First Total Syntheses and Antimicrobial Evaluation of Penicimonoterpene, a Marine-Derived Monoterpenoid, and Its Various Derivatives

The first total synthesis of marine-derived penicimonoterpene (±)-1 has been achieved in four steps from 6-methylhept-5-en-2-one using a Reformatsky reaction as the key step to construct the basic carbon skeleton. A total of 24 new derivatives of 1 have also been designed and synthesized. Their structures were characterized by analysis of their 1H NMR, 13C NMR and HRESIMS data. Some of them showed significant antibacterial activity against Aeromonas hydrophila, Escherichia coli, Micrococcus luteus, Staphylococcus aureus, Vibrio anguillarum, V. harveyi and/or V. parahaemolyticus, and some showed activity against plant-pathogenic fungi (Alternaria brassicae, Colletotrichum gloeosporioides and/or Fusarium graminearum). Some of the derivatives exhibited antimicrobial MIC values ranging from 0.25 to 4 μg/mL, which were stronger than those of the positive control. Notably, Compounds 3b and 10 showed extremely high selectively against plant-pathogenic fungus F. graminearum (MIC 0.25 μg/mL) and pathogenic bacteria E. coli (MIC 1 μg/mL), implying their potential as antimicrobial agents. SAR analysis of 1 and its derivatives indicated that modification of the carbon-carbon double bond at C-6/7, of groups on the allylic methylene unit and of the carbonyl group at C-1, effectively enhanced the antimicrobial activity.


Introduction
In recent years, there has been increasing interest in research on marine natural products, since an enormous range of chemically diverse biologically active metabolites have been discovered from marine organisms [1][2][3][4]. Recently, marine-derived fungi have triggered our interest, due to their ability to produce structurally interesting bioactive secondary metabolites [5][6][7][8][9].
Herein, we report a short synthesis of (±)-1 featuring a Reformatsky reaction as a key step. This approach has the merits of low cost, mild reaction conditions and easy access to diversity-oriented derivatives for potential structure-activity relationship (SAR) investigation. In antimicrobial assays, penicimonoterpene (±)-1 not only displayed antifungal activity against A. brassicae, but also showed potent antibacterial activity against marine bacteria, including Aeromonas hydrophila, Vibrio harveyi, and V. parahaemolyticus. Therefore, we became interested in designing and synthesizing diverse antibacterial inhibitors using (±)-1 as a model compound. Modifications were focused on variation of the substituents at the eight-position (Section A in Figure 1), the carbon-carbon double bond at C-6/7 (Section B) and carboxyl substituents at C-1 (Section C).
In Route I, selective oxidation of 6-methylhept-5-en-2-one with SeO 2 in CH 2 Cl 2 [16][17][18] yielded allylic alcohol 7, which was converted to methyl ester 3a by a Reformatsky reaction using 2.0 eq of BrCH 2 COOCH 3 in the presence of 2.0 eq of Zn powder in refluxing THF. Methyl ester 3a was then hydrolyzed with KOH to afford key intermediate 5. However, this route was not considered satisfactory, because the yield of the Reformatsky reaction step was only 4%.
In Route II, the Reformatsky reaction of 6-methylhept-5-en-2-one with BrCH 2 COOCH 3 yielded 2a in a good yield (85%). After refluxing with 2.2 eq of KOH in a mixture of MeOHH 2 O (v/v, 3/1), Compound 2a was hydrolyzed in excellent yield (94%) to produce Compound 6, which was subsequently oxidized to give 5 with 11% yield. Unfortunately, Compound 5 proved to be difficult to purify from by-products formed in the reaction, leading to the poor yield obtained for this step. In Route III, treatment of Compound 2a with 3.0 eq of t-BuOOH in the presence of 0.1 eq of SeO 2 afforded a chromatographically separable mixture of aldehyde 4a (25%) and alcohol 3a (29%). The former could be converted into the latter (69%) by reduction with NaBH 4 in MeOH. In an effort to improve the efficiency of this process, we studied the conditions for the selective oxidation of the allylic methyl groups of 2a. A microwave irradiation method using SeO 2 and t-BuOOH adsorbed on SiO 2 [19] also afforded 4a, but in low yield (13%). After optimization by extensive variation of the number of equivalents of SeO 2 and the reaction time, it was found that the best results were obtained with 0.1 eq of SeO 2 , and 3.0 eq t-BuOOH in CH 2 Cl 2 , reacting for 19 h at room temperature, yielded Compound 3a in 29% yield and Compound 4a in 25% yield. The key intermediate, 5, was obtained in 85% yield by saponification of 3a with no protection of the tertiary hydroxyl group. Treatment of intermediate 5 with 1.5 eq of Ac 2 O, a catalytic amount of 4-dimethylaminopyridine (DMAP) and 1.5 eq of Et 3 N at room temperature for 12 h gave 1 in 55% yield.
As expected, based on the selectivity rules and site for the SeO 2 -mediated oxidation reaction [20], as well as the mechanism of this process [21], the Δ 6,7 -double bond in alcohol 3a was found to have the E-configuration on the basis of a NOESY correlation between H-6 (δ 5.39) and H 2 -8 (δ 3.98). The 1 H NMR, 13 C NMR, MS and HRMS data for synthetic (±)-1 were identical to those of reported natural penicimonoterpene [15].

