Syntheses and properties of water-soluble Nile Red derivatives

Article abstract of DOI:10.1021/jo061369v

(Chemical Equation Presented) Nile Red (compound A) fluoresces at about 530 nm with good quantum yields in apolar solvents. In more polar ones its fluorescence emission shows a dramatic, and potentially useful, shift to about 640 nm, but its quantum yield

Full text of DOI:10.1021/jo061369v

Syntheses and Properties of Water-Soluble Nile Red Derivatives
Jiney Jose and Kevin Burgess*
Department of Chemistry, Texas A&M UniVersity, Box 30012, College Station, Texas 77841
burgess@tamu.edu
ReceiVed July 1, 2006
Nile Red (compound A) fluoresces at about 530 nm with good quantum yields in apolar solvents. In
more polar ones its fluorescence emission shows a dramatic, and potentially useful, shift to about 640
nm, but its quantum yield is significantly reduced. Further, Nile Red has a very poor solubility in aqueous
media. The hypothesis tested in this paper is that Nile Red derivatives that incorporate water-solubilizing
groups will tend to fluoresce with good quantum yields in aqueous media, and in the more useful
wavelength range around 640 nm. Thus three Nile Red derivatives, 1-3, were prepared. Compound 1
had three hydroxyl groups more than Nile Red, but was surprisingly insoluble in aqueous media. However,
the dicarboxylic acid 2 and carboxylic/sulfonic acid derivative 3 showed excellent water solubilities.
Spectral data for 2 and 3 showed that they do indeed fluoresce with good quantum yields in the 640 nm
region in aqueous media. These properties of compounds 2 and 3 might be useful in the development of
fluorescent probes for biotechnology.
Introduction
development of probes for intracellular imaging, for instance,
where emissions become increasingly more detectable with
wavelengths longer than that associated with autofluorescence
in the cell (typically above 550 nm).
Nile Red, compound A, is a well-known fluorescent dye with
remarkable solvatochromic properties.1-3 In apolar solvents it
fluoresces with a high quantum yield in the region of 530 nm,
but in polar solvents the quantum yield is dramatically reduced
and the emission maximum shows bathochromic shifts of
approximately 100 nm.4 This red-shift characteristic has been
used extensively as a fluorescent reporter on the environment
around molecules labeled with this dye. In other contexts,
however, it would be desirable to use Nile Red derivatives as
labels for applications in biotechnology,5 especially if the large
bathochromic shift could be retained without significantly
reducing the quantum yield. This is particularly true for
Unfortunately, Nile Red itself has very poor solubility in
aqueous media,1 so it is not a particularly useful dye for labeling
most biomolecules. There has been some effort to prepare related
water-soluble derivatives of benzo[a]phenoxazinium, “Nile
Blue”, systems B6,7 but very little consideration has been devoted
to development of Nile Red derivatives that could be used in
aqueous media.8 The closest published research that we are
aware of in this area is that by Briggs and co-workers at
Amersham Co. featuring the 2-hydroxy Nile Red derivatives C
and D.9,10 Their studies did not focus on the properties of these
dyes in water (the data given were in methanol), so questions
(1) Greenspan, P.; Fowler, S. D. J. Lipid Res. 1985, 26, 781-789.
(2) Deda, M. L.; Ghedini, M.; Aiello, I.; Pugliese, T.; Barigelletti, F.;
Accorsi, G. J. Organomet. Chem. 2005, 690, 857-861.
(3) Datta, A.; Mandal, D.; Pal, S. K.; Bhattacharyya, K. J. Phys. Chem.
B 1997, 101, 10221-10225.
(4) Golini, C. M.; Williams, B. W.; Foresman, J. B. J. Fluoresc. 1998,
8, 395-404.
(5) Okamoto, A.; Tainaka, K.; Saito, I. Tetrahedron Lett. 2003, 44, 6871-
6874.
