Introduction

Pyripyropene (PP) analogs were first isolated as inhibitors toward acyl-CoA:cholesterol O-acyltransferase (ACAT), which is the target of an antilipotropic drug derived from the culture broth of Aspergillus fumigatus FO-1289 by Ōmura et al. at the Kitasato Institute [1,2,3,4,5] and were next isolated from Penicillium coprobium PF1169 as anthelmintic compounds by Meiji Seika Pharma [6]. The structure of PP consists of polyoxygenated mixed polyketide-terpenoid metabolites, which are categorized as meroterpenoids, containing a fused pyridyl α-pyrone moiety and eight contiguous stereocenters [7].

As reported previously, PP derivatives have been investigated as a potent ACAT inhibitor by the Kitasato Institute [8,9,10,11,12,13,14]. PP-A (Fig. 1), which is a natural PP analog, has a high level of insecticidal activity against aphids in insecticidal screening tests of Meiji’s natural product library [15]. Furthermore, PP-A is highly effective by both foliar application and soil drench, having a narrow but unique insecticidal spectrum. Aphids and whiteflies commonly occur and seriously damage a variety of crops. Unlike for lepidopteran pests, few genetically modified crops for controlling these sucking pests are not available, even though some toxins for aphid control have been discovered [16]. Control of these pests has mainly been implemented by chemicals and biological products, such as natural enemies and insecticidal fungi. Recently, because sucking pests are reported to frequently develop resistance to commercial standards, such as neonicotinoid, organophosphate, and synthetic pyrethroid insecticides, new insecticides to control such problematic sucking pests are strongly desired [17, 18]. In the previous toxicological information report for PP-A, no serious issues were observed related to mammalian toxicity [15]. Thus PP-A has promise as a novel insecticide; therefore, its optimization was initiated.

Fig. 1
figure 1

Structure of pyripyropene A (PP-A)

In this report, we investigated the effects on the insecticidal activities caused by chemical modifications at the C-1, C-7, and C-11 positions to determine the derivatives having stronger activities than PP-A against insects. The insecticidal spectrums of the derivatives with high insecticidal activity were also evaluated. Furthermore, we conducted the field trials of the most promising derivative to confirm the efficacy on practical uses in the field. Since a foliar spray cannot often cover the whole leaves and shoots under field conditions, it is commonly crucial for the agrochemicals to exhibit good systemicity on/into the leaves or to the young developing leaves to control sucking pests like aphids that preferably infest on young leaves' crops.

Results and Discussion

First, the evaluation of the insecticidal activities of several PP derivatives (1ag) with chemical modifications at the C-1, C-7, and C-11 positions, which were reported by the Kitasato Institute [9], was conducted, focusing on the insecticidal activity against Myzus persicae, an important aphid in agriculture.

The structures and insecticidal activities of PP derivatives are shown in Table 1.

Table 1 Insecticidal activity of 1 for Myzus percicae

Interestingly, the insecticidal activities of 1f (pyripyropene I, PP-I) [4] and 1g having propionyl and n-butyryl groups, respectively, at the C-1, C-7 and C-11 positions are stronger than that of PP-A. Among these derivatives, 1f (PP-I) had the highest insecticidal activity, at 10 times higher, against aphids. Among the derivatives with other substituent groups at the C-7 position, 1e, having a 3-pyridyl carbonyl group, showed relatively high insecticidal activities, while 1a (hydrogen atom), 1b (propionyl group), 1c (n-butyryl group), and 1d (benzoyl group) showed weak insecticidal activities. Thus the substituent groups at the C-1, C-7, and C-11 positions are important for the high insecticidal activity.

Next, the structural conversion of the propionyl group at the C-7 position of 1f (PP-I) to other acyl groups was attempted to further improve the insecticidal activity. The synthesis of PP derivatives with chemical modifications at the C-7 position, including the key step for regioselective hydrolysis, was reported by our group [8]. The preparation of novel PP derivatives 4an commenced with the hydrolysis of the triacetate of PP-A, followed by tri-propionylation to produce pyripyropene I (1f), which was successively subjected to regioselective hydrolysis at the C-7 position using 1.8-diazabicyclo[5.4.0]-undec-7-ene (DBU) to furnish 7-depropionylpyripyropene I (3). Finally, acylation at the C-7 position using a corresponding carboxylic anhydride with triethylamine or carboxylic acid with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) in the presence of 4-(dimethylamino)pyridine (DMAP) produced 4a–n as shown in Scheme 1.

Scheme 1
scheme 1

Synthesis of compound 4: (a) NaOMe, 50% MeOH aq., 93% yield; (b) propionic anhydride, Et3N, DMAP, DMF, 89% yield; (c) DBU, 80% MeOH aq., 56% yield; (d) R2O, Et3N, DMAP, DMF; (e) ROH, EDCI, DMAP, DMF

The structures and insecticidal activities of 4a–n are shown in Table 2.

Table 2 Structure and insecticidal activity for Myzus percicae of 4

The 4 series were evaluated for insecticidal activity against aphids using the same screening assay.

As a result of this study, 4g (3-pyridylcarbonyl group) showed the highest activity level against aphids. The LC90 value of this compound was lower than 4h (2-pyridylcarbonyl group) and 4i (4-pyridylcarbonyl group) and was similar to that of the lead compound 1f. Interestingly, introducing some substituents (chloro, trifluoromethyl, or methyl group) on the 3-pyridine ring at the C-7 position of 4g, such as 4j, 4k, 4l, 4m, or 4n, resulted in a decreased activity level. The conversion to a hydrogen atom (3), acetyl (4a), i-butyryl (4b), pivaloyl (4c), cyclopropylcarbonyl (4d), cyclobutylcarbonyl (4e), or benzoyl group (4f) at the C-7 position of 1f did not increase the activity compared with that of 1f. Thus the 3-pyridylcarbonyl group could be valuable at the C-7 position in PP derivatives having propionyl groups at the C-1 and C-11 positions.

In parallel with the chemical conversion at the C-7 position of 1f, the novel derivatives 5aj, which have the same acyl group at the C-1, C-7, and C-11 positions, were generated by the treatment of 2 with a corresponding carboxylic anhydride with triethylamine or carboxylic acid with EDCI in the presence of DMAP using the synthetic route shown in Scheme 2.

Scheme 2
scheme 2

Synthesis of compound 5: (a) R2O, Et3N, DMAP, DMF; (b) ROH, EDCI, DMAP, DMF

The structures and insecticidal activities of 5a–j are shown in Table 3.

Table 3 Structure and insecticidal activity for Myzus percicae of 5

The 5 series were evaluated for insecticidal activity against aphids using the same screening assay.

As the result of this study, 5c (cyclopropylcarbonyl group) and 5d (cyclobutylcarbonyl group) showed higher activity levels against aphids than lead compound 1f. In contrast, the derivatives possessing a hydrogen atom (2), i-butyryl (5a), pivaloyl (5b), cyclohexylcarbonyl (5e), benzoyl (5f), or pyridylcarbonyl group (5g, 5h, 5i, or 5j) at the C-1, C-7, and C-11 positions did not increase the activity level compared with lead compound 1f. Thus the cyclopropylcarbonyl or cyclobutylcarbonyl group could be valuable at the C-1, C-7, and C-11 positions in PP derivatives having the same substituents at the C-1, C-7, and C11 positions.

Here 5c showed the highest insecticidal activity against aphids and 5c was over 21 times stronger than PP-A based on the LC90 values.

