
==== Front
J Org Chem
J Org Chem
jo
joceah
The Journal of Organic Chemistry
0022-3263
1520-6904
American Chemical Society

39178144
10.1021/acs.joc.4c01485
Note
Catalyst-Free, Three-Component Synthesis of Amidinomaleimides
https://orcid.org/0009-0006-3166-8224
Swift-Ramirez Wyatt R. ∥
https://orcid.org/0009-0001-1925-7362
Whalen Lindsay A. ∥†
Thompson Lia K. ∥†
Shoemaker Kaylee E. ∥†
Rubio Aris V. ∥
https://orcid.org/0000-0003-0296-9846
Weiss Gregory A. *∥‡§
∥ Department of Chemistry, University of California, Irvine, 1102 Natural Sciences 2, Irvine, California 92697-2025, United States
‡ Department of Molecular Biology and Biochemistry, University of California, Irvine, 3205 McGaugh Hall, Irvine, California 92697-3900, United States
§ Department of Pharmaceutical Sciences, University of California, Irvine, 856 Health Sciences Road, Suite 5400, Irvine, California 92697-3958, United States
* gweiss@uci.edu
23 08 2024
20 09 2024
89 18 1375613761
12 06 2024
19 08 2024
14 08 2024
© 2024 The Authors. Published by American Chemical Society
2024
The Authors
https://creativecommons.org/licenses/by-nc-nd/4.0/ Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/).

Maleimide and amidine functionalities often appear in medicinal and natural product targets. We describe a catalyst-free, three-component coupling reaction for the synthesis of amidinomaleimides. This one-pot reaction fuses a broad range of secondary amines and aldehydes with azidomaleimides. The conditions are mild, simple, modular, high yielding, and amenable to aqueous solvents. Most reaction products can be sufficiently purified without column chromatography. The synthesis creates complex, multifunctional molecules with four different molecules, including a tripeptide, arrayed around an amidinomaleimide core.

U.S. Department of Health and Human Services 10.13039/100000054 2 P30 CA 062203-20 University of California 10.13039/100009819 NA U.S. Department of Energy 10.13039/100006133 DE-AR0001508 document-id-old-9jo4c01485
document-id-new-14jo4c01485
ccc-price
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pmcCompounds with amidine functionalities have diverse biological activities including antibacterial,1,2 anti-inflammatory, analgesic,3,4 and antiplatelet.5,6 Thus, amidines attract significant interest from synthetic chemists.7 Despite their pharmacological applications and convergent syntheses, most amidine forming reactions require metal catalysts (e.g., Scheme 1A).8−11 Recent work focuses on removing metal catalysts12−14 to improve green metrics and simplify their uses in pharmaceutical synthesis.15−19

Scheme 1 Prior Examples of (A) Metal-Catalyzed and (B) Metal-Free Amidine Syntheses; (C) Metal-Free, One-Pot Synthesis of Amidinomaleimides (This Work)

The prior reports of catalyst-free amidine syntheses require high temperatures or strongly electrophilic azides. In 2022, for example, Milo and co-workers described a catalyst-free amidine synthesis using sulfonyl azides, anilines, and ketones; their conditions required high temperatures and molecular sieves to avoid catalytic metals (Scheme 1B.1).12 Houk, Yan, and co-workers also reported a catalyst-free synthesis of amidines by combining highly electron deficient perfluoroaryl azides with aldehydes and secondary amines (Scheme 1B.2). In their experiments, the electrophilic perfluoroaryl group had sufficiently activated the azide as a substitute for metal catalysis.13 Though these examples eliminate metal-based catalysts, their limitations include high temperatures or a restricted scope due to starting material requirements (e.g., perfluoroaryl azides; Scheme 1B.2). To expand the scope of metal-free synthesis of amidines, we envisioned a three-component reaction utilizing azidomaleimides (1) as a 1,3-dipole reacting with enamines, formed in situ from aldehydes (2) and secondary amines (3) (Scheme 1C).

