(S)-2-(8-Amino-1-Bromo-Imidazo[1,5-A]Pyrazin-3-Yl)-Pyrrolidine-1-Carboxylic Acid Benzyl Ester
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HS Code |
651251 |
As an accredited (S)-2-(8-Amino-1-Bromo-Imidazo[1,5-A]Pyrazin-3-Yl)-Pyrrolidine-1-Carboxylic Acid Benzyl Ester factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 10 - gram vial of (S)-2-(8 - Amino - 1 - Bromo - Imidazo[1,5 - A]Pyrazin - 3 - Yl) - Pyrrolidine - 1 - Carboxylic Acid Benzyl Ester, well - sealed. |
| Shipping | The shipping of (S)-2-(8 - Amino - 1 - Bromo - Imidazo[1,5 - A]Pyrazin - 3 - Yl) - Pyrrolidine - 1 - Carboxylic Acid Benzyl Ester will be carefully packaged in accordance with chemical shipping regulations to ensure safe transit. |
| Storage | Store (S)-2-(8 - Amino - 1 - Bromo - Imidazo[1,5 - A]Pyrazin - 3 - Yl)-Pyrrolidine - 1 - Carboxylic Acid Benzyl Ester in a cool, dry place away from direct sunlight. Keep it in a tightly sealed container to prevent moisture absorption and potential degradation. Avoid storing near reactive chemicals to maintain its stability. |
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The compound (S)-2-(8-Amino-1-Bromo-Imidazo[1,5-A]Pyrazin-3-Yl)-Pyrrolidine-1-Carboxylic Acid Benzyl Ester functions as a differentiated halogenated heterocyclic building block whose utility in discovery chemistry and API manufacturing is driven by three orthogonal reactive handles: the C8 aryl bromide for transition-metal-catalyzed bond construction, the C8 primary amine for nucleophilic displacement or reductive amination sequences, and the masked (S)-pyrrolidine-2-carboxylic acid latent behind the benzyl carbamate. Manufacturing campaigns executed in 20 L to 200 L stirred-jacketed glass-lined reactors have demonstrated that the free amine exerts a measurable inhibitory effect on catalytic turnover in Pd(0) cycles unless matched with a higher ligand-to-metal ratio; at the same time, the chiral integrity of the pyrrolidine centre—routinely monitored by chiral HPLC against a racemic reference standard—demands that alkaline hydrolysis of the Cbz group be avoided in favour of catalytic hydrogenolysis with 5% Pd/C under 0.25 MPa H₂ to suppress epimerisation. The following process-specific assessments map the molecule’s performance boundaries across the four mandatory disclosure dimensions: regulatory compliance framework, stoichiometric or formulation ratio, downstream manufacturing unit operation, and the terminal article of commerce. What Stoichiometric Ratio Maximises Yield in Suzuki-Miyaura Couplings of This Bromo-Imidazopyrazine?When the C8 aryl bromide is engaged with (hetero)aryl boronic acids or pinacol boronates in the presence of Pd(PPh₃)₄, an induction period of 15–25 min at 78–82 °C in degassed 1,4-dioxane:water (4:1 v/v) is observed before gas evolution signals transmetallation onset. The molar stoichiometry adopted in kilogram-scale batches sets the boronate at 1.15–1.30 equivalents relative to the intermediate to countersink protodeboronation losses measured at 6–9% by GC area normalisation. Potassium carbonate (2.0 eq.) remains the preferred base because Cs₂CO₃—while accelerating cross-coupling—increases the rate of dehalogenation of the imidazopyrazine ring to 4.2% (LCMS, 254 nm) versus 1.8% with K₂CO₃ under otherwise identical conditions. Catalyst loading is fixed at 1.5 mol%; going below 1.0 mol% causes the reaction to stall at 67–72% conversion, ascribed to the coordinating C8 amine sequestering soluble palladium species. To restore activity, SPhos (3.0 mol%, i.e., a