Guanidium Unmasked: Repurposing Common Amide Coupling Reagents for the Synthesis of Pentasubstituted Guanidine Bases

Abstract

Guanidines make up a class of compounds with important applications in catalysis and medicinal chemistry. In this systematic study, we report on the guanylation of aliphatic amines, anilines, (sulfon)amides, ureas, and carbamates by repurposing HATU, a common amide coupling reagent. The products are 2-substituted 1,1,3,3-tetramethylguanidines (TMGs), a group of sterically hindered superbases. The reaction of a guanidinium salt with aliphatic amines has been regarded as an unwanted side-reaction in amide coupling, yet the exact mechanistic details have been unclear. Our mechanistic investigation shows that the guanylation is highly dependent on the nature of the nitrogen nucleophile. Our findings were applied on two fronts: minimizing guanylation in competing amide coupling reactions as well as maximizing guanylation in a simple one-step synthesis of a broad variety of 2-substituted TMGs, including the late-stage functionalization of pharmaceuticals.

Introduction

The guanidine functionality is an important structural motif present in a plethora of bioactive molecules.^1,2 The moiety is a strong base, and under physiological conditions, it becomes protonated, enhancing protein-interactions and aqueous solubility. Consequently, guanidine functionalities are encountered in a variety of pharmaceuticals with distinct substitution patterns (Scheme 1A).¹⁻⁵ In freebase form, guanidines have diverse uses as superbasic catalysts and stoichiometric reagents.⁶ The guanidine substituents can be tailored to give specific reactivity (Scheme 1B).^7,8 This approach has been applied particularly to 2-substituted 1,1,3,3-tetramethylguanidines (TMGs). For example, 1,3-PhG, TMG, and tBuTMG were evaluated as catalysts in SuFEx click chemistry, but tBuTMG uniquely displayed the desired reactivity.⁹ A recent study surveyed a selection of superbase catalysts in glycidol carboxylation, with tBuTMG possessing optimal sterics and basicity.¹⁰ The sterics and basicity of TMGs play important roles in carbon dioxide-based chemistry.¹¹⁻¹³ Another important application of TMGs is within organometallic chemistry as ligands (Scheme 1C). Copper complexes have been used for enantioselective C–H functionalizations, whereas iron and zinc complexes are used in polymerizations.¹⁴⁻¹⁸

Scheme 1. Selected Examples of Important TMGs (A),¹⁻⁵ Guanidines with Modified Substituents (B),^7,8 Guanidine-based Metal Complexes (C),¹⁴⁻¹⁸ Conventional Synthesis of TMGs (D),¹⁹⁻²¹ Common Guanidinium Reagents (E), and HATU as General Guanylation Reagent (F).

Conventionally, TMGs are accessed from treatment of tetramethylurea with phosgene or oxalyl chloride, followed by addition of the desired nitrogen nucleophile (Scheme 1D).¹⁹⁻²¹ While effective, the hazardous reagents and the requirement for inert conditions limit the synthetic utility of this approach. The quest for alternative routes for guanylation is therefore an active research topic, where recent methods have focused on synthesizing mono- or disubstituted guanidines.²²⁻²⁴ In contrast, we were interested in pentasubstituted guanidines, i.e. TMGs. In search of a more benign protocol in order to overcome the challenges of the conventional phosgene-based method, we were attracted to reports from the field of peptide coupling, where TMGs have been encountered as an undesired side product in amide coupling reactions with guanidium-type coupling reagents HBTU, HATU and HCTU (Scheme 1E).²⁵ In the amide coupling process, the typical desired outcome is that the guanidinium reagent reacts with a carboxylic acid. However, highly nucleophilic amines can attack the coupling reagent directly, leading to the formation of the corresponding guanylated amine. Miller and co-workers used HBTU in the synthesis of peptide-based TMGs for catalytic applications,^14,15,26,27 and initial mechanistic studies found that aliphatic amines are guanylated faster using HATU than HBTU.^3,28 However, it remained unclear if this trend applied to other, less nucleophilic, nitrogen species. Interestingly, weakly nucleophilic sulfonamides were reported to readily undergo guanylation by HBTU.²⁹ This seemed to contradict a more recent report, which found that HATU and HBTU were unreactive toward most nucleophiles.³⁰ Despite this side reaction being well-known, we are not aware of a systematic study surveying the role of the reaction conditions in maximizing the guanylation yield. We sought to answer the question if low-reactivity nitrogen nucleophiles could be activated for guanylation by an appropriate tuning of the reaction conditions.

