Electrophilic Oxidation and [1,2]-Rearrangement of the Biindole Core of Birinapant

Abstract

Birinapant/TL32711 (1) is a bivalent antagonist of the inhibitor of apoptosis (IAP) family of proteins and was designed to mimic AVPI, the N-terminal tetrapeptide of the second mitochondria-derived activator of caspases (Smac/DIABLO). Birinapant bound to the BIR3 domains of cIAP1, cIAP2, and XIAP with K_i values of 1, 36, and 45 nM, respectively. Birinapant-mediated activation of cIAP1 resulted in cIAP1 autoubiquitylation and degradation and correlated with inhibition of TNF-mediated NF-κB activation, induction of tumor cell death in vitro, and tumor regression in vivo. Birinapant is being evaluated in Phase 1/2 trials for the treatment of cancer and hepatitis B virus (HBV) infection. After one year at accelerated storage conditions, a formulation of 1 afforded four degradants in >0.1% abundance by HPLC analysis. The primary degradants (2 and 3) were formed via oxidation of the biindole core, while the secondary degradants (5 and 6) arose via [1,2]-rearrangement of 3 and 2, respectively. Forced degradation conditions were developed, which allowed the isolation of 2 and 3 in multigram quantities. Novel deuterated analogues of 1 were prepared to determine the site of oxidation, and NMR experiments confirmed the chemical structures of 5 and 6. The de novo synthesis of 2, 3, 5, and 6 confirmed these experimental findings.

Birinapant/TL32711 (1) is a small-molecule antagonist of the inhibitor of apoptosis (IAP) family of proteins that is currently in clinical trials for the treatment of cancer and hepatitis B virus (HBV) infection.¹⁻³ Structurally, 1 contains two N(Me)Ala-Abu-Pro peptide chains attached to a central biindole core (Figure 1A).⁴ The discovery and development of birinapant drug substance has been reported.^4,5 Birinapant drug product (1 DP) is currently manufactured as a sterile-filtered, aqueous formulation, which is administered as an intravenous infusion. As part of an ongoing chemical development program, 1 DP has been evaluated for long-term stability at multiple conditions including 25 °C/60% relative humidity (RH) and 40 °C/75% RH. In the course of these investigations, partial degradation (>0.1%) of 1 was observed on prolonged storage of 1 DP at the accelerated storage condition of 40 °C/75% RH (Figure 1B). High performance liquid chromatography (HPLC) and mass spectral (MS) analysis of these samples demonstrated that four degradants (i.e., 2, 3, 5, and 6) were formed by the addition of a single oxygen molecule to 1 (i.e., M + 16); degradants arising from the addition of two oxygen molecules (i.e., 4) were observed in lower quantities.⁶ In order to gain insight into the degradation process, we prepared each oxidative degradant via forced degradation and then confirmed their structures based on independent synthesis.

Figure 1.

(A) Chemical structure of birinapant/TL32711 (1). (B) HPLC profile of 1 DP after extended storage at 40 °C/75% RH indicating the formation of oxidative degradants 2 through 6. Asterisk (*) indicates a process impurity, which is unrelated to the degradation process.

To generate sufficient quantities of the oxidative degradants, air was bubbled through an aqueous solution of 1 containing 0.1% acetic acid (HOAc) for 7 days thus affording a mixture of 2–6 albeit in differing ratios from the degradation of authentic 1 DP (Supplemental Figure S1). Using this process, we were able to generate gram-quantities of 2 and 3, which allowed for their chemical characterization by ¹H and ¹³C NMR spectroscopy and mass spectrometry (MS).

Several possible mechanisms for the oxidation of 1 including amine oxidation,⁷ benzylic oxidation,⁸ and indole oxidation⁹ are supported by literature precedent. We observed that mixtures of 2 and 3 reverted to 1 under standard palladium-catalyzed hydrogenation conditions thus supporting these proposed modes of oxidation (data not shown). The product of N-terminal amine oxidation was prepared by independent synthesis and shown not to correlate with any of the observed degradants by HPLC analysis (Supplemental Scheme S1).

Benzylic oxidation adjacent to an indole moiety is reported to occur under free-radical conditions when DDQ is employed as the oxidant.⁸ In contrast, electrophilic oxidation using dimethyldioxirane (DMDO) is reported to result in the direct oxidation of the indole ring to afford 3H-indol-3-ols.⁹ To distinguish between these two oxidative pathways, we incorporated deuterium atoms at the benzylic positions of 1 (Scheme 1).¹⁰ Reduction of 7 with NaBH₄-d₄ furnished 8, which contained two benzylic deuterium atoms. Following protection of the hydroxy group, deuterated 9 was subjected to TFA-mediated indole dimerization;¹⁰⁻¹² further elaboration using known methods afforded 1-d₄.⁴

Scheme 1. Synthesis of Deuterated Birinapant, 1-d₄.