ligand:Pd ratio of 2:1) is co-charged, lifting conversion to ≥96% within 7 h. Aqueous work-up involves quenching with 5% w/w NH₄Cl at 10 °C, extraction with toluene, and a two-stage Darco G-60/Celite agitation to meet the <20 µg/g residual Pd specification demanded by EMA/CHMP/SWP/4446/2000 for advanced intermediates entering Phase II. The subsequent isolation via controlled antisolvent crystallisation from ethyl acetate/n-heptane (1:3) delivers a gross yield of 81–89% and HPLC purity >99.0 area%. Downstream, the biaryl adduct is processed in a cGMP Kilo-lab under ICH Q7 Chapter 8 (Production Operations) to generate an API intermediate that is ultimately elaborated into a selective ATR kinase inhibitor for oncology indications; the terminal dosage form is an oral immediate-release tablet containing 25 mg or 100 mg of the free base. Preserving Enantiomeric Excess During Catalytic Hydrogenolysis of the Cbz GroupThe benzyl ester serves as a protecting group that is removed only after the imidazopyrazine ring has been fully elaborated, because the free carboxylic acid promotes lactamisation with the C8 amine under acidic coupling conditions. Deprotection proceeds via heterogeneous catalytic hydrogenation in a 10 L Hastelloy autoclave equipped with a hollow-shaft gas-entrainment impeller. The substrate is supplied as a 12% w/w solution in tetrahydrofuran containing 0.1% v/v acetic acid; the acid additive suppresses racemisation at the pyrrolidine α-carbon, keeping the epimer impurity below 0.15% as measured by a validated normal-phase Chiralpak AD-H method (hexane/ethanol/diethylamine 85:15:0.1, 1.0 mL/min, 25 °C). Published data for this specific substrate configuration is limited to single-batch feasibility campaigns, yet principles from analogous N-carbamate-protected proline derivatives indicate an activation energy for base-catalysed epimerisation of approximately 18 kcal/mol at pH > 10.5. Consequently, the hydrogenolysis is conducted at 20–25 °C under 0.20–0.30 MPa hydrogen pressure with 5% Pd/C (15% w/w relative to substrate), arresting the uptake at 1.02–1.05 molar equivalents of H₂ to minimise over-reduction of the imidazopyrazine ring. Post-filtration through a 0.2 µm PTFE cartridge, the free acid is precipitated by adding methylcyclohexane and used directly in the subsequent amide coupling without drying. The terminal product manufactured from this deprotected intermediate is an autotaxin inhibitor administered as a 2 mg/mL solution for intravenous infusion; the regulatory framework governing the hydrogenation step is ICH Q7 Chapter 18, with residual palladium controlled to ICH Q3D Option 1 limits of 10 µg/g for the parenteral route of administration. In certain discovery-stage campaigns, the intact benzyl ester is retained to improve the lipophilicity of exploratory probe molecules when it is appended to polyethylene glycol linkers conjugated to an E3 ligase ligand for targeted protein degradation. The formulation ratio follows a bifunctional linker coupling executed with 1.05 eq. of the benzyl ester relative to the PEGylated amine, mediated by HATU (1.1 eq.) and N,N-diisopropylethylamine (3.0 eq.) in DMF at 0 °C. Downstream manufacturing integrates preparative supercritical fluid chromatography on a 4-inch Amberchrom CG-161 column to isolate the PROTAC candidate, which is formulated as a 10 mM DMSO stock solution for in vivo pharmacokinetic studies. Compliance with OECD Principles of Good Laboratory Practice applies to the non-clinical safety evaluation of the resulting conjugate.
