Nora Chouha

Hussein Abou Hamdan

Hajime Yurugi

Riku Yoshii

Hiromi Ii

Ahmad Najem

Ghanem E Ghanem

Susumu Nakata

Krishnaraj Rajalingam

Yu Peng

Hussein Abou-Hamdan

Dong Wang

Canan G Nebigil

Laurent Désaubry

Development of fluorizoline analogues as prohibitin ligands that modulate C-RAF signaling, p21 expression and melanogenesis

Keywords: Cancer, MAP kinases, p21, melanogenesis, prohibitins, heterocycles

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

In 2014, Albericio, Gil, Lavilla and collaborators disclosed a series of trifluorothiazolines that are cytotoxic in cancer cells [START_REF] Perez-Perarnau | A Trifluorinated Thiazoline Scaffold Leading to Pro-apoptotic Agents Targeting Prohibitins[END_REF]. Fluorizoline (1, Figure 1) was found to be the most active compound of this series, with EC 50 in the low micromolar range in a wide array of cancer cells. Further studies showed that fluorizoline induces in vitro the death of chronic lymphocytic leukemia cells [START_REF] Cosialls | The prohibitin-binding compound fluorizoline induces apoptosis in chronic lymphocytic leukemia cells through the upregulation of NOXA and synergizes with ibrutinib, 5-aminoimidazole-4-carboxamide riboside or venetoclax[END_REF][START_REF] Wierz | The prohibitin-binding compound fluorizoline induces apoptosis in chronic lymphocytic leukemia cells ex vivo but fails to prevent leukemia development in a murine model[END_REF], acute myeloid leukemia cells [START_REF] Pomares | Targeting prohibitins induces apoptosis in acute myeloid leukemia cells[END_REF] and non-small cell lung cancer cells [START_REF] Yurugi | Targeting prohibitins with chemical ligands inhibits KRAS-mediated lung tumours[END_REF]. Using affinity chromatography, these compounds were found to target prohibitins-1 and -2 (PHB1/2), which regulate a multitude of signaling pathways [START_REF] Wang | Prohibitin ligands: a growing armamentarium to tackle cancers, osteoporosis, inflammatory, cardiac and neurological diseases[END_REF], including the activation of the kinase C-RAF (RAF1) by the protooncogene RAS [START_REF] Yurugi | Targeting prohibitins with chemical ligands inhibits KRAS-mediated lung tumours[END_REF]. PHB2 also down-regulates the cyclin-dependent kinase inhibitor p21 Waf1/Cip (p21), which is induced by depletion of γ-glutamylcyclotransferase (GGCT) leading to G0/G1 arrest, cellular senescence and growth inhibition in cancer cells [START_REF] Taniguchi | Prohibitin-2 is a novel regulator of p21WAF1/CIP1 induced by depletion of γglutamylcyclotransferase[END_REF].

Mechanistic investigations demonstrated that fluorizoline induces the death of cancer cells via their direct effect on PHBs through several pathways including an upregulation of the mitochondrial proapoptotic factors NOXA and BIM [START_REF] Moncunill-Massaguer | A novel prohibitin-binding compound induces the mitochondrial apoptotic pathway through NOXA and BIM upregulation[END_REF][START_REF] Cosialls | 13 The BCL-2 family members NOXA and BIM mediate fluorizoline-induced apoptosis in multiple myeloma cells[END_REF][START_REF] Núñez-Vázquez | NOXA upregulation by the prohibitin-binding compound fluorizoline is transcriptionally regulated by integrated stress response-induced ATF3 and ATF4[END_REF], an inhibition of C-RAF activation [START_REF] Yurugi | Targeting prohibitins with chemical ligands inhibits KRAS-mediated lung tumours[END_REF] and an upregulation of p21 (Figure 1) [START_REF] Takagi | Fluorizoline induces p21 via PHB2-GGCT blockade[END_REF]. In addition to its anticancer effects, fluorizoline also induces the biosynthesis of melanin in melanocytes through [START_REF] Djehal | Targeting prohibitin with small molecules to promote melanogenesis and apoptosis in melanoma cells[END_REF]. Indeed, the binding of fluorizoline to PHBs triggers a cascade of events that includes the activation of the autophagy factor LC3-II from LC3-I, leading to an activation of the kinase ERK and MITF, the key transcription factor for melanocyte development and differentiation. 1) in cancer cells and melanocytes. In cancer cells, fluorizoline upregulates the pro-apoptotic factors NOXA and BIM and inhibits the PHB-dependent activation of C-RAF. Fluorizoline also blocks the interaction of PHB2 with γ-glutamylcyclotransferase (GGCT) and its translocation to the nucleus to promote the expression of the cyclin-dependent kinase inhibitor p21. In melanocytes, fluorizoline activates the autophagy factor LC3-I into LC3-II to induce pigmentation of the cells.

During our medicinal chemistry program aiming at developing novel PHB ligands [START_REF] Yurugi | Targeting prohibitins with chemical ligands inhibits KRAS-mediated lung tumours[END_REF][START_REF] Djehal | Targeting prohibitin with small molecules to promote melanogenesis and apoptosis in melanoma cells[END_REF][START_REF] Qureshi | FL3, a synthetic flavagline and ligand of prohibitins, protects cardiomyocytes via STAT3 from doxorubicin toxicity[END_REF][START_REF] Wintachai | Assessment of flavaglines as potential chikungunya virus entry inhibitors[END_REF][START_REF] Elderwish | Discovery of 3,3'-pyrrolidinyl-spirooxindoles as cardioprotectant prohibitin ligands[END_REF], we pursued the exploration of the structural requirements of fluorizoline analogues for the inhibition of C-RAF, the activation of p21 in cancer cells, and the induction of melanogenesis in melanocytes.

Results and discussion

Synthesis

To explore the role of the two fluorine atoms in position 4, we replaced one of them by a methyl (Scheme 1). Decarboxylative arylation of thiazolecarboxylate 3a [START_REF] Li | Design, Synthesis, and Biological Evaluation of Antiviral Agents Targeting Flavivirus Envelope Proteins[END_REF][START_REF] Zhang | Decarboxylative Cross-Coupling of Azoyl Carboxylic Acids with Aryl Halides[END_REF] conveniently afforded the 2,5diarylthiazoles 4a, which was treated by selectfluor to deliver the difluorinated compound 5a. As far as we know, this compound is the first example of a 4,5-difluorothiazoline.

