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. Author manuscript; available in PMC: 2015 Dec 7.
Published in final edited form as: Bioorg Med Chem Lett. 2011 Jun 17;21(15):4397–4399. doi: 10.1016/j.bmcl.2011.06.044

Nothospondin, a new AP-1 inhibitory quassinoid from the Cameroonian plant Nothospondias staudtii

Thushara Diyabalanage a, Ranjala Ratnayake a, Jennifer A Wilson a, Curtis J Henrich a,b, John A Beutler a, Nancy H Colburn c, James B McMahon a, Kirk R Gustafson a,*
PMCID: PMC4671370  NIHMSID: NIHMS260111  PMID: 21733691

Abstract

A high throughput screen for inhibitors of the oncogenic transcription factor activator protein-1 (AP-1) was applied to the NCI repository of natural product extracts. The liphophilic extract of the plant Nothospondias staudtii (Simaroubaceae) displayed significant AP-1 inhibition. Bioassay-guided fractionation of the extract lead to a new quassinoid named nothospondin (1), and the known compound glaucarubinone (2). The structure of 1 was elucidated by spectroscopic methods. Compounds 1 and 2 showed potent, dose-dependent AP-1 inhibition at noncytotoxic concentrations.

Keywords: Quassinoid, Nothospondias staudtii, Simaroubaceae, Transcription factor, AP-1

Oncogenic transcription factors help regulate the transcription of numerous genes associated with cell growth and cell cycle progression, and an increase in their transcriptional activity is required to support the transformation and unrestrained growth of cancer cells. Activator protein-1 (AP-1) is a transcription factor that resides within the nucleus and regulates various cellular processes including differentiation, proliferation, and apoptosis.1 AP-1 is activated by signal transduction through the mitogen activated protein kinase (MAPK) cascade that can be induced by extracellular stimuli such as growth factors or cytokines.2 Carcinogenesis is a multi-step process comprised of tumor initiation, promotion, and progression. Activation of AP-1 is required for tumor promotion, and elevated AP-1 activity is believed to play a pivotal role in tumor progression.3 Since the rate limiting stages of carcinogenesis occur during tumor promotion and progression, small molecule inhibitors of AP-1 function could serve as potential therapeutic agents. AP-1 exists as a dimer of Jun proteins (c-Jun, JunB, JunD) or as a heterodimer of Jun and Fos proteins, and dimerization is required for activation and DNA binding.4 Inhibition of AP-1 by the dominant negative c-Jun mutant TAM67 prevented cellular transformation but it did not significantly diminish in vitro cell survival, and it effectively blocked tumorigenesis in vivo.5 AP-1 inhibitors that can mimic the effect of TAM67 could have utility for the prevention and treatment of cancer. As part of an ongoing molecularly targeted discovery effort, extracts from the NCI Natural Products Repository were screened in a high throughput assay for AP-1 inhibitors.6 The lipophilic extract of the tropical plant Nothospondias staudtii (Simaroubaceae),7,8 collected in Cameroon, showed potent inhibition of AP-1 activity. Bioactivity-directed fractionation of this extract afforded a new quassinoid,9 nothospondin A (1), and glaucarubinone (2).10

