Design, Synthesis, and Conformational Analysis of

Article pubs.acs.org/joc

Design, Synthesis, and Conformational Analysis of Trispyrimidonamides as α‑Helix Mimetics Lukas Spanier,†,§ Emanuele Ciglia,†,§ Finn K. Hansen,† Krystina Kuna,† Walter Frank,‡ Holger Gohlke,† and Thomas Kurz*,† †

Institut für Pharmazeutische und Medizinische Chemie, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany ‡ Institut für Anorganische Chemie und Strukturchemie, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany S Supporting Information *

ABSTRACT: The straightforward synthesis of trispyrimidonamides as a new class of α-helix mimetics is reported. Because of the versatility of our synthetic protocol, a variety of side chains including aliphatic, basic, aromatic, and heteroaromatic residues were included. A comprehensive conformational analysis revealed that in polar solvents a trimeric compound adopts conformations that can lead to i, i + 4, i + 8, or i, i + 8 patterns of side chain orientation. This suggests that trispyrimidonamides could be promising α-helix mimetics to target hot spots that are distributed over a wider angular range of an α-helix interface than in the classical i, i + 4, i + 7 case.

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INTRODUCTION Protein−protein interactions (PPIs) are involved in the regulation of numerous essential cellular processes1−7 and play a central role in disease development.8−10 Consequently, modulating PPIs is a promising concept to interfere with physiological and pathophysiological processes.1,7,11−13 Small molecule protein−protein interaction modulators (PPIMs) that efficiently and selectively affect protein−protein binding are one way of interference.14−18 The most frequently occurring recognition motifs in PPIs are α-helices.19,20 This and the fact that usually only a few key residues (so-called “hot spots”) in a protein−protein interface account for the majority of the binding affinity21−23 have fueled the development of small molecules mimicking α-helices (Scheme 1).24−32 Such small-molecule α-helix mimetics have proven valuable as biophysical probes or modulators of protein−protein interactions.10,27,33 Compared to other α-helix mimetics, e.g., β-peptides, aza- and aminoxypeptides, γ-peptides, and peptoids, small-molecule α-helix mimetics promise to have more favorable pharmacokinetic properties.34 The development of small-molecule α-helix mimetics started almost 30 years ago with the synthesis of 1,6-disubstituted indanes where the substituents mimic the i and i + 1 residues of an α-helix.35,36 A pioneering advancement was the synthesis of tris-orthosubstituted terphenyl derivatives by Hamilton and co-workers;37 the substituents of this extended scaffold mimic the i, i + 4, and i + 7 residues of an α-helix. In search for smallmolecule α-helix mimetics that have more hydrophilic and/or © 2014 American Chemical Society

Scheme 1. (I) Terphenyl, (II) Trispyridylamide, and (III) Trispyrimidonamidea

a

Scaffold rings of III are labeled A, B, C.

amphiphilic scaffolds than the terphenyls, terpyridines, trisbenzamides, terephthalamides, trispyridylamides, phenylenaminones, pyridazine, 1,4-benzodiazine-2,5-diones, trisubstituted imidazoles, benzylidenechalcones, and trans-fused polyReceived: October 22, 2013 Published: January 22, 2014 1582

