Design and Biological Evaluation of HeterocyclicQuinolones Targeting Plasmodium FalciparumType II NADH:Quinone Oxidoreductase (PfNDH2)

NH

O

N

OCF3SL-2-25

Peter Gibbons

Introduction to the WT Project.

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• Urgent need for new antimalarial drugs with novel mechanisms of action todeliver effective control and eradication programmes.

• Parasite resistance to all existing antimalarial classes, including artemisinins,has been reported during their clinical use.

• Failure to develop new antimalarials with novel mechanisms of action that circumvent the current resistance challenges will contribute to a resurgence in the disease.

• New antimalarials with dual mechanism of action against two respiratoryenzymes (PfNDH2 and bc1 ) would be advantageous against multi-drug resistant P. falciparum parasites.

• Known inhibitor of PfNDH2 target, hydroxy-2-dodecyl-4-(1H)-quinolone (HDQ).

Why target PfNDH2?

• Novel mechanism of action ?(i)Mitochondria is a proven drug target (e.g. atovaquone) with curative

activity against circulating intra-erythrocytic parasites, Curative activity against liver stage parasites for prophylaxis and Curative activity against gametocytes reducing transmission (at least against stages I-III).

(ii)PfNDH2 is key electron donor for the respiratory chain

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NADHDihroorotate

G-3-PSuccinate

Malate

PfNDH2

N

PΔΨm

+e- III

H+

e-c

QH2 ETCDHOD

G3PD

SQR

MQO

>90 %

<<1%

~ 1%

~1%

<<1%

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Mono aryl Quinolones identified from HTS and initial SAR work.

• Structural modifications (e.g. Cl at 7 position of A ring and methyl substituent at 3-position) led to generation of 60 compounds including lead compound CK-2-68, 31 nM against 3D7 and 16 nM against PfNDH2. • ClogP needed to be reduced and aqueous solubility enhanced in order to administer drug in suitable vehicle, without the need for a pro-drug approach. • Incorporation of a pyridine group reduces ClogP, improves aqueous solubility, and possible salt formation.

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Medicinal Chemistry Strategy for the Discovery of SL-2-25 and SL-2-64

Chemistry GoalsMedicinal Chemistry

• Focus is to enhance inherent antimalarial potency whilst increasingsolubility

• Incorporation of heterocycles to reduce ClogP/logD, disruption of planarity and introduction of solubilising groups

• 2-Aryl Series central focus

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OCF3

6a 14a

General Synthesis of Quinolone Target Molecules.

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• Heterocycle incorporation to reduce ClogP. Chemistry easier with no O or CH2 linker between thetwo rings within the side chain. Would activity be maintained?

• Pyridine ring incorporated into side chain, optimal A ring and terminal aryl ring substituents investigated.

• Methodology allows for the rapid synthesis of Quinolone analogues to probe the SAR.

Yields for the Synthesis of Target Quinolones.

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Target Quinolones continued.

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Synthesis of Hydroxyl Quinolones in A ring and side chain.

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• Presence of OH in both the A ring of the Quinolone core and terminal aryl group investigated. • Possible attachment position for Pro-drug strategy if aqueous solubility of parentQuinolone not satisfactory.

Substitution at 3-position of Quinolone core with Esters andmethyl alcohol.

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• 3-Methyl alcohol Quinolones prepared with pro-drug strategy in mind, however inherentlyunstable at 3-position of quinolone core.

3-Chloro Quinolones.

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NH

O

N

Cl

MeO

OCF3

NH

O

N

MeO

OCF3

Sodium dichloroisocyanurate (0.5 eq.)

MeOH, 2M NaOH, H2O2 hr, 42 %

NH

O

N

MeO

OCF3

MeO

Ac

NH

O

N

OCF3

KOtBu (3.5 eq.), tBuOH

75 0C, 16 hrs, 71 %

MeO

Ac

NH

O

N

OCF3

O

N

OCF3

HO MeO

Ac

NH2+ 1. (COCl)2, DMF, DCM

2.NEt3, THF, 16hr, 73 %

IC50 = 27 nM (3D7)

Incorporation of Morpholine group and Aza Quinolones.

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• Enhance aqueous solubility and allow possibility of salt formation.

Extended side chain Morpholine Quinolones and piperazine linker analogues..

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Phosphate and Morpholine Pro-drugs.

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NH

O

N

OCF3

N

O

N

OCF3

N

O

N

N NCl

O1.

2. KOtBu, THF, 1hr, 40 %

SL-2-25

In Vitro Antimalarial of Bisaryl Quinolones vs 3D7

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• No linker is well tolerated• High degree of substitution in side chain close to the quinolone core is poorly tolerated. (8a, 940 nM, flexibility of side chain key to activity?)• 4-OCF3 group optimal terminal group (12a, 59 nM)• 3-position methyl group favoured (Me > CH2OH > CO2Et)

In Vitro Antimalarial Activities of Bicyclic Quinolones vs 3D7

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• Small X groups e.g. Cl, F, OH well tolerated, larger groups e.g CF3, OCF3, SO2Me not toleratede.g. (8h 75 nM vs 8l > 1000 nM). Hydroxyl Y group not tolerated (11a and 11b). Sub at 4 position favouredover 2 and 3 position.

