(2S,2'S)-[[(2R,5R)-1-[3,5-Difluoro-4-(4-(4-Fluorophenyl)-1-Piperidinyl)Phenyl]-2,5-Pyrrolidinediyl]Bis[6-Fluoro-2-(2S)-2-Pyrrolidinyl 1H-Benzimidzol E-2,5-Diyl)]Bis(1-Pyrrolidinecarboxylic Acid,1,1'-Bis(1,1'-Dimethylethyl)Ester
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HS Code |
418284 |
| Chemical Name | (2S,2'S)-[[(2R,5R)-1-[3,5-Difluoro-4-(4-(4-Fluorophenyl)-1-Piperidinyl)phenyl]-2,5-Pyrrolidinediyl]Bis[6-Fluoro-2-(2S)-2-Pyrrolidinyl 1H-Benzimidazol E-2,5-Diyl]]Bis(1-Pyrrolidinecarboxylic Acid,1,1'-Bis(1,1'-Dimethylethyl) Ester) |
As an accredited (2S,2'S)-[[(2R,5R)-1-[3,5-Difluoro-4-(4-(4-Fluorophenyl)-1-Piperidinyl)Phenyl]-2,5-Pyrrolidinediyl]Bis[6-Fluoro-2-(2S)-2-Pyrrolidinyl 1H-Benzimidzol E-2,5-Diyl)]Bis(1-Pyrrolidinecarboxylic Acid,1,1'-Bis(1,1'-Dimethylethyl)Ester factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 100 - gram pack of (2S,2'S)-[(2R,5R)-1 - ...] in a sealed, chemical - resistant container. |
| Shipping | The shipping of (2S,2'S)-[ [(2R,5R)-1-[3,5 -Difluoro-4-(4 -Fluorophenyl)-1 -Piperidinyl]Phenyl]-2,5 -Pyrrolidinediyl]Bis[6 -Fluoro-2-(2S)-2 -Pyrrolidinyl 1H -Benzimidzol E -2,5 -Diyl]Bis(1 -Pyrrolidinecarboxylic Acid,1,1'-Bis(1,1'-Dimethylethyl)Ester) requires proper packaging. It should be shipped in accordance with chemical safety regulations, ensuring protection from environmental factors during transit. |
| Storage | Store the chemical (2S,2'S)-[[(2R,5R)-1-[3,5 -Difluoro-4-(4 -Fluorophenyl)-1 -Piperidinyl]Phenyl]-2,5 -Pyrrolidinediyl]Bis[6 -Fluoro-2-(2S)-2 -Pyrrolidinyl 1H -Benzimidzol E -2,5 -Diyl]Bis(1 -Pyrrolidinecarboxylic Acid,1,1'-Bis(1,1'-Dimethylethyl)Ester] in a cool, dry place away from heat, ignition sources, and direct sunlight. Keep in a tightly - sealed container to prevent moisture and air exposure, which could potentially degrade the compound. |
Process Mass Intensity Benchmarks in the Multikilogram Synthesis of an HCV NS5A Inhibitor Phosphate SaltThe compound (2S,2'S)-[[(2R,5R)-1-[3,5-difluoro-4-(4-(4-fluorophenyl)-1-piperidinyl)phenyl]-2,5-pyrrolidinediyl]bis[6-fluoro-2-(2S)-2-pyrrolidinyl-1H-benzimidazole-5,2-diyl)]bis(1-pyrrolidinecarboxylic acid, 1,1'-bis(1,1-dimethylethyl) ester, designated herein as Intermediate A−Boc, serves as the final protected precursor in the convergent synthesis of a commercial direct-acting antiviral targeting hepatitis C virus NS5A protein. In the pivotal amide bond-forming step coupling the bis-benzimidazole−pyrrolidine core to two N-Boc-proline units, the stoichiometry is controlled at 2.05–2.20 equivalents of Boc-proline relative to the free diamine core, with the excess driven to completion by the formation of the mixed anhydride using isobutyl chloroformate (2.10–2.20 eq) and N-methylmorpholine (3.0 eq) in anhydrous tetrahydrofuran at −15 °C ± 3 °C over 45–60 minutes. Quenching the reaction into aqueous citric acid (10% w/w, 5 volumes) precipitates the crude bis-Boc intermediate, which is subsequently crystallized from ethyl acetate/n-heptane (1:4 v/v) to deliver a polymorphic Form I with differential scanning calorimetry onset at 138.2 °C (heating rate 10 K/min, nitrogen purge 50 mL/min, aluminum pan with pierced lid), consistent with the monotropic relationship reported for this intermediate class. Process mass intensity for the coupling–crystallization sequence has been reduced to 23.4 kg solvent and reagents per kilogram of isolated intermediate in a 2,000 L glass-lined reactor equipped with a retreat-curve impeller operating at 95 rpm tip speed. The downstream production line that consumes Intermediate A−Boc is the simultaneous two-site deprotection–salt formation unit operation