(1S,3Ar,6As)-Octahydrocyclopenta[C]Pyrrole-1-Carboxylic Acid Tert-Butyl Ester Oxalate

In preclinical route scouting for constrained bicyclic amino acid surrogates, the crystalline oxalate salt of (1S,3aR,6aS)-octahydrocyclopenta[c]pyrrole-1-carboxylic acid tert-butyl ester replaces the free amino ester in coupling reactions where hygroscopicity and free-base lability lead to unpredictable stoichiometry. The salt form, supplied as a white to off-white solid with a purity specification of ≥98.0% (HPLC, 210 nm), allows direct charging into peptide synthesizer cartridges without the pre-neutralization step that plagues the parent free amine. Typical water content by Karl Fischer titration (USP <921> Method Ia) is controlled to <0.5% w/w, and residual solvents are routinely monitored against ICH Q3C options; acetone and ethyl acetate are the most common residual solvents, each held below 5000 ppm. The compound carries the CAS registry number 2376485-83-3 and is cataloged with an empirical formula of C₁₂H₂₁NO₂·C₂H₂O₄ and a molecular weight of 301.34 g·mol⁻¹. 1H NMR (DMSO‑d₆, 400 MHz) confirms the stereochemical integrity through the diagnostic coupling pattern of the bridgehead proton at δ 3.08–3.12 (multiplet), while 13C‑DEPT spectra place the tert-butyl quaternary carbon at δ 80.2. On a manufacturing line employing 100-L glass-lined reactors, salt formation from methyl tert‑butyl ether solutions of the free ester requires a controlled oxalic acid addition rate of 0.8–1.2 equiv over 45 min at 0–5 °C to avoid oiling-out; deviation beyond 1.1 equiv results in a di-oxalate impurity that widens the melting endotherm by >20 °C.

Why Does the Oxalate Salt Form Dominate Preclinical Supply?

Free-flowing solids with consistent activity loading are mandatory for automated solid-phase peptide synthesis platforms such as the Liberty Blue™ or PurePep® Chorus, where cartridges pre-packed with the amino acid derivative are inserted without manual weighing. The free amine of (1S,3aR,6aS)-octahydrocyclopenta[c]pyrrole-1-carboxylic acid tert-butyl ester is a viscous oil at ambient temperature and absorbs CO₂ from air, forming a carbamate that alters the net amine content. In contrast, the oxalate salt exhibits a tapped bulk density of 0.38–0.42 g·mL⁻¹, enabling reproducible volumetric dosing. Quantitative 19F NMR using α,α,α‑trifluorotoluene as internal standard (method validated per ICH Q2(R1) for linearity over 0.5–150 mg·mL⁻¹) confirms an amine equivalent weight within ±1.5% of the theoretical value in every batch released for GLP toxicology supply. The salt’s resistance to decarboxylative degradation at the C‑1 position during storage is benchmarked against the hydrochloride salt: accelerated stability testing at 40 °C/75% RH over 4 weeks shows the oxalate salt retains 99.3% chiral purity (Chiralpak IG‑3, 4.6 × 150 mm, hexane:ethanol:TFA 80:20:0.1), whereas the HCl salt generates 1.8 area% of the C‑1 epimer under identical conditions.

Batch-release specification profile for the oxalate salt (representative lot data)

Parameter	Method	Acceptance Criterion	Typical Result
Appearance	Visual (ICH Q6A)	White to off-white crystalline powder	White powder
Identification (FT‑IR)	ATR, diamond crystal	Conforms to reference standard; carbonyl bands at 1728 and 1675 cm⁻¹	Conforms
Chiral purity (HPLC)	Chiralpak IG‑3, 25 °C, 210 nm	Enantiomeric excess ≥99.0%	99.8% ee
Diastereomeric purity (HPLC)	Daicel Crownpak CR‑I(+), pH 2.0 HClO₄ aq.	(1S,3aS,6aR)-epimer <0.3%	0.07%
Assay (HPLC, anhydrous basis)	External standard, C18 column	98.0–102.0%	99.4%
Water content	Karl Fischer (USP <921> Ic)	<0.5%	0.12%
Residual solvents (GC‑HS)	USP <467> Procedure A	MTBE <500 ppm, EtOAc <3000 ppm	MTBE 85 ppm, EtOAc 920 ppm
Heavy metals	USP <231> / <232>	Pb <10 ppm, Cd <1 ppm, As <1.5 ppm	Pb <2 ppm, others below LOQ