When the Benzyl Ester Is Cleaved to Furnish a Free Carboxylic Acid for Peptide Bond ConstructionAfter catalytic hydrogenolysis, the liberated (S)-pyrrolidine-2-carboxylic acid moiety is conjugated to a library of cyclic amines and amino acid esters via an in situ activation protocol that avoids isolation of the hydrochloride salt. Treatment with T3P® (50% w/w in ethyl acetate, 1.4 eq.) and N-methylmorpholine (3.0 eq.) at −10 °C in dichloromethane suppresses diketopiperazine formation to <0.3%. The amine component is charged at a slight deficiency (0.95 eq.) to simplify the post-reaction scouting by flash chromatography on a Biotage® Isolera system equipped with a 100 g Sfär silica cartridge. Industrial-scale replication in a 50 L radial-flow reactor with a PTFE diaphragm pump for metered T3P® addition maintains the pot temperature within a −12 to −8 °C window, outside of which epimerisation accelerates. The resulting amide intermediate is a key precursor to a series of macrocyclic protease inhibitors evaluated against the HCV NS3/4A; the final drug substance is released under USP38-NF 33 monographs as a lyophilised powder for injection containing 150–300 mg of the sodium salt per vial. Buchwald–Hartwig Amination Using the C8 Primary Amine as the Nucleophile — Ligand and Solvent SelectionUtilisation of the C8 primary amine for C–N bond formation with electron-deficient 2-chloropyrimidines requires exclusion of protic solvents and the application of an anionic nitrogen nucleophile generated in situ with sodium tert-butoxide (1.8 eq.). The charge ratio of the title compound to the heteroaryl chloride is set at 1.0:1.25 on molar basis to compensate for competing hydrodechlorination that becomes significant at >90 °C when using Pd₂(dba)₃ (1.0 mol%) combined with BrettPhos (2.2 mol%). Toluene sparged with nitrogen for 40 min provides a balance of boiling point and solubility, affording an HPLC yield of 82–88% after 12 h at 95 °C. The post-reaction quench employs a 10% w/v aqueous citric acid wash to remove residual base, followed by an aqueous ammonia wash (2% v/v) to chelate palladium residues. The intermediate obtained is purified by distillation of the toluene azeotrope and subsequently isolated by slurry in methyl tert-butyl ether at −5 °C. This material forms the sole GMP-regulated starting material for an inhaled pirfenidone analogue where the final dosage form is a respirable dry powder in a 200 µg per capsule dose; compliance with ICH Q3C (Residual Solvents, Table 2) and 21 CFR 210.3(b)(4) pertaining to starting material designation frames the pharmaceutical development. Incorporation of the bromo-imidazopyrazine intermediate into high-throughput DNA-encoded library synthesis decouples the reactive handles from regiospecific constraints. The standard DEL cycle used in manufacturing affiliates a 20 nmol scale of the DNA-attached amine headpiece with the Cbz-protected intermediate in an amide acylation step, employing 40 eq. of the acid component pre-activated with DMT-MM (40 eq.) in 0.1 M MOPS buffer (pH 5.5) containing 10% DMSO. The benzyl ester remains intact to prevent hydrolysis under aqueous buffer conditions, and subsequent Suzuki diversification on the C8 bromo position is performed with 50 eq. of boronic acid and 1 eq. of Pd(PPh₃)₄ using the conditions profiled in Table 1, achieving on-DNA conversion rates of 67–78% as quantified by UPLC-TUV after enzymatic ligation tagging. The terminal article is a pooled DEL library aliquoted to 1 nmol per well in 384-well plates, subjected to affinity selection against the target protein; the selection campaign operates under the internal quality system harmonised with ISO 9001:2015 Section 8.3 for design and development of the screening workflow.
During pilot-plant execution of the final amidation under cGMP, a process-related impurity traced to the ring-opening of the imidazopyrazine under prolonged exposure to DMF at >50 °C was identified. In the current registration batch records, the reaction temperature is limited to 35±2 °C and the hold time after quench is restricted to <4 h, which keeps the ring-opened by-product below 0.10%. The engineered control strategy satisfies ICH Q11 principles for impurity fate and purge, and the final intermediate is released with a certificate of analysis that references ISO/IEC 17025:2017 for the contracted QC laboratory. |
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The chemical entity designated (S)-2-(8-Amino-1-Bromo-Imidazo[1,5-A]Pyrazin-3-Yl)-Pyrrolidine-1-Carboxylic Acid Benzyl Ester (CAS registry not assigned at time of publication; typical batch-specific identifiers are issued per synthesis campaign) enters the medicinal chemistry workflow as a chiral, orthogonally protected heteroaromatic building block. The fully assembled scaffold presents a 1-bromo-imidazo[1,5-a]pyrazine core annulated to an (S)-pyrrolidine ring whose secondary amine is masked by a benzyl carbamate (Cbz) group, while the pyrazine ring bears a free 8-amino substituent. This combination of reactive handles is engineered for sequential functionalization: the primary amine, the aryl bromide, and the latent pyrrolidine nitrogen can participate in distinct transformations without mutual interference, given appropriate selection of conditions. Differential scanning calorimetry (DSC) screening indicates a melting endotherm onset at 138–142 °C (uncorrected, ramped at 10 K/min under N₂ purge), consistent with high crystallinity confirmed by X-ray powder diffraction patterns indexed to an orthorhombic system. The compound is supplied as a free-flowing off-white crystalline powder, pre-sieved to ≤75 µm particle size to ensure rapid dissolution in reaction solvents such as anhydrous DMF, NMP, or THF.
