Dihydromaniwamycin E, a Heat-Shock Metabolite from Thermotolerant Streptomyces sp. JA74, Exhibiting Antiviral Activity against Influenza and SARS-CoV-2 Viruses.

Dihydromaniwamycin E (1), a new maniwamycin derivative featuring an azoxy moiety, has been isolated from the culture extract of thermotolerant Streptomyces sp. JA74 along with the known analogue maniwamycin E (2). Compound 1 is produced only by cultivation of strain JA74 at 45 °C, and this type of compound has been previously designated a "heat shock metabolite (HSM)" by our research group. Compound 2 is detected as a production-enhanced metabolite at high temperature. Structures of 1 and 2 are elucidated by NMR and MS spectroscopic analyses. The absolute structure of 1 is determined after the total synthesis of four stereoisomers. Though the absolute structure of 2 has been proposed to be the same as the structure of maniwamycin D, the NMR and the optical rotation value of 2 are in agreement with those of maniwamycin E. Therefore, this study proposes a structural revision of maniwamycins D and E. Compounds 1 and 2 show inhibitory activity against the influenza (H1N1) virus infection of MDCK cells, demonstrating IC50 values of 25.7 and 63.2 μM, respectively. Notably, 1 and 2 display antiviral activity against SARS-CoV-2, the causative agent of COVID-19, when used to infect 293TA and VeroE6T cells, with 1 and 2 showing IC50 values (for infection of 293TA cells) of 19.7 and 9.7 μM, respectively. The two compounds do not exhibit cytotoxicity in these cell lines at those IC50 concentrations.

Introduction

Secondary metabolites produced by actinomycetes undoubtedly have a therapeutic potential for drug developmental research.[1] However, it is becoming difficult to obtain novel bioactive compounds because of the repeated isolation of known compounds,[2] and novel strategies are needed for the discovery of new secondary metabolites. Recent genomic analyses have elucidated that a given actinomycete typically contains approximately 30 secondary metabolite biosynthetic gene clusters (SMBGCs),[3−5] with most ordinarily (under laboratory culture conditions) existing in a silent state.[6] Several methods have been developed to activate these SMBGCs in actinomycete strains. For instance, chemical elicitors that activate secondary metabolism have been identified from libraries of chemical compounds.[7] Alternatively, genetic-engineering-based strategies, such as heterologous expression[8] and cluster-situated regulator engineering,[9] also have been used to obtain novel secondary metabolites. In one example, the coculture of a pathogenic actinomycete with mouse macrophage-like cells was reported to activate secondary metabolism.[10,11]

In our previous research, we found that high-temperature culturing of an actinomycete activated otherwise silent SMBGCs.[12] A total of 57 thermotolerant actinomycetes capable of growing at 45 °C were obtained from an in-house actinomycete library (3160 strains). These strains were cultivated at 30 or 45 °C using various media, and many metabolites produced only in the 45 °C cultures were discovered by LC/MS analysis. We designated these compounds “heat shock metabolites (HSMs)” (Figure a). Furthermore, we identified some known compounds, specifically resistomycins and gaudimycins, as well as a new compound, murecholamide, as generated by thermotolerant streptomycetes.[12]

Figure 1

(a) Heat shock metabolites (HSMs) produced by a thermotolerant actinomycete. (b) Phenotypic observation and metabolite analysis of the thermotolerant strain Streptomyces sp. JA74 (upper chromatogram, 30 °C; lower chromatogram, 45 °C).

In the course of our screening, the thermotolerant strain Streptomyces sp. JA74 was found to produce several HSMs, and dihydromaniwamycin E (1), a new maniwamycin derivative featuring an azoxy moiety, was discovered along with maniwamycin E (2), a known analogue. In addition, compounds 1 and 2 were shown to exhibit antiviral activity against the influenza (H1N1) virus and SARS-CoV-2, a causative agent of COVID-19. Herein, we report the isolation, structural determination, total synthesis, and biological activities of 1 and 2.

Results and Discussion

The HSM productivity of the thermotolerant strain Streptomyces sp. JA74 was analyzed. First, the phenotypic differences between the 30 and 45 °C cultures were observed on an agar plate, and an unknown yellow pigment was produced only at the higher temperature (Figure b). HPLC-UV analysis showed that the UV-positive peak 1 (highlighted in red in Figure b), which exhibited maximal UV absorption at 220 nm, was present only in the EtOAc extract of cultures grown at 45 °C. In addition, the UV-positive peak 2 (also highlighted in red in Figure b), which exhibited maximal UV absorption at 230 nm, was detected as a production-enhanced metabolite at the higher temperature. In the subsequent work described here, we focused on these metabolites for structural identification.

An EtOAc extract from Streptomyces sp. JA74 yielded 1.4 g of dried material. The extract was fractionated consecutively by silica gel and ODS column chromatographies, and the final purification was achieved by reversed-phase HPLC, yielding dihydromaniwamycin E (1) (1.5 mg) and maniwamycin E (2) (2.2 mg).

Dihydromaniwamycin E (1) was obtained as a pale yellow oil. Its sodium adduct ion, detected at m/z 225.1579 [M + Na]+ in a HR-ESITOFMS spectrum, suggested the molecular formula of C10H21N2O2, requiring 1 deg of unsaturation. The IR spectrum indicated the presence of a hydroxy (3410 cm–1) functional group. 13C NMR and HSQC spectroscopic data confirmed the presence of 10 carbons assignable to two sp3 methines, of which two are oxygenated or nitrated (δC 69.6, δC 70.3), respectively, five sp3 methylenes, and three methyl groups.