Maleimides are also prevalent in natural products,20−22 medicinal chemistry syntheses, and biological therapeutics.23−25 They can provide a valuable route for bioconjugation with peptides and proteins through the well-established thiol-maleimide click reaction.26 Subsequent investigations have uncovered the reversibility of the resultant thiol-maleimide bioconjugate in serum, prompting the development of new maleimide variants sometimes referred to as next generation maleimides.27−30

Here we explore the reactions of azidomaleimides (1) with aldehydes (2) and secondary amines (3) to yield amidinomaleimide products in a one-pot, one-step synthesis. We present optimized conditions (Table 1, entries 4 and 7) and substrate scope (Table 2) including the yields for each substrate relative to the azidomaleimide (1).

Table 1 Optimization of the Catalyst-Free, Three-Component Synthesis of Amidinomaleimidesa

Entry	Solvent	Temp (°C)	Time (h)	% Yield	
1b	MeOH/EtOAc	25	18	75	
2	MeOH/ACN	40	18	76	
3	MeOH/DCM	40	18	79	
4	MeOH/EtOAc	40	18	88	
5	MeOH/EtOAc	40	2	72	
6	MeOH/EtOAc	50	1	77	
7	MeOH/EtOAc	60	0.5	88	
a Conditions: 1a (0.11 mmol), 2a (1.25 equiv), 3a (1.25 equiv) in MeOH (100 μL)/cosolvent (250 μL) mixture.

b 1a (0.11 mmol), 2a (1.125 equiv), 3a (1.125 equiv) in a MeOH (1.25 mL)/EtOAc (1.25 mL) mixture.

Table 2 Substrate Scope of Secondary Amines, Aldehydes, and Maleimides for Amidinomaleimide Formationa,b,c

a Color coding indicates divergent starting materials for amidinomaleimide formation from 4a: aldehydes (blue) or secondary amines (red). Prior to this reaction, the imide starting material can be formed from various primary amines (pink).

b Reaction conditions: 1 (0.11 mmol), 2 (1.25 equiv), 3 (1.25 equiv) in MeOH (100 μL)/EtOAc (250 μL) mixture at 40 °C for 18 h.

c Isolated yield.

d 60 °C over 30 min was employed.

e Postpreparatory TLC yield.

f 1a (2 equiv), 2a (2 equiv), PGP (0.05 mmol) in MeOH (100 μL)/DMSO (min.) mixture at 40 °C for 18 h. Cleaved with HFIP (3.5 mL), DCM (11.5 mL) for 1 h.

g Percent conversion of PGP to 4r determined by LC-MS.

As reviewed above, catalyst-free amidine synthesis can be achieved by installing electron withdrawing groups adjacent to azides. Such modifications can activate the azide by lowering the energy of its LUMO.14,31,32 We speculated that an azidomaleimide could also serve as an electrophilic azide in this reaction. Specifically, the resonance structures of azidomaleimides (Scheme 2) could encourage the azide-enamine cycloaddition by withdrawing electron density from the azide to the maleimide. Resonance structure 7 does not participate directly in this reaction but increases the electrophilicity of the azide’s terminal nitrogen and encourages the reactive resonance structure 6.

Scheme 2 Azide Resonance Structures Driving Amidine Formation

A mechanism (Scheme 3) for this one-pot, enamine-azide cycloaddition is based upon similar reactions applying a concerted mechanism.14,32 After the formation of the enamine338, the electrophilic azide 1 can proceed through a 1,3 dipolar cycloaddition, forming the triazoline intermediate 9. Next, a ring-opening reaction of the triazoline can create amidine product 4. The expulsion of nitrogen gas likely drives the reaction to completion.

Scheme 3 Proposed Mechanism for One-Pot Multicomponent Amidine Formation

The 1H NMR spectra of the reaction products (4a through 4q) display homogeneous signals for the protons arrayed around the amidine product. This observation suggests that the reaction is stereoselective in its formation of its E/Z stereoisomer. Computational calculations, using Spartan’24, of the enthalpy (ΔH) for the two possible stereoisomers of 4a offer an explanation. The Z stereoisomer was determined to be 3.17 kcal/mol higher in energy than the E stereoisomer (Figure S1). This energy difference is likely due to the increased strain energy between the morpholine ring and the imide carbonyl. Therefore, we believe that the E amidinomaleimide is preferentially formed.