Scheme 1. Synthesis of trifluorothiazoline 5a.

The synthesis of 4,4,5-trifluoro-2,5-diaryl-4,5-thiazolines 5b/c began with the PdCl 2 (bipy)-catalyzed C-H arylation of thiazoles 3b/c to afford 2,5-diarylthiazoles 4b/c following Itami's conditions (Scheme 2) [START_REF] Tani | Programmed synthesis of arylthiazoles through sequential C-H couplings[END_REF]. Perfluorination of these adducts according to Lavilla's protocol gave access to trifluorothiazolines 5b/c. Scheme 2. Synthesis of trifluorothiazolines 5b/c. Next, to explore the importance of the orientation of the dipole S-C=N, we performed the first synthesis of a 4,5,5-trifluoro-4,5-thiazoline (Scheme 3). Scheme 3. Synthesis of 4,5,5-trifluoro-4,5-thiazoline 5d.

Trifluorothiazolines 5e-k were prepared by acylation of 2-aminoacetophenones 7a/b followed by ring closure of amides 8e-k by using Lawesson reagent and a treatment with selectfluor [START_REF] Thomsen | Studies on organophosphorus compounds. XLIV. Novel and convenient methods for the preparation of substituted thiophenes, thiazoles, and 1,3,4-thiadiazole-2(3H)-thiones from bifunctional substrates[END_REF] (Scheme 4). Scheme 4. Synthesis of trifluorothiazolines 5e-k.

Biological results

Inhibition of C-RAF

We previously showed that fluorizoline (1) and rocaglamide, a natural product, inhibit the activation of the kinases C-RAF and MEK1/2 [START_REF] Yurugi | Targeting prohibitins with chemical ligands inhibits KRAS-mediated lung tumours[END_REF]. Indeed, the interaction of C-RAF with PHBs at the plasma membrane is essential for C-RAF activation, and the binding of fluorizoline or rocaglamide to PHBs prevents this interaction, leading to an inhibition of the C-RAF-MEK-ERK pathway (Figure 2E). Here, we found that the replacement of a fluorine atom in position 4 by a methyl (5a), or the inversion of the dipole S-C=N (5d) promote the phosphorylation of C-RAF at Ser338, instead of inhibiting it (Figure 2A). Accordingly, 5d, but not 5a, enhanced the phosphorylation of MEK1.

The replacement of the 4-chlorophenyl by a 4-tolyl in position 5 (5c) increased the inhibition of C-RAF and MEK, and the deletion of the chlorine atom on the other aryl moiety (5b) abolished this activity. Interestingly the azaisostere 5e displayed an enhanced anti-C-RAF (but not anti-MEK) activity compared to fluorizoline. The deletion of the two chlorine atoms on this azaisostere (5f) maintained the inhibition of C-RAF, which is surprising considering that the suppression of these two atoms in fluorizoline has been reported to be deleterious for cytotoxicity [START_REF] Perez-Perarnau | A Trifluorinated Thiazoline Scaffold Leading to Pro-apoptotic Agents Targeting Prohibitins[END_REF]. The reintroduction of a chlorine atom (5h) didn't modify the anti-C-RAF and anti-MEK activities. The replacement of the 2-pyridyl by a 2-pyrimidinyl (5i) was tolerated, but it was not the case for a pyrazinyl (5g), which was found inactive. The benzophenone derivative 5j maintained an anti-C-RAF, suggesting that this compound could be used as a photoactivatable probe [START_REF] Dormán | The Life of Pi Star: Exploring the Exciting and Forbidden Worlds of the Benzophenone Photophore[END_REF] to explore the binding domain of fluorizoline in PHBs. The replacement of the 2-chlorophenyl by a 2-bromophenyl (5k) was well tolerated for the inhibition of C-RAF.

Cytotoxicity in HeLa cells

Next, we performed MTT assays to determine whether these compounds inhibit the growth of HeLa cells (Figure 3). After a 24h h incubation, fluorizoline (1) and its analogues 5b, 5c, 5e and 5k (10 µM) greatly decreased the numbers of viable cells in a dose-dependent manner, reflecting their anti-MEK activity. We recently demonstrated that fluorizoline upregulates the cyclin-dependent kinase inhibitors p21 in several human cancer cell lines, including MCF7 breast cancer cells [START_REF] Takagi | Fluorizoline induces p21 via PHB2-GGCT blockade[END_REF]. In line with these studies, we found that two compounds substituted in position 2 by a 2-pyrazinyl (5g) or a 4-bromophenyl (5k) were more potent than fluorizoline to lower the viability of MCF7 cells and increase the expression of p21. 1) and its analogues 5b-5k at 1 µM concentration. A. The number of trypan blue-negative viable cells is shown (* p<0.05,** p<0.01, *** p<0.001, T test vs DMSO). B. Expression of p21 analyzed by Western blot. MCF7 cells were treated with perfluorothiazolines (1, 5a-k, 1 µM) for 72 h. The cells were counted with a trypan blue staining and p21 expression was analyzed by Western blot analysis. C and D. p21 knockdown restores cell proliferation suppressed by 5b and 5g. MCF7 cells were transfected with siRNA targeting p21 or non-target control siRNA and treated with 1 µM of 5b and 5g for 48 h. C. The cells were counted with a trypan blue staining. (*P < 0.001 by 2-way repeated-measures ANOVA) D. The expression levels of p21 protein were analyzed by Western blot analysis. β-actin is shown as a loading control.

Stimulation of cell pigmentation

We previously found that fluorizoline (1 and 10 µM) significantly promotes the pigmentation of HBL human melanoma cells by measuring the extent of pigment formation [START_REF] Djehal | Targeting prohibitin with small molecules to promote melanogenesis and apoptosis in melanoma cells[END_REF]. In this assay, the only analog that exhibited significantly superior effects to fluorizoline was 5b, substituted with a phenyl and a tolyl group at the 2 and 5 positions (Figure 5). The analogues 5e, 5i and 5j, substituted by a phenyl group and a tolyl group in position 2 by a 5-chloro-2-pyridinyl, 2-pyrimidinyl, and 4-benzophenone, exhibited an activity similar to that of fluorizoline. The other compounds displayed a reduced activity. In contrast to the previous assays, 5d behaved similarly to the other analogues: it slightly stimulated pigmentation instead of inhibiting melanogenesis as expected. 1) and its analogs (0.1 and 1 µM). All experiments were undertaken independently in duplicate. The melanin content was expressed as relative increase compared to vehicletreated HBL cells.

