Synthesis and hepatic transport of strongly fluorescent cholephilic dipyrrinones

Article abstract of DOI:10.1021/jo0511041

A new class of highly fluorescent (φ_F 0.3-0.8) low molecular weight water-soluble cholephilic compounds has been synthesized in two steps from dipyrrinones. The dipyrrinone nitrogens are first bridged by reaction with 1,1′-carbonyldiimidazole t

Full text of DOI:10.1021/jo0511041

Synthesis and Hepatic Transport of Strongly Fluorescent
Cholephilic Dipyrrinones
Zachary R. Woydziak,† Stefan E. Boiadjiev,† Wilma S. Norona,‡ Antony F. McDonagh,‡ and
David A. Lightner*,†
Department of Chemistry, University of Nevada, Reno, Nevada 89557, and Division of Gastroenterology
and the Liver Center, University of California, San Francisco, California 94143-0538
lightner@scs.unr.edu
Received June 1, 2005
A new class of highly fluorescent (φF 0.3-0.8) low molecular weight water-soluble cholephilic
compounds has been synthesized in two steps from dipyrrinones. The dipyrrinone nitrogens are
first bridged by reaction with 1,1′-carbonyldiimidazole to form an N,N′-carbonyldipyrrinone (3H,5H-
dipyrrolo[1,2-c:2′,1′-f]pyrimidine-3,5-dione) nucleus, and a sulfonic acid group is then introduced
at C(8) by reaction with concd H2SO4. The resulting sulfonated N,N′-carbonyl-bridged dipyrrinones
(“sulfoglows”) are isolated as their sodium salts. When the alkyl substituents of the lactam ring
are lengthened from ethyl to decyl, sulfoglows become increasingly lipophilic while maintaining
water solubility. Low molecular weight sulfoglows were rapidly excreted intact in both bile and
urine after intravenous infusion into rats, but higher molecular weight sulfoglows were excreted
more selectively in bile. Hepatobiliary excretion of sulfoglows was partially, but not completely,
blocked in mutant rats deficient in the multidrug-resistance associated transport protein Mrp2
(ABCC2). These observations point to the feasibility of developing simple sulfoglows with clinical
diagnostic potential that are normally excreted in bile but appear in urine when hepatic elimination
is impaired by cholestatic liver disease.
Introduction
into bile) in normal human metabolism, except following
enzymic conjugation with glucuronic acid (Figure 1B).1,4
Accumulation of bilirubin and its glucuronides leading
to jaundice is a well-known sign of liver disease.1 In
contrast to bilirubin, xanthobilirubic acid5 (Figure 1C),
a polar, but water-insoluble synthetic analogue for one-
half of bilirubin, is readily excreted intact in bile without
the need for glucuronidation.6 Both xanthobilirubic acid
and bilirubin are essentially nonfluorescent at room
temperature, e.g., with fluorescence quantum yields φF
<10-3 because of rapid Z f E isomerization in the excited
states.2,7 In earlier work, we demonstrated a dramatic
Bilirubin (Figure 1A), the lipophilic yellow pigment of
neonatal jaundice1 is comprised of two Z-dipyrrinone
chromophores.2 In the absence of light-promoted Z f E
photoisomerization,3,4 it is unexcretable (across the liver
* To whom correspondence should be addressed.
† University of Nevada, Reno.
‡ University of California, San Francisco.
(1) Chowdhury, J. R.; Wolkoff, A. W.; Chowdhury, N. R.; Arias, I.
M. Hereditary jaundice and disorders of bilirubin metabolism. In The
Metabolic and Molecular Bases of Inherited Disease; Scriver, C. R.,;
Beaudet, A. L., Sly, W. S., Valle, D., Eds.; McGraw-Hill, Inc.: New
York, 2001; Vol. II, pp 3063-3101.
(2) (a) Falk, H. The Chemistry of Linear Oligopyrroles and Bile
Pigments; Springer-Verlag: Wien, 1989. (b) Lightner, D. A. Structure,
photochemistry and organic chemistry of bilirubin. In Bilirubin;
Heirwegh, K. P. M., Brown, S. B., Eds.; CRC Press: Boca Raton, FL,
1982; Vol. 1, pp 1-58.
(5) (a) Grunewald, J. O.; Cullen, R.; Bredfeldt, J.; Strope, E. R. Org.
Prep. Proced. Int. 1975, 7, 103-110. (b) Lightner, D. A.; Ma, J. S.;
Adams, T. C.; Franklin, R. W.; Landen, G. L. J. Heterocycl. Chem. 1984,
21, 139-144. (c) Shrout, D. P.; Lightner, D. A. Synthesis 1990, 1062-
1065.
(6) McDonagh, A. F.; Lightner, D. A. The importance of molecular
structure in bilirubin metabolism and excretion. In Hepatic Metabolism
and Disposition of Endo and Xenobiotics; Bock, K. W., Gerok, W.,
Matern, S., Eds.; Falk Symposium No. 57; Kluwer: Dordrecht, The
Netherlands, 1991; Chapter 5, pp 47-59.
(3) McDonagh, A. F.; Palma, L. A.; Trull, F. R.; Lightner, D. A. J.
Am. Chem. Soc. 1982, 104, 6865-6867.