(6) Ho, N.-H.; Weissleder, R.; Tung, C.-H. Tetrahedron 2006, 62, 578-
585.
(7) Xiongwei Yan; Yuan, P. M., U.S. Patent 6465644, 2002.
(8) Long, J.; Wang, Y. M.; Meng, J. B. Chin. Chem. Lett. 1999, 10,
659-660.
(9) Briggs, M. S. J.; Bruce, I.; Miller, J. N.; Moody, C. J.; Simmonds,
A. C.; Swann, E. J. Chem. Soc., Perkin Trans. 1 1997, 7, 1051-1058.
10.1021/jo061369v CCC: $33.50 © 2006 American Chemical Society
Published on Web 09/02/2006
J. Org. Chem. 2006, 71, 7835-7839
7835

Jose and Burgess
“through-bond energy transfer cassettes”,12-14 water-soluble Nile
Red derivatives could be useful acceptors for syntheses of
donor-acceptor cassettes.
Results and Discussion
relating to bathochromic shifts and reduction of quantum yields
in aqueous media were not addressed. Our own unpublished
observations (made in the course of this study), however, show
that one of these derivatives, 2-hydroxy Nile Red, is not
significantly soluble in aqueous media.
Synthesis of Nile Red Derivatives 1-3. The synthesis
developed for target dye 1 is shown in Scheme 1. 3-Amino-
phenol is known to undergo a facile Michael addition with
acrylic acid to give a product that crystallizes out of the reaction
mixture. This reaction gives good quality material on a
multigram scale without necessitating chromatography. Esteri-
fication of the diacid formed in this way gives the diester 4,
which was isolated in good yields by an extraction procedure.
Lithium aluminum hydride reduction of 4 gave the correspond-
ing diol 5 that was then nitrosylated under aqueous conditions.
The nitroso product 6 proved to be somewhat unstable so it
was used without further purification. Assembly of the benzo-
[a]phenoxazinone skeleton was achieved via condensation of
the nitroso compound 6 with 1,6-dihydroxynaphthalene. The
product 1 was isolated via flash chromatography. This was the
only chromatographic step used in the synthesis so it proved
convenient to make this product on a several gram scale.
Scheme 2 shows the synthesis that was developed for the
target dye 2. Nitrosylation of the diester 4 (see below) afforded
7, which was used without purification due to stability and
hygroscopicity issues. Condensation of this nitroso compound
with 1,6-dihydroxynaphthalene gave benzo[a]phenoxazinone 8.
This compound was isolated via flash chromatography (the first
and only one in the sequence). Aqueous hydrolysis of the diester
functionalities of 8 gave the corresponding diacid 2 that could
be isolated via a simple acid base extraction procedure.
This paper concerns development of Nile Red derivatives that
have (i) water solubility, (ii) significant quantum yields in
aqueous media, (iii) fluoresce over 600 nm (the more useful
region for imaging in cells and tissues), and (iv) functional
groups that facilitate attachment to biomolecules. Water solubil-
ity was identified as the critical factor to reach most of these
goals, for the following reason. It is generally accepted that the
reduced quantum yields of Nile Red in polar media are due to
aggregation of the hydrophobic, flat, benzo[a]phenoxazinone
core structures.11 Logically, this type of aggregation could be
suppressed or prevented by introducing water-solubilizing
groups into the dye structure. In the context of this research,
that might impart water solubility, increase the quantum yields,
and facilitate the attachment of these dyes to water-soluble
biomolecules. Effects on fluorescence emission maxima that
occur by making the Nile Red scaffold water-soluble were
unknowns that had to be tested, but we speculated that the
desirable red-shift in the fluorescence maxima that is observed
for Nile Red in polar media was less likely to be due to an
aggregation effect than to stabilization of a relatively polarized
excited state.
