Patent Application
20250163088
Metal based drugs used in cancer therapy are square planar platinum(II) complexes, e. g. cisplatin (compound 1), oxaliplatin (2) and carboplatin (3) being registered worldwide. Although Pt complexes are versatile tools of the trade, their applicability has shortcomings, such as the development of platinum resistance in tumors (Holditch et al., 2019; Manohar and Leung, 2018; McMullen et al., 2020; Mukherjea et al., 2020; Yu et al., 2020), the latter two being very characteristic for cisplatin.
According to Ndagi Umar et al. (Ndagi et al., 2017), in anti-cancer Pt complexes, conjugation with sugars can result in a number of improved properties, e.g. improved solubility, reduction of adverse side effects, and improved uptake by cells.
Ruthenium complexes can be promising alternatives to platinum complexes due to their similar chemical characteristics in terms of ligand exchange (Kenny and Marmion, 2019). Importantly, multiple studies have pointed out the reduced toxicity for Ru complexes as compared to Pt complexes both in cellular and animal models (Gano et al., 2019; Liu et al., 2019; Mello-Andrade et al., 2018; Mihajlovic et al., 2020).
Among the Ru(III)- and Ru(II)-based complexes a great number of derivatives were tested as anticancer metallodrugs and two of them, the imidazolium salt of tetrachlorido(dimethylsulfoxide)(imidazole)ruthenium(III) (NAMI-A) (4) and sodium tetrachloridobis(indazole)ruthenium(III) (NKP1339) (5) reached clinical trials (Burris et al., 2016; Fernandes, 2019; Kenny and Marmion, 2019; Meier-Menches et al., 2018; Zeng et al., 2017). In good agreement with that NAMI-A (Leijen et al., 2015), further compounds KP1019/1339 (IT-139, BOLD100) (Burris et al., 2016) or TLD-1433 (Kulkarni et al., 2022) are also already in different phases of clinical trials against neoplastic diseases as bladder or lung cancer. Of note, NAMI-A and KP-1019 are monodentate (similar to cisplatin), while TLD-1433 is a bidentate complex.
The half-sandwich type Ru(II)-arene organometallics (Melchart and Sadler, 2006), due to a high structural variability, represent one of the most widely investigated compound classes in the development of new candidates of multi-targeted metallodrugs (Bononi et al., 2020; Kenny and Marmion, 2019). The presence of a η6-arene or η5-arenyl residue in the coordination sphere contributes to the stabilization of the +2 oxidation state of the metal ion and to the maintenance of the hydrophilic/lipophilic balance of the whole molecule. The remaining three coordination sites of the Ru(II) ion are usually occupied by at least one leaving group and mono- or bidentate ligands, as it can be seen in RAPTA-T and —C(6) and RM175 (7), respectively, which among others, have become leads for Ru(II)-based complexes (Fernandes, 2019; Habtemariam et al., 2006; Melchart and Sadler, 2006).
Due to the biological relevance of sugars, the incorporation of a carbohydrate-containing ligand into platinum group metal complexes in general (Hartinger et al., 2008; Kenny and Marmion, 2019), and into Ru(II)-arene/arenyl complexes in particular (Fernandes, 2019), seems a rational choice for further drug design. Thus, several functional features of carbohydrates, such as their contribution to different cellular phenomena (e.g. to cell-cell recognition and adhesion), their crucial role in cellular energy supply, their binding capacities to carbohydrate specific proteins (e.g. lectins, glucose transporters and glycoenzymes) can be exploited to obtain new platinum group metal complexes with anticancer potential (Fernandes, 2019; Hartinger et al., 2008).
For several sugar containing half-sandwich Ru(II) complexes, such as RAPTA analogues with glycofuranose-based phosphite ligands (Fernandes, 2019) (compound 8) and Ru(II) complexes having 2,3-diamino-2,3-dideoxy-hexopyranoside (Böge et al., 2015) (compound 9) or 1,4-bis(β-D-glycopyranosyl)tetrazene-type N,N-chelating ligands (Hamala et al., 2020) (compound 10), the antiproliferative activity has already been justified. Hamala and colleagues (Hamala et al., 2020) showed that the inhibitory activity of carbohydrate-based ruthenium complexes was enhanced when the hydroxyl groups of the sugar units were protected by esters and an increasing length of the acyl chain improved the inhibitory efficacy (acetyl <propionyl <butyryl). However, their compounds were non-selective and induced apoptotic ccll death at low micromolar concentrations.
In addition, certain cyclopentadienyl-ruthenium(II) complexes with sugar-based heterocyclic mono-(compound 11, compound 12) or bidentate ligands (compound 13) have also been shown to display low micromolar cytotoxic activity in human cervical carcinoma (HeLa) and colon cancer HCT116 cell lines (Florindo et al., 2014; Florindo et al., 2016; Florindo et al., 2015).
More closely, the compounds disclosed in (25) Florindo et al. (2016) comprise methyl-α-D-mannopyranoside and methyl-α-D-glucopyranoside as a monosaccharide unit without a protecting group (compound 13).
Xiao, N. et al. (Xiao et al., 2017) have synthesized homoleptic and heteroleptic complexes of Ru(TAGP-tapy)3Cl2 (Xiao et al., 2017-tetra-O-acetyl-β-D-glucopyranosyl)-1H-1,2,3-triazol-4-yl]-pyridine) bearing a clustered glucose-derived ligand and Ru(bpy)2(TAGP-tapy)Cl2 (bpy is 2,2′-bipyridine). One of the compounds [Ru(bpy)2(TAGP-tapy)Cl2, compound 14] comprises a triazole ring and a sugar unit with acetyl protecting groups. The authors do not mention that they would expect an antitumor effect from the synthesized compounds.
Similar ligands have been prepared and studied in the art.
Bokor E. et al. (Bokor et al., 2010) have searched for effective glycogen phosphorylase inhibitors, as glycogen phosphorylase is a validated drug target for type 2 diabetes mellitus and thus has the potential to provide new therapies for the disease. The authors used a copper(I)-catalyzed azide-alkyne cycloaddition reaction to produce O-acetyl- or O-benzoyl-protected 1-D-glucopyranosyl-1,2,3-triazoles. In the introduction, they describe that the use of the 1,2,3-triazole ring as an amide substitute is becoming more common because it is stable under many chemical conditions. It is mentioned that N—(β-D-glucopyranosyl)-4-phenyl-1,2,3-triazol-1-yl-acetamide is also a glycogen phosphorylase inhibitor. It is also noted in the introduction that 1-glycosyl-1,2,3-triazole compounds have also been investigated as anti-cancer agents. By removing the protecting groups, the authors obtained test compounds that were tested for their glycogen phosphorylase-b inhibitory activity. 1-(β-D-glucopyranosyl)-1,2,3-triazoles proved to be the best inhibitors from several series of compounds, their inhibition equilibrium constants fell in the lower regions of the micromolar range. The 1-(β-D-glucopyranosyl)-1,2,3-triazoles and N-acyl-β-D-glucopyranosylamines were similar in their inhibitory properties (Chrysina et al., 2009). The authors note that the higher activity of β-anomers than α-anomers is consistent with the literature. However, the authors did not use a nitrogen-containing heterocycle as the triazole-linked R group.
Mohammed, A. I. et al. (Mohamed, 2017) have prepared peracetylated D-glucose-based bis-1,2,3-triazoles and found that they have a mild antibacterial effect only. The strength of the antibacterial effect primarily did not depend on the presence or removal of the protecting group, but on the other chemical properties of the compound.
Samsulová, V. (Šamšulová et al., 2019) has prepared compounds comprising substituted pyridine and a triazole ring and the author has observed that these have an antibacterial activity. However, neither Mohammed A. I. et al., nor Samsulová, V. used metal complexes.
Unlike organic drug molecules the biological effects of metal complexes can be modulated by a wider array of parameters including size and charge of the species, hard-soft character of the metal ion, stability, inertness and geometry of the complex, just to mention a few. In this regard the present inventors have carried out a comprehensive study of Ru-sugar-conjugate based complexes, in which A) the role of the metal chelating part of the ligand, B) the basicity and binding strength of the coordinating donor atoms and C) the effect of the lipophilic/hydrophilic character of the complex, tuned by the presence/absence of various protecting groups at the sugar moiety were explored, may provide with a more detailed outlook to the structure-activity relationship (SAR) of these types of complexes.
To the best of the inventors' knowledge, no real C- and N-glycopyranosyl heterocyclic ligands as potential bidentate chelators have so far been used to obtain VIIIB group transition metal arene/arenyl complexes. In the present invention the inventors disclose a set of C- and N-glycopyranosyl azoles and their half-sandwich metal-complexes and synthesis thereof. Anticancer potential of the above ligands and their complexes have been studied. The compounds of the invention have also been tested for their antimicrobial effect.
The invention relates to a half-sandwich type compound of general formula (I)
Thus, the meaning of M is a transition metal ion selected from the group consisting of group VIIIA (in more recent nomenclature groups 8-10) of 4d and 5d series of the periodic table, i.e. Ru, Os, Rh, Ir, Pd and Pt. Preferably the transition metal ion has the following oxidation states: Ru(II), Os(II), Rh(III), Ir(III), Pd(II) and Pt(II),
Preferably the invention relates to a half-sandwich type compound of general formula (I) for use in a neoplastic disease or in a bacterial disease.
Preferably one or more, preferably at least two of R1, R2, R3 and R4 is selected from C6-C10 aryl, C4-C20 alkyl, C4-C20 alkenyl, C4-C20 alkynyl, C5-C20 alkyl-carbonyl, C5-C20 alkenyl-carbonyl, C5-C20 alkynyl-carbonyl, C7-C20 aralkyl (including alkylaryl), C8-C20 aralkyl-carbonyl and C7-C11 aryl-carbonyl (aroyl), in particular C7-C11 aryl-carbonyl (aroyl).
In an embodiment Het2 comprises a heterocycle selected from the group consisting of the benzologues of the 6 membered aromatic N-heterocycle, preferably selected from the group consisting of quinoline, izoquinolin and 2-izoquinoline.
Preferably
Preferably at least one or more preferably at least two of R1, R2, and R3 is/are selected from C6-C10 aryl, C4-C20 alkyl, C4-C20 alkenyl, C4-C20 alkynyl, C5-C20 alkyl-carbonyl, C5-C20 alkenyl-carbonyl, C5-C20 alkynyl-carbonyl, C7-C20 aralkyl, C8-C20 aralkyl-carbonyl, C7-C11 aryl-carbonyl (aroyl),
Preferably at least one or more preferably at least two of R1, R2, and R3, preferably at least two, in particular at least three of R1, R2, R3 and R4 is/are selected from C5-C20 (preferably C10) alkyl-carbonyl, C5-C20 (preferably C10) alkenyl-carbonyl, C5-C20 (preferably C10) alkynyl-carbonyl, C8-C20 (preferably C15) aralkyl-carbonyl, C7-C11 aryl-carbonyl (aroyl), in particular C7-C11 aryl-carbonyl (aroyl), preferably C7 aryl-carbonyl,
In a particular embodiment said compound has a distribution coefficient (log D) of at least 1.4, preferably at least 1.6, more preferably at least 1.8, highly preferably at least 2.0. In a particular embodiment the distribution coefficient (log D) is not higher than 4.0, in particular 3.5 or 3.0. In a particular embodiment the log D is 1.4 to 4, preferably 1.6 or 1.8 to 4, in particular 1.6 or 1.8 to 3.5, in particular 2 to 4 or 2 to 3.5. In particular, log D is measured between n-octanol saturated with aqueous PBS solution (pH=7.40), and aqueous PBS solution, preferably as disclosed herein.
In a particular embodiment in formula (I),
In a particular embodiment the compound of the invention has general formula (II)
In a preferred embodiment the invention relates to the complex or any complex for use in a disease as disclosed herein in a particular a bacterial disease or a neoplastic disease as detailed below.
In a preferred embodiment the invention relates to the complex or any complex for use in a disease as disclosed herein in a particular a bacterial disease or a neoplastic disease as detailed below.
In a further particularly preferred embodiment said compound has general formula (II.2)
In a preferred variant of this embodiment said compound has formula (III)
In a particularly preferred embodiment the compound of the invention has formula (III.1)
In a particularly preferred embodiment the compound of the invention has formula (III.1.b1), (III.1.b2) or (III.1.b3), in particular (II.1.b1)
In a further particular embodiment the invention relates to a compound having formula (IV)
In a further particular embodiment the invention relates to a compound having formula (IV.b1), (IV.b2) or (IV.b3), in particular formula (IV.b1),
Said compound preferably has a formula preferably selected from the group of formulae (IV.2.b1), (IV.2.b2) and (IV.2.b3), in particular (IV.2.b1),
In a further particular embodiment the invention relates to a compound having formula (V)
Said compound has a formula preferably selected from the group of formulae (V.1) and (V.2)
In a further particular embodiment the invention relates to a compound having formula (V.b1), (V.b2) or (V.b3), in particular formula (V.b1),
Said compound preferably has a formula preferably selected from the group of formulae (IV.1.b1), (IV.1.b2) or (IV.1.b3), in particular (IV.1.b1),
Said compound preferably has a formula preferably selected from the group of formulae (IV.2.b1), (IV.2.b2) and (IV.2.b3), in particular (IV.2.b1),
Preferably, in any of formulae (I) to (V), including preferred and particular options
Particularly preferably said compound has a distribution coefficient (log D) of at least 1.4, preferably at least 1.6, more preferably at least 1.8, highly preferably at least 2.0. In a particular embodiment the distribution coefficient (log D) is not higher than 4.0, in particular 3.5 or 3.0. In a particular embodiment the log D is 1.4 to 4, preferably 1.6 or 1.8 to 4, in particular 1.6 or 1.8 to 3.5, in particular 2 to 4 or 2 to 3.5. In particular, log D is measured between n-octanol saturated with aqueous PBS solution (pH=7.4), and aqueous PBS solution, preferably as disclosed herein.
Highly preferably log D is measured with the method described by Kozsup et al. (Kozsup et al., 2020).
Highly preferably, the compound is selected from the group consisting of
Highly preferably, the compound is selected from the group consisting of
In a particularly preferred embodiment the C7¬ C11 aryl ¬ carbonyl (aroyl) is benzoyl.
In a further aspect of the present invention the compound is for use as a medicament, preferably in human medicine or in veterinary medicine.
Preferably the complex, any indication is as defined herein is any of the complexes listed above in any of formulae above, eg. formulae (I) to (V) and variants as given above.
In a particularly preferred embodiment of the present invention the compound(s) in the medicament or used for treatment is/are selected from compounds defined by formula (II), in particular for formula (III), in particularly preferred embodiment by formulae (IV) and (V) including preferred formulae. Preferred formulae include, unless specifically defined differently, compounds identified both by roman and arab numbers, like (II.1, II.2, III.1, IV.1, V.2) etc.
In particular, the compound is used in human medicine or for use in human medicine.
In particular, the compound for use as a veterinary agent or is used as a veterinary agent.
In an embodiment the compound is used in a combination therapy with an other antineoplastic or antibacterial agent. In a particular embodiment the combination therapy is with eg. PARP inhibitors.
In a particular embodiment the complexes are administered together with
It is contemplated that the compounds may potentiate the effects of the elements of chemoradiotherapy.
The compound of the invention preferably has a non toxic character in particular wherein the R1, R2, R3 and/or R4 or each of them are selected from C8¬ C11 aralkyl¬ carbonyl, C7¬ C11 aryl¬ carbonyl, in particular C7¬ C11 aryl¬ carbonyl, preferably C7 aryl¬ carbonyl, in particular benzoyl.
In particular, the compound is used in a vertebrate. Preferably, the compound is used in a mammalian subject. In a preferred method the mammalian subject is selected from livestock animals and/or from companion animal species. In particular the mammalian subject is bovine, pig, a rodent e.g. murine, rabbit, cavy, guinea pig, etc., sheep, horse, a camelid, a felid, like cat, a canine, like dog etc. and also includes wild animals.
The compound may also be used in fishes, amphibians, reptiles and birds.
In particular, birds include poultry like chicken, turkey, duck, goose etc. and also include wild birds.
In a particularly preferred embodiment of the present invention the compound is for use as a medicament is selected from compounds defined by formula (II), in particular for formula (III), in particularly preferred embodiment by formulae (IV) and (V) including preferred formulae. Preferred formulae include, unless specifically defined differently, compounds identified both by roman and arab numbers, like (II.1, II.2, III.1, IV.1, V.2) etc.
In a further aspect of the present invention the compound is for use as an anti-neoplastic agent in a subject having a neoplasm or a patient endangered by development of a neoplasm or recurrence of a neoplasm wherein preferably the subject is a mammalian, highly preferably a human subject.
Preferably the compound is used as an anti-cancer agent in a patient having cancer or a patient endangered by development of cancer (e.g. recurrence of cancer).
In particular, the compound for use is for use in the treatment of a neoplasm (or cancer) selected from a carcinoma and a sarcoma. In particular the carcinoma is adenocarcinoma or squamous cell carcinoma. In particular, the sarcoma is selected from osteosarcoma or osteogenic sarcoma, chondrosarcoma, leiomyosarcoma, rhabdomyosarcoma, mesothelial sarcoma or mesothelioma, fibrosarcoma, angiosarcoma or hemangioendothelioma, liposarcoma, glioma or astrocytoma, myxosarcoma, and mesenchymous or mixed mesodermal tumors.
The neoplasm, without limitation may be eg. an ovarian cancer, a breast cancer, a skin cancer, a pancraetic cancer, a glioblastoma, etc.
In an embodiment the neoplasm may be a hematologic neoplasm.
In an embodiment the neoplasm is any neoplasm listed herein or listed in (Kumar et al., 2017).
In a further aspect of the present invention the compound is for use against pathogenic bacterial infection in a subject, wherein preferably the subject is a mammalian subject, highly preferably a human subject. In particular, the pathogenic bacteria are Gram-positive bacteria. In a particular embodiment the Gram-positive bacteria are selected from cocci, in particular the group consisting of Streptococcus, Staphylococcus, Enterococcus, in particular Staphylococcus and Enterococcus. In a particular embodiment the Gram-positive bacteria are selected from bacilli, in particular Clostridium, Corynebacterium, Listeria, Bacillus.
In a particular embodiment the Gram-positive bacteria are antibiotic resistant, preferably methicillin-resistant or vancomycin resistant, more preferably a methicillin-resistant Staphylococcus or a vancomycin-resistant Enterococcus.
In a particular embodiment the Gram-positive bacteria, are clinical antibiotic resistant bacteria, e.g. of a clinical resistant strain. In particular embodiment the clinical resistant bacterium is a methicillin-resistant strain or a vancomycin resistant strain.
The invention also relates to a method for treatment wherein a medicament comprising the compound of the invention or comprising the complex of the invention is administered to a patient in need of said medicament. Said patient in need may need prevention of a condition as defined herein or may have a condition or disease as defined herein e.g. a neoplastic disease or a fungal disease.
The compound of the invention is any of the complexes as given above e.g. for any of the formulae (I) to (V) and variants as given above.
In a particularly preferred embodiment of the present invention the compound(s) in the medicament or used for treatment is/are selected from compounds defined by formula (II), in particular for formula (III), in particularly preferred embodiment by formulae (IV) and (V) including preferred formulae. Preferred formulae include, unless specifically defined differently, compounds identified both by roman and arab numbers, like (II.1, II.2, III.1, IV.1, V.2) etc.
The invention also relates to the use of the compound of the invention in a non-therapeutic method. For example the compound may be used in experimental e.g. laboratory animals e.g. for research or drug testing.
The invention also relates to the use of the compound of the invention as an antibacterial agent in a non-therapeutic method.
In a particularly preferred embodiment of the present invention non-therapeutic antibacterial compounds are selected from compounds defined by formula (II), in particular for formula (III), in particularly preferred embodiment by formulae (IV) and (V) including preferred formulae. Preferred formulae include, unless specifically defined differently, compounds identified both by roman and arab numbers, like (II.1, II.2, III.1, IV.1, V.2) etc.
The invention also relates to the use of the compound of the invention as an antibacterial agent in vitro.
The compound of the invention is any of the complexes as given above e.g. for any of the formulae (I) to (V) and variants as given above.
In particular embodiment the compound of the invention is used as antibacterial cleaning agent.
In particular embodiment the compound of the invention is used as aseptic or disinfecting agents e.g. in aseptic or disinfecting compositions, e.g. in sanitizers for artificial and/or biological surfaces or e.g. in skin sanitizers including sanitizer of hand.
In particular embodiment the compound of the invention is used as preservative for preservation of biological material in the laboratory or in the medical field including organs or tissues or cultures.
In particular embodiment the compound of the invention is used as anti-bacterial agent e.g. as preservative, e.g. in food or animal feed. Preferably, the food industry is dairy industry or dry production industry.
In particular embodiment the compound of the invention is used as anti-bacterial agent in agriculture, e.g. as bacterial agent against bacterial pests, e.g. as antibacterial wash, e.g. for antibacterial protection of plants or crops, etc.
In a particular embodiment the invention relates to a composition comprising the compound of the invention as an active agent and at least one further substance. Preferably if the use so requires, the further substance is a biologically acceptable carrier, formulation agent or excipients.
In a particular embodiment the invention relates to a pharmaceutical composition comprising the compound of the invention as an active agent and at least one pharmaceutically acceptable carrier, formulation agent or excipient.
In preferred embodiments the pharmaceutical composition is formulated as disclosed hereinbelow in the chapter pharmaceutical preparations.
In a further aspect the invention relates to a method for synthesis of a heterocyclic N,N-chelating ligand wherein the compound of formula (XI)
Y-Het2 (xii)
wherein
In a particular embodiment the invention relates to a method for synthesis of a cationic half sandwich transition metal complexes as defined herein wherein the
In particular,
A “subject” as used herein is an individual of an animal species, preferably a vertebrate, more preferably a mammalian or avian species, in particular a mammalian species, highly preferably the individual is a primate, a hominid or a human.
A “patient” is a subject who is or intended to be under medical or veterinarian observation, supervision, diagnosis or treatment.
A “treatment” of a subject refers to any process, action, application, therapy, or the like, wherein the subject or patient is under aid, in particular medical or veterinarian aid with the object of improving the subject's or patient's condition, either directly or indirectly. Improving the subject's condition may include improving an aesthetic condition (cosmetic treatment) and/or may include, in particular, restoring or maintaining normal function of an organ or tissue, preferably at least partly restoring or maintaining health (medical or veterinarian treatment).
Treatment typically refers to the administration of an effective amount of a compound or composition described herein. Unless specified differently, a therapeutic treatment includes both medical or veterinarian treatment and prevention (or prophylaxis) i.e. prevention of the onset of a disease as well.
A “composition” of the invention is a composition of matter which comprises at least one compound of the invention as an active agent and at least one further substance. Preferably the compound of the invention is present in an effective amount. Compositions may also comprise further biologically active substances useful e.g. in a combination therapy. Furthermore, the compositions may comprise biologically acceptable carriers, formulation agents, excipients etc. which may be known in the art.
The term “effective amount” qualifies the amount of a compound required to exert the effect of the active agent in a composition. A “therapeutically effective amount” is sufficient to relieve or prevent (or prevent worsening of) one or more of the symptoms or characteristic parameters of a condition, e.g. a disorder or disease.
A “group” (or “moiety”) is used herein as a part of a molecule which can be derived in principle by removing another part, like a hydrogen atom.
As used herein, the term “alkyl” alone or in combinations means a straight or branched-chain (if appropriate) hydrocarbon group containing preferably from 1 to 20, preferably 1 to 15, 1 to 10 or 1 to 8 carbon atom(s) or 1 to 6 or 1 to 4, 1 to 3 or 1 to 2 carbon atom(s) [i.e. “C1-20”, “C1-15”, “C1-10” “C1-8”, “C1-6” or “C1-4”, “C1-3” or “C1-2” alkyl groups, respectively], such as methyl, ethyl, propyl, isopropyl, butyl, sec-butyl and t-butyl. As an example “C1-6alkyl” includes methyl group, ethyl group, isopropyl group, butyl group, n-butyl group, isobutyl group, sec-butyl group, t-butyl group, pentyl group, isopentyl group, 2,3-dimethylpropyl group, hexyl group etc.
As used herein, the term “alkoxy” means an alkyl-O— group in which the alkyl group is as previously described. The bond to the rest of the molecule or complex, i.e. the parent moiety is through the oxygen (if to a carbon atom, ether oxygen).
The term “alkoxy alkyl” means an alkyl group which is substituted by an alkoxy group, i.e. an alkyl-O— group as previously described. The bond to the alkyl moiety is through the oxygen, i.e. it is an ether oxygen.
As used herein, the terms “alkyl-carbonyl”, “alkenyl-carbonyl” and “alkynyl-carbonyl” mean a moiety having carbonyl group substituted with an alkyl group, alkenyl group and alkynyl group, respectively, wherein bond to the parent moiety is through the carbon of the carbonyl group. An “alkyl-carbonyl”, “alkenyl-carbonyl” and “alkynyl-carbonyl” are also called alkanoyl, alkenoyl and alkynoyl, respectively.
An “alkenyl” as used herein, alone or in combinations, means a straight or branched-chain unsaturated hydrocarbon group containing at least one carbon-carbon double bond, said hydrocarbon group containing preferably from 2 to 20, preferably 2 to 15, 2 to 10 or 2 to 8 carbon atoms or 2 to 6, 2 to 4, 2 to 3 or 2 carbon atoms [i.e. “C2-20”, “C2-15”, “C2-10”, “C2-8”, “C2-6” or “C2-4”, “C2-3” or “C2” alkyl groups, respectively].
An “alkynyl” as used herein, alone or in combinations, means a straight or branched-chain unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, said hydrocarbon group containing preferably from 2 to 20, preferably 2 to 15, 2 to 10 or 2 to 8 carbon atoms or 2 to 6, 2 to 4, 2 to 3 or 2 carbon atoms [i.e. “C2-20”, “C2-15”, “C2-10”, “C2-8”, “C2-6” or “C2-4”, “C2-3” or “C2” alkyl groups, respectively].
The term “cycloalkyl” as used herein is a non-aromatic carbon-based alkyl ring composed of at least three carbon atoms.
The meaning of “straight chain alkyl” is an alkyl in which each non-terminal and non-first carbon atom of the group binds covalently to two other carbon atoms. In other words a “straight chain alkyl” forms a single central carbon chain and does not comprise any carbon atom binding to the central carbon chain by replacing a H of said chain.
The meaning of “branched chain alkyl” is an alkyl in which there is at least one non-terminal and non-first carbon atom of the group which binds covalently to three or four other carbon atoms. In other words a “branched chain alkyl” has a central carbon chain which is the longest chain of consecutive carbon atoms in the group and comprises at least one carbon atom of an alkyl substituent binding to the central carbon chain.
A “heterocyclic” ring as used herein is a cyclic moiety that has, besides carbon atom(s), atoms of at least one non-carbon element as member(s) of its ring(s). A heterocycle may comprise multiple rings, e.g. it may comprise an aromatic heterocycle and, fused to the aromatic heterocycle another ring which may or may not be aromatic; i.e. if it is not aromatic it may form a cyclic substituent of the aromatic heterocycle. In a preferred embodiment, if the heteroaryl comprises multiple, in particular two fused rings, both rings are aromatic. Preferably the ring(s) of the heterocyclic moiety is/are 5 to 6 membered ring(s).
An “N-heterocyclic” ring as used herein is a heterocyclic ring comprising at least one N as a ring-forming heteroatom. The “N-heterocyclic” ring may comprise other heteroatoms, typically N, S and O atoms as well.
The term “heterocycloalkyl” refers to a “heterocyclic” ring which is derivable from cycloalkyl group as defined above, wherein at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen or oxygen.
An “aromatic” moiety as used herein can be described as a planar cyclic moiety (a ring) wherein the single bonds (called σ-bonds) between the ring-forming atoms are formed from overlap of hybridized atomic sp2-orbitals in line between the carbon nuclei, wherein a system of delocalized π-bonds are formed from overlap of atomic p-orbitals of each of the ring forming atoms above and below the plane of the ring and wherein the number of π electrons, which is provided by the ring-forming atoms, participates in according to molecular orbital theory, must be equal to 4n+2 (Hückel's rule), in which n=1, 2, 3, etc., preferably 1 or 2, for a single ring with six π electrons, n =1. The ring-forming atoms typically provide one or two π electrons to the delocalized π electron system.
The term “heteroaryl” is defined herein as a group or molecule that contains an aromatic heterocycle, preferably a moiety that has at least one heteroatom, as “member”, incorporated within an aromatic ring. Examples of heteroatoms include nitrogen, oxygen and sulfur, preferably nitrogen and oxygen. In an embodiment a heteroaryl may comprise an aromatic heterocycle and, fused to the aromatic heterocycle another ring which may or may not be aromatic; i.e. if it is not aromatic it may form a cyclic substituent of the aromatic heterocycle. In a preferred embodiment, if the heteroaryl comprises multiple, in particular two fused rings, both rings are aromatic. Members of a heteroaryl relate to the ring-forming atoms, either carbon atom(s) or heteroatom(s).
The term “aryl” as used herein is a group that contains any carbon-based aromatic ring which is preferably a mono- or bicyclic group, wherein the bicyclic group is preferably comprises two fused rings. In a preferred embodiment the aryl group consists of carbon as ring atoms, i.e. “members” only. In a broader meaning the term aryl also includes optionally “heteroaryl”. Optionally, the term “aryl” is limited to non-heteroaryl which is also included into the term aryl and defines a group that contains an aromatic group that does not contain a heteroatom.
An aryl group may be substituted or unsubstituted (i.e. optionally substituted). If the aryl group is substituted it may be substituted with any substituent, and examples of the substituent include C1-4 alkyl, C2-4 alkenyl, C1-3 alkyloxy, C1-3 alkanoyl, C1-3 alkylamine, C1-3 alkylamide, halogen, etc.
“N-heteroaryl” as used herein is an aromatic N-heterocycle, i.e. an aromatic heterocycle having at least one ring N. Optionally, an N-heteroaryl comprises further heteroatoms e.g. nitrogen, oxygen and sulfur, preferably nitrogen and oxygen.
The term “aralkyl” as used herein refers to an aryl alkyl group which is linked to the parent molecule through the alkyl group, which may be further optionally substituted with one or more, preferably one to three or one to two alkyl substituents. Thus, the aryl group may be substituted with an alkyl substituent, preferably each substituent being not larger than a C1-4 alkyl.
“Aryl” or “heteroaryl” may comprise a monocyclic ring, a condensed ring, or a polycyclic ring in which a single ring is bounded by a single bond, preferably a monocyclic or bicyclic ring.
An “aryl-carbonyl” is a moiety having carbonyl group substituted with an aryl group, wherein bond to the parent moiety is through the carbon of the carbonyl group. An “aryl-carbonyl” may be also called aroyl.
An “aralkyl-carbonyl” is a moiety having carbonyl group substituted with an aralkyl group, wherein binding to the parent moiety is through the carbon of the carbonyl group. An “aryl-carbonyl” may be also called aralkanoyl.
A “cap” or “capping” is used herein as a moiety of half-sandwich type metal comprising organometallic compounds which have an aromatic group in the coordination sphere which contributes to the stabilization of the metal ion and to the maintenance of the hydrophilic/lipophilic balance of the whole molecule. Cap molecules having an aromatic ring are preferably a η6-arene or η5-arenyl residues. In a preferred embodiment the half-sandwich type metal comprising organometallic compounds have a bidentate chelating ligand coordinated to the metal ion.
A “chelate” comprises at least two “coordinate bond” or “dative bond”, i.e. a two-center, two-electron bond in which the two electrons derive from the same atom, typically a non-binding electron pair is coordinated to a metal ion. Such a bond is marked in an undulating line in the formulae.
As used herein, the term “fused ring” means that the ring is fused with at least one other ring to form a group of a compound which comprises two or more rings wherein a single bond between two member atoms of the rings is, together with said two members, common in, i.e. shared by the two rings. An example of fused rings is a polycyclic aryl. A polycyclic aryl is understood herein as a group that contains multiple rings of a carbon-based group among which at least one ring is an aryl and which optionally may also comprise a cycloalkyl and/or a heterocycloalkyl.
A “substituted” moiety comprises a substituent selected from the groups and moieties as defined herein; however a substituent is preferably smaller, i.e. shorter, i.e. consists of not more, preferably less atoms than the moiety which is/are substituted thereby. In the present invention, “optionally substituted”, i.e. “unsubstituted or substituted” means that it may be substituted with any substituent.
The singular forms “a”, “an” and “the”, or at least “a”, “an”, include plural reference unless the context clearly dictates otherwise.
The term “comprises” or “comprising” or “including” are to be construed here as having a non-exhaustive meaning and allow the addition or involvement of further features or method steps or components to anything which comprises the listed features or method steps or components. “Comprising” can be substituted by “including” if the practice of a given language variant so requires or can be limited to “consisting essentially of” if other members or components are not essential to reduce the invention to practice.
The compounds that were used in the assay are indicated on
The compounds that were used in the assay are indicated on
The compounds that were used in the assay are indicated on
The compounds that were used in the assay are indicated on
Half-sandwich type organometallic complexes having a transition metal ion and bidentate ligands comprising a carbohydrate-based group linked to an azole ring and another nitrogen-comprising aromatic heterocycle were synthesized and found to have a cytostatic and cytotoxic effects in several in vitro tumor models, while lacking or having low level cytotoxicity towards non-transformed fibroblasts. The complexes also showed an antibiotic effect towards Gram-positive bacteria.
The experimental data suggest that the complexes may act by generating oxidative stress in a membrane dependent mechanism.
The present inventors have prepared a set of half-sandwich type organometallic complexes having a transition metal ion of the VIIIb group of 4d and 5d series, preferably Ru, Os, Rh, Ir. The bidentate ligands in these complexes comprise a carbohydrate-based group (preferably a monosaccharide-based group characterized by a pyranose type sugar ring) and a group with two nitrogen-comprising aromatic heterocycles the one linked to the carbohydrate-based group being an azole ring.
The compounds of the invention were identified to display long-term cytostatic effects, but little rapid toxicity on two different ovarian cancer cell lines. These compounds were not toxic towards primary human fibroblasts. The practical absence of toxicity on non-transformed fibroblasts is surprising in view of the high cytostatic effect even if certain other ruthenium complexes were found to be non-toxic (Gano et al., 2019; Liu et al., 2019; Mello-Andrade et al., 2018; Mihajlovic et al., 2020). The ligands themselves had no biological activity and the presence of the transition metal ion (ruthenium(II), osmium(II), iridium(III) or rhodium(III)) was necessary. The IC50 values of the active compounds were comparable or superior to the currently applied platinum compounds (cisplatin, oxaliplatin and carboplatin) and other sugar-containing ruthenium complexes (Berger et al., 2008; Florindo et al., 2014; Hamala et al., 2020; Hanif et al., 2013) of the art. The compounds were found to be active upon long term application, in SRB assays, similar to platinum compounds.
Initially, a set of exemplary Ru(II) compounds were analysed as shown in Table 1 and on
To this end, 4-(pyridin-2-yl)- and 4-(quinolin-2-yl)-1-(β-D-glucopyranosyl)-1,2,3-triazoles (N-glycosyl derivatives) and 2-(β-D-glycosyl)-5-(pyridin-2-yl)-1,3,4-oxadiazoles (C-glycosyl derivatives) were prepared by 1,3-dipolar cycloaddition reactions. Treatment of these N,N-chelators with dichloro(η6-p-cymene)ruthenium(II) dimer ([(η6-p-cym)RuCl2]2) in the presence of TIPF6 yielded the expected Ru(II)-centered complexes with the general formula [(η6-p-cym)RuII(N—N)Cl] PF6 as mixtures of diastereomers.
The compounds were tested in methylthiazolyldiphenyl-tetrazolium bromide (MTT) reduction assay, to assess short term toxicity and a sulphorhodamine B (SRB) proliferation assay for measuring long term cytostasis or cytotoxicity on A2780 human ovarian carcinoma cells.
