pubs.acs.org/joc
Characterization of the Two Fundamental Conformations of Benzoylureas and Elucidation of the Factors That Facilitate Their Conformational Interchange Guillaume Lessene,† Brian J. Smith,† Robert W. Gable,‡ and Jonathan B. Baell*,† †
The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia., and ‡School of Chemistry, The University of Melbourne, Victoria 3010, Australia
[email protected] Received April 28, 2009
The ability of the benzoylurea core to mimic R helices relies on its ability to form an intramolecular hydrogen bond. The conformational behavior of benzoylureas is investigated in depth in this study via the use of NMR, IR, X-ray, and computational analysis. The results show that the closed conformation maintained by an intramolecular hydrogen bond is favored in most of the cases studied except when steric and electronic effects combined with a solvent possessing a high hydrogen bond accepting ability, such as DMSO, are involved. The study highlights the propensity for benzoylureas to switch conformation depending on the environment of the molecule for a particular set of substituents. We anticipate that our summary of the phenomenon of internal hydrogen bonding and its analysis may further serve as a useful reference source for future workers in this area.
Introduction The discovery of organic scaffolds that mimic targeted binding epitopes is directly relevant to the development of small molecule inhibitors of protein-protein interactions. We have previously reported on our successful efforts in the design of small molecule structural and functional mimetics of pharmacologically active toxins.1-6 Our current interest is the design of R-helical mimetics as inhibitors of those (1) Baell, J. B.; Duggan, P. J.; Forsyth, S. A.; Lewis, R. J.; Lok, Y. P.; Schroeder, C. I. Bioorg. Med. Chem. 2004, 12, 4025–4037. (2) Baell, J. B.; Duggan, P. J.; Lok, Y. P. Aust. J. Chem. 2004, 57, 179–185. (3) Baell, J. B.; Forsyth, S. A.; Gable, R. W.; Norton, R. S.; Mulder, R. J. J. Comput.-Aided Mol. Des. 2001, 15, 1119–1136. (4) Baell, J. B.; Harvey, A. J.; Norton, R. S. J. Comput.-Aided Mol. Des. 2002, 16, 245–262. (5) Harvey, A. J.; Gable, R. W.; Baell, J. B. Bioorg. Med. Chem. Lett. 2005, 15, 3193–3196. (6) Norton, R. S.; Baell, J. B.; Angus, J. A. In Calcium channel pharmacology; McDonough, S. I., Ed.; Kluwer/Academic/Plenum Press: New York, 2003; pp 143-179.
DOI: 10.1021/jo900871a r 2009 American Chemical Society
Published on Web 08/07/2009
protein-protein interactions that comprise a helical epitope in the binding interface.7,8 We identified the benzoylurea moiety to be particularly suitable for this task as it contains heteroatoms that introduce a certain degree of amphiphilicity within the scaffold without being excessively hydrophobic.9 Furthermore, we envisaged this system to be synthetically versatile such that, when adequately functionalized, it could act as a R-helical protein mimetic. In particular, we were interested in the topographical similarity between the benzoylphenylurea and the terphenyl system, already shown by Hamilton and co-workers to be able to (7) Baell, J. B.; Huang, D. C. Biochem. Pharmacol. 2002, 64, 851–863. (8) Lessene, G.; Czabotar, P. E.; Colman, P. M. Nat. Rev. Drug Discovery 2008, 7, 989–1000. (9) Moreover, benzoylureas have a good track record in marketed pharmaceuticals. For example, see: Ware, G.; Whitcare, D. The Pesticide Book, 6th ed.; Meister Media Worldwide, Willoughby, OH, 2004.Okada, H.; Kato, M.; Koyanagi, T.; Mizuno, K. Chem. Pharm. Bull. 1999, 47, 430–433. Li, J.-N.; Song, D.-Q.; Lin, Y.-H.; Hu, Q.-Y.; Yin, L.; Bekesi, G.; Holland, J. F.; Jiang, J.-D. Biochem. Pharmacol. 2003, 65, 1691–1699.
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Lessene et al. CHART 1. Classification of Benzoylurea Derivatives Considered in the Present Study
FIGURE 1. Comparison of (a) the benzoylphenylurea core and (b) Hamilton’s terphenyl system.
