1,2,3-Triazoles derived from olanzapine: their synthesis via an ultrasound assisted CuAAC method and evaluation as inhibitors of PDE4B

Abstract

The antipsychotic drug olanzapine which does not inhibit PDE4 can be converted into the inhibitor of PDE4B via linking its N-10 position with an appropriately N-substituted 1,2,3-triazole moiety through a methylene linker. All these compounds were conveniently prepared by a CuAAC method under ultrasound irradiation at room temperature and evaluated for their PDE4 inhibitory potential in vitro. Three of them were identified as selective inhibitors of PDE4B (IC₅₀ ∼ 5–6 μM) over PDE4D. Overall, the present research reports one of the few examples of an ultrasound assisted CuAAC method used in medicinal chemistry.

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Characterized by abnormalities in the perception of reality,¹ schizophrenia is a complex neuropsychiatric disorder that affects approximately 1% of the adult population worldwide.^2,3 The treatment of most schizophrenic patients generally includes the use of antipsychotic medications that are classified as conventional (typical) and novel (atypical) neuroleptic drugs. These drugs work via suppressing dopamine and occasionally serotonin receptor activities. Among the commonly used antipsychotics,⁴ a dibenzepine based antipsychotic drug e.g. clozapine (A, Fig. 1) (which belongs to the atypical class) has been found to be superior over other drugs. Studies have indicated that its beneficial effects were due to its binding to the D-2, D-4, 5-HT_2A and 5-HT_2C along with D-1, α-1, α-2, M-1 and H-1 receptors.⁵ Another drug olanzapine (zyprexa) (B, Fig. 1) that belongs to the thienobenzodiazepine class and structurally similar to clozapine has also been developed and marketed for the treatment of schizophrenia and bipolar disorder.⁶ In spite of usefulness of these currently available antipsychotic medications in treating schizophrenia several drugs have shown some limitations and therefore adoption of alternative drug strategies is necessary. Recently, studies have indicated that members of the phosphodiesterase (PDE) gene family may play a role in the treatment of schizophrenia.^7a For example the antagonism (blockade) of D2 dopamine receptors caused by the antipsychotic drugs result an increase level of cAMP, an effect also caused by the inhibition of PDEs, a family of enzymes that degrade cyclic nucleotides. Indeed, another study has shown that the disrupted in schizophrenia 1 (DISC1) gene and PDE4B are interacting genetic factors in schizophrenia that regulate cAMP signaling.^7b Thus, linkage between dopamine D2 receptors and PDE activity via cAMP is the basis for a possible therapeutic potential for PDE inhibitors in schizophrenia.^7c Notably, among the four subtypes (e.g. PDE4A, B, C and D) only PDE4B is expressed in the locus coeruleus region in the brain that is rich in nonadrenergic neurons and mediates some anti-depressant effects.^7d The other subtype PDE4D is expressed in the area postrema and nucleus of the solitary tract^7e,f that are responsible for emesis. Thus development of PDE4B selective inhibitors can be beneficial for treating the positive, negative or cognitive symptoms associated with schizophrenia.

Fig. 1 Clozapine (A) and olanzapine (B).

Since the atypical antipsychotics including olanzapine (B, Fig. 1) have been used as a first line therapy to treat schizophrenia, a strategy based on structural modifications of olanzapine has been explored by us for the identification of new PDE4 inhibitors (C, Fig. 2) earlier.⁸ In further continuation of this research we became interested in the synthesis and evaluation of olanzapine based 1,2,3-triazole derivatives (D, Fig. 2) as a new class of PDE4B selective inhibitors. Our focus on 1,2,3-triazole moiety was aided by the fact that various 1,2,3-triazole derivatives have been reported to inhibit PDE4B earlier.⁹ While a diverse class of compounds has been explored for the discovery of novel PDE4 inhibitors^10,11 the use of 1,2,3-triazole-olanzapine based template D for the identification of new inhibitors is not known.

Fig. 2 Design of olanzapine (B) based new PDE4 inhibitors (D) via reported inhibitors C.

