The work presented in this Master’s thesis had the main objective of expanding the chiral spiro-β-lactam family through the diversification of two synthetic platforms: the phosphine-catalysed formal [3+2] cycloaddition, previously established for the synthesis of spirocyclopentenepenicillanates, and an alternative tertiary amine-catalysed strategy explored for the first time towards heterocyclic, oxygen-containing spiro-β-lactam scaffolds. The motivation for this work stems from the therapeutic potential of spiropenicillanates as broad-spectrum antimicrobial agents, first evidenced by the lead compound BSS-730A, which displayed excellent nanomolar activity against multiple strains of HIV-1 and HIV-2, including multi-resistant strains. BSS-730A also showed antiparasitic activity against both the hepatic and erythrocytic stages of Plasmodium infection. Building on the extensive family of spirocyclopentenepenicillanates derived from this lead molecule, two complementary synthetic approaches were pursued to further expand the chemical space of chiral spiropenicillanates. Firstly, the implemented synthetic route for the synthesis of spirocyclopentenepenicillanates’ precursors was optimised at key steps, leading to the development of a greener, more scalable synthesis of diphenyldiazomethane and to a novel biphasic diazotisation methodology for the synthesis of the 6-diazopenicillanate intermediate. Additionally, this family of chiral spiropenicillanates was expanded through the structural diversification of positions 1’ and 2’ of the spirocyclopentene ring. Two new 6-(Z)-alkylidenepenicillanates bearing thienoyl and N-benzylpyrroyl substituents were synthesised and subjected to phosphine-catalysed formal [3+2] cycloadditions with a series of monosubstituted allenoates, affording sixteen novel heteroaryl-substituted spirocyclopentene-β-lactams. The exploratory structural variation of the allenoate-derived ester function at position 2’ was also investigated, leading to the synthesis of the first spiro-β-lactams of this class bearing a non-ester substituent at this position. Secondly, an alternative Lewis base catalytic strategy was explored through the tertiary amine-catalysed formal [4+2] cycloaddition of 6-alkylidenepenicillanates with allenoates, leading to the first report of heterocyclic six-membered spiro-β-lactams. 1,4-Diazabicyclo[2.2.2]octane catalysis consistently and diastereoselectively afforded the formal [4+2] cycloadduct, spiropyranpenicillanate, together with the competing Rauhut-Currier adduct, an allene-bearing β-lactam. In contrast, 4-(dimethylamino)pyridine catalysis proved selective for the exclusive, albeit lower-yielding, formation of the spiropyran cycloadduct. Extension of the optimised conditions to a small library of aryl- and heteroaryl-substituted 6-(Z)-alkylidenepenicillanate substrates afforded five new chiral spiropyranpenicillanates and five new chiral allene-bearing β-lactams, with structures and stereochemistry established through extensive nuclear magnetic resonance analysis and corroborated by density functional theory calculations. The developed methodology was further transposed to continuous-flow conditions, affording comparable isolated yields to the batch process while delivering a 6- to 8-fold increase in production rate. Taken together, the synthetic strategies and novel chiral spiro-β-lactams described in this thesis establish a diversified structural platform, encompassing both carbocyclic and, for the first time, O-heterocyclic spiro-β-lactam scaffolds, laying the groundwork for the future biological evaluation of these compounds and for further structure-activity relationship studies within the chiral penicillanates chemical space.

Introduction

Influenza, respiratory syncytial virus (RSV), and SARS-CoV-2 are high-burden respiratory pathogens that disrupt healthcare systems and devastate economies. The development of broad-spectrum antivirals that can overcome resistance is a high priority and will make a major contribution to the treatment of infections caused by RNA viruses. Recently, our research team has unveiled a series of spiropenicillanates with potent in vitro activity against HIV, influenza and SARS-CoV-2.

Objectives

This work aims to expand the chemical space of bioactive spiro-β-lactams through the development of new synthetic methodologies leading to structurally diverse chiral spiropenicillanates with potential application as new broad-spectrum antivirals.

Methods

The novel chiral penicillanates were obtained by carrying out the reaction of 6-alkylidenepenicillanates with allenoates catalysed by nitrogen-containing bases using batch and continuous flow techniques. Preliminary results have shown that both DABCO and DMAP lead to the efficient synthesis of spiropyran-penicillanates and spirodihydropiran-penicillanates, respectively. It is also noteworthy that DABCO-catalysed reactions offer a pathway for the synthesis of chiral β-lactams bearing an allenoate moiety obtained via the Rauhut-Currier reaction. It should be noted that a wide range of natural compounds bearing allenic groups have been shown to possess biological activity, highlighting the potential of these newly synthesized derivatives. The antiviral activity of the new penicillanates and further details of this study will be disclosed.

