Candidate Selection and Preclinical Evaluation of - ACS Publications

Feb 17, 2009 - Department of Chemistry, UniVersity of LiVerpool, LiVerpool, L69 7ZD, United Kingdom, UniVersity of LiVerpool, MRC Centre for Drug Safe...
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1408

J. Med. Chem. 2009, 52, 1408–1415

Candidate Selection and Preclinical Evaluation of N-tert-Butyl Isoquine (GSK369796), An Affordable and Effective 4-Aminoquinoline Antimalarial for the 21st Century Paul M. O’Neill,*,†,‡ B. Kevin Park,‡ Alison E. Shone,‡ James L. Maggs,‡ Phillip Roberts,‡ Paul A. Stocks,† Giancarlo A. Biagini,§ Patrick G. Bray,§ Peter Gibbons,† Neil Berry,† Peter A. Winstanley,‡ Amira Mukhtar,† Richard Bonar-Law,† Stephen Hindley,† Ramesh B. Bambal,| Charles B. Davis,| Martin Bates,⊥ Timothy K. Hart,# Stephanie L. Gresham,∇ Ron M. Lawrence,⊥ Richard A. Brigandi,O Federico M. Gomez-delas-Heras,[ Domingo V. Gargallo,[ and Stephen A. Ward§ Department of Chemistry, UniVersity of LiVerpool, LiVerpool, L69 7ZD, United Kingdom, UniVersity of LiVerpool, MRC Centre for Drug Safety Science, Department of Pharmacology, School of Biomedical Sciences, UniVersity of LiVerpool, LiVerpool L69 3GE, United Kingdom, LiVerpool School of Tropical Medicine, Pembroke Place, LiVerpool L3 5QA, United Kingdom, Drug Metabolism and Pharmacokinetics, GlaxoSmithKline Drug DiscoVery, 1250 South CollegeVille Road, CollegeVille, PennsylVania 19426, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, SteVenage, Hertfordshire SG1 2NY, United Kingdom, Safety Assessment, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PennsylVania 19406, Safety Assessment, GlaxoSmithKline, The Frythe, Welwyn, Hertfordshire AL6 9AR, United Kingdom, DiscoVery Medicine Infectious Diseases CEDD, DDW DPU, GlaxoSmithKline, 1250 South CollegeVille Road, 4-4238, CollegeVille, PennsylVania 19426, GlaxoSmithKline, S.A., Parque Tecnolo´gico de Madrid, SeVero Ochoa 2, 28760 Tres Cantos, Madrid, Spain ReceiVed October 6, 2008

N-tert-Butyl isoquine (4) (GSK369796) is a 4-aminoquinoline drug candidate selected and developed as part of a public-private partnership between academics at Liverpool, MMV, and GSK pharmaceuticals. This molecule was rationally designed based on chemical, toxicological, pharmacokinetic, and pharmacodynamic considerations and was selected based on excellent activity against Plasmodium falciparum in vitro and rodent malaria parasites in vivo. The optimized chemistry delivered this novel synthetic quinoline in a two-step procedure from cheap and readily available starting materials. The molecule has a full industry standard preclinical development program allowing first into humans to proceed. Employing chloroquine (1) and amodiaquine (2) as comparator molecules in the preclinical plan, the first preclinical dossier of pharmacokinetic, toxicity, and safety pharmacology has also been established for the 4-aminoquinoline antimalarial class. These studies have revealed preclinical liabilities that have never translated into the human experience. This has resulted in the availability of critical information to other drug development teams interested in developing antimalarials within this class. Introduction The developing world urgently needs new, safe, effective, and affordable antimalarial drugs;1 this has been the case since the demise of chloroquine (1)2 (Figure 1) due to Plasmodium falciparum chloroquine resistance transporter (PfCRTa)2 mediated parasite resistance emergence in the 1960s. As a result of this resistance, several groups have set out to develop analogues of chloroquine that circumvent the resistance mechanism and these include chain shortened analogues of chloroquine such as AQ13, ferroquine, and the bisquinoline piperaquine.3,4Using the related 4-aminoquinoline amodiaquine (2) (Figure 1) as our template,4 we have developed simple synthetic routes to novel, rationally designed analogues that are highly effective against chloroquine-resistant parasites and against parasites obtained * To whom correspondence should be addressed. Phone: +44 (0)151 794 3553. Fax: +44 (0)151 794 3588. E-mail: [email protected]. † Department of Chemistry, University of Liverpool. ‡ University of Liverpool, Department of Pharmacology, MRC Centre for Drug Safety Science. § Liverpool School of Tropical Medicine. | Drug Metabolism and Pharmacokinetics, GlaxoSmithKline Drug Discovery, Collegeville, Pennsylvania. ⊥ GlaxoSmithKline, Medicines Research Centre, Stevenage, Hertfordshire. # Safety Assessment, GlaxoSmithKline, King of Prussia, Pennsylvania. ∇ Safety Assessment, GlaxoSmithKline, Welwyn, Hertfordshire. O Discovery Medicine Infectious Diseases CEDD, DDW DPU, GlaxoSmithKline, Collegeville, Pennsylvania. [ GlaxoSmithKline, S.A, Madrid, Spain. a Abbreviations: PfCRT, Plasmodium falciparum chloroquine resistance transporter; MMV, Medicines for Malaria Venture, GSK, GlaxoSmithKline, TPP, target product profile.