The MTT assay is a colorimetric assay for measuring cellular metabolic activity, wherein the mitochondrial metabolic activity is assessed by the activity of NAD(P)H-dependent cellular oxidoreductase enzymes, which can reduce the tetrazolium dye (MTT) to its insoluble formazan while its color is changed. The assay is suitable for measuring the number of viable cells, thereby short term cytotoxicity or cytostatic activity (shift from proliferation to quiescence).
The sulforhodamine B (SRB) assay was developed to measure drug-induced cytotoxicity and cytostasis or proliferation for drug-screening applications during a longer time-span e.g. two days. SRB is a protein dye that can form an electrostatic complex with the basic amino acid residues of proteins under moderately acidic conditions, which provides a sensitive linear response with cell number and/or cellular protein measured. The SRB assay possesses a colorimetric end point and the color development is rapid and stable. In addition, this assay does not depend on the metabolic activity of the cells and therefore shows less interference with the testing compounds.
In the first set of experiments four Ru complexes, Ru-2a, Ru-4, Ru-6, Ru-8 were found to have cytostatic properties both in long-term SRB and short-term MTT assays on A2780 cells (
In a more detailed study these four active complexes and the corresponding ligands were tested on two ovarian cancer cell lines (A2780 and ID8) and on human primary skin fibroblasts to assess anti-neoplastic effect and toxicity on non-neoplastic cells. As a brief summary, the four active complexes, Ru-2a, Ru-4, Ru-6, Ru-8 proved to have different effect in MMT assays Ru-2a and Ru-8 being more effective on neoplastic (A2780 and ID8 cells) cells than Ru-4, Ru-6 whereas none of the complexes had any effect on non-neoplastic primary fibroblasts.
As to long-term SRB assays all complexes were effective on A2780 and ID8 cells (
The possibility that long-term effect was due to enhanced cell-death has been excluded by Annexin V-propidium iodide (PI) double staining cell death measurements, a method for detecting viable, apoptotic or necrotic cells. Specifically, the Annexin V corresponding signal provides a very sensitive method for detecting cellular apoptosis, while the propidium iodide (PI) is used to detect necrotic or late apoptotic cells. Viable cells remain unstained.
The above results plausibly indicate an antineoplastic effect of the compound with a non-toxic profile on non-transformed/primary cells.
With the above measurements a relationship between the biological activity and the structure of the active molecules has been assessed. For sake of illustration all complexes in this exemplary series have been compared to Ru-4 (see
It could be concluded that an important structural feature, that plays a key role in the biological activity of the molecules, is the presence and size of the protecting groups on the hydroxyl groups of the carbohydrate moieties. All bioactive molecules (i.e. Ru-2a, Ru-4, Ru-6, Ru-8 in the first set of experiments) have large apolar group or groups, exemplified by O-benzoyl groups on the sugar moiety (
In good agreement with that, the replacement of the protected monosaccharide in the molecule with a single phenyl group lowered the cytostatic capacity of the ruthenium complex (Ru-13) or fully abrogated it (Ru-14). This finding also corroborates the inevitable role of the sugar or sugar analogue moiety in determining the biological activity, while also supported the view that apolar or aromatic groups other than O-benzoyl groups also work. The lipophilic character has been assessed by measuring distribution between n-octanol and PBS buffer wherein n-octanol was saturated with aqueous PBS solution and vice versa wherein the distribution coefficient (D) was calculated. Apparently, increasing the lipophilic character of the compounds improves the cytostatic properties of sugar-based ruthenium complexes. This is underlined by the log D values in Table 1 and also in Table 4 (see below in Example 2.5 Inhibitory properties and cooperative effect of Ru complexes and known platinum compounds) to
As to the mechanism of action, it has been assessed whether oxidative stress had a role in the cytostatic effects of the compounds of the invention (see Example 3). Apparently, mitochondria are not the source of the reactive oxygen species, as tested by using MitoTEMPO, a mitochondrially targeted antioxidant that can efficiently detoxify mitochondria-derived reactive oxygen species (Hegedűs et al., 2021; Sári et al., 2020). MitoTEMPO did not modulate the effects of Ru-2a, Ru-4, Ru-6 and Ru-8 (
Cytostatic effects of exemplary ruthenium complexes Ru-2a, Ru-4, Ru-6 and Ru-8 have been reverted by strong reductants. However, excess amounts of GSH or NAC, soft Lewis bases with thiol groups, can lead to the disassembly of the inventive complexes (which are soft Lewis acids).
Therefore, to bypass that possibility, vitamin E and a derivative of vitamin E lacking the apolar phytyl chain, Trolox, were applied as antioxidants without thiol group. Interestingly, application of vitamin E, similarly to GSH and NAC, attenuated the cytostatic effects (
Thus it can be plausibly assumed that large, apolar protective groups could cause changes in the biophysical properties of the membranes. Without being bound by theory this can facilitate the membrane permeability or membrane entry of these compounds. The fact that Trolox, a derivative of vitamin E that lacks the long apolar phytyl chain, was ineffective in protecting cells against cytostasis induced by ruthenium compounds supports that apolar groups of the sugar or sugar-analogue and in turn membrane binding plays a role in the effect of the compounds. Taken together these findings point to the causative role of reactive oxygen species production in cytostasis and suggest that oxidative stress in apolar, lipid-containing compartments contribute to the activity of the compounds of the invention as also discussed below.
These facts provide support for claiming that compounds having apolar groups linked to the sugar or sugar analogue moiety and having a high hydrophobicity are effective in the present invention.
Furthermore, it has been found that two other structural features have impact on bioactivity: A) modification of the carbohydrate moiety from glucose to xylose by a formal removal of the hydroxymethyl group at position 5 (Ru-6) or changing the configuration of the C-4 centre (glucose to galactose as in Ru-8) and B) replacement of the 1,3,4-oxadiazole ring by 1,2,3-triazole (Ru-2a). While all these compounds were proved to be active, these changes increased the rapid toxicity of the molecules or even rendered the molecules toxic on primary cells (
It was also a surprising observation that the binding of the active compounds showed high level of cooperativity as suggested by the value of the Hill slope, being 2-3 for Ru-2a, Ru-4, Ru-6, Ru-8.
Cooperativity is a feature which normally cannot be predicted, in particular without detailed information on the structures and binding. A study of Hanif et al. (Hanif et al., 2013) assessing RAPTA-analogs (general formula 8) illustrates this by showing that a single compound was identified with a steep inhibitory curve suggesting cooperative binding whereas cooperative binding was not characteristic for other sugar-containing ruthenium complexes. The Hill equation reflects the binding of ligands to macromolecules as a function of the ligand concentration, whereby the Hill slope (or coefficient; i.e. the slope of the response curve) is a measure of ultrasensitivity and correlates with the cooperativity of the binding between the molecules under examination.
This suggests that the binding of the complexes of the invention facilitates the binding of the subsequent molecules. The Hill slope was also determined for the reference platinum-based drugs, however, these molecules did not show signs of cooperative binding as the Hill slope was ˜1. Apparently, the binding and, probably, the mode of action of the ruthenium complexes identified in this study differs from those of the reference platinum compounds.
This cooperativity is an unforeseen feature and may well contribute the activity of the compounds of the invention.
To test whether the observations in accordance with the invention apply to the group of transition metals analogous to Ru, specifically the 4d and 5d transition metals of the VIIIb group in further preferred embodiments a series of half-sandwich complexes of with real glycosyl azole type bidentate ligands and with Ru(II), Os(II), Rh(III) and Ir(III) were synthesized.
In this further series of experiments each of the ligands L-2a, L-4, L-6 and L-8 were used with each of Os, Rh and Ir, plus Ru, and the obtained complexes were compared in MTT and SRB assays on ovarian adenocarcinoma cell line A2780 as well as on primary fibroblasts with the above described Ru complexes (see
In SRB assays each of the complexes with L-2a ligands proved to be active, with L-4 ligands Rh-4 was found to be slightly active only, with L-6 ligands Rh-6 and Ir-6 were less active than Ru-6 and Os-6, whereas the same two metals with L-8 (Rh-6 and Ir-6) was found to be essentially inactive under measurement conditions while the activity of Ru-8 and Os-8 has been maintained.
As to healthy cells essentially none of the metal ion complexes were proved to be active on primary fibroblasts (except perhaps Ru-2a in SRB-assay and Ru-2a and Os-2a in MTT assay at high concentrations, in 1-2-fold higher concentrations above the IC50 values). Each of them was much more active in SRB on neoplastic cells than on primary fibroblasts.
As to the results of the MTT assay, it can be said that the most of the compounds were found to be inactive in this short-term measurement. Ru-2a was clearly active and perhaps Ir-2a and Ru-8 showed some activity on A2780 cells, whereas no compound has shown activity on primary fibroblasts, except maybe some uncertain and weak activity by Ru-2a in both MTT and SRB assay and Ir-2a in MTT assay between 10 μM and 100 μM. Neither the dimer complexes without ligands nor the ligands themselves provided any significant activity in any assays, which fact reflexes the unique and specific nature of the compounds of the invention.
The general message here again is that the half-sandwich complexes with a bulky apolar group on the monosaccharide or monosaccharide analog unit have cytostatic activity in neoplastic model whereas they are essentially non-toxic in short term model or on healthy cells.
There is an apparent difference in the activity of the complexes with various transition metals. In the experiment Ru and Os have proven to be superior in most cases, which is an unforeseen result. In certain examples Os was observed to be somewhat less MTT-positive, which, unexpectedly, suggests a lower toxicity. Both Ru and Os had a high cytostatic activity. Complexes with Ir and Rh are typically less active Ir being generally more cytostatic than Rh. However, it is apparent that with each transition metal we find complexes of the structure of the invention which have cytostatic effect against neoplastic cells and there are promisingly active compounds in each group which makes it plausible that valuable compounds can be found throughout the scope of the invention.
Complexes with Quinoline Het2 Heterocycle
In a still further set of experiments, the quinoline complexes (Ru-2b, Os-2b, Ir-2b, Rh-2b) were tested on A2780 and ID8 ovarian cancer cells, and on non-transformed primary human fibroblasts, used as controls, in a concentration range up to 33.3 μM (
In somewhat more detail, in the experiments shown on
Also the toxic effects of the active Ru, Os and Ir complexes were verified by Annexin V-FITC propidium iodide (PI) double staining. The complexes were applied in concentrations corresponding to their respective IC50 values and we have not detected increases in the apoptotic (Annexin V positive population) or the necrotic (PI positive and Annexin V—PI double positive populations) as opposed to the hydrogen peroxide-treated cells used as positive control (
In more detail, in the experiments shown on
Based on these results, Rh-2b complex was omitted from further experiments.
Thus, in summary, quinoline-containing complexes are active in cisplatin-resistant cells.
As noted above, one of the major drawbacks of platinum-based drugs is due to cisplatin resistance. We tested the three complexes with efficient cytostatic properties (Ru-2b, Os-2b and Ir-2b) on a cisplatin-resistant A2780 cell line. The complexes did not exert direct toxicity in MTT assays on the cisplatin-resistant cells (
Nevertheless, we have observed important differences in cell proliferation. The IC50 value of cisplatin was 1.21 μM in our previous study on cisplatin-sensitive A2780 cells [16]. The IC50 value increased to 16.47 μM in the cisplatin resistant cell line (13.6 fold increase) in SRB assays. In contrast to that, the IC50 value of Ru-2b (0.8466 μM vs. 1.183 μM, 1.40 fold change) and Ir-2b (0.891 μM vs. 1.535 μM, 1.72 fold change) increased, although not to the same extent as for cisplatin. Furthermore, the IC50 value of Os-2b (0.5777 μM vs. 0.476 μM) was left technically unchanged when comparing the cisplatin sensitive and cisplatin resistant cell lines (
Complexes with Alkanoyl Substituted Carbohydrate Moiety
In a FOURTH set of experiments a set of osmium complexes (similar to Ia [17] in
The compounds had high log D value indicating a strong apolar character up to the point that the free ligand L-16 with the longest C7H15—CO alkanoyl protective groups proved to be insoluble, hence, it was not suitable for testing. All complexes Os-C3-Os-C7, but not the free ligands L-C3-L-C6, exerted rapid toxicity on A2780 cells in MTT assays (
Using alkanoyl protective groups instead of benzoyl groups we published earlier (showed that the aliphatic acyl protection significantly deteriorated the biological properties of the complexes, namely, the compounds exerted rapid toxicity and their IC50 values were higher than even Os-2a (0.73 μM for Os-2a vs. 2.104 μM for Os-C4. Therefore, these complexes, while showing somewhat lower activity, were omitted from further testing.
Taken together, the present inventors have carried out structure-activity relationship investigations to assess whether the structure of the protective groups or modification of the heterocycle distal to the carbohydrate moiety affected the biological activity of the complexes. Namely, the replacement of the O-benzoyl protecting groups of the complexes with pyridine, in particular the most effective complex Os-2a, identified above, by aliphatic acyl protecting groups with homologously increasing chain-length has been accomplished (
In the first stage of this study the present inventors have identified a set of carbohydrate-based half sandwich-type complexes with platinum-group metal ions, such as ruthenium(II), osmium(II), iridium(III) and rhodium(III). A main feature driving the biological activity of these complexes was their apolar nature (log D >+2). The O-benzoyl protective groups on the carbohydrate moiety proved to be particularly preferred in bringing about the apolar character and biological activity of the complexes, whereas the removal of the O-Bz protective groups or their replacement with 0-acetyl groups abolished the biological activity of the complexes.
In a further stage of the study the present inventors have assessed a further set of compounds where the hydroxyl groups of the carbohydrate moiety were protected by aliphatic acyl groups with increasing chain length (C3→C7—CO). Increasing the length of the acyl chain up to 4 carbons (C3—CO) improved the biological activity of the complexes (butyryl<pentanoyl). Ru(II) complexes, with similar structure to those evaluated in this manuscript, with acetyl protective groups were biologically inactive in the same model system. However, when the acyl chain contained 5 or more carbons (C≥5-CO) the IC50 values increased above that of the C4—CO-protected complex (pentanoyl<hexanoyl<heptanoyl), despite the continuous increase in the log D value. Likely, increasing length of the acyl chain increased the apolar character of the complexes and thus decreased their solubility in cell medium impairing the cytostatic activity.
Furthermore, in contrast to the complexes with O-Bz protective groups, the O-alkanoyl-protected complexes exerted toxicity to the cells and their IC50 values were inferior to those of the complexes with O-Bz-protective groups. Apparently, the nature of the protective groups strongly influences the biological activity of the complexes. Thus, surprisingly aroyl residues provide superior performance over the alkanoyl groups.
In a still further stage the present inventors have prepared another ligand, where the pyridine moiety was replaced with a larger quinoline moiety. They have synthesized Ru(II), Os(II), Ir(III) and Rh(III) complexes of that free ligand. The IC50 values of the quinoline complexes were, surprisingly, superior to the pyridine containing complexes. The pyridine-containing complexes had higher IC50 values on ID8 ovarian cancer cells than on A2780 ovarian cancer cells, however, the quinoline-containing complexes had similar submicromolar IC50 values on both ovarian cancer cell lines further supporting their superior biological activity.
Unexpectedly, none of the complexes was toxic or cytostatic on primary, non-transformed human dermal fibroblasts pointing out the selectivity of these compounds to cancer cells. Selectivity is not a common feature of Ru(II) or Os(II) complexes.
In addition to the selectivity of the quinoline-containing complexes towards transformed cancer cells apparently the complexes have widespread cytostatic activity among other carcinomas (as Capan2 cells), sarcomas (as SAOS cells) or hematological malignancies (as L428 cells). These observations are similar in case of both pyridine-containing osmium, ruthenium and iridium complexes and the quinoline-containing complexes. While other Ru complexes have also been found active in the art, a selective cytostatic and not cytotoxic character seems to be rather unique.
Cisplatin resistance is a major drawback for the use of the currently registered platinum-based drugs (Mukherjea et al., 2020; Yu et al., 2020, Sipos et al., 2021). We provided evidence that the quinoline-containing compounds, we describe hereby, have IC50 values in cisplatin resistant A2780 cells similar to the control, cisplatin-sensitive A2780 cells. Importantly, complex Os-7 had the same IC50 value on both cell lines. Structurally unrelated Ru(II) and Os(II) complexes [40,41] can overcome cisplatin resistance in A2780 and SKOV3 cell models, similar to our compounds.
In addition to the selective cytostatic activity, the complexes of the invention in particular the pyridine and quinoline complexes, the latter being particularly preferred, exerted bacteriostatic activity. Interestingly, the Ir-2b complex had no bacteriostatic activity, although, iridium complexes comprising pyridine proved to be active. The MIC values of the Ru-2b and Os-2b complexes were in the low micromolar range similar to their pyridine-containing counterparts both the VRE and the MRSA isolates. VRE isolates were more susceptible to Ru-7 and Os-7 than MRSA isolates that aligns well with our previous observation. Importantly, the complexes are active on multiresistant clinical isolates suggesting that these compounds represent a novel class of antibiotics.
As to the mechanism of action, without being bound by theory, the cytostatic activity of the complexes were dependent on the generation of reactive oxygen species (ROS). In fact, ROS production was evidenced among other platinum group metal complexes (Fernandes, 2019; Mihajlovic et al., 2020; Xu et al., 2018). These and other studies report that ROS production in tumor cells is limited and even minute increases in oxidative stress leads to cytostasis. The pyridine-containing complexes of similar structure have been found to induce oxidative stress and oxidative stress has central role in their cytostatic activity. In the case of the quinoline-based complexes, vitamin E protected cells against the cytostatic effects of the osmium, ruthenium and iridium complexes that underlines the pivotal role of oxidative stress elicited by the complexes. Of note, vitamin E has a long, apolar phytyl chain; if this phytyl chain is removed the protective capacity was lost in the case of ruthenium complexes of similar structure as the ones we report here. This observation together with the apolar nature of the complexes suggest that the complexes likely target apolar compartments in cells.
Based on the inhibitory effect of vitamin E and the fact that we showed increases in 4HNE levels in A2780 cells upon treatment by the complexes we assessed the timecourse of 4HNE production. We used two osmium complexes with different chemical structure Os-2a, a triazole and Os-4, an oxadiazole complex. In both cases 4HNE production was induced early, already 30 minutes post-treatment. These data suggest that ROS production in probably a primary event upon the use of the complexes and not secondary (e.g. due to mitochondrial damage, etc.).
NRF2 is a key transcriptional regulator of the cellular antioxidant defense system (Smolkova et al., 2020). Treatment of cells with an NRF2 inhibitor (ML385) potentiated the effects of the complexes that is in good correlation with the ROS production mechanism of cytostasis.
Oxidative stress frequently impacts on cellular metabolism (Bai et al., 2015; Hill et al., 2018; Modis et al., 2012) and cellular metabolism is linked to cancer cell behavior (Hanahan and Weinberg, 2000, 2011), therefore, we assessed the interaction between treatment with the complexes and core cellular metabolic pathways. Treatment with the complexes induced the extracellular acidification rate that is a proxy readout for glycolysis. Furthermore, treatment with the complexes decreased the OCR/ECAR values that correspond to the ratio between the mitochondrial oxidation and glycolysis. As next step, we blocked glycolysis with 2-deoxyglucose (2DG) that supported the inhibitory effect of Os-2a on cell proliferation. This suggests that the complexes rearrange cellular intermediary metabolism.
The above results may be rationalized by taking into consideration the most labile character of the organorhodium complexes among the studied metal compounds. This may lead to dissociation of the Rh(III) complex before reaching the target organ in the cells while for the rest of the studied metal complexes the intact species can act due to the significantly slower ligand exchange reactions. Another point to be considered is that the appropriate organoosmium and -iridium complexes exhibit in general lower redox stability than the half-sandwich type analogous Ru or Rh complexes. The more facile oxidation might provide support to their higher biological activity in the biological assays and to the assumption that their effect is connected to ROS generation. Furthermore, when the Ru/Os and Rh/Ir pairs are compared the pentahapto bound Cp* ligand forms in general more stable M-C bonds than the hexahapto p-cymene making the half-sandwich type organometallic complexes more resistant to redox reactions for the latter pair of metals.
As a brief conclusion, in the biological study of the complexes in ovarian adenocarcinoma cells antineoplastic properties characterized by little acute toxicity but rather long term cytostasis were identified. The bioactive ruthenium, osmium and iridium complexes had micromolar IC50 values on A2780 cells (and in case of ruthenium on ID8 cells), whereas some rhodium complexes showed weaker activity, while the compounds had little or no activity on primary, non-transformed human fibroblasts highlighting the low toxicity and selectivity of these compounds towards transformed cancer cells.
The presence of the sugar moiety equipped with large hydrophobic protective groups on the hydroxyl groups was found to be important for the biological activity. Furthermore, replacing the O-benzoyl protective groups of the carbohydrate moiety to straight chain O-acyl groups worsened the cytostatic ability of the complexes and rendered them toxic. The replacement of the pyridine substituent with a quinoline moiety improved the IC50 value of the complexes. The complexes were active in a wide variety of carcinoma, sarcoma and lymphoma cell lines. Furthermore, it has been shown that ROS production plays a central role in the biological activity of the complexes. Importantly, the bioactive derivatives were active on platinum resistant cells suggesting that these complexes may be able to overcome cisplatin resistance in vivo.
The bioactive transition metal complexes, identified hereby, show a cooperative binding to yet unidentified cellular target(s) and induce oxidative stress, as supported by our findings, in a membrane dependent manner.
The present inventors provided evidence that complexes of the invention proved to be active in cell models of ovarian cancer, glioblastoma, breast cancer and pancreatic adenocarcinoma, although some preference towards ovarian cancer cells could be observed, suggesting the anti-neoplastic effect is general.
The measured effect and facts about mechanism make it plausible that the complexes of the invention provide a general anti-neoplastic effect.
The invention relates to the compounds disclosed according to the present invention for use in the treatment of neoplasms. Neoplasm can be a carcinoma or a sarcoma.
A “neoplasm” is a type of abnormal and excessive growth of tissue. (The process that occurs to form or produce a neoplasm is called “neoplasia”.) The growth of a neoplasm is uncoordinated with that of the normal surrounding tissue, and persists in growing abnormally, even if the original trigger is removed. This abnormal growth usually forms a mass, when it may be called a tumor.
A number of neoplasms of each type are listed in (Kumar et al., 2017).
Below we provide a few examples without limitation.
A “carcinoma” is a neoplasm which arises from transformed cells of epithelial origin, including epithelial tissue of the skin, or the tissue that lines internal organs, such as the liver or kidneys. Carcinomas may be confined to the primary location of origin; however, they may also spread to other parts of the body.
Carcinomas are divided into two major subtypes: adenocarcinomas, which develop in an organ or gland, and squamous cell carcinomas, which originate in the squamous epithelium. Treatments against carcinomas and in particular adenocarcinomas are particularized herein and can form preferred embodiments.
A “sarcoma” is a neoplasm which arises from transformed cells of mesenchymal, in particular connective tissue origin. Connective tissue includes bone, cartilage, tendon, muscle, fat, vascular or hematopoietic tissues, and sarcomas can arise, among others, in any of these types of tissues.
Sarcomas include osteosarcoma or osteogenic sarcoma, chondrosarcoma, leiomyosarcoma, rhabdomyosarcoma, mesothelial sarcoma or mesothelioma, fibrosarcoma, angiosarcoma or hemangioendothelioma, liposarcoma, glioma or astrocytoma, myxosarcoma, and mesenchymous or mixed mesodermal tumors.
Neoplasms include neoplasms of brain or central nervous system; neoplasms of haematopoietic or lymphoid tissues; malignant neoplasms; in situ neoplasms; benign neoplasms; and neoplasms of uncertain behavior.
The neoplasms of the central nervous system, preferably the brain, include primary neoplasms of brain (including gliomas of the brain, astroblastoma of the brain); primary neoplasms of meninges (including meningiomas); primary neoplasm of spinal cord, cranial nerves or remaining parts of central nervous system (including gliomas). Anti-neoplastic effect against neoplasms of the CNS, in particular CNS-cancers is particular or even preferred.
In situ neoplasms comprise in situ carcinomas of the oral cavity, esophagus, stomach, digestive organs, respiratory system, breast, genital organs, bladder, urinary organs, eye or endocrine glands; skin, and melanomas.
Anti-neoplastic effect in these organs are particular or even preferred embodiment of the invention whereas treatments against neoplasms or cancers of the respiratory system or the breast, urogenital organs, particularly the female reproductive organs are more preferred and anti ovarian cancer effect is particularly preferred.
Neoplastic diseases of the digestive system and those of the endocrine system also provide a serious health problem. Treatments against such neoplasms, in particular if malignant, are also particular embodiments.
“Hematologic neoplasms” (or neoplasms of the hematopoietic and/or lymphoid tissues), in particular malignancies are neoplasms that affect the blood, bone marrow and the lymphatic system or which are of hematopoietic and/or lymphoid origin. Hematologic neoplasms fall into three categories: leukemia, lymphoma, and myeloma.
The neoplasms of haematopoietic or lymphoid tissues include myeloproliferative neoplasms (including mastocytosis); myelodysplastic syndromes (including refractory anemia, refractory thrombocytopenia, refractory neutropenia); myelodysplastic and myeloproliferative neoplasms (including chronic myelomonocytic leukemia); myeloid and lymphoid neoplasms; acute myeloid leukemias; acute leukemias; precursor lymphoid neoplasms (including precursor B-lymphoblastic neoplasms, precursor T-lymphoblastic neoplasms); mature B-cell neoplasms (including follicular lymphoma, diffuse large B-cell lymphoma, plasma cell neoplasms, B-cell lymphomas); mature T-cell or NK-cell neoplasms; Hodgkin lymphoma; histiocytic or dendritic cell neoplasms (including histiocytic sarcoma, follicular dendritic cell sarcoma); and immunodeficiency-associated lymphoproliferative disorders.
Leukemias are cancers of the bone marrow. Examples of leukemia include acute myeloid leukemia, chronic myeloid leukemia, acute or chronic lymphoid leukemia, lymphocytic leukemia, and lymphoblastic leukemia.
Lymphomas develop in the glands or nodes of the lymphatic system. Lymphomas may also occur in specific organs such as the stomach, breast or brain. The lymphomas are subclassified into two categories: Hodgkin lymphoma and non-Hodgkin lymphoma.
The experiments and data on the mechanism provided herein a broad anti-neoplastic effect of the compounds of the invention.
To assess the biological activity of the compounds from a different point of view, antimicrobial effect of the compounds has been tested in multiple models.
The bacterial species used for testing are briefly reviewed hereinbelow.
Such exemplary models were Staphylococcus aureus (ref. strain ATCC43300) and Enterococcus faecalis (ref. strain ATCC29122), clinical isolates (6 methicillin-sensitive Staphylococcus aureus MSSA isolates and 6 methicillin-resistant Staphylococcus aureus (MRSA) isolates as well as 8 clinical isolates of Enterococcus faecium. In a further experiment clinical isolates of vancomycin-sensitive Enterococcus (VSE) and vancomycin-resistant Enterococcus (VRE) were tested against the complexes tested above.
Gram-negative bacteria subjected to examination with the compositions of the invention include Escherichia coli, Acinetobacter baumannii and Pseudomonas aeruginosa (data not shown).
Enterococcus is a large genus of lactic acid bacteria. Two species are common commensal organisms in the intestines of humans: E. faecalis and E. faecium. Enterococcus species can also cause infections such as urinary tract infections, bacteremia, bacterial endocarditis, diverticulitis, meningitis, and bacterial peritonitis. From a medical standpoint, an important feature of this genus is the high level of intrinsic antibiotic resistance. Some enterococci are intrinsically resistant to β-lactam-based antibiotics (penicillins, cephalosporins, carbapenems), as well as many aminoglycosides. In the last two decades, particularly virulent strains of Enterococcus that are resistant to vancomycin (vancomycin-resistant Enterococcus, or VRE) have emerged in nosocomial infections of hospitalized patients.
Enterococcus faecium is a Gram-positive, non-hemolytic bacterium in the genus Enterococcus. It can be commensal (innocuous, coexisting organism) in the gastrointestinal tract of humans and animals, but it may also be pathogenic, causing diseases such as wound infection, sepsis or urinary tract infection. There are strains of this bacterium that have developed multi-drug antibiotic resistance. Enterococcus faecium has been a leading cause of multi-drug resistant enterococcal infections worldwide. Approximately 40% of medical intensive care units reportedly found that the majority of device-associated infections (namely, infections due to central lines and urinary drainage catheters) were due to vancomycin- and ampicillin-resistant E. faecium. It is difficult to fight infections caused by E. faecium since not many antimicrobial solutions are available.
Enterococcus faecalis is a Gram-positive, commensal bacterium inhabiting the human gastrointestinal tract. Like other species in the genus Enterococcus, E. faecalis is found in healthy humans. However, as an opportunistic pathogen, E. faecalis can cause life-threatening infections (such as endocarditis, sepsis, urinary tract infections), especially in the nosocomial environment, where the naturally high levels of antibiotic resistance found in E. faecalis contribute to the burden caused.
The genus Staphylococcus includes at least 40 species. Many of these species are non-pathogenic and reside normally on the skin of humans and other animals. Human pathogenic Staphylococcus species include Staphylococcus aureus (including methicillin-resistant Staphylococcus aureus, or MRSA, and vancomycin-intermediate/vancomycin-resistant S. aureus, or VISA/VRSA), Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus lugdunensis; animal pathogenic Staphylococcus species include Staphylococcus schleiferi, Staphylococcus pseudintermedius and Staphylococcus caprae.
Staphylococcus aureus is a Gram-positive bacterium, and is a usual member of the microbiota of the human body, frequently found in the upper respiratory tract and on the skin. S. aureus can also become a pathogen, being a common cause of skin infections including abscesses, respiratory infections such as pneumonia, and food poisoning. Pathogenic strains often promote infections by producing virulence factors such as potent protein toxins, and the expression of a cell-surface protein that binds and inactivates antibodies. The emergence of antibiotic-resistant strains of S. aureus such as methicillin-resistant S. aureus (MRSA) is a worldwide problem in clinical medicine. S. aureus can cause a range of illnesses, from minor skin infections, such as pimples, impetigo, boils, cellulitis, folliculitis, carbuncles, scalded skin syndrome, and abscesses, to life-threatening diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome, bacteremia, and sepsis. It is still one of the five most common causes of hospital-acquired infections and is often the cause of wound infections following surgery.
Bacteria constitute a large domain of prokaryotic microorganisms. Broadly speaking, there are two different types of cell wall in bacteria that classify bacteria into Gram-positive and Gram-negative bacteria. Gram-positive bacteria possess a thick cell wall that contains many layers of peptidoglycan and lipotheichoic acids. In contrast, Gram-negative bacteria have a relatively thin cell wall consisting of a few layers of peptidoglycan surrounded by a second lipid membrane containing lipopolysaccharides and lipoproteins. These differences in structure can produce differences in antibiotic susceptibility.
Gram-positive bacteria have a thick but porous peptidoglycan layer in the cell wall (thicker than the Gram-negative bacteria), but they do not have an outer membrane whereas Gram-negative bacteria have. This is why Gram-positive bacteria usually allow a more easy access to their cell membrane than Gram-negative bacteria.
The results of the experiments with species and isolates mentioned above are shown on
The measurements were carried out by broth microdilution method.
Broth microdilution is the reference method for antimicrobial susceptibility testing of rapidly growing aerobic bacteria, except for mecillinam and fosfomycin, where agar dilution is the reference method.
EUCAST (EUROPEAN COMMITTEE ON ANTIMICROBIAL SUSCEPTIBILITY TESTING, European Society of Clinical Microbiology and Infectious Diseases; see EUCAST reading guide for broth microdilution, Version 3.0 January 2021, also available at https://eucast.org/ast_of_bacteria/mic_determination/) recommends testing according to the International Standard ISO 20776-1.
Results are recorded as the lowest concentration of antimicrobial agent that inhibits visible growth of a microorganism, the Minimum Inhibitory Concentration (MIC), expressed in mg/L or μg/mL.
The bioactive complexes were also surprisingly bacteriostatic on MRSA and VRE clinical isolates with low micromolar MIC values.
While most complexes tested have turned out to show some activity, below the inevitably anti-bacterial complexes are showed by underlining and the most active ones in bold for each strain/isolates type. Complexes marked as (−) have not been tested in the experiment.
Staphylococcus aureus Reference Strains
We may conclude that Ru, Os and Ir complexes are preferred, wherein complexes with ligand 2a have shown a superior activity (see
Our results show that each of the tested complexes has shown some activity on both the reference strains and on clinical isolates.
Taken together, here again, rhodium complexes were less active than others. Complexes with ligands L-6 and L8 also provided generally a high antibacterial activity whereas those with L-4 might be sometimes less active but this difference may be incidental or sporadic.
As explained above the compounds of the invention were ineffective against a plurality of Gram-negative bacteria.
Moreover the compounds have been tested and found inactive on clinical Candida isolates (anti-fungal effect; data not shown). Exemplary Candida strains used in the experiment were Candida albicans (SC5314) and Candida auris (ATCC21092).
As a general observation, the exemplary complexes of the invention have turned out to be active against the Gram-positive bacteria used in the experiment. We have duly noted the fact that the complexes of the invention proved to be active against Gram-positive bacteria only. Against tested Gram-negative bacteria experiments provided negative results, i.e. the complexes of the invention had no bacteriocidal or bacteriostatic effect in these bacteria having different cell wall structure.
It is to be mentioned here that the complexes of the invention also proved to be ineffective against Candida, which have a different cell wall composition (mostly contain chitin and β-D-glucan). It may well be that these differences in cell wall structures, in case of Gram-negative bacteria and yeasts, can protect the cells from the complexes of the invention which cannot reach the cytoplasma membrane.
The skilled person will understand that the above results support that the complexes of the invention are plausibly effective in a broad spectrum of Gram-positive bacteria a few further example of which are listed below for sake of illustration.
Gram-positive bacteria that are pathogenic to humans include bacteria from the Streptococcus, Staphylococcus, Corynebacterium, Listeria, Bacillus, Clostridium, and Enterococcus genera, and other bacteria from the phyla Firmicutes and Actinobacteria.
Streptococcus species are responsible for many cases of streptococcal pharyngitis, pink eye, meningitis, bacterial pneumonia, endocarditis, erysipelas, and necrotizing fasciitis. Human pathogenic Streptococcus species include S. pyogenes, S. agalactiae, S. dysgalactiae, S. gallolyticus, S. anginosus, S. sanguinis, S. mitis, S. mutans, and S. pneumoniae.
Bacteria from the genus Corynebacterium can also cause human disease, for example Corynebacterium diphtheriae can cause diphtheria.
From the Bacillus genus, two species are medically significant from a human point of view: Bacillus anthracis (which causes anthrax) and Bacillus cereus (which causes food poisoning).
The Clostridium genus includes several significant human pathogens, such as Clostridium difficile (which was later reclassified into the Clostridioides genus as Clostridioides difficile), Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Clostridium sordellii, Clostridium novyi and Clostridium welchii.
Gram-positive bacterial infections include, without limitation, infections by Gram-positive cocci, like Toxic shock syndrome and infections caused by Staphylococcus and Streptococcus, as well as Gram-positive bacilli, like Diphtheria, Anthrax, Erysipelothricosis, Listeriosis and various enterococcal infections.
Thus, the compounds of the invention and compositions comprising them are provided for use against the bacterial infections and in the treatment of the related diseases, as far as regulations allow.
Gram-positive bacteria are usual contaminants in the food industry. Such bacteria are common in dairies and in dry production environments. Cross-contamination may affect food quality. Pathogenic bacteria present in food processing environments may cause food spoilage and also health risk for consumers both humans and non-human animals. The invention also relates to use of the inventive compounds in food industry including animal feed industry.
Gram-positive bacteria are often cause infections in patients with cancer (Shelburne and Musher, 2011). Presently an increased number of finding of Gram-positive bacteria in cancer patients have lead therapeutic suggestions. Thus, in a particular embodiment the compounds of the present invention are used against Gram-positive bacterial infections in cancer patients.
