Volume 30, Issue 66 e202401844
Research Article
Open Access

Uncovering the Potent Antiviral Activity of the Sesterterpenoids from the Sponge Ircinia Felix Against Human Adenoviruses: from the Natural Source to the Total Synthesis

Ana Ruiz-Molina

Ana Ruiz-Molina

Unidad Clínica de Enfermedades Infecciosas, Microbiología y Parasitología, Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain

Instituto de Biomedicina de Sevilla (IBiS), Hospitales Universitarios Virgen del Rocío y Virgen Macarena/CSIC/Universidad de Sevilla, Sevilla, Spain

These authors contributed equally to this work

Contribution: ​Investigation (supporting)

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Dawrin Pech-Puch

Dawrin Pech-Puch

CICA-Centro Interdisciplinar de Química e Bioloxía, Departamento de Química, Facultade de Ciencias, Universidade da Coruña, A Coruña, 15071, Spain

Departamento de Biología Marina, Universidad Autónoma de Yucatán (UADY), Carretera Mérida-Xmatkuil, km. 15.5, A.P. 4–116 Itzimná, Mérida, CP 97100 Mexico

Escuela Nacional de Estudios Superiores Unidad Mérida (ENES Mérida), Universidad Nacional Autónoma de México (UNAM), Carretera Mérida-Tetiz, km 4.5, Tablaje, Catastral No. 6998, Ucú CP, 97357 Mexico

These authors contributed equally to this work

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Ramón E. Millán

Ramón E. Millán

CICA-Centro Interdisciplinar de Química e Bioloxía, Departamento de Química, Facultade de Ciencias, Universidade da Coruña, A Coruña, 15071, Spain

These authors contributed equally to this work

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Lucía Ageitos

Lucía Ageitos

CICA-Centro Interdisciplinar de Química e Bioloxía, Departamento de Química, Facultade de Ciencias, Universidade da Coruña, A Coruña, 15071, Spain

Contribution: ​Investigation (supporting)

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Harold Villegas-Hernández

Harold Villegas-Hernández

Departamento de Biología Marina, Universidad Autónoma de Yucatán (UADY), Carretera Mérida-Xmatkuil, km. 15.5, A.P. 4–116 Itzimná, Mérida, CP 97100 Mexico

Escuela Nacional de Estudios Superiores Unidad Mérida (ENES Mérida), Universidad Nacional Autónoma de México (UNAM), Carretera Mérida-Tetiz, km 4.5, Tablaje, Catastral No. 6998, Ucú CP, 97357 Mexico

Contribution: Resources (supporting)

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Jerónimo Pachón

Jerónimo Pachón

Instituto de Biomedicina de Sevilla (IBiS), Hospitales Universitarios Virgen del Rocío y Virgen Macarena/CSIC/Universidad de Sevilla, Sevilla, Spain

Departamento de Medicina, Facultad de Medicina, Universidad de Sevilla, 41009 Sevilla, Spain

Contribution: Supervision (supporting)

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José Pérez Sestelo

Corresponding Author

José Pérez Sestelo

CICA-Centro Interdisciplinar de Química e Bioloxía, Departamento de Química, Facultade de Ciencias, Universidade da Coruña, A Coruña, 15071, Spain

Contribution: Conceptualization (lead), Funding acquisition (supporting), Supervision (equal), Writing - review & editing (equal)

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Javier Sánchez-Céspedes

Corresponding Author

Javier Sánchez-Céspedes

Unidad Clínica de Enfermedades Infecciosas, Microbiología y Parasitología, Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain

Instituto de Biomedicina de Sevilla (IBiS), Hospitales Universitarios Virgen del Rocío y Virgen Macarena/CSIC/Universidad de Sevilla, Sevilla, Spain

CIBERINFEC, ISCIII - CIBER de Enfermedades Infecciosas, Instituto de Salud Carlos III, Madrid, Spain

Contribution: Conceptualization (equal), Funding acquisition (supporting), Writing - review & editing (supporting)

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Jaime Rodríguez

Corresponding Author

Jaime Rodríguez

CICA-Centro Interdisciplinar de Química e Bioloxía, Departamento de Química, Facultade de Ciencias, Universidade da Coruña, A Coruña, 15071, Spain

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Carlos Jiménez

Corresponding Author

Carlos Jiménez

CICA-Centro Interdisciplinar de Química e Bioloxía, Departamento de Química, Facultade de Ciencias, Universidade da Coruña, A Coruña, 15071, Spain

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First published: 20 September 2024

Graphical Abstract

Isolation, structure elucidation, biological activity, and total synthesis of ircinialactam J, a novel sesterterpene lactam produced by the sponge Ircinia felix, is presented. Ircinialactam J displays a significant antiviral activity against Human Adenoviruses (11 times more active than cidofovir®) without cytotoxicity. This work reports the first synthesis of a lactamic sesterterpene.

