Bioorganic and Medicinal Chemistry 23 (2015) 2680-2694

Lead-oriented synthesis: Investigation of organolithium-mediated routes to 3-D scaffolds and 3-D shape analysis of a virtual lead-like library Monique Lüthy a , Mary C. Wheldon a , Chehasnah Haji-Cheteh a , Masakazu Atobe a,b , Paul S. Bond a , Peter O’Brien a,⇑ , Roderick E. Hubbard a,c , Ian J. S. Fairlamb a aDepartment of Chemistry, University of York, Heslington, York YO10 5DD, UK b Asahi Kasei Pharma Corporation, 632-1 Mifuku, Izunokuni-shi, Shizuoka 410-2321, Japan c Vernalis (R&D) Ltd, Granta Park, Cambridge CB21 6GB, UK article info Article history: Received 9 February 2015 Revised 2 April 2015 Accepted 3 April 2015 Available online 11 April 2015 Keywords: Lead-like library Lead-oriented synthesis 3-D shape analysis Nitrogen heterocycles Organolithium-mediated synthesis abstract Synthetic routes to six 3-D scaffolds containing piperazine, pyrrolidine and piperidine cores have been developed. The synthetic methodology focused on the use of N-Boc a-lithiation-trapping chemistry. Notably, suitably protected and/or functionalised medicinal chemistry building blocks were synthesised via concise, connective methodology. This represents a rare example of lead-oriented synthesis. A virtual library of 190 compounds was then enumerated from the six scaffolds. Of these, 92 compounds (48%) fit the lead-like criteria of: (i) 1 6 AlogP 6 3; (ii) 14 6 number of heavy atoms 6 26; (iii) total polar surface area P 50 Å2 . The 3-D shapes of the 190 compounds were analysed using a triangular plot of normalised principal moments of inertia (PMI). From this, 46 compounds were identified which had lead-like properties and possessed 3-D shapes in under-represented areas of pharmaceutical space. Thus, the PMI analysis of the 190 member virtual library showed that whilst scaffolds which may appear on paper to be 3-D in shape, only 24% of the compounds actually had 3-D structures in the more interesting areas of 3-D drug space. 2015 Elsevier Ltd. All rights reserved. 1. Introduction It is now widely acknowledged that the success of compounds through the drug discovery process is strongly associated with molecular and physical properties.1–5 In 2012, Nadin et al. at GlaxoSmithKline challenged the synthetic community with the task of developing synthetic methodology that would be better suited to the preparation of lead-like molecules.6 Their concept, termed ‘lead-oriented synthesis’, highlighted that ‘lead-oriented synthesis must be able to deliver molecules with specific molecular properties with utility in the drug discovery process’ and that ‘lead-oriented syntheses need to pay particular attention to the physicochemical and functional group properties of the target molecules while also maintaining synthetic efficiency’. With this in mind, and building on Lipinski’s rules,7 lead-oriented synthesis was defined with the following guidelines: (i) lipophilicity should be in the range 1 6 c logP 6 3; (ii) molecular size should be in the range 14 6 number of heavy atoms 6 26 (corresponding to a molecular weight range of 200–350 Da); (iii) molecules with chemically active, electrophilic or redox active functional groups should be avoided; and (iv) molecules with a lower degree of aromatic character and/or more 3-D shape should be prioritised. Additional recommendations on the associated synthetic chemistry focused, amongst other things, on efficiency, cost, suitability for array synthesis and compatibility with polar functional groups. The lead-oriented synthesis guidelines bring together several aspects that have been highlighted as key to drug discovery. The lead optimisation process generally leads to increases in lipophilicity and molecular complexity (and hence the associated molecular weight).8,9 Setting lower target ranges for these properties at the outset is thus considered important. Support is also developing for the view that too many aromatic rings adversely affects the lipophilicity of drug compounds10 (although data analysis is far from straighforward11) and that 3-D shape in drug compounds may give a better profile through the drug development process.12 In this context, Nelson and co-workers have recently reported the lead-oriented synthesis and evaluation of two virtual libraries of lead-like compounds based on a range of drug-relevant scaffolds.13 http://dx.doi.org/10.1016/j.bmc.2015.04.005 0968-0896/ 2015 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. Tel.: +44 1904 322535; fax: +44 1904 322516. E-mail address: [email protected] (P. O’Brien). Bioorganic & Medicinal Chemistry 23 (2015) 2680–2694 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc

Our long-standing interest in the asymmetric synthesis of saturated nitrogen heterocycles14–19 and the growing interest in 3-D lead-like12 and fragment20,21 compounds led us to consider whether our organolithium-based methodology is suitable for the lead-oriented synthesis of 3-D scaffolds. In this paper, we describe concise, connective synthetic methodology for the construction of suitably protected building blocks which are precursors to 3-D scaffolds 1–6 (Fig. 1). These scaffolds incorporate piperazines, pyrrolidines or piperidines which are amongst the most common ring systems found in approved drugs.22,23 Scaffolds 1–6 were specifically selected due to their anticipated 3-D shape (vide infra), the presence of only one (1–4) or two (5–6) stereogenic centres, potential ease of synthesis via established organolithium chemistry and the fact that they had two or three points of diversification (different heteroaromatic groups and/or different O- and Nfunctionality). Furthermore, by choosing a pyridine group as a representative, electron deficient heteroaromatic substituent, it was felt that lead-like compounds of typical polarity would challenge the methodology. The synthetic approach to scaffolds 1–4 involved use of Beak’s N-Boc a-lithiation methodology,24 trapping with a heterocyclic ketone and subsequent cyclisation to the carbamate. In contrast, for the synthesis of scaffolds 5 and 6, an N-Boc a-lithiation-ring expansion approach was adopted.15 Thus, starting from N-Boc pyrrolidine, a-lithiation and trapping with an aldehyde would deliver an amino alcohol, which would be ring expanded via an aziridinium ion,25,26 to give the a-aryl, b-hydroxy piperidine motif.27 In addition to the lead-oriented synthetic methodology, we also describe herein the lead-like and 3-D shape analysis of a virtual library of 190 compounds derived from scaffolds 1–6. For the 3- D shape analysis, we elected to use a triangular plot of normalised principal moments of inertia (PMI) as introduced by Sauer and Schwarz.28 In particular, this analysis allowed us to address the issue of whether scaffolds like 1–6 which appear on paper to be 3-D in shape can actually generate compounds with appropriate lipophilicity/molecular size and that occupy new areas of 3-D pharmaceutical space. Herein, we present our results. 2. Results and discussion 2.1. Development of lithiation-trapping methodology for the synthesis of 3-D scaffolds 1–6 The planned synthetic approach to scaffolds 1–4 involved the alithiation of N-Boc heterocycles and trapping with suitable heterocyclic N-protected amino-ketones. In order to evaluate this methodology, a model system was explored first. Thus, N-Boc pyrrolidine 7 was lithiated a to the nitrogen using our diaminefree protocol29 (1.0 equiv s-BuLi, THF, 78 C, 1 h) and trapped using tetrahydro-4H-pyranone. In this example, the lithium alkoxide intermediate was quenched at 78 C to prevent cyclisation onto the carbamate. This generated two major products which were isolated after chromatography (Scheme 1): the desired alcohol 8 (66% yield) and the aldol self-condensation by-product 9 (11% yield). It seems likely that enolisation of some of the tetrahydro4H-pyranone by the lithiated N-Boc pyrrolidine occurs as a side-reaction. Apparently, there is a fine balance between the basicity and the nucleophilicity of the lithiated N-Boc pyrrolidine in reactions with enolisable heterocyclic ketones. There was a slight reduction in yield if 1.3 equiv of s-BuLi was used (63% of 8 and 6% of 9). Despite the occurrence of the aldol side-reaction to give aldol 9, we were able to use this connective reaction to generate suitable quantities of alcohol 8 for further synthetic studies. For example, alcohol 8 was converted into two novel, 3-D fragments 10 and 11. Treatment with KHMDS at reflux led to cyclisation to form carbamate 10 in 54% yield. Alternatively, Boc deprotection using TFA and acylation delivered amide 11 in 51% yield (Scheme 2). After these successful model studies, our attention switched to the synthesis of piperazine-based scaffolds 1 and 2 starting from NBoc piperazine 12. Diamine-free lithiation with 1.3 equiv s-BuLi was followed by trapping with N-Boc piperidin-4-one or N-Boc azetidin-4-one. In these cases, in order to differentiate between the two Boc groups in the trapped products, the reaction mixtures were allowed to warm to room temperature and stirred for 24 h. This allowed cyclisation of the alkoxide onto the Boc group and directly gave carbamates 13 (64% yield) and 14 (42% yield), isolated after chromatography (Scheme 3). Aldol self-condensation was also noticed but not quantified in these two reactions. With the preparation of 13 and 14, a concise synthesis of scaffolds 1 and 2 has been achieved. Notably, variation of the lithiated N-Boc heterocycle would allow a wider range of 3-D scaffolds related to 1 and 2 to be generated: the synthesis of 13 and 14 represents proof of concept for this general synthetic approach. Carbamates 13 and 14 are attractive medicinal chemistry building blocks with orthogonally protected functionality, allowing stepwise functionalisation of each of the amines. To demonstrate the synthetic potential of 13 and 14 for lead-like library synthesis, they were differentially deprotected. Boc deprotection of 13/14 N N O O N N N O O N N O N O N O 1 2 Het Ar N O O Het Ar N 4 3 Het Ar 5 N O Het Ar 6 Figure 1. Structures of piperazine-, pyrrolidine- and piperidine-based 3-D scaffolds 1–6 with highlighted points of structural diversification (HetAr = heteroaromatic). N Boc 1. 1.0 eq. sBuLi, THF –78 °C, 1 h 2. O O 3. NH4Cl(aq), –78 °C N Boc O OH O OH O O + 7 8 (66%) 9 (11%) Scheme 1. Lithiation of N-Boc pyrrolidine 7 and ketone trapping. N Boc O OH KHMDS, THF reflux, 18 h N O O O 1. TFA, CH2Cl2 rt, 18 h 2. Ac2O, Et3N CH2Cl2, rt, 18 h N O OH O 10 (54%) 8 11 (51%) Scheme 2. Conversion of 8 into 3-D fragments. N N Boc Bn N N Bn O O N Boc N N Bn O O N Boc or 1. 1.3 eq. sBuLi THF, –78 °C, 1 h 2. N O 3. rt, 24 h Boc N O Boc or 12 13 (64%) 14 (42%) Scheme 3. Lithiation of N-Boc piperazine 12 and amino-ketone trapping. M. Lüthy et al. / Bioorg. Med. Chem. 23 (2015) 2680–2694 2681

