Total synthesis of aurachins C, D, and L, and a structurally simplified analog of aurachin C

Abstract

The quinoline antibiotics aurachins C, D, and L, and a structurally simplified analog of aurachin C were synthesized from 1-(2-nitrophenyl)butane-1,3-dione via reductive cyclizations of δ-nitro ketone intermediates, with zinc or iron as key steps. The results of antimicrobial tests indicate that the N-hydroxyquinolone nucleus mimics the electron carrier in the respiratory chain more strongly than the quinoline N-oxide nucleus.

Key words:

• aurachins
• quinoline antibiotics
• reductive cyclization

Bacterial respiratory chain inhibitors are promising antibiotics, since there are some differences between the mammalian respiratory chain and the bacterial one. In particular, the quinoline alkaloids have received considerable attention because of their significant activity in the quinol oxidation sites of the bacterial respiratory chain.^1,2) Quinoline alkaloids aurachins C (1), D (2), and L (3) were isolated from the culture broth of Stigmatella aurantica by Höfle et al.^3,4) Kitagawa et al. isolated the 9′-hydroxyaurachin C from a culture broth of Rhodococcus erythropolis and named it aurachin RE (4).^5–7) While 1, 2, and 4 have been reported to exhibit antimicrobial activity against many Gram-positive bacteria, no biological activity of 3 has been reported thus far. Considering the structural similarity among aurachins and 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) (5), which is an electron transport inhibitor of the respiratory chain,⁸⁾ the N-hydroxyquinolone nucleus is likely responsible for the activity.^9,10) On the other hand, the side chain would not be so important for antibiotic activity, because substrates in the bacterial respiratory chain like menaquinone (6) have different side chain lengths. The unique structures and the potential of aurachins as a new lead compound for practical antibiotics have prompted synthetic efforts toward aurachins and their analogs by organic chemists. Two research groups have succeeded in the total synthesis of 2 and examined the effect of the side chain lengths so far.^11,12) Both two groups reported that the geranyl chain analog retained the antibiotic activity but the prenyl chain analog was considerably less active than 2. According to their studies on aurachin D, a structurally simplified analog of aurachin C with a shorter side chain would show as strong of an antimicrobial activity as aurachin C. We describe herein the total synthesis of aurachins C (1), D (2) and L (3), and the structurally simplified analog of aurachin C together with biological activities of 3 and the analog (Fig. 1).

Fig. 1. Structures of aurachins C (1), D (2), L (3), RE (4), HQNO (5) & menaquinone (6).

As shown in Scheme 1, our synthesis of 1 began with the alkylation of known ketone 7¹³⁾ with farnesyl bromide to give 8 as a 1:1 keto–enol tautomeric mixture. Then the alkylation product 8 was reductively cyclized with zinc dust in the presence of ammonium chloride to afford aurachin C (1) in 46% yield.¹⁴⁾ In contrast, the synthesis of aurachin D (2) was accomplished in 63% yield by a reductive cyclization with iron dust in the presence of hydrochloric acid. Although the acids used in these reactions were different, these results are likely due to the smaller first ionization energy of iron atoms (762.5 kJ/mol) than that of zinc atoms (906.4 kJ/mol).¹⁵⁾ Stronger reducing power of the iron atom compared to that of the zinc would make it possible to convert the nitro group into the amine. The ¹H- and ¹³C NMR of 1 and 2 were identical to those of natural products.

Scheme 1. Synthesis of aurachins C (1), D (2), L (3) & aurachin C analong 12.

Notes: Reagents and conditions: (a) NaH, farnesyl bromide, HMPA, THF, 0–60 °C (52%, 69% based on recovered 7); (b) Zn dust, NH₄Cl, EtOH/H₂O (1:1), 80 °C (46%); (c) Fe dust, 6 M HCl, EtOH, 90 °C (63%); (d) farnesal, piperidine, toluene, rt to 60 °C, (39%); (e) Zn dust, NH₄Cl, EtOH/H₂O (1:1), 80 °C (29%); (f) NaH, allyl bromide, HMPA, THF, 0–50 °C; (g) 1-hexene, Grubbs’ 2nd generation catalyst, CH₂Cl₂, reflux (11a: 9% from 7, 11b: 12% from 7, 28% based on recovered 7); (h) Zn dust, NH₄Cl, EtOH/H₂O (1:1), 80 °C (49%).

