Synthesis of carbohydrate-based naphthoquinones and their substituted phenylhydrazono derivatives as anticancer agents

Abstract

A novel series of carbohydrate-based 1,2-naphthoquinones 13a–c and their substituted phenylhydrazono derivatives 14a–l were synthesized and evaluated for cytotoxicity against HL-60, MDA-MB435, HCT-116 and SF-295 cancer cell lines. The compounds 9a–c showed the best cytotoxicity profile (IC₅₀ below 2 μM) against HL-60 and MDA-MB 435 human cells, while the hydrazone derivative 14i (IC₅₀ HL-60 1.65 μM) was selective for leukemia when compared to the reference drug doxorubicin. None of the compounds exhibited lytic effects against mouse erythrocytes. Characterization of all compounds was confirmed by one- and two-dimensional NMR techniques (¹H, ¹³C-APT, COSY-¹H × ¹H and HETCOR ¹J_CH) and by elemental analysis.

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Introduction

The conjugation of carbohydrates with homo/heteroaromatic compounds has lead to the development of very effective anticancer drugs including the well-known clinical anticancer nucleoside analogues¹ cladribine² (1), clofarabine^2–4 (2), fludarabine^2,5 (3), gemcitabine^2,3 (4) and decitabine^2,6 (5) (Fig. 1). Nucleoside derivatives can exert their cytotoxicity using a range of mechanisms of action^1,2 that result in inhibition of DNA synthesis and apoptotic cell death.

Other examples of carbohydrate-based compounds (Fig. 1) that exhibit anticancer activity include bleomycin^7,8 (BLM A5, 6), a drug clinically used in the treatment of squamous cell carcinomas and malignant lymphomas,⁹ and the naturally occurring alkaloid¹⁰ rebeccamycin (7), a DNA-damaging agent that inhibits the topoisomerase I enzyme. BLM A5 is a glycopeptide⁷ antibiotic that induces breakage of DNA molecules by oxidation of the deoxyribose moiety in the presence of oxygen and a metal ion, e.g., Fe or Co.¹¹

Fig. 1 Examples of carbohydrate-based compounds 1–7 with anticancer activity.

Many quinone derivatives are clinically important drugs used in the treatment of cancer.^12,13 They also possess a variety of pharmacological effects¹⁴ including antimalarial,¹⁵ antifungal,¹⁶ and molluscicidal,¹⁷ as well as demonstrate significant activity against Trypanosoma cruzi^18,19 and tuberculosis.²⁰

Three major mechanisms have been proposed for the cytotoxic action of quinone derivatives in a variety of biological systems.¹² These normally involve the generation of active oxygen species (e.g. superoxide radical anions, hydrogen peroxide and hydroxyl radicals) by redox cycling, intercalation in the DNA double helix or alkylation of biomolecules.¹² The redox cycling of quinone derivatives may be initiated by either a one- or two-electron reduction. They can also be reduced by one-electron reductases to semiquinones (Q˙⁻), which can then undergo redox cycling in the presence of oxygen, generating superoxide anion radical (O₂˙⁻). Superoxide can be converted to hydrogen peroxide (H₂O₂) via a superoxide dismutase (SOD)-catalysed reaction, followed by the formation of a hydroxyl radical (HO) by the iron-catalyzed reduction of peroxide via the Fenton reaction (Scheme 1). All of these oxygen intermediates or reactive oxygen species (ROS) may react directly with DNA or other biomolecules, such as lipids and proteins, leading to cell damage.^12,21

Scheme 1 Formation of reactive oxygen species by redox cycling.^12,21

Natural compounds containing quinone and carbohydrate moieties 8–12 possess significant antibiotic and antitumor activities (Fig. 2).^22–26 In particular, anthracycline antibiotics 10–12 are known to be some of the most effective drugs against a wide variety of solid tumors in human patients. The mechanisms^26–29 of activity of these clinically active drugs interferes with cell growth and proliferation, resulting in an inhibition of DNA synthesis and apoptotic cell death. The sugar substructure attached to the quinone ring is an important structural requirement for the bioactivity of these anthracycline antibiotics;^30–33 the moiety participates in molecular recognition of the main cellular target DNA and topoisomerase II, forming a DNA–drug–topoisomerase II ternary complex in which the enzyme is covalently linked to the broken DNA strand. This event is critical for inducing apoptosis and cell death.^30–33

Quinones such as shikonin, an active component isolated from the traditional medicinal herb Lithospermium erythrorhizon, exhibits anticancer activity by inhibiting telomerase and DNA topoisomerase I/II, and cancer cell growth. Recently, shikonin derivatives attached to different carbohydrate groups were reported to exhibit superior cytotoxicity and solubility when compared with unglycosylated drug.³⁴

Schiff bases containing azomethine-NHN=CH protons have been used in the preparation of many potential drugs and exhibit a broad spectrum of biological activities such as antiviral,³⁵ antibacterial,³⁶ anti-inflammatory,³⁷ antiplatelet³⁸ and anticancer³⁹ activities. Notably, the introduction of a hydrazone group onto the naphthoquinone^40,41 and aryl⁴² nucleus has led to promising anticancer agents.

Fig. 2 Natural sugar-containing quinones 8–12 with anticancer activity.

In this context, the synthesis of carbohydrate-containing quinone derivatives is currently an important field of pharmacy and chemistry. Herein, we report the synthesis and in vitro antitumor activity of a new family of sugar-based 1,2-naphthoquinones 13a–c (Scheme 2). We also investigate the contribution of different hydrazone-substituted phenyl rings attached to the quinone moiety, probing the biological activity of a new series of hydrazone-containing naphthoquinone derivatives 14a–l.

Scheme 2 Strategy for the synthesis of compounds 13a–c and 14a–l.

The cytotoxicity of the new compounds 13a–c and 14a–l was evaluated against human cancer cell lines including leukemia (HL-60), melanoma (MDA-MB 435), colon (HCT-116) and glioblastoma (SF-295).

Results and discussion

Methyl 5-amino-5-deoxy-1,2-O-isopropylidene-β-D-ribofuranoside 15a, 6-amino-1,2 : 3,4-di-O-isopropylidene-α-D-galactopyranose 15b and 5-amino-5-deoxy-1,2-O-isopropylidene-α-D-xylofuranose 15c were prepared from the corresponding commercially available reagents D-ribose, D-xylose and D-galactose using known methods of carbohydrate derivatization.¹⁴ The reaction of sodium 1,2-naphthoquinone-4-sulfonate (16) with aminocarbohydrates 15a–c led to the formation of the 4-amino-1,2-naphthoquinone derivatives, as illustrated in Scheme 3. Table 1 shows the melting points of these compounds and the reaction yields.

Scheme 3 Synthetic pathways used to prepare 13a–c and 14a–l.

Table 1 Yields of the reactions and mp of the quinone derivatives 13a–cc and 14a–l

Compound	R	R1	Mp/°C	Yield (%)
13a	Ribofuranosidyl	—	195–198	65
13b	Galactopyranosyl	—	215–218	50
13c	Xylofuranosyl	—	208–210	55
14a	Ribofuranosidyl	H	170–172	55
14b	Ribofuranosidyl	Me	155–157	60
14c	Ribofuranosidyl	Br	201–203	60
14d	Ribofuranosidyl	CN	247–249	55
14e	Galactopyranosyl	H	95–97	55
14f	Galactopyranosyl	Me	105–107	50
14g	Galactopyranosyl	Br	110–112	55
14h	Galactopyranosyl	CN	223–225	50
14i	Xylofuranosyl	H	205–207	55
14j	Xylofuranosyl	Br	220–221	50
14l	Xylofuranosyl	CN	230–232	40

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The structures of 4-aminonaphthoquinones 13a–c were confirmed by one- and two-dimensional NMR techniques (¹H, ¹³C-APT, COSY-¹H × ¹H and HETCOR ¹J_CH) as well as elemental analysis. The envelope (E) and twist (T) conformations for compounds 13a and 13c, respectively, were established based on the proton coupling constants (J) of the hydrogen signals of the furan rings and by X-ray crystallographic data of the molecular structures of compounds 14c and 14j (Fig. 3 and 4).

