WO2010060891A1 - Rocaglaol derivatives as cardioprotectant agents and as antineoplastic agents - Google Patents

Rocaglaol derivatives as cardioprotectant agents and as antineoplastic agents Download PDF

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Publication number
WO2010060891A1
WO2010060891A1 PCT/EP2009/065698 EP2009065698W WO2010060891A1 WO 2010060891 A1 WO2010060891 A1 WO 2010060891A1 EP 2009065698 W EP2009065698 W EP 2009065698W WO 2010060891 A1 WO2010060891 A1 WO 2010060891A1
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hydrogen
methoxy
group
methyl
bromine
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PCT/EP2009/065698
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French (fr)
Inventor
Laurent Desaubry
Gunsel Canan Desaubry
Thierry Cresteil
Gülen TÜRKERI
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Universite De Strasbourg
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Priority to JP2011537954A priority Critical patent/JP2012509923A/en
Priority to EP09756321A priority patent/EP2358694A1/en
Priority to US13/130,140 priority patent/US20120101153A1/en
Priority to CA2744309A priority patent/CA2744309A1/en
Publication of WO2010060891A1 publication Critical patent/WO2010060891A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D307/93—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems condensed with a ring other than six-membered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention relates to the prevention of antineoplastic agent cardiotoxicity by using new cardioprotectant agents.
  • the present invention also relates to new antineoplastic agents.
  • Cardiotoxicity is a major side effect of various antineoplastic agents including, for instance, anthracyclines, cyclophosphamide, fluorouracil, rituximab, cisplatine, gleevec or herceptin (Pai et al, 2000).
  • anthracyclines are a class of chemotherapeutic agents which is used to treat a wide range of cancers, including, for instance, leukemias, lymphomas, and breast, uterine, ovarian, and lung cancers.
  • anthracycline anticancer agents especially doxorubicin
  • doxorubicin anthracycline anticancer agents
  • This cardiotoxicity often presents as electrocardiogram changes and arrhythmias, or as a cardiomyopathy leading to congestive heart failure, sometime many years after treatment (Steinherz et al., 1995), and is related to a patient's cumulative lifetime dose.
  • Some recent studies have revealed that when the cumulative dose of doxorubicin reaches 700 mg/m 2 , the risks of developing congestive heart failure dramatically increase to 48% (Swain et al., 2003).
  • Dexrazoxane is then usually used in conjunction with doxorubicin as a cardioprotectant agent to reduce the risk of cardiotoxicity side effects of anthracycline chemotherapy.
  • doxorubicin As a derivative of EDTA, dexrazoxane chelates iron, but the precise mechanism by which it protects the heart is not known.
  • dexrazoxane does not provide a full protection against heart damages and some studies revealed that patients treated with it might have a lower anti-tumour response rate to the anthracycline treatment (Van Dalen, et al., 2005; Van Dalen, et al., 2008). Moreover, the effectiveness of dexrazoxane in children is not established yet. Therefore, there is a strong need for cardioprotectant agents that can efficiently prevent the cardiotoxicity of anthracyclines or other antineoplastic agents without having any deleterious effect on the anti-tumour response. This need is still stronger for children who are more susceptible to cardiotoxicity of these agents.
  • the object of the present invention is to provide new cardioprotectant agents.
  • the inventors have shown that rocaglaol and some of its derivatives are able to prevent or limit apoptosis induced by the cardiotoxicity of antineoplastic agents, without any deleterious effect on the anti-tumor activity of these agents.
  • the invention concerns a rocaglaol derivative of the formula (II)
  • R 1 ! is alkoxy, optionally substituted;
  • R 12 is hydrogen; or R 11 and R 12 together form a -0-CH 2 -O- unit;
  • R 13 is selected from hydrogen and (Ci-C3)-alkoxy
  • R 15 is selected from hydrogen, -COOR 21 , -CONR 22 R 23 and -C0NH(CH 2 ) n 0H, wherein n is 2, 3 or 4, wherein
  • R 21 and R 23 are independently selected from hydrogen and (Ci-C 3 )-alkyl and R 22 is selected from the group consisting of (Ci-C3)-alkoxy, (Ci-C3)-alkyl and hydrogen, or
  • R 22 and R 23 together form a 5 atom heterocycle, optionally substituted by a group NHCO-(Ci-C 4 )-alkyl; or R 14 , R 15 and carbons ( ⁇ ) and ( ⁇ ) bearing R 14 and R 15 together form wherein R 24 and R 25 are independently selected from the group consisting of (C 1 -C 3 )- alkyl and hydrogen, preferably from hydrogen and methyl; R 16 is selected from hydrogen, (Ci-C3)-alkoxy and halogen; R 17 is in ortho-position to Ri 6 and is selected from hydrogen, (Ci-C3)-alkoxy and hydroxyl or form together with R 17 a -0-CH 2 -O- unit;
  • R 18 and R 19 are independently selected from the group consisting of hydrogen and halogen, preferably from hydrogen, F, Br and Cl, more preferably are hydrogen or F; or any pharmaceutically acceptable salt thereof, for use for preventing or limiting the cardiotoxicity of an antineoplastic agent, preferably an anthracycline, more preferably doxorubicin.
  • the invention also concerns a method for preventing or limiting the cardiotoxicity of an antineoplastic agent in a subject comprising administering a therapeutically effective amount of a rocaglaol derivative of the formula (II) as described above or any pharmaceutically acceptable salt thereof to said subject.
  • the present invention further concerns the use of a rocaglaol derivative of the formula (II) as described above or any pharmaceutically acceptable salt thereof for preparing a medicament for preventing or limiting the cardiotoxicity of an antineoplastic agent, preferably an anthracycline, more preferably doxorubicin.
  • the rocaglaol derivative of the formula (II) has one or several of the following features: a) R 12 is H and R 11 is a (Ci-C3)-alkoxy, preferably selected from methoxy and ethoxy, more preferably R 11 is methoxy; b) R 13 is selected from hydrogen, methoxy and ethoxy, more preferably R 13 is hydrogen or methoxy; c) R 14 is selected from hydroxyl, -OCOR 20 with R 20 being selected from hydrogen, methyl and ethyl, -NH-(CH 2 ) m -R 27 , wherein m is 0 and R 27 is -SO 2 Me or -COR 28 with R 28 being H or (Ci-C 3 )-alkyl, preferably H, methyl or ethyl, more preferably H or methyl; d) R 15 is selected from hydrogen, -COOR 21 , and -CONR 22 R 23 , wherein R 21 and R 23 are independently
  • R 14 is selected from hydroxyl and -OCOR 20 with R 20 being selected from hydrogen, methyl and ethyl.
  • R 11 is alkoxy, especially methoxy or ethoxy, more preferably methoxy
  • R 12 is hydrogen
  • R 13 is selected from hydrogen, methoxy and ethoxy, more preferably R 13 is hydrogen or methoxy
  • R 15 is selected from hydrogen, COOMe, CONH 2 , CONHMe and CONMe 2
  • R 16 is selected from the group consisting of hydrogen, chlorine, bromine, fluorine and methoxy, more preferably of bromine and methoxy
  • R 18 is in para and is H or fluorine
  • R 17 and R 19 are hydrogen.
  • the rocaglaol derivative has the formula (lib) and R 14 is -NH-(CH 2 ) m -R 27 , wherein m is O and R 27 is -SO 2 Me or -COR 28 with R 28 being H or (Ci-C 3 )-alkyl, preferably H or methyl.
  • R 11 is alkoxy, especially methoxy or ethoxy, more preferably methoxy;
  • R 12 is hydrogen;
  • R 13 is selected from hydrogen, methoxy and ethoxy, more preferably R 13 is hydrogen or methoxy, still more preferably R 13 is methoxy;
  • R 15 is selected from hydrogen, COOMe, CONH 2 , CONHMe and CONMe 2 , more preferably is hydrogen;
  • R 16 is selected from the group consisting of hydrogen, chlorine, bromine, fluorine and methoxy, more preferably is chlorine, bromine, or fluorine, still more preferably is bromine; and, R 17 , R 18 and R 19 are hydrogen.
  • the rocaglaol derivative is selected from the group consisting of FLl, FL3, FL5, FL6, FL7, FL8, FL9, FLlO, FL12, FL13, FL14, FL15, FL16, FL17, FL18, FL19, FL20, FL21, FL22, FL23, FL24 and FL25, more preferably from the group consisting of FLl, FL3, FL5, FL8, FL9, FLlO, FL12, FL13, FL14, FL15, FL16, FL17, FL18, FL19, FL20, FL21, FL22, FL23 and FL25.
  • the invention provides new rocaglaol derivatives of the formula (I)
  • R 3 is -CONH 2 ;
  • R 1 is alkoxy, optionally substituted, preferably selected from the group consisting of methoxy and a group -O-(CH 2 ) n -R 10 wherein n is 1, 2, 3 or 4 and R 10 is selected from the group consisting of hydroxyl, -NMe 2 , -OCONMe 2 , -OCONH 2 and morpholine;
  • R 2 is selected from the group consisting of hydrogen and (Ci-C3)-alkoxy, preferably from hydrogen and methoxy; and, R 4 is halogen, preferably bromine, chloride, iodide, more preferably bromine, or,
  • R 1 is alkoxy, optionally substituted, preferably selected from the group consisting of methoxy and a group -O-(CH 2 ) n -R 10 wherein n is 1, 2, 3 or 4 and R 10 is selected from the group consisting of hydroxyl, -NMe 2 , -OCONMe 2 , -OCONH 2 and morpholine;
  • R 2 is hydrogen
  • R 4 is selected from the group consisting of hydrogen and alkoxy, or, R 1 is substituted alkoxy, preferably a group -O-(CH 2 ) n -R 10 wherein n is 1, 2, 3 or
  • R 4 and R 10 is selected from the group consisting of hydroxyl, -NMe 2 , -OCONMe 2 , - OCONH 2 and morpholine;
  • R 2 is (Ci-C3)-alkoxy, preferably methoxy; and
  • R 4 is selected from the group consisting of hydrogen and alkoxy, or wherein
  • R 3 is -COOR 5 wherein R 5 is (Ci-C 3 )-alkyl, preferably methyl;
  • R 1 is alkoxy, optionally substituted, preferably selected from the group consisting of methoxy and a group -O-(CH 2 ) n -R 10 wherein n is 1, 2, 3 or 4 and R 10 is selected from the group consisting of hydroxyl, -NMe 2 , -OCONMe 2 , -OCONH 2 and morpholine;
  • R 2 is hydrogen
  • R 4 is selected from the group consisting of hydrogen, methoxy and halogen; or wherein
  • R 1 is methoxy, and R 4 is bromine, and wherein
  • R 2 is (Ci-C3)-alkoxy, preferably methoxy, and, R 3 is hydrogen, or
  • R 2 is hydrogen, and, R 3 is selected from the group consisting of hydrogen, -COOR 5 and -CONR 6 R 7 , wherein R 5 is (Ci-C 3 )-alkyl, preferably methyl, and R 6 and R 7 are independently selected from the group consisting of hydrogen and (Ci-C 3 )-alkyl, preferably from hydrogen and methyl; or any pharmaceutically acceptable salt thereof.
  • R 1 is selected from the group consisting of methoxy
  • R 2 is hydrogen
  • R 3 is selected from the group consisting of -COOR 5 and -CONH 2 , wherein R 5 is methyl
  • R 4 is halogen or methoxy, preferably bromine or methoxy.
  • R 1 is methoxy; R 2 is selected from the group consisting of methoxy or hydrogen; R 3 is -CONH 2 ; and, R 4 is halogen, preferably bromine. In a further preferred embodiment, R 1 and R 2 are methoxy, R 3 is hydrogen and R 4 is bromine. In an additional preferred embodiment, R 1 is methoxy; R 2 is hydrogen; R 3 is selected from the group consisting of hydrogen, -COO(CH 3 ) and -CON(CH 3 ) 2 ; and, R 4 is bromine. In another preferred embodiment, R 3 is COOMe, R 1 is methoxy, R 2 is hydrogen and R 4 is selected from the group consisting of hydrogen, methoxy and bromine, preferably is methoxy or bromine.
  • R 1 is methoxy
  • R 4 is bromine
  • R 2 is methoxy and R 3 is hydrogen
  • R 2 is hydrogen and R 3 is selected in the group consisting of hydrogen, -COO(CH 3 ), -CONH 2 , -CONH(CH 3 ) and -CON(CH 3 ) 2 .
  • the rocaglaol derivative is selected from the group consisting of FL3, FL5, FL6, FL7, FL8, FL9 and FL14.
  • the present invention further provides a new rocaglaol derivative the formula (II), wherein R 11 is alkoxy, optionally substituted; R 12 is hydrogen; or R 11 and R 12 together form a -O-CH 2 -O-unit; R 13 is selected from hydrogen and (Ci-C 3 )-alkoxy;
  • R 14 is -NH-R 27 , wherein R 27 is selected from -SO 2 Me, -COR 28 with R 28 being H or (Ci- C 3 )-alkyl;
  • R 15 is selected from hydrogen, -COOR 21 , -CONR 22 R 23 and -C0NH(CH 2 ) n 0H, wherein n is 2, 3 or 4, wherein
  • R 21 and R 23 are independently selected from hydrogen and (Ci-C 3 )-alkyl and R 22 is selected from the group consisting of (Ci-C 3 )-alkoxy, (Ci-C 3 )-alkyl and hydrogen, or R 22 and R 23 together form a 5 atom heterocycle, optionally substituted by a group
  • R 24 and R 25 are independently selected from the group consisting of (C 1 -C 3 )- alkyl and hydrogen, preferably from hydrogen and methyl;
  • R 16 is selected from hydrogen, (Ci-C 3 )-alkoxy and halogen
  • R 17 is in ortho-position to Ri 6 and is selected from hydrogen, (Ci-C 3 )-alkoxy and hydroxyl or form together with R 17 a -O-CH 2 -O-unit;
  • R 18 and R 19 are independently selected from the group consisting of hydrogen and halogen, preferably from H, F, Br and Cl, more preferably H or F; or any pharmaceutically acceptable salt thereof.
  • the new rocaglaol derivative of the formula (II) has one or several of the following features: a) R 11 is alkoxy and R 12 is hydrogen; b) R 13 is selected from hydrogen and methoxy; c) R 14 is -NH-R 27 , wherein R 27 is selected from -SO 2 Me, -COR 28 with R 28 being H, methyl or ethyl; d) R 15 is selected from hydrogen, -COOR 21 , and -CONR 22 R 23 , wherein R 21 and R 23 are independently selected from hydrogen and methyl, and R 22 is selected from the group consisting of hydrogen, methyl and methoxy; more preferably R 15 is selected from hydrogen, COOMe, CONH 2 , CONHMe and CONMe 2 ; e) R 16 is selected from hydrogen, (Ci-C3)-alkoxy and halogen, more preferably from hydrogen, chlorine, bromine, fluorine and methoxy, still more preferably from bromine and methoxy;
  • the rocaglaol derivative may present the formula (Ha) or (lib). More preferably, the rocaglaol derivative has the formula (lib) and R 14 is -NH-R 27 , wherein R 27 is -SO 2 Me or -
  • R 11 is alkoxy, especially methoxy or ethoxy, more preferably methoxy
  • R 12 is hydrogen
  • R 13 is selected from hydrogen, methoxy and ethoxy, more preferably R 13 is hydrogen or methoxy, still more preferably R 13 is methoxy
  • R 15 is selected from hydrogen, COOMe, CONH 2 , CONHMe and CONMe 2 , more preferably is hydrogen
  • R 16 is selected from the group consisting of hydrogen, chlorine, bromine, fluorine and methoxy, more preferably is chlorine, bromine, or fluorine, still more preferably is bromine
  • R 17 is selected from the group consisting of hydrogen, chlorine, bromine, fluorine and methoxy, more preferably is chlorine, bromine, or fluorine, still more preferably is bromine; and, R 17 ,
  • R 18 and R 19 are hydrogen.
  • the rocaglaol derivative is selected from the group consisting of
  • FL22, FL23, FL24 and FL25 preferably is FL23 or FL25.
  • the present invention provides a new rocaglaol derivative the formula (Ha), wherein
  • R 12 is H and R 11 is selected from methoxy and ethoxy, more preferably R 11 is methoxy;
  • R 13 is selected from hydrogen, methoxy and ethoxy, more preferably R 13 is hydrogen or methoxy;
  • R 14 is -OCOR 20 , R 20 being selected from hydrogen, methyl and ethyl; R 15 is selected from hydrogen, -COOR 21 , and -CONR 22 R 23 , wherein R 21 and R 23 are independently selected from hydrogen and methyl, and R 22 is selected from the group consisting of hydrogen, methyl and methoxy; more preferably R 15 is selected from hydrogen,
  • R 15 is hydrogen
  • Rl 6 is chlorine, bromine, or fluorine, preferably bromine
  • R 17 is hydrogen
  • R 18 is in para and is H or fluorine and R 19 is hydrogen; or any pharmaceutically acceptable salt thereof.
  • R 11 and R 13 are methoxy
  • R 12 , R 15 , R 18 and R 19 are hydrogen
  • R16 is bromine
  • the rocaglaol derivative is selected from the group consisting of
  • the present invention provides a new rocaglaol derivative the formula (Ha), wherein R 12 is H and R 11 is selected from methoxy and ethoxy, more preferably R 11 is methoxy;
  • R 13 is selected from hydrogen, methoxy and ethoxy, more preferably R 13 is hydrogen or methoxy;
  • R 14 is hydroxyl
  • R 18 is in para and is fluorine and R 19 is hydrogen, and wherein
  • R 15 is selected from hydrogen, -COOR 21 , and -CONR 22 R 23 , wherein R 21 and R 23 are independently selected from hydrogen and methyl, and R 22 is selected from the group consisting of hydrogen, methyl and methoxy; more preferably R 15 is selected from hydrogen, COOMe, CONH 2 , CONHMe and CONMe 2 ; R 16 is bromine and R 17 is hydrogen; or
  • R 15 is selected from -COOR 21 , and -CONR 22 R 23 , wherein R 21 and R 23 are independently selected from hydrogen and methyl, and R 22 is selected from the group consisting of hydrogen, methyl and methoxy; more preferably R 15 is selected from hydrogen, COOMe, CONH 2 , CONHMe and CONMe 2 ; and,
  • R 16 is methoxy and R 17 is hydrogen; or any pharmaceutically acceptable salt thereof.
  • R 11 and R 13 are methoxy, R 12 is hydrogen, R 16 is bromine and R 15 is selected from hydrogen, COOMe, CONH 2 , CONHMe and CONMe 2 .
  • R 11 and R 13 are methoxy, R 12 is hydrogen, R 16 is methoxy and R 15 is selected from COOMe, CONH 2 , CONHMe and CONMe 2 .
  • R 11 and R 13 are methoxy, R 12 is hydrogen, and R 15 is selected from COOMe, CONH 2 , CONHMe and CONMe 2 .
  • the rocaglaol derivative is FL16, FL17 or FL18.
  • the present invention concerns a new rocaglaol derivative according to the invention as a medicament.
  • the present invention concerns a pharmaceutical composition comprising a new rocaglaol derivative according to the present invention and a pharmaceutically acceptable carrier and/or excipient.
  • the present invention also concerns a pharmaceutical composition comprising a new rocaglaol derivative according to the present invention and an antineoplastic agent, preferably an anthracycline.
  • the present invention further concerns a product containing a new rocaglaol derivative according to the present invention and an antineoplastic agent, preferably an anthracycline, as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer.
  • antineoplastic agent preferably an anthracycline
  • Figure 1 shows a schematic representation of the synthesis of racemic bromo-demethoxy- rocaglaol (FL3).
  • Figure 2 shows a schematic representation of the synthesis of racemic rocaglaol (FLl) and its analogues (FL2 to FL4).
  • Figure 3 shows a schematic representation of the synthesis of racemic flavaglines FL5 to
  • FIG. 4 shows a schematic representation of the synthesis of racemic flavaglines FL5 to
  • Figure 5 shows a schematic representation of the synthesis of racemic flavaglines FL 19 to
  • Figure 6 shows a schematic representation of the synthesis of racemic flavaglines FL22 to
  • Figure 7 is a graph that shows the inhibition of cell proliferation of HepG2 cells exposed to FLl and FL3 with or without 150 nM doxorubicin. HepG2 cells were treated with FLl ( ) or
  • Figures 8A and 8B show the cardioprotective effect of FLl (Figure 8A) and FL3 ( Figure 8B) on apoptosis induced by doxorubicin (l ⁇ M) in H9c2 cardioblast cells. These two graphs represent the relative total apoptotic cells obtained with doxorubicin alone or in combination with FLl ( Figure 8A) or FL3 ( Figure 8B), in comparison with the results obtained with the vehicle.
  • Figure 9 shows the cardioprotective effect of FL5, FL6, FL7 and FL8 on apoptosis induced by doxorubicin (1 ⁇ M) in H9c2 cells at different concentrations.
  • Figure IQA shows a Western blot analysis demonstrating that doxorubicin induces caspase-3 activity (line 2) that is blocked by FL3 (1 nM) (line 3). Note that FL3 (line 4) and vehicle
  • Figure IQB is a histogram of caspase-3 activity according to Western blot quantification and normalization with GAPDH.
  • FIG 11 shows the protection by FL3 of H9c2 cardio myocytes against serum starvation.
  • H9c2 were cultured in 1% serum in the presence of vehicle or FL3 (20 nM). Apoptosis was measured by FACS assay.
  • Figure 12 Figure 12A) FL3 prevents doxorubicin and KRIBB3 induced apoptosis. H9c2 cardiomyocytes were treated with doxorubicin (l ⁇ M), or the HSP27 inhibitor KRIBB3 (l ⁇ M) in presence or absence of FL3 (100 nM) for 1 h and then labeled with annexin V and PI for FACS analysis.
  • Figure 12B Western blott analyses revealed that that FL3 maximally induces
  • HSP27 phosphorylation (upper bands) without altering total HSP27 levels (lower band) in 1 h in H9c2 cells.
  • Figure 13 shows the cardioprotective effect of FLl-IO and FL 12-18 at 10 (left) or 100 (right) nM on apoptosis induced by doxorubicin (1 mM) in H9c2 cells.
  • flavaglines in particular rocaglaol and some of its derivatives, may act as cardioprotectant agents. These compounds are found to be able to prevent or limit the apoptosis induced by the cardio toxicity of antineoplastic agents such as anthracyclines, while potentiating the anti-tumor efficacy of these molecules.
  • Flavaglines are a family of natural compounds extracted from Asian plants of the genus Aglaia (Kim et al., 2006 and Proksch et al., 2001), comprising, for instance, rocaglaol, rocaglamide or silvestrol.
  • Some of these molecules are known to inhibit the proliferation of tumor cells in a low nanomolar range, either by cytostatic (Hausott et al., 2004) or cytotoxic effects (Kim et al., 2007 and Mi et al., 2006), without displaying any significant toxicity on normal cells.
  • flavaglines have only been known to exhibit anticancer, immunosuppressive, neuroprotective (Fahrig et al., 2005), antifungal insecticide and anti- inflammatory (EP 1 693 059) properties.
  • the present invention provides new cardioprotectant rocaglaol derivatives of the formula (I)
  • R 1 is alkoxy, optionally substituted, preferably selected from the group consisting of methoxy and a group -O-(CH 2 ) n -R 10 wherein n is 1, 2, 3 or 4 and R 10 is selected from the group consisting of hydroxyl, -NMe 2 , -OCONMe 2 , -OCONH 2 and morpholin;
  • R 2 is selected from the group consisting of hydrogen and (Ci-C3)-alkoxy, preferably from hydrogen and methoxy;
  • R 3 is selected from the group consisting of -COOR 5 and -CONH 2 , wherein R 5 is (C1-C3)- alkyl, preferably methyl; and
  • R 4 is halogen, preferably bromine, chloride, iodide, more preferably bromine, or wherein,
  • R 1 is alkoxy, optionally substituted, preferably selected from the group consisting of methoxy and a group -O-(CH 2 ) n -R 10 wherein n is 1, 2, 3 or 4 and R 10 is selected from the group consisting of hydroxyl, -NMe 2 , -OCONMe 2 , -OCONH 2 and morpholin;
  • R 2 is hydrogen
  • R 3 is selected from the group consisting of -COOR 5 and -CONH 2 , wherein R 5 is (C1-C3)- alkyl, preferably methyl; and
  • R 4 is selected from the group consisting of hydrogen, halogen and alkoxy, preferably hydrogen or alkoxy, or wherein,
  • R 1 is substituted alkoxy, preferably a group -O-(CH 2 ) n -R 10 wherein n is 1, 2, 3 or 4 and R 10 is selected from the group consisting of hydroxyl, -NMe 2 , -OCONMe 2 , -OCONH 2 and morpholine.
  • R 2 is selected from the group consisting of hydrogen and (Ci-C3)-alkoxy, preferably from hydrogen and methoxy; more preferably R 2 is Ci-C3)-alkoxy, preferably methoxy;
  • R 3 is selected from the group consisting of -COOR 5 and -CONH 2 , wherein R 5 is (C 1 -C 3 )- alkyl, preferably methyl, and R 4 is selected from the group consisting of hydrogen, halogen and alkoxy, more preferably preferably hydrogen or alkoxy, or any pharmaceutically acceptable salt thereof.
  • R 1 is selected from the group consisting of methoxy or 0-(CH 2 ) n -0H wherein n is 1, 2 or 3, preferably R 1 is a methoxy; R 2 is hydrogen; R 3 is selected from the group consisting of -COOR 5 and -CONH 2 , wherein R 5 is methyl; and, R 4 is halogen or methoxy, preferably bromine or methoxy.
  • the rocaglaol derivative may be FL5, FL7, and FL9.
  • R 1 is a substituted alkoxy -O-(CH 2 ) n -R 10 wherein n is 1, 2, 3 or 4 and R 10 is selected from the group consisting of hydroxyl, -NMe 2 , -OCONMe 2 , -OCONH 2 and morpholine, preferably R 10 is hydroxyl; R 2 is hydrogen or methoxy, preferably hydrogen; R 3 is selected from the group consisting of -COOR 5 and -CONH 2 , wherein R 5 is (Ci-C 3 )-alkyl, preferably methyl; and, R 4 is halogen or methoxy, preferably bromine or methoxy.
  • R 1 is a substituted alkoxy -0-(CH 2 ) J1 -R 1 ° wherein n is 2 and R 10 is hydroxyl; R 2 is hydrogen; R 3 is -COOR 5 , wherein R 5 is methyl; and R 4 is methoxy.
  • R 1 is a substituted alkoxy -0-(CH 2 ) J1 -R 1 ° wherein n is 3 and R 10 is hydroxyl; R 2 is hydrogen; R 3 is -COOR 5 , wherein R 5 is methyl; and R 4 is methoxy.
  • R 3 is -CONH 2 .
  • R 1 is alkoxy, optionally substituted, preferably selected from the group consisting of methoxy and a group -0-(CH 2 ) n - R 10 wherein n is 1, 2, 3 or 4 and R 10 is selected from the group consisting of hydroxyl, -NMe 2 , -OCONMe 2 , -OCONH 2 and morpholin;
  • R 2 is selected from the group consisting of hydrogen and (Ci-C3)-alkoxy, preferably from hydrogen and methoxy; and, R 4 is halogen, preferably bromine, chloride, iodide, more preferably bromine; or 2)
  • R 1 is alkoxy, optionally substituted, preferably selected from the group consisting of methoxy and a group -0-(CH 2 )D-R 1 ° wherein n is 1, 2, 3 or 4 and R 10 is selected from the group consisting of hydroxyl, -NMe 2 , - OCONMe 2 , -OCONH 2 and morpholin
  • the rocaglaol derivative may be FL9 and FL14.
  • R 3 is -COOR 5 with R 5 is (Ci-C 3 )-alkyl, preferably methyl.
  • R 1 is alkoxy, optionally substituted, preferably selected from the group consisting of methoxy and a group -O-(CH 2 ) n -R 10 wherein n is 1, 2, 3 or 4 and R 10 is selected from the group consisting of hydroxyl, -NMe 2 , -OCONMe 2 , -OCONH 2 and morpholine;
  • R 2 is hydrogen; and, R 4 is selected from the group consisting of hydrogen, methoxy and halogen.
  • R 3 is COOMe
  • R 1 is methoxy
  • R 2 is hydrogen
  • R 4 is selected from the group consisting of hydrogen, methoxy and bromine, preferably is methoxy or bromine.
  • the rocaglaol derivative may be FL5 or FL7.
  • the present invention concerns a compound of formula (I) wherein R 1 is methoxy; R 2 is selected from the group consisting of methoxy or hydrogen; R 3 is selected from the group consisting of -COOR 5 , and -CONH 2 , wherein R 5 is methyl; and, R 4 is halogen, preferably bromine.
  • R 1 is methoxy
  • R 2 is hydrogen
  • R 3 is -CONH 2
  • R 4 is bromine
  • R 1 is methoxy; R 2 is hydrogen; R 3 is -COOR 5 , wherein R 5 is methyl; and, R 4 is halogen or methoxy, preferably bromine or methoxy.
  • R 1 is methoxy; R 2 is hydrogen; R 3 is -COOR 5 , wherein R 5 is methyl; and R 4 is bromine (FL5).