Results

The reactions enabled the formation of structurally diverse β-lactam derivatives through divergent reaction pathways. Depending on the catalyst employed, either spirocyclic products arising from formal [4+2] annulation reactions or chiral allene-bearing penicillanates generated via Rauhut–Currier processes were obtained. DABCO promoted both pathways, yielding mixtures of spirocyclic and allenic products, whereas DMAP selectively afforded spirocyclic derivatives. Optimization studies demonstrated that reaction conditions significantly influence product distribution and yield. Continuous flow synthesis provided comparable selectivity while considerably reducing reaction times.

Discussion and Conclusions

These results demonstrate that nitrogen-base-catalysed annulation reactions constitute an efficient strategy for generating structurally diverse chiral spiropenicillanates. The methodology expands the accessible chemical space of spiro-β-lactams and provides new scaffolds for future evaluation as potential broad-spectrum antivirals targeting RNA virus infections.

Influenza, respiratory syncytial virus, and SARS-CoV-2 are high-burden respiratory pathogens that disrupt healthcare systems and devastate economies. Most of the scarcely available drugs for these RNA viruses are virus-specific, leading to drug resistance. The development of broad-spectrum host-directed antivirals offers a promising solution to this challenge, exemplified by the recent discovery of novel spiropenicillanates with a disruptive therapeutic profile by our research team. This work focuses on expanding the chemical space of chiral spiro-β-lactams derived from 6-alkylidenepenicillanates. Triphenylphosphine-catalysed formal [3+2] cycloaddition of allenyl ketones and tetrazolyl-allenes afforded spirocyclopentene-β-lactams with a novel substitution pattern. The use of nitrogen-containing bases allowed for divergent annulation pathways, enabling access to previously unexplored spiropyran, spirodihydropyran, and allene-bearing penicillanates.

Influenza, respiratory syncytial virus (RSV), and SARS-CoV-2 are high-burden respiratory pathogens that disrupt healthcare systems and devastate economies. Few effective drugs are currently available for these RNA viruses, and most are virus-specific, leading to drug resistance. The development of broad-spectrum antivirals that can overcome resistance is a high priority and will make a major contribution to the treatment of infections caused by RNA viruses, thereby improving preparedness for pandemic threats posed by these viruses. Recently, our research team has unveiled a series of spiropenicillanates with potent in vitro activity against HIV, influenza and SARS-CoV-2. The current study goes further on penicillanates’ research and drug development as potential new broad spectrum antivirals by exploring different methodologies to expand the chemical space. The novel chiral penicillanates were obtained by carrying out the reaction of 6-alkylidenepenicillanates with allenoates catalysed by nitrogen-containing bases using batch and continuous flow techniques. Preliminary results have shown that both DABCO and DMAP lead to the efficient synthesis of spiropyran-penicillanates and spirodihydropiran-penicillanates. It is also noteworthy that DABCO-catalysed reactions offer a pathway for the synthesis of chiral β-lactams bearing an allenoate moiety obtained via the Rauhut-Currier reaction. It should be noted that a wide range of natural compounds bearing allenic groups have been shown to possess biological activity, highlighting the potential of these newly synthesized derivatives. The antiviral activity of the new penicillanates and further details of this study will be disclosed.

Spirocyclic β-lactams are privileged scaffolds in medicinal chemistry due to their rigid three-dimensional architecture, high fraction of sp³-hybridized carbons, and favourable pharmacokinetic profiles. These features contribute to enhanced bioavailability, metabolic stability, and target selectivity. Our group has previously developed spiro-β-lactam derivatives, including the lead compound BSS-730A, which displays potent, broad-spectrum host-directed antiviral activity. The urgent need for broad-spectrum antivirals (BSAs) is underscored by the recurring emergence of RNA viruses. Traditional antiviral therapies typically target virus-specific proteins and are susceptible to rapid resistance development. In contrast, host directed antivirals (HDAs) offer a promising alternative, acting on conserved host cell pathways essential for viral replication.

Influenza, respiratory syncytial virus (RSV), and SARS-CoV-2 are high-burden respiratory pathogens that disrupt healthcare systems and devastate economies. Few effective drugs are currently available for these RNA viruses, and most are virus-specific, leading to drug resistance. The development of broad-spectrum antivirals that can overcome resistance is a high priority and will make a major contribution to the treatment of infections caused by RNA viruses, thereby improving preparedness for pandemic threats posed by these viruses. Recently, our research team has unveiled a series of spiropenicillanates with potent in vitro activity against HIV, influenza and SARS-CoV-2. The current study goes further on penicillanates’ research and drug development as potential new broad spectrum antivirals by exploring different methodologies to expand the chemical space. The novel chiral penicillanates were obtained by carrying out the reaction of 6-alkylidenepenicillanates with allenoates catalysed by nitrogen-containing bases using batch and continuous flow techniques. Preliminary results have shown that both DABCO and DMAP lead to the efficient synthesis of spiropyran-penicillanates and spirodihydropiran-penicillanates. It is also noteworthy that DABCO-catalysed reactions offer a pathway for the synthesis of chiral β-lactams bearing an allenoate moiety obtained via the Rauhut-Currier reaction. It should be noted that a wide range of natural compounds bearing allenic groups have been shown to possess biological activity, highlighting the potential of these newly synthesized derivatives. The antiviral activity of the new penicillanates and further details of this study will be disclosed.