In Vitro Antimalarial Activities of Other Bicyclic Quinolones vs 3D7

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Continued.

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• One pyridine ring 8b (54 nM) favoured over two pyridine rings 8v (370 nM)• OCF3 optimal terminal substituent e.g. 8w (40 nM) vs 8x (279 nM)• Incorporation of morpholine/piperazine group generally leads to a loss of activity.

In vivo activity

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In Vivo Peters Standard 4-day test- Oral Administration

• SL-2-25 some solubility issues in SSV, compound dosed as suspension and reduced %parasite clearance observed. Fully dissolved in DET (proof of concept) 100 % parasite killAchieved at 20 mg/Kg.• Phosphate salt of SL-2-25 and morpholine Pro-drug 100 % parasite kill in SSV. • Phosphate Pro-drug of SL-2-25 (compound 55) dosed in sodium carbonate solution with100 % parasite kill at 20 mg/Kg.

In vivo activity

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• Peters standard 4 day test performed in Liverpool – oral dosing once daily

Initial PK data consistent with once daily oral dosing

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SL-2-25S (20 mg/kg) Cmax 3.7 µg/mL, Tmax 7.0 h,T ½ 9.9 h, Vd 3970.8 mL/kg, AUC0-t 69.3 µg/h/mL and a ClT of 276.3 mL/h/kg. SL2-99 (20 mg/kg) Cmax 8.1 µg/mL, Tmax 7.0 h, half life (T ½) of 20.3 h, a volume of distribution Vd of 2875.6 mL/kg, an area under the curve of AUC0-t 167.2 µg/h/mL and a calculated total clearance ClT was 98.0 mL/h/kg.

NH

O

N

O

FF

F

.H3PO4

FIGURE Plasma Concentration-Time profile of SL-2-25 in male Wistar rats after administration of a single oral dose of SL-2-25-S (5 mg/kg (n=4))

SL-2-25-SCmax (µg/ml) 3.1Tmax (h) 7AUC0-t (µg.h/ml) 57.9T1/2 (h) 10.6Vd (ml/kg) 1261.4ClT (ml/h/kg) 82.1

Pharmacokinetic parameters calculated using Pk solutions 2.0 software

SL-2-25; Oral Profile in Rats following 5 mg/kg (po)

TABLE

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Solubility

Compound Structure Solubility (µM)pH=7.4 pH=4.5 pH=1

Atovaquone <0.01 <0.01

1st generation Pyridone (GW844520)

0.02 0.2

2nd generation Pyridone (GSK9321121A)

1.0 2.7

SL-2-25(Sl-2-25.H3PO4)

0.04(0.08)

0.08(0.12)

18(42)

WDH-1U-4 <0.01 0.3

OOH

Cl

O

NH

OO

OCF3Cl

NH

OO

OCF3Cl

OH

NH

O

N

OCF3

Kinetic Solubility Assay Performed at Biofocus

NH

O

OCF3

Conclusions

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• 4-6 step synthesis of a range of heterocyclic quinolones with potent antimalarial activity bothin vitro and in vivo.

• Several compounds within the series proven to be potent against novel PfNDH2 enzymatic target.

• Lead compound SL-2-25 demonstrates outstanding antimalarial activity, reduced ClogP, and improved solubility.

• SL-2-25 has antimalarial activity of 54 nM vs 3D7, PfNDH2 activity of 15 nM and an ED50 / ED90 of 1.87 / 4.72 mg / Kg when formulated as the phosphoric acid salt.

Future Work: SAR Development of Pyrazole and Pyridoxyl Series- Solubility/ Activity Improvements

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NH

O

NN

OCF3

WDH-1W-5

NH

O

N O

OCF3

CK-3-22

H, Me or Cl optimal?

Alternative heterocycles to be investigated

Alternative linker?Heterocycles to beincorporated andsubstituents investigated

Is the methyl group optimal? Could Cl be used?

Are other heterocycles tolerated?

Heterocycles to be incorporated and substituents investigated

Alternative linker?

R

R

H-bond donating:OH, CH2OH, NHCHOH-bond accepting:F, OMe, OCF2H

H-bond donating:OH, CH2OH, NHCHOH-bond accepting:F, OMe, OCF2H

• Optimisation of side chain to improve solubility and drug delivery is key.• Initially SAR around leads CK-3-22 and WDH-1W-5 will be explored.

• CH2 linker in WDH-1W-5 is a possible site of metabolism alternatives including CH2CH2, C=O, CF2, oxygen and no linker will be investigated.

Future Work: Further Lead Optimisation-Solubility

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• Side Chain Modifications – to reduce/optimise CLogP and enhance solubility.

•Three Proof of Principle Examples Now Established