with TFA delivered amines 15/16 and hydrogenolysis of the N-benzyl group (H2, 10% Pd/C) in 13/14 gave amines 17/18, all reactions proceeding quantitatively (Scheme 4). Furthermore, de novo 3-D fragments were synthesised from amines 15/16. Hydrogenolysis (H2, 10% Pd/C) of 15/16 gave diamines 19 and 20 in 87% and 99% yield respectively (Scheme 5). A related lithiation approach was utilised to provide access to pyrrolidine-derived scaffolds 3 and 4. We selected the 3-pyridyl group as a representative heteroaromatic substituent and the pyrrolidine ring system as depicted in pyridinyl N-Boc pyrrolidine 22. Pyrrolidine 22 was synthesised in two steps (Scheme 6). Reductive amination of 3-chloropropylamine and 3-pyridine carboxaldehyde gave 21 (29% yield). Beak and co-workers have previously described organolithium base-mediated cyclisations of NBoc benzylamines similar to 21. 30 In our hands, an efficient lithiation-cyclisation process was achieved using 1.3 equiv n-BuLi in THF at 78 C for 3 h which gave pyridinyl N-Boc pyrrolidine 22 in 73% yield on a 1.3 g scale. Using this approach, variation of the heteroaromatic group would be possible starting with different heterocyclic aldehydes. Alternatively, Campos at Merck31 and work in our own group17,29 has shown that a range of heteroaryl substituted N-Boc pyrrolidines can be readily prepared from N-Boc pyrrolidine via a-lithiation, transmetallation to an organozinc reagent and subsequent Negishi coupling. Next, we planned to carry out the benzylic a-lithiation-trapping of pyridinyl N-Boc pyrrolidine 22. Such benzylic lithiation-trapping of a-arylated N-Boc pyrrolidines was first reported by Xiao, Lavey et al.32 and has since been further investigated by ourselves18 and Gawley.33 In these types of lithiations, temperatures higher than 78 C need to be employed so that the N-Boc rotamers can interconvert facilitating higher conversion to trapped products. The weaker base, n-BuLi, can be used to selectively deprotonate the more acidic benzylic protons. Lithiation of pyridinyl N-Boc pyrrolidine 22 using 1.3 equiv n-BuLi in THF at 50 C for 30 min was followed by trapping with N-benzyl piperidin-4-one or NBoc azetidin-4-one. After stirring at room temperature to allow cyclisation onto the Boc groups and purification by chromatography, carbamates 23 (62% yield) and 24 (30% yield) were obtained (Scheme 7). N-Benzyl piperidin-4-one was used rather than the Boc-protected version as it gave a product that was more easily separated from the aldol self-condensation byproduct. Interestingly, there was evidence of rotamers in the 1 H NMR spectrum of carbamate 23 (e.g., one of the aromatic protons appeared as two 0.5 H doublets, separated by 80 Hz) which we believe is due to restricted rotation about the sterically congested C-pyridinyl bond. In order to show that carbamates 23 and 24 could be used in library synthesis, their N-protecting groups were removed. Cleavage of the N-benzyl group in 23 using standard hydrogenolysis conditions (H2, 10% Pd/C, MeOH, room temperature) was unsuccessful. Fortunately, more forcing conditions employing Pearlman’s catalyst (Pd(OH)2/C) and ammonium formate under transfer hydrogenolysis conditions in refluxing EtOH delivered piperidine 25 in 77% yield. Boc deprotection of 24 with TFA proceeded smoothly and gave azetidine 26 in 79% yield (Scheme 7). Finally, N-Boc a-lithiation chemistry was employed in accessing piperidine-based scaffolds 5 and 6. 2-Pyridinyl and 3-pyridinyl groups were selected as representative electron deficient heteroaromatic substituents. In previous work, our group had reported a 5-step synthesis of the neurokinin-1 receptor antagonist L-733,060 using a 5- to 6-ring expansion process15 (via an aziridinium ion intermediate25,26). We hypothesised expansion of this methodology to include pyridinyl substituents. To start with, lithiation of N-Boc pyrrolidine 7 was accomplished using our high temperature, diamine-free protocol (1.3 equiv s-BuLi, THF 30 C, 5 min).29 Subsequent trapping with 3-pyridine carboxaldehyde gave a mixture of diastereomeric N-Boc amino alcohols syn-27 and anti-28. Unfortunately, the diastereomeric products were inseparable by chromatography: after purification, we isolated a 43% yield of a 70:30 mixture (by 1 H NMR spectroscopy) of syn27 and anti-28 diastereomeric products (Scheme 8). The assignment of relative stereochemistry in syn-27 and anti-28 by 1 H NMR spectroscopic analysis is described later (vide infra). N N Bn O O N Boc ( )n ( )n 13 n = 1, piperidine 14 n = 0, azetidine N N Bn O O NH ( )n ( )n N H N O O N Boc ( )n ( )n 15 n = 1 (quant.) 16 n = 0 (quant.) 17 n = 1 (quant.) 18 n = 0 (quant.) TFA CH2Cl2 rt, 2 h 10% Pd/C H2, MeOH rt, 18 h Scheme 4. Investigation of deprotection methods. N H N O O NH 19 (87%) N N Bn O O NH ( )n ( )n 15 n = 1, piperidine 16 n = 0, azetidine 10% Pd/C H2, MeOH rt, 18 h N H N O O NH or 20 (99%) Scheme 5. Conversion of 15/16 into 3-D fragments. N Boc N N O H + Cl NH2 •HCl 1. NaBH(OAc)3 ClCH2CH2Cl rt, 3 h 2. Boc2O CH2Cl2 rt, 16 h N N Boc Cl 21 (29%) 1.3 eq. nBuLi 22 (73%) THF, –78 °C 3 h Scheme 6. Synthesis of pyridinyl N-Boc pyrrolidine 22. N Boc N N O NH O N N O O N NH or 1. 1.3 eq. nBuLi THF, –50 °C, 30 min 2. N O 3. rt, 24 h Bn N O Boc or 23 (62%) 24 (30%) 22 N O N O N Bn N O O N N Boc 20% Pd(OH)2/C NH4 +HCO2 – EtOH, reflux, 2 h 23 24 TFA CH2Cl2 rt, 2 h 25 (77%) 26 (79%) Scheme 7. Lithiation of pyridinyl N-Boc pyrrolidine 22 and amino-ketone trapping. N Boc N Boc N OH H N Boc N OH H syn-27 anti-28 1. sBuLi, THF, –30 °C, 5 min 2. N O H 3. NH4Cl(aq) rt, 2 h (43%) 70:30 TFA CH2Cl2 rt, 3 h N H N OH H 70:30 syn:anti NaBH(OAc)3 0.2 eq. AcOH PhCHO ClCH2CH2Cl rt, 16 h N Bn N OH H N Bn N OH H + 7 + syn-29 (30%) anti-30 (30%) Scheme 8. Preparation of N-benzyl amino alcohols syn-29 and anti-30. 2682 M. Lüthy et al. / Bioorg. Med. Chem. 23 (2015) 2680–2694

The aziridinium ion-based ring expansion process requires a nucleophilic N-alkyl substituent and therefore the N-Boc group was transformed into a N-benzyl substituent. TFA deprotection of the 70:30 mixture of syn-27 and anti-28 was followed by reductive amination using benzaldehyde and NaBH(OAc)3. This gave N-benzyl amino alcohols syn-29 (30% yield) and anti-30 (30% yield) over the two steps, which were now, usefully, separable by chromatography. To achieve the desired 5- to 6-ring expansion process, each of syn-29 and anti-30 was subjected to trifluoracetylation (trifluoroacetic anhydride, Et3N) and ring expansion over 48 h at reflux. The initially formed trifluoroacetates were hydrolysed using aqueous NaOH to give the ring-expanded hydroxy-piperidines syn-31 (76% yield) and anti-32 (79%) (Scheme 9). These reactions proceed via ring-opening of the aziridinium ion depicted in Scheme 9. Hydroxy-piperidines syn-31 and anti-32 were characterised fully and detailed analysis of the coupling constants in their 1 H NMR spectra allowed assignment of relative stereochemistry. A diagnostic signal in the 1 H NMR spectrum of anti-32 in CDCl3 was a doublet (J = 9.0 Hz) at dH 2.99 assigned to the benzylic proton a to the nitrogen. The magnitude of this 3 J trans-diaxial coupling confirms that the pyridinyl and hydroxyl groups are trans and equatorially disposed. The corresponding signal in syn-31 (in CDCl3) appeared as a doublet (J = 1.5 Hz) at dH 3.39. Furthermore, these ring expansion reactions were stereospecific: syn-29 gave syn-31 only and anti-30 gave anti-32 only. Taking this together with the known15,25,26 stereochemical features of the ring expansions, we were able to confidently assign the relative stereochemistry in syn-29 and anti-30 to be that as depicted. This allowed us to carry out two further reactions to establish the stereochemistry of N-Boc amino alcohols syn-27 and anti-28. Thus, hydrogenolysis of the benzyl group in N-benzyl amino alcohol syn-29 and subsequent Boc protection gave N-Boc amino alcohol syn-27 in 41% yield (Scheme 10). The same process converted anti-30 into anti-28 (see Section 4). Comparison of the 1 H NMR spectra with those of the inseparable mixture of syn-27 and anti-28 enabled the diastereoselectivity of the initial aldehyde trapping reaction to be determined. We have also verified that the presence of a benzylic pyridinyl C–N bond does not cause a problem in the deprotection of the Nbenzyl piperidine amino alcohols syn-31 and anti-32. Standard hydrogenolysis conditions efficiently delivered deprotected amino alcohols syn-33 (82% yield) and anti-34 (92% yield) (Scheme 11). Amino alcohols syn-33 and anti-34 represent additional novel 3- D fragments and are ready for further N- or O-functionalisation to deliver lead-like library members. As part of this study, we decided to carry out a similar synthetic sequence to complete the 2-pyridinyl series. This would demonstrate the utility of this approach to deliver scaffolds with different heteroaromatic substituents. The reactions used were identical and the only significant difference was that trapping the lithiated NBoc pyrrolidine with 2-pyridine carboxaldehyde was anti-selective (Scheme 12). Presumably, the proximity of the potentially Lichelating 2-pyridinyl substituent interferes with the transition state that generally leads to syn selectivity.15 Thus, we have developed a convenient, connective route to amino alcohols syn-31/39 and anti-32/40 which are suitable protected versions of piperidine-based scaffolds 5 and 6. 2.2. Lead-like and 3-D shape analysis of a virtual 190-member library derived from scaffolds 1–6 Having developed useful synthetic methodology for the synthesis of 3-D scaffolds, the medicinal chemistry properties and 3-D shape of lead-like compounds derived from scaffolds 1–6 was evaluated. To provide a representative set of data, potentially lead-like compounds derived from six of the synthesised compounds (19, 20, 25, 26, syn-33 and anti-34), corresponding to scaffolds 1–6, were enumerated to generate a library of 190 members (Fig. 2; full details in the Supporting information). For each of the three scaffolds 1/2, 3/4 and 5/6, an appropriate set of N-, O- and heteroaromatic groups was selected to decorate the scaffolds and hence to N Bn N OH H syn-29 N Bn OH N N Ar H H Bn O2CCF3 1. (CF3CO)2O, THF –78 °C, 1 h 2. Et3N, –78 °C, 1 h 3. reflux, 48 h 4. NaOH(aq), rt, 2 h via: syn-31 (76%) N Bn N OH H anti-30 N Bn OH N 3. reflux, 48 h 4. NaOH(aq), rt, 2 h anti-32 (79%) 1. (CF3CO)2O, THF –78 °C, 1 h 2. Et3N, –78 °C, 1 h Scheme 9. Ring expansion of pyrrolidines to piperidines. N Bn N OH H syn-29 1. 20% Pd(OH)2/C NH4 +HCO2 – EtOH, reflux, 2 h 2. Boc2O, EtOH 50 °C, 16 h N Boc N OH H syn-27 (41%) Scheme 10. Synthesis of syn-27. N H OH N 20% Pd(OH)2/C NH4 +HCO2 – EtOH, reflux, 16 h syn-31 or anti-32 syn-33 (82%) or N H OH N anti-34 (92%) Scheme 11. Investigation of N-benzyl deprotection. N Boc N Boc N OH H N Boc N OH H syn-35 anti-36 1. sBuLi, THF, –30 °C, 5 min 2. N O H 3. NH4Cl(aq) rt, 2 h (69%) 40:60 TFA CH2Cl2 rt, 3 h N H N OH H 40:60 syn:anti NaBH(OAc)3 0.2 eq. AcOH PhCHO ClCH2CH2Cl rt, 16 h N Bn N OH H N Bn N OH H + 7 + syn-37 (11%) anti-38 (28%) syn-37 or anti-38 N Bn OH N 1. (CF3CO)2O, THF –78 °C, 1 h 2. Et3N, –78 °C, 1 h 3. reflux, 48 h 4. NaOH(aq), rt, 2 h syn-39 (46%) N Bn OH N anti-40 (69%) or Scheme 12. Synthesis of 2-pyridinyl hydroxy piperidines. M. Lüthy et al. / Bioorg. Med. Chem. 23 (2015) 2680–2694 2683