The considerations discussed above led us to attempt syn-
theses of the Nile Red derivatives 1-3, and investigate their
fluorescence properties in aqueous media (if possible). As stated
above, the immediate objectives of this study were to identify
water-soluble Nile Red derivatives that exhibit fluorescence
emission maxima above 600 nm and significant quantum yields
in aqueous media; dyes of this type could have general
applications in biotechnology. In the more focused context of
our research on fluorescent dyes, i.e., development of the
Preparation of the final target, the sulfonic acid 3, is outlined
in Scheme 3. Michael addition of acrylic acid with 3-aminophenol
in near stoichiometric amounts gave the monoadduct 9. Reaction
of this amine with 1,3-propane sultone gave the ring-opened
product 10. This was nitrosylated to give the very unstable nitro-
syl compound 11, which was then condensed as above without
(12) Burgess, K.; Burghart, A.; Chen, J.; Wan, C.-W. In Proc. SPIE-
Int. Soc. Opt. Eng. 2000.
(13) Burghart, A.; Thoresen, L. H.; Chen, J.; Burgess, K.; Bergstro¨m,
F.; Johansson, L. B.-A. Chem. Commun. 2000, 2203-2204.
(14) Jiao, G.-S.; Thoresen, L. H.; Kim, T. G.; Haaland, W. C.; Gao, F.;
Topp, M. R.; Hochstrasser, R. M.; Metzker, M. L.; Burgess, K. Chem. Eur.
J. 2006, in press.
(10) Simmonds, A. C.; Miller, J. N.; Moody, C. J.; Swann, E.; Briggs,
M. S. J.; Bruce, I., Patent WO 9729154, 1997.
(11) Valdes-Aguilera, O.; Neckers, D. C. Acc. Chem. Res. 1989, 22, 171-
177.
7836 J. Org. Chem., Vol. 71, No. 20, 2006

Water-Soluble Nile Red DeriVatiVes
SCHEME 1
SCHEME 3
TABLE 1. Spectroscopic Properties of the Nile Red Derivatives
1-3
λabs
(nm)
ꢀ
λem
(nm)
fwhm
(nm)
dye
(M-1 cm-1
)
Φa
solvent
1
2
2
2
3
3
3
542
520
560
556
548
558
556
24854
4984
7529
3400
5283
7876
6209
631
632
648
647
632
652
650
60
59
63
70
56
60
66
0.56
0.43
0.33
0.07
0.42
0.37
0.18
EtOH
EtOH
b
c
EtOH
b
c
SCHEME 2
a Measured as specified previously.16 b pH 7.4 phosphate buffer. c pH
9.0 borate buffer. Standards used for quantum yield measurement of
rhodamine B (Φ ) 0.97 in ethanol) and rhodamine 101 (Φ ) 1.0 in ethanol).
Spectroscopic Properties of Nile Red Derivatives 1-3.
In water, the Nile Red derivative 1 showed hardly any solu-
bility at all. Surprisingly, despite having a phenolic-OH, this
compound was still insoluble in pH 9 borate buffer (1 M) or
in sodium carbonate solution. Consequently, spectroscopic data
for this compound were only recorded in EtOH (Table 1).
Compound 1 was shown to have a similar absorption and
emission wavelength maxima, and a slightly better quantum
yield than Nile Red A or the hydroxy Nile Red derivative C
in EtOH.
Compound 2 was more interesting for the purposes of this
study. This dye has good solubility (for spectroscopic purposes)
in EtOH, water, 1 M phosphate buffer at pH 7.4, and 1 M borate
buffer at pH 9.0. In EtOH, its spectral properties are similar to
those of compounds A, C, and 1 except that the molar
absorptivity was measured to be approximately one-fifth that
of 1, and the Stokes shift was slightly greater (2, 110 nm; 1, 90
nm). In aqueous buffers, however, the Stokes shifts for 1 and 2
were very similar. The quantum yield of 2 at pH 7.4 was quite
good (0.33), but at pH 9 it reduced to 0.07. This appears to
correlate with deprotonation of the phenolic hydroxyl, possibly
delay. Unfortunately, flash chromatography was used in each
step of this procedure but, even so, the target material 3 was
isolated on about a 250-mg scale. Repetition of the sequence
on a larger scale was not attempted here, but we suspect that
1-3 g of material could easily be made via this approach.
