METHOD FOR PREPARING MARMYCIN A AND ANALOGUES THEREOF, AND ALSO USES THEREOF
20170260210 · 2017-09-14
Inventors
- Raphaël RODRIGUEZ (GIF SUR YVETTE, FR)
- Tatiana CANEQUE (GIF SUR YVETTE, FR)
- Filipe GOMES DA CRUZ (CHILLY MAZARIN, FR)
- Trang MAI (GIF SUR YVETTE, FR)
- Max MALACRIA (PARIS, FR)
- Giovanni MAESTRI (Mantova, IT)
Cpc classification
C07D309/14
CHEMISTRY; METALLURGY
C07D519/00
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
C07D519/00
CHEMISTRY; METALLURGY
G01N33/50
PHYSICS
Abstract
The present invention relates to a method for preparing marmycin A and analogues thereof, to novel marmycin A analogues, and also to the use of these compounds as an organelle marker and in pharmacy, in particular as antibiotics, anticancer agents and antimalarials.
Claims
1. A method for preparing a compound of formula (II): ##STR00040## where R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, Ra, Rb, Rc, Rd and Re are each independently an atom or group of atoms, n represent the number of Ra, Rb, Rd and Re radicals and are equal to 2; said method comprising a coupling step B of a compound of formula (III): ##STR00041## where the radicals R.sub.1 to R.sub.6 are such as defined for the formula (II) compound and Rg is a reactive group with the NH.sub.2 group of the compound of formula (IV), with a compound of formula (IV): ##STR00042## where the radicals Ra to Re and n are such as defined for the formula (II) compound, said coupling step being conducted in the presence of a copper-containing compound.
2. The method according to claim 1, characterized in that the coupling step B is conducted in the presence of a copper-containing compound of formula Cu—X, X being selected from the group formed by Cl, Br, I, SO.sub.4 and OAc.
3. The method according to claim 1, characterized in that the coupling step B is conducted in the presence of 5 mol % to 25 mol %, of copper-containing compound.
4. The method according to claim 1, characterized in that coupling is conducted in the presence of a base e.g. K.sub.2CO.sub.3.
5. The method according to claim 1, further comprising a coupling step A of a compound of formula (V): ##STR00043## where R.sub.1 and R.sub.2 are such as defined in claim 1, Y being a reactive group with the carbon atom carrying the R.sub.3 group of the formula (VI) compound and Rh being a protective group, with a compound of formula (VI): ##STR00044## to obtain a compound of formula (III′): ##STR00045## where the radicals R.sub.1 to R.sub.6 are such as defined in claim 1, in the presence of toluene or xylene.
6. A method for preparing a pentacyclic structure, comprising: preparation of a formula (II) compound according to claim 1, and a cyclisation step C of said formula (II) compound.
7. A method for preparing a compound of following formula (Ia): ##STR00046## where the radicals R.sub.1 to R.sub.6 and Ra to Re and n are such as defined in claim 1, said method comprising a cyclisation step Ca via C—C glycosylation of a formula (II) compound such as obtained according to claim 1, in the presence of HBF.sub.4 to form the compound of formula (Ia).
8. A method for preparing a compound of following formula (Ib): ##STR00047## where the radicals R.sub.1 to R.sub.6 and Ra to Re and n are such as defined in claim 1, said method comprising a cyclisation step Cb via formation of an O—C bond of a compound of formula (II) such as obtained according to claim 1, in a basic medium.
9. A method for preparing a compound of formula (IaD) such as defined below, said method comprising an addition step D of a compound of formula (Ia): ##STR00048## where the radicals R.sub.1 to R.sub.6, Ra to Re and n are such as defined in claim 1, and wherein at least one of the radicals Ra to Re is an OH group, with a compound of following formula:
Rx-L-Rz (Ie) where: Rx is a reactive group allowing reaction with an OH function; L is a group of atoms called a <<spacer group>>; and Rz is an optionally substituted (C.sub.3-C.sub.20)heterocycle; said addition step D resulting in the formation of a compound of following formula (IaD): ##STR00049## where the radicals R.sub.1 to R.sub.6 and n are such as defined in claim 1, and wherein the radicals Ra1 to Re1 have the same definitions as the radicals Ra to Re defined for formula (Ia) respectively, among which at least one of the radicals Ra1 to Re1 represents the group resulting from the reaction between the groups OH and Rx, bonded covalently to L-Rz.
