Construction of 7-Diethylaminocoumarins Promoted by an Electron-Withdrawing Group

Abstract

The coumarin skeleton has been a focus of attention for many years, and its fluorescence properties vary depending on the substituents. Fluorescent coumarin derivatives are useful tools for many strategies have been developed for their synthesis. Although 7-diethylaminocoumarin has excellent fluorescence properties, it is unstable. We have developed a facile strategy for the synthesis of 7-diethylaminocoumarin derivatives by increasing the electrophilicity of the ynone moiety to promote nucleophilic addition reactions and cyclization. The reaction tolerates a variety of substitutions at the 4-position.

Introduction

Coumarin derivatives show diverse biological activities, including anticoagulant, anti-human immunodeficiency virus (HIV) and antimicrobial activities,^1–3) and are used as fluorescent probes. Fluorescent probes are used in basic biological research such as cell imaging, enzyme activity measurements, and protein and nucleic acid detection. Many synthetic strategies for coumarin have been reported, such as by the Perkin,⁴⁾ Pechmann,^5,6) Knoevenagel,⁷⁾ Morita–Baylis–Hillman,⁸⁾ and Mizoroki–Heck reactions.^9,10) However, most of these reactions involve C–C bonds, reaction under strongly acidic conditions, or the need for high temperature or metal catalysts, making knowledge of organic chemistry a requirement to synthesize a probe containing coumarin.

We recently reported a novel synthetic approach for 7-methoxycoumarin (7-OMe coumarin)¹¹⁾ using the coumarin precursor that we named the turn-on fluorescent probes mediated by conjugate addition and cyclization (TCC probe)¹²⁾ via a conjugate addition reaction to ynone and cyclization to construct the lactone moiety. The advantages of this reaction are mild conditions, and ease of attaching the coumarin moiety to ligands via a nucleophile as the final step.

The 7-OMe coumarin precursor 1 is stable but the fluorescence intensity of synthesized 7-OMe coumarin is low and thus not useful (Figs. 1, 2). 7-Diethylaminocoumarin (7-NEt₂ coumarin) has a high fluorescence quantum yield¹³⁾ making its synthesis desirable, but the corresponding precursor is unstable because of the tertiary amine. Indeed, the yield of 7-NEt₂ coumarin from 2 was low.¹²⁾ Additionally, we obtained a byproduct where phenol was involved in the reaction. Therefore, the phenol may also be a factor that prevents the reaction from proceeding as desired.¹¹⁾ Here, we describe the synthesis of the 7-NEt₂ coumarin unit in a one-pot reaction using tert-butyldimethylsilyl (TBS)-ether 3¹²⁾ by conjugated addition from nucleophiles 4–8. Furthermore, we developed a coumarin precursor with higher reactivity than 3, and synthesized 4-oxycoumarins^14,15) that could not be synthesized using 7-OMe coumarin precursor 1.

Fig. 1. Structures of 7-OMe and 7-NEt₂ Coumarin

Fig. 2. Structures of the Reported Coumarin Precursor and Nucleophiles

Results and Discussion

We investigated appropriate reaction conditions using precursor 3 and nucleophiles 4–8. Precursor 2 was synthesized based on a previously reported synthetic scheme. First, we examined the conjugate addition of secondary amine 4 to precursor 3 (Chart 1). Adduct 9 was afforded in 15% yield, 77% of precursor 3 was recovered, and the corresponding 7-methoxy 11 was obtained in 70% yield¹¹⁾ (Chart 2).

Chart 1. Synthesis of 10

Chart 2. Synthesis of 7-OMe Coumarin 11¹¹⁾

We considered that the reactivity of β-alkyne 3 is lower than that of 1 due to substitution of the electron-donating groups at the 7-position and the TBS protecting group, and thus attempted to synthesize the 7-NEt₂ coumarin precursor with improved reactivity by substituting the ethyl ester 3 with trifluoroethyl ester, an electron-withdrawing group (EWG) (12; Fig. 3). Precursor 12 was synthesized from alkyne 13 in one step (Chart 3).

Fig. 3. Structure of Precursors 3 and 12

Chart 3. Synthesis of Precursor 12 from Alkyne 13

Coumarin precursor 12 was conjugated using nucleophiles containing a secondary amine 4 and afforded 10 in sufficient yield (74%, Table 1). We investigated the synthesis of 7-NEt₂ coumarin using other nucleophiles, thiol 5, primary amine 6, alcohol 7 and phenol 8, and obtained 14 (80%), 15 (44%) with along with amide 18 (22%), 16 (48%) along with adduct of trifluoroethanol 19 (37%), 17 (50%), respectively. found that 7-NEt₂ coumarin could be synthesized with all nucleophiles (Table 1).

