Synthesis of Rhodamines and Rosamines using 3,6-Difluoroxanthone as a Common Intermediate

Abstract

Rhodamines and structurally similar rosamines are some of the most highly utilized tools for molecular imaging experiments. We report a general and high yielding route to produce 18 examples of rhodamines and rosamines, including tetramethyl rhodamine, rhodamine B, and Janelia Fluor 549 from a single xanthone intermediate, 3,6-difluoroxanthone. Spectroscopic studies revealed trends in fluorophore efficiency based on substitution patterns at the 3′, 6′, and 9′ positions, providing insights to aid future designs of rhodamines/rosamines.

INTRODUCTION

Rhodamines are a class of xanthene-based fluorophores that generally absorb and emit light in the 500–700 nm region of the electromagnetic spectrum. The relatively long absorption/emission wavelengths of rhodamines have led to the extensive use of these fluorophores in bioimaging processes, as light in this region is associated with low phototoxicity¹ and deeper tissue penetration.² Indeed, rhodamine-based fluorophores such as tetramethylrhodamine (TMR), rhodamine B (RhB), rhodamine 123, rhodamine 6G (Rh6G), Texas Red, Alexa 546/633, the Janelia Fluor (JF) series, as well as others, are routinely used in fluorescence microscopy,^{3, 4} fluorescence correlation spectroscopy (FCS),^{5, 6} flow cytometry,^{7, 8} and immunoassays.^{9, 10} Rhodamines are also commonly used as inks,¹¹ laser dyes,^{12, 13} ion sensors,^{14, 15} and tracer dyes.^{16, 17}

Rosamines, or analogs of rhodamines that lack a carboxylic acid at the 2 position, possess similar photophysical properties to rhodamines, but are unable to intramolecularly lactonize to nonfluorescent tautomeric forms (Figure 1). In this respect, rhodamines and rosamines share a similar relationship to fluorescein (Fl) and Tokyo Green (TG)¹⁸ fluorophore systems. While rosamines have been synthesized since the late nineteenth century,¹⁹ this class of fluorophores has recently gained attention due to a growing demand for fluorescent probes with specialized functions: rosamines are commonly used as mitochondrial dyes,^{20, 21} photosensitisers,^{22, 23} probes for measuring transmembrane potential,^{24, 25} among other applications.^26–28

Figure 1.

Structures of rhodomines, rosamines, fluorescein, and Tokyo Green fluorophores.

Both rhodamines and rosamines are traditionally synthesized through a condensation of 3-aminophenols with phthalic anhydrides (rhodamines) or benzaldehydes (rosamines) as illustrated in Scheme 1A.^{19, 29, 30} This approach, however, is limited by the number of readily accessible aminophenols and phthalic anhydrides/benzaldehydes, and for the synthesis of rosamines is often not very efficient (typically yields are <40% and purification from side products is often problematic). A more modern method involves a nucleophilic addition of ArLi/ArMgX to the carbonyl group of 3,6-diaminoxanthones (3,6-DAXs), which upon treatment with acidic conditions eliminates to the rosamine (Scheme 1B).^{31, 32} Synthesis of rosamines through this fashion offers the ability to vary substitution at the 9′-position, but is restricted by the number of accessible 3,6-DAXs. The inability to vary 3′,6′-amine substitution is limiting as these positions are known to influence photophysical properties, particularly the quantum yield and molar absorptivity for both rhodamines and rosamines.^{33, 34} To provide access to a larger range of 3,6-DAXs, Burgess et al. developed a S_NAr method enabling the substitution of 3,6-ditriflate xanthone (2) with 5 or 6 cyclic amine systems, which were further elaborated into rosamines and pyronins.³⁴

Scheme 1.

Traditional and proposed synthetic routes to rhodamines and rosamines. (A) Acid catalyzed Friedel Crafts reactions of 3-aminophenol with phthalic anhydrides or benzaldehydes. (B) Synthesis of rosamines via aryllithium or aryl magesium addition to 3,6-DAXs. (C) Proposed access to rhodamines and rosamines via S_NAr amination of xanthone 1 followed by aryllithium or arylmagesium

We became interested in Burgess’s method of developing 3,6-DAXs as means to provide access to azetidine-substituted rhodamines and rosamines. Our attempts to synthesize 3,6-di(azetidin-1-yl)-xanthone (3) from azetidine and 2, however, mirrored Burgess et al. reported synthesis of 3,6-di(pyrrolidin-1-yl)-xanthone (4) in terms of low yields (17% for 3 versus 21% for 4, Scheme 2), despite altering reaction conditions (higher temperature and different solvents) and attempting palladium coupling conditions. Similar substitution attempts using acyclic amines gave even lower yields, suggesting that 6-membered amine ring systems are optimal for disubstitution of 2. Our analysis of this reaction revealed that while monosubstitution of 2 occurs rapidly at 90 °C, disubstitution is much slower due to a decrease in electrophilicity from N lone pair donation. This loss of electrophilicity results in competitive amine attack on the triflyl ether to produce detriflated (S-OAr bond cleavage) side products. Comparable aryl detriflation with amines has been described for similar substitution reactions^{35, 36} and for low yielding palladium-catalyzed coupling attempts.³⁷

Scheme 2.

Low yielding S_NAr amination attempts using ditriflate xanthone 2.

In an attempt to overcome the complications associated with diamino substitution of 2, we envisioned developing 3,6-difluoroxanthone (1) as an intermediate that could readily undergo S_NAr with primary or secondary amines, but avoids detriflation. We rationalized that 3,6-diamino substitution of 1, using readily available amines, would provide access to a pool of 3,6-DAXs that greatly exceeds the number of aminophenols, from which most rhodamines/rosamines are currently synthesized. The 3,6-DAXs could then be converted to rhodamines and rosamines using the ArLi/ArMgX addition/elimination approach (Scheme 1B), providing control over substitution of the 9-position, which is also known to modulate the chemical and photophysical properties of rosamines.³⁴ In using a diaminosubstitution followed by ArLi/ArMgX addition/elimination approach, a wide range of novel rhodamines and rosamines with differing physical, photophysical, and biological properties to those already known, can all be potentially accessed from 1 (Scheme 1C). Additionally, several existing rosamines, which suffer from low yields and competing side reactions, could be more efficiently synthesized.

RESULTS AND DISCUSSION

To evaluate our proposed strategy, difluoroxanthone 1 was prepared in approximately 10 g batches from commercial materials through four transformations as summarized in Scheme 3. Using metal-halogen exchange, 5 was converted into a Grignard intermediate and added to aldehyde 6 to produce alcohol 7 in 93% yield. The alcohol group was oxidized to a ketone using a TEMPO-NaOCl reaction to provide 8 in excellent yield. Demethylation of 8 with BCl₃ followed by deprotonation of the resulting phenol, facilitated intramolecular S_NAr to construct the central xanthone ring of 1 in 89% over two steps. Xanthone 1 is highly crystalline in nature and can be efficiently purified by recrystallization; the 89% yield includes purification by recrystallization.

Scheme 3.

Synthesis of 3,6-difluoroxanthone (1) from commercially available materials.

