The synthesis of non-steroidal anti-inflammatory drug (NSAID)–lantadene prodrugs as novel lung adenocarcinoma inhibitors via the inhibition of cyclooxygenase-2 (COX-2), cyclin D1 and TNF-α-induced NF-κB activation

Abstract

The ester conjugates of potent nuclear factor-kappa B (NF-κB) inhibitors with cyclooxygenase (COX) inhibiting non-steroidal anti-inflammatory drugs (NSAIDs) present a novel approach towards cancer treatment. The ester prodrugs of pentacyclic triterpenoid 3β,22β-dihydroxy-olean-12-en-28-oic acid (4) with different NSAIDs were synthesized for the dual inhibition of NF-κB and COX-2. The results indicated that the lead compound 14 suppressed the tumor necrosis factor-alpha-induced (TNF-α-induced) activation of NF-κB by inhibiting the inhibitor of the nuclear factor-kappa B kinase (IKK) activation, the inhibitor of the nuclear factor-kappa B alpha (IκBα) degradation and at the same time, it down-regulated the NF-κB mediated protein expression of COX-2 and cyclin D1. Furthermore, compound 14 inhibited lung adenocarcinoma A549 cell proliferation in a dose dependent manner and was found to be 50 folds more active than cisplatin in terms of IC₅₀. Compound 14 was also found to be stable in an acidic pH, while it hydrolyzed readily in human plasma to release the active promoieties. From the results it can be inferred that the lantadene–NSAID prodrugs are promising anticancer candidates against lung cancer with a dual inhibition capability against both NF-κB and COX-2.

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1. Introduction

Chronic inflammation plays a major role in lung carcinogenesis and there are a number of evidences from preclinical and clinical studies which showed that persistent inflammation can cause normal cells to change into cancerous cells.¹ The inflammation may be involved in several stages of carcinogenesis, from tumor initiation to tumor promotion and even in the metastatic progression through various mechanisms involving genomic instability, epigenetic modifications, localized immunosuppression, and angiogenesis.² NF-κB is one of the important targets of anticancer drugs currently being developed. It regulates an inflammatory response and apoptotic pathways and remains inactive in the cytoplasm because of its complexation with IκBα.^3–6 In response to inflammatory stimuli, IKK phosphorylates IκBα, leading to its degradation and the release of NF-κB from the NF-κB–IκBα complex.^4–6 The free NF-κB then migrates to the nucleus and binds with specific sequences of DNA, thereby regulating the transcription of target genes.^4–6 The expression of the majority genes that are involved in inflammation (e.g. COX-2) or in cellular proliferation (e.g. cyclin D1) are regulated by NF-κB. In response to various external stimuli, such as pro-inflammatory cytokines, bacterial lipopolysaccharides, ultraviolet rays, reactive oxygen species and phorbol esters, COX-2 becomes elevated in certain tissues.^7,8 Abnormally elevated COX-2 causes the promotion of cellular proliferation, the suppression of apoptosis, the enhancement of angiogenesis, and invasiveness, which leads to oncogenesis.⁹ These observations collectively identify the combination therapy of NF-κB and COX-2 inhibitors as a logical and promising therapeutic strategy against various inflammatory diseases and cancer.

In the last decade NSAIDs have been extensively studied for anticancer and chemopreventive activity against various types of cancers.¹⁰ NSAIDs appear to act via the depression of prostaglandin synthesis through the inhibition of COX-2, which results in the suppression of proliferation, possibly through the enhancement of apoptosis.¹¹ Recently, pentacyclic triterpenoids lantadene A (1) and B (2) isolated from the leaves of the weed Lantana camara L. (Verbenaceae) have attracted lots of interest because of their anticancer properties.^12–15 These compounds along with 3β,22β-dihydroxy-olean-12-en-28-oic acid (4) showed potent cytotoxic effects in the antitumor screening launched by the National Cancer Institute, USA¹⁶ and were found to be potent NF-κB inhibitors.^12,15,17 The conjugation of the two molecular entities, each having its own distinct mechanism of action, into a single novel chemical entity, via a prodrug approach, can impart enhanced physicochemical properties or efficacy and may improve the therapeutic index. In this direction, we have synthesized the prodrugs of different NSAIDs with the NF-κB inhibitor 3β,22β-dihydroxy-olean-12-en-28-oic acid (4). The ester prodrugs were evaluated for their ability to suppress the TNF-α induced NF-κB activation, the IκBα degradation, the expression of cyclin D1 and COX-2 and the antiproliferative effect against lung adenocarcinoma A549 cells. Furthermore, we also studied the physicochemical properties of the lead prodrug (14).

2. Results

2.1. Chemistry

The sequence of steps involved in the synthesis of the lantadene–NSAID prodrugs (5–14) are summarized in Scheme 1. The synthesis of the target compounds commenced with the isolation of the pentacyclic triterpenoids lantadene A (1) and lantadene B (2) from the leaves of the weed Lantana camara Linn. Lantadene A (1) and B (2) both share an olean-12-ene-28-oic acid template and structurally differ from each other via a C-22 ester linkage. The 22β-angeloyloxy side chain with E conformation is present in lantadene A (1), while the 22β-senecioyloxy side chain of Z conformation is present in lantadene B (2). The structures of the isolated compounds were established by the combined use of spectroscopy and elemental analyses. Compound 3 was synthesized by the alkali hydrolysis of 1 and 2 using 10% ethanolic potassium hydroxide, whereas compound 4 was synthesized by the selective reduction of the keto function of 3 by using sodium borohydride as a reducing agent and a methanol (MeOH)–tetrahydrofuran (THF) mixture as the solvent. Compounds 5–14 are the ester conjugates of 3β,22β-dihydroxy-olean-12-en-28-oic acid (4) and different NSAIDs, and were synthesized via a two-step process. In step 1, the reaction was carried out at a carboxylic group of the NSAID to convert it into anhydride, by reacting the NSAID with acetyl chloride in the presence of pyridine (ESI: Scheme S1†). In the subsequent step, the anhydride derivatives of the respective NSAIDs were refluxed for 10–14 h in pyridine with compound 4 in the presence of 4-dimethylaminopyridine (4-DMAP) to yield a mixture of 3β-substituted and 3β,22β-disubstituted prodrugs. The mixture of prodrugs was separated by means of column chromatography using silica gel (100–200 mesh) as an adsorbent and varying the ratio of hexane–ethyl acetate as a mobile phase to yield prodrugs 5–14 in pure form.

Scheme 1 The synthesis of the lantadene–NSAID hybrid compounds 5–14. Reagents and conditions: (a) 10% ethanolic KOH, reflux 6 h; (b) NaBH₄, MeOH–THF, stir 7 h; (c) R–CO–O–CO–CH₃, 4-DMAP, pyridine, reflux 92–95 °C, 10–14 h.

2.2. The in vitro inhibition of TNF-α-induced NF-κB activation in the A549 lung adenocarcinoma cell line

The human lung adenocarcinoma A549 cell line was transiently co-transfected with NF-κB-luc and was used to monitor the effects of the lantadene–NSAID prodrugs on TNF-α induced NF-κB activation. The compounds (1–14) were evaluated in a dose-dependent manner to determine the concentration needed to inhibit 50% of TNF-α-induced NF-κB activation (IC₅₀). Compounds 1–3 showed inhibition of TNF-α-induced NF-κB activation in the range of 6.42 to 1.06 μmol, whereas compound 4 showed IC₅₀ > 10 μmol. The conjugation of diclofenac at the C-3 and C-22 positions of 4 led to a marked increase in the activity. The conjugated compounds 13 and 14 showed IC₅₀ values of 0.96 and 0.64 μmol, respectively. The results of the inhibition of TNF-α-induced NF-κB activation in the A549 lung adenocarcinoma cell line by compounds 1–14 are shown in Table 1.