dx.doi.org/10.1021/jo402353z | J. Org. Chem. 2014, 79, 1582−1593

The Journal of Organic Chemistry

Article

Scheme 2. Retrosynthesis of the Trispyrimidonamide Scaffold

Scheme 3. Synthesis of Monomers 3 and 5-Aminopyrimidones 5a,b

structural diversity for our desired trimeric α-helix mimetics. The synthesis of polysubstituted pyrimidines is usually accomplished by (i) cyclization, (ii) ring transformation, (iii) aromatization as well as (iv) substituent modification, and various efficient preparations have been published.54 We aimed at the synthesis of N3-substituted pyrimidin-4(3H)-ones with a protected amino group in 5-position as well as a carboxylic acid ester in 2-position as suitable monomers. Our retrosynthetic analysis (Scheme 2) led to 4-(ethoxymethylidene)-2-phenyl1,3-oxazol-5(4H)-one 155 and N-substituted amidines56 of type 2 as starting materials. Azlactone 1 deserves particular interest as a building block in heterocyclic chemistry because of its ambivalent behavior toward nucleophiles and has been utilized in various ring transformation reactions for the preparation of 5-benzoylaminopyrimidin-4(3H)-ones.55,57−60 The desired 5-benzoylaminopyrimidin-4(3H)-ones 3a−m were obtained from readily available amidine hydrochloride derivatives 2a−m and azlactone 1 by treatment with triethylamine in acetonitrile under reflux conditions (22−81% yield, Scheme 3, Table 1). As a complementary route for the preparation of N3substituted monomers of type 3, the pyrimidone 3a (R = H) was treated with sodium hydride and alkylating agents to provide the N3-substituted derivatives 3m,n (32% and 64% yield, Scheme 4, Table 1). In all cases, the N3-alkylated products were obtained as the major regioisomers. An authentic sample of 3m obtained by azlactone rearrangement was used for structural assignment. Utilizing these protocols allows the

cyclic ethers were synthesized.38−49 For several of these scaffolds, only n-alkyl, i-alkyl, or arylalkyl substituents have been reported43,48−50 likely because of synthetic difficulties when introducing polar and/or charged substituents. As a consequence, only hydrophobic or aromatic side chains of hot spots can be mimicked. This may lead to a restricted applicability if the frequently occurring hot spots Trp, Tyr, and Arg are to be addressed.51,52 This provided the incentive for us to design and synthesize previously unreported trispyrimidonamides as a new class of αhelix mimetics and investigate by 2D NMR, X-ray crystallography, and molecular modeling their conformational properties. Regarding the synthesis, our approach provides a general toolbox that allows generating trispyrimidonamides with hydrophobic and differently functionalized (aliphatic, aromatic, heteroaromatic, polar, and basic) side chains. That way, a variety of potential hot spots can be mimicked by the R-groups of our trispyrimidonamide scaffold. The 2D NMR experiments together with modeling studies suggest that trispyrimidonamide derivatives adopt conformations in polar solvents that can orient R groups in such a way that they mimic amino acid side chains at positions i, i + 4, i + 8 or i, i + 8 of an α-helix. The i, i + 4, i + 8 pattern is currently targeted by just one class of mimetics.53

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RESULTS AND DISCUSSION Chemistry. We decided to pursue a monomer approach in order to develop a synthetic protocol that allows a high 1583



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an excess of methylamine as nucleophile to furnish a stable Cterminus (Scheme 3). The preparation of the unprotected 5aminopyrimidones 5a,b was achieved by saponification of the benzamide group of 4a,b through treatment with sodium hydroxide (3 equiv) in methanol under reflux conditions. The attempted conversion of the alkyl ester moiety of 3 into the free carboxylic acid resulted again in isolation of the corresponding decarboxylated product. A literature survey revealed that only few pyrimidone derivatives are known with a carboxamide moiety in 2-position. These compounds were either obtained by direct treatment of an ester with a strong nucleophile or under trimethylaluminium-mediated coupling conditions. Our efforts to obtain a dimeric compound by Me3Al-mediated coupling of the ester 3d with the free amine 5b failed. As a consequence, we developed a straightforward protocol for preparation of oligomeric pyrimidone compounds. The hydrolysis of 3c−f provided the corresponding lithium salts. Next, we investigated the formation of an amide bond in order to obtain dimeric pyrimidone derivatives. Several wellestablished coupling agents were screened using 5-aminopyrimidone 5b and the corresponding lithium salt of 3d as a model system. In all cases the progress of the reaction was monitored by HPLC (see Supporting Information for details). The results gathered from this screening were particularly interesting. The reaction was slow and we were unable to obtain a conversion of more than 50% after 24 h utilizing PyBop, HBTU, and DIC/Oxyma as coupling agents. The HATU-mediated amide coupling accomplished a conversion of 78% after 24 h, and the model dimer 6a was obtained in 55% yield after purification by flash column chromatography. Best results were achieved using COMU as a coupling agent. In this case, the reaction was essentially completed after 3 h, and we were able to isolate the desired dimeric compound 6a in 85% yield (see Supporting Information). Having an efficient monomer synthesis and an optimized coupling reaction in hand, we turned our attention toward the preparation of dimeric and trimeric N3-substituted pyrimidonamides as α-helix mimetics. The COMU-mediated coupling reactions of 5-aminopyrimidones 5a and 5b with the corresponding lithium salts of 3c and 3d provided smoothly the benzoyl-protected dimers 6a and 6b in a batch scale (75 and 76% yield, Scheme 5, Table 2). The benzoyl-protecting groups were then cleaved by treatment of 6a and 6b with sodium hydroxide to afford the deprotected dimers 7a and 7b

