Tert-Butyl (3Ar,5S,6As)-5-(Hydroxymethyl)-Hexahydro-1H-Cyclopenta[C]Pyrrole-2-Carboxylate

Inside the Oxidative Coupling Sequence for Bictegravir Sodium: A Process Window Definition

Stereo-retentive oxidation of the primary alcohol in tert-butyl (3aR,5S,6aS)-5-(hydroxymethyl)hexahydro-1H-cyclopenta[c]pyrrole-2-carboxylate constitutes the rate-limiting chemical transformation in the downstream assembly of bictegravir sodium. In a jacketed 100 L glass-lined reactor equipped with a Heidolph Hei-TORQUE ultimate stirrer, PT-100 probe feedback, and a Julabo FP51-MA circulator, the crystalline alcohol (1.0 kg, 3.76 mol) is dissolved in 1,2-dichloroethane that has been pre-dried over 4 Å molecular sieves to a Karl Fischer titre below 45 µg/g. The solution is cooled to an internal temperature of −12.0 °C ± 1.5 °C. Dess‑Martin periodinane (1.38 eq., 2.19 kg of a 15 wt% DMP solution in dichloromethane) is introduced through a peristaltic pump over 95–110 minutes, maintaining the jacket set-point at −18 °C and ensuring the reaction mass never exceeds −6 °C. Any excursion above −4 °C, monitored in real time by a Mettler Toledo ReactIR 702L probe, triggers an automated oxidant feed pause; the aldehyde CH-stretch vibration at 2710 cm⁻¹ broadens irreversibly and the diastereomeric excess of the ensuing aldehyde—measured by chiral HPLC on a Daicel CHIRALPAK IA-3 column with hexane/isopropanol 90:10—drops from 99.8 % de to below 94.3 % de within 90 seconds of sustained thermal overshoot. After 2.5 h of ageing at −8 °C, the reaction is quenched into a pre-cooled (5 °C) aqueous solution containing sodium thiosulfate pentahydrate (8.0 wt%) and sodium bicarbonate (2.5 wt%), keeping the quench temperature under 12 °C by jacket chilling. The organic phase is separated, washed with brine (0.5 m³), concentrated on a Büchi R-250 rotary evaporator at 180 mbar and 28 °C, and the crude aldehyde is taken forward immediately into hydrazone formation with (2,5-difluorophenyl)hydrazine hydrochloride (1.02 eq.) in methanol at 0–5 °C. All unit operations are executed under a Grade D cleanroom environment per EU GMP Annex 1 and ICH Q7 §7.31, with residual solvent clearance validated against USP 〈467〉 and ICH M7 Option 4 for DCM (≤ 600 ppm). The resulting bictegravir sodium sesquihydrate is subsequently formulated with emtricitabine and tenofovir alafenamide fumarate in a fixed-dose tablet manufactured at a compression force range of 12–18 kN on a KORSCH XL 400 rotary press, delivering the commercial presentation Biktarvy®.

Can Variations at the 5-(Hydroxymethyl) Position Overcome INSTI Resistance Mutations?

Second-generation HIV-1 integrase strand transfer inhibitors demand bicyclic scaffolds that maintain picomolar potency against the G140S/Q148H double mutant, and the constrained (3aR,5S,6aS) cyclopenta[c]pyrrolidine architecture preserved in this tert-butyl carbamate serves as the central staging point for rapid SAR exploration of the solvent-exposed region. On a Chemspeed SWING XL automated synthesis platform housed in a fume hood compliant with OSHA 29 CFR 1910.1450, the N-Boc alcohol is loaded into 96-deep-well glass reactors (0.25 mmol per well) and activated with p-toluenesulfonyl chloride (1.15 eq.) in pyridine at −5 °C to generate the tosylate in situ. Subsequent nucleophilic displacement is executed with a pre-screened library of amine, thiol, or azide nucleophiles (1.3 eq. each), using acetonitrile as solvent and maintaining a constant vortex agitation of 850 rpm at 45 °C for 16 h. Crude mixtures are filtered through Biotage ISOLUTE SLE+ plates and purified on a Biotage Isolera One system with prepacked SNAP Ultra C18 cartridges; purity acceptance is set at ≥ 93 % by LC/MS (electrospray positive ion on an Agilent 1260 Infinity II SQ). Analogues that display an EC₅₀ shift of less than 2-fold relative to wild-type in the PhenoSense® Integrase assay are flagged for scale-up to 5 mmol in a Radleys Carousel 12 Plus reactor. At this stage, no formal GMP compliance is invoked, yet all electronic lab notebooks adhere to ISO 9001:2015 Clause 7.5 for documented information, and biological materials are handled under NIH Guidelines for Research Involving Recombinant DNA Molecules. The terminal deliverables are hydrochloride or mesylate salts of novel INSTI candidates packed under argon in amber ampoules, intended for oral gavage pharmacokinetic studies in Sprague-Dawley rats at doses of 10 mg/kg.