Moreover, in evaluating the insecticidal spectra of the derivatives with higher activity than PP-A of 1f, 4g, and 5c against other important sucking pests, 5c exhibited higher insecticidal activities against Aphis gossypii and Trialeurodes vaporariorum than PP-A (Table 4). Interestingly, the efficacies of 1f and 4g against A. gossypii and T. vaporariorum did not show comparable increases. However, the activity was also not increased against Frankliniella occidentalis.

Table 4 Insecticidal activities of PP-A, 1f, 4g, and 5c against agricultural sucking pests

After finding the promising candidate, 5c, in the screening test, we conducted field trials against aphids to evaluate the effectiveness of this compound.

In the field trial against B. brassicae by a foliar application (Fig. 2), the wettable powder (WP) formulation of 5c had an excellent efficacy similar to the market standard, pymetrozine, which is a controlling agent for sucking pests on vegetables and fruits [19]. In the field trial against A. gossypii on potato (Fig. 3), 5c WP showed a good efficacy even when there was a high infestation level at application time but it was slightly lower than that of pymetrozine.

Fig. 2
figure 2

Efficacy of 5c wettable powder (WP) formulation against Brevicoryne brassicae on cotton using a foliar application

Fig. 3
figure 3

Efficacy of 5c wettable powder (WP) formulation against Aphis gossypii on potato using a foliar application

In the preliminary field trial, 5c exhibited a good insecticidal activity against aphids, especially on developed leaves, but the efficacy was slightly less efficient on upper young leaves compared with the market standard. Under such conditions, with young leaves expanding, the efficacy was not improved, even at high dose of 75 g a.i./ha (data not shown). While the trial against Brevicoryne brassicae was conducted under low aphid infestation conditions and a low level of new entries from surrounding untreated plots, the trial against A. gossypii was conducted under higher infestation conditions and a lot of new entries were also observed.

To improve the insecticidal activity, we synthesized PP derivatives with chemical modifications at the C-1, C-7, or C-11 positions and evaluated their insecticidal activities against M. persicae.

Among these PP derivatives, 1f, 4g, 5c, and 5d showed higher insecticidal activities against the aphids than PP-A. Interestingly, the aphicidal activity of 1f is stronger than that of PP-A, while, as previously reported by the Kitasato Institute, 1f showed a significant lower activity compared with PP-A with respect to ACAT-inhibiting activity (IC50 values of the ACAT-inhibiting activities of PP-A and 1f, which were 0.058 μM [1] and 2.45 μM [4], respectively). Thus there may be no correlation between structure and insecticidal and ACAT-inhibiting activities.

Among these derivatives (1f, 4g, 5c, and 5d), 5c had the highest insecticidal activities against M. persicae. Based on the aphicidal spectrum, the derivatives that showed higher activities against M. persicae were active against A. gossypii, another important pest. Furthermore, the highly aphicidal compounds exhibited preferable activities against whiteflies, which are important target pests worldwide, especially on greenhouse crops. Unfortunately, another important pest on vegetables and fruits, thrips, were not target pests. However, the relationship between the insecticidal activities against M. persicae and A. gossypii were not in proportion. While PP-A showed equivalent activities against both aphids, derivatives, like 1f, 4g, and 5c, that were highly active against M. persicae tended to be lower than expected against A. gossypii. This might result from the low systemicity into the leaves of these derivatives because the level is more important for a good activity in the assay against A. gossypii where the compounds were sprayed only the leaves. On the other hand, since the compounds were sprayed directly onto aphid bodies and the leaves in the assay against M. persicae, the compounds possibly exhibit high activities without good systemicity. Furthermore, 5c exhibited a good efficacy against aphids in field trials against B. brassicae. However, this compound was not superior to a market standard, pymetrozine, in the potato trial. Furthermore, the efficacy did not improve even at high dose, which indicated that systemicity was necessary for a good residual efficacy in potato in which young leaves develop rapidly after application. These improved compounds did not show high acute toxicities against mammalian animals, rat, or mouse at 1000 mg/kg body weight using an oral application (data not shown). In summary, based on the strength of the insecticidal activity, the preferable insecticidal spectrum, the highly aphicidal compound 5c is very promising, but suitable systemic properties such as those of PP-A could be important for a better field efficacy.

Experimental procedures

General methods

Reagents were obtained from commercial suppliers and were used without purification. 1H and 13C nuclear magnetic resonance (NMR) spectra were measured on JEOL Lambda 400 MHz and BRUKER Ascend 400 MHz and 500 MHz spectrometers in CDCl3. Mass spectra were obtained on JEOL JMS-FABmate spectrometer or JEOL JMS-700 mass spectrometer or Agilent Technologies 6530-Q-TOF LC/MS mass spectrometer. All infrared spectra were measured on a Horiba FT-210 spectrometer. Optical rotations were measured by using JASCO P-1010 polarimeter. Melting points were measured on OptiMelt (Stanford Research Systems) apparatus. Column chromatography was carried out on silica gel (Varian: Mega Bond Elut) and preparative thin-layer chromatography (PTLC) (Merck: Silica Gel 60 F254 0.5 mm).

1, 7, 11-Tri-deacetyl-1, 11-di-O-propionylpyripyropene A (3)

To a solution of trideacetyl-1,7,11-tri-O-propionylpyripyropene A (1f, PP-I) (890 mg, 1.42 mmol), synthesized by the method previously reported [9], in an 80% aqueous MeOH solution (40 mL) was added 1,8-diazabicyclo[5.4.0]-undec-7-ene (216 mg, 1.42 mmol) and stirred at room temperature for 1.5 h. The reaction mixture was quenched with AcOH, and the mixture was concentrated under reduced pressure and diluted with CHCl3. The organic layer was washed with water and brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The resulting residue was purified by chromatography on silica gel (acetone: hexane = 1: 1) to afford 3 (451 mg, 0.793 mmol) as a solid in 56% yield. 1H NMR (CDCl3) δ 0.91 (s, 3H), 1.13 (t, J = 5.1 Hz, 3H), 1.14 (t, J = 5.1 Hz, 3H), 1.26 (s, 1H), 1.32–1.40 (m, 1H), 1.42 (s, 3H), 1.45 (d, J = 2.7 Hz, 1H), 1.49–1.51 (m, 2H), 1.66 (s, 3H), 1.81–1.91 (m, 2H), 2.13–2.18 (m, 1H), 2.24–2.37 (m, 4H), 2.90 (m, 1H), 3.79 (m, 3H), 4.80 (dd, J = 7.6, 3.5 Hz, 1H), 4.99–5.00 (m, 1H), 6.52 (s, 1H), 7.42 (dd, J = 5.4, 3.5 Hz, 1H), 8.11 (dt, J = 5.4, 1.4 Hz, 1H), 8.70 (d, J = 2.4 Hz, 1H), 9.00 (s, 1H); mass spectrometry (MS) (fast atom bombardment (FAB)) m/z 570 (M+H)+; high-resolution mass spectrometry (HRMS) (electrospray ionization (ESI)) m/z calcd. for C31H40NO9 570.2703, found 570.2701 (M+H)+.

