Ruthenium Arene Complexes with α-Aminoacidate Ligands:
New Insights into Transfer Hydrogenation Reactions
and Cytotoxic Behaviour
Lorenzo Biancalana,a,b Issam Abdalghani,c Federica Chiellini,a Stefano Zacchini,d,b Guido
Pampaloni,a,b Marcello Crucianelli,c,* Fabio Marchetti,a,b,*
a Department of Chemistry and Industrial Chemistry, University of Pisa, Via Moruzzi 13, I-56124 Pisa,
Italy.
b CIRCC, Via Celso Ulpiani 27, I-70126 Bari, Italy.
c Department of Physical and Chemical Sciences, University of L’Aquila, Via Vetoio, I-67100 L’Aquila,
Italy
d Department of Industrial Chemistry “Toso Montanari”, University of Bologna, Viale Risorgimento 4,
I-40136 Bologna, Italy
Corresponding Authors
E-mail: marcello.crucianelli@univaq.it, tel.: (+39) 0862-433308 (M. Crucianelli). E-mail:
fabio.marchetti1974@unipi.it, Tel.: (+39) 050-2219245, webpage: www.dcci.unipi.it/~fabmar/ (F. Marchetti).
Abstract
The Ru(II) arene complexes [Ru(η6-p-cymene)(κ2N,O-aa)Cl] (aa = L-glutaminato, 1; L-homoserinato, 2;
L-tyrosinato, 3; L-serinato, 4; L-prolinato, 5; trans-4-hydroxyl-L-prolinato, 6; L-threoninato, 7; glycinato, 8) were synthesized in high yields by the reactions of [Ru(η6-p-cymene)Cl
2]2 with aa/MOH
(M = Na, K). The salt [Ru(η6-p-cymene)(κ2N,O-L-serinate)(PPh
3)][CF3SO3], 9[CF3SO3], was prepared
in 68% yield from 4 and AgSO3CF3/PPh3. All the products were fully characterized by analytical and
spectroscopic methods, moreover the X-ray structures of 1 and 2 were elucidated by X-ray diffraction. Compounds 1-7 and 9[CF3SO3] were studied as catalytic precursors for the transfer hydrogenation
reaction (THR) of acetophenone, using isopropanol or sodium formate (in water) as hydrogen sources, under variable experimental conditions. The catalytic process was investigated also under microwave irradiation. In general, all the catalytic reactions selectively afforded 1-phenylethanol, and the best results were achieved with 5 and 6, in terms of both activity and enantioselectivity (enantiomeric excess up to 78%). The in vitro cytotoxicity of 1, 4, 5 and 9[CF3SO3] was assessed towards the human
pancreatic cancer cell line BxPC3 and the mouse embryo fibroblast Balb/3T3 Clone A31 cell line, all of the investigated compounds showing to be substantially inactive. The catalytic and biological results will be discussed in relation to the relevant chemical behaviour of the compounds in water solution.
Keywords: ruthenium(II) arene complexes; transfer hydrogenation; anticancer activity.
Introduction
Ruthenium(II) arene compounds, bearing a typical three leg piano stool structure, have aroused a huge interest for their possible catalytic1 and biological applications.2 In particular, they have been
intensively investigated in the last decade as catalytic precursors for hydrogenation reactions,3
culminating with the Nobel Prize award.4 On the other hand, RAPTA compounds, incorporating the
amphiphilic 1,3,5-triaza-7-phosphaadamantane (pta) ligand (Figure 1b),5 and RAED compounds,
containing a bidentate ethylenediamine ligand (Figure 1c),6 exhibit promising anticancer properties,
that have stimulated the attention on many other analogous complexes.
Figure 1. Prominent Ruthenium(II) arene compounds: Noyori’s hydrogenation catalysts (a); RAPTA (b) and
RAED (c) anticancer compounds.
Among the ligands that can be coordinated to the ruthenium arene frame, naturally occurring α-amino acids constitute a class of attracting compounds, in view of their easy availability and low toxicity,7 and
the presence of a stereogenic centre, which may play some role in asymmetric reactions.8
Various complexes of the type [Ru(η6-arene)(2N,O-aa)Cl] (aa = α-amino acidate ligand) have
appeared in the literature. Carmona, Oro and co-workers prepared a series of diastereomeric complexes by addition of homochiral α-amino acids (including alanine, valine, leucine, phenylalanine, L-proline and L-hydroxyL-proline) to the acetylacetonato precursor [Ru(η6-p-cymene)(acac)Cl].9
Alternatively, some [Ru(η6-arene)(2N,O-aa)Cl] complexes were synthesized from [Ru(η6-arene)Cl 2]2
and the appropriate α-amino acid (e.g. L-serine, L-threonine, glycine), in the presence of a base.10
[Ru(η6-p-cymene)(2N,O-L-glutammato)Cl] 11 as well as [Ru(η6-arene)(2N,O-aa)Cl] complexes (arene
= C6H6, C6Me6, p-cymene; aaH = L-proline, piperidine-2-carboxylate, N-methyl-L-phenylalanine) and
the corresponding cationic trimers [Ru(η6-arene)(aa)]
3[BF4]312 were found to be effective catalysts for
the transfer hydrogenation of acetophenone and some related substrates, in isopropanol. [Ru(η6
did not display any cytotoxicity against human prostate cancer cell lines (PC-3 and DU-145).Error: Reference source not found Other [Ru(η6-cymene)(2N,O-aa)Cl] complexes (aaH = glycine, proline, or D-alanine,
L-or D-phenylalanine) revealed to be inactive towards A2780 human ovarian cancer cells.13 Despite these
discouraging results, the possible biological activity of ruthenium arene/α-amino acidate systems still remains controversial. In fact, Furrer et al. found high levels of cytotoxicity, generally superior to cisplatin, against A2780 and A2780CisR cancer cells provided by mixing aqueous solutions of several α-amino acids with [Ru(η6-p-cymene)Cl
2]2 in the presence of AgCF3SO3. The silver salt is an effective
Cl-abstractor, promoting the binding of the amino acidate moiety to ruthenium.14 Interestingly, [Ru(η6
-cymene)(2N,O-aa)Cl] complexes with the amino acidate group N-substituted with a ferrocenyl
pendant displayed moderate cytotoxicity against breast cancer MCF7 cell line.15 In addition, an older
report claimed the possible in vivo activity exerted by [Ru(η6-C
6H6)(2N,O-L-prolinato)Cl] against P388
leukemia cells.16
Herein, we describe the synthesis of three unprecedented [Ru(η6-p-cymene)(2N,O-aa)Cl] complexes,
including the X-ray characterization of two of them, and of the ionic triphenylphosphine derivative [Ru(η6-p-cymene)(κ2N,O-L-serinato)(PPh
3)][CF3SO3]. The new compounds, together with some already
known [Ru(η6-p-cymene)(2N,O-aa)Cl] species (Scheme 1), for which detailed synthetic procedures
and more extensive spectroscopic characterization are presented here,17 have been studied as catalytic
precursors in the THR of acetophenone, in both isopropanol and water. In general, the hydrogenation of carbonyl and imino functions have established to be among the most strategic processes in fine chemistry industry,18 and THR represents a powerful alternative to the use of gaseous hydrogen, due to
safety concerns and easy handling, availability and low cost of the hydrogen donors.19 In the last two
decades, intensive research on THR has been focused on the development of new tailored catalysts working in organic solvents or in water, even if the latter modality remains less explored.20
Furthermore, in view of the specific action exerted by different natural α-amino acids in cancer metabolism, particularly L-glutamine,21 L-serine22 and, to a minor extent, L-proline,23 a selection of the
compounds have been tested for the antiproliferative activity against the human pancreatic cancer cell line BxPC3 and the mouse embryo fibroblasts Balb/3T3 Clone A31 cell line, the latter acting as a model for normal cells.
Results and discussion
1. Synthesis and characterization.
Yellow complexes [Ru(η6-p-cymene)(2N,O-aa)Cl] (aa = glutaminato, 1; homoserinato, 2;
L-tyrosinato, 3; L-serinato, 4; L-prolinato, 5; trans-4-hydroxy-L-prolinato, 6; L-threoninato, 7; glycinato,
8) were obtained in 75-97% yield from the straightforward reaction of [Ru(η6-p-cymene)Cl
2]2 with the
appropriate α-amino acid in methanol, in the presence of NaOH or KOH (Scheme 1a). Ruthenium complexes bearing polar groups on the α-amino acid side chain (1: –CONH2; 2-4, 6, 7: –OH) are
scarcely soluble in common organic solvents such as acetone and dichloromethane, and only soluble in water and alcohols, where the NaCl/KCl by-product is soluble. Therefore, purification of these complexes was achieved by silica chromatography or reflux extraction with CH2Cl2. Also [Ru(η6
-p-cymene)(2N,O-L-serinato)(PPh
3)][CF3SO3], 9[CF3SO3], was prepared, in order to see the effect on
catalytic and cytotoxic performances of the modification of the Ru(II)-aminoacidate frame, through the introduction of a lipophilic group and a net positive charge. Compound 9[CF3SO3] was obtained as a
yellow solid in 68% yield by Cl/PPh
Scheme 1. Synthesis of Ruthenium-p-cymene α-aminoacidate complexes 1-8 (a) and 9[CF3SO3] (b).