Table 1 The TNF-α-induced NF-κB activation inhibitory activities (IC₅₀ in μmol) of the parent compounds (1–4), prodrugs (5–14) and NSAIDs^a

Compound	IC50	Compound	IC50
a NT is not tested; the results are given as the mean of at least three independent experiments with triplicates in each experiment.
1	1.06 ± 0.46	14	0.64 ± 0.02
2	1.56 ± 0.04	Aspirin	>100
3	6.42 ± 1.24	Naproxen	>100
4	>10	Diclofenac	>100
5	4.20 ± 1.20	Ibuprofen	>100
6	3.92 ± 0.42	Ketoprofen	>100
7	2.60 ± 0.30	Cisplatin	NT
8	1.92 ± 0.62
9	>10
10	>10
11	>10
12	>10
13	0.96 ± 0.06

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2.3. The inhibition of TNF-α-dependent IκBα degradation and IKK activity

The activation of NF-κB requires the phosphorylation and degradation of IκBα, the natural inhibitor of NF-κB.¹⁸ Compound 14, which showed a marked inhibition of TNF-α-induced NF-κB activation in the A549 lung adenocarcinoma cell line, was selected for further study. To determine whether the inhibition of TNF-α-induced NF-κB activation was due to the inhibition of IκBα degradation, the cells were treated with various concentrations of 14 for 8 h and then exposed to 0.1 nmol TNF-α for 30 min. The cell extracts were then examined for IκBα status in the cytoplasm by Western blot analysis. TNF-α-induced the IκBα degradation in the control cells, whereas in the cells pretreated with compound 14, TNF-α failed to induce the degradation of IκBα (Fig. 1a and b). The IκBα degradation requires IKK activation; therefore, we also examined the effect of compound 14 on the TNF-α-induced IKK activity. The cells were treated with 14, followed by the treatment with 1 nmol TNF-α for 5 min. The extracts of the whole-cells were prepared, immune precipitated with anti-IKK-α antibody and then subjected to an IKK kinase assay. The results indicated that TNF-α-activated the IKK, whereas compound 14 inhibited the TNF-α-induced IKK activity (Fig. 1c and d).

Fig. 1 The effect of compound 14 on TNF-α-induced IκBα degradation and IKK activation. The cells were pretreated with various concentrations of compound 14 for 8 h and then exposed to 0.1 nmol TNF-α for 30 min. Western blot analysis was performed using anti-IκBα and anti-β-actin antibodies. (a) Western blot analysis describing the effect of compound 14 on TNF-α-induced IκBα degradation; (b) the densitometry analysis of the Western blots shows the relative intensity of IκBα/β-actin. The results shown are the average ± SEM of three separate experiments. A P value < 0.05 was considered significant. 1 vs. 0 (# not significant), 0.5 TNF-α treated vs. 0 TNF-α treated ns/not significant, 1 TNF-α treated vs. 0 TNF-α treated **P < 0.01; (c) Western blot analysis describing the effect of compound 14 on IKK activation; (d) the densitometry analysis of the Western blots shows the fold increase in the IKK activity The results shown are the average ± SEM of three separate experiments. A P value < 0.05 was considered significant. 1 vs. 0 ns/not significant, 0 TNF-α treated vs. 0 ****P < 0.0001, 1 TNF-α treated vs. 1 ****P < 0.0001, 1 TNF-α treated vs. 0 TNF-α treated ns/not significant.

2.4. The evaluation of the COX-2 activity by the quantitation of PGE₂

The COX-2 inhibitory activity of the parent compounds, NSAIDs and prodrugs (1–14) was determined by measuring PGE_2. The selected NSAIDs (aspirin, ibuprofen, ketoprofen, naproxen, and diclofenac) showed varying degrees of COX-2 inhibition. Aspirin showed the weakest COX-2 inhibition activity with IC₅₀ > 100 μmol, whereas diclofenac showed the most promising COX-2 inhibitory activity with IC₅₀ of 0.038 μmol. The parent compounds (1–4) showed IC₅₀ > 100 μmol, whereas the prodrugs of compounds 3 and 4 with diclofenac (13 and 14) showed IC₅₀ of 1.20 and 0.76 μmol, respectively. The results of the inhibition of COX-2 by compounds 1–14 are shown in Table 2.

Table 2 The COX-2 inhibitory activities (IC₅₀ in μmol) of the parent compounds (1–4), prodrugs (5–14) and NSAIDs^a

Compound	IC50	Compound	IC50
a NT is not tested; the results are given as the mean of at least three independent experiments with triplicates in each experiment.
1	>100	14	0.76 ± 0.06
2	>100	Aspirin	>100
3	>100	Naproxen	28.2 ± 8.24
4	>100	Diclofenac	0.038 ± 0.02
5	>100	Ibuprofen	7.60 ± 2.30
6	>100	Ketoprofen	9.20 ± 3.40
7	7.20 ± 2.20	Cisplatin	NT
8	5.60 ± 2.60
9	9.64 ± 1.64
10	8.42 ± 2.62
11	15.62 ± 4.42
12	12.40 ± 6.20
13	1.20 ± 0.20

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2.5. The inhibition of the TNF-α-induced and NF-κB-regulated protein expressions of COX-2 and cyclin D1

TNF-α induced NF-κB activation is necessary for the initiation of cyclin D1 and COX-2, which possess NF-κB-binding sites in their promoters.^19–21 The suppression of COX-2 is essential for anti-inflammatory effects, whereas the suppression of cyclin D1 is responsible for the antiproliferative properties of the NSAIDs. To determine whether compound 14 inhibited the TNF-α-induced cyclin D1 and COX-2 expression, the cells were pretreated with 14, and were then exposed to TNF-α. The results indicated that TNF-α induced the cyclin D1 expression, while compound 14 blocked the TNF-α-induced expression of this gene product in a dose-dependent manner (Fig. 2a and b). Similarly, TNF-α also up-regulated the COX-2 protein expression, whereas the prodrug 14 down-regulated the TNF-α-induced protein expression of COX-2 in a dose-dependent manner (Fig. 2c and d).

Fig. 2 (a) The effect of compound 14 on TNF-α-induced cyclin D1 expression; (b) the densitometry analysis of the Western blots shows the quantitation of the cyclin D1 levels. The results shown are the average ± SEM of three separate experiments. A P value < 0.05 was considered significant. 1 vs. 0 # not significant, 0 TNF-α treated vs. 0 ***P < 0.001, 0.5 TNF-α treated vs. 0 TNF-α treated ns/not significant, 1.0 TNF-α treated vs. 0 TNF-α treated **P < 0.01; (c) the effect of compound 14 on TNF-α-induced COX-2 expression; (d) the densitometry analysis of the Western blots shows the quantitation of the COX-2 levels. The results shown are the average ± SEM of three separate experiments. A P value < 0.05 was considered significant. 1.0 vs. 0 # not significant, 0 TNF-α treated vs. 0 ****P < 0.0001, 0.5 TNF-α treated vs. 0 TNF-α treated ns/not significant, 1.0 TNF-α treated vs. 0 TNF-α treated ***P < 0.001.

2.6. The inhibition of TNF-α-induced PGE₂ synthesis

The prostaglandin E₂ (PGE₂) is formed via the COX-mediated conversion of arachidonic acid.²² Furthermore, we examined whether the suppression of COX-2 expression by compound 14 correlates with the suppression of PGE₂ synthesis. The mouse macrophages were pretreated with different concentrations of 14 for 4 h, followed by the stimulation with 1 nmol TNF-α for 12 h. Thereafter, culture media were collected and analyzed for PGE₂ secretion. The results showed that TNF-α induced the PGE₂ secretion, while compound 14 suppressed it in a dose-dependent manner (Fig. 3).

