The Hybrid Antimalarial Approach. Targeting Multiple stages of Life-cycle of Malaria Parasite

This presentation illustrates the use of hybrid approach in crearting drug molecules acting on more than one biological targets.

1. Prof. Kamaljit Singh Formerly Professor, Department of Chemistry UGC-Centre for Advanced Studies-II Guru Nanak Dev University Amritsar - 143005 India k [email protected] , [email protected] The Hybrid Antimalarial Approach. Targeting Multiple stages of Life-cycle of Malaria Parasite 1

2. Malaria is a protozoan infectious disease caused by the parasites of the genus Plasmodium (200 spp approx ). It is transmitted by the bite of female Anopheles mosquitoes. Causative agents are 6 species of Plasmodium P. falciparum (most virulent) P. vivax P. ovale (2) P. malariae P. knowlesi Malaria Disease relapse 2

3. 3 World Malaria Report 2024, WHO Malaria Facts 263 Million Cases in 2023, 11 million more than 2022 597000 Deaths in 2023 95% deaths only in African region alone. The children <5 years of age accounted for nearly 76.1% of all malaria deaths worldwide. many at risk still lack access to the services they need to prevent, detect and treat the disease 3 Efforts to eradicate malaria were impacted by the COVID-19 pandemic, resulting in an estimated additional 13.4 million cases during that period

4. Liver/hepatic stage (asymptomatic) Erythrocytic/blood stage (symptomatic) Sexual erythrocytic stage (transmission stage) Sexual Vector stages of parasite (sporogony) Nat. Rev. Dis. Primers 2017 , 3, 17050. Life cycle of Plasmodium parasite Asexual vertebrate host stages of parasite (schizogony) Gametes Ookinete Oocyst Sporozoites Via portal vein 4

5. Drugs for the asymptomatic Liver/hepatic stage Drugs for Symptomatic Erythrocytic/blood stage Drugs for Sexual transmission stage Nat. Rev. Dis. Primers 2017 , 3, 17050. Drugs for tackling different stages of infection Insecticides for the vector control 5

6. P rimaquine 1 and Tafenoquine 2 (for hypnozoites) & transmission stage (1) In addition to 1 and 2 , pyrimethamine 3 , cycloguanil 4 (metabolite of proguanil 5 ), and atovaquone 6 (for the replicating liver stage) Artemisinin 7 and its derivatives (artesunate 8 , artemether 9 and dihydroartemisinin 10 ). L umefantrine 11 , Piperaquine 12 , (For the blood stage) Antimalarial drugs targeting different stages of the parasite life cycle 6

7. P yronaridine 13 , amodiaquine 14 , mefloquine 15 , sulfadoxine 16 – pyrimethamine 3 combination, quinine 17 and chloroquine 18 (CQ) (For the blood stage) Antimalarial drugs targeting different stages of the parasite life cycle 7

8. Due to the development of resistance of P. falciparum to most available drugs, and loss of efficacy of insecticides , there is a great need to develop novel antimalarial agents 8 The 8-aminoquinolines (Primaquine) cause dose-dependent haemolysis in glucose-6-phosphate dehydrogenase deficiency (G6PD)

9. The Molecular Hybrid Approach 1. K. Singh et al The Hybrid Antimalarial Approach. Medicinal Chemistry Approaches to Malaria and Other Tropical Diseases. The Annual Reports In Medicinal Chemistry (ARMC) Series (volume Editor: Kelly Chibale), Elsevier (USA) 2019, 53, 73-105. (invited) 2. K. Singh ibid ARMC, volume 62. Special roadmap volume, 2024, 62, 1-18 (Chapter 1) (Invited) 9

10. 10 Conjugate hybrids Covalent linker unrelated to any of the Pharmacophores

11. Multistage action: Active against liver and blood stage infection in vivo Incorporation of resistance reversal agents (RAs): Imipramine Artemisinin Primaquine Antimicrob . Agents Chemother . 2017 , 61, e00249-e317. CQ sub structure Inhibition of Pf CRT 11