Difluoroxanthone 1 was converted into six different 3,6-DAXs (9–14) as illustrated in Table 1. Reaction conditions involved mixing 1 with 20 equiv. of amine at temperatures of 150 °C (cyclic amines) or 200 °C (acyclic amines) in a sealed vessel. In most of our examples, amines were used neat in the reaction effectively carrying out solvolysis; however, for the synthesis of 9 and 13, dimethylamine was generated via KOH decomposition of DMF (for 9)^{38, 39} and azetidine hydrochloride was deprotonated prior to use in DMSO (for 13). We employed two different types of pressure vessel systems: a traditional thick-walled glass pressure vessel heated in a silicone oil bath and a PTFE-lined stainless steel tank reactor (heated in an explosion proof convection oven). While the later method generally gave higher yields and lower reaction times, likely due to more uniform heating, we realize that this system is not as accessible to most labs without a significant monetary investment, and therefore, illustrated that all reactions can be carried out in good yields with common glass pressure vessels. It is notable that product 13 was only obtained via the glass vessel method, as stirring of the reaction appears to be critical to preventing competing polymerization processes.

Table 1.

Conditions and yields for the synthesis of 3,6-DAXs 9–14 from 1.

HNR2a	Time (h)	Temp (°C)	Product	Yield (%)
HNMe2b	24e/20f	200	9	91e/97f
HNEt2c	72e/20f	200	10	81e/93f
HNiPrMec	24e/20f	200	11	78e/92f
HNMeEtc	24e/20f	200	12	75e/98f
Azetidined	24e/-g	150	13	76e/-g
Pyrrolidinec	1e/1f	150	14	85e/99f

^a20 equivalents of amine was used for each reaction.

^bDimethylamine was generated via KOH decomposition of DMF.

^cAmine was used neat in the reaction.

^dDMSO was used as a solvent.

^eReaction was run in pressure vessel heated with an oil bath.

^fReaction was run in a steel high pressure vessel within a convection oven.

^gReaction run in a steel high pressure vessel within a convection oven resulted in polymerization within an hour.

Xanthones 9–14 underwent a reaction with phenyl, 2-carboxyphenyl, or 2-methylphenyl Grignard reagents to generate alkoxide intermediates, which upon acidic workup, underwent elimination to provide TMR, RhB, JF₅₄₉, and rhodamines/rosamines 15–29 (Figure 2 and Table 2). Moderate to good isolated yields (60–92%) were obtained for all rhodamines/rosamines, though longer reaction times were required for complete conversion to rhodamines and 2 methyl rosamines, likely as result of steric hindrance encountered from the attack of tolyl Grignard reagents upon the xanthone carbonyl carbon. Decreased solubility of xanthone 14 in THF also resulted in longer reaction times for 27 and 29.

Figure 2.

Structures of TMR, RhB, JF₅₄₉ and rhodomines/rosamines 15–29 synthesized from 9–14.

Table 2.

Reaction conditions, yields, photophysical properties^a and calculated Log D values for the synthesis and characterization of TMR, RhB, JF₅₄₉, and 15–29.

	Time (h)	Yield (%)b	λmax (nm)	εmax (cm−1 M−1)	λem (nm)	Φf	cLog Dc
TMR	48	92	546	84,100	563	0.59	0.91
15	24	80	550	79,300	571	0.87	0.49
16	48	80	550	106,700	570	0.80	1.00
RhB	48	91	552	104,400	565	0.65	2.34
17	24	86	552	102,600	574	0.59	1.91
18	48	87	555	106,900	572	0.48	2.43
19	48	92	549	94,500	563	0.57	1.63
20	24	76	555	91,500	576	0.83	1.20
21	48	87	552	114,700	570	0.76	1.71
22	48	83	553	92,400	566	0.41	2.46
23	24	86	555	85,500	575	0.44	2.03
24	48	81	554	93,100	574	0.50	2.55
JF 549	48	89	549	102,500	571	0.86	0.65
25	24	87	551	116,500	572	0.85	0.22
26	48	84	549	121,700	570	0.79	0.74
27	72	88d	545	76,900	567	0.75	1.69
28	24	63	551	107,600	573	0.68	1.26
29	72	60	556	110,400	574	0.80	1.77

^aPhotophysical properties were measured using ethanol as a solvent.

^bIsolated Yield after column chromatography.

^ccLog D was calculated using a ChemAxon plugin in MarvinSketch software.

^dHMPA was added as a cosolvent.

All rhodamines/rosamines possessed similar values for λ_max, near 550 nm, though molar absorptivity (ε_max) and quantum yields (Φ_f) varied considerably with 3,6-diaminosubstitution (Table 2 and Figure 3). As observed previously with the JF series,⁴⁰ azetidinyl substitution resulted in the bright fluorophore systems exhibiting high molar absorptivities (>100,000 cm⁻¹ M⁻¹) and quantum yields (0.79–0.86). Additionally, 3,6-bis(dimethylamino)rosamine 16, 3′,6′-bis(ethylmethylamino)rosamine 21, and 3′,6′-bis(pyrrolidinylamino)rosamine 29 all displayed ε_max >100,000 cm⁻¹ M⁻¹ and Φ_f > 0.75 as well, suggesting that these too are efficient fluorophore systems. Some additional trends can be noted when comparing ε_max and the maximum emission wavelength (λ_em) between rhodamines, rosamines, and 2 methyl rosamines within the same 3′,6′-substitution pattern family. In general, ε_max are largest for 2 methyl rosamines followed by rhodamines and then rosamines, suggesting that substitution at the 2 position may partially contribute to chromophore performance. Additionally, rosamines and 2 methyl rosamines, with the exception of 25 and 26, possess 6–13 nm redshifted maximum emission wavelengths (λ_em); this is a trend which can be observed for some of the previously reported rosamine systems^{34, 41, 42} and was noted for 18 by Peterson et al.⁴³ Interestingly, both 25 and 26, which are both within the azetidine-1-yl series, do not possess a significant red-shifted λ_em in comparison to JF₅₄₉.

Figure 3.

Normalized absorbance and emission spectra for: (A) TMR, RhB, and 15–29; (B) JF₅₄₉ and 15–29.

The library of rhodamine/rosamine compounds illustrates a considerable deviation from quantum yield trends observed in the fluorescein-based systems, where high quantum yields are dependent on substitution at the 2 position (Figure 4). It is believed that 2 substitution in fluorophores such as fluorescein or Tokyo Green, prevents a non-radiative decay process by restricting free rotation of the 9’-position aryl group, as fluorescein derivatives without 2 substitution (i.e. 30) possess much lower quantum yields.^{18, 44} Our measured quantum yields for TMR, RhB, JF₅₄₉, and 15–29 would suggest that rhodamine/rosamine based systems do not undergo the same type of 9-aryl rotational non-radiative decay process as fluoresceins, as Φ_f ranges for rosamines (0.44 – 0.87) are similar to those of 2-methylrosamines (0.48 – 0.80) and rhodamines (0.41 – 0.86). Instead, 3,6-disubstitution patterns, likely due to the potential of forming twisted internal charge transfer states^{40, 45} and/or aggregation,^{46, 47} have a larger influence on the observed quantum yields. This observation may be an important consideration for planning future rosamine designs, particularly where 2 substitution is not desired or is problematic for synthetic access.

Figure 4.

Influence of free and restricted rotation of the 9-position aryl group upon quantum yields in Fl TG, carboxy-free fluorescein derivative 30, and TMR, RhB, JF₅₄₉, and 15–29.