(4) (a) McDonagh, A. F.; Lightner, D. A. Semin. Liver Dis. 1988, 8,
272-283. (b) McDonagh, A. F.; Lightner, D. A. Pediatrics 1985, 75,
443-455.
10.1021/jo0511041 CCC: $30.25 © 2005 American Chemical Society
Published on Web 08/23/2005
J. Org. Chem. 2005, 70, 8417-8423
8417

Woydziak et al.
might be similarly excreted, we synthesized the first
“sulfoglow” (1a),7c a xanthoglow analogue with the C(8)
propionic acid replaced by sulfonic acid. When injected
intravenously into rats it was rapidly excreted intact into
bile and urine, which became highly fluorescent.10 These
preliminary studies suggested that it might be possible
to develop highly fluorescent cholephilic (“bile-loving”)
compounds that are excreted in urine only when hepatic
elimination is impeded. Such compounds could provide
a novel method for detecting cholestatic liver disorders
by injecting a bolus dose of the fluorophore intravenously
and examining urine visually or instrumentally for
fluorescence. To test this hypothesis, we required more
lipophilic analogues of 1a that would normally be ex-
creted preferentially and nearly exclusively by the hepa-
tobiliary route but that would be excreted in urine when
normal hepatic excretion is impaired (cholestasis). In the
following text, we describe the syntheses, spectroscopic
properties, and metabolic disposition of cholephilic sul-
foglow sodium salts 1b-e with alkyl groups of varying
lengths on the lactam ring to mitigate the intrinsic high
water solubility of the sulfonate group and modulate the
balance of hepatic vs renal excretion. The synthetic study
is the first part of a more comprehensive investigation
of cholephilic fluorescent pharmacophores for detecting
cholestatic disorders, particularly in the newborn.
Results and Discussion
Syntheses. The syntheses of 1a-e are diagrammed
in Figure 2A. The key starting materials are dipyrrinones
3a-e, which are readily prepared by base-catalyzed
condensation of 3,5-dimethylpyrrole-2-aldehyde (5)11 with
pyrrolinones 4a-e. Three of the latter (4a,12 4b,13 and
4c13) were available from Barton-Zard pyrrole synthe-
ses14 followed by appropriate oxidation steps, as previ-
ously published. The syntheses of 4d and 4e followed
closely the method of synthesis of pyrroles with long alkyl
chains,15 followed by oxidation. Thus, as outlined in
Figure 2B, 1-octanal or 1-undecylic aldehyde and nitro-
ethane were condensed to afford the â-hydroxynitro
product (10d or 10e) in nearly quantitative yield, which
(after acetylation to 9d or 9e) was reacted with p-
toluenesulfonylmethyl isocyanide (TosMIC)16 in the pres-
ence of tetramethylguanidine to afford tosylpyrrole 8d
FIGURE 1. (A) Bilirubin and (B) one of its two monoglucu-
ronides. (C) Xanthobilirubic acid, a dipyrrinone model for
bilirubin, and its sulfonic acid analogue, xanthosulfonic acid.
(D) A highly fluorescent (φF 0.80, cyclohexane) N,N′-carbonyl-
bridged analogue of xanthobilirubic acid (xanthoglow).
increase of dipyrrinone fluorescence (to φF ∼1) by bridging
the two nitrogens of the dipyrrinone.7 The easiest bridge
to build, and most effective in enhancing fluorescence,
is the carbonyl, from treatment of the dipyrrinone with
1,1′-carbonyldiimidazole (CDI) in CH2Cl2. Thus, xantho-
bilirubic acid was easily converted to xanthoglow (Figure
1D), with φF ∼0.80 in cyclohexane at 25 °C for λexc 410
nm and λem 473 nm.7c,8
Seeking a water-soluble analogue of xanthobilirubic
acid, we prepared xanthosulfonic acid (Figure 1C),9 which
was found to be excreted intact by the liver in rats
following intravenous injection of the pigment.10 To
examine whether a water-soluble fluorescent analogue
(7) (a) Ma, J. S.; Lightner, D. A. Tetrahedron 1991, 47, 3719-3726.
(b) Hwang, K. O.; Lightner, D. A. Tetrahedron 1994, 50, 1955-1966.
(c) Brower, J. O.; Lightner, D. A. J. Org. Chem. 2002, 67, 2713-2717.
(d) Pavlopoulos, T. G.; Lightner, D. A.; Brower, J. O. Applied Optics -
LP 2003, 42, 3555-3557.
(8) Boiadjiev, S. E.; Lightner, D. A. J. Phys. Org. Chem. 2004, 17,
675-679.
(9) Boiadjiev, S. E.; Lightner, D. A. Monatsh. Chem. 2001, 132,
1201-1212.
(10) McDonagh, A. F.; Lightner, D. A.; Boiadjiev, S. E.; Brower, J.
O.; Norona, W. S. Bioorg. Biomed. Chem. Lett. 2002, 12, 2483-2486.
(11) (a) de Groot, J. A.; Gorter-LaRoy, G. M.; van Koeveringe, J. A.;
Lugtenburg, J. Org. Prep. Proced. Int. 1981, 13, 97-101. (b) Smith,
K. M.; Langry, K. C.; Minnetian, O. M. J. Org. Chem. 1984, 49, 4602-
4609.
