1. Design and Biological Evaluation of Heterocyclic
Quinolones Targeting Plasmodium Falciparum
Type II NADH:Quinone Oxidoreductase (PfNDH2)
Peter Gibbons

2. Introduction to the WT Project.
2
• Urgent need for new antimalarial drugs with novel mechanisms of action to
deliver 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 respiratory
enzymes (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).

3. 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
3
NADH
Dihroorotate
G-3-P
Succinate
Malate
PfNDH2
N
P
ΔΨm
+
e- III
H+
e-
c
QH2
ETC
DHOD
G3PD
SQR
MQO
>90 %
<<1%
~ 1%
~1%
<<1%

4. 4
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.

5. 5
Medicinal Chemistry Strategy for the Discovery of
SL-2-25 and SL-2-64

6. Chemistry Goals
Medicinal Chemistry
• Focus is to enhance inherent antimalarial potency whilst increasing
solubility
• Incorporation of heterocycles to reduce ClogP/logD, disruption of
planarity and introduction of solubilising groups
• 2-Aryl Series central focus
6
OCF3
6a 14a

7. General Synthesis of Quinolone Target Molecules.
7
• Heterocycle incorporation to reduce ClogP. Chemistry easier with no O or CH2 linker between the
two 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.

8. Yields for the Synthesis of Target Quinolones.
8

9. Target Quinolones continued.
9

10. Synthesis of Hydroxyl Quinolones in A ring and side chain.
10
• 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 parent
Quinolone not satisfactory.

11. Substitution at 3-position of Quinolone core with Esters and
methyl alcohol.
11
• 3-Methyl alcohol Quinolones prepared with pro-drug strategy in mind, however inherently
unstable at 3-position of quinolone core.

12. 3-Chloro Quinolones.
12

13. Incorporation of Morpholine group and Aza
Quinolones.
13
• Enhance aqueous solubility and allow possibility of salt formation.

14. Extended side chain Morpholine Quinolones and
piperazine linker analogues..
14

15. Phosphate and Morpholine Pro-drugs.
15

16. In Vitro Antimalarial of Bisaryl Quinolones vs 3D7
16
• 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)

17. In Vitro Antimalarial Activities of Bicyclic
Quinolones vs 3D7
17
• Small X groups e.g. Cl, F, OH well tolerated, larger groups e.g CF3, OCF3, SO2Me not tolerated
e.g. (8h 75 nM vs 8l > 1000 nM). Hydroxyl Y group not tolerated (11a and 11b). Sub at 4 position favoured
over 2 and 3 position.

18. In Vitro Antimalarial Activities of Other Bicyclic
Quinolones vs 3D7
18

19. Continued.
19
• 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.

20. In vivo activity
20
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 kill
Achieved 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 with
100 % parasite kill at 20 mg/Kg.

21. In vivo activity
21
• Peters standard 4
day test performed in
Liverpool – oral dosing
once daily

22. Initial PK data consistent with once daily oral
dosing
22
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.

23. 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-S
Cmax (µg/ml) 3.1
Tmax (h) 7
AUC0-t (µg.h/ml) 57.9
T1/2 (h) 10.6
Vd (ml/kg) 1261.4
ClT (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

24. 24
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
Kinetic Solubility Assay Performed at Biofocus

25. Conclusions
25
• 4-6 step synthesis of a range of heterocyclic quinolones with potent antimalarial activity both
in 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.

26. Future Work: SAR Development of Pyrazole and
Pyridoxyl Series
- Solubility/ Activity Improvements
26
• 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.
N
H
O
N
Cl
MeO
OCF3
PG227
IC50 (3D7) = 27 nM

27. Future Work: Further Lead Optimisation-Solubility
27
N
H
O
N
N
OCF3
WDH-1W-5
N
H
O
N
N
OCF3
WDH-2Q-3
IC50 (3D7) = 148 nM
IC50 (PfNDH2) = IP
IC50 (3D7) = 74 nM
IC50 (PfNDH2) = 49.1 nM
Solubility =0.01 uM
Solubility = 0.4 uM @ pH = 7.4
Solubility = 21 uM @ pH =7.4
2 x solubility of GSK pyridone at pH = 7.4
105 x more soluble at pH =1
20 x more soluble than GSK second generation
pyridone at pH = 1
N
H
O
N
OCF3
SL-2-25
IC50 (3D7) = 54 nM
N
H
O
N
CF3
Solubility = 0.04 uM @ pH = 7.4
Solubility = 18 uM @ pH = 1
Solubility = 1 uM @ pH = 7.4
Solubility = > 44 uM @ pH = 1
25 x increase in solubility
at pH =
IC50 (3D7) = 109 nM
7.4
1000 x increase
in solubility at pH = 1
@ pH = 7.4
40 x increase in
solubility at pH = 7.4
2100 x increase
in solubility at pH = 1
Pyrazole
isomer
3-Me
N
H
O
O
N
O
5-Membered Heterocyles Torsion Angle Effect in C/D Ring Extended Solubilising Group
2.4 uM @ pH =7.4
42 uM @ pH = 1
IC50 (3D7) = 300 nM
• Side Chain Modifications
– to reduce/optimise CLogP
and enhance solubility.
•Three Proof of Principle
Examples Now Established
Solubility = 0.4 uM @ pH = 7.4
Solubility = 21 uM @ pH = 1