Under forced oxidation conditions (i.e., 0.1% aqueous HOAc, 7 days), 1-d₄ afforded the same mixture of oxidative degradants by HPLC analysis (Supplemental Figure S2); the observed molecular ion for each degradant confirmed the retention of all four deuterium atoms. Thus, benzylic oxidation, which would have resulted in the loss of one deuterium atom, was eliminated as an operative oxidative pathway.

To confirm direct oxidation of the biindole core, we subjected 1 to electrophilic oxidation using DMDO.⁹ Unfortunately, this reaction was unsuccessful likely due to the presence of the two basic nitrogen atoms of 1. Thus, we performed the oxidation on fully protected intermediate 10 (Scheme 2). Treatment of 10 with DMDO, generated in situ by the combination of oxone and acetone, provided a mixture of two diastereomeric hydroxyindolenines (11), each bearing four deuterium atoms further confirming the biindole moiety as the site of oxidation of 1. Removal of the Boc and acetyl protective groups afforded 2-d₄ and 3-d₄, which coeluted with authentic 2 and 3 from the 1 DP stability study. The absolute stereochemistry at the C3-indole position of 2-d₄ and 3-d₄ remains unknown and thus has been assigned arbitrarily.

Scheme 2. Preparation of Synthetic Oxidative Degradants 2-d₄ and 3-d₄ from 10.

These results were confirmed by independent synthesis of 2 and 3 from the Boc-Abu-coupled intermediate (12, Scheme 3). As expected, oxidation of 12, a previously reported intermediate in the synthesis of 1,^4,5 afforded the hydroxyindolenines 13 and 14 as a separable mixture of stereoisomers. Independent elaboration of 13 or 14 afforded synthetic 2 or 3, respectively, which coeluted with authentic 2 and 3 derived from the degradation of 1 DP (Supplemental Figure S3) and were identical by ¹H and ¹³C NMR and MS analysis to 2 and 3 formed via forced degradation of 1. Taken together, these results demonstrated that oxidative degradants 2 and 3 were formed via the biindole oxidation of 1.

Scheme 3. Preparation of Oxidative Degradants 2 and 3 from 12.

Movassaghi et al. reported that 2-aryl-substituted hydroxyindolenines like 2 and 3 rearranged to either 1H-[2,2′]-biindolyl-3-ones or 1′,3′-dihydro-1H-[2,3′]biindolyl-2′-ones depending upon the reaction conditions and that these two regioisomeric rearrangement products could be distinguished by examining the chemical shift of the nascent carbonyl resonance in the ¹³C NMR spectra.⁹ More recently, Qi et al.¹³ and Han and Movassaghi¹⁴ demonstrated that biindole-derived hydroxyindolenines rearranged under thermal or acidic conditions to afford 1H-[2,2′]-biindolyl-3-ones (infra).

To address whether 2 or 3 would undergo this rearrangement, we warmed an aqueous solution of 2 containing 0.1% HOAc for 24 h at 95 °C. Under this condition, 2 readily rearranged to 6 (Supplemental Figure S5). In a separate experiment, we observed that 3 rearranged to 5 under identical reaction conditions. Taken together, these data demonstrated that oxidation of 1 occurred at the biindole core to afford hydroxyindolenines 2 and 3, which further rearranged to 6 and 5, respectively. However, whether 2 or 3 rearranged to provide 1H-[2,2′]-biindolyl-3-ones or the isomeric 1′,3′-dihydro-1H-[2,3′]biindolyl-2′-ones remained unresolved (Supplemental Figure S4).

Owing to the structural complexity of 5 and 6, we performed the oxidation/rearrangement on a simpler biindole substrate in order to independently confirm the chemical structures of 5 and 6. Biindole 15 is employed in the synthesis of 1.^4,5 Treatment of 15 under modified DMDO-mediated oxidation conditions provided a mixture of hydroxyindolenines 16 and 17 (Scheme 4). As with 2 and 3 (supra), the absolute chemistry at the C3-indole positions of 16 and 17 is unknown and therefore has been assigned arbitrarily. This mixture was then subjected to modified rearrangement conditions (0.1% HOAc in toluene at 100 °C) to afford a separable mixture of 18 and 19. The observed ¹³C chemical shifts for the nascent carbonyl groups of 18 and 19 were δ 200.4 and δ 199.6, respectively, thus suggesting that hydroxyindolenines 16 and 17 rearranged to 1H-[2,2′]-biindolyl-3-ones 18 and 19 and not to the structurally related 1′,3′-dihydro-1H-[2,3′]biindolyl-2′-ones (not shown). In a separate series of experiments, we demonstrated that, in fact, exposure of 16 to the rearrangement conditions exclusively afforded 18, while independent treatment of 17 provided 19 thereby demonstrating the stereospecific nature of the [1,2]-rearrangement process (data not shown). Importantly, the absolute stereochemistry at the quaternary centers of 18 and 19 is unresolved and has thus been assigned arbitrarily based on the assigned configurations of 16 and 17, respectively.