Figure 1. Structures of chloroquine (1), amodiaquine (2), isoquine (3), 4, desethyl isoquine (5), and fluoro analogue (FAQ-4, (6)).

from clinical failures after treatment with amodiaquine.5 Equally important has been rational drug design based on metabolic considerations. Specifically, we have designed molecules that are not metabolized to products that are substrates for the chloroquine resistant mechanism and that cannot generate the reactive electrophilic quinoneimine metabolites6 associated with the drug-induced hepatotoxicity,7 agranulocytosis, and lifethreatening idiosyncratic toxicity seen in humans taking amodiaquine.8

10.1021/jm8012618 CCC: $40.75  2009 American Chemical Society Published on Web 02/17/2009

N-tert-Butyl Isoquine (GSK369796)

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1409

Table 1. In Vitro and in Vivo Antimalarial Activity of 4 and Control 4-Aminoquinoline Analoguesa in vitro antimalarial activity vs Plasmodium falciparum strainsb

in vivo antimalarial activity vs Plasmodium berghei ANKA infection in mice

compd

3D7c

HB3c

K1d

ED50 (mg/kg)

95% confidence interval

ED90 (mg/kg)

95% confidence interval

chloroquine (1) amodiaquine (2) isoquine (3) 4 (GSK369796) desethyl isoquine (5)

19.4 ( 1.9 11.2 ( 2.8 11.6 ( 2.3 11.2 ( 2.2 9.2 ( 3.1

14.9 ( 3.9 9.6 ( 3,7 12.6 ( 4.7 12.6 ( 5.3 14.2 ( 3.5

183.2 ( 11.1 15.5 ( 9.4 17.6 ( 7.0 13.2 ( 3.2 17.6 ( 5.7

2.1 3.7 2.8 5.3

2.0-2.2 3.4-3.9 2.3-3.3 4.1-6.8

3.2 5.3 4.7 7.3

2.9-3.4 4.3-6.6 4.1-5.5 6.4-8.4

a IC50 values are expressed in nM. In vivo activity measured against Plasmodium berghei ANKA in standard 4-day test. b Results are the mean ( SD of three independent experiments. c P. falciparum 3D7 and HB3 strains are chloroquine-sensitive. d P. falciparum K1 strain is chloroquine-resistant.