While our rhodamine/rosamine library represents only a small subset of the potential fluorophores that can be accessed, there are several novel compounds produced within this library, which have attractive properties for the development of fluorescent molecular probes or labels. Rhodamine 19 and rosamines 20 and 21 represent the first set of “hybrid” TMR and RhB fluorophores. Both 20 and 21 possess relatively high quantum yields/molar absorptivities and bridge the range of lipophilicities between that of TMR and RhB, which is a trend we simulated using calculated Log D values at pH 7.4 (Table 2).⁴⁸ JF₅₄₉ analog 25 is the brightest fluorophore in our library (ε_max = 116,500, Φ_f = 0.85) and is also calculated to be the most hydrophylic in nature (cLog D = 0.22), and may have applications in probe/label designs where efficient fluorescence is desired and/or water solubility is critical. Rosamine 24, while only moderately bright in comparison to other fluorophores in the library, has comparable lipophilicity to rhodamine 101 (cLog D = 2.58), but with considerably less steric bulk. Additionally, rosamines 16,^{41, 42} 18,⁴³ 26,⁴⁹ 28,⁵⁰ and 29,³⁴ while previously reported, are produced in higher overall yields and without significant side products using our xanthone-based route.

CONCLUSIONS

In summary, we have developed a unified methodology that offers a versatile and efficient approach to access numerous rhodamines and rosamines from a single common intermediate 1. In this study alone, we successfully generated 18 examples, which span a broad range of lipophilicities (cLog D = 0.22 – 2.55) while retaining moderate to high molar absorptivities and quantum yields. The ability to access structurally diverse rhodamines/rosamines is important for the development of novel molecular probes, particularly for studying druglike molecules where pairing a fluorescent tag that maintains the overall lipophilicity is critical for preserving biological, pharmacokinetic, and metabolic properties. Additionally, this process provides a means of producing an assortment of rosamines. Rosamines, unlike rhodamines, are unable to lactonize and thus remain highly fluorescent at much larger ranges of pH, but have been traditionally difficult to synthesize due to low yielding preparations with competing side reactions. The complete series of rhodamines/rosamine also offers a set of tends that may of use in future fluorophore design. Both azetidinyl and pyrrolidinyl substitution at the 3′ and 6′ positions consistently produce bright fluorescent systems, although, dimethylamino and ethylmethylamino substitution can also provide efficient fluorophores with alternative hydrophobicities. While diethylamino and isopropylmethylamino 3′ and 6′ substitution patterns result in lowered ε_max and Φ_f, these substituents offer much higher levels of lipophilicity with moderately intense fluorophore brightness which could be attractive to certain applications. Overall, this technology offers the ability to rapidly assemble and optimize photophysical and chemical properties of rhodamines and rosamines for specific uses, through the use of a robust, effectual, and divergent synthetic route.

EXPERIMENTAL SECTION.

General.

¹H (400 MHz) and ¹³C (101 MHz) NMR spectra were acquired on a Varian 400 MHz instrument at the University of Las Vegas, Nevada. Chemical shifts are reported in ppm (δ) and are referenced to CDCl₃ (7.27 ppm for ¹H and 77.0 ppm for ¹³C{¹H}). Coupling constants J_HH are in hertz and are reported as follows: chemical shift, multiplicity (br = broad, s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, hept = heptet m = multiplet, dd = doublet of doublets, dt = doublet of triplets, app = apparent), coupling constant, and integration. Melting points were acquired using a Stuart SMP10 Digital Melting Point Apparatus and are uncorrected. Infrared spectra (IR) were recorded with a Shimadzu IRAffinity-1 FT-IR spectrophotometer. UV/Vis measurements employed a Beckman Coulter DU-800 spectrophotometer. Fluorescence measurements used a Horiba Scientific FluoroMax-4 spectrofluorometer. High-resolution mass spectra were obtained at the Mass Spectrometry Facility at the University of California, Irvine. Thin layer chromatography (TLC) used aluminum-backed silica plates (0.20 mm, 60 F-254). Column chromatography and silica plugs were performed using Silicycle Siliaflash P60 silica. Plates were visualized by UV light. Commercial reagents were used as received unless otherwise noted. Yields are reported based on isolated material.

(2,4-difluorophenyl)(4-fluoro-2-methoxyphenyl)methanol (7).

To a −78 °C solution of 1-bromo-2,4-difluorobenzene (10.8 mL, 95.6 mmol) in anhydrous THF (60 mL) was added isopropylmagnesium chloride lithium chloride complex solution (1.3 M, 73.4 mL, 96.0 mmol) dropwise under Ar in a flame dried round bottom flask. The resulting mixture stirred at −78 °C for 10 min, was warmed to 0 °C and stirred for 30 min. A mixture of 4-fluoro-2-methoxybenzaldehyde (14.8 g, 96.0 mmol) in anhydrous THF (20.0 mL) was added dropwise at −78 °C. The resulting mixture was warmed to room temperature and stirred overnight. The reaction mixture was quenched using 1M HCl (60 mL) and stirred for 10 min. The resulting phases were separated and the organic phase was washed with Et₂O (3 × 40 mL), dried over anhydrous Na₂SO₄, and concentrated under reduced pressure to yield 7 (23.9 g, 93%) as a pale yellow solid. m.p. 85–88 °C; ¹H NMR (CDCl₃, 400 MHz) δ 7.30 (td, J = 8.5, 6.5 Hz, 1H), 7.12 – 7.00 (m, 1H), 6.77 (td, J = 8.5, 2.6, 1H), 6.69 (ddd, J = 10.3, 8.9, 2.5 Hz, 1H), 6.63 – 6.46 (m, 2H), 6.25 – 6.09 (m, 1H), 3.72 (s, 3H), 2.91 (brs, 1H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 163.5 (s), 161.4 (dd, J = 232, 12 Hz), 161.0 (s), 159.9 (dd, J = 234, 12 Hz), 156.7 (d, J = 10 Hz), 128.1 (dd, J = 10, 6.0 Hz), 127.2 (d, J = 8.8 Hz), 124.7 (dd, J = 14, 4.0 Hz), 110.0 (dd, J = 19, 3.2 Hz), 105.8 (d, J = 19), 110.0 (t, J = 26 Hz), 98.1 (d, J = 26 Hz), 64.2 (s), 54.7 (s); IR (thin film) 3341, 3271, 3078, 1604, 1497, 1411, 1258 cm⁻¹; HRMS (ESI) m/z: [M]⁺ Calcd for C₁₄H₁₁F₃O₂ 268.0711; Found: 268.0706.

(2,4-difluorophenyl)(4-fluoro-2-methoxyphenyl)methanone (8).

To a solution of 7 (13.7 g, 51.1 mmol) in CH₂Cl₂ (350 mL) was added KBr (1.21 g, 10.2 mmol), TEMPO (400 mg, 2.56 mmol, 5 mol%), NaHCO₃ (6.37 g, 75.8 mmol) and saturated aq. NaCl (210 mL). The biphasic mixture was vigorously stirred and aq. 5.25 % w/w NaOCl (210 mL, 148 mmol) was added in one portion. The resulting bright orange mixture was stirred for 1 h, and during this timeframe the color faded to light yellow. The biphasic layers were separated, the aqueous layer was extracted with CH₂Cl₂ (3 × 100 mL), and the combined organic fractions were dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to give a crude light yellow solid. The solid was purified through a column of silica gel using CH₂Cl₂ as an eluent to remove TEMPO and inorganic impurities. The filtrate was concentrated to yield 8 as a colorless solid (12.5 g, 92%). m.p. 63–65 °C; ¹H NMR (CDCl₃, 400 MHz) δ 7.63 (td, J = 8.5, 6.5 Hz, 1H), 7.51 (dd, J = 8.6, 6.7 Hz, 1H), 6.86 (ddd, J = 8.7, 7.8, 2.4 Hz, 1H), 6.79 – 6.62 (m, 2H), 6.57 (dd, J = 10.8, 2.3 Hz, 1H), 3.59 (s, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 188.6 (s), 166.4 (s), 164.2 (dd, J = 243, 13 Hz), 163.9 (s), 160.7 (dd, J = 258, 13 Hz), 159.2 (d, J = 11 Hz), 131.5 (dd, J = 10, 4.0 Hz), 124.4 (d, J = 3.0 Hz), 123.9 (dd, J = 12, 4.0 Hz), 110.6 (dd, J = 21, 13 Hz), 106.6 (d, J = 22), 103.0 (t, J = 26 Hz), 98.6 (d, J = 26 Hz), 54.8 (s); IR (thin film) 3062, 2956, 1651, 1605, 1265 cm⁻¹; HRMS (ESI) m/z: [M]⁺ Calcd for C₁₄H₉F₃O₂ 266.0555; Found: 266.0547.