Compounds 1-3 and 9[CF3SO3] are novel, while 4-8 were previously reported.Error: Reference source
not found,Error: Reference source not founda-b,Error: Reference source not found However, all the products have been fully
characterized by analytical and spectroscopic methods (it should be noted that IR and 13C NMR data
are generally missing in the literature for this family of compounds). The salient spectroscopic data of
1-8 and 9[CF3SO3] are comparatively shown in Table 1. The IR spectra (solid state) clearly exhibit the
absorptions due to the amine (3100-3300 and ca. 3450 cm-1 for NH
2 and NH groups, respectively) and
carboxylate groups (1620-1600 and 1390-1370 cm-1 for the antisymmetric and symmetric vibration
modes, respectively). The wavenumber separation between the two modes (Δνa-s) range from 210 to
255 cm-1, pointing a monodentate coordination of the carboxylate group.24 The amide group in 1 is
characterized by a strong band at 1670 cm-1 (1683 cm-1 in L-glutamine) while the hydroxyl group in 2-4, 6, 7, 9 gives sharp or broad absorptions around 3500-3300 cm-1.
Table 1. Selected IR and NMR data of 1-8 and 9[CF3SO3].
Compd
IR: ῦ/cm-1a
NMR: δ/ppm b
Major diastereomer; data related to minor diastereomer in parenthesis
ν(OH) ν(NH2)/
ν(NH) δ(NH2) νasym(CO2) νsym(CO2) Δνa-s c 13C (CO2) 13C (CαH) 1H (CαH) 1H (NH2)
1 d - 3338m-sh 3284m 1590s 1624m-sh 1394m 230 (178.7)180.3 (56.0) 54.1 (2.94)2.66 (6.73,2.94)6.10, 4.69 2 3427m 3326w 3292w 3212w-br 1580s-sh 1595s 1392s 210 (183.5)184.9 (57.1)55.8 (3.37)3.22 (6.84, 3.43)6.09, 4.63 3 3300w-br3560- 3252m3212m 1590s 1623s 1385s 238 (183.3)184.3 (59.3)57.0 (3.37)3.24 n.d 4 3200w-br3500- 3292m3247m 1575m-sh 1620s 1395s 225 (181.5)183.4 (60.5)58.4 (3.37)3.08 (6.83, 3.37)5.77, 4.59 5 - 3450w-br - 1608s-br 1380s-sh 228 186.4(n.d) (n.d)63.4 (3.47)3.47 n.d 6 3318w-br 3423w-br - 1604s-br 1381m 223 (185.7)186.1 (62.8)62.3 (3.85)3.66 n.d; 3.73 7 3381m 3253w3215w 1578s-sh 1623s 1371s-br 252 (181.4)183.7 (64.0)61.7 (3.18)2.84 5.47, 4.68(6.69) 8 -3543w 3501w 3287w-sh 3231m 1566s 1622s 1367s 255 184.4 45.9 3.07 6.28, 4.17 9[OTf] 3420w-br 3296w-br3241w-br 1588m-sh 1633s-br 1391m-sh 242 (180.1)182.8 (63.2)58.4 (1.99)1.99 (6.62, 2.81)6.12, 3.95 a Solid-state. b CD
3OD solution unless otherwise specified. c Δνa-s = νasym(CO2) νsym(CO2). d DMSO-d6 solution.
n.d. = not detected.
Well-resolved NMR spectra of 1-8 and 9[CF3SO3] were recorded on CD3OD or DMSO-d6 solutions, in
part due to solubility issues but also because the formation of intermolecular aggregates takes place in other solvents (CH2Cl2, CHCl3, acetone, isopropanol).Error: Reference source not founda,25 In fact,
broad 1H resonances were observed for 5 and 8 in CDCl
3 even at high dilution (≈ 2∙10-3 M).
Concerning CD3OD or DMSO-d6 solutions, 1H, 13C and 31P NMR spectra of 1-7 and 9[CF3SO3] contain
two sets of signals, due to the presence of two diastereomeric forms; all the resonances related to the distinct forms could be assigned with the assistance of 2D NMR experiments. Compound 5, containing the L-prolinato ligand, displayed the highest diastereomeric ratio (0.86), the ratio related to 1-4 and 6-7 ranging in between 0.60 and 0.69, in accordance with previously observed trends.Error: Reference source not founda,Error: Reference source not foundc The replacement of a chloride ligand with triphenylphosphine (4
Reference source not foundb In the 13C spectra of 1-8 and 9[CF
3SO3], the resonance due to the
carboxylate group was found in the 186-180 ppm region, while the resonance of the amino acidate α-carbon occurred at 59 ± 5 ppm, except for the CH2 group of 8 (45.9 ppm). In the 1H spectra of
freshly-prepared CD3OD solutions of 1-8 and 9[CF3SO3], the signals related to the NH2/NH groups were
identified, ranging from 6.7 to 2.9 ppm. The two diastereomers of 9[CF3SO3] are characterized by 31P
NMR resonances at 32.6 and 34.5 ppm, respectively. The CF3SO3 anion in the same compound was
detected as a quartet at 121.7 ppm (1J
CF = 319 Hz) and a singlet at −79.6 ppm, respectively in the 13C
and 19F spectra. A weak 35Cl NMR signal was found in CD
3OD solutions of 2 and 5-8, approximately in
the same spectral region where ionic chlorides (i.e., NaCl and [Et3NH]Cl) display a sharp resonance
(around −25 ppm).26 This feature suggests that fast Cl−/solvent exchange occurs, in accordance with
conductivity measurements on other Ru(p-cymene)(aminoacidate) complexes in methanol solution.Error: Reference source not foundb
Apart from possible chloride/solvent exchange, 1-8 and 9[CF3SO3] resulted indefinitely stable in
methanol and DMSO solutions, with no variation in the diastereomeric ratio (d.r.). Previous studies on M(ηn-ring)(2N,O-α-aminocarboxylate) complexes (M = Ir, Ru, Rh; ring = arene or Cp) indicated that
the epimerization process at the metal centre is fast on the NMR timescale at ambient temperature and thus the diastereomeric composition of equilibrium is quickly reached upon dissolution.27a,b
Accordingly, addition of AgNO3 and then NaCl to a methanolic solution of 4 did not modify the
diastereomeric ratio (see Experimental for details). On the other hand, the increase in d.r. observed on going from 4 to 9[CF3SO3] is presumably related to the stereoselective attack of the entering ligand
(PPh3) to the intermediate species generated by treatment with Ag+/MeOH, as previously suggested for
[Ru(η6-p-cymene)(2N,O-aa)(PPh
3)] (aaH = L-alanine, L-proline).Error: Reference source not foundb
X-ray quality crystals of 1 and 2 could be collected, therefore their structures were ascertained by single crystal X-ray diffraction. Views of the ORTEP molecular structures are shown, respectively, in
Figures 2 and 3, while relevant bonding parameters are given in Tables S1 and S3, supplied as Supporting Information.
Compounds 1 and 2 exhibit the expected three-leg piano-stool geometry typical of Ru(II)-arene compounds,28 and the bonding parameters around the Ru(II) centres are similar to those reported for
other [Ru(p-cymene)(α-aminoacidate)Cl] structures.Error: Reference source not found,Error: Reference source not foundb The α-amino acidate ligands retain the absolute S configuration at the C-atom, and both
compounds crystallize in the chiral space group P21. Compound 2 revealed the presence of the two
diastereomers in 1:1 ratio within the asymmetric unit, while a single diastereomer with SCSRu
configuration29 was found in the crystal structure of 1. The N- and O-bonded hydrogens of 1 and 2 are
involved in H-bonds as summarized in Tables S2 and S4.
Figure 2. View of the structure of 1. Displacement ellipsoids are at the 50% probability level. Hydrogen atoms, except those bonded to N-atoms, have been omitted for clarity.
Figure 3. View of the structure of 2. Displacement ellipsoids are at the 50% probability level. Hydrogen atoms, except those bonded to N-and O-atoms, have been omitted for clarity. The two independent molecules present within the unit cell are represented.
Speciation and stability of the complexes in water.
The behaviour of 4-6 and 9[CF3SO3] in D2O solution was assessed by means of 1H, 13C, 31P and 35Cl
NMR spectroscopy. In 4-6, Chloride/water exchange quickly occurred upon dissolution, as indicated by the appearance of a sharp resonance in the 35Cl NMR spectra close to the 0 ppm reference given by
NaCl.
The 1H NMR spectra allowed the identification of the original compounds (4-6) and the corresponding
aquo-derivatives [Ru(η6-p-cymene)(2N,O-aa)(H
2O)]+ (4W, 5W and 6W, respectively; Scheme 2a).Error:
Reference source not found Addition of NaCl to these solutions shifted back the equilibrium to 4-6, thus making unambiguous the assignment of the two sets of signals. In the case of the L-serinato 4, five Ru species were initially detected in D2O, and two of them were attributed to the non dissociated
resulted in disappearance of the Ru-Cl species in the 1H spectrum. Two of the remaining three sets of
resonances were assigned to the aquo-complex (4W, two diastereomers) and the other one, featuring an
unusually high-field shifted resonance for the methylene group (ΔδH = −0.5 ppm vs. 4), can be
tentatively associated to the tridentate chelate compound [Ru(η6-p-cymene)(κ3N,O,O’-L-serinato)]+ (4T;
Scheme 2b) as a single diastereomer.30 Remarkably, previous studies on the interaction of the Ru(η6
-p-cymene)2+ moiety with L-/DL-serine in aqueous solution indicated the involvement of the amino acid
side-chain in the coordination.Error: Reference source not found,31
The 1H spectra of 4-6 in D
2O were unchanged after 24 hours at 40°C, thus suggesting that an
equilibrium was reached, with the aquo-complexes 4W-6W accounting for ≈ 50% of the species in
solution, in addition to ≈ 25% of 4T for the L-serine system.