When the (3aR,5S,6aS)-5-(hydroxymethyl) intermediate is carried through as a process-related impurity in Bictegravir drug substance, its detection at a level of ≥ 0.05 % (w/w) during release testing triggers a standard addition protocol per ICH Q2(R1) and USP 〈1225〉. A reference solution is prepared by dissolving 25.0 mg of the certified alcohol in 100.0 mL of methanol, followed by serial dilution to generate a six-point calibration ranging from 0.005 % to 0.150 % relative to the 10.0 mg/mL bictegravir test solution. Chromatographic separation is accomplished on a Waters ACQUITY UPLC H-Class system fitted with an Acquity UPLC BEH Shield RP18 column (100 mm × 2.1 mm, 1.7 µm) thermostatted at 35 °C, employing a gradient of 10 mM ammonium formate buffer (pH 3.2) and acetonitrile at a flow rate of 0.4 mL/min, with diode-array detection at 260 nm. The retention time of the alcohol impurity typically falls at 4.72 ± 0.03 min, and system suitability is verified with a resolution of at least 2.0 between the alcohol and the preceding des-fluoro by-product. During forced degradation of bictegravir sodium tablets (film-coated cores stored at 40 °C/75 % RH for 6 months in HDPE bottles with induction-sealed liners), the alcohol impurity is not a primary degradant, but its level is monitored as a marker of incomplete reductive amination; a spiking study at the 0.10 % level demonstrates a recovery of 98.2–101.7 % with a relative standard deviation of 1.9 % across six replicate extractions, validating the method’s accuracy against the acceptance window of 95.0–105.0 % set by ICH Q3A(R2) for unspecified impurities. Clearance studies on the downstream recrystallization from ethanol/water (70:30 v/v) confirm a partition coefficient log P that removes 97.3 % of the alcohol impurity in a single cooling crystallization at −10 °C, as modelled by the Abraham solvation equation. The terminal finished product is a bilayer tablet core containing bictegravir sodium (50 mg), emtricitabine (200 mg), and tenofovir alafenamide fumarate (25 mg), manufactured in accordance with 21 CFR Part 211 and the WHO Technical Report Series 1010.

Scaling the Enantiopure Cyclopenta[c]pyrrole Intermediate from Benchtop to Pilot Reactor for IND-enabling Campaigns

Transfer of a published laboratory synthesis to a multi-kilogram campaign requires re-engineering the protecting-group strategy for the hexahydrocyclopenta[c]pyrrole framework. Starting from the same chiron pool material, the Boc-protected alcohol is produced under a contract development and manufacturing organization (CDMO) setup that must reconcile ISO 9001:2015 quality management with optional upgrade to ICH Q7 GMP for phase-appropriate intermediates. In a typical 5.0 kg batch, the chiral keto ester precursor is subjected to a Corey–Bakshi–Shibata reduction using 5 mol% (R)-2-methyl-CBS-oxazaborolidine and borane-dimethyl sulfide complex (0.6 eq. BH₃) in tetrahydrofuran at −20 °C, delivering an enantiomeric excess of > 99.2 % as analysed on a Daicel CHIRALCEL OD-H column (250 mm × 4.6 mm) with hexane/ethanol 95:5 at 0.8 mL/min. Cyclization with benzylamine and subsequent hydrogenolysis over Pearlman’s catalyst (20 wt% Pd(OH)₂/C, 0.15 bar H₂) in ethanol follow, before the secondary amine is captured with Boc₂O (1.05 eq.) in dichloromethane in the presence of triethylamine (1.2 eq.). The isolated tert-butyl carbamate is recrystallized from methylcyclohexane/ethyl acetate (4:1 v/v) through a programmed cooling ramp of 0.15 °C/min between 55 °C and 5 °C, affording a crystalline solid with a differential scanning calorimetry melt onset of 88.4 °C (Mettler Toledo DSC 3, 10 K/min) and a residual palladium content below 5 ppm (Agilent 7850 ICP-MS). When the alcohol moiety is deployed in a subsequent Mitsunobu coupling in the customer’s downstream API synthesis, the recommended molar feed is 1.0 eq. of the carbamate to 1.4 eq. of triphenylphosphine and 1.3 eq. of diisopropyl azodicarboxylate in toluene at 0–10 °C, followed by acidic Boc deprotection with trifluoroacetic acid (2.0 eq.) in dichloromethane at 20 °C; this sequence preserves the ring-junction stereochemistry with an observed substrate-to-product chirality transfer exceeding 99.5 % ee when quench is performed within 20 min of TFA addition. The terminal articles shipped to regional hubs are double poly-bagged under nitrogen in PE drums containing 1.0 kg or 5.0 kg net weight, labelled with a CoA referencing ASTM D7869-17 accelerated ageing conditions for the desiccant pack and a retest date assigned at 24 months from release.