3,6-difluoroxanthone (1).

To a −78 °C solution of 8 (12.7 g, 47.5 mmol) in CH₂Cl₂ under an Ar atmosphere, was added BCl₃ (1M in THF, 91.0 mL, 91.0 mmol) dropwise. The resulting mixture was allowed to warm to room temperature and stirred for an additional 75 min. The mixture was quenched with saturated aq. NaHCO₃ (100 mL), then saturated aq. citric acid was added until the mixture was mildly acidic (as indicated by litmus paper). The resulting mixture was extracted with CH₂Cl₂ (3 × 100 mL), dried over with anhydrous Na₂SO₄, and concentrated under reduced pressure to yield (11.6 g, 96%) of phenol intermediate that was directly used in the subsequent reaction. mp 61–66 °C; ¹H NMR (CDCl₃, 400 MHz) δ 12.20 (s, 1H), 7.56 – 7.35 (m, 2H), 7.04 (td, J = 7.6, 2.3 Hz, 1H), 6.95 (td, J = 8.6, 2.4 Hz, 1H), 6.73 (dd, J = 10.3, 2.0 Hz, 1H), 6.67 – 6.52 (m, 1H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 196.0 (s), 169.2 (s), 166.6 (s), 165.5 (d, J = 14 Hz), 164.7 (dd, J = 257, 12 Hz), 159.7 (dd, J = 255, 12 Hz), 135.6 (d, J = 8.0 Hz), 131.4 (dd, J = 9.6, 6.2 Hz), 122.5 (dd, J = 16, 4.0 Hz), 112.1 (dd, J = 22, 4.0 Hz), 107.6 (d, J = 23), 105.0 (d, J = 24 Hz), 104.8 (t, J = 34 Hz); IR (thin film) 3078, 2924, 2854, 1589, 1497, 1257, 779 cm⁻¹; HRMS (ESI) m/z: [M+H]⁺ Calcd for C₁₃H₈F₃O₂ 253.0476; Found: 253.0487.

A mixture of phenol intermediate (11.4 g, 45.3 mmol) and Hunig’s base (23.0 mL, 132 mmol) in Acetonitrile (86.0 mL) was heated to 90 °C and refluxed for 1 h. The resulted mixture was concentrated under reduced pressure, diluted in CH₂Cl₂ (100 mL) and treated with 1M HCl (120 mL). The biphasic layers were separated, the aqueous layer was extracted with CH₂Cl₂ (3 × 100 mL), and the combined organic fractions were dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to yield 1 (9.79 g, 93%; 89% over 2 steps from 8). mp 175–179 °C; ¹H NMR (CDCl₃, 400 MHz) δ 8.32 (dd, J = 8.8, 6.5 Hz, 1H), 7.21 – 7.05 (m, 4H); ¹³C{¹H} NMR (126 MHz, CDCl₃) δ 175.0 (s), 166.4 (d, J = 257 Hz), 157.3 (s), 129.4 (s), 118.6 (s), 113.1 (d, J = 12 Hz), 104.5 (d, J = 26 Hz); IR (thin film) 3086, 2931, 1605, 1435, 1257 cm⁻¹; HRMS (ESI) m/z: [M]⁺ Calcd for C₁₃H₆F₂O₂ 232.0336; Found: 232.0334.

General Procedure A.

A mixture of 1 and corresponding amine were heated in a sealed tube in an oil bath at the specified temperature. After the allotted time, the reaction mixture was allowed to slowly cool to room temperature. Any crystallized product was filtered by vacuum filtration and the crystals were removed of inorganic impurities by rinsing with water (3 × 30 mL) while performing vacuum filtration. Reaction mixtures/filtrate containing uncrystallized product were brought up with CH₂Cl₂ (50 mL) and washed with 1M HCl (3 × 25mL), dried with anhydrous Na₂SO₄, concentrated under reduced pressure, and combined with any crystalline product obtained. If impurities were evident, the products were purified via recrystallization or by silica gel chromatography.

General Procedure B.

3,6-difluoroxanthone (1) and each corresponding amine were added to a PTFE-lined stainless steel high-pressure autocleave reactor (10 MPa, 260 °C). The sealed reactor was then heated in an explosion-proof convection oven at the listed temperatures and timeframes. The sealed tank reactor was removed from the oven and allowed to fully cool to r.t. and monitored for completion (via TLC). Upon completion of the reaction, any solid crystalline product was collected via filtration, and removed of inorganic impurities by rinsing with water (3 × 30 mL) while performing vacuum filtration. Uncrystallized product mixtures were brought up with CH₂Cl₂ (50 mL) and washed with 1M HCl (3 × 25mL), dried with anhydrous Na₂SO₄, concentrated under reduced pressure, and combined with any crystalline product obtained. If impurities were evident, the products were purified via recrystallization or by silica gel chromatography.

3,6-bis(dimethylamino)-9H-xanthen-9-one (9).

Method 1.

Using General Procedure A, 1 (500 mg, 2.15 mmol) was heated at 200 °C for 24 h with DMF (4.30 mL, 55.6 mmol) and aqueous KOH (10.0 M, 4.30 mL, 43.0 mmol) to afford 9 (552 mg, 91%) as fine yellow needles. ¹H NMR (CDCl₃, 400 MHz) δ 8.12 (d, J = 8.9 Hz, 2H), 6.67 (dd, J = 8.9 Hz, 2.6 Hz, 2H), 6.46 (d, J = 2.6 Hz, 2H), 3.06 (s, 12H).⁵¹

Method 2.

Using General Procedure B at 200 °C for 20 h, 3,6-difluoroxanthone (1, 500 mg, 2.15 mmol) was heated with DMF (4.30 mL, 55.6 mmol) and aqueous KOH (10.0 M, 4.30 mL, 43.0 mmol) to afford 9 (589 mg, 97%) as fine yellow needles. Product 9 when analyzed matched the ¹H NMR spectra from method 1.

3,6-bis(diethylamino)-9H-xanthen-9-one (10).

Method 1.

Using General Procedure A, 1 (500 mg, 2.15 mmol) was heated at 200 °C for 48 h with HNEt₂ (4.95 mL, 47.9 mmol) to afford 10 (590 mg, 81%) as fine light yellow needles. ¹H NMR (CDCl₃, 400 MHz) δ 8.11 (d, J = 9.0 Hz, 2H), 6.66 (dd, J = 9.0 Hz, 2.1 Hz, 2H), 6.45 (d, J = 2.1 Hz, 2H), 3.46 (q, J = 7.2 Hz, 8H), 1.25 (t, J = 7.2 Hz, 12H).⁵²

Method 2.