Generally, Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) is considered as the most effective and convenient tool for accessing 1,4-disubstituted 1,2,3-triazole derivatives.¹² The required Cu(I) catalyst is usually generated in situ via the reduction of a Cu(II) salt with sodium ascorbate. Since ultrasound mediated reactions cause a significant reduction in energy consumption thereby fulfilling green chemistry requirements¹³ hence ultrasound promoted CuAAC protocols have attracted considerable interest.¹⁴ Moreover, these reactions are remarkably faster than the conventional methods. Thus a variety of compounds have been synthesized using these non-classical reaction conditions.^14b Due to our interest in CuAAC approaches¹⁵ and ultrasound assisted methodologies¹⁶ we adopted a ultrasound promoted CuAAC strategy for the synthesis of our target compounds based on D or 3 (Scheme 1) via the reaction of alkyne 1 with azide 2. While the CuAAC strategy has been widely used in various areas of science¹² (e.g. bioconjugation, oligonucleotide synthesis, construction of bolaamphiphilic structures, DNA labeling, and drug discovery) only few reports are available on the use of ultrasound assisted CuAAC in medicinal chemistry/drug discovery.^14d,e

Scheme 1 Synthesis of new olanzapine based 1,2,3-triazole derivatives via CuAAC method.

The required terminal alkyne 1 was prepared via the reaction of olanzapine (B) with propargyl bromide in the presence of NaH in THF.¹⁷ Some limited studies were performed in order to establish the effective and general reaction conditions for the synthesis of compound 3. Thus, the alkyne 1 was reacted with (azidomethyl)benzene (2a) initially under conventional conditions (entry 1, Table 1) when the desired product^18a 3a was obtained after 90 min. The reaction was performed at 80 °C. In order to shorten the reaction time as well as to use a milder condition the reaction was then performed at room temperature (25 °C) under ultrasound irradiation using a laboratory ultrasonic bath SONOREX SUPER RK 510H model producing irradiation of 35 kHz. The product 3a was obtained in relatively lower yields after 5 and 15 min but in good yield after 20 min (entries 2, 3 & 4, Table 1). Reactions were appeared to be cleaner in these cases. All these reactions were performed in MeCN. The use of other solvents such as 1,4-dioxane, DMF and aqueous DMF (1 : 9) was also examined and found to be effective (entries 5–7, Table 1). However, yields of 3a were decreased in these cases. While N,N-diisopropylethylamine (DIPEA) was used as a base in all these reactions its role was also examined. Indeed, a slower reaction was observed in the absence of DIPEA (entry 8, Table 1). Overall, the condition of entry 4 of Table 1 was chosen for the preparation of other analogues of 3a.

Table 1 Effect of reaction conditions on coupling of terminal alkyne 1 with 2a^a

Entry	Solvent	Temp (°C)	Time (min)	Yield^b (%)
a All the reactions were carried out using alkyne 1 (1.0 mmol), azide 2a (1.2 mmol), CuI (10 mol%) and DIPEA (2.0 mmol) under ultrasound irradiation (35 kHz) at room temperature.b Isolated yield.c The reaction was carried out in the absence of ultrasound irradiation.d The reaction was carried out in the absence of DIPEA.
1	MeCN	80	90	65^c
2	MeCN	25	5	19
3	MeCN	25	15	60
4	MeCN	25	20	89
5	1,4-Dioxane	25	35	77
6	DMF	25	40	70
7	H2O : DMF (1 : 9)	25	40	67
8	MeCN	25	180	65^d

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Using the optimized conditions we then prepared a library of compounds (Table 2, see also Table S-1 in ESI†). A number of organic azides (2) were coupled with alkyne 1 to give the desired products 3 in acceptable to good yield. Both alkyl (2a–g & 2p–r) and aryl azides (2h–o) were employed in the present reaction to give a variety of desired products.^18b The benzene ring present in these azides may carry a electron donating or withdrawing group or may contains mono or disubstitutions. The reaction proceeded well in all these cases irrespective of nature of substituent(s) present in azides employed. Based on the fact that catalysts are generally activated by ultrasound a probable mechanism can be proposed^12b (Scheme 2) involving the pre-activation of the CuI by forming a copper–acetylene complex (E-1) in the presence of DIPEA and ultrasound. The E-1 then generates a copper-azide–acetylide complex (E-2) that undergoes cyclization (via E-3) followed by protonation (via E-4) to give 3. While the overall rate of the reaction was enhanced by the ultrasound it appeared that ultrasound might have played a key role in some particular steps.¹⁹