(12) (a) Kinoshita, H.; Hayashi, Y.; Murata, Y.; Inomata, K. Chem.
Lett. 1993, 1437-1440. (b) Bobal, P.; Lightner, D. A. J. Heterocycl.
Chem. 2001, 30, 527-530.
(13) Brower, J. O.; Lightner, D. A.; McDonagh, A. F. Tetrahedron
2000, 56, 7869-7883.
(14) Barton, D. H. R.; Kervagoret, J.; Zard, S. Z. Tetrahedron 1990,
46, 7587-7598.
(15) Ono, N.; Maruyama, K. Bull. Chem. Soc. Jpn. 1988, 61, 4470-
4472.
8418 J. Org. Chem., Vol. 70, No. 21, 2005

Synthesis and Metabolism of Fluorescent Dipyrrinones
FIGURE 2. (A) Reaction scheme for the syntheses of dipyrrinones 3a-e from pyrrole aldehyde 5 and pyrrolinones 4a-e, conversion
of 3a-e to their N,N′-carbonyl-bridged analogues 2a-e, and sulfonation of the latter to afford sulfoglows 1a-e. (B) Synthesis
scheme for 4a-e. (C) Scrambling of 8b and conversion to 7c. Synthetic conditions: (i) KOH, CH3CH2OH; (ii) CDI, DBU in CH2-
Cl2; (iii) concd H2SO4, then Na2CO3; (iv) KF/CH3CH2OH or DBU; (v) Ac2O, pyridine; (vi) TosMIC, TMG; (vii) H2O2, or (a)
phenyltrimethylammonium tribromide (PTT) then (b) aq TFA; (viii) NaBH4; (ix) TFA; (x) PTT.
or 8e in 45% and 61% yield. The latter was converted to
tosylpyrrolinone 6d or 6e by R-bromination of the pyrrole,
followed by acid-catalyzed hydrolysis, and the tosyl group
was cleaved smoothly by reduction with NaBH4 to afford
pyrrolinone 4d or 4e.
they fluoresce intensely with a yellow-green color. They
could be sulfonated selectively at C(8) by reaction with
concentrated H2SO4 to produce the desired sulfoglows
1a-e, which, like 2a-e, are also intensely fluorescent.
The increase in lipophilicity from 1a to 1b and 1c to 1d
and 1e is very noticeable in the workup and manipulation
of the sulfonic acid sodium salts. Thus, in the aqueous
workup followed by evaporation of water, solid 1a-c were
separated from inorganic salts by washing into absolute
ethanol. Pigments 1b and 1c were more soluble than 1a
in ethanol and could be further purified by radial
chromatography. In contrast, 1d and 1e could be ex-
tracted directly into CHCl3 from aqueous solution. Com-
pared with 1a-c,d,e are more amenable to chromatog-
raphy and crystallization.
Barton-Zard-type syntheses13-15 of tosylpyrroles
8a,b,d,e proceeded smoothly from inexpensive starting
materials; nitroethane and propionaldehyde (to give 8a),
valeraldehyde (to 8b), caprylaldehyde (to 8d), or 1-un-
decylic aldehyde (to 8e), as outlined in Figure 2B. A
similar route to 8c required more expensive nitropentane,
Dipyrrinone 3a was known from earlier studies.17
Dipyrrinones 3b-e are new and were prepared in 48,
44, 42, and 68% yields, respectively, by condensing
pyrrolinones 4b-e with pyrrole aldehyde 511 in the
presence of ethanolic KOH. Dipyrrinones 3a-e are bright
yellow nonfluorescent pigments in organic solvents at
room temperature. They were converted in excellent yield
to their N,N′-carbonyl-bridged analogues (2a-e) by reac-
tion with CDI in CH2Cl2 in the presence of DBU. The
bridged dipyrrinones (2a-e) are much more soluble in
organic solvents than their dipyrrinone precursors, and
(16) (a) Chen, Q.; Huggins, M. T.; Lightner, D. A.; Norona, W.;
McDonagh, A. F. J. Am. Chem. Soc. 1999, 121, 9253-9264. (b)
Hoogenboom, B. E.; Oldenziel, O. H.; van Leusen, A. M. Organic
Syntheses; Wiley: New York, 1988; Collect. Vol. VI, pp 987-990.
(17) Falk, H., Leodolter, A.; Schade, G. Monatsh. Chem. 1978, 109,
183-192.
J. Org. Chem, Vol. 70, No. 21, 2005 8419

Woydziak et al.
TABLE 1. Fluorescencea of N,N′-Carbonyl-Bridged
Dipyrrinones 1 and 2 at ∼10-6 M Concentration
φF ) 0.90 ( 0.02,19 were typically very large (φF 0.5-0.8)
in organic solvents, but in water they were approximately
halved (φF ∼0.3). The strong fluorescence is consistent
with radiative de-excitation being the dominant relax-
ation pathway for return to the ground state because
nonradiative pathways cannot be accessed, e.g., photoi-
somerization from 4Z to 4E. The small values of φF in
nonpolar solvents may be caused by dimer formation in
these rather polar derivatives of N,N′-carbonyl dipyr-
rinones, which exhibited far greater solubility in all
solvents tested and very high φF values (>0.65) in
benzene and in cyclohexane than the analogous xantho-
glows with carboxylic side chains.