J. Org. Chem, Vol. 71, No. 20, 2006 7837

Jose and Burgess
FIGURE 1. Absorption at 10-3 M (dashed lines) and fluorescence at 10-5 M (solid lines) of dyes 1-3 (a) in ethanol and dyes 2-3 (b) in phosphate
buffer (pH 7.4) and (c) in borate buffer (pH 9.0).
corresponding to quenching of the fluorescence via charge
transfer.15 The full width at half-maximum height (fwhm) of
the emission for 2 is 70 nm or less. Sharp fluorescence emissions
facilitate resolution in experiments involving more than one
probe. The fwhm for 2 compares favorably with fwhm reported
for the water soluble Nile Blue derivatives mentioned earlier
(they have fwhm in the range of 86-93 nm).6
The sulfonic acid dye 3 also had good solubility in the three
media studied (Table 1). In EtOH its spectroscopic properties
were unexceptional, and the quantum yield was quite good
(0.42). In both the neutral and basic buffers the absorption and
emission maxima were red-shifted but by only 18 nm or less
relative to ethanolic solutions of the dye. Interestingly, the
quantum yield of 3 was comparable in EtOH or pH 7.4 buffer,
and still quite good (0.18) at pH 9, and almost double that of 2
in this medium. Moreover, the fwhm of the fluorescence
emission peaks for the aqueous buffers were slightly lower than
those for compound 2.
Experimental Section
Syntheses of Compounds 1-11: 3-[(2-Carboxyethyl)(3-hy-
droxyphenyl)amino]propionic Acid.17 A solution of 3-amino-
phenol (109.0 g, 1 mol) in acrylic acid (185 mL, 3 mmol) and
water (90 mL) was heated to 70 °C for 3 h. The reaction mixture
was cooled and ethanol (180 mL) was added and kept at 5 °C for
12 h. The white precipitate that formed was filtered, washed with
ethanol (50 mL), and dried to obtain dicarboxylic acid (200.0 g,
80%). Mp 153-154 °C (lit. mp 149-150 °C). 1H NMR (300 MHz,
acetone-d6) δ 7.02-6.98 (m, 1H), 6.28-6.24 (m, 2H), 6.19-6.17
(m, 1H), 3.64 (t, 4H, J ) 4.5 Hz), 2.59 (t, 4H, J ) 4.5 Hz). 13C
NMR (75 MHz, acetone-d6) δ 172.6, 158.6, 148.6, 130.0, 104.0,
104.2, 99.6, 46.8, 31.7. MS (ESI): 253.04 (M+).
Compound 4. A solution of the dicarboxylic acid as prepared
above (10.0 g, 39.5 mmol) in methanol (500 mL) along with HCl
(10.0 M, 1 mL) was refluxed for 12 h. The reaction mixture was
cooled and the MeOH was evaporated under reduced pressure. The
residue was dissolved in EtOAc (100 mL) and the organic layer
was washed with water (5 × 20 mL). The organic layer was
evaporated to dryness under reduced pressure to yield 4 as a yellow
semisolid (7.0 g, 63%). Rf 0.7 (50% EtOAc/hexane). 1H NMR (300
MHz, CD3OD) δ 7.05-6.95 (m, 1H), 6.25-6.15 (m, 3H), 3.65 (s,
6H), 3.59 (t, 4H, J ) 7.2 Hz), 2.58 (t, 4H, J ) 7.2 Hz). 13C NMR
(75 MHz, CD3OD) δ 173.3, 158.4, 148.5, 130.1, 104.6, 104.2, 99.9,
51.1, 46.9, 32.1. MS (ESI): 281.36 (M+). IR (neat) 3442, 2960,
Conclusions
All three of the dyes that are reported here are conveniently
accessible via syntheses that involve about 5 steps. Compounds
1 and 2 can be produced on multigram scales since each
synthesis only involves one chromatographic purification. The
route to dye 3 that was developed here is less amenable to scale-
up than the syntheses of the other two dyes, but, nevertheless,
usable quantities are accessible.
