Sulfonamide Prodrugs with a Two-Stage Release Mechanism for the Efficient Delivery of the TLR4 Antagonist TAK-242

Abstract

We previously demonstrated that the potent TLR4 inhibitor TAK-242 could be covalently conjugated to pancreatic islets using a linker that afforded an effective sustained delivery of the active drug after transplant. This drug-eluting tissue achieved local inhibition of TLR4-linked inflammation and proved beneficial to the islet graft survival. Here, we describe a new family of prodrugs with a modular design featuring a self-immolative para-aminobenzyl spacer bonded directly to the TAK-242 sulfonamide nitrogen, a tether for bioconjugation, and a β-eliminative arylsulfone “trigger”. The inclusion of the para-aminobenzyl spacer affords a more stable prodrug which exhibits complex drug-release kinetics due to a two-stage release mechanism. This manuscript reports the preparation and characterization of several TAK-242 prodrugs fitted with different triggers and linkers and demonstrates that these second-generation prodrugs effectively release TAK-242 while avoiding nonproductive sulfonamide hydrolysis.

Our group is exploring strategies to protect transplant tissue by the localized and sustained delivery of small-molecule immunomodulators. The delivery of the potent toll-like receptor 4 (TLR4) signaling inhibitor TAK-242 (1, Figure 1)¹ is of particular interest, as adverse effects from the early post-transplantation activation of TLR4 have been demonstrated in a wide range of transplants including kidney,² heart,³ lung,⁴ bone,⁵ islets,⁶ liver,⁷ and stem cell.⁸ Using a murine model of pancreatic islet transplantation, we demonstrated that free TAK-242 reduced sterile inflammation-mediated stress in islets during isolation and promoted successful transplant outcomes.⁹ In order to evaluate advantages of localized delivery, we developed a TAK-242 prodrug (2)¹⁰ that featured an arylsulfone β-elimination release mechanism that has been extensively exploited by Santi et al. for the delivery of amine-linked prodrugs (and the dissolution of hydrogels) with tunable half-lives.¹¹ A bioorthogonal strain-promoted alkyne/azide “click” reaction¹² was used ex vivo to conjugate this prodrug (2) to the surface of murine pancreatic islets that had been acylated with a dibenzocyclooctyne N-hydroxysuccinimide ester. The prodrug-modified islets thus prepared remained viable and functional and slowly released active TAK-242 to the local environment after transplantation (Scheme 1), suppressing innate inflammation and significantly improving transplantation outcomes.¹⁰

Figure 1.

Sulfonamide toll-like receptor 4 inhibitor TAK-242 (1) and our previously reported prodrug (2).

Scheme 1. Pancreatic Islets Covalently Modified with Prodrug 2 Slowly Release Active TAK-242 after Transplantation.

While those studies provided proof of principle for the potential of functionalized drug-eluting transplants, the efficiency of the first generation prodrug design was compromised by an undesirable decomposition of prodrug 2 (Scheme 2). Our work with TAK-242 has revealed that while the TAK-242 sulfonamide bond is relatively stable, introducing a carbamate linker results in a significant destabilization of the sulfonamide bond. Fortunately, our recent studies of TAK-242 prodrugs activated by enzymatic or Pd(II) catalysis¹³ revealed that this sulfonamide drug is a reasonable leaving group and can be directly released from sp³ carbons (Scheme 3A). While the 1,6-elimination of TAK-242 from the p-aminobenzyl group was sluggish, the benzyl substituent did not activate the sulfonamide to hydrolysis, and so we chose to utilize this strategy for the development of a “generation 2” TAK-242 prodrug 4 for bioconjugation to transplant tissue (Scheme 3B).

Scheme 2. In Buffer, Prodrug 2 Undergoes Unproductive Sulfonamide Hydrolysis That Competes with the Desired β-Elimination and Drug Release.

Scheme 3. (A) Recently Reported Nitrobenzyl Prodrug (3) Releases Free TAK-242 by 1,6-Elimination after Reduction of Nitro Group and (B) Prodrug Design Reported Herein (4) Involves β-Elimination To Form Intermediate 5 Which Subsequently Undergoes 1,6-Elimination To Release TAK-242.