Metabolism. The biliary excretion of small (∼0.25 mg)
intravenous bolus doses of 1a-d was studied in normal
(Sprague-Dawley) male rats and in homozygous male
TR- and Gunn rats. TR- rats are a mutant strain that
lacks the canalicular membrane transporter Mrp2
(ABCC2),20,21 which is required for efficient excretion of
bilirubin glucuronides in bile. Biliary excretion of biliru-
bin glucuronides and other endogenous cholephiles is
markedly impaired in these animals compared to normal
rats. Gunn rats are a mutant strain that is deficient in
the UGT1A family of glucuronosyl transferases, including
UGT1A1, the specific isozyme that catalyzes bilirubin
glucuronidation.22 Their bile is devoid of bilirubin glu-
curonides, and we used them for these studies to simplify
the appearance and analysis of HPLC chromatograms
of bile measured with diode-array detection in the 400-
450 nm range. In terms of Mrp2 and canalicular trans-
port they are not significantly different from normal rats.
Biliary excretion of each compound was studied in
detail in four Mrp2-normal rats (including at least one
Gunn and one Sprague-Dawley rat) and at least two TR-
rats. Urine was collected for HPLC analysis during the
experiments at irregular intervals whenever the animal
spontaneously micturated and in some animals by aspi-
ration of the urinary bladder at the end of the experi-
ment.
C6H6
CHCl3
CH3OH (CH3)2SO
H2O
bridged
dipyrrinone φF λem φF λem φF λem φF λem φF λem
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
0.54 487 0.48 494 0.30 517 0.51 502 0.30 529
0.67 503 0.43 489 0.41 516 0.67 503 0.29 528
0.81 504 0.55 493 0.48 515 0.81 504 0.27 523
0.80 502 0.47 493 0.46 515 0.80 502 0.26 528
0.50 482 0.49 492 0.45 517 0.74 503 0.24 527
0.82 502 0.87 491 0.46 502 0.82 503
0.90 502 0.91 491 0.55 524 0.90 502
0.87 502 0.90 491 0.50 522 0.87 502
0.87 502 0.86 489 0.49 524 0.87 502
0.74 472 0.66 490 0.36 523 0.67 501
b
b
b
b
b
a λexc ) excitation wavelength, varying from 409 to 420 nm; λem
) emission wavelength in nm; φF ) fluorescence quantum yield.
b Insoluble.
plus acetaldehyde.13 A less expensive route was suggested
in the work of Kohori et al.:18 that tosylpyrrole 8b might
be equilibrated to a mixture of 8b and 8c, catalyzed by
TFA (Figure 2C). In our hands, such isomerization gave
a 90% recovery of isomerized tosylpyrroles in a 4:1 ratio
favoring 8c. The reaction darkens rapidly, producing
byproducts, including a dark side product that is not
easily removed by chromatography. An oily reaction
mixture of crude 8b + 8c was clarified by repeated
filtration chromatography and then brominated with
N,N,N-trimethylphenylammonium tribromide (PTT) at
the free R-site of the tosylpyrroles. The resulting bromo-
tosylpyrroles (7b + 7c) were crystalline, and pure 7c was
obtained by fractional crystallization in 28% overall yield
from pure 8b. Treatment of 7c with TFA-H2O afforded
the tosylpyrrolinone 6c in 39% yield, which gave 4c, as
previously described.13 Unfortunately, this isomerization
procedure, when applied to 8d did not lead to crystalline
products at the various reaction stages and so the exo
analogue of 1d was not prepared, nor was the exo
analogue of 1e.
Structures and Spectroscopy. The structures of
1a-e follow logically from their synthetic precursors:
2a-e and 3a-e, and from characterization by NMR.
Especially noticeable in converting 3a-e into 2a-e is
the disappearance of the lactam and pyrrole NH signals
Figure 3 shows HPLC chromatograms of bile from a
Gunn rat, a Sprague-Dawley rat, and a TR- rat before
and after intravenous injection of a bolus dose of 1b,
which has a butyl substituent at C(3). The chromato-
grams show clearly that the compound is taken up
rapidly by the liver and excreted in bile predominantly
in unchanged form. Compounds 1a and 1c-e behaved
similarly. Metabolites of 1a-c were not observed in bile
or urine, but very minor amounts of more polar metabo-
lites of the heptyl- and decyl-substituted analogues (1d
and 1e) were detected. These were not identified, but
were clearly not UGT1A-formed glucuronides since they
were produced in both Gunn and Sprague-Dawley rats.
The biliary excretion profile for 1c, with an exo n-butyl
substituent at C(2), in Gunn/Sprague-Dawley rats,
1
from 2a-e in the H NMR, and the appearance in the
13C NMR of a characteristic new signal at ∼143 ppm from
the new N,N′-imide carbonyl.7c,8 But the most striking
visible manifestation of the conversion of 3 f 2 is the
emergence of strong fluorescence during the course of the
reactionsan event that characteristically signals resis-
tance to isomerization about the dipyrrinone C(4)-C(5)
double bond.