The original lead compound 3 (Figure 1), the direct regioisomer of amodiaquine (2), was selected in order to eliminate the para-aminophenol metabolic alert present in 2. As anticipated, 3 was a potent antimalarial, highly active against a broad range of chloroquine-resistant parasites in vitro. This subtle modification completely abolished the potential for quinoneimine formation (catalyzed by either P450 enzymes or myeloperoxidase). However, the molecule was subject to unacceptably high first pass metabolism to dealkylated metabolites in four animal species, which complicated the development process and also compromised activity against chloroquineresistant parasites. Molecule 3 served as a starting point for lead optimization, and through an iterative process of synthesis, activity testing, and drug metabolism pharmacokinetic (DMPK) evaluation, the ultimate candidate N-tert-butyl isoquine (4) was identified. This molecule conforms to the target product profile (TPP) for an orally active antimalarial as set out by Medicines for Malaria Venture (MMV, Geneva) and the World Health Organization (WHO/TDR). Following demonstration that the N-tert-butyl function of a fluorinated derivative of AQ, 6, was significantly more resistant to metabolic cleavage, we predicted that the N-tert-butyl functional group would provide a significant advantage over the N-diethylamino group present in original lead compound 3. It was hoped that this manipulation would result in a much-simplified metabolic profile and enhanced oral bioavailability. Results and Discussion Antimalarial Activity. Analogue 4 has been evaluated against a large number of isolates of P. falciparum in vitro and against rodent parasites in vivo. The molecule displays excellent low nM activity against chloroquine-resistant and sensitive parasites (Table 1). In vivo 4 has oral activity equivalent to amodiaquine (Table 1) but with a better antimalarial exposure profile, as measured by the nonrecrudescence dose level (NRL) compared to amodiaquine or chloroquine (Table 2). During the lead optimization process, field reports of amodiaquine failure in Africa raised significant doubt about the eventual clinical utility of an amodiaquine variant.9 Unfortunately, the parasites responsible for these failures were phenotypically and genotypically distinct from laboratory isolates. The development team undertook a series of clinical audits of amodiaquine efficacy and collected parasites predose and at the time of recrudesence. After culture adaptation a selection of these were tested for activity against 4 (Figure 2). In parasites originating from three sites on two continents, 4 was also superior to both chloroquine and the active metabolite of amodiaquine, desethyl amodiaquine, in vitro. Chemistry. A critical element of the development plan was the need to identify a scalable synthetic route capable of producing a final product with a cost of goods equivalent or

Table 2. Therapeutic Efficacy of Isoquine Derivatives against Plasmodium yoelii 17X Infection in Micea compd

test

parameter

mean (mg/kg)

IC 95% b (mg/kg)

chloroquine (1)

4-day

amodiaquine (2)

4-day

4 (GSK369796)

4-day

ED50c ED90 NRLd ED50 ED90 NRL ED50 ED90 NRL

3.3 4.4 >40e 2.6 3.7 >40e 3.8 5.4 20

2.8-3.7 4.0-4.9 NAf 2.1-3.2 3.3-4.1 NA 3.3-4.3 4.8-6.1 NA

a Standard 4-day test assay. Chloroquine (1) and amodiaquine (2) were included as control compounds. b Interval of confidence of the mean. c Dose at which a 50% reduction of parasitaemia in peripheral blood is achieved at day 4 after infection. d Nonrecrudescence level (NRL) is defined as the minimum dose at which no recrudescence is observed. Highest dose deployed in this study was 40 mg/kg. e NRL level is only defined for treatments which make parasitaemia undetectable by flow cytometry at day 4 in the “4-day test”. At the highest dose deployed chloroquine, amodiaquine, isoquine, desethyl isoquine, and FAQ4 (6) (40 mg/kg) were unable to render parasitaemia undetectable, therefore it is not possible to evaluate NRL level for these compounds. For compound 4, parasitaemias in peripheral blood were measured 96 h after infection (day 4) and then every 2-3 days until day 23 after infection in order to assess recrudescence after treatment. f Not applicable.

less than that for chloroquine. For an antimalarial targeted toward resource poor populations, affordability is almost as important as effectiveness and safety. The original route, based on a two-step synthesis, each with a flash column chromatography step, was already relatively simple, but the driver to reduce overall costs was such that we needed to simplify this even further. By coupling the 3-aminophenol unit to the quinoline nucleus prior to the Mannich reaction, the target intermediate material was produced in a quantitative yield of purity >98% (Scheme 1). The second stage of the process, addition of the Mannich side chain, proved to be more problematic. Early scale up indicated poor regioselectivity, formation of dimannich impurities, and significant and costly isolation and purification requirements. Using a statistical design approach to the processing window for stage 2, we were able to judiciously select optimum solvent, time, and temperature conditions, producing a route with enhanced regioselectivity, reduced degradation, and which on addition of water in the final step caused the benzoxazine to precipitate with a purity of >98% by HPLC. Treatment of this with dilute HCl in s-butanol produced the dihydrochloride salt at 99% purity. The chemistry has since been scaled up to multikilogram scale up and produces 4 in 59% yield and >98% purity. The achievements of the process development campaign enabled this candidate to meet the TPP criteria for an affordable antimalarial. Mechanism of Action. Degradation of hemoglobin by malaria parasites releases ferriprotoporphyrin IX (FP) that is detoxified by crystallization into hemozoin (HZ).10 Antimalarial drugs such as chloroquine and amodiaquine bind to FP and

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Figure 2. Sensitivity assays of chloroquine (CQ), desethyl amodiaquine (DAQ), and 4 (796) on Thai, Kenyan, and Rwandan Plasmodium falciparum isolates.