Scheme 4. Synthesis and [1,2]-Rearrangement of Hydroxyindolenines 16 and 17.

Final elucidation of the degradation pathway came from the conversion of 18 into 6 using our standard HATU-mediated peptide coupling strategy (Scheme 5).⁴ Hydrogenolytic removal of the two Cbz protective groups of 18 afforded the bis-pyrrolidine (20), which was then coupled to Cbz-Abu-OH. Further elaboration of the peptide chains provided 6 which was identical by HPLC, MS, ¹H, and ¹³C NMR analyses to authentic 6 formed either via the degradation of 1 DP (Supplemental Figure S5) or via acid-mediated rearrangement of 2 (Supplemental Figure S4).

Scheme 5. Transformation of 1H-[2,2′]-Biindolyl-3-one 18 into Oxidative Degradant 6.

In an analogous fashion, 1H-[2,2′]-biindolyl-3-one 19 was converted in several steps to 5, which was identical to the authentic standard (data not shown). Interestingly, during the Cbz group removal from 19, an azepine-containing product was isolated in modest yield together with the desired bis-pyrrolidine (Supplemental Scheme S2). No effort was made to optimize this reaction; however, the azepine was likely formed via the intramolecular reductive amination of the intermediate bis-pyrrolidine. A similar intramolecular cyclization was recently described in the synthesis of trigonoliimine C.¹³⁻¹⁵

Birinapant drug product (1 DP) is a sterile, aqueous solution, which is administered in the clinic by intravenous infusion. During the investigation of long-term storage conditions for 1 DP, we observed the formation of four oxidative degradants at >0.1% occurrence by HPLC analysis. To clarify the degradation process and support further drug product development, we isolated 2, 3, 5, and 6 following forced degradation of 1 and determined their chemical structures via independent synthesis.

This investigation demonstrated that the central biindole core of 1 reacted with atmospheric oxygen (or a transition metal oxo species) to afford two primary oxidative degradants 2 and 3, which were diastereomeric at the indole C3 position.¹⁴ Forced degradation of 1-d₄ confirmed that oxidation did not occur at the benzylic positions of 1. Both hydroxyindolenines 2 and 3 could be prepared in gram-quantities by forced degradation of 1 and these synthetic products were identical by HPLC and mass spectral analysis to those formed on extended storage of 1 DP at ≥40 °C.

The observation that an acidic solution of hydroxyindolenine 2 transformed with heating to 1H-[2,2′]-biindolyl-3-one 6 further corroborated these results. Notably, diastereomer 3 was shown to convert to 5 under identical conditions. Contemporaneous with this investigation, Qi et al.¹³ and Han and Movassaghi¹⁴ demonstrated a similar Wagner–Meerwein-type [1,2]-rearrangement of hydroxyindolenines derived from 2,2′-bis-tryptamines en route to the total syntheses of the trigonoliimines. Thus, the oxidative pathway for the degradation of 1 DP at elevated temperature is summarized in Figure 2.

Figure 2.

Proposed pathway for the oxidative degradation of 1.

The ultimate goal of our drug development program was to develop a formulation of 1, which achieved ambient temperature stability on extended storage. To further aid in this process, a thorough understanding of the oxidizing species, i.e., molecular oxygen, peroxides, etc., effect of the container closure system, i.e., composition of glass vial, and the application of pharmaceutically acceptable antioxidants, i.e., BHT, tocopherol, etc., may be required. The insights gained from this study will aid in the development of stabilized formulations of 1.

Experimental Procedures

Chemistry

Birinapant (1), 12, and 15 were prepared following reported procedures.^4,5 Deuterated intermediates 8, 9, 10, and 1-d₄ were prepared using standard modifications of those methods.¹⁰¹H and ¹³C NMR and mass spectral characterization of 2, 3, 5, 6, 16, 17, 18, and 19 are provided in the Supporting Information.