As observed previously for N,N′-carbonyl-bridged syn-
dipyrrinones,7c,8 sulfonated analogues 1a-e gave pro-
nounced hypochromicity and a bathochromically shifted
λmax of the long-wavelength UV-vis transition relative
to unbridged dipyrrinones 3, with only a small influence
due to changes in solvent type and polarity (Table S-1,
Supporting Information). Solutions of 1 were visibly
strongly fluorescent (Table 1). Excitation of the long
wavelength band (409-420 nm) produced intense fluo-
rescence between 490 and 530 nm, with a large Stokes
shift. The relative fluorescence quantum yields at room
temperature in C6H6, CHCl3, CH3OH, DMSO, and H2O
determined8 versus 9,10-diphenylanthracene standard,
(18) Kohori, K.; Kinoshita, H.; Inomata, K. Chem. Lett. 1995, 799-
800.
(19) Eaton, D. F. Luminescence spectroscopy. In Handbook of
Organic Photochemistry; Scaiano, J., Ed.; CRC Press: Boca Raton, FL,
1989; Vol. 1, Chapter 8.
(20) Jansen, P. L.; Peters, W. H.; Lamers, W. H. Hepatology 1985,
5, 573-579.
(21) Fardel, O.; Jigorel, E.; Le Vee, M.; Payen, L. Biomed. Pharma-
cother. 2005, 59, 104-114.
(22) Clarke, D. J.; Keen, J. N.; Burchell, B. FEBS Lett. 1992, 299,
183-186.
8420 J. Org. Chem., Vol. 70, No. 21, 2005

Synthesis and Metabolism of Fluorescent Dipyrrinones
FIGURE 3. HPLC chromatograms of bile from rats before (0
min) and at indicated times (3-120 min) after intravenous
injection of 0.25 mg of 1b: (a) Gunn rat; (b) Sprague-Dawley
rat; (c) TR- rat. The inset in each panel shows a chromatogram
of the serum solution of 1b that was injected into the animal.
Peaks eluting after the main 1b peak in b are di- and
monoglucuronides of bilirubin; those eluting after the main
1b peak in c are acyl-migration isomers of bilirubin glucu-
ronides. HPLC conditions used for each panel were similar but
not identical.
FIGURE 4. Biliary excretion profiles of 1a-e in rats. Panel
a: Mean data for the excretion of 1c in three Gunn and two
Sprague-Dawley rats; error bars show standard deviation.
Panel b: Mean data for the excretion of 1c in two TR- rats.
Panel c: Comparative biliary excretion profiles of 1a, 1b, 1d,
and 1e in Gunn/Sprague-Dawley rats. Each line represents
mean data from at least four experiments. Error bars omitted
for clarity.
value of 0.6 ( 0.2. In TR- rats the kinetic profiles of
biliary excretion (e.g., Figure 4b) were similar to those
in Gunn and Sprague-Dawley rats, but the fraction of
the injected dose that was excreted was reduced to a
mean value of 0.3 ( 0.1.
For compounds 1a-c, with ethyl and butyl substitu-
ents, substantial amounts of unchanged compound were
detected by HPLC in urine collected during or at the end
of the experiments. In contrast, compounds 1d and 1e,
with heptyl and decyl substituents, were undetectable
or present in only trace amounts in urine following
intravenous injection.
The in vivo experiments show that 1a-e are taken up
efficiently by the liver and excreted rapidly in bile
following intravenous injection. Their fractional excretion
in bile was greatly diminished in the Mrp2-deficient TR-
animals, but this membrane protein is clearly not es-
based on the HPLC data, is shown in Figure 4a and for
all four endo-alkyl-substituted compounds (1a, 1b, 1d,
and 1e) in Figure 4c. The biliary excretion profiles for
the four compounds were similar, with rapid appearance
of each compound in bile within minutes of injection and
complete disappearance within ∼3 h. As expected, excre-
tion of the endo-ethyl congener (1a) was fastest and
excretion of the endo-n-decyl analogue (1e) slowest, with
the endo-n-butyl (1b) and endo-n-heptyl (1d) derivatives
intermediate. However, counterintuitively, the endo-n-
heptyl compound (1d) appeared to be excreted somewhat
faster than the endo-n-butyl derivative (1b). Excretion
of the latter was slightly slower than its exo-substituted
counterpart (1c). The fraction of the injected dose ex-
creted in bile in 4 h in Gunn and Sprague-Dawley rats
was similar for all five compounds studied with a mean
J. Org. Chem, Vol. 70, No. 21, 2005 8421

Woydziak et al.