  • the present invention also concerns a new rocagloal derivative of the formula (I), wherein
  • R 1 is methoxy
  • R 2 is selected from the group consisting of hydrogen and (Ci-C3)-alkoxy, preferably from hydrogen and methoxy;
  • R 3 is selected from the group consisting of hydrogen, -COOR 5 and -CONR 6 R 7 , wherein
  • R 5 is (Ci-C 3 )-alkyl, preferably methyl, and
  • R 6 and R 7 are independently selected from the group consisting of hydrogen and (C1-C3)- alkyl, preferably from hydrogen and methyl; and R 4 is bromine, or any pharmaceutically acceptable salt thereof.
  • R 1 is methoxy; and R 4 is bromine, and wherein R 2 is (Ci-C3)-alkoxy, preferably methoxy, and,
  • R 3 is hydrogen, or
  • R 2 is hydrogen
  • R 3 is selected from the group consisting of hydrogen, -COOR 5 and -CONR 6 R 7 , wherein R 5 is (Ci-C 3 )-alkyl, preferably methyl, and R 6 and R 7 are independently selected from the group consisting of hydrogen and (Ci-C 3 )-alkyl, preferably from hydrogen and methyl; or any pharmaceutically acceptable salt thereof.
  • R 1 and R 2 are methoxy, R 3 is hydrogen and R 4 is bromine (FL3). In a further preferred embodiment, R 1 and R 2 are methoxy, R 3 is -CONH 2 , and R 4 is bromine
  • R 1 is methoxy;
  • R 2 is hydrogen;
  • R 3 is selected from the group consisting of hydrogen, -COO(ClHb) and -CON(CF ⁇ ) 2 , preferably of hydrogen and -
  • R 1 is methoxy
  • R 2 is hydrogen
  • R 3 is -CON(CF ⁇ ) 2 and R 4 is bromine (FL8).
  • R 1 is methoxy
  • the present invention also concerns a new rocagloal derivative of the formula (II), wherein R 1 ' is alkoxy, optionally substituted;
  • R 12 is hydrogen; or R 11 and R 12 together form a -O-CH 2 -O-unit;
  • R 13 is selected from hydrogen and (Ci-C3)-alkoxy;
  • R 14 is -NH-R 27 , wherein R 27 is selected from -SO 2 Me, -COR 28 with R 28 being H or (Ci- C 3 )-alkyl;
  • R 15 is selected from hydrogen, -COOR 21 , -CONR 22 R 23 and -C0NH(CH 2 ) n 0H, wherein n is 2, 3 or 4, wherein
  • R 21 and R 23 are independently selected from hydrogen and (Ci-C 3 )-alkyl and R 22 is selected from the group consisting of (Ci-C3)-alkoxy, (Ci-C3)-alkyl and hydrogen, or
  • R 22 and R 23 together form a 5 atom heterocycle, optionally substituted by a group
  • R 24 and R 25 are independently selected from the group consisting of (C1-C3)- alkyl and hydrogen, preferably from hydrogen and methyl;
  • R 16 is selected from hydrogen, (Ci-C3)-alkoxy and halogen;
  • R 17 is in ortho-position to Ri 6 and is selected from hydrogen, (Ci-C3)-alkoxy and hydroxyl or form together with R 17 a -O-CH 2 -O-unit;
  • R 18 and R 19 are independently selected from the group consisting of hydrogen and halogen, preferably from H, F, Br and Cl, more preferably H or F; or any pharmaceutically acceptable salt thereof.
  • the rocaglaol derivative of the formula (II) has preferably one or several of the following features: a) R 11 is alkoxy and R 12 is hydrogen; b) R 13 is selected from hydrogen and methoxy; c) R 14 is -NH-R 27 , wherein R 27 is selected from -SO 2 Me, -COR 28 with R 28 being H, methyl or ethyl; d) R 15 is selected from hydrogen, -COOR 21 , and -CONR 22 R 23 , wherein R 21 and R 23 are independently selected from hydrogen and methyl, and R 22 is selected from the group consisting of hydrogen, methyl and methoxy; more preferably R 15 is selected from hydrogen, COOMe, CONH 2 , CONHMe and CONMe 2 ; e) R 16 is selected from hydrogen, (Ci-C3)-alkoxy and halogen, more preferably from hydrogen, chlorine, bromine, fluorine and methoxy, still more preferably from bromine and methoxy;
  • the rocaglaol derivative of the formula (II) meets one feature, two features [for instance a) and b); a) and c); a) and d); a) and e); a) and f); a) and g); b) and c); b) and d); b) and e); b) and f); b) and g); c) and d); c) and e); c) and f); c) and g); d) and e); d) and f); d) and g); e) and f); e) and g); f) and g)], three features [for instance a), b) and c); a), b) and d); a), b) and e); a), b) and f); a), b) and f); a), b) and f); a), b) and g); a), c) and
  • the rocaglaol derivative may present the formula (Ha) or (lib), the definition of RI l, R12, R13, R14, R15, R16, R17, R18 and R19 is the same as above.
  • the rocaglaol derivative has the formula (lib) and R 14 is -NH-R 27 , wherein R 27 is -SO 2 Me or -COR 28 with R 28 being H or (Ci-C 3 )-alkyl, preferably H or methyl.
  • R 11 is alkoxy, especially methoxy or ethoxy, more preferably methoxy
  • R 12 is hydrogen
  • R 13 is selected from hydrogen, methoxy and ethoxy, more preferably R 13 is hydrogen or methoxy, still more preferably R 13 is methoxy
  • R 15 is selected from hydrogen, COOMe, CONH 2 , CONHMe and CONMe 2 , more preferably is hydrogen
  • R 16 is selected from the group consisting of hydrogen, chlorine, bromine, fluorine and methoxy, more preferably is chlorine, bromine, or fluorine, still more preferably is bromine
  • R 19 are hydrogen.
  • the rocaglaol of formula (lib) is such as R 11 and R 13 are methoxy, R 15 is hydrogen, R 16 is bromine, and, R 17 , R 18 and R 19 are hydrogen.
  • R 14 can be -NHCHO (FL23) or -NHSO 2 Me (FL25).
  • the rocaglaol of formula (Ha) is such as R 11 and R 13 are methoxy, R 15 is hydrogen, R 16 is bromine, R 17 , R 18 and R 19 are hydrogen, and R 14 can be -NHCHO (FL22) or
  • the present invention also concerns a new rocagloal derivative of the formula (II), wherein
  • R 12 is H and R 11 is selected from methoxy and ethoxy, more preferably R 11 is methoxy;
  • R 13 is selected from hydrogen, methoxy and ethoxy, more preferably R 13 is hydrogen or methoxy;
  • R 14 is -OCOR 20 , R 20 being selected from hydrogen, methyl and ethyl;
  • R 15 is selected from hydrogen, -COOR 21 , and -CONR 22 R 23 , wherein R 21 and R 23 are independently selected from hydrogen and methyl, and R 22 is selected from the group consisting of hydrogen, methyl and methoxy; more preferably R 15 is selected from hydrogen, COOMe, CONH 2 , CONHMe and CONMe 2 ; still more preferably R 15 is hydrogen;
  • Rl 6 is chlorine, bromine, or fluorine, preferably bromine
  • R 17 is hydrogen
  • R 18 is in para and is H or fluorine and R 19 is hydrogen; or any pharmaceutically acceptable salt thereof. More preferably, R 11 and R 13 are methoxy, R 12 , R 15 , R 18 and R 19 are hydrogen, and R16 is bromine. Still preferably, R 14 is -OCHO (FL 19), -OCOMe (FL20) or -OCOEt (FL21).
  • the present invention finally provides a new rocaglaol derivative the formula (Ha), wherein R 12 is H and R 11 is selected from methoxy and ethoxy, more preferably R 11 is methoxy; R 13 is selected from hydrogen, methoxy and ethoxy, more preferably R 13 is hydrogen or methoxy; R 14 is hydroxyl;
  • R 18 is in para and is fluorine and R 19 is hydrogen, and wherein R 15 is selected from hydrogen, -COOR 21 , and -CONR 22 R 23 , wherein R 21 and R 23 are independently selected from hydrogen and methyl, and R 22 is selected from the group consisting of hydrogen, methyl and methoxy; more preferably R 15 is selected from hydrogen, COOMe, CONH 2 , CONHMe and CONMe 2 ; R 16 is bromine and R 17 is hydrogen; or
  • R 15 is selected from -COOR 21 , and -CONR 22 R 23 , wherein R 21 and R 23 are independently selected from hydrogen and methyl, and R 22 is selected from the group consisting of hydrogen, methyl and methoxy; more preferably R 15 is selected from hydrogen, COOMe, CONH 2 , CONHMe and CONMe 2 ; and,
  • R 16 is methoxy and R 17 is hydrogen; or any pharmaceutically acceptable salt thereof.
  • R 11 and R 13 are methoxy, R 12 is hydrogen, R 16 is bromine and R 15 is selected from hydrogen, COOMe, CONH 2 , CONHMe and CONMe 2 . Still more preferably, R 15 is COOMe (FL 17) or CONH 2 (FL 18). In an alternative preferred embodiment, R 11 and R 13 are methoxy, R 12 is hydrogen, R 16 is methoxy and R 15 is selected from COOMe, CONH 2 , CONHMe and CONMe 2 . Still more preferably, R 15 is COOMe
  • R 11 and R 13 are methoxy
  • R 12 is hydrogen
  • R 15 is selected from COOMe, CONH 2 , CONHMe and CONMe 2 .
  • a "cardioprotectant agent” is an agent that protects cardio myocytes from damages, in particular from apoptosis.
  • alkoxy corresponds to an alkyl group bonded to the molecule by an -O- (ether) bond.
  • (Ci-C3)-alkoxy groups include methoxy, ethoxy, propyloxy, and isopropyloxy.
  • alkyl refers to a univalent radical containing only carbon and hydrogen atoms arranged in a chain. (Ci-C 3 )-alkyl groups include methyl, ethyl, propyl, or isopropyl.
  • Me refers to a methyl group.
  • Et refers to an ethyl group.
  • heterocycle refers to a group containing a ring structure comprising one or more heteroatoms, such as sulfur, oxygen or nitrogen. 5 atom heterocycles include, for example, furan, pyrrole and oxazole.
  • pharmaceutically acceptable salt refers to salts which are non-toxic for a patient and suitable for maintaining the stability of a therapeutic agent and allowing the delivery of said agent to target cells or tissue.
  • Pharmaceutically acceptable salts are well known in the art (Berge et al., 1977). These salts can be prepared in situ during the final isolation and purification of the rocaglaol derivatives, or separately by reacting the free base function with a suitable organic acid.
  • Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid
  • organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid.
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate,
  • the present invention also concerns a rocaglaol derivative of the formulae (I), (II), (Ha) and (lib) according to the invention, as described above, as a medicament, in particular as cardioprotectant agent, and more particularly as cardioprotectant agent against cardiotoxicity of an antineoplastic agent.
  • the derivative is selected in the group consisting of FL3, FL5, FL6, FL7, FL8, FL9, FL14, FL16, FL17, FL18, FL19, FL20, FL21, FL22, FL23, FL24 and FL25. Those derivatives have also demonstrated advantageous cytotoxicity against tumoral cells.
  • the present invention further concerns a rocaglaol derivative of the formulae (I), (II), (Ha) and (lib) according to the invention for use for treating cancer. It also concerns the use of a rocaglaol derivative of the formulae (I), (II), (Ha) and (lib) according to the invention for preparing a medicament for treating cancer. It finally concerns a method for treating a cancer in a subject in need thereof, comprising administering a therapeutically active amount of a rocaglaol derivative of the formulae (I), (II), (Ha) and (lib) according to the invention.
  • This particular aspect also concerns the preferred embodiments disclosed above for the rocaglaol derivatives of the formulae (I), (II), (Ha) and (lib), and in particular a derivative selected in the group consisting of FL3, FL5, FL6, FL7, FL8, FL9, FL14, FL16, FL17, FL18, FL 19, FL20, FL21, FL22, FL23, FL24 and FL25.
  • rocaglaol derivatives can be synthesized by any process known by the man skilled in the art. Such processes are described, for example, in the patent applications DE 19934952 and WO 2005/092876 or in the articles of Diedrichs et al. (Diedrichs et al, 2005), Gerard et al. (Gerard et al., 2004) and Dobler et al. (Dobler et al., 2001).
  • the present invention also concerns a pharmaceutical composition
  • a pharmaceutical composition comprising a cardioprotectant rocaglaol derivative of formulae (I), (II), (Ha) and (lib) according to the present invention and a pharmaceutically acceptable carrier and/or excipient.
  • This particular aspect also concerns the preferred embodiments disclosed above for the rocaglaol derivatives of the formulae (I), (II), (Ha) and (lib), and in particular a derivative selected in the group consisting of FL3, FL5, FL6, FL7, FL8, FL9, FL 14, FL 16, FL 17, FL 18, FL 19, FL20, FL21, FL22, FL23, FL24 and FL25.
  • the pharmaceutical composition comprising the rocaglaol derivative of formulae (I), (II), (Ha) and (lib) according to the present invention, including their preferred embodiments and the compounds FL3, FL5, FL6, FL7, FL8, FL9, FL14, FL16, FL17, FL18, FL19, FL20, FL21, FL22, FL23, FL24 and FL25, is formulated in accordance with standard pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York) known by a person skilled in the art.
  • compositions include those suitable for oral, rectal, topical (including transdermal, buccal and sublingual), or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration.
  • parenteral including subcutaneous, intramuscular, intravenous and intradermal
  • conventional excipient can be used according to techniques well known by those skilled in the art.
  • the compositions for parenteral administration are generally physiologically compatible sterile solutions or suspensions which can optionally be prepared immediately before use from solid or lyophilized form.
  • Adjuvants such as a local anesthetic, preservative and buffering agents can be dissolved in the vehicle and a surfactant or wetting agent can be included in the composition to facilitate uniform distribution of the active ingredient.
  • the composition can be formulated into conventional oral dosage forms such as tablets, capsules, powders, granules and liquid preparations such as syrups, elixirs, and concentrated drops.
  • Non toxic solid carriers or diluents may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, magnesium, carbonate, and the like.
  • binders which are agents which impart cohesive qualities to powdered materials are also necessary.
  • starch, gelatine, sugars such as lactose or dextrose, and natural or synthetic gums can be used as binders.
  • Disintegrants are also necessary in the tablets to facilitate break-up of the tablet.
  • Disintegrants include starches, clays, celluloses, algins, gums and crosslinked polymers.
  • lubricants and glidants are also included in the tablets to prevent adhesion to the tablet material to surfaces in the manufacturing process and to improve the flow characteristics of the powder material during manufacture.
  • Colloidal silicon dioxide is most commonly used as a glidant and compounds such as talc or stearic acids are most commonly used as lubricants.
  • the composition can be formulated into ointment, cream or gel form and appropriate penetrants or detergents could be used to facilitate permeation, such as dimethyl sulfoxide, dimethyl acetamide and dimethylformamide.
  • nasal sprays, rectal or vaginal suppositories can be used.
  • the active compound can be incorporated into any of the known suppository bases by methods known in the art. Examples of such bases include cocoa butter, polyethylene glycols (carbowaxes), polyethylene sorbitan monostearate, and mixtures of these with other compatible materials to modify the melting point or dissolution rate.
  • the pharmaceutical composition of the invention is suitable for parenteral administration.
  • composition according to the invention may be formulated to release the active drug substantially immediately upon administration or at any predetermined time or time period after administration.
  • Pharmaceutical composition according to the invention can comprise one or more cardioprotectant rocaglaol derivatives associated with pharmaceutically acceptable excipients and/or carriers. These excipients and/or carriers are chosen according to the form of administration as described above.
  • Other active compounds can also be associated with rocaglaol derivatives of the present invention such as other cardioprotectant molecules or antineoplastic agents, such as anthracycline, preferably doxorubicin.
  • the present invention also concerns a product containing a rocaglaol derivative of formulae (I), (II), (Ha) and (lib) according to the present invention, including their preferred embodiments and the compounds FL3, FL5, FL6, FL7, FL8, FL9, FL14, FL16, FL17, FL18, FL19, FL20, FL21, FL22, FL23, FL24 and FL25, and an antineoplastic agent, preferably an anthracycline, as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer.
  • antineoplastic agent preferably an anthracycline
  • R 1 ! is alkoxy, optionally substituted
  • R 12 is hydrogen; or R 11 and R 12 together form a -O-CH 2 -O-unit; R 13 is selected from hydrogen and (Ci-C3)-alkoxy;
  • R 21 and R 23 are independently selected from hydrogen and (Ci-C 3 )-alkyl and R 22 is selected from the group consisting of (Ci-C3)-alkoxy, (Ci-C3)-alkyl and hydrogen, or
  • R 22 and R 23 together form a 5 atom heterocycle, optionally substituted by a group NHCO- (Ci-C 4 )-alkyl; or R 14 , R 15 and carbons ( ⁇ ) and ( ⁇ ) bearing R 14 and R 15 together form wherein R 24 and R 25 are independently selected from the group consisting of (C 1 -C 3 )- alkyl and hydrogen, preferably from hydrogen and methyl;
  • R 16 is selected from hydrogen, (Ci-C3)-alkoxy and halogen;
  • R 17 is in ortho-position to Ri 6 and is selected from hydrogen, (Ci-C3)-alkoxy and hydroxyl or form together with R 17 a -O-CH 2 -O-unit;
  • R 18 and R 19 are independently selected from the group consisting of hydrogen and halogen, preferably from H, F, Br and Cl, more preferably H or F; or any pharmaceutically acceptable salt thereof, for use for preventing or limiting the cardiotoxicity of an antineoplastic agent, preferably an anthracycline, more preferably doxorubicin.
  • the rocaglaol derivative of the formula (II) has one or several of the following features : a) R 12 is H and R 11 is selected from methoxy and ethoxy, more preferably R 11 is methoxy; b) R 13 is selected from hydrogen, methoxy and ethoxy, more preferably R 13 is hydrogen or methoxy; c) R 14 is selected from hydroxyl,-OCOR 20 , R 20 being selected from hydrogen, methyl and ethyl, -NH-(CH 2 ) m -R 27 , wherein m is 0 and R 27 is -SO 2 Me or -COR 28 with R 28 being H or (Ci-C 3 )-alkyl, preferably H or methyl; d) R 15 is selected from hydrogen, -COOR 21 , and -CONR 22 R 23 , wherein R 21 and R 23 are independently selected from hydrogen and methyl, and R 22 is selected from the group consisting of hydrogen, methyl and methoxy; more
  • the rocaglaol derivative of the formula (II) meets one feature, two features [for instance a) and b); a) and c); a) and d); a) and e); a) and f); a) and g); b) and c); b) and d); b) and e); b) and f); b) and g); c) and d); c) and e); c) and f); c) and g); d) and e); d) and f); d) and g); e) and f); e) and g); f) and g)], three features [for instance a), b) and c); a), b) and d); a), b) and e); a), b) and f); a), b) and f); a), b) and f); a), b) and g); a), c) and
  • R 18 is a halogen and is in position para, ortho or meta and R 19 is hydrogen.
  • R 18 is F, Cl or Br, more preferably F or Br, still more preferably is F.
  • R 18 is in para.
  • R 18 and R 19 are both halogens, preferably the same halogen, and R 18 and R 19 are in para and ortho, in ortho and meta, in para and meta, or both in ortho.
  • R 18 and R 19 are both F, Cl or Br, more preferably F.
  • the rocaglaol derivative presents the formula (Ha) or (lib)
  • R 14 is selected from hydroxyl and - OCOR 20 , R 20 being selected from hydrogen, methyl and ethyl.
  • R 1 ⁇ is alkoxy, especially methoxy or ethoxy, more preferably methoxy;
  • R 12 is hydrogen;
  • R 13 is selected from hydrogen, methoxy and ethoxy, more preferably R 13 is hydrogen or methoxy;
  • R 15 is selected from hydrogen, COOMe, CONH 2 , CONHMe and CONMe 2 ;
  • R 16 is selected from the group consisting of hydrogen, chlorine, bromine, fluorine and methoxy, more preferably of bromine and methoxy;
  • R 18 is in para and is H or fluorine; and, R 17 and R 19 are hydrogen.
  • the rocaglaol derivative has the formula (III)
  • R 11 is alkoxy, optionally substituted; and R 12 is hydrogen; or R 11 and R 12 together form a -O-CH 2 -O-unit;
  • R 13 is selected from the group consisting of hydrogen and (Ci-C3)-alkoxy, preferably from hydrogen and methoxy;
  • R 14 is selected from the group consisting of hydroxyl, oxo group and -OCOR 20 , R 20 being selected from (Ci-C 3 )-alkyl and hydrogen, preferably from hydrogen and methyl; R 15 is selected from the group consisting of hydrogen, -COOR 21 , -CONR 22 R 23 and -
  • R 21 and R 23 are independently selected from hydrogen and (Ci-C 3 )-alkyl, preferably from hydrogen and methyl, and R 22 is selected from the group consisting of (Ci-C3)-alkoxy, (C 1 - C 3 )-alkyl and hydrogen, preferably from hydrogen, methyl and methoxy, or
  • R 22 and R 23 together form a 5 atom heterocycle, optionally substituted by a group NHCO-(Ci-
  • R 24 and R 25 are independently selected from the group consisting of (Ci-C 3 )-alkyl and hydrogen, preferably from hydrogen and methyl;
  • R 16 is selected from the group consisting of hydrogen, (Ci-C3)-alkoxy and halogen;
  • R 17 is in ortho-position to Ri 6 and is selected from the group consisting of hydrogen, (C1-C3)- alkoxy and hydroxyl or form together with R 17 a -O-CH 2 -O-unit; or any pharmaceutically acceptable salt thereof.
  • the rocaglaol derivative has the formula (lib) and R 14 is -NH-(CH 2 ) m -R 27 , wherein m is 0 and R 27 is -SO 2 Me or -COR 28 with R 28 being H or (Ci- C 3 )-alkyl, preferably H or methyl.
  • R 1 ⁇ is alkoxy, especially methoxy or ethoxy, more preferably methoxy;
  • R 12 is hydrogen
  • R 13 is selected from hydrogen, methoxy and ethoxy, more preferably R 13 is hydrogen or methoxy, still more preferably R 13 is methoxy;
  • R 15 is selected from hydrogen, COOMe, CONH 2 , CONHMe and CONMe 2 , more preferably is hydrogen;
  • R 16 is selected from the group consisting of hydrogen, chlorine, bromine, fluorine and methoxy, more preferably is chlorine, bromine, or fluorine, still more preferably is bromine; and,
  • R 17 , R 18 and R 19 are hydrogen.
  • suitable rocaglaol derivatives of the formula (II) are disclosed in the following documents: WO07/139749; EP1693059; WO05/113529; WO05/092876; WO03/045375; US 6,518,274; US 6,420,393; DE19934952.
  • the present invention also concerns a rocaglaol derivative of the formulae (I), (II), (Ha), (lib) and (III) as described above or any pharmaceutically acceptable salt thereof, for preventing or limiting the cardiotoxicity of an antineoplastic agent.
  • this particular aspect also concerns the preferred embodiments disclosed above for the rocaglaol derivatives of the formulae (I), (II), (Ha) and (lib), and in particular a derivative selected in the group consisting of FLl, FL3, FL5, FL6, FL7, FL8, FL9, FLlO, FL12, FL13, FL14, FL15, FL16, FL17, FL18, FL 19, FL20, FL21, FL22, FL23, FL24 and FL25, more preferably from the group consisting of FLl, FL3, FL5, FL8, FL9, FLlO, FL12, FL13, FL14, FL15, FL16, FL17, FL18, FL19, FL20, FL21, FL22, FL23 and FL25.
  • the present invention further concerns a method for preventing or limiting the cardiotoxicity of an antineoplastic agent in a subject comprising administering a therapeutically effective amount of a rocaglaol derivative of the formulae (I), (II), (Ha), (lib) and (III) as described above or any pharmaceutically acceptable salt thereof to said subject.
  • This particular aspect also concerns the preferred embodiments disclosed above for the rocaglaol derivatives of the formulae (I), (II), (Ha) and (lib), and in particular a derivative selected in the group consisting of FLl, FL3, FL5, FL6, FL7, FL8, FL9, FLlO, FL12, FL13, FL14, FL15, FL16, FL17, FL18, FL 19, FL20, FL21, FL22, FL23, FL24 and FL25, more preferably from the group consisting of FLl, FL3, FL5, FL8, FL9, FLlO, FL12, FL13, FL14, FL15, FL16, FL17, FL18, FL19, FL20, FL21, FL22, FL23 and FL25.
  • the method of the invention for preventing or limiting the cardiotoxicity of an antineoplastic agent comprises administering 0.01 to 10 mg / kg of body weight / day of a rocaglaol derivative of the formulae (I), (II), (Ha), (lib) and (III) as described above or any pharmaceutically acceptable salt thereof to said subject.
  • the method of the invention comprises administering 0.1 to 5 mg / kg of body weight / day of a rocaglaol derivative of the formulae (I), (II), (Ha), (lib) and (III) as described above or any pharmaceutically acceptable salt thereof to said subject.
  • This particular aspect also concerns the preferred embodiments disclosed above for the rocaglaol derivatives of the formulae (I), (II), (Ha) and (lib), and in particular a derivative selected in the group consisting of FLl, FL3, FL5, FL6, FL7, FL8, FL9, FLlO, FL12, FL13, FL14, FL15, FL16, FL17, FL18, FL19, FL20, FL21, FL22, FL23, FL24 and FL25, more preferably from the group consisting of FLl, FL3, FL5, FL8, FL9, FLlO, FL12, FL13, FL14, FL15, FL16, FL17, FL18, FL19, FL20, FL21,
  • cardiotoxicity means having a toxic effect on the heart, which includes cardiac events, such as but not limited to, mild blood pressure changes, thrombosis, electrocardiographic (ECG) changes, arrthymias, myocarditis, pericarditis, myocardial infarction (MI), cardiomyopathy, cardiac failure (left ventricular dysfunction or failure) and congestive heart failure (CHF).
  • cardiac events such as but not limited to, mild blood pressure changes, thrombosis, electrocardiographic (ECG) changes, arrthymias, myocarditis, pericarditis, myocardial infarction (MI), cardiomyopathy, cardiac failure (left ventricular dysfunction or failure) and congestive heart failure (CHF).
  • ECG electrocardiographic
  • MI myocardial infarction
  • CHF congestive heart failure
  • an “antineoplastic agent” is an agent with anti-cancer activity that inhibits or halts the growth of cancerous cells or immature pre-cancerous cells, kills cancerous cells or immature precancerous cells, increases the susceptibility of cancerous or pre-cancerous cells to other antineoplastic agents, and/or inhibits metastasis of cancerous cells.
  • agents may include chemical agents as well as biological agents.
  • antineoplastic agents can cause cardiovascular toxicity such as anthracyclines, cyclophosphamide, paclitaxel, fluorouracil, rituximab, arsenic trioxide, trastuzumab, thalidomide, etoposide, vinca alkaloids, pentastatin, cytarabine, interferons, busulfan and cisplatin.
  • the antineoplastic agent belongs to the group of anthracyclines.
  • anthracyclines include but are not limited to, doxorubicin, daunorubicin, idarubicin, detorubicin, carminomycin, epirubicin, morpholinodoxorubicin, morpholinodaunorubicin, methoxymorpholinyldoxorubicin, substitutes, derivatives, and one or more combinations thereof. More preferably, the anthracycline molecule is doxorubicin.
  • the term "to prevent or to limit the cardiotoxicity” means inhibiting or reducing cardiotoxic side effects of the antineoplastic agent administered to the subject, in particular, inhibiting or reducing the apoptosis of cardiomyocytes.
  • the effect of the rocaglaol derivative on the apoptosis of cardiomyocytes can be assessed by any method known by the skilled person.
  • the effects of compounds according to invention are assessed by measuring and comparing the apoptosis of H9c2 cardiomyocytes in absence and in presence of an antineoplastic agent and with a combination of an antineoplastic agent and a rocaglaol derivative of the invention. Results are expressed in percentage of apoptotic cells.
  • the apoptosis of cardiomyocyte in presence of a rocaglaol derivative of the invention is reduced by at least 5%, more preferably by at least 10% and the most preferably by at least 25%.
  • the present invention also concerns the use of a rocaglaol derivative of the formulae (I), (II), (Ha), (lib) and (III) as described above, or any of its pharmaceutically acceptable salts, as cardioprotectant agent.
  • This particular aspect also concerns the preferred embodiments disclosed above for the rocaglaol derivatives of the formulae (I), (II), (Ha) and (lib), and in particular a derivative selected in the group consisting of FLl, FL3, FL5, FL6, FL7, FL8, FL9, FLlO, FL12, FL13, FL14, FL15, FL16, FL17, FL18, FL19, FL20, FL21, FL22, FL23, FL24 and FL25, more preferably from the group consisting of FLl, FL3, FL5, FL8, FL9, FLlO, FL12, FL13, FL14, FL15, FL16, FL17, FL18, FL19, FL20, FL21, FL22, FL23 and FL25.
  • the rocaglaol derivative is one of the formulae (I), (II), (Ha), (lib) according to the invention, as described herein.