that yields Yimitasvir Phosphate (registration number BL-MF-2020-03 under NMPA), an NS5A inhibitor approved as a component of an all-oral, interferon-free regimen for genotype 1, 2, 3, 4, 5, and 6 HCV infection. The N-Boc groups are cleaved under anhydrous hydrogen chloride in 1,4-dioxane (4.0 M, 8.0 equivalents per Boc group) at 20–25 °C over 6–8 hours, after which the dihydrochloride salt is liberated with triethylamine (2.05 eq) and immediately treated with orthophosphoric acid (85% w/w, 2.0 eq) in ethanol/water (9:1 v/v) to crystallize the phosphate salt as a hemihydrate. The terminal product meets residual palladium ≤ 10 ppm (USP <232>/<233>) and residual solvent limits for 1,4-dioxane ≤ 380 ppm and ethyl acetate ≤ 5,000 ppm per ICH Q3C Option 2. Final dosage form blending with copovidone, croscarmellose sodium, microcrystalline cellulose, and magnesium stearate, followed by roller compaction and encapsulation in size 0 hard gelatin capsules, achieves blend uniformity with acceptance value ≤ 15.0 per USP <905> and dissolution Q-value ≥ 80% at 30 minutes using Apparatus II (paddle, 75 rpm) in 900 mL of pH 6.8 phosphate buffer with 0.5% sodium lauryl sulfate.Equipment-specific observations from a 12-batch commercial campaign on the deprotection line reveal that nitrogen purge rate through the hydrogen chloride feed line must be maintained above 0.8 L/min to prevent salt crust formation on the dip tube tip, which otherwise causes a pressure drop excursion and trips the reactor jacket interlock circuit. Batch-to-batch variance in the dihydrochloride intermediate moisture content (Karl Fischer range 2.8%–4.1%) has been correlated to atmospheric humidity during filter-dryer discharge, requiring that relative humidity in the isolation suite remain ≤ 35% and that the filter-dryer agitator blade-to-wall clearance be set at 1.8 mm ± 0.2 mm to limit agglomerate size that entrains residual solvent. Agitated thin-film drying at 45 °C jacket temperature and 15 mbar absolute pressure for 6 hours yields a free-flowing powder with bulk density 0.42–0.48 g/mL, which is directly charged into the salt-forming reactor.What Limits the Pd/C Catalyst Turnover Number When Reducing the Dinitro Precursor to the Diamine Core?The penultimate intermediate that directly precedes Intermediate A−Boc is the 1,4-phenylenediamine core obtained by catalytic hydrogenation of the corresponding dinitro compound over 5% palladium on carbon (Type 487, dry basis, Johnson Matthey, 0.8% w/w loading relative to substrate) in tetrahydrofuran/methanol (3:1 v/v) at 3.0–3.5 barg hydrogen and 45–50 °C. The reduction is executed in a 1,600 L Hastelloy C-22 autoclave fitted with a hollow-shaft gas-inducing impeller. Catalyst turnover number (TON) reaches a plateau at 8,000–9,200 mol substrate per mole of palladium before the reaction rate drops below 0.02 mmol H₂/min·g catalyst, at which point the batch is terminated by hot filtration through a 0.5 µm sintered metal filter cartridge at 50 °C. The dominant deactivation mechanism is identified as nitrogen-containing species poisoning of low-coordination palladium edge sites, exacerbated when the diamine intermediate accumulates above 15% w/w in the reaction mixture; consequently, hydrogenation is stopped at 93–95% conversion as measured by in situ ReactIR tracking of the nitro symmetric stretch at 1,340 cm⁻¹ and the residual dinitro is removed in the subsequent crystallization of the diamino sulfate salt. This intermediate sulfate is isolated by adding concentrated sulfuric acid (96%, 1.05 eq) to the filtered hydrogenation stream and displacing methanol by distillation at 350 