On the other hand, 9[CF3SO3] was considerably less soluble in water (S = 3.5∙10-3 M, as ascertained by 1H NMR spectroscopy). Two sets of signals, related to the diastereomers of 9+, were identified in the 1H
and 31P NMR spectra and no variation occurred during the following 24 hours at 40°C, even in the
presence of 0.1 M NaCl. Uncoordinated PPh3 was not found in the organic phase after D2O/CDCl3
extraction. Therefore, at variance to [Ru(η6-p-cymene)(2N,O-aa)Cl] compounds, complex 9+ in
aqueous solution does not undergo appreciable ligand/solvent or ligand/chloride exchange.
Scheme 2. NMR detected species in D2O solutions of compounds 4-6.
The complexes 1-7 and 9[CF3SO3] were used for catalytic studies in the transfer hydrogenation reaction
(THR) of acetophenone as a model substrate, with the aim of evaluating their effective applicability. Complex 8, lacking of a chiral centre on the aminoacidate moiety, was not included in this study. We started this study by checking the activity of a selection of complexes, i.e. 4, 5, 6 and 7. Benchmark conditions typical for this type of processes were adopted [2-propanol (IPA)/KOH, ambient temperature, 24 hours], in order to compare the activity of the complexes with those previously claimed for 4 and 5 in the reduction of acetophenone. It is noteworthy that, in the literature case, 4 and 5 were prepared by mixing the corresponding α-amino acidate potassium salts and [Ru(p-cymene)Cl2]2 in
water, and used in admixture with the side product KCl without any purification.32 The present catalytic
reactions with 4 and 5 afforded results comparable to those already published, in terms of conversion of starting material (s.m.) and stereoselectivity in the formation of the product 1-phenylethanol, the (R) configuration being detected for the main enantiomer (Table 2, entries 5 and 6, conditions A).Error: Reference source not found,Error: Reference source not founda Only moderate activity and stereoselectivity were
observed using 7 (Table 2, entry 8, conditions A), containing the L-threoninate ligand, while much better results were achieved with 5 and 6 (Table 2, entries 6 and 7, conditions A). This fact outlines the important role exerted by the rigid skeleton of, respectively, L-prolinate and 4-hydroxyl-L-prolinate ligands.Error: Reference source not founda Indeed, it has been documented that good results in terms of
yield and stereoselectivity, in asymmetric THRs catalyzed by various Ru(II) arene complexes, can be obtained using proline derivatives as chiral ligands.33
Stimulated by our preliminary results, we moved to investigate the catalytic activity of all ruthenium compounds containing a chiral α-aminocarboxylate ligand, namely 1-7 and 9[CF3SO3], by changing
some parameters, otherwise working under similar conditions as above described. Thus, a lower amount of catalyst was employed [substrate (s.m.)/Catalyst/Base (S/C/B) ratio of 200:1:2.5 rather than 100:1:1], and the reaction mixtures were heated at 80 °C for 4 hours. The results are reported in Table 2
(entries 1-9, conditions B). Under the new experimental conditions, all catalysts were active and completely selective toward the formation of 1-phenylethanol as the only recovered product, with yields ranging from moderate (30%) to high (90%). Nevertheless, the heating revealed to be detrimental to the stereoselectivity of the reaction, as clearly found using 5, 6 and 7 (compare entries 6-8, conditions B vs. A, in Table 2). Experimental evidences have been reported that slower THR experiments, conducted at lower temperatures, may provide a higher optical yield.Error: Reference source not founda,34 Actually, at the early stage of the reaction (after 1 hour), the enantiomeric excess
(ee) checked with 5 was higher (42%) than that observed at the end (22%), thus confirming that this reaction basically undergoes equilibrium.Error: Reference source not foundb For this reason, the role
exerted by the reverse process, i.e. the dehydrogenation of 1-phenylethanol, toward the observed decreasing of the ee, should not be ruled out.
Table 2. THRs of Acetophenone with IPA/KOH and complexes 1-7 and 9[CF3SO3].
Entry Complex Reaction Conditions a A B Conv. (%) b (%) eec Conv.(%) b (%) eec 1 - - - 0 n.d. 2 [Ru(p-cymene)(Gln)Cl] 1 - - 80 n.d. 3 [Ru(p-cymene)(Hom)Cl] 2 - - 55 n.d. 4 [Ru(p-cymene)(Tyr)Cl] 3 - - 45 n.d. 5 [Ru(p-cymene)(Ser)Cl] 4 8 n.d. 85 0 6 [Ru(p-cymene)(Pro)Cl] 5 77 80 (38)62d (78)22d 7 [Ru(p-cymene)(Hyp)Cl] 6 70 77 54 23 8 [Ru(p-cymene)(Thr)Cl] 7 49 58 70 34 9 [Ru(p-cymene)(Ser)(PPh3)]+ 9+ - - 90 0
a A: Catalyst = 0.0025 mmol; s.m. = 0.25 mmol; KOH = 0.0025 mmol; S/C/B = 100:1:1; 2.5 mL of IPA; 24 hours; ambient temperature. B: Catalyst: 0.0025 mmol; s.m. = 0.5 mmol; KOH = 0.0063 mmol; S/C/B = 200:1:2.5; 5.0 mL of IPA; 4.0 hours; 80 °C. b Calculated by 1H-NMR integration. c Determined by HPLC using a Chiralcel OD-H column; the main phenylethanol enantiomer being always the (R) isomer. d MW-assisted reaction: catalyst =
0.0025 mmol; s.m. = 0.25 mmol; KOH = 0.005 mmol; S/C/B = 100:1:2; 2.0 mL of IPA; T = 40 °C; 30 min. n.d. = not determined.
In principle, the use of microwave irradiation as source of energy for THR is ideal because 2-propanol is a high absorbance solvent that is very efficient for transferring microwave energy, thus taking advantages, in terms of stereoselectivity control, also from the possibility of shortened reaction times. As matter of fact, the microwave-assisted Ru-catalyzed asymmetric THR of acetophenone in 2-propanol was recently investigated.35 Moreover, shorter reaction times reduce energy demands and
hence make the reaction system pointing toward “green” catalysis. In light of this, we decided to evaluate the activity of the L-proline derived catalyst 5, as a selected probe for the hydrogenation of acetophenone under MW irradiation, otherwise employing the experimental conditions adopted for the thermal reaction (Table 2, entry 6, “d” note). In effect, the MW-assisted THR of acetophenone at 40 °C afforded the product with an acceptable stereoselectivity (78%), at all comparable with that observed at ambient temperature but after only 30 minutes, although with a moderate conversion value (Table 2, entry 6, compare the values of “d” note vs. conditions A). The repetition of the same reaction in a second cycle by MW heating did not substantially improve the results in terms of yield (up to 48%) nor for the ee (dropped to 70%). On the other hand, increasing the reaction temperature (up to 80 °C), always by MW heating, while reducing the reaction time (15 min.), afforded the expected increase of yield of 1-phenylethanol (up to 70%), but produced worse enantiomeric excess (30-35%).
Once proved that 1-7 and 9+ were active as catalytic precursors for the THR of acetophenone under
traditional IPA/KOH conditions, either at ambient temperature or above, we decided to investigate the behaviour of the same complexes in aqueous medium in the absence of a base.
It has been well established that aqueous phase THRs of ketones represent a very effective and green approach to chiral alcohols, affording the corresponding products with limited reaction times, high productivity and high selectivity. Noticeably, the reaction can be readily accomplished with tailor-made
catalysts by using mild and readily available formate salts as reductant and avoiding organic solvents.36
Even though the use of green solvents such as water is preferable,Error: Reference source not foundc,37
frequently observed solubility concerns of either substrate or catalyst oblige to use IPA or a mixture of formic acid and triethylamine (HCO2H:Et3N) as best reagents.
In view of the water solubility of our ruthenium complexes, we studied their catalytic activity in acetophenone THR in water medium using sodium formate as hydrogen source. The main results obtained with catalysts 1-7 and 9[CF3SO3], under optimized conditions, are reported in Table 3
(reaction conditions A). Again, the best outcomes in terms of yield (82-85 %) and stereoselectivity (35, 52 %) were observed for the L-proline and trans-4-hydroxy-L-proline compounds 5 and 6, followed by L-glutamine, L-homoserine and L-theonine complexes (1-3), providing high conversion (72-83%) but low stereoselectivity (< 20%). At the opposite end, 4, 7 and 9+, containing L-serinato and L-threoninato
ligands, gave a very low activity (12-34%) with no stereoselectivity in the alcohol.
Table 3. Aqueous THR of Acetophenone with HCO2Na and complexes 1-7 and 9[CF3SO3].
Entry Complex
Reaction Conditions a
A B C D
Conv.