10. The method according to claim 1, wherein: R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are each independently selected from the group formed by: H, OH, halogen, C(O)OH, ═O, (C.sub.1-C.sub.10)alkyl, O(C.sub.1-C.sub.10)alkyl, OC(O)(C.sub.1-C.sub.10)alkyl, (C.sub.2-C.sub.10)alkenyl, C(O)O(C.sub.1-C.sub.10)alkyl, NH.sub.2, NH(C.sub.1-C.sub.10)alkyl, N[(C.sub.1-C.sub.10)alkyl].sub.2, NHC(O)(C.sub.1-C.sub.10)alkyl, N(C.sub.1-C.sub.10)alkyl-C(O)(C.sub.1-C.sub.10)alkyl, C(O)NH.sub.2, C(O)N(C.sub.1-C.sub.10)alkyl, C(O)N[(C.sub.1-C.sub.10)alkyl].sub.2, oses and epoxy groups, wherein said alkyls and/or said oses can be substituted; or R.sub.1 with R.sub.2 and/or R.sub.3 with R.sub.4 and/or R.sub.4 with R.sub.5 and/or R.sub.5 with R.sub.6, together with the carbon atoms to which they are attached, form a (C.sub.3-C.sub.10)cycloalkyl or (C.sub.6-C.sub.10)aryl group, said cycloalkyl or aryl groups optionally being substituted, and wherein at least one of the carbon atoms may optionally be replaced by a heteroatom; the radicals Ra are each independently selected from the group formed by: H, OH, halogen, C(O)OH, ═O, (C.sub.1-C.sub.10)alkyl, O(C.sub.1-C.sub.10)alkyl, OC(O)(C.sub.1-C.sub.10)alkyl, (C.sub.2-C.sub.10)alkenyl, C(O)O(C.sub.1-C.sub.10)alkyl, NH.sub.2, NH(C.sub.1-C.sub.10)alkyl, N[(C.sub.1-C.sub.10)alkyl].sub.2 NHC(O)(C.sub.1-C.sub.10)alkyl, N(C.sub.1-C.sub.10)alkyl-C(O)(C.sub.1-C.sub.10)alkyl, C(O)NH.sub.2, C(O)N(C.sub.1-C.sub.10)alkyl, C(O)N[(C.sub.1-C.sub.10)alkyl].sub.2, and the reactive groups allowing the formation of a glycosidic C—C bond, wherein said alkyls can be substituted; the radicals Rb, Rc and Re are each independently selected from the group formed by: H, OH, halogen, C(O)OH, ═O, (C.sub.1-C.sub.10)alkyl, O(C.sub.1-C.sub.10)alkyl, OC(O)(C.sub.1-C.sub.10)alkyl, (C.sub.2-C.sub.10)alkenyl, C(O)O(C.sub.1-C.sub.10)alkyl, NH.sub.2, NH(C.sub.1-C.sub.10)alkyl, N[(C.sub.1-C.sub.10)alkyl].sub.2, NHC(O)(C.sub.1-C.sub.10)alkyl, N(C.sub.1-C.sub.10)alkyl-C(O)(C.sub.1-C.sub.10)alkyl, C(O)NH.sub.2, C(O)N(C.sub.1-C.sub.10)alkyl and C(O)N[(C.sub.1-C.sub.10)alkyl].sub.2, wherein said alkyls can be substituted; the radicals Rd are each independently selected from the group formed by: H, OH, halogen, C(O)OH, ═O, (C.sub.1-C.sub.10)alkyl, O(C.sub.1-C.sub.10)alkyl, OC(O)(C.sub.1-C.sub.10)alkyl, (C.sub.2-C.sub.10)alkenyl, C(O)O(C.sub.1-C.sub.10)alkyl, NH.sub.2, NH(C.sub.1-C.sub.10)alkyl, N[(C.sub.1-C.sub.10)alkyl].sub.2, NHC(O)(C.sub.1-C.sub.10)alkyl, N(C.sub.1-C.sub.10)alkyl-C(O)(C.sub.1-C.sub.10)alkyl, C(O)NH.sub.2, C(O)N(C.sub.1-C.sub.10)alkyl, C(O)N[(C.sub.1-C.sub.10)alkyl].sub.2, (C.sub.1-C.sub.10)alkyl-COO.sup.−, methoxymethyloxygen, and the reactive groups allowing the formation of an O—C bond such as AcO, wherein said alkyls can be substituted.
11. A compound of following formula (Ia): ##STR00050## where the radicals R.sub.1 to R.sub.6 and Ra to Re and n are such as defined in claim 1, with the exception of the compounds where R.sub.3 and R.sub.4, together with the carbon atoms to which they are attached, form a phenyl group, and with the exception of the following compounds: ##STR00051##
12. A compound of following formula (Ib): ##STR00052## where the radicals R.sub.1 to R.sub.6 and Ra to Re and n are such as defined in claim 1.
13. A compound of following formula (IaD): ##STR00053## where the radicals R.sub.1 to R.sub.6 are each independently an atom or group of atoms, and n represents the number of Ra, Rb, Rd and Re radicals and are equal to 2, and the radicals Ra.sub.1 to Re.sub.1 are such as defined in claim 9.
14. A compound of following formula (IaD): ##STR00054##
15. A pharmaceutical composition comprising a compound of formula (Ia), (Ib), (IaD) or (II) such as defined in claim 1.
16. (canceled)
17. (canceled)
18. (canceled)
19. A method for treating and/or preventing a disease selected among a cancer, bacterial infection and malaria, comprising administering to a mammal in need thereof a therapeutically effective amount of a compound of formula (Ia), (Ib), (IaD) or (II) such as defined in claim 1.
