[Show abstract][Hide abstract] ABSTRACT: The cyclin-dependent kinase (CDK) inhibitor seliciclib (1, CYC202) is in phase II clinical development for the treatment of cancer. Here we describe the synthesis of novel purines with greater solubility, lower metabolic clearance, and enhanced potency versus CDKs. These compounds exhibit novel selectivity profiles versus CDK isoforms. Compound αSβR-21 inhibits CDK2/cyclin E with IC(50)=30 nM, CDK7-cyclin H with IC(50)=1.3 μM, and CDK9-cyclinT with IC(50)=0.11 μM; it (CCT68127) inhibits growth of HCT116 colon cancer cells in vitro with GI(50)=0.7 μM; and shows antitumour activity when dosed p.o. at 50mg/kg to mice bearing HCT116 solid human tumour xenografts.
[Show abstract][Hide abstract] ABSTRACT: Extensive evidence implicates activation of the lipid phosphatidylinositide 3-kinase (PI3K) pathway in the genesis and progression of various human cancers. PI3K inhibitors thus have considerable potential as molecular cancer therapeutics. Here, we detail the pharmacologic properties of a prototype of a new series of inhibitors of class I PI3K. PI103 is a potent inhibitor with low IC50 values against recombinant PI3K isoforms p110alpha (2 nmol/L), p110beta (3 nmol/L), p110delta (3 nmol/L), and p110gamma (15 nmol/L). PI103 also inhibited TORC1 by 83.9% at 0.5 micromol/L and exhibited an IC50 of 14 nmol/L against DNA-PK. A high degree of selectivity for the PI3K family was shown by the lack of activity of PI103 in a panel of 70 protein kinases. PI103 potently inhibited proliferation and invasion of a wide variety of human cancer cells in vitro and showed biomarker modulation consistent with inhibition of PI3K signaling. PI103 was extensively metabolized, but distributed rapidly to tissues and tumors. This resulted in tumor growth delay in eight different human cancer xenograft models with various PI3K pathway abnormalities. Decreased phosphorylation of AKT was observed in U87MG gliomas, consistent with drug levels achieved. We also showed inhibition of invasion in orthotopic breast and ovarian cancer xenograft models and obtained evidence that PI103 has antiangiogenic potential. Despite its rapid in vivo metabolism, PI103 is a valuable tool compound for exploring the biological function of class I PI3K and importantly represents a lead for further optimization of this novel class of targeted molecular cancer therapeutic.
Cancer Research 07/2007; 67(12):5840-50. DOI:10.1158/0008-5472.CAN-06-4615 · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: CCT018159 was recently identified as a novel inhibitor of heat shock protein (Hsp) 90, a promising target for cancer therapy. Pharmacokinetic and metabolic properties are likely to be important for efficacy and need to be optimized during drug development. Here, we define the preclinical metabolism and pharmacokinetics of CCT018159 and some early derivatives. In addition, we assess in vitro metabolic stability screening and in vivo cassette dosing (simultaneous administration of several compounds to a single animal) as approaches to investigate these compounds. The plasma clearance following individual i.v. administration to mice was rapid (0.128-0.816 L/h), exceeding hepatic blood flow. For CCT066950 and CCT066952, this could be attributed in part to extensive (>80%) blood cell binding. Oral bioavailability ranged from 1.8% to 29.6%. Tissue distribution of CCT066952 was rapid and moderate, and renal excretion of the compounds was minimal (<1% of dose excreted). Compounds underwent rapid glucuronidation both in vivo and following incubation with mouse liver microsomes. However, whereas CCT066965 was metabolized to the greatest extent in vitro, this compound displayed the slowest plasma clearance. The rank order of the compounds from the highest to lowest area under the curve was the same following discrete and cassette dosing. Furthermore, pharmacokinetic variables were similar whether the compounds were dosed alone or in combination. We conclude that the pharmacokinetics of CCT018159 are complex. Cassette dosing is currently the best option available to assess the pharmacokinetics of this promising series of compounds in relatively high throughput and is now being applied to identify compounds with optimal pharmacokinetic properties during structural analogue synthesis.
