Modular assembly of cytotoxic acetogenin mimetics by click linkage with nitrogen functionalities

Chan Mao; Bing Han; Li-Shun Wang; Shaozhong Wang; Zhu-Jun Yao

doi:10.1039/C1MD00108F

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DOI: 10.1039/C1MD00108F (Concise Article) Med. Chem. Commun., 2011, 2, 918-922

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Modular assembly of cytotoxic acetogenin mimetics by click linkage with nitrogen functionalities†

Chan Mao ^a, Bing Han ^b, Li-Shun Wang *^b, Shaozhong Wang ^a and Zhu-Jun Yao *^a
^aState Key Laboratory of Coordination Chemistry, Nanjing National Laboratory of Microstructures, Institute of Chemical Biology and Drug Innovation, School of Chemistry and Chemical Engineering, Nanjing University, 22 Hankou Road, Nanjing, 210093, China. E-mail: yaoz@nju.edu.cn; Fax: +86-25-83593732; Tel: +86-25-83583732
^bKey Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, EISU Chemical Biology Division, 280 South Chongqing Road, Shanghai, 200025, China. E-mail: jywangls@shusmu.edu.cn

Received 23rd April 2011 , Accepted 10th June 2011

First published on 6th July 2011

Abstract

Linear annonaceous acetogenin mimetic AA005 has been found to exhibit potent antitumor activity and significant selectivity between human normal and cancerous cells. Utilizing the concept of Click chemistry, a convergent modular fragment-assembly approach has been newly developed and successfully applied to the synthesis of three representative AA005-like molecules via formation of small nitrogen-containing heterocycles or amide bond. These nitrogen-containing analogues were found to exhibit low micromolar inhibitory activities against the growth of several cancer cell lines.

Introduction

Annonaceous acetogenins have been attracting worldwide attention for more than 20 years because of their potent bioactivities, especially as potent growth inhibitors of certain tumor cells^1a–1f and environmental neurotoxins.^1g Mechanism studies mentioned that this family of plant polyketides might target Complex I of mitochondria during their actions,¹ and binding of calcium was proposed as a putative mechanism for cytotoxicity.^1f In our previous studies,² a bullatacin (an annonaceous acetogenin) mimicking compound AA005 has been successfully developed and found to exhibit potent antitumor activity and significant selectivity between human normal and cancerous cells (Fig. 1).^2b,2c,2h Click chemistry is a modern chemical philosophy introduced by Sharpless in 2001, describing the tailored chemistry to generate substances quickly and reliably by joining small units together,³ and it has been widely applied in organic synthesis and drug discovery. Considering the linear character of the AA005 skeleton, we wish to explore the application of Click chemistry to the development of new anticancer acetogenins mimetics. Practically, generation of a proper small heterocycle or amide bond (serving as the linking moiety) in the middle region of AA005-like molecules using two pre-functionalized fragments would provide a new access to this class of anticancer compounds convergently. Unlike our previous exploration by altering the activity-essential ether region of AA005 with nitrogenated functionalities,^2j this work chose a non-functionalized middle region (hydrocarbon chain) for the modular linkage. Such a proposal is also different from our previous synthetic protocol, in which epoxide-opening by acetylene was used as the major C–C bond formation manner in the synthesis of AA005 and its analogues.^1d,2,4


	Fig. 1 Three newly proposed AA005-like molecules 1–3.

Our previous works also have mentioned that both the terminal unsaturated γ-lactone and the middle ethylene glycol ether moiety are essential functionalities for the bioactivity of AA005-like analogues.² The hydrocarbon chain between these two moieties is capable of diverse modifications. In drug studies, introduction of nitrogen atom has been commonly adopted and it will not increase any additional stereogenic center(s) into the products. Advantages of nitrogen-functionality introduction also include providing an additional element of diversity into acetogenin mimetics and its potential capability serving as new harbor to host other functional groups or molecular tools in biological studies. In order to explore these considerations, three new representative AA005-like molecules with different nitrogen-containing moieties in the middle hydrocarbon region were thus designed (Fig. 1). In one of these representative compounds (Fig. 1, 3), we introduced a piperidine moiety, which has been widely applied in pharmaceutical compounds to increase the oral bioavailability.

Retrosynthetic analysis of these AA005-like derivatives is illustrated in Fig. 2. The typical mimetic structure can be broken down into two pre-functionalized fragments. Each of them can be synthesized individually starting from common intermediates 8 and 12, which are equipped with the biologically essential functionalities, unsaturated lactone and glycol ether, respectively. By varying the combination, these three analogues of AA005 could be synthesized by modular assembly of the pre-functionalized fragments in convergent fashions, using Huisgen [1,3]-dipolar cycloaddition, amide bond formation and reductive amination under proper conditions, respectively.


	Fig. 2 Retrosynthesis of AA005-like molecules 1–3.