Table 1. Synthesis of Compounds 10, 14–17

Nucleophile	R	X	Product (yield)	Byproduct (yield)
4	BnCH2	NMe	10 (74%)	—
5	BnCH2	S	14 (80%)	—
6	BnCH2	NH	15 (44%)	18 (22%)
7	BnCH2	O	16 (48%)	19 (37%)
8	Ph	O	17 (50%)	—

Reagents and Conditions: Reagents 4–8 (4–7: 1.5 equivalent (equiv.) or 8: 3.2 equiv.), Et₃N (14–17: 2.0 equiv. or 10: 5.0 equiv.) in N,N-dimethylformamide (DMF) (0.1 M). Tetrabutylammonium fluoride (TBAF) was added after conjugate reaction which was monitored by TLC. See Supplementary Materials for detailed reaction conditions.

We were previously unable to obtain 7-OMe coumarin, the conjugate adducts of alcohol 7 and phenol 8¹¹⁾ and thus applied the EWG strategy to construct 7-OMe coumarin. We synthesized 7-methoxy precursor 21 bearing a trifluoroethyl ester (Chart 4). As expected, 7-OMe coumarins 22 and 23 were successfully synthesized using alcohol or phenol as a nucleophile (Charts 5, 6). Compounds 22 and 23 are volatile and thus the isolated yields were low (21 and 23%). It should be emphasized that this result was achieved using the EWG strategy.

Chart 4. Synthesis of Precursor 21 from Alkyne 20

Chart 5. Synthesis of 22

Chart 6. Synthesis of 23

Finally, we attempted to attach the coumarin to a steroid skeleton to demonstrate our strategy (Chart 7). Steroid (cholane-3,24-diol) 24 with a protected alcohol was reacted with precursor 21, affording coumarin-conjugated cholane 25 in 42% yield in a one-pot reaction.

Chart 7. Incorporation of 7-NEt₂ Coumarin into a Steroid Skeleton

The fluorescence spectra of coumarins 10, 14–17, 22 and 23 were measured to evaluate their fluorescence properties in CH₂Cl₂, tetrahydrofuran (THF), MeOH, and H₂O (Table 2). The fluorescence spectra were different depending on the conjugating heteroatoms at the 4-position. We also measured the fluorescence spectra of 7-OMe coumarin (Table 2). Both compounds 22 and 23 have an oxygen atom at the 4-position and their fluorescence spectra were the same.

Table 2. Fluorescence Properties of Compounds 10, 14–17, 22 and 23

Compound	λ (nm)	CH2Cl2	THF	MeOH	H2O
10	λmaxabs	357	354	357	359
	λmaxf	423	415	449	467
14	λmaxabs	379	372	382	396
	λmaxf	460	453	489	467
15	λmaxabs	351	345	348	352
	λmaxf	395	385	410	440
16	λmaxabs	367	360	363	385
	λmaxf	426	413	463	445
17	λmaxabs	361	357	362	365
	λmaxf	424	418	449	468
22	λmaxabs	312	312	310	310
	λmaxf	353	346	373	392
23	λmaxabs	311	311	310	311
	λmaxf	355	345	377	389
25	λmaxabs	365	360	363	374
	λmaxf	424	410	461	437

Finally, we compared the quantum yields of 7-NEt₂ 10 and 7-OMe 11, the compounds we had particularly focused on.¹¹⁾ As expected, the results were better for 10, with a quantum yields 200 times higher than for 11 (Table 3).

Table 3. The Fluorescence Quantum Yields of 7-OMe and 7-NEt₂ Coumarin Analogs

Compound	Φ
	CH2Cl2	THF	MeOH	H2O
10	0.37	0.13	0.37	0.11
11	0.0041	0.0032	0.0018	0.0005

The fluorescence quantum yields of coumarin analogs were calculated by using quinine sulfate (Φ = 0.577 in 0.1 M H₂SO₄) as a reference standard.¹⁶⁾

Conclusion

We constructed 7-NEt₂ coumarin using stable TBS-protected 3 instead of unstable 7-NEt₂ coumarin precursor 2. TBS-protected 3 has low reactivity and thus we converted the ethyl ester to trifluoroethyl ester to provide efficient coumarin precursor 12. We synthesized 7-NEt₂ coumarins, that possess better fluorescent property, using precursor 12.

Acknowledgments

We are grateful to Showa Pharmaceutical University for financial support.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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