Half-Sandwich Type Platinum-Group Metal Complexes of C-Glucosaminyl Azines: Synthesis and Antineoplastic and Antimicrobial Activities

Abstract

While platinum-based compounds such as cisplatin form the backbone of chemotherapy, the use of these compounds is limited by resistance and toxicity, driving the development of novel complexes with cytostatic properties. In this study, we synthesized a set of half-sandwich complexes of platinum-group metal ions (Ru(II), Os(II), Ir(III) and Rh(III)) with an N,N-bidentate ligand comprising a C-glucosaminyl group and a heterocycle, such as pyridine, pyridazine, pyrimidine, pyrazine or quinoline. The sugar-containing ligands themselves are unknown compounds and were obtained by nucleophilic additions of lithiated heterocycles to O-perbenzylated 2-nitro-glucal. Reduction of the adducts and, where necessary, subsequent protecting group manipulations furnished the above C-glucosaminyl heterocycles in their O-perbenzylated, O-perbenzoylated and O-unprotected forms. The derived complexes were tested on A2780 ovarian cancer cells. Pyridine, pyrazine and pyridazine-containing complexes proved to be cytostatic and cytotoxic on A2780 cells, while pyrimidine and quinoline derivatives were inactive. The best complexes contained pyridine as the heterocycle. The metal ion with polyhapto arene/arenyl moiety also impacted on the biological activity of the complexes. Ruthenium complexes with p-cymene and iridium complexes with Cp* had the best performance in ovarian cancer cells, followed by osmium complexes with p-cymene and rhodium complexes with Cp*. Finally, the chemical nature of the protective groups on the hydroxyl groups of the carbohydrate moiety were also key determinants of bioactivity; in particular, O-benzyl groups were superior to O-benzoyl groups. The IC₅₀ values of the complexes were in the low micromolar range, and, importantly, the complexes were less active against primary, untransformed human dermal fibroblasts; however, the anticipated therapeutic window is narrow. The bioactive complexes exerted cytostasis on a set of carcinomas such as cell models of glioblastoma, as well as breast and pancreatic cancers. Furthermore, the same complexes exhibited bacteriostatic properties against multiresistant Gram-positive Staphylococcus aureus and Enterococcus clinical isolates in the low micromolar range.

1. Introduction

Registered platinum complexes (cisplatin, oxaliplatin and carboplatin) constitute the backbone of modern oncological chemotherapy in multiple solid tumors with poor prognosis, including a large set of carcinomas, such as ovarian cancer, sarcomas and hematological malignancies [1,2]. The applicability of platinum compounds is limited by platinum resistance and toxicity [3,4,5,6,7,8].

Novel organometallic compounds of other platinum-group metals, such as complexes of ruthenium [2,9,10,11,12,13,14,15], osmium [9,11,12,14,16,17,18,19], iridium [11,14,17,20,21] or rhodium [11,14,20,22], are being developed for the replacement of platinum drugs and have been reported to have better toxicity profiles than platinum-based drugs [23,24,25,26]. The anticancer potential of such platinum-group metal complexes is also supported by three ruthenium derivatives, i.e., NAMI-A [27], KP1019/1339 (IT-139, BOLD100) [28] and TLD-1433 [29], which are in different phases of clinical trials against neoplastic diseases such as bladder or lung cancer.

In the quest for potential substitutes for platins, the half-sandwich type complexes of platinum-group metal ions (e.g., Ru(II), Os(II), Ir(III) and Rh(III)) have emerged as a promising compound class, with a large number of representatives displaying anticancer potencies [14,15,19,21,22]. In addition to the antineoplastic effects, several of these piano-stool complexes have different antimicrobial (e.g., antibacterial [30,31,32,33,34,35,36,37,38,39], antiparasitic [40,41,42], antiviral [30,43] and antifungal [44]) properties.

We recently reported a series of half-sandwich complexes with five-membered chelate rings constructed with the use of N- and C-glycopyranosyl heterocyclic N,N-bidentate ligands (Figure 1, I) [32,45,46]. Several representatives of I displayed low micromolar or, in certain cases, submicromolar (e.g., Ia) cytostatic activities against cancer cells, in addition to proving to be selective for such cells. The antiproliferative potency of these complexes is thought to be related to reactive oxygen species production [32,45,46]. It is worth mentioning that the complexes with antineoplastic activities (e.g., Ia) were also shown to be effective against Gram-positive multiresistant bacteria [31,32]. A short summary of the structure–activity relationships (SARs) of these complexes is presented in Figure 1, while for a more detailed explanation of the SARs, the reader is referred to our previous publications [31,32,45,46]. One of the most important structural motifs related to biological efficacy is the presence of the sugar moiety O-protected with large hydrophobic acyl, preferably with benzoyl groups. This feature contributes, to a large extent, to the favorably increased lipophilic character of the biologically active complexes [32,45,46].

Apart from the above complexes, there are only two more literature examples of half-sandwich complexes with sugar-based N,N-chelators. 1,4-Bis(β-d-glycopyranosyl)tetrazenes [47] (e.g., II) and methyl 2,3-diamino-2,3-dideoxy-hexopyranosides [48] (e.g., III) were incorporated into the coordination sphere of the reported complexes. The antineoplastic effects of these organometallics were also studied, some of which were found to be cytotoxic at low micromolar concentrations against various cancer cells (II and III represent the most efficient compounds of the respective series) [47,48].

Based on the structures of the sugar-derived ligands of complexes I and III, we considered that C-glucosaminyl N-heterocycles, with an N-donor atom in the glycon and another in the heterocyclic aglycon part, are capable of forming half-sandwich type complexes with a six-membered chelate ring. In the present study, the preparation of a series of C-glucosaminyl azines and their incorporation as N,N-bidentate ligands into type IV complexes were envisaged. Biological studies were also conducted to reveal the anticancer and antibacterial potencies of the new organometallic compounds. With the exception of the glycosyl heterocyclic ligand, the main structural elements of the new complexes as depicted in formula IV were designed to be identical to those of type I complexes with biological activities. In addition, the replacement of the O-benzoyl groups of the monosaccharide unit by O-benzyl groups was envisaged in order to examine the effect of ether-type protection on the biological efficiency of the complexes.

2. Results

2.1. Chemistry

For the preparation of the planned C-glucosaminyl azine-type N,N-chelators, 3,4,6-tri-O-benzyl-2-nitro-d-glucal [49] (1) was used as the starting material with the expectation that a common general procedure can be elaborated with additions to the double bond of organometallic nucleophiles derived from the respective heterocycles [50]. In line with these plans, nitro-Michael addition [51,52,53] of 1 with lithiated six-membered heterocycles preformed from the corresponding halogenated heterocycles with n-butyl lithium or generated in situ (

Table 1. Synthesis of C-(2′-deoxy-2′-nitro-3′,4′,6′-tri-O-benzyl-β-d-glucopyranosyl)azines and their transformation into C-(2′-amino-2′-deoxy-3′,4′,6′-tri-O-benzyl-β-d-glucopyranosyl)azines.

Conditions: (i) 1.2–2.0 equiv. of Het-Hlg (Hlg = Br, I), 2.5 M solution of n-Bu-Li in hexane (1.2–2.0 equiv.), dry THF, −78 °C; (ii) Zn powder, ccHCl or 2M aq. HCl, THF-H2O (2:1), r.t.
Het		Conditions and Yields (%)
			2		3
a		i	52	ii	64
b		i	13	ii	NI *
c		i	71	ii	NI *
d		i	63	ii	7
e		i	60	ii	NI *

* Could not be isolated.

, i) resulted in a set of C-(2′-deoxy-2′-nitro-3′,4′,6′-tri-O-benzyl-β-d-glucopyranosyl)azines (2a–e) in good to acceptable yields.

In the next step, the reduction of the nitro group of compounds 2 showed much less uniform behavior. To achieve O-perbenzylated C-glucosaminyl azines, reduction of the nitro group of 2a–e by Zn-HCl (ii) was investigated first. This transformation of 2a to the expected 2-glucosaminyl pyridine (3a) was smoothly accomplished in good yield. However, similar reactions of nitro derivatives 3b–e led to multicomponent reaction mixtures, from which only the 2-glucosaminyl pyrazine 3d could be isolated in low yield. Unfortunately, our further attempts to obtain the glucosamine derivatives 3d,e from 2d,e under different reductive conditions also failed; either no reaction took place (Fe, ccHCl, THF-H₂O 1:1, 0 °C; SnCl₂, dry EtOH, reflux; SiCl₃H, DIPEA, dry CH₃CN, 0 °C; B₂(OH)₄, THF-H₂O 1:1, 80 °C) or formation of inseparable product mixtures (Sn, ccHCl, THF-H₂O 1:1, 0 °C) was observed.

Next, the synthesis of O-unprotected C-glucosaminyl azines was examined starting from compounds 2a–e and 3a (

Table 2. Synthesis of C-(2′-amino-2′-deoxy-β-d-glucopyranosyl)azines.

Conditions: (i) 1M solution of BCl3 in CH2Cl2 (5 equiv.), dry CH2Cl2, −78 °C; (ii) 1M solution of BCl3 in CH2Cl2 (4 equiv.), pentamethylbenzene (9 equiv.), dry CH2Cl2, −78 °C; (iii) H2, Pd(C), dry EtOH, reflux; (iv) Sn powder, ccHCl, THF-H2O (2:1), r.t.
Het		Conditions and Yields (%)
			4		5
a		i	85	i	42 (from 3a)
				ii	95 (from 3a)
				iii	NI * (from 4a)
b		i	89	iii	NI * (from 4b)
				iv	NI * (from 4b)
c		i	98	iii	38 (from 4c)
d		i	78	iii	66 (from 4d)
e		i	76	iii	NI * (from 4e)
				iv	29 (from 4e)

* Formation of the expected product was detected, but the compound could not be isolated in a pure state.

). A BCl₃-mediated O-debenzylation of the pyridine derivative 3a afforded the O-unprotected analog 5a in moderate yield (Table 2, i). This reaction was repeated in the presence of the cation scavenger pentamethylbenzene [54] (ii) to afford compound 5a in excellent yield.

Attempted transformation of compounds 2a–e into 5a–e in one step via simultaneous O-debenzylation and reduction of the nitro group by catalytic hydrogenation (Pd(C) or Pd(OH)₂, cat. ccHCl, EtOH, reflux,) was unsuccessful; in each case, decomposition of the starting materials (2a–e) was observed. Therefore, the synthesis of the glucosamine derivatives 5a–e from 2a–e was performed in a two-step procedure. First, the O-benzyl-protecting groups of 2a–e were removed by BCl₃ (i) to afford C-(2′-deoxy-2′-nitro-β-d-glucopyranosyl)azines 4a–e in high yields. Reduction of the nitro group of 4a–e was then examined under two conditions. Catalytic hydrogenation of 4c and 4d (iii) afforded the desired pyrimidine- and pyrazine-containing C-glucosaminyl derivatives 5c and 5d, respectively, in acceptable yields. The same conditions (iii) applied to compounds 4a,b,e resulted in complex product mixtures, in which the desired C-glucosaminyl pyridine 5a, pyridazine 5b and quinoline 5e were detected by TLC analysis; however, they could not be separated in a pure state. Treatment of 4e with Sn powder in the presence of ccHCl (iv) was also carried out to afford the target 5e in acceptable yield.

In order to obtain O-perbenzoylated C-glucosaminyl azines, a direct exchange of the O-benzyl protecting groups with benzoyl groups by a Zn(OTf)₂-mediated reaction [55] of compounds 2a–d with benzoyl chloride (

Table 3. Synthesis of C-(2′-amino-2′-deoxy-3′,4′,6′-tri-O-benzoyl-β-d-glucopyranosyl)azines.

Conditions: (i) 6 equiv. of benzoyl chloride, 2 equiv. of Zn(OTf)2, dry ClCH2CH2Cl, r.t.; (ii) Zn powder, ccHCl or 2M aq. HCl, THF-H2O (2:1), r.t.; (iii) H2, Pd(C), dry EtOH, reflux; (iv) 2 equiv. of Boc2O, 1,4-dioxane-H2O (1:1), r.t.; (v) 7.2 equiv. of benzoyl chloride, dry pyridine, 60 °C; (vi) 2 equiv. of CF3COOH, dry CH2Cl2, r.t.
Het		Conditions and Yields (%)
			6		7		8		9
a		i	88	ii	38 (from 6a)	-	-	-	-
b		i	97	ii	NI * (from 6b)	iv	NI *	iii-v	27 ** (from 4b)
				vi	88 (from 9b)
c		i	45	ii	NI * (from 6c)	iv	67	v	80 (from 8c)
				vi	96 (from 9c)
d		i	82	ii	NI * (from 6d)	iv	67	v	71 (from 8d)
				vi	96 (from 9d)
e		i	-	vi	84 (from 9e)	iv	80	v	81 (from 8e)

* Could not be isolated. ** Overall yield for three steps.

, i) was performed to afford C-(2′-deoxy-2′-nitro-3′,4′,6′-tri-O-benzoyl-β-d-glucopyranosyl)azines 6a–d in good to excellent yields. Subsequent reduction of the nitro group of the pyridine derivative 6a by Zn-HCl (ii) afforded the O-perbenzoylated glucosamine derivative 7a in moderate yield. Analogous reactions (ii) carried out with compounds 6b–d led to complex reaction mixtures, from which the desired C-glucosaminyl heterocycles 7b–d could not be isolated. For the transformation of 6b–d into 7b–d, further experiments were conducted under various reductive conditions (e.g., H₂, Pd(C) or Pd(OH)₂, dry EtOH, reflux; SnCl₂, dry EtOH, reflux; Sn, ccHCl, THF-H₂O 1:1, 0 °C; B₂(OH)₄, THF-H₂O 1:1, 80 °C); however, none of these experiments was successful.

Due to the above difficulties, another three-step procedure starting from 5c-e was applied to obtain the planned 7c–e (Table 3). Thus, the NH₂ group of 5c-e was protected first as a carbamate using Boc₂O (iv), and the resulting 8c-e were O-perbenzoylated upon treatment with benzoyl chloride (v) to afford the O- and N-protected glucosaminyl derivatives 9c–e. Finally, acid-mediated liberation of the NH₂ group in 9c–e (vi) was carried out, providing the final products 7c-e in high yields.

As mentioned earlier, the 3-(2′-amino-2′-deoxy-β-d-glucopyranosyl)pyridazine 5b could not be obtained in a pure state from 4b (Table 2). In order to obtain the O-perbenzoylated 9b, a consecutive three-step procedure starting from 4b was conducted to avoid the need to use pure intermediate 5b (Table 3). Thus, the NO₂→NH₂ transformation was carried out by catalytic hydrogenation of 4b (Table 3, iii), followed by the Boc protection of the amino group of intermediate 5b (iv); subsequent O-perbenzoylation of the resulting 8b (v) furnished the desired C-glucosaminyl pyridazine 9b in acceptable overall yields (27% for three steps). Then, standard N-Boc deprotection (vi) afforded the desired 7b in high yield.

Next, the newly prepared heterocyclic glucosamine derivatives were used as N,N-bidentate ligands in the formation of platinum-group metal half-sandwich complexes.

Treatment of O-perbenzylated 2-glucosaminyl pyridine with dichloro(η⁶-p-cymene)ruthenium(II) and -osmium(II) and dichloro(pentamethylcyclopentadienyl)iridium(III) and -rhodium(III) dimers (Ru-/Os-/Ir-/Rh-dimer) in the presence of the halide abstractor TlPF₆ afforded the expected cationic complexes Ru-3a, Os-3a, Ir-3a and Rh-3a, respectively, with six-membered chelate rings (

Table 4. Synthesis of half-sandwich platinum-group metal complexes with the O-perbenzylated and O-unprotected C-glucosaminyl azines.

Entry	Ligand	R	X	M	Product	Yield (%) *
1	3a	Bn	CH	Ru(II)	Ru-3a	74
2				Os(II)	Os-3a	64
3				Ir(III)	Ir-3a	93
4				Rh(III)	Rh-3a	83
5	3d	Bn	N	Ru(II)	Ru-3d	33
6		Bn	N	Os(II)	Os-3d	47
7	5a	H	CH	Ru(II)	Ru-5a	43

* Each complex was isolated as a single diastereoisomer.

, entries 1–4). Analogous Ru(II) and Os(II) complexes Ru-3d and Os-3d with the pyrazine derivative 3d were also obtained under similar conditions (entries 5 and 6, respectively).

Our previous studies [45,46] on other series of half-sandwich complexes constructed with glycopyranosyl azole ligands revealed that the O-protection of the hydroxyl groups of the sugar moiety by large, apolar protecting groups played a pivotal role in achieving significant biological effects. Complexes containing O-unprotected monosaccharide-based ligands proved to be biologically inactive. Nevertheless, for a comparative study of the new set of platinum-group metal complexes presented here, compound Ru-5a incorporating O-deprotected 2-glucosaminyl pyridine 5a was also synthesized (Table 4, entry 7).

Complexations of the O-perbenzoylated C-glucosaminyl azines 7a–e with the dimeric chloro-bridged platinum-group metal complexes Ru-dimer, Os-dimer, Ir-dimer and Rh-dimer were also performed under the same conditions as described above to afford the expected half-sandwich type complexes Ru-7a–Ru-7e, Os-7a–Os-7e, Ir-7a–Ir-7e and Rh-7a–Rh-7e, respectively, in good to excellent yields (

Table 5. Synthesis of half-sandwich platinum-group metal complexes with the O-perbenzoylated C-glucosaminyl azines.

Entry	Ligand	Het	M	Product	Yield (%)	Number of Isomers
1	7a		Ru(II)	Ru-7a	81	1
2			Os(II)	Os-7a	43	1
3			Ir(III)	Ir-7a	76	1
4			Rh(III)	Rh-7a	86	1
5	7b		Ru(II)	Ru-7b	88	2 (d.r. = 2:1) *
6			Os(II)	Os-7b	73	2 (d.r. = 5:4) *
7			Ir(III)	Ir-7b	69	2 (d.r. = 9:1 ) *
8			Rh(III)	Rh-7b	86	2 (d.r. = 5:1) *
9	7c		Ru(II)	Ru-7c	99	1
10			Os(II)	Os-7c	98	1
11			Ir(III)	Ir-7c	99	1
12			Rh(III)	Rh-7c	99	1
13	7d		Ru(II)	Ru-7d	91	1
14			Os(II)	Os-7d	91	1
15			Ir(III)	Ir-7d	99	1
16			Rh(III)	Rh-7d	96	1
17	7e		Ru(II)	Ru-7e	82	1
18			Os(II)	Os-7e	96	1
19			Ir(III)	Ir-7e	88	1
20			Rh(III)	Rh-7e	90	1

* Diastereomeric ratio (d.r.).

).

In most of the complexations presented in Table 4 and Table 5, a single diastereoisomer of the complexes was formed. As an exception, the reactions of 7b yielded complexes Ru-7b, Os-7b, Ir-7b and Rh-7b as mixtures of two diastereoisomers (Table 5, entries 5–8).

A single crystal of complex Ru-3a was obtained by slow evaporation of a CHCl₃-MeOH solvent mixture. A search of the Cambridge Structural Database (Ver 5.43, Update November 2021) [56] resulted in 98 hits for similar Ru·Cl·η⁶··NH₂··N coordination. However, our structure is unique, as the Ru-Cl distance is the shortest in this family of compounds by 2.374(5) Å (average: 2.416(15) Å), while the angle of the arene ring and the N-N-Ru plane is rather high, at 60.7° (average: 57(2)°). Moreover, none of the hits contains a pyranose ring attached to one coordinated amino nitrogen atom in any position. A more detailed search for Ru·Cl·η⁶··NH₂ coordination revealed more than 200 hits; the Ru-Cl distance was also in the very short region, with an average of 2.42 Å. Ring puckering analysis [57] indicates that the C1-O5 ring has a chair conformation (Θ = 19.6(18)°, Φ = 275(5)°), which is in agreement with the NMR data related to the coupling constants of the proton resonances of the sugar skeleton.

X-ray crystallography analysis of Ru-3a provided unequivocal evidence of the existence of a six-membered chelate ring and revealed the spatial arrangement of structural elements in the coordination sphere of the Ru(II) ion (Figure 2). Thus, following the general convention [58], the absolute configuration of the stereogenic Ru(II) was assigned as R. The absolute configuration is confirmed by the analysis of the anomalous dispersion data as the Flack parameter [59] (Table S3).

For structural elucidation of the prepared compounds, ¹H and ¹³C NMR measurements were also performed. Comparison of the ¹H NMR spectroscopic data of the complexes to those of the starting Ru/Os/Ir/Rh dimers and the C-glucosaminyl heterocyclic ligands revealed several significant changes in the chemical shifts, some of which are representatively highlighted by the superposition of the ¹H NMR spectra of Ru-dimer, 3a and Ru-3a (Figure 3).

As a consequence of the complexation, the H-2’ signal of the sugar skeleton of 3a shifted upfield by 1.2 ppm (B), while the H-5′ resonance showed a 0.3 ppm downfield shift (C). Such changes in the chemical shifts of H-2′ and H-5′ are characteristic not only of Ru-3a but also of all complexes isolated as a single isomer (Os/Ir/Rh-3a, Ru/Os-3d, Ru-5a, Ru/Os/Ir/Rh-7a and Ru/Os/Ir/Rh-7c-d) and the major component of the diastereomeric mixtures of Ru/Os/Ir/Rh-7b (Δ = δ_complex−δ_ligand = (−1.3)–(−0.5) ppm for H-2′ and (+0.3)–(+0.6) ppm for H-5′; Table S1). It should be noted that the formation of the minor stereoisomers of Ru/Os/Ir/Rh-7b from ligand 7b resulted in practically no (for 7b → Ir/Rh-7b) or less significant changes (+0.15 ppm for 7b → Ru/Os-7b) in the chemical shift of the H-5′ signal (Table S1).

The transformation of Ru-dimer into Ru-3a significantly affected the proton resonances of the p-cymene moiety. For example, the signal of CH₃ attached to the benzene ring displayed remarkable upfield shifts (0.46 ppm) as a result of the complexation (E). A similar trend in the appearance of this CH₃ signal was observed for all single isomeric Ru(II) and Os(II) complexes (Os/Ir/Rh-3a, Ru/Os-3d, Ru-5a, Ru/Os/Ir/Rh-7a and Ru/Os/Ir/Rh-7c-d) and for the main components of complexes Ru-7b and Os-7b (Δ = δ_complex − δ_dimer = (−0.3)–(−0.6) ppm for C₆H₄-CH₃, Table S1). In the case of the minor isomers of Ru-7b and Os-7b, the same signal indicated a slight downfield shift (Δ = δ_complex − δ_dimer = ~+0.1 ppm, Table S1) relative to that of the corresponding Ru-dimer and Os-dimer, respectively.

These data strongly suggest that in each complex obtained as a single isomer (Os/Ir/Rh-3a, Ru/Os-3d, Ru-5a, Ru/Os/Ir/Rh-7a and Ru/Os/Ir/Rh-7c-d) and in the major component of Ru/Os/Ir/Rh-7a, the absolute configuration of the metal center is identical to that of the reference complex, Ru-3a.

A more detailed collection of the comparative spectroscopic data are presented in Table S1 in the Supplementary Materials.

2.2. Cell Biology

2.2.1. C-Glucosaminyl Azines Exert Cytostatic Activity

The complexes described above are intended to replace registered platinum complexes. Platinum complexes constitute the core of the chemotherapy regimen used in ovarian cancer [8]; therefore, we used a cellular model of ovarian cancer, A2780, and primary human dermal fibroblasts as models of non-transformed cells (controls). For the characterization of the complexes, we used an MTT assay after 4 h of treatment for the detection of early toxicity and an SRB assay 48 h after treatment for the detection of cytostasis [60,61,62].

First, we assessed the complexes of ligands 7a–e. The complexes of the pyridine- and pyridazine-containing ligands 7a and 7b had superior bioactivity relative to that of the complexes of ligands 7c, 7d and 7e, with pyrimidine, pyrazine and quinoline aglycon parts, respectively (Figure 4 and Figure 5,

Table 6. Kinetic and logD values of the complexes assessed in the present manuscript.