Abstract

Human Adenovirus (HAdV) infections in immunocompromised patients can result in disseminated diseases with high morbidity and mortality rates due to the absence of available treatments for these infections. The sponge Ircinia felix was selected for the significant anti-HAdV activity displayed by its organic extracts. Its chemical analysis yielded three novel sesterterpene lactams, ircinialactams J−L, along with three known sesterterpene furans which structures were established by a deep spectrometric analysis. Ircinialactam J displayed significant antiviral activity against HAdV without significant cytotoxicity, showing an effectiveness 11 times greater than that of the standard treatment, cidofovir®. Comparison of the antiviral evaluation results of the isolated compounds allowed us to deduce some structure-activity relationships. Mechanistic assays suggest that ircinialactam J targets an early step of the HAdV replicative cycle before HAdV genome reaches the nucleus of the host cell. The first total synthesis of ircinialactam J was also accomplished to prove the structure and to provide access to analogues. Key steps are a regio- and stereoselective construction of the trisubstituted Z-olefin at Δ7 by iron-catalyzed carbometallation of a homopropargylic alcohol, a stereoselective methylation to generate the stereogenic center at C18, and the formation of the (Z)-Δ20 by stereoselective aldol condensation to introduce the tetronic acid unit. Ircinialactam J is a promising chemical lead to new potent antiviral drugs against HAdV infections.

Introduction

Human Adenoviruses (HAdV) are double-stranded DNA viruses, non-enveloped, and with an icosahedral capsid.1 According to the Human Adenovirus Working Group, there are currently more than 100 HAdV genotypes grouped into seven species (HAdVA−G).[2],[3] While in healthy individuals HAdV infections are usually mild and self-limited,[4],[5] in immunocompromised patients, these infections show a wide clinical symptomatology that can severely affect this population. For instance, these infections can result in disseminated diseases with high morbidity and mortality rates, especially in pediatric units.6 Additionally, the use of molecular techniques of diagnosis have led to the increasing identification of HAdV as the causative agent of occasional cases and outbreaks of community-acquired pneumonia (CAP).7

Currently, no specific drug has been approved for the treatment of HAdV infections, and only broad-spectrum antiviral drugs such as ribavirin, ganciclovir, or cidofovir have demonstrated in vitro efficacy against HAdV. However, the treatment of patients with these antivirals revealed a very variable efficacy, in addition to having severe secondary effects8 and an unfavorable pharmacokinetic profile that may limit their use. The lipidic conjugate of cidofovir®, brincidofovir (CMX001) (Figure 1), is a potential alternative to be used for the treatment of HAdV infections. This drug showed promising results in phase II and III clinical trials (NCT01231344 and NCT02087306); however, its use is also associated with long-term gastrointestinal disorders which limit its potential clinical use in this setting.[9], [10] Given this situation, the development of new drugs with enhanced anti-HAdV efficacy is of current interest.

Details are in the caption following the image

Antiviral marine natural product derivative Ara-A and other antiviral drugs.

Marine organisms are a rich source of bioactive molecules, including potential novel antiviral candidates.11 For instance, spongothymidine and spongouridine are two marine natural products that were extracted from the Caribbean marine sponge Cryptotethia crypta.12 Both arabino-nucleosides were the basis for the development of vidarabine (ara-A), a synthetic antiviral drug (Figure 1), which was the first FDA-approved marine-derived antiviral product used to treat herpes simplex virus type 1 and 2 infections for many years. Additionally, they are being considered as the basis for the development of other well-known antiviral nucleosides such as azidothymidine (AZT) and acyclovir.13

As part of our ongoing efforts to find novel natural products from marine organisms[14],[15],[16] and more specifically from sponges,17 with pharmacological activity, including antiviral,18 sixty-five marine organisms (51 sponges, 13 ascidians, and 1 gorgonian), were collected from the coast of Yucatan Peninsula (Mexico). From the biological evaluation against human adenovirus type 5 (HAdV5) of their corresponding organic extracts,19 the sponge Ircinia felix was selected for the significant anti-HAdV activity displayed by its organic extract. The sponge I. felix is an important source of marine sesterterpenoids,20 such as the furanosesterterpene tetronic acids, the linear C21 furanoterpenes, and the scalarane-type sesterterpenoids.21 They display a wide range of biological activities including antibacterial, antiviral, cytotoxic, antinociceptive, antitumoral, and anti-inflammatory.22

Herein, we report the discovery of three new sesterterpene lactams, ircinialactam J−L, along with three known sesterterpene furans, from Ircinia felix. Chemical structures were elucidated through deep spectral analysis and biological assays uncovered their potent antiviral activity against HAdV. Specifically, this activity was observed in two types of sesterterpenes: one belonging to the known sesterterpene furans category while the other being a novel sesterterpene, named ircinialactam J. The total stereoselective synthesis of ircinialactam J was also accomplished to confirm its structure and the antiviral activity found in the isolated natural product.