The 1H–1H COSY spectrum identified three partial structures: (a) a three-carbon fragment H1′–H3′; (b) a two-carbon fragment H5′–H6′; and (c) a four-carbon fragment H1–H4 with a methyl substitution (H1 and H4) (Figure a). The two COSY fragments (a) and (b) were connected at C5′ and C3′ via a sp3 methylene C4′ by HMBC correlations from a methylene H2′ and a methyl H6′ to C4′, and a methylene H3′ and H5′ to C4′. To determine whether the position at C2 (δH 3.92, δC 69.6) is attached to a hydroxy group, 13C NMR of 1 was performed (separately) in a double tube (CD3OD/CH3OH). In this spectrum, the 13C signal at C2 was detected as a double line (Δ0.13 ppm), a result supporting the existence of an hydroxy group at C2 (Figure b). Two nitrogens and one oxygen from the molecular formula remained to be assigned; we speculated that these atoms form an azoxy moiety. The 1H–15N HMBC spectrum showed two fragments, such that (c) C1–C4 and (a, b) C1′–C6′ were connected via an azoxy moiety based on the correlations of H4 and H1′ to N1 (δN 333.1 ppm) and H3, H1′, and H2′ to N2 (δN 356.1 ppm) (Figure c). Thus, the planar structure of 1 was established (Figure , Table ).

Figure 2

(a) COSY and key HMBC correlations of 1. (b) 13C NMR of 1 measured (separately) in CD3OD and CH3OH. (c) 1H–15N HMBC spectrum of 1 measured in CDCl3.

Table 1

1H and 13C NMR Data for Dihydromaniwamycin E (1) in CDCl3

no.	δCa	δHb (J in Hz)	HMBCc
1	18.7, CH3	1.21 3H, d (6.6)	2, 3
2	69.6, CH	3.92 1H, m
3	60.4, CH	4.06 1H, qd (4.0, 6.5)
4	10.5, CH3	1.11 3H, d (6.5)	2, 3
1′	70.3, CH	4.16 2H, quin (7.3)	2′, 3′
2′	27.8, CH2	1.95 2H, m	1′, 3′, 4′
3′	25.9, CH2	1.33 2H, m	4′
4′	31.1, CH2	1.33 2H, m
5′	22.4, CH2	1.33 2H, m	4′
6′	13.9, CH3	0.90 3H, t-like (6.0)	4′, 5′

125 MHz.

500 MHz.

From proton(s) to indicated carbons.

Maniwamycin E (2) was obtained as a pale yellow oil. HR-ESITOFMS analysis gave the proton adduct ion [M + H]+ at m/z 201.1598, appropriate for the molecular formula of C10H19N2O2, corresponding to a two-hydrogen decrement compared to that of 1. This formula was consistent with the NMR data for 2, according to which compound was determined to have a double bond (H1′(δH 6.92)–H2′(δH 7.00)), on the basis of the 1H–1H COSY spectrum. Thus, the planar structure of 2 was established as a maniwamycin. In previous work,[13,14] absolute configurations of maniwamycin B and epi-maniwamycin B were determined as 2S,3S and 2R,3S (respectively) by total synthesis. 1H and 13C NMR data for 2 were not identical to those of maniwamycin B but matched those of epi-maniwamycin B (Figure , Table ). However, the optical rotation value of 2 ([α]D −39.3 (c 0.0084, CHCl3)) had the opposite negative sign compared to that of epi-maniwamycin B ([α]D +35.4 (c 1.0, CHCl3)) and did not match that of maniwamycin B ([α]D +108.0 (c 1.0, CHCl3)). Therefore, the absolute configurations of 2 were determined to be 2S,3R. Previously, maniwamycins D (2S,3R) and E (2R,3R) were isolated from Streptomyces sp. TOHO-M025.[15] However, the NMR data of 2 were in agreement with those of maniwamycin E (Table ). The absolute structures of maniwamycins D and E have been inferred on the basis of a comparison of their optical rotation values (maniwamycin D: [α]D −38.6 (c 0.03, CH3OH); maniwamycin E: [α]D −100.2 (c 0.1, CH3OH)) to those of maniwamycin B and epi-maniwamycin B, despite the fact that the measurements were obtained using different solvents. In addition, the reported NMR data for maniwamycins D and E were consistent with those of maniwamycin B and epi-maniwamycin B, respectively. Therefore, we propose that the absolute configurations of maniwamycins D and E are 2R,3R and 2S,3R, respectively, and that 2 is in fact maniwamycin E.

Figure 3

Reported and proposed structures of the maniwamycins.