Table 1. Monomers Synthesized

a

entry

alkyl

R

yield [%]

3a 3b 3c 3d 3e 3f 3g 3h 3i 3j 3k 3l 3m 3n

Me Me Et Et Et Et Et Et Et Et Et Me Me Me

H 3-indolylethyl isobutyl sec-butyl N-Boc-4-aminobutyl 2-propyl 4-hydroxyphenylethyl 4-methoxyphenylethyl 4-hydroxybenzyl 4-methoxybenzyl 3-(benzyloxy)-3-oxopropyl phenylethyl 2-(tert-butyl)-2-oxoethyl 4-methoxybenzyl

50 65 31 22 33 30 55 41 52 81 22 35 49, (32)a (64)a

Yield from N-alkylation.

Scheme 4. Synthesis of Monomers 3m,n by Alkylation

preparation of a variety of monomers with a high structural diversity with regard to the nature of the N3-substitution. Notably, the monomers obtained are capable of mimicking aliphatic, aromatic, heteroaromatic, acidic, and basic side chains of amino acids. After the establishment of efficient methods for preparation of the desired protected monomers 3 we studied the deprotection of both protecting groups. All attempts to deprotect the benzoyl protecting group led to the corresponding decarboxylated product. Consequently, we transformed 3b,c into the N-methyl carboxamide derivatives 4a,b utilizing Scheme 5. Synthesis of Dimers 6a,b

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Table 2. Synthesized Di- and Trimeric Pyrimidones

a

entry

R1a

R2a

R3a

yield [%]

7a 7b 8a 8b 8c

Isobutyl “Leu” 3-Indolylethyl “Trp” Isobutyl “Leu” 3-Indolylethyl “Trp” 3-Indolylethyl “Trp”

sec-Butyl “Ile” Isobutyl “Leu” sec-Butyl “Ile” Isobutyl “Leu” Isobutyl “Leu”

− − 2-Propyl “Val” N-Boc-4-aminobutyl “BOC-Lys” 4-Aminobutyl “Lys”

77 74 55 41 90b

Residues corresponding to mimicked amino acids. bFrom 8b by TFA mediated cleavage of BOC protecting group.