	A2780						Fibroblast						logD
	MTT			SRB			MTT			SRB
	Max Inhibition	IC50	Hill	Max Inhibition	IC50	Hill	Max Inhibition	IC50	Hill	Max Inhibition	IC50	Hill
3a	70.22	ND	ND	>90	36.23	1.33	32.22	ND	ND	75	ND	ND
Ru-3a	>90	8.02	1.68	>90	1.86	2.98	80.22	ND	ND	75	9.62	2.87	2.72
Os-3a	85.65	ND	ND	>90	2.13	2.12	69.36	ND	ND	75	15.08	3.09	1.78
Ir-3a	82.71	ND	ND	>90	1.69	1.41	80.22	ND	ND	75	14.14	2.12	3.18
Rh-3a	25.36	ND	ND	>90	9.81	1.11	37.82	ND	ND	75	ND	ND	1.64
3d	ND	ND	ND	13.07	ND	ND	18.39	ND	ND	13.15	ND	ND
Ru-3d	>90	19.34	2.33	>90	3.77	2.31	80.31	ND	ND	64.44	ND	ND	2.06
Os-3d	86.83	32.34	1.98	>90	6.83	2.52	73.24	ND	ND	30.17	ND	ND	2.64
5a	ND	ND	ND	ND	ND	ND
Ru-5a	ND	ND	ND	ND	ND	ND							−1.91
7a	18.61	ND	ND	76.24	ND	ND	27.02	ND	ND	26.00	ND	ND
Ru-7a	>90	9.15	2.54	>90	4.11	2.46	65.18	ND	ND	56.31	ND	ND	1.15
Os-7a	>90	13.77	ND	>90	8.58	2.26	65.18	ND	ND	49.00	ND	ND	2.15
Ir-7a	>90	11.64	ND	>90	6.20	1.96	65.18	ND	ND	56.31	ND	ND	1.08
Rh-7a	35.97	ND	ND	>90	18.25	1.88	27.02	ND	ND	32.56	ND	ND	1.20
7b	9.12	ND	ND	18.38	ND	ND	11.39	ND	ND	ND	ND	ND
Ru-7b	68.22	ND	ND	>90	17.78	2.57	47.04	ND	ND	ND	ND	ND	1.39
Os-7b	53.39	ND	ND	>90	56.37	1.26	27.22	ND	ND	ND	ND	ND	1.32
Ir-7b	53.39	ND	ND	77.58	ND	ND	27.22	ND	ND	ND	ND	ND	1.17
Rh-7b	62.85	ND	ND	>90	31.98	1.97	11.39	ND	ND	ND	ND	ND	1.41
7c	26.30	ND	ND	79.53	ND	ND
Ru-7c	33.13	ND	ND	66.28	ND	ND							1.31
Os-7c	33.13	ND	ND	66.28	ND	ND							1.59
Ir-7c	41.53	ND	ND	82.52	ND	ND							1.40
Rh-7c	33.13	ND	ND	79.53	ND	ND							1.13
7d	18.08	ND	ND	73.37	ND	ND
Ru-7d	64.04	ND	ND	80.44	ND	ND							1.26
Os-7d	46.72	ND	ND	80.44	ND	ND							1.42
Ir-7d	38.71	ND	ND	80.44	ND	ND							1.30
Rh-7d	18.08	ND	ND	64.76	ND	ND							1.04
7e	13.56	ND	ND	45.20	ND	ND
Ru-7e	27.04	ND	ND	67.50	ND	ND							2.22
Os-7e	27.04	ND	ND	67.50	ND	ND							2.21
Ir-7e	27.04	ND	ND	61.28	ND	ND							1.39
Rh-7e	31.89	ND	ND	67.32	ND	ND							1.66

ND—not detectable.

). Complexes of 7a–e induced early toxicity, as evidenced by the MTT assays. The complexes of the ligand 7a were the most effective in inducing early toxicity, with IC₅₀ values ranging between 9 and 14 µM and achieving more than 90% inhibition (Figure 4, Table 6). Other complexes did not reach over 90% inhibition, although they induced early toxicity (Figure 4 and Figure 5, Table 6). In terms of long-term cytostatic activity, the complexes of 7a and 7b exerted complete inhibition of cell growth in SRB assays, with IC₅₀ values between 4 and 9 µM (Figure 4, Table 6). Complexes of ligands 7c–e did not inhibit cell proliferation fully up to 100 μM (Figure 5, Table 6). The best IC₅₀ values fell into the low micromolar range; Ru-3a and Ir-3a had IC₅₀ values of 1.86 and 1.69 μM, respectively.

In general, the IC₅₀ values of the Ru(II)-containing complexes were lower than those of the Os(II), Ir(III) and Rh(III) analogs constructed with the same ligand (e.g., Ru-7a vs. Ir-7a, Os-7a and Rh-7a; Table 6). In terms of effectiveness, the Ru-containing complexes were followed by the corresponding iridium complexes, then by the osmium and, finally, by the rhodium complexes (Figure 4 and Figure 5, Table 6). Of note, the difference between Ru, Os, Ir and Rh complexes was not as pronounced as we observed in our prior studies with glycosyl azole-type ligands [32,45,46]. Furthermore, the free ligands 7a, 7c, 7d and 7e effectively induced cytostasis (Figure 4 and Figure 5, Table 6).

We further investigated the effects of complexes of 7a and 7b on human primary dermal fibroblasts, as these complexes were efficient in inducing cytostasis. Testing the complexes on primary, non-transformed cells informed us of the selectivity of the complexes between transformed cancer cells modelled by A2780 cells and primary, non-transformed cells modelled by primary fibroblasts. The complexes of 7a and 7b induced early toxicity (10–70% maximal inhibition) and long-term cytostasis in primary dermal fibroblasts (25–60% maximal inhibition) although with lower efficacy than in A2780 cells (Figure 4, Table 6).

2.2.2. O-Benzyl Protective Groups Improve, While the O-Deprotection Abrogates the Cytostatic Activity of the Complexes

Next, we assessed the complexes constructed by the O-perbenzylated C-glucosaminyl heterocycles 3. Due to the synthetic difficulties of this series of ligands, only the complexes of the pyridine and pyrazine derivatives 3a and 3d were available for further analysis.

The complexes of 3a, such as Ru-3a, Os-3a and Ir-3a, exerted considerable early toxicity as judged by MTT assays; Ru-3a induced early toxicity on A2780 cells, with an IC₅₀ value of 8.02 μM (Figure 6, Table 6). Interestingly, Rh-3a-induced early toxicity was negligible on A2780 cells (Figure 6, Table 6). With regard to cytostasis, complexes Ru-3a, Os-3a, Ir-3a and Rh-3a were cytostatic on A2780 cells, with micromolar IC₅₀ values, whereas ligand 3a had an IC₅₀ value above 30 μM (Figure 6A, Table 6). The complexes were active on primary human dermal fibroblasts. Ru-3a, Os-3a and Ir-3a exerted ≥70% inhibition in MTT assays, while 3a and Rh-3a exerted ~30–40% inhibition (Figure 6A, Table 6). In SRB assays, 3a and all corresponding complexes (Ru-3a, Os-3a, Ir-3a and Rh-3a) exerted full inhibition, and for Ru-3a, Os-3a and Ir-3a, it was possible to determine the IC₅₀ values that fell into the range of ~10–15 μM (Figure 6A, Table 6).

Next, we assessed 3d and its ruthenium and osmium complexes, Ru-3d and Os-3d. Ru-3d and Os-3d exerted early toxicity on A2780 and human primary dermal fibroblast cells in low micromolar concentrations (Figure 6B, Table 6). The ligand 3d had no activity in MTT assays on A2780 and human primary dermal fibroblast cells in low micromolar concentrations (Figure 6, Table 6). Similar to their activity in the MTT assay, Ru-3d and Os-3d exerted early toxicity in SRB assays on A2780 cells in low micromolar concentrations, while 3d did not exert considerable activity in SRB assays on A2780 cells (Figure 6B, Table 6). In contrast to their activity in MTT assays, 3d and Os-3d did not inhibit cell proliferation in SRB assays, and Ru-3d had only a minor effect on primary human dermal fibroblasts (Figure 6B, Table 6).

We assessed the effect of the O-deprotection of the carbohydrate moiety, which was shown to abrogate the bioactivity, similar to our previous observations [45,46,47]. We used the free ligand, 5a, which is the deprotected equivalent of 3a and 7a, and its ruthenium complex, Ru-5a. Ligand 5a and its ruthenium complex, Ru-5a, did not exhibit any biological activity on A2780 cells either in MTT or SRB assays (Figure 7, Table 6).

2.2.3. Compound Ru-3a Is Cytostatic in Multiple Carcinoma Cell Lines

Carbohydrate-containing ruthenium, osmium and iridium complexes with similar structures were shown to be effective in a large set of carcinoma, sarcoma and lymphoma cell lines [32,45,46,47,63,64,65,66,67]; therefore, we assessed the bioactivity of these complexes in other carcinoma cell lines. For this assay, we chose Ru-3a and the ligand, 3a, as this complex had one of the best IC₅₀ values in A2780 cells (IC₅₀ = 1.86 μM) and had the best-performing protective group attached.

In agreement with the data presented in Figure 6, Ru-3a inhibited the proliferation of another ovarian cancer cell line (ID8), a glioblastoma cell line (U251), a breast carcinoma cell line (MCF7) and a pancreatic adenocarcinoma cell line (Capan2), with IC₅₀ values in the low micromolar range falling between 2 and 4 μM (Figure 8,

Table 7. The kinetic values of compounds 3a and Ru-3a in cancer cell lines other than A2780.

	ID8			U251			MCF7			Capan2
	SRB			SRB			SRB			SRB
	Max Inhibition	IC50	Hill	Max Inhibition	IC50	Hill	Max Inhibition	IC50	Hill	Max Inhibition	IC50	Hill
3a	>90	13.05	2.10	>90	29.36	1.77	>90	21.33	1.43	>90	14.99	ND
Ru-3a	>90	2.54	3.87	>90	3.97	2.55	>90	2.30	2.02	>90	2.25	2.55

ND—not detectable.

). Importantly, 3a was also active in these cell lines, with IC₅₀ values falling between 10 and 30 μM (Figure 8, Table 7).

2.2.4. Complexes with Cytostatic Properties Are Bacteriostatic on Gram-Positive Multiresistant Staphylococcus aureus and Enterococcus isolates

Prior investigations by us [31,32] and others [30,31,32,33,34,35,36,37,38,39,68,69,70,71,72,73,74,75] showed that complexes of the platinum-group metals (platinum, palladium, ruthenium, osmium, iridium and rhodium) can exert bacteriostatic activity. Furthermore, we showed that those compounds were bacteriostatic and cytostatic in neoplasia models [31,32,45,46]. Therefore, we assessed Ru/Os/Ir/Rh-3a, Ru/Os-3d, Ru/Os/Ir/Rh-7a and Ru/Os/Ir/Rh-7b complexes and the corresponding free ligands. Free ligands and rhodium complexes did not exert bacteriostatic activity on any of the investigated strains or isolates. In contrast, the remaining ruthenium, osmium and iridium complexes (Ru/Os/Ir-3a, Ru/Os-3d, Ru/Os/Ir-7a and Ru/Os/Ir-7b) exhibited bacteriostatic activity on the reference strain of Enterococcus faecalis and Staphylococcus aureus, VRE and MRSA, with the exception of Os-3d on the reference strain of Enterococcus faecalis and Ir-7b on the reference strain of Staphylococcus aureus (Figure 9,

Table 8. The MIC values (μM) of a subset of ligands and complexes.

	3a	Ru-3a	Os-3a	Ir-3a	Rh-3a	3d		Ru-3d	Os-3d
E. faecalis ATCC 29,212	>40	10	10	20	>40	>40		20	>40
27,085 VRE	>40	5	10	10	>40	>40		20	40
25,051 VRE	>40	5	5	10	>40	>40		20	40
25,498 VRE	>40	5	10	10	>40	>40		20	40
S. aureus ATCC 29213	>40	5	5	20	>40	>40		20	40
24,408 MRSA	>40	5	10	10	>40	>40		20	40
24,328 MRSA	>40	5	5	10	>40	>40		20	40
20,426 MRSA	>40	5	5	10	>40	>40		20	40
24,268 MRSA	>40	5	10	5	>40	>40		20	40
24,035 MRSA	>40	5	10	20	>40	>40		20	40
24,272 MRSA	>40	5	10	10	>40	>40		20	40
	7a	Ru-7a	Os-7a	Ir-7a	Rh-7a	7b		Ru-7b	Os-7b	Ir-7b	Rh-7b
E. faecalis ATCC 29,212	>40	5	5	40	>40	>40		10	10	40	>40
27,085 VRE	>40	5	10	40	>40	>40		10	10	40	>40
25,051 VRE	>40	5	5	40	>40	>40		10	10	40	>40
25,498 VRE	>40	5	5	40	>40	>40		10	10	40	>40
S. aureus ATCC 29,213	>40	5	10	20	>40	>40		20	20	>40	>40
24,408 MRSA	>40	5	5	10	>40	>40		10	10	40	>40
24,328 MRSA	>40	5	5	20	>40	>40		20	20	40	>40
20,426 MRSA	>40	5	10	10	>40	>40		10	10	40	>40
24,268 MRSA	>40	5	5	10	>40	>40		10	10	40	>40
24,035 MRSA	>40	5	10	10	>40	>40		10	10	40	>40
24,272 MRSA	>40	5	5	10	>40	>40		20	10	40	>40
	7e	Ru-7e	Os-7e	Ir-7e	Rh-7e		Vehicle Control
E. faecalis ATCC 29.212	>40	>40	>40	>40	>40		>40
27,085 VRE	>40	>40	>40	>40	>40		>40
25,051 VRE	>40	>40	>40	>40	>40		>40
25,498 VRE	>40	>40	>40	>40	>40		>40
S. aureus ATCC 29.213	>40	>40	>40	>40	>40		>40
24,408 MRSA	>40	>40	>40	>40	>40		>40
24,328 MRSA	>40	>40	>40	>40	>40		>40
20,426 MRSA	>40	>40	>40	>40	>40		>40
24,268 MRSA	>40	>40	>40	>40	>40		>40
24,035 MRSA	>40	>40	>40	>40	>40		>40
24,272 MRSA	>40	>40	>40	>40	>40		>40

). Ruthenium and osmium complexes were characterized by the lowest MIC values, followed by iridium complexes. The ruthenium complexes of 3a and 7a had the lowest MIC values on the reference strain and clinical isolates of Staphylococcus aureus and Enterococcus faecalis, highlighting the superior performance of pyridine-containing complexes in terms of their bacteriostatic activity, similar to their higher performance as cytostatic agents.

3. Discussion

In this study, we described a set of half-sandwich type platinum-group metal complexes with O-protected C-glucosaminyl heterocyclic N,N-bidentate ligands. Among these, the pyridine-containing complexes had the lowest IC₅₀ and MIC values, followed by the pyrazine- and pyridazine-containing complexes, while their pyrimidine and quinoline counterparts proved to be inactive (Figure 10A).

Another important structural feature of the complexes is the metal ion with the polyhapto arene/arenyl moiety. In this study, ruthenium complexes with p-cymene and iridium complexes with Cp* achieved the best performance in ovarian cancer cells, followed by osmium complexes with p-cymene and rhodium complexes with Cp* (Figure 10A). This is at odds with our previous observations, as we identified osmium complexes with the most potent cytostatic properties, followed by ruthenium complexes and, with a large gap, iridium complexes [32,45,46]. Furthermore, rhodium complexes were inactive in cellular models of carcinomas [32,45,46] in contrast to the presently reported results. However, the effect of the metal ions with polyhapto arene/arenyl moiety proved to be similar in terms of the bacteriostatic effects as in our previous experience [31,32], where the ruthenium complexes had the lowest MIC values, followed by osmium and iridium complexes (Figure 10A). Rhodium complexes did not exhibit bacteriostatic properties.

Prior studies have shown that the chemical composition of the protective groups of the sugar part plays a key role in the bioactivity of monosaccharide-containing half-sandwich complexes, showing O-benzoyl-protected complexes to be the most effective [32,47]. In this study, besides the O-benzoyl-protected C-glucosaminyl pyridine 7a and pyrazine 7d complexes, their O-benzylated counterparts (complexes 3a and 3d, respectively) were also tested. Importantly, the benzyl-protected compounds had better IC₅₀ values in cancer cell models than the benzoyl-protected compounds (Figure 10B). Interestingly, the bacteriostatic properties of the benzyl/benzoyl-protected compounds did not differ drastically (Figure 9 and Figure 10A).

Based on the positive logD values (Table 6), all complexes with O-benzyl and O-benzoyl protective groups are lipophilic. The lipophilic character of the complexes is a prerequisite of their biological activity [31,32,45,46,47,63]. In fact, among the currently identified complexes, the readout of apolarity (logD) and the IC₅₀ value correlate (Figure 10C). Apparently, increasing the apolar character of the complexes improves the biological effectiveness, which is further strengthened by the fact that when the protective groups are absent, as in Ru-5a or in comparable members of the previously reported series [45,46], the bioactivity of the complexes is lost.

An unexpected observation was that the C-glucosaminyl heterocycles used as ligands, namely pyridines 3a and 7a, pyridazine 7b, pyrimidine 7c, pyrazines 3d and 7d and quinoline 7e, showed cytostatic effects, which proved comparable to those of the respective complexes in A2780 cells. In addition, 3a induced cytostasis in primary human fibroblasts. To the best of our knowledge, such effects have not yet been described with C-glycosyl heterocycles; therefore, this finding deserves further investigation.

The cytostatic compounds identified in this study were active in other carcinoma cell lines (glioblastoma, breast cancer and pancreatic adenocarcinoma), as well as in another cellular model of ovarian cancer, ID8. Prior studies assessing complexes of similar structure underscore the widespread activity of such complexes, evidencing bioactivity in carcinoma cell lines such as MDA-MD-231 and MCF7 breast cancer cells [45,47], colon cancer [63,64,65,66], lung cancer [63], cervical carcinoma (HeLa) cells [67], U251 glioblastoma cells [45] Capan2 pancreatic adenocarcinoma cells [45,46], L428 Hodgkin lymphoma [32,46] and Saos osteosarcoma [32,46], in addition to ovarian cancer. These cell models include a diverse set of carcinomas, hematological malignancies and a sarcoma.

Importantly, the complexes with a cytostatic property were less active on primary, non-transformed human dermal fibroblasts. While this property of the complexes suggests a selectivity for transformed neoplastic cells over non-transformed cells, the anticipated therapeutic window is relatively narrow as compared to previous observations of complexes with ligands of similar [32,45,46] or different structure [76,77].

4. Conclusions

In this study, a set of half-sandwich complexes of platinum-group metal ions (Ru(II), Os(II), Ir(III) and Rh(III)) with six-membered C-glucosaminyl heterocycles were synthesized. These complexes exerted cytostatic properties against a set of carcinomas with low micromolar IC₅₀ values, while they were less active against primary, untransformed human dermal fibroblasts, anticipating a narrow therapeutic window for the compounds. Furthermore, the same complexes had bacteriostatic properties against multiresistant Gram-positive Staphylococcus aureus and Enterococcus clinical isolates in the low micromolar range. The molecular mechanism of the cytostatic and bacteriostatic properties of the compounds is currently under investigation in our laboratory, and the results will be published in due course.

5. Materials and Methods

5.1. Synthesis

5.1.1. General Methods

Optical rotations were determined by a P-2000 polarimeter (Jasco, Easton, MD, USA) at room temperature. The ¹H and ¹³C NMR spectra were recorded with a DRX360 (360/90 MHz for ¹H/¹³C), DRX400 (400/100 MHz for ¹H/¹³C) or Bruker Asvance II 500 (500 for ¹H) spectrometer (Bruker, Karlsruhe, Germany). Chemical shifts are referenced to Me₄Si (¹H-NMR) or to the residual solvent signals (¹³C-NMR). The more detailed proton-signal assignments for compounds 3a, 5a, 7a, Ru-3a, Ru-5a, Ru-7a, Ir-7a, Ru-7b and Os-7b are based on COSY correlations. The HRMS data were determined in positive ionization mode using a Bruker maXis II (ESI-HRMS) spectrometer. DC Kieselgel 60 F₂₅₄ plates (Sigma-Aldrich, Saint Louis, MO, USA) were used for TLC analysis, and the spots on the plates were visualized under UV light and developed by gentle heating. For column chromatographic purification, Kieselgel 60 silica gel (Molar Chemicals, Halásztelek, Hungary, particle size 0.063–0.2 mm) was applied. Among anhydrous solvents, EtOH (VWR Chemicals), pyridine (VWR Chemicals) and 1,2-dichloroethane (Sigma-Aldrich) were purchased from the indicated suppliers, while the others were obtained by applying standard distillation methods. Anhydrous CH₂Cl₂ was prepared by distillation from P₄O₁₀ and stored over 4 Å molecular sieves, while THF was distilled first from sodium benzophenone ketyl and redistilled from LiAlH₄ directly before use. 2-Bromopyridine (TCI), 3-bromopyridazine (Fluorochem), 2-iodopyrimidine (Fluorochem), 2-iodopyrazine (Fluorochem), 2-bromoquinoline (TCI), dichloro(η⁶-p-cymene)ruthenium(II) dimer (Ru-dimer, Strem Chemicals, Newburyport, MA, USA), dichloro(pentamethylcyclopentadienyl)iridium(III) dimer (Ir-dimer, Acros Organics), dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer (Rh-dimer, Alfa Aesar) and TlPF₆ (Strem Chemicals) are commercially available chemicals. Dichloro(η⁶-p-cymene)osmium(II) dimer [78] (Os-dimer) and 3,4,6-tri-O-benzyl-2-nitro-d-glucal [49,79] (1) were synthesized by the adaptation of literature procedures.

5.1.2. General Procedure I for the Preparation of C-(2′-Deoxy-2′-nitro-3′,4′,6′-tri-O-benzyl-β-d-glucopyranosyl)heterocycles 2a–e

Method A: In a dry, round-bottom flask, the corresponding halogenated heterocycle (4.33 mmol, 2 eq.) was dissolved in freshly distilled dry THF (10 mL). The stirred solution was cooled down to −78 °C, and a 2.5 M solution of n-butyllithium in n-hexane (1.74 mL, 4.33 mmol, 2 eq.) was added dropwise over 10 min, with stirring continued for 5 min to form the corresponding lithiated heterocycle. In another dry, round-bottom flask containing activated 4 Å molecular sieves (powder, 0.2 g), 2-nitroglucal 1 (1.0 g, 2.17 mmol) was dissolved in freshly distilled dry THF (10 mL). After cooling this solution to −78 °C, the solution of freshly prepared lithiated heterocycle was added. The reaction mixture was then stirred at −78 °C, and the transformation was monitored by TLC (1:4 EtOAc-hexane). When the TLC indicated complete disappearance of 1, the reaction was quenched by the addition of sat. aq. NH₄Cl solution (100 mL) and allowed to warm to rt. The molecular sieves were then filtered off. The filtrate was diluted with EtOAc (200 mL) and extracted with water (100 mL) and brine (100 mL). The separated organic phase was dried over MgSO₄ and filtered, and the solvents were removed under reduced pressure. The residue was purified by column chromatography.

Method B: In a dry, round-bottom flask, 2-nitroglucal 1 (1.0 g, 2.17 mmol) and the corresponding halogenated heterocycle (2.60 mmol, 1.2 eq.) were dissolved in freshly distilled dry THF (20 mL). The stirred solution was cooled down to −78 °C, and a 2.5 M solution of n-butyllithium in n-hexane (1.04 mL, 2.60 mmol, 1.2 eq.) was added over 15 min by means of a syringe pump. Stirring was continued for an additional 15 min at the same temperature. After completion of the reaction (~0.5 h), as judged by TLC (1:2 EtOAc-hexane), the reaction was quenched by the addition of sat. aq. NH₄Cl solution (100 mL) and allowed to warm to rt. The mixture was diluted with EtOAc (200 mL) and extracted with water (100 mL) and brine (100 mL). The separated organic phase was dried over MgSO₄ and filtered, and the solvents were removed under reduced pressure. The residue was purified by column chromatography.

5.1.3. General Procedure II for Cleavage of the O-Benzyl Protecting Groups of the O-Perbenzylated C-Glycopyranosyl Heterocycle 2a–e, 3a to obtain compounds 4a–e and 5a

A solution of the corresponding O-perbenzylated C-glycopyranosyl heterocycle 2a–e or 3a in dry CH₂Cl₂ (10 mL/100 mg substrate) was cooled down to −78 °C, and a 1 M solution of BCl₃ in CH₂Cl₂ (5 eq.) was added dropwise over 5 min. The reaction mixture was stirred at this temperature until the TLC (9:1 CHCl₃-MeOH) showed the completion of the reaction. Then, the reaction was quenched by the addition of MeOH (10 mL) and allowed to warm to rt. The solvents were then removed under reduced pressure, and the residue was purified by column chromatography.

5.1.4. General Procedure III for the Preparation of C-(2′-Deoxy-2′-nitro-3′,4′,6′-tri-O-benzoyl-β-d-glucopyranosyl)heterocycles 6a–d

To a solution of the corresponding C-(2′-deoxy-2′-nitro-3′,4′,6′-tri-O-benzyl-β-d-glucopyranosyl)heterocycle 2a–d in dry dichloroethane (10 mL/100 mg substrate), Zn(OTf)₂ (2 eq.) and benzoyl chloride (6 eq.) were added, and the reaction mixture was stirred at rt. After completion of the reaction, as judged by TLC (1:2 EtOAc-hexane), the reaction mixture was diluted with CH₂Cl₂ (40 mL) and extracted with sat. aq. NaHCO₃ solution (50 mL), then with water (50 mL). The separated organic phase was dried over MgSO₄ and filtered, and the solvents were removed under diminished pressure. The crude product was purified by column chromatography.

5.1.5. General Procedure IV for the Preparation of C-(2′-(tert-Butoxycarbonyl)amino-2′-deoxy-β-d-glucopyranosyl)heterocycles 8c–e

To a solution of the corresponding C-(2′-amino-2′-deoxy-β-d-glucopyranosyl)azine 5c-e in a 1:1 mixture of water and 1,4-dioxane (5 mL/50 mg substrate), Boc₂O (2 eq.) was added, and the reaction mixture was stirred at rt. When the TLC (9:1 CHCl₃-MeOH) showed complete transformation of the staring material (~1 day), the solvents were removed under reduced pressure. The residue was purified by column chromatography.

5.1.6. General Procedure V for the Preparation of C-(2′-(tert-Butoxycarbonyl)amino-2′-deoxy-3′,4′,6′-tri-O-benzoyl-β-d-glucopyranosyl)heterocycles 9c–e

To a solution of the corresponding C-(2′-(tert-butoxycarbonyl)amino-2′-deoxy-β-d-glucopyranosyl)azine 8c–e in dry pyridine (5 mL/100 mg substrate), benzoyl chloride (1.2 eq./OH group) was added at rt. The reaction mixture was stirred at 60 °C for 1 h. Since the TLC (1:1 EtOAc-hexane) showed the incompleteness of the reaction, an additional portion of benzoyl chloride (1.2 eq./OH group) was added to the reaction mixture, and heating was continued for 1 h. The reaction mixture was allowed to cool to rt and further stirred overnight. The reaction mixture was then diluted with CH₂Cl₂ (50 mL) and extracted with sat. aq. solution of NaHCO₃ (25 mL), then with water (25 mL). The separated organic phase was dried over MgSO₄ and filtered, and the solvents were removed under diminished pressure. The residue was purified by column chromatography.