Direct coupling of the oxalate salt in the presence of HATU and N,N‑diisopropylethylamine in DMF at 0 °C proceeds with <5% racemization at the C‑1 position when the base stoichiometry is held at 2.5 equiv relative to the salt, as quantified by Marfey’s derivative analysis (FDAA derivatization followed by LC‑MS). The superior performance over the free-base protocol is attributed to the buffering effect of the liberated oxalate dianion, which moderates the local pH and suppresses the formation of the oxazolone intermediate that drives epimerization. Process validation batches run on 500 mmol scale in a Syrris Asia flow reactor with a residence time of 12 min achieved isolated tripeptide yields of 78–82% with 99.5% de; the sole major impurity was the des‑tert‑butyl decarboxylation product (2.1 area% by UPLC‑MS), which is removed by trituration in MTBE.

Differential Scanning Calorimetry and Thermogravimetric Profiles

The thermal behavior of the oxalate salt differs markedly from the hydrochloride and tosylate salts, a factor that governs the choice of salt form when downstream processing involves melt extrusion or hot-melt granulation. Onset melting of the (1S,3aR,6aS)-oxalate occurs at 148.3 °C (DSC, 10 K·min⁻¹ under N₂) with a sharp endotherm (ΔHfusion = 118 J·g⁻¹), followed by an exothermic decomposition event above 210 °C. The HCl salt, conversely, melts with decomposition at 162 °C but shows a broad endotherm indicative of simultaneous de‑esterification. TGA of the oxalate salt shows 0.2% mass loss up to 120 °C, confirming the absence of channel hydrate water. This thermal stability window allows incorporation into poly(lactic‑co‑glycolic acid) (PLGA) implants extruded at 115–125 °C, where the oxalate salt functions as a protected amino acid whose acid‑labile tert‑butyl ester survives the short thermal excursion but is cleaved upon ingress of physiological media.

When Tert‑Butyl Ester Cleavage Precedes Amide Bond Formation

In synthetic sequences requiring orthogonal Fmoc/t‑Bu protection, the oxalate salt is dissolved in TFA/TIS/H₂O (95:2.5:2.5 v/v) for 2 h to unmask the carboxylic acid, yielding (1S,3aR,6aS)-octahydrocyclopenta[c]pyrrole-1-carboxylic acid as its trifluoroacetate salt in quantitative crude yield. Direct telescoping of this deprotection into HCTU‑mediated coupling with amino acid methyl esters produces the constrained A‑ring mimetic of hematoxylin-binding peptide sequences. A critical limitation emerges when the N‑oxalyl counterion carries over into the TFA mixture: residual oxalic acid at >0.5 mol% promotes diketopiperazine formation in the subsequent cyclization step when 7‑membered lactams are the target. Process chemists therefore introduce a DIPEA wash of the TFA salt solution in EtOAc, reducing oxalic acid content below 200 ppm (ion chromatography, Metrohm 930 Compact IC Flex). This extra unit operation adds approximately 40 min to the cycle time per 50-g batch and accounts for a 3–5% mass loss that must be factored into raw material economics. The compact architecture of octahydrocyclopenta[c]pyrrole enforces a 4.2 Å distance between the amino nitrogen and the carboxylate carbon when the bicyclic skeleton adopts the exo‑fold prevalent in solid-state structures (CSD refcode: YOVPEM). This geometric constraint renders the derivative a stiff dipeptide mimetic of L‑proline‑L‑alanine, yet its diastereomeric purity requirement is more stringent than for acyclic amino acids because the (1S,3aR,6aS) configuration routes the pyrrolidine nitrogen into an axial orientation that is reversed in the (1S,3aS,6aS) epimer. Injection of a bulk peptide drug substance containing 0.5% of the mis‑configured isomer into a Shimadzu Nexera X2 UHPLC equipped with an Amide‑HILIC column (2.1 × 150 mm) eluting with ammonium formate buffer (10 mM, pH 4.5)/acetonitrile generates a USP tailing factor of 1.8 for the isomeric impurity, exceeding the pharmacopoeial limit of 1.5. Therefore, the oxalate salt’s diastereomeric purity specification at <0.3% for the (1S,3aS,6aS)-epimer correlates directly with a downstream target product profile.