12. Representative Examples from our Group K. Singh et.al. Eur. J. Med. Chem. 2018, 148, 39-53; Eur. J. Med. Chem. 2015, 101, 52-62; Eur. J. Med. Chem. 2015, 101, 266-273. Eur. J. Med. Chem. 2015, 100, 1-9. Eur. J. Med. Chem. 2013, 606, 314-323. ChemMedChem 2022, 17, e202200031; ChemMedChem 2022, 17, e202200414. J. Med. Chem. 2014, 57,435-448...... 12

13. Molecular design and synthesis of ivermectin hybrids targeting hepatic and erythrocytic stages of Plasmodium. 13

14. IVERMECTIN A potent endectocide licensed in 1981 as veterinary drug with broad-spectrum activity against ectoparasites and nematodes. Used in MDA for the treatment of neglected tropical diseases including lymphatic filariasis and onchocerciasis (river blindness). Acts through binding invertebrate Glutamate-gated chloride ( GluCl ) channels causing hyperpolarization of the neurons and muscles leading to the death of the invertebrates. Singh K. et al J. Med. Chem. 2021 , 64, 9711-9731 . Fermentation product of Streptomyces avermitilis 14

15. Diana Fontinha , iMM , Lisboa Miguel Prudencio , iMM , Lisboa Lovepreet Singh, Post-doc at Brain Research Institute, Korea Singh K et al J. Med. Chem. 2020, 63, 1750-1762. The First generation ivermectin hybrids 15

16. The Synthetic challenge oleandrosyl-oleandrosyl disaccharide Hexahydrobenzofuran 6,6-Spiroacetal E,E -Diene 16 membered macrolactone 19 Chiral centres!! 16

17. Alkyne precursors The choice of partner pharmacophores was guided by their established antiplasmodial activity, For example: Triazole tethered IVM hybrids Design Strategy Singh K et al J. Med. Chem. 2020 , 63, 1750-1762. Azide precursor Click reaction 17

18. Synthesis of azide precursor Synthesis of ivermectin aglycon Scheme 1. Synthesis of IVM aglycon 3. 18

19. Scheme 2. Synthesis of azide precursor 6 from 3 . Singh K et al J. Med. Chem. 2020 , 63, 1750-1762. 19

20. Scheme 3 . Synthesis of alkyl 4-methyl-6-aryl-2-(prop-2-yn-1-yloxy)pyrimidine-5-carboxylates 10a-c . Synthesis of pyrimidine based alkyne precursors Synthesis of alkyne precursors 20

21. Scheme 4. Synthetic routes to ferrocenyl alkynes 13a and 13b. S ynthesis of ethynylferrocene and 4-ethynylphenylferrocene Singh K et al J. Med. Chem. 2020 , 63, 1750-1762. 21

22. Scheme 5. Synthetic routes to IVM hybrids 28-32 . Synthesis of The first Generation IVM hybrids 22

23. Characterization HRMS: m/z [M+H] + for C 55 H 70 N 6 O 16 calculated 1071.4804 observed 1071.4794 Figure 1 . 1 H NMR (500 MHz, CDCl 3 ) spectrum of 32 . 7.79 (Triazole-H) 4.97 (C15-H) 4.59 (C8a-H) 3.93 (OCH 3 ) 4.00 (OCH 3 ) C28-H (1.01) 0.83 (C24a-H) 0.79 (C26a-H) 2.67 (CH 3 ) Singh K et al J. Med. Chem. 2020 , 63, 1750-1762. 23

24. ( H 9 , H 10 ) H 9 H 10 ( H 9 , H 8a ) H 8a ( ArH 1 , Ar H 2 ) ArH 1 ArH 2 H 11 ( H 10 , H 11 ) H 10 H 11 H 15 H 16 ( H 15 , H 16 ) ( H 17 , H 16 ) ( H 25 , H 24 ) ( H 25 , H 26 ) H 25 H 17 H 24 H 26 (A) (B) 29 Figure 2 . 1 H- 1 H COSY (500 MHz, CDCl 3 ) spectrum of 29 . 24

