3-Hydroxycarboplatin, a Simple Carboplatin Derivative Endowed with an Improved Toxicological Profile
3-Hydroxycarboplatin, a Simple Carboplatin Derivative Endowed with an Improved Toxicological Profile
3-Hydroxycarboplatin, a simple carboplatin derivative, was synthesised using a novel method, characterised and evaluated for its anticancer activity in vitro and in vivo. It shows comparable antitumour activity to that of carboplatin but has much lower toxicity particularly with respect to myelosuppression, revealing great potential for development as a new antitumour platinum drug to replace carboplatin.
cis-Diammine(1,1-cyclobutanedicarboxylato)platinum(II) (carboplatin) is a second generation Pt anticancer drug following cis-diamminedichloroplatinum(II) (cisplatin). It shows the same level of activity as cisplatin in treating some kinds of cancers, but is much less nephrotoxic and emetic than cisplatin (1, 2). Carboplatin has gained worldwide marketing approval and is currently used as standard therapy in ovarian cancer patients.
Compared with cisplatin, carboplatin causes substantial myelosuppression, principally thrombocytopenia – a dose-limiting side effect that has prompted a continuing search for new, potent Pt complexes possessing lower toxicity. Indeed, the past decade has witnessed a shift in focus toward nonclassical Pt compounds represented by picoplatin, polynuclear complexes, trans-Pt complexes and Pt(IV) complexes (3). Unfortunately, the outcomes of clinical trials of these complexes have failed to meet expectations, and none of these complexes has been approved for clinical application (4). However, direct modification of clinically established Pt drugs remains an effective way to create new derivatives with improved toxicological profiles (5, 6). 3-Hydroxycarboplatin (Figure 1), first reported in 2004 by G. Bernhardt et al. (7), is a direct and simple derivative of carboplatin in which the cyclobutane ring is substituted with OH at position 3. As part of a drug development programme beginning in 2001 aimed at improving the pharmacological profile of carboplatin, we designed and prepared a series of carboplatin derivatives, including 3-hydroxycarboplatin. Following extensive biological evaluation, we found that 3-hydroxycarboplatin showed anticancer activity similar to that of carboplatin, but exhibited an improved toxicological profile, suggesting great promise for further development. In the present article, we report the synthesis, characterisation and biological effects of 3-hydroxycarboplatin in vitro as well as in vivo using two animal models.
2. Synthesis and Characterisation
2.1 Preparation of 3-Hydroxy-1,1-cyclobutanedicarboxylic Acid
3-Hydroxy-1,1-cyclobutanedicarboxylic acid was prepared according to Scheme I in a similar method to that reported earlier (7) with epichlorohydrin as the starting chemical. First, epichlorohydrin was treated with benzyl bromide in the presence of mercuric chloride and heated at 160°C to give the benzyl ether 5 with a yield of 70%. Then, the addition of diethyl malonate on derivative 5 gave the cyclobutane 6 with 51% yield. The hydrolysis of 6 yielded the potassium salt 7 with 69% yield. Hydrogenolysis was then used for the cleavage of 7 forming the desired 3-hydroxy-1,1-cyclobutanedicarboxylic acid 8 in 60% yield. The overall yield for the formation of 8 was about 15% (melting point 156–158°C). The product 8 was characterised by elemental analysis and proton nuclear magnetic resonance spectroscopy (1H-NMR), and the data are consistent with its composition and structure and are as reported in the literature (7).
Found (% calculated for C6H8O5): C, 44.8 (45.0); H, 5.05 (5.00).
1H-NMR (D2O, 500.1 MHz, δ (ppm)): 4.27 (1H, CHOH), 2.77 (2H, CH2), 2.37 (2H, CH2).