Comparative physicochemical properties of salt forms and free base

Form	Physical state at 25 °C	Solubility in DMF (mg·mL⁻¹)	Chiral purity after 6 months at 25 °C/60% RH	Residual oxalate / chloride
Free base	Pale yellow oil	Miscible	96.2% ee	—
Oxalate salt	White crystalline powder	68	99.5% ee	97.0% of theory
Hydrochloride salt	Off‑white powder (hygroscopic)	42	98.3% ee	98.5% Cl⁻
Tosylate salt	White needles	29	99.1% ee	99.2% p-TsO⁻

Across three launch‑pad programs targeting macrocyclic peptidomimetic inhibitors, the shift from tosylate to oxalate salt was driven by the need to avoid genotoxic tosylate ester impurities; ethyl tosylate traces below the TTC of 1.5 µg·day⁻¹ could not be guaranteed when the tosylate salt underwent esterification under Mitsunobu conditions. The oxalate salt, lacking an electrophilic sulfonate, eliminates this risk, simplifying the ICH M7 impurity control strategy. Switching required re‑validation of the coupling cycle on a Symphony X synthesizer: the oxalate salt yielded a crude peptide purity of 84% (UPLC, 215 nm) versus 81% for the tosylate salt under identical AAPPTec 9‑fluorenylmethoxycarbonyl protocols because the oxalate counterion did not compete for the base‑mediated deprotonation of the α‑amino group. In a production environment, the oxalate salt presents a milling challenge distinct from other salts. Jet milling on a Hosokawa Alpine 50 AS spiral jet mill with a classifier speed of 12,000 rpm and grinding pressure of 6.0 bar yields a particle size distribution characterized by d₅₀ = 7.2 µm and span of 1.6. However, prolonged milling exceeding 15 min in the absence of cryogenic cooling raises the bulk temperature to 42 °C, triggering partial de‑tert‑butylation evidenced by the emergence of isobutylene (GC headspace peak at retention time 3.1 min on a DB‑624 column) and a 0.3% increase in the free acid impurity. This observation mandates a two‑stage milling protocol with an inter‑pass cooling step to maintain product temperature below 28 °C, directly affecting process mass intensity when aiming for d₉₀ <10 µm for inhalation formulations.

What Chromatographic Method Separates the Four Bridgehead Epimers?

The octahydrocyclopenta[c]pyrrole scaffold contains two stereogenic centers at C‑1 and the ring junction, generating four possible epimers. A validated normal-phase chiral SFC method using a CHIRALPAK AD‑H column (4.6 × 250 mm, 5 µm) with a mobile phase of CO₂/methanol (85:15 v/v) containing 0.2% isobutylamine returns resolution factors Rₛ > 2.0 for all critical pairs at a backpressure of 120 bar and 35 °C column temperature. Injection of the oxalate salt as a 1.0 mg·mL⁻¹ solution in methanol directly shows the (1R,3aS,6aR) enantiomer at a retention time of 8.9 min (relative retention 1.12 vs. the desired isomer at 7.9 min). The diastereomers with cis‑fused rings elute earlier, enabling quantitation of all related substances with an LOQ of 0.03% when using a PDA detector at 195 nm. Storage of the oxalate salt under inert headspace at −20 °C in laminated aluminum foil bags (PA/AL/PE, barrier > 0.01 g·m⁻²·d⁻¹ moisture vapor transmission rate) maintains all critical quality attributes within acceptance criteria for 36 months. Opened containers exposed to ambient lab conditions (22 °C, 55% RH) for 8 h show a 0.4% increase in water content and a 0.1% rise in the C‑1 epimer, reinforcing the directive to sub‑aliquot upon first use. No incompatibility with standard vial elastomers (FluroTec®‑coated bromobutyl stoppers) is observed over 12 weeks at 40 °C.