25. Hepatic stage Antiplasmodial activity Compound IC 50 (µM) P. berghei a 28 0.990 ± 0.068 30 0.911 ± 0.076 31 0.990 ± 0.050 32 0.503 ± 0.002 3 4.557 ± 1.981 IVM 1.639 ± 0.189 Atovaquone 0.0007 ± 0.05 a Results are represented as mean ±SD, n≥2. IVM hybrids 23-25, 28-32 were tested against hepatic infection of P. berghei . 23-25 (with TBDMS) failed to exhibit any antiplasmodial activity at 1 µM and 10 µM. In general, pyrimidine-based hybrids 30-32 were more active than ferrocene counterparts 28. Hybrid 32 was found to be the most active (IC 50 = 0.503 µM) member of the series and was 3-fold more active than the IVM. S ubstituents at the aromatic ring of the pyrimidine subunit significantly modulate the antiplasmodial activity. The highest antiplasmodial activity shown by compound 32 bearing 2-nitro-4,5-dimethoxy substituted aromatic ring at the C-4 position of the pyrimidine subunit. P. berghei hepatic infection Table 1. In vitro antiplasmodial activity of hybrids 28, 30-32. 3 fold 25

26. Compound IC 50 (nM) a PfNF54 b 28 256.7 ± 46.6 30 161.2 ± 40.9 31 110.5 ± 64 32 50.2 ± 24.5 3 - IVM 519.6 ± 175.4 Chloroquine 23.7 ± 175.4 P. falciparum erythrocytic infection Compounds 28 , 30 - 32 were tested for their in vitro effects against CQ S NF54 strain of P. falciparum . IVM and chloroquine were employed as controls. Compounds showed significantly enhanced ( IC 50 = 50-256 nM ) antiplasmodial activity than IVM ( IC 50 = 519 nM ). Compound 32 ( IC 50 = 50.2 nM ) was most active and was ~11-fold more active than IVM. a Results are represented as mean ±SD, n≥2. b Chloroquine -sensitive (CQ S ) strain. Table 2 . In vitro antiplasmodial activity of hybrids 28, 30-32. Blood stage antiplasmodial activity 11 fold 26

27. 27 Thus, modification of IVM by linking pyrimidine and ferrocene subunits through triazole linker at the C-13 position of the IVM and retaining free C5-OH leads constitutes a good strategy.

28. Insecticidal activity Anopheles stephensi mosquitoes were allowed to feed on mice treated with 1 mg/kg IVM, IVM aglycon and hybrid 32. IVM aglycon and 32 failed to show any detrimental effects on the survivorship of the mosquitoes. The lack of substantial insecticidal effect may be due to the absence of the disaccharide unit at C-13 of the macrolide. Lit.: IVM aglycon displayed ~10 4 -fold reduced insecticidal effects than parent IVM. T he advantage gained in terms of increased antiplasmodial effects was offset by the significantly reduced insecticidal effects. Mosquito survival (%) Days elapsed Ivermectin Compound 32 DMSO Aglycon 3 28

29. The pyrimidine subunit is oriented outwards; no interaction with the channel Glide Score: -12.024 Glide Energy: -135.48 kcal/mol The result suggested that compound 32 may modulate the ion-conductive pathway, similar to IVM. The in silico molecular docking analysis revealed that hybrid 32 showed key contacts with the specific amino acid residues in a manner similar to the IVM. Singh K et al J. Med. Chem. 2020 , 63, 1750-1762. Poor Insecticidal activity: Does it bind to GluCl ? Docking of IVM in Glu-Cl 29

30. Significant enhancement ( IC 50 = 50-256 nM ) of the blood stage antiplasmodial activity compared to IVM ( IC 50 = 519 nM ). Significant enhancement ( IC 50 = 0.503-0.990  M ) of the hepatic stage antiplasmodial activity compared to IVM ( IC 50 = 1.639  M ). Structure modification of IVM is a good strategy. However, significantly reduced insecticidal activity compared to pristine IVM. Hybrids do bind to Glu-Cl similar to IVM. Conclusions 30

