical currents played a role in cellular division; he was growing E. coli cells in an ammonium chloride buffer and he applied a current to the cells through platinum electrodes immersed in the buffer. Platinum hydrolysis products – including cisplatin – were building up on the “inert” platinum electrodes (4), ultimately causing the inhibition of cellular division of the bacteria and resulting in the bacteria growing to 300 times its normal length (6). Because of this inhibition, Rosenberg proposed that this cisplatin platinum complex (as well as several other cis platinum coordination complexes) might be effective as an anticancer agent; each platinum complex was tested with human leukemia cells, as well as on sarcomas implanted in rats. The most effective anticancer agent of the test group was cis-PtCl2(NH3)2 – cisplatin (7).
Cisplatin was approved for clinical use in the United States by the Food and Drug Association in 1978 (8).

How Does Cisplatin Work?
The mechanism of cisplatin is widely believed to include an interaction with DNA leading to apoptosis, or programmed death of the cell. Cisplatin is directly administered into the bloodstream of the patient, whereupon the drug interacts with a high concentration (about 100 mM) of blood plasma chloride that prevents aquation of the drug molecule; the chloride concentration is high enough to hinder cisplatin’s chloride ligands from being replaced by water molecules. But the drug molecule undergoes attack from thiol-containing blood plasma proteins, including cysteine (amino acid) and human serum albumin. As soon as one day after the drug is administered, 65-98% of the platinum is protein bound in the blood plasma (8, 9); this protein binding may be responsible for deactivation of the cisplatin drug and also some of the side effects caused by the drug (10, 11). (Side effects may be quite severe, including nausea, vomiting, hearing difficulty, toxicity to the kidneys and nervous system, among others) (5). The remaining part of the cisplatin molecule may either diffuse through the cell membrane of the tumor cells or may be actively transported across the tumor cell membrane via copper transporting proteins. Inside the tumor cell, the chloride concentration is considerably lower than in the blood plasma (at most 20 mM); thus at this point one of the chloro ligands on the drug molecule is aquated, that is, one chloro ligand is replaced by a water molecule. This monoaquation forms a positively-charged and highly reactive species that is unable to leave the tumor cell; studies in vitro show that this charged species causes 98% of platinum binding to cellular DNA in the nucleus (12) by reacting with one of the DNA bases, usually guanine. This forms a DNA 1,2-adduct that is monofunctional, i.e. it has one reactive site. A bifunctional (i.e. having two reactive sites) 1,3-adduct is formed via ring closure, either directly by way of the monofunctional adduct, or by aquation of the second chloro ligand followed by ring closure (13). These bifunctional adducts include guanine-guanine and guanine-adenine, distorting the DNA in a way that it can be recognized by one or more binding proteins, which then either begin DNA damage repair or begin the process of apoptosis (14).
The success of the cisplatin drug is greatly hindered by tumor resistance. Tumor cells related to cancers such as colon cancer and non-small-cell lung cancer are inherently resistant to cisplatin; tumor cells related to cancers such as ovarian cancer and small-cell lung cancer acquire resistance to cisplatin over a certain period of time. Both forms of resistance are due to decreased cellular drug accumulation; increased capability of cells to repair DNA damage caused by the drug; and / or increased levels of thiol-containing blood plasma proteins that cause deactivation of the drug (13).

Syntheses of Cisplatin
Early methods of synthesizing cisplatin were vastly unreliable, yielding impure products. The first adequate synthesis (i.e. good product purity, high overall yield, and low reaction time) was presented in 1970 by S.C. Dhara in the Indian Journal of Chemistry; “A rapid method for the synthesis of cis-[PtCl2(NH3)2],” shown in Scheme I, presented the framework for the majority of subsequent syntheses of cisplatin. In Dhara’s procedure, the starting material potassium tetrachloroplatinate, K2[PtCl4], is first converted to its tetraiodo counterpart, tetraiodoplatinate, K2[PtI4], via addition of a saturated potassium iodide (KI) solution. This initial step will later ensure the retrieval of the desired cis product with no contamination by the Magnus’ Green salt, [Pt(NH3)4][PtCl4]. The second step involves addition of ammonia (NH3), which forms a yellow compound, cis-[PtI2(NH3)2]. After this product is collected and dried, the addition of aqueous silver nitrate (AgNO3) causes precipitation of insoluble silver iodide (AgI), which can be easily filtered off and discarded. The filtrate contains the desired cis-[Pt(OH2)2(NH3)2]2+, which is then treated with potassium chloride (KCl), yielding the precipitation of a yellow powder, cis-[PtCl2(NH3)2], cisplatin (4).