(%) b (%) eec Conv.(%) b (%) eec Conv.(%) b (%) eec Conv.(%) b (%) eec
1 - 0 n.d. - - - - 0 n.d. 2 [(p-cymene)Ru(Gln)Cl] 1 75 7 90 15 - - 90 8 3 [(p-cymene)Ru(Hom)Cl] 2 72 6 74 13 - - 70 5 4 [(p-cymene)Ru(Tyr)Cl] 3 83 19 - - - - 60 17 5 [(p-cymene)Ru(Ser)Cl] 4 34 0 - - 39 7 18 0 6 [(p-cymene)Ru(Pro)Cl] 5 85 56 83 68 (40)91d (68)40d 7 [(p-cymene)Ru(Hyp)Cl] 6 82 35 80 58 92 38 8 [(p-cymene)Ru(Thr)Cl] 7 30 0 38 8 19 0 9 [(p-cymene)Ru(Ser)(PPh3)]+ 9+ 12 0 22 6 25 0
a A: Catalyst = 0.0025 mmol; s.m. = 0.25 mmol; S/C = 100:1; HCO
2Na = 5.0 equiv.; 2.0 mL of water; 2.0 hours; 80 °C. B: Reaction performed at 40°C for 12 hours, otherwise working under A conditions. C: Reaction performed with 2.0 mol% of SDS, otherwise working under A conditions. D: MW-assisted reaction, reaction
time = 10 min, otherwise working under A conditions. b Calculated by 1H-NMR integration. c Determined by HPLC using a Chiralcel OD-H column; the main phenylethanol enantiomer being always the (R) isomer. d Reaction performed at 40 °C, for 30 min., otherwise working under D conditions. n.d. = not determined.
We decided then to test the selected catalysts 1-2 and 5-6 at a lower temperature (40 °C). As expected, the kinetic was slower, and acceptable yields were observed only after 12 hours. Nevertheless, higher stereoselectivities were observed with all the studied catalysts (Table 3, entries 2-3 and 6-7, conditions B vs. A), the L-prolinate derivative 5 remaining the best one (ee = 68%).
Since it has been shown that surfactants may furnish advantages in terms of yield and enantioselectivity during THRs,Error: Reference source not foundb,38 we performed the catalytic experiments with 4, 7
and 9[SO3CF3] in the presence of sodium dodecyl sulfate (SDS, 2.0 mol%). Unfortunately, no
significant improvement was observed (see Table 3, entries 5, 8 and 9, conditions C vs A, respectively).
Finally, with the idea to further investigate the potentiality of our catalysts in water, we tried to exploit the microwave irradiation as energy source. It is noteworthy that, despite THRs of ketones have been very well investigated under traditional thermal conditions, only a few articles were so far published concerning microwave assisted conditions in aqueous medium.39 At first, we optimized the reaction
conditions for the MW-assisted reduction of acetophenone using 5 as a selected catalyst; the results are compiled in Table 4. We observed that different amounts of either sodium formate or catalyst, while slightly affecting the total yield of 1-phenylethanol, did not exert a relevant role on the stereoselectivity of the reaction, leaving the ee substantially unchanged (Table 4, entries 1-2 or 3-4, respectively).
Table 4. Optimization setup of MW-assisted aqueous THR of acetophenone a
Entry HCO2Na
(equiv.) Cat. 5 (mol %) Conv.
b ee (%) c
1 2 1.0 65 38
3 5 0.5 70 39
4 5 1.5 88 41
a Reaction conditions: 2.0 mL of water; s.m.: 0.25 mmol, 10 min; 80 °C. b Calculated by 1H-NMR integration. c Determined by HPLC using a Chiralcel OD-H column; the main 1-phenylethanol enantiomer being always the (R) isomer.
Afterwards, we evaluated the catalytic activity of all Ru compounds under optimized MW-assisted conditions: the results are shown in Table 3 (Entries 1-9, conditions D). During a blank test, no activity was observed in the absence of Ru catalyst. Instead, 1-phenylethanol was obtained with various yields with all Ru catalysts during a reaction time of 10 minutes. The pattern of catalytic activity emerged among the Ru compounds resembles that observed with the thermal reactions (see above). Indeed, the L-prolinate and trans-4-hydroxy-L-prolinate catalysts 5 and 6 performed as the best ones (> 90% conversion, ee = 40%), while L-serine and L-threonine complexes (4, 7 and 9+) afforded again a low
conversion (18-25 %) and a racemic product. According to what previously observed in 2-propanol (Table 2, entry 6, “d” note), also in aqueous phase under MW irradiation the decrease of temperature allowed to reach a better stereoselectivity, even if with a slower kinetic, which implies a reduction in the substrate conversion (Table 3, entry 6, “d” note). This fact confirms the crucial role exerted by the temperature on the final value of ee, under variable conditions. Anyhow, in terms of stereoselectivity, the results obtained in the IPA/KOH medium are slightly better than those observed in the aqueous phase.
THR in water: mechanistic considerations.
Bifunctional Noyori’s concerted mechanism40 has been invoked in previous papers dealing with the
THR of ketones catalyzed by [M(η6-ring)Cl(α-amino acidate)] compounds (M = Ir, Ru, Rh, Os; ring =
arene or Cp).Error: Reference source not founda,c In agreement with such mechanism (Scheme 3), the
ruthenium formate complex (intermediate A). The formation of the ruthenium hydride B, through CO2
elimination, is expected to be the key species forming the chiral alcohol upon reaction with the keto substrate. The resultant ruthenium species, after reaction with sodium formate and water, will be able to regenerate the formate complex A, thus allowing to restart a new catalytic cycle.Error: Reference source not found
Scheme 3. Proposed mechanism of the catalytic cycle for the aqueous THR with Ru-α-aminoacidate complexes.
To probe the formation of possible hydride intermediate, complex 5 was dissolved in D2O and the
appropriate amount of sodium formate was added. After heating at 60 °C for 20 minutes, resonances attributable to ruthenium-hydride species were 1H NMR detected, in the typical up-field region of the
spectrum (−to − ppm), see Experimental.41 Consistently with the proposed mechanism (Scheme 3),
these resonances disappeared after addition of acetophenone and further heating at 60 °C for 20 minutes, giving way to the bearing of the signals of the alcohol product.
Analogous experiments were carried out on compounds 4 and 9[CF3SO3], aiming to rationalize their
poor THR catalytic activity in water. Thus, 4 reacted quantitatively with one equivalent of Na(HCO2) at
up to −20 ppm) was detected, instead the cleanly formed Ru species, 10, displayed a set of signals indicative of a single diastereomer. The same derivative (10) was also obtained from the reaction of 4 and KOH in D2O, therefore we propose 10 to be the tridentate complex [Ru(η6-p-cymene)(3
N,O,O’-OCH2CH(NH2)CO2)] (Scheme 4a), resulting from deprotonation of the hydroxyl group belonging to the
α-amino acidic side chain. Compound 10, compared to the protonated counterpart 4T (see Scheme 2),
featured downfield-shifted 1H resonances for the aromatic protons (Δδ
H ≈ − 0.3 ppm) and the OCH2 and
NCH groups within the tridentate ligand (ΔδH = − 0.18 and − 0.10 ppm, respectively). In the 13C NMR
spectrum, the salient resonances of the tridentate ligand were found at 182.4 (C=O), 63.5 (CH2O) and
60.9 (CHN) ppm.
Scheme 4. Formation of 10 from the reactions of 4 (a) and 9[CF3SO3] (b) with Na(HCO2) in D2O.
Even in the presence of a large excess of Na(HCO2), 4 was cleanly converted into 10. Addition of
acetophenone and subsequent heating at 80°C for 45' caused only minor variations in the 1H spectrum;
the formation of 1-phenylethanol and the appearance of Ru-H signals (δH = – 8.26, – 13.41) were
observed after 14 hours heating. The presumable generation of 10 during the catalytic process is likely to inhibit the crucial formation of ruthenium-hydride intermediates, thus slowing down the hydrogenation reaction. Compound 9[CF3SO3] revealed to be even more inert under the same
investigated conditions: a considerable fraction (ca. 1/3) of 9+ persisted in solution after heating in the
by 1H NMR (Scheme 4b), indicating that 4 and 9+ yield the same intermediate species, upon reaction
with Na(HCO2) and loss of their monodentate ligand (Cl– or PPh3, respectively). To this end, a
prominent role may be attributed to the higher stabilization of 9[CF3SO3] compared to 4, due to the
presence in the former of a PPh3 ligand rather than a chloride ligand.42 Subsequent formation of
1-phenylethanol and very weak signals for Ru-H species (the same as 4) were observed after 14 hours at 80°C.
In the light of these findings, the catalytic activity of [Ru(η6-p-cymene)(2N,O-aa)Cl] complexes for
THR in aqueous medium appears to be related to the specific nature of the α-amino acidate ligands. Compound 5, containing a rigid bicyclic structure provided by the coordination of L-prolinate, is an effective catalyst which readily generates ruthenium hydride derivatives in the presence of Na(HCO2),
presumably following Noyori’s pathway (Scheme 3). On the other hand, L-serine compounds 4 and 9+
favourably form a stable tridentate chelate derivative (10) upon side-chain coordination, thus retarding the successive formation of Ru-hydride intermediates enabling the reduction of acetophenone. Similar considerations are fit to explain the good catalytic performance of the trans-4-hydroxy-L-proline compound 6 and the poor activity of the L-threonine one 7 (threonine contains a potentially coordinating hydroxyl group within the α-amino acidic side chain).
3. Cytotoxicity studies.
Compounds 1, 4, 5 and 9[CF3SO3] were selected for a preliminary cytotoxicity study by virtue of the
peculiar biological role associated to the corresponding α-amino acid (proline, serine and L-glutamine, see Introduction). The ability of the Ru complexes to inhibit cell growth was evaluated in
vitro against the human pancreatic cancer cell line BxPC3 and the mouse embryo fibroblast Balb/3T3
Clone A31 cell line, the latter representing a model for non-tumour cells. In general, the compounds did not show an appreciable cytotoxic activity on both cell lines (IC50 > 250 μM).
However, it is worthy of notice that a relative decrease in cell viability was observed for the tumour cell line BxPC3 treated with 125 and 250 μM of 9[CF3SO3] and 250 μM of 1, while at the same
concentrations cell viability of the normal cell line Balb3T3 CloneA31 was comparable to the untreated control (Figure 4).