Two of the three dyes, i.e., 2 and 3, have solubilities that are
satisfactory for applications as biological markers. They both
have carboxylic acid functionalities, and these could be activated
and used to conjugate the dyes to biomolecules. The fluores-
cence of dye 2 is significantly diminished under basic conditions,
but this effect is less for 3. Applications in biotechnology involve
near-neutral pH values much more frequently than basic ones
so this characteristic is not a serious drawback for dye 2. Under
near-neutral conditions, the quantum yields for 2 and 3 are good
(>0.3), and they emit fluorescence as reasonably sharp peaks.
Overall, dyes 2 and 3 retain the desirable bathochromic shift
that Nile Red exhibits in alcoholic media relative to apolar ones,
except that they can be dissolved in aqueous salt solutions.
Further, the undesirable reduction of quantum yield that is
observed for Nile Red on moving to more polar media is not a
significant issue for the water-soluble dyes.
2362, 1741 cm-1
.
Compound 5. A solution of 4 (1.0 g, 3.6 mmol) in THF (15
mL) was added dropwise at 0 °C under vigorous stirring to a
solution of LiAlH4 (820 mg, 21.6 mmol) in THF (10 mL) in a
three-necked flask fitted with a reflux condenser (the reaction is
exothermic); a thick white precipitate was formed almost im-
mediately, and TLC (1:1 hexane/EtOAc) after 2 h showed complete
disappearance of 4. The reaction mixture was quenched with water
and filtered to remove the solid residues. The resulting filtrate was
concentrated under reduced pressure to yield 5 (730 mg, 90%) as
a white solid. Rf 0.2 (1:1hexane/EtOAc). 1H NMR (300 MHz, CD3-
OD) δ 6.31 (t, 1H, J ) 8.1 Hz), 5. 62 (s, 1H), 5.56 (d, 1H, J ) 7.2
Hz), 5.52 (d, 1H, J ) 8.1 Hz) 3.12 (t, 4H, J ) 6.0 Hz), 2.81-2.73
(m, 4 H), 1.33 (t, 4 H, J ) 6.3 Hz). 13C NMR (75 MHz, CD3OD)
δ 167.5, 149.9, 128.9, 108.9, 105.1, 101.5, 60.0, 48.7, 30.3. MS
(ESI): 225.13 (M+). IR (neat) 3350, 2921, 2854 cm-1
.
Compound 6. Sodium nitrite (0.92 g, 13.3 mmol) in water (11.2
mL) was added, over a period of 1 h, via a syringe pump at the
rate of 0.2 mL per min, to a solution of 5 (2.0 g, 8.9 mmol) in HCl
(9.0 mL, 10.0 M) and water (4.5 mL) at 0 °C. The mixture was
stirred for 2.5 h at 0 °C and filtered to remove residual impurities.
(16) Williams, A. T. R.; Winfield, S. A.; Miller, J. N. Analyst 1983,
108, 1067-1071.
(15) Nagy, K.; Gokturk, S.; Biczok, L. J. Phys. Chem. A 2003, 107,
8784-8790.
(17) Mitskyavichyus, V. Y. Chem. Heterocycl. Compd. 1996, 32, 456-
458.
7838 J. Org. Chem., Vol. 71, No. 20, 2006

Water-Soluble Nile Red DeriVatiVes
The filtrate was evaporated under reduced pressure, the residue
dissolved in methanol, dried with magnesium sulfate, and filtered,
and methanol was evaporated to yield 6 (1.54 g, 74%). This
somewhat unstable, hygroscopic nitroso compound was used in the
next step without further purification. Rf 0.4 (90% EtOAc/MeOH).