Using General Procedure B at 200 °C for 20 h, 3,6-difluoroxanthone (1, 500 mg, 2.15 mmol) was heated with HNEt₂ (4.95 mL, 47.9 mmol) to afford 10 (678 mg, 93%) as fine light yellow needles. Product 10 when analyzed matched the ¹H NMR spectra from method 1.

3,6-bis(isopropyl(methyl)amino)-9H-xanthen-9-one (11).

Method 1.

Using General Procedure A, 1 (500 mg, 2.15 mmol) was heated at 200 °C for 72 h with N-isopropylmethylamine (5.00 mL, 47.9 mmol) to afford 11 (567 mg, 78%) as yellow-orange needles after crystallization in CH₂Cl₂/hexanes. m.p. 95–98 °C; ¹H NMR (CDCl₃, 400 MHz) δ 8.10 (d, J = 9.0 Hz, 2H), 6.74 (dd, J = 9.1, 2.5 Hz, 2H), 6.53 (d, J = 2.4 Hz, 2H), 4.21 (hept, J = 6.6 Hz, 2H), 2.84 (s, 6H), 1.22 (d, J = 6.6 Hz, 12H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 174.9, 158.3, 153.9, 127.5, 111.9, 109.3, 97.3, 48.6, 30.0, 19.6; IR (thin film) 3080, 2927, 2897, 1589, 1437, 1117 cm⁻¹; HRMS (ESI) m/z: [M+H]⁺ Calcd for C₂₁H₂₇N₂O₂ 339.2072; Found: 339.2086.

Method 2.

Using General Procedure B at 200 °C for 20 h, 3,6-difluoroxanthone (1, 500 mg, 2.15 mmol) was heated with N-isopropylmethylamine (5.00 mL, 47.9 mmol) to afford 11 (672 mg, 92%) as a light yellow powder after column chromatography (2% MeOH in CH₂Cl₂). The obtained product when analyzed matched the m.p. and ¹H NMR spectra from method 1.

3,6-bis(ethyl(methyl)amino)-9H-xanthen-9-one (12).

Method 1.

Using General Procedure A, 1 (500 mg, 2.15 mmol) was heated at 200 °C for 72 h with N-ethylmethylamine (4.10 mL, 47.9 mmol) to afford 12 (501 mg, 75%) as brown cubic crystals after crystallization in CH₂Cl₂/hexanes. m.p. 119–122 °C; ¹H NMR (CDCl₃, 400 MHz) δ 8.09 (d, J = 9.1 Hz, 2H), 6.63 (dt, J = 9.1, 2.6 Hz, 2H), 6.42 (t, J = 2.6 Hz, 2H), 3.44 (q, J = 7.0 Hz, 4H), 2.98 (s, 6H), 1.16 (t, J = 7.1 Hz, 6H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 174.9, 158.3, 153.0, 127.6, 111.7, 108.7, 96.7, 46.7, 37.5, 11.6; IR (thin film) 3082, 2968, 2924, 1591, 1435, 1113 cm⁻¹; HRMS (ESI) m/z: [M+H]⁺ Calcd for C₁₉H₂₃N₂O₂ 311.1760; Found: 311.1754.

Method 2.

Using General Procedure B at 200 °C for 20 h, 3,6-difluoroxanthone (1, 1.00 g, 4.30 mmol) was heated with N-ethylmethylamine (8.20 mL, 95.8 mmol) to afford 12 (1.31 g, 98%) as a light yellow powder after column chromatography (3% MeOH in CH₂Cl₂). The obtained product when analyzed matched the m.p. and ¹H NMR spectra from method 1.

3,6-di(azetidin-1-yl)-9H-xanthen-9-one (13).

A mixture of azetidine hypochloride (1.86 g, 19.9 mmol) and aqueous KOH (10.0 M, 2.00 mL, 20.0 mmol) were heated to 70 °C and stirred vigorously for 1 h in a sealed tube. 3,6-difluoroxanthone (1, 500 mg, 2.15 mmol) was added to the reaction mixture and heated at 150 °C for 48 h. The reaction mixture was allowed to cool to room temperature and was treated with cold water (100 mL) to generate a yellow precipitate which was collected via vacuum filtration, rinsed with cold water, and dried overnight under high vacuum to give 13 (501 mg, 76%). m.p. 254–258 °C; ¹H NMR (10% CD₃OD in CDCl₃, 400 MHz) δ 7.94 (d, J = 8.7 Hz, 2H), 6.26 (dd, J = 8.7, 2.2 Hz, 2H), 6.04 (d, J = 2.1 Hz, 2H), 3.92 (t, J = 7.4 Hz, 8H), 2.34 (p, J = 7.4 Hz, 4H); ¹³C{¹H} NMR (101 MHz, 10% CD₃OD in CDCl₃) δ 175.0, 157.2, 154.4, 126.5, 111.0, 107.1, 94.5, 50.5, 15.4; IR (thin film) 2953, 2884, 1618, 1530, 1464, 1352, 1252, 775 cm⁻¹; HRMS (ESI) m/z: [M+H]⁺ Calcd for C₁₉H₁₈N₂O₂ 307.1447; Found: 307.1446.

3,6-di(pyrrolidin-1-yl)-9H-xanthen-9-one (14).

Method 1.

Using General Procedure A, 1 (500 mg, 2.15 mmol) was heated at 150 °C for 1 h with pyrrolidine (4.00 mL, 47.9 mmol) to afford 14 (610 mg, 85%) as yellow crystals. ¹H NMR (CDCl₃, 400 MHz) δ 8.06 (d, J = 8.9 Hz, 2H), 6.48 (dd, J = 8.9, 2.1 Hz, 2H), 6.26 (d, J = 2.2 Hz, 2H), 3.33 (t, J = 6.6 Hz, 8H), 2.00 (t, J = 6.6 Hz, 8H).³⁴

Method 2.

Using General Procedure B at 150 °C for 20 h, 3,6-difluoroxanthone (1, 500 mg, 2.15 mmol) was heated with pyrrolidine (4.00 mL, 47.9 mmol) to afford 14 (712 mg, 99%) as yellow crystals. The obtained product when analyzed matched the ¹H NMR spectra from method 1.

General Procedure C.

Intermediate xanthones (9–14) were added to an oven-dried round bottom flask, then sealed and placed under an Ar atmosphere. The corresponding Grignard reagent (phenylmagnsium chloride or tolylmagnesium chloride), was added at 0 °C in one portion then stirred for the allotted time at room temperature. The resulting mixture was cooled to 0 °C then quenched with MeOH (5.00 mL), concentrated under reduced pressure, and then treated with 5.00 mL 1:11 HCl in MeOH and stirred for 10 min. The acidified mixture was reconcentrated under reduced pressure and purified by column chromoatography (10% MeOH in CH₂Cl₂).

General Procedure D.