Figure 4. Cell viability determined by WST-1 assay of BxPC3 (left) and Balb3T3 CloneA31 (right) cell lines
exposed to compounds 1, 4, 5 and 9[CF3SO3]. Results are expressed as % with respect to the control.
Our data on pancreatic cancer cells agree with those previously published by Sadler et al.,Error: Reference source not found indicating that [Ru(p-cymene)(aa)Cl] complexes [from aaH = glycine (8), L-alanine, L-phenylalanine, D-alanine, -alanine] do not possess a meaningful cytotoxicity against ovarian cancer cells (see Introduction). This fact was explained on the basis of the rapid and extensive formation of the aquo species, [Ru(η6-cymene)(2N,O-aa)(H
2O)]+, which was supposed to be
deactivated by coordination to biological components before reaching the tumour targets. The possible intramolecular replacement of water by a donor atom within the amino acidate side chain, as it has been ascertained in the case of 4 (Scheme 2), might retard reactions with biological substrates but might also represent an alternative mechanism of inhibition. In this regard, it should be mentioned that compounds [Ru(η6-p-cymene)(κ2N,O,S-aa)]X (aa = L/DL-methionine, X = Cl, NO
3), containing a tridentate α-amino
acidate ligand, were found to be ineffective against A2780 cancer cells.Error: Reference source not found On the other hand, the absence of cytotoxicity has been observed here even when a triphenylphosphine co-ligand is introduced, affording a relatively inert cationic complex (9+). This fact
suggests that the intrinsic nature of the amino acidate skeleton, other than considerations on the rate of chloride dissociation (see above), could play some critical role to render the complexes non cytotoxic. This idea is corroborated by previous results pointing out that the coordination of a triphenylphosphine ligand to the general [Ru(p-cymene)Cl] frame usually determines an increased antiproliferative effect.Error: Reference source not found,43,44 Such effect has been attributed to the enhanced
lipophilicity supplied by the phosphine group, favouring the cellular uptake of the resulting compounds.
As the story of NAMI-A and RAPTA-C teaches, a poor cytotoxicity in vitro does not mean a poor anticancer activity.Error: Reference source not found,45 Due to their solubility and stability in aqueous
medium (besides possible Cl─/water exchange), Ru(II) arene α-amino acidate complexes may be
suitable candidates for in vivo investigation. This idea is reinforced by the relative, slight selectivity displayed by 1 and 9[CF3SO3] at high concentrations (see above).
Conclusions
We have reported the synthesis and the extensive characterization of a series of Ru(II) arene complexes containing α-amino acidate ligands (from naturally occurring α-amino acids). Although compounds belonging to this family already appeared in the literature and studied for their catalytic and anticancer applications, herein we have provided new insights in these two directions. Thus, the Ru(II) α-amino acidate compounds have been investigated as catalytic precursors in the asymmetric THR of acetophenone, including under microwave-assisted aqueous conditions, that were scarcely explored to date. Excellent results have been achieved in terms of selectivity, working in isopropanol under benchmark conditions. The catalysts are generally effective even in water medium in the absence of a base, either under MW irradiation or with conventional heating. Some crucial role exerted by the α-amino acidate structure seems to be relevant to the catalytic results. More precisely, the best outcomes,
in terms of activity and enantioselectivity, have been achieved with those complexes containing a bicyclic skeleton, derived from coordination of L-prolinate or trans-4-hydroxy-L-prolinate. On the other hand, the presence of potential donor atoms in the amino acidic side chain determines a worse performance, which has been related to the possibility of the ligand to behave as a tridentate one. This feature disfavours the availability of a vacant coordination site at the metal centre, which is necessary to the critical generation of hydride species.
A selection of Ru(II) -amino acidate compounds, chosen for a preliminary cytotoxicity study, did not show appreciable in vitro activity against pancreatic cancer cells, regardless the nature of the -amino acidate ligand. This outcome is in agreement with previous tests performed using analogous compounds on other cancer cell lines, and might be ascribable to structural features other than the fast deactivation of the complexes via Ru-Cl hydrolytic cleavage, as previously hypothesized. Anyway, on account of some interesting properties (water solubility, stability, presence of a naturally derived ligand, selectivity towards cancer cells compared to normal cells at high concentrations), this class of compounds could be considered for future in vivo investigations.
Experimental section
1) General experimental details.
[Ru(η6-p-cymene)Cl
2]2 was prepared according to the literature from RuCl3·xH2O (99.9%, Alfa Aesar)
and α-phellandrene (Sigma Aldrich).46 The other reactants and solvents were obtained from Alfa Aesar,
Sigma Aldrich or TCI Europe and were of the highest purity available. 1.0 M NaOH solution in water was prepared from Normex solution (Carlo Erba) and standardized by potassium hydrogen phthalate titration before use. All operations were carried out in air with common laboratory glassware. All isolated Ru complexes were air-stable; nevertheless storage under N2 is recommended for long periods
DRX400 instrument equipped with a BBFO broadband probe. Chemical shifts (expressed in parts per million) are referenced to the residual solvent peaks (1H, 13C) 47 or to external standard (19F to CFCl
3, 31P to 85% H
3PO4, 35Cl to 1 M NaCl in D2O). Spectra were assigned with the assistance of DEPT-135, 1H-1H (COSY) and 1H-13C (gs-HSQC and gs-HMBC) correlation experiments.48 Diastereomeric ratios
were calculated based on 1H NMR spectroscopy. NMR resonances related to minor diastereomer and
superimposed to those belonging to the major diastereomer are reported in brackets. Infrared spectra were recorded on a Perkin Elmer Spectrum One FT-IR spectrometer, equipped with a UATR sampling accessory. IR spectra were processed with Spectragryph software.49 Carbon, hydrogen and nitrogen
analyses were performed on a Vario MICRO cube instrument (Elementar). Melting points and decomposition temperatures were determined on a STMP3 Stuart scientific instrument with a capillary apparatus.
2) Synthesis and characterization of compounds.
Synthesis of [Ru(η6-p-cymene)(κ2N,O-L-glutaminato)Cl], 1 (Scheme 5).
Scheme 5. Structure of 1 (numbering refers to carbon atoms).
A red suspension of [Ru(η6-p-cymene)Cl
2]2 (252 mg, 0.412 mmol) and L-glutamine (120 mg, 0.823
mmol) in MeOH (5 mL) was treated with 1.0 M NaOH (0.85 mL, 0.85 mmol). The resulting orange opalescent solution was stirred at ambient temperature for 14 hours. The progress of reaction was
checked by TLC (silica gel/MeOH) therefore volatiles were removed under vacuum. The residue was suspended in boiling MeOH, loaded on a short silica column and an orange band was eluted with boiling MeOH. The eluted solution was taken to dryness under vacuum, then the residue was re-dissolved in boiling EtOH and filtered. Volatiles were removed under vacuum from the filtrate and the resulting orange solid was washed with Et2O and dried under vacuum (50°C). Yield: 270 mg, 79%. The
title compound is soluble in DMSO and H2O, moderately soluble in MeOH and hot EtOH and insoluble
in other common organic solvents. Orange, needle-like crystals suitable for X-ray analysis were obtained from a DMSO:MeOH (1:1 v/v) solution of 1 layered with iPrOH (or acetone) and settled aside at -20°C. Anal. calcd. for C15H23ClN2O3Ru: C, 43.32; H, 5.57; N, 6.74. Found: C, 43.45; H, 5.48; N,
6.62. Mp: decomp. at 220-225°C (black liquid). IR (solid state): ῦ/cm-1 = 3338m-sh (ν
NH2), 3284m
(νNH2), 3210m, 3163m, 3136m, 2957w, 2923w, 2874w, 1670m (νC12=O), 1624m-sh (νasym,CO2), 1590s-br
(δNH2), 1502w, 1466w, 1437m, 1417m, 1394s (νsym,CO2), 1343m, 1318m, 1281m, 1269m, 1188m,
1165m, 1139m, 1103m, 1092m, 1063w, 1040m, 1003w, 926w-sh, 918m, 889w, 868w, 853m, 805m, 779w, 708w, 679w, 666w. Diastereomeric ratio: 65:35 (1H NMR, DMSO-d
6); the d.r. was consistent
among distinct preparations of compound 1. Major diastereomer. 1H NMR (DMSO-d
6): δ/ppm = 7.42 (s, 1H, C12-NH), 6.81 (s, 1H, C12-NH’), 6.10 (t, 2J HH = 3JHH = 8.5 Hz, 1H, C9-NH), 5.64 (d, 3JHH = 4.9 Hz, 1H, C4’-H), 5.54 (d, 3J HH = 5.6 Hz, 1H, C4-H), 5.43 (d, 3JHH = 5.5 Hz, 1H, C3-H), 5.30 (d, 3JHH = 5.6 Hz, 1H, C3’-H), 4.69 (t, 2J HH = 3JHH = 8.5 Hz, 1H, C9-NH’), 2.73 (hept, 3JHH = 6.5 Hz, 1H, C6-H), 2.69–2.64 (m, 1H, C9-H), 2.20 (t, 3J HH = 7.3 Hz, 2H, C11-H), 2.06 (s, 3H, C1-H), 1.91–1.75 (m, 1H, C10-H), 1.68–1.57 (m, 1H, C10-H’), 1.24–1.16 (m, 6H, C7-H + C7’-H). 13C{1H} NMR (DMSO-d 6): δ/ppm = 180.3 (C8), 174.6 (C12), 99.4 (C5), 95.0 (C2), 82.2 (C4), 80.0 (C4’), 79.9 (C3’), 79.0 (C3), 54.1 (C9), 31.7 (C11), 30.2 (C6), 28.7 (C10), 22.4, 22.0 (C7 + C7’), 17.6 (C1). 13C{1H} NMR (CH3OH/C6D6 coaxial): δ/ppm = 184.3 (C8), 178.2 (C12), 101.7 (C5), 96.9 (C2); 83.7, 81.4, 81.1, 80.6 (C3/C3’/C4/C4’); 57.7 (C9), 32.5 (C11), 30.1 (C6), 29.0 (C10); 22.7, 22.1 (C7/C7’); 18.0 (C1). Minor
diastereomer. 1H NMR (DMSO-d 6) : δ/ppm = 7.38 (s, 1H, C12-NH), 7.20 (s, 1H, C12-NH’), 6.73 (s, 1H, C9-NH), 5.70 (d, 3J HH = 5.6 Hz, 1H, C4-H), 5.68–5.65 (m, 1H, C4’-H), 5.47–5.44 (m, 2H, C3-H + C3’-H), 2.99-2.88 (m, 1H, C9-H + C9-NH’), {2.73 (C6-H)}, 2.14 (t, 3J HH = 7.4 Hz, 2H, C11-H), 2.03 (s, 3H, C1-H), {1.91–1.75 (C10-H)}, 1.56–1.44 (m, 1H, C10-H’), {1.24–1.16 (C7-H + C7’-H)}. 13C{1H} NMR (DMSO-d 6): δ/ppm = 178.7 (C8), 174.3 (C12), 99.5 (C5), 95.4 (C2), 82.0 (C4’), 80.0 (C4), 79.7 (C3’), 79.2 (C3), 56.0 (C9), 31.4 (C11), {30.2 (C6)}, 30.0 (C10), {22.4 (C7)}, 21.8 (C7’), 17.7 (C1). 13C{1H} NMR (CH 3OH/C6D6 coaxial): δ/ppm = 101.8 (C5), 97.2 (C2); 83.5, 81.3, 81.0, {80.6} (C3/C3’/C4/C4’); 56.1 (C9), 32.0 (C11), 30.5 (C10), {30.1 (C6)}; 22.6, 22.0 (C7/C7’); 17.9 (C1). No variation in the 1H spectrum of 1 in DMSO-d
6 was observed after 120 days at ambient
temperature.
