Thermal Stability and Utility of Dienes as Protecting Groups for Acrylamides

Abstract

Acrylamides are privileged electrophiles used in targeted covalent therapies, often installed at the end of a synthetic sequence due to their reactive nature. Herein, we report several diene-acrylamide adducts with a range of thermal stabilities toward retro-Diels–Alder deprotection of the acrylamide, enabling this masked functionality to be introduced early in a synthetic route and deprotected in a specific temperature range. Through kinetic studies, we identify solvent and structural trends that impact the stability of trimethylsilyl cyclopentadiene (TMS-CP) acrylamide adducts. TMS-CP protected acrylamides were installed on several amine-containing drugs, whose acrylamides were thermally unveiled (T = 160 °C, time ≤ 1 h) in moderate to high yields. We also showcase the potential utility of this protection strategy by improving the yield of a base-promoted S_NAr reaction when the acrylamide is masked.

Small molecules which covalently bind to biological targets are an important therapeutic class. Within the past 15 years, there has been an increase in the number of drug discovery programs rationally targeting a specific amino acid for covalent modification.¹ Many amino acids have been targeted in chemical biology and medicinal chemistry applications, with cysteine being the most common residue due to its high and unique nucleophilicity.² Various electrophilic warheads have been developed to selectively react with cysteine;³ however, α,β-unsaturated amides are the most commonly employed electrophile to target noncatalytic cysteine residues.⁴ Acrylamides, such as those found in Zanubrutinib 1, Osimertinib 2, and Dacomitinib 3 (Figure 1), have had great clinical success in recent years as cysteine targeting warheads.⁵

Figure 1.

Examples of acrylamide-containing FDA-approved drugs.

There is a large body of literature precedent for the protection of electron deficient alkenes, such as maleimides, α,β-unsaturated ketones, and α,β-unsaturated esters, as thermally reversible retro-Diels–Alder (DA) adducts.^6,7 There are fewer examples of such a strategy being used to mask α,β-unsaturated amides.⁸⁻¹⁰ Though less reactive than their keto or ester counterparts, acrylamides can react via undesired pathways depending on the reaction conditions as well as their electronic and steric environments. Basic and/or nucleophilic conditions such as those in nucleophilic aromatic substitution or palladium mediated cross couplings can be particularly problematic for acrylamides.¹¹⁻¹³ Most often, this motif is installed at the end of a synthetic sequence to avoid these problems. Notably, there are several examples where a β-chloro or sulfonyl propionamide 4 serves as a masked acrylamide, and the olefin 5 is generated by treatment with strong base (Figure 2A).^12,14 To our knowledge, there is a single report disclosing a Diels–Alder adduct as a masked acrylamide in a medicinal chemistry setting.¹⁵ As shown in Figure 2B, a patent application from Sandoz Pharmaceuticals describes a synthesis of Ibrutinib 7, in which the acrylamide is protected as the cyclopentadiene (CP) adduct 6. In the final step, the acrylamide is unveiled by heating to 250 °C in diphenyl ether.

Figure 2.

Protecting group strategies for acrylamides.

While the protecting group strategies described above will have settings in which they work, there is a need to develop other protecting groups for acrylamides to address the unique challenges faced by each medicinal chemistry team. As such, it would be advantageous to have a protecting group that would unveil the acrylamide at a lower temperature. We explored the thermal stabilities of several diene-acrylamide DA adducts, enabling this sensitive functionality to be installed early in a synthesis and unmasked within a specific temperature range.

We began by synthesizing model acrylamides protected with various dienes and determining their relative thermal stabilities when heating in Dowtherm A¹⁶ (Table 1). Conversion was determined based upon the loss of starting DA-adduct rather than product formation. Conversion to the acrylamide was in some cases accompanied by the formation of unidentified side-products, possibly due to undesired reaction of the reactive acrylamide with the released diene at these elevated temperatures.

Table 1. Thermal Stabilities of Diene-Acrylamide Adducts^a in Dowtherm A^b.