20. A method for treating and/or preventing a resistant cancer, in particular a cancer having cancerous stem cells, comprising administering to a mammal in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a compound of formula (Ia), (Ib), (IaD) or (II) such as defined in claim 1.
21. A method to detect at least one lysosome, comprising the contacting of at least one cell, with a compound of formula (Ia), (Ib), (IaD) or (II) such as defined in claim 1.
22. A method for the marking of at least one lysosome comprising the contacting of at least one cell preferably of eukaryote type, with a compound of formula (Ia), (Ib), (IaD) or (II) such as defined in claim 1.
Description
DESCRIPTION OF THE FIGURES
[0179]
[0180]
[0181]
[0182]
[0183]
[0184]
[0185]
[0186]
[0187]
[0188]
[0189]
[0190]
EXAMPLES
Example 1: Synthesis of Compounds of Formulas (V) and (VI) of the Invention
[0191] The synthesis of dienophile 5 (compound of formula (V)) is performed in accordance with Scheme 1. Compound 8 (supplier, Aldrich) is protected under conventional conditions in the form of diacetylated naphthalene 9 which is then brominated in an acid medium (see Kitani, Y.; Morita, A.; Kumamoto, T.; Ishikawa, T. Helvetica Chimica Acta 2002, 85, 1186 Carreno, M. C. et al Chem. Eur. J. 2000, 6, 906 Shis, C.; Swenton, J. S. J. Org. Chem. 1982, 47, 2825).
##STR00029##
[0192] The diene 15 (compound of formula (VI)) is prepared from compound 10 that is commercially available (supplier, Aldrich) according to Scheme 2. A first bromination step is first carried out. Compound 11 is protected as an acetal 12 in the presence of ethylene glycol and a catalytic amount of acid. The reaction is conducted at a concentration of 0.4 M in under 2 hours under reflux with benzene. Compound 12 is converted to 13 via a palladium-catalysed coupling step to graft the vinyl chain thereupon.
[0193] Deprotection of 13 to 14 is obtained in a mild acid medium to prevent isomerisation of the double bond and the formation of an unsaturated α-β ketone. The action of methylmagnesium bromide in the presence of cerium chloride under cold conditions allows the racemic formation of the desired diene 15.
##STR00030##
Example 2: Coupling Step a and Preparation of a Formula (III) Compound of the Invention
[0194] Compound 16 (compound of formula (III′)) is prepared according to following Scheme 3:
##STR00031##
[0195] Compounds 5 and 15 are brought under reflux with toluene for 16 hours, after which the medium is subjected to slow evaporation for about 1 hour with a rotary evaporator in a bath at 60° C. The medium is re-dissolved in MeOH and left under agitation in the dark for 4 hours. 3 equivalents of potassium carbonate are then added to (compound of formula (III)).
Example 3: Synthesis of Aminopyranose
[0196] Following the procedure described in the literature (cf. Raymond N. Russell, Theresa M. Weigel, Oksoo Han, Hung-wen Liu. Carbohydrate Research, Volume 201, Issue 1, 15 Jun. 1990, Pages 95-114 (b) Brimacombe, J. S.; Hanna, R.; Saeed, M. S.; Tucker, L. C. N. J. Chem. Soc., Perkin Trans. 1 1982, 2583-2587.) compound 4 is prepared from L-rhamnopyranose 7 (supplier, Alfa Aeser).
[0197] After protection under standard conditions with benzylidene at position 2 and 3, compound 17 is again protected via action of MOMCl (chloromethyl methyl ether) at position 4. The use of a strong base on compound 18 allows selective deprotection to form the ketone 19 at position 3. Compound 19 reacts with O-benzylhydroxylamine in the presence of sodium acetate to form the intermediate 20. In the presence of cerium chloride and methyllithium, compound 20 is selectively methylated to 21 which undergoes hydrogenolysis to obtain the desired product 4, in accordance with Scheme 4 below.
##STR00032##
Example 4: Coupling Step B, Preparation of Formula (II) Compounds of the Invention
Example 4.1
[0198] Coupling B of the invention was conducted a first time according to following Scheme 5:
##STR00033##
[0199] Coupling B was performed in the presence of K.sub.2CO.sub.3 in toluene under reflux with 20 mol % Cu—I, 2 eq. of K.sub.2CO.sub.3, 3 eq. of 4 in toluene at 160° C. for 72 hours. A yield of 33% was obtained.
[0200] Different operating conditions were also tested as shown in Table 1 below.
TABLE-US-00002 TABLE 1 Catalyst Base (mol %) Equiv. 4 (equiv.) Solvent T° C. t (h) Yield (%) Cul 10 2 K.sub.2CO.sub.3 2 Toluene 140 72 33 Cul 20 3 K.sub.2CO.sub.3 2 Toluene 140 24 22-32 Cul 20 1 (2 eq K.sub.2CO.sub.3 2 Toluene 150 24 14 23) Cul 20 3 K.sub.2CO.sub.3 2 Toluene 150 24 33 Cul 20 2 K.sub.2CO.sub.3 3 Toluene 150 24 18 Cul 10 3 K.sub.2CO.sub.3 1.4 CH.sub.3CN 150 24 3 Cul 20 5 K.sub.2CO.sub.3 2 Toluene 160 72 33
Example 4.2
[0201] Coupling B of the invention was also performed according to following Scheme 6.
