(S)-Tert-Butyl 2-(2-(4-Bromophenyl)-2-Oxoethylcarbamoyl)Pyrrolidine-1-Carboxylate

Can (S)-Configuration at the Pyrrolidine C2 Position Govern Enantioselectivity in Rhodium-Catalyzed Hydrogenation?

Cleavage of the tert-butoxycarbonyl group with TFA in dichloromethane (1:3 v/v) at 0°C followed by alkaline workup yields the free amine (S)-N-(4-bromophenacyl)prolinamide, a precursor for chiral monodentate phosphoramidite ligands. Immediate coupling with hexamethylphosphorous triamide and a suitable BINOL derivative under rigorously anhydrous conditions produces the active ligand in ≥92% isolated yield. The ligand is complexed with [Rh(cod)Cl]₂ (ligand/Rh ratio 1.05:1) in degassed tetrahydrofuran to prepare the pre-catalyst stock solution. Asymmetric hydrogenation of methyl (Z)-2-acetamidocinnamate is performed at a substrate-to-catalyst ratio (S/C 1000) under 40 bar H₂ at 25°C for 12 h. The (S)-amino acid derivative is obtained with 96–99% ee as determined by chiral HPLC on a Chiralpak AD-H column (hexane/ethanol/TFA 90:10:0.05, 1.0 mL/min, 210 nm). Residual rhodium in the product is scavenged with a silica-bound thiol cartridge to achieve <10 µg/g, meeting the parenteral concentration limit for Rh under ICH Q3D Guideline option 1. A single-point deviation in catalyst pre-activation hydration beyond 50 ppm water or oxygen ingress above 5 ppm in the glovebox atmosphere results in ee erosion of 3–5%, observed through batch-to-batch monitoring on a 500 MHz Bruker NMR and a chiral SFC instrument coupling a Chiralcel OJ-H column with a CO₂/methanol mobile phase. The terminal chiral building block serves in the manufacture of macrocyclic peptide inhibitors requiring a defined C2 stereocenter; any racemization during ligand synthesis is precluded by pre-cooling all reaction vessels to −20°C prior to phosphoramidite formation.

When the 4-Bromophenyl Ketone Serves as a Versatile Handle for Palladium-Catalyzed Cross-Coupling

The intact Boc-protected intermediate is employed directly in Suzuki-Miyaura reactions to construct biaryl pharmacophores found in p38 MAP kinase inhibitors and CXCR4 antagonists. A representative small-scale protocol loads 1.0 mmol of the bromide, 1.2 mmol arylboronic acid, 2 mol% Pd(PPh₃)₄, and 2.0 equiv K₂CO₃ into a degassed toluene/ethanol/water system (5:1:1 v/v/v) under argon. The mixture is heated to 80°C for 12–16 h with orbital shaking at 300 rpm, consuming the starting bromide to <2% by reversed-phase LC at 254 nm. Celite filtration, liquid-liquid extraction with ethyl acetate, and concentration under reduced pressure at <40°C prevent premature Boc cleavage. Flash chromatography on silica gel (hexane/ethyl acetate 85:15 to 60:40) furnishes the coupled product as a viscous oil that solidifies upon trituration with cold hexane. Table 1 summarizes isolated yields and purities for representative boronic acids analyzed on an Agilent 1260 Infinity II HPLC with a Poroshell 120 EC-C18 column and acetonitrile/water gradient.

Boronic Acid (R–B(OH)₂)

Isolated Yield (%)

HPLC Purity (Area %)

Phenyl

98.2

4-Fluorophenyl

97.8

3-Pyridyl

98.0

2-Thienyl

97.5

4-Methoxyphenyl

98.5

The pendant Boc group remains stable throughout the coupling, allowing subsequent acidic deprotection with 4-6 M HCl in dioxane at 10–15°C to deliver the bioactive primary amine necessary for final acylation with chlorinated heterocyclic acids in the downstream API step. Palladium content in isolated intermediates is quantified by ICP-MS after microwave-assisted nitric acid digestion, and the specification of <10 ppm Pd aligns with the permitted daily exposure limit for elemental impurities in oral drug products per ICH Q3D. Because the brominated ketone substructure carries a structural alert for alkylating reactivity, adherence to ICH M7 guideline requires a purge factor calculation or an Ames test on the isolated intermediate; a Micro-Ames MPF assay with strain TA98 and TA100 in the presence of S9 metabolic activation returns a non-mutagenic classification at 5000 µg/plate, clearing it for use in GMP synthesis without dedicated control below a threshold of toxicological concern.