An unexplored reactivity of 6-(Z)-alkylidenepenicillanates was unveiled, describing the synthesis of compounds having a cyclopropane ring spiro-fused to the penicillanic core. This was achieved via the in situ generation of sulfur ylide intermediates from the corresponding sulfur salts, which react with 6-(Z)-alkylidenepenicillanates, leading to spirocyclopropanepenicillanates. The formal [2 + 1] cycloaddition, which involved the creation of three new chiral centers, proved to be diastereoselective, affording the new chiral spiropenicillanates in good yields. Notably, one spiro-β-lactam exhibited excellent anti-HIV-1 activity.

Background

Despite advances in therapeutics and prophylaxis, SARS-CoV-2 and influenza viruses continue to pose a global threat, leading to millions of infections and deaths. The rapid mutation of these viruses and emergence of new variants demand innovative treatment and prevention solutions. Previously, we identified spiro-β-lactams with potent activity against HIV and Plasmodium spp. In this study, we evaluate a new set of spiro-β-lactams against HIV, SARS-CoV-2 and influenza, seeking to understand their efficacy and underlying mechanisms.

Methods

The five novel spiro-β-lactam compounds were tested against a range of seasonal influenza viruses (AH1N1pdm09, AH3N2, oseltamivir-resistant AH1N1pdm09, and B/Yamagata), SARS-CoV-2 Omicron BA.1, and HIV. Influenza activity was measured using neuraminidase-based assays in MDCK-SIAT1 and Calu-3 cells, while SARS-CoV-2 efficacy was determined via plaque assays in Calu-3 cells. HIV activity was assessed with a luciferase reporter assay in TZM-bl cells. To explore the mechanism of action of these compounds, time-of-addition studies, electron microscopy (EM), and single-cell mRNA sequencing were conducted.

Results

All tested compounds were active against the viruses, with IC50 values ranging from 0.23 to 2.3 µM for influenza, 0.05 to 0.7 µM for SARS-CoV-2, and 0.01 to 0.09 µM for HIV. Compounds affect HIV at different stages of its life cycle. The lead compound BSS-730A caused an increase in cell phagocytic/autophagic vacuoles. These morphological changes were reversible eight hours after the compound were removed. Single-cell transcriptome analysis in human peripheral blood mononuclear cells treated with BSS-730A showed an up-regulation of FTL (ferritin light chain) and FTH1 (ferritin heavy chain 1) gene expression, suggesting an antiviral mechanism of action involving iron regulation and oxidative stress, likely linked to ferroptosis.

Conclusions

This study identifies spiro-β-lactams as promising, potent antivirals against SARS-CoV-2, influenza, and HIV. Their host-centered mechanism, potentially involving ferroptosis, could offer a novel strategy to combat viral mutations. Future research will focus on in vivo validation and the potential of integrating spiro-β-lactams into combination therapies to enhance antiviral effectiveness. These findings lay the foundation for a new class of broad-spectrum antivirals capable of addressing both current and emerging viral threats.

Integrase inhibitors (INIs) are an important class of drugs for treating HIV-2 infection, given the limited number of drugs active against this virus. While the clinical efficacy of raltegravir and dolutegravir is well established, the clinical efficacy of bictegravir for treating HIV-2 infected patients has not been determined. Little information is available regarding the activity of bictegravir against HIV-2 isolates from patients failing raltegravir-based therapy. In this study, we examined the phenotypic and matched genotypic susceptibility of HIV-2 primary isolates from raltegravir-naïve and raltegravir-failing patients to raltegravir, dolutegravir, and bictegravir, and to the new spiro-β-lactam BSS-730A. The instantaneous inhibitory potential (IIP) was calculated to help predict the clinical activity of bictegravir and BSS-730A. Isolates from raltegravir-naïve patients were highly sensitive to all INIs and BSS-730A. Combined integrase mutations E92A and Q148K conferred high-level resistance to raltegravir, and E92Q and T97A conferred resistance to raltegravir and dolutegravir. The antiviral activity of bictegravir and BSS-730A was not affected by these mutations. BSS-730A displayed strong antiviral synergism with raltegravir. Mean IIP values at Cmax were similar for all INIs and were not significantly affected by resistance mutations. IIP values were significantly higher for BSS-730A than for INIs. The high IIP values of bictegravir and BSS-730A for raltegravir-naïve and raltegravir-resistant HIV-2 isolates highlight their potential value for treating HIV-2 infection. Overall, the results are consistent with the high clinical efficacy of raltegravir and dolutegravir for HIV-2 infection and suggest a promising clinical profile for bictegravir and BSS-730A.

