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Synthesis, purification and evaluation of new indolylaryl-sulfones as broad spectrum HIV-1 non-nucleoside reverse

transcriptase inhibitorsAäron Despeghel

KU Leuven promotor: Prof. Dr. D. CabooterSapienza University promotor: Prof. Dr. R. Silvestri

Background

HIV/AIDS: pandemic since early 1980’s World’s leading infectious killer Globally: 35 million infected Since 1981: 39 million deaths Every year: 2 million new infections

Current treatment plan: highly active antiretroviral therapy (HAART) => combination of multiple anti-retroviral drugs

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HIV-1 replication cycle

Source image: Laskey SB, Siliciano RF. A mechanistic theory to explain the efficacy of antiretroviral therapy. Nat Rev Micro. 2014;12(11):772-80.

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Reverse transcriptase

Integrase

Protease

Reverse transcriptase (RT)

Most important drug target No proofreading ability High mutation rate → decreased drug susceptibility Development of resistance

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Non-nucleoside RT inhibitors (NNRTIs)

Bind non-competitively in hydrophobic pocket Conformational changes Inhibition polymerase active site Reduction in viral replication

Nevirapine, the first approved NNRTI 5

Indolylarylsulfones (IASs)A region

C region B region

L-737,126 (Merck, 1992)

Active against HIV-1 RT Bioavailability Heterocyclic rings in B

region Water solubility HIV-1 K103N activity

Compound 0 (Silvestri et al., 2014)

(Highly) active against HIV-1 WT and mutant strains K103N Y181C Y188L

Low cytotoxicity

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Aim of study: new IASs

Compound 1 Compound 2

Compound 37

Aim of study Synthesis and purification of compound 1 - 3 Evaluation

Activity against HIV-1 WT NL4-3CytotoxicitySelectivity index (SI)Activity against HIV-1 mutant strains

K103N Y188L Y181C K103N – Y181C

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Synthesis and purification Oxidation of thiol to disulfide via 1,3‐dibromo‐5,5‐dimethylhydantoin

Silica gel column chromatography (petroleum ether/dichloromethane, 7:3 v/v) Yield: 99%

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Synthesis and purification (cont’d) Deprotonation of amine by NaH Nucleophilic attack of 3’ indole on partially positively charged sulfur atom Reaction under argon pressure

No further purification (difficult, low yield) Indicative yield: 88% 10

Synthesis and purification (cont’d) Activation of carboxylic acid by derivation to acyl chloride Nucleophilic addition of ethanol to carbonyl carbon with elimination of HCl

Silica gel column chromatography (ethyl acetate/n-hexane 1:4 v/v) Indicative yield: 57% 11

Synthesis and purification (cont’d) Double oxidation: from sulfide to sulfoxide to sulfonylgroup Oxidant: m-chloroperoxybenzoic acid (mCPBA) is reduced to m-chlorobenzoic acid

Unsuccessful separation with TLC => trituration with n-hexane Yield: 68%

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Synthesis and purification (cont’d) Hydroxide-ion-promoted hydrolysis Nucleophilic attack of hydroxide ion on carbonyl carbon with ethoxide elimination

Trituration with n-hexane and acetonitrile Yield: 93%

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Synthesis and purification (cont’d)

2-aminophenol

Coupling reaction using 1,1-carbonyldiimidazole (CDI) after failure of PyBOP Transformation to imidazole ester before carboxamide formation

Silica gel column chromatography (ethyl acetate/n-hexane 1:2 v/v) Yield: 21% - 37% - 45% Cumulative yield: 7% - 12% - 14%14

Yield of synthesis steps

Synthesis stepAbsolute yield

(mg)Relative yield

(%)Cumulative relative yield

(%)

A region 3902 99 99

Joining indole – A region

2119a 88a 87a

Esterification 1150b 57b 50b

Oxidation 826 68 34

Hydrolysis 609 93 31

Coupling: 2-aminophenol

51.6 21 7

Coupling: 3-aminophenol

90.9 37 12

Coupling: 4-aminophenol

110.5 45 14a: indicative yield, based on unpurified residueb: indicative yield, based on unpurified starting product

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Evaluation of compound 1 - 3Activity against HIV-1 WT NL4-3 and mutant strains

Lymphoid MT-4 cell line (infected with appropriate HIV-1 strain)Different drug concentrationsMTT methodExpressed by 50% effective concentration (EC50)

CytotoxicityLymphoid MT-4 cell line (mock-infected)Different drug concentrationsMTT methodExpressed by 50% cytotoxic concentration (CC50)

Selectivity index (SI)CC50 / EC50

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Anti-HIV-1 WT activity and cytotoxicity

Compound CC50 (nM) EC50 ± SD (nM) SI

1 19717 ± 703 ˂ 0.7 ˃ 28167

2 24619 ± 4792 ˂ 0.7 ˃ 35170

3 18072 ± 6860 ˂ 0.7 ˃ 25817

NVP ˃ 18776 112.4 ± 74.9 ˃ 167

EFV ˃ 15839 15.9 ± 12.7 ˃ 996

AZT ˃ 30595 3.7 ± 3.7 ˃ 8269

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Activity against HIV-1 mutant strains  Mutant HIV-1 strain (EC50)

Compound K103N(nM)

Y181C(nM)

Y188L(nM)

K103N - Y181C(nM)

1 < 0.7 20 ± 8 4814 ± 857 ˃ 19718

2 < 0.7 44 ± 22 455 ± 85 6089 ± 6949

3 < 0.7 < 0.7 989 ± 132 967 ± 154

NVP ˃ 3756 ˃ 3756 ˃ 3756 ˃ 3756

EFV 130 ± 180 160 ± 180 760 ± 630 ˃ 317

AZT 16 ± 12 6.0 ± 3.4 33 ± 18 16 ± 13

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Conclusion Successful synthesis and purification of compound 1 – 3 Biological assay

Cytotoxicity: all compounds ✔✔✔ Activity against HIV-1

Wild type: all compounds ✔✔✔ K103N: all compounds ✔✔✔ Y181C: compound 3 ✔✔✔; compound 1 & 2 ✔ Y188L: micromolar concentration range K103N - Y181C: micromolar concentration range

Further testing to determine more specific activity Narrower compound concentrations

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Prospective Testing of compounds against other mutant strains

G190A V106M

ADME properties Water solubility Predictions concerning metabolism with Metasite software Parallel artificial membrane permeability assay (PAMPA)

Further exploration of B region To obtain stronger antiretroviral activity

Against Y188LAgainst K103N-Y181C 20

Acknowledgements

Prof. Dr. D. Cabooter Prof. Dr. R. Silvestri Dr. Valeria Famiglini, PhD Lab 5 at Sapienza University

Collaborations Prof. Dr. J. Este, Barcelona, Spain Prof. Dr. D. Schols, Leuven, Belgium Dr. A. Brancale, PhD, Cardiff, United Kingdom Prof. Dr. G. Maga, Pavia, Italy

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