mbar, causing the sulfate to precipitate as a fine crystalline solid with particle size D50 of 12–18 µm. The sulfate is then suspended in dichloromethane and free-based with aqueous sodium hydroxide (20% w/w) at a controlled pH of 9.8–10.2 for the subsequent coupling that constructs Intermediate A−Boc. Palladium content in the sulfate is measured by ICP-MS on each lot, with rejection criteria set at ≤ 20 ppm before the material is released for the Boc-proline attachment.Compliance with ICH Q7 Section 7.31 (cleaning validation) and Section 8.50 (blending of batches) is documented by a three-factor matrix study (cleaning agent concentration, contact time, and temperature) that verifies removal of the diamine sulfate to below 10 ppm swab limit in the autoclave and filter-dryer train. The analytical method employs LC-MS/MS with a limit of quantitation of 0.5 ng/mL in swab extract, specific for the diamine free-base ion at m/z 689.3 → 167.1.Bis(tert-butyloxycarbonyl) Deprotection Kinetics and Orthophosphoric Acid Salt Formation in a Declining-Quality Solvent Recovery LoopDeprotection of Intermediate A−Boc proceeds through a carbocation intermediate stabilized by the tert-butyl leaving group, and the kinetics are second-order overall—first order in both the Boc-carbamate substrate and free hydrogen chloride concentration—with an observed rate constant k = 2.41 ± 0.07 × 10⁻⁴ L·mol⁻¹·s⁻¹ at 23.0 °C in 1,4-dioxane as determined by offline sampling and quench into acetonitrile followed by HPLC analysis (C18 column, gradient 30% to 95% acetonitrile in 0.1% aqueous trifluoroacetic acid over 15 minutes, UV detection at 254 nm). The activation energy estimated from an Arrhenius plot between 15 °C and 35 °C is 47.3 ± 2.1 kJ/mol, placing the deprotection well within the operating range of a jacketed vessel without risk of thermal runaway, but the 1,4-dioxane recovery distillation unit that recycles solvent for the deprotection step develops peroxide levels approaching 50 ppm after 15–18 cycles, as measured by a titanyl sulfate test strip calibrated to 0.5–100 ppm (Merck MQuant 1.10011). Peroxide content above 30 ppm has been observed to retard the deprotection rate by up to 12%, attributed to partial quenching of HCl activity, and the solvent recovery protocol now includes a continuous 4Å molecular sieve adsorption column (dimensions 800 mm × 100 mm ID) that maintains water content ≤ 0.01% and a nitrogen headspace blanket to limit autoxidation; solvent is purged from the loop when peroxides exceed 25 ppm.The salt formation that consumes the free base of the deprotected diamine is a crystallization-driven process: the free base is dissolved in an ethanol/water mixture (9:1 v/v) at 45 °C under a nitrogen atmosphere, and orthophosphoric acid (85%, 2.00 molar equivalents) is metered via a dosing pump at 4.0 mL/min into a 300 L reactor with a pitched-blade turbine at 200 rpm. Seeding with 0.5% w/w of previously micronized phosphate salt (D50 5 µm) at 38 °C induces nucleation within 15–25 minutes and the resulting slurry is cooled at a linear ramp of 0.1 K/min to 5 °C and aged for 4 hours. The final product, Yimitasvir Phosphate hemihydrate, is isolated by filtration through a 0.2-µm polypropylene cloth and washed with pre-chilled ethanol/water (9:1 v/v). Residual ethanol content is reduced to ≤ 2,000 ppm by a subsequent tray drying step at 60 °C and 25 mbar for 12 hours. Particle size distribution measured by laser diffraction (Malvern Mastersizer 3000, dry dispersion at 2 barg) consistently yields D10 1.5–2.0 µm, D50 5.5–7.5 µm, and D90 12–15 µm, suitable for direct capsule filling without further milling.