2-iodobenzoic acid (1.86 g, 7.50 mmol) was added to an oven-dried round-bottom flask, then sealed and placed under Ar atmosphere. Anhydrous THF (4.00 mL) was added via syringe and the resulting solution was cooled to −78 °C. Isopropylmagnesium chloride lithium chloride complex solution (1.3 M, 11.5 mL, 15.0 mmol) was then added to the flask dropwise, and the resulting mixture was stirred at −78 °C for 30 min. The resulting solution was warmed to 0 °C then was added to corresponding intermediate (9–14) under an Ar environment via syringe. The resulting mixture was stirred at 0 °C for 30 min and then gradually warmed to r.t. and allowed to react for the allotted time period. The resulting mixture was cooled to 0 °C then quenched with MeOH (5.00 mL), concentrated under reduced pressure, and then treated with 5.00 mL 1:11 aqueous HCl (12 M) in MeOH and stirred for 10 min. The acidified mixture was reconcentrated under reduced pressure, dissolved into CH₂Cl₂ (50 mL), treated with aqueous 5.0 M NaOH to premote the more hydrophobic lactone form. The resulting slurry was filtered and remaining solid was washed with 50 mL of CH₂Cl₂. The organic layer of the filtrate was extracted with aqueous 5.0 M NaOH (30 mL × 3) to remove any remaining benzoic acid, reprotonated with 1M HCl (10% in MeOH, 15 mL), and concentrated in vacuo. The resulting oil was purified via column chromoatography (10% MeOH in CH₂Cl₂).

Tetramethylrhodamine chloride (TMR).

Using General Procedure D with a reaction time of 48 h, 3,6-bis(dimethylamino)-9H-xanthen-9-one (9, 212 mg, 0.75 mmol) reacted to produce TMR (292 mg, 92%) after column chromatography as an iridescent dark film; an analytical sample was prepared by dissolving in a minimal amount of EtOH, crystallizing in Et₂O, and filtering the resulting dark iridescent green-purple crystals. ¹H NMR (CDCl₃, 400 MHz) δ 8.21 (d, J = 7.3 Hz, 1H), 7.80 – 7.65 (m, 2H), 7.21 (d, J = 9.2 Hz, 1H), 7.02 (dd, J = 9.1, 2.5 Hz, 2H), 6.92 (d, J = 2.5 Hz, 2H), 3.28 (s, 12 H).

N-(6-(dimethylamino)-9-phenyl-3H-xanthen-3-ylidene)-N-methylmethanaminium chloride (15).

Using General Procedure C with a reaction time of 24 h, 9 (212 mg, 0.75 mmol) was reacted with phenylmagnesium chloride (2.0 M in THF, 1.88 mL, 3.75 mmol) to afford 15 (227 mg, 80%) as an iridescent dark red film that when scraped from the flask forms a dark red powder. ¹H NMR (CDCl₃, 400 MHz) δ 7.78 – 7.56 (m, 3H), 7.52 – 7.25 (m, 4H), 7.36 (d, J = 2.4 Hz, 1H), 7.00 – 6.82 (m, 3H), 3.37 (s, 12H).

N-(6-(dimethylamino)-9-(o-tolyl)-3H-xanthen-3-ylidene)-N-methylmethanaminium chloride (16).

Using General Procedure C with a reaction time of 48 h, 9 (212 mg, 0.75 mmol) was reacted with 2-methylphenylmagnesium chloride (1.0 M in THF, 7.50 mL, 7.50 mmol) to afford 16 (234 mg, 80%) as an iridescent deep purple film that when scraped from the flask forms a dark powder. ¹H NMR (CD₃OD, 400 MHz) δ 7.63 – 7.40 (m, 3H), 7.35 – 7.18 (m, 3H), 7.10 (dd, J = 9.5, 2.5 Hz, 2H), 6.97 (d, J = 2.4 Hz, 2H), 3.32 (s, 12 H), 2.06 (s, 3H).⁴¹

Rhodamine B chloride (RhB).

Using General Procedure D with a reaction time of 48 h, 3,6-bis(diethylamino)-9H-xanthen-9-one (10, 254 mg, 0.75 mmol) reacted to produce RhB (327 mg, 91%) after column chromatography as a dark red film; an analytical sample was prepared by dissolving in a minimal amount of EtOH, crystallizing in Et₂O, and filtering the resulting dark iridescent green-purple crystals. ¹H NMR (DMSO-d₆, 400 MHz) δ 8.23 (d, J = 6.9 Hz, 1H), 7.88 – 7.81 (m, 2H), 7.48 (d, J = 7.4 Hz, 1H), 7.13 – 6.99 (m, 4H), 6.97 (d, J = 2.4 Hz, 2H), 3.65 (q, J = 7.2 Hz, 8 H), 1.22 (t, J = 7.2 Hz, 6H).

N-(6-(diethylamino)-9-phenyl-3H-xanthen-3-ylidene)-N-ethylethanaminium chloride (17).

Using General Procedure C with a reaction time of 24 h, 10 (254 mg, 0.75 mmol) was reacted with phenylmagnesium chloride (2.0 M in THF, 1.88 mL, 3.75 mmol) to afford 17 (281 mg, 86%) as an iridescent dark green film that when scraped from the flask forms a dark powder. ¹H NMR (CDCl₃, 400 MHz) δ 7.66 – 7.60 (m, 3H), 7.39 – 7.35 (m, 2H), 7.36 (d, J = 9.6, 2H), 6.94 (dd, J = 9.6, 2.5 Hz, 2H), 6.86 (d, J = 2.5 Hz, 2H), 3.70 (q, J = 7.2 Hz, 8H), 1.34 (t, J = 7.2 Hz, 12H).⁵³

N-(6-(diethylamino)-9-(o-tolyl)-3H-xanthen-3-ylidene)-N-ethylethanaminium chloride (18).

Using General Procedure C with a reaction time of 48 h, 10 (254 mg, 0.75 mmol) was reacted with 2-methylphenylmagnesium chloride (1.0 M in THF, 7.50 mL, 7.50 mmol) to afford 18 (293 mg, 87%) as an iridescent dark purple film that when scraped from the flask forms a dark powder. ¹H NMR (CDCl₃, 400 MHz) δ 7.55 – 7.39 (m, 3H), 7.17 – 7.12 (m, 3H), 6.86 – 6.77 (m, 4H), 3.61 (q, J = 7.2 Hz, 8H), 2.05 (s, 3H), 1.32 (t, J = 7.1 Hz, 12H).⁴³

N-(9-(2-carboxyphenyl)-6-(ethyl(methyl)amino)-3H-xanthen-3-ylidene)-N-methylethanaminium chloride (19).

Using General Procedure D with a reaction time of 48 h, 3,6-bis(ethyl(methyl)amino)-9H-xanthen-9-one (12, 233 mg, 0.75 mmol) reacted to produce 19 (311 mg, 92%) after column chromatography as an iridescent dark film; an analytical sample was prepared by dissolving in a minimal amount of EtOH, crystallizing in Et₂O, and filtering the resulting fine burgundy colored crystals. ¹H NMR (10% CD₃OD in CDCl₃, 400 MHz) δ 8.33 (apps, 1H), 7.90 – 7.66 (m, 2H), 7.30 (apps, 1H), 7.13 (s, 2H), 7.03 – 6.70 (m, 4H), 3.68 (brq, J = 7.2 Hz, 4H), 3.28 (s, 6H), 1.32 (brt, J = 7.2 Hz, 4H); ¹³C{¹H} NMR (101 MHz, 10% CD₃OD in CDCl₃) δ 166.4, 160.3, 157.8, 156.4, 133.6, 132.7, 131.7, 131.4, 131.2, 130.4, 130.1, 114.2, 113.8, 96.6, 48.3, 39.2, 12.3; IR (thin film) 3371, 3062, 2970, 2931, 1705, 1589, 1334, 1180 cm⁻¹; HRMS (ESI) m/z: [M]⁺ Calcd for C₂₆H₂₇N₂O₃ 415.2022; Found: 415.2026.