Results and Discussion

Isolation and Structural Identification of Variabilins 1–3 and Ircinialactams 4–6

Specimens of the sponge I. felix were extracted using MeOH to produce an organic extract with anti-HAdV activity. This organic extract was partitioned using the modified Kupchan23 to yield a n-butanol fraction enriched in sesterterpenes which was submitted to a solid phase extraction (SPE)-C18 using a H2O/MeOH/CH2Cl2 gradient system. Fractions containing the sesterterpenes were then separated by reversed-phase HPLC to afford pure compounds 16.

Analysis of the NMR data of compounds 13 and comparison with those reported in the literature allowed us to establish their planar structures as (7Z,12Z,20Z)-variabilin,24 (7E,12E,20Z)-variabilin,25 and (12E,20Z)-8-hydroxyvariabilin,26 respectively. Moreover, comparison of the optical rotation data of 2 and 3 with the values reported for their enantiomers allowed us to determine the R configuration at C18 position in both compounds, as shown in Figure 2.27 On the other hand, compound 1 was already reported from Ircinia dendroides; however, the configuration at C18 and the optical rotation value were not provided.24 In our case, the optical rotation value of [α]25D =+ 39.9 found for 1 suggested an 18 R configuration. Next, comparison the experimental electronic circular dichroism (ECD) spectrum of 1 and the simulated ECD spectrum generated by DFT calculations using the HSH1PBE/cc-pVDZ///PBEPBE/6–311++(3d,2p) combination on the two potential enantiomers (18 R)-1 and (18S)-1 (as shown in Figure S38) confirmed the proposed 18R configuration. In this way, the structure of 1 was established as depicted in Figure 2 and named as (+)-(7Z,12Z,18 R,20Z)-variabilin.

Details are in the caption following the image

Sesterterpenes 16 isolated from Ircinia felix.

(+)-HRESIMS and 13C NMR of 4, isolated as a white powder, allowed us to establish its molecular formula as C33H43NO4 (13 degrees of unsaturation). The NMR data of 4 (Table 1, S2), very similar to those of 1 (Table S1), show the presence of a tetronic acid moiety at one end, and a linear polyprenyl chain with three double bonds at positions Δ7, Δ12 and Δ20 like (7Z,12Z,18 R,20Z)-variabilin (1). The characteristic 13C chemical shifts of C-9 (δC 23.8), C-14 (δC 23.7), and C-20 (δC 115.0) along with the NOESY correlations (Figure S37) between the olefinic proton H-7 at δH 5.07 and the CH3–9 protons at δH 1.68, and between the olefinic proton H-12 at δH 5.11 m and the CH3-14 protons at δH 1.64 (Figure S18), confirmed the Z geometry of the three double bonds in 4.27

Table 1. 13C NMR (125 MHz) and 1H NMR (500 MHz) data of 46 in CDCl3.

δC in ppm, type

δ in ppm, mult. (J in Hz)

Position

4

5

6

4

5

6

1

52.3, CH

173.4, C

51.4, CH

3.73, m

3.92, m

2

136.5, CH

120.5, CH

136.2, CH

6.65, t (1.4)

5.79, m

6.74, m

3

139.3, C

161.5, C

139.3, C

4

172.7, C

56.3, CH2

172.5, C

3.73, m

5

26.3, CH2

29.7, CH2

26.8, CH2

2.24, m

2.31, m

2.30, m

6

25.6, CH2

25.9, CH2

25.6, CH2

2.23 & 2.17, m

2.25, m

2.15, m

7

123.5, CH

123.1, CH

123.4, CH

5.07, m

5.03, m

5.08, m

8

137.0, C

137.5, C

137.0, C

9

23.8, CH3

23.6, CH3

23.8, CH3

1.68, d (1.3)

1.69, m

1.68, d (1.4)

10

32.8, CH2

32.1, CH2

32.7, CH2

1.97, m

1.98, m

1.95, m

11

26.8, CH2

26.4, CH2

26.3, CH2

2.03, m

2.01, m

2.09, m

12

125.0, CH

124.6, CH

125.0, CH

5.11 m

5.11 m

5.11 m

13

135.9, C

136.2, C

135.9, C

14

23.7, CH3

23.6, CH3

23.7, CH3

1.64, d (1.2)

1.66, m

1.64, d (1.6)