To optimize this reaction pathway, we first developed a facile route to the metal-free synthesis of azidomaleimide (1) (Scheme 4). Bromomethyl maleimides were synthesized through a previously reported route.34 Then, this component was readily converted to the desired azide in one step in good yields. The resulting material was pure following extraction, as determined by NMR, and was used directly in the following synthesis. Overall yields for the three steps to construct the azidomaleimide core ranged between 32% and 49%.

Scheme 4 Synthesis of 2-Azido-3-methyl-maleimides

We next investigated the reaction conditions for the amidine forming reaction involving 3-azido-4-methyl maleimide (1a), morpholine (2a), and butyraldehyde (3a) to yield amidine 4a (Table 1). Starting with the conditions for the similar reaction shown in Scheme 1B.2,13 we obtained a 75% yield of the amidine product after 18 h (entry 1). We next varied the substrate concentrations, solvents, and temperatures. As suggested by prior reports,13 acetone was initially chosen as a cosolvent; however, this solvent reacts with the enamine intermediate (6) creating an aldol-like byproduct along with the desired product, as confirmed by MS (Figure S2). Polar aprotic solvents yielded good results; EtOAc provided the highest yield (entry 4). The rate of the reaction, as expected, increased with the temperature (entries 5–7). Running the reaction at 60 °C for 30 min (entry 7) or at 40 °C overnight (entry 4) resulted in similar yields. Thus, conducting the reaction at 40 °C overnight (entry 4) yielded the best results while maintaining mild conditions. This procedure was used on a 2 mmol scale, resulting in an 81% yield of the amidine product.

To isolate this amidine product effectively, a two-stage extraction first utilized a conventional ethyl acetate–water extraction followed by washes with brine. Next, the organic layer was dried under a vacuum before being dissolved in acetonitrile. This solution was then washed with hexanes, which were discarded, before drying the acetonitrile layer to yield the final product. The first extraction removes excess amine, and the second extraction then eliminates the excess enamine byproduct and remaining aldehyde starting material. This two-stage extraction obviated the need for column chromatography for approximately 80% of the compounds described here, as assessed by NMR. However, some aldehydes have solubilities similar to the desired product in acetonitrile (Table 2, 4e, 4g, 4h); these products could not be purified via this extraction method.

For the handful of amidinomaleimides resistant to extraction techniques, flash chromatography adversely affected isolated yields. Specifically, flash chromatography led to the formation of a heterogeneous mixture of maleimide ring-opened products, preventing their ready isolation. Neutralizing the silica gel with a base proved unsuccessful. We hypothesize that the mild Lewis acidity of silica gel can activate the imide and encourage its hydrolysis. This degradation unexpectedly complicates attempts to isolate the desired product for compounds resistant to the two-stage extraction method. For these compounds, preparatory thin-layer chromatography (TLC) was used successfully for isolation of product, though with a decrease in product yield (13–18% recovered). Notably, in the absence of Bronsted or Lewis acids, the reported amidinomaleimide (4a) appears quite stable. When stored at 25 °C as a solution in DMSO, the compound showed no significant decay after 4 months, as determined by NMR (Figure S3).

Precedent from reactions involving electrophilic azides suggested that greater electron withdrawing potential conjugated to the azide could lead to higher yields and accelerated reactions.14 However, when electron-deficient, aniline-based azidomaleimides (1b, 1c) were used in the optimized conditions (Table 1, entry 4), the resulting products (4b, 4c) exhibited greater heterogeneity. A shorter time frame (Table 1, entry 7) increased product homogeneity, albeit with lower yields (54–58%). When dinitroaniline (Table 3) was used, the amidine product could not be isolated. We suspect that strongly electron withdrawing anilines increase the maleimide’s susceptibility to undergo hydrolysis. Under longer reaction conditions, the maleimide is more prone to hydrolysis and creates a heterogeneous product mixture which cannot be separated by extraction. Nonaromatic maleimides also readily undergo this coupling, offering additional functionality at the imide site. For example, an azidomaleimide with an ester (1d) was combined with the other reagents to form the desired product (4d) in good yields (86%). Such maleimides could allow for further modification through transesterification or other reactions.35

Table 3 Incompatible Substrates for Amidinomaleimide Formationa

a Color coding as shown in Table 2. N.d. = Not detected. Yield approximated as only detected by HRMS.