It will readily be understood by a person skilled in the art that the antibacterial effect of the inventive complexes gives rise to both medical uses, like antibiotics or medical disinfection agents, and non-medical antibacterial uses as well.
In a therapeutic treatment the compounds of the invention can be used as an antibiotic against bacteria, preferably against Gram-positive bacteria.
Antibiotic resistance is a major health problem today and this is a particular danger in nosocomial infections. As shown herein, the present inventive compounds have proved to be active against a number of antibiotic resistant strains and hospital isolates.
Antibiotics can be administered in various routes.
Oral administration has been known as the safest and most convenient route. In the present invention due to hydrophobicity limited aqueous solubility may be associated with poor bioavailability when administered orally.
There are several ways to improve solubility in oral preparations including e.g. drug-loaded microbial biopolymeric nanocarrier or inclusion complexes like those with cyclodextrines etc.
Such preparations may also be administered via the parenteral route which is quite often the route of choice due to manageable dosage, bioavailability and absence of first-pass metabolism.
Advanced technology formulation methods may be used here as well. Such methods include nanoparticle formulations like nanostructured lipid carriers, inclusion complexes may be used here as well to increase solubility in the bloodstream etc.
Transdermal delivery systems have also been developed to deliver antibiotics to the systemic circulation. This way may also help to overcome the antimicrobial resistance. Microemulsions may be an appropriate choice of getting the molecules through the epithelial barrier. Moreover, microneedles may also be utilized for a similar purpose.
The inhalation route may be the preferred option in case of respiratory tract and organ infections. The compounds of the invention are not volatile in themselves and would require formulation into solution or suspension which can be administered by nebulizers or metered-dose inhalers. Alternatively, preparing dry particles is also an option which may require dry powder inhalers to administer into the respiratory system. Excipients in aerosols may include usual additives like L-leucine, lactose, magnesium stearate or poly(lactic-acid) or poly(lactic-co-glycolid acid) microspheres.
In bacterial diseases of the skin and skin-structures the topical route may be preferred. Topical formulations may include various copolymers of poly(lactic-acid), poly(ethylene-glycol), cationic polycarbonate etc. Well known formulations of liposomes, polymers, micelles, etc. have been developed by way of nanotechnology in recent times.
The complexes of the invention may also be used as antibacterial agent in cavities of the body like in the oral cavity, in the nose, wherein liquids, e.g. rinses or washes or inhalation as described above is a preferred route. Moreover, the cavities of the urogenital system like in the vagina can be subject to antibacterial treatment e.g. in the form of suppositories or lotions or washes.
Selective antibacterial treatment may also be applied in the bowels or in the gut wherein administration routes may include oral administration, however, anal administration may also be applied. In the latter case suppositories or capsules may be applied.
Other methods are also well known in the art.
The above treatments and therapeutic methods both in respect of neoplasms and against Gram-positive bacteria are contemplated as relating to the inventions.
Well known and recent delivery methods of antibiotics are described by Nainu F et al. (Nainu et al., 2021) and by Vassallo et al. (Vassallo et al., 2020).
As to non-medical antibacterial use of the complexes of the invention the following exemplary uses are listed herein.
The complexes of the invention may be used in veterinary medicine to treat cancers or infections caused by Gram-positive bacteria in various food and companion animal species.
In an embodiment the complexes can be used as antibacterial cleaning agent, for example for cleaning surfaces. In this application they can be added to cleaning compositions as a further active agent. Typically in such application other cleaning agents like surfactants are added to the composition. Preparation of such antibacterial cleaning compositions are well known in the art.
In another embodiment the complexes can be used as aseptic or disinfecting agents in such aseptic or disinfecting compositions. For example antibacterial aseptic compositions may be used for disinfecting laboratory or medical tools or apparatuses. Furthermore, the complexes of the invention can be used in sanitizers for artificial and/or biological surfaces e.g. in skin sanitizers including sanitizer of hand.
Another field of application is that of preservatives. In this embodiment the complexes of the invention may be used for preservation of biological material in the laboratory or in the medical field including organs or tissues or cultures.
Alternatively, preservative effect of the agents may be utilized in the food industry. Cleaning or sanitizing apparatus or tools is also a way of utilization here. Moreover, the antibacterial agents which are safe for human or animal health may be used as preservatives in food or animal feed.
The skilled person will understand that these are examples and will be able to identify other fields of use.
As a brief summary, apparently, the mode of action of the biologically active complexes of the invention to induce cytostasis differs for different classes of ruthenium complexes (Fernandes, 2019). Carbohydrate-ruthenium complexes were shown to induce apoptosis that the present inventors have not detected for our compounds in concentrations corresponding to the IC50 values (Hamala et al., 2020) and other complexes of the series have a similar effect.
Rather, the active compounds identified in the present invention led to oxidative stress marked by increased 4HNE expression, a marker for lipid peroxidation. The functional role of reactive oxygen species production was confirmed by using vitamin E. The source of reactive oxygen species was other than the mitochondria. Reactive oxygen species production was shown to be cytostatic in numerous carcinomas (Bakewell et al., 2020; Fernandes, 2019; Mihajlovic et al., 2020; Smolkova et al., 2020; Xu et al., 2018). The compounds of the invention appear to act through a membrane dependent mechanism as supported by the importance of the hydrophobicity of the sugar or sugar-analogue, the sensitivity to the apolar chain in case of the counter-effect of vitamin A.
While the oxidative stress appeared to be an essential part of the mode of action in this invention, the fact that only complexes with hydrophobic moieties and/or with high hydrophobicity were active plausibly suggest a membrane dependent mechanism which, however, is independent of mitochondria. This is in good agreement with the experiments that thiol-free antioxidant vitamin E abrogated the effect whereas Trolox, a derivative of vitamin E lacking the apolar phytyl chain did not.
The complexes may potentiate the effects of the elements of chemoradiotherapy that:
For the formation of the transition metal complexes of the invention the sugar-based heterocyclic N,N-chelating ligands were prepared first.
The synthesis of 1-(β-D-glucopyranosyl)-4-hetaryl-1,2,3-triazoles was accomplished by the well-known copper(I)-catalyzed azide-alkyne cycloaddition (Meldal and Tornoe, 2008; Rostovtsev et al., 2002) (CuAAc). Thus, the easily available 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl azide (Györgydeák and Thiem, 2006; Paulsen et al., 1974) (1) was treated with ethynyl heterocycles a and b in the presence of bis-triphenylphosphano-copper(I)-butyrate (Bokor et al., 2012; Gonda and Novak, 2010) to give the expected O-peracetylated 1-(β-D-glucopyranosyl)-4-(pyridin-2-yl)- and -4-(quinolin-2-yl)-1,2,3-triazoles (L-1a,b) in high yields (Scheme Table 1). Removal of the O-acetyl protecting groups of L-1a,b was effected by the Zemplén method resulting in the unprotected derivatives L-3a,b in good yields. O-Perbenzoylation of compound L-3a was then also carried out to give the 1-(2′,3′,4′,6′-tetra-O-benzoyl-β-D-glucopyranosyl)-4-(pyridin-2-yl)-1,2,3-triazole (L-2a) in excellent yield.
The preparation of the sugar-based 5-(pyridin-2-yl)-1,3,4-oxadiazoles was carried out by the ring-transformation of the corresponding 5-substituted-tetrazoles following our previously reported procedure (Tóth et al., 2009). Thus, tetrazoles 2-5 were reacted with 2-picolinic acid in the presence of DCC under heating to furnish the desired 1,3,4-oxadiazoles L-4, L-6, L-7, L-9 in moderate yields (Table 2). The O-deprotected derivatives L-10-L-12 were then obtained upon treatment of L-4, L-6, L-7, respectively, with sodium methoxide in methanol. Under these conditions the open-chain sugar derivative L9 did not furnish the expected O-deacetylated derivative. This might be due to the ring-opening of the oxadiazole, as it was demonstrated for 2-(D-arabino-1,2,3,4-tetraacetoxybutyl)-5-methyl-1,3,4-oxadiazole (Viana et al., 2008). Acetylation of L-10 and benzoylation of L-12 by using standard methods afforded the expected O-peracetylated 2-glucosyl-1,3,4-oxadiazole L-5 and the 0-perbenzoylated 2-galactosyl-1,3,4-oxadiazole L-8, respectively, in high yields.
To get the desired cationic half-sandwich Ru(II) complexes, the above heterocyclic monosaccharide derivatives were reacted with the commercially available dichloro(η6-p-cymene)ruthenium(II) dimer ([(η6-p-cym)RuCl2]2, Ru-dimer).
The complexation reactions of the Ru-dimer with an equimolar amount or a slight excess of the 0-peracylated (L-1 and L-2) and the 0-unprotected (L-3) 1-(β-D-glucopyranosyl)-4-hetaryl-1,2,3-triazoles in the presence of the halide abstraction reagent TIPF6 in a CH2Cl2-MeOH solvent mixture were smoothly accomplished at room temperature to give the PF6 salts of the expected [(η6-p-cym)RuII(N—N)Cl]+ complexes Ru-1-Ru-3 in excellent yields (Scheme 1). The complexes containing the O-peracylated glucosyl-1,2,3-triazole ligands (Ru-1a,b and Ru-2a) were stable and inert enough to be purified by column chromatography on silica gel, while the isolation of the highly polar complexes having the 0-deprotected heterocyclic chelators (Ru-3a,b) could be effected by crystallisation. Due to the formation of a new stereogenic centre on the metal ion and the chiral nature of the glucose unit diastereomers of the complexes were formed in each case, whose separation could be achieved neither by column chromatography nor by crystallisation.
The skilled person will understand that the counter ion would not affect the activity of the compounds of the invention and any such ion of appropriate solubility may be used in the invention.
Complexation of the Ru-dimer with the monosaccharide-based 5-(pyridin-2-yl)-1,3,4-oxadiazoles (L-4-L-12) was also performed by applying the same procedure resulting in diastereomeric mixtures of the corresponding [(η6-p-cym)RuII(N—N)Cl]PF6 half-sandwich type complex molecules (Table 3). Column chromatographic purification for compounds Ru-4-Ru-9 and recrystallization for Ru-10-Ru-12 furnished the test molecules in moderate to high yields.
It is to be noted that the compounds of the invention have proved to be stable which is a feature not evident in case of half-sandwich complexes of transition metals with heterocycle ligands e.g. with sugar or sugar-analogue moieties.
The procedures for the formation of the [(η6-p-cym)MII(N—N))Cl]PF6 (M=Ru, Os) and [(η5-Cp*)MIII(N—N))Cl]PF6 (M=Ir, Rh) type complexes containing O-peracylated glycosyl azole ligands were highly analogous to those for the preparation of the Ru complexes. Briefly, to a solution of the respective dimers ([(η6-p-cym)MIICl2]2(M=Ru, Os) or [(η5-Cp*)MIIICl2]2(M=Ir, Rh)) in the corresponding O-peracylated glycosyl azole (2 eq.) and TIPF6 (2 eq.) were added. Appropriate reaction time and solvent was applied to remove precipitate (here in the example TICI). While counter ions other than PF6− can also be used, clearly an inert ion is preferred and this one is particularly preferred. The reactions were carried out up to the disappearance of the half-sandwich dimer and after the complexation reaction the crude complex was purified, washed and dried to give the [(η6-p-cym)MII(N—N))Cl]PF6 (M=Ru, Os) or [(η5-Cp*)MIII(N—N))Cl]PF6 (M=Ir, Rh) type complex, respectively. The crude product can be purified by column chromatography as well (9:1 or 95:5 CHCl3-MeOH), but appears to be more time and solvent consuming and less pure.
The procedure for the formation of the [(η6-p-cym)MII(N—N))Cl]PF6 (M=Ru, Os) and [(η5-Cp*)MIII(N—N))Cl] PF6 (M=Ir, Rh) type complexes containing unprotected glycosyl azole ligands were also analogous in case of all metal ions as follows.
To a solution of dimer ([(η6-p-cym)MIICl2]2 (M=Ru, Os) or [(η5-Cp*)MIIICl2]2 (M=Ir, Rh)) the corresponding unprotected glycosyl azole (2 eq.) and TIPF6 (2 eq.) were combined. Appropriate reaction time and solvent was applied to remove precipitate (here in the example TICI). After a sufficient level or total disappearance of the half-sandwich dimer compound and completion of the complexation reaction precipitate was removed and then the crude complex obtained after evaporation and purified to give the desired products [(η6-p-cym)MII(N—N))Cl]PF6 (M=Ru, Os) or [(η5-Cp*)MIII(N—N))Cl]PF6 (M=Ir, Rh) type complex.
As to the synthesis of the quinoline derivatives, specifically of the ligand 1-(2′,3′,4′,6′-tetra-O-benzoyl-D-D-glucopyranosyl)-4-(quinolin-2-yl)-1,2,3,-triazole (L-17) was carried out starting from the 0-unprotected derivative L-3b by using standard O-perbenzoylation conditions (Scheme Table 4). p-Cymene-containing Ru(II) and Os(II) and pentamethylcyclopentadienyl containing Ir(III) and Rh(III) cationic half-sandwich complexes with PF6 counter ion were then prepared by the treatment of L-17 (L2-b,) with the appropriate dimeric platinum-group metal chloride precursors (Ru/Os-dimer and Ir/Rh-dimer, respectively) in the presence of TIPF6 (Scheme Table 4).
As to the synthesis of a complexes wherein the sugar moiety is substituted with C3-C7 alkyl chains, a series of O-peracylated 1-(β-D-glucopyranosyl)-4-(pyridin-2-yl)-1,2,3,-triazoles was prepared. Gentle heating of the corresponding unprotected glucosyl heterocycle 11, as described in the Examples, with aliphatic carboxylic acid chlorides in pyridine furnished the desired triazoles L-C3-L-C7 in good yields (Scheme Table 5). These compounds were then incorporated as N,N-bidentate ligands into [(η6-p-cym)OsII(N—N)Cl]PF6 type half-sandwich complexes by their reactions with dichloro(η6-p-cymene)osmium(II) dimer ([(η6-p-cym)OsCl2]2, Os-dimer) and TIPf6. Similar to our previous studies on the synthesis of analogous half-sandwich complexes, the new test compounds L-C3-L-C7 were obtained in good to excellent yields as mixtures of two diastereoisomers (Table 1).
The skilled person will understand that analogous structures can be obtained by methods analogous to those above.
For example, other known bidentate heterocycle comprising ligands within the scope of the invention can be used whereas the sugar or sugar-analogue moiety with sufficiently bulky hydrophobic substituent can be used in the complexation reaction. Finding appropriate solvent, e.g. solvent of appropriate polarity, if needed solvent mixtures, as well as reaction temperatures and time are within the skills of a person skilled in the art of organic or metallo-organic chemistry.
Other reaction conditions like stirring as well as monitoring the procedure of the reaction are also a relatively easy task for the skilled person starting from the teaching provided herein and using her/his general knowledge. Specifically, a plethora of methods like TLC or spectroscopic methods, like MS, if needed combined with GC or other chromatography, and/or spectrophotometric method are at hand to monitor the progress of the reaction.
Isolation or purification methods include traditional methods in chemistry like evaporation, recrystallization, precipitation filtering, washing of crystals in appropriate non-solving solvents, etc. are very well known and applicable here.
If needed preparatory chromatographic methods can also be applied to purify the products of the invention.
As shown in the examples the products are relatively stable in solution which facilitates these procedures. It will be apparent for a person skilled in the art that analogues of monosacharides as defined in the appended claims also belong to the invention. In preferred embodiments relating to therapeutic as well as non-therapeutic uses of the complexes of the invention the skilled person is able to define functional activity of the compound based on the teaching provided herein. For example testing anti-neoplastic effect or anti-bacterial effect of any claimed compound is well at hand of the skilled person. Also defining hydrophobicity of the compounds is a task which can be easily accomplished by the skilled person based on the teaching provided herein. The methods disclosed herein can be used for such testing.
Also defining the contribution of a moiety to the hydrophobicity of the compounds of the invention is within the skills of a person skilled in the art. This can be done by synthesizing the molecules and measuring the hydrophobicity or by calculating properties, e.g. dipole moment or hydrophobic surface area of the moiety in question.
Other cap molecules, typically arenes can also be used as the appropriate dimer complexes can be prepared with the metal ions. It is well within the skills of the person skilled in the art to calculate appropriate oxidation state vs. charge requirements and find the appropriate compounds.
Also the selection of the halogen ion forming part of the complex and testing it by experiments are tasks at hand of a person skilled in the art.
The skilled person, in the knowledge of chemical properties of the complexes of the invention as well as the desired purpose of pharmaceutical application will be able to define and prepare the appropriate formulation. By way of illustration several examples of such formulation are given below.
As to the excipients the relatively highly apolar nature of the inventive complexes have to be considered and possibly emulsifiers or stabilizers added.
They are more readily absorbed than the solid formulations and can be administered by various routes like:
1. Oral preparations—Oral preparations are easier to swallow and administer medicines to children and old-age patients. Flavourings and sugar are added to some liquids to make them palatable. They are available as solutions, suspensions, or emulsions and must be shaken well before use.
2. Topical Preparations—The application of a drug to an area of the body for direct treatment is called topical application. It includes but not limited to:
3. Sublingual and Buccal Administration—It is useful for drugs which are active in very low concentration in the blood. Such drugs are administered as tablets under the tongue or between the cheek and the gum and allowed to dissolve. In this manner, the drug directly enters the bloodstream, bypassing the digestive tract and acts faster.
4. Rectal Administration
5. Parental Drug Administration—is drug administration outside the GI tract of the patient. Drugs can be inserted anywhere with the help of injections.
6. Suppositories—Basic excipient may be a poly-alcohol like glycerine plus additives providing appropriate solution or suspension to provide the release at the site of administration with the desired speed.
7. Injections—injections are typically solvent form, however, in case of injections at the site of the neoplasm or bacterial infection may be a slow release solid or suspension form which may be used more often in this invention than intravenous injection.
Below the invention is described in more detail by way of examples which are non-limiting and the skilled person will be able to consider and use the whole teaching of the specification, including the document cited herein and information known in the given technical field as well as her/his general knowledge to reduce the invention into practice in the entire scope claimed.
The formation of the complexes and the existence of the diastereomeric pairs were confirmed by 1H and 13C NMR spectroscopy in each case. As a representative, the superposition of the 1H and 13C NMR spectra of Ru-7, the free ligand L-7 and the Ru-dimer, respectively, are presented in
The aqueous stability of the complexes was also studied over time. As an example, time dependence of the NMR spectra of Ru-3a are shown in
For comparative biological studies additional two Ru(II) complexes containing non-sugar based ligands (Scheme 2, Ru-13 and Ru-14) were also synthesized starting from Ru-dimer with 1-phenyl-4-(pyridine-2-yl)-1,2,3-triazole (Tawfiq et al., 2014) (L-13) and 2-phenyl-5-(pyridine-2-yl)-1,3,4-oxadiazole (Wei et al., 2010; Weiss et al., 2020) (L-14), respectively.
2.1 Screening of Ruthenium Compounds with Antineoplastic Properties
We screened 14 ligands and their ruthenium complexes bringing up the number of compounds to 28 in a concentration range of 100 μM-0.0017 μM. The compounds were tested in an assay aiming to assess short term toxicity (methylthiazolyldiphenyl-tetrazolium bromide (MTT) reduction assay, 4 hours) and a long term cytostasis or cytotoxicity (sulphorhodamine B (SRB) proliferation assay, 48 hours) on A2780 human ovarian carcinoma cells. Initially, four Ru complexes, Ru-2a, Ru-4, Ru-6, Ru-8 were found to have cytostatic properties both in long-term SRB and short-term MTT assays (
On
Next, we assessed the four active complexes, Ru-2a, Ru-4, Ru-6, Ru-8 and the corresponding ligands, L-2a, L-4, L-6, L-8 in detail. All compounds were tested on two ovarian cancer cell lines (A2780 and ID8) and on human primary skin fibroblasts (non-transformed, primary cells) in MTT and SRB assays. MTT assays were performed 4 hours post-treatment and indicated rapid toxicity of the compounds, while SRB assays were performed 2 days post-treatment and represent long term cytostasis or toxicity.
On
None of the free ligands exerted either rapid toxicity or rapid cytostatic effect on any of the cell lines in short-term MTT assays (
On
Ruthenium-complexes Ru-2a, Ru-4, Ru-6, Ru-8 were cytostatic, while none of the corresponding ligands (L-2a, L-4, L-6, L-8) had cytostatic properties in long-term SRB assays on A2780 and ID8 cells (
Cytostasis on the long term can be due to enhanced cell death. To exclude that possibility, we performed Annexin V-propidium iodide (PI) double staining. Treating A2780 cells with Ru-2a, Ru-4, Ru-6 and Ru-8 did not increase largely the proportions of PI positive, Annexin V positive and double positive cells in contrast to hydrogen peroxide that was used as positive control either at 2 hours, 4 hours or 48 hours post treatment (
Thus, cytostatic effect of the Ru complexes is not caused by an enhanced cell death of A2780 cells.
Since ruthenium complexes are regarded as alternatives to platinum-centered drugs (Kenny and Marmion, 2019), we used the currently therapeutically available platinum-based drugs, cisplatin (1), oxaliplatin (2) and carboplatin (3) as reference compounds and tested them on A2780 and ID8 cells as well as on primary human fibroblasts. Platinum drugs had no effect in MTT assays (
Thus, it appears that typically the compounds of the invention may have even a somewhat better inhibitory potential and less toxicity than the examined platinum compounds of the art.
To compare the inhibitory properties of Ru-2a, Ru-4, Ru-6, Ru-8, we performed a nonlinear regression of the long-term cytostatic curves (SRB curves) (
Besides the IC50 value, regression analysis yielded the Hill slope. Hill slope is a readout, characterized by the slope of the curve, that provides information on the binding characteristics of a compound (Gesztelyi et al., 2012). The Hill slope of platinum compounds on A2780 and ID8 cells was in the range of 0.6-1.6. In stark contrast to that, the Hill slope of the ruthenium complexes was in the range of 1.5-3.7 (Table 4). Higher Hill slope suggests higher cooperativity upon the binding of the ruthenium complexes to target biomolecules than in the case of platinum compounds.
In comparison with these complexes Os and Ir analogues were also measured in cysplatin resistant A2780 cells as described herein. The results are shown on
The result of the SRB assay is also given in Table 5
As can be seen the IC50 values are roughly similar showing the activity of the compounds in cisplatin resistant cells.
Currently available ruthenium complexes with antineoplastic activity have diverse modes of action involving (mitochondrial) reactive oxygen species production (Bakewell et al., 2020; Fernandes, 2019; Mihajlovic et al., 2020; Xu et al., 2018) and the induction of DNA damage (Parveen et al., 2019; Spillane et al., 2008). We detected an increase in the expression of an oxidative stress marker, 4-hydroxy-nonenal (4HNE) at the level of the whole lane, as well as, when a specific band was assessed, in A2780 cells treated with the active ruthenium complexes Ru-2a, Ru-4, Ru-6, Ru-8 corresponding to the IC50 values (
Osmium complexes also induced reactive oxygen species production (
To further study whether cytostasis by the present compounds is ROS-related the ROS production mechanism, the interrelation between Nrf2, a key transcriptional regulator of the cellular antioxidant defense system (Smolková et al., 2020), and ruthenium/osmium complexes was studied.
A2780 cells were treated with Nrf2 inhibitor ML385 at 1.2 μM concentration in a combination with a concentration series of Os-2a for 48 hours, then SRB assay was performed. Data is represented as average ±SEM, from two biological replicates. Nonlinear regression was performed to obtain the IC50 values. Values were normalized for vehicle treated cells. It has been found that treatment of cells with an NRF2 inhibitor (ML385) potentiated the effects of the complexes that is in good correlation with the ROS production mechanism of cytostasis.
We assessed whether oxidative stress had a role in the cytostatic effects exerted by Ru-2a, Ru-4, Ru-6 and Ru-8. To that end, we tried to revert the cytostatic effects of ruthenium complexes by strong reductants as reduced glutathione (GSH) and N-acetyl-cysteine (NAC). Furthermore, we also tested MitoTEMPO, a mitochondrially targeted antioxidant that can efficiently detoxify mitochondria-derived reactive oxygen species (Bai, 2015; Hegedus et al., 2021; Sári et al., 2020). GSH and NAC co-treatment attenuated the cytostatic effects induced by Ru-2a, Ru-4, Ru-6 and Ru-8 (
As thiols are soft Lewis bases and ruthenium is a soft Lewis acid, excess amounts of GSH or NAC can lead to the disassembly of ruthenium complexes. To provide evidence against this scenario, we applied another antioxidant, vitamin E that does not have thiol groups. The application of vitamin E, similar to GSH and NAC, attenuated the cytostatic effects of the bioactive ruthenium complexes (
Interestingly, treatment of cells with Trolox, a derivative of vitamin E lacking the apolar phytyl chain, did not provide protection against ruthenium compounds (
The effect of vitamin E, a lipid soluble antioxidant has also been studied with the quinoline containing compounds and has been found that it blocks the cytostatic activity of the bioactive complexes (
Vitamin E treatment increased the IC50 values of the complexes in all cases (
Reactive oxygen species production leads to DNA damage and poly(ADP-ribose) polymerase (PARP) activation (Bai, 2015; Curtin and Szabo, 2020). Certain ruthenium complexes were shown to potentiate the effects of PARP inhibitors (Bakewell et al., 2020; de Camargo et al., 2019; Xu et al., 2018; Yusoh et al., 2020). We assessed whether Ru-2a, Ru-4, Ru-6 and Ru-8 have similar properties by treating cells with 3 μM rucaparib, a clinically available potent PARP inhibitor.
These results are illustrated on
Rucaparib reduced cell proliferation in agreement with previous publications (e.g. (Zanjirband et al., 2017)) (
In an embodiment the compounds of the invention can be administered together with PARP inhibitors.
The question has been raised whether the sugar-based ruthenium complexes of the invention may be active in neoplastic diseases in general. This has been tested on other cell lines which are models for other neoplastic diseases. Breast cancer (modelled by MCF7 cells) has been selected as an option as prior art data (Hamala et al., 2020) suggested the potential effectiveness of other ruthenium complexes. Pancreatic adenocarcinoma (modelled by Capan2 cells) and glioblastoma (modelled by U251 cells), similar to ovarian cancer, are usually malign diseases where treatment options are limited (Kiss et al., 2020; Sipos et al., 2021; Tykocki and Eltayeb, 2018).
All active complexes (Ru-2a, Ru-4, Ru-6 and Ru-8) exerted cytostatic effects on all cell lines in proliferation assays (SRB) (see
To assess the role of the monosaccharide moiety in the bioactive compounds we synthesized and assessed two molecules, where the monosaccharide unit was substituted with a phenyl group (Ru-13/L-13 and Ru-14/L-14). None of the free ligands, L-13 or L-14 had cytostatic activity on A2780 cells in SRB assays (
The triazole-containing Ru-13 was cytostatic on A2780 cells with an IC50 value of ˜12 μM that is higher by one order of magnitude than the IC50 value of the sugar-containing molecule Ru-2a (
In a still further set of experiments, the quinoline complexes (Ru-2b, Os-2b, Ir-2b, Rh-2b) were tested on A2780 and ID8 ovarian cancer cells, and on non-transformed primary human fibroblasts, used as controls, in a concentration range up to 33.3 μM (
In these experiments shown on
All of the complexes and the free ligand exerted little rapid cytotoxicity in MTT assays, however, in SRB assays that detects cytostasis, the Ru-2b, Os-2b and Ir-2b complexes completely blocked cell proliferation with submicromolar or low micromolar IC50 values both in A2780 and ID8 cells
Quinoline-containing complexes Ru-2b, Os-2b, Ir-2b have been assessed for cytostatic activity on other cancer cell lines, too. 3×103 Capan2 cells, 2×103 Saos cells and 8×103 L428 were plated to 96 well plates. Cells were treated with the compounds in the concentrations for 48 hours. Then for Capan2 and Saos SRB assay was performed, L428 cells were counted using a Burker chamber. Data is represented as average ±SD, from three biological replicates; individual assays were performed in duplicates. Values were normalized for vehicle treated cells, absorbance for vehicle treated cells equals to 1. Normality was checked using the Shapiro-Wilk test. The Saos Os-7 dataset was normalized using the Box-Cox method, other datasets had normal distribution. Each complex was individually assessed for statistical significance using One-way ANOVA test followed by Dunnett's post-hoc test; all values were compared to the values of the lowest concentration. Nonlinear regression was performed on the Capan2 and Saos datasets (
The data are presented in Table 6.
Also the toxic effects of the active Ru, Os and Ir complexes were verified by Annexin V-FITC propidium iodide (PI) double staining.
In the experiments shown on
As noted above, one of the major drawbacks of platinum-based drugs is due to cisplatin resistance. We tested the three complexes with efficient cytostatic properties (Ru-2b, Os-2b and Ir-2b) on a cisplatin-resistant A2780 cell line.
In this experiment, 6×103 cisplatin-resistant A2780 cells were plated to 96 well plates. Cells were treated with the compounds in the concentrations indicated for either 4 hours for an MTT assay or for 48 hours for an SRB assay. Data is represented as average ±SD, from three biological replicates; individual assays were performed in duplicates. Values were normalized for vehicle treated cells, absorbance for vehicle treated cells equals to 1. Each compound was assessed individually. Normality was assessed using the Shapiro-Wilk test. Normality was achieved using the Box-Cox transformation in the case of Os-2b and Ir-2b datasets, while Ru-2b dataset had normal distribution. Statistical significance was assessed using One-way ANOVA test comparing all points to the smallest concentration. Nonlinear regression was performed on the results of the SRB assay datasets.
The complexes did not exert direct toxicity in MTT assays on the cisplatin-resistant cells (
Nevertheless, we have observed important differences in cell proliferation. The IC50 value of cisplatin was 1.21 μM in our previous study on cisplatin-sensitive A2780 cells. The IC50 value increased to 16.47 μM in the cisplatin resistant cell line (13.6 fold increase) in SRB assays. In contrast to that, the IC50 value of Ru-2b (0.8466 μM vs. 1.183 μM, 1.40 fold change) and Ir-2b (0.891 μM vs. 1.535 μM, 1.72 fold change) increased, although not to the same extent as for cisplatin. Furthermore, the IC50 value of Os-2b (0.5777 μM vs. 0.476 μM) was left technically unchanged when comparing the cisplatin sensitive and cisplatin resistant cell lines (
The case is similar with the complexes of the first stage of the study as shown by the IC50 values in Table 5 in comparison with the corresponding values with the non-cisplatin resistant A2780 strain.
In a further set of experiments a set of osmium complexes and their sugar-derived ligands, in which the benzoyl protective groups on the carbohydrate moiety were replaced by aliphatic acyl groups of different chain length (straight chain C3-C7—CO).
Cells were treated with the compounds in the concentrations indicated for either 4 hours for an MTT assay or for 48 hours for an SRB assay. Data is represented as average ±SD, from three biological replicates; individual assays were performed in duplicates. Correlation between the log D and the IC50 values in alkyl-protected complexes is shown on
Values were normalized for vehicle treated cells, absorbance for vehicle treated cells equals to 1. The MTT dataset for Os-C4+L-C4 and the SRB datasets for Os-2+L-2, Os-3+L-3 showed normal distribution, the MTT datasets for Os-C3+L-C3, Os-C5+L-C5, Os-C6+L-C6, Os-C7 and the SRB dataset for Os-C5+L-C5, Os-C6+L-C6, Os-C7 was normalized using the Box-Cox method. Except for the Os-C7 complex, Two-way ANOVA test was performed and all values were compared with each other (Tukey's post-hoc test). For Os-C7 One-way ANOVA was performed on Box-Cox normalized values followed by Dunnett's post-hoc comparing all values to the smallest treatment concentration. *, ** and *** indicate statistically significant differences between vehicle-treated (control) and the cells treated with a compound at p<0.05, p<0.01 and p<0.001, respectively. ## and ### indicate statistically significant differences between the free ligand and the corresponding complex at p<0.01 and p<0.001, respectively. Nonlinear regression was performed on the data (
The compounds had high log D value indicating a strong apolar character up to the point that the free ligand L-C7 with the longest C7H15—CO alkanoyl protective groups proved to be insoluble, hence, it was not suitable for testing. All complexes Os-C3-Os-C7, but not the free ligands L-C3-L-C6, exerted rapid toxicity on A2780 cells in MTT assays (
Antimicrobial effect of the complexes has been examined in accordance with the microdilution method based on the proposals of the EUCAST (European Committee on Antimicrobial Susceptibility Testing).
While most complexes tested have turned out to show some activity, below the inevitably anti-bacterial complexes are showed by underlining and the most active ones in bold for each strain/isolates type. Complexes marked as (−) have not been tested in the experiment.
In case of pyridine-containing, benzoyl-substituted compounds ruthenium, osmium, iridium and rhodium complexes, having similar structure to the quinoline-based compounds identified in this study, have bacteriostatic activity on multiresistant Enterococcus and Staphylococcus aureus clinical isolates in low micromolar or submicromolar concentrations.
In a further stage of the experiments the bioactive Ru-2b, Os-2b and Ir-2b complexes for bacteriostatic activity have been tested.
Ru-2b, Os-2b and Ir-2b complexes exerted bacteriostatic activity on reference strains and clinical VRE and MRSA isolates. The MIC values of the complexes were determined on the reference strains of S. aureus (ATCC11007) and E. faecalis. (ATCC29112) and on clinical VRE and MRSA isolates were determined by microdilution assays (repeated at least twice in duplicates) as described in Materials and Methods. The numbers indicate how many isolates were susceptible to the compound out of those tested; i.e. 1/6 stands for 1 isolate was susceptible out of 6 tested (see
The Ru-2b complex had a MIC value of 5 μM on the reference Enterococcus faecalis (ATCC29112) strain and a MIC of 10 μM on the Staphylococcus aureus (ATCC11007) reference strain. The Ru-7 complex was bacteriostatic on all of the multiresistant clinical VRE isolates with a MIC value of 5 μM, while inhibited the growth of all multiresistant clinical MRSA isolates with MIC values of 5 to 40 μM (
The Os-2b complex had a MIC value of 10 μM on the reference Enterococcus faecalis (ATCC29112) strain and a MIC value of 20 μM on the Staphylococcus aureus (ATCC11007) reference strain. The Os-7 complex was bacteriostatic on all of the multiresistant clinical VRE isolates with a MIC value of 10 μM, while inhibited the growth of 1 out of the 6 multiresistant clinical MRSA isolates with a MIC value of 5 μM (
The Ir-2b complex had no bacteriostatic activity on either reference strains or on the clinical isolates (
E. faecalis
S. aureus
Antimicrobial effect of complexes Os-C3 to Os-C6 is tested as described for Ru-2b, Os-2b and Ir-2b complexes.
Optical rotations were measured on a Jasco P-2000 polarimeter (Jasco, Easton, MD, USA) at rt, and the data referred to the average of three parallel measurements. The 1H and 13C NMR spectra of the newly synthesized compounds were recorded with Bruker DRX360 (360/90 MHz for 1H/13C) or Bruker DRX400 (400/100 MHz for 1H/13C) spectrometers. Chemical shifts are referenced to Me4Si (1H-NMR) or to the residual solvent signals (13C-NMR). Assignments of the proton- and carbon signals of the new compounds were based on COSY and HSQC correlations.
MS spectra were obtained by a Bruker maXis II (ESI-HRMS) spectrometer.
TLC analysis were carried out by using DC Kieselgel 60 F254 plates (Sigma-Aldrich) and the spots were visualized under UV light and by gentle heating.
For column chromatographic purification Kieselgel 60 (Molar Chemicals, particle size 0.063-0.2 mm) silica gel was applied. Anhydrous pyridine was purchased from VWR Chemicals. Anhydrous solvents were obtained by using standard distillation procedures. Anhydrous CH2Cl2, CHCl3 and toluene were produced by distillation from P4O10 and then stored over 4 Å molecular sieves (CH2Cl2, CHCl3) or sodium wires (toluene). MeOH was dried by distillation over Mg turnings and iodine.