Conclusion

While the structure-activity relationships (SAR) of several classes of PHB ligands are well documented [START_REF] Wang | Prohibitin ligands: a growing armamentarium to tackle cancers, osteoporosis, inflammatory, cardiac and neurological diseases[END_REF], no advance on the SAR of trifluorothiazolines has been reported since their seminal disclosure in 2014 [START_REF] Perez-Perarnau | A Trifluorinated Thiazoline Scaffold Leading to Pro-apoptotic Agents Targeting Prohibitins[END_REF]. Therein, we identified in particular 5c that displays an enhanced anti-MEK activity and a higher cytotoxicity in HeLa cells compared to fluorizoline, paving the road for the development of new drugs against cancers and RASopathies [2-5, 8, 22].

We also uncovered that the isomer 5d, characterized by an inverted S-C=N dipole, stimulates the activation of C-RAF and MEK instead of inhibiting them. As far as we know, this is the first PHB ligand that induces C-RAF activation. Such a reversal of activity is not unprecedented. Indeed, in our previous work on triazines, we developed PHB ligands that promote melanogenesis and other ones that inhibit it [START_REF] Djehal | Targeting prohibitin with small molecules to promote melanogenesis and apoptosis in melanoma cells[END_REF]. Paradoxically activating C-RAF signaling has been shown to induce the death of the Merkel cell carcinoma (MCC) cell line UISO [START_REF] Houben | Activation of the MAP kinase pathway induces apoptosis in the merkel cell carcinoma cell line UISO[END_REF], suggesting that C-RAF activators, such as 5d, hold some therapeutic potential to treat this highly aggressive type of cancer. In a more general manner, PHBs represent therapeutic targets for an ever-increasing number of diseases [START_REF] Wang | Prohibitin ligands: a growing armamentarium to tackle cancers, osteoporosis, inflammatory, cardiac and neurological diseases[END_REF], and in this context, the discovery of new classes of PHB ligands may provide the means to use targeted approaches for disorders involving PHBs.

Experimental part

Chemistry

Detailed synthetic methods can be found in SI Materials and Methods.

Biology

Cells

HeLa S3 (DSMZ) was authenticated by Eurofin genomics and cultured in DMEM (10 % heat inactivated FBS). HeLa cells were seeded at 2x10 5 cells/ml in 12 well cell culture plate and cultured for 1 day. HeLa cells were starved in the serum free medium with compounds for 1h followed by the stimulation with EGF (100 ng/ml) for 30 min. After stimulation, 150 µl of Laemmli buffer was added to the well and the sample were used for SDS-PAGE. MCF7 human breast cancer cells were obtained from RIKEN BioResource Center (Tsukuba, Japan) and cultured in DMEM supplemented with 10% fetal bovine serum (HyClone, South Logan, UT, USA) and 1% penicillin and streptomycin. Cells were maintained in 5% CO2 at 37°C. MCF7 cells were seeded at 5x10ˆ4 cells/well in 6 well cell culture plates and cultured for 1 day. Then, cells were treated with perfluorothiazolines (1, 5a-k, 1 µM) at 1 µM for 72h. A trypan blue dye exclusion test was performed using 0.4% trypan blue solution (Wako Pure Chemical Industries, Osaka, Japan) and a Countess II automated cell counter (Thermo Fisher Scientific, Waltham, MA, USA).

HBL melanoma cells (LOCE-MM001) were established in the LOCE (Laboratory of Oncology and Experimental Surgery) at the Institut J. Bordet-Université Libre de Bruxelles. HBL cells were grown in HAM's F10 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), and with Lglutamine, penicillin, and streptomycin at standard concentrations (Thermo Fisher Scientific) [START_REF] Wouters | Robust gene expression programs underlie recurrent cell states and phenotype switching in melanoma[END_REF][START_REF] Najem | Tyrosine-Dependent Phenotype Switching Occurs Early in Many Primary Melanoma Cultures Limiting Their Translational Value[END_REF].

SDS-PAGE and Western blotting

The samples were subjected to 10% SDS-PAGE followed by Western blotting. After transfer, the membrane was incubated with 3% BSA/TBST (20mM Tris-HCl, pH 7.5, 150mM NaCl, 0.05% Tween-20) for 1 h at room temperature followed by the incubation with primary antibody for overnight at 4˚C. After the overnight incubation, the membrane was washed with TBST 5 times and incubated with HRPconjugated secondary antibody in TBST for 1 h at room temperature. After the secondary antibody treatment, the membrane was washed and the signal was detected by chemiluminescence substrate (Millipore) and Chemi Doc touch (Bio-Rad). A list of antibodies is provided in SI Materials and Methods.

MTT assay

HeLa cells were seeded in 96 well plates at a concentration of 1x10 5 cells/ml. 100 µl cell suspension was added into 96 well cell culture plates with compounds (1.25-10 µM). 10 µl of MTT solution was added to the wells after 24 h and incubated for 4 h. After incubation with MTT, 100 µl solubilisation buffer was added and incubated overnight. MTT was measured at OD 570 nm.

Transfection of siRNA targeting p21

Synthesized siRNAs were purchased from RNAi (Tokyo, Japan) or Gene Design (Osaka, Japan). The siRNA sequences were as follows: non-targeting, 5'-GUACCGCACGUCAUUCGUAUC -3' (sense) and 5'-UACGAAUGACGUGCGGUACGU -3' (anti-sense); p21, 5'-CUGUACUGUUCUGUGUCUU -3' (sense) and 5'-AAGACACAGAACAGUACAG -3' (anti-sense). Transient transfection of siRNA targeting p21 was performed using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's protocol. Briefly, MCF7 cells were seeded in 6-well plates (5 × 10 4 cells / well), cultured for 24 h, and transfected with siRNAs at a concentration of 10 nM. At 24 h post-transfection, the cells were treated with 5b 1 μM or 5g 1 μM for 72 h, and then collected and analyzed with Western blot and a trypan blue dye exclusion test was performed using 0.4% trypan blue solution (Wako Pure Chemical Industries, Osaka, Japan) and a Countess II automated cell counter (Thermo Fisher Scientific, Waltham, MA, USA).