15

32.0, CH2

32.9, CH2

32.0, CH2

2.00, m

1.97, m

1.95, m

16

26.4, CH2

26.3, CH2

26.4, CH2

1.44, m

2.01, m

1.26, m

1.28, m

1.43, m

17

37.5, CH2

37.7, CH2

37.5, CH2

1.30, m

1.30, m

1.26, m

1.43, m

1.43, m

1.43, m

18

31.1, CH

31.2, CH

31.1, CH

2.83, m

2.81, m

2.82, m

19

21.2, CH3

21.1, CH3

21.2, CH3

1.05, d (6.7)

1.04, m

1.05, d (6.7)

20

115.0, CH

114.4, CH

115.0, CH

5.49, d (10.1)

5.31, m

5.48, d (10.2)

21

143.7, C

143.9, C

143.6, C

22

162.8, C

162.8, C

162.8, C

23

99.6, C

99.4, C

99.4, C

24

171.8, C

171.8, C

171.8, C

25

6.4, CH3

6.4, CH3

6.4, CH3

1.83, s

1.80, s

1.82, s

1’

44.8, CH2

44.1, CH2

41.3, CH2

3.72, m

3.71, m

3.51, m

2’

35.1, CH2

35.1, CH2

37.5, CH2

2.91, t (7.3)

2.91, t (7.3)

1.48, m

3’

138.4, C

138.4, C

26.0, CH

1.57, m

4’

128.9, CH

128.9, CH

22.5, CH3

7.29, m

7.31, m

0.94, d (6.5)

5’

128.8, CH

128.7, CH

22.6, CH3

7.19, m

7.21, m

0.94, d (6.5)

6’

126.9, CH

126.9, CH

7.21, m

7.25, m

7’

128.8, CH

128.7, CH

7.19, m

7.19, m

8’

128.9, CH

128.9, CH

7.29, m

7.29, m

The presence of a phenyl ring in 4 was deduced by the characteristic NMR carbon and proton signals in the δC 127–140 region and around δH 7.20 and by analysis of its 1H-1H COSY (Figure S16) and DEPT-135 edited HSQC (Figure S15) spectra. Moreover, the diagnostic NMR resonances for 13C at δC 52.3 (C-1), 136.5 (C-2), 139.3 (C-3), and 172.7 (C-4) and for protons at δH 3.73 m (H-1) and 6.65 (H-2) indicated the presence of an α,β-unsaturated-γ-lactam instead of a furan ring in 1. The connection between the α,β-unsaturated-γ-lactam unit and the phenyl ring through the nitrogen via two methylene groups was established by a combination of 2D-NMR experiments. Long-range HMBC correlations from H-1 at δH 3.73 to C-2 and C-3, from H-2 at δH 6.65 to C-4, and from H-1’ at δH 3.72 to C-1, C-4 and C-2’ (δC 35.1) confirmed the existence of a phenethylamine lactam moiety in 4. Furthermore, the link between the polyprenyl segment and the phenethylamine lactam group was established by the HMBC correlation between H-6 at δH 2.17 and C-3 at δC 139.3. The γ-lactam regiochemistry in 4 was deduced by the diagnostic proton chemical shift of the olefinic H-2 at δH 6.65 and by a crucial HMBC correlation between H-5 at δH 2.24 and C-4 at δC 172.7 (Figure S17).

As in 1, the 18R configuration in 4 was suggested by the positive [α]25D + 9.5 and it was consistent with the comparison of the experimental and calculated ECD spectrum generated by TDDFT on the two possible enantiomers (18R)-4 and (18S)-4.28 The initial systematic conformation search with the Maestro program for 4 gave 11 conformers within a 3.0 kcal/mol window, which were geometrically optimized at DFT level using the combination HSEH1PBE/cc-pVDZ. All the 11 conformers of each enantiomer were subjected to theoretical calculation of ECD using a combination TD-SCF-PBEPBE functional and 6–311 G++(3 d,2p) basis set in chloroform as solvent. The simulated electronic circular dichroism (ECD) curve of the 18 R enantiomer showed a good agreement with the experimental one, whereas the 18S enantiomer had an opposite sign and a different shape (Figure S39). Therefore, we confirmed that 4 had the 18R configuration. Based on these spectroscopic data and other analytical results, we established the chemical structure of compound 4 as shown in Figure 2. This new member of the ircinialactam family was named (+)-ircinialactam J. The planar structure of ircinialactam J (4) is the same as ircinialactam I,29 but they differ in the configuration of Δ7 and Δ12 double bonds and that of Me group at C-18.