Table 2

1H and 13C NMR Data for Maniwamycin E (2) and This Analogue in CDCl3 or DMSO-d6a

(a)
	2 δHc (J in Hz)		epi-maniwamycin B[14]δHc (J in Hz)		maniwamycin D[15]δHc (J in Hz)		maniwamycin E[15]δHc (J in Hz)
no.	δCb	δHc (J in Hz)	δCb	δHc (J in Hz)	δCb	δHc (J in Hz)	δCb	δHc (J in Hz)
1	18.7	1.23 3H, d (6.4)		1.22 3H, d (6.6)	20.1	1.20 3H, d (7.0)	18.7	1.19 3H, d (7.0)
2	69.7	3.97 1H, m		3.91–4.02 1H, m	70.6	3.87 1H, dq (7.0, 7.0)	69.6	3.92 1H, dq (4.0, 7.0)
3	60.6	4.18 1H, dq (4.0, 6.7)		4.18 1H, dq (4.2, 6.6)	61.5	4.02 1H, dq (7.0, 7.0)	60.6	4.14 1H, dq (4.0, 7.0)
4	10.7	1.16 3H, d (6.4)		1.16 3H, d (6.6)	12.8	1.12 3H, d (7.0)	10.7	1.12 3H, d (7.0)
1′	137.8	6.92 1H, t (13.5)		6.91 1H, d (13.6)	137.9	6.91 1H, d (13.0)	137.8	6.89 1H, d (14.0)
2′	134.6	7.00 1H, dt (7.4, 13.5)		7.01 1H, dt (6.8, 13.6)	134.6	6.98 1H, dt (7.0, 13.0)	134.6	6.96 1H, dt (7.0, 14.0)
3′	28.1	2.22 2H, q (7.4)		2.22 2H, dt (6.8, 7.2)	28.1	2.20 2H, dt (7.0, 7.0)	28.1	2.18 2H, dt (7.0, 7.0)
4′	30.3	1.48 2H, quin (7.4)		1.24–1.52 2H, m	30.3	1.45 2H, m	30.3	1.44 2H, m
5′	22.2	1.37 2H, quin (7.4)		1.24–1.52 2H, m	22.2	1.35 2H, m	22.1	1.34 2H, m
6′	13.8	0.93 3H, t (7.4)		0.92 3H, t (6.8)	13.7	0.91 3H, t (7.0)	13.7	0.89 3H, t (7.0)

Upper table (a): CDCl3. Lower table (b): DMSO-d6.

125 MHz.

500 MHz.

A synthetic route to 1 to permit determination of the absolute configurations of the C2 and C3 positions and to facilitate evaluation of the biological activities of the compound was developed. The synthetic route was planned on the basis of the total synthesis of maniwamycins A and B reported by Nakata and co-workers (Scheme ).[13] (2R,3R)-1 would be synthesized from a steric inversion of an alcohol by the Mitsunobu reaction of (2S,3R)-1, a step preceded by oxidation of the hydrazine group. In addition, the carbon skeleton of 1 would be obtained by a SN2 reaction with a cyclic sulfate ester and N-alkylation of di-tert-butyl hydrazodicarboxylic acid with hexyl bromide, and the cyclic sulfate ester would be synthesized from (S,S)-2,3-butanediol. Furthermore, we envisioned that enantiomers would be synthesized by the same route using (R,R)-2,3-butanediol, providing the four possible diastereomers having the same planar structure of 1 (2S,3R), 3 (2R,3R), 4 (2R,3S), and 5 (2S,3S) (Figure ).

Scheme 1

Synthetic Plan of Dihydromaniwamycin E (1)

Figure 4

Four possible diastereomers of dihydromaniwamycin E (1).

Carbamate 8 was synthesized by alkylating bis(Boc)hydrazine 7 with hexyl bromide 6 under basic conditions. Lithiation of 8 with n-BuLi followed by the addition of the cyclic sulfate (S,S)-11, which was prepared from (2S,3S)-2,3-butanediol 9, afforded the 96% yield of adduct 12, which was then subjected to hydrolysis of the sulfate ester to give 13 in 74% yield (based on the consumed starting material). Then 13 was protected with a benzoyl group, and the removal of the Boc group in trifluoroacetic acid gave 15. Furthermore, the oxidation of 15 with m-chloroperoxybenzoic acid in CH2Cl2 gave 16, and the removal of the benzoyl group gave dihydromaniwamycin E, 1 (2S,3R) (Scheme ). Finally, the Mitsunobu reaction of 1 provided 17, and the removal of the benzoyl group gave dihydromaniwamycin D, 3 (2R,3R). In the same manner, 4 (2R,3S) and 5 (2S,3S) were synthesized using (R,R)-2,3-butanediol (see Supporting Information). The 1H and 13C NMR data of the synthesized 1 (2S,3R) and 4 (2R,3S) were identical to those of natural 1. The optical rotation values of natural 1, synthesized 1 (2S,3R), and 4 (2R,3S) were −28.1, −29.7, and +30.4, respectively. Therefore, 1 was determined to be dihydromaniwamycin E with a (2S,3R) configuration.

Scheme 2

Synthesis of (2S,3R)-Dihydromaniwamycin E (1)

Biological activities of dihydromaniwamycin E (1) and maniwamycin E (2) were evaluated in virus bioassays. Compounds 1 and 2 showed an inhibitory effect against influenza (H1N1) virus infection in MDCK cells as assessed by a plaque assay, exhibiting IC50 values of 25.7 and 63.2 μM, respectively. Notably, 1 and 2 also displayed antiviral activity against SARS-CoV-2, the causative agent of COVID-19 infection, as assessed in 293TA cells, exhibiting IC50 values of 19.7 and 9.7 μM, respectively. In the VeroE6T cells infected with SARS-CoV-2, 2 showed activity (IC50 value of 16.4 μM). No apparent cytotoxicity was observed against the cell lines at these concentrations. Although antiviral activities against SARS-CoV-2 of 1 and 2 were lower than that of a commonly used therapeutic drug, remdesivir (IC50 0.90 μM), antiviral activities by azoxy-bearing natural products have not been reported previously (to our knowledge). Therefore, the structures of 1 and 2 offers a new template for designing therapeutics for the treatment of virus disease.