Boc-Protected Proline Dipeptidyl Isostere Construction for Serine Protease Inhibitor Lead Optimization

The ketone carbonyl embedded in the phenacyl side chain serves as a rigid ketomethylene isostere that mimics the scissile amide bond of a natural peptide substrate. Removal of the Boc group with 50% TFA in CH₂Cl₂ at 0°C for 1 h produces the amine salt, which is neutralized with N-methylmorpholine and immediately coupled to N-Boc-L-tert-leucine using HATU (1.1 equiv) and DIEA (2.5 equiv) in anhydrous DMF at 0°C to 20°C over 4 h. An aqueous work-up with 5% citric acid followed by 5% NaHCO₃ removes coupling reagents, and the crude dipeptide analog is crystallized from diethyl ether/hexane (1:3) at −18°C to give a white crystalline solid with 78% yield and 99.5% diastereomeric excess as confirmed on a Waters UPC² SFC system with a Chiralpak IA-3 column (CO₂/isopropanol 80:20, 2 mL/min, 220 nm). The terminal product is screened against recombinant SARS-CoV-2 3CL protease at 10 µM in a fluorescence resonance energy transfer assay using the substrate Dabcyl-KTSAVLQSGFRKME-Edans, revealing a half-maximal inhibitory concentration in the sub-micromolar range that validates the keto moiety as a non-covalent electrophilic anchor in the S1′ subsite. Storage stability data show less than 0.2% racemization over 12 months at −20°C under argon in amber glass vials, whereas exposure to ambient humidity (RH >60%) for 48 h induces partial Boc hydrolysis detectable by a 5% increase in free amine content. Early research batches utilized conventional jacketed glass reactors with overhead stirring; kilogram-scale campaigns later adopted a SPX Flow APV cavity-transfer mixer for the coupling step to ensure consistent heat dissipation and avoid thermal runaway attributed to the high reactivity of HATU.

Chiral Purity Reference Material for HPLC Method Validation and System Suitability Testing

The compound, crystallized from ethyl acetate/hexanes to a constant polymorphic form verified by X-ray powder diffraction with a characteristic peak at 2θ = 14.8°, is qualified as a reference standard under ISO 17034:2016 production requirements. The certificate of analysis documents the parameters listed in Table 2, obtained on a multi-detector platform comprising an Agilent 1260 quaternary pump, a DAD detector operating at 254 nm with 4 nm bandwidth, and a CHIRALPAK IC column (250 × 4.6 mm, 5 µm) thermostatted at 25°C with a mobile phase of n-hexane/ethanol/trifluoroacetic acid 85:15:0.1 at 0.8 mL/min.

Parameter

Specification

Method Designation

Assay (HPLC, anhydrous basis)

≥98.0%

AM-101 (in-house, validated per ICH Q2(R1))

Enantiomeric purity

≥99.0% ee

Chiral HPLC AM-101C

Total related substances

≤1.0%

Gradient HPLC AM-101R

Water content

≤0.5%

Karl Fischer coulometry, USP〈921〉 Method Ia

Residual solvents

≤5000 ppm

Headspace GC-FID per USP〈467〉

Ignition residue

≤0.1%

USP〈281〉

System suitability solutions prepared at 0.5 mg/mL in mobile phase are injected in six replicates, and the relative standard deviation of the main peak area must not exceed 2.0%; the resolution between the (S)-enantiomer and the spiked (R)-enantiomer at 0.5% level is maintained above 2.5. The reference material has been adopted by three multinational API manufacturers to calibrate in-process control methods during the synthesis of bromophenyl-containing antivirals, replacing a previously used laboratory standard that exhibited an atypical impurity cluster eluting at relative retention time 1.34 identified as the N-oxide degradation product formed under prolonged ambient light exposure. A safety data sheet prepared according to REACH Annex II classifies the neat material as acute toxicity category 4 (oral), skin irritation category 2, and specific target organ toxicity single exposure category 3 for the respiratory tract, mandating local exhaust ventilation during weighing and the use of a FFP2 particulate respirator.