generate compounds which could possess lead-like properties. All 190 library members would be synthetically viable using simple functionalisation of suitably protected derivatives of scaffolds 1– 6. The enumeration is shown schematically in Figure 2 and is illustrated with L-11, L-96, L-120 and L-184, four potential lead-like compounds. The following properties were calculated for compounds L-1 to L-190: AlogP, 34 molecular weight, number of heavy atoms, polar surface area, number of hydrogen bond acceptors/donors, number of rotatable bonds and number of aromatic rings. Full details are reported in the Supporting information. Using the criteria proposed by Nadin et al.,6 lead-like compounds should have 1 6 AlogP 6 3 and molecular size in the range 14 6 number of heavy atoms 6 26. Of the 190 library members, 150 (79%) have an AlogP value in the suggested range and 137 (72%) have 14 6 number of heavy atoms 6 26. An additional criterion to consider is the total polar surface area and values P50 Å2 are useful for lead-like compounds: 160 (84%) library members satisfy this criterion. Taking all of these parameters together, we found that 92 compounds (48%) of the 190 virtual library members could be considered lead-like in terms of these three properties. Next, analysis of the 3-D shape of the 190 members of the virtual library was carried out. There are a number of methods that have been used to characterise the 3-D shape of molecules including principal moments of inertia (PMI) plots,28 molecular globularity35 and determination of the plane of best fit.36 Of these, we opted to analyse the 3-D shapes using the triangular plot of normalised principal moments of inertia (PMI) for the molecular mechanics-generated lowest energy conformation, as introduced by Sauer and Schwarz.28 The three vertices of these plots correspond to rod, disc and spherical shapes. In general, there is a proliferation of fragment and drug molecules with flat and disc shapes (i.e. lowest energy conformations that lie close to the rod-disc axis).13,20,21,36 Molecules which occupy areas of the PMI plot distant from this well-populated area will be of interest. To start with, the molecular-mechanics derived lowest energy conformations were generated for compounds in the virtual library which correspond to scaffolds 1–6 (compounds 19, 20, 25, 26, syn33 and anti-34 shown in Figure 3) as described in the Section 4 and Supporting information. The PMI values for these conformations are plotted in Figure 4. For our analysis, an ‘attractive area’ of the PMI plot has been defined where compounds have a NPR1 value P0.35 and a NPR2 value P0.7. This area of the PMI plot corresponds to an under-represented region of 3-D pharmaceutical space. Despite the fact that all six compounds 19, 20, 25, 26, syn-33 and anti-34 appear 3-D on paper, only three (20, 25 and 26) occupy this central, under-represented, attractive part of the plot. However, decoration of the scaffolds that are initially outside this area and lie closer to the roddisc axis (19, syn-33 and anti-34) with substituents generates lead-like compounds which are then in the attractive 3-D area of the PMI plot (vide infra). Given the developing interest in 3-D N H O O S O O F F N HetAr O H O O N N N N N N O O N ( )n ( )n 1 n = 1 2 n = 0 Scaffold N-Substituents N O N O HetAr ( )n ( )n 3 n = 1 4 n = 0 Scaffold N H O O S O O F F N-Substituents N N N N N O S N Heteroaromatics 50 compounds 50 compounds 5 cis 6 trans 90 compounds Scaffold N H O O S O O F F N-Substituents O-Substituents Heteroaromatics N N O O NH N O O NH N OH S O N O F F S N O Virtual lead-like compounds N O O O N N L-11 L-96 L-120 L-184 Figure 2. Enumeration of a virtual library with potential lead-like properties. N H N O O NH 19 (L-1) N H N O O NH 20 (L-26) N O NH O N N O O N NH 25 (L-51) 26 (L-76) N H OH N syn-33 (L-101) N H OH N anti-34 (L-146) MW 211 AlogP –3.5 MW 183 AlogP –2.4 MW 273 AlogP –0.4 MW 245 AclogP 0.7 MW 178 AlogP –1.0 MW 178 AlogP –1.0 Figure 3. Structures of 3-D scaffolds (fragments) synthesised in this study. Figure 4. PMI plot of compounds 19 (L-1), 20 (L-26), 25 (L-51), 26 (L-76), syn-33 (L101) and anti-34 (L-146); NPR is the normalised principal moment of inertia (see Section 4.3). 2684 M. Lüthy et al. / Bioorg. Med. Chem. 23 (2015) 2680–2694

fragments,20,21 it should be highlighted that compounds 19, 20, 25, 26, syn-33 and anti-34 can be classified as fragments. Five of these fragments (19, 20, 26, syn-33 and anti-34) satisfy typical fragment criteria with molecular weight in the range 150–250 (Fig. 3) 37 but only two (20 and 26) have interesting 3-D shapes based on our definition of the attractive area of the PMI plot. The 3-D shape of the whole 190-member virtual library was then evaluated using a PMI plot (Fig. 5). Of note, the PMI plot shows that this library contains a wide range of 3-D shapes and shows excellent coverage of 3-D chemical space. However, a significant number of compounds lie close to the unattractive rod-disc axis. Using our defined selection criterion of compounds with a NPR1 value P0.35 and a NPR2 value P0.7, there are only 77 (40%) of compounds in the under-represented area. This is perhaps surprising since, on paper at least, all six scaffolds and the derived virtual library members appear significantly 3-D in structure (see Supporting information). The PMI plots for the potentially lead-like compounds derived from each of scaffolds 1–6 are depicted in Figure 6 and are particularly revealing. All of the compounds enumerated from the spirocyclic fused piperazine scaffolds 1 and 2 lie close to the unattractive rod-disc axis of the PMI plot. Indeed, only 3 out of 50 compounds have a NPR1 value P0.35 and a NPR2 value P0.7. This clearly emphasises that structures with apparently interesting 3-D shapes (at least on paper and to the eye) may not actually have low energy conformations in interesting areas of 3-D chemical space. In contrast, the other four scaffolds (3–6) generate a greater proportion of compounds with a NPR1 value P0.4 and a NPR2 value P0.7. Of these four scaffolds, the simplest piperidine scaffolds 5 and 6 show the most significant 3-D shape diversity and Figure 5. PMI plot of 190 members of the virtual library. Figure 6. PMI plot of all members of the virtual library by scaffold type (1–6). M. Lüthy et al. / Bioorg. Med. Chem. 23 (2015) 2680–2694 2685

have the highest proportions of compounds in the more interesting areas of 3-D chemical space. Finally, the lead-like property analysis described earlier was combined with a 3-D PMI-based shape analysis. There are 92 compounds (48%) out of the 190 virtual library members that fit the lead-like criteria of: (i) 1 6 AlogP 6 3; (ii) 14 6 number of heavy atoms 6 26; (iii) total polar surface area P50 Å2 . The PMI plot of these 92 compounds is shown in Figure 7. Clearly, these compounds show good 3-D shape diversity. Focusing in on the attractive 3-D shape region of the PMI plot, there are 46 (24%) which satisfy the 3-D shape criterion of a NPR1 value P0.35 and a NPR2 value P0.7. These compounds can be considered lead-like in their properties and with interesting 3-D shape. A PMI plot of only these 46 compounds is shown in Figure 8. Such a plot could potentially be used to select compounds for synthesis from this virtual library. The four compounds that are most spherical in 3-D shape based on the PMI analysis are L-91, L-109, L-114 and L128 (Fig. 9). Although all four compounds are spherical in shape, it should be emphasised that each presents different types of protein-binding vectors that are oriented in different directions. 3. Conclusion In conclusion, we have demonstrated that new examples of NBoc a-lithiation synthetic chemistry can be used to provide access to some structurally diverse scaffolds 1–6 based on piperazine, pyrrolidine and piperidine cores. A key feature of the approach to scaffolds 1–4 is its connectivity and, as a result, it could be expanded to include different N-Boc heterocycles and different electrophiles. In addition, the routes to scaffolds 3–6 were validated using different 2- and 3-pyridinyl substituents to show that the methodology was compatible with representative electron deficient heteroaromatic substituents. Our synthetic approach fits most of the criteria of lead-oriented synthesis.6,38 Starting from scaffolds 1–6, a virtual library of 190 compounds was enumerated. All of the compounds would be synthetically accessible based on the methodology developed herein. Evaluation of the lead-like properties and 3-D shape of the 190 library members showed that 46 compounds could be considered both lead-like and with interesting 3-D shape. With regards to the PMI plot, we have defined compounds with a NPR1 value P0.35 and a NPR2 value P0.7 as being of particular interest as this represents an under-populated area of drug-space. One aspect of the PMI-based 3-D shape analysis that our work reveals is that structures which are apparently 3-D on paper do not necessarily lead to 3-D shapes in new areas of pharmaceutical space. For example, of the four compounds (L-11, L-96, L-120 and L-184) presented in Figure 2 as representative members of the virtual library, only L-96 and L-120 fit our 3-D shape criteria. Our study also showed that whilst the scaffold itself may not occupy the interesting area of the PMI plot, lead-like compounds derived from it could do so. We believe that a more detailed analysis of 3-D shape is useful when considering lead-like properties of virtual libraries. We also suggest that PMI plots of virtual libraries could be used to select compounds for synthesis. In this regard, from our data, compounds L-91, L-109, L-114 and L-128 have lead-like properties and populate a rarely occupied region of pharmaceutical space, as they are the most spherical in 3-D shape. 4. Experimental section 4.1. General All-non aqueous reactions were carried out under oxygen-free Ar using flame-dried glassware. THF was freshly distilled from sodium and benzophenone. s-BuLi and n-BuLi were titrated against N-benzylbenzamide before use.39 Petrol refers to the fraction of petroleum ether boiling in the range of 40–60 C and was purchased in Winchester quantities. Brine refers to a saturated solution. Water is distilled water. Flash column chromatography was carried out using Fluka Chemie GmbH silica (220–440 mesh). Thin layer chromatography was carried out using commercially available Merk F254 aluminium backed silica plates. Proton (400 MHz) and carbon (100.6 MHz) NMR spectra were recorded on a Jeol ECX-400 instrument using an internal deuterium lock. For samples recorded in CDCl3, chemical shifts are quoted on parts per million relative to CHCl3 (dH 7.26) and CDCl3 (dC 77.0, central line of triplet). For samples recorded in CD3OD, chemical shifts are quoted on parts per million relative to dH 4.78 and CD3OD (dC 49.15, central line of septet). Carbon NMR spectra were recorded with broad band decoupling and assigned using DEPT experiments. Coupling constants (J) are quoted in Hertz. Melting points were Figure 7. PMI plot of 92 lead-like compounds. Figure 8. PMI plot of 46 lead-like compounds with a NPR1 value P0.35 and a NPR2 value P0.7. N O O NH N O N O N L-91 L-109 O N O N L-114 O O N O N L-128 O O Figure 9. Members of the virtual library that satisfy lead-like properties and are most spherical in 3-D shape. 2686 M. Lüthy et al. / Bioorg. Med. Chem. 23 (2015) 2680–2694

