Synthesis of Structurally Diverse N‐Substituted Quaternary‐Carbon‐Containing Small Molecules from α,α‐Disubstituted Propargyl Amino Esters

Abstract N‐containing quaternary stereocenters represent important motifs in medicinal chemistry. However, due to their inherently sterically hindered nature, they remain underrepresented in small molecule screening collections. As such, the development of synthetic routes to generate small molecules that incorporate this particular feature are highly desirable. Herein, we describe the diversity‐oriented synthesis (DOS) of a diverse collection of structurally distinct small molecules featuring this three‐dimensional (3D) motif. The subsequent derivatisation and the stereoselective synthesis exemplified the versatility of this strategy for drug discovery and library enrichment. Chemoinformatic analysis revealed the enhanced sp3 character of the target library and demonstrated that it represents an attractive collection of biologically diverse small molecules with high scaffold diversity.

Diversity-oriented synthesis strategy used to access compounds 2 -33 Scheme S1. Diversity-oriented synthesis strategy used to access compounds 2 -33 (cont.) Figure S1. The 27 molecular frameworks featured within DOS library.

GENERAL REMARKS
All non-aqueous reactions were performed in dry glassware under a stream of Argon using anhydrous solvents. Tetrahydrofuran was dried over sodium wire and distilled from a mixture of lithium aluminium hydride and calcium hydride with triphenylmethane as the indicator.
Dichloromethane, toluene and methanol were all distilled from calcium hydride. Petroleum ether was distilled before use and refers to the fraction between 40-60 ºC; anhydrous DMF and DCE were purchased from Aldrich and used as received. All reagents were obtained from commercial suppliers and used without further purification. Reactions were carried out at ambient temperature unless otherwise stated. All temperatures below 0 ºC are achieved with an external bath: those of 0 ºC were maintained using an ice/water bath, those of lower temperatures using a dry ice/ DMF bath.
Yields refer to chromatographically and spectroscopically pure compounds unless otherwise Assignments are supported by either chemical shift, APT/DEPT, two dimensional experiments (HMBC and HMQC) or by analogy to fully interpreted spectra for related compounds. High resolution mass spectrometry (HRMS), measurements were carried out on a Micromass LCT Premier spectrometer using electron spray ionisation (ESI) techniques.
Masses are quoted within the error limits of ±5ppm mass units.

Synthesis of methyl 2-amino-2-methylpent-4-ynoate (1)
Following a modified version of a reported procedure, 1 methyl pyruvate (4.85 mL, 53.6 mmol) and p-anisidine (4.40 g, 35.7 mmol) were dissolved in MeCN (35 mL) and the mixture was heated at 50 ºC for 1 hour. Then, the solvent was removed under reduced pressure and the crude product was dissolved in a mixture of petroleum ether/CH2Cl2 (10:1). The grey precipitate formed was removed by filtration and the filtrate was concentrated in vacuo. The resulting green oil was dissolved in DMF (158 mL) and the mixture was cooled to 0 ºC.
Propargyl bromide (80% wt. % in toluene, 4.77 mL, 42.9 mmol) and activated zinc powder (3.5 g, 53.6 mmol) were added. The reaction mixture was then warmed to room temperature and then heated to 60 ºC. After 1 hour the medium was cooled to 0 °C, hydrolysed with a saturated aqueous solution of NH4Cl and extracted with EtOAc (3x). The combined organic layers were washed with brine, dried with Na2SO4, filtered, and the solvent evaporated. The residue was purified by flash column chromatography (silica gel; petroleum ether/EtOAc, 5:1) to afford 5.65 g of S1 (64% yield) as a yellow oil. The spectroscopic data are in agreement with those previously reported in the literature. A solution of CAN (6.20 g, 12.4 mmol) in H2O (39 mL) was added drop-wise over 15 minutes to a stirred solution of S1 (1.50 g, 6.20 mmol) in MeCN (39 mL) cooled to 0 ºC. After 2 hours stirring, the resulting solution was treated with 2N HCl to achieve pH 1. The aqueous phase was washed with EtOAc (3x) and basified to pH 10-12 by the addition of Na2CO3. The resulting suspension was extracted with CH2Cl2 (3x), dried over Na2SO4, filtered, and the solvents evaporated under reduced pressure to yield 570 mg of 1 (65% yield) as an orange oil without further purification. The spectroscopic data are in agreement with those previously reported in the literature. 2 HRMS (ESI): m/z calcd for C7H12NO2 [M+H] + : 142.0863; found: 142.0865.