cisplatindharasynth.JPG
Scheme I: the Dhara synthesis of cisplatin (4).

Dhara’s procedure for the synthesis of cisplatin is highly successful due to the trans effect. Chernyaev introduced the trans effect in 1926 in order to explain how the rate of substitution of a ligand in a square planar or octahedral metal complex is primarily dependent on the group trans (opposite) to it, considerably more so than the groups cis (adjacent) to it. Looking at the intermediate K[PtI3(NH3)] group in Scheme I, the iodo ligand that is trans to another iodo group is much more easily displaced than the iodo ligand trans to the ammonia group; this is dictated by Chernyaev’s trans effect, which shows how the initial conversion from potassium tetrachloroplatinate to potassium tetraiodoplatinate yields the final desired cis configuration of the cisplatin platinum complex, due to the stronger trans effect provided by iodo ligands as opposed to chloro ligands (4).
Transplatin, the trans isomer of cisplatin, was first synthesized in 1844 by J. Reiset (15); however the most commonly used synthesis of transplatin was introduced in 1963 by G.B. Kauffman and D.O. Cowan in Inorganic Synthesis. This common procedure is shown in Scheme II. The same starting material is used as in Dhara’s cisplatin synthesis; potassium tetrachloroplatinate is first heated while ammonia is added, resulting in the conversion to [Pt(NH3)4]Cl2 salt. The solution undergoes evaporation, and then hydrochloric acid (HCl) is added and evaporation is performed again, resulting in precipitation of a yellow powder, trans-[PtCl2(NH3)2], transplatin. This synthesis of transplatin is also largely driven by the trans effect; looking at the intermediate [PtCl(NH3)3]+ group in Scheme II, the ammonia group trans to the chloro ligand is the most easily displaced, thus yielding the desired transplatin molecule (4). Transplatin, and most trans compounds in general, are much more reactive than their cis counterparts, reacting at a much faster rate with ammonia, water, and glutathione in red blood cells; the transplatin drug’s fast reactivity is due to the trans effect. Also, trans compounds are less biologically active than their cis counterparts because of the trans compounds’ inability to form 1,2-adducts, and the 1,3-adducts that the trans compounds are able to form are much more rapidly repaired by DNA binding proteins (16, 17).

transplatinsynth.JPG
Scheme II: synthesis of transplatin (4).

In 1894, N.S. Kurnakow introduced a means for distinguishing between cis and trans isomers of square planar complexes. When reacted with thiourea [SC(NH2)2], cisplatin will yield a dark yellow solution containing [Pt(Th)4]2+ (Th = thiourea), while transplatin will yield a white insoluble compound, trans-[Pt(NH3)2(Th)2]Cl2. This example gives straightforward visual evidence regarding which isomer of the square planar complex is being dealt with. Several other tests to distinguish between cisplatin and transplatin also exist; however the Kurnakow test is the most commonly utilized (18).