Table 2 Synthesis of olanzapine based 1,2,3-triazole derivatives (3) via CuAAC method^a (see also Table S-1 in ESI)

Entry	Azide (2); R=	Product (3)	Time (min)	Yield^b (%)
a All the reactions were carried out using alkyne 1 (1.0 mmol), azide 2 (1.2 mmol), CuI (10 mol%) and DIPEA (2.0 mmol) under ultrasound irradiation (35 kHz) at room temperature.b Isolated yield.
1	2a; –CH2Ph	3a	20	89
2	2b; –CH2C6H4Cl-p	3b	25	83
3	2c; –CH2C6H4OMe-p	3c	20	85
4	2d; –CH2C6H4NO2-m	3d	25	77
5	2e; –CH2C6H4Cl-o	3e	30	79
6	2f; –CH2C6H3(Cl-o)(CF3-p)	3f	35	73
7	2g; –CH2C6H3(OMe)2-m,p	3g	30	84
8	2h; –Ph	3h	25	78
9	2i; –C6H4Me-p	3i	25	81
10	2j; –C6H4F-p	3j	30	70
11	2k; –C6H4Br-p	3k	30	68
12	2l; –C6H4OMe-p	3l	30	82
13	2m; –C6H4NO2-p	3m	30	72
14	2n; –C6H4CN-p	3n	35	78
15	2o; –C6H4Me2-o,p	3o	35	69
16	2p; –CH2CH=CHPh	3p	30	74
17	2q; –CH2CO2Et	3q	25	81
18	2r; –(CH2)3CO2Et	3r	30	87

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Scheme 2 Proposed reaction mechanism.

The PDE4B inhibitory potential of all the synthesized compounds were assessed in vitro at 10 μM by using a PDE4B enzyme assay²⁰ (Table 3 and Fig. 3). Rolipram²¹ a well known PDE4 inhibitor was used as a reference compound in this assay. Compounds that showed inhibition greater than 50% were considered as reasonable inhibitors of PDE4B.

Table 3 Inhibition of PDE4B by compound 3 at 10 μM^a

Entry	Compounds (3); R=	Average % inhibition	SD
a SD = standard deviation.
1	3a; –CH2Ph	72.82	6.3
2	3b; –CH2C6H4Cl-p	55.41	1.9
3	3c; –CH2C6H4OMe-p	18.56	17.0
4	3d; –CH2C6H4NO2-m	34.38	17.3
5	3e; –CH2C6H4Cl-o	61.17	2.5
6	3f; –CH2C6H3(Cl-o)(CF3-p)	75.37	3.9
7	3g; –CH2C6H3(OMe)2-m,p	66.36	1.5
8	3h; –Ph	64.33	4.2
9	3i; –C6H4Me-p	55.92	11.2
10	3j; –C6H4F-p	74.83	5.1
11	3k; –C6H4Br-p	54.29	8.2
12	3l; –C6H4OMe-p	60.07	7.2
13	3m; –C6H4NO2-p	62.48	3.6
14	3n; –C6H4CN-p	73.31	14.6
15	3o; –C6H4Me2-o,p	45.45	5.8
16	3p; –CH2CH=CHPh	65.04	5.8
17	3q; –CH2CO2Et	43.68	12.8
18	3r; –(CH2)3CO2Et	61	15.5
19	Rolipram	78.05	1.0

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Fig. 3 Representation of PDE4B inhibition of by compound 3 at 10 μM.