1H NMR (300 MHz, CD3OD) δ 7.70 (d, 1H, J ) 10.2 Hz), 7.26
(d, 1H, J ) 10.2 Hz), 6.49 (s, 1H), 4.02 (d, 2H, J ) 7.4 Hz), 3.98
131.5, 128.2, 125.0, 124.4, 119.3, 110.9, 108.9, 105.0, 97.5, 47.2,
32.6. MS (ESI): 421.01 (M - H)-. IR (neat) 3425, 3059, 2928,
1637 cm-1
.
Compound 9. 3-Aminophenol (5.0 g, 4.6 mmol) in acrylic acid
(4.7 mL, 6.9 mmol) and water (3 mL) was heated to 70 °C for 12
h. The reaction mixture was cooled to room temperature then the
solvent was evaporated under reduced pressure. The residual
material was purified by flash chromatography eluting with 1:1
hexane/EtOAc to afford 9 as a white semisolid (3.3 g, 40%). Rf
0.3 (EtOAc). 1H NMR (300 MHz, CD3OD) δ 6.93 (s, 1H), 6.21-
6.12 (m, 3H), 4.31-4.27 (m, 1H), 3.34-3.30 (m, 2H), 2.64-2.52
(m, 2H). 13C NMR (75 MHz, CD3OD) δ 175.1, 158.1, 149.6, 129.8,
105.5, 104.8, 100.2, 39.8, 33.5. MS (ESI): 181.08 (M+).
(d, 2H, J ) 7.4 Hz), 3.81-3.66 (br, 4H), 2.02-1.86 (br, 4H). 13
C
NMR (75 MHz, CD3OD) δ 166.1, 163.6, 144.8, 123.4, 120.0, 98.0,
58.3, 51.1, 31.8. MS (ESI): 254.24 (M+).
Compound 1. Compound 6 (1.0 g, 3.5 mmol) was dissolved in
50 mL of dry distilled DMF and solid 1,6-dihydroxynaphthol then
HCl (2 mL, 10.0 M) were added in that order. The reaction mixture
was heated to 130 °C for 5 h and cooled to room temperature, then
the DMF was removed under reduced pressure. The residual mater-
ial was purified by flash chromatography eluting with 1:10 MeOH/
EtOAc to afford 1 as a red solid (740 mg, 54%). Rf 0.3 (90%EtOAc/
MeOH). 1H NMR (300 MHz, DMSO-d6) δ 10.41 (s, 1H), 7.94 (d,
1H, J ) 8.4 Hz), 7.86 (d, 1H, J ) 2.7 Hz), 7.56 (d, 1H, J ) 9.0
Hz), 7.06 (dd, 1H, J ) 6.0, 2.4 Hz), 6.82 (d, 1H, J ) 8.4 Hz), 6.66
(s, 1H), 6.14 (s, 1H), 4.63 (d, 4H, J ) 4.8 Hz), 3.47 (d, 4H, J )
1.5 Hz), 1.66-1.78 (br, 4H). 13C NMR (75 MHz, DMSO-d6) δ
182.3, 161.3, 152.3, 151.9, 147, 139.4, 134.4, 131.4, 128.2, 124.5,
119.1, 110.7, 108.7, 104.8, 97.2, 96.9, 58.9, 48.3, 30.6. MS (ESI):
Compound 10. A solution of 9 (3.0 g, 16.5 mmol) and propane
sultone (5.0 g, 41.2 mmol) in DMF (20 mL) was heated, with
stirring, to 130 °C for 3 h. The solvent was evaporated under
reduced pressure and crude residue purified by flash chromatog-
raphy initially eluting with EtOAc to remove all of the unreacted
sultone and then eluting with 1:1 MeOH:EtOAc to afford 10 (2.4
g, 48%) as a dark brown semisolid. Rf 0.2 (1:1 EtOAc:MeOH). 1H
NMR (300 MHz, CD3OD) δ 8.47 (s, 1H), 7.04-6.94 (m, 1H),
6.31-6.22 (br, 2H), 6.10 (d, 1H, J ) 9.0 Hz), 3.68-3.58 (m, 4H),
2.88 (t, 2H, J ) 6.0 Hz), 2.5 (t, 2H, J ) 6.0 Hz), 2.08-1.96 (br,
3H). 13C NMR (75 MHz, CD3OD) δ 172.8, 159.2, 149.3, 130.0,
104.6, 103.8, 99.8, 60.9, 49.7, 33.8, 28.1, 23.2. MS (ESI): 302.06
395.16 (M + H)+. IR (neat) 3358, 2918, 2847 cm-1
.