N-(6-(ethyl(methyl)amino)-9-phenyl-3H-xanthen-3-ylidene)-N-methylethanaminium chloride (20).

Using General Procedure C with a reaction time of 24 h, 12 (233 mg, 0.75 mmol) was reacted with phenylmagnesium chloride (2.0 M in THF, 1.88 mL, 3.75 mmol) to afford 20 (232 mg, 76%) as an iridescent purple film that when scraped from the flask forms a dark powder. ¹H NMR (CDCl₃, 400 MHz) δ 7.45 – 7.35 (m, 3H), 7.23 – 7.02 (m, 4H), 6.79 (dd, J = 9.6, 2.6 Hz, 2H), 6.61 (d, J = 2.8 Hz, 2H), 3.50 (q, J = 7.8 Hz, 4H), 3.09 (s), 1.09 (t, J = 7.8 Hz, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 157.7, 157.4, 156.2, 131.8, 130.2, 129.20, 129.18, 128.8, 114.2, 113.1, 96.4, 47.9, 38.8, 11.9; IR (thin film) 3317, 3047, 2947, 2870, 1589, 1327, 1150 cm⁻¹; HRMS (ESI) m/z: [M]⁺ Calcd for C₂₅H₂₇N₂O 371.2123; Found: 371.2108.

N-(6-(ethyl(methyl)amino)-9-(o-tolyl)-3H-xanthen-3-ylidene)-N-methylethanaminium chloride (21).

Using General Procedure C with a reaction time of 48 h, 12 (233 mg, 0.75 mmol) was reacted with 2-methylphenylmagnesium chloride (1.0 M in THF, 7.50 mL, 7.50 mmol) to afford 21 (274 mg, 87%) as an iridescent purple film that when scraped from the flask forms a shiny dark powder; ¹H NMR (CDCl₃, 400 MHz) δ 7.51 (td, J = 7.5, 1.4 Hz, 1H), 7.46 – 7.38 (m, 2H), 7.23 – 7.14 (m, 3H), 7.02 (dd, J = 9.5, 2.4 Hz, 2H), 6.87 (d, J = 2.5 Hz, 2H), 3.75 (q, J = 7.2 Hz, 4H), 3.35 (s, 6H), 2.06 (s, 3H), 1.34 (t, J = 7.1 Hz, 6H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 157.8, 157.7, 156.4, 135.6, 131.4, 131.3, 130.7, 130.0, 128.6, 126.0, 114.4, 113.3, 96.5, 48.0, 38.9, 19.5, 12.0; IR (thin film) 3363, 2970, 2924, 1582, 1458, 1335, 1180 cm⁻¹; HRMS (ESI) m/z: [M]⁺ Calcd for C₂₆H₂₉N₂O 385.2280; Found: 385.2286.

N-(9-(2-carboxyphenyl)-6-(isopropyl(methyl)amino)-3H-xanthen-3-ylidene)-N-methylpropan-2-aminium chloride (22).

Using General Procedure D with a reaction time of 48 h, 3,6-bis(isopropyl(methyl)amino)-9H-xanthen-9-one (11, 254 mg, 0.75 mmol) reacted to produce 22 (298 mg, 83%) after column chromatography as a burgundy red film that when scraped from the flask forms a deep red powder; ¹H NMR (CDCl₃, 400 MHz) δ 8.02 (d, J = 7.3 Hz, 1H), 7.60 – 7.46 (m, 2H), 7.11 (d, J = 7.1 Hz, 1H), 6.67 (d, J = 8.9 Hz, 2H), 6.58 – 6.45 (m, 4H), 4.11 (hept, J = 13.2 Hz, 2H), 2.76 (s, 6H), 1.13 (d, J = 6.5 Hz, 12H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 167.9, 153.6, 152.21, 152.18, 132.4, 131.4, 128.7, 128.4, 125.9, 124.7, 109.6, 108.0, 104.0, 97.1, 48.0, 29.2, 18.6; IR (thin film) 3369, 3047, 2970, 1751, 1589, 1335, 1103, 694 cm⁻¹; HRMS (ESI) m/z: [M]⁺ Calcd for C₂₈H₃₁N₂O₃ 443.2335; Found: 443.2331.

N-(6-(isopropyl(methyl)amino)-9-phenyl-3H-xanthen-3-ylidene)-N-methylpropan-2-aminium chloride (23).

Using General Procedure C with a reaction time of 24 h, 3,6-bis(isopropyl(methyl)amino)-9H-xanthen-9-one (11, 254 mg, 0.75 mmol) was reacted with phenylmagnesium chloride (2.0 M in THF, 1.88 mL, 3.75 mmol) to afford 23 (281 mg, 86%) after column chromatography as a deep purple film that when scraped from the flask forms a dark powder; ¹H NMR (10% CD₃OD in CDCl₃, 400 MHz) δ 7.65 – 7.54 (m, 3H), 7.40 – 7.24 (m, 4H), 6.95 (dd, J = 9.7, 2.6 Hz, 2H), 6.84 (d, J = 2.5 Hz, 2H), 4.35 (hept, J = 6.8 Hz, 2H), 3.02 (s, 6H), 1.27 (d, J = 6.8 Hz, 12H); ¹³C{¹H} NMR (101 MHz, 10% CD₃OD in CDCl₃) δ 157.1, 156.8, 155.9, 131.0, 130.7, 129.6, 128.4, 128.0, 113.4, 112.52, 95.9, 49.9, 30.2, 18.7; IR (thin film) 3395, 3021, 2970, 2931, 1589, 1481, 1342, 1111 cm⁻¹; HRMS (ESI) m/z: [M]⁺ Calcd for C₂₇H₃₁N₂O 399.2437; Found: 399.2433.

N-(6-(isopropyl(methyl)amino)-9-(o-tolyl)-3H-xanthen-3-ylidene)-N-methylpropan-2-aminium chloride (24).

Using General Procedure C with a reaction time of 48 h, 3,6-bis(isopropyl(methyl)amino)-9H-xanthen-9-one (11, 254 mg, 0.75 mmol) was reacted with 2-methylphenylmagnesium chloride (1.0 M in THF, 7.50 mL, 7.50 mmol) to afford 24 (273 mg, 81%) after column chromatography as a deep purple film that when scraped from the flask forms a shiny dark powder; ¹H NMR (CDCl₃, 400 MHz) δ 7.51 (dd, J = 7.5, 4.9 Hz, 1H), 7.48 – 7.38 (m, 2H), 7.23 – 7.07 (m, 5H), 6.99 – 6.95 (m, 2H), 4.63 – 4.33 (m, 2H), 3.15 (hept, J = 7.0 Hz, 2H), 2.06 (s, 3H), 1.37 (d, J = 7.0, 12H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 157.8, 157.6, 156.8, 135.6, 131.4, 130.7, 130.0, 128.6, 126.1, 114.6, 113.5, 105.0, 96.8, 50.2, 31.4, 19.7; IR (thin film) 3402, 3078, 2970, 2924, 1589, 1342, 1180, 1119 cm⁻¹; HRMS (ESI) m/z: [M]⁺ Calcd for C₂₈H₃₃N₂O 413.2593; Found: 413.2585.

Janelia Fluor 549 chloride (JF₅₄₉).