The investigation of the substrate scope (Table 2) revealed significant trends for reaction preferences. Moderate to good yields of amidines (62–66%) were obtained from sterically hindered and bulky aldehydes (f-i). Separately, the β-ether substrate (Table 3) does not result in the desired amidine product. This aldehyde forms a vinyl ether-based enamine, which can form resonance structures in conflict with the enamine, reducing its dipolarophile quality.

A wide range of secondary amines can readily participate in this reaction. For example, heterocyclic secondary amines exhibit good yields (68–89%). Furthermore, secondary amines, including sterically bulky groups and reactive functionalities (4j-4m, 4q, 4r), are tolerated. However, unprotected carboxylic acids (Table 3) failed to form the desired product. This result is not unexpected due to the product’s sensitivity to acid. The reaction worked well when applied to acyclic secondary amines (4n–p), as indicated by the good yields achieved (80–84%). Inspired by previous reports,13 attempts to replace the secondary amine with anilines (Table 3) were unsuccessful. The reaction yielded minimal amidine product (<1%, detected by HRMS). Though anilines can form enamines, the electron withdrawing by the aromatic ring decreases the nucleophilicity of the aniline-based enamine.36

Given the success of pyrrolidine as a substrate (4q), we investigated the three-component reaction with a larger tripeptide. Thus, a peptide with an N-terminal proline was next tested for compatibility with the amidine forming reaction. The Pro-Gly-Pro (PGP) tripeptide was synthesized via SPPS.37 The resin-bound tripeptide was suspended in ethyl acetate and subsequently reacted with 1a and butyraldehyde to yield 4r. The product was cleaved using a cleavage solution of 1,1,1,3,3,3-hexafluoroisopropanol in DCM to avoid degradation in TFA; percent conversion was quantified using LC-MS analysis (Figure S4) revealing between 87% to 91% conversion of PGP to 4r given excess azidomaleimide (1a) and butyraldehyde.

This reaction platform offers both disadvantages and advantages for the synthesis of complex maleimides. Preliminarily, the amidinomaleimides presented here do not appear compatible with the thiol-maleimide click reaction (data not shown) as precedented with disubstituted maleimides.29 However, this convergent reaction can provide improved yields for trifunctionalized maleimides compared to linear, synthetic routes.38 The most challenging component from a synthetic and safety standpoint, azide 1, appears on a central, generalizable core, which can be readily synthesized in high yields (Scheme 4). Large quantities (g) of the core azide can be made in advance from relatively inexpensive starting materials. The required functionalities for components joined in the reaction (i.e., secondary amines and aldehydes) can typically be accessed with minimal effort.

The results reported here show generality for the multicomponent coupling of up to four different molecules (R1 through R4) in one step. The maleimide could tolerate diverse primary amines in the R1 position. The R2 position becomes set by the aldehyde; quite a few aldehydes, including bulky examples, work in this reaction. The tolerance for asymmetric secondary amines, including sterically hindered amines, delivers two more molecules (R3 and R4) in one convergent step. Thus, the resultant amidinomaleimides can be densely arrayed and rich in functional capabilities.

Data Availability Statement

The data underlying this study are available in the published article and its Supporting Information.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.joc.4c01485.Methodology, compound characterization, including 1H NMR and 13C NMR, and Supplemental Data are included. (PDF)

FAIR data, including the primary NMR FID files, for compounds 1a–1d and 4a–4q (ZIP)

Supplementary Material

jo4c01485_si_001.zip

jo4c01485_si_002.pdf

Author Contributions

† L.A.W., L.K.T., and K.E.S. are equal contributors.

The authors declare no competing financial interest.

Acknowledgments

We gratefully acknowledge the support of ARPA-E of the DOE (DE-AR0001508), an Anti-Cancer Challenge pilot project grant from the University of California, Irvine Chao Family Comprehensive Cancer Center, and the NCI of the NIH (2 P30 CA 062203-20). We wish to thank the UCI Mass Spectrometry Facility and its staff (Drs. Felix Grun and Ben Katz) for expert assistance with small molecule analyses. We thank Prof. Scott Rychnovsky for helpful conversations and computational modeling.
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