Herein, we report a remarkably simple procedure to access a variety of 2-substituted TMGs from nitrogen nucleophiles, including weakly nucleophilic anilines and amides, previously thought to be unreactive (Scheme 1F).³⁰ Our mechanistic results provide strategies for maximizing guanylation, as well as minimizing it in conventional amide coupling.

Results and Discussion

We began our study by choosing 4-fluoroaniline 1a as a moderately nucleophilic model substrate. The guanidium-type reagents HBTU, HCTU, and HATU were selected as the tetramethylguanidine donor. Previous literature reports, employing aliphatic amines as substrates, had indicated that HATU forms the guanylated products faster than HBTU in DMF.²⁸ Applying literature conditions, we found the reported trend to also apply for 1a (Table 1, entries 1–4). Guanylation using HATU was largely unaffected by the choice of solvent, whereas HBTU reacted much more slowly in acetonitrile (ACN). The use of triethylamine (TEA) as a base was critical, as the yields were significantly decreased in its absence. An exception to this general pattern was HATU in DMF (Table 1, entry 2), which did not seem to require the addition of base. Subsequently, we proceeded to study HCTU (entries 5 and 6), which behaved similarly to HATU (entries 1 and 2). This suggests that the superiority of HATU and HCTU over HBTU is likely electronic in origin. For a detailed list of screened conditions, including solvent screening, see Supporting Information. Next, we proceeded to further optimize the guanylation yield using HATU in ACN. Surprisingly, a catalytic amount of triethylamine (Table 1, entry 7) and a stoichiometric amount (1.0 equiv; Table 1, entry 8) gave a similar result. These results suggest that TEA has an activating effect in the reaction, possibly on nucleophile 1a. However, an excess of TEA seemed to be beneficial (Table 1, entry 1). The influence of water on the reaction was confirmed by a stoichiometric amount of HATU being consumed (Table 1, entry 9). A further increase in the amount of HATU finally resulted in nearly quantitative yields (Table 1, entries 10 and 11).

Table 1. Guanylation Optimization Using p-Fluoroaniline 1a^a.

entry	reagentb	solvent	TEA (equiv)	yield of 2a (%)
1	HATU	ACN	2.0	77 (9)c
2	HATU	DMF	2.0	78 (81)c
3	HBTU	ACN	2.0	17 (0)c
4	HBTU	DMF	2.0	59 (18)c
5	HCTU	ACN	2.0	68 (0)c
6	HCTU	DMF	2.0	83 (62)c
7	HATU	ACN	0.2	70
8	HATU	ACN	1.0	71
9	HATU	ACN	2.0	0d
10	HATU (1.2)	ACN	2.0	81
11	HATU (1.5)	ACN	2.0	99

^aYields determined by GC-FID using mesitylene as an internal standard.

^b1.0 equiv, unless mentioned otherwise.

^cIn the absence of TEA.

^dWith 2.0 equiv water.

We proceeded to screen whether the reaction could be extended to weaker nucleophiles. To this end, 4-nitroaniline 1b was chosen as the model substrate (Table 2). Indeed, 1b showed zero guanylation using TEA (entries 1 and 2). Several nonionic organic bases were screened, but these gave only trace yields at best (see Supporting Information). Gratifyingly, KOtBu in DMF was a major breakthrough (entry 3). Increasing the amount of KOtBu proved to be highly beneficial (Table 2, entry 4), yet, performing the reaction in ACN resulted in a complex mixture (Table 2, entry 5). As KOtBu did not fully dissolve in DMF, 18-crown-6 was trialed in a stoichiometric amount, but it did not affect the reactivity (see Supporting Information). Increasing the amount of HATU to 1.5 equiv resulted in a lower yield (entry 6), however, this could be compensated for by a further increase of the amount of KOtBu, which gave the best result (entry 7). Organic base DBU gave only trace amounts of 2b (entry 8). Likewise, KHMDS was ineffective (entry 9), however, NaH gave 2b in a good yield (entry 10). Considering the safety issues associated with NaH in DMF, we advocate for the use of KOtBu.³¹

Table 2. Guanylation Optimization Using Nitroaniline 1b^a.

entry	solvent	baseb	yield of 2b (%)
1	ACN	TEA (2.0)	0
2	DMF	TEA (2.0)	0
3	DMF	KOtBu (1.0)	9
4	DMF	KOtBu (2.0)	63
5	ACN	KOtBu (2.0)	40c
6	DMFd	KOtBu (2.0)	45
7	DMFd	KOtBu (3.0)	70
8	DMF	DBU (2.0)	3
9	DMF	KHMDS (2.0)	0
10	DMF	NaH (2.0)	68

^aYields determined by GC-FID using mesitylene as an internal standard. 1.0 equiv HATU, unless stated otherwise.