Synthesis of [Ru(η6-p-cymene)(κ2N,O-L-homoserinato)Cl], 2 (Scheme 6).
Scheme 6. Structure of 2 (numbering refers to carbon atoms).
The synthesis was performed as described for 1, using [Ru(η6-p-cymene)Cl
2]2 (254 mg, 0.415 mmol),
L-homoserine (100 mg, 0.839 mmol) and 1.0 M NaOH (0.85 mL, 0.85 mmol) in MeOH (5 mL). After 3.5 hours, the progress of reaction was checked by TLC (silica gel/MeOH) and volatiles were removed under vacuum. The residue was reflux extracted with CH2Cl2 and volatiles were removed from the
resulting solution. The yellow residue was washed with Et2O and dried under vacuum (50°C). Yield:
acetone, CH2Cl2 and insoluble in Et2O. Crystals suitable for X-ray analysis were obtained from a
MeOH solution of 2 layered with Et2O (or acetone) and settled aside at -20°C. Anal. calcd. for
C14H22ClNO3Ru: C, 43.24; H, 5.70; N, 3.60. Found: C, 43.10; H, 5.58; N, 3.61. Mp: decomp. at
220-224°C (black liquid). IR (solid state): ῦ/cm-1 = 3427m (ν
OH), 3326w (νNH2), 3292w (νNH2), 3219w-br
(νNH2), 3120w, 2964w, 2927w, 2895w, 2875w, 1595s (νasym,CO2), 1580s-sh (δNH2), 1499w, 1470w, 1392s
(νsym,CO2), 1367s, 1340m, 1281w, 1258w, 1191w, 1165w, 1138w, 1091w, 1072m, 1039s, 1003w, 977w,
956w, 908w, 887w, 868m, 823w, 805m, 795w-sh, 778w, 712w, 672w. Diastereomeric ratio: 65:35 (1H
NMR, CD3OD); the d.r. was consistent among distinct preparations of compound 2. Major
diastereomer. 1H NMR (CD 3OD): δ/ppm = 6.09 (t, 2JHH = 3JHH = 7.6 Hz, 1H, NH), 5.67 (m, 1H, C4’-H), 5.61 (d, 3J HH = 5.8 Hz, 1H, C4-H), 5.51–5.47 (m, 1H, C3-H), 5.40 (d, 3JHH = 5.8 Hz, 1H, C3’-H), 4.63 (t, 2J HH = 3JHH = 7.7 Hz, 1H, NH’), 3.78–3.73 (m, 2H, C11-H), 3.26–3.17 (m, 1H, C9-H), 2.84 (hept, 3J HH = 6.9 Hz, 1H, C6-H), 2.17 (s, 3H, C1-H), 2.02–1.95 (m, 1H, C10-H), 1.85–1.77 (m, 1H, C10-H’), 1.34–1.27 (m, 6H, C7-H + C7’-H). 13C{1H} NMR (CD 3OD): δ/ppm = 184.9 (C8), 102.3 (C5), 97.4 (C2), 83.8 (C4), 81.76 (C3’), 81.7 (C4’), 81.0 (C3), 60.7 (C11), 55.8 (C9), 35.7 (C10), 32.1 (C6), 22.9 (C7/C7’), 22.5 (C7/C7’), 18.3 (C1). 35Cl NMR (CD 3OD): δ/ppm = -25.2 (Δν1/2 = 3.2∙102 Hz). Minor diastereomer. 1H NMR (CD 3OD): δ/ppm = 6.89–6.79 (m, 1H, NH), {5.67 (C4-H + C4’-H)}, {5.51– 5.47 (C3-H + C3’-H)}, 3.73–3.68 (m, 2H, C11-H), 3.46-3.40 (m, 1H, NH’), 3.39–3.35 (m, 1H, C9-H), {2.84 (C6-H)}, 2.15 (s, 3H, C1-H), 2.09–2.02 (m, 1H, C10-H), 1.78–1.70 (m, 1H, C10-H’), {1.34–1.27 (C7-H + C7-H’)}. 13C{1H} NMR (CD 3OD): δ/ppm = 183.5 (C8), 102.2 (C5), 97.5 (C2), 83.9 (C4/C4’), 81.83 (C4/C4’), 81.4 (C3/C3’), 81.1 (C3/C3’), 60.0 (C11), 57.1 (C9), 37.6 (C10), {32.1 (C6)}, {22.9 (C7/C7’)}, 22.4 (C7/C7’), 18.4 (C1). No variation in the 1H spectrum of 2 in CD
3OD was observed
after 73 days at ambient temperature.
Scheme 7. Structure of 3 (numbering refers to carbon atoms).
Compound [Ru(η6-p-cymene)Cl
2]2 (243 mg, 0.397 mmol) was added to a suspension of L-tyrosine (144
mg, 0.795 mmol) and KOH (45 mg, 0.80 mmol) in H2O:MeOH (1:4 v/v, 10 mL). The resulting yellow
ochre suspension was stirred at ambient temperature for 2.5 hours and the progress of reaction was checked by TLC (silica gel/MeOH). Volatiles were then removed under vacuum (40 °C). The residue was suspended in boiling MeOH, loaded on a short silica column and an orange band was eluted with boiling MeOH. The eluted solution was taken to dryness under vacuum and the residue was re-dissolved in boiling EtOH and filtered. Volatiles were removed under vacuum from the filtrate and the resulting golden yellow solid was washed with Et2O and dried under vacuum (50°C). Yield: 270 mg,
75%. The compound is soluble in H2O, MeOH and hot EtOH, poorly soluble in acetone, CH2Cl2 and
insoluble in Et2O. Anal. calcd. for C19H24ClNO3Ru: C, 50.61; H, 5.36; N, 3.11. Found: C, 50.47; H,
5.44; N, 3.18. Mp: decomp. at 235-245°C (black liquid). IR (solid state): ῦ/cm-1 = 3560-3300w-br
(νOH), 3252m (νNH2), 3212m (νNH2), 3134m, 3072m, 2965m, 2928m, 2875m, 2816w, 2689w, 1623s
(νasym,CO2), 1610s, 1590s (δNH2), 1514s, 1468m, 1464m, 1453m, 1445m, 1437m, 1385s (νsym,CO2), 1347m,
1326m, 1265s, 1239s, 1200m-sh, 1172m, 1144m, 1106m, 1088m, 1056m, 1033w, 1005w, 948w, 911w, 869m, 848m, 826m, 807m, 751w, 729w, 720w, 691w, 673w, 654w. Diastereomeric ratio: 61:39 (1H NMR, CD
3OD). Major diastereomer. 1H NMR (CD3OD): δ/ppm = 7.15 (d, 3JHH = 8.1 Hz, 2H,
C12-H), 6.81 (d, 3J
HH = 8.2 Hz, 2H, C13-H), 5.53 (d, 3JHH = 5.7 Hz, 1H, C4’-H), 5.47 (d, 3JHH = 5.9 Hz,
1H,C4-H), 5.32 (d, 3J
C9-H), 2.96 (dd, 2J
HH = 14.6 Hz, 3JHH = 6.7 Hz, 1H, C10-H), 2.90 (dd, 2JHH = 14.5 Hz, 3JHH = 4.9 Hz, 1H,
C10-H’), 2.71–2.62 (m, 1H, C6-H), 2.02 (s, 3H, C1-H), 1.27–1.19 (m, 6H, C7-H + C7’-H). *partially superimposed with CHD2OD signal. 13C{1H} NMR (CD3OD): δ/ppm = 184.3 (C8), 157.9 (C14), 131.8
(C12), 128.4 (C11), 116.8 (C13), 102.3 (C5), 97.5 (C2), 83.6 (C4), 81.7 (C3’/C4’), 81.6 (C3’/C4’), 80.7 (C3), 57.0 (C9), 38.6 (C10), 32.0 (C6), 22.9 (C7), 22.3 (C7’), 18.1 (C1). Minor diastereomer. 1H NMR (CD3OD): δ/ppm = 7.05 (d, 3JHH = 8.1 Hz, 2H, C12-H), 6.74 (d, 3JHH = 8.2 Hz, 2H, C11-H), 5.63 (pseudo-t, 3J HH = 4.2 Hz, 2H, C4-H + C4’-H), 5.46–5.41 (m, 2H, C3-H + C3’-H), 3.39–3.36* (m, C9-H), 3.08 (dd, 2J HH = 14.4 Hz, 3JHH = 3.8 Hz, 1H, C10-H), 2.81–2.69 (m, 2H, C10-H’), 2.82–2.73 (m,
1H, C6-H), 2.12 (s, 3H, C1-H), {1.27–1.19 (C7-H + C7’-H)}. *partially superimposed with CHD2OD
signal. 13C{1H} NMR (CD
3OD): δ/ppm = 183.3 (C8), 157.7 (C14), 131.6 (C12), 128.7 (C11), 116.7
(C13), {102.3 (C5)}, 97.7 (C2), 83.7 (C4), 81.9 (C3), 81.5 (C4’), 80.8 (C3’), 59.3 (C9), 40.1 (C10), 32.1 (C6), 22.8 (C7), 22.4 (C7’), 18.3 (C1). No variation in the 1H spectrum of 3 in CD
3OD was
observed after 23 days at ambient temperature.