Fig. 3 Molecular structure of compound 14c. Displacement ellipsoids for non-hydrogen atoms are drawn at the 30% probability level.

Fig. 4 Molecular structure of compound 14j, with disorder in the acetonide rings. Displacement ellipsoids for non-hydrogen atoms are drawn at the 30% probability level.

The twist-boat conformation of the D-galactose ring in 13b was confirmed by the ¹H–¹H vicinal coupling constant values J_{H-1′,H-2′}, J_{H-2′,H-3′}, J_{H-3′,H-4′} and J_{H-4′,H-5′} (5.0, 2.1, 7.8 and 1.5 Hz, respectively) and by hydrogen chemical shift comparison with literature data for the analogous carbohydrates.⁴³

The reaction of 1,2-naphthoquinone derivatives 13a–c with substituted phenylhydrazine hydrochlorides 17a–d led to the corresponding hydrazone derivatives, as illustrated in Scheme 1. Although the possibility of the nucleophilic attack of hydrazine derivatives on the carbonyl carbon atom C-1 or C-2 of 13a–c, only hydrazone compounds 14a–l were isolated, resulting from the nucleophilic addition to the more electrophilic carbonyl group (C-1).

In this type of reaction, E- and Z-hydrazones are in equilibrium through the intermediate hemiaminals 18a–l (Scheme 4).

Scheme 4 Equilibrium of E- and Z-hydrazones through intermediate hemiaminals 18a–l.

The ¹H NMR spectra of 14a–l indicate that only one of two possible isomers was formed. The (Z)-stereochemistry for the C=N imine double bond in the series of the hydrazono derivatives 14a–l was assigned on the basis of X-ray crystallographic analysis of the molecular structures of 14c and 14j (Fig. 3 and 4). The (Z)-conformation of 14c and 14j was established by the intramolecular N(2)–H(2A)⋯O(1) six-membered hydrogen-bonded ring and the π-electron delocalization in the adjacent aromatic systems. Furthermore, the bond length C(1)–N(1) is the shortest distance between the C and N atoms in 14c and 14j, indicating a double bond. In both structures, the hydrazone chain and quinone ring form a planar system, with bond distances typical of delocalized aromatic compounds.

The carbohydrate chain in compound 14c adopts an envelope conformation in which the O(11) atom is located at 0.300(3) Å, out of the plane of the other four atoms C(12), C(13), C(14) and C(15), with Cremer–Pople puckering parameters q₂ and ϕ₂ of 0.22(1) Å and 27(2)°, respectively. The furan sugar ring of 14j has a twist conformation, ϕ₂ = 20(2)°; q₂ = 0.39(1) Å, according to Cremer–Pople puckering parameters, in which two adjacent C(12) and C(13) carbon atoms lie above [0.313(16) Å] and below [−0.340(17) Å] the plane defined by the remaining three atoms, respectively.

Structurally important interactions are summarized in Table 2. For example, in both crystals, N(2)–H(2A)⋯O(1) and N(3)–H(3A)⋯O(12) intramolecular hydrogen bonds are observed. Analysis of the crystal packing for compounds 14c and 14j reveals a net of weak C–H⋯O hydrogen bonds and π⋯π stacking interactions. Additionally, intermolecular hydroxyl group hydrogen bonds in 14j form an chain along the b-axis.

Table 2 Relevant hydrogen-bonding interactions (d/Å, θ/°) in compounds 14c and 14j

	D–H⋯A	d(D–H)	d(H⋯A)	d(D⋯A)	θ(D–H⋯A)	Symmetry code
14c	N(2)–H(2A)⋯O(1)	0.88	1.86	2.552(2)	135	—
	N(3)–H(3A)⋯O(12)	0.88	2.15	2.952(3)	151	—
	C(7)–H(7)⋯O(14)^i	0.95	2.40	3.251(3)	149	(i) 1 − x, ½ − y, 1½ + z
	C(11)–H(11B)⋯O(1)^ii	0.98	2.47	3.212(3)	132	(ii) ½ + x, ½ − y, 2 − z
	C(17)–H(17B)⋯O(11)^iii	0.98	2.43	3.399(3)	169	(iii) −1 + x, y, z
14j	N(2)–H(2A)⋯O(1)	0.88	1.83	2.536(7)	136	—
	N(3)–H(3A)⋯O(12)	0.88	2.03	2.713(7)	133	—
	O(12)–H(12O)⋯O(1)^iv	0.84	1.84	2.645(6)	160	(iv) −x, −½ + y, ½ − z
	C(7)–H(7)⋯O(12)^iv	0.98	2.54	3.276(8)	136	(v) 1 − x, −½ + y, ½ − z
	C(15)–H(15)⋯O(14)^iii	0.98	2.34	3.155(7)	141	(iii) 1 + x, y, z

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Biological assay

The in vitro anticancer activities of the new carbohydrate-based naphthoquinones 13a–c and the phenylhydrazono derivatives 14a–l were assessed against four human cancer cell lines in comparison to doxorubicin (positive control) using the MTT assay. The concentrations that induce 50% inhibition of cell growth (IC₅₀) are reported in Table 3 in μg mL⁻¹ (μM).

Table 3 Cytotoxicity of compounds 13a–c and 14a–l for cancer cell lines (data presented as IC₅₀/μg mL⁻¹ (IC₅₀/μM))^a

	Cancer cell line
Compound	HL-60	MDA-MB-435	HCT-116	SF-295
a 95% confidence intervals were obtained by nonlinear regression for all cell lines from three independent experiments (IC50/μg mL^−1). Doxorubicin (DOX) was used as positive control. Nd– not determined.
13a	1.27 (3.53)	0.87 (2.42)	4.00 (11.13)	2.65 (7.37)
	0.73–2.19^a	0.69–1.08^a	3.66–4.37^a	2.18–3.22^a
13b	1.83 (4.40)	1.13 (2.72)	>5 (12.03)	2.17 (5.22)
	1.01–3.30^a	0.86–1.48^a		1.88–2.49^a
13c	1.29 (3.73)	1.97 (5.70)	3.22 (9.32)	2.34 (6.77)
	0.64–2.59^a	1.52–2.54^a	2.66–3.89^a	1.89–2.90^a
14a	>5 (11.12)	>5 (11.12)	>5 (11.12)	>5 (11.12)
14b	>5 (10.79)	>5 (10.79)	>5 (10.79)	>5 (10.79)
14c	>5 (9.46)	>5 (9.46)	>5 (9.46)	>5 (9.46)
14d	>5 (10.54)	>5 (10.54)	>5 (10.54)	>5 (10.54)
14e	>5 (9.89)	>5 (9.89)	>5 (9.89)	>5 (9.89)
14f	>5 (9.62)	>5 (9.62)	>5 (9.62)	>5 (9.62)
14g	>5 (8.55)	>5 (8.55)	>5 (8.55)	>5 (8.55)
14h	>5 (9.42)	>5 (9.42)	>5 (9.42)	>5 (9.42)
14i	1.65 (3.79)	>5	>5 (11.48)	4.48 (10.29)
	1.03–2.65^a			3.78–5.30^a
14j	>5 (9.72)	>5 (9.72)	>5 (9.72)	>5 (9.72)
14l	>5 (9.42)	>5 (9.42)	>5 (9.42)	>5 (9.42)
Doxorubicin	0.03 (0.5)	0.88 (1.52)	Nd	0.41 (0.71)
	0.02–0.04^a	0.62–1.21^a		0.29–0.44^a

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The results indicate that naphthoquinone derivatives 13a–c, which were attached to various carbohydrate groups, exhibited a considerable cytotoxic effect against all cancer cell lines and without any lytic effect against erythrocytes. These results are in accordance with National Cancer Institute (NCI) protocols, where compounds exhibiting IC₅₀ values <4 μg ml⁻¹ and below 10 μM are considered active.⁴⁴

The data collected from three independent experiments, in triplicate, reveal that 13a, 4-aminonaphthalene-1,2-dione, is the most active derivative, with IC₅₀ value of 0.87 μg ml⁻¹ against the melanoma cell line (MDA-MB-435). The enhanced anticancer activity of 13a can be related to the chemical structure (e.g., conformation and intermolecular interactions) of the pyranose ring. We also speculate that cellular permeability of the 1,2-naphthoquinonic system can be improved with the construction of the new compounds 13a–c possessing a carbohydrate chain at C-4 position of the quinone moiety.