Several compounds such as 3a, 3b, 3e, 3f, 3g, 3h, 3j, 3l, 3m, 3n and 3p showed more than 50% inhibition of PDE4B and compounds 3a, 3f and 3j were identified as superior among them. While rolipram showed ∼78% inhibition the parent compound olanzapine however did not show any PDE4 inhibitory properties when tested at concentrations up to 30 μM. Thus a non PDE4 inhibitor olanzapine has been converted into PDE4 inhibitors via linking its N-10 position with an appropriately N-substituted 1,2,3-triazole moiety through a methylene linker. Moreover, 3a, 3f and 3j showed 42–44% inhibition of PDE4D compared to rolipram's >90% inhibition when tested at 10 μM indicating their selectivity towards PDE4B over D. Though the SAR (Structure Activity Relationship) was not clearly understood in case of N-benzyl series (3a–g) it appeared that the unsubstituted benzene ring (e.g. 3a) was more effective than the monosubstituted one (e.g. 3b–e). However, presence of two substituents on the benzene ring (3f and 3g) was found to be almost equally effective like 3a. Indeed, 3f was emerged as an initial hit molecule. In case of N-aryl series (3h–o) the SAR appeared to be better understandable. For example, no group (3h) or a fluoro substituent (3j) at the para-position of the benzene ring was found to be beneficial for PDE4B inhibition. Other groups such as Me, Br, OMe, and NO₂ were moderately effective and the corresponding compounds showed some inhibition of PDE4B. The presence of CN (3n) however appeared to be more effective whereas a disubstitution on the benzene ring (3o) was less effective. Among the compounds (3p–r) having a linear chain at their N-10 position, the compound 3p showed moderate activity whereas others inhibited PDE4B but were less attractive. In a dose response study the compound 3a, 3f and 3j showed inhibitory activities across all the dose tested (Fig. 4) with IC₅₀ values 5.01, 6.23 and 5.60 μM respectively.

Fig. 4 Dose dependent inhibition of PDE4B by 3a, 3f and 3j.

In conclusion, the antipsychotic drug olanzapine that does not inhibit PDE4 has been converted into the inhibitor of PDE4B via linking its N-10 position with an appropriately N-substituted 1,2,3-triazole moiety through a methylene linker. All these compounds were conveniently prepared by CuAAC method under ultrasound irradiation at room temperature in good yields. The use of ultrasound allowed a cleaner and quicker access to these compounds than the conventional thermal process. These compounds were evaluated for their PDE4 inhibitory potential in vitro. Three of them showed selectivity towards PDE4B over PDE4D with IC₅₀ in the range of 5–6 μM for PDE4B inhibition. Overall, the present research reports one of the few examples of ultrasound assisted CuAAC method used in medicinal chemistry. Moreover, our efforts on exploration of olanzapine derivatives for the discovery of new PDE4 inhibitors may be beneficial in finding the potential cure of positive, negative or cognitive symptoms associated with schizophrenia.

Acknowledgements

The authors thank management of DRILS and DBT (Grant BT/PR4286/BRB/10/1012/2011) for support. SBN thanks DBT, India for a Research Fellowship.