Scheme 1. Two-Step Optimized Synthesis of Candidate 4 (GSK369796)

Table 3. Equilibrium Constants for the Binding of Antimalarials with Heme in DMSO Solutions compd

solvent

log K1

log K2

chloroquine (1) 40% DMSO 5.30 ((0.1) 6.20 ((0.1) amodiaquine (2) 40% DMSO 4.40 ((0.1) 6.20 ((0.1) 4 (GSK369796) 40% DMSO 5.23 ((0.1) 5.36 ((0.1)

corrupt this process.10 To study the interaction of 4 with FP, we have applied a UV-visible spectroscopic method for determining accurate binding equilibrium constants. Titrations were carried out in a mixed aqueous/organic solvent to minimize porphyrin aggregation effects and m-oxo dimer formation.11 Buffered 40% DMSO was used to provide a strictly monomeric haem species in solution.12 The mode of action for antimalarial binding was investigated, and the processes 1 and 2, shown in Figure 3, were mathematically modeled and examined as possible best fits for the titrations. In model 1, there is a stepwise bonding of two equiv of drug to one molecule of heme. In model 2, there is a stepwise addition of two additions of heme to 1 equiv of drug. The UV-visible spectrum obtained after each titrated addition was analyzed and stacked against the corresponding absorbances. The data was transferred for analysis using the Pro-Fit nonlinear curve fitting program licensed from Quansoft. The data was fitted to achieve χ2 at a minima to produce K1 and K2 fitted parameters for both models. This was initially done at one wavelength and then simultaneously at 10-15 wavelengths to give a more unbiased approach to fitting and more accurate values in comparison to fitting at just a single

Figure 3. Potential binding interactions for drug (L) with heme (P). After the first association of porphyrin (P) with drug (L), there are two distinct binding events in which either another drug or porphyrin molecule can coordinate to the intermediate adduct.

best-fit model 2 2 2

wavelength. Table 3 below shows the K1 and K2 values obtained from this method for CQ, AQ, and 4 with the best-fitted model shown. An important observation from the curve fitting procedure is that the binding stoichiometries for each of the 4-aminoquinolines studies are similar. As for chloroquine and amodiaquine, the second binding event in the titrations of 4 involves the addition of another molecule of haem and this is in line with earlier studies derived from NMR experiments. On the basis of these results, we performed molecular modeling of the interaction of the drug target with 4, where the drug is complexed with hematin (defined by K1).13 The most favorable complex (Figure 4) shows the aromatic quinoline ring parallel to the edge of the hematin, consistent with a π-π stacking interaction.4 There is also a favorable hydrogen-bonding network between the carboxylate groups of the hematin and the alcohol and protonated amine of the Mannich side chain. DMPK Studies. The pharmacokinetics of isoquine (3), desethyl isoquine (5), and 4 (Figure 1) were each investigated in detail, following single intravenous and oral administration to the mouse, rat, dog, and/or monkey14,15 (Table 4). Protein binding and blood cell association were investigated in the plasma or whole blood from preclinical species and humans,

Figure 4. A model of the most favorable porphyrin-N-tert-butyl isoquine complex (carbon, gray; hydrogen, white; oxygen, red; nitrogen, blue; chlorine, green; iron, orange). Hydrogen bonds indicated with black dashed lines. For clarity, only hydrogens involved in hydrogen bonds are shown.

N-tert-Butyl Isoquine (GSK369796)

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Table 4. Comparative Pharmacokinetics of 4 (GSK369796), Isoquine (3), and Desethyl Isoquine (5) in Animalsa parameter

species

NTBI

DE-isoquine

isoquine

blood CL mL/min/kg

mouse rat dog monkey mouse rat dog monkey

17 26 ( 5 6.3 ( 1.6 14.5 ( 0.7 ∼100 89 ( 12 68 ( 18 ∼100

44 62 ( 22 12 ( 3 49 ( 3 ∼100 60 ( 26 NE 40 ( 14

219 89 ( 4 151 ( 24 ND 21 17 ( 4 NQ ND

oral F, %b

a Data are expressed as mean ( SD (n ) 3); ND: no data (given poor PK profile in mouse, rat, and dog, isoquine was not studied in the monkey); NE: not estimated (emesis observed in the oral leg may have resulted in decreased oral exposure); NQ; nonquantifiable (solution dose of 3 mg/kg); CL, clearance. b F ) % oral bioavailability.