1, 11-Di-deacetyl-1, 11-di-O-propionylpyripyropene A (4a)

To a solution of 3 (30 mg, 0.0527 mmol) in anhydrous N,N-dimethylformamide (DMF) (1 ml) were added triethylamine (Et3N) (88 μl, 0.632 mmol), 4-(dimethylamino)pyridine (DMAP) (13 mg, 0.105 mmol) and acetic anhydride (31 μl, 0.316 mmol) and stirred at room temperature for 30 min. The reaction mixture was poured into water and extracted with EtOAc. The organic layer was washed with water and brine, dried over MgSO4, filtered, and concentrated in vacuo. The resulting residue was purified by PTLC (acetone: hexane = 1:1) to afford 4a (14 mg, 0.0222 mmol) as a solid in 42% yield. 1H NMR (CDCl3) δ 0.90 (s, 3H), 1.12 (t, J = 7.8 Hz, 3H), 1.13 (t, J = 7.8 Hz, 3H), 1.19 (s, 1H), 1.25–1.34 (m, 1H), 1.44 (s, 3H), 1.53–1.63 (m, 3H), 1.69 (s, 3H), 1.73–1.90 (m, 2H), 2.10 (m, 1H), 2.16 (s, 3H), 2.33 (dq, J = 7.6, 2.4 Hz, 2H), 2.36 (dq, J = 7.6, 3.2 Hz, 2H), 2.87 (m, 1H), 3.72 (m, 2H), 4.81 (dd, J = 11.6, 4.6 Hz, 1H), 4.97–5.00 (m, 2H), 6.46 (s, 1H), 7.40 (dd, J = 8.1, 4.6 Hz, 1H), 8.10 (m, 1H), 8.69 (d, J = 4.9 Hz, 1H), 9.00 (s, 1H); MS (ESI) m/z 612 (M+H)+; HRMS (ESI) m/z calcd. for C33H42NO10 612.2809, found 612.2801 (M+H)+.

1, 7, 11-Tri-deacetyl-7-O-isobutyryl-1, 11-di-O-propionylpyripyropene A (4b)

Reaction of 3 (30 mg, 0.0527 mmol) with isobutyric anhydride (53 μl, 0.316 mmol) gave 4b (11 mg, 0.0172 mmol) as a solid in 33% yield by a similar procedure to 4a. 1H NMR (CDCl3) δ 0.90 (s, 3H), 1.13 (t, J = 7.6 Hz, 6H), 1.19 (s, 1H), 1.24 (d, J = 4.6 Hz, 3H), 1.26 (d, J = 4.6 Hz, 3H), 1.33–1.38 (m, 1H), 1.45 (s, 3H), 1.54 (d, J = 3.8 Hz, 1H), 1.60–1.64 (m, 2H), 1.67 (s, 3H), 1.75–1.90 (m, 2H), 2.15–2.19 (m, 1H), 2.32 (q, J = 7.6 Hz, 2H), 2.38 (q, J = 7.6 Hz, 2H), 2.65 (quint., J= 7.6 Hz, 1H), 2.88 (d, J = 1.6 Hz, 1H), 3.68 (d, J = 12.4 Hz, 1H), 3.83 (d, J = 11.9 Hz, 1H), 4.80 (dd, J = 11.3, 4.9 Hz, 1H), 5.00 (m, 2H), 6.38 (s, 1H), 7.40 (dd, J = 8.1, 4.6 Hz, 1H), 8.09 (dt, J = 8.1, 1.9 Hz, 1H), 8.69 (dd, J = 4.6, 1.6 Hz, 1H), 9.00 (d, J = 1.6 Hz, 1H); MS (ESI) m/z 640 (M+H)+; HRMS (ESI) m/z calcd. for C35H46NO10 640.3122, found 640.3130 (M+H)+.

1, 7, 11-Tri-deacetyl-7-O-pivaloyl-1, 11-di-O-propionylpyripyropene A (4c)

Reaction of 3 (30 mg, 0.0527 mmol) with pivalic anhydride (64 μl, 0.316 mmol) gave 4c (23 mg, 0.0358 mmol) as a solid in 68% yield by a similar procedure to 4a. 1H NMR (CDCl3) δ 0.91 (s, 3H), 1.13 (t, J= 7.8 Hz, 3H), 1.16 (t, J= 7.8 Hz, 3H), 1.25 (s, 1H), 1.28 (s, 9H), 1.30–1.40 (m, 1H), 1.45 (s, 3H), 1.54 (d, J = 3.8 Hz, 1H), 1.60–1.66 (m, 2H), 1.71 (s, 3H), 1.75–1.90 (m, 2H), 2.15–2.19 (m, 1H), 2.32 (q, J = 7.6 Hz, 2H), 2.38 (q, J = 7.6 Hz, 2H), 2.89 (s, 1H), 3.66 (d, J = 11.6 Hz, 1H), 3.83 (d, J = 11.6 Hz, 1H), 4.79 (dd, J = 11.3, 5.4 Hz, 1H), 4.97–5.00 (m, 2H), 6.34 (s, 1H), 7.40 (dd, J = 8.4, 4.9 Hz, 1H), 8.09 (dt, J = 8.4, 2.2 Hz, 1H), 8.69 (d, J= 4.9 Hz, 1H), 9.00 (d, J = 2.2 Hz, 1H); MS (ESI) m/z 654 (M+H)+; HRMS (ESI) m/z calcd. for C36H48NO10 654.3278, found 654.3278 (M+H)+.

1, 7, 11-Tri-deacetyl-1, 11-di-O-propionyl-7-O-(3-pyridylcarbonyl)pyripyropene A (4g)

To a solution of 3 (30 mg, 0.0527 mmol) and nicotinic acid (13 mg, 0.105 mmol) in anhydrous DMF (3 ml) were added 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (15 mg, 0.0791 mmol) and DMAP (6.4 mg, 0.0527 mmol) and stirred at room temperature for 4.5 h. The reaction mixture was poured into water and then extracted with EtOAc. The organic layer was washed with water and brine, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The resulting residue was purified by PTLC (acetone: hexane = 1: 1) to give 4g (27 mg, 0.0402 mmol) as a solid in 76% yield. 1H NMR (CDCl3) δ 0.92 (s, 3H), 1.14 (t, J = 7.6 Hz, 3H), 1.20 (t, J = 7.6 Hz, 3H), 1.26 (s, 1H), 1.42–1.50 (m, 1H), 1.59 (s, 3H), 1.61–1.83 (m, 3H), 1.85 (s, 3H), 1.83–2.00 (m, 2H), 2.18–2.23 (m, 1H), 2.33 (q, J = 7.6 Hz, 2H), 2.43 (q, J = 7.6 Hz, 2H), 2.94 (m, 1H), 3.72 (d, J = 11.9 Hz, 1H), 3.82 (d, J = 12.7 Hz, 1H), 4.83 (dd, J = 11.3, 4.9 Hz, 1H), 5.03–5.06 (m, 1H), 5.27 (dd, J = 11.3, 4.9 Hz, 1H), 6.42 (s, 1H), 7.38 (dd, J = 8.1, 4.9 Hz, 1H), 7.45 (dd, J = 8.1, 4.9 Hz, 1H), 8.07 (dt, J = 8.1, 2.2 Hz, 1H), 8.36 (dt, J = 8.1, 1.9 Hz, 1H), 8.67 (dd, J = 5.1, 1.9 Hz, 1H), 8.83 (dd, J = 4.9, 1.9 Hz, 1H), 8.97 (d, J = 1.9 Hz, 1H), 9.30 (d, J = 1.9 Hz, 1H); MS (FAB) m/z 675(M+H)+; HRMS (ESI) m/z calcd. for C37H43N2O10 675.2918, found 675.2919 (M+H)+.