The 2-ethynylpyridine (TCI Chemicals) and the dichloro(η6-p-cymene)ruthenium(II) dimer (Ru-dimer, Strem Chemicals), dichloro(pentamethylcyclopentadienyl)iridium(III) dimer (Ir-dimer, Acros Organics), dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer (Rh-dimer, Alfa Aesar) and TIPF6 (Strem Chemicals) were purchased from the indicated suppliers.
The 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl-azide (Györgydeák and Thiem, 2006; Paulsen et al., 1974) (1), the 5-(2′,3′,4′,6′-tetra-O-benzoyl-β-D-glucopyranosyl)-tetrazole (Hadady et al., 2004; Kun et al., 2011) (2), the 5-(2′,3′,4′-tri-O-benzoyl-D-D-xylopyranosyl)-tetrazole (Somsak et al., 2014) (3), the 5-(2′,3′,4′,6′-tetra-O-acetyl-D-D-galactopyranosyl)-tetrazole (Farkas et al., 1977) (4), the 2-(L-arabino-1′,2′,3′,4′-tetraacetoxybutyl)-tetrazole (Viana et al., 2008) (5), the 2-ethynylquinoline (Son et al., 2013), 1-phenyl-4-(pyridine-2-yl)-1,2,3-triazole (Wei et al., 2010) (Tawfiq et al., 2014) (L-13), 2-phenyl-5-(pyridine-2-yl)-1,3,4-oxadiazole (Wei et al., 2010; Weiss et al., 2020) (L-14) and the dichloro(η6-p-cymene)osmium(II) dimer (Os-dimer) (Godó et al., 2012) were synthesized according to literature procedures.
The 1-(β-D-glucopyranosyl)-4-(pyridin-2-yl)- and -(quinolin-2-yl)-1,2,3-triazoles (1 and 2, respectively) were synthesized according to our earlier described procedures [16].
To a solution of 1-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-azide (Györgydeák and Thiem, 2006; Paulsen et al., 1974) (1) in CH2Cl2 (1 mL/50 mg azide) the appropriate 2-ethynylated heterocycle (1 eq.) and the C3H7COOCu(PPh3)2 catalyst (Bokor et al., 2012) (3 mol %) were added. The reaction mixture was stirred at rt for 1 day, and the completion of the reaction was judged by TLC (1:1 EtOAc-hexane). The solvent was then removed under diminished pressure and the residue was purified by column chromatography.
The corresponding O-peracylated 5-glycosyl-tetrazole (2-5) was dissolved in dry toluene (1 mL/100 mg substrate), then 2-picolinic acid (2 eq.) and DCC (2 eq.) were added. The reaction mixture was stirred at boiling temperature until the TLC showed total consumption of the tetrazole. The insoluble materials were filtered off, washed with CH2Cl2 and the filtrate was evaporated under reduced pressure. The crude product was purified by column chromatography and crystallisation.
An O-acyl protected glycosyl-azole was dissolved in a 1:1 mixture of dry MeOH and dry CHCl3 (1 mL/25 mg substrate) and a few drops of ˜1 M solution of NaOMe in MeOH was added (pH=8-9). The reaction mixture was left at rt until the TLC indicated total conversion of the starting material. The neutralisation of the solution was carried out by the addition of a cation exchange resin (Amberlyst 15, H+ form). The resin was then filtered off and the solution was evaporated under reduced pressure. The crude product was purified by column chromatography or crystallisation.
To a solution of dimer ([(η6-p-cym)MIICl2]2(M=Ru, Os) or [(η5-Cp*)MIIICl2]2(M=Ir, Rh)) in CH2Cl2 (10 mg/1 mL) the corresponding O-peracylated glycosyl azole (2 eq.) and TIPF6 (2 eq.) were added. Under stirring the same volume of methanol was added to this reaction mixture in order to accelerate the precipitation of the TICI. The reaction mixture was then stirred at rt for an hour, and the total disappearance of the half-sandwich dimer compound was judged by TLC (9:1 CHCl3-MeOH). After completion of the complexation reaction the precipitated TICI was filtered off by membrane filter and the solution was evaporated in vacuo. The crude complex was purified by dissolving the compound in CHCl3 (10 mg/mL) and the product was precipitated by the addition of Et2O (CHCl3:Et2O=1:2 or 1:4 ratio), then the product was washed with CHCl3-Et2O (1:1 or 1:2) solvent mixture (30 mg/mL) and dried in vacuo affording [(η6-p-cym)MII(N—N))Cl]PF6 (M=Ru, Os) or [(η5-Cp*)MIII(N—N))Cl]PF6 (M=Ir, Rh) type complex. The crude product can be purified by column chromatography as well (9:1 or 95:5 CHCl3-MeOH), but appears to be more time and solvent consuming and less pure.
To a solution of dimer ([(η6-p-cym)MIICl2]2(M=Ru, Os) or [(η5-Cp*)MIIICl2]2(M=Ir, Rh)) in CH2Cl2 (10 mg/1 mL) the corresponding unprotected glycosyl azole (2 eq.) and TIPF6 (2 eq.) were suspended. Under stirring the same volume of methanol was added to this reaction mixture in order to dissolve the heterocyclic sugar derivative and accelerate the precipitation of the TICI. The reaction mixture was then stirred at rt for an hour, and the total disappearance of the half-sandwich dimer compound was judged by TLC (9:1 CHCl3-MeOH). After completion of the complexation reaction the precipitated TICI was filtered off by membrane filter and the solution was evaporated in vacuo. The crude complex was purified by crystallisation from pure iPrOH or iPrOH: Et2O=2:5 solvent mixture affording [(η6-p-cym)MII(N—N))Cl]PF6 (M=Ru, Os) or [(η5-Cp*)MIII(N—N))Cl]PF6 (M=Ir, Rh) type complex.
A solution of the appropriate 1-(β-D-glucopyranosyl)-4-hetaryl-1,2,3-triazole (1 or 2) in anhydrous pyridine (4 mL/50 mg substrate) was cooled down in an ice bath and the corresponding carboxylic acid chloride (4.8 equiv.) was added under stirring. The reaction mixture was then heated at 60° C. and monitored by TLC (1:1 CHCl3-MeOH and 1:2 EtOAc-hexane). If the TLC showed incomplete conversion after one hour, an additional portion of acid chloride (4.8 equiv.) was added to the mixture. After completion of the reaction the pyridine was removed in vacuo. The residue was dissolved in CHCl3 (30 mL) and extracted with sat. aq. solution of NaHCO3 (2×30 mL) and with water (35 mL). The separated organic phase was dried over MgSO4, filtered and evaporated. The residual crude product was purified by column chromatography.
The corresponding O-peracylated 1-(β-D-gucopyranosyl)-4-hetaryl-1,2,3-triazole (L-2-L-7, 2.0 or 2.1 equiv.), the complex dimer (Ru—/Os-/Ir-/Rh-dimer, 1 equiv.) and TIPF6 (2 equiv.) were dissolved in a 1:1 mixture of anhydrous CH2Cl2 and MeOH (1-1 mL/10 mg dimer). The reaction mixture was vigorously stirred until the TLC (95:5 CHCl3-MeOH) showed complete disappearance of the starting dimer complex. After completion of the reaction, the precipitated TICI was filtered off and the solvents were removed. The residual crude product was purified by crystallization or by column chromatography.
Prepared from azide (Györgydeák and Thiem, 2006; Paulsen et al., 1974) 1 (100 mg, 0.27 mmol) and 2-ethynylpyridine (28 μL, 0.27 mmol) according to general procedure I. Purified by column chromatography (1:1→2:1 EtOAc-hexane) to give 121 mg white amorphous solid (95%). Rf=0.18 (1:1 EtOAc-hexane); [α]D=−55 (c 0.21, CHCl3). 1H NMR (360 MHz, CDCl3) δ (ppm): 8.61 (1H, d, J=4.8 Hz, Py-H-6), 8.42 (1H, s, Tria-H-5), 8.15 (1H, d, J=7.8 Hz, Py-H-3), 7.78 (1H, dt, J=7.8, 1.6 Hz, Py-H-4), 7.25 (1H, m, Py-H-5), 5.94 (1H, d, J=8.8 Hz, H-1′), 5.51 (1H, pt, J=9.5, 9.5 Hz, H-2′), 5.46 (1H, pt, J=9.5, 9.5 Hz, H-3′), 5.28 (1H, pt, J=9.5, 9.5 Hz, H-4′), 4.32 (1H, dd, J=12.6, 4.8 Hz, H-6′a), 4.17 (1H, dd, J=12.6, 1.7 Hz, H-6′b), 4.04 (1H, ddd, J=9.5, 4.8, 1.7 Hz, H-5′), 2.10, 2.08, 2.05, 1.90 (4×3H, 4 s, 4×CH3); 13C NMR (90 MHz, CDCl3) δ (ppm): 170.6, 170.1, 169.4, 168.9 (4×C═O), 149.8, 149.2 (Tria-C-4, Py-C-2), 149.7 (Py-C-6), 137.0 (Py-C-4), 123.3 (Py-C-5), 120.5 (Py-C-3), 120.7 (Tria-C-5), 86.0 (C-1′), 75.3 (C-5′), 72.8 (C-3′), 70.7 (C-2′), 67.8 (C-4′), 61.7 (C-6′), 20.8, 20.7 (2), 20.3 (4×CH3). ESI-HRMS positive mode (m/z): calcd for C21H25N4O9+ [M+H]+ 477.1616; C21H24N4NaO9+ [M+Na]+ 499.1435. Found: [M+H]+ 477.1614; [M+Na]+ 499.1432.
Prepared from azide (Györgydeák and Thiem, 2006; Paulsen et al., 1974) 1 (1.00 g, 2.68 mmol) and 2-ethynylquinoline (Son et al., 2013) (0.41 g, 2.68 mmol) according to general procedure I. Purified by column chromatography (1:1 EtOAc-hexane) to yield 1.20 g white amorphous solid (85%). Rf=0.23 (1:1 EtOAc-hexane); [α]D=−90 (c 0.20, CHCl3). 1H NMR (360 MHz, CDCl3) δ (ppm): 8.63 (1H, s, Tria-H-5), 8.31 (1H, d, J=8.6 Hz, Qu-H-3), 8.24 (1H, d, J=8.6 Hz, Qu-H-4), 8.08 (1H, d, J=8.5 Hz, Qu-H-5 or Qu-H-8), 7.82 (1H, d, J=8.0 Hz, Qu-H-5 or Qu-H-8), 7.72 (1H, pt, J=7.9, 7.6 Hz, Qu-H-6 or Qu-H-7), 7.53 (1H, pt, J=7.5, 7.4 Hz, Qu-H-6 or Qu-H-7), 5.99 (1H, d, J=9.2 Hz, H-1′), 5.59 (1H, pt, J=9.4, 9.2 Hz, H-2′), 5.48 (1H, pt, J=9.5, 9.4 Hz, H-3′), 5.31 (1H, pt, J=9.7, 9.5 Hz, H-4′), 4.35 (1H, dd, J=12.6, 4.8 Hz, H-6′a), 4.18 (1H, dd, J=12.6, 2.1 Hz, H-6′b), 4.07 (1H, ddd, J=9.7, 4.8, 2.1 Hz, H-5′), 2.11, 2.09, 2.05, 1.91 (4×3H, 4 s, 4×CH3); 13C NMR (90 MHz, CDCl3) δ (ppm): 170.6, 170.1, 169.4, 169.0 (4×C═O), 149.8, 149.4, 148.2 (Tria-C-4, Qu-C-2, Qu-C-8a), 137.0 (Qu-C-4), 129.9 (Qu-C-6 or Qu-C-7), 129.3 (Qu-C-5 or Qu-C-8), 128.0 (Qu-C-4a), 127.8 (Qu-C-5 or Qu-C-8), 126.6 (Qu-C-6 or Qu-C-7), 121.4 (Tria-C-5), 118.7 (Qu-C-3), 86.0 (C-1′), 75.3 (C-5′), 72.9 (C-3′), 70.7 (C-2′), 67.8 (C-4′), 61.7 (C-6′), 20.8, 20.6 (2), 20.3 (4×CH3). ESI-HRMS positive mode (m/z): calcd for C25H27N4O9+ [M+H]+ 527.1773; C25H26N4NaO9+ [M+Na]+ 549.1592. Found: [M+H]+ 527.1773; [M+Na]+549.1593.
The 1-(β-D-glucopyranosyl)-4-(pyridin-2-yl)-1,2,3-triazole (L-3a, 20.0 mg, 0.065 mmol) was suspended in dry pyridine (0.5 mL) and benzoyl chloride (36 μL, 0.310 mmol) was added. The reaction mixture was stirred at 60° C. until the TLC (3:2 EtOAc-hexane) showed complete disappearance of the starting material (1 h). The solvent was removed under diminished pressure. The residue was dissolved in CH2Cl2 (20 mL) and extracted with sat. aq. NaHCO3 (10 mL) and then with water (10 mL). The organic phase was dried over MgSO4, filtered and evaporated. Purification by column chromatography (3:2 EtOAc-hexane) yielded 41 mg of white amorphous solid (87%). Rf=0.30 (3:2 EtOAc-hexane); [α]D=−75 (c 0.20, CHCl3). 1H NMR (400 MHz, CDCl3) δ (ppm): 8.61 (2H, 1 signal, Tria-H-5, Py-H-6), 8.17-7.21 (23H, m, Ar, Py-H-3-Py-H-5), 6.34 (1H, d, J=9.2 Hz, H-1′), 6.16 (1H, pt, J=9.6, 9.5 Hz, H-3′) 6.07 (1H, pt, J=9.5, 9.2 Hz, H-2′), 5.90 (1H, pt, J=9.6, 9.6 Hz, H-4′), 4.69-4.49 (3H, m, H-5′, H-6′a, H-6′b); 13C NMR (100 MHz, CDCl3) δ (ppm): 166.2, 165.8, 165.2, 164.7 (4×C═O), 149.7, 149.0 (Tria-C-4, Py-C-2), 149.6 (Py-C-6), 137.1 (Py-C-4), 133.8, 133.7, 133.6, 133.5, 133.4, 130.2-128.2 (Ar), 123.3 (Py-C-5), 121.0 (Tria-C-5), 120.6 (Py-C-3), 86.4 (C-1′), 75.7 (C-5′), 73.2 (C-3′), 71.3 (C-2′), 69.0 (C-4′), 62.8 (C-6′). ESI-HRMS positive mode (m/z): calcd for C41H32N4NaO9+ [M+Na]+ 747.2061. Found: 747.2041.
Prepared from compound L-1a (0.81 g, 1.70 mmol) according to general procedure III. Purified by column chromatography (7:2 CHCl3-MeOH) to give 0.40 g white amorphous solid (76%). Rf=0.26 (7:2 CHCl3-MeOH); [α]D=−12 (c 0.20, MeOH). 1H NMR (400 MHz, CD3OD) δ (ppm): 8.63 (1H, s, Tria-H-5), 8.59 (1H, d, J=4.3 Hz, Py-H-6), 8.09 (1H, d, J=7.9 Hz, Py-H-3), 7.92 (1H, pt, J=7.9, 7.8 Hz, Py-H-4), 7.38 (1H, m, Py-H-5), 5.70 (1H, d, J=9.2 Hz, H-1′), 3.96 (1H, pt, J=9.1, 9.0 Hz, H-2′), 3.90 (1H, dd, J=12.2, 1.3 Hz, H-6′a), 3.74 (1H, dd, J=12.2, 5.3 Hz, H-6′b), 3.64-3.58 (2H, m, H-3′ or H-4′, H-5′), 3.54 (1H, pt, J=9.2, 9.1 Hz, H-3′ or H-4′); 13C N M R (90 MHz, CD3OD) δ (ppm): 150.9, 148.6 (Tria-C-4, Py-C-2), 150.5 (Py-C-6), 138.9 (Py-C-4), 124.6 (Py-C-5), 123.4 (Tria-C-5), 121.7 (Py-C-3), 89.8 (C-1′), 81.2 (C-5′), 78.5 (C-3′ or C-4′), 74.1 (C-2′), 70.9 (C-3′ or C-4′), 62.4 (C-6′). ESI-HRMS positive mode (m/z): calcd for C13H16N4NaO5+ [M+Na]+ 331.1013; C26H32N8NaO10+ [2M+Na]+ 639.2134. Found: [M+Na]+ 331.1012; [2M+Na]+639.2135.
Prepared from compound L-1b (500 mg, 0.95 mmol) according to general procedure III. The crude product was recrystallised from MeOH to yield 290 mg of white amorphous solid (85%). Rf=0.44 (7:2 CHCl3-MeOH); [α]D=−4 (c 0.20, DMSO). 1H NMR (400 MHz, DMSO-d6+1-2 drops of D2O) δ (ppm): 8.99 (1H, s, Tria-H-5), 8.51 (1H, d, J=8.6 Hz, Qu-H-4), 8.25 (1H, d, J=8.6 Hz, Qu-H-3), 8.06-8.01 (2H, m, Qu-H-5, Qu-H-8), 7.82 (1H, pt, J=7.8, 7.4 Hz, Qu-H-6 or Qu-H-7), 7.63 (1H, pt, J=7.6, 7.4 Hz, Qu-H-6 or Qu-H-7), 5.68 (1H, d, J=9.2 Hz, H-1′), 3.90 (1H, pt, J=9.2, 9.1 Hz, H-2′), 3.77-3.72 (1H, m, H-6′a), 3.56-3.49 (2H, m, H-5′, H-6′b), 3.46 (1H, pt, J=9.1, 9.0 Hz, H-3′), 3.34 (1H, pt, J=9.2, 9.0 Hz, H-4′); 13C NMR (90 MHz, DMSO-d6) δ (ppm): 150.1, 147.5, 147.3 (Tria-C-4, Qu-C-2, Qu-C-8a), 137.3 (Qu-C-4), 130.1 (Qu-C-6 or Qu-C-7), 128.6, 128.1 (Qu-C-5, Qu-C-8), 127.3 (Qu-C-4a), 126.5 (Qu-C-6 or Qu-C-7) 123.3 (Tria-C-5), 118.3 (Qu-C-3), 87.8 (C-1′), 80.0 (C-5′), 76.8 (C-3′), 72.2 (C-2′), 69.5 (C-4′), 60.8 (C-6′). ESI-HRMS positive mode (m/z): calcd for C17H19N4O5+ [M+H]+ 359.1350; C17H18N4NaO5+ [M+Na]+ 381.1169; C34H36N8NaO10+ [2M+Na]+739.2447. Found: [M+H]+ 359.1349; [M+Na]+ 381.1168; [2M+Na]+ 739.2448.
Prepared from tetrazole (Hadady et al., 2004; Kun et al., 2011) 2 (5.00 g, 7.71 mmol) and 2-picolinic acid (1.90 g, 14.43 mmol) according to general procedure II. Reaction time: 5 hours. Purification by column chromatography (1:1 EtOAc-hexane) and crystallisation from EtOH gave 1.96 g white solid (35%). Rf=0.28 (1:1 EtOAc-hexane). [α]D=−92 (c 0.20, CHCl3). 1H NMR (400 MHz, CDCl3) δ (ppm): 8.81 (1H, d, J=4.8 Hz, Py-H-6), 8.21 (1H, d, J=7.8 Hz, Py-H-3), 8.03-7.81, 7.56-7.27 (22H, m, Ar, Py-H-4, Py-H-5), 6.10 (1H, pt, J=9.4, 9.4 Hz, H-3′), 6.05 (1H, pt, J=9.4, 9.4 Hz, H-2′), 5.86 (1H, pt, J=9.4, 9.4 Hz, H-4′), 5.28 (1H, d, J=9.4 Hz, H-1′), 4.67 (1H, dd, J=12.4, 2.3 Hz, H-6′a), 4.54 (1H, dd, J=12.4, 5.5 Hz, H-6′b), 4.37 (1H, ddd, J=9.4, 5.5, 2.3 Hz, H-5′); 13C NMR (90 MHz, CDCl3) δ (ppm): 166.2, 165.8, 165.2, 165.1, 164.9, 162.2 (4×C═O, OD-C-2, OD-C-5), 150.5 (Py-C-6), 143.2 (Py-C-2), 137.3 (Py-C-4), 133.7, 133.6, 133.5, 133.2, 130.0-128.4 (Ar), 126.2 (Py-C-5), 123.5 (Py-C-3), 77.3 (C-5′), 73.8 (C-3′), 72.1 (C-1′), 70.6 (C-2′), 69.2 (C-4′), 63.3 (C-6′). ESI-HRMS positive mode (m/z): calcd for C41H31N3NaO10+ [M+Na]+748.1902. Found: 748.1907.
To a solution of the 2-(β-D-glucopyranosyl)-5-(pyridin-2-yl)-1,3,4-oxadiazole (L-10, 20 mg, 0.065 mmol) in dry pyridine (0.5 mL) acetic anhydride (0.06 mL, 0.635 mmol) was added and the mixture was stirred at 60° C. After one hour the TLC (1:1 EtOAc-hexane) showed total consumption of L-10. The solvent was removed under reduced pressure and the residue was purified by column chromatography (1:1 EtOAc-hexane). White amorphous solid, yield: 28 mg (90%). Rf=0.21 (1:1 EtOAc-hexane); [α]D=−61 (c 0.19, CHCl3). 1H NMR (400 MHz, CDCl3) δ (ppm): 8.82 (1H, d, J=4.4 Hz, Py-H-6), 8.26 (1H, d, J=7.9 Hz, Py-H-3), 7.91 (1H, dt, J=7.9, 1.1 Hz, Py-H-4), 7.49 (1H, m, Py-H-5), 5.56 (1H, pt, J=9.8, 9.7 Hz, H-2′), 5.40 (1H, pt, J=9.4, 9.3 Hz, H-3′), 5.24 (1H, pt, J=9.8, 9.7 Hz, H-4′), 4.92 (1H, d, J=10.1 Hz, H-1′), 4.30 (1H, dd, J=12.6, 5.1 Hz, H-6′a), 4.18 (1H, dd, J=12.6, 2.2 Hz, H-6′b), 3.91 (1H, ddd, J=9.7, 5.1, 2.2 Hz, H-5′), 2.09, 2.07, 2.04, 1.94 (4×3H, 4 s, 4×CH3); 13C NMR (100 MHz, CDCl3) δ (ppm): 170.7, 170.3, 169.4, 169.3 (4×C═O), 165.1, 162.1 (OD-C-2, OD-C-5), 150.6 (Py-C-6), 143.2 (Py-C-2), 137.4 (Py-C-4), 126.3 (Py-C-5), 123.6 (Py-C-3), 76.9 (C-5′), 73.5 (C-3′), 71.6 (C-1′), 69.7 (C-2′), 68.0 (C-4′), 62.0 (C-6′), 20.8, 20.7 (2), 20.5 (4×CH3). ESI-HRMS positive mode (m/z): calcd for C21H23N3NaO10+ [M+Na]+ 500.1276. Found: 500.1275.
Prepared from tetrazole (Somsak et al., 2014) 3 (3.00 g, 5.83 mmol) and 2-picolinic acid (1.42 g, 11.53 mmol) according to general procedure II. Reaction time: 5 hours. Purification by column chromatography (2:1 EtOAc-hexane) and crystallisation from EtOH yielded 2.00 g white solid (58%). Rf=0.35 (4:1 EtOAc-hexane); [α]D=−123 (c 0.20, CHCl3). 1H NMR (360 MHz, CDCl3) δ (ppm): 8.80 (1H, d, J=4.8 Hz, Py-H-6), 8.21 (1H, d, 7.9 Hz, Py-H-3), 7.99-7.83, 7.57-7.30 (17H, m, Ar, Py-H-4, Py-H-5), 6.05 (1H, pt, J=9.2, 9.2 Hz, H-3′), 5.96 (1H, pt, J=9.2, 9.2 Hz, H-2′), 5.57 (1H, ddd, J=10.1, 9.2, 5.3 Hz, H-4′), 5.15 (1H, d, J=9.2 Hz, H-1′), 4.62 (1H, dd, J=11.4, 5.3 Hz, H-5′eq), 3.80 (1H, pt, J=11.4, 10.1 Hz, H-5′ax); 13C NMR (90 MHz, CDCl3) δ (ppm): 165.8, 165.6, 165.1 (2), 162.5 (3×C═O, OD-C-2, OD-C-5), 150.6 (Py-C-6), 143.3 (Py-C-2), 137.3 (Py-C-4), 133.7, 133.6, 133.5, 130.0-128.5 (Ar), 126.2 (Py-C-5), 123.5 (Py-C-3), 72.9 (C-3′), 72.5 (C-1′), 70.4 (C-2′), 69.6 (C-4′), 67.5 (C-5′). ESI-HRMS positive mode (m/z): calcd for C33H25N3NaO8+ [M+Na]+ 614.1534. Found: 614.1535.
Prepared from tetrazole (Farkas et al., 1977) 4 (0.50 g, 1.25 mmol) and 2-picolinic acid (0.31 g, 2.50 mmol) according to general procedure II. Reaction time: 2 hours. Purification by column chromatography (1:1 EtOAc-hexane) and crystallisation from EtOH yielded 0.30 g white solid (50%). Rf=0.17 (1:1 EtOAc-hexane); [α]D=−41 (c 0.21, CHCl3). 1H NMR (360 MHz, CDCl3) δ (ppm): 8.82 (1H, ddd, J=4.8, 1.8, 0.9 Hz, Py-H-6), 8.27 (1H, dd, J=7.9, 0.9 Hz, Py-H-3), 7.91 (1H, dt, J=7.9, 1.8 Hz, Py-H-4), 7.50 (1H, ddd, J=7.7, 4.8, 1.1 Hz, Py-H-5), 5.67 (1H, pt, J=10.1, 10.0 Hz, H-2′), 5.56 (1H, d, J=3.4 Hz, H-4′), 5.24 (1H, dd, J=10.1, 3.4 Hz, H-3′), 4.89 (1H, d, J=10.0 Hz, H-1′), 4.23-4.11 (3H, m, H-5′, H-6′a, H-6′b), 2.23, 2.06, 2.02, 1.96 (4×3H, 4 s, 4×CH3); 13C NMR (90 MHz, CDCl3) δ (ppm): 170.4, 170.3, 170.0, 169.4, 165.0, 162.3 (4×C═O, OD-C-2, OD-C-5), 150.5 (Py-C-6), 143.2 (Py-C-2), 137.3 (Py-C-4), 126.2 (Py-C-5), 123.6 (Py-C-3), 75.6 (C-5′), 72.2 (C-1′), 71.4 (C-3′), 67.3 (C-4′), 66.9 (C-2′), 61.6 (C-6′), 20.8, 20.7, 20.6, 20.5 (4×CH3). ESI-HRMS positive mode (m/z): calcd for C21H24N3O10+ [M+H]+ 478.1456; C21H23N3NaO10+ [M+Na]+500.1276; C42H46N6NaO20+ [2M+Na]+ 977.2659. Found: [M+H]+ 478.1454; [M+Na]+ 500.1274; [2M+Na]+ 977.2653.
The 2-(β-D-galactopyranosyl)-5-(pyridin-2-yl)-1,3,4-oxadiazole (L-10, 20.0 mg, 0.065 mmol) was suspended in dry pyridine (0.5 mL) and benzoyl chloride (36 μL, 0.310 mmol) was added. The reaction mixture was stirred at 60° C. until the TLC (3:2 EtOAc-hexane) showed complete disappearance of the starting material (1 h). The solvent was removed under diminished pressure. The residue was dissolved in CH2Cl2 (20 mL) and extracted with sat. aq. NaHCO3 (10 mL) and then with water (10 mL). The organic phase was dried over MgSO4, filtered and evaporated. Purification by column chromatography (3:2 EtOAc-hexane) yielded 38 mg of white amorphous solid (81%). Rf=0.27 (3:2 EtOAc-hexane); [α]D=+9 (c 0.20, CHCl3). 1H NMR (400 MHz, CDCl3) δ (ppm): 8.81 (1H, d, J=4.8 Hz, Py-H-6), 8.21 (1H, d, J=7.9 Hz, Py-H-3), 8.17-7.23 (22H, m, Ar, Py-H-4, Py-H-5), 6.32 (1H, pt, J=10.1, 10.0 Hz, H-2′), 6.16 (1H, d, J=3.3 Hz, H-4′), 5.84 (1H, dd, J=10.1, 3.3 Hz, H-3′), 5.30 (1H, d, J=10.0 Hz, H-1′), 4.68 (1H, dd, J=11.1, 6.4 Hz, H-6′a), 4.58 (1H, pt, J=6.4, 5.9 Hz, H-5′), 4.49 (1H, dd, J=11.1, 5.9 Hz, H-6′b); 13C NMR (100 MHz, CDCl3) δ (ppm): 166.2, 165.7, 165.6, 165.1, 165.0, 162.4 (4×C═O, OD-C-2, OD-C-5), 150.5 (Py-C-6), 143.3 (Py-C-2), 137.3 (Py-C-4), 133.8, 133.6, 133.5, 133.4, 130.2-130.0, 129.9-128.5 (Ar), 126.2 (Py-C-5), 123.6 (Py-C-3), 76.1 (C-5′), 72.5, 72.3 (C-1′, C-3′), 68.4 (C-4′), 67.9 (C-2′), 62.3 (C-6′). ESI-HRMS positive mode (m/z): calcd for C41H31N3NaO10+ [M+Na]+ 748.1902. Found: 748.1901.
Prepared from tetrazole (Viana et al., 2008) 5 (3.50 g, 9.77 mmol) and 2-picolinic acid (2.41 g, 19.58 mmol) according to general procedure II. Reaction time: 5 hours. Purification by column chromatography (1:1→2:1→3:1 EtOAc-hexane) yielded 0.57 g of white amorphous solid (13%). Rf=0.27 (4:1 EtOAc-hexane); [α]D=+6 (c 0.20, CHCl3). 1H NMR (400 MHz, CDCl3) δ (ppm): 8.79 (1H, d, J=4.4 Hz, Py-H-6), 8.26 (1H, d, J=7.9 Hz, Py-H-3), 7.90 (1H, dt, J=7.8, 1.4 Hz, Py-H-4), 7.49 (1H, m, Py-H-5), 6.35 (1H, d, J=2.2 Hz, H-1′), 5.69 (1H, dd, J=9.4, 2.2 Hz, H-2′), 5.36 (1H, ddd, J=9.4, 3.9, 2.3 Hz, H-3′), 4.35 (1H, dd, J=12.6, 2.3 Hz, H-4′a), 4.23 (1H, dd, J=12.6, 3.9 Hz, H-4′b), 2.22, 2.11, 2.10, 2.06 (4×3H, 4 s, CH3); 13C NMR (100 MHz, CDCl3) δ (ppm): 170.6, 169.7, 169.6 (2) (4×C═O), 164.5, 162.6 (OD-C-2, OD-C-5), 150.4 (Py-C-6), 143.1 (Py-C-2), 137.4 (Py-C-4), 126.3 (Py-C-5), 123.5 (Py-C-3), 68.7 (C-2′), 67.8 (C-3′), 64.4 (C-1′), 61.5 (C-4′), 20.8, 20.7, 20.5 (2) (4×CH3). ESI-HRMS positive mode (m/z): calcd for C19H22N3O9 [M+H]+ 436.1351; C19H21N3NaO9+ [M+Na]+ 458.1170. Found: [M+H]+ 436.1349; [M+Na]+ 458.1170.
Prepared from compound L-4 (0.60 g, 0.83 mmol) according to general procedure III. Purified by column chromatography (9:1 CHCl3: MeOH) to give 0.22 g white amorphous solid (87%). Rf=0.29 (8:2 CHCl3-MeOH); [α]D=+17 (c 0.20, MeOH). 1H NMR (360 MHz, CD3OD) δ (ppm): 8.75 (1H, ddd, J=4.9, 1.7, 0.9 Hz, Py-H-6), 8.25 (1H, dd, J=7.9, 1.1 Hz, Py-H-3), 8.07 (1H, dt, J=7.9, 1.7 Hz, Py-H-4), 7.64 (1H, ddd, J=7.9, 4.9, 1.1 Hz, Py-H-5), 4.68 (1H, d, J=9.9 Hz, H-1′), 3.90 (1H, dd, J=12.2, 1.9 Hz, H-6′a), 3.84 (1H, dd, J=9.9, 8.7 Hz, H-2′), 3.70 (1H, J=12.2, 5.4 Hz, H-6′b), 3.54 (1H, pt, J=8.9, 8.6 Hz, H-3′), 3.54-3.50 (1H, m, H-5′), 3.46 (1H, pt, J=9.3, 8.8 Hz, H-4′); 13C NMR (90 MHz, CD3OD) δ (ppm): 166.4, 165.7 (OD-C-2, OD-C-5), 151.4 (Py-C-6), 144.0 (Py-C.2), 139.3 (Py-C-4), 127.9 (Py-C-5), 124.5 (Py-C-3), 82.9 (C-5′), 79.1 (C-3′), 74.7 (C-1′), 73.4 (C-2′), 71.3 (C-4′), 62.8 (C-6′). ESI-HRMS positive mode (m/z): calcd for C13H15N3NaO6+ [M+Na]+ 332.0853. Found: 332.0844.
Prepared from compound L-6 (500 mg, 0.85 mmol) according to general procedure III. Purified by column chromatography (9:1 CHCl3-MeOH) to give 82 mg white amorphous solid (35%). Rf=0.27 (9:1 CHCl3-MeOH); [α]D=−44 (c 0.20, MeOH). 1H NMR (360 MHz, CD3OD) δ (ppm): 8.74 (1H, ddd, J=4.9, 1.6, 0.9 Hz, Py-H-6), 8.24 (1H, ddd, J=7.9, 1.1, 0.9 Hz, Py-H-3), 8.06 (1H, dt, J=7.9, 1.6 Hz, Py-H-4), 7.63 (1H, ddd, J=7.7, 4.9, 1.1 Hz, Py-H-5), 4.59 (1H, d, J=9.8 Hz, H-1′), 4.03 (1H, dd, J=11.1, 5.4 Hz, H-5′eq), 3.84 (1H, pt, J=9.8, 9.1 Hz, H-2′), 3.66 (1H, td, J=10.1, 9.1, 5.4 Hz, H-4′), 3.47 (1H, pt, J=9.1, 9.1 Hz, H-3′), 3.41 (1H, pt, J=11.1, 10.1 Hz, H-5′ax); 13C NMR (90 MHz, CD3OD) δ (ppm): 166.4, 165.7 (OD-C-2, OD-C-5), 151.4 (Py-C-6), 144.0 (Py-C-2), 139.3 (Py-C-4), 127.9 (Py-C-5), 124.5 (Py-C-3), 79.2 (C-3′), 75.4 (C-1′), 73.4 (C-2′), 71.7 (C-5′), 71.0 (C-4′). ESI-HRMS positive mode (m/z): calcd for C12H13N3NaO5+ [M+Na]+ 302.0747; C24H26N6NaO10+ [2M+Na]+ 581.1603. Found: [M+Na]+ 302.0747; [2M+Na]+ 581.1604.
Prepared from compound L-7 (350 mg, 0.73 mmol) according to general procedure III. Purified by column chromatography (7:2 CHCl3-MeOH) to give 177 mg white amorphous solid (78%). Rf=0.33 (7:2 CHCl3-MeOH); [α]D=+25 (c 0.21, MeOH). 1H NMR (400 MHz, CD3OD) δ (ppm): 8.77 (1H, d, J=4.8 Hz, Py-H-6), 8.27 (1H, d, J=7.9 Hz, Py-H-3), 8.08 (1H, dt, J=7.9, 1.5 Hz, Py-H-4), 7.65 (1H, m, Py-H-5), 4.63 (1H, d, J=9.8 Hz, H-1′), 4.22 (1H, pt, J=9.8, 9.6 Hz, H-2′), 4.01 (1H, d, J=3.2 Hz, H-4′), 3.84-3.73 (3H, m, H-5′, H-6′a, H-6′b), 3.67 (1H, dd, J=9.6, 3.2 Hz, H-3′); 13C NMR (90 MHz, CD3OD) δ (ppm): 166.5, 165.7 (OD-C-2, OD-C-5), 151.4 (Py-C-6), 144.1 (Py-C-2), 139.3 (Py-C-4), 127.9 (Py-C-5), 124.5 (Py-C-3), 81.7 (C-5′), 75.8 (C-3′), 75.1 (C-1′), 70.7 (C-4′), 70.2 (C-2′), 62.8 (C-6′). ESI-HRMS positive mode (m/z): calcd for C13H16N3O6+ [M+H]+ 310.1034; C13H15N3NaO6+ [M+Na]+ 332.0853; C26H30N6NaO12+ [2M+Na]+ 641.1814. Found: [M+H]+ 310.1035; [M+Na]+ 332.0852; [2M+Na]+ 641.1815.