^aAmide adducts were synthesized through HATU coupling of 4-phenylpiperidine with racemic diene-acrylic acid adducts.

^bAmide adducts (0.03 mmol) and an inert internal standard (0.03 mmol) were diluted in Dowtherm A (0.4 mL) in a vial under a slight positive pressure of nitrogen equipped with a vent needle. The mixtures were heated to the shown temperature for 30 min. Conversion was determined by LC analysis based upon the loss of starting material relative to internal standard.

^cFor compounds whose diene was not volatile at the shown temperature, and there was no significant loss of starting material, the reaction was run a second time in the presence of a nucleophilic scavenger and loss of starting material was quantified, in case the forward Diels–Alder was confounding the results (see the SI for details).

^dFor compounds whose Diels–Alder adduct eluted at the same retention time as the acrylamide, conversion was quantified by NMR.

As expected, CP adduct 9 was very stable, yielding 21% conversion at 200 °C. While kinetically less stable, anthracene is not volatile and we observed no significant product formation upon heating 10 in Dowtherm A. The retro-DA was inferred by adding a nucleophilic scavenger and looking for the disappearance of starting material. Exo and endo fulvene adducts 11 and 12 were significantly less stable, giving conversions of 15% and 18%, respectively, at 120 °C. When furan was used as a masking group, the adducts showed conversion at 80 °C (14 and 15). Dimethyl substituted furan adduct 16 was the least stable of all substrates, with 28% conversion at 80 °C. There are a range of thermal stabilities that can be achieved for the protected acrylamides through selection of the diene. The desired stability range can be fine-tuned through selection of the endo or exo adduct or by placing substituents at the bridgeheads of the adducts.¹⁷

While the thermal stability ranges of both the fulvene and furan adducts would be useful for protecting some acrylamides, we were interested in developing adducts with good stability up to 100 °C. This would allow the adduct to stay intact under many standard chemical transformations involving elevated temperatures. Toward that goal, we investigated trimethylsilyl-cyclopentadiene (TMS-CP), an alkene protecting group first introduced by the Magnus laboratory in their synthesis of aspidosperma-type indole alkaloids.^18,19 Relative to unsubstituted CP, adducts with TMS-CP will more readily undergo a retro-DA reaction due to a well oriented σ_C–Si → σ*_C–C donation into the breaking σ-bonds of the DA adduct.²⁰ TMS-CP adduct 13 was more stable than the fulvene substrates, as evidenced by the reduced conversion at 120 and 160 °C. Based on the preliminary ranking of stabilities of adducts in Table 1, we wanted to more closely investigate the deprotection kinetics of TMS-CP adducts.

We synthesized several sterically and electronically differentiated masked acrylamides and determined their stabilities through ¹H NMR kinetic experiments (Table 2). There are several points that merit discussion. Most importantly, adducts displayed good to excellent stabilities at 100 °C in d₇-DMF (t_1/2 = 28–140 h). As expected, the secondary and primary aliphatic amine derived endo adducts (13 and 17, respectively) were more stable than the relatively more electron deficient aniline derived endo adducts (18 and 19).²¹ Based on our observations from Table 1, we synthesized the exo adducts (20 and 21) of our less stable aniline derived substrates and determined that the half-life can be modestly increased. Secondary amide substrates were more stable than tertiary amide substrates. Finally, though the effects were minor and did not hold true for all substrates, these protected acrylamides were generally most stable in the nonpolar o-xylene.^7a,22 Armed with a better understanding of the factors influencing the stability of these adducts, we wanted to verify these deprotections would proceed on more medicinally relevant molecules.

Table 2. Thermal Stabilities of Diene-Acrylamide Adducts in Dowtherm A^a.

^aAmide adducts, thiophenol (3 equiv), tributylamine (3.3 equiv), and dibenzylether (1 equiv, internal standard) were dissolved in the indicated deuterated solvent and placed in a preheated NMR spectrometer. ¹H spectra were collected at regular time intervals, and half-lives were calculated based upon the first order loss of starting material.