carried out on a Gallenkamp melting point apparatus. Infrared spectra were recorded on an ATI Mattson Genesis FT-IR spectrometer. Electrospray high and low resonance mass spectra were recorded at room temperature on a Bruker Daltronics micrOTOF spectrometer. N-Boc pyrrolidine 7 and N-Boc piperazine 12 were synthesised according to the published procedures.29 4.1.1. General procedure A: hydrogenolysis using 10% Pd/C and H2 10% Pd/C (0.03 mmol, 0.1 equiv) was added to a stirred solution of the carbamate (0.32 mmol, 1.0 equiv) in MeOH–EtOAc (4:1, 2.5 mL) or MeOH (2.5 mL) at rt. Then, the reaction flask was evacuated under reduced pressure and back-filled with Ar three times. After a final evacuation, a balloon of H2 was attached and the reaction mixture was stirred vigorously at rt under H2 for 18 h. Then, the solids were removed by filtration through Celite and washed with MeOH (15 mL). The filtrate was evaporated under reduced pressure to give the crude depotected amine. 4.1.2. General procedure B: hydrogenolysis using 20% Pd(OH)2/C and ammonium formate Pd(OH)2/C (8 mg, 20 wt.%, 0.05 mmol, 0.1 equiv) and ammonium formate (96 mg, 1.50 mmol, 5.0 equiv) were added to a stirred solution of the N-benzyl amine (40 mg, 0.15 mmol, 1.0 equiv) in EtOH (10 mL) at rt under Ar. The resulting mixture was stirred and heated at reflux for 2 or 16 h. Then, the reaction mixture was allowed to cool to rt and the solids were removed by filtration through a plug of Celite and washed with EtOH (15 mL). The filtrate was evaporated under reduced pressure to give the crude deprotected amine. 4.1.3. General procedure C: ring expansion of pyrrolidine to piperidine Trifluoroacetic anhydride (0.25 mL, 1.38 mmol, 3.0 equiv) was added to a stirred solution of the N-benzyl hydroxy pyrrolidine (0.46 mmol, 1.0 equiv) in THF (10 mL) at 78 C under Ar. The resulting solution was stirred at 78 C for 1 h and then Et3N (3.0 equiv) was added dropwise. The resulting solution was stirred at 78 C for 1 h. Then, the resulting mixture was allowed to warm to rt. The reaction mixture was then stirred and heated at reflux for 48 h. The reaction was allowed to cool to rt and 20% NaOH(aq) (10 mL) was added dropwise. Then, the resulting mixture was stirred at rt for 2 h. CH2Cl2 (10 mL) was added and the two layers were separated. The aqueous layer was extracted with CH2Cl2 (3  10 mL) and the combined organics were dried (MgSO4) and evaporated under reduced pressure to give the crude product. 4.1.4. General procedure D: hydrogenolysis-Boc protection 20% Pd(OH)2/C (12 mg, 0.08 mmol, 0.3 equiv) and ammonium formate (5.0–15.0 equiv) were added to a stirred solution of the N-benzyl amine (62 mg, 0.23 mmol, 1.0 equiv) in EtOH (10 mL) at rt under Ar. The resulting mixture was stirred and heated at reflux for 2–5 h. Then, the reaction mixture was allowed to cool to 50 C and Boc2O (55 mg, 0.25 mmol, 1.1 equiv) was added. The reaction mixture was stirred and heated at 50 C for 16 h. After cooling to rt, EtOH (10 mL) was added. The solids were removed by filtration through a plug of Celite and washed with EtOH (15 mL). The filtrate was evaporated under reduced pressure to give the crude product. 4.2. Experimental procedures and characterisation data 4.2.1. tert-Butyl 2-(4-hydroxytetrahydro-2H-pyran-4- yl)pyrrolidine-1-carboxylate 8 and 40 -hydroxyhexahydro2H,20 H-3,40 -bipyran-4(3H)-one 9 s-BuLi (0.76 mL of a 1.3 M solution in hexane, 1.0 mmol, 1.0 equiv) was added dropwise to a stirred solution of N-Boc pyrrolidine 7 (0.18 mL, 1.0 mmol, 1.0 equiv) in THF (7 mL) at 78 C under Ar. The resulting yellow solution was stirred at 78 C for 1 h. Then, tetrahydro-4H-pyran-4-one (0.20 mL, 2.0 mmol, 2.0 equiv) was added. The resulting solution was stirred at 78 C for 10 min and saturated NH4Cl(aq) (5 mL) was added. The resulting mixture was then allowed to warm to rt over 1 h. The mixture was extracted with Et2O (3 x 10 mL). The combined organic extracts were dried (MgSO4) and evaporated under reduced pressure to give the crude product. Purification by flash chromatography on silica with 35:65, 25:75 pet. ether–EtOAc and EtOAc as eluent gave amino alcohol 8 (171 mg, 66%) as a pale yellow oil, Rf (40:60 pet. ether–EtOAc) 0.5; IR (ATR) 3375 (OH), 2959, 2872, 1660 (C@O), 1392, 1366, 1160, 1096, 955, 843, 538 cm1 ; 1 H NMR (400 MHz, CDCl3) d 3.89–3.73 (m, 7H, NCH, OCH), 3.67 (br s, 1H, OH), 3.18–3.08 (m, 1H, CH), 2.09–1.93 (m, 1H, CH), 1.84 (s, 1H, CH), 1.78–1.51 (m, 5H, CH), 1.46 (s, 9H, CMe3); 13C NMR (101.6 MHz, CDCl3) d 157.7 (C@O), 80.7 (CMe3), 75.9 (OCH2), 71.8 (OCH2), 63.7 (COH), 63.4 (NCH), 48.3 (NCH2), 35.8 (CH2), 28.4 (CMe3), 28.3 (CH2), 24.4 (CH2), 23.8 (CH2); MS (ESI) m/z 294 [(M+Na)+ , 100]; HMRS (ESI) m/z calcd for C14H25NO4 (M+Na)+ 294.1676, found 294.1678 (0.7 ppm error) and aldol 9 (22 mg, 11%) as a pale yellow oil, Rf (40:60 pet. ether–EtOAc) 0.2; IR (ATR) 3417 (OH), 2970, 2862, 1727 (C@O), 1396, 1226, 1100, 1060, 937, 772, 577 cm1 ; 1 H NMR (400 MHz, CDCl3) d 4.26 (ddd, J = 11.5, 6.0, 1.5 Hz, 1H, OCH), 4.17 (dddd, J = 11.0, 6.5, 3.0, 1.5 Hz, 1H, OCH), 3.85–3.65 (m, 6H, OCH), 3.40 (br s, 1H, OH), 2.68–2.55 (m, 2H, CH), 2.47–2.39 (m, 1H, CH), 1.75–1.53 (m, 4H, CH); 13C NMR (101.6 MHz, CDCl3) d 210.1 (C@O), 69.0 (OCH2), 68.9 (COH), 68.5 (OCH2), 63.1 (OCH2), 63.0 (OCH2), 59.7 (CH), 44.0 (CH2), 36.5 (CH2), 34.5 (CH2); MS (ESI) m/ z 223 [(M+Na)+ , 100]; HMRS (ESI) m/z calcd for C10H16O4 (M+Na)+ 223.0941, found 223.0937 (+1.3 ppm error). 4.2.2. Octahydro-30 H-spiro[pyran-4,10 -pyrrolo[1,2-c]oxazol]-30 - one 10 KHMDS (0.81 mL of a 0.5 M solution in toluene, 0.405 mmol, 1.1 equiv) was added dropwise to a stirred solution of amino alcohol 8 (100 mg, 0.368 mmol, 1.0 equiv) in THF (5 mL) at 78 C under Ar. The resulting solution was stirred at 78 C for 20 min before being warmed to rt and stirred at rt for 16 h. Saturated NH4Cl(aq) (10 mL) was added and the mixture was extracted with Et2O (3  5 mL). The combined organic extracts were dried (MgSO4) and evaporated under reduced pressure to give the crude product. Purification by flash chromatography on silica with 50:50 EtOAc–pet. ether as eluent gave carbamate 10 (39 mg, 54%) as a colourless oil, Rf (1:1 EtOAc–pet. ether) 0.1; IR (ATR) 2960, 2870, 1738 (C@O), 1395, 1226, 1102, 1029, 914, 771, 547 cm1 ; 1 H NMR (400 MHz, CDCl3) d 3.90–3.76 (m, 4H, OCH), 3.62 (ddd, J = 11.0, 8.0, 8.0 Hz, 1H, NCH), 3.52 (dd, J = 10.5, 5.5 Hz, 1H, NCH), 3.19 (ddd, J = 11.0, 9.0, 3.0 Hz, 1H, NCH), 2.15–2.06 (m, 1H, CH), 2.03–1.95 (m, 1H, CH), 1.94–1.74 (m, 5H, CH), 1.51 (dddd, J = 11.0, 11.0, 11.0, 8.0 Hz, 1H, CH); 13C NMR (101.6 MHz, CDCl3) d 160.4 (C@O), 78.3 (CO), 68.6 (NCH), 64.2 (OCH2), 64.0 (OCH2), 45.3 (NCH2), 38.0 (CH2), 32.6 (CH2), 25.5 (CH2), 25.4 (CH2); MS (ESI) m/z 220 [(M+Na)+ , 100], 198 [M+H)+ , 5]; HMRS (ESI) m/z calcd for C10H15NO3 (M+Na)+ 220.0944, found 220.0944 (0.0 ppm error). 4.2.3. 1-(2-(4-Hydroxytetrahydro-2H-pyran-4-yl)pyrrolidin-1- yl)ethanone 11 TFA (1 mL, 13.1 mmol) was added to a stirred solution of amino alcohol 8 (100 mg, 0.369 mmol, 1.0 equiv) in CH2Cl2 (1 mL) at rt under Ar. The resulting solution was stirred at rt for 18 h. The solvent was then evaporated under reduced pressure to give the crude deprotected amino alcohol. Acetic anhydride (0.097 mL, 1.03 mmol, 2.7 equiv) was added to a stirring solution of crude deprotected amino alcohol (118 mg) and Et3N (0.14 mL, M. Lüthy et al. / Bioorg. Med. Chem. 23 (2015) 2680–2694 2687

1.03 mmol, 2.7 equiv) in CH2Cl2 (5 mL) at 0 C under Ar. The resulting solution was stirred at 0 C for 3 h before being allowed to warm to rt for 2 h and then stirred at rt for 16 h. Water (10 mL) was added and the mixture was extracted with CH2Cl2 (3  10 mL). The combined organic extracts were dried (MgSO4) and evaporated under reduced pressure to give crude product. Purification by flash chromatography on silica with 90:10 EtOAc– pet. ether as eluent gave acetamide 11 (40 mg, 51%) as a pale orange solid, mp 75–79 C; Rf (90:10 EtOAc–pet. ether) 0.2; IR (ATR) 3273 (OH), 2948, 2870, 1612 (C@O), 1496, 1136, 1087, 1017, 846, 531 cm1 ; 1 H NMR (400 MHz, CDCl3) d 4.11 (dd, J = 7.5, 7.5 Hz, 1H, NCH), 3.90–3.81 (m, 2H, OCH), 3.80–3.71 (m, 2H, OCH), 3.67–3.61 (m, 1H, NCH), 3.41–3.30 (m, 1H, NCH), 2.15 (s, 3H, Me), 2.14–1.83 (m, 2H, CH), 1.87–1.74 (m, 2H, CH), 1.68 (ddd, J = 12.5, 12.5, 5.5 Hz, 1H, CH), 1.57 (ddd, J = 12.5, 12.5, 5.5 Hz, 1H, CH), 1.42 (dddd, J = 12.5, 2.0, 2.0, 2.0 Hz, 1H, CH), 1.21 (dddd, J = 12.5, 2.0, 2.0, 2.0 Hz, 1H, CH); 13C NMR (101.6 MHz, CDCl3) d 172.8 (C@O), 71.4 (COH), 68.0 (NCH), 63.5 (CH2), 63.4 (CH2), 49.9 (CH2), 35.9 (CH2), 31.2 (CH2), 28.2 (CH2), 24.6 (CH2), 23.4 (Me); MS (ESI) m/z 236 [(M+Na)+ , 100]; HMRS (ESI) m/z calcd for C11H19NO3 (M+Na)+ 236.1257, found 236.1264 (3.1 ppm error). 