Using General Procedure D with a reaction time of 48 h, 3,6-di(azetidin-1-yl)-9H-xanthen-9-one (13, 230 mg, 0.75 mmol) reacted to produce JF₅₄₉ (298 mg, 89%) after column chromatography as an iridescent dark film; an analytical sample was prepared by dissolving in a minimal amount of EtOH, crystallizing in Et₂O, and filtering the resulting deep red crystals. ¹H NMR (CDCl₃, 400 MHz) δ 8.01 (d, J = 7.4 Hz, 1H), 7.68 – 7.58 (m, 2H), 7.16 (d, J = 7.4 Hz, 1H), 6.62 (d, J = 8.4 Hz, 2H), 6.24 (s, 2H), 6.15 (d, J = 8.4 Hz, 2H), 3.98 – 3.87 (m, 8H), 2.48 – 2.31 (m, 4H).⁵⁴

1-(6-(azetidin-1-yl)-9-phenyl-3H-xanthen-3-ylidene)azetidin-1-ium chloride (25).

Using General Procedure C with a reaction time of 24 h, 3,6-di(azetidin-1-yl)-9H-xanthen-9-one (13, 230 mg, 0.75 mmol) was reacted with phenylmagnesium chloride (2.0 M in THF, 1.88 mL, 3.75 mmol) to afford 25 (263 mg, 87%) after column chromatography as a red iridescent film that when scraped from the flask forms a dark powder; ¹H NMR (CDCl₃, 400 MHz) δ 7.63 (apps, 3H), 7.35 (apps, 2H), 7.27 (d, J = 9.4 Hz, 2H), 6.60 (d, J = 9.2 Hz, 2H), 6.44 (s, 2H), 4.51 – 4.17 (m, 8H), 2.75 – 2.45 (m, 4H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 157.5, 157.3, 156.1, 131.76, 131.75, 130.1, 129.1, 128.8, 113.2, 112.4, 94.3, 51.8, 15.9; IR (thin film) 3317, 2980, 1597, 1489, 1296, 1180 cm⁻¹; HRMS (ESI) m/z: [M]⁺ Calcd for C₂₅H₂₃N₂O 367.1810; Found: 367.1794.

1-(6-(azetidin-1-yl)-9-(o-tolyl)-3H-xanthen-3-ylidene)azetidin-1-ium chloride (26).

Using General Procedure C with a reaction time of 48 h, 3,6-di(azetidin-1-yl)-9H-xanthen-9-one (13, 230 mg, 0.75 mmol) was reacted with 2-methylphenylmagnesium chloride (1.0 M in THF, 7.50 mL, 7.50 mmol) to afford 26 (263 mg, 84%) after column chromatography as a deep purple iridescent film that when scraped from the flask forms a dark powder. ¹H NMR (CD₃OD, 400 MHz) δ 7.57 (td, J = 7.4, 1.3 Hz, 2H), 7.52 – 7.40 (m, 2H), 7.22 (dd, J = 7.5, 1.3 Hz, 1H), 7.11 (d, J = 8.9 Hz, 2H), 6.64 (dd, J = 8.9, 2.2 Hz, 2H), 6.57 (d, J = 2.2 Hz, 2H), 4.40 – 4.28 (m, 8H), 2.63 – 2.49 (m, 4H), 2.05 (s, 3H).⁴⁹

1-(9-(2-carboxyphenyl)-6-(pyrrolidin-1-yl)-3H-xanthen-3-ylidene)pyrrolidin-1-ium chloride (27).

2-iodobenzoic acid (1.86 g, 7.50 mmol) was added to an oven-dried round-bottom flask, then sealed and placed under Ar atmosphere. Anhydrous THF (4.00 mL) was added via syringe and the resulting solution was cooled to −78 °C. Isopropylmagnesium chloride lithium chloride complex solution (1.3 M, 11.5 mL, 15.0 mmol) was then added to the flask dropwise, and the resulting mixture was stirred at −78 °C for 30 min. The resulting solution was warmed to 0 °C then was added via syringe to 3,6-di(pyrrolidin-1-yl)-9H-xanthen-9-one (14, 251 mg, 0.75 mmol) in 4.0 mL of HMPA under an Ar environment. The resulting mixture was stirred at 0 °C for 30 min and then gradually warmed to r.t. and allowed to react for 72 h. The resulting mixture was cooled to 0 °C then quenched with MeOH (5.00 mL), concentrated under reduced pressure (rotoevaporation at 5 torr followed by 0.1 torr to remove HMPA), and then treated with 5.00 mL 1:11 aqueous HCl (12 M) in MeOH and stirred for 10 min. The acidified mixture was reconcentrated under reduced pressure, dissolved into CH₂Cl₂ (50 mL), treated with aqueous 5.0 M NaOH to premote the more hydrophobic lactone form. The resulting slurry was filtered and remaining solid was washed with 50 mL of CH₂Cl₂. The organic layer of the filtrate was extracted with aqueous 5.0 M NaOH (30 mL × 3) to remove any remaining benzoic acid, reprotonated with 1M HCl (10% in MeOH, 15 mL), and concentrated in vacuo. The resulting semi-solid material was purified via column chromoatography (10% MeOH in CH₂Cl₂) to afford 27 (314 mg, 88%) as an iridescent dark film dark purple film; an analytical sample was prepared by dissolving in a minimal amount of EtOH, crystallizing in Et₂O, and filtering the resulting burgundy colored crystals. ¹H NMR (CD₃OD, 400 MHz) δ 8.12 (d, J = 7.2 Hz, 1H), 7.70 – 7.63 (m, 2H), 7.31 – 7.20 (m, 3H), 6.82 (dd, J = 9.3, 2.1 Hz, 2H), 6.76 (d, J = 2.2 Hz, 2H), 3.65 – 3.49 (m, 8H), 2.19 – 2.06 (m, 8H).⁵⁵

1-(9-phenyl-6-(pyrrolidin-1-yl)-3H-xanthen-3-ylidene)pyrrolidin-1-ium chloride (28).

Using General Procedure C with a reaction time of 24 h, 14 (251 mg, 0.75 mmol) was reacted with phenylmagnesium chloride (2.0 M in THF, 1.88 mL, 3.75 mmol) to afford 28 (203 mg, 63%) after column chromatography as a purple-green iridescent film that when scraped from the flask forms a shiny dark powder. ¹H NMR (CDCl₃, 400 MHz) δ 7.48 – 7.37 (m, 3H), 7.29 – 7.12 (m, 4H), 6.93 (dd, J = 9.5, 2.3 Hz, 2H), 6.79 (d, J = 2.3 Hz, 2H), 3.39 – 3.30 (m, 8H), 2.00 (t, J = 7.0 Hz, 8H).⁵⁰

1-(6-(pyrrolidin-1-yl)-9-(o-tolyl)-3H-xanthen-3-ylidene)pyrrolidin-1-ium chloride (29).

Using General Procedure C with a reaction time of 72 h, 14 (251 mg, 0.75 mmol) was reacted with 2-methylphenylmagnesium chloride (1.0 M in THF, 7.50 mL, 7.50 mmol) to afford 29 (200 mg, 60%) after column chromatography as a purple-green iridescent film that when scraped from the flask forms a shiny dark powder. ¹H NMR (CD₃OD, 400 MHz) δ 7.58 – 7.40 (m, 3H), 7.26 (d, J = 9.5 Hz, 3H), 6.94 (d, J = 9.5, 2.1 Hz, 2H), 6.85 (d, J = 2.1 Hz, 2H), 3.65 – 3.58 (m, 8H), 2.20 – 2.13 (m, 8H), 2.05 (s, 3H).³⁴