in vitro. The routes and rates of metabolism were studied in animal and human liver microsomes and hepatocytes, as was concentration-dependent human cytochrome P450 inhibition. These studies were essential to the development and selection of the lead compounds and ultimately the choice of 4 for progression into definitive safety assessment studies and clinical investigation. In preclinical species, blood clearance was lowest for N-tertbutyl isoquine, highest for isoquine, and intermediate for desethyl isoquine (see Table 4). Oral bioavailability was best for N-tert-butyl isoquine (g68%) and worst for isoquine (e21%). In rodents, following oral administration of isoquine, exposure to desethyl isoquine exceeded that of isoquine substantially (metabolic ratio g10), suggesting that isoquine behaves as a pro-drug as has been shown for AQ. Steady-state distribution volume for N-tert-butyl isoquine was high in animals, exceeding total body water in all species, suggesting there is extensive tissue penetration. Elimination half-lives were moderate: 3 h in the mouse and 11 h in the monkey. Substantial partitioning into red blood cells was observed, particularly in the dog and monkey, with much less in rodent and in normal human blood in vitro. Human blood partitioning in vitro was similar to desethyl amodiaquine. Protein binding was somewhat higher for 4 compared to desethyl amodiaquine in the mouse (93 vs 74%) but similar in human (88 vs 86%). Activity of these molecules is similar in mouse infection models (similar in vitro potency and exposure as well), suggesting high protein binding does not limit the pharmacological activity. In all cases studied, there was in vitro accumulation within red blood cells with a blood to plasma ratio ranging from 1.2fold in human to 15-fold in dog blood. In infected mice blood exposure profiles were substantially higher than those in noninfected animals (see profiles in Figure 5). Rational metabolic drug design was central to the project, and it was found that in vitro and in vivo metabolism studies in a range of species confirmed theoretical predictions. Examination of the metabolism of 4 and desethyl isoquine (5) following incubation with liver microsomes and hepatocytes from mouse, rat, dog, and human did not show any evidence of glutathione conjugation or reactive metabolite formation via the quinoneimine pathway that occurs extensively with AQ. 4 and desethyl isoquine (5) were metabolized by multiple metabolic pathways in vitro. In human and animal liver microsomes, mono-oxygenation metabolites (M2, M3, M4), aldehyde (M5), carboxylic acid (M6), and glucuronides of parent, M5 and M6 were detected for both compounds (Figure 6). Although the N-dealkylated metabolite (M1) was detected for 5, it was not detected for 4 in vitro as predicted. Ketone metabolite (M7) was detected for 4 in all species. However, 5