Molecular Cancer Therapeutics 07/2006; 5(6):1628-37. DOI:10.1158/1535-7163.MCT-06-0041 · 6.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study we investigated the in vitro time dependence of radiosensitisation, pharmacokinetics and metabolism of NU7026, a novel inhibitor of the DNA repair enzyme DNA-dependent protein kinase (DNA-PK). At a dose of 10 muM, which is nontoxic to cells per se, a minimum NU7026 exposure of 4 h in combination with 3 Gy radiation is required for a significant radiosensitisation effect in CH1 human ovarian cancer cells. Following intravenous administration to mice at 5 mg kg(-1), NU7026 underwent rapid plasma clearance (0.108 l h(-1)) and this was largely attributed to extensive metabolism. Bioavailability following interperitoneal (i.p.) and p.o. administration at 20 mg kg(-1) was 20 and 15%, respectively. Investigation of NU7026 metabolism profiles in plasma and urine indicated that the compound undergoes multiple hydroxylations. A glucuronide conjugate of a bis-hydroxylated metabolite represented the major excretion product in urine. Identification of the major oxidation site as C-2 of the morpholine ring was confirmed by the fact that the plasma clearance of NU7107 (an analogue of NU7026 methylated at C-2 and C-6 of the morpholine ring) was four-fold slower than that of NU7026. The pharmacokinetic simulations performed predict that NU7026 will have to be administered four times per day at 100 mg kg(-1) i.p. in order to obtain the drug exposure required for radiosensitisation.
British Journal of Cancer 11/2005; 93(9):1011-8. DOI:10.1038/sj.bjc.6602823 · 4.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Biliary excretion is a significant component in the metabolism of many drugs, but remains difficult to detect and characterise non-invasively. A previous publication recently described the detection of metabolites of ifosfamide in gall bladder in a guinea pig model using in vivo 1H-decoupled 31P 3-D magnetic resonance spectroscopic imaging and a clinical 1.5 T MR scanner.. Here high-resolution 31P magnetic resonance spectroscopy (MRS) of extracted bile identifies peaks as parent ifosfamide (1.19+/-1.47 mM; mean+/-sd), carboxyifosfamide (2.04+/-1.04 mM) and a major contribution from a previously unreported peak at 16.0 ppm (4.05+/-2.38 mM). The unknown resonance was identified using liquid chromatography-mass spectrometry (LCMS) as the glutathione conjugate of ifosfamide (MW=531). This was confirmed by analysing products from the reaction of glutathione with ifosfamide using LCMS and MRS. These results demonstrate how combined in vivo and analytical MRS, together with mass spectrometry, can help identify visceral routes of drug metabolism, thereby aiding understanding of +/-drug disposition and mechanisms of action and toxicity. In particular, the distribution of ifosfamide and its metabolites into bile may be related to oxazophosphorine-related cholecystitis reported in patients.
Cancer Chemotherapy and Pharmacology 11/2005; 56(4):409-14. DOI:10.1007/s00280-005-1023-2 · 2.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: R-roscovitine (seliciclib, CYC202) is a cyclin-dependent kinase inhibitor currently in phase II clinical trials in patients with cancer. Here, we describe its mouse metabolism and pharmacokinetics as well as the identification of the principal metabolites in hepatic microsomes, plasma, and urine. Following microsomal incubation of R-roscovitine at 10 microg/mL (28 micromol/L) for 60 minutes, 86.7% of the parent drug was metabolized and 60% of this loss was due to formation of one particular metabolite. This was identified as the carboxylic acid resulting from oxidation of the hydroxymethyl group of the amino alcohol substituent at C2 of the purine core present in R-roscovitine. Identification was confirmed by chemical synthesis and comparison of an authentic sample of the R-roscovitine-derived carboxylate metabolite (COOH-R-roscovitine). Other minor metabolites were identified as C8-oxo-R-roscovitine and N9-desisopropyl-R-roscovitine; these accounted for 4.9% and 2.6% of the parent, respectively. The same metabolic pattern was observed in vivo, with a 4.5-fold lower AUC(infinity) for R-roscovitine (38 micromol/L/h) than for COOH-R-roscovitine (174 micromol/L/h). Excretion of R-roscovitine in the urine up to 24 hours post-dosing accounted for an average of only 0.02% of the administered dose of 50 mg/kg, whereas COOH-R-roscovitine represented 65% to 68% of the dose irrespective of the route of administration (i.v., i.p., or p.o.). A partially deuterated derivative (R-roscovitine-d9) was synthesized to investigate if formation of COOH-R-roscovitine could be inhibited by replacement of metabolically labile protons with deuterium. After 60 minutes of incubation of R-roscovitine-d9 or R-roscovitine with mouse liver microsomes, formation of COOH-R-roscovitine-d9 was decreased by approximately 24% compared with the production of COOH-R-roscovitine. In addition, the levels of R-roscovitine-d9 remaining were 33% higher than those of R-roscovitine. However, formation of several minor R-roscovitine metabolites was enhanced with R-roscovitine-d9, suggesting that metabolic switching from the major carbinol oxidation pathway had occurred. Synthetic COOH-R-roscovitine and C8-oxo-R-roscovitine were tested in functional cyclin-dependent kinase assays and shown to be less active than R-roscovitine, confirming that these metabolic reactions are deactivation pathways.
