Study of Aromatic Derivatization of Carnosic Acid: Synthesis and Antiproliferative Evaluation of Methyl 11,12-Dimethoxy-14-Acetyl Carnosate ^†

Abstract

Over the last decade, plant-derived diterpenes have attracted considerable attention as a rich source of structurally diverse bioactive compounds with significant potential for drug discovery. Among them, abietane-type diterpenes represent one of the most extensively investigated families due to their broad spectrum of biological activities, including antioxidant, antimicrobial, anti-inflammatory, neuroprotective, and antitumor effects. The availability of the chiral natural (+)-carnosic acid at reasonable price from some providers encouraged us to use it as starting material. In this communication, we report the synthesis of methyl 11,12-dimethoxy-14-acetyl carnosate, a novel derivative of the natural product carnosic acid. The target compound was prepared from methyl 11,12-dimethoxycarnosate through a key Friedel–Crafts acylation. The structural characterization was unambiguously established by extensive ¹H, ¹³C, and 2D-NMR analysis. It is interesting to note that the proximity of the acetyl group at the C14 position to the characteristic isopropyl moiety at C13 of the abietane skeleton induced an atypical pattern of spectral signals in ¹H and ¹³C NMR spectra. Preliminary biological evaluation in a panel of tumor cell lines revealed promising antiproliferative activity for this derivative, highlighting its potential as a scaffold for further medicinal chemistry optimization.

1. Introduction

Abietane diterpenoids constitute a major class of naturally occurring terpenes characterized by a tricyclic carbon skeleton. Among them, the group of aromatic abietanes represent a diverse family of plant secondary metabolites, with more than 200 compounds identified to date possessing an aromatic C-ring [1]. These metabolites are widely distributed in plants, particularly within the Lamiaceae, Cupressaceae, and Pinaceae families, where they exhibit remarkable structural diversity resulting from variations in oxidation patterns and functional group substitution. This diversity is closely associated with a broad range of biological activities and ecological functions. Among aromatic abietanes, carnosic acid (CA) (Figure 1) is one of the most abundant and biologically significant members. It is a major natural constituent of rosemary (Rosmarinus Officinalis) and serves as a key biosynthetic precursor of several oxidized diterpenoids, including carnosol, rosmanol, and related derivatives.

Figure 1. Carnosic acid (CA).

The biological relevance of CA is closely related to its unique molecular architecture. The combination of a highly lipophilic abietane skeleton and a redox-active catechol moiety confers a broad spectrum of pharmacological activities, including antioxidant, anti-inflammatory, antimicrobial, antiviral, antiparasitic, neuroprotective, and antiproliferative effects [2]. Owing to its potent antioxidant properties, CA has also been widely exploited in the food, nutraceutical, and cosmetic industries, where it is used as a natural preservative and functional ingredient. The increasing interest in carnosic acid has also been driven by its potential as a versatile scaffold for medicinal chemistry. Accordingly, numerous semisynthetic derivatives have been synthesized to enhance potency, improve selectivity, and optimize pharmacokinetic behavior while providing valuable insights into structure–activity relationships. In particular, modifications involving the C12 hydroxyl group, the C20 carboxylic acid, and other reactive positions have generated promising analogues with improved biological profiles, highlighting carnosic acid as an excellent precursor for the design of new bioactive molecules with potential therapeutic applications.

2. Results and Discussion

2.1. Synthesis

Compound 1 was synthesized from carnosic acid by adapting the literature procedure described by Zhou et al., [3] employing 3.3 equivalents of dimethyl sulfate (Me₂SO₄) and 3.5 equivalents of potassium carbonate (K₂CO₃). Compound 1a was obtained according to the synthetic route shown in Scheme 1, which was adapted from a reported ipso Friedel–Crafts protocol [4]. Although the reaction was initially designed to achieve the targeted ipso Friedel–Crafts substitution on C-13, the desired product was not isolated. Instead, compound 1a was formed under the reaction conditions and was isolated as the main product. Owing to its novel structure, this compound was fully characterized and subsequently evaluated for its antiproliferative activity.

Scheme 1. Synthesis of methyl 11,12-dimethoxy-14-acetyl carnosate (1a) with AlCl₃ and AcCl at 0 °C to room temperature (rt) in DCM.

2.2. Structural Elucidation

Methyl 11,12-dimethoxy-14-acetyl carnosate (1a) was analyzed by ¹H, ¹³C, DEPT135, DEPT90, and 2D-NMR to elucidate its structure (Figure 2, Table 1).

Figure 2. Structural elucidation of compound 1a.