FIGURE 2. Two different benzoylurea conformations represented in the Cambridge Crystallographic Database.
geometrically and functionally mimic biologically active R-helical binding epitopes (Figure 1).10-13 The pivotal feature of the benzoylureas is the central acylurea group that is required to exist in the intended “closed” conformation necessary to project functional groups appropriately (A, Figure 2), forming a pseudo-6membered ring with an intramolecular hydrogen bond and a central Z amide bond (E and Z geometry will refer to the position of the R1 and R2 groups in relation to their neighboring carbonyl group). Preliminary molecular mechanics modeling used in our design process confirmed this hypothesis. However, alternative conformations can be postulated: formally there exist eight possible conformations through rotation about three bonds. A search of the Cambridge Crystallographic Database (CCD) confirmed that many benzoylureas exist in this pseudo-6-membered ring conformation. However, there are some benzoylureas for which the C1-N1 bond adopts a Z conformation14 as shown by B in Figure 2. This conformation is not predicted by molecular mechanics to occupy a global (10) Orner, B. P.; Ernst, J. T.; Hamilton, A. D. J. Am. Chem. Soc. 2001, 123, 5382–5383. (11) Kutzki, O.; Park, H. S.; Ernst, J. T.; Orner, B. P.; Yin, H.; Hamilton, A. D. J. Am. Chem. Soc. 2002, 124, 11838–11839. (12) Yin, H.; Lee, G.-i.; Sedey, K. A.; Kutzki, O.; Park, H. S.; Orner, B. P.; Ernst, J. T.; Wang, H.-G.; Sebti, S. M.; Hamilton, A. D. J. Am. Chem. Soc. 2005, 127, 10191–10196. (13) This work as well as applications to generate biologically active, BclxL-binding, R helical mimetics was presented in part at the 20th Royal Australian Chemical institute Organic Chemistry Conference, Cairns, Australia, 2004, abstract P(RACIOC) 40 and at Connect 2005, Sydney, Australia, 2005, Abstracts 248 and 364. The worldwide patent disclosing these structures was published in 2006 (Lessene G.; Baell, J. B. R-Helical Mimetics, PCT/AU2005/000968). This system has apparently been “rediscovered” in the interim and published ( Rodriguez, J. M.; Hamilton, A. D. Angew. Chem., Int. Ed. 2007, 46, 8614–8617. Rodriguez, J. M.; Ross, N. T.; Katt, W. P.; Dhar, D.; Lee, G.-i.; Hamilton, A. D. ChemMedChem 2009, 4, 649–656). (14) Of 25 structures in version 5.30 of CCD, three were in this alternative conformation. (15) Maximin2 using the Tripos Forcefield as implemented in Sybyl7.01 (Tripos Associates, MO); a third, extended (EEZ) form has been identified as a putative biologically active conformation using the Catalyst/HIPHOP software program but in light of the likely generic inability of molecular mechanics to properly analyse benzoylurea conformation, this finding is questionable (see: Kurogi, Y.; Miyata, K.; Okamura, T.; Hashimoto, K.; Tsutsumi, K.; Nasu, M.; Moriyasu, M. J. Med. Chem. 2001, 44, 2304-2307).
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or even local energy minimum.15 Since appropriate scaffold conformation is clearly critical to mimetic design, we undertook a conformational analysis of model compounds under a variety of experimental conditions and support our findings with selected use of high-level density functional theory computational calculations. We describe here for the first time why benzoylureas can adopt two distinct conformations;the “closed” form A or the “twisted” form B. We show that the preferred conformation adopted for a given benzoylurea depends on the nature of substituent groups and can be logically explained by a subtle interplay of electrostatic and steric effects.16 We further elucidate why benzoylureas are better described structurally as bis-amides rather than as acylureas; conformationally, they behave as two distinct amide units without any urea character whatsoever. This structural characteristic enables strengthening of an internal hydrogen bond that stabilizes the closed form, which is otherwise inherently sterically disfavored. This hydrogen bond is not a resonance-assisted hydrogen bond as defined by Gilli et al., but despite this, is shown by gas-phase calculations to remain very strong.17-19 Results and Discussion Compounds Studied. Compounds of interest in this study are shown in Chart 1, divided into four classes characterized by the nature of the substituents R1 and R2. A nonbranched R1 substituent is defined as any alkyl group that comprises a methylene moiety R to the N1 nitrogen atom, such as methyl or ethyl, whereas we define this substituent as “branched” when the carbon R to the nitrogen is branched, such as for R1 = isopropyl, sec-butyl, tert-butyl, etc. Thus, we define (16) Kohmoto et al. have reported the closed form of a benzoylureas (Kohmoto, S.; Iwasaki, N.; Fukui, D.; Nishio, T.; Iida, I.; Kishikawa, K.; Yamamoto, M.; Yamada, K. J. Chem. Soc., Perkin Trans. 2 1996, 985-988) and analyzed a twisted form of an alkylacylurea ( Kohmoto, S.; Kasimura, H.; Nishio, T.; Iida, I.; Kishikawa, K.; Yamamoto, M.; Yamada, K. J. Chem. Soc., Perkin Trans. 2 1994, 1565-1568) but have not extended this to an understanding of conversion between these two forms. (17) Gilli, G.; Gilli, P. J. Mol. Struct. 2000, 552, 1–15. (18) Gilli, P.; Bertolasi, V.; Pretto, L.; Lycka, A.; Gilli, G. J. Am. Chem. Soc. 2002, 124, 13554–13567. (19) Gilli, P.; Bertolasi, V.; Ferretti, V.; Gilli, G. J. Am. Chem. Soc. 2000, 122, 10405–10417.