Compound 7. Sodium nitrite (1.32 g, 19.2 mmol) in water (10
mL) was added over a period of 50 min with a syringe pump at
the rate of 0.2 mL per minute to a solution of 4 (5.0 g, 17.8 mmol)
in HCl (6.0 mL, 10.0 M) and water (3 mL) at 0 °C. The mixture
was stirred for 2.5 h at 0 °C and filtered to remove solid impurities.
The filtrate was evaporated under reduced pressure to yield 7 (4.2
g, 74%) after drying. This somewhat unstable nitroso compound
was directly used for the next step. Rf 0.4 (90% EtOAc/MeOH).
1H NMR (300 MHz, CD3OD) δ 7.60 (d, 1H, J ) 9 Hz), 7.15(d,
1H, J ) 12.0 Hz), 6.34(s, 1H), 4.09(d, 4H, J ) 27.0 Hz), 3.54(s,
6H), 2.81-2.73 (m, 4H). IR (neat) 3383, 2967, 2362, 1729 cm
-1.13C NMR (75 MHz, acetone-d6) δ 182.1, 163.1, 157.5, 134.3,
104.8, 104.7, 99.7, 44.3, 39.5, 22.6. MS (ESI): 311.23 (M + H)+.
Compound 8. 1,6-Dihydroxynaphthol (1.85 g, 11.6 mmol) with
HCl (2.0 mL, 10.0 M) was added to a solution of 7 (4.0 g, 11.6
mmol) in MeOH (100 mL) all in one portion. The reaction mixture
was refluxed for 5 h. The solvent was evaporated and the residue
was purified by flash chromatography with 90% EtOAc/MeOH
eluant to afford 8 as a red solid (2.8 g, 54%). Rf 0.7 (90%EtOAc/
(M - H)-. IR (neat) 3403, 2910, 1626 cm-1
.
Compound 11. To a solution of 10 (0.5 g, 1.6 mmol) in HCl
(2.0 mL, 10.0 M) and water (1 mL) at 0 °C was added sodium
nitrite (0.13 g, 1.8 mmol) dissolved in water (2 mL) over a period
of 10 min with a syringe pump at the rate of 0.2 mL per min. The
mixture was stirred for 3 h at 0 °C and filtered to remove residual
impurities. The filtrate was evaporated under reduced pressure to
yield 11 (0.4 g, 74%). The crude product was very moisture
sensitive; it was therefore used directly without further purification
for the next step.