Table 1. Carbon and proton numbering scheme for compound 1a.

NO.	δH (J Hz)	δC
1α	1.16–1.08 m	35.2 b,d
1β	3.34 dt, (13.5, 4.6)
2α	1.55–1.49 m	20.0 b
2β	2.30–2.19 m
3 α	1.33–1.27 m	40.8 b
3β	1.55–1.49 m
4	-	33.5
5	1.44 dd (12.5, 1.4)	53.3
6 α	1.79–1.75 m	17.9 b
6β	2.19–2.09 m
7(α + β)	2.67–2.54 m c	29.1 b,d
8	-	127.1
9	-	135.7
10	-	48.2
11	-	153.0
12	-	150.5
13	-	134.6
14	-	137.5
15	2.64 sept (7.0)	31.3
16 17	1.33–1.27 m	22.0 a 21.7 a
18	0.93	32.5
19	0.75	19.6
20	-	176.3
21	-	209.1
22	2.45 s	33.1
11-OMe	3.58 s	58.8
12-OMe	3.76 s	59.5
20-OMe	3.65 s	51.4

^a Signals may be interchanged. ^b Signal assignments based on HSQC experiments. ^c Overlapping signals. ^d Low-intensity and broadened signals in the ¹³C NMR spectrum.

HMBC analysis: The assignments of the aromatic carbon signals of ring C in the ¹³C NMR spectrum were established based on the observed correlations between H-6α and C8, as well as between H-16/H-17 and C13. The positions of the C11-OMe and C12-OMe substituents were further confirmed by their corresponding ¹H NMR signals and the correlations with the adjacent aromatic carbons. Additional correlations not discussed above are shown in Figure 3.

Figure 3. COSY and HMBC key interactions of compound 1a.

COSY analysis. The ¹H–¹H COSY spectrum established the expected scalar coupling network within ring A. H-1β showed correlations with H-1α, H-2α, and H-2β, while H-2β correlated with H-1α, H-2α, H-3β, and H-1β, confirming the connectivity among H-1, H-2, and H-3. Additional mutual correlations were observed between H-5, H-6, and H-7, supporting the proton connectivity within ring B. The isopropyl methine proton (H-15) displayed strong correlations with both methyl groups (H-16 and H-17), consistent with the expected spin system of the isopropyl substituent.

NOESY analysis. The NOESY spectrum provided key through-space correlations supporting the relative stereochemistry of compound 1a. H-1β showed cross-peaks with H-1α, H-2α, and H-2β, whereas H-1α correlated with H-2α. H-2β exhibited NOE interactions with H-2α and H-1α/H-1β, while H-3α correlated with H-3β. In ring B, H-5 showed correlations with H-18, H-6α, H-6β, and H-7, and H-6β displayed additional NOESY with H-6α and H-7. H-7 was also spatially correlated with H-5 and H-6α. The methyl ketone group (H-22) exhibited NOESY with H-7, H-15, H-16, and H-17 while H-15 shows correlation with both methyl groups (H-16 and H-17). In turn, H-16 and H-17 each showed correlations with MeO-12 and H-15. Finally, H-18 displayed NOESY with H-3α, H-5, H-6α, and H-19, while H-19 correlated with H-2β, H-3β, H-6β, H-18, and H-21. Collectively, COSY, NOESY, and HMBC data are consistent with the proposed structure and absolute stereochemistry of compound 1a.

The ¹³C NMR signals assigned to C1 and C7 in Table 1 exhibited low intensity and broad line shapes. Additionally, the ¹H NMR signals of the isopropyl group exhibited reduced resolution and lower intensity after the introduction of the C14 methyl ketone moiety. These observations could be related to the influence of neighboring functional groups. The signal of C1 may be affected by the adjacent ester moiety, while the broadening observed for C7 could be from the presence of the neighboring methyl ketone group on C14. Additionally, the proximity of the methyl ketone and isopropyl substituents on C13 may generate steric hindrance, limiting rotational freedom around the adjacent C–C bond and thereby contributing to the reduced signal intensity and broadened resonance.

2.3. Antiproliferative Assay

The biological activity of compound 1a was evaluated against six different human cancer cell lines (Table 2). GI₅₀ (Growth Inhibition 50) is the concentration of a compound required to inhibit 50% of net cell growth compared with untreated control cells. Among them, the SW1573 lung cancer cell line exhibited the highest sensitivity. Compared with carnosic acid (GI₅₀ ≈ 20 μM) and standard chemotherapeutics such as etoposide (GI₅₀ ≈ 15 µM) and cisplatin (GI₅₀ ≈ 2.7 µM), compound 1a displayed markedly enhanced antiproliferative activity.