The (+)-HRESIMS of the fifth sesterterpene, named ircinialactam K (5), showed the same molecular formula C33H43NO4 as ircinialactam J (4). Except for the chemical shifts corresponding to the γ-lactam moiety, the NMR data of 5 (Table S3) are very similar to those of ircinialactam J (4). The diagnostic proton chemical shift of the olefinic H-2 in 5 at δH 5.79 instead of that in 4 at δH 6.65 along with the key NOESY correlation between H-4 at δH 3.73 and H-6 at δH 2.25 found in 5 (Figure S26) allowed us to determine the regiochemistry of the γ-lactam ring as shown in Figure 2. This was confirmed by the characteristic NMR carbon resonances at δC 173.4 (C-1), 120.5 (C-2), 161.5 (C-3), and 56.3 (C-4), which are similar to those reported for ircinialactam B and G isolated from Sarcotragus sp. and Psammocinia sp.,29 and sarcotrine D, isosarcotrine E, and isosarcotrine F from an Sarcotragus sp.[30],[31] In this way, 5 resulted to be a γ-lactam regioisomer of ircinialactam J (4). Based on its positive optical rotation value of [α]25D + 6.4 and comparing the ECD spectrum of 5 with that of 4 (Figure S39), the absolute configuration of 5 at C-18 was deduced as R.

The molecular formula of the last sesterterpene isolated from the sponge, compound 6, was deduced as C30H45NO4 from the [M+Na]+ ion peak at m/z 506.3248 observed in its (+)-HRESIMS. The 1H and 13C NMR spectral data of 6 (Table S4) resemble those of ircinialactam J (4). More specifically, COSY, HSQC, and HMBC correlations observed for the tetronic acid, the polyprenyl chain, and the γ-lactam ring in 6 are the same as those in 4. However, the main structural difference deduced by NMR analysis between both compounds was located at the N substituent on the γ-lactam ring: an N-isopentenyl group in 6 instead of the N-phenethyl in ircinialactam J (4).

Thus, the presence of an isopentenyl group in 6 was deduced from the chemical shift signals assigned to two methyl groups at δC 22.5 (C-4’) and 22.6 (C-5’), both at δH 0.94 (d, J =6.5 Hz), linked to a methine group at δH 1.57 m (H-3’)/δC 26.0 (C-3’) and this in turn to a CH2CH2 group at δH 1.48 m (H-2’)/ δC 37.5 (C-2’) and δH 3.51 m (H-1’)/ δC 41.3 (C-1’), observed in its 1D and 2D NMR experiments. The link of the isopentenyl group to the γ-lactam ring through the nitrogen atom was deduced from the HMBC correlations from H-1’ at δH 3.51 to C-1 at δC 51.4, C-4 at δC 172.5 and C-2’ at δC 37.5 (Figure S33). Key HMBC correlation from H-5 at δH 2.30 to C-4 at δC 172.5 along with the diagnostic proton chemical shift of the olefinic H-2 at δH 6.74 allowed us to determine the γ-lactam regiochemistry in 6 as shown in Figure 2. The isopentenyl chain linked to a γ-lactam ring in 6 was also found in previous reported sesterptepenes as sarcotrine A and its epimer isolated from a marine sponge Sarcotragus sp.30

Again, the 18R absolute configuration as in 15 was deduced for 6 from its positive optical rotation value of [α]25D + 3.4 (c 0.04, MeOH) and by comparison of the ECD spectrum of 6 with those of ircinialactam J (4) and ircinialactam K (5) (Figure S39). Therefore, compound 6 was named (+)-ircinialactam L.

In relation to the biosynthesis of the lactam ring in this type of compounds, a putative biosynthetic pathway was proposed where this moiety would derive from the furan ring as precursor. Accordingly, the oxidation of the furan ring affords a mono-epoxide intermediate that after several transformations yields a lactone, which can further react with the suitable free amino acids producing the corresponding γ-lactam.29 The biocatalytic transformation of a furan ring to γ-lactam unit in the furanosesterterpene palinurin by a fungus Cunninghamella sp., was previously reported.32

Biological Activity

Considering the antiviral activity against human adenovirus type 5 shown by the organic extracts from this sponge,19 the isolated sesterterpenes 15 were submitted to the corresponding biological assays. Antiviral assays showed that (7Z,12Z,18 R,20Z)-variabilin (1) and ircinialactam J (4) exhibited significant inhibition of HAdV5 (species C) infection in a dose-dependent manner in human embryonic kidney 293β5 cells when evaluated at low multiplicity of infection (MOI) (Figure 3A and B). The IC50 values of 1 and 4, 6.30±0.70 μM and 5.23±3.77 μM, respectively, were significantly lower than those shown by cidofovir® (24.36±6.09 μM), the drug of choice to treat HAdV infections.33 Additionally, (7Z,12Z,18 R,20Z)-variabilin (1) and ircinialactam J (4) did not significantly alter cell viability at concentrations <50 μM when their cytotoxicity was evaluated using the Alamar Blue Cell Viability Assay. Their cytotoxic concentration 50 % (CC50) was >200 μM for 1 and 139.45±36.46 μM for 4, in both cases higher than their IC50 and like the CC50 obtained for cidofovir® (181.24±35.10 μM). Thus, sesterterpenes 1 and 4 showed selective indexes (SI) of >31.75 and 26.66, respectively, improving in both cases the SI of cidofovir® (7.44).