  • the rocaglaol derivative is selected from the group consisting of FLl, FL3, FL5, FL6, FL7, FL8, FL9, FLlO, FL12, FL13, FL14, FL15, FL16, FL17, FL18, FL 19, FL20, FL21, FL22, FL23, FL24 and FL25, more preferably from the group consisting of FLl, FL3, FL5, FL8, FL9, FLlO, FL12, FL13, FL14, FL15, FL16, FL17, FL18, FL19, FL20, FL21, FL22, FL23 and FL25.
  • the amount of cardioprotectant rocaglaol derivative of the invention to be administered has to be determined by standard procedure well known by those of ordinary skill in the art. Physiological data of the patient (e.g. age, size, and weight) and the routes of administration have to be taken into account to determine the appropriate dosage.
  • the cardioprotectant rocaglaol derivative of the invention may be administered as a single dose or in multiple doses.
  • the pharmaceutical composition comprises a therapeutically effective amount of the rocaglaol derivative of the invention.
  • a “therapeutically effective amount” is intended an amount of cardioprotectant rocaglaol derivative of the invention administered to a patient that is sufficient to provide a cardioprotective effect, i.e. that is sufficient to protect cardiomyocytes from damages, in particular from apoptosis.
  • This therapeutically effective amount comprises a blood plasma concentration in a range of from 1 nM to 1 ⁇ M, preferably of from 15 nM to 100 nM or from 18 nM to 90 nM, more preferably of from 20 nM to 70 nM, most preferably of from 23 nM to 50 nM, and even most preferably of from 25 nM to 30 nM.
  • the total amount of the rocaglaol derivative of the invention may vary depending on the volume of blood plasma of the patient. Suitable means and measures to determine the therapeutically effective amount are available to the person skilled in the art.
  • the pharmaceutical composition according to the invention comprises 0.1 mg to 1 g of the rocaglaol derivative of the invention. In another particular embodiment, the pharmaceutical composition according to the invention comprises 0.7 to 700 mg of the rocaglaol derivative of the invention, preferably 0.7 to 350 mg of the rocaglaol derivative of the invention.
  • Cardioprotectant rocaglaol derivatives of the invention can be used in combination with other cardioprotective molecules or with antineoplastic agents.
  • rocaglaol derivatives and the other active molecules can be administered simultaneously or consecutively.
  • the present invention discloses a pharmaceutical composition
  • a pharmaceutical composition comprising a rocaglaol derivative of formula (II), preferably of formula (I), and an antineoplastic agent. It also concerns a product containing a rocaglaol derivative of formula (II), preferably of formula (I) and an antineoplastic agent as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer.
  • the antineoplastic agent is an anthracycline, in particular doxorubicin.
  • Example 3 Synthesis and NMR characterization of rocaglaol derivatives FL6 to FL25 Synthesis of FLlO Methyl 2-(4-bromophenyl)-6, 8-dimethoxy-5-hydroxy-2 , 5-methano-10-oxo-3-phenyl-2, 3, 4, 5- tetrahydro-l-benzoxepin-4-carboxylate (4a of Figure 4).
  • NMR 13 C (CDCl 3 ): 51.9, 52.4, 53.5, 54.6, 55.4, 55.6, 56.0, 56.3, 57.2, 62.3, 81.0, 81.3, 83.8, 87.5, 92.9, 93.9, 94.6, 98.0, 103.7, 104.7, 121.9, 122.1, 126.9, 127.5, 128.1, 128.5, 128.9, 129.0, 129.3, 129.8, 130.4, 130.5, 130.7, 131.3, 132.6, 134.8, 137.5, 139.4, 152.7, 153.5, 158.2, 158.6, 161.5, 162.2, 170.6, 171.7, 202.9.
  • Glacial acetic acid (51 ⁇ L, 0.89 mmol) was added to a solution Of Me 4 NBH(OAc) 3 (140 mg, 0.53 mmol) in CH 3 CN (3 mL). After stirring for 5 min at room temperature, a solution of the previous compound (40 mg, 0.09 mmol) in CH 3 CN (2 mL) was added dropwise. The resulting mixture was stirred for 3 h at rt, successively quenched with saturated aqueous NH 4 Cl (15 niL) and a 3 M aqueous solution of sodium/potassium tartrate (3 mL) and stirred for 30 min. The aqueous solution was extracted with AcOEt (2 x 30 mL).
  • ester 6b of Figure 4 70 mg, 0.14 mmol
  • the solution was then heated at 100 0 C for 36 h. After cooling to rt, the solution was concentrated in vacuo.
  • a pure sample of amide 9b of Figure 4 15 (16 mg, 16%) was isolated by RP-HPLC (Symetry shield RP 18, 7 ⁇ m, 19x300 mm, with a flow rate of 10 niL/min using a 50 min gradient from water (0.1% TFA) to CH 3 CN (0.1%
  • Methyl 3 a- (4-bromophenyl) - 1, 8b-dihydroxy- 6, 8-dmethoxy- 1 -oxo-3- (3-fluoroph enyl) -2,3- 3a,8b-tetrahydro-cyclopenta[b]benzofuran-2-carboxylate (FL 17, 6d of Figure 4).
  • Glacial acetic acid (520 ⁇ L, 4.33 mmol) was added to a solution Of Me 4 NBH(OAc) 3 (1.42 g, 5.38 mmol) in CH 3 CN (80 mL).
  • N-methylmorpholine (114 ⁇ L, 10.4 mmol) was added at 0 0 C to a solution of amines 11a and lib of Figure 6 (100 mg) in CH 2 Cl 2 (2 mL). After 10 min at 0 0 C, mesyl chloride (64 ⁇ L, 0.82 mmol) was added dropwise and the solution was then stirred for 4 h at rt. The mixture was quenched with 5 mL of HCl 1 N, and extracted with EtOAc (2x10 mL). The organic layer was washed with a solution of Na 2 CO 3 (10 mL), brine (10 mL), dried over MgSO 4 , and concentrated under reduced pressure. The crude product was purified by flash chromatography on silica gel (CH 2 CVEt 2 O, 95:5) to give 12c of Figure 6 (FL24, 5 mg, 4%) and 12d of Figure 6 (FL25, 70 mg, 60%).
  • Example 4 Effect of rocaglaol and its derivatives on human cancer cell viability and on doxorubicin cardiotoxicity
  • Racemic rocaglaol (FLl) and its derivatives (FL2-4) were synthesised by the approach developed by Dobler and collaborators (Dobler et al, 2001) ( Figures 2 and 3).
  • the synthesis of FL3 and FL5 is detailed in examples 1 and 2.
  • the synthesis of FL6 to FL23 is detailed in example 3.
  • Rocaglaol derivatives FL5-11 were synthesized according to the strategy of Porco and collaborators (Gerard et al., 2004) ( Figure 3).
  • Rocaglaol derivatives FL5 to FLlO and FL 12 to FL 18 were synthesized according to the strategy of Figure 4.
  • Rocaglaol derivatives FL 19 to FL25 were synthesized according to the strategy of Figures 5 and 6.
  • KRIBB3 and doxorubicin were purchased from Sigma-Aldrich (Sigma-Aldrich, Saint- Quentin Fallavier, France).
  • Rabbit polyclonal anti-phospho-HSP27 (ser 82) antibody, rabbit polyclonal anti-HSP27 antibody and horseradish peroxidase-conjugated secondary goat anti- rabbit IgG were purchased from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
  • Enzyme-linked chemiluminescence were purchased from Amersham (Amersham Biosciences, Indianapolis, IN, USA). All others chemicals were purchased from Becton Dickinson (Becton Dickinson Biosciences, Le Pont De Claix, France).
  • Cell lines were purchased from Sigma-Aldrich (Sigma-Aldrich, Saint- Quentin Fallavier, France).
  • the human cell lines KB (epidermoid carcinoma) and HepG2 (hepatocarcinoma) were obtained from ECACC (Salisbury, UK) and grown in D-MEM medium supplemented with 10% fetal calf serum, in the presence of penicilline, streptomycine and fungizone in 75 cm 2 flask under 5% CO2.
  • the human cell lines HCTl 16 (colon adenocarcinoma), PC3 (prostate adenocarcinoma) and HCT 15 (colon adenocarcinoma) were obtained from ECACC (Wiltshire, UK).
  • the human cell lines HL60 promyeocytic leukaemia
  • MDA435 breast ductal carcinoma; ATCC reference HBT 129
  • MDA231 breast adenocarcinoma; ATCC reference HBT26
  • MCF7 cowast adenocarcinoma
  • SK-OV3 cells or OV3, ATCC reference HTB77 were kindly provided by NCI (Frederick, MD).
  • the cell lines HCTl 16, HCT15, MCF7, MDA435, MDA231, PC3, OV3, HL60R and HL60 were grown in RPMI medium, in particular supplemented with 10% fetal calf serum, in the presence of penicillin, streptomycin and fungizone in 75 cm 2 flask under 5% CO2. Resistant
  • HL60 cells were obtained by prolonged treatment with doxorubicin (50 ⁇ g/L medium).
  • Resistant MCF7 (MCF7R) cells were obtained by prolonged treatment with doxorubicin.
  • H9c2 cells rat heart myoblast
  • D-MEM medium supplemented with 10% foetal calf serum at 37°C in 5% CO 2 . The medium was changed every 2-3 days.
  • H9c2 cells were plated for 24h in 100 mm Petri dishes at 0,7 X 10 4 cells/cm 2 .
  • the cells were washed and cultured for 12h in glucose-free medium (Gibco, DMEM w- L-glutamine, w/o D-glucose, sodium pyruvate) supplemented with only 1% fetal calf serum.
  • Cells were then treated with flavaglines derivatives or their vehicle for 12h and then were treated with doxorubicin for an additional 14h.
  • Kribb3 (1 ⁇ M) was preincubated for Ih before flavaglines and doxorubicin treatment in cardiomyocytes. Cells were washed, and subsequently FACS analyses were performed.
  • Apoptosis was analysed by fluorescence-activated cell sorting analysis (FACS-Calibur, Becton-Dickinson Biosciences). 0,5 X 10 6 cells were harvested, washed with Annexin Binding Buffer (0,01 M HEPES, 0,14 M NaCl, 2,5 mM CaCl 2 ) and labeled with annexin V (dilution 1 :50) and propidium iodide (6,7 ⁇ g/ml). All assays were performed at least in triplicate, and the results were analyzed by BD Cell Quest Pro software (Becton-Dickinson Biosciences, Le Pont De Claix, France).
  • Taxotere 0.17 0.5 531 0.52 0.25 0.018 0.51 1.4
  • the inventors observed that FL3 maintained its cytotoxicity on HL60 and MCF7 cell lines, even when they became resistant to doxorubicin.
  • the inventors started their structure-activity relationship investigation by introducing an ester or amide moiety on position 2 to FL3.
  • Methyl ester FLlO exhibited a slightly reduced cytotoxicity than did compound FL3.
  • introduction of a tertiary amide (FL 12) significantly enhanced cytotoxicity. This effect was more pronounced with secondary amide FL 13 and primary amide FL 14 on most cell lines.
  • both FL 13 and FL 14 were less active that reference compound FL3 on HL60R cells, which have developed resistance to chemotherapy by overexpressing the P-glycoprotein (P-gp), a plasma membrane protein encoded by the multidrug resistance (MDRl) gene.
  • P-gp P-glycoprotein
  • MDRl multidrug resistance
  • 8-Demethoxy compounds FL6, FL5, FL8 and FL9 were significantly less active (ED50 4 to 20 times higher) than cognate compounds FL3, FLlO, FL12 and FL13, indicating a preference but not an absolute requirement of a methoxy group in position 8 for cytotoxicity.
  • this 8-demethoxy series the introduction of an amide at C-2 diminished also the cytotoxicity on HL60R.
  • HepG2 cells of hepatocellular carcinoma which display a low sensitivity toward clinically used anticancer drugs.
  • Apoptosis was induced in H9c2 cardiomyocytes by doxorubicin and detected by FACS analysis after labeling the cells with annexin and PI.
  • These cardiomyoblasts derived from rat heart represent an established model of doxorubicin cardiotoxicity in vitro. Incubation of the cells with 1 ⁇ M doxorubicin for 14h induced 32% apoptosis (total apoptotic cells). It was observed that pre-incubation of H9c2 cells with different concentrations of rocaglaol and its derivatives significantly reduced the apoptosis induced by doxorubicin ( Figures 8 and 9).
  • FL3 protects cardiomvocvtes against serum starvation
  • the inventors examined whether FL3 could protect H9c2 cardiomyocytes from stress due to serum starvation (1% serum).
  • FL3 (20 nM) strongly reduced the apoptosis induced by a 72 h serum starvation treatment ( Figure 11).
  • FL3 alleviates doxorubicin cardiotoxicity in mice
  • FL3 displays some cardioprotection in vivo.
  • Administration of FL3 (0.1 mg/kg i.p., 2x/day for 4 days) protected mice from the cardiotoxicity induced by doxorubicine (20 mg/kg i.p.): they observed a 70% survival rate for the mice that received FL3.
  • the survival rate for the mouse who received the vehicle was only 30% (see Table 3). This data indicates also that FL3 displays a proper bioavailabity.
  • Doxorubicin (1 x, 20 mg/kg i.p.) 30 %
  • Doxorubicin (1 x, 20 mg/kg i.p.) + 7n 0/ FL3 (8 x, 0.1 mg/kg i.p.)
  • KRIBB3 a synthetic molecule that inhibits HSP25/27 phosphorylation.
  • KRIBB3 a synthetic molecule that inhibits HSP25/27 phosphorylation.
  • a 1 h treatment of H9c2 cardiomyocytes with KRIBB3 (1 ⁇ M) induced a cytotoxicity that was significantly alleviated by a co-treatment with FL3 (100 nM), indicating that FL3 effects are opposed to those of KRIBB3.
  • doxorubicin induces an oxidative stress, while serum starvation blocks growth factors signaling that is necessary to cell survival.
  • Anthracyclines such as doxorubicin, epirubicin, and daunorubicin are among the most anticancer drugs used in chemotherapy, in spite of their acute and chronic cardiotoxicities, which are dose related, cumulative and irreversible. Eventually, these myocardial dysfunctions can lead to congestive heart failure. Fortunately, the anticancer effects and the cardiotoxicity are not mediated by the same mechanisms.
  • the former is due to an inhibition of topoisomerase II, while the latter is caused by reactive reactive oxygen species (ROS) that are generated by a redox cycling that involves the reversible reduction of the anthracycline by NADPH and a reoxidation by oxygen.
  • ROS reactive reactive oxygen species
  • the iron chelator dexrazoxane is the only cardioprotective agents with proven efficacy in cancer patients receiving anthracycline chemotherapy. However this efficacy is limited and effects on survival failed to be established.
  • the relative order of activity (FL2 ⁇ FL4 « FLl ⁇ FL3) for cardioprotection were similar to those for cytotoxicity for cancer cells, suggesting that these compounds probably act through the same molecular target.

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Abstract

The present invention discloses new rocaglaol derivatives and the use of rocaglaol derivatives to prevent or to limit the cardiotoxicity of an antineoplastic agent, in particular to prevent or to limit the apoptosis of cardiomyocytes induced by such agent.

Description

Rocaglaol derivatives as cardioprotectant agents and as antineoplastic agents
Field of the Invention
The present invention relates to the prevention of antineoplastic agent cardiotoxicity by using new cardioprotectant agents. In addition, the present invention also relates to new antineoplastic agents.
Background of the Invention
Cardiotoxicity is a major side effect of various antineoplastic agents including, for instance, anthracyclines, cyclophosphamide, fluorouracil, rituximab, cisplatine, gleevec or herceptin (Pai et al, 2000). Among them, anthracyclines are a class of chemotherapeutic agents which is used to treat a wide range of cancers, including, for instance, leukemias, lymphomas, and breast, uterine, ovarian, and lung cancers. Nevertheless, the clinical utility of anthracycline anticancer agents, especially doxorubicin, is limited by a progressive toxic cardiomyopathy linked to mitochondrial damage and cardiomyocyte apoptosis (Swain et al., 2003). This cardiotoxicity often presents as electrocardiogram changes and arrhythmias, or as a cardiomyopathy leading to congestive heart failure, sometime many years after treatment (Steinherz et al., 1995), and is related to a patient's cumulative lifetime dose. Some recent studies have revealed that when the cumulative dose of doxorubicin reaches 700 mg/m2, the risks of developing congestive heart failure dramatically increase to 48% (Swain et al., 2003). Then, this dose is calculated during treatment, and anthracycline treatment is usually stopped or re-evaluated upon reaching the maximum cumulative dose of the particular anthracycline. Important researches have been carried out to identify methods or drugs which are able to prevent or limit the cardiotoxicity of anthracyclines (Wounters et al., 2005). Several strategies have been explored (e.g. US 2007-834799, US 2007-711490, WO 2006/063091 or WO 2007/101925), but the use of dexrazoxane (Zinecard®, Cardioxane®) remains the only one with a clinical proven efficacy. Dexrazoxane is then usually used in conjunction with doxorubicin as a cardioprotectant agent to reduce the risk of cardiotoxicity side effects of anthracycline chemotherapy. As a derivative of EDTA, dexrazoxane chelates iron, but the precise mechanism by which it protects the heart is not known.
Nevertheless, the use of dexrazoxane does not provide a full protection against heart damages and some studies revealed that patients treated with it might have a lower anti-tumour response rate to the anthracycline treatment (Van Dalen, et al., 2005; Van Dalen, et al., 2008). Moreover, the effectiveness of dexrazoxane in children is not established yet. Therefore, there is a strong need for cardioprotectant agents that can efficiently prevent the cardiotoxicity of anthracyclines or other antineoplastic agents without having any deleterious effect on the anti-tumour response. This need is still stronger for children who are more susceptible to cardiotoxicity of these agents.
Summary of the Invention
The object of the present invention is to provide new cardioprotectant agents. The inventors have shown that rocaglaol and some of its derivatives are able to prevent or limit apoptosis induced by the cardiotoxicity of antineoplastic agents, without any deleterious effect on the anti-tumor activity of these agents.
In a first aspect, the invention concerns a rocaglaol derivative of the formula (II)
Figure imgf000003_0001
wherein
R1 ! is alkoxy, optionally substituted; R12 is hydrogen; or R11 and R12 together form a -0-CH2-O- unit;
R13 is selected from hydrogen and (Ci-C3)-alkoxy;
R is selected from hydroxyl, oxo group, -OCOR , R being selected from hydrogen and (Ci-C3)-alkyl, =N-OR26 with R26 being H or methyl, -NH-(CH2)m-R27, wherein m is 0 or 1 and R27 is selected from H, -OH, -SO2Me, -COR28 with R28 being H or (Ci-C3)- alkyl;
R15 is selected from hydrogen, -COOR21, -CONR22R23 and -C0NH(CH2)n0H, wherein n is 2, 3 or 4, wherein
R21 and R23 are independently selected from hydrogen and (Ci-C3)-alkyl and R22 is selected from the group consisting of (Ci-C3)-alkoxy, (Ci-C3)-alkyl and hydrogen, or
R22 and R23 together form a 5 atom heterocycle, optionally substituted by a group NHCO-(Ci-C4)-alkyl; or R14, R15and carbons (α) and (β) bearing R14 and R15 together form
Figure imgf000004_0001
wherein R24 and R25 are independently selected from the group consisting of (C1-C3)- alkyl and hydrogen, preferably from hydrogen and methyl; R16 is selected from hydrogen, (Ci-C3)-alkoxy and halogen; R17 is in ortho-position to Ri6 and is selected from hydrogen, (Ci-C3)-alkoxy and hydroxyl or form together with R17 a -0-CH2-O- unit;
R18 and R19 are independently selected from the group consisting of hydrogen and halogen, preferably from hydrogen, F, Br and Cl, more preferably are hydrogen or F; or any pharmaceutically acceptable salt thereof, for use for preventing or limiting the cardiotoxicity of an antineoplastic agent, preferably an anthracycline, more preferably doxorubicin.
In another aspect, the invention also concerns a method for preventing or limiting the cardiotoxicity of an antineoplastic agent in a subject comprising administering a therapeutically effective amount of a rocaglaol derivative of the formula (II) as described above or any pharmaceutically acceptable salt thereof to said subject. The present invention further concerns the use of a rocaglaol derivative of the formula (II) as described above or any pharmaceutically acceptable salt thereof for preparing a medicament for preventing or limiting the cardiotoxicity of an antineoplastic agent, preferably an anthracycline, more preferably doxorubicin. Preferably, the rocaglaol derivative of the formula (II) has one or several of the following features: a) R12 is H and R11 is a (Ci-C3)-alkoxy, preferably selected from methoxy and ethoxy, more preferably R11 is methoxy; b) R13 is selected from hydrogen, methoxy and ethoxy, more preferably R13 is hydrogen or methoxy; c) R14 is selected from hydroxyl, -OCOR20 with R20 being selected from hydrogen, methyl and ethyl, -NH-(CH2)m-R27, wherein m is 0 and R27 is -SO2Me or -COR28 with R28 being H or (Ci-C3)-alkyl, preferably H, methyl or ethyl, more preferably H or methyl; d) R15 is selected from hydrogen, -COOR21, and -CONR22R23, wherein R21 and R23 are independently selected from hydrogen and methyl, and R22 is selected from the group consisting of hydrogen, methyl and methoxy; more preferably R15 is selected from hydrogen, COOMe, CONH2, CONHMe and CONMe2; e) R16 is selected from hydrogen, (Ci-C3)-alkoxy and halogen, more preferably from hydrogen, chlorine, bromine, fluorine and methoxy, still more preferably from bromine and methoxy; f) R17 is hydrogen; g) R18 is in para and is H or fluorine and R19 is hydrogen. In a particular embodiment, the rocaglaol derivative presents the formula (Ha) or (lib)
Figure imgf000005_0001
wherein the definition of RI l, R12, R13, R14, R15, R16, R17, R18 and R19 is the same as above. More particularly, the rocaglaol derivative has the formula (Ha) and R14 is selected from hydroxyl, -OCOR20 with R20 being selected from hydrogen and (Ci-C3)-alkyl, and =N-OR26 with R26 being H or methyl. Preferably, R14 is selected from hydroxyl and -OCOR20 with R20 being selected from hydrogen, methyl and ethyl. More preferably, R11 is alkoxy, especially methoxy or ethoxy, more preferably methoxy; R12 is hydrogen; R13 is selected from hydrogen, methoxy and ethoxy, more preferably R13 is hydrogen or methoxy; R15 is selected from hydrogen, COOMe, CONH2, CONHMe and CONMe2; R16 is selected from the group consisting of hydrogen, chlorine, bromine, fluorine and methoxy, more preferably of bromine and methoxy; R18 is in para and is H or fluorine; and, R17 and R19 are hydrogen. Alternatively, the rocaglaol derivative has the formula (lib) and R14 is -NH-(CH2)m-R27, wherein m is O and R27 is -SO2Me or -COR28 with R28 being H or (Ci-C3)-alkyl, preferably H or methyl. In particular, R11 is alkoxy, especially methoxy or ethoxy, more preferably methoxy; R12 is hydrogen; R13 is selected from hydrogen, methoxy and ethoxy, more preferably R13 is hydrogen or methoxy, still more preferably R13 is methoxy; R15 is selected from hydrogen, COOMe, CONH2, CONHMe and CONMe2, more preferably is hydrogen; R16 is selected from the group consisting of hydrogen, chlorine, bromine, fluorine and methoxy, more preferably is chlorine, bromine, or fluorine, still more preferably is bromine; and, R17, R18 and R19 are hydrogen.
In a particularly preferred embodiment, the rocaglaol derivative is selected from the group consisting of FLl, FL3, FL5, FL6, FL7, FL8, FL9, FLlO, FL12, FL13, FL14, FL15, FL16, FL17, FL18, FL19, FL20, FL21, FL22, FL23, FL24 and FL25, more preferably from the group consisting of FLl, FL3, FL5, FL8, FL9, FLlO, FL12, FL13, FL14, FL15, FL16, FL17, FL18, FL19, FL20, FL21, FL22, FL23 and FL25.
In a second aspect, the invention provides new rocaglaol derivatives of the formula (I)
OH
Figure imgf000006_0001
wherein
R3 is -CONH2; and wherein
R1 is alkoxy, optionally substituted, preferably selected from the group consisting of methoxy and a group -O-(CH2)n-R10 wherein n is 1, 2, 3 or 4 and R10 is selected from the group consisting of hydroxyl, -NMe2, -OCONMe2, -OCONH2 and morpholine;
R2 is selected from the group consisting of hydrogen and (Ci-C3)-alkoxy, preferably from hydrogen and methoxy; and, R4 is halogen, preferably bromine, chloride, iodide, more preferably bromine, or,
R1 is alkoxy, optionally substituted, preferably selected from the group consisting of methoxy and a group -O-(CH2)n-R10 wherein n is 1, 2, 3 or 4 and R10 is selected from the group consisting of hydroxyl, -NMe2, -OCONMe2, -OCONH2 and morpholine;
R2 is hydrogen; and,
R4 is selected from the group consisting of hydrogen and alkoxy, or, R1 is substituted alkoxy, preferably a group -O-(CH2)n-R10 wherein n is 1, 2, 3 or
4 and R10 is selected from the group consisting of hydroxyl, -NMe2, -OCONMe2, - OCONH2 and morpholine; R2 is (Ci-C3)-alkoxy, preferably methoxy; and R4 is selected from the group consisting of hydrogen and alkoxy, or wherein
R3 is -COOR5 wherein R5 is (Ci-C3)-alkyl, preferably methyl;
R1 is alkoxy, optionally substituted, preferably selected from the group consisting of methoxy and a group -O-(CH2)n-R10 wherein n is 1, 2, 3 or 4 and R10 is selected from the group consisting of hydroxyl, -NMe2, -OCONMe2, -OCONH2 and morpholine;
R2 is hydrogen; and
R4 is selected from the group consisting of hydrogen, methoxy and halogen; or wherein
R1 is methoxy, and R4 is bromine, and wherein
R2 is (Ci-C3)-alkoxy, preferably methoxy, and, R3 is hydrogen, or
R2 is hydrogen, and, R3 is selected from the group consisting of hydrogen, -COOR5 and -CONR6R7, wherein R5 is (Ci-C3)-alkyl, preferably methyl, and R6 and R7 are independently selected from the group consisting of hydrogen and (Ci-C3)-alkyl, preferably from hydrogen and methyl; or any pharmaceutically acceptable salt thereof. In a preferred embodiment, R1 is selected from the group consisting of methoxy; R2 is hydrogen; R3 is selected from the group consisting of -COOR5 and -CONH2, wherein R5 is methyl; and, R4 is halogen or methoxy, preferably bromine or methoxy.
In another preferred embodiment, R1 is methoxy; R2 is selected from the group consisting of methoxy or hydrogen; R3 is -CONH2; and, R4 is halogen, preferably bromine. In a further preferred embodiment, R1 and R2 are methoxy, R3 is hydrogen and R4 is bromine. In an additional preferred embodiment, R1 is methoxy; R2 is hydrogen; R3 is selected from the group consisting of hydrogen, -COO(CH3) and -CON(CH3)2; and, R4 is bromine. In another preferred embodiment, R3 is COOMe, R1 is methoxy, R2 is hydrogen and R4 is selected from the group consisting of hydrogen, methoxy and bromine, preferably is methoxy or bromine.
In a further preferred embodiment, R1 is methoxy, and R4 is bromine, and wherein either R2 is methoxy and R3 is hydrogen, or R2 is hydrogen and R3 is selected in the group consisting of hydrogen, -COO(CH3), -CONH2, -CONH(CH3) and -CON(CH3)2.
In particular, the rocaglaol derivative is selected from the group consisting of FL3, FL5, FL6, FL7, FL8, FL9 and FL14.
The present invention further provides a new rocaglaol derivative the formula (II), wherein R11 is alkoxy, optionally substituted; R12 is hydrogen; or R11 and R12 together form a -O-CH2-O-unit; R13 is selected from hydrogen and (Ci-C3)-alkoxy;
R14 is -NH-R27, wherein R27 is selected from -SO2Me, -COR28 with R28 being H or (Ci- C3)-alkyl;
R15 is selected from hydrogen, -COOR21, -CONR22R23 and -C0NH(CH2)n0H, wherein n is 2, 3 or 4, wherein
R21 and R23 are independently selected from hydrogen and (Ci-C3)-alkyl and R22 is selected from the group consisting of (Ci-C3)-alkoxy, (Ci-C3)-alkyl and hydrogen, or R22 and R23 together form a 5 atom heterocycle, optionally substituted by a group
NHCO-(Ci-C4)-alkyl; or R14, R15and carbons (α) and (β) bearing R14 and R15 together form
wherein R24 and R25 are independently selected from the group consisting of (C1-C3)- alkyl and hydrogen, preferably from hydrogen and methyl;
R16 is selected from hydrogen, (Ci-C3)-alkoxy and halogen;
R17 is in ortho-position to Ri6 and is selected from hydrogen, (Ci-C3)-alkoxy and hydroxyl or form together with R17 a -O-CH2-O-unit;
R18 and R19 are independently selected from the group consisting of hydrogen and halogen, preferably from H, F, Br and Cl, more preferably H or F; or any pharmaceutically acceptable salt thereof.