7-O-Cyclopropylcarbonyl-1, 7, 11-tri-deacetyl-1, 11-di-O-propionylpyripyropene A (4d)

Reaction of 3 (30 mg, 0.0527 mmol) with cyclopropanecarboxylic acid (25 μl, 0.310 mmol) gave 4d (18 mg, 0.0286 mmol) as a solid in 54% yield by a similar procedure to 4g. 1H NMR (CDCl3) δ 0.90 (s, 3H), 0.93 (d, J = 2.7 Hz, 2H), 0.96 (d, J = 2.7 Hz, 2H), 1.03–1.19 (m, 6H), 1.26 (s, 1H), 1.32–1.39 (m, 1H), 1.45 (s, 3H), 1.52 (d, J = 3.8 Hz, 1H), 1.61–1.69 (m, 3H), 1.71 (s, 3H), 1.73–1.94 (m, 2H), 2.14–2.19 (m, 1H), 2.24–2.40 (m, 4H), 2.95 (m, 1H), 3.68 (d, J = 11.9 Hz, 1H), 3.81 (d, J = 11.9 Hz, 1H), 4.79 (dd, J = 11.3, 5.4 Hz, 1H), 4.96–5.00 (m, 2H), 6.45 (s, 1H), 7.40 (dd, J = 8.1, 4.6 Hz, 1H), 8.10 (dt, J = 8.1, 1.9 Hz, 1H), 8.68 (m, 1H), 9.01 (m, 1H); MS (FAB) m/z 638 (M+H)+; HRMS (ESI) m/z calcd. for C35H44NO10 638.2965, found 638.2968 (M+H)+.

7-O-Cyclobutylcarbonyl-1, 7, 11-tri-deacetyl-1, 11-di-O-propionylpyripyropene A (4e)

Reaction of 3 (30 mg, 0.0527 mmol) with cyclobutanecarboxylic acid (29 μl, 0.310 mmol) gave 4e (15 mg, 0.0229 mmol) as a solid in 43% yield by a similar procedure to 4g. 1H NMR (CDCl3) δ 0.90 (s, 3H), 1.13 (t, J = 7.6 Hz, 3H), 1.17 (t, J = 7.6 Hz, 3H), 1.26 (s, 1H), 1.34–1.40 (m, 1H), 1.44 (s, 3H), 1.54 (d, J = 4.3 Hz, 1H), 1.61–1.67 (m, 2H), 1.69 (s, 3H), 1.72–2.42 (m, 12H), 2.91 (m, 1H), 3.23 (quint., J = 8.1 Hz, 1 H), 3.69 (d, J= 11.9 Hz, 1H), 3.81 (d, J = 11.9 Hz, 1H), 4.80 (dd, J = 11.3, 4.9 Hz, 1H), 4.99–5.04 (m, 2H), 6.40 (s, 1H), 7.39 (dd, J = 8.1, 4.9 Hz, 1H), 8.09 (dt, J = 8.1, 1.6 Hz, 1H), 8.69 (dd, J = 4.6, 1.6 Hz, 1H), 9.01 (d, J = 1.6 Hz, 1H); MS (ESI) m/z 652 (M+H)+; HRMS (ESI) m/z calcd. for C36H45NO10 652.3077, found 652.3125 (M+H)+.

7-O-Benzoyl-1, 7, 11-tri-deacetyl-1, 11-di-O-propionylpyripyropene A (4f)

Reaction of 3 (30 mg, 0.0527 mmol) with benzoic acid (85 mg, 0.696 mmol) gave 4f (34 mg, 0.0505 mmol) as a solid in 95% yield by a similar procedure to 4g. 1H NMR (CDCl3) δ 0.92 (s, 3H), 1.14 (t, J = 7.6 Hz, 3H), 1.20 (t, J = 7.6 Hz, 3H), 1.26 (s, 1H), 1.37–1.46 (m, 1H), 1.51 (s, 3H), 1.62 (d, J = 3.8 Hz, 1H), 1.68–1.82 (m, 2H), 1.87 (s, 3H), 1.91–2.00 (m, 2H), 2.18–2.23 (m, 1H), 2.33 (q, J = 7.6 Hz, 2H), 2.43 (dq, J = 7.6, 1.4 Hz, 2H), 2.97 (s, 1H), 3.70 (d, J = 11.9 Hz, 1H), 3.84 (d, J = 11.9 Hz, 1H), 4.83 (dd, J = 11.1, 5.1 Hz, 1H), 5.05 (d, J = 4.3 Hz, 1H), 5.27 (dd, J = 11.1, 4.6 Hz, 1H), 6.45 (s, 1H), 7.39–7.66 (m, 4H), 8.05–8.13 (m, 3H), 8.70 (d, J = 4.6 Hz, 1H), 9.00 (s, 1H); MS (FAB) m/z 674 (M+H)+; HRMS (ESI) m/z calcd. for C38H44NO10 674.2965, found 674.2958 (M+H)+.

1, 7, 11-Tri-deacetyl-1, 11-di-O-propionyl-7-O-(2-pyridylcarbonyl)pyripyropene A (4h)

Reaction of 3 (30 mg, 0.0527 mmol) with picolinic acid (13 mg, 0.105 mmol) gave 4h (40 mg, 0.0446 mmol) as a solid in 85% yield by a similar procedure to 4g. 1H NMR (CDCl3) δ 0.91 (s, 3H), 1.13 (t, J = 7.6 Hz, 3H), 1.20 (t, J = 7.6 Hz, 3H), 1.26 (s, 1H), 1.37–1.46 (m, 1H), 1.50 (s, 3H), 1.63–1.75 (m, 2H), 1.87 (s, 3H), 1.83–1.96 (m, 2H), 2.13–2.23 (m, 1H), 2.32 (q, J = 7.6 Hz, 2H), 2.41 (dq, J = 7.6, 1.4 Hz, 2H), 2.99 (m, 1H), 3.67 (d, J = 11.9 Hz, 1H), 3.83 (d, J = 11.9 Hz, 1H), 4.83 (dd, J = 11.3, 5.4 Hz, 1H), 4.98–5.06 (m, 1H), 5.38 (dd, J = 10.8, 5.4 Hz, 1H), 6.43 (s, 1H), 7.35–7.44 (m, 1H), 7.50–7.55 (m, 1H), 7.89 (dt, J = 7.6, 1.6 Hz, 1H), 8.07 (dt, J = 8.1, 1.6 Hz, 1H), 8.18 (d, J = 7.6 Hz, 1H), 8.67 (dd, J = 4.9, 1.6 Hz, 1H), 8.82–8.84 (m, 1H), 8.97 (d, J = 2.4 Hz, 1H); MS (FAB) m/z 675 (M+H)+; HRMS (ESI) m/z calcd. for C37H43N2O10 675.2918, found 675.2911 (M+H)+.

1, 7, 11-Tri-deacetyl-1, 11-di-O-propionyl-7-O-(4-pyridylcarbonyl)pyripyropene A (4i)

Reaction of 3 (30 mg, 0.0527 mmol) with isonicotinic acid (13 mg, 0.105 mmol) gave 4i (15 mg, 0.0225 mmol) as a solid in 43% yield by a similar procedure to 4g. 1H NMR (CDCl3) δ 0.92 (s, 3H), 1.14 (t, J = 7.6 Hz, 3H), 1.20 (t, J = 7.6 Hz, 3H), 1.26 (s, 1H), 1.38–1.42 (m, 1H), 1.50 (s, 3H), 1.64–1.78 (m, 3H), 1.85 (s, 3H), 1.88–2.05 (m, 2H), 2.17–2.23 (m, 1H), 2.33 (q, J = 7.6 Hz, 2H), 2.42 (dq, J = 7.6, 1.1 Hz, 2H), 2.99 (m, 1H), 3.72 (d, J = 12.4 Hz, 1H), 3.81 (d, J = 11.5 Hz, 1H), 4.83 (dd, J = 11.5, 4.9 Hz, 1H), 5.03–5.05 (m, 1H), 5.25 (dd, J = 11.5, 5.4 Hz, 1H), 6.41 (s, 1H), 7.37 (dd, J = 8.1, 5.2 Hz, 1H), 7.91 (dd, J = 4.6, 1.6 Hz, 2H), 8.07 (dt, J = 8.1, 1.6 Hz, 1H), 8.67 (dd, J = 4.9, 1.9 Hz, 1H), 8.83 (dd, J = 4.3, 1.6 Hz, 2H), 8.97 (d, J = 1.6 Hz, 1H); MS (FAB) m/z 675 (M+H)+; HRMS (ESI) m/z calcd. for C37H42N2O10 674.2839, found 674.2841 (M)+.