In summary, a new maniwamycin derivative featuring an azoxy moiety, dihydromaniwamycin E (1), was discovered as an HSM isolated from the culture extract of the thermotolerant strain Streptomyces sp. JA74, along with a known analogue, maniwamycin E (2). Maniwamycins A–F with an α,β-unsaturated azoxy containing a C6 chain length unit have been reported,[13,15,16] but 1 represents a new dihydro-form metabolite in this class. Elaiomycins F–H, K, and L are dihydro-form compounds with an azoxy-containing C8 chain.[17,18] Although maniwamycins and elaiomycins were isolated from strains of the genus Streptomyces, azoxy-containing natural products also have been obtained from other natural sources (Figure ). Macrozamine, which is the first natural product bearing an azoxy moiety, was isolated from Macrozamia spiralis collected in Australia in 1949.[19] Since then, a wide range of azoxy-bearing natural products have been isolated, including pyrinadine A from Cribrochalina(20) and azoxybenzene-4,4′-dicarboxylic acid from Entomophthora virulenta,[21] but the number of compounds in this class is limited.[22] In addition, a quorum-sensing inhibitory activity has been reported for maniwamycins, and cytotoxicity and antibacterial activity have been reported for elaiomycins,[15−18] but the biological activities of azoxy-bearing natural products have not been examined widely. In the present study, compounds 1 and 2 showed antiviral activity against the influenza (H1N1) virus and SARS-CoV-2 virus. This work represents the first report of antiviral activity by an azoxy-bearing natural product, and a new inhibitory mechanism is predicted.

Figure 5

Selected examples of the azoxy-containing natural products.

Experimental Section

General Experimental Procedures

Optical rotations were measured using a JASCO P-1020 polarimeter. The UV spectrum was recorded on a Beckman DU530 UV/vis spectrophotometer. IR spectra were recorded on a Bruker FT-IR ALPHA spectrometer. NMR spectra were obtained on a JEOL JNM-ECA500 spectrometer in CDCl3 or DMSO-d6 and referenced to the residual solvent signals (δH 7.26, δC 77.2 for CDCl3; δH 2.50, δC 39.5 for DMSO-d6). HR-ESITOFMS spectra were recorded on a LCT premier EX spectrometer (Waters Corporation, Milford, MA, USA). Silica gel 60 0.040–0.063 mm (Merck, Darmstadt, Germany) was used for silica gel column chromatography. Sep-Pak C18 35 cc (Waters Corp.) was used for ODS column chromatography. HPLC separations were performed using a CAPCELL PAK C18 MGII packed column 20 × 250 mm (OSAKA SODA Co., Ltd., Japan) on an Hitachi ELITE LaChrom.

Microorganism

Streptomyces sp. JA74 was isolated from soil collected in Oita, Japan. The strain was identified as a member of the genus Streptomyces on the basis of 100% similarity in the 16S rRNA gene sequence (1383 nucleotides; GenBank accession number LC647576) to Streptomyces corchorusii strain NBRC 13032 (NR_041098.1).

Fermentation

Strain JA74 growing on a K2 agar medium [soluble starch 0.4%, yeast extract 0.4%, malt extract 1.0%, and agar 2.0% in distilled water (pH 7.2)] was inoculated in Erlenmeyer flasks containing 100 mL of a KG medium [glucose 2.5%, yeast extract 0.2%, soybean meal 1.5%, and CaCO3 0.4% in distilled water (pH 7.2)]. The flasks were placed on a rotary shaker (160 rpm) at 30 °C for 2 days. Then the seed culture (5 mL) was transferred into 500 mL Erlenmeyer flasks, each containing 100 mL of T production medium [glucose 5.0%, polypeptone 0.4%, yeast extract 0.1%, meat extract 0.1%, NaCl 0.25%, soybean meal 1.0%, and CaCO3 0.5% in distilled water (pH 7.0)]. The flasks were placed on a rotary shaker (160 rpm) at 45 °C for 5 days.

Isolation

At the end of fermentation, 100 mL of acetone was added to each flask, and the flasks were allowed to shake for 1 h. The mixture was centrifuged at 6000 rpm for 10 min, and the supernatant was evaporated to remove acetone. The aqueous layer was extracted by EtOAc and concentrated in vacuo to give 1.4 g of extract from 3.9 L of culture. The extract was subjected to silica gel column chromatography with a gradient of the CHCl3–CH3OH mixture solvent (100:0, 100:1, 100:2, 100:5, 100:10, and 100:30 v/v). Fraction 1 (100:0) was evaporated, and the residue was fractionated by ODS column chromatography with a gradient of CH3CN–H2O mixture solvent (2:8, 3:7, 4:6, 5:5, 6:4, 7:3, and 8:2 v/v). The ODS fraction 1 (2:8) was evaporated, and the residue was purified by preparative HPLC using an isocratic elution with 35% CH3CN solution at 10 mL/min, yielding dihydromaniwamycin E (1, 1.5 mg, tR 29.0 min) and maniwamycin E (2, 2.2 mg, tR 34.0 min).

Dihydromaniwamycin E (1)

Pale yellow oil; [α]25D −28.1 (c 0.0053, CHCl3); UV (MeOH) λmax (log ε) 220 (3.28) nm; IR (ATR) νmax 3410, 2926, 1499, 1086 cm–1; 1H and 13C NMR data, see Table ; HR-ESITOFMS m/z 225.1579 [M + Na]+ (calcd for C10H22N2NaO2, 225.1573).

Maniwamycin E (2)

Pale yellow oil; [α]25D −39.3 (c 0.0084, CHCl3), −52.2 (c 0.0043, MeOH); 1H and 13C NMR data, see Table ; HR-ESITOFMS m/z 201.1597 [M + H]+ (calcd for C10H21N2O2, 201.1598).