Molecular Cancer Therapeutics 02/2005; 4(1):125-39. · 6.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: R-roscovitine (seliciclib, CYC202; hereafter ROS) is a CDK inhibitor currently in Phase II clinical trials in patients with cancer. Here we describe its mouse metabolism and pharmacokinetics, as well as the identification of the principal metabolites in hepatic microsomes, plasma, and urine. Following microsomal incubation of ROS at 10 mg/mL, (28 mM) for 60 min, 86.7 % of the parent drug was metabolised and 60 % of this loss was due to formation of one particular metabolite. This was identified as the carboxylic acid resulting from oxidation of the hydroxymethyl group of the amino alcohol substituent at C2 of the purine core present in ROS. This was confirmed by chemical synthesis and comparison of an authentic sample of the ROS-derived carboxylate metabolite (COOH-ROS). Other minor metabolites were identified as C8-oxo-ROS and N9-desisopropyl-ROS; these accounted for 4.9 % and 2.6 % of the parent, respectively. The same metabolic pattern was observed in vivo, with a 4.5-fold lower AUC8 for ROS (38 µM.h) than for COOH-ROS (184 µM.h). Excretion of ROS in the urine up to 24 h post dosing accounted for an average of only 0.02 % of the administered dose of 50 mg/kg, whilst COOH-ROS represented 65-68 % of the dose, irrespective of the route of administration (i.v., i.p., or p.o.). A partially deuterated derivative (ROS-d9) was synthesized to investigate if formation of COOH-ROS could be inhibited by replacement of metabolically labile protons with deuterium. After 60 min incubation of ROS-d9 or ROS with mouse liver microsomes, formation of COOH-ROS-d9 was decreased by approximately 24 % compared with the production of COOH-ROS. In addition, the levels of ROS-d9 remaining were 33 % higher than those of ROS. However, formation of a number of minor ROS metabolites was enhanced with ROS-d9, suggesting that metabolic switching from the major carbinol oxidation pathway had occurred. Synthetic COOH-ROS and C8-oxo-ROS were tested in functional CDK kinase assays and shown to be less active than ROS, confirming that these metabolic reactions are deactivation pathways.
Molecular Cancer Therapeutics 01/2005; 4(1):125-139. · 6.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There is currently much interest in developing analogues of the benzoquinone ansamycin geldanamycin that may overcome the limitations of 17-(allylamino)-17-demethoxygeldanamycin (17AAG), which is the first known inhibitor of heat shock protein 90 (Hsp90) to enter clinical trials. Studies were performed to assess whether cassette dosing, the coadministration of several compounds to a single animal, is a suitable approach to evaluate the preclinical pharmacokinetics of geldanamycin analogues in high throughput.
Five geldanamycin analogues (17AAG, NSC 255110, NSC 682300, NSC 683661, NSC 683663) were administered intravenously to mice in combination at 5 mg/kg each and as single agents at 5 mg/kg and 50 mg/kg, or 12.5 mg/kg for NSC 682300. The compounds were also incubated with mouse liver microsomes individually and in combination at 15 microM each. Quantitative analysis was performed by LC/MS/MS. Plasma and tissue pharmacokinetic parameters were evaluated by non-compartmental analysis. In vitro metabolic stability was assessed by monitoring disappearance of the parent compound.