31. Synthesis and antiplasmodial activity of the Second-generation dual acting ivermectin hybrids. Isolation of regioisomers and epimers. 31

32. Diana Fontinha , iMM , Lisboa Miguel Prudencio , iMM , Lisboa Parth, GNDU, Amritsar Singh K et al Scientific Reports (Nature) . 2022, 12, 564 32

33. Design strategy Introduction of chloroquine analogue and primaquine at the C 13 -OH position of aglycon of IVM. Carbonyl transfer reagent carbonyl diimidazole ( CDI ) was utilized as a coupling agent, which displays excellent reactivity with amines and alcohols. Traditional coupling agents are limited by the several disadvantages such as toxicity and instability under the reaction conditions. 33

34. Synthesis of Ivermectin precursors Singh K et al Scientific Reports (Nature) . 2022, 12, 564 34

35. Synthesis of IVM-CQ hybrids ( Regioisomers ) 35

36. Compound t R (minutes) % Area 6a′ (R = H) 30.73 0.13 6a (R = Me) 39.00 32.05 6b′ (R = H) 42.93 0.47 6b (R = Me) 48.65 66.82 Figure 1. Reverse-Phase HPLC chromatogram of 6. Table 1 . Retention time (t R ) and % area of isomers of 6 . Isolation and purification HPLC analysis of 6 revealed the presence of a mixture of four (6a  /6b  , R = H; 6a and 6b, R = Me) components in the ratio specified in (Table 1). Mobile Phase: mixture of acetonitrile-methanol-water (49.2:32.8:18 v/v/v) Singh K et al Scientific Reports. (Nature) 2022, 12, 564. 36

37. Figure 2. HPLC (A) chromatogram of a mixture of isomers of IVM-CQ ( 6 ). UPLC chromatograms of individual isomers ( 6a : B; 6b : C). UPLC-MS chromatograms of 6a (D) and 6b (E). 98.41% purity 99.58% purity T he mixture of 6 was subjected to preparative HPLC using reverse phase (X Select CSH C18) column and the two major components ( 6 a and 6 b , Figure 2) were isolated. 6a and 6b we re obtained in 25% and 71% isolated yields , respectively. 37

38. 1.87 (C4a-H) 5.42 (C3-H) 4.30 (C5-H) 3.98 (C6-H) 3.27 (C2-H) HRMS: m/z [M+H] + for C 47 H 61 ClN 2 O 10 calculated 849.4087 observed 849.4038 Quinoline ring (6.43-8.51) Characterization Figure 3. 1 H NMR (500 MHz, CDCl 3 ) spectrum of 6 a. 4.36 (OCH 2 ) 2.30 (CH 2 ) 3.52 (NHCH 2 ) 38

39. Figure 4. 1 H NMR (500 MHz, CDCl 3 ) spectrum of 6 a and 6b. 4.30 (C5-H) 5.42 (C3-H) 3.27 (C2-H) 1.86 (C4a-H) 1.22 (C4a-H) 6.15 (C3-H) 3.61 (C5-H) 2.54 (C4-H) Regioisomers 6a 6b 39

40. H 16 H 17 H 6 H 5 H 18b H 12a H 12 (A) H 4a H 4 ( H 6 , H 5 ) ( H 17 , H 16 ) ( H 17 , H 18b ) ( H 4 , H 4a ) ( H 12 , H 12a ) H 4 H 12 H 16 H 3 H 11 H 15 H 13 (B) ( H 4 , H 3 ) ( H 12 , H 11 ) ( H 12 , H 13 ) ( H 16 , H 15 ) Figure 5. 1 H- 1 H COSY (500 MHz, CDCl 3 ) spectrum of 6 b. Singh K et al Scientific Reports. 2022, 12, 564 (Nature) 40