Compound 3. A solution of crude 11 (0.39 g, 1.1 mmol) and
1,6-dihydroxynaphthol (0.2 g, 1.2 mmol) in DMF (15 mL) was
heated to reflux for 5 h. The DMF was evaporated under reduced
pressure and the residual material was purified by flash chroma-
tography; eluting with EtOAc removed any unreacted 1,6-dihy-
droxynaphthol and then eluting with 1:1 EtOAc:MeOH afforded 3
(0.25 g, 53%) as a red solid. Rf 0.15 (1:1 EtOAc: MeOH). 1H NMR
(500 MHz, CD3OD) δ 8.07 (d, 1H, J ) 8.5 Hz), 8.00 (s, 1H), 7.65
(d, 1H, J ) 9.0 Hz), 7.10 (d, 1H, J ) 8.5 Hz), 6.97 (d, 1H, J ) 9.0
Hz), 6.76 (s, 1H), 6.24 (s, 1H), 3.83 (t, 2H, J ) 7 Hz), 3.69 (t, 2H,
J ) 8.0 Hz), 2.92-2.89 (m, 2H), 2.68 (t, 2H, J ) 7.5 Hz), 2.14 (t,
2H, J ) 9.0 Hz). 13C NMR (75 MHz, CD3OD) δ 184.2, 174.3,
161.3, 152.9, 151.6, 146.9, 139.6, 134.6, 131.2, 127.6, 125.4, 124.2,
118.1, 111.2, 108.6, 103.8, 96.8, 50.6, 49.6, 31.8, 22.8. MS (ESI):
471.06 (M - H)-.
1
MeOH). H NMR (300 MHz, DMSO-d6) δ 7.94 (d, 1H, J ) 9
Hz), 7.86 (s, 1H), 7.58 (d, 1H, J ) 9 Hz), 7.09-7.05 (m, 1H),
6.81-6.78 (m, 1H), 6.69 (s, 1H), 6.13 (s, 1H), 3.70 (t, 4H, J ) 6.0
Hz), 3.60 (s, 6H), 2.65-2.60 (t, 4H, J ) 6.0 Hz). 13C NMR (75
MHz, DMSO-d6) δ 182.4, 172.4, 161.4, 152.1, 150.9, 146.8, 140.7,
134.3, 131.4, 128.2, 124.9, 124.5, 119.3, 110.7, 108.9, 105.1, 97.7,
52.2, 46.9, 32.1. MS (ESI): 450.14 (M+). IR (neat) 3422, 2955,
1734 cm-1
.
Acknowledgment. We thank Dr. Michael J. Collins and
CEM Corporation for their support with microwave technologies
(which was used in exploratory studies) and the TAMU/LBMS-
Applications Laboratory directed by Dr. Shane Tichy for
assistance with mass spectrometry. Support for this work was
provided by The National Institutes of Health (GM72041) and
by The Robert A. Welch Foundation.
Compound 2. A solution of K2CO3 (276 mg, 2 mmol) dissolved
in 5 mL of water was added to a solution of 8 (100 mg, 0.2 mmol)
in MeOH/water (1:1). The reaction mixture was heated to 40 °C
for 36 h. The solvent was evaporated under reduced pressure; the
crude mixture was then dissolved in water (10 mL) and washed
with EtOAc (3 × 5 mL). The aqueous layer was acidified with
HCl (4-5 drops, 10.0 M) to pH 4-5. This aqueous layer was
extracted with CH2Cl2/2-propanol (3 × 5 mL, 1:1) and the organic
layer was evaporated under reduced pressure to give 2 (60 mg,
Supporting Information Available: General experimental
conditions and characterization data for compounds 1-11. This
material is available free of charge via the Internet at http://
pubs.acs.org.
1
60%) as a blue solid. H NMR (300 MHz, DMSO-d6) δ 7.95 (d,
1H, J ) 9.0 Hz), 7.88-7.87 (m, 1H), 7.62 (d, 1H, J ) 9 Hz), 7.09
(dd, 1H, J ) 8.7, 2.7 Hz), 6.82 (d, 1H, J ) 9.3 Hz), 6.69 (s, 1H),
6.16 (s, 1H), 3.76-3.65 (br, 4H), 2.58-2.51 (br, 4H). 13C (75 MHz,
DMSO-d6) δ 182.3, 173.5, 161.5, 152.2, 151.1, 146.9, 140.4, 134.3,
JO061369V
J. Org. Chem, Vol. 71, No. 20, 2006 7839