Of the compounds that were detectable following individual administration at 5 mg/kg, 17AAG and NSC 683661 exhibited nonlinear pharmacokinetics. In addition, the plasma area under the curve (AUC) and the half-life of these compounds was greater following cassette dosing at 5 mg/kg compared to single administration at the same dose. When pharmacokinetic parameters were calculated up to the same time point following cassette and individual administration at the higher dose, three of the compounds displayed non-linear increases in AUC and slower clearances following cassette compared to single compound dosing. When all measurable concentrations at the higher dose were included, the half-life of NSC 683663 was nine-fold longer following individual compared to cassette administration. 17AAG displayed the highest AUC following cassette dosing, whereas NSC 683663 displayed the highest AUC following single-compound dosing. Excluding NSC 683663, the rank order from the highest to the lowest AUC was the same; however, NSC 682300, which ranked fifth, was administered at a four-fold lower individual dose than the other compounds. Exposure of the liver and kidneys to the compounds was greater than that of plasma. Despite being administered at a lower dose, NSC 682300 displayed the highest kidney AUC of the five compounds. The same ranking was maintained between cassette and single compound dosing in the kidney. With the exception of NSC 682300, in vitro metabolic stability was predictive of in vivo pharmacokinetics in the plasma and liver. The extent of metabolism of four of the five compounds was lower following microsomal incubation in combination compared to incubation alone, suggestive of likely drug-drug interaction in the cassette. However, for 17AAG this may be partly due to metabolism of NSC 683661 and NSC 683663 to this compound.
Whilst cassette dosing has advantages for use in drug discovery, it is probably unsuitable to evaluate the pharmacokinetics of geldanamycin analogues due to non-linear pharmacokinetics and drug-drug interaction. The issues identified for this compound series should also be considered in assessing the suitability of cassette dosing for other chemotypes.
Cancer Chemotherapy and Pharmacology 01/2005; 54(6):475-86. DOI:10.1007/s00280-004-0853-7 · 2.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The acquisition of resistance to apoptosis, the cell's intrinsic suicide program, is essential for cancers to arise and progress and is a major reason behind treatment failures. We show in this article that small molecule antagonists of the sigma-1 receptor inhibit tumor cell survival to reveal caspase-dependent apoptosis. sigma antagonist-mediated caspase activation and cell death are substantially attenuated by the prototypic sigma-1 agonists (+)-SKF10,047 and (+)-pentazocine. Although several normal cell types such as fibroblasts, epithelial cells, and even sigma receptor-rich neurons are resistant to the apoptotic effects of sigma antagonists, cells that can promote autocrine survival such as lens epithelial and microvascular endothelial cells are as susceptible as tumor cells. Cellular susceptibility appears to correlate with differences in sigma receptor coupling rather than levels of expression. In susceptible cells only, sigma antagonists evoke a rapid rise in cytosolic calcium that is inhibited by sigma-1 agonists. In at least some tumor cells, sigma antagonists cause calcium-dependent activation of phospholipase C and concomitant calcium-independent inhibition of phosphatidylinositol 3'-kinase pathway signaling. Systemic administration of sigma antagonists significantly inhibits the growth of evolving and established hormone-sensitive and hormone-insensitive mammary carcinoma xenografts, orthotopic prostate tumors, and p53-null lung carcinoma xenografts in immunocompromised mice in the absence of side effects. Release of a sigma receptor-mediated brake on apoptosis may offer a new approach to cancer treatment.