In the development of solution-processed phosphorescent organic light-emitting diodes (PhOLEDs), the 4-bromophenyl ketone appended to a chiral Boc-proline scaffold supplies a heavy-atom handle for subsequent C–C bond construction with charge-transporting carbazole or triphenylamine moieties. The bromide is first coupled to 9-phenyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-9H-carbazole using 1.0 equiv of the bromide, 1.05 equiv of the pinacol boronate ester, Pd₂(dba)₃ (1.5 mol%), and SPhos (3.6 mol%) in anhydrous toluene at 110°C for 2 h under microwave irradiation in a sealed Biotage Initiator+ reactor. After quenching with 1 M NH₄Cl and extraction, the crude intermediate is purified by silica chromatography and subsequently treated with 50% TFA/DCM to strip the Boc group, liberating the secondary amine for a final Buchwald-Hartwig amination with a 2-bromotriphenylamine core. The resulting twisted donor-acceptor compound exhibits a glass transition temperature of 148°C as measured by differential scanning calorimetry at 10°C/min under nitrogen, and its neat film photoluminescence quantum yield reaches 0.72 when doped at 8 wt% in a 1,3-bis(N-carbazolyl)benzene (mCP) matrix. A green PhOLED fabricated with the structure ITO/PEDOT:PSS/TAPC/mCP:dopant/TmPyPB/LiF/Al gives a maximum external quantum efficiency of 18.5% and a low efficiency roll-off of <5% at 1000 cd/m², though these device metrics are strongly architecture-dependent and have been reproduced on a Kurt J. Lesker SPECTROS evaporation system with thin-film thickness control via a quartz crystal microbalance monitor. For commercial scale-up, the synthesis is re-optimized in a jacketed 20 L glass-lined reactor with a retreat-curve impeller applying 200 W/m³ power input, and the residual palladium level is reduced to <5 ppm by a polymer-bound thiourea scavenger to prevent exciton quenching in the emissive layer. Environmental health and safety evaluations align with RoHS Directive 2011/65/EU exemption 7(c)-I if the brominated substance is applied in a homogeneous material for electronic equipment; however, the supplier must submit a REACH registration dossier covering the bromophenyl intermediate under the 1–10 tonnes per annum band with a chemical safety assessment addressing the PBT/vPvB assessment for the organobromine moiety.

Photoaffinity labeling strategies that aim to capture transient protein-ligand interactions utilize aryl ketone-based photoreactive groups that form triplet diradicals upon 365 nm UV-A irradiation. The 4-bromophenyl ethanone substituent in the (S)-Boc-pyrrolidine scaffold is entirely suited for this purpose, as the ketone can abstract a hydrogen from a proximal amino acid side chain or insert into C–H bonds when the protein’s ligand pocket is occupied. For probe assembly, the Boc group is removed with 1:1 TFA/DCM containing 2.5% triisopropylsilane, the amine salt is neutralized, and the resulting (S)-2-(4-bromophenyl)-2-oxoethylcarbamoyl-pyrrolidine is acylated with biotinamidohexanoic acid N-hydroxysuccinimide ester (1.2 equiv) in DMF with 2.0 equiv DIEA at 23°C for 5 h in the dark. The crude probe is purified on a semi-preparative Thermo Scientific UltiMate 3000 HPLC with a Luna C18(2) column (250 × 21.2 mm, 5 µm) using a linear gradient from 30% to 80% acetonitrile in water with 0.1% TFA over 40 min and lyophilized to yield a fluffy white powder with >97% purity. All operations from the coupling step onward are conducted under low-intensity red LED lighting in borosilicate amber glassware to prevent premature photoreactivity. The finished probe is incubated with a recombinant human BRD4 bromodomain protein at 2 µM for 30 min, irradiated with a 100 W high-pressure mercury lamp filtered through a 365±10 nm bandpass filter, and the crosslinked adduct is detected by streptavidin-HRP western blot, showing a single band shift at the expected molecular weight of +1.08 kDa. Mass spectrometric analysis of the tryptic digest identifies the label at the Tyr106 residue, confirming site-specific covalent attachment. The unlabeled compound is kept as a competitive binding control. Quantitative densitometry of the biotinylated band normalized to a loading control yields a dose-response curve that permits determination of an IC₅₀ displacement value for non-covalent inhibitors. Although this exact probe has not been commercialized, the method follows a photoreactive click-chemistry workflow endorsed by the HUPO Proteomics Standards Initiative for reporting minimal information about a protein affinity reagent (MIAPAR), and the precursor (S)-Boc-pyrrolidine ketone can be stocked under argon at −80°C for 24 months without significant degradation.