^bEquivalents in parentheses.

^cComplex mixture.

^dHATU 1.5 equiv.

The optimization results above were elucidated further by a kinetic study (Figure 1), revealing that the two methods proceeded at vastly different rates. The KOtBu-mediated reaction reached full conversion in 60 min. In contrast, the TEA-based reaction took substantially longer, with full conversion taking up to 16 h. The TEA-mediated reaction had a short induction period caused by residual moisture reacting with HATU.

Figure 1.

Guanidine formation over time. Conditions: 1a (0.5 mmol), TEA (2.0 equiv), HATU (1.0 equiv) in ACN, or 1b (0.5 mmol), KOtBu (2.0 equiv), HATU (1.0 equiv) in DMF. Yields determined by GC-FID using mesitylene as an internal standard.

We then proceeded to explore the scope of the reaction using various anilines (Scheme 2). The TEA-promoted reaction (condition A) of 1a readily provided guanidine 2a in a good isolated yield, whereas electron-deficient 1b failed to form 2b. In contrast, the KOtBu-promoted reaction (condition B) readily delivered 2b. Electron-rich anilines reacted promptly using condition A, providing products 2c-e in excellent yields. Concurrently, electron-deficient anilines failed to react using condition A, but condition B facilitated the formation of guanidines 2f-i in moderate to good yield. Interestingly, guanidine 2j was only obtained under condition A, likely due to interference between the boronate ester and KOtBu in condition B. We were pleased to find that sterically hindered and electron-deficient 1k, a challenging combination, readily formed 2k, a derivative of the adrenoceptor agonist clonidine. Condition B was also well-compatible with the heterocyclic aniline analogue 1l, yielding 2l in 63%. We extended our studies to aliphatic amine nucleophiles. Although aliphatic amines are known to react with HATU,^3,28,30 we would like to underline that guanylation sensitivity to electronics and sterics has not been studied, to the best of our knowledge. In this regard, p-fluorobenzylamine 1m reacted readily in the presence of triethylamine in ACN, as did significantly more electron-deficient 1n. While the sterically more demanding cyclohexylamine 1o still reacted smoothly, the method reached its limit with the bulky tert-butylamine 1p where no reaction could be observed. Furthermore, both heterocyclic 1q as well as the aliphatic fluorinated 1r and 1s turned out to be well-compatible with guanylation condition A. Attempts to produce bis-guanylated 2t from the corresponding 1,2-diamine failed, and instead, the bicyclic 2u was obtained. This likely occurred through initial guanylation, followed by an intramolecular attack of the neighboring amine (Supporting Information, Section S11). Next, we explored the other end of the nucleophilicity scale with very weak nitrogen-based nucleophiles. Benzamides 1v–x readily reacted under condition B to give the corresponding N-acylated guanidines in good yields. Trifluoroacetamide 1y could only be isolated in low yield, possibly due to decomposition during the workup. Carbamates 1z and 1aa were also smoothly guanylated using condition B. On the other hand, sulfonamides 1ab and 1ac did not require strong base and excellent yields of the N-sulfonylated guanidines were achieved with TEA. Monosubstituted (thio)ureas 1ad and 1ae gave no product, most likely due to decomposition (Supporting Information, Section S12). Delightfully, 1,1-disubstituted (thio)ureas 1af and 1ag were readily guanylated under condition B.

Scheme 2. Substrate Scope of the Guanylation Reaction.

Yields (%) refer to isolated product on 2 mmol scale, except for compounds listed as <5%. For these, trace amounts were observed by GC-MS, but isolation was not attempted.

In DMF.

Most basic guanidines were easy to purify by precipitation, where the crude nitrogen base was added to oxalic acid in Et₂O (see Supporting Information). Subsequent back extraction of the oxalate salt from aqueous sodium hydroxide provided the products in a high NMR purity (>95%) without the need for recrystallization or even chromatographic separation. Lastly, as the guanylation reactions seemed efficient and products were easy to purify, late-stage functionalization was attempted on a selection of more complex amine-containing pharmaceuticals (Scheme 3). We obtained the corresponding TMG derivatives in moderate to good yields, clearly illustrating the suitability of the system for late-stage functionalization in the context of medicinal chemistry.