Analysis of cell visible pigmentation

A macroscopic evaluation of cell pigmentation revealed that eumelanogenesis was stimulated by fluorizoline analogues after ten days of treatment of HBL cells. Pellets consisting of cell debris and dark pigment were washed twice with PBS and were centrifuged again at 1200 g. The intensity of cell pigmentation was assessed using ImageJ.

Declaration of competing interest

Figure 1 .

 1 Figure 1. Mechanism of action of fluorizoline (1) in cancer cells and melanocytes. In cancer cells, fluorizoline upregulates the pro-apoptotic factors NOXA and BIM and inhibits the PHB-dependent activation of C-RAF. Fluorizoline also blocks the interaction of PHB2 with γ-glutamylcyclotransferase (GGCT) and its translocation to the nucleus to promote the expression of the cyclin-dependent kinase inhibitor p21. In melanocytes, fluorizoline activates the autophagy factor LC3-I into LC3-II to induce pigmentation of the cells.

Figure 2 .

 2 Figure 2. Effect of fluorizoline (1) and its analogues (5a-k) on RAS-MAPK activation. HeLa cells were treated with perfluorothiazolines (1, 5a-k, 10 µM) for 1 h and stimulated with EGF for 30 min. The cells were collected and analyzed by western blot to monitor the phosphorylation of C-RAF and MEK1/2. a No

Figure 3 .

 3 Figure 3. Cytotoxicity of fluorizoline (1) and its analogues (5a-k). HeLa cells were treated with perfluorothiazolines (1, 5a-k) for 24 h and harvested for MTT assay. Dot plots show the relative values of compounds treated cells against DMSO treated cells from 3 independent experiments. The bars represent mean ± SEM.

Figure 4 .

 4 Figure 4. Inhibition of MCF7 proliferation and induction of p21 expression by fluorizoline (1) and its analogues 5b-5k at 1 µM concentration. A. The number of trypan blue-negative viable cells is shown (* p<0.05,** p<0.01, *** p<0.001, T test vs DMSO). B. Expression of p21 analyzed by Western blot. MCF7 cells were treated with perfluorothiazolines (1, 5a-k, 1 µM) for 72 h. The cells were counted with a trypan blue staining and p21 expression was analyzed by Western blot analysis. C and D. p21 knockdown restores cell proliferation suppressed by 5b and 5g. MCF7 cells were transfected with siRNA targeting p21 or non-target control siRNA and treated with 1 µM of 5b and 5g for 48 h. C. The cells were counted with a trypan blue staining. (*P < 0.001 by 2-way repeated-measures ANOVA) D. The expression levels of p21 protein were analyzed by Western blot analysis. β-actin is shown as a loading control.

Figure 5 .

 5 Figure 5. Effect of fluorizoline analogs on cell pigmentation. HBL human melanoma cells were treated for ten days with fluorizoline (1) and its analogs (0.1 and 1 µM). All experiments were undertaken independently in duplicate. The melanin content was expressed as relative increase compared to vehicletreated HBL cells.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

Part of this work is supported through a grant (RA1739/7-1) from the DFG and CRC1292 to KR.


Dong Wang

Mustafa Tezeren

Hussein Abou-Hamdan

Peng Yu

Canan G Nebigil

Laurent Désaubry
email: desaubry@unistra.fr

Flavaglines: Discovery From Plants Used in Traditional Chinese Medicine, Synthesis and Drug Development against Cancer and Immune Disorders

Keywords: Natural products, Traditional Chinese Medicine, eIF4A, prohibitins, cancer, cardioprotection, total synthesis, medicinal chemistry

Flavaglines, a family of compounds coming from plants used in Traditional Chinese Medicine, exhibit a broad range of biological effects including anticancer, antiviral, cardioprotectant and anti-inflammatory activities. They exert their action by targeting the scaffold proteins called prohitins-1 and-2, and the mRNA helicases eIF4A and DDX3. Flavaglines are densely functionalized cyclopenta [b]benzofurans that have attracted the attention of some of the most eminent organic chemists. This review provides an overview of the biosynthesis, total synthesis and pharmacological activities of flavaglines, which recently culminated with the entrance of a synthetic derivative, Zotatifin, into clinical trials against advanced solid tumors refractory or intolerant to standard treatments.

INTRODUCTION

The plant genus Aglaia found in the tropical and subtropical rainforest of southern China, the Indo-Malayan region, some Pacific Islands and Northern Australia is characterized by benzo[b]oxepines, cyclopenta[b] benzofurans (also called flavaglines) and cyclopenta [bc] benzopyrans [START_REF] Harneti | Phytochemistry and biological activities of Aglaia species[END_REF]. These trees have a long history in traditional medicine. In China, the branches and leaves of Aglaia odorata are often decocted or made into externally applied ointment to treat pain in rheumatic joints, injuries from falls, superficial infections and toxic swellings. Its flowers are recommended against distress of chest and diaphragm, common cold and cough [START_REF] Heyne | The Useful Indonesian Plants, Research and Development Agency[END_REF]. The bark decoction of Aglaia elliptica, is used to treat tumors, whereas its flowers are applied to wounds [START_REF] Lemmens | Plant Resources of South-East Asia[END_REF].

In India, Aglaia roxburghiana is traditionally used in Ayurvedic medicine against inflammation, leprosy, throat infections, bilious and febrile complaint, and also to treat snake and scorpion bites [START_REF] Janaki | Anti-inflammatory activity of Aglaia roxburghiana var. beddomei extract and triterpenes roxburghiadiol A and B[END_REF][START_REF] Ayyanar | Traditional knowledge of Kani tribals in Kouthalai of Tirunelveli hills, Tamil Nadu, India[END_REF]. Aglaia elaeagnoidea and Aglaia Lawii are used to treat diarrhea, skin diseases, bacterial infection, headaches, liver diseases and tumours [START_REF] Khare | Indian Herbal Remedies[END_REF].

In Thailand, Aglaia odorata is used as a tonic, febrifuge and a remedy for menorrhagia during the menopause [START_REF] Division | Traditional medicine textbook (Thai drug)[END_REF].