41. Figure 10. Possible transformations of 11. (A) (B) Figure 11. NOESY (500 MHz, CDCl 3 ) spectrum of epimers 11 a and 11 b and corresponding DFT optimized structures of ivermectin aglycon. 11a : [  ] D 25 +26.667 ° ( c 0.0015, CH 3 OH) 11b : [  ] D 25 +29.412 ° ( c 0.0017, CH 3 OH) Singh K et al Scientific Reports. 2022, 12, 564 (Nature) 41

42. Scheme 3. Synthetic routes to the synthesis of IVM-PQ hybrid 11. Synthesis of IVM-PQ hybrids 42

43. Isolation and purification Compound t R (minutes) % Area 11a′ + 11b′ (R = H) 29.25 1.76 11a (R = Me) 35.46 48.31 11b (R = Me) 36.82 49.34 Table 2. Retention time (t R ) and % area of isomers of 11 . Figure 6. Reverse-Phase HPLC chromatogram of 11. Mobile Phase: mixture of acetonitrile-methanol-water (49.2:32.8:18 v/v/v) HPLC analysis of 11 revealed the presence of a mixture of four ( 11a  / 11b  , R = H; 11a and 11b, R = Me ) components in the ratio specified in (Table 2). 43

44. Figure 7. HPLC (A) chromatogram of a mixture of isomers of IVM- P Q ( 11 ). UPLC chromatograms of individual isomers ( 11 a : B; 11 b : C). UPLC-MS chromatograms of 11 a (D) and 11 b (E). 98.95% purity 97.30% purity The mixture of 11 was subjected to preparative HPLC using chiral column ( Chiralpak -IC ) and the two major components ( 11a and 11b , Figure 7) were isolated. 11a and 11b are obtained in 51% and 49%, respectively. 44

45. 1.87 (C4a-H) 5.04 (C13-H, C15-H) 4.30 (C5-H) 3.97 (C6-H) Aromatic protons (PQ, 6.18-to 8.53) 3.88 (OCH 3 ) (3.64) (CH (CH 3 ) 1.35 CH (CH 3 ) Figure 8. 1 H NMR (500 MHz, CDCl 3 ) spectrum of 11 a. 1 H NMR spectrum of 11 b was identical with 11 a Characterization HRMS: m/z [M+H] + for C 50 H 69 N 3 O 10 calculated 872.5055 observed 872.5011 45

46. ( H 5 , H 6 ) ( H 17 , H 16 ) ( H 12 , H 12a ) H 5 H 6 H 17 H 16 H 12 H 12a H 8a H 9 ( H 9 , H 8a ) ( H 11 , H 12 ) H 11 ( H 3 , H 2 ) ( H 13 , H 12 ) ( H 15 , H 16 ) H 13 H 15 H 2 H 12 H 16 H 3 A) B) Figure 9 . 1 H- 1 H COSY (500 MHz, CDCl 3 ) spectrum of 11 a. 46

47. Compound IC 50 (µM) a P. berghei 6a 0.274 ± 0.103 6b 0.186 ± 0.025 6a+6b 0.317 ± 0.035 11a 1.291 ± 0.042 11b 2.057 ± 0.159 11a+11b 1.360 ± 0.006 Ivermectin 1.321 ± 0.011 IVM-aglycon 6.375 ± 0.909 Primaquine 8.428 ± 3.389 Chloroquine nd Antiplasmodial activity In vitro activity against P. berghei hepatic infection of mice IVM-CQ hybrids displayed the highest activity with IC 50 values ranging from 0.186 to 0.317 µM. Hybrid 6b (IC 50 = 0.186 µM ) is the most active and is 7-folds more active than pristine IVM as well as primaquine. Hybrid 6b is ~3-fold more active than the most potent member (IC 50 = 0.503 µM) of the first-generation hybrids. a Results are represented as mean ±SD, n≥2 . nd : not determined Singh K et al Scientific Reports. 2022, 12, 564 (Nature) 7 fold 45 fold 47