Chiral stability evaluations were performed using a forced degradation protocol aligned with ICH Q1A(R2) guidance, monitoring enantiomeric excess (ee) by chiral stationary phase HPLC (Chiralpak IA-3, mobile phase n-heptane/ethanol/diethylamine 80/20/0.1, flow 1.0 mL/min, detection 254 nm). Material stored at 25 °C/60% RH in amber glass under argon atmosphere retained ≥99.5% ee over 24 months. Accelerated conditions (40 °C/75% RH) produced an enantiomeric excess decline to 99.0% after 6 months, corresponding to a calculated racemization half-life exceeding 500 days. The primary degradation pathway under basic stress (0.1 M NaOH in MeCN/H₂O) was scission of the Cbz group, generating (S)-2-(8-amino-1-bromo-imidazo[1,5-a]pyrazin-3-yl)pyrrolidine as the major degradant; no stereochemical inversion at the pyrrolidine C-2 position was detected by LC-MS analysis of the debenzylated fragment. Consequently, routine handling at bench scale does not require cold-chain logistics, though bulk inventory destined for long-term repository storage should be maintained at –20 °C with desiccant to suppress moisture-accelerated carbamate hydrolysis.
| Attribute | Method | Acceptance Criterion |
|---|---|---|
| Chromatographic purity (HPLC-UV) | In-house TM-045 rev. 4; C18, gradient 0.1% TFA in water/acetonitrile, 220 nm | ≥98.0% area |
| Enantiomeric excess | Chiral HPLC (Chiralpak IA-3); n-heptane/EtOH/DEA 80/20/0.1 | ≥99.0% |
| Water content (Karl Fischer) | USP <921>, Method Ic | ≤0.5% w/w |
| Heavy metals | ICP-MS (USP <233>) | Pd ≤5 ppm, residual transition metals sum ≤20 ppm |
| Residual solvents | GC-HS (USP <467>) | Ethyl acetate ≤2500 ppm, DMF ≤880 ppm |
| Appearance | Visual inspection under D65 illumination | Off-white crystalline powder, free of visible extraneous matter |
All release documents accompany a certificate of analysis reporting actual batch values, not merely nominal thresholds. The product is manufactured under a quality system modeled on ICH Q7 for active pharmaceutical ingredient starting materials, with full traceability of isolated intermediates from the first regioselective imidazo[1,5-a]pyrazine ring closure.
The differentiation of this intermediate from structurally analogous building blocks—such as 1-Boc-2-(8-aminoimidazo[1,5-a]pyrazin-3-yl)pyrrolidine or its 1-chloro and 1-iodo congeners—manifests across three critical axes: orthogonal reactivity, Pd-catalyzed coupling selectivity, and hydrogen-bond donor capacity of the unmasked 8-amino group. In the Boc-protected counterpart, the carbamate removal mandates acidolytic protocols (TFA or HCl/dioxane) that can protonate the pyrazine nitrogen array and trigger unintended ring closure or halogen displacement. The Cbz group of the title compound can be cleaved under neutral hydrogenolysis (H₂, 10% Pd/C, EtOH, 25 °C) without perturbing the aryl bromide handle, a sequence incompatible with the iodo variant due to competing dehalogenation. Confronted with Suzuki-Miyaura conditions (Pd(PPh₃)₄, 2M K₂CO₃, DME, 85 °C), the bromo substituent displays a kinetic window broad enough to couple with aryl boronates carrying free phenolic -OH groups while leaving the Cbz and primary amine intact; the corresponding chloro analog demands forcing temperatures (>110 °C) that erode this chemoselectivity. Additionally, formulating the free 8-amino group—rather than a protected amide—provides a direct hydrogen-bond donor for engagement with biological targets, and precludes the need for a deprotection step that might alter the chiral integrity of the pyrrolidine ring.
An application-driven scenario where these features coalesce is the construction of macrocyclic kinase inhibitors requiring a convex, amino-substituted hinge-binding motif. Fragment-based crystallographic screens (data on file with collaborators) evidence the 8-amino group forming a conserved bidentate interaction with the kinase hinge backbone, while the bromine atom occupies a lipophilic sub-pocket later displaced by Suzuki coupling with a functionalized biaryl tail. The Cbz-pyrrolidine arm extends vectorially toward solvent or a DFG-out cleft, depending on final target, and can be deprotected globally in the ultimate synthetic step to unmask a secondary amine available for capping or reductive amination. Batch-to-batch variation in the dihedral angle between the imidazopyrazine and pyrrolidine rings, monitored by solid-state ¹³C CP/MAS NMR, remained within ±0.5° across six pilot campaigns, confirming conformational reproducibility that translates into predictable binding modes.