Notes and references

1. C. Andreasen, Positive and Negative Symptoms: Historical and Conceptual Aspects, in Schizophrenia: Positive and Negative Symptoms and Syndromes, ed. C. Andreasen and A. G. Karger, Iowa city, 1990; vol. 24, pp. 1–42.
2. D. B. Alison and D. E. Casey, J. Clin. Psychiatry, 2001, 62, 22.
3. P. A. Arguello and J. A. Gogos, Drug Discovery Today: Dis. Models, 2006, 3, 319.
4. J. M. Kane, Psycpharmacologic Approaches to the treatment of Schizophrenia: Practical Aspect in Advances in the Neurobiology of Schizophrenia, ed. J. A. D. Boer, H. G. M. westenberg and H. M. V. Praag, John Wiley & Sons, Chichester, 1995, vol. 1, pp. 245–263.
5. C. R. Ashby and R. Y. Wang, Synapse, 1996, 24, 349.
6. F. P. Bymaster, D. L. Nelson, N. W. Delapp, J. F. Falcone, K. Eckols, L. L. Truex and M. M. Foreman, Schizophr. Res., 1999, 37, 107.
7. (a) J. A. Siuciak, CNS Drugs, 2008, 22, 983,  DOI:10.2165/0023210-200822120-00002; (b) J. K. Millar, B. S. Pickard, S. Mackie, R. James, S. Christie, S. R. Buchanan, M. P. Malloy, J. E. Chubb, E. Huston, G. S. Baillie, P. A. Thomson, E. V. Hill, N. J. Brandon, J. C. Rain, L. M. Camargo, P. J. Whiting, M. D. Houslay, D. H. Blackwood, W. J. Muir and D. J. Porteous, Science, 2005, 310, 1187; (c) T. B. Halene and S. J. Siegel, J. Pharmacol. Exp. Ther., 2008, 326, 230; (d) J. A. Cherry and R. L. Davis, J. Comp. Neurol., 1999, 407, 287; (e) P. S. Torres, X. Miro, J. M. Palacios, R. Cortes, P. Puigdomenech and G. Mengod, J. Chem. Neuroanat., 2000, 20, 349; (f) S. Lamontagne, E. Meadows, P. Luk, D. Normandin, E. Muise, L. Bou-let, D. J. Pon, A. Robichaud, G. S. Robertson, K. M. Metters and F. Nantel, Brain Res., 2001, 920, 84.
8. For our earlier effort in this area, see: (a) D. R. Gorja, S. Mukherjee, C. L. T. Meda, G. S. Deora, K. L. Kumar, A. Jain, G. H. Chaudhari, K. S. Chennubhotla, R. K. Banote, P. Kulkarni, K. V. L. Parsa, K. Mukkanti and M. Pal, Org. Biomol. Chem., 2013, 11, 2075; (b) P. V. Babu, D. R. Gorja, C. L. T. Meda, G. S. Deora, S. K. Kolli, K. V. L. Parsa, K. Mukkanti and M. Pal, Tetrahedron Lett., 2014, 55, 3176.
9. (a) K. S. S. Praveena, S. Durgaprasad, N. S. Babu, A. Akkenapally, C. G. Kumar, G. S. Deora, N. Y. S. Murthy, K. Mukkanti and S. Pal, Bioorg. Chem., 2014, 53, 8; (b) J. Mareddy, S. B. Nallapati, J. Anireddy, Y. P. Devi, L. N. Mangamoori, R. Kapavarapu and S. Pal, Bioorg. Med. Chem. Lett., 2013, 23, 6721.
10. For an overview, see: A. Kodimuthali, S. Jabaris and M. Pal, J. Med. Chem., 2008, 51, 5471.
11. For our earlier effort on PDE4 inhibitors, see: (a) K. S. Kumar, P. M. Kumar, K. A. Kumar, M. Sreenivasulu, A. A. Jafar, D. Rambabu, G. R. Krishna, C. M. Reddy, R. Kapavarapu, K. S. Kumar, K. K. Priya, K. V. L. Parsa and M. Pal, Chem. Commun., 2011, 47, 5010; (b) G. R. Reddy, T. R. Reddy, S. C. Joseph, K. S. Reddy, L. S. Reddy, P. M. Kumar, G. R. Krishna, C. M. Reddy, D. Rambabu, R. Kapavarapu, C. Lakshmi, T. Meda, K. K. Priya, K. V. L. Parsa and M. Pal, Chem. Commun., 2011, 47, 7779; (c) P. M. Kumar, K. S. Kumar, P. K. Mohakhud, K. Mukkanti, R. Kapavarapu, K. V. L. Parsa and M. Pal, Chem. Commun., 2012, 48, 431; (d) R. Sunke, R. Adepu, R. Kapavarapu, S. Chintala, C. L. T. Meda, K. V. L. Parsa and M. Pal, Chem. Commun., 2013, 49, 3570; (e) B. Prasad, B. Y. Sreenivas, G. R. Krishna, R. Kapavarapu and M. Pal, Chem. Commun., 2013, 49, 6716.
12. (a) H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem., Int. Ed., 2001, 40, 2004; (b) C. W. Tornøe, C. Christensen and M. Meldal, J. Org. Chem., 2002, 67, 3057; (c) V. V. Rostovtsev, L. G. Green, V. V. Fokin and K. B. Sharpless, Angew. Chem., Int. Ed., 2002, 41, 2596; (d) M. Meldal and C. W. Tornøe, Chem. Rev., 2008, 108, 2952; (e) For a recent review, see: E. Haldón, M. C. Nicasio and P. J. Pérez, Org. Biomol. Chem., 2015, 13, 9528.