Fig. 3 The effect of compound 14 on TNF-α-induced PGE₂ secretion. The results shown are the average ± SEM of three separate experiments. A P value < 0.05 was considered significant. 2.5 vs. 0 ****P < 0.0001, 0.5 TNF-α treated vs. 0 TNF-α treated ****P < 0.0001, 1.0 TNF-α treated vs. 0 TNF-α treated ****P < 0.0001, 2.5 TNF-α treated vs. 0 TNF-α treated ****P < 0.0001.

2.7. The evaluation of the cytotoxic activity

Chronic inflammation of the lungs results in lung carcinoma via the activation of NF-κB which leads to the over-expression of COX-2.²³ Since the lantadene–NSAID prodrugs showed a marked inhibition of NF-κB and COX-2, they were further evaluated for their in vitro cytotoxicity against A549 lung cancer cells. The cytotoxicity profiles of the parent compounds (1–4) and prodrugs (5–14) are reported in Table 3. The parent pentacyclic triterpenoids 1, 2, 3 and 4 showed cytotoxicity against A549 lung cancer cells with IC₅₀ values of 2.84, 1.19, >10, and >10 μmol, respectively. The prodrugs of compound 4 with various NSAIDs at the C-3 and C-22 positions showed marked cytotoxicities (IC_50s) in the range of 6.74 to 0.42 μmol (Table 3). Compounds 13 and 14 showed the most promising cytotoxicity against A549 cells with IC₅₀ values of 0.84 and 0.42 μmol, respectively.

Table 3 The in vitro cytotoxicity profiles (IC₅₀ in μmol) of the parent compounds (1–4), prodrugs (5–14) and NSAIDs against the A549 cell line^a

Compound	IC50	Compound	IC50
a The results are given as the mean of at least three independent experiments with triplicates in each experiment.
1	2.84 ± 0.72	14	0.42 ± 0.02
2	1.19 ± 0.28	Aspirin	>10
3	>10	Naproxen	>10
4	>10	Diclofenac	>10
5	5.64 ± 1.32	Ibuprofen	>10
6	3.40 ± 1.20	Ketoprofen	>10
7	1.74 ± 0.24	Cisplatin	21.3 ± 3.62
8	0.92 ± 0.02
9	6.74 ± 2.24
10	5.60 ± 2.20
11	2.68 ± 0.18
12	1.92 ± 0.02
13	0.84 ± 0.04

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2.8. The chemical hydrolysis of lead prodrug in acidic pH

The ideal prodrug is one that remains stable in the acidic pH of the stomach and should undergo rapid hydrolysis at the site of action to release the active promoieties. To assess the chemical stability of 14, it was exposed to the simulated gastric fluid of pH 2 for 0, 2, 5, 8 and 12 h. The progress of the hydrolysis was monitored by HPLC. The results of the chemical hydrolysis study revealed that only 0.00, 2.39, 6.76, 10.43 and 15.28% of 14 was hydrolyzed after the exposure periods of 0, 2, 5, 8 and 12 h, respectively (Table 4). On account of the results obtained in the chemical stability studies, it can be inferred that the lead prodrug candidate 14 showed an ample resistance towards acidic hydrolysis and survived the stomach pH conditions.

Table 4 Chemical stability of prodrugs (14) in simulated gastric fluid of pH 2

Time	Percentage of prodrug remaining in simulated gastric fluid
	Compound 14
0 h	100
2 h	97.61
5 h	93.24
8 h	89.57
12 h	84.72

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2.9. The enzymatic hydrolysis of the lead prodrug in human plasma

To assess the susceptibility of the lead prodrug 14 towards human blood plasma esterases, it was exposed to 80% human plasma for periods of 0, 15, 30, 60 and 120 min. The level of prodrug that underwent hydrolysis in human plasma was examined by using HPLC. The results of the plasma hydrolysis study indicated that 0.00, 19.13, 34.52, 43.91 and 55.65% of 14 was hydrolyzed after the exposure time of 0, 15, 30, 60 and 120 min, respectively (Table 5). The rate of hydrolysis in 80% human plasma was a significantly higher rate than the rate of hydrolysis observed in the acidic media. Based on the comparison of the HPLC data obtained in both the chemical and metabolic stability studies, it can be inferred that the prodrug 14 was hydrolyzed at a slower rate in the simulated gastric fluid, while in the human blood plasma it underwent rapid hydrolysis to release the parent drug moieties to reach the site of action.

Table 5 Metabolic stability of prodrugs (14) in human plasma

Time	Percentage of prodrug remaining in human plasma
	Compound 14
0 min	100
15 min	80.87
30 min	65.48
60 min	56.09
120 min	44.35

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3. Discussion

A strong relationship has been found between inflammation and cancer, suggesting that inflammation can lead to cancer.²⁴ A wide array of DNA-binding proteins are activated in response to inflammatory stimuli, which can lead to an inappropriate induction of various proinflammatory genes in tumor cells and in the surrounding host tissues. Different transcription factors are abnormally turned on or switched off in various human malignancies and among these, NF-κB has been most extensively investigated because of its ubiquitous presence and multiple functions.²⁵ Nuclear factor NF-κB is an important target that has been shown to mediate inflammation and suppress apoptosis, and it is commonly over-expressed in cancer cells.³ Similarly, COX-2, an inducible form of cyclooxygenase, serves as an interface between inflammation and cancer. The aberrant induction of COX-2 has been implicated in the pathogenesis of various types of malignancies. COX-2 promotes the breakdown of arachidonic acid to produce a series of prostaglandins, which are key mediators of inflammatory responses.⁹ Some pro-inflammatory prostaglandins such as PGE₂ have been reported to play vital roles in carcinogenesis.^26,27

The pentacyclic triterpenoid lantadenes and their congeners showed potent cytotoxic effects in antitumor screening launched by the National Cancer Institute, USA;¹⁶ in particular, against the panel of lung cancer cell lines. In our previous studies, we identified that pentacyclic triterpenoid lantadenes and their congeners showed a marked inhibition of NF-κB in tumors.^12,15,17 Recently, a number of NSAIDs have attracted considerable interest because of their chemopreventive potential against various types of cancer. It has been well reported that the ability of NSAIDs to prevent cancer is due to their potential to inhibit COX-2 and NF-κB. The conjugation of two active chemical entities via a prodrug approach having complimentary pharmacological targets is an attractive way to enhance the physicochemical properties or efficacy and it may also improve the therapeutic index. This study was designed to synthesize and evaluate the pentacyclic triterpenoid lantadene–NSAID conjugates for their potential to inhibit NF-κB activation, NF-κB-regulated protein expressions and cell proliferation events. The results indicated that the lead compound 14 can suppress the TNF-α-induced activation of NF-κB by inhibiting IKK activation and IκBα degradation. Compound 14 also inhibited the NF-κB regulated protein expression of COX-2, which regulates inflammation and cyclin D1, which in turn regulate the proliferation. Compound 14 inhibited the proliferation of lung adenocarcinoma A549 cells in a dose dependent manner. For the development of prodrug as a drug candidate, it is important to have optimum bioavailability; the prodrug should be sufficiently stable chemically and bio-labile to allow conversion into the parent drug molecules, once absorbed. For a better absorption from the stomach, the prodrug should be stable at gastric pH (pH 1–2). To assess these requirements, hydrolysis studies were carried out in HCl buffer at pH 2.0. The hydrolysis rate was monitored and prodrug 14 was found to be 84.72% intact at the end of 12 h. On the other hand prodrug 14 was readily hydrolyzed in 80% human plasma to release the parent drug molecules, which makes it suitable for the optimum delivery of 3β,22β-dihydroxy-olean-12-en-28-oic acid and an NSAID.

4. Conclusion

The present study provides evidence that lantadene–NSAID prodrugs can be promising drug candidates against lung cancer. The lead prodrug 14 showed a marked cytotoxicity against lung adenocarcinoma cells A549 and was found to be 50 folds more active than cisplatin. The lead prodrug 14 exerted its effect by the dual inhibition of NF-κB and COX-2 and warrants further investigations.