Representative procedures for the forced degradation of 1, the DMDO-mediated oxidation of [2,2′]-biindoles, and rearrangement to the resultant 1H-[2,2′]-biindolyl-3-ones are provided below.

Isolation of 2 and 3 Following the Forced Degradation of Birinapant (1)

Birinapant (1, 5.0 g, 6.2 mmol) was dissolved in water containing 0.1% HOAc (250 mL). Air was gently bubbled through the colorless solution at ambient temperature. After 7 days, HPLC analysis revealed 85% conversion of 1 to afford 2 (41 area %), 3 (25 area %), 4 (mixture of isomers, 6 area %), 5 (5 area %), and 6 (2 area %). The yellow-colored solution was concentrated, and the residue was purified by RP-HPLC to provide 1.4 g of 2 (27%) and 0.7 g of 3 (14%) as yellow-colored solids.

Preparation of 6 via the [1,2]-Rearrangement of 2

Primary degradant 2 (140 mg, 0.17 mmol) was dissolved in water containing 0.1% HOAc (10 mL). The resulting yellow-colored solution was warmed to 95 °C for 24 h at which time HPLC analysis revealed >95% conversion of 2 to 6. The reaction mixture was concentrated, and the residue was purified by RP-HPLC to afford 110 mg of 6 (79%) as a yellow-colored solid.

Preparation of 16 and 17 via DMDO-Mediated Oxidation of 15

To suppress overoxidation of the biindole moiety, a biphasic modification of the DMDO-mediated oxidation was applied.⁹ To a heterogeneous mixture of saturated aqueous NaHCO₃ (30 mL) and acetone (30 mL) was slowly added oxone (3.6 g, 5.8 mmol) in deionized water (50 mL) at 0 °C. The resulting homogeneous mixture was stirred at 0 °C for 1 h followed by the slow addition of 15 (4.3 g, 5.3 mmol) in DCM (100 mL). After 1 h at 0 °C, the reaction mixture was diluted with deionized water (100 mL) and DCM (100 mL). The organic layer was separated, and the aqueous phase was extracted with DCM (2 × 50 mL). The combined organic extracts were washed with brine and dried over anhydrous Na₂SO₄, filtered, and concentrated. The crude residue was purified by RP-HPLC to provide 1.1 g (26%) of 16 (early eluting product) and 2.7 g (63%) of 17 (late eluting product) as yellow-colored solids.

Preparation of 18 via [1,2]-Rearrangement of 16

A solution containing 16 (1.1 g, 1.31 mmol) in toluene (40 mL) and HOAc (0.5 mL) was stirred at 100 °C. After 4 h, HPLC analysis revealed complete conversion to 18. The solvent was removed in vacuo, and the crude residue was purified by RP-HPLC to yield 950 mg of 18 (86%) as a yellow-colored solid.

Glossary

ABBREVIATIONS USED

Abu

α-aminobutyric acid

AVPI

alanine-valine-proline-isoleucine tetrapeptide

BHT

butylated hydroxytoluene

BIR3

baculovirus IAP repeat domain 3

Cbz

benzyloxycarbonyl

cIAP

cellular IAP1 or cIAP2

DDQ

2,3-dichloro-5,6-dicyano-1,4-benzoquinone

DIABLO

direct IAP binding protein with low pI (also, Smac)

DMDO

dimethyldioxirane

DMSO

dimethyl sulfoxide

DP

drug product

HPLC

high-performance liquid chromatography

HOAc

acetic acid

IAP

inhibitor of apoptosis proteins

LC/MS

liquid chromatography coupled to mass spectrometry

ML-IAP

melanoma IAP

MS

mass spectrum or spectrometry

NaBH₄

sodium borohydride

NMR

nuclear magnetic resonance

[O]

oxidation

oxone

potassium peroxymonosulfate

RH

relative humidity

Smac

second mitochondria-derived activator of caspases (also, DIABLO)

TFA

trifluoroacetic acid

TNF

tumor necrosis factor

TNFR1

TNF receptor 1

XIAP

X chromosome-linked IAP

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmedchemlett.5b00461.

• NMR (¹H, ¹³C) and mass spectral characterization of 1-d₄, 2, 3, 5, 6, 16, 17, 18, and 19 (PDF)

Author Present Address

^† Department of Chemistry, Chemical Methodologies and Library Development, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United States.

Author Present Address

^‡ Celgene Corp., 10300 Campus Point Drive, Suite 100, San Diego, California 92121, United States.

Author Contributions

Y.D., T.H., M.G.L., S.R.R., M.D.A., P.T.K., M.S.H., Y.-H.L., and S.M.C. contributed to the design and synthesis of compounds.

The authors declare no competing financial interest.