13. S. Marullo, F. D'Anna, C. Rizzo and R. Noto, Ultrason. Sonochem., 2015, 23, 317.
14. (a) A. Barge, S. Tagliapietra, A. Binello and G. Cravotto, Curr. Org. Chem., 2011, 15, 189; (b) C. O. Kappe and E. van der Eycken, Chem. Soc. Rev., 2010, 39, 1280; (c) P. Cintas, A. Barge, S. Tagliapietra, L. Boffa and G. Cravotto, Nat. Protoc., 2010, 5, 607; (d) M. F. Mady, G. E. Awad and K. B. Jørgensen, Eur. J. Med. Chem., 2014, 84, 433; (e) G. B. da Silva, B. M. Guimarães, S. P. O. Assis, V. L. M. Lima and R. N. de Oliveira, J. Braz. Chem. Soc., 2013, 24, 914; (f) G. Cravotto, V. V. Fokin, D. Garella, A. Binello, L. Boffa and A. Barge, J. Comb. Chem., 2010, 12, 13.
15. (a) P. V. Babu, S. Mukherjee, D. R. Gorja, S. Yellanki, R. Medisetti, P. Kulkarni, K. Mukkanti and M. Pal, RSC Adv., 2014, 4, 4878; (b) A. R. Ellanki, A. Islam, V. S. Rama, R. P. Pulipati, D. Rambabu, G. R. Krishna, C. M. Reddy, K. Mukkanti, G. R. Vanaja, A. M. Kalle, K. S. Kumar and M. Pal, Bioorg. Med. Chem. Lett., 2012, 22, 3455.
16. For selected references, see: (a) N. Mulakayala, G. P. Kumar, D. Rambabu, M. Aeluri, M. V. B. Rao and M. Pal, Tetrahedron Lett., 2012, 53, 6923; (b) D. Rambabu, S. K. Kumar, B. Y. Sreenivas, S. Sandra, A. Kandale, P. Misra, M. V. B. Rao and M. Pal, Tetrahedron Lett., 2013, 54, 495; (c) R. G. Chary, G. R. Reddy, Y. S. S. Ganesh, K. Vara Prasad, S. K. P. Chandra, S. Mukherjee and M. Pal, RSC Adv., 2013, 3, 9641; (d) S. K. Kumar, D. Rambabu, C. V. Kumar, B. Y. Sreenivas, K. R. S. Prasad, M. V. Basaveswara Rao and M. Pal, RSC Adv., 2013, 3, 24863; (e) P. R. K. Murthi, D. Rambabu, M. V. B. Rao and M. Pal, Tetrahedron Lett., 2014, 55, 507; (f) M. S. Rao, M. Haritha, N. Chandrasekhar, M. V. B. Rao and M. Pal, Tetrahedron Lett., 2014, 55, 1660.
17. J. Fairhurst, T. M. Hotten, D. E. Tupper and D. T. Wong, US Pat., Application No US006034078A, March 7, 2000.
18. (a) The compound 3a was characterized by spectral data (see ESI†). Some of its ¹HNMR (red) and ¹³CNMR (blue) signals obtained are shown below; (b) All the products were characterized by spectral data. For HSQC and DEPT of compound 3c see the ESI†.
19. Cavitation (known to be the origin of sonochemical effects) involves the rapid growth of microbubbles in a liquid when a large negative pressure (e.g. the acoustic pressure on the rarefaction cycle) is applied to it. These microcavities collapse violently in the subsequent compression cycles. As a result enough kinetic energy is released to break chemical bonds. This perhaps favours the faster conversion of alkyne 1 to E-1, E-3 to E-4 and E-4 to 3 under ultrasound irradiation (Scheme 2). For further information, see: (a) K. S. Suslick, Science, 1990, 247, 1439; (b) K. S. Suslick, D. A. Hammerton and R. E. Cline Jr, J. Am. Chem. Soc., 1986, 108, 5641; (c) E. B. Flint and K. S. Suslick, Science, 1991, 253, 1397; (d) K. S. Suslick and S. J. Doktycz, Adv. Sonochem., 1990, 1, 197.
20. P. Wang, J. G. Myers, P. Wu, B. Cheewatrakoolpong, R. W. Egan and M. M. Billah, Biochem. Biophys. Res. Commun., 1997, 19, 320.
21. J. Demnitz, L. LaVecchia, E. Bacher, T. H. Keller, T. Muller, F. Schurch, H. P. Weber and E. Pombo-Villar, Molecules, 1998, 3, 107.

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Footnote

† Electronic supplementary information (ESI) available: Experimental procedures, copies of the ¹H and ¹³C NMR spectra and HPLC. See DOI: 10.1039/c5ra20380e

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