In order to further characterise the product, single crystals suitable for X-ray analysis were selected from its zinc salt which was prepared in a two-step procedure. 3-Hydroxy-1,1-cyclobutanedicarboxylic acid was mixed with an excess of zinc hydroxide (Zn(OH)2) in water at 45°C for 2 h and then the remaining Zn(OH)2 was removed by filtration. The resulting filtrate was evaporated under reduced pressure to give rise to the corresponding zinc salt in a white crystalline form. A molecular plot of the chemical structure determined by X-ray analysis, depicted in Figure 2, demonstrates that the anion is 3-hydroxy-1,1-cyclobutanedicarboxylate.
Treatment of 3-hydroxy-1,1-cyclobutanedicarboxylic acid with sodium hydrogen carbonate (NaHCO3) in water provided the Na salt of 3-hydroxy-1,1-cyclobutanedicarboxylic acid, which was then reacted with silver nitrate (AgNO3) to produce the insoluble Ag salt as an intermediate with a yield of 96% (Scheme I).
Found (% calculated for Ag2C6H6O5): C, 19.4 (19.2); H, 1.65 (1.60); Ag, 57.5 (57.8).
Infrared (IR) (KBr, cm−1): 1720 (s, νasCOO−), 1357 (s, νsCOO−).
2.2 Preparation of 3-Hydroxycarboplatin
3-Hydroxycarboplatin was synthesised in a three-step reaction route (Scheme II) starting from commercially available potassium tetrachloroplatinate (K2PtCl4). K2PtCl4 was first converted in situ to potassium tetraiodoplatinate (K2PtI4), followed by addition of ammonia to produce insoluble cis-diamminediiodoplatinum(II) (cis-[Pt(NH3)2I2]). The quantitative reaction in water with the Ag salt of 3-hydroxy-1,1-cyclobutanedicarboxylic acid yielded the target complex (Scheme II). Recrystallisation from a solution of water and ethanol (1:1) was required to obtain samples for structural characterisation and biological tests. The overall yield was about 76% and the purity, determined by an established reversed-phase high-performance liquid chromatography (RP-HPLC) method (8), was >98.5%.
The target complex was structurally characterised by elemental analysis, Fourier transform infrared (FTIR) spectroscopy, 1H-/carbon nuclear magnetic resonance (13C-NMR) spectroscopy, fast atomic bombardment mass spectrometry (FAB+-MS) (Table I) and X-ray crystallography. The results agree well with the literature (7).
|Composition, %||Found: C, 18.4; N, 7.14; H, 3.17; Pt, 50.2|
|Calculated for C6H12N2O5Pt: C, 18.6; N, 7.23; H, 3.10; Pt, 50.4|
|FTIRa, KBr, cm−1||3296 (s, νN–H), 3118–2953 (w, νC–H), 1641–1609 (s, νasCOO−), 1391–1354 (s, νsCOO−), 1103–1049 (m, νC–O), 449 (w, νPt–N)|
|FAB+-MSb, cm/z, intensity||110 (100%) [C6H5O2]+, 202 (35%) [C6H5O2 + gly]+, 230 (12%) [Pt(NH3)2]+, 355 (8%) [C6H6O5Pt]+, 388 (92%) M+, 480 (28%) [M + gly]+|
|1H-NMRd, e, DMSO-d6, δ ppm||4.15 (qi, 1H, CH, J = 7.2 Hz), 3.27 (t, 2H, CH2, J = 2.6 Hz), 2.55 (t, 2H, CH2, J = 2.6 Hz)|
|13C-NMR, DMSO-d6, δ ppm||182.1 (C-1), 181.8 (C-3), 61.6 (C-6), 42.2 (C-2), 17.5 (C-4,5)|
The elemental analysis data are in good agreement with the calculated values of 3-hydroxycarboplatin. It exhibited three typical protonated molecular ion peaks reflecting the platinum isotopes: 194Pt (33%), 195Pt (34%) and 196Pt (25%) (9). A strong peak with a relative intensity of 92% at m/z 388, corresponding to M+, was observed in its mass spectrum. The IR spectrum showed the characteristic absorption for stretching bands of N–H near 3296 cm−1, C–H around 2900 cm−1 and Pt–N at 449 cm−1. The νas(COO)-νs(COO) value was larger than 200 cm−1, suggesting that COO− acts as a monodentate ligand (10). The 1H-NMR spectra are all consistent with the corresponding protons both in the chemical shifts and the number of hydrogens. Two protons of CH2 split into two bands near 3.27 ppm and 2.55 ppm, probably due to different spatial orientations in a chair configuration of the cyclobutane ring after the introduction of OH at position 3 (11). The 13C-NMR spectra conform to the expected carbon chemical shifts in the 3-hydroxycarboplatin molecule.