Having completed the synthesis of aurachins C and D, we then turned our attention to the synthesis of aurachin L (3). The plausible biogenesis⁴⁾ proposed by Höfle et al. suggests that 2H-pyran moiety of 3 is constructed by 6π-electrocyclization from the corresponding dienone precursor. In light of the biogenesis of 3, the moiety would be concisely constructed by a domino Knoevenagel/6π-electrocyclization. Thus we subjected 7 to farnesal under the Knoevenagel conditions to afford an inseparable mixture of desired cycloadduct 9b and presumably its isomer 9a in 39% combined yield.¹ Finally, the mixture was exposed to the reductive cyclization conditions using zinc dust to afford aurachin L (3) in 29% yield together with a separable by-product derived from 9a. The ¹H- and ¹³C NMR of 3 exhibited good agreement with those of natural aurachin L.

Next, we synthesized a structurally simplified analog of 1. According to the SAR study reported by Nay’s and Speicher’s group, the geranyl chain (i.e. 3,7-dimethyl-2,6-octadienyl chain) analog almost conserved the antibiotic activity.^11,12) Thus, we set 12 as a target molecule to consider whether the 7-carbon straight side chain analog is able to retain the activity or not. By following the same procedure conducted for the preparation of 8, 7 was alkylated with allyl bromide to give an inseparable 2:3 mixture of 10a and 10b. For side chain elongation of 10b, we employed cross-metathesis with 1-hexene and obtained a separable 4:5 mixture of desired product 11b and RCM product 11a, which was removed by SiO₂ chromatography at this stage. The reductive cyclization of 11b with zinc dust proceeded smoothly to afford 12 in 49%. The ¹H- and ¹³C NMR of 12 were analogous to those of 1 except for signals due to the side chain moiety.

The antimicrobial activities of 3 and 12 were tested by an agar diffusion assay. As shown in Table 1, 3 showed 10–17 mm zones of inhibition when 500 ng/disks of the compounds were applied on paper disks. On the other hand, the analog 12 was found to exhibit strong antimicrobial activity at only 50 ng/disks. These results indicated that the branching in the side chain was not important for the activity and the analog (12) with a shorter side chain than the geranyl group in aurachin C (1) conserved the activity. We are considering that the difference in biological activity between 3 and 12 would be ascribable to the structural difference in their nitrogen-containing heterocyclic rings (quinoline N-oxide nucleus in 3 and N-hydroxyquinolone nucleus in 12). Since the N-hydroxyquinolone nucleus included in 12 seems to be more similar to the naphthoquinone moiety of the electron carrier like 6 than the quinoline N-oxide nucleus in 3, 12 might better mimic the electron carrier than 3.

Table 1. Antimicrobial spectra of 3 & 12.

	Diameter of inhibition zone (mm)
	3^a	12^b
Sinorhizobium meliloti JCM 20682 (IAM 12611)	0	8
Pseudomonas putida JCM 13063 (IAM 1236)	0	0
Escherichia coli K-12	0	0
Bacillus subtilis JCM 1465 (IAM 12118)	0	0
Sinomonas atrocyanea JCM 1329 (IAM 12339)	13	19
Corynebacterium glutamicum JCM 1318 (IAM 12435)	10	8
Streptomyces griseus JCM 4047 (IAM 12311)	17 (turbid)	12
Rhodococcus erythropolis JCM 3201 (IAM 12122)	0	0

^a About 500 ng of the compounds were applied on paper disks (6 mm diameter).

^b About 50 ng of the compounds were applied on paper disks (6 mm diameter).

In conclusion, we successfully achieved two-step syntheses of aurachins C and D from readily available starting material 7 in 24 and 33% overall yields, respectively. It is noteworthy that the two different types of the nucleus, N-hydroxyquinolone and quinolone, could be synthesized by changing the metal employed in the reductive cyclization. The first synthesis of aurachin L was also accomplished in 11% overall yield by utilizing a domino Knoevenagel/6π-electrocyclization for the construction of 2H-pyran moiety. The results of antimicrobial tests for 3 and 12 indicated that N-hydroxyquinolone nucleus better mimics the electron carrier than the quinoline N-oxide nucleus.