7-O-(6-Chloro-3-pyridylcarbonyl)-1, 7, 11-tri-deacetyl-1,11-di-O-propionylpyripyropene A (4j)

Reaction of 3 (30 mg, 0.0527 mmol) with 6-chloronicotinic acid (16 mg, 0.105 mmol) gave 4j (31 mg, 0.0431 mmol) as a solid in 82% yield by a similar procedure to 4g. 1H NMR (CDCl3) δ 0.92 (s, 3H), 1.14 (t, J = 7.6 Hz, 3H), 1.20 (t, J = 7.6 Hz, 3H), 1.26 (s, 1H), 1.38–1.46 (m, 1H), 1.50 (s, 3H), 1.61 (m, 1H), 1.66–1.78 (m, 2H), 1.84 (s, 3H), 1.87–1.99 (m, 2H), 2.12–2.23 (m, 1H), 2.31 (q, J = 7.6 Hz, 2H), 2.41 (q, J = 7.6 Hz, 2H), 2.95 (m, 1H), 3.73 (d, J= 11.9 Hz, 1H), 3.81 (d, J = 11.9 Hz, 1H), 4.83 (dd, J = 11.3, 4.9 Hz, 1H), 5.04 (m, 1H), 5.25 (dd, J = 11.3, 4.9 Hz, 1H), 6.40 (s, 1H), 7.38 (dd, J = 7.8, 4.6 Hz, 1H), 7.47 (d, J = 8.1 Hz, 1H), 8.06 (dt, J = 7.8, 1.6 Hz, 1H), 8.30 (dd, J = 8.1, 2.4 Hz, 1H), 8.67 (dd, J = 4.6, 1.4 Hz, 1H), 8.97 (d, J = 2.4 Hz, 1H), 9.06 (d, J = 2.7 Hz, 1H); MS (FAB) m/z 709(M+H)+; HRMS (ESI) m/z calcd. for C37H42ClN2O10 709.2528, found 709.2524 (M+H)+.

1, 7, 11-Tri-deacetyl-1, 11-di-O-propionyl-7-O-(6-trifluoromethyl-3-pyridylcarbonyl)pyripyropene A (4k)

Reaction of 3 (30 mg, 0.0527 mmol) with 6-(trifluoromethyl)nicotinic acid (30 mg, 0.158 mmol) gave 4k (35 mg, 0.0477 mmol) as a solid in 90% yield by a similar procedure to 4g. 1H NMR (CDCl3) δ 0.92 (s, 3H), 1.14 (t, J = 7.6 Hz, 3H), 1.21 (t, J = 7.6 Hz, 3H), 1.26 (s, 1H), 1.44 (m, 1H), 1.50 (s, 3H), 1.57–1.62 (m, 1H), 1.67–1.80 (m, 2H), 1.85 (s, 3H), 1.91–1.95 (m, 2H), 2.17–2.24 (m, 1H), 2.33 (q, J= 7.6 Hz, 2H), 2.42 (q, J = 7.6 Hz, 2H), 2.92 (m, 1H), 3.74 (d, J = 11.9 Hz, 1H), 3.81 (d, J = 11.9 Hz, 1H), 4.84 (dd, J = 11.1, 4.9 Hz, 1H), 5.04 (m, 1H), 5.27 (dd, J = 11.1, 4.9 Hz, 1H), 6.40 (s, 1H), 7.38 (dd, J = 8.1, 4.9 Hz, 1H), 7.84 (d, J = 8.4 Hz, 1H), 8.05–8.08 (m, 1H), 8.54 (d, J = 8.1 Hz, 1H), 8.67 (d, J = 4.6 Hz, 1H), 8.96 (d, J = 2.2 Hz, 1H), 9.38 (s, 1H); MS (FAB) m/z 743 (M+H)+; HRMS (ESI) m/z calcd. for C38H42F3N2O10 743.2792, found 743.2794 (M+H)+.

1, 7, 11-Tri-deacetyl-1, 11-di-O-propionyl-7-O-(4-trifluoromethyl-3-pyridylcarbonyl)pyripyropene A (4l)

Reaction of 3 (30 mg, 0.0527 mmol) with 4-(trifluoromethyl)nicotinic acid (30 mg, 0.158 mmol) gave 4l (19 mg, 0.0257 mmol) as a solid in 49% yield by a similar procedure to 4g. 1H NMR (CDCl3) δ 0.94 (s, 3H), 1.14 (t, J = 7.6 Hz, 3H), 1.19 (t, J = 7.6 Hz, 3H), 1.26 (s, 1H), 1.38–1.47 (m, 1H), 1.48 (s, 3H), 1.57–1.71 (m, 3H), 1.75 (s, 3H), 1.83–1.97 (m, 2H), 2.10–2.22 (m, 1H), 2.33 (q, J = 7.6 Hz, 2H), 2.41 (dq, J = 7.6, 1.6 Hz, 2H), 2.96 (m, 1H), 3.74–3.80 (m, 2H), 4.83 (dd, J = 11.6, 5.7 Hz, 1H), 5.02–5.03 (m, 1H), 5.28 (dd, J = 11.6, 5.4 Hz, 1H), 6.41 (s, 1H), 7.40 (dd, J= 7.6, 5.4 Hz, 1H), 7.69 (d, J = 5.4 Hz, 1H), 8.08 (dt, J = 8.1, 2.2 Hz, 1H), 8.69 (dd, J= 4.9, 1.6 Hz, 1H), 8.97 (d, J= 4.6 Hz, 1H), 9.00 (d, J = 2.4 Hz, 1H), 9.16 (s, 1H); MS (FAB) m/z 743 (M+H)+; HRMS (ESI) m/z calcd. for C38H42F3N2O10 743.2792, found 743.2789 (M+H)+.

1, 7, 11-Tri-deacetyl-7-O-(6-methyl-3-pyridylcarbonyl)-1, 11-di-O-propionylpyripyropene A (4m)

Reaction of 3 (20 mg, 0.0351 mmol) with 6-methylnicotinic acid (29 mg, 0.210 mmol) gave 4m (15 mg, 0.0219 mmol) as a solid in 63% yield by a similar procedure to 4g. 1H NMR (CDCl3) δ 0.92 (s, 3H), 1.12 (t, J = 7.8 Hz, 3H), 1.15 (t, J = 7.7 Hz, 3H), 1.26 (s, 1H), 1.39–1.47 (m, 1H), 1.50 (s, 3H), 1.61 (d, J = 2.4 Hz, 1H), 1.69–1.81 (m, 2H), 1.85 (s, 3H), 1.90–1.99 (m, 2H), 2.18–2.21 (m, 1H), 2.33 (dq, J= 7.7, 1.2 Hz, 2H), 2.41 (dq, J = 7.6 Hz, 2.7 Hz, 2H), 2.66 (s, 3H), 2.96 (m, 1H), 3.72 (d, J = 11.7 Hz, 1H), 3.83 (d, J = 12.0 Hz, 1H), 4.83 (dd, J = 11.4, 4.9 Hz, 1H), 5.04 (m, 1H), 5.25 (dd, J = 11.7, 5.3 Hz, 1H), 6.41 (s, 1H), 7.30 (d, J = 8.0 Hz, 1H), 7.38 (dd, J = 8.1, 4.9 Hz, 1H), 8.07 (dt, J = 8.1, 2.2 Hz, 1H), 8.24 (dd, J = 8.0, 2.2 Hz, 1H), 8.67 (dd, J = 4.9, 1.5 Hz, 1H), 8.97 (d, J = 2.2 Hz, 1H), 9.18 (d, J = 2.2 Hz, 1H); MS (FAB) m/z 689 (M+H)+; HRMS (ESI) m/z calcd. for C38H44N2O10 688.2996, found 688.2994 (M)+.