did not form M7. The exact position of oxygenation for M2, M3, and M4 could not be ascertained due to the low sensitivity in the MS spectrum. Also for M6, due to the very low extent of formation, the nature of glucuronide, -acyl versus -hydroxyl, could not be determined. The in vitro metabolism of 5 herein reported is consistent with urinary and biliary metabolites described previously following administration of tritiated isoquine (3) to rats. All metabolites observed in vitro were similarly detected in the urine of mice administered 4 or 5 orally, with the exception that M1 was detected for 4 and M7 was detected for 5. To assess the relative importance of N-dealkylation in the in vivo biotransformation of these compounds following oral administration of 4 or 5 to the mouse, systemic exposure to the N-dealkylation metabolite (M1) was compared. Dose-normalized AUC for M1 following administration of desethyl isoquine was >1000 times the dose-normalized AUC for M1 following administration of 4. Total urinary excretion of M1 was >60fold higher for 5 compared to 4 in the mouse. The studies described above demonstrate that replacement of the N-ethyl group with an N-tert-butyl substituent substantially reduced the rate of N-dealkylation of 4 compared to isoquine (3) and desethyl isoquine (5). This conclusion is supported by the lower blood clearance of 4 in mouse, rat, dog, and monkey and the very substantial differences in systemic exposure and urinary excretion of the N-dealkylation metabolite (M1) following oral administration of 4 or desethyl isoquine (5) to the mouse. The potential for inhibition of the major human cytochrome P450 isozymes was compared for 4, 3, 5, 2, and desethyl amodiaquine using recombinant isozymes and fluorescent probe substrates (Table 5). All compounds inhibited 2D6, with IC50s ranging from 3 to 7 µM across the series. For 4, mean IC50 values were g23 µM for all other isozymes studied, suggesting limited potential for inhibition of 1A2, 2C8, 2C9, 2C19, and 3A4 (Table 5). Generally, the potential for inhibition of 1A2, 2C9, 2C19, and 3A4 was modest across this series. However, desethyl isoquine had a substantially lower IC50 against CYP1A2 (8 uM) compared to 4 (29 uM). Activation of human CYP3A4 was observed for N-tert-butyl isoquine and isoquine (3-fold stimulation of activity for both). Amodiaquine was a potent 2C8 inhibitor, while the IC50 for desethyl AQ was much higher and similar to both desethyl isoquine (23 uM) and 4 (23 uM) (Table 5). Formation of desethyl amodiaquine is thought to be mediated by human cytochrome P450 2C8.16 The studies described above were performed to identify potentially important clinical drug-drug interactions and to assist in the selection of the optimal combinant such as an artemisinin derivative or synthetic endoperoxide. Preclinical Toxicology Assessment. Antimalarial 4 is a derivative of compounds for which there is human experience of millions of clinical exposures, i.e., chloroquine and amodiaquine. These are drugs that were developed half a century ago, long before the thalidomide disaster17 prompted the introduction of rigorous preclinical safety evaluations for all new drugs. Preliminary studies with 4 suggested some unexpected preclinical safety findings that required detailed interrogation and generation of comparable data for chloroquine and amodiaquine for comparison given the absence of preclinical safety data for these agents. The development plan for 4 included evaluations in four preclinical species standardly used for toxicology studiess mouse, rat, dog, and nonhuman primate.18,19 Following exploratory studies in mice and dogs, the rat and nonhuman primate were considered the most appropriate species for use in the GLP

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Figure 5. Peripheral blood and serum levels of 4-aminoquinoline analogues after single 10 mg/kg oral administration to CD1 mice as solutions in sterile saline. Results obtained in noninfected animals are shown in (A) (compounds levels in serum) and (C) (compounds levels in total blood). Results obtained in infected animals (10% P. yoelii parasitemia) are shown in (B) (compounds levels in serum) and (D) (compounds levels in total blood). NON INF ) noninfected, INF ) infected.

Figure 6. Metabolites of 4 and 5 following incubation with animal and human liver microsomes and hepatocytes in vitro. For both compounds, glucuronidation metabolites of parent, M5 and M6, were detected in liver microsomes and hepatocytes. M1 was detected only with desethyl isoquine, and M7 was detected only with 4.

preclinical toxicology assessments to support progression of this drug into humans. The goal was to achieve systemic drug exposure levels that were substantially higher than those predicted for (first time in humans) FTIH plasma drug levels in terms of safety and tolerability. 4-Aminoquinoline 4 did not cause gene mutations or chromosomal damage in vitro in a bacterial mutation assay and mouse lymphoma assay or in an in vivo mouse micronucleus

assay. Investigations of the potential for adverse CNS activity via in vitro off-target receptor profiling (CEREP screen)20 with 4 showed a similar profile to chloroquine and amodiaquine with antagonistic activity of muscarinic and 5HT receptors. Adverse dose-related CNS effects (respiratory depression, tremors, and convulsions) were evident in preclinical safety pharmacology and toxicity studies. Similar effects occurred in animals with

N-tert-Butyl Isoquine (GSK369796)

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1413

Table 5. Comparative P450 Inhibition Potential of N-tert-Butyl Isoquine (4) and other 4-Aminoquinoline Derivativesa compd

3A4 DEF

3A4 PPR

1A2

2D6

2C8

2C9

2C19

amodiaquine (2) isoquine (3) 4 (GSK369796) desethyl isoquine (5) desethyl amodiaquine

28 ( 6 Ab Ab >100 24 ( 3

>100 >100 >100 g55 >100

15 ( 5 23 ( 8 29 ( 6 8(3 28 ( 11

6.1 ( 0.5 7.0 ( 0.2 3.0 ( 0.5 3.4 ( 0.1 6.4 ( 0.1

2.7 ( 1.4 13 ( 2 23 ( 2 23 ( 7 20 ( 4

62 ( 9 75 ( 8 g90 >100 58 ( 2

g85 39 ( 11 24 ( 10 62 ( 16 54 ( 11

a

Mean and standard deviation of three independent IC50 (uM) determinations reported. b Activation observed.