4.2.4. tert-Butyl 7-benzyl-3-oxohexahydrospiro[oxazolo[3,4- a]pyrazine-1,40 -piperidine]-10 -carboxylate 13 s-BuLi (1.0 mL of a 1.3 M solution in hexanes, 1.3 mmol, 1.3 equiv) was added dropwise to a stirred solution of N-Boc-N0 - benzyl piperazine 12 (276 mg, 1.0 mmol, 1.0 equiv) in THF (9 mL) at 78 C under Ar. The resulting solution was stirred at 78 C for 60 min. Then, a solution of N-Boc-piperidin-4-one (399 mg, 2.0 mmol, 2.0 equiv) in THF (1 mL) was added dropwise. The resulting solution was stirred at 78 C for 10 min. The reaction mixture was allowed to warm to rt over 24 h. Then, saturated NH4Cl(aq) (1 mL) was added and the two layers were separated. The aqueous layer was extracted with Et2O (3  20 mL). The combined organic layers were dried (MgSO4) and evaporated under reduced pressure to give the crude product. Purification by flash column chromatography on silica with 4:1 EtOAc–pet. ether as eluent gave carbamate 13 (256 mg, 64%) as a white solid, mp 103– 105 C, Rf (EtOAc) 0.5; IR (ATR) 1761 (C@O), 1682 (C@O) 1397, 1158, 735, 697 cm1 ; 1 H NMR (400 MHz, CDCl3) d 7.35–7.25 (m, 5H, Ph), 4.11 (br s, 2H, NCH), 3.76 (dd, J = 13.0, 2.5 Hz, 1H, NCH), 3.57 (d, J = 13.0 Hz, 1H, NCHAHBPh), 3.49 (d, J = 13.0 Hz, 1H, NCHAHBPh), 3.41 (dd, J = 11.0, 3.0 Hz, 1H, NCH), 3.13–3.03 (m, 3H, NCH), 2.77 (dd, J = 11.0, 3.5 Hz, 1H, NCH), 2.72 (dd, J = 11.0, 2.5 Hz, 1H, NCH), 2.09 (ddd, J = 11.5, 11.5, 3.5 Hz, 1H, CH), 1.97 (dd, J = 11.0, 11.0 Hz, 1H, CH), 1.85 (br d, J = 13.0 Hz, 1H, CH), 1.70–1.57 (m, 1H, CH), 1.53–1.46 (m, 2H, CH), 1.44 (s, 9H, CMe3); 13C NMR (100.6 MHz, CDCl3) d 155.9 (C@O), 154.4 (C@O), 137.3 (ipso-Ph), 129.0 (Ph), 128.6 (Ph), 127.6 (Ph), 79.9 (CO), 78.8 (CMe3), 63.2 (NCH2), 61.7 (NCH), 52.7 (NCH2), 51.8 (NCH2Ph), 41.3 (NCH2), 41.2 (NCH2), 36.3 (CCH2), 30.6 (CCH2), 28.5 (CMe3); MS (ESI) m/z 424 [(M+Na)+ ], 401 [(M+H)+ ]; HRMS m/z calcd for C22H32N3O4 (M+H)+ 402.2387, found 402.2381 (+2.5 ppm error). 4.2.5. tert-Butyl 70 -benzyl-30 -oxohexahydrospiro[azetidine-3,10 - oxazolo[3,4-a]pyrazine]-1-carboxylate 14 s-BuLi (2.0 mL of a 1.3 M solution in hexanes, 2.6 mmol, 1.3 equiv) was added dropwise to a stirred solution of N-Boc-N0 - benzyl piperazine 12 (552 mg, 2.0 mmol, 1.0 equiv) in THF (18 mL) at 78 C under Ar. The resulting solution was stirred at 78 C for 60 min. Then, a solution of N-Boc-azetidin-3-one (684 mg, 4.0 mmol, 2.0 equiv) in THF (2 mL) was added dropwise. The resulting solution was stirred at 78 C for 10 min. The reaction mixture was allowed to warm to rt over 24 h. Then, saturated NH4Cl(aq) (1 mL) was added and the two layers were separated. The aqueous layer was extracted with Et2O (3  20 mL). The combined organic layers were dried (MgSO4) and evaporated under reduced pressure to give the crude product. Purification by flash column chromatography on silica with 4:1 EtOAc–pet. ether as eluent gave carbamate 14 (317 mg, 42%) as a white solid, mp 122–125 C, Rf (EtOAc) 0.5; IR (ATR) 1759 (C@O), 1684 (C@O), 1396, 1084, 739 cm1 ; 1 H NMR (400 MHz, CDCl3) d 7.37–7.27 (m, 5H, Ph), 4.19 (d, J = 10.0 Hz, 1H, NCH), 4.06 (d, J = 10.5 Hz, 1H, NCH), 3.98 (d, J = 10.5 Hz, 1H, NCH), 3.96 (d, J = 10.0 Hz, 1H, NCH), 3.81–3.72 (m, 2H, NCH), 3.58 (d, J = 13.0 Hz, 1H, NCHAHBPh), 3.51 (d, J = 13.0 Hz, 1H, NCHAHBPh), 3.08 (ddd, J = 13.0, 13.0, 3.5 Hz, 1H, NCH), 2.98 (dd, J = 11.5, 3.5 Hz, 1H, NCH), 2.78 (dd, J = 11.5, 3.0 Hz, 1H, NCH), 2.03 (ddd, J = 12.0, 12.0, 3.5 Hz, 1H, NCH), 1.83 (dd, J = 11.0, 11.0 Hz, 1H, NCH), 1.41 (s, 9H, CMe3); 13C NMR (100.6 MHz, CDCl3) d 155.9 (C@O), 155.1 (C@O), 137.3 (ipso-Ph), 129.0 (Ph), 128.6 (Ph), 127.6 (Ph), 80.6 (CO), 75.3 (CMe3), 63.0 (NCH2), 59.4 (NCH), 53.8 (NCH2), 51.5 (NCH2Ph), 41.2 (NCH2), 28.4 (NCH2), 28.3 (CMe3); MS (ESI) m/z 396 [(M+Na)+ ], 374 [(M+H)+ ]; HRMS m/z calcd for C20H28N3O4 (M+H)+ 374.2074, found 374.2065 (+2.9 ppm error). 4.2.6. 7-Benzyltetrahydrospiro[oxazolo[3,4-a]pyrazine-1,40 - piperidin]-3(5H)-one 15 TFA (5.0 mL, 65 mmol, 60 equiv) was added to a stirred solution of carbamate 13 (441 mg, 1.1 mmol, 1.0 equiv) in CH2Cl2 (5 mL) at rt. The resulting solution was stirred at rt for 2 h. Then, NH4OH(aq) (10 mL) was added and the mixture was extracted with CH2Cl2 (3  20 mL). The combined organic layers were dried (MgSO4) and evaporated under reduced pressure to give amine 15 (340 mg, quant.) as a white solid, mp 182–185 C, Rf (100:9:1 EtOAc–MeOH–NH4OH) 0.4; IR (ATR) 3316 (NH), 2923, 2813, 1730 (C@O), 1421, 741, 699 cm1 ; 1 H NMR (400 MHz, CDCl3) d 7.35– 7.23 (m, 5H, Ph), 3.76 (dd, J = 13.0, 2.5 Hz, 1H, NCH), 3.56 (d, J = 13.5 Hz, 1H, NCHAHBPh), 3.52 (d, J = 13.5 Hz, 1H, NCHAHBPh), 3.41 (dd, J = 11.0, 3.5 Hz, 1H, NCH), 3.09–2.91 (br m, 4H, NCH2), 2.78–2.75 (m, 2H, NCH), 2.05–1.79 (m, 6H, CH and NH), 1.72 (ddd, J = 13.5, 11.5, 4.5 Hz, 1H, CH), 1.60 (ddd, J = 13.5, 11.5, 4.5 Hz, 1H, CH); 13C NMR (100.6 MHz, CDCl3) d 156.1 (C@O), 137.4 (ipso-Ph), 129.1 (Ph), 128.6 (Ph), 127.6 (Ph), 79.1 (CO), 63.2 (NCH2), 61.9 (NCH), 52.8 (NCH2), 51.8 (NCH2Ph), 42.3 (NCH2), 42.2 (NCH2), 41.2 (NCH2), 37.0 (CCH2), 31.4 (CCH2); MS (ESI) m/z 324 [(M+Na)+ ], 302 [(M+H)+ ]; HRMS m/z calcd for C17H24N3O2 (M+H)+ 302.1863, found 302.1859 (+1.3 ppm error). 4.2.7. 70 -Benzyltetrahydrospiro[azetidine-3,10 -oxazolo[3,4- a]pyrazin]-30 (50 H)-one 16 TFA (1.0 mL, 13 mmol, 60 equiv) was added to a stirred solution of carbamate 14 (79 mg, 0.21 mmol, 1.0 equiv) in CH2Cl2 (1 mL) at rt. The resulting solution was stirred at rt for 2 h. Then, NH4OH(aq) (2 mL) was added and the mixture was extracted with CH2Cl2 (3  20 mL). The combined organic layers were dried (MgSO4) and evaporated under reduced pressure to give amine 16 (59 mg, quant.) as a colourless oil, Rf (100:9:1 EtOAc–MeOH–NH4OH) 0.3; IR (ATR) 3331 (NH), 2929, 2867, 2812, 1748 (C@O) cm1 ; 1 H NMR (400 MHz, CDCl3) d 7.37–7.22 (m, 5H, Ph), 4.02 (d, J = 10.0 Hz, 1H, NCH), 3.93–3.87 (m, 2H, NCH), 3.77–3.67 (m, 3H, NCH), 3.55 (s, 2H, NCH2Ph), 3.44–3.04 (m, 4H, NCH), 2.77–2.72 (m, 1H, NCH), 1.99 (dddd, J = 11.0, 11.0, 3.5, 1.0 Hz, 1H, NCH), 1.88 (dd, J = 11.0, 11.0 Hz, 1H, NCH); 13C NMR (100.6 MHz, CDCl3) d 155.4 (C@O), 137.2 (ipso-Ph), 129.0 (Ph), 128.5 (Ph), 127.5 (Ph), 79.3 (CO), 63.0 (NCH2), 60.1 (NCH2), 59.8 (NCH), 54.2 (NCH2), 53.4 (NCH2), 51.5 (NCH2), 41.2 (NCH2); MS (ESI) m/z 296 [(M+Na)+ ], 274 [(M+H)+ ]; HRMS m/z calcd for C15H20N3O2 (M+H)+ 274.1550, found 274.1540 (+3.6 ppm error). 2688 M. Lüthy et al. / Bioorg. Med. Chem. 23 (2015) 2680–2694

4.2.8. tert-Butyl 3-oxohexahydrospiro[oxazolo[3,4-a]pyrazine1,40 -piperidine]-10 -carboxylate 17 Using general procedure A, 10% Pd/C (53 mg, 0.05 mmol, 0.1 equiv) and carbamate 13 (200 mg, 0.50 mmol, 1.0 equiv) in MeOH–EtOAc (4:1, 5 mL). gave amine 17 (162 mg, quant.) as a colourless oil, Rf (100:9:1 EtOAc–MeOH–NH4OH) 0.4; IR (ATR) 3333 (NH), 2976, 2953, 2930, 1742 (C@O), 1674 (C@O), 1422, 1158, 727 cm1 ; 1 H NMR (400 MHz, CDCl3) d 3.93 (br s, 2H, NCH), 3.75–3.71 (m, 1H, NCH), 3.33–3.29 (m, 1H, NCH), 3.22– 2.82 (m, 5H, NCH), 2.65–2.55 (m, 2H, NCH), 1.88–1.76 (m, 2H, CH), 1.67–1.51 (m, 1H, CH), 1.41 (s, 9H, CMe3), 1.41–1.35 (m, 1H, CH); 13C NMR (100.6 MHz, CDCl3) (rotamers) d 155.8 (C@O), 154.5 (C@O), 79.9 (CO), 79.0 (CMe3), 78.7 (CMe3), 62.0 (NCH), 61.5 (NCH), 50.1 (NCH2), 45.7 (NCH2), 44.5 (NCH2), 41.8 (NCH2), 41.0 (NCH2), 36.1 (CCH2), 30.6 (CCH2), 28.4 (CMe3); MS (ESI) m/z 334 [(M+Na)+ ], 312 [(M+H)+ ], 256 [(M – CMe3) + ]; HRMS m/z calcd for C15H25N3O4 (M+Na)+ 334.1737, found 334.1731 (+1.7 ppm error). 4.2.9. tert-Butyl 30 -oxohexahydrospiro[azetidine-3,10 -oxazolo- [3,4-a]pyrazine]-1-carboxylate 18 Using general procedure A, 10% Pd/C (17 mg, 0.03 mmol, 0.1 equiv) and carbamate 14 (120 mg, 0.32 mmol, 1.0 equiv) in MeOH–EtOAc (4:1, 2.5 mL) gave amine 18 (94 mg, quant.) as a colourless amorphous solid, Rf (100:9:1 EtOAc–MeOH–NH4OH) 0.1; IR (ATR) 3346 (NH), 2976, 2880, 1755 (C@O), 1694 (C@O), 1391, 1161, 1079, 727 cm1 ; 1 H NMR (400 MHz, CDCl3) d 4.20– 4.16 (m, 1H, NCH), 4.09–3.97 (m, 3H, NCH), 3.73–3.70 (m, 2H, NCH), 3.20–3.16 (m, 1H, NCH), 3.01–2.90 (m, 2H, NCH), 2.62– 2.56 (m, 1H, NCH), 2.49–2.43 (m, 1H, NCH), 1.39 (s, 9H, CMe3); 13C NMR (100.6 MHz, CDCl3) d 155.9 (C@O), 155.0 (C@O), 80.5 (CO), 75.5 (CMe3), 59.8 (NCH), 47.4 (NCH2), 44.4 (NCH2), 41.9 (NCH2), 28.2 (CMe3) (two NCH2 resonances are not resolved); MS (ESI) m/z 306 [(M+Na)+ ], 284 [(M+H)+ ], 228 [(M – CMe3) + ]; HRMS m/z calcd for C13H22N3O4 (M+H)+ 284.1605, found 284.1609 (0.8 ppm error). 4.2.10. Tetrahydrospiro[oxazolo[3,4-a]pyrazine-1,40 -piperidin]- 3(5H)-one 19 Using general procedure A, 10% Pd/C (27 mg, 0.03 mmol, 0.1 equiv) and benzylamine 15 (77 mg, 0.254 mmol, 1.0 equiv) in MeOH (2.5 mL) gave amine 19 (47 mg, 87%) as a colourless oil, Rf (100:9:1 EtOAc–MeOH–NH4OH) 0.1; IR (ATR) 3355 (NH), 2928, 2852, 2484, 1737 (C@O), 1654, 1428, 976, 500 cm1 ; 1 H NMR (400 MHz, CD3OD) d 3.57 (dd, J = 13.5, 3.5 Hz, 1H, NCH), 3.34 (dd, J = 11.5, 3.5 Hz, 1H, NCH), 3.08–2.23 (m, 7H, NCH), 2.54–2.44 (m, 2H, NCH), 1.88–1.71 (m, 4H, CH); 13C NMR (100.6 MHz, CD3OD) d 157.0 (C@O), 79.3 (CO), 62.2 (NCH), 45.4 (NCH2), 44.4 (NCH2), 42.2 (NCH2), 41.7 (NCH2), 41.6 (NCH2), 34.8 (CCH2), 29.6 (CCH2); MS (ESI) m/z 234 [(M+Na)+ ], 212 [(M+H)+ ]; HRMS m/z calcd for C10H18N3O2 (M+H)+ 212.1394, found 212.1395 (0.7 ppm error). 4.2.11. Tetrahydrospiro[azetidine-3,10 -oxazolo[3,4-a]pyrazin]- 30 (50 H)-one 20 Using general procedure A, 10% Pd/C (9 mg, 0.01 mmol, 0.1 equiv) and carbamate 16 (24 mg, 0.088 mmol, 1.0 equiv) in MeOH (1 mL) gave amine 20 (16 mg, 99%) as a colourless oil, Rf (100:9:1 EtOAc–MeOH–NH4OH) 0.1; IR (ATR) 3041 (NH), 2917, 2851, 1758 (C@O), 1660, 1432, 1178, 1124, 837, 798, 722 cm1 ; 1 H NMR (400 MHz, CD3OD) d 4.24 (dd, J = 11.0, 11.0 Hz, 2H, NCH), 4.09 (d, J = 13.0 Hz, 1H, NCH), 4.02 (d, J = 12.0 Hz, 1H, NCH), 3.95 (dd, J = 12.0, 3.5 Hz, 1H, NCH), 3.61 (dd, J = 13.5, 3.5 Hz, 1H, NCH), 3.38 (dd, J = 12.0, 3.5 Hz, 1H, NCH), 3.09–2.95 (m, 2H, NCH2), 2.68–2.57 (m, 2H, NCH2); 13C NMR (100.6 MHz, CD3OD) d 155.5 (C@O), 79.3 (CO), 59.5 (NCH2), 57.8 (NCH), 54.6 (NCH2), 45.3 (NCH2), 43.7 (NCH2), 40.3 (NCH2); MS (ESI) m/z 206 [(M+Na)+ ], 184 [(M+H)+ ]; HRMS m/z calcd for C8H14N3O2 (M+H)+ 184.1081, found 184.1081 (0 ppm error). 4.2.12. tert-Butyl 3-chloropropyl-pyridin-3-ylmethyl-carbamate 21 NaBH(OAc)3 (1.59 g, 7.52 mmol, 1.4 equiv) was added to a stirred solution of 3-chloropropylamine hydrochloride (500 mg, 5.37 mmol, 1.0 equiv) and 3-pyridine carboxaldehyde (575 mg, 0.50 mL, 5.37 mmol, 1.0 equiv) in 1,2-dichloroethane (21 mL) at rt. The resulting mixture was stirred at rt for 3 h. Saturated NaHCO3(aq) (30 mL) was added and the two layers were separated. The aqueous layer was extracted with CH2Cl2 (3  30 mL) and the combined organic layers were dried (MgSO4) and evaporated under reduced pressure to give the crude amine. The amine was dissolved in CH2Cl2 (5 mL) and added to a stirred solution of ditert-butylcarbonate (1.17 g, 5.37 mmol, 1.0 equiv) in CH2Cl2 (15 mL) at 0 C. The resulting mixture was evaporated under reduced pressure to give the crude product. Purification by flash column chromatography on silica with 90:10 CHCl3–MeOH as eluent gave N-Boc propylamine 21 (358 mg, 29%) as a colourless oil, Rf (90:10 CHCl3–MeOH) 0.6; IR (ATR) 2975, 1685 (C@O), 1411, 1365, 1248, 1158, 773 cm–1; 1 H NMR (400 MHz, CDCl3) 8.60–8.47 (m, 2H, Ar), 7.68–7.51 (m, 1H, Ar), 7.32–7.26 (m, 1H, Ar), 4.46 (s, 2H, NCH2), 3.54 (br s, 2H, NCH2), 3.44–3.24 (m, 2H, CH2Cl), 2.08–1.88 (m, 2H, CH2), 1.49 (br s, 9H, CMe3); 13C NMR (100.6 MHz, CDCl3) (rotamers) d 155.8 (C@O), 155.3 (C@O), 149.0 (Ar), 148.8 (Ar), 135.4 (Ar), 134.7 (Ar), 133.8 (ipso-Ar), 123.4 (Ar), 80.4 (CMe3), 48.9 (NCH2), 48.0 (NCH2), 44.7 (NCH2), 44.1 (NCH2), 42.2 (CH2Cl), 31.1 (CH2), 28.3 (CMe3); MS (ESI) m/z 285 [(M+H)+ , 100], 229 (500); HRMS (ESI) m/z calcd for C14H21 35ClN2O2 (M+H)+ 285.1364, found 285.1361 (+1.0 ppm error). 