Prepared from complex Ru-dimer (50 mg, 0.082 mmol), compound L-1a (86 mg, 0.181 mmol, 2.2 eq.) and TIPF6 (57 mg, 0.163 mmol) according to general procedure IV. Purified by column chromatography (9:1 CHCl3-MeOH) to give 143 mg (98%) yellow powder. Rf: 0.32 (9:1 CHCl3-MeOH). Diastereomeric ratio: 1:1. 1H NMR (400 MHz, CDCl3) δ (ppm): 9.25, 9.22 (2×1H, 2 d, J=5.5 Hz in each, 2×Py-H-6), 8.92, 8.79 (2×1H, 2 s, 2×Tria-H-5), 7.96 (2H, t, J=7.6 Hz, 2×Py-H-4), 7.89 (2H, d, J=7.8 Hz, 2×Py-H-3), 7.57-7.52 (2H, m, 2×Py-H-5), 6.00, 5.99 (2×1H, 2 d, J=9.4 Hz in each, 2×H-1′), 5.95-5.83, 5.73-5.67 (10H, m, 2×4×p-cym-CHAr, 2×H-2′), 5.47, 5.46 (2×1H, 2 pt, J=9.4, 9.2 Hz in each, 2×H-3′), 5.35, 5.33 (2×1H, 2 pt, J=9.7, 9.7 Hz in each, 2×H-4′), 4.38, 4.32 (2×1H, 2 dd, J=12.8, 4.7 Hz in each, 2×H-6′a), 4.26-4.15 (4H, m, 2×H-5′, 2×H-6′b), 2.79, 2.74 (2×1H, 2 hept, J=6.9 Hz in each, 2×i-Pr—CH), 2.22, 2.20 (2×3H, 2 s, 2×C6H4—CH3), 2.10, 2.09, 2.05, 1.95, 1.93 (24H, singlets, 2×4×COCH3), 1.17-1.12 (12H, m, 2×2×i-Pr—CH3); 13C NMR (90 MHz, CDCl3) δ (ppm): 170.8, 170.7, 170.0, 169.9, 169.6, 169.5, 169.4, 169.2 (2×4×C═O), 155.5, 155.4 (2×Py-C-6), 147.6, 147.5, 147.1, 146.7 (2×Tria-C-4, 2×Py-C-2), 140.3, 140.2 (2×Py-C-4), 127.1, 126.9 (2×Py-C-5), 125.4, 125.3 (2×Tria-C-5), 122.9 (2) (2×Py-C-3), 106.3, 105.5, 103.1, 101.8 (2×2×p-cym-CqAr), 86.8, 86.7 (2×C-1′), 86.4, 85.4, 85.3, 85.0, 84.8, 83.9, 83.9, 83.1 (2×4×p-cym-CHAr), 75.5, 75.3 (2×C-5′), 73.1, 73.0 (2×C-3′), 70.2, 69.8 (2×C-2′), 67.6, 67.5 (2×C-4′), 61.6, 61.5 (2×C-6′), 31.1, 31.0 (2×i- Pr—CH), 22.5, 22.2, 22.1, 21.7 (2×2×i-Pr—CH3), 20.8-20.3 (2×4×COCH3), 18.7 (2) (2×C6H4—CH3). ESI-HRMS positive mode (m/z): calcd for C31H38ClN4O9Ru+ [M−PF6]+ 747.1371. Found: 747.1370.
Prepared from complex Ru-dimer (50 mg, 0.082 mmol), compound L-1b (95 mg, 0.180 mmol, 2.2 eq.) and TIPF6 (57 mg, 0.163 mmol) according to general procedure IV. Purified by column chromatography (9:1 CHCl3-MeOH) to give 145 mg (96%) yellow powder. Rf: 0.55 (9:1 CHCl3-MeOH). Diastereomeric ratio: 2:1. 1H NMR (400 MHz, CDCl3) δ (ppm): 9.24 (s, minor Tria-H-5), 9.03 (s, major Tria-H-5), 8.75 (d, J=8.8 Hz, major Qu-H-8), 8.68 (d, J=8.7 Hz, minor Qu-H-8), 8.39 (d, J=8.6 Hz, major Qu-H-4), 8.37 (d, J=8.9 Hz, minor Qu-H-4), 8.04-7.89, 7.75-7.70 (2 m, minor and major Qu-H-3, Qu-H-5-Qu-H-7), 6.11 (d, J=9.4 Hz, minor H-1′), 6.08 (d, J=9.2 Hz, major H-1′), 6.03 (pt, J=9.2, 9.0 Hz, major H-2′), 5.97 (pt, J=9.4, 9.0 Hz, minor H-2′), 5.96, 5.95 (2 d, J=6.0 Hz in each, major p-cym-CHAr) 5.88, 5.84 (2 d, J=6.0 Hz in each, minor p-cym-CHAr) 5.83-5.80 (m, minor and major p-cym-CHAr), 5.78 (d, J=6.0 Hz, minor p-cym-CHAr), 5.67 (d, J=5.9 Hz, major p-cym-CHAr), 5.52 (pt, J=9.2, 9.2 Hz, major H-3′), 5.49 (pt, J=9.5, 9.4 Hz, minor H-3′), 5.39 (pt, J=10.2, 9.7 Hz, minor H-4′), 5.34 (pt, J=10.0, 9.7 Hz, major H-4′), 4.44 (dd, J=12.7, 4.7 Hz, minor H-6′a), 4.33 (dd, J=12.7, 5.1 Hz, major H-6′a), 4.32-4.17 (m, minor and major H-5′, H-6′b), 2.57 (hept, J=6.9 Hz, major i-Pr—CH), 2.53 (hept, J=6.9 Hz, minor i-Pr—CH), 2.15 (s, minor C6H4—CH3), 2.13, 2.09 (singlets, COCH3), 2.07 (s, major C6H4—CH3), 2.06, 2.02, 1.96 (singlets, COCH3), 1.07, 1.04 (2 d, J=6.9 Hz in each, 2×major i-Pr—CH3), 1.02, 1.00 (2 d, J=6.9 Hz in each, 2×minor i-Pr—CH3); 13C NMR (90 MHz, CDCl3) δ (ppm): 170.9, 170.0, 169.6, 169.4 (minor 4×C═O), 170.8, 170.1, 169.8, 169.7 (major 4×C═O), 149.6, 148.4, 147.5 (major Tria-C-4, Qu-C-2, Qu-C-8a), 149.2, 148.4, 148.9 (minor Tria-C-4, Qu-C-2, Qu-C-8a), 141.3 (minor Qu-C-4), 141.0 (major Qu-C-4), 133.1, 129.6, 129.2, 129.1, 129.1 (minor Qu-C-4a, Qu-C-5-Qu-C-8), 132.7, 129.5, 129.1, 129.1, 129.0 (major Qu-C-4a, Qu-C-5-Qu-C-8), 127.6 (major Tria-C-5), 127.5 (minor Tria-C-5), 119.1 (major Qu-C-3), 118.8 (minor Qu-C-3), 106.3, 102.1 (minor p-cym-CqAr), 105.4, 102.7 (major p-cym-CqAr), 88.1, 86.7, 86.5, 86.4, 85.7, 85.1, 84.6, 84.5, 84.3, 84.0 (minor and major p-cym-CHAr, minor and major C-1′), 75.6 (minor C-5′), 75.2 (major C-5′), 73.2 (minor C-3′), 73.0 (major C-3′), 70.2 (major C-2′), 69.8 (minor C-2′), 67.5 (major C-4′), 67.7 (minor C-4′), 61.6 (major C-6′), 61.5 (minor C-6′), 31.3 (minor i-Pr—CH), 31.1 (major i-Pr—CH), 22.7, 21.6 (major i-Pr—CH3), 22.4, 21.8 (minor i-Pr—CH3), 20.8, 20.7, 20.7, 20.6, 20.4 (2×4×COCH3), 18.7 (minor C6H4—CH3), 18.5 (major C6H4—CH3). ESI-HRMS positive mode (m/z): calcd for C35H40ClN4O9Ru+ [M−PF6]+ 797.1528. Found: 797.1531.
Prepared from complex Ru-dimer (10.0 mg, 0.0163 mmol), compound L-2a (24.9 mg, 0.0344 mmol, 2.1 eq.) and TIPF6 (11.4 mg, 0.0326 mmol, 2 eq.) according to general procedure IV. Purified by column chromatography (95:5 CHCl3-MeOH) to give 34.6 mg (93%) orange powder. Rf: 0.46 (95:5 CHCl3-MeOH). Diastereomeric ratio: 1:1. 1H NMR (400 MHz, CDCl3) δ (ppm): 9.23, 9.20 (2×1H, 2 d, J=5.6 Hz in each, 2×Py-H-6), 9.02, 8.98 (2×1H, 2 s, 2×Tria-H-5), 8.11-7.75, 7.62-7.24 (46H, m, 2×20×Ar, 2×Py-H-3-Py-H-5), 6.60, 6.10 (2×1H, 2 pt, J=9.4, 9.3 Hz in each, 2×H-2′), 6.50, 6.43 (2×1H, 2 d, J=9.3 Hz in each, 2×H-1′), 6.22, 6.15 (2×1H, 2 pt, J=9.6, 9.4 Hz in each, 2×H-3′), 5.97, 5.94 (2×1H, 2 pt, J=9.7, 9.6 Hz in each, 2×H-4′), 5.81, 5.65 (2), 5.57 (2), 5.53, 5.47, 5.44 (2×4H, 2×4 d, J=6.1 Hz in each, 2×4×p-cym-CHAr), 4.78-4.52 (2×3H, m, 2×H-5′, 2×H-6′a, 2×H-6′b), 2.50, 2.36 (2×1H, 2 hept, J=6.9 Hz in each, 2×i-Pr—CH), 2.08, 2.00 (2×3H, 2 s, 2×C6H4—CH3), 0.94, 0.91, 0.79, 0.74 (2×2×3H, 2×2 d, J=6.9 Hz in each 2×2×i-Pr—CH3); 13C NMR (100 MHz, CDCl3) δ (ppm): 166.3, 166.2, 165.6, 165.5, 165.2 (2), 164.9, 164.8 (2×4×C═O), 155.7, 155.5 (2×Py-C-6), 147.3, 147.2, 147.0, 146.6 (2×Tria-C-4, 2×Py-C-2), 140.1, 140.0 (2×Py-C-4), 134.4, 134.2, 133.9, 133.8, 133.7 (2), 133.6, 133.5, 133.3, 130.3-128.0 (Ar), 127.2, 126.9 (2×Py-C-5), 126.2, 123.8 (2×Tria-C-5), 122.7 (2×Py-C-3), 105.8, 105.3, 103.9, 102.6 (2×2×p-cym-CqAr), 87.2, 86.7 (2×C-1′), 86.4, 85.8, 85.2, 85.0, 84.0, 83.9, 83.2, 82.5 (2×4×p-cym-CHAr), 76.1, 75.6 (2×C-5′), 73.6, 72.7 (2×C-3′), 71.7, 70.4 (2×C-2′), 68.7, 68.6 (2×C-4′), 62.8, 62.7 (2×C-6′), 31.0 (2) (2×i-Pr—CH), 22.5, 22.3, 21.7, 21.4 (2×2×i-Pr—CH3), 18.8, 18.6 (2×C6H4—CH3). ESI-HRMS positive mode (m/z): calcd for C51H46ClN4O9Ru+ [M−PF6]+ 995.2002. Found: 995.1994.
Prepared from complex Ru-dimer (50.0 mg, 0.082 mmol), compound L-3a (50.4 mg, 0.163 mmol, 2 eq.) and TIPF6 (57.0 mg, 0.163 mmol) according to general procedure V. Yield: 107 mg (90%). An analytically pure sample was obtained by recrystallisation from iPrOH to give 20 mg orange powder. Diastereomeric ratio: 1:1. 1H NMR (400 MHz, CD3OD) δ (ppm): 9.42 (2H, d, J=5.4 Hz, 2×Py-H-6), 9.22, 9.21 (2×1H, 2 s, 2×Tria-H-5), 8.19 (2H, pt, J=7.8, 7.7 Hz, 2×Py-H-4), 8.10 (2H, d, J=7.8 Hz, 2×Py-H-3), 7.69-7.66 (2H, m, 2×Py-H-5), 6.12-6.05 (4H, m, 2×p-cym-CHAr), 5.90-5.84 (4H, m, 2×p-cym-CHAr), 5.88 (2H, d, 2×H-1′), 3.95-3.89 (4H, m, 2×H-2′, 2×H-6′a), 3.77 (2H, dd, J=12.1, 5.3 Hz, 2×H-6′b), 3.69 (2H, ddd, J=9.5, 5.3, 1.9 Hz, 2×H-5′), 3.64 (2H, pt, J=9.5, 8.9 Hz, 2×H-3′), 3.56 (2H, pt, J=9.3, 9.1 Hz, 2×H-4′), 2.79-2.71 (2H, hept, J=6.7 Hz, 2×i-Pr—CH), 2.22 (6H, s, 2×C6H4—CH3), 1.18-1.09 (12H, m, 2×2×i-Pr—CH3); 13C NMR (100 MHz, CD3OD) δ (ppm): 156.8 (2) (2×Py-C-6), 149.6 (2), 148.2, 148.1 (2×Py-C-2, 2×Tria-C-4), 141.5 (2) (2×Py-C-4), 127.6 (2) (2×Py-C-5), 125.5, 125.2 (2×Tria-C-5), 123.6 (2) (2×Py-C-3), 106.8, 106.6, 104.1, 103.8 (2×2×p-cym-CqAr), 91.3, 91.2 (2×C-1′), 87.3, 87.2, 86.3, 86.1, 85.6, 85.5, 84.8, 84.6 (2×4×p-cym-CHAr), 81.7, 81.6 (2×C-5′), 78.2, 78.1 (2×C-3′), 74.5, 74.3 (2×C-2′), 70.8, 70.7 (2×C-4′), 62.3, 62.2 (2×C-6′), 32.3, 32.2 (2×i-Pr—CH), 22.6 (2), 22.0, 21.9 (2×2×i-Pr—CH3), 18.8, 18.7 (2×C6H4—CH3). ESI-HRMS positive mode (m/z): calcd for C23H30ClN4O5Ru+ [M−PF6]+ 579.0946. Found: 579.0946.
Prepared from complex Ru-dimer (50.0 mg, 0.082 mmol), compound L-3b (58.8 mg, 0.164 mmol, 2 eq.) and TIPF6 (57 mg, 0.163 mmol) according to general procedure V. Yield: 121 mg (96%). An analytically pure sample was obtained by recrystallisation from iPrOH to give 34 mg orange powder. Diastereomeric ratio: 1:1. 1H NMR (400 MHz, CD3OD) δ (ppm): 9.42, 9.41 (2×1H, 2 s, 2×Tria-H-5), 8.80 (2H, d, J=8.8 Hz, 2×Qu-H-8), 8.70, 8.68 (2×1H, 2 d, J=8.5 Hz in each, 2×Qu-H-4), 8.15-8.09, 7.88-7.84 (2×4H, m, Qu-H-3, Qu-H-5-Qu-H-7), 6.13-5.96 (2×4H, m, 2×p-cym-CHAr), 5.95, 5.93 (2×1H, 2 d, J=9.1 Hz in each, 2×H-1′), 4.00, 3.99 (2×1H, 2 pt, J=9.2, 9.1 Hz in each, 2×H-2′), 3.97, 3.95 (2×1H, 2 dd, J=12.1, 2.1 Hz in each, 2×H-6′a), 3.80 (2H, dd, J=12.1, 5.3 Hz, 2×H-6′b), 3.74, 3.71 (2×1H, ddd, J=9.3, 5.3, 2.1 Hz in each, 2×H-5′), 3.68, 3.67 (2×1H, 2 pt, J=9.0, 8.9 Hz in each, 2×H-3′), 3.60, 3.59 (2×1H, 2 pt, J=9.3, 9.2 Hz in each, 2×H-4′), 2.48, 2.46 (2×1H, 2 hept, J=6.9 Hz in each, 2×i-Pr—CH), 2.20 (6H, s, 2×C6H4—CH3), 1.00, 0.98, 0.92 (2) (2×2×3H, 4 d, J=6.9 Hz in each, 2×2×i-Pr—CH3); 13C NMR (90 MHz, CD3OD) δ (ppm): 151.0 (2), 149.7 (2), 149.1 (2), (2×Tria-C-4, 2×Qu-C-2, 2×Qu-C-8a), 142.6 (2×Qu-C-4), 134.0, 130.7, 130.6 (3), 130.5, 130.1 (2×Qu-C-4a, 2×Qu-C-5-Qu-C-8), 127.1, 126.9 (2×Tria-C-5), 119.5, 119.4 (2×Qu-C-3), 106.5, 106.3, 104.9, 104.7 (2×2×p-cym-CqAr), 91.4, 91.2 (2×C-1′), 88.6, 88.5, 86.5, 86.3, 86.2, 85.8, 84.6, 84.3 (2×4×p-cym-CHAr), 81.8, 81.7 (2×C-5′), 78.2, 78.1 (2×C-3′), 74.4, 74.3 (2×C-2′), 70.7 (2) (2×C-4′), 62.3, 62.20 (2×C-6′), 32.3, 32.2 (2×i-Pr—CH), 22.7, 22.6, 21.6, 21.5 (2×2×i-Pr—CH3), 18.8, 18.7 (2×C6H4—CH3). ESI-ESI-HRMS positive mode (m/z): calcd for C27H32ClN4O5Ru+ [M−PF6]+: 629.1103. Found: 629.1103.
Prepared from complex Ru-dimer (10.0 mg, 0.016 mmol), compound L-4 (24.9 mg, 0.034 mmol, 2.1 eq.) and TIPF6 (11.4 mg, 0.033 mmol) according to general procedure IV. After filtration and removal of the solvent the residue was dissolved in CHCl3 (3 mL) and Et2O (6 mL) was added. The precipitated product was filtered off to give 28.4 mg (76%) yellow powder. The product can also be obtained by column chromatographic purification (9:1 CHCl3-MeOH), albeit in lower yield (52%). Rf: 0.38 (9:1 CHCl3-MeOH). Diastereomeric ratio: 2:1. 1H NMR (400 MHz, CDCl3) δ (ppm): 9.50 (s, minor Py-H-6), 9.26 (s, major Py-H-6), 8.18-7.70, 7.60-7.28 (m, minor and major Ar, Py-H-3-Py-H-5), 6.19 (pt, J=9.8, 9.7 Hz, minor H-3′), 6.16 (pt, J=9.8, 9.5 Hz, major H-2′), 6.08 (pt, J=9.5, 9.5 Hz, major H-3′), 5.88 (pt, J=9.7, 9.5 Hz, major H-4′), 5.86 (pt, J=9.7, 9.6 Hz, minor H-4′), 5.90-5.84 (m, 2×minor p-cym-CHAr), 5.76-5.72 (m, major p-cym-CHAr, minor H-2′), 5.68-5.65 (m, 2×minor p-cym-CHAr), 5.53-5.51 (m, major p-cym-CHAr), 5.46 (d, J=9.8 Hz, major H-1′), 5.35 (d, J=9.8 Hz, minor H-1′), 5.31-5.29 (m, 2×major p-cym-CHAr), 4.74-4.68 (m, minor and major H-6′a), 4.57-4.52 (m, minor and major H-6′b), 4.50-4.42 (m, minor and major H-5′), 2.78 (hept, J=6.8 Hz, major i-Pr—CH), 2.72 (hept, J=6.8 Hz, minor i-Pr—CH), 2.02 (s, minor C6H4—CH3), 1.95 (s, major C6H4—CH3), 1.16, 1.15, 1.12, 1.08 (4 d, J=6.8 Hz in each, minor and major 2×i-Pr—CH3); 13C NMR (90 MHz, CDCl3) δ (ppm): 166.2, 165.8, 165.7, 165.7, 165.3, 165.2, 165.2, 164.9, 164.8, 164.7 (minor and major C═O, OD-C-2, OD-C-5), 158.3 (minor Py-C-6), 156.6 (major Py-C-6), 140.3 (minor Py-C-4), 140.2 (major Py-C-2), 140.1 (major Py-C-4), 139.1 (minor Py-C-2), 134.6, 134.0, 133.9, 133.8, 133.7, 133.5, 133.4, 131.1, 130.6, 130.2-128.5, 127.8 (minor and major Ar, Py-C-5), 125.3 (minor Py-C-3), 125.2 (major Py-C-3), 107.0, 102.3 (minor p-cym-CqAr), 104.8, 102.1 (major p-cym-CqAr), 88.7, 84.6, 83.2 (2) (major p-cym-CHAr), 85.5, 85.2, 83.8, 83.6 (minor p-cym-CHAr), 77.8 (minor C-5′), 77.4 (major C-5′), 73.8, 71.5, 69.8 (major C-1′-C-3′), 72.8, 71.9, 71.4 (minor C-1′-C-3′), 68.8 (major C-4′), 68.7 (minor C-4′), 63.0 (minor C-6′), 62.6 (major C-6′), 31.3 (minor i-Pr—CH), 31.1 (major i-Pr—CH), 23.1, 21.3 (major i-Pr—CH3), 22.2, 22.1 (minor i-Pr—CH3), 18.8 (minor C6H4—CH3), 18.1 (major C6H4—CH3). ESI-HRMS positive mode (m/z): calcd for C51H45ClN3O10Ru+ [M−PF6]+ 996.1843; C53H52N3O12Ru+ [M−PF6−Cl+OMe+MeOH]+ 1024.2604. Found: [M−PF6]+ 996.1861; [M−PF6−Cl+OMe+MeOH]+ 1024.2598.
Prepared from complex Ru-dimer (10.0 mg, 0.0163 mmol), compound L-5 (17.9 mg, 0.0375 mmol, 2.3 eq.) and TIPF6 (11.4 mg, 0.0326 mmol) according to general procedure IV. Purified by column chromatography (9:1 CHCl3-MeOH) to give 21.7 mg (74%) yellow powder. Rf: 0.54 (9:1 CHCl3-MeOH). Diastereomeric ratio: 4:3. 1H NMR (400 MHz, CDCl3) δ (ppm): 9.45 (d, J=3.9 Hz, minor Py-H-6), 9.30 (d, J=4.4 Hz, major Py-H-6), 8.23-8.08 (m, minor and major Py-H-3, Py-H-4), 7.88 (m, minor Py-H-5), 7.80 (m, major Py-H-5), 6.01-5.96, 5.82-5.69 (m, minor and major p-cym-CHAr), 5.75 (pt, J=9.7, 9.6 Hz, major H-2′), 5.44, (pt, J=9.3, 9.2 Hz, minor H-2′), 5.40 (pt, J=9.4, 9.0 Hz, minor and major H-3′), 5.24 (pt, J=9.8, 9.2 Hz, minor H-4′), 5.23 (pt, J=9.8, 9.8 Hz, major H-4′), 5.03 (d, J=10.0 Hz, major H-1′), 5.00 (d, J=8.7 Hz, minor H-1′), 4.30 (dd, J=12.8, 4.8 Hz, minor and major H-6′a), 4.17 (dd, J=12.8, 2.4 Hz, minor and major H-6′b), 3.99 (ddd, J=10.2, 4.8, 2.4 Hz, minor and major H-5′), 3.03 (hept, J=6.8 Hz, major i-Pr—CH), 2.91 (hept, J=6.8 Hz, minor i-Pr—CH), 2.23, 2.17, 2.10-2.02 (singlets, minor and major C6H4—CH3, COCH3), 1.36 (d, J=6.8 Hz, major 2×i-Pr—CH3), 1.30, 1.25 (2 d, J=6.8 Hz in each, minor 2×i-Pr—CH3); 13C NMR (90 MHz, CDCl3) δ (ppm): 170.7, 170.6, 170.1, 170.0, 169.8, 169.6, 169.4 (2×4×C═O), 164.9, 164.6 (major OD-C-2, OD-C-5), 164.9, 164.7 (minor OD-C-2, OD-C-5), 157.6 (minor Py-C-6), 156.4 (major Py-C-6), 140.5 (minor Py-C-4), 140.3 (major Py-C-4), 140.3, 139.5 (minor and major Py-C-2), 130.8 (minor Py-C-5), 130.0 (major Py-C-5), 125.5 (minor Py-C-3), 125.4 (major Py-C-3), 106.4 (minor p-cym-CqAr), 105.0 (major p-cym-CqAr), 102.7 (minor p-cym-CqAr), 102.4 (major p-cym-CqAr), 88.2, 86.0, 85.6, 83.5, 83.2, 83.1 (minor and major p-cym-CHAr), 77.2 (minor C-5′), 77.0 (major C-5′), 73.7, 70.9 (major C-1′ and C-3′), 73.0, 71.2, 70.0 (minor C-1′-C-3′), 68.8 (major C-2′), 67.7 (minor C-4′), 67.6 (major C-4′), 61.9 (minor C-6′), 61.7 (major C-6′), 31.3 (minor i-Pr—CH), 31.2 (major i-Pr—CH), 22.9, 21.7 (major i-Pr—CH3), 22.4, 22.0 (minor i-Pr—CH3), 20.8, 20.7, 20.6, 20.6, 20.5 (2×4×COCH3), 18.8 (minor C6H4—CH3), 18.3 (major C6H4—CH3). ESI-HRMS positive mode (m/z): calcd for C31H37ClN3O10Ru+ [M−PF6]+ 748.1211. Found: 748.1212.
Prepared from complex Ru-dimer (50 mg, 0.082 mmol), compound L-6 (97 mg, 0.164 mmol, 2 eq.) and TIPF6 (57 mg, 0.163 mmol) according to general procedure IV. Purified by column chromatography (9:1 CHCl3-MeOH) to give 140 mg (85%) yellow powder. Rf: 0.67 (9:1 CHCl3-MeOH). Diastereomeric ratio: 3:2. 1H NMR (400 MHz, CDCl3) δ (ppm): 9.46 (d, J=4.1 Hz, minor Py-H-6), 9.26 (d, J=4.2 Hz, major Py-H-6), 8.18-7.73, 7.60-7.32 (m, minor and major Ar, Py-H-3-Py-H-5), 6.11 (pt, J=9.1, 9.1, major H-2′), 6.06-6.02 (m, major and minor H-3′), 5.86 (d, J=5.4 Hz, minor p-cym-CHAr), 5.80-5.76 (m, minor and major p-cym-CHAr), 5.74 (pt, J=9.3, 9.1 Hz, minor H-2′), 5.65, 5.63 (2 d, J=6.1 Hz in each, minor p-cym-CHAr), 5.61-5.53 (m, major and minor H-4′), 5.53 (d, J=5.4 Hz, major p-cym-CHAr), 5.37-5.35 (m, major p-cym-CHAr), 5.28 (d, J=9.1 Hz, major H-1′), 5.24 (d, J=9.1 Hz, minor H-1′), 4.64 (dd, J=11.8, 5.1 Hz, minor H-5′eq), 4.60 (dd, J=11.8, 5.4 Hz, major H-5′eq), 3.87 (pt, J=10.9, 10.1, minor and major H-5′ax), 2.80 (hept, J=6.9 Hz, major i-Pr—CH), 2.75 (hept, J=6.9 Hz, minor i-Pr—CH), 2.04 (s, minor C6H4—CH3), 1.99 (s, major C6H4—CH3), 1.19-1.11 (m, minor and major 2×i-Pr—CH3); 13C NMR (90 MHz, CDCl3) δ (ppm): 165.7, 165.6 (2), 165.5, 165.4, 165.3, 165.2, 164.9, 164.7, 164.6 (minor and major C═O, OD-C-2, OD-C-5), 157.9 (minor Py-C-6), 157.0 (major Py-C-6), 140.4 (minor Py-C-4), 140.3 (major Py-C-4), 140.0 (major Py-C-2), 139.3 (minor Py-C-2), 134.4, 133.9, 133.8 (2), 133.7, 133.5, 130.8-128.0 (minor and major Ar, Py-C-5), 125.2 (minor Py-C-3), 125.1 (major Py-C-3), 106.3, 104.9, 102.4, 102.1 (minor and major p-cym-CqAr), 88.4, 86.0, 85.1, 84.3, 83.5, 83.4, 83.3, 83.1 (minor and major p-cym-CHAr), 73.1, 72.1, 71.9, 71.8, 70.5, 69.7, 69.5, 69.0 (minor and major C-1′-C-4′), 67.8, 67.4 (minor and major C-5′), 31.2, 31.0 (minor and major i-Pr—CH), 23.1, 22.4, 21.8, 21.1 (minor and major i-Pr—CH3), 18.6, 18.1 (minor and major C6H4—CH3). ESI-HRMS positive mode (m/z): calcd for C43H39ClN3O8Ru+ [M−PF6]+ 862.1473; C45H46N3O10Ru+ [M−PF6−Cl+OMe+MeOH]+ 890.2234. Found: [M−PF6]+ 862.1470; [M−PF6−Cl+OMe+MeOH]+ 890.2231.
Prepared from complex Ru-dimer (50 mg, 0.082 mmol), compound L-7 (78 mg, 0.163 mmol, 2 eq.) and TIPF6 (57 mg, 0.163 mmol) according to general procedure IV. Purified by column chromatography (9:1 CHCl3-MeOH) to give 108 mg (74%) yellow powder. Rf: 0.54 (9:1 CHCl3-MeOH). Diastereomeric ratio: 2:1. 1H NMR (400 MHz, CDCl3) δ (ppm): 9.46 (d, J=4.4 Hz, minor Py-H-6), 9.34 (d, J=4.4 Hz, major Py-H-6), 8.24-8.10 (m, minor and major Py-H-4, Py-H-3), 7.87 (m, minor Py-H-5), 7.80 (m, major Py-H-5), 6.01-5.95 (m, minor and major p-cym-CHAr), 5.85 (pt, J=10.2, 10.0 Hz major H-2′), 5.82-5.71 (m, minor and major p-cym-CHAr), 5.57-5.55 (m, minor and major H-4′), 5.47 (pt, J=10.0, 10.0 Hz minor H-2′), 5.29 (dd, J=10.0, 3.2 Hz, minor H-3′), 5.25 (dd, J=10.0, 3.2 Hz, major H-3′), 5.01 (d, J=10.2 Hz, major H-1′), 4.98 (d, J=10.0 Hz, minor H-1′), 4.23-4.10 (m, minor and major H-5′, H-6′a, H-6′b), 3.01 (hept, J=6.8 Hz, major i-Pr—CH), 2.89 (hept, J=6.8 Hz, minor i-Pr—CH), 2.24-2.23, 2.17, 2.07-2.04 (singlets, minor and major C6H4—CH3, COCH3), 1.34 (d, J=6.8 Hz, 2×major i-Pr—CH3), 1.28, 1.24 (2 d, J=6.8 Hz in each, 2×minor i-Pr—CH3); 13C NMR (90 MHz, CDCl3) δ (ppm): 170.3, 170.2, 170.1, 170.0, 169.8, 169.7 (2×4×C═O), 164.8, 164.7, 164.5 (2) (minor and major OD-C-2, OD-C-5), 157.4 (minor Py-C-6), 156.5 (major Py-C-6), 140.3 (minor Py-C-4), 140.1 (major Py-C-4), 139.9 (major Py-C-2), 139.2 (minor Py-C-2), 130.5 (minor Py-C-5), 129.9 (major Py-C-5), 125.3 (minor Py-C-3), 125.2 (major Py-C-3), 106.2, 102.6 (minor p-cym-CqAr), 105.1, 102.2 (major p-cym-CqAr), 87.6, 85.0, 83.1, 82.9 (major p-cym-CHAr), 85.7, 85.2, 83.3, 83.0 (minor p-cym-CHAr), 75.6 (minor C-5′), 75.4 (major C-5′), 71.4, 71.0, 67.0 (major C-1′, C-3′, C-4′), 71.3, 70.7, 67.1, 66.9 (minor C-1′-C-4′), 65.8 (major C-2′), 61.4 (minor C-6′), 61.3 (major C-6′), 31.1 (minor i-Pr—CH), 31.0 (major i-Pr—CH), 22.6, 21.4 (major i-Pr—CH3), 22.2, 21.8 (minor i-Pr—CH3), 20.6, 20.6, 20.5, 20.5, 20.4, 20.4 (2×4×COCH3), 18.5 (minor C6H4—CH3), 18.1 (major C6H4—CH3). ESI-HRMS positive mode (m/z): calcd for C33H44N3O12Ru+ [M−PF6−Cl+OMe+MeOH]+ 776.1973. Found: 776.1973.
Prepared from complex Ru-dimer (10.0 mg, 0.0163 mmol), compound L-8 (24.9 mg, 0.0343 mmol, 2.1 eq.) and TIPF6 (11.4 mg, 0.0326 mmol) according to general procedure IV. Purified by column chromatography (95:5 CHCl3-MeOH) to give 30.3 mg (81%) yellow powder. Rf: 0.49 (95:5 CHCl3-MeOH). Diastereomeric ratio: 5:4. 1H NMR (400 MHz, CDCl3) δ (ppm): 9.49 (d, J=4.7 Hz, minor Py-H-6), 9.26 (d, J=5.1 Hz, major Py-H-6), 8.21-7.25 (m, minor and major Ar, Py-H-3-Py-H-5), 6.43 (pt, J=10.1, 10.0 Hz, major H-2′), 6.20 (d, J=3.3 Hz, major H-4′), 6.16 (d, J=2.6 Hz, minor H-4′), 6.02-5.94 (m, minor H-2′ and H-3′), 5.84-5.75 (m, minor and major p-cym-CHAr, major H-3′), 5.67, 5.64 (2 d, J=6.1 Hz in each, minor p-cym-CHAr) 5.52 (d, J=6.1 Hz, major p-cym-CHAr), 5.47 (d, J=10.1 Hz, major H-1′), 5.37 (d, J=8.7 Hz, minor H-1′), 5.32, 5.27 (2 d, J=6.1 Hz in each, major p-cym-CHAr), 4.72-4.62 (m, minor and major H-5′, H-6′a), 4.53 (dd, J=10.3, 4.1 Hz, minor H-6′b), 4.44 (dd, J=10.3, 4.0 Hz, major H-6′b), 2.80 (hept, J=6.9 Hz, major i-Pr—CH), 2.73 (hept, J=6.9 Hz, minor i-Pr—CH), 2.03 (s, minor C6H4—CH3), 1.94 (s, major C6H4—CH3), 1.17, 1.15 (2 d, J=6.9 Hz in each, 2×major i-Pr—CH3), 1.14, 1.09 (2 d, J=6.9 Hz in each, 2×minor i-Pr—CH3); 13C NMR (100 MHz, CDCl3) δ (ppm): 166.2, 166.1, 165.9, 165.7, 165.5 (2), 165.4, 165.3, 164.9, 164.8 (2), 164.7 (2×4×C═O, 2×OD-C-2, 2×OD-C-5), 157.9 (minor Py-C-6), 156.3 (major Py-C-6), 140.4 (major Py-C-2), 140.3 (minor Py-C-4), 140.2 (major Py-C-4), 139.3 (minor Py-C-2), 134.5, 134.2, 133.9, 133.8 (2), 133.6, 133.5 (2), 130.9-128.1 (minor and major Ar, Py-C-5), 125.3 (minor Py-C-3), 125.2 (major Py-C-3), 106.8, 104.7, 102.3, 102.0 (minor and major p-cym-CqAr), 88.8, 85.4 (2), 84.5, 83.6, 83.5, 83.0 (2) (minor and major p-cym-CHAr), 76.7 (minor C-5′), 76.3 (major C-5′), 72.5 (major C-3′), 72.2 (minor C-1′), 71.7 (major C-1′), 71.3, 68.7 (minor C-2′, C-3′), 68.3 (major C-4′), 68.2 (minor C-4′), 66.8 (major C-2′), 62.4 (minor C-6′), 62.1 (major C-6′), 31.3 (minor i-Pr—CH), 31.1 (major i-Pr—CH), 23.2, 21.3 (major i-Pr—CH3), 22.2, 22.0 (minor i-Pr—CH3), 18.7 (minor C6H4—CH3), 18.0 (major C6H4—CH3). ESI-HRMS positive mode (m/z): calcd for C51H45ClN3O10Ru+ [M−PF6]+ 996.1843. Found: 996.1842.