Table 6. hERG Inhibition and Clinical Effects compd

IC50 ( SE (µM)

v QTc in clinic

chloroquine (1) amodiaquine (2) desethyl amodiaquine isoquine (3) 4 (GSK369796) halofantrine

2.5 2.4 ( 0.3 9.5 ( 0.5 3.9 ( 0.3 7.5 ( 0.8 0.04

yes unknown unknown not tested not tested yes

chloroquine that are also seen in man, particularly in overdose conditions.21 In dose range finding studies in the rat, mouse, dog, and primate, there was macroscopic and microscopic evidence of changes related to phospholipidosis that have been well documented and studied for this drug class and are the result of the weak base properties of these drugs. These effects did not affect normal organ function, were generally reversible, and seen at exposures comparable to those predicted for human efficacy. In definitive studies of 4-14 days duration in rats, high doses of 4, chloroquine, and amodiaquine caused cardiovascular toxicity, principally myocardial necrosis, skeletal muscle myopathy, and hepatic effects (hypertrophy, transaminase release, and apoptosis) along with CNS effects. On the basis of drug exposure data from these studies and related toxicological and pathological findings, 4 appears to have a safer profile than chloroquine and a profile no worse than amodiaquine. The exposures at the doses tested were 2-5-fold higher than the predicted human plasma Cmax of 0.1-0.3 µg/mL and AUC of 16 µg · h/mL. In repeat dose toxicity studies in monkeys, dose-limiting CNS effects were observed at approximately plasma drug levels 3-fold higher than the clinical target. At lower doses, in addition to phospholipidosis in multiple tissues, only mild hepatocellular hypertrophy and mild serum transaminase increases (up to 5-fold) were observed in monkeys. 4 inhibited cloned hERG potassium ion channel repolarization with an IC50 comparable to other antimalarial agents in this class (Table 6). In monkeys, mild, transient increases in QTc intervals were observed along with some ECG rhythm abnormalities. Similar cardiac effects have been reported in man following CQ administration.22 However, a review of clinical data for antimalarials in this class suggests that using the ratio of hERG IC50:free drug level is not a reliable predictor of human risk.23,24 In general, the incidence and magnitude of the pathologies seen with 4 were related to duration of dosing, plasma halflife, and tissue accumulation, and these observations were also seen with chloroquine, amodiaquine, and the related drug piperaquine (data not shown). Short-term dosing in rats for 4 days at tolerated doses, which is believed to be more representative of clinical therapeutic use (3 days dosing), failed to demonstrate the cardiac effects with 4 although they were still apparent with chloroquine. The safety profile observed for 4, in the absence of any prior human experience, might have precluded the further development of any 4-aminoquinoline and indicates limitations of our current preclinical testing strategies to accurately predict human risk for this therapeutic indication.