1, 7, 11-Tri-deacetyl-7-O-(4-methyl-3-pyridylcarbonyl)-1, 11-di-O-propionylpyripyropene A (4n)

Reaction of 3 (20 mg, 0.0351 mmol) with 4-methylnicotinic acid hydrochloride (36 mg, 0.210 mmol) gave 4n (16 mg, 0.0232 mmol) as a solid in 66% yield by a similar procedure to 4g. 1H NMR (CDCl3) δ 0.93 (s, 3H), 1.14 (t, J = 7.6 Hz, 3H), 1.20 (t, J = 7.6 Hz, 3H), 1.26 (s, 1H), 1.33–1.44 (m, 1H), 1.50 (s, 3H), 1.61 (m, 1H), 1.68–1.77 (m, 2H), 1.84 (s, 3H), 1.91–1.99 (m, 2H), 2.17–2.23 (m, 1H), 2.32 (q, J = 7.6 Hz, 2H), 2.43 (dq, J = 7.6, 3.0 Hz, 2H), 2.69 (s, 3H), 2.96 (m, 1H), 3.75 (d, J = 12.2 Hz, 1H), 3.80 (d, J = 12.2 Hz, 1H), 4.48 (dd, J = 11.1, 5.1 Hz, 1H), 5.04 (d, J = 4.1 Hz, 1H), 5.23 (dd, J = 10.8, 5.4 Hz, 1H), 6.42 (s, 1H), 7.24 (d, J= 5.9 Hz, 1H), 7.39 (dd, J = 8.1, 4.9 Hz, 1H), 8.08 (d, J = 8.4 Hz, 1H), 8.61 (d, J= 5.1 Hz, 1H), 8.67 (d, J= 3.5 Hz, 1H), 8.98 (s, 1H), 9.17 (s, 1H); MS (FAB) m/z 689 (M+H)+; HRMS (ESI) m/z calcd. for C38H44N2O10 688.2996, found 688.2992 (M)+.

1, 7, 11-Tri-O-cyclopropylcarbonyl-1, 7, 11-tri-deacetylpyripyropene A (5c)

To a solution of 2 (30 mg, 0.0656 mmol), which was synthesized by the method previously reported [9], and cyclopropanecarboxylic acid (103 μl, 1.31 mmol) in anhydrous DMF (2 mL) were added 1-ethyl-3-(3-dimethylformamide)-carbodiimide hydrochloride (76 mg, 0.394 mmol) and DMAP (32 mg, 0.262 mmol) and stirred at room temperature for 68 h. The reaction mixture was then poured into water and extracted with EtOAc. The organic layer was washed with water and brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The resulting residue was purified by PTLC (acetone: hexane = 1:1) to afford 5c (34 mg, 0.0510 mmol) as a solid in 78% yield. 1H NMR (CDCl3) δ 0.83–1.12 (m, 12H), 0.91 (s, 3H), 1.26 (s, 1H), 1.33–1.41 (m, 1H), 1.45 (s, 3H), 1.52–1.69 (m, 6H), 1.71 (s, 3H), 1.81–1.93 (m, 2H), 2.14–2.18 (m, 1H), 2.92 (m, 1H), 3.72 (d, J = 11.9Hz, 1H), 3.82 (d, J = 11.9 Hz, 1H), 4.80 (dd, J = 11.4, 4.9 Hz, 1H), 4.99–5.04 (m, 2H), 6.46 (s, 1H), 7.41 (dd, J = 8.3, 4.9 Hz, 1H), 8.10 (dt, J = 8.3, 1.7 Hz, 1H), 8.69 (dd, J = 4.9, 1.5 Hz, 1H), 9.01 (d, J = 1.4 Hz, 1H); MS (FAB) m/z 662 (M+H)+; HRMS (ESI) m/z calcd. for C37H43NNaO10 684.2785, found 684.2778 (M+Na)+.

1, 7, 11-Tri-deacetyl-1, 7, 11-tri-O-pivaloylpyripyropene A (5b)

To a solution of 2 (30 mg, 0.0656 mmol) in anhydrous DMF (2 ml) were added Et3N (60 mg, 0.590 mmol), DMAP (8 mg, 0.00656 mmol), and pivalic anhydride (239 μl, 1.18 mmol) and stirred at room temperature for 16 h. The reaction mixture was added to water and extracted with EtOAc. The organic layer was washed with water and brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The resulting residue was purified by PTLC (acetone: hexane = 1:1) to give 5b (28 mg, 0.0390 mmol) as a solid in 60% yield. 1H NMR (CDCl3) δ 0.93 (s, 3H), 1.19 (s, 9H), 1.24 (s, 9H), 1.26 (s, 1H), 1.28 (s, 9H), 1.37–1.40 (m, 1H), 1.46 (s, 3H), 1.50–1.66 (m, 3H), 1.72 (s, 3H), 1.81–1.89 (m, 2H), 2.16–2.21 (m, 1H), 3.68 (d, J = 12.0 Hz, 1H), 3.74 (d, J = 12.0 Hz, 1H), 4.77 (dd, J = 11.2, 5.2 Hz, 1H), 4.92–4.97 (m, 1H), 5.01 (d, J = 4.0 Hz, 1H), 6.35 (s, 1H), 7.40 (dd, J = 8.0, 4.0 Hz, 1H), 8.09 (dt, J = 8.0, 1.6 Hz, 1H), 8.69 (dd, J = 4.8, 1.6 Hz, 1H), 9.00 (d, J = 2.0 Hz, 1H); MS (FAB) m/z 710 (M+H)+; HRMS (ESI) m/z calcd. for C40H56NO10 710.3904, found 710.3906 (M+H)+.

1, 7, 11-Tri-O-cyclobutylcarbonyl-1, 7, 11-tri-deacetylpyripyropene A (5d)

Reaction of 2 (30 mg, 0.0656 mmol) with cyclobutanecarboxylic acid (124 μl, 1.31 mmol) gave 5d (29 mg, 0.0411 mmol) as a solid in 63% yield by a similar procedure to 5c. 1H NMR (CDCl3) δ 0.90 (s, 3H), 1.26 (s, 1H), 1.32–1.41 (m, 1H), 1.44 (s, 3H), 1.51–1.63 (m, 3H), 1.69 (s, 3H), 1.79–2.04 (m, 8H), 2.17–2.40 (m, 13H), 2.89 (m, 1H), 3.08–3.26 (m, 3H), 3.67 (d, J= 11.9 Hz, 1H), 3.78 (d, J = 11.9 Hz, 1H), 4.79 (dd, J = 11.1, 5.4 Hz, 1H), 4.97–5.00 (m, 2H), 6.41 (s, 1H), 7.41 (dd, J = 8.1, 4.9 Hz, 1H), 8.09 (dt, J = 8.4, 1.9 Hz, 1H), 8.68 (m, 1H), 9.00 (m, 1H); MS (FAB) m/z 704 (M+H)+; HRMS (ESI) m/z calcd. for C40H50NO10 704.3435, found 704.3429 (M+H)+.