5. Experimental

5.1. Materials and general experimental methods

All chemicals and solvents were purchased from Spectrochem, SD fine chemicals limited, Loba Chemie, HiMedia, Finar chemicals, Merck, and Sigma-Aldrich, India. Antibodies to IκBα, IKK-α, COX-2, and cyclin D1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-β-actin antibody was purchased from Sigma Chemicals (St Louis, MO, USA). The progress of the reactions was monitored on Merck TLC plates, silica gel 60 F₂₅₄ (Merck, Germany). For the purification of the compounds, glass columns of appropriate sizes were used. The melting points were determined on the digital melting point apparatus (Indo Sati Scientific Lab Equipments, India) and were uncorrected. The FT-IR spectra of the isolated and synthesized compounds were recorded on a PerkinElmer spectrum 400 FT-IR and FT-NIR spectrophotometer using potassium bromide pellets. NMR spectra of the compounds were recorded with a Bruker AVANCE II 400 NMR spectrometer using CDCl₃, DMSO-d₆, and a mixture of CDCl₃ and DMSO-d₆ as solvents, and the chemical shifts were presented in parts per million (δ). Tetramethylsilane was used as an internal standard in NMR analysis. The ESI-MS spectra of the compounds were recorded on a Waters Micromass Q-T of micro mass spectrometer using electrospray ionization at 70 eV. The elemental analyses of the compounds were carried out with a 2400 CHN analyzer (PerkinElmer, USA). For HPLC analysis, a Waters HPLC system comprised of a Waters 717plus autosampler, Waters 515 HPLC pumps, a Waters Spherisorb ODS2 (80 Å, 5 μm, 4.6 × 250 mm) C18 column, a Waters 2996 PDA detector and the empower software system 2.1 was used.

5.2. Plant materials

Leaves of the weed Lantana camara L. were collected in September 2010 from Palampur (HP), India. The plant material was collected from private land and we confirm that the owner of the land gave their permission for us to collect plant material from his site. We further confirm that the plant material collected was not an endangered or protected species. The leaves were shade-dried and powdered. The plant material was taxonomically identified and authenticated by Dr Sunil Dutta, Scientist, National Medicinal Plant Board, Ayush, New Delhi, India. A voucher specimen (LC; 097 JUIT) had been deposited in the Herbarium of Jaypee University of Information Technology, Waknaghat, India.

5.3. The extraction and isolation of lantadene A (1) and B (2)

The extraction and isolation of lantadene A (1) and B (2) were performed as per our previously reported protocol with slight modifications (ESI: page 5–6†).^28,29

5.4. The synthesis of 22β-hydroxy-3-oxo-olean-12-en-28-oic acid (3)

Compound 3 was synthesized from compounds 1 and 2 as per our previously reported protocol with slight modifications (Scheme 1) (ESI: page 6–7†).^29,30

5.5. The synthesis of 3β,22β-dihydroxy-olean-12-en-28-oic acid (4)

Compound 4 was obtained as per the method of selective reduction of the keto group reported by us previously (ESI: page 7–8†).^17,29 Briefly, the keto group of compound 3 was reduced into the hydroxyl group of 4, employing sodium borohydride as the selective reducing agent and an MeOH–THF mixture as the solvent.

5.6. The synthesis of 3β-substituted and 3β,22β-disubstituted olean-12-en-28-oic acids (5–14)

The synthesis of the 3β-substituted (5, 7, 9, 11 and 13) and 3β,22β-disubstituted (6, 8, 10, 12 and 14) olean-12-en-28-oic acid prodrugs was carried out in two steps. In step 1, the carboxylic function of the NSAIDs was converted into the anhydride function by the base catalyzed reaction of an acid and acyl halide (ESI: Scheme S1†). The NSAIDs with an equimolar amount of acetyl chloride, in the presence of pyridine, were refluxed in dichloromethane (ibuprofen, ketoprofen, and naproxen)/tetrahydrofuran (aspirin and diclofenac) for 4–5 h. The organic solvent was removed in a rotary evaporator and the reaction mixture was washed with chloroform (100 ml × 3) under reduced pressure at 60–65 °C to yield solid to semisolid anhydride products of the respective NSAIDs, which were used in the subsequent step without additional purification.

In step 2, 3β,22β-dihydroxy substituted compound 4 and the anhydride derivatives of the respective NSAIDs, in the presence of 4-DMAP, were refluxed in pyridine for 10–14 h (Scheme 1). At the end of the reaction, the reaction mixture was transferred to the 10% HCl solution and the precipitated product was extracted with dichloromethane and washed for a further three times with a 10% HCl solution (100 ml × 3). The organic layer was removed under reduced pressure and the crude product obtained was chromatographed over silica gel (100–200 mesh) and eluted with varying ratios of hexane–ethyl acetate to yield the final purified products (5–14).

5.6.1. 3β-(2-Acetoxybenzoyloxy)-22β-hydroxy-olean-12-en-28-oic acid (5). Yield: 23.47%, mp: 181–182 °C. Anal. calcd for C₃₉H₅₄O₇ (634.39): %C, 73.78; H, 8.57. Found: %C, 73.82; H, 8.56. IR (KBr, cm⁻¹): 3429, 3355 (O–H), 2990, 2950, 2924, 2876, 2847 (C–H), 1731 (C=O), 1614, 1584 (C=C). ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.82–7.84 (1H, dd, J = 8.00, 1.68 Hz, C-7′-Ar-H), 7.43–7.47 (1H, dt, C-5′-Ar-H), 6.97–6.99 (1H, dd, J = 8.40, 0.8 Hz, C-4′-Ar-H), 6.86–6.90 (1H, dt, C-6′-Ar-H), 5.34–5.36 (1H, t, J = 3.38 Hz, C-12-H), 4.48–4.52 (1H, t, J = 7.92 Hz, C-3-H), 3.90–3.92 (1H, t, J = 2.94 Hz, C-22-H), 2.99–3.03 (1H, dd, J = 13.76, 4.12 Hz, C-18-H), 2.35 (3H, s, C-9′-H), 1.15 (3H, s, CH₃), 1.02 (3H, s, CH₃), 0.95 (3H, s, CH₃), 0.89 (3H, s, CH₃), 0.87 (3H, s, CH₃), 0.86 (3H, s, CH₃), 0.77 (3H, s, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 180.05 (C-28), 171.09 (C-1′), 169.72 (C-8′), 149.63 (C-3′), 142.88 (C-13), 135.51 (C-5′), 129.74 (C-7′), 125.61 (C-6′), 123.92 (C-4′), 123.85 (C-2′), 122.67 (C-12), 80.86 (C-3), 76.15 (C-22), 55.25 (C-5), 50.54 (C-17), 47.52 (C-9), 45.77 (C-19), 41.81 (C-14), 39.24 (C-8), 38.15 (C-18), 38.09 (C-1), 37.68 (C-4), 37.68 (C-21), 36.93 (C-10), 33.69 (C-29), 32.56 (C-7), 30.01 (C-20), 28.02 (C-15), 27.56 (C-23), 26.29 (C-27), 25.86 (C-30), 23.91 (C-16), 23.50 (C-2), 23.44 (C-11), 21.11 (C-9′), 18.10 (C-6), 17.04 (C-26), 16.67 (C-24), 15.49 (C-25). ESI-MS (negative-ion mode, m/z): 634.50 (M⁻), 633.50 (M⁻ − 1).