Results and discussion

To examine the above proposed modular approach two major fragments are crucial for the syntheses of targets 1–3. The first segment 11, which was previously used by us in the synthesis of AA005,^2h was synthesized from diol 4 accordingly (Scheme 1). Selective protection of the secondary hydroxyl of 4 afforded methoxymethyl (MOM) ether 5,^2c whose remaining primary hydroxyl was further converted into the mesylate 6. Formation of ethylene glycol ether was accomplished by successive O-alkylations with mesylate 6 and (R)-(-)-epichlorohydrin in the presence of phase transfer catalyst. Epoxide opening of the resulting epoxide 10 with monotrimethylsilyl acetylene lithium followed by deprotections of TMS and MOM provided the first segment, the terminal acetylene 11.


	Scheme 1 Reagents and conditions: (a) i) HC(OCH₃)₃, (D)-CSA, CH₂Cl₂, rt, 2 h; ii) 1M Dibal-H in toluene, CH₂Cl₂, −78 °C to rt, 91% over 2 steps; (b) MsCl, Et₃N, CH₂Cl₂, 0 °C to rt, 3 h, 93%; (c) ethylene glycol, NaH, DMF, 130 °C, 10 h, 77%; (d) 50% NaOH, Bu₄NHSO₄, (R)-epichlorohydrin, rt, 8 h, 81%; (e) i) trimethylsilyacetylene, n-BuLi, BF₃·Et₂O, then 10, THF, −78 °C, 2 h; ii) MOMCl, iPr₂NEt, CH₂Cl₂, 0 °C to rt, 5 h; iii) TBAF, THF, 0 °C, 3 h, 65% over 3 steps; (f) i) 1M BH₃·THF in THF, 0 °C, 3 h; ii) 15% NaOH, 30% H₂O₂, rt, 2.5 h, 60% over 2 steps; (g) Et₃N, MsCl, CH₂Cl₂, 0 °C to rt, 3.5 h, 90%; (h) NaN₃, ethanol, 80 °C, 12 h, 86%.

The other terminal azide 15 was synthesized from a previously used lactone 12.⁴Alcohol 13 was prepared in good chemo- and regioselectivity from olefin-lactone 12 by reaction with 1 M BH₃·THF complex in THF at 0 °C followed by oxidation with H₂O₂ in the presence of aqueous NaOH.⁵ The resulting primary alcohol 13 was then converted into the corresponding azide 15 in a two-step fashion, viamesylate 14, in good overall yield.^6,7

With the above two fragments in hand, modular generation of AA005-like molecule 1 was performed under the standard Sharpless Click conditions. The terminal alkyne 16 (obtained by deprotection of 11) was treated with azide 15 (1.1 equiv.) at room temperature in the presence of catalytic amount of copper sulfate and sodium ascorbate in THF and water.⁸ The biphasic reactions were completed after 12 h, affording the corresponding triazole analogue 1 in 68% isolated yield by simple extraction (Scheme 2).


	Scheme 2 Reagents and conditions: (a) HCl, CH₃OH, rt, 12 h, 88%; (b) CuSO₄·5H₂O (10 mol%), sodium ascorbate (20 mol%), THF/H₂O (1/1), rt, 12 h, 68%.

Synthesis of the amide analogue 2 also began with the terminal alkyne 16 and the azide 15 (Scheme 3). Protection of diol 16 with Ac₂O followed by oxidative cleavage of the terminal alkyne functionality afforded the corresponding acid 18.⁹ In a parallel reaction, azide 15 was reduced to the corresponding amine 19 with triphenylphosphine in THF.¹⁰ Coupling reaction of acid 18 with amine 19 was accomplished under EDCI conditions.¹¹ Final removal of two acetates from the resulting amide provided the second target analogue 2.


	Scheme 3 Reagents and conditions: (a) Ac₂O, pyridine, DMAP, CH₂Cl₂, 0 °C to rt, 4 h, 91%; (b) KMnO₄, HOAc, THF/H₂O, 0 °C to rt, 8 h, 71%; (c) Ph₃P, THF, rt, 20 h, 68%; (d) 18, Et₃N, EDCI, HOBT, DMF, rt, 24 h, 60%; (e) NH₂NH₂·H₂O, CH₃OH, 8 h, 81%.

To synthesize the third piperidine analogue 3, 4-(bromomethyl)cyclopent-1-ene (23) was firstly prepared from the commercially available 3-cyclopentene-1-carboxylic acid¹²via the bromination of tosylate 22 with lithium bromide in dry acetone (Scheme 4). Treatment of the commonly used epoxide 10 with freshly prepared (cyclopent-3-enylmethyl)magnesium bromide (in diethyl ether) provided alcohol 25 in the presence of CuI.¹³ Protection of alcohol 25 with MOMCl followed by oxidative double-bond cleavage with Lemieux–Johnson conditions¹⁴ provided an unstable dialdehyde. Immediate reductive amination of the resulting crude dialdehyde with amine 19 followed by full deprotection of MOM ethers afforded the piperidine analogue 3.