Details are in the caption following the image

Inhibitory activity of (7Z,12Z,18 R,20Z)-variabilin (1) and ircinialactam J (4). The dose-dependent activity of 1 (A) and 4 (B) at low MOI on HAdV5-GFP in a plaque assay using the 293β5 cell line. Antiviral effects of 1 (C) and 4 (D) at high MOI in a single round infection assay using HAdV5-GFP on A549 cell line. For all panels, the negative control (−) is non-infected cells, while the positive control (+) is cells infected at the same MOI but in the absence of the compound. Data are presented as the mean ± SD from triplicate assays.

In a single round infection assay in A549 cells, compounds 1 and 4 inhibited HAdV infection in a dose-dependent manner at high MOI, with IC50 values of 35.50 μM and 11.49 μM, respectively (Figure 3C and 3D). Our results suggest that the mechanism of action may be related to the early steps of the HAdV replication cycle, that span from the attachment of the viral particle to its cellular receptors to the introduction of the viral genome into the cell nucleus. Our entry assay showed that the treatment with sesterterpenes 1 and 4 inhibited the expression of the HAdV5-GFP transgene, suggesting inhibition of an early event before transcription and replication of HAdV DNA.34

To gain some knowledge regarding their potential mechanism of action, (7Z,12Z,18 R,20Z)-variabilin (1) and ircinialactam J (4) were further evaluated for their impact on the de novo HAdV DNA synthesis. A quantitative real-time PCR to evaluate the DNA replication was performed in the presence of compounds 1 and 4 in a 24 h assay to avoid the effect of newly generated viruses from subsequent rounds of infection. Compounds 1 and 4 at a concentration of 50 μM inhibited HAdV5 replication by 91.26 % and 99.79 %, respectively (Figure 4A). This inhibition of the HAdV DNA replication suggested two possibilities. Compounds 1 and 4 could block the accessibility of HAdV genomes to the nucleus after endosomal escape, or they could block HAdV replication by interfering with specific proteins involved in this process, such as the DNA polymerase, or alternatively blocking HAdV transcription of E1A and E1B early genes, a prerequisite for DNA replication. To identify the step of the HAdV replicative cycle in which 1 and 4 were directing their activity, we evaluated whether the presence of these compounds affected nuclear accessibility of HAdV genomes. As shown in Figure 4B, the amount of HAdV5 DNA that reached the cell nucleus in cells treated with 1 and 4 was lower compared to those treated with DMSO. Thus, our mechanistic assays suggested that the antiviral activity of both compounds, 1 and 4, was directed to an early step of the HAdV replicative cycle before HAdV genomes reached the cell nucleus.

Details are in the caption following the image

Inhibition of an early step of the HAdV replicative cycle. Compounds 1 and 4 significantly reduced de novo production of HAdV5 DNA copies compared to a positive control 24 h post-infection in a quantitative PCR assay (A). The presence of 1 and 4 significantly reduced the access of HAdV5 genomes to the nucleus (B). For all panels, the negative control (−) is non-infected cells, while the positive control (+) is cells infected at the same MOI but in the absence of the compound. Data are presented as the mean ± SD from triplicate assays.

The absence of antiviral against HAdV5 in compound 2, when compared to 1, suggests that the Z configuration of the Δ7 and Δ12 plays a pivotal role in the antiviral activity, since they only differ in the configuration of those double bonds. Interestingly, the active ircinialactam J (4) also bears the same Z configuration. On the other hand, the position of the carbonyl group in the γ-lactam ring for the ircinialactams seems to also play a significant role since the regioisomer ircinialactam K (5) is devoid of antiviral activity.

Total Synthesis of Ircinialactam J (4)

To confirm the structure and antiviral activity, and opening access to valuable derivatives, the total synthesis of ircinialactam J (4) was undertaken. Sarcotragins A and B from a Sarcotragus sp. sponge were the first reported trisnorpyrrolosesterterpenes.35 More examples of this rare class of sesterterpene lactams have been reported from sponges belonging to Sarcotragus,[29],[30],[31],[36] Ircinia,27 Psammocina,[37],[29] and Cacospongia38 genus. Some of them displayed interesting pharmacological activities such as antiprotozoal,28 while others exhibited selective modulatory properties against α1 and α3 GlyR isoforms. Although compounds with these modulatory properties can inspire the development of therapeutics to treat a wide array of GlyR mediated diseases and disorders (chronic inflammatory pain, epilepsy, spasticity and hyperekplexia),27, 37 the synthesis of these sesterterpene-lactams has not been reported yet.