48. In vitro activity against P. falciparum erythrocytic infection (CQ S Pf NF54) Compound IC 50 (nM) a PfNF54 b 6a 48.2 ± 3.1 6b 64.8 ± 27.5 6a + 6b 74.3 ± 33.0 11a nd 11b nd 11a + 11b nd Ivermectin 359.6 ± 65.7 IVM-aglycon nd Primaquine nd Chloroquine 23.7 ± 10.1 IVM-CQ hybrids (6a and 6b) displayed the highest erythrocytic activity with IC 50 values ranging from 48.2 to 74.3 n M. Compound 6 a is the most active compound and is nearly 7.5-fold more active than IVM, however, is less active than chloroquine. IVM-aglycon, IVM-PQ hybrids and PQ lacked substantial antiplasmodial activity in the preliminary screens, thus, IC 50 values were not determined. Individual isomers are more active than the mixture of isomers. a Results are represented as mean ±SD, n≥2. b Chloroquine -sensitive (CQ S ) strain. nd : not determined. 7.5 fold 48

49. Conclusions Different types of IVM hybrids using 4- and 8-aminoquinolines were synthesized. Regioisomers (6a and 6b) and epimers (11a and 11b) were isolated using preparative HPLC and are convincingly characterized using 2D NMR techniques. IVM-CQ hybrid 6b (IC 50 = 0.186 µM) was the most active compound against P. berghei hepatic stage infection in vitro and is 7-fold more active than IVM. IVM-PQ hybrids 11a and 11b showed comparable hepatic stage antiplasmodial activity with IVM. Against the erythrocytic infection, 6a (IC 50 = 48.2 nM) is the most active compound. IVM-CQ hybrids are more active than the most active member (IC 50 = 0.503 µM, hepatic stage; IC 50 = 0.50 nM; erythrocytic stage) of the first-generation IVM hybrids. 49

50. Synthesis and antiplasmodial activity of the Third-generation dual acting ivermectin hybrids. Singh K et al (2024, Submitted) 50

51. Diana Fontinha , iMM , Lisboa Miguel Prudencio , iMM , Lisboa Parth, GNDU, Amritsar 51

52. Design strategy Respecting the nature! 52

53. Synthesis of Ivermectin azide precursor Scheme 2. Synthesis of azide precursor 15 from IVM . 53

54. Synthesis of Ivermectin Hybrid Scheme 3. Synthetic routes to IVM hybrids 17-18 . 54

55. HPLC Analysis (17) (18) t R (min) Area Area % 8.40819 0.21078 1.1 9.92345 0.22928 1.2 11.79815 9.612 50.20 13.26459 0.37139 1.95 14.31819 8.72253 45.55 Total 19.14598 100 t R (min) Area Area % 8.40239 12.56988 34.72 9.23457 2.04844 5.82 9.88101 20.57091 58.46 Total 35.18933 100 Table 1 . Retention time (t R ) and % area of isomers of 17 . Table 2 . Retention time (t R ) and % area of isomers of 18 . Singh K et al (2024, Submitted) 55

56. Characterization Figure 2. 1 H- 1 H COSY (500 MHz, CDCl 3 ) spectrum of 17 ( Regioisomers ) . 56

57. Characterization Figure 3. 1 H- 1 H COSY (500 MHz, CDCl 3 ) spectrum of 18 ( Regioisomers ) . 57

58. Figure 4. 1 H NMR (500 MHz, CDCl 3 ) spectrum of 17. Characterization HRMS: m/z [M+H] + for C 55 H 70 N 6 O 16 calculated 1235.5526 observed 1237.5470 Isomerism 1.83 2.49 1.14 Singh K et al (2024, Submitted) 58

59. Figure 5. 1 H NMR (500 MHz, CDCl 3 ) spectrum of 18. Characterization HRMS: m/z [M+H] + for C 69 H 94 N 6 O 22 calculated 1359.6493 observed 1359.3913 Isomerism 2.45 1.88 1.14 59