	Scheme 4 Reagents and conditions: (a) LiAlH₄, THF, 0 °C to rt, 6h, 80%; (b) TsCl, Pyridine, DMAP, CH₂Cl₂, 0 °C to rt, 84%; (c) Acetone, LiBr, reflux, 74%; (d) 23, CuI, Et₂O, −40 °C, 85%; (e) iPr₂NEt, MOMCl, DCM, 0 °C to rt, 88%; (f) NaIO₄, cat. K₂OsO₄, rt; (g) i) 19, NaBH₃CN, AcOH, CH₂Cl₂, rt; ii) HCl, CH₃OH, rt, 8h, 26% over three steps.

After completion of the synthesis of all three newly proposed AA005 analogues 1–3, we evaluated their in vitro cytotoxicities using AA005 as the positive control. All four compounds inhibited cell growth in a concentration-dependent manner for all tested cell lines, which were determined by the growth inhibition analysis with Cell Counting Kit-8 assay (Fig. 3 and Fig. 4).^15,16 Compared with the growth inhibition between leukemic cell lines and adherent cell lines, the inhibitory effect of four compounds was more significant in the former ones, especially in APL cell line NB4 cells. Among the adherent cell lines, MCF-7 and BT-474 cell lines were less sensitive to all tested compounds. In general, AA005 and the piperidine analogue 3 are more potent than other two analogues 1 and 2 in all the tested cell lines.


	Fig. 3 Leukemic cells of U937, NB4, K562, Kasumi-1, HL-60 and Jurkat were treated with annonaceous acetogenin mimic AA005 (A0), new analogues 1 (A1), 2 (A2) and 3 (A3) as indicated concentration for 48 h. Each column represents the mean + S.D. of triplicates in an independent experiment.


	Fig. 4 Adherent cells of breast cancer cell lines MCF-7, MDA-MB-435S, BT-549, BT-474, SK-BR-3 and colorectal adenocarcinoma cell line SW620 were treated with annonaceous acetogenin mimic AA005 (A0), new analogues 1 (A1), 2 (A2) and 3 (A3) as indicated concentration for 48 h. Each column represents the mean + S.D. of triplicates in an independent experiment.

At 48 h of treatment, 48.6 ± 0.8% growth inhibition of NB4 cells was achieved by the treatment of 0.2 μM of AA005, while only 6.2 ± 0.8% inhibition was achieved with 0.2 μM of 3. Under higher concentrations (5 μM and 25 μM), both AA005 and 3 resulted in more than 70% inhibition (Fig. 3). Similar results were observed in other leukemic cell lines and some breast cancer cell lines, such as BT-549. IC₅₀ of the tested compounds was calculated according to the growth inhibition by analysis with Cell Counting Kit-8 assay and summarized in Table 1.

Table 1 IC₅₀ of annonaceous acetogenin mimic AA005 and new analogues 1–3.^a

Cancer cell lines	IC₅₀ (μM)
Cancer cell lines	AA005	1	2	3
a IC₅₀ of annonaceous acetogenin mimic AA005 and new AA005-like analogues 1–3 in leukemic cells, breast cancer cells and colorectal adenocarcinomic cells were calculated based on the growth inhibition determined by CCK-8 assay.
U937	1.2	>25	5.2	4.4
NB4	0.7	3.2	4.5	1.9
K562	2.7	8.5	15	3.1
Kasumi-1	1.1	17.8	16.7	3.9
HL-60	>25	>25	7.4	>25
Jurkat	2.6	>25	14	1.2
MCF-7	>25	>25	>25	>25
MDA-MB-435S	>25	>25	>25	5
BT-549	3.3	>25	>25	2.2
BT-474	>25	>25	>25	>25
SK-BR-3	22.1	>25	>25	8
SW620	>25	>25	>25	8.1

Conclusion

In summary, a modular assembly protocol for convergent synthesis of linear annonaceous acetogenin mimetics has been developed and successfully applied to the synthesis of three representative analogues of AA005. Nitrogen functionality was employed as the linkage in this new methodology under the concept of Click chemistry. Two individual fragments bearing pre-equipped terminals and biofunctionally essential moieties successfully served as the common intermediates in the synthesis. Biological evaluation of these newly synthesized analogues indicates that embedment of nitrogen functionality to the middle region of AA005 does not significantly affect the bioactivity. The piperidine-containing analogue 3 exhibited low micromolar inhibitory activities against the growth of several cancer cell lines. The newly developed methodology in this study may potentially be applicable to the synthesis of additional diverse analogues of this family, as well as other more complex bioactive compounds.

Acknowledgements

Financial supports from NSFC (Nos. 21032002, 81071668), Ministry of Health (Nos. 2009ZX09103-101, 2009ZX09501), 973 Program (No. 2010CB833200) and the Fundamental Research Funds for the Central Universities (No. 1082020502) are greatly appreciated.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Experimental details and characterizations of new compounds, NMR copies of new compounds, and experimental procedure of biological screenings. See DOI: 10.1039/c1md00108f

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