The Oak Ridge Thermal Ellipsoid Plot (ORTEP) drawing of the complex depicted along with its atomic numbering scheme is shown in Figure 3 and the selected bond distances and bond angles are listed in Table II. The Pt(II) centre has the expected square planar geometry exhibiting the usual structure parameters. The basal square plane is constituted by two NH3 and two COO− moieties of 3-hydroxy-1,1-cyclobutanedicarboxylate. As shown in Table II, Pt–N and Pt–O distances and coordinate bond angles of N–Pt–N and O–Pt–O are in the normal range. Similar to the carboplatin molecule (12), the cyclobutane ring adopts a chair configuration and is nearly perpendicular to the Pt(II) coordination plane.
2.3 Physicochemical Properties of 3-Hydroxycarboplatin
The solubility of 3-hydroxycarboplatin and carboplatin in water and in octanol was measured at 25°C (Table III). As expected, 3-hydroxycarboplatin is nearly twice as soluble in water as carboplatin. Surprisingly, it is also much more soluble in octanol than carboplatin, probably because the lattice energy is decreased with the reduction in symmetry from C2v of carboplatin to Cs of 3-hydroxycarboplatin (13). Therefore, 3-hydroxycarboplatin has a more favourable oil/water partition coefficient (logP).
| Solubility at 25°C, mg ml−1
|Compound||In water||In octanol||Partition coefficient, logP|
The aquation rate constant, which is an important parameter in judging the stability of Pt anticancer compounds, was determined by using a previously reported conductivity method (14). The observed aquation rate constant (kobs) of 3-hydroxycarboplatin at 25°C under an N2 atmosphere was 2.9 × 10−6 min−1, less than kobs of carboplatin (3.8 × 10−6 min−1) under the same conditions, suggesting that 3-hydroxycarboplatin is slightly more stable in water than carboplatin itself.
3. Biological Evaluation
3.1 In Vitro Anticancer Activity
3-Hydroxycarboplatin and carboplatin were assayed in vitro against carboplatin-sensitive human cancer cell lines including the non-small cell lung cancer cell line A549 and the ovarian cancer cell lines SK-OV-3 and COCI. Cellular survival was evaluated by the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method (15, 16). Fifty percent inhibitory concentration (IC50) values were calculated from curves constructed by plotting cell survival (%) versus compound concentration (in μg ml−1).
As shown in Table IV, the activity of 3-hydroxycarboplatin against A549, SK-OV-3 and COCI cancer cells was comparable to that of carboplatin. This indicates that introduction of the OH group into carboplatin does not reduce cytotoxicity.
3.2 Acute Toxicity
Acute toxicity is an adverse, non-specific effect that occurs in a healthy animal within 2 weeks of intravenous (iv) injection of a single dose of the drug. Acute toxicity tests of 3-hydroxycarboplatin and carboplatin were carried out in healthy Institute of Cancer Research (ICR) mice according to standard procedures (16). Toxicity, measured as the LD50 value (i.e., the dose that causes the death of 50% of tested animals), was 275 mg kg−1 for 3-hydroxycarboplatin and 148 mg kg−1 for carboplatin, indicating that 3-hydroxycarboplatin is less toxic than carboplatin in animals following iv administration. Histological postmortem examinations of the mice suggested that these Pt complexes caused death primarily through myelosuppression.