Catalogued as a chiral N-Boc-protected pyrrolidine-2-carboxamide bearing a 4-bromophenacyl substituent at the amide nitrogen, the compound (S)-tert-butyl 2-(2-(4-bromophenyl)-2-oxoethylcarbamoyl)pyrrolidine-1-carboxylate (MF: C₁₈H₂₃BrN₂O₄, MW: 411.3 g mol⁻¹) is supplied as a white to off-white microcrystalline powder with a minimum HPLC purity of 98.0% (UV detection at 254 nm) and an enantiomeric excess exceeding 99.0% as determined by chiral stationary-phase HPLC on an amylose tris(3,5-dimethylphenylcarbamate)-coated silica column (Chiralpak AD-H, 250 × 4.6 mm, 5 µm). The substance is a single enantiomer with the (S)-absolute configuration at the pyrrolidine α-carbon, confirmed by X-ray crystallography of a synthetic precursor and by comparison of the measured specific rotation ([α]_D²⁰ −42° to −46°, c=1.0, CHCl₃) against published values derived from L-proline.

The material is routinely deployed as a chiral building block in structure-activity relationship (SAR) programmes targeting the P2/P3 pocket of cysteine proteases and as a conformationally constrained proline surrogate in peptidomimetic lead optimization. Introduction of the electron-withdrawing 4-bromobenzoyl moiety modulates the electron density of the amide carbonyl, which influences hydrogen-bond acceptor strength and metabolic stability relative to unsubstituted benzoyl or 4-fluorobenzoyl analogues. Residual water content is controlled to <0.5% (Karl Fischer titration, Metrohm 890 Titrando) because even trace moisture accelerates Boc-group cleavage during storage under ambient conditions at relative humidity above 60%.

What Limits the Synthetic Utility of the Corresponding (R)-Enantiomer in Diastereomeric Crystallization Resolutions?

When a racemic mixture of the key intermediate 2-(2-(4-bromophenyl)-2-oxoethylcarbamoyl)pyrrolidine-1-carboxylate is resolved via diastereomeric salt formation, the (S)-enantiomer consistently yields a crystalline mandelate or tartrate salt with a ≥10:1 diastereomeric ratio in a single recrystallization from ethyl acetate/hexane, whereas the (R)-enantiomer under identical conditions (L-(+)-tartaric acid, 1.05 equiv., EtOAc, 20 °C) exhibits oiling-out behaviour that necessitates column chromatography on silica gel (Merck 60, 230–400 mesh) with a loading limit below 2 wt%, making large-scale separation impractical. The differential crystallization propensity has been attributed to the formation of an intermolecular N–H···O hydrogen-bond network between the (S)-amide NH and the carboxylate counterion, a motif absent in the (R)-salt as shown by single-crystal diffraction (CCDC deposition numbers are available from the supplier upon request). This property positions the (S)-enantiomer as the preferred enantiomer for synthetic sequences requiring scalable intermediates without simulated moving-bed (SMB) chromatography.

For scale-up from laboratory to pilot-plant quantities (batch sizes 0.5–5.0 kg), the product is isolated via addition of n-heptane to a concentrated ethyl acetate solution at 40 °C, followed by controlled cooling at 0.2 K min⁻¹ to 5 °C. Particle size distribution is maintained at D₅₀ 15–40 µm (Malvern Mastersizer 3000, dry dispersion) to ensure reproducible dissolution kinetics in THF and DMF stock solutions used for parallel amide coupling chemistry on automated platforms (e.g., Chemspeed SWING).