From a structural point of view, the synthesis of ircinialactams requires the stereoselective construction of two trisubstituted alkenes and the careful incorporation of the sensitive γ-lactam and the tetronic units. Taking these features into account, our retrosynthetic analysis for ircinialactam J (4) devised disconnections at both ends of the molecule to selectively incorporate the lactam ring and tetronic unit in a late step (Scheme 1). The synthesis of central fragment B was devised from natural terpene nerol, which incorporates the required Z-trisubstituted olefin at C12-C13 positions. Key steps are the stereoselective α-carbonyl methylation at C18 and a regio- and stereoselective alkyne carbometallation for the formation of the (Z)-trisubstituted olefin at C7-C8.

Details are in the caption following the image

Retrosynthetic analysis of ircinialactam J (4).

The synthesis started with the preparation of aldehyde 7 by chemoselective oxidative cleavage of the terminal olefin of nerol (Scheme 2).39 Carbon homologation of 7 through stereoselective Horner-Wadsworth-Emmons reaction, followed by 1,4-hydride reduction of the corresponding α,β-unsaturated ester, and hydrolysis of the obtained ester, gave the carboxylic acid 8 in 58 % overall yield. Stereoselective enolate methylation of a chiral oxazolidinone derivative of 8 proceeded with complete diastereoselectivity, as shown by 1H NMR (see section 5.4.2 in the SI). Further reduction of the resulting product with LAH gave alcohol 9 as a single enantiomer.40 The enantiomeric excess and the R configuration of the new stereogenic center in 9 were determined by 1H NMR analysis of the R/S methoxyphenylacetic acid derivatives (see sections 4.9, 4.0, and 5.5.1 in the SI).41 At this point, the enantiomerically pure alcohol 9 was converted into the p-methoxybenzyl ether 10. The orthogonal cleavage of the silyl ether afforded an allylic alcohol that was converted to the corresponding bromide. Then, the propyne unit was incorporated by allylic substitution reaction of the previously prepared bromide using the primary propargyl anion of 1-(trimethylsilyl)propyne. After removal of the TMS group, the terminal alkyne 11 was deprotonated using n-BuLi and the reaction with the tetrahydropyran ether of 2-iodoetanol afforded the homopropargylic alcohol 12 after THP cleavage with Amberlyst® 15 resin (Scheme 2).

Details are in the caption following the image

Synthesis of homopropargylic alcohol 12.

With the homopropargylic alcohol 12 in hand, the stereoselective synthesis of (Z)-trisubstituted olefin was achieved through a regio- and stereoselective alkyne carbometallation. After considering different alternatives,42 iron(III)-catalyzed carbometallation of homopropargylic alcohols reaction using Fe(acac)3 (20 mol %) and MeMgBr proceeded smoothly at room temperature to give the desired (Z)-alkene 13 as a single diastereoisomer in 60 % yield (Scheme 3).43 Afterwards, the homoallylic alcohol 13 was converted to the corresponding iodide 14 (91 %) and the lactam was introduced through enolate alkylation. In this endeavour, we used the γ-ethoxy substituted lactam 15 from the N-homobenzyl succinimide.44 Gratifyingly, treatment of 15 with LDA at −78 °C and addition of iodide 14 afforded the desired product 16 in excellent yield (82 %) following Hiemstra procedure.[45],[46] Posterior reaction with of 16 with trifluoroacetic acid afforded the α,β-unsaturated lactam 17 in 76 % yield after acidic β-elimination and isomerization (Scheme 3).

Details are in the caption following the image

Final steps in the total synthesis of ircinialactam J (4).

With the γ-lactam 17 in hand, we tackled the incorporation of the tetronic unit by stereoselective aldol condensation. Toward this end, the benzyl ether in 17 was cleaved by oxidation with DDQ and Swern oxidation allowed the synthesis of the enantiomerically pure chiral aldehyde 18 in 73 % yield. Treatment of the aldehyde 18 with the anion of methyl tetronate 19, followed by trapping the β-hydroxyketone with mesyl chloride and β-elimination using DBU as base, gave the corresponding aldol condensation product 21 in 49 % overall yield (3 steps) as a mixture of geometric isomers (E : Z =1 : 9) separable by column chromatography.