5.1.7. General Procedure VI for the Preparation of C-(2′-Amino-2′-deoxy-3′,4′,6′-tri-O-benzoyl-β-d-glucopyranosyl)heterocycles 7b–e from Compounds 9b–e

The corresponding C-(2′-(tert-butoxycarbonyl)amino-2′-deoxy-3′,4′,6′-tri-O-benzoyl-β-d-glucopyranosyl)azine 9b–e was dissolved in dry CH₂Cl₂ (5 mL/100 mg substrate), and trifluoroacetic acid (2 eq.) was added. The reaction mixture was stirred at rt until the TLC (95:5 CHCl₃-MeOH or 1:1 EtOAc-hexane) indicated complete disappearance of the starting material (~1 h). The solvent and the excess CF₃COOH were then removed under reduced pressure. The residue was dissolved in CH₂Cl₂ (50 mL) and extracted with sat. aq. solution of NaHCO₃ (25 mL) and with water (25 mL). The separated organic phase was dried over MgSO₄ and filtered, and the solvent was removed under diminished pressure. The residue was purified by column chromatography.

5.1.8. General Procedure VII for the Synthesis of Half-Sandwich Platinum-Group Metal Complexes

To a solution of the corresponding complex dimer (Ru-dimer, Os-dimer ([(η⁶-p-cym)M^IICl₂]₂ (M = Ru, Os) or Ir-dimer, Rh-dimer [(η⁵-Cp*)M^IIICl₂]₂ (M = Ir, Rh)) in CH₂Cl₂ (1 mL/10 mg dimer), the appropriate C-glucosaminyl azine (1.9–2.3 eq.) and TlPF₆ (2 eq.) were added. To this stirred reaction mixture, MeOH (1 mL/10 mg dimer) was added at rt in order to accelerate the precipitation of the TlCl. The heterogeneous mixture was then continued further stirred at rt, and the completion of the reaction was monitored by TLC (95:5 CHCl₃-MeOH). When TLC showed total disappearance of the starting dimer (~1 h), the precipitated TlCl was filtered off. The resulting solution was evaporated under diminished pressure. The remaining crude complex was purified by column chromatography and/or crystallization.

5.1.9. Synthesis and Characterization of the New Compounds

2-(2′-Deoxy-2′-nitro-3′,4′,6′-tri-O-benzyl-β-d-glucopyranosyl)pyridine (2a)

Prepared from 2-nitroglucal 1 (0.50 g, 1.08 mmol) and 2-bromopyridine (0.21 mL, 0.34 g, 2.16 mmol, 2 eq.) according to general procedure I, method A. Reaction time: 1 h. Purified by column chromatography (1:4 EtOAc-hexane) to afford 0.30 g (52%) of a colorless syrup. R_f = 0.18 (1:4 EtOAc-hexane). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.57 (1H, ddd, J = 4.9, 1.8, 0.9 Hz, H-6), 7.71 (1H, td, J = 7.8, 1.8 Hz, H-4), 7.42 (1H, ddd, J = 7.8, 1.8, 0.9 Hz, H-3), 7.32–7.18 (16H, m, Ar, H-5), 4.95 (1H, pt, J = 9.8, 9.2 Hz, H-2′ or H-3′ or H-4′), 4.91 (1H, d, J = 9.7 Hz, H-1′), 4.83, 4.61 (2 × 1H, 2 d, J = 10.6 Hz in both, PhCH₂), 4.81, 4.63 (2 × 1H, 2 d, J = 10.9 Hz in both, PhCH₂), 4.59, 4.53 (2 × 1H, 2 d, J = 12.1 Hz in both, PhCH₂), 4.46 (1H, pt, J = 8.6, 8.5 Hz, H-2′ or H-3′ or H-4′), 3.82 (1H, pt, J = 9.5, 8.5 Hz, H-2′ or H-3′ or H-4′), 3.81–3.77 (3H, m, H-5′, H-6′a,b); ¹³C NMR (90 MHz, CDCl₃) δ (ppm): 154.9 (C-2), 149.6 (C-6), 137.1 (C-4), 138.0, 137.7, 137.4, 128.6–127.9 (Ar), 124.1, 122.4 (C-3, C-5), 90.0, 83.2, 80.0, 77.9 (2) (C-1′–C-5′), 75.7, 75.4, 73.7 (3 × PhCH₂), 68.7 (C-6′). ¹H and ¹³C NMR data correspond to those reported in [50].

3-(2′-Deoxy-2′-nitro-3′,4′,6′-tri-O-benzyl-β-d-glucopyranosyl)pyridazine (2b)

Prepared from 2-nitroglucal 1 (2.00 g, 4.33 mmol) and 3-bromopyridazine (0.83 g, 5.20 mmol, 1.2 eq.) according to general procedure I, method B. Purification of the crude product by column chromatography (1:1 EtOAc-hexane) afforded a syrup, which was triturated in a solvent mixture of EtOAc (0.5 mL) and diisopropyl ether (15 mL). The precipitated product was filtered off and washed with diisopropyl ether to afford 0.30 g (13%) of a white, amorphous solid. R_f = 0.21 (1:1 EtOAc-hexane). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.19 (1H, dd, J = 5.0, 1.7 Hz, H-6), 7.65 (1H, dd, J = 8.5, 1.7 Hz, H-4), 7.53 (1H, dd, J = 8.5, 5.0 Hz, H-5), 7.35–7.19 (15H, m, Ar), 5.19 (1H, d, J = 10.0 Hz, H-1′), 4.95 (1H, pt, J = 10.0, 9.9 Hz, H-2′ or H-3′ or H-4′), 4.85, 4.63 (2 × 1H, 2 d, J = 10.8 Hz in both, PhCH₂), 4.83, 4.65 (2 × 1H, 2 d, J = 10.4 Hz in both, PhCH₂), 4.58, 4.52 (2 × 1H, 2 d, J = 12.2 Hz in both, PhCH₂), 4.51 (1H, pt, J = 9.8, 8.3 Hz, H-2′ or H-3′ or H-4′), 3.86 (1H, pt, J = 9.4, 8.3 Hz, H-2′ or H-3′ or H-4′), 3.83–3.74 (3H, m, H-5′, H-6′a,b); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 158.0 (C-3), 151.9 (C-6), 137.9, 137.6, 137.2, 128.7–127.9 (Ar), 127.3, 125.5 (C-4, C-5), 89.5, 82.8, 80.0, 78.5, 77.6 (C-1′–C-5′), 75.9, 75.4, 73.7 (3 × PhCH₂), 68.5 (C-6′). ESI-HRMS positive mode (m/z): calcd for C₃₁H₃₂N₃O₆⁺ [M+H]⁺ 542.2286; C₃₁H₃₁N₃O₆Na⁺ [M+Na]⁺ 564.2105; C₆₂H₆₂N₆O₁₂Na⁺ [2M+Na]⁺ 1105.4318. Found: [M+H]⁺ 542.2291; [M+Na]⁺ 564.2106; [2M+Na]⁺ 1105.4318.

2-(2′-Deoxy-2′-nitro-3′,4′,6′-tri-O-benzyl-β-d-glucopyranosyl)pyrimidine (2c)

Prepared from 2-nitroglucal 1 (1.00 g, 2.17 mmol) and 2-iodopyrimidine (0.54 g, 2.60 mmol, 1.2 eq.) according to general procedure I, method B. Purified by column chromatography (1:2 EtOAc-hexane) to afford 0.83 g (71%) of a white, amorphous solid. R_f = 0.29 (1:2 EtOAc-hexane). ¹H NMR (360 MHz, CDCl₃) δ (ppm): 8.77 (2H, d, J = 4.9 Hz, H-4, H-6), 7.35–7.15 (16H, m, Ar, H-5), 5.24 (1H, pt, J = 10.1, 10.0 Hz, H-2′ or H-3′ or H-4′), 5.07 (1H, d, J = 10.0 Hz, H-1′), 4.82, 4.63 (2 × 1H, 2 d, J = 10.6 Hz in both, PhCH₂), 4.82, 4.58 (2 × 1H, 2 d, J = 10.6 Hz in both, PhCH₂), 4.58,4.49 (2 × 1H, 2 d, J = 12.2 Hz in both, PhCH₂), 4.46 (1H, pt, J = 9.9, 9.5 Hz, H-2′ or H-3′ or H-4′), 3.86 (1H, pt, J = 9.9, 8.5 Hz, H-2′ or H-3′ or H-4′), 3.84–3.74 (3H, m, H-5′, H-6′a,b); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 163.6 (C-2), 157.8 (C-4, C-6), 137.8, 137.6, 137.3, 128.7–127.9 (Ar), 121.3 (C-5), 88.4, 83.0, 80.6, 80.2, 77.7 (C-1′–C-5′), 75.7, 75.3, 73.6 (3 × PhCH₂), 68.4 (C-6′). ESI-HRMS positive mode (m/z): calcd for C₃₁H₃₂N₃O₆⁺ [M+H]⁺ 542.2286; C₃₁H₃₁N₃O₆Na⁺ [M+Na]⁺ 562.2105. Found: [M+H]⁺ 542.2288; [M+Na]⁺ 562.2105.

2-(2′-Deoxy-2′-nitro-3′,4′,6′-tri-O-benzyl-β-d-glucopyranosyl)pyrazine (2d)

Prepared from 2-nitroglucal 1 (1.00 g, 2.17 mmol) and 2-iodopyrazine (0.54 g, 2.60 mmol, 1.2 eq.) according to general procedure I, method B. Purified by column chromatography (1:3 EtOAc-hexane) to afford 0.74 g (63%) of a white, amorphous solid. R_f = 0.54 (1:2 EtOAc-hexane). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.75 (1H, d, J = 1.4 Hz, H-3), 8.58 (1H, d, J = 2.5 Hz, H-6), 8.52 (1H, dd, J = 2.5, 1.4 Hz, H-5), 7.35–7.19 (15H, m, Ar) 5.00 (1H, d, J = 9.9 Hz, H-1′), 4.94 (1H, pt, J = 9.9, 9.7 Hz, H-2′ or H-3′ or H-4′), 4.84, 4.62 (2 × 1H, 2 d, J = 10.8 Hz in both, PhCH₂), 4.82, 4.63 (2 × 1H, 2 d, J = 10.6 Hz in both, PhCH₂), 4.59, 4.53 (2 × 1H, 2 d, J = 12.1 Hz in both, PhCH₂), 4.45 (1H, pt, J = 8.7, 8.7 Hz, H-2′ or H-3′ or H-4′), 3.83 (1H, pt, J = 9.4, 8.7 Hz, H-2′ or H-3′ or H-4′), 3.82–3.76 (3H, m, H-5′, H-6′a,b); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 150.7 (C-2), 145.3, 144.2, 143.9 (C-3, C-5, C-6), 137.8, 137.6, 137.2, 128.7–127.9 (Ar), 89.2, 83.0, 80.1, 77.8, 77.6 (C-1′–C-5′), 75.9, 75.4, 73.7 (3 × PhCH₂), 68.5 (C-6′). ESI-HRMS positive mode (m/z): calcd for C₃₁H₃₁N₃O₆Na⁺ [M+Na]⁺ 564.2105. Found: 564.2107.

2-(2′-Deoxy-2′-nitro-3′,4′,6′-tri-O-benzyl-β-d-glucopyranosyl)quinoline (2e)

Prepared from 2-nitroglucal 1 (2.00 g, 4.33 mmol) and 2-bromoquinoline (1.83 g, 8.80 mmol, 2 eq.) according to general procedure I, method A. Reaction time: 2 h. Purified by column chromatography (1:9 EtOAc-hexane) to afford 1.53 g (60%) of a colorless syrup. R_f = 0.35 (1:4 EtOAc-hexane). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.18 (1H, d, J = 8.5 Hz, H-3 or H-4), 8.04 (1H, dd, J = 8.5, 1.0 Hz, H-5 or H-8), 7.79 (1H, dd, J = 8.2, 1.4 Hz, H-5 or H-8), 7.69 (1H, ddd, J = 8.5, 7.0, 1.4 Hz, H-6 or H-7), 7.57 (1H, d, J = 8.5 Hz, H-3 or H-4), 7.53 (1H, ddd, J = 8.2, 7.0, 1.0 Hz, H-6 or H-7), 7.35–7.20 (15H, m, Ar), 5.13 (1H, d, J = 9.7 Hz, H-1′), 5.10 (1H, pt, J = 9.8, 8.0 Hz, H-2′ or H-3′ or H-4′), 4.86, 4.65 (2 × 1H, 2 d, J = 10.9 Hz in both, PhCH₂), 4.84, 4.64 (2 × 1H, 2 d, J = 10.6 Hz in both, PhCH₂), 4.61, 4.53 (2 × 1H, 2 d, J = 12.2 Hz in both, PhCH₂), 4.50 (1H, pt, J = 8.5, 8.4 Hz, H-2′ or H-3′ or H-4′), 4.88 (1H, pt, J = 9.5, 8.4 Hz, H-2′ or H-3′ or H-4′), 3.86–3.80 (3H, m, H-5′, H-6′a,b); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 155.0, 147.3 (C-2, C-8a), 138.0, 137.7, 137.3, 137.4, 130.0, 129.9, 128.7–127.8, 127.6, 127.2, 119.4 (Ar, C-3–C-8, C-4a), 89.3, 83.3, 80.1, 80.0, 77.8 (C-1′–C-5′), 75.8, 75.4, 73.7 (3 × PhCH₂), 68.6 (C-6′). ESI-HRMS positive mode (m/z): calcd for C₃₆H₃₄N₂O₆Na⁺ [M+Na]⁺ 613.2309. Found: 613.2309.

2-(2′-Amino-2′-deoxy-3′,4′,6′-tri-O-benzyl-β-d-glucopyranosyl)pyridine (3a)

Compound 2a (0.19 g, 0.35 mmol) and Zn powder (0.69 g, 10.55 mmol, 30 eq.) were suspended in a solvent mixture of THF (10 mL) and water (5 mL). This suspension was cooled down in an ice bath, and ccHCl solution was added (0.7 mL, 8.14 mmol, 23 eq.). The reaction mixture was stirred at rt until the TLC (1:1 EtOAc-hexane) showed total consumption of the starting material (1 h). The reaction was quenched by the addition of sat. aq. NaHCO₃ solution (50 mL). The insoluble inorganic salts and the rest of the Zn were filtered off, and the remaining solution was extracted with CH₂Cl₂ (2 × 50 mL). The combined organic phase was extracted with water (50 mL), then with brine (50 mL), dried over MgSO₄ and filtered. The solvent was removed under reduced pressure. The residue was purified by column chromatography (95:5 CHCl₃-MeOH) to afford114 mg (64%) of a pale yellow amorphous solid. R_f = 0.34 (95:5 CHCl₃-MeOH); [α]_D = +30 (c 0.5, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.56 (1H, ddd, J = 4.9, 1.8, 1.0 Hz, H-6), 7.70 (1H, td, J = 7.7, 1.8 Hz, H-4), 7.47 (1H, ddd, J = 7.7, 1.8, 1.0 Hz, H-3), 7.37–7.20 (16H, m, Ar, H-5), 5.01, 4.80 (2 × 1H, 2 d, J = 11.4 Hz in both, PhCH₂), 4.84, 4.63 (2 × 1H, 2 d, J = 10.7 Hz in both, PhCH₂), 4.62, 4.56 (2 × 1H, 2 d, J = 12.2 Hz in both, PhCH₂), 4.27 (1H, d, J = 9.6 Hz, H-1′), 3.80–3.75 (3H, m, H-4′, H-6′a,b), 3.70 (1H, m, H-5′), 3.62 (1H, pt, J = 9.2, 9.2 Hz, H-3′), 3.22 (1H, pt, J = 9.7, 9.6 Hz, H-2′), 1.65 (2H, s, NH₂); ¹³C NMR (90 MHz, CDCl₃) δ (ppm): 158.7 (C-2), 148.9 (C-6), 138.7, 138.3, 138.2 (Ar), 137.0 (C-4), 128.6–127.7 (Ar), 123.2, 122.7 (C-3, C-5), 87.0, 83.0, 79.8, 78.9 (C-1′, C-3′–C-5′), 75.5, 75.0, 73.6 (3 × PhCH₂), 69.4 (C-6′), 57.2 (C-2′). ESI-HRMS positive mode (m/z): calcd for C₃₂H₃₄N₂O₄Na⁺ [M+Na]⁺ 533.2410. Found: 533.2411.

2-(2′-Amino-2′-deoxy-3′,4′,6′-tri-O-benzyl-β-d-glucopyranosyl)pyrazine (3d)

Compound 2d (0.10 g, 0.19 mmol) and Zn powder (0.12 g, 1.84 mmol, 10 eq.) were suspended in a solvent mixture of THF (5 mL) and water (2.5 mL). To this stirred mixture, a 1 M aq. solution of HCl (1.5 mL, 1.50 mmol, 8 eq.) was added dropwise over 1 h using a syringe pump. The reaction mixture was further stirred at rt until TLC (95:5 CHCl₃-MeOH) indicated the completion of the reaction (2 h). The reaction was quenched by the addition of sat. aq. NaHCO₃ solution (25 mL). The insoluble inorganic salts and the rest of the Zn were filtered off, and the remaining solution was extracted with CH₂Cl₂ (2 × 25 mL). The combined organic phase was extracted with water (25 mL), dried over MgSO₄ and filtered. The solvent was removed under reduced pressure. Column chromatographic purification of the residue (95:5 CHCl₃-MeOH) afforded 6.8 mg (7%) of a pale yellow syrup. R_f = 0.23 (95:5 CHCl₃-MeOH); [α]_D = +19 (c 0.1, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.77 (1H, d, J = 1.0 Hz, H-3), 8.53–8.52 (2H, m, H-5, H-6), 7.35–7.21 (15H, m, Ar), 5.03, 4.78 (2 × 1H, 2 d, J = 11.4 Hz in both, PhCH₂), 4.85, 4.64 (2 × 1H, 2 d, J = 10.8 Hz in both, PhCH₂), 4.61, 4.56 (2 × 1H, 2 d, J = 12.3 Hz in both, PhCH₂), 4.31 (1H, d, J = 9.7 Hz, H-1′), 3.81–3.73 (2H, m, H-6′a,b), 3.77 (1H, pt, J = 9.8, 8.5 Hz, H-3′ or H-4′), 3.71 (1H, m, H-5′), 3.60 (1H, pt, J = 9.2, 9.0 Hz, H-3′ or H-4′), 3.25 (1H, pt, J = 9.7, 9.6 Hz, H-2′), 1.68 (2H, br s, NH₂); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 154.2 (C-2), 145.0, 144.3, 143.5 (C-3, C-5, C-6), 138.6, 138.2, 138.1, 129.9–127.8 (Ar), 86.9, 81.4, 80.0, 78.8 (C-1′, C-3′–C-5′), 75.6, 75.0, 73.7 (3 × PhCH₂), 69.2 (C-6′), 56.8 (C-2′). ESI-HRMS positive mode (m/z): calcd for C₃₁H₃₄N₃O₄⁺ [M+H]⁺ 512.2544; C₃₁H₃₃N₃O₄Na⁺ [M+Na]⁺ 534.2363. Found: [M+H]⁺ 512.2541, [M+Na]⁺ 534.2359.

2-(2′-Deoxy-2′-nitro-β-d-glucopyranosyl)pyridine (4a)

Prepared from compound 2a (0.30 g, 0.55 mmol) according to general procedure II. Reaction time: 0.5 h. Purification by column chromatography (9:1 CHCl₃-MeOH) yielded 128 mg (85%) of a white, amorphous solid. R_f = 0.53 (4:1 CHCl₃-MeOH). ¹H NMR (400 MHz, CD₃OD) δ (ppm): 8.53 (1H, d, J = 4.4 Hz, H-6), 7.85 (1H, t, J = 7.7 Hz, H-4), 7.54 (1H, d, J = 7.8 Hz, H-3), 7.40 (1H, m, H-5), 4.95 (1H, d, J = 9.7 Hz, H-1′), 4.74 (1H, pt, J = 10.1, 9.9 Hz, H-2′ or H-3′ or H-4′), 4.21 (1H, pt, J = 9.1, 8.7 Hz, H-2′ or H-3′ or H-4′), 3.91 (1H, m, H-6′a), 3.76 (1H, dd, J = 12.0, 4.2 Hz, H-6′b), 3.62–3.55 (2H, m, H-2′ or H-3′ or H-4′, H-5′); ¹³C NMR (90 MHz, CD₃OD) δ (ppm): 156.4 (C-2), 150.2 (C-6), 138.9 (C-4), 125.6, 124.4 (C-3, C-5), 92.7, 82.7, 80.7, 76.4, 71.1 (C-1′–C-5′), 62.4 (C-6′). ESI-HRMS positive mode (m/z): calcd for C₁₁H₁₄N₂O₆Na⁺ [M+Na]⁺ 293.0744. Found: 293.0744.

3-(2′-Deoxy-2′-nitro-β-d-glucopyranosyl)pyridazine (4b)

Prepared from compound 2b (0.30 g, 0.55 mmol) according to general procedure II. Reaction time: 0.5 h. Purification by column chromatography (9:1 CHCl₃-MeOH) yielded 133 mg (89%) of a white, amorphous solid. R_f = 0.39 (4:1 CHCl₃-MeOH). ¹H NMR (360 MHz, CD₃OD) δ (ppm): 9.17 (1H, d, J = 4.9 Hz, H-6), 8.00 (1H, dd, J = 8.6, 1.4 Hz, H-4), 7.79 (1H, dd, J = 8.6, 4.9 Hz, H-5), 5.22 (1H, d, J = 10.0 Hz, H-1′), 4.82 (1H, pt, J = 10.1, 10.0 Hz, H-2′ or H-3′ or H-4′), 4.25 (1H, pt, J = 10.0, 9.9 Hz, H-2′ or H-3′ or H-4′), 3.94 (1H, dd, J = 12.2, 2.1 Hz, H-6′a), 3.77 (1H, dd, J = 12.2, 5.3 Hz, H-6′b), 3.67 (1H, ddd, J = 9.8, 5.3, 2.1 Hz, H-5′), 3.57 (1H, pt, J = 9.8, 9.5 Hz, H-2′ or H-3′ or H-4′); ¹³C NMR (90 MHz, CD₃OD) δ (ppm): 160.3 (C-3), 153.0 (C-6), 129.7, 128.2 (C-4, C-5), 91.9, 82.9, 79.2, 76.3, 71.0 (C-1′–C-5′), 62.4 (C-6′). ESI-HRMS positive mode (m/z): calcd for C₁₀H₁₃N₃O₆Na⁺ [M+Na]⁺ 294.0697. Found: 294.0698.

2-(2′-Deoxy-2′-nitro-β-d-glucopyranosyl)pyrimidine (4c)

Prepared from compound 2c (0.10 g, 0.18 mmol) according to general procedure II. Reaction time: 0.5 h. Purification by column chromatography (9:1 CHCl₃-MeOH) yielded 49 mg (98%) of a white, amorphous solid. R_f = 0.52 (2:1 CHCl₃-MeOH). ¹H NMR (360 MHz, CD₃OD) δ (ppm): 8.99 (2H, d, J = 5.0 Hz, H-4, H-6), 7.70 (1H, t, J = 5.0 Hz, H-5), 5.24 (1H, d, J = 10.1 Hz, H-1′), 4.85 (1H, pt, J = 10.1, 10.0 Hz, H-2′ or H-3′ or H-4′), 4.20 (1H, pt, J = 10.0, 9.9 Hz, H-2′ or H-3′ or H-4′), 3.96 (1H, dd, J = 12.2, 2.1 Hz, H-6′a), 3.76 (1H, dd, J = 12.2, 5.5 Hz, H-6′b), 3.68 (1H, ddd, J = 9.5, 5.5, 2.1 Hz, H-5′), 3.52 (1H, pt, J = 9.9, 9.5 Hz, H-2′ or H-3′ or H-4′); ¹³C NMR (90 MHz, CD₃OD) δ (ppm): 165.2 (C-2), 159.0 (C-4, C-6), 122.8 (C-5), 91.2, 82.9, 80.8, 76.3, 71.0 (C-1′–C-5′), 62.4 (C-6′). ESI-HRMS positive mode (m/z): calcd for C₁₀H₁₃N₃O₆Na⁺ [M+Na]⁺ 294.0697. Found: 294.0698.

2-(2′-Deoxy-2′-nitro-β-d-glucopyranosyl)pyrazine(4d)

Prepared from compound 2d (0.30 g, 0.55 mmol) according to general procedure II. Reaction time: 0.5 h. Purification by column chromatography (9:1 CHCl₃-MeOH) yielded 117 mg (78%) of a white, amorphous solid. R_f = 0.48 (4:1 CHCl₃-MeOH). ¹H NMR (400 MHz, CD₃OD) δ (ppm): 8.83 (1H, d, J = 1.5 Hz, H-3), 8.60 (1H, d, J = 2.6 Hz, H-6), 8.57 (1H, dd, J = 2.6, 1.5 Hz, H-5), 5.10 (1H, d, J = 9.9 Hz, H-1′), 4.81 (1H, pt, J = 10.0, 10.0 Hz, H-2′ or H-3′ or H-4′), 4.21 (1H, pt, J = 10.0, 9.7 Hz, H-2′ or H-3′ or H-4′), 3.93 (1H, dd, J = 12.2, 2.1 Hz, H-6′a), 3.76 (1H, dd, J = 12.2, 5.4 Hz, H-6′b), 3.64 (1H, ddd, J = 10.0, 5.4, 2.1 Hz, H-5′), 3.54 (1H, pt, J = 9.4, 9.3 Hz, H-2′ or H-3′ or H-4′); ¹³C NMR (90 MHz, CD₃OD) δ (ppm): 152.9 (C-2), 146.2, 145.5, 145.2 (C-3, C-5, C-6), 91.6, 82.9, 78.6, 76.5, 71.1 (C-1′–C-5′), 62.4 (C-6′). ESI-HRMS positive mode (m/z): calcd for C₁₀H₁₃N₃O₆Na⁺ [M+Na]⁺ 294.0697. Found: 294.0698.