Scheme 3. Late-stage Functionalization of Active Pharmaceutical Ingredients and Pharma Precursors.

Yields (%) refer to isolated product on 2 mmol scale. See Scheme 2 for conditions A and B.

Having explored the substrate scope, we decided to benchmark our method against conventional synthetic methods.^7,19−21 We chose the substrates 4-nitroaniline 1b, fluorinated benzylamine 1n, and 4-fluorobenzamide 1x. Tetramethylurea was first treated with oxalyl chloride. Then the nucleophile (1.0 equiv) was added alone or in the presence of TEA (nucleophile 1.0 eq and TEA 1.0 equiv). Only fluorinated benzylamine 1n gave guanidine products (Supporting Information, Section S10). Guanidine 2b has previously been synthesized via the conventional route.^7,21 This suggests our method is more robust and has better reproducibility. In particular, our method is better suited for weakly nucleophilic species, such as benzamides and electron-deficient anilines.

The kinetic studies (Figure 1) and the reaction scope (Scheme 2) suggested that conditions A and B did not share a common reaction mechanism, an apparent fact that was further investigated by competition studies of 4-substituted anilines using Hammett σ parameters (Figures 2 and 3).³²⁻³⁴ Under condition A, a negative slope (ρ = −1.77) was recorded, which suggests a buildup of partial positive charge at the aniline nitrogen atom in the transition state (TS). The absolute value of ρ is consistent with an associative mechanism, where the base activates the aniline for nucleophilic attack.¹¹ In contrast, under condition B, a positive slope (ρ = +2.36) was obtained, which implies that significant negative charge forms at the aniline nitrogen atom in the TS. The large absolute value of ρ suggests that the charge is localized close to the aromatic ring; hence, the substituents exert a larger influence. The presence of a large negative charge is further supported by good correlation with σ^– values (R² = 0.93, see Supporting Information). It should be noted that 2b slightly deviates from the general trend, but this may be due to secondary equilibrium effects exerted by the nitro-substituent.³²

Figure 2.

Hammett competition study of 4-substituted anilines under condition A. Reaction conditions: aniline (1.0 equiv), 4-substituted aniline (1.0 equiv), TEA (2.0 equiv), HATU (1.0 equiv), ACN (2.0 mL), 0.5 mmol scale; stirred at room temperature for 16 h.

Figure 3.

Hammett competition study of 4-substituted anilines under condition B. Reaction conditions: aniline (1.0 equiv), 4-substituted aniline (1.0 equiv), KOtBu (4.0 equiv), HATU (1.0 equiv), DMF (2.0 mL), 0.5 mmol scale; stirred at room temperature for 60 min.

The proposed mechanisms are listed in Scheme 4. Under condition A, triethylamine associates to the aniline, activating it for nucleophilic attack. The rate-determining step (RDS) is the addition to HATU, which is likely to occur in a concerted manner with the deprotonation.¹¹ The activating effect of triethylamine is further supported by the fact that a catalytic amount of the base is sufficient for synthetically useful yields (Table 1, entry 7). Under condition A, the hydrogen-bonded complex of TEA and an electron-deficient aniline is not sufficiently nucleophilic to directly react with HATU. In contrast, under condition B, deprotonation of the aniline is the RDS. The anion is significantly more nucleophilic, and once formed, it will rapidly add to HATU under substitution of the oxyazabenzotriazole anion (OAt).

Scheme 4. Mechanistic Proposal of the Base-Dependent Guanylation Reactions.

Having studied factors that promote guanylation, we pondered if reaction conditions could be tuned to suppress guanylation in amide couplings, a common side reactivity and source of impurities of this industrially highly relevant C–N bond forming reaction. Considering that condition A involves a base-assisted addition of aniline to HATU (Scheme 4), we were curious whether alternative bases with more bulk than TEA could prevent guanylation. In this regard, DIPEA, DIPA, TMP, and PMP were tested (Table 3, entries 2–5). These bases have pK_BH values similar to those of TEA (ca. 18 in ACN) but are sterically more hindered. We found that the guanylation yield was only marginally affected by the increase of the sterics of the base. Less basic pyridine was explored next, and we observed that guanylation yield was reduced by nearly an order of magnitude (entry 6), and the more hindered 2,6-lutidine and 2,6-ditert-butylpyridine inhibited guanylation further (entries 7 and 8). In contrast, DMAP, similar in basicity to TEA, gave a high guanylation yield (entry 9). During optimization, we observed that guanylation proceeded without an external base, although in low yield, suggesting that aniline 1a may act as a base (Table 1, entry 1). Consequently, we also studied 4-methoxy-N,N-dimethylaniline, similar in basicity to pyridine, but more hindered than 1a (Table 3, entry 10). In this case, no guanylation was observed. In summary, our results imply that sterically hindered weaker bases (pK_BH ≤ 14) do not mediate guanylation and could therefore be a smart choice in HATU-induced coupling reactions to avoid guanidine-related impurities.