Synthesis of methyl 2-acetamido-2-methylpent-4-ynoate (S6)
To a solution of 1 (50 mg, 0.35 mmol) in CH2Cl2 (1 mL) cooled to 0 ºC, was added acetyl chloride (0.037 mL, 0.53 mmol) and Et3N (0.10 mL, 0.70 mmol) and the mixture warmed to room temperature and stirred for 16 hours. Saturated aqueous solution of NH4Cl was added and the organic layer was separated. The aqueous layer was extracted with CH2Cl2 (2x) and the combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo.
The mixture was stirred at room temperature overnight and then the organic solvent was removed under reduced pressure.

Synthesis of 7a-methyl-5,7a-dihydro-1H,3H-pyrrolo[1,2-c]oxazol-3-one (33)
InCl3 (62 mg, 0.28 mmol) was introduced into 5 mL flask and heated with a heat gun (150 ºC) under vacuum for 2 min. After being allowed to cool to room temperature, THF (0.8 mL) was added under Argon. The mixture was stirred at room temperature for 10 min and cooled added. The mixture was stirred at 85 ºC for 2 hours (in a sealed tube). Then, the reaction was cooled to room temperature, diluted with CH2Cl2 (5 mL) and washed with water (5 mL).
The aqueous layer was extracted with CH2Cl2 (3x) and the combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. The crude product was purified by flash column chromatography (silica gel; EtOAc/petroleum ether 2:1) to give 7 mg of 33

STEREOCHEMICAL AND STRUCTURAL ASSIGNMENTS Compounds 2 and 3
The position of the phenyl substituent present in 2 and 3 was established by means of nOe studies. Thereby, in the nOe spectrum of compound 2 the aromatic proton (Ha) at the α -position to the nitrogen showed an interaction with the methylene group (2Hc) adjacent to the NH ( Figure S3). As expected, the nOe spectrum of 3 showed an interaction between the aromatic proton (Ha) at the α-position to the nitrogen and the methylene group (2Hb) adjacent to the quaternary stereocenter ( Figure S4).

Compound 14
Attempts to determine the relative position of the substituents present within 14 were carried out. Unfortunately, nOe experiments were not conclusive however, further analysis is being carried out to determine the relative configuration of 14.

Compound 16
The relative position of the substituents present in 16 was established by means of nOe studies. Thereby, in the nOe spectrum of compound 16, the alkene proton (Ha) showed an interaction with the NH proton (Hb) ( Figure S6). Furthermore, an interaction could also be seen between the alkene proton (Ha) and the t-butyl group methyl groups Hc).

Conformation Limit 100
Only the conformer with the lowest energy was retained for principal moment of inertia (PMI) calculations. Normalized PMI ratios (I1/I3 and I2/I3) of these conformers were obtained from MOE and then plotted on a triangular graph, with the canonical coordinates (0,1), (0.5,0.5) and (1,1) representing a perfect rod, disc and sphere respectively (Figure 3).          Figure S9 shows that the synthesized DOS Library displays a high level of molecular shape diversity, covering a broad area of molecular shape space. Notable differences in the molecular shape space distribution can be observed between the DOS library and the DOS Library Ph, with a shift towards the right-hand side of the plot. In comparison to the Maybridge 'Ro3' core fragment collection, the DOS library presented in this work shows a broader coverage of the 3D molecular space shape, whilst the Maybridge library is densely populated around the 'rod-' and 'disk-' like areas of the plot. Furthermore, a higher percentage of the DOS Library and DOS Library Ph occupy the right-hand side of the plot suggesting more 3D character, when compared to the Maybridge library.

General details
Computational analysis was carried out using the same MOE software package and settings described above in section A. The DOS library compounds were analysed for the for the

Multi-Dimensional Scaling (MDS) analysis
2,000 ChEMBL compounds with IC50, EC50 and Ki values ≤ 10 µM were randomly selected from each bioactivity class for MDS analysis. In addition, compounds synthesized via two different targeted approaches were used as a benchmark, namely those previously presented by Hergenrother (88 compounds), 11 and Kiessling (9 compounds). 12 For this purpose, the SDF files of all compounds belonging to the different bioactivity classes as well as the synthetic libraries and the DOS library presented were standardized using the