Compounds 13a,b were slightly less active than the clinically anticancer agent doxorubicin against all cancer cell lines tested. Although doxorubicin be considered a mainstay of cancer chemotherapy,⁴⁵ it has several clinical limitations such as cardiotoxic effects and a high incidence of multi-drug resistance.

Among the hydrazones 14a–l, only the unsubstituted hydrazone derivative 14i showed significant activity against leukemia cells lines. A dramatic loss of cytotoxicity for these compounds compared to the cytotoxic activity of their precursors 13 can be related to the absence of the redox center group C=O, which was derivatized as a hydrazone moiety (–C=N–NH–), leading to lower toxicity.

Non-substituted hydrazone compound 14i showed selective cytotoxicity against HL-60 cell lines, with IC₅₀ value of 1.65 μg ml⁻¹, whereas IC₅₀ values over 5 μg ml⁻¹ were observed against all other cell lines. These results suggest that the compound 14i can act as a prodrug of 13c improving its antitumor selectivity. Studies have been reported the use of hydrazone prodrugs as an important approach for enhancing the antitumor selectivity and decreasing severe side effect of antitumor agents.⁴⁶ The hydrazone linker is relatively stable at neutral pH, but labile under acidic conditions.⁴⁷ According to literature, this class of the compound can be metabolized in vitro and in vivo to biologically active carbonyl compounds.⁴⁶

The mechanical stability of red blood cells is a good parameter for in vitro screening of unspecific cytotoxicity because erythrocyte membranes can tolerate significant structural change.⁴⁸ Assays of compounds 13a–c and 14a–l showed no hemolytic activity (EC > 250 mg mL⁻¹).We therefore suggest that the toxicity mechanism involved is not related to nonspecific membrane damage (Table 3).

Conclusion

In summary, a new series of naphthoquinones 13a–c and their corresponding phenylhydrazones 14a–l have been synthesized and evaluated for anticancer activity against human cancer cell lines including leukemia (HL-60), melanoma (MDA-MB-435), colon (HCT-116) and central nervous system (SF-295). Compounds 13a–c exhibited considerable cytotoxic activity. The compound 4-((methyl-5′-deoxy-2′,3′-O-isopropylidene-β-D-methylfuranosid-5′-yl)methylamino)naphthalene-1,2-dione (13a) was the most active of this series, with IC₅₀ values below 2 μg ml⁻¹ against leukemia and melanoma cell lines.

Except for compound (Z)-4-((5′-deoxy-1,2-O-isopropylidene-α-D-xylofuranosid-5′-yl)methylamino)-1-(2-phenyl)hydrazono)naphthalene-2(1H)-one (14i), the introduction of a hydrazone pharmaphoric group at the C-1 position of naphthoquinone ring led to a dramatic drop in antitumor activity against all tested cancer cell lines. Compound 14i displayed an IC₅₀ of 1.65 μg ml⁻¹ against leukemia cells (HL-60), whereas doxorubicin was not selective against any cancer cell line. These pharmacology results suggest that the compound 14i can act as an anticancer prodrug of 13c improving its antitumor selectivity. No compounds exhibited lytic effects on mouse erythrocytes.

Due to the in vitro anticancer activity of these novel naphthoquinone derivatives 13a–c and 14i, we believe that these compounds are attractive for further structural modifications contributing to the design of new antiproliferative agents.

Experimental

Chemical reagents and solvents used in this study were purchased from Merck AG (Darmstadt, Germany) and VETEC LTDA. The synthesis of new compounds 13a–c and 14a–l was performed in a 40-kHz ultrasonic bath (USC 1400, ULTRASONIC CLEANER, UNIQUE, Indaiatuba, São Paulo, Brazil). Melting points were determined with a Fisher-Johns instrument and are uncorrected. Infrared (IR) spectra were recorded on a Perkin-Elmer FT-IR 1600 spectrophotometer using KBr pellets. NMR spectra, unless otherwise stated, were obtained in deuterated DMSO and CDCl₃ using Varian Unity Plus 300 and 500 MHz spectrometers. Chemical shifts (δ) are expressed in ppm and coupling constants (J) in Hertz. Optical rotation was recorded with a Perkin-Elmer 243B Polarimeter (sodium lamp at 589 nm). Microanalyses were performed on a Perkin-Elmer 2400 instrument, and all reported values were within ±0.4% of the calculated compositions.

The progress of all reactions was monitored by TLC performed on 2.0 cm × 6.0 cm aluminium sheets pre-coated with silica gel 60 (HF-254, E. Merck) to a thickness of 0.25 mm. The developed chromatograms were viewed under ultraviolet light at 254 nm. Merck silica gel (60–200 mesh) was used for column chromatography.

4.1. General procedure for the preparation of the 4-aminonaphthalene-1,2-dione derivatives 13a–c

A solution of 1,2-naphthoquinone-4-sulfonic acid sodium salt (16, 1.0 mmol) and aminocarbohydrate 15a–c (1.0 mmol) in 10 mL EtOH–H₂O (1 : 1) was sonicated for 1 h at room temperature. The solvent was removed under reduced pressure. The residue was purified by flash column chromatography (gradient elution, 10–30% EtOAc in hexane) to afford the desired compounds 13a–c.

4-((Methyl-5′-deoxy-2′,3′-O-isopropylidene-β-D-methylfuranosid-5′-yl)methylamino)naphthalene-1,2-dione (13a). The reaction between naphthoquinone 16 (260 mg, 1 mmol) and aminocarbohydrate 15a (203 mg, 1 mmol) yielded 13a (230 mg, 0.65 mmol, 65%) as an orange solid: mp 195–198 °C; [α]_D²⁰ −18 (c 0.002, CH₂Cl₂); IR (KBr) ν/cm⁻¹: 3281 (N–H); 1592 and 1694 (C=O).

¹H NMR (300.00 MHz, DMSO) δ 1.25 (s, 3H, CH₃), 1.37 (s, 3H, CH₃), 3.28 (s, 3H, OCH₃), 3.46–3.52 (m, 2H, H-5′ and H-5′′), 4.43 (dd, 1H, J = 6.9 and 7.8 Hz, H-4′), 4.66 (d, 1H, J = 6.0 Hz, H-2′), 4.83 (d, 1H, J = 6.0 Hz, H-3′), 4.98 (s, 1H, H-1′), 5.67 (s, 1H, H-3), 7.70 (td, J = 1.5 and 7.8 Hz, H-6), 7.84 (td, J = 1.5 and 7.8 Hz, H-7), 8.0 (dd, J = 1.5 and 7.8 Hz, H-8), 8.15 (dd, J = 1.5 and 7.8 Hz, H-5), 8.40 (br s, 1H, N–H).