In a preferred embodiment, the new rocaglaol derivative of the formula (II) has one or several of the following features: a) R11 is alkoxy and R12 is hydrogen; b) R13 is selected from hydrogen and methoxy; c) R14 is -NH-R27, wherein R27 is selected from -SO2Me, -COR28 with R28 being H, methyl or ethyl; d) R15 is selected from hydrogen, -COOR21, and -CONR22R23, wherein R21 and R23 are independently selected from hydrogen and methyl, and R22 is selected from the group consisting of hydrogen, methyl and methoxy; more preferably R15 is selected from hydrogen, COOMe, CONH2, CONHMe and CONMe2; e) R16 is selected from hydrogen, (Ci-C3)-alkoxy and halogen, more preferably from hydrogen, chlorine, bromine, fluorine and methoxy, still more preferably from bromine and methoxy; f) R17 is hydrogen; g) R18 is in para and is H or fluorine and R19 is hydrogen.
In particular, the rocaglaol derivative may present the formula (Ha) or (lib). More preferably, the rocaglaol derivative has the formula (lib) and R14 is -NH-R27, wherein R27 is -SO2Me or -
COR28 with R28 being H or (Ci-C3)-alkyl, preferably H or methyl. In a preferred embodiment, R11 is alkoxy, especially methoxy or ethoxy, more preferably methoxy; R12 is hydrogen; R13 is selected from hydrogen, methoxy and ethoxy, more preferably R13 is hydrogen or methoxy, still more preferably R13 is methoxy; R15 is selected from hydrogen, COOMe, CONH2, CONHMe and CONMe2, more preferably is hydrogen; R16 is selected from the group consisting of hydrogen, chlorine, bromine, fluorine and methoxy, more preferably is chlorine, bromine, or fluorine, still more preferably is bromine; and, R17,
R18 and R19 are hydrogen.
In a particular embodiment, the rocaglaol derivative is selected from the group consisting of
FL22, FL23, FL24 and FL25, preferably is FL23 or FL25.
In addition, the present invention provides a new rocaglaol derivative the formula (Ha), wherein
R12 is H and R11 is selected from methoxy and ethoxy, more preferably R11 is methoxy;
R13 is selected from hydrogen, methoxy and ethoxy, more preferably R13 is hydrogen or methoxy;
R14 is -OCOR20, R20 being selected from hydrogen, methyl and ethyl; R15 is selected from hydrogen, -COOR21, and -CONR22R23, wherein R21 and R23 are independently selected from hydrogen and methyl, and R 22 is selected from the group consisting of hydrogen, methyl and methoxy; more preferably R 15 is selected from hydrogen,
COOMe, CONH2, CONHMe and CONMe2; still more preferably R15 is hydrogen; Rl 6 is chlorine, bromine, or fluorine, preferably bromine;
R17 is hydrogen;
R18 is in para and is H or fluorine and R19 is hydrogen; or any pharmaceutically acceptable salt thereof.
More preferably, R11 and R13 are methoxy, R12, R15, R18 and R19 are hydrogen, and R16 is bromine.
In a particular embodiment, the rocaglaol derivative is selected from the group consisting of
FL 19, FL20 and FL21.
Furthermore, the present invention provides a new rocaglaol derivative the formula (Ha), wherein R12 is H and R11 is selected from methoxy and ethoxy, more preferably R11 is methoxy;
R13 is selected from hydrogen, methoxy and ethoxy, more preferably R13 is hydrogen or methoxy;
R14 is hydroxyl;
R18 is in para and is fluorine and R19 is hydrogen, and wherein
R15 is selected from hydrogen, -COOR21, and -CONR22R23, wherein R21 and R23 are independently selected from hydrogen and methyl, and R22 is selected from the group consisting of hydrogen, methyl and methoxy; more preferably R15 is selected from hydrogen, COOMe, CONH2, CONHMe and CONMe2; R16 is bromine and R17 is hydrogen; or
R15 is selected from -COOR21, and -CONR22R23, wherein R21 and R23 are independently selected from hydrogen and methyl, and R22 is selected from the group consisting of hydrogen, methyl and methoxy; more preferably R15 is selected from hydrogen, COOMe, CONH2, CONHMe and CONMe2; and,
R16 is methoxy and R17 is hydrogen; or any pharmaceutically acceptable salt thereof.
More preferably, R11 and R13 are methoxy, R12 is hydrogen, R16 is bromine and R15 is selected from hydrogen, COOMe, CONH2, CONHMe and CONMe2. In an alternative preferred embodiment, R11 and R13 are methoxy, R12 is hydrogen, R16 is methoxy and R15 is selected from COOMe, CONH2, CONHMe and CONMe2. Optionally, R11 and R13 are methoxy, R12 is hydrogen, and R15 is selected from COOMe, CONH2, CONHMe and CONMe2.
In a particular embodiment, the rocaglaol derivative is FL16, FL17 or FL18. In a second aspect, the present invention concerns a new rocaglaol derivative according to the invention as a medicament.
In another aspect, the present invention concerns a pharmaceutical composition comprising a new rocaglaol derivative according to the present invention and a pharmaceutically acceptable carrier and/or excipient. The present invention also concerns a pharmaceutical composition comprising a new rocaglaol derivative according to the present invention and an antineoplastic agent, preferably an anthracycline.
The present invention further concerns a product containing a new rocaglaol derivative according to the present invention and an antineoplastic agent, preferably an anthracycline, as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer.
Legends of the Figures
Figure 1 shows a schematic representation of the synthesis of racemic bromo-demethoxy- rocaglaol (FL3).
Figure 2 shows a schematic representation of the synthesis of racemic rocaglaol (FLl) and its analogues (FL2 to FL4).
Figure 3 shows a schematic representation of the synthesis of racemic flavaglines FL5 to
FLI l. Figure 4 shows a schematic representation of the synthesis of racemic flavaglines FL5 to
FLlO, and FL12 to FL18. Reactants and conditions: (a) hv, (E)-PhCH=CHCOOMe, CH2Cl2-
MeOH, 0 0C, 15 h; (b) MeONa, MeOH, 60 0C, 20 min; (c) LiCl, H2O, DMSO, 100 0C, 12 h;
(d) Me4NBH(OAc)3, AcOH, CH3CN; (e) (i) KOH, MeOH, 45 0C, 12 h, (ii) Me2MLHCl or
MeNH2-HCl, ECDI, HOBT, DIPEA, CH2Cl2, rt, 12 h; f) NH3, MeOH, 100 0C, 36 h; (g) hn, CH2Cl2-MeOH, 0 0C, 15 h; (h) hv, CH2Cl2-MeOH, 0 0C, 15 h.
Figure 5 shows a schematic representation of the synthesis of racemic flavaglines FL 19 to
FL23. Reactants and conditions: (i) DCC, DMAP, HCOOH, CH2Cl2, rt, 36 h; (j) (R3CO)2O,
DMAP, pyr, rt, 6 h; (k) H2NOKHCl, pyr, EtOH, 70 0C, 4 h; (1) LiAlH4, THF, 45°C, 3 h; (m)
HCOOEt, THF, reflux, 12 h. Figure 6 shows a schematic representation of the synthesis of racemic flavaglines FL22 to
FL25. Reactants and conditions: (k) H2N0Me.HCl, pyr, EtOH, 70 0C, 4 h; (1) LiAlH4, THF,
45°C, 3 h; (m) HCOOEt, THF, reflux, 12 h or MeSO2Cl, N-methylmorpholine , CH2Cl2, rt.
Figure 7 is a graph that shows the inhibition of cell proliferation of HepG2 cells exposed to FLl and FL3 with or without 150 nM doxorubicin. HepG2 cells were treated with FLl ( ) or
FL3 (O) alone or with doxorubicin (FLl : ■ , FL3: *) for 72 h hours, and cell proliferation was measured by MTS assay. Experiments were performed in duplicate (n=3).
Figures 8A and 8B show the cardioprotective effect of FLl (Figure 8A) and FL3 (Figure 8B) on apoptosis induced by doxorubicin (lμM) in H9c2 cardioblast cells. These two graphs represent the relative total apoptotic cells obtained with doxorubicin alone or in combination with FLl (Figure 8A) or FL3 (Figure 8B), in comparison with the results obtained with the vehicle.
Figure 9 shows the cardioprotective effect of FL5, FL6, FL7 and FL8 on apoptosis induced by doxorubicin (1 μM) in H9c2 cells at different concentrations. Figure IQA shows a Western blot analysis demonstrating that doxorubicin induces caspase-3 activity (line 2) that is blocked by FL3 (1 nM) (line 3). Note that FL3 (line 4) and vehicle
(line 1) do not induce caspase-3 activity. Figure IQB is a histogram of caspase-3 activity according to Western blot quantification and normalization with GAPDH.
Figure 11 shows the protection by FL3 of H9c2 cardio myocytes against serum starvation. H9c2 were cultured in 1% serum in the presence of vehicle or FL3 (20 nM). Apoptosis was measured by FACS assay.
Figure 12 : Figure 12A) FL3 prevents doxorubicin and KRIBB3 induced apoptosis. H9c2 cardiomyocytes were treated with doxorubicin (lμM), or the HSP27 inhibitor KRIBB3 (lμM) in presence or absence of FL3 (100 nM) for 1 h and then labeled with annexin V and PI for FACS analysis. Figure 12B) Western blott analyses revealed that that FL3 maximally induces
HSP27 phosphorylation (upper bands) without altering total HSP27 levels (lower band) in 1 h in H9c2 cells.
Figure 13 shows the cardioprotective effect of FLl-IO and FL 12-18 at 10 (left) or 100 (right) nM on apoptosis induced by doxorubicin (1 mM) in H9c2 cells.
Detailed description of the invention
The inventors have surprisingly found that flavaglines, in particular rocaglaol and some of its derivatives, may act as cardioprotectant agents. These compounds are found to be able to prevent or limit the apoptosis induced by the cardio toxicity of antineoplastic agents such as anthracyclines, while potentiating the anti-tumor efficacy of these molecules. Flavaglines are a family of natural compounds extracted from Asian plants of the genus Aglaia (Kim et al., 2006 and Proksch et al., 2001), comprising, for instance, rocaglaol, rocaglamide or silvestrol. Some of these molecules are known to inhibit the proliferation of tumor cells in a low nanomolar range, either by cytostatic (Hausott et al., 2004) or cytotoxic effects (Kim et al., 2007 and Mi et al., 2006), without displaying any significant toxicity on normal cells. Up to now, flavaglines have only been known to exhibit anticancer, immunosuppressive, neuroprotective (Fahrig et al., 2005), antifungal insecticide and anti- inflammatory (EP 1 693 059) properties.
The present invention provides new cardioprotectant rocaglaol derivatives of the formula (I)
OH
Figure imgf000013_0001
wherein
R1 is alkoxy, optionally substituted, preferably selected from the group consisting of methoxy and a group -O-(CH2)n-R10 wherein n is 1, 2, 3 or 4 and R10 is selected from the group consisting of hydroxyl, -NMe2, -OCONMe2, -OCONH2 and morpholin;
R2 is selected from the group consisting of hydrogen and (Ci-C3)-alkoxy, preferably from hydrogen and methoxy;
R3 is selected from the group consisting of -COOR5 and -CONH2, wherein R5 is (C1-C3)- alkyl, preferably methyl; and
R4 is halogen, preferably bromine, chloride, iodide, more preferably bromine, or wherein,
R1 is alkoxy, optionally substituted, preferably selected from the group consisting of methoxy and a group -O-(CH2)n-R10 wherein n is 1, 2, 3 or 4 and R10 is selected from the group consisting of hydroxyl, -NMe2, -OCONMe2, -OCONH2 and morpholin;
R2 is hydrogen; R3 is selected from the group consisting of -COOR5 and -CONH2, wherein R5 is (C1-C3)- alkyl, preferably methyl; and
R4 is selected from the group consisting of hydrogen, halogen and alkoxy, preferably hydrogen or alkoxy, or wherein,
R1 is substituted alkoxy, preferably a group -O-(CH2)n-R10 wherein n is 1, 2, 3 or 4 and R10 is selected from the group consisting of hydroxyl, -NMe2, -OCONMe2, -OCONH2 and morpholine. R2 is selected from the group consisting of hydrogen and (Ci-C3)-alkoxy, preferably from hydrogen and methoxy; more preferably R2 is Ci-C3)-alkoxy, preferably methoxy; R3 is selected from the group consisting of -COOR5 and -CONH2, wherein R5 is (C1-C3)- alkyl, preferably methyl, and R4 is selected from the group consisting of hydrogen, halogen and alkoxy, more preferably preferably hydrogen or alkoxy, or any pharmaceutically acceptable salt thereof.
In one embodiment, R1 is selected from the group consisting of methoxy or 0-(CH2)n-0H wherein n is 1, 2 or 3, preferably R1 is a methoxy; R2 is hydrogen; R3 is selected from the group consisting of -COOR5 and -CONH2, wherein R5 is methyl; and, R4 is halogen or methoxy, preferably bromine or methoxy. In particular, the rocaglaol derivative may be FL5, FL7, and FL9.
In another embodiment, R1 is a substituted alkoxy -O-(CH2)n-R10 wherein n is 1, 2, 3 or 4 and R10 is selected from the group consisting of hydroxyl, -NMe2, -OCONMe2, -OCONH2 and morpholine, preferably R10 is hydroxyl; R2 is hydrogen or methoxy, preferably hydrogen; R3 is selected from the group consisting of -COOR5 and -CONH2, wherein R5 is (Ci-C3)-alkyl, preferably methyl; and, R4 is halogen or methoxy, preferably bromine or methoxy. In a particular embodiment, R1 is a substituted alkoxy -0-(CH2)J1-R1 ° wherein n is 2 and R10 is hydroxyl; R2 is hydrogen; R3 is -COOR5, wherein R5 is methyl; and R4 is methoxy. In another particular embodiment, R1 is a substituted alkoxy -0-(CH2)J1-R1 ° wherein n is 3 and R10 is hydroxyl; R2 is hydrogen; R3 is -COOR5, wherein R5 is methyl; and R4 is methoxy. In a particular embodiment, R3 is -CONH2. In this embodiment, 1) R1 is alkoxy, optionally substituted, preferably selected from the group consisting of methoxy and a group -0-(CH2)n- R10 wherein n is 1, 2, 3 or 4 and R10 is selected from the group consisting of hydroxyl, -NMe2, -OCONMe2, -OCONH2 and morpholin; R2 is selected from the group consisting of hydrogen and (Ci-C3)-alkoxy, preferably from hydrogen and methoxy; and, R4 is halogen, preferably bromine, chloride, iodide, more preferably bromine; or 2) R1 is alkoxy, optionally substituted, preferably selected from the group consisting of methoxy and a group -0-(CH2)D-R1 ° wherein n is 1, 2, 3 or 4 and R10 is selected from the group consisting of hydroxyl, -NMe2, - OCONMe2, -OCONH2 and morpholin; R2 is hydrogen; and R4 is selected from the group consisting of hydrogen, halogen and alkoxy, preferably hydrogen or alkoxy; or, 3) R1 is substituted alkoxy, preferably a group -O-(CH2)n-R10 wherein n is 1, 2, 3 or 4 and R10 is selected from the group consisting of hydroxyl, -NMe2, -OCONMe2, -OCONH2 and morpholine; R2 is selected from the group consisting of hydrogen and (Ci-C3)-alkoxy, preferably from hydrogen and methoxy; more preferably R2 is Ci-C3)-alkoxy, preferably methoxy; and, R4 is selected from the group consisting of hydrogen, halogen and alkoxy, more preferably hydrogen or alkoxy. In particular, the rocaglaol derivative may be FL9 and FL14. In another particular embodiment, R3 is -COOR5 with R5 is (Ci-C3)-alkyl, preferably methyl. In this embodiment, R1 is alkoxy, optionally substituted, preferably selected from the group consisting of methoxy and a group -O-(CH2)n-R10 wherein n is 1, 2, 3 or 4 and R10 is selected from the group consisting of hydroxyl, -NMe2, -OCONMe2, -OCONH2 and morpholine; R2 is hydrogen; and, R4 is selected from the group consisting of hydrogen, methoxy and halogen. Preferably, R3 is COOMe, R1 is methoxy, R2 is hydrogen and R4 is selected from the group consisting of hydrogen, methoxy and bromine, preferably is methoxy or bromine. In particular, the rocaglaol derivative may be FL5 or FL7.
In a more preferred embodiment, the present invention concerns a compound of formula (I) wherein R1 is methoxy; R2 is selected from the group consisting of methoxy or hydrogen; R3 is selected from the group consisting of -COOR5, and -CONH2, wherein R5 is methyl; and, R4 is halogen, preferably bromine.
In a particular embodiment, R1 is methoxy, R2 is hydrogen, R3 is -CONH2 and R4 is bromine
(FL9).
In another preferred embodiment, R1 is methoxy; R2 is hydrogen; R3 is -COOR5, wherein R5 is methyl; and, R4 is halogen or methoxy, preferably bromine or methoxy. In a particular embodiment, R1 is methoxy; R2 is hydrogen; R3 is -COOR5, wherein R5 is methyl; and R4 is bromine (FL5).
In another particular embodiment, R1 is methoxy; R2 is hydrogen; R3 is -COOR5, wherein R5 is methyl; and R4 is methoxy (FL7). In another aspect, the present invention also concerns a new rocagloal derivative of the formula (I), wherein
R1 is methoxy;
R2 is selected from the group consisting of hydrogen and (Ci-C3)-alkoxy, preferably from hydrogen and methoxy;
R3 is selected from the group consisting of hydrogen, -COOR5 and -CONR6R7, wherein
R5 is (Ci-C3)-alkyl, preferably methyl, and
R6 and R7 are independently selected from the group consisting of hydrogen and (C1-C3)- alkyl, preferably from hydrogen and methyl; and R4 is bromine, or any pharmaceutically acceptable salt thereof.
Preferably, it concerns a rocagloal derivative of the formula (I), wherein
R1 is methoxy; and R4 is bromine, and wherein R2 is (Ci-C3)-alkoxy, preferably methoxy, and,
R3 is hydrogen, or
R2 is hydrogen, and,
R3 is selected from the group consisting of hydrogen, -COOR5 and -CONR6R7, wherein R5 is (Ci-C3)-alkyl, preferably methyl, and R6 and R7 are independently selected from the group consisting of hydrogen and (Ci-C3)-alkyl, preferably from hydrogen and methyl; or any pharmaceutically acceptable salt thereof.
In a preferred embodiment, R1 and R2 are methoxy, R3 is hydrogen and R4 is bromine (FL3). In a further preferred embodiment, R1 and R2 are methoxy, R3 is -CONH2, and R4 is bromine
(FL14).
In another embodiment, R1 is methoxy; R2 is hydrogen; R3 is selected from the group consisting of hydrogen, -COO(ClHb) and -CON(CF^)2, preferably of hydrogen and -
CON(CF^)2; and R4 is bromine. In a preferred embodiment, R1 is methoxy; R2 is hydrogen; R3 is -CON(CF^)2 and R4 is bromine (FL8). In another preferred embodiment, R1 is methoxy;
R2 is hydrogen; R3 is hydrogen and R4 is bromine (FL6).
In a further aspect, the present invention also concerns a new rocagloal derivative of the formula (II), wherein R1 ' is alkoxy, optionally substituted;
R12 is hydrogen; or R11 and R12 together form a -O-CH2-O-unit; R13 is selected from hydrogen and (Ci-C3)-alkoxy; R14 is -NH-R27, wherein R27 is selected from -SO2Me, -COR28 with R28 being H or (Ci- C3)-alkyl;
R15 is selected from hydrogen, -COOR21, -CONR22R23 and -C0NH(CH2)n0H, wherein n is 2, 3 or 4, wherein
R21 and R23 are independently selected from hydrogen and (Ci-C3)-alkyl and R22 is selected from the group consisting of (Ci-C3)-alkoxy, (Ci-C3)-alkyl and hydrogen, or
R22 and R23 together form a 5 atom heterocycle, optionally substituted by a group
NHCO-(Ci-C4)-alkyl; or R14, R15and carbons (α) and (β) bearing R14 and R15 together form
Figure imgf000017_0001
wherein R24 and R25 are independently selected from the group consisting of (C1-C3)- alkyl and hydrogen, preferably from hydrogen and methyl; R16 is selected from hydrogen, (Ci-C3)-alkoxy and halogen;
R17 is in ortho-position to Ri6 and is selected from hydrogen, (Ci-C3)-alkoxy and hydroxyl or form together with R17 a -O-CH2-O-unit; R18 and R19 are independently selected from the group consisting of hydrogen and halogen, preferably from H, F, Br and Cl, more preferably H or F; or any pharmaceutically acceptable salt thereof.
In this embodiment, the rocaglaol derivative of the formula (II) has preferably one or several of the following features: a) R11 is alkoxy and R12 is hydrogen; b) R13 is selected from hydrogen and methoxy; c) R14 is -NH-R27, wherein R27 is selected from -SO2Me, -COR28 with R28 being H, methyl or ethyl; d) R15 is selected from hydrogen, -COOR21, and -CONR22R23, wherein R21 and R23 are independently selected from hydrogen and methyl, and R22 is selected from the group consisting of hydrogen, methyl and methoxy; more preferably R15 is selected from hydrogen, COOMe, CONH2, CONHMe and CONMe2; e) R16 is selected from hydrogen, (Ci-C3)-alkoxy and halogen, more preferably from hydrogen, chlorine, bromine, fluorine and methoxy, still more preferably from bromine and methoxy; f) R17 is hydrogen; g) R18 is in para and is H or fluorine and R19 is hydrogen.
Optionally, the rocaglaol derivative of the formula (II) meets one feature, two features [for instance a) and b); a) and c); a) and d); a) and e); a) and f); a) and g); b) and c); b) and d); b) and e); b) and f); b) and g); c) and d); c) and e); c) and f); c) and g); d) and e); d) and f); d) and g); e) and f); e) and g); f) and g)], three features [for instance a), b) and c); a), b) and d); a), b) and e); a), b) and f); a), b) and g); a), c) and d); a), c) and e); a), c) and f); a), c) and g); a), d) and e); a), d) and f); a), d) and g); a), e) and f); a), e) and g); b), c) and d); b), c) and e); b), c) and f); b), c) and g); c), d) and e); c), d) and f); c), d) and g); d), e) and f); d), e) and g); e), f) and g)], four features [a), b), c) and d); a), b), c) and e); a), b), c) and f); a), b), c) and g); a), b), d) and e); a), b), d) and f); a), b), d) and g); a), b), e) and f); a), b), e) and g); a), b), f) and g); a), c), d) and e); a), c), d) and f); a), c), d) and g); a), c), e) and f), a), c), e) and g); a), d), e) and f); a), d), e) and g); a), d), f) and g); a), e), f) and g); b), c), d) and e); b), c ),d) and f); b), c), d) and g); b), d), e) and f); b), d), e) and g); b), e), f) and g); c), d), e) and f); c), d), e) and g); c), e), f) and g); d), e), f) and g)], five features [for instance a), b), c), d) and e); a), b), c), d) and f); a), b), c), d) and g); a), b), c), e) and f); a), b), c), e) and g); a), b), c), f) and g); a), b), d), e) and f); a), b), d), e) and g); a), b), d), f) and g); a), b), e), f) and g); a), c), d), e) and f); a), c), d), e) and g); a), c), d), f) and g); a), c), e), f) and g); a), d), e), f) and g); b), c), d), e) and f); b), c), d), e) and g); b), c), e), f) and g); b), d), e), f) and g); c), d), e), f) and g)], six features [a), b), c), d), e) and f); a), b), c), d), e) and g); a), b), c), d), f) and g); a), b), c), e), f) and g); a), b), d), e), f) and g); a), c), d), e), f) and g); b), c), d), e), f) and g)] or all the seven features a), b), c), d), e), f) and g).
In particular, the rocaglaol derivative may present the formula (Ha) or (lib), the definition of RI l, R12, R13, R14, R15, R16, R17, R18 and R19 is the same as above. In a preferred embodiment, the rocaglaol derivative has the formula (lib) and R14 is -NH-R27, wherein R27 is -SO2Me or -COR28 with R28 being H or (Ci-C3)-alkyl, preferably H or methyl. More preferably, R11 is alkoxy, especially methoxy or ethoxy, more preferably methoxy; R12 is hydrogen; R13 is selected from hydrogen, methoxy and ethoxy, more preferably R13 is hydrogen or methoxy, still more preferably R13 is methoxy; R15 is selected from hydrogen, COOMe, CONH2, CONHMe and CONMe2, more preferably is hydrogen; R16 is selected from the group consisting of hydrogen, chlorine, bromine, fluorine and methoxy, more preferably is chlorine, bromine, or fluorine, still more preferably is bromine; and, R17, R18 and
R19 are hydrogen. In a preferred embodiment, the rocaglaol of formula (lib) is such as R11 and R13 are methoxy, R15 is hydrogen, R16 is bromine, and, R17, R18 and R19 are hydrogen. In this particular preferred embodiment, R14 can be -NHCHO (FL23) or -NHSO2Me (FL25).
Alternatively, the rocaglaol of formula (Ha) is such as R11 and R13 are methoxy, R15 is hydrogen, R16 is bromine, R17, R18 and R19 are hydrogen, and R14 can be -NHCHO (FL22) or
-NHSO2Me (FL24).
In a further aspect, the present invention also concerns a new rocagloal derivative of the formula (II), wherein
R12 is H and R11 is selected from methoxy and ethoxy, more preferably R11 is methoxy;
R13 is selected from hydrogen, methoxy and ethoxy, more preferably R13 is hydrogen or methoxy;
R14 is -OCOR20, R20 being selected from hydrogen, methyl and ethyl;
R15 is selected from hydrogen, -COOR21, and -CONR22R23, wherein R21 and R23 are independently selected from hydrogen and methyl, and R22 is selected from the group consisting of hydrogen, methyl and methoxy; more preferably R15 is selected from hydrogen, COOMe, CONH2, CONHMe and CONMe2; still more preferably R15 is hydrogen;
Rl 6 is chlorine, bromine, or fluorine, preferably bromine;
R17 is hydrogen;
R18 is in para and is H or fluorine and R19 is hydrogen; or any pharmaceutically acceptable salt thereof. More preferably, R11 and R13 are methoxy, R12, R15, R18 and R19 are hydrogen, and R16 is bromine. Still preferably, R14 is -OCHO (FL 19), -OCOMe (FL20) or -OCOEt (FL21).
The present invention finally provides a new rocaglaol derivative the formula (Ha), wherein R12 is H and R11 is selected from methoxy and ethoxy, more preferably R11 is methoxy; R13 is selected from hydrogen, methoxy and ethoxy, more preferably R13 is hydrogen or methoxy; R14 is hydroxyl;
R18 is in para and is fluorine and R19 is hydrogen, and wherein R15 is selected from hydrogen, -COOR21, and -CONR22R23, wherein R21 and R23 are independently selected from hydrogen and methyl, and R22 is selected from the group consisting of hydrogen, methyl and methoxy; more preferably R15 is selected from hydrogen, COOMe, CONH2, CONHMe and CONMe2; R16 is bromine and R17 is hydrogen; or
R15 is selected from -COOR21, and -CONR22R23, wherein R21 and R23 are independently selected from hydrogen and methyl, and R22 is selected from the group consisting of hydrogen, methyl and methoxy; more preferably R15 is selected from hydrogen, COOMe, CONH2, CONHMe and CONMe2; and,
R16 is methoxy and R17 is hydrogen; or any pharmaceutically acceptable salt thereof.
More preferab >liyy,, in this embodiment, R11 and R13 are methoxy, R12 is hydrogen, R16 is bromine and R15 is selected from hydrogen, COOMe, CONH2, CONHMe and CONMe2. Still more preferably, R15 is COOMe (FL 17) or CONH2 (FL 18). In an alternative preferred embodiment, R11 and R13 are methoxy, R12 is hydrogen, R16 is methoxy and R15 is selected from COOMe, CONH2, CONHMe and CONMe2. Still more preferably, R15 is COOMe
(FL 16).
Optionally, R11 and R13 are methoxy, R12 is hydrogen, and R15 is selected from COOMe, CONH2, CONHMe and CONMe2.
As used herein, a "cardioprotectant agent" is an agent that protects cardio myocytes from damages, in particular from apoptosis.
The term "alkoxy", as used herein, corresponds to an alkyl group bonded to the molecule by an -O- (ether) bond. (Ci-C3)-alkoxy groups include methoxy, ethoxy, propyloxy, and isopropyloxy.
The term "alkyl" as used herein, refers to a univalent radical containing only carbon and hydrogen atoms arranged in a chain. (Ci-C3)-alkyl groups include methyl, ethyl, propyl, or isopropyl. The term "Me" as used herein, refers to a methyl group. The term "Et" as used herein, refers to an ethyl group.
The term "morpholin" as used herein, refers to a heterocycle of formula -O(CH2CH2)2NH with both amine and ether functional groups. The term "heterocycle" as used herein, refers to a group containing a ring structure comprising one or more heteroatoms, such as sulfur, oxygen or nitrogen. 5 atom heterocycles include, for example, furan, pyrrole and oxazole.