Prepared from complex Ru-dimer (50 mg, 0.082 mmol), compound L-9 (78 mg, 0.179 mmol, 2.2 eq.) and TIPF6 (57 mg, 0.163 mmol) according to general procedure IV. Purified by column chromatography (9:1 CHCl3-MeOH) to give 130 mg (94%) yellow powder. Rf: 0.54 (9:1 CHCl3-MeOH). Diastereomeric ratio: 1:1. 1H NMR (400 MHz, CDCl3) δ (ppm): 9.33 (2H, d, J=4.2 Hz, 2×Py-H-6), 8.15 (2H, t, J=7.4 Hz, 2×Py-H-4), 8.07 (2H, d, J=7.4 Hz, 2×Py-H-3), 7.79 (2H, m, 2×Py-H-5), 6.52, 6.46 (2×1H, 2 d, J=2.3 Hz in each, 2×H-1′), 6.01-5.95 (4H, m, 2×p-cym-CHAr), 5.84-5.71 (6H, m, 2×p-cym-CHAr, 2×H-2′), 5.34, 5.30 (2×1H, 2 ddd, J=8.9, 4.1, 2.9 Hz in each, 2×H-3′), 4.36, 4.35 (2×1H, 2 dd, J=12.5, 2.9 Hz in each, 2×H-4′a), 4.21, 4.20 (2×1H, 2 dd, J=12.5, 4.1 in each, 2×H-4′b), 2.98, 2.97 (2×1H, 2 hept, J=6.9 Hz in each, 2×i-Pr—CH), 2.29, 2.24, 2.21, 2.18, 2.13, 2.12, 2.11, 2.10, 2.08, 2.08 (30H, singlets, 2×i-Pr—CH3, 2×4×COCH3), 1.33-1.29 (12H, m, 2×2×i-Pr—CH3); 13C NMR (90 MHz, CDCl3) δ (ppm): 170.7, 170.6, 169.9, 169.8, 169.6, 169.5, 169.4, 169.1 (2×4×C═O), 165.2, 165.1, 164.5 (2) (2×OD-C-2, 2×OD-C-5), 156.9, 156.8 (2×Py-C-6), 140.4 (2) (2×Py-C-4), 139.8, 139.7 (2×Py-C-2), 130.3 (2) (2×Py-C-5), 125.4, 125.3 (2×Py-C-3), 105.9, 105.7, 102.4, 102.3 (2×2×p-cym-CqAr), 87.2, 86.6, 85.4, 85.1, 83.5 (2), 83.4, 83.3 (2×4×p-cym-CHAr), 68.9, 68.7 (2×C-2′), 68.0, 67.8 (2×C-3′), 64.3, 63.7 (2×C-1′), 61.5 (2) (2×C-4′), 31.3 (2) (2×i-Pr—CH), 22.8, 22.6, 21.9, 21.8 (2×2×i-Pr—CH3), 21.0, 20.9 (2), 20.8, 20.7, 20.6, 20.5 (2) (2×4×COCH3), 18.5, 18.4 (2×C6H4—CH3). ESI-HRMS positive mode (m/z): calcd for C29H35ClN3O9Ru+ [M−PF6]+ 706.1105; C31H42N3O11Ru+ [M−PF6−Cl+OMe+MeOH]+ 734.1866. Found: [M−PF6]+ 706.1106; [M−PF6−Cl+OMe+MeOH]+ 734.1868.
Prepared from complex Ru-dimer (39 mg, 0.064 mmol), compound L-10 (39 mg, 0.126 mmol, 2 eq.) and TIPF6 (44 mg, 0.126 mmol) according to general procedure V. The crude product was triturated with Et2O to give 80 mg (87%) orange powder. Diastereomeric ratio: 1:1. 1H NMR (400 MHz, CD3OD) δ (ppm): 9.54 (2H, d, J=5.5 Hz, 2×Py-H-6), 8.40-8.36 (4H, m, 2×Py-H-3, 2×Py-H-4), 7.98-7.93 (2H, m, 2×Py-H-5), 6.19-6.13 (2×2H, m, 2×p-cym-CHAr), 5.94-5.92 (2×2H, m, 2×p-cym-CHAr), 4.85, 4.83 (2×1H, 2 d, J=9.8 Hz in each, 2×H-1′), 3.92, 3.91 (2×1H, 2 dd, J=12.1, 6.1 Hz in each, 2×H-6′a), 3.83, 3.81 (2×1H, 2 pt, J=9.5, 9.3 Hz in each, 2×H-2′), 3.71, 3.70 (2×1H, 2 dd, J=12.1, <1 Hz in each, 2×H-6′b), 3.59-3.54 (4H, m, 2×H-3′, 2×H-5′), 3.45, 3.45 (2×1H, 2 pt, J=9.8, 9.5 Hz in each, 2×H-4′), 2.91, 2.90 (2×1H, 2 hept, J=6.9 Hz in each, 2×i-Pr—CH), 2.23 (6H, s, 2×C6H4—CH3), 1.28-1.23 (12H, m, 2×2×i-Pr—CH3); 13C NMR (90 MHz, CD3OD) δ (ppm): 168.5 (2), 166.0 (2) (2×OD-C-2, 2×OD-C-5), 158.3, 158.3 (2×Py-C-6), 142.0 (2) (2×Py-C-4), 141.6, 141.5 (2×Py-C-2), 131.1 (2) (2×Py-C-5), 126.6, 126.5 (2×Py-C-3), 107.3, 107.2, 103.5, 103.4 (2×2×p-cym-CqAr), 87.4, 87.2, 85.9, 85.8, 85.1, 85.0, 84.9, 84.8 (2×4×p-cym-CHAr), 83.2, 83.1 (2×C-5′), 78.8, 78.7 (2×C-3′), 74.6 (2) (2×C-1′), 73.3, 73.2 (2×C-2′), 71.1 (2) (2×C-4′), 62.6 (2) (2×C-6′), 32.4 (2) (2×i-Pr—CH), 22.8, 22.7, 22.1, 22.0 (2×2×i-Pr—CH3), 18.8 (2) (2×C6H4—CH3). ESI-HRMS positive mode (m/z): calcd for C23H29ClN3O6Ru+ [M−PF6]+ 580.0786; C25H36N3O8Ru+ [M−PF6−Cl+OMe+MeOH]+ 608.1548. Found: [M−—PF6]+ 580.0787; [M−PF6−Cl+OMe+MeOH]+ 608.1545.
Prepared from complex Ru-dimer (10.0 mg, 0.016 mmol), compound L-11 (9.1 mg, 0.033 mmol, 2 eq.) and TIPF6 (11.4 mg, 0.033 mmol) according to general procedure V. Purified by recrystallisation from an iPrOH-Et2O solvent mixture (2 mL and 5 mL, respectively) to give 9.5 mg (42%) yellow powder. Diastereomeric ratio: 1:1. 1H NMR (400 MHz, CD3OD) δ (ppm): 9.53 (2H, d, J=5.4 Hz, 2×Py-H-6), 8.38-8.34 (4H, m, 2×Py-H-3, 2×Py-H-4), 7.95-7.92 (2H, m, 2×Py-H-5), 6.18-6.12 (2×2H, m, 2×p-cym-CHAr), 5.94-5.90 (2×2H, m, 2×p-cym-CHAr), 4.77, 4.76 (2×1H, 2 d, J=9.6 Hz in each, 2×H-1′), 4.07, 4.06 (2×1H, 2 dd, J=11.1, 5.3 Hz in each, 2×H-5′eq), 3.81, 3.80 (2×1H, 2 pt, J=9.6, 9.0 Hz in each, 2×H-2′), 3.68, 3.65 (2×1H, 2 ddd, J=10.5, 8.9, 5.3 Hz in each, 2×H-4′), 3.51 (2H, pt, J=9.0, 8.9 Hz, 2×H-3′), 3.47, 3.46 (2×1H, 2 pt, J=11.1, 10.5 Hz in each, 2×H-5′ax), 2.91 (2H, 2 hept, J=6.8 Hz, 2×i-Pr—CH), 2.22 (6H, s, 2×C6H4—CH3), 1.28-1.23 (12H, m, 2×2×i-Pr—CH3); 13C NMR (100 MHz, CD3OD) δ (ppm): 168.8, 168.6, 166.1, 166.0 (2×OD-C-2, 2×OD-C-5), 158.2 (2) (2×Py-C-6), 142.0 (2) (2×Py-C-4), 141.6, 141.5 (2×Py-C-2), 131.1 (2) (2×Py-C-5), 126.6, 126.5 (2×Py-C-3), 107.4 (2), 103.5, 103.4 (2×2×p-cym-CqAr), 87.4, 87.3, 86.0, 85.9, 85.0 (2), 84.9, 84.8 (2×4×p-cym-CHAr), 79.0, 78.9 (2×C-3′), 75.4, 75.3 (2×C-1′), 73.5, 73.3, (2×C-2′), 71.8, 71.7 (2×C-5′), 70.8 (2) (2×C-4′), 32.4 (2) (2×i-Pr—CH), 22.7 (2), 22.0 (2) (2×2×i-Pr—CH3), 18.7 (2) (2×C6H4—CH3). ESI-HRMS positive mode (m/z): calcd for C22H27ClN3O5Ru+ [M−PF6]+ 550.0680; C24H34N3O7Ru+ [M−PF6−Cl+OMe+MeOH]+ 578.1442. Found: [M−PF6]+ 550.0672; [M−PF6−Cl+OMe+MeOH]+ 578.1432.
Prepared from complex Ru-dimer (10.0 mg, 0.016 mmol), compound L-12 (10.1 mg, 0.033 mmol, 2 eq.) and TIPF6 (11.4 mg, 0.033 mmol) according to general procedure V. Purified by recrystallisation from an iPrOH-Et2O solvent mixture (2 mL and 5 mL, respectively) to give 11.9 mg (50%) yellow powder. Diastereomeric ratio: 1:1. 1H NMR (360 MHz, CD3OD) δ (ppm): 9.55 (2H, d, J=5.6 Hz, 2×Py-H-6), 8.40-8.35 (4H, m, 2×Py-H-3, 2×Py-H-4), 7.96-7.93 (2H, m, 2×Py-H-5), 6.19-6.13 (2×2H, m, 2×p-cym-CHAr), 5.94-5.92 (2×2H, m, 2×p-cym-CHAr), 4.78, 4.76 (2×1H, 2 d, J=9.8 Hz in each, 2×H-1′), 4.18, 4.15 (2×1H, 2 pt, J=9.8, 9.4 in each, 2×H-2′), 4.01 (2H, d, J=3.2 Hz, 2×H-4′), 3.84-3.73 (6H, m, 2×H-5′, 2×H-6′a, 2×H-6′b), 3.69, 3.68 (2×1H, 2 dd, J=9.4, 3.2 in each, 2×H-3′), 2.91 (2H, 2 hept, J=6.9 Hz, 2×i-Pr—CH), 2.23 (6H, s, 2×C6H4—CH3), 1.27-1.23 (12H, m, 2×2×i-Pr—CH3); 13C NMR (90 MHz, CD3OD) δ (ppm): 168.7, 168.6, 166.0, 165.9 (2×OD-C-2, 2×OD-C-5), 158.1 (2) (2×Py-C-6), 141.9 (2) (2×Py-C-4), 141.6, 141.5 (2×Py-C-2), 131.1 (2) (2×Py-C-5), 126.5, 126.4 (2×Py-C-3), 107.4, 107.3, 103.5, 103.4 (2×2×p-cym-CqAr), 87.2, 87.1, 86.0, 85.8, 85.0 (2), 84.8, 84.7 (2×4×p-cym-CHAr), 82.0, 81.9 (2×C-5′), 75.6, 75.5, 75.1, 75.0 (2×C-1′, 2×C-3′), 70.6, 70.5, 70.2, 70.0 (2×C-2′, 2×C-4′), 62.7 (2) (2×C-6′), 32.4 (2) (2×i-Pr—CH), 22.7, 22.6, 22.1, 22.0 (2×2×i-Pr—CH3), 18.7 (2) (2×C6H4—CH3). ESI-HRMS positive mode (m/z): calcd for C23H29ClN3O6Ru+ [M−PF6]+ 580.0786; C25H36N3O8Ru+ [M−PF6−Cl+OMe+MeOH]+ 608.1548. Found: [M−PF6]+ 580.0792; [M−PF6−Cl+OMe+MeOH]+ 608.1553.
Prepared from complex Ru-dimer (20.0 mg, 0.033 mmol), 1-phenyl-4-(pyridine-2-yl)-1,2,3-triazole (Tawfiq et al., 2014) (L-13, 14.5 mg, 0.065 mmol, 2 eq.) and TIPF6 (22.8 mg, 0.065 mmol) according to general procedure V. Purified by recrystallisation from a CHCl3-Et2O solvent mixture (3 mL and 6 mL, respectively) to give 36.8 mg (88%) yellow powder. Racemic mixture. 1H NMR (400 MHz, acetone-d6) δ (ppm): 9.62 (1H, s, Tria-H-5), 9.57 (1H, ddd, J=5.6, 1.4, 0.9 Hz, Py-H-6), 8.29 (1H, dt, J=7.6, 1.4 Hz, Py-H-4), 8.25 (1H, ddd, J=7.9, 1.9, 0.9 Hz, Py-H-3), 8.12-8.09 (2H, m, Ph), 7.79-7.68 (4H, m, Ph, Py-H-5), 6.27, 6.24, 6.04, 5.99 (4×1H, 4 d, J=6.2 Hz in each, 4×p-cym-CHAr), 2.91 (1H, hept, J=6.9 Hz, i-Pr—CH), 2.29 (3H, s, C6H4—CH3), 1.23, 1.19 (2×3H, 2 d, J=6.9 Hz in each, 2×i-Pr—CH3); 13C NMR (90 MHz, acatone-d6) δ (ppm): 156.6 (Py-C-6), 149.2, 148.2 (Tria-C-4, Py-C-2), 141.1 (Py-C-4), 131.6, 131.2, 127.3, 124.0, 123.3, 121.9 (Ph, Py-C-3, Py-C-5, Tria-C-5), 106.2, 103.4 (2×p-cym-CqAr), 87.4, 85.8, 85.1, 84.5 (4×p-cym-CHAr), 31.9 (i-Pr—CH), 22.6, 21.9 (2×i-Pr—CH3), 18.7 (C6H4—CH3). ESI-HRMS positive mode (m/z): calcd for C23H24ClN4Ru+ [M−PF6]+: 493.0730. Found: 493.0709.
Prepared from complex Ru-dimer (20.0 mg, 0.033 mmol), 2-phenyl-5-(pyridine-2-yl)-1,3,4-oxadiazole (Wei et al., 2010; Weiss et al., 2020) (L-14, 14.6 mg, 0.065 mmol, 2 eq.) and TIPF6 (22.8 mg, 0.065 mmol) according to general procedure V. Purified by recrystallisation from a CHCl3-Et2O solvent mixture (3 mL and 6 mL, respectively) to give 34.9 mg (84%) yellow powder. Racemic mixture. 1H NMR (360 MHz, acatone-d6) δ (ppm): 9.69 (1H, ddd, J=5.6, 1.3, 0.8 Hz, Py-H-6), 8.54 (1H, ddd, J=7.8, 1.6, 0.8 Hz, Py-H-3), 8.48 (1H, dt, J=7.8, 1.3 Hz, Py-H-4), 8.33-8.29 (2H, m, Ph), 8.01 (1H, ddd, J=7.8, 5.6, 1.6 Hz, Py-H-5), 7.85-7.72 (3H, m, Ph), 6.30-6.27, 6.07-6.04 (2×2H, 2 m, 4×p-cym-CHAr), 3.03 (1H, hept, J=6.9 Hz, i-Pr—CH), 2.31 (3H, s, C6H4—CH3), 1.32, 1.30 (2×3H, 2 d, J=6.9 Hz in each, 2×i-Pr—CH3); 13C NMR (90 MHz, acatone-d6) δ (ppm): 168.2, 164.7 (OD-C-2, OD-C-5), 157.8 (Py-C-6), 141.6 (Py-C-4), 141.5 (Py-C-2), 134.9, 130.7, 130.3, 128.5, 125.9, 122.7 (Ph, Py-C-3, Py-C-5), 106.7, 102.9 (2×p-cym-CqAr), 86.9, 85.6, 84.7, 84.5 (4×p-cym-CHAr), 32.0 (i-Pr—CH), 22.7, 22.0 (2×i-Pr—CH3), 18.8 (C6H4—CH3). ESI-HRMS positive mode (m/z): calcd for C23H23ClN3ORu+ [M−PF6]+: 494.0556. Found: 494.0553.
Prepared from complex Os-dimer (20.0 mg, 0.0253 mmol), compound L-2a (36.7 mg, 0.0506 mmol, 2.0 eq.) and TIPF6 (17.6 mg, 0.0504 mmol) according to general procedure IV. After filtration and removal of the solvent the residue was dissolved in CHCl3 (5 mL) and Et2O (10 mL) was added. The precipitated product was filtered off, washed with a solvent mixture of CHCl3-Et2O=1:1 (2 mL) to give 52.9 mg (85%) yellow powder. Rf: 0.57 (9:1 CHCl3-MeOH). Diastereomeric ratio: 1:1. 1H NMR (400 MHz, CDCl3) δ (ppm): 9.19 (2H, d, J=5.3 Hz, 2×Py-H-6), 9.07, 9.05 (2×1H, 2 s, 2×Tria-H-5), 8.05-7.75, 7.53-7.23 (46H, m, 2×20×Ar, 2×Py-H-3-Py-H-5), 6.58, 6.24-5.60 (14H, m, 2×H-2′, 2×H-3′, 2×H-4′, 2×4×p-cym-CHAr), 6.49, 6.43 (2×1H, 2 d, J=9.2 and 9.0 Hz, respectively, 2×H-1′), 4.79-4.53 (6H, m, 2×H-5′, 2×H-6′a, 2×H-6′b), 2.35, 2.16 (2×1H, 2 hept, J=6.9 Hz in each, 2×i-Pr—CH), 2.14, 2.07 (2×3H, 2 s, 2×C6H4—CH3), 0.89, 0.86, 0.75, 0.63 (2×2×3H, 2×2 d, J=6.9 Hz in each, 2×2×i-Pr—CH3); 13C NMR (100 MHz, CDCl3) δ (ppm): 166.3, 166.2, 165.6, 165.5, 165.2 (2), 164.9, 164.8 (2×4×C═O), 155.8, 155.7 (2×Py-C-6), 148.4, 148.1, 148.0, 147.9 (2×Tria-C-4, 2×Py-C-2), 140.3, 140.2 (2×Py-C-4), 134.3, 134.2, 133.9, 133.8, 133.7, 133.6, 133.5, 133.3, 130.2-128.4, 128.0, 127.9, 127.7, 127.6 (Ar, 2×Py-C-5, 2×Tria-C-5), 122.5, 122.4 (2×Py-C-3), 97.0, 96.4, 96.1, 95.2 (2×2×p-cym-CqAr), 87.2, 86.7 (2×C-1′), 78.2, 77.7, 76.9, 76.8, 76.1, 75.7, 74.9, 74.5, 73.7, 73.6, 72.8, 72.7, 71.7, 70.4, 68.7, 68.6 (2×4×p-cym-CHAr, 2×C-1′-C-5′), 62.8, 62.7 (2×C-6′), 31.1 (2) (2×i-Pr—CH), 22.8, 22.6, 21.9, 21.5 (2×2×i-Pr—CH3), 18.6, 18.5 (2×C6H4—CH3). ESI-HRMS positive mode (m/z): calcd for C51H46ClN4O9Os+ [M−PF6]+ 1085.2566. Found: 1085.2555.
Prepared from complex Os-dimer (10.0 mg, 0.0126 mmol), compound L-4 (18.4 mg, 0.0254 mmol, 2.0 eq.) and TIPF6 (8.8 mg, 0.0252 mmol) according to general procedure IV. After filtration and removal of the solvent the residue was dissolved in CHCl3 (3 mL) and Et2O (12 mL) was added. The precipitated product was filtered off, washed with a solvent mixture of CHCl3-Et2O=1:2 (1 mL) to give 22.1 mg (71%) orange powder. Rf: 0.44 (95:5 CHCl3-MeOH). Diastereomeric ratio: 7:6. 1H NMR (400 MHz, CDCl3) δ (ppm): 9.45 (d, J=5.6 Hz, major Py-H-6), 9.18 (d, J=5.6 Hz, minor Py-H-6), 8.18-7.28 (m, minor and major Ar, Py-H-3-Py-H-5), 6.21-6.05, 5.90-5.84, 5.77-5.72 (m, J=9.8, 9.7 Hz, minor and major H-2′, H-3′, H-4′, 4×major p-cym-CHAr, 2×minor p-cym-CHAr), 5.57, 5.51 (2 d, J=5.7 Hz in each, 2×minor p-cym-CHAr), 5.46 (d, J=9.8 Hz, minor H-1′), 5.35 (d, J=9.9 Hz, major H-1′), 4.73, 4.72 (2 dd, J=12.6, 2.6 Hz in each, minor and major H-6′a), 4.56, 4.55 (2 dd, J=12.6, 5.6 Hz in each, minor and major H-6′b), 4.48, 4.45 (2 ddd, J=9.9, 5.6, 2.6 Hz in each, minor and major H-5′), 2.63 (hept, J=6.9 Hz, minor i-Pr—CH), 2.57 (hept, J=6.9 Hz, major i-Pr—CH), 2.09 (s, major C6H4—CH3), 2.01 (s, minor C6H4—CH3), 1.14, 1.10, 1.05, 1.04 (4 d, J=6.9 Hz in each, minor and major 2×i-Pr—CH3); 13C NMR (100 MHz, CDCl3) δ (ppm): 169.1, 168.8, 166.3, 165.8, 165.7, 165.3, 165.2 (2), 164.9, 164.8 (minor and major C═O, OD-C-2, OD-C-5), 158.3 (minor Py-C-6), 156.5 (major Py-C-6), 140.3 (major Py-C-4), 140.1 (minor Py-C-2), 140.1 (minor Py-C-4), 139.0 (major Py-C-2), 134.6, 134.0 (2), 133.8, 133.7, 133.5 (2), 133.4, 131.8-127.7 (minor and major Ar, Py-C-5), 125.3 (2) (minor and major Py-C-3), 98.2, 94.9 (major p-cym-CqAr), 95.5, 93.7 (minor p-cym-CqAr), 81.0, 77.8, 77.7, 77.5, 77.4, 76.7, 75.4, 74.9, 74.8, 74.4, 73.8, 72.8, 71.8, 71.4 (2), 69.7, 68.8, 68.7 (minor and major p-cym-CHAr, C-1′-C-5′), 63.0 (major C-6′), 62.6 (minor C-6′), 31.4 (major i-Pr—CH), 31.1 (minor i-Pr—CH), 23.5, 21.3 (minor i-Pr—CH3), 22.5, 22.2 (major i-Pr—CH3), 18.7 (major C6H4—CH3), 18.0 (minor C6H4—CH3). ESI-HRMS positive mode (m/z): calcd for C51H45ClN3O10Os+ [M−PF6]+ 1086.2406. Found: [M−PF6]+ 1086.2391.
Prepared from complex Os-dimer (10.0 mg, 0.0126 mmol), compound L-6 (15.0 mg, 0.0254 mmol, 2.0 eq.) and TIPF6 (8.8 mg, 0.0252 mmol) according to general procedure IV. After filtration and removal of the solvent the residue was dissolved in CHCl3 (3 mL) and Et2O (12 mL) was added. The precipitated product was filtered off, washed with a solvent mixture of CHCl3-Et2O=1:2 (2 mL) to give 23.3 mg (84%) orange powder. Rf: 0.34 (95:5 CHCl3-MeOH). Diastereomeric ratio: 7:5. 1H NMR (400 MHz, CDCl3) δ (ppm): 9.40 (d, J=5.6 Hz, major Py-H-6), 9.22 (d, J=5.6 Hz, minor Py-H-6), 8.17-7.32 (m, minor and major Ar, Py-H-3-Py-H-5), 6.11 (pt, J=9.0, 9.0 Hz, major H-2′ or H-3′), 6.08-6.03 (m, major and minor H-2′ or H-3′ and p-cym-CHAr), 5.91 (d, J=5.8 Hz, minor p-cym-CHAr), 5.86, 5.84 (2 d, J=6.2 Hz in each, 2×major p-cym-CHAr), 5.76 (pt, J=9.1, 9.0 Hz, minor H-2′ or H-3′), 5.75 (m, major p-cym-CHAr), 5.64 (d, J=5.8 Hz, minor p-cym-CHAr), 5.62-5.51 (m, major and minor H-4′, minor p-cym-CHAr), 5.30 (d, J=9.7 Hz, minor H-1′), 5.25 (d, J=9.0 Hz, major H-1′), 4.66 (dd, J=11.7, 5.2 Hz, major H-5′eq), 4.60 (dd, J=11.4, 5.5 Hz, minor H-5′eq), 3.88 (pt, J=11.4, 9.7 Hz, major H-5′ax), 3.87 (pt, J=11.4, 10.4 Hz, minor H-5′ax), 2.63 (hept, J=6.9 Hz, minor i-Pr—CH), 2.60 (hept, J=6.9 Hz, major i-Pr—CH), 2.11 (s, major C6H4—CH3), 2.04 (s, minor C6H4—CH3), 1.14, 1.12, 1.06, 1.05 (4 d, J=6.9 Hz in each, minor and major 2×i-Pr—CH3); 13C NMR (100 MHz, CDCl3) δ (ppm): 167.0, 168.8, 165.8, 165.7 (2), 165.6, 165.5, 165.4, 165.3, 165.1 (minor and major C═O, OD-C-2, OD-C-5), 158.0 (major Py-C-6), 156.7 (minor Py-C-6), 140.3 (major Py-C-4), 140.2 (minor Py-C-4), 140.0 (minor Py-C-2), 139.2 (major Py-C-2), 134.5, 134.0, 133.9 (2), 133.8, 133.6, 131.5-128.0 (minor and major Ar, Py-C—S), 125.3 (2) (minor and major Py-C-3), 97.7, 94.9 (major p-cym-CqAr), 95.7, 93.9 (minor p-cym-CqAr), 80.7, 78.3, 77.3, 76.7, 75.2, 74.9, 74.8, 74.4, 73.0, 72.1, 71.9, 71.8, 70.7, 69.7, 69.6, 69.0 (minor and major C-1′-C-4′), 67.9 (minor C-5′), 67.5 (major C-5′), 31.3 (major i-Pr—CH), 31.1 (minor i-Pr—CH), 23.4, 21.4 (minor i-Pr—CH3), 22.7, 22.1 (major i-Pr—CH3), 18.6 (major C6H4—CH3), 18.0 (minor C6H4—CH3). ESI-HRMS positive mode (m/z): calcd for C43H39ClN3O8Os+ [M−PF6]+ 952.2037. Found: [M−PF6]+ 952.2025.
Prepared from complex Os-dimer (20.0 mg, 0.0253 mmol), compound L-8 (36.7 mg, 0.0506 mmol, 2.0 eq.) and TIPF6 (17.6 mg, 0.0504 mmol) according to general procedure IV. After filtration and removal of the solvent the residue was dissolved in CHCl3 (5 mL) and Et2O (10 mL) was added. The precipitated product was filtered off, washed with a solvent mixture of CHCl3-Et2O=1:2 (2 mL) to give 48.4 mg (78%) orange powder. Rf: 0.69 (9:1 CHCl3-MeOH). Diastereomeric ratio: 3:2. 1H NMR (400 MHz, CDCl3) δ (ppm): 9.43 (d, J=5.6 Hz, major Py-H-6), 9.22 (d, J=5.6 Hz, minor Py-H-6), 8.21-7.24 (m, minor and major Ar, Py-H-3-Py-H-5), 6.42 (pt, J=10.1, 10.0 Hz, minor H-2′), 6.20 (d, J=3.4 Hz, minor H-4′), 6.17 (d, J=2.6 Hz, major H-4′), 6.06, 6.04 (2 d, J=5.8 Hz in each, 2×major p-cym-CHAr), 6.03-5.95 (m, major H-2′ and H-3′), 5.88-5.84 (m, minor and 2×major p-cym-CHAr), 5.80 (dd, J=10.1, 3.4 Hz, minor H-3′), 5.76, 5.56, 5.53 (3 d, J=5.8 Hz in each, 3×minor p-cym-CHAr), 5.48 (d, J=10.2 Hz, minor H-1′), 5.39 (d, J=9.7 Hz, major H-1′), 4.73-4.63 (m, minor and major H-5′, H-6′a), 4.54 (dd, J=10.3, 4.1 Hz, major H-6′b), 4.48 (m, minor H-6′b), 2.63 (hept, J=6.9 Hz, minor i-Pr—CH), 2.57 (hept, J=6.9 Hz, major i-Pr—CH), 2.10 (s, major C6H4—CH3), 2.00 (s, minor C6H4—CH3), 1.13, 1.06 (2 d, J=6.9 Hz in each, 2×minor i-Pr—CH3), 1.10, 1.04 (2 d, J=6.9 Hz in each, 2×major i-Pr—CH3); 13C N M R (100 MHz, CDCl3) δ (ppm): 169.0, 168.8, 166.2, 166.1, 165.9, 165.7, 165.6, 165.5, 165.4, 165.3, 165.0, 164.9 (minor and major C═O, OD-C-2, OD-C-5), 158.1 (major Py-C-6), 156.6 (minor Py-C-6), 140.4 (major Py-C-4), 140.2 (minor Py-C-4), 140.1 (minor Py-C-2), 139.1 (major Py-C-2), 134.5, 134.2, 133.9, 133.8 (2), 133.6, 133.5 (2), 131.6-128.0 (minor and major Ar, Py-C-5), 125.4 (major Py-C-3), 125.3 (minor Py-C-3), 97.9, 94.9 (major p-cym-CqAr), 95.6, 93.7 (minor p-cym-CqAr), 80.9, 78.0, 77.3, 76.7, 76.6, 76.3, 75.3, 74.9 (2), 74.5, 72.4, 72.1, 71.7, 71.3, 68.7, 68.4, 68.3, 66.8 (minor and major p-cym-CHAr, C-1′-C-5′), 62.4 (major C-6′), 62.2 (minor C-6′), 31.3 (major i-Pr—CH), 31.0 (minor i-Pr—CH), 23.5, 21.3 (minor i-Pr—CH3), 22.6, 22.1 (major i-Pr—CH3), 18.6 (major C6H4—CH3), 17.9 (minor C6H4—CH3). ESI-HRMS positive mode (m/z): calcd for C51H45ClN3O10Os+ [M−PF6]+ 1086.2406. Found: [M−PF6]+ 1086.2390.
Prepared from complex Ir-dimer (20.0 mg, 0.0251 mmol), compound L-2a (36.4 mg, 0.0502 mmol, 2.0 eq.) and TIPF6 (17.5 mg, 0.0501 mmol) according to general procedure IV. After filtration and removal of the solvent the residue was dissolved in CHCl3 (5 mL) and Et2O (10 mL) was added. The precipitated product was filtered off, washed with a solvent mixture of CHCl3-Et2O=1:1 mixture (2 mL) to give 51.9 mg (84%) yellow powder. Rf: 0.58 (9:1 CHCl3-MeOH). Diastereomeric ratio: 2:1. 1H NMR (400 MHz, CDCl3) δ (ppm): 9.20 (s, minor Tria-H-5), 9.13 (s, major Tria-H-5), 8.68 (1 signal, minor and major Py-H-6), 8.05-7.82, 7.58-7.23 (m, minor and major Ar, Py-H-3-Py-H-5), 6.53 (d, J=9.3 Hz, major H-1′), 6.48 (d, J=8.5 Hz, minor H-1′), 6.46 (pt, J=9.4, 9.2 Hz, major H-2′ or H-3′ or H-4′), 6.22 (pt, J=9.2, 9.2 Hz, minor H-2′ or H-3′ or H-4′), 6.19 (pt, J=9.2, 8.7 Hz, minor H-2′ or H-3′ or H-4′), 6.13 (pt, J=9.5, 9.5 Hz, major H-2′ or H-3′ or H-4′), 5.98 (pt, J=9.5, 9.5 Hz, minor H-2′ or H-3′ or H-4′), 5.92 (pt, J=9.9, 9.7 Hz, major H-2′ or H-3′ or H-4′), 4.78-4.54 (minor and major H-5′, H-6′a, H-6′b), 1.62 (s, major Cp*—CH3), 1.57 (s, minor Cp*—CH3); 13C NMR (100 MHz, CDCl3) δ (ppm): 166.3, 165.6, 165.2, 164.6 (minor C═O), 166.2, 165.5, 165.3, 164.7 (major C═O), 151.5 (2) (minor and major Py-C-6), 148.5, 147.8 (minor Tria-C-4, Py-C-2), 148.1, 147.9 (major Tria-C-4, Py-C-2), 140.6 (minor Py-C-4), 140.5 (major Py-C-4), 134.1, 134.0, 133.8, 133.7, 133.6, 133.5, 133.4, 133.3, 130.2-127.8 (minor and major Ar, Py-C-5, Tria-C-5), 123.0 (minor Py-C-3), 122.9 (major Py-C-3), 89.6 (major Cp*), 89.5 (minor Cp*), 87.3 (minor C-1′), 87.0 (major C-1′), 76.1, 72.9, 71.4, 68.7 (minor C-2′-C-5′), 75.8, 73.6, 70.3, 68.6 (major C-2′-C-5′), 62.8 (minor C-6′), 62.7 (major C-6′), 8.7 (minor and major Cp*—CH3). ESI-HRMS positive mode (m/z): calcd for C51H47ClN4O9Ir+ [M−PF6]+ 1087.2655. Found: [M−PF6]+ 1087.2647.