2-(2′-Deoxy-2′-nitro-β-d-glucopyranosyl)quinoline (4e)

Prepared from compound 2e (83 mg, 0.14 mmol) according to general procedure II. Reaction time: 1 h. Purification by column chromatography (9:1 CHCl₃-MeOH) yielded 31 mg (76%) of a white, amorphous solid. R_f = 0.19 (9:1 CHCl₃-MeOH). ¹H NMR (400 MHz, CD₃OD) δ (ppm): 8.36, 7.70 (2 × 1H, 2 d, J = 8.5 Hz in both, H-3, H-4), 8.00, 7.93 (2 ×1H, 2 d, J = 7.9 Hz in both, H-5, H-8), 7.77, 7.61 (2 × 1H, 2 t, J = 7.9 Hz in both, H-6, H-7), 5.14 (1H, d, J = 9.9 Hz, H-1′), 4.90 (1H, pt, J = 10.1, 10.0 Hz, H-2′ or H-3′ or H-4′), 4.27 (1H, pt, J = 9.7, 9.0 Hz, H-2′ or H-3′ or H-4′), 3.96 (1H, dd, J = 12.2, < 1Hz, H-6′a), 3.80 (1H, dd, J = 12.2, 4.7 Hz, H-6′b), 3.67 (1H, m, H-5′), 3.61 (1H, pt, J = 9.2, 9.1 Hz, H-2′ or H-3′ or H-4′); ¹³C NMR (90 MHz, CD₃OD) δ (ppm): 157.2, 148.3 (C-2, C-8a), 138.9, 131.2, 129.7, 129.0, 128.4, 121.2 (C-3–C-8), 129.5 (C-4a), 92.3, 82.9, 81.1, 76.6, 71.2 (C-1′–C-5′), 62.5 (C-6′). ESI-HRMS positive mode (m/z): calcd for C₁₅H₁₆N₂O₆Na⁺ [M+Na]⁺ 343.0901. Found: 343.0900.

2-(2′-Amino-2′-deoxy-β-d-glucopyranosyl)pyridine (5a)

Method A: Prepared from compound 3a (65 mg, 0.13 mmol) according to general procedure II. Reaction time: 0.5 h. Purification by column chromatography (7:3 CHCl₃-MeOH) yielded 15 mg (42%) of a white, amorphous solid.

Method B: Compound 3a (0.22 g, 0.43 mmol) and pentamethylbenzene (0.58 g, 3.88 mmol, 9 eq.) were dissolved in dry CH₂Cl₂ (22 mL), and the solution was cooled down to −78 °C. To this solution, a 1 M solution of BCl₃ in CH₂Cl₂ (1.72 mL, 1.72 mmol, 4 eq.) was added dropwise over 5 min. The reaction mixture was stirred at this temperature until the TLC (9:1 CHCl₃-MeOH) showed the completion of the reaction (0.5 h). Then, the reaction was quenched by the addition of MeOH (10 mL) and allowed to warm to rt. The solvents were then removed under reduced pressure. Purification of the residue by column chromatography (7:3 CHCl₃-MeOH) yielded 113 mg (95%) of a white, amorphous solid. R_f = 0.23 (7:3 CHCl₃-MeOH); [α]_D = +84 (c 0.5, MeOH). ¹H NMR (400 MHz, CD₃OD) δ (ppm): 8.57 (1H, d, J = 4.5 Hz, H-6), 7.90 (1H, td, J = 7.8, 1.7 Hz, H-4), 7.72 (1H, d, J = 7.8 Hz, H-3), 7.41 (1H, dd, J = 7.5, 5.0 Hz, H-5), 4.57 (1H, d, J = 10.0 Hz, H-1′), 3.94 (1H, dd, J = 12.1, 1.8 Hz, H-6′a), 3.76 (1H, dd, J = 12.1, 4.9 Hz, H-6′b), 3.66 (1H, pt, J = 9.0, 8.9 Hz, H-3′), 3.54–3.45 (2H, m, H-4′, H-5′), 3.21 (1H, pt, J = 10.0, 9.9 Hz, H-2′); ¹³C NMR (90 MHz, CD₃OD) δ (ppm): 158.7 (C-2), 149.6 (C-6), 139.1 (C-4), 125.1, 123.9 (C-3, C-5), 82.5, 78.7, 76.4, 71.6 (C-1′, C-3′–C-5′), 62.7 (C-6′), 57.6 (C-2′). ESI-HRMS positive mode (m/z): calcd for C₁₁H₁₇N₂O₄⁺ [M+H]⁺ 241.1183; C₁₁H₁₆N₂O₄Na⁺ [M+Na]⁺ 263.1001. Found: [M +H]⁺ 241.1183; [M+Na]⁺ 263.1002.

2-(2′-Amino-2′-deoxy-β-d-glucopyranosyl)pyrimidine (5c)

A degassed, vigorously stirred suspension of 10% Pd(C) (56 mg) in dry EtOH (11 mL) was saturated with H₂, and compound 4c (0.11 g, 0.41 mmol) was added. The reaction mixture was heated at reflux temperature until the TLC (3:2 CHCl₃-MeOH) indicated complete conversion of the starting material. After completion of the reaction (2 h), the catalyst was filtered off through a pad of celite and washed with MeOH. The resulting solution was then evaporated under reduced pressure. Purification of the remaining crude product by column chromatography (3:2 CHCl₃-MeOH) yielded 37 mg (38%) of a white, amorphous solid. R_f = 0.10 (3:2 CHCl₃-MeOH); [α]_D = +22 (c 0.1, MeOH). ¹H NMR (400 MHz, CD₃OD) δ (ppm): 8.85 (2H, d, J = 4.9 Hz, H-4, H-6), 7.48 (1H, t, J = 4.9 Hz, H-5), 4.42 (1H, d, J = 9.7 Hz, H-1′), 3.87 (1H, dd, J = 12.1, 1.5 Hz, H-6′a), 3.73 (1H, dd, J = 12.1, 4.5 Hz, H-6′b), 3.50 (1H, pt, J = 9.5, 9.0 Hz, H-3′ or H-4′), 3.50–3.45 (1H, m, H-5′), 3.44 (1H, pt, J = 9.2, 9.0 Hz, H-3′ or H-4′), 3.08 (1H, pt, J = 9.5, 9.4 Hz, H-2′); ¹³C NMR (90 MHz, CD₃OD) δ (ppm): 167.7 (C-2), 158.8 (C-4, C-6), 122.2 (C-5), 83.8, 82.6, 79.3, 71.4 (C-1′, C-3′–C-5′), 62.8 (C-6′), 57.6 (C-2′). ESI-HRMS positive mode (m/z): calcd for C₁₀H₁₅N₃O₄Na⁺ [M+Na]⁺ 264.0955. Found: 264.0957.

2-(2′-Amino-2′-deoxy-β-d-glucopyranosyl)pyrazine (5d)

A degassed, vigorously stirred suspension of 10% Pd(C) (60 mg) in dry EtOH (12 mL) was saturated with H₂, and compound 4d (0.12 g, 0.43 mmol) was added. The reaction mixture was heated at reflux temperature until the TLC (3:2 CHCl₃-MeOH) indicated complete conversion of the starting material. After completion of the reaction (6 h), the catalyst was filtered off through a pad of celite and washed with MeOH. The resulting solution was then evaporated under reduced pressure. Purification of the remaining crude product by column chromatography (7:3 CHCl₃-MeOH) yielded 68 mg (66%) of a white, amorphous solid. R_f = 0.15 (3:2 CHCl₃-MeOH); [α]_D = +41 (c 0.5, MeOH). ¹H NMR (400 MHz, CD₃OD) δ (ppm): 8.79 (1H, d, J = 1.3 Hz, H-3), 8.62 (1H, dd, J = 2.4, 1.3 Hz, H-5), 8.57 (1H, d, J = 2.4 Hz, H-6), 4.38 (1H, d, J = 9.7 Hz, H-1′), 3.90 (1H, dd, J = 12.1, 1.7 Hz, H-6′a), 3.73 (1H, dd, J = 12.1, 5.0 Hz, H-6′b), 3.51–3.40 (3H, m, H-3′, H-4′, H-5′), 3.00 (1H, pt, J = 9.5, 9.4 Hz, H-2′); ¹³C NMR (90 MHz, CD₃OD) δ (ppm): 155.4 (C-2), 146.0, 145.4, 145.1 (C-3, C-5, C-6), 82.6, 81.4, 79.2, 71.6 (C-1′, C-3′–C-5′), 62.8 (C-6′), 58.0 (C-2′). ESI-HRMS positive mode (m/z): calcd for C₁₀H₁₆N₃O₄Na⁺ [M+H]⁺ 242.1135. Found: 242.1133.

2-(2′-Amino-2′-deoxy-β-d-glucopyranosyl)quinoline (5e)

Compound 4e (0.10 g, 0.33 mmol) and tin powder (1.17 g, 9.82 mmol, 30 eq.) were suspended in a solvent mixture of THF (5 mL) and water (2.5 mL). This heterogenous mixture was cooled down in an ice bath, and ccHCl solution (0.85 mL, 9.88 mmol 30 eq.) was added. The reaction mixture was then stirred at rt. When the TLC (4:1 CHCl₃-MeOH) showed complete conversion of 4e (1 d), a 2 M aq. solution of NaOH was added to the reaction mixture to obtain a slightly basic solution, which was then neutralized by the addition of sat. aq. NH₄Cl solution. The solvents were removed under diminished pressure. The residue was treated with MeOH (20 mL), and the inseparable inorganic salts and the excess of the unreacted Sn were filtered off. The resulting solution was evaporated in vacuo. Column chromatographic purification of the residue (9:1 CHCl₃-MeOH) yielded 28 mg (29%) of a pale yellow, amorphous solid. R_f = 0.11 (4:1 CHCl₃-MeOH); [α]_D = −11 (c 0.1, MeOH). ¹H NMR (400 MHz, CD₃OD) δ (ppm): 8.34 (1H, d, J = 8.5 Hz, H-4), 8.06 (1H, dd, J = 8.5, 1.2 Hz, H-8), 7.92 (1H, dd, J = 8.2, 1.4 Hz, H-5), 7.76 (1H, ddd, J = 8.5, 6.8, 1.4 Hz, H-7), 7.71 (1H, d, J = 8.5 Hz, H-3), 7.59 (1H, ddd, J = 8.2, 6.8, 1.2 Hz, H-6), 4.50 (1H, d, J = 9.7 Hz, H-1′), 3.94 (1H, dd, J = 12.2, 1.3 Hz, H-6′a), 3.79 (1H, ddd, J = 12.2, 3.2, 1.3 Hz, H-5′), 3.56–3.50 (3H, m, H-3′, H-4′, H-6′b), 3.12 (1H, pt, J = 9.5, 9.4 Hz, H-2′); ¹³C NMR (90 MHz, CD₃OD) δ (ppm): 160.1, 148.3 (C-2, C-8a), 138.7, 131.0, 129.6, 129.0, 128.0, 121.7 (C-3–C-8), 129.4 (C-4a), 83.3, 82.6, 79.1, 71.7 (C-1′, C-3′–C-5′), 62.9 (C-6′) 58.3(C-2′). ESI-HRMS positive mode (m/z): calcd for C₁₅H₁₈N₂O₄Na⁺ [M+Na]⁺ 313.1159; C₃₀H₃₆N₄O₈Na⁺ [2M+Na]⁺ 603.2425. Found: [M+Na]⁺ 313.1158; [2M+Na]⁺ 603.2425.

2-(2′-Deoxy-2′-nitro-3′,4′,6′-tri-O-benzoyl-β-d-glucopyranosyl)pyridine (6a)

Prepared from compound 2a (95 mg, 0.18 mmol) and benzoyl chloride (0.13 mL, 1.12 mmol, 6 eq.) according to general procedure III. Reaction time: 5 d. Purified by column chromatography (1:4 EtOAc-hexane) yielded 90 mg (88%) of a white, amorphous solid. R_f = 0.38 (1:2 EtOAc-hexane). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.60 (1H, ddd, J = 4.9, 1.5, 0.9 Hz, H-6), 8.14–7.29 (18H, m, Ar, H-3, H-4, H-5), 6.39 (1H, pt, J = 10.0, 9.7 Hz, H-2′ or H-3′ or H-4′), 5.76 (1H, pt, J = 9.8, 9.8 Hz, H-2′ or H-3′ or H-4′), 5.46 (1H, pt, J = 10.1, 10.1 Hz, H-2′ or H-3′ or H-4′), 5.28 (1H, d, J = 9.9 Hz, H-1′), 4.66 (1H, dd, J = 12.4, 3.1 Hz, H-6′a), 4.53 (1H, dd, J = 12.4, 5.3 Hz, H-6′b), 4.41 (1H, ddd, J = 10.1, 5.3, 3.1 Hz, H-5′); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 166.2, 165.4, 165.2 (3 × C=O), 153.8 (C-2), 149.7 (C-6), 137.4 (C-4), 133.8 (2), 133.3, 130.1–128.4 (Ar), 124.6, 123.1 (C-3, C-5), 87.2, 79.5, 76.9, 73.3, 69.4 (C-1′–C-5′), 63.3 (C-6′). ESI-HRMS positive mode (m/z): calcd for C₃₂H₂₇N₂O₉⁺ [M+H]⁺ 583.1711; C₃₂H₂₆N₂O₉Na⁺ [M+Na]⁺ 605.1531. Found: [M+H]⁺ 583.1713; [M+Na]⁺ 605.1532.

3-(2′-Deoxy-2′-nitro-3′,4′,6′-tri-O-benzoyl-β-d-glucopyranosyl)pyridazine (6b)

Prepared from compound 2b (50 mg, 0.092 mmol) and benzoyl chloride (64 μL, 0.55 mmol, 6 eq.) according to general procedure III. Reaction time: 10 d. Purification by column chromatography (1:1 EtOAc-hexane) yielded 52 mg (97%) of a white, amorphous solid. R_f = 0.32 (1:1 EtOAc-hexane). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.21 (1H, d, J = 5.0 Hz, H-6), 8.01–7.33 (17H, m, Ar, H-4, H-5), 6.45 (1H, pt, J = 9.8, 9.2 Hz, H-2′ or H-3′ or H-4′), 5.79 (1H, pt, J = 9.5, 9.5 Hz, H-2′ or H-3′ or H-4′), 5.60–5.49 (2H, m, H-1′, H-2′ or H-3′ or H-4′), 4.73–4.43 (3H, m, H-5′, H-6′a,b); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 166.2, 165.3, 165.2 (3 × C=O), 157.0 (C-3), 152.1 (C-6), 133.9, 133.4, 130.1–129.8, 129.4, 128.6–128.5, 128.4, 128.2 (Ar), 127.5, 126.2 (C-4, C-5), 86.4, 78.0, 77.0, 73.0, 69.0 (C-1′–C-5′), 62.9 (C-6′). ESI-HRMS positive mode (m/z): calcd for C₃₁H₂₅N₃O₉Na⁺ [M+Na]⁺ 606.1483. Found: 606.1479.

2-(2′-Deoxy-2′-nitro-3′,4′,6′-tri-O-benzoyl-β-d-glucopyranosyl)pyrimidine (6c)

Prepared from compound 2c (0.50 g, 0.92 mmol) and benzoyl chloride (0.65 mL, 5.60 mmol, 6 eq.) according to general procedure III. Reaction time: 30 d. Purification by column chromatography (1:2 EtOAc-hexane) yielded 0.24 g (45%) of a white, amorphous solid. R_f = 0.34 (1:2 EtOAc-hexane). ¹H NMR (360 MHz, CDCl₃) δ (ppm): 8.81 (2H, d, J = 4.8 Hz, H-4, H-6), 7.98–7.33 (16H, m, Ar, H-5), 6.39 (1H, pt, J = 10.0, 9.7 Hz, H-2′ or H-3′ or H-4′), 5.80 (1H, pt, J = 9.7, 9.6 Hz, H-2′ or H-3′ or H-4′), 5.68 (1H, pt, J = 10.2, 10.2 Hz, H-2′ or H-3′ or H-4′), 5.42 (1H, d, J = 10.0 Hz, H-1′), 4.65 (1H, dd, J = 12.3, 3.0 Hz, H-6′a), 4.52 (1H, dd, J = 12.3, 5.0 Hz, H-6′b), 4.44 (1H, ddd, J = 10.0, 5.0, 3.0 Hz, H-5′); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 166.2, 165.3 (2), 162.8 (3 × C=O, C-2), 157.9 (C-4, C-6), 133.8, 133.2, 130.1–128.4 (Ar), 121.5 (C-5), 85.8, 80.4, 77.2, 73.0, 69.2 (C-1′–C-5′), 63.2 (C-6′). ESI-HRMS positive mode (m/z): calcd for C₃₁H₂₅N₃O₉Na⁺ [M+Na]⁺ 606.1483. Found: 606.1483.

2-(2′-Deoxy-2′-nitro-3′,4′,6′-tri-O-benzoyl-β-d-glucopyranosyl)pyrazine (6d)

Prepared from compound 2d (50 mg, 0.092 mmol) and benzoyl chloride (64 μL, 0.55 mmol, 6 eq.) according to general procedure III. Reaction time: 5 d. Purification by column chromatography (1:2 EtOAc-hexane) yielded 44 mg (82%) of a white, amorphous solid. R_f = 0.14 (1:2 EtOAc-hexane). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.83 (1H, d, J = 1.5 Hz, H-3), 8.62 (1H, d, J = 2.5 Hz, H-6), 8.55 (1H, dd, J = 2.5, 1.5 Hz, H-5), 8.01–7.35 (15H, m, Ar), 6.38 (1H, pt, J = 9.4, 9.3 Hz, H-2′ or H-3′ or H-4′), 5.74 (1H, pt, J = 9.7, 9.7 Hz, H-2′ or H-3′ or H-4′), 5.43 (1H, pt, J = 9.9, 9.5 Hz, H-2′ or H-3′ or H-4′), 5.39 (1H, d, J = 9.8 Hz, H-1′), 4.68 (1H, dd, J = 12.3, 2.9 Hz, H-6′a), 4.52 (1H, dd, J = 12.3, 5.4 Hz, H-6′b), 4.28 (1H, ddd, J = 10.0, 5.4, 2.9 Hz, H-5′); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 166.2, 165.3, 165.2 (3 × C=O), 149.7 (C-2), 145.7, 144.7, 144.0 (C-3, C-5, C-6), 133.9, 133.4, 130.1–129.9, 129.5, 128.7–128.6, 128.5, 128.3 (Ar), 86.3, 77.4, 77.1, 73.1, 69.1 (C-1′–C-5′), 63.0 (C-6′). ESI-HRMS positive mode (m/z): calcd for C₃₁H₂₆N₃O₉⁺ [M+H]⁺ 584.1664; C₃₁H₂₅N₃O₉Na⁺ [M+Na]⁺ 606.1483. Found: [M+H]⁺ 584.1659; [M+Na]⁺ 606.1477.

2-(2′-Amino-2′-deoxy-3′,4′,6′-tri-O-benzoyl-β-d-glucopyranosyl)pyridine (7a)

Compound 6a (0.10 g, 0.17 mmol) and Zn powder (0.11 g, 1.71 mmol, 10 eq.) were suspended in a solvent mixture of THF (10 mL) and water (5 mL). To this stirred mixture, a 2 M aq. solution of HCl was added (2.6 mL, 5.14 mmol, 30 eq.). The reaction mixture was further stirred at rt until the TLC (95:5 CHCl₃-MeOH) showed total consumption of the starting material (5 h). The reaction was quenched by the addition of sat. aq. NaHCO₃ solution (50 mL). The insoluble inorganic salts and the rest of the Zn were filtered off, and the remaining solution was extracted with CH₂Cl₂ (2 × 50 mL). The combined organic phase was extracted with water (50 mL), then with brine (50 mL), dried over MgSO₄ and filtered. The solvent was removed under reduced pressure. The residue was purified by column chromatography (100:1 CHCl₃-MeOH) to yield 36 mg (38%) of a white, amorphous solid. R_f = 0.47 (50:1 CHCl₃-MeOH); [α]_D = −16 (c 0.5, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.60 (1H, ddd, J = 4.9, 1.9, 0.9 Hz, H-6), 8.01–7.25 (18H, m, Ar, H-3, H-4, H-5), 5.71 (1H, pt, J = 9.5, 9.5 Hz, H-4′), 5.66 (1H, pt, J = 9.5, 9.5 Hz, H-3′), 4.63 (1H, dd, J = 12.2, 3.1 Hz, H-6′a), 4.53 (1H, d, J = 9.5 Hz, H-1′), 4.52 (1H, dd, J = 12.2, 5.3 Hz, H-6′b), 4.25 (1H, ddd, J = 9.4, 5.3, 3.1 Hz, H-5′), 3.58 (1H, pt, J = 9.7, 9.6 Hz, H-2′), 1.71 (2H, s, NH₂); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 166.6, 166.3, 165.7 (3 × C=O), 157.7 (C-2), 149.1 (C-6), 137.2 (C-4), 133.4, 133.3, 133.1, 129.9–128.4 (Ar), 123.6, 122.9 (C-3, C-5), 83.9, 77.7, 76.6, 70.3 (C-1′, C-3′–C-5′), 64.0 (C-6′), 56.7 (C-2′). ESI-HRMS positive mode (m/z): calcd for C₃₂H₂₉N₂O₇⁺ [M+H]⁺ 553.1969; C₃₂H₂₈N₂O₇Na⁺ [M+Na]⁺ 575.1789. Found: [M+H]⁺ 553.1970; [M+Na]⁺ 575.1789.

3-(2′-Amino-2′-deoxy-3′,4′,6′-tri-O-benzoyl-β-d-glucopyranosyl)pyridazine (7b)

Prepared from compound 9b (105 mg, 0.16 mmol) according to general procedure VI. Purification by column chromatography (95:5 CHCl₃-MeOH) yielded 78 mg (88%) of a white, amorphous solid. R_f = 0.31 (95:5 CHCl₃-MeOH); [α]_D = +7 (c 0.5, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.17 (1H, d, J = 5.0 Hz, H-6), 8.02–7.32 (17H, m, Ar, H-4, H-5), 5.70 (2H, m, H-3′, H-4′), 4.82 (1H, d, J = 9.7 Hz, H-1′), 4.65 (1H, dd, J = 12.2, 2.9 Hz, H-6′a), 4.52 (1H, dd, J = 12.2, 5.2 Hz, H-6′b), 4.32–4.27 (1H, m, H-5′), 3.55 (1H, pt, J = 9.7, 9.6 Hz, H-2′), 1.78 (2H, s, NH₂); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 166.6, 166.3, 165.6 (3 × C=O), 160.6 (C-3), 151.5 (C-6), 133.5, 133.4, 133.2, 129.9–129.8, 129.7, 129.3, 129.0, 128.5–128.4 (Ar), 127.5, 125.8 (C-4, C-5), 82.3, 77.4, 76.7, 69.9 (C-1′, C-3′–C-5′), 63.6 (C-6′), 56.7 (C-2′). ESI-HRMS positive mode (m/z): calcd for C₃₁H₂₈N₃O₇⁺ [M+H]⁺ 554.1922; C₃₁H₂₇N₃O₇Na⁺ [M+Na]⁺ 576.1741. Found: [M+H]⁺ 554.1922; [M+Na]⁺ 576.1740.

2-(2′-Amino-2′-deoxy-3′,4′,6′-tri-O-benzoyl-β-d-glucopyranosyl)pyrimidine (7c)

Prepared from compound 9c (90 mg, 0.14 mmol) according to general procedure VI. Purification by column chromatography (95:5 CHCl₃-MeOH) yielded 73 mg (96%) of a white, amorphous solid. R_f = 0.33 (95:5 CHCl₃-MeOH); [α]_D = −15 (c 0.5, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.84 (2H, d, J = 4.9 Hz, H-4, H-6), 7.99–7.30 (16H, m, Ar, H-5), 5.74 (1H, pt, J = 9.7, 9.6 Hz, H-3′ or H-4′), 5.65 (1H, pt, J = 9.8, 9.6 Hz, H-3′ or H-4′), 4.67 (1H, d, J = 9.9 Hz, H-1′), 4.60 (1H, dd, J = 12.2, 3.2 Hz, H-6′a), 4.51 (1H, dd, J = 12.2, 5.4 Hz, H-6′b), 4.29 (1H, ddd, J = 9.2, 5.4, 3.2 Hz, H-5′), 3.88 (1H, pt, J = 10.0, 9.8 Hz, H-2′), 1.46 (2H, s, NH₂); ¹³C NMR (90 MHz, CDCl₃) δ (ppm): 166.8, 166.3, 165.9, 165.6 (3 × C=O, C-2), 157.7 (C-4, C-6), 133.4, 133.3, 133.0, 130.0–129.8, 129.4, 129.2, 128.5–128.3 (Ar), 120.9 (C-5), 85.2, 77.8, 77.0, 70.4 (C-1′, C-3′–C-5′), 64.2 (C-6′), 55.3 (C-2′). ESI-HRMS positive mode (m/z): calcd for C₃₁H₂₈N₃O₇⁺ [M+H]⁺ 554.1922; C₃₁H₂₇N₃O₇Na⁺ [M+Na]⁺ 576.1741. Found: [M+H]⁺ 554.1916; [M+Na]⁺ 576.1734.