##STR00034##
[0202] A yield of 33% was also obtained.
[0203] In a dry tube with septum the following were mixed under argon: triflate 3 (34.00 mg, 0.081 mmol), the catalyst CuI (3.08 mg, 0.016 mmol, 20% mol) and K.sub.2CO.sub.3 (22.3 mg, 0.162 mmol, 2 equiv.), after which the amine 4 was added (88.64 mg, 0.405 mmol, 5 equiv.) dissolved in 4 mL of dry toluene.
[0204] The septum was then replaced on the stopper under argon and the reaction left to take place at 160° C. for 72 h. The medium was cooled, washed in saturated NaHCO.sub.3 solution (5 mL), and extracted with DCM (3×5 mL). The organic layers were dried over Na.sub.2SO.sub.4 and the solvent removed in vacuo. Purification by flash-chromatography on silica gel (heptane/AcOEt, 8:2) was finally carried out.
[0205] 13.05 mg of compound 27 (33%, 8-(((2S,3R,4R,6R)-6-methoxy-3-(methoxymethoxy)-2,4-dimethyltetrahydro-2H-pyran-4-yl)amino)-3-methyltetraphene-7,12-dione) were obtained in the form of a purple solid.
[0206] .sup.1H NMR (300 MHz, CDCl.sub.3) δ 10.56 (s, 1H), 8.35 (d, J=7.5 Hz, 1H), 8.29-8.17 (m, 1H), 7.81-7.62 (m, 2H), 7.57 (d, J=7.1 Hz, 1H), 7.46 (t, J=8.0 Hz, 1H), 7.35 (d, J=8.6 Hz, 1H), 4.89 (q, J=6.8 Hz, 2H), 4.63 (d, J=3.2 Hz, 1H), 4.31 (dq, J=9.4, 6.2 Hz, 1H), 3.48 (s, 3H), 3.28 (d, J=9.4 Hz, 1H), 3.14 (s, 3H), 2.77 (d, J=14.7 Hz, 1H), 1.82 (dd, J=14.8, 4.2 Hz, 1H), 1.65 (s, 3H), 1.39 (d, J=6.3 Hz, 3H). .sup.13C NMR (126 MHz, CDCl.sub.3) b 187.33, 185.15, 151.03, 138.46, 137.16, 136.42, 135.50, 134.22, 134.20, 131.96, 128.82, 128.58, 128.45, 127.74, 123.06, 120.54, 115.62, 113.84, 99.09, 97.56, 87.49, 64.48, 56.81, 55.75, 54.90, 38.38, 26.54, 21.73, 18.70. HRMS (ESI-TOF) calculated for C.sub.29H.sub.32NO.sub.6 [M+H].sup.+ 490.2224; found: 490,2225.
Example 5: Cyclisation Step Ca, Preparation of Formula (Ia) Compounds of the Invention
Example 5.1
[0207] Compounds 30 and 31 were placed in the presence of 1.5 eq HBF.sub.4 (50% w/w in aqueous solution) under reflux in CH.sub.3CN. The cyclisation product 1 was obtained with a yield of 17%.
[0208] Therefore, in a single step, both cyclisation and MOM deprotection are carried out to obtain the natural product (Scheme 7).
##STR00035##
[0209] Several tests were carried out as shown in Table 2 below.
TABLE-US-00003 TABLE 2 Acid Equiv. Solvent T° C. t (h) Yield (%) HBF.sub.4OEt.sub.2 1 CH.sub.3CN reflux 4 12 HBF.sub.4 50% 1.5 CH.sub.3CN reflux 24 7 m/m aqueous sol. HBF.sub.4 50% 1 CH.sub.3CN reflux 7 11 m/m aqueous sol. HBF.sub.4 50% 60 mol % CH.sub.3CN reflux 7 12 m/m aqueous sol. HCl 2 CH.sub.3CN reflux 8 8
[0210] In particular, with 60 mol % HBF.sub.4 under reflux with acetonitrile, product 1 was obtained with a yield of 13%.
Example 5.2
[0211] A solution of compound 27 (21.00 mg, 0.0429 mmol) and HBF.sub.4 (5 μL, 0.0429 mmol, 1 equiv.) in 2 mL of CH.sub.3CN was placed under reflux for 8 h.
[0212] The mixture was then cooled, diluted in dichloromethane (5 mL) and washed with saturated NaHCO.sub.3 solution (3×5 mL). The organic phase was dried over Na.sub.2SO.sub.4 and the solvent evaporated in vacuo. Thin layer preparative chromatography using heptane/AcOEt (1:1) as eluent allowed 2.3 mg of Marmycin A to be obtained with a yield of 13%
[0213] Recrystallization by evaporation of a CH.sub.2Cl.sub.2:Heptane mixture allowed red crystal needles to be obtained.