4.2.13. 2-Pyridin-3-ylpyrrolidine-1-carboxylic acid tert-butyl ester 22 n-BuLi (2.37 mL of a 2.5 M solution in hexane, 5.93 mmol, 1.3 equiv) was added dropwise to a stirred solution of N-Boc propylamine 21 (1.30 g, 4.56 mmol, 1.0 equiv) in THF (46 mL) at 78 C under Ar. The resulting solution was stirred at 78 C for 3 h. Then, saturated NH4Cl(aq) (6 mL) was added and the two layers were separated. The aqueous layer was extracted with Et2O (3  40 mL). The combined organic layers were dried (MgSO4) and evaporated under reduced pressure to give the crude product. Purification by flash column chromatography on silica with 90:10 CH2Cl2–MeOH as eluent gave pyridinyl N-Boc pyrrolidine 22 (823 mg, 73%) as a colourless oil, Rf (90:10 CH2Cl2–MeOH) 0.5; 1 H NMR (400 MHz, CDCl3) (65:35 mixture of rotamers) d 8.51– 8.41 (m, 2H, Ar), 7.49 (d, J = 8.0 Hz, 1H, Ar), 7.23 (dd, J = 8.0, 5.0 Hz, 1H, Ar), 4.95 (br s, 0.35H, NCH), 4.77 (br s, 0.65H, Ar), 3.68–3.44 (m, 2H, NCH), 2.44–2.23 (m, 1H, CH), 1.98–1.75 (m, 3H, CH), 1.44 (s, 3.15H, CMe3), 1.18 (s, 5.85H, CMe3); 13C NMR (100.6 MHz, CDCl3) (rotamers) d 154.2 (C@O), 148.0 (Ar), 147.7 (Ar), 147.2 (Ar), 140.3 (Ar), 133.2 (ipso-Ar), 133.0 (ipso-Ar), 123.2 (Ar), 79.6 (CMe3), 59.1 (NCH), 58.7 (NCH), 47.2 (NCH2), 47.0 (NCH2), 35.8 (CH2), 34.5 (CH2), 28.4 (CMe3), 28.1 (CMe3), 23.5 (CH2), 23.2 (CH2). Spectroscopic data consistent with those reported in the literature.31 4.2.14. 1-Benzyl-7a0 -(pyridin-3-yl)tetrahydro-30 H-spiro [piperidine-4,10 -pyrrolo[1,2-c]oxazol]-30 -one 23 n-BuLi (0.52 mL of a 2.5 M solution in hexanes, 1.3 mmol, 1.3 equiv) was added dropwise to a stirred solution of pyridinyl N-Boc-pyrrolidine 22 (250 mg, 1.0 mmol, 1.0 equiv) in THF (10 mL) at 50 C under Ar. The resulting solution was stirred at 50 C for 30 min. Then, N-benzyl-4-piperidone (371 lL, 2.0 mmol, 2.0 equiv) was added dropwise. The resulting solution was stirred at 50 C for 10 min. The reaction mixture was allowed to warm to M. Lüthy et al. / Bioorg. Med. Chem. 23 (2015) 2680–2694 2689

rt over 24 h. Then, saturated NH4Cl(aq) (1 mL) was added and the two layers were separated. The aqueous layer was extracted with Et2O (3  20 mL). The combined organic layers were dried (MgSO4) and evaporated under reduced pressure to give the crude product. Purification by flash column chromatography on silica with 100:9:1 CH2Cl2–MeOH–NH4OH as eluent gave carbamate 23 (225 mg, 62%) as an amorphous solid, Rf (10:1 CH2Cl2–MeOH) 0.5; IR (ATR) 3028, 2925, 2813, 2772, 1753 (C@O), 1708, 1588, 1573, 736, 699 cm1 ; 1 H NMR (400 MHz, CDCl3) (50:50 mixture of rotamers) d 8.72–8.59 (m, 2H, Ar), 7.78 (d, J = 7.5 Hz, 0.5H, Ar), 7.58 (d, J = 7.5 Hz, 0.5H, Ar), 7.37–7.27 (m, 6H, Ar), 3.84–3.79 (m, 1H, NCH), 3.46–3.42 (m, 2H, NCH2Ph), 3.18 (dd, J = 10.0, 10.0 Hz, 0.5H, NCH), 3.08 (dd, J = 10.0, 10.0 Hz, 0.5H, NCH), 2.86–2.82 (m, 1H, NCH), 2.61–2.51 (m, 1H, NCH), 2.39 (dd, J = 11.5, 11.5 Hz, 1H, NCH), 2.28–1.90 (m, 6H, CH), 1.41–1.30 (m, 2H, CH), 0.91 (ddd, 1H, J = 13.0, 13.0, 4.5 Hz, CH); 13C NMR (100.6 MHz, CDCl3) (rotamers) d 161.1 (C@O), 149.8 (Ar), 149.4 (Ar), 148.0 (Ar), 147.2 (Ar), 138.1 (ipso-Ar), 137.8 (ipso-Ar), 134.2 (Ar), 133.9 (ipso- Ar), 133.1 (Ar), 129.1 (Ar), 128.3 (Ar), 127.2 (Ar), 123.8 (Ar), 123.5 (Ar), 81.8 (C), 81.6 (C), 62.9 (NCH2), 49.5 (NCH2), 48.8 (NCH2), 45.1 (NCH2), 35.7 (CH2), 32.5 (CH2), 29.7 (CH2), 23.6 (CH2); MS (ESI) m/z 364 [(M+H)+ ]; HRMS m/z calcd for C22H26N3O2 (M+H)+ 364.2020, found 364.2019 (+0.4 ppm error). 4.2.15. tert-Butyl 30 -oxo-7a0 -(pyridin-3-yl)tetrahydro-30 Hspiro[azetidine-3,10 -pyrrolo[1,2-c]oxazole]-1-carboxylate 24 n-BuLi (0.52 mL of a 2.5 M solution in hexanes, 1.3 mmol, 1.3 equiv) was added dropwise to a stirred solution of pyridinyl N-Boc-pyrrolidine 22 (250 mg, 1.0 mmol, 1.0 equiv) in THF (9 mL) at 50 C under Ar. The resulting solution was stirred at 50 C for 30 min. Then, a solution of N-Boc-4-azetidinone (342 mg, 2.0 mmol, 2.0 equiv) in THF (1 mL) was added dropwise. The resulting solution was stirred at 50 C for 10 min. The reaction mixture was allowed to warm to rt over 24 h. Then, saturated NH4Cl(aq) (1 mL) was added and the two layers were separated. The aqueous layer was extracted with Et2O (3  20 mL). The combined organic layers were dried (MgSO4) and evaporated under reduced pressure to give the crude product. Purification by flash column chromatography on silica with 4:1 EtOAc–pet. ether as eluent gave carbamate 24 (103 mg, 30%) as a yellow oil, Rf (9:1 CHCl3– MeOH) 0.7; IR (ATR) 2974, 2930, 2881, 1769 (C@O), 1697 (C@O), 1391, 1366, 1162, 729 cm1 ; 1 H NMR (400 MHz, CDCl3) d 8.68– 8.66 (m, 2H, Ar), 7.74–7.70 (m, 1H, Ar), 7.41–7.40 (m, 1H, Ar), 4.36 (d, J = 11.0 Hz, 1H, NCH), 4.31 (d, J = 11.0 Hz, 1H, NCH), 3.93–3.83 (m, 1H, NCH), 3.85 (d, J = 10.0 Hz, NCH), 3.24 (d, J = 10.0 Hz, NCH), 3.23–3.17 (m, 1H, NCH), 2.43 (dd, J = 12.0, 7.0 Hz, 1H, CH), 2.15–2.05 (m, 1H, CH), 1.86–1.77 (m, 1H, CH), 1.62–1.53 (m, 1H, CH), 1.37 (s, 9H, CMe3); 13C NMR (100.6 MHz, CDCl3) d 159.9 (C@O), 155.9 (C@O), 150.3 (Ar), 147.0 (Ar), 139.9 (ipso-Ar), 133.2 (Ar), 124.0 (Ar), 80.6 (CO), 79.7 (CMe3), 74.9 (NC), 46.3 (NCH2), 34.5 (CH2), 28.1 (CMe3), 23.7 (CH2); MS (ESI) m/z 364 [(M+H)+ ]; HRMS m/z calcd for C18H26N2O4 (M+H)+ 346.1761, found 346.1767 (2.0 ppm error). 4.2.16. 7a0 -(Pyridin-3-yl)tetrahydro-30 H-spiro[piperidine-4,10 - pyrrolo[1,2-c]oxazol]-30 -one 25 Using general procedure B, 20% Pd(OH)2/C (2 mg, 0.0125 mmol, 0.1 equiv), N-benzyl-piperidine 23 (46 mg, 0.127 mmol, 1.0 equiv) and ammonium formate (40 mg, 0.635 mmol, 5.0 equiv) in EtOH (1.3 mL) for 2 h gave the crude product. Purification by flash column chromatography on silica with 100:9:1 CH2Cl2–MeOH– NH4OH as eluent gave amine 25 (27 mg, 77%) as a yellow oil, Rf (100:9:1 CH2Cl2–MeOH–NH4OH) 0.1; IR (ATR) 3317 (NH), 1746 (C@O) cm1 ; 1 H NMR (400 MHz, CD3OD) d 8.56–8.47 (m, 2H, Ar), 7.83 (d, J = 8.0 Hz, 1H, Ar), 7.50–7.34 (m, 1H, Ar), 3.77–3.57 (m, 1H, NCH), 3.19–2.96 (m, 2H, NCH), 2.87 (ddd, 1H, J = 13.5, 13.5, 3.0 Hz, CH), 2.77–2.72 (m, 2H, CH), 2.27–1.83 (m, 5H, CH), 1.40– 1.27 (m, 2H, CH), 0.80 (ddd, 1H, J = 13.5, 13.5, 8.0 Hz, CH); 13C NMR (100.6 MHz, CD3OD) (rotamers) d 162.6 (C@O), 150.4 (Ar), 150.0 (Ar), 148.3 (Ar), 148.1 (Ar), 136.7 (Ar), 136.1 (Ar), 135.9 (ipso-Ar), 135.2 (ipso-Ar), 125.8 (Ar), 125.3 (Ar), 83.1 (C), 78.5 (C), 46.4 (NCH2), 46.2 (NCH2), 43.1 (NCH2), 42.5 (NCH2), 36.4 (NCH2), 36.3 (NCH2), 33.4 (CH), 32.0 (CH2), 24.6 (CH2); MS (ESI) m/z 274 [(M+H)+ ]; HRMS m/z calcd for C15H20N3O2 (M+H)+ 274.1550, found 274.1545 (+1.7 ppm error). 4.2.17. 7a0 -(Pyridin-3-yl)tetrahydro-30 H-spiro[azetidine-3,10 - pyrrolo[1,2-c]oxazol]-30 -one 26 TFA (1.0 mL, 13 mmol, 140 equiv) was added to a stirred solution of carbamate 24 (30 mg, 0.09 mmol, 1.0 equiv) in CH2Cl2 (1 mL) at rt. The resulting solution was stirred at rt for 2 h. Then, NH4OH(aq) (2 mL) was added and the mixture was extracted with CH2Cl2 (3  20 mL). The combined organic layers were dried (MgSO4) and evaporated under reduced pressure to give the crude product. Purification by flash column chromatography on silica with 10:9:1 EtOAc–MeOH–NH4OH as eluent gave amine 26 (17 mg, 79%) as a yellow oil, Rf (10:9:1 EtOAc–MeOH–NH4OH) 0.3; IR (ATR) 3074 (NH), 2850, 1764, 1663 (C@O), 1434, 1181, 1124, 839, 800, 518 cm1 ; 1 H NMR (400 MHz, CD3OD) d 8.65– 8.63 (m, 1H, Ar), 8.52–8.50 (m, 1H, Ar), 7.96–7.93 (m, 1H, Ar), 7.48–7.45 (m, 1H, Ar), 4.09 (d, J = 10.5 Hz, 1H, NCH), 4.09 (d, J = 10.5 Hz, 1H, NCH), 3.78 (dd, J = 10.5, 1.0 Hz, 1H, NCH), 3.67 (ddd, J = 11.5, 8.5, 8.5 Hz, 1H, NCH), 3.32 (dd, J = 10.5, 1.0 Hz, 1H, NCH), 3.22–3.20 (m, 1H, NCH), 3.11–3.05 (m, 1H, NCH), 2.95 (d, J = 10.5 Hz, 1H, NCH), 2.49 (dd, J = 12.5, 7.0 Hz, 1H, NCH), 2.07– 1.98 (m, 1H, CH), 1.77 (ddd, J = 12.0, 12.0, 8.5 Hz, 1H, NCH), 1.51– 1.42 (m, 1H, NCH), 1.30–1.18 (m, 2H, NCH); 13C NMR (100.6 MHz, CD3OD) d 161.3 (C@O), 149.8 (Ar), 147.4 (Ar), 135.6 (Ar), 135.4 (ipso-Ar), 125.1 (Ar), 86.6 (C), 75.8 (C), 56.0 (NCH2), 55.2 (NCH2), 46.3 (NCH), 33.9 (NCH2), 24.5 (NCH2); MS (ESI) m/z 246 [(M+H)+ ]; HRMS m/z calcd for C13H16N3O2 (M+H)+ 246.1237, found 246.1243 (2.0 ppm error). 4.2.18. tert-Butyl-2-(hydroxy(pyridine-3-yl)methyl)pyrrolidine1-carboxylate syn-27 and anti-28 s-BuLi (18.0 mL of a 1.3 M solution in hexanes, 22.8 mmol, 1.3 equiv) was added dropwise to a stirred solution of N-Boc pyrrolidine 7 (3.0 g, 17.5 mmol, 1.0 equiv) in THF (150 mL) at 30 C under Ar. The resulting yellow solution was stirred at 30 C for 5 min. Then, 3-pyridine-carboxaldehyde (4.87 g, 4.30 mL, 45.6 mmol, 2.6 equiv) was added dropwise and the resulting solution was stirred at 30 C for 10 min and allowed to warm to rt over 2 h. Water (150 mL) was added and the two layers were separated. The aqueous layer was extracted with Et2O (3  50 mL) and the combined organics were dried (MgSO4) and evaporated under reduced pressure to give the crude product as a yellow oil. Purification by flash chromatography on silica with 97:3 CH2Cl2– MeCN as eluent gave a 70:30 mixture (by 1 H NMR spectroscopy) of hydroxy pyrrolidines syn-27 and anti-28 (2.07 g, 43%) as a yellow oil. Characterisation of syn-27/anti-28 is described below. 4.2.19. 1-Benzylpyrrolidin-2-yl(pyridin-3-yl)methanol syn-29 and anti-30 TFA (0.48 mL, 2.82 mmol, 4.0 equiv) was added to a stirred solution of a 70:30 mixture of hydroxy pyrrolidines syn-27 and anti-28 (196 mg, 0.71 mmol, 1.0 equiv) in CH2Cl2 (3 mL) at 0 C under Ar. The resulting solution was allowed to warm to rt and then stirred at rt for 3 h. 20% NaOH(aq) (2 mL) was added and the two layers were separated. The aqueous layer was extracted with CH2Cl2 (3  10 mL) and the combined organics were dried (MgSO4) and evaporated under reduced pressure to give the crude amines (108 mg, 0.61 mmol). Benzaldehyde (0.13 mL, 0.67 mmol, 2690 M. Lüthy et al. / Bioorg. Med. Chem. 23 (2015) 2680–2694