Synthesis of [Ru(η6-p-cymene)(κ2N,O-L-serinato)Cl], 4 (Scheme 8).Error: Reference source not
foundb
Scheme 8. Structure of 4 (numbering refers to carbon atoms).
The title compound was prepared as described for 2, using [Ru(η6-p-cymene)Cl
2]2 (439 mg, 0.717
Reaction time: 2 h. Alternatively, the compound can be purified by silica chromatography (Eluent: EtOH:MeOH 1:1 v/v). Yellow solid. Yield: 495 mg, 92%. The title compound is soluble in H2O,
DMSO, MeOH, less soluble in EtOH, poorly soluble in iPrOH, CHCl
3, CH2Cl2 and insoluble in Et2O.
Anal. calcd. for C13H20ClNO3Ru: C, 41.66; H, 5.38; N, 3.74. Found: C, 41.50.; H, 5.49; N, 3.81. Mp:
decomp. at 200°C (black solid), 217°C (black liquid). IR (solid state): ῦ/cm-1 = 3500-3200w-br (ν OH),
3292m (νNH2), 3247m (νNH2), 3059m, 2958m, 2965m-sh, 2932m, 2899w, 2872w, 1620s (νasym,CO2),
1575m-sh (δNH2), 1502w, 1472m, 1453m, 1439m-sh, 1395s (νsym,CO2), 1378m-sh, 1359m, 1346m,
1282m, 1223w, 1199w, 1164m, 1116w, 1094m, 1054s, 1043m-sh, 1010w, 991m, 962w, 915m, 874m, 857m, 818w, 807w, 675w. Diastereomeric ratio: 59:41 (1H NMR, CD
3OD and DMSO-d6); the d.r. was
consistent among distinct preparations of compound 4. Major diastereomer. 1H NMR (CD
3OD): δ/ppm = 5.77 (m, 1H, NH), 5.69 (d, 3J HH = 6.1 Hz, 1H, C4’-H), 5.65 (d, 3JHH = 5.9 Hz, 1H, C4-H), 5.48 (d, 3JHH = 5.9 Hz, 1H, C3-H), 5.44 (d, 3J HH = 5.8 Hz, 1H, C3’-H), 4.59 (m, 1H, NH’), 3.80 (dd, 2JHH = 11.3 Hz, 3J HH = 4.8 Hz, 1H, C10-H), 3.69 (dd, 2JHH = 11.2 Hz, 3JHH = 3.6 Hz, 1H, C10-H’), 3.08 (t, 3JHH = 4.3 Hz, 1H, C9-H), 2.85 (hept, 3J HH = 7.6 Hz, 1H, C6-H), 2.18 (s, 3H, C1-H), 1.33–1.28 (m, 6H, C7-H + C7’-H). 1H NMR (DMSO-d 6): δ/ppm = 5.91 (t, 2JHH = 3JHH = 9.0, NH), 5.70 (d, 3JHH = 5.5 Hz, 1H, C4-H/C4’-H), 5.58 (d, 3J HH = 5.8 Hz, 1H, C4-H/C4’-H), 5.45 (m, C3-H/C3’-H), 5.35 (d, 3JHH = 5.7 Hz, 1H, C3-H/C3’-H), 4.48 (t, 2J HH = 3JHH = 9.0 Hz, NH’), 3.76 (dd, 2JHH = 10.9 Hz, 3JHH = 3.1 Hz, 1H, C10-H), 3.63 (dd, 2J HH = 10.9 Hz, 3JHH = 6.6, 1H, C10-H’), 3.16 (s, 1H, OH), 3.09–3.02 (m, 1H, C9-H), 2.73 (hept, 3J HH = 6.8 Hz, 1H, C6-H), 2.05 (s, 3H, C1-H), 1.24–1.16 (m, 6H, C7-H + C7’-H). 13C{1H} NMR (CD3OD): δ/ppm = 183.4 (C8), 102.2 (C5), 97.5 (C2), 83.7 (C4), 82.1 (C3’), 81.7 (C4’), 80.8 (C3), 63.3 (C10), 58.4 (C9), 32.0 (C6), 22.8 (C7), 22.5 (C7’), 18.3 (C1). 13C{1H} NMR (DMSO-d 6): δ/ppm = 179.2 (C8), 99.4 (C5), 95.1 (C2); 82.1, 80.2, 80.1, 79.1 (C3/C3’/C4/C4’); 61.9 (C10), 56.8 (C9), 30.18 (C6), 22.4 (C7), 22.0 (C7’), 17.6 (C1). Minor diastereomer. 1H NMR (CD 3OD): δ/ppm = 6.83 (m, 1H, NH), 5.73–5.70 (m, 2H, C3-H + C3’-H), 5.53 (m, 2H, C4-H + C4’-H), 3.70 (d, 3J HH = 4.7 Hz, 2H,
C10-H + C10-C10-H’), 3.40–3.35 (m, 2C10-H, C9-C10-H + NC10-H’), {2.85 (C6-C10-H)}, 2.15 (s, 3C10-H, C1-C10-H), {1.33-1.28 (C7-C10-H + C7’-H)}. 1H NMR (DMSO-d 6): δ/ppm = 7.04 (t, 2JHH = 3JHH = 7.7 Hz, 1H, NH), {5.70 (C4-H/C4’-H)}, 5.64 (d, 3J HH = 5.9 Hz, C4-H/C4’-H), 5.47 (d, 3JHH = 6.0 Hz, C3-H/C3’-H), {5.45 (C3-H/C3’-H)}, {3.16 (OH)}, {2.73 (C6-H)}, 3.01–2.94 (m, 1H, NH’), 2.02 (s, 3H, C1-H), {1.24–1.16 (C7-H + C7’-H)}. 13C{1H} NMR (CD 3OD): δ/ppm = 181.5 (C8), 102.1 (C5), 97.7 (C2), 84.2 (C4), 81.6 (C3), 81.2 (C3’+C4’), 63.7 (C10), 60.5 (C9), 32.1 (C6), 23.0 (C7), 22.3 (C7’), {18.3 (C1)}. 13C{1H} NMR (DMSO-d6): δ/ppm = 177.6 (C8), {99.4 (C5)}, 95.6 (C2); 82.5, 79.9, 79.6, 79.3 (C3/C3’/C4/C4’); 62.7
(C10), 58.6 (C9), 30.22 (C6), 22.5 (C7), 21.8 (C7’), {17.6 (C1)}. No variation in the 1H spectrum of 4
in CD3OD was observed after 81 days at ambient temperature. The 1H spectrum of 4 in DMSO-d6 after
40 days at ambient temperature showed no variation in the diastereomeric ratio but the release of a minor amount (≈ 7%) of p-cymene. 1H NMR (DMSO-d
6): δ/ppm = 7.08 (pseudo-q, 3JHH = 8.4 Hz, 4H),
2.82 (hept, 3J
HH = 7.0 Hz, 1H), 2.24 (s, 3H), 1.16 (d, 3JHH = 6.9 Hz, 6H)]. Complete dissociation of
p-cymene with formation of a brown color occurred upon heating the solution to 120°C for 1.5 hours.
Alternative preparation of 4: attempts to modify the diastereomeric ratio.