5.6.2. 3β,22β-Di(2-acetoxybenzoyloxy)-olean-12-en-28-oic acid (6). Yield: 17.19%, mp: 172–173 °C. Anal. calcd for C₄₈H₆₀O₁₀ (796.42): %C, 72.34; H, 7.59. Found: %C, 72.31; H, 7.57. IR (KBr, cm⁻¹): 3358.44 (O–H), 2949.33, 2926.35, 2877.47 (C–H), 1730.16 (C=O), 1614.61 (C=C). ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.75–7.84 (2H, m, C-7′ & C-7′′-Ar-H), 7.43–7.52 (2H, m, C-5′ & C-5′′-Ar-H), 6.86–7.09 (4H, m, C-4′, C-6′ & C-4′′, C-6′′-Ar-H), 5.35–5.37 (1H, t, J = 3.32 Hz, C-12-H), 5.01–5.02 (1H, t, J = 2.86 Hz, C-22-H), 4.48–4.52 (1H, t, J = 7.92 Hz, C-3-H), 2.99–3.03 (1H, dd, J = 13.76, 4.28 Hz, C-18-H), 2.36 (3H, s, C-9′-H), 2.21 (3H, s, C-9′′-H), 1.15 (3H, s, CH₃), 1.02 (3H, s, CH₃), 0.95 (3H, s, CH₃), 0.90 (3H, s, CH₃), 0.87 (3H, s, CH₃), 0.86 (3H, s, CH₃), 0.77 (3H, s, CH₃). ESI-MS (negative-ion mode, m/z): 795.60 (M⁻).

5.6.3. 3β-((RS)-2-(4-Isobutylphenyl)propanoyloxy)-22β-hydroxy-olean-12-en-28-oic acid (7). Yield: 39.49%, mp: 109–111 °C. Anal. calcd for C₄₃H₆₄O₅ (660.48): %C, 78.14; H, 9.76. Found: %C, 78.18; H, 9.74. IR (KBr, cm⁻¹): 2953, 2870 (C–H), 1733, 1713 (C=O). ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.08–7.14 (2H, m, C-5′and C-9′-Ar-H), 6.98–7.02 (2H, m, C-6′ and C-8′-Ar-H), 5.25–5.27 (1H, t, J = 2.86 Hz, C-12-H), 4.33–4.37 (1H, m, C-3-H), 3.85–3.87 (1H, t, J = 2.58 Hz, C-22-H), 3.53–3.62 (1H, m, C-2′-H), 2.85–2.90 (1H, dd, J = 13.64, 3.72 Hz, C-18-H), 2.34–2.37 (2H, m, C-10′-H), 1.18 (3H, s, CH₃), 1.04 (3H, s, CH₃), 0.81 (3H, s, CH₃), 0.80 (6H, s, 2× CH₃), 0.79 (3H, s, CH₃), 0.67 (3H, s, CH₃), 0.62 (3H, s, CH₃), 0.46 (3H, s, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 176.11 (C-28), 172.35 (C-1′), 141.90 (C-13), 139.44 (C-7′), 137.11 (C-4′), 128.06 (C-5′ & C-9′), 126.43 (C-6′ & C-8′), 121.53 (C-12), 79.84 (C-3), 75.56 (C-22), 54.19 (C-5), 49.23 (C-17), 46.44 (C-9), 44.60 (C-19), 44.42 (C-2′), 43.97 (C-10′), 40.83 (C-14), 38.14 (C-8), 37.33 (C-18), 37.02 (C-4), 36.84 (C-1), 36.72 (C-21), 35.84 (C-10), 32.49 (C-29), 31.54 (C-7), 29.18 (C-11′), 28.84 (C-20), 26.51 (C-15), 26.44 (C-23), 24.95 (C-27), 24.65 (C-30), 23.09 (C-16), 22.46 (C-11), 22.37 (C-2), 21.30 (C-12′ & C-13′), 16.83 (C-6), 16.25 (C-3′), 15.81 (C-26), 15.41 (C-24), 14.37 (C-25). ESI-MS (negative-ion mode, m/z): 659.60 (M⁻).

5.6.4. 3β,22β-Di((RS)-2-(4-isobutylphenyl)propanoyloxy)-olean-12-en-28-oic acid (8). Yield: 30.85%, mp: 99–100 °C. Anal. calcd for C₅₆H₈₀O₆ (848.60): %C, 79.20; H, 9.50. Found: %C, 79.23; H, 9.51. IR (KBr, cm⁻¹): 2952.20, 2876.35 (C–H), 1738.17 (C=O), 1613.56, 1585.61 (C=C). ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.04–7.21 (8H, m, C-5′, C-9′, C-6′, C-8′, & C-5′′, C-9′′, C-6′′, C-8′′-Ar-H), 5.32–5.34 (1H, t, J = 3.16 Hz, C-12-H), 5.01–5.02 (1H, t, J = 2.52 Hz, C-22-H), 4.40–4.44 (1H, m, C-3-H), 3.58–3.72 (2H, m, C-2′-H & C-2′′-H), 2.92–2.97 (1H, dd, J = 13.64, 4.12 Hz, C-18-H), 2.41–2.45 (4H, m, C-10′-H & C-10′′-H), 1.11 (3H, s, CH₃), 0.91 (3H, s, CH₃), 0.88 (3H, s, CH₃), 0.88 (3H, s, CH₃), 0.87 (3H, s, CH₃), 0.87 (3H, s, CH₃), 0.86 (3H, s, CH₃), 0.82 (3H, s, CH₃), 0.75 (3H, s, CH₃), 0.68 (3H, s, CH₃), 0.53 (3H, s, CH₃). ESI-MS (negative-ion mode, m/z): 848.80 (M⁻), 847.70 (M⁻ − 1).

5.6.5. 3β-((RS)-2-(3-Benzoylphenyl)propanoyloxy)-22β-hydroxy-olean-12-en-28-oic acid (9). Yield: 31.03%, mp: 110–112 °C. Anal. calcd for C₄₆H₆₀O₆ (708.44): %C, 77.93; H, 8.53. Found: %C, 77.91; H, 8.54. IR (KBr, cm⁻¹): 3479.44 (O–H), 3061.34, 2945.16, 2876.26 (C–H), 1732.10 (C=O), 1658.12 (C=O keto), 1597.28, 1580.34 (C=C). ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.78–7.84 (3H, m, C-12′, C-16′, & C-5′-Ar-H), 7.67–7.69 (1H, m, C-7′-Ar-H), 7.55–7.61 (2H, m, C-14′ & C-9′-Ar-H), 7.42–7.50 (3H, m, C-13′, C-15′, & C-8′-Ar-H), 5.30–5.32 (1H, t, J = 3.26 Hz, C-12-H), 4.45–4.49 (1H, t, J = 8.06 Hz, C-3-H), 3.96–3.97 (1H, t, J = 3.00 Hz, C-22-H), 3.79–3.85 (1H, m, C-2′-H), 2.95–3.00 (1H, dd, J = 13.72, 4.32 Hz, C-18-H), 1.14 (3H, s, CH₃), 0.98 (3H, s, CH₃), 0.94 (3H, s, CH₃), 0.89 (3H, s, CH₃), 0.88 (3H, s, CH₃), 0.78 (3H, s, CH₃), 0.77 (3H, s, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 196.43 (C-10′), 179.12 (C-28), 172.81 (C-1′), 142.99 (C-13), 140.23 (C-6′), 137.91 (C-4′), 137.41 (C-11′), 132.54 (C-9′), 131.62 (C-14′), 130.09 (C-12′ & C-16′), 129.33 (C-5′), 129.24 (C-8′), 128.57 (C-7′), 128.31 (C-13′ & C-15′), 122.54 (C-12), 79.10 (C-3), 77.55 (C-22), 55.23 (C-5), 50.05 (C-17), 47.64 (C-9), 45.41 (C-19), 45.13 (C-2′), 41.94 (C-14), 39.23 (C-8), 38.73 (C-18), 38.46 (C-4), 38.32 (C-1), 37.78 (C-21), 37.02 (C-10), 33.68 (C-29), 32.74 (C-7), 30.05 (C-20), 28.07 (C-15), 27.56 (C-23), 26.12 (C-27), 25.77 (C-30), 24.06 (C-16), 23.47 (C-11), 23.42 (C-2), 18.31 (C-6), 18.17 (C-3′), 17.48 (C-26), 16.90 (C-24), 15.57 (C-25). ESI-MS (negative-ion mode, m/z): 708.30 (M⁻), 707.30 (M⁻ − 1).