Prepared from complex Ir-dimer (20.0 mg, 0.0251 mmol), compound L-4 (36.4 mg, 0.0502 mmol, 2.0 eq.) and TIPF6 (17.5 mg, 0.0501 mmol) according to general procedure IV. Purified by column chromatography (95:5 CHCl3-MeOH) to give 49.3 mg (80%) yellow powder. Rf: 0.32 (95:5 CHCl3-MeOH). Diastereomeric ratio: 5:2. 1H NMR (400 MHz, CDCl3) δ (ppm): 8.95 (d, J=5.5 Hz, minor Py-H-6), 8.78 (d, J=5.5 Hz, major Py-H-6), 8.22-7.78, 7.57-7.26 (m, minor and major Ar, Py-H-3-Py-H-5), 6.32 (pt, J=9.7, 9.6 Hz, major H-2′ or H-3′ or H-4′), 6.19 (pt, J=9.7, 9.5 Hz, minor H-2′ or H-3′ or H-4′), 6.04 (pt, J=9.5, 9.5 Hz, major H-2′ or H-3′ or H-4′), 5.87 (pt, J=9.8, 9.7 Hz, minor H-2′ or H-3′ or H-4′), 5.86 (pt, J=9.8, 9.7 Hz, major H-2′ or H-3′ or H-4′), 5.78 (pt, J=9.7, 9.7 Hz, minor H-2′ or H-3′ or H-4′), 5.51 (d, J=10.2 Hz, major H-1′), 5.42 (d, J=9.9 Hz, minor H-1′), 4.72 (dd, J=12.8, 2.4 Hz, minor H-6′a), 4.67 (dd, J=12.6, 2.4 Hz, major H-6′a), 4.57 (dd, J=12.8, 5.6 Hz, minor H-6′b), 4.53 (dd, J=12.6, 4.3 Hz, major H-6′b), 4.47 (ddd, J=9.8, 4.3, 2.4 Hz, major H-5′), 4.46 (m, minor H-5′), 1.61 (s, minor Cp*—CH3), 1.50 (s, major Cp*—CH3); 13C NMR (90 MHz, CDCl3) δ (ppm): 169.4, 166.2, 165.8, 165.3, 165.2, 165.0 (major C═O, OD-C-2, OD-C-5), 169.2, 166.2, 165.8, 165.7, 165.6, 165.4 (minor C═O, OD-C-2, OD-C-5), 153.8 (minor Py-C-6), 152.5 (major Py-C-6), 140.8 (minor Py-C-4), 140.4 (major Py-C-4), 140.0 (major Py-C-2), 138.9 (minor Py-C-2), 134.4, 133.9, 133.8, 133.7, 133.6, 133.5, 133.4, 133.3, 132.0, 130.9, 130.5, 130.1-127.8 (minor and major Ar, Py-C-5), 125.8 (major Py-C-3), 125.7 (minor Py-C-3), 90.3 (minor Cp*), 90.0 (major Cp*), 77.7, 72.9, 71.8, 71.3, 68.8 (minor C-1′-C-5′), 77.3, 74.2, 71.2, 69.1, 68.7 (major C-1′-C-5′), 63.0 (minor C-6′), 62.6 (major C-6′), 8.8 (minor Cp*—CH3), 8.5 (major Cp*—CH3). ESI-HRMS positive mode (m/z): calcd for C53H53N3O12Ir+ [M−PF6−Cl+OMe+MeOH]+ 1116.3253. Found: [M−PF6−Cl+OMe+MeOH]+ 1116.3248.
Prepared from complex Ir-dimer (20.0 mg, 0.0251 mmol), compound L-6 (29.7 mg, 0.0502 mmol, 2.0 eq.) and TIPF6 (17.5 mg, 0.0501 mmol) according to general procedure IV. After filtration and removal of the solvent the residue was dissolved in CHCl3 (5 mL) and Et2O (10 mL) was added. The precipitated product was filtered off, washed with a solvent mixture of CHCl3-Et2O=1:2 (1 mL) to give 34.6 mg (72%) yellow powder. Rf: 0.44 (95:5 CHCl3-MeOH). Diastereomeric ratio: 3:1. 1H NMR (400 MHz, CDCl3) δ (ppm): 8.93 (d, J=5.5 Hz, minor Py-H-6), 8.80 (d, J=5.5 Hz, major Py-H-6), 8.23-7.80, 7.57-7.30 (m, minor and major Ar, Py-H-3-Py-H-5), 6.21 (pt, J=9.7, 9.5 Hz, major H-2′ or H-3′), 6.11 (pt, J=9.2, 9.2 Hz, minor H-2′ or H-3′), 6.00 (pt, J=9.6, 9.5 Hz, major H-2′ or H-3′), 5.78 (pt, J=9.2, 9.2 Hz, minor H-2′ or H-3′), 5.59 (ddd, J=10.1, 9.6, 5.4 Hz, minor H-4′), 5.51 (ddd, J=10.4, 9.9, 5.4 Hz, major H-4′), 5.33 (d, J=10.0 Hz, major H-1′), 5.29 (d, J=9.3 Hz, minor H-1′), 4.65 (dd, J=11.6, 5.4 Hz, minor H-5′eq), 4.57 (dd, J=11.3, 5.4 Hz, major H-5′eq), 3.87 (pt, J=11.3, 10.4 Hz, major H-5′ax), 3.86 (pt, J=11.6, 10.1 Hz, minor H-5′ax), 1.63 (s, minor Cp*—CH3), 1.52 (s, major Cp*—CH3); 13C NMR (90 MHz, CDCl3) δ (ppm): 169.4, 169.1, 165.8 (2), 165.7, 165.6 (3), 165.5, 165.1 (minor and major C═O, OD-C-2, OD-C-5), 153.5 (minor Py-C-6), 152.4 (major Py-C-6), 140.8 (minor Py-C-4), 140.5 (major Py-C-4), 140.1 (major Py-C-2), 139.2 (minor Py-C-2), 134.3, 133.8, 133.7, 133.5, 131.7, 130.8-128.1 (minor and major Ar, Py-C-5), 125.9 (major Py-C-3), 125.6 (minor Py-C-3), 90.3 (minor Cp*), 90.0 (major Cp*), 73.5, 71.7, 69.5, 69.0 (major C-1′-C-4′), 72.3, 72.2, 70.8, 69.2 (minor C-1′-C-4′), 67.8 (major C-5′), 67.7 (minor C-5′), 8.8 (minor Cp*—CH3), 8.6 (major Cp*—CH3). ESI-HRMS positive mode (m/z): calcd for C45H47ClN3O10Ir+ [M−PF6]+ 954.2128. Found: [M−PF6]+ 954.2121.
Prepared from complex Ir-dimer (20.0 mg, 0.0251 mmol), compound L-8 (36.7 mg, 0.0506 mmol, 2.0 eq.) and TIPF6 (17.5 mg, 0.0501 mmol) according to general procedure IV. After filtration and removal of the solvent the residue was dissolved in CHCl3 (5 mL) and Et2O (10 mL) was added. The precipitated product was filtered off, washed with a solvent mixture of CHCl3-Et2O=1:1 (2 mL) to give 47.5 mg (77%) yellow powder. Rf: 0.60 (9:1 CHCl3-MeOH). Diastereomeric ratio: 2:1. 1H NMR (400 MHz, CDCl3) δ (ppm): 8.94 (d, J=5.5 Hz, minor Py-H-6), 8.79 (d, J=5.5 Hz, major Py-H-6), 8.22-7.79, 7.69-7.24 (m, minor and major Ar, Py-H-3-Py-H-5), 6.54 (pt, J=10.2, 10.1 Hz, major H-2′), 6.19 (dd, J=3.3, 1.0 Hz, major H-4′), 6.17 (dd, J=3.3, 1.0 Hz, minor H-4′), 6.05 (pt, J=10.1, 9.8 Hz, minor H-2′), 5.94 (dd, J=10.1, 3.3 Hz, minor H-3′), 5.75 (dd, J=10.1, 3.3 Hz, major H-3′), 5.50 (d, J=10.2 Hz, major H-1′), 5.44 (d, J=9.8 Hz, minor H-1′), 4.74-4.42 (m, minor and major H-5′, H-6′a, H-6′b), 1.62 (s, minor Cp*—CH3), 1.51 (s, major Cp*—CH3); 13C NMR (100 MHz, CDCl3) δ (ppm): 169.4, 166.1, 165.7, 165.5, 165.3, 165.1 (major C═O, OD-C-2, OD-C-5), 169.2, 166.2, 165.7, 165.6, 165.5, 165.4 (minor C═O, OD-C-2, OD-C-5), 153.5 (minor Py-C-6), 152.3 (major Py-C-6), 140.8 (minor Py-C-4), 140.5 (major Py-C-4), 140.2 (major Py-C-2), 139.2 (minor Py-C-2), 134.3, 134.2, 133.8 (2), 133.7, 133.6, 133.5, 133.4, 131.7, 130.7-128.2 (minor and major Ar, Py-C-5), 125.9 (major Py-C-3), 125.7 (minor Py-C-3), 90.3 (minor Cp*), 90.0 (major Cp*), 76.6, 72.1, 71.5, 68.5, 68.2 (minor C-1′-C-5′), 76.2, 72.8, 71.4, 68.3, 66.1 (major C-1′-C-5′), 62.4 (minor C-6′), 62.2 (major C-6′), 8.8 (minor Cp*—CH3), 8.5 (major Cp*—CH3). ESI-HRMS positive mode (m/z): calcd for C51H46ClN3O10Ir+ [M−PF6]+ 1088.2491. Found: [M−PF6]+ 1088.2492.
Prepared from complex Rh-dimer (20.0 mg, 0.0324 mmol), compound L-2a (46.9 mg, 0.0647 mmol, 2.0 eq.) and TIPF6 (22.6 mg, 0.0647 mmol) according to general procedure IV. After filtration and removal of the solvent the residue was dissolved in CHCl3 (6 mL) and Et2O (12 mL) was added. The precipitated product was filtered off, washed with a solvent mixture of CHCl3-Et2O=1:1 (2 mL) to give 67.5 mg (91%) orange powder. Rf: 0.33 (9:1 CHCl3-MeOH). Diastereomeric ratio: 2:1. 1H NMR (400 MHz, CDCl3) δ (ppm): 9.06 (s, minor Tria-H-5), 9.03 (s, major Tria-H-5), 8.67 (1 signal, minor and major Py-H-6), 8.04-7.82, 7.60-7.22 (m, minor and major Ar, Py-H-3-Py-H-5), 6.53-6.46, 6.25-6.11, 5.99-5.90 (minor and major H-1′-H-4′), 4.77-4.52 (minor and major H-5′, H-6′a, H-6′b), 1.62 (s, major Cp*—CH3), 1.55 (s, minor Cp*—CH3); 13C NMR (100 MHz, CDCl3) δ (ppm): 166.3, 165.6, 165.2, 164.6 (minor C═O), 166.2, 165.5, 165.2, 164.7 (major C═O), 151.5 (2) (minor and major Py-C-6), 146.9, 146.8, 146.2 (2) (minor and major Tria-C-4, Py-C-2), 140.4 (minor Py-C-4), 140.2 (major Py-C-4), 134.3, 134.1, 133.9, 133.8, 133.7, 133.5, 133.4, 133.3, 130.2-128.5, 127.9, 127.8, 127.5, 127.4 (minor and major Ar, Py-C-5, Tria-C-5), 122.9 (minor Py-C-3), 122.8 (major Py-C-3), 97.5 (minor Cp*), 97.4 (major Cp*), 87.3 (minor C-1′), 86.8 (major C-1′), 76.1, 72.8, 71.6, 68.7 (minor C-2′-C-5′), 75.7, 73.7, 70.3, 68.6 (major C-2′-C-5′), 62.9 (minor C-6′), 62.7 (major C-6′), 9.0 (minor and major Cp*—CH3). ESI-HRMS positive mode (m/z): calcd for C51H47ClN4O9Rh+ [M−PF6]+ 997.2081. Found: [M−PF6]+ 997.2068. 11.40 Complex Rh-4 Prepared from complex Rh-dimer (10.0 mg, 0.0162 mmol), compound L-4 (23.5 mg, 0.0324 mmol, 2.0 eq.) and TIPF6 (11.3 mg, 0.0323 mmol) according to general procedure IV. After filtration and removal of the solvent the residue was dissolved in CHCl3 (3 mL) and Et2O (12 mL) was added. The precipitated product was filtered off, washed with a solvent mixture of CHCl3-Et2O=1:2 (4 mL) to give 32.6 mg (88%) orange powder. Rf: 0.62 (95:5 CHCl3-MeOH). Diastereomeric ratio: 3:1. 1H NMR (400 MHz, CDCl3) δ (ppm): 8.96 (d, J=5.4 Hz, minor Py-H-6), 8.76 (d, J=5.4 Hz, major Py-H-6), 8.22-7.79, 7.59-7.28 (m, minor and major Ar, Py-H-3-Py-H-5), 6.33 (pt, J=10.0, 9.7 Hz, major H-2′ or H-3′ or H-4′), 6.18 (pt, J=9.6, 9.6 Hz, minor H-2′ or H-3′ or H-4′), 6.04 (pt, J=9.5, 9.4 Hz, major H-2′ or H-3′ or H-4′), 5.85 (2 pt, J=9.7, 9.7 Hz in each, minor and major H-2′ or H-3′ or H-4′), 5.74 (pt, J=9.9, 9.7 Hz, minor H-2′ or H-3′ or H-4′), 5.48 (d, J=10.1 Hz, major H-1′), 5.37 (d, J=10.0 Hz, minor H-1′), 4.73-4.66 (m, minor and major H-6′a), 4.57-4.51 (m, minor and major H-6′b), 4.48-4.40 (m, minor and major H-5′), 1.61 (s, minor Cp*—CH3), 1.52 (s, major Cp*—CH3); 13C NMR (100 MHz, CDCl3) δ (ppm): 166.3, 166.2, 165.8, 165.7, 165.3, 165.2, 165.1, 165.0, 164.9 (minor and major C═O, OD-C-2, OD-C-5), 153.6 (minor Py-C-6), 152.1 (major Py-C-6), 140.6 (minor Py-C-4), 140.3 (major Py-C-4), 140.1 (major Py-C-2), 139.0 (minor Py-C-2), 134.6, 133.9, 133.8 (2), 133.7, 133.5, 133.4, 133.3, 131.4-127.8 (minor and major Ar, Py-C-5), 125.5 (major Py-C-3), 125.2 (minor Py-C-3), 98.1, 98.0 (minor Cp*), 97.8, 97.7 (major Cp*), 77.7, 72.8, 71.9, 71.3, 68.8 (minor C-1′-C-5′), 77.2, 74.2, 71.2, 69.1, 68.7 (major C-1′-C-5′), 63.0 (minor C-6′), 62.5 (major C-6′), 9.1 (minor Cp*—CH3), 8.8 (major Cp*—CH3). ESI-HRMS positive mode (m/z): calcd for C51H46ClN3O10Rh+ [M−PF6]+ 998.1921. Found: [M−PF6]+ 998.1905.
Prepared from complex Rh-dimer (10.0 mg, 0.0162 mmol), compound L-6 (19.1 mg, 0.0323 mmol, 2.0 eq.) and TIPF6 (11.3 mg, 0.0323 mmol) according to general procedure IV. After filtration and removal of the solvent the residue was dissolved in CHCl3 (3 mL) and Et2O (12 mL) was added. The precipitated product was filtered off, washed with a solvent mixture of CHCl3-Et2O=1:1 (2 mL) to give 28.3 mg (87%) orange powder. Rf: 0.34 (95:5 CHCl3-MeOH). Diastereomeric ratio: 5:2. 1H NMR (400 MHz, CDCl3) δ (ppm): 8.95 (d, J=5.3 Hz, minor Py-H-6), 8.80 (d, J=5.3 Hz, major Py-H-6), 8.21-7.82, 7.59-7.31 (m, minor and major Ar, Py-H-3-Py-H-5), 6.22 (pt, J=9.6, 9.7 Hz, major H-2′ or H-3′), 6.12 (pt, J=9.3, 9.3 Hz, minor H-2′ or H-3′), 6.00 (pt, J=9.4, 9.3 Hz, major H-2′ or H-3′), 5.75 (pt, J=9.4, 9.3 Hz, minor H-2′ or H-3′), 5.59 (ddd, J=10.7, 9.7, 5.4 Hz, minor H-4′), 5.51 (ddd, J=10.7, 9.7, 5.5 Hz, major H-4′), 5.31 (d, J=10.0 Hz, major H-1′), 5.25 (d, J=9.4 Hz, minor H-1′), 4.64 (dd, J=11.6, 5.4 Hz, minor H-5′eq), 4.56 (dd, J=11.3, 5.5 Hz, major H-5′eq), 3.87 (pt, J=11.3, 10.7 Hz, major H-5′ax), 3.84 (pt, J=11.6, 10.7 Hz, minor H-5′ax), 1.63 (s, minor Cp*—CH3), 1.53 (s, major Cp*—CH3); 13C NMR (100 MHz, CDCl3) δ (ppm): 165.8, 165.7, 165.6 (2), 165.5 (2), 165.2, 165.1, 164.9 (2) (minor and major C═O, OD-C-2, OD-C-5), 153.5 (minor Py-C-6), 152.2 (major Py-C-6), 140.6 (minor Py-C-4), 140.4 (major Py-C-4), 140.1 (major Py-C-2), 139.2 (minor Py-C-2), 134.5, 133.9, 133.8, 133.7, 133.5, 131.2-128.0 (minor and major Ar, Py-C-5), 125.5 (major Py-C-3), 125.1 (minor Py-C-3), 98.1, 98.0 (minor Cp*), 97.8, 97.7 (major Cp*), 73.5, 71.7, 69.6, 69.0 (major C-1′-C-4′), 72.4, 72.2, 70.8, 69.2 (minor C-1′-C-4′), 67.7 (2) (minor and major C-5′), 9.1 (minor Cp*—CH3), 8.8 (major Cp*—CH3). ESI-HRMS positive mode (m/z): calcd for C43H40ClN3O8Rh+ [M−PF6]+ 864.1553. Found: [M−PF6]+ 864.1552.
Prepared from complex Rh-dimer (20.0 mg, 0.0324 mmol), compound L-8 (47.0 mg, 0.0648 mmol, 2.0 eq.) and TIPF6 (22.6 mg, 0.0647 mmol) according to general procedure IV. After filtration and removal of the solvent the residue was dissolved in CHCl3 (6 mL) and Et2O (12 mL) was added. The precipitated product was filtered off, washed with a solvent mixture of CHCl3-Et2O=1:2 (2 mL) to give 63.0 mg (85%) orange powder. Rf: 0.29 (95:5 CHCl3-MeOH). Diastereomeric ratio: 9:2. 1H NMR (400 MHz, CDCl3) δ (ppm): 8.95 (d, J=5.4 Hz, minor Py-H-6), 8.81 (d, J=5.3 Hz, major Py-H-6), 8.22-7.24 (m, minor and major Ar, Py-H-3-Py-H-5), 6.55 (pt, J=10.2, 10.1 Hz, major H-2′), 6.19 (d, J=3.4 Hz, major H-4′), 6.16 (d, J=3.3 Hz, minor H-4′), 6.02 (pt, J=10.1, 9.7 Hz, minor H-2′), 5.94 (dd, J=10.1, 3.3 Hz, minor H-3′), 5.76 (dd, J=10.1, 3.4 Hz, major H-3′), 5.47 (d, J=10.2 Hz, major H-1′), 5.40 (d, J=9.7 Hz, minor H-1′), 4.71-4.40 (m, minor and major H-5′, H-6′a, H-6′b), 1.61 (s, minor Cp*—CH3), 1.52 (s, major Cp*—CH3); 13C NMR (100 MHz, CDCl3) δ (ppm): 166.2, 165.8, 165.6, 165.4 (2), 164.9 (minor C═O, OD-C-2, OD-C-5), 166.1, 165.7, 165.5, 165.3, 165.2, 165.0 (major C═O, OD-C-2, OD-C-5), 153.5 (minor Py-C-6), 152.2 (major Py-C-6), 140.6 (minor Py-C-4), 140.4 (major Py-C-4), 140.1 (major Py-C-2), 139.2 (minor Py-C-2), 134.5, 134.2, 133.8 (2), 133.6, 133.5 (2), 131.2-128.1 (minor and major Ar, Py-C-5), 125.5 (major Py-C-3), 125.2 (minor Py-C-3), 98.1, 98.0 (minor Cp*), 97.8, 97.7 (major Cp*), 76.6, 72.2, 71.5, 68.5, 68.3 (minor C-1′-C-5′), 76.1, 72.8, 71.4, 68.3, 66.2 (major C-1′-C-5′), 62.4 (minor C-6′), 62.1 (major C-6′), 9.0 (minor Cp*—CH3), 8.8 (major Cp*—CH3). ESI-HRMS positive mode (m/z): calcd for C51H46ClN3O10Rh+ [M−PF6]+ 998.1921. Found: [M−PF6]+ 998.1920.
Prepared from triazole 11[[?]](0.050 g, 0.16 mmol) and butyryl chloride (2×81 μL, 2×0.78 mmol) according to general procedure I. Reaction time: 3 h. Purified by column chromatography (1:3 EtOAc-hexane) to give 82 mg (87%) white amorphous solid. Rf=0.47 (1:2 EtOAc-hexane). 1H NMR (360 MHz, CDCl3) δ (ppm): 8.61 (1H, d, J=4.7 Hz, Py-H-6), 8.43 (1H, s, Tria-H-5), 8.13 (1H, d, J=7.9 Hz, Py-H-3), 7.77 (1H, t, J=7.6 Hz, Py-H-4), 7.25 (1H, dd, J=7.6, 4.7 Hz, Py-H-5), 5.97 (1H, d, J=8.7 Hz, H-1′), 5.54, 5.50, 5.32 (3×1H, 3 pt, J=9.6, 9.5 Hz in each, H-2′, H-3′, H-4′), 4.30 (1H, dd, J=12.6, 4.8 Hz, H-6′a), 4.18 (1H, dd, J=12.6, <1 Hz, H-6′b), 4.05 (1H, ddd, J=9.6, 4.8, <1 Hz, H-5′), 2.36-2.23 (6H, m, 3×CH2), 2.11 (2H, t, J=7.4 Hz, CH2), 1.70-1.53 (6H, m, 3×CH2), 1.41 (2H, sext, J=7.4 Hz, CH2), 0.96-0.88 (9H, m, 3×CH3), 0.72 (3H, t, J=7.4 Hz, CH3); 13C N M R (90 MHz, CDCl3) δ (ppm): 173.0, 172.3, 171.8, 171.3 (4×C═O), 149.5, 148.8 (Tria-C-4, Py-C-2), 149.5 (Py-C-6), 136.8, 123.0, 120.2 (Py-C-3, Py-C-4, Py-C-5), 120.5 (Tria-C-5), 85.9 (C-1′), 75.2, 72.2, 70.1, 67.3 (C-2′-C-5′), 61.2 (C-6′), 35.7 (2), 35.6, 35.3 (4×CH2), 18.1, 18.0 (2), 17.9 (4×CH2), 13.5 (2), 13.4, 13.2 (4×CH3). ESI-HRMS positive mode (m/z): cald for: C29H40N4O9Na+ [M+Na]+ 611.2686. Found: 611.2687.
Prepared from triazole 11[[?]](0.050 g, 0.16 mmol) and valeryl chloride (2×95 μL, 2×0.78 mmol) according to general procedure I. Reaction time: 1.5 h. Purified by column chromatography (1:3 EtOAc-hexane) to give 57 mg (55%) pale yellow amorphous solid. Rf=0.58 (3:7 EtOAc-hexane). 1H NMR (360 MHz, CDCl3) δ (ppm): 8.61 (1H, d, J=4.5 Hz, Py-H-6), 8.41 (1H, s, Tria-H-5), 8.14 (1H, d, J=7.9 Hz, Py-H-3), 7.78 (1H, dt, J=7.9, 1.5 Hz, Py-H-4), 7.25 (1H, ddd, J=7.9, 4.5, 1.5 Hz, Py-H-5), 5.95 (1H, d, J=8.7 Hz, H-1′), 5.53, 5.49, 5.31 (3×1H, 3 pt, J=9.5, 9.4 Hz in each, H-2′, H-3′, H-4′), 4.28 (1H, dd, J=12.6, 4.8 Hz, H-6′a), 4.18 (1H, dd, J=12.6, 1.2 Hz, H-6′b), 4.03 (1H, ddd, J=9.6, 4.8, 1.2 Hz, H-5′), 2.38-2.24 (6H, m, 3×CH2), 2.13 (2H, t, J=7.4 Hz, CH2), 1.64-1.24 (14H, m, 7×CH2), 1.36 (2H, sext, J=7.3 Hz, CH2), 0.94-0.87 (9H, m, 3×CH3), 0.75 (3H, t, J=7.3 Hz, CH3); 13C NMR (90 MHz, CDCl3) δ (ppm): 173.3, 172.6, 172.0, 171.6 (4×C═O), 149.5, 148.9 (Tria-C-4, Py-C-2), 149.4 (Py-C-6), 136.8, 123.1, 120.2 (Py-C-3, Py-C-4, Py-C-5), 120.5 (Tria-C-5), 85.9 (C-1′), 75.2, 72.2, 70.2, 67.3 (C-2′-C-5′), 61.3 (C-6′), 33.6, 33.5 (2), 33.3 (4×CH2), 26.7 (3), 26.5 (4×CH2), 22.1 (3), 21.8 (4×CH2), 13.6, 13.5 (2), 13.4 (4×CH3). ESI-HRMS positive mode (m/z): cald for: C33H48N4O9Na+ [M+Na]+ 667.3313. Found: 667.3311.
Prepared from triazole 11[[?]](0.050 g, 0.16 mmol) and hexanoyl chloride (2×110 μL, 2×0.78 mmol) according to general procedure I. Reaction time: 3 h. Purified by column chromatography (1:4 EtOAc-hexane) to give 80 mg (71%) pale yellow amorphous solid. Rf=0.69 (3:7 EtOAc-hexane). 1H NMR (360 MHz, CDCl3) δ (ppm): 8.61 (1H, d, J=4.9 Hz, Py-H-6), 8.40 (1H, s, Tria-H-5), 8.14 (1H, d, J=7.9 Hz, Py-H-3), 7.78 (1H, dt, J=7.7, 1.7 Hz, Py-H-4), 7.25 (1H, ddd, J=7.7, 4.9, 1<Hz, Py-H-5), 5.94 (1H, d, J=8.9 Hz, H-1′), 5.52, 5.47, 5.29 (3×1H, 3 pt, J=9.6, 9.5 Hz in each, H-2′, H-3′, H-4′), 4.27 (1H, dd, J=12.6, 4.7 Hz, H-6′a), 4.18 (1H, dd, J=12.6, 1.9 Hz, H-6′b), 4.03 (1H, ddd, J=9.6, 4.7, <1 Hz, H-5′), 2.36-2.23 (6H, m, 3×CH2), 2.12 (2H, t, J=7.5 Hz, CH2), 1.65-1.20, (20H, m, 10×CH2), 1.19-1.11 (2H, m, CH2), 1.07-1.01 (2H, m, CH2), 0.92-0.86 (9H, m, 3×CH3), 0.75 (3H, t, J=7.3 Hz, CH3); 13C NMR (90 MHz, CDCl3) δ (ppm): 173.3, 172.6, 172.0, 171.6 (4×C═O), 149.6, 148.9 (Tria-C-4, Py-C-2), 149.5 (Py-C-6), 136.8, 123.1, 120.3 (Py-C-3, Py-C-4, Py-C-5), 120.5 (Tria-C-5), 85.9 (C-1′), 75.3, 72.3, 70.2, 67.4 (C-2′-C-5′), 61.3 (C-6′), 33.9, 33.8 (2), 33.5 (4×CH2), 31.2 (2), 31.1, 30.8 (4×CH2), 24.3 (3), 24.2 (4×CH2), 22.2 (2), 22.1, 22.0 (4×CH2), 13.9, 13.8, 13.7, 13.6 (4×CH3). ESI-HRMS positive mode (m/z): cald for: C37H56N4O9Na+ [M+Na]+ 723.3940. Found: 723.3937.
Prepared from triazole 11[[?]](0.050 g, 0.16 mmol) and heptanoyl chloride (2×110 L, 2×0.71 mmol) according to general procedure I. Reaction time: 2 h. Purified by column chromatography (2:7 EtOAc-hexane) to give 97 mg (80%) yellow amorphous solid. Rf=0.51 (3:7 EtOAc-hexane). 1H NMR (360 MHz, CDCl3) δ (ppm): 8.61 (1H, d, J=4.2 Hz, Py-H-6), 8.42 (1H, s, Tria-H-5), 8.14 (1H, d, J=7.8 Hz, Py-H-3), 7.77 (1H, t, J=7.8 Hz, Py-H-4), 7.26-7.23 (1H, m, Py-H-5), 5.97 (1H, d, J=8.6 Hz, H-1′), 5.54, 5.49, 5.31 (3×1H, 3 pt, J=9.5, 9.4 Hz in each, H-2′, H-3′, H-4′), 4.29 (1H, dd, J=12.6, 4.7 Hz, H-6′a), 4.17 (1H, dd, J=12.6, <1 Hz, H-6′b), 4.07-4.04 (1H, m, H-5′), 2.37-2.24 (6H, m, 3×CH2), 2.12 (2H, t, J=7.4 Hz, CH2), 1.61-0.87 (41H, m, 16×CH2, 3×CH3), 0.78 (3H, t, J=7.3 Hz, CH3); 13C NMR (90 MHz, CDCl3) δ (ppm): 173.2, 172.5, 171.9, 171.5 (4×C═O), 149.5, 148.8 (Tria-C-4, Py-C-2), 149.4 (Py-C-6), 136.7, 123.0, 120.2 (Py-C-3, Py-C-4, Py-C-5), 120.4 (Tria-C-5), 85.8 (C-1′), 75.2, 72.2, 70.1, 67.3 (C-2′-C-5′), 61.2 (C-6′), 33.9, 33.8 (2), 33.5 (4×CH2), 31.3 (3), 31.1 (4×CH2), 28.6 (3), 28.3 (4×CH2), 24.6, 24.5 (2), 24.4 (4×CH2), 22.3, 22.3 (2), 22.2 (4×CH2), 13.9 (3), 13.8 (4×CH3). ESI-HRMS positive mode (m/z): cald for: C41H64N4O9Na+ [M+Na]+ 779.4566. Found: 779.4560.
Prepared from triazole 11[[?]](0.051 g, 0.17 mmol) and octanoyl chloride (2×135 μL, 2×0.78 mmol) according to general procedure I. Reaction time: 2.5 h. Purified by column chromatography (1:9 EtOAc-hexane) to give 105 mg (81%) pale yellow amorphous solid. Rf=0.45 (3:7 EtOAc-hexane). 1H NMR (360 MHz, CDCl3) δ (ppm): 8.62 (1H, dd, J=4.9, 1.6 Hz, Py-H-6), 8.46 (1H, s, Tria-H-5), 8.16 (1H, d, J=7.9 Hz, Py-H-3), 7.78 (1H, dt, J=7.9, 1.6 Hz, Py-H-4), 7.26 (1H, ddd, J=7.5, 4.9, <1 Hz, Py-H-5), 5.95 (1H, d, J=8.9 Hz, H-1′), 5.54, 5.48, 5.30 (3×1H, 3 pt, J=9.9, 9.5 Hz in each, H-2′, H-3′, H-4′), 4.28 (1H, dd, J=12.6, 4.9 Hz, H-6′a), 4.18 (1H, dd, J=12.6, 1.8 Hz, H-6′b), 4.04 (1H, ddd, J=9.9, 4.9, <1 Hz, H-5′), 2.40-2.23 (6H, m, 3×CH2), 2.12 (2H, t, J=7.4 Hz, CH2), 1.71-0.78 (60H, m, 24×CH2, 4×CH3); 13C NMR (90 MHz, CDCl3) δ (ppm): 173.3, 172.6, 171.9, 171.5 (4×C═O), 149.4, 148.5 (Tria-C-4, Py-C-2), 149.2 (Py-C-6), 137.0, 123.1, 120.4 (Py-C-3, Py-C-4, Py-C-5), 120.7 (Tria-C-5), 85.9 (C-1′), 75.3, 72.3, 70.1, 67.3 (C-2′-C-5′), 61.3 (C-6′), 34.0, 33.9, 33.8, 33.5 (4×CH2), 31.6, 31.5 (2), 31.4 (4×CH2), 29.0 (3), 28.8 (3), 28.7, 28.6 (8×CH2), 24.7, 24.6, 24.6, 24.5 (4×CH2), 22.5 (3), 22.4 (4×CH2), 14.0 (3), 13.9, (4×CH3). ESI-HRMS positive mode (m/z): cald for: C45H72N4O9Na+ [M+Na]+ 835.5192. Found: 835.5192.
Prepared from triazole 12 [[?42)]](100 mg, 0.279 mmol) and benzoyl chloride (157 μL, 1.35 mmol) according to general procedure I. Reaction time: 2 h. Purified by column chromatography (2:3 EtOAc-hexane) to give 207 mg (96%) white amorphous solid. Rf=0.40 (2:3 EtOAc-hexane). 1H NMR (400 MHz, CDCl3) δ (ppm): 8.80 (H, s, Tria-H-5), 8.27-7.26 (26H, m, Ar, Qu), 6.37 (1H, d, J=8.8 Hz, H-1′), 6.17 (1H, pt, J=9.5, 9.2 Hz, H-2′ or H-3′ or H-4′), 6.11 (1H, pt, J=9.4, 9.0 Hz, H-2′ or H-3′ or H-4′), 5.92 (1H, pt, J=9.5, 9.4 Hz, H-2′ or H-3′ or H-4′), 4.72-4.50 (3H, m, H-5′, H-6′a, H-6′b); 13C NMR (100 MHz, CDCl3) δ (ppm): 166.2, 165.8, 165.3, 164.8 (4×C═O), 149.9, 149.4, 148.2 (Tria-C-4, Qu-C-2, Qu-C-8a), 137.1, 133.8, 133.7, 133.6, 133.4, 130.3-128.5, 128.1, 128.0, 127.8, 126.7, 121.7, 118.9 (Tria-C-5, Qu-C-3-Qu-C-8, Qu-C-4a, Ar), 86.5 (C-1′), 75.8, 73.2, 71.4, 69.0 (C-2′-C-5′), 62.9 (C-6′). ESI-HRMS positive mode (m/z): calcd for: C45H35N4O9+ [M+H]+ 775.2399; C45H34N4O9Na+ [M+Na]+ 797.2218. Found: [M+H]+ 775.2395; [M+Na]+ 797.2211.
Prepared from ligand L-C3 (30.4 mg, 0.052 mmol), Os-dimer (21.0 mg, 0.026 mmol) and TIPF6 (17.7 mg, 0.051 mmol) according to general procedure II. Reaction time: 1 h. Purified by column chromatography (95:5 CHCl3-MeOH) to yield 56 mg (96%) yellow syrup. Diastereomeric ratio: 1:1. Rf=0.34 (95:5 CHCl3-MeOH). 1H NMR (400 MHz, CDCl3) δ (ppm): 9.25, 9.23 (2×1H, 2 d, J=6.5 Hz in each, 2×Py-H-6), 9.02, 8.89 (2×1H, 2 s, 2×Tria-H-5), 8.04-8.01 (2H, m, 2×Py-H-3), 7.99-7.94 (2H, m, 2×Py-H-4), 7.55, 7.52 (2×1H, 2 dd, J=7.4, 6.5 Hz, 2×Py-H-5), 6.20-5.86 (12H, m, 2×H-1′, 2×(H-2′ or H-3′ or H-4′), 2×4×p-cym-CH), 5.50, 5.48, 5.39, 5.37 (2×2H, 2×2 pt, J=9.4, 9.2 Hz in each, H-2′ and/or H-3′ and/or H-4′), 4.38-4.14 (6H, m, 2×H-5′, 2×H-6′a, 2×H-6′b), 2.63, 2.59 (2×1H, 2 hept, J=6.9 Hz in each, 2×i-Pr—CH), 2.37-0.76 (74H, m, 16×CH2, 14×CH3); 13C NMR (90 MHz, CDCl3) δ (ppm): 173.3, 173.2, 172.4, 172.3, 172.1, 172.0, 171.9, 171.6 (2×4×C═O), 155.7, 155.6 (2×Py-C-6), 148.4, 148.3 (2), 147.9 (2×Tria-C-4, 2×Py-C-2), 140.5, 140.4 (2×Py-C-4), 129.8 (2) (2×Tria-C-5), 127.7, 127.6 (2×Py-C-5), 122.6 (2) (2×Py-C-3), 96.9, 96.5, 95.7, 94.4 (2×2×p-cym-CqAr), 87.0, 86.6 (2×C-1′), 78.4, 77.2, 77.0, 76.8, 75.7, 75.6, 75.5, 74.8, 74.4, 73.6, 72.9, 72.6, 70.1, 69.3, 67.3, 67.1 (2×(C-2′-C-5′), 2×4×p-cym-CHAr), 61.3, 61.2 (2×C-6′), 35.9 (4), 35.8 (2), 35.7, 35.4 (2×4×CH2), 31.2, 31.1 (2×i-Pr—CH), 22.9, 22.5, 22.3, 21.9 (2×2×i-Pr—CH3), 18.6, 18.5 (2×C6H4—CH3), 18.4, 18.3 (4), 18.2, 18.1 (2) (2×4×CH2), 13.7 (3), 13.6 (3), 13.6, 13.5 (2×4×CH3). ESI-HRMS positive mode (m/z): cald for: C39H54ClN4O9Os+ [M−PF6]+ 949.3180. Found: 949.3180.