The term "pharmaceutically acceptable salt" refers to salts which are non-toxic for a patient and suitable for maintaining the stability of a therapeutic agent and allowing the delivery of said agent to target cells or tissue. Pharmaceutically acceptable salts are well known in the art (Berge et al., 1977). These salts can be prepared in situ during the final isolation and purification of the rocaglaol derivatives, or separately by reacting the free base function with a suitable organic acid. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like.
The present invention also concerns a rocaglaol derivative of the formulae (I), (II), (Ha) and (lib) according to the invention, as described above, as a medicament, in particular as cardioprotectant agent, and more particularly as cardioprotectant agent against cardiotoxicity of an antineoplastic agent. In particular, the derivative is selected in the group consisting of FL3, FL5, FL6, FL7, FL8, FL9, FL14, FL16, FL17, FL18, FL19, FL20, FL21, FL22, FL23, FL24 and FL25. Those derivatives have also demonstrated advantageous cytotoxicity against tumoral cells. Accordingly, the present invention further concerns a rocaglaol derivative of the formulae (I), (II), (Ha) and (lib) according to the invention for use for treating cancer. It also concerns the use of a rocaglaol derivative of the formulae (I), (II), (Ha) and (lib) according to the invention for preparing a medicament for treating cancer. It finally concerns a method for treating a cancer in a subject in need thereof, comprising administering a therapeutically active amount of a rocaglaol derivative of the formulae (I), (II), (Ha) and (lib) according to the invention.
This particular aspect also concerns the preferred embodiments disclosed above for the rocaglaol derivatives of the formulae (I), (II), (Ha) and (lib), and in particular a derivative selected in the group consisting of FL3, FL5, FL6, FL7, FL8, FL9, FL14, FL16, FL17, FL18, FL 19, FL20, FL21, FL22, FL23, FL24 and FL25.
These rocaglaol derivatives can be synthesized by any process known by the man skilled in the art. Such processes are described, for example, in the patent applications DE 19934952 and WO 2005/092876 or in the articles of Diedrichs et al. (Diedrichs et al, 2005), Gerard et al. (Gerard et al., 2004) and Dobler et al. (Dobler et al., 2001). As illustrative example, the synthesis of twenty three of these rocaglaol derivatives, including bromo-demethoxy- rocaglaol (FL3) and methyl bromo-didemethoxy-rocaglate (FL5), is detailed in the present application in examples 1, 2 and 3.
The present invention also concerns a pharmaceutical composition comprising a cardioprotectant rocaglaol derivative of formulae (I), (II), (Ha) and (lib) according to the present invention and a pharmaceutically acceptable carrier and/or excipient. This particular aspect also concerns the preferred embodiments disclosed above for the rocaglaol derivatives of the formulae (I), (II), (Ha) and (lib), and in particular a derivative selected in the group consisting of FL3, FL5, FL6, FL7, FL8, FL9, FL 14, FL 16, FL 17, FL 18, FL 19, FL20, FL21, FL22, FL23, FL24 and FL25. The pharmaceutical composition comprising the rocaglaol derivative of formulae (I), (II), (Ha) and (lib) according to the present invention, including their preferred embodiments and the compounds FL3, FL5, FL6, FL7, FL8, FL9, FL14, FL16, FL17, FL18, FL19, FL20, FL21, FL22, FL23, FL24 and FL25, is formulated in accordance with standard pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York) known by a person skilled in the art.
Possible pharmaceutical compositions include those suitable for oral, rectal, topical (including transdermal, buccal and sublingual), or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. For these formulations, conventional excipient can be used according to techniques well known by those skilled in the art. The compositions for parenteral administration are generally physiologically compatible sterile solutions or suspensions which can optionally be prepared immediately before use from solid or lyophilized form. Adjuvants such as a local anesthetic, preservative and buffering agents can be dissolved in the vehicle and a surfactant or wetting agent can be included in the composition to facilitate uniform distribution of the active ingredient. For oral administration, the composition can be formulated into conventional oral dosage forms such as tablets, capsules, powders, granules and liquid preparations such as syrups, elixirs, and concentrated drops. Non toxic solid carriers or diluents may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, magnesium, carbonate, and the like. For compressed tablets, binders, which are agents which impart cohesive qualities to powdered materials are also necessary. For example, starch, gelatine, sugars such as lactose or dextrose, and natural or synthetic gums can be used as binders. Disintegrants are also necessary in the tablets to facilitate break-up of the tablet. Disintegrants include starches, clays, celluloses, algins, gums and crosslinked polymers. Moreover, lubricants and glidants are also included in the tablets to prevent adhesion to the tablet material to surfaces in the manufacturing process and to improve the flow characteristics of the powder material during manufacture. Colloidal silicon dioxide is most commonly used as a glidant and compounds such as talc or stearic acids are most commonly used as lubricants.
For transdermal administration, the composition can be formulated into ointment, cream or gel form and appropriate penetrants or detergents could be used to facilitate permeation, such as dimethyl sulfoxide, dimethyl acetamide and dimethylformamide. For transmucosal administration, nasal sprays, rectal or vaginal suppositories can be used. The active compound can be incorporated into any of the known suppository bases by methods known in the art. Examples of such bases include cocoa butter, polyethylene glycols (carbowaxes), polyethylene sorbitan monostearate, and mixtures of these with other compatible materials to modify the melting point or dissolution rate. In a preferred embodiment, the pharmaceutical composition of the invention is suitable for parenteral administration.
Pharmaceutical composition according to the invention may be formulated to release the active drug substantially immediately upon administration or at any predetermined time or time period after administration. Pharmaceutical composition according to the invention can comprise one or more cardioprotectant rocaglaol derivatives associated with pharmaceutically acceptable excipients and/or carriers. These excipients and/or carriers are chosen according to the form of administration as described above. Other active compounds can also be associated with rocaglaol derivatives of the present invention such as other cardioprotectant molecules or antineoplastic agents, such as anthracycline, preferably doxorubicin.
The present invention also concerns a product containing a rocaglaol derivative of formulae (I), (II), (Ha) and (lib) according to the present invention, including their preferred embodiments and the compounds FL3, FL5, FL6, FL7, FL8, FL9, FL14, FL16, FL17, FL18, FL19, FL20, FL21, FL22, FL23, FL24 and FL25, and an antineoplastic agent, preferably an anthracycline, as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer. The present invention also concerns the use of a rocaglaol derivative of the formula (II)
Figure imgf000024_0001
wherein
R1 ! is alkoxy, optionally substituted;
R12 is hydrogen; or R11 and R12 together form a -O-CH2-O-unit; R13 is selected from hydrogen and (Ci-C3)-alkoxy;
R is selected from hydroxyl, oxo group, -OCOR , R being selected from hydrogen and (Ci-C3)-alkyl, =N-OR26 with R26 being H or methyl, -NH-(CH2)m-R27, wherein m is 0 or 1 and R27 is selected from H, -OH, -SO2Me, -COR28 with R28 being H or (Ci-C3)- alkyl; R15 is selected from hydrogen, -COOR21, -CONR22R23 and -C0NH(CH2)n0H, wherein n is 2, 3 or 4, wherein
R21 and R23 are independently selected from hydrogen and (Ci-C3)-alkyl and R22 is selected from the group consisting of (Ci-C3)-alkoxy, (Ci-C3)-alkyl and hydrogen, or
R22 and R23 together form a 5 atom heterocycle, optionally substituted by a group NHCO- (Ci-C4)-alkyl; or R14, R15and carbons (α) and (β) bearing R14 and R15 together form
Figure imgf000025_0001
wherein R24 and R25 are independently selected from the group consisting of (C1-C3)- alkyl and hydrogen, preferably from hydrogen and methyl;
R16 is selected from hydrogen, (Ci-C3)-alkoxy and halogen; R17 is in ortho-position to Ri6 and is selected from hydrogen, (Ci-C3)-alkoxy and hydroxyl or form together with R17 a -O-CH2-O-unit;
R18 and R19 are independently selected from the group consisting of hydrogen and halogen, preferably from H, F, Br and Cl, more preferably H or F; or any pharmaceutically acceptable salt thereof, for use for preventing or limiting the cardiotoxicity of an antineoplastic agent, preferably an anthracycline, more preferably doxorubicin.
Preferably, the rocaglaol derivative of the formula (II) has one or several of the following features : a) R12 is H and R11 is selected from methoxy and ethoxy, more preferably R11 is methoxy; b) R13 is selected from hydrogen, methoxy and ethoxy, more preferably R13 is hydrogen or methoxy; c) R14 is selected from hydroxyl,-OCOR20, R20 being selected from hydrogen, methyl and ethyl, -NH-(CH2)m-R27, wherein m is 0 and R27 is -SO2Me or -COR28 with R28 being H or (Ci-C3)-alkyl, preferably H or methyl; d) R15 is selected from hydrogen, -COOR21, and -CONR22R23, wherein R21 and R23 are independently selected from hydrogen and methyl, and R22 is selected from the group consisting of hydrogen, methyl and methoxy; more preferably R15 is selected from hydrogen, COOMe, CONH2, CONHMe and CONMe2; e) R16 is selected from hydrogen, (Ci-C3)-alkoxy and halogen, more preferably from hydrogen, chlorine, bromine, fluorine and methoxy, still more preferably from bromine and methoxy; f) R17 is hydrogen; g) R18 is in para and is H or fluorine and R19 is hydrogen. Optionally, the rocaglaol derivative of the formula (II) meets one feature, two features [for instance a) and b); a) and c); a) and d); a) and e); a) and f); a) and g); b) and c); b) and d); b) and e); b) and f); b) and g); c) and d); c) and e); c) and f); c) and g); d) and e); d) and f); d) and g); e) and f); e) and g); f) and g)], three features [for instance a), b) and c); a), b) and d); a), b) and e); a), b) and f); a), b) and g); a), c) and d); a), c) and e); a), c) and f); a), c) and g); a), d) and e); a), d) and f); a), d) and g); a), e) and f); a), e) and g); b), c) and d); b), c) and e); b), c) and f); b), c) and g); c), d) and e); c), d) and f); c), d) and g); d), e) and f); d), e) and g); e), f) and g)], four features [a), b), c) and d); a), b), c) and e); a), b), c) and f); a), b), c) and g); a), b), d) and e); a), b), d) and f); a), b), d) and g); a), b), e) and f); a), b), e) and g); a), b), f) and g); a), c), d) and e); a), c), d) and f); a), c), d) and g); a), c), e) and f), a), c), e) and g); a), d), e) and f); a), d), e) and g); a), d), f) and g); a), e), f) and g); b), c), d) and e); b), c ),d) and f); b), c), d) and g); b), d), e) and f); b), d), e) and g); b), e), f) and g); c), d), e) and f); c), d), e) and g); c), e), f) and g); d), e), f) and g)], five features [for instance a), b), c), d) and e); a), b), c), d) and f); a), b), c), d) and g); a), b), c), e) and f); a), b), c), e) and g); a), b), c), f) and g); a), b), d), e) and f); a), b), d), e) and g); a), b), d), f) and g); a), b), e), f) and g); a), c), d), e) and f); a), c), d), e) and g); a), c), d), f) and g); a), c), e), f) and g); a), d), e), f) and g); b), c), d), e) and f); b), c), d), e) and g); b), c), e), f) and g); b), d), e), f) and g); c), d), e), f) and g)], six features [a), b), c), d), e) and f); a), b), c), d), e) and g); a), b), c), d), f) and g); a), b), c), e), f) and g); a), b), d), e), f) and g); a), c), d), e), f) and g); b), c), d), e), f) and g)] or all the seven features a), b), c), d), e), f) and g).
Optionally, R18 is a halogen and is in position para, ortho or meta and R19 is hydrogen. Preferably, R18 is F, Cl or Br, more preferably F or Br, still more preferably is F. Preferably, R18 is in para. Optionally, R18 and R19 are both halogens, preferably the same halogen, and R18 and R19 are in para and ortho, in ortho and meta, in para and meta, or both in ortho. Preferably, R18 and R19 are both F, Cl or Br, more preferably F.
In a particular embodiment, the rocaglaol derivative presents the formula (Ha) or (lib)
Figure imgf000026_0001
(lib) wherein the definition of RI l, R12, R13, R14, R15, R16, R17, R18 and R19 is the same than for formula (II) and the same preferred features also apply to the compounds of formula (Ha) or (lib).
In a particular preferred embodiment, the rocaglaol derivative has the formula (Ha) and R14 is selected from hydroxyl, -OCOR20, R20 being selected from hydrogen and (Ci-C3)-alkyl, and =N-OR26 with R26 being H or methyl. Preferably, R14 is selected from hydroxyl and - OCOR20, R20 being selected from hydrogen, methyl and ethyl. In a preferred embodiment of this particular embodiment,
R1 λ is alkoxy, especially methoxy or ethoxy, more preferably methoxy; R12 is hydrogen;
R13 is selected from hydrogen, methoxy and ethoxy, more preferably R13 is hydrogen or methoxy;
R15 is selected from hydrogen, COOMe, CONH2, CONHMe and CONMe2; R16 is selected from the group consisting of hydrogen, chlorine, bromine, fluorine and methoxy, more preferably of bromine and methoxy;
R18 is in para and is H or fluorine; and, R17 and R19 are hydrogen.
Optionally, the rocaglaol derivative has the formula (III)
R14
Figure imgf000027_0001
wherein
R11 is alkoxy, optionally substituted; and R12 is hydrogen; or R11 and R12 together form a -O-CH2-O-unit;
R13 is selected from the group consisting of hydrogen and (Ci-C3)-alkoxy, preferably from hydrogen and methoxy;
R14 is selected from the group consisting of hydroxyl, oxo group and -OCOR20, R20 being selected from (Ci-C3)-alkyl and hydrogen, preferably from hydrogen and methyl; R15 is selected from the group consisting of hydrogen, -COOR21, -CONR22R23 and -
C0NH(CH2)n0H, wherein n is 2, 3 or 4, wherein
R21 and R23 are independently selected from hydrogen and (Ci-C3)-alkyl, preferably from hydrogen and methyl, and R22 is selected from the group consisting of (Ci-C3)-alkoxy, (C1- C3)-alkyl and hydrogen, preferably from hydrogen, methyl and methoxy, or
R22 and R23 together form a 5 atom heterocycle, optionally substituted by a group NHCO-(Ci-
C4)-alkyl; or R14, R15and carbons (α) and (β) bearing R14 and R15 together form
Figure imgf000028_0001
wherein R24 and R25 are independently selected from the group consisting of (Ci-C3)-alkyl and hydrogen, preferably from hydrogen and methyl;
R16 is selected from the group consisting of hydrogen, (Ci-C3)-alkoxy and halogen; R17 is in ortho-position to Ri6 and is selected from the group consisting of hydrogen, (C1-C3)- alkoxy and hydroxyl or form together with R17 a -O-CH2-O-unit; or any pharmaceutically acceptable salt thereof.
In another particular preferred embodiment, the rocaglaol derivative has the formula (lib) and R14 is -NH-(CH2)m-R27, wherein m is 0 and R27 is -SO2Me or -COR28 with R28 being H or (Ci- C3)-alkyl, preferably H or methyl.
In a preferred embodiment of this particular embodiment, R1 λ is alkoxy, especially methoxy or ethoxy, more preferably methoxy;
R12 is hydrogen;
R13 is selected from hydrogen, methoxy and ethoxy, more preferably R13 is hydrogen or methoxy, still more preferably R13 is methoxy;
R15 is selected from hydrogen, COOMe, CONH2, CONHMe and CONMe2, more preferably is hydrogen;
R16 is selected from the group consisting of hydrogen, chlorine, bromine, fluorine and methoxy, more preferably is chlorine, bromine, or fluorine, still more preferably is bromine; and,
R17, R18 and R19 are hydrogen. Examples of suitable rocaglaol derivatives of the formula (II) are disclosed in the following documents: WO07/139749; EP1693059; WO05/113529; WO05/092876; WO03/045375; US 6,518,274; US 6,420,393; DE19934952.
The present invention also concerns a rocaglaol derivative of the formulae (I), (II), (Ha), (lib) and (III) as described above or any pharmaceutically acceptable salt thereof, for preventing or limiting the cardiotoxicity of an antineoplastic agent. Preferably, this particular aspect also concerns the preferred embodiments disclosed above for the rocaglaol derivatives of the formulae (I), (II), (Ha) and (lib), and in particular a derivative selected in the group consisting of FLl, FL3, FL5, FL6, FL7, FL8, FL9, FLlO, FL12, FL13, FL14, FL15, FL16, FL17, FL18, FL 19, FL20, FL21, FL22, FL23, FL24 and FL25, more preferably from the group consisting of FLl, FL3, FL5, FL8, FL9, FLlO, FL12, FL13, FL14, FL15, FL16, FL17, FL18, FL19, FL20, FL21, FL22, FL23 and FL25.
The present invention further concerns a method for preventing or limiting the cardiotoxicity of an antineoplastic agent in a subject comprising administering a therapeutically effective amount of a rocaglaol derivative of the formulae (I), (II), (Ha), (lib) and (III) as described above or any pharmaceutically acceptable salt thereof to said subject. This particular aspect also concerns the preferred embodiments disclosed above for the rocaglaol derivatives of the formulae (I), (II), (Ha) and (lib), and in particular a derivative selected in the group consisting of FLl, FL3, FL5, FL6, FL7, FL8, FL9, FLlO, FL12, FL13, FL14, FL15, FL16, FL17, FL18, FL 19, FL20, FL21, FL22, FL23, FL24 and FL25, more preferably from the group consisting of FLl, FL3, FL5, FL8, FL9, FLlO, FL12, FL13, FL14, FL15, FL16, FL17, FL18, FL19, FL20, FL21, FL22, FL23 and FL25. The subject is any mammal, preferably a human being. In a particular embodiment, the method of the invention for preventing or limiting the cardiotoxicity of an antineoplastic agent comprises administering 0.01 to 10 mg / kg of body weight / day of a rocaglaol derivative of the formulae (I), (II), (Ha), (lib) and (III) as described above or any pharmaceutically acceptable salt thereof to said subject. Preferably, the method of the invention comprises administering 0.1 to 5 mg / kg of body weight / day of a rocaglaol derivative of the formulae (I), (II), (Ha), (lib) and (III) as described above or any pharmaceutically acceptable salt thereof to said subject. This particular aspect also concerns the preferred embodiments disclosed above for the rocaglaol derivatives of the formulae (I), (II), (Ha) and (lib), and in particular a derivative selected in the group consisting of FLl, FL3, FL5, FL6, FL7, FL8, FL9, FLlO, FL12, FL13, FL14, FL15, FL16, FL17, FL18, FL19, FL20, FL21, FL22, FL23, FL24 and FL25, more preferably from the group consisting of FLl, FL3, FL5, FL8, FL9, FLlO, FL12, FL13, FL14, FL15, FL16, FL17, FL18, FL19, FL20, FL21,
FL22, FL23 and FL25.
The term "cardiotoxicity", "cardiovascular toxicity" or "cardiotoxic side effect" means having a toxic effect on the heart, which includes cardiac events, such as but not limited to, mild blood pressure changes, thrombosis, electrocardiographic (ECG) changes, arrthymias, myocarditis, pericarditis, myocardial infarction (MI), cardiomyopathy, cardiac failure (left ventricular dysfunction or failure) and congestive heart failure (CHF). Considering the cardiotoxicity of antineoplastic agents, these side effects are essentially due to apoptosis of cardiomyocytes induced by such agents.
An "antineoplastic agent" is an agent with anti-cancer activity that inhibits or halts the growth of cancerous cells or immature pre-cancerous cells, kills cancerous cells or immature precancerous cells, increases the susceptibility of cancerous or pre-cancerous cells to other antineoplastic agents, and/or inhibits metastasis of cancerous cells. These agents may include chemical agents as well as biological agents. Various antineoplastic agents can cause cardiovascular toxicity such as anthracyclines, cyclophosphamide, paclitaxel, fluorouracil, rituximab, arsenic trioxide, trastuzumab, thalidomide, etoposide, vinca alkaloids, pentastatin, cytarabine, interferons, busulfan and cisplatin. Preferably, the antineoplastic agent belongs to the group of anthracyclines. In the present document, "anthracyclines" include but are not limited to, doxorubicin, daunorubicin, idarubicin, detorubicin, carminomycin, epirubicin, morpholinodoxorubicin, morpholinodaunorubicin, methoxymorpholinyldoxorubicin, substitutes, derivatives, and one or more combinations thereof. More preferably, the anthracycline molecule is doxorubicin. As used herein, the term "to prevent or to limit the cardiotoxicity" means inhibiting or reducing cardiotoxic side effects of the antineoplastic agent administered to the subject, in particular, inhibiting or reducing the apoptosis of cardiomyocytes. The effect of the rocaglaol derivative on the apoptosis of cardiomyocytes can be assessed by any method known by the skilled person. For example, in the experimental section, the effects of compounds according to invention are assessed by measuring and comparing the apoptosis of H9c2 cardiomyocytes in absence and in presence of an antineoplastic agent and with a combination of an antineoplastic agent and a rocaglaol derivative of the invention. Results are expressed in percentage of apoptotic cells. Preferably, the apoptosis of cardiomyocyte in presence of a rocaglaol derivative of the invention is reduced by at least 5%, more preferably by at least 10% and the most preferably by at least 25%. The present invention also concerns the use of a rocaglaol derivative of the formulae (I), (II), (Ha), (lib) and (III) as described above, or any of its pharmaceutically acceptable salts, as cardioprotectant agent. This particular aspect also concerns the preferred embodiments disclosed above for the rocaglaol derivatives of the formulae (I), (II), (Ha) and (lib), and in particular a derivative selected in the group consisting of FLl, FL3, FL5, FL6, FL7, FL8, FL9, FLlO, FL12, FL13, FL14, FL15, FL16, FL17, FL18, FL19, FL20, FL21, FL22, FL23, FL24 and FL25, more preferably from the group consisting of FLl, FL3, FL5, FL8, FL9, FLlO, FL12, FL13, FL14, FL15, FL16, FL17, FL18, FL19, FL20, FL21, FL22, FL23 and FL25.
Preferably, the rocaglaol derivative is one of the formulae (I), (II), (Ha), (lib) according to the invention, as described herein.
In a particular embodiment, the rocaglaol derivative is selected from the group consisting of FLl, FL3, FL5, FL6, FL7, FL8, FL9, FLlO, FL12, FL13, FL14, FL15, FL16, FL17, FL18, FL 19, FL20, FL21, FL22, FL23, FL24 and FL25, more preferably from the group consisting of FLl, FL3, FL5, FL8, FL9, FLlO, FL12, FL13, FL14, FL15, FL16, FL17, FL18, FL19, FL20, FL21, FL22, FL23 and FL25.
The amount of cardioprotectant rocaglaol derivative of the invention to be administered has to be determined by standard procedure well known by those of ordinary skill in the art. Physiological data of the patient (e.g. age, size, and weight) and the routes of administration have to be taken into account to determine the appropriate dosage. The cardioprotectant rocaglaol derivative of the invention may be administered as a single dose or in multiple doses. In a preferred embodiment, the pharmaceutical composition comprises a therapeutically effective amount of the rocaglaol derivative of the invention.
By a "therapeutically effective amount" is intended an amount of cardioprotectant rocaglaol derivative of the invention administered to a patient that is sufficient to provide a cardioprotective effect, i.e. that is sufficient to protect cardiomyocytes from damages, in particular from apoptosis. This therapeutically effective amount comprises a blood plasma concentration in a range of from 1 nM to 1 μM, preferably of from 15 nM to 100 nM or from 18 nM to 90 nM, more preferably of from 20 nM to 70 nM, most preferably of from 23 nM to 50 nM, and even most preferably of from 25 nM to 30 nM. Then, it is to be understood that the total amount of the rocaglaol derivative of the invention may vary depending on the volume of blood plasma of the patient. Suitable means and measures to determine the therapeutically effective amount are available to the person skilled in the art.
In a particular embodiment, the pharmaceutical composition according to the invention comprises 0.1 mg to 1 g of the rocaglaol derivative of the invention. In another particular embodiment, the pharmaceutical composition according to the invention comprises 0.7 to 700 mg of the rocaglaol derivative of the invention, preferably 0.7 to 350 mg of the rocaglaol derivative of the invention.
Cardioprotectant rocaglaol derivatives of the invention can be used in combination with other cardioprotective molecules or with antineoplastic agents. In this case, rocaglaol derivatives and the other active molecules can be administered simultaneously or consecutively.
Accordingly, the present invention discloses a pharmaceutical composition comprising a rocaglaol derivative of formula (II), preferably of formula (I), and an antineoplastic agent. It also concerns a product containing a rocaglaol derivative of formula (II), preferably of formula (I) and an antineoplastic agent as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer. In a preferred embodiment, the antineoplastic agent is an anthracycline, in particular doxorubicin. The following examples are given for purposes of illustration and not by way of limitation.
Examples
Example 1: Synthesis of bromo-demethoxy-rocaglaol (FL3)
The synthesis of bromo-demethoxy-rocaglaol (FL3) is represented on Figure 1. The different steps are described below. (S)-3-((R)-2,3-dihvdro-4,6-dimethoxy-2-(4-bromophenyl)-3-oxobenzofuran-2-yl)-3- phenylpropanal (3c)
A suspension of benzofuranone Ic (4.7 g, 13.5 mmol) in t-BuOH (300 ml) was heated to 500C under argon. Benzyltrimethylammonium hydroxide in MeOH (40%, 306 μL, 0.73 mmol) and, immediately after, cinnamaldehyde 2a (3.40 ml, 27.0 mmol), were added. The mixture was stirred for 2 h at 500C, cooled to room temperature (rt), concentred and acidified with HCl 1 M (30 ml), extracted with CH2Cl2, dried over MgSO4 and concentrated to dryness. Purification of the resulting yellow solid residue by chromatography (Et2O-Pentane 6:4) yielded 1.94 g (33%) of ketoaldehyde 3c as a white solid: Rt 0.4 (Et2O/ Hept 9:1). NMR 1U (300 MHz, CDCl3): 2.61 (IH, ddd, J = 0.9, 4.0, 17.3 Hz), 3.07 (IH, ddd, J= 2.30, 10.9, 17.3 Hz), 3.70 (3H, s), 3.85 (3H, s), 4.19 (IH, dd, J= 4.0, 10.9 Hz), 5.81 (IH, d, J= 1.8 Hz), 6.21 (IH, d, J = 1.8 Hz), 7.08-7.17 (3H, m), 7.29-7.32 (2H, m), 7.49, 7.63 (4H, AA'BB', J = 8.7
Hz), 9.41 (IH, d, J= 1.1 Hz). NMR 13C (75 MHz, CDCl3): 44.0; 46.8; 55.9; 55.9; 88.4; 93.0; 103.7; 122.6; 126.9; 127.5; 128.2; 129.5; 131.8; 132.0; 135.6; 136.3; 159.1; 169.9; 174.1; 194.1; 200.0. IR (thin film): 2725, 1720, 1699, 1617, 1590, 1154, 747 cm"1. HR-MS calcd for 5 C25H2IBrLiO5 : 487.0732; found : 487.0727 (-0.02 ppm).
(S)-3-((R)-2,3-dihvdro-4,6-dimethoxy-2-(4-bromophenyl)-3-oxobenzofuran-2-yl)-3-phenyl-2- fftrimethylsilvDoxyJ-propanenitrile (4c)
Acetonitrile (12 mL) was added at room temperature under argon to aldehyde 3c (1.2 g, 2.5 mmol). Trimethylsilyl cyanide (744 mg, 7.5 mmol) was added dropwise and immediately
10 after zinc iodide (10 mg) was added at room temperature under argon. The resulting mixture was stirred for 1 hour, filtered and concentred. Cyanohydrin 4c (1.74 g) was used in the next step without purification. i?/0.5 (Et2O/Hept 8:2). NMR 1H (300 MHz, CDCl3): 7.47-7.60 (4H, m); 7.13-1.23 (5H, m) ; 6.21 (IH, t, J= 2.3 Hz); 5.79 (IH, t, J= 2.3 Hz); 3.87-3.89 (IH, m); 3.83 (3H, s); 3.70-3.76 (IH, m); 3.66 (3H, s); 2.30-2.38 (2H, m); -0.01 (9H, d, J= 4.5 Hz).