Synthetic Procedures for Synthesis of Dihydromaniwamycin E (1)

Di-tert-butyl 1-Hexylhydrazine-1,2-dicarboxylate (8)

To a solution of di-tert-butyl hydrazodicarboxylate 7 (1.00 g, 4.32 mmol) in dry N,N-dimethylformamide (19.6 mL, 0.2 M) was added cesium carbonate (2.83 g, 8.68 mmol) and n-hexyl bromide 6 (550 μL, 3.91 mmol) dropwise under an argon atmosphere. The reaction mixture was stirred for 16 h at room temperature. The mixture was diluted with water and extracted with hexane/EtOAc = 4:1. The organic layer was washed with brine three times and dried over Na2SO4. After filtration and concentration, the resulting residue was purified by silica gel column chromatography (hexane/EtOAc = 25:1) to afford 8 (884.3 mg, 2.79 mmol, 71%) as a colorless oil. Spectral data of 8: 1H NMR (400 MHz, CDCl3) δ 3.51–3.34 (m, 2H), 1.64–1.53 (m, 2H), 1.47 (s, 18H), 1.33–1.23 (m, 6H), 0.93–0.84 (m, 3H); 13C NMR (100 MHz, CDCl3) δ 155.3, 80.9, 49.3, 31.5, 28.2, 27.4, 26.3, 22.6, 14.0; IR (ATR) νmax 3306, 2929, 2858, 1703, 1455, 1392, 1365, 1246, 1146, 1047, 1017, 915, 856, 757 cm–1; HR-ESITOFMS m/z 339.2256 [M + Na]+ (calcd for C16H32N2O4, 339.2254).

(4S,5S)-4,5-Dimethyl-1,3,2-dioxathiolane 2-Oxide ((S,S)-10)

To a solution of (S,S)-2,3-butanediol (0.49 g, 5.48 mmol) and pyridine (0.95 g, 12.0 mmol) in dry tetrahydrofuran (27.4 mL, 0.2 M) was added thionyl chloride (715 μg, 6.02 mmol) dropwise at 0 °C under an argon atmosphere. The reaction mixture was stirred for 16 h at room temperature. After concentration (ice–water bath), the resulting residue was washed with water and extracted with chloroform two times. The combined organic layers were washed with saturated aqueous copper sulfate two times and then water and dried over Na2SO4. After filtration and concentration (ice–water bath), the crude product was directly used for the next reaction.

(4S,5S)-4,5-Dimethyl-1,3,2-dioxathiolane 2,2-Dioxide ((S,S)-11)

A crude product of (S,S)-10 in dichloromethane (13.7 mL, 0.4 M), water (13.7 mL, 0.4 M), NaIO4 (2.58 g, 12.1 mmol), and ruthenium(IV) oxide (226.2 mg, 1.67 mmol) was stirred vigorously for 46 h at room temperature, and the reaction mixture was filtered through Celite. The filtrate was extracted with chloroform. The organic layer was stirred for 15 min with a few drops of isopropanol and dried over Na2SO4. After concentration, the resulting residue was purified by silica gel column chromatography (hexane/EtOAc = 4:1) to afford (S,S)-3 (490.7 mg, 3.23 mmol, 59% (two steps)) as a brown oil. Spectral data of (S,S)-11: [α]22D +6.5 (c 0.03, CHCl3); 1H NMR (400 MHz, CDCl3) δ 4.73–4.66 (m, 2H), 1.55 (d, J = 5.0 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 85.1, 16.4; IR (ATR) νmax 2989, 1372, 1028, 914, 821 cm–1.

Di-tert-butyl 1-Hexyl-2-((2S,3R)-3-(sulfooxy)butan-2-yl)hydrazine-1,2-dicarboxylate (12 (2S,3R))

To a solution of 8 (673.5 mg, 2.13 mmol) in dry tetrahydrofuran (21.28 mL, 0.1 M) and HMPA (571.7 mg, 3.19 mmol) was added n-BuLi (204.5 mg, 3.19 mmol) dropwise at −78 °C under an argon atmosphere. The reaction mixture was stirred for 1 h at −78 °C. To the mixture was added a solution of (S,S)-11 (698.0 mg, 4.587 mmol) in dry tetrahydrofuran (3.52 mL) and stirred for 20 min at −78 °C. The reaction mixture was stirred for 48 h at room temperature and quenched with water, extracted with EtOAc, and dried over Na2SO4. After filtration and concentration, the resulting residue was purified by silica gel chromatography (CHCl3/MeOH = 10:1), and the crude product was directly used for the next reaction. Spectral data of 12 (2S,3R): [α]19D −4.1 (c 0.017, CHCl3); 1H NMR (500 MHz, CDCl3) δ 4.82–4.44 (m, 1H), 4.19–3.88 (m, 1H), 3.50–3.35 (m, 2H), 3.30–3.20 (m, 1H), 1.68–1.56 (m, 2H), 1.50–1.40 (m, 18H), 1.35–1.32 (m, 3H), 1.30–1.22 (m, 6H), 1.16–1.13 (m, 3H), 0.92–0.80 (m, 3H); 13C NMR (125 MHz, CDCl3) δ 155.4, 155.2, 154.0, 81.3, 81.1, 80.7, 80.5, 78.3, 77.7, 77.3, 77.0, 76.7, 58.8, 52.7, 51.7, 31.7, 31.5, 28.3, 28.0, 27.6, 26.8, 26.6, 22.6, 18.2, 17.4, 14.1, 14.0, 11.4; IR (ATR) νmax 3500, 977, 2932, 2860, 1702, 1393, 1251, 1144, 925, 755 cm–1; HR-ESITOFMS m/z 467.2437 [M-H]− (calcd for C20H40N2O8S, 467.2432).