2-(2′-Amino-2′-deoxy-3′,4′,6′-tri-O-benzoyl-β-d-glucopyranosyl)pyrazine (7d)

Prepared from compound 9d (0.12 g, 0.18 mmol) according to general procedure VI. Purification by column chromatography (95:5 CHCl₃-MeOH) yielded 98 mg (96%) of a white, amorphous solid. R_f = 0.32 (95:5 CHCl₃-MeOH); [α]_D = −7 (c 0.5, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.82 (1H, d, J = 1.2 Hz, H-3), 8.58–8.56 (2H, m, H-5, H-6), 8.01–7.31 (15H, m, Ar), 5.71 (1H, pt, J = 9.7, 9.4 Hz, H-3′ or H-4′), 5.68 (1H, pt, J = 9.7, 9.4 Hz, H-3′ or H-4′), 4.65 (1H, dd, J = 12.3, 3.0 Hz, H-6′a), 4.62 (1H, d, J = 9.8 Hz, H-1′), 4.51 (1H, dd, J = 12.3, 5.3 Hz, H-6′b), 4.28 (1H, ddd, J = 8.9, 5.3, 3.0 Hz, H-5′), 3.64 (1H, pt, J = 9.7, 9.7 Hz, H-2′), 1.82 (2H, s, NH₂); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 166.6, 166.2, 165.6 (3 × C=O), 153.1 (C-2), 145.1, 144.6, 143.6 (C-3, C-5, C-6), 133.5, 133.4, 133.1, 129.9–129.8, 129.7, 129.2, 129.0, 128.5–128.4 (Ar), 81.9, 77.4, 76.7, 70.0 (C-1′, C-3′–C-5′), 63.7 (C-6′), 56.1 (C-2′). ESI-HRMS positive mode (m/z): calcd for C₃₁H₂₈N₃O₇⁺ [M+H]⁺ 554.1922; C₃₁H₂₇N₃O₇Na⁺ [M+Na]⁺ 576.1741. Found: [M+H]⁺ 554.1916; [M+Na]⁺ 576.1735.

2-(2′-Amino-2′-deoxy-3′,4′,6′-tri-O-benzoyl-β-d-glucopyranosyl)quinoline (7e)

Prepared from compound 9e (0.12 g, 0.16 mmol) according to general procedure VI. Purification by column chromatography (1:1 EtOAc-hexane) yielded 83 mg (84%) of a white, amorphous solid. R_f = 0.18 (1:1 EtOAc-hexane); [α]_D = +30 (c 0.5, CHCl₃). ¹H NMR (500 MHz, CDCl₃) δ (ppm): 8.22–7.31 (21H, m, Ar, H-3–H-8), 5.77–5.71 (2H, m, H-3′, H-4′), 4.73 (1H, d, J = 9.5 Hz, H-1′), 4.66 (1H, dd, J = 12.2, 2.8 Hz, H-6′a), 4.54 (1H, dd, J = 12.2, 5.4 Hz, H-6′b), 4.31 (1H, ddd, J = 9.1, 5.4, 2.8 Hz, H-5′), 3.69 (1H, pt, J = 9.5, 9.1 Hz, H-2′), 1.83 (2H, br s, NH₂); ¹³C NMR (90 MHz, CDCl₃) δ (ppm): 166.6, 166.3, 165.7 (3 × C=O), 157.9, 147.2 (C-2, C-8a), 137.4, 133.4, 133.3, 133.1, 130.0–129.6, 129.5, 129.2, 128.5–128.4, 128.0, 127.7, 127.0, 119.9 (Ar, C-3–C-8, C-4a), 84.2, 77.6, 76.7, 70.3 (C-1′, C-3′–C-5′), 63.9 (C-6′), 56.5 (C-2′). ESI-HRMS positive mode (m/z): calcd for C₃₆H₃₁N₂O₇⁺ [M+H]⁺ 603.2126. Found: 603.2123.

2-(2′-(tert-Butoxycarbonyl)amino-2′-deoxy-β-d-glucopyranosyl)pyrimidine (8c)

Prepared from compound 5c (60 mg, 0.25 mmol) and Boc₂O (0.11 g, 0.50 mmol) according to general procedure IV. Purification by column chromatography (9:1 CHCl₃-MeOH) yielded 57 mg (67%) of a white, amorphous solid. R_f = 0.37 (4:1 CHCl₃-MeOH). ¹H NMR (400 MHz, D₂O) δ (ppm): 8.81 (2H, d, J = 5.0 Hz, H-4, H-6), 7.55 (1H, t, J = 5.0 Hz, H-5), 4.51 (1H, d, J = 9.7 Hz, H-1′), 3.94 (1H, dd, J = 12.4, 1.9 Hz, H-6′a), 3.86 (1H, dd, J = 12.4, 4.6 Hz, H-6′b), 3.74–3.61 (4H, m, H-2′–H-5′), 1.21 (6H, s, 2 × CH₃), 1.10 (3H, s, CH₃); ¹³C NMR (100 MHz, D₂O + 2 drops of CD₃OD) δ (ppm): 165.0 (C-2), 157.7 (C-4, C-6), 156.8 (C=O), 121.6 (C-5), 81.2, 79.7, 74.5, 69.8 (C-1′, C-3′–C-5′), 80.9 (C(CH₃)₃), 60.9 (C-6′), 56.8 (C-2′), 27.6 (3 × CH₃). ESI-HRMS positive mode (m/z): calcd for C₁₅H₂₃N₃O₆Na⁺ [M+Na]⁺ 364.1479; C₃₀H₄₆N₆O₁₂Na⁺ [2M+Na]⁺ 705.3066. Found: [M+Na]⁺ 364.1474; [2M+Na]⁺ 705.3057.

2-(2′-(tert-Butoxycarbonyl)amino-2′-deoxy-β-d-glucopyranosyl)pyrazine (8d)

Prepared from compound 5d (0.10 g, 0.42 mmol) and Boc₂O (0.18 g, 0.83 mmol) according to general procedure IV. Purification by column chromatography (9:1 CHCl₃-MeOH) yielded 94 mg (67%) of a white, amorphous solid. R_f = 0.37 (4:1 CHCl₃-MeOH). ¹H NMR (400 MHz, CD₃OD) δ (ppm): 8.72 (1H, d, J = 1.5 Hz, H-3), 8.56 (1H, dd, J = 2.7, 1.5 Hz, H-5), 8.53 (1H, d, J = 2.7 Hz, H-6), 4.41 (1H, d, J = 9.1 Hz, H-1′), 3.90 (1H, dd, J = 12.1, 2.2 Hz, H-6′a), 3.77 (1H, dd, J = 12.1, 5.1 Hz, H-6′b), 3.62–3.52 (3H, m, H-2′, H-3′, H-4′), 3.44 (1H, ddd, J = 9.0, 5.1, 2.2 Hz, H-5′), 1.22 (9H, s, 3 × CH₃); ¹³C NMR (100 MHz, CD₃OD) δ (ppm): 157.5, 155.3 (C=O, C-2), 145.0, 144.9, 144.8 (C-3, C-5, C-6), 82.3, 81.3, 76.6, 71.8 (C-1′, C-3′–C-5′), 79.9 (C(CH₃)₃), 62.7 (C-6′), 58.6 (C-2′), 28.6 (3 × CH₃). ESI-HRMS positive mode (m/z): calcd for C₁₅H₂₃N₃O₆Na⁺ [M+Na]⁺ 364.1479. Found: 364.1471.

2-(2′-(tert-Butoxycarbonyl)amino-2′-deoxy-β-d-glucopyranosyl)quinoline (8e)

Prepared from compound 5e (25 mg, 0.086 mmol) and Boc₂O (37.6 mg, 0.172 mmol) according to general procedure IV. Purification by column chromatography (9:1 CHCl₃-MeOH) yielded 27 mg (80%) of a white, amorphous solid. R_f = 0.35 (4:1 CHCl₃-MeOH). ¹H NMR (360 MHz, CD₃OD) δ (ppm): 8.29 (1H, d, J = 8.5 Hz, H-4), 8.05 (1H, dd, J = 8.5, 1.2 Hz, H-8), 7.90 (1H, dd, J = 8.1, 1.4 Hz, H-5), 7.75 (1H, ddd, J = 8.5, 6.9, 1.5 Hz, H-7), 7.69 (1H, d, J = 8.5 Hz, H-3), 7.58 (1H, ddd, J = 8.1, 6.9, 1.2 Hz, H-6), 4.49 (1H, d, J = 9.4 Hz, H-1′), 3.94 (1H, dd, J = 12.1, 2.3 Hz, H-6′a), 3.82 (1H, dd, J = 12.1, 5.2 Hz, H-6′b), 3.70–3.56 (3H, m, H-2′, H-3′, H-4′), 3.49 (1H, ddd, J = 9.5, 5.2, 2.3 Hz, H-5′), 1.00 (6H, s, 2 × CH₃), 0.79 (3H, s, CH₃); ¹³C NMR (90 MHz, CD₃OD) δ (ppm): 160.2, 157.4, 148.0 (C=O, C-2, C-8a,), 138.2, 130.9, 129.2, 128.9, 127.8, 121.7 (C-3–C-8), 129.4 (C-4a), 83.6, 82.3, 77.0, 72.0 (C-1′, C-3′–C-5′), 79.8 (C(CH₃)₃), 62.9 (C-6′) 58.8 (C-2′), 28.4 (3 × CH₃). ESI-HRMS positive mode (m/z): calcd for C₂₀H₂₆N₂O₆Na⁺ [M+Na]⁺ 413.1683. Found: 413.1683.

3-(2′-(tert-Butoxycarbonyl)amino-2′-deoxy-3′,4′,6′-tri-O-benzoyl-β-d-glucopyranosyl)pyridazine (9b)

A degassed, vigorously stirred suspension of 10% Pd(C) (65 mg) in dry EtOH (13 mL) was saturated with H₂, and compound 4b (0.13 g, 0.48 mmol) was added. The reaction mixture was heated at reflux temperature until the TLC (3:2 CHCl₃-MeOH) indicated complete conversion of the starting material. After completion of the reaction (3 h), the catalyst was filtered off through a pad of celite and washed with EtOH. The resulting solution was then evaporated under reduced pressure. Purification of the residue by column chromatography (3:2 CHCl₃-MeOH) yielded 100 mg of a white, amorphous solid containing the desired 3-(2′-amino-2′-deoxy-β-d-glucopyranosyl)pyridazine 5b, along with unidentified impurities. This mixture was dissolved in a solvent mixture of water (5 mL) and 1,4-dioxane (5 mL), and Boc₂O (0.21 g, 0.96 mmol) was added. The reaction mixture was stirred at rt until the TLC (9:1 CHCl₃-MeOH) showed complete transformation of 5b (1 day). Then, the solvents were removed under reduced pressure. Column chromatographic purification of the residue (9:1 CHCl₃-MeOH) resulted in 70 mg of 3-(2′-(tert-butoxycarbonyl)amino-2′-deoxy-β-d-glucopyranosyl)pyridazine (8b) contaminated with inseparable impurities. To a solution of the resulting 8b in dry pyridine (5 mL), benzoyl chloride (0.2 mL, 1.72 mmol) was added at rt. The reaction mixture was stirred at 60 °C for 1 h. Since the TLC (1:1 EtOAc-hexane) showed incompleteness of the reaction, an additional portion of benzoyl chloride (0.2 mL, 1.72 mmol) was added to the reaction mixture, and heating was continued for 1 h. The reaction mixture was allowed to cool to rt and further stirred overnight. The reaction mixture was then diluted with CH₂Cl₂ (50 mL) and extracted with sat. aq. solution of NaHCO₃ (25 mL), then with water (25 mL). The separated organic phase was dried over MgSO₄ and filtered, and the solvents were removed under diminished pressure. The residue was purified by column chromatography (1:1 EtOAc-hexane) to afford the title compound 9b (83 mg, 27% for 3 steps) as a white, amorphous solid. R_f = 0.28 (1:1 EtOAc-hexane. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.12 (1H, dd, J = 4.9, 1.7 Hz, H-6), 8.04–7.32 (17H, m, Ar, H-4, H-5), 5.82 (1H, pt, J = 9.3, 9.3 Hz, H-3′ or H-4′), 5.79 (1H, pt, J = 9.7, 9.5 Hz, H-3′ or H-4′), 5.12 (1H, d, J = 9.9 Hz, NH), 5.02 (1H, d, J = 10.3 Hz, H-1′), 4.67 (1H, dd, J = 12.3, 2.8 Hz, H-6′a), 4.50 (1H, dd, J = 12.3, 4.8 Hz, H-6′b), 4.30 (1H, ddd, J = 9.5, 4.8, 2.8 Hz, H-5′), 4.28 (1H, pt, J = 10.1, 10.0 Hz, H-2′), 1.05 (9H, s, 3 × CH₃); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 166.7, 166.2, 165.4 (3 × C=O), 159.6, 155.0 (C=O, C-3), 151.4 (C-6), 133.6, 133.4, 133.2, 130.0–129.8, 129.7, 129.1, 128.9, 128.6–128.4 (Ar), 127.3, 125.7 (C-4, C-5), 81.3, 76.7, 74.3, 69.8 (C-1′, C-3′–C-5′), 80.0 (C(CH₃)₃), 63.3 (C-6′), 55.9 (C-2′), 27.9 (3 × CH₃). ESI-HRMS positive mode (m/z): calcd for C₃₆H₃₅N₃O₉Na⁺ [M+Na]⁺ 676.2266; C₇₂H₇₀N₆O₁₈Na⁺ [2M+Na]⁺ 1329.4639. Found: [M+Na]⁺ 676.2256; [2M+Na]⁺ 1329.4641.

2-(2′-(tert-Butoxycarbonyl)amino-2′-deoxy-3′,4′,6′-tri-O-benzoyl-β-d-glucopyranosyl)pyrimidine (9c)

Prepared from compound 8c (60 mg, 0.18 mmol) and benzoyl chloride (0.15 mL, 1.29 mmol, 7.2 eq.) according to general procedure V. Purification by column chromatography (1:1 EtOAc-hexane) yielded 92 mg (80%) of a white, amorphous solid. R_f = 0.22 (1:1 EtOAc-hexane). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.82 (2H, d, J = 4.9 Hz, H-4, H-6), 7.98–7.27 (16H, m, Ar, H-5), 5.86 (1H, pt, J = 9.9, 9.6 Hz, H-3′ or H-4′), 5.80 (1H, pt, J = 9.6, 9.4 Hz, H-3′ or H-4′), 4.98 (1H, d, J = 9.5 Hz, NH), 4.90 (1H, d, J = 10.2 Hz, H-1′), 4.63 (1H, dd, J = 12.3, 3.5 Hz, H-6′a), 4.58 (1H, dd, J = 12.3, 5.4 Hz, H-6′b), 4.55 (1H, pt, J = 10.2, 9.7 Hz, H-2′), 4.30 (1H, ddd, J = 9.2, 5.4, 3.5 Hz, H-5′), 1.09 (9H, s, 3 × CH₃); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 166.7, 166.3, 165.4, 165.2 (3 × C=O, C-2), 154.6 (C=O), 157.4 (C-4, C-6), 133.4, 133.3, 133.0, 130.0–129.8, 129.7, 129.2, 129.0, 128.4–128.2 (Ar), 120.7 (C-5), 82.8, 76.7, 74.6, 70.2 (C-1′, C-3′–C-5′), 79.6 (C(CH₃)₃), 64.1 (C-6′), 55.2 (C-2′), 28.0 (3 × CH₃). ESI-HRMS positive mode (m/z): calcd for C₃₆H₃₅N₃O₉Na⁺ [M+Na]⁺ 676.2266. Found: 676.2256.

2-(2′-(tert-Butoxycarbonyl)amino-2′-deoxy-3′,4′,6′-tri-O-benzoyl-β-d-glucopyranosyl)pyrazine (9d)

Prepared from compound 8d (90 mg, 0.26 mmol) and benzoyl chloride (0.22 mL, 1.89 mmol, 7.2 eq.) according to general procedure V. Purification by column chromatography (1:1 EtOAc-hexane) yielded 122 mg (71%) of a white, amorphous solid. R_f = 0.36 (1:1 EtOAc-hexane). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.83 (1H, s, H-3), 8.55–8.53 (2H, m, H-5, H-6), 8.02–7.30 (15H, m, Ar), 5.88 (1H, d, J = 9.8, 9.6 Hz, H-3′ or H-4′), 5.83 (1H, d, J = 9.5, 9.3 Hz, H-3′ or H-4′), 5.15 (1H, d, J = 9.3 Hz, NH), 4.83 (1H, d, J = 10.2 Hz, H-1′), 4.68 (1H, dd, J = 12.3, 2.9 Hz, H-6′a), 4.53 (1H, dd, J = 12.3, 4.9 Hz, H-6′b), 4.35 (1H, pt, J = 9.8, 9.7 Hz, H-2′), 4.34–4.29 (H, m, H-5′), 1.09 (9H, s, 3 × CH₃); ¹³C NMR (90 MHz, CDCl₃) δ (ppm): 166.8, 166.3, 165.4 (3 × C=O), 154.8, 152.4 (C=O, C-2), 144.6 (2), 143.4 (C-3, C-5, C-6), 133.5, 133.4, 133.2, 130.0–129.8, 129.7, 129.1, 129.0, 128.5–128.4 (Ar), 80.7, 76.8, 74.3, 70.0 (C-1′, C-3′–C-5′), 79.9 (C(CH₃)₃), 63.5 (C-6′), 55.9 (C-2′), 28.0 (3 × CH₃). ESI-HRMS positive mode (m/z): calcd for C₃₆H₃₅N₃O₉Na⁺ [M+Na]⁺ 676.2266. Found: 676.2260.

2-(2′-(tert-Butoxycarbonyl)amino-2′-deoxy-3′,4′,6′-tri-O-benzoyl-β-d-glucopyranosyl)quinoline (9e)

Prepared from compound 8e (50 mg, 0.13 mmol) and benzoyl chloride (0.11 mL, 0.95 mmol, 7.2 eq.) according to general procedure V. Purification by column chromatography (1:2 EtOAc-hexane) yielded 73 mg (81%) of a white, amorphous solid. R_f = 0.21 (1:2 EtOAc-hexane). ¹H NMR (500 MHz, CDCl₃) δ (ppm): 8.23–7.32 (21H, m, Ar, H-3–H-8), 5.84 (1H, pt, J = 9.6, 9.5 Hz, H-3′ or H-4′), 5.77 (1H, dd, J = 10.0, 9.8 Hz, H-3′ or H-4′), 4.98 (1H, d, J = 9.8 Hz, NH), 4.89 (1H, d, J = 10.2 Hz, H-1′), 4.67 (1H, dd, J = 12.2, 2.9 Hz, H-6′a), 4.52 (1H, dd, J = 12.2, 4.8 Hz, H-6′b), 4.36 (1H, q, J = 10.2 Hz, H-2′), 4.28 (1H, ddd, J = 9.7, 4.8, 2.9 Hz, H-5′), 0.86 (9H, s, 3 × CH₃); ¹³C NMR (90 MHz, CDCl₃) δ (ppm): 170.8, 166.4, 165.5 (3 × C=O), 157.4, 155.1, 146.5 (C=O, C-2, C-8a), 137.9, 133.5, 133.3, 133.2, 130.3–129.8, 129.4, 129.1, 128.5–128.3, 128.2, 127.9, 127.0, 119.5 (Ar, C-3–C-8, C-4a), 82.5, 76.6, 74.7, 70.1 (C-1′, C-3′–C-5′), 79.5 (C(CH₃)₃), 63.5 (C-6′), 56.1 (C-2′), 27.7 (3 × CH₃). ESI-HRMS positive mode (m/z): calcd for C₄₁H₃₈N₂O₉Na⁺ [M+Na]⁺ 725.2470. Found: 725.2473.

Complex Ru-3a

Prepared from compound 3a (47 mg, 0.092 mmol, 1.9 eq.), Ru-dimer (30 mg, 0.049 mmol) and TlPF₆ (34 mg, 0.097 mmol) according to general procedure VII. Purification by column chromatography (95:5 CHCl₃-MeOH) yielded 63 mg (74%) of a yellow solid. R_f = 0.50 (95:5 CHCl₃-MeOH). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.02 (1H, d, J = 5.5 Hz, H-6), 7.86 (1H, dd, J = 8.1, 1.8 Hz, H-3), 7.82 (1H, td, J = 8.1, 1.5 Hz, H-4), 7.40–7.26 (16H, m, Ar, H-5), 5.84, 5.80, 5.59, 5.13 (4 × 1H, 4 d, J = 5.9 Hz in each, 4 × p-cym-CH_Ar), 5.34 (1H, pt, J = 10.8 Hz, NH₂), 4.89, 4.59 (2 × 1H, 2 d, J = 12.1 Hz in both, PhCH₂), 4.78, 4.56 (2 × 1H, 2 d, J = 12.3 Hz in both, PhCH₂), 4.75, 4.65 (2 × 1H, 2 d, J = 11.3 Hz in both, PhCH₂), 4.54 (1H, d, J = 10.2 Hz, H-1′), 4.23 (1H, pt, J = 8.9, 8.8 Hz, H-3′), 3.99 (1H, ddd, J = 9.4, 5.6, 2.6, H-5′), 3.84–3.77 (2H, m, H-6′a,b), 3.53 (1H, pt, J = 9.1, 9.0 Hz, H-4′), 3.20 (1H, dd, J = 10.8, 5.3 Hz, NH₂), 2.58 (1H, hept, J = 6.9 Hz, i-Pr-CH), 2.03–1.94 (1H, m, H-2′), 1.70 (3H, s, C₆H₄-CH₃), 1.16, 1.02 (2 × 3H, 2 d, J = 6.9 Hz in both, 2 × i-Pr-CH₃); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 162.0 (C-2), 156.8 (C-6), 139.4 (C-4), 138.4, 138.1 (2), 129.1–127.8 (Ar), 124.5, 123.2 (C-3, C-5), 104.9, 98.7 (2 × p-cym-C_qAr), 86.6, 84.8, 83.8, 83.4, 82.9, 77.7, 76.8, 76.0 (4 × p-cym-CH_Ar, C-1′, C-3′–C-5′), 75.1, 74.2, 73.5 (3 × PhCH₂), 68.7 (C-6′), 53.6 (C-2′), 31.0 (i-Pr-CH), 23.2, 21.5 (2 × i-Pr-CH₃), 17.7 (C₆H₄-CH₃). ESI-HRMS positive mode (m/z): calcd for C₄₂H₄₈ClN₂O₄Ru⁺ [M-PF₆]⁺ 781.2349. Found: 781.2346.

Complex Os-3a

Prepared from compound 3a (12.3 mg, 0.024 mmol, 1.9 eq.), Os-dimer (10.0 mg, 0.013 mmol) and TlPF₆ (8.7 mg, 0.025 mmol) according to general procedure VII. After purification by column chromatography (95:5 CHCl₃-MeOH), the complex was dissolved in CHCl₃ (1 mL), and diisopropyl ether (8 mL) was added. The precipitated product was filtered off, then washed with CHCl₃-diisopropyl ether (1:8, 1 mL) to yield 15.6 mg (64%) of a dark purple solid. R_f = 0.58 (95:5 CHCl₃-MeOH). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.87 (1H, dd, J = 6.0, 1.5 Hz, H-6), 7.90 (1H, d, J = 7.8 Hz, H-3), 7.81 (1H, td, J = 7.8, 1.5 Hz, H-4), 7.41–7.26 (16H, m, Ar, H-5), 6.09, 6.08, 5.82, 5.27 (4 × 1H, 4 d, J = 5.7 Hz in each, 4 × p-cym-CH_Ar), 5.90–5.76 (1H, broad signal, NH₂), 4.91, 4.77 (2 × 1H, 2 d, J = 12.3 Hz in both, PhCH₂), 4.76, 4.67 (2 × 1H, 2 d, J = 11.5 Hz in both, PhCH₂), 4.59, 4.55 (2 × 1H, 2 d, J = 12.2 Hz in both, PhCH₂), 4.50 (1H, d, J = 10.2 Hz, H-1′), 4.22 (1H, pt, J = 9.0, 8.8 Hz, H-3′ or H-4′), 4.00–3.98 (1H, m, H-5′), 3.84–3.77 (2H, m, H-6′a,b), 3.67 (1H, dd, J = 11.4, 4.6 Hz, NH₂), 3.57 (1H, pt, J = 9.2, 9.1 Hz, H-3′ or H-4′), 2.47 (1H, hept, J = 7.0 Hz, i-Pr-CH), 2.32–2.23 (1H, m, H-2′), 1.75 (3H, s, C₆H₄-CH₃), 1.19, 0.98 (2 × 3H, 2 d, J = 6.9 Hz in both, 2 × i-Pr-CH₃); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 161.3 (C-2), 157.7 (C-6), 139.5 (C-4), 138.4, 138.1 (2), 129.9–127.8 (Ar), 124.9, 122.5 (C-4, C-5), 95.0, 89.9 (2 × p-cym-C_qAr), 82.7, 78.3, 77.7, 76.8, 76.3, 75.6, 75.4, 73.6 (4 × p-cym-CH_Ar, C-1′, C-3′–C-5′), 75.2, 74.2, 73.5 (3 × PhCH₂), 68.7 (C-6′), 53.6 (C-2′), 31.1 (i-Pr-CH), 23.6, 21.6 (2 × i-Pr-CH₃), 17.6 (C₆H₄-CH₃). ESI-HRMS positive mode (m/z): calcd for C₄₂H₄₇N₂O₄Os⁺ [M-HCl-PF₆]⁺ 835.3148. Found: 835.3143.