[0214] .sup.1H NMR (950 MHz, CDCl.sub.3) δ 9.59 (s, 1H), 9.55 (d, J=8.8 Hz, 1H), 8.31 (d, J=8.5 Hz, 1H), 8.08 (d, J=8.5 Hz, 1H), 7.67 (s, 1H), 7.60 (d, J=7.3 Hz, 1H), 7.57 (dd, J=8.8, 1.7 Hz, 1H), 7.53 (d, J=7.4 Hz, 1H), 4.85-4.80 (m, 1H), 3.22 (t, J=9.4 Hz, 1H), 3.17 (dq, J=9.2, 6.0 Hz, 1H), 2.56 (s, 1H), 2.19 (ddd, J=13.2, 3.2, 1.2 Hz, 1H), 1.87 (dd, J=13.2, 1.8 Hz, 1H), 1.55 (s, 1H), 1.27 (d, J=6.0 Hz, 1H). .sup.13C NMR (239 MHz, CDCl.sub.3) δ 186.53, 185.77, 148.75, 138.81, 136.56, 136.47, 136.15, 134.72, 134.65, 132.21, 128.82, 128.54, 128.31, 127.80, 127.42, 122.34, 116.13, 111.35, 79.16, 69.36, 69.33, 51.76, 35.05, 25.03, 21.73, 18.43. HRMS (ESI-TOF) calculated for C.sub.26H.sub.24NO.sub.4 [M+H].sup.+ 414.1700; found: 414.1703.
Example 6: Cyclisation Step Cb, Preparation of Formula (Ib) Compounds of the Invention
[0215] Compounds of formula (Ib) were also obtained from compounds 28 and 32. In a strong basic medium, the products 33 (oxamarmycin) and 34 were obtained with respective yields of 52% and 65%, as shown in Scheme 8 below.
##STR00036##
[0216] A solution of compound 28 (5.00 mg, 0.0112 mmol) in 2 mL of dry DMF was cooled to 00° C. and added to NaH (2.70 mg, 0.1123 mmol, 10 equiv.):
after 2 h at this temperature, the reaction was halted with MeOH. The solvent was evaporated in vacuo and preparative thin layer chromatography using petroleum ether/AcOEt (8:2) as eluent allowed 2.54 mg of O-Marmycin to be obtained with a yield of 52% (purple solid).
[0217] .sup.1H NMR (300 MHz, CDCl.sub.3) δ 9.65 (d, J=8.9 Hz, 1H), 9.40 (s, 1H), 8.37 (d, J=8.6 Hz, 1H), 8.07 (d, J=8.7 Hz, 1H), 7.66 (s, 1H), 7.58 (m, 2H), 7.06 (d, J=8.4 Hz, 1H), 4.72 (d, J=4.3 Hz, 1H), 3.94 (dq, J=12.1, 6.1 Hz, 1H), 3.81 (dd, J=9.8, 1.3 Hz, 1H), 3.28 (s, 3H), 2.55 (s, 3H), 2.28 (d, J=14.8 Hz, 1H), 2.06 (dd, J=14.6, 4.7 Hz, 1H), 1.38 (s, 3H), 1.31 (d, J=6.2 Hz, 3H). .sup.13C NMR (126 MHz, CDCl.sub.3) δ 186.01, 185.61, 145.39, 138.58, 136.73, 136.58, 135.08, 134.08, 131.92, 129.62, 129.38, 128.90, 128.72, 127.77, 122.59, 119.66, 117.69, 112.88, 97.52, 79.61, 62.13, 55.58, 48.38, 41.89, 28.49, 21.72, 17.45. HRMS (ESI-TOF) calculated for C.sub.27H.sub.26NO.sub.5 [M+H].sup.+ 444.1805; found: 444.1826.
Comparative Example 7: Coupling as Per Buchwald-Hartwig Reaction
Example 7.1
[0218] Compound 22 was obtained under the conditions described in Scheme 3 from commercially available alcohol, and the amine 23 is also commercially available (supplier, Aldrich). The reaction was carried out according to the following Scheme:
##STR00037##
[0219] Different catalysts, ligands and solvents and different bases and temperatures were tested (cf. Table 3). In the best case, only 13% of product 24 was observed. The remainder was a hydrolysis product 25 and reagent 22 which had not reacted.