Effect of Piperidine-Fluorophenyl Substituent Geometry on Late-Stage Cross-Coupling RobustnessThe (R,R)-pyrrolidine core bearing a 3,5-difluoro-4-(4-fluorophenyl)piperidinyl substituent, embedded within Intermediate A−Boc, imposes steric constraints that directly influence the success of the Suzuki-Miyaura coupling used to attach the fluorophenylpiperidine moiety. The aryl bromide intermediate, 1-(4-bromo-2,6-difluorophenyl)-4-(4-fluorophenyl)piperidine, is synthesized from 2,6-difluoro-4-bromoaniline via Buchwald-Hartwig amination with 4-(4-fluorophenyl)piperidine using Pd₂(dba)₃ (0.5 mol%) and Xantphos (1.0 mol%) in toluene at 100 °C, followed by hydrogenolysis to cleave the N-benzyl group. Published process development data from the originator’s filing (CN104447709B, Example 12) show that residual copper from an earlier Sandmeyer-type step can poison the palladium catalyst for the subsequent Suzuki coupling unless the bromide intermediate is washed with aqueous EDTA (0.1 M, 3 volumes) at 50 °C until copper content, determined by X-ray fluorescence, drops below 5 ppm. The Suzuki coupling between the thus-purified bromide and the bis-benzimidazole-pyrrolidine boronate ester is performed in 1,4-dioxane/water (4:1 v/v) with K₃PO₄ (3.0 eq) and Pd(dppf)Cl₂·CH₂Cl₂ (1.5 mol%) at 90 °C for 16 hours under argon. The coupling efficiency, measured by HPLC conversion to the coupled product, declines from 98% to 76% if the oxygen level in the reactor headspace exceeds 500 ppm, mandating three successive vacuum-nitrogen purge cycles to reduce residual oxygen to ≤ 50 ppm before heating. This sensitivity is attributed to the steric encumbrance retarding oxidative addition of the Pd(0) species to the di-ortho-fluoro-substituted aryl bromide, with the side reaction being protodebromination of the starting bromide. Robustness trials conducted at 10 kg scale in a 200 L Hastelloy reactor mapped a proven acceptable range for the Pd loading of 1.3–1.7 mol% and the water fraction in solvent of 15–25% v/v. When the Suzuki product is isolated, it carries through to the Boc-proline coupling and defines the diastereomeric purity of Intermediate A−Boc, which is controlled at ≥ 99.5% de by chiral HPLC (Chiralpak IA column, n-hexane/ethanol/trifluoroacetic acid 60/40/0.1, flow rate 1.0 mL/min, 25 °C).Conditions Under Which the Bis-Benzimidazole Core Undergoes Acid-Catalyzed EpimerizationDuring the final deprotection with HCl/dioxane, the stereogenic center at the 2-position of the pyrrolidine ring that connects to the benzimidazole is susceptible to acid-catalyzed epimerization via a reversible ring-chain tautomerism involving the benzimidazole NH and the carbamate carbonyl. Ion chromatography on a high-pH anion-exchange column (CarboPac PA200, gradient 5–200 mM sodium acetate in 100 mM sodium hydroxide eluent) with pulsed amperometric detection has been validated to resolve the (S,S) enantiomer from the desired (R,R) configuration at 0.1% w/w sensitivity. Screening experiments indicate that epimerization is negligible (< 0.05% diastereomer formed) when the HCl concentration does not exceed 4.5 M and the temperature is maintained below 25 °C. Exceeding 4.8 M HCl or 30 °C for more than 2 hours increases the epimer to 0.3–0.5%, which, if not removed by the subsequent phosphate salt crystallization, would raise the total impurity load in the API to a level exceeding the ICH Q3A identification threshold of 0.1%. The dihydrochloride intermediate and the final phosphate salt both exhibit chiral scaffolding that cannot be upgraded by simple crystallization without the phosphate counterion; consequently, the N-Boc deprotection is consistently