Analogs of Cisplatin
Research is constantly underway to improve the clinical performance of cisplatin, because of cisplatin’s unfortunate side effects (i.e. nausea, vomiting, hearing loss, and toxicity to the kidneys and nervous system) (5). However, for a platinum-complex drug to reach clinical trials, the drug must have at least one major advantage in comparison to cisplatin; examples of major advantages may be activity against cancers with tumor resistance to cisplatin, reduced side effects, reduced toxicity, or oral administration (instead of intravenous injection) (7). Cleare and Hoeshcele performed numerous studies before reaching a list of criteria, dubbed the “structure-activity relationships,” detailing the structural features necessary for a platinum complex to demonstrate anti-cancer properties. Several of these necessary structural features include: two leaving groups, with cis geometry, that are moderately easy to remove (the reactivity of the leaving groups influences the activity and also the toxicity of the drug, which is why the leaving groups should only be “moderately easy” as opposed to “very easy” to remove); two amine groups with cis geometry; neutral charge; few alkyl substituents on the amine ligands, and at least one proton per amine ligand; and finally the general structure of a platinum complex is shown in Figure 1, where X = leaving groups (such as chloro groups) and R = protons or alkyl substituents. The majority of anti-cancer platinum complexes follow all of these “structure-activity” criteria (19, 20).

cisplatinplatinumcomplex.JPG
Figure 1: general structure of a platinum complex for an analog of cisplatin (4).

Barnett Rosenberg, who made the connection between cisplatin and anti-cancer treatment, was also the first to investigate into cisplatin analogs with greater biological activity. Rosenberg’s research group, in collaboration with the Institute of Cancer Research in London, is responsible for carboplatin, cis-diammine(cyclobutane-1,1-dicarboxylate-O,O')platinum(II), the second drug (after cisplatin) to gain clinical approval from the US Food and Drug Administration in 1989 (2). The leaving group on the carboplatin drug, which is a bidentate cyclobutanedicarboxylate group, is a more stable leaving group than the chloride groups present on the cisplatin drug, which causes a slower reaction of the carboplatin drug within the molecule, and which ultimately results in lower toxicity than cisplatin. Carboplatin has fewer and much less severe side effects; kidney toxicity is completely eliminated as a side effect, and nausea and vomiting are considerably lessened when compared to cisplatin treatment (2, 5). Also, the carboplatin drug may be used against certain cancers that are resistant to cisplatin treatment, such as small-cell and non-small-cell lung cancers, ovarian cancer, bladder cancer, and acute leukemia, each of which is either inherently resistant to cisplatin or builds up a resistance to the cisplatin drug over time (2).
Yoshinori Kidani discovered [(1R,2R)-cyclohexane-1,2-diamine](ethanedioato-O,O')platinum(II), also known as oxaliplatin, in 1976. Kidani received a U.S. patent for oxaliplatin in 1979, but oxaliplatin was not approved until 1996 in Europe, and eventually in 2002 the U.S. Food and Drug Administration granted it clinical approval as well. Oxaliplatin is approved in Europe and the U.S. specifically as an advanced colorectal cancer agent; it is the only drug thus far to have displayed anti-cancer activity specifically against colorectal cancer. It is a unique platinum complex because it features a bidentate 1,2-aminocyclohexane ligand instead of the two amine ligands present on the cisplatin drug and the carboplatin drug (3).
Drug molecules have also been designed specifically to overcome cisplatin resistance. An example is picoplatin, cis-ammine dichloro(2-methyl pyridine) platinum(II). As previously discussed, cisplatin seems to be deactivated after reacting with thiol-containing compounds in the blood plasma; the picoplatin molecule contains a bulky 2-methylpyridine group that prevents thiol binding to the platinum center. Picoplatin entered clinical trials in 1997 for the treatment of solid tumors, and displayed significant activity toward small-cell lung cancer, ovarian cancer, colorectal cancer, and hormone-refractory prostate cancer in Phase I and Phase II clinical trials. In Phase III clinical trials picoplatin was aimed specifically toward advanced small-cell lung cancer, but the drug failed to meet expectations; thus the picoplatin drug is now undergoing Phase III clinical trials specifically for metastatic colorectal cancer (21).
The majority of anti-cancer platinum research has been based on platinum(II) complexes; however, Barnett Rosenberg’s experiment that connected cisplatin with anti-cancer activity also showed that platinum(IV) species might possess anti-cancer activity. Research involving platinum(IV) complexes has recently picked up due to high biological activity, low toxicity, and the possibility of oral administration, which is typically preferred when compared to intravenous injection (22). Typically platinum(IV) complexes are inert, and are considered prodrugs, which ultimately must be reduced to platinum(II) analogs in order to increase reactivity and display anti-cancer activity. Thus the platinum(IV) complexes tend to lose two axial ligands, changing the complex from the octahedral platinum(IV) configuration to the square planar platinum(II) configuration (4). Several platinum(IV) drugs have entered clinical trials, including iproplatin, tetraplatin / ormaplatin, and sarbaplatin. Iproplatin, (cis,trans,cis-[PtCl2](OH2)(isopropyl amine)2], was selected for clinical trials due to unique structure, high solubility, high biological activity, and reduced toxicity in relation to cisplatin, but ultimately iproplatin displayed no major advantages over carboplatin and was not tested any further (4). Ormaplatin, also known as tetraplatin, or [PtCl4(D,L-cyclohexane-1,2-diamine)], was selected for clinical trials because animal studies provided evidence for lower kidney toxicity than cisplatin, but ultimately ormaplatin caused severe toxicity to the nervous system and was not tested any further (5). Satraplatin, cis,trans-[PtCl2(acetato)2(NH3)(cyclohexylamine)], was selected for clinical trials because it displayed high biological activity in cancers that were resistant to cisplatin, as well as displaying minimal toxicity in animals in terms of the kidneys and nervous system; also, perhaps most impressive, the satraplatin drug’s biological activity was still comparable to those of carboplatin and cisplatin when administered orally – one of the most desired qualities of an anti-cancer agent. Thus satraplatin has progressed to Phase III clinical trials, specifically for hormone-refractory prostrate cancer.
Several novel drugs are being investigated that directly violate one or more of the aforementioned “structure-activity” criteria proposed by Cleare and Hoeshcele; these are referred to as polynuclear compounds due to their numerous platinum centers (23). One polynuclear platinum compound, known as BBR 3464, was selected for clinical trials, due to high biological activity, both in cancers that are resistant to cisplatin as well as being generally more effective than cisplatin. BBR 3464 is still currently in Phase II clinical trials (24).