¹³C NMR (75.0 MHz, DMSO) δ 24.6 (CH₃), 26.1 (CH₃), 46.0 (C-5′), 54.4 (OCH₃), 81.7 (C-2′), 82.9 (C-4′), 84.4 (C-3′), 98.2 (C-3), 108.7 (C-1′), 111.5 (-OCO_-), 123.3 (C-8), 127.9 (C-5), 130.0 (C-4a), 131.3 (C-6), 131.0 (C-8a), 134.2 (C-7), 154.7 (C-4), 174.5 (C-2), 181.5 (C-1).

Anal. Calc. for C₁₉H₂₁NO₆: C, 63.50; H, 5.89; N, 3.90. Found: C, 62.92; H, 6.04; N, 3.91%.

4-((6′-Deoxy-1′,2′ : 3′,4′-di-O-isopropylidene-D-galactopiranos-6′-yl)methylamino)naphthalene-1,2-dione (13b). The reaction between naphthoquinone 16 (260 mg, 1 mmol) and aminocarbohydrate 15b (259 mg, 1.0 mmol) yielded 13b (207 mg, 0.5 mmol, 50%) as an orange solid: mp 215–218 °C; [α]_D²⁰ −15 (c 0.002, CH₂Cl₂); IR (KBr) ν/cm⁻¹: 3305 (N–H), 1694 and 1591 (C=O).

¹H NMR (300.00 MHz, DMSO) δ 1.21 (s, 3H, CH₃), 1.28 (s, 3H, CH₃), 1.32 (s, 3H, CH₃), 1.41 (s, 3H, CH₃), 3.76–3.86 (m, 2H, H-6′ and H-6′′), 4.10–4.13 (m, 1H, H-5′), 4.34 (dd, 1H, J = 2.1 and 5.0 Hz, H-2′), 4.40 (dd, 1H, J = 1.5 and 7.8 Hz, H-4′), 4.63 (dd, 1H, J = 2.1 and 7.8 Hz, H-3′), 5.45 (d, 1H, J = 5.0 Hz, H-1′), 5.77 (s, 1H, H-3), 7.68 (td, 1H, J = 1.5 and 7.5 Hz, H-6), 7.82 (td, 1H, J = 1.5 and 7.5 Hz, H-7), 7.98 (dd, 1H, J = 1.5 and 7.5 Hz, H-5), 8.11 (dd, 1H, J = 1.5 and 7.5 Hz, H-8), 8.56 (m, 1H, N–H).

¹³C NMR (75.0 MHz, DMSO) δ 24.2 (CH₃), 24.8 (CH₃), 25.6 (CH₃), 26.0 (CH₃), 42.9 (C-6′), 64.3 (C-5′), 70.1 (C-3′), 70.5 (C-4′), 79.8 (C-2′), 95.6 (C-1′), 98.5 (C-3), 108.0 (C-7′), 108.5 (C-8′), 123.1 (C-8), 127.8 (C-5), 130.8 (C-4a), 131.1 (C-8a), 131.3 (C-6), 134.3 (C-7), 154.9 (C-4), 174.7 (C-2), 181.8 (C-1).

Anal. Calc. for C₂₂H₂₅NO₇: C, 63.60; H, 6.07; N, 3.37. Found: C, 63.14; H, 6.19; N, 3.33%.

4-((5′-Deoxy-1,2-O-isopropylidene-α-D-xylofuranosid-5′-yl)methylamino)naphthalene-1,2-dione (13c). The reaction between naphthoquinone 16 (260 mg, 1 mmol) and aminocarbohydrate 15c (189 mg, 1.0 mmol) yielded 13c (190 mg, 0.55 mmol, 55%) as an orange solid: mp 208–210 °C; [α]_D²⁰ −40 (c 0.01, MeOH); IR (KBr) ν/cm⁻¹: 3254 (N–H), 1687 and 1592 (C=O).

¹H NMR (300.00 MHz, DMSO) δ 1.25 (s, 3H, CH₃), 1.36 (s, 3H, CH₃), 3.50–3.58 (m, 1H, H-5′′), 3.64–3.74 (m, 1H, H-5′), 4.12–4.16 (m, 1H, H-3′), 4.34–4.40 (m, 1H, H-4′), 4.46 (d, 1H, J = 3.6 Hz, H-2′), 5.54 (1H; d; J = 4.8 Hz, OH), 5.77 (s, 1H, H-3), 5.87 (d, 1H, J = 3.6 Hz, H-1′), 7.67 (td, J = 1.2 and 7.5 Hz, H-6), 7.80 (td, J = 1.2 and 7.5 Hz, H-7), 7.97 (dd, J = 1.2 and 7.5 Hz, H-5), 8.10 (dd, J = 1.2 and 7.5 Hz, H-8), 8.40 (dd, 1H, J = 8.4 Hz, N–H).

¹³C NMR (75.0 MHz, DMSO) δ 25.9 (CH₃), 26.6 (CH₃), 42.2 (C-5′), 77.8 (C-4′), 76.6 (C-3′), 85.0 (C-2′), 98.2 (C-3), 104.2 (C-1′), 110.6 (C-6′), 123.3 (C-8), 127.8 (C-5), 130.7 (C-4a), 131.1 (C-8a), 131.3 (C-6), 134.2 (C-7), 155.0 (C-4), 174.9 (C-2), 181.5 (C-1).

Anal. Calc. for C₁₈H₁₉NO₆: C, 62.60; H, 5.55; N, 4.06. Found: C, 61.94; H, 6.21; N, 4.53%.

4.2. General procedure for the preparation of the phenylhydrazone derivatives 14a–l

A mixture of 4-aminocarbohydrate-1,2-naphthoquinone (1.0 mmol, 13a–c) and phenylhydrazine hydrochloride (1.0 mmol, 17a–d) in MeOH (10 mL) was stirred for 24 h at room temperature. The reaction mixture was concentrated under reduced pressure and the resulting residue was purified by column chromatography using silica gel and ethyl acetate–hexane (3 : 7) as the eluent to give the pure phenylhydrazone derivatives 14a–l.

(Z)-1-(2-(Phenyl)hydrazono)-4-((methyl-5′-deoxy-2′,3′-O-isopropylidene-β-D-methylfuranosid-5′-yl)methylamino)naphthalene-2(1H)-one (14a). The reaction between naphthoquinone 13a (100 mg, 0.28 mmol) and phenylhydrazine hydrochloride 17a (40 mg, 0.28 mmol) yielded 14a (68 mg, 0.15 mmol, 55%) as an orange solid: 170–172 °C; IR (KBr) ν/cm⁻¹: 1500 (C=O), 3431 (N–H) and 1600 (C=N).

¹H NMR (300.00 MHz, CDCl₃) δ 1.33 (s, 3H, CH₃), 1.52 (s, 3H, CH₃), 3.44 (s, 3H, OCH₃), 3.46–3.49 (m, 2H, H-5′ and H-5′′), 4.68 (td, 1H, J = 0.6 and 4.5 Hz, H-4′), 4.72 (d, 1H, J = 6.0 Hz, H-2′), 4.77 (dd, 1H, J = 0.6 and 6.0 Hz, H-3′), 5.12 (s, 1H, H-1′), 5.82 (s, 1H, H-3), 6.32 (br s, 1H, N–H), 7.10 (tt, 1H, J = 1.2 and 7.8 Hz, H-4′′), 7.36–7.41 (m, 3H, H-3′′, H-5′′ and H-6), 7.51–7.56 (m, 3H, H-2′′, H-6′′ and H-7), 7.60 (dd, 1H, J = 1.2 and 7.8 Hz, H-5), 8.51 (dd, 1H, J = 1.2 and 7.8 Hz, H-8).