TABLE-US-00004 TABLE 3 Catalyst Base Yield (mol %) Ligand (equiv.) Solvent T° C. t (h) (%) Pd(dba).sub.2.sup.a BINAP NaO.sup.tBu Toluene 110 16 h — Pd(OAc).sub.2.sup.a BINAP NaO.sup.tBu Toluene 110 17 h traces Pd(OAc).sub.2.sup.b DPPF Cs.sub.2CO.sub.3 Toluene 110 20 h — [PdCl(allyl)].sub.2.sup.a DPPF NaO.sup.tBu Toluene 110 20 h — Pd(OAc).sub.2.sup.c BINAP Cs.sub.2CO.sub.3 Toluene 110 22 h — Pd(OAc).sub.2.sup.c BINAP Cs.sub.2CO.sub.3 CH.sub.3CN Reflux 48 h — Pd(OAc).sub.2.sup.c BINAP Cs.sub.2CO.sub.3 DMF Reflux 8 h traces Pd(OAc).sub.2.sup.a P(Ph.sub.3).sub.3 Cs.sub.2CO.sub.3 Toluene 110 48 h — Pd(OAc).sub.2.sup.c BINAP NaO.sup.tBu THF Reflux 48 h — Pd(OAc).sub.2.sup.d BINAP Cs.sub.2CO.sub.3 DMF 150 30 min traces Pd(OAc).sub.2.sup.d BINAP Cs.sub.2CO.sub.3 Toluene 160 30 min traces Pd(OAc).sub.2.sup.e BINAP NaO.sup.tBu Toluene 180 35 min 13 Pd(OAc).sub.2.sup.f BINAP NaO.sup.tBu Toluene 110 35 min 4 .sup.a5% Pd, 5% L, 1.4 eq. base, 1.2 eq. LiCl .sup.b10% Pd, 15% L, 2 eq. Base, 2 eq. LiCl .sup.c5% Pd, 10% L, 2 eq. Base, 2 eq. LiCl .sup.d5% Pd, 10% L, 1 eq. base, 1 eq. LiCl .sup.e15% Pd, 15% L, 1.2 eq. base; .sup.f15% Pd, 15% L, 1.2 eq. base, 1.4 eq. LiCl.
Example 7.2
[0220] Coupling was also performed between triflate 22 and aminopyranose 4. The yield dropped to 3% and much hydrolysis product 25 was observed (Scheme 10).
##STR00038##
[0221] As a result, coupling B of the invention allows a yield of the formula (II) compound of at least 30% to be obtained, much higher than the yield obtained with coupling of Buchwald-Hartwig type.
Comparative Example 8: Cyclisation Test Performed in the Presence of an Acid
[0222] The pathway of intramolecular C-glycosylation promoted by a Lewis or Brønsted acid was tested. An extensive study was conducted by varying the acid (BF.sub.3 Et.sub.2O, TMSOTf, ScOTf, InOTf, PPTS, APTS, Cp.sub.2HfCl.sub.2/AgClO.sub.4) without any conclusive results. Only deprotection of compound 27 to 28, and the formation of amino-anthraquinone 29 were observed.
##STR00039##
[0223] Contrary to the operation conditions for cyclisation Ca and Cb of the invention, cyclisation in the presence of an acid did not allow the desired compounds to be obtained.
Example 9: Cytotoxic Activity of the Compounds of the Invention
[0224] Cell proliferation assays were carried out in the following manner to demonstrate the cytotoxic activity of the compounds of the invention:
[0225] U2OS and A2780 cells purchased from ATCC were held in McCoy's 5A or RPMI 1640 medium respectively, supplemented with 10% foetal bovine serum (FBS)) and 1× Antibiotic-Antimycotic (all from Gibco®) at 37° C. with 5% CO.sub.2. Measurement of cell viability was carried out by seeding 2000 cells per well in a 96-well plate. N-Acetyl Cystein (NAC, A9165 Sigma) or Pan-caspase zVAD-FMK inhibitor (550377 by BD pharmingen) were pre-treated for 1 hour or 30 minutes respectively before treatment with Marmycin A. The reagent <<CellTiter-Blue®>> Reagent) (20 μl/well) was added after 24, 48, or 72 treatment hours, and the cells were incubated for one hour before fluorescence detection (560(20)Ex/590(10)Em) on a Perkin Elmer Wallac 1420 Victor2 Microplate Reader.
[0226] The same protocol was followed to evaluate cell viability of HT1080 cells in the presence of compound 33 of Example 6 (called Oxamethoxymarmycin), doxorubicin or etoposide.
[0227] The results are given in
[0228] In
[0229] In
[0230] In
Example 10: Cell Location of the Compounds of the Invention
[0231] U2OS cells cultured to less than 40% confluence were treated for 24 hours with 10 μM of compound unless otherwise indicated. The marker LysoTracker® Blue DND-22 (L7525, Molecular Probes@) was added 30 minutes before fixing. GFP-Lamp1 was transiently transfected following the manufacturer's instructions. To summarise, 5 mL of CellLight® Lysosomes-GFP BacMam 2.0 (C10596, Life Technologies) were mixed with 200 μM of U2OS culture medium and added to each well of a 24-well plate. After overnight incubation (16 hours), the cells were washed and treated with Marmycin A as indicated in Example 9. For analysis by immunofluorescence, the cells were fixed 12 minutes in 2% formaldehyde/PBS, permeabilised 10 min with 0.1% Triton X/PBS and fixed for 1 h with 5% BSA, 0.2% Tween-20/PBS (blocking buffer). Cover glasses were incubated with anti-LAMP1, p62, antibodies, diluted to 1/100 in the blocking buffer overnight at 4° C. The cells were then washed 3 times with 0.2% Tween-20/PBS, and incubated as described above with anti-mouse Alexa 488-conjugated secondary antibody (A11029, Invitrogen) diluted to 1/500 in blocking buffer. The cover glasses were washed as described above and mounted with VectaShield® mounting medium for analysis by fluorescence with or without DAPI (Vector Laboratories Ltd). The images were obtained with a Leica microscope (Zeiss) and analysed with ImageJ software.