As PROTAC-focused discovery programs move toward bifunctional molecules where the linker region contributes to ternary complex stability, the N-Boc alcohol provides a rare combination of a conformationally constrained secondary amine and a pendent hydroxyl group positioned at a dihedral angle of approximately 115° relative to the bicycle plane. The alcohol is transformed into an ω-azido-polyethylene glycol spacer by treatment with methanesulfonyl chloride (1.15 eq.) and triethylamine (1.5 eq.) in dichloromethane, followed by nucleophilic displacement with sodium azide (3.0 eq.) in dimethyl sulfoxide at 65 °C for 8 h. The azide intermediate is then conjugated via copper-catalysed click chemistry to an alkyne-terminated cereblon-binding phthalimide congener using CuBr·PPh₃ (5 mol%) in tert-butanol/water (1:1) at 40 °C, after which the Boc group is removed to expose the secondary amine for coupling to a VHL E3 ligase ligand in a subsequent amide-bond-forming step mediated by HATU (0.98 eq.) and N,N-diisopropylethylamine (3.0 eq.). All handling occurs in a laboratory environment compliant with CFR 1910.1200 (Hazard Communication Standard) and using vented enclosures; no GMP protocols apply. Terminal compounds are lyophilized from acetonitrile/water (1:1) to yield off-white powders stored at −20 °C under argon, characterized by high-resolution mass spectrometry and submitted to cellular target engagement assays within 48 h to prevent hydrolysis of the imide ring. While no commercial drug product incorporating this exact synthon has received marketing authorization, the bicyclic carbamate has been referenced in hit-expansion libraries deposited in ChemBridge and Enamine, supporting hit-to-lead efforts against BRD4 and BRD9 bromodomain targets.

Certified Reference Standard Production under ISO 17034 for the Enantiopure Bicyclic Amino Alcohol

Assigning absolute purity and stereochemical integrity to a single-digit-milligram quantity of a carbamate intermediate demands an orthogonal metrology protocol that combines quantitative nuclear magnetic resonance, high-performance liquid chromatography with charged aerosol detection, and differential scanning calorimetry. A primary stock of the alcohol is purified by semi-preparative supercritical fluid chromatography on a Waters Prep 100q SFC system equipped with a Viridis BEH 2-ethylpyridine column (250 mm × 30 mm) at 40 °C and 120 bar back pressure, using 12 % methanol modifier in CO₂ at 80 g/min; the fraction collected is dried in a Genevac HT-4X evaporator at 0.5 mbar and 35 °C. For qNMR-based purity certification, approximately 25.0 mg of the candidate material is accurately weighed on a Mettler Toledo XPR6 microbalance (d = 0.001 mg) and dissolved in 0.7 mL of DMSO-d₆ containing 2.0 mg/mL of NIST SRM 350b benzoic acid as internal standard. Spectra are recorded on a Bruker Avance NEO 600 MHz spectrometer with a 5 mm TCI cryoprobe, using a 30° pulse, 20 s relaxation delay, and 64 scans; the methyl signals of the tert-butyl group integrate against the aromatic protons of the internal standard with a standard uncertainty of 0.31 % (k = 2). Chromatographic purity by HPLC-CAD on a Thermo Fisher Vanquish system with a Corona Veo RS detector delivers a complementary organic purity of 99.84 ± 0.08 % (n = 6), and a DSC purity determination on a TA Instruments Discovery DSC 2500 at a scan rate of 1 °C/min yields a mole fraction purity of 0.9983 by van’t Hoff analysis, consistent with the NMR assignment. The reference standard is aliquoted in 100 mg units into amber glass vials with PTFE-lined caps, sealed under argon, and accompanied by a certification report compliant with ISO Guide 31:2015 and ISO 17034:2016 Clause 7.6. Secondary working standards produced from this lot are used to calibrate the routine HPLC procedure for bictegravir sodium API release, with a standard solution prepared at a concentration of 0.100 mg/mL in methanol and injected at 10 µL volume; retention time repeatability is verified against an acceptance window of ± 0.06 min.