A solution of [Ru(η6-p-cymene)Cl
2]2 (66 mg, 0.11 mmol) and L-serine (25 mg, 0.24 mmol) in MeOH (5
mL) was stirred at ambient temperature for 2 hours and then at reflux for 2 hours. Under these conditions no reaction took place and only the two reagents were identified in solution by NMR analysis. Therefore the solution was cooled to -15°C and 1.0 M NaOH (0.25 mL, 0.25 mmol) was added dropwise. The resulting yellow solution was allowed to warm up to ambient temperature then volatiles were removed under vacuum. The yellow residue was dissolved in CD3OD. The 1H NMR
spectrum indicated the formation of 4 with a 58:42 diastereomeric ratio. Therefore, the solid was dissolved in MeOH and AgNO3 (37 mg, 0.22 mmol) was added. The resulting suspension was stirred at
ambient temperature for 2 hours under protection from the light then KCl (17 mg, 0.23) was added at 0°C. The resulting yellow suspension was allowed to heat to ambient temperature, stirred for 2 hours
and then filtered. Volatiles were removed under vacuum from the filtrate solution and the resulting yellow solid was dissolved in CD3OD. The NMR spectrum indicated no variation in the diastereomeric
ratio of 4.
Synthesis of [Ru(η6-p-cymene)(κ2N,O-L-prolinato)Cl], 5 (Scheme 9).Error: Reference source not
found,Error: Reference source not founda
Scheme 9. Structure of 5 (numbering refers to carbon atoms).
The synthesis was performed as described for 1, using [Ru(η6-p-cymene)Cl
2]2 (248 mg, 0.405 mmol),
L-proline (94 mg, 0.816 mmol) and 1.0 M NaOH (0.82 mL, 0.82 mmol) in MeOH (5 mL). After 1.5 hours, the progress of reaction was checked by TLC (silica gel/MeOH) and volatiles were removed under vacuum. The residue was suspended in CH2Cl2 and the suspension was filtered on celite. The
filtrate was taken to dryness under vacuum and the resulting yellow solid was washed with Et2O and
dried under vacuum (40°C). Yield: 302 mg, 97%. The compound is soluble in H2O, MeOH and
CH2Cl2, moderately soluble in THF, acetone, iPrOH and insoluble in Et2O. Anal. calcd. for
C15H22ClNO2Ru: C, 46.81; H, 5.76; N, 3.64. Found: C, 46.60; H, 5.89; N, 3.45. Mp: decomp. at
160-170°C (orange liquid + evolution of gas), 211-217°C (black liquid). IR (solid state): ῦ/cm-1 = 3450w-br
(νNH), 3180w-br, 3059w, 2962m, 2928m-sh, 2871m, 1608s-br (νasym,CO2), 1566m-sh, 1531w, 1497w,
1470m, 1447m, 1380s-sh (νsym,CO2), 1361s-br, 1317m, 1299m, 1200w, 1114w, 1089w, 1056m, 1036m,
CD3OD); the d.r. was consistent among distinct preparations of compound 5. Major diastereomer. 1H NMR (CD3OD): δ/ppm = 5.71 (d, 3JHH = 5.9 Hz, 1H, C4-H), 5.56 (s, 2H, C3’-H + C4’-H), 5.40 (d, 3JHH = 5.9 Hz, 1H, C3-H), 3.95 (dd, 2J HH = 11.0 Hz, 3JHH = 5.7 Hz, 1H, C12-H), 3.51–3.43 (m, 1H, C9-H), 3.09 (td, 2J HH = 11.3 Hz, 3JHH = 5.8 Hz, 1H, C12-H’), 2.84 (hept, 3JHH = 7.0 Hz, 1H, C6-H), 2.17 (s, 3H, C1-H), 2.16–2.11 (m, 1H, C10-H’), 1.96–1.90 (m, 1H, C11-H), 1.79–1.67 (m, 2H, C10-H + C11-H’), 1.35 (d, 3J HH = 6.9 Hz, 3H, C7’-H), 1.30 (d, 3JHH = 6.9 Hz, 3H, C7-H). 13C{1H} NMR (CD3OD): δ/ppm = 186.4 (C8), 102.2 (C5), 96.6 (C2), 84.8 (C4’), 84.1 (C3), 80.9 (C3’), 80.5 (C4), 63.4 (C9), 58.3 (C12), 32.2 (C6), 30.1 (C10), 27.9 (C11), 22.9 (C7), 22.4 (C7’), 18.3 (C1). 35Cl NMR (CD 3OD): δ/ppm
= ─28.0 (Δν1/2 = 3.0∙102 Hz). Minor diastereomer. 1H NMR (CD3OD): δ/ppm = 5.71–5.68 (m, 1H,
C4-H), 5.62 (d, 3J
HH = 5.3 Hz, 1H, C3’-H/C4’H), 5.48 (d, 3JHH = 5.7 Hz, 1H, C3’-H/C4’H), 5.22 (d, 3JHH =
5.6 Hz, 1H, C3-H), 3.65–3.47 (m, 1H, C12-H), {3.51–3.43 (C9-H)}, 3.24–3.17 (m, 1H, C12-H’), {2.84 (C6-H)}, {2.16–2.11 (m, 5H, C1-H + C10-H’ + C11-H)}, {1.79–1.67 (m, 2H, C10-H + C11-H’)}, 1.32–1.28 (m, 6H, C7-H + C7’-H). No variation in the 1H spectrum of 5 in CD
3OD was observed after
47 days at ambient temperature. NMR spectra of 5 in CDCl3 displayed broad resonances even at low
concentration (≈ 2∙10-3 M) due to association phenomena. 1H NMR (CDCl
3): δ/ppm = 8.14 (m-br, 0.3H, NH); 5.76, 5.64, 5.60, 5.55, 5.38, 5.29 (m-br, 4H, C3-H + C3’-H + C4-H + C4’-H), 4.65 (s, 0.4H, NH); 4.00, 3.82, 3.59, 3.32, 3.00 (m-br, 5H, C9-H + C10-H + C12-H); 2.86 (m-br, 1H, C6-H); 2.19, 2.14 (s, 3H, C1-H); 1.89, 1.78 (m-br, 2H, C11-H), 1.31-1.22 (m, 6H, C7-H + C7’-H). 13C{1H} NMR (CDCl 3): δ/ppm = 183.5-183.4 (br, C8); 101.7, 100.8 (C5); 95.3, 95.0 (C2); 83.5, 83.2, 82.5, 81.9, 80.7, 79.7, 79.0 (C3 + C3’ + C4 + C4’); 62.2 (C9); 57.3, 52.2 (C12); 31.0-30.9 (br, C6); 30.2, 29.0 (C10); 27.5, 27.2 (C11); 22.7, 22.6, 22.4, 22.3 (C7 + C7’); 18.4, 18.3 (C1).
Synthesis of [Ru(η6-p-cymene)(κ2N,O-L-hydroxyprolinato)Cl], 6 (Scheme 10).Error: Reference
Scheme 10. Structure of 6 (numbering refers to carbon atoms).
The synthesis was performed as described for 1, using [Ru(η6-p-cymene)Cl
2]2 (74 mg, 0.12 mmol),
trans-4-hydroxy-L-proline (33 mg, 0.25 mmol) and 1.0 M NaOH (0.25 mL, 0.25 mmol) in MeOH (3 mL). After 2 hours, the progress of reaction was checked by TLC (silica gel/MeOH) and volatiles were removed under vacuum (40°C). The residue was suspended in EtOH, loaded on top of a short silica column and an orange band was eluted with boiling EtOH. The eluted solution was taken to dryness under vacuum and the resulting yellow solid was washed with Et2O and dried under vacuum (40°C).
Yield: 91 mg, 94%. The compound is soluble in H2O, MeOH and EtOH, poorly soluble in acetone,
CH2Cl2, CHCl3 and insoluble in Et2O. Anal. calcd. for C15H22ClNO3Ru: C, 44.94; H, 5.53; N, 3.49.
Found: C, 45.02; H, 5.50; N, 3.40. Mp: decomp. at 206°C (black solid), 236°C (black liquid). IR (solid state): ῦ/cm-1 = 3423w-br (ν NH), 3318w-br (νOH), 3207w, 3160w, 3061w, 3041w, 2997w, 2959m, 2924w, 2869w, 1604s (νasym,CO2), 1497w, 1470w-sh, 1454m, 1435m, 1421m, 1381m (νsym,CO2), 1361m, 1324m, 1308m, 1278w, 1209m, 1190w-sh, 1138m, 1113w, 1090w, 1052s, 1069m-sh, 1062m-sh, 1052m, 992w, 954w, 929m, 903w, 866m, 855m, 804m, 788w, 761w, 723w, 687w, 670w. Diastereomeric ratio: 67:33 (1H NMR, CD
3OD). Major diastereomer. 1H NMR (CD3OD): δ/ppm =
5.73 (d, 3J HH = 5.8 Hz, 1H, C4-H), 5.57 (pseudo-t, 3JHH = 5.8 Hz, 2H, C3’-H + C4’-H), 5.42 (d, 3JHH = 5.9 Hz, 1H, C3-H), 4.42-4.38 (m, 1H, C11-H), 3.87 (d, 2J HH = 11.7 Hz, 1H, C12-H), 3.66 (t, 3JHH = 8.8 Hz, 1H, C9-H), 3.22 (dd, 2J HH = 12.0 Hz, 3JHH = 2.3 Hz, 1H, C12-H’), 2.84 (hept, 3JHH = 6.7 Hz, 1H, C6-H), 2.18 (s, 3H, C1-H), 2.14–2.09 (m, 1H, C10-H), 1.92 (ddd, J = 18.1, 8.8, 4.5 Hz, 1H, C10-H’), 1.34 (d, 3J HH = 6.9 Hz, 3H, C7-H), 1.32–1.28 (m, 3H, C7’-H). 13C{1H} NMR (CD3OD): δ/ppm = 186.1