Scheme 6. Synthesis of Trispyrimidonamides 8a,b

in 77 and 74% yield, respectively (Scheme 6, Table 2). Subsequently, the reaction of 7a and 7b with corresponding lithium salts of 3f and 3e in the presence of COMU led to the corresponding trispyrimidonamides 8a and 8b (55% and 41% yield, Scheme 6, Table 2). Finally, the Boc-protecting group in 8b was removed by acidolysis to furnish trimer 8c (Scheme 7, 90% yield) with unprotected 4-aminobutyl and 3-indolylethyl side chains, thus highlighting the versatility of our synthetic protocol in regard to side chain tolerance. The conformational properties of our synthesized trispyrimidonamides were studied by X-ray analysis of trimer 8a and 2D NMR spectroscopy (Figure 1). Diffraction quality crystals were obtained by vapor diffusion (see Supporting Information). The X-ray structure of 8a (Figure 1A) shows the trimer adopting a conformation C1 in which only R1 and R3 are oriented on the same side, whereas R2 points in the opposite direction. In order to study the properties of 8a in solution, a ROESY experiment was performed in CDCl3 (see Figure 1B and Figure S1, Supporting Information). All relevant signals were assigned by HSQC and HMBC spectra. Key ROE correlations are shown as arrows in Figure 1B. We observed ROE cross peaks between the pyrimidone proton of ring B with the methine and methyl protons of the isopropyl side chain. We reasoned that these ROE correlations would only be observable for a conformation C1 (Figure 1B), which is in a good agreement with the X-ray structure of 8a (Figure 1A). Unfortunately, 8a−c were not sufficiently soluble to perform a 2D NMR study in D2O. However, we were able to study the

Scheme 7. Deprotection of 8b to the Corresponding Amine

conformational properties of the TFA salt of 8c in methanol-d4. We observed ROE cross signals between the isobutyl side chain and the pyrimidone protons of the two terminal rings (rings A and C), which indicates the presence of a conformation C1, analogous to the conformation observed in CDCl3 (Figure 2 and Figures S2 and S3, Supporting Information). Interestingly, our ROESY analysis revealed an additional ROE cross peak between the methyl protons of the isobutyl side chain and the 1585



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Figure 1. Structural and conformational study of compound 8a. (A) Solid state structure. (B) 1H−1H-ROESY spectra (region between 9.18 and 8.84 Hz, 600 MHz, CDCl3) and suggested conformational structure; key ROEs are marked in red.

in the crystal structure of 8a (Figure S4B, Supporting Information). During the MD simulations, frequent rotations around the ϕ and ψ torsion angles are observed (data not shown), demonstrating that the sampled conformations are in equilibrium. This is also confirmed by the symmetry of the maps of the relative free energy of IV as a function of the orientation of the methyl substituents of rings A and B (torsion angle Ω1; Figure 3A), and B and C (torsion angle Ω2; Figure 3A) (Figure 3B). The relative free energy map for the simulation of IV in chloroform reveals a preferred conformation C1 (Ω1 ≈ ± 165°; Ω2 ≈ ± 155°; Figure 3B,C). The alternative conformation C2 (Ω1 ≈ ± 55°; Ω2 ≈ ± 55°) is energetically disfavored by ΔG ≈ 2.5 kcal mol−1 in chloroform. This relates to a ratio of 98.5/1.5 for the populations of C1 versus C2 in the conformational ensemble at 300 K. In contrast, the simulation of IV in methanol reveals C2 as the preferred conformation (Figure 3B,D) whereas C1 is now disfavored by ΔG ≈ 1.0 kcal mol−1. This relates to a ratio of 19/81 for the populations of C1 versus C2 in the conformational ensemble at 300 K. These results are in excellent agreement with those from the 2D-NMR studies, which identified only C1 as the preferred conformation of 8a in chloroform but suggested that 8c can adopt both conformations C1 and C2 in methanol. In addition, the MD simulations confirm for C1 an almost coplanar orientation of the pyrimidone rings, as found in the crystal structure of 8a, but suggest for C2 that neighboring rings are rotated by 55° with respect to each other. In all, these results validate our setup of the MD simulations and the quality of the force-field parameters used for the solute and solvent. As no experimental information is available for the conformational preference of trispyrimidonamides in water, we investigated by MD simulations in explicit water the conformational preference of IV using the same setup as above. The MD simulations suggest that C2 becomes even more preferred over C1 than in methanol (Figure 3B,D), with C1 being disfavored by ΔG ≈ 3.0 kcal mol−1 equivalent to a ratio of

Design, Synthesis, and Conformational Analysis of

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