1, 7, 11-Tri-O-cyclohexylcarbonyl-1, 7, 11-tri-deacetylpyripyropene A (5e)

Reaction of 2 (20 mg, 0.0436 mmol) with cyclohexanecarboxylic acid (109 μl, 0.871 mmol) gave 5e (22 mg, 0.0273 mmol) as a solid in 63% yield by a similar procedure to 5c. 1H NMR (CDCl3) δ 0.91 (s, 3H), 1.45 (s, 3H), 1.70 (s, 3H), 1.10–2.05 (m, 37H), 2.14–2.49 (m, 3H), 3.04 (s, 1H), 3.65 (d, J = 11.3 Hz, 1H), 3.77 (d, J = 11.9 Hz, 1H), 4.78 (dd, J = 10.8, 5.4 Hz, 1H), 4.97–5.01 (m, 2H), 6.41 (s, 1H), 7.42 (dd, J= 8.1, 4.9 Hz, 1H), 8.11 (dd, J = 8.1, 1.9 Hz, 1H), 8.69 (d, J = 4.3 Hz, 1H), 9.01 (s, 1H); MS (FAB) m/z 788 (M+H)+; HRMS (ESI) m/z calcd. for C46H62NO10 788.4374, found 788.4362 (M+H)+.

1, 7, 11-Tri-O-benzoyl-1, 7, 11-tri-deacetylpyripyropene A (5f)

Reaction of 2 (30 mg, 0.0656 mmol) with benzoic acid (160 mg, 1.31 mmol) gave 5f (38 mg, 0.0494 mmol) as a solid in 75% yield by a similar procedure to 5c. 1H NMR (CDCl3) δ 1.17 (s, 3H), 1.26 (s, 1H), 1.57 (s, 3H), 1.65 (m, 1H), 1.77–1.82 (m, 2H), 1.88 (s, 3H), 1.94–2.05 (m, 3H), 2.13–2.31 (m, 1H), 2.95 (m, 1H), 4.16 (s, 2H), 5.06 (dd, J = 6.5, 2.4 Hz, 1H), 5.17–5.32 (m, 2H), 6.42 (s, 1H), 7.34–7.64 (m, 10H), 8.01–8.12 (m, 7H), 8.66 (dd, J = 5.1, 1.6 Hz, 1H), 8.97 (d, J = 1.9 Hz, 1H); MS (FAB) m/z 770 (M+H)+; HRMS (ESI) m/z calcd. for C46H44NO10 770.2965, found 770.2952 (M+H)+.

1, 7, 11-Tri-deacetyl-1, 7, 11-tri-O-(3-pyridylcarbonyl)pyripyropene A (5g)

Reaction of 2 (30 mg, 0.0656 mmol) with nicotinic acid (161 mg, 1.31 mmol) gave 5g (34 mg, 0.0443 mmol) as a solid in 68% yield by a similar procedure to 5c. 1H NMR (CDCl3) δ 1.17 (s, 3H), 1.25 (s, 1H), 1.47–1.55 (m, 1H), 1.60 (s, 3H), 1.66 (d, J = 4.0 Hz, 1H), 1.81 (d, J = 11.5 Hz, 1H), 1.90 (s, 3H), 1.90–1.98 (m, 1H), 2.02–2.15 (m, 1H), 2.20–2.24 (m, 1H), 2.29–2.32 (m, 1H), 3.02 (s, 1H), 4.12 (d, J = 12.0 Hz, 1H), 4.25 (d, J = 12.0 Hz, 1H), 5.07 (d, J = 3.7 Hz, 1H), 5.18–5.29 (m, 2H), 6.42 (s, 1H), 7.36–7.47 (m, 4H), 8.07 (dt, J = 8.0, 2.0 Hz, 1H), 8.28 (dd, J = 8.0, 2.0 Hz, 1H), 8.32–8.36 (m, 2H), 8.67 (dd, J = 4.8, 1.6 Hz, 1H), 8.79 (dt, J = 4.8, 1.6 Hz, 2H), 8.83 (dd, J = 4.8, 1.6 Hz, 1H), 8.98 (dd, J = 2.0, 0.8 Hz, 1H), 9.22 (dd, J = 2.0, 0.8 Hz, 1H), 9.30 (ddd, J = 5.2, 2.0, 0.8 Hz, 2H); MS (ESI) m/z 773 (M+H)+; HRMS(ESI) m/z calcd. for C43H41N4O10 773.2823, found 773.2825 (M+H)+.

1, 7, 11-Tri-deacetyl-1, 7, 11-tri-O-(2-pyridylcarbonyl)pyripyropene A (5h)

Reaction of 2 (20 mg, 0.0437 mmol) with picolinic acid (32 mg, 0.262 mmol) gave 5h (28 mg, 0.0366 mmol) as a solid in 84% yield by a similar procedure to 5c. 1H NMR (CDCl3) δ 1.20 (s, 3H), 1.26 (s, 1H), 1.54–1.55 (m, 1H), 1.59 (s, 3H), 1.70 (d, J = 4.3 Hz, 1H), 1.89 (s, 3H), 1.92–1.94 (m, 2H), 1.98–2.13 (m, 1H), 2.16–2.20 (m, 1H), 2.29–2.33 (m, 1H), 2.93 (s, 1H), 4.14 (d, J = 12.0 Hz, 1H), 4.26 (d, J = 12.0 Hz, 1H), 5.08 (d, J = 3.9 Hz, 1H), 5.33 (dd, J = 7.6, 5.2 Hz, 1H), 5.45 (dd, J = 7.6, 5.2 Hz, 1H), 6.42 (s, 1H), 7.38 (ddd, J = 8.0, 4.8, 0.8 Hz, 1H), 7.45–7.46 (m, 2H), 7.51 (ddd, J = 8.0, 4.8, 1.2 Hz, 1H), 7.80–7.85 (m, 2H), 7.88 (dt, J = 8.0, 2.0 Hz, 1H), 8.04–8.08 (m, 2H), 8.09–8.11 (m, 1H), 8.15–8.18 (m, 1H), 8.67 (dd, J = 4.8, 1.6 Hz, 1H), 8.73–8.77 (m, 2H), 8.80–8.82 (m, 1H), 8.97 (d, J = 1.7 Hz, 1H); MS (ESI) m/z 773 (M+H)+; HRMS (ESI) m/z calcd. for C43H41N4O10 773.2823, found 773.2823 (M+H)+.