Rocaglamide (1, Figures 1 and2) was isolated in 1982 from a plant used in Traditional Chinese Medicine (TCM), Aglaia elliptifolia by Taiwanese scientists, King and collaborators [START_REF] King | X-ray crystal structure of rocaglamide, a novel antileukemic 1Hcyclopenta[b]benzofuran from Aglaia elliptifolia[END_REF]. These authors identified the structure of rocaglamide and demonstrated its anticancer activity in a murine model of leukemia. Since then, more than 100 flavaglines have been isolated from Aglaia species, and recently from trees of the gender Dysoxylum [START_REF] Harneti | Phytochemistry and biological activities of Aglaia species[END_REF][START_REF] Greger | Comparative phytochemistry of flavaglines (= rocaglamides), a group of highly bioactive flavolignans from Aglaia species (Meliaceae)[END_REF][START_REF] Yang | Discovery and synthesis of rocaglaol derivatives inducing apoptosis in HCT116 cells via suppression of MAPK signaling pathway[END_REF][START_REF] Schulz | Rocaglamide and silvestrol: a long story from anti-tumor to anti-coronavirus compounds[END_REF][START_REF] Pan | Rocaglamide, silvestrol and structurally related bioactive compounds from Aglaia species[END_REF]. In the 1980's, this compound and some of its natural analogs were found to delay tumor growth in mice at the National Cancer Institute. But, as they did not eradicate these tumors, this investigation was interrupted.

In 2004, Marian and collaborators dubbed "flavaglines" this family of flavonol-cinnamate-derived cyclopenta[b] benzofurans as they stem for the metabolism of flavonoids in plants of the genus Aglaia. Importantly, this team discovered that flavaglines induce the death of cancer cells without affecting normal cells [START_REF] Hausott | Flavaglines: a group of efficient growth inhibitors block cell cycle progression and induce apoptosis in colorectal cancer cells[END_REF].

Thereafter, flavaglines were found to display antiinflammatory, neuro-and cardioprotectant activities, suggesting that they not only display anticancer properties but also protect the organism against the adverse effect of chemotherapies.

The discovery in 2008 and 2012 that these drugs target the initiation factor of protein synthesis eIF4A [START_REF] Bordeleau | Therapeutic suppression of translation initiation modulates chemosensitivity in a mouse lymphoma model[END_REF][START_REF] Cencic | Antitumor activity and mechanism of action of the cyclopenta[b]benzofuran, silvestrol[END_REF][START_REF] Chambers | Synthesis of biotinylated Episilvestrol: highly selective targeting of the translation factors eIF4AI/II[END_REF][START_REF] Sadlish | Evidence for a functionally relevant rocaglamide binding site on the eIF4A-RNA complex[END_REF] and prohibitins (PHBs) [START_REF] Polier | The Natural Anticancer Compounds Rocaglamides Inhibit the Raf-MEK-ERK Pathway by Targeting Prohibitin 1 and 2[END_REF] greatly stimulated this field of research, leading to the advancement in 2019 of a synthetic flavagline, zotatifin (also called eFT226) into of phase 1/2 clinical trial against advanced solid tumors refractory or intolerant to standard treatments [START_REF] Ernst | Design of Development Candidate eFT226, a First in Class Inhibitor of Eukaryotic Initiation Factor 4A RNA Helicase[END_REF]. Next year flavaglines were found to also target the mRNA helicase DDX3 [START_REF] Chen | Dual targeting of DDX3 and eIF4A by the translation inhibitor rocaglamide A[END_REF].

BIOSYNTHESIS

The coexistence of cinnamic amides 12, aglaforbesins 15, aglains 16 and flavaglines in the plants of the genus Aglaia, led Proksch and coll. to propose the biosynthetic pathway presented in Scheme 1 [START_REF] Nugroho | An insecticidal rocaglamide derivatives and related compounds from Aglaia adorata (Meliaceae)[END_REF]. A [3 + 2] condensation between a hydroxyflavone 11 and a cinnamic amide 12 generates benzo[b]oxepines 13, which can be reduced to diols 16 or undergo acyloin rearrangement to afford flavaglines 17. The presence of flavaglines in these plants probably stems from their potent insecticide activity [START_REF] Nugroho | An insecticidal rocaglamide derivatives and related compounds from Aglaia adorata (Meliaceae)[END_REF][START_REF] Ishibashi | Instecticidal 1H-cyclopenta[b]benzofurans from Aglaia odorata[END_REF][START_REF] Nugroho | Insecticidal rocaglamide derivatives from Aglaia duppereana[END_REF][START_REF] Greger | Insecticidal flavaglines and other compounds from Fijian Aglaia species[END_REF]. Scheme 1. Biosynthesis of flavaglines proposed by Proksch and coll. [START_REF] Nugroho | An insecticidal rocaglamide derivatives and related compounds from Aglaia adorata (Meliaceae)[END_REF].

TOTAL SYNTHESES

The densely functionalized tricyclic core of flavaglines represents a considerable challenge that was first taken up by Barry Trost [START_REF] Trost | An unusual oxidative cyclization. A synthesis and absolute stereochemical assignment of (-)rocaglamide[END_REF]. Since then, other approaches have been described and recently reviewed [START_REF] Zhao | Recent Advances in the Synthesis of Flavaglines, a Family of Potent Bioactive Natural Compounds Originating from Traditional Chinese Medicine[END_REF]. However, only two strategies have been extensively used in medicinal chemistry Scheme 2. Dobler's racemic synthesis of flavaglines [START_REF] Dobler | Total synthesis of (+/-)-rocaglamide and some aryl analogues[END_REF].

programs. The first of these particularly practical approaches was originally developed by Richard Taylor and improved by Markus Dobler (Scheme 2) [START_REF] Dobler | Total synthesis of (+/-)-rocaglamide and some aryl analogues[END_REF][START_REF] Davey | A novel 1,3-dithianebased cyclopenta-annellation procedure: Synthesis of the rocaglamide skeleton[END_REF][START_REF] Davey | Synthesis of the novel anti-leukaemic tetrahydrocyclopenta[b]benzofuran, rocaglamide and related synthetic studies[END_REF]. It is based on a Michael reaction between a benzofuranone 18 and cinnamaldehyde, followed by the formation of a cyanohydrin 21 that can be deprotonated to react with the ketone and provide the flavagline backbone.

