Structural optimization of a retrograde trafficking inhibitor that protects cells from infections by human polyoma- and papillomaviruses

Abstract

Human polyoma- and papillomaviruses are non-enveloped DNA viruses that cause severe pathologies and mortalities. Under circumstances of immunosuppression, JC polyomavirus causes a fatal demyelinating disease called progressive multifocal leukoencephalopathy (PML) and the BK polyomavirus is the etiological agent of polyomavirus-induced nephropathy and hemorrhagic cystitis. Human papillomavirus type 16, another non-enveloped DNA virus, is associated with the development of cancers in tissues like the uterine cervix and oropharynx. Currently, there are no approved drugs or vaccines to treat or prevent polyomavirus infections. We recently discovered that the small molecule Retro-2(cycl), an inhibitor of host retrograde trafficking, blocked infection by several human and monkey polyomaviruses. Here, we report diversity-oriented syntheses of Retro-2(cycl) and evaluation of the resulting analogs using an assay of human cell infections by JC polyomavirus. We defined structure-activity relationships and also discovered analogs with significantly improved potency as suppressors of human polyoma- and papillomavirus infection in vitro. Our findings represent an advance in the development of drug candidates that can broadly protect humans from non-enveloped DNA viruses and toxins that exploit retrograde trafficking as a means for cell entry.

Keywords: Dihydroquinazolinone; Papillomavirus; Polyomavirus; Retrograde trafficking; SAR.

Figure 1

Inhibition of JCPyV infectivity in SVG-A Cells and HPV16 infectivity in HelaM Cells: Cells were pre-incubated with increasing concentrations of Retro-2^cycl or DHQZ 36 prior to inoculation with virus.

Figure 2

HPV16 and JCPyV Proximity Ligation Assay: Cell nuclei are indicated by blue fluorescence. Green fluorescence indicates co-localization of target proteins: JCPyV – capsid protein VP1 and ER protein PDI; HPV16 – capsid protein L1 and Golgi protein TGN 46.

Scheme 1

Diversity oriented synthesis of Retro-2^cycl analogs. a) R₂NH₂, dicyclohexylcarbodiimide, 4-dimethylaminopyridine, dichloromethane, room temperature, 16 hours. b) 10% Pd/C, ammonium formate, methanol. c) R₁CHO, Sc(OTf)₃, methanol, MW 100°C, 1 h. d) R₂NH₂, tetrahydrofuran, reflux, then R₁CHO, Sc(OTf)₃.

Scheme 2

Synthesis and activity of n-butylamino dihydroquinazolinone a) n-butylamine, triethylamine, dimethoxyethane, room temperature, 16 h, 64%. b) 10% Pd/C, ammonium formate, methanol, room temperature, 2 h. c) 5-ethylthiophene-2-carboxaldehyde, Sc(OTf)₃, methanol, reflux, 2 h, 54% over 2 steps.

Scheme 3

Synthesis and activity of quinazolinone and N-methyldihydroquinazolinone analogs. a) 4-fluorobenzylamine, ethanol, reflux, then 5-ethylthiophenal, CuCl₂, 94%. b) 4-fluorobenzylamine, tetrahydrofuran, reflux, then 5-ethylthiophenal, Sc(OTf)₃, 88%.