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Lessene et al. SCHEME 1.
Retrosynthesis of Benzoylureas
four distinct classes of compounds: class I compounds (1) possess a nonbranched N1 substituent (ethyl) and a phenyl ring on N2; class II compounds (2a-c) possess a nonbranched N1 substituent (ethyl) and a varying alkyl R2 group; class III compounds (3) possess a branched N1 substituent (isopropyl) and a phenyl ring on N2; class IV compounds (4a-c) possess a branched N1 substituent (isopropyl) and an alkyl R2 group.20 Synthesis of Benzoylureas. Although a large number of benzoylureas are known, the chemistry surrounding this group of compounds is rather disparate, especially when R1 is not a hydrogen atom. Methods using carbodiimides21,22 or ureas23 are known and furnish “symmetrical” benzoylureas (R1 = R2). Examples where R1 and R2 are different alkyl groups have been presented in an Eli Lilly patent and in a study of semisynthetic β-lactam antibiotics.24,25 The key feature in these two preparations is the formation of an acylcarbamoyl chloride from reaction of a chlorocarbonyl donor with an activated secondary amide. Subsequent reaction with amines furnishes differentially substituted benzoylureas (Scheme 1). In the Eli Lilly report,24 the amide was activated by generation of the anion via the use of butyllithium, whereas Ohi et al.25 achieved activation through silylation with trimethylsilyl choride (TMSCl). We attempted the butyllithium-mediated method first, but this was not successful with amides bearing a branched R1 substituent and so we turned to the silylation-based approach. We noted that a milder silylation-based route to the synthesis of acylcarbamoyl chlorides than that used by Ohi et al. has also been reported, using trimethylsilyl triflate (TMSOTf) as the silylating agent and phosgene as the chlorocarbonyl donor.26,27 This synthetic route has not been applied to the synthesis of benzoylureas, but we reasoned that it should be possible to do so by reacting the acylcarbamoyl chloride intermediate with either an aniline using triethylamine as an acid scavenger or with 2 equiv of an alkylamine. (20) All classes are drawn as extended, conformationally “neutral” forms, indicative of neither the closed nor twisted conformations. (21) Kishikawa, K.; Yamamoto, M.; Kohmoto, S.; Yamada, K. J. Org. Chem. 1989, 54, 2428–2432. (22) Lee, W. W.; Martinez, A. P.; Goodman, L. J. Med. Chem. 1974, 17, 326–330. (23) Smith, T. D.; Jones, P. G.; Schmutzler, R. Z. Naturforsch. B 1992, 47, 526–532. (24) Cooper, R. D. G.; Herron, D. K. Eli Lilly Co.: France, 1975. (25) Ohi, N.; Aoki, B.; Shinozaki, T.; Moro, K.; Kuroki, T.; Noto, T.; Nehashi, T.; Matsumoto, M.; Okazaki, H.; Matsunaga, I. Chem. Pharm. Bull. 1987, 35, 1903–1909. (26) Horner, J. H.; Musa, O. M.; Bouvier, A.; Newcomb, M. J. Am. Chem. Soc. 1998, 120, 7738–7748. (27) Esker, J. L.; Newcomb, M. Tetrahedron Lett. 1992, 33, 5913–5916.
SCHEME 2.