The second approach, inspired by the biosynthesis of flavaglines in plants, was developed by John Porco (Scheme 3) [START_REF] Gerard | A biomimetic approach to the rocaglamides employing photogeneration of oxidopyryliums derived from 3hydroxyflavones[END_REF][START_REF] Stone | Biomimetic kinetic resolution: highly enantio-and diastereoselective transfer hydrogenation of aglain ketones to access flavagline natural products[END_REF][START_REF] Wang | Synthesis of aza-rocaglates via ESIPT-Mediated [3+2] photocycloaddition[END_REF][START_REF] Yueh | A photochemical flow reactor for large scale syntheses of aglain and rocaglate natural product analogues[END_REF]. UV irradiation of a 3-hydroxyflavone 11 in the presence of a cinnamic ester or amide generates a photoexcited triplet biradical 24 capable of performing a [3 + 2] cycloaddition, to afford the aglain backbone [START_REF] Trost | An unusual oxidative cyclization. A synthesis and absolute stereochemical assignment of (-)rocaglamide[END_REF]. In the presence of a base, this adduct undergoes an α-acyloin rearrangement to generate the flavagline 26. Scheme 3. Porco's biomimetic synthesis of flavaglines [START_REF] Stone | Biomimetic kinetic resolution: highly enantio-and diastereoselective transfer hydrogenation of aglain ketones to access flavagline natural products[END_REF].

Ragot and coll. at Bayer developed also a short synthesis of bis-demehoxyrocaglaol 31 that uses the intramolecular ring closure of epoxide 30 as a key step (Scheme 4) [START_REF] Thede | Stereoselective Synthesis of (±)-Rocaglaol Analogues[END_REF].

Unfortunately, this compound is not pharmacologically active, and the synthesis of rocaglaol (2) from cyclopentanone 29 was not reported. Scheme 4. Bayer synthesis of bis-demethoxyrocaglaol 31, a pharmacologically inactive analogue of rocaglaol (2) [START_REF] Thede | Stereoselective Synthesis of (±)-Rocaglaol Analogues[END_REF].

As part of our medicinal program to develop flavaglines with improved pharmacological properties, we considered using this strategy to prepare new derivatives. For this purpose, we developed two efficient syntheses of the cyclopentenone 37 substituted by the different aryl moieties necessary for the pharmacological activity (Scheme 5). The first approach lied on a gold(I)-catalyzed cyclopentanone formation that was originally described with tertiary silyl ethers [START_REF] An | Gold(I)-Catalyzed Synthesis of Highly Substituted 2-Cyclopentenones from 5-Siloxypent-3en-1-ynes[END_REF]. This synthesis began with a Perkin condensation of acid 32 and benzaldehyde followed by the conversion to an acyl chloride and a Sonogashira coupling that afforded ketone 33 as a sole E isomer [START_REF] Basmadjian | Exploratory studies toward a synthesis of flavaglines. A novel access to a highly substituted cyclopentenone intermediate[END_REF]. Condensation with lithiated trimethoxybenzene, molybdenum (VI)-catalyzed transposition and etherification of allylic alcohol 34 conveniently furnished the allylic ether 35. Gratifyingly, gold(I)-catalyzed annulation of alkyne 36 afforded the cyclopentenone 37 in 63% yield.

During the development of this synthesis, we serendipitously found that Re 2 O 7 efficiently catalyzes the formation of cyclopentenone 37 directly from styryl-propargyl alcohol 34 [START_REF] Basmadjian | Exploratory studies toward a synthesis of flavaglines. A novel access to a highly substituted cyclopentenone intermediate[END_REF]. This type of reaction has not been described before. It represents an interesting alternative to the Rautenstrauch rearrangement. Selective monodemethylation using BBr 3 efficiently afforded phenol 38 in a 75% yield (Scheme 5). Unfortunately, all our attempts to construct the flavagline scaffold from this compound, including variation around the strategy developed by Ragot and coll. were unsuccessful. This explains also why these authors didn't use this method to synthesize compounds substituted with the methoxys groups necessary for a pharmacological activity. Scheme 5. Gold and rhenium-catalyzed formation of cyclopentanone 38 and failed attempt to prepare rocaglaol (2) [START_REF] Basmadjian | Exploratory studies toward a synthesis of flavaglines. A novel access to a highly substituted cyclopentenone intermediate[END_REF].

These two methoxys were also found to modulate the chemical reactivity of flavaglines. Thus, we found that acylation of amine 39 with dimethylcarbamoyl chloride at reflux in dichloromethane afforded a mixture of carbamates 45 and 46 in a 83/17 ratio (Scheme 6) [START_REF] Abou-Hamdan | Unexpected Inversion of Configuration During the Carbamoylation of 1-Azaflavaglines[END_REF]. This unusual inversion of configuration of an aminoalcohol was not observed with flavagline 47. The mechanism of this reaction probably involves the benzylic carbocation 40, which may either afford the styrylurea 43 or undergo a ring closure to form the oxazolidinone dimethyliminium salt 41. The latter may also undergo a rearrangement into 43. Upon protonation and hydration 43 may generate the hydroxyureas 45 and 46 via the carbocations 42 and 44, with a strong preference for the thermodynamically more stable isomer 45.

This possibility to generate a stabilized carbocation was exploited to introduce an azide in position 1a from the cyclic sulfite 50 (Scheme 7) [START_REF] Zhao | Bioisosteric modification of flavaglines[END_REF]. Reduction and acylation thereof provided the formamide 52 and the sulfonamide 53, which, unfortunately, were found to be pharmacologically inactive. Scheme 6. Carbamoylation of flavaglines 39 and 47 [START_REF] Zhao | Bioisosteric modification of flavaglines[END_REF] . Scheme 7. Synthesis of 1-azaisosteres 52 and 53 [START_REF] Zhao | Bioisosteric modification of flavaglines[END_REF].

MECHANISM OF ACTION AND PHARMACOLOGICAL ACTIVITIES

Flavaglines mediate their biological activities by inhibiting the mRNA helicases eIF4A and DDX3 and also by modulating the activity of the scaffold proteins prohibitins-1 and [2 (PHB1/2) (Figure 3). eIF4A is a helicase that unwinds strongly structured mRNA. Only a small number of mRNAs, which mainly code for proteins involved in cancer, need this helicase for their translation. Flavaglines stabilize eIF4A complexed to mRNAs, which prevents the liberation and recycling of eIF4A, leading to an inhibition of its activity [START_REF] Chu | Rocaglates induce gain-of-function alterations to eIF4A and eIF4[END_REF]. Importantly, this inhibition seems to affect every cancer cell type, including persister cells that usually tolerate anticancer drugs and are at the origin of acquired resistance and cancer relapse. The inhibition of eIF4A also relieves the resistance to BRAF and MEK inhibitors in a murine model of metastatic melanoma, prevents the immune escape of tumor cells by blocking inhibitory checkpoints, abrogate taxane resistance in prostate cancer and synergizes with MEK inhibitors to kill NRAS-mutant cancer cell lines.