solution of 10% TFA in CH2Cl2 (v/v). The resulting solution
was stirred 24 h before being diluted with an additional 100
mL of CH2Cl2, and then it was washed with 4 × 200 mL of
water, 1 × 200 mL of saturated aqueous Na2CO3, and 1 × 200
mL of brine solution. The dark organic layer was then dried
over anhyd Na2SO4, and the solvent was removed by roto-
evaporation to give a crude deep-blue oil. The oil (8b + 8c)
was rectified by repeatedly passing it through a small column
of TLC-grade silica under reduced pressure (using CH2Cl2 as
an eluent). After purification, a light yellow oil (3.63 g, 12.3
mmol) was obtained and taken up in 32 mL of CH2Cl2. The
stirred solution was chilled to 0 °C, and then a solution of 6.93
g (18.5 mmol, 1.5 equiv) of phenyl-N,N,N-trimethylammonium
tribromide (PTT) in 60 mL of CH2Cl2 was added dropwise. The
resulting deep brown solution was stirred at 0 °C for 2 h before
being brought up to a total volume of 100 mL by adding CH2-
Cl2. It was then washed with Na2S2O3 solution (2 × 100 mL),
water (1 × 100 mL), and brine solution (1 × 100 mL). The
organic layer was dried over anhyd Na2SO4, and after removal
of the solvent the resulting dark brown oil was purified by
filtration through TLC-grade silica. The purified oil (7b + 7c)
crystallized with the addition of hexane to give grayish crystals
that could be recrystallized from CH2Cl2-hexane to give 1.75
g (4.73 mmol) of pure bromopyrrole (7c) (28%): crystals; mp
sential for their biliary excretion since substantial biliary
excretion was observed in TR-rats. In this respect, they
behave somewhat differently from xanthosulfonic acid10
(Figure 1C) and the ditaurine conjugate of bilirubin,23
which are excreted almost as efficiently in TR-rats as in
normal rats or Gunn rats and do not require Mrp2 for
efficient efflux from liver to bile. The Mrp2-independent
pathway by which these sulfonated pigments are secreted
into bile is not known; possibilities are active transport
by Bsep (the bile salt export pump) or Bcrp1 (breast-
cancer resistance protein 1) which are present in the
canalicular membrane of the liver.24 The mechanism of
uptake of these compounds into the liver, and the identity
of any transport proteins involved, is also unknown.
On the basis of the limited number of in vivo experi-
ments reported here, increasing the length of the endo-
alkyl substituent from ethyl to n-decyl did not markedly
increase the fraction of the dose eliminated through the
liver into bile. However, from HPLC analysis of urine
samples, renal excretion of the n-heptyl and n-decyl
compounds (1d and 1e) was negligibly low compared to
that of the ethyl- and butyl-substituted analogues. In
preliminary experiments, we have observed that renal
excretion of the decyl derivative 1e remains barely
detectable in rats with acute experimental cholestasis
caused by ligation of the common bile duct, whereas, in
contrast, renal excretion of the heptyl sulfoglow 1d, and
more polar fluorescent metabolites, was readily detect-
able under the same conditions. Thus, 1d appears to be
on the border between cholephilic sulfoglows that un-
dergo some renal excretion and those that do not, and in
its behavior close to the desired goal of a fluorescent probe
that shows renal elimination only when hepatic elimina-
tion is blocked.
1
174-176 °C (lit.13 mp 177-178 °C); H NMR (CDCl3) δ 0.89
(3H, t, J ) 7.3 Hz), 1.26-1.50 (4H, m), 2.17 (3H, s), 2.30 (2H,
t, J ) 7.3 Hz), 2.41 (3H, s), 7.30 (2H, d, J ) 8.4 Hz), 7.76 (2H,
d, J ) 8.4 Hz), 8.78 (1H, br s) ppm; 13C NMR (CDCl3) δ 9.9,
14.0, 21.7, 22.6, 24.7, 32.1, 104.9, 125.6, 126.1, 126.9, 130.0,
140.0, 144.0 ppm. Bromopyrrole 7c was converted to tosylpyr-
rolinone 6c and pyrrolinone 4c as reported previously.13
General Procedure for Condensation to Dipyrrinone.
A solution of 2.0 mmol of pyrrolinone 4a-e and 2.5 mmol of
3,5-dimethyl-2-formyl-1H-pyrrole (5)11 in 8 mL of ethanol and
2.5 mL of 4 M KOH was heated at reflux for 1-2 days. The
mixture was cooled and poured into 200 mL of ice-water. The
precipitated yellow product was collected by filtration, washed
with water (3 × 50 mL), and dried under vacuum (P2O5) to
give crude dipyrrinones 3. Alternatively, 3d,e were extracted
into CH2Cl2. The extracts were washed with H2O, and after
removal of solvent, the residue was purified by radial chro-
matography. The products 3a-e were recrystallized from CH3-
OH or CH2Cl2-hexane to give pure dipyrrinones.
3-n-Butyl-2,7,9-trimethyl-(10H)-dipyrrin-1-one (3b). The
general procedure gave the desired dipyrrinone from 0.25 g
(1.63 mmol) of 4-n-butyl-3-methylpyrrolin-2-one (4b) in 48%
yield (0.20 g, 0.78 mmol): mp 140-141 °C; IR (thin film) ν
3349, 2929, 1671, 1636, 1582, 1477, 1380, 1264 cm-1; 1H NMR
(CDCl3) δ 0.95 (3H, t, J ) 7.3 Hz), 1.39 (2H, m), 1.55 (2H, m),
1.94 (3H, s), 2.18 (3H, s), 2.45 (3H, s), 2.54 (2H, t, J ) 7.3 Hz),
5.83 (1H, s), 6.13 (1H, s), 10.48 (1H, br.s), 11.35 (1H, br s) ppm;
13C NMR (CDCl3) δ 8.6, 11.4, 13.4, 13.8, 22.5, 24.3, 32.6, 101.3,
110.1, 123.1, 123.2, 126.3, 126.3, 127.6, 134.2, 147.0, 174.1
ppm. Anal. Calcd for C16H22N2O (258.4): C, 74.38; H, 8.56; N,
10.84. Found: C, 74.02; H, 8.36; N, 10.37.
General Procedure for Inserting Bridging Carbonyl.