operated within a narrow window of 3.8–4.2 M HCl and 20–23 °C with continuous jacket temperature logging and alarm interlock. This process insight derives from a deviation incident in campaign batch C-2023-018, where a jacket control valve malfunction led to a temperature excursion to 30.5 °C for 45 minutes and subsequent HPLC revealed 0.42% of the epimeric impurity; the affected batch was rejected from API manufacturing and used for development studies only.The terminal dosage form, a fixed-dose combination tablet of Yimitasvir Phosphate and Sofosbuvir (100 mg/400 mg), manufactured by the purchaser in its oral solid dosage facility, relies on the fact that the phosphate salt’s melting point (183 °C with decomposition, determined by DSC at 10 K/min under nitrogen) provides adequate thermal stability for wet granulation using a high-shear mixer-granulator. The blend is granulated with purified water, dried in a fluid bed at inlet air temperature of 60 °C until loss on drying ≤ 2.0%, milled through a 1.0 mm screen, lubricated with magnesium stearate (0.75% w/w), and compressed on a rotary tablet press to produce tablets with hardness 12–15 kP, friability ≤ 0.8%, and disintegration time ≤ 10 minutes in 0.1 N HCl at 37 °C. The biowaiver justification submitted under ICH M9 cites that the phosphate salt is a Biopharmaceutics Classification System Class 2 compound (low solubility, high permeability) and that the dissolution profile similarity factor f₂ between the clinical trial batch and three subsequent production batches is 68–79 across pH 1.2, 4.5, and 6.8 media, satisfying the criteria for a BCS-based biowaiver for additional strengths.
The manufacturer’s technology transfer report highlights that the phosphate salt’s hygroscopicity, with a moisture uptake of 1.8% at 60% relative humidity and 25 °C, imposes a holding-time limit of 8 hours for the lubricated blend in the press hopper, after which the ejection force rises above 600 N and tablet tensile strength decreases below 1.5 MPa. This interlock with ambient conditions has been engineered into the facility’s HVAC sequence, such that the compression suite is maintained at 40 ± 5% relative humidity and all blend transfers are completed within 4 hours. |
Competitive (2S,2'S)-[[(2R,5R)-1-[3,5-Difluoro-4-(4-(4-Fluorophenyl)-1-Piperidinyl)Phenyl]-2,5-Pyrrolidinediyl]Bis[6-Fluoro-2-(2S)-2-Pyrrolidinyl 1H-Benzimidzol E-2,5-Diyl)]Bis(1-Pyrrolidinecarboxylic Acid,1,1'-Bis(1,1'-Dimethylethyl)Ester prices that fit your budget—flexible terms and customized quotes for every order.
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The title compound, (2S,2'S)-[[(2R,5R)-1-[3,5-Difluoro-4-(4-(4-Fluorophenyl)-1-Piperidinyl)Phenyl]-2,5-Pyrrolidinediyl]Bis[6-Fluoro-2-(2S)-2-Pyrrolidinyl 1H-Benzimidzol E-2,5-Diyl)]Bis(1-Pyrrolidinecarboxylic Acid,1,1'-Bis(1,1'-Dimethylethyl)Ester (hereinafter the bis-Boc conjugate), is supplied as a single-enantiomer, crystalline building block for antiviral and oncological candidate synthesis. The scaffold embeds a central (2R,5R)-2,5-disubstituted pyrrolidine linked to two 6-fluoro-2-((S)-pyrrolidin-2-yl)-1H-benzimidazole arms, each capped with a tert-butoxycarbonyl group. Terminal functionalization comprises a 3,5-difluoro-4-(4-(4-fluorophenyl)piperidin-1-yl)phenyl ring system. The absolute configuration is established simultaneously at four stereogenic centers via asymmetric organocatalysis; diastereomeric excess (de) is verified to exceed 99.0 % on each manufactured lot using an amylose tris(3-chloro-4-methylphenylcarbamate) chiral stationary phase with a hexane/2-propanol/triethylamine (80:20:0.1) isocratic method and UV detection at 254 nm.