1, 7, 11-Tri-deacetyl-1, 7, 11-tri-O-(6-trifluoromethyl-3-pyridylcarbonyl)pyripyropene A (5i)

Reaction of 2 (30 mg, 0.0656 mmol) with 6-(trifluoromethyl)nicotinic acid (250 mg, 1.31 mmol) gave 5i (31 mg, 0.0318 mmol) as a solid in 48% yield by a similar procedure to 5c. 1H NMR (CDCl3) δ 1.18 (s, 3H), 1.26 (s, 1H), 1.51–1.54 (m, 1H), 1.60 (s, 3H), 1.64 (d, J = 4.0 Hz, 1H), 1.76–1.79 (m, 1H), 1.90 (s, 3H), 1.90–2.00 (m, 1H), 2.10–2.12 (m, 1H), 2.23–2.26 (m, 1H), 2.32–2.35 (m, 1H), 2.96 (d, J = 1.6 Hz, 1H), 4.16 (d, J = 12.0 Hz, 1H), 4.29 (d, J = 12.0 Hz, 1H), 5.06 (dd, J = 3.9, 2.4 Hz, 1H), 5.17–5.26 (m, 2H), 6.41 (s, 1H), 7.38 (ddd, J = 8.1, 4.8, 0.8 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.84 (d, J = 8.0 Hz, 2H), 8.07 (dt, J = 8.0, 1.6 Hz, 1H), 8.47 (dd, J = 8.1, 1.5 Hz, 1H), 8.54 (dd, J = 8.2, 1.9 Hz, 2H), 8.68 (dd, J = 4.9, 1.6 Hz, 1H), 8.97 (d, J = 1.6 Hz, 1H), 9.30 (d, J = 1.8 Hz, 1H), 9.37–9.39 (m, 2H); MS (ESI) m/z 977 (M+H)+; HRMS (ESI) m/z calcd. for C46H38F9N4O10 977.2444, found 977.2443 (M+H)+.

1, 7, 11-Tri-deacetyl-1, 7, 11-tri-O-(4-trifluoromethyl-3-pyridylcarbonyl)pyripyropene A (5j)

Reaction of 2 (30 mg, 0.0656 mmol) with 4-(trifluoromethyl)nicotinic acid (250 mg, 1.31 mmol) gave 5j (36 mg, 0.0370 mmol) as a solid in 56% yield by a similar procedure to 5c. 1H NMR (CDCl3) δ 1.06 (s, 3H), 1.26 (s, 1H), 1.45 (dt, J = 13.0, 4.2 Hz, 1H), 1.54 (s, 3H), 1.58 (d, J = 4.0 Hz, 1H), 1.77 (s, 3H), 1.77–1.79 (m, 1H), 1.85–1.97 (m, 1H), 2.04–2.09 (m, 2H), 2.26–2.29 (m, 1H), 2.98 (brs, 1H), 4.10 (d, J = 12.1 Hz, 1H), 4.29 (d, J = 12.1 Hz, 1H), 5.02 (d, J = 2.9 Hz, 1H), 5.19 (dd, J = 11.7, 5.0 Hz, 1H), 5.29 (dd, J = 11.7, 5.0 Hz, 1H), 6.42 (s, 1H), 7.40 (ddd, J = 8.1, 4.8, 0.8 Hz, 1H), 7.66 (d, J = 5.2 Hz, 2H), 7.69 (d, J = 5.2 Hz, 1H), 8.07–8.10 (m, 1H), 8.69 (dd, J = 4.8, 1.6 Hz, 1H), 8.95 (d, J = 5.2 Hz, 2H), 8.97 (d, J = 5.2 Hz, 1H), 9.00 (d, J = 1.6 Hz, 1H), 9.11 (s, 1H), 9.17 (s, 1H), 9.25 (s, 1H); MS (ESI) m/z 977 (M+H)+; HRMS (ESI) m/z calcd. for C46H38F9N4O10 977.2444, found 977.2433 (M+H)+.

Insecticidal screening against agricultural pests

Pyripyropene derivatives were evaluated by an insecticidal screening against green peach aphid (M. persicae) following the method described in our former report [15]. The screening against cotton aphid (A. gossypii), greenhouse whitefly (T. vaporariorum), and western flower thrips (F. occidentalis) were conducted by the method previously reported [20].

Field efficacy of compound 5c in a foliar application against cabbage aphid (B. brassicae) on cabbage

The field trial was conducted in a cabbage field of Odawara City, Kanagawa Prefecture in Japan using one of general formulation types, WP. This formulation, including 5% (w/w) active ingredient, was prepared following a preparation method reported previously [20]. The determined amount of diluted solution of 5c WP in water was applied to cabbage infested with cabbage aphids. Before application and at 1, 5, 7, 13 and 20 days after application, the numbers of aphids were counted in each plot. The corrected density index was calculated as follows:

$$\begin{array}{*{20}{l}} {{\mathrm{Corrected}}\,{\mathrm{density}}\,{\mathrm{index}}} \hfill & \\ \quad{\mathrm{ = }} {\left[ {{\textstyle{{\left( {{\mathrm{the}}{\kern 1pt} {\mathrm{number}}{\kern 1pt} {\mathrm{of}}{\kern 1pt} {\mathrm{aphids}}{\kern 1pt} {\mathrm{in}}{\kern 1pt} {\mathrm{treated}}{\kern 1pt} {\mathrm{plot}}{\kern 1pt} {\mathrm{at}}{\kern 1pt} {X}{\kern 1pt} {\mathrm{days}}{\kern 1pt} {\mathrm{after}}{\kern 1pt} {\mathrm{application}}} \right)} \over {\left( {{\mathrm{the}}{\kern 1pt} {\mathrm{number}}{\kern 1pt} {\mathrm{of}}{\kern 1pt} {\mathrm{aphids}}{\kern 1pt} {\mathrm{in}}{\kern 1pt} {\mathrm{treated}}{\kern 1pt} {\mathrm{plot}}{\kern 1pt} {\mathrm{before}}{\kern 1pt} {\mathrm{application}}} \right)}}}} \right]} \hfill \\ {} \hfill & \qquad{} {{\mathrm{ \times }}\left[ {{\textstyle{{\left( {{\mathrm{the}}{\kern 1pt} {\mathrm{number}}{\kern 1pt} {\mathrm{of}}{\kern 1pt} {\mathrm{aphids}}{\kern 1pt} {\mathrm{in}}{\kern 1pt} {\mathrm{untreated}}{\kern 1pt} {\mathrm{plot}}{\kern 1pt} {\mathrm{before}}{\kern 1pt} {\mathrm{application}}} \right)} \over {\left( {{\mathrm{the}}{\kern 1pt} {\mathrm{number}}{\kern 1pt} {\mathrm{of}}{\kern 1pt} {\mathrm{aphids}}{\kern 1pt} {\mathrm{in}}{\kern 1pt} {\mathrm{untreated}}{\kern 1pt} {\mathrm{plot}}{\kern 1pt} {\mathrm{at}}{\kern 1pt} {X}{\kern 1pt} {\mathrm{days}}{\kern 1pt} {\mathrm{after}}{\kern 1pt} {\mathrm{application}}} \right)}}}} \right]} \hfill \\ \qquad{{\mathrm{ \times 100}}} \hfill \end{array}$$
(1)

Then, compared with untreated plot, the control percentage was calculated as follows:

$${\mathrm{\% }}{\kern 1pt} {\mathrm{control = 100}}-\left( {{\mathrm{the}}\,{\mathrm{corrected}}\,{\mathrm{density}}\,{\mathrm{index}}} \right){\mathrm{.}}$$
(2)

Field efficacy of 5c in a foliar application against cotton aphid (A. gossypii) on potato

The field trial was conducted in a potato field of Misawa City, Aomori Prefecture in Japan as a non-disclosed trial conducted by the Institute of Japan Plant Protection Association. The WP formulation, including 5% (w/w) active ingredient, was prepared and used in the same way as the field trial against B. brassicae. The determined amount of diluted solution of 5c WP in water was applied to potatoes infested with cotton aphids. Before application and at 2, 7 and 14 days after application, the numbers of aphids were counted in each plot. The corrected density index and percentage of control were calculated by the same formulae as the field trial against cabbage aphid.