Dipyrrinone 3a-e (0.50 mmol) was dissolved in 35 mL of dry
CH2Cl2. To this solution was added 0.41 g (2.50 mmol, 5 equiv)
of 1,1′-carbonyldiimidazole, 0.38 g (2.50 mmol, 5 equiv) of DBU,
and 0.50 g of 4 Å molecular sieves. The solution was allowed
to reflux with magnetic stirring for 19 h before being cooled
and filtered to remove the molecular sieve debris. The filtered
solution was then washed sequentially with water (2 × 50 mL)
and brine (50 mL) and then dried over anhyd MgSO4. The
organic solvent was removed (rotovap) to give a crude brownish
oil, which was purified by radial chromatography (using 1%
MeOH in CH2Cl2 as eluent) to give bright yellow crystals of
the pure product.
Experimental Section
For general procedures, see refs 8-10 and 13. Di-n-octyl-
amine, used for preparing HPLC solvent, was obtained from
a commercial supplier. Fluorescence measurements were done
on solutions prepared as reported previously.7c,8 Fluorescence
quantum yields at 20 °C were determined as reported8 by
relating the quantum yield of the sample to that of a reference
standard, 9,10-diphenylanthracene (φF ) 0.90 ( 0.02 in
cyclohexane19). The equation used to relate these quantum
yields is given by
2
2
φs ) [(ArFsns )/(AsFrnr )] φr
where the subscript s refers to the sample and the subscript
r refers to the reference standard; φ is quantum yield, A is
the absorbance at the excitation wavelength, F is the inte-
grated emission band, and n is the index of refraction (at the
sodium D line) of the solvent containing the sample and the
reference standard.
Pyrrolinones 4a-c,12,13 aldehyde 5,11 dipyrrinone 3a,17 N,N′-
carbonyl-bridged dipyrrinone 2a,7c and its sulfonated deriva-
tive 1a7c were synthesized according to previously published
methods. See the Supporting Information for the syntheses of
compounds 1c, 2c, and 3c, 1d; 2d, 3d, 4d, 6d, 7d, 8d, 9d,
10d; and 1e, 2e, 3e, 4e, 6e, 7e, 8e, 9e, and 10e.
5-Bromo-4-n-butyl-3-methyl-2-p-toluenesulfonyl-1H-
pyrrole (7c).13 3-Butyl-4-methyl-2-p-toluenesulfonyl-1H-pyr-
role (8b) (5.00 g, 17.2 mmol) was dissolved into 100 mL of a
3-n-Butyl-2,7,9-trimethyl-N,N′-carbonyl-(10H)-dipyrrin-
1-one (2b). Using the general procedure for inserting a
bridging carbonyl, 0.25 g (1.00 mmol) of 3b gave 0.18 g (0.63
mmol, 63%) of a product (2b): mp 150-152 °C; IR (thin film)
(23) Jansen, P. L. M.; Vanklinken, J. W.; Vangelder, M.; Ottenhoff,
R.; Oude Elferink, R. P. J. Am. J. Physiol. 1993, 265, G445-G452.
(24) Chandra, P.; Brouwer, K. L. Pharm. Res. 2004, 21, 719-735.
8422 J. Org. Chem., Vol. 70, No. 21, 2005

Synthesis and Metabolism of Fluorescent Dipyrrinones
ν 2927, 1760, 1684, 1521, 1308 cm-1; 1H NMR (CDCl3) δ 0.97
(3H, t, J ) 7.3 Hz), 1.25-1.60 (4H, m), 1.96 (3H, s), 2.15 (3H,
s), 2.52 (2H, t, J ) 7.3 Hz), 2.68 (3H, s), 5.83 (1H, s), 6.12 (1H,
s) ppm; 13C NMR (CDCl3) δ 10.2, 13.5, 13.8, 15.6, 22.5, 23.3,
30.6, 97.2, 117.2, 120.8, 126.9, 131.4, 131.5, 135.0, 140.7, 143.5,
167.3 ppm. Anal. Calcd for C17H20N2O2 (284.4): C, 71.81; H,
7.09; N, 9.85. Found: C, 71.89; H, 7.09; N, 9.48.
General Procedure for Sulfonation to 1b-e. Finely
powdered solid bridged dipyrrinone 2b-e (0.40 mmol) was
placed in a flask immersed in a cooling bath at 0 °C, and 5
mL of concd H2SO4 (precooled to 0 °C) was added. The solid
slowly dissolved to give a magenta solution, which was stirred
for 2 h at 0 °C. The temperature was then lowered to -5 to
-10 °C, and the solution was slowly neutralized with saturated
aqueous Na2CO3 while introducing a stream of air in order to
reduce foaming. After dilution with water to ∼150 mL,
extraction with CH2Cl2 resulted in removal of the starting
material in the case of 1b and 1c. In the case of 1d and 1e,
the product was also extracted. The latter two, after evapora-
tion of CH2Cl2, were purified by radial chromatography. In the
case of 1b and 1c, the yellow alkaline aqueous solution was
evaporated to dryness and the pigment was removed from the
solid mixture with Na2SO4 by extraction with absolute EtOH.
After evaporation of the ethanol, the residue was purified by
radial chromatography.