15 Tricyclic Ketone (5c)
LDA (2.7 mmol, 0.6 M) was added dropwise at -78°C under argon to a solution of protected cyanohydrin 4c (1.43 g, 2.46 mmol) in dry THF (12 mL). After stirring for 2 hours at -78°C the solution was heated to -500C for 10 min. The reaction was quenched by the addition of saturated aqueous ammonium chloride (15 mL). Standard extractive work-up (CH2Cl2) gave a
20 yellow solid (1.49 g).This solid was directly treated with tetra-n-butylammonium fluoride (2.7 mL, IM in THF) added dropwise at room temperature in dry THF (10 mL). The solution was stirred for 4 hours and she was quenched by addition of methanol. Standard extractive workup (AcOEt) and purification by flash chromatography (Et2O) afforded tricyclic ketone 5c (240 mg, 20 %) as a white solid: tf/ 0.35 (Et2O/Hept 8:2). NMR 1H (300 MHz, CDCl3): 2.96-3.09
25 (2H, m), 3.21 (IH, br s), 3.81 (3H, s), 3.84 (3H, s), 3.90 (IH, dd, J= 10.2, 12.1 Hz), 6.1 (IH, d, J= 1.9 Hz), 6.33 (IH, d, J= 1.9 Hz), 6.89-6.94 (4H, m), 7.10-7.12 (3H, m), 7.24-7.27 (2H, m). NMR 13C (75 MHz, CDCl3): 39.6; 48.6; 55.6; 55.7; 88.6; 89.7; 92.8; 100.8; 106.2; 121.7; 127.1; 127.8; 128.0; 128.3; 130.7; 133.0; 136.6; 158.4; 160.9; 164.8; 210.3. IR (thin film): 3477, 2942, 2841, 1750, 1621, 1597, 1149 cm"1. HR-MS calcd for C25H2iBriNai06 :
30 503.0470; found : 503.0465 (-0.99 ppm). Racemic bromo-demethoxy-rocaglaol (FL3)
Acetonitrile (1.8 mL) was added at room temperature under argon to tetramethylammonium triacetoxyborohydride (828 mg, 3.15 mmol) followed by the addition of glacial acetic acid (1.8 mL). After stirring for 30 min at room temperature a solution of ketone 5c (170 mg, 0.354 mmol) in dry acetonitrile (4.5 rnL) was added dropwise. The resulting mixture was stirred overnight at room temperature. The reaction was quenched by the addition of saturated aqueous ammonium chloride (40 mL). Standard extractive work-up (AcOEt) and purification by flash chromatography (Et2O) afforded FL3 (103 mg, 60 %) as a white solid: Rf .25 (Et2O/ 5 Hept 8:2).
Example 2: Synthesis of methyl bromo-didemethoxy-rocaglate (FL5)
The synthesis of methyl bromo-didemethoxy-rocaglate (FL5) represented on Figures 3 and 4 is based on Porco's Strategy (Gerard et al, 2004). The different steps are described below.
10 (E)-3-(4-bromophenyl)-l-(2-hvdroxy-4-methoxyphenyl)prop-2-en-l-one (Figure 3 (8): R1 = H. R2 = Me. R3 = Br)
To a solution of 10.2 g (0.062 mol) of 2'-hydroxy-4'-methoxyacetophenone in 150 mL of methanol, were successively added 13.8 g (4 eq, 0.25 mol) of potassium hydroxide and 11.4 g (1 eq, 0.062 mol) of 4-bromobenzaldehyde. The solution was heated overnight at 600C,
15 cooled to 00C in an ice bath and acidified to pH 2 with concentrated HCl. The precipitate was filtered, washed with 100 mL of water and dried in vacuo to afford 20.4 g (99%) of chalcone (8 of Figure 3). NMR 1H (300 MHz, CDCl3): 3.83 (3H, s,), 6.45 (2H, m), 7.50 (5H, m), 7.77 (2H, m), 13.33 (IH, s). NMR 13C (75 MHz, CDCl3): 55.8, 101.3, 108.1, 114.2 , 121.1, 125.1, 130.0, 131.4, 132.4, 133.9, 143.1, 166.6, 167.0, 191.7.
20 2-(4-bromophenyl)-3-hvdroxy-7-methoxy-4H-chromen-4-one (Figure 3 (9): R1 = H, R2 = Me, R3 = Br)
To a solution of 10.0 g (1 eq, 0.030 mol) of chalcone (8 of Figure 3) in 100 mL of methanol, were added 10.1 g (6 eq, 0.18 mol) of potassium hydroxide. The solution was heated at 600C, and when it became red, 12.3 mL (4 eq, 0.12 mol) of hydrogene peroxide were slowly added.
25 The solution was stirred for 15 min at r.t and cooled to 00C in an ice bath. The mixture was then acidified to pH 2 with concentrated HCl. The precipitate was filtered, washed with 100 mL of water and dried in vacuo to afford 7.30 g (70%) of 3 -hydroxy flavone (9 of Figure 3) as a yellow solid. NMR 1H (300 MHz, CDCl3): 3.92 (3H, S2), 6.92 (IH, d, J = 2.2 Hz), 6.98 (IH, dd, J = 2.2; 8.8 Hz), 7.63 (2H, d, J = 8.8 Hz), 8.11 (3H, m). NMR 13C (75 MHz, CDCl3):
30 56.0, 100.2, 114.7, 115.1, 122.9, 126.0, 129.1, 130.6, 131.4, 139.1, 143.2, 156.4, 163.7, 172.3. Cvclopentafbcjbenzopyran (Figure 3 (10): R1 = H, R2 = Me, R3 = Br) or Methyl 2-(4- bromophenyl)-5-hydroxy-2 , 5-methano-8-methoxy-l 0-oxo-3-phenyl-2, 3, 4, 5-tetrahydro-l- benzoxepin-4-carboxylate (4b of Figure 4)
A solution of 3 -hydroxy flavone (9 of Figure 3 or 3b of Figure 4) (1 g, 1 eq, 3 mmol) and 5 methyl cinnamate (5.15 g, 10 eq, 30 mmol) in 90 mL of a mixture of dichloromethane/methanol (3/1) in a pyrex tube was degassed with argon for 5 min, and irradiated for 15h at 00C using an Iwasaki 400 W mercury lamp. The solution was concentrated in vacuo and purified by flash chromatography (40/60 heptane/ AcOEt) to afford 560 mg of cyclopenta[δc]benzopyrans as a white solid, which was heated at 65 0C for 4h in
10 20 mL of AcOEt. The solution was concentrated in vacuo to afford 560 mg (38%) of cyclopenta[6c]benzopyran 10 as a white solid. NMR 1H (300 MHz, CDCl3): 3.80 (3H, s), 3.82 (3H, m), 3.70 (IH, d, J = 8.3 Hz), 4.65 (IH, d, J = 8.3 Hz), 6.56 (2H, m), 7.19-7.40 (9H, m), 7.52 (IH, d, J = 8.4 Hz). NMR 13C (75 MHz, CDCl3): 52.8, 54.4, 55.8, 60.9, 88.6, 97.8, 97.3, 101.9, 108.4, 117.0, 122.5, 125.7, 127.4, 128.5, 130.0, 130.8, 134.7, 139.3, 152.6, 161.7,
15 171.5, 191.5.
Cyclopenta[&]tetrahydrobenzofuran (Figure 3 (11):, R1 = H, R2 = Me, R3 = Br) or Methyl 3a-
(4-bromophenyl)-8b-hydroxy-8-methoxy-l-oxo-3-phenyl-2,3-3a,8b-tetrahydro- cyclopentafbJbenzofuran-2-carboxylate (5b of Figure 4)
To a solution of cyclopenta[δc]benzopyran (10 of Figure 3 or 4b of Figure 4) (400 mg, 1 eq, 0 0.8 mmol) in MeOH (75 mL) were added 78 mg (2,5 eq, 2.0 mmol) of NaH in MeOH (5 mL) at 00C. The resulting solution was stirred for 20 min at 600C. After quenching the reaction with saturated aqueous NH4Cl, 40 mL of AcOEt was then added, and the organic layer was washed with water (2x20 mL) and brine (20 mL). The organic layer was dried over MgSO4 and concentrated in vacuo to afford 370 mg of crude ketol (93%) as a white solide which was 5 used without further purification. NMR 1H (300 MHz, CDCl3): 3.67 (3H, s), 3.87 (3H, s), 4.11 (IH, s), 4.54 (IH, s, H6), 6.64 (IH, dd, J = 2.3, 8.4 Hz), 6.72 (IH, d, J = 2.3 Hz), 6.90- 7.40 (1OH, m). NMR 13C (CDCl3): 51.8, 53.2, 55.8, 55.9, 87.7, 96.9, 100.6, 108.4, 115.3, 118.0, 126.0, 127.8, 128.3, 128.4, 128.7, 129.0, 132.8, 138.5, 145.0, 159.9, 172.2, 203.9. Methyl bromo-didemethoxy-rocaglate or Methyl 3a-(4-bromophenyl)-l, 8b-dihydroxy-8-
30 methoxy-l-oxo-3-phenyl-2, 3-3a, 8b-tetrahydro-cyclopentafbJbenzofuran-2-carboxylate (6b of Fieure 4) (FL5)
To a solution of 620 mg (6 eq, 2.35 mmol) of Me4NBH(OAc)3 and 0.23 mL (10 eq, 3.93 mmol) of acetic acid in 20 mL of CH3CN was added a solution of 200 mg (1 eq, 0.39 mmol) of the crude ketol product (5b of Figure 4) in 10 mL of CH3CN. The resulting solution was stirred for 3 h at rt before being quenched with 20 rnL of saturated aqueous NH4Cl. The solution was then treated with 10 rnL of a 3 M aqueous solution of sodium/potassium tartrate and stirred for 30 min. The aqueous solution was extracted with CH2Cl2 (2x30 rnL). The combined organic layers were washed with brine, dried over MgSO4 and concentrated in vacuo. HPLC purification afforded 100 mg (50%) of the corresponding endo product as a white solid.
Example 3: Synthesis and NMR characterization of rocaglaol derivatives FL6 to FL25 Synthesis of FLlO Methyl 2-(4-bromophenyl)-6, 8-dimethoxy-5-hydroxy-2 , 5-methano-10-oxo-3-phenyl-2, 3, 4, 5- tetrahydro-l-benzoxepin-4-carboxylate (4a of Figure 4).
A solution of hydroxyflavone (3a of Figure 4) (1.0 g, 2.7 mmol) and methyl cinnamate (3.43 g, 21 mmol) in 90 mL Of CH2CVMeOH (3:1) was degassed with argon for 10 min in a pyrex tube. This mixture was then irradiated (450 W Iwasaki UV lamp) for 30 h at 0 0C under an argon atmosphere. The solution was concentrated in vacuo, purified by flash chromatography (heptane/AcOEt 8:2 to 4:6), heated to reflux in EtOAc (20 mL) for 4 h and concentrated in vacuo to give 400 mg (29%) of 4a of Figure 4 as a white solid. NMR 1H (CDCl3): 3.56 (3H, s), 3.76 (3H, s), 3.83 (3H, s), 4.17 (IH, d, J= 9.1 Hz), 4.49 (IH, d, J= 9.1 Hz), 6.09 (IH, d, J = 2.1 Hz), 6.19 (IH, d, J= 2.1 Hz), 6.90-7.20 (5H, m), 7.21 (2H, d, J= 9.1 Hz), 7.50 (2H, d, J = 9.1 Hz). NMR 13C (CDCl3): 51.9, 52.4, 53.5, 54.6, 55.4, 55.6, 56.0, 56.3, 57.2, 62.3, 81.0, 81.3, 83.8, 87.5, 92.9, 93.9, 94.6, 98.0, 103.7, 104.7, 121.9, 122.1, 126.9, 127.5, 128.1, 128.5, 128.9, 129.0, 129.3, 129.8, 130.4, 130.5, 130.7, 131.3, 132.6, 134.8, 137.5, 139.4, 152.7, 153.5, 158.2, 158.6, 161.5, 162.2, 170.6, 171.7, 202.9. Methyl 3a-(4-bromophenyl)-6,8-dimethoxy-8b-hydroxy-l-oxo-3-phenyl-2,3-3a,8b-tetrahydro- cyclopenta[b]benzofuran-2-carboxylate (5a of Figure 4)
To a solution of 4a of Figure 4 (120 mg, 0.2 mmol) in MeOH (15 mL) was added a solution of NaOMe in MeOH (0.1 M, 5 mL) at 0 0C. The resulting solution was stirred for 20 min at 60 0C, cooled to rt, and quenched with saturated NH4C1, extracted with AcOEt (20 mL), and the organic layer was washed with water (2x20 mL) and brine (20 mL), dried over MgSO4 and concentrated in vacuo to afford 120 mg of crude β-ketoester 5a of Figure 4 as white solid which was used without further purification.. NMR 1H (CDCl3): 3.65 (3H, s), 3.77 (3H, s), 3.84 (3H, s), 4.05 (IH, d, J = 12.7 Hz), 4.24 (IH, d, J = 12.7 Hz), 4.47 (IH, s), 6.10 (IH, d, J = 2.0 Hz), 6.33 (IH, d, J = 2.0 Hz), 6.90-7.20 (5H, m), 7.27 (2H, d, J = 8.7 Hz), 7.50 (2H, d, J = 8.7 Hz). NMR 13C (CDCl3): 51.9, 53.1, 55.7, 55.8, 56.9, 88.5, 89.2, 92.7, 99.1, 105.9, 122.0,
127.0, 127.8, 128.2, 128.5, 130.9, 134.3, 135.1, 158.7, 160.7, 165.1, 170.0, 203.1. Methyl 3a-(4-bromophenyl)-l,8b-dihydroxy-6,8-dmethoxy-l-oxo-3-phenyl-2,3-3a,8b- tetrahydro-cyclopenta[b]benzofuran-2-Carboxylate (FLlO or 6a of Figure 4). Glacial acetic acid (128 mL, 2.22 mmol) was added to a solution OfMe4NBH(OAc)3 (351 mg, 1.33 mmol) in CH3CN (3 mL). After stirring for 5 min at room temperature, a solution of 5a of Figure 4 (120 mg, 0.22 mmol) in CH3CN (10 mL) was added dropwise. The resulting mixture was stirred was stirred for 3 h at rt, successively quenched with saturated aqueous NH4Cl (15 mL) and a 3 M aqueous solution of sodium/potassium tartrate (3 mL) and stirred for 30 min. The aqueous solution was extracted with AcOEt (2x30 mL). The combined organic layers were washed with brine, dried over MgSO4 and concentrated in vacuo. Purification by HPLC (Symetry shield RP 18, 7 μm, 19x300 mm, with a flow rate of 10 niL/min using a 50 min gradient from water (0.1% TFA) to CH3CN (0.1% TFA)) yielded diol 6a of Figure 4 (60 mg, 50%) as a white solid. NMR data : see below.
Synthesis ofFL6 (Ib of Figure 4)
3a-(4-Bromophenyl)-8b-hydroxy-8-methoxy-3-phenyl-2,3-3a,8b-tetrahydro- cyclopenta[b]benzofuran-l-one.
Lithium chloride (13 mg, 0.3 mmol) and water (11 μL, 0.6 mmol) were added to a solution of 5b of Figure 4 (100 mg, 0.2 mmol) in DMSO. The mixture was stirred at 100 0C for 12 h, cooled to room temperature, diluted with 10 mL of water and extracted with AcOEt (3 x 10 mL). The combined organic layers were washed with brine, dried over MgSO4 and concentrated. The white solid was purified by flash chromatography (heptane/ AcOEt 9:1 to 4:6), and concentrated in vacuo to give 40 mg (22%) of the title ketone as a white solid. 1H NMR (CDCl3): 3.05 (2H, m), 3.80 (IH, m), 3.90 (3H, s), 6.67 (IH, dd, J= 2.3, 8.5 Hz), 6.75 (IH, d, J = 2.3 Hz), 6.87-6.94 (4H, m), 7.10-7.14 (3H, m), 7.24-7.27 (3H, m). 13C NMR (CDCl3): 39.5, 52.8, 55.6, 88.7, 90.1, 92.6, 100.8, 106.2, 121.4, 126.5, 126.8, 127.8, 128.0, 128.8, 130.7, 133.8, 137.3, 160.9, 164.8, 210.5. 3a-(4-Bromophenyl)-8-methoxy-3-phenyl- 1 ,2 ,3 ,3a-tetrahydro-cyclopenta[b] benzofuran- l,8b(lH)-diol (FL6, Ib of Figure 4).
Glacial acetic acid (51 μL, 0.89 mmol) was added to a solution Of Me4NBH(OAc)3 (140 mg, 0.53 mmol) in CH3CN (3 mL). After stirring for 5 min at room temperature, a solution of the previous compound (40 mg, 0.09 mmol) in CH3CN (2 mL) was added dropwise. The resulting mixture was stirred for 3 h at rt, successively quenched with saturated aqueous NH4Cl (15 niL) and a 3 M aqueous solution of sodium/potassium tartrate (3 mL) and stirred for 30 min. The aqueous solution was extracted with AcOEt (2 x 30 mL). The combined organic layers were washed with brine, dried over MgSO4 and concentrated in vacuo. Purification by HPLC (Symetry shield RPl 8, 7 μm, 19 x 300 mm, with a flow rate of 10 5 niL/min using a 50 min gradient from water (0.1% TFA) to CH3CN (0.1% TFA)) yielded diol Ib of Figure 4 (FL6) (20 mg, 50%) as a white solid. NMR data : see below.
Synthesis of FL 7 (6c of Figure 4)
Methyl 5-hydroxy-2, 5-methano-8-methoxy-2-(4-methoxyphenyl)- 10-oxo-2 ' , 3, 4, 5-tetrahydro-l-
10 benzoxepin-4-carboxylate (4c of Figure 4).
A solution of hydroxyflavone 3c of Figure 4(1.0 g, 3.4 mmol) and methyl cinnamate (5.15 g, 34 mmol) in 90 mL of CH2CVMeOH (3:1) was degassed with argon for 10 min in a pyrex tube. This mixture was then irradiated (450 W Iwasaki UV lamp) for 15 h at 0 0C under an argon atmosphere. The solution was concentrated in vacuo, purified by flash chromatography
15 (heptane/AcOEt 4:6), heated to reflux in EtOAc (20 mL) for 4 h and concentrated in vacuo to give 440 mg (29%) of adduct 4c of Figure 4 as a white solid. 1H NMR (CDCl3): 3.62 (IH, d, J = 8.1 Hz), 3.68 (3H, s), 3.73 (3H, s), 3.83 (3H, s), 4.65 (IH, d, J = 8.1 Hz), 6.60 (2H, m), 7.10-7.40 (8H, m), 7.56 (2H, d, J = 8.9 Hz). 13C NMR (CDCl3): 50.7, 52.8, 54.4, 55.8, 55.9, 88.4, 98.0, 101.7, 108.2, 117.3, 125.5, 127.3, 128.6, 130.2, 130.9, 134.6, 139.1, 152.8, 161.6,
20 162.6, 171.4, 191.4.
Methyl 8b-hydroxy-8-methoxy-3a-(4-methoxyphenyl)-l-oxo-3-phenyl-2,3-3a,8b-tetrahydro- cyclopenta[b]benzofuran-2-carboxylate (5c of Figure 4).
To a solution of aglain 4c of Figure 4 (900 mg, 0.8 mmol) in MeOH (100 mL) was added a solution of NaOMe in MeOH (0.4 M, 5 mL) at 0 0C. The resulting solution was stirred for 20
25 min at 60 0C, cooled to rt, and quenched with saturated NH4Cl, extracted with AcOEt (40 mL), and the organic layer was washed with water (2 x 20 mL) and brine (20 mL), dried over MgSO4 and concentrated in vacuo to afford 370 mg of crude β-ketoester 5c of Figure 4 as a white solid which was used without further purification.. 1H NMR (CDCl3): 3.69 (3H, s), 3.77 (3H, s), 3.91 (3H, s), 4.16 (IH, s), 4.57 (IH, s), 6.75 (2H, m), 6.90-7.20 (8H, m), 7.56 (2H, d,
30 J = 8.4 Hz). 13C NMR (CDCl3): 50.9, 51.9, 53.4, 55.6, 56.0, 87.6, 96.7, 100.9, 108.6, 115.5, 117.9, 127.8, 128.2, 128.3, 128.8, 129.1, 132.7, 138.3, 145.3, 160.1, 162.5, 172.2, 203.9. Methyl l,8b-dihydroxy-8-methoxy-3a-(4-methoxyphenyl)-l-oxo-3-phenyl-2,3-3a,8b- tetrahydro-cyclopenta[b]benzofuran-2-carboxylate (FL7, 6c of Figure 4). Glacial acetic acid (248 mL, 4.33 mmol) was added to a solution OfMe4NBH(OAc)S (686 mg,
2.61 mmol) in CH3CN (30 mL). After stirring for 5 min at room temperature, a solution of ketone 5c of Figure 4 (200 mg, 0.43 mmol) in CH3CN (10 mL) was added dropwise. The resulting mixture was stirred for 3 h at rt, successively quenched with saturated aqueous NH4Cl (20 mL) and a 3 M aqueous solution of sodium/potassium tartrate (10 mL) and stirred for 30 min. The aqueous solution was extracted with CH2Cl2 (2 x 30 mL). The combined organic layers were washed with brine, dried over MgSO4 and concentrated in vacuo.
Purification by HPLC (Symetry shield RPl 8, 7 μm, 19 x 300 mm, with a flow rate of 10 niL/min using a 50 min gradient from water (0.1% TFA) to CH3CN (0.1% TFA)) yielded diol 6c of Figure 4 (100 mg, 50%) as a white solid. NMR data : see below.
Synthesis ofFL9, FL12, FL 13 and FLU (7a, 8a, 9a and 9b of Figure 4)
3a-(4-Bromophenyl)-l,8b-dihydroxy-6,8-dimethoxy-N,N-dimethyl-l-oxo-3-phenyl-2,3-3a,8b- tetrahydro-cyclopenta[b]benzofuran-2-carboxamide (FL12, 7a of Figure 4). To a solution of acid 6a of Figure 4 (30 mg, 0.06 mmol) in CH2Cl2 (3 mL) was added dimethylamine hydrochloride (6 mg, 0.07 mmol) and DMAP (8 mg, 0.07 mmol). At 0 0C, EDCI (13 mg, 0.07 mmol) was added portionwise over a 5 min period. The mixture was stirred for 30 min at 0 0C and then triethylamine (9 μL, 0.07 mmol) was added. The solution was stirred 1 h at 0 0C and 15 h at rt, quenched with 1 mL of IN HCl and diluted with 5 mL of water. The aqueous layer was extracted with CH2Cl2 (3 x 15 mL). The collected organic layers were washed with brine, dried over MgSθ4,concentrated, purified by flash chromatography (CH2Cl2/ AcOEt/Et3N 78:20:2 to 58:40:2), and concentrated in vacuo to afford 20 mg (63%) of amide 7a of Figure 4 (FL12) as a white solid. NMR data : see below. 3a-(4-Bromophenyl)-l,8b-dihydroxy-6,8-dimethoxy-N-methyl-l-oxo-3-phenyl-2,3-3a,8b- tetrahydro-cyclopenta[b]benzofuran-2-carboxamide (FL13, 8a of Figure 4).
To a solution of acid 6a of Figure 4 (30 mg, 0.06 mmol) in CH2Cl2 (3 mL) was added methylamine hydrochloride (5 mg, 0.07 mmol) and DMAP (8 mg, 0.07 mmol). At 0 0C, EDCI (13 mg, 0.07 mmol) was added portionwise over a 5 min period. The mixture was stirred for 30 min at 0 0C and then triethylamine (9 μL, 0.07 mmol) was added. The solution was stirred 1 h at 0 0C and 15 h at rt. The mixture was quenched with 1 mL of IN HCl and diluted with 5 mL of water. The aqueous layer was extracted with CH2Cl2 (3 x 15 mL). The collected organic layer was washed with brine (20 mL), dried over MgSO4, concentrated, purified by flash chromatography (CH2Cl2/ AcOEt/Et3N 68:30:2 to 58:40:2), and concentrated in vacuo to afford 26 mg (84%) of amide 8a of Figure 4 (FL13) as a white solid. NMR data : see below.
3a-(4-Bromophenyl)-l,8b-dihydroxy-6,8-dimethoxy-l-oxo-3-phenyl-2,3-3a,8b-tetrahydro- cyclopenta[b]benzofuran-2-carboxamide (FL14, 9a of Figure 4). 5 Ester 6a of Figure 4 (50 mg, 0.09 mmol) was added to a saturated solution of ammoniac in methanol (5 mL) in a sealed tube. The solution was then heated at 100 0C for 36 h, cooled to rt, and concentrated in vacuo. The crude product was purified by flash chromatography
(CH2Cl2/Et0H 95:5) to give 15 mg (30%) of amide 9a of Figure 4 (FL14) as a white solid.
NMR data : see below. 10 3a-(4-Bromophenyl)-l,8b-dihydroxy-8-methoxy-l-oxo-3-phenyl-2,3-3a,8b-tetrahydro- cyclopenta[b]benzofuran-2-carboxamide (FL9, 9b of Figure 4).
To a saturated solution of 5 mL of ammoniac in MeOH was added ester 6b of Figure 4 (70 mg, 0.14 mmol) in a sealed tube. The solution was then heated at 100 0C for 36 h. After cooling to rt, the solution was concentrated in vacuo. A pure sample of amide 9b of Figure 4 15 (16 mg, 16%) was isolated by RP-HPLC (Symetry shield RP 18, 7 μm, 19x300 mm, with a flow rate of 10 niL/min using a 50 min gradient from water (0.1% TFA) to CH3CN (0.1%
TFA)). NMR data : see below.
Synthesis ofFL8 (7b of Figure 4)
20 Methyl 2-(4-bromophenyl)-5-hydroxy-2 , 5-methano-8-methoxy-l 0-oxo-2, 3, 4, 5-tetrahydro-l- benzoxepin-4-carboxylate (11 of Figure 4).
A solution of hydroxyflavone 3b of Figure 4 (1.0 g, 3 mmol) and dimethyl cinnamamide 10 of Figure 4 (5.10 g, 30 mmol) in 90 mL of CH2Cl2/Me0H (3:1) was degassed with argon for 10 min in a pyrex tube. This mixture was then irradiated (450 W Iwasaki UV lamp) for 25 h at 0
25 0C under an argon atmosphere. The solution was concentrated in vacuo, purified by flash chromatography (heptane/AcOEt 4:6), heated to reflux in EtOAc (20 mL) for 4 h and concentrated in vacuo to give 560 mg (18%) of adduct 11 of Figure 4 as a white solid. 1H NMR (CDCl3): 2.87 (3H, s), 3.23 (3H, s), 3.80 (3H, m), 3.90 (IH, d, J= 7.7 Hz), 4.99 (IH, d, J = 7.7 Hz), 6.55 (2H, m), 6.80-7.20 (1OH, m). 13C NMR (CDCl3): 36.4, 37.8, 51.4, 55.6,
30 60.6, 88.3, 97.9, 101.3, 108.1, 118.2, 121.1, 125.1, 127.6, 128.2, 130.0, 130.9, 134.6, 137.5, 152.8, 161.9, 171.9, 191.7. Methyl 3a-(4-bromophenyl)-8b-hydroxy-8-methoxy-l-oxo-3-phenyl-2,3-3a,8b-tetrahydro- cyclopenta[b]benzofuran-2-carboxylate.
To a solution of aglain 11 of Figure 4 (150 mg, 0.3 mmol) in MeOH (15 rnL) was added a solution of NaOMe in MeOH (0.4 M, 2 mL) at 0 0C. The resulting solution was stirred for 20 min at 60 0C, cooled to rt, and quenched with saturated NH4Cl, extracted with AcOEt (15 mL). The organic layer was washed with water (2 x 20 mL) and brine (20 mL), dried over MgSO4 and concentrated in vacuo to afford 140 mg of the title β-ketoester as a white solid which was used in the next step without further purification. 1H NMR (CDCI3): 2.88 (3H, s), 3.22 (3H, s), 3.83 (4H, m), 4.37 (IH, s), 6.58 (IH, dd, J = 2.2, 8.5 Hz), 6.67 (IH, d, J = 2.2 Hz), 6.83 (2H, m), 7.10 (3H, m), 7.30 (5H, m). 13C NMR (CDCl3): 36.5, 37.4, 51.6, 55.5, 56.1, 88.3, 96.7, 100.8, 108.4, 115.6, 118.5, 126.1, 127.3, 128.4, 128.5, 128.9, 129.3, 133.0, 138.8, 160.2, 172.3, 203.6.
3a-(4-Bromophenyl)-l,8b-dihydroxy-8-methoxy-N,N-dimethyl-l-oxo-3-phenyl-2,3-3a,8b- tetrahydro-cyclopenta[b]benzofuran-2-carboxamide (FL8, 7b of Figure 4). Glacial acetic acid (142 mL, 2.49 mmol) was added to a solution OfMe4NBH(OAc)3 (390 mg, 1.49 mmol) in CH3CN (15 mL). After stirring for 5 min at room temperature, a solution of ketone 5b of Figure 4 (130 mg, 0.25 mmol) in CH3CN (5 mL) was added dropwise. The resulting mixture was stirred for 3 h at rt, successively quenched with saturated aqueous NH4Cl (20 mL) and a 3 M aqueous solution of sodium/potassium tartrate (10 mL) and stirred for 30 min. The aqueous solution was extracted with CH2Cl2 (2 x 30 mL). The combined organic layers were washed with brine, dried over MgSO4 and concentrated in vacuo. Purification by HPLC (Symetry shield RPl 8, 7 μm, 19 x 300 mm, with a flow rate of 10 niL/min using a 50 min gradient from water (0.1% TFA) to CH3CN (0.1% TFA)) yielded diol 7b of Figure 4 (60 mg, 46%) as a white solid. NMR data : see below. Methyl 2-(4-bromophenyl)-6,8-dimethoxy-5-hydroxy-2,5-methano-10-oxo-3-(3-fluorophenyl)- 2,3,4,5-tetrahydro-l-benzoxepin-4-carboxylate (4d of Figure 4).