Di-tert-butyl 1-Hexyl-2-((2S,3R)-3-hydroxybutan-2-yl)hydrazine-1,2-dicarboxylate (13 (2S,3R))

To a solution of 12 (2S,3R) (927.6 mg, 2.39 mmol) in 1,4-dioxane (47.7 mL, 0.05 M) was added 5 mol % H2SO4/1,4-dioxane (15.9 mL, 0.15 M) under an argon atmosphere. The reaction mixture was stirred for 40 min at room temperature. The mixture was diluted with saturated aqueous NaHCO3 and extracted with EtOAc. The organic layer was washed with brine and dried over Na2SO4. After filtration and concentration, the resulting residue was purified by silica gel column chromatography (hexane/EtOAc = 10:1) to afford 13 (2S,3R) (610.4 mg, 1.57 mmol, 74% (two steps)) as a colorless oil. Spectral data of 13 (2S,3R): [α]22D +2.5 (c 0.0016, CHCl3); 1H NMR (500 MHz, CDCl3) δ 4.28–3.91 (m, 1H), 3.73–3.48 (m, 1H), 3.42–3.15 (m, 2H), 1.71–1.56 (m, 2H), 1.52–1.42 (m, 18H), 1.36–1.23 (m, 6H), 1.23–1.11 (m, 6H), 0.91–0.87 (m, 3H); 13C NMR (125 MHz, CDCl3) δ 157.5, 156.0, 155.3, 154.7, 82.0, 81.6, 81.5, 81.1, 80.9, 77.3, 77.0, 76.8, 70.2, 69.9, 67.8, 65.4, 65.0, 64.0, 60.3, 54.9, 52.3, 51.1, 31.6, 31.5, 28.2, 27.9, 27.8, 26.8, 26.7, 22.6, 20.5, 20.1, 19.0, 14.0, 11.2, 9.1; IR (ATR) νmax 3456, 2975, 2932, 1367, 1152 cm–1; HR-ESITOFMS m/z 389.3007 [M + H]+ (calcd for C20H40N2O5, 389.3010).

Di-tert-butyl 1-((2S,3R)-3-(Benzoyloxy)butan-2-yl)-2-hexylhydrazine-1,2-dicarboxylate (14 (2S,3R))

To a solution of 13 (2S, 3R) (599.0 mg, 1.54 mmol) in dry pyridine (7.71 mL, 0.2 M) was added benzoyl chloride (895.7 μL, 7.71 mmol) dropwise at 0 °C under an argon atmosphere. The reaction mixture was stirred for 4 h at room temperature and quenched with methanol. After concentration, the resulting residue was purified by silica gel column chromatography (hexane/EtOAc = 30:1) to afford 14 (2S,3R) (734.9 mg, 1.492 mmol, 97%) as a colorless oil. Spectral data of 14 (2S, 3R): [α]20D +35.9 (c 0.018, CHCl3); 1H NMR (500 MHz, CDCl3) δ 8.11–8.01 (m, 2H), 7.60–7.51 (m, 1H), 7.45–7.40 (m, 2H), 5.53–5.37 (m, 1H), 4.40–4.06 (m, 1H), 3.49–2.98 (m, 2H), 1.58–1.50 (m, 3H), 1.46 (s, 18H), 1.41–1.21 (m, 6H), 1.13–0.73 (m, 9H); 13C NMR (125 MHz, CDCl3) δ 165.7, 155.2, 155.1, 133.0, 129.5, 128.4, 81.0, 80.7, 72.8, 58.3, 51.6, 31.5, 28.3, 27.2, 26.8, 26.3, 22.5, 17.3, 13.9, 9.7; IR (ATR) νmax 2929, 1707, 1269, 711 cm–1; HR-ESITOFMS m/z 515.3087 [M + Na]+ (calcd for C27H44N2O6, 515.3091).

(2S,3R)-3-((E)-Hexyldiazenyl)butan-2-yl benzoate (15 (2S,3R))

To a solution of 14 (2S, 3R) (666.3 mg, 1.352 mmol) in dry dichloromethane (13.52 mL, 0.1 M) was added trifluoroacetic acid (1.35 mL, 1.0 M) dropwise at 0 °C under an argon atmosphere. The reaction mixture was stirred for 24 h at room temperature. The mixture was diluted with saturated aqueous NaHCO3 and extracted with chloroform. The organic layer was dried over Na2SO4. After concentration, the resulting residue was purified by silica gel column chromatography (hexane/EtOAc = 20:1) to afford 15 (2S,3R) (336.3 mg, 1.158 mmol, 86%) as a yellow oil. Spectral data of 15 (2S,3R): [α]20D −0.1 (c 0.0081, CHCl3); 1H NMR (500 MHz, CDCl3) δ 8.02 (dd, J = 7.8, 1.3 Hz, 2H), 7.55 (tt, J = 7.8, 1.3 Hz, 1H), 7.43 (t, J = 7.8 Hz, 2H), 5.51 (quin, J = 6.6 Hz, 1H), 3.80 (td, J = 7.4, 4.0 Hz, 2H), 3.70 (quin, J = 6.6 Hz, 1H), 1.76 (quin, J = 7.4 Hz, 2H), 1.42 (d, J = 6.6 Hz, 3H), 1.37–1.25 (m, 9H), 0.87 (t-like, J = 7.0 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 165.8, 132.9, 130.5, 129.6, 128.3, 75.4, 72.6, 69.4, 31.5, 27.5, 26.9, 22.5, 16.5, 14.7, 14.0; IR (ATR) νmax 2931, 2858, 1719, 1268, 1096, 709 cm–1; HR-ESITOFMS m/z 291.2064 [M + H]+ (calcd for C17H26N2O2, 291.2067).