Recently, using the O-nitrobenzoxadiazole-conjugated derivative of rocaglamide 54, Iwasaki and coll. found that flavaglines also target the helicase DDX3 [START_REF] Chen | Dual targeting of DDX3 and eIF4A by the translation inhibitor rocaglamide A[END_REF]. Indeed this probe was found to perform a proximity-specific fluorescence labeling of both eIF4A and DDX3 (Figure 4). Like eIF4A, DDX3 belongs to the DEAD-box helicase family, which is quite conservative, explaining why flavaglines inhibit both of these proteins that are attractive targets against cancers.

In addition to these helicases, flavaglines also target prohibitins-1 and -2 (PHB1/2). These scaffold proteins exists as two isoforms in the animal kingdom where they regulate the activity of a myriad of signaling proteins [START_REF] Hernando-Rodríguez | Mitochondrial quality control mechanisms and the PHB (Prohibitin) complex[END_REF][41][42][START_REF] Mishra | Prohibitin -At the crossroads of obesity-linked diabetes and cancer[END_REF].

PHBs are characterized by a stomatin/prohibitin/flotillin/ HflK/HflC (SPFH) domain that is present in an evolutionarily conserved family of proteins in archaea, bacteria and eukaryotes [START_REF] Wang | SFPH proteins as therapeutic targets for a myriad of diseases[END_REF]. Thus, through their actions on PHBs, flavaglines inhibit the activation of KRAS and C-RAF, to block the C-RAF/MEK/ERK pathway critical to the survival of many cancer cell types [START_REF] Yurugi | Targeting prohibitins with chemical ligands inhibits KRAS-mediated lung tumours[END_REF][START_REF] Yurugi | A subset of flavaglines inhibits KRAS nanoclustering and activation[END_REF]. In bladder cancer, flavaglines inhibit PHB1 phosphorylation by Akt, leading to removal of PHB1 in mitochondria and also upregulate GADD45α to arrest cell cycle progression at the G2/M phase [START_REF] Yuan | Flavagline analog FL3 induces cell cycle arrest in urothelial carcinoma cell of the bladder by inhibiting the Akt/PHB interaction to activate the GADD45α pathway[END_REF]. They also promote the expression of Axin 1 to inhibit Wnt/β-catenin signaling and combat intestinal tumorigenesis [START_REF] Jackson | The Synthetic Small Molecule FL3 Combats Intestinal Tumorigenesis via Axin1-Mediated Inhibition of Wnt/beta-Catenin Signaling[END_REF]. Moreover, flavaglines block mitophagy and energy productions by inhibiting the effects of PHB2 on the mitochondrial inner protease PARL and LC3-II and by disrupting the complex PHB/SH2D4/STAT3 in mitochondria [START_REF] Ploeger | Prohibitin, STAT3 and SH2D4A physically and functionally interact in tumor cell mitochondria[END_REF]. Indeed, they prevent the interleukine-6induced phosphorylation of STAT3 leading to a reduction STAT3 transcriptional activity and HIF1α stabilization in hepatocellular carcinoma.

Flavaglines can also induce apoptosis of cancer cells by activating AIF, and caspase-8 through yet uncharacterized mechanisms.

In addition to their antitumor effects, flavaglines protect the heart of mice against the adverse effects of doxorubicin, a widely used anticancer medicine that induces a severe cardiotoxicity in patients. This cardioprotective effect is also mediated by prohibitins: flavaglines induce a cardioprotection by inducing a translocation of PHB1 and STAT3 to mitochondria, a phosphorylation of HSP27 and an overexpression of PHB1 in cardiomyocytes [START_REF] Ribeiro | Flavaglines as Potent Anticancer and Cytoprotective Agents[END_REF][START_REF] Qureshi | FL3, a Synthetic Flavagline and Ligand of Prohibitins, Protects Cardiomyocytes via STAT3 from Doxorubicin Toxicity[END_REF] Altogether, these data suggest that flavaglines could improve the efficacy of chemotherapies and alleviate in the same time their adverse effects.

In line with the use of Aglaia extracts in traditional Chinese medicine to treat inflammatory diseases, flavaglines protect mice in a model of Crohn's disease in which inflammation is driven by epithelial ulceration of the colon [START_REF] Han | Flavaglines Ameliorate Experimental Colitis and Protect Against Intestinal Epithelial Cell Apoptosis and Mitochondrial Dysfunction[END_REF]. Indeed, FL3 (6) decreased the inflammation of the colon and in the same time promoted the survival of the intestinal epithelial cells against the inflammatory stress.

Through their effects on eIF4A and/or PHBs, flavaglines have been shown to display potent antiviral activities against several types of viruses, including Chikungunya virus and coronaviruses [START_REF] Taroncher-Oldenburg | Targeting the DEAD-Box RNA helicase eIF4A with rocaglates-A pan-antiviral strategy for minimizing the impact of future RNA virus pandemics[END_REF][START_REF] Müller | The rocaglate CR-31-B (-) inhibits SARS-CoV-2 replication at noncytotoxic, low nanomolar concentrations in vitro and ex vivo[END_REF][START_REF] Mueller | Comparison of broad-spectrum antiviral activities of the synthetic rocaglate CR-31-B (-) and the eIF4A-inhibitor Silvestrol[END_REF][START_REF] Liu | A Novel Class of Small Molecule Compounds that Inhibit Hepatitis C Virus Infection by Targeting the Prohibitin-CRaf Pathway[END_REF][START_REF] Zhang | Chemical Synthesis Enables Structural Reengineering of Aglaroxin C Leading to Inhibition Bias for Hepatitis C Viral Infection[END_REF][START_REF] Zhou | Requirement of the eukaryotic translation initiation factor 4F complex in hepatitis E virus replication[END_REF].