The molecular architecture of spirocyclic compounds has been widely explored within the medicinal chemistry field to obtain new compounds with singular three-dimensional pharmacophoric features and improved bioactivity. Herein, the synthesis of 68 new spirocyclopentene-β-lactams is described, resulting from a rational drug design and structural modulation of a highly promising lead compound BSS-730A, previously identified as having dual antimicrobial activity associated with a novel mechanism of action. Among this diverse library of new compounds, 22 were identified as active against HIV-1, with eight displaying an IC50 lower than 50 nM. These eight compounds also showed nanomolar activity against HIV-2, and six of them displayed micromolar antiplasmodial activity against both the hepatic and the blood stages of infection by malaria parasites, in agreement with the lead molecule’s bioactivity profile. The spirocyclopentene-β-lactams screened also showed low cytotoxicity against TZM-bl and Huh7 human cell lines. Overall, a family of new spirocyclopentene penicillanates with potent activity against HIV and/or Plasmodium was identified. The present structure–activity relationship open avenues for further development of spirocyclopentene-β-lactams as multivalent, highly active broad spectrum antimicrobial agents.

The synthesis and antimicrobial activity of new spiro-b-lactams is reported. The design of the new molecules was based on the structural modulation of two previously identified lead spiro-penicillanates with dual activity against HIV and Plasmodium. The spiro-b-lactams synthesized were assayed for their in vitro activity against HIV-1, providing relevant structure-activity relationship information. Among the tested compounds, two spirocyclopentenyl-b-lactams were identified as having remarkable nanomolar activity against HIV-1. Additionally, the same molecules showed promising antiplasmodial activity, inhibiting both the hepatic and blood stages of Plasmodium infection.

The high burden of malaria and HIV/AIDS prevents economic and social progress in developing countries. A continuing need exists for development of novel drugs and treatment regimens for both diseases in order to address the tolerability and long-term safety concerns associated with current treatment options and the emergence of drug resistance. We describe new spiroβ-lactam derivatives with potent (nM) activity against HIV and Plasmodium and no activity against bacteria and yeast. The best performing molecule of the series, BSS-730A, inhibited both HIV-1 and HIV-2 replication with an IC50 of 13 ± 9.59 nM and P. berghei hepatic infection with an IC50 of 0.55 ± 0.14 μM with a clear impact on parasite development. BSS-730A was also active against the erythrocytic stages of P. falciparum, with an estimated IC50 of 0.43 ± 0.04 μM. Time-of-addition studies showed that BSS-730A potentially affects all stages of the HIV replicative cycle, suggesting a complex mechanism of action. BSS-730A was active against multidrug-resistant HIV isolates, with a median 2.4-fold higher IC50 relative to control isolates. BSS-730A was equally active against R5 and X4 HIV isolates and displayed strong synergism with the entry inhibitor AMD3100. BSS-730A is a promising candidate for development as a potential therapeutic and/or prophylactic agent against HIV and Plasmodium.

Structural modulation of previous identified lead spiro-β-lactams with antimicrobial activity was carried out. The target chiral spiro-γ-lactams were synthesized via 1,3-dipolar cycloaddition reaction of a diazo-γ-lactam with electrondeficient dipolarophiles. In vitro activity against HIV and Plasmodium of a wide range of spiro-β-lactams and spiro-γ-lactams was evaluated. Among these compounds, one derivative with good anti-HIV activity and two with promising antiplasmodial activity (IC50 < 3.5 μM) were identified.

Objective

The main objective of this work was to synthesize and evaluate the biologic activity of novel spiro-lactams based on previous identified lead compounds with antimicrobial activity.

Results

A novel synthetic route to chiral spiro-γ-lactams has been established. The studied β- and γ-lactams were not cytotoxic, and three compounds with promising antimicrobial activity were identified, whose structural modulation may lead to new and more potent drugs.

Conclusion

The designed structural modulation of biological active spiro-β-lactams involved the replacement of the four-membered β-lactam ring by a five membered γ-lactam ring. Although, conformational and superimposition computational studies revealed no significant differences between β- and γ-lactam pharmacophoric features, the studied structural modulation did not lead to compounds with similar biological profile. The observed results suggest that the β-lactamic core is a requirement for the activity against both HIV and Plasmodium.