5.6.6. 3β,22β-Di((RS)-2-(3-benzoylphenyl)propanoyloxy)-olean-12-en-28-oic acid (10). Yield: 24.86%, mp: 107–108 °C. Anal. calcd for C₆₂H₇₂O₈ (944.52): %C, 78.78; H, 7.68. Found: %C, 78.83; H, 7.70. IR (KBr, cm⁻¹): 3445.45 (O–H), 3061.38, 2946.13, 2875.25 (C–H), 1732.70 (C=O ester), 1660.13 (C=O keto), 1597.31, 1580.37 (C=C). ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.75–7.86 (6H, m, C-12′, C-16′, C-5′, & C-12′′, C-16′′, C-5′′-Ar-H), 7.35–7.69 (12H, m, C-7′, C-14′, C-9′, C-13′, C-15′, C-8′, & C-7′′, C-14′′, C-9′′, C-13′′, C-15′′, C-8′′-Ar-H), 5.36–5.38 (1H, t, J = 3.10 Hz, C-12-H), 5.01–5.02 (1H, t, J = 2.38 Hz, C-22-H), 4.46–4.49 (1H, t, J = 6.80 Hz, C-3-H), 3.68–3.84 (2H, m, C-2′-H & C-2′′-H), 2.96–3.01 (1H, dd, J = 13.84, 3.44 Hz, C-18-H), 1.15 (3H, s, CH₃), 0.96 (3H, s, CH₃), 0.89 (3H, s, CH₃), 0.87 (3H, s, CH₃), 0.80 (3H, s, CH₃), 0.71 (3H, s, CH₃), 0.62 (3H, s, CH₃). ESI-MS (negative-ion mode, m/z): 944.40 (M⁻ − 1), 943.50 (M⁻ − 2).

5.6.7. 3β-((+)-(S)-2-(6-Methoxynaphthalen-2-yl)propanoyloxy)-22β-hydroxy-olean-12-en-28-oic acid (11). Yield: 41.61%, mp: 169–170 °C. Anal. calcd for C₄₄H₆₀O₆ (684.44): %C, 77.16; H, 8.83. Found: %C, 77.21; H, 8.84. IR (KBr, cm⁻¹): 3422 (O–H), 2947, 2874 (C–H), 1733 (C=O), 1634, 1606 (C=C). ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.65–7.70 (3H, m, C-7′, C-12′, & C-5′-Ar-H), 7.40–7.42 (1H, dd, J = 8.52, 1.72 Hz, C-13′-Ar-H), 7.07–7.14 (2H, m, C-8′ & C-10′-Ar-H), 5.31–5.33 (1H, t, J = 3.38 Hz, C-12-H), 4.44–4.48 (1H, t, J = 7.94 Hz, C-3-H), 3.90 (3H, s, C-14′-H), 3.81–3.88 (2H, m, C-22-H & C-2′-H), 2.93–2.98 (1H, dd, J = 13.96, 4.52 Hz, C-18-H), 1.10 (3H, s, CH₃), 0.89 (3H, s, CH₃), 0.83 (3H, s, CH₃), 0.76 (3H, s, CH₃), 0.73 (3H, s, CH₃), 0.70 (3H, s, CH₃), 0.55 (3H, s, CH₃). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 179.94 (C-28), 174.30 (C-1′), 157.68 (C-9′), 142.91 (C-13), 136.00 (C-4′), 133.80 (C-11′), 129.28 (C-7′), 128.28 (C-6′), 127.23 (C-12′), 126.39 (C-5′), 126.11 (C-13′), 122.57 (C-12), 119.05 (C-8′), 105.55 (C-10′), 81.01 (C-3), 76.51 (C-22), 55.28 (C-14′), 55.26 (C-5), 50.60 (C-17), 47.45 (C-9), 45.56 (C-19), 45.16 (C-2′), 41.83 (C-14), 39.19 (C-8), 38.22 (C-18), 38.05 (C-4), 37.84 (C-1), 37.67 (C-21), 36.85 (C-10), 33.49 (C-29), 32.51 (C-7), 29.78 (C-20), 28.07 (C-15), 27.65 (C-23), 27.52 (C-27), 25.75 (C-30), 24.10 (C-16), 23.49 (C-2), 23.40 (C-11), 18.27 (C-6), 18.13 (C-3′), 16.71 (C-26), 16.54 (C-24), 15.40 (C-25). ESI-MS (negative-ion mode, m/z): 684.50 (M⁻), 683.50 (M⁻ − 1).

5.6.8. 3β,22β-Di((+)-(S)-2-(6-methoxynaphthalen-2-yl)propanoyloxy)-olean-12-en-28-oic acid (12). Yield: 32.21%, mp: 135–136 °C. Anal. calcd for C₅₈H₇₂O₈ (896.52): %C, 77.64; H, 8.09. Found: %C, 77.58; H, 8.07. IR (KBr, cm⁻¹): 3437.54 (O–H), 3058.48, 2944.32 (C–H), 1725.29, 1709.28 (C=O), 1633.46, 1606.36 (C=C). ¹H NMR (400 MHz, CDCl₃, δ ppm): 7.55–7.70 (6H, m, C-7′, C-12′, C-5′, & C-7′′, C-12′′, C-5′′-Ar-H), 7.29–7.42 (2H, m, C-13′ & C-13′′-Ar-H), 7.08–7.14 (4H, m, C-8′, C-10′ & C-8′′, C-10′′-Ar-H), 5.27–5.28 (1H, t, J = 3.20 Hz, C-12-H), 4.98–4.99 (1H, t, J = 2.38, C-22-H), 4.44–4.48 (1H, t, J = 7.96 Hz, C-3-H), 3.91 & 3.90 (6H (3H + 3H), singlet each, C-14′-H & C-14′′-H), 3.77–3.86 & 3.63–3.68 (2H (1H + 1H), multiplet each, C-2′-H & C-2′′-H), 2.79–2.84 (1H, dd, J = 13.64, 4.00 Hz, C-18-H), 1.13 (3H, s, CH₃), 0.93 (3H, s, CH₃), 0.80 (3H, s, CH₃), 0.74 (3H, s, CH₃), 0.72 (3H, s, CH₃), 0.69 (3H, s, CH₃), 0.55 (3H, s, CH₃). ESI-MS (negative-ion mode, m/z): 895.60 (M⁻ − 1).