Ideas & Recommendations
As has just been demonstrated in the above discussion of cisplatin analogs, much research has been undertaken regarding improving the platinum-based anti-cancer agent. There are still several avenues yet to be explored. It is highly preferred for these anti-cancer agents to be orally administered as opposed to intravenous injection, and of the drugs previously discussed, only sarbaplatin displays high anti-cancer activity when administered orally, and sarbaplatin is still in clinical trials. One definite opportunity for further research would involve the development of drugs specifically designed for oral administration. Also, the last drug mentioned in the previous section, known as BBR 3464, is said to be in direct violation of two of the “structure-activity” criteria put forth by Cleare and Hoeshcele. Another area that may be a good opportunity for further research would involve investigating other compounds that violate one or more of these “structure-activity relationships,” since BBR 3464 has proven to be so much more effective than cisplatin thus far through Phase I and Phase II clinical trials. Possible “structure-activity violations” may include polynuclear platinum compounds, as mentioned above; or varying the alkyl substituents bound to the amine groups on the compound. A third opportunity for further research may involve the basic idea of varying the ligands bound to the platinum center. The most work in terms of cisplatin analog development seems to be along these lines, varying the ligands on the platinum center, but there are still possibilities available for trial, particularly in the spectrum of bidentate ligands.

Conclusions
Cisplatin has been a leading anti-cancer agent for over thirty years and is still largely popular due to its quick and relatively simple synthesis, its overall effectiveness, and its capacity to fight a variety of different types of cancers. However, the downfalls of cisplatin are serious, especially the toxicity to the kidneys and nervous system and permanent hearing loss; these intimidating side effects have led to an outburst of research and development in the area of cisplatin analogs, i.e. cis isomers of square planar platinum(II) complexes. Several novel analogs have been clinically approved for anti-cancer use, including carboplatin and oxaliplatin; and several more novel analogs are undergoing clinical trials, such as picoplatin, satraplatin, and the compound BBR 3464. Unfortunately there is still no “miracle cure” or even “miracle treatment” for cancers, thus there is still a vast array of research currently underway, as well as a wide-open future in terms of anti-cancer platinum-based drug development.


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