Complex Ir-3a

Prepared from compound 3a (12.2 mg, 0.024 mmol, 1.9 eq.), Ir-dimer (10.0 mg, 0.013 mmol) and TlPF₆ (8.8 mg, 0.025 mmol) according to general procedure VII. Purification by column chromatography (95:5 CHCl₃-MeOH) yielded 22.7 mg (93%) of a yellow solid. R_f = 0.51 (95:5 CHCl₃-MeOH). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.58 (1H, d, J = 5.8 Hz, H-6), 7.92–7.87 (2H, m, H-3, H-4), 7.42–7.27 (16H, m, Ar, H-5), 4.83, 4.70 (2 × 1H, 2 d, J = 12.6 Hz in both, PhCH₂), 4.78, 4.69 (2 × 1H, 2 d, J = 11.4 Hz in both, PhCH₂), 4.60, 4.56 (2 × 1H, 2 d, J = 12.1 Hz in both, PhCH₂), 4.25 (1H, d, J = 10.0 Hz, H-1′), 4.12–4.05 (2H, m, H-5′, NH₂), 4.08 (1H, pt, J = 8.1, 8,0 Hz, H-3′ or H-4′), 3.95 (1H, dd, J = 11.5, 5.5 Hz, NH₂), 3.85 (1H, dd, J = 10.8, 4.1 Hz, H-6′a), 3.79 (1H, dd, J = 10.8, 3.0 Hz, H-6′b), 3.69 (1H, pt, J = 8.0, 7.9 Hz, H-3′ or H-4′), 2.55–2.47 (1H, m, H-2′), 1.41 (15H, s, Cp*-CH₃); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 160.8 (C-2), 155.3 (C-6), 139.9 (C-4), 138.3, 138.1, 137.8, 129.9, 129.3–127.9 (Ar), 126.0, 123.0 (C-4, C-5), 88.1 (Cp*), 81.5, 77.5, 77.4, 77.1 (C-1′, C-3′–C-5′), 74.3, 74.1, 73.5 (3 × PhCH₂), 69.0 (C-6′), 54.1 (C-2′), 8.5 (Cp*-CH₃). ESI-HRMS positive mode (m/z): calcd for C₄₂H₄₉ClN₂O₄Ir⁺ [M-PF₆]⁺ 873.2999. Found: 873.2997.

Complex Rh-3a

Prepared from compound 3a (15.7 mg, 0.031 mmol, 1.9 eq.), Rh-dimer (10.0 mg, 0.016 mmol) and TlPF₆ (11.3 mg, 0.032 mmol) according to general procedure VII. After purification by column chromatography (95:5 CHCl₃-MeOH), the complex was dissolved in CHCl₃ (1 mL), and diisopropyl ether (8 mL) was added. The precipitated product was filtered off, then washed with CHCl₃-diisopropyl ether (1:8, 1 mL) to yield 23.6 mg (83%) of an orange solid. R_f = 0.30 (95:5 CHCl₃-MeOH).¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.61 (1H, dd, J = 5.7, 1.7 Hz, H-6), 7.94–7.85 (2H, m, H-3, H-4), 7.46–7.28 (16H, m, Ar, H-5), 4.81, 4.68 (2 × 1H, 2 d, J = 12.5 Hz in both, PhCH₂), 4.77, 4.70 (2 × 1H, 2 d, J = 11.2 Hz in both, PhCH₂), 4.61, 4.58 (2 × 1H, 2 d, J = 12.8 Hz in both, PhCH₂), 4.26 (1H, d, J = 9.9 Hz, H-1′), 4.03 (1H, ddd, J = 7.6, 4.1, 3.2 Hz, H-5′), 3.96 (1H, pt, J = 7.7, 7.6 Hz, H-3′ or H-4′), 3.85 (1H, dd, J = 10.8, 4.1 Hz, H-6′a), 3.79 (1H, dd, J = 10.8, 3.2 Hz, H-6′b), 3.72 (1H, pt, J = 7.7, 7.7 Hz, H-3′ or H-4′), 3.47 (1H, dd, J = 11.1, 5.9 Hz, NH₂), 3.24 (1H, pt, J = 10.2 Hz, NH₂), 2.40–2.32 (1H, m, H-2′), 1.43 (15H, s, Cp*-CH₃); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 160.8 (C-2), 154.2 (C-6), 139.8 (C-4), 138.2, 138.1, 137.7, 129.9, 129.2–127.9 (Ar), 125.6, 123.2 (C-3, C-5), 96.5, 96.4 (Cp*), 82.3, 77.7, 77.6, 76.9 (C-1′, C-3′–C-5′), 74.3, 74.0, 73.6 (3 × PhCH₂), 69.0 (C-6′), 54.4 (C-2′), 8.9 (Cp*-CH₃). ESI-HRMS positive mode (m/z): calcd for C₄₂H₄₉ClN₂O₄Rh⁺ [M-PF₆]⁺ 783.2430. Found: 783.2430.

Complex Ru-3d

Prepared from compound 3d (16.7 mg, 0.033 mmol, 2 eq.), Ru-dimer (10.0 mg, 0.016 mmol) and TlPF₆ (11.4 mg, 0.033 mmol) according to general procedure VII. After purification by column chromatography (100:1 CHCl₃-MeOH), the complex was dissolved in CHCl₃ (1.5 mL), and diisopropyl ether (12 mL) was added. The precipitated product was filtered off, then washed with diisopropyl ether (1 mL) to yield 10.0 mg (33%) of a brown solid. R_f = 0.31 (95:5 CHCl₃-MeOH). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.09 (1H, s, H-3), 8.94 (1H, dd, J = 3.2, 1.1 Hz, H-5), 8.65 (1H, d, J = 3.2 Hz, H-6), 7.43–7.26 (15H, m, Ar), 5.89, 5.85, 5.54, 5.15 (4 × 1H, 4 d, J = 6.0 Hz in each, 4 × p-cym-CH_Ar), 5.22–5.16 (1H, broad signal, NH₂), 4.92, 4.77 (2 × 1H, 2 d, J = 12.3 Hz in both, PhCH₂), 4.76, 4.66 (2 × 1H, 2 d, J = 11.3 Hz in both, PhCH₂), 4.67 (1H, d, J = 10.3 Hz, H-1′), 4.56 (2H, s, PhCH₂), 4.20 (1H, pt, J = 9.1, 8.9 Hz, H-3′ or H-4′), 3.99–3.94 (1H, m, H-5′), 3.81–3.77 (2H, m, H-6′a,b), 3.56 (1H, pt, J = 9.2, 9.1 Hz, H-3′ or H-4′), 3.16 (1H, dd, J = 11.1, 5.1 Hz, NH₂), 2.56 (1H, hept, J = 6.9 Hz, i-Pr-CH), 2.04–1.95 (1H, m, H-2′), 1.76 (3H, s, C₆H₄-CH₃), 1.16, 1.03 (2 × 3H, 2 d, J = 6.9 Hz in both, 2 × i-Pr-CH₃); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 156.5 (C-2), 149.9, 145.3, 145.2 (C-3, C-5, C-6), 138.4, 138.0, 137.9, 129.1–127.9 (Ar), 105.7, 98.9 (2 × p-cym-C_qAr), 86.8, 85.2, 84.1, 84.0, 83.3, 77.5, 77.2, 75.1 (4 × p-cym-CH_Ar, C-1′, C-3′–C-5′), 75.3, 74.4, 73.5 (3 × PhCH₂), 68.5 (C-6′), 53.2 (C-2′), 31.1 (i-Pr-CH), 23.0, 21.6 (2 × i-Pr-CH₃), 17.8 (C₆H₄-CH₃). ESI-HRMS positive mode (m/z): calcd for C₄₁H₄₇ClN₃O₄Ru⁺ [M-PF₆]⁺ 782.2307. Found: 782.2315.

Complex Os-3d

Prepared from compound 3d (13.0 mg, 0.025 mmol, 2 eq.), Os-dimer (10.0 mg, 0.013 mmol) and TlPF₆ (8.7 mg, 0.025 mmol) according to general procedure VII. After purification by column chromatography (100:1 CHCl₃-MeOH), the complex was dissolved in CHCl₃ (1.5 mL), and diisopropyl ether (12 mL) was added. The precipitated product was filtered off, then washed with diisopropyl ether (1 mL) to yield 12.0 mg (47%) of a greenish–brown solid. R_f = 0.27 (95:5 CHCl₃-MeOH). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.10 (1H, s, H-3), 8.76 (1H, dd, J = 3.3, 1.1 Hz, H-5), 8.56 (1H, d, J = 3.3 Hz, H-6), 7.42–7.27 (15H, m, Ar), 6.12, 6.11, 5.80, 5.29 (4 × 1H, 4 d, J = 5.8 Hz in each, 4 × p-cym-CH_Ar), 5.90–5.77 (1H, broad signal, NH₂), 4.92, 4.75 (2 × 1H, 2 d, J = 12.3 Hz in both, PhCH₂), 4.77, 4.68 (2 × 1H, 2 d, J = 11.4 Hz in both, PhCH₂), 4.70–4.65 (1H, broad signal, NH₂), 4.62 (1H, d, J = 10.4 Hz, H-1′), 4.56 (2H, s, PhCH₂), 4.23 (1H, pt, J = 9.1, 9.0 Hz, H-3′ or H-4′), 3.99–3.95 (1H, m, H-5′), 3.83–3.76 (2H, m, H-6′a,b), 3.59 (1H, pt, J = 9.3, 9.2 Hz, H-3′ or H-4′), 2.46 (1H, hept, J = 6.9 Hz, i-Pr-CH), 2.33–2.24 (1H, m, H-2′), 1.80 (3H, s, C₆H₄-CH₃), 1.18, 0.99 (2 × 3H, 2 d, J = 6.9 Hz in both, 2 × i-Pr-CH₃); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 155.4 (C-2), 150.4, 146.0, 144.8 (C-3, C-5, C-6), 138.4, 138.0, 137.9, 129.1–127.9 (Ar), 96.2, 90.3 (2 × p-cym-C_qAr), 82.5, 78.5, 77.5, 77.1, 76.2, 76.0, 75.3, 74.7 (4 × p-cym-CH_Ar, C-1′, C-3′–C-5′), 75.3, 74.4, 73.5 (3 × PhCH₂), 68.4 (C-6′), 53.0 (C-2′), 31.1 (i-Pr-CH), 23.4, 21.7 (2 × i-Pr-CH₃), 17.6 (C₆H₄-CH₃). ESI-HRMS positive mode (m/z): calcd for C₄₁H₄₇ClN₃O₄Os⁺ [M-PF₆]⁺ 872.2861. Found: 872.2867.

Complex Ru-5a

Prepared from compound 5a (26 mg, 0.094 mmol, 2.3 eq.), Ru-dimer (25 mg, 0.041 mmol) and TlPF₆ (62 mg, 0.176 mmol, 4.3 eq.) according to general procedure VII. Column chromatographic purification (95:5 CHCl₃-MeOH) yielded 23 mg (43%) of a yellow solid. R_f = 0.50 (7:3 CHCl₃-MeOH). ¹H NMR (400 MHz, CD₃OD) δ (ppm); 9.09 (1H, ddd, J = 5.8, 1.4, 0.8 Hz, H-6), 8.07–8.00 (2H, m, H-3, H-4), 7.53–4.49 (1H, ddd, J = 7.8, 5.8, 2.0 Hz, H-5), 5.96, 5.94, 5.81, 5.64 (4 × 1H, 4 d, J = 6.0 Hz in each, 4 × p-cym-CH_Ar), 5.70 (1H, dd, J = 11.8, 5.6 Hz, NH₂), 4.41 (1H, d, J = 10.1 Hz, H-1′), 4.01 (1H, dd, J = 12.2, 2.1 Hz, H-6′a), 3.87–3.79 (1H, broad signal, NH₂), 3.84 (1H, pt, J = 9.6, 8.9 Hz, H-3′), 3.82 (1H, dd, J = 12.2, 5.6 Hz, H-6′b), 3.67 (1H, ddd, J = 9.5, 5.6, 2.1 Hz, H-5′), 3.39 (1H, pt, J = 9.3, 9.2 Hz, H-4′), 2.83 (1H, hept, J = 6.9 Hz, i-Pr-CH), 2.22–2.18 (1H, m, H-2), 1.91 (3H, s, CH₃), 1.28, 1.23 (2 × 3H, 2 d, J = 6.9 Hz in both, 2 × i-Pr-CH₃); ¹³C NMR (100 MHz, CD₃OD) δ (ppm): 161.8 (C-2), 158.1 (C-6), 140.9 (C-4), 125.8, 123.6 (C-3, C-5), 106.0, 100.8 (2 × p-cym-C_qAr), 87.8, 85.2, 84.1, 83.4, 81.8, 80.2, 77.5, 71.3 (4 × p-cym-CH_Ar, C-1′, C-3′–C-5′), 62.6 (C-6′), 56.1 (C-2′), 32.3 (i-Pr-CH), 23.3, 21.9 (2 × i-Pr-CH₃), 18.0 (C₆H₄-CH₃). ESI-HRMS positive mode (m/z): calcd for C₂₁H₃₀ClN₂O₄Ru⁺ [M-PF₆]⁺ 511.0935. Found: 511.0934.

Complex Ru-7a

Prepared from compound 7a (18.0 mg, 0.033 mmol, 2 eq.), Ru-dimer (10.0 mg, 0.016 mmol) and TlPF₆ (11.4 mg, 0.033 mmol) according to general procedure VII. The crude product was dissolved in CHCl₃ (3 mL), and diisopropyl ether (12 mL) was added. The precipitation was filtered off, then washed with CHCl₃-diisopropyl ether (1:2, 1 mL) to yield 25.5 mg (81%) of a yellow solid. R_f = 0.49 (95:5 CHCl₃-MeOH). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.02 (1H, d, J = 6.1 Hz, H-6), 8.12–7.31 (18H, m, Ar, H-3, H-4, H-5), 6.68–6.59 (1H, broad signal, NH₂), 6.15 (1H, d, J = 5.9 Hz, p-cym-CH_Ar), 6.10 (1H, pt, J = 9.4, 9.3 Hz, H-3′), 6.03, 6.00 (2 × 1H, 2 d, J = 6.0 Hz in both, 2 × p-cym-CH_Ar), 5.75 (1H, pt, J = 9.8, 9.6 Hz, H-4′), 5.47 (1H, d, J = 6.0 Hz, p-cym-CH_Ar), 5.01–4.97 (2H, m, H-1′, H-6′a), 4.86 (1H, ddd, J = 10.0, 3.4, 2.8 Hz, H-5′), 4.49 (1H, dd, J = 12.7, 3.4 Hz, H-6′b), 3.39 (1H, dd, J = 11.2, 7.3 Hz, NH₂), 2.89 (1H, hept, J = 6.9 Hz, i-Pr-CH), 2.65–2.57 (1H, m, H-2′), 1.81 (3H, s, C₆H₄-CH₃), 1.25, 1.23 (2 × 3H, 2 d, J = 6.9 Hz in both, 2 × i-Pr-CH₃); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 168.7, 166.3, 165.6 (3 × C=O), 160.2 (C-2), 156.7 (C-6), 140.0 (C-4), 134.4, 133.8, 133.3, 130.4–130.0, 129.9, 128.7–128.6, 127.8 (Ar), 125.3, 122.9 (C-3, C-5), 104.3, 100.7 (2 × p-cym-C_qAr), 88.0, 84.5, 82.6, 82.3, 77.9, 77.2, 74.4, 67.6 (4 × p-cym-CH_Ar, C-1′, C-3′–C-5′), 62.1 (C-6′), 54.3 (C-2′), 31.0 (i-Pr-CH), 22.8, 22.2 (2 × i-Pr-CH₃), 17.9 (C₆H₄-CH₃). ESI-HRMS positive mode (m/z): calcd for C₄₂H₄₂ClN₂O₇Ru⁺ [M-PF₆]⁺ 823.1727. Found: 823.1727.

Complex Os-7a

Prepared from compound 7a (14.0 mg, 0.025 mmol, 2 eq.), Os-dimer (10.0 mg, 0.013 mmol) and TlPF₆ (8.7 mg, 0.025 mmol) according to general procedure VII. After purification by column chromatography (95:5 CHCl₃-MeOH), the complex was dissolved in CHCl₃ (2 mL), and diisopropyl ether (16 mL) was added. The precipitated product was filtered off, then washed with CHCl₃-diisopropyl ether (1:4, 1 mL) to yield 11.5 mg (43%) of a dark purple solid. R_f = 0.45 (95:5 CHCl₃-MeOH). ¹H NMR (500 MHz, CDCl₃) δ (ppm): 8.86 (1H, dd, J = 5.9, 1.6 Hz, H-6), 8.15–7.34 (18H, m, Ar, H-3, H-4, H-5), 7.24–7.16 (1H, broad signal, NH₂), 6.52, 6.38, 6.20, 5.66 (4 × 1H, 4 d, J = 5.6 Hz in each, 4 × p-cym-CH_Ar), 5.96 (1H, pt, J = 9.3, 9.1 Hz, H-3′ or H-4′), 5.78 (1H, pt, J = 9.7, 9.6 Hz, H-3′ or H-4′), 4.99 (1H, dd, J = 12.7, 2.1 Hz, H-6′a), 4.88 (1H, d, J = 10.1 Hz, H-1′), 4.84 (1H, ddd, J = 10.2, 3.3, 2.1 Hz, H-5′), 4.49 (1H, dd, J = 12.7, 3.3 Hz, H-6′b), 4.24 (1H, dd, J = 11.6, 7.2 Hz, NH₂), 2.87–2.78 (2H, m, H-2′, i-Pr-CH), 1.85 (3H, s, C₆H₄-CH₃), 1.27, 1.26 (2 × 3H, 2 d, J = 6.9 Hz in both, 2 × i-Pr-CH₃); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 168.8, 166.3, 165.6 (3 × C=O), 159.6 (C-2), 156.8 (C-6), 140.2 (C-4), 134.5, 133.8, 133.3, 130.4–130.0, 129.9, 128.8–128.6, 127.7 (Ar), 125.5, 122.4 (C-3, C-5), 94.3, 92.2 (2 × p-cym-C_qAr), 80.6, 79.0, 77.1, 76.2, 74.3, 73.2, 73.1, 67.3 (4 × p-cym-CH_Ar, C-1′, C-3′–C-5′), 62.0 (C-6′), 54.2 (C-2′), 31.2 (i-Pr-CH), 23.1, 22.6 (2 × i-Pr-CH₃), 17.8 (C₆H₄-CH₃). ESI-HRMS positive mode (m/z): calcd for C₄₂H₄₂ClN₂O₇Os⁺ [M-PF₆]⁺ 913.2292. Found: 913.2288.

Complex Ir-7a

Prepared from compound 7a (13.9 mg, 0.025 mmol, 2 eq.), Ir-dimer (10.0 mg, 0.013 mmol) and TlPF₆ (8.8 mg, 0.025 mmol) according to general procedure VII. The crude product was dissolved in CHCl₃ (3 mL), and diisopropyl ether (12 mL) was added. The precipitation was filtered off, then washed with CHCl₃-diisopropyl ether (1:1, 1 mL) to yield 20.1 mg (76%) of a yellow solid. R_f = 0.36 (95:5 CHCl₃-MeOH). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.67 (1H, ddd, J = 5.8, 1.6, 0.7 Hz, H-6), 8.14–7.36 (18H, m, Ar, H-3, H-4, H-5), 6.08 (1H, dd, J = 11.9, 6.0 Hz, NH₂), 5.82 (1H, pt, J = 9.9, 9.7 Hz, H-4′), 5.69 (1H, pt, J = 9.6, 9.5 Hz, H-3′), 5.08 (1H, dd, J = 12.8, 2.2 Hz, H-6′a), 4.78 (1H, ddd, J = 10.0, 3.2, 2.2 Hz, H-5′), 4.53 (1H, dd, J = 12.8, 3.2 Hz, H-6′b), 4.49 (1H, d, J = 10.5 Hz, H-1′), 4.22 (1H, dd, J = 11.9, 8.1 Hz, NH₂), 3.09–3.00 (1H, m, H-2′), 1.76 (15H, s, Cp*-CH₃); ¹³C NMR (100 MHz, acetone-d₆) δ (ppm): 167.9, 166.5, 166.0 (3 × C=O), 158.9 (C-2), 156.3 (C-6), 141.4 (C-4), 134.7, 134.6, 134.2, 130.8, 130.7–130.4, 130.0, 129.9, 129.5–129.4 (Ar), 127.2, 123.4 (C-3, C-5), 89.0 (Cp*), 81.8, 76.4, 76.3, 70.1 (C-1′, C-3′–C-5′), 63.3 (C-6′), 54.8 (C-2′), 9.2 (Cp*-CH₃). ESI-HRMS positive mode (m/z): calcd for C₄₂H₄₃ClN₂O₇Ir⁺ [M-PF₆]⁺ 915.2377. Found: 915.2381.

Complex Rh-7a

Prepared from compound 7a (17.9 mg, 0.032 mmol, 2 eq.), Rh-dimer (10.0 mg, 0.016 mmol) and TlPF₆ (11.3 mg, 0.032 mmol) according to general procedure VII. The crude product was dissolved in CHCl₃ (3 mL), and diisopropyl ether (12 mL) was added. The precipitation was filtered off, then washed with CHCl₃-diisopropyl ether (1:1, 0.5 mL) to yield 27.1 mg (86%) of an orange solid. R_f = 0.32 (95:5 CHCl₃-MeOH). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.68 (1H, d, J = 5.3 Hz, H-6), 8.13–7.43 (18H, m, Ar, H-3, H-4, H-5), 5.81 (1H, pt, J = 9.8, 9.7 Hz, H-3′ or H-4′), 5.66 (1H, pt, J = 9.8, 9.6 Hz, H-3′ or H-4′), 5.15 (1H, dd, J = 11.0, 5.0 Hz, NH₂), 5.06 (1H, dd, J = 12.9, 2.2 Hz, H-6′a), 4.72 (1H, ddd, J = 10.2, 3.1, 2.2 Hz, H-5′), 4.53 (1H, d, J = 10.0 Hz, H-1′), 4.51 (1H, dd, J = 12.9, 3.1 Hz, H-6′b), 3.51 (1H, pt, J = 10.2, 9.8 Hz, NH₂), 2.88–2.80 (1H, m, H-2′), 1.76 (15H, s, Cp*-CH₃); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 169.3, 166.2, 165.6 (3 × C=O), 158.0 (C-2), 153.8 (C-6), 140.4 (C-4), 134.6, 133.8, 133.3, 130.5–130.0, 129.9, 128.8–128.7, 128.6, 127.6 (Ar), 126.0, 123.4 (C-3, C-5), 97.4, 97.3 (Cp*), 79.1, 78.3, 75.3, 67.2 (C-1′, C-3′–C-5′), 62.0 (C-6′), 55.0 (C-2′), 9.3 (Cp*-CH₃). ESI-HRMS positive mode (m/z): calcd for C₄₂H₄₃ClN₂O₇Rh⁺ [M-PF₆]⁺ 825.1808. Found: 825.1807.

Complex Ru-7b

Prepared from compound 7b (18.5 mg, 0.033 mmol, 2.05 eq.), Ru-dimer (10.0 mg, 0.016 mmol) and TlPF₆ (11.4 mg, 0.033 mmol) according to general procedure VII. The crude product was dissolved in CHCl₃ (3 mL), and diisopropyl ether (12 mL) was added. The precipitation was filtered off, then washed with CHCl₃-diisopropyl ether (1:2, 2 mL) to yield 28.0 mg (88%) of a brownish–orange solid. R_f = 0.51 (95:5 CHCl₃-MeOH). Diastereomeric ratio = 2:1. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.18 (dd, J = 4.9, 2.0 Hz, major H-6), 9.08 (dd, J = 4.8, 2.0 Hz, minor H-6), 8.21 (dd, J = 8.5, 2.0 Hz, major H-4), 8.19 (dd, J = 8.4, 2.0 Hz, minor H-4), 8.08–7.25 (m, minor and major Ar), 7.79 (dd, J = 8.5, 4.9 Hz, minor H-5), 7.77 (dd, J = 8.5, 4.9 Hz, major H-5), 6.33 (pt, J = 10.6 Hz, major NH₂), 6.09 (pt, J = 9.2, 9.0 Hz, major H-3′), 6.06 (pt, J = 9.5, 9.1 Hz, minor H-3′), 6.03–5.96 (broad signal, minor NH₂), 6.00, 5.94, 5.76, 5.42 (4 d, J = 6.0 Hz in each, 4 × major p-cym-CH_Ar), 5.82, 5.80, 5.77, 5.69 (4 d, J = 6.0 Hz in each, 4 × minor p-cym-CH_Ar), 5.65 (pt, J = 9.9, 9.6 Hz, major H-4′), 5.57 (pt, J = 9.2, 9.1 Hz, minor H-4′), 5.23 (d, J = 10.3 Hz, major H-1′), 5.18 (d, J = 10.5 Hz, minor H-1′), 4.97 (dd, J = 12.7, 2.2 Hz, major H-6′a), 4.72–4.68 (m, minor H-6′a or H-6′b), 4.68 (ddd, J = 10.1, 3.4, 2.2 Hz, major H-5′), 4.49–4.44 (m, minor H-6′a or H-6′b), 4.46 (dd, J = 12.7, 3.4 Hz, major H-6′b), 4.44–4.39 (m, minor H-5′), 4.13 (dd, J = 11.3, 4.9 Hz, major NH₂), 3.79 (pt, J = 11.9 Hz, minor NH₂), 3.22–3.15 (m, minor H-2′), 3.01 (hept, J = 6.9 Hz, minor i-Pr-CH), 2.93–2.85 (m, major H-2′), 2.80 (hept, J = 6.9 Hz, major i-Pr-CH), 2.28 (s, minor C₆H₄-CH₃), 1.85 (s, major C₆H₄-CH₃), 1.28, 1.27 (2 d, J = 6.9 Hz in both, 2 × minor i-Pr-CH₃), 1.22, 1.17 (2 d, J = 6.9 Hz in both, 2 × major i-Pr-CH₃); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 167.7, 166.4, 165.4, 165.3 (3 × major C=O, major C-3), 166.9, 166.3, 165.3, 164.1 (3 × minor C=O, minor C-3), 152.3 (major C-6), 151.1 (minor C-6), 134.1, 133.9, 133.8, 133.7, 133.5 (2), 130.3–127.8 (minor and major Ar, C-4, C-5), 105.7, 100.5 (2 × minor p-cym-C_qAr), 104.9, 100.5 (2 × major p-cym-C_qAr), 88.5, 85.7, 85.5, 83.2 (4 × major p-cym-CH_Ar), 86.6, 86.5, 86.0, 83.3 (4 × minor p-cym-CH_Ar), 77.4, 75.0, 74.4, 67.7 (major C-1′, C-3′–C-5′), 76.3, 75.7, 73.3, 69.2 (minor C-1′, C-3′–C-5′), 62.7 (minor C-6′), 61.9 (major C-6′), 53.6 (minor C-2′), 53.6 (major C-2′), 30.9 (minor i-Pr-CH), 30.8 (major i-Pr-CH), 22.9, 21.8 (2 × major i-Pr-CH₃), 22.5, 22.3 (2 × minor i-Pr-CH₃), 18.2 (minor C₆H₄-CH₃), 17.9 (major C₆H₄-CH₃); ESI-HRMS positive mode (m/z): calcd for C₄₁H₄₁ClN₃O₇Ru⁺ [M-PF₆]⁺ 824.1679. Found: 824.1674.