5.6.9. 3β-(2-(2-(2,6-Dichlorophenylamino)phenyl)acetoyloxy)-22β-hydroxy-olean-12-en-28-oic acid (13). Yield: 19.58%, mp: 153–154 °C. Anal. calcd for C₄₄H₅₇Cl₂NO₅ (749.36): %C, 70.38; H, 7.65. Found: %C, 70.33; H, 7.66. IR (KBr, cm⁻¹): 3387.23, 3259.24 (N–H & O–H), 3079.34, 2949.29, 2877.39 (C–H), 1704.37 (C=O), 1574.12 (C=C). ¹H NMR (400 MHz, CDCl₃ + DMSO-d₆ mixture, δ ppm): 9.72 (1H, s, N-9′-H), 7.35–7.37 (2H, d, J = 8.04 Hz, C-12′ & C-14′-Ar-H), 7.10–7.12 (1H, d, J = 8.68 Hz, C-8′-Ar-H), 6.98–7.02 (1H, t, J = 8.02 Hz, C-13′-Ar-H), 6.90–6.95 (1H, t, J = 8.38 Hz, C-6′-Ar-H), 6.73–6.77 (1H, t, J = 7.84 Hz, C-7′-Ar-H), 6.30–6.33 (1H, d, J = 8.56 Hz, C-5′-Ar-H), 5.25–5.27 (1H, t, J = 3.22 Hz, C-12-H), 4.39–4.43 (1H, m, C-3-H), 3.87–3.88 (1H, t, J = 2.76 Hz, C-22-H), 3.81 (1H, s (br), C-22-OH), 3.52 (2H, s, C-2′-H), 2.94–2.98 (1H, dd, J = 13.80, 3.84 Hz, C-18-H), 1.15 (3H, s, CH₃), 1.00 (3H, s, CH₃), 0.93 (3H, s, CH₃), 0.88 (3H, s, CH₃), 0.84 (3H, s, CH₃), 0.84 (3H, s, CH₃), 0.80 (3H, s, CH₃). ¹³C NMR (100 MHz, CDCl₃ + DMSO-d₆ mixture, δ ppm): 175.97 (C-28), 169.81 (C-1′), 143.39 (C-13), 143.14 (C-4′), 138.11 (C-10′), 129.23 (C-8′), 128.98 (C-11′ & C-15′), 128.60 (C-12′ & C-14′), 127.97 (C-6′), 125.57 (C-3′), 123.23 (C-13′), 121.12 (C-12), 119.75 (C-5′), 115.75 (C-7′), 79.97 (C-3), 75.87 (C-22), 54.72 (C-5), 49.51 (C-17), 46.98 (C-9), 45.63 (C-19), 43.71 (C-2′), 41.52 (C-14), 38.79 (C-8), 38.11 (C-18), 37.64 (C-4), 37.39 (C-1), 37.16 (C-21), 36.45 (C-10), 33.42 (C-29), 32.26 (C-7), 29.63 (C-20), 27.69 (C-15), 27.11 (C-23), 26.09 (C-27), 25.41 (C-30), 23.55 (C-16), 23.09 (C-11), 22.92 (C-2), 17.73 (C-6), 16.60 (C-26), 16.42 (C-24), 15.06 (C-25). ESI-MS (negative-ion mode, m/z): 749.20 (M⁻).

5.6.10. 3β,22β-Di(2-(2-(2,6-dichlorophenylamino)phenyl)acetoyloxy)-olean-12-en-28-oic acid (14). Yield: 13.60%, mp: 143–144 °C. Anal. calcd for C₅₈H₆₆Cl₄N₂O₆ (1026.37): %C, 67.70; H, 6.47. Found: %C, 67.64; H, 6.49. IR (KBr, cm⁻¹): 3355.18 (N–H/O–H), 2990.30, 2950.24, 2925.26, 2877.35 (C–H), 1729.11 (C=O), 1604.43 (C=C). ¹H NMR (400 MHz, CDCl₃ + DMSO-d₆ mixture, δ ppm): 7.31–7.35 (4H, m, C-12′, C-14′ & C-12′′, C-14′′-Ar-H), 7.11–7.17 (2H, m, C-8′ & C-8′′-Ar-H), 6.88–7.04 (4H, m, C-13′, C-6′ & C-13′′, C-6′′-Ar-H), 6.71–6.79 (2H, m, C-7′ & C-7′′-Ar-H), 6.33–6.37 (2H, m, C-5′ & C-5′′-Ar-H), 5.27–5.29 (1H, t, J = 3.24 Hz, C-12-H), 4.91–4.92 (1H, t, J = 2.78 Hz, C-22-H), 4.40–4.44 (1H, t, J = 8.00 Hz, C-3-H), 3.56 & 3.51 (4H (2H + 2H), singlet each, C-2′-H & C-2′′-H), 2.96–3.01 (1H, dd, J = 13.80, 3.96 Hz, C-18-H), 1.15 (3H, s, CH₃), 1.01 (3H, s, CH₃), 0.93 (3H, s, CH₃), 0.88 (3H, s, CH₃), 0.85 (3H, s, CH₃), 0.84 (3H, s, CH₃), 0.81 (3H, s, CH₃). ¹³C NMR (100 MHz, CDCl₃ + DMSO-d₆ mixture, δ ppm): 176.70, 172.82, 171.94, 143.07, 142.70, 142.32, 138.10, 137.04, 130.92, 130.52, 129.33, 129.02, 128.75, 128.58, 127.83, 127.50, 124.53, 124.34, 123.89, 123.51, 122.61, 121.20, 119.76, 117.00, 115.87, 80.11, 75.93, 54.77, 49.59, 47.02, 45.86, 45.60, 43.58, 41.49, 38.78, 38.07, 37.42, 37.41, 37.15, 36.43, 33.41, 32.22, 29.60, 27.65, 27.12, 26.02, 25.40, 23.53, 23.04, 22.89, 17.71, 16.53, 16.33, 15.03. ESI-MS (m/z): 1025.21 (M⁻ − 1).

5.7. In vitro cell culturing and the cytotoxicity assay

The A549 lung cancer cell line was procured from the American Type Culture Collection (ATCC, USA) and was grown in RPMI medium supplemented with 5% fetal bovine serum and 1% penicillin–streptomycin (Gibco, Invitrogen, USA). The A549 cells (4000 cells per well) were seeded into 96-well plates and incubated overnight for cell attachment. For treatment, the compounds were added at concentrations ranging from 0.01 to 100 μmol and incubated for 48 h. At the end of the incubation, 20 μl per well of 5 mg ml⁻¹ 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Amresco, USA) was added and the cells were further incubated for 4 h. The supernatant was discarded and the purple formazan complex formed was dissolved using 100 μl of DMSO (Fisher Scientific, UK). The absorbance was read at 570 nm using a Spectra Max M4 microplate reader (Molecular Devices Inc., USA).

5.8. Cell viability assay

Cells were seeded at 10 × 10³ cells per well, 100 μl, in 96-well plates, 24 h before experimental treatments. The cells were treated with compounds in different concentrations in triplicates and incubated for 48 h. The cell viability was measured by measuring the metabolic conversion (by viable cells) of the dye MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] from a Cell Titer 96 AQueous One Solution Cell Proliferation Assay kit (Promega, Southampton, United Kingdom). In each well of a 96-well plate, 20 μl of MTS was added, and the plates were incubated for 4 h in a cell culture incubator. The MTS assay results were read in a 96-well format plate reader by measuring the absorbance at 490 nm.

5.9. The in vitro inhibition of TNF-α-induced NF-κB activation in A549 lung cells

The A549 cells were cultured in 12-well plates and transiently co-transfected with 0.2 μg of a pNF-κB-Luc vector (Stratagene, La Jolla, CA) and 0.2 μg of the pSV-β-galactosidase dissolved in 3 μl Lipofectamine™ or Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA) as the internal control. The plasmids were transfected according to the manufacturer's instructions. After 6 h, the medium was changed and the cells were cultured for an additional 6 h. The cells were then treated with TNF-α (15 ng ml⁻¹) and the test compounds simultaneously for 8 h. The A549 cells treated with TNF-α alone served as positive controls, while the cells without TNF-α treatment served as negative controls. The luciferase activities from these cells were then measured by using the Bright-Glo Luciferase Assay kit from Promega (Madison, WI), following the manufacturer's protocol. The relative NF-κB activities of the cells treated with the test compounds were obtained as the ratio of its luciferase activity to that from the positive controls, both of which had been corrected with background (signals from negative controls) and cell viability. In these experimental conditions, none of the test compounds induced significant toxicity to the A549 cells (<5% reduction of cell viability). The IC₅₀ of each fraction was determined by fitting the relative NF-κB activity to the drug concentration by using a sigmoidal dose–response model of varied slope in GraphPad Prism 6.0. The IC₅₀ reported herein is the average of at least three replicates.

5.10. Western blotting

The cytoplasmic protein (30–35 mg) or whole-cell extract was prepared as described previously³¹ and resolved by employing SDS-polyacrylamide gel electrophoresis (PAGE).³¹ Then, the proteins were electrotransferred to a nitrocellulose membrane, blocked with 5% nonfat dry milk and probed with primary antibodies against IκBα, cyclin D1 and COX-2 for 2 h at 40 °C. The blotting membrane was washed, exposed to horseradish peroxidase-conjugated secondary antibodies for 1 h and the blots were finally detected by chemiluminescence.