Nothospondin (1, Fig. 1, Table 1)11 provided a [M+H]+ ion at m/z 393.1927 in the HREIMS consistent with a molecular formula of C21H28O7. Analysis of the 13C and gHSQC NMR spectra revealed that the 21 carbon resonances consisted of 4 methyls, 2 methylenes, 7 methines, 7 quaternary carbons, and one methoxyl group. An oxymethine doublet assigned to H-2 (δH 4.71) showed HMBC correlations with a ketone at C-1 (δC 209.1) and an oxymethine at C-3 (δC 83.7), and it had a 8.7 Hz coupling to H-3 (δH 2.93) that indicated a diaxial orientation for these two protons. Assignment of the H3-18 doublet (δH 1.04) was based on COSY coupling to H-4 and HMBC correlations with C-3, C-4 (δC 34.7), and C-5 (δC 41.6). Substitution of H3-19 (δH 1.46) on C-10 was established by HMBC correlations with C-1, C-5, and C-10 (δC 48.1). These data and COSY correlations between H-3/H-4 (δH 1.96) and H-4/H-5 (δH 1.30) were consistent with the structure proposed for the A ring. Assignment of ring B was facilitated by the HMBC correlations of H-9 (δH 3.15) with C-8 (δC 36.9), C-10, C-17 (δC 23.2), and C-19 (δC 15.6). In addition, HMBC correlations of H-7 (δH 4.29) with C-5 and C-9 (δC 47.5) and COSY correlations between H-5/H-6b (δH 2.10) and H-6b/H-7 unambiguously established the connectivity from C-5 through C-7. Ring C was revealed by a carbonyl at δC 190.8 (C-11) consistent with a conjugated ketone and HMBC correlations from H-9 to C-11, from H-14 (δH 2.42) to C-12 (δC 148.4), C-13 (δC 140.5), and C-20 (δC 16.0), and from the methoxyl group (δH 3.67) to C-12. Finally, the presence of a six-membered lactone ring that encompassed an ester carbonyl at C-16 (δC 169.0) linked to the oxygen at C-7 was deduced from the low field shift of H-7 (δH 4.29) and HMBC correlations from H-15a (δH 2.55) to C-13, C-14 (δC 47.3), and C-16, and from H-15b (δH 2.97) to C-8, C-14, and C-16. The relative stereochemistry of 1 was established from a series of selective 1D ROESY experiments. Irradiation of H3-19 produced ROESY enhancements in H-2, H-4, H-6a (δH 1.82) and H3-17 (δH 1.20) that indicated these groups are located at the top (β) face of the molecule (Fig. 2). In addition, H3-17 had ROESY interactions with H-7 and H-14 that established these protons as β as well. Substituents on the bottom (α) face of 1 were defined by ROESY interactions between H3-18/H-3, H3-18/H-6b and H-5/H-9. These data allowed the structural and relative configurational assignment of nothospondin (1) as a new tetracyclic quassinoid. The identity of compound 2 was established by comparison of its 1H and 13C data with published values for glaucarubinone.10

Figure 1.

Figure 1

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Structure of nothospondin (1) and glaucarubinone (2).

Table 1.

NMR data (CDCl3) for nothospondin (1)a

No. δ C δH (J in Hz) gHMBC
1 209.1
2 76.9 4.71 d (8.7) 1, 3, 19
3 83.7 2.93 dd (10.1, 8.7) 2, 4, 18
4 34.7 1.96 m 3, 5, 18
5 41.6 1.30 m 19
6a 25.7 1.82 ddd (14.8, 12.4, 2.1) 5, 10
6b 2.10 dt (14.8, 3.6) 4, 5, 8, 10
7 82.1 4.29 brt (2.9) 5, 9
8 36.9
9 47.5 3.15 s 1, 7, 8, 10, 11, 17, 18
10 48.1
11 190.8
12 148.4
13 140.5
14 47.3 2.42 dd (12.1, 7.0) 8, 9, 12, 13, 17, 20
15a 31.7 2.55 dd (18.6, 12.1) 12, 13, 14
15b 2.97 dd (18.6, 7.0) 8, 9, 16
16 169.0
17 23.2 1.20 s 7, 8, 9, 14
18 14.9 1.04 d (6.5) 3, 4, 5
19 15.6 1.46 s 1, 5, 9, 10
20 16.0 1.89 s 12, 13, 14
OCH3 60.0 3.67 s 12
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a

Data were acquired with a Bruker Avance 111 600 MHz spectrometer; chemical shifts are in ppm and referenced to the residual solvent signal; J in Hz; HMBC correlations are from the proton(s) stated to the indicated carbon.

Figure 2.

Figure 2

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Key ROESY correlations for nothospondin (1).

The AP-1 inhibitory activity of compounds 1 and 2 was determined by a β-lactamase driven reporter assay using fluorescence resonance energy transfer (FRET) technology, followed by an XTT assay to test for cytotoxicity.12 Glaucarubinone (2) showed the most potent AP-1 inhibition with an EC50 of 0.13 μM and it was noncytotoxic at a high-test concentration of 80 μM. Nothospondin (1) was less potent against AP-1 (EC50 1.49 μM) and it showed some cytotoxicity (IC50 approximately 10 μM). The potent AP-1 inhibitory activity of 2, which has an ether bridge between C-17 and C-11, was consistent with a prior structure–activity study in which all of the AP-1 active quassinoids had C-17/C-11 or C-17/C-13 ether bridges.13 Nothospondin (1) is the first quassinoid without an ether link that can inhibit AP-1, albeit at significantly reduced potency.

Acknowledgments

We thank D. Newman (NCI) and T. McCloud (SAIC-Frederick) for the plant extract and M. Dyba and S. Terasova (Biophysics Resource, SBL, NCI-Frederick) for assistance with the HRLCMS studies. This research was supported in part by the Intramural Research Program of NIH, NCI, CCR. This project was also funded in part with Federal funds from the NCI, NIH, under Contract HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government.

References and notes

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