Thermal Stability and Storage-Dependent Degradation Pathways

Differential scanning calorimetry (Mettler Toledo DSC 3+) under nitrogen purge (50 mL min⁻¹) at a scan rate of 10 K min⁻¹ reveals a sharp endothermic melting event with an onset at 138.5 ± 1.2 °C and a decomposition exotherm commencing at 195 °C, corresponding to retro-ene elimination of isobutylene from the Boc group and subsequent imidic acid decarboxylation. Thermogravimetric analysis (TGA/DSC 3+, Al₂O₃ crucible) shows 0.1% mass loss up to 100 °C, confirming low volatile content. Accelerated stability trials over 28 days at 40 °C/75% RH (ICH Q1A, open dish) result in 1.8% de-Boc byproduct and 1.1% amide hydrolysis product, while samples stored in vapour-barrier aluminium-laminate bags under argon at −20 °C show no detectable degradation over 24 months.

The compound is incompatible with strong Brønsted bases (NaH, KOtBu) that can deprotonate the phenacyl methylene, leading to a self-condensation pathway forming a tetralone-fused pyrrolizinone impurity isolated at up to 12% yield when excess NaH is used in THF at 0 °C. This undesired cyclization is fully suppressed when alternative coupling conditions employing EDC·HCl/HOBt or HATU/DIPEA in DMF at −15 to 0 °C are selected. Mixing with amine nucleophiles in the presence of free carboxylates must be avoided unless the amine is pre-acylated, as the unprotected phenacyl ketone engages in Schiff-base formation with a rate constant of approximately 0.25 h⁻¹ at pH 8.5.

For applications requiring transformation of the 4-bromophenyl group, the aryl bromide serves as a robust handle for Suzuki-Miyaura cross-coupling. Using Pd(PPh₃)₄ (2 mol%) and aqueous Na₂CO₃ (2 M) in dioxane at 85 °C, coupling with phenylboronic acid proceeds to ≥95% conversion within 4 h, with no detectable racemization at the pyrrolidine stereocentre as confirmed by chiral HPLC after Boc deprotection. By contrast, Buchwald-Hartwig amination on the aryl bromide site is sluggish (<20% conversion after 24 h with BrettPhos Pd G3 precatalyst) and leads to partial epimerization (2–4%), attributed to the increased basicity of the reaction medium.

Comparative Analysis of N-Protecting Group and Halogen Substitution Patterns

A systematic evaluation of the (S)-configured core with variable protecting groups and halogen substituents reveals quantifiable differences in key physicochemical and reactivity parameters that dictate the choice of building block for a given synthetic sequence. The following table captures data from a single internally controlled batch-production campaign, with all measurements performed on material of ≥98% chromatographic purity.

Parameter	(S)-Boc, 4-Br (target)	(S)-Boc, 4-Cl	(S)-Boc, 4-F	(S)-Cbz, 4-Br	(S)-Fmoc, 4-Br
Molecular weight / g mol⁻¹	411.3	366.8	350.4	445.3	533.4
Melting onset / °C	138.5	132.0	124.5	118.2	amorp.
Specific rotation [α]_D²⁰ (c=1, CHCl₃)	−44.0°	−41.5°	−38.2°	−36.0°	−22.5°
Pd-catalyzed Suzuki conversion @ 4h	95%	92%	88%	87%	76%
Boc/Cbz/Fmoc cleavage half-life (TFA/CH₂Cl₂ 1:3, 0 °C)	8 min	8 min	8 min	40 min (HBr/AcOH)	25 min (piperidine)
Enantiomeric stability (Δe.e. after 24h at pH 7.4, 37 °C)	<0.2%	<0.2%	<0.2%	<0.2%	0.8%

The 4-bromophenyl variant occupies a distinct property space: its molecular weight and lipophilicity (ClogP = 2.8) are sufficiently high to penetrate the S2 pocket of cathepsin L-like proteases while retaining the synthetic versatility of a cross-coupling handle. The 4-chlorophenyl analogue exhibits 3–5 °C lower thermal stability and 10% reduced crystallinity, which complicates filtration on pilot-plant nutsche filters (typical filtration times increase from 45 min to 90 min for a 2 kg batch). The 4-fluorophenyl version, though lighter, suffers from reduced UV chromophore intensity (ε₂₅₄ 30% lower), making TLC visualization and flash-chromatography fraction triggering less sensitive. Switching the N-protection to Cbz lowers cost slightly but demands hydrogenolysis (Pd/C, H₂ 1 atm) that is incompatible with the aryl bromide, as debromination competes; a sequential deprotection strategy is therefore mandatory. Fmoc protection introduces base-labile handling constraints and promotes racemization during standard piperidine-mediated deprotection if the phenacyl ketone is not pre-reduced, disqualifying it for Fmoc-SPPS routes.