A solution of hydroxyflavone 3a of Figure 4 (1.0 g, 3.7 mmol) and methyl (E)-3- fluorocinnamate (2.58 g, 16 mmol) in 90 mL Of CH2Cl2ZMeOH (3:1) was degassed with argon for 10 min in a pyrex tube. This mixture was then irradiated (450 W Iwasaki UV lamp) for 40 h at 0 0C under an argon atmosphere. The solution was concentrated in vacuo, purified by flash chromatography (heptane/AcOEt 8:2 to 6:4), heated to reflux in EtOAc (20 mL) for 4 h and concentrated in vacuo to give 225 mg (16%) of adduct 4d of Figure 4 as a white solid. 1H NMR (CDCl3): 3.56 (3H, s), 3.61 (IH, d, J = 9.4 Hz), 3.72 (3H, s), 3.81 (3H, s), 4.15 (IH, d, J = 9.4 Hz), 6.07 (IH, d, J= 2.2 Hz), 6.15 (IH, d, J= 2.2 Hz), 6.68 (IH, m), 6.90 (3H, m), 7.23 (2H, d, J= 8.7 Hz), 7.51 (2H, d, J= 8.7 Hz). 13C NMR (CDCl3): 51.7, 54.3, 55.1, 55.4, 56.0,
88.5, 92.5, 94.9, 98.5, 104.1, 112.6, 114.0, 116.7, 125.6, 127.8, 129.1-129.2 (d, 7.9 Hz), 130.5, 142.5-142.6 (d, 7.5 Hz), 153.9, 158.5, 158.9, 166.1-164.3 (d, 245.0 Hz), 161.3, 171.3, 172.2. Methyl 3a-(4-bromophenyl)-6,8-dimethoxy-8b-hydroxy-l-oxo-3-(3-fluorophenyl)-2,3-3a,8b- tetrahydro-cyclopenta[b]benzofuran-2-carboxylate (5d of Figure 4).
To a solution of aglain 4d of Figure 4 (500 mg, 0.9 mmol) in MeOH (50 mL) was added a solution of NaOMe in MeOH (0.4 M, 5 mL) at 0 0C. The resulting solution was stirred for 20 min at 60 0C, cooled to rt, and quenched with saturated NH4Cl, extracted with AcOEt (40 mL), and the organic layer was washed with water (2 x 20 mL) and brine (20 mL), dried over MgSO4 and concentrated in vacuo to afford 370 mg of crude β-ketoester 5d of Figure 4 as a white solid which was used without further purification.. 1H NMR (CDCI3): 3.67 (3H, s), 3.79 (3H, s), 3.84 (3H, s), 3.99 (IH, d, J = 13.2 Hz), 4.23 (IH, d, J = 13.2 Hz), 6.10 (IH, d, J= 2.0 Hz), 6.34 (IH, d, J= 2.0 Hz), 6.66 (IH, m), 6.92 (2H, d, J= 8.6 Hz), 7.10 (3H, m), 7.28 (2H, d, J = 8.6 Hz). 13C NMR (CDCl3): 51.8, 53.4, 55.9, 56.0, 56.3, 88.6, 89.4, 90.2, 93.5, 105.7, 114.2, 115.4, 120.7, 128.5, 129.3, 129.9-130.0 (d, 8.1 Hz), 131.2, 132.5, 137.8-137.9 (d, 7.3 Hz), 158.2, 160.6-162.9 (d, 232.9 Hz), 164.1, 165.4, 168.1, 202.7.
Methyl 3 a- (4-bromophenyl) - 1, 8b-dihydroxy- 6, 8-dmethoxy- 1 -oxo-3- (3-fluoroph enyl) -2,3- 3a,8b-tetrahydro-cyclopenta[b]benzofuran-2-carboxylate (FL 17, 6d of Figure 4). Glacial acetic acid (520 μL, 4.33 mmol) was added to a solution Of Me4NBH(OAc)3 (1.42 g, 5.38 mmol) in CH3CN (80 mL). After stirring for 5 min at room temperature, a solution of ketone 5d of Figure 4 (500 mg, 0.90 mmol) in CH3CN (10 mL) was added dropwise. The resulting mixture was stirred for 3 h at rt, successively quenched with saturated aqueous NH4Cl (30 mL) and a 3 M aqueous solution of sodium/potassium tartrate (20 mL) and stirred for 30 min. The aqueous solution was extracted with CH2Cl2 (2 x 40 mL). The combined organic layers were washed with brine, dried over MgSO4 and concentrated in vacuo. The crude product was purified by flash chromatographie (CH2Cl2ZEt2O 95:5) to give diol 6d of Figure 4 (265 mg, 53%) as a white solid. 1H NMR (CDCl3): 3.56 (IH, s, OH), 3.66 (3H, s), 3.81 (3H, s), 3.8 (4H, m), 4.33 (IH, d, J= 14.1 Hz), 4.98 (IH, d, J= 6.4 Hz), 6.11 (IH, d, J = 2.0 Hz), 6.28 (IH, d, J = 2.0 Hz), 6.66 (2H, m), 6.77 (IH, m), 7.03 (IH, m), 7.08 (IH, d, J = 8.6 Hz), 7.31 (IH, d, J = 8.6 Hz). 13C NMR (CDC13):5O.5, 52.2, 54.8, 55.8, 79.6, 89.6, 93.0, 93.8, 101.5, 107.3, 113.7-113.9(d, J = 21.3 Hz), 114.9-115.1 (d, J = 21.6 Hz), 121.9, 123.2, 129.4-129.5 (d, J = 8.8 Hz), 129.5, 130.5, 133.7, 139.4-139.5 (d, J = 7.3 Hz), 157.0, 160.6, 161.3-163.7 (d, J = 245.4 Hz), 164.3, 170.2. 3a-(4-Bromophenyl)-l, 8b-dihydroxy-6, 8-dimethoxy-l-oxo-3-(3-fluorophenyl)-2, 3-3a, 8b- tetrahydro-cyclopenta[b]benzofuran-2-carboxamide (FLl 8).
Ester (50 mg, 0.09 mmol) was added to a saturated solution of ammoniac in methanol (5 mL) in a sealed tube. The solution was then heated at 100 0C for 36 h, cooled to rt, and 5 concentrated in vacuo. The crude product was purified by flash chromatography (AcOEt) to give 7 mg (14%) of amide FL18 as a white solid. NMR data : see below. Methyl 2-(4-methoxyphenyl)-6,8-dimethoxy-5-hydroxy-2,5-methano-10-oxo-3-(3- fluorophenyl)-2,3,4,5-tetrahydro-l-benzoxepin-4-carboxylate (4e of Figure 4). A solution of hydroxyflavone 3d of Figure 4 (1.0 g, 3.0 mmol) and methyl (E)-3-
10 fluorocinnamate (3.29 g, 18 mmol) in 90 mL Of CH2CVMeOH (3:1) was degassed with argon for 10 min in a pyrex tube. This mixture was then irradiated (450 W Iwasaki UV lamp) for 40 h at 0 0C under an argon atmosphere. The solution was concentrated in vacuo, purified by flash chromatography (heptane/AcOEt 7:3 to 6:4), heated to reflux in EtOAc (20 mL) for 4 h and concentrated in vacuo to give 400 mg (26%) of adduct 4e of Figure 4 as a white solid. 1H
15 NMR (CDCl3): 3.57 (3H, s), 3.63 (IH, d, J = 9.2 Hz), 3.68 (3H, s), 3.74 (3H, s), 3.82 (3H, s), 4.18 (IH, d, J = 9.2 Hz), 6.09 (IH, d, J = 2.2 Hz), 6.18 (IH, d, J = 2.2 Hz), 6.66 (3H, m), 6.98 (3H, m), 7.58 (2H, d, J= 8.9 Hz). 13C NMR (CDCl3): 52.0, 54.3, 55.1, 55.4, 56.0, 62.8, 87.7, 92.8, 94.6, 98.1, 103.6, 112.9, 113.4-113.7 (d, 21.0 Hz), 116.5-116.8 (d, 22.1 Hz), 125.8, 127.7, 129.2-129.3 (d, 7.7 Hz), 130.2, 142.8-142.9 (d, 7.7 Hz), 153.7, 158.6, 158.9, 160.9-
20 164.1 (d, 244.4 Hz), 161.4, 171.3, 171.8.
Methyl 3a-(4-methoxyphenyl)-6,8-dimethoxy-8b-hydroxy-l-oxo-3-(3-fluorophenyl)-2,3-3a,8b- tetrahydro-cyclopenta[b]benzofuran-2-carboxylate (5e of Figure 4).
To a solution of aglain 4e of Figure 4 (300 mg, 0.6 mmol) in MeOH (30 mL) was added a solution of NaOMe in MeOH (0.4 M, 5 mL) at 0 0C. The resulting solution was stirred for 20
25 min at 60 0C, cooled to rt, and quenched with saturated NH4Cl, extracted with AcOEt (3 x 20 mL), and the organic layer was washed with water (2 x 20 mL) and brine (20 mL), dried over MgSO4 and concentrated in vacuo to afford 300 mg of crude β-ketoester 5e of Figure 4 as a white solid which was used without further purification.. 1H NMR (CDCl3): 3.63 (3H, s), 3.64 (3H, s), 3.74 (3H, s), 3.79 (3H, s), 3.99 (IH, d, J = 13.3 Hz), 4.19 (IH, d, J = 13.3 Hz), 6.06
30 (IH, A, J = 1.9 Hz), 6.31 (IH, A, J = 1.9 Hz), 6.51 (2H, d, J= 8.9 Hz), 6.66 (3H, m), 6.80 (IH, m), 6.93 (2H, d, J= 8.9 Hz). 13C NMR (CDCl3): 51.8, 53.1, 55.2, 55.7, 55.8, 56.4, 88.6, 90.0, 93.1, 99.3, 112.3, 113.4, 114.1-114.3 (d, 21.6 Hz), 115.0-115.2 (d, 22.4 Hz), 125.3, 127.9, 128.1, 129.5-129.6 (d, 9.2 Hz), 138.3-138.4 (d, 6.6 Hz), 158.7, 159.5-161.9 (d, 244.7 Hz), 161.0, 163.8, 165.1, 167.2, 202.7. Methyl 3a-(4-methoxyphenyl)-l, 8b-dihydroxy-6, 8-dimethoxy-l-oxo-3-(3-fluorophenyl)-2, 3-
3a,8b-tetrahydro-cyclopenta[b]benzofuran-2-carboxylate (FL 16, 6e of Figure 4). Glacial acetic acid (110 μL, 2.0 mmol) was added to a solution Of Me4NBH(OAc)S (310 mg, 1.18 mmol) in CH3CN (20 mL). After stirring for 5 min at room temperature, a solution of ketone 5e of Figure 4 (100 mg, 0.20 mmol) in CH3CN (5 mL) was added dropwise. The resulting mixture was stirred for 3 h at rt, successively quenched with saturated aqueous NH4Cl (20 mL) and a 3 M aqueous solution of sodium/potassium tartrate (10 mL) and stirred for 30 min. The aqueous solution was extracted with CH2Cl2 (2 x 20 mL). The combined organic layers were washed with brine, dried over MgSO4 and concentrated in vacuo. The crude product was purified by flash chromatographie (CH2Cl2ZEt2O 95:5) to give diol 6 (FL) (85 mg, 85%) as a white solid. NMR data : see below.
3a-(4-methoxyphenyl)-8b-hydroxy-6,8-dimethoxy-3-(3-fluorophenyl)-2,3-3a,8b-tetrahydro- cyclopenta[b]benzofuran-l-one. Lithium chloride (13 mg, 0.3 mmol) and water (11 μL, 0.6 mmol) were added to a solution of keto-ester 5e of Figure 4 (100 mg, 0.2 mmol) in DMSO (2 mL). The mixture was stirred at 100 0C for 12 h, cooled to room temperature, diluted with 10 mL of water and extracted with AcOEt (3 x 10 mL). The combined organic layers were washed with brine, dried over MgSO4 and concentrated to give 75 mg (84%) of the title ketone as a white solid which was used without further purification. 1H NMR (CDCl3): 2.95 (2H, m), 3.67 (3H, m), 3.78 (3H, s), 3.81 (4H, m), 6.07 (1H, d, J= 1.9 Hz), 6.31 (IH, d, J= 1.9 Hz), 6.65 (2H, d, J= 8.8 Hz), 6.75 (3H, m), 6.93 (2H, d, J = 8.8 Hz), 7.10 (IH, m). 13C NMR (CDCl3): 41.0, 48.4, 55.3, 55.7, 55.9, 88.9, 89.9, 92.9, 101.2, 106.5, 113.4, 113.7-113.9 (d, J = 26.9 Hz), 115.1-115.3 (d, J = 22.0 Hz), 123.6, 125.7, 128.2, 129.4-129.5 (d, J = 7.7 Hz), 140.1-140.2 (d, J = 7.3 Hz),158.6, 159.0, 161.2, 161.4-163.8 (d, J = 245.0 Hz), 164.9, 210.2. 3a-(4-methoxyphenyl)-6,8-dimethoxy-3-(3-fluorophenyl)-l,2,3,3a-tetrahydro- cyclopenta[b]benzofuran-l,8b(lH)-diol (FL 15).
Glacial acetic acid (90 μL, 1.55 mmol) was added to a solution Of Me4NBH(OAc)3 (245 mg, 0.93 mmol) in CH3CN (8 mL). After stirring for 5 min at room temperature, a solution of the previous compound (70 mg, 1.55 mmol) in CH3CN (2 mL) was added dropwise. The resulting mixture was stirred for 3 h at rt, successively quenched with saturated aqueous NH4Cl (15 mL) and a 3 M aqueous solution of sodium/potassium tartrate (3 mL) and stirred for 30 min. The aqueous solution was extracted with AcOEt (2 x 30 mL). The combined organic layers were washed with brine, dried over MgSO4 and concentrated in vacuo. The crude product was purified by flash chromatographie (CH2Cl2ZEt2O 95:5) to give diol FL15
(35 mg, 50%) as a white solid. NMR data : see below.
Synthesis ofFL19, FL20 and FL21 3a-(4-Bromophenyl)-6,8-dimethoxy-3-phenyl- 1 ,2 ,3 ,3a-tetrahydro-cyclopenta[b] benzofuran- l,8b(lH)-diol 1-formate (FLl 9, 8a of Figure 5).
To a solution of FL3 (50 mg, 0.10 mmol) in anhydrous CH2Cl2 (1 rnL) was added successively DCC (24 mg, 0.11 mmol) and DMAP (1 mg, 0.01 mmol). After 5 min of stirring, formic acid (4 μL, 0.11 mmol) was added slowly at 0 0C. The solution was stirred 36 h at rt. Then, the reaction was diluted with pentane and filtered. The solid was washed with of pentane (2 x 5 mL) and the filtrate was concentrated in vacuo. The crude product was purified by flash chromatography (CH2Cl2) to give 15 mg of formic ester 8a of Figure 5 (FL 19). NMR data : see below. 3a-(4-Bromophenyl)-6,8-dimethoxy-3-phenyl- 1,2,3, 3a-tetrahydro-cyclopenta[b]benzofuran- l,8b(lH)-diol 1-acetate (FL20, 8b of Figure 5).
To a solution of FL3 (50 mg, 0.10 mmol) in anhydrous pyridine (1 mL), was added DMAP (3 mg, 0.03 mmol) and acetic anhydride (67 μL, 0.50 mmol). The solution was sonicated until complete solubilisation and stirred at rt for 6 h. The mixture was then concentrated in vacuo and purified by flash chromatography (CH2Cl2) to give 42 mg (84%) of ester 8b of Figure 5 (FL20). NMR data : see below.
3a-(4-Bromophenyl)-6,8-dim,ethoxy-3-phenyl- 1 ,2 ,3 ,3a-tetrahydro-cyclopenta[b] benzofuran- l,8b(lH)-diol 1-propionate (FL21, 8c of Figure 5).
To a solution of FL3 (50 mg, 0.10 mmol) in anhydrous pyridine (1 mL), was added DMAP (3 mg, 0.03 mmol) and propionic anhydride (67 μL, 0.50 mmol). The solution was sonicated until complete solubilisation and stirred at rt for 6 h. The mixture was then concentrated in vacuo and purified by flash chromatography (CH2Cl2) to give 49 mg (98%) of ester 8c of Figure 5 (FL21). NMR data : see below.
Synthesis ofFL22 and FL23 (12a and 12b of Figure 5) 3a-(4-Bromophenyl)-6, 8-dimethoxy-8b-hydroxy-3-phenyl-2, 3-3a, 8b-tetrahydro- cyclopentafb] benzofuran- 1 -one oxime (10 of Figure 5).
A solution of ketone 9 of Figure 5 (375mg, 0.78 mmol) and hydroxylammonium chloride (270 mg, 3.89 mmol) in 10 mL of pyridine-EtOH (1 :1) was stirred at 700C for 4 hours. After concentration in vacuo, the residue was dissolved in ethyl acetate. The organic phase was successively washed with 1 N HCl, saturated Na2CO3 solution and brine, then dried over magnesium sulfate, filtered, concentrated and purified on silica gel 60 (ether) to yield 330 mg of ketone 10 of Figure 5 as a white solid. 1H NMR (400MHz, DMSO-d6): 11.15 (s, IH), 7.34 (d, J=8.5Hz, 2H), 7.12-7.06 (m, 3H), 6.99-6.97 (m, 4H), 6.39 (d, J=2Hz, IH), 6.18 (d, J=2Hz, IH), 5.35 (s, IH), 3.80 (s, 3H), 3.75 (s, 3H), 3.54 (dd, J=12.4Hz and J=9.8Hz, IH), 3.07-2.90 (m, 2H).13C NMR (100MHz, DMSO-d6): 163.2, 159.5, 158.4 (X2), 137.9, 134.8, 130.0, 128.8, 127.7, 127.6, 126.5, 120.0, 109.2, 101.3, 92.7, 89.6, 86.8, 55.5, 55.4, 48.8, 29.0. LC- MS (ESI) : exact mass (C25H22BrNO5) = 495.07 ; found : 496.0 [M+H+]. 3a-(4-Bromophenyl)-6,8-dimethoxy-8b-hydroxy-3-phenyl-l,2,3,3a-tetrahydro- cyclopenta[b]benzofuran-l,8b(lH)-formamide (FL22 and FL23, 12a and 12b)
A solution of oxime 10 of Figure 5 (280 mg, 0.564 mmol) in THF (3 mL) is added to a solution of LiAlH4 (65 mg, 1.69 mmol) in THF (5 mL). The mixture was stirred for 3 h at 45°C, cooled to 00C, diluted with EtOAc (10 mL), and quenched with the dropwise addition of 1 M NaOH (10 mL), stirred for 10 min, extracted with EtOAc, washed with brine, dried over MgSO4 and concentrated to dryness. Purification by chromatography on silica gel 60 (Et2O/MeOH 95:5 to 0:100) yielded 100 mg of the expected primary as a white solid. One drop of acetic acid was added to a solution of this amine (40 mg, 0.083 mmol) in ethyl formate (1.1 mL) and THF (2 mL). The mixture was stirred for 2 h at reflux overnight, concentred and purified by chromatography (Et2O) to yield formamides 12a of Figure 5 (FL22, 10 mg) and 12b (FL23, 23 mg). These compounds were further purified by HPLC (Symetry shield RP 18, 7 μm, 19x300 mm, with a flow rate of 10 mL/min using a 50 min gradient from water (0.1% TFA) to CH3CN (0.1% TFA) to give pure samples of formamides 12a of Figure 5 (Rt = 25.5 min) and 12b of Figure 5 (Rt = 28 min).
Alternative synthesis of FL22 and FL23 (12a and 12b of Figure 6) and Synthesis of FL24 and FL25 (12c and 12d of Figure 6)
3a-(4-Bromophenyl)-6, 8-dimethoxy-8b-hydroxy-3-phenyl-2, 3-3a, 8b-tetrahydro- cyclopentafbjbenzofuran-l-(θ-methyloxime) (10 of Figure 6).
A solution of ketone 9 of Figure 6 (500 mg, 1.04 mmol) and O-methylhydroxylamine hydrochloride (430 mg, 5.2 mmol) in 40 mL of pyridine/EtOH (1 :1) was stirred for 3 hours at 700C. After concentration in vacuo, the residue was dissolved in EtOAc. The organic phase was successively washed with 1 N HCl, aq Na2CO3 and brine, dried over MgSO4, filtered and concentrated to quantitatively give 538 mg of oxime 10 of Figure 6 as a slightly yellow solid. 3a-(4-Bromophenyl)-6,8-dimethoxy-8b-hydroxy-3-phenyl-l,2,3,3a-tetrahydro- cyclopenta[b]benzofuran-l,8b(lH)-formamide (FL22 and FL23, 12a and 12b of Figure 6) Oxime 10 of Figure 6 (538 mg, 1.04 mmol) was dissolved in a solution of BH3 (1 M, 20 rnL) in THF at 00C. The mixture was stirred under reflux for 4 hours, and stirred overnight at rt. The reaction was quenched by a dropwise addition of 3 M NaOH (30 mL). The aqueous phase was extracted twice with CH2Cl2 and the organic phase was successively washed with water and brine, dried over MgSO4 and concentrated to dryness. Purification by chromatography on silica gel (Et2O/MeOH 70:30) yielded 221 mg of a mixture of amines 11a and lib of Figure 6 as a white solid (44%). One drop of acetic acid was added to a solution of this mixture of amines 11a and lib of Figure 6 (130 mg, 0.269 mmol) in ethyl formate (0.35 mL, 4.31 mmol) and THF (6 mL). The mixture was stirred at reflux overnight, concentrated and purified several times by chromatography on silica gel (Et2O/ EtOAc, 60:40) to yield formamides 12a of Figure 6 (FL22, 11 mg) and 12b of Figure 6 (FL23, 60 mg). 3a-(4-Bromophenyl)-6,8-dimethoxy-8b-hydroxy-3-phenyl- 1,2,3, 3a-tetrahydro- cyclopenta[b]benzofuran-l,8b(lH)-methanesulfonamide (FL24 and FL25, 12c and 12d)
N-methylmorpholine (114 μL, 10.4 mmol) was added at 00C to a solution of amines 11a and lib of Figure 6 (100 mg) in CH2Cl2 (2 mL). After 10 min at 00C, mesyl chloride (64 μL, 0.82 mmol) was added dropwise and the solution was then stirred for 4 h at rt. The mixture was quenched with 5 mL of HCl 1 N, and extracted with EtOAc (2x10 mL). The organic layer was washed with a solution of Na2CO3 (10 mL), brine (10 mL), dried over MgSO4, and concentrated under reduced pressure. The crude product was purified by flash chromatography on silica gel (CH2CVEt2O, 95:5) to give 12c of Figure 6 (FL24, 5 mg, 4%) and 12d of Figure 6 (FL25, 70 mg, 60%).
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Example 4 : Effect of rocaglaol and its derivatives on human cancer cell viability and on doxorubicin cardiotoxicity
Synthesis ofracemic rocaglaol and its derivatives Racemic rocaglaol (FLl) and its derivatives (FL2-4) were synthesised by the approach developed by Dobler and collaborators (Dobler et al, 2001) (Figures 2 and 3). The synthesis of FL3 and FL5 is detailed in examples 1 and 2. The synthesis of FL6 to FL23 is detailed in example 3. Rocaglaol derivatives FL5-11 were synthesized according to the strategy of Porco and collaborators (Gerard et al., 2004) (Figure 3). Rocaglaol derivatives FL5 to FLlO and FL 12 to FL 18 were synthesized according to the strategy of Figure 4. Rocaglaol derivatives FL 19 to FL25 were synthesized according to the strategy of Figures 5 and 6. Materials
KRIBB3 and doxorubicin were purchased from Sigma-Aldrich (Sigma-Aldrich, Saint- Quentin Fallavier, France). Rabbit polyclonal anti-phospho-HSP27 (ser 82) antibody, rabbit polyclonal anti-HSP27 antibody and horseradish peroxidase-conjugated secondary goat anti- rabbit IgG were purchased from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Enzyme-linked chemiluminescence were purchased from Amersham (Amersham Biosciences, Indianapolis, IN, USA). All others chemicals were purchased from Becton Dickinson (Becton Dickinson Biosciences, Le Pont De Claix, France). Cell lines
The human cell lines KB (epidermoid carcinoma) and HepG2 (hepatocarcinoma) were obtained from ECACC (Salisbury, UK) and grown in D-MEM medium supplemented with 10% fetal calf serum, in the presence of penicilline, streptomycine and fungizone in 75 cm2 flask under 5% CO2. The human cell lines HCTl 16 (colon adenocarcinoma), PC3 (prostate adenocarcinoma) and HCT 15 (colon adenocarcinoma) were obtained from ECACC (Wiltshire, UK). The human cell lines HL60 (promyeocytic leukaemia), MDA435 (breast ductal carcinoma; ATCC reference HBT 129) and MDA231 (breast adenocarcinoma; ATCC reference HBT26) were obtained from ATCC. MCF7 (breast adenocarcinoma) were obtained from Dr Kassack from the University of Bonn in Germany. SK-OV3 cells (or OV3, ATCC reference HTB77) were kindly provided by NCI (Frederick, MD).
The cell lines HCTl 16, HCT15, MCF7, MDA435, MDA231, PC3, OV3, HL60R and HL60 were grown in RPMI medium, in particular supplemented with 10% fetal calf serum, in the presence of penicillin, streptomycin and fungizone in 75 cm2 flask under 5% CO2. Resistant
HL60 cells were obtained by prolonged treatment with doxorubicin (50μg/L medium). Resistant MCF7 (MCF7R) cells were obtained by prolonged treatment with doxorubicin. H9c2 cells (rat heart myoblast) were obtained from ATCC and grown in D-MEM medium supplemented with 10% foetal calf serum at 37°C in 5% CO2. The medium was changed every 2-3 days.
Method for measuring inhibition of cell proliferation: MTS assay
Cells were plated in 96-well tissue culture plates in 200 μl medium and cultured during 24h before to be treated with compounds dissolved in DMSO (compound concentrations at a range of 0.5 nM to 10 μM) using a Biomek 2000 or 3000 (Beckman). Controls received the same volume of DMSO (1% final volume). After a 72h exposure, MTS reagent (Promega) was added and incubated for 3h at 37°C. The absorbance was then monitored at 490 nm and results were expressed as the value [1-(OD490 treated/OD490 control)χ100] corresponding to the percentage of inhibition of cell proliferation. For IC50 determinations (50% of inhibition of cell proliferation), experiments were performed in separate duplicate. Method for measuring cardiotoxicitv
H9c2 cells were plated for 24h in 100 mm Petri dishes at 0,7 X 104 cells/cm2. Next, the cells were washed and cultured for 12h in glucose-free medium (Gibco, DMEM w- L-glutamine, w/o D-glucose, sodium pyruvate) supplemented with only 1% fetal calf serum. Cells were then treated with flavaglines derivatives or their vehicle for 12h and then were treated with doxorubicin for an additional 14h. Kribb3 (1 μM) was preincubated for Ih before flavaglines and doxorubicin treatment in cardiomyocytes. Cells were washed, and subsequently FACS analyses were performed. Apoptosis was analysed by fluorescence-activated cell sorting analysis (FACS-Calibur, Becton-Dickinson Biosciences). 0,5 X 106 cells were harvested, washed with Annexin Binding Buffer (0,01 M HEPES, 0,14 M NaCl, 2,5 mM CaCl2) and labeled with annexin V (dilution 1 :50) and propidium iodide (6,7 μg/ml). All assays were performed at least in triplicate, and the results were analyzed by BD Cell Quest Pro software (Becton-Dickinson Biosciences, Le Pont De Claix, France). Western Blotting - H9C2 cells were plated for 24h on 60 mm petrish dishes at 1,5 X 104 cells/cm2. Next, cells were washed and cultured for 12h in glucose-free medium (Gibco, DMEM w- L-glutamine, w/o D-glucose, sodium pyruvate) supplemented with only 1% fetal calf serum. Cells were then incubated with FL3 or vehicle for Ih or 4h and after appropriate treatment washed twice with Phosphate-Buffered Saline (PBS). Cells were harvested with lysis buffer (50 mM Tris-Hcl, 1 niM EDTA, 100 niM NaCl, 0,1% SDS, 1% NP-40, ImM
Na3VO4, 1 ug/mL aprotinin, 1 ug/mL pepstatin, 1 ug/mL leupeptin, pH = 7). Whole cell lysates were centrifugated at 12000 x g for 15 min at 4°C. The supernatant (20 ug) were mixed with a loading buffer, denatured at 1000C for 3 min, electrophoresed on 12% SDS- PAGE under denaturing conditions, and transferred to a polyvinylidene difluoride membrane. Blots were then incubated with a blocking solution containing 5% fat-free milk powder in PBS plus Tween (0.5% Tween 20) (PBS-T) at room temperature for Ih. After three washes with PBS-T for 10-min intervals, blots were incubated overnight at 4°C under gentle shacking with respective primary antibody (rabbit polyclonal anti-phospho-HSP27 (1:500); rabbit polyclonal anti-HSP27 (1 :500)) diluted in PBS-T containig 0,5% fat-free milk powder. After three washes with PBS-T, membrane was incubated for Ih at room temperature under gentle shacking with a horseradish peroxidase-conjugated secondary goat anti-rabbit IgG (1 :1,000 dilution) in PBS-T containig 0,5% fat-free milk powder. The expected bands were visualized after incubation by enzyme-linked chemiluminescence for 5 min and quantified by scanning laser densitometry, normalizing to total amounts of the corresponding proteins.