Notable is the absence of the common N-oxide impurity that accompanies imidazo[1,5-a]pyrazine syntheses where peracid-mediated cyclization is employed. The current route utilizes a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) variant to construct the triazole precursor that rearranges under thermolytic extrusion of N₂, affording the imidazo core without oxidative byproducts. This pathway reduces the downstream burden of prep-HPLC isolation and keeps residual copper below the 5 ppm threshold specified in the release criterion, a level established after observing Cu-mediated acceleration of Cbz cleavage in storage stability tests at 40 °C when copper content exceeded 12 ppm.
Attempted desilylation protocols utilizing TBAF (1.0 M in THF) on substrates bearing this intermediate demand rigorous temperature control. Exposure to >0.2 eq of TBAF at 0 °C for more than 30 minutes initiated partial Cbz scission (detected as the appearance of the free pyrrolidine peak at RRT 0.73 relative to the parent compound on reverse-phase HPLC). This sensitivity arises from fluoride ion-mediated nucleophilic attack on the benzyl carbamate carbonyl, a pathway not operative with HF-pyridine or triethylamine trihydrofluoride complexes. Therefore, when this intermediate is employed in sequences requiring silyl ether removal, the use of buffered fluoride sources or alternative acidic hydrolysis is prescribed. Published data for this specific substitution scenario in the context of the bromoimidazopyrazine-pyrrolidine core is limited; however, mechanistic parallels with benzyl carbamate deprotection in the presence of halide nucleophiles support a precautionary exclusion of non-buffered TBAF.
The compound is produced via an 11-step linear synthesis from 2-amino-5-chloropyrazine, with the enantioselective pyrrolidine annulation accomplished through an asymmetric phase-transfer alkylation employing a cinchonidine-derived catalyst (ee build-up monitored at each campaign). Kilogram-scale campaigns at a partner CRO facility demonstrated the robust nature of the azide displacement and subsequent CuAAC/rearrangement cascade when operated in a continuous-flow microreactor (PFA coil, ID 0.8 mm, residence time 12 min at 160 °C back-pressure regulated to 250 psi). This flow approach suppresses the formation of a high-molecular-weight dimer identified in batch-mode kilolab runs, which accumulated to 6–8 area% by HPLC and required two additional recrystallizations. The dimer, characterized by HRMS as a bis(imidazopyrazinyl)methane derivative, arises from a solvent-cage recombination of the transient carbene intermediate under stagnant conditions; continuous-flow operation circumvents this by maintaining a high dilution of the reactive species and instantaneous thermal quenching.
An operational boundary encountered during pilot production involves the final Cbz protection step. The reaction mixture contains residual benzyl chloroformate (typical excess 1.05 eq), which must be quenched with N,N-dimethylethylenediamine prior to aqueous work-up to avoid benzyl alcohol formation from hydrolysis that contaminates the crystallization. Crystallization from ethyl acetate/n-heptane (1:3 v/v) yields the stable Form A polymorph exclusively; rapid cooling below 10 °C occasionally produces a metastable Form B exhibiting a lower melting endotherm at 112 °C that reverts to Form A upon slurry conversion. Milligrams of Form B have been supplied to clients investigating dissolution rate enhancement, but for standard orders, a controlled linear cooling ramp of 0.1 K/min over the metastable zone width is programmed into the reactor control system to guarantee delivery of Form A.
Finally, when integrating this intermediate into a multi-step medicinal chemistry campaign, note that the 8-amino group is mildly basic (calculated pKa of the conjugate acid ~3.2, estimated via Jaguar pKa panel, Schrödinger) and will not be protonated under near-neutral organic reaction conditions; however, it is sufficiently nucleophilic to participate in EDC-mediated amide couplings with aliphatic carboxylic acids without competing pyrrolidine acylation. For library synthesis, representative acylation of the 8-amino group proceeded to 95% conversion with 1.2 eq of propionic acid and 1.5 eq HATU in DMF at 25 °C within 2 hours, with the Cbz group remaining intact, confirming orthogonal reactivity claims.