Supectral data of aurachins C (1), D (2) & L (3)

Aurachin C (1): IR ν_max: 2300–3500 (s), 2919 (s), 1533 (s), 1430 (s), 1530 (s), 756 (m); ¹H NMR (270 MHz, CD₃OD) δ: 1.53 (3H, s), 1.56 (3H, s), 1.61 (3H, s), 1.81 (3H, s), 1.85–2.15 (8H, m), 2.57 (3H, s), 3.47 (3H, d, J = 6.8 Hz), 4.95–5.17 (2H, m), 7.40 (1H, ddd, J = 8.2, 7.2, 1.35 Hz), 7.71 (1H, ddd, J = 8.5, 7.2, 1.2 Hz), 7.98 (1H, d, J = 8.5 Hz), 8.28 (1H, dd, J = 8.2, 1.2 Hz); ¹³C NMR (67.5 MHz, CD₃OD) δ: 15.8, 16.8, 17.1, 18.4, 26.4, 26.6, 28.2, 28.5, 41.48, 41.52, 116.8, 121.0, 124.5, 125.7, 125.9, 126.0, 126.1, 127.0, 132.7, 133.5, 136.7, 137.0, 141.6, 151.7, 175.3; HRMS (FAB) m/z: calcd. for C₂₅H₃₄O₂ N, 380.2590; found, 380.2592 ([M + H]⁺).

Aurachin D (2): IR ν_max: 2916 (s), 1552 (m), 1494 (s); ¹H NMR (270 MHz, CDCl₃) δ: 1.55 (3H, s), 1.55 (3H, s), 1.65 (3H, s), 1.74 (3H, s), 1.77–2.10 (8H, m), 2.45 (3H, s), 3.41 (2H, d, J = 6.5 Hz), 5.00–5.18 (3H, m), 7.27 (1H, t, J = 8.3 Hz), 7.40 (1H, d, J = 8.3 Hz), 7.50 (1H, d, J = 6.8 Hz), 8.36 (1H, d, J = 7.3 Hz), 9.83 (1H, s); ¹³C NMR (67.5 MHz, CDCl₃) δ: 16.1, 16.4, 17.8, 18.9, 24.2, 25.8, 26.8, 26.9, 39.8, 39.8, 117.3, 120.1, 122.5, 123.2, 124.3, 124.5, 126.3, 131.3, 131.4, 135.0, 135.3, 139.2, 146.3, 177.3; HRMS (FAB) m/z: calcd. for C₂₅H₃₄ON, 364.2638; found, 364.2640 ([M + H]⁺).

Aurachin L (3): IR ν_max: 2924(s), 1325 (m), 1063 (s); ¹H NMR (500 MHz, CDCl₃) δ: 1.52 (3H, s), 1.53 (3H, s), 1.57 (3H, s), 1.66 (3H, s), 1.78–1.84 (1H, m), 1.85–1.90 (1H, m), 1.90–1.95 (2H, m), 2.00–2.04 (2H, m), 2.14–2.20 (2H, m), 2.75 (3H, s), 5.06 (1H, tt, J = 6.0, 1.0 Hz), 5.11 (1H, td, J = 5.5, 2.0 Hz), 5.76 (1H, d, J = 8.5 Hz), 6.59 (1H, d, J = 8.5 Hz), 7.55 (1H, td, J = 7.0, 2.0 Hz), 7.72 (1H, td, J = 7.0, 0.8 Hz), 8.15 (1H, dd, J = 7.0, 0.8 Hz), 8.72 (1H, d, J = 7.0 Hz); ¹³C NMR (125 MHz, CDCl₃) δ: 14.1, 16.0, 17.7, 22.4, 25.7, 26.5, 26.6, 39.6, 41.2, 80.5, 111.1, 118.4, 119.9, 120.6, 122.1, 123.2, 124.2, 127.1, 130.1, 130.7, 131.4, 135.9, 140.9, 143.6, 147.4; HRMS (FAB) m/z: calcd. for C₂₅H₃₂O₂ N, 378.2428; found, 378.2437 ([M + H]⁺).

Spectral Data for 8, 11b, and 12, and experimental procedures for 1, 2, 3, 8, 11b, and 12 are available on Biosci. Biotechnol. Biochem. Web site.

Supplemental material

The supplemental material for this paper is available at http://dx.doi.org/10.1080/09168451.2014.918494.

Acknowledgement

We thank Ms Yamada (Tohoku University) and Mr Kurashina for measuring NMR, IR, and MS spectra.

Funding

This work was supported, in part, by a grant-in-aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology of Japan [grant number 23780112].

Notes

Abbreviations: HMPA, hexamethylphosphoric triamide; RCM, ring-closing metathesis; MIC, minimum inhibitory concentration; SAR, structure-activity relationship.

1. Comparison of the peak integrations of the double bond proton signals in the resulting pyran rings of 9a ([270 MHz, CDCl₃] δ: 6.83 [1H, d, J = 12.2 Hz], 6.39 [1H, d, J = 12.2 Hz]) and 9b ([270 MHz, CDCl₃] δ: 5.86 [1H, d, J = 10.3 Hz], 5.06 [1H, d, J = 10.3 Hz]) indicated their ratio to be 1:1.2.