Effect of rocaglaol (FLl) and its derivatives (FL2, FL, 3 and FL4) on human cancer cell viability
The effect of rocaglaol and its derivatives was determined on human cancer cell lines by MTS assay after a 72 h treatment. As shown in table 1, FLl (racemic rocaglaol) reduced the cell proliferation and viability more effectively than the anticancer drug doxorubicin on a panel of human cancer cell lines. Effects were observed in the low nanomolar range. Removing the methoxy group present on ring A decreased potency more than 1 000 times (compound FL2). Conversely, the replacement of the methoxy group on ring A by a bromine atom improved the cytotoxicity on all these cancer cell lines (compound FL3). The rank of activity (H «MeO<Br) suggests a preference for an hydrophobic substituent in the para position of ring A. On contrary, introducing a methoxy on the para position of ring B was detrimental for the cytotoxicity (FL4). Table 1 : Inhibition of cell proliferation by flavaglines FLl to FL4 on various human cancer cell lines (IC50, nM).
Cell lines FLl FL2 FL3 FL4 Doxorubicin
KB 2 2500 <1 >10000 1
MCF7 4 10000 1 NI 12
MCF7R 4 10000 <1 >10000 58
HCTl 16 6 6000 <1 >10000 6
HCT 15 6 8000 1 NI 81
HepG2 70 >10000 4 NI 240
HL60 3 6000 <1 >10000 13
In addition to FL3 compound, the in vitro cytotoxicity of the new synthesized flavaglines, FL5-16 and FL 18 was also evaluated on a variety of human cancer cell lines from nasopharynx (KB), neutrophil (HL60 and HL60R), colon (HCTl 16), breast (MDA435 and MDA231), ovary (O V3), and prostate (PC3)-derived neoplasms by the MTS assay. Results are summarized in Table 2 with the values of taxotere, doxorubicine and vinblastine shown for comparison.
Table 2 : Cytotoxicity of flavaglines analogs against human cancer cell lines (IC50, nM)
Compound KB HL60 HL60R HCTl 16 MDA435 MDA231 OV3 PC3
FL3 15 5.5 4.5 7.6 8.7 3.8 8.5 17
FL5 165 65 44 96 65 27 101 174
FL6 255 115 67 108 205 48 251 224
FL7 340 195 121 201 298 51 259 273
FL8 35 15 28 13 13.4 5.7 37 33
FL9 22 16 78 15 16.3 10 29 31
FLlO 24 14 14 18 15 8.7 21 29
FLI l 1 710 1 120 1280 1420 1110 1070 1460 2 520
FL12 7 4 12 5.9 3.2 2.7 6.7 7.2
FL13 4 2 16 3.8 2.6 1.9 3.8 3.3
FL14 2 1 18 1.8 1.5 1.0 2.1 3.7
FL15 60
FL16 28 FLl 8 14
FL19 7
FL20 40
FL21 50
FL22 44
FL23 2
FL24 580
FL25 16
Doxorubicin 40 30 990 22 3.5 0.047 41 186
Taxotere 0.17 0.5 531 0.52 0.25 0.018 0.51 1.4
Vinblastin 0.8 1.11 >100 1.42 1.24 0.63 2.7 5.5
The inventors observed that FL3 maintained its cytotoxicity on HL60 and MCF7 cell lines, even when they became resistant to doxorubicin.
The inventors started their structure-activity relationship investigation by introducing an ester or amide moiety on position 2 to FL3. Methyl ester FLlO exhibited a slightly reduced cytotoxicity than did compound FL3. On the opposite, introduction of a tertiary amide (FL 12) significantly enhanced cytotoxicity. This effect was more pronounced with secondary amide FL 13 and primary amide FL 14 on most cell lines. However both FL 13 and FL 14 were less active that reference compound FL3 on HL60R cells, which have developed resistance to chemotherapy by overexpressing the P-glycoprotein (P-gp), a plasma membrane protein encoded by the multidrug resistance (MDRl) gene.
Next, the inventors examined the requirement of the 8-methoxy group for the cytotoxicity on cancers cells. 8-Demethoxy compounds FL6, FL5, FL8 and FL9 were significantly less active (ED50 4 to 20 times higher) than cognate compounds FL3, FLlO, FL12 and FL13, indicating a preference but not an absolute requirement of a methoxy group in position 8 for cytotoxicity. In this 8-demethoxy series, the introduction of an amide at C-2 diminished also the cytotoxicity on HL60R.
Replacement of the bromine in 4 '-position of FLlO by a methoxy (FL7) was detrimental to the cytotoxicity activity, as it was previously observed with FL3 and rocaglaol. Rocaglaol (FLl) and rocaglaol derivatives (FL2, FL3 and FL4) do not have any deletetious effect on the doxorubicin cytotoxicity
To test the cytotoxic effect of combination of FLl or FL3 with doxorubicin, the inventors selected HepG2 cells of hepatocellular carcinoma which display a low sensitivity toward clinically used anticancer drugs.
The inhibition of HepG2 cell proliferation with FLl, FL3 or doxorubicin alone and with doxorubicin associated with FLl or FL3 is shown Figure 7. These results demonstrated that rocaglaol or rocaglaol derivatives do not have any deleterious effect on doxorubicin cytotoxicity, on contrary, they strengthen this activity on cancer cells. Effect of rocaglaol and its derivatives on doxorubicin cardiotoxicitv
Apoptosis was induced in H9c2 cardiomyocytes by doxorubicin and detected by FACS analysis after labeling the cells with annexin and PI. These cardiomyoblasts derived from rat heart represent an established model of doxorubicin cardiotoxicity in vitro. Incubation of the cells with 1 μM doxorubicin for 14h induced 32% apoptosis (total apoptotic cells). It was observed that pre-incubation of H9c2 cells with different concentrations of rocaglaol and its derivatives significantly reduced the apoptosis induced by doxorubicin (Figures 8 and 9). The most active compound, FL3, inhibited doxorubicin- induced apoptosis in a concentration- dependent manner reaching its maximum at 1 nM (Figure 8B). At this concentration, FL3 diminished by 70 % the apoptosis induced by 1 μM doxorubicin (Figure 10A). Figure 13 shows the cardioprotective effect of FLl-IO and FL 12- 18 on apoptosis induced by doxorubicin in H9c2 cells at different concentrations at 10 or 100 nM of rocaglaol derivatives. Western blot analysis with antibody specific for the active form of caspase 3 (Chemicon International, ref AB3623) revealed that FL3 reduced doxorubicin-mediated caspase-3 activity. Indeed, H9c2 cardiomyocytes incubated for 14h with 1 μM doxorubicin plus 1 nM FL3 failed to activate the cleavage of pro-caspase 3 in caspase 3 (Figure 10B). FL3 may induce the death of cancer cells via activation of the AIF and caspase- 12 pathways (Thuaud et al, 2009, J. Med. Chem, 52, 5176-5187). Therefore, it offers important advantages as antineoplastic agents since it acts synergistically with anthracyclines independently of caspases-3, -7, -8 and -9, suggesting that it would retain its activity in cells refractory to activation of these caspases. Considering that drug resistance and side effects are the two major obstacles limiting the efficacy of cancer chemotherapy, rocaglaol derivatives of the invention hold substantial potential for the treatment of cancers. FL3 protects cardiomvocvtes against serum starvation To further explore the scope of flavagline cardioprotection, the inventors examined whether FL3 could protect H9c2 cardiomyocytes from stress due to serum starvation (1% serum). FL3 (20 nM) strongly reduced the apoptosis induced by a 72 h serum starvation treatment (Figure 11). FL3 alleviates doxorubicin cardiotoxicity in mice
The inventors explored whether FL3 displays some cardioprotection in vivo. Administration of FL3 (0.1 mg/kg i.p., 2x/day for 4 days) protected mice from the cardiotoxicity induced by doxorubicine (20 mg/kg i.p.): they observed a 70% survival rate for the mice that received FL3. The survival rate for the mouse who received the vehicle was only 30% (see Table 3). This data indicates also that FL3 displays a proper bioavailabity.
Tableau 3: in vivo cardioprotection Treatment Survival rate
Doxorubicin (1 x, 20 mg/kg i.p.) 30 %
Doxorubicin (1 x, 20 mg/kg i.p.) + 7n 0/ FL3 (8 x, 0.1 mg/kg i.p.)
FL3 (8 x, 0.1 mg/kg i.p.) 100 %
FLS cardioprotection is mediated through HSP27 phosphorylation Phosphorylation of the small heat shock protein HSP27 (or its murine homo log HSP25) induces a cascade of events that protect cardiomyocytes from many stresses, including doxorubicin cardiotoxicity. To examine whether this protein is involved in FL3 cardioprotection, the inventors tested whether this effect could be blocked by KRIBB3, a synthetic molecule that inhibits HSP25/27 phosphorylation. Interestingly, a 1 h treatment of H9c2 cardiomyocytes with KRIBB3 (1 μM) induced a cytotoxicity that was significantly alleviated by a co-treatment with FL3 (100 nM), indicating that FL3 effects are opposed to those of KRIBB3. As expected KRIBB3 worsened doxorubicin cytotoxicity, but again, this effect was reversed by FL3: cells treated with doxorubicin, KRIBB3 and FL3 all together displayed the same level of apoptosis as those treated with doxorubicin alone. These results prompted the inventors to examine whether FL3 induces HSP27 phosphorylation (Fig. 12B). Western blot analysis showed indeed a high level of phosphorylation after a 1 h treatment, which significantly decreased after 4h. Conclusion These experiments demonstrated that rocaglaol and its derivatives according to the present invention are able to prevent or limit cardiomyocyte apoptosis, in particular doxorubicin- induced apoptosis.
Indeed, the inventors have shown that rocaglaol and its derivatives protect H9c2 cardiomyocytes from apoptosis induced by doxorubicin and serum starvation. Interestingly these two stresses are of different nature: doxorubicin induces an oxidative stress, while serum starvation blocks growth factors signaling that is necessary to cell survival. Anthracyclines such as doxorubicin, epirubicin, and daunorubicin are among the most anticancer drugs used in chemotherapy, in spite of their acute and chronic cardiotoxicities, which are dose related, cumulative and irreversible. Eventually, these myocardial dysfunctions can lead to congestive heart failure. Fortunately, the anticancer effects and the cardiotoxicity are not mediated by the same mechanisms. The former is due to an inhibition of topoisomerase II, while the latter is caused by reactive reactive oxygen species (ROS) that are generated by a redox cycling that involves the reversible reduction of the anthracycline by NADPH and a reoxidation by oxygen. The iron chelator dexrazoxane is the only cardioprotective agents with proven efficacy in cancer patients receiving anthracycline chemotherapy. However this efficacy is limited and effects on survival failed to be established. Interestingly, the relative order of activity (FL2 ~ FL4 « FLl < FL3) for cardioprotection were similar to those for cytotoxicity for cancer cells, suggesting that these compounds probably act through the same molecular target. Remarkably, 50 nM FL3 alleviated by 61% the apoptosis induced by 20 times superior concentrations of doxorubicin. This effect was accompanied by a concomitant suppression of the induction of caspase-3 by doxorubicine. Same tendencies have been observed for the other rocaglaol derivatives. Indeed, FL5, FL8-10 and FL 12- 18 showed activity for cytotoxicity and cardioprotection, whereas FL6, FL7 and FLl 1 showed no or weak such activity (Figure 13).
The inventors showed that this cardioprotection is mediated by HSP27 phosphorylation. The search for cardioprotective agents that alleviate anthracyclines cardiotoxicity in the last 30 years has led to drugs with limited, if any, efficacy in clinic. Considering that anthracyclines are the most widely used anticancer drug, the discovery of a new class of cardioprotective agents is of paramount importance in clinic. The benefit of an association of rocaglaol derivatives to anthracyclines might be triple: flavaglines could enhance the tumoricide effects of anthracyclines, delay chemoresistance evolution and alleviate the main adverse effects of anthracyclines, cardiotoxicity. A drug with all these attributes would greatly improve current treatments. References
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Claims

Claims
1- A rocaglaol derivative of the formula (II)
Figure imgf000061_0001
wherein
R1 ! is alkoxy, optionally substituted;
R , 12 is hydrogen; or R11 and R12 together form a -O-CH2-O-unit;
R13 is selected from hydrogen and (Ci-C3)-alkoxy; R is selected from hydroxyl, oxo group, -OCOR , R being selected from hydrogen and (Ci-C3)-alkyl, =N-OR26 with R26 being H or methyl, -NH-(CH2)m-R27, wherein m is 0 or 1 and R27 is selected from H, -OH, -SO2Me, -COR28 with R28 being H or (Ci-C3)- alkyl;
R15 is selected from hydrogen, -COOR21, -CONR22R23 and -C0NH(CH2)n0H, wherein n is 2, 3 or 4, wherein
R21 and R23 are independently selected from hydrogen and (Ci-C3)-alkyl and R22 is selected from the group consisting of (Ci-C3)-alkoxy, (Ci-C3)-alkyl and hydrogen, or
R22 and R23 together form a 5 atom heterocycle, optionally substituted by a group NHCO-
(Ci-C4)-alkyl; or R14, R15and carbons (α) and (β) bearing R14 and R15 together form
Figure imgf000061_0002
wherein R24 and R25 are independently selected from the group consisting of (C1-C3)- alkyl and hydrogen, preferably from hydrogen and methyl; R16 is selected from hydrogen, (Ci-C3)-alkoxy and halogen; R17 is in ortho-position to Ri6 and is selected from hydrogen, (Ci-C3)-alkoxy and hydroxyl or form together with R17 a -O-CH2-O-unit;
R18 and R19 are independently selected from the group consisting of hydrogen and halogen, preferably from H, F, Br and Cl, more preferably H or F; or any pharmaceutically acceptable salt thereof, for use for preventing or limiting the cardio toxicity of an antineoplastic agent, preferably an anthracycline, more preferably doxorubicin.
2- Rocaglaol derivative according to claim 1, wherein the rocaglaol derivative of the formula (II) has one or several of the following features : a) R12 is H and R11 is selected from methoxy and ethoxy, more preferably R11 is methoxy; b) R13 is selected from hydrogen, methoxy and ethoxy, more preferably R13 is hydrogen or methoxy; c) R14 is selected from hydroxyl,-OCOR20, R20 being selected from hydrogen, methyl and ethyl, -NH-(CH2)m-R27, wherein m is 0 and R27 is -SO2Me or -COR28 with R28 being H or (Ci-C3)-alkyl, preferably H or methyl; d) R15 is selected from hydrogen, -COOR21, and -CONR22R23, wherein R21 and R23 are independently selected from hydrogen and methyl, and R22 is selected from the group consisting of hydrogen, methyl and methoxy; more preferably R15 is selected from hydrogen, COOMe, CONH2, CONHMe and CONMe2; e) R16 is selected from hydrogen, (Ci-C3)-alkoxy and halogen, more preferably from hydrogen, chlorine, bromine, fluorine and methoxy, still more preferably from bromine and methoxy; f) R17 is hydrogen; g) R18 is in para and is H or fluorine and R19 is hydrogen.
3- Rocaglaol derivative according to claim 1 or 2, wherein the rocaglaol derivative presents the formula (Ha) or (lib)
Figure imgf000063_0001
wherein the definition of RI l, R12, R13, R14, R15, R16, R17, R18 and R19 is the same as claim 1 or 2.
4- Rocaglaol derivative according to any one of claims 1-3, wherein the rocaglaol derivative has the formula (Ha) and R14 is selected from hydroxyl, -OCOR20, R20 being selected from hydrogen and hydrogen, methyl and ethyl, and =N-OR26 with R26 being H or methyl.
5- Rocaglaol derivative according to claim 4, wherein R11 is alkoxy, especially methoxy or ethoxy, more preferably methoxy; R12 is hydrogen; R13 is selected from hydrogen, methoxy and ethoxy, more preferably R13 is hydrogen or methoxy; R15 is selected from hydrogen, COOMe, CONH2, CONHMe and CONMe2; R16 is selected from the group consisting of hydrogen, chlorine, bromine, fluorine and methoxy, more preferably of bromine and methoxy; R18 is in para and is H or fluorine; and, R17 and R19 are hydrogen.
6- Rocaglaol derivative according to any one of claims 1-3, wherein the rocaglaol derivative has the formula (lib) and R14 is -NH-(CH2)m-R27, wherein m is 0 and R27 is -SO2Me or - COR28 with R28 being H or (Ci-C3)-alkyl, preferably H or methyl.
7- Rocaglaol derivative according to claim 6, wherein R11 is alkoxy, especially methoxy or ethoxy, more preferably methoxy; R12 is hydrogen; R13 is selected from hydrogen, methoxy and ethoxy, more preferably R13 is hydrogen or methoxy, still more preferably R13 is methoxy; R15 is selected from hydrogen, COOMe, CONH2, CONHMe and CONMe2, more preferably is hydrogen; R16 is selected from the group consisting of hydrogen, chlorine, bromine, fluorine and methoxy, more preferably is chlorine, bromine, or fluorine, still more preferably is bromine; and, R17, R18 and R19 are hydrogen. 8- Rocaglaol derivative according to any one of claims 1-3, wherein the rocaglaol derivative is selected from the group consisting of FLl, FL3, FL5, FL6, FL7, FL8, FL9, FLlO, FL12, FL13, FL14, FL15, FL16, FL17, FL18, FL19, FL20, FL21, FL22, FL23, FL24 and FL25, more preferably from the group consisting of FLl, FL3, FL5, FL8, FL9, FLlO, FL12, FL13, FL14, FL15, FL16, FL17, FL18, FL19, FL20, FL21, FL22, FL23 and FL25.
9- Rocaglaol derivative of the formula (I)
OH
Figure imgf000064_0001
wherein R3 is -CONH2; and wherein
R1 is alkoxy, optionally substituted, preferably selected from the group consisting of methoxy and a group -0-(CH2)D-R10 wherein n is 1, 2, 3 or 4 and R10 is selected from the group consisting of hydroxyl, -NMe2, -OCONMe2, -OCONH2 and morpholine;
R2 is selected from the group consisting of hydrogen and (Ci-C3)-alkoxy, preferably from hydrogen and methoxy; and,
R4 is halogen, preferably bromine, chloride, iodide, more preferably bromine, or, R1 is alkoxy, optionally substituted, preferably selected from the group consisting of methoxy and a group -O-(CH2)n-R10 wherein n is 1, 2, 3 or 4 and R10 is selected from the group consisting of hydroxyl, -NMe2, -OCONMe2, -OCONH2 and morpholine;
R2 is hydrogen; and, R4 is selected from the group consisting of hydrogen and alkoxy, or, R1 is substituted alkoxy, preferably a group -O-(CH2)n-R10 wherein n is 1, 2, 3 or
4 and R10 is selected from the group consisting of hydroxyl, -NMe2, -OCONMe2, - OCONH2 and morpholine; R2 is (Ci-C3)-alkoxy, preferably methoxy; and R4 is selected from the group consisting of hydrogen and alkoxy, or wherein
R3 is -COOR5 wherein R5 is (Ci-C3)-alkyl, preferably methyl;
R1 is alkoxy, optionally substituted, preferably selected from the group consisting of methoxy and a group -O-(CH2)n-R10 wherein n is 1, 2, 3 or 4 and R10 is selected from the group consisting of hydroxyl, -NMe2, -OCONMe2, -OCONH2 and morpholine;
R2 is hydrogen; and
R4 is selected from the group consisting of hydrogen, methoxy and halogen; or wherein
R1 is methoxy, and R4 is bromine, and wherein
R2 is (Ci-C3)-alkoxy, preferably methoxy, and, R is hydrogen, or
R2 is hydrogen, and, R3 is selected from the group consisting of hydrogen, -COOR5 and -CONR6R7, wherein R5 is (Ci-C3)-alkyl, preferably methyl, and R6 and R7 are independently selected from the group consisting of hydrogen and (Ci-C3)-alkyl, preferably from hydrogen and methyl; or any pharmaceutically acceptable salt thereof. - The rocaglaol derivative according to claim 9, wherein R1 is methoxy;
R2 is hydrogen;
R3 is selected from the group consisting of -COOR5 and -CONH2, wherein R5 is methyl; and
R4 is halogen or methoxy, preferably bromine or methoxy. - The rocaglaol derivative according to claim 9, wherein R1 is methoxy; R2 is selected from the group consisting of methoxy or hydrogen;
R3 is-CONH2; and
R4 is halogen, preferably bromine.
5 12- The rocaglaol derivative according to claim 9, wherein R1 and R2 are methoxy, R3 is hydrogen and R4 is bromine.
13- The rocaglaol derivative according to claim 9, wherein R1 is methoxy; R2 is hydrogen; R3 is selected from the group consisting of hydrogen, -COO(CH3) and -CON(CH3)2; and, R4 is
10 bromine.
14- The rocaglaol derivative according to claim 9, wherein R3 is COOMe, R1 is methoxy, R2 is hydrogen and R4 is selected from the group consisting of hydrogen, methoxy and bromine, preferably is methoxy or bromine.
15
15- The rocaglaol derivative according to claim 9, wherein R1 is methoxy, and R4 is bromine, and wherein either R2 is methoxy and R3 is hydrogen, or R2 is hydrogen and R3 is selected in the group consisting of hydrogen, -COO(CH3), -CONH2, -CONH(CH3) and -CON(CH3)2.
20 16- The rocaglaol derivative according to claim 9, wherein the rocaglaol derivative is selected from the group consisting of FL3, FL5, FL6, FL7, FL8, FL9 and FL14.
17- A rocaglaol derivative of the formula (II)
Figure imgf000066_0001
(π)
25 wherein
R11 is alkoxy, optionally substituted;
R12 is hydrogen; or R11 and R12 together form a -O-CH2-O-unit; R13 is selected from hydrogen and (Ci-C3)-alkoxy;
R14 is -NH-R27, wherein R27 is selected from -SO2Me, -COR28 with R28 being H or (Ci- C3)-alkyl;
R15 is selected from hydrogen, -COOR21, -CONR22R23 and -CONH(CH2)nOH, wherein n is 2, 3 or 4, wherein
R21 and R23 are independently selected from hydrogen and (Ci-C3)-alkyl and R22 is selected from the group consisting of (Ci-C3)-alkoxy, (Ci-C3)-alkyl and hydrogen, or R22 and R23 together form a 5 atom heterocycle, optionally substituted by a group NHCO-(Ci-C4)-alkyl; or R14, R15and carbons (α) and (β) bearing R14 and R15 together form
Figure imgf000067_0001
wherein R24 and R25 are independently selected from the group consisting of (C1-C3)- alkyl and hydrogen, preferably from hydrogen and methyl; R16 is selected from hydrogen, (Ci-C3)-alkoxy and halogen; R17 is in ortho-position to Ri6 and is selected from hydrogen, (Ci-C3)-alkoxy and hydroxyl or form together with R17 a -O-CH2-O-unit;
R18 and R19 are independently selected from the group consisting of hydrogen and halogen, preferably from H, F, Br and Cl, more preferably H or F; or any pharmaceutically acceptable salt thereof.
18- The rocaglaol derivative according to claim 17, wherein the rocaglaol derivative of the formula (II) has one or several of the following features: a) R11 is alkoxy and R12 is hydrogen; b) R13 is selected from hydrogen and methoxy; c) R14 is -NH-R27, wherein R27 is selected from -SO2Me, -COR28 with R28 being H, methyl or ethyl; d) R15 is selected from hydrogen, -COOR21, and -CONR22R23, wherein R21 and R23 are independently selected from hydrogen and methyl, and R22 is selected from the group consisting of hydrogen, methyl and methoxy; more preferably R15 is selected from hydrogen, COOMe, CONH2, CONHMe and CONMe2; e) R16 is selected from hydrogen, (Ci-C3)-alkoxy and halogen, more preferably from hydrogen, chlorine, bromine, fluorine and methoxy, still more preferably from bromine and methoxy; f) R17 is hydrogen; g) R18 is in para and is H or fluorine and R19 is hydrogen.
19- The rocaglaol derivative according to claim 17 or 18, wherein the rocaglaol derivative presents the formula (Ha) or (lib)
Figure imgf000068_0001
wherein the definition of RI l, R12, R13, R14, R15, R16, R17, R18 and R19 is the same as claim 15 or 16.
20- The rocaglaol derivative according to any one of claims 17-19, wherein the rocaglaol derivative has the formula (lib) and R14 is -NH-R27, wherein R27 is -SO2Me or -COR28 with R28 being H or (Ci-C3)-alkyl, preferably H or methyl.
21- The rocaglaol derivative according to any one of claims 17-20, wherein R11 is alkoxy, especially methoxy or ethoxy, more preferably methoxy; R12 is hydrogen; R13 is selected from hydrogen, methoxy and ethoxy, more preferably R13 is hydrogen or methoxy, still more preferably R13 is methoxy; R15 is selected from hydrogen, COOMe, CONH2, CONHMe and CONMe2, more preferably is hydrogen; R16 is selected from the group consisting of hydrogen, chlorine, bromine, fluorine and methoxy, more preferably is chlorine, bromine, or fluorine, still more preferably is bromine; and, R17, R18 and R19 are hydrogen.
22- The rocaglaol derivative according to claim 17, wherein the rocaglaol derivative is selected from the group consisting of FL22, FL23, FL24 and FL25, preferably is FL23 or FL25. 23- A rocaglaol derivative of the formula (Ha)
Figure imgf000069_0001
wherein
R12 is H and R11 is selected from methoxy and ethoxy, more preferably R11 is methoxy; R13 is selected from hydrogen, methoxy and ethoxy, more preferably R13 is hydrogen or methoxy;
R14 is -OCOR20, R20 being selected from hydrogen, methyl and ethyl;
R15 is selected from hydrogen, -COOR21, and -CONR22R23, wherein R21 and R23 are independently selected from hydrogen and methyl, and R22 is selected from the group consisting of hydrogen, methyl and methoxy; more preferably R15 is selected from hydrogen,
COOMe, CONH2, CONHMe and CONMe2; still more preferably R15 is hydrogen;
Rl 6 is chlorine, bromine, or fluorine, preferably bromine;
R17 is hydrogen;
R18 is in para and is H or fluorine and R19 is hydrogen; or any pharmaceutically acceptable salt thereof.
24- The rocaglaol derivative according to claim 23, wherein R11 and R13 are methoxy, R12, R15, R18 and R19 are hydrogen, and R16 is bromine.
25- The rocaglaol derivative according to claim 23, wherein the rocaglaol derivative is selected from the group consisting of FL 19, FL20 and FL21.
26- A rocaglaol derivative of the formula (Ha) R14
Figure imgf000070_0001
wherein
R12 is H and R11 is selected from methoxy and ethoxy, more preferably R11 is methoxy; R13 is selected from hydrogen, methoxy and ethoxy, more preferably R13 is hydrogen or methoxy; R14 is hydroxyl;
R18 is in para and is fluorine and R19 is hydrogen, and wherein
R15 is selected from hydrogen, -COOR21, and -CONR22R23, wherein R21 and R23 are independently selected from hydrogen and methyl, and R22 is selected from the group consisting of hydrogen, methyl and methoxy; more preferably R15 is selected from hydrogen, COOMe, CONH2, CONHMe and CONMe2; and, R16 is bromine and R17 is hydrogen; or R15 is selected from -COOR21, and -CONR22R23, wherein R21 and R23 are independently selected from hydrogen and methyl, and R22 is selected from the group consisting of hydrogen, methyl and methoxy; more preferably R15 is selected from hydrogen, COOMe, CONH2, CONHMe and CONMe2; and, R16 is methoxy and R17 is hydrogen; or any pharmaceutically acceptable salt thereof.
27- The rocaglaol derivative according to claim 26, wherein R11 and R13 are methoxy, R12 is hydrogen, R16 is bromine and R15 is selected from hydrogen, COOMe, CONH2, CONHMe and CONMe2.
28- The rocaglaol derivative according to claim 26, wherein R11 and R13 are methoxy, R12 is hydrogen, R16 is methoxy and R15 is selected from COOMe, CONH2, CONHMe and CONMe2. 29- The rocaglaol derivative according to claim 28, wherein the rocaglaol derivative is FL16, FL17 or FL18.
5 30- The rocaglaol derivative according to anyone of claims 9 to 29 as a medicament.
31- A pharmaceutical composition comprising a rocaglaol derivative according to anyone of claims 9 to 29 and a pharmaceutically acceptable carrier and/or excipient.
10 32- A pharmaceutical composition comprising a rocaglaol derivative according to anyone of claims 9 to 29 and an antineoplastic agent, preferably an anthracycline.
33- Product containing a rocaglaol derivative according to anyone of claims 9 to 29 and an antineoplastic agent, preferably an anthracycline, as a combined preparation for simultaneous, 15 separate or sequential use in the treatment of cancer.
34-The rocaglaol derivative according to claim 1, wherein the rocaglaol derivative is a rocaglaol derivative according to anyone of claims 9 to 29.
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