1.1 equiv) was added to a stirred solution of crude amines (108 mg, 0.61 mmol), NaBH(OAc)3 (272 mg, 1.22 mmol) and AcOH (0.01 mL, 0.12 mmol) in 1,2-dichloroethane (4 mL) at rt. The resulting solution was stirred at rt for 16 h. CH2Cl2 (10 mL) and saturated NaHCO3(aq) (10 mL) were added to the resulting mixture and the two layers were separated. The aqueous layer was extracted with CH2Cl2 (3  10 mL) and the combined organics were dried (MgSO4) and evaporated under reduced pressure to give the crude product. Purification by flash column chromatography on silica with 96:4 CH2Cl2–MeOH as eluent gave N-benzyl amino alcohol anti-30 (57 mg, 30%) as a yellow oil, Rf (96:4 CH2Cl2–MeOH) 0.2; IR (ATR) 3226 (OH), 2963, 2797, 1453, 1164, 1111, 1091, 1025 cm1 ; 1 H NMR (400 MHz, CDCl3) d 8.52 (br s, 1H, Ar), 8.42 (d, J = 3.0 Hz, 1H, Ar), 7.72 (br d, J = 8.0 Hz, 1H, Ar), 7.34–7.33 (m, 4H, Ar), 7.29–7.23 (m, 2H, Ar), 4.86 (d, J = 3.5 Hz, 1H, OCH), 4.12 (d, J = 13.0 Hz, 1H, PhCHAHBN), 3.49 (d, J = 13.0 Hz, 1H, PhCHAHBN), 3.04 (ddd, J = 9.0, 5.0, 5.0 Hz, 1H, NCH), 2.88 (ddd, J = 9.0, 6.0, 3.5 Hz, 1H, NCH), 2.35 (ddd, J = 9.0, 9.0, 9.0 Hz, NCH), 1.69–1.59 (m, 3H, CH), 1.38–1.31 (m, 1H, CH); 13C NMR (100.6 MHz, CDCl3) d 148.4 (Ar), 147.6 (Ar), 138.5 (ipso-Ar), 137.0 (ipso-Ar), 133.5 (Ar), 129.0 (Ar), 128.7 (Ar), 127.5 (Ar), 123.4 (Ar), 69.0 (OCH or NCH), 68.6 (OCH or NCH), 58.4 (PhCH2N), 54.9 (NCH2), 24.2 (CH2), 23.3 (CH2); MS (ESI) m/z 269 [(M+H)+ , 100]; HRMS (ESI) m/z calcd for C17H20N2O (M+H)+ 269.1648, found 269.1645 (+1.1 ppm error) and N-benzyl amino alcohol syn-29 (58 mg, 30%) as a yellow oil, Rf (96:4 CH2Cl2–MeOH) 0.1, IR (ATR) 3205 (OH), 2962, 2795, 1452, 1191, 1117, 1061, 1026 cm1 ; 1 H NMR (400 MHz, CDCl3) d 8.58 (d, J = 1.5 Hz, 1H, Ar), 8.45 (dd, J = 4.5, 1.5 Hz, 1H, Ar), 7.69 (ddd, J = 7.5, 1.5, 1.5 Hz, 1H, Ar), 7.34–7.21 (m, 6H, Ar), 4.41 (d, J = 5.5 Hz, 1H, OCH), 3.70 (d, J = 13.5 Hz, 1H, PhCHAHBN), 3.44 (d, J = 13.5 Hz, PhCHAHBN), 3.06 (ddd, J = 9.0, 5.5, 3.5 Hz, 1H, NCH), 3.00–2.94 (m, 1H, NCH), 2.46– 2.40 (m, 1H, NCH), 1.97–1.86 (m, 1H, CH), 1.79–1.65 (m, 3H, CH); 13C NMR (100.6 MHz, CDCl3) d 148.8 (Ar), 148.3 (Ar), 138.7 (ipsoAr), 138.1 (ipso-Ar), 134.2 (Ar), 129.0 (Ar), 128.6 (Ar), 127.6 (Ar), 123.5 (Ar), 73.1 (OCH or NCH), 70.3 (NCH or OCH), 61.3 (PhCH2N), 54.2 (NCH2), 28.9 (CH2), 24.2 (CH2); MS (ESI) m/z 269 [(M+H)+ , 100]; HRMS (ESI) m/z calcd for C17H20N2O (M+H)+ 269.1648, found 269.1659 (3.8 ppm error). 4.2.20. 1-Benzyl-2-(pyridin-3-yl)piperidin-3-ol syn-31 Using general procedure C, N-benzyl pyrrolidine syn-29 (86 mg, 0.32 mmol), trifluoroacetic anhydride (0.20 mL, 0.96 mmol), Et3N (0.20 mL, 0.96 mmol) in THF (10 mL) and 20% NaOH(aq) (10 mL) gave the crude product. Purification by flash column chromatography on silica with 1:1 CH2Cl2–EtOAc and then EtOAc as eluent gave 3-hydroxy piperidine syn-31 (65 mg, 76%) as a yellow oil, Rf (EtOAc) 0.1; IR (ATR) 3277 (OH), 2924, 2858, 2804, 1422, 1134, 1028, 1015, 715 cm1 ; 1 H NMR (400 MHz, CDCl3) d 8.62 (d, J = 1.5 Hz, 1H, Ar), 8.50 (dd, J = 5.0, 1.5 Hz, 1H, Ar), 7.88 (br d, J = 8.0 Hz, 1H, Ar), 7.33–7.22 (m, 6H, Ar), 3.75 (d, J = 14.0 Hz, 1H, PhCHAHBN), 3.74 (br s, 1H, OCH), 3.39 (d, J = 1.5 Hz, 1H, NCH), 3.01 (br d, J = 11.5 Hz, 1H, NCH), 2.91 (d, J = 14.0 Hz, 1H, PhCHAHBN), 2.67 (br s, 1H, OH), 2.08–1.99 (m, 2H, CH), 1.93 (ddddd, J = 13.5, 13.5, 13.5, 4.0, 4.0 Hz, 1H, CH), 1.66 (dddd, J = 13.5, 13.5, 2.5, 2.5 Hz, 1H, CH), 1.56–1.53 (m, 1H, CH); 13C NMR (100.6 MHz, CDCl3) d 149.9 (Ar), 148.9 (Ar), 138.5 (ipso-Ar), 136.8 (ipso-Ar), 136.4 (Ar), 128.7 (Ar), 128.4 (Ar), 127.1 (Ar), 123.4 (Ar), 70.2 (OCH or NCH), 69.8 (OCH or NCH), 59.5 (PhCH2N), 53.4 (NCH2), 31.7 (CH2), 19.8 (CH2); MS (ESI) m/z 269 [(M+H)+ , 100]; HRMS (ESI) m/z calcd for C17H20N2O (M+H)+ 269.1652, found 269.1648 (1.4 ppm error). 4.2.21. 1-Benzyl-2-(pyridin-3-yl)piperidin-3-ol anti-32 Using general procedure C, N-benzyl pyrrolidine anti-30 (123 mg, 0.46 mmol), trifluoroacetic anhydride (0.25 mL, 1.38 mmol), Et3N (0.25 mL, 1.38 mmol) in THF (10 mL) and 20% NaOH(aq) (10 mL) gave the crude product. Purification by flash column chromatography on silica with EtOAc as eluent gave 3- hydroxy piperidine anti-32 (98 mg, 79%) as a yellow oil, Rf (EtOAc) 0.1; IR (ATR) 3164 (OH), 2931, 2854, 2792, 1429, 1253, 1110, 964, 715 cm1 ; 1 H NMR (400 MHz, CDCl3) d 8.58 (br s, 1H, Ar), 8.25 (d, J = 3.5 Hz, 1H, Ar), 7.85 (br d, J = 8.0 Hz, 1H, Ar), 7.28–7.18 (m, 6H, Ar), 3.65 (ddd, J = 11.5, 9.0, 5.0 Hz, 1H, OCH), 3.58 (d, J = 13.5 Hz, 1H, PhCHAHBN), 3.25 (br s, 1H, OH), 2.99 (d, J = 9.0 Hz, 1H, NCH), 2.92 (br d, J = 12.5 Hz, 1H, NCH), 2.88 (d, J = 13.5 Hz, 1H, PhCHAHBN), 2.17–2.13 (m, 1H, NCH), 1.97 (ddd, J = 11.5, 11.5, 3.5 Hz, 1H, CH), 1.71–1.63 (m, 2H, CH), 1.43 (dddd, J = 12.0, 12.0, 12.0, 5.0 Hz, 1H, CH); 13C NMR (100.6 MHz, CDCl3) d 150.5 (Ar), 148.8 (Ar), 139.0 (ipsoAr), 137.7 (ipso-Ar), 136.5 (Ar), 128.5 (Ar), 128.3 (Ar), 126.9 (Ar), 123.8 (Ar), 73.7 (OCH or NCH), 73.6 (OCH or NCH), 59.5 (PhCH2N), 52.5 (NCH2), 33.3 (CH2), 23.4 (CH2); MS (ESI) m/z 269 [(M+H)+ , 100]; HRMS (ESI) m/z calcd for C17H20N2O (M+H)+ 269.1648, found 269.1651 (1.1 ppm error). 4.2.22. tert-Butyl-2-(hydroxy(pyridine-3-yl)methyl)pyrrolidine1-carboxylate syn-27 Using general procedure D, 20% Pd(OH)2/C (12 mg, 0.08 mmol), ammonium formate (145 mg, 2.30 mmol), N-benzyl hydroxy pyrrolidine syn-29 (62 mg, 0.23 mmol) in EtOH (10 mL) and Boc2O (55 mg, 0.25 mmol) gave the crude product. Purification by flash column chromatography on silica with 50:50 CH2Cl2–MeCN as eluent gave N-Boc hydroxy pyrrolidine syn-27 (26 mg, 41%) as a colourless oil, Rf (50:50 CH2Cl2– MeCN) 0.4; IR (ATR) 3332 (OH), 2974, 2931, 2886, 1687, 1668 (C@O), 1578, 1390, 1365, 1160, 1115, 772, 717 cm1 ; 1 H NMR (400 MHz, CDCl3) d 8.53–8.51 (m, 2H, Ar), 7.73 (d, J = 7.0 Hz, 1H, Ar), 7.28 (dd, J = 7.0, 5.0 Hz, 1H, Ar), 6.13 (br s, 1H, OH), 4.59 (d, J = 8.0 Hz, 1H, OCH), 4.08 (ddd, J = 8.0, 8.0, 4.0 Hz, 1H, NCH), 3.48 (ddd, J = 7.5, 3.5, 3.5 Hz, 1H, NCH), 3.35–3.33 (m, 1H, NCH), 1.73–1.56 (m, 4H, CH), 1.50 (s, 9H, CMe3); 13C NMR (100.6 MHz, CDCl3) d 158.3 (C@O), 149.3 (Ar), 149.0 (Ar), 138.1 (ipso-Ar), 134.8 (Ar), 123.7 (Ar), 81.2 (CMe3), 76.9 (OCH), 64.1 (NCH), 47.9 (NCH2), 28.8 (CH2), 28.6 (CMe3), 23.9 (CH2); MS (ESI) m/z 279 [(M+H)+ , 100]; HRMS (ESI) m/z calcd for C15H22N2O3 (M+H)+ 279.1703, found 279.1696 (+2.6 ppm error). 4.2.23. tert-Butyl-2-(hydroxy(pyridine-3-yl)methyl)pyrrolidine1-carboxylate anti-28 Using general procedure D, 20% Pd(OH)2/C (12 mg, 0.08 mmol), ammonium formate (217 mg, 3.45 mmol), N-benzyl hydroxy pyrrolidine anti-30 (62 mg, 0.23 mmol) in EtOH (10 mL) and Boc2O (55 mg, 0.25 mmol) gave the crude product as a yellow oil. Purification by flash column chromatography on silica with 50:50 CH2Cl2–MeCN as eluent gave N-Boc hydroxy pyrrolidine anti-28 (24 mg, 37%) as a colourless oil, Rf (50:50 CH2Cl2–MeCN) 0.3; IR (ATR) 3332 (OH), 2973, 2926, 2878, 1668 (C@O), 1579, 1391, 1365, 1162, 1111, 771, 715 cm1 ; 1 H NMR (400 MHz, CDCl3) (80:20 mixture of rotamers) d 8.55–8.49 (m, 2H, Ar), 7.71–7.63 (m, 1H, Ar), 7.27–7.24 (m, 1H, Ar), 5.95 (br s, 1H, OH), 5.16 (br s, 0.2H, OCH), 4.86 (br s, 0.8H, OCH), 4.33 (br s, 0.8H, NCH), 3.97 (br s, 0.2H, NCH), 3.54 (br s, 0.2H, NCH), 3.34–3.28 (m, 1H, NCH), 2.82–2.77 (m, 0.8H, NCH), 2.04–1.65 (m, 3H, CH), 1.49 (s, 9H, CMe3), 1.20–1.08 (m, 1H, CH); 13C NMR (100.6 MHz, CDCl3) d 157.6 (C@O), 148.9 (Ar), 148.6 (Ar), 136.8 (ipso-Ar), 134.9 (Ar), 123.3 (Ar), 80.9 (CMe3), 75.2 (OCH), 63.2 (NCH), 48.0 (NCH2), 28.6 (CMe3), 27.7 (CH2), 23.6 (CH2); MS (ESI) m/z 279 [(M+H)+ , 100]; HRMS (ESI) m/z calcd for C15H22N2O3 (M+H)+ 279.1703, found 279.1702 (+0.3 ppm error). M. Lüthy et al. / Bioorg. Med. Chem. 23 (2015) 2680–2694 2691

4.2.24. 2-(Pyridin-3-yl)piperidin-3-ol syn-33 Using general procedure B, 20% Pd(OH)2/C (15 mg, 0.10 mmol, 0.6 equiv), N-benzyl piperidine syn-31 (40 mg, 0.15 mmol, 1.0 equiv) and ammonium formate (144 mg, 2.28 mmol, 15.0 equiv) in EtOH (10 mL) for 16 h gave hydroxy piperidine syn-33 (22 mg, 82%) as a yellow oil, IR (ATR) 3235 (OH or NH), 2929, 2849, 1423, 1181, 1087, 997, 802, 714 cm1 ; 1 H NMR (400 MHz, CDCl3) d 8.52 (s, 1H, Ar), 8.44 (dd, J = 5.0, 1.5 Hz, 1H, Ar), 7.69 (dd, J = 8.0, 1.5 Hz, 1H, Ar), 7.26–7.22 (m, 1H, Ar), 3.81 (s, 1H, CHO), 3.77 (s, 1H, CHN), 3.18 (dddd, J = 11.5, 2.0, 2.0, 2.0 Hz, 1H, NCH), 2.78 (ddd, J = 11.5, 11.5, 2.5 Hz, 1H, NCH), 2.01 (br d, J = 13.5 Hz, 1H, CH), 1.84 (dddd, J = 13.5, 13.5, 13.5, 3.5, 3.5 Hz, 1H, CH), 1.75–1.65 (m, 1H, CH), 1.50 (br d, J = 13.5 Hz, 1H, CH); 13C NMR (100.6 MHz, CDCl3) d 148.7 (Ar), 148.6 (Ar), 137.7 (ipso-Ar), 134.7 (Ar), 123.4 (Ar), 68.5 (OCH), 63.1 (NCH), 47.5 (NCH2), 32.1 (CH2), 19.8 (CH2); MS (ESI) m/z 179 [(M+H)+ , 100]; HRMS (ESI) m/z calcd for C10H14N2O (M+H)+ 179.1179, found 179.1178 (+0.6 ppm error). 4.2.25. 2-(Pyridin-3-yl)piperidin-3-ol anti-34 Using general procedure B, 20% Pd(OH)2/C (8 mg, 0.05 mmol, 0.3 equiv), N-benzyl piperidine anti-32 (40 mg, 0.15 mmol, 1.0 equiv) and ammonium formate (96 mg, 1.50 mmol, 10.0 equiv) in EtOH (10 mL) for 16 h gave hydroxy piperidine anti-34 (25 mg, 92%) as a yellow oil, IR (ATR) 3266 (OH or NH), 3130 (OH or NH), 2923, 2852, 1422, 1169, 1098, 1078, 799, 717 cm1 ; 1 H NMR (400 MHz, CDCl3) d 8.57 (d, J = 2.0 Hz, 1H, Ar), 8.41 (dd, J = 5.0, 2.0 Hz, 1H, Ar), 7.75 (ddd, J = 8.0, 2.0, 2.0 Hz, 1H, Ar), 7.23 (dd, J = 8.0, 5.0 Hz, 1H, Ar), 3.54 (ddd, J = 10.5, 9.0, 4.5 Hz, 1H, CHO), 3.36 (d, J = 9.0 Hz, 1H, CHN), 3.10–3.06 (m, 1H, CH), 2.70 (ddd, J = 11.5, 11.5, 3.0 Hz, 1H, CH), 2.24–2.15 (m, 1H, CH), 1.82–1.77 (m, 1H, CH), 1.69 (ddddd, J = 13.0, 13.0, 13.0, 4.0, 4.0 Hz, 1H, CH), 1.51–1.43 (m, 1H, CH); 13C NMR (100.6 MHz, CDCl3) d 149.9 (Ar), 149.1 (Ar), 137.6 (ipso-Ar),, 135.7 (Ar), 123.6 (Ar), 72.7+ (OCH), 67.1 (NCH), 46.8 (NCH2), 33.9 (CH2), 25.4 (CH2); MS (ESI) m/z 179 [(M+H)+ , 100]; HRMS (ESI) m/z calcd for C10H14N2O (M+H)+ 179.1179, found 179.1176 (+1.6 ppm error). 4.2.26. tert-Butyl-2-(hydroxy(pyridin-2-yl)methyl)pyrrolidine1-carboxylate syn-35 and anti-36 s-BuLi (2.0 mL of a 1.3 M solution in hexanes, 2.6 mmol, 1.3 equiv) was added dropwise to a stirred solution of N-Boc pyrrolidine 7 (342 mg, 2.0 mmol, 1.0 equiv) in THF (14 mL) at 30 C under Ar. The resulting yellow solution was stirred at 30 C for 5 min. Then, 2-pyridine-carboxaldehyde (429 mg, 4.0 mmol, 2.0 equiv) was added dropwise and the resulting solution was stirred at 30 C for 10 min and allowed to warm to rt over 2 h. Water (15 mL) was added and the two layers were separated. The aqueous layer was extracted with Et2O (3  5 mL) and the combined organics were dried (MgSO4) and evaporated under reduced pressure to give the crude product as a yellow oil. Purification by flash chromatography on silica with 98:2 CH2Cl2/MeOH as eluent gave a 40:60 mixture of hydroxy pyrrolidines syn-35 and anti-36 (384 mg, 69%) as a yellow oil. Characterisation of syn-35/anti-36 is described below. 4.2.27. 