Compound 4 is a modular prodrug design comprised of a tether R (typically terminated with an azide group for click conjugation), an arylsulfone substituted carbamate trigger that deactivates the aniline toward 1,6-elimination, a self-immolative aniline spacer, and the sulfonamide drug. Two sequential reactions are required for the release of free TAK-242 from 4: a sulfone β-elimination affording an aniline (that remains connected to the tether), followed by a 1,6-elimination of the revealed aniline to release the active drug. For ease of synthesis we chose to adjust the drug release kinetics by incorporating a variety of aryl groups into the β-eliminative “trigger”, as these reactions are well characterized and can provide a wide range of reaction rates. This design allowed the use of simple arylthio-substituted ethanols as precursors to the trigger component, with oxidation to the sulfone delayed until the desired compound is assembled, avoiding premature β-elimination. Accordingly, we first synthesized a series of hydroxyethylaryl sulfides in good yield (Scheme 4). These compounds incorporated variously substituted aryl groups (and an ethylene that can be deuterated¹⁴), providing a range of rates for the β-eliminative trigger removal.

Scheme 4. Preparation of Trigger Alcohols with Various Aryl Substituents.

7a, Ar = C₆H₅- (84%); 7b, Ar = 4-CF₃C₆H₄- (81%); 7c, Ar = 4-ClC₆H₄- (98%); 7d, Ar = 4-CH₃C₆H₄- (91%); 7e, Ar = 4-CH₃OC₆H₄- (95%); 7f, Ar = 2,5-(CH₃)₂C₆H₃- (96%); 7g, Ar = 4-CH₃C₆H₄-, d₄, (84%); 7h, Ar = 4-CH₃OC₆H₄-, d₄, (72%).

With the trigger alcohols in hand, the prodrug core was assembled by adding azide-terminated linkers and the various trigger alcohols to a protected 4-aminbenzyl alcohol core. In one approach (Scheme 5A), aniline 9 was alkylated with 6-azidohexyl bromide, and the resulting amine 10 is treated with crude preformed chloroformates 8, derived from the various trigger alcohols, to afford compounds 12. In a second, less convergent (but higher-yielding) approach (Scheme 5B), anilines 9 were first converted to carbamates 11 by reaction with crude chloroformates 8. The resulting carbamates 11 were efficiently alkylated with either a simple alkyl tether or a more hydrophilic tether derived from tetraethylene gylcol. Overall, 12 prodrug core compounds 12 (alkyl tether) and 12′ (hydrophilic tether) were prepared. In preparation for preliminary studies of the sulfone elimination kinetics, the silyl protecting groups of 12 and 12′ were removed (either AcOH or TBAF) and the sulfides oxidized to produce 12 sulfones 13 and 13′.

Scheme 5. Assembly of Prodrug Core Incorporating Trigger Sulfones (Overall Yields for Four Steps).

R₁ = N₃(CH₂)₆-, R₂ = N₃(CH₂CH₂O)₃CH₂CH₂-; 13a (Ar = C₆H₅-) route A 19%, route B 29%; 13b (Ar = 4-CF₃C₆H₄-) route A 7%; 13c (Ar = 4-ClC₆H₄-) route A 15%; 13′c (Ar = 4-ClC₆H₄-) route B 40%; 13d (Ar = 4-CH₃C₆H₄-) route A 8%, route B 18%; 13′d (Ar = 4-CH₃C₆H₄-) route B (22%); 13e (Ar = 4-CH₃OC₆H₄-) route A (19%); 13′e (Ar = 4-CH₃OC₆H₄-) route B (53%); 13f (Ar = 2,5-(CH₃)₂C₆H₃-) route A (21%); 13′g (Ar = CH₃C₆H₄-, d₄) route B (59%); 13h (Ar = CH₃OC₆H₄-, d₄) route A (2%); 13′h (Ar = CH₃OC₆H₄-, d₄) route B (49%).