Synthesis of Diverse Derivatives of (±)-1
In order to investigate the influence of structural changes on the antimicrobial activities of 1, a series of derivatives of 1 were synthesized by modification of three main structural features of 1, including the 8-acetoxy group, the C-6/7 double bond and the carboxyl group at C-1. The syntheses of these derivatives are summarized in Scheme II and Table 1.
In an attempt to enhance the antibacterial activities, we envisaged modification at C-8 by introducing electron-withdrawing groups, such as Cl and Br (Scheme II). Chlorine-substituted products 9a and 9b were successfully prepared in 69%-77% yield by using 1.5 eq of NCS (N-chlorosuccinimide) as the chlorinating reagent in the presence of PPh 3 at 0 °C overnight. Unfortunately, NBS (N-bromosuccinimide) treatment failed to give the targeted bromination products, possibly because NBS had a tendency to be more reactive than NCS, leading to the bromine-substituted products being less stable, or because other products were formed.
Hydrogenation of 1, 2a-b, 3a-b, 5, 6, 8a-c, 9a-b, 10 and 18 proceeded with H 2 using 10% Pd/C as the catalyst to give corresponding products, as shown in Table 1. The Pd-catalyzed isomerization of primary allylic alcohols [23], 3a, 3b, and 5, into the corresponding saturated aldehydes have been achieved at room temperature. Another interesting result was that allylic ester groups (e.g., in 8b and 8c) were easily removed by catalytic hydrogenation in addition to the reduction of the olefin unit at room temperature with excellent yields. It is worth noting that ester substrates 1, 8a-c and 10, prepared by acylation of the corresponding alcohols, as well as chlorine-substituted products 9a and 9b were transformed into related methyl products 11, 13, 14 and 15 by hydrogenolysis using 10% Pd/C as the catalyst with good yields (76%-96%), while subjecting the parent allylic alcohols, 3a, 3b and 5, to the same conditions did not lead to the corresponding methyl products. These results indicated that chloro, as well as ester groups, might be as valuable as protecting groups, since they could be easily removed by catalytic hydrogenation in high yields.
In summary, we have efficiently synthesized penicimonoterpene (±)-1 for the first time, as well as 24 racemic derivatives via simple routes and at low cost. Of 24 derivatives, Compounds 3a-b, 4a-b, 8a-c, 9a-b, 10, 14 and 17-20 were new, and these products were fully characterized by NMR and HRESIMS (see Supplementary Information).

Antibacterial Activity and SAR Analysis
Synthetic Compounds 1-20 were tested in vitro for antibacterial activity against two Gram-positive bacteria (Micrococcus luteus and Staphylococcus aureus) and five Gram-negative bacteria, including Escherichia coli and the marine bacteria, Aeromonas hydrophila, Vibrio anguillarum, V. harveyi and V. parahaemolyticus. Many of these derivatives exhibited antibacterial effects. Compounds 13-17 showed significant activity, with MIC values ranging from 0.25 to 64 μg/mL, as listed in Table 2.
Several observations were made regarding structure-activity relationships among these compounds. Reduction of the carbon-carbon double bond at C-6/7, significantly increasing the antibacterial activity (e.g., 13 vs. 2b, 14 vs. 18, 15 vs. 2a, 16 vs. 12, except for Compounds 19 and 20, which contain an aldehyde group). For compounds containing the double bond at C-6/7, the general order of antibacterial potency for substituents at C-1 was COOH > COOCH 2 CH 3 > COOCH 3 > COOCOCH 3 (except in the case of E. coli, for which Compound 10 showed better selective activity). Hydroxylation at C-8 (as in 3a and 3b) resulted in loss of activity.
Notably, hydrogenated Compounds 13-17 and aldehyde 4b showed inhibitory activity to all tested bacterial strains, and in many cases, the activity was stronger than or comparable to the corresponding positive controls (Table 2). For V. parahaemolyticus, Compounds 9b and 12 showed activity similar to that of the positive control (the common antibacterial agent, chloramphenicol). Compound 10 exhibited better selective activity against E. coli with a MIC value of 1 μg/mL, which is 16-fold more potent than that of 1 and two-fold more active than chloramphenicol. Compound 11 exhibited an MIC value of 4 μg/mL against S. aureus, making it more potent than chloramphenicol in the assay (MIC = 8 μg/mL).