¹³C NMR (75.0 MHz, CDCl₃) δ 24.8 (CH₃), 26.4 (CH₃), 46.1 (C-5′), 55.6 (OCH₃), 82.1 (C-2′), 85.6 (C-3′), 85.9 (C-4′), 100.0 (C-3), 110.1 (C-1′), 112.8 (C-6′), 115.8 (C-2′′ and C-6′′), 120.2 (C-5), 122.9 (C-4a), 123.0 (C-8), 124.0 (C-4′′), 126.1 (C-6), 128.1 (C-1), 129.4 (C-3′′ and C-5′′), 129.5 (C-7), 134.7 (C-8a), 143.1 (C-1′′), 153.6 (C-4), 179.4 (C-2).

Anal. Calc. for C₂₅H₂₇N₃O₅: C, 66.80; H, 6.05; N, 9.35. Found: C, 66.81; H, 6.12; N, 9.03%.

(Z)-1-(2-(4′′-Methylphenyl)hydrazono)-4-((methyl-5′-deoxy-2′,3′-O-isopropylidene-β-D-methylfuranosid-5′-yl)methylamino)naphthalene-2(1H)-one (14b). The reaction between naphthoquinone 13a 100 mg, 0.28 mmol) and phenylhydrazine hydrochloride 17b (38 mg, 0.28 mmol) yielded 14b (77 mg, 0.17 mmol, 60%) as an orange solid: 155–157 °C; IR (KBr) ν/cm⁻¹: 1501 (C=O), 3430 (N–H) and 1602 (C=N).

¹H NMR (500.00 MHz, CDCl₃) δ 1.33 (s, 3H, CH₃), 1.52 (s, 3H, CH₃), 2.36 (s, 3H, Ar–CH₃), 3.45 (s, 3H, OCH₃), 3.49–3.52 (m, 2H, H-5′ and H-5′′), 4.66–4.69 (m, 1H, H-4′), 4.72 (d, 1H, J = 6.0 Hz, H-2′), 4.77 (d, 1H, J = 6.0 Hz, H-3′), 5.13 (s, 1H, H-1′), 5.96 (s, 1H, H-3), 6.40 (br s, 1H, N–H), 7.20 (d, 2H, J = 8.0 Hz, H-3′′ and H-5′′), 7.40 (td, 1H, J = 1.5 and 8.0 Hz, H-6), 7.44 (d, 2H, J = 8.0 Hz, H-2′′ and H-6′′), 7.55 (td, 1H, J = 1.5 and 8.0 Hz, H-7), 7.61 (dd, 1H, J = 1,5 and 8.0 Hz, H-5), 8.51 (dd, 1H, J = 1.5 and 8.0 Hz, H-8), 16.0 (s, 1H, N–N–H).

¹³C NMR (125.0 MHz, CDCl₃) δ 20.9 (Ar–CH₃), 24.8 (CH₃), 26.4 (CH₃), 46.1 (C-5′), 55.6 (OCH₃), 82.2 (C-2′), 85.6 (C-3′), 85.7 (C-4′), 100.0 (C-3), 110.1 (C-1′), 112.8 (C-6′), 115.8 (C-2′′ and C-6′′), 120.2 (C-5), 122.7 (C-4a), 123.0 (C-8), 125.8 (C-6), 128.1 (C-1), 129.4 (C-7), 130.0 (C-3′′ and C-5′′), 134.0 (C-4′′), 134.7 (C-8a), 140.7 (C-1′′), 153.4 (C-4), 178.7 (C-2).

Anal. Calc. for C₂₆H₂₉N₃O₅: C, 67.37; H, 6.31; N, 9.07. Found: C, 67.16; H, 6.44; N, 8.88%.

(Z)-1-(2-(4′′-Bromophenyl)hydrazono)-4-((methyl-5′-deoxy-2′,3′-O-isopropylidene-β-D-methylfuranosid-5′-yl)methylamino)naphthalene-2(1H)-one (14c). The reaction between naphthoquinone 13a (100 mg, 0.28 mmol) and phenylhydrazine hydrochloride 17c (64 mg, 0.28 mmol) yielded 14c (88 mg, 0.16 mmol, 60%) as an orange solid: 201–203 °C; IR (KBr) ν/cm⁻¹: 1500 (C=O), 3340 (N–H) and 1591 (C=N).

¹H NMR (300.00 MHz, CDCl₃) δ 1.33 (s, 3H, CH₃), 1.53 (s, 3H, CH₃), 3.45 (s, 3H, OCH₃), 3.47–3.50 (m, 2H, H-5′ and H-5′′), 4.68 (td, 1H, J = 0.6 and 4.0 Hz, H-4′), 4.72 (d, 1H, J = 6.0 Hz, H-2′), 4.76 (dd, 1H, J = 0.6 and 6.0 Hz, H-3′), 5.12 (s, 1H, H-1′), 5.81 (s, 1H, H-3), 6.37 (br s, 1H, N–H), 7.37–7.43 (m, 1H, H-6), 7.39 (d, 2H, J = 8.4 Hz, H-2′′ and H-6′′), 7.49 (d, 2H, J = 8.4 Hz, H-3′′ and H-5′′), 7.58 (td, 1H, J = 1.2 and 8.1 Hz, H-7), 7.60 (dd, 1H, J = 1.2 and 8.1 Hz, H-5), 8.46 (dd, 1H, J = 1.2 and 8.1 Hz, H-8).

¹³C NMR (75.0 MHz, CDCl₃) δ 24.8 (CH₃), 26.4 (CH₃), 46.2 (C-5′), 55.6 (OCH₃), 82.1 (C-2′), 85.4 (C-4′), 85.5 (C-3′), 99.8 (C-3), 110.1 (C-1′), 112.9 (C-6′), 116.5 (C-4′′), 117.3 (C-2′′ and C-6′′), 120.4 (C-5), 123.1 (C-4a), 123.2 (C-8), 126.6 (C-6), 128.7 (C-1), 129.9 (C-7), 132.4 (C-3′′ and C-5′′), 134.0 (C-8a), 142.0 (C-1′′), 154.5 (C-4), 178,3 (C-2).

Anal. Calc. for C₂₅H₂₆BrN₃O₅: C, 56.83; H, 4.96; N, 7.95. Found: C, 56.81; H, 5.41; N, 7.56%.

(Z) 4-(2-(4-((Methyl-5′′-deoxy-2′′,3′′-O-isopropylidene-β-D-methylfuranosid-5′′-yl)methylamino)-2-oxonaphthalene-1(2H)-ylidene)hydrazinyl)benzonitrile (14d). The reaction between naphthoquinone 13a (100 mg, 0.28 mmol) and phenylhydrazine hydrochloride 17d (47 mg, 0.28 mmol) yielded 14d (66 mg, 0.14 mmol, 50%) as an orange solid: 247–250 °C; IR (KBr) ν/cm⁻¹: 1503 (C=O), 3380 (N–H), 1594 (C=N) and 2221 (C≡N).

¹H NMR (300.00 MHz, CDCl₃) δ 1.33 (s, 3H, CH₃), 1.52 (s, 3H, CH₃), 3.46 (s, 3H, OCH₃), 3.52–3.55 (m, 2H, H-5′ and H-5′′), 4.67–4.69 (m, 1H, H-4′), 4.72 (d, 1H, J = 6.0 Hz, H-2′), 4.75 (d, 1H, J = 6.0 Hz, H-3′), 5.13 (s, 1H, H-1′), 5.98 (s, 1H, H-3), 6.71 (br s, 1H, N–H), 7.46 (td, 1H, J = 1.0 and 8.0 Hz, H-6), 7.53 (d, 2H, J = 9.0 Hz, H-2′′ and H-6′′), 7.59 (td, 1H, J = 1.2 and 8.0 Hz, H-7), 7.61 (dd, 1H, J = 1.0 and 8.0 Hz, H-5), 7.65 (d, 2H, J = 9.0 Hz, H-3′′ and H-5′′), 8.46 (dd, 1H, J = 1.0 and 8.0 Hz, H-8), 15.72 (N–N–H).