Table 3. Base Screening in the Guanylation of 1a Using HATU in ACN.

entry	base	pKBH in ACNa	yield of 2a (%)
1	triethylamine	18.83	77
2	N,N-diisopropylethylamine	18.60	64
3	N,N-diisopropylamine	18.81	52
4	2,2,6,6-tetramethylpiperidine	18.64	44
5	1,2,2,6,6-pentamethylpiperidine	18.62	54
6	pyridine	12.53	17
7	2,6-lutidine	14.16	11
8	2,6-di(tBu)pyridine	8.05	0
9	4-(N,N-dimethylamino)pyridine	17.95	47
10	4-methoxy-N,N-dimethylaniline	12.72	0

^aRefs.^35–40

In order to validate this side observation, the results of the base screen were applied in an amide coupling study using 4-fluorobenzoic acid 3a (Table 4). The intermolecular competition between carboxylic acid 3a and aniline 1a was probed with both HATU and HBTU. Using TEA, trace amounts of guanylated byproduct 2a were observed (entries 1 and 2) while switching to 2,6-lutidine as base effectively suppressed guanylation while preserving very high amidation yields (entries 3 and 4).

Table 4. Intermolecular Competition Studies on the Effect of Base in Amidation Reactions^a.

entry	coupling reagent	base	4a (%)	2a (%)
1	HATU	triethylamine	86	<1
2	HBTU	triethylamine	84	4
3	HATU	2,6-lutidine	87	0
4	HBTU	2,6-lutidine	83	0

^aYields determined by GC-FID using mesitylene as an internal standard.

We then proceeded to a more challenging scenario of both intermolecular and intramolecular competition with carboxylic acid 3b (Table 5). Previous work had reported 3b to undergo exhaustive guanylation of the sulfonamide using HBTU and N,N-diisopropylethylamine, similar in its basicity as TEA but a more hindered base, in CH₂Cl₂, producing 6b as the main product.²⁹ In our hands, HATU and TEA did yield the desired amide 4b, yet with small amounts of guanidine 2a (entry 1). Quantitative ¹⁹F{¹H} NMR showed an additional two minor peaks at −118.32 and −118.59 ppm. The former and more abundant one was assigned to 6b based on literature reactivity.²⁹ While we cannot rule out formation of 5b, it is clear from ¹H NMR results that 5b is not formed in significant quantities (see Supporting Information, Section 16). Similar to the simple amide coupling test, changing the base to 2,6-lutidine not only improved amidation yield but also eliminated the impurities at −118.32 ppm (6b) and −118.59 ppm (entry 2).

Table 5. Intramolecular and Intermolecular Competition Studies on the Effect of Base in Amidation reactions^a.

entry	base	4b (%)	2a (%)	6b (%)	unknown (%)
1	triethylamine	23	8	3	2
2	2,6-lutidine	44	5	0	0

^aYield was determined with ¹⁹F{¹H}-NMR using α,α,α-trifluorotoluene as an internal standard.

Conclusion

In summary, we have shown HATU to be an efficient guanylation reagent. Two complementary methods were developed to form 2-substituted TMGs with a wide substrate scope and good isolated yields under mild conditions. Our HATU-based system was shown to be well-suited for late-stage functionalization of pharmaceuticals, possessing superior reactivity over conventional methods. The appropriate base must be chosen by taking the nature of the nucleophile into account. Strong nucleophiles are optimally paired with mildly basic TEA and react via uncharged intermediates. In contrast, weak nucleophiles are not sufficiently activated by TEA, but deprotonation using KOtBu, a strong base, results in fast guanylation. The lessons learned in the optimization of the guanylation reaction were applied to improve the chemoselectivity of amide couplings when using guanidium-based coupling reagents.

Data Availability Statement

The data underlying this study are available in the published article and its Supporting Information, and openly available in Zenodo at 10.5281/zenodo.14652609.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.joc.4c02645.

• The electronic Supporting Information provide general information, general experimental procedures, reaction optimization, synthetic methods, compound characterization, and NMR spectra (PDF)

Author Contributions

All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.