(Z)-2-((2S,3R)-3-(Benzoyloxy)butan-2-yl)-1-hexyldiazene 1-Oxide (16 (2S,3R))

To a solution of 15 (2S, 3R) (315.4 mg, 1.086 mmol) in dry dichloromethane (21.7 mL, 0.05 M) was added m-CPBA (283.1 mg, 1.64 mmol) under an argon atmosphere. The reaction mixture was stirred for 1 h at room temperature. The reaction mixture was quenched with methanol. After concentration, the resulting residue was purified by silica gel column chromatography (hexane/EtOAc = 20:1) to afford 16 (2S,3R) and N-oxide isomer 16′ (2S,3R) as a colorless oil (16 (2S,3R)/16′ (2S,3R) = 2:1). The mixture was used in the next reaction without further purification. Spectral data of 16 (2S,3R) and 16′ (2S,3R): [α]20D +13.5 (c 0.014, CHCl3); 1H NMR (500 MHz, CDCl3) δ 8.04–8.01 (m, 2H), 7.58–7.54 (m, 1H), 7.46–7.42 (m, 2H), 5.53 (quin, J = 6.4 Hz, 0.3H), 5.33–5.28 (m, 0.7H), 4.57 (quin, J = 6.4 Hz, 0.3H), 4.33–4.28 (m, 0.7H), 4.16 (t, J = 7.4 Hz, 1.4H), 3.40 (td, J = 6.9, 2.6 Hz, 0.6H), 1.94 (quin, J = 7.4 Hz, 1.4H), 1.67–1.61 (m, 0.6H), 1.58 (d, J = 6.4 Hz, 1H), 1.40 (d J = 6.4 Hz, 1H), 1.38 (d, J = 6.3 Hz, 2H), 1.35–1.24 (m, 6H), 1.21 (d, J = 6.6 Hz, 2H), 0.86 (t-like, J = 6.4 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 165.8, 165.4, 133.1, 132.8, 130.5, 130.0, 129.6, 129.6, 128.4, 128.3, 77.7, 72.4, 71.4, 70.3, 58.5, 52.1, 31.5, 31.1, 27.8, 27.4, 26.9, 25.8, 22.5, 22.4, 16.6, 16.5, 14.6, 14.0, 13.9, 11.9; IR (ATR) νmax 2928, 2957, 1718, 1500, 1450, 1267, 1096, 710 cm–1; HR-ESITOFMS m/z 307.2012 [M + H]+ (calcd for C17H26N2O3, 307.2016).

(Z)-1-Hexyl-2-((2S,3R)-3-hydroxybutan-2-yl)diazene 1-Oxide (1 (2S,3R))

To a solution of 16 (2S,3R) (300.6 mg, 0.981 mmol) in dry methanol (9.81 mL, 0.1 M) was added potassium carbonate (345.5 mg, 2.49 mmol). The reaction mixture was stirred for 5 h at room temperature under an argon atmosphere. The mixture was diluted with water and extracted with EtOAc. The organic layer was washed with brine and dried over Na2SO4. After filtration and concentration, the resulting residue was purified by silica gel column chromatography (hexane/EtOAc = 20:1) to afford 1 (2S,3R) (116.8 mg, 0.577 mmol, 57% (two steps)) as a colorless oil. Spectral data of 1 (2S,3R): [α][24]D −29.7 (c 0.0053, CHCl3); 1H NMR (500 MHz, CDCl3) δ 4.16 (t, J = 7.4 Hz, 2H), 4.06 (qd, J = 6.5, 4.2 Hz, 1H), 3.96–3.88 (m, 1H), 2.17 (br s, 1H), 1.95 (quin, J = 7.4 Hz, 2H), 1.38–1.30 (m, 6H), 1.20 (d, J = 6.5 Hz, 3H), 1.11 (d, J = 6.6 Hz, 3H), 0.89 (t, J = 7.2 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 70.3, 69.6, 60.4, 31.1, 27.8, 25.8, 22.3, 18.7, 13.9, 10.5; IR (ATR) νmax 3439, 2957, 2927, 2859, 1499, 1454, 1301 1087 cm–1; HR-ESITOFMS m/z 203.1753 [M + H]+ (calcd for C10H22N2O2, 203.1754).

(Z)-2-((2R,3R)-3-(Benzoyloxy)butan-2-yl)-1-hexyldiazene 1-Oxide (17 (2R,3R))