¹³C NMR (75.0 MHz, CDCl₃) δ 24.8 (CH₃), 26.4 (CH₃), 46.1 (C-5′), 54.8 (OCH₃), 82.1 (C-2′), 85.5 (C-4′), 85.6 (C-3′), 99.7 (C-3), 105.7 (CN), 110.1 (C-1′), 112.9 (C-6′), 115.5 (C-2′′ and C-6′′), 119.4 (C-4′′), 120.4 (C-5), 123.6 (C-8), 123.8 (C-4a), 127.2 (C-6), 130.0 (C-7), 130.8 (C-1), 133.6 (C-3′′ and C-5′′), 134.1 (C-8a), 146.6 (C-1′′), 154.6 (C-4), 179.9 (C-2).

Anal. Calc. for C₂₆H₂₆N₄O₅: C, 65.81; H, 5.52; N, 11.81. Found: C, 65.98; H, 6.27; N, 11.47%.

(Z)-4-((6′-Deoxy-1′,2′:3′,4′-di-O-isopropylidene-D-galactopiranos-6′-yl)methylamino)-1-(2-phenyl)hydrazono)naphthalene-2(1H)-one (14e). The reaction between naphthoquinone 13b (100 mg, 0.24 mmol) and phenylhydrazine hydrochloride 17a (35 mg, 0.24 mmol) yielded 14e (66 mg, 0.13 mmol, 55%) as an orange solid: 95–97 °C; IR (KBr) ν/cm⁻¹: 1501 (C=O), 3423 (N–H) and 1593 (C=N).

¹H NMR (500.00 MHz, CDCl₃) δ 1.35 (s, 3H, CH₃), 1.39 (s, 3H, CH₃), 1.50 (s, 3H, CH₃), 1.53 (s, 3H, CH₃), 3.50–3.62 (m, 2H, H-6′ and H-6′′), 4.15-4.17 (m, 1H, H-5′), 4.33 (dd, 1H, J = 2.5 and 8.0 Hz, H-4′), 4.37 (dd, 1H, J = 2.5 and 4.5 Hz, H-2′), 4.67 (dd, 1H, J = 2.5 and 8.0 Hz, H-3′), 5.60 (d, 1H, J = 4.5 Hz, H-1′), 5.87 (s, 1H, H-3), 5.98 (m, 1H, N–H), 7.10 (tt, 1H, J = 1.2 and 7.5 Hz, H-4′′), 7.37–7.40 (m, 3H, H-6, H-3′′ and H-5′′), 7.48–7.55 (m, 4H, H-5, H-7, H-2′′ and H-6′′), 8.50 (dd, 1H, J = 1.5 and 8.0 Hz, H-8).

¹³C NMR (125.0 MHz, CDCl₃) δ 24.2 (CH₃), 24.9 (CH₃), 26.0 (CH₃), 26.1 (CH₃), 44.1 (C-6′), 65.1 (C-5′), 70.6 (C-2′), 70.9 (C-3′), 72.0 (C-4′), 96.4 (C-1′), 99.0 (C-3), 108.9 (C-7′), 109.7 (C-8′), 115.8 (C-2′′ and C-6′′), 120.2 (C-5), 122.6 (C-4a), 123.1 (C-8), 124.1 (C-4′′), 126.1 (C-6), 127.8 (C-1), 129.4 (C-3′′ and C-5′′), 129.7 (C-7), 134.7 (C-8a), 143.0 (C-1′′), 153.7 (C-4), 178.1 (C-2).

Anal. Calc. for C₂₈H₃₁N₃O₆: C, 66.52; H, 6.18; N, 8.31. Found: C, 66.58; H, 6.35; N, 7.71%.

(Z)-4-((6′-Deoxy-1′,2′:3′,4′-di-O-isopropylidene-D-galactopiranos-6′-yl)methylamino)-1-(2-(4′′-methylphenyl)hydrazono)naphthalene-2(1H)-one (14f). The reaction between naphthoquinone 13b (100 mg, 0.24 mmol) and phenylhydrazine hydrochloride 17b (38 mg, 0.24 mmol) yielded 14f (63 mg, 0.12 mmol, 50%) as an orange solid: 105–108 °C; IR (KBr) ν/cm⁻¹: 1502 (C=O), 3420 (N–H) and 1591 (C=N).

¹H NMR (300.00 MHz, CDCl₃) δ 1.34 (s, 3H, CH₃), 1.40 (s, 3H, CH₃), 1.50 (s, 3H, CH₃), 1.53 (s, 3H, CH₃), 2.35 (s, 3H, Ar–CH₃), 3.60–3.65 (m, 2H, H-6′ and H-6′′), 4.14–4.18 (m, 1H, H-5′), 4.34 (dd, 1H, J = 2.1 and 7.8 Hz, H-4′), 4.37 (dd, 1H, J = 2.4 and 5.1 Hz, H-2′), 4.67 (dd, 1H, J = 2.4 and 7.8 Hz, H-3′), 5.60 (d, 1H, J = 5.1 Hz, H-1′), 5.97 (s, 1H, H-3), 6.07 (s, 1H, N–H), 7.20 (d, 2H, J = 8.4 Hz, H-3′′ and H-5′′), 7.40 (td, 1H, J = 1.5 and 8.0 Hz, H-6), 7.42 (d, 2H, J = 8.4 Hz, H-2′′ and H-6′′), 7.48–7.56 (m, 2H, H-5 and H-7), 8.50 (dd, 1H, J = 1.5 and 8.4 Hz, H-8).

¹³C NMR (75.0 MHz, CDCl₃) δ 21.0 (Ar-CH₃), 23.7 (CH₃), 24.3 (CH₃), 25.5 (CH₃), 25.7 (CH₃), 44.2 (C-6′), 65.1 (C-5′), 70.6 (C-2′), 70.9 (C-3′), 72.0 (C-4′), 96.4 (C-1′), 98.9 (C-3), 108.9 (C-7′), 109.7 (C-8′), 115.9 (C-2′′ and C-6′′), 120.1 (C-5), 122.3 (C-4a), 123.0 (C-8), 125.9 (C-6), 127.5 (C-1), 129.6 (C-7), 130.0 (C-3′′ and C-5′′), 134.2 (C-4′′), 134.7 (C-8a), 140.7 (C-1′′), 153.7 (C-4), 178.1 (C-2).

Anal. Calc. for C₂₉H₃₃N₃O₆: C, 67.04; H, 6.40; N, 8.09. Found: C, 67.05; H, 6.88; N, 7.73%.

(Z)-1-(2-(4′′-Bromophenyl)hydrazono))-4-((6′-deoxy-1′,2′:3′,4′-di-O-isopropylidene-D-galactopiranos-6′-yl)methylamino)naphthalene-2(1H)-one (14g). The reaction between naphthoquinone 13b (100 mg, 0.24 mmol) and phenylhydrazine hydrochloride 17c (55 mg, 0.24 mmol) yielded 14f (77 mg, 0.13 mmol, 55%) as an orange solid: 110–112 °C; IR (KBr) ν/cm⁻¹: 1504 (C=O), 3422 (N–H) and 1588 (C=N).

¹H NMR (500.00 MHz, CDCl₃) δ 1.34 (s, 3H, CH₃), 1.39 (s, 3H, CH₃), 1.50 (s, 3H, CH₃), 1.53 (s, 3H, CH₃), 3.60–3.70 (m, 2H, H-6′ and H-6′′), 4.15–4.17 (m, 1H, H-5′), 4.34 (dd, 1H, J = 2.0 and 8.0 Hz, H-4′), 4.37 (dd, 1H, J = 2.5 and 4.5 Hz, H-2′), 4.67 (dd, 1H, J = 2.5 and 8.0 Hz, H-3′), 5.60 (d, 1H, J = 4.5 Hz, H-1′), 6.03 (s, 1H, H-3), 6.33 (m, 1H, N–H), 7.46–7.50 (m, 1H, H-5), 7.46 (d, 2H, J = 8.5 Hz, H-3′′ and H-5′′), 7.36 (d, 2H, J = 8.5 Hz, H-2′′ and H-6′′), 7.40 (td, 1H, J = 1.5 and 7.5 Hz, H-6), 7.55 (td, 1H, J = 1.5 and 7.5 Hz, H-7), 8.44 (dd, 1H, J = 1.5 and 7.5 Hz, H-8), 15.60 (N–N–H).