Antifungal Activity and SAR Analysis
The inhibitory effects of Compounds 1-20 against three plant-pathogenic fungi are summarized in Table 3. Compound 1 exhibited activity against A. brassicae with an MIC value of 64 μg/mL .  Compounds 4b, 11, 13-15 and 19 showed activity against C. gloeosporioides, and the activity of Compound 14 (MIC = 8 μg/mL) in this assay was twice as potent as that of the positive control (amphotericin B, MIC = 16 μg/mL). In assays against F. graminearum, eight compounds, including 1, 3b, and 11-16, displayed activity, with MIC values ranging from 0.25 to 64 μg/mL. Among them, Compound 3b showed particularly noteworthy activity (MIC = 0.25 μg/mL), as it was 128-fold more potent than amphotericin B (MIC = 32 μg/mL) in this assay. Compound 3b showed extremely high selectively against F. graminearum and might have potential as an antifungal agent. Four other compounds (11)(12)(13)(14)(15) were also more potent (two-to eight-fold) than the positive control. Table 3. Minimum inhibition concentration (MIC) of 1 and its derivatives (μg/mL) against three plant-pathogenic fungi a .

Compounds
A. brassicae C. gloeosporioides F. graminearum Based on the above data, newly synthesized Compounds 3b, 11, 14 and 15 were found to be the most effective, especially against F. graminearum. From an SAR standpoint, it is noteworthy that the oxidation of the methyl to a hydroxymethyl group at C-8 (2b vs. 3b) or replacement of the methyl ester group at C-1 by an ethyl ester (3a vs. 3b) significantly increased the antifungal activity. Compounds with a reduced double bond at C-6/7 (11 and 13-15) also showed better inhibitory activities against C. gloeosporioides and F. graminearum (except for those containing an aldehyde group, such as 17, 19 and 20).

General
Chemicals and instruments: 2-Methyl-2-hepten-6-one was purchased from TCI Company. t-Butyl hydroperoxide (70% in water), BrCH 2 COOCH 3 and BrCH 2 COOCH 2 CH 3 were purchased from J&K Company (Qingdao, China). THF was dried over LiAlH 4 and distilled prior to use. CH 2 Cl 2 was distilled over CaH 2 . The reagents were all analytically or chemically pure. All solvents and liquid reagents were dried by standard methods in advance and distilled before use according to Perrin and Armarego [24]. Thin-layer chromatography (TLC) was performed using silica gel GF-254 plates (Qing-Dao Chemical Company, Qingdao, China) with detection by UV (254 nm). NMR spectra were recorded on Bruker Advance 500 spectrometers with tetramethylsilane (TMS) as an internal standard. Chemical shifts were reported in parts per million (ppm, δ) downfield from tetramethylsilane. Proton coupling patterns were described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m) and broad (br). Mass spectra were recorded by electrospray ionization (ESI) using a VG Autospec 3000 mass spectrometer.
Plant-pathogenic fungi: All strains of fungi were provided by Qindao Agricultural University. The strains were retrieved from a storage tube and incubated in potato dextrose agar media (PDA) at 28 °C for a week to get new mycelia for the antifungal assay.
Bacterial strains: The strains were provided by the Institute of Oceanology, Chinese Academy of Sciences, and cultured at 28 or 37 °C in nutrient agar (NA).

Reduction of Aldehyde 4a to Alcohol 3a
NaBH 4 (117 mg, 3.1 mmol) was added to a stirred solution of aldehyde 4a (840 mg, 3.1 mmol) in MeOH (50 mL). The mixture was stirred for 1 h at room temperature, then concentrated under reduced pressure. The residue thus obtained was treated with EtOAc (20 mL) and saturated NH 4 Cl solution (50 mL); then, the separated aqueous phase was extracted with EtOAc (3 × 30 mL). The combined organic phases were dried (Na 2 SO 4 ), filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (EtOAc-petroleum ether, 1:5) on silica gel to yield a colorless oil, 3a (460 mg, 69%).