Cancer Research 08/2004; 64(14):4875-86. DOI:10.1158/0008-5472.CAN-03-3180 · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A series of three dose escalating studies were conducted to investigate the ability of the 17alpha-hydroxylase/C(17,20)-lyase inhibitor abiraterone acetate, to cause maximum suppression of testosterone synthesis when delivered to castrate and noncastrate males with prostate cancer. Study A was a single dose study in castrate males. Study B was a single dose study in noncastrate males and study C was a multiple dose study in noncastrate males. The drug was given orally in a once-daily dose and blood samples taken to assess pharmacokinetic (PK) parameters and hormone levels in all patients. The study drug was well tolerated with some variability in PKs. Suppression of testosterone levels to <0.14 nmol l(-1) was seen in four out of six castrate males treated with a single dose of 500 mg. At 800 mg given days 1-12 in noncastrate males, target suppression was achieved in three out of three patients, but a two- to three-fold increase of Luteinising Hormone (LH) levels in two out of three patients overcame suppression within 3 days. All patients in the multiple dose study developed an abnormal response to a short Synacthen test by day 11, although baseline cortisol levels remained normal. This is the first report of the use of a specific 17alpha-hydroxylase/(17,20)-lyase inhibitor in humans. Repeated treatment of men with intact gonadal function with abiraterone acetate at a dose of 800 mg can successfully suppress testosterone levels to the castrate range. However, this level of suppression may not be sustained in all patients due to compensatory hypersecretion of LH. The enhanced testosterone suppression achieved in castrate men merits further clinical study as a second-line hormonal treatment for prostate cancer. Adrenocortical suppression may necessitate concomitant administration of replacement glucocorticoid.
British Journal of Cancer 07/2004; 90(12):2317-25. DOI:10.1038/sj.bjc.6601879 · 4.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Idoxifene is a novel selective oestrogen receptor modulator (SERM) which had greater binding affinity for the oestrogen receptor (ER) and reduced agonist activity compared with tamoxifen in preclinical studies. In a randomized phase II trial in 56 postmenopausal patients with progressive locally advanced/metastatic breast cancer we assessed whether idoxifene showed evidence of activity compared with an increased 40 mg/day dose of tamoxifen in patients who had previously demonstrated resistance to the standard 20 mg/day dose of tamoxifen. Of 47 patients eligible for response (25 idoxifene, 22 tamoxifen), two partial responses and two disease stabilizations (SD) for >6 months were seen with idoxifene (overall clinical benefit rate 16%, 95% CI 4.5-36.1%). The median duration of clinical benefit was 9.8 months. In contrast, no objective responses were seen with the increased 40 mg/day dose of tamoxifen, although two patients had SD for 7 and 14 months (clinical benefit rate 9%, 95% CI 1.1-29.2%). Idoxifene was well tolerated and the reported possible drug-related toxicities were similar in frequency to those with tamoxifen (hot flushes 13% vs 15%, mild nausea 20% vs 15%). Endocrine and lipid analysis in both groups showed a similar significant fall in serum follicle-stimulating hormone and luteinizing hormone after 4 weeks, together with a significant rise in sex hormone binding globulin levels and 11% reduction in serum cholesterol levels. In conclusion, while idoxifene was associated with only modest evidence of clinical activity in patients with tamoxifen-resistant breast cancer, its toxicity profile and effects on endocrine/lipid parameters were similar to those of tamoxifen.
Cancer Chemotherapy and Pharmacology 05/2004; 53(4):341-8. DOI:10.1007/s00280-003-0733-6 · 2.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Determination of pharmacokinetic properties in the intact animal remains a major bottleneck in drug discovery. Cassette dosing involves administration of a cocktail of drugs to individual animals. Here we describe the cassette dosing properties of a 107-membered library of 2,6,9-trisubstituted purine cyclin-dependent kinase 2 (CDK2) inhibitors. A three-step parallel synthesis approach produced compounds with purity ranging from 63% to 100%. Cassette dosing was validated by comparing the pharmacokinetic parameters obtained following i.v. administration of a mixture of olomoucine, R-roscovitine (CYC202), and bohemine, each at 16.6 mg/kg, with results for administration of single agents at 50 mg/kg. No significant difference was observed between the pharmacokinetic parameters of agents when dosed in combination compared with those of individual compounds. CYC202 showed the highest area under the curve (AUC) and the longest elimination half-life (t(1/2)). Further cassettes evaluated the library of trisubstituted purines with CYC202 and purvalanol A included as pharmacokinetic standards in a validated limited sampling strategy. The ratios of pharmacokinetic parameters to that of CYC202 [AUC, maximum concentration (C(max)), and t(1/2)] remained similar when compounds were tested in two different cassettes or as individual compounds. Following dosing of the same cassette on three different days, there was less than 20% variation in pharmacokinetic parameters between days. The structure-pharmacokinetics relationship showed that the favored purine substituents are benzylamine and veratrylamine at position 6, amino-2 propanol at position 2, and methylpropyl or hydroxyethyl at position 9. Without cassette dosing, this study would have used 3 times as many animals and would have taken 4 times longer, illustrating the power of this method in lead optimization.