¹³C NMR (125.0 MHz, CDCl₃) δ 24.2 (CH₃), 24.9 (CH₃), 26.0 (CH₃), 26.1 (CH₃), 44.3 (C-6′), 65.0 (C-5′), 70.6 (C-2′), 70.9 (C-3′), 72.1 (C-4′), 96.4 (C-1′), 99.7 (C-3), 108.9 (C-7′), 109.7 (C-8′), 116.6 (C-4′′), 117.2 (C-2′′ and C-6′′), 120.3 (C-5), 122.6 (C-4a), 123.2 (C-8), 126.5 (C-6), 127.9 (C-1), 130.0 (C-7), 132.4 (C-3′′ and C-5′′), 134.4 (C-8a), 142.1 (C-1′′), 154.3 (C-4), 179.9 (C-2).

Anal. Calc. for C₂₈H₃₀BrN₃O₆: C, 57.54; H, 5.17; N, 7.19. Found: C, 55.78; H, 5.26; N, 6.53%.

(Z) 4-(2-(4-((6′′-Deoxy-1′,2′:3′,4′-di-O-isopropylidene-D-galactopiranos-6′-yl)methylamino)-2-oxonaphthalene-1(2H)-ylidene)hydrazinyl)benzonitrile (14h). The reaction between naphthoquinone 13b (100 mg, 0.24 mmol) and phenylhydrazine hydrochloride 17d (40 mg, 0.24 mmol) yielded 14f (70 mg, 0.12 mmol, 50%) as an orange solid: 223–226 °C; IR (KBr) ν/cm⁻¹: 1500 (C=O), 3400 (N–H), 1595 (C=N) and 2220 (C≡N).

¹H NMR (300.00 MHz, CDCl₃) δ 1.35 (s, 3H, CH₃), 1.40 (s, 3H, CH₃), 1.51 (s, 3H, CH₃), 1.54 (s, 3H, CH₃), 3.58–3.72 (m, 2H, H-6′ and H-6′′), 4.14–4.17 (m, 1H, H-5′), 4.34 (dd, 1H, J = 1.5 and 7.5 Hz, H-4′), 4.38 (dd, 1H, J = 2.4 and 5.0 Hz, H-2′), 4.68 (dd, 1H, J = 2.4 and 7.5 Hz, H-3′), 5.60 (d, 1H, J = 5.0 Hz, H-1′), 5.92 (s, 1H, H-3), 6.33 (m, 1H, N–H), 7.43 (td, 1H, J = 1.5 and 7.0 Hz, H-6), 7.48–7.55 (m, 1H, H-5), 7.51 (d, 2H, J = 9.0 Hz, H-2′′ and H-6′′), 7.59 (td, 1H, J = 1.5 and 7.0 Hz, H-7), 7.64 (d, 2H, J = 9.0 Hz, H-3′′ and H-5′′), 8.44 (dd, 1H, J = 1.5 and 7.0, Hz, H-8), 15.81 (N–N–H).

¹³C NMR (75.0 MHz, CDCl₃) δ 24.2 (CH₃), 24.8 (CH₃), 26.0 (CH₃), 26.1 (CH₃), 44.1 (C-6′), 65.0 (C-5′), 70.5 (C-2′), 70.9 (C-3′), 72.0 (C-4′), 96.4 (C-1′), 98.9 (C-3), 105.5 (CN), 108.9 (C-7′), 109.7 (C-8′), 115.9 (C-2′′ and C-6′′), 119.4 (C-4′′), 120.3 (C-5), 123.4 (C-4a), 123.5 (C-8), 127.2 (C-6), 128.5 (C-1), 129.9 (C-7), 133.6 (C-3′′ and C-5′′), 134.1 (C-8a), 147.7 (C-1′′), 154.1 (C-4), 179.6 (C-2).

Anal. Calc. for C₂₉H₃₀N₄O₆: C, 65.65; H, 5.70; N, 10.56. Found: C, 65.64; H, 5.83; N, 10.27%.

(Z)-4-((5′-Deoxy-1,2-O-isopropylidene-α-D-xylofuranosid-5′-yl)methylamino)-1-(2-phenyl)hydrazono)naphthalene-2(1H)-one (14i). The reaction between naphthoquinone 13c (100 mg, 0.31 mmol) and phenylhydrazine hydrochloride 17a (44 mg, 0.31 mmol) yielded 14i (70 mg, 0.17 mmol, 55%) as an orange solid: 205–207 °C; IR (KBr) ν/cm⁻¹: 1505 (C=O), 3340 (N–H), 1595 (C=N).

¹H NMR (300.00 MHz, CDCl₃) δ 1.33 (s, 3H, CH₃), 1.51 (s, 3H, CH₃), 3.66–3.82 (m, 2H, H-5′ and H-5′′), 4.41 (d, 1H, J = 2.4 Hz, H-3′), 4.45–4.47 (m, 1H, H-4′), 4.63 (d, 1H, J = 3.3 Hz, H-2′), 5.90 (s, 1H, H-3), 6.03 (d, 1H, J = 3.3 Hz, H-1′), 6.40 (m, 1H, N–H), 7.08–7.12 (m, 1H, H-4′′), 7.24-7.27 (m, 1H, H-6), 7.41–7.45 (m, 1H, H-7), 7.35–7.39 (m, 2H, H-3′′ and H-5′′), 7.41–7.45 (m, 2H, H-2′′ and H-6′′), 7.49 (dd, J = 0.9 and 8.0 Hz, H-5), 8.31 (dd, J = 0.9 and 7.8 Hz, H-8), 15.52 (N–N–H).

¹³C NMR (75.0 MHz, CDCl₃) δ 26.1 (CH₃), 26.7 (CH₃), 42.6 (C-5′), 75.6 (C-3′), 77.5 (C-4′), 85.6 (C-2′), 98.4 (C-3), 104.8 (C-1′), 111.9 (C-6′), 115.8 (C-2′′ and C-6′′), 120.4 (C-5), 122.2 (C-4a), 122.9 (C-8), 124.3 (C-4′′), 126.2 (C-6), 127.6 (C-1), 129.7 (C-7), 129.4 (C-3′′ and C-5′′), 134.4 (C-8a), 142.8 (C-1′′), 154.3 (C-4), 174.4 (C-2).

Anal. Calc. for C₂₄H₂₅N₃O₅: C, 66.19; H, 5.79; N, 9.65. Found: C, 66.43; H, 5.93; N, 9.29%.

(Z)-1-(2-(4′′-Bromophenyl)hydrazono))-4-((5′-deoxy-1,2-O-isopropylidene-α-D-xylofuranosid-5′-yl)methylamino)naphthalene-2(1H)-one (14j). The reaction between naphthoquinone 13c (100 mg, 0.31 mmol) and phenylhydrazine hydrochloride 17c (70 mg, 0.31 mmol) yielded 14j (82 mg, 0.17 mmol, 55%) as an orange solid: 220–221 °C; IR (KBr) ν/cm⁻¹: 1502 (C=O), 3350 (N–H) and 1592 (C=N).