60. Synthesis of Ivermectin Hybrid Scheme 4. Synthetic routes to IVM hybrids 19-20 and 23-24 . 60

61. Antiplasmodial & Insecticidal Activity Compound IC 50 ( μM ) P. Berghei IC 50 (nM) Pf. NF54 Insecticidal a (Survival at day 14) 1 1.503 ± 0.528 519.6 ± 175.4 0 % A-glycon 5.686 ± 0.111 - 55 % 12 5.632 ± 1.263 - 7 % 13a 2.911 ± 1.344 717.4 ± 50.9 71 % 13b 3.694 ± 1.080 701.3 ± 50.9 94 % 14a - - 62 % 14b - - 65 % 19 - - 72 % 20 - 304.6 ± 20.3 0 % 61

62. Antiplasmodial & Insecticidal Activity First -generation IVM hybrids Second -generation IVM hybrids Third- generation IVM hybrids Compound IC 50 ( μM ) P. Berghei IC 50 (nM) Pf. NF54 Insecticidal a (Survival at day 14) First -generation IVM hybrids 2 0.990 ± 0.068 256.7 ± 46.6 - 3 0.911 ± 0.076 161.2 ± 40.9 - 4 0.503 ± 0.002 50.2 ± 24.5 - 5 0.990 ± 0.050 110.5 ± 64 - Second -generation IVM hybrids 6a 0.274 ± 0.103 48.2 ± 3.1 ND 6b 0.186 ± 0.025 64.8 ± 27.5 ND 6a + 6b 0.317 ± 0.035 74.3 ± 33.0 ND 7a 1.291 ± 0.042 ND ND 7b 2.057 ± 0.159 ND ND 7a + 7b 1.360 ± 0.006 ND ND Third -generation IVM hybrids 17a + 17b 1.539 ± 0.475 106.2 ± 24.6 33 % 18a + 18b 2.329 ± 1.462 34.7 ± 7.0 96 % 23 5.699 ± 0.798 - 0 % 24 8.454 ± 0.25 - 83 % PQ 8.428 ± 3.389 ND ND 62

63. Docking Studies The in silico molecular docking analysis revealed that IVM intermediate 19 showed key contacts with the specific amino acid residue in a manner similar to the IVM. Glide Score: -11.2026 Glide Energy: -74.8466 kcal/mol Results suggested that the docking of 19 in GluCl -Fab may induce global conformational changes resulting in the modulation of the ion conductive pathway. 63

64. Conclusion Different types of IVM hybrids using pyrimidine based motif were synthesized. Hybrid 17 (IC 50 1.539 ± 0.475 μM ) showed activity comparable to the parent IVM (IC 50 1.503 ± 0.528 μM ) against the hepatic stage of P. berghei . All these hybrids were more active compared to PQ. Compound 23 and 20 recorded 0 % mosquito survival after 14 days and its insecticidal activity matched that of the parent 2. 64

65. ………… and the Quest continues THANK YOU! 65

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68. Extensive and overuse of drugs in monotherapy (also Intermittent preventive treatment) Incomplete therapeutic treatments of infected patients Lack of compliance Unchecked use of counterfeit drugs deficient in std. dose Parasite’s adaptability to generate resistance (Genetic and metabolic basis) Uncontrolled rates of reproduction and dispersal of resistant strains Causes of Drug Resistance The development of resistant parasite strains primarily arising from genetic mutations . 68

69. Sites of Action of antimalarial drugs Dihydrofolate reductase (DFHR) Inhibitors Dihydroorotate dehydrogenase (DHODH) Inhibitors Cytochrome bc1 Inhibitors Hemozoin Inhibitors Drugs targeting multiple stages Malar. J. 2019 , 18, 93 69

70. An important step forward in malaria prevention and control was the introduction of the first malaria vaccine recommended by WHO, RTS, S/AS01, which targets the most abundant protein on the P. falciparum sporozoite surface, the Circumsporozoite protein. A second vaccine, R21/Matrix-M , was approved by the WHO in 2023. These vaccines are recommended for children living in regions with moderate to high malaria transmission 70