Molecular Cancer Therapeutics 04/2004; 3(3):353-62. · 6.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Compounds of formula 1 one of R R alkyl, or aryl, and wherein at least one of R R cycloalkyl group, each of which may be optionally substituted with one or more OH groups; R, COOH, CONH. A further aspect of the invention relates to pharmaceutical compositions comprising compounds of formula 1, and the use of said compounds in treating proliferative disorders, viral disorders, CNS disorders, diabetes, stroke, alopecia or neurodegenerative disorders.
[Show abstract][Hide abstract] ABSTRACT: The use of pentafluoronitrobenzene as a scaffold for solid-phase synthesis of 2,4,6-substituted-3,5-difluoronitrobenzenes is described. The scaffold is amenable to the synthesis of structurally diverse combinatorial libraries. Primary and secondary amines can be introduced to the scaffold via three successive nucleophilic aromatic substitutions under increasingly forcing conditions. The synthesis of a 36-member validation library is described as follows. Displacement of the para-fluorine was achieved in solution with a set of primary and secondary amines. Following purification, the para-substituted scaffold was attached to an amino acid-loaded hydroxymethylbenzyloxypolystyrene resin via a second substitution of one of the ortho-fluorines. The final reactive ortho-fluorine was then displaced by a second set of amines. After cleavage from the solid support the library was furnished in good overall purity, as determined by LCMS.
[Show abstract][Hide abstract] ABSTRACT: Roscovitine (R, 2-[1-(hydroxymethyl)propylamino]-6-benzylamino-9-isopropylpurine) is a selective inhibitor of cyclin dependent kinase 2, an important enzyme complex involved in control of cell division and a potential target for the design of anti-cancer drugs. We have previously shown that, in the Balb C- mouse, the major metabolic pathway of R was conversion of the hydroxymethyl moiety of the C2 sidechain to a carboxymethyl group (RCOOH). This metabolite was the major product detected in plasma and accounted for some 69% of metabolites in 24 h urine. Other, minor, pathways included C8 hydroxylation of the purine nucleus and N-dealkylation of the N9 isopropyl or the 1-(hydroxy-methyl)propylamino sidechains. We have used a selectively deuterated analogue of roscovitine to explore the possibility of retarding certain oxidative pathways via the deuterium isotope effect. Roscovitine was synthesised with all hydrogens in the N6 isopropyl group and the two hydrogens alpha to the hydroxyl moiety of the 1-(hydroxymethyl)propylamino side-chain replaced by deuterium (d9-roscovitine, D9R). R and D9R were co-administered (1:1) at a total dose of 50 mg/kg i.v. Plasma samples were taken at 0.25, 0.5, 1, 2, 4, 6 and 24 h post administration, three animals were used at each timepoint. R, D9R, RCOOH and the deuterated carboxylic acid metabolite D7RCOOH were quantified by LCMSMS. There was a slight increase in D9R plasma levels compared with R and a slight decrease in D7RCOOH levels compared with RCOOH. However, the results were not statistically significant and there were no differences in calculated pharmacokinetic parameters (AUC, Cmax, Tmax, Cl and T1/2) between either R and D9R or RCOOH and D7RCOOH. However, deuteration in the isopropyl sidechain inhibited formation of metabolites derived from either N-dealkylation or hydroxylation of this group. There was conversely an approximate 2 fold increase in C8 hydroxylation of the purine nucleus of D9R compared with R. These results show that replacement of specific hydrogen atoms in R with deuterium can have the effect of altering the involvement of different metabolic pathways involved of R. However, these changes are insufficient to have a significant effect on the pharmacokinetic behaviour of R or its major metabolite RCOOH.