5.11. IKK assay

The IKK assay was performed as per the method described previously.³² Briefly, IKK complexes from whole-cell extracts were precipitated with antibody against IKK-α, followed by treatment with protein A/G-Sepharose beads (Pierce, Rockford, USA). After 2 h of incubation, the beads were washed with lysis buffer and then assayed in a kinase assay mixture containing 50 mmol HEPES (pH 7.4), 20 mmol MgCl₂, 2 mmol DTT, 20 μCi [γ-³²P] ATP, 10 mmol unlabeled ATP and 2 mg of substrate GST-IκBα. After incubation at 30 °C for 30 min, the reaction was terminated by boiling with SDS sample buffer for 5 min. Finally, the protein was resolved on 10% SDS-PAGE, the gel was dried, and the radioactive bands were visualized by PhosphorImager. To determine the total amounts of IKK-α and IKK-β in each sample, 50 mg of the whole-cell protein was resolved on 7.5% SDS-PAGE, electrotransferred to a nitrocellulose membrane, and then blotted with either anti-IKK-α or anti-IKK-β antibodies.

5.12. The evaluation of COX-2 activity by the quantitation of PGE₂

The effect of the test compounds on the COX activity was determined by measuring PGE₂ production as described previously.³³ Briefly, the reaction mixtures were prepared in 100 mmol Tris–HCl buffer (pH 8.0) containing 1 μmol heme, 500 μmol phenol, 300 μmol epinephrine, sufficient amounts of COX-2 to generate 150 ng of PGE₂ per ml and various concentrations of test compounds. The reaction was initiated by the addition of arachidonic acid (final concentration, 10 μmol) and incubated for 10 min at room temperature (final volume 200 μl). The reaction was then terminated by adding 20 μl of the reaction mixture to 180 μl of 27.8 μmol indomethacin, and PGE₂ was quantitated by an ELISA method. The samples were diluted to the desired concentration with 100 mmol potassium phosphate buffer (pH 7.4) containing 2.34% NaCl, 0.1% bovine serum albumin, 0.01% sodium azide, and 0.9 mmol Na₄EDTA. Following transfer to a 96-well plate (Nunc-Immuno Plate Maxisorp, Fisher Scientific, Pittsburgh, PA) coated with a goat anti-mouse IgG (Jackson Immuno Research Laboratories, West Grove, PA), the tracer (PGE₂-acetylcholinesterase, Cayman Chemical, Ann Arbor, MI) and primary antibody (mouse anti-PGE₂, Monsanto, St. Louis, MO) were added. The plates were then incubated overnight at room temperature, the reaction mixtures were removed, and the wells were washed with a solution of 10 mmol potassium phosphate buffer (pH 7.4) containing 0.01% sodium azide and 0.05% Tween 20. Ellman's reagent (200 μl) was added to each well and the plate was incubated at 37 °C for 3–5 h, until the control wells yielded an optical density of 0.5–1.0 at 412 nm. A standard curve with PGE₂ (Cayman Chemical, Ann Arbor, MI) was generated on the same plate, which was used to quantify the PGE₂ levels produced in the presence of the test samples. The results were expressed as a percentage, relative to the control (solvent-treated) samples, and dose–response curves were constructed for the determination of the IC₅₀ values. The IC₅₀ values were generated from the results of four serial dilutions of test compounds and are the mean of three different experiments.

5.13. PGE₂ secretion

The effect of compound 14 on the PGE₂ secretion was studied. The murine macrophage cell line RAW 264.7 was procured from the American Type Culture Collection (ATCC, USA). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; GIBCO, Inc., NY, USA) supplemented with 100 U ml⁻¹ of penicillin, 100 μg ml⁻¹ of streptomycin and 10% fetal bovine serum (FBS; GIBCO, Inc., NY, USA). The cells were incubated in an atmosphere of 5% CO₂ at 37 °C and were subcultured every 3 days. One million murine macrophage cells were seeded into a six-well plate, pretreated with the different concentrations of compound 14 for 4 h, then stimulated with 1 nmol TNF-α for 12 h. The culture media were collected and the concentration of PGE₂ was determined using a PGE₂ ELISA kit purchased from R&D Systems (Minneapolis, MN, USA).

5.14. HPLC analysis

The reversed-phase high-performance liquid chromatography (HPLC) was used for the analysis of chemical and metabolic stability, and the chromatographic purity of compounds. The isocratic solvent systems comprising of methanol–acetonitrile–water–acetic acid (68 : 20 : 12 : 0.01) and methanol–acetonitrile–water–acetic acid (68 : 22 : 10 : 0.01) were used as a mobile phase. The mobile phase constitutes for HPLC analysis were mixed (v/v) and filtered through a 0.45 μm Millipore membrane filter. The injection volume was 10 μl and the flow-rate was kept at 1 ml min⁻¹. The peak areas showed a good reproducibility with a relative standard deviation of 0.5%.

5.15. The in vitro chemical stability of the lead prodrug 14 in simulated gastric fluid

The lead prodrug 14 was assessed for its stability against chemical hydrolysis in a simulated gastric fluid of pH 2. The reaction was initiated by mixing 200 μl stock solution of compound 14 (5 mg ml⁻¹ in THF) to 1.80 ml of the HCl buffer of pH 2 in a screw-capped glass vial. The reaction mixture was incubated at 37 °C in a water bath and samples (200 μl) were withdrawn at a specific time intervals, diluted with 800 μl ACN, and analyzed by using HPLC. The extent of the prodrug that remained in the unhydrolyzed form was calculated as: % remaining = (peak area at the respective time (min)/peak area at 0 min) × 100.

5.16. The in vitro metabolic stability of the lead prodrug 14 in human plasma

The lead prodrug 14 was further evaluated against enzymatic hydrolysis in 80% human plasma of pH 7.4. To assess the hydrolysis, a 50 μl solution of compound 14 (5 mg ml⁻¹ in THF) was added to 450 μl of the diluted plasma and the solution was incubated in a water bath at 37 °C. The samples (50 μl) were withdrawn at specific time intervals, diluted with 1950 μl of ACN, and were subjected to centrifugation for 5 min at 7000 rpm. The supernatant obtained on centrifugation was analyzed by using HPLC. The level of prodrug that remained in the unhydrolyzed form was determined as: % remaining = (peak area at the respective time (min)/peak area at 0 min) × 100.

Statistical analysis

The results are expressed as the mean of at least three values and were analyzed by one way ANOVA followed by Tukey's multiple comparisons test using GraphPad Prism 6.0. The statistical significance was set at the P < 0.05 level.

Conflict of interest

The author(s) confirm that this article content has no conflict of interest.

Abbreviations

TNF-α	Tumor necrosis factor-alpha
NF-κB	Nuclear factor-kappa B
IKK	Inhibitor of nuclear factor-kappa B kinase
IκBα	Inhibitor of nuclear factor-kappa B alpha
COX-2	Cyclooxygenase-2
PGE2	Prostaglandin E2
NSAIDs	Non-steroidal anti-inflammatory drugs
4-DMAP	4-Dimethylaminopyridine
DCC	N,N′-Dicyclohexylcarbodiimide
HPLC	High-performance liquid chromatography
MTT	3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide

Acknowledgements

The authors are thankful to the Department of Science and Technology, Government of India for providing financial assistance to this research work. We are also thankful to Sophisticated Analytical Instrumentation Facility (SAIF), Panjab University, Chandigarh, India for spectral analyses.

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Footnote

† Electronic supplementary information (ESI) available: the isolation and synthesis protocols, the spectral data of compounds 1–4, the synthetic scheme of anhydrides, FT-IR, ¹H NMR, ¹³C NMR, mass spectra, and HPLC purity chromatograms of all compounds (1–14) are provided (ESI Fig. 1–66). See DOI: 10.1039/c4ra00280f

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