Complex Os-7b

Prepared from compound 7b (14.2 mg, 0.026 mmol, 2.05 eq.), Os-dimer (10.0 mg, 0.0126 mmol) and TlPF₆ (8.7 mg, 0.025 mmol) according to general procedure VII. The crude product was dissolved in CHCl₃ (3 mL), and diisopropyl ether (6 mL) was added. The precipitation was filtered off, then washed with CHCl₃-diisopropyl ether (1:2, 3 mL) to yield 19.6 mg (73%) of a dark green solid. R_f = 0.38 (95:5 CHCl₃-MeOH). Diastereomeric ratio = 5:4. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.08 (dd, J = 4.9, 2.0 Hz, major H-6), 8.93 (dd, J = 4.8, 2.0 Hz, minor H-6), 8.30 (dd, J = 8.7, 2.0 Hz, minor H-4), 8.24 (dd, J = 8.6, 2.0 Hz, major H-4), 8.12–7.31 (m, minor and major Ar), 7.85 (dd, J = 8.7, 4.8 Hz, minor H-5), 7.69 (dd, J = 8.6, 4.9 Hz, major H-5), 6.89 (pt, J = 11.0 Hz, major NH₂), 6.72–6.65 (broad signal, minor NH₂), 6.27, 6.22, 6.19, 5.70 (4 d, J = 5.8 Hz in each, 4 × major p-cym-CH_Ar), 6.08, 6.04, 6.03, 5.92 (4 d, J = 5.7 Hz in each, 4 × minor p-cym-CH_Ar), 5.94 (pt, J = 9.0, 9.0 Hz, major H-3′), 5.88 (pt, J = 9.2, 8.9 Hz, minor H-3′), 5.73 (pt, J = 9.9, 9.8 Hz, major H-4′), 5.63 (pt, J = 9.4, 9.3 Hz, minor H-4′), 5.49 (d, J = 10.5 Hz, minor H-1′), 5.12 (d, J = 10.3 Hz, major H-1′), 4.98 (dd, J = 12.7, 2.2 Hz, major H-6′a), 4.73 (dd, J = 12.5, 2.5 Hz, minor H-6′a), 4.70 (ddd, J = 10.1, 3.4, 2.2 Hz, major H-5′), 4.64 (dd, J = 11.7, 4.9 Hz, major NH₂), 4.52–4.44 (m, minor and major H-6′b), 4.44–4.34 (m, minor H-5′, NH₂), 3.38–3.27 (m, minor H-2′), 3.13–3.05 (m, major H-2′), 2.88 (hept, J = 6.9 Hz, minor i-Pr-CH), 2.77 (hept, J = 6.9 Hz, major i-Pr-CH), 2.26 (s, minor C₆H₄-CH₃), 1.93 (s, major C₆H₄-CH₃), 1.26–1.20 (m, 2 × minor and major i-Pr-CH₃); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 167.9, 167.0, 166.4, 166.3, 165.4, 165.3, 164.4, 163.6 (3 × minor and major C=O, minor and major C-3), 152.7 (major C-6), 152.0 (minor C-6), 134.2, 133.9, 133.8, 133.7, 133.5, 133.4, 130.9–127.9 (minor and major Ar, C-4, C-5), 96.5, 94.2 (2 × minor p-cym-C_qAr), 94.9, 92.5 (2 × major p-cym-C_qAr), 80.3, 79.8 (2), 78.3 (2), 77.0, 76.5, 75.2 (2), 75.9, 75.8, 74.3, 73.6, 73.3, 69.0, 67.4 (4 × minor and major p-cym-CH_Ar, minor and major C-1′, C-3′–C-5′), 62.6 (minor C-6′), 61.8 (major C-6′), 54.5 (minor C-2′), 53.5 (major C-2′), 31.0 (minor i-Pr-CH), 30.9 (major i-Pr-CH), 22.4, 21.9 (2 × major i-Pr-CH₃), 22.7, 22.6 (2 × minor i-Pr-CH₃), 18.2 (minor C₆H₄-CH₃), 17.9 (major C₆H₄-CH₃). ESI-HRMS positive mode (m/z): calcd for C₄₁H₄₁ClN₃O₇Os⁺ [M-PF₆]⁺ 914.2234. Found: 914.2239.

Complex Ir-7b

Prepared from compound 7b (14.2 mg, 0.026 mmol, 2.05 eq.), Ir-dimer (10.0 mg, 0.013 mmol) and TlPF₆ (8.8 mg, 0.025 mmol) according to general procedure VII. The crude product was dissolved in CHCl₃ (3 mL), and diisopropyl ether (6 mL) was added. The precipitation was filtered off, then washed with CHCl₃-diisopropyl ether (1:1, 4 mL) to yield 18.5 mg (69%) of a yellow solid. R_f = 0.33 (95:5 CHCl₃-MeOH). Diastereomeric ratio = 9:1. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.09 (dd, J = 4.9, 2.0 Hz, major H-6), 8.96 (dd, J = 4.8, 2.0 Hz, minor H-6), 8.34 (dd, J = 8.7, 2.0 Hz, major H-4), 8.29 (dd, J = 8.7, 2.0 Hz, minor H-4), 8.12–7.34 (m, minor and major Ar), 7.86 (dd, J = 8.7, 4.8 Hz, minor H-5), 7.78 (dd, J = 8.7, 4.9 Hz, major H-5), 5.96 (d, J = 10.2 Hz, minor H-1′), 5.87 (pt, J = 9.1, 9.0 Hz, major H-3′ or H-4′), 5.81 (pt, J = 9.8, 9.3 Hz, minor H-3′ or H-4′), 5.78 (pt, J = 9.9, 9.5 Hz, major H-3′ or H-4′), 5.66 (pt, J = 9.2, 9.1 Hz, minor H-3′ or H-4′), 5.59 (pt, J = 10.6 Hz, major NH₂), 5.36–5.29 (m, minor NH₂), 5.12–5.05 (m, minor NH₂), 4.99 (dd, J = 12.7, 2.2 Hz, major H-6′a), 4.89 (d, J = 10.3 Hz, major H-1′), 4.75 (ddd, J = 10.3, 3.6, 2.2 Hz, major H-5′), 4.77–4.66 (m, major NH₂, minor H-6′a), 4.54 (dd, J = 12.5, 5.2 Hz, minor H-6′b), 4.48 (dd, J = 12.7, 3.6 Hz, major H-6′b), 4.29 (ddd, J = 10.0, 5.2, 2.7 Hz, minor H-5′), 3.75–3.67 (m, minor H-2′), 3.44–3.35 (m, major H-2′), 1.69 (s, major Cp*-CH₃), 1.57 (s, minor Cp*-CH₃); ¹³C NMR (100 MHz, CDCl₃) δ (ppm); only the major isomer can be clearly assigned: 168.4, 166.4, 165.5, 163.7 (3 × C=O, C-3), 153.5 (C-6), 134.3, 133.8, 133.4, 130.5–130.0, 129.8, 129.6–127.6, 127.9 (Ar, C-4, C-5), 89.3 (Cp*), 78.0, 76.4, 74.6, 67.3 (C-1′, C-3′–C-5′), 61.9 (C-6′), 54.3 (C-2′), 8.8 (Cp*-CH₃). ESI-HRMS positive mode (m/z): calcd for C₄₁H₄₂ClN₃O₇Ir⁺ [M-PF₆]⁺ 916.2329. Found: 916.2322.

Complex Rh-7b

Prepared from compound 7b (9.2 mg, 0.017 mmol, 2.05 eq.), Rh-dimer (5.0 mg, 0.008 mmol) and TlPF₆ (5.6 mg, 0.016 mmol) according to general procedure VII. The crude product was dissolved in CHCl₃ (1.5 mL), and diisopropyl ether (3 mL) was added. The precipitation was filtered off, then washed with CHCl₃-diisopropyl ether (1:1, 2 mL) to yield 13.5 mg (86%) of an orange solid. R_f = 0.38 (95:5 CHCl₃-MeOH). Diastereomeric ratio = 5:1. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.17 (dd, J = 4.9, 1.9 Hz, major H-6), 9.10 (dd, J = 4.8, 1.9 Hz, minor H-6), 8.28 (dd, J = 8.7, 1.9 Hz, major H-4), 8.17 (dd, J = 8.7, 1.9 Hz, minor H-4), 8.12–7.32 (m, minor and major Ar), 7.80 (dd, J = 8.7, 4.8 Hz, minor H-5), 7.77 (dd, J = 8.7, 4.9 Hz, major H-5), 6.01 (d, J = 10.0 Hz, minor H-1′), 5.89 (pt, J = 9.2, 9.1 Hz, major H-3′ or H-4′), 5.79 (pt, J = 9.7, 9.6 Hz, minor H-3′ or H-4′), 5.74 (pt, J = 9.8, 9.6 Hz, major H-3′ or H-4′), 5.61 (pt, J = 9.6, 9.4 Hz, minor H-3′ or H-4′), 5.07 (d, J = 10.3 Hz, major H-1′), 4.95 (dd, J = 12.7, 2.2 Hz, major H-6′a), 4.92–4.87 (broad signal, minor NH₂), 4.81 (pt, J = 10.6 Hz, major NH₂), 4.73 (ddd, J = 10.2, 3.7, 2.2 Hz, major H-5′), 4.63 (dd, J = 12.4, 2.6 Hz, minor H-6′a), 4.50 (dd, J = 12.4, 5.4 Hz, minor H-6′b), 4.48 (dd, J = 12.7, 3.7 Hz, major H-6′b), 4.37 (pt, J = 9.1 Hz, minor NH₂), 4.25 (dd, J = 10.6, 4.4 Hz, major NH₂), 4.21 (ddd, J = 10.1, 5.4, 2.6 Hz, minor H-5′), 3.57–3.47 (m, minor H-2′), 3.30–3.21 (m, major H-2′), 1.73 (s, major Cp*-CH₃); 1.65 (s, minor Cp*-CH₃); ¹³C NMR (90 MHz, CDCl₃) δ (ppm): 170.0, 165.1, 164.5, 162.7 (3 × minor C=O, minor C-3), 168.3, 166.4, 165.5, 164.5 (3 × major C=O, major C-3), 152.8 (major C-6), 151.4 (minor C-6), 134.5, 134.2, 133.7 (2), 133.4, 133.3, 130.5–128.1 (minor and major C-4, C-5, Ar), 98.0, 97.9 (minor Cp*), 97.5, 97.4 (major Cp*), 79.1, 76.4, 74.3, 68.9 (minor C-1′, C-3′–C-5′), 78.9, 75.5, 74.6, 67.5 (major C-1′, C-3′–C-5′), 63.0 (minor C-6′), 62.0 (major C-6′), 54.8 (minor C-2′), 52.6 (major C-2′), 9.0 (major Cp*-CH₃), 8.7 (minor Cp*-CH₃). ESI-HRMS positive mode (m/z): calcd for C₄₁H₄₂ClN₃O₇Rh⁺ [M-PF₆]⁺ 826.1761. Found: 826.1757.

Complex Ru-7c

Prepared from compound 7c (18.5 mg, 0.033 mmol, 2.05 eq.), Ru-dimer (10.0 mg, 0.016 mmol) and TlPF₆ (11.4 mg, 0.033 mmol) according to general procedure VII. The crude product was dissolved in CHCl₃ (3 mL), and diisopropyl ether (12 mL) was added. The precipitation was filtered off, then washed with CHCl₃-diisopropyl ether (1:2, 2 mL) to yield 31.2 mg (99%) of a brownish–orange solid. R_f = 0.44 (95:5 CHCl₃-MeOH). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.33, 8.93 (2 ×1H, 2 dd, J = 5.8, 2.2 Hz and 4.7, 2.2 Hz, respectively, H-4, H-6), 8.02–7.21 (16H, m, Ar, H-5), 6.19 (1H, dd, J = 11.6, 8.3 Hz, NH₂), 6.11 (1H, pt, J = 9.2, 9.2 Hz, H-3′ or H-4′), 6.01, 5.91, 5.89, 5.40 (4 × 1H, 4 d, J = 6.0 Hz in each, 4 × p-cym-CH_Ar), 5.71 (1H, pt, J = 9.7, 9.6 Hz, H-3′ or H-4′), 5.11 (1H, d, J = 10.3 Hz, H-1′), 4.82 (1H, dd, J = 12.7, 2.4 Hz, H-6′a), 4.73 (1H, m, H-5′), 4.60 (1H, dd, J = 12.7, 3.6 Hz, H-6′b), 3.67 (1H, dd, J = 11.6, 6.2 Hz, NH₂), 2.94–2.86 (1H, m, H-2′), 2.75 (1H, hept, J = 6.9 Hz, i-Pr-CH), 1.72 (3H, s, C₆H₄-CH₃), 1.23, 1.09 (2 × 3H, 2 d, J = 6.9 Hz in both, 2 × i-Pr-CH₃); ¹³C NMR (90 MHz, CDCl₃) δ (ppm): 168.5, 166.4, 166.2, 165.2 (3 × C=O, C-2), 165.1 (C-6), 159.4 (C-4), 134.4, 133.8, 133.2, 130.3–128.5, 127.4 (Ar), 122.1 (C-5), 105.4, 100.8 (2 × p-cym-C_qAr), 86.2, 84.2, 83.5, 82.6 (4 × p-cym-CH_Ar), 78.0, 77.4, 74.3, 68.2 (C-1′, C-3′–C-5′), 62.7 (C-6′), 54.2 (C-2′), 31.0 (i-Pr-CH), 23.0, 21.8 (2 × i-Pr-CH₃), 18.0 (C₆H₄-CH₃). ESI-HRMS positive mode (m/z): calcd for C₄₁H₄₁ClN₃O₇Ru⁺ [M-PF₆]⁺ 824.1679. Found: 824.1681.

Complex Os-7c

Prepared from compound 7c (14.2 mg, 0.026 mmol, 2.05 eq.), Os-dimer (10.0 mg, 0.013 mmol) and TlPF₆ (8.7 mg, 0.025 mmol) according to general procedure VII. The crude product was dissolved in CHCl₃ (3 mL), and diisopropyl ether (6 mL) was added. The precipitation was filtered off, then washed with CHCl₃-diisopropyl ether (1:1, 2 mL) to yield 26.3 mg (98%) of a dark green solid. R_f = 0.27 (95:5 CHCl₃-MeOH). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.21, 8.86 (2 ×1H, 2 dd, J = 5.9, 2.2 Hz and 4.7, 2.2 Hz, respectively, H-4, H-6), 8.03–7.21 (16H, m, Ar, H-5), 6.86 (1H, dd, J = 12.0, 8.5 Hz, NH₂), 6.23, 6.18, 6.14, 5.57 (4 × 1H, 4 d, J = 5.7 Hz in each, 4 × p-cym-CH_Ar), 6.07 (1H, pt, J = 9.2, 9.2 Hz, H-3′ or H-4′), 5.74 (1H, pt, J = 9.8, 9.6 Hz, H-3′ or H-4′), 5.03 (1H, d, J = 10.3 Hz, H-1′), 4.82 (1H, dd, J = 12.7, 2.4 Hz, H-6′a), 4.71 (1H, ddd, J = 10.2, 3.5, 2.4 Hz, H-5′), 4.60 (1H, dd, J = 12.7, 3.5 Hz, H-6′b), 4.43 (1H, dd, J = 12.0, 6.0 Hz, NH₂), 3.19–3.10 (1H, m, H-2′), 2.68 (1H, hept, J = 6.9 Hz, i-Pr-CH), 1.75 (3H, s, C₆H₄-CH₃), 1.23, 1.06 (2 × 3H, 2 d, J = 6.9 Hz in both, 2 × i-Pr-CH₃); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 168.4, 166.2, 165.6, 165.2 (3 × C=O, C-2), 165.5 (C-6), 159.2 (C-4), 134.5, 133.9, 133.2, 130.3–130.0, 129.8, 128.8–128.5, 127.3 (Ar), 122.4 (C-5), 96.0, 92.5 (2 × p-cym-C_qAr), 78.3, 77.9, 77.0, 75.8, 74.5, 74.2, 73.5, 68.0 (4 × p-cym-CH_Ar, C-1′, C-3′–C-5′), 62.6 (C-6′), 53.9 (C-2′), 31.1 (i-Pr-CH), 23.4, 21.9 (2 × i-Pr-CH₃), 17.9 (C₆H₄-CH₃). ESI-HRMS positive mode (m/z): calcd for C₄₁H₄₁ClN₃O₇Os⁺ [M-PF₆]⁺ 914.2234. Found: 914.2234.

Complex Ir-7c

Prepared from compound 7c (14.2 mg, 0.026 mmol, 2.05 eq.), Ir-dimer (10.0 mg, 0.013 mmol) and TlPF₆ (8.8 mg, 0.025 mmol) according to general procedure VII. The crude product was dissolved in CHCl₃ (3 mL), and diisopropyl ether (6 mL) was added. The precipitation was filtered off, then washed with CHCl₃-diisopropyl ether (1:1, 4 mL) to yield 26.4 mg (99%) of a yellow solid. R_f = 0.30 (95:5 CHCl₃-MeOH). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.98, 8.84 (2 ×1H, 2 dd, J = 4.8, 2.2 Hz and 5.9, 2.2 Hz, respectively, H-4, H-6), 8.01–7.29 (16H, m, Ar, H-5), 5.94 (1H, dd, J = 11.8, 5.2 Hz, NH₂), 5.83 (1H, pt, J = 9.6, 9.6 Hz, H-3′ or H-4′), 5.66 (1H, pt, J = 9.5, 9.6 Hz, H-3′ or H-4′), 4.81–4.56 (4H, m, H-1′, H-5′, H-6′a,b), 4.29 (1H, dd, J = 11.8, 8.2 Hz, NH₂), 3.37–3.29 (1H, m, H-2′), 1.74 (15H, s, Cp*-CH₃); ¹³C NMR (90 MHz, CDCl₃) δ (ppm): 169.3, 166.3, 165.5, 164.1 (3 × C=O, C-2), 162.3, 160.5 (C-4, C-6), 134.8, 133.8, 133.2, 130.6–130.0, 129.8, 128.9–128.5, 127.5 (Ar), 123.2 (C-5), 89.4 (Cp*), 80.5, 77.9, 75.2, 67.7 (C-1′, C-3′–C-5′), 62.8 (C-6′), 54.9 (C-2′), 9.1 (Cp*-CH₃). ESI-HRMS positive mode (m/z): calcd for C₄₁H₄₂ClN₃O₇Ir⁺ [M-PF₆]⁺ 916.2329. Found: 916.2324.

Complex Rh-7c

Prepared from compound 7c (9.2 mg, 0.017 mmol, 2.05 eq.), Rh-dimer (5.0 mg, 0.008 mmol) and TlPF₆ (5.6 mg, 0.016 mmol) according to general procedure VII. The crude product was dissolved in CHCl₃ (1.5 mL), and diisopropyl ether (3 mL) was added. The precipitation was filtered off, then washed with CHCl₃-diisopropyl ether (1:1, 4 mL) to yield 15.5 mg (99%) of an orange solid. R_f = 0.24 (95:5 CHCl₃-MeOH). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.02, 8.92 (2 ×1H, 2 dd, J = 4.9, 2.3 Hz and 5.7, 2.3 Hz, respectively, H-4, H-6), 8.03–7.29 (16H, m, Ar, H-5), 5.84 (1H, pt, J = 9.6, 9.6 Hz, H-3′ or H-4′), 5.67 (1H, pt, J = 9.7, 9.6 Hz, H-3′ or H-4′), 5.15 (1H, dd, J = 12.1, 3.7 Hz, NH₂), 4.85–4.58 (3H, m, H-5′, H-6′a,b), 4.72 (1H, d, J = 10.2 Hz, H-1′), 3.46 (1H, pt, J = 10.0 Hz, NH₂), 3.21–3.13 (1H, m, H-2′), 1.81 (15H, s, Cp*-CH₃); ¹³C NMR (90 MHz, CDCl₃) δ (ppm): 169.4, 166.3, 165.5, 164.7 (3 × C=O, C-2), 161.7, 160.4 (C-4, C-6), 134.8, 133.8, 133.2, 130.6–130.0, 129.9, 128.9–128.5, 127.5 (Ar), 122.8 (C-5), 97.9, 97.8 (Cp*), 80.0, 78.4, 75.4, 67.8 (C-1′, C-3′–C-5′), 62.8 (C-6′), 55.3 (C-2′), 9.5 (Cp*-CH₃). ESI-HRMS positive mode (m/z): calcd for C₄₁H₄₂ClN₃O₇Rh⁺ [M-PF₆]⁺ 826.1761. Found: 826.1757.

Complex Ru-7d

Prepared from compound 7d (18.5 mg, 0.033 mmol, 2.05 eq.), Ru-dimer (10.0 mg, 0.016 mmol) and TlPF₆ (11.4 mg, 0.033 mmol) according to general procedure VII. The crude product was dissolved in CHCl₃ (3 mL), and diisopropyl ether (6 mL) was added. The precipitation was filtered off, then washed with CHCl₃-diisopropyl ether (1:2, 6 mL) to yield 28.8 mg (91%) of a brown solid. R_f = 0.35 (95:5 CHCl₃-MeOH). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.09 (1H, s, H-3), 9.02 (1H, dd, J = 3.2, 1.1 Hz, H-5), 8.76 (1H, d, J = 3.2 Hz, H-6), 8.08–7.26 (15H, m, Ar), 6.26 (1H, dd, J = 11.5, 8.3 Hz, NH₂), 6.07 (1H, pt, J = 9.3, 9.2 Hz, H-3′ or H-4′), 6.01–5.96 (3H, m, 3 × p-cym-CH_Ar), 5.72 (1H, pt, J = 9.7, 9.7 Hz, H-3′ or H-4′), 5.44 (1H, d, J = 6.0 Hz, p-cym-CH_Ar), 5.03 (1H, d, J = 10.2 Hz, H-1′), 4.98 (1H, dd, J = 12.8, 2.2 Hz, H-6′a), 4.76 (1H, ddd, J = 10.2, 3.4, 2.2 Hz, H-5′), 4.48 (1H, dd, J = 12.8, 3.4 Hz, H-6′b), 3.54 (1H, dd, J = 11.5, 6.7 Hz, NH₂), 2.81 (1H, hept, J = 6.9 Hz, i-Pr-CH), 2.73–2.64 (1H, m, H-2′), 1.77 (3H, s, C₆H₄-CH₃), 1.23, 1.16 (2 × 3H, 2 d, J = 6.9 Hz in both, 2 × i-Pr-CH₃); ¹³C NMR (90 MHz, CDCl₃) δ (ppm): 168.4, 166.3, 165.4 (3 × C=O), 154.7 (C-2), 150.1, 146.3, 144.6 (C-3, C-5, C-6), 134.4, 133.9, 133.4, 130.3–130.0, 129.7, 128.8–128.6, 127.6 (Ar), 105.4, 101.0 (2 × p-cym-C_qAr), 87.4, 84.9, 83.4, 83.3 (4 × p-cym-CH_Ar), 77.0, 76.9, 74.6, 67.7 (C-1′, C-3′–C-5′), 61.9 (C-6′), 53.8 (C-2′), 31.0 (i-Pr-CH), 22.9, 21.9 (2 × i-Pr-CH₃), 17.9 (C₆H₄-CH₃). ESI-HRMS positive mode (m/z): calcd for C₄₁H₄₁ClN₃O₇Ru⁺ [M-PF₆]⁺ 824.1679. Found: 824.1673.