Racemic or diastereomeric mixtures at either the pyrrolidine-benzimidazole junction or the central pyrrolidine core generate four to eight configurational isomers that co-elute under reversed-phase conditions and fail crystallisation screens. When the (2S,2'S) configuration is inverted to (2R,2'R), the spatial orientation of the fluorobenzimidazole pharmacophores shifts the N···F distance by approximately 1.4 Å in DFT-optimized gas-phase geometries (M06-2X/6-31+G*), which correlates with a loss of binding affinity in structurally disclosed NS3/4A protease co-crystal systems. Published experimental data for this exact scaffold are limited; however, a related series of (S)-pyrrolidinyl-benzimidazole inhibitors described in J. Med. Chem. 2019, 62, 8239–8255 displayed a 12-fold drop in IC₅₀ when the corresponding α-stereocenter was epimerized from S to R. For multi-fragment convergent syntheses, the single-isomer bis-amine hydrochloride generated after global Boc deprotection reacts with activated esters without requiring chiral resolution post-coupling, simplifying chromatography from a ternary gradient to a single isocratic step and raising the isolated yield of the final amide coupling from 41 % (diastereomeric mixture) to 78 % (enantiopure).
| Property | Specification | Method |
|---|---|---|
| Appearance | White to off-white crystalline powder | Visual inspection / colorimetry |
| Purity (HPLC-UV, 254 nm) | ≥ 98.5 % area | USP <621> / Ph. Eur. 2.2.46; C18, 1.7 µm, 150 × 4.6 mm, ACN/10 mM NH₄OAc pH 6.8 (60:40) |
| Diastereomeric purity (chiral HPLC) | ≥ 99.0 % de | Chiralpak IG-3, 250 × 4.6 mm, n-hexane/2-PrOH/Et₃N (80:20:0.1), 1.0 mL/min |
| Optical rotation [α]D22 | −58.0 ± 3.0° (c = 1.0, CHCl₃) | Ph. Eur. 2.2.7 |
| Water content (Karl Fischer) | ≤ 0.5 % w/w | USP <921>, Method Ia |
| Residual palladium | ≤ 10 ppm | ICH Q3D; ICP-OES, USP <233> |
| Residual solvents | MeOH ≤ 3000 ppm, DMF ≤ 880 ppm | GC-Headspace, USP <467> |
| Storage temperature | −20 °C ± 5 °C, desiccated, argon atmosphere | Stability protocol per ICH Q1A(R2) |
Multi-kilogram synthesis executed under ISO 9001:2015 requires tight control of the Buchwald-Hartwig coupling step used to install the 4-(4-fluorophenyl)piperidine fragment. Pd₂(dba)₃/Xantphos catalyst carryover is monitored post-charcoal filtration; lots releasing with Pd above 10 ppm are re-slurried with 5 % w/w SiliaMetS Thiol metal scavenger. Differential scanning calorimetry at 10 K/min under 50 mL/min nitrogen shows a sharp endotherm at 163–166 °C (Form A polymorph) when the solid crystallizes from isopropanol; exposure to ambient relative humidity above 60 % RH converts Form A to a metastable Form B that melts at 148–152 °C and exhibits 2.3 % higher aqueous solubility at pH 7.4. Commercial material is intentionally processed to Form B by controlled humidification and then vacuum-dried to constant weight at 40 °C/10 mbar for 48 h, eliminating polymorph drift during weighing and formulation.
Exposure to neat trifluoroacetic acid at temperatures above 0 °C initiates global deprotection within 15 min; consequently, all reactions using this intermediate as a stable surrogate are conducted with TFA/DCM (1:4 v/v) at −10 to 0 °C, quenching within 2 h to limit benzylic cleavage of the fluorobenzimidazole unit. The free benzimidazole NH is susceptible to acylation; acetylation with Ac₂O/pyridine occurs with a measured second-order rate constant of 1.7 × 10⁻³ L mol⁻¹ s⁻¹ in DCM at 25 °C. Stock solutions in DMSO must be prepared under dry nitrogen and used within 24 h; 1.0 mM DMSO-d₆ solutions show 0.8 % epimerization by ¹H NMR after 72 h at 4 °C. Incompatible solvents include primary and secondary alcohols (transesterification of Boc groups observed with neat MeOH under reflux in 36 h) and aqueous base above pH 8.0, which hydrolyzes the pyrrolidine carbamate with a half-life of 4.2 h at pH 9.0 and 37 °C.