Benzoylurea Preparation
We applied this approach to the benzoylureas and were pleased to see that this procedure furnished all the desired compounds in high yields and with great ease of purification (Scheme 2). Spectroscopic Study. A clear feature that distinguishes between the closed and twisted benzoylurea conformations in Figure 2 is the respective presence or absence of an intramolecular hydrogen bond. IR and 1H NMR spectroscopy are classical techniques used to analyze hydrogen-bonding behavior.28 IR spectroscopy results are relatively straightforward to interpret. Hydrogen bonding can be intermolecular, involving the solvent or another molecule of solute, or can be intramolecular. Assignment of an intramolecular hydrogen bond by IR spectroscopy, therefore, is clearer in a non-hydrogenbonding solvent (or very weak hydrogen-bonding solvent), such as chloroform, with dilute conditions (to reduce the potential for dimer formation of the solute), and for solutes with only a limited number of amide NH groups (to minimize band overlap).28-31 The time scale in IR spectroscopy is fast compared with conformational changes and allows the observation of the two characteristic bands of N-H bond vibration. In non-hydrogen-bonding solvents, a broadband at longer wavelengths (usually around 3200-3340 cm-1) corresponds to a hydrogen-bonded NH, while a sharp band at shorter wavelengths (usually around 3400-3450 cm-1) corresponds to a solvent-exposed NH. Caution must be used in the use of IR spectra to estimate conformer ratios since a hydrogen-bonded conformation might have a distinctly different absorption coefficient from a non-hydrogen-bonded conformation. In this study, we used IR spectroscopy to qualitatively assess the presence of hydrogen-bonded versus non-hydrogen-bonded conformations to support quantitative interpretation of data obtained via 1H NMR spectroscopy. NMR has advantages over IR as a spectroscopic technique since data from experiments in polar solvents as well as nonpolar solvents can be readily and meaningfully interpreted.32-34 If the benzoylurea interconverts rapidly on an NMR time scale between two different conformations, A and B, and if the limiting chemical shift values of a given proton signal of each form are known, the equilibrium (28) Aaron, H. S. In Topics in Stereochemistry; Allinger, N. L., Eliel, E. L., Eds.; John Wiley & Sons: New York, 1979; Vol. 11, pp 1-52. (29) Gellman, S. H.; Dado, G. P.; Liang, G. B.; Adams, B. R. J. Am. Chem. Soc. 1991, 113, 1164–1173. (30) Stevens, E. S.; Sugawara, N.; Bonora, G. M.; Toniolo, C. J. Am. Chem. Soc. 1980, 102, 7048–7050. (31) Analogous arguments apply to OH, SH, and amino NH groups, but our focus here is the amide NH. (32) Baxter, N. J.; Williamson, M. P. J. Biomol. NMR 1997, 9, 359–369. (33) Cierpicki, T.; Otlewski, J. J. Biomol. NMR 2001, 21, 249–261. (34) Kessler, H. Angew. Chem., Int. Ed. 1982, 21, 512–523.
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Lessene et al. TABLE 1. Interpretation of NMR Spectroscopic Data in Terms of Conformational Behavior (These Rules Are a Synthesis of Findings from Refs 29 and 30 in Chloroform and Ref 34 in DMSO)a
a This summary relates mainly to a two-state system, which applies to our case. Other more complex behaviors can be observed with molecules inherently more flexible or having more than one hydrogen bond donor or acceptor. For example, a small positive NTCDS has been observed for a non-hydrogen-bonded NH becoming increasingly hydrogen bonded in an enthalpically less favored conformation with increasing temperature. From our reading it is clear that these rules are still not fully worked out and our interpretations are only intended to be guides.42 bAmounts of water can influence the results of the NMR analysis.29,54 Dry CDCl3 was used for this study. cTo put this into context, it has been stated in one particular case that a large enthalpic difference of around 6 kJ/mol in favor of A over B could correspond to a NTDCS of -10 ppb/K. The proportion of B in this case increases slightly with increasing temperature.29 dThe degree of enthalpic difference has been related to the how close the geometry of the hydrogen bond in A is to the optimum linear geometry, but this can be readily counteracted by steric strain, for example.45
an internally hydrogen-bonded conformation and non-hydrogen-bonded conformation where the two conformations are of similar enthalpy; (ii) a proton mostly engaged in an intramolecular hydrogen bond whose conformational environment is invariant over the course of the experiment; or (iii) a proton mostly not engaged in an intramolecular hydrogen bond whose conformational environment does not change over the course of the experiment. On the other hand, a large NH proton NTDCS (