In conclusion, 4-aminoquinoline 4 represents a rationally designed antimalarial that is highly effective in killing malaria parasites, possesses a metabolic profile in line with its original rational design, has a better overall preclinical safety profile than chloroquine or amodiaquine, and can be synthesized in a scalable and cost-effective way. The antimalarial activity profiles for this molecule are superior to other 4-aminoquinolines under development, including AQ13 and piperaquine, and 4 may have a lower cost of goods than metallocene based 4-aminoquinolines such as ferroquine.3 We look forward to reporting on the clinical profile of this exciting new antimalarial as it progresses through the clinical development program. Experimental Section Parasitology. Cultures of parasites were grown in flasks containing human erythrocytes (2-5%) with parasitemia in the range of 1-10% suspended in RPMI 1640 medium supplemented with 25 mM HEPES and 32 mM NaHCO3 and 10% human serum (complete medium). Cultures were gassed with a mixture of 3% O2, 4% CO2. and 93% N2. In Vitro Antimalarial Assays. Antimalarial activity was assessed with an adaption of the 48 h sensitivity assay of Desjardins et al.26 using [3H]-hypoxanthine incorporation as an assessment of parasite growth. Stock drug solutions were prepared in 100% dimethylsulfoxide (DMSO) and diluted to the appropriate concentration using complete medium. Assays were performed in sterile 96-well microtiter plates, each plate contained 200 mL of parasite culture (2% parasitemia, 0.5% hematocrit) with or without 10 mL drug dilutions. Each drug was tested in triplicate and parasite growth compared to control wells (which constituted 100% parasite growth). After 24 h incubation at 37 °C, 0.5 mCi hypoxanthine was added to each well. Cultures were incubated for a further 24 h before they were harvested onto filter mats, dried for 1 h at 55 °C, and counted using a Wallac 1450 Microbeta Trilux liquid scintillation and luminescence counter. IC50 values were calculated by interpolation of the probit transformation of the log dose-response curve.27 Parasite field isolates were collected from patients failing treatment with amodiaquine alone (Kilifi, Kenya) or amodiaquine plus SP (sulfadoxine/pyrimethamine) (Rukara, Rwanda) and were culture adapted using previously established techniques.26 Briefly, parasitized blood was washed three times in RPMI 1640 and the resulting washed pellet was diluted to a hematocrit of 0.5% in complete culture medium containing 10% human AB serum. After 48 h, hematocrit was increased to ∼2% with O+ human erythrocytes and parasites were cultured for a further 4-5 days. Following stable growth for 4 to 5 days, parasites were stored under liquid N2 for subsequent drug sensitivity testing. Approval for the use of these parasite field isolates was obtained from local and Liverpool School of Tropical Medicine Ethics Committees. In Vivo Antimalarial Activity. The efficacy of selected 4-aminoquinolines was measured against P. yoelii or P. berghei in a 4-day test (Peters et al.).28 Cohorts of age-matched female CD1 mice were infected iv with 6.4 × 106 or 10.0 × 106 parasites obtained from infected donors, and the mice were randomly distributed in groups of n ) 5 mice · group-1 (day 0). Treatments were administered from day 0 (one hour after infection) until day 3. The percentage of YOYO-1+ murine erythrocytes in peripheral blood at day 4 after infection was measured30 and recrudescence up to day 22-25 if the parasitemias were below our detection limit (0.01%) at day 4

1414 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5

after infection. The therapeutic efficacy of compounds was expressed as the effective dose (mg · hg mg/kg-1) that reduces parasitemia by 50% (ED50) and 90% (ED90) with respect to vehicle treated groups (ED90) and the dose that achieved eradication of parasitemia until day 23 after infection (NRL). All compounds and corresponding vehicles were administered orally at 20 mg · kg-1 or subcutaneously at 10 mg · kg-1, as appropriate. Chloroquine or amodiaquine were included as assay quality control for each in vivo assay of P. yoelii or P. berghei, respectively. Chemistry. Details for the optimized multikilo synthesis of 4 (in >98% a/a purity and in 57% overall yield in two steps) has been reported by Lawrence and co-workers.29 Hematin Binding Studies and Molecular Modeling. Details of equilibrium binding studies are contained in the Supporting Information. A conformational search, allowing rotations about all rotable bonds, was performed on protonated molecule 4 with a molecule of heme (with both carboxylate groups deprotonated and one hydroxide moiety coordinated to the iron(III) center) using a Monte Carlo method with the MMFF94 force field. A maximum energy at which a trial conformer was saved in the database was10 kcal/mol and a maximum number of diverse conformers retained set at 100 (Spartan’04, Wave function, Inc., Irvine, CA). Drug Metabolism and Pharmacokinetic Studies. In vivo studies were approved by the appropriate institutional animal care and use committee. Pharmacokinetic, in vitro human cytochrome P450 inhibition, protein binding, blood partitioning, intrinsic clearance, and qualitative biotransformation studies were performed in a manner similar to that described by Xiang et al., 2006.15 Details of additional experiments performed on isoquine and related antimalarials can be found in Davis et al. 2008.14 Safety Pharmacology. All safety pharmacology, genetic toxicology, and general toxicology studies were conducted in accordance with the International Conference on Harmonization (ICH) Safety Guidelines.18 For general toxicology studies, study designs and end points followed current guidances for drug safety testing.19 All in vivo studies were approved by the appropriate institutional animal care and use committee. Studies to assess the off-target activity were performed at Cerep, Inc. (Seattle, WA) using their established methods.20

Acknowledgment. This work was supported by GlaxoSmithkline (GSK), MMV,25 and the Engineering and Physical Sciences Research Council (EPSRC) (DTA to A.M.). We would like to thank Dr. S. Borrmann and Dr. A. Nzila and the KEMRI-Wellcome Trust Research Programme, Kilifi, Kenya, and Prof. U. D’Allesandro and the Rukara Medical Centre for their roles in the amodiaquine clinical audits. Supporting Information Available: Details of equilibrium binding studies. This material is available free of charge via the Internet at http://pubs.acs.org.

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