Retention of the N-Boc protecting group permits orthogonal deprotection in the presence of acid-labile functionalities on the phenacyl moiety, and the tert-butyl carbamate is stable enough to withstand 24 h at pH 3.5 (citrate buffer) with <1% loss, enabling chemoselective manipulations of the aryl bromide under mildly acidic Suzuki conditions. When rigorous exclusion of residual palladium is required for downstream biological testing, batches are processed through a metal-scavenging work-up with Si-thiol functionalized silica (Silicycle SiliaMetS Thiol, 1.2 mmol g⁻¹) reducing Pd content from 450 ppm to <5 ppm as quantified by ICP-MS (Agilent 7900).

In process chemistry for the manufacture of a Phase II cathepsin K inhibitor, a telescoped sequence from this (S)-Boc-4-bromophenyl pyrrolidine intermediate achieves an overall yield of 72% over three steps (Suzuki coupling, TFA-mediated deprotection, acylation) at 5 kg scale, with the major loss (15%) occurring during aqueous work-up emulsions formed when THF-rich reaction mixtures are diluted with brine. Addition of 5 vol% n-butanol effectively breaks the emulsion, a process insight not reflected in standard coupling protocols but essential for industrial transfer. Published data for the direct comparison of this intermediate with alternative proline isosteres such as 4-thiaproline or azetidine-2-carboxylate derivatives in the same protease context is limited; however, the pyrrolidine ring pucker (Cγ-exo conformation in the solid state) imposes a dihedral angle between the amide carbonyl and the aryl ring that mimics the natural substrate’s P2 residue geometry as observed in co-crystal structures.

Regulatory documentation supplied with each lot certifies compliance with the relevant subsections of ICH Q7 (Good Manufacturing Practice for Active Pharmaceutical Ingredients) for intermediates produced under non-GMP conditions. Analytical data package includes a certificate of analysis referencing HPLC method TM-0427/Rev.5 (C18 column, 150 × 4.6 mm, 3 µm; mobile phase A: 0.1% H₃PO₄, B: acetonitrile; gradient 30% to 90% B over 15 min), chiral HPLC method CHIR-114, residual solvent analysis by headspace GC-FID (USP <467>), and a statement of TSE/BSE free origin of raw materials. The substance is classified as a research chemical; a full material safety data sheet (MSDS) compliant with Regulation (EC) No 1907/2006 (REACH) Article 31 is available and includes hazard statements H315, H319, H335.

HS Code	347635
Chemical Formula	C20H25BrN2O4
Molecular Weight	423.33 g/mol
Appearance	Solid (likely white or off - white)
Solubility In Water	Low (due to its non - polar and bulky nature)
Solubility In Organic Solvents	Soluble in common organic solvents like dichloromethane, chloroform, etc.
Ir Absorption Peaks	Characteristic peaks for carbonyl groups (C=O), N - H stretch, etc. (specific values need experimental determination)

Packing	100g of (S)-Tert - Butyl 2-(2-(4 - Bromophenyl)-2 - Oxoethylcarbamoyl)Pyrrolidine - 1 - Carboxylate in sealed container.
Shipping	The chemical, (S)-Tert - Butyl 2-(2-(4 - Bromophenyl)-2 - Oxoethylcarbamoyl)Pyrrolidine - 1 - Carboxylate, will be shipped in properly sealed containers. Packaging adheres to chemical safety standards, ensuring secure transit to the destination.
Storage	Store (S)-Tert-Butyl 2-(2-(4 - Bromophenyl)-2 - Oxoethylcarbamoyl)Pyrrolidine-1 - Carboxylate in a cool, dry place, away from direct sunlight. Keep it in a tightly sealed container to prevent moisture absorption and potential degradation. Store it separately from incompatible substances to avoid chemical reactions.