3.3 Antitumour Activity
The antitumour activity of 3-hydroxycarboplatin and carboplatin was compared in mouse Lewis lung tumour and human ovarian carcinoma (3AO cell line) xenograft models, using well-established methods (15–19). Based on previous studies, the maximum tolerated dose (MTD) is 90 mg kg−1 for 3-hydroxycarboplatin and 60 mg kg−1 for carboplatin in mice treated with drugs every three days (Q3D) three times each. Tumour-bearing mice were intravenously given 3-hydroxycarboplatin and carboplatin at the MTD. As shown in Table V, treatment with 3-hydroxcarboplatin following tumour implantation caused a dose-dependent reduction of tumour weight in both Lewis lung tumour and 3AO xenograft mice. Notably, the potency of 3-hydroxcarboplatin with respect to inhibition of tumour growth was comparable or superior to that of carboplatin, consistent with the results observed in in vitro cytotoxicity tests.
3.4 Myelosuppression Toxicity
To further explore the potential advantage of 3-hydroxycarboplatin over carboplatin, a repeated-dosing toxicity study was conducted (20). ICR mice were iv administered either 3-hydroxycarboplatin or carboplatin on days 1, 3, 5, and 7. On day 8, all mice were anesthetised and blood samples were collected for blood cell analysis; bone marrow cells were also extracted for proliferation tests. Blood cell counts are a good indicator of bone marrow cell proliferation, and thus myelosuppression, a major side effect of carboplatin. The decrease in blood cell counts associated with this myelosuppression is especially prominent for white blood cell and platelet numbers. As shown in Figures 4–6, both 3-hydroxycarboplatin and carboplatin exerted myelosuppressive effects, as evidenced by lower blood cell counts compared with the control group. However, the effects were much more pronounced with carboplatin, which caused very severe thrombocytopenia.
Consistent with the results of peripheral blood cell counts, mouse bone marrow cell proliferation was suppressed to a much lesser extent by 3-hydroxycarboplatin than by carboplatin (Table VI). 3-Hydroxycarboplatin at a dose of 60 mg kg−1 did not significantly suppress bone marrow cell proliferation, with myeloproliferation ranging from extremely to moderately active. However, the degree of myeloproliferation was low or extremely low in bone marrow from mice treated with the same dose of carboplatin (60 mg kg−1). Collectively, these data indicate that the myelosuppressive side effects of 3-hydroxycarboplatin are much lower than those of carboplatin.
| Relative myeloproliferative indexa
|3-Hydroxycarboplatin||60 mg kg−1||2/10||7/10||1/10||0/10||0/10|
| 90 mg kg−1
|Carboplatin||60 mg kg−1||0/10||1/10||2/10||4/10||3/10|
|90 mg kg−1||0/9||0/9||0/9||1/9||8/9|
3-Hydroxycarboplatin, a simple carboplatin derivative, shows antitumour activity comparable to that of carboplatin but has much lower toxicity, particularly with respect to myelosuppression, reflecting an improved toxicological profile. It also exhibits desirable physicochemical properties. Therefore, 3-hydroxycarboplatin has extremely high potential for development as a clinically useful anticancer agent to replace carboplatin.
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Weiping Liu is a Research Professor at Kunming Institute of Precious Metals, Yunnan, China and also the Chief Scientist of Chemistry and Pharmacy Division under the institute, focusing on the development of platinum compounds for cancer therapy as well as homogeneous catalysis.
Liguang Lou is a Professor of Pharmacology at Shanghai Institute of Materia Medica, Chinese Academy of Sciences, China, with interests in the discovery of new anticancer agents with novel mechanisms of action.
Xizhu Chen is a Senior Engineer at Kunming Institute of Precious Metals, specialising in the synthesis of platinum anticancer compounds.
Qingsong Ye is an Assistant Research Fellow at Kunming Institute of Precious Metals, working in the platinum group metals chemistry group with interests in chemical synthesis.
Shuqian Hou is the former President of Kunming Institute of Precious Metals. His main interests are related to the applications of platinum compounds. He is now the Vice Head of the Science and Technology Department of the Yunnan Provincial Government, China.
Chengying Xie is an Associate Professor at Shanghai Institute of Materia Medica, Chinese Academy of Sciences, with research in the field of oncological pharmacology.