The first stage of drug release (β-elimination of the trigger sulfones to afford the parent aniline) was initially evaluated using the simple benzyl alcohols 13 (Scheme 6). Accordingly, compounds 13a–f,h were incubated at 37 °C in a 9:1 mixture of phosphate buffered saline (PBS; pH 7.4) and DMSO, and the sulfone elimination was monitored by HPLC. As anticipated, simple changes to the aryl substituents provided a broad range of trigger half-lives (Supporting Information Figure S1). Unfortunately, attempts to characterize drug release kinetics for the completed prodrugs 4 were problematic due to poor solubility. We therefore pivoted to the preparation and evaluation of compounds with a more hydrophilic tether, and four intermediates (13′c–e,h), providing a range of trigger half-lives, were selected for characterization. Incubation of these compounds (20% DMSO in PBS pH 7.4, 37 °C) revealed trigger half-lives ranging from 1.4 to 38.5 h (Scheme 6).

Scheme 6. Kinetics of Trigger Release from Oxidized Precursors 13′ (R = N₃(CH₂CH₂O)₃CH₂CH₂-).

PBS (pH 7.4) with 20% DMSO at 37 °C. Data for compounds 13 (R = N₃(CH₂)₆-) in Supporting Information Figure S1.

With the prodrug cores and trigger kinetics in hand, full TAK-242 prodrugs were prepared (Scheme 7). Starting with a selection of eight compounds 12 and 12′, benzyl alcohol protecting groups were removed and the resulting alcohols converted to bromides by treatment with PBr₃. These crude benzyl bromides were reacted with the racemic sulfonamide drug racTAK-242 under weakly basic conditions, and the trigger sulfides were oxidized to sulfones in a final step. This oxidation was saved for last in order to avoid premature β-elimination of the trigger moieties. Eight representative racTAK-242 prodrugs 4a–d and 4′c–e,h were prepared by this route in reasonable yields from the prodrug cores, providing candidates with a range of trigger half-lives as well as containing tethers of varying hydrophilicity (Scheme 7).

Scheme 7. Loading of TAK-242 on Prodrug Cores 12 and 12′.

As noted above, while the limited aqueous solubility of prodrugs 4 precluded an effective analysis of the overall kinetics of drug release, the more hydrophilic prodrugs 4′ could be thoroughly evaluated in buffer containing 20% DMSO. Under these conditions compounds 4′ and all intermediates and products remained soluble over the course of the HPLC analysis. This allowed both the initial trigger removal (forming intermediate 5′) and the subsequent 1,6-elimination (releasing free racTAK-242) to be monitored (Scheme 8). Compound 4′c exhibited a rapid loss of the starting prodrug (anticipated trigger half-life of ∼1.4 h) with a rapid accumulation of the aniline intermediate 5′, followed by a slow 1,6-elimination releasing free TAK-242 (rac-1). On the other hand, the less reactive prodrug 4′h (anticipated trigger half-life of ∼38.5 h) slowly built up and sustained an accumulation of the aniline intermediate 5′ which steadily released the free drug. Compounds 4′d and 4′e provided intermediate drug release rates.

Scheme 8. HPLC Analysis of Kinetics of Two-Step TAK-242 Release in PBS (pH 7.4) with 20% DMSO at 37 °C (R = N₃(CH₂CH₂O)₃CH₂CH₂-).

Overall, we were pleased to find that these new prodrugs functioned as desired; they released TAK-242 cleanly, with no sulfonamide hydrolysis or other side reactions observed in the kinetics experiments. Additionally, while the two-step kinetics were more complex, drug-release could be modulated by selecting different β-elimination triggers, providing a range of half-lives. Further tuning of release kinetics could potentially be approached by modifying the aniline linker to adjust the 1,6-elimination kinetics. For bioconjugation applications, the tether linkage to the aniline core ensures that the prodrug remains immobilized until final release of the active component. Current studies are examining the use of these novel prodrugs in various transplantation models, optimizing the prodrug solubility, and evaluating this strategy for the development of slow-release TAK-242 derivatives suitable for systemic application.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.2c00492.

• Full experimental details for the synthesis of compounds 4–16 and HPLC analyses of prodrug hydrolysis and release of parent drug; preparation of several related compounds; pH studies of β-elimination for 4′d (PDF)

Author Contributions

J.H.K. synthesized and characterized the prodrugs, performed the kinetic analyses, and produced the manuscript. R.R.K. guided the experimentation and edited the manuscript. A.T.L., C.A.S., and M.R.S. assisted with compound syntheses and characterizations. All authors have given approval to the final version of the manuscript.

Research reported in this publication was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIH) under Award R15DK127340.

The authors declare no competing financial interest.