Table 2. GI₅₀ on different cell lines for compound 1a.

Cell Line	GI50 (µM)
A549	3.50 ± 0.42
HeLa	4.00 ± 0.10
MIA PaCa-2	3.20 ± 0.92
SW1573	1.20 ± 0.45
T-47D	6.90 ± 1.90
WiDr	5.50 ± 0.76

3. Materials and Methods

3.1. Materials and Equipment

Carnosic acid (98%) was purchased from BLDPharm (Shanghai, China). Acetyl chloride (98%) and AlCl₃ were obtained from Merck Life Science S.L.U. (St. Louis, MO, USA). TLC was performed on silica gel 60 F254 plates (Merck Life Science S.L.U., St. Louis, MO, USA) and detected with UV light at 254 nm, visualized by immersion in a 10% sulfuric acid solution and heating with a heat gun. Column chromatography was carried out on silica gel 60 (0.040–0.063 mm, Scharlab, S.L., Barcelona, Spain).

Analytical data were obtained using the following instruments: NMR spectra were recorded on a Bruker Ascend 400 (9.4T) NMR spectrometer (Bruker, Billerica, MA, USA). All spectra were recorded in CDCl₃ as solvents. Cole-Parmer MP-800D melting point apparatus (Cole-Parmer, Vernon Hills, IL, USA), AB Sciex QTOF 6600+ mass spectrometer (SCIEX, Framingham, MA, USA) and JASCO P2000 polarimeter (JASCO Inc, Easton, MD, USA).

The antiproliferative activity was assessed against a panel of human cancer cell lines using the standard National Cancer Institute (NCI) sulforhodamine B (SRB) assay [5]. The cell lines used in this study were A549 (lung carcinoma), HeLa (cervical adenocarcinoma), MIA PaCa-2 (pancreatic adenocarcinoma), SW1573 (lung carcinoma), T-47D (breast carcinoma), and WiDr (colorectal adenocarcinoma).

3.2. Synthesis of Methyl 11,12-Dimethoxy-14-Acetyl Carnosate (1a)

To a solution of methyl 11–12 dimethoxycarnosate (1) (954 mg, 2.55 mmol) in anhydrous DCM (10.0 mL) at 0 °C, acetyl chloride (98%, 1.63 mL, 22.92 mmol, 9.0 Eq.) was added dropwise. With vigorous stirring, anhydrous AlCl₃ (2.04 g, 15.28 mmol, 6.0 Eq.) was added in small portions during 5 min. Then, the reaction mixture was gradually allowed to warm to rt and left to react for 3 h. The reaction was quenched with 10% aqueous HCl (12.0 mL) at 0 °C. The reaction mixture was transferred to a separatory funnel and the layers were separated. The aqueous layer was extracted with Et₂O (3 × 20.0 mL), and the combined organic layers were washed with saturated aqueous NaHCO₃ (12.0 mL) and brine (10.0 mL), then dried over Na₂SO₄. The solvent was removed under reduced pressure to afford an orange oil. The crude product was subsequently purified by column chromatography on silica gel (n-hexane/EtOAc, 9:1) to give methyl 11,12-dimethoxy-14-acetyl carnosate (1a) as a yellowish solid (179.3 mg, yield = 17%, 56% based on recovered starting material), along with the recovery of 666.8 mg of unreacted starting material (1). [α]²⁵_D = +108.2° (c 0.37, DCM). ¹H NMR (400 MHz) and ¹³C NMR (100 MHz) (see Table 1). Melting point (m. p.): 150–152 °C, HRMS (ESI) m/z 417.2636 [M + H]⁺, calcd for C₂₅H₃₇O₅: 417.2620; Anal. calcd. for C₂₅H₃₆O₅: C, 71.8; H, 8.7; Found: C, 72.1; H, 8.7.

4. Conclusions

Although the synthetic yield of compound 1a was relatively low, its biological evaluation revealed a marked improvement in antiproliferative activity compared with the starting material, carnosic acid. In particular, 1a exhibited enhanced potency across the tested cancer cell lines, with the strongest activity observed against the SW1573 lung cancer cell line (GI₅₀ ≈ 1.2 μM). These findings suggest that the structural modification successfully increased the biological efficacy of the parent scaffold, highlighting compound 1a as a promising lead for further optimization. Furthermore, the unusual NMR behavior is noteworthy and requires further investigation to fully elucidate the structural and dynamic factors involved.