Several TMS-CP-protected acrylamides were installed on marketed drugs bearing aliphatic amines which were then heated to unveil the resulting acrylamides (Scheme 1). We reasoned that since these adducts were generally more stable in o-xylene, the deprotections would be faster in more polar 1,2,4-trichlorobenzene.²³ We obtained reasonable yields for many of the initially tested substrates; however, the electron deficient Sitagliptin analogue 29 was produced in only 32% yield. To improve the yield for the deprotection of Sitagliptin adduct 29 and other substrates, we performed a short optimization study using the more electron deficient aniline derived 18 (Table 3).

Scheme 1. Substrate Scope^,

Adducts were synthesized through HATU coupling of amines with racemic TMS-CP Diels–Alder adduct (see the SI for details).

Adducts (0.025 M) were heated to 160 °C in Dowtherm A in a vial under a slight positive pressure of nitrogen equipped with a vent needle. Isolated yields after purification are reported.

Reactions were run in 1,2,4-trichlorobenzene for 12 h.

Table 3. Optimization of Retro-Diels–Alder.

solvent	time (min)	conc (M)	relative conversiona	yieldb
1,2,4-trichlorobenzene	90	0.05	1	N/D
	30	0.05	1.21	N/D
	30	0.025	1.27	N/D
Dowtherm A	30	0.025	1.41	74%
	30	0.1	N/D	69%

^a18 was diluted in the indicated solvent in a vial under a slight positive pressure of nitrogen equipped with a vent needle. The solutions were heated to 160 °C for the indicated reaction time. An internal standard was added, and the amount of product formed, relative to the internal standard, was determined by LC analysis. Relative conversion is defined as the ratio of product to internal standard, divided by that ratio for the first table entry.

^bReactions were performed on a 1 mmol scale.

When adduct 18 was heated in trichlorobenzene for 90 min, LCMS analysis of the mixture showed product acrylamide 32 as well as several overlapping peaks with the same mass and approximate retention time of the starting material. We believe that these peaks correspond to DA isomers in which the TMS group is not at its original position and thus would not have the same rate enhancement for the retro-DA reaction that the parent isomer does.²⁴ We speculate that the unproductive forward-DA reaction with more electron deficient acrylamides is competitive with the evaporation of TMS-CP under these reaction conditions.²⁵ Based upon our kinetic studies, we thought the retro-DA would achieve near full conversion sooner than 90 min and prolonged reaction times would not be beneficial. This was borne out experimentally, as the relative formation of 32 increased when the reaction was run for 30 min. There was a small increase in product formation running the reaction under more dilute conditions. Switching the solvent from trichlorobenzene back to Dowtherm A gave an increase in the amount of acrylamide generated. Though we do not fully understand the reasons why this switch improved our relative conversions, decreasing the amount of byproducts isomeric to the starting material, we were happy to exploit these findings for our deprotections. When run on the 1 mmol scale using optimized conditions, we obtained an isolated yield of 74%. Lastly, it is worth noting that this deprotection was run at a higher concentration (0.1 M) on the 1 mmol scale, giving only a minor decrease in yield.

When subjecting previous substrates to our newly optimized conditions, we found yields to improve overall. Most notably, the yield to unveil Sitagliptin acrylamide 29 improved from 32% to 89%. We also examined these deprotections on two aniline containing drugs. While the deprotection of a lenalidomide derivative gave a reasonable yield of acrylamide 30, sulfamethoxazole acrylamide 31 was produced in just 33%.²⁶ Thus, this strategy will not be as effective for very electron deficient anilinic acrylamides.