Prepared from ligand L-C4 (33.1 mg, 0.052 mmol), Os-dimer (20.0 mg, 0.025 mmol) and TIPF6 (17.6 mg, 0.050 mmol) according to general procedure II. Reaction time: 1 h. Purified by column chromatography (95:5 CHCl3-MeOH) to yield 52 mg (88%) yellow syrup. Diastereomeric ratio: 4:3. Rf=0.38 (95:5 CHCl3-MeOH). 1H NMR (400 MHz, CDCl3) δ (ppm): 9.25, 9.23 (2×1H, 2 d, J=6.6 Hz in each, 2×Py-H-6), 9.03, 8.90 (2×1H, 2 s, 2×Tria-H-5), 8.04-8.01 (2H, m, 2×Py-H-3), 7.99-7.94 (2H, m, 2×Py-H-4), 7.55-7.50 (2H, m, 2×Py-H-5), 6.20-5.86 (12H, m, 2×H-1′, 2×(H-2′ or H-3′ or H-4′), 2×4×p-cym-CH), 5.51, 5.47, 5.40, 5.35 (2×2H, 2×2 pt, J=9.4, 9.2 Hz in each, H-2′ and/or H-3′ and/or H-4′), 4.38-4.14 (6H, m, 2×H-5′, 2×H-6′a, 2×H-6′b), 2.67-2.55 (2H, m, 2×i-Pr—CH), 2.40- 0.77 (90H, m, 24×CH2, 14×CH3); 13C NMR (90 MHz, CDCl3) δ (ppm): 173.4, 173.3, 172.5, 172.4, 172.2, 172.1, 172.0, 171.7 (2×4×C═O), 155.7, 155.5 (2×Py-C-6), 148.3, 148.2 (2), 147.8 (2×Tria-C-4, 2×Py-C-2), 140.4, 140.3 (2×Py-C-4), 129.8, 129.6 (2×Tria-C-5), 127.6, 127.5 (2×Py-C-5), 122.6 (2) (2×Py-C-3), 96.8, 96.4, 95.8, 94.3 (2×2×p-cym-CqAr), 86.9, 86.5 (2×C-1′), 78.4, 76.9 (3), 75.7 (2), 75.6, 74.7, 74.3, 73.4, 72.8, 72.5, 70.0, 69.3, 67.2, 67.0 (2×(C-2′-C-5′), 2×4×p-cym-CHAr), 61.3, 61.1 (2×C-6′), 33.7 (3), 33.6 (2), 33.5, 33.3 (2) (2×4×CH2), 31.1, 31.0 (2×i-Pr—CH), 26.8 (2), 26.8 (2), 26.7 (2), 26.6, 26.6 (2×4×CH2), 22.9, 22.4, 22.3, 21.8 (2×2×i-Pr—CH3), 22.2 (4), 22.1 (2), 22.0 (2) (2×4×CH2), 18.6 (2) (2×C6H4—CH3), 13.8 (3), 13.7 (3), 13.6 (2), (2×4×CH3). ESI-HRMS positive mode (m/z): cald for: C43H62ClN4O9Os+ [M−PF6]+ 1005.3807. Found: 1005.3806.
Prepared from ligand L-C5 (18.6 mg, 0.027 mmol), Os-dimer (10.0 mg, 0.013 mmol) and TIPF6 (8.8 mg, 0.025 mmol) according to general procedure II. Reaction time: 1 h. Purified by column chromatography (95:5 CHCl3-MeOH) to yield 25 mg (80%) yellow syrup. Diastereomeric ratio: 5:4. Rf=0.40 (95:5 CHCl3-MeOH). 1H NMR (400 MHz, CDCl3) δ (ppm): 9.22, 9.20 (2×1H, 2 d, J=6.4 Hz in each, 2×Py-H-6), 8.99, 8.86, (2×1H, 2 s, 2×Tria-H-5), 8.03-8.00 (2H, m, 2×Py-H-3), 7.97-7.93 (2H, m, 2×Py-H-4), 7.53-7.48 (2×1H, m, 2×Py-H-5), 6.18-5.86 (12H, m, 2×H-1′, 2×H-2′ or H-3′ or H-4′, 2×4×p-cym-CH), 5.49, 5.47 (2×1H, 2 pt, J=9.1, 9.1 Hz in each, H-2′ or H-3′ or H-4′), 5.37, 5.35 (2×1H, 2 pt, J=10.1, 10.1 Hz in each, H-2′ or H-3′ or H-4′), 4.39-4.13 (6H, m, 2×H-5′, 2×H-6′a, 2×H-6′b), 2.69-2.56 (2H, m, 2×i-Pr—CH), 2.38-0.78 (106H, m, 32×CH2, 14×CH3); 13C NMR (90 MHz, CDCl3) δ (ppm): 173.5, 173.4, 172.6, 172.4, 172.3, 172.2, 172.1, 171.8 (2×4×C═O), 155.6, 155.4 (2×Py-C-6), 148.4 (3), 147.8 (2×Tria-C-4, 2×Py-C-2), 140.5, 140.4 (2×Py-C-4), 127.7, 127.5 (2×Py-C-5), 126.1, 125.8 (2×Tria-C-5), 122.8, 122.7 (2×Py-C-3), 97.0, 96.5, 95.7, 94.3 (2×2×p-cym-CqAr), 87.1, 86.7 (2×C-1′), 78.5, 77.2, 77.0, 76.8, 75.9, 75.8, 75.7, 74.9, 74.5, 73.6, 72.9, 72.6, 70.1, 69.4, 67.3, 67.2 (2×(C-2′-C-5′), 2×4×p-cym-CHAr), 61.4, 61.2 (2×C-6′), 34.1 (4), 34.9 (2), 34.8, 33.6 (2×4×CH2), 31.4 (4), 31.3 (3), 31.2 (2×4×CH2), 31.2 (2) (2×i-Pr—CH), 24.5 (3), 24.4 (2). 24.3 (3) (2×4×CH2), 22.9, 22.5, 22.4, 21.9 (2×2×i-Pr—CH3), 22.4 (4), 22.3 (2), 22.2 (2) (2×4×CH2), 18.7, 18.6 (2×C6H4—CH3), 14.0 (2), 13.9 (4), 13.8 (2) (2×4×CH3). ESI-HRMS positive mode (m/z): cald for: C47H70ClN4O9Os+ [M−PF6]+ 1061.4433. Found: 1061.4432.
Prepared from ligand L-C6 (38.4 mg, 0.051 mmol), Os-dimer (20.2 mg, 0.025 mmol) and TIPF6 (17 mg, 0.049 mmol) according to general procedure II. Reaction time: 1 h. Purified by column chromatography (95:5 CHCl3-MeOH) to yield 34 mg (53%) yellow syrup. Diastereomeric ratio: 9:7. Rf=0.48 (95:5 CHCl3-MeOH). 1H NMR (400 MHz, CDCl3) δ (ppm): 9.23, 9.21 (2×1H, 2 d, J=6.6 Hz in each, 2×Py-H-6), 9.02, 8.88 (2×1H, 2 s, 2×Tria-H-5), 8.03, 8.01 (2×1H, 2 d, J=7.3 Hz, 2×Py-H-3), 7.95, 7.95 (2×1H, 2 t, J=7.7 Hz, 2×Py-H-4), 7.53-7.49 (2H, m, 2×Py-H-5), 6.18-5.85 (12H, m, 2×H-1′, 2×(H-2′ or H-3′ or H-4′), 2×4×p-cym-CH), 5.50, 5.45 (2×1H, 2 pt, J=9.1, 9.1 Hz in each, H-2′ or H-3′ or H-4′), 5.39, 5.34 (2×1H, 2 pt, J=10.1, 10.1 Hz in each, H-2′ or H-3′ or H-4′), 4.39-4.13 (6H, m, 2×H-5′, 2×H-6′a, 2×H-6′b), 2.69-2.56 (2×1H, m, 2×i-Pr—CH), 2.38-0.78 (122H, m, 40×CH2, 14×CH3); 13C NMR (90 MHz, CDCl3) δ (ppm): 173.4, 173.3, 172.5, 172.4, 172.2, 172.1, 172.1, 171.7 (2×4×C═O), 155.5, 155.3 (2×Py-C-6), 148.3, 148.2, 147.7 (2) (2×Tria-C-4, 2×Py-C-2), 140.4, 140.3 (2×Py-C-4), 129.8, 129.7 (2×Tria-C-5), 127.5, 127.4 (2×Py-C-5), 122.7, 122.6 (2×Py-C-3), 96.9, 96.4, 95.6, 94.2 (2×2×p-cym-CqAr), 87.0, 86.6 (2×C-1′), 78.4, 76.9, 75.8, 75.7, 75.7, 75.6, 74.8, 74.4, 73.5, 72.8, 72.6 (2), 70.0, 69.3, 67.2, 67.0 (2×(C-2′-C-5′), 2×4×p-cym-CHAr), 61.3, 61.1 (2×C-6′), 34.0 (3), 33.9 (2), 33.8, 33.6 (2) (2×4×CH2), 31.5 (2), 31.4 (4), 31.4, 31.3 (2×4×CH2), 31.1, 31.0 (2×i-Pr—CH), 28.8 (2), 28.8 (4), 28.7, 28.6 (2×4×CH2), 24.8 (2), 24.7, 24.7 (2), 24.6, 24.6, 24.5 (2×4×CH2), 22.9, 22.3 (2), 21.9 (2×2×i-Pr—CH3), 22.5 (4), 22.4 (4), (2×4×CH2), 18.6 (2) (2×C6H4—CH3), 14.1 (3), 14.0 (4), 14.0 (2×4×CH3). ESI-HRMS positive mode (m/z): cald for: C51H78ClN4O9Os+ [M−PF6]+ 1117.5060. Found: 1117.5059.
Prepared from ligand L-C7 (42.2 mg, 0.052 mmol), Os-dimer (20.0 mg, 0.025 mmol) and TIPF6 (17.0 mg, 0.049 mmol) according to general procedure II. Reaction time: 1 h. Purified by column chromatography (95:5 CHCl3-MeOH) to yield 48 mg (72%) yellow syrup. Diastereomeric ratio: 5:4. Rf=0.38 (95:5 CHCl3-MeOH). 1H NMR (400 MHz, CDCl3) δ (ppm): 9.24, 9.23 (2×1H, 2 d, J=6.2 Hz in each, 2×Py-H-6), 9.03, 8.90 (2×1H, 2 s, 2×Tria-H-5), 8.04-8.01 (2H, m, 2×Py-H-3), 7.95, 7.95 (2×1H, 2 pt, J=7.4 Hz in each, 2×Py-H-4), 7.54-7.49 (2H, m, 2×Py-H-5), 6.19-5.86 (12H, m, 2×H-1′, 2×(H-2′ or H-3′ or H-4′), 2×4×p-cym-CH), 5.50, 5.46 (2×1H, 2 pt, J=9.4, 9.5 Hz in each, H-2′ or H-3′ or H-4′), 5.39, 5.34 (2×1H, 2 pt, J=9.9, 10.0 Hz in each, H-2′ or H-3′ or H-4′), 4.40-4.15 (6H, m, 2×H-5′, 2×H-6′a, 2×H-6′b), 2.62, 2.60 (2×1H, 2 hept, J=6.9 Hz in each, 2×i-Pr—CH), 2.38-0.81 (138H, m, 48×CH2, 14×CH3); 13C NMR (90 MHz, CDCl3) δ (ppm): 173.4, 173.3, 172.5, 172.4, 172.2, 172.1, 172.0, 171.8 (2×4×C═O), 155.7, 155.6 (2×Py-C-6), 148.3, 148.2 (2), 147.8 (2×Tria-C-4, 2×Py-C-2), 140.4, 140.3 (2×Py-C-4), 129.8, 129.7 (2×Tria-C-5), 127.6, 127.5 (2×Py-C-5), 122.7, 122.6 (2×Py-C-3), 96.9, 96.4, 95.8, 94.3 (2×2×p-cym-CqAr), 87.0, 86.6 (2×C-1′), 78.4, 77.0, 76.9, 76.7, 75.7 (2), 75.6, 74.8, 74.4, 73.4, 72.8, 72.6, 70.0, 69.3, 67.2, 67.0 (2×(C-2′-C-5′), 2×4×p-cym-CHAr), 61.3, 61.1 (2×C-6′), 34.0 (2), 34.0 (2), 33.9 (2), 33.8, 33.6 (2×4×CH2), 31.7 (2), 31.7 (4), 31.6, 31.6 (2×4×CH2), 31.1, 31.0 (2×i-Pr—CH), 29.1, 29.1 (2), 29.0, 29.0, 28.9, 28.9, 28.8 (2×4×CH2), 24.8 (2), 24.8 (2), 24.7, 24.7, 24.6, 24.6 (2×4×CH2), 22.9, 22.4 (2), 22.3, 21.9 (2×2×i-Pr—CH3), 22.6 (5), 22.6, 22.5 (2×4×CH2), 18.6 (2) (2×C6H4—CH3), 14.1 (8) (2×4×CH3). ESI-HRMS positive mode (m/z): cald for: C55H86ClN4O9Os+ [M−PF6]+ 1173.5686. Found: 1173.5687.
Prepared from ligand L-7 (26.6 mg, 0.0343 mmol, 2.1 eq.), Ru-dimer (10 mg, 0.0163 mmol) and TIPF6 (11.4 mg, 0.0326 mmol) according to general procedure II. After filtration and removal of the solvent, the residue was dissolved in CHCl3 (3 mL) and Et2O (6 mL) was added. The precipitated product was filtered off, then washed with a solvent mixture of CHCl3-Et2O=1:1 (1 mL) to give 26.2 mg (67%) brown powder. Diastereomeric ratio: 2:1. Rf=0.42 (95:5 CHCl3-MeOH). 1H NMR (400 MHz, CDCl3) δ (ppm): 9.32 (s, minor Tria-H-5), 9.28 (s, major Tria-H-5), 8.63 (d, J=8.8 Hz, major Qu-H-8), 8.60 (d, J=8.9 Hz, minor Qu-H-8), 8.27 (d, J=8.5 Hz, major Qu-H-4), 8.26 (d, J=8.4 Hz, minor Qu-H-4), 8.07-7.28 (m, minor and major Qu-H-3, Qu-H-5-Qu-H-7, Ar), 6.66 (pt, J=9.3, 9.3 Hz, major H-2′ or H-3′ or H-4′), 6.57 (d, J=9.3 Hz, major H-1′), 6.53 (d, J=8.8 Hz, minor H-1′), 6.28 (pt, J=9.5, 9.5 Hz, minor H-2′ or H-3′ or H-4′), 6.17 (pt, J=9.5, 9.4 Hz, major H-2′ or H-3′ or H-4′), 6.16 (pt, J=9.5, 9.2 Hz, minor H-2′ or H-3′ or H-4′), 6.01 (pt, J=10.0, 9.6 Hz, minor H-2′ or H-3′ or H-4′), 5.95 (pt, J=10.0, 9.6 Hz, major H-2′ or H-3′ or H-4′), 5.72- 5.45 (m, minor and major 4×p-cym-CHAr), 4.83-4.55 (m, minor and major H-5′, H-6′a, H-6′b), 2.35 (hept, J=6.9 Hz, minor i-Pr—CH), 2.12 (hept, J=6.9 Hz, major i-Pr—CH), 2.10 (s, major C6H4—CH3), 2.02 (s, minor C6H4—CH3), 0.85, 0.81 (2 d, J=6.9 Hz in each, 2×minor i-Pr—CH3), 0.69, 0.60 (2 d, J=6.9 Hz in each, 2×major i-Pr—CH3); 13C NMR (100 MHz, CDCl3) δ (ppm): 166.3, 165.6, 165.2, 165.0 (minor 4×C═O), 166.2, 165.5, 165.3, 165.2 (major 4×C═O), 149.2, 148.4, 147.5 (major Tria-C-4, Qu-C-2, Qu-C-8a), 149.1, 148.3, 147.8 (minor Tria-C-4, Qu-C-2, Qu-C-8a), 141.1, 134.3, 133.8 133.5, 133.3, 133.0, 130.6-128.0, 118.8, 118.7 (minor and major Tria-C-5, Qu-C-5-Qu-C-8, Qu-C-4a, Ar), 105.6, 103.0 (minor p-cym-CqAr), 105.6, 103.6 (major p-cym-CqAr), 87.3, 87.2, 87.0, 86.7, 85.1, 85.0, 84.5, 83.4, 82.8 (minor and major p-cym-CHAr, C-1′), 76.2, 72.7, 72.2, 68.8 (minor C-2′-C-5′), 75.8, 73.6, 70.5, 68.6 (major C-2′-C-5′), 62.9 (minor C-6′), 62.7 (major C-6′), 31.1 (minor i-Pr—CH), 31.0 (major i-Pr—CH), 22.5, 21.4 (minor i-Pr—CH3), 22.3, 21.4 (major i-Pr—CH3), 18.8 (major C6H4—CH3), 18.7 (minor C6H4—CH3). ESI-HRMS positive mode (m/z): calcd for: C55H48ClN4O9Ru+ [M−PF6]+ 1045.2165. Found: 1045.2163.
Prepared from ligand L-2b (20.6 mg, 0.0266 mmol, 2.1 eq.), Os-dimer (10.0 mg, 0.0126 mmol) and TIPF6 (8.8 mg, 0.0252 mmol) according to general method II. After filtration and removal of the solvent, the residue was dissolved in CHCl3 (3 mL) and Et2O (6 mL) was added. The precipitated product was filtered off, then washed with a solvent mixture of CHCl3-Et2O=1:1 (2 mL) to give 21.7 mg (67%) brown powder. Diastereomeric ratio: 10:9. Rf=0.38 (95:5 CHCl3-MeOH). 1H NMR (400 MHz, CDCl3) δ (ppm): 9.36, 9.26 (2×1H, 2 s, 2×Tria-H-5), 8.52, 8.50 (2×1H, 2 d, J=8.9 Hz in both, 2×Qu-H-8), 8.24, 8.22 (2×1H, 2 d, J=8.9 Hz in both, 2×Qu-H-4), 8.08-7.28 (48H, m, 2×20×Ar, 2×Py-H-3-Py-H-5), 6.55, 6.51 (2×1H, 2 d, J=9.3 Hz in each, 2×H-1′), 6.65, 6.26, 6.18, 6.17, 6.03-5.93 (2×3H, 4 pt, m, J=9.4 Hz in each, 2×H-2′, H-3′, H-4′), 6.03-5.93, 5.82, 5.79, 5.74, 5.71, 5.69, 5.66 (2×4H, m, 6 d, J=5.7 Hz in each, 2×4×p-cym-CHAr), 4.84-4.55 (2×3H, m, 2×H-5′, 2×H-6′a, 2×H-6′b), 2.22, 2.13 (2×3H, 2 s, 2×C6H4—CH3), 2.20, 1.91 (2×1H, 2 hept, J=6.9 Hz in each, 2×i-Pr—CH), 0.80, 0.73, 0.54, 0.51 (2×2×3H, 2×2 d, J=6.9 Hz in each 2×2×i-Pr—CH3); 13C NMR (100 MHz, CDCl3) δ (ppm): 166.4, 166.2, 165.6, 165.4, 165.3, 165.2, 165.1, 164.9 (2×4×C═O), 149.8, 148.9, 148.6, 148.1 (2×Tria-C-4, Qu-C-2, Qu-C-8a), 141.5, 141.3 (2×Qu-C-2), 134.3, 134.2, 133.8, 133.6, 133.5, 133.4, 133.3, 133.2, 130.4-127.9 (2×Qu-C-4a, Qu-C-5-Qu-C-8, Tria-C-5, Ar), 118.5, 118.4, (2×Qu-C-3), 97.2, 96.7, 96.6, 96.1 (2×2×p-cym-CqAr), 87.3, 86.8 (2×C-1′), 78.9, 78.7, 76.7, 76.2, 75.8, 75.4, 75.2, 74.1, 73.5, 73.2, 72.7, 72.0, 70.4, 68.8, 68.6 (2×4×p-cym-CHAr, 2×C-2-C-5′), 62.8, 62.7 (2×C-6′), 31.2, 31.1 (2×i-Pr—CH), 22.9, 22.6, 21.7, 21.6 (2×2×i-Pr—CH3), 18.7, 18.6 (2×C6H4—CH3). ESI-HRMS positive mode (m/z): calcd for: C55H48ClN4O9Os+ [M−PF6]+ 1135.2725. Found: 1135.2724.
Prepared from ligand L-2b (20.4 mg, 0.0264 mmol, 2.1 eq.), Ir-dimer (10.0 mg, 0.0126 mmol) and TIPF6 (8.7 mg, 0.0249 mmol) according to general method II. After filtration and removal of the solvent, the residue was dissolved in CHCl3 (3 mL) and Et2O (6 mL) was added. The precipitated product was filtered off, then washed with a solvent mixture of CHCl3-Et2O=1:1 (1 mL) to give 19.7 mg (61%) yellow powder. Diastereomeric ratio: 5:3. Rf=0.41 (95:5 CHCl3-MeOH). 1H NMR (400 MHz, CDCl3) δ (ppm): 9.41 (s, minor Tria-H-5), 9.34 (s, major Tria-H-5), 8.38-7.24 (m, minor and major Qu-H-3-Qu-H-8, Ar), 6.61 (d, J=9.3 Hz, major H-1′), 6.58 (d, J=9.9 Hz, minor H-1′), 6.46 (pt, J=9.4, 9.4 Hz, major H-2′ or H-3′ or H-4′), 6.29-6.23 (m, minor H-2′ and/or H-3′ and/or H-4′), 6.15 (pt, J=9.4, 9.4 Hz, major H-2′ or H-3′ or H-4′), 6.02 (pt, J=9.8, 9.7 Hz, minor H-2′ or H-3′ or H-4′), 5.93 (pt, J=9.8, 9.7 Hz, minor H-2′ or H-3′ or H-4′), 4.81-4.54 (m, minor and major H-5′, H-6′a, H-6′b), 1.52 (s, major Cp*—CH3), 1.48 (s, minor Cp*—CH3); 13C NMR (100 MHz, CDCl3) δ (ppm): 166.3, 165.5, 165.3, 165.0 (minor and major 4×C═O), 149.7, 148.9, 145.7, (minor Tria-C-4, Qu-C-2, Qu-C-8a), 149.3, 149.1, 145.6 (major Tria-C-4, Qu-C-2, Qu-C-8a), 141.8, 141.6, 134.2, 134.1, 134.0, 133.8, 133.5, 133.4, 133.3, 132.7, 132.6, 130.3-127.9, 127.3, 118.8 (minor and major Tria-C-5, Qu-C-3-Qu-C-8, Qu-C-4a, Ar), 90.0 (major Cp*), 89.9 (minor Cp*), 87.4 (minor C-1′), 87.2 (major C-1′), 76.1, 75.9, 73.7, 72.9, 71.7, 70.5, 68.7, 68.5 (minor and major C-2′-C-5′), 62.8 (major C-6′), 62.7 (minor C-6′), 9.1 (minor and major Cp*—CH3). ESI-HRMS positive mode (m/z): calcd for: C55H49ClN4O9Ir+ [M−PF6]+ 1137.2813. Found: [M−PF6]+ 1137.2810.
Prepared from ligand L-2b(26.3 mg, 0.0340 mmol, 2.1 eq.), Rh-dimer (10.0 mg, 0.0162 mmol) and TIPF6 (11.3 mg, 0.0323 mmol) according to general method II. After filtration and removal of the solvent, the residue was dissolved in CHCl3 (3 mL) and Et2O (6 mL) was added. The precipitated product was filtered off, then washed with a solvent mixture of CHCl3-Et2O=1:1 (2 mL) to give 26.2 mg (80%) orange powder. Diastereomeric ratio: 5:3. Rf=0.38 (95:5 CHCl3-MeOH). 1H NMR (400 MHz, CDCl3) δ (ppm): 9.33 (s, minor Tria-H-5), 9.28 (s, major Tria-H-5), 8.40-7.22 (m, minor and major Qu-H-3-Qu-H-8, Ar), 6.61 (d, J=9.3 Hz, major H-1′), 6.57 (d, J=8.6 Hz, minor H-1′), 6.52 (pt, J=9.3, 9.2 Hz, major H-2′ or H-3′ or H-4′), 6.28 (pt, J=9.6, 9.4 Hz, minor H-2′ or H-3′ or H-4′), 6.23 (pt, J=9.5, 9.0 Hz, minor H-2′ or H-3′ or H-4′), 6.17 (pt, J=9.5, 9.4 Hz, major H-2′ or H-3′ or H-4′), 6.02 (pt, J=9.5, 9.5 Hz, minor H-2′ or H-3′ or H-4′), 5.94 (pt, J=9.8, 9.7 Hz, major H-2′ or H-3′ or H-4′), 4.80-4.54 (m, minor and major H-5′, H-6′a, H-6′b), 1.51 (s, major Cp*—CH3), 1.44 (s, minor Cp*—CH3); 13C NMR (100 MHz, CDCl3) δ (ppm): 166.2 (2), 165.7, 165.5 156.3, 165.2, 165.0, 164.7 (minor and major 4×C═O), 148.1, 148.0, 147.9, 147.5, 145.9 (2) (minor and major Tria-C-4, Qu-C-2, Qu-C-8a), 141.3, 141.2, 134.1, 133.8, 133.7, 133.6, 133.5, 133.4, 133.3, 133.2, 130.3-127.8, 127.3, 119.0 (minor and major Tria-C-5, Qu-C-3-Qu-C-8, Qu-C-4a, Ar), 97.9, 97.8 (minor and major Cp*), 87.5 (minor C-1′), 87.0 (major C-1′), 76.2, 75.8, 73.7, 72.8, 71.8, 70.6, 68.7, 68.6 (minor and major C-2′-C-5′), 62.8 (minor C-6′), 62.7 (major C-6′), 9.3 (major Cp*—CH3), 9.2 (minor Cp*—CH3). ESI-HRMS positive mode (m/z): calcd for: C55H49ClN4O9Rh+ [M−PF6]+ 1047.2243. Found: [M−PF6]+ 1047.2240.
Prior the experiments n-octanol was saturated with aqueous PBS solution (pH=7.40) and vice versa. The corresponding complex (approximately 0.2-0.3 mg) was dissolved in a mixture of 2.50 mL pre-saturated n-octanol and 2.50 mL pre-saturated PBS buffer, and the mixture was vigorously stirred for 3 days. Based on the NMR stability measurements, this time was necessary to reach equilibrium between the various ionic complex species. Due to the lipophilic/hydrophilic character of the complexes those with benzoyl protection or the non-sugar derivative containing ones could mostly be found in the n-octanol, while the acetyl- and non-protected complexes in the aqueous PBS phase. The appropriate separated solution was centrifuged (ScanSpeed 406G instrument, 4000 RPM for 5 minutes) and the absorption of the “stock solution” was measured (VWR UV-1600PC Spectrophotometer, 270-420 nm). Then 2.00 mL stock solution was stirred vigorously with 16.00 mL pre-saturated, clean n-octanol or PBS solution. After one day the phases were separated, centrifuged and absorption of the solution was measured again. From the absorption difference of the stock solutions the distribution coefficient (DPH, here measured at pH 7.) was calculated according to the previously described formulae (Kozsup et al., 2020).
After phase separation, log D is calculated from the absorbance values of the UV-Vis spectrum and the volume of the phases as required.
Carboplatin, oxaliplatin, cisplatin, rucaparib, Trolox and MitoTEMPO were from Sigma-Aldrich (St. Louis, MO, USA). All other materials for cellular experiments were from Sigma-Aldrich, unless otherwise stated.
Cells were cultured under standard cell culture conditions, 37° C., 5% CO2, humidified atmosphere.
A2780 cells were cultured in RMPI 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, 1% penicillin-streptomycin (Sigma-Aldrich).
ID8 cells were cultured in high glucose DMEM (4.5 g/L glucose) medium supplemented with 4% fetal calf serum, 2 mM glutamine, 1% penicillin-streptomycin (Sigma-Aldrich), 1% ITS supplement (Sigma-Aldrich I3146).
U251 cells were maintained in MEM (Sigma-Aldrich), 10% fetal bovine serum (Sigma-Aldrich), 1% Penicillin/Streptomycin (Invitrogene), 2 mM Glutamine.
MCF7 cells were maintained in MEM (Sigma-Aldrich), 10% fetal bovine serum (Sigma-Aldrich), 1% Penicillin/Streptomycin (Invitrogene), 2 mM Glutamine.
Capan2 cells were maintained in MEM (Sigma-Aldrich), 10% fetal bovine serum (Sigma-Aldrich), 1% Penicillin/Streptomycin (Invitrogene), 2 mM Glutamine.
Human primary dermal fibroblasts were cultured in low glucose DMEM (1 g/L glucose) medium supplemented with 20% fetal calf serum, 2 mM glutamine, 1% penicillin-streptomycin (Sigma-Aldrich).
L428 cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, 1% penicillin-streptomycin.
Saos cells were maintained in DMEM (4.5 g/L glucose) medium supplemented with 10% fetal calf serum, 2 mM glutamine, 1% penicillin-streptomycin.
Cisplatin resistant A2780 cells were grown in RMPI 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, 1% penicillin-streptomycin. Cisplatin resistant cells underwent selection (1 μM cisplatin) once a week for 3 days before plating for any assay.
Microdilution experiments were performed according to the standards of EUCAST.
(EUCAST (EUROPEAN COMMITTEE ON ANTIMICROBIAL SUSCEPTIBILITY TESTING, European Society of Clinical Microbiology and Infectious Diseases; see EUCAST reading guide for broth microdilution, Version 3.0 January 2021, also available at https://eucast.org/ast_of_bacteria/mic_determination/) recommends testing according to the International Standard ISO 20776-1. See: “MIC determination of non-fastidious and fastidious organisms.”)
The bacterial isolates to be tested were grown on Mueller-Hinton agar plates. Inoculum density of bacteria was set at 5.0×105 CFU/mL in microtiter plates in a final volume of 200 μL Mueller-Hinton broth. Tested concentration range was 0.08-40 μM (10 concentrations, two-fold serial dilutions), drug-free growth control and inoculum-free negative control were included. The inoculated plates were incubated for 24 hours at 37° C. then were assessed visually. Minimum inhibitory concentration (MIC) was defined as the lowest concentration with 50%<inhibitory effect compared to the growth control. All experiments were performed at least twice in duplicates.
Results are recorded as the lowest concentration of antimicrobial agent that inhibits visible growth of a microorganism, the Minimum Inhibitory Concentration (MIC), expressed in mg/L or μg/mL.
The examined reference strains are the following:
S. aureus
S. aureus
S. aureus
S. aureus
S. aureus
S. aureus
S. aureus
S. aureus
S. aureus
S. aureus
S. aureus
S. aureus
E. faecium
E. faecium
E. faecium
E. faecium
E. faecium
E. faecium
E. faecium
E. faecium
The concentrations were identical in case of each microorganism tested:
From each test compounds 160 μM concentrations were prepared in 2 mL end volume in Mueller-Hinton broth, from which 100 μL was also measured into the first wells of each row of wells. Then up to the tenth well of each row 2× dilution was carried out with Mueller-Hinton broth to obtain the above described concentrations.
Thereafter the diluted bacterium suspensions were measured into the wells so as to obtain the advised CFU except in case of the negative control. The ready-made plates were incubated for 24 h at 37° C. and then read. Upon comparison the Growth control was used as a base. Each well wherein an at least 50% growth inhibition was found has been selected and marked.
Examination was made at least in two parallels in each case.
MTT reduction assay assesses the activity of mitochondrial complex I and was used to assess rapid toxicity. MTT reduction assay performed similarly to (Bakondi et al., 2003) as follows. Cells were seeded in 96-well plates. The next day cells were treated with the compounds for 4 hours in the concentrations indicated in a cell incubator. At the end of treatment MTT was added in 0.5 mg/ml final concentration and cells were incubated at 37° C. in a cell incubator. Then culture media was removed and the reduced MTT dye was resolved in dimethyl-sulfoxide (DMSO) and plates were measured in a plate photometer (Thermo Scientific Multiscan GO spectrophotometer, Waltham, MA, USA) at 540 nm. On each plate wells were designed to contain untreated/vehicle-treated cells. In calculations the readings for these wells was considered as 1 and all readings were expressed relative to these values.
SRB accumulation assay assesses protein content in a sample and was used to assess cell proliferation. SRB accumulation assay was performed similarly to (Fodor et al., 2016) as follows. Cells were seeded in 96-well plates. The next day cells were treated with the compounds for 48 hours in the concentrations indicated in a cell incubator. At the end of treatment cells were fixed with 10% trichloroacetic acid (TCA). Fixed cells were stained with SRB (0.4 m/V % dissolved in 1% acetic acid) for 60 minutes. Fixed cells were washed in 1% acetic acid 3 times; acetic acid was removed and cells were left to dry. Protein-bound SRB was released by adding 100 μl 10 mM Tris base. Plates were measured in a plate photometer (Thermo Scientific Multiscan GO spectrophotometer, Waltham, MA, USA) at 540 nm. On each plate wells were designed to contain untreated cells. In calculations the readings for these wells was considered as 1 and all readings were expressed relative to these values.
AnnexinV-PI double staining was applied to assess apoptotic and necrotic cell death similar to (Bai et al., 2001; Virag et al., 1998a; Virag et al., 1998c). A2780 cells were treated with the indicated compounds at the concentration corresponding to their IC50 value for 2, 4 and 48 hours. The 4 and 48 hours time point corresponds to the time points for the MTT and SRB assays, while the early 2 hours time point reflects an optimum time point for the detection of apoptotic or necrotic cell death (Bai et al., 2001; Virag et al., 1998a; Virag et al., 1998c). Quadrants were set based on the FITC and PI values observed for the vehicle-treated cells.
The preparation of protein extracts, the separation of protein extracts in SDS-polyacrylamide gel electrophoresis and the subsequent Western blotting was performed as described in (Marton et al., 2018) using the antibodies in Table 6. Enhanced chemiluminescence was developed using ChemiDoc Imager, (Bio-Rad, Hercules, California, USA). Densitometry was performed using Image Lab Touch Software, Bio-Rad, Hercules, California, USA)
Statistical analysis was performed using 8.0.1 version of Graphpad Prism. Values were tested for normal distribution using the D'Agostino and Pearson normality test. When necessary, values were log normalized or were normalized using the Box-Cox normalization method (Box and Cox, 1964), as indicated in the figure captions. The following statistical test, post hoc test and the level of significance is indicated in the figure captions. Nonlinear regression was performed using the built-in “[Inhibitor] vs. response—Variable slope (four parameters), least square fit” utility of Graphpad that yielded IC50 and Hill slope values.
General consideration for Seahorse oximetry can be found at (Miko et al., 2017) and at https://www.agilent.com/en/product/cell-analysis/real-time-cell-metabolic-analysis/xf-analyzers.
A2780 cells were plated into specialized Seahorse cell plates and were subjected to oximetry. First, baseline oxygen consumption was recorded. Then cells received the complexes in concentrations corresponding to their respective IC50 values and OCR and ECAR values were collected every 30 minutes. At the end of the experiment cells received antimycin.
The first step in calculation was to subtract antimycin-resistant respiration rates from the baseline or complex-induced respiration. The first two baseline respiration (OCR) and extracellular acidification rates (ECAR) rates were omitted. All respiration and ECAR value was expressed as a fold change compared to controls.
Cells were seeded in 96 well plates the day before the assay. Cells were treated with the compounds for 96 hours. Cells were counted using a Burker chamber. On each plate wells were designed to contain untreated cells. In calculations the readings for these wells was considered as 1 and all readings were expressed relative to these values.
| Number | Date | Country | Kind |
|---|---|---|---|
| P2100326 | Sep 2021 | HU | national |
This is the national stage of International Application PCT/HU2022/050067, filed Sep. 19, 2022. The invention relates to Half-sandwich transition metal complexes and uses thereof.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/HU2022/050067 | 9/19/2022 | WO |