Sodium 3-n-Butyl-2,7,9-trimethyl-N,N′-carbonyl-(10H)-
dipyrrin-1-one-8-sulfonate (1b). Following the general pro-
cedure, tricyclic 2b (100 mg, 0.35 mmol) was converted to 88
mg (65%) of yellow salt 1b: mp >300 °C; IR (thin film) ν 2927,
1769, 1684, 1317 cm-1; 1H NMR (DMSO-d6) δ 0.87 (3H, t, J )
7.5 Hz), 1.25-1.60 (4H, m), 1.81 (3H, s), 2.26 (3H, s), 2.58 (2H,
t, J ) 7.5 Hz), 2.77 (3H, s), 6.96 (1H, s) ppm; 13C NMR (DMSO-
d6) δ 8.3, 10.1, 13.5, 13.8, 22.0, 23.5, 31.0, 98.3, 120.0, 125.6,
125.7, 130.8, 131.5, 134.4, 142.9, 145.9, 166.9 ppm; HRMS
(FAB, 3-NBA) calcd 365.1171 (C17H21N2O5S, M + H+), found
365.1186, ∆ ) 1.5 mDa, error 4.1 ppm.
Metabolism Studies. Sprague-Dawley rats were from
local suppliers and Gunn and TR- rats from our own colonies.
For excretion studies, the femoral vein and common bile duct
of an adult male (>250 g) were cannulated under ketamine
anesthesia and a liposomal solution containing phosphatidyl-
choline (1.5 g), cholesterol (62 mg), sodium cholate (12.95 g),
and taurine (3.75 g) in 1 L of water was infused (2 mL/h)
through the femoral catheter to maintain hydration and bile
flow. The total length of the biliary cannula was 7.5 cm. The
animal was placed in a restraining cage under an infrared
heating lamp and, once bile flow and body temperature were
stable (∼30-60 min), pigment (0.25 mg), dissolved in normal
rat serum (1 mL) with the aid of a small volume (0.1 mL) of
DMSO, was infused via the femoral vein as a bolus over a
period of 20-45 s. Bile was collected in 20-µL aliquots into
micropipets from the tip of the bile duct cannula immediately
before injection of pigment, 3 min after injection, and at
frequent intervals thereafter. Samples were flash frozen
immediately in dry ice and then kept at -70 °C until analyzed
by HPLC. Collection of each bile sample took <1 min for
Sprague-Dawley and Gunn rats but often exceeded 1 min for
TR- rats because of their relatively slow bile flow rates.
Consequently, the biliary excretion curves for experiments in
TR- rats are less precise than those for the other two strains.
Bile flow rates were measured gravimetrically by periodically
collecting timed 5-min aliquots of bile into tared Pasteur tubes.
Urine samples were collected in 20-µL aliquots into micropi-
pets from the tip of the penis or by aspiration of the urinary
bladder at the end of each experiment. For HPLC, frozen bile
samples (20 µL) were mixed with 80 µL of ice-cold 0.1 M
methanolic di-n-octylamine acetate, microfuged for 30 s, and
the supernate (20 µL) was injected onto the column. Isocratic
HPLC analyses were run using a Beckman-Altex Ultrapshere-
IP 5 µm C-18 ODS column (25 × 0.46 cm) fitted with a
similarly packed precolumn (4.5 × 0.46 cm) and Hewlett-
Packard multiwavelength diode array detector. Compounds
were monitored at their absorbance maxima and peak areas
measured using HP ChemStation software. The elution solvent
was 0.1 M di-n-octylamine acetate in 5-8% aqueous methanol,
flow rate 0.75 mL/min, and column temperature 35-38 °C.
Biliary excretion curves were derived by plotting integrated
HPLC peak areas normalized to the maximum peak area. The
fraction of the injected dose excreted was estimated by
comparing the area under the biliary excretion curve (HPLC
peak area versus time), adjusted for total bile volume excreted,
with the HPLC peak area of the pigment in a 20-µL sample of
the original serum solution injected into the rat. Areas under
biliary excretion curves were determined using Un-Scan-It
software (Silk Scientific, Inc., Orem, UT). Except for the animal
surgery, metabolic procedures were done under orange or red
safelights in a darkroom.
Acknowledgment. This work was supported by the
U.S. National Institutes of Health (HD17779 and
DK26307). Z.R.W. was an R. C. Fuson Undergraduate
Research Fellow. He thanks the University of Nevada,
Reno, for an Undergraduate Research Grant (2002-
2003), the EPSCoR BRIN Undergraduate Research
Opportunities Program for a 2003 summer Research
Fellowship, and the Undergraduate NIH Biomedical
Research Fellowship Program for a 2003-2004 fellow-
ship. S.E.B. is on leave from the Institute of Organic
Chemistry, Sofia, Bulgaria. We thank Dr. Q. Chen for
the syntheses of 6d-10d. We thank also the Nebraska
Center for Mass Spectrometry for the HRMS spectra of
1a-e.
Supporting Information Available: The syntheses of
1c-3c, 1d-4d, 6d-10d, 1e-4e, and 6e-10e are described.
UV-vis spectral data from compounds 1-3 are available in
Table S-1. This material is available free of charge via the
Internet at http://pubs.acs.org.
JO0511041
J. Org. Chem, Vol. 70, No. 21, 2005 8423