[0232]
[0233]
[0234]
Example 11: Western Blot
[0235] The cells treated as indicated in Example 10 were washed twice in PBS and lysed with Laemmli buffer 2×. The cell extracts were brought to the boil for five minutes at 100° C. and quantified with a Nanodrop 2000 device (Thermal Scientific). 100 μg of protein lysate were separated with 4-20% Mini-PROTEAN® TGX Stain-Free™ gel (BioRad) and transferred onto a nitrocellulose membrane (Amersham). Stains were detected with different antibodies such as anti-β-actin (ab8226, Abcam), anti-p62 (610833, BD Transduction Laboratories™), anti-H2AX (PA1-14198, Thermal Scientific), anti-gH2AX (Ser139) (#2577, Cell Signaling), anti-p21, anti-p53 (1C12) (#2524, Cell Signaling), anti-p-p53, anti-LC3B (#2775, Cell Signaling), anti-BID, diluted to 1/1000 in 5% BSA, 0.1% Tween-20/TBS.
[0236] Western Blot analysis specifically showed that Marmycin A (<<1 !>>) induces a response that only scarcely damages caspase-dependent DNA, which tallies with the absence of direct marking of the genome, whereas Doxorubicin (<<4 !>>) generates a DNA-damage response (phosphorylation of H2AX on ser139, phosphorylation of P53, induction of P21).
Example 12: Preparation of Formula (IaD) Compounds, Addition Step D of the Invention
[0237] To a mixture of marmycin A (2.3 mg, 0.0055 mmol), artesunate (2.1 mg, 0.0056 mmol) and 4-dimethylaminopyridine (DMAP) (0.7 mg, 0.0006 mmol, 10 mol %) in dry dichloromethane (0.3 mL) the addition was made of N,N′-dicyclohexylcarbodiimide solution (1.1 mg, 0.0055 mmol) in dichloromethane (0.2 mL) at 0° C. The resulting solution was mixed overnight at ambient temperature and concentrated to a dry residue.
[0238] The reaction product was purified by thin layer chromatography (heptane/AcOEt, 1:1) to obtain a red solid called artesumycin (1.8 mg, 42%).
[0239] .sup.1H NMR (500 MHz, CDCl.sub.3) δ 9.65 (s, 1H), 9.59 (d, J=9.0 Hz, 1H), 8.39 (d, J=8.5 Hz, 1H), 8.11 (d, J=9.0 Hz, 1H), 7.69 (s, 1H), 7.63-7.56 (m, 2H), 7.50 (d, J=7.5 Hz, 1H), 5.80 (d, J=9.5 Hz, 1H), 5.41 (s, 1H), 4.83 (s, 1H), 4.79 (d, J=9.5 Hz, 1H), 3.44 (dq, J=12.0, 6.0 Hz, 1H), 2.79-2.74 (m, 4H), 2.58-2.54 (m, 4H), 2.36 (td, J=14.0, 4.0 Hz, 1H), 2.25 (dd, J=13.0, 2.0 Hz, 1H), 2.02 (ddd, J=7.5, 4.0, 3.0 Hz, 1H), 1.89-1.86 (m, 2H), 1.75 (dd, J=13.5, 4.0 Hz, 2H), 1.67 (dd, J=13.0, 3.0 Hz, 1H), 1.62-1.58 (m, 2H), 1.44-1.41 (m, 4H), 1.42 (s, 3H), 1.35 (dd, J=13.0, 3.0 Hz, 1H), 1.09 (d, J=6.0 Hz, 3H), 1.95-1.02 (m, 1H), 0.93 (d, J=6.0 Hz, 3H), 0.86 (d, J=7.0 Hz, 3H). .sup.13C NMR (151 MHz, CDCl.sub.3) b 186.6, 185.6, 172.1, 171.2, 148.6, 138.7, 136.5, 135.9, 134.9, 134.6, 132.2, 128.8, 128.6, 128.3, 127.8, 126.7, 122.5, 115.9, 111.0, 104.6, 92.3, 91.5, 80.2, 79.6, 69.3, 66.4, 51.5, 51.1, 45.2, 37.3, 36.2, 35.0, 34.1, 31.9, 29.8, 29.1, 28.9, 26.1, 25.1, 24.6, 22.1, 21.7, 20.3, 18.0, 12.2. HRMS (ESI-TOF) calculated for C.sub.45H.sub.49NNaO.sub.11.sup.+ [M+Na].sup.+ 802.3198; found: 802.3204. [α].sub.D.sup.20+253 (c 0.015, tetrahydrofuran).