To demonstrate the utility and advantage of this protecting group strategy, we synthesized both protected and deprotected acrylamides, 33 and 34 respectively, and carried out a base-mediated S_NAr (Scheme 2). When we used 2 equiv of amine 35, unprotected acrylamide 34 underwent the desired displacement to give acrylamide 37 in only 4%, in contrast to the 77% yield for protected 36. While the yield could be improved by using 1.1 equiv of amine 35, there will be cases in which the desired reaction will be unable to outcompete side reactivity with the acrylamide, highlighting the need for a protection strategy. Since Boc protecting groups are known to be thermally labile and DA adducts are prone to retro-DA under acidic conditions, we demonstrated that each could be selectively deprotected in the presence of the other giving 38 and 37, respectively.

Scheme 2. Synthetic Utility of TMS-CP Protecting Group.

Reaction employed 1.1 equiv of N-Boc-piperazine (35).

Reaction employed 2.0 equiv of N-Boc-piperazine (35).

Though an unsubstituted acrylamide is often the targeted warhead, substitutions on the acrylamide are frequently explored. For example, a β-methyleneamine substitution is found in the marketed EGFR, covalent inhibitors Afatinib,²⁷ Dacomitinib 3,^5b and Neratinib.²⁸ We wanted to determine how this substitution would affect the thermal stability of our TMS-CP protected acrylamides. As shown in Scheme 3, we synthesized the protected acrylic acid derivative 40 in 3 steps. A Lewis-acid-catalyzed DA reaction between TMS-CP and bromo-crotonate 39, followed by displacement of the bromide with piperidine and then saponification delivered protected acid 40 in 23% overall yield. Acid 40 was coupled to 4-phenylpiperidine to give the TMS-CP protected acrylamide 41. Utilizing our optimized conditions for deprotection, we were able to unveil acrylamide 42 in 75% yield. Pleasingly, substituted adduct 41 shows similar thermal stability to unsubstituted adduct 13 in d₇-DMF.

Scheme 3. Thermal Stability and Deprotection of TMS-CP-Masked, Substituted Acrylamide.

We would like to acknowledge two caveats regarding the use of these masked acrylamides. The first is that the corresponding protected acrylic acid is not commercially available. However, we were able to make endo-TMS-CP protected acrylic acid on the multigram scale in two steps from commercial materials.²⁹ The second is that we have only described the synthesis and use of the racemic carboxylic acids. While the ultimate acrylamides have no stereocenters, when coupled with chiral amines, diastereomeric products are generated. We would point out that resolutions of racemic carboxylic acids³⁰ and enantioselective DA reactions³¹ are well established in the literature and either strategy would likely be able to produce enantioenriched protected acids.

In summary, we explored the thermal stability of several diene-acrylamide adducts and determined that TMS-CP protected acrylamides are relatively stable up to 100 °C and can be deprotected in 1 h or less at 160 °C. Optimized conditions gave moderate to high yields for the deprotection of acrylamides on several complex drugs, though the yield was reduced for a very electron poor anilinic acrylamide containing substrate. We showed an example where protection of the acrylamide can lead to higher yields for an S_NAr reaction and demonstrated protecting group orthogonality to a Boc group. Finally, a TMS-CP protected acrylamide with a β-methyleneamine substitution was shown to have similar thermal stability to the analogous unsubstituted acrylamide. It is our hope that the study described herein will be useful to both the medicinal chemistry and chemical biology communities, shortening drug development timelines and improving access to acrylamide containing probes.

Glossary

Abbreviations

Boc

tert-butoxycarbonyl

CP

cyclopentadiene

DA

Diels–Alder

DCM

dichloromethane

DIPEA

N,N-diisopropylethylamine

DMF

dimethylformamide

EGFR

epidermal growth factor receptor

HATU

1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

LCMS

liquid chromatography mass spectrometry

NMR

nuclear magnetic resonance

S_NAr

nucleophilic aromatic substitution

TFA

trifluoroacetic acid

TMS-CP

trimethyl-silyl cyclopentadiene

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.2c00075.

• Detailed experimental procedures, spectroscopic data, and ¹H and ¹³C spectra (PDF)

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.