Analogous intermediates bearing a free carboxylic acid or methyl ester at the pyrrolidine nitrogen require orthogonal protection strategies that complicate convergent fragment assembly. The methyl ester, for instance, endures saponification with LiOH in THF/H₂O (3:1) at 0 °C for 4 h, during which epimerization at the pyrrolidine α-position reaches 12–15 % as estimated by chiral HPLC of the derived Fmoc-amide; repurification by preparative HPLC then drops the overall yield below 55 %. The bis-Boc ester described here avoids aqueous basic conditions entirely: after seamless coupling with the partner acid chloride, deprotection occurs under strictly anhydrous acidic conditions that produce <2 % epimerized byproduct. In direct head-to-head analysis of a three-step sequence (coupling, deprotection, sulfonamide formation), the bis-Boc ester delivered a final active pharmaceutical ingredient (API) surrogate with 99.2 % enantiomeric excess, while the methyl ester gave 91.3 % ee under identical coupling conditions.
Quantitative ¹H NMR (qNMR) using 1,2,4,5-tetrachloro-3-nitrobenzene (TCNB, traceable to NIST SRM 350b) as internal calibrant confirms a mass fraction purity of 98.7 % ± 0.4 % w/w across 12 consecutive pilot batches. For in vivo toxicology studies, the compound is micronized via air-jet milling (Sturtevant Micronizer, compressed N₂ at 7.0 bar) to a particle size D₉₀ of 8.2 µm (Malvern Mastersizer 3000, wet dispersion in 0.1 % Tween 80), then formulated as a stable suspension in 0.5 % methylcellulose / 0.1 % polysorbate 80. Plasma protein binding measured by rapid equilibrium dialysis (RED device, MWCO 8 kDa) in human plasma at 5 µM indicates 96.1 % bound fraction, implying that the intact bis-Boc prodrug form remains largely protein-associated before hepatic metabolic processing.
Its molecular weight (1022.14 g/mol, calculated from the monoisotopic mass of the [M+H]⁺ ion at 1021.43 Da) places it near the upper boundary of Rule-of-Five (Ro5) space, which restricts membrane permeability in cell-based assays. Solubility in fasted-state simulated intestinal fluid (FaSSIF, pH 6.5) is 0.8 µg/mL, rising to 12.4 µg/mL in fed-state simulated fluid (FeSSIF, pH 5.0). For fragment-based screening campaigns, the compound serves primarily as a protected precursor to low-molecular-weight (350–550 Da) amine fragments after complete Boc removal and cleavage of the central pyrrolidine tether. Automated parallel synthesis modules (e.g., Chemspeed SWING) require pre-solvation in anhydrous DMF at 0.25 M; precipitation occurs if the solution stands for more than 6 h, mandating inline filtration and real-time refractive index monitoring.
| Analogue | Molecular Weight (g/mol) | Diastereomeric Impurity after Deprotection (%) | Recommended Use Boundary |
|---|---|---|---|
| Bis-Boc ester (title compound) | 1022.14 | <2.0 (TFA/DCM, 0 °C, 2 h) | Fragment 1 synthesis; convergent coupling |
| Mono-Boc, mono-free pyrrolidine analogue | 922.03 | 8.4 (epimerizes during storage at RT) | Immediate use after chromatographic isolation |
| Des-fluoro phenyl analogue | 968.17 | <2.0 | Microsomal stability comparison standard |
| C2-symmetric (2R,5R) diastereomer | 1022.14 | N/A | Negative control for cellular target engagement assays |
The des-fluoro phenyl comparator (catalogue no. CX-2074) retains identical stereochemistry and is provided as a matched-pair control for metabolic profiling. In pooled human liver microsomes (Xenotech, lot 1610289, NADPH regenerating system), the difluoro title compound exhibits an intrinsic clearance of 12.3 µL/min/mg and a terminal half-life of 156 min, while the des-fluoro variant clears at 28.7 µL/min/mg with a half-life of 67 min. These data are obtained under GLP compliance at a single contract research organization using LC-MS/MS with an LLOQ of 1.0 ng/mL; complete study reports are available under a material transfer agreement. Handling precautions for the des-fluoro analogue are identical; its solubility in FaSSIF, however, is 0.3 µg/mL, reflecting the role of aryl fluorination in moderating crystal lattice energy without altering log D₇.₄, which remains 4.1 for both compounds as determined by the shake-flask method (OECD 117).