Example 13: Cell Location of Formula (IaD) Compounds of the Invention
[0240] U2OS cells cultured to less than 40% confluence were treated for 24 hours with 10 μM artesumycin. The marker LysoTracker® Blue DND-22 (L7525, Molecular Probes®) was added 30 minutes before fixing. GFP-Lamp1 was transiently transfected following the manufacturer's instructions. To summarise, 5 mL of CellLight® Lysosomes-GFP BacMam 2.0 (C10596, Life Technologies) were mixed with 200 μM of U2OS culture medium and added to each well of a 24-well plate. After overnight incubation (16 hours) the cells were washed and treated with artesumycin as indicated in Example 9. Immunofluorescence analysis was carried out in the same manner as in Example 10.
[0241]
Example 14: Cytotoxic Activity of the Formula (IaD) Compounds of the Invention
[0242] Cell proliferation assays were performed in similar manner to those in Example 9 to demonstrate the cytotoxic activity of the formula (IaD) compounds of the invention.
[0243] MDA-MB-231 cells (human breast cancer cells) purchased from ATCC were held in McCoy's 5A medium, supplemented with 10% foetal bovine serum (FBS)) and 1× Antibiotic-Antimycotic (all from Gibco®) at 37° C. with 5% CO.sub.2.
[0244] Cell viability was measured by seeding 2000 cells per well in a 96-well plate. N-Acetyl Cystein (NAC, A9165 Sigma) or Pan-caspase zVAD-FMK inhibitor (550377 from BD pharmingen) were pre-treated for 1 hour or 30 minutes respectively before treatment with the assayed compounds. The reagent <<CellTiter-Blue® Reagent>> (20 μl/well) was added after a treatment time of 24, 48 or 72 hours, and the cells were incubated for one hour before fluorescence detection (560(20)Ex/590(10)Em) on a Perkin Elmer Wallac 1420 Victor2 Microplate Reader.
[0245] The assayed compounds were doxorubicin, marmycin A, artesunate, artesumycin and a combination of marmycin A and artesunate (cf.
[0246]
[0247] A synergic effect is therefore observed for artesumycin compared with the activity of the marmycin A and artesunate combination.
Example 15: Cytotoxic Activity of the Formula (IaD) Compounds of the Invention
[0248] Cell viability: cell viability was assessed using the following protocol:
[0249] Seeding 1000 cells/well in a 96-well plate. The cells were treated for 72 hours. After a treatment time of 72 hours the reagent CellTiter-Blue® Reagent (G8081, Promega) was added and the cells incubated for one hour before recording fluorescence intensities (Excitation, 560/20 nm; Emission, 590/10 nm) using a Perkin Elmer Wallac 1420 Victor2 Microplate Reader.
[0250] The results are given in
[0251] Cell culture: the following material was used: saline buffer <<Dulbecco's Phosphate-Buffered Saline>> (14190-094, 500 mL, Gibco), DMEM/F12 (31331-028, 500 mL, Gibco), DMEM high-glucose with UltraGlutamine (BE12-604F/U1, BioWhittaker, Lonza), McCoy's 5A medium (Modified) (26600-023, Gibco), RPMI 1640 with L-glutamine (BE12-702F/U1, BioWhittaker, Lonza), Foetal Bovine Serum—FBS, 10270-106, Gibco), Hydrocortisone (H0888, Sigma), Insulin (10516, Sigma or 19278, Sigma), BD Epidermal growth factor human recombinant (hEGF, 354052, BD Biosciences), PEN-STREP (DE17-602E, BioWhittaker, Lonza), puromycin dihydrochloride (A11138-02, 460 Life Technologies).
[0252] The human mammary epithelial cell line was infected with a retrovirus carrying hTERT, SV40 and the oncogenic allele HrasV12, called HMLER CD44high/CD24 low cells, not expressing E-cadherin and Vimentin (reference HMLER CD24low or HMLER CD44+/CD24−), courteously offered by A. Puisieux (INSERM).
[0253] The line referenced HMLER ID2 is a hTert, SV40, HRasV12 transformed line, isogenic but non-stem.
[0254] The HMLER CD44.sup.high/CD24.sup.low cells (Cancer stem cells—CSCs), HMLER CD44.sup.high/CD24.sup.high (non-CSCs) were cultured in DMEM/F12 supplemented with 10% FBS, 10 μg/mL insulin, 0.5 μg/mL hydrocortisone, 10 ng/mL hEGF and 0.5 μg/mL of puromycin.
[0255] A mycoplasma assay was performed using a mycoplasma PCR detection kit (G238, 470 Applied Biological Materials) confirming the absence of cell contamination.
[0256] The assayed compounds were doxorubicin, artesunate and artesumycin (TC5) (cf.
[0257]
[0258]
[0259] These results illustrate the efficacy of artesumycin on human malignant cell lines resistant to conventional therapies such as taxol, whether they be stem or non-stem.