71. Every year on April 25th World Malaria Day is recognized as an occasion to reflect, raise awareness, and highlight the need for continued investment in Malaria Research that can contribute to the control and, ultimately, the elimination of the disease. With half of the world’s population at risk, continued investment and sustained political commitment are crucial to effectively manage this public health issue. 71

72. As part of the worldwide effort to control and eradicate malaria, it is crucial to identify new compounds that are effective against multidrug-resistant P. falciparum strains, ideally active against multiple stages of the parasite life cycle, preventing transmission and occurrence of relapse 72

73. Hybrid compounds comprise two or more pharmacophores that are covalently linked in a single molecule. E xtension of the concept of a fixed-dose combination of two or more drugs in a single formulation type. D istinct biological targets, attributing a dual mode of action corresponding to their respective targets. Covalent bitherapy or double drugs. Delay in development of resistance. Lower risk of Drug-Drug interaction. Matched pharmacokinetics. Active against both CQ R and CQ S strains. Good drug profile: solubility, absorption, metabolism, safety parameters The Molecular Hybrid Approach 1. K. Singh et al The Hybrid Antimalarial Approach. Medicinal Chemistry Approaches to Malaria and Other Tropical Diseases. The Annual Reports In Medicinal Chemistry (ARMC) Series (volume Editor: Kelly Chibale), Elsevier (USA) 2019, 53, 73-105. (invited) 2. K. Singh ibid ARMC, volume 62. Special roadmap volume, 2024 (Invited) 73

74. Human Stages of Parasite Vector Control IVM as an Antimalarial Mosquitocidal Acts on GluCl channels in Anopheles . Acts on Zoophagic vectors (mainly feeding on bovines, therefore n ot targeted by ITNs or IRS ). Vectors carrying P. vivax parasites. Inhibitory effects against reproductive health of the vector as well as the sporogonic development in vector host . Inhibits Plasmodium infection of human hepatoma cells in vitro ( IC 50 = 2.1 µM ). ~80% inhibition of liver infection is observed upon treatment with 10 mg/kg of IVM in infected mice. Hepatic stage Asexual erythrocytic stage In vitro activity ( IC 50 = ~100 nM ) have been reported. Showed in vitro gametocidal activity ANTIMALARIAL EFFECTS OF IVERMECTIN Singh K. et al J. Med. Chem. 2021 , 64, 9711-9731 . 74

75. Safety Doses higher than FDA approved doses (150-200 µg/kg) are safe. Safe and efficacious in combination with ACTs and other antimalarial drugs. Artemether-Lumefantrine Dihydroartemisinin-Piperaquine Primaquine Current Status Two dosing regimens are being used in MDA programme: i ) 1 x 400 µg/kg per Month (>60,000 doses administered) ii) 3 x 300 µg/kg per Month + ACTs (safe in a clinical trial) Development of improved formulations of ivermectin to prolong and enhance the mosquito killing effect with acceptable safety profile is currently underway 75

76. William C. Campbell and Satoshi Ōmura discovered Avermectin , the derivatives of which have radically lowered the incidence of River Blindness and Lymphatic Filariasis, as well as showing efficacy against an expanding number of other parasitic diseases. Tu Youyou discovered Artemisinin , the drug that has significantly reduced the mortality rates for patients suffering from Malaria. The Nobel Prize in Physiology or Medicine 2015 Prize share: 1/4 Prize share: 1/4 Prize share: 1/2 76

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78. OH Me H H H H H H Me 78

79. The oxahydrindene part of the macrolide ring of the IVM hybrids having two hydroxyl groups adjacent to sp 3 hybridized carbons is prone to transformation into the benzenoid structure through double β-elimination of water, which, upon prototropic shift of a 8a-H, would result in an aromatic benzofuran ring (B). Interestingly, the spiroketal moiety of the macrolide remained intact. 79