¹H NMR (500.00 MHz, DMSO) δ 1.22 (s, 3H, CH₃), 1.35 (s, 3H, CH₃), 3.47–3.52 (m, 1H, H-5′), 3.60–3.66 (m, 1H, H-5′′), 4.10 (d, 1H, J = 4.0 Hz, H-3′), 4.34–4.40 (m, 1H, H-4′), 4.46 (d, 1H, J = 4.0 Hz, H-2′), 5.75 (s, 1H, H-3), 5.87 (d, 1H, J = 4.0 Hz, H-1′), 7.45–7.48 (m, 1H, H-6), 7.46 (d, 2H, J = 9.0 Hz, H-2′′ and H-6′′), 7.55 (d, 2H, J = 9.0 Hz, H-3′′ and H-5′′), 7.58 (td, J = 0.9 and 7.5 Hz, H-7), 7.90 (dd, 1H, J = 5.5 Hz, N–H), 8.05 (dd, J = 0.9 and 7.5 Hz, H-5), 8.36 (dd, J = 0.9 and 7.5 Hz, H-8), 15.81 (N–N–H).

¹³C NMR (125.0 MHz, DMSO) δ 26.1 (CH₃), 26.7 (CH₃), 41.9 (C-5′), 73.7 (C-3′), 77.9 (C-4′), 85.1 (C-2′), 97.6 (C-3), 104.3 (C-1′), 110.7 (C-6′), 115.1 (C-4′′), 117.1 (C-2′′ and C-6′′), 122.4 (C-5), 122.5 (C-8), 123.3 (C-4a), 126.7 (C-6), 128.5 (C-1), 130.0 (C-7), 132.4 (C-3′′ and C-5′′), 133.5 (C-8a), 142.3 (C-1′′), 154.2 (C-4), 178.3 (C-2).

Anal. Calc. for C₂₄H₂₄BrN₃O₅: C, 56.04; H, 4.70; N, 8.17. Found: C, 56.74; H, 5.02; N, 7.71%.

(Z) 4-(2-(4-((5′′-Deoxy-1,2-O-isopropylidene-α-D-xylofuranosid-5′′-yl)methylamino)-2-oxonaphthalene-1(2H)-ylidene)hydrazinyl)benzonitrile (14l). The reaction between naphthoquinone 13c (100 mg, 0.31 mmol) and phenylhydrazine hydrochloride 17d (52 mg, 0.31 mmol) yielded 14l (53 mg, 0.12 mmol, 40%) as a red solid: 230–233 °C; IR (KBr) ν/cm⁻¹: 1501 (C=O), 3365 (N–H), 1592 (C=N) and 2224 (C≡N).

¹H NMR (300.00 MHz, DMSO) δ 1.23 (s, 3H, CH₃), 1.36 (s, 3H, CH₃), 3.63–3.71 (m, 1H, H-5′′), 3.63–3.71 (m, 1H, H-5′), 4.12 (d, 1H, J = 2.7 Hz, H-3′), 4.34–4.39 (m, 1H, H-4′), 4.47 (d, 1H, J = 4.0 Hz, H-2′), 5.77 (s, 1H, H-3), 5.87 (d, 1H, J = 4.0 Hz, H-1′), 7.51 (td, 1H, J = 1.2 and 8.1 Hz, H-6), 7.58–7.64 (m, 1H, H-7), 7.62 (d, 2H, J = 8.7 Hz, H-2′′ and H-6′′), 7.79 (d, 2H, J = 8.7 Hz, H-3′′ and H-5′′), 8.0 (dd, 1H, J = 5.0 Hz, N–H), 8.08 (dd, 1H, J = 1.2 and 8.1 Hz, H-5), 8.40 (dd, 1H, J = 1.2 and 8.1 Hz, H-8).

¹³C NMR (75.0 MHz, DMSO) δ 26.0 (CH₃), 26.7 (CH₃), 42.0 (C-5′), 73.6 (C-3′), 77.9 (C-4′), 85.0 (C-2′), 97.6 (C-3), 104.2 (CN), 104.3 (C-1′), 110.6 (C-6′), 115.3 (C-2′′ and C-6′′), 119.4 (C-4′′), 121.8 (C-5), 122.6 (C-8), 123.9 (C-4a), 126.4 (C-6), 130.1 (C-7), 130.3 (C-1), 133.9 (C-3′′ and C-5′′), 134.0 (C-8a), 146.6 (C-1′′), 154.7 (C-4), 178.6 (C-2).

Anal. Calc. for C₂₅H₂₄N₄O₅: C, 65.21; H, 5.25; N, 12.17. Found: C, 65.19; H, 5.47; N, 11.64%.

X-Ray determination

X-Ray diffraction was performed using an Oxford Diffraction Xcalibur Atlas Gemini ultradiffractometer at 150 K with Mo-Kα (14c) and Cu-Kα (14j) radiation. The collection and reduction of data was performed with CrysalisPro software. The structures (Table 4) were solved by direct methods and refined by full-matrix least squares on F² with the SHELX-97 software package.⁴⁹ CCDC reference number 889649 for 14c and 889650 for 14j.

Table 4 X-Ray crystallographic data for compounds

—	14c	14j
Chemical formula	C25H26O5N3Br	C24H24O5N3Br
M r	528.4	514.37
Crystal size/mm	0.39 × 0.14 × 0.07	0.25 × 0.15 × 0.03
Crystal color, habit	Purple, needle	Purple, needle
Cell setting	Orthorhombic	Orthorhombic
Space group	P212121	P212121
a/Å	5.8640(2)	5.1446(3)
b/Å	15.2666(18)	13.3386(10)
c/Å	26.181(2)	33.974(3)
V/Å^3	2343.8(4)	2331.3(3)
Z	4	4
μ/mm^−1	1.80	2.75
T min, Tmax	0.651, 0.894	0.664, 1
R int	0.042	0.086
R[F^2 > 2σ(F^2)], wR(F^2), S	0.029, 0.058, 0.96	0.053, 0.133, 1.10
No. reflections	4782	2404
No. parameters	310	316
Δρmax, Δρmin/e Å^−3	0.43, −0.33	0.70, −0.57

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Cytotoxicity against cancer cell lines

Compounds (0.009–5 μg mL⁻¹) were tested for cytotoxic activity against four cancer cell lines: SF-295 (glioblastoma), HCT-116 (colon), MDA-MB-435 (melanoma), HL-60 (leukemia) (National Cancer Institute, Bethesda, MD). All cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin, and 100 μg mL⁻¹ streptomycin at 37 °C with 5% CO₂. Each compound was dissolved with DMSO to obtain a stock solution of 1 mg mL⁻¹. The final concentration of DMSO in the culture medium was kept constant below 0.1% (v/v) and the negative control received the same amount of DMSO. Compounds were incubated with cells for 72 h. Doxorubicin (0.18–1.06 μM) was used as a positive control. Cell viability was determined by reduction of the yellow dye 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to a blue formazan product as described by Mosmann.⁵⁰

Cell membrane disruption

The test was performed in 96-well plates using a 2% mouse erythrocyte suspension in 0.85% NaCl containing 10 mM CaCl₂. The diluted compounds were tested at 200 μg mL⁻¹ as mentioned above. DMSO was used as a negative control and Triton X-100 (1%) was used as positive control. After incubation at room temperature for 1 h followed by centrifugation, the supernatant was removed, the liberated hemoglobin was measured spectrophotometrically at 540 nm and EC₅₀ was calculated. EC₅₀ is the calculated effective concentration that induced 50% of the lysis generated with Triton X-100.

Acknowledgements

This work was supported by the Brazilian agency FAPERJ-PRONEX. Fellowships granted to U. F. F., by CAPES, CNPq, CNPq-PIBIC are gratefully acknowledged. We would like to thank the LabCri/UFMG for the use of X-ray facilities.

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Footnote

† CCDC reference numbers 889649 and 889650. For crystallographic data in CIF or other electronic format see DOI: 10.1039/c2ra21514d

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