DEVELOPMENT OF ZOTATIFIN (EFT226)

Founded in 2012 in California, eFFECTOR Therapeutics is a pharmaceutical company that developed the first flavagline to enter clinical trials [START_REF] Ernst | Design of Development Candidate eFT226, a First in Class Inhibitor of Eukaryotic Initiation Factor 4A RNA Helicase[END_REF]. When this project started, many patents had been filed by academic laboratories and several pharmaceutical companies (Bayer and its spin-off, IMD Natural Solutions (Germany), Infinity Pharmaceuticals (USA) and Pierre Fabre (France)) [START_REF] Fahrig | A synthetic derivative of the natural product rocaglaol is a potent inhibitor of cytokine-mediated signaling and shows neuroprotective activity in vitro and in animal models of Parkinson's disease and traumatic brain injury[END_REF][START_REF] Michael | Fox Foundation for Parkinson's Research. IMD-026259: an innovative drug for diseasemodifying treatment of Parkinson's disease[END_REF][START_REF] Liu | Synthetic Silvestrol Analogues as Potent and Selective Protein Synthesis Inhibitors[END_REF][START_REF] Marion | Flavagline derivatives[END_REF]. To generate some intellectual property, the chemists at eFFECTOR came up with the idea of replacing one of the carbons in the flavagline backbone with a nitrogen. In the course of their investigations, they noticed that compounds with a 40-degree aryl-aryl torsion as a preferred low-energy conformation tend to display higher potency (Figure 5). This observation has been used prospectively to select the compounds to synthesize. The isostere 56 with a nitrogen in position 8 was found to be 25-fold less cytotoxic than rocaglamide in MDA-MB-231 breast cancer cells (Figure 6). In accordance with a previous report by another biotech company, Infinity Pharmaceuticals [START_REF] Liu | Synthetic Silvestrol Analogues as Potent and Selective Protein Synthesis Inhibitors[END_REF], the replacement of the methoxy in position 4' by a nitrile (57) enhanced the activity. Shifting the nitrogen and the methoxy (57→58) improved further the cytotoxicity, but at the expense of CACO permeability and solubility. These two problems could be overcome by reducing the tertiary amide to a dimethylamine 59. By shifting the nitrogen to position 7 and reinstalling the methoxy that had been removed, a compound as cytotoxic as rocaglamide with improved solubility and permeability was identified. This compound, eFT226 [START_REF] Yang | Discovery and synthesis of rocaglaol derivatives inducing apoptosis in HCT116 cells via suppression of MAPK signaling pathway[END_REF], has successfully passed US regulatory preclinical requirements and entered phase 1/2 clinical trial against advanced solid tumors refractory or intolerant to standard treatments, under the name zotatifin [START_REF] Ernst | Design of Development Candidate eFT226, a First in Class Inhibitor of Eukaryotic Initiation Factor 4A RNA Helicase[END_REF]. To develop this clinical candidate, more than 20 000 flavagline derivatives were synthesized.

Thanks to the basic dimethylamino side chain, eFT226 is much more soluble than rocaglamide (>20 mg/mL), which greatly facilitates intravenous administration. eFT226 (1 mg/kg, intravenous administration) inhibited tumour growth in a MDA-MB-231 triple negative breast cancer orthotopic xenograft model treated once weekly for 2 weeks. Biophysical studies demonstrated that eFT226 enhances the binding of polypurine RNA motifs to eIF4A independently of ATP similarly to eIF4A1. The mutation of phenylalanine 163 to leucine, which inhibits the binding of eIF4A to flavaglines, reduced the antiproliferative effects of eFT226 by a factor of 60, demonstrating that eIF4A is the main target to mediate its anticancer properties. Up to now, the effect of this compound on PHB signaling has not been reported. Zotatifin is not expected to be efficient against every type of tumors. eFFECTOR's scientist found that this drug is particularly effective in cancers where the kinase mTOR is driving mRNA translation, such as diffuse large B-cell lymphoma (DLBCL), and Burkitt lymphoma. In mouse models of these two types of lymphoma, eFT226 displayed impressive anticancer activities at low doses (1 mg/kg/week i.v.) [START_REF] Thompson | Targeting Oncogene mRNA Translation in B-Cell Malignancies with eFT226, a Potent and Selective Inhibitor of eIF4A[END_REF].

The synthesis of zotatifin was achieved using Porco's biomimetic synthesis to construct the flavagline scaffold prepared by lithiation of pyridine derivative 61 using classical reactions.

Conclusion and perspective

Since their discovery in 1982; flavaglines have attracted the attention of many organic chemists due to the challenge posed by the complexity of their structure. So far only two Scheme 8. Synthesis of zotatifin, ((-)-eFT226) [START_REF] Ernst | Design of Development Candidate eFT226, a First in Class Inhibitor of Eukaryotic Initiation Factor 4A RNA Helicase[END_REF]. methods are used in medicinal chemistry: that of Taylor and Dobbler, and that of Porco.

Regarding the pharmacological studies of these compounds, these are dominated by their anticancer effects, with as a culmination the entry of Zotatifin in clinical trials against advanced solid tumors refractory or intolerant to standard treatments. In this area, the major obstacle is to identify how to combine flavaglines with other drugs to optimize their anticancer effects in patients. Although in vivo cytoprotective, antiviral, anti-inflammatory and immunosuppressive effects have been demonstrated, the exploration of these effects should increase in the coming years, even if it remains marginal for the moment compared to studies in oncology.

In addition to their potential, flavaglines have been shown to be useful as chemical tools to explore the function of prohibitins and eIF4A. To our knowledge, the involvement of these targets in the mechanism of action of traditional medicine preparations incorporating Aglaia extract has not yet been reported.

Figure 2 .

 2 Figure 2. Representative examples of natural (1-5) and synthetic (6-10) flavaglines.

Figure 1 .

 1 Figure 1. Major milestones achieved in the development of flavaglines as clinical candidates.

Figure 4 .

 4 Figure 4. Proximity-specific fluorescence labeling of the mRNA helicases eIF4A and DDX3 [20].

Figure 5 .

 5 Figure 5. Conformational analysis. At the lowest energy conformation, ϴ = 40° is optimal for the inhibition of eIF4A [19].

Figure 6 .

 6 Figure 6. Summary of the development of eFT226 (Zotatifin) from rocaglamide by eFFECTOR scientists [19].

ACKNOWLEDGEMENTS

We acknowledge the financial support from the National Natural Science Foundation of China (No. 81673296).

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.