Encouraged by these results, we undertook the final methyl ether cleavage for the total synthesis of ircinialactam J (4). Following a previous synthesis of (18S)-variabilin,47 O-methylircinialactam J (21) was treated with sodium n-propanethiolate48 in DMF at room temperature, however the complex mixture of reaction products made impossible to get pure ircinialactam J (4). Alternatively, we tested the alternative methoxymethyl ether (MOM) of the tetronic acid. Accordingly, the aldol reaction of aldehyde 18 with the anion of methoxymethyl ether tetronate 20 using the previous developed procedure, gave MOM ether ircinialactam J (22) in 47 % overall yield (3 steps) as a mixture of geometric isomers (E : Z=1 : 9). In this case, we were pleased to find that treatment of 22 with a methanolic solution of HCl at room temperature provided the natural iricinialactam J (4) as a single product in 44 % yield (27 steps, 0.15 % overall yield). The NMR analysis of the synthetic natural product showed identical 1H NMR, 13C NMR, and circular dichroism spectra (see Supporting Information) along with a positive optical rotation which was slightly larger than the natural one. In addition, biological analyses with the synthetic sample showed inhibition of HAdV5 with an IC50 value of 2.27±0.31 μM (almost 11 times more active than cidofovir®), CC50 value >200 μM and a SI >88.1.

Conclusions

Novel sesterterpene lactams (ircinialactams J−L, 46) and known sesterterpene furans (variabilins 13), were isolated from the organic extract of the marine sponge Ircinia felix. Biological evaluation showed that (7Z,12Z,18R,20Z)-variabilin (1) and ircinialactam J (4) exhibited significant anti-HAdV activity. Specifically, ircinialactam J displayed antiviral activity against HAdV at low micromolar concentrations (IC50 =2.27 μM) without cytotoxicity (>200 μM), showing higher activity than the standard cidofovir® (IC50 =24.36 μM/CC50 =181.24 μM), the unspecific drug of choice in infections by HAdV. The mechanism of action by which 1 and 4 block HAdV infections seems to be related with the entrance of the virus into the cell, although additional studies are necessary to verify the mode of action. The Z configuration of the Δ7 and Δ12 in this type of sesterterpene seems to play a key role in the antiviral activity as it was deduced from the antiviral evaluation results of the isolated compounds.

The first total synthesis of a sesterterpene lactam, ircinialactam J (4), was completed, confirming the structural assignment. Key steps in the synthesis were a regio- and stereoselective iron(III)-catalyzed carbometallation of a homopropargylic alcohol to form the Δ7 with Z configuration, a stereoselective enolate methylation to generate the stereogenic center at C18, and the formation of the (Z)-Δ20 by diastereoselective aldol condensation using a tetronic acid derivative.

These new molecules may be used as prototypes for the optimization of a new set of antiviral molecules that could lead to the clinical development of a new anti-HAdV drug with high efficacy and low toxicity to be used for treatment of infections by this pathogen. Moreover, these results support the importance of further research of marine organisms for the discovery of new bioactive molecules with antiviral properties among others.

Acknowledgments

We gratefully acknowledge the help of colleagues, Daniel Catzim Pech, Carlos González-Salas, Gabriel González Mapen, Jorge Peniche Pérez, Melissa llanes López and Rodrigo Garcia Uribe for collecting the marine samples. We thank Patricia Gomez (ICMyL-UNAM) for helping with taxonomic identification. J.R. and C.J. acknowledge Xunta de Galicia and CESGA for the computational resources. Funding for open access charge: Universidade da Coruña/CISUG. This work was supported by grants PID2021-122732OB−C22 from MCIN/AEI/10.13039/ 501100011033/ FEDER “A way to make Europe” (AEI, Spanish State Agency for Research and FEDER Program from the European Union) and by Plan Nacional de I+D+i 2013–2016 and Instituto de Salud Carlos III, Proyectos de Desarrollo Tecnológico en Salud (DTS20/00010). Work in University of A Coruña was also supported by grant ED431 C 2022/39 from Xunta de Galicia. JS−C (CB21/13/00006) was supported by CIBERINFEC - Consorcio Centro de Investigación Biomédica en Red, Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea – NextGenerationEU. J.S.C. is also supported by the program “Nicolás Monardes” (C-0059–2018) Servicio Andaluz de Salud, Junta de Andalucía. DP−P received a postdoctoral fellowship from the National Council of Humanities, Sciences and Technologies (CONAHCYT) of Mexico. L.A. thanks Xunta de Galicia (Spain) for a post-doctoral fellowship, ref: ED481B-2024-076.

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    Conflict of Interests

    The authors declare no conflict of interest.

    Data Availability Statement

    The data that support the findings of this study are available in the supplementary material of this article.