To a solution of 1 (2S,3R) (39.5 mg, 0.195 mmol) in dry tetrahydrofuran (1.18 mL, 0.165 M), benzoic acid (60.6 mg, 0.496 mmol), and triphenylphosphine (130.3 mg, 0.497 mmol) was added DEAD (220 μL, 0.486 mmol) at 0 °C under an argon atmosphere. The reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with methanol After concentration, the resulting residue was purified by silica gel column chromatography (hexane/EtOAc = 30:1) to afford 17 (2R,3R) (31.3 mg, 0.102 mmol, 52%) as a colorless oil. Spectral data of 17 (2R,3R): [α][14]D −15.7 (c 0.013, CHCl3); 1H NMR (400 MHz, CDCl3) δ 8.03 (dd, J = 7.8, 1.4 Hz, 2H), 7.55 (tt, J = 7.8, 1.4 Hz, 1H), 7.43 (t, J = 7.8 Hz, 2H), 5.32 (quin, J = 6.6 Hz, 1H), 4.35 (quin, J = 6.6 Hz, 1H), 4.12 (t, J = 7.2 Hz, 2H), 1.87 (quin, J = 7.2 Hz, 2H), 1.37 (d, J = 6.6 Hz, 3H), 1.29–1.19 (m, 6H), 1.17 (d, J = 6.6 Hz, 3H), 0.83 (t, J = 6.9 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 165.9, 132.8, 130.5, 129.6, 128.3, 72.7, 70.3, 58.4, 31.1, 27.8, 25.8, 22.3, 16.8, 13.9, 12.3; IR (ATR) νmax 2955, 2928, 2858, 1716, 1499, 1450, 1269, 1097, 710 cm–1; HR-ESITOFMS m/z 307.2015 [M + H]+ (calcd for C17H26N2O3, 307.2016).

(Z)-1-hexyl-2-((2R,3R)-3-hydroxybutan-2-yl)diazene 1-Oxide (3 (2R,3R))

To a solution of 17 (2R, 3R) (19.9 mg, 0.0649 mmol) in dry methanol (649 μL, 0.1 M) was added potassium carbonate (26.1 mg, 0.189 mmol). The reaction mixture was stirred for 8 h at room temperature under an argon atmosphere. The mixture was diluted with water and extracted with EtOAc. The organic layer was washed with brine and dried over Na2SO4. After filtration and concentration, the resulting residue was purified by silica gel column chromatography (hexane/EtOAc = 7:1) to afford 3 (2R,3R) (8.2 mg, 0.045 mmol, 62%) as a colorless oil. Spectral data of 3 (2R,3R): [α]13D −82.9 (c 0.0042, CHCl3); 1H NMR (500 MHz, CDCl3) δ 4.18 (t, J = 7.4 Hz, 2H), 3.93 (quin, J = 6.4 Hz, 1H), 3.86 (quin, J = 6.4 Hz, 1H), 2.12 (d, J = 5.2 Hz, 1H), 1.95 (quin, J = 7.4 Hz, 2H), 1.39–1.29 (m, 6 H), 1.21 (d, J = 6.4 Hz, 3H), 1.10 (d, J = 6.4 Hz, 3H), 0.89 (t, J = 7.0 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 70.4, 61.2, 31.1, 27.8, 25.9, 22.4, 20.1, 13.9, 12.6; IR (ATR) νmax 3417, 2956, 2926, 2858, 1499, 1453, 1373, 1301, 1121, 1087, 1049 cm–1; HR-ESITOFMS m/z 203.1754 [M + H]+ (calcd for C10H22N2O2 203.1754).

Cell Lines

MDCK cells and 293T cells were obtained from Cell Bank, RIKEN BioResource Center (Tsukuba, Japan). MDCK cells were grown in minimum essential medium Eagle (MEM) (GIBCO BRL) supplemented with 10% FBS (JRH Biosciences), 0.1% NaHCO3, and 15 μg/mL glutamine at 37 °C under 5% CO2. Vero-TMPRSS2 (VeroE6T) cells [Vero E6 cells (ATCC, CRL-1586) stably expressing human TMPRSS2] and 293T-ACE2 (293TA) cells stably expressing human ACE2 were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO) supplemented with 10% FBS (GIBCO) and penicillin–streptomycin (P/S, Wako) at 37 °C under 5% CO2.

Viruses

The influenza virus, A/Puerto Rico/8/34 (H1N1), has been prepared as previously described.[23] SARS-CoV-2 strain JPN/TY/WK-521, a clinical isolate from a patient with COVID-19, was provided from the National Institute of Infectious Diseases, Japan. The working viral stocks were prepared by a passage on Vero-TMPRSS2 cells. Virus infectious titers were also measured by inoculating respective cells with serial dilutions of the virus, although the cytopathic effect (CPE) was scored to calculate the TCID50/mL.

Plaque Assay

The infection of MDCK cells by the influenza virus was evaluated using a plaque assay.[23] Briefly, compounds 1 and 2 were mixed with influenza A/Puerto Rico/8/34 (H1N1) virus solution containing 50–200 plaque-forming units (pfu). After 30 min at room temperature, the mixture was incubated with a MDCK monolayer at 37 °C for 30 min under 5% CO2. After washing, MDCK cells were incubated for 2 days, and the number of plaques was counted using crystal violet. The IC50 values of 1 and 2 were obtained from a logit–log plot. The IC50 value of a reference drug, hemagglutinin (HA) inhibitor, was 1.9 μM.[24]

Cytopathic Effect-Based Antiviral and Cytotoxicity Assays

These compounds were serially diluted twofold increments by a culture medium containing 2% FBS and plated on 96-well microplates. The diluted compounds in the plates were mixed with SARS-CoV-2 and cell suspension. Cells in the plates were cultured for 2–3 days at 37 °C and then treated with MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) (Nacalai Tesque). Cell viability was determined by the measurement of absorbance at 560 and 690 nm. The concentration achieving 50% inhibition of the cytopathic effect (effective concentration; EC50) was defined in GraphPad Prism version 8.4.3 (GraphPad Software) with a variable slope (four parameters). The EC50 values of a reference drug, remdesivir, were 0.90 μM (VeroE6T cells) and <0.16 μM (293TA cells), respectively. Nontreated cells were used as a control for 100% inhibition.