1-Benzylpyrrolidin-2-yl(pyridin-2-yl)methanol syn-37 and anti-38 TFA (2.05 mL, 12.85 mmol, 4.0 equiv) was added to a stirred solution of a 40:60 mixture of hydroxy pyrrolidines syn-35 and anti-36 (894 mg, 3.21 mmol, 1.0 equiv) in CH2Cl2 (15 mL) at 0 C under Ar. The resulting solution was allowed to warm to rt and then stirred at rt for 3 h. 20% NaOH(aq) (10 mL) was added and the two layers were separated. The aqueous layer was extracted with CH2Cl2 (3  30 mL) and the combined organics were dried (MgSO4) and evaporated under reduced pressure to give the crude amines (419 mg, 2.35 mmol). Benzaldehyde (0.50 mL, 2.59 mmol) was added to a stirred solution of crude amines (108 mg, 0.61 mmol), NaBH(OAc)3 (1.05 g, 4.70 mmol) and AcOH (0.04 mL, 0.47 mmol) in 1,2-dichloroethane (14 mL) at rt. The resulting solution was stirred at rt for 16 h. CH2Cl2 (15 mL) and saturated NaHCO3(aq) (15 mL) were added to the resulting mixture and the two layers were separated. The aqueous layer was extracted with CH2Cl2 (3  15 mL) and the combined organics were dried (MgSO4) and evaporated under reduced pressure to give the crude product. Purification by flash column chromatography on silica with 96:4 CH2Cl2-MeOH as eluent gave N-benzyl amino alcohol anti-38 (178 mg, 28%) as a yellow oil, Rf (96:4 CH2Cl2–MeOH) 0.2; IR (ATR) 3362 (OH), 2977, 2883, 2797, 1436, 1116, 1028, 750, 698 cm1 ; 1 H NMR (400 MHz, CDCl3) d 8.51 (br d, J = 5.0 Hz, 1H, Ar), 7.69 (ddd, J = 8.0, 8.0, 2.0 Hz, 1H, Ar), 7.56 (d, J = 8.0 Hz, 1H, Ar), 7.39–7.32 (m, 4H, Ar), 7.29–7.25 (m, 1H, Ar), 7.16 (ddd, J = 8.0, 5.0, 1.0 Hz, 1H, Ar), 4.97 (d, J = 3.5 Hz, 1H, OCH), 4.75 (br s, 1H, OH), 4.26 (d, J = 13.0 Hz, 1H, NCHAHBPh), 3.52 (d, J = 13.0 Hz, 1H, NCHAHBPh), 3.31 (ddd, J = 9.5, 6.0, 3.5 Hz, 1H, NCH), 3.06 (ddd, J = 9.5, 5.0, 5.0 Hz, 1H, NCH), 2.39 (ddd, J = 9.0, 9.0, 9.0 Hz, 1H, NCH), 1.68–1.57 (m, 3H, CH), 1.44–1.37 (m, 1H, CH)); 13C NMR (100.6 MHz, CDCl3) d 161.1 (ipso-Ar), 148.7 (Ar), 138.2 (ipso-Ar), 136.8 (Ar), 129.0 (Ar), 128.6 (Ar), 127.5 (Ar), 122.2 (Ar), 120.8 (Ar), 71.1 (OCH), 68.4 (NCH), 58.4 (PhCH2N), 54.5 (NCH2), 24.3 (CH2), 23.2 (CH2); (MS (ESI) m/z 269 [(M+H)+ , 100]; HRMS (ESI) m/z calcd for C17H20N2O (M+H)+ 269.1648, found 269.1658 (3.5 ppm error) and N-benzyl amino alcohol syn-37 (91 mg, 11%) as a yellow oil, Rf (96:4 CH2Cl2–MeOH) 0.1, IR (ATR) 3396 (OH), 2960, 2797, 1435, 1209, 1102, 1070, 749, 699 cm1 ; 1 H NMR (400 MHz, CDCl3) d 8.55 (ddd, J = 4.0, 1.5, 1.5 Hz, 1H, Ar), 7.69–7.63 (m, 2H, Ar), 7.31–7.20 (m, 5H, Ar), 7.15 (ddd, J = 7.0, 5.0, 1.5 Hz, 1H, Ar), 4.64 (d, J = 3.5 Hz, 1H, OCH), 4.13 (br s, 1H, OH), 3.49 (d, J = 13.0 Hz, 1H, NCHAHBPh), 3.43 (ddd, J = 9.5, 4.5, 3.5 Hz, NCH), 3.27 (d, J = 13.0 Hz, 1H, NCHAHBPh), 2.96 (ddd, J = 9.5, 7.0, 3.5 Hz, 1H, NCH), 2.37 (ddd, J = 9.5, 9.5, 6.5 Hz, 1H, NCH), 2.14–2.04 (m, 1H, CH), 1.92–1.85 (m, 1H, CH), 1.75–1.59 (m, 2H, CH); 13C NMR (100.6 MHz, CDCl3) d 162.2 (ipso-Ar), 148.8 (Ar), 138.6 (ipso-Ar), 136.8 (Ar), 128.9 (Ar), 128.5 (Ar), 127.4 (Ar), 122.4 (Ar), 121.2 (Ar), 74.4 (OCH), 68.5 (NCH), 60.4 (PhCH2N), 54.6 (NCH2), 29.4 (CH2), 24.3 (CH2); MS (ESI) m/z 291 [(M+Na)+ , 100]; HRMS (ESI) m/z calcd for C17H20N2NaO (M+Na)+ 291.1468, found 291.1462 (+2.2 ppm error). 4.2.28. 1-Benzyl-2-(pyridin-2-yl)piperidin-3-ol syn-39 Using general procedure C, N-benzyl pyrrolidine syn-37 (159 mg, 0.59 mmol), trifluoroacetic anhydride (0.33 mL, 1.77 mmol), Et3N (0.33 mL, 1.77 mmol) in THF (15 mL) and 20% NaOH(aq) (15 mL) gave the crude product. Purification by flash column chromatography on silica with 98:2 and 96:4 EtOAc–MeOH as eluent gave 3-hydroxy piperidine syn-39 (72 mg, 46%) as a brown oil, Rf (95:5 EtOAc–MeOH) 0.3; IR (ATR) 3362 (OH), 3060, 3029, 2936, 2794, 1433, 1119, 1015, 749, 696 cm1 ; 1 H NMR (400 MHz, CDCl3) d 8.57 (br d, J = 5.5 Hz, 1H, Ar), 7.68 (ddd, J = 7.5, 7.5, 2.0 Hz, 1H, Ar), 7.50 (d J = 7.5 Hz, 1H, Ar), 7.29–7.18 (m, 6H, Ar), 4.22 (br s, 1H, OH), 3.97 (br s, 1H, OCH), 3.70 (d, J = 14.0 Hz, 1H, PhCHAHBN), 3.56 (d, J = 2.0 Hz, 1H, NCH), 3.09 (d, J = 14.0 Hz, 1H, PhCHAHBN), 2.95 (dddd, J = 13.0, 3.5, 3.5, 1.5 Hz, 1H, NCH), 2.13 (ddd, J = 13.0, 13.0, 3.0 Hz, 1H, NCH), 2.00–1.93 (m, 2H, CH), 1.67–1.62 (m, 1H, CH), 1.52–1.48 (m, 1H, CH); 13C NMR (100.6 MHz, CDCl3) d 161.0 (ipso-Ar), 149.0 (Ar), 138.8 (ipso-Ar), 136.7 (Ar), 128.9 (Ar), 128.2 (Ar), 127.0 (Ar), 124.1 (Ar), 122.6 (Ar), 71.1 (OCH), 68.7 (NCH), 59.1 (PhCH2N), 52.3 (NCH2), 31.4 (CH2), 19.8 (CH2); MS (ESI) m/z 269 [(M+H)+ , 100]; HRMS (ESI) m/z calcd for C17H20N2O (M+H)+ 269.1648, found 269.1654 (2.2 ppm error). 2692 M. Lüthy et al. / Bioorg. Med. Chem. 23 (2015) 2680–2694

4.2.29. 1-Benzyl-2-(pyridin-2-yl)piperidin-3-ol anti-40 Using general procedure C, N-benzyl pyrrolidine anti-38 (168 mg, 0.63 mmol), trifluoroacetic anhydride (0.35 mL, 1.89 mmol), Et3N (0.35 mL, 1.89 mmol) in THF (15 mL) and 20% NaOH(aq) (15 mL) gave the crude product. Purification by flash column chromatography on silica with 95:5 CH2Cl2–MeOH as eluent gave 3-hydroxy piperidine anti-40 (116 mg, 69%) as a brown oil, Rf (95:5 CH2Cl2–MeOH) 0.3; IR (ATR) 3328 (OH), 2934, 2798, 1433, 1112, 1073, 969, 776, 698 cm1 ; 1 H NMR (400 MHz, CDCl3) d 8.55 (br d, J = 5.0 Hz, 1H, Ar), 7.71 (ddd, J = 8.0, 8.0, 2.0 Hz, 1H, Ar), 7.67 (d, J = 8.0 Hz, 1H, Ar), 7.30–7.22 (m, 5H, Ar), 7.20 (ddd, J = 8.0, 5.0, 2.0 Hz, 1H, Ar), 3.75 (ddd, J = 11.0, 9.0, 4.5 Hz, 1H, OCH), 3.60 (d, J = 14.0 Hz, 1H, PhCHAHBN), 3.23 (d, J = 9.0 Hz, 1H, NCH), 3.04 (br s, 1H, OH), 3.02 (d, J = 14.0 Hz, 1H, PhCHAHBN), 2.93 (ddd, J = 12.0, 3.0, 3.0 Hz, 1H, NCH), 2.19–2.13 (m, 1H, NCH), 2.06–1.99 (m, 1H, NCH), 1.70–1.64 (m, 2H, CH), 1.49–1.39 (m, 1H, CH); 13C NMR (100.6 MHz, CDCl3) d 162.0 (ipso-Ar), 148.9 (Ar), 138.9 (ipso-Ar), 137.1 (Ar), 128.6 (Ar), 128.2 (Ar), 126.8 (Ar), 122.8 (Ar), 122.6 (Ar), 75.9 (OCH), 73.0 (NCH), 59.3 (PhCH2N), 52.0 (NCH2), 33.3 (CH2), 23.0 (CH2); MS (ESI) m/z 269 [(M+H)+ , 100]; HRMS (ESI) m/z calcd for C17H20N2O (M+H)+ 269.1648, found 269.1655 (2.5 ppm error). 4.2.30. tert-Butyl-2-(hydroxy(pyridin-2-yl)methyl)pyrrolidine1-carboxylate syn-35 Using general procedure D, 20% Pd(OH)2/C (17 mg, 0.12 mmol), ammonium formate (107 mg, 1.70 mmol), N-benzyl hydroxy pyrrolidine syn-37 (90 mg, 0.34 mmol) in EtOH (12 mL) and Boc2O (81 mg, 0.37 mmol) gave the crude product as a yellow oil. Purification by flash column chromatography on silica with 98:2 CH2Cl2–MeOH as eluent gave N-Boc hydroxy pyrrolidine syn-35 (64 mg, 68%) as a colourless oil, Rf (98:2 CH2Cl2–MeOH) 0.35; IR (ATR) 3384 (OH), 2974, 2931, 2882, 1687 (C@O), 1591, 1570, 1391, 1365, 1161, 1112, 1062, 764 cm1 ; 1 H NMR (400 MHz, CDCl3) (60:40 mixture of rotamers) d 8.51 (br s, 1H, Ar), 7.65– 7.63 (m, 1H, Ar), 7.42–7.40 (m, 0.6H, Ar), 7.18–7.12 (m, 1.4H, Ar), 5.76 (br s, 0.6H, OH), 5.05 (br s, 0.4H, OH), 4.87 (br s, 0.4H, OCH), 4.79 (br s, 0.6H, OCH), 4.19–4.14 (m, 1H, NCH), 3.35–3.25 (m, 1.6H, NCH), 2.95 (br s, 0.4H, NCH), 1.80 (br s, 1.6H, CH), 1.63 (br s, 2H, CH), 1.48 (s, 9H, CMe3), 0.58 (br s, 0.4H, CH); 13C NMR (100.6 MHz, CDCl3) d 161.4 (ipso-Ar or C@O), 157.5 (ipso-Ar or C@O), 148.3 (Ar), 136.6 (Ar), 122.7 (Ar), 121.9 (Ar), 80.5 (CMe3), 77.5 (OCH), 63.4 (NCH), 47.7 (NCH2), 28.6 (CMe3), 27.2 (CH2), 23.9 (CH2); MS (ESI) m/z 279 [(M+H)+ , 100]; HRMS (ESI) m/z calcd for C15H22N2O3 (M+H)+ 279.1703, found 279.1694 (+3.5 ppm error). 4.2.31. tert-Butyl-2-(hydroxy(pyridin-2-yl)methyl)pyrrolidine1-carboxylate anti-36 Using general procedure D, 20% Pd(OH)2/C (11 mg, 0.08 mmol), ammonium formate (69 mg, 1.10 mmol), N-benzyl hydroxy pyrrolidine anti-38 (60 mg, 0.22 mmol) in EtOH (10 mL) and Boc2O (53 mg, 0.24 mmol) gave the crude product as a yellow oil. Purification by flash column chromatography on silica with 98:2 CH2Cl2–MeOH as eluent gave N-Boc hydroxy pyrrolidine anti-36 (56 mg, 91%) as a colourless oil, Rf (98:2 CH2Cl2–MeOH) 0.3; IR (ATR) 3261 (OH), 2970, 2929, 2881, 1688 (C@O), 1593, 1569, 1394, 1365, 1162, 1100, 769 cm1 ; 1 H NMR (400 MHz, CDCl3) (65:35 mixture of rotamers) d 8.53–8.50 (m, 1H, Ar), 7.65 (dd, J = 7.0, 7.0 Hz, 1H, Ar), 7.39 (d, J = 7.0 Hz, 0.65H, Ar), 7.22–7.16 (m, 1.35H, Ar), 5.15 (br s, 0.65H, OCH), 5.05 (br s, 0.35H, OCH), 4.64 (br s, 1H, OH), 4.19 (br s, 0.65H, NCH), 3.99 (br s, 0.35H, NCH), 3.55–3.29 (m, 1.35H, NCH), 3.14–3.08 (m, 0.65H, NCH), 2.02–1.59 (m, 4H, CH), 1.46 (s, 5.85H, CMe3), 1.43 (s, 3.15H, CMe3); 13C NMR (100.6 MHz, CDCl3) (rotamers) d 160.1 (ipso-Ar or C@O), 159.4 (ipso-Ar or C@O), 156.7 (ipso-Ar or C@O), 156.1 (ipso-Ar or C@O), 148.1 (Ar), 147.8 (Ar), 136.7 (Ar), 136.6 (Ar), 122.6 (Ar), 122.4 (Ar), 121.5 (Ar), 121.2 (Ar), 79.8 (CMe3), 79.6 (CMe3), 74.6 (OCH), 73.2 (OCH), 63.2 (NCH), 63.1 (NCH), 47.6 (NCH2), 47.0 (NCH2), 29.8 (CH2), 28.7 (CMe3), 28.3 (CMe3), 25.8 (CH2), 24.3 (CH2), 23.7 (CH2); MS (ESI) m/z 279 [(M+H)+ , 100]; HRMS (ESI) m/z calcd for C15H22N2O3 (M+H)+ 279.1703, found 279.1692 (+4.2 ppm error). 4.3. 3-D shape analysis 3-D structures were generated Pipeline Pilot 8.5.0.200, 2011, Accelrys Software Inc. Prior to conformer generation a wash step was performed, which involved ionising the molecule at pH 7.4, adding explicit hydrogens and outputting the canonical tautomer. Conformers were generated using the BEST method in Catalyst with a maximum relative energy threshold of 20 kcal mol1 . These conformations were then minimised using 1000 steps of Steepest Descent with a RMS gradient tolerance of 3 and 200 steps of Conjugate Gradient with an RMS gradient tolerance of 0.1. Minimisation was performed using the CHARMm forcefield with Momany–Rone partial charge estimation and a Generalised Born implicit solvent model. The lowest energy conformer was selected. The generated conformations were used to generate the three Principal Moments of Inertia (I1, I2 and I3) which were then normalised by dividing the two lower values by the largest (I1/I3 and I2/I3) using Pipeline Pilot built-in components. Full details are provided in the Supporting information. Acknowledgments We thank the EPSRC (DTA to M.C.W. and Institutional Award to POB/REH/ML), the Wellcome Trust (Chemical Biology Centre via C2D2, PSB), the Royal Thai Government (scholarship to C.H.C.) and Asahi Kasei Pharma Corporation (secondment of MA to the University of York) for funding. We also thank Accelrys for provision of Pipeline Pilot and Dr Steve Roughley of Vernalis for advice on 3-D shape analysis. Supplementary data Supplementary data (copies of 1 H NMR and 13C NMR spectra and analysis of the virtual library) associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. bmc.2015.04.005. References and notes 1. Gleeson, M. P. J. Med. Chem. 2008, 51, 817. 2. Hughes, J. D.; Blagg, J.; Price, D. A.; Bailey, S.; DeCrescenzo, G. A.; Devraj, R. V.; Ellsworth, E.; Fobian, Y. M.; Gibbs, M. E.; Gilles, R. W.; Greene, N.; Huang, E.; Krieger-Burke, T.; Loesel, J.; Wager, T.; Whiteley, L.; Zhang, Y. Bioorg. Med. Chem. Lett. 2008, 18, 4872. 3. 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