Complex Os-7d

Prepared from compound 7d (14.2 mg, 0.026 mmol, 2.05 eq.), Os-dimer (10.0 mg, 0.013 mmol) and TlPF₆ (8.7 mg, 0.025 mmol) according to general procedure VII. The crude product was dissolved in CHCl₃ (3 mL), and diisopropyl ether (6 mL) was added. The precipitation was filtered off, then washed with CHCl₃-diisopropyl ether (1:2, 3 mL) to yield 24.4 mg (91%) of a brown solid. R_f = 0.29 (95:5 CHCl₃-MeOH). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.09 (1H, s, H-3), 8.87 (1H, dd, J = 3.2, 1.1 Hz, H-5), 8.69 (1H, d, J = 3.2 Hz, H-6), 8.09–7.28 (15H, m, Ar), 6.96 (1H, dd, J = 11.9, 8.4 Hz, NH₂), 6.27–6.24 (2H, m, 2 × p-cym-CH_Ar), 6.20 (1H, d, J = 5.7 Hz, p-cym-CH_Ar), 6.03 (1H, pt, J = 9.2, 9.2 Hz, H-3′ or H-4′), 5.75 (1H, pt, J = 9.7, 9.6 Hz, H-3′ or H-4′), 5.62 (1H, d, J = 5.7 Hz, p-cym-CH_Ar), 4.99 (1H, dd, J = 12.8, 2.2 Hz, H-6′a), 4.94 (1H, d, J = 10.2 Hz, H-1′), 4.75 (1H, ddd, J = 10.2, 3.4, 2.2 Hz, H-5′), 4.49 (1H, dd, J = 12.8, 3.4 Hz, H-6′b), 4.28 (1H, dd, J = 11.9, 6.4 Hz, NH₂), 2.97–2.88 (1H, m, H-2′), 2.74 (1H, hept, J = 6.9 Hz, i-Pr-CH), 1.80 (3H, s, C₆H₄-CH₃), 1.23, 1.14 (2 × 3H, 2 d, J = 6.9 Hz in both, 2 × i-Pr-CH₃); ¹³C NMR (90 MHz, CDCl₃) δ (ppm): 168.5, 166.3, 165.3 (3 × C=O), 153.6 (C-2), 150.4, 147.1, 144.3 (C-3, C-5, C-6), 134.5, 133.9, 133.4, 130.3–130.0, 129.7, 128.8–128.6, 127.5 (Ar), 95.9, 92.7 (2 × p-cym-C_qAr), 79.5, 77.6, 76.7, 76.6, 74.6, 74.3, 74.1, 67.5 (4 × p-cym-CH_Ar, C-1′, C-3′–C-5′), 61.8 (C-6′), 53.6 (C-2′), 31.1 (i-Pr-CH), 23.3, 22.1 (2 × i-Pr-CH₃), 17.7 (C₆H₄-CH₃). ESI-HRMS positive mode (m/z): calcd for C₄₁H₄₁ClN₃O₇Os⁺ [M-PF₆]⁺ 914.2234. Found: 914.2233.

Complex Ir-7d

Prepared from compound 7d (14.2 mg, 0.026 mmol, 2.05 eq.), Ir-dimer (10.0 mg, 0.013 mmol) and TlPF₆ (8.8 mg, 0.025 mmol) according to general procedure VII. The crude product was dissolved in CHCl₃ (3 mL), and diisopropyl ether (6 mL) was added. The precipitation was filtered off, then washed with CHCl₃-diisopropyl ether (1:1, 4 mL) to yield 26.5 mg (99%) of a yellow solid. R_f = 0.24 (95:5 CHCl₃-MeOH). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.18 (1H, s, H-3), 8.76 (1H, d, J = 3.2 Hz, H-6), 8.54 (1H, dd, J = 3.2, 1.2 Hz, H-5), 8.10–7.32 (15H, m, Ar), 5.94 (1H, dd, J = 11.8, 6.4 Hz, NH₂), 5.84 (1H, pt, J = 9.8, 9.7 Hz, H-3′ or H-4′), 5.68 (1H, pt, J = 9.6, 9.5 Hz, H-3′ or H-4′), 5.03 (1H, dd, J = 12.9, 2.2 Hz, H-6′a), 4.74 (1H, ddd, J = 10.1, 3.0, 2.2 Hz, H-5′), 4.56 (1H, d, J = 10.1 Hz, H-1′), 4.50 (1H, dd, J = 12.9, 3.0 Hz, H-6′b), 4.27 (1H, dd, J = 11.8, 7.9 Hz, NH₂), 3.17–3.09 (1H, m, H-2′), 1.73 (15H, s, Cp*-CH₃); ¹³C NMR (90 MHz, CDCl₃) δ (ppm): 169.3, 166.2, 165.5 (3 × C=O), 152.1 (C-2), 147.8, 147.5, 145.5 (C-3, C-5, C-6), 134.8, 133.8, 133.3, 130.6–130.0, 129.8, 128.8–128.6, 128.5, 127.5 (Ar), 89.7 (Cp*), 78.9, 77.7, 75.4, 67.1 (C-1′, C-3′–C-5′), 61.8 (C-6′), 54.5 (C-2′), 8.9 (Cp*-CH₃). ESI-HRMS positive mode (m/z): calcd for C₄₁H₄₂ClN₃O₇Ir⁺ [M-PF₆]⁺ 916.2329. Found: 916.2331.

Complex Rh-7d

Prepared from compound 7d (9.2 mg, 0.017 mmol, 2.05 eq.), Rh-dimer (5.0 mg, 0.008 mmol) and TlPF₆ (5.6 mg, 0.016 mmol) according to general procedure VII. The crude product was dissolved in CHCl₃ (1.5 mL), and diisopropyl ether (3 mL) was added. The precipitation was filtered off, then washed with CHCl₃-diisopropyl ether (1:1, 4 mL) to yield 15.1 mg (96%) of an orange solid. R_f = 0.21 (95:5 CHCl₃-MeOH). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.19 (1H, s, H-3), 8.83 (1H, d, J = 3.1 Hz, H-6), 8.61 (1H, dd, J = 3.1, 1.2 Hz, H-5), 8.11–7.32 (15H, m, Ar), 5.83 (1H, pt, J = 9.8, 9.7 Hz, H-3′ or H-4′), 5.69 (1H, pt, J = 9.7, 9.6 Hz, H-3′ or H-4′), 5.13 (1H, dd, J = 11.4, 6.1 Hz, NH₂), 5.05 (1H, dd, J = 12.9, 2.2 Hz, H-6′a), 4.75 (1H, ddd, J = 10.2, 2.9, 2.2 Hz, H-5′), 4.69 (1H, d, J = 10.1 Hz, H-1′), 4.51 (1H, dd, J = 12.9, 2.9 Hz, H-6′b), 3.54 (1H, pt, J = 9.9 Hz, NH₂), 3.02–2.93 (1H, m, H-2′), 1.78 (15H, s, Cp*-CH₃); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 169.4, 166.2, 165.5 (3 × C=O), 152.6 (C-2), 147.1, 146.8, 145.6 (C-3, C-5, C-6), 134.8, 133.8, 133.3, 130.6–130.0, 129.8–128.7, 128.6, 127.5 (Ar), 98.1, 98.0 (Cp*), 78.3, 78.2, 75.5, 67.1 (C-1′, C-3′–C-5′), 61.7 (C-6′), 54.9 (C-2′), 9.3 (Cp*-CH₃). ESI-HRMS positive mode (m/z): calcd for C₄₁H₄₂ClN₃O₇Rh⁺ [M-PF₆]⁺ 826.1761. Found: 826.1754.

Complex Ru-7e

Prepared from compound 7e (21.2 mg, 0.035 mmol, 2.15 eq.), Ru-dimer (10.0 mg, 0.016 mmol) and TlPF₆ (11.4 mg, 0.033 mmol) according to general procedure VII. The crude product was dissolved in CHCl₃ (3 mL), and diisopropyl ether (12 mL) was added. The precipitation was filtered off, then washed with CHCl₃-diisopropyl ether (1:2, 2 mL) to yield 27.2 mg (82%) of an orange solid. R_f = 0.66 (95:5 CHCl₃-MeOH). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.38 (1H, d, J = 9.3 Hz, H-3 or H-4 or H-5 or H-8), 8.37 (1H, d, J = 8.7 Hz, H-3 or H-4 or H-5 or H-8), 8.16–7.34 (19H, m, Ar, H-3 and/or H-4 and/or H-5 and/or H-8, H-6, H-7), 6.58 (1H, dd, J = 11.4, 6.3 Hz, NH₂), 6.22–6.18 (3H, m, 3 × p-cym-CH_Ar), 6.04 (1H, pt, J = 9.5, 9.2 Hz, H-3′ or H-4′), 5.83 (1H, pt, J = 9.7, 9.7 Hz, H-3′ or H-4′), 5.51 (1H, d, J = 6.1 Hz, p-cym-CH_Ar), 5.14 (1H, d, J = 10.1 Hz, H-1′), 5.05 (1H, dd, J = 12.7, 2.1 Hz, H-6′a), 4.93 (1H, ddd, J = 9.8, 3.3, 2.1 Hz, H-5′), 4.53 (1H, dd, J = 12.7, 3.3 Hz, H-6′b), 3.61 (1H, dd, J = 11.4, 7.6 Hz, NH₂), 2.89 (1H, hept, J = 6.9 Hz, i-Pr-CH), 2.82–2.74 (1H, m, H-2), 1.59 (3H, s, C₆H₄-CH₃), 1.26, 1.19 (2 × 3H, 2 d, J = 6.9 Hz in both, 2 × i-Pr-CH₃); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 168.9, 166.3, 165.7, 163.5, 149.3 (3 × C=O, C-2, C-8a), 141.3, 134.4, 133.8, 133.3, 131.4, 131.3, 130.4–130.0, 129.9, 129.3, 129.0–128.6, 127.8, 120.0 (Ar, C-3–C-8, C-4a), 104.4, 100.6 (2 × p-cym-C_qAr), 87.3, 85.2, 83.3, 82.3, 79.9, 77.4, 74.6, 67.3 (4 × p-cym-CH_Ar, C-1′, C-3′–C-5′), 62.1 (C-6′), 54.6 (C-2′), 31.0 (i-Pr-CH), 22.8, 21.7 (2 × i-Pr-CH₃), 17.7 (C₆H₄-CH₃). ESI-HRMS positive mode (m/z): calcd for C₄₆H₄₄ClN₂O₇Ru⁺ [M-PF₆]⁺ 873.1885. Found: [M-PF₆]⁺ 873.1876.

Complex Os-7e

Prepared from compound 7e (15.6 mg, 0.026 mmol, 2.05 eq.), Os-dimer (10.0 mg, 0.013 mmol) and TlPF₆ (8.7 mg, 0.025 mmol) according to general procedure VII. The crude product was dissolved in CHCl₃ (1 mL), and diisopropyl ether (8 mL) was added. The precipitation was filtered off, then washed with CHCl₃-diisopropyl ether (1:4, 2 mL) to yield 26.9 mg (96%) of a dark green solid. R_f = 0.58 (95:5 CHCl₃-MeOH). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.18 (1H, d, J = 9.1 Hz, H-3 or H-4 or H-5 or H-8), 8.29 (1H, d, J = 8.7 Hz, H-3 or H-4 or H-5 or H-8), 8.16–7.33 (19H, m, Ar, H-3 and/or H-4 and/or H-5 and/or H-8, H-6, H-7), 7.25 (1H, dd, J = 11.7, 6.9 Hz, NH₂), 6.54–6.51 (2H, m, 2 × p-cym-CH_Ar), 6.48 (1H, d, J = 5.7 Hz, p-cym-CH_Ar), 5.96 (1H, pt, J = 9.2, 9.1 Hz, H-3′ or H-4′), 5.83 (1H, pt, J = 9.8, 9.6 Hz, H-3′ or H-4′), 5.71 (1H, d, J = 5.7 Hz, p-cym-CH_Ar), 5.04 (1H, dd, J = 12.7, 2.3 Hz, H-6′a), 4.99 (1H, d, J = 10.2 Hz, H-1′), 4.90 (1H, ddd, J = 10.0, 3.4, 2.3 Hz, H-5′), 4.58 (1H, dd, J = 11.9, 7.3 Hz, NH₂), 4.52 (1H, dd, J = 12.7, 3.4 Hz, H-6′b), 3.03–2.95 (1H, m, H-2′), 2.82 (1H, hept, J = 6.9 Hz, i-Pr-CH), 1.63 (3H, s, C₆H₄-CH₃), 1.28, 1.18 (2 × 3H, 2 d, J = 6.9 Hz in both, 2 × i-Pr-CH₃); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 168.9, 166.3, 165.7, 163.2, 149.4 (3 × C=O, C-2, C-8a), 141.6, 134.4, 133.8, 133.3, 132.5, 131.4, 130.5–130.0, 129.9, 129.4, 129.0–128.6, 127.8, 119.5 (C-3–C-8, C-4a, Ar), 95.0, 92.4 (2 × p-cym-C_qAr), 81.0, 79.3, 77.4, 76.8, 75.2, 74.5, 73.5, 67.2 (4 × p-cym-CH_Ar, C-1′, C-3′–C-5′), 62.1 (C-6′), 54.8 (C-2′), 31.1 (i-Pr-CH), 23.1, 21.9 (2 × i-Pr-CH₃), 17.6 (C₆H₄-CH₃). ESI-HRMS positive mode (m/z): calcd for C₄₆H₄₄ClN₂O₇Os⁺ [M-PF₆]⁺ 963.2439. Found: 963.2432.

Complex Ir-7e

Prepared from compound 7e (15.5 mg, 0.026 mmol, 2.05 eq.), Ir-dimer (10.0 mg, 0.013 mmol) and TlPF₆ (8.8 mg, 0.025 mmol) according to general procedure VII, with a slight modification. During the reaction, the desired product Ir-7e also precipitated from the reaction mixture. Therefore, after completion of the reaction, the solvents were removed under reduced pressure. Then, the residue was treated with CH₃CN (10 mL), and the insoluble TlCl was filtered off. The resulting solution was evaporated in vacuo. The residue was dissolved in a mixture of CH₃CN (0.1 mL) and CHCl₃ (2 mL), and diisopropyl ether (8 mL) was added. The precipitated product was filtered off, then washed with CHCl₃-diisopropyl ether (1:2, 2 mL) to yield 24.6 mg (88%) of a dark yellow solid. R_f = 0.41 (95:5 CHCl₃-MeOH). ¹H NMR (400 MHz, CD₃CN) δ (ppm): 8.72 (1H, d, J = 9.0 Hz, H-3 or H-4 or H-5 or H-8), 8.60 (1H, d, J = 8.7 Hz, H-3 or H-4 or H-5 or H-8), 8.16–7.43 (19H, Ar, H-3 and/or H-4 and/or H-5 and/or H-8, H-6, H-7), 5.80 (1H, pt, J = 9.8, 9.4 Hz, H-3′ or H-4′), 5.73 (1H, pt, J = 9.6, 9.3 Hz, H-3′ or H-4′), 5.27 (1H, d, J = 12.9, NH₂), 4.80 (1H, dd, J = 12.4, 2.2 Hz, H-6′a), 4.82–4.73 (1H, broad signal, NH₂), 4.63 (1H, dd, J = 12.4, 3.9 Hz, H-6′b), 4.62–4.56 (1H, m, H-5′), 4.55 (1H, d, J = 10.5 Hz, H-1′), 3.50–3.52 (1H, m, H-2′), 1.70 (15H, s, Cp*-CH₃); ¹³C NMR (90 MHz, CD₃CN) δ (ppm): 168.1, 166.8, 166.2, 162.6, 147.1 (3 × C=O, C-2, C-8a), 142.9, 134.9, 134.8, 134.4, 132.6, 131.9, 130.6–130.0, 129.9, 129.6–129.4, 120.4 (Ar, C-3–C-8, C-4a), 89.7 (Cp*), 84.3, 76.4 (2), 70.0 (C-1′, C-3′–C-5′), 63.3 (C-6′), 54.4 (C-2′), 9.9 (Cp*-CH₃). ESI-HRMS positive mode (m/z): calcd for C₄₆H₄₆N₂O₇Ir⁺ [M+H-Cl-PF₆]⁺ 931.2929. Found: 9631.2920.

Complex Rh-7e

Prepared from compound 7e (20.0 mg, 0.033 mmol, 2.05 eq.), Rh-dimer (10.0 mg, 0.016 mmol) and TlPF₆ (11.3 mg, 0.032 mmol) according to general procedure VII, with a slight modification. During the reaction, the desired product, Rh-7e, also precipitated from the reaction mixture. Therefore, after completion of the reaction, the solvents were removed under reduced pressure. Then, the residue was treated with CH₃CN (10 mL), and the insoluble TlCl was filtered off. The resulting solution was evaporated in vacuo. The residue was dissolved in a mixture of CH₃CN (0.1 mL) and CHCl₃ (2 mL), and diisopropyl ether (8 mL) was added. The precipitated product was filtered off, then washed with CHCl₃-diisopropyl ether (1:4, 2 mL) to yield 29.9 mg (90%) of a red solid. R_f = 0.58 (95:5 CHCl₃-MeOH). ¹H NMR (400 MHz, CD₃CN) δ (ppm): 8.86 (1H, d, J = 8.9 Hz, H-3 or H-4 or H-5 or H-8), 8.61 (1H, d, J = 8.7 Hz, H-3 or H-4 or H-5 or H-8), 8.12–7.43 (19H, m, Ar, H-3 and/or H-4 and/or H-5 and/or H-8, H-6, H-7), 5.83 (1H, dd, J = 10.2, 9.3 Hz, H-3′ or H-4′), 5.68 (1H, pt, J = 9.8, 9.4 Hz, H-3′ or H-4′), 4.80 (1H, dd, J = 12.6, 2.6 Hz, H-6′a), 4.71 (1H, d, J = 10.3 Hz, H-1′), 4.64 (1H, dd, J = 12.6, 4.0 Hz, H-6′b), 4.63–4.58 (1H, m, H-5′), 4.49 (1H, d, J = 12.6 Hz, NH₂), 3.95 (1H, pt, J = 10.4 Hz, NH₂), 3.24–3.16 (1H, m, H-2′), 1.68 (15H, s, Cp*-CH₃); ¹³C NMR (90 MHz, CD₃CN) δ (ppm): 168.2, 166.9, 166.3, 162.7, 147.2 (3 × C=O, C-2, C-8a), 142.3, 134.9, 134.8, 134.4, 132.0, 131.6, 130.8, 130.7–130.5, 130.0, 129.9, 129.8–129.3, 120.8 (Ar, C-3–C-8, C-4a), 98.1, 98.0 (Cp*), 82.1, 77.3, 76.3, 70.0 (C-1′, C-3′–C-5′), 63.4 (C-6′), 54.4 (C-2′), 10.0 (Cp*-CH₃). ESI-HRMS positive mode (m/z): calcd for C₄₆H₄₄ClN₂O₇Rh⁺ [M-PF₆]⁺ 875.1965. Found: 875.1953.

5.2. X-ray Crystallography

X-ray-quality crystals of Ru-3a were grown by slow evaporation of a CHCl₃-MeOH solvent mixture. A crystal well-looking in a polarized light microscope was fixed under a microscope onto a Mitegen loop using high-density oil. Diffraction intensity data were collected at room temperature using a Bruker-D8 Venture diffractometer (Bruker AXS GmbH, Karlsruhe, Germany) equipped with INCOATEC IμS 3.0 (Incoatec GmbH, Geesthacht, Germany) dual (Cu and Mo) sealed-tube micro sources and a Photon ii charge-integrating pixel array detector (Bruker AXS GmbH, Karlsruhe, Germany) using Mo Kα (λ = 0.71073 Å) radiation. High-multiplicity data collection and integration were performed using APEX4 software (version 2021–4.0, Bruker AXS Inc., 2021, Madison, WI, USA). Data reduction and multiscan absorption correction were performed using SAINT (version 8.40B, Bruker AXS Inc., 2019, Madison, USA). The structure was solved using direct methods and refined on F² using the SHELXL program [80] incorporated into the APEX4 suite. Refinement was performed anisotropically for all non-hydrogen atoms. Hydrogen atoms were placed into geometric positions, except the amino protons, which could be found on the difference electron density map, with the respective N-H distances restrained. Multiscan absorption correction had to be applied because of the presence of heavy atoms and due to the shape of the crystal. Further experimental details are shown in Table S3. The CIF file was manually edited using Publcif software [81], while graphics were prepared using the Mercury program [82]. The results for the X-ray diffraction structure determinations were acceptable according to the Checkcif functionality of PLATON software (Utrecht University, Utrecht, The Netherlands) [83].

Structural parameters such as bond length and angle data were in the expected range (Table S5), except the short Ru-Cl distance. The solid-state structure was stabilized by strong N-H..Cl and N-H..F, as well as weak C-H..Cl hydrogen bonds (Figure S2 and Table S4).

5.3. Determination of the Distribution Coefficients (logD)

The logD values of the newly synthesized complexes were determined according to a procedure described in our previous publications [45].

5.4. Chemicals for Biology Experiments

All chemicals used in the cell biology and biochemistry assays were obtained from Sigma-Aldrich unless otherwise stated. The free ligands and complexes investigated in this study were dissolved in dimethyl-sulfoxide for biology experiments, and 0.1% dimethyl-sulfoxide was used as a vehicle control.

5.5. Cell Lines

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 and 1% penicillin-streptomycin.

ID8 cells were cultured in high-glucose DMEM (4.5 g/L glucose) supplemented with 4% fetal calf serum, 2 mM glutamine, 1% penicillin-streptomycin and 1% ITS supplement (I3146, Sigma-Aldrich).

Capan2 cells were maintained in MEM, 10% fetal bovine serum, 1% penicillin-streptomycin and 2 mM glutamine.

Human primary dermal fibroblasts were cultured in low-glucose DMEM (1 g/L glucose) supplemented with 20% fetal calf serum, 2 mM glutamine and 1% penicillin-streptomycin.

U251 cells were maintained in MEM, 10% fetal bovine serum, 1% penicillin-streptomycin and 2 mM glutamine.

MCF7 cells were maintained in MEM, 10% fetal bovine serum and 1% penicillin-streptomycin, 2 mM Glutamine.

5.6. Bacterial Reference Strains

The reference strains of Staphylococcus aureus (ATCC29213) and Enterococcus faecalis (ATCC29212) were purchased from the ATCC (Manassas, VA, USA).

5.7. Clinical Isolates of S. aureus and E. faecium

We used a set of clinical isolates of S. aureus and E. faecium that were collected at the Medical Center of the University of Debrecen (Hungary) between 1 January 2018. and 31 December 2020. The isolates were reported in [31] and are presented in

Table 9. The clinical isolates used in the study. VRE—vancomycin-resistant Enterococcus, MRSA—methicillin-resistant Staphylococcus aureus.

Species and Strain Identity		Sample	Year
Reference	E. faecalis
VRE	25,051	Nephrostoma	2018
VRE	27,085	Wound	2018
VRE	25,498	Rectal swab to screen for multiresistant pathogens	2018
Reference	S. aureus
MRSA	24,272	Throat	2018
MRSA	24,408	Bronchial	2018
MRSA	20,426	Blood	2020
MRSA	24,035	Wound	2018
MRSA	24,328	Throat	2018
MRSA	24,268	Throat	2018

. The clinical isolates were identified using a Microflex MALDI-TOF mass spectrometer (Bruker, Billerica, MA, USA). The antibiotic susceptibility of the isolates was tested following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines, which were valid at the time of collection.

5.8. Methylthiazolyldiphenyl-Tetrazolium Bromide (MTT) Reduction Assay

An MTT reduction assay was measures the activity of mitochondrial complex I and can be used to detect toxicity [60,61]. The assay was performed in a manner similar to that described in [84]. Briefly, cells were plated in 96-well plates the day before the assay. Cells were treated with the compounds for 4 h; then, MTT was added to a 0.5 mg/mL final concentration, and cells were incubated at 37 °C in a cell incubator for 40–60 min as a function of the cell line being assessed. Culture medium was removed, the reduced MTT dye was dissolved in dimethyl-sulfoxide, 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 vehicle-treated cells. In calculations, the readings for these wells were considered to 1, and all readings were expressed relative to these values.

5.9. Sulforhodamine B (SRB) Binding Assay

An SRB assay measures protein content of cells in correlation with the cell number in an assay well and can therefore be used to assess cell proliferation or long-term cytostasis [62]. Cells were seeded in 96-well plates the day before the assay. Cells were treated with the compounds for 48 h. Then medium was removed, and cells were fixed with 10% trichloroacetic acid. Fixed cells were washed in distilled water 3 times, followed by staining with SRB (0.4 m/V% dissolved in 1% acetic acid) for 10 min. Stained cells were washed in 1% acetic acid 5 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 vehicle-treated cells. In calculations, the readings for these wells were considered to be 1, and all readings were expressed relative to these values.

5.10. Broth Microdilution

Microdilution experiments were performed according to the standards of EUCAST [85]. The bacterial isolates to be tested were grown on Mueller–Hinton agar plates. The 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. The tested concentration range was 0.08–40 μM (10 concentrations, twofold serial dilutions), and a drug-free growth control and an inoculum-free negative control were included. The inoculated plates were incubated for 24 h at 37 °C, then visually assessed. Minimum inhibitory concentration (MIC) was defined as the lowest concentration with a 50%≤ inhibitory effect compared to the growth control. All experiments were performed at least twice in duplicate.

5.11. Statistical Evaluation

Statistical analysis was performed using version 8.0.1 of GraphPad Prism. Values were tested for normal distribution using the Shapiro–Wilk normality test. When necessary, values were log-normalized or normalized using the Box–Cox normalization method [86] as indicated in the figure captions. The following statistical test, post hoc test and the level of significance are 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, which yielded IC₅₀ and Hill slope values if the sigmoid curves reached a plateau of inhibition and there was no decrease between two subsequent data points or when inhibition was over 90%. In other cases, the percentage of inhibition was taken for the maximum concentration (100 μM).